Novel compounds and their use

Abstract

This specification defines a compound of formula (I) as defined herein, JPEG2026527672000078.jpg54170In formula, R a , R b , R c Compounds having the meanings given herein, A, and p, as well as pharmaceutically acceptable salts and solvates thereof, are provided, which are useful in the treatment of diseases in which total immunoproteasome inhibition is desirable or required, particularly in the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to novel compounds, compositions comprising such compounds, and the use of such compounds and compositions in medicine. In particular, this invention relates to the use of such compounds and compositions in methods for the treatment or prevention of diseases or conditions in which total immunoproteasome inhibition is desirable or required, such as proliferative disorders and autoimmune diseases. [Background technology]

[0002] Proteasomes are essential for maintaining protein homeostasis and are involved in various important cellular processes, including cell division, cell signaling, and antigen processing (Coux, O.; Tanaka, K.; Goldberg, AL, Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 1996, 65, 801-47). In mammals, the major types of proteasomes are constitutive proteasomes, which are present in all cells throughout the body, and immunoproteasomes, which are mainly expressed in hematopoietic cells (Kniepert, A.; Groettrup, M., The unique functions of tissue-specific proteasomes. Trends in biochemical sciences 2014, 39(1), 17-24). Both constitutive proteasomes and immunoproteasomes possess a 20S core particle (CP; cCP in the case of constitutive proteasomes, iCP in the case of immunoproteasomes), which is composed of 14 α-type subunit proteins and 14 β-type subunit proteins, α 1-7 β 1-7 β 1-7 α 1-7The arrangement in this configuration forms a hollow cylindrical structure. The β1, β2, and β5 subunits possess protease activity and hydrolyze polypeptides supplied to the CP barrel into oligopeptides of 8 to 10 amino acids. The main structural difference between cCP and iCP lies in the catalytic active sites and their substrate binding channels. cCP incorporates the β1c (caspase-like activity (CL)), β2c (trypsin-like activity (TL)), and β5c (chymotrypsin / elastase-like activity (ChT-L activity)) subunits, whereas in iCP, these are replaced by β1i (ChT-L), β2i (TL), and β5i (ChT-L), respectively.

[0003] Research into proteasome inhibition began in the early 1990s, leading to the approval of bortezomib (2003), carfilzomib (2012), and ixazomib (2015) as treatments for multiple myeloma (Manasanch, EE; Orlowski, RZ, Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol 2017, 14(7), 417-433). These compounds primarily target β5c of cCP and β5i of iCP, respectively. However, depending on the dose, they can also inhibit other catalytic activities simultaneously. Constitutive proteasomes are present in all healthy tissues and are classified as off-target when considering proteasome inhibition from a clinical perspective. Selective inhibition of immunoproteasomes has been explored over the past decade to minimize side effects, reduce cytotoxicity, and explore potential anti-inflammatory applications. However, it has become clear that co-inhibition of multiple subunits is necessary to obtain appropriate therapeutic effects (Johnson, HWB; et al., Required Immuno-proteasome Subunit Inhibition Profile for Anti-Inflammatory Efficacy and Clinical Candidate KZR-616((2S,3R)-N-((S)-3-(Cyclo-pent-1-en-1-yl)-1-((R)-2-methyl-oxiran-2-yl)-1-oxo-propan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholino-acetamido)propanamido)-propenamide).J Med Chem 2018,61(24),11127-11143.).

[0004] Proteasome inhibitors such as carfilzomib and bortezomib have been shown to induce resistance pathways, and as a result, proteasome inhibition as a treatment is known to be ineffective in these hematological malignancies. One possibility for overcoming the resistance observed in hematological malignancies and the cytotoxicity caused by constitutive proteasome inhibition is to develop proteasome inhibitors that inhibit only the immunoproteasome, which are total immunoproteasome inhibitors.

[0005] While compounds targeting at least two immune subunits with good affinity have already been reported (ONX0914 (Muchamuel, T.; et al., A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat. Med. 2009, 15(7), 781-7), KZR-616 (Johnson, HWB; et al., ibid.), and LU-005i (see below)), a true global immunoproteasome inhibitor that inhibits all three immunoproteasome subunits with equally high potency while remaining inactive on the constitutive subunits does not yet exist. [ka] Surprisingly, it has now been discovered that certain compounds can act as immunoproteasome inhibitors, particularly global immunoproteasome inhibitors, which may be useful in the treatment or prevention of diseases or conditions where global immunoproteasome inhibition is desirable or necessary, such as proliferative disorders and autoimmune diseases.

[0006] Any list or discussion of previously published documents in this specification should not be construed as an endorsement that such documents are part of cutting-edge technology or common general knowledge. [Overview of the project]

Means for Solving the Problem

[0007] In a first aspect of the present invention, a compound of formula (I),

Chemical formula

[0008] The compounds of formula (I), including their pharmaceutically acceptable salts and solvates, are referred to herein as "compounds of the present invention."

[0009] To avoid any doubt, those skilled in the art will understand that any reference herein to a particular embodiment of the present invention (such as the first embodiment of the present invention, i.e., a compound of formula (I) as defined in the first embodiment of this specification) includes references to all embodiments and their specific features, and that further embodiments and features of the present invention may be formed by combining those embodiments and specific features.

[0010] Examples of pharmaceutically acceptable salts include acid addition salts and base salts. Such salts may be formed by conventional means, for example, by reacting the free acid or free base form of the compound of the present invention with one equivalent or more of a suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (e.g., by lyophilization under reduced pressure or by filtration). Salts may also be prepared using techniques known to those skilled in the art, for example, by exchanging the counterions of the compound of the present invention in salt form with other counterions using a suitable ion exchange resin.

[0011] Examples of specific acid addition salts include carboxylates (for example, formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, caprate, caprylate, stearate, acrylate, caproate, propiolate, ascorbate, citrate, glucuronate, glutamate, glycolate, gluconate, α-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate) Examples include salts (severate, sebacate, fumarate, malate, maleate, hydroxymaleate, hyperinate, phthalate or terephthalate), halide salts (e.g., chloride salts, bromide salts or iodide salts), sulfonates (e.g., benzenesulfonate, methyl-, bromo-, or chlorobenzenesulfonate, xylenesulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, hydroxyethanesulfonate, 1- or 2-naphthalenesulfonate, or 1,5-naphthalenedisulfonate), or sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, or nitrates.

[0012] Specific examples of base salts include those formed from alkali metals (such as Na and K salts), alkaline earth metals (such as Mg and Ca salts), organic bases (such as dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, and lysine), and inorganic bases (such as ammonia and aluminum hydroxide). More specifically, examples of base addition salts include Mg salts, Ca salts, and especially K salts and Na salts.

[0013] Specific examples of salts include trifluoroacetate and dicyclohexylamine salts.

[0014] To avoid any doubt, it should be added that the compounds of the present invention may exist as solids, and therefore the scope of the invention includes all of their amorphous, crystalline, and partially crystalline forms, and may also exist as oils. When the compounds of the present invention exist in crystalline and partially crystalline forms, such forms may include solvates, which are also included in the scope of the invention.

[0015] A "solvate" refers to a solid form in which a related compound (for example, the compound of formula (I)) is bonded with one or more solvent molecules. The term solvate includes hydrates and other solvates of pharmaceutically acceptable solvents. Water and DMSO are preferred solvents for solvate formation.

[0016] To avoid any doubt, it should be added that the compounds of the present invention may also exist in solution (i.e., in solution in a suitable solvent). For example, the compounds of the present invention may exist in aqueous solution, in which case the compounds of the present invention may exist in the form of their hydrates.

[0017] The compounds of the present invention may contain double bonds and, therefore, unless otherwise indicated, may exist as E (entgegen) and Z (zusammen) geometric isomers for each individual double bond. Unless otherwise specified, all such isomers and mixtures thereof are included within the scope of the present invention.

[0018] The compounds of the present invention may also exhibit tautomerism. All tautomers and mixtures thereof (in particular those that are stable enough to allow their isolation) are included within the scope of the present invention.

[0019] "Amino acids" and "residues" (for example, phenylalanine "residues") refer to the dehydrated portions of amino acids present in a polypeptide chain, represented by the following formula. [ka] In the formula, SC represents the amino acid side chain. To avoid any ambiguity, the term "amino acid" includes non-protein amino acids unless otherwise specified.

[0020] "Amino acid side chain" or "side chain of an amino acid" refers to the group attached to the carboxyl group and amino group at the α-position in an α-amino acid, and includes non-proteinogenic α-amino acids, and especially proteinogenic amino acids. Those skilled in the art will understand that the most common natural amino acids are known by their common names and will be aware of the side chain groups present in these amino acids.

[0021] "Protein" amino acids are the 22 amino acids that can be naturally encoded or found in the genetic code of any organism. "Non-protein" amino acids are those that are not naturally encoded or found in the genetic code of any organism. The set of non-protein amino acids is generally considered to include all organic compounds in which an amine (-NH2) functional group and a carboxylic acid (-COOH) functional group are linked via a single additional carbon atom, to which side chains and hydrogens are attached, but excludes selenocysteine, pyrrolicin, and the 20 standard amino acids that are incorporated into proteins during translation. Non-protein amino acids include amino acids that are intermediates in biosynthesis, amino acids that are produced post-translation in proteins, and amino acids that have physiological roles (e.g., components of bacterial cell walls, neurotransmitters, toxins, etc.).

[0022] The compounds of the present invention may also contain at least one chiral carbon atom and therefore may exhibit optical isomerism and / or diastereoisomerism (i.e., may exist in the form of enantiomers or diastereomers). Diastereomers may be separated using conventional techniques, such as chromatography or fractional crystallization. Various stereoisomers (i.e., enantiomers) may be isolated by separating racemic or other mixtures of the compound using conventional techniques, such as fractional crystallization or HPLC. Alternatively, the desired enantiomer or diastereomer may be obtained from a appropriately optically active starting material (i.e., the "chiral pool" method) under conditions that will not cause racemization or epimerization, by derivatization (i.e., decomposition including dynamic decomposition, e.g., treatment with a homochiral acid followed by separation of the diastereomer derivative by conventional means such as chromatography) by reaction with a suitable starting material and a "chiral auxiliary" which can then be removed in a suitable step, or by reaction with a suitable chiral reagent or chiral catalyst, all of which may be carried out under conditions known to those skilled in the art. Unless otherwise specified, all stereoisomers and mixtures thereof are included within the scope of the present invention.

[0023] Unless otherwise specified, C as defined herein 1-z Alkyl groups (where z is the upper limit of the range) may be linear, branched, and / or cyclic (and thus C) if there are a sufficient number of carbon atoms (i.e., at least two or three as needed). 3-z (to form a cycloalkyl group). If a sufficient number (i.e., at least four) carbon atoms are present, such a group is also a subcyclic (and therefore C 4-zThey may also form a partial cycloalkyl group. For example, possible cycloalkyl groups include cyclopropyl, cyclopentyl, and cyclohexyl. Similarly, a possible partial cyclic alkyl group (which may also be called a “partially cycloalkyl” group) is cyclopropylmethyl. To avoid any doubt, specific alkyl groups that may be listed include linear (i.e., not branched and / or cyclic) alkyl groups.

[0024] Unless otherwise specified, C as defined herein 2-z The alkenyl group (where z is the upper limit of the range) may be linear, branched if there are a sufficient number of carbon atoms (i.e., at least 3), and / or cyclic (and therefore C 4-z (They may also form a cycloalkenyl group.) If there are a sufficient number of carbon atoms (i.e., at least five), such a group may also be a subcyclic. To avoid any doubt, specific examples of alkenyl groups include straight-chain (i.e., not branched and / or cyclic) alkenyl groups.

[0025] As used herein, the term aryl means C 6-14 (For example, C6- 10 ) This may refer to an aromatic group. Such a group may be monocyclic or bicyclic, and if bicyclic, it may be entirely or partially aromatic. C 6-10 Examples of aryl groups include phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, and indanyl (for example, phenyl, naphthyl, etc.). To avoid any ambiguity, it should be added that the substituent on the aryl group may be attached via any suitable carbon atom of the ring system.

[0026] To avoid any doubt, it should be added that those skilled in the art will understand that the aryl groups that can form part of the compounds of the present invention are chemically obtainable, as is known to those skilled in the art. Specific examples of aryl groups include phenyl and naphthyl.

[0027] The present invention also includes isotope-labeled compounds of the present invention in which one or more atoms are actually replaced by atoms having atomic masses or mass numbers different from those commonly found in nature (or most abundantly found in nature), but which are identical to those described herein. All isotopes of any particular atom or element specified herein are intended to be within the range of compounds of the present invention. Accordingly, the compounds of the present invention also include deuterium compounds, i.e., compounds of the present invention in which one or more hydrogen atoms are replaced by hydrogen isotopes of mass.

[0028] To avoid any doubt, it should be added that if two or more substituents in the compound of the present invention may be identical, the actual identity of each substituent is never interdependent. For example, two or more Z 1 In situations where the element exists, those Z 1 The bases may be the same or different. Similarly, two or more Zs 2 There are groups, each being -C(O)-C 1-4 When representing alkyl, the problematic -C(O)-C 1-4 The alkyl groups may be the same or different.

[0029] Furthermore, to avoid any ambiguity, where terms such as "1-4" are used herein, it will be understood by those skilled in the art to mean 1, 2, 3, and 4 (including both ends). Unless otherwise specified, the same reasoning will apply to other terms used herein.

[0030] Furthermore, to avoid any ambiguity, it should be added that the substituent itself is explicitly stated to be optionally substituted by one or more substituents (for example, -Z 1 -Z2 C is optionally substituted by one or more groups independently selected from 1-6 These substituents (alkyl) may, if possible, be located on the same or different atoms. Such optional substituents may be present in any preferred number (for example, the group in question may be substituted with one or more such substituents, such as one such substituent).

[0031] To avoid any doubt, if a group is referred to herein as being optionally substituted, it is particularly intended that such optional substituents may be absent (i.e., references to such optional substituents may be omitted), in which case the optionally substituted group may be referred to as unsubstituted.

[0032] To avoid any doubt, it should be added that those skilled in the art will understand that the compounds of the present invention, which are the subject of this invention, include those that are readily available, i.e., those that can be prepared in a stable form. That is, the compounds of the present invention include compounds that are robust enough to survive isolation, for example, isolation from a reaction mixture to a useful purity.

[0033] In the compound of formula (I), the following portion of the molecule may be called the P1' portion (or P1' region, etc.). [ka]

[0034] In a particular embodiment (i.e., a particular embodiment of the first aspect of the present invention), R b represents H. To avoid any ambiguity, R a In such compounds, -[CH2] m -X represents X

[0035] In another embodiment of the present invention, R a or R b (especially R) a ) is -[CH2] m-X represents, and m is 0 or 1. In certain embodiments, m is 0.

[0036] In a further embodiment, X represents -OC(O)-Y, -C(O)-Y, or -OY. In particular, X may represent -OC(O)-Y. Each of the foregoing groups is R a or R b may form part of, but preferably forms part of R a . In such compounds, it is also preferred that m is 0.

[0037] In a further embodiment, Y represents -C 3-5 alkyl (optionally substituted by -Z 1 -Z 2 ) or -CH(R y )-(Z 1 -Z 2 ) n .

[0038] Specific compounds of the present invention include those containing an amino acid (or a protected derivative thereof) as part of R a or R b (preferably R a ). Such compounds include those in which X represents -OC(O)-Y, -C(O)-Y, or OY, Y represents -CH(R y )-(Z 1 -Z 2 ), and -Z 1 - represents -NH- or -N(R z )-. Thus, in certain embodiments, the R a or R b portion may terminate with an amino acid group or a protected derivative thereof. These compounds of formula (I) may alternatively have one of R a and R b represent H (preferably R b is H), and the other represents either -[CH2] m -O-T or -[CH2] m -T, where T is an oxygen atom or [CH 2]mIt can be defined as a compound representing an amino acid group or a protected derivative thereof bonded to a base. T structurally is -C(O)-CH(R y )-NH-Z 2 or -C(O)-CH(R y )-N(R z )-Z 2 and can be represented by any of them. Thus, such a compound where -Z 2 is -C(O)O-tert-butyl is a Boc-protected amino acid. Other protecting groups known to those skilled in the art for the protection of amines may alternatively be used for -Z 2 , for example, carbobenzyloxy (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bzl), and benzyl (Bn).

