Amino acid having functional group capable of intermolecular hydrogen bonding, peptide compound containing amino acid, and method for production thereof

Abstract

To provide an amino acid capable of improving the membrane permeability of peptide compounds, as well as a peptide compound including the amino acid.SOLUTION: The present invention provides an amino acid having a side chain capable of forming at least one intramolecular hydrogen bond.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to amino acids capable of improving the membrane permeability of peptide compounds, peptide compounds containing such amino acids, and methods for producing them. [Background technology]

[0002] Compared to small molecular weight compounds, medium molecular weight compounds (molecular weight: 500-2000) may be superior in accessing tough targets, such as inhibiting protein-protein interactions. Furthermore, compared to antibodies, medium molecular weight compounds may also be superior in terms of their ability to be transported into cells. Among physiologically active medium molecular weight compounds, peptide drugs are highly valuable molecular species, with over 40 types already on the market (Non-Patent Document 1).

[0003] Representative examples of peptide drugs include cyclosporin A and polymyxin B. Focusing on their structures, their distinctive features include the fact that they contain several unnatural amino acids, such as N-methylamino acids, or that they are cyclic peptides. Unnatural amino acids are amino acids that are not naturally encoded on mRNA. It is of great interest that naturally occurring cyclosporin A and polymyxin B contain unnatural amino acids, and that these unnatural structural and cyclic structures interact with the site of action in the body and contribute to pharmacokinetics, resulting in pharmacological activity.

[0004] In recent years, various conditions have been reported for improving membrane permeability (conditions necessary for achieving drug-likeness) that can contribute to improved pharmacokinetics of medium-sized peptides, such as the presence of a cyclic moiety, the number of N-substituted amino acids, the range of amino acid residues, and lipophilicity (Patent Document 1). Furthermore, a report has been published focusing on unnatural N-methyl amino acids and producing an N-methyl peptide library by translational synthesis (Patent Document 2). Furthermore, interactions between a target and a drug are important for forming a complex, and hydrogen bonds are an important example of such interactions (Non-Patent Document 3). Proton donors are essential for hydrogen bond formation, but they can have a negative impact on membrane permeability. Indeed, N-methylation of amino acids is known to be effective as a method for improving membrane permeability (Patent Document 1). In addition to N-methylation of amino acids, the effect of masking protons using amide bonds in the main chain on membrane permeability has also been investigated (Non-Patent Document 2).

[0005] On the other hand, there have been no reports of improving the membrane permeability of peptides by designing unnatural amino acids with protons and functional groups capable of masking protons in the amino acid side chains and using the proton-masking function. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2013 / 100132 [Patent Document 2] International Publication No. 2012 / 033154 [Non-patent literature]

[0007] [Non-Patent Document 1] Future Med. Chem. 2009, 1,1289-1310. [Non-patent document 2] Angew. Chem. Int. Ed. 2018, 57,14414-14438. [Non-patent document 3] Latest Medicinal Chemistry, Vol. 1, p.87 Summary of the Invention [Problem to be solved by the invention]

[0008] Improving the interaction between a target molecule and a peptide is essential for creating peptides useful in medicine. To improve the interaction between a target molecule and a peptide, it is even more important that not only the main chain of the amino acid but also the side chain structure interact with the target molecule. To improve the interaction with a target molecule, it is more advantageous to incorporate unnatural amino acids designed to enhance the interaction with the target molecule into the peptide, rather than just using peptides composed of the 20 natural amino acids. While the methods described in Patent Documents 1 and 2 provide suggestions from the perspectives of N-methylation of the amide bond site of the amino acid, which is the constituent unit of the peptide, the number of amino acid residues, and lipophilicity, they do not consider the structural characteristics of when heteroatoms, such as oxygen atoms or nitrogen atoms, are introduced into the amino acid side chain, which are necessary for forming hydrogen bonds that are thought to be advantageous for binding to the target molecule. While Non-Patent Document 1 describes examples of methods for cyclizing peptide drugs and cyclic peptides, it does not suggest a correlation between membrane permeability and amino acid structure. Non-Patent Document 2 gives examples of peptide drugs, but does not suggest that structural changes in the amino acids that make up the peptides are linked to their usefulness as medicines. It merely considers known compounds, and its versatility as medicines is limited.

[0009] The present invention was made in view of the current situation in which research into the design of peptide compounds, particularly their effects on membrane permeability, has been conducted without focusing on the structural characteristics of each amino acid, and an objective of the present invention is to provide amino acids that improve the membrane permeability of peptide compounds, and peptide compounds containing such amino acids. [Means for solving the problem]

[0010] The present inventors have discovered the following as a means to solve the above problems and have completed the invention. Specifically, they designed amino acid side chains that have heteroatoms necessary for improving interaction with target molecules and that allow protons on the amino acid side chains to be masked by intramolecular hydrogen bonding within the same amino acid side chain. They then produced peptide compounds containing these amino acids and evaluated their membrane permeability. Surprisingly, they found that peptide compounds containing these amino acids have superior membrane permeability compared to peptide compounds containing amino acids that do not form intramolecular hydrogen bonds. Furthermore, they found that amino acids with side chains that form intramolecular hydrogen bonds are effectively designed so that the proton donor and proton acceptor can form pseudocyclic structures, preferably 4-, 5-, 6-, or 7-membered rings. They also found the optimal number of amino acid residues, the number of N-substituted amino acid residues, the proportion of N-substituted amino acid residues in the peptide, the cLogP range, and the number of aromatic rings constituting a peptide for membrane permeation of peptide compounds containing these amino acids.

[0011] In one non-limiting specific embodiment, the present invention includes the following. [1] A peptide compound in which two or more amino acids are linked together, at least one of which is capable of forming a hydrogen bond within its side chain. [2] The peptide compound according to [1], wherein the amino acid capable of forming a hydrogen bond in the side chain is capable of forming a pseudo 4- to 7-membered ring in the side chain. [3] The peptide compound according to [1] or [2], wherein the amino acid capable of forming a hydrogen bond in the side chain is represented by the following formula A: [ka] During the ceremony, R1 is hydrogen, C1-C6 alkyl, or a group represented by formula 1 or formula 2, where R 2A and R 2B is R 2A is hydrogen, C1-C6 alkyl, or a group represented by formula 1 or formula 2, and R 2Bis hydrogen or C1-C6 alkyl, or R 2A and R 2B together with the carbon atoms to which they are attached form a 4- to 6-membered ring, or R1 is the nitrogen atom to which R1 is attached, R 2A , and R 2A is bonded to the carbon atom to form a 4- to 6-membered heterocyclic ring, and the heterocyclic ring may have one or more substituents selected from the group consisting of a group represented by formula 1 or formula 2, —OH, and an alkoxy group, in which case R 2B is hydrogen or C1-C6 alkyl, R3 is a single bond or -CHR4-; R4 is hydrogen, C1-C4 alkyl, or a group represented by formula 1 or formula 2; Formula 1 and Formula 2 are respectively represented by the following formulas: [ka] wherein: * indicates the point of attachment Q1 and Q2 are independently a single bond, a C1-C4 alkylene, or a C2-C4 heteroalkylene containing one oxygen atom; A1 is -O- or -S-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl group, and oxo (=O); A2 is a single bond, —O—, or —S—; L2 is a single bond or a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl group, and oxo (=O); X is -OH, -NR Z1 R Z2 , or -CONR Z1 R Z2or a 5-6-membered saturated or unsaturated heterocyclyl containing 1 to 3 heteroatoms and optionally substituted by oxo or one or more halogens; R Z1 and R Z2 is independently selected from the group consisting of hydrogen, —OH, C1-C4 alkyl, and C1-C4 alkylsulfonyl; Y is -OH, C1-C4 alkylsulfonylamino, -NR Z3 R Z4 , or -CONR Z3 R Z4 or a 5- to 6-membered saturated or unsaturated heterocyclyl containing 1 to 3 heteroatoms and optionally substituted by oxo or halogen; R Z3 and R Z4 is independently selected from the group consisting of hydrogen, —OH, C1-C4 alkyl, and C1-C4 alkylsulfonyl; Z is hydrogen or C1-C4 alkyl; However, when A2 is -O- or -S-, Z is not hydrogen, Here, the amino acid represented by formula A contains at least one group represented by formula 1 or formula 2. [4] R1 is a group represented by formula 1 or formula 2, R 2A and R 2B are independently hydrogen or C1-C6 alkyl, or R 2A and R 2B form a 4- to 6-membered ring together with the carbon atoms to which they are attached, R3 is a single bond or -CHR4-; R4 is hydrogen or C1-C4 alkyl The peptide compound according to [3]. [5] R1 is a nitrogen atom to which R1 is bonded, R 2A , and R 2A forms a 4- to 6-membered heterocyclic ring together with the carbon atom to which it is bonded, and the heterocyclic ring has a group represented by formula 1 or formula 2, R2B is hydrogen or C1-C6 alkyl; R3 is a single bond or -CHR4-; R4 is hydrogen or C1-C4 alkyl The peptide compound according to [3]. [6] R1 is hydrogen or C1-C6 alkyl R 2A is a group represented by formula 1 or formula 2, R 2B is hydrogen or C1-C6 alkyl, R3 is a single bond or -CHR4-; R4 is hydrogen or C1-C4 alkyl The peptide compound according to [3]. [7] R1 is hydrogen or C1-C6 alkyl; R 2A and R 2B are independently hydrogen or C1-C6 alkyl, or R 2A and R 2B form a 4- to 6-membered ring together with the carbon atoms to which they are attached, R3 is -CHR4-, R4 is a group represented by formula 1 or formula 2; The peptide compound according to [3]. [8] Q1 is C1-C4 alkylene, A1 is -O- or -S-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); X is -OH; The peptide compound according to any one of [3] to [7]. [9] Q1 is C1-C4 alkylene, A1 is -O- or -S-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); X is -CONR Z1 R Z2 and R Z1 is C1-C4 alkyl, R Z2 is hydrogen, The peptide compound according to any one of [3] to [7].

[10] Q1 is C1-C4 alkylene, A1 is -O- or -S-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); X is a 5-6 membered saturated or unsaturated heterocyclyl containing 1 to 3 heteroatoms and optionally substituted by oxo or one or more halogens; The peptide compound according to any one of [3] to [7].

[11] Q1 is a single bond, A1 is -O- or -S-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); X is -OH; The peptide compound according to any one of [3] to [7].

[12] The peptide compound according to any one of [3] to

[11] , wherein Q1 is CH2-.

[13] The peptide compound according to any one of [3] to

[12] , wherein A1 is -O-.

[14] The peptide compound according to any one of [3] to

[13] , wherein L1 is selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH(CH3)-, -CH2CH(CF3)-, -CH2C(CH3)2-, -(CH2)2CH(CH3)-, -CH2C(CH3)2CH2-, -(CH2)2C(CH3)2-, and -CH2CO-.

[15] Q2 is C1-C4 alkylene; L2 is a single bond, Y-CONR Z3 R Z4 and A2 is -O- or -S-; Z is C1-C4 alkyl; The peptide compound according to any one of [3] to [7].

[16] Q2 is C1-C4 alkylene; L2 is a single bond, Y-CONR Z3 R Z4 and A2 is a single bond, Z is hydrogen; The peptide compound according to any one of [3] to [7].

[17] Q2 is C1-C4 alkylene; L2 is a single bond, Y is C1-C4 alkylsulfonylaminocarbonyl; A2 is -O- or -S-; Z is C1-C4 alkyl; The peptide compound according to any one of [3] to [7].

[18] Q2 is C1-C4 alkylene; L2 is a single bond, Y is —OH; A2 is -O- or -S-; Z is C1-C4 alkyl; The peptide compound according to any one of [3] to [7].

[19] The peptide compound according to any one of [3] to [7] and

[16] to

[18] , wherein Q2 is -CH2-.

[20] The peptide compound according to any one of [3] to [7],

[15] , and

[19] , wherein Y is methylaminocarbonyl, -CON(OH)Me, or -CONH(OH).

[21] The peptide compound according to any one of [3] to [7],

[16] , and

[19] , wherein Y is —CON(OH)Me.

[22] The peptide compound according to any one of [3] to [7],

[15] , and

[17] to

[21] , wherein A2 is -O-.

[23] The peptide compound according to any one of [3] to [7],

[15] , and

[17] to

[22] , wherein Z is methyl.

[24] The amino acid represented by formula A is a group consisting of: [ka] [ka] [ka] [ka] The peptide compound according to [3], selected from the group consisting of:

[25] The peptide compound according to any one of [1] to

[24] , which is composed of 5 to 30 amino acids.

[26] The peptide compound according to any one of [1] to

[25] , which is cyclic.

[27] The peptide compound according to

[26] , which comprises a cyclic portion consisting of 2 to 15 amino acids.

[28] The peptide compound according to any one of [1] to

[27] , which comprises 2 to 30 N-substituted amino acids.

[29] The peptide compound according to any one of [1] to

[28] , wherein the ratio of the number of N-substituted amino acids to the total number of amino acids is 30% or more.

[30] The peptide compound according to any one of [1] to

[29] , having a ClogP of 4.0 to 18.

[31] 1.0 x 10 -7 P over cm / sec app The peptide compound according to any one of [1] to

[30] , wherein

[32] A library comprising the peptide according to any one of [1] to

[31] and / or a nucleic acid encoding said peptide.

[33] An amino acid represented by formula A according to any one of [3] to

[23] .

[34] The group consisting of: [ka] [ka] [ka] The amino acid according to

[33] , selected from the group consisting of:

[35] A protected amino acid in which the amino group and / or carboxyl group contained in the amino acid according to

[33] or

[34] is protected with a protecting group.

[36] The amino-protecting group is selected from the group consisting of Fmoc, Boc, Cbz, Alloc, nosyl, dinitronosyl, t-Bu, trityl, and cumyl groups, and / or The protected amino acid according to

[35] , wherein the carboxyl-protecting group is selected from the group consisting of a methyl group, an allyl group, a t-Bu group, a trityl group, a cumyl group, a methoxytrityl group, and a benzyl group. [Effects of the Invention]

[0012] According to the present invention, it is possible to search for peptide pharmaceuticals, specifically to provide amino acids that have excellent membrane permeability and specifically bind to target molecules, and peptides containing such amino acids, or these amino acids and peptides. [Brief explanation of the drawings]

[0013] [Figure 1-1]FIG. 1 shows the relationship between ClogP / total AA and Papp when a population of cyclic peptide compounds not having a Trp side chain is used. [Figure 1-2] FIG. 1 shows the relationship between ClogP / total AA and Papp when a population of cyclic peptide compounds having a Trp side chain is used. [Figure 2] FIG. 1 shows the relationship between the ARC of a cyclic peptide compound and cell membrane permeability. [Figure 3-1] For example, this is a diagram of the potential energy surface for the side chain partial structure of Ser(NMe-Aca). [Figure 3-2] For example, this is a diagram showing the conformation distribution of crystal structures using the results of X-ray structural analysis registered in CSD for the side chain partial structure of Ser(NMe-Aca). [Figure 4-1] For example, this is a diagram showing the potential energy curves for the side chain partial structures of Ser(EtOH), bAla(3R-MeOEtOH), and bAla(2S-MeOEtOH). [Figure 4-2] For example, this figure shows the conformation distribution of crystal structures using the results of X-ray structural analysis registered in CSD for the side chain partial structures of Ser(EtOH), bAla(3R-MeOEtOH), and bAla(2S-MeOEtOH). DETAILED DESCRIPTION OF THE INVENTION

[0014] Preferred, non-limiting embodiments of the present invention are described below.

