PRMT5 inhibitor and application 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). The compound can be used for preparing a pharmaceutical composition for treating diseases related to PRMT5 activity. # imgabs0 #

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 or deuterated product thereof:

[0008]

[0009] in,

[0010] Ra is selected from the group consisting of:

[0011] W is O or S;

[0012] X1 and X2 are each independently selected from the following group: CR, N;

[0013] Ring A is selected from the group consisting of a substituted or unsubstituted 5-6 membered monoheterocyclic ring, a substituted or unsubstituted 7-12 membered spiroheterocyclic ring;

[0014] R8 is selected from the group consisting of:

[0015] Benzene ring, pyridine ring,

[0016] Wherein, the C ring is selected from the following group: substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered heteroaromatic ring; and the R8 is substituted by one or more substituents selected from the following group: CF3, cyclopropyl, vinyl, difluorocyclobutyl, S(O)2CH3, B(OH)2;

[0017] R2 is selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl;

[0018] R3 is selected from the group consisting of H, halogen, cyano, amino, nitro, hydroxyl, thiol, aldehyde, carboxyl, sulfonyl, substituted or unsubstituted C1-C6 alkyl;

[0019] or R2 located on two adjacent ring atoms and the ring atoms connected thereto together constitute a structure selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6-membered heteroaromatic ring, a substituted or unsubstituted 5-6-membered heterocycle (including a saturated or partially unsaturated heterocycle);

[0020] R is H, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl;

[0021] Unless otherwise specified, in the above formulae, the substitution refers to the substitution of hydrogen atoms on the corresponding groups by one or more substituents selected from the following groups: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, benzyl, SF5, C1-C 12 Alkoxycarbonyl, C1-C6 aldehyde, amino, C1-C6 amide, nitro, cyano, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C3-C8 cycloalkyl, C2-C 10 Alkenyl, C1-C6 alkoxy, C1-C6 alkyl-amino, C6-C 10 aryl, five-membered or six-membered heteroaryl, five-membered or six-membered non-aromatic heterocyclic group, -O-(C6-C 10 aryl), -O-(five-membered or six-membered heteroaryl), C1-C 12 Alkylaminocarbonyl, unsubstituted or halogenated C2-C 10Acyl, sulfonyl (-SO2-OH), phosphoryl (-PO3-OH), unsubstituted or halogenated C1-C4 alkyl-S(O)2-, unsubstituted or halogenated C1-C4 alkyl-SO-;

[0022] Unless otherwise specified, the carbocyclic ring or heterocyclic ring may be a saturated or partially unsaturated non-aromatic ring, and may be a monocyclic ring, a bridged ring, a spirocyclic ring or a fused ring; when the carbocyclic ring or heterocyclic ring is a fused ring, the carbocyclic ring or fused ring may be partially aromatic, such as an aromatic ring and a saturated ring.

[0023] In another preferred embodiment, the Ra is selected from the following group:

[0024]

[0025] Wherein, the R9 is selected from the following group: deuterium, tritium, halogen, hydroxyl, carboxyl, unsubstituted or halogenated C1-C6 alkyl.

[0026] In another preferred embodiment, the A ring is selected from the following group:

[0027]

[0028] Any hydrogen atom on the ring may be lost to form a chemical bond, or may be replaced by one or more substituents.

[0029] In another preferred embodiment, the formula I has a structure shown in the following formula IV:

[0030]

[0031] Wherein, Q is O, NH, NR8, CH2, CH2CH2, C(CH2-CH2), or a chemical bond (i.e., is a five-membered ring);

[0032] R8 is defined as above;

[0033] The R 8a and R 8b Each independently selected from the following group: H; or said R 8a and R 8b and the carbon atoms connected thereto together form a 4-7 membered carbocyclic or heterocyclic ring;

[0034] or R8 and R located on two adjacent ring atoms 8a and the ring atoms connected thereto together form a structure selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6-membered heteroaromatic ring, a substituted or unsubstituted 5-6-membered heterocyclic ring (including a saturated or partially unsaturated heterocyclic ring);

[0035] And when the R 8aand R 8b When each is independently H, the R 8a or R 8b may be optionally substituted by R8; when said R 8a and R 8b When the carbon atoms connected to it together form a 4-7 membered carbocyclic or heterocyclic ring, the R8 can be located on the carbocyclic or heterocyclic ring.

[0036] In another preferred embodiment, the formula I has a structure as shown below:

[0037]

[0038] wherein the C ring is selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring; and said R8' is one or more substituents selected from the group consisting of CF3, cyclopropyl, vinyl, difluorocyclobutyl, S(O)2CH3, B(OH)2;

[0039] Wherein Ra is selected from:

[0040]

[0041] Wherein X can be O, NR2, CHR2; wherein R2 is as defined above.