[0039] R y represents a methylbiphenyl or the side chain of a proteinogenic amino acid, optionally in a form where the side chain is chemically protected. Amino acid side chains containing a primary amine or amide functional group (for example, the side chain of lysine) can be protected using any one of Boc, Cbz, Fmoc, Ac, Bzl, and Bn, or other suitable protecting groups known to those skilled in the art such as allyloxycarbonyl (Alloc). Side chains containing a carboxylate group (for example, the side chain of aspartic acid or glutamic acid) can be protected by conversion to an ester (for example, Bn ester, trityl (Trt) ester, C 1-6 alkyl ester, fluorenylmethyl (Fm) ester or silyl ester) or other suitable protecting groups known to those skilled in the art. Side chains containing an alcohol functional group (for example, the side chain of serine or threonine) are C 1-4Alkyl ethers, Bn, Ac, Bzl, triphenylmethyl (Trt), tetrahydropyranyl (Thp), and silyl ethers (such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), and tert-butyldimethylsilyl (TBDMS)), or other suitable protecting groups known to those skilled in the art may be protected. Side chains containing thiol functional groups (e.g., the side chain of cysteine) may be protected with Trt, Bn, tert-butyl, or other suitable protecting groups known to those skilled in the art. Side chains containing guanidine / guanidinium functional groups (e.g., the side chain of arginine) may be protected with Cbz and 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf), or other suitable protecting groups known to those skilled in the art.

[0040] In a particular embodiment, R y This represents a methyl biphenyl (BipA side chain), a protein amino acid side chain, or a chemically protected form of a protein amino acid side chain, the chemical protection involving the attachment of a moiety selected from the group consisting of benzoyl, benzyl, and trityl groups.

[0041] A preferred compound of the present invention is one in which n is 1. Other preferred compounds are one in which m is 0 or 1, and n is optionally 1.

[0042] In the compound of formula (I), R c The position where R is bonded can be referred to as the "P3" position. In particular, when the ring at A is relatively small (i.e., when it contains almost no substituents other than hydrogen), structural changes at the P3 position are generally well tolerated. Therefore, in the compound of the present invention, R c -C is optionally substituted with H or one or more Q substituents. 1-4 It can represent alkyl, and Q is -OR d , -NHR e , or -C(O)NHR f Represents R d , Re , and R f However, H, -C 1-4 Alkyl (optionally substituted with phenyl or methylphenyl), -C(O)-C 1-4 Alkyl, -C(O)OC 1-4 Alkyl, -C(O)-C 1-4 Alkenyl, -C(O)OC 1-4 It can represent an alkenyl, -C(=N)-NH2, -C(=N)-NH- (protecting group), or pyrimidinyl. The types of protecting groups described above for the P1' position can also be applied to the P3 position. In particular, these include Cbz and Pbf when Q represents a guanidinyl group.

[0043] R c Specific groups that may be mentioned in relation to Q include hydrogen bond acceptors (for example, those containing an N or O atom with at least one lone pair of electrons). In one embodiment, Q represents -NH2, -NH-C(O)-CH3, -NH(Alloc), -NH-C(=N)-NH2, -NH-C(=N)-NH-(protecting group), -C(O)-NH2, -OH, -O-benzyl, -O-xylyl, or -NH(pyrimidinyl).

[0044] R c Specific groups that can be mentioned in relation to this include the side chains of proteinogenic amino acids and their protective derivatives. Side chains containing at least one hydrogen bond acceptor or their protective derivatives have been shown to be particularly effective, with oxygen-containing side chains (and their protective derivatives) exhibiting the greatest efficacy. Therefore, in another embodiment, R c is -C substituted with one Q substituent 1-3 Q represents alkyl, and Q is -OH and -OC 1-4 Selected from the group consisting of alkyl groups (optionally substituted with phenyl or methylphenyl).

[0045] Compounds that have been shown to be particularly effective as pan-immunoproteasome inhibitors include R cSome of these represent the side chain of serine or its protective derivatives. Protected derivatives of serine that can be cited in this context include serine protected with Bn, ethyl, or propyl, and the total immunoproteasome efficacy of compounds containing serine protected with P3 is such that this is a ring lacking a bulky side chain at the A position (i.e., R h or R i It has been found that the effect is enhanced when combined with H, -OH, or -CH3.

[0046] R c The compounds of the present invention, in which methyl is represented, have also been found to be effective as panimmunoproteasome inhibitors.

[0047] In the compound of formula (I), the position where ring A is attached can be called the "P4" position. Compounds in which ring A contains a Boc substituent have been shown to have enhanced global immunoproteasome activity. While we do not wish to be bound by theory, substituents containing a tert-butyl group can undergo hydrophobic interactions at target sites due to their bulky and hydrophobic properties, and therefore, especially when the substituent is located at the 4th position of the 6-membered ring represented by A in formula (I), it is thought to show similar potential.

[0048] R c In other embodiments, such as when representing a protected amino acid side chain, ring A does not have a bulky side chain (i.e., R h and R i (where represents H, -OH, or -CH3).

[0049] Specific examples of group A include morpholinyl, 4-methylpiperidinyl, 4-(t-butyloxycarbonyl)-piperidinyl, piperidinyl, and 4-hydroxycyclohexyl. In one embodiment, A represents a morpholinyl group.

[0050] R cThe carbon atom to which the compound is bonded is a chiral center. The compounds of the present invention may have either a D configuration or an L configuration at that position, or may relate to a mixture of both configurations at that position. Compounds of formula (I) in which the P3 region corresponds to a D-amino acid are thought to have particularly improved β5i selectivity.

[0051] The compounds of the present invention include other chiral centers, including the binding site of an essential cyclohexylmethyl group ("P1" position) and the binding site of an essential O-methyltyrosine side chain ("P2" position). Thus, the P1 and P2 regions represent covalently bonded amino acids. The compounds of the present invention may independently have either a D configuration or an L configuration at each position, or they may relate to a mixture of both configurations at each position.

[0052] It is preferable that both the P1 and P2 regions have the L configuration. References to the “L configuration” herein (including elsewhere) include a product in which substantially no compounds having one or more D configurations in that region exist (and vice versa). Those skilled in the art will understand that absolute purity is impossible in this regard. Therefore, the present invention relates to compounds in which, with respect to any given chiral center, the enantiomer excess with respect to the desired enantiomer is at least 50%, preferably at least 80%, for example, at least 90%.

[0053] Specific compounds of the present invention that may be exemplified include compounds and their pharmaceutically acceptable salts as described in the examples provided herein. Therefore, specific compounds of the present invention include: [ka] [ka] [ka] [ka] [ka] [ka] [ka] Furthermore, there are pharmaceutically acceptable salts of these.

[0054] medical use As described herein, the compounds of the present invention and compositions containing them are useful as pharmaceuticals.

[0055] Accordingly, according to a second aspect of the present invention, compounds of the present invention as defined herein above (i.e., compounds as defined in the first aspect of the present invention, including all embodiments and specific features thereof) are provided for use as pharmaceuticals (or for use in medical care).

[0056] To avoid any doubt, references to compounds defined in the first embodiment of the present invention include references to compounds of formula (I) (including all embodiments thereof) and their pharmaceutically acceptable salts and solvates.

[0057] The compounds of the present invention may have pharmacological activity on their own, or certain pharmaceutically acceptable (e.g., “protective”) derivatives of the compounds of the present invention, which may not have such activity, may be present or prepared, and may be administered parenterally or orally and subsequently metabolized in the body to form the compounds of the present invention. Accordingly, such compounds (which may have some pharmacological activity, but such activity is considerably lower than that of the active compound after metabolism) may be described as “prodrugs” of the compounds of the present invention.

[0058] As used herein, a reference to a prodrug will include a compound that forms an experimentally detectable amount of the compound of the present invention within a predetermined time after enteral or parenteral administration (e.g., oral or parenteral administration). All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0059] Furthermore, certain compounds of the present invention may have no pharmacological activity or only minimal pharmacological activity on their own, but may be administered parenterally or orally and subsequently metabolized in the body to form compounds of the present invention that have pharmacological activity on their own. Therefore, such compounds (which may have some pharmacological activity, but such activity is considerably lower than that of the active compounds of the present invention from which they are metabolized) may be described as “prodrugs.”

[0060] To avoid any doubt, it should be added that the compounds of the present invention are useful because they have pharmacological activity and / or are metabolized in the body after oral or parenteral administration to form compounds that have pharmacological activity.

[0061] As described herein, the compounds of the present invention may be particularly useful for the treatment and / or prevention of diseases or conditions in which total immunoproteasome inhibition is desirable or required. Accordingly, in a third aspect of the present invention, the compounds of the present invention as defined above are provided for use in the treatment or prevention of diseases or conditions in which total immunoproteasome inhibition is desirable or required. Use in the treatment of diseases or conditions in which total immunoproteasome inhibition is desirable or required is particularly preferred.

[0062] In an alternative third embodiment of the present invention, a method is provided for treating or preventing a disease or condition in which total immunoproteasome inhibition is desirable or required, the method comprising administering a therapeutically effective amount of the compound of the present invention, as defined above, to a patient in need thereof.

[0063] In a further alternative third embodiment of the present invention, the use of the compounds of the present invention as defined above is provided for the manufacture of pharmaceuticals for the treatment or prevention of diseases or conditions in which total immunoproteasome inhibition is desired or required.

[0064] Those skilled in the art will understand that references to the treatment of a particular condition (or, similarly, the act of treating that condition) have their usual meanings in the medical field. In particular, the term may refer to achieving a reduction in the severity and / or frequency of one or more clinical symptoms associated with a condition, as determined by a physician examining a patient who has or is susceptible to such symptoms. For example, in the case of multiple myeloma, it may refer to an extension of the patient's progression-free survival.

[0065] As used herein, the terms prevention (and similarly, the act of prevention) include references to prophylaxis of disease or disorder (and vice versa). Thus, references to prevention may also include references to prophylaxis, and vice versa. In particular, such terms may refer to achieving a reduction in the likelihood that a patient (or a healthy subject) will develop a disease (e.g., a reduction of at least 10%, e.g., a reduction of at least 20%, 30%, or 40%, e.g., a reduction of at least 50%). (This is understood to mean that the patient's condition changes and the patient is diagnosed by a physician, for example, with the relevant disease or disorder and is deemed to require treatment).

[0066] As used herein, references to a patient (or more patients) refer to a living subject receiving treatment, including mammalian patients (e.g., humans). In particular, references to a patient refer to a human patient. To avoid any doubt, the compounds of the present invention may also be used for the treatment of non-human animals.

[0067] Those skilled in the art will understand that such treatment or prevention should be performed for patients (or subjects) who require it. The need for such treatment or prevention for a patient (or subject) can be assessed by those skilled in the art using routine techniques. In this specification, references to patients requiring prophylactic therapy also include references to patients who are susceptible to diseases or conditions for which total immunoproteasome inhibition is desirable or required, but who have not currently been diagnosed with such a disease.

[0068] As used herein, the terms disease and disorder may be used interchangeably.

[0069] As used herein, the term “effective dose” refers to the amount of a compound that confers a therapeutic effect to a patient receiving treatment. Such effect may be observed in an objective manner (i.e., measurable by several tests or markers) or subjective manner (i.e., the subject suggests and / or feels such effect). In particular, such effect may be observed in an objective manner (e.g., by measurement) using appropriate tests known to those skilled in the art.

[0070] In a particular embodiment (i.e., a particular embodiment of a third aspect of the present invention), the disease or condition is a hematological malignancy, a solid tumor, an autoimmune disease, or an inflammatory disease.

[0071] As described herein, compounds of the first aspect of the present invention may be particularly useful in hematological malignancies selected from the group consisting of leukemia, lymphoma, myeloma (including multiple myeloma), myelodysplastic syndromes, and myeloproliferative syndromes.

[0072] Those skilled in the art will understand that references to solid cancers that can be treated or prevented with the compounds of the present invention include prostate cancer, breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, osteosarcoma, and colitis-related cancers.

[0073] Certain inflammatory diseases that can be treated or prevented using the compounds of the present invention include encephalitis, viral myocarditis, inflammatory bowel disease, arthritis, polymyositis, dermatomyositis, autoimmune hepatitis, and lupus nephritis.

[0074] The compounds of the present invention may also be used to treat or prevent diseases or conditions selected from the group consisting of Alzheimer's disease, angiogenesis, acute kidney injury, ischemic stroke, premature birth, abdominal aortic aneurysm, atherosclerosis, cardiac remodeling, and graft-versus-host disease (GvHD).

[0075] Pharmaceutical composition As described herein, the compounds of the present invention are useful as pharmaceuticals. Such compounds may be administered alone or via known pharmaceutical compositions / formulations.

[0076] In a fourth aspect of the present invention, a pharmaceutical composition is provided comprising a compound of the present invention as defined herein and one or more pharmaceutically acceptable excipients of an optional nature.

[0077] As used herein, the term pharmaceutically acceptable excipient includes references to vehicles, adjuvants, carriers, diluents, pH adjusters, and buffers, tonicity adjusters, stabilizers, wetting agents, and the like. In particular, such excipients may include adjuvants, diluents, or carriers.

[0078] To avoid any doubt, it should be added that any reference herein to the compounds of the present invention for specific uses (as well as, similarly, to uses and methods of use relating to the compounds of the present invention) may also be applied to pharmaceutical compositions comprising the compounds of the present invention as described herein.

[0079] Accordingly, a fifth aspect of the present invention is provided, a pharmaceutical composition as defined in the fourth aspect of the present invention, used for the treatment or prevention of a disease or condition in which total immunoproteasome inhibition is desirable or required (as defined herein by reference to the third aspect of the present invention and all its embodiments).

[0080] Those skilled in the art will understand that the compounds of the present invention may act systemically and / or topically (i.e., on specific sites) and therefore may be administered as appropriate using suitable techniques known to those skilled in the art.

[0081] Those skilled in the art will understand that the compounds and compositions described herein are typically administered in pharmaceutically acceptable dosage forms by oral, intravenous, subcutaneous, buccal, rectal, skin, nasal cavity, trachea, bronchi, sublingual, intranasal, topical, any other parenteral route, or by inhalation.

[0082] The pharmaceutical compositions described herein may include compositions in the form of tablets, capsules, or elixirs for oral administration, suppositories for rectal administration, or sterile solutions or suspensions for parenteral or intramuscular administration. Alternatively, the pharmaceutical compositions may be formulated for topical administration, particularly when such compounds of the present invention act topically.

[0083] Accordingly, in certain embodiments, the pharmaceutical formulations are provided in pharmaceutically acceptable dosage forms, including tablets or capsules, liquids administered orally or by injection, suppositories, creams, gels, effervescent agents, inhalants (e.g., applied intranasally), or forms suitable for topical administration. To avoid doubt, in such embodiments, the compounds of the present invention may exist in other forms, such as solids (e.g., solid dispersions), liquids (e.g., in solutions), or micelles.

[0084] For example, in the preparation of pharmaceutical formulations for oral administration, the compound may be mixed with a solid powder component such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, or another suitable component, and with disintegrants and lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, and polyethylene glycol wax. The mixture may then be processed into granules or compressed into tablets.

[0085] Soft gelatin capsules can be prepared using capsules containing one or more active compounds (e.g., compounds of the first, and by extension, second and third embodiments of the present invention, and optionally additional therapeutic agents) together with, for example, vegetable oil, fat, or other vehicles suitable for soft gelatin capsules. Similarly, hard gelatin capsules can contain such compounds(s) in combination with solid powder components such as lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin.

[0086] Dosage units for rectal administration may be prepared in the form of (i) suppositories containing the compound(s) mixed with a neutral lipid base, (ii) gelatin rectal capsules containing the active substance in a mixture with vegetable oil, paraffin oil, or other vehicle suitable for gelatin rectal capsules, (iii) ready-made microenemas, or (iv) dried microenemas that are reconstituted in a suitable solvent immediately before administration.

[0087] Liquid preparations for oral administration may be prepared in the form of a solution or suspension containing, for example, a syrup or suspension, the compound(s), a sugar or sugar alcohol, and the remainder of a formulation consisting of a mixture of ethanol, water, glycerol, propylene glycol, and polyethylene glycol. If desired, such liquid preparations may contain colorants, flavorings, saccharin, and carboxymethylcellulose or other thickeners. Liquid preparations for oral administration may also be prepared in the form of a dry powder that is reconstituted with a suitable solvent before use.

[0088] Solutions for parenteral administration may be prepared as a solution of the compound(s) in a pharmaceutically acceptable solvent. These solutions may also contain stabilizing and / or buffering components and are dispensed into unit doses in the form of ampoules or vials. Solutions for parenteral administration may also be prepared as a dry preparation that is reconstituted improvisationally with a suitable solvent before use.

[0089] For example, depending on the efficacy and physical properties of the compound (i.e., the active ingredient) of the present invention, the pharmaceutical formulations that can be listed include those in which the active ingredient is present in an amount of at least 1% by weight (or at least 10% by weight, at least 30% by weight, or at least 50% by weight). That is, the ratio of the active ingredient to the other components of the pharmaceutical composition (i.e., the addition of adjuvants, diluents, and carriers) is at least 1:99 by weight (or at least 10:90, at least 30:70, or at least 50:50).

