A class of PRMT5-MTA inhibitors

Abstract

The present invention provides a class of compounds having PRMT5-MTA inhibitory activity. Specifically, the present invention provides a compound having PRMT5-MTA inhibitory activity represented by formula (I). The compound can be used to prepare a pharmaceutical composition for treating diseases related to PRMT5-MTA 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-MTA, 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 (MTAP WT Targeting PRMT5 with an MTA-coordinated small molecule inhibitor could preferentially target the MTA-bound state of PRMT5, which is enriched in MTAP-deficient tumor cells, while providing a superior therapeutic index 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] Ring A is a substituted or unsubstituted 5-membered heterocyclic ring or a 5-membered heteroaromatic ring;

[0011] R1 is selected from the group consisting of unsubstituted or halogenated C1-C6 alkyl;

[0012] R2 is selected from the group consisting of H, unsubstituted or halogenated C1-C6 alkyl;

[0013] R3 is selected from the group consisting of H, unsubstituted or halogenated C1-C6 alkyl;

[0014] 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.

[0015] In another preferred embodiment, the is a structure selected from the group consisting of: .

[0016] In another preferred embodiment, the R1 is a C1-C6 alkyl group.

[0017] In another preferred embodiment, the R2 is H or C1-C6 alkyl.

[0018] In another preferred embodiment, the R3 is selected from the following group: H, C1-C6 alkyl.

[0019] In another preferred embodiment, the is a structure selected from the group consisting of:

[0020] .

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

[0022] .

[0023] 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.

[0024] 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).

[0025] 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.

[0026] 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.

[0027] 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).

[0028] 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.

[0029] 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

[0030] 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.

[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 various abbreviations are shown in Table 1 below:

[0051] Table 1

[0052]

[0053] The raw materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0054] 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.

[0055] 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).

[0056] 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).

[0057] Embodiment Synthesis General Method:

[0058]

[0059] General Method: Synthesis of Intermediate B2

[0060] Synthesis route:

[0061]

[0062] Step 1: Preparation of 2-dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine

[0063] A mixture of 5-(trifluoromethyl)pyridin-2-amine (1) (30 g, 185 mmol, 1.0 eq), chlorobenzene (450 mL) and 1,1,3-trichloro-2-propanone (45 g, 277 mmol, 1.5 eq) was reacted at 135°C for 4 hours. The target product was detected by LCMS. The reaction solution was adjusted to pH 8 with sodium carbonate, extracted with ethyl acetate (500 mL * 3), the organic phases were combined and dried over magnesium sulfate, the filtrate was filtered and concentrated to dryness under reduced pressure, and purified by column chromatography (silica, 15% ethyl acetate in petroleum ether) to obtain yellow solid 2-dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 60% yield). 1 H NMR (400 MHz, DMSO- d 6) d ppm: 9.24 (s, 1H), 8.27 (s, 1H), 7.79 (d, J=9.68 Hz, 1H), 7.65 (s, 1H), 7.56 (dd, J=1.65, 9.57 Hz, 1H).

[0064] Step 2: Preparation of 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde

[0065] 2-Dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 1.0 eq), water (600 mL), calcium carbonate (33 g, 334 mmol, 3.0 eq) were heated to 100°C and reacted for 2 hours. LCMS detected the formation of the target product. Celite and ethyl acetate (600 mL) were added to the reaction solution, stirred at room temperature for 30 minutes, filtered, and extracted with ethyl acetate (600 mL*2). The combined organic phases were dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a brown solid 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (35 g), which was used directly in the next step without further purification. 1 HNMR (400 MHz, CDCl3) d ppm: 10.09-10.29 (m, 1H), 8.59 (s, 1H), 8.27 (s, 1H), 7.82 (br d, J=9.46 Hz, 1H), 7.44 (br d, J=9.02 Hz, 1H).

