CLASS OF PRMT5 INHIBITORS AND THEIR APPLICATION

Abstract

Provided is a class of compounds with methyltransferase inhibitory activity. Specifically, provided is a class of compounds with PRMT5 inhibitory activity. The compounds can be used for preparing a pharmaceutical composition for treating PRMT5 activity-related diseases. (I)

Description

A class of PRMT5 inhibitors and uses thereof Technical Field

[0001] The present invention relates to the field of pharmaceutical compounds. Specifically, the present invention provides a class of compounds for inhibiting PRMT5, and 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 a significant pathogenic role in many human diseases.

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

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

[0005] Therefore, there is a need in the art to provide new small molecule compounds that target PRMT5 in MTAP-null tumors.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a new class of small molecule compounds targeting PRMT5 in MTAP-null tumors.

[0008] The first aspect of the present invention provides a compound represented by the following formula I, or a pharmaceutically acceptable stereoisomer, salt or deuterated product thereof:

[0009] in,

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

[0011] W is O or S;

[0012] X1 and X2 are each independently selected from the following group: CR, N; X3 is N;

[0013] The L1 is selected from the following group: chemical bond, -O-, -CHR-, -C(R)R-;

[0014] Ring A is selected from the following group: substituted or unsubstituted 8-12 membered fused bicyclic heterocyclic group (including carbocyclic or heterocyclic ring, preferably pentahedral and hexacyclic ring), substituted or unsubstituted 7-10 membered fused bicyclic heteroaryl group (preferably pentahedral and hexacyclic ring);

[0015] R8 is selected from the group consisting of H, halogen, cyano, amino, nitro, hydroxy, thiol, aldehyde, carboxyl, C2-C6 alkynyl, SF5, substituted or unsubstituted or halogenated C1-C6 alkyl, or R8 is

[0016] The L3 is selected from the following group: chemical bond, -O-, -CHR-, -C(R)R-, carbonyl, S, -NH-;

[0017] Ring B is selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring, a substituted or unsubstituted C3-C6 carbocyclic ring (including saturated or partially unsaturated rings), and a substituted or unsubstituted 3-7 membered heterocyclic ring (including saturated or partially unsaturated rings).

[0018] R2 is selected from the following groups: R7, -L2R7; wherein, said L2 is selected from the following groups: -O-, -CHR-, -C(R)R-, carbonyl; wherein R7 is selected from the following groups: hydrogen or none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C 6-10 aromatic ring, substituted or unsubstituted 5-12 membered heteroaromatic ring, substituted or unsubstituted C3-C 10 Carbocycle (including saturated or partially unsaturated, including monocyclic, condensed, spirocyclic or bridged rings), substituted or unsubstituted 3-10 membered heterocycle (including saturated or partially unsaturated, including monocyclic, condensed, spirocyclic or bridged rings);

[0019] R3 is selected from the group consisting of H, halogen, cyano, substituted or unsubstituted C1-C6 alkyl;

[0020] R4 and R5 are each independently selected from the group consisting of H, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 carbocycle (including saturated or partially unsaturated), substituted or unsubstituted 3-6 membered heterocycle; or R4 and R5 together with the connected ring atoms form a 5-12 membered saturated or unsaturated ring, and the ring may be substituted or unsubstituted;

[0021] R is H, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl;

[0022] Unless otherwise specified, in the above formulae, the substitution refers to the substitution of hydrogen atoms on the corresponding groups by one or more substituents selected from the following groups: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, benzyl, C1-C 12 Alkoxycarbonyl, C1-C6 aldehyde, amino, C1-C6 amide, nitro, cyano, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C3-C8 cycloalkyl, C2-C 10 Alkenyl, C1-C6 alkoxy, C1-C6 alkyl-amino, C6-C 10 Aryl, five-membered or six-membered heteroaryl, five-membered or six-membered non-aromatic heterocyclic group, -O-(C6-C 10 aryl), -O-(five-membered or six-membered heteroaryl), C1-C 12 Alkylaminocarbonyl, unsubstituted or halogenated C2-C 10 Acyl, sulfonyl (-SO2-OH), phosphoryl (-PO3-OH), unsubstituted or halogenated C1-C4 alkyl-S(O)2-, unsubstituted or halogenated C1-C4 alkyl-SO-.

[0023] In another preferred embodiment, the A ring has a structure selected from the following group:

[0024] In another preferred embodiment, the Ra is selected from the following group:

[0025] Wherein, R9 is selected from the following group: deuterium, tritium, halogen, hydroxyl, carboxyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, unsubstituted or substituted C1-C6 alkyl-OH, -NH(unsubstituted or halogenated C1-C6 alkyl), -N(unsubstituted or halogenated C1-C6 alkyl)2; m is selected from 0, 1, 2 or 3.

[0026] In another preferred embodiment, the L1 is -CH2-, -CH(CH3)-; and the A ring is selected from the following group:

[0027] Wherein, the C ring is selected from the following groups: a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring, a substituted or unsubstituted C3-C6 carbon ring (including saturated or partially unsaturated), and a substituted or unsubstituted 3-6 membered heterocyclic ring (including saturated or partially unsaturated).

[0028] In another preferred embodiment, R2 is an ortho-substituted 5- or 6-membered heteroaromatic ring, as shown in the following formula:

[0029] Ortho substituent R 10 It is hydrogen, deuterium, halogen, halogenated or unhalogenated C1-C3 alkyl, halogenated or unhalogenated C1-C3 alkoxy.

[0030] Preferably, the D ring is selected from the following groups: a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring, more preferably, the D ring is selected from the following groups:

[0031] The A ring is selected from the following groups: substituted or unsubstituted 8-12-membered fused bicyclic heterocyclic group (including carbocyclic or heterocyclic ring, preferably pentahedral and hexacyclic ring), substituted or unsubstituted 7-10-membered fused bicyclic heteroaryl group (preferably pentahedral and hexacyclic ring). Preferably, the A ring is selected from the following groups:

[0032] Wherein, the C ring is selected from the following group: a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring; and R8 is CF3.

[0033] In another preferred embodiment, the R2 is selected from the following groups: R7, -L2R7; wherein, the L2 is selected from the following groups: -O-, -CHR-, carbonyl, S, -NH-; wherein R7 is selected from the following groups: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C 6-10 an aromatic ring, or a substituted or unsubstituted 5-12 membered heteroaromatic ring.

[0034] In another preferred embodiment, the R7 is selected from the following group: a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted benzene ring, and a substituted or unsubstituted 5-7 membered heteroaromatic ring.

[0035] In another preferred embodiment, R2 is selected from the following group: R7, -(CHR)R7; wherein R7 is selected from the following group: substituted or unsubstituted C 6-10 an aromatic ring, or a substituted or unsubstituted 5-12 membered heteroaromatic ring.

[0036] In another preferred embodiment, the R2 is a substituted or unsubstituted 5-7 membered heteroaromatic ring; and the A ring is selected from the following group: a substituted or unsubstituted 7-10 membered fused bicyclic heteroaromatic group; and the R8 is CF3.

[0037] In another preferred embodiment, the Ra has a structure shown in the following formula:

[0038] The second aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound as described in any of the preceding aspects, one or more of its pharmaceutically acceptable salts, racemates, optical isomers, stereoisomers or tautomers, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0039] The third aspect of the present invention provides a compound as described in any of the preceding aspects, its racemate, optical isomer or pharmaceutically acceptable salt for the preparation of a drug for treating or preventing diseases associated with abnormal gene levels or abnormal expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fusion or overexpression of the methyltransferase).

[0040] In another preferred embodiment, the disease is selected from the following group: the disease or condition is ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, gastric cancer, pancreatic cancer or bladder cancer.

[0041] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION

[0042] Through extensive and in-depth research, the present inventors unexpectedly discovered for the first time a class of compounds that have PRMT5 regulatory effects, and completed the present invention on this basis.

[0043] the term

[0044] In the present invention, the halogen is F, Cl, Br or I.

[0045] 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 formulae are intended to encompass any possible optical or geometric isomers (e.g., R-type, S-type or racemate, or cis-trans isomers of olefins, etc.).

[0046] In the present invention, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

[0047] In the present invention, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like.

[0048] In the present invention, the term "C2-C6 alkenyl" refers to a straight chain or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl.

[0049] In the present invention, the term "C2-C6 alkynyl" refers to a straight chain or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl.

[0050] In the present invention, the term "C3-C10 cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclodecyl. The terms "C3-C8 cycloalkyl," "C3-C7 cycloalkyl," and "C3-C6 cycloalkyl" have similar meanings.

[0051] In the present invention, the term "C3-C10 cycloalkenyl" refers to a cyclic alkenyl group having 3 to 10 carbon atoms in the ring, including but not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and cyclodecyl. The term "C3-C7 cycloalkenyl" has a similar meaning.

[0052] In the present invention, the term "C1-C12 alkoxycarbonyl" refers to an alkoxycarbonyl group having 1 to 12 carbon atoms in the alkyl chain, including but not limited to methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl and the like.

[0053] In the present invention, the term "C1-C12 alkylaminocarbonyl" refers to an alkylaminocarbonyl group having 1 to 12 carbon atoms in the alkyl chain, including but not limited to methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl, isopropylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, dimethylaminocarbonyl and the like.

[0054] In the present invention, the terms "aromatic ring" and "aryl group" have the same meaning. Preferably, "aryl group" is "C6-C12 aryl group" or "C6-C10 aryl group." The term "C6-C12 aryl group" refers to an aromatic ring group having 6 to 12 carbon atoms and no heteroatoms in the ring, such as phenyl and naphthyl. The term "C6-C10 aryl group" has a similar meaning.

[0055] In the present invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning and refer to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to herein include oxygen, sulfur, and nitrogen. Examples include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0056] In the present invention, the term "3-12 membered heterocyclic group" refers to a saturated or unsaturated 3-12 membered ring group containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, such as dioxolanyl. The term "3-7 membered heterocyclic group" has a similar meaning.

[0057] In the present invention, the term "substituted" refers to the replacement of one or more hydrogen atoms on a specific group with a specific substituent. The specific substituent is the substituent described above, or the substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable site of the group, and the substituent may be the same or different at each position. A cyclic substituent, such as a heterocycloalkyl, may be connected to another ring, such as a cycloalkyl, to form a spirobicyclic system, for example, the two rings having a common carbon atom. It will be understood by those skilled in the art that the combinations of substituents contemplated by the present invention are those that are stable or chemically feasible. The substituents include, but are not limited to, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, 3- to 12-membered heterocyclic groups, aryl, heteroaryl, halogen, hydroxyl, carboxyl (-COOH), C1-8 aldehyde, C2-10 acyl, C2-10 ester, C1-C12 alkoxycarbonyl, amino, alkoxy, C1-10 sulfonyl, etc.

[0058] When a term such as "C1-8" or similar is used, it means that the group can have 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0059] When, for example, "3-12 membered" or similar expressions are used, it means that the group may have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms or heteroatoms as ring atoms.

[0060] PRMT5 modulator compounds

[0061] The present invention provides a class of compounds having PRMT5 regulatory activity:

[0062] in,

[0063] Ra is selected from the group consisting of:

[0064] W is O or S;

[0065] X1 and X2 are each independently selected from the following group: CR, N; X3 is N;

[0066] The L1 is selected from the following group: chemical bond, -O-, -CHR-, -C(R)R-;

[0067] Ring A is selected from the following group: substituted or unsubstituted 8-12 membered fused bicyclic heterocyclic group (including carbocyclic or heterocyclic ring, preferably pentahedral and hexacyclic ring), substituted or unsubstituted 7-10 membered fused bicyclic heteroaryl group (preferably pentahedral and hexacyclic ring);

[0068] R8 is selected from the group consisting of H, halogen, cyano, amino, nitro, hydroxy, thiol, aldehyde, carboxyl, C2-C6 alkynyl, SF5, substituted or unsubstituted or halogenated C1-C6 alkyl, or R8 is

[0069] The L3 is selected from the following group: chemical bond, -O-, -CHR-, -C(R)R-, carbonyl, S, -NH-;

[0070] Ring B is selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring, a substituted or unsubstituted C3-C6 carbocyclic ring (including saturated or partially unsaturated rings), and a substituted or unsubstituted 3-7 membered heterocyclic ring (including saturated or partially unsaturated rings).