[0090] Those skilled in the art will understand that the compounds of the present invention may be administered in variable doses (for example, as the formulations described above), and that preferred doses can be easily determined by those skilled in the art. Oral and topical doses (as well as subcutaneous doses, although these doses may be relatively lower) may range from about 0.01 μg / kg body weight / day (μg / kg / day) to about 200 μg / kg / day, preferably about 0.01 to about 10 μg / kg / day, and more preferably about 0.1 to about 5.0 μg / kg / day. For example, when administered orally, treatment with such compounds may involve the administration of formulations typically containing about 0.01 μg to about 2000 mg, for example, about 0.1 μg to about 500 mg, or 1 μg to about 100 mg (for example, about 20 μg to about 80 mg) of the active ingredient(s). For intravenous administration, the most preferred dose is in the range of about 0.001 to about 10 μg / kg / hour during constant-rate infusion. Advantageously, treatment may involve administering such compounds and compositions once daily, or the total daily dose may be divided into two, three, or four doses per day (for example, twice daily in relation to the doses described herein, e.g., doses of 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, or 200 mg twice daily).

[0091] Where used herein in relation to a specific value (e.g., a quantity), the term “about” (or similar terms such as “approximately”) is understood to indicate that such value may vary by up to 10% of the defined value (in particular, up to 5%, e.g., up to 1%). In each case, such term may be replaced by notation such as “±10%” (or by indicating the variation of a particular quantity calculated based on the relevant value). In each case, it is also intended that such term may be deleted.

[0092] To avoid any doubt, it should be added that a person skilled in the art (e.g., a physician) can determine the most appropriate actual dosage for an individual patient, which is likely to vary depending on the route of administration, the type and severity of the condition being treated, as well as the species, age, weight, sex, renal function, hepatic function, and response of the particular patient being treated. The above dosages are examples for the average case, and naturally, there may be individual cases where a higher or lower dosage range is appropriate, and such doses are within the scope of the present invention.

[0093] Preparation of compounds / compositions Such formulations may be prepared in accordance with standard and / or acceptable pharmaceutical practices.

[0094] Accordingly, in a further aspect of the present invention, a process for preparing a pharmaceutical formulation is provided, as defined above, which includes associating a compound of the present invention with one or more pharmaceutically acceptable excipients, as defined above.

[0095] As used herein, the reference to “bringing into association” means that the two components are made suitable for concomitant administration with respect to one another.

[0096] The compounds of the present invention described herein may be prepared according to techniques known to those skilled in the art, as described in the examples provided below.

[0097] According to a sixth aspect of the present invention, a process for preparing the compounds of the present invention as defined above, comprising the step of reacting a compound of formula (II) with a compound of formula (III), [ka] In equation (II), A, p and R c However, as defined above, [ka] In equation (III), Y is as defined above, The above step is carried out in the presence of a suitable coupling reagent (for example, O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 1,1′-carbonyldiimidazole, N,N′-dicyclohexylcarbodiimide, or similar), under standard conditions known to those skilled in the art (for example, optionally, in the presence of a suitable solvent, a suitable base, and / or in an inert atmosphere), under reaction conditions known to be used in, for example, the Steglig esterification reaction.

[0098] The compounds of formulas (II) and (III) can be obtained from readily available starting materials using appropriate reagents and reaction conditions, either commercially available, known in the literature, or by analogy using the processes described herein, or by conventional synthetic procedures, according to standard techniques. In this regard, those skilled in the art may refer in particular to "Comprehensive Organic Synthesis" by BMTrost and I. Fleming, Pergamon Press, 1991. Further references include "Heterocyclic Chemistry," 3rd edition, by J.A. Joule, K. Mills and G.F. Smith, published by Chapman & Hall; "Comprehensive Heterocyclic Chemistry II," by A.A. Katritzky, C.W. Reees and E.F. V. Criven, Pergamon Press, 1996; and "Science of Synthesis," vols. 9-17 (Hetarenes and Related Ring Systems), Georg Thieme Verlag, 2006.

[0099] In particular, the compound of formula (III) may be prepared by reacting the compound of formula (IV) with the compound of formula (V). [ka] In equation (IV), A, p, and R c This is defined as above, [ka] The compound of formula (IV) is first reacted with hydrazine hydrate under suitable conditions known to those skilled in the art, such as in the presence of a polar solvent, to form a peptide hydrazide, and then further reacted with tert-butyl nitrite in a non-aqueous solvent under suitable conditions such as low temperature before contact with the compound of formula (V).

[0100] Other specific transformation steps that may be used (including those that may be employed to form the compound of formula (I)) include the following: (i) Oxidation, for example, oxidizing a moiety containing an alkene group (e.g., -CH=CH2) to an epoxide in the presence of a suitable oxidizing agent such as MnO2 or mcpba. (ii) Formation of amides. For example, by a reaction between an acid chloride and an amine, or by an amide coupling reaction, i.e., a reaction that forms an amide from a carboxylic acid (or its ester), for example, -C(O)OH (or its ester) to -C(O)N(R 1 )R 2 Base (here, R 1 and R 2Reactions to convert the compound to a hydrogen or carbon-containing group include, for example, in the case of -COOH, the reaction may be carried out in the presence of a suitable coupling reagent (e.g., O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, 1,1′-carbonyldiimidazole, N,N′-dicyclohexylcarbodiimide, or similar), or in the case of an ester (e.g., -C(O)OCH3, or -C(O)OCH2CH3), the reaction may be carried out in the presence of trimethylaluminum, for example, alternatively, the -C(O)OH group may be first activated to the corresponding acyl halide (e.g., -C(O)Cl) by treating it with, for example, oxalyl chloride, thionyl chloride, phosphorus pentachloride, phosphorus oxychloride, or similar, and in either case, the compound is of the chemical formula HN(R 1 )R 2 (Here, R 1 and R 2 The compound (as defined above) is reacted with the compound under standard conditions known to those skilled in the art (for example, optionally, in the presence of a suitable solvent, a suitable base, and / or in an inert atmosphere). (iii) Converting the methoxy group to a hydroxyl group by reaction in the presence of a suitable reagent, such as a boron fluoride-dimethyl sulfide complex or BBr3 (for example, in the presence of a suitable solvent such as dichloromethane). (iv) Alkylation or acylation reactions that can be carried out in the presence of a base and a solvent (such as those described herein). (v) Specific deprotection steps. For example, deprotection of an N-Boc protecting group by reaction in the presence of an acid, or a hydroxyl group protected as a silyl ether (e.g., a tert-butyl-dimethylsilyl protecting group) can be deprotected by reaction with a fluoride ion source, for example, by the use of the reagent tetrabutylammonium fluoride (TBAF).

[0101] Similarly, the compounds of formula (IV) and formula (V) can be obtained from readily available starting materials using appropriate reagents and reaction conditions, either commercially available, known in the literature, or by analogy using the processes described herein, or by conventional synthetic procedures, according to standard techniques.

[0102] Those skilled in the art will understand that the substituents as defined herein, and substituents on them, can be modified one or more times after or during the above-described process for the preparation of the compounds of the present invention by methods known to those skilled in the art. Examples of such methods include substitution, reduction, oxidation, dehydrogenation, alkylation, dealkylation, acylation, hydrolysis, esterification, etherification, halogenation, and nitration. The precursor group can be changed at any point in the reaction sequence to a different such group or to the group defined by formula (I). Those skilled in the art may also refer to “Comprehensive Organic Functional Group Transformations” by AR Katritzky, O. Meth-Cohn and CWRees, Pergamon Press, 1995, and / or “Comprehensive Organic Transformations” by RC Larock, Wiley-VCH, 1999.

[0103] The compounds of the present invention can be isolated from the reaction mixture and, if necessary, purified using prior art known to those skilled in the art. Accordingly, the process for preparing the compounds of the present invention described herein may, as a final step, include isolation and, optionally, purification of the compounds of the present invention.

[0104] Those skilled in the art will understand that in the processes described above and below, it may be necessary to protect the functional groups of the intermediate compound with protecting groups. Protection and deprotection of functional groups may be carried out before or after the reaction in the scheme described above.

[0105] Protecting groups can be applied and removed according to techniques known to those skilled in the art and techniques such as those described below. For example, the protected compounds / intermediates described herein can be chemically converted to unprotected compounds using standard deprotection techniques. The type of chemical reaction involved will determine the need and type of protecting group, as well as the sequence to achieve the synthesis. The use of protecting groups is fully described in "Protective Groups in Organic Synthesis," 3rd edition, TW Greene & P. ​​G.M. Wutz, Wiley-Interscience (1999), the contents of which are incorporated herein by reference.

[0106] While we do not wish to be constrained by theory, the compounds of the present invention have an inhibitory profile that is more favorable to iCP compared to LU-005i, and therefore are considered to have potential as global immunoproteasome selective inhibitors. By inhibiting only the immunoproteasome, global immunoproteasome inhibitors may be able to overcome the resistance observed in hematological cancers and the cytotoxicity caused by constitutive proteasome inhibition.

[0107] The compounds of the present invention may have advantages over compounds known in the prior art, whether for use in the indications described above, such as being more potent, less toxic, longer-acting, more potent, having fewer side effects, being more readily absorbed, and / or having a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), as well as / or other useful pharmacological, physical, or chemical properties. In particular, the compounds of the present invention may have the advantage of being more potent and / or exhibiting advantageous properties in vivo. [Brief explanation of the drawing]

[0108] [Figure 1] The inhibition profiles of P1′N-Boc amino acids and P1′ amino acids (2-24) in the LU-005i(1) skeleton are shown as an IC50 (μM) heatmap. [Figure 2] The inhibition profiles of P1′N-Boc esters (25-27) in the LU-005i(1) skeleton are shown as an IC50 (μM) heatmap. [Figure 3] The inhibition profiles of P3-P4 skeleton screening (28-57) based on the LU-005i(1) skeleton are shown as an IC50 (μM) heatmap. [Figure 4] The inhibition profiles of secondary P3-P4 screening (29, 30, 34, 35, 58-73) based on the LU-005i(1) framework are shown as an IC50 (μM) heatmap. [Figure 5] For a series of compounds (2, 3, 14, 65, 68, 74-76) with mutations at the P3 and P1′ positions, the inhibition profiles in Raji lysates are shown as heatmaps of IC50 (μM). [Figure 6] For a series of compounds (2, 3, 14, 65, 68, 74-76) with mutations at the P3 and P1′ positions, the inhibition profiles in the AMO native lysate are shown as an IC50 (μM) heatmap.

[0109] To avoid any ambiguity, it should be added that the numbers used in the figure descriptions refer to the compound numbers of the examples described herein. [Examples]

[0110] The present invention will be further described with reference to the following embodiments, which are not intended to limit the scope of the invention.

[0111] In the event of any inconsistency between the nomenclature and any compound depicted in the figures, the latter shall prevail (unless it contradicts any experimental details that may be provided and / or is evident from the context). The starting materials and intermediates used in the synthesis of the compounds described herein are commercially available or can be prepared by the methods described herein or by methods known in the art.

[0112] Commercially available reagents and solvents were used as received. Reactions sensitive to H2O and oxygen were carried out under an N2 atmosphere. Solvents used in synthesis were dried as needed and stored on 4 Å molecular sieves. However, pyridine, DIPEA, and TEA were stored on KOH pellets. TLC analysis was performed using aluminum sheets pre-coated with silica gel (Merck, TLC silica gel 60 F254). Compounds were analyzed by UV absorption (λ=254 nm) or by ninhydrin (50 g / L in n-butanol) or cerium molybdate (25 g / L in 10% H2SO4 aqueous solution ((NH4)6Mo7O) 24 The reaction was visualized by spraying with 10 g / L NH4Ce(SO4)4·H2O) and, if necessary, by carbonization at 150°C. Column chromatography was performed using silica gel from Screening Devices bv (particle size 40-63 μm, pore size 60 Å). If necessary, a Celite Highflow Supercell (Merck) was used for impregnation of the reaction mixture before silica gel chromatography. 1H, 13C APT, 1H Cosy, and HSQC spectra were recorded using a brucker AV-400 (400 / 100 MHz) and AV-500 (500 / 125 MHz) spectrometer. Chemical shifts were reported as ∂ values ​​(ppm), relative to TMS (∂=0.00 ppm) or residual solvent peaks. J-bond constants were reported in Hz. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using a Finnigan Surveyor HPLC system equipped with a Nucleodur C18 Gravity 3μm 50×4.60 mm column (detection wavelength 200–500 nm), connected to either a Finnigan LCQ Advantage Max mass spectrometer with ESI or a Thermo LCQ Fleet ion mass spectrometer with ESI. Typical conditions ranged from 10–90%, with a total runtime of 13.5 minutes.

[0113] High-resolution mass spectra were acquired using a mass spectrometer equipped with a positive-ion mode electrospray ion source (Q Exactive HF hybrid quadrupole-Orbitrap) by direct injection (H2O / MeCN=1:1, 1.0 μM solution in 0.1% formic acid) (source voltage 3.5 kV, sheath gas flow rate 10, capillary temperature 275 °C). Resolution was set to R=240,000 at m / z 400 (mass range m / z=160-2000), and an external lock mass was used. The high-resolution mass spectrometer was calibrated using a calibration mixture (Thermo Finnigan) before measurement.

[0114] In synthesis referring to general procedures, reaction conditions (such as reaction length or temperature) may vary. Generally, the reaction was tracked by thin-layer chromatography or LC-MS, and workup was performed where appropriate. Purification may vary between experiments. Generally, the solvent and the ratio of solvent used for elution / gradient were selected to provide appropriate Rf and / or retention times.

[0115] General procedure General chemical synthesis of LU-005i-OH and structural variants: Epoxy ketone 83 was prepared as follows (Scheme 1). Weinreb amide 78 was obtained by condensation of the Weinreb salt with N-Boc cyclohexylalanine 77 using HCTU. Dimethylmethyl phosphonate was deprotonated using n-BuLi to generate a phosphorus ylide, which was then added to Weinreb amide 78 to form phosphonate 79. In a two-step one-pot reaction, phosphonate 83 was deprotonated to form a phosphorus ylide intermediate. This intermediate then underwent a Horner-Wadsworth-Emmons reaction with formaldehyde to produce enone intermediate 80. This intermediate 80 immediately underwent a Baylis-Hillman type reaction with formaldehyde to form allyl alcohol 81. Next, allyl alcohol 81 was epoxidized using hydrogen peroxide by nucleophilic epoxidation, and after separation of the diastereomer, epoxy ketone 82 was obtained with the desired stereochemistry. Next, the N-Boc protecting group in 82 was removed using TFA in DCM, resulting in the epoxy ketone construction block 83. [ka] Scheme 1. Synthesis of epoxy ketone 83. Reagents and conditions: (a) HCl·NMeOMe, HCTU, DiPEA, DCM, rt, 91%; (b) Dimethylmethylphosphonate, n-BuLi, THF, -78°C; (c) Formaldehyde, K2CO3, H2O / MeOH, 0°C, 51%; (d) H2O2, DiPEA, benzonitrile, 0°C, 29%; (e) TFA, DCM, rt, quantitative.

[0116] Next, both the phenolic alcohol and carboxylate of N-Boc tyrosine were methylated using iodomethane and potassium carbonate to form 4-methoxyphenylalanine ester 85 (Scheme 2). Then, the N-Boc protecting group of 4-methoxyphenylalanine ester 85 was removed, and the resulting amine was condensed with N-Boc alanine using HCTU to form dipeptide 86. Following N-Boc deprotection of 86 (treatment with TFA), the NH2-dipeptide intermediate was condensed with 2-morpholinoacetic acid to obtain methyl ester 87. Methyl ester 87 was converted to acyl hydrazide 88, which was then converted to acyl azide 89 using tert-butyl nitrite under acidic anhydride conditions. Subsequently, acyl azide 89 was reacted with NH2-epoxy ketone 83 to form LU-005i-OH2. Next, the OH group in compound 2 was functionalized by Steglig esterification reactions with a wide range of carboxylic acids to obtain a library of esterified LU-005i-OH derivatives 3-13 and 25-26. Finally, the N-Boc and O-TBS protecting groups of compounds 2-12 and 26 were removed by treatment with TFA to obtain NH2-amino acid ester derivatives 14-24 and OH-lactic acid ester derivative 27. [ka] Scheme 2. Synthesis of LU-005i-OH2 and esterified LU-005i-OH derivatives 3-27. Reagents and conditions: (a) MeI, K2CO3, DMF rt, quantitative; (b) i: TFA, DCM, rt, ii: NH2-Ala-OMe, HCTU, DiPEA, DCM, rt, 91%; (c) i: TFA, DCM, ii: Morpholinoacetic acid, HCTU, DiPEA, DCM, rt, 80%; (d) Hydrazine hydrate, MeOH, rt; (e) i: tBuONO, HCl, DMF, -30°C, ii: 83, DiPEA, DMF, -30°C->rt, 69%; (f) RCO2H, DIC, DMAP, DCM, rt, 34-99%; (g) TFA, DCM, rt.

