PRMT5 Inhibitor and Its Use

Abstract

Provided are compounds having methyltransferase inhibitory activity, specifically, compounds having PRMT5 inhibitory activity, and said compounds can be used in the preparation of pharmaceutical compositions for treating PRMT5 activity-related diseases. Formula I.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical compounds, and more particularly, the present invention provides compounds that inhibit PRMT5 and their use in pharmaceutical compositions.

Background Art

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

[0003] Epigenetic regulation involves genetically modifying genetic material without altering the nucleotide sequence. Usually, epigenetic regulation is mediated by selective and reversible modifications (e.g., methylation) of DNA and proteins (e.g., histones), and these modifications control the structural transitions between the transcriptionally active and inactive states of chromatin. These covalent modifications are controlled by enzymes such as methyltransferases (e.g., PRMT5), where many are associated with specific genetic mutations that can cause human diseases. PRMT5 plays a role in diseases such as proliferative diseases, metabolic diseases, and blood diseases.

[0004] PRMT5 is a known essential cell gene, and 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, sarcopenia, cardiac hypertrophy). Therefore, there is a need for new strategies to exploit such metabolic vulnerabilities and preferentially target PRMT5 in MTAP-null tumors (MTAPWT) while sparing PRMT5 in normal tissues. Targeting PRMT5 with MTA-competitive small molecule inhibitors preferentially targets the MTA-binding state of PRMT5, provides a better therapeutic index in MTAP-null tumor cells that are abundant and have low MTA levels without compromising MTAP in normal cells.

[0005] Therefore, in the art, there is a need to provide a novel small molecule compound that targets PRMT5 in MTAP-null tumors. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] An object of the present invention is to provide a novel small molecule compound that targets PRMT5 in MTAP-null tumors. MEANS FOR SOLVING THE PROBLEMS

[0007] A first aspect of the present invention provides a compound as shown in the following formula I, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof, [Chemical formula] wherein, Ra is [Chemical formula] selected from the group consisting of, W is O or S, X1 and X2 are each independently selected from the group consisting of CR and N, and X3 is N, L1 is selected from the group consisting of a chemical bond, -O-, -CHR-, -C(R)R-, The A ring is selected from the group consisting of a substituted or unsubstituted 8- to 12-membered fused bicyclic heterocyclic group (including a carbocyclic or heterocyclic ring, preferably a 5-membered fused 6-membered ring), a substituted or unsubstituted 7- to 10-membered fused bicyclic heteroaryl group (preferably a 5-membered fused 6-membered ring), R8 is selected from the group consisting of H, halogen, cyano group, amino group, nitro group, hydroxy group, thiol group, aldehyde group, carboxy group, C2-C6 alkynyl group, SF5, a substituted or unsubstituted or halogenated C1-C6 alkyl group, or R8 is [Chemical formula] wherein, The L3 is selected from the group consisting of a chemical bond, -O-, -CHR-, -C(R)R-, a carbonyl group, S, and -NH-; The B ring is selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5- to 6-membered heteroaromatic ring, a substituted or unsubstituted C3-C6 carbocyclic ring (including the case of being saturated or partially unsaturated), and a substituted or unsubstituted 3- to 7-membered heterocyclic ring (including the case of being saturated or partially unsaturated); R2 is selected from the group consisting of R7 and -L2R7, where the L2 is selected from the group consisting of -O-, -CHR-, -C(R)R-, and a carbonyl group, and where R7 is hydrogen or absent, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C 6-10 aromatic ring, a substituted or unsubstituted 5- to 12-membered heteroaromatic ring, a substituted or unsubstituted C3-C 10 carbocyclic ring (including the case of being saturated or partially unsaturated, and including a monocyclic ring, a fused ring, a spiro ring, or a bridged ring), and a substituted or unsubstituted 3- to 10-membered heterocyclic ring (including the case of being saturated or partially unsaturated, and including a monocyclic ring, a fused ring, a spiro ring, or a bridged ring), and is selected from the group consisting of; R3 is selected from the group consisting of H, a halogen, a cyano group, and a substituted or unsubstituted C1-C6 alkyl group; R4 and R5 are each independently selected from the group consisting of H, a halogen, a cyano group, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C1-C6 alkoxy group, a substituted or unsubstituted C3-C6 carbocyclic ring (including the case of being saturated or partially unsaturated), and a substituted or unsubstituted 3- to 6-membered heterocyclic ring, or R4 and R5 together with the ring atoms to which they are attached form a 5- to 12-membered saturated or unsaturated ring, and the ring may or may not be substituted; R is H, a halogen, a substituted or unsubstituted C1-C4 alkyl group, a substituted or unsubstituted C1-C4 alkoxy group, or a substituted or unsubstituted C3-C6 cycloalkyl group; Unless otherwise specified, in the above formulas, the substitution means that a hydrogen atom on the corresponding group is deuterium, tritium, a halogen, a hydroxy group, a carboxy group, a thiol group, a benzyl group, C1-C 12An alkoxycarbonyl group, a C1-C6 aldehyde group, an amino group, a C1-C6 amide group, a nitro group, a cyano group, an unsubstituted or halogenated C1-C6 alkyl group, an unsubstituted or halogenated C3-C8 cycloalkyl group, a C2-C 10 alkenyl group, a C1-C6 alkoxy group, a C1-C6 alkyl-amino group, a C6-C 10 aryl group, a five- or six-membered heteroaryl group, a five- or six-membered non-aromatic heterocyclic group, -O-(C6-C 10 aryl), -O-(five- or six-membered heteroaryl), C1-C 12 alkylaminocarbonyl group, an unsubstituted or halogenated C2-C 10 acyl group, sulfonyl (-SO2-OH), phosphoryl (-PO3-OH), an unsubstituted or halogenated C1-C4 alkyl-S(O)2-, an unsubstituted or halogenated C1-C4 alkyl-SO-, and is substituted by one or more substituents selected from the group consisting of.

[0008] In another preferred example, the A ring has a structure selected from the group consisting of the following.

Chemical formula

[0009] In another preferred example, the Ra is selected from the group consisting of the following,

Chemical formula

Chemical formula

[0010] In another preferred example, L1 is -CH2- or -CH(CH3)-, and the A ring is selected from the group consisting of the following:

Chemical formula

[0011] In another preferred example, R2 is an ortho-substituted 5- or 6-membered heteroaromatic ring as shown in the following formula:

Chemical formula

[0012] Preferably, the D ring is selected from the group consisting of a substituted or unsubstituted benzene ring and a substituted or unsubstituted 5- to 6-membered heteroaromatic ring. More preferably, the D ring is

Chemical formula

Chemical formula

[0013] In another preferred example, the R2 is selected from the group consisting of R7 and -L2R7, where the L2 is selected from the group consisting of -O-, -CHR-, a carbonyl group, S, and -NH-, and where the R7 is a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C 6-10 is selected from the group consisting of an aromatic ring, a substituted or unsubstituted 5- to 12-membered heteroaromatic ring.

[0014] In another preferred example, the R7 is selected from the group consisting of a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted benzene ring, and a substituted or unsubstituted 5- to 7-membered heteroaromatic ring.

[0015] In another preferred example, R2 is selected from the group consisting of R7 and -(CHR)R7, where R7 is a substituted or unsubstituted C 6-10 is selected from the group consisting of an aromatic ring, a substituted or unsubstituted 5- to 12-membered heteroaromatic ring.

[0016] In another preferred example, the R2 is a substituted or unsubstituted 5- to 7-membered heteroaromatic ring, and the A ring is selected from the group consisting of substituted or unsubstituted 7- to 10-membered fused bicyclic heteroaryl groups, and the R8 is CF3.

[0017] In another preferred example, the Ra has a structure as shown in the following formula. [Chemical formula]

[0018] The second aspect of the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of one or more compounds according to any of the foregoing aspects, pharmaceutically acceptable salts, racemic compounds, optical isomers, stereoisomers or tautomers thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary substances and / or diluents.

[0019] The third aspect of the present invention provides the use of a compound, a racemic compound, an optical isomer or a pharmaceutically acceptable salt thereof as described in any of the foregoing aspects in the preparation of a drug for treating or preventing a disease associated with gene level abnormality or expression abnormality of PRMT5 (for example, corresponding nucleic acid mutation, deletion, or methyltransferase generation ectopia or fusion or overexpression).

[0020] In another preferred example, the disease is selected from the group consisting of the disease or symptomatic ovarian cancer, lung cancer, lymphoma, glioblastoma, colorectal cancer, melanoma, gastric cancer, pancreatic cancer or bladder cancer.

Advantages of the Invention

[0021] It should be understood that within the scope of the present invention, new or preferred technical solutions can be constituted by combining each of the above technical features of the present invention with each of the technical features specifically described below (for example, in the examples). Due to space limitations, it will not be repeated here.

Modes for Carrying Out the Invention

[0022] Through extensive and meticulous research, the inventors of the present invention unexpectedly discovered a compound having a PRMT5 regulatory effect for the first time. Based on this, the present invention was completed.

[0023] Term

[0024] In the present invention, the halogen is F, Cl, Br or I.

[0025] In the present invention, unless otherwise specified, the terms used have the ordinary meanings known to those skilled in the art. In the present invention, unless otherwise specified, all chemical formulas include all possible optical isomers or geometric isomers (for example, R-type, S-type or racemate, or cis-trans isomers of alkenes, etc.).

[0026] In the present invention, the term "C1-C6 alkyl group" refers to a linear or branched alkyl group having 1 to 6 carbon atoms, and includes, without limitation, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, an s-butyl group, a t-butyl group, a pentyl group, a hexyl group, etc., preferably including an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, an s-butyl group, and a t-butyl group.

[0027] In the present invention, the term "C1-C6 alkoxy group" refers to a linear or branched alkoxy group having 1 to 6 carbon atoms, and includes, without limitation, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, etc.

[0028] In the present invention, the term "C2-C6 alkenyl group" refers to a linear or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, and includes, without limitation, a vinyl group, a propenyl group, a butenyl group, an isobutenyl group, a pentenyl group, a hexenyl group, etc.

[0029] In the present invention, the term "C2-C6 alkynyl group" refers to a linear or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, and includes, without limitation, an ethynyl group, a propynyl group, a butynyl group, an isobutynyl group, a pentynyl group, a hexynyl group, etc.

[0030] In the present invention, "C3-C 10 cycloalkyl group" refers to a cyclic alkyl group having 3 to 10 carbon atoms in the ring, and includes, without limitation, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclodecyl group, etc. The terms "C3-C8 cycloalkyl group", "C3-C7 cycloalkyl group", and "C3-C6 cycloalkyl group" have the same meaning.

[0031] In the present invention, "C3-C 10The term "cycloalkenyl group" refers to a cyclic alkenyl group having 3 to 10 carbon atoms in the ring, and includes, without limitation, cyclopropenyl group, cyclobutenyl group, cyclopentenyl group, cyclohexenyl group, cycloheptenyl group, cyclooctenyl group, octadecene group, etc. The term "C3-C7 cycloalkenyl group" has the same meaning.

[0032] In the present invention, "C1-C 12 The term "alkoxycarbonyl group" refers to an alkoxycarbonyl group having 1 to 12 carbon atoms on the alkyl chain, and includes, without limitation, methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, isopropoxycarbonyl group, t-butoxycarbonyl group, benzyloxycarbonyl group, etc.

[0033] In the present invention, "C1-C 12 The term "alkylaminocarbonyl group" refers to an alkylaminocarbonyl group having 1 to 12 carbon atoms on the alkyl chain, and includes, without limitation, methylaminocarbonyl group, ethylaminocarbonyl group, propylaminocarbonyl group, isopropylaminocarbonyl group, t-butylaminocarbonyl group, benzylaminocarbonyl group, dimethylaminocarbonyl group, etc.

[0034] In the present invention, the terms "aromatic ring" or "aryl group" have the same meaning. Preferably, the "aryl group" is a "C6-C 12 aryl group" or a "C6-C 10 aryl group". The term "C6-C 12 aryl group" refers to an aromatic ring group having 6 to 12 carbon atoms without heteroatoms in the ring such as phenyl group, naphthyl group, etc. The term "C6-C 10 aryl group" has the same meaning.

[0035] In the present invention, the terms "aromatic heterocyclic ring" or "heteroaryl group" have the same meaning and refer to a heteroaromatic group containing one or more heteroatoms. The heteroatoms referred to in this specification include oxygen, sulfur, and nitrogen. For example, a furyl group, a thienyl group, a pyridyl group, a pyrazolyl group, a pyrrolyl group, an N-alkylpyrrolyl group, a pyrimidinyl group, a pyrazinyl group, an imidazolyl group, a tetrazolyl group, etc. The heteroaryl group ring can be condensed with an aryl group, a heterocyclic group, or a cycloalkyl group ring, where the ring bonded to the parent structure is a heteroaryl group ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0036] In the present invention, the term "3- to 12-membered heterocyclic group" refers to a saturated or unsaturated 3- to 12-membered ring group containing 1 to 3 heteroatoms selected from oxygen, sulfur, and nitrogen, such as a dioxolanyl group. The term "3- to 7-membered heterocyclic group" has the same meaning.