[0042] In another preferred embodiment, the formula I has a structure as shown below:

[0043] X is selected from the group consisting of O, CHR2, NR2; wherein R2 is as defined above;

[0044] X1 and X2 are each independently selected from the group consisting of CH, N, and CF;

[0045] R8' is selected from the following group:

[0046] Benzene ring, pyridine ring,

[0047] Wherein, the C ring is selected from the following group: a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring; and the R8 is substituted by one or more substituents selected from the following group: CF3, cyclopropyl, vinyl, difluorocyclobutyl, S(O)2CH3, B(OH)2.

[0048] In another preferred embodiment, R3 is selected from the following group: H, halogen, cyano, amino, nitro, hydroxyl, thiol, aldehyde, carboxyl, sulfonyl, substituted or unsubstituted C1-C6 alkyl.

[0049] In another preferred embodiment, the R3 is an unsubstituted C1-C6 alkyl.

[0050] In another preferred embodiment, the R3 is a halogenated C1-C6 alkyl group.

[0051] In another preferred embodiment, the formula I has a structure shown in the following formula V:

[0052]

[0053] In another preferred embodiment, the compound has a structure selected from the following table: In another preferred embodiment, the compound has a structure selected from the following table:

[0054]

[0055]

[0056] The absolute stereochemistry of the above compounds is arbitrarily assigned (chiral assignments are random based on the order of chiral SFC separations). Compounds with stereogenic centers where the configuration is not indicated in the depicted structure and not assigned are mixtures of enantiomers at that center.

[0057] 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.

[0058] 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).

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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

[0064] The present inventors, through extensive and in-depth research, unexpectedly discovered for the first time a class of compounds with PRMT5 regulatory effects, and completed the present invention on this basis.

[0065] the term

[0066] In the present invention, the halogen is F, Cl, Br or I.

[0067] 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.).

[0068] 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.

[0069] In the present invention, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like.

[0070] In the present invention, the term "C2-C6 alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl.

[0071] In the present invention, the term "C2-C6 alkynyl" refers to a straight or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl.

[0072] In the present invention, the term "C3-C10 cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclodecyl, etc. The terms "C3-C8 cycloalkyl", "C3-C7 cycloalkyl", and "C3-C6 cycloalkyl" have similar meanings.

[0073] In the present invention, the term "C3-C10 cycloalkenyl" refers to a cyclic alkenyl group having 3 to 10 carbon atoms in the ring, including but not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and cyclodecyl, etc. The term "C3-C7 cycloalkenyl" has a similar meaning.

[0074] In the present invention, the term "C1-C12 alkoxycarbonyl" refers to an alkoxycarbonyl group having 1 to 12 carbon atoms in the alkyl chain, including but not limited to methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl and the like.

[0075] In the present invention, the term "C1-C12 alkylaminocarbonyl" refers to an alkylaminocarbonyl group having 1 to 12 carbon atoms in the alkyl chain, including but not limited to methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, dimethylaminocarbonyl and the like.

[0076] In the present invention, the term "C5-C9 furanosyl" refers to a furanosyl group having 5 to 9 carbon atoms, wherein the 1-position of the sugar group is connected to the main chain, including but not limited to ribofuranosyl, deoxyribofuranosyl, galactofuranosyl and the like.

[0077] In the present invention, the term "C5-C9 pyranosyl" refers to a pyranosyl group having 5 to 9 carbon atoms, wherein position 1 of the saccharide group is connected to the main chain, including but not limited to pyranosyl glucopyranosyl, pyranosyl glucuronide, pyranosyl rhamnosyl, pyranosyl galactosyl, pyranosyl mannosyl, pyranosyl xylopyranosyl and the like.

[0078] In the present invention, the term "aromatic ring" or "aryl" has the same meaning, preferably "aryl" is "C6-C12 aryl" or "C6-C10 aryl". The term "C6-C12 aryl" refers to an aromatic ring group having 6 to 12 carbon atoms without heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C6-C10 aryl" has a similar meaning.

[0079] 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.

[0080] In the present invention, the term "3-12 membered heterocyclic group" refers to a saturated or unsaturated 3-12 membered ring group containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen in the ring, such as dioxolanyl, etc. The term "3-7 membered heterocyclic group" has a similar meaning.