[0015] All elements described in the following examples are intended to be considered to be equivalently described in this "Form for Carrying Out the Invention" without being bound by any patent practice, custom, laws, regulations, etc. in any country in which the patent application is intended to be granted that may attempt to restrictively interpret the contents described in the examples.

[0016] Any combination of part or all of one or more elements described anywhere in this specification is intended to be included in this specification, unless there is a technical contradiction based on the common technical knowledge of a person skilled in the art, and is described as being naturally understood by a person skilled in the art.

[0017] The following abbreviations are used herein: Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), and Val (valine). In addition to these, the abbreviations listed in the abbreviation table and Table 1 below are also used.

[0018] (Definition of functional groups, etc.) As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. The alkyl group includes linear and branched ones. The alkyl group may have 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q" means that the number of carbon atoms is p to q.) and preferred examples include a C1-C6 alkyl group, a C1-C5 alkyl group, a C1-C4 alkyl group, and a C1-C3 alkyl group. Specific examples of the alkyl group include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, tert-butyl group, sec-butyl group, 1-methylpropyl group, 1,1-dimethylpropyl group, 2,2-dimethylpropyl, 1,2-dimethylpropyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, isopentyl, and neopentyl.

[0019] As used herein, the term "heteroalkyl" refers to a group containing preferably 1 to 5 heteroatoms in the skeleton of the "alkyl", and is preferably C2-C 10 Examples of heteroalkyl include -CH2OCH3, -CH2OCH2CH3, -CH(CH3)OCH3, and -CH2CH2N(CH3)2.

[0020] As used herein, the term "halogen atom" includes F, Cl, Br, and I, with F or Cl being preferred.

[0021] As used herein, "fluoroalkyl" refers to a group in which one or more hydrogen atoms of the aforementioned "alkyl" have been substituted with fluorine atoms, and preferred examples include C1-C6 fluoroalkyl, C1-C4 fluoroalkyl, and C1-C2 fluoroalkyl. Specific examples of fluoroalkyl include trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 2,2,3,3-tetrafluoropropyl, heptafluoropropyl, trifluoromethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, 2,2,3,3-tetrafluoropropoxy, and heptafluoropropoxy.

[0022] As used herein, "alkylsulfonyl" refers to a sulfonyl group to which the "alkyl" is bonded (i.e., -SO2-alkyl). The alkylsulfonyl is preferably C1-C6 alkylsulfonyl, C 1- Examples thereof include C4 alkylsulfonyl, specifically methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, i-propylsulfonyl, etc.

[0023] As used herein, "alkylsulfonylamino" refers to a group in which one hydrogen atom of an amino group (-NH2) is substituted with the above-mentioned "alkylsulfonyl". As the alkylsulfonylamino, preferred are C1-C6 alkylsulfonylamino, C 1- C4 alkylsulfonylamino and the like, specifically methylsulfonylamino, ethylsulfonylamino, n-propylsulfonylamino, i-propylsulfonylamino and the like.

[0024] As used herein, an "alkenyl group" is a monovalent group having at least one double bond (two adjacent sp2 carbon atoms). Depending on the configuration of the double bond and substituents (if present), the geometry of the double bond can be in an entgegen (E) or zusammen (Z) configuration, a cis or trans configuration. Alkenyl groups include linear or branched chains, including linear chains containing internal olefins. Preferably, C2-C 10 Alkenyl groups are preferred, and C2-C6 alkenyl groups are more preferred. Specific examples of such alkenyl groups include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl groups.

[0025] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). It includes straight-chain or branched-chain alkynyl groups, including internal alkylene. It is preferably C2-C6. 10 Alkynyl groups are preferred, and C2-C6 alkynyl groups are more preferred. Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, and hexynyl groups.

[0026] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. Preferably, the term "cycloalkyl" refers to a C3-C 10 Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[2.2.1]heptyl. The cycloalkyl group may be partially unsaturated. Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[2.2.1]heptyl.

[0027] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C 10Specific examples of the aryl include phenyl and naphthyl.

[0028] As used herein, the term "heteroaryl" refers to a monovalent aromatic ring containing preferably 1 to 5 heteroatoms among the atoms constituting the ring (also referred to herein as "within the ring"), and may be partially saturated. The ring may be a monocycle or two condensed rings (for example, a bicyclic heteroaryl condensed with benzene or a monocyclic heteroaryl). The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl). Specific examples of heteroaryl include imidazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, oxadiazolonyl, thiadiazolonyl, tetrazolyl, pyridyl, and indolyl.

[0029] As used herein, "having a heteroatom in the ring" means that a heteroatom is contained in the atoms constituting the ring, and examples of such rings include aromatic heterocycles such as pyridine, and non-aromatic heterocycles such as piperidine, morpholine, pyrrolidine, and azetidine. When the heteroatom is an oxygen atom, the expression "having an oxygen atom in the ring" is used.

[0030] As used herein, the term "heterocyclic group" refers to a group having at least one heteroatom (e.g., N, O, S, etc.) in the ring, and is also referred to as a saturated heterocyclyl group or an unsaturated heterocyclyl group. The heteroatom is preferably N or O, the number of heteroatoms is preferably 1 or 2, and the ring is preferably a 4- to 6-membered ring. The heterocyclic group may be substituted with an alkyl group, a fluoroalkyl group, an oxo group, or a halogen atom. Preferred examples of the heterocyclic group include a pyridyl group, a piperidino group, a morpholino group, a pyrrolidino group, an oxadiazolonyl group, and an azetidinyl group.

[0031] As used herein, the term "arylalkyl (aralkyl)" refers to a group containing both aryl and alkyl, for example, a group in which at least one hydrogen atom of the alkyl is substituted with an aryl, and preferably refers to a group in which "C5-C 10 aryl C1-C6 alkyl". For example, benzyl.

[0032] As used herein, "alkylene" refers to a divalent group derived by further removing one optional hydrogen atom from the aforementioned "alkyl." Preferred examples of alkylene include C1-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, and C1-C6 alkylene. Specific examples of alkylene include -CH2-, -(CH2)2-, -(CH2)3-, CH(CH3)CH2-, -C(CH3)2-, -(CH2)4-, CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, -(CH2)5-, and -(CH2)6-.

[0033] As used herein, "heteroalkylene" refers to a divalent group derived by further removing one optional hydrogen atom from the "heteroalkyl," and examples include C2-C6 heteroalkylene, C2-C5 heteroalkylene, C2-C4 heteroalkylene, C2-C3 heteroalkylene, C2 heteroalkylene, etc. Specific examples of heteroalkylene containing an oxygen atom as a heteroatom in the group include -CHO-, -OCH2-, -CHOCH2-, -OCHCH2-, -CHCHO-, etc.

[0034] In this specification, "arylene" means a divalent group derived from the aryl by further removing one arbitrary hydrogen atom. The arylene may be a single ring or a condensed ring. The number of atoms constituting the ring is not particularly limited, but is preferably 6 to 10 (C6-C 10 Specific examples of arylene include phenylene.

[0035] As used herein, the term "heteroarylene" refers to a divalent group derived from the heteroaryl by further removing any one hydrogen atom. The heteroarylene may be a single ring or a fused ring. The number of atoms constituting the ring is not particularly limited, but is preferably 5 to 10 (5- to 10-membered heteroarylene). Specific examples of heteroarylene include imidazolediyl, pyridinediyl, oxadiazolediyl, thiazolidinyl, and thiadiazolediyl.

[0036] As used herein, the term "fused ring structure" refers to a cyclic structure in a cyclic compound having two or more rings, in which the rings share two or more atoms. The term "fused ring structure of two or more aromatic rings" refers to a cyclic structure in a cyclic compound having two or more aromatic rings, in which the aromatic rings share two or more atoms. Examples of fused ring structures include, but are not limited to, an indole skeleton, a benzofuran skeleton, a benzimidazole skeleton, a quinoline skeleton, and bicyclo[4.4.0]decane.

[0037] In the present specification, when the modifier "optionally substituted" is used, examples of the substituent include an alkyl group, a fluoroalkyl group, an alkoxy group, a fluoroalkoxy group, an alkenyl group, an alkenyloxy group, an alkynyl group, an alkynyloxy group, a cycloalkyl group, an aryl group, a heteroaryl group, a heterocyclyl group, an arylalkyl group, a heteroarylalkyl group, a halogen atom, a nitro group, an amino group, a monoalkylamino group, a dialkylamino group, a cyano group, a carboxyl group, an alkoxycarbonyl group, and a formyl group.

[0038] As used herein, the term "heterocycle" refers to a non-aromatic monovalent or divalent heterocycle containing preferably 1 to 5 heteroatoms among the atoms constituting the ring. The heterocycle may have a double and / or triple bond within the ring, and a carbon atom within the ring may be oxidized to form a carbonyl. The heterocycle may be a monocycle, a fused ring, or a spirocycle. The number of atoms constituting the ring is preferably 3 to 12 (3- to 12-membered heterocycle), more preferably 4 to 7 (4- to 7-membered heterocycle), and even more preferably 5 to 6 (5- or 6-membered heterocycle). Specific examples of the heterocycle include piperazine, pyrrolidine, piperidine, morpholine, homomorpholine, (R)-hexahydropyrrolo[1,2-a]pyrazine, (S)-hexahydropyrrolo[1,2-a]pyrazine, 3-oxopiperazine, 2-oxopyrrolidine, azetidine, 2-oxoimidazolidine, oxetane, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, tetrahydropyridine, thiomorpholine, pyrazolidine, imidazoline, oxazolidine, isoxazolidine, thiazolidine, imidazolidine, isothiazolidine, thiadiazolidine, oxazolidone, benzodioxane, benzoxazoline, dioxolane, dioxane, and tetrahydrothiopyran.

[0039] (peptide compounds) In one aspect, the present invention relates to a peptide compound comprising two or more amino acids linked together, at least one of which (specifically, for example, one, two, three, four, or more) is an amino acid capable of forming a hydrogen bond within its side chain.

[0040] Peptide Compound Structure The term "peptide compound" as used herein includes linear or cyclic peptide compounds in which two or more amino acids are linked together. Note that a cyclic peptide compound is synonymous with a "peptide compound having a cyclic portion."

[0041] As used herein, "amino acid" includes natural amino acids and unnatural amino acids. As used herein, "natural amino acids" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Examples of unnatural amino acids include, but are not limited to, β-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains differ from those of natural amino acids, and hydroxycarboxylic acids. As used herein, amino acids may have any configuration. The side chain of an amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, cycloalkyl groups, spiro-linked cycloalkyl groups, and the like. Each of these may have a substituent, and the substituents are not limited and may be independently selected from any substituents containing, for example, a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. Examples of such substituents include optionally substituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. Furthermore, the amino acids herein may have a side chain "capable of forming an intramolecular hydrogen bond," as described below. In a non-limiting embodiment, the amino acids herein may be compounds having a carboxy group and an amino group in the same molecule (even in this case, imino acids such as proline and hydroxyproline are also included in the amino acids).

[0042] Halogen-derived substituents include fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), and the like.

[0043] Substituents derived from O atoms include hydroxyl (-OH), oxy (-OR), carbonyl (-C=OR), carboxyl (-COH), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio group (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO2-R), aminosulfonyl (-SO2-NHR), sulfamoylamino (-NH-SO2-NHR), thiocarboxyl (-C(=O)-SH), and carboxylcarbonyl (-C(=O)-COH).

[0044] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, and the like.

[0045] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0046] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like. (-C=O-OR)

[0047] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0048] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0049] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0050] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl, cycloalkylaminocarbonyl, alkenylaminocarbonyl, alkynylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, aralkylaminocarbonyl, etc. In addition to these, compounds in which the H atom bonded to the N atom in -C=O-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0051] Examples of carbonylamino (-NH-C=OR) include alkylcarbonylamino, cycloalkylcarbonylamino, alkenylcarbonylamino, alkynylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0052] Examples of oxycarbonylamino (-NH-C=O-OR) include alkoxycarbonylamino, cycloalkoxycarbonylamino, alkenyloxycarbonylamino, alkynyloxycarbonylamino, aryloxycarbonylamino, heteroaryloxycarbonylamino, aralkyloxycarbonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-C=O-OR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0053] Examples of sulfonylamino (-NH-SO2-R) include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, etc. In addition to these, compounds in which the H atom bonded to the N atom in -NH-SO2-R is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0054] Examples of aminosulfonyl (-SO2-NHR) include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, etc. In addition to these, compounds in which the H atom bonded to the N atom in -SO2-NHR is further substituted with alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0055] Examples of sulfamoylamino (-NH-SO-NHR) include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, etc. Furthermore, the two H atoms bonded to the N atom in -NH-SO-NHR may be substituted with substituents independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, and these two substituents may form a ring.

[0056] Substituents derived from S atoms include thiol (-SH), thio (-SR), sulfinyl (-S=OR), sulfonyl (-S(O)2-R), and sulfo (-SO3H).

[0057] Examples of thio (-SR) are selected from alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0058] Examples of sulfinyl (-S=OR) include alkylsulfinyl, cycloalkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, arylsulfinyl, heteroarylsulfinyl, aralkylsulfinyl, and the like.

[0059] Examples of sulfonyl (-S(O)2-R) include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0060] Substituents derived from N atoms include azido (-N3, also referred to as "azido group"), cyano (-CN), primary amino (-NH2), secondary amino (-NH-R), tertiary amino (-NR(R')), amidino (-C(=NH)-NH2), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH2), substituted guanidino (-NR-C(=NR''')-NR'R"), and aminocarbonylamino (-NR-CO-NR'R").

[0061] Examples of secondary amino (-NH-R) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0062] Examples of tertiary amino (—NR(R′)) include an amino group having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., such as alkyl(aralkyl)amino, and these two substituents may form a ring.

[0063] Examples of substituted amidino (-C(=NR)-NR'R") include groups in which the three substituents R, R', and R" on the N atom are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, such as alkyl(aralkyl)(aryl)amidino.

[0064] Examples of substituted guanidino (-NR-C(=NR''')-NR'R") include groups in which R, R', R", and R''' are each independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0065] Examples of aminocarbonylamino (-NR-CO-NR'R") include groups in which R, R', and R" are each independently selected from a hydrogen atom, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl, or groups in which these groups form a ring.

[0066] Substituents derived from the B atom include boryl (-BR(R')) and dioxyboryl (-B(OR)(OR')). These two substituents R and R' are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., or may form a ring.

[0067] Generally, an amino acid refers to a molecule having one or more amino groups and one or more carboxyl groups within the molecule. However, in this specification, hydroxycarboxylic acids having one or more hydroxyl groups and one or more carboxyl groups within the molecule can also be included in the amino acids of the present invention. In this specification, hydroxycarboxylic acids are sometimes referred to as hydroxyamino acids.