[0066] Step 3: Preparation of 1-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2)

[0067] To a solution of 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (4) (100 mg, 467 μmol, 1.0 eq) in DCM (2.00 mL) was added KOAc (91.7 mg, 934 μmol, 2.0 eq) and 1-methyl-1H-pyrazol-4-amine (45.4 mg, 467 μmol, 1.0 eq) at -5°C and the reaction mixture was stirred at -5°C for 1 hour. Then NaBH(OAc)3 (198 mg, 934 μmol, 2.0 eq) was added and stirred at -5°C for another 3 hours. LCMS showed complete consumption of the starting material and several new peaks were shown on LCMS. The reaction mixture was diluted with saturated Na2CO3 aqueous solution (3.00 mL) and extracted with dichloromethane (2.00 mL×4). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (ethyl acetate / methanol = 8 / 1) to give 1-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2) (80.0 mg, 271 μmol, 58.0% yield) as a yellow solid. 1 HNMR (400 MHz, CDCl3) d ppm: 8.47 (s, 1H), 7.69 (d, J = 9.5 Hz, 1H), 7.64 (s,1H), 7.35 (d, J = 1.5, 9.5 Hz, 1H), 7.30 (s, 2H), 6.97 (s, 1H), 4.39 (s, 2H), 3.81 (s, 3H); LC-MS, [MH] + 217.0.

[0068] General Method: Synthesis of Intermediate B3

[0069] Synthesis route:

[0070]

[0071] Step 1: Preparation of 1,3-dimethyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B3)

[0072] 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (10 g, 46 mmol, 1.0 eq) and 1,3-dimethylpyrazol-4-amine (6 g, 56 mmol, 1.2 eq) were dissolved in dichloromethane (150 mL), acetic acid (3 g, 56 mmol, 3 mL, 1.2 eq) was added, the reaction solution was reacted at 25°C for 1 hour, sodium triacetoxyborohydride (25 g, 117 mmol, 2.5 eq) was added, the reaction solution was reacted at 25°C for 3 hours, and the target product was detected by LCMS. The reaction solution was quenched with 200 mL of sodium bicarbonate and extracted with ethyl acetate (150 mL*2). The combined organic phases were washed with 400 mL of saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, 35% ethyl acetate:ethanol (3:1) in petroleum ether) afforded 1,3-dimethyl-N-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B3) (14 g, 45 mmol, 97% yield) as a brown solid. 1 HNMR (400 MHz, DMSO- d 6) d ppm:9.19 (s, 1H), 7.92 (s, 1H), 7.67 (d, J=9.46 Hz, 1H), 7.41 (dd, J=1.76, 9.46Hz, 1H), 6.93 (s, 1H), 4.56 (br s, 1H), 4.17 (br d, J=3.96 Hz, 2H), 3.52-3.61(m, 3H), 2.04 (s, 3H). LCMS: [MH] + 310.3, RT= 0.64 min.

[0073] General method: Synthesis of compound 1

[0074]

[0075] Step 1: Preparation of ethyl 4-amino-1H-pyrrole-2-carboxylate

[0076] Under N2, Pd / C (2.89 g, 2.72 mmol, 10% purity, 0.05 eq.) was added to a solution of 4-nitro-1H-pyrrole-2-carboxylic acid ethyl ester (a) (10.0 g, 54.3 mmol, 1.0 eq.) in MeOH (150 mL). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) for 16 hours at 25 °C. TLC (commercial hexane / ethyl acetate = 2 / 1, Rf = 0.4) showed that the starting material was completely consumed and the desired compound was detected. The filtrate was filtered and concentrated. The crude product can be used in the next step without further purification. 4-amino-1H-pyrrole-2-carboxylic acid ethyl ester (b) (8.30 g, crude product) was obtained as a black oil.

[0077] 1 H NMR (400 MHz, CDCl3) δ (ppm) 9.19 (br s, 1H), 6.49 (dt, J = 1.9,7.2 Hz, 2H), 4.29 (q, J = 7.0 Hz, 2H), 3.10 (br s, 2H), 1.34 (t, J = 7.0 Hz,3H).

[0078] Step 2: Preparation of ethyl 4-((tert-butoxycarbonyl)amino)-1H-pyrrole-2-carboxylate

[0079] (2 batches) To a solution of ethyl 4-amino-1H-pyrrole-2-carboxylate (b) (22.0 g, 143 mmol, 1.0 eq) in DCM (250 mL) was added Et3N (43.3 g, 428 mmol, 59.6 mL, 3.0 eq) and (Boc)2O (46.7 g, 214 mmol, 49.2 mL, 1.5 eq). The mixture was stirred at 25 °C for 16 hours. LCMS showed that the starting material was completely consumed and the desired compound was detected. 500 mL of H2O was added, extracted with DCM (300 mL*3), and the combined organic phases were washed with brine (500 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was triturated with DCM / commercial hexane = 1 / 5 (500 mL) to give ethyl 4-((tert-butoxycarbonyl)amino)-1H-pyrrole-2-carboxylate (c) (68.0 g, 267 mmol, yield 93.7%) as an off-white solid.