[0071] R2 is selected from the following groups: R7, -L2R7; wherein, said L2 is selected from the following groups: -O-, -CHR-, -C(R)R-, carbonyl; wherein R7 is selected from the following groups: hydrogen or none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C 6-10 aromatic ring, substituted or unsubstituted 5-12 membered heteroaromatic ring, substituted or unsubstituted C3-C 10 Carbocycle (including saturated or partially unsaturated, including monocyclic, condensed, spirocyclic or bridged rings), substituted or unsubstituted 3-10 membered heterocycle (including saturated or partially unsaturated, including monocyclic, condensed, spirocyclic or bridged rings);

[0072] R3 is selected from the group consisting of H, halogen, cyano, substituted or unsubstituted C1-C6 alkyl;

[0073] R4 and R5 are each independently selected from the group consisting of H, halogen, cyano, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 carbocycle (including saturated or partially unsaturated), substituted or unsubstituted 3-6 membered heterocycle; or R4 and R5 together with the connected ring atoms form a 5-12 membered saturated or unsaturated ring, and the ring may be substituted or unsubstituted;

[0074] R is H, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl;

[0075] Unless otherwise specified, in the above formulae, the substitution refers to the substitution of hydrogen atoms on the corresponding groups by one or more substituents selected from the following groups: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, benzyl, C1-C 12 Alkoxycarbonyl, C1-C6 aldehyde, amino, C1-C6 amide, nitro, cyano, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C3-C8 cycloalkyl, C2-C 10 Alkenyl, C1-C6 alkoxy, C1-C6 alkyl-amino, C6-C 10 Aryl, five-membered or six-membered heteroaryl, five-membered or six-membered non-aromatic heterocyclic group, -O-(C6-C 10 aryl), -O-(five-membered or six-membered heteroaryl), C1-C 12 Alkylaminocarbonyl, unsubstituted or halogenated C2-C 10 Acyl, sulfonyl (-SO2-OH), phosphoryl (-PO3-OH), unsubstituted or halogenated C1-C4 alkyl-S(O)2-, unsubstituted or halogenated C1-C4 alkyl-SO-.

[0076] Pharmaceutical compositions and methods of administration

[0077] Since the compounds of the present invention have excellent methyltransferase inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate related diseases caused by abnormal activity or expression of methyltransferases (such as PRMT5).

[0078] The pharmaceutical composition of the present invention comprises a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0079] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0080] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration routes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0081] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0082] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0083] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0084] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0085] Suspensions, in addition to the active compounds, may contain 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.

[0086] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0087] Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0088] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In some preferred embodiments, the compounds of the present invention can be administered together with other small molecule compounds to form PROTACs, or together with other macromolecular compounds such as monoclonal antibodies to form ADCs.

[0089] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0090] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0091] The definitions of each abbreviation are as follows:

[0092] The starting materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0093] Purification of intermediates and compounds is performed using standard chemical laboratory procedures, such as normal-phase or reverse-phase chromatography or recrystallization. Normal-phase chromatography utilizes prepacked silica gel columns or preparative thin-layer chromatography. Silica gel columns are typically glass columns or flash chromatography instruments. For normal-phase chromatography, the mobile phase is selected from petroleum ether / ethyl acetate, dichloromethane / methanol, or other suitable solvents, and the resulting mixture is prepared and eluted. Reverse-phase preparative liquid chromatography utilizes a C18 column and is performed using a preparative liquid chromatograph or flash chromatography instrument, with detection at 214 nM and 254 nM or using preparative liquid chromatography-mass spectrometry. Gradient elution is performed using the mobile phases of water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile, water / acetonitrile containing 0.1% ammonium bicarbonate, water / acetonitrile containing 0.1% formic acid, 0.1% ammonia / acetonitrile, water / acetonitrile containing 0.1% trifluoroacetic acid, or other suitable solvent systems.

[0094] The structures of intermediates and compounds were characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS). NMR spectrometers used were Bruker Ascend 400, Varian 400, ZKNJ BIXI-1 300 MHz, Bruker Avance III 400 MHz, or Bruker AVANCE Neo 400 MHz. Solvents used were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, or other deuterated solvents as noted. Spectral data are reported in terms of chemical shift δ (peak splitting number, coupling constant J (Hz), and number of hydrogen atoms). Tetramethylsilane was used as an internal standard for chemical shifts, and its chemical shift was set to zero (δ, 0 ppm). Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad).

[0095] Representative liquid chromatography-mass spectrometry (LCMS) methods for the structural characterization of intermediates and compounds are as follows:

[0096] Method 1: On an Agilent LC1260 system coupled to a 6120 single quadrupole mass spectrometer

[0097] Column: Waters CORTECS C-18, 2.7 μm, 4.6 x 30 mm. Solvent A: 0.05% formic acid in water, Solvent B: 0.05% formic acid in acetonitrile, 5% acetonitrile to 95% acetonitrile over 1 minute, hold for 1 minute, for a total of 2.5 minutes; Flow rate: 1.8 mL / min; Column temperature: 40°C.

[0098] Column: XSelect CSH C18, 3.5 μm, 4.6 x 50 mm. Solvent A: 0.05% ammonia in water, Solvent B: 0.05% ammonia in acetonitrile, 5% acetonitrile to 95% acetonitrile over 1 minute, hold for 1 minute, for a total of 2.5 minutes; Flow rate: 1.8 mL / min; Column temperature: 40°C.

[0099] Method 2: performed on an Agilent LC / MSD 1200 system coupled with a quadrupole mass spectrometer.

[0100] Column: ODS2000 (50×4.6 mm, 5 μm) (ES (+) or (-) ionization mode), temperature 30°C; flow rate 1.5 mL / min.

[0101] Example Synthesis General Method:

[0102] General Method: Synthesis of Intermediate A1

[0103] Synthesis route:

[0104] Step 1: N-(4-bromophenyl)-2-oxocyclopentane-1-carboxamide (2)

[0105] 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) was stirred at 110 ° C for 12 hours. The mixture was then added to a 2M aqueous HCl solution (100 mL) and diluted with water (300 mL). The reaction was extracted with ethyl acetate (300 mL × 3). The organic layer was washed with brine (200 mL × 3) and dried over anhydrous sodium sulfate. The organic phase was then filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography using an EA / PE solution eluted from 0% to 30% in 20 minutes to give N-(4-bromophenyl)-2-oxocyclopentane-1-carboxamide (25 g, 21% yield) as a light 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

[0106] Step 2: 8-Bromo-2,3-dihydro-1H-cyclopentadienyl[c]quinolin-4-ol (3)

[0107] A mixture of N-(4-bromophenyl)-2-oxocyclopentane-1-carboxamide (25.0 g, 0.088 mol) in concentrated sulfuric acid (100 mL) was stirred at 100 ° C for 12 hours. The mixture was poured into ice water (500 mL). NaHCO 3 was added until pH 7-8. The precipitate formed was filtered and washed with some cold methanol (50 mL). The filtrate was filtered. The combined solids were dried and recrystallized from ethanol (20 mL) to obtain 8-bromo-2,3-dihydro-1H-cyclopentadienyl[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

[0108] Step 3: 4-Hydroxy-2,3-dihydro-1H-cyclopentadienyl[c]quinoline-8-carboxylic acid methyl ester (4)

[0109] 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) were stirred at 100°C under a CO atmosphere for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with MeOH in DCM from 0% to 10% over 20 minutes, 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

[0110] Step 4: Methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-cyclopentadienyl[c]quinoline-8-carboxylate (5)

[0111] 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) was added trifluoromethanesulfonic anhydride (21.6 g, 0.076 mol) at 0°C. The mixture was then stirred at 20°C for 12 hours. The reaction was quenched with NaHCO3(aq.) (200 mL) and then extracted with DCM (200 mL×3). The organic solution was washed with brine (200 mL). The organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with EA in PE from 0% to 30% over 20 min to give methyl 4-(((trifluoromethyl)sulfonyl)oxy)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylate (11.0 g, 77% yield) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ8.57(d,J=2.0Hz,1H),8.30(dd,J=8.8,2.0Hz,1H),8.08(d,J=8.8Hz,1H),4.01(s,3H),3.43(t,J =8.0Hz,2H),3.21(t,J=8.0Hz,2H),2.55–2.32(m,2H).LC-MS: Rt=1.544min, (ESI)m / z.376.1[M+H] + .C 15 H 12 F3NO5S 375.04.

[0112] Step 5: 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopentadienyl[c]quinoline-8-carboxylic acid methyl ester (6)

[0113] 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) was added CsCO (28.6 g, 88 mmol), Pd(dba) (2.7 g, 2.93 mmol), Xantphos (3.4 g, 5.86 mmol), and PMBNH (6.0 g, 0.044 mmol) at 20°C. The reaction mixture was stirred at 110°C under an N atmosphere for 12 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with MeOH in DCM from 0% to 5% over 20 minutes to afford methyl 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopentadi[c]quinoline-8-carboxylate (8.1 g, 76% yield) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ8.33(d,J=2.0Hz,1H),8.11(dd,J=8.8,2.0Hz,1H),7.79(d,J=8.8Hz,1H),7.36(d,J=8.8Hz,2H),6.89(d,J=8.8Hz,2H),4.7 9(s,2H),3.95(s,3H),3.80(s,3H),3.24(t,J=8.0Hz,2H),2.80(t,J=8.0Hz,2H),2.36–2.19(m,2H).LC-MS: Rt=1.036min, (ESI)m / z.363.1[M+H] + .C 22 H 22 N2O3 362.16.

[0114] Step 6: 4-Amino-2,3-dihydro-1H-cyclopentadienyl[c]quinoline-8-carboxylic acid methyl ester (7)

[0115] A solution of methyl 4-((4-methoxybenzyl)amino)-2,3-dihydro-1H-cyclopentadien[c]quinoline-8-carboxylate (8.1 g, 20 mmol) in CF3COOH (50 mL) was stirred at 70°C for 12 hours. The solvent was then removed and the residue was dissolved in DCM (200 mL). The organic phase was washed with NaHCO3(aq.) (300 mL) and dried over Na2SO4. The mixture was then filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with MeOH in DCM from 0% to 5% over 20 minutes, to give methyl 4-amino-2,3-dihydro-1H-cyclopentadien[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.

[0116] Step 7: 4-Amino-2,3-dihydro-1H-cyclopentadienyl[c]quinoline-8-carboxylic acid (Intermediate A1)

[0117] 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) was stirred at 95°C for 12 hours. The solvent was then removed to give 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxylic acid (500 mg, 88% yield) as a white solid, which was used in the next step without further purification. 1 H NMR (400MHz, DMSO-d6) δ8.65(s,2H),8.31(s,1H),8.22(d,J=8.0Hz,1H),7.83(d,J=8.0Hz,2H),3.32( t,J=8.0Hz,2H),2.95(t,J=8.0Hz,2H),2.34–2.20(m,2H).LC-MS: Rt=0.763min, (ESI)m / z.229.1[M+H] + .C 13 H 12 N2O2 228.09.