[0037] In the present invention, the term "substituted" refers to the substitution of one or more hydrogen atoms of a specific group by a specific substituent. The specific substituent is the corresponding substituent described above, or the substituent appearing in each example. Unless otherwise specified, a certain substituent can have a substituent selected from a specific group at any substitutable position of the group, and the substituents may be the same or different at each position. A cyclic substituent such as a heterocycloalkyl group can be bonded to another ring such as a cycloalkyl group to form a spiro ring system, for example, two rings having a common carbon atom. Those skilled in the art will understand that the combinations of substituents contemplated by the present invention are stable or chemically feasible. The substituents are, for example, C 1-8 alkyl group, C 2-8 alkenyl group, C 2-8 alkynyl group, C 3-8 cycloalkyl group, 3- to 12-membered heterocyclic group, aryl group, heteroaryl group, halogen, hydroxyl group, carboxyl group (-COOH), C 1-8 aldehyde group, C 2-10 acyl group, C2-10 An ester group, C1-C 12 An alkoxycarbonyl group, an amino group, an alkoxy group, C 1-10 A sulfonyl group or the like (not limited to these).

[0038] For example, when an expression such as "C 1-8 " or a similar expression is used, it means that the group can have 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0039] For example, when an expression such as "3 to 12 members" or a similar expression is used, it means that the group can have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms or heteroatoms as ring atoms.

[0040] PRMT5 regulator compound

[0041] The present invention provides a compound having PRMT5 regulating activity,

Chemical formula

Chemical formula

[0042] Pharmaceutical Compositions and Administration Methods

[0043] Since the compounds of the present invention have excellent methyltransferase inhibitory activity, the compounds of the present invention and their various crystalline 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 for treating, preventing, and alleviating related diseases caused by abnormal activity or expression level of methyltransferase (e.g., PRMT5).

[0044] The pharmaceutical composition of the present invention contains a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or carrier within the range of a safe and effective amount. Here, "safe and effective amount" refers to the amount of the compound sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1 to 2000 mg of the compound / dose of the present invention, more preferably 1 to 200 mg of the compound / dose of the present invention. Preferably, the said "1 dose" is one capsule or tablet.

[0045] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" means that each component of the composition can be blended with the compounds of the present invention and among them without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween (registered trademark)), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0046] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and typical administration methods include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0047] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as, for example, sodium citrate or dicalcium phosphate, or is mixed with components such as (a) fillers or solubilizers such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as hydroxy methylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants such as glycerin, (d) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) retarders such as paraffin, (f) absorption promoters such as quaternary amine compounds, (g) wetting agents such as cetyl alcohol and glycerol 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. In capsules, tablets, and pills, the dosage form can also include buffering agents.

[0048] Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coating and shell materials such as enteric coatings and other materials known in the art. They can include opacifying agents, and the release of the active compound or compounds of such compositions can be delayed in a specific part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. Optionally, the active compound can form microcapsules with one or more of the above excipients.

[0049] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may include inert diluents conventionally used in the art such as water or other solvents, and solubilizing and emulsifying agents such as, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0050] In addition to these inert diluents, the compositions may also include adjuvants such as, for example, wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

[0051] In addition to the active compound, the suspension may include suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

[0052] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or non - aqueous 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.

[0053] Dosage forms of the compounds of the present invention for topical administration 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 as necessary.

[0054] 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 also form PROTACs together with other small molecule compounds or form ADCs for administration together with other macromolecular compounds such as monoclonal antibodies.

[0055] When a pharmaceutical composition is used, a safe and prevalent amount of the compound of the present invention is applied to a mammal (e.g., human) in need of treatment, where the dosage at the time of administration is the effective dosage to be considered. For a person weighing 60 kg, the daily dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage needs to take into account factors such as the administration route and the health status of the patient, and all of these are within the scope of the skills of a skilled physician.

[0056] Hereinafter, the present invention will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without indicating specific conditions usually follow conventional conditions or conditions proposed by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0057] The definitions of each abbreviation are as follows.

Table 1

[0058] The raw materials can be obtained through commercial channels or prepared by methods already known or published in the art.

[0059] Purification of intermediates and compounds is carried out by the operations of conventional chemical experiments such as normal-phase or reverse-phase chromatography or recrystallization. Normal-phase chromatography is preparative silica gel chromatography column or preparative thin-layer chromatography. The silica gel chromatography column is mainly a glass column or high-speed preparative chromatography. The mobile phase of normal-phase chromatography is selected from petroleum ether / ethyl acetate, dichloromethane / methanol or other suitable solvents and eluted in proportion. Reverse-phase preparative liquid chromatography employs a C18 column, detects using preparative liquid chromatography or high-speed preparative chromatography, 214 nM and 254 nM or preparative liquid chromatography-mass spectrometer, and gradient elutes 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% aqueous ammonia, water / acetonitrile containing 0.1% trifluoroacetic acid or other suitable solvent systems as the mobile phase.

[0060] Characterization of the structures of intermediates and compounds uses the methods of nuclear magnetic resonance (NMR) and mass spectrometry (LCMS). The NMR spectrometers used in nuclear magnetic resonance are Bruker Ascend 400 or Varian 400 or ZKNJ BIXI-1 300 MHz or Bruker Avance III 400 MHz or Bruker AVANCE Neo 400 MHz. The solvents used are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol or other marked deuterated solvents. The spectral data are reported in the mode of chemical shift δ (number of peak splits, coupling constant J (Hz), number of hydrogens). Tetramethylsilane is used as an internal standard substance for chemical shift, and its chemical shift is set to zero (δ, 0 ppm). The meanings of some abbreviations are s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak).

[0061] Typical methods of liquid chromatography-mass spectrometry (LCMS) in the characterization of the structures of intermediates and compounds are as follows.

[0062] Method 1: It is carried out on an Agilent LC1260 system coupled to a 6120 single quadrupole mass spectrometer. Column: Waters CORTECS C-18, 2.7 μm, 4.6 × 30 mm. Solvent A: 0.05% aqueous formic acid solution, Solvent B: acetonitrile solution of 0.05% formic acid, from 5% acetonitrile to 95% acetonitrile in 1 minute, maintained for 1 minute, total 2.5 minutes, flow rate: 1.8 mL / min, column temperature 40 °C. Column: XSelect CSH C18, 3.5 μm, 4.6 × 50 mm. Solvent A: 0.05% aqueous ammonia solution, Solvent B: acetonitrile solution of 0.05% ammonia, from 5% acetonitrile to 95% acetonitrile within 1 minute, maintained for 1 minute, total 2.5 minutes, flow rate: 1.8 mL / min, column temperature 40 °C. Method 2: It is carried out on an Agilent LC / MSD 1200 system coupled to a quadrupole mass spectrometer. Column: ODS 2000 (50 × 4.6 mm, 5 μm) (ES(+) or (-) ionization mode), temperature 30 °C, flow rate 1.5 mL / min.

[0063] General method for the synthesis of the examples:

Chemical formula

[0064] General method: Synthesis of intermediate A1 Synthesis route:

Chemical formula

[0065] Step 1: N-(4-Bromophenyl)-2-oxocyclopentane-1-carboxamide (2) A mixture of methyl 2-oxocyclopentane-1-carboxylate (50 g, 0.35 mol) and 4-bromoaniline (121 g, 0.70 mol) in toluene (300 mL) is stirred at 110 °C for 12 h. Next, the mixture is added to 2 M aqueous HCl (100 mL) and diluted with water (300 mL). The reaction mixture is extracted with ethyl acetate (300 mL × 3). The organic layer is washed with brine (200 mL × 3) and dried over anhydrous sodium sulfate. Next, the organic phase is filtered and concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with an EA / PE solution from 0% to 30% over 20 min to give N-(4-bromophenyl)-2-oxocyclopentane-1-carboxamide (25 g, 21% yield) as a pale yellow hard solid. LC-MS: Rt = 1.257 min, (ESI) m / z. [M+H] + 283.0, [M+2+H] + 285.0; C 12 H 12 BrNO2

[0066] Step 2: 8-Bromo-2,3-dihydro-1H-cyclopenta[c]quinolin-4-ol (3) A mixture of N-(4-bromophenyl)-2-oxocyclopentane-1-carboxamide (25.0 g, 0.088 mol) in concentrated sulfuric acid (100 mL) is stirred at 100 °C for 12 h. The mixture is poured into ice water (500 mL). NaHCO3 is added until pH 7 - 8. The precipitate formed is filtered and washed with some cold methanol (50 mL). The filtrate is filtered. The combined solids are dried and recrystallized from ethanol (20 mL) to give 8-bromo-2,3-dihydro-1H-cyclopenta[c]quinolin-4-ol (6 g, 26% yield) as a light brown solid. LC-MS: Rt = 1.299 min, (ESI) m / z. [M+H] + 264.0; [M+2+H] + 266.0, C 12 H 10 BrNO

[0067] Step 3: Methyl 4-hydroxy-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (4) 8-Bromo-2,3-dihydro-1H-cyclopenta[c]quinolin-4-ol (6.0 g, 23 mmol), Et3N (6.9 g, 68 mmol) and Pd(dppf)Cl2 (3.3 g, 4.40 mmol in MeOH (100 mL)) are stirred at 100 °C under a CO atmosphere for 12 h. The mixture is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a DCM solution of MeOH from 0% to 10% over 20 min to afford methyl 4-hydroxy-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (3 g, 54% yield) as a brown solid. LC-MS: Rt = 1.183 min, (ESI) m / z. [M+H] + 244.09,C 14 H 13 NO3

[0068] Step 4: Methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (5) To a solution of methyl 4-hydroxy-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (9.3 g, 0.038 mol) and pyridine (9.1 g, 0.11 mmol) in DCM (200 mL) is added trifluoromethanesulfonic anhydride (21.6 g, 0.076 mol) at 0 °C. The mixture is then stirred at 20 °C for 12 h. The reaction is quenched with NaHCO3(aq.) (200 mL) and then extracted with DCM (200 mL × 3). The organic solution is washed with brine (200 mL). The organic phase is dried over Na2SO4 and filtered. The filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a PE solution of EA from 0% to 30% over 20 min to afford methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (11.0 g, 77% yield) as a yellow solid. 11H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 2.0 Hz, 1H), 8.30 (dd, J = 8.8, 2.0 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 4.01 (s, 3H), 3.43 (t, J = 8.0 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 2.55 - 2.32 (m, 2H). LC-MS: Rt = 1.544 min, (ESI) m / z 376.1 [M+H] + . C 15 H 12 F3NO5S 375.04.

[0069] Step 5: Methyl 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (6) To a stirred solution of methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (11.0 g, 29 mmol) in dioxane (100 mL) are added Cs2CO3 (28.6 g, 88 mmol), Pd2(dba)3 (2.7 g, 2.93 mmol), Xantphos (3.4 g, 5.86 mmol), and PMBNH2 (6.0 g, 0.044 mmol) at 20 °C. The reaction mixture is stirred at 110 °C for 12 h under a N2 atmosphere. The mixture is filtered and the filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a DCM solution of MeOH from 0% to 5% over 20 min to give methyl 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (8.1 g, 76% yield) as a yellow solid. 11H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 2.0 Hz, 1H), 8.11 (dd, J = 8.8, 2.0 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.79 (s, 2H), 3.95 (s, 3H), 3.80 (s, 3H), 3.24 (t, J = 8.0 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H), 2.36 - 2.19 (m, 2H). LC-MS: Rt = 1.036 min, (ESI) m / z 363.1 [M + H] + . C 22 H 22 N2O3 362.16

[0070] Step 6: Methyl 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (7) A solution of methyl 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopenta[d]quinoline-8-carboxylate (8.1 g, 20 mmol) in CF3COOH (50 mL) is stirred at 70 °C for 12 h. Then the solvent is removed and the residue is dissolved in DCM (200 mL). The organic phase is washed with NaHCO3(aq.) (300 mL) and dried over Na2SO4. Then the mixture is filtered and the filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a DCM solution of MeOH from 0% to 5% over 20 min to give methyl 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (4.6 g, 85% yield) as a yellow solid. LC-MS: Rt = 0.921 min, (ESI) m / z 243.1 [M + H] + . C 14 H 14 N2O 242.11.

[0071] Step 7: 4-Amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylic acid (Intermediate A1) A solution of methyl 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (0.60 g, 2.31 mmol) in HCl (4 mol / L in H2O, 20 mL) is stirred at 95 °C for 12 h. The solvent is then removed and the residue is 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylic acid as a white solid (500 mg, 88% yield), which is used in the next step without further purification. 1 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 2H), 8.31 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 3.32 (t, J = 8.0 Hz, 2H), 2.95 (t, J = 8.0 Hz, 2H), 2.34 - 2.20 (m, 2H). LC-MS: Rt = 0.763 min, (ESI) m / z 229.1 [M+H] + . C 13 H 12 N2O2 228.09.