[0080] LC-MS: Rt = 0.768 min, (ESI) m / z. [M+H-56] + 199.1.

[0081] 1H NMR (400 MHz, CDCl3) δ (ppm) 8.98 (br s, 1H), 7.12-7.25 (m, 1H), 6.67 (br s, 1H), 6.33 (br s, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.49-1.56 (m,9H), 1.36 (t, J = 7.2 Hz, 3H).

[0082] Step 3: Preparation of ethyl 5-bromo-4-(tert-butoxycarbonylamino)-1H-pyrrole-2-carboxylate

[0083] NBS (21.0 g, 118 mmol, 1.0 eq) was added to a DCM (300 mL) solution of ethyl 4-(tert-butoxycarbonylamino)-1H-pyrrole-2-carboxylate (c) (30.0 g, 118 mmol, 1.0 eq). The mixture was stirred at 25 ° C for 16 hours. LC-MS showed that the starting material was completely consumed and the desired compound was detected. Filter and wash the filter with saturated NaHCO3 aqueous solution (300 mL*2). The organic phase was washed with brine (200 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was triturated with DCM / commercial hexane = 1 / 5 (200 mL) to give ethyl 5-bromo-4-(tert-butoxycarbonylamino)-1H-pyrrole-2-carboxylate (d) (35.0 g, 105 mmol, yield 89.0%) as a white solid.

[0084] LC-MS: Rt = 1.443 min, (ESI) m / z. [M-56] + 276.9.

[0085] 1 H NMR (400 MHz, CDCl3) δ (ppm) 9.38 (br s, 1H), 7.17-7.28 (m, 1H), 6.14 (br s, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.54 (s, 9H), 1.36 (t, J = 7.2 Hz,3H).

[0086] Step 4: Preparation of ethyl 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylate

[0087] To a solution of ethyl 5-bromo-4-(tert-butoxycarbonylamino)-1H-pyrrole-2-carboxylate (d) (4.0 g, 12.0 mmol, 1.0 equiv) in THF (60 mL) was added KOAc (3.53 g, 36.0 mmol, 3.0 equiv), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolan (6.10 g, 24.0 mmol, 2.0 equiv), XPhos (572 mg, 1.20 mmol, 0.1 equiv) and Pd2(dba)3 (550 mg, 600 μmol, 0.05 equiv) under N2. The mixture was stirred at 75 °C for 3 h. LCMS showed that the starting material was completely consumed and the desired compound was detected. Filter and concentrate in vacuo. The crude product was used in the next step without further purification. Ethyl 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylate (e) (11.0 g, crude) was obtained as a red solid.

[0088] LC-MS: Rt = 1.862 min, (ESI) m / z. [M+H-56] + 325.1.

[0089] Step 5: Preparation of ethyl 8-amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7-tetraene-4-carboxylate

[0090] To a solution of ethyl 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylate (e) (11.0 g, 11.9 mmol, 1.0 equiv) in dioxane (120 mL) and H2O (24 mL) were added K2CO3 (4.92 g, 35.6 mmol, 3.0 equiv), (4-cyano-2,5-dihydrofuran-3-yl) trifluoromethanesulfonate (5.19 g, 21.4 mmol, 1.8 equiv) and Pd(dppf)Cl2 (434 mg, 593 μmol, 0.05 equiv) under N2 protection. The mixture was stirred at 80 °C for 16 h. LCMS showed that the starting material was completely consumed and the desired compound was detected. Add 100 mL H2O, extract with EtOAc (100 mL*3), wash the combined organic phase with brine (100 mL), dry over anhydrous MgSO4, filter and concentrate in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® silica gel flash chromatography column, eluent 0~10% ethyl acetate / commercial hexane gradient elution @ 80 mL / min, commercial hexane / ethyl acetate, Rf = 0.23). 8-Amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7-tetraene-4-carboxylic acid ethyl ester (f) (2.4 g, crude product) was obtained as a gray solid and 4-((tert-butoxycarbonyl)amino)-5-(4-cyano-2,5-dihydrofuran-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (f-1) (1.5 g, 2.72 mmol, yield 22.9%, purity 63%) was obtained as a yellow solid.