[0118] General Method: Synthesis of Intermediate A2

[0119] Synthesis route:

[0120] Step 1: Methyl 2,5-difluoro-4-nitrobenzoate (2)

[0121] 2,5-Difluoro-4-nitrobenzoic acid (1) (50 g, 246.18 mmol, 1 eq) was dissolved in methanol (500 mL), and then thionyl chloride (43.93 g, 369.28 mmol, 26.79 mL, 1.5 eq) was added at 0 °C. The reaction mixture was reacted at 40 °C for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated to dryness under reduced pressure. After dilution with 300 mL of water, the mixture was extracted three times with 1 L of ethyl acetate. The organic phase was washed with saturated brine (400 mL), dried over anhydrous magnesium sulfate, and the filtrate was filtered and concentrated to dryness under reduced pressure. The crude product was slurried with petroleum ether at 25 °C for 60 minutes to obtain methyl 2,5-difluoro-4-nitrobenzoate (2) (103 g, 474.38 mmol, 96.35% yield) as a white solid. HNMR: ES23714-64-P1A, 1H NMR (400MHz, CHLOROFORM-d) δppm 7.89 (td, J=9.51, 5.63Hz, 2H) 4.00 (s, 3H). 19F NMR(376MHz,CHLOROFORM-d)δppm-110.27(s,1F)-121.56(m,1F)

[0122] Step 2: Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3)

[0123] Methyl 2,5-difluoro-4-nitrobenzoate (2) (80 g, 368.45 mmol, 1 eq) and 2-methyl-1H-imidazole (36.30 g, 442.14 mmol, 1.2 eq) were dissolved in dimethyl sulfoxide (1.2 L). The reaction mixture was reacted at 50 °C for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was diluted with 4 L of water and extracted with 4.5 L of ethyl acetate. The organic phase was washed with saturated brine (3 L), dried over anhydrous magnesium sulfate, and the filtrate was filtered and concentrated to dryness under reduced pressure. The crude product was slurried with methyl tert-butyl methyl paraffin at 25 °C for 60 minutes (25.5 g). (2). The mother liquor was purified by column chromatography (silica, 50% tetrahydrofuran in petroleum ether) to give a yellow liquid which was then slurried at 25 degrees Celsius for 60 minutes to give a yellow solid (8.23 g). Yellow solid 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoic acid methyl ester (3) (25.5 g, 91.32 mmol, 24.79% yield) 2,5-difluoro-4-nitrobenzoic acid methyl ester (2) (raw material recovery) (20.34 g, 93.68 mmol, 25.43% yield). Yellow solid 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoic acid methyl ester (3) (8.2 g, 29.07 mmol, 7.89% yield, 99% purity) H NMR: ES23714-67-P1A1, 1H 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)

[0124] Step 3: Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4)

[0125] Methyl 2-fluoro-5-(2-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3) (13.9 g, 49.78 mmol, 1 eq) was dissolved in tetrahydrofuran (300 ml) and palladium hydroxide on carbon (2.8 g, 49.78 mmol, 20% purity, 1 eq) was added under a hydrogen atmosphere. The reaction system was replaced with hydrogen three times. The reaction solution was heated to 50 degrees Celsius under a hydrogen (1 eq) (50 psi) atmosphere for 32 hours. LCMS showed that the reaction was complete. The reaction solution was filtered through celite 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). 1H NMR (400 MHz, DMSO-d6) Shift 7.46 (d, J = 7.63 Hz, 1H), 7.07 (d, J = 1.38 Hz, 1H), 6.93 (d, J = 1.25 Hz, 1H), 6.59 (d, J = 13.51 Hz, 1H), 6.15 (br s, 2H), 3.68-3.77 (m, 3H), 2.01-2.12 (m, 3H). 19F NMR (376 MHz, DMSO-d6) Shift -109.31-108.47 (m, 1F).

[0126] Step 4: 7-Fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5)

[0127] Methyl 4-amino-2-fluoro-5-(2-methyl-1H-imidazol-1-yl)benzoate (4) (12 g, 48.15 mmol, 1 eq) was added to 1-methyl-2-pyrrolidone at 25°C, followed by 1,1-carbonyldiimidazole (19.52 g, 120.37 mmol, 2.5 eq). The reaction mixture was heated to 115°C for 16 hours. LCMS indicated the reaction was complete. The two batches of reaction mixture were combined. 600 mL of ethyl acetate and 600 mL of water were added to the reaction mixture and the mixture was slurried at 25°C for 16 hours. The slurry was filtered under reduced pressure, and the filter cake was washed with 100 mL of ethyl acetate. The solid was concentrated under reduced pressure to give methyl 7-fluoro-1-methyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (24.1 g, 87.56 mmol, 86.60% yield) as a gray solid. The crude product was used directly in the next reaction. 1H NMR (400 MHz, DMSO-d6) Shift 11.71 (br s, 1H), 8.40 (d, J = 6.38 Hz, 1H), 7.76 (s, 1H), 7.08 (d, J = 11.38 Hz, 1H), 3.88 (s, 3H), 2.89 (s, 3H). 19F NMR (376 MHz, DMSO-d6) Shift -111.43--110.58 (m, 1F)

[0128] Step 5: Methyl 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6)

[0129] 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) were added to acetonitrile (240 ml). Benzotriazole-1-oxo-tris(dimethylaminophosphonium)hexafluorophosphonium salt (25.07 g, 56.68 mmol, 1.3 eq) was added portionwise at 15-20°C. The reaction solution was slightly exothermic, became homogeneous, and solids precipitated. The reaction mixture was reacted at 15-20 degrees Celsius for 16 hours under nitrogen protection. LCMS showed that the starting material was completely consumed and the target compound was detected. The reaction suspension was filtered under reduced pressure and the filter cake was washed with 100 ml of acetonitrile. The solid was collected and dried under reduced pressure to obtain off-white solid 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) (15.3 g, 36.05 mmol, 82.68% yield). LCMS ES15882-1146-P1A: (ESI) m / z = 425.3 [M+1] +; RT = 1.721 min. 1H NMR (400 MHz, DMSO-d6) Shift 8.49(d,J=7.00Hz,1H),8.45(t,J=5.57Hz,1H),7.95(s,1H),7.23(d,J=12.51Hz,1H),7.18(d,J=8.38Hz,1H),6.58(d,J=2 .38Hz,1H),6.47(dd,J=2.38,8.38Hz,1H),4.66(d,J=5.25Hz,2H),3.88(s,3H),3.82(s,3H),3.73(s,3H),2.93(s,3H).19F NMR(376.5MHz,DMSO-d6)Shift-113.02,,,

[0130] Step 6: 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (7)

[0131] 4-((2,4-dimethoxybenzyl)amino)-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) (16.3 g, 38.40 mmol, 1.0 eq) was added to dichloromethane (50 mL), followed by the addition of trifluoroacetic acid (250 mL). The reaction mixture was heated to 50°C for 16 hours. LCMS showed that the starting material was completely consumed and the target compound was detected. The reaction mixture was concentrated to dryness under reduced pressure to give a purple solid 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (7) (28.3 g, crude). The crude product was used directly in the next reaction. LCMS ES15882-1150-P1A: (ESI) m / z = 275.3 [M+1] +; RT = 0.607 min

[0132] Step 7: 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2)

[0133] Methyl 4-amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (7) (crude product from the previous step) (28.3 g, 38.70 mmol, 1 eq) was added to tetrahydrofuran (80 mL) and methanol (80 mL). Sodium hydroxide (7.74 g, 193.48 mmol, 5 eq) was dissolved in water (80 mL) and added to the reaction mixture. The reaction mixture was heated to 50°C and reacted for 4 hours. The desired compound was detected by LCMS. The reaction mixture was cooled to 20°C and concentrated under reduced pressure to remove the organic solvent. The residue was diluted with 10:1 water to methanol (300 mL) and filtered through celite. The filter cake was washed three times with 10:1 water to methanol (300 mL). All filtrates were combined and concentrated under reduced pressure to remove the methanol. The pH of the residue was adjusted to 5-6 with acetic acid. The resulting slurry was stirred at 15-20°C for 12 hours. The resulting solid was filtered under reduced pressure and washed with water. The collected solid was freeze-dried to yield a white solid. 4-Amino-7-fluoro-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A2) (9.9 g, 37.55 mmol, 97.05% yield, 98.71% purity). LCMS ES15882-1154-P1C: (ESI) m / z = 261.1 [M+1]+; RT = 0.422 min. 1H NMR (400MHz, DMSO-d6)Shift 13.15(br s,1H),8.53(d,J=7.04Hz,1H),7.85(s,1H),7.68(s,2H),7.16(d,J=12.10Hz,1H),2.94(s,3H)

[0134] General Method: Synthesis of Intermediate A3

[0135] Synthesis route:

[0136] Step 1: 1-(5-Bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2)

[0137] 1-Bromo-2-chloro-5-fluoro-4-nitrobenzene (1) (3 g, 11 mmol, 1 eq) was added to a solution of 2-methyl-1H-imidazole (1.2 g, 14 mmol, 1.2 eq) in acetonitrile (50 mL). Potassium carbonate (4 g, 29 mmol, 2.5 eq) was then added to the reaction mixture. The mixture was heated to 80°C and stirred for 16 hours. The reaction mixture was detected to be completely reacted with the desired product. The acetonitrile was removed by concentration under reduced pressure, water (40 mL) was added, and the mixture was extracted with ethyl acetate (3 x 50 mL). The mixture 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 afford 1-(5-bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2) (4 g) as a white solid. H NMR: 1H NMR (400MHz, DMSO-d6) δ8.55 (s, 1H), 8.32 (s, 1H), 7.22 (d, J = 1.32Hz, 1H), 6.91 (d, J = 1.32Hz, 1H), 2.08-2.23 (m, 3H).

[0138] Step 2: 4-Bromo-5-chloro-2-(2-methylimidazol-1-yl)aniline (3)

[0139] 1-(5-Bromo-4-chloro-2-nitro-phenyl)-2-methyl-imidazole (2) (3.5 g, 11 mmol, 1 eq) was dissolved in a mixture of water (8 mL), ethanol (16 mL) and tetrahydrofuran (16 mL). Ammonium chloride (8.9 g, 166 mmol, 15 eq) was added to the reaction solution. The temperature was raised to 70°C and iron powder (2.5 g, 44 mmol, 4 eq) was added to the reaction solution. The reaction was stirred at 90°C for 2 hours. LC-MS analysis showed that the reaction of the starting material was complete and the target product was produced. The reaction solution was filtered through celite and washed with ethyl acetate (40 mL x 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: 1H NMR (400MHz, DMSO-d6) δ7.41(s,1H),7.12(s,1H),7.07(s,1H),6.99(s,1H),5.45(s,2H),2.13(s,3H).

[0140] Step 3: 8-Bromo-7-chloro-1-methyl-5-hydrogen-imidazo[1,5-a]quinoxaline-4-one (4)

[0141] 4-Bromo-5-chloro-2-(2-methylimidazol-1-yl)aniline (3) (3 g, 10.5 mmol, 1 eq) and 1,1-carbonyldiimidazole (2.6 g, 15.7 mmol, 1.5 eq) were dissolved in 1,2-dichlorobenzene (30 mL) in sequence. The reaction mixture was stirred at 130 °C for 16 h. LC-MS confirmed the complete reaction of the starting materials and the formation of the target product. The reaction mixture was stirred in a solution of ethyl acetate and water (2 / 1 15 mL) for 30 min and filtered to obtain a filter cake. The filter cake was concentrated under vacuum to obtain a black solid 8-bromo-7-chloro-1-methyl-5-hydrogen-imidazo[1,5-a]quinoxalin-4-one (4) (2.2 g, 7 mmol, 67% yield). H NMR: 1H NMR (400MHz, DMSO-d6) δ8.04 (s, 1H), 7.33 (d, J = 3.30Hz, 2H), 2.83 (s, 3H)

[0142] Step 4: 7-Chloro-1-methyl-4-oxo-5H-imidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5)

[0143] 8-Bromo-7-chloro-1-methyl-5-hydrogen-imidazo[1,5-a]quinoxaline-4-one (4) (500 mg, 1.6 mmol, 1 eq) was dissolved in ethanol (5 mL). 1,8-diazabicyclo[5.4.0]undecane-7-ene (365 mg, 2.4 mmol, 362 μL, 1.5 eq) was added. The nitrogen atmosphere was replaced three times. Tributylphosphine tetrafluoroborate (46 mg, 160 μM, 0.1 eq), molybdenum hexacarbonyl (232 mg, 880 μM, 118 μL, 0.55 eq) and palladium acetate (36 mg, 160 μM, 0.1 eq) were added. The reaction was stirred at 90°C for two hours. LC-MS analysis showed that the reaction of the starting material was complete and the target product was produced. The ethanol was removed from the reaction solution under reduced pressure, water (10 mL) was added, and the mixture was extracted with 30 mL of ethyl acetate (10 mL * 3). The residue 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 methyl 7-chloro-1-methyl-4-oxo-5H-imidazole[1,5-a]quinoxaline-8-carboxylate (5) (500 mg) as a yellow solid.

