Compounds and uses

Abstract

Provided herein are compounds of formula (I) wherein Ra, Rb, Rc, A and p have the meanings given in the specification, as defined in formula (I), as well as pharmaceutically acceptable salts and solvates thereof, which are useful in the treatment of diseases in which pan-immune proteasome inhibition is desired or desired, and in particular 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. Specifically, this invention relates to the use of such compounds and compositions in methods for treating or preventing diseases or conditions in which pan-immunoproteasome inhibition is desired or required, such as proliferative disorders and autoimmune diseases. Background Technology

[0002] Proteasomes are essential for maintaining protein homeostasis and participate in a variety of key 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). The dominant proteasome types in mammals are constitutive proteasomes, present in all somatic cells, and immunoproteasomes, primarily expressed by hematopoietic cells (Kniepert, A.; Groettrup, M., The unique functions of tissue-specific proteasomes. Trends in biochemicalsciences 2014, 39(1), 17-24.). Both constitutive and immunoproteasomes are characterized by a 20S core particle (CP; cCP represents constitutive proteasomes and iCP represents immunoproteasomes), which consists of 14 α- and 14 β-subunits, which are expressed in α-... 1-7 β 1-7 β 1-7 α 1-7 The components are arranged in a manner that forms a hollow cylinder. The β1, β2, and β5 subunits possess proteolytic activity and hydrolyze peptides fed into the CP tank into oligopeptides of eight to ten amino acids. The main structural difference between cCP and iCP lies in the catalytic active site and its substrate binding channel. cCP incorporates β1c (cystase-like activity (CL)), β2c (trypsin-like activity (TL)), and β5c (chymotrypsin / elastase-like (ChT-L activity)) subunits, which are replaced in iCP by β1i (ChT-L), β2i (TL), and β5i (ChT-L).

[0003] Research into proteasome inhibition began in the early 1990s and culminated in the approval of bortezomib (2003), carfilzomib (2012), and ixazomib (2015) for the treatment of multiple myeloma (Manasanch, EE; Orlowski, RZ, Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol 2017, 14 (7), 417-433.). These compounds primarily target the β5c and β5i of cCP and iCP, respectively. However, depending on the dose, other catalytic activities can also be co-inhibited. Constitutive proteasomes are present in all healthy tissues and are classified as off-target when examining proteasome inhibition in a clinical setting. Selective inhibition of the immunoproteasome has been sought over the past decade to minimize side effects, reduce cytotoxicity, and explore potential anti-inflammatory applications. However, it is evident that the combined inhibition of several subunits is required to produce a treatment-related effect (Johnson, HWB, et al., Required Immuno-proteasome SubunitInhibition 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-epoxyethylene-2-yl)-1-oxo-propane-2-yl)-3-hydroxy-3-(4-methoxy-phenyl)-2-((S)-2-(2-morpholino-acetamido)propamido)-acrylamide). J Med Chem 2018, 61 (24), 11127-11143.).

[0004] Proteasome inhibitors (i.e., carfilzomib and bortezomib) have also been shown to induce resistance pathways, thus rendering proteasome inhibition unusable as a treatment for these hematologic cancers. One possible approach to overcome the resistance and cytotoxicity caused by constitutive proteasome inhibition observed in hematologic cancers is to develop proteasome inhibitors that specifically inhibit the immunoproteasome, a pan-immunoproteasome inhibitor.

[0005] Although compounds targeting at least two immune subunits with good affinity have been reported (ONX 0914 (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 pan-proteasome inhibitor that blocks all three immune subunits with the same potency while leaving the c-subunit unaffected remains lacking.

[0006]

[0007] It has now been surprisingly discovered that certain compounds can act as inhibitors of the immunoproteasome, particularly pan-immunoproteasome inhibitors, which may be used to treat or prevent diseases or conditions in which pan-immunoproteasome inhibition is desired or required (such as proliferative diseases and autoimmune diseases).

[0008] The enumeration or discussion of previously published documents in this specification should not necessarily be regarded as an admission that such documents are part of the prior art or common general knowledge. Summary of the Invention

[0009] In a first aspect of the invention, a compound of formula (I) is provided.

[0010]

[0011] in:

[0012] R a and R b One of them represents H, and the other represents –[CH2]. m -X;

[0013] X represents -OC(O)-Y, -C(O)-Y, -OH, or OY;

[0014] Y represents -C 1-6 Alkyl (optionally -Z) 1 -Z 2 (replace) or -CH(R) y )-(Z 1 -Z 2 ) n ;

[0015] R y The side chain represents a methyl biphenyl or a proteogenic amino acid, optionally wherein the side chain is in a chemically protected form;

[0016] Each Z 1 Independently -NH-, -N(R) z - or -O-;

[0017] R z If it exists, it is related to R. y Combined to form a proline ring;

[0018] Each Z 2 Independently H, -C(O)-C 1-4 Alkyl, -C(O)OC 1-4 Alkyl or -Si(C) 1-4 Alkyl)3;

[0019] m is 0, 1, or 2;

[0020] n is 1 or 2;

[0021] p is 0 or 1;

[0022] R c -C represents H or -C optionally substituted with one or more Q substituents. 1-4 alkyl;

[0023] Q means -OR d -NHR e or -C(O)NHR f ;

[0024] R d R e and R f Indicates H, -C 1-4 Alkyl groups (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 Alkenyl, -C(=N)-NH2, -C(=N)-NH- (protecting group), or pyrimidinyl;

[0025] A represents the circular portion selected from the following groups:

[0026]

[0027] R h Represents H and C 1-4 Alkyl or -C(O)OC 1-4alkyl;

[0028] R i Represents H and C 1-4 Alkyl, -N(R) j (R) k ) or -OH;

[0029] R j and R k Independently represent H or C 1-4 alkyl;

[0030] Or its pharmaceutically acceptable salts or solvates.

[0031] Compounds of formula (I) (including their pharmaceutically acceptable salts and solvates) are referred to herein as “compounds of the present invention”.

[0032] For the avoidance of doubt, those skilled in the art will understand that references herein to compounds of a particular aspect of the invention (such as the first aspect of the invention, i.e., relating to compounds of formula (I) as defined in the first aspect of the invention) will include references to all embodiments thereof and their particular features, which may be combined to form further embodiments and features of the invention.

[0033] Pharmaceutically acceptable salts include acid addition salts and base salts. Such salts can be formed by conventional means, for example by reacting the free acid or free base form of the compounds of the present invention with one or more equivalents of a suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques (e.g., under vacuum, by freeze-drying, or by filtration). Salts can also be prepared using techniques known to those skilled in the art, such as by exchanging the counter ion of the compounds of the present invention in salt form with another counter ion (e.g., using a suitable ion exchange resin).

[0034] Specific acid addition salts that may be mentioned include carboxylates (e.g., formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, caprate, octanoate, stearate, acrylate, hexanoate, propargylate, 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, carboxylates, etc.). Cinnamates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, malates, maleates, hydroxymaleates, hippurates, phthalates, or terephthalates), halide salts (e.g., chlorides, bromides, or iodides), sulfonates (e.g., benzenesulfonates, methylbenzenesulfonates, bromobenzenesulfonates, or chlorobenzenesulfonates, xylenesulfonates, methanesulfonates, ethanesulfonates, propanesulfonates, hydroxyethanesulfonates, 1- or 2-naphthalenesulfonates, or 1,5-naphthalenedisulfonates), or sulfates (e.g., dihydrogen sulfates), pyrosulfates, hydrogen sulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, or nitrates, etc.

[0035] Specific alkali salts that may be mentioned 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, alkali addition salts that may be mentioned include Mg salts, Ca salts, and most particularly K and Na salts.

[0036] More specific salts that can be mentioned include trifluoroacetate and dicyclohexylamine.

[0037] To avoid ambiguity, the compounds of the present invention may exist as solids, and therefore the scope of the invention includes all 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 included within the scope of the invention.

[0038] "Solvate" refers to a solid form in which the associated compound (e.g., compound of formula (I)) is associated with one or more solvent molecules. The term solvate includes hydrates and other solvates in pharmaceutically acceptable solvents. Preferred solvents for forming solvates are water and DMSO.

[0039] To avoid ambiguity, the compounds of the present invention may also exist in solution (i.e., in a solution in a suitable solvent). For example, the compounds of the present invention may exist in aqueous solution form, in which case they may exist as hydrates.

[0040] The compounds of this invention may contain double bonds, and unless otherwise stated, may therefore exist as E (entgegen) and Z (zusammen) geometric isomers with respect to each individual double bond. Unless otherwise stated, all such isomers and mixtures thereof are included within the scope of this invention.

[0041] The compounds of this invention can also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of this invention (especially those with sufficient stability to allow for their isolation).

[0042] "Amino acid" and "residue" (e.g., phenylalanine "residue") refer to the dehydrated portion of an amino acid present in a polypeptide chain and represented by the following formula.

[0043] Where SC represents the amino acid side chain. To avoid ambiguity, the term "amino acid" includes non-proteinogenic amino acids unless otherwise stated.

[0044] "Amino acid side chain" or "side chain of an amino acid" refers to a group attached to the position of the carboxyl and amino groups in an α-amino acid, including 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 unassuming names and will be aware of the side chain groups present in these amino acids.

[0045] "Proteinogenic" amino acids are the 22 amino acids that are naturally encoded or naturally present in the genetic code of an organism. "Non-proteinogenic" amino acids are those that are not naturally encoded or present in the genetic code of any organism. The non-proteinogenic amino acid group is generally considered to include all organic compounds having an amine (-NH2) or carboxylic acid (-COOH) functional group linked via a single extra carbon atom, along with a side chain and hydrogen atom bound to that extra carbon atom, but excluding selenocysteine, pyrrolidone, and the 20 standard amino acids incorporated into proteins during translation. Non-proteinogenic amino acids include those that are intermediates in biosynthesis, those that are formed post-translationally in proteins, and those that have physiological functions (e.g., components of bacterial cell walls, neurotransmitters, and toxins).

[0046] The compounds of the present invention contain at least one asymmetric carbon atom and therefore can exhibit optical and / or diastereomeric phenomena (i.e., exist in enantiomer or diastereomeric form). Diastereomers can be separated using conventional techniques (e.g., chromatography or stepwise crystallization). Various stereoisomers (i.e., enantiomers) can be separated by using conventional techniques (e.g., stepwise crystallization or HPLC) to separate racemic mixtures or other mixtures of the compounds. Alternatively, the desired enantiomer or diastereomer can be obtained from a suitable starting material by reacting it with a chiral auxiliary under conditions that do not cause racemization or epimerization (i.e., the "chiral pool" method). This chiral auxiliary can then be separated at a suitable stage by derivatization (i.e., resolution, including dynamic resolution; for example, with a pure chiral acid), followed by conventional methods such as chromatography, or by reaction with a suitable chiral reagent or chiral catalyst. All methods and processes can be carried out under conditions known to those skilled in the art. Unless otherwise stated, all stereoisomers and mixtures thereof are included within the scope of this invention.

[0047] Unless otherwise specified, C as defined in this document 1-z The alkyl group (where z is the upper limit of the range) can be straight-chain, or branched and / or cyclic (thus forming C) when there are a sufficient number (i.e., at least two or three, if appropriate) of carbon atoms. 3-z Cycloalkyl groups). Such groups can also be partially cyclic when a sufficient number (i.e., at least four) of carbon atoms are present (thus forming C1646). 4-z Partially cycloalkyl groups). For example, cycloalkyl groups that may be mentioned include cyclopropyl, cyclopentyl, and cyclohexyl. Similarly, partially cycloalkyl groups that may be mentioned (which may also be referred to as “partially cycloalkyl” groups) include cyclopropylmethyl. To avoid ambiguity, specific alkyl groups that may be mentioned include straight-chain (i.e., unbranched and / or cyclic) alkyl groups.

[0048] Unless otherwise specified, C as defined herein 2-z The alkenyl group (where z is the upper limit of the range) can be linear, or branched and / or cyclic (thus forming C when a sufficient number (i.e., at least three) of carbon atoms are present) 4-z Cyclic alkenyl groups). Such groups can also be partially cyclic when a sufficient number (i.e., at least five) of carbon atoms are present. To avoid ambiguity, specific alkenyl groups that may be mentioned include straight-chain (i.e., unbranched and / or cyclic) alkenyl groups.

[0049] As may be used in this article, the term aryl can refer to C 6-14 (e.g., C) 6-10Aromatic groups. These groups can be monocyclic or bicyclic, and when bicyclic, they can be wholly or partially aromatic. C may be mentioned. 6-10 Aryl groups include phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, indanyl, etc. (e.g., phenyl, naphthyl, etc.). To avoid ambiguity, the substituent can be attached to the aryl group via any suitable carbon atom in the ring system.

[0050] For the avoidance of doubt, those skilled in the art will understand that the aryl groups that can form part of the compounds of the present invention are those that can be obtained by chemical methods as known to those skilled in the art. Specific aryl groups that may be mentioned include phenyl and naphthyl groups, such as phenyl.

[0051] This invention also includes compounds of the invention labeled with the same isotopes as those described herein, but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature (or the most abundant kind found in nature). All isotopes of any particular atom or element specified herein are contemplated within the scope of the compounds of the invention and their uses. Therefore, the compounds of the invention also include deuterated compounds, i.e., compounds of the invention in which one or more hydrogen atoms are replaced by the hydrogen isotope deuterium.

[0052] To avoid ambiguity, in compounds of the present invention where the identity of two or more substituents may be identical, the actual identity of the corresponding substituents does not depend on each other in any way. For example, in the presence of two or more Z... 1 In the case of groups, those Z 1 The groups can be the same or different. Similarly, when there are two or more Z groups... 2 Groups and each representing -C(O)-C 1-4 In the case of alkyl groups, the -C(O)-C discussed 1-4 The alkyl groups can be the same or different.

[0053] Similarly, to avoid ambiguity, when terms such as “1 to 4” are used herein, they will be understood by those skilled in the art to mean 1, 2, 3, and 4, including end values. Unless otherwise stated, the same reasoning will apply to other such terms used herein.

[0054] Furthermore, to avoid ambiguity, when the specified substituent is optionally substituted by one or more substituents (e.g., optionally substituted by one or more independently selected from -Z), 1 -Z 2 C groups substituted 1-6Alkyl groups, which may be located on the same or different atoms where possible. Such optional substituents may be present in any suitable amount (e.g., the relevant group may be substituted by one or more such substituents, such as one such substituent).

[0055] To avoid ambiguity, where groups are referred to herein as optionally substituted, it is specifically envisioned that such optional substituents may be absent (i.e., the reference to such optional substituents may be removed), in which case the optionally substituted group may be referred to as unsubstituted.

[0056] To avoid ambiguity, those skilled in the art will recognize 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. In other words, the compounds of the present invention include those that are robust enough to survive separation, for example, from reaction mixtures to usable purity.

[0057] In the compound of formula (I), the molecules... The portion may be referred to as the P1' portion (or P1' region, or similar).

[0058] In a particular embodiment (i.e., a particular embodiment of the first aspect of the invention), R b H is used to represent R. To avoid ambiguity, R is used in such compounds. a Indicates -[CH2] m -X.

[0059] In another embodiment of the invention, R a Or R b (especially R) a ) indicates –[CH2] m -X, where m is 0 or 1. In a specific embodiment, m is 0.

[0060] In another embodiment, X represents -OC(O)-Y, -C(O)-Y, or -OY. Most particularly, X can represent -OC(O)-Y. Each of the groups can form R. a Or R b It is part of, but preferably they form R a Part of it. For such compounds, it is also preferred that m is 0.

[0061] In another embodiment, Y represents -C 3-5 Alkyl (optionally -Z) 1 -Z 2 (replace) or -CH(R) y )-(Z 1 -Z 2 ) n.

