Peptide epoxyketone compound, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are a peptide epoxyketone compound, a preparation method therefor, and a use thereof. The structural formula of the peptide epoxyketone compound is as shown in I. The compound or a pharmaceutically acceptable salt thereof can selectively inhibit the activity of an immunoproteasome, and thus can be used for developing drugs for treating various autoimmune diseases in humans and animals.

Description

Peptide epoxy ketone compound and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a peptide epoxy ketone derivative or a pharmaceutically acceptable salt thereof and application thereof in pharmacodynamics. BACKGROUND

[0002] The ubiquitin proteasome system (UPS) is the major pathway for specific protein degradation in eukaryotes and is involved in almost all life processes in cells. The proteasome is a highly complex supramolecular complex composed of 20S catalytic core particles (CPs) and 19S regulatory particles (RPs). The 20S proteasome is a 700 kDa cylindrical-shaped multi-catalytic protease complex containing 28 subunits organized into four rings. In yeast and other eukaryotes, seven different a subunits form the outer ring and seven different β subunits make up the inner ring. The a subunits serve as binding sites for the 19S (PA700) and 11S (PA28) regulatory complexes, as well as a physical barrier to the internal proteolytic chamber formed by the two β subunit rings. Thus, in vivo, the proteasome is thought to exist as a 26S particle (26S proteasome). The 20S complex contains three active subunits (β1 with chymotrypsin-like activity, β2 with trypsin-like activity, and β5 with chymostatin-like activity). In vivo experiments have shown that inhibition of the β5 subunit of the 20S complex of the proteasome can easily be correlated with inhibition of the 26S proteasome. Cleavage of the amino-terminal prosequences of the active site β subunits during particle formation exposes the amino-terminal threonine residue that serves as a catalytic nucleophile. The subunits responsible for catalytic activity in the proteasome thus have an amino-terminal nucleophilic residue, and these subunits belong to the N-terminal nucleophile hydrolase family (where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosyl asparaginases. In mammals, most cells and tissues express a "constitutive proteasome". When cells are stimulated by IFN-γ, TNF-α, oxidative stress, inflammatory insults, etc., the β1i / low molecular weight protein (LMP2), β2i / multi-catalytic endopeptidase complex 1 (MECL-1), and β5i / LMP7 subunits are induced intracellularly, replacing the β1, β2, and β5 subunits in the constitutive proteasome, and assembling into a new proteasome. Because the β1i, β2i, and β5i subunits enhance the ability of the proteasome to generate peptides that bind to major histocompatibility complex 1 (MHC-1), the newly assembled proteasome is called the immunoproteasome. This substitution of subunits also changes the cleavage specificity of the 20S proteasome, with decreased caspase-like activity and increased chymotrypsin- and trypsin-like activities of the immunoproteasome, resulting in more peptides that can bind to MHC-1.

[0003] The basic expression of immunoproteasome in most non-immune cells is very low, and the expression is disordered in various autoimmune diseases, such as systemic lupus erythematosus and lupus nephritis. Immunoproteasome inhibitors selectively inhibit the activity of immunoproteasome, and play a therapeutic role by affecting antigen presentation function, inhibiting T cell activity and related cytokine secretion to selectively block the expression of immunoproteasome subunits, so that immunoproteasome is considered as a therapeutic target for autoimmune diseases. ONX-0914 is the first reported tripeptide epoxy ketone β5i selective immunoproteasome inhibitor. Cells exposed to ONX0914 for a long time can inhibit the expression of LMP2 and LMP7, and block autoimmunity. KZR-616 is a selective immunoproteasome inhibitor developed on the basis of ONX0914, and has better solubility than ONX-0914. It is the only immunoproteasome inhibitor for injection currently entering clinical trials. Immunoproteasome inhibitors are expected to become a new choice for the treatment of autoimmune diseases. Therefore, it is particularly urgent to develop immunoproteasome inhibitors with higher selectivity as less toxic therapeutic drugs. SUMMARY

[0004] In a first aspect, the present application provides a peptide epoxy ketone compound of formula I or a pharmaceutically acceptable salt thereof:

[0005] wherein:

[0006] P is unsubstituted or substituted with R a a 4-10 membered saturated or partially unsaturated heterocyclic ring containing 1 or 2 heteroatoms selected from nitrogen, oxygen and sulfur; R a is one or more, R a is selected from C 1-6 alkyl;

[0007] R 1 is hydrogen, C 1-6 alkyl, or R 1 together with the carbon to which it is attached forms a cycloalkyl; wherein R 1 is unsubstituted or substituted with R b , R b is one or more, R b is selected from OR 6 , SR 6 , N(R 6 )2 or CN;

[0008] R 2 is -CONH2, -SO2NH2, -SO2Me, -COOH, -OH, -CN, halogen, C 1-6 alkyl or C 1-6 alkoxy;

[0009] R3 For hydrogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 3-8 Cycloalkenyl, aryl, or 5-6 heteroaryl groups comprising 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, or sulfur; R 3 Not replaced or R c Replace, R c There are one or more, the R c Selected from C 1-6 Alkyl, halogen, C 1-6 Alkoxy, OR 6 SR 6 and N(R) 6 )2;

[0010] R 4 It is hydrogen, methyl, ethyl, or hydroxymethyl; preferably, R 4 It is methyl;

[0011] R 5 It is hydrogen or hydroxyl; preferably, R 5 It is a hydroxyl group;

[0012] R 6 For H or C 1-6 alkyl,

[0013] m is an integer between 0 and 4.

[0014] In some embodiments, R 3 Selected from hydrogen, C 1-6 Alkyl, C 2-6 alkenyl or C 2-6 Alkyne group. Preferably, in some embodiments, R 3 Selected from C 2-6 Alkenyl group.

[0015] In some embodiments, R 3 Selected from Further preferred, R 3 Selected from: Further optimization, R 3 Selected from: The optimal choice, R 3 Selected as

[0016] In some embodiments, P is selected from: More preferably, in some embodiments, P is selected from...

[0017] In some embodiments, R1 C1-C6alkyl. Preferably, in some embodiments, R 1-6 C1-C6alkyl. Preferably, in some embodiments, R 1 C1-C6alkyl. Preferably, in some embodiments, R 1-3 C1-C6alkyl. Preferably, in some embodiments, R 1 C1-C6alkyl. Preferably, in some embodiments, R

[0018] C1-C6alkyl. Preferably, in some embodiments, R 2 C1-C6alkyl. Preferably, in some embodiments, R 1-3 C1-C6alkyl. Preferably, in some embodiments, R 1-3 C1-C6alkyl. Preferably, in some embodiments, R 2 C1-C6alkyl. Preferably, in some embodiments, R 2 C1-C6alkyl. Preferably, in some embodiments, R

[0019] In some embodiments, the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof is represented by any one of the following structures:

[0020] In some embodiments, the peptide epoxy ketone compound of Formula I has the following stereochemical configuration:

[0021] wherein P, R 1 , R 2 , R 3 , R 4 , R 5 , and m are as described above.

[0022] Preferably, in some embodiments, the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof is represented by any one of the following structures:

[0023] Preferably, the pharmaceutically acceptable salt described herein is a salt of the peptide epoxy ketone compound with an acid selected from any one of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0024] Preferably, the pharmaceutically acceptable salt described herein is a salt of the peptide epoxy ketone compound with an acid selected from any one of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0025] In another aspect of the present application, a method for preparing the epoxy ketone compound of Formula I is provided, wherein the synthesis of the compound of Formula I-a specifically comprises the following steps:

[0026] The compound of Formula H-8 is condensed with the compound of Formula F-5 after removing the Boc protecting group to obtain the compound of Formula I-a.

[0027] In another aspect of the present application, a pharmaceutical composition is provided, comprising the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-18, and a pharmaceutically acceptable carrier.

[0028] In another aspect of the present application, the use of the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described above in the manufacture of a medicament for reducing the activity of immunoproteasome or in the manufacture of a medicament for treating a disease associated with abnormal activity of immunoproteasome is provided. In some embodiments, the medicament is suitable for oral administration, parenteral administration, injection administration, inhalation administration, transdermal administration or transmucosal administration. In some embodiments, the administration is via oral administration.

[0029] In another aspect of the present application, the medicament is used for treating autoimmune diseases, wherein the disease associated with abnormal activity of immunoproteasome is an autoimmune disease. In some embodiments, the autoimmune disease comprises systemic lupus erythematosus, ulcerative colitis, rheumatoid arthritis.

[0030] Advantages of the present application: The present application provides a new structure of immunoproteasome inhibitor, which has higher selectivity, less toxicity, fewer adverse reactions and is suitable for oral administration. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the in vivo efficacy of some compounds in treating ulcerative colitis in mice;

[0032] Figure 2 is the in vivo efficacy of compound 37 in treating ulcerative colitis in mice by oral administration;

[0033] Figure 3 is the in vivo efficacy of compound 37 in treating systemic lupus erythematosus in mice by oral administration; DETAILED DESCRIPTION

[0034] DEFINITIONS

[0035] Unless otherwise indicated, the following terms and phrases used herein have the following meanings. A particular term or phrase should not be construed as being indefinite or unclear if it is not specifically defined, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0036] As used herein, C m-n means that the moiety has m to n carbon atoms. For example, "C 1-6 "alkyl" means that the alkyl group has 1 to 6 carbon atoms.

[0037] As used herein, a numerical range refers to each integer within the given range. For example, "C 1-4 " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms.

[0038] The term "halo" or "halogen" means fluorine, chlorine, bromine, or iodine.

[0039] The term "cyano" refers to a -CN group.

[0040] The term "alkyl" refers to a straight or branched chain, saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, attached to the rest of the molecule by a single bond. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, or t-butyl, and the like.

[0041] The term "alkoxy" refers to an "-O-alkyl" group.

[0042] The term "alkenyl" refers to a straight or branched chain, unsaturated aliphatic hydrocarbon group containing a carbon-carbon double bond, consisting of carbon and hydrogen atoms. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, 2-propenyl, and the like.

[0043] The term "alkyne" refers to a straight or branched chain, unsaturated aliphatic hydrocarbon group containing a carbon-carbon triple bond, consisting of carbon and hydrogen atoms. Non-limiting examples of alkyne groups include, but are not limited to, ethynyl, propynyl, and the like.

[0044] The term "heterocycle" generally represents a ring or ring system in which at least one of the atoms that form the ring backbone is not carbon (e.g., is nitrogen, oxygen, or sulfur). Typically, a heterocycle contains no more than four nitrogen, no more than two oxygen, and no more than two sulfur atoms. A heterocycle can be further defined; unless otherwise specified, a heterocycle can be a saturated, partially unsaturated, or fully unsaturated ring. When a fully unsaturated heterocycle satisfies Huckel's rule, then the ring can also be referred to as a "heteroaromatic" or "aromatic heterocycle." Unless otherwise specified, a heterocycle and ring system can be attached via any available carbon or nitrogen.

[0045] The term "aryl" includes a 5-7 membered substituted or unsubstituted monocyclic aromatic group, in which each atom of the ring is carbon. The term "aryl" also includes polycyclic systems having two or more rings, in which two or more carbons are common to two connecting rings, in which at least one of the rings is aromatic, for example, the other cyclic ring can be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, and the like. In some embodiments, the aryl ring can be substituted with a halogen, such as fluorine.

[0046] The term "heteroaryl" includes substituted or unsubstituted aromatic 5-7 membered ring structures, e.g., 5-6 membered rings, whose ring structure includes 1-4 heteroatoms. The term "heteroaryl" includes polycyclic ring systems having two or more rings, where two or more carbons are common to two connecting rings, where at least one is a heteroaryl, including, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. In some embodiments, the heteroaryl ring can be substituted with a halogen atom, such as fluorine.

[0047] The term "cycloalkyl" as used herein refers to a 3- to 7-membered saturated substituted or unsubstituted ring, where each atom of the ring is carbon. The term "cycloalkyl" also includes polycyclic ring systems having two or more rings, where one or more carbon atoms are common to two adjacent rings, where at least one of the rings is a cycloalkyl.

[0048] The term "cycloalkenyl" as used herein refers to a 3- to 7-membered substituted or unsubstituted ring, where each atom of the ring is carbon. The ring has one or more unsaturated bonds such that the ring remains non-aromatic. The term "cycloalkenyl" also includes polycyclic ring systems having two or more rings, where one or more carbon atoms are common to two adjacent rings, where at least one of the rings is a cycloalkenyl.

[0049] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0050] As the pharmaceutically acceptable salt, for example, a salt with an inorganic acid, a salt with an organic acid, a salt with an acidic amino acid, and the like.

[0051] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains an acidic or basic moiety by conventional chemical methods. Generally, the salt preparation methods are by reacting the free base form of these compounds in water or in an organic solvent, or in a mixture of both, with a chemical equivalent of the appropriate acid.

[0052] Certain compounds of the present application can exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present application. Certain compounds of the present application can exist in multiple crystalline or amorphous forms.

[0053] In the present text, the bonds represented by simple lines can be intended to include all the possible stereoisomeric forms of the compounds, including mixtures, racemates, diastereoisomers, geometric isomers and single isomers. The absolute configuration of a stereogenic center represented by a bond containing a dotted line and a bold line is indicated.

[0054] The compounds of the present application can contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds can contain deuterium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C), etc. All isotopic variations of the compounds of the present application, whether radioactive or not, are encompassed within the scope of the present application.

[0055] The term "pharmaceutically acceptable carrier" means a carrier that does not cause an appreciable level of irritation to an organism and does not abrogate the biological activity and properties of the active compound. The term "pharmaceutically acceptable carrier" means an inert material with which the active ingredient is administered, which is advantageous for the administration of the active ingredient, including, but not limited to, any of the excipients, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, disintegrating agents, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the State Food and Drug Administration for use in humans or animals (e.g., domestic animals). Non-limiting examples of the carriers include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols, etc. For further information on carriers, see

[0056] The present application is further illustrated by the following specific examples. It is to be understood that these examples are merely for the purpose of illustration and are not to be construed as limiting the scope of the present application. The chemical reactions described in the examples of the preparation can be readily adapted to prepare a number of other compounds of the present application, and alternative methods for preparing the compounds of the present application are deemed to be within the scope of the present application. Unless otherwise defined, the terms used in the present text are the same as those familiar to one skilled in the art. Furthermore, any method and material similar or equivalent to those described can be used in the present application.

[0057] In particular, the compounds of the present application, which are represented by the partial Formula I (compounds described by Formula 1-a) are synthesized by the following Route 1.

[0058] The Route 1 specifically includes the following steps:

[0059] Step 1): Preparation of the compound represented by Formula H-8

[0060] Compound of formula H-2 is prepared from compound of formula H-1 and commercially available (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-iodopropionate. (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-iodopropionate is reacted with zinc dust to form an active zinc reagent intermediate which is then coupled with compound of formula H-1 in the presence of a palladium catalyst to form H-2. Compound of formula H-2 is saponified in the presence of a base to form compound of formula H-3, preferably lithium hydroxide in methanol / water. Compound of formula H-3 is condensed with hydroxylamine hydrochloride to form compound of formula H-4 which is reacted with a Grignard reagent to form compound of formula H-5, preferably EDCI / HOBT as the condensing agent. The carbonyl group of compound of formula H-5 is reduced to form the hydroxyl group to form compound of formula H-6, preferably dicyclohexylborane complex as the reducing agent. Compound of formula H-6 is double bond epoxidized to form compound of formula H-7, preferably tert-butyl hydroperoxide / acetylacetone vanadium as the oxidizing condition. The hydroxyl group of compound of formula H-7 is oxidized to form the carbonyl group to form compound of formula H-8, preferably Dess-Martin periodinane as the oxidizing agent.