[0072] General method: Synthesis of Intermediate A2 Synthetic route: [Chemical formula]

[0073] Step 1: Methyl 2,5-difluoro-4-nitrobenzoate (2) 2,5-Difluoro-4-nitrobenzoic acid (1) (50 g, 246.18 mmol, 1 equiv) is dissolved in methanol (500 mL), and thionyl chloride (43.93 g, 369.28 mmol, 26.79 mL, 1.5 equiv) is added at 0 °C. The reaction mixture is reacted at 40 °C for 16 h. Completion of the reaction is indicated by LCMS. The reaction mixture is concentrated to dryness under reduced pressure. After dilution by adding 300 mL of water, it is extracted three times with 1 L of ethyl acetate. The organic phase is washed with saturated brine (400 mL), dried over anhydrous magnesium sulfate, the filtrate is filtered, and concentrated to dryness under reduced pressure. The crude product is slurried with petroleum ether at 25 °C for 60 min to obtain methyl 2,5-difluoro-4-nitrobenzoate (2) as a white solid (103 g, 474.38 mmol, 96.35% yield). HNMR: ES23714-64-P1A, 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.89 (td, J = 9.51, 5.63 Hz, 2H) 4.00 (s, 3H). 19F NMR (376 MHz, CHLOROFORM-d) δ ppm -110.27 (s, 1F) -121.56 (m, 1F)

[0074] Step 2: Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) Methyl 2,5-difluoro-4-nitrobenzoate (2) (80 g, 368.45 mmol, 1 equiv), 2-methyl-1H-imidazole (36.30 g, 442.14 mmol, 1.2 equiv) are dissolved in dimethyl sulfoxide (1.2 L). The reaction solution is reacted at 50 °C for 16 h. Completion of the reaction is indicated by LCMS. After adding 4 L of water to dilute the reaction solution, it is extracted with 4.5 L of ethyl acetate. The organic phase is washed with saturated brine (3 L), dried over anhydrous magnesium sulfate, the filtrate is filtered, and concentrated to dryness under reduced pressure. The crude product is slurried with methyl t-butyl ether at 25 °C for 60 min (25.5 g). (2) The mother liquor is purified by column chromatography (silica, 50% tetrahydrofuran in petroleum ether) to obtain a yellow liquid, which is slurried with methyl t-butyl ether at 25 °C for 60 min to obtain a yellow solid (8.23 g). Yellow solid 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) (recovery of raw material) (20.34 g, 93.68 mmol, 25.43% yield). Yellow solid methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) (8.2 g, 29.07 mmol, 7.89% yield, 99% purity). HNMR:ES23714-67-P1A1, 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.39 (d, J = 9.90 Hz, 1H) 8.08 - 8.20 (m, 1H) 7.23 (d, J = 1.32 Hz, 1H) 6.92 (s, 1H) 3.91 (s, 3H) 2.12 (s, 3H). 19F NMR (376 MHz, DMSO-d6) δ ppm -105.45 (br s, 1F)

[0075] Step 3: Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4) 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 on carbon hydroxide (2.8 g, 49.78 mmol, 20% purity, 1 eq) was added under a hydrogen atmosphere. The reaction system was replaced with hydrogen gas three times. The reaction solution was heated to 50 °C under a hydrogen gas (1 eq) (50 psi) atmosphere and reacted for 32 hours. Completion of the reaction was indicated by LCMS. 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 obtain 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). 1 H 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).

[0076] Step 4: Methyl 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) At 25 °C, methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4) (12 g, 48.15 mmol, 1 equiv) is added to 1-methyl-2-pyrrolidone, and 1,1-carbonyldiimidazole (19.52 g, 120.37 mmol, 2.5 equiv) is added. The reaction mixture is heated to 115 °C and reacted for 16 h. Completion of the reaction is indicated by LCMS. The reaction mixtures of two batches are combined and processed. 600 mL of ethyl acetate and 600 mL of water are added to the reaction mixture, and it is slurried at 25 °C for 16 h. The slurry is filtered under reduced pressure, and the filter cake is washed with 100 mL of ethyl acetate. The solid is concentrated under reduced pressure to obtain 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) as a gray solid. The crude product is used directly in the next-step reaction. 1 H 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)

[0077] Step 5: Methyl 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) 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 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) are added to acetonitrile (240 mL). Benzotriazol-1-oxo-tris(dimethylaminophosphine) hexafluorophosphate (25.07 g, 56.68 mmol, 1.3 eq) is added in one batch at 15 - 20 °C. The reaction mixture exotherms slightly, becomes homogeneous, and a solid precipitates. The reaction mixture is reacted at 15 - 20 °C for 16 h under nitrogen gas protection. LCMS indicates complete consumption of the starting material and detection of the target compound. The reaction suspension is filtered under reduced pressure, and the filter cake is washed with 100 mL of acetonitrile. The solid is collected and dried by suction under reduced pressure to obtain the off-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). LCMSES15882-1146-P1A: (ESI) m / z = 425.3 [M+1] + ; RT = 1.721 min. 1 H NMR (400 MHz, DMSO-d6) Shift 8.49 (d, J = 7.00 Hz, 1H), 8.45 (t, J = 5.57 Hz, 1H), 7.95 (s, 1H), 7.23 (d, J = 12.51 Hz, 1H), 7.18 (d, J = 8.38 Hz, 1H), 6.58 (d, J = 2.38 Hz, 1H), 6.47 (dd, J = 2.38, 8.38 Hz, 1H), 4.66 (d, J = 5.25 Hz, 2H), 3.88 (s, 3H), 3.82 (s, 3H), 3.73 (s, 3H), 2.93 (s, 3H). 19F NMR (376.5 MHz, DMSO-d6) Shift -113.02.

[0078] Step 6: Methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7) 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 eq) is added to dichloromethane (50 mL), and trifluoroacetic acid (250 mL) is added. The reaction solution is heated to 50 °C and reacted for 16 hours. LCMS indicates complete consumption of the starting material and detection of the target compound. The reaction solution is concentrated to dryness under reduced pressure to obtain purple solid methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7) (28.3 g, crude product), and the crude product is used directly in the next step reaction. LCMSES15882-1150-P1A: (ESI) m / z = 275.3 [M+1] + ; RT = 0.607 min

[0079] Step 7: 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2) 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 equivalent) is added to tetrahydrofuran (80 mL) and methanol (80 mL). Sodium hydroxide (7.74 g, 193.48 mmol, 5 equivalents) is dissolved in water (80 mL) and then added to the reaction solution. The reaction solution is heated to 50 °C and reacted for 4 hours. The required compound is detected by LCMS. After the reaction solution is cooled to 20 °C, it is concentrated under reduced pressure to remove the organic solvent, and the residue is diluted with 10 to 1 water to methanol (300 mL), then filtered through diatomaceous earth, and the filter cake is washed repeatedly 3 times with 10 to 1 water to methanol (300 mL). All the filtrates are combined and concentrated under reduced pressure to remove methanol. The pH of the residue is adjusted to 5 - 6 with acetic acid. The resulting slurry is stirred at 15 - 20 °C for 12 hours and filtered under reduced pressure, and the obtained solid is washed with water. The solid is collected and lyophilized 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). LCMSES15882-1154-P1C: (ESI) m / z = 261.1 [M + 1] + ; RT = 0.422 min. 1 H NMR (400 MHz, DMSO-d6) Shift 13.15 (br s, 1H), 8.53 (d, J = 7.04 Hz, 1H), 7.85 (s, 1H), 7.68 (s, 2H), 7.16 (d, J = 12.10 Hz, 1H), 2.94 (s, 3H)

[0080] General method: Synthesis of Intermediate A3 Synthetic route:

Chemical formula

[0081] Step 1: 1-(5-Bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2) 1-Bromo-2-chloro-5-fluoro-4-nitrobenzene (1) (3 g, 11 mmol, 1 equiv) was added to a solution of 2-methyl-1H-imidazole (1.2 g, 14 mmol, 1.2 equiv) in acetonitrile (50 mL), and then potassium carbonate (4 g, 29 mmol, 2.5 equiv) was added to the reaction mixture. The temperature was raised to 80 °C and the mixture was stirred for 16 h. Completion of the reaction of the starting materials was detected and the target product was formed. The mixture was concentrated under reduced pressure to remove acetonitrile, water (40 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The extract was dried over anhydrous magnesium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by column chromatography (silica, 35% tetrahydrofuran in petroleum ether) to obtain 1-(5-bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2) (4 g) as a white solid. H NMR: 1 H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.32 (s, 1H), 7.22 (d, J = 1.32 Hz, 1H), 6.91 (d, J = 1.32 Hz, 1H), 2.08 - 2.23 (m, 3H).

[0082] Step 2: 4-Bromo-5-chloro-2-(2-methylimidazol-1-yl)aniline (3) 1-(5-Bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2) (3.5 g, 11 mmol, 1 equiv) was dissolved in a mixed solution of (8 mL), ethanol (16 mL) and tetrahydrofuran (16 mL), ammonium chloride (8.9 g, 166 mmol, 15 equiv) was added to the reaction mixture, the temperature was raised to 70 °C, and iron powder (2.5 g, 44 mmol, 4 equiv) was added to the reaction mixture. The reaction mixture was stirred at 90 °C for 2 h. Completion of the reaction of the starting materials was detected by LC-MS and the target product was formed. The reaction mixture was filtered through diatomaceous earth and washed with ethyl acetate (40 mL × 3). The solvent was removed under reduced pressure to obtain 4-bromo-5-chloro-2-(2-methylimidazol-1-yl)aniline (3) (3.1 g, 10.8 mmol, 98% yield) as a black solid. H NMR: 11H NMR (400 MHz, DMSO-d6) δ 7.41 (s, 1H), 7.12 (s, 1H), 7.07 (s, 1H), 6.99 (s, 1H), 5.45 (s, 2H), 2.13 (s, 3H).

[0083] Step 3: 8-Bromo-7-chloro-1-methyl-5H-imidazo[1,5-a]quinoxalin-4-one (4) 4-Bromo-5-chloro-2-(2-methylimidazol-1-yl)aniline (3) (3 g, 10.5 mmol, 1 equiv) and 1,1-carbonyldiimidazole (2.6 g, 15.7 mmol, 1.5 equiv) are successively dissolved in a solution of 1,2-dichlorobenzene (30 mL). The reaction solution is stirred at 130 °C for 16 h. Completion of the reaction of the raw materials is detected by LC-MS, and the target product is formed. The reaction solution is stirred with a solution of ethyl acetate and water (2 / 1 15 mL) for 30 min and then filtered to obtain a filter cake. The filter cake is concentrated under vacuum to obtain a black solid, 8-bromo-7-chloro-1-methyl-5H-imidazo[1,5-a]quinoxalin-4-one (4) (2.2 g, 7 mmol, 67% yield). 1H NMR: 1 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 7.33 (d, J = 3.30 Hz, 2H), 2.83 (s, 3H)

[0084] Step 4: Methyl 7-chloro-1-methyl-4-oxo-5H-imidazo[1,5-a]quinoxaline-8-carboxylate (5) 8-Bromo-7-chloro-1-methyl-5H-imidazo[1,5-a]quinoxalin-4-one (4) (500 mg, 1.6 mmol, 1 equiv) was dissolved in ethanol (5 mL) solution, 1,8-diazabicyclo[5.4.0]undec-7-ene (365 mg, 2.4 mmol, 362 μL, 1.5 equiv) was added, the nitrogen gas was replaced three times, tributylphosphonium tetrafluoroborate (46 mg, 160 μmol, 0.1 equiv), molybdenum hexacarbonyl (232 mg, 880 μmol, 118 μL, 0.55 equiv) and palladium acetate (36 mg, 160 μmol, 0.1 equiv). The reaction mixture was stirred at 90 °C for 2 h. The completion of the reaction of the raw material was detected by LC-MS, and the target product was generated. Ethanol was removed under reduced pressure from the reaction solution, water (10 mL) was added, and the mixture was extracted with ethyl acetate 30 mL (10 mL×3). It was dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, 30% tetrahydrofuran in petroleum ether) gave 7-chloro-1-methyl-4-oxo-5H-imidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5) (500 mg) as a yellow solid.

[0085] Step 5: Methyl 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) Methyl 7-chloro-1-methyl-4-oxo-5H-imidazo[1,5-a]quinoxaline-8-carboxylate (5) (480 mg, 1.6 mmol, 1 equiv) was dissolved in acetonitrile (5 mL), and 2,4-dimethoxybenzylamine (341 mg, 2 mmol, 306 μL, 1.3 equiv), benzotriazol-1-oxo-tris(dimethylaminophosphine) hexafluorophosphate (1 g, 2.4 mmol, 1.5 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.2 g, 7.9 mmol, 1.2 mL, 5 equiv) were added. The reaction mixture was stirred at room temperature for 16 h. Completion of the reaction of the starting material was detected by LC-MS, and the desired product was formed. Acetonitrile was removed under reduced pressure from the reaction solution, water (8 mL) was added, and the mixture was extracted with ethyl acetate (30 mL, 10 mL×3). The extract was dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, 30% tetrahydrofuran in petroleum ether) gave the yellow solid methyl 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (500 mg, 1.1 mmol, 70% yield).

[0086] Step 6: 7-Chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3) Methyl 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (500 mg, 1.1 mmol, 1 equiv) is dissolved in water (5 mL) and ethanol (5 mL), and sodium hydroxide (132 mg, 3.3 mmol, 3 equiv) is added. The reaction mixture is stirred at 50 °C for 5 h. Completion of the reaction of the starting material is detected by LCMS, and the target product is formed. 6 mol / L hydrochloric acid (0.5 mL) is added to the reaction mixture, and it is concentrated to dryness under reduced pressure. The crude product is used directly in the next step to obtain the white solid 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3) (350 mg, 820 μmol, 74.6% yield).