[0144] Step 5: 7-Chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) 7-Chloro-1-methyl-4-oxo-5H-imidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5) (480 mg, 1.6 mmol, 1 eq) was dissolved in acetonitrile (5 mL) and 2,4-dimethoxybenzylamine (341 mg, 2 mmol, 306 μL, 1.3 eq), benzotriazole-1-oxo-tris(dimethylaminophosphine)hexafluorophosphine salt (1 g, 2.4 mmol, 1.5 eq) and 1,8-diazabicyclo[5.4.0]undecane-7-ene (1.2 g, 7.9 mmol, 1.2 mL, 5 eq) were added. The reaction was stirred at room temperature for 16 h. LC-MS confirmed that the reaction of the starting materials was complete and the target product was produced. The reaction mixture was decompressed to remove acetonitrile, and water (8 mL) was added and extracted with 30 mL of ethyl acetate (10 mL * 3). The mixture 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 methyl 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (500 mg, 1.1 mmol, 70% yield) as a yellow solid.

[0145] Step 6: 7-Chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3)

[0146] Methyl 7-chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (500 mg, 1.1 mmol, 1 eq) was dissolved in water (5 mL) and ethanol (5 mL), and sodium hydroxide (132 mg, 3.3 mmol, 3 eq) was added. The reaction mixture was stirred at 50°C for 5 hours. LCMS analysis showed that the reaction of the starting material was complete and the target product was produced. The reaction mixture was added with 6 M hydrochloric acid (0.5 mL) and concentrated to dryness under reduced pressure. The crude product was used directly in the next step to obtain 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) as a white solid.

[0147] General Method: Synthesis of Intermediate A4

[0148] Synthesis route:

[0149] Step 1: Methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate

[0150] Methyl 3-fluoro-4-nitrobenzoate (1) (2.00 g, 10.04 mmol, 1 eq) was added to acetonitrile (40 eq) and potassium carbonate (4.16 g, 30.13 mmol, 3 eq) and 2,4-dimethyl-1H-imidazole (2) (965 mg, 10.04 mmol, 1 eq). The reaction mixture was reacted at 85°C for 16 hours. LC-MS showed that the starting material was completely consumed and the target product was produced. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was diluted with dichloromethane (80 eq) and filtered. The filtrate was dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate (3) (2.6 g, 9.45 mmol, 94.05% yield) as a yellow solid. The crude product was used directly in the next step without further purification.

[0151] Step 2: Methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate

[0152] Methyl 3-(2,4-dimethylimidazol-1-yl)-4-nitrobenzoate (3) (2.5 g, 9.08 mmol, 1 eq) was dissolved in ethanol (20 eq), tetrahydrofuran (20 eq), and water (10 eq). Iron powder (5.07 g, 90.82 mmol, 10 eq) and ammonium chloride (2.43 g, 45.41 mmol, 5 eq) were added at room temperature. The reaction mixture was reacted at 90°C for 16 hours. LC-MS showed that the starting material was completely consumed and the desired product was produced. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was diluted with water (50 equivalents) and extracted with ethyl acetate (40 equivalents * 3). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate (4) (1.9 g, 6.13 mmol, 67.49% yield, 79.13% purity) as a yellow solid. The crude product was 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%

[0153] Step 3: 1,3-Dimethyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester

[0154] Methyl 4-amino-3-(2,4-dimethylimidazol-1-yl)benzoate (4) (500 mg, 2.04 mmol, 1 eq) and 1,1-carbonyldiimidazole (495 mg, 3.06 mmol, 1.5 eq) were dissolved in 1,2-dichlorobenzene (10 eq). The reaction mixture was reacted at 120°C under nitrogen for 16 h. LC-MS showed that the starting material was completely consumed and the target product was produced. The reaction mixture was filtered, the filter cake was slurried with water, and dried under reduced pressure to obtain 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). The crude product was used directly in the next step without further purification.

[0155] LCMS: ES19974-392-P1B1, (ESI) m / z=272.0[M+1]+; RT=0.61min, purity:95.55%

[0156] Step 4: Methyl 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylate

[0157] Methyl dimethyl-4-oxo-4,5-dihydroimidazo[1,5-a]quinoxaline-8-carboxylate (5) (700 mg, 2.58 mmol, 1 eq) was dissolved in acetonitrile (25 eq). Benzotriazole-1-oxo-tris(dimethylaminophosphonium)hexafluorophosphonium salt (1.83 g, 4.13 mmol, 1.6 eq) and 1,8-diazabicyclo[5.4.0]undecane-7-ene (1.96 g, 12.90 mmol, 1.94 eq, 5 eq) were added. The reaction mixture was reacted at room temperature for 0.5 h. (2,4-Dimethoxyphenyl)methylamine (647.2 mg, 3.87 mmol, 583.1 μL, 1.5 eq) was added to the reaction mixture, and the reaction mixture was reacted at 50°C for 15.5 h. LC-MS showed that the starting material was completely consumed and the target product was produced. The reaction mixture was filtered, and the filter cake was dried under reduced pressure to obtain a brown solid of 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). The crude product was used directly in the next step without further purification. LCMS: ES19974-397-P1B1, (ESI) m / z = 421.1 [M+1] +; RT = 0.78 min, purity: 97.13%

[0158] Step 5: 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylic acid

[0159] Methyl 4-((2,4-dimethoxybenzyl)amino)-1,3-dimethylimidazo[1,5-a]quinoxaline-8-carboxylate (6) (850 mg, 2.02 mmol, 1 eq) was dissolved in methanol (10 eq), tetrahydrofuran (10 eq), and water (5 eq) and lithium hydroxide (424.2 mg, 10.11 mmol, 5 eq) was added. The reaction mixture was reacted at 50°C for 16 hours. LC-MS indicated complete consumption of the starting material and the formation of the desired product. The reaction solution was concentrated to dryness under reduced pressure, and the residue was adjusted to pH 6-7 with acetic acid, filtered, and the filter cake was dried under reduced pressure to give 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. The crude product was used directly in the next step without further purification.

[0160] General Method: Intermediate A5 and Intermediate A5b

[0161] Intermediate A5 is a known compound. Intermediate A5 was separated by SFC (conditions: chromatographic column: DAICEL CHIRALPAK IC (250 mm x 30 mm, 10 μm); mobile phase: [MeOH (0.1% IPAm)]; B%: 42%-42%, 15 min) to afford Intermediate A5a (600 mg, 2.46 mmol, 40.00% yield) and Intermediate A5b (600 mg, 2.46 mmol, 40.00% yield) as white solids. Note: The chiral center is not an absolute configuration and is designated according to the order of SFC separation.

[0162] Intermediate A5a: 1 H NMR (400MHz, DMSO-d6) δ8.07(d,J=1.9Hz,1H),7.98(dd,J=1.9,8.8Hz,1H),7.54(d,J=8.8Hz ,1H),6.74(s,2H),5.47-5.35(m,2H),5.34-5.25(m,1H),1.41(d,J=6.0Hz,3H).LC-MS,[MH] + 245.0

[0163] Intermediate A5b: 1H NMR (400MHz, DMSO-d6) δ8.05(d,J=1.8Hz,1H),7.98(dd,J=1.9,8.8Hz,1H),7.52(d,J=8.8H z,1H),6.68(s,2H),5.46-5.35(m,2H),5.33-5.26(m,1H),1.41(d,J=6.1Hz,3H)LC-MS,[MH] + 245.0

[0164] General Method: Synthesis of Intermediate A6

[0165] Synthesis route:

[0166] Step 1: N-(4-Bromo-2-fluorophenyl)-4-methyl-1H-pyrazole-5-carboxamide (3)

[0167] To 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 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) showed complete consumption of the starting material and a major peak with the desired m / z was detected. The reaction mixture was quenched with ice water (200 mL) and then extracted with ethyl acetate (60 mL x 6). The combined organic layers were washed with brine (30.0 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 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) (7.09 g, 23.8 mmol, 78.2% yield) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ13.26(br s,1H)9.52(s,1H)7.92(t,J=8.52Hz,1H)7.70(s,1H)7.62(dd,J=10.31,1.85Hz,1H)7.41(br d,J=8.70Hz,1H)2.25(s,3H).LC-MS;[MH] + 298.0

[0168] Step 2: 8-Bromo-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (4)

[0169] 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) was added NaH (1.43 g, 35.7 mmol, 60% purity, 1.5 eq). The mixture was stirred at 120 ° C for 16 hours. LC-MS showed complete consumption of the starting material and one major peak with the desired m / z. The reaction mixture was quenched with saturated ammonium chloride (300 mL), and the precipitate was collected and washed with water (50 mL), then concentrated under reduced pressure to give 8-bromo-3-methylpyrazolo[1,5-a]quinoxaline-4(5H)-one (4) (6.50 g, crude) as a white solid. 1 [MH] + 278.0

[0170] Step 3: 8-Bromo-N-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrazolo[1,5-a]quinoxalin-4-amine (5)

[0171] To a solution of 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4(5H)-one (4) (1.00 g, 3.60 mmol, 1 eq) in MeCN (10.0 mL) was added PMBNH2 (1.23 g, 8.99 mmol, 1.16 mL, 2.5 eq), BOP (3.18 g, 7.19 mmol, 2 eq) and DBU (2.74 g, 18.0 mmol, 2.71 mL, 5 eq). The mixture was stirred at 50 ° C for 16 hours. LC-MS showed complete consumption of the starting material and a major peak with the desired m / z. The reaction mixture was diluted with saturated ammonium chloride (10.0 mL) and ethanol (5.00 mL). The precipitate was collected and washed with water (10.0 mL), then concentrated under reduced pressure to give 8-bromo-N-(4-methoxybenzyl)-3-methyl-4,5-dihydropyrazolo[1,5-a]quinoxalin-4-amine (5) (1.09 g, crude) as a yellow solid. 1H NMR(400MHz, DMSO-d6)δ8.20(d,J=1.88Hz,1H)7.95(s,1H),7.44-7.48(m,2H)7.38(d,J=8.63Hz,2H)7.28(br s,1H)6.87(d,J=8.75Hz,2H)4.70(d,J=5.88Hz,2H)3.71(s,3H)2.53(s,3H).LC-MS; [MH] + 399.0

[0172] Step 4: 8-Bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6)

[0173] 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) was stirred at 60°C for 12 hours. LC-MS showed complete consumption of the starting material and a major peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to afford compound 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6) (400 mg, crude) as a yellow solid. LC-MS: [MH] + 277.0

[0174] Step 5: Ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7)

[0175] To a solution of 8-bromo-3-methylpyrazolo[1,5-a]quinoxalin-4-amine (6) (400 mg, 1.08 mmol, 1 eq) in EtOH (4 mL) was added Mo(CO)6 (143 mg, 541 umol, 72.9 uL, 0.5 eq), DBU (659 mg, 4.33 mmol, 652 uL, 4 eq), (t-Bu)3PBF4 (94.2 mg, 325 umol, 0.3 eq) and Pd(OAc)2 (36.5 mg, 162 umol, 0.15 eq). The mixture was stirred at 90 ° C for 12 hours. LC-MS (ET63399-28-R1A1) showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was diluted with saturated ammonium chloride (10 mL) and ethanol (10 mL). The precipitate was collected and washed with water (10.0 mL), then concentrated under reduced pressure to give ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7) (250 mg, 925 umol, 85.4% yield) as a yellow solid. LC-MS; [MH] + 271.1

[0176] Step 6: 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A6)

[0177] To a solution of ethyl 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylate (7) (250 mg, 925 umol, 1 eq) in EtOH (3 mL) and H2O (1 mL) was added LiOH·H2O (116 mg, 2.77 mmol, 3 eq). The mixture was stirred at 25 °C for 12 h. LC-MS (ET63399-32-R1A1) showed complete consumption of the starting material and the desired mass was detected. The reaction mixture was concentrated, diluted with water (10 mL), and washed with DCM (10.0 mL × 3) to remove impurities. The aqueous phase was adjusted to pH = 5 with 1 M HCl, and the precipitate was collected and purified by preparative HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [water (FA)-ACN]; B%: 1%-35%, 8 minutes, UV 220nm and 254nm) to give 4-amino-3-methylpyrazolo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A6) (60.0 mg, 248umol, 26.8% yield) as a yellow solid. 1 H NMR(400MHz, DMSO-d6)δ12.94(br s,1H)8.67(d,J=1.96Hz,1H)7.96(s,1H)7.89(dd,J=8.44,1.96Hz,1H)7.52(d,J=8.44Hz,1H)7.19(br s,2H)2.49(br s,3H).LC-MS,[MH]+243.1