[0081] In the present invention, the term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is a substituent described above, or a substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, and the substituent may be the same or different at each position. A cyclic substituent, such as a heterocycloalkyl, may be connected to another ring, such as a cycloalkyl, to form a spiro bicyclic system, for example, two rings having a common carbon atom. It will be appreciated by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically feasible. The substituents include, but are not limited to, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, 3- to 12-membered heterocyclic groups, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C1-8 aldehyde, C2-10 acyl, C2-10 ester, C1-C12 alkoxycarbonyl, amino, alkoxy, C1-10 sulfonyl, etc.

[0082] Pharmaceutical compositions and methods of administration

[0083] 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).

[0084] 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.

[0085] "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 ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] The definitions of each abbreviation are as follows:

[0098]

[0099]

[0100] The raw materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0101] The purification of intermediates and compounds is carried out by normal phase or reverse phase chromatography or conventional chemical experimental operations such as recrystallization. Normal phase chromatography is pre-filled with silica gel chromatographic columns or preparative thin layer chromatography. Silica gel chromatographic columns are mainly glass columns or rapid preparative chromatographs. The mobile phase of normal phase chromatography is selected and proportioned from petroleum ether / ethyl acetate, dichloromethane / methanol or other suitable solvents for elution. Reverse phase preparative liquid chromatography uses a C18 column, and is carried out using a preparative liquid chromatograph or a rapid preparative chromatograph, 214nM and 254nM or preparative liquid chromatography-mass spectrometry instrument detection, and uses water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile, water / acetonitrile containing 0.1% ammonium bicarbonate, water / acetonitrile containing 0.1% formic acid, 0.1% ammonia water / acetonitrile, 0.1% trifluoroacetic acid water / acetonitrile or other suitable solvent systems as mobile phases for gradient elution.

[0102] The 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

[0103] 300MHz or Bruker Avance III 400MHz or Bruker AVANCE Neo 400MHz. The solvents used were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol or other deuterated solvents as noted. Spectral data are reported in the mode: chemical shift δ (peak splitting number, coupling constant J (Hz), number of hydrogens). Tetramethylsilane was used as an internal 15 standard for chemical shift and its chemical shift was set to zero (δ, 0ppm). The meanings of some abbreviations are: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak).

[0104] Representative methods for liquid chromatography-mass spectrometry (LCMS) in the structural characterization of intermediates and compounds are as follows:

[0105] Method 1: Agilent LC1260 system coupled to a 6120 single quadrupole mass spectrometer

[0106] Column: Waters CORTECS C-18, 2.7 μm, 4.6*30 mm. Solvent A: 0.05% formic acid in water, solvent 20B: 0.05% formic acid in acetonitrile, 5% acetonitrile to 95% acetonitrile in one minute, hold for one minute, 2.5 minutes in total; flow rate: 1.8 mL / min; column temperature 40°C.

[0107] Column: XSelect CSH C18, 3.5μm, 4.6*50mm. Solvent A: 0.05% ammonia solution, solvent B: 0.05% ammonia in acetonitrile solution, 5% acetonitrile to 95% acetonitrile within one minute, hold for one minute, a total of 2.5 minutes; flow rate: 1.8mL / min; column temperature 40℃.

[0108] Method 2: performed on an Agilent LC / MSD 1200 system coupled to a quadrupole mass spectrometer.

[0109] Column: ODS2000 (50×4.6 mm, 5 μm) (ES (+) or (-) ionization mode), temperature 30° C.; flow rate 1.5 mL / min.

[0110] Representative SFC (supercritical fluid chromatography) chiral separation and chiral compound characterization equipment and methods:

[0111] Method 3:

[0112] Chromatographic column: DAICEL CHIRALPAK IC (250mm*30mm, 10um

[0113] Mobile phase: A:CO2 B:[MeOH(0.1%IPAM)]

[0114] Elution gradient: 40%

[0115] Method 4:

[0116] Chromatographic column: DAICEL CHIRALPAK AD (250mm*30mm, 10um);

[0117] Mobile phase: [ACN / EtOH (0.1% NH3H2O]; B%: 69%, 20 min, isocratic elution)

[0118] Method 5:

[0119] Chromatographic column: DAICEL CHIRALPAK AD (250mm*50mm, 10um);

[0120] Mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 55%, isocratic elution mode)

[0121] Representative preparative HPLC (high performance liquid chromatography) method:

[0122] Method 6:

[0123] Chromatographic column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [H2O(10mM NH4HCO3)-ACN]; B%: 25%-55%, 8.0 minutes, UV 220&254nm.

[0124] Method 7:

[0125] Chromatographic column: Waters Xbridge BEH C18 250*50mm*10μm; mobile phase: [water (NH4HCO3 10mM)-ACN]; B%: 20%-45%, 10 minutes, UV 220&254nm.