[0087] General method: Synthesis of Intermediate A4 Synthetic route:

Chemical formula

[0088] Step 1: Methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate Methyl 3-fluoro-4-nitrobenzoate (1) (2.00 g, 10.04 mmol, 1 equiv) is added to acetonitrile (40 equiv), potassium carbonate (4.16 g, 30.13 mmol, 3 equiv) and 2,4-dimethyl-1H-imidazole (2) (965 mg, 10.04 mmol, 1 equiv) are added, and the reaction mixture is reacted at 85 °C for 16 h. Complete consumption of the starting material is indicated by LC-MS, and the target product is formed. The reaction mixture is concentrated to dryness under reduced pressure, the residue is diluted with dichloromethane (80 equiv), filtered, the filtrate is dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the yellow solid methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate (3) (2.6 g, 9.45 mmol, 94.05% yield), which is used directly in the next step without further purification as the crude product.

[0089] Step 2: Methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate Methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate (3) (2.5 g, 9.08 mmol, 1 eq) is dissolved in ethanol (20 eq), tetrahydrofuran (20 eq) and water (10 eq), and iron powder (5.07 g, 90.82 mmol, 10 eq) and ammonium chloride (2.43 g, 45.41 mmol, 5 eq) are added at room temperature. The reaction mixture is reacted at 90 °C for 16 h. LC-MS shows complete consumption of the starting material and formation of the desired product. The reaction mixture is filtered, concentrated to dryness under reduced pressure, the residue is diluted with water (50 eq), extracted with ethyl acetate (40 eq × 3), the organic phases are combined, dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure to give yellow solid methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate (4) (1.9 g, 6.13 mmol, 67.49% yield, 79.13% purity), and the crude product is used directly in the next step without further purification. LCMS: ES19974-375-P1C2, (ESI) m / z = 246.1 [M+1] + ; RT = 0.61 min, purity: 79.13%

[0090] Step 3: Methyl 1,3-dimethyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate Methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate (4) (500 mg, 2.04 mmol, 1 equiv) and 1,1-carbonyldiimidazole (495 mg, 3.06 mmol, 1.5 equiv) were dissolved in 1,2-dichlorobenzene (10 equiv), and the reaction mixture was reacted at 120 °C for 16 h under nitrogen gas protection. Complete consumption of the starting material was indicated by LC-MS, and the desired product was formed. The reaction mixture was suction filtered, the filter cake was slurried with water, and dried under reduced pressure to obtain a brown solid, methyl 1,3-dimethyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (460 mg, 1.62 mmol, 79.48% yield, 95.55% purity), which was used in the next step without further purification as the crude product as it was. LCMS:ES19974-392-P1B1,(ESI)m / z=272.0[M+1] + ;RT=0.61min,purity:95.55%

[0091] Step 4: Methyl 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylate Methyl dimethyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (700 mg, 2.58 mmol, 1 equiv) was dissolved in acetonitrile (25 equiv), and benzotriazol-1-oxo-tris(dimethylaminophosphine) hexafluorophosphate (1.83 g, 4.13 mmol, 1.6 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.96 g, 12.90 mmol, 1.94 equiv, 5 equiv) were added. The reaction mixture was reacted at room temperature for 30 minutes, and (2,4-dimethoxyphenyl)methylamine (647.2 mg, 3.87 mmol, 583.1 μL, 1.5 equiv) was added to the reaction mixture. The reaction mixture was reacted at 50 °C for 15.5 hours. Complete consumption of the starting material was indicated by LC-MS, and the target product was formed. The reaction mixture was filtered, and the filter cake was dried under reduced pressure to obtain a brown solid, methyl 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (900 mg, 2.08 mmol, 80.57% yield, 97.13% purity). Without further purification, the crude product was used directly in the next step. LCMS: ES19974-397-P1B1, (ESI) m / z = 421.1 [M+1] + ; RT = 0.78 min, purity: 97.13%

[0092] Step 5: 4-((2,4-Dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylic acid Methyl 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (850 mg, 2.02 mmol, 1 equiv) was dissolved in methanol (10 equiv), tetrahydrofuran (10 equiv) and water (5 equiv), and lithium hydroxide (424.2 mg, 10.11 mmol, 5 equiv) was added. The reaction mixture was reacted at 50 °C for 16 h. LC-MS indicated complete consumption of the starting material and the formation of the desired product. The reaction mixture was concentrated to dryness under reduced pressure, acetic acid was added to the residue to adjust the pH to 6 - 7, filtered, and the filter cake was dried under reduced pressure to obtain 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A4) (800 mg, 1.97 mmol, 97.37% yield) as a brown solid, and the crude product was used directly in the next step without further purification.

[0093] General method: Intermediate A5 and Intermediate A5b [Chemical formula]

[0094] Intermediate A5 is a known compound. Intermediate A5 was separated by SFC (conditions: chromatography column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm), mobile phase: [MeOH (0.1% IPAm)], B%: 42% - 42%, 15 min) to obtain Intermediate A5a (600 mg, 2.46 mmol, 40.00% yield) as a white solid and Intermediate A5b (600 mg, 2.46 mmol, 40.00% yield) as a white solid. Note: The chiral center is not in an absolute configuration and is designated in the order of SFC separation.

[0095] Intermediate A5a: 11H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J = 1.9 Hz, 1H), 7.98 (dd, J = 1.9, 8.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 6.74 (s, 2H), 5.47 - 5.35 (m, 2H), 5.34 - 5.25 (m, 1H), 1.41 (d, J = 6.0 Hz, 3H). LC-MS, [M+H] + 245.0

[0096] Intermediate A5b: 1 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 1.8 Hz, 1H), 7.98 (dd, J = 1.9, 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6.68 (s, 2H), 5.46 - 5.35 (m, 2H), 5.33 - 5.26 (m, 1H), 1.41 (d, J = 6.1 Hz, 3H) LC-MS, [M+H] + 245.0

[0097] General method: Synthesis of Intermediate A6 Synthesis route: [Chemical formula]

[0098] Step 1: N-(4-Bromo-2-fluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide (3) A solution of 4-bromo-2-fluoroaniline (5.78 g, 30.4 mmol, 1 eq) and 4-methyl-1H-pyrazole-5-carboxylic acid (4.60 g, 36.5 mmol, 1.2 eq) in Py (120 mL) was added with POCl3 (4.66 g, 30.4 mmol, 2.82 mL, 1 eq), and stirred at 0 °C for 1 h. LC-MS (ET63399-4-R1A1) indicated the complete consumption of the starting materials and detected the major peak with the required m / z. The reaction mixture was quenched with ice water (200 mL), then extracted with ethyl acetate (60 mL × 6). The combined organic layers were washed with brine (30.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was triturated with ethyl acetate (50.0 mL) to give N-(4-bromo-2-fluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide (3) as a white solid (7.09 g, 23.8 mmol, 78.2% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.26 (br s, 1H) 9.52 (s, 1H) 7.92 (t, J = 8.52 Hz, 1H) 7.70 (s, 1H) 7.62 (dd, J = 10.31, 1.85 Hz, 1H) 7.41 (br d, J = 8.70 Hz, 1H) 2.25 (s, 3H). LC-MS; [MH] + 298.0

[0099] Step 2: 8-Bromo-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (4) NaH (1.43 g, 35.7 mmol, 60% purity, 1.5 eq) was added to a solution of N-(4-bromo-2-fluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide (3) (7.09 g, 23.8 mmol, 1 eq) in DMA (70 mL). The mixture was stirred at 120 °C for 16 h. LC-MS indicated the complete consumption of the starting materials and the major peak had the required m / z. The reaction mixture was quenched with saturated ammonium chloride (300 mL), the precipitate was collected, washed with water (50 mL), and then concentrated under reduced pressure to give 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (4) as a white solid (6.50 g, crude product).1 1H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 2.00 Hz, 1H) 7.91 (s, 1H) 7.52 (dd, J = 8.63, 2.00 Hz, 1H) 7.28 (d, J = 8.63 Hz, 1H) 2.42 (s, 3H). LC-MS; [M+H] + 278.0

[0100] Step 3: 8-Bromo-N-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrazolo[1,5-a]quinoxalin-4-amine (5) To a solution of 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (4) (1.00 g, 3.60 mmol, 1 equiv) in MeCN (10.0 mL) is added PMBNH2 (1.23 g, 8.99 mmol, 1.16 mL, 2.5 equiv), BOP (3.18 g, 7.19 mmol, 2 equiv)) and DBU (2.74 g, 18.0 mmol, 2.71 mL, 5 equiv). The mixture is stirred at 50 °C for 16 h. LC-MS indicates complete consumption of the starting material and one major peak with the required m / z. The reaction mixture is diluted with saturated ammonium chloride (10.0 mL) and ethanol (5.00 mL). The precipitate is collected, washed with water (10.0 mL) and then concentrated under reduced pressure to afford 8-bromo-N-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrazolo[1,5-a]quinoxalin-4-amine (5) (1.09 g, crude product) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 1.88 Hz, 1H) 7.95 (s, 1H), 7.44 - 7.48 (m, 2H) 7.38 (d, J = 8.63 Hz, 2H) 7.28 (br s, 1H) 6.87 (d, J = 8.75 Hz, 2H) 4.70 (d, J = 5.88 Hz, 2H) 3.71 (s, 3H) 2.53 (s, 3H). LC-MS; [M+H] + 399.0

[0101] Step 4: 8-Bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6) A solution of 8-bromo-N-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrazolo[1,5-a]quinoxalin-4-amine (5) (500 mg, 1.26 mmol, 1 equiv) in TFA (5 mL) is stirred at 60 °C for 12 h. Complete consumption of the starting material is indicated by LC-MS, and the required m / z is present in one major peak. The reaction mixture is concentrated under reduced pressure to afford the compound 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6) (400 mg, crude product) as a yellow solid. LC-MS: [MH] + 277.0

[0102] Step 5: Ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7) To a solution of 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6) (400 mg, 1.08 mmol, 1 equiv) in EtOH (4 mL) are added Mo(CO)6 (143 mg, 541 μmol, 72.9 μL, 0.5 equiv), DBU (659 mg, 4.33 mmol, 652 μL, 4 eq), (t-Bu)3PBF4 (94.2 mg, 325 μmol, 0.3 eq) and Pd(OAc)2 (36.5 mg, 162 μmol, 0.15 eq). The mixture is stirred at 90 °C for 12 h. Complete consumption of the starting material is indicated by LC-MS (ET63399-28-R1A1), and the required mass is detected. The reaction mixture is diluted with saturated ammonium chloride (10 mL) and ethanol (10 mL). The precipitate is collected, washed with water (10.0 mL), and then concentrated under reduced pressure to afford ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7) (250 mg, 925 μmol, 85.4% yield) as a yellow solid. LC-MS; [MH] + 271.1

[0103] Step 6: 4-Amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A6) To a solution of ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7) (250 mg, 925 μmol, 1 equiv) in EtOH (3 mL) and H2O (1 mL) is added LiOH·H2O (116 mg, 2.77 mmol, 3 equiv). The mixture is stirred at 25 °C for 12 h. Complete consumption of the starting material is indicated by LC-MS (ET63399-32-R1A1), and the required mass is detected. The reaction mixture is concentrated, diluted with water (10 mL), and washed with DCM (10.0 mL×3) to remove impurities. The aqueous phase is adjusted to pH = 5 with 1M HCl, the precipitate is collected, and purified by preparative HPLC (column: Phenomenex Luna C18 75×30 mm×3 μm, mobile phase: [water (FA)-ACN], B%: 1% - 35%, 8 min, UV220 nm and 254 nm) to give 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A6) as a yellow solid (60.0 mg, 248 μmol, 26.8% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 12.94 (br s, 1H) 8.67 (d, J = 1.96 Hz, 1H) 7.96 (s, 1H) 7.89 (dd, J = 8.44, 1.96 Hz, 1H) 7.52 (d, J = 8.44 Hz, 1H) 7.19 (br s, 2H) 2.49 (br s, 3H). LC-MS, [M+H] + 243.1

[0104] General method: Synthesis of Intermediate A7 Synthetic route:

Chemical Structure

[0105] Step 1: Methyl 2-fluoro-5-(4-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) A mixture of methyl 2,5-difluoro-4-nitrobenzoate (20.0 g, 92.1 mmol, 1 equiv) and 5-methyl-1H-imidazole (7.56 g, 92.1 mmol, 1 equiv) in DMSO (200 mL) is stirred at 50 °C for 12 h. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The residue is diluted with H2O (400 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers are washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue is purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 - 0 / 1) to give methyl 2-fluoro-5-(4-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (9.30 g, 33.3 mmol, 34.2% yield) as a yellow solid. LC-MS (ESI) m / z = 280.1 [M+H] +

[0106] Step 2: Methyl 4-amino-2-fluoro-5-(4-methyl-1H-imidazol-1-yl)benzoate (4) Iron powder (7.44 g, 133 mmol, 4 equiv) is added to a solution of methyl 2-fluoro-5-(4-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (9.30 g, 33.3 mmol, 1 equiv) and NH4Cl (26.7 g, 499 mmol, 15 equiv) in EtOH (90.0 mL) / THF (90.0 mL) / H2O (45.0 mL). The mixture is stirred at 80 °C for 2 h. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The reaction mixture is filtered, the filtrate is diluted with water (300 mL), and extracted with EtOAc (200 mL × 3). The combined organic layers are washed with brine (100 mL), dried over Na2SO4, and concentrated under reduced pressure to give methyl 4-amino-2-fluoro-5-(4-methyl-1H-imidazol-1-yl)benzoate (8.00 g, 32.1 mmol, 95.2% yield) as a yellow solid. LC-MS (ESI) m / z = 250.0 [M+H] +

