PRMT5 inhibitors and uses thereof

Abstract

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

Description

Technical Field

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

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

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

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

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

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

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

[0008]

[0009] in,

[0010] Ra is selected from the following group:

[0011] W represents O or S;

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

[0013] Ring A is selected from the following group: substituted or unsubstituted 5-6 member monoheterocycles, substituted or unsubstituted 7-12 member spiroheterocycles;

[0014] R8 is selected from the following group:

[0015] Benzene ring, pyridine ring,

[0016] Wherein, the C ring is selected from the group consisting of: substituted or unsubstituted benzene rings, substituted or unsubstituted 5-6 membered heteroaromatic rings; and the R8 is substituted by one or more substituents selected from the group consisting of: 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 groups;

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

[0019] Or, R2 located on two adjacent ring atoms and the ring atoms connected to them together form a structure selected from the following group: substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered heteroaromatic ring, substituted or unsubstituted 5-6 membered heterocycle (including saturated or partially unsaturated heterocycles).

[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 formulas, substitution refers to the substitution of a hydrogen atom on the corresponding group by one or more substituents selected from the group consisting of: 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- or six-membered heteroaryl, five- or six-membered non-aromatic heterocyclic group, -O-(C6-C 10 aryl), -O- (five- 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 carbon ring or heterocycle may be a saturated or partially unsaturated non-aromatic ring, and may be a monocyclic, bridged, spirocyclic, or fused ring; when the carbon ring or heterocycle is a fused ring, the carbon ring or fused ring may be partially aromatic, such as an aromatic ring or a saturated ring.

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

[0024]

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

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

[0027]

[0028] In this process, any hydrogen atom on the ring can be lost to form a chemical bond, or it can be replaced by one or more substituents.

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

[0030]

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

[0032] The definition of R8 is as described above;

[0033] The R mentioned 8a and R 8b Each is independently selected from the following group: H; or the aforementioned R. 8a and R 8b Together with the carbon atoms attached to them, they form 4-7 member carbon rings or heterocycles;

[0034] Or R8 and R located on two adjacent ring atoms 8a Together with the ring atoms attached to it, they form a structure selected from the following group: substituted or unsubstituted benzene rings, substituted or unsubstituted 5-6 membered heteroaromatic rings, and substituted or unsubstituted 5-6 membered heterocycles (including saturated or partially unsaturated heterocycles).

[0035] And when the R mentioned 8aand R 8b When each is independently H, the R mentioned 8a or R 8b It can be optionally replaced by R8; when the R 8a and R 8b When the carbon atoms connected to it together form a 4-7 member carbon ring or heterocycle, the R8 can be located on the carbon ring or heterocycle.

[0036] In another preferred embodiment, formula I has the structure shown in the following formula:

[0037]

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

[0039] Ra is selected from:

[0040]

[0041] Where X can be O, NR2, or CHR2; and R2 is defined as described above.

[0042] In another preferred embodiment, formula I has the structure shown in the following formula:

[0043] X is selected from the following group: O, CHR2, NR2; where R2 is defined as described above;

[0044] X1 and X2 are each independently selected from the following groups: CH, N, CF;

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

[0046] Benzene ring, pyridine ring,

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

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

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

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

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

[0054]

[0055]

[0056] The absolute stereochemistry of the above compounds is arbitrarily specified (chirality is specified randomly based on the order of separation of chiral SFCs). Compounds with a stereoisomeric center (where the configuration is not indicated in the described structure) and which are not specified are mixtures of enantiomers at that center.

[0057] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a therapeutically effective amount of one or more of the compound as described in the first aspect, a pharmaceutically acceptable salt thereof, a racemic mixture, an optical isomer, a stereoisomer, or a tautomer thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, excipients, and / or diluents.

[0058] In a third aspect of the invention, the use of the compound, racemate, optical isomer, or pharmaceutically acceptable salt thereof as described in the first aspect in the preparation of a medicament for treating or preventing diseases associated with abnormalities at the gene level or expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferases).