[0062] Specific compounds of the present invention include those comprising an amino acid (or a protected derivative thereof) as R. a Or R b (Preferably R) a Those that are part of a group. Such compounds include those where X represents -OC(O)-Y, -C(O)-Y, or OY, and Y represents -CH(R). y )-(Z 1 -Z 2 And -Z 1 - indicates -NH- or -N(R) z Those of )-. Therefore, in a particular embodiment, R a Or R b Some may terminate with an amino acid group or its protected derivative. Alternatively, these compounds of formula (I) can be defined as having R a and R b One of them represents H (preferably, R). b (H) and the other represents –[CH2). m -OT or –[CH2] m Compounds with a -T group, where T represents the carbonyl group of the amino acid and an oxygen atom or [CH2]. m A group of amino acid groups or their protected derivatives bound together. T can be structurally derived from -C(O)-CH(R) y )-NH-Z 2 or -C(O)-CH(R) y )-N(R z )-Z 2 This indicates that -Z is the value of -Z. 2 Compounds of this type with the -C(O)O-tert-butyl group are Boc-protected amino acids. Alternatively, they can be -Z 2 Other protecting groups known to those skilled in the art for protecting amines, such as carbyloxy (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bzl), and benzyl (Bn), are used.

[0063] R y The side chain represents a methylbiphenyl or proteogenic amino acid, optionally wherein the side chain is in a chemically protected form. Amino acid side chains containing primary amine or amide functional groups (e.g., the side chain of lysine) can be protected using any of Boc, Cbz, Fmoc, Ac, Bzl, and Bn, or any other suitable protecting group known to those skilled in the art (such as allyloxycarbonyl (Alloc)). Side chains containing carboxylic acid ester groups (e.g., the side chain of aspartic acid or glutamic acid) can be protected by conversion to esters (e.g., Bn ester, triphenylmethyl (Trt) ester, C...1-6 Alkyl esters, fluorenylmethyl (Fm) esters, or silyl esters, or protected with any other suitable protecting group known to those skilled in the art. Side chains containing alcohol functional groups (e.g., serine or threonine side chains) can be protected using C 1-4 Alkyl ethers, Bn, Ac, Bzl, triphenylmethyl (Trt), tetrahydropyranyl (Thp), and silyl ethers (such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), and tert-butyldimethylsilyl (TBDMS)) or any other suitable protecting group known to those skilled in the art. Side chains containing thiol functional groups (e.g., cysteine ​​side chains) can be protected using any of Trt, Bn, and tert-butyl or any other suitable protecting group known to those skilled in the art. Side chains containing guanidine / guanidinium functional groups (e.g., arginine side chains) can be protected using any of Cbz and 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf) or any other suitable protecting group known to those skilled in the art.

[0064] In a particular embodiment, R y This indicates a chemically protected form of a methyl biphenyl (BipA side chain), a proteomic amino acid side chain, or a proteomic amino acid side chain, wherein the chemical protection involves the linkage of a portion selected from the group consisting of benzoyl, benzyl, and triphenylmethyl groups.

[0065] Preferred compounds of the present invention include those in which n is 1. Other preferred compounds include those in which m is 0 or 1, and optionally n is 1.

[0066] In the compound of formula (I), R c The binding site can be referred to as the "P3" position. It has been found that structural variations at the P3 position are generally well-tolerated, especially when the ring at A is relatively small (i.e., it contains very few non-hydrogen substituents, if any). Therefore, in the compounds of the present invention, R... c It can represent H or -C optionally substituted with one or more Q substituents. 1-4 Alkyl group, where Q represents -OR d -NHR e or -C(O)NHR f And R d R e and R f Indicates H, -C 1-4 Alkyl groups (optionally substituted with phenyl or methylphenyl), -C(O)-C 1-4 Alkyl, -C(O)OC 1-4 Alkyl, -C(O)-C1-4 alkenyl, -C(O)OC 1-4 Alkenyl, -C(=N)-NH2, -C(=N)-NH- (protecting group), or pyrimidinyl. The same protecting groups described above for the P1' position also apply to the P3 position. When Q represents a guanidino group, this specifically includes Cbz and Pbf.

[0067] Regarding R c Specific groups that may be mentioned include those that are hydrogen bond acceptors (e.g., they include N or O atoms having 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).

[0068] Regarding R c Other specific groups that may be mentioned are the side chains of proteogenic amino acids, and protected derivatives of those side chains. Side chains containing at least one hydrogen bond acceptor, or their protected derivatives, have proven particularly effective, with oxygen-containing side chains (and their protected derivatives) exhibiting the greatest efficacy. Therefore, in another embodiment, R c -C represents a group substituted by a Q substituent. 1-3 Alkyl groups, wherein Q is selected from -OH and -OC. 1-4 The group consisting of alkyl groups (optionally substituted with phenyl or methylphenyl).

[0069] Compounds that have shown particular efficacy as pan-immunoproteasome inhibitors include R. c Those referring to the side chains of serine or its protected derivatives. Protected derivatives of serine that may be mentioned in this context include serine protected with Bn, ethyl, or propyl groups, and it has been found that compounds containing protected serine at P3 exhibit enhanced pan-immunopioid efficacy (i.e., R) when combined with a ring lacking any bulky side chain at the A position. h Or R i (Indicates H, -OH, or -CH3).

[0070] It was also discovered that R c The compounds of the present invention, which represent methyl groups, can be effectively used as pan-immunoproteasome inhibitors.

[0071] In compounds of formula (I), the position where ring A binds can be referred to as the "P4" position. Compounds in which ring A contains a Boc substituent have been shown to have a high probability of enhancing pan-immunoplasmic proteasome activity. Not wishing to be bound by theory, it is believed that substituents containing a tert-butyl group will show similar potential, as their bulky and hydrophobic nature allows the substituent to undergo hydrophobic interactions at the target site, especially if the substituent is located at the 4 position of the six-membered ring represented by A in formula (I).

[0072] In other embodiments, such as when R c When representing protected amino acid side chains, ring A lacks any bulky side chains (i.e., R). h and R i (Indicates H, -OH, or -CH3).

[0073] Therefore, specific A groups that can be mentioned are morpholino, 4-methylpiperidinyl, 4-(tert-butoxycarbonyl)-piperidinyl, piperidinyl, and 4-hydroxycyclohexyl. In one embodiment, A represents a morpholino group.

[0074] R c The bound carbon atom is a chiral center. The compounds of the present invention may have a D- or L-configuration at this position, or may involve a mixture of both configurations at this position. Compounds of formula (I) in which the P3 region corresponds to a D-amino acid are believed to have particularly enhanced β5i selectivity.

[0075] The compounds of the present invention contain additional chiral centers, including a binding site for a forced cyclohexylmethyl group (“P1” position) and a forced O-methyltyrosine side chain (“P2” position). Therefore, the P1 and P2 regions represent covalently linked amino acids. The compounds of the present invention may independently have a D- or L-configuration at each position, or may involve a mixture of the two configurations at each position.

[0076] Preferably, both P1 and P2 regions have an L-configuration. References to "L-configuration" herein and elsewhere include the presence (and vice versa) of compounds in these regions that substantially do not carry one or more D-configurations. Those skilled in the art will recognize that absolute purity in this respect is impossible. Therefore, the present invention relates to compounds in which, for any given chiral center, the enantiomer excess is at least 50%, preferably at least 80%, such as at least 90%, of the desired enantiomer.

[0077] Specific compounds of the present invention that may be mentioned include those compounds as described in the examples provided herein and their pharmaceutically acceptable salts. Therefore, specific compounds of the present invention that may be mentioned include:

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] And its pharmaceutically acceptable salts.

[0086] Pharmaceutical uses

[0087] As indicated herein, the compounds of the present invention and compositions comprising them can be used as pharmaceuticals.

[0088] Therefore, according to a second aspect of the invention, compounds of the invention as defined above (i.e., compounds as defined in the first aspect of the invention, comprising all embodiments and specific features thereof) are provided for use as a medicament (or for use in pharmaceuticals).

[0089] To avoid ambiguity, references to compounds as defined in the first aspect of the invention will include references to compounds of formula (I) (including all embodiments thereof) and their pharmaceutically acceptable salts and solvates.

[0090] Although the compounds of the present invention may themselves possess pharmacological activity, certain pharmaceutically acceptable (e.g., “protected”) derivatives of the compounds of the present invention may exist or be prepared to not possess such activity, but may be administered parenterally or orally and then metabolized in vivo to form the compounds of the present invention. Such compounds (which may possess some pharmacological activity, provided that such activity is significantly lower than that of the active compounds they metabolize) can therefore be described as “prodrugs” of the compounds of the present invention.

[0091] As used herein, references to prodrugs will include compounds which, after administration into the enteric or parenteral route (e.g., oral or parenteral), form an experimentally detectable amount of the compounds of the present invention within a predetermined time. All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0092] Furthermore, some compounds of the present invention may have little or no pharmacological activity, but may be administered parenterally or orally and then metabolized in vivo to form the pharmacologically active compounds of the present invention. Such compounds (which also include compounds that may have some pharmacological activity but which is significantly less than the activity of the active compounds of the present invention that they metabolize) may also be described as “prodrugs”.

[0093] To avoid ambiguity, the compounds of the present invention are useful because they have pharmacological activity and / or are metabolized in vivo after oral or parenteral administration to form pharmacologically active compounds.

[0094] As described herein, the compounds of the present invention can be particularly used to treat and / or prevent diseases or conditions in which pan-immunoproteasome inhibition is desired or required. Therefore, in a third aspect of the invention, compounds of the present invention as defined above are provided for the treatment or prevention of diseases or conditions in which pan-immunoproteasome inhibition is desired or required. Use for the treatment of diseases or conditions in which pan-immunoproteasome inhibition is desired or required is particularly preferred.

[0095] In an alternative third aspect of the invention, a method is provided for treating or preventing diseases or symptoms in which pan-immunoproteasome inhibition is desired or required, the method comprising administering to a patient in need a therapeutically effective amount of a compound of the invention as defined above.

[0096] In a further alternative third aspect of the invention, use is provided of the compounds of the invention as defined above in the preparation of medicaments for treating or preventing diseases or symptoms in which pan-immunoproteasome inhibition is desired or required.

[0097] Technicians will understand that references to treatment of a specific symptom (or similarly, treatment of the symptom) will take on their normal meaning in the medical field. Specifically, the term may refer to the achievement of a reduction in the severity and / or frequency of one or more clinical symptoms associated with the symptom, as determined by a physician treating a patient who has or is susceptible to such symptoms. For example, in the case of multiple myeloma, the term may refer to an extension of the patient's progression-free survival.

[0098] As used herein, the term prevention (and similarly, prevention) will include references to the prevention of a disease or condition (and vice versa). Therefore, references to prevention can also refer to prevention of prophylaxis, and vice versa. Specifically, such terms can refer to a reduction in the likelihood of a patient (or healthy subject) developing a symptom (which can be understood as a change in the patient's symptom that would lead a physician to diagnose the patient with, for example, a disease or disorder requiring treatment) (e.g., a reduction of at least 10%, such as a reduction of at least 20%, 30%, or 40%, such as a reduction of at least 50%).

[0099] As used herein, references to patients (or patients in general) will refer to living individuals being treated, including mammalian (e.g., human) patients. Specifically, references to patients will refer to human patients. To avoid ambiguity, the compounds of the present invention can also be used to treat non-human animals.

[0100] Those skilled in the art will understand that this treatment or prevention will be administered to patients (or subjects) who require it. Those skilled in the art can use standard techniques to assess a patient's (or subject's) need for such treatment or prevention. Patients mentioned herein as requiring prophylactic therapy include those who are susceptible to diseases or conditions in which pan-immunoproteasome inhibition is expected or required but are not currently diagnosed with such a disease.

[0101] As used herein, the terms disease and symptom are used interchangeably.

[0102] As used herein, the term effective amount will refer to the amount of compound that imparts a therapeutic effect to the treated patient. Effects can be observed in an objective manner (i.e., measurable by some test or biomarker) or subjectively (i.e., by the subject giving indications of the effect and / or feeling the effect). In particular, effects can be observed (e.g., measured) objectively using appropriate tests as known to those skilled in the art.

[0103] In specific embodiments (i.e., certain embodiments of the third aspect of the invention), the disease or symptom is a hematologic malignancy, a solid tumor, an autoimmune disease, or an inflammatory disease.

[0104] As described herein, the compounds of the first aspect of the invention can be particularly used for hematologic malignancies selected from the group consisting of leukemia, lymphoma, myeloma (including multiple myeloma), myelodysplastic syndromes, and myeloproliferative syndromes.

[0105] Those skilled in the art will understand that solid cancers that can be treated or prevented by the compounds of the present invention include prostate cancer, breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, ovarian cancer, osteosarcoma, and inflammatory bowel cancer.

[0106] Specific inflammatory diseases that can be treated or prevented using the compounds of this invention include encephalitis, viral myocarditis, inflammatory bowel disease, arthritis, polymyositis, dermatomyositis, autoimmune hepatitis, and lupus nephritis.

[0107] The compounds of the present invention can 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).

[0108] Pharmaceutical Composition

[0109] As described herein, the compounds of the present invention can be used as pharmaceuticals. Such compounds can be administered alone or in the form of known pharmaceutical compositions / formulations.

[0110] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising the compounds of the invention as defined herein and optionally one or more pharmaceutically acceptable excipients.

[0111] As used herein, pharmaceutically acceptable excipients include references to mediators, adjuvants, carriers, diluents, pH adjusters and buffers, tension modifiers, stabilizers, wetting agents, etc. Specifically, such excipients may include adjuvants, diluents, or carriers.

[0112] To avoid any doubt, the compounds of the present invention mentioned herein for a particular purpose (and similarly, the uses and methods of use of the compounds of the present invention mentioned herein) may also be applied to pharmaceutical compositions comprising the compounds of the present invention as described herein.

[0113] Therefore, in a fifth aspect of the invention, a pharmaceutical composition as defined in the fourth aspect of the invention is provided for treating or preventing diseases or conditions in which pan-immunoproteasome inhibition is desired or required (as defined herein, refer to the third aspect of the invention and all embodiments thereof).

[0114] Those skilled in the art will understand that the compounds of the present invention can act systemically and / or locally (i.e. at a specific site), and therefore can be applied accordingly using suitable techniques known to those skilled in the art.

[0115] Those skilled in the art will understand that the compounds and compositions as defined herein will generally be administered orally, intravenously, subcutaneously, buccally, rectally, transdermally, nasally, tracheally, bronchially, sublingually, intranasally, topically, via any other parenteral route, or by inhalation in pharmaceutically acceptable dosage forms.

[0116] The pharmaceutical compositions described herein will include compositions in the form of tablets, capsules, or elixirs for oral administration, suppositories for rectal administration, and sterile solutions or suspensions for parenteral or intramuscular administration. Alternatively, particularly where such compounds of the present invention act locally, the pharmaceutical compositions may be formulated for local application.

[0117] Therefore, in certain embodiments, the pharmaceutical formulation is provided in a pharmaceutically acceptable dosage form, including tablets or capsules, oral or injectable liquid forms, suppositories, creams, gels, foams, inhalers (e.g., intranasal application), or forms suitable for topical application. For the avoidance of doubt, in such embodiments, the compounds of the present invention may be present in solid form (e.g., solid dispersions), liquid form (e.g., solutions), or other forms (such as micelles).

[0118] For example, in preparing pharmaceutical formulations for oral administration, compounds can be mixed with solid powdered ingredients (such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, or other suitable ingredients), as well as with disintegrants and lubricants (such as magnesium stearate, calcium stearate, sodium stearate fumarate, and polyethylene glycol wax). The mixture can then be processed into granules or compressed into tablets.

[0119] Soft gelatin capsules can be prepared using capsules containing one or more active compounds (e.g., compounds of the first aspect and therefore the second and third aspects of the invention, and optionally additional therapeutic agents) and carriers such as vegetable oils, fats, or other suitable for soft gelatin capsules. Similarly, hard gelatin capsules can contain such compounds in combination with solid powder ingredients such as lactose, sucrose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin.