[0068] An α-amino acid refers to an amino acid molecule in which the amino group and carboxyl group are attached to the same carbon atom, and the substituent on this carbon atom is called the amino acid side chain. The amino group, carboxyl group, and the series of carbon atoms to which they are attached are called the amino acid backbone.

[0069] The main chain amino group of an amino acid may be unsubstituted (NH group) or substituted (i.e., -NHR group: R represents alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline). Amino acids in which such main chain amino groups are substituted are referred to herein as "N-substituted amino acids." Preferred examples of "N-substituted amino acids" herein include, but are not limited to, N-alkyl amino acids, N-C-C alkyl amino acids, N-C-C alkyl amino acids, N-methyl amino acids, and N-substituted amino acids having a side chain "capable of forming an intramolecular hydrogen bond."

[0070] The "amino acids" constituting the peptide compounds herein include all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes contained in the "amino acids" constituting the peptide compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Includes Cl etc.

[0071] The "linear peptide compound" of the present invention is formed by linking natural amino acids and / or unnatural amino acids via amide bonds or ester bonds, and is not particularly limited as long as it is a compound that does not have a cyclic moiety. The total number of natural or unnatural amino acids constituting the linear peptide compound can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, with preferred ranges being 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, and 9 to 13.

[0072] The "cyclic peptide compound" of the present invention is formed by linking natural amino acids and / or unnatural amino acids via amide bonds or ester bonds, and is not particularly limited as long as it is a compound having a cyclic moiety. The total number of natural or unnatural amino acids constituting the cyclic peptide compound can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, with preferred ranges being 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, and 9 to 13.

[0073] As used herein, the "cyclic portion" of a peptide compound refers to a cyclic portion formed by linking two or more amino acid residues. Furthermore, as used herein to refer to a partial structure of a cyclic peptide compound, the "linear portion" refers to a portion that is not included in the main chain structure of the cyclic portion and has at least one amide bond and / or ester bond in the chain of the linear portion.

[0074] The number of amino acids constituting the cyclic portion of the cyclic peptide compound herein is not limited, and examples include 2 or more, 3 or more, 4 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 30 or less, 20 or less, 18 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In consideration of both membrane permeability and metabolic stability, the number of amino acids constituting the cyclic portion is preferably 2 to 30, 2 to 15, or 5 to 15, more preferably 5 to 14, 7 to 14, or 8 to 14, even more preferably 8 to 13, 9 to 13, 8 to 12, 8 to 11, or 9 to 12, and particularly preferably 9 to 11.

[0075] In a non-limiting embodiment, the number of amino acids (number of units) in the linear portion is preferably 0 to 8, more preferably 0 to 5, and more preferably 0 to 3. Note that in a non-limiting embodiment, the "linear portion" herein may include natural amino acids and unnatural amino acids (including chemically modified or backbone-converted amino acids).

[0076] In a non-limiting embodiment, the peptide compound or cyclic peptide compound herein preferably does not have an ester bond in the cyclic or linear portion.

[0077] In a non-limiting embodiment, the cyclic peptide compound herein may be a cyclic peptide compound that does not have at least one selected from the group consisting of the following (A) to (C) in the side chain of the peptide moiety in the cyclic portion or the linear portion: (A) Indole skeleton; (B) A fused ring structure consisting of two or more aromatic rings; (C) Unsubstituted hydroxyphenyl group. Among these, preferred are cyclic peptide compounds that do not have the above (A) or (C) in the side chain of the cyclic moiety, and more preferred are cyclic peptide compounds that do not have the above (B) or (C) in the side chain of the cyclic moiety. The "fused ring structure of two or more aromatic rings" may also be a "fused ring structure."

[0078] In a non-limiting embodiment, the molecular weight of the cyclic peptide compound herein may be 500-2,000.

[0079] In a non-limiting embodiment, the cyclic peptide compound herein may be a cyclic peptide compound that does not have at least one, or both, selected from the group consisting of (A) a methylthio group and (B) a thiol group in the side chain of the peptide portion of the cyclic portion or linear portion.

[0080] In the present invention, when a peptide compound is formed by an amide bond between two amino acids, the OH group of the carboxyl group in the main chain of the first amino acid is substituted with the nitrogen atom of the amino group in the main chain of the second amino acid, thereby forming the peptide compound.

[0081] In the present invention, when a peptide compound is formed by an ester bond between two amino acids, the OH group of the carboxyl group in the main chain of the first amino acid is substituted with the oxygen atom of the hydroxyl group in the main chain of the second hydroxyamino acid to form the peptide compound.

[0082] In the present invention, when a peptide compound is formed by linking natural amino acids and / or unnatural amino acids via amide bonds or ester bonds, the peptide compound is formed by linking two or more amino acids consecutively via amide bonds and / or ester bonds.

[0083] In the present specification, the portion of a peptide compound linked by an amide bond and / or an ester bond may be referred to as a "peptide portion." However, when the peptide compound is cyclic, the bonding mode of the cyclization portion is not limited to an amide bond or an ester bond. Examples of the bonding mode of the cyclization portion include covalent bonds such as an amide bond, a carbon-carbon bond, a disulfide bond, an ester bond, a thioester bond, a thioether bond, a lactam bond, a bond via a triazole structure, and a bond via a fluorophore structure. Among these, an amide bond is preferred because of its high metabolic stability. That is, in one aspect, the cyclic peptide compound of the present specification preferably has an amide bond at the cyclization portion. The "bonding mode of the cyclization portion" refers to the bonding mode at the portion cyclized by the cyclization reaction.

[0084] The "peptide compound" of the present invention may be a linear or cyclic peptide that contains at least two N-substituted amino acids (preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30, particularly preferably 5, 6, or 7, with preferred ranges being 2 to 30, 3 to 30, 6 to 20, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 8 to 14, or 9 to 13), in addition to or solely within the above-mentioned total number of natural and unnatural amino acids, and at least one non-N-substituted amino acid. Examples of "N-substitution" include, but are not limited to, substitution of a hydrogen atom bonded to an N atom with a methyl group, ethyl group, propyl group, butyl group, or hexyl group. N-substituted amino acids preferably include amino acids in which the amino group contained in a natural amino acid has been N-methylated, N-ethylated, N-propylated, N-butylated, or N-pentylated, and are referred to as N-methylamino acids, N-ethylamino acids, N-propylamino acids, N-butylamino acids, and N-pentylamino acids. Converting an N-unsubstituted amino acid into an N-substituted amino acid is referred to as N-substitution, and is sometimes referred to as N-alkylation, N-methylation, or N-ethylation. The proportion of N-substituted amino acids contained in the peptide compound of the present invention is, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the total number of amino acids constituting the peptide compound.

[0085] In a non-limiting embodiment, the number of N-substituted amino acids contained in the peptide moiety of a cyclic peptide compound herein is preferably 2 or more or 3 or more, more preferably 4 or more, 5 or more, or 6 or more, even more preferably 7 or more, and particularly preferably 8 or more, and is preferably 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, or 9 or less. The number of N-substituted amino acids contained in a cyclic peptide compound herein is, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more of the number of amino acids constituting the cyclic portion. The N-substituted amino acids herein may preferably be N-alkylamino acids, more preferably N-methylamino acids. That is, in a non-limiting embodiment, the number of N-substituted amino acids is, for example, the number of N-alkylamino acids or the number of N-methylamino acids. When the number of amino acids constituting the cyclic portion is 8, the number of N-substituted amino acids is preferably 3 to 7. When the number of amino acids constituting the cyclic portion is 9, the number of N-substituted amino acids is preferably 3 to 8. When the number of amino acids constituting the cyclic portion is 10, the number of N-substituted amino acids is preferably 3 to 8. When the number of amino acids constituting the cyclic portion is 11, the number of N-substituted amino acids is preferably 4 to 9. When the number of amino acids constituting the cyclic portion is 12, the number of N-substituted amino acids is preferably 4 to 10. When the number of amino acids constituting the cyclic portion is 13, the number of N-substituted amino acids is preferably 4 to 11. When the number of amino acids constituting the cyclic portion is 14, the number of N-substituted amino acids is preferably 5 to 12.

[0086] In this specification, the "amino acids" that constitute a peptide compound may be referred to as "amino acid residues."

[0087] The "peptide compound" herein may include pharmaceutically acceptable salts thereof or solvates thereof.

[0088] As used herein, the term "side chain" is used in the context of the side chain of an amino acid or the side chain of the cyclic portion of a cyclic peptide compound, and refers to a portion that is not included in the main chain structure of each.

[0089] As used herein, the term "number of amino acids" refers to the number of amino acid residues (amino acid units) that constitute a peptide compound, and refers to the number of amino acid units that are generated when the amide bond, ester bond, and bond at the cyclization site that link the amino acids are cleaved.

[0090] Membrane permeability of peptide compounds In a non-limiting embodiment, the peptide compound or cyclic peptide compound herein may be a cyclic peptide compound that does not have a functional group that is highly ionized at neutral (e.g., pH = 7.0) in order to achieve high membrane permeability. As used herein, pKa refers to the measured pKa unless otherwise specified. Furthermore, the pKa value determined using ADMETPredictor, which will be described later, is referred to as the calculated pKa. As used herein, basic pKa refers to the measured basic pKa unless otherwise specified. Furthermore, the basic pKa value determined using ADMETPredictor, which will be described later, is referred to as the calculated basic pKa.

[0091] The pKa and basic pKa can be determined by conventional methods. For example, they can be measured by the method described in Experimental Chemistry Lectures 5, "Thermal Measurement and Equilibrium," p. 460 (edited by the Chemical Society of Japan, published by Maruzen Co., Ltd.). More specifically, they can be measured by the method described in Reference Example 3-1. Furthermore, when the pKa value of the side chain and basic pKa value of the amino acid to be measured are difficult to determine due to the influence of other functional groups, the other functional groups can be appropriately protected with protecting groups or the like so that only the pKa and basic pKa of the functional group of interest can be measured.

[0092] As used herein, an "acidic side chain" refers to a side chain with a pKa of 10 or less, and a "basic side chain" refers to a side chain with a basic pKa of 4 or more. As used herein, a side chain with a pKa of more than 10 or a side chain with a basic pKa of less than 4 is defined as a neutral side chain.

[0093] In a non-limiting embodiment, the peptide compound or cyclic peptide compound described herein may contain, in addition to amino acids having side chains capable of forming intramolecular hydrogen bonds, amino acids having acidic side chains that do not form intramolecular hydrogen bonds. In this case, the pKa of the acidic side chain may be 3.5 to 10. The pKa of the acidic side chain is preferably 3.5 or higher, more preferably 3.9 or higher, more preferably 4.5 or higher, more preferably 5.0 or higher, more preferably 5.5 or higher, and more preferably 5.7 or higher. The basic pKa is preferably 10 or lower. Preferred examples of the pKa range include 3.5 to 10, 3.9 to 10, 4.5 to 10, 5.0 to 10, 5.5 to 10, and 5.7 to 10. When these pKa values ​​are expressed as calculated values, the calculated pKa is preferably 3.5 or higher, more preferably 4.5 or higher, 5.0 or higher, 5.4 or higher, 8.0 or higher, and 8.3 or higher. The calculated pKa is preferably 10 or lower. The range of the calculated pKa is preferably 3.5 to 10, 4.5 to 10, 5.0 to 10, 5.4 to 10, 8.0 to 10, or 8.3 to 10, for example.

[0094] In a non-limiting embodiment, the peptide compound or cyclic peptide compound herein may contain an amino acid having a basic side chain that does not form an intramolecular hydrogen bond, in addition to an amino acid having a side chain capable of forming an intramolecular hydrogen bond. In this case, the basic pKa of the basic side chain may be 4.0 to 10. The basic pKa of the basic side chain is preferably 10 or less, more preferably 9.5 or less, 9.0 or less, 8.5 or less, 7.5 or less, or 7.2 or less, and particularly preferably 6.5 or less. The basic pKa is preferably 4.0 or more. Preferred examples of the basic pKa range include 4.0 to 10, 4.0 to 9.5, 4.0 to 9.0, 4.0 to 8.5, 4.0 to 7.5, 4.0 to 7.2, and 4.0 to 6.5. When these basic pKa values ​​are expressed as calculated values, the basic calculated pKa is preferably 10 or less, and more preferably 9.5 or less, 9.0 or less, 8.8 or less, 8.6 or less, 8.5 or less, 7.5 or less, or 6.5 or less. The basic calculated pKa is preferably 4.0 or more. Preferred examples of the basic calculated pKa range include 4.0 to 10, 4.0 to 9.5, 4.0 to 9.0, 4.0 to 8.8, 4.0 to 8.6, 4.0 to 8.5, 4.0 to 7.5, and 4.0 to 6.5.

[0095] Although not intended to be limiting, as shown in the reference examples below, the number or proportion of aromatic rings contained in the side chain of the peptide moiety can affect the membrane permeability of a cyclic peptide compound. If the number or proportion of aromatic rings contained in the side chain of the peptide moiety exceeds a certain number, the probability of obtaining a compound with high membrane permeability may decrease. In other words, the number of aromatic rings may have a negative effect on membrane permeability.

[0096] In this specification, the term "negative impact" as used in the context of the effects of an invention refers to an impact that negates the effects of the invention. For example, if the effect of the invention that would have been achieved is 100%, a "negative impact" can be said to exist if the effect is 30%, 20%, 10%, or 5% or less.

[0097] In a non-limiting embodiment, for high membrane permeability, the number of aromatic rings contained in the side chain of the peptide moiety of the cyclic peptide compound herein is preferably 5 or less, preferably 0, 1, 2, or 3, with preferred ranges being 0 to 3 and 1 to 3. Furthermore, the ratio of the number of aromatic rings contained in the side chain of the peptide moiety to the number of amino acids constituting the peptide moiety is preferably 40% or less, preferably 35% or less, 30% or less, 27% or less, 25% or less, or 20% or less.

[0098] As used herein, the term "aromatic ring count" (ARC) refers to the number of aromatic rings contained in the side chain of the peptide moiety of a cyclic peptide compound. For example, a phenol group is counted as one, a bicyclic fused ring such as an indole skeleton is counted as two, and a tricyclic fused ring such as anthracene is counted as three.

[0099] In a non-limiting embodiment, while the number of aromatic rings that may be contained in the cyclic peptide compound herein is limited for high membrane permeability, it is preferable that amino acids containing side chains capable of forming intramolecular hydrogen bonds be present in the cyclic portion, which is a site that can contribute to binding with a target molecule. That is, in one embodiment, when the peptide compound herein is a cyclic peptide compound and the cyclic portion of the cyclic peptide compound contains an amino acid containing an aromatic ring in a side chain capable of forming an intramolecular hydrogen bond, the number of aromatic rings contained in the cyclic portion of the peptide compound is 1, 2, or 3, and the range of the number of aromatic rings is 1 to 3 or 2 to 3. Furthermore, when the peptide compound herein is a cyclic peptide compound and the cyclic portion of the cyclic peptide compound contains an amino acid containing an aromatic ring in a side chain capable of forming an intramolecular hydrogen bond, the ratio of the number of aromatic rings contained in the cyclic portion to the total number of aromatic rings contained in the peptide compound is 30% or more, 40% or more, 60% or more, 80% or more, or 100%.