[0126] Method 8:

[0127] Chromatographic column: Welch Xtimate C18 250*70mm#10μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 20%-50%, 20 minutes, UV 220&254nm.

[0128] Embodiment Synthesis general method:

[0129]

[0130] General Method: Synthesis of Intermediate A2

[0131]

[0132] Step 1: Methyl 2,5-difluoro-4-nitrobenzoate (2)

[0133] 2,5-Difluoro-4-nitrobenzoic acid (1) (50 g, 246.18 mmol, 1 eq) was dissolved in methanol (500 ml) and then thionyl chloride (43.93 g, 369.28 mmol, 26.79 ml, 1.5 eq) was added at 0 °C. The reaction solution was reacted at 40 °C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. After dilution with 300 ml of water, it was extracted three times with 1 liter of ethyl acetate. The organic phase was washed with saturated brine (400 ml), dried over anhydrous magnesium sulfate, and the filtrate was concentrated to dryness under reduced pressure. The crude product was slurried with petroleum ether at 25 °C for 60 minutes to obtain white solid methyl 2,5-difluoro-4-nitrobenzoate (2) (103 g, 474.38 mmol, 96.35% yield). HNMR: ES23714-64-P1A, 1H NMR (400MHz, CHLOROFORM-d) δppm 7.89 (td, J=9.51, 5.63Hz, 2H) 4.00 (s, 3H). 19F NMR(376MHz,CHLOROFORM-d)δppm-

[0134] 110.27(s,1F)-121.56(m,1F)

[0135] Step 2: Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3)

[0136] Methyl 2,5-difluoro-4-nitrobenzoate (2) (80 g, 368.45 mmol, 1 eq.) and 2-methyl-1H-imidazole (36.30 g, 442.14 mmol, 1.2 eq.) were dissolved in dimethyl sulfoxide (1.2 L). The reaction solution was reacted at 50 °C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with 4 L of water and extracted with 4.5 L of ethyl acetate. The organic phase was washed with saturated brine (3 L), dried over anhydrous magnesium sulfate, and the filtrate was filtered and concentrated to dryness under reduced pressure. The crude product was slurried with methyl tert-butyl ether at 25 °C for 60 minutes (25.5 g). (2). The mother liquor was purified by column chromatography (silica, 50% tetrahydrofuran in petroleum ether) to obtain a yellow liquid, which was then slurried at 25 degrees Celsius for 60 minutes to obtain a yellow solid (8.23 grams). Yellow solid 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoic acid methyl ester (3) (25.5 grams, 91.32 mmol, 24.79% yield) 2,5-difluoro-4-nitrobenzoic acid methyl ester (2) (raw material recovery) (20.34 grams, 93.68 mmol, 25.43% yield). Yellow solid 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoic acid methyl ester (3) (8.2 grams, 29.07 mmol, 7.89% yield, 99% purity)

[0137] HNMR:ES23714-67-P1A1,1H NMR(400MHz,DMSO-d6)δppm 8.39(d,J=9.90Hz,1H)8.08-8.20(m,1H)7.23(d,J=1.32Hz,1H)6.92(s,1H)3.91(s,3H)2.12(s,3H). 19F NMR(376MHz,DMSO-d6)δppm-105.45(br s,1F)

[0138] Step 3: Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4)

[0139] Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) (13.9 g, 49.78 mmol, 1 eq) was dissolved in tetrahydrofuran (300 ml), and palladium hydroxide on carbon (2.8 g, 49.78 mmol, 20% purity, 1 eq) was added under a hydrogen atmosphere. The reaction system was replaced with hydrogen three times. The reaction solution was heated to 50 degrees Celsius under a hydrogen (1 eq) (50 psi) atmosphere for 32 hours. LCMS showed that the reaction was complete. The reaction solution was filtered on diatomaceous earth, and the filter cake was washed four times with 300 ml of ethyl acetate. The filtrate was concentrated to dryness under reduced pressure to obtain a gray solid. The crude product was directly used for the next step. 4-Amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoic acid methyl ester (4) (12 g, 48.15 mmol, 96.72% yield). 1H NMR (400 MHz, DMSO-d6) Shift 7.46 (d, J = 7.63 Hz, 1H), 7.07 (d, J = 1.38 Hz, 1H), 6.93 (d, J = 1.25 Hz, 1H), 6.59 (d, J = 13.51 Hz, 1H), 6.15 (br s, 2H), 3.68-3.77 (m, 3H), 2.01-2.12 (m, 3H). 19F NMR (376 MHz, DMSO-d6) Shift -109.31--108.47 (m, 1F).