[0091] LC-MS: Rt = 0.847 min, (ESI) m / z. [M+H] + 248.2.

[0092] LC-MS: Rt = 1.449 min, (ESI) m / z. [M+H-56] + 292.2.

[0093] Step 6: Preparation of 8-amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7-tetraene-4-carboxylic acid

[0094] To a solution of ethyl 8-amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7-tetraene-4-carboxylate (f) (0.6 g, 2.43 mmol, 1.0 eq) in THF (6 mL), MeOH (1.2 mL) and H2O (1.2 mL) was added LiOH.H2O (153 mg, 3.64 mmol, 1.5 eq). The mixture was stirred at 50 °C for 3 h. LCMS showed complete consumption of the starting material and the desired compound was detected. The mixture was concentrated in vacuo to remove the organic phase. 10 mL of H2O was added, the aqueous phase was filtered and the insoluble material was discarded. The aqueous phase was acidified to pH 5 with 2 M HCl aq, filtered and the grey solid was collected. The crude product was used in the next step without further purification. 8-Amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7-tetraene-4-carboxylic acid (g) was obtained as a gray solid (180 mg, 772 μmol, yield 31.8%, purity 94%).

[0095] LC-MS: Rt = 0.487 min, (ESI) m / z. [M+H] + 220.2.

[0096] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 11.55 (br s, 1H), 6.71 (s, 1H), 5.73 (br s, 2H), 5.14 (br s, 2H), 4.89-4.95 (m, 2H).

[0097] Step 7: Preparation of 5-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-6,8-dihydro-1H-furo[3,4-d]pyrrolo[3,2-b]pyridine-2-carboxamide (Compound 1)

[0098] To a solution of 8-amino-11-oxa-3,7-diazatricyclo[7.3.0.02,6]dodeca-1,4,6,8-tetraene-4-carboxylic acid (g) (70 mg, 319 μmol, 1.0 eq) in DMF (1.4 mL) were added DIEA (124 mg, 958 μmol, 167 μL, 3.0 eq), 1,3-dimethyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (98.8 mg, 319 μmol, 1.0 eq) and HATU (146 mg, 383 μmol, 1.2 eq). The mixture was stirred at 25° C. for 16 hours. LCMS showed that the desired compound was detected. After filtration, the mixture was purified by preparative HPLC (basic conditions) (column: WePure Biotech XP tC18 250*70*10um; mobile phase: [H2O (0.05% NH3H2O+10mM NH4HCO3)-ACN]; gradient: 25%-45% B, 10.0 minutes). 5-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-6,8-dihydro-1H-furano[3,4-d]pyrrolo[3,2-b]pyridine-2-carboxamide (Compound 1) (45 mg, 86.4 μmol, yield 27.0%, purity 98%) was obtained as an off-white solid.

[0099] LC-MS: Rt = 0.958 min, (ESI) m / z. [M+H] + 511.4.

[0100] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 11.60 (br s, 1H), 9.20 (s, 1H), 7.99(s, 1H), 7.71 (br d, J = 9.7 Hz, 1H), 7.68 (s, 1H), 7.45 (dd, J =1.5, 9.5 Hz, 1H), 5.51 (s, 2H), 4.86-5.30 (m, 7H), 3.78 (s, 3H), 1.77 (s, 3H).

[0101] 19 F NMR (376 MHz, DMSO-d6) δ (ppm) -60.38 (br s, 3F).

[0102] To a solution of 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]propan-1-amine (b) (50 mg, 191 umol, 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 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 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 2) (52 mg, 103.26umol, yield 53.98%, purity 100%) was obtained as a white solid.

[0103] LC-MS: Rt = 1.712 min, (ESI) m / z . [M+H] + 504.2.

[0104] 1 H NMR (400 MHz, DMSO- d 6) d (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).

[0105] 19 F NMR (376.5 MHz, DMSO- d 6) d (ppm) -115.68, -116.40.