[0178] General Method: Synthesis of Intermediate A7

[0179] Synthesis route:

[0180] Step 1: Methyl 2-fluoro-5-(4-methyl-1H-imidazol-1-yl)-4-nitrobenzoate (3)

[0181] A mixture of 2,5-difluoro-4-nitrobenzoic acid methyl ester (20.0 g, 92.1 mmol, 1 equivalent) and 5-methyl-1H-imidazole (7.56 g, 92.1 mmol, 1 equivalent) in DMSO (200 mL) was stirred at 50 ° C for 12 hours. LCMS showed that the remaining starting material was detected and the required mass was detected. The residue was diluted with H o (400 mL) and extracted with EtOAc (200 mL × 3). The combined organic layer was washed with brine (200 mL), dried over Na sO , filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO , petroleum ether / ethyl acetate = 20 / 1 to 0 / 1) to obtain compound 2-fluoro-5- (4-methyl -1H- imidazole -1- bases) -4-nitrobenzoic acid methyl ester (9.30 g, 33.3 mmol, 34.2% yield) as a yellow solid. LC-MS (ESI) m / z = 280.1 [M+H] +

[0182] Step 2: Methyl 4-amino-2-fluoro-5-(4-methyl-1H-imidazol-1-yl)benzoate (4)

[0183] To a solution of 2-fluoro-5-(4-methyl-1H-imidazol-1-yl)-4-nitrobenzoic acid methyl ester (9.30 g, 33.3 mmol, 1 equivalent) and NH4Cl (26.7 g, 499 mmol, 15 equivalents) in EtOH (90.0 mL) / THF (90.0 mL) / H2O (45.0 mL) was added Fe powder (7.44 g, 133 mmol, 4 equivalents). The mixture was stirred at 80 ° C for 2 hours. LCMS showed that the starting material had been consumed and the desired mass was detected. The reaction mixture was filtered, the filtrate was diluted with water (300 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with brine (100 mL), dried over Na 2 SO 4 , and concentrated under reduced pressure to afford 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] +

[0184] Step 3: 7-Fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (5)

[0185] 4-Amino-2-fluoro-5-(4-methyl-1H-imidazol-1-yl)benzoic acid methyl ester (500 mg, 2.01 mmol, 1 eq) and CDI (487 mg, 2.41 mmol, 1.2 eq) were placed in 1,2-dichlorobenzene (20.0 mL) in a microwave tube. The sealed tube was heated at 150 ° C for 3 hours under microwave. 11 parallel reactions were performed. LCMS showed that the starting material had been consumed and the required mass was detected. The reaction mixture was filtered and the filter cake was dried under reduced pressure to obtain a residue. The crude product was ground with MTBE (20.0 mL) at 25 ° C for 30 minutes to obtain 7-fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6.00 g, 21.80 mmol, 85.0% yield) as a yellow solid. LC-MS (ESI) m / z = 276.0 [M + H] +

[0186] Step 4: 7-fluoro-4-((4-methoxybenzyl)amino)-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6) To a solution of 7-fluoro-4-hydroxy-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester 5 (6.00 g, 21.8 mmol, 1 eq) in MeCN (120 mL) was added BOP (19.2 g, 43.6 mmol, 2 eq), DBU (16.5 g, 109 mmol, 16.4 mL, 5 eq) and PMBNH2 (7.48 g, 54.5 mmol, 7.05 mL, 2.5 eq). The mixture was stirred at 70° C. for 16 h. LCMS showed complete consumption of the starting material and the desired mass was detected. The reaction mixture was diluted with saturated ammonium chloride (20.0 mL). The filter cake was 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] +

[0187] Step 5: 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester (6)

[0188] A solution of 7-fluoro-4-((4-methoxybenzyl)amino)-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester 6 (2.00 g, 5.07 mmol, 1 equivalent) in TFA (20.0 mL) was stirred at 75 ° C for 16 hours. LCMS showed that the starting material had been consumed and the required mass was detected. The reaction mixture was concentrated under reduced pressure to give 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester 7 (1.39 g, crude product) as a yellow solid. LC-MS (ESI) m / z = 275.0 [M + H] +

[0189] Step 6: 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A7)

[0190] To a solution of 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid methyl ester 7 (1.39 g, 5.07 mmol, 1 equivalent) in EtOH (15 mL) / H2O (5.00 mL) was added LiOH (638 mg, 15.2 mmol, 3 equivalents). The mixture was stirred at 25 ° C for 12 hours. LCMS showed that the starting material had been consumed and a main peak with the desired mass was detected. The reaction mixture was diluted with H2O (20.0 mL) and extracted with (DCM 10.0 mL × 2) to remove impurities. The aqueous layer was adjusted to pH = 6 with 2M HCl, the precipitate was collected and dried under reduced pressure to give 4-amino-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (intermediate A7) (1.30 g, 5.00 mmol, 98.5% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ13.15(br s,1H),9.12(s,1H),8.56(br d,J=6.4Hz,1H),7.35(br s,2H),7.13(br d,J=12.1Hz,1H),2.62(s,3H).LC-MS(ESI)m / z=261.0[M+H] +

[0191] General Method: Synthesis of Intermediate B1

[0192] Synthesis route:

[0193] Step 1: N-methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (1)

[0194] To a DMF (100 mL) solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (2.0 g, 12.3 mmol) and HATU (7.0 g, 18.5 mmol) was added Et3N (6.2 g, 61.7 mmol) and methoxy(methyl)amine (1.9 g, 30.83 mmol) at 25°C. The mixture was then stirred at 25°C for 12 hours. The reaction was quenched with water (100 mL) and then extracted with EA (100 mL × 3). The organic solution was washed with brine (100 mL). The organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with a solution of MeOH in DCM from 0% to 10% in 20 minutes to give N-methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (2.01 g, 79% yield) as a yellow solid. LC-MS: Rt=1.049min, (ESI)m / z.[M+H] + 206.1; C10H11N3O2.

[0195] Step 2: Pyrazolo[1,5-a]pyridine-2-carbaldehyde (3)

[0196] To a solution of N-methoxy-N-methylpyrazolo[1,5-a]pyridine-2-carboxamide (1.5 g, 7.31 mmol) in THF (20 mL) was added LiAlH4 (439 mg, 10.96 mmol) at -60 ° C. The mixture was then stirred at -60 ° C for 2 hours. The reaction was quenched with water (40 mL) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with MeOH in DCM from 0% to 10% in 20 minutes to give pyrazolo[1,5-a]pyridine-2-carboxaldehyde (450 mg, 42% yield) as a yellow oil. LC-MS: Rt = 1.071 min, (ESI) m / z. [M + H] + 147.1; C8H6N2O

[0197] Step 3: N-(Pyrazolo[1,5-a]pyridin-2-ylmethyl)-1-(pyrimidin-2-yl)ethan-1-amine (Intermediate B1)

[0198] 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)ethanamine (126 mg, 1.03 mmol) and NaBH3CN (86 mg, 1.37 mmol) and reacted at 25°C. The mixture was then stirred at 25°C for 2 hours. 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 MeOH in DCM from 0% to 5% in 20 minutes to give N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-1-(pyrimidin-2-yl)ethanamine (40 mg, 23% yield) as a yellow oil. LC-MS: Rt=0.521 min, (ESI) m / z.[M+H]+254.2; C14H15N5

[0199] General Method: Synthesis of Intermediate B2

[0200] Synthesis route:

[0201] Step 1: 2-Dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3)

[0202] A mixture of 5-(trifluoromethyl)pyridin-2-amine (1) (30 g, 185 mmol, 1 eq), chlorobenzene (450 mL) and 1,1,3-trichloro-2-propanone (45 g, 277 mmol, 1.5 eq) was reacted at 135°C for 4 hours. LCMS confirmed the formation of the desired product. The reaction mixture was adjusted to pH 8 with sodium carbonate and extracted with ethyl acetate (500 mL x 3). The combined organic phases were dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The mixture was purified by column chromatography (silica, 15% ethyl acetate in petroleum ether) to afford 2-dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 60% yield) as a yellow solid. H NMR: ES19506-784-P1A, 1H NMR (400MHz, DMSO-d6) δ9.24 (s, 1H), 8.27 (s, 1H), 7.79 (d, J = 9.68Hz, 1H), 7.65 (s, 1H), 7.56 (dd, J = 1.65, 9.57Hz, 1H).

[0203] Step 2: 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (4)

[0204] 2-Dichloromethyl-6-trifluoromethylimidazo[1,2-a]pyridine (3) (30 g, 111 mmol, 1 eq), water (600 ml), and calcium carbonate (33 g, 334 mmol, 3 eq) were heated to 100°C and reacted for 2 hours. LCMS detected the formation of the target product. Celite and ethyl acetate (600 ml) were added to the reaction solution, stirred at room temperature for 30 minutes, filtered, and extracted with ethyl acetate (600 ml*2). The combined organic phases were dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a brown solid 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxaldehyde (4) (35 g). The crude product was used directly in the next step without further purification. H NMR: ES19506-789-P1A1, 1H NMR (400MHz, CHLOROFORM-d) δ 10.09-10.29 (m, 1H), 8.59 (s, 1H), 8.27 (s, 1H), 7.82 (br d, J=9.46Hz, 1H), 7.44 (br d,J=9.02Hz,1H).

[0205] Step 3: 1-Methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2)

[0206] To a solution of 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (4) (100 mg, 467 umol, 1 eq) in DCM (2.00 mL) was added KOAc (91.7 mg, 934 umol, 2 eq) and 1-methyl-1H-pyrazol-4-amine (45.4 mg, 467 umol, 1 eq) at -5°C and the reaction mixture was stirred at -5°C for 1 hour. NaBH(OAc)3 (198 mg, 934 umol, 2 eq) was then added and stirred at -5°C for another 3 hours. LCMS (ET63219-45-P1A1) showed complete consumption of Cpd.4 and several new peaks were observed on the LCMS. The reaction mixture was diluted with saturated aqueous Na2CO3 (3.00 mL) and extracted with dichloromethane (2.00 mL x 4). The combined organic layers were dried over NaSO and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (ethyl acetate / methanol=8 / 1) to give 1-methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B2) (80.0 mg, 271 umol, 58.0% yield) as a yellow solid. 1H NMR (400MHz, CDCl3) δ8.47(s,1H),7.69(d,J=9.5Hz,1H),7.64(s,1H),7.35(d,J= 1.5,9.5Hz,1H),7.30(s,2H),6.97(s,1H),4.39(s,2H),3.81(s,3H); LC-MS,[MH] + 217.0.

[0207] General Method: Synthesis of Intermediate B3

[0208] Synthesis route:

[0209] Step 1: 1,3-Dimethyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)-1H-pyrazol-4-amine (Intermediate B3)

[0210] 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (4) (10 g, 46 mmol, 1 eq) and 1,3-dimethylpyrazol-4-amine (6 g, 56 mmol, 1.2 eq) were dissolved in dichloromethane (150 ml), and acetic acid (3 g, 56 mmol, 3 ml, 1.2 eq) was added. The reaction solution was reacted at 25°C for 1 hour. Sodium triacetoxyborohydride (25 g, 117 mmol, 2.5 eq) was added, and the reaction solution was reacted at 25°C for 3 hours. LCMS detected the production of the target product. The reaction solution was quenched with 200 ml of sodium bicarbonate and extracted with ethyl acetate (150 ml*2). The combined organic phases were washed with 400 ml of saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, 35% ethyl acetate:ethanol (3:1) in petroleum ether) 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,1H NMR (400MHz, DMSO-d6) δ9.19(s,1H),7.92(s,1H),7.67(d,J=9.46Hz,1H),7.41(dd,J=1.76,9.46Hz,1H),6.93(s,1H),4.56(br s,1H),4.17(br d, J=3.96Hz, 2H), 3.52-3.61 (m, 3H), 2.04 (s, 3H). LCMS: ES19506-795-P1B, (ESI) m / z=310.3(M+1)+, RT=0.64min.