[0120] Dosage units for rectal administration can be prepared as: (i) suppositories containing one or more compounds mixed with a neutral fat matrix; (ii) gelatin rectal capsules containing active material mixed with vegetable oil, paraffin oil, or other mediators suitable for gelatin rectal capsules; (iii) ready-made microenemas; or (iv) dried microene preparations reconstituted in a suitable solvent just before administration.

[0121] Liquid formulations for oral administration can be prepared in the form of syrups or suspensions (e.g., solutions or suspensions) containing the compound and the remainder of the formulation consisting of sugars or sugar alcohols and a mixture of ethanol, water, glycerol, propylene glycol, and polyethylene glycol. Such liquid formulations may, if desired, contain colorants, flavoring agents, saccharin, and carboxymethyl cellulose or other thickeners. Liquid formulations for oral administration can also be prepared in the form of dry powders, reconstituted with a suitable solvent prior to use.

[0122] Solutions for parenteral administration can be prepared as solutions of the compound in pharmaceutically acceptable solvents. These solutions may also contain stabilizing and / or buffering components and are dispensed as unit doses in ampoules or vials. Solutions for parenteral administration can also be prepared as dry formulations, temporarily reconstituted with a suitable solvent prior to use.

[0123] Depending on the potency and physical properties of compounds (i.e., active ingredients) such as those of the present invention, pharmaceutical formulations that may be mentioned 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 of the pharmaceutical composition to other components (i.e., added 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).

[0124] Those skilled in the art will understand that the compounds of the present invention can be administered in different doses (e.g., as formulations as described above), wherein a suitable dose can be readily determined by those skilled in the art. Oral and topical doses (and subcutaneous doses, although these doses may be relatively low) can range from about 0.01 μg / kg body weight / day (μg / kg / day) to about 200 μg / kg / day, preferably from about 0.01 μg / kg / day to about 10 μg / kg / day, and more preferably from about 0.1 μg / kg / day to about 5.0 μg / kg / day. For example, when administered orally, treatment with such compounds may include administration of a formulation typically containing one or more active ingredients between about 0.01 μg and about 2000 mg, such as between about 0.1 μg and about 500 mg, or between 1 μg and about 100 mg (e.g., about 20 μg to about 80 mg). When administered intravenously, the most preferred dose range during constant-rate infusion will be from about 0.001 μg / kg / hour to about 10 μg / kg / hour. Advantageously, treatment may include administration of such compounds and compositions in a single daily dose, or the total daily dose may be administered in fractions twice, three or four times daily (e.g., twice daily, such as doses of 5 mg, 10 mg, 25 mg, 50 mg, 100 mg or 200 mg twice daily, referring to the doses described herein).

[0125] When used in this document with regard to specific values ​​(such as quantities), the term “about” (or similar terms such as “approximately”) will be understood to indicate that the variation of such values ​​may be up to 10% of the defined value (specifically, up to 5%, such as up to 1%). It is noted that in each case, such terms can be replaced by symbols such as “±10%” (or by indicating the variance of a specific quantity calculated based on the relevant values). It is also noted that in each case, such terms may be omitted.

[0126] To avoid ambiguity, a person skilled in the art (such as a physician) will be able to determine the actual dosage best suited for an individual patient, which may vary depending on the route of administration, the type and severity of the condition to be treated, and the species, age, weight, sex, renal function, liver function, and response of the specific patient being treated. While the dosages mentioned above are exemplary in general, higher or lower dosage ranges may be required in individual cases, and such dosage ranges are all within the scope of this invention.

[0127] Preparation of compounds / compositions

[0128] Pharmaceutical formulations can be prepared according to standards and / or recognized pharmaceutical practices.

[0129] Therefore, in another aspect of the invention, a method for preparing a pharmaceutical formulation as defined above is provided, the method comprising associating a compound of the invention as defined above with one or more pharmaceutically acceptable excipients.

[0130] As used herein, references to making...associated will mean making the two components suitable for combined administration.

[0131] The compounds of the present invention as described herein can be prepared according to techniques well known to those skilled in the art, such as those described in the examples provided below.

[0132] According to a sixth aspect of the invention, a method for preparing compounds of the invention as defined above is provided, the method comprising the steps of: causing the compound of formula (II) to react in the presence of a suitable coupling agent (e.g., O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, 1,1'-carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide, etc.) under standard conditions known to those skilled in the art (e.g., optionally in a suitable solvent, in the presence of a suitable base and / or in an inert atmosphere), for example under reaction conditions known for Steglich esterification reactions.

[0133]

[0134] Among them, A, p and R c As defined above, compounds of formula (III)

[0135] (III)

[0136] Where Y is the reaction as defined above.

[0137] The compounds of formulas (II) and (III) are commercially available, known in the literature, or can be obtained from available starting materials by means of processes similar to those described herein or by conventional synthetic procedures, according to standard techniques, using appropriate reagents and reaction conditions. In this regard, those skilled in the art may in particular refer to BM Trost and I. Fleming, “Comprehensive Organic Synthesis,” Pergamon Press, 1991. Other applicable references include JA Joule, K. Mills, and GF Smith, “Heterocyclic Chemistry,” 3rd edition. Published by Chapman & Hall; AR Katritzky, CW Rees and EFV Scriven, “Comprehensive Heterocyclic Chemistry II”, Pergamon Press, 1996; and “Science of Synthesis”, Volumes 9–17 (Hetarenes and Related Cyclic Systems), Georg Thieme Verlag, 2006.

[0138] Specifically, compound (III) can be prepared by the following reaction: compound (IV),

[0139]

[0140] Among them, A, p and R c As defined above, compound (V)

[0141]

[0142] 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 peptidylhydrazine, and then further reacted with tert-butyl nitrite under suitable conditions (such as at low temperature in a non-aqueous solvent), followed by contact with the compound of formula (V).

[0143] Other specific transformation steps that may be mentioned (including those that can be used to form compounds of formula (I)) include:

[0144] (i) For example, in the presence of a suitable oxidizing agent (e.g., MnO2 or mcpba, etc.), a portion containing an alkenyl group (e.g., -CH=CH2) is oxidized to an epoxide;

[0145] (ii) The formation of amides, for example by the reaction of acyl chlorides with amines or by amide coupling reactions, i.e., the formation of amides from carboxylic acids (or their esters) (e.g., -C(O)OH (or their esters)), can be converted to -C(O)N(R) 1 )R 2 Group (where R) 1 and R 2(The reaction is a hydrogen or carbon-containing species), and the reaction can be carried out (e.g., for -COOH) in the presence of a suitable coupling agent (e.g., O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, 1,1'-carbonyldiimidazole, N,N'-dicyclohexylcarbodiimide, etc.), or in the case of an ester (e.g., -C(O)OCH3 or -C(O)OCH2CH3), in the presence of, for example, trimethylaluminum, or alternatively, the -C(O)OH group can first be activated to the corresponding acyl halide (e.g., -C(O)Cl, by treatment with oxaloyl chloride, thionyl chloride, phosphorus pentachloride, phosphorus oxychloride, etc.), and in all cases, the relevant compound is reacted with the formula HN(R 1 )R 2 Compound (where R) 1 and R 2 The reaction is carried out under standard conditions known to those skilled in the art (e.g., optionally in a suitable solvent, in the presence of a suitable base, and / or in an inert atmosphere) as defined above.

[0146] (iii) The methoxy group is converted into a hydroxy group by reacting with a suitable reagent (such as boron fluoride-dimethyl sulfide complex or BBr3) in the presence of a suitable solvent such as dichloromethane.

[0147] (iv) Alkylation or acylation reactions, which can be carried out in the presence of a base and a solvent (such as those described above);

[0148] (v) Specific deprotection steps, such as deprotecting the N-Boc protecting group by reacting in the presence of an acid, or deprotecting the hydroxyl group to a silyl ether (e.g., tert-butyl-dimethylsilyl protecting group) by reacting with a fluoride ion source (e.g., by using the reagent tetrabutylammonium fluoride (TBAF)).

[0149] Similarly, the compounds of formulas (IV) and (V) are commercially available, known in the literature, or can be obtained from available starting materials by means of processes similar to those described herein or by conventional synthetic procedures, according to standard techniques, using appropriate reagents and reaction conditions.

[0150] Those skilled in the art will understand that substituents as defined herein and substituents thereof may be modified once or more during or after the processes described above for the preparation of the compounds of the present invention by methods well known to those skilled in the art. Examples of such methods include substituenting, reduction, oxidation, dehydrogenation, alkylation, dealkylation, acylation, hydrolysis, esterification, etherification, halogenation, and nitration. At any time during the reaction sequence, the precursor group may be changed to a different such group or to a group defined by formula (I). Those skilled in the art may also refer to AR Katritzky, O. Meth-Cohn, and CW Rees, “Comprehensive Organic Functional Group Transformations,” Pergamon Press, 1995, and / or RC Larock, “Comprehensive Organic Transformations,” Wiley-VCH, 1999.

[0151] The compounds of the present invention can be isolated from their reaction mixture and, if necessary, purified using conventional techniques known to those skilled in the art. Therefore, methods for preparing the compounds of the present invention as described herein may comprise the isolation and, optionally, purification of the compounds of the present invention as a final step.

[0152] Those skilled in the art will understand that, in the processes described above and below, the functional groups of the intermediate compounds may need to be protected by protecting groups. Protection and deprotection of the functional groups can occur before or after the reactions in the schemes mentioned above.

[0153] Protecting groups may be applied or removed using techniques well known to those skilled in the art or as described below. For example, standard deprotection techniques can be used to chemically convert a protected compound / intermediate described herein into an unprotected compound. The type of chemistry involved will determine the need for and type of protecting groups, as well as the sequence of synthesis. The use of protecting groups is fully described in “Protective Groups in Organic Synthesis,” 3rd edition, TW Greene & PGM Wutz, Wiley-Interscience (1999), the contents of which are incorporated herein by reference.

[0154] Without being bound by theory, the compounds of the present invention are believed to have an iCP-favorable inhibitory profile relative to LU-005i, and therefore have the potential to serve as selective inhibitors of the pan-immunoproteasome. By specifically inhibiting the immunoproteasome, pan-immunoproteasome inhibitors may be able to overcome the resistance observed in hematologic cancers and the cytotoxicity caused by constitutive proteasome inhibition.

[0155] The compounds of the present invention may have the following advantages: whether used for the aforementioned indications or others, they may be more effective, less toxic, have a longer duration of action, be more potent, produce fewer side effects, be more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other available pharmacological, physical, or chemical properties than compounds known in the prior art. Specifically, the compounds of the present invention may have the following advantages: they are more effective in vivo and / or exhibit advantageous properties. Attached Figure Description

[0156] Figure 1 The P1' N-Boc amino acids and P1' amino acids (2-24) on the LU-005i (1) scaffold are shown in IC50. 50 Inhibition profile in (µM) heatmap;

[0157] Figure 2 The P1' N-Boc ester (25-27) on the LU-005i (1) scaffold is shown in IC 50 Inhibition profile in (µM) heatmap;

[0158] Figure 3 This demonstrates the P3-P4 stent screening (28-57) based on the LU-005i (1) stent in IC 50 Inhibition profile in (µM) heatmap;

[0159] Figure 4 The secondary P3-P4 screening (29, 30, 34, 35, 58-73) based on the LU-005i (1) stent is shown in IC 50 Inhibition profile in (µM) heatmap;

[0160] Figure 5 Raji cleavages of a group of compounds (2, 3, 14, 65, 68, 74-76) exhibiting variations at the P3 and P1' positions are shown in IC50. 50 Inhibition profile in (µM) heatmap;

[0161] Figure 6The AMO wild-type lysates of a group of compounds (2, 3, 14, 65, 68, 74-76) with variations at the P3 and P1' positions are shown in IC50. 50 Inhibition profile in (µM) heatmap.

[0162] To avoid ambiguity, the numbers used in the illustrations refer to the numbers of compounds as provided in the examples herein. Detailed Implementation

[0163] The invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention.

[0164] If there is a difference between the name and any compound depicted graphically, the latter shall prevail (unless it contradicts any experimental details that may be given, or unless it is clearly apparent from the context).

[0165] 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.

[0166] Commercially available reagents and solvents were used as is. H2O and oxygen-sensitive reactions were carried out under a N2 atmosphere. Solvents used in the synthesis were dried and stored on 4 Å molecular sieves, except for pyridine, DIPEA, and TEA stored on KOH aggregates, if necessary. TLC analysis was performed using aluminum sheets pre-coated with silica gel (Merck, TLC silica gel 60 F254). Analysis was performed by UV absorption (λ = 254 nm) or with ninhydrin (50 g / L in n-butanol) or cerium molybdate (25 g / L (NH4)6Mo7O). 24The compounds were visualized by spraying with 10 g / L (NH4Ce(SO4)4•H2O in 10% H2SO4 aqueous solution) and subsequently carbonized at 150 °C when appropriate. Column chromatography was performed on bv silica gel (particle size 40–63 μm, pore size 60 Å) in a screening apparatus. The reaction mixture was impregnated with diatomaceous earth hyflo supercell (Merck) prior to silica gel chromatography when appropriate. 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) and referenced to TMS (∂ = 0.00 ppm) or residual solvent peaks. J coupling was reported in Hz. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed on a Finnigan surveyor HPLC system equipped with a nucleodur C18 Gravity 3 μm 50 x 4, 60 mm column (detection at 200–500 nm), coupled to a Finnigan LCQ advantage max mass spectrometer with ESI or a Thermo LCQ Fleet ion mass spectrometer with ESI. General methods used ranged from 10% to 90%, with a total run time of 13.5 minutes.

[0167] High-resolution mass spectra are reported by direct injection (1.0 μM H₂O / MeCN 1:1 and 0.1% formic acid solution) on a Q Exactive HF hybrid quadrupole-orbital trap mass spectrometer equipped with a positive-mode electrospray ionization source (source voltage 3.5 kV, sheath gas flow rate 10, capillary temperature 275 °C), achieving a resolution R = 240,000 and externally locked mass at m / z 400 (mass range m / z = 160–2000). The high-resolution mass spectrometer was calibrated using a calibration mixture (Thermo Finnigan) prior to measurements.

[0168] For synthesis following a general procedure, reaction conditions (such as reaction length or temperature) may vary. Generally, thin-layer chromatography or LC-MS analysis is performed after the reaction, and post-processing is carried out when appropriate. Purification may vary between experiments: generally, the solvent and solvent ratio used for the eluent / gradient are selected to provide an appropriate Rf and / or retention time.

[0169] General Procedure

[0170] General chemical synthesis of LU-005i-OH and structural variants:

[0171] Epoxyketone 83 was prepared as follows (Scheme 1). The condensation of a Weinreb salt with N-Boc cyclohexylalanine 77 using HCTU provided Weinreb amide 78. Deprotonation of dimethyl phosphonate using n-BuLi resulted in the formation of a phosphorus ylide, which was then added to Weinreb amide 78 to form phosphonate 79. Phosphonate 83 was deprotonated in a two-step one-pot reaction to form a phosphorus ylide intermediate, which, in a Horner-Wozworth-Emmons reaction with formaldehyde, yielded an enone intermediate 80, which immediately reacted with formaldehyde in a Bayes-Hillman type reaction to form allyl alcohol 81. Allyl alcohol 81 was then epoxidized using hydrogen peroxide in a nucleophilic epoxidation process to provide the desired stereochemical epoxyketone 82 after separation of diastereomers. The N-Boc protecting group in 82 was then removed using a TFA-containing DCM to yield the epoxyketone structural unit 83.

[0172]

[0173] Scheme 1. Synthesis of epoxy ketone 83. Reagents and conditions: (a) HCl•NMeOMe, HCTU, DiPEA, DCM, rt, 91%; (b) Methyl dimethylphosphonate, n-BuLi, THF, -78℃; (c) Formaldehyde, K2CO3, H2O / MeOH, 0℃, 51%; (d) H2O2, DiPEA, benzyl nitrile, 0℃, 29%; (e) TFA, DCM, rt, quantitative.

