Squaraine-based proteasome inhibitors

Abstract

The present application belongs to the field of pharmaceutical chemistry, and specifically discloses a square amide proteasome inhibitor, a preparation method thereof and pharmaceutical application thereof.

Description

Technical Field

[0001] This invention relates to squaramide proteasome inhibitors, their preparation methods, and their pharmaceutical applications. Background Technology

[0002] In eukaryotes, protein degradation is primarily mediated via the ubiquitin pathway, in which the targeted protein is linked to a 76-amino acid polypeptide called ubiquitin. Once targeted, the ubiquitinated protein then acts as a substrate for the 26S proteasome, a multi-catalytic protease that cleaves proteins into short peptides through its three main proteolytic activities. While playing a general role in intracellular protein turnover, proteasome-mediated degradation also plays a crucial role in numerous processes, such as major histocompatibility complex (MHC) antigen presentation (MHC), apoptosis, cell growth regulation, NF-κB activation, antigen processing, and pro-inflammatory signal transduction.

[0003] The 20S proteasome is a 700 kDa cylindrical, multi-catalytic protease complex comprising 28 subunits forming four rings. In yeast and other eukaryotes, seven distinct α-subunits form the outer ring, while seven distinct β-subunits form the inner ring. The α-subunits act as binding sites for the 19S (PA700) and 11S (PA28) regulatory complexes and also as physical barriers within the inner proteolytic chamber formed by the two β-subunit rings. Therefore, in living organisms, the protease is considered to be a 26S particle (26S proteasome). Experiments in living organisms have shown that inhibition of the 20S proteasome can be readily correlated with inhibition of the 26S proteasome. During particle formation, the cleavage of the original N-terminal sequence of the β-subunit exposes the N-terminal threonine residue, which acts as a catalytic nucleophile. Therefore, the subunits responsible for catalytic activity in proteases have nucleophilic residues at their amino-terminus, and these subunits belong to the N-terminal nucleophilic (Ntn) family of hydrolases (where the nucleophilic N-terminal residues are, for example, cysteine, serine, threonine (Cys, Ser, Thr) families and other nucleophilic groups). This family includes, for example, penicillin G acyltransferase (PGA), penicillin V acyltransferase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosyl asparaginase. In addition to the universally expressed β subunit, higher vertebrates also possess three interferon-γ-inducible β subunits (LMP7, LMP2, and MECL1), which replace the normal counterparts β5, β1, and β7, respectively, thereby altering the catalytic activity of the proteasome. By using different peptide matrices, three major proteolytic activities of the eukaryotic 20S protease have been identified as: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (TL), which cleaves after basic residues; and gamma-glutamyl transferase activity (PGPH), which cleaves after acidic residues. Two additional, less characteristic activities have also been attributed to the protease: BrAAP activity, which cleaves after branched-chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids. The major protease proteolytic activities appear to originate from different catalytic sites, as inhibitors, point mutations in the β-subunit, and interferon-gamma-induced β-subunit exchange alterations modify these activities to varying degrees.

[0004] Patent documents such as WO2014 / 152127 and WO2014 / 152134 disclose a series of protease inhibitors that can be used to treat autoimmune diseases. Summary of the Invention

[0005] On the one hand, the present invention provides compounds having the structure of formula (I), or pharmaceutically acceptable salts thereof:

[0006]

[0007] Where R 1 For A—(L)n—; L is C 1-3 Alkyl group, A is selected from: C 1-6 Alkylene-R 10 C 2-6 imide-R 10 OC 1-6 Alkylene-R 10 C 0-6 Alkylene N(R) 11 2. Aryl, heteroaryl, cycloalkyl, heterocyclic or C 3-6 Cycloalkenyl, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 SR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic groups for substitution;

[0008] R 2 R 3 R 5 R 7 and R 9 Independently selected from hydrogen or C 1-3 Alkyl groups;

[0009] R 4 Selected from hydrogen, halogen, hydroxyl, C 1-6 alkyl groups, wherein the C 1-6 The alkyl group is optionally surrounded by one or more halogens, hydroxyl groups, or C. 1-3 Alkyl substitution;

[0010] R 6 and R 8 Independently selected from C 1-3 Alkylene—G, wherein G is selected from: C 1-6 alkyl, C 3-7 cycloalkenyl, aryl or heteroaryl, wherein the C 1-6 alkyl, C 3-7 Cycloalkenyl, aryl, and heteroaryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be substituted with one or more halogens or hydroxyl groups;

[0011] R 10 Selected from H, CF3, OR 11 Or aryl;

[0012] X is selected from O or NR 11 ;

[0013] R 11 Selected from H or C 1-6 Alkyl groups; and

[0014] n is selected from 0, 1, 2 or 3.

[0015] In some implementations, n is 0. In some implementations, n is 1.

[0016] In some implementation schemes, A is selected from: C 1-6 Alkylene-R 10 C 6-10 Aryl, 5-14 heteroaryl, C 3-8 Cycloalkyl or 3-10-membered heterocyclic group, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

[0017] In some implementation schemes, A is selected from: C 1-6 Alkylene-R 10 C 6-10 Aryl, 5-6 quinone heteroaryl, C 3-8 Cycloalkyl or 3-6 membered heterocyclic group, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

[0018] In some embodiments, A is selected from: methyl, ethyl, propyl, isopropyl, butyl, neopentyl, phenyl, naphthyl, indene, oxazolyl, isoxazolyl, 1,2,4-oxadiazolyl, thiazolyl, furanyl, tetrahydrofuranyl, pyrroleyl, thiopheneyl, pyrazolyl, imidazolyl, pyridyl, pyranyl, pyrimidinyl, pyrazinyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.2]octyl, morpholinyl, piperazine, oxacyclobutyl, ethylene oxide, or cyclothioethane, wherein A is optionally selected by one or more of C 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

[0019] In some embodiments, A is selected from: neopentyl, phenyl, 1,2,4-oxadiazolyl, thiazolyl, furanyl, pyridyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.2]octyl, oxacyclobutyl, morpholinyl, or tetrahydro-2H-pyranyl, wherein A is optionally selected by one or more of C 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

[0020] In some embodiments, A is selected from: phenyl, pyridyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein A is optionally selected from one or more of C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

[0021] In some specific implementation schemes, R 1 Selected from:

[0022]

[0023] In some specific implementation schemes, R 1 Selected from:

[0024] In some more specific implementation schemes, R 2 R 3 R 5 and R 7 It is hydrogen.

[0025] In some more specific implementation schemes, R 9 It is a methyl group.

[0026] In some implementation schemes, R 4 Selected from C 1-6 alkyl groups, wherein the C 1-6 The alkyl group is optionally surrounded by one or more halogens, hydroxyl groups, or C. 1-3 Alkyl substitution.

[0027] In some implementation schemes, R 4 The methyl group is methyl, wherein the methyl group is optionally converted by one or more halogens, hydroxyl groups, or C.1-3 Alkyl substitution.

[0028] In some more specific implementation schemes, R 4 It is either methyl or -CH2-OH.

[0029] In some implementation schemes, R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: aryl, wherein the aryl group may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more halogens or hydroxyl groups.

[0030] In some implementation schemes, R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: aryl, wherein the aryl group may optionally be converted by one or more ORs. 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more hydroxyl groups.

[0031] In some implementation schemes, R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: phenyl, wherein the phenyl group may optionally be converted by one or more ORs. 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more hydroxyl groups.

[0032] In some more specific implementation schemes, R 6 Selected from

[0033] In some implementation schemes, R 8 Selected from C 1-3 Alkylene—G, wherein G is selected from: C 1-6 alkyl, C 3-7 Cycloalkenyl or C 3-7 Aryl, wherein the C 1-6 alkyl, C 3-7 The cycloalkenyl and aryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more halogens or hydroxyl groups.

[0034] In some implementation schemes, R 8 Selected from C 1-3Alkylene—G, wherein G is selected from: isopropyl, cyclopentene, or phenyl, wherein C 1-6 alkyl, C 3-7 The cycloalkenyl and aryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more halogens or hydroxyl groups.

[0035] In some implementation schemes, R 8 Selected from C 1-3 Alkylene—G, wherein G is selected from isopropyl, cyclopentene, or phenyl.

[0036] In some implementation schemes, R 8 Selected from

[0037] Furthermore, the present invention provides compounds having the structure of formula (II), or pharmaceutically acceptable salts thereof:

[0038]

[0039] Among them, R 1 R 4 and R 8 As defined in compound I, R 12 It is selected from hydrogen, halogen or hydroxyl.

[0040] In some specific implementation schemes, R 12 Selected from hydrogen or hydroxyl.

[0041] Furthermore, the present invention provides compounds having the structure of formula (III), or pharmaceutically acceptable salts thereof:

[0042]

[0043] Among them, R 1 R 4 and R 8 As defined in compound I, R 12 It is selected from hydrogen, halogen or hydroxyl.

[0044] In some specific implementation schemes, R 12 Selected from hydrogen or hydroxyl.

[0045] In some more specific embodiments, the present invention provides compounds with the following structures, or pharmaceutically acceptable salts thereof:

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] In some more specific embodiments, the present invention provides compounds with the following structures, or pharmaceutically acceptable salts thereof:

[0053]

[0054]

[0055]

[0056]

[0057] In some more specific embodiments, the present invention provides compounds with the following structures, or pharmaceutically acceptable salts thereof:

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] The compounds disclosed in this invention are inhibitors of the immunoproteasome (iP). In some cases, the compounds disclosed in this invention can inhibit the iP subunit LMP7. The activity of LMP7 can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, as measured by the proteasome subunit assay described in the examples below. One or more other iP subunits can be inhibited by the compounds disclosed in this invention, such as LMP2, MECL-1, β1, β2, and β5. In various embodiments, the compounds disclosed in this invention can inhibit LMP7 and one or both of LMP2 and MECL-1. The compounds disclosed in this invention can reduce the activity or expression of cytokines, such as one or more of IL-2, MHC-1, IL-6, TNF-α, and IFN-β. Therefore, the present invention provides methods in which compounds such as those disclosed herein can inhibit the expression or activity of one or more of IL-2, MHC-1, IL-6, TNF-α and IFN-β by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, as determined by the proteasome subunit assay method described in the examples below.

[0064] The biological consequences of proteasome inhibition are multifaceted. Proteasome inhibition has been suggested as a preventative and / or therapeutic approach for a variety of autoimmune diseases, including, but not limited to, neurotoxic / degenerative diseases, Alzheimer's disease, ischemic diseases, inflammation, HIV / AIDS, organ transplant rejection, septic shock, suppression of antigen presentation, reduction of viral gene expression, parasitic infections, acidosis-related conditions, macular degeneration, lung diseases, wasting diseases, fibrotic diseases, and diseases of bone and hair growth. Therefore, pharmaceutical formulations of proteasome-specific compounds, such as epoxides, provide a means of administering drugs to patients and treating these diseases.

[0065] This invention also provides a method for treating autoimmune diseases in patients, comprising administering to a patient a therapeutically effective amount of a compound or composition disclosed herein. As used herein, "autoimmune disease" refers to a disease or condition arising from and affecting an individual's own tissues. Such immune-related diseases include, but are not limited to, rheumatoid arthritis, lupus, inflammatory bowel disease, multiple sclerosis, or Crohn's disease.

[0066] Pharmaceutical Composition

[0067] The methods provided by this invention include the manufacture and use of pharmaceutical compositions, wherein the pharmaceutical compositions comprise one or more compounds provided by this invention. Furthermore, the pharmaceutical compositions typically contain a pharmaceutically acceptable carrier.

[0068] The term “pharmaceutically acceptable” is used herein to refer to ligands, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a commensurate and reasonable benefit / risk ratio.

[0069] As used in this article, "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials.

[0070] The term "pharmaceutically acceptable salt" refers to the relatively non-toxic addition salts of inorganic and organic acids of the compounds provided in this invention. These salts can also be prepared during the final isolation and purification of the compounds, or solely by reacting the purified compounds in their free base form with a suitable acid.

[0071] The actual dosage of the active ingredient in the pharmaceutical composition provided by this invention can be changed to achieve an effective therapeutic response for a specific patient, composition, and administration method without toxicity.

[0072] In pharmaceutically acceptable mixtures, the concentrations of the compounds provided herein will depend on several factors, including the dosage of the compound, the pharmacokinetic characteristics of the compound used, and the route of administration. In some embodiments, when used for parenteral administration, the compositions provided herein are aqueous solutions and may contain about 0.1 to 10% by weight / volume of the compounds provided herein. Typical dosage ranges can be from about 0.01 to about 50 mg / kg body weight daily, administered in 1-4 fractions. Each fraction may contain the same or different compounds. This dosage is a therapeutically effective amount and depends on several factors, including the patient's overall health condition and the route of administration of the chosen compound.

[0073] Dosage forms or compositions can be prepared containing 0.005% to 100% of the compound described in this invention and a non-toxic carrier. Methods for preparing these compositions are well known to those skilled in the art. The compositions considered may contain 0.001% to 100% of the active ingredient, 0.1% to 95% in one example and 75% to 85% in another. Although the dosage varies depending on the patient's symptoms, age, weight, and the nature and severity of the condition to be treated or prevented, generally, the route of administration and form of the medicine for adult patients is a daily dose containing 0.01 to 2000 mg of the compound, which may be administered in a single dose or in fractions. The amount of the active ingredient forming a single dosage form with the carrier is typically the amount of the compound that produces the therapeutic effect.

[0074] definition

[0075] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art.

[0076] The term “substituted” or “replaced by” refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the substituted compound is stable.

[0077] The term "optional" or "optionally" means that the event or situation subsequently described may or may not occur, including both the occurrence and non-occurrence of the event or situation. The phrase "optionally replaced by..." as used in this invention includes both "replaced" and "not replaced".

[0078] In this article, "one or more" refers to an integer from one to ten. For example, "one or more" means one, two, three, four, five, six, seven, eight, nine, or ten; further, it can be one, two, three, four, or five; and even further, it can be one, two, or three; but the chemical valence bond requirement must be met.