[0061] Step 2) Preparation of compound of formula J-11

[0062] Compound of formula J-1 is amide condensed with compound of formula J-2 to form compound of formula J-3, preferably isobutyl chloroformate, N-methylmorpholine in tetrahydrofuran as the reaction condition. Compound of formula J-3 is deprotected to form compound of formula J-4, preferably trifluoroacetic acid in dichloromethane or HCl in ethyl acetate as the condition. Compound of formula J-4 is nucleophilically substituted with benzyl bromide to form compound of formula J-5, preferably TEA or DIPEA as the base, THF / H2O as the solvent. Compound of formula J-5 is then reacted with glycine and nickel nitrate under basic condition to form nickel complex of formula J-6. Nickel complex of formula J-6 is reacted with aldehyde of formula J-12 under strong base condition to form compound of formula J-7 via Aldol reaction. Compound of formula J-7 is hydrolyzed under acidic condition to form compound of formula J-8. The amino group of compound of formula J-8 is reacted with Boc20 to form compound of formula J-9. Compound of formula J-9 is then reacted with benzyl bromide to form benzyl ester of formula J-10. Finally, benzyl ester of formula J-10 is deprotected to form compound of formula J-11.

[0063] Step 3) Synthesis of compound of formula I

[0064] The compound of Formula F-1 is condensed with an amino acid benzyl ester having a different side chain group to give a compound of Formula F-2. The compound of Formula F-2 is deprotected of the benzyl protecting group under catalysis of Pd / C to give a compound of Formula F-3. The compound of Formula F-3 is condensed with a compound of Formula J-11 to give a compound of Formula F-4, preferably using HATU as the condensing agent. The compound of Formula F-4 is deprotected of the benzyl protecting group under catalysis of Pd / C to give a compound of Formula F-5. The compound of Formula H-8 is condensed with the compound of Formula F-5 after removal of the Boc protecting group to give a compound of Formula I.

[0065] In another specific embodiment, compounds of Formula I (Formula I-b compounds) are synthesized by Route Two using standard methods in the art.

[0066] Route Two specifically includes the following steps:

[0067] Step 1): Same as Step 1) of Route One.

[0068] Step 2): Synthesis of Formula I-b compounds

[0069] The compound of Formula F-1 is condensed with an amino acid benzyl ester having a different side chain group to give a compound of Formula F-2. The compound of Formula F-2 is deprotected of the benzyl protecting group under catalysis of Pd / C to give a compound of Formula F-3. The compound of Formula F-3 is condensed with L-tyrosine benzyl ester p-toluenesulfonate to give a compound of Formula Q-4, preferably using HATU as the condensing agent. Subsequently, the compound of Formula Q-4 is deprotected of the benzyl protecting group under catalysis of Pd / C to give a compound of Formula Q-5. The compound of Formula H-8 is condensed with the compound of Formula Q-5 after removal of the Boc protecting group to give a compound of Formula I-b.

[0070] Example 1: (2S,3R)-3-Hydroxy-3-(4-methoxyphenyl)-N-((S)-5-methyl-l-((R)-2- methyloxiranyl-2-yl)-l-oxo-4-en-2-yl)-2-((S)-2-(2-(piperazin-l-yl)acetamido) propanamido) propanamide (37)

[0071] Step 1): Preparation of tert-butyl ((R)-5-methyl-l-((S)-2-methyl oxiranyl-2-yl)-l- oxohex-4-en-2-yl)carbamate (10)

[0072] Step a): Isobutyraldehyde 1 (15 g, 208 mmol) and 2,6-di-tert-butyl-4-methylpyridine (44.8 g, 218.4 mmol) were dissolved in DCM, triflic anhydride was added under ice-bath stirring, and the reaction was continued for 10 min after the addition was completed. Then the reaction was carried out at 60 °C for 9 h. After the reaction was completed, the reaction was reduced to room temperature, 150 mL of n-hexane was added, the pH was adjusted to 3-4 with concentrated hydrochloric acid, and the filter cake was dried by suction filtration. The filtrate was dried, and the organic phase was rotary evaporated to obtain a black liquid. The 44 °C fraction was collected by vacuum distillation to obtain colorless liquid product 3 (23 g, yield 54%). 1 H NMR (400 MHz, Chloroform-d) δ 6.63-6.31 (m, 1H), 1.76 (dd, J = 23.4, 1.6 Hz, 6H).

[0073] Step b): Zinc powder (57 g, 882 mmol) was weighed into a 250 mL tomato flask, 100 mL of DMF was added, and TMSCl (15.8 g, 147 mmol) was slowly added. After the addition was completed, the reaction was carried out at room temperature for 45 min. The supernatant was discarded, and the zinc powder was washed with DMF (60 mL x 3), and finally 100 mL of DMF was added. (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-iodopropionate 2 (43.5 g, 132.3 mmol) was dissolved in 80 mL of DMF, and the above zinc powder solution was added, and the reaction was carried out at room temperature for 2 h. Then the supernatant of the reaction liquid was transferred to a new reaction flask, Pd(dppf)Cl2 (5.36 g, 7.35 mmol) and intermediate 3 were added. Replace Ar, and react at 50 °C for 12 h. After the reaction was completed, water (500 mL) was added to the reaction liquid, and DCM was extracted, then the DCM was washed once with acid water at pH = 5, dried with Na2SO4, and rotary evaporated, and purified by silica gel column to obtain 4 (19 g, yield 56%). 1 H NMR (400 MHz, Chloroform-d) δ 5.06-5.00 (m, 1H), 4.35 (dt, J = 8.3, 5.6 Hz, 1H), 3.74 (s, 3H), 2.48 (ddt, J = 49.9, 14.7, 6.9 Hz, 2H), 1.67 (d, J = 40.9 Hz, 6H), 1.45 (s, 9H). MS (ESI) m / z 258.15 [M-H] + ;

[0074] Step c): Compound 4 (19 g, 73.9 mmol) was dissolved in tetrahydrofuran (100 mL) and stirred in an ice bath. Lithium hydroxide monohydrate (4.5 g, 103.4 mmol) was dissolved in 50 mL of water and slowly added to the reaction. The reaction was then allowed to proceed at room temperature for 3 h. After the reaction was complete, most of the tetrahydrofuran solution was removed by rotary evaporation. The pH was adjusted to 5 with 0.4 N hydrochloric acid, and the solution was extracted with ethyl acetate (10 mL x 3). The organic phases were combined and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain compound 5 (19 g). MS (ESI) m / z 243.1 [M-H] + ;

[0075] Step d): Compound 5 (19 g, 78.14 mmol) was dissolved in THF (200 mL) and stirred to 0 °C. HOBt (15.8 g, 117.2 mmol) was added and stirred for 15 min. EDCI (22.4 g, 117.2 mmol) was added and stirred for 15 min. Dimethylhydroxylamine hydrochloride (8 g, 82 mmol) was added and stirred for 15 min. DIPEA (30.2 mL, 234.4 mmol) was slowly added. The reaction was allowed to proceed at room temperature for 4 h. After the reaction was complete, the solution was poured into ice water and extracted with ethyl acetate. The organic phases were combined and washed with 0.4 M dilute hydrochloric acid, 5% sodium bicarbonate, and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain compound 6 (22 g). 1 H NMR (400 MHz, Chloroform-d) δ 5.11 (m, 1H), 4.70 (s, 1H), 3.78 (s, 3H), 3.21 (s, 3H), 2.39 (m, 2H), 1.72 (d, J = 1.4 Hz, 3H), 1.62 (d, J = 1.4 Hz, 3H), 1.45 (s, 9H). MS (ESI) m / z 287.15 [M+H] + .

[0076] Step e): Compound 6 (22 g, 76.9 mmol) was placed in a three-necked flask, 200 mL of tetrahydrofuran was added, and nitrogen was replaced. The solution was stirred at 0 °C for 10 min, and isopropenyl magnesium bromide (1 M / 269 mL, 269 mmol) was slowly added. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 h. After the reaction was complete, the solution was quenched with an ammonium chloride solution, and an appropriate amount of water was added. The solution was extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. Compound 7 (13 g, total yield 65.9% for three steps) was obtained by silica gel column chromatography purification. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (d, J = 7.9 Hz, 1H), 6.06 (s, 1H), 5.88 (d, J = 1.8 Hz, 1H), 5.06 (td, J = 6.9, 4.2 Hz, 1H), 4.69 (td, J = 8.2, 5.3 Hz, 1H), 2.23 (ddt, J = 57.3, 14.9, 6.9 Hz, 2H), 1.78 (s, 3H), 1.65 - 1.50 (m, 7H), 1.36 (s, 9H). MS (ESI) m / z 268.2 [M+H] + .

[0077] Step f): Dicyclohexylamine (6.2 g, 34.2 mmol) was added to n-hexane (50 mL) and borane dimethyl sulfide complex (2 M / 18.65 mL, 37.3 mmol) was added under ice bath stirring for 1 h. Intermediate 7 (8.3 g, 31.08 mmol) was dissolved in toluene and the solution from the first step was added under stirring at -15 °C and the reaction was continued at -15 °C for 4 h. After the reaction was completed, the pH was adjusted to 3 with hydrochloric acid, suction filtered, partitioned, the aqueous phase was extracted with ethyl acetate twice, the organic phases were combined, rotary evaporated and purified by column to give compound 8 (6.7 g, yield 80%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 5.16 (ddt, J = 8.4, 7.0, 1.4 Hz, 1H), 5.04 (s, 1H), 4.97 (q, J = 1.5 Hz, 1H), 4.76 - 4.66 (m, 1H), 4.18 (s, 1H), 3.78 (s, 1H), 2.26 - 2.10 (m, 2H), 1.79 (s, 2H), 1.72 (d, J = 1.4 Hz, 3H), 1.62 (d, J = 1.3 Hz, 2H), 1.46 (s, 7H). MS (ESI) m / z 270.1 [M-H] + .

[0078] Step g): Intermediate 8 (7.35 g, 27.3 mmol) was dissolved in DCM (60 mL) under ice bath, water (15 mL) was added, vanadium acetylacetonate (220 mg) was added under vigorous stirring. tert-Butyl hydroperoxide (70% / 7.5 g, 57.4 mmol) was added slowly dropwise and the reaction was continued at room temperature for 1 h, then 50 mL of water was added, extracted with DCM and purified by column to give 2.3 g of product 9 as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 5.22 - 5.10 (m, 1H), 4.90 (d, J = 9.3 Hz, 1H), 3.93 - 3.77 (m, 2H), 2.99 (d, J = 4.7 Hz, 1H), 2.65 (d, J = 4.8 Hz, 1H), 2.20 (q, J = 6.0, 4.5 Hz, 2H), 1.75 - 1.59 (m, 6H), 1.46 (s, 8H), 1.27 (s, 2H). MS (ESI) m / z 286.15 [M-H] + .

[0079] Step h): Compound 9 (2.3 g, 8.1 mmol) was dissolved in DCM (20 mL), NaHC03(3.4 g, 40.35 mmol) was added, then Des-Martin periodinane (4.45 g, 10.5 mmol) was added slowly, and the reaction was carried out at room temperature for 1 h. After the reaction was detected to be complete, water was added for quenching, then DCM was added for extraction, and after rotary evaporation, the product 10 was obtained in the form of a light yellow oil after purification by silica gel column. 1 H NMR (400 MHz, Chloroform-d) δ 5.00 (q, J = 7.7, 6.9 Hz, 1H), 4.43 - 4.27 (m, 1H), 3.24 (d, J = 5.0 Hz, 1H), 2.88 (d, J = 5.0 Hz, 1H), 2.59 - 2.43 (m, 1H), 2.25 (dt, J = 14.5, 7.1 Hz, 1H), 1.71 (d, J = 1.7 Hz, 3H), 1.61 (d, J = 1.4 Hz, 3H), 1.50 (s, 3H), 1.41 (s, 9H).

[0080] Step 2) Preparation of (2S, 3R)-benzyl 2-amino-3-hydroxy-3-(4-methoxyphenyl) propanoate hydrochloride

[0081] Step a): Boc-L-proline 21 (20 g, 93 mmol) was dissolved in THF (150 mL), stirred at -5 °C, protected by Ar gas, then N-methyl morpholine was added. Isobutyl chloroformate (14 g, 111.6 mmol) was dissolved in THF (50 mL) and added to the reaction, after the addition was completed, the reaction was carried out at room temperature for 1 h. Then, 2-aminobenzophenone 20 (11 g, 55.8 mmol) was added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, water was added, EA was extracted 3 times, and after the EA was combined and dried, rotary evaporation was performed to obtain the crude intermediate 22 (32 g), which was directly used in the next step. MS (ESI) m / z 395.1 [M-H] + .

[0082] Step b): The product crude 22 (32 g) in step a) was dissolved in EA (30 mL), then HCl / EA (4 M, 70 mL) was added, and the mixture was stirred at room temperature for 3-4 h. After the reaction was completed, the solvent was removed by rotary evaporation to obtain crude 23, which was directly used in the next step. MS (ESI) m / z 295.05 [M-H] + .

[0083] Step c): The product crude 23 in step b) was dissolved in a mixture of THF (100 mL) and water (100 mL), and DIPEA (14.5 g, 112 mmol) was added under ice bath. Then benzyl bromide (14.4 g, 84 mmol) was slowly added dropwise, and the mixture was stirred under ice bath for 6 h. After the reaction was completed, water was added, and EA was used for extraction. The crude product 24 g was obtained by rotary evaporation. The crude product was added with 96 mL of water and 120 mL of EA, and the pH was adjusted to 3 with concentrated hydrochloric acid. A large amount of white solid was precipitated. After stirring for 3 h, the mixture was filtered, and the filter cake was washed with a small amount of EA and dried to obtain the white solid product 24 (18.2 g, total yield of 3 steps 77%). 1 H NMR (400 MHz, DMSO-d6) δ 7.74 - 7.58 (m, 4H), 7.51 (t, J = 7.5 Hz, 2H), 7.39 (ddt, J = 10.2, 7.4, 3.6 Hz, 7H), 7.22 (d, J = 8.0 Hz, 1H), 4.25 (ddd, J = 19.2, 10.5, 6.6 Hz, 3H), 3.41 (s, 1H), 3.29 - 3.15 (m, 1H), 2.18 (p, J = 8.1 Hz, 1H), 1.96 (s, 1H), 1.65 (q, J = 13.8, 10.9 Hz, 1H), 1.27 (d, J = 22.9 Hz, 1H). MS (ESI) m / z 385.1 [M-H] + .