[0174] Next, both the phenolic alcohol and the carboxylic ester of N-Boc tyrosine were methylated using iodomethane and potassium carbonate to form 4-methoxyphenylalanine ester 85 (Scheme 2). The N-Boc protecting group of 85 was then removed, and the resulting amine was condensed with N-Boc alanine using HCTU to form a dipeptide 86. De-N-Bocization of 86 (treated with TFA), followed by condensation of the NH2-dipeptide intermediate with 2-morpholinoacetic acid, provided methyl ester 87. Methyl ester 87 was converted to hydrazide 88, which was then converted to acyl azide 89 under anhydrous acidic conditions using tert-butyl nitrite. Acyl azide 89 was subsequently reacted with NH2-epoxy ketone 83 to form LU-005i-OH2. The OH group in 2 was then functionalized by Streglich esterification with a wide range of carboxylic acids to form a library of esterified LU-005i-OH derivatives 3-13 and 25-26. Finally, the corresponding N-Boc and O-TBS protecting groups in compounds 2-12 and 26 were removed by TFA treatment to obtain a set of NH2-amino acid ester derivatives 14-24 and OH-lactic acid ester derivative 27.

[0175]

[0176] Scheme 2. Synthesis of LU-005i-OH 2 and esterified LU-005i-OH derivative 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℃, ii: 83, DiPEA, DMF, -30℃ -> rt, 69%; (f) RCO2H, DIC, DMAP, DCM, rt, 34-99%; (g) TFA, DCM, rt.

[0177] Program A: Boc unprotection

[0178] The Boc-protected compound was dissolved in anhydrous DCM (0.3 M), and TFA was added to the solution to achieve a TFA:DCM ratio of 1:4 (v:v). The reaction mixture was stirred for 1 to 2 hours and monitored by TLC. After complete conversion, the reaction mixture was concentrated under vacuum and co-evaporated with toluene 3x.

[0179] Procedure B: Esterification

[0180] LU-005i-OH was dissolved in anhydrous DCM (0.2 M) and purged with N2. DIC (2.0 eq.), amino acids (1.1 eq.), and DMAP (1.1 eq.) were added to the solution. The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum, and the title compound was obtained by silica gel column chromatography using DCM:MeOH.

[0181]

[0182] Boc-Tyr(OMe)-OMe (90)

[0183] Boc-Tyr(OMe)-OH (5 g; 16.9 mmol, 1.0 eq.) 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 eq.) and K2CO3 (3.27 g; 23.66 mmol; 1.5 eq.) were added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with EtOAc, washed with NaS2O3 (sat., aq.), H2O, and brine (NaCl sat., aq.), dried over MgSO4, filtered, and concentrated under vacuum. The title compound (6.06 g; 19.6 mmol; quantified) was obtained without further purification. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.53 min (ESI-MS (m / z): 310.16 (M+H) + )).

[0184]

[0185] H2N-Tyr(OMe)-OMe (85)

[0186] Boc-Tyr(OMe)-OMe was deprotected at a scale of 19.6 mmol according to procedure A, yielding the title compound as a white solid (5.49 g; 19.6 mmol; quantified). LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.25 min (ESI-MS (m / z): 210.11 (M+H) + )).

[0187]

[0188] Boc-Ala-Tyr(OMe)-OMe (86)

[0189] H₂N-Tyr(OMe)-OMe (4.10 g; 19.6 mmol; 1.0 eq.) was co-evaporated twice with toluene, then dissolved in anhydrous DMF (0.2 M) and purged with N₂. HCTU (12.16 g; 29.4 mmol; 1.5 eq.) and Boc-Ala-OH (7.42 g; 39.2 mmol; 2.0 eq.) were added to this solution. When completely dissolved, DiPEA (13.7 ml; 78.4 mmol; 4.0 eq.) was added dropwise to the reaction mixture and stirred for 16 h. The reaction mixture was washed with HCl (1.0 M, aq.), NaHCO₃ (sat., aq.), and brine (NaCl sat., aq.), dried over MgSO₄, and concentrated under vacuum. Silica gel column chromatography (0:100 -> 2:100 MeOH:DCM) yielded the title compound as a colorless oil (6.79 g; 17.8 mmol; 91%). f :0.2, in 2:100 MeOH:DCM. 1 H NMR (400 MHz, CDCl3) δ 7.05 – 7.00 (m,2H), 6.90 (d, J = 8.0 Hz, 1H), 6.82 – 6.78 (m, 2H), 5.39 (d, J = 7.6 Hz, 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 = 7.1 Hz, 3H). 13 C NMR (101MHz, CDCl3) δ 172.5, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.71 min (ESI-MS (m / z): 380.80 (M+H) + )).

[0190]

[0191] H2N-Ala-Tyr(OMe)-OMe (91)

[0192] Boc-Ala-Tyr(OMe)-OMe was deprotected at the mmol scale according to procedure A, yielding the title compound as a yellow oil (2.24 g; 8.0 mmol; quantified). LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.03 min (ESI-MS (m / z): 281.00 (M+H) + )).

[0193]

[0194] Morph-Ala-Tyr(OMe)-OMe (87)

[0195] NH₂-Ala-Tyr(OMe)-OMe (2.24 g; 8.0 mmol; 1.0 eq.) was co-evaporated twice with toluene, then dissolved in anhydrous DMF (0.2 M) and purged with N₂. PyBOP (4.03 g; 9.6 mmol; 1.2 eq.) and Morph-COOH (1.28 g; 8.8 mmol; 1.1 eq.) were added to this solution. When completely dissolved, DiPEA (4.89 mL; 28.0 mmol; 3.5 eq.) was added dropwise to the reaction mixture and stirred for 16 h. The reaction mixture was washed with HCl (1.0 M, aq.), NaHCO₃ (sat., aq.), and brine (NaCl sat., aq.), dried over MgSO₄, and concentrated under vacuum. Silica gel column chromatography (0:100 -> 2:100 MeOH:DCM) yielded the title compound (2.61 g, 6.4 mmol; 80%) as a pale yellow oil. f :0.3, in 2:100 MeOH:DCM. 1H NMR (400 MHz, CDCl3) δ7.55 (t, J = 7.9 Hz, 1H), 7.11 – 6.93 (m, 2H), 6.88 – 6.75 (m, 2H), 6.68 (d,J = 7.8 Hz, 1H), 4.77 (ddd, J = 7.9, 6.7, 5.5, 1H), 4.51 (p, J = 7.1 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 3H), 3.70 (t, J = 4.7 Hz, 4H), 3.14 – 2.89 (m, 4H), 2.49 (t, J = 4.6 Hz, 4H), 1.36 (d, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 171.9, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.29 min (ESI-MS (m / z): 408.07 (M+H) + )).

[0196]

[0197] Morph-Ala-Tyr(OMe)-NHNH2(88)

[0198] Morph-Ala-Tyr(OMe)-OMe (0.8 g; 2.0 mmol; 1.0 eq.) was dissolved in MeOH (0.1 M), and hydrazine hydrate (65% by mass; 1.88 ml; 60.0 mmol; 30.0 eq.) was added to the solution. The resulting reaction mixture was stirred at rt for 16 h. The resulting reaction mixture was concentrated under vacuum and co-evaporated three times with toluene. This yielded a white solid, which was further used as a crude product for the following reaction. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 3.03 min (ESI-MS (m / z): 408.20 (M+H + )).

[0199]

[0200] Boc-Cha-N(OCH3)CH3(78)

[0201] Boc-Cha-OH (10.0 g; 36.9 mmol; 1.0 eq.) was dissolved in anhydrous DMF (0.1 M) and purged with N2. HCTU (22.87 g, 55.3 mmol, 1.5 eq.) and N,O,-dimethylhydroxylamine (7.19 g; 73.7 mmol; 2.0 eq.) were added to this solution. When the reagents were completely dissolved, DiPEA (25.7 ml; 147 mmol; 4.0 eq.) 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, aq.), washed with HCl (1.0 M, aq.), NaHCO3 (sat., aq.), and brine (sat. NaCl, aq.), dried over MgSO4, and concentrated under vacuum. Silica gel column chromatography (1:20 -> 1:4 EtOAc:PE) yielded the title compound (10.43 g, 33.2 mmol; 90%) as a clear oil. f :0.3, in 1:4 EtOAc:PE. 1 H NMR (400 MHz, CDCl3) δ5.02 (d, J = 9.2 Hz, 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 C NMR (101 MHz, CDCl3) δ 155.82, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 8.40 min (ESI-MS (m / z): 314.80 (M+H + )).

[0202]

[0203] Boc-Cha-CH2-P(=O)OMe(OMe) (79)

[0204] Dimethyl methylphosphonate (7.48 ml; 70 mmol; 4.0 eq) 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 eq) 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 eq.) was co-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 solution containing phosphorus ylide. The resulting reaction mixture was stirred at -78 °C under N2 for 3 hours. Next, the reaction was quenched with NH4Cl (100 ml; sat., aq.), and the reaction mixture was then heated to room temperature. The reaction mixture was then diluted with EtOAc, and the resulting organic and aqueous layers were separated. The aqueous layer was extracted three times with EtOAc, and the combined EtOAc was washed with H2O and brine (NaCl sat., aq.), dried over MgSO4, and concentrated under vacuum. The resulting crude oil was ready for use in the next reaction without further purification. 1 H NMR (400 MHz, CDCl3) δ5.24 (d, J = 26.3 Hz, 1H), 4.36 (d, J = 11.5 Hz, 1H), 3.80 (d, J = 3.9 Hz,3H), 3.78 (d, J = 3.8 Hz, 4H), 3.75 (s, 6H), 3.73 (d, J = 2.4 Hz, 6H), 3.34 (dd, J = 22.5, 14.2 Hz, 1H), 3.10 (dd, J = 22.0, 14.2 Hz, 1H), 1.90 – 1.58(m, 8H), 1.45 (s, 15H), 1.41 – 1.31 (m, 3H), 1.29 – 1.10 (m, 5H), 1.03 – 0.81 (m, 4H). LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.48 min (ESI-MS (m / z): 377.67 (M+H) + )).

[0205]

[0206] Boc-Cha-C(CH2OH)=CH2 (81)

[0207] Boc-Cha-CH2-P(=O)OMe-OMe (5.9 g; 17.5 mmol; 1.0 eq.) was dissolved in a 1:1 ratio of THF and H2O (0.1 M). K2CO3 (7.26 g; 52.5 mmol; 3.0 eq.) and formaldehyde (37% by mass in H2O; 3.91 ml; 52.5 mmol; 3.0 eq.) were added to the solution. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with EtOAc and acidified with HCl (1.0 M, aq.). The organic and aqueous layers were separated, and the aqueous layer was extracted three times with EtOAc. The resulting EtOAc was washed with H2O and brine (NaCl sat., aq.), dried over MgSO4, filtered, and concentrated under vacuum. Silica gel column chromatography (1:20 -> 1:4 EtOAc:PE, v:v) was performed. The procedure produced the title compound (2.42 g; 8.93 mmol; 51%). 1 ¹H NMR (400 MHz, CDCl₃) δ 6.21 (d, J = 33.7 Hz, 2H), 5.57 (d, J = 8.8 Hz, 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 to 90% MeCN / H₂O, 0.1% TFA, 12.5 min) Rt (min): 7.81 min (ESI-MS (m / z): 311.60 (M+H) + )).

[0208]

[0209] Boc-Cha-EK-CH3OH (82)

[0210] Boc-Cha-C(CH3OH)=CH2 (3.4 g; 11 mmol; 1.0 eq.) was dissolved in MeOH (0.2 M), cooled to 0 °C, and hydrogen peroxide (3.14 ml; 55 mmol; 5.0 eq.), benzonitrile (5.66 ml; 55 mmol; 5.0 eq.), and DiPEA (9.58 ml; 55 mmol; 5.0 eq.) were added. The resulting reaction mixture was stirred at 0 °C for 16 hours. The reaction mixture was diluted with EtOAc and acidified with HCl (1.0 M, aq.). The organic and aqueous layers were separated, and the aqueous layer was extracted three times with EtOAc. The resulting EtOAc layer was dried over Mg2SO4, filtered, and concentrated under vacuum. The crude product contained the product as a racemic mixture. The desired S isomer was separated by a slow and thorough separation, achieved by increasing the eluent concentration by one percentage point from 10% EtOAc / PE. Finally, purification by rapid column chromatography (5% -> 15% (v / v) EtOAc:PE) yielded the title compound (1.1 g; 3.22 mmol; 29%). 1 H NMR (400 MHz, CDCl3) δ 4.91 (d, J = 8.6 Hz, 1H), 4.39 – 4.28 (m, 1H), 4.19 (dd, J = 12.7,4.6 Hz, 1H), 3.76 (dd, J = 12.8, 5.5 Hz, 1H), 3.33 (d, J = 5.0 Hz, 1H), 3.09 (d, J = 5.0 Hz, 1H), 2.37 (t, J = 6.3 Hz, 1H), 1.86 (d, J = 10.9 Hz, 1H), 1.77 – 1.53 (m, 6H), 1.42 (s, 12H), 1.33 – 1.07 (m, 6H), 1.05 – 0.86 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ 208.7, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.65 min (ESI-MS (m / z): 327.53 (M+H+ )).

[0211]

[0212] H2N-Cha-EK-CH3OH (83)

[0213] Deprotection of Boc-Cha-EK-CH3OH was performed at the mmol scale according to procedure A, yielding the title compound as a yellow oil (0.38 g; 1.08 mmol; quantified). LC-MS (linear gradient 10 to 50% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.43 min (ESI-MS (m / z): 228.07 (M+H) + )).

[0214]

[0215] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (2)

[0216] Morph-Ala-Tyr(OMe)-NHNH2 (0.81 g; 2.0 mmol; 1.2 eq.) was co-evaporated twice with toluene, dissolved in anhydrous DMF (0.1 M), purged with N2, and cooled to -40 °C. tBuONO (0.438 mL; 3.67 mmol; 2.2 eq.) and HCl (4.0 M in dioxane; 2.33 mL; 9.33 mmol; 5.6 eq.) were added to this solution. The resulting reaction mixture was stirred at -40 °C for four hours. After four hours, the HCl was quenched with DiPEA (1.16 mL; 6.67 mmol; 4.0 eq.), and a solution of NH2-Cha-EK-CH3OH (0.379 g; 1.70 mmol; 1.0 eq.) in anhydrous DMF (0.2 M) was added dropwise. The resulting reaction mixture was stirred for 16 hours while being heated to room temperature during this period. The reaction mixture was diluted with H₂O and EtOAc, washed with NaHCO₃ (sat., aq.), H₂O, and brine (sat. NaCl, aq.), dried over MgSO₄, and concentrated under vacuum. Silica gel column chromatography (0:100 -> 10:100 MeOH:DCM) yielded the title compound (0.71 g, 1.17 mmol; 69%) as a clear oil. f: 0.35 5:100 MeOH:DCM. H NMR (400MHz, CDCl3) δ 7.47 (d, J = 7.5 Hz, 1H), 7.16 – 7.06 (m, 2H), 6.86 (d, J = 7.7Hz, 1H), 6.83 – 6.76 (m, 2H), 6.50 (d, J = 7.7 Hz, 1H), 4.63 – 4.51 (m, 2H), 4.44 (p, J = 7.1 Hz, 1H), 4.17 (d, J = 12.6 Hz, 1H), 3.76 (s, 3H), 3.73 (d, J= 5.1 Hz, 1H), 3.70 (t, J = 4.6 Hz, 4H), 3.29 (d, J = 5.0 Hz, 1H), 3.07 (d, J= 5.0 Hz, 1H), 3.00 (d, J = 6.9 Hz, 2H), 2.95 (s, 1H), 2.89 (s, 1H), 2.46 (q,J = 4.2 Hz, 5H), 1.78 (d, J = 13.0 Hz, 1H), 1.72 – 1.49 (m, 5H), 1.35 (d, J =7.0 Hz, 3H), 1.28 – 1.09 (m, 6H), 1.01 – 0.82 (m, 2H). 13 C NMR (101 MHz, 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 (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.12 min (ESI-MS (m / z): 603.27 (M+H) + HRMS (ESI) m / z:C 31 H 46 N4O8 [M+H] + Calculated value: 603.33884; Experimental value: 603.33823

[0217]

[0218] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NHBoc (3)

[0219] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol; 1.0 eq.) and Boc-Ala-OH (12 mg; 0.066 mmol; 2.0 eq.) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (18 mg; 0.023 mmol; 70%) as a lyophilized white powder. 1 H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 6.7 Hz, 3H), 6.42 (d, J = 7.5 Hz, 1H), 5.05 (d, J = 7.7 Hz, 1H), 4.89 (d, J = 12.2 Hz, 1H), 4.62 – 4.49 (m, 2H), 4.41 (p, J = 7.1 Hz, 1H), 4.30 (d, J = 7.5 Hz, 1H), 4.13 (d, J = 12.2 Hz, 2H), 3.83 (q, J = 6.4 Hz, 2H), 3.77 (s, 3H), 3.70 (t, J = 4.7 Hz, 4H), 3.37 (d, J = 4.9 Hz, 1H), 3.06 (d, J = 4.9 Hz, 1H), 3.02 – 2.98 (m, 2H), 2.96 (s, 1H), 2.88 (d, J = 16.4 Hz, 1H), 2.48 (tq, J = 11.8, 6.9, 5.6 Hz, 4H), 1.80 – 1.52 (m, 6H), 1.45 (s, 9H), 1.41 – 1.33 (m, 6H), 1.29 – 1.17 (m, 5H), 0.98 – 0.83 (m, 2H). 13C NMR (126MHz, CDCl3) δ 205.4, 172.7, 172.0, 171.0, 170.3, 158.7, 157.0, 130.5, 128.3,114.1, 66.9, 63.3, 61.7, 59.8, 55.3, 54.3, 53.8, 50.1, 49.4, 49.2, 48.5,42.3, 37.6, 36.7, 34.3, 34.0, 31.9, 29.5, 28.4, 26.4, 26.2, 27.0, 23.6, 18.8,17.7. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.61 min (ESI-MS (m / z): 774.20 (M+H) + )).