[0079] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by two Rs, each R has an independent option.

[0080] The range of numbers in this article refers to the integers within a given range. For example, "C 0-6 "" means that the group can have 0, 1, 2, 3, 4, 5, or 6 carbon atoms; "C 3-6 "" means that the group can have 3, 4, 5 or 6 carbon atoms.

[0081] The term "membered ring" refers to the number of skeletal atoms or groups of atoms that make up the ring. For example, groups like C=O, S(=O)2, or S(=O) have skeletal atoms C, S, and S respectively, and are represented as unary. "5-7 membered rings" indicates that the number of skeletal atoms or groups of atoms in the ring is 5, 6, or 7. For example, pyridine and piperidine are six-membered rings, while thiazoles and pyrroles are five-membered rings.

[0082] In This indicates the point where the group is chemically bonded to the rest of the molecule.

[0083] The term "effective dose" or "therapeutic effective dose" refers to a sufficient amount of a drug or agent that is non-toxic but achieves the desired effect.

[0084] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of a particular compound as a free acid or base without any adverse biological effects. Examples include acid (including organic and inorganic acids) addition salts or base addition salts (including organic and inorganic bases).

[0085] The term "pharmaceutically acceptable carrier" refers to carriers that do not cause significant irritation to the body and do not impair the biological activity and properties of the active compound. This includes, but is not limited to, diluents, disintegrants, binders, flow aids, and wetting agents.

[0086] The compounds of this invention contain asymmetric centers that may lead to stereoisomerism, as well as other chemical structures; therefore, this invention also includes these stereoisomers and mixtures thereof. Since the compounds of this invention (or pharmaceutically acceptable salts thereof) can exist in diastereomer or enantiomeric forms, or mixtures thereof, all optical isomers (e.g., enantiomers and diastereomers), racemic mixtures of said isomers, diastereomer mixtures, and other mixtures are within the scope of this invention. The compounds of this invention can be prepared using diastereomers, enantiomers, or racemic mixtures as starting materials. Optically active pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0087] This application also includes compounds of this application that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that may be incorporated into compounds of this application include isotopes of hydrogen, carbon, oxygen, nitrogen, and sulfur, such as... 2 H, 3 H, 11 C 13 C 14 C 15 O、 17 O、 18 O、 35 S, etc. Certain isotope-labeled compounds of this application (e.g., using...) 3 H and 14 Those labeled with C can be used in the analysis of compound and / or substrate tissue distribution. Deuteration (i.e. 2 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 11C can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of this application can typically be prepared by replacing unlabeled reagents with isotopically labeled reagents using a procedure similar to those disclosed in the schemes and / or examples below. Furthermore, heavier isotopes (such as deuterium) can be used. 2 H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, where deuterium substitution can be partial or complete, with partial deuterium substitution referring to at least one hydrogen being replaced by at least one deuterium.

[0088] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which can be straight-chain or branched. For example, the term "C 1-6 "Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, etc. The term "C"... 1-6 "alkyl" can further be "C 1-4 Alkyl", C 1-3 Alkyl, C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl.

[0089] The term "alkylene" refers to a divalent alkyl group, wherein the alkyl group, as defined above, has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C2). 1-20 Alkylene). The alkylene preferably has 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms (i.e., C). 1-12 Alkylenes, more preferably alkylenes having 1 to 6 carbon atoms (i.e., C16-164 ... 1-6 Alkylenes). Non-limiting examples of alkylenes include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), etc.

[0090] The term "C1-C6 alkoxy" refers to "C1-C6 alkyl-O-", where "C1-C6 alkyl" is as defined above. Non-limiting examples of C1-C6 alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, 1-butoxy, 2-methyl-1-propoxy, 2-butoxy, 2-methyl-2-propoxy, 1-pentoxy, 2-pentoxy, or 3-pentoxy. The "C1-C6 alkoxy" of this invention can further be "C1-C4 alkoxy" or "C1-C3 alkoxy".

[0091] The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0092] The term "hydroxyl group" refers to the -OH group.

[0093] The term "aryl" refers to an aromatic ring group consisting of an all-carbon monocyclic or fused polycyclic aromatic ring with a conjugated π-electron system. The "C6-C" group described in this invention... 10 "Aryl" can include, but is not limited to, phenyl, naphthyl, anthracene, etc.

[0094] The term "heteroaryl" refers to an aromatic monocyclic or fused polycyclic group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur, for example, one, two, or three. The nitrogen, carbon, or sulfur atom in the heteroaryl group may optionally be oxidized, such as sulfur, to form a sulfone or sulfoxide. The "5-14-membered heteroaryl" described in this invention can further be "5-12-membered heteroaryl," "5-10-membered heteroaryl," "5-9-membered heteroaryl," or "5-6-membered heteroaryl." Non-limiting examples of heteroaryl groups include, but are not limited to: furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrrolyl, pyridinyl, pyrimidinyl, pyridazinyl, thiazolyl, thiophene, pyrazolyl, pyridinone, oxadiazolone, indolone, benzimidazolone, benzopyrrole, etc.

[0095] The term "cycloalkyl" refers to a saturated carbon ring, including monocyclic, bicyclic, or polycyclic fused, bridged, or spirocyclic rings. For example, the term "C3-C..." 10 "Cycloalkyl" refers to a ring containing 3 to 10 carbon atoms, such as 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered or 10-membered ring atoms. It can further be C3-C8 cycloalkyl or C3-C6 cycloalkyl. Non-limiting examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, indane, etc.

[0096] The term "cycloalkenyl" refers to a group having at least one double bond in the aforementioned cycloalkyl group. For example, it can be a "3-12 membered cycloalkenyl," meaning it can have 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cyclic carbon atoms. Unless otherwise specified, a membered cycloalkenyl includes all possible monocyclic and fused rings (including fused in fused, spiro, or bridged forms). Cycloalkenyl can be 3-12 membered cycloalkenyl, 3-8 membered cycloalkenyl, 3-7 membered cycloalkenyl, 4-6 membered cycloalkenyl, 5-6 membered cycloalkenyl, 5-7 membered cycloalkenyl, 7-11 membered spirocycloalkenyl, 7-11 membered fused cycloalkenyl, 6-11 membered bridged cycloalkenyl, etc. Examples of cycloalkenyl groups include cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadien-1-yl, cycloheptenyl, 1,4-cycloheptadien-1-yl, cyclooctenyl, 1,5-cyclooctadien-1-yl, etc., but are not limited to these.

[0097] Term "C" 3-7 "Cycloalkenyl" refers to a group that has at least one double bond in a 3-7 membered cycloalkyl group, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc.

[0098] The term "heterocyclic group" refers to a saturated or partially saturated non-aromatic ring, in which at least one ring system contains one or more heteroatoms. It can be a monocyclic, bicyclic, polycyclic, spirocyclic, or bridged ring, i.e., containing a monocyclic heterocyclic group, a fused heterocyclic group, a spirocyclic group, or a bridged heterocyclic group. The "3-12 membered heterocyclic group" described in this invention refers to a cyclic group containing one, two, three, four, or five heteroatoms and containing 3-12 (e.g., 5, 6, 5-7, 3-8, 5-12, 3-10, or 8-10) ring atoms, wherein the heteroatoms are selected from nitrogen, oxygen, and sulfur. The nitrogen, carbon, or sulfur atoms in the heterocyclic group may optionally be oxidized, such as the "-CH2-" group of the heterocyclic group, which may optionally be oxidized to form -C(=O)-; or the sulfur may optionally be oxidized to form a sulfone or sulfoxide. Non-limiting examples of heterocyclic groups include, but are not limited to: pyrrolyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydropyridyl, tetrahydropyrrolyl, azacyclic butyl, thiazolyl, azoleyl, piperidinyl, etc.

[0099] The term "Threotype," also known as the Thustic configuration, refers to the concepts of erythrotype and threotype configurations used in organic chemistry when dealing with stereochemical problems involving two chiral carbon compounds in a molecule. A threotype refers to two identical atoms or groups located on opposite sides of the Fisher projection, while an erythrotype refers to two identical atoms or groups located on the same side of the Fisher projection. (See the example below.) For example, methyl 2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate (Threo-) refers to a mixture of methyl (2S,3R)-2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate and methyl (2R,3S)-2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate.

[0100] HEX: n-Hexane

[0101] DEA: Diethylamine

[0102] EtOH: Ethanol

[0103] FA: Formic acid

[0104] MeCN: Acetonitrile

[0105] Example Section

[0106] The following examples and test cases illustrate the present invention in detail, but they do not limit the scope of the invention, and variations can be made without departing from the scope of the invention.

[0107] The compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art.

[0108] Preparation of Intermediate 1

[0109]

[0110] a) Preparation of cyclopent-1-en-1-yl trifluoromethanesulfonate

[0111] Cyclopentanone (25 g), sodium carbonate (47.25 g, 445.8 mmol, 1.5 eq), and dichloromethane were added to a 2 L three-necked flask. Trifluoromethanesulfonic anhydride (83.8 g) was slowly added dropwise at -20 °C over approximately 1.6 hours. The mixture was then slowly brought to room temperature and reacted overnight. The reaction was quenched by slowly adding water (400 mL) in an ice-water bath. The mixture was extracted with dichloromethane (300 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was directly concentrated to obtain 39.4 g of the title compound.

[0112] b) Preparation of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(cyclopent-1-en-1-yl)propionate

[0113] Preparation of zinc reagent: Add zinc powder (81.6 g) to a 2 L three-necked flask, N,N-dimethylformamide (350 mL), purge with nitrogen, then add dibromoethane (11.73 g), and react at 60 °C for 30 minutes; cool to room temperature and add trimethylchlorosilane (1.36 g), stir at room temperature for 30 minutes, then add (R)-2-((tert-butoxycarbonyl)amino)-3-iodopropionate methyl ester (45.6 g) in N,N-dimethylformamide (50 mL) solution under ice-water bath, react at 60 °C for 2 hours, and cool for later use;

[0114] Cyclopent-1-en-1-yl trifluoromethanesulfonate (30.0 g) was added to a 250 mL two-necked flask, followed by 1,1-bis(diphenylphosphine)diferropalladium dichloride (3.37 g) and N,N-dimethylformamide (80 mL). After purging with nitrogen, the mixture was added to a zinc reagent reaction flask and reacted overnight at 60 °C. The reaction was quenched with water (200 mL), extracted with ethyl acetate (3 × 200 mL), and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to prepare slurry. The filtrate was purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 9 / 1 (V / V)) to give 15.2 g of the title compound.

[0115] c) Preparation of (S)-2-((tert-butoxycarbonyl)amino)-3-(cyclopent-1-en-1-yl)propionic acid

[0116] Methyl (S)-2-((tert-Butoxycarbonyl)amino)-3-(cyclopent-1-en-1-yl)propionate (15.2 g), water (150 mL), and methanol (75 mL) were added to a 1 L three-necked flask. Lithium hydroxide monohydrate (4.74 g) was then added. After nitrogen purging, the mixture was stirred overnight at room temperature and concentrated to dryness under reduced pressure. Then, dichloromethane (200 mL) and water (200 mL) were added, and the pH was adjusted to 3-4. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 14.49 g of the title compound.

[0117] Preparation of d)(S)-(3-(cyclopent-1-en-1-yl)-1-(methoxy(methyl)amino)-1-oxopropyl-2-yl)carbamate tert-butyl

[0118] (S)-2-((tert-Butoxycarbonyl)amino)-3-(cyclopent-1-en-1-yl)propionic acid (14.5 g) was added to a 250 mL two-necked flask under ice-water bath conditions. Dichloromethane (120 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.32 g), and 1-hydroxybenzotriazole (7.67 g) were added. After nitrogen purging, N,O-dimethylhydroxylamine hydrochloride (5.54 g) and N,N-diisopropylethylamine (18.34 g) were added. The mixture was slowly heated to room temperature and stirred overnight. The reaction was quenched with water (200 mL), and the mixture was extracted with dichloromethane (3 × 200 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 9 / 1 (V / V)) to give 15.3 g of the title compound.

[0119] Preparation of e)(S)-(1-(cyclopent-1-en-1-yl)-4-methyl-3-oxopent-4-en-2-yl)carbamate tert-butyl

[0120] In a three-necked flask (500 mL) containing 8.0 g of (S)-(3-(cyclopent-1-en-1-yl)-1-(methoxy(methyl)amino)-1-oxopropyl-2-yl)carbamate tert-butyl ester, tetrahydrofuran (80 mL) was added, and the mixture was purged with nitrogen. Propylene-1-en-2-ylmagnesium bromide (1 M, 107.2 mL) was slowly added dropwise over an ice bath, completing the addition in approximately 1.5 hours. The reaction was carried out over an ice-water bath for 3 hours. The reaction was quenched with saturated brine, extracted with ethyl acetate (3 × 100 mL), and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 9 / 1 (V / V)) to give 5.04 g of the title compound.

[0121] Preparation of f)((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)carbamate tert-butyl

[0122] (S)-(1-(cyclopent-1-en-1-yl)-4-methyl-3-oxopent-4-en-2-yl) tert-butyl carbamate (5.0 g) and N,N-dimethylformamide (30 mL) were added to a 250 mL two-necked flask. 10% sodium hypochlorite solution (26.64 g) was added dropwise at -20 °C. The reaction was carried out in an ice-water bath for 2 hours. The reaction was quenched with water (20 mL), and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 9 / 1 (V / V)) to give 1.53 g of the title compound.

[0123] 1H NMR (400MHz, DMSO-d6) δ5.39(s,1H),4.83(d,J=7.3Hz,1H),4.34(td,J=8.1,4.8Hz,1H),3.21(d,J=4.8Hz,1H ),2.82(d,J=5.0Hz,1H),2.52–2.39(m,1H),2.30–2.13(m,5H),1.88–1.69(m,2H),1.44(s,3H),1.34(s,9H).

[0124] Preparation of g)(S)-2-amino-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)prop-1-one trifluoroacetate

[0125] 400 mg of ((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)carbamate tert-butyl ester was added to a 50 mL single-necked flask, followed by 4 mL of dichloromethane and then 4 mL of trifluoroacetic acid. The mixture was reacted at room temperature for 30 minutes. The reaction solution was then concentrated under reduced pressure to obtain 500 mg of the title compound.