[0107] Step 3: Methyl 7-fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate (5) Methyl 4-amino-2-fluoro-5-(4-methyl-1H-imidazol-1-yl)benzoate (500 mg, 2.01 mmol, 1 eq) and CDI (487 mg, 2.41 mmol, 1.2 eq) are placed in 1,2-dichlorobenzene (20.0 mL) in a microwave tube. The sealed tube is heated at 150 °C for 3 h under microwave. 11 parallel reactions are carried out. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The reaction mixture is filtered and the filter cake is dried under reduced pressure to give a residue. The crude product and MTBE (20.0 mL) are triturated at 25 °C for 30 min to give methyl 7-fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate as a yellow solid (6.00 g, 21.80 mmol, 85.0% yield). LC-MS (ESI) m / z = 276.0 [M+H] +

[0108] Step 4: Methyl 7-fluoro-4-((4-methoxybenzyl)amino)-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) A solution of methyl 7-fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate 5 (6.00 g, 21.8 mmol, 1 equiv) in MeCN (120 mL) is added with BOP (19.2 g, 43.6 mmol, 2 equiv), DBU (16.5 g, 109 mmol, 16.4 mL, 5 equiv)), and PMBNH2 (7.48 g, 54.5 mmol, 7.05 mL, 2.5 equiv). The mixture is stirred at 70 °C for 16 h. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The reaction mixture is diluted with saturated ammonium chloride (20.0 mL). The filter cake is washed with water (10.0 mL) and concentrated under reduced pressure to give methyl 7-fluoro-4-((4-methoxybenzyl)amino)-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate 6 (5.00 g, 12.6 mmol, 50.0% yield) as a yellow solid. LC-MS (ESI) m / z = 395.1 [M+H] +

[0109] Step 5: Methyl 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) A solution of methyl 7-fluoro-4-((4-methoxybenzyl)amino)-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate 6 (2.00 g, 5.07 mmol, 1 equiv) in TFA (20.0 mL) is stirred at 75 °C for 16 h. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The reaction mixture is concentrated under reduced pressure to give methyl 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate 7 (1.39 g, crude product) as a yellow solid. LC-MS (ESI) m / z = 275.0 [M+H] +

[0110] Step 6: 4-Amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A7) To a solution of methyl 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylate 7 (1.39 g, 5.07 mmol, 1 equiv) in EtOH (15 mL) / H2O (5.00 mL) is added LiOH (638 mg, 15.2 mmol, 3 equiv). The mixture is stirred at 25 °C for 12 h. LCMS shows complete consumption of the starting material and one major peak with the required mass is detected. The reaction mixture is diluted with H2O (20.0 mL) and extracted with (DCM 10.0 mL × 2) to remove impurities. The aqueous layer is adjusted to pH = 6 with 2M HCl, the precipitate is collected and dried under reduced pressure to give 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A7) as a white solid (1.30 g, 5.00 mmol, 98.5% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.15 (br s, 1H), 9.12 (s, 1H), 8.56 (br d, J = 6.4 Hz, 1H), 7.35 (br s, 2H), 7.13 (br d, J = 12.1 Hz, 1H), 2.62 (s, 3H). LC-MS (ESI) m / z = 261.0 [M+H] +

[0111] General method: Synthesis of Intermediate B1 Synthetic route:

Chemical Structure

[0112] Step 1: N-Methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (1) A solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (2.0 g, 12.3 mmol) and HATU (7.0 g, 18.5 mmol) in DMF (100 mL) is added with Et3N (6.2 g, 61.7 mmol) and methoxy(methyl)amine (1.9 g, 30.83 mmol) at 25 °C. Next, the mixture is stirred at 25 °C for 12 h. The reaction is quenched with water (100 mL), and then extracted with EA (100 mL × 3). The organic solution is washed with brine (100 mL). The organic phase is dried over Na2SO4 and filtered. The filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a DCM solution of MeOH from 0% to 10% over 20 min to give N-methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (2.01 g, 79% yield) as a yellow solid. LC-MS: Rt = 1.049 min, (ESI) m / z. [M+H] + 206.1; C 10 H 11 N3O2。

[0113] Step 2: Pyrazolo[1,5-a]pyridine-2-carboxaldehyde (3) To a solution of N-methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (1.5 g, 7.31 mmol) in THF (20 mL) at -60 °C is added LiAlH4 (439 mg, 10.96 mmol). Next, the mixture is stirred at -60 °C for 2 h. The reaction is quenched with water (40 mL) and filtered. The filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with a MeOH in DCM solution from 0% to 10% over 20 min to give pyrazolo[1,5-a]pyridine-2-carboxaldehyde as a yellow oil (450 mg, 42% yield). LC-MS: Rt = 1.071 min, (ESI) m / z. [M+H] + 147.1; C8H6N2O。

[0114] Step 3: N-(Pyrazolo[1,5-a]pyridin-2-ylmethyl)-1-(pyrimidin-2-yl)ethan-1-amine (Intermediate B1) To a solution of pyrazolo[1,5-a]pyridine-2-carboxaldehyde (100 mg, 0.68 mmol) in MeOH (5 mL) were added 1-(pyrimidin-2-yl)ethylamine (126 mg, 1.03 mmol) and NaBH3CN (86 mg, 1.37 mmol), and the reaction was carried out at 25 °C. Next, the mixture was stirred at 25 °C for 2 h. The reaction was quenched with water (1 mL), and the solvent was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with a DCM solution of MeOH from 0% to 5% over 20 min to give N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-1-(pyrimidin-2-yl)ethylamine (40 mg, 23% yield) as a yellow oil. LC-MS: Rt = 0.521 min, (ESI) m / z [M + H]. + 254.2; C 14 H 15 N5.

[0115] General method: Synthesis of intermediate B2 Synthetic route:

Chemical formula

[0116] Step 1: 2-Dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) A mixture of 5-(trifluoromethyl)pyridin-2-amine (1) (30 g, 185 mmol, 1 equiv), chlorobenzene (450 mL), and 1,1,3-trichloro-2-propanone (45 g, 277 mmol, 1.5 equiv) was reacted at 135 °C for 4 h. The formation of the target product was detected by LCMS. The pH of the reaction solution was adjusted to about 8 with sodium carbonate, extracted with ethyl acetate (500 mL × 3), the organic phases were combined, dried over magnesium sulfate, the filtrate was filtered, concentrated to dryness under reduced pressure, and purified by column chromatography (silica, 15% ethyl acetate in petroleum ether) to give the yellow solid 2-dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 60% yield). H NMR: ES19506-784-P1A 11H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.27 (s, 1H), 7.79 (d, J = 9.68 Hz, 1H), 7.65 (s, 1H), 7.56 (dd, J = 1.65, 9.57 Hz, 1H).

[0117] Step 2: 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) 2-Dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 1 equiv), water (600 mL), calcium carbonate (33 g, 334 mmol, 3 equiv) are heated to 100 °C and reacted for 2 h. The formation of the target product is detected by LCMS. Diatomaceous earth and ethyl acetate (600 mL) are added to the reaction solution, stirred at room temperature for 30 min, filtered, and extracted with ethyl acetate (600 mL × 2). The organic phases are combined, dried over magnesium sulfate, filtered, and the filtrate is concentrated to dryness under reduced pressure to obtain 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (35 g), a brown solid, which is used in the next step without further purification. 1H NMR: ES19506-789-P1A1, 1 1H NMR (400 MHz, CHLOROFORM-d) δ 10.09 - 10.29 (m, 1H), 8.59 (s, 1H), 8.27 (s, 1H), 7.82 (br d, J = 9.46 Hz, 1H), 7.44 (br d, J = 9.02 Hz, 1H).

[0118] Step 3: 1-Methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2) A solution of 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (100 mg, 467 μmol, 1 equiv) in DCM (2.00 mL) was added with KOAc (91.7 mg, 934 μmol, 2 equiv) and 1-methyl-1H-pyrazol-4-amine (45.4 mg, 467 μmol, 1 equiv) at -5 °C, and the reaction mixture was stirred at -5 °C for 1 h. Next, NaBH(OAc)3 (198 mg, 934 μmol, 2 equiv) was added and the mixture was stirred at -5 °C for 3 h. LCMS (ET63219-45-P1A1) indicated complete consumption of Cpd.4 and showed several new peaks by LCMS. The reaction mixture was diluted with saturated aqueous Na2CO3 solution (3.00 mL) and extracted with dichloromethane (2.00 mL × 4). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (ethyl acetate / methanol = 8 / 1) to afford 1-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2) as a yellow solid (80.0 mg, 271 μmol, 58.0% yield). 1 H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.69 (d, J = 9.5 Hz, 1H), 7.64 (s, 1H), 7.35 (d, J = 1.5, 9.5 Hz, 1H), 7.30 (s, 2H), 6.97 (s, 1H), 4.39 (s, 2H), 3.81 (s, 3H), LC-MS, [MH] + 217.0.

[0119] General method: Synthesis of Intermediate B3 Synthetic route:

Chemical formula

[0120] Step 1: 1,3-Dimethyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B3) 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (10 g, 46 mmol, 1 equiv) and 1,3-dimethylpyrazol-4-amine (6 g, 56 mmol, 1.2 equiv) were dissolved in dichloromethane (150 mL), acetic acid (3 g, 56 mmol, 3 mL, 1.2 equiv) was added, and the reaction mixture was reacted at 25 °C for 1 h. Sodium triacetoxyborohydride (25 g, 117 mmol, 2.5 equiv) was added, and the reaction mixture was reacted at 25 °C for 3 h. The formation of the target product was detected by LCMS. The reaction mixture was quenched with 200 mL of sodium bicarbonate and extracted with ethyl acetate (150 mL × 2). The combined organic phases were washed with 400 mL of saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, 35% ethyl acetate in petroleum ether:ethanol (3:1)) gave 1,3-dimethyl-N-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B3) (14 g, 45 mmol, 97% yield) as a brown solid. H NMR: ES19506-795-P1A, 1 H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 7.92 (s, 1H), 7.67 (d, J = 9.46 Hz, 1H), 7.41 (dd, J = 1.76, 9.46 Hz, 1H), 6.93 (s, 1H), 4.56 (br s, 1H), 4.17 (br d, J = 3.96 Hz, 2H), 3.52 - 3.61 (m, 3H), 2.04 (s, 3H). LCMS: ES19506-795-P1B, (ESI) m / z = 310.3 (M + 1)+, RT = 0.64 min.

[0121] General method: Synthesis of Intermediate B4 Synthetic route:

Chem.

[0122] Step 1: t-Butyl (thiazolo[4,5-c]pyridin-2-ylmethyl)carbamate (3) A solution of 4-iodopyridine-3-amine (20.0 g, 90.9 mmol, 1 equiv) and t-butyl(2-amino-2-thioethyl)carbamate (20.7 g, 109 mmol, 1.2 equiv) in MeCN (200 mL) was added with Pd2(dba)3 (4.16 g, 4.55 mmol, 0.05 equiv), DPPF (10.1 g, 18.2 mmol, 0.2 equiv) and CaO (10.2 g, 182 mmol, 3.09 mL, 2 equiv), and the mixture was stirred at 80 °C for 16 h under N2. Complete consumption of the starting material was indicated by LCMS and the required mass was detected. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 30 / 1~1 / 1) to afford t-butyl(thiazolo[4,5-c]pyridin-2-ylmethyl)carbamate (3) (17 g, 64.0 mmol, 70.5% yield) as a brown solid. 1 H NMR(400MHz,CHLOROFORM-d)δ 9.28(s,1H),8.53(d,J=5.4Hz,1H),7.84(d,J=5.4Hz,1H),5.41(br s,1H),4.77(br d,J=5.8Hz,2H),1.50(s,9H)、LC-MS、[MH] + 266.1

[0123] Step 2: Thiazolo[4,5-c]pyridin-2-ylmethylamine hydrochloride (4) HCl / EtOAc (4M, 15 mL) was added to a solution of t-butyl(thiazolo[4,5-c]pyridin-2-ylmethyl)carbamate (3) (3 g, 11.3 mmol, 1 equiv) in DCM (15 mL). The mixture was stirred at 25 °C for 16 h. Complete consumption of the starting material was indicated by LCMS (ET63218-11-P1A2) and the required mass was detected. The reaction mixture was concentrated under reduced pressure to afford thiazolo[4,5-c]pyridin-2-ylmethylamine hydrochloride (4) (2 g, crude product, HCl) as a pale yellow solid. LC-MS, [MH] + 166.1