[0117] Procedure A: Boc Deprotection The Boc-protected compound was dissolved in anhydrous DCM (0.3M), and TFA was added to this solution in a TFA:DCM ratio of 1:4 (v:v). The reaction mixture was stirred for 1-2 hours and monitored by TLC. After complete conversion, the reaction mixture was concentrated under reduced pressure and evaporated three times with toluene.

[0118] Procedure B: Esterification LU-005i-OH was dissolved in anhydrous DCM (0.2 M) and purged with N2. DIC (2.0 equivalents), amino acids (1.1 equivalents), and DMAP (1.1 equivalents) were added to this solution. The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the title compound was obtained by silica gel column chromatography using DCM:MeOH.

[0119] Boc-Tyr(OMe)-OMe(90) [ka] Boc-Tyr(OMe)-OH (5 g, 16.9 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (0.1 M), purged with N2, and cooled to 0°C. MeI (1.27 ml, 20.3 mmol, 1.2 equivalents) and K2CO3 (3.27 g, 23.66 mmol, 1.5 equivalents) were added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with SiO2, washed with NaS2O3 (saturated aqueous solution), washed with H2O and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, filtered, and concentrated under reduced pressure. The title compound was obtained without further purification (6.06 g, 19.6 mmol, quantitative). In LC-MS (straight gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 mins), Rt (min): 6.53 mins was observed (ESI-MS (m / z): 310.16 (M+H + ))

[0120] H2N-Tyr(OMe)-OMe(85) [ka] Deprotection of Boc-Tyr(OMe)-OMe was carried out according to Procedure A on a 19.6 mmol scale, yielding the title compound as a white solid (5.49 g, 19.6 mmol, quantitative). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 6.25 min (ESI-MS(m / z): 210.11(M+H) + ))

[0121] Boc-Ala-Tyr(OMe)-OMe(86) [ka] H2N-Tyr(OMe)-OMe (4.10 g, 19.6 mmol, 1.0 equivalent) was evaporated twice with toluene, then dissolved in anhydrous DMF (0.2 M) and purged with N2. To this solution, HCTU (12.16 g, 29.4 mmol, 1.5 equivalents) and Boc-Ala-OH (7.42 g, 39.2 mmol, 2.0 equivalents) were added. Once completely dissolved, DiPEA (13.7 ml, 78.4 mmol, 4.0 equivalents) was added dropwise to the reaction mixture and stirred for 16 hours. The reaction mixture was washed with HCl (1.0 M aqueous solution), NaHCO3 (saturated aqueous solution) and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, and concentrated under reduced pressure. The title compound was obtained as a colorless oil by silica gel column chromatography (0:100 → 2:100 MeOH:DCM) (6.79 g, 17.8 mmol, 91%). f :2:100 MeOH: 0.2 under DCM conditions. 1 H NMR(400MHz,CDCl3)δ7.05-7.00(m,2H),6.90(d,J=8.0Hz,1H),6.82-6.78(m,2H),5.39(d,J=7.6Hz,1H),4.79(q,J=6 .4,1H),4.26-4.18(m,1H),3.75(s,3H),3,68(s,3H),3.04(qd,J=14.0,6.0,2H),1.43(s,9H),1.30(d,J=7.1Hz,3H). 13¹³C NMR (101MHz, CDCl3) δ172.5, 171.8, 158.5, 155.3, 130.2, 127.7, 113.8, 79.7, 55.0, 53.3, 52.1, 49.9, 36.9, 28.2, 18.3. LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 6.71 min (ESI-MS (m / z): 380.80 (M+H + ))

[0122] H2N-Ala-Tyr(OMe)-OMe(91) [ka] Deprotection of Boc-Ala-Tyr(OMe)-OMe was carried out on an mmol scale according to procedure A, yielding the title compound as a yellow oily substance (2.24 g, 8.0 mmol, quantitative). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.03 min (ESI-MS(m / z): 281.00(M+H + ))

[0123] Morph-Ala-Tyr(OMe)-OMe(87) [ka] NH2-Ala-Tyr(OMe)-OMe (2.24 g, 8.0 mmol, 1.0 equivalent) was evaporated twice with toluene, then dissolved in anhydrous DMF (0.2 M) and purged with N2. To this solution, PyBOP (4.03 g, 9.6 mmol, 1.2 equivalents) and Morph-COOH (1.28 g, 8.8 mmol, 1.1 equivalents) were added. Once completely dissolved, DiPEA (4.89 ml, 28.0 mmol, 3.5 equivalents) was added dropwise to the reaction mixture and stirred for 16 hours. The reaction mixture was washed with HCl (1.0 M aqueous solution), NaHCO3 (saturated aqueous solution) and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, and concentrated under reduced pressure. The title compound was obtained as a yellowish oily substance by silica gel column chromatography (0:100 → 2:100 MeOH:DCM) (2.61 g, 6.4 mmol, 80%). f :2:100 MeOH: 0.3 under DCM conditions. 1 H NMR(400MHz,CDCl3)δ7.55(t,J=7.9Hz,1H),7.11-6.93(m,2H),6.88-6.75(m,2H),6.68(d,J=7.8Hz,1H),4.77(ddd,J=7.9,6.7,5.5,1H) ,4.51(p,J=7.1Hz,1H),3.77(s,3H),3.73(s,3H),3.70(t,J=4.7Hz,4H),3.14-2.89(m,4H),2.49(t,J=4.6Hz,4H),1.36(d,J=7.0Hz,3H). 13 ¹³C NMR (101MHz, CDCl3) δ171.9, 171.9, 169.9, 158.7, 130.3, 127.7, 114.0, 66.9, 61.7, 55.3, 53.8, 53.5, 52.5, 36.9, 18.2. LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.29 min (ESI-MS (m / z): 408.07 (M+H + ))

[0124] Morph-Ala-Tyr(OMe)-NHNH2(88) [ka] Morph-Ala-Tyr(OMe)-OMe (0.8 g, 2.0 mmol, 1.0 equivalent) was dissolved in MeOH (0.1 M), and hydrazine hydrate (65% by mass, 1.88 ml, 60.0 mmol, 30.0 equivalents) was added to the solution. The resulting reaction mixture was stirred under reduced pressure for 16 hours. The resulting reaction mixture was concentrated under reduced pressure and evaporated three times with toluene. A white solid was obtained, which was further used as the crude product for the next reaction. LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt (min): 3.03 min (ESI-MS (m / z): 408.20 (M+H + ))

[0125] Boc-Cha-N(OCH3)CH3(78) [ka] Boc-Cha-OH (10.0 g, 36.9 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (0.1 M) and purged with N2. To this solution, HCTU (22.87 g, 55.3 mmol, 1.5 equivalents) and N,O,-dimethylhydroxylamine (7.19 g, 73.7 mmol, 2.0 equivalents) were added. Once the reagents were completely dissolved, DiPEA (25.7 ml, 147 mmol, 4.0 equivalents) was added dropwise, and the resulting yellow reaction mixture was stirred for 16 hours. The reaction mixture was then acidified with HCl (1.0 M aqueous solution), washed with HCl (1.0 M aqueous solution), washed with NaHCO3 (saturated aqueous solution) and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, and concentrated under reduced pressure. The title compound was obtained as a clear oily substance by silica gel column chromatography (1:20 → 1:4 ¼:PE) (10.43 g, 33.2 mmol, 90%). f :1:4 siRNA:0.3 under PE conditions. 1H NMR(400MHz,CDCl3)δ5.02(d,J=9.2Hz,1H),4.87-4.55(m,1H),3.78(s,3H),3.20(s,3H),1.91(d,J=12.6 Hz,1H),1.82-1.55(m,5H),1.54-1.46(m,1H),1.45-1.32(m,10H),1.31-1.06(m,3H),1.04-0.82(m,2H). 13 ¹³C NMR (101MHz, CDCl3) δ155.82, 155.79, 79.62, 61.74, 48.47, 40.67, 34.19, 34.12, 32.36, 32.28, 28.50, 26.62, 26.42, 26.20. LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 8.40 min (ESI-MS (m / z): 314.80 (M+H + ))

[0126] Boc-Cha-CH2-P(=O)OMe(OMe)(79) [ka] Dimethylmethylphosphonate (7.48 ml, 70 mmol, 4.0 equivalents) was dissolved in anhydrous THF (0.1 M), purged with N2, and cooled to -78°C. n-BuLi (2.5 M in hexane, 28.0 ml, 70.0 mmol, 4.0 equivalents) was added dropwise to the solution. The resulting reaction mixture was stirred at -78°C under N2 for 2 hours. Boc-Cha-N(OCH3)CH3 (5.5 g, 17.5 mmol, 1.0 equivalent) was evaporated twice with toluene, dissolved in anhydrous THF, and purged with N2. The THF solution containing Boc-Cha-N(OCH3)CH3 was added dropwise to the phosphorus ylide-containing solution. The resulting reaction mixture was stirred at -78°C under an N2 atmosphere for 3 hours. Next, the reaction was quenched with NH4Cl (100 ml, saturated aqueous solution), and the reaction mixture was subsequently heated to room temperature. Next, the reaction mixture was diluted with RINKAN, and the resulting organic and aqueous layers were separated. The aqueous layer was extracted three times with RINKAN, and the combined RINKAN was washed with H2O and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, and concentrated under reduced pressure. The resulting crude oil was used in the next reaction without further purification. 1 H NMR(400MHz,CDCl3)δ5.24(d,J=26.3Hz,1H),4.36(d,J=11.5Hz,1H),3.80(d,J= 3.9Hz,3H),3.78(d,J=3.8Hz,4H),3.75(s,6H),3.73(d,J=2.4Hz,6H),3.34(dd,J =22.5,14.2Hz,1H),3.10(dd,J=22.0,14.2Hz,1H),1.90-1.58(m,8H),1.45(s,1 5H),1.41-1.31(m,3H),1.29-1.10(m,5H),1.03-0.81(m,4H).LC-MS(Linear gradient 10~90% In a test using MeCN / H2O, 0.1% TFA, at 12.5 mins, Rt (min): 7.48 mins was observed (ESI-MS (m / z): 377.67 (M+H + ))

[0127] Boc-Cha-C(CH2OH)=CH2(81) [ka] Boc-Cha-CH2-P(=O)OMe-OMe (5.9 g, 17.5 mmol, 1.0 equivalent) was dissolved in THF and H2O (0.1 M) in a 1:1 ratio. K2CO3 (7.26 g, 52.5 mmol, 3.0 equivalent) and formaldehyde (37% by mass in H2O, 3.91 ml, 52.5 mmol, 3.0 equivalent) were added to the solution. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ELISA and acidified with HCl (1.0 M aqueous solution). The organic layer and aqueous layer were separated, and the aqueous layer was extracted three times with ELISA. The obtained ELISA was washed with H2O and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, filtered, and concentrated under reduced pressure. The title compound was obtained by silica gel column chromatography (1:20->1:4 SiO:PE, v:v) (2.42 g, 8.93 mmol, 51%). 1 ¹H NMR (400MHz, CDCl3) showed δ6.21 (d,J=33.7Hz,2H), 5.57 (d,J=8.8Hz,1H), 5.12-5.03 (m,1H), 4.39-4.23 (m,2H), 1.76-1.50 (m,7H), 1.42 (s,13H), 1.37-1.06 (m,6H), 1.02-0.81 (m,3H). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 7.81 min (ESI-MS (m / z): 311.60 (M+H + ))

[0128] Boc-Cha-EK-CH3OH(82) [ka] Boc-Cha-C(CH3OH)=CH2 (3.4 g, 11 mmol, 1.0 equivalent) was dissolved in MeOH (0.2 M), cooled to 0°C, and hydrogen peroxide (3.14 ml, 55 mmol, 5.0 equivalent), benzonitrile (5.66 ml, 55 mmol, 5.0 equivalent), and DiPEA (9.58 ml, 55 mmol, 5.0 equivalent) were added. The resulting reaction mixture was stirred at 0°C for 16 hours. The reaction mixture was diluted with HCl and acidified with HCl (1.0 M aqueous solution). The organic and aqueous layers were separated, and the aqueous layer was extracted three times with HCl. The resulting HCl layer was dried over Mg2SO4, filtered, and concentrated under reduced pressure. The crude product contained the product as a racemic mixture. The target S isomer was isolated by slow, thorough separation achieved by starting at 10% HCl / PE and increasing the eluate concentration by 1 percentage point increments. Finally, the title compound was obtained by purification using flash column chromatography (5%->15% (v / v) ¼:PE) (1.1 g, 3.22 mmol, 29%). 1 H NMR(400MHz,CDCl3)δ4.91(d,J=8.6Hz,1H),4.39-4.28(m,1H),4.19(dd,J=12.7,4.6Hz,1H),3.76(dd,J=12.8,5.5Hz,1H),3.33(d,J=5.0Hz,1H) ),3.09(d,J=5.0Hz,1H),2.37(t,J=6.3Hz,1H),1.86(d,J=10.9Hz,1H), 1.77-1.53(m,6H),1.42(s,12H),1.33-1.07(m,6H),1.05-0.86(m,3H). 13 ¹³C NMR (101MHz, CDCl3) δ208.7, 155.7, 79.9, 62.0, 61.1, 60.4, 53.2, 51.3, 49.2, 37.9, 34.3, 34.0, 31.8, 28.3, 26.4, 26.2, 25.9, 21.0, 14.2. LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 7.65 min (ESI-MS (m / z): 327.53 (M+H + ))

[0129] H2N-Cha-EK-CH3OH(83) [ka] Deprotection of Boc-Cha-EK-CH3OH was carried out on an mmol scale according to procedure A, yielding the title compound as a yellow oily substance (0.38 g, 1.08 mmol, quantitative). LC-MS (linear gradient 10-50% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.43 min (ESI-MS(m / z): 228.07(M+H) + ))

[0130] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH(2) [ka] Morph-Ala-Tyr(OMe)-NHNH2 (0.81 g, 2.0 mmol, 1.2 equivalents) was evaporated twice with toluene, dissolved in anhydrous DMF (0.1 M), purged with N2, and cooled to -40°C. To this solution, tBuONO (0.438 ml, 3.67 mmol, 2.2 equivalents) and HCl (4.0 M in dioxane, 2.33 ml, 9.33 mmol, 5.6 equivalents) were added. The resulting reaction mixture was stirred at -40°C for 4 hours. After 4 hours, the HCl was quenched with DiPEA (1.16 ml, 6.67 mmol, 4.0 equivalents), and a solution of NH2-Cha-EK-CH3OH in anhydrous DMF (0.2 M) (0.379 g, 1.70 mmol, 1.0 equivalent) was added dropwise. The resulting reaction mixture was stirred for 16 hours, during which time it was heated to return to room temperature. Reaction fluid H2O と The solution was diluted with dimethyl acetate, washed with NaHCO3 (saturated aqueous solution), H2O, and saline solution (saturated aqueous solution of NaCl), dried over MgSO4, and concentrated under reduced pressure. The title compound was obtained as a clear oily substance by silica gel column chromatography (0:100->10:100 MeOH:DCM) (0.71 g, 1.17 mmol, 69%). f:5:100 MeOH:DCM under conditions of 0.35. ¹H NMR (400MHz, CDCl₃) δ 7.47 (d, J = 7.5Hz, 1H), 7.16–7.06 (m, 2H), 6.86 (d, J = 7.7Hz, 1H), 6.83–6.76 (m, 2H), 6.50 (d, J = 7.7Hz, 1H), 4.63–4.51 (m, 2H), 4.44 (p, J = 7.1Hz, 1H), 4.17 (d, J = 12.6Hz, 1H), 3.76 (s, 3H), 3.73 (d, J = 5.1Hz, 1H), 3.70 (t ,J=4.6Hz,4H),3.29(d,J=5.0Hz,1H),3.07(d,J=5.0Hz,1H),3.00(d,J=6.9Hz,2H),2.95(s,1H),2.89(s,1H),2.46(q, J=4.2Hz,5H),1.78(d,J=13.0Hz,1H),1.72-1.49(m,5H),1.35(d,J=7.0Hz,3H),1.28-1.09(m,6H),1.01-0.82(m,2H). 13 C NMR (101MHz, CDCl3) δ 207.6, 206.1, 172.0, 170.9, 170.3, 158.6, 130.4, 128.2, 114.0, 66.9, 62.6, 62.0, 61.6, 61.4, 55.2, 54.3, 53.7, 52.1, 51.7, 50.4, 49.3, 48.4, 37.7, 36.7, 34.3, 33.8, 31.8, 26.3, 26.2, 25.9, 17.8. LC-MS (linearly coupled with 10~90% MeCN / H2O, 0.1%...) TFA, 12.5 points), Rt (minutes): 5.12 points, ESI-MS (m / z): 603.27 (M+H + )). HRMS(ESI)m / z:[M+H + ]について、C 31 H 46 The calculated value of N4O8 is 603.33884, and the measured value is 603.33823.