[0140] Step 4: 7-Fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5)

[0141] Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4) (12 g, 48.15 mmol, 1 eq) was added to 1-methyl-2-pyrrolidone at 25°C, and then 1,1-carbonyldiimidazole (19.52 g, 120.37 mmol, 2.5 eq) was added. The reaction solution was heated to 115°C for 16 hours. LCMS showed that the reaction was complete. The two batches of reaction solution were combined and processed. 600 ml of ethyl acetate and 600 ml of water were added to the reaction solution and slurried at 25°C for 16 hours. The slurry was filtered under reduced pressure and the filter cake was washed with 100 ml of ethyl acetate. The solid was concentrated under reduced pressure to give a gray solid 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5) (24.1 g, 87.56 mmol, 86.60% yield). The crude product was directly used for the next reaction. 1H NMR (400 MHz, DMSO-d6) Shift 11.71 (br s, 1H), 8.40 (d, J = 6.38 Hz, 1H), 7.76 (s, 1H), 7.08 (d, J = 11.38 Hz, 1H), 3.88 (s, 3H), 2.89 (s, 3H). 19F NMR (376 MHz, DMSO-d6) Shift -111.43--110.58 (m, 1F)

[0142] Step 5: 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6)

[0143] 7-Fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5) (12.0 g, 43.60 mmol, 1.0 eq), 2,4-dimethoxybenzylamine (10.9 g, 65.19 mmol, 9.82 ml, 1.50 eq) and 1.8-diazabicyclo[5.4.0]undec-7-ene (19.92 g, 130.80 mmol, 19.72 ml, 3.0 eq) were added to acetonitrile (240 ml). Benzotriazole-1-oxo-tris(dimethylaminophosphine)hexafluorophosphine salt (25.07 g, 56.68 mmol, 1.3 eq) was added in batches at 15-20 degrees Celsius. The reaction solution was slightly exothermic, the reaction solution became homogeneous, and solid precipitated. The reaction mixture was reacted at 15-20 degrees Celsius for 16 hours under nitrogen protection. LCMS showed that the starting material was completely consumed and the target compound was detected. The reaction suspension was filtered under reduced pressure and the filter cake was washed with 100 ml of acetonitrile. The solid was collected and dried under reduced pressure to obtain an off-white solid 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) (15.3 g, 36.05 mmol, 82.68% yield). LCMS ES15882-1146-P1A: (ESI) m / z = 425.3 [M+1] +; RT = 1.721 min. 1H NMR (400 MHz, DMSO-d6) Shift 8.49(d,J=7.00Hz,1H),8.45(t,J=5.57Hz,1H),7.95(s,1H),7.23(d,J=12.51Hz,1H),7.18(d,J=8.38Hz,1H),6.58(d,J=2 .38Hz,1H),6.47(dd,J=2.38,8.38Hz,1H),4.66(d,J=5.25Hz,2H),3.88(s,3H),3.82(s,3H),3.73(s,3H),2.93(s,3H).19F NMR(376.5MHz,DMSO-d6)Shift-113.02.

[0144] Step 6: 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (7)

[0145] 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) (16.3 g, 38.40 mmol, 1.0 eq) was added to dichloromethane (50 ml), and trifluoroacetic acid (250 ml) was added. The reaction solution was heated to 50 degrees Celsius for 16 hours. LCMS showed that the starting material was completely consumed and the target compound was detected. The reaction solution was concentrated to dryness under reduced pressure to obtain a purple solid 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (7) (28.3 g, crude product). The crude product was directly used for the next step reaction. LCMS ES15882-1150-P1A: (ESI) m / z = 275.3 [M + 1] +; RT = 0.607 min

[0146] Step 7: 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2)

[0147] Methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7) (the crude product of the previous step) (28.3 g, 38.70 mmol, 1 eq) was added to tetrahydrofuran (80 ml) and methanol (80 ml). Sodium hydroxide (7.74 g, 193.48 mmol, 5 eq) was dissolved in water (80 ml) and added to the reaction solution. The reaction solution was heated to 50 degrees Celsius and reacted for 4 hours. The desired compound was detected by LCMS. After the reaction solution was cooled to 20 degrees Celsius, it was concentrated under reduced pressure to remove the organic solvent. The residue was diluted with water to methanol 10 to 1 (300 ml) and filtered on diatomaceous earth. The filter cake was repeatedly washed three times with water to methanol 10 to 1 (300 ml). All the filtrates were combined and concentrated under reduced pressure to remove methanol. The residue was adjusted to pH 5-6 with acetic acid. The obtained slurry was stirred at 15-20 degrees Celsius for 12 hours, and the obtained solid was filtered under reduced pressure and washed with water. The collected solid was freeze-dried to obtain a white solid. 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2) (9.9 g, 37.55 mmol, 97.05% yield, 98.71% purity). LCMS ES15882-1154-P1C: (ESI) m / z=261.1[M+1]+; RT=0.422 min. 1HNMR(400MHz,DMSO-d6)Shift 13.15(br s,1H),8.53(d,J=7.04Hz,1H),7.85(s,1H),7.68(s,2H),7.16(d,J=12.10Hz,1H),2.94(s,3H)