[0124] Step 3: 1-(Pyrimidin-2-yl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)ethan-1-amine (Intermediate B4) To a solution of thiazolo[4,5-c]pyridin-2-ylmethylamine hydrochloride (4) (2 g, 9.92 mmol, 1 eq) and 1-(pyrimidin-2-yl)ethan-1-one (1.21 g, 9.92 mmol, 1 eq) in DCM (20 mL) was added KOAc (1.17 g, 11.9 mmol, 1.2 eq). The mixture was stirred at 25 °C for 30 min, then at 25 °C, NaBH(OAc)3 (2.73 g, 12.9 mmol, 1.3 eq) was added to the above mixture and the mixture was stirred at 25 °C for 2 h. LCMS indicated complete consumption of the starting material and the required mass was detected. The mixture was adjusted to pH = 8 - 9 with saturated NaHCO3 and then extracted with DCM (20 mL × 3). The organic phase was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane / methanol = 50 / 1 - 10 / 1) to afford 1-(pyrimidin-2-yl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)ethan-1-amine (Intermediate B4) as a brown oil (320 mg, 1.18 mmol, 11.9% yield). 1 H NMR (400 MHz, CHLOROFORM-d) δ=9.22(s,1H),8.74(d,J=4.9Hz,2H),8.50(d,J=5.4Hz,1H),7.85(d,J=5.4Hz,1H),7.22(t,J=4.9Hz,1H),4.27(d,J=16.4Hz,1H),4.16 - 4.04(m,2H),1.54(d,J=6.9Hz,3H)、LC-MS、[MH] + 272.0

[0125] General method: Synthesis of Intermediate B5 Synthetic route:

Chemical Structure

[0126] Step 1: Pyrazolo[1,5-a]pyridine-2-carboxylic acid (2) To a solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (10.0 g, 61.67 mmol, 1 equiv), HATU (28.14 g, 74.01 mmol, 1.2 equiv), and DIEA (31.88 g, 246.69 mmol, 4 equiv) in DCM (500 mL) is added N,O-dimethylhydroxylamine hydrochloride (12.03 g, 123.35 mmol, 2 equiv). The mixture is stirred at 25 °C for 16 h. LCMS (ET63565-18-P1A) indicates complete consumption of the starting material and detection of the product. The reaction mixture is concentrated under reduced pressure to afford a residue. The residue is purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give the compound pyrazolo[1,5-a]pyridine-2-carboxylic acid (2) (12.1 g, 58.9 mmol, 95.6% yield) as a white solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.49 (d, J = 7.1 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.15 (dd, J = 7.3, 8.4 Hz, 1H), 7.00 (s, 1H), 6.85 (t, J = 6.9 Hz, 1H), 3.79 (s, 3H), 3.49 (s, 3H), LC-MS, [MH] + 206.22.

[0127] Step 2: 1-(Pyrazolo[1,5-a]pyridin-2-yl)ethan-1-one (3) To a solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (2) (2 g, 9.75 mmol, 1 equiv) in THF (20.0 mL) at -60 °C is added MeLi (11.70 mL, 1.2 equiv), and then the mixture is stirred at 25 °C for 16 h. N2 under hr. LCMS (ET63565-13-P1A1) indicates complete consumption of the starting material and detection of the product. The residue is purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1 to 3 / 1) to give 1-(Pyrazolo[1,5-a]pyridin-2-yl)ethan-1-one (3) (277 mg, 1.73 mmol, 17.7% yield) as a white solid. LC-MS, [MH] + 161.1.

[0128] Step 3: N-Ethyl-1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-amine (Intermediate B5) A mixture of 1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-one (3) (0.1 g, 624.33 μmol, 1 eq) and ethylamine (112.58 mg, 2.50 mmol, 163.39 μL, 4 eq) in DCM (2 mL) was stirred at -60 °C for 30 minutes, then NaBH4 (264.64 mg, 1.25 mmol, 2 eq) was added to the mixture at 0 °C, and the mixture was stirred at 25 °C for 16 hours under a N2 atmosphere. Complete consumption of the starting material was indicated by LCMS (ET63565-9-P1A2), and the product was detected. The reaction mixture was quenched with H2O (5 mL) and diluted with DCM (5 mL × 3), and the combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (DCM:MeOH = 10:1) to give N-Ethyl-1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-amine (Intermediate B5) as a white solid (50 mg, 264 μmol, 42.3% yield). 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.39 (br d, J = 6.9 Hz, 1H), 7.46 (br d, J = 8.5 Hz, 1H), 7.12 - 7.03 (m, 1H), 6.70 (br t, J = 6.8 Hz, 1H), 6.44 (s, 1H), 4.13 (br d, J = 6.8 Hz, 1H), 3.32 - 3.21 (m, 1H), 2.77 - 2.56 (m, 2H), 1.53 (d, J = 6.6 Hz, 4H), 1.17 - 1.14 (m, 3H): LC-MS, [MH] + 190.1.

[0129] General Method: Synthesis of Intermediate B6 Synthetic Route:

Chemical Structure

[0130] Step 1: 2-(Dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3) A mixture of 4-(trifluoromethyl)benzene-1,2-diamine (5 g, 28.3 mmol, 1 eq) and dichloroacetic acid (7.32 g, 56.7 mmol, 4.66 mL, 2 eq) in HCl (125 mL) (4 M) is stirred at 100 °C for 10 minutes. For 16 hours. Consumption of Cpd.1 is shown by LCMS (ET60224-68-P1A) and the required mass is detected. The reaction mixture is filtered and the filter cake is washed with water. The combined filtrate is extracted with DCM (20 ml×3). The combined organic layers are washed with brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give a residue. The residue is purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give the yellow oily compound 2-(dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3) (4.4 g, 16.3 mmol, 57.6% yield). 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.01(s,1H),7.79(d,J=8.6Hz,1H),7.66(d,J=8.2Hz,1H),7.26(s,1H);LCMS:[M+H] + 268.9

[0131] Step 2: 6-(Trifluoromethyl)-1H-benzo[d]imidazole-2-carboxaldehyde (4) Calcium carbonate (1.12 g, 11.1 mmol, 3 eq) is added to a suspension of 2-(dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3) (1 g, 3.72 mmol, 1 eq) in H2O (20 mL). The mixture is stirred at 100 °C for 8 hours. Consumption of Cpd.3 is shown by LCMS (ET60224-74-P1B) and the required mass is detected. The reaction mixture is diluted with H2O (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers are concentrated under reduced pressure to give 6-(trifluoromethyl)-1H-benzo[d]imidazole-2-carboxaldehyde (4) as a white solid (310 mg, 1.45 mmol, 38.9% yield). 11H NMR (400 MHz, DMSO-d6) δ 14.28 - 13.59 (m, 1H), 10.02 (s, 1H), 8.40 - 8.30 (m, 2H), 8.23 - 8.05 (m, 3H), 8.00 (dd, J = 5.4, 8.5 Hz, 1H), 7.89 (br s, 1H), 7.83 - 7.61 (m, 3H), 7.34 (br d, J = 7.9 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H); LCMS: [M+H] + 215.2。

[0132] Step 3: 2-Methyl-N-((6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)propan-1-amine (Intermediate B6) At 25 °C, KOAc (170 mg, 1.74 mmol, 1.2 eq) is added to a solution of 6-(trifluoromethyl)-1H-benzo[d]imidazole-2-carboxaldehyde (4) (0.31 g, 1.45 mmol, 1 eq) and 2-methylpropan-1-amine (105 mg, 1.45 mmol, 143 μL, 1 eq) in DCM (6.2 mL). The mixture is stirred at 25 °C for 30 minutes, then NaBH(OAc)3 (398 mg, 1.88 mmol, 1.3 eq) is added to the above mixture at 25 °C, and the mixture is stirred at 25 °C for 15.5 hours. Consumption of Cpd.4 is indicated by LCMS (ET60224-77-P1A), and the required mass is detected. The reaction mixture is diluted with H2O (10 mL) and extracted with DCM (2 mL × 3). The combined organic layers are washed with brine (10 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue is purified by preparative TLC (petroleum ether / ethyl acetate = 0 / 1) to give 2-methyl-N-((6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)propan-1-amine (Intermediate B6) as a colorless oil (100 mg, 368 μmol, 25.4% yield, 100% purity). 11H NMR (400 MHz, CHLOROFORM-d) δ 7.80 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 4.07 (s, 2H), 2.45 (d, J = 6.8 Hz, 2H), 1.82 - 1.68 (m, 1H), 0.89 (d, J = 6.6 Hz, 6H); LCMS: [M+H] + 272.0

[0133] General method: Synthesis of Intermediate B7

Chemical formula

[0134] Step: 2-Methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)propan-1-amine (Intermediate B7) To a solution of 2-methylpropan-1-amine (68.3 mg, 933 μmol, 92.8 μL, 1 equiv) and 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (0.2 g, 933 μmol, 1 equiv) in DCM (4 mL) was added KOAc (109 mg, 1.12 mmol, 1.2 equiv), and the mixture was stirred at 25 °C for 30 min at 25 °C. Then, NaBH(OAc)3 (257 mg, 1.21 mmol, 1.3 equiv) was added to the above mixture at 25 °C, and the mixture was stirred at 25 °C for 15.5 h. Consumption of Cpd.4 was indicated by LCMS (ET60224-75-P1A), and the required mass was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (2 mL × 3) to remove impurities. The aqueous layer was basified to pH = 8 with saturated Na2CO3 and then extracted with DCM (10 mL × 3). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give colorless oily Cpd.A6 (108 mg, 398 μmol, 42.6% yield, 100% purity). 11H NMR (400 MHz, CHLOROFORM-d) δ 8.49 (s, 1H), 7.70 - 7.62 (m, 2H), 7.33 (br d, J = 9.2 Hz, 1H), 4.01 (s, 2H), 2.54 (d, J = 6.7 Hz, 2H), 1.84 (quind, J = 6.6, 13.3 Hz, 1H), 0.97 (d, J = 6.6 Hz, 6H). LC-MS, [M+H] + 272.0.

[0135] Synthesis of Example 1

Chemical Structure

[0136] Step 1: 4-Amino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-N-(1-(pyrimidin-2-yl)ethyl)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxamide (Example 1) A solution of 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylic acid (80 mg, 0.35 mmol) in SOCl2 (2 mL) is stirred at 70 °C for 12 h. The solvent is concentrated to dryness and used directly in the next step of the reaction. 4-Amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carbonyl chloride (80 mg, 0.32 mmol) is added to a solution of N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-1-(pyrimidin-2-yl)ethan-1-amine (35 mg, 0.14 mmol) and Et3N (140 mg, 1.38) in THF (15 mL) at 0 °C. The mixture is then stirred at 25 °C for 12 h. The reaction is quenched with NaHCO3(aq.) (20 mL) and then extracted with EA (20 mL × 3). The organic solution is washed with brine (20 mL). The organic phase is dried over Na2SO4 and filtered. The filtrate is concentrated under reduced pressure. The residue is purified by silica gel chromatography, eluting with MeOH in DCM from 0% to 5% over 20 min to give 4-amino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-N-(1-(pyrimidin-2-yl)ethyl)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxamide (2.3 mg, 4% yield) as a yellow solid (Example 1). 1 H NMR (400 MHz, MeOD) δ 8.75 (s, 2H), 8.40 (s, 2H), 7.87 - 7.61 (m, 2H), 7.55 (d, J = 8.8 Hz, 1H), 7.32 (s, 1H), 7.18 (s, 1H), 6.83 (s, 1H), 6.51 (s, 1H), 5.68 (s, 1H), 5.40 (s, 1H), 4.73 - 4.53 (m, 2H), 3.24 (s, 2H), 2.94 (s, 2H), 2.41 - 2.13 (m, 2H), 1.84 - 1.65 (m, 3H). LC-MS: Rt = 0.981 min, (ESI) m / z. 464.2 [M + H] + C 27 H 25 N7O

[0137] Synthesis of Example 3

Chemical Structure

[0138] Step: 4-Amino-N-isobutyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 3) To a mixture of 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A14) (30 mg, 130 μmol, 1 eq) and 2-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)propan-1-amine (Intermediate B7) (35.3 mg, 130 μmol, 1 eq) in DMF (2 mL) were added TCFH (43.8 mg, 156 μmol, 1.2 eq) and NMI (32.1 mg, 390 μmol, 31.1 μL, 3 eq), and the mixture was stirred at 20 °C for 16 h under a N2 atmosphere. LC-MS indicated almost complete consumption of the starting materials and the required m / z was detected. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm, mobile phase: [water (NH4HCO3)-ACN], B%: 30% - 60%, 8 min, UV220&254 nm) to give 4-amino-N-isobutyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 3) as a white solid (37 mg, 76.5 μmol, 58.7% yield, 100% purity). 1 1H NMR (400 MHz, DMSO-d6) δ = 9.20 (br s, 1H), 7.96 (br s, 1H), 7.91 - 7.63 (m, 2H), 7.48 (br d, J = 9.4 Hz, 2H), 7.56 (br d, J = 6.4 Hz, 1H), 6.66 (br s, 2H), 5.27 (br d, J = 0.9 Hz, 2H), 5.00 (br s, 2H), 4.86 - 4.51 (m, 2H), 3.30 - 3.19 (m, 2H), 2.16 - 1.90 (m, 1H), 1.00 - 0.61 (m, 6H), LC-MS, [MH] + 484.2

[0139] Synthesis of Example 13 [Chemistry]

