Compounds for inhibiting prmt5 and pharmaceutical compositions thereof

Abstract

The present application provides a kind of compound with methyltransferase inhibitory activity, specifically, the present application provides a kind of compound with PRMT5 inhibitory activity shown in formula I.The compound can be used to prepare the pharmaceutical composition for treating the disease related to PRMT5 activity.I.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical compounds, and more specifically, it provides a class of compounds for inhibiting PRMT5 and their use in pharmaceutical compositions. Background Technology

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

[0003] Epigenetic regulation involves heritable modifications of genetic material without altering its nucleotide sequence. Typically, epigenetic regulation is mediated by selective and reversible modifications (e.g., methylation) of DNA and proteins (e.g., histones), which 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 can lead to human diseases. PRMT5 plays a role in diseases such as proliferative disorders, metabolic disorders, and blood disorders.

[0004] PRMT5 is a known essential cellular gene. Conditional PRMT5 knockout and siRNA knockout studies have shown that PRMT5 inhibition in normal tissues is associated with a range of diseases (e.g., pancytopenia, infertility, skeletal muscle loss, and cardiac hypertrophy). Therefore, novel strategies are needed to exploit this metabolic vulnerability and preferentially target PRMT5 in MTAP-deficient tumors while preserving PRMT5 in normal tissues (MTAPWT). Targeting PRMT5 with MTA-co-located small molecule inhibitors can preferentially target the MTA-binding state of PRMT5 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 field to provide novel small molecule compounds that target PRMT5 in MTAP-deficient tumors. Summary of the Invention

[0006] The purpose of this invention is to provide a new class of small molecule compounds that target PRMT5 in MTAP-deficient tumors.

[0007] A first aspect of the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in,

[0010] Ring A is a substituted or unsubstituted 5-membered heterocyclic ring or a 5-membered heteroaromatic ring;

[0011] R1 is selected from the following group: unsubstituted or halogenated C1-C6 alkyl groups, substituted or unsubstituted -(CH2). m -Ar; and the Ar is selected from the following group: benzene ring, 5-6 membered heteroaryl ring; m is 0, 1 or 2;

[0012] R2 is selected from the following group: substituted or unsubstituted benzene rings, substituted or unsubstituted 5-6 membered heteroaromatic rings;

[0013] R3 is selected from the following group: H, unsubstituted or halogenated C1-C6 alkyl groups;

[0014] R4 is selected from the following group: H, halogens;

[0015] Unless otherwise specified, in the above formulas, substitution refers to the replacement of the hydrogen atom on the corresponding group by one or more substituents selected from the group consisting of halogens, unsubstituted or halogenated C1-C6 alkyl groups.

[0016] In another preferred embodiment, the described The structure is selected from the following group: , .

[0017] In another preferred embodiment, the Ar is selected from the group consisting of: .

[0018] In another preferred embodiment, R2 is selected from the group consisting of: substituted or unsubstituted benzene rings, substituted or unsubstituted benzene rings, etc. Replaced or not replaced .

[0019] In another preferred embodiment, R4 is H or F.

[0020] In another preferred example, m is 1.

[0021] In another preferred embodiment, the compound is selected from the group consisting of:

[0022] , , , , , , .

[0023] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising 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, excipients and / or diluents.

[0024] In a third aspect of the invention, there is provided the use of the compound as described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing diseases associated with abnormalities at the gene level or expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferases).

[0025] In another preferred embodiment, the disease is a malignant tumor or cancer, selected from the group consisting of: 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; e.g., 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, cholangiosarcoma, and 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.

[0026] A fourth aspect of the present invention provides a pharmaceutical composition comprising 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, excipients and / or diluents.

[0027] A fifth aspect of the present invention provides the use of a compound as described in any of the foregoing aspects or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing diseases associated with abnormalities at the gene level or expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferases).

[0028] In another preferred embodiment, the disease is selected from the group consisting of: ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, stomach cancer, pancreatic cancer, or bladder cancer.

[0029] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0030] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that the cell proliferation inhibition effect of PRMT5 regulator compounds is significantly enhanced and the selectivity is improved when a specific substituent (biaryl substituent) is introduced into the compounds. Based on this, the inventors completed the present invention.

[0031] the term

[0032] In this invention, the halogen is F, Cl, Br or I.

[0033] In this invention, unless otherwise specified, the terms used have their general meanings known to those skilled in the art. In this invention, unless otherwise specified, all chemical formulas are intended to cover any possible optical or geometric isomers (e.g., R-type, S-type, or racemic, or cis-trans isomers of alkenes, etc.).