[0126] Preparation of intermediate 2

[0127]

[0128] a) Preparation of 2-amino-3-hydroxy-3-(4-methoxyphenyl)propionic acid (threo-)

[0129] Glycine (50 g) and p-methoxybenzaldehyde (139 g) were added to a reaction flask, along with anhydrous ethanol (1.5 L) and potassium hydroxide (91.6 g). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure until no droplets remained. The residue was dissolved in 500 mL of water and the pH was adjusted to 5 with dilute hydrochloric acid (4 N). The mixture was extracted with ethyl acetate (1 L x 3), and the aqueous layer was concentrated to approximately 400 mL. The mixture was filtered, and the filter cake was washed with (100 mL x 2) of water and dried under vacuum to give 48 g of the title compound (threo-).

[0130] 1 H NMR (400MHz, D2O) δ7.42(d,J=8.7Hz,2H),7.06(t,J=8.4Hz,2H),5.25(d,J=4.6Hz,1H),3.91(d,J=4.7Hz,1H),3.86(d,J=3.1Hz,3H).

[0131] ESI-MS(m / z) = 212[M+1] + .

[0132] b) Preparation of methyl 2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-)

[0133] Anhydrous methanol (60 mL) was added to a three-necked flask, and the mixture was cooled to 0–5 °C. Thionyl chloride (3.38 g) was added dropwise, while maintaining the temperature below 10 °C. 2-Amino-3-hydroxy-3-(4-methoxyphenyl)propionic acid (threo-) (2 g) was added. The mixture was stirred at room temperature for 1 hour, then refluxed for 4 hours. After cooling the reaction system to room temperature, 30 mL of water was added to quench the reaction. The organic solvent was removed by concentration under reduced pressure. The pH was adjusted to 8–9 with a saturated sodium carbonate aqueous solution. The mixture was extracted six times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 1.6 g of the title compound.

[0134] 1 H NMR (400MHz, DMSO) δ8.42 (s, 2H), 7.36-7.24 (m, 2H), 6.95 (dd, J = 12.2, 10.3Hz, 2H), 6.46 (d, J = 2.8Hz, 1H), 4. 99(dd,J=20.5,3.8Hz,1H),4.10(d,J=5.7Hz,1H),3.76(d,J=5.7Hz,3H),3.62(s,3H).ESI-MS(m / z)=226[M+1] + .

[0135] c) Preparation of methyl 2-(tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-)

[0136] Methyl 2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-) (1 g) was dissolved in tetrahydrofuran (20 mL) and added to a 50 mL single-necked flask. Di-tert-butyl dicarbonate (1.16 g) and triethylamine (900 mg) were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain sand, and purified by column chromatography (mobile phase: dichloromethane / methanol = 95 / 5 (V / V)) to obtain 1 g of the title compound.

[0137] 1 H NMR (400MHz, DMSO) δ7.25(d,J=8.6Hz,2H),6.87(d,J=8.6Hz,2H),6.58(d,J=9.0Hz,1H),5.59(s,1H),4.95(t ,J=10.0Hz,1H),4.29-4.13(m,1H),3.72(s,3H),3.59(s,3H),1.24(d,J=45.2Hz,9H).ESI-MS(m / z)=326[M+1] + .

[0138] d) Preparation of 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionic acid (threo-)

[0139] Methyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-) (1 g) was dissolved in tetrahydrofuran / water = 3 / 1 (40 mL), cooled to 0 °C in an ice-water bath, and then lithium hydroxide monohydrate (310 mg) and hydrogen peroxide (30%, 2.51 g) were added to the reaction flask. The mixture was stirred at 0 °C for 1 hour, and then slowly heated to room temperature for 3 hours. The reaction solution was quenched with sodium sulfite aqueous solution, and extracted and separated by ethyl acetate / water (30 mL / 40 mL). The aqueous phase was collected, acidified to pH = 5 with dilute hydrochloric acid, and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 1.2 g of the title compound.

[0140] ESI-MS(m / z) = 312[M+1] + .

[0141] e) Preparation of 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionate (threotype)

[0142] 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionic acid (threo-) (1.2 g) and cesium carbonate (1.26 g) were added to a reaction flask, followed by N,N-dimethylformamide (25 mL). The mixture was cooled to 0 °C in an ice-water bath, and benzyl bromide (1.32 g) was added dropwise at 0 °C. The reaction was brought to room temperature and stirred for 1 hour. The mixture was then quenched with water, extracted three times with ethyl acetate (30 mL), and the organic phases were combined. The mixture was washed three times with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain sintered precipitate. The sintered precipitate was purified by column chromatography (mobile phase: dichloromethane / methanol = 95 / 5 (V / V)) to give 1.05 g of the title compound.

[0143] 1 H NMR (400MHz, DMSO) δ7.37-7.31(m,3H),7.27(dd,J=10.3,5.2Hz,4H),6.86(d,J=8.6Hz,2H),6.70(d,J=8.8Hz,1H),5.63(d, J=6.5Hz,1H),5.07(s,2H),4.97(dd,J=13.2,7.8Hz,1H),4.29(dd,J=8.7,4.5Hz,1H),3.73(s,3H),1.27(d,J=25.7Hz,9H).

[0144] ESI-MS(m / z) = 402[M+1] + .

[0145] f) Preparation of 2-amino-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-)

[0146] 1.05 g of benzyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propionate (threotype) was added to 10 mL of dichloromethane and cooled to 0 °C in an ice-water bath. 5 mL of trifluoroacetic acid was added, and the mixture was stirred at 0 °C for 3 hours. The reaction was diluted with 20 mL of dichloromethane and quenched with saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 910 mg of the title compound.

[0147] ESI-MS(m / z) = 302[M+1] + .

[0148] Example 1:

[0149] (S)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopen-2-yl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0150]

[0151] a) Preparation of 3-ethoxy-4-((2-morpholinoethyl)amino)cyclobut-3-ene-1,2-dione

[0152] Diethyl squaric acid (1.5 g) was dissolved in ethanol (5 mL), triethylamine (303 mg) was added, and N-(2-aminoethyl)morpholine (318.8 mg) was added dropwise. The mixture was stirred overnight at room temperature, and the reaction was quenched by adding water (20 mL). The mixture was extracted with dichloromethane (3 × 20 mL), and the organic phases were combined. The organic phases were washed with water (2 × 40 mL) and saturated sodium chloride solution (2 × 40 mL). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to prepare slag, and purified by column chromatography (mobile phase: dichloromethane / methanol = 15 / 1 (V / V)) to give 385 mg of the title compound.

[0153] b) Preparation of benzyl propionate (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)

[0154] 180 mg of 3-ethoxy-4-((2-morpholinoethyl)amino)cyclobut-3-ene-1,2-dione was dissolved in ethanol (2 mL), and triethylamine (72 mg) was added. Then, a solution of (S)-2-((S)-2-aminopropamido)-3-(4-methoxyphenyl)propionate benzyl ester (250 mg) dissolved in ethanol (2 mL) was added dropwise to the reaction mixture, and the mixture was stirred overnight at room temperature. The reaction was quenched with water (15 mL), extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The organic phase was washed with water (2 × 20 mL), washed with saturated sodium chloride solution (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain slag. The filtrate was purified by column chromatography (mobile phase: dichloromethane / methanol = 15 / 1 (V / V)) to give 210 mg of the title compound.

[0155] Preparation of (c)-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)propionic acid

[0156] Benzyl propionate (100 mg) of (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-(((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamido)propionate was mixed with tetrahydrofuran (1 mL), purged with hydrogen using palladium / carbon (10%, 20 mg), stirred overnight at room temperature, filtered with diatomaceous earth as an aid, filtered under vacuum, and the mother liquor was concentrated under reduced pressure to obtain 80 mg of the title compound.

[0157] Preparation of (d)(S)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0158] Add (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)propionic acid to a 50 mL reaction flask, then add benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (60.0 mg), 1-hydroxybenzotriazole (23.0 mg), and N,N-dimethylformamide (2 mL). Stir for 10 minutes in an ice bath, then add N,N-diisopropylethylamine (45.3 mg) and (S)-4-methyl- 1-((R)-2-methylethyleneoxy-2-yl)-1-oxopentan-2-amine-2,2,2-trifluoroacetate (48.5 mg) was reacted with water (10 mL) at room temperature for 1 hour. The reaction was quenched by adding water and extracted with dichloromethane (3 × 10 mL). The organic phases were combined and washed with water (2 × 15 mL) and saturated sodium chloride solution (2 × 15 mL). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to prepare slag and purified by column chromatography (mobile phase: dichloromethane / methanol = 15 / 1 (V / V)) to give 33.9 mg of the title compound.

[0159] 1 H NMR(300MHz,Chloroform-d)δ8.78(s,1H),8.32(s,2H),7.92-7.67(m,1H),6.96-6.70(m,2 H),6.52(d,J=7.8Hz,2H),5.36(s,1H),5.04(s,1H),4.61(s,1H),3.97(s,1H),3.76-3.55(m ,5H),3.49(d,J=3.0Hz,3H),3.07(d,J=7.9Hz,1H),2.67(s,2H),2.49(s,4H),2.02(d,J=25 .0Hz,4H),1.68(d,J=11.1Hz,3H),1.50-1.35(m,2H),1.34-1.20(m,4H),1.00-0.82(m,5H).

[0160] ESI-MS (m / z) = 628.33 [M+1] + .

[0161] Example 2

[0162] (S)-3-(4-methoxyphenyl)-N-((S)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxo-3-phenylprop-2-yl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0163]

[0164] Referring to the preparation method of Example 1, the (S)4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopentan-2-amine-2,2,2-trifluoroacetate in step d) can be replaced with (S)-2-amino-1-((R)-2-methylepoxyethylene-2-yl)-3-phenylprop-1-one.

[0165] 1 H NMR(300MHz,Chloroform-d)δ8.45-8.16(m,1H),8.11-7.75(m,2H),7.71-7.52(m,1H),7 .26-7.20(m,1H),7.18-7.02(m,4H),6.91-6.80(m,2H),6.70-6.52(m,2H),5.21-4.67(m ,3H),3.71-3.66(m,4H),3.61(s,3H),3.07(d,J=12.6Hz,1H),2.98-2.85(m,2H),2.65(d ,J=6.6Hz,3H),2.53(s,4H),2.00-1.94(m,5H),1.39-1.32(m,3H),1.29(d,J=9.6Hz,3H).

[0166] ESI-MS (m / z) = 662.26 [M+1] + .

[0167] Example 3

[0168] (S)-2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0169]

[0170] The preparation method in Example 1 can be used as a reference, except that N-(2-aminoethyl)morpholine in step a) can be replaced with benzylamine.

[0171] 1H NMR(300MHz, DMSO-d6)δ8.63-8.20(m,2H),7.97(s,1H),7.67(d,J=8.1Hz,1H),7.44-7.25(m,5H), 7.20-7.08(m,2H),6.87-6.71(m,2H),4.71(d,J=6.4Hz,2H),4.68-4.58(m,1H),4.57-4.43(m,1H), 4.43-4.31(m,1H),3.69(d,J=3.4Hz,3H),3.25-3.15(m,1H),3.04-2.97(m,1H),2.96-2.85(m,1H), 2.71-2.57(m,1H),1.44-1.39(m,3H),1.38-1.22(m,5H),1.07(d,J=6.8Hz,1H),0.91-0.78(m,8H).

[0172] ESI-MS (m / z) = 605.57 [M+1] + .

[0173] Example 4

[0174] (S)-2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxo-3-phenylprop-2-yl)propamidyl)propamid

[0175]

[0176] Referring to the preparation method of Example 1, N-(2-aminoethyl)morpholine in step a) is replaced with phenylethylamine, and (S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopentan-2-amine-2,2,2-trifluoroacetate in step d) is replaced with (S)-2-amino-1-((R)-2-methylepoxyethylene-2-yl)-3-phenylprop-1-one.

[0177] 1H NMR(300MHz,DMSO-d6)δ8.90-8.59(m,1H),8.52-8.24(m,1H),7.98(s,1H),7.70(s,1H),7.43- 7.22(m,9H),7.21-7.09(m,2H),6.87-6.75(m,2H),4.72(d,J=6.2Hz,2H),4.70-4.51(m,2H),4. 45(ddd,J=17.0,8.7,5.2Hz,1H),3.69(t,J=3.3Hz,3H),3.62(d,J=4.4Hz,2H),2.99(ddd,J=14. 2,9.3,4.4Hz,2H),2.82-2.58(m,1H),1.44-1.27(m,3H),1.26-1.08(m,3H),0.90-0.77(m,2H).

[0178] ESI-MS (m / z) = 639.61 [M+1] + .

[0179] Example 5

[0180] (S)-2-((S)-2-((3,4-dioxo-2-(anilino)cyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0181]

[0182] The preparation method in Example 1 can be used as a reference, except that N-(2-aminoethyl)morpholine in step a) can be replaced with aniline.

[0183] 1H NMR(300MHz, DMSO-d6)δ9.90(s,1H),8.44(d,J=8.3Hz,1H),8.33(t,J=11.0Hz,1H),8.00(d,J=8.2Hz,1H),7.51-7.30(m,4H),7.2 2-7.10(m,2H),7.07-6.94(m,1H),6.84-6.71(m,2H),4.73(t,J=7.5Hz,1H),4.52(td,J=9.2,3.8Hz,1H),4.41(ddd,J=11.9,7.6,4 .2Hz,1H),3.72-3.56(m,3H),3.19(t,J=6.1Hz,1H),3.03(d,J=5.2Hz,1H),2.95(dd,J=14.0,4.1Hz,1H),2.72-2.60(m,1H),1.76- 1.57(m,1H),1.42(s,3H),1.38(d,J=4.3Hz,1H),1.33(d,J=6.7Hz,3H),1.26(s,1H),0.92(d,J=6.5Hz,3H),0.86(d,J=6.6Hz,3H).