[0140] Step 1: (N-Imidazo[1,2-a]pyridin-2-ylmethyl)cyclobutylamine (2) Imidazo[1,2-a]pyridine-2-carboxaldehyde (1) (300 mg, 2.05 mmol, 1 equiv), cyclobutylamine (300 mg, 4.22 mmol, 361.45 μL, 2.05 equiv) were dissolved in methanol (5 mL), stirred at room temperature for 12 h, sodium borohydride (118 mg, 3.12 mmol, 1.52 equiv) was added, and stirred at room temperature for 4 h. Complete consumption of the starting material was detected by LCMS, and the target product was formed. Acetic acid (60 μL) was added to the reaction solution to quench it, concentrated to dryness under reduced pressure, 10% aqueous sodium carbonate solution (25 mL) and dichloromethane / ethanol (10:1, 25 mL) were added. The layers were separated, the aqueous phase was extracted with dichloromethane / ethanol (10:1, 25 mL × 3), the organic phases were combined, dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain a brown syrup of (N-Imidazo[1,2-a]pyridin-2-ylmethyl)cyclobutylamine (2) (401 mg, crude product). LCMSES15882-916-P1A: (ESI) m / z = 202.2 [M+1] + ; RT = 1.249 min 1 H NMR (400 MHz, CHLOROFORM-d) Shift 8.02 - 8.09 (m, 1H), 7.46 - 7.57 (m, 2H), 7.13 (ddd, J = 1.22, 6.79, 8.99 Hz, 1H), 6.73 (dt, J = 0.98, 6.79 Hz, 1H), 3.88 (s, 2H), 3.29 - 3.47 (m, 1H), 2.15 - 2.27 (m, 2H), 1.57 - 1.84 (m, 4H)

[0141] Step 2: 4-Amino-N-cyclobutyl-7-fluoro-N-(imidazo[1,2-a]pyridin-2-ylmethyl)-1-methylpyrazolo[4,3-c]quinoline-8-carboxamide (Example 13) (N-Imidazo[1,2-a]pyridin-2-ylmethyl)cyclobutylamine (2) (60 mg, 298.11 μmol, 1 eq) and N-ethyl-N-isopropyl-2-propylamine (193 mg, 1.50 mmol, 261.29 μL, 5.03 eq) were dissolved in tetrahydrofuran (5 mL), and 4-amino-7-fluoro-1-methylpyrazolo[4,3-c]quinoline-8-carbonyl chloride (104 mg, 295.80 μmol, 9.92e-1 eq, dihydrochloride) was added. The reaction mixture was stirred at room temperature for 16 h. Complete consumption of the starting material was detected by LCMS, and the desired product was formed. The reaction mixture was concentrated to dryness under reduced pressure, diluted with dimethyl sulfoxide, and purified by reverse-phase preparative liquid chromatography (basic conditions, Boston Prime C18 column 150×30 mm×5 μm, mobile phase: [water (ammonium hydroxide v / v)-acetonitrile], B% gradient: 28% - 48%, 9 min) to obtain the white solid 4-amino-N-cyclobutyl-7-fluoro-N-imidazo[1,2-a]pyridin-2-ylmethyl)-1-methylpyrazolo[4,3-c]quinoline-8-carboxamide (Example 13) (72 mg, 161.69 μmol, 54.24% yield, 99.59% purity). 1 H NMR (400 MHz, DMSO-d6) Shift 8.40 - 8.61 (m, 1H), 8.11 - 8.38 (m, 2H), 7.73 - 7.90 (m, 1H), 7.44 - 7.58 (m, 1H), 7.13 - 7.42 (m, 4H), 6.87 (t, J = 6.65 Hz, 1H), 3.86 - 5.01 (m, 6H), 2.04 - 2.31 (m, 3H), 1.78 - 1.96 (m, 1H), 1.26 - 1.68 (m, 2H). 19F NMR (376.5 MHz, DMSO-d6) Shift -116.26, -116.59.

[0142] Synthesis of Example 49 [Chemical Structure]

[0143] Step: (S)-4-Amino-3-methyl-N-(1-methyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 49) To a solution of 1-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2) (500 mg, 1.69 mmol, 1.00 eq) and (S)-4-amino-3-methyl-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A5b) (434 mg, 1.78 mmol, 1.05 eq) in DMF (5.00 mL) are added TCFH (713 mg, 2.54 mmol, 1.50 eq) and NMI (695 mg, 8.47 mmol, 675 μL, 5.00 eq). The mixture is stirred at 25 °C for 16 h. Retention of A29 is shown by LC-MS (ET68149-2-P1A1) and the required mass is detected. The solution is purified by preparative HPLC (chromatography column: Waters Xbridge BEH C18 250×50 mm×10 μm, mobile phase: [water (NH4HCO3 10 mM)-ACN], B%: 20% - 45%, 10 min, UV220 & 254 nm) to give (S)-4-amino-3-methyl-N-(1-methyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 49) as a white solid (285 mg, 535 μmol, 31.6% yield, 97.9% purity). T 1 1H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.03 (s, 1H), 7.73 (d, J = 9.5 Hz, 1H), 7.69 - 7.49 (m, 2H), 7.46 (dd, J = 1.3, 9.4 Hz, 2H), 7.42 - 6.94 (m, 2H), 6.61 (s, 2H), 5.43 - 5.35 (m, 1H), 5.27 - 5.19 (m, 1H), 5.18 - 5.10 (m, 1H), 5.05 (s, 2H), 3.66 (br s, 3H), 1.38 (d, J = 6.3 Hz, 3H), LC-MS, [MH]+ 522.2。

[0144] Synthesis of Example 90

Chemical Structure

[0145] Step: (S)-4-Amino-N-(1,3-dimethylpyrazol-4-yl)-3-methyl-N-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 90) (S)-4-Amino-3-methyl-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A5b) (3.12 g, 12.76 mmol, 1 equivalent), 1,3-dimethyl-N-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B1) (3.95 g, 12.77 mmol, 1.0 equivalent) and 1-methylpyrrolidin-2-one (30 mL) were added to a mixture of N,N,N,N-tetramethylchloroformamidinium hexafluorophosphate (4.30 g, 15.32 mmol, 1.2 equivalents) and N-methylimidazole (3.14 g, 38.29 mmol, 3.05 mL, 3.0 equivalents), and the reaction mixture was stirred at room temperature for 16 hours. Complete consumption of the starting materials was detected by LCMS, and the desired product was formed. The reaction mixture was diluted with acetonitrile (10 mL) and water (10 mL), filtered, and the filtrate was purified by reverse-phase preparative liquid chromatography (column: C18 150×40 mm, mobile phase: [water (ammonium hydroxide + ammonium bicarbonate)-acetonitrile], gradient: 17% - 57%). The fractions were lyophilized to obtain the white solid (S)-4-amino-N-(1,3-dimethylpyrazol-4-yl)-3-methyl-N-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 90) (100% purity). HNMR: ES13683-1863-P1A, 11H NMR (400 MHz, DMSO-d6) Shift 9.21 (s, 1H), 8.05 (br s, 1H), 7.71 (br d, J = 9.24 Hz, 1H), 7.59 (s, 1H), 7.28 - 7.57 (m, 4H), 6.63 (s, 2H), 5.33 - 5.49 (m, 1H), 5.07 - 5.30 (m, 2H), 4.99 (br s, 2H), 3.59 (s, 3H), 1.66 (br s, 3H), 1.38 (d, J = 6.16 Hz, 3H). 19F NMR: 19F NMR (376 MHz, DMSO-d6) Shift -60.41 (br s, 1F). LCMS: ES13683-1863-P1C, (ESI) m / z = 536.3 [M+1] + , RT = 0.802 min

[0146] Synthesis of Example 119 [Chemical Structure]

[0147] Step: 4-Amino-N-ethyl-N-(1-(pyrazolo[1,5-a]pyridin-2-yl)ethyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 119) A solution of N-ethyl-1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-amine (Intermediate B5) (50 mg, 264.19 μmol, 1 eq) in THF (1 mL) at 0 °C was added with DIEA (136.58 mg, 1.06 mmol, 184.06 μL, 4 eq) and 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carbonyl chloride (65.69 mg, 264.19 μmol, 1 eq) at 0 °C. Next, the mixture was stirred at 0 °C for 1 h under a N2 atmosphere. LCMS indicated complete consumption of the starting material and the product was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm, mobile phase: [water (NH4HCO3)-ACN], B%: 10% - 50%, 8 min, UV220&254 nm) to give 4-amino-N-ethyl-N-(1-(pyrazolo[1,5-a]pyridin-2-yl)ethyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 119) (41.9 mg, 104 μmol, 39.5% yield) as a white solid. T 1 H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 7.0 Hz, 1H), 7.82 - 7.50 (m, 4H), 7.36 - 7.14 (m, 1H), 6.88 (s, 1H), 6.69 (s, 3H), 5.42 - 5.18 (m, 3H), 5.02 (br s, 2H), 1.82 - 1.54 (m, 3H), 1.18 - 0.83 (m, 3H). LC-MS, [MH] + 402.1.

[0148] Synthesis of Example 210

Chemical formula

[0149] Step 1: 7-Chloro-4-[(2,4-dimethoxyphenyl)methylamino]-1-methyl-N-(1-methylpyrazol-4-yl)-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]imidazo[1,5-a]quinoxaline-8-carboxamide (3) 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3) (145 mg, 339 μmol, 1 equiv) was added to a solution of 1-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-4-amine (Intermediate B2) (100 mg, 339 μmol, 1 equiv) in acetonitrile (2 mL). After dissolution, N,N,N,N-tetramethylchloroformamidinium hexafluorophosphate (285 mg, 1 mmol, 3 equiv) and N-methylimidazole (139 mg, 1.7 mmol, 135 μL, 5 equiv) were sequentially added. The reaction mixture was stirred at 50 °C for 16 h. Completion of the reaction of the starting materials was detected by LC-MS, and the desired product was formed. After filtration of the reaction solution, it was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography (silica, 10% methanol in dichloromethane) to give 7-chloro-4-[(2,4-dimethoxyphenyl)methylamino]-1-methyl-N-(1-methylpyrazol-4-yl)-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]imidazo[1,5-a]quinoxaline-8-carboxamide (3) as a yellow oil (130 mg, 185 μmol, 54.5% yield).

[0150] Step 2: 4-Amino-7-chloro-1-methyl-N-(1-methyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl])methyl)imidazo[1,5-a]quinoxaline-8-carboxamide (Example 210) 7-chloro-4-[(2,4-dimethoxyphenyl)methylamino]-1-methyl-N-(1-methyl-1H-pyrazol-4-yl)-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]imidazo[1,5-a]quinoxaline-8-carboxamide (3) (120 mg, 170 μmol, 1 equiv) was added to a mixed solution of trifluoroacetic acid (0.4 mL) and dichloromethane (1 mL) and dissolved. The reaction mixture was stirred at 50 °C for 16 h. Complete reaction of the starting material was indicated by LC-MS and the desired product was formed. The reaction mixture was concentrated to dryness under reduced pressure. Purification by reverse phase preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water and acetonitrile with decreasing polarity as the eluent) gave white 4-amino-7-chloro-1-methyl-N-(1-methyl-1H-pyrazol-4-yl)-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl])methyl)imidazo[1,5-a]quinoxaline-8-carboxamide (Example 210) (30 mg, 53 μmol, 31% yield, 98.% purity). H NMR: 1 H NMR (400 MHz, DMSO-d6) δ 9.15 - 9.31 (m, 1H), 8.33 (s, 1H), 8.07 - 8.16 (m, 1H), 7.92 - 8.03 (m, 1H), 7.76 - 7.86 (m, 1H), 7.58 - 7.75 (m, 2H), 7.41 - 7.55 (m, 3H), 7.18 - 7.33 (m, 1H), 4.71 - 5.17 (m, 2H), 3.53 - 3.83 (m, 3H), 2.80 - 2.97 (m, 3H). LCMS: (ESI) m / z = 554.2 (M+1)+, RT = 1.544 min, purity of 98.7%.