[0034] In this invention, the term "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and includes, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

[0035] In this invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning, referring to a heteroaryl group containing one or more heteroatoms. Heteroatoms referred to herein include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0036] Pharmaceutical Compositions and Administration

[0037] Because 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 diseases caused by abnormal activity or expression of methyltransferases (e.g., PRMT5).

[0038] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 5-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0039] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that 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 and with the compounds of the present invention without significantly reducing the efficacy of the compounds. 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0040] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0041] 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 components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0042] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0043] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0044] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0045] In addition to the active compound, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0046] Compositions for parenteral injection may comprise 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.

[0047] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0048] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In some preferred embodiments, the compounds of this invention can be administered together with other small molecule compounds to form a PROTAC, or together with other large molecule compounds such as monoclonal antibodies to form an ADC.

[0049] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0050] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0051] The definitions of each abbreviation are shown in Table 1 below:

[0052] Table 1

[0053]

[0054] The raw materials can be obtained commercially or prepared using methods known or disclosed in the art.

[0055] Purification of intermediates and compounds is performed using conventional chemical experimental procedures such as normal-phase or reverse-phase chromatography or recrystallization. Normal-phase chromatography uses pre-packed silica gel columns or preparative thin-layer chromatography. Silica gel columns are primarily glass columns or rapid preparative chromatographs. The mobile phase for normal-phase chromatography is selected and proportioned from petroleum ether / ethyl acetate, dichloromethane / methanol, or other suitable solvents for elution. Reversed-phase preparative liquid chromatography uses a C18 column and is performed using a preparative liquid chromatograph or a rapid preparative chromatograph, with detection at 214 nM and 254 nM or using preparative liquid chromatography-mass spectrometry. Gradient elution is performed using 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 containing 0.1% trifluoroacetic acid, or other suitable solvent systems as the mobile phase.

[0056] The structures of intermediates and compounds were characterized using nuclear magnetic resonance (NMR) and LCMS. The NMR spectrometers used were a Bruker Ascend 400, Varian 400, ZKNJ BIXI-1 300 MHz, Bruker Avance III 400 MHz, or Bruker AVANCE Neo 400 MHz. The solvents used were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, or other labeled deuterated solvents. Spectral data were reported in mode: chemical shift δ (number of peak splits, coupling constant J (Hz), number of hydrogen atoms). Tetramethylsilane was used as an internal standard for the chemical shift, and its chemical shift was set to zero (δ, 0 ppm). Some abbreviations are: s (singleton), d (doublet), t (triplet), q (quartet), m (multiplex), br (broad peak).

[0057] LCMS spectra were acquired using an Agilent 1260–6120 system equipped with an SQ mass spectrometer detector, using a Waters CORTEC SC-18, 2.7 μm, 4.6 x 30 mm column (solvent A: 0.05% formic acid (FA) aqueous solution; solvent B: 0.05% FA acetonitrile (ACN) solution; change from 5% ACN to 95% ACN in 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 CSHC 18, 3.5 μm, 4.6 x 50 mm column (solvent A: 0.05% NH3 aqueous solution; solvent B: 0.05% NH3 acetonitrile solution; change from 5% ACN to 95% ACN in 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) aqueous solution; solvent B: 0.05% TFA acetonitrile solution; equilibration from 5% acetonitrile to 95% acetonitrile takes 2.0 min, hold for 0.5 min, and reequilibration to 5% CAN takes 2.7 min; flow rate: 0.5 mL / min; column temperature: 45 °C).

[0058] General synthesis method for the examples:

[0059]

[0060] Example 1: Synthesis of Compound 6

[0061]

[0062] Step 1: Preparation of 4-[1-(trifluoromethyl)pyrazol-4-yl]thiazole-2-carboxaldehyde

[0063] 4-Bromothiazol-2-carboxaldehyde (a) (135 mg, 703 μmol, 1 eq) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1-(trifluoromethyl)pyrazole (202 mg, 773 μmol, 1.1 eq) were dissolved in THF (2 mL) and H₂O (0.4 mL), followed by the addition of K₃PO₄ (448 mg, 2.11 mmol, 3 eq), degassing, and purging three times with N₂. XPhosPdG₃ (59.5 mg, 70.3 μmol, 0.1 eq) was then added, and 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 20 mL of H₂O and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with 15 mL of brine, dried over MgSO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel rapid chromatography column, elution with a gradient of 0–7.5% ethyl acetate / petroleum ether, flow rate 30 mL / min). 4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-carboxaldehyde (b) (75 mg, 300 μmol, yield 42.73%, purity 99%) was obtained as a white solid.