[0148] General Method: Synthesis of Intermediate B1

[0149]

[0150] Step 1: (2R)-tert-butyl 2-(2-chloroacetyl)piperidine-1-carboxylate (2)

[0151] Solution A: To a solution of N-isopropylpropan-2-amine (1.46 g, 14.3 mmol, 2.03 mL, 5.0 eq) in THF (20 mL) was added n-butyllithium (2.5 M, 5.75 mL, 5.0 eq) dropwise at 0°C -70°C, then the reaction was stirred at -70°C for 30 min, warmed to 20°C and stirred at 20°C for 30 min.

[0152] A solution of 1-(tert-butyl)2-methyl (R)-piperidine-1,2-dicarboxylate (1) (700 mg, 2.88 mmol, 1.0 eq) and chloro(iodo)methane (2.03 g, 11.5 mmol, 835 μL, 4.0 eq) was added dropwise at -70°C to solution A in THF (10 mL), and the reaction mixture was then stirred at -70°C for 30 minutes. The reaction was quenched with AcOH without monitoring (for the next post-treatment). An acetic acid solution (2.5 mL THF and 2.5 mL AcOH) was added dropwise, keeping the internal temperature of the reaction below -70°C, and stirred at -70°C for 30 minutes. Warm to room temperature, add H2O (40 mL), extract with EtOAc (30 mL×3), and the combined organic layers were dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by flash silica gel chromatography ( 20g Silica Flash Column, eluent: 0-15% ethyl acetate / petroleum ether gradient @ 40 mL / min, petroleum ether / ethyl acetate = 4 / 1, Rf = 0.61 (I2)). (2R)-tert-butyl 2-(2-chloroacetyl)piperidine-1-carboxylate (2) (516 mg, 1.97 mmol, 68.5% yield) was obtained as a yellow oil. 1 H NMR (400MHz, CDCl3) δ (ppm) 4.80-5.04 (m, 1H), 3.87-4.23 (m, 3H), 2.62-3.14 (m, 1H), 2.16 (br d,J=13.1Hz,1H),1.63-1.73(m,3H),1.40-1.47(m,12H).

[0153] Step 2: (2R)-tert-butyl 2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidine-1-carboxylate (3)

[0154] To a solution of (2R)-2-(2-chloroacetyl)piperidine-1-carboxylic acid tert-butyl ester (2) (416 mg, 1.59 mmol, 1.0 eq) in DME (8 mL) was added 5-(trifluoromethyl)pyridine-2-amine (309 mg, 1.91 mmol, 1.2 eq). The mixture was stirred at 90 ° C for 3 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove DME. The residue was diluted with NaHCO3 (30 mL) and extracted with DCM (15 mL×3). The combined organic layers were washed with brine (30 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( 20g Silica Flash Column, eluent: 0-18% ethyl acetate / petroleum ether gradient @30 mL / min, petroleum ether / ethyl acetate = 3 / 1, Rf = 0.26). (2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidine-1-carboxylic acid tert-butyl ester (3) (20 mg, 42.7 μmol, 2.69% yield, 79% purity) was obtained as a yellow oil. LC-MS: Rt = 0.82 min, (ESI) m / z. [M+H] + 370.1;

[0155] Step 3: 2-[(2R)-2-Piperidinyl]-6-(trifluoromethyl)imidazo[1,2-a]pyridine (Intermediate B1)

[0156] A mixture of tert-butyl (2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidine-1-carboxylate (3)

[0157] (32 mg, 86.6 μmol, 1.0 eq) was dissolved in HCl / dioxane (1.5 mL). The mixture was then stirred at 20 ° C for 2 hours. LC-MS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove HCl / dioxane. The crude product was used in the next step without further purification. 2-[(2R)-2-piperidinyl]-6-(trifluoromethyl)imidazo[1,2-a]pyridine (Intermediate B) (39 mg, crude, 2HCl) was obtained as a yellow oil. LC-MS: Rt = 0.64 min, (ESI) m / z. [M+H] + 270.1.