[0151] Synthesis of Example 241

Chemical Structure

[0152] Step 1: 4-Fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (3) 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (1) (700 mg, 3.27 mmol, 1 equiv) was dissolved in methanol (14 mL), 4-fluoro-1-methyl-1H-pyrazol-5-amine (2) (402.61 mg, 3.50 mmol, 1.07 equiv) and glacial acetic acid (255.18 mg, 4.25 mmol, 243.26 μL, 1.3 equiv) were added, and the reaction mixture was stirred at 25 °C for 1 h. Sodium cyanoborohydride (616.24 mg, 9.81 mmol, 3 equiv) was added and the mixture was stirred at 25 °C for 15 h. Completion of the reaction of the starting materials was detected by LC-MS, and the target product was formed. The reaction solution was concentrated and spin-dried, 14 mL of 2 mol / L aqueous sodium carbonate solution and 14 mL of ethyl acetate were added, the layers were separated, and the mixture was extracted with ethyl acetate (14 mL × 3). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification was carried out by column chromatography (12 g + 4 g silica gel column, eluent 0 - 30% ethyl acetate:ethanol 3:1 / petroleum ether, flow rate 30 mL / min), and concentrated under reduced pressure to give the pale yellow solid 4-fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (3) (970 mg, 3.10 mmol, 94.73% yield). LCMS: ES13685-1213-P1A, (ESI) m / z = 314.0 [M+1] + ; RT = 1.537 min. NMR: ES13685-1213-R2A, 1 1H NMR (400 MHz, CHLOROFORM-d) 7.11 (d, J = 4.38 Hz, 1H), 3.57 (s, 3H), 3.24 (br s, 2H). 19F NMR (376 MHz, CHLOROFORM-d) -185.48 (s, 1F)

[0153] Step 2: 4-Fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazole-3-amine (Example 241) Reaction solution: 4-Amino-7-fluoro-methyl-imidazo[1,5-a]quinoxaline-8-carboxylic acid (500 mg, 1.92 mmol, 1 equiv), 4-Fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (3) (601.89 mg, 1.92 mmol, 1 equiv), and N,N-diisopropylethylamine (993.30 mg, 7.69 mmol, 1.34 mL, 4 equiv) are dissolved in 1-methyl-2-pyrrolidone (10 mL), and 2-chloro-1,3-dimethyl-4,5-dihydroimidazol-1-chloride (487.23 mg, 2.88 mmol, 1.5 equiv) is added. The reaction mixture is stirred at 50 °C for 16 h. 47% residual raw material is detected by LC-MS, and 36% of the target product is formed. 2-Chloro-1,3-dimethyl-4,5-dihydroimidazol-1-chloride (74.71 mg, 441.93 μmol, 0.23 equiv) is added. The reaction mixture is stirred at 50 °C for 16 h, and 49% residual raw material is detected by LC-MS, and 38% of the target product is formed. Purification is carried out by reverse-phase preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.05% formic acid) and acetonitrile (20%-40%) with decreasing polarity as the eluent) to obtain a pale yellow solid. It is detected by NMR that some formate salts are formed, and the crude product is purified by reverse-phase preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using a mixture of water (containing 0.05% ammonium hydroxide) and acetonitrile (32% - 52%) with decreasing polarity as the eluent) to obtain the white solid 4-Amino-7-fluoro-N-(4-fluoro-2-methyl-pyrazol-3-yl)-1-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]imidazo[1,5-a]quinoxaline-8-carboxamide (Example 241) (155 mg, 276.26 μmol, 14.38% yield). LCMS: ES13685-1217-P1D1, (ESI) m / z = 556.1 [M+1] +; RT = 1.929 min NMR: ES13685 - 1217 - P1B, 1 1H NMR (400 MHz, DMSO - d6) δ 9.28 (br s, 1H), 8.10 (br s, 1H), 7.92 (br s, 1H), 7.82 (s, 1H), 7.73 (d, J = 9.76 Hz, 1H), 7.55 (s, 2H), 7.47 (br d, J = 8.63 Hz, 1H), 7.29 (br s, 1H), 7.11 (br d, J = 10.38 Hz, 1H), 5.19 (br s, 1H), 5.04 - 5.14 (m, 1H), 3.59 (s, 3H), 2.86 (br s, 3H). NMR: ES13685 - 1217 - P1B, 19F NMR (376 MHz, DMSO - d6) δ - 60.47 (s, 3F), - 118.15 (s, 1F), - 173.54 (s, 1F)

[0154] Synthesis of Example 285

Chemical Structure

[0155] Step 1: (E) - 4 - ((((dimethylamino)methylene)amino) - 7 - fluoro - 3 - methylimidazo[1,5 - a]quinoxaline - 8 - carbonyl chloride (Intermediate A7 - 1) To a stirred solution of 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A7) (270 mg, 1.04 mmol, 1 equiv) in DCM (3.00 mL) is added HCl / dioxane (4 M, 778 uL, 3 equiv). The mixture is stirred at 25 °C for 30 minutes. The reaction mixture is then concentrated and evaporated to dryness with toluene (10 mL × 3), the crude product is dissolved in DCM (3.00 mL) and cooled to 0 °C, and oxalyl chloride (790 mg, 6.23 mmol, 544 uL, 6 equiv is added dropwise at 0 °C) and DMF (75.8 mg, 1.04 mmol, 79.8 uL, 1 equiv) are added. The mixture is stirred at 25 °C for 12 hours. Complete consumption of the starting material is shown by LCMS and one major peak with the required mass is detected. The reaction mixture is concentrated, boiled with n-hexane (10 mL × 3) and dried under reduced pressure to give (E)-4-(((dimethylamino)methylene)amino)-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carbonyl chloride (Intermediate A7-1) (340 mg, crude product). LC-MS (ESI) m / z = 330.0 [M+H] +

[0156] Step 2: 4-Amino-7-fluoro-N-(1-methoxypropan-2-yl)-3-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)imidazo[1,5-a]quinoxaline-8-carboxamide (Example 285) At 0 °C, to a solution of 1-methoxy-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)propan-2-amine (50.0 mg, 174 μmol, 1 eq) and DIEA (89.9 mg, 696 μmol, 121 μL, 4 eq) in THF (1.00 mL) was added intermediate A7-1 (63.9 mg, 191 μmol, 1.1 eq). The mixture was stirred at 25 °C for 3 h. At 25 °C, MeOH (1.00 mL) was added to quench the reaction mixture and concentrated under reduced pressure to give a residue. The residue was dissolved in MeOH (1 mL) and NH3 / MeOH (7M, 1 mL). The mixture was stirred at 70 °C for 2 h. LCMS indicated complete consumption of the starting material and one major peak with the required mass was detected. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 μm, mobile phase: [water (NH4HCO3)-ACN], B%: 30%-60%, 8 min, UV220 nm & 254 nm) to give 4-amino-7-fluoro-N-(1-methoxypropan-2-yl)-3-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)imidazo[1,5-a]quinoxaline-8-carboxamide (Example 285) as a yellow solid (58.1 mg, 109 μmol, 64.2% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.29 - 8.54 (m, 2H), 8.18 - 7.78 (m, 2H), 7.66 (br s, 1H), 7.38 (br d, J = 7.9 Hz, 1H), 7.13 (br s, 1H), 6.72 (br s, 2H), 4.95 - 4.39 (m, 2H), 4.22 - 3.82 (m, 1H), 3.52 (br s, 2H), 3.31 - 3.10 (m, 3H), 2.63 (s, 3H), 1.21 (br s, 3H) LC-MS (ESI) m / z = 530.2 [M + H] +

[0157] Synthesis of Example 304

Chemical Structure

[0158] Step: 4-Amino-N-(1-(pyrimidin-2-yl)ethyl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 304) At 0 °C, to a solution of 1-(pyrimidin-2-yl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)ethan-1-amine (Intermediate B4) (27 mg, 99.6 μmol, 1 eq) in THF (1 mL) are added DIEA (51.5 mg, 398 μmol, 69.4 μL, 4 eq) and 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carbonyl chloride hydrochloride (28.4 mg, 99.6 μmol, 1 eq, HCl). The mixture is stirred at 25 °C for 2 h. Complete consumption of the starting material is indicated by LCMS and the required mass is detected. The mixture is quenched with MeOH (2 mL) and concentrated under reduced pressure to give a residue. The residue is purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm, mobile phase: [water (NH4HCO3)-ACN], B%: 15%-55%, 8 min, UV 220 nm & 254 nm) to afford 4-amino-N-(1-(pyrimidin-2-yl)ethyl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Example 304) as a white solid (19 mg, 38.9 μmol, 39.0% yield, 98.9% purity). 1 H NMR (400 MHz, DMSO-d6) δ 9.26 - 9.11 (m, 1H), 8.81 (d, J = 4.9 Hz, 2H), 8.49 (br d, J = 1.1 Hz, 1H), 8.13 (br d, J = 5.1 Hz, 1H), 7.74 - 7.57 (m, 3H), 7.42 (t, J = 4.9 Hz, 1H), 6.73 (br s, 2H), 5.50 - 5.09 (m, 4H), 5.00 (br s, 2H), 4.96 - 4.87 (m, 1H), 1.68 (br d, J = 7.0 Hz, 3H). LC-MS, [M+H] + 484.2

[0159] The following compounds were synthesized based on general methods, and the corresponding structures of the products are shown in Table 1, and the characterization is shown in Table 2.

Table 2-1

[0160]

Table 2-2

[0161]

Table 2-3

[0162]

Table 2-4

[0163]

Table 2-5

[0164]

Table 2-6

[0165]

Table 2-7

[0166]

Table 2-8

[0167]

Table 2-9

[0168]

Table 2-10

[0169]

Table 2-11

[0170]

Table 2-12

[0171]

Table 2-13

[0172]

Table 2-14

[0173]

Table 2-15

[0174]

Table 2-16

[0175]

Table 2-17

[0176]

Table 2-18

[0177]

Table 2-19

[0178]

Table 2-20

[0179]

Table 2-21

[0180]

Table 2-22

[0181]

Table 2-23

[0182]

Table 2-24

[0183]

Table 2-25

[0184]

Table 2-26

[0185]

Table 2-27

[0186]

Table 2-28

[0187] Biological Test Example 1. PRMT5 In Vitro Inhibition Activity Experiment

[0188] Experimental Materials PRMT5 (Active Motif, Catalog No. 31921), 3H]-SAM (Perkin Elmer, catalog number NET155V001MC), SAM (Sigma, catalog number A7007), MTA (Sigma, catalog number D5011), SAH (Sigma, catalog number A9384), 384-well plate (Perkin Elmer, catalog number 6007299), Echo 550 (manufacturer: Labcyte, model: Echo 550), 384-well flash plate (manufacturer: Perkin Elmer, model: SMP410A001PK)

[0189] Experimental method 1. Enzyme reaction process (1) Place 1x assay buffer (modified Tris Buffer). (2) Dilution of the compound: Dissolve the compound in 100% DMSO, and use the Echo 550 to add the compound solution to the 384-well plate. (3) Preparation of the enzyme solution: Add PRMT5 to 1x assay buffer to prepare enzyme solution 1, and add PRMT5 and MTA to 1x assay buffer to prepare enzyme solution 2. (4) Preparation of the substrate solution: Add the peptide section and [3H]-SAM to 1x assay buffer. (5) Add 15 μL of the enzyme solution to the 384-well plate, add 15 μL of 1x assay buffer to the negative control wells, and incubate at room temperature for 30 minutes. (6) Add 15 μL of the substrate solution to each well and incubate at room temperature for 90 minutes. (7) Preparation of the stop reaction solution: Add pre-cooled SAM to 1x assay buffer. (8) Add 10 μL of the stop reaction solution to each well to stop the reaction. (9) Transfer 25 μL / well of the mixed solution to the flash plate and incubate at room temperature for 1 hour. (10) Wash the flash plate 3 times with dH2O + 0.1% Tween-20 solution. (11) Read the radioactivity value using Microbeta.

[0190] 2. Data analysis (1) Convert the original data to % inhibition according to Equation 1. Equation 1: % inhibition = (Max - Signal) / (Max - Min) × 100 (2) Substitute the % inhibition data into XL-Fit Equation 2 to obtain the IC 50 value. Equation 2: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X) × HillSlope) Here, Y is the % inhibition and X is the concentration of the compound.

[0191] The biological activities of several compounds were measured by experimental methods and are shown in Table 3.

Table 3

[0192] Biological test example 2. In vitro inhibitory growth experiment of HCT116 and HCT116-MTAP-KO cells

[0193] Experimental materials The HCT116 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences, and the MTAP gene was knocked out using CRISPR / Cas9 technology to obtain the HCT116-MTAP-KO cell line. McCoy's 5A medium (Gibco, catalog number 16600082), fetal bovine serum (Gibco, catalog number 10099141C), penicillin-streptomycin double antibody (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo detection kit (Promega, catalog number G7572), 384-well clear flat-bottom black-wall cell culture plate (Corning, catalog number 3764), ultra-micro pipetting device (Tecan, catalog number D300e), multi-functional microplate reader (Biotek, catalog number SynergyHTX)

[0194] Experimental method 1. Cell culture: The culture conditions for HCT116 cells and HCT116-MTAP-KO cells are McCoy's 5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody, and it is always ensured that the cells are in the logarithmic growth phase and the cell viability exceeds 95%. 2. Preparation of compound concentration gradient: The test compound is added to a 384-well plate using an ultra-micro pipetting device, starting from 30 μM (for HCT116 cells) or 3 μM (for HCT116-MTAP-KO cells), diluted 3-fold with DMSO, with a total of nine concentrations, and three duplicate wells are set up. 3. Compound-treated cells: The trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension is added to the 384-well plate spotted with the test compound at 40 μL per well, that is, each well contains 100 cells, and the final DMSO concentration is 0.4%. The cell culture plate is placed in a 37°C, 5% carbon dioxide incubator and cultured for 6 days. 4. Detection: Add 20 μL of CellTiter-Glo reagent per well to the cell culture plate, and incubate for 30 minutes at room temperature with shaking. The luminescence signal at 578 nm is detected using a multi-functional microplate reader. 5. Data analysis: Using GraphPad Prism 8.0 software, the data was fitted using a four-parameter inhibitor-response model to obtain the IC 50 value (50% inhibitory concentration) of the test compound.

[0195] The biological activities of several compounds were measured by the experimental method, where "A" represents IC 50 (nm) < 100, "B" represents 100 < IC 50 (nm) < 1000, "C" represents 1000 < IC 50 (nm) < 10000, as shown in Table 4, where the first column is the cell growth inhibition rate HCT116 MTAP WT IC 50 (nm), and the second column is the cell growth inhibition rate HCT116 - MTAP null IC 50 (nm).

[0196]

Table 4-1

[0197]

Table 4-2

[0198]

Table 4-3

[0199]

Table 4-4

[0200] All documents mentioned in the present invention are incorporated by reference in this application as if each document was individually cited as a reference. Further, 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 are also included within the scope defined by the appended claims of this application.

Claims

[Claim 1] The invention described herein.