[0059] In another preferred embodiment, the disease is a malignant tumor or cancer, selected from the group consisting of: ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, malignant peripheral nerve sheath tumor (MPNST), esophageal cancer (e.g., esophageal squamous cell carcinoma or esophageal adenocarcinoma), bladder cancer (e.g., bladder cancer, urothelial carcinoma), mesothelioma, non-small cell lung cancer (NSCLC; e.g., lung squamous cell carcinoma or lung adenocarcinoma), astrocytoma, undifferentiated pleomorphic sarcoma, diffuse large B-cell lymphoma (DLBCL), leukemia, head and neck cancer, gastric adenocarcinoma, myxofibrosarcoma, cholangiosarcoma, and brain cancer, gastric cancer, kidney cancer, breast cancer, endometrial cancer, urinary tract cancer, liver cancer, soft tissue cancer, pleural cancer, and colorectal cancer or sarcoma.

[0060] A fourth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds described in any of the preceding aspects, pharmaceutically acceptable salts, racemates, optical isomers, stereoisomers, or tautomers thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, excipients, and / or diluents.

[0061] A fifth aspect of the present invention provides the use of a compound, its racemic mixture, optical isomer, or pharmaceutically acceptable salt as described in any of the foregoing aspects in the preparation of a medicament for treating or preventing diseases associated with abnormalities at the gene level or expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferases).

[0062] In another preferred embodiment, the disease is selected from the group consisting of: ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, stomach cancer, pancreatic cancer, or bladder cancer.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0064] Through extensive and in-depth research, the inventors unexpectedly discovered a class of compounds with PRMT5-regulating effects for the first time. This invention was completed based on this discovery.

[0065] the term

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

[0067] In this invention, unless otherwise specified, the terms used have their general meanings known to those skilled in the art. In this invention, unless otherwise specified, all chemical formulas are intended to cover any possible optical or geometric isomers (e.g., R-type, S-type, or racemic, or cis-trans isomers of alkenes, etc.).

[0068] In this invention, the term "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and includes, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

[0069] In this invention, the term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 6 carbon atoms, and includes, without limitation, methoxy, ethoxy, propoxy, isopropoxy, and butoxy.

[0070] In this invention, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms and containing a double bond, and includes, without limitation, vinyl, propenyl, butenyl, isobutenyl, pentenyl, and hexenyl groups.

[0071] In this invention, the term "C2-C6 ynyl" refers to a straight-chain or branched ynyl group having 2 to 6 carbon atoms and containing a triple bond, and includes, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentylyl, and hexynyl.

[0072] In this invention, the term "C3-C10 cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms on a ring, and non-limitingly includes 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 this invention, the term "C3-C10 cycloalkenyl" refers to a cyclic alkenyl group having 3 to 10 carbon atoms on a ring, and non-limitingly includes cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and cyclodecylene, etc. The term "C3-C7 cycloalkenyl" has a similar meaning.

[0074] In this invention, the term "C1-C12 alkoxycarbonyl" refers to an alkoxycarbonyl group having 1 to 12 carbon atoms on an alkyl chain, and includes, without limitation, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc.

[0075] In this invention, the term "C1-C12 alkylaminocarbonyl" refers to an alkylaminocarbonyl group having 1 to 12 carbon atoms on an alkyl chain, and includes, without limitation, methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, dimethylaminocarbonyl, etc.

[0076] In this invention, the term "C5-C9 furanose" refers to a furanose having 5 to 9 carbon atoms, wherein the 1-position of the glycosyl group is connected to the main chain, and includes, without limitation, furanose ribosyl, furanose deoxyribosyl, furanose galactosyl, etc.

[0077] In this invention, the term "C5-C9 pyranosyl" refers to a pyranosyl group having 5 to 9 carbon atoms, wherein the 1-position of the glycosyl group is connected to the main chain, and includes, without limitation, pyranoglucosyl, pyranoglucuronic acid glycosyl, pyranrhamnosyl, pyrangalactosyl, pyranmannosyl, pyranxylxyl, etc.

[0078] In this invention, the terms "aromatic ring" and "aryl" have the same meaning, and preferably "aryl" refers to "C6-C12 aryl" or "C6-C10 aryl". The term "C6-C12 aryl" refers to an aromatic cyclic group with 6 to 12 carbon atoms that does not contain heteroatoms on the ring, such as phenyl or naphthyl. The term "C6-C10 aryl" has a similar meaning.