[0084] Step d): Glycine (10.4 g, 139.25 mmol) and KOH (12.6 g, 222.8 mmol) were dissolved in methanol (90 mL), and the mixture was stirred at 45 °C until the reaction solution was clear. Nickel nitrate hexahydrate (16.2 g, 55.7 mmol), 24 (10.7 g, 27.8 mmol) were added, and the mixture was stirred at 65 °C for 3 h. After the reaction was completed, the reaction solution was cooled to room temperature, and the reaction solution was poured into acetic acid water (13.4 g, 222.8 mmol, 5% aqueous solution), and the mixture was stirred for 30 min. Red solid was precipitated, and the mixture was filtered to obtain a red solid. The solid was dissolved in DCM, dried, and rotary evaporated to obtain a red solid 25 (10 g), which was directly used in the next step. MS (ESI) m / z 498.1 [M-H] + .

[0085] Step e): 25 (10 g, 20.12 mmol) was dissolved in methanol (30 mL), sodium methoxide (5.4 M / 10 mL, 54.33 mmol) was added, and stirred for 10 min. p-Methoxybenzaldehyde (5.5 g, 40.24 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 h. Acetic acid (3.26 g, 54.33 mmol) was prepared as a 5% aqueous solution, added to the reaction to quench, extracted with DCM, and the crude product 26 (18.4 g) was obtained as a red solid after rotary evaporation. MS (ESI) m / z 300.1 [M-H] + .

[0086] Step f): The product 26 from step e) was dissolved in methanol (50 mL) and stirred at room temperature. Concentrated hydrochloric acid (9 mL) was diluted in water (40 mL) and added to the reaction, which was allowed to proceed at room temperature for 4 h until 26 was completely reacted. At this time, the solution changed from red to blue, and intermediate 24 solid precipitated. The mixture was suction filtered, the filter cake was washed with EA, and the filter cake was recovered as intermediate 24. The filtrate was extracted with 50 mL of EA, and the aqueous phase was separated and extracted twice with DCM. The final aqueous phase was added to 1-pyrrolidine dithioacid ammonium (6.6 g, 40.24 mmol), stirred at room temperature for 2 h, suction filtered, and the filter cake was washed with a small amount of water. The aqueous phase was the solution of intermediate 27, which was used directly in the next step. MS (ESI) m / z 212.05 [M-H] + .

[0087] Step g): To the aqueous solution of intermediate 27 was added dioxane (50 mL) and (Boc)20 (6.6 g, 30.2 mmol), and then TEA was slowly added dropwise to adjust the pH to 7-8. The reaction was allowed to proceed at room temperature for 2 h, and if a small amount of starting material remained, (Boc)20 and TEA were added. After the reaction was complete, most of the dioxane was removed by rotary evaporation, 50 mL of water was added, and the pH was adjusted to 10 with a sodium carbonate solution. The aqueous phase was extracted with EA, and the EA was washed with a small amount of 5% sodium carbonate solution after the phases were separated. The aqueous phases were combined, the pH was adjusted to 3, and the mixture was extracted with EA three times. After the EA was rotary evaporated, intermediate 28 (3.6 g, total yield 41.6% over three steps) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.38 - 7.15 (m, 2H), 6.90 - 6.83 (m, 2H), 6.27 (d, J = 9.2 Hz, 1H), 5.00 (d, J = 3.6 Hz, 1H), 4.13 (dd, J = 9.2, 3.5 Hz, 1H), 3.72 (s, 3H), 1.29 (s, 9H). MS (ESI) m / z 310.1 [M-H] - .

[0088] Step h): Intermediate 28 (3.6 g, 11.58 mmol) was dissolved in DMF (20 mL), and cesium carbonate (4.2 g, 12.74 mmol) was added under ice bath, then benzyl bromide (2.97 g, 11.37 mmol) was added, after stirring for 30 min, the reaction was carried out at room temperature for 1.5 h. After the reaction was completed, water was added, extracted with EA for 3 times, the organic phase was combined, and silica gel column purification was carried out to obtain intermediate 29 (4.5 g, yield 97%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.39 - 7.32 (m, 4H), 7.30 - 7.24 (m, 4H), 6.96 - 6.83 (m, 2H), 5.64 (d, J = 4.1 Hz, 1H), 5.00 (d, J = 4.1 Hz, 2H), 4.30 (dd, J = 8.8, 4.5 Hz, 1H), 3.73 (s, 3H), 1.30 (s, 9H). MS (ESI) m / z 402.15 [M-H] + .

[0089] Step i): Intermediate 29 (3 g, 7.5 mmol) was dissolved in EA (10 mL). HCl / EA (4 M / 9 mL, 37.4 mmol) was added under ice bath, after 15 min, the reaction was carried out at room temperature for 4-5 h, and a large amount of white solid was precipitated. 9 mL of petroleum ether was added and stirred for 30 min, and then filtered to obtain white solid powder 30 (2 g, yield 79%). MS (ESI) m / z 302.1 [M-H] + .

[0090] Step 3): Preparation of (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-5-methyl-1-((R)-2- methyloxirane-2-yl)-1-oxo-4-en-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido) propanamide

[0091] Step a): L-alanine benzyl ester hydrochloride 31 (12 g, 93.0 mmol) was dissolved in DCM (150 mL), then DIPEA (48.0 g, 372 mmol) was added, stirred for 15 min under -20 °C, then chloroacetyl chloride (7.9 g, 115.1 mmol) was slowly added dropwise, and the reaction was continued for 4 h. After the reaction was completed, saturated aqueous ammonium chloride solution was added to quench the reaction, water (150 mL) was added, extracted with DCM (50 mL x 3), the combined organic phase was washed with saturated sodium chloride and dried over anhydrous Na2SO4. After filtration and evaporation, the crude product was obtained, which was purified by silica gel column chromatography to obtain the product (2-chloroacetyl)-L-alanine benzyl ester (19.3 g, yield 81%). MS (ESI) m / z 256.83 [M+H]+ .

[0092] Step b): Compound 32 (5 g, 19.6 mM), morpholine (2.1 g, 23.5 mM) were dissolved in THF (50 mL), then KI (813 mg, 4.9 mM) and NaHC03(4.1 g, 49 mM) were added, the reaction was stirred at room temperature for 24 h. The reaction solution was filtered, and the filtrate was directly concentrated by rotary evaporation, and purified by silica gel column to obtain (2-morpholinoacetyl)-L-alanine benzyl ester 33 (4.2 g, yield 70%). 1 H NMR (400 MHz, Chloroform-d) δ 7.41 - 7.33 (m, 1H), 5.24 - 5.08 (m, 2H), 4.68 (p, J = 7.4 Hz, 1H), 3.72 (t, J = 4.6 Hz, 4H), 3.14 - 3.00 (m, 2H), 2.68 - 2.48 (m, 4H), 1.43 (d, J = 6.6 Hz, 3H). MS (ESI) m / z 307.3 [M+H] + .

[0093] Step c): (2-morpholinoacetyl)-L-alanine benzyl ester 33 (2.8 g, 9.15 mmol) was dissolved in 50 mL of methanol, Pd / C (10%, 0.28 g) was added, and stirred at 50°C under hydrogen atmosphere for 2 h. After the reaction was completed, the solvent was distilled. (2-morpholinoacetyl)-L-alanine 34 (1.7 g, yield 86%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 4.25 (p, J = 7.3 Hz, 1H), 3.61 (t, J = 4.6 Hz, 4H), 2.97 (q, J = 15.3 Hz, 2H), 2.46 (q, J = 5.3 Hz, 4H), 1.29 (d, J = 7.3 Hz, 3H). MS (ESI) m / z 215.3 [M-H] + .

[0094] Step d): Compound (2-morpholinoacetyl)-L-alanine 34 (1.7 g, 7.87 mmol) was dissolved in dry DMF (20 mL) and stirred for 10 min, (2S,3R)-benzyl 2-amino-3-hydroxy-3-(4- methoxyphenyl)propanoate 30 (2.37 g, 7.87 mmol) was added at 0 °C, stirred for 10 min, HATU (4.49 g, 11.81 mmol) was added and stirred for 10 min, DIPEA (2.03 g, 15.74 mmol) was added dropwise slowly. Stirred for 30 min, then the reaction was allowed to warm to room temperature. After completion of the reaction, it was poured into water and extracted with EA. The organic phases were combined and washed with dilute hydrochloric acid, sodium bicarbonate solution, saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness. Purification by column chromatography gave compound 35 (2.12 g, 54% yield). MS (ESI) m / z 500.1 [M+H] + .

[0095] Step e): Compound 35 (2.12 g, 4.25 mmol) was dissolved in methanol (50 mL), 10% Pd / C (212 mg) was added and stirred at 50 °C under hydrogen atmosphere for 2 h. After completion of the reaction, it was filtered under suction and the filtrate was evaporated to dryness to give compound 36 (1.6 g, 91.8% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.07 (dd, J = 34.7, 9.1 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.3 Hz, 2H), 6.82 (t, J = 7.1 Hz, 2H), 5.21 - 5.01 (m, 1H), 4.41 (dt, J = 15.1, 7.5 Hz, 2H), 3.62 - 3.48 (m, 4H), 2.96 - 2.76 (m, 2H), 2.44 - 2.21 (m, 4H), 1.06 (dd, J = 79.5, 7.0 Hz, 3H). MS (ESI) m / z 410.3 [M+H] + .

[0096] Step f): Compound 10 (140 mg, 0.49 mmol) was dissolved in DCM (1 mL), TFA (1 mL) was added in ice bath for 10 min, the solvent was removed by rotary evaporation to give the trifluoroacetate salt of 2-amino-5-methyl-l-((S)-2-methyloxirane-2-yl)hex-4-en-l-one, which was dissolved in DMF (3 mL) and stirred in ice bath for 10 min. Compound 36 (200 mg, 0.49 mmol) and HATU (242 mg, 0.64 mmol) were added to the reaction and stirred for 10 min, then DIPEA (126 mg, 0.98 mmol) was added dropwise slowly. After stirring for 30 min, the reaction was allowed to warm to room temperature. After the reaction was completed, it was poured into water and extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, rotary evaporated and purified by silica gel column to give compound 37 (183 mg, yield 65%). 1 H NMR (400 MHz, Chloroform-d) δ 7.34 - 7.24 (m, 2H), 6.86 (d, J = 8.8 Hz, 2H), 5.31 (d, J = 2.7 Hz, 1H), 5.11 - 5.04 (m, 1H), 4.61 (dd, J = 8.2, 2.7 Hz, 1H), 4.49 (ddd, J = 8.0, 6.8, 5.1 Hz, 1H), 4.38 (p, J = 7.0 Hz, 1H), 3.80 (s, 3H), 3.72 (t, J = 4.9 Hz, 4H), 3.31 (d, J = 5.1 Hz, 1H), 3.00 (d, J = 6.0 Hz, 2H), 2.89 (d, J = 5.1 Hz, 1H), 2.53 (m, 5H), 2.30 (dt, J = 14.6, 7.4 Hz, 1H), 1.73 (d, J = 1.5 Hz, 3H), 1.65 (d, J = 1.4 Hz, 3H), 1.48 (s, 3H), 1.22 (d, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 208.17, 172.81, 170.40 (d, J = 5.9 Hz), 159.19, 135.97, 131.43, 127.00, 117.83, 113.74, 71.62, 66.93, 61.76, 59.52, 58.07, 55.27, 53.77, 52.85, 52.50, 48.95, 29.03, 25.87, 17.85 (d, J = 6.7 Hz), 16.62. MS (ESI) m / z 575.4 [M+H] + .

[0097] Preparation of maleate salt of compound 37

[0098] Compound 37 (100 mg) was first dissolved in ethyl acetate, and maleic acid (about 20 mg, 1 : 1 molar ratio) was added to the reaction. The suspension was stirred at about 600 rpm on a magnetic plate at room temperature. White solid was precipitated after stirring, then about 4.0 mL of ethyl acetate was added to the suspension, the supernatant was stirred overnight, and then separated by suction filtration, and the residue was dried under vacuum at 50 °C for 2 hours.

[0099] 1 H NMR (400 MHz, MeOD) δ 7.29 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.27 (s, 2H), 5.08 (t, J = 7.2 Hz, 1H), 5.03 - 4.95 (m, 2H), 4.58 (t, J = 5.0 Hz, 1H), 4.51 (t, J = 6.5 Hz, 1H), 4.42 (q, J = 7.0 Hz, 1H), 3.96 - 3.80 (m, 4H), 3.81 - 3.69 (m, 5H), 3.31 (dd, J = 9.2, 7.8 Hz, 2H), 3.06 (d, J = 5.0 Hz, 1H), 2.89 (d, J = 5.0 Hz, 1H), 2.56 - 2.35 (m, 1H), 2.34 - 2.13 (m, 1H), 1.68 (s, 3H), 1.62 (s, 3H), 1.41 (s, 3H), 1.33 (d, J = 7.1 Hz, 3H). 13 CNMR (101 MHz, MeOD) δ 207.28, 173.01, 170.02, 169.04, 165.23, 159.31, 135.00, 134.21, 132.67, 127.47, 117.90, 113.16, 72.58, 64.08, 59.01, 58.51, 57.82, 54.28, 52.69, 51.32, 51.06, 49.14, 29.55, 24.63, 16.70, 16.60, 15.18. MS (ESI) m / z 575.3 [M+H] + .

[0100] Example 2: Preparation of (2S,3R)-2-((S)-2-(2-((1R,5S)-3-oxa-8- azabicyclo[3.2.1]octan-8-yl)acetamido)propanamido)-N-((S)-3-(cyclopent-1-en-1- yl)-1-((R)-2-methyloxetan-2-yl)-1-oxopropan-2-yl)-3-hydroxy-3-(4- methoxyphenyl)propanamide (38)

[0101] Step 1): Preparation of compound tert-butyl ((R)-3-(cyclopent-l-en-l-yl)-l-((S)-2- methyloxirane-2-yl)-l-oxopropan-2-yl)carbamate (11)

[0102] The title compound 11 was synthesized in a similar manner as described in Example 1, Step 1 by replacing compound 3 with cyclopent-l-en-l-yl trifluoromethanesulfonate.

[0103] 1 H NMR (400 MHz, Chloroform-d) δ 5.44 (s, 1H), 4.39 (m, 1H), 3.27 (d, J = 5.0 Hz, 1H), 2.87 (d, J = 5.0 Hz, 1H), 2.63 - 2.43 (m, 1H), 2.25 (m, 5H), 1.85 (p, J = 7.4 Hz, 2H), 1.49 (s, 3H), 1.39 (s, 9H).

[0104] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2.

[0105] Step 3): The title compound 38 was synthesized in a similar manner as described in Example 1, Step 3 by replacing morpholine with 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride and compound 10 with compound 11.