[0100] The membrane permeability of the peptide compound of the present invention can be measured by the method for measuring membrane permeability described in WO 2018 / 124162.

[0101] In one non-limiting embodiment, P of the peptide compound of the invention app is 1.0 x 10 -7 cm / sec or more, 5.0×10 -7 cm / sec or more, 8.0×10 -7 cm / sec or more is preferable, 9.0 × 10 -7 cm / sec or more is more preferable, and 1.0 × 10 -6 More preferably, 3.0 × 10 cm / sec or more -6 cm / sec or more is particularly preferred. In this specification, the "membrane permeability coefficient (P app )" means a value measured using the measurement method (improved method) described in WO 2018 / 124162, unless otherwise specified.

[0102] In a non-limiting embodiment, the ClogP of the cyclic peptide compound herein is preferably 4 or more, more preferably 5 or more, even more preferably 6 or more, particularly preferably 8 or more, and is preferably 18 or less, 17 or less, or 16 or less, for example, 4 to 18, 5 to 17, or 6 to 16. The ClogP herein is a partition coefficient calculated by a computer, and can be calculated using Daylight Version 4.9 from Daylight Chemical Information Systems, Inc.

[0103] In a non-limiting embodiment, the lower limit of ClogP / total aa for a cyclic peptide compound herein is preferably 1.0 or more, more preferably 1.1 or more, and even more preferably 1.2 or more. The upper limit of ClogP / total aa is preferably 1.8 or less, 1.7 or less, 1.6 or less, or 1.5 or less. Examples of ranges for ClogP / total aa include 1.0 to 1.8, 1.0 to 1.7, 1.1 to 1.6, and 1.1 to 1.5. As used herein, "total aa" (also referred to as "total AA") refers to the number of amino acids constituting the peptide portion of a peptide compound. For example, the total aa of a cyclic peptide compound consisting of 10 amino acids and 1 amino acid in the linear portion is 11. As used herein, ClogP / total aa is calculated by dividing ClogP by total aa.

[0104] Metabolic stability of peptide compounds In a non-limiting embodiment, the peptide compounds herein preferably have good metabolic stability. To achieve good metabolic stability, the number of amino acids contained in the peptide compound is preferably 8 or more, more preferably 9 or more, and even more preferably 11 or more. In one embodiment, it is preferable that the peptide compound does not have a thioether bond that may be easily oxidized. In another embodiment, it is preferable that the peptide compound does not have a methylthio group, as this is easily oxidized and may interfere with metabolic stability.

[0105] (amino acids that can form intramolecular hydrogen bonds) In one non-limiting embodiment, the peptide compounds of the present invention contain at least one amino acid, specifically, for example, one, two, three, four, or more, that can form hydrogen bonds (i.e., intramolecular hydrogen bonds) within their side chains.

[0106] As used herein, the side chain of an amino acid includes a chain bonded to a carbon atom (e.g., an α-, β-, or γ-carbon atom) contained in the amino acid, and a chain bonded to a nitrogen atom. The length of the side chain of an amino acid can be determined by the method described in Reference Example 1. Specifically, the N-terminus of the amino acid unit is capped with an acetyl group and the C-terminus with a methylamino group, conformations are generated using LowModeMD in the molecular modeling software MOE (Chemical Computing Group), and the distance from the atom to which the side chain moiety is bonded (the α-carbon atom (Cα carbon) in the case of a natural amino acid) to the farthest atom (excluding hydrogen atoms) in the same side chain can be determined.

[0107] As used herein, the term "long side chain" refers to a side chain having a length of 5.4 angstroms or more. Long side chains of amino acids are preferred for intramolecular hydrogen bonding. The length of a long side chain is preferably 5.4 angstroms or more, more preferably 5.6 angstroms or more, even more preferably 5.8 angstroms or more, and particularly preferably 6.0 angstroms or more. The upper limit of the length of the side chain is not particularly limited, but examples include 20 angstroms or less, 15 angstroms or less, 13 angstroms or less, 12 angstroms or less, 11 angstroms or less, 10 angstroms or less, 9.0 angstroms or less, 8.8 angstroms or less, 8.5 angstroms or less, and 8.0 angstroms or less. Examples of the range of the length of the long side chain include 5.4 to 20 angstroms, 6.0 to 20 angstroms, 6.0 to 15 angstroms, 6.0 to 13 angstroms, and 6.0 to 10 angstroms.

[0108] In a non-limiting embodiment, the "long side chain" is preferably (i) a side chain that does not contain an amide bond or that contains one amide bond in the side chain, or (ii) a side chain that does not contain an amide bond in the side chain. That is, in a non-limiting embodiment, a preferred example is a cyclic peptide compound having, in the cyclic portion, an amino acid that does not contain two or more amide bonds in the side chain and has a long side chain of 6.0 to 11 angstroms in length. In the present specification, the long side chain may or may not contain an aromatic ring.

[0109] In one non-limiting embodiment, amino acids capable of forming intramolecular hydrogen bonds are preferably amino acids with long side chains.

[0110] The presence or absence of intramolecular hydrogen bonds can be determined, for example, by X-ray structural analysis. 1 This can be determined by the chemical shift measured by H NMR. Generally, if the temperature-dependent chemical shift change is less than 2 ppb / K, it is considered to be a clear hydrogen bond, and if it is more than 4 ppb / K, it is considered to be no hydrogen bond. Conformational analysis by computational chemistry, as exemplified in Example 3, is also used to determine the possibility of intramolecular hydrogen bonding.

[0111] In one aspect, the peptide compound of the present invention contains at least one amino acid capable of forming a pseudo-W-membered ring in its side chain, specifically, for example, one, two, three, four, or more.

[0112] As used herein, the term "pseudo W-membered ring" refers to a ring that does not have a cyclic structure formed by a covalent bond, but forms a pseudo-cyclic structure by fixing the conformation through intramolecular hydrogen bonds. "W" represents the size of the ring and is a natural number of 3 or greater. The atoms constituting the pseudo-cyclic portion necessarily include three atoms: a hydrogen atom serving as a proton donor, an atom directly bonded to that hydrogen atom, and a proton acceptor capable of forming a hydrogen bond with the proton donor. W, representing the ring size of the pseudo-cyclic structure, may be 3 or greater, preferably 4 to 7, or 5 to 7, and more preferably 5 to 6. In the case of the side chain partial structure of Ser(nPrOH) shown below as an example, the hydrogen atom of the hydroxyl group serving as the proton donor, the oxygen atom of the hydroxyl group to which that hydrogen atom is directly bonded, and the oxygen atom of the ether group serving as the proton acceptor each correspond to the three atoms, forming a pseudo-6-membered ring. Furthermore, intramolecular hydrogen bonds are characterized by their ability to convert between cyclized and non-cyclized forms depending on the surrounding environment. In hydrophobic environments such as those found in biological membranes, a cyclized structure that masks the proton donor can be expected to improve the membrane permeability of compounds containing functional groups that form intramolecular hydrogen bonds. On the other hand, when a compound binds to a target molecule as an inhibitor of the target molecule, the compound containing functional groups that form intramolecular hydrogen bonds can adopt a non-cyclized structure, which is expected to allow both or either the proton donor and the proton acceptor to bind to the target molecule, thereby enabling more efficient intermolecular hydrogen bonding between the compound and the target molecule. As an example, the following diagram shows a schematic diagram of a portion of a structure capable of forming intramolecular hydrogen bonds forming intermolecular bonds with the amide moiety of the target molecule (* indicates the α-carbon of Ser(nPrOH), and ** and *** indicate the binding points of the target molecule to the protein, respectively). [ka]

[0113] In a non-limiting embodiment, an amino acid having a side chain capable of forming an intramolecular hydrogen bond may form a pseudo 4- to 7-membered ring in the partial structure that forms the intramolecular hydrogen bond, preferably a pseudo 5- to 7-membered ring, and particularly preferably a pseudo 5- to 6-membered ring.

[0114] In a non-limiting embodiment, the following structures are exemplified herein as structures capable of forming an intramolecular hydrogen bond (* represents the point of attachment to Q1 in formula 1 or Q2 in formula 2). [ka] [ka] [ka]

[0115] In one non-limiting embodiment, amino acids capable of forming hydrogen bonds within their side chains can be represented by Formula A below. [ka]

[0116] In one aspect, in formula (A), R1 is hydrogen, C1-C6 alkyl, or a group represented by formula 1 or formula 2, in which case (a) R 2A and R 2B is R 2A is hydrogen, C1-C6 alkyl, or a group represented by formula 1 or formula 2, and R 2B is hydrogen or C1-C6 alkyl, or (b) R 2A and R 2B form a 4- to 6-membered ring together with the carbon atoms to which they are attached. When R1 is C1-C6 alkyl, R1 is preferably methyl. 2B is C1-C6 alkyl, R 2B is preferably methyl. 2Bis C1-C6 alkyl, R 2A is preferably a group represented by formula 1 or formula 2, or a C1-C6 alkyl. 2A and R 2B However, when they are taken together with the carbon atom to which they are bonded to form a 4- to 6-membered ring, the 4- to 6-membered ring is preferably cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0117] In another aspect, in formula (A), R is a nitrogen atom to which R is attached, R 2A , and R 2A together with the carbon atom to which it is attached to form a 4- to 6-membered heterocyclic ring, in which case R 2B is hydrogen or C1-C6 alkyl. 2B is C1-C6 alkyl, R 2B is preferably methyl. The 4- to 6-membered heterocycle may have one or more substituents selected from the group consisting of a group represented by formula 1 or formula 2, —OH, and an alkoxy group. Furthermore, the 4- to 6-membered heterocycle is preferably pyrrolidine (i.e., a proline-like structure).

[0118] R1, R 2A , R 2B As a combination of 2A is a group represented by formula 1 or formula 2, R1 is hydrogen or C1-C6 alkyl, and R 2B is preferably hydrogen or C1-C6 alkyl, and the C1-C6 alkyl is preferably methyl. When R1 is a group represented by formula 1 or formula 2, R 2A is hydrogen or C1-C6 alkyl, and R 2B is hydrogen, C1-C6 alkyl, or R 2A and R 2B preferably combine with each other to form a 4- to 6-membered ring, and the C1-C6 alkyl is preferably methyl.

[0119] In formula (A), R3 is a single bond or -CHR4-, where R4 is hydrogen, C1-C4 alkyl, or a group represented by formula 1 or formula 2. When R3 is -CHR4-, R4 is preferably hydrogen or a group represented by formula 1 or formula 2.

[0120] Formula (A) may contain two or more groups represented by formula 1 and / or formula 2, but preferably contains only one of the groups represented by formula 1 or formula 2. Specific examples of embodiments containing one of the groups represented by formula 1 or formula 2 include the following. (i) R1 is a group represented by formula 1 or formula 2, and R 2A is hydrogen or C1-C6 alkyl, preferably hydrogen, and R 2B is hydrogen or C1-C6 alkyl, preferably hydrogen; R3 is a single bond or -CHR4-; R4 is hydrogen or C1-C4 alkyl, preferably hydrogen. (ii) R1 is hydrogen or C1-C6 alkyl, and R 2A is a group represented by formula 1 or formula 2, and R 2B is hydrogen or C1-C6 alkyl, preferably hydrogen; R3 is a single bond or -CHR4-, preferably a single bond; and R4 is hydrogen or C1-C4 alkyl, preferably hydrogen. (iii) R1 is hydrogen or C1-C6 alkyl, and R 2A is hydrogen or C1-C6 alkyl, preferably hydrogen, and R 2B is hydrogen or C1-C6 alkyl, preferably hydrogen; R3 is -CHR4-; and R4 is a group represented by formula 1 or formula 2. (iv) R1, the nitrogen atom to which R1 is bonded, R 2A , and R 2A are bonded together to form a 4- to 6-membered heterocycle (preferably a pyrrolidine ring), and the heterocycle has a group represented by formula 1 or formula 2, and R 2Bis hydrogen or C1-C6 alkyl, preferably hydrogen; R3 is a single bond or -CHR4-, preferably a single bond; and R4 is hydrogen or C1-C4 alkyl, preferably hydrogen.

[0121] Formula 1 and Formula 2 are respectively represented by the following formulas: [ka] where * indicates the point of attachment. The amino acid represented by formula A contains at least one group represented by formula 1 or formula 2. For example, the amino acid represented by formula A can contain either a group represented by formula 1 or formula 2, in which case any one of R1, R2, or R4 in formula A is a group represented by formula 1 or formula 2.

[0122] In Formula 1, Q1 is a single bond, C1-C4 alkylene, or C2-C4 heteroalkylene containing one oxygen atom. When Q1 is C1-C4 alkylene, Q1 is preferably -CH2- or -(CH2)2-.

[0123] In formula 1, A1 is —O— or —S—, preferably —O—.

[0124] In Formula 1, L1 is a linear C1-C3 alkylene optionally substituted with one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl group, and oxo (=O). Specific examples of L1 include -CH2-, -(CH2)2-, -(CH2)3-, -CH2CH(CH3)-, -CH2CH(CF3)-, -CH2C(CH3)2-, -(CH2)2CH(CH3)-, -CH2C(CH3)2CH2-, -(CH2)2C(CH3)2-, and -CH2CO-. In one aspect, it is preferred that L1 is not unsubstituted -(CH2)2-.

[0125] In Formula 1, X represents -OH, -NR Z1 R Z2 , or -CONRZ1 R Z2 or a 5-6 membered saturated or unsaturated heterocyclyl containing 1-3 heteroatoms and optionally substituted by oxo or one or more halogens, wherein R Z1 and R Z2 is independently selected from the group consisting of hydrogen, —OH, C1-C4 alkyl, and C1-C4 alkylsulfonyl. Z1 R Z2 When X is -CONR, it is preferred that X is -NH, -NHMe, -NHEt, -NH(nPr), -NH(iPr), or -NH(tBu). Z1 R Z2 In the above formula, X is preferably -CONH2, -CONHMe, -CONHEt, -CONH(nPr), -CONH(iPr), or -CONH(tBu). In addition, when X is oxo or a 5- to 6-membered saturated or unsaturated heterocyclyl optionally substituted with one or more halogens, the saturated or unsaturated heterocyclyl is preferably 4,5-dihydro-1,2,4-oxadiazolyl, 4,5-dihydro-1,2,4-thiadiazolyl, or pyrrolidyl, and X is preferably one of the following groups: [ka] is preferred.

[0126] Specific preferred combinations of -Q1-A1-L1-X include the following: (i) Q1 is C1-C4 alkylene, preferably -CH2-; A1 is -O- or -S-, preferably -O-; L1 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); and X is -OH. (ii) Q1 is C1-C4 alkylene, preferably -CH2-; A1 is -O- or -S-, preferably -O-; L1 is a linear C1-C3 alkylene, preferably -CH2-, optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl groups, and oxo (=O); and X is -NR Z1 R Z2 and R Z1 is C1-C4 alkyl, and R Z2 is hydrogen. (iii) Q1 is C1-C4 alkylene, preferably -CH2-; A1 is -O- or -S-, preferably -O-; L1 is a linear C1-C3 alkylene, preferably -CH2-, optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl, and oxo (=O); and X is -CONR Z1 R Z2 and R Z1 is C1-C4 alkyl, and R Z2 is hydrogen. (iv) Q1 is C1-C4 alkylene, preferably -CH2-; A1 is -O- or -S-, preferably -O-; L1 is straight-chain C1-C3 alkylene, preferably -CH2-, optionally substituted by one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl, and oxo (=O); and X is a 5- to 6-membered saturated or unsaturated heterocyclyl containing 1 to 3 heteroatoms and optionally substituted by oxo or one or more halogens (preferably fluorine). (v) Q1 is a single bond, A1 is -O- or -S-, preferably -O-, L1 is a straight-chain C1-C3 alkylene optionally substituted with one or more substituents selected from the group consisting of C1-C2 alkyl groups, C1-C2 fluoroalkyl, and oxo (=O), and X is -OH.