[0184] ESI-MS (m / z) = 590.93 [M+1] + .

[0185] Example 6

[0186] (S)-2-((S)-2-((3,4-dioxo-2-((pyridin-4-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0187]

[0188] The preparation method in Example 1 can be used as a reference, except that N-(2-aminoethyl)morpholine in step a) can be replaced with 4-pyridinemethylamine hydrochloride.

[0189] 1H NMR (300MHz, DMSO-d6) δ8.60-8.53(m,2H),8.40(d,J=7.6Hz,1H),8.28(d,J=7.4Hz,1H),8.03(s,1H),7.76(s,1H),7. 37-7.28(m,2H),7.19-7.09(m,2H),6.84-6.74(m,2H),4.76(d,J=6.2Hz,2H),4.71-4.60(m,1H),4.59-4.44(m,1H),4. 44-4.31(m,1H),3.69(d,J=3.4Hz,3H),3.25-3.15(m,1H),3.02(d,J=5.2Hz,1H),2.98-2.86(m,1H),2.65(t,J=12.1Hz ,1H),1.77-1.56(m,1H),1.53-1.42(m,1H),1.41(s,3H),1.38-1.33(m,1H),1.29(d,J=6.8Hz,3H),0.92-0.78(m,6H).

[0190] ESI-MS (m / z) = 606.48 [M+1] + .

[0191] Example 7

[0192] (S)-2-((S)-2-((3,4-dioxo-2-(pyridin-3-ylamino)cyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0193]

[0194] The preparation method in Example 1 can be used as a reference, except that N-(2-aminoethyl)morpholine in step a) can be replaced with pyridine-3-amine.

[0195] 1H NMR(300MHz,DMSO-d6)δ10.08(d,J=7.9Hz,1H),8.74-7.89(m,6H),7.49-7.11(m,3H),6.94 -6.67(m,2H),4.85-4.67(m,1H),4.66-4.47(m,1H),4.47-4.28(m,1H),3.68(d,J=16.6Hz,3 H),3.25-3.17(m,1H),3.08-2.99(m,1H),2.99-2.84(m,1H),2.76-2.59(m,1H),1.75-1.48 (m,1H),1.41(d,J=4.3Hz,3H),1.37-1.30(m,3H),1.27(d,J=5.2Hz,1H),0.94-0.79(m,6H).

[0196] ESI-MS (m / z) = 592.46 [M+1] + .

[0197] Example 8

[0198] (S)-2-((S)-2-((3,4-dioxo-2-(pyridin-2-ylamino)cyclobut-1-en-1-yl)amino)propamidyl)-3-(4-methoxyphenyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0199]

[0200] Prepared according to the preparation method of Example 1, except that N-(2-aminoethyl)morpholine in step a) is replaced with pyridine-2-amine.

[0201] 1H NMR(300MHz,DMSO-d6)δ10.87(s,1H),9.32-9.22(m,1H),8.43-8.36(m,1H),8.33-8.26(m,1H),8.20-8.1 5(m,1H),7.82-7.71(m,1H),7.27-7.20(m,1H),7.18-7.12(m,2H),7.05-6.98(m,1H),6.79-6.71(m,2H),4 .74-4.66(m,1H),4.57-4.48(m,2H),4.42(s,3H),3.72-3.65(m,1H),3.27-3.16(m,2H),3.04-2.92(m,2H ),2.75-2.60(m,1H),1.68(dt,J=13.7,6.7Hz,1H),1.41(s,3H),1.38(d,J=6.8Hz,3H),0.93-0.86(m,6H).

[0202] ESI-MS (m / z) = 592.31 [M+1] + .

[0203] Example 9

[0204] (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-((4-methoxyphenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamidyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0205]

[0206] Prepared according to the preparation method of Example 1, except that N-(2-aminoethyl)morpholine in step a) is replaced with p-methoxyaniline.

[0207] 1H NMR(300MHz, DMSO-d6)δ9.80(s,1H),8.41(d,J=8.4Hz,1H),8.30(d,J=7.4Hz,1H),7.91(d,J=8.1Hz,1H),7.39-7.33 (m,2H),7.18-7.12(m,2H),6.95-6.89(m,2H),6.81-6.74(m,2H),4.77-4.66(m,1H),4.56-4.47(m,1H),4.43(d,J=4 .3Hz,1H),3.73(s,3H),3.65(s,3H),3.21-3.18(m,1H),3.06-3.00(m,1H),2.99-2.90(m,1H),2.72-2.59(m,1H),1. 75-1.62(m,1H),1.42(s,3H),1.40-1.34(m,2H),1.32(d,J=6.9Hz,3H),0.92(d,J=6.6Hz,3H),0.86(d,J=6.5Hz,3H).

[0208] ESI-MS (m / z) = 621.48 [M+1] + .

[0209] Example 10

[0210] (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-(((3-methoxyphenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamid)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamid

[0211]

[0212] Prepared according to the preparation method of Example 1, except that N-(2-aminoethyl)morpholine in step a) is replaced with 3-methoxyaniline.

[0213] 1H NMR(300MHz,Chloroform-d)δ9.92(s,1H),8.97(s,1H),7.93(s,1H),7.60-7 .35(m,2H),7.23-7.01(m,2H),6.77(d,J=8.4Hz,1H),6.65(d,J=7.6Hz,2H), 6.44(d,J=7.8Hz,2H),5.51(s,1H),5.07(s,1H),4.58(s,1H),3.92-3.70(m, 6H),3.49(s,1H),3.06(s,3H),1.65(s,3H),1.28(s,6H),0.90-0.77(m,6H).

[0214] ESI-MS (m / z) = 621.3 [M+1] + .

[0215] Example 11

[0216] (S)-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-methoxyphenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamidyl)-N-((S)-4-methyl-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopent-2-yl)propamidyl)propamid

[0217]

[0218] The preparation method in Example 1 can be used as a reference, except that N-(2-aminoethyl)morpholine in step a) can be replaced with 2-methoxyaniline.

[0219] 1 H NMR(300MHz,Chloroform-d)δ9.03(s,1H),8.69(s,1H),7.98(s,1H),7.67(s,1H),7.22-6.9 8(m,2H),7.01-6.82(m,3H),6.75(d,J=8.0Hz,2H),6.58(d,J=7.9Hz,1H),5.48-4.94(m,2H) ,4.74-4.41(m,1H),3.87-3.71(m,1H),3.55-3.36(m,3H),3.28(s,3H),3.04(s,1H),2.97-2 .78(m,1H),2.39(s,1H),1.98(s,2H),1.77-1.54(m,4H),1.35(s,3H),0.89(d,J=5.3Hz,6H).

[0220] ESI-MS (m / z) = 621.3 [M+1]+ .

[0221] Example 12

[0222] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0223] a) Preparation of 3-methoxy-4-(((5-methylfuran-2-yl)methyl)amino)cyclobut-3-ene-1,2-dione

[0224] (5-methylfuran-2-yl)methylamine (410 mg) and 3,4-dimethoxycyclobut-3-ene-1,2-dione (500 mg) were added to a 50 mL two-necked flask, followed by methanol (30 mL). The mixture was reacted at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure to obtain sand, which was then purified by column chromatography (mobile phase: dichloromethane / methanol = 10 / 1 (V / V)) to give 743 mg of the title compound.

[0225] b) Preparation of 2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-L-alanine benzyl ester

[0226] 3-Methoxy-4-(((5-methylfuran-2-yl)methyl)amino)cyclobut-3-ene-1,2-dione (743 mg), ethanol (40 mL), and L-alanine benzyl ester (602 mg) were added to a 100 mL single-necked flask and reacted at room temperature for 16 h. A solid precipitated out. The solid was filtered, and the filter cake was collected to give 364 mg of the title compound.

[0227] ESI-MS (m / z) = 369.1 [M+1] + .

[0228] c) Preparation of 2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-L-alanine

[0229] (2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-L-alanine benzyl ester (364 mg) was added to a reaction flask, followed by the addition of tetrahydrofuran (40 mL), and hydrogen purging with palladium / carbon (10%, 36 mg). The mixture was stirred overnight at room temperature, filtered with diatomaceous earth as an aid, and the solution was concentrated under reduced pressure to obtain 294 mg of the title compound.

[0230] d) Preparation of 3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)benzyl propionate (threo-)

[0231] (2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)-L-alanine (110 mg) was added to a 50 mL reaction flask, followed by N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (151.4 mg) and N,N-dimethylformamide (10 mL). The mixture was stirred in an ice bath for 10 minutes, and then N,N-diisopropylethylamine (85.8 mg) and 2-amino-3-hydroxylamine were added. 100 mg of benzyl-3-(4-methoxyphenyl)propionate (threo-) was reacted with water (10 mL) to quench the reaction in ethyl acetate (3 × 10 mL). The organic phases were combined, washed with water (2 × 15 mL), washed with saturated sodium chloride solution (2 × 15 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to form sand, and purified by column chromatography (mobile phase: dichloromethane / methanol = 10 / 1 (V / V)) to give 122 mg of the title compound.

[0232] ESI-MS (m / z) = 560.2 [M-1] -

[0233] e) Preparation of 3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)propionic acid (threo-)

[0234] 122 mg of 3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamido)benzyl propionate (threo-) was added to a reaction flask, followed by the addition of tetrahydrofuran (10 mL), and hydrogen purging with palladium / carbon (10%, 13 mg). The mixture was stirred overnight at room temperature, filtered with diatomaceous earth as an aid, and the solution was concentrated under reduced pressure to obtain 120 mg of the title compound.

[0235] ESI-MS (m / z) = 470.1 [M-1] -

[0236] f) Preparation of N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-)

[0237] 100 mg of 3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamido)propionic acid (threo-) was added to a 50 mL reaction flask. Then, 121 mg of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and 10 mL of N,N-dimethylformamide were added. The mixture was stirred in an ice bath for 10 minutes. Finally, 137.1 mg of N,N-diisopropylethylamine and (S)- 2-Amino-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)prop-1-one trifluoroacetate (98.4 mg) was reacted with water (10 mL) to quench the reaction, extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined. The organic phases were washed with water (2 × 15 mL) and saturated sodium chloride solution (2 × 15 mL). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to prepare slag, and purified by column chromatography (mobile phase: dichloromethane / methanol = 10 / 1 (V / V)) to give 52.1 mg of the title compound.

[0238] g)(2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(( The preparation of 2-(((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide was performed by chiral column resolution of N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((5-methylfuran-2-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-), yielding peaks -1,tr = 3.847 min and -2,tr = 10.183 min. (Separation method: Column: CHIRALPAK-IA 2*25cm; Mobile phase A: HEX (0.1% DEA); Mobile phase B: EtOH:DCM = 1:1; A:B = 65:35; Flow rate: 70ml / min; UV: 220nm).

[0239] Peak-1, tr = 3.847 min

[0240] 11H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.82 (s, 1H), 7.58 (s, 1H), 7.30 - 7.17 (m, 2H), 6.75 - 6.69 (m, 2H), 6.21 (d, J = 3.1 Hz, 1H), 6.05 - 6.00 (m, 1H), 5.52 (d, J = 4.4 Hz, 1H), 5.40 (s, 1H), 4.90 (t, J = 4.1 Hz, 1H), 4.78 (s, 1H), 4.67 (s, 2H), 4.57 (q, J = 7.2 Hz, 1H), 4.37 (dd, J = 9.2, 3.6 Hz, 1H), 3.67 (s, 3H), 3.13 (d, J = 5.2 Hz, 1H), 2.96 (d, J = 5.1 Hz, 1H), 2.41 (dd, J = 14.6, 5.8 Hz, 1H), 2.29 - 2.21 (m, 1H), 2.24 (s, 6H), 2.21 (s, 1H), 1.78 (p, J = 7.5 Hz, 2H), 1.35 (s, 3H), 1.26 (t, J = 8.2 Hz, 3H).

[0241] LCMS m / z = 649.25 [M + 1] + 。

[0242] Peak - 2, tr = 10.183 min

[0243] 1H NMR (400MHz, DMSO-d6) δ8.31(s,1H),8.04(d,J=7.4Hz,1H),7.87(s,1H),7.58(s,1H),7.31-7.24(m,2H),6.86-6.78(m,2H),6.19(d,J=3. 1Hz,1H),6.01(dq,J=2.2,1.0Hz,1H),5.47(d,J=4.9Hz,1H),5.33(s,1H),4.98(dd,J=5.0,3.3Hz,1H),4.77(s,1H),4.64(qd,J=15.2,5.8 Hz,2H), δ4.49(td,J=8.3,5.0Hz,1H),4.43(dd,J=9.2,3.1Hz,1H),3.70(s,3H),3.28(d,J=5.3Hz,1H),2.99(d,J=5.2Hz,1H),2.36(dd,J= 14.6, 4.8Hz, 1H), 2.23 (d, J = 1.0Hz, 3H), 2.19 (s, 1H), 2.16 (s, 3H), 2.14 (s, 1H), 1.73 (p, J = 7.5Hz, 2H), 1.39 (s, 3H), 1.05 (d, J = 6.8Hz, 3H).

[0244] LCMS m / z = 649.25 [M+1] + .

[0245] Example 13

[0246] (2S,3R)-2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamid)-N-((S)-3-(cyclopent-1-en-1-alkyl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propamid and (2R,3S)-2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamid)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propamid

[0247]

[0248] Prepared according to the preparation method of Example 12, except that (5-methylfuran-2-yl)methylamine in step a) is replaced with benzylamine, to obtain 2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamido)-N-((S)-3-(cyclopent-1-en-1-alkyl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-).

[0249] Chiral resolution of 2-((S)-2-((2-(benzylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propamido)-N-((S)-3-(cyclopent-1-en-1-alkyl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) yielded peak -1, tr = 4.607 min and peak -2, tr = 10.207 min. (Resolution method: Column: CHIRALPAK-IA 2*25cm; Mobile phase A: HEX (0.1% DEA); Mobile phase B: EtOH:DCM = 1:1; A:B = 65:35; Flow rate: 20 ml / min; UV: 220 nm).