[0106] 1 H NMR (400 MHz, Chloroform-d) δ 7.69 - 7.42 (m, 1H), 7.20 (d, J = 9.0 Hz, 2H), 6.80 (dt, J = 20.4, 6.6 Hz, 2H), 5.53 - 5.04 (m, 1H), 4.41 (dt, J = 21.6, 7.1 Hz, 1H), 4.11 (q, J = 7.1 Hz, 1H), 3.75 (dh, J = 9.1, 4.6 Hz, 3H), 3.36 - 3.10 (m, 4H), 2.79 - 2.14 (m, 10H), 1.83 (dd, J = 15.6, 8.4 Hz, 2H), 1.57 - 1.39 (m, 3H), 1.35 - 1.01 (m, 7H) 13C NMR (101 MHz, Chloroform-d) δ 208.17, 172.81, 170.40, 159.19, 138.84, 131.50, 128.55, 128.47, 127.21, 127.00, 113.71, 71.62, 70.86, 62.58, 61.83, 59.54, 59.27, 58.21, 57.46, 55.25, 52.54, 52.30, 51.38, 50.55, 48.86, 48.30, 34.76, 32.35, 23.51, 17.80, 16.61. HRMS calcd for C 32 H 44 N4O7[M+H] + ,613.3231.Found 613.3228.

[0107] Example 3: (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2- methyloxetan-2-yl)-1-oxopentan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido) propanamide (39)

[0108] Step 1): Preparation of compound tert-butyl ((R)-4-methyl-1-((S)-2-methyl oxetan-2-yl)-1- oxopentan-2-yl)carbamate (16)

[0109] The title compound 16 was synthesized in a similar manner as described in Example 1, Step 1, directly replacing compound 5 with commercially available Boc-L-leucine.

[0110] 1 H NMR (400 MHz, Chloroform-d) δ 4.87 (s, 4H), 4.06 (dd, J = 10.0, 2.7 Hz, 1H), 3.31 (p, J = 1.7 Hz, 1H), 3.14 (d, J = 4.7 Hz, 1H), 3.03 (d, J = 4.6 Hz, 1H), 1.75 (m, 2H), 1.53 (s, 3H), 1.37 (s, 9H), 1.00 (m, 6H).

[0111] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2).

[0112] Step 3): In a similar manner as described in Example 1, Step 3), compound 10 was replaced with compound 16, using the same procedure to synthesize compound 39.

[0113] 1 H NMR (400 MHz, Chloroform-d) δ 7.53 (d, J = 7.0 Hz, 1H), 7.41 (dd, J = 21.3, 7.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.14 (dt, J = 8.7, 4.3 Hz, 2H), 6.82 (d, J = 5.3 Hz, 2H), 5.23 (d, J = 32.1 Hz, 1H), 4.72 (dtd, J = 29.5, 8.1, 4.6 Hz, 1H), 4.59 (dt, J = 7.2, 3.4 Hz, 1H), 4.38 (dt, J = 11.2, 6.9 Hz, 1H), 3.77 (d, J = 3.9 Hz, 3H), 3.69 (q, J = 5.3 Hz, 4H), 3.37 - 3.21 (m, 1H), 3.13 - 2.70 (m, 5H), 2.53 (dt, J = 9.7, 4.4 Hz, 1H), 2.09 (m, 4H), 1.49 (d, J = 14.4 Hz, 3H), 1.34 - 1.13 (m, 6H). HRMS calcd for C 28 H 42 N4O8 13 C NMR (101 MHz, Chloroform-d) δ 208.76, 172.70, 170.5, 159.24, 159.08, 131.75, 131.27, 127.76, 127.27, 127.00, 113.73, 113.66, 71.60, 66.88, 61.62, 59.37, 59.08, 58.24, 57.71, 55.22, 53.73, 52.56, 50.94, 49.54, 48.36, 38.43, 37.20, 34.12, 31.79, 29.69, 26.27, 25.89, 17.68, 16.80. [M+H] + ,563.3075. Found 563.3070.

[0114] Example 4: (2S,3R)-2-((S)-2-(2-((1R,4R)-2-Oxa-5-azabicyclo[2.2.1]heptan-5-yl)acetamido) propanamido)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)-3- hydroxy-3-(4-methoxyphenyl)propanamide (40)

[0115] Step 1): Compound 11 was synthesized using the same procedure as in Example 2, Step 1).

[0116] Step 2): Compound 30 was synthesized using the same method as described in Step 2) of Example 1.

[0117] Step 3): Using the same method as described in Step 3) of Example 1, the starting morpholine was replaced with (1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptane, compound 10 was replaced with compound 11, compound 40 was synthesized using the same method.

[0118] 1 H NMR (400 MHz, Chloroform-d) δ 7.61 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.28 - 7.22 (m, 2H), 7.10 (dt, J = 9.9, 5.3 Hz, 1H), 6.85 (t, J = 9.1 Hz, 2H), 5.47 (s, 1H), 5.38 - 5.12 (m, 1H), 4.61 (tt, J = 16.9, 13.2, 5.5 Hz, 2H), 4.42 (q, J = 8.5 Hz, 2H), 4.00 - 3.89 (m, 1H), 3.79 (d, J = 3.3 Hz, 3H), 3.66 (t, J = 7.2 Hz, 1H), 3.27 - 3.19 (m, 2H), 2.70 - 2.15 (m, 10H), 1.84 (ddt, J = 29.4, 18.9, 8.5 Hz, 4H), 1.58 - 1.47 (m, 3H), 1.39 - 1.14 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 208.67, 172.31, 169.40, 158.99, 138.84, 131.50, 128.51, 127.21, 127.00, 113.71, 71.62, 70.86, 62.58, 61.83, 59.41, 58.21, 57.46, 55.25, 52.47, 51.38, 50.55, 48.58, 35.43, 34.76, 32.65, 32.04, 23.51, 17.80, 16.61. HRMS calcd for C 31 H 42 N4O8[M+H] + ,599.3075. Found 599.3067.

[0119] Example 5: (2S, 3R)-3-Hydroxy-3-(4-methoxyphenyl)-N-((S)-1-((R)-2- methyloxirane-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido) propanamido) propanamide (41)

[0120] Step 1): Preparation of compound tert-butyl ((S)-1-((S)-2-methyloxirane-2-yl)-1- oxo-3-phenylpropan-2-yl)carbamate (12)

[0121] The title compound 12 was synthesized by replacing compound 5 with commercially available Boc-L-phenylalanine directly in a similar manner as described in Example 1, Step 1.

[0122] 1 H NMR (400 MHz, Chloroform-d) δ 7.35 - 7.15 (m, 5H), 4.98 (d, J = 8.4 Hz, 1H), 4.60 (m, 1H), 3.31 (d, J = 5.0 Hz, 1H), 3.12 (dd, J = 13.9, 5.0 Hz, 1H), 2.92 (d, J = 5.0 Hz, 1H), 2.75 (dd, J = 13.9, 7.8 Hz, 1H), 1.54 (s, 3H), 1.38 (s, 9H).

[0123] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2).

[0124] Step 3): Compound 41 was synthesized by replacing compound 10 with compound 12 in a similar manner as described in Example 1, Step 3).

[0125] 1H NMR (400 MHz, Chloroform-d) δ 7.33 - 7.18 (m, 5H), 7.15 - 7.02 (m, 2H), 6.82 (dtd, J = 13.6, 11.5, 10.5, 7.6 Hz, 2H), 5.28 - 5.15 (m, 1H), 4.85 (ddq, J = 12.4, 8.1, 4.9, 3.6 Hz, 1H), 4.73 (ddd, J = 10.2, 7.6, 3.5 Hz, 1H), 4.59 (ddd, J = 11.0, 8.0, 2.4 Hz, 1H), 4.42 - 4.31 (m, 1H), 3.79 (t, J = 6.1 Hz, 3H), 3.71 (tt, J = 9.5, 3.9 Hz, 4H), 3.39 - 3.20 (m, 1H), 3.20 - 3.07 (m, 1H), 3.04 - 2.84 (m, 3H), 2.80 (ddd, J = 14.5, 9.8, 5.3 Hz, 1H), 2.49 (ddt, J = 24.8, 10.0, 4.9 Hz, 4H), 1.57 - 1.41 (m, 3H), 1.32 - 1.12 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 207.23, 172.55, 170.57, 169.93, 135.78, 131.34, 129.42, 129.22, 128.67, 128.62, 128.53, 127.77, 127.18, 127.06, 126.95, 113.71, 71.53, 66.90, 58.17, 55.31, 53.74, 52.81, 52.45, 48.55, 36.91, 17.56, 16.52, 12.44. HRMS calcd for C 31 H 40 N4O8[M + H] + ,597.2918. Found 597.2924.

[0126] Example 6: (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-1-((R)-2-methoxy-2-yl)-1- oxo-3-(pyridin-2-yl)propan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamide)propanamide (42)

[0127] Step 1): Preparation of compound tert-butyl ((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3- (pyridin-3-yl)propan-2-yl)carbamate (17)

[0128] The title compound 17 was synthesized by replacing compound 5 with commercially purchased N-tert-butoxycarbonyl-3-pyridyl-L-alanine in a similar manner as described in Step 1 of Example 1.

[0129] 1 H NMR (400 MHz, Chloroform-d) δ 8.50 (dd, J = 4.8, 1.7 Hz, 1H), 8.40 (d, J = 2.3 Hz, 1H), 7.52 (m, 1H), 7.24 (dd, J = 7.9, 5.0 Hz, 1H), 5.01 (d, J = 8.7 Hz, 1H), 4.57 (m, 1H), 3.26 (d, J = 4.8 Hz, 1H), 3.12 (dd, J = 14.1, 4.8 Hz, 1H), 2.94 (t, J = 5.6 Hz, 1H), 2.71 (dd, J = 14.1, 7.9 Hz, 1H), 1.51 (s, 3H), 1.35 (s, 9H).

[0130] Step 2): Compound 30 was synthesized using the same procedure as Step 2) of Example 1.

[0131] Step 3): Compound 42 was synthesized by replacing compound 10 with compound 17 in a similar manner as described in Step 3) of Example 1.

[0132] 1 H NMR (400 MHz, Chloroform-d) 1 H NMR (400 MHz, Chloroform-d) δ 8.45 - 8.39 (m, 1H), 7.66 - 7.55 (m, 1H), 7.48 (dt, J = 22.5, 7.4 Hz, 1H), 7.27 - 7.18 (m, 2H), 7.12 (td, J = 10.8, 7.4 Hz, 1H), 6.91 - 6.75 (m, 2H), 5.36 - 5.16 (m, 1H), 4.81 (ddt, J = 12.0, 8.1, 4.4 Hz, 1H), 4.75 - 4.64 (m, 1H), 4.56 (ddd, J = 8.1, 5.4, 2.7 Hz, 1H), 4.34 (m, 1H), 3.78 (t, J = 3.5 Hz, 3H), 3.70 (dq, J = 14.7, 5.7, 5.3 Hz, 4H), 3.35 - 2.92 (m, 4H), 2.92 - 2.75 (m, 2H), 2.51 (m, 4H), 1.59 - 1.46 (m, 3H), 1.34 - 1.23 (m, 3H). 13C NMR (101 MHz, Chloroform-d) δ 207.23, 172.55, 170.36, 169.93, 135.78, 131.34, 129.42, 129.22, 128.67, 128.56, 127.77, 127.18, 127.06, 126.95, 113.71, 71.53, 66.92, 59.18, 58.17, 57.84, 55.27, 53.74, 52.81, 52.51, 52.38, 48.55, 37.10, 36.71, 17.56, 16.51, 12.44. HRMS calcd for C 30 H 39 N5O8[M+H] + ,598.2871.Found 598.2864.

[0133] Example 7: (S)-3-(4-hydroxyphenyl)-2-((S)-3-methoxy-2-(2-morpholinoacetamido)propanamido)-N-((S)-1-(R)-2-methoxy-2-yl)-1-oxo-3-phenylpropanamido-2-yl)propanamide (43)

[0134] Step 1): Compound 12 was synthesized using the same procedure as described in Step 1) of Example 3.

[0135] Step 2): Using a similar procedure as described in Step 3) of Example 1, replacing the starting material L-alanine benzyl hydrogen chloride salt 31 with the commercially available compound (S)-benzyl 2-amino-3-methoxypropanoate, compound 30 with the commercially available L-tyrosine benzyl ester p-toluenesulfonate salt, and compound 10 with compound 12, compound 43 was synthesized.

[0136] 1 H NMR (400 MHz, Chloroform-d) δ 7.97 - 7.77 (m, 1H), 7.37 - 7.11 (m, 5H), 7.02 - 6.86 (m, 2H), 6.69 (ddq, J = 15.0, 8.5, 3.0 Hz, 2H), 4.90 - 4.74 (m, 1H), 4.68 - 4.54 (m, 1H), 4.54 - 4.44 (m, 1H), 3.70 (ddt, J = 20.0, 9.6, 4.2 Hz, 5H), 3.52 - 3.22 (m, 5H), 3.12 - 2.99 (m, 3H), 2.97 - 2.87 (m, 2H), 2.86 - 2.65 (m, 1H), 2.52 (ddd, J = 9.4, 6.5, 3.5 Hz, 5H), 1.48 (t, J = 2.8 Hz, 3H).13 C NMR (101 MHz, Chloroform-d) δ 207.33, 170.91, 170.45, 169.74, 155.49, 130.46, 129.32, 128.61, 127.19, 127.11, 115.51, 71.40, 66.89, 61.59, 59.18, 54.31, 53.66, 53.09, 52.64, 52.39, 51.98, 37.18, 36.92, 29.70, 16.53, 16.46. HRMS calcd for C31H40N4O8 [M+H]+, 597.2918. Found 597.2914.

[0137] Example 8: (S)-N-((S)-3-cyclopentyl-l-((R)-2-methoxy-2-yl)-l-oxopropan-2-yl)-3- (4-hydroxyphenyl)-2-((S)-3-methoxy-2-(2-morpholinoacetamido)propanamide)propanamide (44)

[0138] Step 1): Preparation of compound tert-butyl ((R)-3-cyclopentyl-l-((S)-2- methyloxirane-2-yl)-l-oxopropan-2-yl)carbamate (13)

[0139] The title compound 13 was synthesized by replacing compound 5 with commercially available Boc-L-cyclopentylalanine directly in a similar manner as described in Example 1, Step 1).

[0140] 1 H NMR (400 MHz, Chloroform-d) δ 4.91 (d, J = 8.9 Hz, 1H), 4.30 (td, J = 9.2, 3.4 Hz, 1H), 3.30 (d, J = 5.0 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 1.97 - 1.73 (m, 4H), 1.64 (s, 3H), 1.54 (s, 6H), 1.43 (s, 9H).

[0141] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2).

[0142] Step 3): The title compound 44 was synthesized by replacing starting material L-alanine benzyl ester hydrochloride 31 with commercially available compound (S)-2-amino-3- methoxypropanoic acid benzyl ester, compound 30 with commercially available L- tyrosine benzyl ester p-toluenesulfonic acid salt, and compound 10 with compound 13 in a similar manner as described in Example 1, Step 3).