[0158] Example 1: Synthesis of Compound 1

[0159]

[0160] Step 1: (4-amino-7-fluoro-3-methyl-imidazo[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone (Compound 1)

[0161] 2-Chloro-1,3-dimethyl-4,5-dihydroimidazol-1-ium chloride (DMC) (46 mg, 272 μmol, 1.4 eq) was added to a solution of (3S)-3-[4-(trifluoromethyl)phenyl]morpholine (1) (45 mg, 194 μmol, 1.0 eq), 4-amino-7-fluoro-3-methyl-imidazole [1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3) (55 mg, 211 μmol, 1.09 eq) and DIEA (100 mg, 775 μmol, 135 μL, 3.98 eq) in NMP (1 mL). The reaction mixture was stirred at 50 ° C for 5 hours. LC-MS showed that the starting material was consumed and the desired compound was detected. The reaction mixture was purified by preparative HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 38%-58% B over 10 minutes). The solutions of the desired compound were combined and lyophilized to give (4-amino-7-fluoro-3-methyl-imidazole [1,5-a] quinoxaline-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone (Compound 1) (62 mg, 131 μmol, 67.29% yield, 100% purity) as an off-white solid. 1 HNMR(400MHz,DMSO-d6)δ(ppm)8.73-9.23(m,1H),8.09-8.50(m,1H),7.46-7.93(m,4H),7.15-7.34(m,1H),7.07(s,2H),5.78(br s,0.7H),4.88(br s,0.2H),4.21-4.62(m,1H),3.68-4.03(m,2H),3.49-3.68(m,1H),3.37-3.45(m,1H),2.95-3.32(m,1H),2.63(s,3H). 19 F NMR(376.5MHz,DMSO-d6)δ(ppm)-60.919,-118.933. LC-MS: Rt=0.990min, (ESI)m / z.[M+H] + 474.1; Chiral SFC: Rt=2.814min,ee%=99.28%

[0162] Example 2: Synthesis of Compound 2

[0163]

[0164] Step 1: (4-amino-7-fluoro-1-methyl-imidazo[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl]methanone (Compound 2)

[0165] To a solution of 4-amino-7-fluoro-1-methyl-imidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2) (50 mg, 192 μmol, 1.0 eq) and (3S)-3[4-(trifluoromethyl)phenyl]morpholine (1) (46.6 mg, 201 μmol, 1.05 eq) in NMP (1 mL) was added DIEA (124 mg, 961 μmol, 167 μL, 5 eq) and 2-chloro-1,3-dimethyl-4,5-dihydroimidazol-1-ium chloride (DMC) (48.7 mg, 288 μmol, 1.5 eq), and the reaction was stirred at 50° C. for 16 hours. LC-MS showed that the desired product was detected. The reactant was diluted with CH3CN (1 mL) and H2O (1 mL) and purified by preparative HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 35%-55% B over 10 minutes). The solutions of the desired product were combined and lyophilized to give (4-amino-7-fluoro-1-methyl-imidazole [1,5-a] quinoxaline-8-yl) -[(3S) -3- [4- (trifluoromethyl) phenyl] morpholin-4-yl]methanone (Compound 2) (65 mg, 135 μmol, 70.38% yield, 98.5% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ(ppm)7.67-8.27(m,5H),7.38-7.64(m,3H),7.24(br d,J=10.8Hz,1H),4.24-6.03(m,2H),3.49-4.09(m,3H),3.38-3.47(m,2H),2.84-3.29(m,3H). 19 F NMR(376MHz, DMSO-d6)δ(ppm)-60.91(br s,3F),-119.00(brs,1F).LC-MS:Rt=2.268min,(ESI)m / z.[M+H] + 474.0; SFC:Rt=

[0166] 1.557min,ee%=100%.

[0167] Example 3: Synthesis of Compound 3

[0168]

[0169] Step 1: [(3R)-4-amino-3-methyl-1,3-dihydrofuran[3,4-c]quinolin-8-yl]-[(2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]-1-piperidinyl]methanone (Compound 3)