[0127] In formula 2, Q2 is a single bond, C1-C4 alkylene, or C2-C4 heteroalkylene containing one oxygen atom. When Q2 is C1-C4 alkylene, Q2 is preferably -CH2- or -(CH2)2-.

[0128] In Formula 2, L2 is a single bond or a linear C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl, and oxo (=O). Specific examples of L2 include a single bond, -CH2-, -CO-, -(CH2)2-, -(CH2)3-, -CH2CH(CH3)-, -CH2CH(CF3)-, -CH2C(CH3)2-, -(CH2)2CH(CH3)-, -CH2C(CH3)2CH2-, -(CH2)2C(CH3)2-, and -CH2CO-.

[0129] In Formula 2, Y represents -OH, C1-C4 alkylsulfonylamino, -NR Z3 R Z4 , or -CONR Z3 R Z4 or a 5-6-membered saturated or unsaturated heterocyclyl containing 1-3 heteroatoms and optionally substituted by oxo or halogen, wherein R Z3 and R Z4 is independently selected from the group consisting of hydrogen, —OH, C1-C4 alkyl, and C1-C4 alkylsulfonyl. Z3 R Z4 When Y is C1-C4 alkylsulfonylamino, Y is preferably -NH2, NHMe, NHEt, NH(nPr), NH(iPr), or NH(tBu). When Y is C1-C4 alkylsulfonylamino, Y is preferably methylsulfonylamino. Furthermore, when Y is C1-C4 alkylsulfonylamino, L2 is preferably -CO-.

[0130] In formula 2, A2 is a single bond, -O-, or -S-, and is preferably -O-.

[0131] In formula 2, Z is hydrogen or C1-C4 alkyl, and when Z is C1-C4 alkyl, the C1-C4 alkyl is preferably methyl.

[0132] In formula 2, when A2 is -O- or -S-, Z is not hydrogen. That is, specific examples of -A2-Z include A2 being a single bond and Z being hydrogen, A2 being -O- and Z being C1-C4 alkyl, or A2 being -S- and Z being C1-C4 alkyl.

[0133] Specific preferred combinations of -Q2-, -L2-Y, and -A2-Z include the following: (i) Q2 is C1-C4 alkylene, preferably -CH2-; L2 is a linear C1-C3 alkylene, preferably -CO-, optionally substituted by one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl, and oxo (=O); and Y is -NR Z3 R Z4 , preferably methylamino, —N(OH)Me, or —NH(OH), A2 is —O—, or —S—, preferably —O—, and Z is C1-C4 alkyl, preferably methyl. (ii) Q2 is C1-C4 alkylene, preferably -CH2-, L2 is a single bond, and Y is -CONR Z3 R Z4 , preferably methylaminocarbonyl or -CON(OH)Me, or -CONH(OH), A2 is -O-, or -S-, preferably -O-, and Z is C1-C4 alkyl, preferably methyl. (iii) Q2 is C1-C4 alkylene, preferably -CH2-; L2 is a linear C1-C3 alkylene, preferably -CO-, optionally substituted by one or more substituents selected from the group consisting of fluorine, C1-C2 alkyl group, C1-C2 fluoroalkyl, and oxo (=O); and Y is -NR Z3 R Z4, preferably -N(OH)Me, A2 is a single bond, and Z is hydrogen. (iv) Q2 is C1-C4 alkylene, preferably -CH2-, L2 is a single bond, and Y is -CONR Z3 R Z4 , preferably -CON(OH)Me, A2 is a single bond, and Z is hydrogen. (v) Q2 is C1-C4 alkylene, preferably -CH2-; L2 is a straight-chain C1-C3 alkylene optionally substituted by one or more substituents selected from the group consisting of fluorine, a C1-C2 alkyl group, a C1-C2 fluoroalkyl, and oxo (=O), preferably -CO-; Y is C1-C4 alkylsulfonylamino, preferably methylsulfonylamino; A2 is -O- or -S-, preferably -O-; and Z is C1-C4 alkyl, preferably methyl. (vi) Q2 is C1-C4 alkylene, preferably —CH2—, L2 is a single bond, Y is C1-C4 alkylsulfonylaminocarbonyl, preferably methylsulfonylaminocarbonyl, A2 is —O— or —S—, preferably —O—, and Z is C1-C4 alkyl, preferably methyl. (vii) Q2 is C1-C4 alkylene, preferably —CH2—, L2 is a single bond, Y is —OH, A2 is —O— or —S—, preferably —O—, and Z is C1-C4 alkyl, preferably methyl.

[0134] More specific examples of such amino acids capable of forming hydrogen bonds in their side chains and / or amino acids capable of forming pseudo 4- to 7-membered rings in their side chains include the following amino acids. [ka] [ka] [ka] [ka]

[0135] In a non-limiting embodiment, when an amino acid represented by formula A of the present invention is contained in a peptide compound, it is preferably linked to an adjacent amino acid via the amino group and carboxyl group of the main chain of the amino acid. In this case, the amino acid represented by formula (A) can also be represented as follows (* represents the point of attachment to the adjacent amino acid in the peptide compound): [ka]

[0136] In a non-limiting embodiment, the present invention relates to a protected amino acid in which the amino group and / or carboxyl group of the amino acid capable of forming an intramolecular hydrogen bond is protected with a protecting group. Examples of protecting groups for the amino group of the protected amino acid include Fmoc, Boc, Cbz, Alloc, nosyl, dinitronosyl, t-Bu, trityl, and cumyl groups. Examples of protecting groups for the carboxyl group of the protected amino acid include methyl, allyl, t-Bu, trityl, cumyl, methoxytrityl, and benzyl groups. For example, these protecting groups can be introduced using the method described in Greene's "Protective Groups in Organic Synthesis" (5th ed., John Wiley & Sons, 2014).

[0137] Without intending to be bound by any particular theory, the present inventors believe as follows. When an amide bond in the peptide backbone is used as a means of masking a proton on the side chain of an amino acid in a peptide by a hydrogen bond, the formation of a hydrogen bond that masks the proton on the side chain must rely on chance because the conformation of each peptide varies. Therefore, it is difficult to always mask the side chain proton by forming a hydrogen bond. Furthermore, when an amide bond in the backbone of an adjacent amino acid is used to form a hydrogen bond that masks the side chain proton, the variety of side chains that can be adopted is limited. In the case of an amino acid that is expected to form a hydrogen bond with an amide bond in the backbone of an adjacent amino acid, the position of the proton donor is limited to a position close to the amide bond in the backbone that can form a hydrogen bond. On the other hand, in a non-limiting embodiment, the amino acids described herein can form hydrogen bonds "within their side chains," which may increase the flexibility of side chain design. For example, it is possible to lengthen the side chain while maintaining the formation of a hydrogen bond that can mask the proton, which may increase the possibility that the proton on the side chain is available for interaction with a target.

[0138] (General manufacturing method) Next, a general method for producing the amino acid capable of forming a hydrogen bond in the side chain and the peptide compound of the present invention will be described.

[0139] (Method for producing amino acids capable of forming intramolecular hydrogen bonds) In the production of amino acids having an intramolecular hydrogen bond in the side chain, amino acids having an ether bond in the side chain can be produced by the following two synthetic methods.

[0140] Method 1: A compound containing an aziridine in which the amino and carboxylic acid groups are protected is reacted with an alcohol having the corresponding appropriate protecting groups in the presence of a Lewis acid to perform a ring-opening reaction, followed by deprotection of unnecessary protecting groups. In the following general formula, P1 can be a Cbz group, Fmoc group, Alloc group, or nosyl (Ns) group, and P2 can be a Me group, allyl group, or benzyl group. A catalytic amount of BF3·OEt2 is preferred as the Lewis acid used in the ring-opening reaction. Dichloromethane is a preferred solvent, but the alcohol (ROH) used as a reagent can also be used as the solvent. If R contains a functional group that may be involved in the reaction, it is preferable to protect the functional group with a protecting group that is stable in the presence of a Lewis acid. For example, if R contains a hydroxyl group, it is preferable to protect the hydroxyl group with a benzyl group, etc., and if R contains a secondary amino group, it is preferable to protect the amino group with an Alloc group, etc. After constructing the basic amino acid skeleton by the ring-opening reaction, the desired amino acid can be obtained by appropriately converting the protecting group (an amino acid protected by a Cbz group, Fmoc group, Boc group, Alloc group, nosyl group (Ns group), etc. at P3, or an unprotected amino acid where P3 is hydrogen). [ka]

[0141] Method 2: The synthesis shown in the following scheme can be carried out by alkylating an amino acid having a hydroxyl group in the side chain and a protected amino group with an α-halocarbonyl compound such as 2-bromo-N-methylacetamide or bromoacetonitrile, followed by appropriate functional group conversion. In the following general formula, P1 is preferably a protecting group that is stable under basic conditions, such as a Cbz group, a Boc group, or a Trt group. As the α-halocarbonyl compound, R aPreferred examples of X1 are NMe and NtBu. X1 is a halogen, preferably a bromo group. Examples of bases used in the alkylation reaction include NaH, NaOtBu, and NaOt-Pent. Using two or more equivalents of this base for an amino acid with a hydroxyl group in the side chain and a protected amino group can selectively alkylate the hydroxyl group of the amino acid. Preferred examples of solvents used include DMF and DMI. After alkylation using bromoacetonitrile, the amino acid backbone can be constructed by constructing a heterocycle based on the resulting nitrile group. The following scheme illustrates the construction of an oxadiazolone ring by reacting the resulting nitrile group with hydroxylamine and then cyclizing it with CDI. After constructing the amino acid backbone, the desired amino acid can be obtained by appropriate conversion of the protecting group (an amino acid protected by Cbz, Fmoc, Boc, Alloc, or nosyl (Ns) group, or an unprotected amino acid where P3 is hydrogen). [ka]

[0142] Method 3: Phenylanine derivatives can be produced using methods such as the Jackson-Negishi coupling method (Journal of Organic Chemistry, 2010, 75, 245) or the asymmetric benzylation of benzophenone imine from glycine tert-butyl ester using a phase-transfer catalyst (J. Am. Chem. Soc. 1999, 121, 6519). Alternatively, they can be produced by a cross-coupling reaction involving decarboxylation using an N-hydroxyphthalimide ester (NHPI ester) and an aromatic iodine compound or an aromatic bromine compound, as shown in the following general formula: In the following general formula, n represents the number of carbon atoms. While not particularly limited, n = 1 or 2 is preferred. P1 is a group stable under cross-coupling conditions, preferably an Fmoc group. R4-X1 is an aromatic halide compound, and R4 represents an aromatic moiety. If R4 contains a functional group that can be converted under cross-coupling reaction conditions, it is preferably protected with a protecting group stable under cross-coupling reaction conditions. For example, when R4 contains a hydroxyl group, it is preferable to protect the hydroxyl group with a tetrahydropyranyl (THP) group or the like. X1 is a halogen, preferably a bromo (Br) group or an iodo (I) group. Nickel and zinc are preferably used as catalysts for the cross-coupling reaction, and 4,4'-di-tert-butyl-2,2'-bipyridyl (dtbbpy) is preferably used as the catalyst ligand. Dimethylacetamide is preferably used as the solvent. After constructing the basic skeleton of an amino acid by a cross-coupling reaction accompanied by decarboxylation, the amino acid can be converted to the desired amino acid (an amino acid protected at P3 with a Cbz group, Fmoc group, Boc group, Alloc group, nosyl group (Ns group), or an unprotected amino acid where P3 is hydrogen) by appropriate conversion of the protecting group. [ka] As shown below, amino acids in which the side chain site is on the N atom of the amino acid can also be produced by a similar reaction. [ka]

[0143] Method 4: The corresponding β-amino acids can be produced using the Arndt-Eistert synthesis (organic synthesis strategies based on named reactions, p. 18). [ka]

[0144] Method 5: β-amino acids can be produced by ring-opening an epoxide having a corresponding functional group using ammonia, introducing a protecting group into the amine moiety of the resulting compound, converting the resulting secondary alcohol into a leaving group, converting it into a nitrile group with stereoinversion by a nucleophilic substitution reaction, and then converting it into a carboxylic acid. In the general formula below, if R contains a functional group involved in the reaction, it is preferable to protect it with an appropriate protecting group. For example, if R contains a hydroxyl group, it is preferable to protect the hydroxyl group with a Trt group. Preferred examples of P1, the protecting group for the amino group obtained after the ring-opening reaction, include a Cbz group, a Boc group, and an Alloc group. The resulting secondary alcohol is converted into a leaving group X2 (preferably, a mesyl group (OMs group) is exemplified as X2). The resulting leaving group is converted into a cyanohydrin (CN - The basic amino acid skeleton can be constructed by converting the nitrile group to a carboxylic acid through a nucleophilic substitution reaction with methyl group, accompanied by stereoinversion, and then converting it to a carboxylic acid. The nitrile group can be converted to a carboxylic acid, for example, by hydrolysis under acidic conditions. After constructing the basic amino acid skeleton, the desired amino acid can be obtained by appropriately replacing the protecting group (amino acid protected with Cbz, Fmoc, Boc, Alloc, or nosyl (Ns) groups at P3, or unprotected amino acid with hydrogen at P3). [ka]

[0145] Recipe 6 A cyclic amino acid having a hydroxyl group and a protected amino group and a halogen compound are subjected to an alkylation reaction using a base, followed by appropriate conversion of the functional group, to produce a cyclic amino acid, as shown in the following scheme. The starting cyclic amino acid is preferably a 4- to 8-membered amino acid, and a 5-membered amino acid is exemplified in the following scheme. The position of the hydroxyl group present in the cyclic moiety is not particularly limited, but as shown in the general formula, it is preferably located at a position two or more atoms away from the N atom of the amino acid. In the general formula below, P1 can be a Cbz group, a Boc group, an Alloc group, a nosyl group (Ns group), or the like. A preferred example of the base used in the alkylation reaction is sodium hydride (NaH), and a preferred example of the solvent is dimethylformamide. In the alkylation reaction, if the halogen compound (RX) has a functional group on R that may be involved in the reaction, it is preferable to protect the functional group with a protecting group that is stable under basic conditions. For example, when R contains a hydroxyl group, it is preferable to protect the hydroxyl group with a silyl ether such as a tetrahydropyranyl (THP) group, a benzyl (Bn) group, or a t-butyldimethylsilyl (TBDMS) group. Furthermore, X1 is a halogen, examples of which include a chloro (Cl) group, a bromo (Br) group, and an iodo (I) group. After constructing the basic skeleton of an amino acid by an alkylation reaction, the amino acid can be converted into the desired amino acid (an amino acid protected with a Cbz group, an Fmoc group, a Boc group, an Alloc group, a nosyl group (Ns) group, or an unprotected amino acid in which P3 is hydrogen) by appropriate conversion of the protecting group. [ka]