[0220]

[0221] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NHBoc (4)

[0222] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (10 mg; 0.016 mmol) was esterified with Boc-Val-OH (7.2 mg; 0.033 mmol) according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (8.0 mg; 0.010 mmol; 60%) as a lyophilized white powder. 1H NMR (500 MHz, CDCl3) δ 7.13 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.55 (dd, J =29.2, 7.9 Hz, 1H), 4.39 (dt, J = 14.0, 7.1 Hz, 1H), 4.24 (s, 8H), 4.18 – 4.04(m, 1H), 3.84 (q, J = 6.6 Hz, 8H), 3.77 (d, J = 4.6 Hz, 4H), 3.71 (s, 2H), 2.61 (s, 3H), 2.49 (s, 1H), 2.05 (s, 1H), 1.82 – 1.52 (m, 5H), 1.46 (d, J = 11.1 Hz, 11H), 1.36 (d, J = 7.1 Hz, 2H), 1.26 (s, 20H), 1.14 (d, J = 6.5 Hz, 52H), 0.96 (d, J = 6.8 Hz, 1H), 0.92 – 0.82 (m, 7H). LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 10.42 min (ESI-MS (m / z): 802.22 (M+H) + )).

[0223]

[0224] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NHBoc (5)

[0225] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Ile-OH (15 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (17 mg; 0.020 mmol; 60%) as a lyophilized white powder. 11H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 7.5 Hz, 1H), 6.31 (d, J = 7.4 Hz, 1H), 5.04 (d, J = 8.9 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.61 – 4.48 (m, 2H), 4.41 (p, J = 7.1 Hz, 1H), 4.26 (dd, J = 9.0, 4.6 Hz, 1H), 4.04 (d, J = 12.2 Hz, 1H), 3.78 (s, 3H), 3.70 (t, J = 4.7 Hz, 4H), 3.38 (d, J = 4.9 Hz, 1H), 3.06 (d, J = 4.9 Hz, 1H), 3.03 – 2.97 (m, 2H), 2.95 (s, 1H), 2.87 (d, J = 16.5 Hz, 1H), 2.46 (q, J = 4.8 Hz, 4H), 1.82 (s, 4H), 1.78 – 1.53 (m, 7H), 1.45 (s, 10H), 1.37 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 35.3 Hz, 7H), 0.92 (dd, J = 9.3, 7.1 Hz, 9H). 13 13C NMR(101 MHz, CDCl3) δ 205.2, 171.9, 170.9, 170.3, 158.6, 130.4, 128.2, 114.0, 79.9, 66.9, 63.3, 61.6, 59.7, 57.8, 55.2, 54.2, 53.7, 50.0, 49.5, 48.4, 42.3, 38.2, 37.5, 37.0, 34.3, 34.0, 31.8, 29.6, 28.3, 26.3, 26.2, 25.9, 25.1, 23.5, 17.5, 15.4, 11.8. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.28 min (ESI-MS (m / z): 816.33 (M+H + ))

[0226]

[0227] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NHBoc (6)

[0228] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Phe-OH (18 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (17 mg; 0.020 mmol; 61%) as a lyophilized white powder. 1 H NMR (400 MHz, CDCl3)δ 7.47 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 11.6 Hz, 5H), 7.13 (d, J = 8.7 Hz,4H), 6.81 (d, J = 8.5 Hz, 3H), 6.42 (d, J = 7.5 Hz, 1H), 4.96 (d, J = 8.3 Hz,1H), 4.90 (d, J = 12.2 Hz, 1H), 4.62 – 4.48 (m, 3H), 4.42 (p, J = 7.1 Hz,1H), 4.16 (s, 2H), 4.05 (d, J = 12.1 Hz, 1H), 3.84 (dq, J = 13.4, 6.7 Hz, 2H), 3.77 (s, 3H), 3.70 (t, J = 4.7 Hz, 4H), 3.35 (d, J = 4.9 Hz, 1H), 3.12 (dd, J = 13.9, 5.7 Hz, 1H), 3.05 (d, J = 5.8 Hz, 1H), 3.03 – 2.97 (m, 3H), 2.96 (s, 1H), 2.87 (d, J = 16.4 Hz, 1H), 2.47 (q, J = 4.6 Hz, 4H), 1.80 –1.51 (m, 7H), 1.41 (d, J = 5.6 Hz, 8H), 1.37 (d, J = 7.1 Hz, 4H), 1.25 (s,6H), 0.96 – 0.81 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 205.3, 171.9, 171.1,171.0, 158.6, 156.9, 154.9, 135.7, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.34 min (ESI-MS (m / z): 850.33 (M+H + )).

[0229]

[0230] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NHBoc (7)

[0231] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Phe(Phe)-OH (23 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (33 mg; 0.035 mmol; quantified) as a lyophilized white powder. 1H NMR (400MHz, 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 = 7.1Hz, 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 = 4.9Hz, 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)。 13C NMR (101 MHz, CDCl3) δ205.2, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 8.00 min (ESI-MS (m / z): 926.40 (M+H + )).

[0232]

[0233] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NHBoc (8)

[0234] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (10 mg; 0.017 mmol) was esterified with Boc-Asn-OH (7.7 mg; 0.0332 mmol) according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (4.7 mg; 0.006 mmol; 34%) as a lyophilized white powder. 1H NMR (500 MHz, CDCl3) δ 7.43 (s, 1H), 7.18 (d, J = 7.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 7.8 Hz, 3H), 6.72 (d, J = 11.8 Hz, 1H), 6.49 (s, 1H), 5.84 (s,1H), 5.75 (d, J = 8.5 Hz, 2H), 4.84 – 4.69 (m, 1H), 4.54 (t, J = 7.1 Hz, 4H), 4.38 (p, J = 7.0 Hz, 2H), 4.27 (s, 1H), 3.78 (s, 4H), 3.71 (s, 5H), 3.34 (d,J = 5.0 Hz, 1H), 3.09 (d, J = 4.8 Hz, 1H), 3.04 – 2.93 (m, 4H), 2.86 (d, J =16.0 Hz, 2H), 2.71 (d, J = 11.2 Hz, 1H), 2.46 (s, 5H), 2.36 (s, 2H), 1.87 –1.51 (m, 17H), 1.45 (d, J = 1.1 Hz, 13H), 1.36 (d, J = 7.0 Hz, 6H), 1.26 (s,45H), 0.99 – 0.79 (m, 14H). LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.31 min (ESI-MS (m / z): 817.25 (M+H) + )).

[0235]

[0236] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NHBoc (9)

[0237] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Asp(OBzl)-OH (21 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (17 mg; 0.019 mmol; 57%) as a lyophilized white powder.1 1H NMR (300 MHz, CDCl3) δ 7.43 (d, J = 7.4 Hz, 1H), 7.35 (t, J = 2.2 Hz, 5H), 7.11 (d, J = 8.6 Hz, 2H), 6.85 – 6.77 (m, 2H), 6.74 (d, J = 7.6 Hz, 1H), 6.34 (d, J = 7.5 Hz, 1H), 5.48 (d, J = 8.6 Hz, 1H), 4.89 (d, J = 12.2 Hz, 1H), 4.62 – 4.46 (m, 3H), 4.41 (p, J = 7.1 Hz, 1H), 4.07 (d, J = 12.3 Hz, 1H), 3.77 (s, 3H), 3.70 (t, J = 4.6 Hz, 4H), 3.48 (s, 2H), 3.32 (d, J = 5.0 Hz, 1H), 3.06 – 2.92 (m, 5H), 2.89 (d, J = 6.3 Hz, 2H), 2.84 (d, J = 5.7 Hz, 1H), 2.45 (q, J = 4.0 Hz, 4H), 1.89 – 1.49 (m, 11H), 1.44 (s, 10H), 1.36 (d, J = 7.0 Hz, 4H), 1.30 – 1.03 (m, 7H), 1.00 – 0.77 (m, 3H). 13 13C NMR (75 MHz, CDCl3) δ 205.2, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 6.56 min (ESI-MS (m / z): 908.50 (M+H + )).

[0238]

[0239] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NHBoc (10)

[0240] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Glu(OBzl)-OH (21 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (17 mg; 0.019 mmol; 57%) as a lyophilized white powder. 1 H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.4 Hz, 1H), 7.40 – 7.29 (m, 5H), 7.12 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 3H), 6.44 (d, J = 7.1 Hz, 1H), 5.12 (s, 3H), 4.93(s, 1H), 4.62 – 4.48 (m, 2H), 4.41 (p, J = 7.1 Hz, 1H), 4.33 (d, J = 5.6 Hz,1H), 4.19 (s, 3H), 4.10 (d, J = 12.2 Hz, 1H), 3.91 – 3.79 (m, 4H), 3.77 (s,3H), 3.70 (t, J = 4.7 Hz, 4H), 3.37 (d, J = 4.9 Hz, 1H), 3.06 (d, J = 4.8 Hz,1H), 3.00 (d, J = 7.2 Hz, 2H), 2.94 (s, 1H), 2.87 (d, J = 16.4 Hz, 1H), 2.44 (dd, J = 9.0, 4.8 Hz, 6H), 2.21 (dd, J = 13.5, 7.2 Hz, 1H), 2.03 – 1.83 (m,4H), 1.80 – 1.50 (m, 6H), 1.43 (s, 9H), 1.36 (d, J = 7.1 Hz, 3H), 1.32 – 1.16 (m, 5H), 1.02 – 0.81 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 205.3, 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 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.44 min (ESI-MS (m / z): 922.33 (M+H + )).

[0241]

[0242] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NHBoc (11)

[0243] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Ser(OBn)-OH (21 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (33 mg; 0.037 mmol; quantified) as a lyophilized white powder. 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. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 7.45 min (ESI-MS (m / z): 880.27 (M+H + )).

[0244]

[0245] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Cys(Trt)-NHBoc (12)

[0246] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) and Boc-Cys(Trt)-OH (30 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (39 mg; 0.037 mmol; quantified) as a lyophilized white powder. 1 H NMR (400MHz, CDCl3) δ 7.42 (dd, J = 19.6, 7.2 Hz, 9H), 7.30 (d, J = 7.1 Hz, 6H), 7.25– 7.19 (m, 4H), 7.11 (d, J = 8.7 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.40 (d,J = 7.5 Hz, 1H), 4.97 (d, J = 8.2 Hz, 1H), 4.77 (d, J = 12.3 Hz, 1H), 4.58(q, J = 7.0 Hz, 1H), 4.54 – 4.46 (m, 1H), 4.41 (p, J = 7.1 Hz, 1H), 4.23 (d,J = 7.2 Hz, 7H), 3.84 (dq, J = 13.0, 6.5 Hz, 6H), 3.76 (s, 3H), 3.73 – 3.64(m, 4H), 3.29 (d, J = 5.0 Hz, 1H), 3.03 – 2.96 (m, 2H), 2.94 (s, 1H), 2.86(d, J = 16.5 Hz, 1H), 2.61 – 2.41 (m, 6H), 1.78 – 1.48 (m, 6H), 1.43 (s,10H), 1.41 (s, 3H), 1.36 (d, J = 7.0 Hz, 4H), 1.31 – 1.16 (m, 7H), 1.03 –0.79 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 171.9, 170.9, 170.3, 158.6, 157.0,144.2, 130.4, 129.5, 128.1, 128.0, 126.9, 126.8, 114.0, 66.9, 61.6, 59.5,55.2, 54.2, 53.8, 52.4, 49.9, 48.4, 42.1, 37.6, 36.5, 34.3, 34.0, 33.8, 31.7,30.0, 28.3, 27.9, 26.3, 26.0, 23.5, 17.6. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 8.78 min (ESI-MS (m / z): 1048.27 (M+H) + )).

[0247]

[0248] Morph-Ala-Tyr(OMe)-Cha-EK- OC(=O)-Pro-NHBoc (13)

[0249] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (20 mg; 0.033 mmol) was esterified with Boc-Pro-OH (14 mg; 0.066 mmol) according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (22 mg; 0.027 mmol; 84%) as a lyophilized white powder. 1H NMR (400 MHz, CDCl3)δ 7.42 (t, J = 8.1 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.6 Hz,2H), 6.72 (d, J = 7.1 Hz, 1H), 6.31 (d, J = 7.4 Hz, 1H), 4.85 (dd, J = 50.5,12.2 Hz, 1H), 4.62 – 4.47 (m, 2H), 4.41 (td, J = 7.2, 4.6 Hz, 1H), 4.25 –4.17 (m, 1H), 3.99 (d, J = 12.2 Hz, 1H), 3.90 – 3.79 (m, 1H), 3.77 (s, 3H),3.70 (t, J = 4.6 Hz, 4H), 3.54 – 3.41 (m, 2H), 3.35 (dd, J = 25.1, 4.9 Hz,1H), 3.06 (dd, J = 14.3, 4.9 Hz, 1H), 3.02 – 2.96 (m, 2H), 2.94 (d, J = 3.3Hz, 1H), 2.86 (dd, J = 16.5, 7.2 Hz, 1H), 2.53 – 2.38 (m, 4H), 2.29 – 2.12(m, 1H), 1.97 (ddd, J = 12.6, 6.2, 4.3 Hz, 1H), 1.88 (p, J = 6.7, 6.2 Hz,2H), 1.82 – 1.52 (m, 8H), 1.45 (d, J = 19.4 Hz, 9H), 1.39 – 1.19 (m, 7H),1.06 – 0.82 (m, 3H)。 13C NMR (101 MHz, CDCl3) δ 205.3, 172.4, 171.9, 170.9,170.4, 158.6, 130.4, 128.2, 114.0, 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 (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt(min): 6.80 min (ESI-MS (m / z): 800.13 (M+H + )).