[0143] 1 H NMR (400 MHz, Chloroform-d) δ 7.89 (d, J = 7.5 Hz, 1H), 7.09 - 6.96 (m, 2H), 6.79 - 6.70 (m, 2H), 4.71 - 4.58 (m, 1H), 4.57 - 4.49 (m, 2H), 3.73 (dq, J = 10.5, 6.1, 4.7 Hz, 4H), 3.54 (dd, J = 9.4, 7.4 Hz, 1H), 3.48 - 3.41 (m, 1H), 3.35 (d, J = 8.6 Hz, 3H), 3.27 (t, J = 4.9 Hz, 1H), 3.01 (dddd, J = 22.2, 14.2, 8.8, 5.2 Hz, 3H), 2.90 (d, J = 5.0 Hz, 1H), 2.52 (dp, J = 14.0, 5.1, 4.7 Hz, 4H), 2.04 - 1.57 (m, 6H), 1.52 (s, 3H), 1.44 - 0.98 (m, 5H). 13 C NMR (101 MHz, Chloroform-d) δ 208.20, 170.67, 169.61, 130.56, 127.45, 127.31, 115.56, 71.49, 66.92, 61.63, 59.11, 54.33, 53.71, 52.36, 51.35, 37.49, 36.76, 33.16, 31.96, 29.70, 25.22, 24.97, 16.67 HRMS calcd for C 30 H 44 N4O8[M+H] + , 589.3231. Found 589.3222.

[0144] Example 9: (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-3-(4- methoxyphenyl)-l-((R)-2-methoxy-2-yl)-l-oxopropan-2-yl)-2-((S)-2-(2- morpholinoacetamido)propanamide (45)

[0145] Preparation of compound tert-butyl (S)-3-(4-methoxyphenyl)-l-((S)-2- methyloxirane-2-yl)-l-oxopropan-2-yl)carbamate (14)

[0146] Step 1): The title compound 14 was synthesized by replacing compound 5 with commercially available Boc-O-methyl-L-tyrosine in a similar manner as described in Example 1, Step 1).

[0147] 1 H NMR (400 MHz, Chloroform-d) δ 7.15 - 7.01 (m, 2H), 6.92 - 6.84 (m, 2H), 3.81 (s, 3H), 3.30 (d, J = 5.0 Hz, 1H), 3.07 (dd, J = 14.1, 5.1 Hz, 1H), 2.92 (d, J = 5.0 Hz, 1H), 2.72 (dd, J = 14.1, 7.6 Hz, 1H), 1.52 (s, 3H), 1.39 (s, 9H).

[0148] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2).

[0149] Step 3): The title compound 45 was synthesized by replacing compound 10 with compound 14 in a similar manner as described in Example 1, Step 3).

[0150] 1 H NMR (400 MHz, Chloroform-d) δ 7.25 - 7.14 (m, 2H), 7.10 - 6.97 (m, 2H), 6.93 - 6.76 (m, 4H), 5.24 (dd, J = 27.6, 2.8 Hz, 1H), 4.83 - 4.65 (m, 1H), 4.63 - 4.55 (m, 1H), 4.37 (dt, J = 11.0, 7.1 Hz, 1H), 3.78 (d, J = 3.7 Hz, 6H), 3.70 (ddd, J = 9.3, 6.1, 2.9 Hz, 4H), 3.30 (dd, J = 29.8, 4.9 Hz, 1H), 3.14 - 2.87 (m, 4H), 2.82 - 2.71 (m, 1H), 2.61 - 2.40 (m, 4H), 1.50 (d, J = 16.4 Hz, 3H), 1.33 - 1.18 (m, 3H). 13C NMR (101 MHz, Chloroform-d) δ 207.95, 170.56, 170.32, 169.99, 158.72, 131.21, 130.33, 128.21, 127.64, 127.14, 126.96, 114.02, 113.96, 113.69, 71.46, 66.92, 61.65, 59.38, 57.80, 55.23, 54.17, 53.74, 52.69, 49.02, 48.23, 36.23, 35.54, 17.60, 16.59. HRMS calcd for C 32 H 42 N4O8[M+H] + , 627.3024. Found 627.3017.

[0151] Example 10: (2S,3R)-3-Hydroxy-N-((S)-3-methoxy-l-((R)-2-methoxy-2-yl)- l-oxopropan-2-yl)-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamide) propanamide (46)

[0152] Preparation of compound tert-butyl (R)-3-methoxy-l-((S)-2-methyloxetan-2-yl)-l- oxopropan-2-yl)carbamate (15)

[0153] Step 1): The title compound 15 was synthesized by replacing compound 5 with commercially available N-Boc-O-methyl-L-serine directly in a similar manner as described in Example 1, Step 1).

[0154] 1 H NMR (400 MHz, Chloroform-d) δ 4.46 (m, 1H), 3.86 (dd, J = 9.9, 3.9 Hz, 1H), 3.60 (dd, J = 9.9, 3.6 Hz, 1H), 3.35 (s, 3H), 2.98 (s, 1H), 2.90 (s, 1H), 1.55 (s, 3H), 1.44 (s, 9H).

[0155] Step 2): Compound 30 was synthesized using the same procedure as described in Example 1, Step 2).

[0156] Step 3): Compound 46 was synthesized by replacing compound 10 with compound 15 in a similar manner as described in Example 1, Step 3) using the same procedure.

[0157] 1 H NMR (400 MHz, Chloroform-d) δ 7.67 - 7.42 (m, 1H), 7.36 - 7.23 (m, 2H), 7.16 - 7.04 (m, 1H), 6.93 - 6.75 (m, 2H), 6.03 (d, J = 4.4 Hz, 1H), 5.47 - 5.20 (m, 1H), 4.83 - 4.58 (m, 1H), 4.59 - 4.35 (m, 1H), 3.79 (dd, J = 5.8, 2.8 Hz, 3H), 3.75 - 3.65 (m, 4H), 3.40 - 3.25 (m, 2H), 3.25 - 3.10 (m, 1H), 3.07 - 2.85 (m, 2H), 2.67 - 2.37 (m, 4H), 2.08 - 1.81 (m, 4H), 1.60 - 1.33 (m, 3H), 1.29 - 1.10 (m, 3H). NMR (101 MHz, Chloroform-d) δ 208.76, 172.73, 170.69, 170.19, 159.14, 131.55, 127.52, 127.00, 113.706, 71.60, 66.88, 61.62, 59.22, 58.24, 57.71, 55.24, 53.73, 52.53, 50.94, 49.53, 48.36, 38.43, 17.80. C 26 H 38 N4O8[M+H] + , 551.2711. Found 511.2711.

[0158] Example 11: N-(2S)-1-((3R)-1-(((S)-3-(Cyclopent-1-enyl-1-yl)-1-((R)-2-methoxy-2-yl)-1- oxopropan-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-yl)amino-1- oxopropan-2-yl)-1H-indazole-3-carboxamide (47)

[0159] Step 1): Compound 11 was synthesized using the same procedure as Example 2, Step 1).

[0160] Step 2): Compound 30 was synthesized using the same procedure as Example 1, Step 2).

[0161] Step 3): The following intermediate was obtained by condensation of indazole-3-carboxylic acid with L-alanine benzyl ester hydrochloride.

[0162] The specific method is as follows: compound indazole-3-carboxylic acid (1.5 g, 9.3 mmol) is dissolved in DMF (20 mL) and stirred for 10 min, L-alanine benzyl hydrochloride (2.0 g, 9.3 mmol) is added at 0 °C, stirred for 10 min, HATU (5.3 g, 13.9 mmol) is added and stirred for 10 min, and then DIPEA (2.4 g, 18.6 mmol) is slowly added dropwise. After stirring for 30 min, the reaction is transferred to room temperature. After the reaction is completed, it is poured into water and extracted with EA. After the organic phases are combined, they are washed with dilute hydrochloric acid, sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, the solvent is evaporated, and column chromatography is used for purification to obtain compound (1H-indazole-3-carbonyl) alanine benzyl ester (1.7 g, yield 56%). MS (ESI) m / z 324.5 [M+H] + .

[0163] In a similar manner to that described in Example 1, step 3), intermediate 33 is replaced with the compound (1H-indazole-3-carbonyl) alanine benzyl ester synthesized above, compound 10 is replaced with compound 11, and the same method is used to synthesize compound 47.

[0164] 1 H NMR (400 MHz, DMSO-d6) δ 8.37 - 8.02 (m, 2H), 8.00 - 7.80 (m, 1H), 7.67 - 7.60 (m, 1H), 7.43 (ddq, J = 8.2, 6.7, 1.4 Hz, 1H), 7.27 - 7.17 (m, 2H), 6.72 - 6.58 (m, 2H), 5.57 (t, J = 5.0 Hz, 1H), 5.47 - 5.32 (m, 1H), 4.95 (dt, J = 15.5, 3.9 Hz, 1H), 4.71 - 4.53 (m, 2H), 4.52 - 4.30 (m, 1H), 3.75 - 3.41 (m, 3H), 3.30 - 2.89 (m, 3H), 2.40 (dd, J = 13.5, 7.1 Hz, 1H), 2.19 (dt, J = 23.6, 7.8 Hz, 5H), 1.74 (tt, J = 14.1, 7.1 Hz, 2H), 1.52 - 1.34 (m, 3H), 1.30 - 1.18 (m, 5H). 13C NMR (101 MHz, DMSO-d6) δ 208.01, 158.69, 141.62, 139.86, 138.22, 128.00, 127.81, 127.29, 127.06, 122.65, 121.97, 113.34, 111.23, 72.30, 59.48, 58.99, 55.25, 49.91, 34.95, 32.48, 23.41, 22.56, 18.60, 16.62. MS (ESI) m / z 604.9 [M+H] + .

[0165] Example 12: (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-1-((R)-2-methoxy-2-yl)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamide)propanamide (48)

[0166] Step 1): Preparation of compound tert-butyl ((R)-1-((S)-2-methyloxiran-2-yl)-3-(naphthalen-2-yl)-1-oxopropan-2-yl)carbamate (18)

[0167] The title compound 18 was synthesized by replacing compound 5 with commercially available Boc-3-(2-naphthyl)-D-alanine directly in a similar manner as described in Example 1, Step 1).

[0168] 1 H NMR (400 MHz, Chloroform-d) δ 7.91 - 7.67 (m, 3H), 7.61 - 7.34 (m, 3H), 7.25 - 7.13 (m, 1H), 6.04 (s, 1H), 5.84 (d, J = 1.8 Hz, 1H), 5.46 - 5.19 (m, 2H), 3.18 (m, 2H), 1.86 (s, 3H), 1.39 (s, 9H).

[0169] Step 2): Compound 30 was synthesized using the same procedure as in Example 1, Step 2).

[0170] Step 3): Compound 48 was synthesized by replacing compound 10 with compound 18 in a similar manner as described in Example 1, Step 3).

[0171] 1H NMR (400 MHz, Chloroform-d) δ 7.86 - 7.76 (m, 3H), 7.59 - 7.41 (m, 2H), 7.40 - 7.14 (m, 3H), 7.09 - 6.99 (m, 1H), 6.85 - 6.67 (m, 2H), 5.21 (dd, J = 22.7, 3.0 Hz, 1H), 5.02 - 4.76 (m, 1H), 4.58 (ddd, J = 13.2, 8.0, 2.8 Hz, 1H), 4.45 - 4.15 (m, 1H), 3.89 - 3.70 (m, 2H), 3.69 - 3.61 (m, 4H), 3.43 - 3.23 (m, 2H), 3.05 - 2.85 (m, 2H), 2.40 (pt, J = 10.0, 5.7 Hz, 4H), 1.55 - 1.40 (m, 3H), 1.27 - 1.06 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 208.11, 172.70, 170.57, 159.05, 134.02, 133.35, 132.46, 131.41, 128.12, 127.73, 127.62, 127.28, 126.94, 126.25, 125.82, 113.65, 71.53, 66.87, 61.54, 59.44, 58.03, 55.35, 54.13, 53.55, 52.65, 49.10, 48.34, 43.47, 37.13, 36.32, 17.48, 16.62. HRMS calcd for C 35 H 42 N4O8[M+H] + , 647.3075. Found 647.3065.

[0172] Example 13: (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-1-((R)-2-methoxy-2-yl)-1- oxo-3-(m-tolyl)propan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamide)propanamide (49)

[0173] Preparation of compound tert-butyl ((R)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-(m- tolyl)propan-2-yl)carbamate (18)

[0174] Step 1): The title compound 18 was synthesized by direct replacement of compound 5 with commercially available Boc-L-3-methylphenylalanine in a similar manner as described in Step 1) of Example 1, Step 1).

[0175] 1 H NMR (400 MHz, Chloroform-d) δ 7.19 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 4.93 (d, J = 8.3 Hz, 1H), 4.55 (m, 3.29 (d, J = 5.0 Hz, 1H), 3.07 (dd, J = 13.9, 4.9 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 2.68 (dd, J = 13.9, 7.9 Hz, 1H), 2.33 (s, 3H), 1.50 (s, 3H), 1.37 (s, 9H).

[0176] Step 2): Compound 30 was synthesized using the same procedure as described in Step 2) of Example 1.

[0177] Step 3): Compound 49 was synthesized using the same procedure as described in Step 3) of Example 1, replacing compound 10 with compound 19.

[0178] 1 H NMR (400 MHz, Chloroform-d) δ 7.19 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 4.93 (d, J = 8.3 Hz, 1H), 4.55 (m, 3.29 (d, J = 5.0 Hz, 1H), 3.07 (dd, J = 13.9, 4.9 Hz, 1H), 2.90 (d, J = 5.0 Hz, 1H), 2.68 (dd, J = 13.9, 7.9 Hz, 1H), 2.33 (s, 3H), 1.50 (s, 3H), 1.37 (s, 9H). 13CNMR (101 MHz, Chloroform-d) δ 208.18, 172.60, 170.48, 159.11, 135.66, 130.01, 128.46, 127.90, 127.06, 126.29, 113.68, 71.51, 66.90, 61.61, 57.88, 55.43, 54.17, 53.73, 52.82, 52.46, 49.09, 48.36, 43.59, 36.96, 36.21, 21.39, 17.56, 16.59, 12.50. HRMS calcd for C32H42N4O8[M+H] + , 611.3075. Found 611.3065.

[0179] Example 14: (2S,3R)-N-((S)-3-(Cyclopent-l-en-l-yl)-l-((R)-2-methoxy-2-yl)-l- oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-(piperazin-l- yl)acetamido)propanamide (50)

[0180] Step 1): Compound 11 was synthesized using the same method as described in Example 2, Step 1).

[0181] Step 2): Compound 30 was synthesized using the same method as described in Example 1, Step 2).

[0182] Step 3): Using the same method as described in Example 1, Step 3), with the raw material morpholine replaced by N-BOC-piperazine, compound 11 replaced by compound 30, the same method was used for synthesis and Boc was removed at the end using EA / HCl to obtain compound 50.

[0183] 1H NMR (400 MHz, Chloroform-d) δ 7.55 (dt, J = 47.4, 7.5 Hz, 1H), 7.34 - 7.21 (m, 2H), 7.21 - 7.14 (m, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.91 - 6.71 (m, 2H), 5.45 (s, 1H), 5.35 - 5.16 (m, 1H), 4.70 - 4.52 (m, 2H), 4.44 (dq, J = 13.7, 7.0 Hz, 1H), 3.77 (d, J = 5.8 Hz, 3H), 3.68 (dd, J = 11.2, 6.0 Hz, 4H), 3.38 - 3.21 (m, 1H), 3.08 - 2.77 (m, 3H), 2.67 - 2.11 (m, 10H), 1.85 (ddt, J = 12.4, 8.0, 4.7 Hz, 2H), 1.60 - 1.41 (m, 4H), 1.36 - 1.23 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 208.08, 169.50, 159.38, 138.41, 128.88, 128.50, 127.69, 126.99, 113.71, 74.19, 61.95, 58.13, 55.68, 55.27, 52.43, 51.05, 50.78, 48.43, 34.73, 34.48, 32.37, 25.09, 23.49, 17.54, 16.61. MS (ESI) m / z 585.8 [M+H] + .