[0079] In this invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning, referring to a heteroaryl group containing one or more heteroatoms. Heteroatoms referred to herein include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

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

[0081] In this invention, the term "substitution" refers to the replacement of one or more hydrogen atoms on a specific group by a specific substituent. The specific substituent is either the substituent described accordingly above or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Cyclic substituents, such as heterocyclic alkyl groups, may be attached to another ring, such as a cycloalkyl group, thereby forming a spirobicyclic system, for example, where the two rings share a common carbon atom. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible. The substituents are, for example (but 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 Administration

[0083] Because the compounds of the present invention have excellent methyltransferase inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases caused by abnormal activity or expression of methyltransferases (e.g., PRMT5).

[0084] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 5-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0085] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0086] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

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

[0088] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0089] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0090] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0091] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0092] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0093] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0094] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In some preferred embodiments, the compounds of this invention can be administered together with other small molecule compounds to form a PROTAC, or together with other large molecule compounds such as monoclonal antibodies to form an ADC.

[0095] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0096] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

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

[0098]

[0099]

[0100] The raw materials can be obtained commercially or prepared using methods known or disclosed in the art.

[0101] Purification of intermediates and compounds is performed using conventional chemical experimental procedures such as normal-phase or reverse-phase chromatography or recrystallization. Normal-phase chromatography uses pre-packed silica gel columns or preparative thin-layer chromatography. Silica gel columns are primarily glass columns or rapid preparative chromatographs. The mobile phase for normal-phase chromatography is selected and proportioned from petroleum ether / ethyl acetate, dichloromethane / methanol, or other suitable solvents for elution. Reversed-phase preparative liquid chromatography uses a C18 column and is performed using a preparative liquid chromatograph or a rapid preparative chromatograph, with detection at 214 nM and 254 nM or using preparative liquid chromatography-mass spectrometry. Gradient elution is performed using water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile, water / acetonitrile containing 0.1% ammonium bicarbonate, water / acetonitrile containing 0.1% formic acid, water / acetonitrile containing 0.1% ammonia, water / acetonitrile containing 0.1% trifluoroacetic acid, or other suitable solvent systems as the mobile phase.

[0102] The structures of intermediates and compounds were characterized using nuclear magnetic resonance (NMR) and LCMS. The NMR spectrometers used were either a Bruker Ascend 400, a Varian 400, or a ZKNJ BIXI-1.

[0103] 300MHz or Bruker Avance III 400MHz or Bruker Avance Neo 400MHz. The solvent used is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, or other labeled deuterated solvents. Spectral data are reported in mode: chemical shift δ (number of peaks split, coupling constant J (Hz), number of hydrogens). Tetramethylsilane is used as the inner standard for the chemical shift, and its chemical shift is set to zero (δ, 0ppm). Some abbreviations are: s (singleton), d (doublet), t (triplet), q (quartet), m (multiplex), br (broad peak).

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

[0105] Method 1: Performed on an Agilent LC1260 system coupled with a 6120 single quadrupole mass spectrometer.

[0106] Column: Waters CORTECS C-18, 2.7 μm, 4.6 x 30 mm. Solvent A: 0.05% formic acid aqueous solution; Solvent B: 0.05% formic acid in acetonitrile solution; concentration was increased from 5% acetonitrile to 95% acetonitrile in one minute, held for one minute, for a total of 2.5 minutes; 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, from 5% acetonitrile to 95% acetonitrile in one minute, held for one minute, for 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 with a quadrupole mass spectrometer.

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

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

[0111] Method 3:

[0112] Column: DAIICEL CHIRALPAK IC (250mm*30mm, 10µm)

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

[0114] Elution gradient: 40%

[0115] Method 4:

[0116] Column: DAIICEL CHIRALPAK AD (250mm*30mm, 10um);

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

[0118] Method 5:

[0119] Column: DAIICEL 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) methods:

[0122] Method Six:

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

[0124] Method Seven:

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

[0126] Method Eight:

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

[0128] General synthesis method for the examples:

[0129]

[0130] General method: Synthesis of intermediate A2

[0131]