[0250]

[0251] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NH2•TFA (14)

[0252] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ala-NHBoc was performed at a scale of 0.013 mmol according to procedure A, yielding the title compound (9.2 mg; 0.012 mmol;) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.80 min (ESI-MS (m / z): 674.33 (M+H) + HRMS (ESI) m / z: C 34 H 51 N5O9's [M+H] + Calculated value: 674.37595; Experimental value: 674.37600

[0253]

[0254] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NH2•TFA (15)

[0255] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Val-NHBoc was performed at a scale of 0.012 mmol according to procedure A, yielding the title compound (5.25 mg; 0.006 mmol) as a lyophilized white powder. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.22 min (ESI-MS (m / z): 702.33 (M+H) + HRMS (ESI) m / z: C 36 H 55 N5O9's [M+H] + Calculated value: 702.41581, Experimental value: 702.40814

[0256]

[0257] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NH2•TFA (16)

[0258] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ile-NHBoc was performed at a scale of 0.011 mmol according to procedure A, yielding the title compound (8.7 mg; 0.010 mmol;) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.21 min (ESI-MS (m / z): 716.27 (M+H) + HRMS (ESI) m / z: C 37 H 57 N5O9's [M+H] + Calculated value: 716.42882, Experimental value: 716.42241

[0259]

[0260] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NH2•TFA (17)

[0261] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe-NHBoc was performed at a scale of 0.012 mmol according to procedure A, yielding the title compound (9.8 mg; 0.011 mmol;) as a lyophilized white powder. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.34 min (ESI-MS (m / z): 750.33 (M+H) + HRMS (ESI) m / z: C 40 H 55 N5O9's [M+H] + Calculated value: 750.40725, Experimental value: 750.40657

[0262]

[0263] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NH2•TFA (18)

[0264] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Phe(Phe)-NHBoc was performed at a scale of 0.018 mmol according to procedure A, yielding the title compound (17 mg; 0.018 mmol;) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.97 min (ESI-MS (m / z): 826.33 (M+H) + HRMS (ESI) m / z: C 46 H 59 N5O9's [M+H] + Calculated value: 826.43855; Experimental value: 826.43831

[0265]

[0266] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NH2•TFA (19)

[0267] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asn-NHBoc was performed at a scale of 0.012 mmol according to procedure A, yielding the title compound (4.03 mg; 0.0048 mmol) as a lyophilized white powder. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 3.80 min (ESI-MS (m / z): 717.25 (M+H) + HRMS (ESI) m / z: C 35 H 52 N6O 10 [M+H] + Calculated value: 717.38177, Experimental value: 717.38202

[0268]

[0269] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NH2•TFA (20)

[0270] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Asp(OBzl)-NHBoc was performed at a scale of 0.011 mmol according to procedure A, yielding the title compound (14 mg; 0.015 mmol) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.51 min (ESI-MS (m / z): 808.40 (M+H) + HRMS (ESI) m / z: C 42 H 57 N5O 11 [M+H] + Calculated value: 808.41273; Experimental value: 808.41203

[0271]

[0272] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NH2•TFA (21)

[0273] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Glu(OBzl)-NHBoc was performed at a scale of 0.011 mmol according to procedure A, yielding the title compound (12 mg; 0.012 mmol) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.47 min (ESI-MS (m / z): 822.33 (M+H) + HRMS (ESI) m / z: C 43 H 59 N5O 11 [M+H] + Calculated value: 822.42838; Experimental value: 822.42800

[0274]

[0275] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NH2•TFA (22)

[0276] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Ser(OBn)-NHBoc was performed at a scale of 0.011 mmol according to procedure A, yielding the title compound (8.7 mg; 0.010 mmol) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.51 min (ESI-MS (m / z): 780.40 (M+H) + HRMS (ESI) m / z: C 41 H 57 N5O 10 [M+H] + Calculated value: 780.41782, Experimental value: 780.41751

[0277]

[0278] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Cys(Trt)-NH2•TFA (23)

[0279] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Cys(Trt)-NHBoc was performed at a scale of 0.009 mmol according to procedure A, yielding the title compound (24 mg; 0.0226 mmol) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.88 min (ESI-MS (m / z): 948.27 (M+H) + HRMS (ESI) m / z: C 53 H 65 N5O9S [M+H] + Calculated value: 948.45758; Experimental value: 948.45715

[0280]

[0281] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Pro-NH2•TFA (24)

[0282] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-Pro-NHBoc was performed at a scale of 0.013 mmol according to procedure A, yielding the title compound (11 mg; 0.013 mmol) as a white powder after lyophilization. LC-MS (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 4.84 min (ESI-MS (m / z): 700.33 (M+H) + HRMS (ESI) m / z: C 36 H 52 N4O9 [M+H] + Calculated value: 700.39160, Experimental value: 700.39127

[0283]

[0284] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)CHCH3(CH3) (25)

[0285] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (15.1 mg, 0.025 mmol) was co-evaporated twice with toluene and subsequently dissolved in anhydrous DCM (0.1 M) and purged with N2. Isobutyric anhydride (8.3 µL, 0.05 mmol, 2.0 eq.) and DMAP (3.0 mg, 0.025 mmol, 1.0 eq.) were added to this solution. The resulting reaction mixture was stirred for two hours and then concentrated under vacuum. Silica gel column chromatography (0% -> 3% (v / v) MeOH / DCM) yielded the title compound (7.03 mg, 10.46 µmol) as a lyophilized white powder. HRMS (ESI) m / z: C 35 H 52 N4O9 [M+H] + Calculated value: 673.38071; Experimental value: 673.38071

[0286]

[0287] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OTBS (26)

[0288] Morph-Ala-Tyr(OMe)-Cha-EK-CH3OH (22.9 mg, 0.038 mmol) was Streglich-esterified with lactic acid-OTBS (15.5 mg, 0.076 mmol) according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (15.46 mg, 0.020 mmol) as a lyophilized white powder. HRMS (ESI) m / z:C 40 H 64 N4O 10 Si of [M+H] + Calculated value: 789.44645; Experimental value: 789.44607

[0289]

[0290] Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OH (27)

[0291] Deprotection of Morph-Ala-Tyr(OMe)-Cha-EK-OC(=O)-LA-OTBS was performed at a scale of 0.013 mmol according to procedure A, yielding the title compound (9.64 mg, 0.014 mmol) as a white powder after lyophilization. HRMS (ESI) m / z: C 34 H 50 N4O 10 [M+H] + Calculated value: 675.35997; Experimental value: 675.35945

[0292]

[0293] Mor-Ala-Tyr(OMe)-Cha-EK-Me-Oε-Ahx-Boc (92)

[0294] Morph-Ala-Tyr(OMe)-Cha-EK-Me-OH (20 mg; 0.033 mmol) and Boc-e-Ahx-OH (15 mg; 0.066 mmol) were esterified by Streglich according to procedure B. Silica gel column chromatography (3% (v / v) MeOH / DCM) yielded the title compound (21 mg; 0.025 mmol; 78%) as a lyophilized white powder. 1H NMR (400 MHz, CDCl3) δ 7.43 15 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 7.7 Hz, 2H), 6.81 (dd, J =17.1, 8.1 Hz, 3H), 6.71 (s, 1H), 5.03 (d, J = 12.2 Hz, 1H), 4.62 (d, J = 8.4Hz, 2H), 4.54 (d, J = 8.6 Hz, 1H), 4.41 (p, J = 7.1 Hz, 1H), 4.13 (d, J = 7.4Hz, 2H), 3.91 – 3.79 (m, 3H), 3.77 (s, 3H), 3.69 (t, J = 4.6 Hz, 4H), 3.37(d, J = 4.9 Hz, 1H), 3.18 (p, J = 7.0 Hz, 1H), 3.04 (d, J = 4.8 Hz, 2H), 3.00(d, J = 7.2 Hz, 1H), 2.95 (s, 1H), 2.87 (d, J = 16.4 Hz, 1H), 2.45 (q, J 20 =4.1 Hz, 4H), 2.33 (q, J = 8.6, 8.0 Hz, 2H), 1.84 (d, J = 19.8 Hz, 3H), 1.76 –1.55 (m, 7H), 1.45 (s, 12H), 1.35 (d, J = 7.0 Hz, 4H), 1.33 – 1.16 (m, 8H), 1.08 – 0.80 (m, 3H).13C NMR (101 MHz, CDCl3) δ 172.7, 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 to 90% MeCN / H2O, 25 0.1% TFA, 12.5 min) Rt (min): 7.05 min (ESI-MS (m / z): 816.20 (M+H+)).

[0295]

[0296] Mor-Ala-Tyr(OMe)-Cha-EK-Me-Oε-Ahx-NH2 (93)

[0297] According to procedure A, Morph-Ala-Tyr(OMe)-Cha-EK-COO-Me-Oe-Ahx-NHBoc was deprotected at a scale of 0.025 mmol to produce the title compound (12 mg; 0.014 mmol; 56%) as a white powder after lyophilization. LC-MS 5 (linear gradient 10 to 90% MeCN / H2O, 0.1% TFA, 12.5 min) Rt (min): 5.10 min (ESI-MS(m / z): 716.40 (M+H+)).

[0298] Example 2 - Proteasome Inhibition Assay Based on Competitive Activity Proteomic Analysis

[0299] The bioactivity of compounds 1-27 was assessed using a competitive activity-based proteomic assay.

[0300] Over the past three decades, activity-based proteomic analysis (ABPP) has been used to detect and identify enzymes from complex biological samples. In ABPP, an activity-based probe (ABP) containing a substrate-like element (in most cases) linked to an electrophile and having a reporter group (biotin, fluorophore, or bioorthogonal tag) reacts within the active site of the target enzyme or enzyme family to form a covalent and irreversible bond. Several proteasome inhibitors (peptide vinyl sulfone, peptide epoxide) react in a mechanism-based manner, making them a good starting point for designing proteasome-targeting ABPs. A set of three ABPs was eventually developed that, after running ABP-treated cell lysates on an SDS-PAGE gel and subsequently performing in-gel fluorescence detection, resolved all six human cCP and iCP active subunits. This ABPP assay allows for rapid analysis of the composition of cCP and iCP catalytically active components in cell lysates. For example, lysates of Raji cells (a constitutively proteasome isoform of B-cell lymphoma cells) treated with three ABPS followed by SDS PAGE and in-gel fluorescence detection produced six bands corresponding to all six iCP and cCP active sites.

[0301] Competitive ABPP complements comparative ABPP and allows for the identification of active compounds, as well as their selectivity and potency, in a library of putative proteasome inhibitors. Since most proteasome inhibitors designed in the literature, and the examples described in this document, are peptide-based electrophiles that react within the proteasome active site to form covalent and irreversible bonds or durable complexes, in competitive ABPP, various inhibitors are added to cell lysates (or cells, in the case of studying cell permeability) at different concentrations and at different times before adding three ABPs, SDS-PAGE, and in-gel fluorescence scanning. In this manner, the selectivity and activity of the set of putative proteasome inhibitors depicted in Example 1 are determined. Cell lysates were prepared by adding two volumes of lysis buffer containing 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) digitalis saponins, and 25 U / ml benzonase. The mixture was incubated on ice for one hour, followed by centrifugation at 22,000 rcf for 15 minutes at 15°C. Protein concentrations were then determined using the Bradford assay, followed by dilution of the lysate with assay buffer.

[0302] Cell lysates were diluted to 1.4–2.0 µg / µL total protein in 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 in DMSO) at 37°C for one hour. Additionally, the inhibited cell lysates were exposed to a probe mixture (Cy2: BODIPY(FL)-LU-112, Cy3: BODIPY(TMR)-NC-005VS, Cy5: Cy5-NC-001) at 37°C for one hour. Next, reducing gel loading buffer was added to denature the cell lysates, and the mixture was boiled at 95°C for four minutes. Subsequently, the denatured cell lysates were loaded onto a 12.5% ​​SDS-PAGE gel and fractionated by gel electrophoresis at 160 V for 90 minutes. Immediately after electrophoresis, the lysates were transferred to a ChemiDoc gel with Cy5, Cy3, and Cy2 channels. TM Multiplex fluorescence detection of probes based on residual activity was performed on the MP system. The gel was then fixed and stained overnight in Coomassie blue, followed by destaining in demineralized water for two days. Similarly, in ChemiDoc... TM Coomassie blue detection was performed on the MP system and transformed using ImageLab. Adjusted fluorescence band intensities were transformed using ImageLab. Normalized adjusted volumes were plotted relative to inhibitor concentrations, and then IC50 for each inhibitor was calculated using GraphPad Prism 9.0 software (using a nonlinear regression model of the [inhibitor] response relative to normalized response with a variable slope). 50 value.

[0303] For N-Boc amino acid ester 3-13, weak or no inhibition was observed at any of the cCP active sites. Comparison of compound 3-13 with LU-005i revealed that LU-005i was significantly more effective against β5c compared to other proteasome subunits. Figure 1 Among the compounds discovered, the observed low potency of β1c and β2c was confirmed by data found during the development of LU-005i, where the combination of P1 and P2 residues was determined to inhibit the affinity for β1c and β2c; Tyr(OMe)-Cha. Compared to LU-005i 1, Figure 1All N-Boc esters shown exhibited at least a 1.5-fold reduction in potency towards β5c. Furthermore, LU005i-OH2, 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-NH2 12, valine-NH2 13, isoleucine-NH2 16, aspartic acid-NH2 20, and cysteine-NH2 23 all demonstrated iCP selectivity. Of the proteasome inhibitors listed above, LU-005i-OH 2, N-Boc alanine 3, N-Boc aspartic acid 9, N-Boc glutamate 10, alanine-NH2 14, aspartic acid-NH2 20, serine-NH2 22, and cysteine-NH2 23 are of the most interest because they all exhibit selectivity ratios of two to more for iCPs compared to cCPs (highest cCP potency relative to lowest iCP potency).

[0304] Compounds characterized by aliphatic and large amino acid esters (N-Boc valine 4, N-Boc isoleucine 5, N-Boc phenylalanine 6, N-Boc diphenylalanine 7, and N-Boc-S-Trt cysteine ​​8) showed no efficacy against β1i and β2i, suggesting that the size of the amino acid residues at P1' negatively impacts the affinity for these subunits. Coincidentally, this makes these compounds highly selective β5i inhibitors. Compared to N-Boc phenylalanine 6, N-Boc diphenylalanine 7, and N-Boc-S-Trt cysteine ​​8, N-Boc aspartic acid 9, N-Boc glutamate 10, and N-Boc serine 11 were shown to be potent inhibitors of β1i and β2i. Possibly, the rotational flexibility and hydrogen bonding capacity found in the benzylated carboxylic acids of N-Boc aspartic acid 9 and N-Boc glutamate 10, and the benzylated hydroxyl group of N-Boc serine 11, counteract the conflict in the S' bag. Furthermore, increased potency of β1i, β2i, and β5c was observed when N-Boc serine 11 was compared to N-Boc aspartic acid 9, and further increased potency of β1i, β2i, and β5c was observed when N-Boc aspartic acid 9 was compared to N-Boc glutamate 10. Interestingly, a similar inhibitory profile was observed when N-Boc aspartic acid 9 was compared to N-Boc asparagine 7. Among N-Boc amino acid ester proteasome inhibitors, N-Boc alanine 3 was shown to be the only inhibitor exhibiting potency of 10 µM or higher against each of the cCP active sites, while showing potency of 4 µM or lower against the iCP active site. Figure 1When N-Boc alanine 2 is compared with branched alkyl derivatives N-Boc valine 4 and N-Boc isoleucine 5, it can be inferred that such residues reduce the affinity for β1i and β2i subunits.