[0184] Example 15: (2S,3R)-2-((S)-2-(2-((1R,5S)-3-oxa-8-azabicyclo[3.2.1]octan-8- yl)acetamido)propanamido)-3-hydroxy-3-(4-methoxyphenyl)-N-((S)-5-methyl-1- ((R)-2-methoxy-2-yl)-1-oxohex-4-en-2-yl)propanamide (51)

[0185] Step 1): Compound 10 was synthesized using the same procedure as described in Step 1) of Example 1.

[0186] Step 2): Compound 30 was synthesized using the same procedure as described in Step 2) of Example 1.

[0187] Step 3): Compound 51 was synthesized using the same procedure as described in Step 3) of Example 1, replacing the starting material morpholine with 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride.

[0188] 1H NMR (400 MHz, Chloroform-d) δ 7.29 - 7.23 (m, 2H), 6.89 - 6.84 (m, 2H), 5.38 - 5.23 (m, 1H), 5.08 - 4.88 (m, 2H), 4.66 (qdd, J = 8.3, 6.1, 2.3 Hz, 1H), 4.53 - 4.29 (m, 2H), 3.80 (q, J = 3.2, 2.8 Hz, 3H), 3.74 - 3.54 (m, 4H), 3.40 - 3.15 (m, 2H), 3.05 - 2.80 (m, 6H), 1.77 - 1.68 (m, 3H), 1.68 - 1.58 (m, 3H), 1.57 - 1.45 (m, 4H), 1.42 - 1.34 (m, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 208.17, 172.81, 170.40, 159.19, 136.11, 127.22, 127.00, 113.76, 113.68, 73.26, 62.02, 61.82, 59.24, 57.68, 55.28, 55.21, 51.97, 29.70, 25.89, 25.12, 17.89, 17.67, 16.56. MS (ESI) m / z 601.8 [M+H] + .

[0189] Example 16: (2S,3R)-3-Hydroxy-3-(4-methoxyphenyl)-N-((S)-5-methyl-1-((R)-2- methoxy-2-yl)-1-oxa-4- en-2-yl)-2-((S)-2-(2-(piperazin-1-yl)acetamido)propanamide (52)

[0190] Step 1): Compound 10 was synthesized using the same procedure as described in Step 1) of Example 1.

[0191] Step 2): Compound 30 was synthesized using the same procedure as described in Step 2) of Example 1.

[0192] Step 3): In a similar manner as described in Step 3) of Example 1, the starting morpholine was replaced with N-BOC-piperazine and Boc was removed with EA / HCl at the end to give compound 52.

[0193] 1H NMR (400 MHz, Chloroform-d) δ 7.20 - 7.05 (m, 2H), 6.83 (dq, J = 10.0, 7.8, 6.9 Hz, 2H), 5.00 - 4.86 (m, 1H), 4.66 - 4.49 (m, 2H), 4.47 - 4.37 (m, 1H), 4.37 - 4.21 (m, 1H), 3.81 - 3.77 (m, 3H), 3.76 - 3.67 (m, 3H), 3.57 (tdd, J = 10.6, 6.1, 3.6 Hz, 2H), 1.70 (dd, J = 3.8, 1.5 Hz, 2H), 1.59 (dd, J = 13.5, 1.3 Hz, 2H), 1.55 - 1.52 (m, 1H), 1.51 - 1.43 (m, 6H), 1.40 (dt, J = 7.1, 2.9 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 208.22, 171.34, 170.82, 168.83, 158.98, 134.43, 132.51, 128.79, 128.52, 119.40, 114.50, 113.35, 72.75, 61.36, 55.47, 53.13, 51.96, 51.63, 51.28, 47.76, 47.57, 29.49, 26.03, 19.39, 18.78, 18.08, 16.72. MS (ESI) m / z 574.7 [M+H] + .

[0194] Example 17: (S)-2-((S)-2-(2-((1R,5S)-3-oxo-8-azabicyclo[3.2.1]octan-8-yl)acetamido) propanamido)-3-(4-methoxyphenyl)-N-(S)-5-methyl-1-((R)-2-methoxy-2-yl)-1-oxohexan-4- en-2-yl)propanamide (53)

[0195] Step 1): Compound 10 was synthesized using the same procedure as described in Step 1) of Example 1.

[0196] Step 2): Compound 53 was synthesized using the same procedure as described in Step 3) of Example 1, replacing the starting material morpholine with 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride and replacing compound 30 with O-methyl-L-tyrosine benzyl ester hydrochloride.

[0197] 1H NMR (400 MHz, Chloroform-d) δ 7.20 - 7.05 (m, 2H), 6.83 (dq, J = 10.0, 7.8, 6.9 Hz, 2H), 5.00 - 4.86 (m, 1H), 4.66 - 4.49 (m, 2H), 4.47 - 4.37 (m, 1H), 4.37 - 4.21 (m, 1H), 3.81 - 3.77 (m, 3H), 3.76 - 3.67 (m, 3H), 3.57 (tdd, J = 10.6, 6.1, 3.6 Hz, 2H), 1.70 (dd, J = 3.8, 1.5 Hz, 2H), 1.59 (dd, J = 13.5, 1.3 Hz, 2H), 1.55 - 1.52 (m, 1H), 1.51 - 1.43 (m, 6H), 1.40 (dt, J = 7.1, 2.9 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 171.86, 158.58, 130.40, 130.29, 128.28, 117.45, 113.96, 61.98, 59.20, 55.54, 55.19, 54.29, 53.97, 51.94, 48.43, 43.51, 37.08, 25.84, 17.86, 17.68, 16.55, 12.46. MS (ESI) m / z 585.8 [M+H] + .

[0198] Example 18: (2S,3R)-3-Hydroxy-3-(4-methoxyphenyl)-N-((S)-5-methyl-1-((R)-2- methoxy-2-yl)-1-oxohexan-4-en-2-yl)-2-((S)-2-(2-(4-(methyl-d3)piperazin-1-yl)acetamido) propanamide (54)

[0199] Step 1): Compound 10 was synthesized using the same procedure as described in Step 1) of Example 1.

[0200] Step 2): Compound 30 was synthesized using the same procedure as described in Step 2) of Example 1.

[0201] Step 3): Compound 54 was synthesized using the same procedure as described in Step 3) of Example 1, replacing the starting morpholine with 1-(methyl-d3)piperazine.

[0202] 1H NMR (400 MHz, DMSO-d6) δ 8.56 - 8.21 (m, 1H), 8.19 - 7.94 (m, 1H), 7.72 - 7.56 (m, 1H), 7.35 - 7.16 (m, 2H), 6.81 (ddt, J = 10.8, 4.0 Hz, 2H), 5.23 - 4.95 (m, 1H), 4.68 - 4.53 (m, 1H), 4.54 - 4.45 (m, 1H), 4.43 - 4.32 (m, 1H), 4.19 (dq, J = 10.8, 7.0 Hz, 1H), 3.70 (d, J = 2.7 Hz, 3H), 3.40 (s, 3H), 3.06 - 2.92 (m, 2H), 2.90 - 2.77 (m, 2H), 2.50 - 2.15 (m, 8H), 1.74 - 1.60 (m, 3H), 1.60 - 1.48 (m, 3H), 1.49 - 1.27 (m, 3H), 1.07 (dd, J = 7.0, 2.8 Hz, 1H), 0.75 (dd, J = 6.9, 2.0 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 208.22, 171.34, 170.82, 168.83, 158.98, 134.43, 132.51, 128.79, 128.52, 119.50, 119.35, 114.50, 113.35, 72.75, 61.36, 55.74, 55.41, 55.03, 53.13, 51.63, 47.66, 29.49, 26.04, 19.39, 18.78, 18.08, 16.72. MS (ESI) m / z 591.9 [M+H] + .

[0203] Example 19: Bioactivity test

[0204] I. Proteasome inhibitory activity test

[0205] (I) Intracellular immunoproteasome inhibitory activity test scheme

[0206] The immunoproteasome activity fluorescent detection kit (purchased from UBP Bio immunoproteasome activity fluorescent detection kit II (Cat. # J4170)) was used to determine the β5i, β2i inhibitory activity of intracellular immunoproteasome, containing the required fluorescent polypeptide substrate Ac-ANW-AMC (chymotrypsin-like), Ac-KQL-AMC (trypsin-like) and proteasome assay buffer, and the cells used were human non-small cell lung cancer cells A549.

[0207] 1. Cell sample preparation

[0208] Human non-small cell lung cancer A549 cells were purchased from ATCC (American Type Culture Collection) and cultured in F-12k complete medium containing 10% FBS at 37°C, 5% CO2.

[0209] 1) Cells were cultured to the logarithmic growth phase, inoculated in 100 mm dishes and incubated overnight. Recombinant Human IFN-γc (Ref. 300-02, PeproTech) was added to a final concentration of 10 ng / mL for 24 h, and then the cells were collected.

[0210] 2) The cell pellet was resuspended in pre-cooled lysis buffer (40 mM Tris, pH 7.2, 50 mM NaCl, 2 mM βME, 2 mM ATP, 5 mM MgCl2, 10% glycerol) and sonicated 3 times. The cell lysate was centrifuged at 17000 g for 20 min at 4°C, and the supernatant was collected.

[0211] 3) Take 25 μL of protein sample in a 96-well transparent plate, add 200 μL of BCA working solution, and mix well. Incubate in a 37°C incubator for 30 min, and measure the absorbance value of the sample well with a microplate reader. According to the linear equation of the standard sample, calculate the protein concentration of the sample well, adjust the protein working solution concentration to 5 mg / mL, and store at -80°C for later use.

[0212] 2. Preparation of substrate

[0213] Prepare 2X fluorescent substrate in buffer. Dilute 20X buffer with water to 1X working solution, and heat in a 37°C water bath for 10 min. Add 2 μL of Ac-ANW-AMC substrate (1000X) to 998 μL of preheated buffer, vortex to dissolve, prepare 100 μM (2X) substrate working solution, and treat in a 37°C water bath for 10 min. Prepare 100 μM Ac-KQL-AMC in the same way.

[0214] Prepare an appropriate amount of substrate according to the number of sample wells, 50 μL (2X) of substrate is required for each reaction well.

[0215] 3. Drug configuration and preparation of test wells

[0216] Drug preparation for testing the inhibition rate of the test compound at a concentration of 1 μM: weigh the drug, dissolve in DMSO to a concentration of 10 mM. Take 10 μL of the compound and add to 40 μL of DMSO to prepare a 2 mM (200X) working solution. Dilute 10 concentration points (0-2 mM) by 2 times. Add 0.625 μL of the compound at different concentration points to 100 μL of 1X buffer, vortex, and then add 25 μL of cell lysate, so that the final concentration in the reaction system is 0-10 μM. Heat at 37°C for 10 min.

[0217] IC50 value of the test compound 50 Drug preparation for testing the IC50 value of the test compound: weigh the drug, dissolve in DMSO to a concentration of 10 mM. Take 10 μL of the compound and add to 40 μL of DMSO to prepare a 2 mM (200X) working solution. Dilute 10 concentration points (0-2 mM) by 2 times. Add 0.625 μL of the compound at different concentration points to 100 μL of 1X buffer, vortex, and then add 25 μL of cell lysate, so that the final concentration in the reaction system is 0-10 μM. Heat at 37°C for 10 min.

[0218] 4. Preparation of the reaction system

[0219] Take 50 μL of the cell lysate treated with the compound (or untreated cell lysate) and add to a 96-well black transmittance plate. Then add 50 μL of the substrate, shake at 37°C for 30 s, and immediately detect the fluorescence value using a 360 nm / 460 nm fluorescence enzyme marker (BMG LABTECH POLARstar OPTIMA Microplate Reader). Continuously detect for 15 min and record the fluorescence value.

[0220] 5. Data processing

[0221] Calculate the fluorescence value of the product obtained under the action of the drug at different concentrations after deducting the background. Calculate the inhibition rate of LMP7 (β5i) subunit enzyme activity at a concentration of 1 μM. For the calculation of IC50 value, use GraphPad Prism software to calculate the IC 50 concentration of the drug for inhibition of the immunoproteasome.

[0222] (B) Test scheme for the inhibition activity of the tissue-type proteasome

[0223] The present application uses the fluorescent polypeptide substrate Suc-Leu-Leu-Val-Tyr-AMC (purchased from Enzo, BML-P802-0005) to determine the β5 subunit activity of proteasome, and uses the fluorescent polypeptide substrate Ac-Arg-Leu-Arg-AMC (purchased from Enzo, BML-AW9785-0005) to determine the β2 subunit activity of proteasome. The proteasome is human red blood cell 20S proteasome, which is purchased from Enzo (BML-PW8720-0050). The buffer solution is purchased from Enzo (BML-KI340-0020). The experimental system is 16 μL, in which the substrate is 8 μL, the proteasome is 4 μL (0.8 ng), the final concentration is 50 μM, the drug (inhibitor) is 4 μL, and the final concentration is 1 × 10 -5 M, and the last concentration is 0 M, and the actual configuration concentration is 4 × 10 -9 M. The specific experimental process is as follows: -5 M ~ 9.75 × 10 -9 M.

[0224] 1. Drug configuration

[0225] The drug is weighed and dissolved in DMSO to a concentration of 10 -2 M. 2 μL is taken by a pipette and added to 98 μL of DMSO to obtain 2 × 10 -4 M, and then 40 μL of the 2 × 10 -4 M drug is taken and added to 160 μL of H2O to obtain 4 × 10 -5 M, and the same method is used to obtain 1 × 10 -5 M, 2.5 × 10 -6 M, 6.25 × 10 -7 M, 1.56 × 10 -7 M, 3.9 × 10 -8 M, 9.75 × 10 -9 M, and the last concentration 0 M is no drug.

[0226] 2. Substrate preparation

[0227] 25 mg of the fluorescent polypeptide substrate is dissolved in 654 μL of DMSO to obtain a 50 mM stock solution, which is stored at -20°C. When used, it is diluted 500 times, and 8 μL is added to each sample, so that the final substrate concentration in the reaction system is 50 μM.

[0228] 3. Preparation of reaction system

[0229] The 20S proteasome was diluted from 2 ng / μL to 8 ng / μL with a buffer solution, added to a 384-well fluorescent enzyme plate, 4 μL per well, and then 4 μL of the sample to be tested was added to each well. The marketed drug Ixazomib was used as a positive control drug, and the reaction was carried out at 37°C for 15 min. After the reaction was completed, 8 μL of fluorescent substrate was added to each well, and the reaction was carried out at 37°C for 1 hour in the dark. The fluorescence value was detected using a 360 nm / 460 nm fluorescent enzyme reader (BMG LABTECH POLARstar OPTIMA Microplate Reader).