[0250] Peak-1, tr = 4.607 min

[0251] 1 H NMR(400MHz, DMSO--d6)δ8.20(s,1H),7.94(d,J=8.8Hz,2H),7.64(s,1H),7.50-7.24(m,5H),7.24-7.08(m,2H),6 .80-6.63(m,2H),5.52(d,J=4.4Hz,1H),5.40(s,1H),4.90(t,J=4.1Hz,1H),4.85-4.64(m,3H),4.57(q,J=7.4Hz,1 H),4.37(dd,J=9.1,3.7Hz,1H),3.67(s,3H),3.13(d,J=5.2Hz,1H),2.95(d,J=5.1Hz,1H),2.90-2.76(m,1H),2.4 1(dd,J=14.9,5.8Hz,1H),2.23(dt,J=12.9,6.8Hz,4H),1.78(p,J=7.5Hz,2H),1.35(s,3H),1.27(d,J=6.8Hz,3H).

[0252] LCMS m / z = 645.0 [M+1] + .

[0253] Peak-2, tr = 10.207 min

[0254] 1 H NMR (400MHz, DMSO-d6) δ8.45-8.14(m,1H),8.05(d,J=7.4Hz,1H),7.93(s,1H),7.62(s,1H),7.48 -7.11(m,7H),6.87-6.75(m,2H),5.47(d,J=5.0Hz,1H),5.33(s,1H),4.98(dd,J=5.1,3.1Hz,1H) ,4.85-4.60(m,3H),4.53-4.35(m,2H),3.70(s,3H),3.27(d,J=5.3Hz,1H),2.99(d,J=5.3Hz,1H) ,2.43-2.29(m,1H),2.28-2.04(m,5H),1.72(p,J=7.5Hz,2H),1.39(s,3H),1.05(d,J=6.8Hz,3H).

[0255] LCMS m / z = 645.0 [M+1] + .

[0256] Example 14

[0257] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0258]

[0259] a) Preparation of 3-methoxy-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-3-ene-1,2-dione

[0260] (Tetrahydro-2H-pyran-4-yl)methylamine (1g) and 3,4-dimethoxycyclobut-3-ene-1,2-dione (1.23g) were added to a 100mL two-necked flask, followed by ethanol (20mL). The mixture was reacted overnight at room temperature and filtered under reduced pressure to obtain a filter cake, which was 810mg of the title compound.

[0261] b) Preparation of benzyl (3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)-L-propionate

[0262] 400 mg of 3-methoxy-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-3-ene-1,2-dione, 10 mL of ethanol, and 318.2 mg of L-alanine benzyl ester were added to a 100 mL single-necked flask and reacted overnight at room temperature. A solid precipitated out. The solid was filtered and the filter cake was collected to give 116 mg of the title compound.

[0263] ESI-MS (m / z) = 369.1 [M+1] + .

[0264] Preparation of (c) (3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)-L-alanine

[0265] Benzyl(3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)-L-propionate (116 mg) was added to a reaction flask, followed by tetrahydrofuran (20 mL), and hydrogen purging with palladium / carbon (10%, 25 mg). The mixture was stirred overnight at room temperature, filtered with diatomaceous earth as an aid, and the mother liquor was concentrated under reduced pressure to obtain 17 mg of the title compound.

[0266] d) Preparation of benzyl 2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propionamidyl)-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-)

[0267] (3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)-L-alanine (17 mg) was added to a 50 mL reaction flask, followed by N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (34.3 mg) and N,N-dimethylformamide (4 mL). The mixture was stirred in an ice bath for 10 minutes, and then N,N-diisopropylethylamine (23.3 mg) and 2-amino-3-hydroxylamine were added. Benzyl-3-(4-methoxyphenyl)propionate (threo-) (18.2 mg) was reacted with water (10 mL) to quench the reaction, extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined. The organic phases were washed with water (2 × 15 mL) and saturated sodium chloride solution (2 × 15 mL). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to prepare slag, and purified by column chromatography (mobile phase: dichloromethane / methanol = 10 / 1 (V / V)) to give 67 mg of the title compound.

[0268] ESI-MS (m / z) = 566.2 [M-1] - .

[0269] Preparation of e)2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propamido)-3-hydroxy-3-(4-methoxyphenyl)propionic acid (threo-)

[0270] 67 mg of benzyl 2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propamido)-3-hydroxy-3-(4-methoxyphenyl)propionate (threo-) was added to a reaction flask, followed by the addition of tetrahydrofuran (10 mL), and hydrogen purging with palladium / carbon (10%, 30 mg). The mixture was stirred overnight at room temperature, filtered with diatomaceous earth as an aid, and the solution was concentrated under reduced pressure to obtain 36 mg of the title compound.

[0271] ESI-MS (m / z) = 474.1 [M-1] - .

[0272] f)N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide Preparation of (Threo-): 2-((S)-2-((3,4-dioxo-2-(((tetrahydro-2H-pyran-4-yl)methyl)amino)cyclobut-1-en-1-yl)amino)propamido)-3-hydroxy-3-(4-methoxyphenyl)propionic acid (Threo-) (36 mg) was added to a 50 mL reaction flask, followed by the addition of N,N,N′,N′-tetramethyl-O-(7-azabenzene) (43.2 mg)triazol-1-yl)hexafluorophosphate and N,N-dimethylformamide (4 mL) were stirred in an ice bath for 10 minutes. Then, N,N-diisopropylethylamine (29.4 mg) and (S)-2-amino-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)prop-1-one trifluoroacetate (14.8 mg) were added. The reaction mixture was moved to room temperature and stirred for 1 hour. The reaction was quenched with water (10 mL), extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined. The organic phase was washed with water (2 × 15 mL) and saturated sodium chloride solution (2 × 15 mL). The mixture was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to prepare slag, and purified by column chromatography (mobile phase: dichloromethane / methanol = 10 / 1 (V / V)) to give 13 mg of the title compound.

[0273] LCMS m / z = 653.1 [M+1] + .

[0274] Example 15

[0275] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-(((4-methyltetrahydro-2H-pyran-4-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-ylamino)propionamide)propionamide (threo-)

[0276]

[0277] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (4-methyltetrahydro-2H-pyran-4-yl)methylamine.

[0278] LCMS m / z = 667.1 [M+1] + .

[0279] Example 16

[0280] 4-(((2-(((2S)-1-(1-((((S)-3-(cyclopent-1-en-1-yl)-1-(R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxypropyl-2-yl)amino)-1-oxopropyl-2-ylamino)-3,4-dioxocyclobut-1-en-1-ylamino)methyl benzoate)(threo-)

[0281]

[0282] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with methyl 4-(aminomethyl)benzoate.

[0283] LCMS m / z = 703.1 [M+1] + .

[0284] Example 17

[0285] 4-((2-(((2S)-1-(1-(((S)-3-(cyclopent-1-en-1-yl)-1-(R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxypropyl-2-yl)amino)-1-oxopropyl-2-ylamino)-3,4-dioxocyclobut-1-en-1-ylamino)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (threo-)

[0286]

[0287] Prepared according to the preparation method of Example 14, except that ((tetrahydro-2H-pyran-4-yl)methylamine in step a) is replaced with methyl 4-aminobicyclo[2.2.2]octane-1-carboxylate.

[0288] LCMS m / z = 721.1[M+1] + .

[0289] Example 18

[0290] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropan-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-3-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-3-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0291]

[0292] Prepared according to the preparation method of Example 12, except that (5-methylfuran-2-yl)methylamine in step a) is replaced with pyridin-3-ylmethylamine to obtain N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-)((pyridin-3-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide enantiomer (threo-).

[0293] The enantiomeric N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-)((pyridin-3-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was then separated by a chiral column, yielding peak -1, tr = 7.753 min and peak -2, tr = 11.817 min. (Separation method: Column: CHIRALPAK-IA 2*25cm; Mobile phase A: HEX (0.1% DEA); Mobile phase B: EtOH:DCM = 1:1; A:B = 50:50; Flow rate: 20 ml / min; UV 220 nm)

[0294] Peak-1, tr = 7.753 min

[0295] 1 H NMR (400MHz, DMSO-d6) δ8.56(d,J=2.3Hz,1H),8.52(dd,J=4.8,1.7Hz,1H),8.20(s,1H),7.93(s,2H),7.73(d,J=7.9Hz,1H),7.66(s, 1H),7.40(dd,J=7.9,4.8Hz,1H),7.27-7.16(m,2H),6.78-6.67(m,2H),5.52(d,J=4.4Hz,1H),5.40(s,1H),4.90(t,J=4.2Hz,1H),4. 76(d,J=6.1Hz,3H),4.57(q,J=7.2Hz,1H),4.38(dd,J=9.2,3.7Hz,1H),3.66(s,3H),3.13(d,J=5.2Hz,1H),2.95(d,J=5.2Hz,1H),2. 85(q,J=7.2Hz,1H),2.41-2.35(m,1H),2.30-2.24(m,2H),2.22-2.16(m,2H),1.83-1.74(m,2H),1.35(s,3H),1.27(d,J=6.8Hz,3H).

[0296] LCMS m / z = 646[M+1] + .

[0297] Peak-2, tr = 11.817 min

[0298] 1H NMR (400MHz, DMSO-d6) δ8.54(d,J=1.8Hz,1H),8.50(dd,J=4.7,1.6Hz,1H),8.31(s,1H),8.04(d,J=7.3Hz,1H),7.95(s,1H),7.71(d ,J=7.6Hz,1H),7.66(s,1H),7.39(dd,J=7.9,4.8Hz,1H),7.27(d,J=8.6Hz,2H),6.82(d,J=8.7Hz,2H),5.46(s,1H),5.33(s,1H),4.9 7(d,J=3.8Hz,1H),4.74(t,J=6.5Hz,3H),4.52-4.40(m,2H),3.70(s,3H),3.27(d,J=5.2Hz,2H),2.99(d,J=5.2Hz,1H),2.70-2.64(m ,1H),2.37-2.29(m,2H),2.14(d,J=6.1Hz,2H),1.72(dd,J=14.8,7.4Hz,2H),1.39(s,3H),1.05(d,J=6.8Hz,3H).LCMSm / z=646[M+1] + .

[0299] Example 19

[0300] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-2-((S)-2-((2-((2-hydroxy-2-methylpropyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide (threo-)

[0301]

[0302] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with 1-amino-2-methylprop-2-ol.

[0303] LCMS m / z = 627.1 [M+1] + .

[0304] Example 20

[0305] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-2-((S)-2-((2-(((4-methoxybenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide (threo-)

[0306]

[0307] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (4-methoxyphenyl)methylamine.

[0308] LCMS m / z = 675.1 [M+1] + .

[0309] Example 21

[0310] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-2-((S)-2-((2-((2-methoxybenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-2-((S)-2-((2-((2-methoxybenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide

[0311]

[0312] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) can be replaced with (2-methoxyphenyl)methylamine to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-3-hydroxy-2-((S)-2-((2-((2-methoxybenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide (threo-).

[0313] Chiral resolution of N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-2-((S)-2-((2-((2-methoxybenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-(4-methoxyphenyl)propionamide (threo-) yielded peaks -1, tr = 9.68 min and -2, tr = 12.95 min. (Resolution method: Column: CHIRALPAK-IC 2*25cm; Mobile phase A: HEX (0.1% DEA); Mobile phase B: EtOH:DCM = 1:1; A:B = 40:60; Flow rate: 20 ml / min; UV: 220 nm)

[0314] Peak-1, tr = 7.68 min

[0315] 1 H NMR (400MHz, DMSO-d6) δ8.19(s,1H),7.93(d,J=7.7Hz,1H),7.76(s,1H),7.66(d,J=8.5Hz,1H),7.36-7.28(m,1H),7.25(d,J=7.7Hz,1 H),7.20(d,J=8.7Hz,2H),7.03(d,J=8.1Hz,1H),6.97-6.90(m,1H),6.75-6.67(m,2H),5.51(d,J=4.4Hz,1H),5.40(s,1H),4.90(t,J= 4.1Hz,1H),4.79(s,1H),4.68(d,J=6.2Hz,2H),4.57(m,1H),4.36(dd,J=9.2,3.7Hz,1H),3.82(s,3H),3.65(s,3H),3.13(d,J=5.2Hz, 1H), 2.95 (d, J = 5.1Hz, 1H), 2.40 (s, 1H), 2.28 (d, J = 7.2Hz, 2H), 2.22 (d, J = 7.4Hz, 3H), 1.78 (m, 2H), 1.35 (s, 3H), 1.26 (d, J = 6.8Hz, 3H).

[0316] LCMS m / z = 675[M+1] + .

[0317] Peak-2, tr = 12.95 min

[0318] 1H NMR (400MHz, DMSO-d6) δ8.29(s,1H),8.04(d,J=7.6Hz,1H),7.80(s,1H),7.67(s,1H),7.29(t,J=9.3Hz,3H),7.22(d,J=6.1Hz,1H), 7.01(d,J=8.2Hz,1H),6.92(t,J=7.2Hz,1H),6.82(d,J=8.7Hz,2H),5.46(d,J=4.8Hz,1H),5.32(s,1H),4.98(s,1H),4.77(s,1H),4. 66(t,J=6.4Hz,2H),4.48(d,J=4.7Hz,1H),4.43(dd,J=9.2,3.0Hz,1H),3.81(s,3H),3.69(s,3H),3.27(d,J=5.3Hz,2H),2.99(d,J= 5.3Hz,1H),2.67(s,1H),2.41-2.30(m,2H),2.14(d,J=7.3Hz,2H),1.70(dd,J=14.8,7.4Hz,2H),1.39(s,3H),1.04(d,J=6.8Hz,3H).

[0319] LCMS m / z = 675[M+1] + .

[0320] Example 22

[0321] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-morpholinoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-)

[0322]

[0323] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with 2-morpholinoethane-1-amine.

[0324] LCMS m / z = 668.1 [M+1] + .