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

[0133] 2,5-Difluoro-4-nitrobenzoic acid (1) (50 g, 246.18 mmol, 1 equivalent) was dissolved in methanol (500 mL), and thionyl chloride (43.93 g, 369.28 mmol, 26.79 mL, 1.5 equivalent) was added at 0 °C. The reaction mixture was reacted at 40 °C for 16 h. LC-MS showed that the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure. After dilution with 300 mL of water, it was extracted three times with 1 L 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 min to give methyl 2,5-difluoro-4-nitrobenzoate (2) (103 g, 474.38 mmol, 96.35% yield) as a white solid. 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-nitrobenzene (2) (80 g, 368.45 mmol, 1 equivalent) and 2-methyl-1H-imidazolium (36.30 g, 442.14 mmol, 1.2 equivalent) were dissolved in dimethyl sulfoxide (1.2 L). The reaction mixture was reacted at 50 °C for 16 h. LC-MS showed that the reaction was complete. The reaction mixture 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 concentrated to dryness under reduced pressure. The crude product was slurried with methyl tert-butyl methyl ether at 25 °C for 60 min (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 with methyl tert-butyl methyl ester at 25°C for 60 minutes to obtain a yellow solid (8.23 g). Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) (25.5 g, 91.32 mmol, 24.79% yield) methyl 2,5-difluoro-4-nitrobenzoate (2) (raw material recovered) (20.34 g, 93.68 mmol, 25.43% yield). Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) (8.2 g, 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 equivalent) was dissolved in tetrahydrofuran (300 mL), and palladium hydroxide on carbon (2.8 g, 49.78 mmol, 20% purity, 1 equivalent) was added under a hydrogen atmosphere. The reaction system was purged with hydrogen three times. The reaction solution was heated to 50°C for 32 h under a hydrogen atmosphere (1 equivalent) (50 psi). LC-MS showed that the reaction was complete. The reaction solution was filtered through 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 give a gray solid. The crude product was used directly in the next step. Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (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: Methyl 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5)

[0141] Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4) (12 g, 48.15 mmol, 1 equivalent) was added to 1-methyl-2-pyrrolidone at 25°C, followed by 1,1-carbonyldiimidazole (19.52 g, 120.37 mmol, 2.5 equivalents). The reaction mixture was heated to 115°C and reacted for 16 hours. LC-MS showed that the reaction was complete. The two batches of reaction mixture were combined and processed. 600 mL of ethyl acetate and 600 mL of water were added to the reaction mixture and stirred 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, methyl 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (24.1 g, 87.56 mmol, 86.60% yield). The crude product was used directly in 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: Methyl 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6)

[0143] Methyl 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (12.0 g, 43.60 mmol, 1.0 equivalent), 2,4-dimethoxybenzylamine (10.9 g, 65.19 mmol, 9.82 mL, 1.50 equivalent), and 1,8-diazabicyclo[5.4.0]undecane-7-ene (19.92 g, 130.80 mmol, 19.72 mL, 3.0 equivalent) were added to acetonitrile (240 mL). Benzotriazole-1-oxo-tris(dimethylaminophosphide)hexafluorophosphate (25.07 g, 56.68 mmol, 1.3 equivalent) was added in portions at 15–20 °C. The reaction mixture was slightly exothermic, became homogeneous, and a solid precipitated. The reaction mixture was reacted at 15–20°C for 16 hours under nitrogen protection. LC-MS showed complete consumption of the starting material and detection of the target compound. 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 give a grayish-white solid methyl 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (15.3 g, 36.05 mmol, 82.68% yield). LC-MS 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: Methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7)

[0145] Methyl 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (16.3 g, 38.40 mmol, 1.0 equivalence) was added to dichloromethane (50 mL), followed by trifluoroacetic acid (250 mL). The reaction mixture was heated to 50°C and reacted for 16 hours. LCMS showed complete consumption of the starting material and detection of the target compound. The reaction mixture was concentrated to dryness under reduced pressure to obtain a purple solid, methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7) (28.3 g, crude). The crude product was directly used for the next 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 from the previous step) (28.3 g, 38.70 mmol, 1 equivalent) was added to tetrahydrofuran (80 mL) and methanol (80 mL). Sodium hydroxide (7.74 g, 193.48 mmol, 5 equivalent) was dissolved in water (80 mL) and added to the reaction mixture. The reaction mixture was heated to 50°C and reacted for 4 hours. The desired compound was detected by LCMS. After cooling the reaction mixture to 20°C, the organic solvent was removed by concentration under reduced pressure. The residue was diluted with water to methanol at a ratio of 10:1 (300 mL) and filtered through diatomaceous earth. The filter cake was washed three times with water to methanol at a ratio of 10:1 (300 mL). All filtrates were combined and concentrated under reduced pressure to remove methanol. The pH of the residue was adjusted to 5–6 with acetic acid. The resulting slurry was stirred at 15–20°C for 12 hours, filtered under reduced pressure, and the resulting solid was washed with water. The collected solid was freeze-dried to obtain a white solid. 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaloline-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)-2-(2-chloroacetyl)piperidine-1-carboxylic acid tert-butyl ester (2)