[0131] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NHBoc(3)

change

[0132] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NHBoc(4)

Chem.

[0133] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NHBoc(5)

Chem.

[0134] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NHBoc(6) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Phe-OH (18 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (17 mg, 0.020 mmol, 61%). 1 H NMR(400MHz,CDCl3)δ7.47(d,J=7.5Hz,1H),7.29(d,J=11.6Hz,5H),7.13(d,J=8.7Hz,4H),6.81(d,J=8.5Hz,3H),6.42(d,J=7.5Hz,1H),4.96(d, J=8.3Hz,1H),4.90(d,J=12.2Hz,1H),4.62-4.48(m,3H),4.42(p,J=7.1Hz,1H),4.16(s,2H),4.05(d,J=12.1Hz,1H),3.84(dq,J=13.4,6.7Hz,2H ),3.77(s,3H),3.70(t,J=4.7Hz,4H),3.35(d,J=4.9Hz,1H),3.12(dd,J=13.9,5.7Hz,1H),3.05(d,J=5.8Hz,1H),3.03-2.97(m,3H),2.96(s,1H) ,2.87(d,J=16.4Hz,1H),2.47(q,J=4.6Hz,4H),1.80-1.51(m,7H),1.41 (d,J=5.6Hz,8H),1.37(d,J=7.1Hz,4H),1.25(s,6H),0.96-0.81(m,3H). 13C NMR(101MHz,CDCl3)δ205.3,171.9,171.1,171.0,158.6,156.9,154.9,13 5.7,130.4,129.4,128.7,128.2,127.1,114.0,80.0,66.9,63.4,61.6,59 .7,55.2,54.3,54.2,53.7,50.0,49.3,48.5,42.2,38.3,37.5,36.6,34.3 ,33.9,31.8,29.7,28.3,26.3,26.2,25.9,23.5,17.6.LC-MS (linear gradient 10~90% In a test using MeCN / H2O, 0.1% TFA, at 12.5 mins, Rt (min): 7.34 mins was observed (ESI-MS (m / z): 850.33 (M+H + ))

[0135] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NHBoc(7) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Phe(Phe)-OH (23 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (33 mg, 0.035 mmol, quantitative). 11H NMR (400 MHz, CDCl3) δ 7.55 (dd, J = 16.5, 8.1 Hz, 4H), 7.44 (d, J = 8.7 Hz, 3H), 7.39 - 7.31 (m, 1H), 7.21 (d, J = 7.9 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H), 6.85 - 6.74 (m, 3H), 6.42 (d, J = 7.4 Hz, 1H), 5.01 (d, J = 8.3 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.66 - 4.58 (m, 1H), 4.58 - 4.49 (m, 2H), 4.41 (p, J = 7.1 Hz, 1H), 4.25 (d, J = 7.9 Hz, 4H), 4.06 (d, J = 12.2 Hz, 1H), 3.91 - 3.79 (m, 5H), 3.77 (s, 3H), 3.69 (t, J = 4.6 Hz, 4H), 3.37 (d, J = 4.9 Hz, 1H), 3.17 (dd, J = 13.8, 5.7 Hz, 1H), 3.09 (dd, J = 13.9, 5.8 Hz, 1H), 3.03 (d, J = 4.9 Hz, 1H), 3.01 - 2.96 (m, 2H), 2.94 (s, 1H), 2.86 (d, J = 16.4 Hz, 1H), 2.45 (q, J = 4.4 Hz, 4H), 1.80 - 1.52 (m, 6H), 1.43 (s, 7H), 1.36 (d, J = 7.0 Hz, 4H), 1.32 - 1.19 (m, 4H), 1.02 - 0.81 (m, 3H). 13 13C NMR (101 MHz, CDCl3) δ 205.2, 171.9, 171.1, 171.0, 170.3, 158.6, 157.0, 140.7, 134.7, 130.4, 129.8, 128.8, 128.2, 127.4, 127.3, 127.0, 114.0, 66.9, 63.6, 61.6, 59.7, 55.2, 54.2, 53.8, 48.4, 42.1, 37.9, 37.5, 36.6, 34.3, 33.9, 31.7, 28.3, 26.2, 25.9, 22.7, 18.2. LC-MS (linear gradient 10~90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt (min): 8.00 min (ESI-MS (m / z): 926.40 (M+H + ))

[0136] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NHBoc(8) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (10 mg, 0.017 mmol) and Boc-Asn-OH (7.7 mg, 0.0332 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (4.7 mg, 0.006 mmol, 34%). 1 H NMR(500MHz,CDCl3)δ7.43(s,1H),7.18(d,J=7.0Hz,1H),7.11(d,J=8.5Hz,2H),6.81(d,J=7.8Hz,3H),6.72(d,J=11.8Hz,1H),6.49(s ,1H),5.84(s,1H),5.75(d,J=8.5Hz,2H),4.84-4.69(m,1H),4.54(t,J=7.1Hz,4H),4.38(p,J=7.0Hz,2H),4.27(s,1H),3.78(s,4H),3. 71(s,5H),3.34(d,J=5.0Hz,1H),3.09(d,J=4.8Hz,1H),3.04-2.93(m,4H),2.86(d,J=16.0Hz,2H),2.71(d,J=11.2Hz,1H),2.46(s,5H) ,2.36(s,2H),1.87-1.51(m,17H),1.45(d,J=1.1Hz,13H),1.36(d,J=7.0Hz,6H),1.26(s,45H),0.99-0.79(m,14H).LC-MS(linear gradient 10~90% In a MeCN / H2O (0.1% TFA, 12.5 min) test, Rt(min): 6.31 min was observed (ESI-MS(m / z): 817.25(M+H + ))

[0137] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NHBoc(9) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Asp(OBzl)-OH (21 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (17 mg, 0.019 mmol, 57%). 1 H NMR(300MHz,CDCl3)δ7.43(d,J=7.4Hz,1H),7.35(t,J=2.2Hz,5H),7.11(d,J=8.6Hz,2H),6.85-6.77(m,2H),6.74(d,J=7.6Hz,1H) ,6.34(d,J=7.5Hz,1H),5.48(d,J=8.6Hz,1H),4.89(d,J=12.2Hz,1H),4.62-4.46(m,3H),4.41(p,J=7.1Hz,1H),4.07(d,J=12.3Hz) ,1H),3.77(s,3H),3.70(t,J=4.6Hz,4H),3.48(s,2H),3.32(d,J=5.0Hz,1H),3.06-2.92(m,5H),2.89(d,J=6.3Hz,2H),2.84(d,J= 5.7Hz,1H),2.45(q,J=4.0Hz,4H),1.89-1.49(m,11H),1.44(s,10H),1.36(d,J=7.0Hz,4H),1.30-1.03(m,7H),1.00-0.77(m,3H). 13 C NMR(75MHz,CDCl3)δ205.2,171.9,170.9,170.3,158.6,135.3,130.4,128.6,128.5,128.4,128.2,127.8,126.7,113.4,80.9,66.9, 62.8,61.6,59.6,55.2,54.2,53.8,50.0,49.2,48.4,37.5,36.8,36.5,34.3,33.8,31.7,28.3,26.2,25.2,17.5.LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% In TFA (12.5 min), Rt (min): 6.56 min (ESI-MS (m / z): 908.50 (M+H + ))

[0138] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NHBoc(10) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Glu(OBzl)-OH (21 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (17 mg, 0.019 mmol, 57%). 1 H NMR(400MHz,CDCl3)δ7.45(d,J=7.4Hz,1H),7.40-7.29(m,5H),7.12(d,J=8.6Hz,2H),6.81(d,J=8.6Hz,3H),6.44(d,J=7.1Hz,1H),5.12(s,3H),4. 93(s,1H),4.62-4.48(m,2H),4.41(p,J=7.1Hz,1H),4.33(d,J=5.6Hz,1H) ,4.19(s,3H),4.10(d,J=12.2Hz,1H),3.91-3.79(m,4H),3.77(s,3H),3.7 0(t,J=4.7Hz,4H),3.37(d,J=4.9Hz,1H),3.06(d,J=4.8Hz,1H),3.00(d, J=7.2Hz,2H),2.94(s,1H),2.87(d,J=16.4Hz,1H),2.44(dd,J=9.0,4.8Hz ,6H),2.21(dd,J=13.5,7.2Hz,1H),2.03-1.83(m,4H),1.80-1.50(m,6H), 1.43(s,9H),1.36(d,J=7.1Hz,3H),1.32-1.16(m,5H),1.02-0.81(m,3H). 13C NMR(101MHz,CDCl3)δ205.3,171.9,171.0,170.3,158.6,157.0,135.7,130.4,128.6,128.3,128.3,114.0,66.9,66.5,63.3,61.6,59.6,55.2, 54.2,53.8,52.8,50.0,49.4,48.4,42.2,37.5,36.6,34.3,33.9,31.7, 30.1,29.7,28.3,27.7,26.3,26.2,25.9,23.5,17.7.LC-MS (linear gradient 10~90% MeCN / H2O, 0.1% In TFA (12.5 min), Rt (min): 7.44 min (ESI-MS (m / z): 922.33 (M+H + ))

[0139] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NHBoc(11) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Ser(OBn)-OH (21 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (33 mg, 0.037 mmol, quantitative). 11H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.4 Hz, 1H), 7.39 - 7.28 (m, 6H), 7.12 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 3H), 6.41 (d, J = 7.5 Hz, 1H), 5.39 (d, J = 8.8 Hz, 1H), 4.89 (d, J = 12.3 Hz, 1H), 4.61 - 4.47 (m, 5H), 4.46 - 4.36 (m, 2H), 4.25 (d, J = 7.9 Hz, 5H), 4.18 (d, J = 12.3 Hz, 1H), 3.84 (dq, J = 12.9, 6.5 Hz, 7H), 3.77 (s, 3H), 3.70 (t, J = 4.7 Hz, 4H), 3.30 (d, J = 4.9 Hz, 1H), 3.03 (d, J = 4.9 Hz, 1H), 3.00 (d, J = 6.8 Hz, 2H), 2.96 - 2.82 (m, 2H), 2.45 (q, J = 4.4 Hz, 4H), 1.78 - 1.49 (m, 6H), 1.45 (s, 10H), 1.36 (d, J = 7.1 Hz, 3H), 1.34 - 1.18 (m, 6H), 1.00 - 0.81 (m, 4H). 13 13C NMR (101 MHz, CDCl3) δ 205.3, 171.9, 170.9, 170.3, 170.0, 158.6, 157.0, 130.4, 128.5, 128.2, 127.9, 127.6, 114.0, 73.4, 69.9, 66.9, 63.0, 61.6, 59.7, 55.2, 54.2, 54.0, 53.8, 48.4, 42.1, 37.4, 36.6, 34.3, 33.9, 31.7, 28.3, 26.3, 26.2, 25.9, 23.5, 17.6. In LC - MS (linear gradient 10 - 90% MeCN / H2O, 0.1% TFA, 12.5 min), Rt (min): 7.45 min was shown (ESI - MS (m / z): 880.27 (M + H + ))。

[0140] Morph - Ala - Tyr(OMe) - Cha - EK - OC(=O) - Cys(Trt) - NHBoc(12)

Chemical Structure

[0141] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Pro-NHBoc(13) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg, 0.033 mmol) and Boc-Pro-OH (14 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (22 mg, 0.027 mmol, 84%). 1 H NMR(400MHz,CDCl3)δ7.42(t,J=8.1Hz,1H),7.12(d,J=8.6Hz,2H),6.80(d,J=8. 6Hz,2H),6.72(d,J=7.1Hz,1H),6.31(d,J=7.4Hz,1H),4.85(dd,J=50.5,12.2Hz, 1H),4.62-4.47(m,2H),4.41(td,J=7.2,4.6Hz,1H),4.25-4.17(m,1H),3.99(d, J=12.2Hz,1H),3.90-3.79(m,1H),3.77(s,3H),3.70(t,J=4.6Hz,4H),3.54-3.41 (m,2H),3.35(dd,J=25.1,4.9Hz,1H),3.06(dd,J=14.3,4.9Hz,1H),3.02-2.96( m,2H),2.94(d,J=3.3Hz,1H),2.86(dd,J=16.5,7.2Hz,1H),2.53-2.38(m,4H),2. 29-2.12(m,1H),1.97(ddd,J=12.6,6.2,4.3Hz,1H),1.88(p,J=6.7,6.2Hz,2H), 1.82-1.52(m,8H),1.45(d,J=19.4Hz,9H),1.39-1.19(m,7H),1.06-0.82(m,3H). 13C NMR(101MHz,CDCl3)δ205.3,172.4,171.9,170.9,170.4,158.6,130.4,128.2,11 4.0,80.1,77.4,77.0,76.7,66.9,63.2,62.7,61.6,59.7,59.1,58.8,55.2,54.3 ,53.8,50.0,49.2,48.4,46.5,46.3,42.2,37.5,36.4,34.4,33.9,31.7,31.0,29.9,29.7,28.5,28.3,26.3,26.0,24.4,23.6,23.5,17.6,17.4.LC-MS (straight gradient 10~90% MeCN / H2O, 0.1% TFA, 12.5 mins) showed Rt(min):6.80 mins (ESI-MS(m / z):800.13(M+H + ))

[0142] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NH2·TFA(14) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NHBoc was carried out according to Procedure A on a 0.013 mmol scale, yielding the title compound as a white powder (after lyophilization) (9.2 mg, 0.012 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.80 min (ESI-MS(m / z): 674.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 34 H 51 The calculated value for N5O9 was 674.37595, and the measured value was 674.37600.

[0143] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NH2·TFA(15) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NHBoc was carried out according to Procedure A on a 0.012 mmol scale, yielding the title compound as a white powder (after lyophilization) (5.25 mg, 0.006 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.22 min (ESI-MS(m / z): 702.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 36 H 55 The calculated value for N5O9 was 702.41581, and the measured value was 702.40814.

[0144] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NH2·TFA(16) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NHBoc was carried out according to Procedure A on a 0.011 mmol scale, yielding the title compound as a white powder (after lyophilization) (8.7 mg, 0.010 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.21 min (ESI-MS(m / z): 716.27(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 37 H 57 The calculated value for N5O9 was 716.42882, and the measured value was 716.42241.

[0145] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NH2·TFA(17) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NHBoc was carried out according to Procedure A on a 0.012 mmol scale, yielding the title compound as a white powder (after lyophilization) (9.8 mg, 0.011 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.34 min (ESI-MS(m / z): 750.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 40 H 55 The calculated value for N5O9 was 750.40725, and the measured value was 750.40657.

[0146] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NH2·TFA(18) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NHBoc was carried out according to Procedure A on a 0.018 mmol scale, yielding the title compound as a white powder (after lyophilization) (17 mg, 0.018 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.97 min (ESI-MS(m / z): 826.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 46 H 59 The calculated value for N5O9 was 826.43855, and the measured value was 826.43831.

[0147] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NH2·TFA(19) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NHBoc was carried out according to Procedure A on a 0.012 mmol scale, yielding the title compound as a white powder (after lyophilization) (4.03 mg, 0.0048 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 3.80 min (ESI-MS(m / z): 717.25(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 35 H 52 N6O 10 The calculated value is 717.38177, and the measured value is 717.38202.

[0148] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NH2·TFA(20) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NHBoc was carried out according to Procedure A on a 0.011 mmol scale, yielding the title compound as a white powder (after lyophilization) (14 mg, 0.015 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.51 min (ESI-MS(m / z): 808.40(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 42 H 57 N5O 11 The calculated value is 808.41273, and the measured value is 808.41203.

[0149] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NH2·TFA(21) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NHBoc was carried out according to Procedure A on a 0.011 mmol scale, yielding the title compound as a white powder (after lyophilization) (12 mg, 0.012 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.47 min (ESI-MS(m / z): 822.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 43 H 59 N5O 11 The calculated value is 822.42838, and the measured value is 822.42800.

[0150] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NH2·TFA(22) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NHBoc was carried out according to Procedure A on a 0.011 mmol scale, yielding the title compound as a white powder (after lyophilization) (8.7 mg, 0.010 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.51 min (ESI-MS(m / z): 780.40(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 41 H 57 N5O 10 The calculated value is 780.41782, and the measured value is 780.41751.

[0151] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Cys(Trt)-NH2·TFA(23) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Cys(Trt)-NHBoc was carried out according to Procedure A on a 0.009 mmol scale, yielding the title compound as a white powder (after lyophilization) (24 mg, 0.0226 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.88 min (ESI-MS(m / z): 948.27(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 53 H 65 The calculated value for N5O9S was 948.45758, and the measured value was 948.45715.

[0152] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Pro-NH2·TFA(24) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Pro-NHBoc was carried out on a 0.013 mmol scale according to procedure A, yielding the title compound as a white powder (after lyophilization) (11 mg, 0.013 mmol). LC-MS (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 4.84 min (ESI-MS(m / z): 700.33(M+H + )) HRMS(ESI)m / z:[M+H + Regarding C 36 H 52 The calculated value for N4O9 was 700.39160, and the measured value was 700.39127.