[0211] General Method: Synthesis of Intermediate B4

[0212] Synthesis route:

[0213] Step 1: tert-Butyl (thiazolo[4,5-c]pyridin-2-ylmethyl)carbamate (3)

[0214] To 4-iodopyridin-3-amine (20.0 g, 90.9 mmol, 1 eq.) and tert-butyl (2-amino-2-thioethyl)carbamate

[0215] To a solution of (20.7 g, 109 mmol, 1.2 equivalents) in MeCN (200 mL) was added Pd2(dba)3(4.16 g, 4.55 mmol, 0.05 equivalent), DPPF (10.1 g, 18.2 mmol, 0.2 equivalents) and CaO (10.2 g, 182 mmol, 3.09 mL, 2 equivalents) and the mixture was stirred at 80 ° C for 16 hours under N2. LCMS showed that the starting material was completely consumed 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 to 1 / 1) to give tert-butyl (thiazole-[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) δ9.28 (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

[0216] Step 2: Thiazolo[4,5-c]pyridin-2-ylmethanamine hydrochloride (4)

[0217] To a solution of tert-butyl (thiazolo[4,5-c]pyridin-2-ylmethyl)carbamate (3) (3 g, 11.3 mmol, 1 equiv) in DCM (15 mL) was added HCl / EtOAc (4 M, 15 mL). The mixture was stirred at 25 °C for 16 hours. LCMS (ET63218-11-P1A2) showed that the starting material had been consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to give thiazolo[4,5-c]pyridin-2-ylmethylamine hydrochloride (4) (2 g, crude, HCl) as a light yellow solid. LC-MS, [MH] + 166.1

[0218] Step 3: 1-(Pyrimidin-2-yl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)ethan-1-amine (Intermediate B4)

[0219] To a solution of thiazolo[4,5-c]pyridin-2-ylmethanamine 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 0.5 h, and then NaBH(OAc) 3 (2.73 g, 12.9 mmol, 1.3 eq) was added to the above mixture at 25 ° C, and the mixture was stirred at 25 ° C for 2 h. LCMS showed that the starting material had been consumed and the desired mass was detected. The mixture was adjusted to pH = 8-9 with saturated NaHCO 3 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 to 10 / 1) to give 1-(pyrimidin-2-yl)-N-(thiazolo[4,5-c]pyridin-2-ylmethyl)ethan-1-amine (Intermediate B4) (320 mg, 1.18 mmol, 11.9% yield) as a brown oil. 1 H NMR(400MHz,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

[0220] General Method: Synthesis of Intermediate B5

[0221] Synthesis route:

[0222] Step 1: Pyrazolo[1,5-a]pyridine-2-carboxylic acid (2)

[0223] To a solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (10.0 g, 61.67 mmol, 1 equivalent) and HATU (28.14 g, 74.01 mmol, 1.2 equivalents), DIEA (31.88 g, 246.69 mmol, 4 equivalents) in DCM (500 mL) was added N, O-dimethylhydroxylamine hydrochloride (12.03 grams, 123.35 mmol, 2 equivalents). The mixture was stirred at 25 ° C for 16 hours. LCMS (ET63565-18-P1A) showed that the starting material was completely consumed and the product was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to obtain 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(400MHz,CHLOROFORM-d)δ8.49(d,J=7.1Hz,1H),7.57(d,J=8.9Hz,1H),7.15(dd,J=7 .3,8.4Hz,1H),7.00(s,1H),6.85(t,J=6.9Hz,1H),3.79(s,3H),3.49(s,3H); LC-MS,[MH] + 206.22.

[0224] Step 2: 1-(Pyrazolo[1,5-a]pyridin-2-yl)ethan-1-one (3)

[0225] 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) was added MeLi (11.70 mL, 1.2 equiv) at -60 ° C, and the mixture was stirred at 25 ° C for 16 hours. hr under N2. LCMS (ET63565-13-P1A1) showed that the starting material was completely consumed and the product was detected. The residue was purified by column chromatography (petroleum ether / ethyl acetate=50 / 1 to 3 / 1) to give 1-(pyrazolo[1,5-a]pyridin-2-yl)ethane-1-one (3) (277 mg, 1.73 mmol, 17.7% yield) as a white solid. LC-MS, [MH] + 161.1.

[0226] Step 3: N-ethyl-1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-amine (Intermediate B5)

[0227] A mixture of 1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-one (3) (0.1 g, 624.33 umol, 1 eq) and ethylamine (112.58 mg, 2.50 mmol, 163.39 uL, 4 eq) in DCM (2 mL) was stirred at -60 ° C for 0.5 h, and then NaBH4 (264.64 mg, 1.25 mmol, 2 eq) was added to the mixture at 0 ° C and stirred at 25 ° C under N2 atmosphere for 16 hours. LCMS (ET63565-9-P1A2) showed that the starting material was completely consumed and the product was detected. The reaction mixture was quenched with H2O (5 mL) and diluted with DCM (5 mL×3). The combined organic layers were concentrated under reduced pressure to obtain 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) (50 mg, 264 umol, 42.3% yield) as a white solid. 1 H NMR(400MHz,CHLOROFORM-d)δ8.39(br d,J=6.9Hz,1H),7.46(br d,J=8.5Hz,1H),7.12-7.03(m,1H),6.70(br t,J=6.8Hz,1H),6.44(s,1H),4.13(br d,J=6.8Hz,1H),3.32-3.21(m,1H),2.77-2.56(m,2H),1.53(d,J=6.6Hz,4H),1.17-1.14(m,3H):LC-MS,[MH] + 190.1.

[0228] General Method: Synthesis of Intermediate B6

[0229] Synthesis route:

[0230] Step 1: 2-(Dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3)

[0231] A mixture of 4-(trifluoromethyl)benzene-1,2-diamine (5 g, 28.3 mmol, 1 eq) and 2,2-dichloroacetic acid (7.32 g, 56.7 mmol, 4.66 mL, 2 eq) in HCl (125 mL) (4 M) was stirred at 100 ° C for 10 minutes. 16 hours. LCMS (ET60224-68-P1A) showed that Cpd.1 was consumed and the desired mass was detected. The reaction mixture was filtered and the filter cake was washed with water. The combined filtrate was extracted with DCM (20 ml * 3). The combined organic layer was washed with brine (100 mL), dried over MgSO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give compound 2-(dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3) (4.4 g, 16.3 mmol, yield 57.6%) as a yellow oil. 1 H NMR (400MHz, CHLOROFORM-d) δ8.01 (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

[0232] Step 2: 6-(Trifluoromethyl)-1H-benzo[d]imidazole-2-carbaldehyde (4)

[0233] To a suspension of 2-(dichloromethyl)-6-(trifluoromethyl)-1H-benzo[d]imidazole (3) (1 g, 3.72 mmol, 1 equiv) in H2O (20 mL) was added CaCO3 (1.12 g, 11.1 mmol, 3 equiv). The mixture was stirred at 100 ° C for 8 hours. LCMS (ET60224-74-P1B) showed that Cpd.3 was consumed and the desired mass was detected. The reaction mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were concentrated under reduced pressure to give 6-(trifluoromethyl)-1H-benzo[d]imidazole-2-carbaldehyde (4) (310 mg, 1.45 mmol, 38.9% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ14.28-13.59(m,1H),10.02(s,1H),8.40-8.30(m,2H),8.23-8.05(m,3H),8.00(dd,J=5.4,8.5Hz,1H),7.89(br s,1H),7.83-7.61(m,3H),7.34(br d,J=7.9Hz,1H),7.27(d,J=7.6Hz,1H); LCMS:[M+H]+ 215.2

[0234] Step 3: 2-Methyl-N-((6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)propan-1-amine (Intermediate B6)

[0235] To a solution of 6-(trifluoromethyl)-1H-benzo[d]imidazole-2-carbaldehyde (4) (0.31 g, 1.45 mmol, 1 eq) and 2-methylpropan-1-amine (105 mg, 1.45 mmol, 143 uL, 1 eq) in DCM (6.2 mL) was added KOAc (170 mg, 1.74 mmol, 1.2 eq) at 25°C. The mixture was stirred at 25°C for 0.5 h, and then NaBH(OAc)3 (398 mg, 1.88 mmol, 1.3 eq) was added to the mixture at 25°C, and the mixture was stirred at 25°C for 15.5 h. LCMS (ET60224-77-P1A) showed that Cpd.4 was consumed and the desired mass was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (2 mL×3). The combined organic layers were washed with brine (10 mL), dried over MgSO , filtered, and concentrated under reduced pressure to give a residue. The residue was 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) (100 mg, 368 μmol, 25.4% yield, 100% purity) as a colorless oil. 1 H NMR(400MHz,CHLOROFORM-d)7.80(s,1H),7.56(d,J=8.4Hz,1H),7.43(d,J=8.5Hz,1H),4 .07(s,2H),2.45(d,J=6.8Hz,2H),1.82-1.68(m,1H),0.89(d,J=6.6Hz,6H); LCMS:[M+H] + 272.0

[0236] General Method: Synthesis of Intermediate B7

[0237] Step: 2-Methyl-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)propan-1-amine (Intermediate B7)

[0238] To a solution of 2-methylpropan-1-amine (68.3 mg, 933 umol, 92.8 uL, 1 eq) and 6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (0.2 g, 933 umol, 1 eq) in DCM (4 mL) was added KOAc (109 mg, 1.12 mmol, 1.2 eq) at 25°C. The mixture was stirred at 25°C for 0.5 h, then NaBH(OAc)3 (257 mg, 1.21 mmol, 1.3 eq) was added to the above mixture at 25°C, and the mixture was stirred at 25°C for 15.5 h. LCMS (ET60224-75-P1A) showed that Cpd.4 was consumed and the desired mass was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (2 mL x 3) to remove impurities. The aqueous layer was basified with saturated Na2CO3 to pH = 8, and then extracted with DCM (10 mL * 3). The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to give Cpd.A6 (108 mg, 398 umol, 42.6% yield, 100% purity) as a colorless oil. 1 H NMR(400MHz,CHLOROFORM-d)δ8.49(s,1H),7.70-7.62(m,2H),7.33(br d,J=9.2Hz,1H),4.01(s,2H),2.54(d,J=6.7Hz,2H),1.84(quind,J=6.6,13.3Hz,1H),0.97(d,J=6.6Hz,6H); LC-MS,[MH] + 272.0.

[0239] Synthesis of Example 1

[0240] Step 1: 4-amino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-N-(1-(pyrimidin-2-yl)ethyl)-2,3-dihydro-1H-cyclopentadieno[c]quinoline-8-carboxamide (Example 1)

[0241] A solution of 4-amino-2,3-dihydro-1H-cyclopentadien[c]quinoline-8-carboxylic acid (80 mg, 0.35 mmol) in SOCl2 (2 mL) was stirred at 70°C for 12 hours. The solvent was concentrated to dryness and used directly in the next reaction. 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, 1.3 g) in THF (15 mL) at 0°C was added 4-amino-2,3-dihydro-1H-cyclopentadien[c]quinoline-8-carbonyl chloride (80 mg, 0.32 mmol). The mixture was then stirred at 25°C for 12 hours. The reaction was quenched with NaHCO3(aq.) (20 mL) and extracted with EA (20 mL x 3). The organic solution was washed with brine (20 mL). The organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with MeOH in DCM from 0% to 5% in 20 minutes to give 4-amino-N-(pyrazolo[1,5-a]pyridin-2-ylmethyl)-N-(1-(pyrimidin-2-yl)ethyl)-2,3-dihydro-1H-cyclopentadiene[c]quinoline-8-carboxamide (2.3 mg, 4% yield) as a yellow solid (Example 1). 1H NMR(400MHz,MeOD)δ8.75(s,2H),8.40(s,2H),7.87–7.61(m,2H),7.55(d,J= 8.8Hz,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=0.981min,(ESI)m / z.464.2[M+H]+.C27H25N7O

[0242] Synthesis of Example 3

[0243] 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)