[0325] Example 23

[0326] (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-(2-methylthiazolyl-5-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide and (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-(2-methylthiazolyl-5-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0327]

[0328] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) is replaced with (2-methylthiazol-5-yl)methylamine to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-(2-(2-methylthiazol-5-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-).

[0329] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-(2-(2-methylthiazolyl-5-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-) was prepared and separated by chiral column chromatography, yielding peak -1, tr = 9.801 min and peak -2, tr = 10.112 min. (Separation method: Column: X-Bridge Prep C) 18 19*250mm; Mobile phase A: H2O (0.1% FA); Mobile phase B: MeCN; A:B = 25:45; Flow rate: 25ml / min; UV 220nm

[0330] Peak-1, tr = 9.801 min

[0331] 11H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.30 (s, 1H), 8.12 (s, 1H), 8.06 (s, 1H), 7.84 (s, 1H), 7.51 (s, 1H), 7.26 (d, J = 7.8 Hz, 2H), 6.81 (d, J = 8.1 Hz, 2H), 5.49 (s, 1H), 5.33 (s, 1H), 4.98 (s, 1H), 4.86 (s, 2H), 4.75 (s, 1H), 4.48 (s, 1H), 4.42 (d, J = 8.0 Hz, 1H), 3.70 (s, 3H), 2.99 (d, J = 4.7 Hz, 1H), 2.67 (s, 1H), 2.60 (s, 3H), 2.33 (s, 1H), 1.73 (d, J = 7.1 Hz, 3H), 1.39 (s, 3H), 1.23 (s, 3H), 1.05 (d, J = 6.0 Hz, 3H).

[0332] LCMS m / z = 666 [M+1] + 。

[0333] Peak - 2, tr = 10.112 min

[0334] 1 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.20 (s, 1H), 7.96 (s, 2H), 7.76 (s, 1H), 7.53 (s, 1H), 7.21 (d, J = 8.3 Hz, 2H), 6.73 (d, J = 8.9 Hz, 2H), 5.53 (s, 1H), 5.40 (s, 1H), 4.89 (s, 2H), 4.79 (s, 1H), 4.56 (s, 1H), 4.38 (s, 1H), 3.67 (s, 3H), 3.12 (s, 1H), 2.96 (s, 1H), 2.67 (s, 1H), 2.61 (s, 3H), 2.33 (s, 1H), 1.99 (s, 1H), 1.78 (s, 2H), 1.35 (s, 3H), 1.24 (s, 6H).

[0335] LCMS m / z = 666 [M+1] + 。

[0336] Example 24

[0337] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-2-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-2-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0338]

[0339] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) can be replaced with pyridin-2-ylmethylamine to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-2-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-).

[0340] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((3,4-dioxo-2-((pyridin-2-ylmethyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated using a chiral column, yielding peaks -1, tr = 8.279 min and -2, tr = 8.540 min. (Separation method: Column: X-Bridge Prep C) 18 19*250mm; Mobile phase A: H2O (0.1% FA); Mobile phase B: MeCN; A:B = 25:45; Flow rate: 25ml / min; UV 220nm

[0341] Peak-1, tr = 8.279 min

[0342] 11H NMR (400 MHz, DMSO-d6) δ 8.55 (d, J = 3.4 Hz, 1H), 8.29 (s, 1H), 8.10 (s, 1H), 8.04 (d, J = 7.1 Hz, 1H), 7.80 (t, J = 6.8 Hz, 2H), 7.40 - 7.32 (m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 5.46 (s, 1H), 5.32 (s, 1H), 4.99 (s, 1H), 4.79 (s, 2H), 4.49 (d, J = 5.2 Hz, 1H), 4.45 (d, J = 9.2 Hz, 1H), 3.70 (s, 3H), 3.62 (s, 1H), 3.27 (d, J = 5.2 Hz, 2H), 2.99 (d, J = 5.3 Hz, 1H), 2.67 (s, 1H), 2.00 (dd, J = 14.3, 6.8 Hz, 2H), 1.78 (dd, J = 15.1, 7.4 Hz, 1H), 1.73 - 1.64 (m, 1H), 1.39 (s, 3H), 1.24 (s, 3H), 1.06 (d, J = 6.8 Hz, 3H).

[0343] LCMS m / z = 646 [M+1] + 。

[0344] Peak - 2, tr = 8.540 min

[0345] 1 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.16 (s, 2H), 7.96 (s, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.40 - 7.30 (m, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 5.53 (d, J = 4.5 Hz, 1H), 5.41 (s, 1H), 4.91 (s, 1H), 4.84 (s, 2H), 4.58 (d, J = 6.4 Hz, 1H), 4.38 (d, J = 5.7 Hz, 1H), 3.69 (d, J = 10.0 Hz, 1H), 3.62 (s, 3H), 3.13 (d, J = 5.0 Hz, 1H), 2.96 (d, J = 5.1 Hz, 1H), 2.67 (s, 1H), 2.40 (s, 2H), 1.99 (s, 1H), 1.86 - 1.71 (m, 2H), 1.35 (s, 3H), 1.28 (d, J = 6.6 Hz, 3H), 1.24 (s, 3H).

[0346] LCMS m / z = 646 [M+1] + 。

[0347] Example 25

[0348] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-(((5-methyl-1,2,4-oxadiazol-3-yl)methyl)amino)-3,4-dioxocyclobut-1-en-1-ylamino)propionamide)propionamide (threo-)

[0349]

[0350] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (5-methyl-1,2,4-oxadiazol-3-yl)methylamine.

[0351] LCMS m / z = 651[M+1] + .

[0352] Example 26

[0353] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-(cyclopropylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0354]

[0355] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with cyclopropylamine.

[0356] LCMS m / z = 595[M+1] + .

[0357] Example 27

[0358] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((2-methoxyphenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-)

[0359]

[0360] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with 2-methoxyaniline.

[0361] LCMS m / z = 661[M+1] + .

[0362] Example 28

[0363] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((4-methoxyphenyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-)

[0364]

[0365] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with 4-methoxyaniline.

[0366] LCMS m / z = 661[M+1] + .

[0367] Example 29

[0368] (2S,3R)-2-((S)-2-((2-((cyclobutylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-2-((S)-2-((2-((cyclobutylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0369]

[0370] Prepared according to the preparation method of Example 12, except that (5-methylfuran-2-yl)methylamine in step a) is replaced with cyclobutylmethylamine to prepare 2-((S)-2-((2-((cyclobutylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-).

[0371] 2-((S)-2-((2-((cyclobutylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated by chiral column, yielding peak -1, tr = 10.00 min and peak -2, tr = 19.73 min. (Separation method: Column: NB-Chiral NX(2), 50*4.6 mm; Mobile phase A: Hex (10 mM NH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40 ml / min; UV 220 nm)

[0372] Peak-1, tr = 10.00 min

[0373] 1 H NMR(400MHz, DMSO-d6)δ8.19(s,1H),7.94(d,J=7.6Hz,1H),7.54(d,J=26.1Hz,2H),7.33-7.09(m,2H),6.87-6.60(m,2H),5 .52(d,J=4.4Hz,1H),5.41(s,1H),4.91(t,J=4.1Hz,1H),4.77(d,J=9.0Hz,1H),4.58(td,J=7.8,5.8Hz,1H),4.37(dd,J=9. 2,3.6Hz,1H),3.69(s,2H),3.55(t,J=6.8Hz,2H),3.13(d,J=5.2Hz,1H),2.96(d,J=5.2Hz,1H),2.48-2.35(m,2H),2.22(tt ,J=15.6,7.8Hz,4H),1.98(td,J=9.2,5.4Hz,2H),1.92-1.75(m,4H),1.74-1.61(m,2H),1.35(s,3H),1.27(d,J=6.7Hz,3H).

[0374] LCMS m / z = 623[M+1] + .

[0375] Peak-2, tr = 19.73 min

[0376] 1 H NMR (400MHz, DMSO-d6) δ8.30(s,1H),8.06(d,J=7.4Hz,1H),7.73-7.47(m,2H),7.28(d,J=8.4Hz,2H),6.82(d ,J=8.4Hz,2H),5.46(d,J=5.0Hz,1H),5.34(s,1H),5.15-4.87(m,1H),4.76(s,1H),4.47(ddd,J=22.7,8.7,4 .2Hz,2H),3.70(s,3H),3.52(q,J=9.2,6.7Hz,2H),3.28(d,J=5.3Hz,1H),3.00(d,J=5.3Hz,1H),2.47-2.31( m,2H),2.17(q,J=7.3,6.3Hz,4H),2.04-1.91(m,2H),1.88-1.60(m,5H),1.39(s,3H),1.05(d,J=6.8Hz,3H).

[0377] LCMS m / z = 623[M+1] + .

[0378] Example 30

[0379] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((oxecyclobutane-3-ylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-)

[0380]

[0381] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with 3-aminomethyloxetane.

[0382] LCMS m / z = 625[M+1] + .

[0383] Example 31

[0384] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((1-methylcyclopropyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((1-methylcyclopropyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide

[0385]

[0386] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) can be replaced with (1-methylcyclopropyl)methylamine to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((1-methylcyclopropyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-).

[0387] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-((2-((((1-methylcyclopropyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)propionamide (threo-) was prepared and separated using a chiral column, yielding peaks -1, tr = 8.26 min and -2, tr = 13.63 min. (Separation method: Column: CHIRAL ART Cellulose-SZ, 3*25cm; Mobile phase A: Hex (10mM NH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40 ml / min; UV 220 nm).

[0388] Peak-1, tr = 8.26 min

[0389] 11H NMR (400 MHz, DMSO-d6) δ 8.23 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.63 (q, J = 8.5, 7.4 Hz, 2H), 7.25 - 7.18 (m, 2H), 6.76 - 6.69 (m, 2H), 5.52 (d, J = 4.4 Hz, 1H), 5.41 (d, J = 2.5 Hz, 1H), 4.91 (t, J = 4.1 Hz, 1H), 4.79 (t, J = 7.6 Hz, 1H), 4.58 (td, J = 7.7, 5.7 Hz, 1H), 4.38 (dd, J = 9.2, 3.6 Hz, 1H), 3.68 (s, 3H), 3.38 (dd, J = 6.6, 3.2 Hz, 2H), 3.13 (d, J = 5.2 Hz, 1H), 2.96 (d, J = 5.1 Hz, 1H), 2.41 (dd, J = 14.5, 5.6 Hz, 1H), 2.22 (tt, J = 15.7, 7.9 Hz, 5H), 1.79 (p, J = 7.5 Hz, 2H), 1.35 (s, 3H), 1.29 (d, J = 6.8 Hz, 3H), 1.05 (s, 3H), 0.44 (q, J = 3.8 Hz, 2H), 0.30 (t, J = 2.8 Hz, 2H).

[0390] LCMS m / z = 623 [M+1] + 。

[0391] Peak - 2, tr = 13.63 min

[0392] 1H NMR (400MHz, DMSO-d6) δ8.32(d,J=9.1Hz,1H),8.07(d,J=7.4Hz,1H),7.69(s,1H),7.62(d,J=7.9Hz,1H),7.31-7.26(m,2H),6.85 -6.80(m,2H),5.47(d,J=5.0Hz,1H),5.36-5.31(m,1H),4.99(dd,J=5.2,3.2Hz,1H),4.77(d,J=7.3Hz,1H),4.52-4.43(m,2H),3.7 1(s,3H),3.40(dd,J=13.5,6.5Hz,1H),3.28(d,J=5.3Hz,2H),3.00(d,J=5.3Hz,1H),2.37(dd,J=14.6,4.9Hz,1H),2.17(td,J=8.5 ,3.3Hz,5H),1.74(p,J=7.5Hz,2H),1.39(s,3H),1.06(d,J=6.8Hz,3H),1.03(s,3H),0.42(q,J=3.8Hz,2H),0.28(q,J=3.8Hz,2H).

[0393] LCMS m / z = 623[M+1] + .

[0394] Example 32

[0395] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(cyclopropylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0396]

[0397] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with cyclopropylmethylamine.

[0398] LCMS m / z = 609[M+1] + .

[0399] Example 33

[0400] tert-Butyl(1-(((2-(((2S)-1-((1-(((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxypropyl-2-yl)amino)-1-oxypropyl-2-yl)amino)-3,4-dioxycyclobut-1-en-1-yl)amino)methyl)cyclobutyl)carbamate (threo-)

[0401]

[0402] Prepared according to the preparation method of Example 14, except that (tetrahydro-2H-pyran-4-yl)methylamine in step a) is replaced with tert-butyl (1-(aminomethyl)cyclobutyl)carbamate.

[0403] LCMS m / z = 738[M+1] + .

[0404] Example 34

[0405] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0406]

[0407] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) can be replaced with cyclopentylmethylamine to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-).

[0408] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated by chiral column, yielding peak -1, tr = 10.00 min and peak -2, tr = 19.73 min. (Separation method: Column: NB-Chiral NX(2), 50*4.6MM; Mobile phase A: Hex (10mMNH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40ml / min; UV 220nm).

[0409] Peak-1, tr = 10.00 min

[0410] 1 H NMR(400MHz, DMSO-d6)δ8.20(s,1H),7.94(d,J=7.6Hz,1H),7.56(d,J=17.1Hz,2H),7.30-7.10(m,2H),6.88-6.57(m, 2H),5.52(d,J=4.4Hz,1H),5.41(s,1H),4.91(t,J=4.0Hz,1H),4.78(s,1H),4.58(q,J=7.1Hz,1H),4.38(dd,J=9.1,3 .6Hz,1H),3.68(s,3H),3.54-3.39(m,2H),3.13(d,J=5.2Hz,1H),2.96(d,J=5.2Hz,1H),2.41(dd,J=14.7,5.7Hz,1H) ,2.34-2.13(m,5H),2.03(p,J=7.4Hz,1H),1.79(p,J=7.5Hz,2H),1.74-1.43(m,6H),1.35(s,3H),1.32-1.09(m,5H).

[0411] LCMS m / z = 637[M+1] + .