[0153] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)CHCH3(CH3)(25) [ka] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (15.1 mg, 0.025 mmol) was co-evaporated twice with toluene, then dissolved in anhydrous DCM (0.1 M) and purged with N2. To this solution, isobutyric anhydride (8.3 μL, 0.05 mmol, 2.0 equivalents) and DMAP (3.0 mg, 0.025 mmol, 1.0 equivalent) were added. The resulting reaction mixture was stirred for 2 hours and then concentrated under reduced pressure. The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (0% → 3% (v / v) MeOH / DCM) (7.03 mg, 10.46 μmol). HRMS(ESI)m / z:[M+H + Regarding C 35 H 52 The calculated value for N4O9 is 673.38071, and the measured value is also 673.38071.

[0154] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OTBS(26) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (22.9 mg, 0.038 mmol) and lactic acid-OTBS (15.5 mg, 0.076 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (15.46 mg, 0.020 mmol). HRMS(ESI)m / z:[M+H + Regarding C 40 H 64 N4O 10 The calculated value of Si is 789.44645, and the measured value is 789.44607.

[0155] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OH(27) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OTBS was carried out according to Procedure A on a 0.013 mmol scale, yielding the title compound as a white powder (after lyophilization) (9.64 mg, 0.014 mmol). HRMS(ESI)m / z:[M+H + Regarding C 34 H 50 N4O 10 The calculated value is 675.35997, and the measured value is 675.35945.

[0156] Mor-Ala-Tyr(OMe)-Cha-EK-Me-Oε-Ahx-Boc(92) [ka] Following procedure B, Steglig esterification was performed between Morph-Ala-Tyr(OMe)-Cha-EK-Me-OH (20 mg, 0.033 mmol) and Boc-e-Ahx-OH (15 mg, 0.066 mmol). The title compound was obtained as a white powder (after lyophilization) by silica gel column chromatography (3% (v / v) MeOH / DCM) (21 mg, 0.025 mmol, 78%). 1H NMR(400MHz,CDCl3)δ7.4315(d,J=7.7Hz,1H),7.10(d,J=7.7Hz,2H),6.81(dd,J=17.1,8.1Hz,3H),6.71(s,1H),5.03(d,J=12.2Hz,1H),4.62(d,J =8.4Hz,2H),4.54(d,J=8.6Hz,1H),4.41(p,J=7.1Hz,1H),4.13(d,J=7.4 Hz,2H),3.91-3.79(m,3H),3.77(s,3H),3.69(t,J=4.6Hz,4H),3.37(d,J =4.9Hz,1H),3.18(p,J=7.0Hz,1H),3.04(d,J=4.8Hz,2H),3.00(d,J=7.2Hz,1H),2.95(s,1H),2.87(d,J=16.4Hz,1H),2.45(q,J20=4.1Hz,4H),2. 33(q,J=8.6,8.0Hz,2H),1.84(d,J=19.8Hz,3H),1.76-1.55(m,7H),1.45 (s,12H),1.35(d,J=7.0Hz,4H),1.33-1.16(m,8H),1.08-0.80(m,3H).13C NMR (101MHz, CDCl3) δ172.7, 171.7, 171.2, 158.6, 157.0, 130.5, 128.0, 113.9, 66.9, 63.1, 61.7, 55.2, 53.8, 49.0, 42.2, 40.5, 36.8, 34.0, 31.7, 30.0, 28.5, 26.3, 26.2, 26.0, 25.9, 24.5, 23.5, 17.8. LC-MS (linear gradient 10-90% MeCN / H2O, 25 0.1% TFA, 12.5 min) showed Rt(min): 7.05 min (ESI-MS (m / z): 816.20 (M+H + ))

[0157] Mor-Ala-Tyr(OMe)-Cha-EK-Me-Oε-Ahx-NH2(93) [ka] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-COO-Me-Oe-Ahx-NHBoc was carried out according to Procedure A on a 0.025 mmol scale, yielding the title compound as a white powder (after lyophilization) (12 mg, 0.014 mmol, 56%). LC-MS 5 (linear gradient 10-90% MeCN / H2O, 0.1% TFA, 12.5 min) showed Rt(min): 5.10 min (ESI-MS(m / z): 716.40(M+H + ))

[0158] Example 2 - Competitive Activity-Based Protein Profiling Proteasome Inhibition Assay The biological activity of compounds 1-27 was evaluated using a competitive activity-based protein profiling assay.

[0159] Activity-based protein profiling (ABPP) has been used for the past 30 years to detect and identify enzymes in complex biological samples. In ABPP, an activity-based probe (ABP) containing a substrate-like element (often linked to an electrophile) and a reporter portion (biotin, fluorescent agent, or bio-orthogonal tag) reacts within the active site of a target enzyme or enzyme family, forming a covalent and irreversible bond. Several proteasome inhibitors (peptide vinyl sulfone, peptide epoxy ketone) react in a mechanism-based manner, making them a good starting point for designing proteasome-targeted ABPs. Ultimately, a set of three ABPs was developed, and combining them made it possible to isolate and analyze all six active subunits of human cCP and iCP after developing ABP-treated cell lysates on an SDS-PAGE gel and performing in-gel fluorescence detection. This ABPP assay allows for rapid analysis of cell lysates regarding their cCP and iCP catalytic activity content. For example, when lysates of Raji cells, a B-cell lymphoma cell line constitutively expressing both proteasome isoforms, are treated with three types of ABPS, followed by SDS-PAGE and gel fluorescence detection, six bands corresponding to all six active sites of iCP and cCP are obtained.

[0160] Competitive ABPP complements comparative ABPP and allows for the identification of active compounds, as well as the elucidation of their selectivity and efficacy, for a library of putative proteasome inhibitors. Most proteasome inhibitors described in the literature, and the examples described in this text, are peptide-based electrophiles that react within the proteasome active site to form irreversible covalent bonds or long-lasting complexes. In competitive ABPP, three different ABPs are added, and individual inhibitors are added to cell lysates (or cells, if cell permeability is being investigated) at various concentrations and for various durations before SDS-PAGE and in-gel fluorescence scanning. In this way, the selectivity and activity of the panel of putative proteasome inhibitors described in Example 1 were determined by the following method. Cell lysates were prepared by adding twice the amount of lysis buffer to the cell pellet. The lysis buffer contained 250 mM sucrose, 50 mM Tris pH 7.5, 2 mM DTT, 5 mM MgCl2, 10% (v:v) glycerol, 2 mM ATP, 0.05% (w:v) digitonine, and 25 U / ml benzonase. After standing on ice for 1 hour, the mixture was centrifuged at 15°C and 22000 rcf for 15 minutes. Protein concentrations were then determined using the Bradford assay, followed by dilution of the cell lysates with assay buffer.

[0161] Cell lysates were diluted to a total protein concentration of 1.4–2.0 μg / μL in an assay buffer containing 50 mM Tris pH 7.5, 2 mM DTT, 5 mM MgCl2, 10% glycerol, and 2 mM ATP. The cell lysates were then exposed to a series of inhibitor dilutions (30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, and 0.003 μM, diluted with DMSO) at 37°C for 1 hour. Furthermore, the inhibited cell lysates were reacted with a probe cocktail (Cy2: BODIPY(FL)-LU-112, Cy3: BODIPY(TMR)-NC-005VS, Cy5: Cy5-NC-001) at 37°C for 1 hour. Finally, the cell lysates were denatured by adding reducing gel loading buffer and boiling at 95°C for 4 minutes. Subsequently, the denatured cell lysates were loaded onto a 12.5% ​​SDS-PAGE gel and fractionated by gel electrophoresis at 160V for 90 minutes. Immediately after electrophoresis, multiple fluorescence detection of remaining activity-based probes was performed using a ChemiDoc® MP System equipped with Cy5, Cy3, and Cy2 channels. The gel was then fixed, stained overnight in Coomassie blue, and decolorized in deionized water for 2 days. Similarly, detection with Coomassie blue was performed using the ChemiDoc® MP System and converted using ImageLab. The corrected intensity of the fluorescence bands was converted using ImageLab. The normalized corrected volume was plotted against the inhibitor concentration, and then IC was calculated for each inhibitor. 50 The values ​​were calculated using GraphPad Prism 9.0 software (nonlinear regression, [inhibitor] vs. normalized response, variable slope model).

[0162] N-Boc amino acid esters 3-13 showed only weak inhibition or no inhibition at all against any cCP active site. Comparison of compounds 3-13 with LU-005i revealed that LU-005i was potent against β5c compared to other proteasome subunits. The low potency against β1c and β2c observed in the compounds shown in Figure 1 was supported by data obtained during the development of LU-005i, confirming that the affinity for β1c and β2c was suppressed by the P1 and P2 residue combination, Tyr(OMe)-Cha. All N-Boc esters shown in Figure 1 showed at least a 1.5-fold decrease in potency against β5c compared to LU-005i 1. Furthermore, LU005i-OH 2, N-Boc alanine 3, N-Boc asparagine 8, N-Boc aspartic acid 9, N-Boc glutamic acid 10, N-Boc serine 11, N-Boc proline 13, alanine-NH212, valine-NH213, isoleucine-NH216, aspartic acid-NH220, and cysteine-NH223 all exhibit iCP selectivity. Of the proteasome inhibitors listed above, LU-005i-OH 2, N-Boc alanine 3, N-Boc aspartic acid 9, N-Boc glutamic acid 10, alanine-NH214, aspartic acid-NH220, serine-NH222, and cysteine-NH223 are the most interesting because their selectivity ratio for iCP is more than twice that of cCP (highest cCP potency vs. lowest iCP potency).

[0163] None of the aliphatic and bulky amino acid ester compounds (N-Boc valine 4, N-Boc isoleucine 5, N-Boc phenylalanine 6, N-Boc diphenylalanine 7, and N-Boc-S-Trt cysteine ​​8) showed efficacy against β1i and β2i. This suggests that the size of the amino acid residue at the P1' position negatively affects the affinity for these subunits. Coincidentally, this makes the above compounds highly selective β5i inhibitors. In contrast to what was found with N-Boc phenylalanine 6, N-Boc diphenylalanine 7, and N-Boc-S-Trt cysteine ​​8, N-Boc aspartic acid 9, N-Boc glutamic acid 10, and N-Boc serine 11 proved to be potent inhibitors against β1i and β2i. It is likely that the rotational flexibility and hydrogen bonding ability of the benzylated carboxylic acids of N-Boc aspartic acid 9 and N-Boc glutamic acid 10, as well as the benzylated hydroxyl group of N-Boc serine 11, cancel out steric collisions within the S' pocket. Furthermore, when comparing N-Boc serine 11 with N-Boc aspartic acid 9, an increase in potency against β1i, β2i, and β5c was observed, and similarly, when comparing N-Boc aspartic acid 9 with N-Boc glutamic acid 10, a further increase in potency against β1i, β2i, and β5c was observed. Interestingly, a similar inhibitory profile was observed when comparing N-Boc aspartic acid 9 with N-Boc asparagine 7. Among N-Boc amino acid ester proteasome inhibitors, N-Boc alanine 3 was found to be the only inhibitor that showed efficacy of 10 μM or more against each active site of cCP and 4 μM or less against the active site of iCP (Figure 1). When N-Boc alanine 2 was compared with the branched alkyl derivatives N-Boc valine 4 and N-Boc isoleucine 5, it is hypothesized that these residues reduce affinity for the β1i and β2i subunits.

[0164] Overall, when all NH2 amino acid esters found in Figure 1 were compared with N-Boc esters, an increase in potency against β1i and β2i was observed (3-13). All NH2 amino acid esters except phenylalanine-NH217, diphenylalanine-NH218, glutamic acid-NH219, serine-NH220, and proline-NH224 showed potency of 10 μM or more against β1c and β2c. Removal of the N-Boc protecting group introduces two structural changes: a significant decrease in steric bulk and the formation of a hydrophilic amine that is protonated at physiological pH. Valine-NH215, isoleucine-NH216, phenylalanine-NH217, and diphenylalanine-NH218 showed increased affinity for β1i, β2c, β2i, and β5c. This suggests that the combination of an N-Boc protecting group with a branched alkyl residue or a bulky aromatic residue eliminates the inhibition of β1i, β2c, β2i, and β5c. When glutamate-NH221 and serine-NH222 were compared to their respective N-Boc protected derivatives, increased potency against β1i, β2c, β2i, β5c, and β5i was observed. Interestingly, serine-NH222 proved to be a potent inhibitor of β1i, β2c, β2i, β5c, and β5i, while its corresponding N-Boc protected derivative was potent only against β5i. Furthermore, glutamate-NH221 showed no iCP selectivity and proved to be a potent inhibitor of β1i, β2i, β5c, and β5i. Aspartate-NH220 was the only inhibitor among the three O-benzyl protection inhibitors (20-22) that showed greater selectivity for iCP than for cCP when compared to the three corresponding N-Boc protected derivatives (9-11).

[0165] Surprisingly, cysteine-NH223, which has a similar side chain length to serine-NH222 and possesses an aromatic protecting group (albeit a much larger protecting group compared to the benzyl protecting group), proved to be an iCP selective inhibitor, whereas its corresponding N-Boc protected derivative (12) was not. Alanine-NH214 showed improved selectivity for iCPs, with a 10-fold increase in potency for both β1i and β2i and a 5-fold increase in potency for β5c compared to N-Boc alanine 3. Taken together, these studies confirmed that alanine-NH214 was the most selective inhibitor of all three iCPs among all the compounds tested.

[0166] Thus, alanine-NH214 emerged as the best iCP-selective proteasome inhibitor. To further investigate this, alanine-NH214 and N-Boc alanine 3 were re-evaluated in a competitive ABPP assay over a wider concentration range (up to 30 μM, Figure 2) and compared with 25, 26, and 27.

[0167] All alanine isomers shown in Figure 2 exhibit improved selectivity for the iCP active subunit compared to the cCP active subunit compared to the inhibitors shown in Figure 1. Comparing O-TBS lactate 26 with N-Boc alanine 14, a decrease in efficacy against β2i was observed in the former, suggesting that the increased steric bulk of the TBS protecting group is unfavorable to β2i. Unlike alanine-NH214, lactate-OH 27 did not show improved efficacy against β2c and β5c. Furthermore, when comparing lactate 27 and alanine-NH214 with their corresponding protected compounds (26 and 3), lactate 27 did not show the increased efficacy against β1i and β2i observed with alanine-NH214. Interestingly, compared to O-TBS lactate 26, lactate 27 showed an eight-fold decrease in efficacy against β1i. Isobutyric acid 25 exhibited a similar inhibition profile to N-Boc alanine 2, showing an iCP-selective inhibition profile. In terms of size, isobutyric acid 25 and lactate 27 were comparable to alanine 13, but their inhibition profiles were not. This suggests that the atomic properties of the N-terminal atom, rather than the steric bulk of the N-terminus, are the primary determinants of potency and selectivity. Overall, no clear trend was observed regarding the size of amine-free isomers.

[0168] Reference Example 3 - Synthesis of Compounds 28-51 (P3 Mutants) Compounds 28 to 51, shown below, were synthesized by the method described in Example 1, and by a method similar to the method described below for compound 28. [ka] The compounds are named according to the abbreviations for the P4 and P3 positions. For example, compound 28 is Mor(P4)-Gly(P3)-Tyr(OMe)(P2)-Cha(P1)-EK and is abbreviated as 28(Mor-Gly).

[0169] Synthesis of compound 28 [ka] a Reagents and conditions: (a) Boc-Gly-OH, HCTU, DiPEA, DCM, 17%; (b) (i) TFA, DCM, quantitative; (ii) Morpholinoacetic acid, HCTU, DiPEA, DCM, 48%; (c) Hydrazine hydrate, MeOH, quantitative; (d) (i) tBuONO, HCl, DMF, -30℃; (ii) 97, DiPEA, 13%.

[0170] Reference Example 4 - Synthesis of Compounds 52-57 (P3 mutants) Compounds 52-57, shown below, were synthesized by the method described in Example 1, and compound 28 was synthesized by a method similar to the method described above. [ka]

[0171] Reference Example 5 - Competitive ABPP assay in Raji cell lysates The biological activity of compounds 28-57 was evaluated using the biological assay described in Example 2. The obtained apparent IC50 was... 50 The values ​​were plotted on the heatmap shown in Figure 3.