[0146] Recipe 7 The above amino acids can be N-methylated by cyclization using paraformaldehyde under acidic conditions, followed by reduction of the resulting imine while opening the ring of the resulting cyclized product under acidic conditions, according to the following scheme: [ka]

[0147] (Method for producing peptide compounds) Chemical synthesis method for peptide compounds Examples of chemical synthesis methods for peptide compounds or cyclic peptide compounds herein include liquid-phase synthesis, solid-phase synthesis using Fmoc synthesis or Boc synthesis, and combinations thereof. In Fmoc synthesis, the base unit is an amino acid in which the main chain amino group is protected with an Fmoc group, and the side chain functional groups are protected as needed with a protecting group that is not cleaved by basicity, such as piperidine, t-Bu, THP, or Trt, and the main chain carboxylic acid is not protected. The base unit is not particularly limited as long as it has an Fmoc-protected amino group and a carboxyl group. For example, a dipeptide may be used as the base unit. The base unit to be placed at the N-terminus may be other than an Fmoc amino acid. For example, it may be a Boc amino acid or a carboxylic acid analog without an amino group. The main chain carboxyl group or the side chain carboxyl group of an amino acid having a carboxyl group in its side chain and whose main chain carboxyl group is protected with an appropriate protecting group is supported on a solid phase by chemical reaction with a functional group on the solid phase support. The Fmoc group is then deprotected using a base such as piperidine or DBU, and the newly generated amino group is condensed with a subsequently added, carboxyl-containing protected amino acid, forming a peptide bond. In the condensation reaction, various combinations of carboxyl group activators are possible, such as DIC and HOBt, DIC and HOAt, or HATU and DIPEA. The desired peptide sequence can be generated by repeating the Fmoc group removal and subsequent peptide bond formation reaction. After the desired sequence is obtained, the peptide is cleaved from the solid phase and, if necessary, the protective groups on the side chain functional groups are deprotected. It is also possible to perform structural transformation or cyclization of the peptide before cleavage from the solid phase. Cleavage from the solid phase and deprotection can be performed under the same conditions, such as 90:10 TFA / HO, or, if necessary, under separate conditions. Cleavage from the solid phase can be achieved with a weak acid such as 1% TFA, or by using a protecting group such as Pd to take advantage of the orthogonal nature of the chemical reaction. Cyclization or other steps can also be performed between or at the end of these steps.For example, a side-chain carboxylic acid can be condensed with an amino group in the N-terminal main chain, or a side-chain amino group can be condensed with a carboxylic acid in the C-terminal main chain. In this case, orthogonality is required between the C-terminal carboxylic acid and the side-chain carboxylic acid to be cyclized, or between the N-terminal main-chain amino group or hydroxy group and the side-chain amino group to be cyclized. As mentioned above, protecting groups are selected with consideration for orthogonality. The resulting reaction product can be purified using a reverse-phase column or molecular sieve column. Details of these methods are described, for example, in the Solid-Phase Synthesis Handbook published by Merck Ltd. on May 1, 2002. Commercially available resins for solid-phase synthesis can be used, such as CTC resin, Wang resin, or SASRIN resin.

[0148] In the preparation of the compounds described herein, if a defined group undergoes undesired chemical transformation under the conditions of the method, the compound can be prepared by, for example, protecting and deprotecting the functional group. The selection and deprotection of protecting groups can be performed using methods described in, for example, Greene's "Protective Groups in Organic Synthesis" (5th ed., John Wiley & Sons 2014), which can be used appropriately depending on the reaction conditions. The order of reaction steps, such as introducing substituents, can also be changed as necessary. Examples of protecting groups for amino groups include Fmoc, Boc, Cbz, and Alloc groups. These carbamate groups can be introduced by reacting the amino group with a carbamating agent in the presence of a base catalyst. Examples of carbamating agents include BocO, BocOPh, FmocOSu, FmocCl, CbzCl, and AllocCl. Examples of base catalysts include lithium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, cesium bicarbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium phosphate, potassium phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, N,N-dimethylaminopyridine, etc. A carbamate group, which is a protecting group for an amino group, can be removed under basic conditions, acidic conditions, or hydrogenolysis conditions.

[0149] Translational synthesis method for peptide compounds As used herein, the translational synthesis method for peptide compounds is exemplified by synthesis methods using a cell-free translation system, including synthesis methods using a reconstituted cell-free translation system. The use of a reconstituted cell-free translation system is preferred because it allows the removal of factors that are to be eliminated.

[0150] In one aspect, the present specification provides a method for translational synthesis of a peptide compound described herein that includes at least one, two or more, or three or more amino acids described herein. Without being limited thereto, such a translational synthesis method may include the following (i) and (ii): (i) preparing a tRNA bound to at least one, two or more, or three or more types of amino acids as defined herein; (ii) A step of translating a nucleic acid containing at least one codon corresponding to the anticodon of the tRNA in a cell-free translation system to obtain the peptide compound. In a non-limiting embodiment, the translational synthesis method herein may be a translational synthesis method for a cyclic peptide compound.

[0151] Cell-free translation system As used herein, the term "cell-free translation system" refers to a combination of ribosomes extracted from cells, protein factors involved in translation, tRNA, amino acids, an energy source such as ATP, and a regeneration system for these. It is not limited to any system capable of translating mRNA into protein. The term "cell-free translation system" as used herein also includes a system in which a translation synthesis reaction is in progress. The cell-free translation system used herein may contain nucleic acids that serve as templates for peptide translation, as well as initiation factors, elongation factors, release factors, aminoacyl-tRNA synthetases, and other factors. These factors can be obtained by purification from extracts of various cells. Examples of cells used to purify factors include prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include Escherichia coli cells, extreme thermophilic bacteria cells, and Bacillus subtilis cells. Examples of eukaryotic cells known include yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, and animal cells. In addition to naturally occurring tRNAs and aminoacyl-tRNA synthetases (ARSs), artificial tRNAs and artificial aminoacyl-tRNA synthetases that recognize unnatural amino acids can also be used. Using artificial tRNAs and artificial aminoacyl-tRNA synthetases allows for the site-specific synthesis of peptides incorporating unnatural amino acids. If necessary, transcription from template DNA can also be performed by adding an RNA polymerase such as T7 RNA polymerase to the cell-free translation system.

[0152] As used herein, the phrase "a cell-free translation system containing a certain substance" also includes embodiments in which the substance is not contained at the start of translation synthesis, but is synthesized and contained within the system during the process of translation synthesis. For example, if an acylated tRNA for an amino acid is synthesized during the process of translation synthesis, the cell-free translation system is understood to contain the aminoacyl-tRNA.

[0153] PURESYSTEM® (BioComber, Japan) is a reconstituted cell-free translation system in which protein factors, energy regeneration enzymes, and ribosomes required for translation in Escherichia coli are extracted and purified, and then mixed with tRNA, amino acids, ATP, GTP, and other components. Not only does it contain few impurities, but because it is a reconstituted system, it is easy to create a system that does not contain protein factors or amino acids that you want to eliminate. ((i) Nat Biotechnol. 2001;19:751-755. Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. (ii) Methods Mol Biol. 2010;607:11-21. PUREtechnology. Shimizu Y, Ueda T.)

[0154] For example, while many methods have been reported that use stop codons to introduce unnatural amino acids, the aforementioned PURESYSTEM can be used to construct a synthesis system that excludes natural amino acids and ARSs. This allows unnatural amino acids to be linked to the codons encoding the natural amino acids to be excluded (J Am Chem Soc. 2005;127:11727-35. Ribosomal synthesis of unnatural peptides. Josephson K, Hartman MC, Szostak JW.). Furthermore, by breaking the codon degeneracy, unnatural amino acids can be added without excluding natural amino acids (Kwon I, et al. Breaking the degeneracy of the genetic code. J Am Chem Soc. 2003, 125, 7512-3.). Peptides containing N-methyl amino acids can be synthesized by ribosomes using cell-free translation systems such as the PURESYSTEM.

[0155] More specifically, the translation synthesis may involve, for example, protein factors necessary for translation in E. coli (methionyl-tRNA transformylase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (select from AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS, GluRS, GlyRS, HisRS, IleRS, LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, and ValRS)), ribosomes, amino acids, creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase, E. coli-derived tRNA, creatine phosphate, potassium glutamate, HEPES-KOH This can be achieved by adding mRNA to a known cell-free translation system such as PURESYSTEM, which contains a pH 7.6 mixture of magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, UTP, and other ingredients. Furthermore, by adding T7 RNA polymerase, coupled transcription and translation from a template DNA containing a T7 promoter can be performed. Furthermore, peptide compounds containing unnatural amino acids can be translationally synthesized by adding desired aminoacyl-tRNAs or unnatural amino acids (e.g., F-Tyr) tolerated by aminoacyl-tRNA synthetase (ARS) to the system (Kawakami T, et al., Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J Am Chem Soc. 2008, 130, 16861-3., Kawakami T, et al., Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol. 2009, 5, 888-90.). Furthermore, peptide compounds containing unnatural amino acids can be translationally synthesized by including a modified ARS in the system in place of or in addition to a natural ARS, and also including a group of unnatural amino acids in the system.Alternatively, the efficiency of translational incorporation of unnatural amino acids can be increased by utilizing mutants of ribosomes or EF-Tu (Dedkova LM, et al. Construction of modified ribosomes for incorporation of D-amino acids into proteins. Biochemistry. 2006, 45, 15541-51; Doi Y, et al. Elongation factor Tu mutants expand aminoacid tolerance of protein biosynthesis system. J Am Chem Soc. 2007, 129, 14458-62; Park HS, et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science. 2011, 333, 1151-4).

[0156] In a non-limiting embodiment, the cell-free translation system herein (also referred to as the "translation system herein") may be a translation system for producing a peptide compound, and is preferably a translation system for producing a cyclic peptide compound.

[0157] In a non-limiting embodiment, the translation system herein may contain 5 to 32, 5 to 28, or 5 to 20 types of unnatural amino acids, preferably 8 to 20, 10 to 20, or 13 to 20. In one embodiment, 50% or more, 60% or more, 70% or more, or 80% or more of the types of amino acids contained in the translation system herein may be unnatural amino acids.

[0158] In a non-limiting embodiment, the translation system herein may contain 5 to 28 or 5 to 20 types of N-substituted amino acids, with 5 to 18 or 5 to 15 being preferred examples. In one embodiment, 40% or more, 50% or more, 60% or more, or 70% or more of the types of amino acids contained in the translation system herein may be N-substituted amino acids. Here, N-substituted amino acids may refer to N-alkyl amino acids or N-methyl amino acids, but even in this case, this does not exclude the inclusion of other N-substituted amino acids in the translation system herein.

[0159] In a non-limiting embodiment, the translation system herein may be adjusted so that the average number of aromatic rings contained in peptide compounds produced using the translation system falls within a certain range. For example, the translation system herein may be adjusted so that the average proportion of amino acids having aromatic rings to the total number of amino acids constituting the cyclic portion of a peptide compound translationally synthesized is 40% or less, 35% or less, 30% or less, 27% or less, 25% or less, or 20% or less, or so that the average number of aromatic rings contained in the side chains of the cyclic portion of a peptide compound having a cyclic portion composed of 8 to 11 amino acids is 0 to 3. The method for adjustment is not particularly limited, and examples include adjusting the number of types of amino acids having aromatic rings to the total number of types of amino acids contained in the translation system.

[0160] In a non-limiting embodiment, the translation system herein may contain amino acids having aromatic rings, and the proportion of the number of types of amino acids having aromatic rings to the total number of types of amino acids contained in the translation system is preferably 40% or less, and preferred examples include 35% or less, 30% or less, 27% or less, 25% or less, and 20% or less.

[0161] In a non-limiting embodiment, the cell-free translation system herein may be a cell-free translation system for producing a peptide compound herein containing at least one, two or more, or three or more amino acids herein. Without being limited thereto, such a cell-free translation system may include the following (i) and (ii): (i) a tRNA having at least one, two or more, or three or more amino acids bound thereto; (ii) a nucleic acid encoding the peptide compound; Here, the nucleic acid may contain at least one codon corresponding to the anticodon of the tRNA.

[0162] tRNA Translational incorporation of unnatural amino acids into peptides requires aminoacylation of orthogonal tRNAs that are efficiently incorporated into ribosomes ((i) Biochemistry. 2003;42:9598-608. Adaptation of anorthogonalar leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. Anderson JC, Schultz PG., (ii) Chem Biol. 2003;10:1077-84. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code. Murakami H, Kourouklis D, Suga H.). The following five methods can be used to aminoacylate tRNA.

[0163] Within cells, aminoacyl-tRNA synthetases (ARSs) are prepared for each amino acid as enzymes for aminoacylation of tRNA. Therefore, the first method is to utilize the fact that certain ARSs can tolerate unnatural amino acids such as N-Me His, or to prepare and use mutant aminoacyl-tRNA synthetases that can tolerate unnatural amino acids ((i) Proc Natl Acad Sci U S A. 2002;99:9715-20. An engineered Escherichia coli methylosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germcell-free system. Kiga D, Sakamoto K, Kodama K, Kigawa T, Matsuda T, Yabuki T, Shirouzu M, Harada Y, Nakayama H, Takio K, Hasegawa Y, Endo Y, Hirao I, Yokoyama S. (ii) Science. 2003;301:964-7. An expanded eukaryotic genetic code. Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG. Chin, JW. (iii) ProcNatlAcad Sci U S A. 2006;103:4356-61. Enzymatic aminoacylation of tRNA with unnatural amino acids. Hartman MC, Josephson K, Szostak JW.) Second, a method can be used in vitro to aminoacylate tRNA and then chemically modify the amino acid (J AmChem Soc. 2008;130:6131-6. Ribosomal synthesis of N-methyl peptides. Subtelny AO, Hartman MC, Szostak JW.).Third, aminoacyl-tRNA can be obtained by removing the CA from the CCA sequence at the 3' end of tRNA and ligating it with separately prepared aminoacylated pdCpA using RNA ligase (Biochemistry. 1984;23:1468-73. T4 RNA ligase-mediated preparation of novel "chemically misacylated" tRNAPheS. Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM.). Aminoacylation can also be achieved using flexizyme, a ribozyme that can attach activated esters of various unnatural amino acids to tRNA (J AmChem Soc. 2002;124:6834-5. Aminoacyl-tRNA synthesis by aresin-immobilized ribozyme. Murakami H, Bonzagni NJ, Suga H.). Fourth, ultrasonic mixing of tRNA and an amino acid activated ester in cationic micelles can also be used (Chem Commun (Camb). 2005;(34):4321-3. Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation. Hashimoto N, Ninomiya K, Endo T, Sisido M.). Fifth, aminoacylation can also be achieved by adding a PNA complementary to the 3' end of tRNA to which an amino acid activated ester is bound to the tRNA (J Am Chem Soc. 2004;126:15984-9. In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid. Ninomiya K, Minohata T, Nishimura M, Sisido M.).