[0412] Peak-2, tr = 19.73 min

[0413] 1H NMR (400MHz, DMSO-d6) δ8.06(d,J=7.4Hz,1H),7.59(s,2H),7.28(d,J=8.6Hz,2H),6.99-6.67(m,2H),5.46 (d,J=4.9Hz,1H),5.34(s,1H),4.98(t,J=4.1Hz,1H),4.77(s,1H),4.61-4.36(m,2H),3.70(s,3H),3.44(q, J=7.5Hz,2H),3.28(d,J=5.3Hz,1H),3.00(d,J=5.3Hz,1H),2.43-2.30(m,1H),2.17(q,J=7.4,6.2Hz,5H), 2.01(p,J=7.4Hz,1H),1.84-1.43(m,8H),1.39(s,3H),1.18(dt,J=12.3,6.7Hz,3H),1.06(d,J=6.8Hz,3H).

[0414] LCMS m / z = 637[M+1] + .

[0415] Example 35

[0416] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0417]

[0418] Referring to the preparation method of Example 12, replace (5-methylfuran-2-yl)methylamine in step a) with 2-(aminomethyl)-N,N-dimethylaniline to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-).

[0419] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated using a chiral column, yielding peaks -1, tr = 10.90 min and -2, tr = 16.50 min. (Separation method: Column: CHIRAL ART Cellulose-SZ, 3*25cm; Mobile phase A: Hex (10mM NH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40 ml / min; UV 220 nm)

[0420] Peak-1, tr = 10.90 min

[0421] 1 H NMR (400MHz, DMSO-d6) δ8.22(s,1H),7.95(s,1H),7.69(d,J=43.8Hz,2H),7.27(t,J=7.8Hz,2H),7.20(dd,J=14.0,8. 1Hz,3H),7.07(d,J=7.2Hz,1H),6.73(d,J=8.7Hz,2H),5.52(d,J=4.2Hz,1H),5.40(s,1H),4.90(s,1H),4.81(s,2H),4 .57(d,J=5.7Hz,1H),4.38(d,J=5.8Hz,1H),3.67(s,3H),3.13(d,J=5.1Hz,1H),2.95(d,J=5.1Hz,1H),2.67(s,1H),2. 63(s,6H),2.40(s,2H),2.33(d,J=1.9Hz,1H),2.23(s,3H),1.78(t,J=7.7Hz,2H),1.35(s,3H),1.28(d,J=6.7Hz,3H).

[0422] LCMS m / z = 688[M+1] + .

[0423] Peak-2, tr = 16.50 min

[0424] 1 H NMR (400MHz, DMSO-d6) δ8.38-8.24(m,1H),8.05(s,1H),7.85-7.74(m,1H),7.70-7.58(m,1H),7.32-7. 24(m,4H),7.17(s,1H),7.06(d,J=7.2Hz,1H),6.82(d,J=8.4Hz,2H),5.47(s,1H),5.33(s,1H),4.98(s, 1H),4.78(s,2H),4.44(d,J=12.4Hz,2H),3.70(s,3H),3.28(d,J=4.6Hz,1H),2.99(d,J=5.0Hz,1H),2.6 7(s,1H),2.61(s,6H),2.33(s,3H),2.16(s,3H),1.78-1.68(m,2H),1.39(s,3H),1.05(d,J=7.2Hz,3H).

[0425] LCMS m / z = 688[M+1] + .

[0426] Example 36

[0427] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((2-cyclopropylbenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0428]

[0429] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (2-cyclopropylphenyl)methylamine.

[0430] LCMS m / z = 685[M+1] + .

[0431] Example 37

[0432] 1-(((2-(((2S)-1-((1-(((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxypropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)methyl)cyclobutane-1-carboxylic acid methyl ester (threo-)

[0433]

[0434] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with methyl 1-(aminomethyl)cyclobutane-1-carboxylic acid.

[0435] LCMS m / z = 681[M+1] + .

[0436] Example 38

[0437] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-((2-(pyrrolidine-1-yl)benzyl)amino)cyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0438]

[0439] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (2-(pyrrolidine-1-yl)phenyl)methylamine.

[0440] LCMS m / z = 714[M+1] + .

[0441] Example 39

[0442] 2-((S)-2-((2-((cyclohexylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0443]

[0444] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with cyclohexylmethylamine.

[0445] LCMS m / z = 651[M+1] + .

[0446] Example 40

[0447] 3-((((2S)-1-(1-(((S)-3-(cyclopent-1-en-1-yl)-1-(R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)amino)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-ylamino)-3,4-dioxocyclobut-1-en-1-ylamino)methyl)bicyclo[1.1.1]amyl formate (threo-)

[0448]

[0449] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with methyl 3-(aminomethyl)bicyclo[1.1.1]pentane-1-carboxylic acid.

[0450] LCMS m / z = 693[M+1] + .

[0451] Example 41

[0452] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-((2-((2-fluorobenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0453]

[0454] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (2-fluorophenyl)methylamine.

[0455] LCMS m / z = 663[M+1] + .

[0456] Example 42

[0457] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((2-fluoro-6-methylbenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)propionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0458]

[0459] Referring to the preparation method of Example 14, the (tetrahydro-2H-pyran-4-yl)methylamine in step a) can be replaced with (2-fluoro-6-methylphenyl)methylamine.

[0460] LCMS m / z = 677[M+1] + .

[0461] Example 43

[0462] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0463]

[0464] Referring to the preparation method of Example 12, the (5-methylfuran-2-yl)methylamine in step a) is replaced with cyclopentylmethylamine, and the L-alanine benzyl ester in step b) is replaced with serine benzyl ester, to prepare the N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide enantiomer (threo-).

[0465] The enantiomer of N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((cyclopentylmethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated by chiral column chromatography, yielding peak -1, tr = 10.90 min and peak -2, tr = 16.50 min. (Separation method: Column: CHIRAL ART Cellulose-SZ, 3*25cm; Mobile phase A: Hex (10mM NH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40 ml / min; UV 220 nm)

[0466] Peak-1, tr = 9.61 min

[0467] 1 H NMR (400MHz, DMSO-d6) δ8.14(d,J=9.1Hz,1H),7.99(d,J=7.4Hz,1H),7.63(d,J=22.8Hz,3H),7.32-7.16(m,2H),6.76(dd,J=8.6,1.7Hz ,2H),5.58(d,J=4.6Hz,1H),5.41(s,1H),5.29(s,1H),5.04(s,1H),4.78(s,1H),4.64-4.50(m,1H),4.48-4.27(m,1H),3.69(d,J=1.6Hz ,4H),3.57(t,J=6.8Hz,1H),3.45(d,J=7.0Hz,2H),3.20(d,J=5.2Hz,1H),2.98(d,J=5.3Hz,1H),2.40(d,J=12.5Hz,3H),2.21(d,J=21. 0Hz,5H),2.09-1.94(m,1H),1.81(q,J=7.4Hz,2H),1.75-1.61(m,2H),1.61-1.45(m,4H),1.37(d,J=1.6Hz,3H),1.22(d,J=15.6Hz,3H).

[0468] LCMS m / z = 653[M+1] + .

[0469] Peak-2, tr = 13.21 min

[0470] 1H NMR (400MHz, DMSO-d6) δ8.06(d,J=6.9Hz,1H),7.97(s,1H),7.70(s,2H),7.23(d,J=8.6Hz,2H),6.79(d,J=8.6Hz, 2H),5.56(s,1H),5.35(s,1H),5.10(s,1H),4.93(s,1H),4.73(s,1H),4.47(s,1H),4.40(d,J=5.3Hz,1H),3.71(s ,3H),3.45(dd,J=15.1,8.3Hz,5H),3.26(d,J=5.2Hz,2H),2.99(d,J=5.1Hz,1H),2.35(d,J=14.6Hz,2H),2.04(dd ,J=14.6,7.8Hz,1H),1.82-1.74(m,2H),1.68(s,2H),1.55(d,J=24.0Hz,5H),1.39(s,3H),1.22(d,J=13.2Hz,3H).

[0471] LCMS m / z = 653[M+1] + .

[0472] Example 44

[0473] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-((2-((2-fluorobenzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-)

[0474]

[0475] The preparation method of Example 14 can be used as follows, except that (tetrahydro-2H-pyran-4-yl)methylamine in step a) is replaced with (2-fluorophenyl)methylamine, and L-alanine benzyl ester in step b) is replaced with serine benzyl ester.

[0476] LCMS m / z = 679[M+1] + .

[0477] Example 45

[0478] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0479]

[0480] The preparation method of Example 12 is followed, except that (5-methylfuran-2-yl)methylamine in step a) is replaced with 2-(aminomethyl)-N,N-dimethylaniline, and L-alanine benzyl ester in step b) is replaced with serine benzyl ester. The resulting N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropamide)-3-hydroxy-3-(4-methoxyphenyl)propamide (threo-).

[0481] The enantiomeric N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((2-(2-(((dimethylamino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated by chiral column chromatography, yielding peak -1, tr = 11.50 min and peak -2, tr = 18.10 min. (Separation method: Column: Lux 5uCellulose-2, 30*250mm; Mobile phase A: Hex (10mM NH3-MeOH); Mobile phase B: EtOH; A:B = 70:30; Flow rate: 40ml / min; UV 280 / 290nm)

[0482] Peak-1, tr = 11.50 min

[0483] 11H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.88 (s, 1H), 7.69 (s, 1H), 7.30 - 7.22 (m, 4H), 7.17 (d, J = 7.5 Hz, 1H), 7.07 (td, J = 7.5, 1.1 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 5.58 (d, J = 4.7 Hz, 1H), 5.41 (s, 1H), 5.29 (t, J = 5.1 Hz, 1H), 5.08 - 4.99 (m, 1H), 4.80 (d, J = 4.2 Hz, 3H), 4.55 (dd, J = 13.6, 7.8 Hz, 1H), 4.38 (dd, J = 9.0, 2.8 Hz, 1H), 3.68 (s, 4H), 3.62 - 3.48 (m, 1H), 3.19 (d, J = 5.2 Hz, 1H), 2.97 (d, J = 5.2 Hz, 1H), 2.62 (s, 6H), 2.40 (dd, J = 14.4, 5.1 Hz, 1H), 2.26 - 2.20 (m, 3H), 1.87 - 1.73 (m, 2H), 1.36 (s, 3H), 1.30 - 1.12 (m, 2H).

[0484] LCMS m / z = 704.4 [M+1] + 。

[0485] Peak - 2, tr = 18.10 min

[0486] 1H NMR (400MHz, DMSO-d6) δ8.06(d,J=7.3Hz,1H),8.01(d,J=8.8Hz,1H),7.92(s,1H),7.78(s,1H),7.26(dd,J=17.5,8.1Hz,4H),7.17(d,J=7.9 Hz,1H),7.06(t,J=7.4Hz,1H),6.78(d,J=8.7Hz,2H),5.55(d,J=4.2Hz,1H),5.35(s,1H),5.11(s,1H),4.93(d,J=3.7Hz,1H),4.89-4.67(m,3 H),4.47(dd,J=12.8,8.1Hz,1H),4.41(dd,J=8.8,3.2Hz,1H),3.69(s,3H),3.50(dd,J=10.4,4.9Hz,1H),3.45-3.38(m,2H),3.25(d,J=5.2Hz ,1H),2.98(d,J=5.2Hz,1H),2.65(d,J=17.0Hz,6H),2.35(d,J=15.2Hz,1H),2.20-2.15(m,2H),1.82-1.68(m,2H),1.39(s,3H),1.24(s,2H).

[0487] LCMS m / z = 704.4 [M+1] + .

[0488] Example 46

[0489] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-(3,4-dioxo-2-(((trifluoromethyl)phenyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropamido-3-hydroxy-3-(4-methoxyphenyl)propionyl Amines and (2R,3S)-N-((S)-3-(cyclopent-1-enyl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropyl-2-yl)-2-((S)-2-(3,4-dioxo-2-(((trifluoromethyl)phenyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropamido-3-hydroxy-3-(4-methoxyphenyl)propamid

[0490]

[0491] Referring to the preparation method of Example 12, replace (5-methylfuran-2-yl)methylamine in step a) with (2-(trifluoromethyl)phenyl)methylamine, and replace L-alanine benzyl ester with serine benzyl ester in step b) to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-(3,4-dioxo-2-(((trifluoromethyl)phenyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropamido-3-hydroxy-3-(4-methoxyphenyl)propamido (threo-).

[0492] N-((S)-3-(cyclopent-1-enyl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxoprop-2-yl)-2-((S)-2-(3,4-dioxo-2-(((trifluoromethyl)phenyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropamido-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was prepared and separated by chiral column chromatography, yielding peak -1, tr = 8.505 min and peak -2, tr = 9.218 min. (Separation method: Column: X-Bridge Prep C) 18 19*250mm; Mobile phase A: H2O (0.1% FA); Mobile phase B: MeCN; A:B = 40:60; Flow rate: 25ml / min; UV 220nm

[0493] Peak-1, tr = 8.505 min

[0494] 11H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 8.6 Hz, 1H), 8.09 - 7.94 (m, 2H), 7.87 - 7.67 (m, 3H), 7.63 (d, J = 7.6 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.1 Hz, 2H), 6.77 (t, J = 9.0 Hz, 2H), 5.57 (t, J = 7.9 Hz, 1H), 5.38 (d, J = 25.4 Hz, 1H), 5.30 (t, J = 5.1 Hz, 1H), 5.05 (t, J = 14.4 Hz, 1H), 4.91 (s, 2H), 4.79 (s, 1H), 4.53 (dt, J = 20.3, 10.1 Hz, 1H), 4.46 - 4.33 (m, 1H), 3.68 (d, J = 7.9 Hz, 3H), 3.53 (ddd, J = 20.9, 11.1, 5.6 Hz, 1H), 3.43 - 3.35 (m, 1H), 3.25 (d, J = 5.3 Hz, 1H), 3.19 (d, J = 5.2 Hz, 1H), 2.98 (t, J = 4.4 Hz, 1H), 2.36 (dd, J = 35.5, 14.1 Hz, 2H), 2.25 - 2.16 (m, 3H), 1.84 - 1.69 (m, 2H), 1.37 (d, J = 9.5 Hz, 3H).