[0106] General method: Synthesis of compound 2

[0107]

[0108] Step 1: Preparation of ethyl 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylate

[0109] 5-Bromo-4-(tert-butoxycarbonylamino)-1H-pyrrole-2-carboxylic acid ethyl ester (a) (9.5 g, 28.5 mmol, 1 eq), (PinB)2 (14.5 g, 57 mmol, 2 eq), Pd2(dba)3 (1.31 g, 1.43 mmol, 0.05 eq), KOAc (8.40 g, 85.6 mmol, 3.00 eq) and XPhos (1.36 g, 2.85 mmol, 0.1 eq) were dissolved in THF (150 mL), degassed and purged with N2 3 times, and then the mixture was stirred at 75 ° C for 3 hours under N2 atmosphere. LC-MS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was used directly in the next step without further purification to give orange solid 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (b) (21.4 g, purity 50%).

[0110] Step 2: Preparation of ethyl 4-(tert-butoxycarbonylamino)-5-(5-cyano-1-methyl-pyrazol-4-yl)-1H-pyrrole-2-carboxylate

[0111] A mixture of 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (b) (9.5 g, 12.5 mmol, 1 eq., purity 50%), 4-bromo-2-methyl-pyrazole-3-carbonitrile (2.32 g, 12.5 mmol, 1 eq.), Pd(dppf)Cl2 (457 mg, 624 μmol, 0.05 eq.), K2CO3 (2.24 g, 16.2 mmol, 1.3 eq.) in dioxane (100 mL) and H2O (20 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 80 °C for 16 h under N2 atmosphere. LC-MS showed the reaction was complete. The reaction mixture was diluted with H2O (80mL) and extracted with EtOAc (100mL*3). The combined organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 120g SepaFlash® silica gel flash chromatography column, eluent 0~3%, MeOH / DCM@100mL / min, commercial hexane / ethyl acetate=2 / 1, Rf=0.27). 4-(tert-butoxycarbonylamino)-5-(5-cyano-1-methyl-pyrazole-4-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (c) (1.68g, 4.67mmol, yield 37.4%) was obtained as a white solid.

[0112] 1 H NMR (400 MHz, DMSO- d 6 ) d (ppm) 12.06 (br s, 1H), 8.50 (br s, 1H),7.86 (s, 1H), 6.78 (br s, 1H), 4.25 (q, J = 7.2 Hz, 2H), 4.02 (s, 3H), 1.41 (brs, 9H), 1.29 (t, J = 7.0 Hz, 3H).

[0113] LC-MS: Rt = 0.858 min, (ESI) m / z . [M-56+H] + 304.0.

[0114] Step 3: Preparation of 8-amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxylic acid methyl ester

[0115] Ethyl 4-(tert-butoxycarbonylamino)-5-(5-cyano-1-methyl-pyrazol-4-yl)-1H-pyrrole-2-carboxylate (c) (1.36 g, 3.78 mmol, 1 eq) was dissolved in HCl / MeOH (15 mL). The mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. K2CO3 (2.62 g, 18.9 mmol, 5 eq) was added to the reaction. The mixture was stirred at 50 °C for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica gel flash chromatography column, SepaFlash® silica gel flash chromatography column, eluent 0-40%, (ethyl acetate / EtOH=3 / 1) / petroleum ether@40 mL / min, commercial hexane / (ethyl acetate / EtOH=3 / 1)=1 / 2, Rf=0.31). 8-Amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxylic acid methyl ester (d) (500 mg, 2.04 mmol, yield 53.8%) was obtained as a brown solid.

[0116] 1 H NMR (400 MHz, DMSO- d 6 ) d (ppm) 12.22 (br s, 1H), 8.10 (s, 1H), 6.91(d, J = 2.1 Hz, 1H), 6.00 (s, 2H), 4.33 (s, 3H), 3.84 (s, 3H).

[0117] LC-MS: Rt = 0.560 min, (ESI) m / z . [M+H] + 246.1.