[0164] More specifically, aminoacyl-tRNA can be prepared using the following methods. A template DNA encoding the desired tRNA sequence, with a T7, T3, or SP6 promoter located upstream, is prepared, and RNA can be synthesized by transcription using an RNA polymerase compatible with the promoter, such as T7 RNA polymerase, T3, or SP6 RNA polymerase. Alternatively, tRNA can be extracted and purified from cells, and the desired tRNA can be extracted using a probe with a sequence complementary to the tRNA sequence. Cells transformed with an expression vector for the desired tRNA can also be used as the source. RNA with the desired sequence can also be synthesized by chemical synthesis. For example, aminoacyl-tRNA can be obtained by ligating the thus-obtained tRNA, in which the CA has been removed from the 3'-terminal CCA sequence, with separately prepared aminoacylated pdCpA or pCpA using RNA ligase (pdCpA method, pCpA method). This tRNA is useful for the production of peptide compounds. Alternatively, aminoacylation can be performed by preparing a full-length tRNA and using flexizyme, a ribozyme that loads activated esters of various unnatural amino acids onto the tRNA. Furthermore, although not intended to be limiting, aminoacyl-tRNAs can also be produced using natural ARSs or their modified forms. When natural ARSs or their modified forms are used, aminoacyl-tRNAs once consumed in the translation system can be regenerated by the natural ARSs or their modified forms, eliminating the need for large amounts of pre-produced aminoacyl-tRNAs in the translation system. Such modified ARSs are described in WO2016 / 148044. These methods for producing aminoacyl-tRNAs can also be combined as appropriate.

[0165] Annulus formation In one non-limiting embodiment, the cyclic portion of the peptide compound is formed by subjecting a linear peptide compound to a cyclization reaction.

[0166] As used herein, the term "cyclization reaction" refers to a reaction that forms a cyclic moiety at the peptide moiety of a peptide compound. In one embodiment, the term "peptide compound" as used herein also includes peptide compounds obtained by further chemically modifying or reacting a peptide compound in which a cyclic moiety has been formed by a cyclization reaction. Chemical modification or the like may also be performed before the cyclization reaction.

[0167] Scheme A shows an example of a cyclization reaction of the peptide portion of a peptide compound herein. The black circle (●) unit (main chain unit), D unit (aspartic acid unit), and triangle (▲) unit (cyclized N-terminal unit) each represent an amino acid residue constituting the peptide portion. The white circle represents a resin for solid-phase synthesis. In Scheme A, the cyclic portion is a portion consisting of one triangle unit, nine black circle units, and one D unit. Each unit may be the same amino acid or different amino acids. The cyclic peptides of the present invention can be produced, for example, by the method described in WO 2013 / 100132. [ka]

[0168] As used herein, the term "unit" refers to any of the amino acids before cyclization after synthesis of a linear peptide compound, the amino acids after cyclization, and the amino acids at the time when chemical modification after cyclization is complete. The amino acid residues at the time when chemical modification after cyclization is complete include, for example, amino acid residues that have been chemically converted or have undergone backbone conversion by chemical modification after production of the peptide compound.

[0169] Library In a non-limiting embodiment, the library of peptide compounds or nucleic acids encoding them in the present disclosure may be a library consisting essentially of the peptide compounds described herein or the nucleic acids encoding them, respectively. The term "library" as used herein includes a library containing the peptide compounds of the present specification and a library containing nucleic acids encoding the peptide compounds of the present specification. The term "library" as used herein includes a library of the peptide compounds of the present specification and a library of peptide compound-nucleic acid complexes, of which libraries of cyclic peptide compounds or cyclic peptide-nucleic acid complexes are preferred, and libraries of cyclic peptide-mRNA complexes are particularly preferred. A preferred library is a display library. Examples of display libraries include libraries that utilize display, of which mRNA display libraries, DNA display libraries, and ribosome display libraries are preferred, with mRNA display libraries being more preferred.

[0170] Without intending to be bound by any particular theory, the present inventors believe as follows. In conventional peptide libraries, masking the proton donor contained in the side chain of an amino acid constituting a peptide by forming an intramolecular hydrogen bond has relied on chance. In particular, in peptide libraries in which constituent amino acids are randomly arranged to create diversity, it has been difficult to form hydrogen bonds between amino acids as intended. Peptide compounds containing the amino acids described herein use amino acids in which hydrogen bonds have already been formed at the proton donor site contained in the side chain, making it possible to intentionally form intramolecular hydrogen bonds at the side chain site of the peptide compound. A library of peptide compounds obtained using the amino acids described herein may have a high frequency of occurrence of peptide compounds that can mask the side chain proton donor by forming intramolecular hydrogen bonds, i.e., may have a high probability of containing peptide compounds with high membrane permeability.

[0171] As used herein, a peptide compound may be a "peptide compound encoded by a nucleic acid." As used herein, the term "peptide compound encoded by a nucleic acid" is not limited to peptide compounds directly synthesized by translation using the nucleic acid as a template, but may also include peptide compounds that can be synthesized through post-translational modification. For example, when a linear peptide compound is synthesized by translating a nucleic acid and a cyclic peptide compound is then synthesized through a cyclization step, the cyclic peptide compound may be referred to as a cyclic peptide compound encoded by the nucleic acid. In one embodiment, a peptide compound translationally synthesized using a nucleic acid as a template, or a complex of a peptide compound that has subsequently undergone post-translational modification and the nucleic acid (peptide compound-nucleic acid complex) are also included in the (cyclic) peptide compound encoded by a nucleic acid.

[0172] In a non-limiting embodiment, the average number of unnatural amino acids, N-substituted amino acids, or amino acids with aromatic rings contained in peptide compounds contained in a library herein or peptide compounds encoded by nucleic acids contained in the library can be adjusted by adjusting the frequency of codons assigned to the amino acids when synthesizing a DNA library. For example, in a peptide library containing 10 variable amino acid residues, if 20 amino acids are selected, 10 of the 20 amino acids can be selected as N-substituted amino acids, allowing synthesis to be performed so that the codon units for N-substituted amino acids account for 10 / 20 (50%) per variable site. In this manner, the average percentage of the number of unnatural amino acids, N-substituted amino acids, aromatic rings, or amino acids with aromatic rings herein may be calculated.

[0173] As used herein, with respect to a library of peptide compounds and / or nucleic acids encoding them, "consisting essentially of" may mean that the ratio of the theoretical number of peptide compounds and / or nucleic acid variations encoding them listed therein to the theoretical total number of peptide compounds and / or nucleic acid variations encoding them contained in the library is 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, but is not limited to these.

[0174] In determining the "theoretical (total) number" of variations of a peptide compound in the library herein, those that cannot actually be produced as a peptide compound are not included in the theoretical (total) number. For example, in the following cases (1) and (2), the corresponding amino acid is not synthesized by translation, and therefore peptide compounds containing such amino acids are not included in the theoretical (total) number: (1) when an amino acid is added to the translation solution as a material for the peptide compound, but a nucleic acid encoding the amino acid is not included as a template; or (2) when a translation solution is used in which the nucleic acid serving as a template for the peptide compound contains a base sequence but does not contain the corresponding amino acid. Furthermore, if there are by-products or unreacted products in the process of producing a cyclic peptide compound, these are not included in the theoretical (total) number.

[0175] For example, when a peptide of 11 residues is translated and synthesized from a base sequence in which the codon units are randomly arranged using a codon table in which a set of codon units corresponds to one type of amino acid, using a total of 20 natural amino acids, and the translation initiation amino acid is fixed to methionine, the theoretical number of variations is 20 10 This becomes:

[0176] In a non-limiting embodiment, the library herein may contain, in addition to peptide compounds and / or nucleic acids encoding them, other components necessary for screening peptide compounds capable of specifically binding to target molecules, as long as they do not negatively affect the effectiveness of the library herein.

[0177] The library herein includes a plurality of peptide compounds or nucleic acids encoding them. As used herein, the number of variations (types) of peptide compounds included in the library is referred to as "diversity." The diversity of peptide compounds included in the library herein or nucleic acids encoding them is not particularly limited, but may be in the range of 10 3 That's it, 10 4 That's it, 10 5 That's it, 10 6 That's it, 10 7 That's it, 10 8 That's it, 10 9 That's it, 10 10 That's it, 10 11 Above, and 10 12 The above are examples. These variations are not limited to actual measurements, but may be theoretical values.

[0178] In a non-limiting embodiment, the average molecular weight of the peptide portion other than the nucleic acid linkage portion of a peptide compound contained in a library herein or encoded by a nucleic acid contained in the library, or the average molecular weight of the cyclic portion of a cyclic peptide compound, may be 500 to 2000.

[0179] Display Library A display library is a library in which a peptide (phenotype) is associated with the RNA or DNA encoding the peptide (genotype). This library can be used to identify peptide compounds that can specifically bind to a target molecule. For example, by contacting the library with a desired immobilized target and washing away molecules that do not bind to the target, it is possible to enrich for peptides that bind to the target (panning method). By analyzing the genetic information associated with the peptides selected through this process, the sequences of the peptides that bound to the target can be determined. For example, methods utilizing the nonspecific binding of the antibiotic puromycin, an aminoacyl-tRNA analog, to proteins during ribosomal mRNA translation elongation have been reported, including mRNA display (Proc Natl Acad SciUSA. 1997;94:12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins. Roberts RW, Szostak JW.) and in vitro virus (FEBS Lett. 1997;414:405-8. In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H.).

[0180] A spacer such as puromycin is attached to the 3' end of an mRNA library transcribed from a DNA library containing a promoter such as the T7 promoter. When the mRNA is translated into protein in a cell-free translation system, the puromycin is mistaken for an amino acid and incorporated into the protein by the ribosome, linking the mRNA and the protein it encodes, creating a library in which mRNA and its product are associated. This process is highly efficient because it does not require transformation of E. coli or other bacteria, making it possible to construct large-scale display libraries. The sequences of the bound proteins can be determined by synthesizing cDNA from mRNA, which is a tag containing genetic information attached to the molecules enriched and selected by panning, amplifying it via PCR, and analyzing the base sequence.

[0181] In addition to mRNA display, cell-free translation system-based display libraries include cDNA display, which is a library consisting of cDNAs encoding peptides bound to a peptide-puromycin complex (Nucleic Acids Res. 2009;37(16):e108. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N.), and ribosome display, which utilizes the relatively stable complex between ribosomes and translation products during mRNA translation (Proc Natl Acad Sci U S A. 1994;91:9022-6. An in vitro polysome display system for identifying ligands from very large peptide libraries. Mattheakis LC, Bhatt RR, Dower WJ.), covalent display, which utilizes the covalent bond formation between bacteriophage endonuclease P2A and DNA (Nucleic Acids Res. 2005;33:e10. Covalent antibody display - an in vitro antibody-DNA library selection system. Reiersen H, Lobersli I, Loset GA, Hvattum E, Simonsen B, Stacy JE, McGregor D, Fitzgerald K, Welschof M, Brekke OH, Marvik OJ.), and CIS display, which utilizes the binding of the microbial plasmid replication initiator protein RepA to the replication origin, ori (Proc Natl Acad Sci U S A. 2004;101:2806-10).CIS display: In vitro selection of peptides from libraries of protein-DNA complexes. Odegrip R, Coomber D, Eldridge B, Hederer R, Kuhlman PA, Ullman C, FitzGerald K, McGregor D.) is known. In vitro compartmentalization (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.), in which a transcription-translation system is encapsulated in a water-in-oil emulsion or liposome for each DNA molecule constituting a DNA library, and the translation reaction is carried out, is also known. The above methods can be implemented using known methods as appropriate.

[0182] Nucleic Acid Library The "nucleic acid" of the present invention can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or nucleotide derivatives containing artificial bases. It can also include peptide nucleic acid (PNA). The nucleic acid of the present invention can be any one of these nucleic acids or a mixture thereof, as long as the desired genetic information is retained. In other words, the nucleic acid of the present invention also includes DNA-RNA hybrid nucleotides and chimeric nucleic acids in which different nucleic acids such as DNA and RNA are linked in a single strand.

[0183] Examples of nucleic acid libraries that serve as templates for peptide compounds included in peptide compound libraries include mRNA libraries and DNA libraries. Nucleic acid libraries can be obtained by synthesizing mixed bases at positions in the peptide sequence where amino acid residues are not fixed. For example, DNA libraries can be synthesized using A, T, G, and C, while RNA libraries can be synthesized using a mixture of four bases (A, U, G, and C) repeated a multiple of three times (N), or by synthesizing codons with N as the first and second letters and a mixture of two bases (W, M, K, S) as the third letter. Furthermore, if the number of amino acid types to be introduced is limited to 16 or less, a single base can be used as the third letter. Furthermore, the frequency of occurrence of amino acid residues can be freely adjusted by preparing codon units corresponding to the three letters of a codon and mixing them in any ratio for synthesis.

[0184] These nucleic acid libraries can be translated using a cell-free translation system. When using a cell-free translation system, it is preferable to include a sequence encoding a spacer downstream of the target nucleic acid. Examples of spacer sequences include, but are not limited to, sequences containing glycine or serine. It is also preferable to include a linker formed from RNA, DNA, or a polymer (e.g., a five-member polymer) of hexaethylene glycol (SPC18) between the nucleic acid library and a compound, such as puromycin or its derivatives, that is incorporated into the peptide during ribosomal translation.

[0185] Library manufacturing method In a non-limiting embodiment, the library of the present disclosure can be produced in accordance with the method for producing peptide compounds of the present disclosure described above, and can be combined with known methods as appropriate. In one embodiment, the library of peptide compounds of the present disclosure can be produced using the cell-free translation system of the present disclosure described above. That is, the method for producing a library of the present disclosure may include a step of synthesizing peptide compounds using the cell-free translation system of the present disclosure. In one embodiment, the examples, preferred ranges, and aspects described for the cell-free translation system of the present disclosure can be applied directly to the method for producing a library of the present disclosure.

[0186] In one non-limiting embodiment, an mRNA display library can be produced as follows: First, a DNA library is chemically synthesized in which a desired sequence is placed downstream of a promoter such as the T7 promoter. This is then converted into double-stranded DNA by a primer extension reaction using this as a template. This is then transcribed into mRNA using an RNA polymerase such as T7 RNA polymerase. A linker (spacer) containing an antibiotic such as puromycin, an aminoacyl-tRNA analog, is attached to the 3' end of this RNA. This is added to a known cell-free translation system such as the PURESYSTEM described above and incubated, whereby the mRNA is translated, and the mRNA and the peptide encoded thereby are linked via a linker containing puromycin or the like. In this way, a display library consisting of complexes of mRNA and its product, in which the mRNA and its product are associated, can be constructed. The linker can further contain a spacer known to those skilled in the art. If necessary, post-translational modifications such as cyclization can be performed using the methods described above or known methods.