[0305] Overall, in the Figure 1 When all the NH2 amino acid esters found were compared with their N-Boc counterparts (3-13), increased potency for β1i and β2i was observed. Except for phenylalanine-NH2 17, diphenylalanine-NH2 18, glutamic acid-NH2 19, serine-NH2 20, and proline-NH2 24, all NH2 amino acid esters showed potency equal to or greater than 10 µM for β1c and β2c. Removal of the N-Boc protecting group introduced two structural changes: a significant reduction in steric volume and the formation of a hydrophilic amine that is protonated at physiological pH. Valine-NH2 15, isoleucine-NH2 16, phenylalanine-NH2 17, and diphenylalanine-NH2 18 showed increased affinity for β1i, β2c, β2i, and β5c. This indicates that the combination of the N-Boc protecting group with branched alkyl or bulky aromatic residues eliminates the inhibition of β1i, β2c, β2i, and β5c. When glutamic acid -NH2 21 and serine -NH2 22 were compared with their N-Boc counterparts, increased potency against β1i, β2c, β2i, β5c, and β5i was observed. Interestingly, serine -NH2 22 proved to be a potent inhibitor of β1i, β2c, β2i, β5c, and β5i, while its N-Boc counterpart was only effective against β5i. Furthermore, glutamic acid -NH2 21 did not exhibit iCP selectivity and ultimately became a potent inhibitor of β1i, β2i, β5c, and β5i. Aspartic acid -NH2 20 was the only inhibitor among the three O-benzyl protecting inhibitors (20-22) to show higher selectivity for iCP than for cCP when the three inhibitors were compared with their N-Boc counterparts (9-11).

[0306] Notably, cysteine-NH2-23, with a side chain length similar to serine-NH2-22 and carrying an aromatic protecting group, did indeed prove to be a selective inhibitor of iCP, even though the protecting group is much larger than that of the benzyl protecting group, unlike its N-Boc counterpart (12). When compared with N-Boc alanine 3, alanine-NH2-14 showed improved selectivity for iCP, with a tenfold increase in potency for both β1i and β2i and a fivefold increase in potency for β5c. In summary, these studies identified alanine-NH2-14 as the most selective inhibitor of all three iCPs among all the compounds tested.

[0307] Therefore, alanine-NH2 14 was identified as the optimal iCP-selective proteasome inhibitor, and to further investigate this, alanine-NH2 14 was re-evaluated against N-Boc alanine 3 and tested in a competitive ABPP assay at a wider concentration range (up to 30 µM). Figure 2 Compare with 25, 26 and 27.

[0308] and Figure 1 Compared to the inhibitors described in the text, Figure 2 All the alanine isomers shown are characterized by higher selectivity for the iCP active subunit than for the cCP active subunit. When comparing O-TBS lactate 26 with N-Boc alanine 14, a decreased potency for β2i was observed, indicating that β2i is detrimental to the increase in the steric volume of the TBS protecting group. Unlike alanine-NH2 14, lactate-OH 27 did not show increased potency for β2c and β5c. Furthermore, when comparing both with their protecting group counterparts (26 and 3), as observed with alanine-NH2 14, lactate 27 did not show increased potency for β1i and β2i. Interestingly, lactate 27 showed an eight-fold decrease in potency for β1i compared to O-TBS lactate 26. Isobutyrate 25 showed a similar inhibition profile to N-Boc alanine 2, exhibiting iCP-selective inhibition. In terms of size, isobutyrate 25 and lactate 27 are comparable to alanine 13, but their inhibition profiles are not. This indicates that the N-terminal space volume is not the primary determinant of efficacy and selectivity, but rather the atomic properties of the N-terminal atom. Overall, no clear trend was observed regarding the size of the amine-free isomers.

[0309] Reference Example 3 – Synthesis of Compounds 28 to 51 (P3 variants)

[0310] Compounds 28-51, as shown below, have been synthesized by methods similar to those described in Example 1 and those described below for compound 28.

[0311]

[0312] Compounds are named according to the abbreviations of the P4 and P3 sites. For example, compound 28 is Mor(P4)-Gly(P3)-Tyr(OMe)(P2)-Cha(P1)-EK, and its abbreviation is 28 (Mor-Gly).

[0313] Synthesis of Compound 28

[0314]

[0315] aReagents and conditions: (a) Boc-Gly-OH, HCTU, DiPEA, DCM, 17%; (b) (i) TFA, DCM, quantitative; (ii) Morpholine acetic acid, HCTU, DiPEA, DCM, 48%; (c) Hydrazine hydrate, MeOH, quantitative; (d) (i) tBuONO, HCl, DMF, -30℃; (ii) 97, DiPEA, 13%.

[0316] Reference Example 4 - Synthesis of compounds 52 to 57 (P3 variants)

[0317] Compounds 52 to 57, as shown below, were synthesized by methods similar to those described in Example 1 and those described above for compound 28.

[0318]

[0319] Reference Example 5 - Competitive ABPP assay in Raji cell lysates

[0320] The bioactivity of compound 28-57 was assessed using the bioassays described in Example 2. The resulting apparent IC50 values... 50 Values ​​plotted on Figure 3 The heatmap shown.

[0321] Based on its moderate pan-immunopiproteasome selectivity, LU-005i 1 was chosen as our lead compound. The C-terminal epoxide, the cyclohexyl-1-alanine (Cha) residue at position P1, and the Tyr (OMe) residue at position P2 were kept fixed in our design, as these structural elements have previously been shown to be accepted by all iCP activities. The P3 and P4 (N-cap) residues were modified to obtain compounds with increased iCP selectivity by increasing activity against iCP, decreasing activity against cCP, or a combination of both. Based on this principle, we created the first group of 24 peptide epoxides (compounds 28-57) with the general structure YX-Cha-Tyr(OMe)-epoxide, where Y is one of the five tested N-caps and X is one of the five selected α-amino acids. Because both the β1i and β5i subunits are characterized by a smaller S3 bag than their constitutive counterparts, four relatively small P3 residues were chosen: Gly, Ala, Ser, and 2-aminoisobutyric acid (Aib). As a control for the effect of small P3 residues on activity and selectivity, we selected large O-benzyl-Ser (Ser(OBn)). For the P4 moiety, we used a morpholine cap (also present in LU-005i 1), piperazine (HPip), N-methylpiperazine (MePip), tert-butoxycarbonyl-protected piperazine (N-BocPip), and 4-hydroxycyclohexyl (HCH). All 24 compounds were synthesized according to previously established protocols. As an example, the synthetic route for compound 28 is described above (see the Experimental section for details on the synthesis and characterization of all compounds). The proteasome inhibitory potency and selectivity of the obtained 24 compounds were evaluated in an activity-based proteomic proteomic analysis (ABPP) assay and compared with LU-005i 1. This technique utilizes three selectively active probes labeled with the β2c / i subunit (shown in green), β5c / i subunit (shown in red), and β1c / i subunit (shown in blue) to assess the inhibitory profile of all catalytic activities of cCP and iCP. Extracts from Raji cells (a human B-cell lymphoma cell line) were used because they express both iCP and cCP, and compounds 28–57 were tested in a dilution series (final concentrations from 0.01 µM to 100 µM). Briefly, cell extracts were treated with inhibitors followed by three ABPs. Samples were then separated by SDS-PAGE, and intragel scanning was performed to detect any remaining fluorescence. The apparent IC50 was then calculated. 50 Values ​​and plotted as a heatmap ( Figure 3The results showed that all compounds were poor inhibitors of β1c and β2c, confirming previous studies that the Tyr(OMe)-Cha-epoxyketone scaffold interferes with β1c and β2c inhibition. Compounds 50 (HCH-Ser(OBn)), 45 (MePip-Ser(OBn)), and 34 (BocPip-Ser) appeared to be the most potent β1i inhibitors, significantly outperforming LU-005i 1 and all other compounds containing Ala, Gly, or Aib at P3. Surprisingly, the large-volume P3-Ser(OBn) characterized by compounds 45 and 50 exhibited high potency, even though we did not initially expect these inhibitors to fit into the small-sized β1i-S3 cavity. Notably, the data also showed that large side chains may be present at P3 or P4, but are unfavorable at both positions. For example, compounds 35 (BocPip-Ser(OBn)) and 40 (HPip-Ser(OBn)) showed lower potency against β1i.

[0322] We then investigated the β2i potency of 24 compounds plus LU-005i (1). The results clearly showed that 34 (BocPip-Ser) was the most potent and selective epoxide ketone in the series, while compounds with Ser(OBn) at P3 proved to be poorer β2i inhibitors compared to their corresponding l-Ser derivatives.

[0323] Finally, the inhibitory potency of the β5c and β5i subunits was analyzed. The most active β5i inhibitors ultimately became compounds 34 (BocPip-Ser) and 35 (BocPip-Ser(OBn)). While compound 35 showed only slightly lower selectivity for β5i (3-fold), compound 34 demonstrated significantly higher selectivity (21-fold). The S3 pocket at the β5i active site is relatively smaller than the pocket in the β5c subunit due to the substitution of Ala27 by Ser (constitutive proteasome versus immunoproteasome), which may explain the better performance of 34 with its less space-demanding Ser residues. In conclusion, in terms of both potency and selectivity, compound 34 proved to be the most effective in this series: it effectively inhibited the activity of each of the immunoproteasome subunits (IC5c of the i-subunit). 50 It has a concentration ≤ 0.84 µM and exhibits considerable selectivity for constitutive proteasome counterparts (IC50). 50 Ratios β1c / β1i: 36, β2c / β2i: 33, β5c / β5i: 7).

[0324] Second Generation Key Document Library

[0325] Based on 34, the next group of compounds (52-57) was designed, synthesized, and evaluated. All compounds in this series share the common structure Boc-Pip-X-Cha-Tyr(OMe)-epoxyketone, combining the C-terminal dipeptide epoxyketone of LU-005i 1 with P4 residues as present in lead compound 12. For P3 substituents, we selected Arg (as in 52), carboxybenzyl (Cbz)-protected Arg 53, and O-p-xylene (OpXyl, 54). Also included are the corresponding P3 diastereomers of 34 and 35, BocPip-d-Ser 55, and BocPip-d-Ser(OBn). 56, because the d-amino acid at P3 has previously been described as enhancing β5i selectivity relative to its l-amino acid counterpart among epoxyketone inhibitors. Additionally, compound 57, carrying a pyrimidine-protected Lys side chain at P3, is included as an arginine mimic. Compounds 52–57 were determined by competitive ABPP, and the activities of all constitutive proteasome and immunoproteasome active sites were scored and plotted as previously described. Although several compounds showed activity against all immunoproteasome activities at low concentrations, most of them concomitantly inhibited the β5c active site, making them less attractive than lead compound 34. As expected, the peptide epoxyketone characterized by the d-amino acid at P3 proved to be a poor β5c inhibitor, but unfortunately, it also ultimately became a weak β1i and β2i inhibitor. This β5i selectivity has been previously noted and is likely due to spatial factors associated with the kink-binding pattern induced by the P3 d-amino acid.

[0326] Reference Example 6 - Synthesis of Compounds 58 to 77 (P3 variants)

[0327] Compounds 58-77, as shown below, were synthesized using methods similar to those described in Examples 1 and 3. Further synthetic details are provided below.

[0328]

[0329] Compounds are named according to the abbreviations of the P4 and P3 sites.

[0330] A novel compound library was designed containing serine at P3 and BocPip or Morph at P4. Modifications were made to improve the selectivity of β5i relative to β5c. The amino acids and their derivatives used included: diaminopropionic acid (Dap), homoserine (Hser), diaminobutyric acid (Dab), asparagine (Asn), pentaserine (HHSer), and glutamine (Gln).

[0331] Figure 4All peptide epoxides presented were characterized by cyclohexylalanine epoxides at P1, and thus a large quantity of 97 was synthesized. The condensation of N-Boc cyclohexylalanine 77 with a Weinreb salt yielded Weinreb amide 78 (Scheme 3). Then, propylene bromide was carbide-lithiated, and the resulting propylene-lithium was added to Weinreb amide 78 to give α,β-unsaturated ketone 98. Nucleophilic epoxidation using hydrogen peroxide produced two diastereomers of epoxide 99, which were separated by silica gel column chromatography to give N-Boc epoxide 99. Finally, the N-Boc protecting group in 99 was removed using TFA to give epoxide 97.

[0332]

[0333] 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.

[0334] With a large quantity of epoxy ketone 97 available, the focus was on constructing various N-terminal P2-P3-P4 fragments that, when combined, would generate the target library. For this, it was necessary to prepare various amino acids characterized by P3 and perform appropriate functionalizations to ensure compatibility with peptide synthesis chemistry. To synthesize partially and orthogonally protected diaminopropionic acid for subsequent incorporation at P3, N-Boc asparagine 100 was rearranged via Hoffmann rearrangement using diacetoxyiodobenzene (PIDA) as the oxidant to form N-Boc diaminopropionic acid (Dap) 102 (Scheme 4). Next, the N-β group of Dap102 was protected with an Alloc group (102 to 104), and then the N-Alloc Dap 104 was coupled with 4-methoxyphenylalanine to generate dipeptide 106. De-N-Bocization of 106, followed by condensation with 2-morpholinoacetic acid, yielded ester 108, which was then converted to acylhydrazine 110, subsequently converted to an acyl azide intermediate under anhydrous acidic conditions using tert-butyl nitrite. After the formation of the acyl azide intermediate, the pH was neutralized using DiPEA by LC-MS, followed by the addition of epoxy ketone 97 to form target compound 58 equipped with P3 N-Alloc Dap. The N-Alloc protecting group in 58 was removed under palladium catalysis using catalytic amounts of Pd(PPh3)4 and phenylsilane as allyl scavengers to generate target compound 59, P3 NH2 Dap. Finally, compound 59 was acetylated with pyridine containing acetic anhydride to produce 60. Starting with glutamine, the synthesis described above was repeated and performed to synthesize diaminobutyric acid (Dab) derivatives 61, 62 and 63 (Scheme 4).

[0335]

[0336] Scheme 4. Synthesis of a proteasome inhibitor based on P3-modified LU-005i. 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, 108:78%, 109:77%; (e) hydrazine hydrate, MeOH, rt, quantified. (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%.

[0337] To synthesize compounds 64-67, isobutyl chloroformate was used to activate the carboxylic acid side chain of aspartic acid 112 in the presence of N-methylmorpholine (pKa 7.38) as a proton scavenger. Using a stronger base as a proton scavenger (such as triethylamine (pKa 10.75)) 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). The benzyl ester in 114 was then saponified with lithium hydroxide to give carboxylic acid 116. This was obtained by sequentially deprotonating the carboxylic acid and alcohol with two equivalents of sodium hydride, followed by the addition of one equivalent of benzyl bromide O-Bn homoserine 118. The carboxylic acid ester 118 was then condensed with methyl 4-methoxyphenylalanine to form N-Boc dipeptide 120. The N-Boc dipeptide 120 was then de-N-Bocized using a TFA-containing DCM, and the resulting free amine was condensed with 2-morpholinoacetic acid to give ester 122. The ester in 122 was then saponified using lithium hydroxide in an equal mixture of H₂O and methanol to produce carboxylic acid 124. Compound 124 was then condensed with epoxide 97 using PyBOP as a peptide coupling agent to form O-Bn HSer compound 64. The O-Bn protecting group was removed by hydrogenation using Pd / carbon under a hydrogen-saturated atmosphere to produce OHHSer compound 65. To synthesize HSer compounds 69 and 70, the N-Boc piperazine group was condensed with the de-N-Bocized dipeptide 120 instead of 2-morpholinoacetic acid, and the synthetic route was then followed as described above. In a similar manner, 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.

[0338]

[0339] Scheme 5. Synthesis of proteasome inhibitors based on P3 and P4 modified LU-005i. Reagents and conditions: (a) i: isobutyl chloroformate, NMM, THF, 0℃; ii: NaBH4, MeOH 0℃ -> rt, 114:81%, 115:71%; (b) LiOH, H2O / MeOH 1:1, rt, quantification. (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 piperazine-acetic acid, 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%.

[0340] To construct the final target compounds of the key library, namely compounds 68 and 73, N-asparagine (Asn) was condensed with 4-methoxyphenylalanine to form Asn dipeptide 132 (Scheme 6). 132 was de-N-Bocized in a TFA-containing DCM, followed by condensation with 2-morpholinoacetic acid to produce methyl ester 134, which was then converted to acyl hydrazine 136 by treatment with hydrazine hydrate. Acyl hydrazine 136 was then converted to the corresponding acyl azide intermediate 138 under anhydrous acidic conditions using tert-butyl nitrite, followed by the addition of epoxide 97 to form P3 Asn derivative 68. Furthermore, the above synthetic route was repeated using N-Boc glutamine (Gln) to obtain P3 Gln derivative 73. Notably, when preparing methyl ester 134 via the condensation of dipeptide 132 with 2-morpholinoacetic acid, successful conversion was observed only when 2-morpholinoacetic acid was pre-activated with HCTU. All other attempts resulted in the formation of a glutamine guanidine adduct as the major product of the reaction.