[0118] Step 4: Preparation of 8-amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7,11-pentaene-4-carboxylic acid

[0119] To a solution of 8-amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxylic acid methyl ester (d) (460 mg, 1.88 mmol, 1 eq) in MeOH (1 mL), THF (1 mL) and H2O (1 mL) was added LiOH.H2O (118. mg, 2.81 mmol, 1.5 eq). The mixture was stirred at 25°C for 16 hours. LC-MS showed that the reaction was complete. The reaction mixture was extracted with DCM (5 mL*3). The pH of the solution was adjusted to 6 by adding 1 M HCl. The mixture was filtered and dried under reduced pressure to obtain a residue. The crude product was used in the next step without further purification. 8-Amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7,11-pentaene-4-carboxylic acid (e) was obtained as a black solid (240 mg, 1.04 mmol, 55.3% yield).

[0120] 1 H NMR (400 MHz, DMSO- d 6 ) d (ppm) 12.11 (br s, 1H), 8.10 (s, 1H), 6.87(d, J = 2.1 Hz, 1H), 6.08 (br s, 2H), 4.33 (s, 3H).

[0121] Step 5: Preparation of 8-amino-N-(1,3-dimethylpyrazol-4-yl)-10-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxamide (Compound 2)

[0122] 1,3-Dimethyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-4-amine (66.8 mg, 216 μmol, 1 eq) and 8-amino-10-methyl-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxylic acid (e) (50 mg, 216 μmol, 1 eq), DIEA (83.8 mg, 648 μmol, 113 μL, 3 eq) were dissolved in DMF (1 mL), and HATU (86.3 mg, 227 μmol, 1.05 eq) was added, and the resulting mixture was stirred at 20° C. for 16 hours. The reaction was complete after LC-MS detection. The reaction solution was filtered to obtain a liquid, and the residue was purified by preparative HPLC (chromatographic column: WePure Biotech XPtC18 150×40 mm, 7 μm; mobile phase: [H2O (0.05% NH3H2O ​​+ 10 mM NH4HCO3)-ACN]; gradient: 25%-45% B, 10.0 min). 8-amino-N-(1,3-dimethylpyrazol-4-yl)-10-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]-3,7,10,11-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,11-pentaene-4-carboxamide (Compound 2) (20 mg, 38.2 μmol, yield 17.7%, purity 100%) was obtained as a white solid.

[0123] 1 H NMR (400 MHz, DMSO- d 6 ) d (ppm) 11.96 (d, J = 1.6 Hz, 1H), 9.20 (s, 1H), 8.15 (s, 1H), 8.00 (s, 1H), 7.70 (d, J = 9.5 Hz, 1H), 7.66 (s, 1H), 7.44 (dd, J =1.8, 9.5 Hz, 1H), 5.79 (s, 2H), 5.30 (br s, 1H), 4.61-5.25 (m, 2H), 4.30 (s, 3H), 3.78 (s, 3H), 1.79 (s, 3H).

[0124] 19 F NMR (376 MHz, DMSO- d 6 ) d (ppm) -60.37 (s, 1F).

[0125] LC-MS: Rt = 1.915 min, (ESI) m / z . [M+H] + 523.2; purity 100%.

[0126] General method: Synthesis of compound 4

[0127]

[0128] Step 1: Preparation of 4-(tert-Butyloxycarbonylamino)-5-(4-cyano-2-methyl-pyrazol-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester

[0129] To a solution of 4-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (a) (8.18 g, 10.7 mmol, 1.0 eq., 50% purity) and 5-bromo-1-methyl-pyrazole-4-carbonitrile (2.00 g, 10.7 mmol, 1.0 eq.) in dioxane (40 mL) and H2O (8 mL) was added K3PO4 (4.57 g, 21.5 mmol, 2 eq.), then the reaction was evacuated and backfilled with N2 three times, then additional Pd(dppf)Cl2 (393 mg, 537 μmol, 0.05 eq.) was added. The reaction was stirred at 80 °C under N2 atmosphere for 16 h. LC-MS showed that the desired product was detected. The reactants were separated, the aqueous layer was extracted with EtOAc (20 mL * 3), the combined organic layers were dried over Na2SO4, filtered and concentrated to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica gel flash chromatography column, eluent 0-20, 35% ethyl acetate / commercial hexane gradient @ 35 mL / min). 4- (tert-butyloxycarbonylamino) -5- (4-cyano-2-methyl-pyrazole-3-yl) -1H-pyrrole-2-carboxylic acid ethyl ester (b) (1.4 g, 2.73 mmol, yield 25.36%, purity 70%) was obtained as a yellow foam.