[0230] 4. Data processing

[0231] The fluorescence values of the products obtained under the action of different concentrations of drugs after deducting the background were calculated, and the GraphPad Prism software was used to calculate the IC 50 values of the proteasome inhibition of the drugs.

[0232] Using (I) intracellular immunoproteasome inhibition activity test scheme, the inhibition rates of compounds 37-54 and KZR-616 (prepared according to the method recorded in the literature: J Med Chem. 2018; 61(24): 11127-11143.) on LMP7 (β5i) subunit enzyme activity at 1 μM concentration were tested, and the results are shown in Table 1. Most of the compounds have strong activity inhibition effect on LMP7 (β5i) subunit at 1 μM concentration.

[0233] Table 1 Inhibition rates of compounds 37-54 and KZR-616 on LMP7 (β5i) subunit enzyme activity

[0234] The key to the development of selective immunoproteasome inhibitors is the high selectivity to immunoproteasome, and the consideration of the activity inhibition of active subunits on immunoproteasome, so as to reduce adverse reactions while achieving good efficacy. Subsequently, using the above (I) intracellular immunoproteasome inhibition activity test scheme, the IC 50 values of compound 37 on LMP7 (β5i) subunit and MECL-1 (β2i) subunit were further tested, and the IC 50 values of compound 37 on β5 and β2 subunit enzyme activity were tested using the above (II) tissue type proteasome inhibition activity test scheme, and the corresponding selectivity (β5 / β5i, β2 / β2i) was calculated. The results are shown in Table 2. Compound 37 has good inhibition effect on LMP7 (β5i) subunit and MECL-1 (β2i) subunit. Compared with KZR-616, compound 37 has lower inhibition activity on β5 and β2 subunits, and shows better selectivity.

[0235] Table 2 Enzymatic inhibition activity IC of compound 37 and KZR-616 on β5i subunit, β2i, β5 and β2 subunit 50

[0236] II. In vivo efficacy of compounds 37-54 in mouse ulcerative colitis

[0237] Ulcerative colitis is a chronic nonspecific, non-infectious, inflammatory intestinal disease mainly involving the colorectal mucosa and submucosa, which can highly affect the patient's life and lead to long-term complications. The dextran sulfate sodium (DSS)-induced mouse intestinal inflammation model is the most widely used chemical-induced mouse ulcerative colitis model. By dissolving DSS in drinking water, acute ulcerative colitis or chronic colitis is induced, mouse intestinal epithelial cells are damaged, non-specific immune cells release cytokines, and ultimately the integrity of the mucosal barrier is destroyed, and animals show obvious weight loss, loose stools, blood in the stool and granulocyte infiltration, which is extremely similar to human ulcerative colitis in clinical symptoms and pathological characteristics.

[0238] 1) In vivo efficacy of compounds 37-54 in mouse ulcerative colitis by intraperitoneal injection

[0239] 6-7 weeks old female C57BL / 6 mice (purchased from Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd.) were randomly divided into groups (6 mice per group), including compound 37 intraperitoneal injection treatment group (15 mg / kg), compound 38 intraperitoneal injection treatment group (15 mg / kg), compound 39 intraperitoneal injection treatment group (15 mg / kg), compound 40 intraperitoneal injection treatment group (15 mg / kg), compound 41 intraperitoneal injection treatment group (15 mg / kg), compound 42 intraperitoneal injection treatment group (15 mg / kg), compound 43 intraperitoneal injection treatment group (15 mg / kg), compound 44 intraperitoneal injection treatment group (15 mg / kg), compound 45 intraperitoneal injection treatment group (15 mg / kg), compound 46 intraperitoneal injection treatment group (15 mg / kg), compound 47 intraperitoneal injection treatment group (15 mg / kg), compound 48 intraperitoneal injection treatment group (15 mg / kg), compound 49 intraperitoneal injection treatment group (15 mg / kg), compound 50 intraperitoneal injection treatment group (15 mg / kg), compound 51 intraperitoneal injection treatment group (15 mg / kg), compound 52 intraperitoneal injection treatment group (15 mg / kg), compound 53 intraperitoneal injection treatment group (15 mg / kg), compound 53 intraperitoneal injection treatment group (15 mg / kg), positive drug Mesalazine oral treatment group (100 mg / kg), positive drug KZR-616 intraperitoneal injection treatment group (15 mg / kg), model (ulcerative colitis model group, DSS), and blank (normal control group).

[0240] Preparation of test compounds and KZR-616: The drug was weighed, dissolved in PEG400, and then physiological saline was added to a concentration of 3 mg / mL (PEG400:H2O = 1:9, V / V). Mesalazine was directly dissolved in physiological saline to a final concentration of 10 mg / mL.

[0241] Each group of mice continuously and freely drank 3% DSS solution to construct an acute ulcerative colitis model, and the treatment group was treated with compounds once a day. The normal control group of mice normally drank sterile water. During the entire experiment, the state and blood in stool of the mice were observed every day. After 9 days, the mice were euthanized, dissected, and the colon tissue was taken to measure the length of the distance from the anus to the ileocecal junction, and the results were recorded and plotted.

[0242] The results showed that the colon length of mice in the acute ulcerative colitis group was significantly shorter than that of the normal control group (Figure 1). This indicates that the mouse model of acute ulcerative colitis was successfully established. Among them, mice in the treatment groups of compounds 39, 41-47, and 53 were in poor condition and died 3-6 days after administration. Mice in the other compound treatment groups were in good condition during the treatment period. The treatment results showed that mice in the treatment groups of compounds 37 and 51 had the longest average colon length, exhibiting superior in vivo efficacy compared to Mesalazine and KZR-616.

[0243] 2) Oral in vivo efficacy of compound 37 in mice with ulcerative colitis

[0244] Six- to seven-week-old female C57BL / 6 mice were randomly divided into four groups (n=6 per group): Modle (ulcerative colitis model group, DSS), 37 (25 mg / kg, po, qd) oral treatment group (25 mg / kg), 37 (50 mg / kg, po, qd) oral treatment group (50 mg / kg), Mesalazine (100 mg / kg, po, qd) positive control group (100 mg / kg), KZR-616 (10 mg / kg, ip, qd) positive control group (10 mg / kg), and Blank (normal control group). Preparation of the test compound: Compound 37 was weighed, dissolved in PEG400, and then physiological saline was added to concentrations of 2.5 mg / mL and 5 mg / mL (PEG400:H2O = 2:8, V / V). Preparation of KZR-616: Weigh the drug, dissolve it in PEG400, and then add physiological saline to a concentration of 2 mg / mL (PEG400:H2O = 1:9, V / V). Preparation of Mesalazine: Dissolve Mesalazine directly in physiological saline to a final concentration of 10 mg / mL.

[0245] In the establishment of an acute ulcerative colitis model, mice were given free access to 3% DSS solution continuously, while the therapeutic compound was administered once daily. Mice in the normal control group received sterile water normally. Throughout the experiment, the mice were weighed daily, and their condition and fecal bleeding were recorded. After 9 days, the mice were euthanized, dissected, and colonic tissue was taken and laid flat on white paper. The distance from the anus to the ileocecal junction was measured, recorded, and plotted.

[0246] The results showed that from day 5 onwards, all mice in the untreated ulcerative colitis group developed severe diarrhea and bloody stools. Simultaneously, the mice exhibited significant lethargy, loss of appetite, drowsiness, and disheveled, dull fur. Compared to the normal control group, the colon length in the acute ulcerative colitis group was significantly shortened (Figure 2). These results indicate that the acute ulcerative colitis mouse model was successfully established.

[0247] As shown in Figure 2, the treatment results show that the treatment group compound 37 has good oral treatment effect, and the colon length of the 50mg / kg dose group shows a pharmacodynamic effect comparable to that of the KZR-616 intraperitoneal injection treatment group at a dose of 50mg / kg, indicating that the compound of the present application can be developed into an oral preparation in the future, which has more important convenience for taking than KZR-616, and has important clinical significance.

[0248] Three, in vivo efficacy of mouse systemic lupus erythematosus

[0249] Systemic lupus erythematosus (SLE) is a complex autoimmune disease, which is more common in women of childbearing age. Its etiology is not very clear, and it is speculated that it may be related to the immune dysfunction caused by the combined action of genetic factors and environmental factors. Its course is repeated and involves various systems of the body, and the most vulnerable is the kidney. Studies have shown that lupus nephritis is the main factor leading to renal failure and death in SLE patients. The commonly used SLE model internationally is female SPF MRL / lpr mice, which is also the most classic animal model.

[0250] MRL / Lpr mice, as a representative animal model for studying the pathogenesis of lupus, spontaneously develop lupus symptoms similar to human disease at about 16 weeks of age. Due to the absence of the Fas gene, these mice have a lymphoproliferative gene, resulting in a decrease in T cell mortality, lymph node enlargement, and failure of autoreactive lymphocytes to be cleared through the apoptosis pathway, thereby producing autoimmune disease symptoms, which are very similar to human SLE. The lupus-like phenotype of the MRL / Lpr model is mainly manifested as lymph node enlargement, splenomegaly, production of autoantibodies, and immune complex-mediated glomerulonephritis, with increased urinary protein and plasma urea nitrogen.

[0251] 1) Experimental scheme

[0252] The systemic lupus erythematosus model mice were MRL / lpr female mice, 4-6 weeks old, 18-20g. The new generation of immunoproteasome inhibitor KZR-616 was used as a positive control. The model mice were divided into 5 groups, 7 in each group. They were model control group, compound 37 oral group (50mg / kg), compound 37 oral group (100mg / kg), compound 37 intraperitoneal injection group (10mg / kg), KZR-616 intraperitoneal injection group (10mg / kg). The body weight, skin lesions and lymph nodes were observed and scored regularly. Urine was collected before and after drug administration to detect urinary protein. After the experiment, the mice were dissected, and the spleen, liver and kidney were weighed to calculate the spleen, liver and kidney indices, and the splenomegaly, hepatomegaly and nephromegaly were investigated.

[0253] 2) Dosing regimen

[0254] The intragastric administration scheme is as follows: a certain amount of compound 37 is accurately weighed into a centrifuge tube, and then dissolved in a certain volume of polyethylene glycol (400), after ultrasonic dissolution and dissolution, normal saline is added (polyethylene glycol: normal saline = 20:80, v:v), the final concentration of the compound in the 50mg / kg group is 5mg / mL, and the final concentration of the compound in the 100mg / kg group is 10mg / mL, and the intragastric administration is 10μL / g of body weight, and the administration frequency is once a day.

[0255] The intragastric administration scheme is as follows: a certain amount of compound 37 is accurately weighed into a centrifuge tube, and then dissolved in a certain volume of polyethylene glycol (400), after ultrasonic dissolution and dissolution, normal saline is added (polyethylene glycol: normal saline = 20:80, v:v), the final concentration of the compound in the 50mg / kg group is 5mg / mL, and the final concentration of the compound in the 100mg / kg group is 10mg / mL, and the intragastric administration is 10μL / g of body weight, and the administration frequency is once a day.

[0256] 3) Body weight change rate

[0257] The body weight change of each group is shown in FIG. 3. As shown in FIG. 3, the body weight of the KZR-616 group decreased significantly, and after 2 times of administration per week for 4 consecutive weeks, in vivo toxicity occurred. The body weight of the compound 37 oral and injection groups decreased slightly. In addition, during the entire treatment period, one mouse in the KZR-616 group died at the 2nd week and the 4th week, respectively, while no death occurred in the compound 37 injection group and the 100mg / kg oral treatment group, and the mice were in good condition. These results show that the compound 37 has good in vivo efficacy and safety compared to KZR-616.

[0258] 4) Skin lesion score

[0259] The scoring system is as follows: 1) skin redness, bleeding; 2) hair loss and skin dryness; 3) edema; 4) epidermal shedding / erosion; 5) lichen sclerosus plaques. The score for each item is as follows: normal = 0 points; mild = 1 point; moderate = 2 points; severe = 3 points. The severity of skin damage is determined according to the total score of each evaluation symptom.

[0260] Skin lesion scores were performed periodically before and after administration, and the results are shown in Table 3. The results indicate that intraperitoneal injection of compound 37 at 10 mg / kg effectively controlled the severity of skin lesions without causing mouse mortality, demonstrating significantly better therapeutic efficacy than the KZR-616 injection group (10 mg / kg). Similarly, the oral administration group also showed significant therapeutic effects, further indicating that the compound described in this invention can be developed into an oral formulation with greater convenience of administration compared to KZR-616, and has significant clinical implications.

[0261] Table 3. Skin lesion scores of compound 37 in a mouse model of systemic lupus erythematosus. Note 1: Compared with the healthy control group: #### P<0.0001; 2. Compared with the model control group: **** P<0.0001.

[0262] 5) Urine protein test

[0263] In acidic media, the pyrogallol red-molybdate complex (CBB) binds to the protonated basic amino groups of proteins, causing a color change from brown to blue, with a maximum absorption peak at 595 nm. The increase in absorbance at 595 nm is directly proportional to the protein concentration in the sample. The protein concentration in the sample is determined based on this absorbance change. The Nanjing Jiancheng Bioengineering Institute urine protein quantification kit (catalog number: C035-2-1, CBB method) was used. The CBB working solution of the kit was diluted: CBB reagent: double-distilled water = 1:4, i.e., a 5-fold dilution. Specific testing methods are shown in Table 4.

[0264] Table 4. Methods for measuring urinary protein

[0265] After each group is prepared, mix thoroughly, let stand for 5 minutes, measure the absorbance of each tube at a wavelength of 595 nm and a light path of 1 cm using double-distilled water, and calculate the urinary protein concentration in the sample according to the following formula:

[0266] The urine protein of each group of mice was determined before and after administration, and the results are shown in Table 5. The results show that at the end of treatment (6W), the urine protein level of the compound 37 injection group (10 mg / kg) was significantly reduced relative to the KZR-616 injection group (10 mg / kg); in addition, unexpectedly, the compound 37 oral group (100 mg / kg) also significantly reduced the urine protein level relative to the KZR-616 injection group (10 mg / kg). And similarly, the above results further indicate that compound 37 has better in vivo efficacy and is expected to be developed into an oral immunological protein inhibitor, which has more important convenience for taking relative to KZR-616, and has important clinical significance.

[0267] Table 5 Urine protein results of compound 37 in the treatment of systemic lupus erythematosus model mice Note: 1. Compared with the healthy control group: #### P <0.0001; 2. Compared with the model control group: *** P <0.001, **** P <0.0001; 3. Compound 37 oral group (100 mg / kg) compared with KZR-616 injection group (10 mg / kg), P <0.0001; 4. Compound 37 injection group (10 mg / kg) compared with KZR-616 injection group (10 mg / kg), P <0.0001.