[0244] To a mixture of 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A14) (30 mg, 130 umol, 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 umol, 1 eq) in DMF (2 mL) was added TCFH (43.8 mg, 156 umol, 1.2 eq) and NMI (32.1 mg, 390 umol, 31.1 uL, 3 eq) and the mixture was stirred at 20 ° C. under N atmosphere for 16 hours. LC-MS showed that most of the starting material had been consumed and the desired 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*30mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-60%, 8 minutes, UV 220&254nm) 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) (37 mg, 76.5umol, 58.7% yield, 100% purity) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ=9.20(br s,1H),7.96(br s,1H),7.91-7.63(m,2H),7.48(br d,J=9.4Hz,2H),7.56(br d,J=6.4Hz,1H),6.66(br s,2H),5.27(br d,J=0.9Hz,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

[0245] Synthesis of Example 13

[0246] Step 1: (N-imidazo[1,2-a]pyridin-2-ylmethyl)cyclobutaneamine (2)

[0247] Imidazolo[1,2-a]pyridine-2-carbaldehyde (1) (300 mg, 2.05 mmol, 1 eq) and cyclobutylamine (300 mg, 4.22 mmol, 361.45 μL, 2.05 eq) were dissolved in methanol (5 mL) and stirred at room temperature for 12 hours. Sodium borohydride (118 mg, 3.12 mmol, 1.52 eq) was added and stirred at room temperature for 4 hours. LCMS confirmed the complete consumption of the starting material and the formation of the desired product. The reaction mixture was quenched with acetic acid (60 μL), concentrated to dryness under reduced pressure, and then added with 10% aqueous sodium carbonate solution (25 mL) and dichloromethane / ethanol (10:1, 25 mL). The mixture was separated, and 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 give a brown syrup of N-imidazo[1,2-a]pyridin-2-ylmethyl)cyclobutanamine (2) (401 mg, crude). LCMS ES15882-916-P1A: (ESI) m / z = 202.2 [M+1] +; RT = 1.249 min

[0248] 1H NMR(400MHz,CHLOROFORM-d)Shift 8.02-8.09(m,1H),7.46-7.57(m,2H),7.13(ddd,J=1.22,6.79,8.99Hz,1H),6.73(dt,J= 0.98,6.79Hz,1H),3.88(s,2H),3.29-3.47(m,1H),2.15-2.27(m,2H),1.57-1.84(m,4H)

[0249] 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)

[0250] N-Imidazolo[1,2-a]pyridin-2-ylmethyl)cyclobutanamine (2) (60 mg, 298.11 μmol, 1 eq) and N-ethyl-N-isopropyl-2-propanamine (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 hours. LCMS analysis showed that the starting material was completely consumed and the target product was produced. 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 x 30 mm x 5 μm; mobile phase: [water (ammonia v / v)-acetonitrile]; B% gradient: 28%-48% over 9 minutes). 4-Amino-N-cyclobutyl-7-fluoro-N-imidazo[1,2-a]pyridin-2-ylmethyl)-1-methylpyrazolo[4,3-c]quinoline-8-carboxamide (Example 13) was obtained as a white solid (72 mg, 161.69 μmol, 54.24% yield, 99.59% purity). 1H NMR(400MHz,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),6.8 7(t,J=6.65Hz,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(376.5MHz,DMSO-d6)Shift-116.26,-116.59

[0251] Synthesis of Example 49

[0252] 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)

[0253] 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) was added TCFH (713 mg, 2.54 mmol, 1.50 eq) and NMI (695 mg, 8.47 mmol, 675 μL, 5.00 eq). The mixture was stirred at 25° C. for 16 hours. LC-MS (ET68149-2-P1A1) showed that A29 was retained and the desired mass was detected. The solution was purified by preparative HPLC (chromatographic column: Waters Xbridge BEH C18 250*50mm*10μm; mobile phase: [water (NH4HCO3 10mM)-ACN]; B%: 20%-45%, 10 minutes, UV 220&254nm) 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) (285 mg, 535μmol, 31.6% yield, 97.9% purity) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.18(s,1H),8.03(s,1H),7.73(d,J=9.5Hz,1H),7.69-7.49(m,2H),7.46(dd,J=1.3,9.4Hz, 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=6.3Hz,3H); LC-MS,[MH] + 522.2.

[0254] Synthesis of Example 90

[0255] 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)

[0256] To a mixture of (S)-4-amino-3-methyl-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (Intermediate A5b) (3.12 g, 12.76 mmol, 1 eq), 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 eq), and 1-methylpyrrolidin-2-one (30 mL) was added N,N,N,N-tetramethylchloroformamidine hexafluorophosphate (4.30 g, 15.32 mmol, 1.2 eq) and N-methylimidazole (3.14 g, 38.29 mmol, 3.05 mL, 3.0 eq). The reaction mixture was stirred at room temperature for 16 hours. LCMS indicated complete consumption of the starting material and the formation of the desired product. The reaction solution 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 (ammonia water + ammonium bicarbonate)-acetonitrile]; gradient: 17%-57%). The fraction was lyophilized to give (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) as a white solid (100% purity). HNMR:ES13683-1863-P1A,1H NMR(400MHz,DMSO-d6)Shift9.21(s,1H),8.05(br s,1H),7.71(br d,J=9.24Hz,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=6.16Hz,3H).FNMR:19F NMR(376MHz,DMSO-d6)Shift-60.41(br s,1F).LCMS:ES13683-1863-P1C,(ESI)m / z=536.3[M+1]+,RT=0.802min

[0257] Synthesis of Example 119

[0258] 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)

[0259] To a solution of N-ethyl-1-(pyrazolo[1,5-a]pyridin-2-yl)ethan-1-amine (intermediate B5) (50 mg, 264.19 umol, 1 eq) in THF (1 mL) was added DIEA (136.58 mg, 1.06 mmol, 184.06 uL, 4 eq) and 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carbonyl chloride (65.69 mg, 264.19 umol, 1 eq) at 0 ° C. The mixture was then stirred at 0 ° C. under a N2 atmosphere for 1 hour. LCMS showed that the starting material was completely consumed 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*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 10%-50%, 8 minutes, UV220 & 254nm) 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, 104umol, 39.5% yield) as a white solid. 1 H NMR(400MHz, DMSO-d6)δ8.69(d,J=7.0Hz,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.

[0260] Synthesis of Example 210

[0261] 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)

[0262] 7-Chloro-4-((2,4-dimethoxybenzyl)amino)-1-methylimidazo[1,5-a]quinoxaline-8-carboxylic acid (Intermediate A3) (145 mg, 339 μmol, 1 eq) was dissolved in 1-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-4-amine (Intermediate B2) (100 mg, 339 μmol, 1 eq) in acetonitrile (2 mL). N,N,N,N-tetramethylchloroformamidine hexafluorophosphate (285 mg, 1 mmol, 3 eq) and N-methylimidazole (139 mg, 1.7 mmol, 135 μL, 5 eq) were then added sequentially. The reaction was stirred at 50°C for 16 hours. LC-MS analysis confirmed the complete reaction of the starting material and the formation of the desired product. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (silica, 10% methanol in dichloromethane) to afford 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).

[0263] 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)

[0264] 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) (120 mg, 170 μmol, 1 eq) was added to a mixture of trifluoroacetic acid (0.4 mL) and dichloromethane (1 mL) and dissolved. The reaction was stirred at 50°C for 16 hours. LC-MS showed that the starting material was completely reacted and the desired product was produced. 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 decreasingly polar mixtures of water and acetonitrile as eluent) gave 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) as white (30 mg, 53 μmol, 31% yield, 98% purity). H NMR: 1H NMR(400MHz,DMSO-d6)δ9.15-9.31(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=1.544min,purity of 98.7%.

[0265] Synthesis of Example 241

[0266] Step 1: 4-Fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (3)

[0267] 6-(Trifluoromethyl)imidazo[1,2-a]pyridine-2-carbaldehyde (1) (700 mg, 3.27 mmol, 1 eq) was dissolved in methanol (14 mL). 4-Fluoro-1-methyl-1H-pyrazol-5-amine (2) (402.61 mg, 3.50 mmol, 1.07 eq) and glacial acetic acid (255.18 mg, 4.25 mmol, 243.26 μL, 1.3 eq) were added. The reaction was stirred at 25°C for 1 hour. Sodium cyanoborohydride (616.24 mg, 9.81 mmol, 3 eq) was then added, and the mixture was stirred at 25°C for 15 hours. LC-MS analysis confirmed the complete reaction of the starting material and the formation of the desired product. The reaction mixture was concentrated to dryness, and 14 mL of 2 mol / L sodium carbonate solution and 14 mL of ethyl acetate were added. The mixture was separated and 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. The mixture was purified 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 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) as a light yellow solid. LCMS: ES13685-1213-P1A, (ESI) m / z = 314.0 [M+1]+; RT = 1.537 min. NMR: ES13685-1213-R2A, 1H NMR (400MHz, CHLOROFORM-d) 7.11 (d, J = 4.38Hz, 1H), 3.57 (s, 3H), 3.24 (br s, 2H). HNMR:ES13685-1213-R2A,19F NMR(376MHz,CHLOROFORM-d)-185.48(s,1F)

[0268] Step 2: 4-Fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (Example 241)

[0269] To a reaction mixture of 4-amino-7-fluoro-methyl-imidazo[1,5-a]quinoxaline-8-carboxylic acid (500 mg, 1.92 mmol, 1 eq), 4-fluoro-2-methyl-N-[[6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl]methyl]pyrazol-3-amine (3) (601.89 mg, 1.92 mol, 1 eq) and N,N-diisopropylethylamine (993.30 mg, 7.69 mol, 1.34 mL, 4 eq) dissolved in 1-methyl-2-pyrrolidone (10 mL) was added 2-chloro-1,3-dimethyl-4,5-dihydroimidazol-1-yl chloride (487.23 mg, 2.88 mmol, 1.5 eq). The reaction was stirred at 50°C for 16 hours. LC-MS analysis revealed 47% starting material remaining and 36% desired product. 2-Chloro-1,3-dimethyl-4,5-dihydroimidazolium-1-chloride (74.71 mg, 441.93 μmol, 0.23 equiv) was then added. The reaction was stirred at 50°C for 16 hours. LC-MS analysis revealed 49% starting material remaining and 38% desired product produced. Purification by reverse-phase preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using decreasingly polar mixtures of water (containing 0.05% formic acid) and acetonitrile (20%-40%) as eluents) afforded a pale yellow solid. NMR analysis revealed partial formation of formate, and the crude product was purified by reverse-phase preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length, using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (32%-52%) as eluents) to afford 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) as a white solid.LCMS: ES13685-1217-P1D1, (ESI) m / z=556.1[M+1]+; RT=1.929minNMR: ES13685-1217-P1B,1H NMR(400MHz, DMSO-d6)9.28(br s,1H),8.10(br s,1H),7.92(br s,1H),7.82(s,1H),7.73(d,J=9.76Hz,1H),7.55(s,2H),7.47(br d,J=8.63Hz,1H),7.29(br s,1H),7.11(br d,J=10.38Hz,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 (376MHz, DMSO-d6) -60.47 (s, 3F), -118.15 (s, 1F), -173.54 (s, 1F).