[0341]

[0342] Scheme 6. Synthesis of P3 analogues of LU-005i. Reagents and conditions: (a) TFA•NH2-Tyr(OMe)-OMe, HCTU, DIPEA, DMF, rt, 132:82%, 133:82%; (b) ii: 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%.

[0343] Reference Example 7 - Competitive ABPP assay in Raji cell lysates

[0344] The bioassays described in Example 2 were used to assess the bioactivity of the compounds in Example 6.

[0345] The resulting apparent IC 50 Values ​​plotted on Figure 4 The heatmap shown.

[0346] Figure 4 The potency and selectivity of all compounds described herein as proteasome inhibitors were determined in Raji cell lysates (B-cell lymphoma cell lines expressing both cCP and iCP) using a competitive ABPP assay. Compounds 58–73 were serially tested at concentrations ranging from 0.003 µM to 30 µM to determine the apparent inhibitory concentration (IC50) for each catalytically active proteasome subunit. 50 The resulting apparent IC 50 The values ​​are plotted as a heatmap, such as Figure 4 As shown. Using heatmaps, the initial structure-activity relationship (SAR) of the LU-005i analogue library and the relationship between the P3 and P4 substituents and the cCP / iCP inhibition profile can be determined.

[0347] In evaluating the inhibitory potency of this group of compounds, two groups were considered of interest: those exhibiting high potency against all three iCP subunits, and those exhibiting less pronounced potency against these activities but high selectivity against the corresponding cCP activities. Morph-HSer derivative 65, Morph-Asn derivative 68, and BocPip-HSer derivative 70 all fit into one of the two categories described above. All three are characterized by a P3 residue with the same functional length from the α-carbon to the side chain. Morph-Asn 68 and BocPip-HSer 70 both exhibited potent potency against the iCP subunits, outperforming 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-HSer 65 was shown to be the proteasome inhibitor with the strongest selectivity against iCP relative to cCP.

[0348] Proteasome inhibitors characterized by Dap or Dab at P3 were neither superior to LU-005i 1 nor improved compared to previously screened inhibitors 28 and 30. Compounds 58 and 60 showed lower potency against β1c and β2c but increased five-fold potency against β5c compared to LU-005i 1, while compounds 59 and 62 proved to be relatively less potent β1i inhibitors. The protonated basic NH2 at pH 7 of Dap 59 and Dab 62 is likely incompatible with the S3 binding bag of β1i. Surprisingly, the relative decrease in inhibitory potency (compared to LU-005i) of 62 did not match that of 65, confirming that the charged amine, rather than the increased carbon length, caused the potency reduction. Compound 60 proved to be a weak inhibitor of both β1 and β2i compared to compound 59, while simultaneously making it a highly selective β5i inhibitor. Fully introducing Dap or Dab onto P3 did not produce compounds characterized by the desired combination of increased β1i and β2i potency and decreased β5c potency.

[0349] Previous structure-activity relationship studies have shown that compound 30 is a poor inhibitor of all proteasome active sites β1i, β1c, β2i, β2c, β5i, and β5c. Perhaps counterintuitively, compared to 28, analogs 64 and 66, characterized by benzylated homoserine instead of benzylated serine (as in 30), have both proven to be more effective inhibitors of β1i, β2i, β5c, and β5i. Furthermore, when comparing benzylated analogs 64 and 66 with their OH counterparts 65 and 67, 70 and 72 have proven to be more effective for β1i, β2i, and β5c. However, when looking at the ratio between the lowest cCP inhibition and the highest iCP inhibition, compound 65 appears to be superior to compound 2. For compounds characterized by asparagine or glutamine at P3, unexpected inhibitory profiles were obtained. Intuitively, asparagine and glutamine would be expected to exhibit similar inhibitory profiles to their carbon side-chain analogs homoserine (65) and pentahomoserine (67). Conversely, compounds 68 and 73, containing asparagine and glutamine, have been shown to be more potent β1i, β2i, and β5c inhibitors than their homoserine (65) and pentaserine (67) counterparts. Similar to the comparisons observed between Morph-Dap 59 and Morph-Dab 62, and between Morph-HSer 65 and Morph-HHSer 67, increasing the length of the carbon side chain reduces the potency of either β1i or β2i, or both.

[0350] Previous analysis showed that when LU-005i 1, characterized by an N-terminal 2-morpholinoacetyl cap, was compared to the same compound characterized by an N-Boc piperazine cap, the latter proved to be more potent for all iCP activities. In contrast, compound 28 proved to be a poor inhibitor of all cCP / iCP active sites, while BocPip-Ser(OBn) 35 inhibited all six cCP / iCP active sites, even with varying potency. One-carbon homologs (relative to the P3 moiety) 69 and 71 exhibited cCP / iCP potency and selectivity largely comparable to compound 34, which has a serine residue at P3. In contrast, compound 36, lacking an O-benzylated P3 residue, proved to be a more potent β1i and β2i inhibitor compared to LU-005i 1. Interestingly, this compound was also shown to be a β2c inhibitor with three times the potency of 35.

[0351] X-ray structure analysis of BocPip-Ser(OBn)35 revealed strong hydrophobic interactions between the N-Boc piperazine cap and the residues in the S4 pouch of yß2 and yß5. This anchors the BocPip P4 residues within the S4 pouch in a manner that smaller, more hydrophilic morpholine caps would not be able to achieve. Figure 5 Apparent ICs found in 50The data confirm this, and it can be concluded that the inhibitory efficacy of β2i in such compounds is independent of the properties of the P3 residues.

[0352] Furthermore, careful study of inhibitors characterized by serine, homoserine, or pentaserine residues at P3 revealed that Morph-HSer 65 is the most potent inhibitor of both β1i and β2i inhibitors, and also showed an optimal ratio of weak cCP inhibition to strong iCP inhibition. In addition, compounds characterized by amine residues at P3 were less potent inhibitors of both β1i and β2i compared to OH analogs (65 and 67) and LU-005i 1.

[0353] Example 8 - Synthesis of compounds 74-76

[0354] Compounds 74-76, as shown below, were synthesized using methods similar to those described in Examples 1, 3, and 6. Further synthetic details are provided below.

[0355] Example 1 describes the synthesis of epoxy ketone 83, and much of the 83 effort at hand focuses on the synthesis of the P2-P3-P4 fragment characterized by homoserine at P3 (Scheme 7). To synthesize the target peptide epoxy ketone 74-76, the carboxylic acid side chain of aspartic acid 112 was activated with isobutyl chloroformate, followed by the addition of sodium borohydride, thereby reducing the anhydride intermediate to homoserine derivative 114. The benzyl ester of 114 was then saponified with lithium hydroxide to form carboxylic acid 116. Next, two equivalents of sodium hydride were added, followed by one equivalent of benzyl bromide, to obtain O-Bn homoserine 118. The sequential condensation of 118 with methyl 4-methoxyphenylalanine yielded N-Boc dipeptide 120. De-N-Bocization of 120 with TFA-containing DCM produced a free amine, which was then condensed with 2-morpholinoacetic acid to produce methyl ester 122. Methyl ester 122 was then converted to hydrazide 140, which could then be converted to an acyl azide intermediate using tert-butyl nitrite under anhydrous acidic conditions. After complete conversion, as monitored by LC-MS, neutralization was performed with DiPEA, followed by the addition of epoxide ketone 83 to form target compound 74. Furthermore, the O-benzyl protecting group in 74 was removed by hydrogenation using Pd / carbon under a hydrogen-saturated atmosphere, yielding the OH HSer target compound 75. The OH group in 74 was then functionalized by Streglich esterification with N-Boc alanine to form N-Boc alanine ester compound 76. Subsequent deprotection of the O-Bn group in 76 was confirmed by palladium / carbon under a hydrogen-saturated atmosphere, but unfortunately, the side reaction of epoxide hydrogenation resulted in CO bond cleavage, forming an alcohol moiety.

[0356]

[0357] Scheme 7. Synthesis of P3 HSer LU-005i-OH analogs 74, 75 and N-Boc alanine ester derivative 76. Reagents and conditions: (a) i: Isobutyl chloroformate, NMM, THF, 0℃; ii: NaBH4, MeOH, 0℃ -> rt, 81%; (b) LiOH, H2O / MeOH 1:1, rt, quantitative; (c) NaH, BnBr, DMF, 0℃ -> 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: tBuONO, HCl, DMF, -30℃; ii: 83, DiPEA, DMF, -30℃ -> rt, 69%; (h) Pd / C, H2, MeOH, rt; (i) NHBoc-Ala-CO2H, DIC, 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.

[0358] Example 9 - Competitive ABPP assay in Raji cell lysates

[0359] The potency and selectivity profiles of the synthesized compounds 74–76, compared to compounds 2, 3, 14, 65, and 68, which are proteasome inhibitors, were determined using a competitive ABPP assay in Raji cell lysates (B-cell lymphoma cell lines expressing both cCP and iCP). To further expand the range of all novel proteasome inhibitors, AMO wild-type cells were selected as additional cell lysates to evaluate the proteasome inhibitor (AMO-wt, a plasmacytoma cell line also expressing both cCP and iCP). Figure 6 ).

[0360] Compounds 74-76 and lead structures 2, 3, 14, 65, and 68 were tested at concentrations ranging from 0.003 µM to 10 µM to determine the concentrations (IC50) of the epigenetic inhibitors in both Raji cell lysates and AMO-wt cell lysates. 50 The resulting apparent IC 50 Values ​​plotted as Figure 5 and 6 The heatmap shown. All compounds were shown to be weak inhibitors of β1c and β2c in Raji cell lysates. Figure 5As shown in Reference Example 7, peptide epoxides characterized by O-benzylhomoserine at the P3 position are potent inhibitors of the β1i, β5c, and β5i active sites. Notably, compounds 74 and 76 were shown to have the same potency for β1i, β5c, and β5i as Morph-HSer(OBn) 65 compared to LU-005i 1, and increased potency for β2i was also observed in these compounds. Furthermore, compound 76 showed a four-fold decrease in potency for β1i and a three-fold decrease in potency for β2i compared to 74. Morph-HSer 65 was the only inhibitor compared to LU-005i 1, showing increased potency for β1i, β2i, and β5i in addition to decreased potency for β5c. Compared to Morph-HSer 65, compound 75, characterized by homoserine at the P3 position and hydroxymethyl at the P1' position, ultimately emerged as a more selective iCP inhibitor. Specifically, when comparing compounds 75 and 65, a more than tenfold increase in potency against β2i was observed, while potency against β5c decreased by a factor of two. When 75 was compared to LU-005i-OH 2, a fourfold increase in potency against β2i, a twofold decrease in potency against β5c, and most interestingly, a fivefold decrease in potency against β1i, were observed.

[0361] Figure 6 The active sites of cCP / iCP (the inhibitory efficacy of compounds 2, 3, 14, 65, and 68 in the AMO-wt cleavages) were depicted. The IC50 values ​​of 1–9 of the Raji cleavages were also analyzed. 50 value( Figure 5 IC50 values ​​of 2, 3, 14, 65, and 68 for the found AMO wild-type lysates 50 value( Figure 6 When compared, the overall inhibition profiles appear very similar. Interestingly, LU-005i-OH 2 was shown to be a more potent inhibitor of both the cCP and iCP active subunits in AMO-wt. Furthermore, the iCP selectivity of 2 in the AMO-wt lysate was the same as that in the Raji lysate, where the potency of the iCP active subunit was at least twice that of the cCP active subunit. When the IC50 of the AMO-wt lysate was compared... 50When compared with values ​​in the Raji lysate, N-Boc alanine ester 3 showed a two-fold decrease in potency against β1i, a ten-fold decrease in potency against β5i, and a six-fold increase in potency against β2i. Surprisingly, alanine-NH2 14 was shown to be a less potent inhibitor of β1i and β2i, eventually becoming a more potent inhibitor of β1c and β2c. In the Raji lysate, 14 was shown to have iCP selectivity, with the iCP active site showing at least a six-fold difference in potency relative to the cCP active site. In the AMO-wt lysate, compound 14 showed low selectivity for the iCP active site when compared with the Raji lysate. On the other hand, when the IC50 values ​​in the Raji lysate were increased... 50 When compared with values ​​in the AMO-wt lysate, compound 75 was shown to have higher selectivity for the iCP active subunit, indicating that the potency against β5c was almost 2-fold reduced in the AMO-wt lysate. In contrast, when the inhibition profile of 75 was compared with that of 65 in the AMO-wt lysate, compound 75 was shown to be a more effective inhibitor of β1i and β2i, in addition to being more selective for the iCP active site.

[0362] The following abbreviations may be used in this article.

[0363]

Claims

1. A compound of formula (I), in R a and R b One of them represents H, and the other represents –[CH2]. m -X; X represents -OC(O)-Y, -C(O)-Y, -OH, or OY; Y represents -C 1-6 Alkyl (optionally -Z) 1 -Z 2 (replace) or -CH(R) y )-(Z 1 -Z 2 ) n ; R y The side chain represents a methyl biphenyl or a proteogenic amino acid, optionally wherein the side chain is in a chemically protected form; Each Z 1 Independently -NH-, -N(R) z - or -O-; R z If it exists, it is related to R. y Combined to form a proline ring; Each Z 2 Independently H, -C(O)-C 1-4 Alkyl, -C(O)OC 1-4 Alkyl or -Si(C) 1-4 Alkyl)3; m is 0, 1, or 2; n is 1 or 2; p is 0 or 1; R c -C represents H or -C optionally substituted with one or more Q substituents. 1-4 alkyl; Q means -OR d -NHR e or -C(O)NHR f ; R d R e and R f Indicates H, -C 1-4 Alkyl groups (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 Alkenyl, -C(=N)-NH2, -C(=N)-NH- (protecting group), or pyrimidinyl; A represents the circular portion selected from the following groups: R h Represents H and C 1-4 Alkyl or -C(O)OC 1-4 alkyl; R i Represents H and C 1-4 Alkyl, -N(R) j (R) k ) or -OH; R j and R k Independently represent H or C 1-4 alkyl; Or its pharmaceutically acceptable salts or solvates.

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

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

4. The compound according to any one of the preceding claims, wherein Y represents -C 3-5 Alkyl (optionally -Z) 1 -Z 2 (replace) or -CH(R) y )-(Z 1 -Z 2 ) n .

5. The compound according to any one of the preceding claims, wherein R y This refers to a chemically protected form of a methyl biphenyl, a side chain of a proteoamino acid, or a side chain of a proteoamino acid, wherein the chemical protection involves the linking of a portion selected from the group consisting of benzoyl, benzyl, and triphenylmethyl groups.

6. The compound according to any one of the preceding claims, wherein: (i) m is 0 or 1; and / or (ii) n is 1.

7. The compound according to any one of the preceding claims, wherein 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).

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

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

10. The compound according to any one of the preceding claims, wherein the compound is selected from the group consisting of:

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

12. A compound as defined in any one of claims 1 to 10 or a pharmaceutical preparation as defined in claim 11, used in a pharmaceutical medicament.

13. A compound as defined in any one of claims 1 to 10 or a pharmaceutical preparation as defined in claim 11, for the treatment or prevention of a disease or condition in which pan-immunoproteasome inhibition is desired or required.

14. The compound or pharmaceutical formulation used according to claim 13, wherein the disease or symptom is a hematologic malignancy, a solid tumor, an autoimmune disease, or an inflammatory disease.

15. The compound or pharmaceutical preparation used 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, brain inflammation, colitis-associated 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 method for preparing a compound of formula (I) as defined in claim 1, the method comprising the steps of: Make compound (II) Among them, A, p and R c As defined in claim 1, and with compounds of formula (III), (III) Wherein Y is the reaction as defined in claim 1.

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