[0170] To a solution of 2-[(2R)-2-piperidinyl]-6-(trifluoromethyl)imidazo[1,2-a]pyridine (Intermediate B1) (34 mg, 85.4 μmol, 1.0 eq, 2HCl), (3S)-4-amino-3-methyl-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A5b) (20.8 mg, 85.4 μmol, 1 eq) in NMP (1 mL) was added DIEA (33.1 mg, 256 μmol, 44.6 μL, 3.0 eq) and NMI (21.0 mg, 256 μmol, 20.4 μL, 3.0 eq) and the mixture was stirred for 1 hour. TCFH (28.7 mg, 102 μmol, 1.2 eq) was then added and the reaction was stirred at 20 °C for 16 hours. LC-MS showed that the reaction was complete. 1 mL of DMSO was added to the reaction mixture and purified by reverse phase HPLC (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 35%-55% B over 10 minutes). The desired product solution was combined and lyophilized to obtain [(3R)-4-amino-3-methyl-1,3-dihydrofuran [3,4-c] quinolin-8-yl] -[(2R)-2-[6-(trifluoromethyl) imidazo [1,2-a] pyridin-2-yl] -1-piperidinyl] ketone (Compound 3) (5 mg, 10.0 μmol, 11.8% yield, 100% purity) as a white solid. 1 HNMR(400MHz,DMSO-d6)δ(ppm)9.17(s,1H),8.06(s,1H),7.47-7.85(m,5H),6.58(br s,2H),4.98-5.50(m,4H),4.46(br s,1H),2.73-3.00(m,1H),1.75-1.97(m,1H),1.20-1.72(m,8H). 19 F NMR (376MHz, DMSO-d6) δ (ppm) -60.55 (br d, 3F, J = 184.0Hz). LC-MS: Rt = 0.775min, (ESI) m / z. [M+H] +410.0; SFC: Rt=0.700min; ee%=98%

[0171] Note: The chirality of intermediate A5b and compound 3 is randomly assigned, and the two compounds may be their stereoisomers.

[0172] Biological test example: HCT116 and HCT116-MTAP-KO cell proliferation inhibition experiment in vitro

[0173] Experimental Materials

[0174] 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.

[0175] McCoy'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)

[0176] Experimental methods

[0177] 1. Cell culture: HCT116 and HCT116-MTAP-KO cells were cultured in McCoy'5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody; they were always in the logarithmic growth phase and the cell viability was greater than 95%.

[0178] 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.

[0179] 3. Compound treatment of cells: Add the trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension to the 384-well plate with the test compound, 40 μL per well, i.e., 100 cells per well, and the final DMSO concentration is 0.4%. The cell culture plate is placed in a 37°C, 5% carbon dioxide incubator for 6 days.

[0180] 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 multifunctional microplate reader to detect the luminescent signal at 578 nm.

[0181] 5. Data Analysis:

[0182] 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)

[0183] Structure of reference compound:

[0184] The examples and compound activity test results are as follows:

[0185] Table 1

[0186]

[0187] *American Association for Cancer Research: Orlando, Florida, April 14-19, 2023, public data

[0188] 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 selected from the group consisting of: in, Ra is selected from the group consisting of: W is O; Ring A is selected from the group consisting of a substituted or unsubstituted 5-6 membered monocyclic heterocyclic ring; R8 is selected from the group consisting of: Benzene ring, pyridine ring, Wherein, the C ring is selected from the following group: a substituted or unsubstituted 5-6 membered heteroaromatic ring; and the R8 is substituted by one or more CF3; R2 is selected from the group consisting of: hydrogen; Unless otherwise specified, in the above formulae, the substitution refers to the replacement of hydrogen atoms on the corresponding groups by one or more substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, unsubstituted or halogenated C1-C6 alkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The Ra is selected from the following group: Wherein, the R9 is selected from the following group: unsubstituted or halogenated methyl.

3. The compound according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The A ring is selected from the following group: Any hydrogen atom on the ring may be lost to form a chemical bond, or may be replaced by one or more substituents.

4. The compound according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The formula I has a structure shown in the following formula IV: Wherein, Q is O or CH2.

5. The compound according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The formula I has the structure shown below: Wherein the C ring is a substituted or unsubstituted benzene ring; and the R8' is one or more substituents selected from the following group: CF3; Wherein Ra is selected from:

6. The compound according to claim 1, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The formula I has the structure shown below: X is selected from the group consisting of O, CH2; X1 and X2 are each independently selected from the group consisting of CH, N, and CF; R8 is a benzene ring; and said R8 is substituted by one or more CF3.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, characterized in that: The compound has a structure selected from the following:

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of a compound as described in any one of claims 1 to 7, 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.

9. Use of a compound according to any one of claims 1 to 7, its racemate, optical isomer or pharmaceutically acceptable salt in the preparation of a drug for treating or preventing a disease associated with abnormal gene level or abnormal expression of PRMT5 (such as corresponding nucleic acid mutation, deletion, or ectopic or fusion or overexpression of the methyltransferase).

10. Use of a compound or composition as claimed in any one of claims 1 to 7, a racemate, a stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing a disease associated with abnormal gene levels or abnormal expression of PRMT5 (such as corresponding nucleic acid mutation, deletion, or abnormal MTAP gene levels, or ectopic or fused or overexpressed methyltransferase); preferably, 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 external 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.

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