[0270] Synthesis of Example 285

[0271] Step 1: (E)-4-(((dimethylamino)methylene)amino)-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carbonyl chloride (Intermediate A7-1)

[0272] 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 eq) in DCM (3.00 mL) was added HCl / dioxane (4 M, 778 uL, 3 eq). The mixture was stirred at 25 ° C for 0.5 hours. The reaction mixture was then concentrated, evaporated and dried with toluene (10 mL x 3), and the crude product was dissolved in DCM (3.00 mL) and cooled to 0 ° C. Oxalyl chloride (790 mg, 6.23 mmol, 544 uL, 6 eq added dropwise at 0 ° C) and DMF (75.8 mg, 1.04 mmol, 79.8 uL, 1 eq) were added. The mixture was stirred at 25 ° C for 12 hours. LCMS showed that the starting material had been consumed and a main peak with the desired mass was detected. The reaction mixture was concentrated, digested with n-hexane (10 mL x 3), and dried under reduced pressure to obtain (E)-4-(((dimethylamino)methylene)amino)-7-fluoro-3-methylimidazo[1,5-a]quinoxaline-8-carbonyl chloride (Intermediate A7-1) (340 mg, crude) as a yellow solid. LC-MS (ESI) m / z = 330.0 [M+H] +

[0273] 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)

[0274] To a solution of 1-methoxy-N-((6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)methyl)propan-2-amine (50.0 mg, 174 umol, 1 eq) and DIEA (89.9 mg, 696 umol, 121 uL, 4 eq) in THF (1.00 mL) at 0°C was added intermediate A7-1 (63.9 mg, 191 umol, 1.1 eq). The mixture was stirred at 25°C for 3 hours. The reaction mixture was quenched by the addition of MeOH (1.00 mL) at 25°C and concentrated under reduced pressure to give a residue. The residue was dissolved in MeOH (1 mL) and NH 3 / MeOH (7 M, 1 mL). The mixture was stirred at 70°C for 2 hours. LCMS indicated that the starting material had been consumed and a major peak of the desired 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*30mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-60%, 8 minutes, UV 220nm&254nm) 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) (58.1 mg, 109umol, 64.2% yield) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.29-8.54(m,2H),8.18-7.78(m,2H),7.66(br s,1H),7.38(br d,J=7.9Hz,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] +

[0275] Synthesis of Example 304

[0276] 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)

[0277] At 0 ° C, DIEA (51.5 mg, 398 umol, 69.4 uL, 4 eq) and 4-amino-1,3-dihydrofuro [3,4-c] quinoline-8-carbonyl chloride hydrochloride (28.4 mg, 99.6 umol, 1 eq, HCl) were added to a solution of 1- (pyrimidin-2-yl) -N- (thiazolo [4,5-c] pyridin-2-ylmethyl) ethyl -1-amine (intermediate B4) (27 mg, 99.6 umol, 1 eq) in THF (1 mL). The mixture was stirred at 25 ° C for 2 hours. LCMS showed that the starting material was completely consumed and the desired mass was detected. The mixture was quenched with MeOH (2 mL) and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 15%-55%, 8 minutes, UV220nm&254nm) to give 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) (19 mg, 38.9umol, 39.0% yield, 98.9% purity) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ9.26-9.11(m,1H),8.81(d,J=4.9Hz,2H),8.49(br d,J=1.1Hz,1H),8.13(br d,J=5.1Hz,1H),7.74-7.57(m,3H),7.42(t,J=4.9Hz,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=7.0Hz, 3H); LC-MS, [MH] + 484.2

[0278] The following compounds were synthesized based on the general method. The corresponding structures of the products are shown in Table 1 and the characterizations are shown in Table 2:

[0279] Table 2

[0280] Biological test example 1 PRMT5 in vitro inhibitory activity experiment

[0281] Experimental Materials

[0282] PRMT5 (Active Motif, catalog number 31921), [ 3 H]-SAM (PerkinElmer, catalog number NET155V001MC), SAM (Sigma, catalog number A7007), MTA (Sigma, catalog number D5011), SAH (Sigma, catalog number A9384), 384-well plate (Perkin Elmer, catalog number 6007299), Echo 550 (manufacturer: Labcyte, model: Echo 550), 384-well Flashplate (manufacturer: Perkin Elmer, model: SMP410A001PK)

[0283] Experimental methods

[0284] 1. Enzyme reaction process

[0285] (1) Configure 1x assay buffer (modified Tris Buffer).

[0286] (2) Compound dilution: The compound was dissolved in 100% DMSO and the compound solution was added to a 384-well plate using Echo 550.

[0287] (3) Prepare enzyme solution: Add PRMT5 to 1x assay buffer to prepare enzyme solution 1; add PRMT5 and MTA to 1x assay buffer to prepare enzyme solution 2.

[0288] (4) Prepare substrate solution: Add peptide and [3H]-SAM to 1x assay buffer.

[0289] (5) Add 15 μL of enzyme solution to a 384-well plate and 15 μL of 1x assay buffer to the negative control wells. Incubate at room temperature for 30 minutes.

[0290] (6) Add 15 μL of substrate solution to each well and incubate at room temperature for 90 minutes.

[0291] (7) Prepare the termination reaction solution: add pre-cooled SAM to 1x assay buffer.

[0292] (8) Add 10 μL of stop reaction solution to each well to terminate the reaction.

[0293] (9) Transfer 25 μL / well of the mixed solution to the Flashplate and incubate at room temperature for 1 hour.

[0294] (10) Wash the Flashplate three times with dH2O + 0.1% Tween-20 solution.

[0295] (11) Use Microbeta to read the radioactivity value.

[0296] 2. Data Analysis

[0297] (1) Convert the raw data into %inhibition according to formula 1:

[0298] Formula 1: %inhibition=(Max-Signal) / (Max-Min)*100

[0299] (2) Substitute the %inhibition data into XL-Fit formula 2 to obtain the IC50 value:

[0300] Formula 2: Y=Bottom+(Top-Bottom) / (1+(IC50 / X)*HillSlope)

[0301] Where Y is %inhibition and X is the compound concentration.

[0302] The biological activities of some compounds were measured by experimental methods, see Table 3

[0303] Table 3

[0304] Biological test example 2 HCT116, HCT116-MTAP-KO cell proliferation inhibition experiment in vitro

[0305] Experimental Materials

[0306] The HCT116 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. The MTAP gene was knocked out using CRISPR / Cas9 technology to obtain the HCT116-MTAP-KO cell line.

[0307] 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 plates (Corning, catalog number 3764), ultra-micropipette reader (Tecan, catalog number D300e), multi-function microplate reader (Biotek, catalog number SynergyHTX)

[0308] Experimental methods

[0309] 1. Cell culture: HCT116 and HCT116-MTAP-KO cells were cultured in McCoy's 5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin. The cells were kept in the logarithmic growth phase and had a cell viability greater than 95%.

[0310] 2. Preparation of compound concentration gradient: The test compound was added to a 384-well plate using an ultra-micropipette, starting from 30 μM (HCT116 cells) or 3 μM (HCT116-MTAP-KO cells), and diluted 3-fold with DMSO for a total of 9 concentrations in triplicate.

[0311] 3. Compound-treated cells: Add 40 μL of the trypsinized HCT116 or HCT116-MTAP-KO cell suspension to a 384-well plate containing the test compound, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. Incubate the cell culture plate in a 37°C, 5% CO2 incubator for 6 days.

[0312] 4. Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate at room temperature with shaking for 30 minutes. Detect the luminescence signal at 578 nm using a multi-function microplate reader.

[0313] 5. Data Analysis:

[0314] GraphPad Prism 8.0 software was used to fit the data using a four-parameter inhibitor-response model to obtain the IC values ​​of the test compounds. 50 value (half maximal inhibitory concentration).

[0315] The biological activities of some compounds were determined by experimental methods. "A" represents IC 50 (nm)<100, “B” means 100 <IC 50 (nm)<1000, “C” means 1000 <IC 50 (nm) <10000, see Table 4, where the first column is the cell proliferation inhibition rate HCT116MTAP WT IC 50 (nm), the second column is the cell proliferation inhibition rate HCT116-MTAP null IC 50 (nm):

[0316] Table 4

[0317] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable stereoisomer or salt thereof, or a deuterated product thereof in which Ra is selected from a group consisting of W represents O or S; X1, X2 are each independently selected from the group consisting of CR and N; L1 represents -CHR- or -C(R)R-; ring A is a substituted or unsubstituted 8-12 membered fused bicyclic heterocyclyl or a substituted or unsubstituted 7-10 membered fused bicyclic heteroaryl, wherein the bicyclic heterocyclyl or fused bicyclic heteroaryl is selected from the group consisting of where ring C is chosen from a group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6-membered heteroaromatic ring, a substituted or unsubstituted C3-C6 carbocycle, and a substituted or unsubstituted 3-6-membered heterocycle; R8 is selected from the group consisting of H, deuterium, halogen, cyano, amino, nitro, hydroxyl, carboxyl, C2-C6 alkynyl, SF5, substituted or unsubstituted or halogenated C1-C6 alkyl and unsubstituted or halogenated C1-C6 alkoxyl or R8 is a L3 is selected from the group consisting of chemical bonds, -O-, -CHR-; cycle B is selected from the group consisting of a substituted or unsubstituted benzene cycle, a substituted or unsubstituted 5-6-membered heteroaromatic cycle, a substituted or unsubstituted C3-C6 carbocycle, a substituted or unsubstituted 3-7-membered heterocycle; R2 is selected from the group consisting of: R7 and -L2R7; where L2 is selected from the group consisting of -O-, -CHR-, and carbonyl; where R7 is selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-10 aromatic ring and substituted or unsubstituted 5-12-membered heteroaromatic ring, substituted or unsubstituted C3-C10 carbocycle and substituted or unsubstituted 3-10-membered heterocycle; R3 is selected from the group consisting of H, deuterium, halogen, cyano, and unsubstituted or halogenated C1-C6 alkyl; R is H, deuterium, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxyl, and substituted or unsubstituted C3-C6 cycloalkyl; R9 is selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxyl; m is 0, 1 or 2; the terms "heteroaromatic ring", "heteroaryl" or "heterocycle" contain one or more heteroatoms selected from oxygen, sulfur and nitrogen; unless otherwise stated, in the above formulas, substituted refers to hydrogen atoms in the corresponding group substituted with one or more substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, C1-C12 alkoxycarbonyl, C1-C6 carbonyl, amino, C1-C6 amide, nitro, cyano, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C3-C8 cycloalkyl, C2-C10 alkenyl, C1-C6 alkoxyl, C1-C6 alkylamino, C1-C12 alkylaminocarbonyl, unsubstituted or halogenated C2-C10 acyl, -SO2-OH, -PO3-OH, unsubstituted or halogenated C1-C4 alkyl-S(O)2-, unsubstituted or halogenated C1-C4 alkyl-SO- and -SF5.

2. The compound according to claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, in which X1, X2 are selected independently of each other from the group consisting of CR; Ra is selected from wherein R9 is selected from the group consisting of deuterium, tritium, unsubstituted or halogenated C1-C6 alkyl.

3. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein L1 is -CH2- or -CH(CH3)-.

4. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein R2 is an ortho-substituted 5-membered or 6-membered heteroaromatic ring, as shown below: in which R10 is a substituent located adjacent to the connecting fragment and selected from the group consisting of hydrogen, deuterium, halogen, unsubstituted or halogenated C1-C3 alkyl and unsubstituted or halogenated C1-C3 alkoxy.

5. The compound according to claim 4 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein the D ring is selected from the group consisting of a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6-membered heteroaromatic ring, more preferably the D ring is selected from the group consisting of 6. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein ring A is selected from the group consisting of wherein the C ring is selected from the group consisting of a substituted or unsubstituted benzene ring and a substituted or unsubstituted 5-6-membered heteroaryl ring and R8 is CF3.

7. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein R2 is selected from the group consisting of R7 and -L2R7, wherein L2 is selected from the group consisting of -O-, -CHR- and carbonyl; wherein R7 is selected from the group consisting of substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C6-10 aromatic ring and substituted or unsubstituted 5-12-membered heteroaromatic ring.

8. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein R7 is selected from the group consisting of: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted 5-7-membered heteroaromatic ring.

9. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein R2 is selected from the group consisting of R7 and -(CHR)R7; wherein R7 is selected from the group consisting of a substituted or unsubstituted C6-10 aromatic ring and a substituted or unsubstituted 5-12-membered heteroaromatic ring.

10. The compound of claim 1 or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein R2 is a substituted or unsubstituted 5-7-membered heteroaromatic ring and R8 is CF3.

11. A compound selected from the following group or a pharmaceutically acceptable stereoisomer or salt or deuterated product thereof, wherein the compound has a structure selected from the following Table 1. Table 1 12. A pharmaceutical composition comprising a therapeutically effective amount of one or more compounds according to any one of claims 1 to 11, a pharmaceutically acceptable stereoisomer or salt, or a deuterated product thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary accessories and / or diluents.

13. The use of a compound according to any one of claims 1 to 11, its pharmaceutically acceptable stereoisomer or salt, or its deuterated product in the production of medicaments for the treatment or prevention of diseases associated with abnormal levels of the MTAP gene.

14. The use according to claim 13, characterized in that the disease is selected from the group comprising ovarian cancer, esophageal cancer, lung cancer, lymphatic system cancer, glioblastoma, colon cancer, melanoma, stomach cancer, pancreatic cancer, or bladder cancer.

Images