[0151] Solution A: To a THF (20 mL) solution of N-isopropylpropyl-2-amine (1.46 g, 14.3 mmol, 2.03 mL, 5.0 eq), n-butyllithium (2.5 M, 5.75 mL, 5.0 eq) was added dropwise. The reaction was then heated to -70 °C and stirred for 30 minutes, followed by warming to 20 °C and stirring for another 30 minutes at 20 °C.

[0152] A solution of 1-(tert-butyl)-2-methyl(R)-piperidine-1,2-dicarboxylic acid ester (1) (700 mg, 2.88 mmol, 1.0 eq) and chloro(iodide)methane (2.03 g, 11.5 mmol, 835 μL, 4.0 eq) was added dropwise to solution A in 10 mL of THF at -70 °C, and the reaction mixture was stirred at -70 °C for 30 min. The reaction was quenched with AcOH without monitoring (for the next post-treatment). 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 min. The mixture was warmed to room temperature, H2O (40 mL) was added, and the mixture was extracted with EtOAc (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give the residue. The residue was purified by rapid silica gel chromatography. 20g The silica flash column was used as the eluent, with a gradient of 0–15% ethyl acetate / petroleum ether at 40 mL / min, where petroleum ether / ethyl acetate = 4 / 1, and Rf = 0.61 (I2)). Tert-butyl (2R)-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)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidin-1-carboxylic acid tert-butyl ester (3)

[0154] Add 5-(trifluoromethyl)pyridine-2-amine (309 mg, 1.91 mmol, 1.2 eq) to a solution of (2R)-2-(2-chloroacetyl)piperidin-1-carboxylic acid tert-butyl ester (2) (416 mg, 1.59 mmol, 1.0 eq) in DME (8 mL). Stir the mixture at 90 °C for 3 hours. LC-MS showed 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 the residue. The residue was purified by rapid silica gel chromatography. 20g Silica Flash Column, eluent gradient of 0–18% ethyl acetate / petroleum ether @ 30 mL / min, petroleum ether / ethyl acetate = 3 / 1, Rf = 0.26). (2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidin-1-carboxylic acid tert-butyl ester (3) (20 mg, 42.7 μmol, 2.69% yield, 79% purity) yielded 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 (2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]piperidine-1-carboxylic acid tert-butyl ester (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 ready for the next step without further purification. A yellow oily 2-[(2R)-2-piperidinyl]-6-(trifluoromethyl)imidazo[1,2-a]pyridine (intermediate B) (39 mg, crude, 2HCl) was obtained. 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-methylimidazol[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl] methyl ketone (Compound 1)

[0161] 2-chloro-1,3-dimethyl-4,5-dihydroimidazolium-1-onium 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-methylimidazolium[1,5-a]quinoxaloline-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 h. 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 min). The solutions of the desired compounds were combined and lyophilized to give (4-amino-7-fluoro-3-methyl-imidazol[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl] methyl ketone (compound 1) (62 mg, 131 μmol, 67.29% yield, 100% purity), as a grayish-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-methylimidazol[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl] methyl ketone (Compound 2)

[0165] DIEA (124 mg, 961 μmol, 167 μL, 5 eq) and 2-chloro-1,3-dimethyl-4,5-dihydroimidazole-1-onium chloride (DMC) (48.7 mg, 288 μmol, 1.5 eq) were added 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 in NMP (1 mL), and the reaction was stirred at 50 °C for 16 h. The desired product was detected by LC-MS. The reactants were 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 for more than 10 minutes). The solutions of the desired products were combined and lyophilized to give (4-amino-7-fluoro-1-methyl-imidazol[1,5-a]quinoxalin-8-yl)-[(3S)-3-[4-(trifluoromethyl)phenyl]morpholin-4-yl] methyl ketone (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] 1.557 min, ee% = 100%.