[0495] LCMS m / z = 729 [M+1] + 。

[0496] Peak - 2, tr = 9.218 min

[0497] 1H NMR (400MHz, DMSO-d6) δ8.06(dd,J=14.5,7.9Hz,4H),7.77(d,J=7.9Hz,1H),7.70(t,J=7.7Hz,1H),7.63(d,J=7.6Hz,1 H),7.54(t,J=7.5Hz,1H),7.24(t,J=7.7Hz,2H),6.76(t,J=8.1Hz,2H),5.57(t,J=7.2Hz,1H),5.34(s,1H),5.16(s,1H ),4.92(s,3H),4.74(s,1H),4.43(ddd,J=12.0,10.8,6.0Hz,2H),3.68(d,J=7.2Hz,3H),3.53-3.38(m,4H),3.25(d,J= 5.2Hz,1H),2.98(d,J=5.2Hz,1H),2.35(d,J=16.0Hz,1H),2.19-2.13(m,3H),1.78-1.69(m,2H),1.37(d,J=8.3Hz,3H).

[0498] LCMS m / z = 729[M+1] + .

[0499] Example 47

[0500] (2S,3R)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-(((3-(trifluoromethyl)pyridin-2-yl)methyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide and (2R,3S)-N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-(((3-(trifluoromethyl)pyridin-2-yl)methyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide

[0501]

[0502] Referring to the preparation method of Example 12, replace (5-methylfuran-2-yl)methylamine in step a) with (3-(trifluoromethyl)pyridin-2-yl)methylamine, and replace L-alanine benzyl ester in step b) with serine benzyl ester to prepare N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-(((3-(trifluoromethyl)pyridin-2-yl)methyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropamide)-3-hydroxy-3-(4-methoxyphenyl)propamide (threo-).

[0503] N-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methylepoxyethylene-2-yl)-1-oxopropane-2-yl)-2-((S)-2-((3,4-dioxo-2-(((3-(trifluoromethyl)pyridin-2-yl)methyl)amino)cyclobut-1-en-1-yl)amino)-3-hydroxypropionamide)-3-hydroxy-3-(4-methoxyphenyl)propionamide (threo-) was separated by chiral column chromatography, yielding peak -1,tr = 9.80 min and peak -2,tr = 12.50 min. (Separation method: Column: XBridge Prep OBD C18 Column 30*150mm; Mobile phase A: Water (10mmol / L NH4HCO3); Mobile phase B: MeCN; A:B=55:45; Flow rate: 60ml / min; UV 220nm)

[0504] Peak-1, tr = 9.80 min

[0505] 11H NMR (400 MHz, DMSO-d6) δ 8.87 (d, J = 4.0 Hz, 1H), 8.23 (d, J = 7.9 Hz, 2H), 8.11 (d, J = 8.9 Hz, 1H), 7.97 (t, J = 10.3 Hz, 2H), 7.61 (dd, J = 7.8, 5.0 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 5.57 (d, J = 4.7 Hz, 1H), 5.42 (s, 1H), 5.27 (t, J = 5.2 Hz, 1H), 5.20 - 4.99 (m, 3H), 4.82 (d, J = 7.5 Hz, 1H), 4.55 (dd, J = 13.6, 7.7 Hz, 1H), 4.39 (dd, J = 9.0, 2.7 Hz, 1H), 3.72 - 3.64 (m, 1H), 3.62 (s, 3H), 3.60 - 3.48 (m, 1H), 3.20 (d, J = 5.2 Hz, 1H), 2.98 (d, J = 5.2 Hz, 1H), 2.41 (dd, J = 14.4, 5.3 Hz, 1H), 2.31 - 2.17 (m, 5H), 1.80 (p, J = 7.5 Hz, 2H), 1.37 (s, 3H).

[0506] LCMS m / z = 730 [M+1] + 。

[0507] Peak - 2, tr = 12.50 min

[0508] 1H NMR (400MHz, DMSO-d6) δ8.87(d,J=4.4Hz,1H),8.29(s,1H),8.23(d,J=7.1Hz,1H),8.10-7.91(m,3H),7.60(dd,J=7.8,4.9Hz, 1H),7.24(d,J=8.7Hz,2H),6.76(t,J=9.2Hz,2H),5.54(d,J=4.6Hz,1H),5.34(s,1H),5.20-5.03(m,3H),4.97-4.90(m,1H),4 .75(s,1H),4.53-4.44(m,1H),4.41(dd,J=8.8,3.3Hz,1H),3.69(s,3H),3.51(dt,J=9.4,4.7Hz,1H),3.46-3.39(m,1H),3.26 (d,J=5.2Hz,2H),2.99(d,J=5.2Hz,1H),2.35(dd,J=16.5,3.2Hz,1H),2.20-2.12(m,4H),1.73(p,J=7.4Hz,2H),1.38(s,3H).

[0509] LCMS m / z = 730[M+1] + .

[0510] Part Two: Activity Assay

[0511] Experimental Example 1: Human 20S Immunoplasmosis LMP7 Subunit Activity Test Method

[0512] As previously described in Parlati et al. Blood (2009) 114:3439-3447, an ELISA-based technique, namely the proteasome constitutive / immunoproteasome subunit enzyme-linked immunosorbent assay (ProCISE), is used to quantitatively assess subunit-specific activity. This assay is used to assess inhibitory activity against LMP7. The specific steps are as follows:

[0513] 1. Human 20S Immunoproteasome Reaction Buffer: 10 mM HEPES (Gibco; 15630-106)

[0514] 2. Preparation and addition of human 20S immunoproteasome: Prepare 20S human immunoproteasome working solution (final concentration 0.1 ng / μL, South Bay Bio, SBB-PP0004) using reaction buffer. Add 10 μL of human 20S immunoproteasome working solution to each well of a 384-well plate using an automated dispensing system (BioTek, MultiFlo FX). Add an equal volume of reaction buffer to the blank control group (i.e., enzyme-free control group).

[0515] 3. Compound Preparation and Loading: The compounds and positive control compounds prepared in the examples were diluted from 10 mM to 1 mM using DMSO (Sigma, D4540-500 mL). The compounds were then loaded using a compound titrator (Tecan, D300e). The concentrations of the compounds and positive control compounds were set as follows: an initial concentration of 3 μM, with eight concentrations set in a 3-fold gradient. The negative control group was added with DMSO (final concentration 0.3%). After centrifugation at 2500 rpm for 30 s, the mixtures were incubated at 25°C for 30 min.

[0516] 4. Preparation and addition of detection reagents: The human 20S immunoproteasome LMP7 subunit was inhibited using Proteasome-Glo TM Assays Kit (Promega; G8622) Suc-LLVY-Glo TM The substrate solution was used for detection. 10 μL of uc-LLVY-Glo was added to each well of the detection plate. TM The substrate solution (final concentration 20 μM) was centrifuged at 2500 rpm for 30 s and then incubated at 25 °C for 30 min.

[0517] 5. The fluorescence value of each well was detected using the Luminescence module of a Decan (SPARK) microplate reader. A dose-response curve of drug concentration and inhibition rate was fitted using GraphPad Prism5 software: log(inhibitor) vs. response-variable slope, to obtain the IC50 of the compound's inhibitory effect on the human 20S immunoproteasome LMP7 subunit activity. 50 value.

[0518] 6. Formula for calculating inhibition rate:

[0519]

[0520] ν i : Compound group luminescence readings

[0521] ν o : Negative control group luminescence readings

[0522] S b : Luminescence readings of the blank control group

[0523] Positive compound: KZR-616

[0524] The activity assay data for the relevant compounds are detailed in the table below:

[0525] Example IC50(nM) Example IC50(nM) 2 156.6 31-1 157.1 3 85.1 34-1 32.8 4 92.1 35-1 29.5 11 171.6 38 126.6 13-1 31.8 39 74.6 18-1 82.7 41 15.5 20 45.0 43-1 16.7 21-1 26.3 44 38.3 24-1 51.9 45-1 23.4 27 99.3 46-1 23.4 28 130.4 46-2 107.7 29-1 38.2 47-1 29.8

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: Where R 1 For A—(L)n—; L is C 1-3 Alkyl group, A is selected from: C 1-6 Alkylene-R 10 C 2-6 imide-R 10 OC 1-6 Alkylene-R 10 C 0-6 Alkylene N(R) 11 2. Aryl, heteroaryl, cycloalkyl, heterocyclic or C 3-6 Cycloalkenyl, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 SR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic groups for substitution; R 2 R 3 R 5 R 7 and R 9 Independently selected from hydrogen or C 1-3 Alkyl groups; R 4 Selected from hydrogen, halogen, hydroxyl, C 1-6 alkyl groups, wherein the C 1-6 The alkyl group is optionally surrounded by one or more halogens, hydroxyl groups, or C. 1-3 Alkyl substitution; R 6 and R 8 Independently selected from C 1-3 Alkylene—G, wherein G is selected from: C 1-6 alkyl, C 3-7 cycloalkenyl, aryl or heteroaryl, wherein the C 1-6 alkyl, C 3-7 Cycloalkenyl, aryl, and heteroaryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be substituted with one or more halogens or hydroxyl groups; R 10 Selected from H, CF3, OR 11 Or aryl; X is selected from O or NR 11 ; R 11 Selected from H or C 1-6 Alkyl groups; and n is selected from 0, 1, 2 or 3.

2. The compound according to claim 1, wherein, n is 0 or n is 1.

3. The compound according to claim 1, wherein, A is selected from: C 1-6 Alkylene-R 10 C 6-10 Aryl, 5-14 heteroaryl, C 3-8 Cycloalkyl or 3-10-membered heterocyclic group, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or substituted with 3-7 membered heterocyclic alkyl groups; Preferably, A is selected from: C 1-6 Alkylene-R 10 C 6-10 Aryl, 5-6 quinone heteroaryl, C 3-8 Cycloalkyl or 3-6 membered heterocyclic group, wherein A is optionally selected from one or more groups chosen from C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or substituted with 3-7 membered heterocyclic alkyl groups; Preferably, A is selected from: methyl, ethyl, propyl, isopropyl, butyl, neopentyl, phenyl, naphthyl, indene, oxazolyl, isoxazolyl, 1,2,4-oxadiazolyl, thiazolyl, furanyl, tetrahydrofuranyl, pyrroleyl, thiopheneyl, pyrazolyl, imidazoleyl, pyridyl, pyranyl, pyrimidinyl, pyrazinyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.2]octyl, morpholinyl, piperazineyl, oxacyclobutyl, ethylene oxide, or cyclothioethane, wherein A is optionally selected from one or more of C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or substituted with 3-7 membered heterocyclic alkyl groups; Preferably, A is selected from: neopentyl, phenyl, 1,2,4-oxadiazolyl, thiazolyl, furanyl, pyridinyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, bicyclo[2.2.2]octyl, oxacyclobutyl, morpholinyl, or tetrahydro-2H-pyranyl, wherein A is optionally selected by one or more of C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or substituted with 3-7 membered heterocyclic alkyl groups; Preferably, A is selected from: phenyl, pyridyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein A is optionally selected from one or more of C. 1-6 Alkyl, halogen, CF3, OR 11 、N(R 11 2. NHR 11 C(O)OR 11 Or 3-7 membered heterocyclic alkyl substitution.

4. The compound according to claim 1, R 1 Selected from: Preferred, R 1 Selected from:

5. The compound according to claim 1, wherein, R 2 R 3 R 5 and R 7 It is hydrogen; R 9 It is methyl; R 4 Selected from C 1-6 alkyl groups, wherein the C 1-6 The alkyl group is optionally surrounded by one or more halogens, hydroxyl groups, or C. 1-3 Alkyl substitution; Preferred, R 4 The methyl group is methyl, wherein the methyl group is optionally converted by one or more halogens, hydroxyl groups, or C. 1-3 Alkyl substitution; Preferred, R 4 It is either methyl or -CH2-OH; R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: aryl, wherein the aryl group may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be substituted with one or more halogens or hydroxyl groups; Preferred, R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: aryl, wherein the aryl group may optionally be converted by one or more ORs. 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more hydroxyl groups; Preferred, R 6 Selected from C 1-3 Alkylene—G, wherein G is selected from: phenyl, wherein the phenyl group may optionally be converted by one or more ORs. 11 Replace, where C 1-3 The alkylene group may optionally be replaced by one or more hydroxyl groups; Preferred, R 6 Selected from R 8 Selected from C 1-3 Alkylene—G, wherein G is selected from: C 1-6 alkyl, C 3-7 Cycloalkenyl or C 3-7 Aryl, wherein the C 1-6 alkyl, C 3-7 The cycloalkenyl and aryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be substituted with one or more halogens or hydroxyl groups; Preferred, R 8 Selected from C 1-3 Alkylene—G, wherein G is selected from: isopropyl, cyclopentene, or phenyl, wherein C 1-6 alkyl, C 3-7 The cycloalkenyl and aryl groups may optionally be converted by one or more halogens, OR 11 C 1-3 Alkyl or SO2R 11 Replace, where C 1-3 The alkylene group may optionally be substituted with one or more halogens or hydroxyl groups; Preferred, R 8 Selected from C 1-3 Alkylene—G, wherein G is selected from: isopropyl, cyclopentene or phenyl; Preferred, R 8 Selected from 6. A compound of formula (II), or a pharmaceutically acceptable salt thereof: in, R 1 R 4 and R 8 As defined in compound I, R 12 Selected from hydrogen, halogen, or hydroxyl, preferably, R 12 Selected from hydrogen or hydroxyl.

7. A compound of formula (III), or a pharmaceutically acceptable salt thereof: in, R 1 R 4 and R 8 As defined in compound I, R 12 Selected from hydrogen, halogen, or hydroxyl, preferably, R 12 Selected from hydrogen or hydroxyl.

8. Compounds with the following structures, or pharmaceutically acceptable salts thereof:

9. Compounds with the following structures, or pharmaceutically acceptable salts thereof:

10. Compounds with the following structures, or pharmaceutically acceptable salts thereof:

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