[0172] Based on moderate global immunoproteasome selectivity, LU-005i 1 was selected as the lead compound. In the design of this study, the C-terminal epoxy ketone, the cyclohexyl-L-alanine (Cha) residue at position P1, and the Tyr(OMe) residue at position P2 were fixed. This is because these structural elements have been shown to be accepted by all iCP activities. The P3 and P4 (N-cap) residues were altered to obtain compounds with improved iCP selectivity by increasing activity for iCP activity, decreasing activity for cCP activity, or a combination of both. Based on this rationale, a first set of 24 peptide epoxy ketones (compounds 28-57) was constructed. These have the general structure of YX-Cha-Tyr(OMe)-epoxyketone, where Y is one of the five N-caps tested and X is one of the five selected α-amino acids. Since both the β1i and β5i subunits have smaller S3 pockets compared to their corresponding constitutive subunits, four relatively small residues were selected as the P3 residue: Gly, Ala, Ser, and 2-aminoisobutyric acid (Aib). A large O-benzylated serine (Ser(OBn)) was selected as a control to evaluate the effect of small residues at the P3 position on activity and selectivity. For the P4 moiety, morpholine (Mor) cap (also present in LU-005i 1), piperazine (HPip), N-methylpiperazine (MePip), tert-butyloxycarbonyl-protected piperazine (N-BocPip), and 4-hydroxylcyclohexyl (HCH) were used. The synthesis of all 24 compounds was carried out according to established protocols. As an example, the synthetic route to compound 28 is shown above (see the Experiments section for details on the synthesis and properties of all compounds). The proteasome inhibitory efficacy and selectivity of the 24 obtained compounds were evaluated in comparison to LU-005i1 using a competitive activity-based protein profiling (ABPP) assay.This technique utilizes three selective activity-based probes labeling the β2c / i subunit (appearing green), β5c / i subunit (appearing red), and β1c / i subunit (appearing blue) to evaluate the inhibition profiles of all catalytic activity of cCP and iCP. Extracts from the human B-cell lymphoma cell line Raji were used because they express both iCP and cCP, and compounds 28–57 were tested in a dilution series (final concentrations 0.01 μM–100 μM). Briefly, cell extracts were treated with the inhibitors, followed by treatment with the three ABPs. The samples were then separated by SDS-PAGE, and residual fluorescence was detected by in-gel scanning. Apparent IC. 50 The values ​​were derived and plotted as a heatmap (Figure 3). The results revealed that all compounds were insufficient as β1c and β2c inhibitors, supporting previous studies showing that the Tyr(OMe)-Cha-epoxyketone skeleton interferes with β1c and β2c inhibition. Compounds 50 (HCH-Ser(OBn)), 45 (MePip-Ser(OBn)), and 34 (BocPip-Ser) were considered the most potent inhibitors against β1i, showing significantly superior performance compared to all other compounds, including LU-005i 1, that have Ala, Gly, or Aib at the P3 position. Initially, it was not expected that these inhibitors would fit the small size of the β1i-S3 cavity, but surprisingly, compounds 45 and 50, with their bulky P3-Ser(OBn), showed high potency. Notably, the data also revealed that a large side chain is possible at either the P3 or P4 position, but undesirable at both positions. For example, compounds 35 (BocPip-Ser(OBn)) and 40 (HPip-Ser(OBn)) had weak effects on β1i.

[0173] Subsequently, the β2i efficacy of 24 compounds and LU-005i(1) was investigated. As a result, compound 34 (BocPip-Ser) was found to be the most effective and selective epoxy ketone among this group of compounds. On the other hand, compounds with Ser(OBn) at the P3 position were found to be insufficient as β2i inhibitors compared to their corresponding l-Ser derivatives.

[0174] Finally, the inhibitory titers against the β5c and β5i subunits were analyzed. Compounds 34 (BocPip-Ser) and 35 (BocPip-Ser(OBn)) were found to be the most active β5i inhibitors. Compound 35 showed only slight selectivity (3x) for β5i, while compound 34 showed much higher selectivity (21x) for β5i. The substitution of Ala27 to Ser (constitutive proteasome vs. immunoproteasome) results in a relatively smaller S3 pocket in the β5i active site compared to that of the β5c subunit, which may explain the superior performance of compound 34, which has a sterically less bulky Ser residue. Overall, in terms of potency and selectivity, compound 34 was found to be the most effective compound in this group. That is, it potently inhibits each activity of the immunoproteasome (ICD against the i subunit). 50 All values ​​were ≤0.84 μM, and they showed considerable selectivity for the corresponding constitutive proteasome activity (IC). 50 ratio β1c / β1i:36, β2c / β2i:33, β5c / β5i:7).

[0175] Second Generation Focused Library Based on compound 34, the following group of compounds was designed, synthesized, and evaluated (52-57). All compounds in this series share the general structure of Boc-Pip-X-Cha-Tyr(OMe)-epoxyketone, combining the C-terminal dipeptide epoxyketone of LU-005i 1 with the P4 residue present in lead compound 12. Other P3 substituents selected included Arg (same as 52), carboxybenzyl (Cbz)-protected Arg 53, and Op-xylol (OpXyl, 54). Furthermore, BocPip-d-Ser 55 and BocPip-d-Ser(OBn) 56, the P3 diastereomers of compounds 34 and 35, respectively, were also included. This is because the d-amino acid at P3 has been previously reported to enhance β5i selectivity for the corresponding l-amino acid in epoxyketone inhibitors. In addition, compound 57, which has a pyrimidine-protected Lys side chain at the P3 position, was included as an arginine mimetic. Compounds 52–57 were assayed with competitive ABPP, and activity was scored and plotted for all constitutive and immunoproteasome active sites, as before. While some compounds showed activity against all immunoproteasome activities at low concentrations, most co-inhibited the β5c active site and were therefore less promising than lead compound 34. As expected, peptide epoxy ketones with a d-amino acid at the P3 position proved insufficient as β5c inhibitors, and unfortunately, they were also found to be weak as β1i and β2i inhibitors. This β5i selectivity has been observed previously and is likely due to steric factors related to the bent binding mode induced by the d-amino acid at the P3 position.

[0176] Reference Example 6 - Synthesis of Compounds 58-77 (P3 mutants) Compounds 58-77, shown below, were synthesized by methods similar to those described in Examples 1 and 3. Further details of the synthesis will be described later. [ka]

[0177] The compounds are named according to the abbreviations for the P4 and P3 positions.

[0178] A library of novel compounds containing serine at the P3 position and BocPip or Morph at the P4 position was designed. Modifications were made to improve the selectivity of β5i over β5c. The amino acids and their derivatives used include: diaminopropionic acid (Dap), homoserine (Hser), diaminobutanoic acid (Dab), asparagine (Asn), pentahomoserine (HHSer), and glutamine (Gln).

[0179] All peptide epoxy ketones shown in Figure 4 have a cyclohexylalanine epoxy ketone at the P1 position, and therefore a considerable amount of epoxy ketone 97 was synthesized. Condensation of N-Boc cyclohexylalanine 77 with the Weinreb salt yielded Weinreb amide 78 (Scheme 3). Next, bromopropene was carbolithiated, and the resulting propenyl lithium was added to Weinreb amide 78 to obtain α,β-unsaturated ketone 98. Nucleophilic epoxidation with hydrogen peroxide yielded both diastereomers of epoxide 99, which were isolated by silica gel column chromatography to obtain N-Boc epoxy ketone 99. Finally, the N-Boc protecting group of epoxy ketone 99 was removed using TFA to obtain epoxy ketone 97. [ka] Scheme 3. Synthesis of cyclohexylalanine epoxy ketone 97. Reagents and conditions: (a) HCl·HNMeOMe, HCTU, DiPEA, DCM, rt, 90%; (b) i: Bromopropene, tBuLi, THF, -78℃; ii: 78, THF, -78℃ 70%; (c) H2O2, DiPEA, benzonitrile, MeOH, 0℃, 50%; (d) TFA, DCM, rt, quantitative.

[0180] Having obtained a large quantity of epoxy ketone 97, we focused on constructing various N-terminal P2-P3-P4 fragments that could be combined to give the desired library. For this purpose, it was necessary to prepare various amino acids to be placed at the P3 position and to functionalize them appropriately to be suitable for peptide synthesis chemistry. To synthesize a partially and orthogonally protected diaminopropionic acid for use in subsequent introduction to the P3 position, N-Boc asparagine 100 was rearranged by Hoffmann rearrangement using diacetoxyiodobenzene (PIDA) as the oxidizing agent to obtain N-Boc diaminopropionic acid (Dap) 102 (Scheme 4). Next, the N-β of Dap 102 was protected with an Alloc group (102-104), and N-Alloc Dap 104 was bonded to 4-methoxyphenylalanine to obtain dipeptide 106. De-N-Bocation of 106 was followed by condensation with 2-morpholinoacetic acid to obtain ester 108. Next, this was converted to acylhydrazide 110, and further converted to an acylazide intermediate using tert-butyl nitrite under acidic anhydride conditions. After the formation of the acylazide intermediate, which was tracked by LC-MS, the pH was neutralized with DiPEA, and then epoxy ketone 97 was added to obtain target compound 58 having an N-Alloc Dap at the P3 position. Using a catalytic amount of Pd(PPh3)4 and phenylsilane as an allyl scavenger, the N-Alloc protecting group of compound 58 was removed with a palladium catalyst to obtain the P3 NH2Dap target compound 59. Finally, compound 59 was acetylated in pyridine with acetic anhydride to obtain compound 60. The synthesis described above was repeated using glutamine as the starting material to synthesize diaminobutanoic acid (Dab) derivatives 61, 62, and 63 (Scheme 4). [ka] Scheme 4. Synthesis of P3-modified LU-005i-based proteasome inhibitors. Reagents and conditions: (a) PIDA, THF:H2O 1:1, 0℃; (b) Alloc-OSu, pyridine, DCM, rt, 10⁴:94%, 10⁵:90%; (c) i: TFA, DCM, rt, ii: TFA·NH2-Tyr(OMe)-OMe, HCTU, DIPEA, DMF, rt, 10⁶:54%, 10⁷:80%; (d) i: TFA, DCM, rt, ii: 2-morpholinoacetic acid, HCTU, DIPEA, DMF, rt, 10⁸:7 8%, 109:77%; (e) Hydrazine hydrate, MeOH, rt, quantitative (f) i:tBuONO, HCl, DMF, -30℃, ii:97, DiPEA, DMF, -30℃->rt, 58:54%, 61:32%; (g) Pd(PPh3)3, phenylsilane, DCM, 0℃, 59:43%, 62:64%; (h) Ac2O, pyridine, DMF, rt, 60:93%, 63:95%.

[0181] In the synthesis of compounds 64-67, the carboxylic acid side chain of aspartic acid 112 was activated with isobutyl chloroformate in the presence of N-methylmorpholine (pKa 7.38) as a proton scavenger. Using a stronger base such as triethylamine (pKa 10.75) as a proton scavenger resulted in the formation of byproducts. After the formation of the isobutyl anhydride intermediate, sodium borohydride was added to reduce the anhydride intermediate to homoserine (HSer) derivative 114 (Scheme 5). Next, the benzyl ester of derivative 114 was saponified with lithium hydroxide to obtain carboxylic acid 116. Subsequently, the carboxylic acid and alcohol were deprotonated with 2 equivalents of sodium hydride, followed by the addition of 1 equivalent of benzyl bromide to obtain O-Bn homoserine 118. Next, carboxylic acid 118 was condensed with 4-methoxyphenylalanine methyl ester to form N-Boc dipeptide 120. Next, N-Boc dipeptide 120 was de-N-Boc'd using TFA in DCM, and the resulting free amine was condensed with 2-morpholinoacetic acid to obtain ester 122. Then, the ester in 122 was saponified with lithium hydroxide in an equal mixture of H2O and methanol to obtain carboxylic acid 124. Next, compound 124 was condensed with epoxy ketone 97 using PyBOP as a peptide coupling reagent to form O-Bn HSer compound 64. Under a hydrogen-saturated atmosphere, the O-Bn protecting group was removed by hydrogenation with Pd on the carbon to obtain OH HSer compound 65. To synthesize HSer compounds 69 and 70, N-Boc piperazinyl was condensed with de-N-Boc'd dipeptide 120 instead of 2-morpholinoacetic acid, and then the synthetic route described above was carried out. Similarly, the above synthetic route was repeated using N-Boc glutamic acid 113 to obtain O-Bn pentahomoserine (HHSer) target compounds 66 and 71, and P3 OH HHSer target compounds 67 and 72. [ka] Scheme 5. Synthesis of P3 and P4 modified LU-005i-based proteasome inhibitors. Reagents and conditions: (a) i: Isobutyl chloroformate, NMM, THF, 0°C; ii: NaBH4, MeOH 0°C->rt, 114:81%, 115:71%; (b) LiOH, H2O / MeOH 1:1, rt, quantitative (c) NaH, BnBr, DMF, 0℃->rt, 118:62%, 119:59%; (d) i: TFA, DCM, rt, ii: TFA·NH2-Tyr(OMe)-OMe, HCTU, DIPEA, DMF, rt, 120:26%, 121:29%; (e): TFA, DCM, rt, ii: 2-morpholinoacetic acid, HCTU, DIPEA, DMF, rt, 122:75%, 123:84%; (f): TFA, DCM, rt, ii: N-Boc piperazinyl acetate, HCTU, DIPEA, DMF, rt, 126:83%, 127:93%; (g) LiOH, H2O / MeOH 1:1, rt, quantitative; (h)97, PyBOP, NMM, DMF, rt, 64:91%, 65:84%, 69:88%, 71:70%; (i) Pd / C, H2, MeOH, rt, 65:85%, 67:84%, 70:75%, 72:95%.

[0182] To construct compounds 68 and 73, the final target compounds of the focused library, N-Boc asparagine (Asn) was condensed with 4-methoxyphenylalanine to obtain Asn dipeptide 132 (Scheme 6). 132 was de-N-Bocified using TFA in DCM, followed by condensation with 2-morpholinoacetic acid to obtain methyl ester 134. This was then converted to acylhydrazide 136 by treatment with hydrazine hydrate. Acylhydrazide 136 was then converted to the corresponding acylazide intermediate 138 using tert-butyl nitrite under acidic anhydride conditions, and then epoxy ketone 97 was added to form P3 Asn derivative 68. Furthermore, the above synthetic route was repeated using N-Boc glutamine (Gln) to obtain P3 Gln derivative 73. Of particular note is that when preparing methyl ester 134 by condensation of dipeptide 132 with 2-morpholinoacetic acid, the conversion was observed to be successful only when 2-morpholinoacetic acid was pre-activated with HCTU. All other attempts resulted in the formation of a glutaming anidinium adduct as the main product of the reaction. [ka] Scheme 6. Synthesis of P3 analog of LU-005i. Reagents and conditions: (a) TFA·NH2-Tyr(OMe)-OMe, HCTU, DIPEA, DMF, rt, 132:82%, 133:82%; (b): TFA, DCM, rt, ii: 2-morpholinoacetic acid, HCTU, DIPEA, DMF, rt, 134:88%, 135:35%; (c) Hydrazine hydrate, MeOH, rt; (d) i: tBuONO, HCl, DMF, -30℃, ii:97%, DiPEA, DMF, -30℃->rt, 68:36%, 73:42%.

[0183] Reference Example 7 - Competitive ABPP assay in Raji cell lysates The biological activity of the compound in Example 6 was evaluated using the biological assay described in Example 2.

[0184] The apparent IC obtained 50 The values ​​were plotted on the heatmap shown in Figure 4. The potency and selectivity profiles of all compounds listed in Figure 4 as proteasome inhibitors were determined using a competitive ABPP assay with Raji cell lysates (a B-cell lymphoma cell line expressing both cCP and iCP). Compounds 58–73 were tested in a concentration series from 0.003 μM to 30 μM, and the apparent inhibitory concentration (IC) against each catalytically active proteasome subunit was determined. 50 ) was determined. The apparent IC obtained 50 The values ​​were plotted as a heatmap, as shown in Figure 4. Using the heatmap, it is possible to determine the initial structure-activity relationship (SAR) for the LU-005i analog library, as well as the relationship between the P3 and P4 substituents and the cCP / iCP inhibition profile.

[0185] In evaluating the inhibitory efficacy of the series of compounds, two groups of compounds were deemed noteworthy: one group exhibiting high efficacy against all three iCP subunits, and another group exhibiting less pronounced efficacy against these activities but high selectivity for the corresponding cCP activity. Morph-HSer derivative 65, Morph-Asn derivative 68, and BocPip-HSer derivative 70 all fall into one of these two groups. All three are characterized by a P3 residue with identical length from the alpha carbon to the side chain function. Morph-Asn 68 and BocPip-HSer 70 both showed strong efficacy against the iCP subunits, surpassing LU-005i 1 in this respect. On the other hand, Morph-HSer 65 outperformed Morph-Ser 28, but showed a similar inhibitory profile compared to LU-005i 1. More importantly, Morph-HSer65 was found to be the proteasome inhibitor with the strongest selectivity for iCPs compared to cCPs.