[0064] LC-MS: Rt = 0.840 min, (ESI) m / z. [M+H] + 247.9.

[0065] Step 2: Preparation of 2-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl-2-yl]methyl]propyl-1-amine

[0066] 4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl-2-carboxaldehyde (b) (75 mg, 303.40 μmol, 1 eq) was dissolved in MeOH (2 mL), and a solution of 2-methylpropyl-1-amine (44.4 mg, 606 μmol, 60.3 μL, 2 eq) 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 in portions over 1 minute at 5–20 °C, 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 adding 1 mL of MeOH at 20 °C, and the reaction mixture was concentrated under vacuum to obtain the residue. The residue was dissolved in DCM (15 mL), washed with NaHCO3 (10 mL), then washed with brine (10 mL), dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by rapid silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel rapid chromatography column, elution with a gradient of 0–20% ethyl acetate / petroleum ether @ 35 mL / min). 2-Methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl-2-yl]methyl]propyl-1-amine (c) (60 mg, 197 μmol, yield 64.98%), as a yellow solid.

[0067] LC-MS: Rt = 0.721 min, (ESI) m / z. [M+H] + 305.1.

[0068] Step 3: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 6)

[0069] DIEA (118 mg, 915 μmol, 159 μL, 5 eq) was added to a THF (2 mL) solution of 2-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl-2-yl]methyl]propyl-1-amine (c) (60 mg, 197 μmol, 1.08 eq) 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carboxyl chloride (64.3 mg, 183 μmol, 1 eq, 2 HCl). 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 reversed-phase HPLC (column: Boston Prime C18150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 37%-67%, 8min) to give 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazolyl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 6) (21.9 mg, 40.0 μmol, yield 21.88%, purity 100%), as a white solid.

[0070] LC-MS: Rt = 2.147 min, (ESI) m / z. [M+H] + 547.2

[0071] 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).

[0072] Example 2 Synthesis of Compound 7

[0073]

[0074] Step 1: Preparation of 4-[6-(trifluoromethyl)-3-pyridyl]thiazole-2-carboxaldehyde

[0075] 4-Bromothiazol-2-carboxaldehyde (a) (1 g, 5.21 mmol, 1 eq) and [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), followed by the addition of K3PO4 (3.32 g, 15.6 mmol, 3 eq), degassing, and purging three times with N2. XPhos Pd G3 (440 mg, 520 μmol, 0.1 eq) was then added, and 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 H2O and extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with 30 mL brine, dried over MgSO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid silica gel chromatography (ISCO®; 12+40g SepaFlash® silica gel rapid chromatography column, elution with a gradient of 0-10.2% ethyl acetate / petroleum ether, flow rate 40 mL / min). 4-[6-(trifluoromethyl)-3-pyridyl]thiazol-2-carboxaldehyde (b) (1.12 g, 4.35 mmol, yield 83.46%) was obtained as a yellow solid.

[0076] LC-MS: Rt = 0.865 min, (ESI) m / z. [M+H] + 258.9;

[0077] 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).

[0078] Step 2: Preparation of 2-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridyl]thiazolyl-2-yl]methyl]propyl-1-amine

[0079] 4-[6-(trifluoromethyl)-3-pyridyl]thiazol-2-carboxaldehyde (b) (200 mg, 774 μmol, 1 eq) was dissolved in MeOH (4 mL) and stirred at 25 °C. A solution of 2-methylpropyl-1-amine (113 mg, 1.55 mmol, 153 μL, 2 eq) was added. After addition, the mixture was stirred at 25 °C for 16 hours. NaBH4 (29.3 mg, 774 μmol, 1 eq) was added in portions over 2 minutes at 5–20 °C, 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 quenched at 20 °C by adding 2 mL of MeOH, and the reaction mixture was concentrated under vacuum to obtain the residue. The residue was dissolved in DCM (30 mL), washed with NaHCO3 (20 mL), then washed with brine (20 mL), dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by rapid silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel rapid chromatography column, elution with a gradient of 0–20% ethyl acetate / petroleum ether @ 35 mL / min). 2-Methyl-N-[[4-[6-(trifluoromethyl)-3-pyridyl]thiazolyl-2-yl]methyl]propyl-1-amine (c) (158 mg, 475 μmol, yield 61.45%, purity 95%), was given as a yellow solid.