[0130] LC-MS: Rt = 0.742 min, (ESI) m / z. [M+H] + 360.1.

[0131] Step 2: Preparation of 4-amino-5-(4-cyano-2-methyl-pyrazol-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester

[0132] 4-(tert-Butyloxycarbonylamino)-5-(4-cyano-2-methyl-pyrazole-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (b) (1.4g, 2.73mmol, 1eq, 70% purity) was dissolved in 1,2-dichlorobenzene (30mL) and stirred at 130°C for 60 hours. HNMR showed that the reaction was complete. The reaction was cooled to room temperature, diluted with commercial hexane (30mL), filtered and washed with EtOAc / commercial hexane (1 / 1, 10*3mL), filtered and collected gray solids. 4-Amino-5-(4-cyano-2-methyl-pyrazole-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (c) (850mg, 3.28mmol, purity 89%) was obtained as a gray solid.

[0133] LC-MS: Rt = 0.590 min, (ESI) m / z. [M+H] + 260.6.

[0134] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 12.58 (s, 1H), 8.56 (s, 1H), 7.29 (d, J = 0.9 Hz, 1H), 4.24-4.48 (m, 7H), 1.38 (t, J = 7.2 Hz, 3H).

[0135] Step 3: Preparation of ethyl 7-amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,10-pentaene-11-carboxylate

[0136] 4-Amino-5-(4-cyano-2-methyl-pyrazol-3-yl)-1H-pyrrole-2-carboxylic acid ethyl ester (c) (850 mg, 3.28 mmol, 1 eq, 89% purity) was dissolved in NMP (10 mL) and stirred at 130 ° C for 16 hours. LC-MS showed that the reaction was complete and the desired product mass was detected. The reaction was cooled to room temperature, diluted with H2O (50 mL), extracted with EtOAc / EtOH (3 / 1, 50 mL*3), and the combined organic layers were washed with H2O (50 mL*2), dried with Na2SO4, filtered and concentrated to obtain a residue. The residue was triturated with MTBE (10 mL), filtered and dried under high vacuum. Ethyl 7-amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,10-pentaene-11-carboxylate (d) (730 mg, 2.82 mmol, yield 85.88%) was obtained as a yellow solid.

[0137] LC-MS: Rt = 2.707 min, (ESI) m / z. [M+H] + 260.0.

[0138] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 12.00 (br s, 1H), 8.21 (s, 1H), 6.90 (s, 1H), 6.38 (s, 2H), 4.32-4.39 (m, 2H), 4.31 (s, 3H), 1.85-1.96 (0.62, 2H, NMP), 1.35 (t, J = 7.1 Hz, 3H).

[0139] Step 4: Preparation of 7-amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7,10-pentaene-11-carboxylic acid

[0140] To a solution of ethyl 7-amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]twelve-1(9),2(6),4,7,10-pentaene-11-carboxylate (d) (730 mg, 2.82 mmol, 1 eq) in MeOH (5 mL), THF (5 mL) and H2O (5 mL) was added LiOH.H2O (236 mg, 5.63 mmol, 2.0 eq), and the reaction was stirred at 50°C for 16 hours. LC-MS showed that the reaction was complete. The reaction was concentrated to give a residue. The pH of the residue was adjusted to 6 with 1M HCl, filtered, the residue was triturated with CH3CN, filtered and the gray solid was collected. 7-Amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7,10-pentaene-11-carboxylic acid (e) (530 mg, 2.29 mmol, yield 81.4%) was obtained as a yellow solid.

[0141] LC-MS: Rt = 0.691 min, (ESI) m / z. [M+H] + 232.1.

[0142] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 13.59 (br s, 1H), 12.80 (s, 1H), 8.74-9.10 (m, 2H), 8.72 (s, 1H), 7.00 (d, J = 1.7 Hz, 1H), 4.28 (s, 3H).