[0268] Four, in vivo efficacy of mice with rheumatoid arthritis

[0269] (I) In vivo efficacy of the compound

[0270] 1. Experimental method

[0271] Chronic inflammatory pain is one of the most common types of clinical pain conditions. Adjuvant arthritis (AA) is an animal experimental model of rheumatoid arthritis (RA) recognized at home and abroad, and is also one of the ideal animal models for studying early immune disorders. The 24-hour AA model can observe obvious symptoms and immune and pathological reactions, such as redness, swelling, heat of the affected joint, and immune dysfunction, etc. The secondary reaction period is caused by immune inflammation caused by immune dysfunction, accompanied by long-term chronic pain. In this experiment, single male Wistar rats were used to subcutaneously inject complete Freund's adjuvant into the right toe to activate the body's specific immune system and cause damage to its own tissues, and to establish a Wistar rat adjuvant arthritis model. After the model was successfully established, rats with a total score of 2 or more on the arthritis index score of both hind limbs were selected as model rats. According to the score, the rats were divided into a model control group, a test drug 37 oral group (50 mg / kg, 100 mg / kg), and a celecoxib group (50 mg / kg). Each group had 8 rats. Oral administration was performed once a day for 14 consecutive days. The toe volume measuring instrument was used to detect the toe swelling degree before administration, 7 days and 14 days after administration (3 hours after administration). The drug efficacy was evaluated by calculating the difference between the rat foot swelling on day 0 and the foot swelling after 7 days and 14 days of administration. The smaller the difference, the lower the degree of foot swelling, and the better the drug efficacy.

[0272] 2、Experimental results

[0273] The results are shown in Table 6. After 7 days of administration, the compound 37 oral 100 mg / kg group can significantly alleviate the swelling degree of the primary lesion right foot, which is comparable to the positive control celecoxib, and also has good therapeutic effect on the secondary lesion left foot. After 14 days of continuous administration, the compound 37 oral 50 mg / kg group and the 100 mg / kg group have significant therapeutic effect on the primary lesion right foot, and the 100 mg / kg group is more effective in relieving foot swelling and better than the celecoxib group. For the secondary lesion left foot, the compound 37 oral 100 mg / kg group also has good therapeutic effect. These results show that compound 37, as a new type of oral immunoproteasome inhibitor, has good therapeutic effect on rheumatoid arthritis, and the high-dose group of compound 37 shows better in vivo efficacy than celecoxib.

[0274] Table 6 Compound 37 treatment of rheumatoid arthritis model mice

[0275] (ii) In vivo efficacy of pharmaceutically acceptable salts

[0276] 1、Model preparation

[0277] Preparation of immunogen: Under sterile conditions at 4℃, Immunization Grade Bovine Type II Collagen (CII, 2 mg / mL) was mixed with an equal volume of Freund's incomplete adjuvant IFA on ice (1:1) to emulsify (i.e. 1 mg CII per 1 mL), and then injected with a syringe double-push for 20 min, and prepared immediately before use.

[0278] The rats were injected intradermally with 0.2 mL of the mixture (0.1 mL of the mixture at the root of each hind limb) at the root of the tail for 2-3 cm, and then immunized again after 7 days. Each rat was injected with 0.2 mL of the 1:1 emulsion of CII and IFA. The blank control group was injected with an equal volume of normal saline.

[0279] 2. Grouping and administration regimen

[0280] After the second immunization, the rats were observed daily for the onset of the disease. Rats with a total score of 2 or more in the hind limbs (about 14 days after the first immunization) were randomly grouped according to the foot volume for administration. After grouping, the rats were immediately administered by gavage or subcutaneous injection, once a day for 14 days, or three times a week for two weeks. The end of the test was 14 days after administration. The grouping and administration information is as follows:

[0281] The structure of the maleic acid salt of compound 37 is as follows, and the specific preparation is as follows: ethyl acetate is used as the solvent, compound 37 (100 mg) is first dissolved in ethyl acetate, maleic acid (about 20 mg, 1:1 molar ratio) is added, and the suspension is stirred at about 600 rpm on a magnetic plate at room temperature. After stirring, white solids are precipitated, about 4.0 mL of ethyl acetate is then added to the suspension, the supernatant is stirred overnight, and then separated by suction filtration, and the residue is dried under vacuum at 50℃ for 2h.

[0282] 2. Drug use basis

[0283] Test drug: determined according to previous pre-experiments.

[0284] Positive control (non-steroidal anti-inflammatory drug for improving symptoms): Celecoxib, instructions: used for relieving the symptoms and signs of osteoarthritis, relieving the symptoms and signs of rheumatoid arthritis in adults, treating acute pain in adults, and relieving the symptoms and signs of ankylosing spondylitis. The dose of celecoxib for treating rheumatoid arthritis is 100 mg to 200 mg, taken orally twice a day. Rat dose: 200 mg / 60 kg x 6.2 ≈ 20 mg / kg.

[0285] 3. Drug preparation

[0286] (1) Maleic acid salt of compound 37

[0287] A. Maleate salt of compound 37 ig: dose 50 mg / kg, administration volume 10 mL / kg, preparation concentration 5 mg / mL, solvent normal saline (NS). Take 50 mg of compound 37 and add to 10 mL of NS.

[0288] B. Maleate salt of compound 37 ig: dose 25 mg / kg, administration volume 10 mL / kg, preparation concentration 2.5 mg / mL, solvent normal saline (NS). Take 50 mg / kg of compound 37 ig and dilute 2 times with NS, take 50 mg of compound 37 and add to 10 mL of NS to 20 mL.

[0289] C. Maleate salt of compound 37 sc: dose 25 mg / kg, administration volume 2 mL / kg, preparation concentration 12.5 mg / mL, solvent normal saline (NS). Take 125 mg of compound 37 and add to 10 mL of NS.

[0290] (2) Celecoxib ig

[0291] Dose 20 mg / kg, administration volume 10 mL / kg, preparation concentration 2 mg / mL, solvent NS.

[0292] Prepare fresh every day, and discard the remaining preparation.

[0293] 4. Test indexes

[0294] (1) Body weight

[0295] Test the body weight before administration, 3 d, 7 d, 10 d, and 14 d after administration.

[0296] (2) Arthritis index (AI)

[0297] Test the arthritis index (AI) before modeling, before administration, 3 d, 7 d, 10 d, and 14 d (2 h after administration) after administration.

[0298] Scoring criteria: score the wrist, metacarpophalangeal joint of the forelimb, and the ankle, toe joint of the hind limb according to the following criteria: 0 points: no redness and swelling; 1 point: toe joint redness or mild swelling, inflammation of a single area of the paw or foot pad; 2 points: mild to moderate joint swelling, inflammation of more than 2 areas of the paw and foot pad or ankle joint; 3 points: moderate to severe swelling, mild dysfunction; 4 points: severe joint redness, stiffness, even deformity, and severe dysfunction. Score each limb separately, for a total of 16 points.

[0299] (3) Foot volume and toe swelling degree

[0300] The foot volume was measured by foot volume meter before modeling, before administration, 3d, 7d, 10d, 14d (2h after administration) and the swelling inhibition rate (%) was calculated according to the following formula: Swelling degree (%) = (swollen foot volume - pre-swollen foot volume) / pre-swollen foot volume x 100%. Swelling inhibition rate (%) = (model group foot swelling degree - administration group foot swelling degree) / model group foot swelling degree x 100%.

[0301] 5. Data statistics and processing

[0302] The test data were expressed as mean ± SD and the differences between groups were analyzed by t test. P < 0.05 was considered as statistically significant difference.

[0303] 6. Experimental results

[0304] (1) Effect on body weight

[0305] Compared with the model control group, the body weight of the rats in the compound 37 maleate salt 50mg / kg group was slightly increased 14d after administration (P < 0.05), and the body weight of the rats in the other groups was not significantly affected (P > 0.05). The results are shown in Tables 7-1 and 7-2.

[0306] Table 7-1 Effect on body weight (g) of CIA rats (mean ± SD) Note: Compared with the model control group: * P < 0.05.

[0307] Table 7-2 Effect on body weight (g) of CIA rats (mean ± SD) Note: Compared with the model control group: * P < 0.05.

[0308] (2) Effect on arthritis index (AI)

[0309] The AI of the rats in the model control group was slightly increased 14d after administration. Compared with the model control group, the AI of the rats in the compound 37 maleate salt 25, 50mg / kg ig group was significantly reduced 7d-14d after administration (P < 0.001-0.01) and showed a dose-dependent manner. The AI of the rats in the compound 37 maleate salt 25mg / kg SC group was significantly reduced 7d-14d after administration (P < 0.001-0.01) and the effect was slightly stronger than that of the compound 37 maleate salt 25, 50mg / kg ig group.

[0310] Compared with the model control group, the AI of the rats in the positive control celecoxib 20mg / kg ig group was significantly improved 3d-14d after administration (P < 0.001-0.05). The results are shown in Table 8.

[0311] Table 8 Effects on AI (mm) of CIA rats Note: Compared with model control group: * P<0.05, ** P<0.01, *** P<0.001.

[0312] (3) Effects on paw swelling degree

[0313] The paw swelling degree of the model control group was progressively increased within 14 days after administration. According to the difference between the paw swelling degree after administration and that before administration, compared with the model control group, the maleate salt of compound 37 at 25, 50 mg / kg ig could significantly reduce the paw swelling degree 7-14 days after administration (P<0.001-0.01), and the swelling inhibition rates were 57.8% and 63.9% respectively 14 days after administration, and there was a dose-dependent effect. The maleate salt of compound 37 at 25 mg / kg SC three times a week could significantly reduce the paw swelling degree 7-14 days after administration (P<0.001-0.01), and the swelling inhibition rate was 70.2% 14 days after administration, which was slightly stronger than that of the maleate salt of compound 37 at 25, 50 mg / kg ig.

[0314] Compared with the model control group, the positive control celecoxib at 20 mg / kg ig could significantly reduce the paw swelling degree 7-14 days after administration (P<0.001-0.01), and the swelling inhibition rates were 65.8% respectively 14 days after administration. The results are shown in Tables 9, 10 and 11.

[0315] Table 9 Effects on paw volume (mL) of CIA rats Note: Compared with model control group: * P<0.05, ** P<0.01, *** P<0.001.

[0316] Table 10 Effects on paw swelling degree (%) of CIA rats Note: Compared with model control group: * P<0.05, ** P<0.01.

[0317] Table 11 Effects on paw swelling inhibition rate (%) of CIA rats 14 days after administration

[0318] In summary, in the rat CIA arthritis model, the maleate salt of compound 37 25, 50 mg / kg ig administration for 14 d and the maleate salt of compound 37 25 mg / kg sc three times a week for two weeks can reduce the arthritis index (AI), reduce the foot volume and reduce the foot swelling degree to different extents, and the effect of sc is better than that of ig.

[0319] Example 20: Solubility test

[0320] 1. Experimental method

[0321] (1) Medium (pH 7.0 aqueous solution) preparation

[0322] 0.2 mol / L potassium dihydrogen phosphate solution: take 27.22 g of potassium dihydrogen phosphate, dissolve and dilute to 1000 mL with water.

[0323] 0.2 mol / L sodium hydroxide solution: take 8.00 g of sodium hydroxide, dissolve and dilute to 1000 mL with water.

[0324] Take 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution and 145.5 mL of 0.2 mol / L sodium hydroxide solution, and mix well.

[0325] (2) Solubility test

[0326] Test method: take 20 mL of pH 7.0 aqueous solution, place it in a 50 mL conical flask with a plug, add compound 37, maleate salt of compound 37, and shake in a 37°C constant temperature water bath for 24 hours, then filter and determine the concentration.

[0327] 2. Experimental results

[0328] Table 1 2 Solubility test results Note: The solubility data of KZR-616 is from CN105143212B.

[0329] As can be seen from Table 12, the solubility of compound 37 is increased by more than 50% compared with KZR-616; and the solubility of the salt of compound 37 is further significantly improved, both of which have the application prospect of being prepared into oral administration preparations, improving the convenience and compliance of patients taking medicine.

Claims

1. A peptide epoxy ketone compound of Formula I: ###0001### or a pharmaceutically acceptable salt thereof. wherein: P is unsubstituted or substituted by R a substituted 4-10 membered saturated or partially unsaturated heterocycle containing 1 or 2 heteroatoms selected from nitrogen, oxygen, and sulfur; R a is one or more, R a selected from C 1-6 alkyl; R 1 is hydrogen, C 1-6 1-6alkyl, or R 1 and the carbon to which it is attached form a cycloalkyl group; wherein R 1 is unsubstituted or substituted with R b , R b is one or more, R b is selected from OR 6 , SR 6 , N(R 6 )2 or CN; R 2 is -CONH2, -SO2NH2, -SO2Me, -COOH, -OH, -CN, halo, C 1-6 alkyl or C 1-6 alkoxy; R 3 is hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl, aryl or a 5-6 membered heteroaryl comprising 1, 2, 3 or 4 heteroatoms selected from nitrogen, oxygen or sulfur; R 3 is unsubstituted or substituted by R c , R c is one or more, said R c is selected from C 1-6 alkyl, halogen, C 1-6 alkoxy, OR 6 , SR 6 or N(R 6 )2; R 4 is hydrogen, methyl, ethyl or hydroxymethyl; preferably, R 4 is methyl; R 5 is hydrogen or hydroxy; preferably, R 5 is hydroxy; R 6 is H or C 1-6 alkyl, m is an integer from 0 to 4.

2. The peptidic epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 3 is selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl; preferably, R 3 is selected from C 2-6 alkenyl; more preferably, R 3 is selected from: further preferably, R 3 is selected from: even further preferably, R 3 is selected from: most preferably, R 3 is selected as 3. The peptidic epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein P is selected from: Preferably, P is selected from 4. The peptidic epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 is C 1-6 alkyl; preferably, R 1 is C 1-3 alkyl; more preferably, R 1 is methyl.

5. The peptidic epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 2 is selected from C 1-3 alkyl or C 1-3 alkoxy; preferably, R 2 is selected from methoxy, ethoxy or propoxy; more preferably, R 2 is methoxy.

6. A peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof represented by any one of the following structures:

7. The compound according to any one of claims 1-6, wherein the compound has the following stereochemical configuration: wherein P, R 1 , R 2 , R 3 , R 4 , R 5 , m are as described in any one of claims 1-6.

8. A peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof represented by any one of the following structures:

9. The peptidic epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The pharmaceutically acceptable salt is a salt of the peptide epoxy ketone compound with any one acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

10. The peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The pharmaceutically acceptable salt is 11. A process for the preparation of an epoxy ketone compound of formula I as claimed in claim 1, wherein, The synthesis of the compound of Formula I-a specifically includes the following steps: The compound shown as formula H-8 is condensed with the compound shown as formula F-5 after removal of the Boc protecting group to obtain the compound shown as formula I-a; wherein P, R 1 , R 2 , R 3 , and m are as described in claim 1.

12. A pharmaceutical composition comprising the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-10, and a pharmaceutically acceptable carrier.

13. Use of the peptide epoxy ketone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-10 or the pharmaceutical composition according to claim 12 in the manufacture of a medicament for reducing the activity of immunoproteasome or in the manufacture of a medicament for treating a disease related to abnormal activity of immunoproteasome.

14. The use according to claim 13, wherein the medicament is suitable for oral administration, parenteral administration, injection administration, inhalation administration, transdermal administration or transmucosal administration; preferably, the medicament is suitable for oral administration.

15. The use according to claim 13, wherein the disease related to abnormal activity of immunoproteasome is an autoimmune disease; preferably, the autoimmune disease comprises systemic lupus erythematosus, ulcerative colitis, rheumatoid arthritis.

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