[0080] LC-MS: Rt = 0.734 min, (ESI) m / z. [M+H] + 316.1

[0081] Step 3: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridyl]thiazolyl-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 7)

[0082] DIEA (92.1 mg, 713 μL, 124 μL, 5 eq) was added to a solution of 2-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridyl]thiazolyl-2-yl]methyl]propyl-1-amine (c) (53.9 mg, 171 μL, 1.2 eq) and 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carboxyl chloride (50.1 mg, 142 μL, 1 eq, 2 HCl) in THF (2 mL). 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 reversed-phase HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 17%-47%, 8min) to give 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridyl]thiazolyl-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 7) (30mg, 53.3umol, yield 37.35%, purity 99%), as a white solid.

[0083] LC-MS: Rt = 2.00 min, (ESI) m / z. [M+H] + 558.2.

[0084] 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).

[0085] 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).

[0086] Example 3: Synthesis of Compound 5

[0087]

[0088] Step 1: Preparation of 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazolyl-2-yl]methyl]propyl-1-amine

[0089] 4-(6-methyl-3-pyridyl)thiazolyl-2-carboxaldehyde (a) (200 mg, 979 μmol, 1 eq) and 2-methylpropyl-1-amine (145 mg, 1.98 mmol, 197 μL, 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 the mixture was 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 μL) 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 yellow solid 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazolyl-2-yl]methyl]propyl-1-amine (b) (276 mg, crude) was obtained and could be used in the next step without further purification.

[0090] LC-MS: Rt = 1.796 min, (ESI) m / z [M+H] + 262.2;

[0091] Step 2: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-(6-methyl-3-pyridyl)thiazolyl-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 5)

[0092] To a solution of 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazolyl-2-yl]methyl]propyl-1-amine (b) (50 mg, 191 μmol, 1 eq) and DIPEA (123 mg, 956 μmol, 166 μL, 5 eq) in THF (5 mL), 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carboxyl chloride (65 mg, 184 μmol, 0.97 eq, 2HCl) was added. The reaction mixture was stirred at 25–30 °C for 16 hours. LC-MS indicated that the starting material had been consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (alkaline conditions; column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 38%-58%, 9min). The fractions of the target compound were combined and lyophilized to give a white solid P1. 4-Amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-(6-methyl-3-pyridyl)thiazolyl-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (compound 5) (52 mg, 103.26 μmol, yield 53.98%, purity 100%) was given as a white solid.

[0093] LC-MS: Rt = 1.712 min, (ESI) m / z [M+H] + 504.2;

[0094] 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)

[0095] 19 F NMR (376.5 MHz, DMSO- d 6) δ (ppm) -115.68, -116.40

[0096] Example 4 Synthesis of Compound 3

[0097]

[0098] Step 1: Preparation of 1,3-dimethyl-N-((4-(6-methylpyridin-3-yl)thiazo-2-yl)methyl)-1H-pyrazole-4-amine

[0099] A mixture of 4-(6-methylpyridin-3-yl)thiazolyl-2-carboxaldehyde (a) (100 mg, 490 μmol, 1.0 eq.), 1,3-dimethyl-1H-pyrazole-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 h. NaBH(OAc)3 (312 mg, 1.47 mmol, 3.01 eq.) was added, and the mixture was stirred at 15–20 °C for 12 h. The reaction mixture was then heated at 40 °C for 16 h. LC-MS indicated that the starting material had been consumed and the desired compound was detected. The reaction mixture was diluted with H2O and CH2Cl2 and alkalized to pH 12 with 2MK2CO3 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 rapid silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel rapid chromatography column, eluent gradient of 0–30% (EtOAc: EtOH = 3:1) / 5% Et₃N in petroleum ether @ 30 mL / min; petroleum ether: (ethyl acetate: EtOH = 3:1), Rf = 0.43). The fractions of the desired compound were combined and concentrated under reduced pressure to give 1,3-dimethyl-N-((4-(6-methylpyridin-3-yl)thiazolyl-2-yl)methyl)-1H-pyrazole-4-amine (c) (117 mg, yield 79.82%) as a brown solid.

[0100] LC-MS: Rt = 0.49 min, (ESI) m / z. [M+H] + 300.1.