[0186] Proteasome inhibitors with Dap or Dab at the P3 position did not outperform LU-005i1, nor did they show any improvement over previously screened inhibitors 28 and 30. Compounds 58 and 60 showed lower efficacy against β1c and β2c compared to LU005i1, but their efficacy against β5c increased fivefold. On the other hand, compounds 59 and 62 were found to be relatively weak β1i inhibitors. The basic NH2 of Dap59 and Dab62 is protonated at pH 7, making them likely incompatible with the S3 binding pocket of β1i. Surprisingly, the decrease in inhibitory efficacy observed in 62 compared to LU-005i was not observed in 65, supporting the idea that the decrease in efficacy is due to the charged amine rather than an increase in carbon chain length. Compound 60 was found to be a weak inhibitor against both β1 and β2i compared to compound 59, while simultaneously being a highly selective β5i inhibitor. Overall, the introduction of Dap or Dab at the P3 position did not result in compounds with the desired combination of increased potency against β1i and β2i and decreased potency against β5c.

[0187] Previous structure-activity relationship studies revealed that compound 30 was an inadequate inhibitor of all proteasome active sites β1i, β1c, β2i, β2c, β5i, and β5c. Contrary to intuition, analogs 64 and 66, which have benzylated homoserine (as in 30) rather than benzylated serine, both showed higher potency as inhibitors of β1i, β2i, β5c, and β5i compared to 28. Furthermore, when comparing the benzylated analogs 64 and 66 with the OH-modified analogs 65 and 67, 70 and 72 proved to be more potent against β1i, β2i, and β5c. However, when considering the ratio of lowest cCP inhibition to highest iCP inhibition, compound 65 appeared to outperform compound 2. Compounds with asparagine or glutamine at the P3 position yielded unexpected inhibition profiles. Intuitively, asparagine and glutamine would be expected to exhibit similar inhibitory profiles to their carbon side-chain analogues, homoserine (65) and pentahomoserine (67). In contrast, compounds 68 and 73, containing asparagine and glutamine, were shown to be significantly potent inhibitors of β1i, β2i, and β5c compared to their respective homoserine (65) and pentahomoserine (67) counterparts. As observed in the comparisons of Morph-Dap59 vs. Morph-Dab62 and Morph-HSer65 vs. Morph-HHSer67, increasing carbon side-chain length resulted in decreased potency against either β1i or β2i, or both.

[0188] Previously analyzed data showed that LU-005i 1, which has a 2-morpholinoacetyl cap at the N-terminus, was more potent against all iCP activity than the same compound with an N-Bocpiperazinyl cap. In contrast, compound 28 was found to be insufficient as an inhibitor against all cCP / iCP active sites, while BocPip-Ser(OBn)35 inhibited all six cCP / iCP active sites, albeit with varying potency. The one-carbon homolog (with respect to the P3 portion) 69 and the two-carbon homolog 71 showed nearly equivalent cCP / iCP potency and selectivity to compound 34, which has serine at P3. In contrast, compound 36, which lacks an O-benzylated P3 residue, was found to be a more potent β1i and β2i inhibitor compared to LU-005i 1. Interestingly, this compound was also proven to be a 3-fold potent β2c inhibitor compared to BocPip-Ser(OBn)35.

[0189] X-ray analysis of BocPip-Ser(OBn)35 revealed that the N-Boc piperazine cap exhibits strong hydrophobic interactions with the yβ2 and yβ5 residues in the S4 pocket. This allows the P4 residues of BocPip to be immobilized in the S4 pocket in a way that is not possible with smaller hydrophilic morpholine caps. The apparent IC is shown in Figure 5. 50 The data supports this, and we can conclude that the β2i inhibitory activity in this class of compounds is independent of the properties of the P3 residue.

[0190] Similarly, a thorough examination of inhibitors with serine, homoserine, or pentahomoserine at P3 revealed that Morph-HSer65 was the strongest inhibitor of β1i and β2i, and also exhibited the best ratio of weak cCP inhibition to strong iCP inhibition. Furthermore, compounds with amine residues at P3 were less potent β1i and β2i inhibitors compared to OH analogs (65 and 67) and LU-005i 1.

[0191] Example 8 - Synthesis of Compounds 74-76 Compounds 74-76, shown below, were synthesized by methods similar to those described in Examples 1, 3, and 6. Further details of the synthesis will be described later.

[0192] In Example 1, the synthesis of epoxy ketone 83 was described. Since a large amount of epoxy ketone 83 was obtained, we focused on the synthesis of a P2-P3-P4 fragment having homoserine at the P3 position (Scheme 7). In the synthesis of target peptide epoxy ketones 74-76, the carboxylic acid side chain of aspartic acid 112 was activated with isobutyl chloroformate, and then sodium borohydride was added to reduce the anhydrous intermediate to homoserine derivative 114. Next, the benzyl ester of derivative 114 was saponified with lithium hydroxide to form carboxylic acid 116. Then, 2 equivalents of sodium hydride were added, followed by 1 equivalent of benzyl bromide to obtain O-Bn homoserine 118. O-Bn homoserine 118 was successively condensed with 4-methoxyphenylalanine methyl ester to obtain N-Boc dipeptide 120. N-Boc deprotection of N-Boc dipeptide 120 using TFA in DCM yielded a free amine, which was then condensed with 2-morpholinoacetic acid to obtain methyl ester 122. Next, methyl ester 122 was converted to acyl hydrazide 140, which could be converted to an acyl azide intermediate using tert-butyl nitrite under acidic anhydride conditions. After monitoring by LC-MS to confirm complete conversion, the reaction was neutralized with DiPEA, followed by the addition of epoxy ketone 83 to form target compound 74. Furthermore, the O-benzyl protecting group of target compound 74 was removed by hydrogenation using palladium on carbon under a hydrogen-saturated atmosphere to obtain OHHSer target compound 75. Next, the OH group of target compound 74 was functionalized by Steglich esterification with N-Boc alanine to form N-Boc alanine ester compound 76. Subsequently, deprotection of the O-Bn group of N-Boc alanine ester compound 76 was successfully achieved using palladium on carbon under a hydrogen-saturated atmosphere, but an unfortunate side reaction of epoxide hydrogenation cleaved the CO bond, forming an alcohol moiety. [ka] Scheme 7.P3 Synthesis of HSer LU-005i-OH analogs 74, 75 and N-Boc alanine ester derivative 76. Reagents and conditions: (a) i: Isobutyl chloroformate, NMM, THF, 0°C, ii: NaBH4, MeOH, 0°C->rt, 81%; (b) LiOH, H2O / MeOH 1:1, rt, quantitative; (c) NaH, BnBr, DMF, 0°C->rt, 62%; (d) i: TFA, DCM, rt, ii: NH2-Tyr(OMe)-OMe, HCTU, DiPEA, DMF, rt; (e) i: TFA, DCM, rt, ii: 2-morpholinoacetic acid, HCTU, DiPEA, DMF, rt, 75%; (f) Hydrazine hydrate, MeOH, rt; (g) i: tB uONO, HCl, DMF, -30℃, ii:83, DiPEA, DMF, -30℃->rt, 69%; (h) Pd / C, H2, MeOH, rt; (i) NHBoc-Ala-CO2H, D IC, DMAP, DCM, rt;(j)i:Pd / C, H2, MeOH, rt, ii:TFA, DCM, rt;(k)i:Pd / C, H2, MeOH, rt, ii:TFA, DCM, rt.

[0193] Example 9 - Competitive ABPP assay in Raji cell lysates The potency and selectivity profiles of the synthesized compounds 74–76, compared to compounds 2, 3, 14, 65, and 68 as proteasome inhibitors, were determined using a competitive ABPP assay in Raji cell lysates (a B-cell lymphoma cell line expressing both cCP and iCP). To further extend the overall applicability of the novel proteasome inhibitors, AMO wild-type cells were selected as an additional cell lysate and used to evaluate the proteasome inhibitors (AMO-wt is a plasmacytoma cell line expressing both cCP and iCP; Figure 6).

[0194] Compounds 74-76 and lead structures 2, 3, 14, 65, and 68 were tested at concentrations ranging from 0.003 μM to 10 μM, and the apparent inhibitor concentration (IC) was determined in both Raji cell lysates and AMO-wt cell lysates. 50 ) was determined. The apparent IC obtained 50The values ​​were plotted as heatmaps, as shown in Figures 5 and 6. All compounds were found to be weak inhibitors of β1c and β2c in Raji cell lysates (Figure 5). As shown in Reference Example 7, peptide epoxy ketones with O-benzyl homoserine at the P3 position are potent inhibitors of the β1i, β5c, and β5i active sites. Surprisingly, compounds 74 and 76 were as potent against β1i, β5c, and β5i as Morph-HSer(OBn)65, and increased potency against β2i was also observed in these compounds compared to LU-005i1. Furthermore, compound 76 showed a 4-fold decrease in potency against β1i and a 3-fold decrease in potency against β2i compared to 74. Compared to the compounds described in Reference Example 7, Morph-HSer65 was the only inhibitor that showed increased potency against β1i, β2i, and β5i and decreased potency against β5c compared to LU-005i1. Compound 75, which has homoserine at the P3 position and hydroxymethyl at the P1′ position, was found to be a more selective iCP inhibitor compared to Morph-HSer65. Specifically, when comparing compounds 75 and 65, the efficacy of β2i increased by more than 10 times, and the efficacy of β5c decreased by 2 times. When 75 was compared with LU-005i-OH 2, the efficacy of β2i increased by 4 times, the efficacy of β5c decreased by 2 times, and the efficacy of β1i decreased by 5 times.

[0195] Figure 6 shows the cCP / iCP active sites (inhibitory titers of compounds 2, 3, 14, 65, and 68 in AMO-wt lysates). IC25 of compounds 1-9 obtained from Raji lysates (Figure 5). 50 The IC values ​​and the IC values ​​of compounds 2, 3, 14, 65, and 68 obtained from AMO wild-type lysates (Figure 6). 50When compared with the values, the overall inhibition profiles appeared to be very similar. Interestingly, LU-005i-OH 2 proved to be a much more potent inhibitor against both the cCP and iCP active subunits of AMO-wt. Furthermore, LU-005i-OH 2 showed iCP selectivity in AMO-wt lysates as well as in Raji lysates, with the potency against the iCP active subunit being at least twofold compared to the potency against the cCP active subunit. N-Boc alanine ester 3 showed IC25 in AMO-wt lysates. 50 Values ​​and IC in Raji dissolved material 50 Comparing the values, the potency against β1i decreased by 2 times, the potency against β5i decreased by 10 times, and the potency against β2i increased by 6 times. Surprisingly, alanine-NH214 was not a very potent inhibitor against β1i and β2i, but it was found to be a more potent inhibitor against β1c and β2c. In the Raji lysate, compound 14 showed iCP selectivity, with at least a 6-fold difference in potency between the iCP active site and the cCP active site. In the AMO-wt lysate, compound 14 was found to be less selective towards the iCP active site compared to the Raji lysate. On the other hand, compound 75 showed IC25 in the Raji lysate. 50 Values ​​and IC in AMO-wt dissolved material 50 Comparing the values, compound 75 was found to be more selective against the iCP active subunit, with the AMO-wt lysate showing nearly a twofold decrease in potency against β5c. In contrast, comparing the inhibitory profiles of compound 75 and 65 in AMO-wt lysates, compound 75 was found to be a much potent inhibitor against β1i and β2i, in addition to being more selective against the iCP active site.

[0196] The following abbreviations may be used in this specification. aq Aqueous (Note: Translated as "aqueous solution" or "water layer" depending on the context) Boc tert-butoxycarbonyl DCM Dichloromethane DIC N,N′-Diisopropylcarbodiimide DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF Dimethylformamide DMSO (Dimethyl Sulfoxide 5) HCl ethyl acetate EtOH Ethanol HCTU O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate MeCN acetonitrile MeOH methanol NMR nuclear magnetic resonance rt room temperature Rt retention time TBS tert-butyldimethylsilyl TFA (Trifluoroacetic Acid) THF (Tetrahydrofuran) TLC (Thin-Layer Chromatography)

Claims

1. A compound of formula (I), 【Chemistry 1】 During the ceremony, R ａ and R ｂ One side represents H, and the other side represents -[CH ２ ] ｍ -X represents, X represents -OC(O)-Y, -C(O)-Y, -OH, or OY, Y is -C １－６ alkyl (-Z １ -Z ２ optionally substituted by) or -CH(R ｙ )- (Z １ -Z ２ ) ｎ represents, R ｙ However, it represents a methyl biphenyl or protein amino acid side chain, and the side chain is optionally chemically protected. Each Z １ However, independently, -NH-, -N(R ｚ ) -, or -O-, R ｚ However, if it exists, R ｙ It combines with to form a proline ring, Each Z ２ However, independently, H, -C(O)-C １－４ Alkyl, -C(O)O-C １－４ Alkyl, or -Si(C １－４ Alkyl) ３ And, m is 0, 1, or 2, n is 1 or 2, p is 0 or 1, R ｃ However, -C is optionally substituted with H or one or more Q substituents. １－４ Represents alkyl, Q is -OR ｄ , - NHR ｅ , or -C(O)NHR ｆ This represents, R ｄ , R ｅ , and R ｆ However, H, -C １－４ Alkyl (optionally substituted with phenyl or methylphenyl), -C(O)-C １－４ Alkyl, -C(O)O-C １－４ Alkyl, -C(O)-C １－４ Alkenyl, -C(O)O-C １－４ Alkenyl, -C(=N)-NH ２ , -C(=N)-NH- (protecting group), or pyrimidinyl, A, 【Chemistry 2】 This represents a ring portion selected from the group consisting of the following: R ｈ However, H, C １－４ Alkyl, or -C(O)O-C １－４ Represents alkyl, R ｉ However, H, C １－４ Alkyl, -N(R ｊ ) (Caution ｋ ), or -OH, R ｊ and R ｋ However, independently H or C １－４ Representing alkyl, A compound, or a pharmaceutically acceptable salt or solvate thereof.

2. R ｂ The compound according to claim 1, wherein represents hydrogen.

3. The compound according to claim 1 or claim 2, wherein X represents -OC(O)-Y, -C(O)-Y, or -OY.

4. Y is -C ３－５ Alkyl (-Z １ -Z ２ (Optionally replaced by) or -CH (R ｙ )-(Z １ -Z ２ ) ｎ A compound according to any one of the above claims, which represents the compound described in any one of the above claims.

5. R ｙ The compound according to any one of the claims, wherein the compound represents methyl biphenyl, a side chain of a proteinogenic amino acid, or a chemically protected form of a side chain of a proteinogenic amino acid, wherein the chemical protection involves the attachment of a moiety selected from the group consisting of a benzoyl group, a benzyl group, and a trityl group.

6. A compound according to any one of the above claims, (i) m is 0 or 1, and / or (ii) A compound in which n' is 1.

7. Q is -NH ２ , -NH-C(O)-CH ３ , -NH(Alloc), -NH-C(=N)-NH ２ , -NH-C(=N)-NH- (protecting group), -C(O)-NH ２ The compound according to any one of the claims, representing -OH, -O-benzyl, -O-xylyl, or -NH (pyrimidinyl).

8. R ｃ The compound according to any one of claims 1 to 6, wherein the compound represents a serine side chain, a protective derivative of a serine side chain, or methyl.

9. The compound according to any one of the claims, wherein A represents a morpholinyl group.

10. A compound according to any one of the above claims, wherein the compound is 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】

11. A pharmaceutical formulation comprising a compound defined in any one of claims 1 to 10 in combination with a pharmaceutically acceptable excipient.

12. A compound defined in any one of claims 1 to 10, or a pharmaceutical formulation defined in claim 11, for use in medical treatment.

13. A compound as defined in any one of claims 1 to 10, or a pharmaceutical formulation as defined in claim 11, for use in the treatment or prevention of diseases or conditions in which total immunoproteasome inhibition is desirable or required.

14. The compound or pharmaceutical formulation for use according to claim 13, wherein the disease or condition is a hematological malignancy, a solid tumor, an autoimmune disease, or an inflammatory disease.

15. The compound or pharmaceutical formulation for use according to claim 13 or claim 14, wherein the disease or condition is selected from the group consisting of leukemia, lymphoma, myeloma (including multiple myeloma), myelodysplastic syndrome, myeloproliferative syndrome, prostate cancer, breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, osteosarcoma, Alzheimer's disease, encephalitis, colitis-related cancer, angiogenesis, viral myocarditis, acute kidney injury, ischemic stroke, premature birth, abdominal aortic aneurysm, atherosclerosis, cardiac remodeling, graft-versus-host disease (GvHD), inflammatory bowel disease, arthritis, polymyositis, dermatomyositis, autoimmune hepatitis, and lupus nephritis.

16. A process for preparing a compound of formula (I) as defined in claim 1, comprising the step of reacting a compound of formula (II) with a compound of formula (III), 【Chemistry 9】 In equation (II), A, p and R ｃ However, as defined in claim 1, 【Chemistry 10】 A process in formula (III), where Y is as defined in claim 1.

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