[0167] Example 3: Synthesis of Compound 3

[0168]

[0169] Step 1: [(3R)-4-amino-3-methyl-1,3-dihydrofuran[3,4-c]quinoline-8-yl]-[(2R)-2-[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]-1-piperidinyl] methyl ketone (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, 2 HCl) and (3S)-4-amino-3-methyl-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylic acid (intermediate A5b) (20.8 mg, 85.4 μmol, 1 eq) in NMP (1 mL), 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) were added, and the mixture was stirred for 1 h. Then TCFH (28.7 mg, 102 μmol, 1.2 eq) was added, and the reaction was stirred at 20 °C for 16 h. LC-MS showed the reaction was complete. 1 mL of DMSO was added to the reaction mixture, and the mixture was purified by reversed-phase HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 35%-55% B for more than 10 minutes). The solutions of the desired products were combined and lyophilized to give [(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] methyl ketone (compound 3) (5 mg, 10.0 μmol, 11.8% yield, 100% purity), 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 chiral types of intermediate A5b and compound 3 are randomly assigned, and the two compounds may be stereoisomers of each other.

[0172] Biological test case: In vitro inhibition of proliferation of HCT116 and HCT116-MTAP-KO cells.

[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 antibiotics (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo assay kit (Promega, catalog number G7572), 384-well clear flat-bottom black-walled cell culture plate (Corning, catalog number 3764), micropipette (Tecan, catalog number D300e), multi-functional microplate reader (Biotek, catalog number SynergyHTX)

[0176] Experimental methods

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

[0178] 2. Preparation of compound concentration gradients: The test compounds were added to 384-well plates using an ultra-micro pipette, starting with 30 μM (HCT116 cells) or 3 μM (HCT116-MTAP-KO cells), and diluted 3-fold with DMSO to obtain a total of 9 concentrations, with triplet wells.

[0179] 3. Cell treatment with compounds: Add 40 μL of trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension to 384-well plates pre-stamped with the test compounds, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. Incubate the cell culture plates at 37°C in a 5% CO2 incubator for 6 days.

[0180] 4. Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate with shaking at room temperature for 30 minutes. Detect the luminescence signal at 578 nm using a multi-mode microplate reader.

[0181] 5. Data Analysis:

[0182] The first column shows the cell proliferation inhibition rate: HCT116 MTAP WT IC 50 (nm), the second column is the cell proliferation inhibition rate HCT116-MTAP null IC 50 (nm)

[0183] Compare the structures of the compounds:

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

[0185] Table 1

[0186]

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

[0188] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof. wherein the compound has a structure selected from the group consisting of:

2. A pharmaceutical composition, characterized by, The pharmaceutical composition contains a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable carriers.

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

4. Use according to claim 3, characterized in that, The abnormal gene level or abnormal expression of PRMT5 includes a corresponding nucleic acid mutation, deletion, or ectopic or fusion or overexpression of the methyltransferase.

5. The use according to claim 3, characterized in that, The disease is a malignant tumor or cancer selected from the group consisting of: ovarian cancer, lung cancer, lymphoma, colon cancer, melanoma, malignant peripheral nerve sheath tumor (MPNST), esophageal cancer, bladder cancer, mesothelioma, undifferentiated pleomorphic sarcoma, diffuse large B-cell lymphoma (DLBCL), leukemia, head and neck cancer, myxofibrosarcoma, cholangiosarcoma, and brain cancer, gastric cancer, kidney cancer, breast cancer, endometrial cancer, liver cancer, soft tissue cancer, pleural cancer and colorectal cancer.

6. The use according to claim 3, characterized in that, The disease is a malignant tumor or cancer selected from the group consisting of: glioblastoma, astrocytoma, non-small cell lung cancer, urinary tract cancer, gastric adenocarcinoma or sarcoma.

7. The use according to claim 3, characterized in that, The disease is selected from the group consisting of: esophageal squamous cell carcinoma, esophageal adenocarcinoma, urothelial carcinoma, lung squamous carcinoma, or lung adenocarcinoma.

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