[0101] Step 2: Preparation of 4-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((4-(6-methylpyridin-3-yl)thiazolyl)methyl)-1,3-dihydrofurano[3,4-c]quinoline-8-carboxamide (compound 3)

[0102] 1,3-Dimethyl-N-((4-(6-methylpyridin-3-yl)thiazolyl-2-yl)methyl)-1H-pyrazole-4-amine (c) (80 mg, 267 μmol, 1.0 eq.), 4-amino-1,3-dihydrofurano[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 h. The reaction was then heated at 40 °C for another 16 h. LC-MS indicated that the starting material had been 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 min). The fractions of the desired compounds were combined and lyophilized to give 4-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((4-(6-methylpyridin-3-yl)thiazolyl)methyl)-1,3-dihydrofurano[3,4-c]quinoline-8-carboxamide (compound 3) (23 mg, yield 16.75%, purity 99.53%), as a white solid.

[0103] LC-MS: Rt = 1.270 min, (ESI) m / z. [M+H] + 512.1.

[0104] 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).

[0105] The following compounds were synthesized using a general method similar to those used in the previous examples, and the characterization results are as follows:

[0106] Table 2

[0107] .

[0108] Biological Test Example 1: PRMT5 In vitro inhibitory activity assay

[0109] Experimental materials

[0110] 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)

[0111] Experimental methods

[0112] 1. Enzyme reaction process

[0113] (1) Configure 1x assay buffer (modified Tris Buffer).

[0114] (2) Diluting the compound: The compound was dissolved in 100% DMSO and the compound solution was added to a 384-well plate using an Echo 550.

[0115] (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.

[0116] (4) Prepare substrate solution: Add peptide and [3H]-SAM to 1x assay buffer.

[0117] (5) Add 15 μL of enzyme solution to a 384-well plate and 15 μL of 1x assay buffer to the negative control well. Incubate at room temperature for 30 minutes.

[0118] (6) Add 15 μL of substrate solution to each well and incubate at room temperature for 90 minutes.

[0119] (7) Prepare the termination reaction solution: Add the pre-cooled SAM to 1x assay buffer.

[0120] (8) Add 10µL of the reaction termination solution to each well to terminate the reaction.

[0121] (9) Transfer 25 µL / well of the mixed solution to the Flashplate and incubate at room temperature for 1 hour.

[0122] (10) Clean the Flashplate three times with dH2O + 0.1% Tween-20 solution.

[0123] (11) Read the radiation value using Microbeta.

[0124] 2. Data Analysis

[0125] (1) Convert the original data to % inhibition according to Formula 1:

[0126] Formula 1: % inhibition = (Max-Signal) / (Max-Min) *100

[0127] (2) Substitute the % inhibition data into XL-Fit formula 2 to obtain the IC50 value:

[0128] Formula 2: Y=Bottom + (Top-Bottom) / (1+(IC50 / X)*HillSlope)

[0129] Where Y represents % inhibition and X represents the compound concentration. The biological activity of some compounds was determined using the above experimental methods.

[0130] Biological test case: In vitro inhibition of proliferation of HCT116 and HCT116-MTAP-KO cells.

[0131] Experimental materials

[0132] 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.

[0133] McCoy's 5A medium (Gibco, catalog number 16600082), fetal bovine serum (Gibco, catalog number 10099141C), penicillin-streptomycin antibiotics (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo assay kit (Promega, catalog number G7572), 384-well clear flat-bottom black-walled cell culture plate (Corning, catalog number 3764), micropipettes (Tecan, catalog number D300e), multi-functional microplate reader (Biotek, catalog number SynergyHTX)

[0134] Experimental methods

[0135] 1. Cell culture: The culture conditions for HCT116 and HCT116-MTAP-KO cells were McCoy's 5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin antibiotics; ensuring that they were always in the logarithmic growth phase and that the cell viability was greater than 95%.

[0136] 2. Preparation of compound concentration gradients: The test compounds were added to 384-well plates using an ultra-micro pipette, starting at 30 μM (HCT116 cells) or 3 μM (HCT116-MTAP-KO cells), and diluted 3-fold with DMSO to obtain a total of 9 concentrations, with triplet wells.

[0137] 3. Cell treatment with compounds: Add 40 μL of trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension to 384-well plates pre-stamped with the test compound, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. Incubate the cell culture plates at 37°C in a 5% CO2 incubator for 6 days.

[0138] 4. Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate with shaking at room temperature for 30 minutes. Detect the luminescence signal at 578 nm using a multi-mode microplate reader.

[0139] 5. Data Analysis:

[0140] The first column shows 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)

[0141] The examples and compound activity test results are shown in Table 3 below:

[0142] Table 3

[0143]

[0144] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound selected from the group consisting of: 、 、 。 2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of malignant tumors or cancer; wherein the malignant tumor or cancer is liver cancer.

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