[0143] Step 5: Preparation of 7-amino-3-methyl-N-(1-methylpyrazol-4-yl)-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,10-pentaene-11-carboxamide (Compound 4)

[0144] To a solution of 7-amino-3-methyl-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodeca-1(9),2(6),4,7,10-pentaene-11-carboxylic acid (a) (50 mg, 216 μmol, 1 eq) and 1-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-4-amine (b) (63.8 mg, 216 μmol, 1 eq) in DMF (1 mL) was added DIEA (83.9 mg, 648 μmol, 113 μL, 3 eq) and HATU (86.3 mg, 227 μmol, 1.05 eq), and the reaction was stirred at 25 °C for 16 h. LC-MS detection showed that the reaction was completed and the target product was detected. The reaction solution was filtered and purified by preparative HPLC (chromatographic column: WePureBiotechXPtC18150×40mm, 7μm; mobile phase: [H2O (0.05% NH3H2O+10mM NH4HCO3)-ACN]; gradient: 25%-45% B, 10.0min), and the target product fractions were combined and lyophilized. 7-Amino-3-methyl-N-(1-methylpyrazol-4-yl)-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]-3,4,8,12-tetraazatricyclo[7.3.0.02,6]dodecan-1(9),2(6),4,7,10-pentaene-11-carboxamide (Compound 4) (30 mg, 59.0 μmol, yield 27.28%) was obtained as an off-white solid.

[0145] LC-MS: Rt = 0.963 min, (ESI) m / z. [M+H] + 509.3.

[0146] 1H NMR (400 MHz, DMSO-d6) δ (ppm) 11.05-12.21 (m, 1H), 9.20 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.85 (br s, 1H), 7.64-7.79 (m, 1H), 7.47 (br d, J= 9.5 Hz, 2H), 6.19 (s, 2H), 5.28-5.90 (m, 1H), 5.11 (br s, 2H), 4.28 (s,3H), 3.83 (s, 3H).

[0147] 19 F NMR (376 MHz, DMSO-d6) δ (ppm) -60.40 (br s, 3F).

[0148] The following compounds were synthesized using a similar general method to that in the previous examples, and the characterization results are as follows:

[0149] Table 2

[0150]

[0151] Biological test example 1 PRMT5 in vitro inhibitory activity experiment

[0152] Experimental Materials

[0153] PRMT5 (ActiveMotif, 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 (PerkinElmer, catalog number 6007299), Echo550 (manufacturer: Labcyte, model: Echo550), 384-well Flashplate (manufacturer: PerkinElmer, model: SMP410A001PK)

[0154] Experimental methods

[0155] 1. Enzyme reaction process

[0156] (1) Configure 1x assay buffer (modified Tris Buffer).

[0157] (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.

[0158] (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.

[0159] (4) Prepare substrate solution: Add peptide and [3H]-SAM to 1x assay buffer.

[0160] (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.

[0161] (6) Add 15 μL of substrate solution to each well and incubate at room temperature for 90 min.

[0162] (7) Prepare the termination reaction solution: add pre-cooled SAM to 1x assay buffer.

[0163] (8) Add 10 µL of stop reaction solution to each well to terminate the reaction.

[0164] (9) Transfer 25 µL / well of the mixed solution to the Flashplate and incubate at room temperature for 1 hour.

[0165] (10) Wash the Flashplate three times with dH2O + 0.1% Tween-20 solution.

[0166] (11) Read the radioactivity value using Microbeta.

[0167] 2. Data Analysis

[0168] (1) Convert the original data into % inhibition according to formula 1:

[0169] Formula 1: % inhibition = (Max-Signal) / (Max-Min) *100

[0170] (2) Substitute the % inhibition data into XL-Fit formula 2 to obtain IC 50 value:

[0171] Formula 2: Y=Bottom + (Top-Bottom) / (1+(IC 50 / X)*HillSlope)

[0172] Where Y is % inhibition, X is the concentration of the compound, and the biological activity of some compounds was measured by experimental methods.

[0173] Table 3

[0174]

[0175] 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: ； I in, for or ; R1 is selected from the group consisting of: unsubstituted C1-C6 alkyl; R2 is selected from the group consisting of H, unsubstituted C1-C6 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The for .

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The R1 is methyl.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The R2 is H or methyl.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The for .

6. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The compound is selected from the following group: , , , .

7. 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.

8. The use according to claim 7, 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.

9. The use according to claim 7, 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).

10. The use according to claim 7, 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.

11. The use according to claim 7, 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.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

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