[0187] In a non-limiting embodiment, the library of the present disclosure may be a peptide compound library containing peptide compounds containing at least one, two or more, or three or more amino acids selected from the group consisting of amino acids described herein. A method for producing such a library may include a step of translationally synthesizing peptide compounds in a cell-free translation system comprising the following steps (i) and (ii): (i) a tRNA bound to at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids described herein; (ii) a nucleic acid library encoding the peptide compound library; Here, the nucleic acid library may contain nucleic acids containing at least one codon corresponding to the anticodon of the tRNA.

[0188] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0189] The present invention is further illustrated by, but not limited to, the following examples.

[0190] The abbreviations used in the examples are listed in the following abbreviation table. [Table 1]

[0191] The reaction solvents used for peptide synthesis and solid-phase synthesis were those for peptide synthesis (purchased from Watanabe Chemical and Wako Pure Chemical Industries). Examples include DCM, DMF, NMP, 2% DBU in DMF, and 20% piperidine in DMF. For reactions that do not use water as a solvent, dehydrated solvents, super-dehydrated solvents, and anhydrous solvents (purchased from Kanto Chemical and Wako Pure Chemical Industries, etc.) were used. The LC / MS analysis conditions are as follows:

[0192] [Table 2]

[0193] Example 1 Chemical Synthesis of Peptide Compounds Peptide elongation was performed according to the Fmoc peptide synthesis method described in WO 2013 / 100132 using the following basic route. Specifically, the five steps consisted of: 1) Fmoc peptide elongation from the N-terminus of Asp, with the carboxylic acid of the Asp side chain supported on 2-chlorotrityl resin; 2) peptide cleavage from the 2-chlorotrityl resin; 3) amide cyclization by condensation of the carboxylic acid of the Asp side chain, generated by cleavage from the 2-chlorotrityl resin during the cleavage process, with the amino group of the N-terminus (triangle unit) of the peptide chain; 4) deprotection of the protecting groups of the side chain functional groups contained in the peptide chain; and 5) purification of the compound by preparative HPLC. Furthermore, for peptide compounds containing amino acids with acidic functional groups in the side chain, a 0.08 M solution of triethylamine hydrochloride in dichloromethane can be used to wash the resin during the peptide elongation reaction to prevent over-elongation of the peptide due to residual deprotecting agent. In this example, peptide compounds were synthesized according to this basic route unless otherwise noted. [ka]

[0194] 1-1. Fmoc-amino acids used in peptide synthesis using a peptide synthesizer In the peptide synthesis described herein, the following Fmoc-amino acids were used in the synthesis using a peptide synthesizer. Amino acids having reactive functional groups in the side chains were protected with appropriate protecting groups. The amino acid abbreviations are listed in Table 3 below. Fmoc-D-Val-OH, Fmoc-MeLeu-OH, Fmoc-MePhe-OH, Fmoc-MeGly-OH, Fmoc-gMeAbu-OH (sometimes written as Fmoc-g-MeAbu-OH), Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-M eAla-OH, Fmoc-MeVal-OH, Fmoc-D-Abu-OH, Fmoc-Val-OH, Fmoc-MeAbu-OH, Fmoc-MeIle-OH, Fmoc-D-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-D-Ala-OH, Fmoc-Tyr (Clt)-OH, Fmoc-Phe-OH, Fmoc-Phe(4-CF3)-OH, Fmoc-Ala-OH, Fmoc-Hph-OH, Fmoc-Gly-OH, Fmoc-b-MeAla-OH, Fmoc-Ala(3-Pyr)-OH, Fmoc-D-MeAla-OH, Fmoc-Phe{#(CH2)2}-OH, Fmoc-Ala(Thz)-OH, Fmoc-D-Pro-OH, Fmoc-Pro-OH, Fmoc-Ser(Bn)-OH, and other reagents were purchased from Watanabe Chemical, Chempep, Chem-Impex, or Bachem. Fmoc-nPrGly-OH, Fmoc-MePhe(3-Cl)-OH, and Fmoc-MeAla(4-Thz)-OH were synthesized according to the method described in WO2013 / 100132. Additionally, Fmoc-Thr(THP)-OH (compound aa01), Fmoc-nBuGly-OH (compound aa02), Fmoc-Ser(iPen)-OH (compound aa04), Fmoc-MeHph-OH (compound a a05), Fmoc-Ser(3-F-5-Me-Pyr)-OH (compound aa12), Fmoc-Ser(Ph-3-Cl)-OH (compound aa16), Fmoc-Nle(6-OTHP)-OH (compound aa17) , Fmoc-Ser(EtOTHP)-OH (compound aa22), Fmoc-MeSer(EtOTHP)-OH (compound aa27), Fmoc-Ser(S-2-PrOTHP)-OH (compound aa34), Fmoc -MeSer(S-2-PrOTHP)-OH (compound aa39), Fmoc-Ser(R-2-PrOTHP)-OH (compound aa46), Fmoc-MeSer(R-2-PrOTHP)-OH (compound aa51),Fmoc-Ser(tBuOTHP)-OH (compound aa54), Fmoc-MeSer(tBuOTHP)-OH (compound aa55), Fmoc-Ser(2-Me-2-BuOTHP)-OH (compound aa62), Fmoc-MeSer(2-Me-2-BuOTHP)-OH (compound aa64), Fmoc-Hnl(7-F3-6-OH)-OH (compound aa68), Fmoc-Ser(1-CF3-EtOH)-OH (compound aa70), Fmoc-MeSer(1-CF3-EtOH)-OH (compound aa71), Fmoc-Ser(1- CF3-EtOTHP)-OH (compound aa72), Fmoc-Gln(Me)-OH (compound aa75), Fmoc-Ser(NtBu-Aca)-OH (compound aa78), Fmoc-MeSer(NtBu-Aca)-OH (compound aa79), Fmoc-Ser(NMe-Aca)-OH (compound aa81), Fmoc-Ser(nPrOTHP)-OH (compound aa85), Fmoc-Ser(2-Me2-PrOTHP)-OH (compound aa89), Fmoc-Ser(S-2-BuOTHP)-OH (compound aa95), Fmoc-S er(R-2-BuOTHP)-OH(compound aa101), Fmoc-Phe(4-OClt-3-OMe)-OH(compound aa10 4), Tyr(3-OMe), Fmoc-Ser(3-Me-5-Oxo- Odz)-OH(compound aa109), Fmoc-bAla(3R-MeOEtOTHP)-OH(compound aa111), Fmoc-bAla(2S-MeOEtOTHP)-OH(compound aa117), Fmoc-Nva(2R-4-F2-Pyrro(N-Alloc)) -OH (compound aa121), Fmoc-Ser(S-4-F2-Pyrro(N-Alloc)-Me)-OH (compound aa126), Fmoc-Phe(3-OMe-4-CONMe)-OH (compound aa127), Fmoc-Phe(4-OMe-3-CONMe)-OH (compound aa128), Fmoc-Phe(3-OMe-4-CONHMs)-OH (compound aa129), Fmoc-Hyp(2-EtOTHP)-OH (compound aa133), Fmoc-(Ph(3-OMe-4-CONMe)Et)Gly-OH (compound 134)Fmoc-Hph(3-OMe-4-CONMe)-OH (compound aa135), Fmoc-D-Hph(3-OMe-4-CONMe)-OH (compound aa136), Fmoc-MeHph(3-OMe-4-CONMe)-OH (compound aa137), Fmoc-Phe(4-CONMeOTHP)-OH (compound aa139), Fmoc-Phe(3-OMe-4-CONHOTHP)-OH (compound aa140), Fmoc-Ser(S-(Alloc)Mor-3-Me)-OH (compound aa141), and the like were synthesized as follows. For sequences containing amino acids with tertiary alcohols in the side chains (e.g., Ser(tBuOH), MeSer(tBuOH), Ser(2-Me-2-BuOH), MeSer(2-Me-2-BuOH), etc.), peptides were successfully elongated using Fmoc amino acids in which the tertiary alcohol moiety in the side chain was not protected with THP (e.g., Fmoc-Ser(tBuOH)-OH (compound aa53), Fmoc-Ser(2-Me-2-BuOH)-OH (compound aa61), etc.). For sequences containing amino acids with a secondary alcohol side chain and a CF3 group attached to the carbon atom to which the alcohol is attached (e.g., Hnl(7-F3-6-OH), Ser(1-CF3-EtOH), etc.), peptides were successfully elongated using Fmoc amino acids in which the secondary alcohol side chain was not protected with THP (e.g., Fmoc-Hnl(7-F3-6-OH)-OH (compound aa68), Fmoc-Ser(1-CF3-EtOH)-OH (compound aa70), etc.).

[0195] [Table 3]

[0196] 1-2. Amino acid synthesis for peptide synthesis using a peptide synthesizer Synthesis of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (compound aa01, Fmoc-Thr(THP)-OH) [ka] Toluene (50 mL) was added to a mixture of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid monohydrate (Fmoc-Thr-OH monohydrate, purchased from Tokyo Chemical Industry Co., Ltd., 5.0 g, 13.9 mmol) and pyridinium p-toluenesulfonate (PPTS) (0.175 g, 0.70 mmol), and the water content was removed by azeotropy by distilling the toluene under reduced pressure. To the resulting residue, ultra-dehydrated tetrahydrofuran (THF) (28 mL) and 3,4-dihydro-2H-pyran (8.8 mL, 97 mmol) were added, and the mixture was stirred at 50°C for 4 hours under a nitrogen atmosphere. After confirming the disappearance of the starting materials by LCMS (SQDFA05), the mixture was cooled to 25°C, and ethyl acetate (30 mL) was added. Subsequently, saturated aqueous sodium chloride solution (30 mL) was added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (30 mL). All the obtained organic layers were combined and washed twice with saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product (9.3 g). Of the resulting crude product, 4.65 g was dissolved in tetrahydrofuran (THF) (30 mL), followed by the addition of 1.0 M phosphate buffer (30 mL) adjusted to pH 8.0. This mixture was stirred at 50°C for 4 hours. After cooling to 25°C, ethyl acetate (30 mL) was added, and the organic and aqueous layers were separated. After extraction with ethyl acetate (30 mL) added to the aqueous layer, all the resulting organic layers were combined and washed twice with saturated aqueous sodium chloride (30 mL). The organic layer was dried over sodium sulfate, the solvent was evaporated under reduced pressure, and the mixture was further dried at 25°C for 30 minutes under reduced pressure using a pump. The resulting residue was dissolved in diethyl ether (50 mL), followed by the addition of heptane (50 mL). Diethyl ether was distilled off under controlled reduced pressure (~100 hPa), and the resulting mixture was filtered to obtain a solid. This heptane washing procedure was repeated twice. The resulting solid was dried under reduced pressure at 25°C for 2 hours to obtain the sodium salt of Fmoc-Thr(THP)-OH (2.80 g, 6.26 mmol). Ethyl acetate (50 mL) and 0.05 M aqueous phosphoric acid (pH 2.1) (140 mL) were added to the entire sodium salt of Fmoc-Thr(THP)-OH obtained, and the mixture was stirred at 25°C for 5 minutes. The organic and aqueous layers were then separated. Ethyl acetate (50 mL) was added to the aqueous layer for extraction, and all the resulting organic layers were combined and washed twice with saturated aqueous sodium chloride (50 mL). The organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was dried under reduced pressure at 25°C for 2 hours. The resulting solid was then dissolved in t-butyl methyl ether (TBME) (50 mL), and the solvent was evaporated under reduced pressure. Further drying at 25°C under reduced pressure with a pump for 1 hour afforded (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (compound aa01, Fmoc-Thr(THP)-OH, 2.70 g, 30 mol% t-butyl methyl ether (TBME) remaining) as a diastereomer derived from the asymmetric carbon on the THP protection. The resulting Fmoc-Thr(THP)-OH was stored in a -25°C freezer. LCMS(ESI)m / z=424.2(M−H)- Retention time: 0.84 minutes, 0.85 minutes (Analysis conditions SQDFA05)

[0197] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-butylglycine (compound aa02, Fmoc-nBuGly-OH) [ka] To a solution of sodium hydride (26 g, 1.08 mol) in tetrahydrofuran (THF) (500 mL), tert-butyl (tert-butoxycarbonyl)glycinate (Boc-Gly-OtBu) (100 g, 432.36 mmol) was added portionwise at room temperature. After stirring for 30 minutes, a solution of 1-iodobutane (239 g, 1.30 mol) in dimethylformamide (DMF) (50 mL) was added dropwise. The reaction mixture was stirred for 16 hours at room temperature, followed by the addition of water. The tetrahydrofuran (THF) was removed under reduced pressure, followed by extraction three times with ethyl acetate. The resulting organic solvent was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (petroleum ether / ethyl acetate) to quantitatively obtain tert-butyl N-(tert-butoxycarbonyl)-N-butylglycinate (Boc-nBuGly-OtBu) (130 g). To a 1,4-dioxane solution (1000 mL) of tert-butyl N-(tert-butoxycarbonyl)-N-butylglycinate (Boc-nBuGly-OtBu) (260 g, 904.68 mmol) obtained by the method described above, concentrated hydrochloric acid (1000 mL) was added dropwise at 0°C. The reaction mixture was warmed to room temperature and then stirred at room temperature for 16 hours. The reaction mixture was concentrated to obtain the hydrochloride salt of butylglycine (H-nBuGly-OH) (200 g) as a crude product. A mixture of the crude product, butylglycine (H-nBuGly-OH) (60 g), potassium carbonate (188.9 g, 1.37 mol), and water / 1,4-dioxane (1:1) (3000 mL) was stirred at room temperature for 30 minutes, and then N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (183.2 g, 543.09 mmol) was added portionwise at room temperature. The reaction mixture was stirred at room temperature for 16 hours and then washed three times with ether. 5M aqueous hydrochloric acid was added until the resulting aqueous layer reached pH 3, followed by extraction three times with ethyl acetate. The organic solvent was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting crude product was washed with ether to yield N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-butylglycine (compound aa02, Fmoc-nBuGly-OH) (121 g). LCMS(ESI)m / z=354 (M+H)+ Retention time: 0.87 minutes (Analysis conditions SQDFA05)

[0198] Fmoc-Ser(iPen)-OH (compound aa04) was synthesized according to the following scheme. [ka]

[0199] Synthesis of N-(tert-butoxycarbonyl)-O-(3-methylbut-2-en-1-yl)-L-serine (compound aa03) [ka] (tert-Butoxycarbonyl)-L-serine (Boc-Ser-OH) (50 g, 234.65 mmol) was dissolved in dimethylformamide (DMF) (250 mL) and added with sodium hydride (22 g, 916.67 mmol, 60% oil dispersion) under ice-cooling (0 °C). After stirring for 30 minutes under ice-cooling, 1-bromo-3-methylbut-2-ene (44 g, 295.24 mmol) was added dropwise. After the addition, the reaction mixture was stirred at 25 °C for 16 hours. The reaction was quenched by adding ice water, and the pH was adjusted to 2-3 with the addition of aqueous hydrochloric acid (5 M). The mixture was then extracted twice with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the ethyl acetate was removed by concentration under reduced pressure to obtain a crude product containing the desired compound. The crude product was purified by normal phase column chromatography (petroleum ether / ethyl acetate = 100 / 0 → 80 / 20) to obtain N-(tert-butoxycarbonyl)-O-(3-methylbut-2-en-1-yl)-L-serine (compound aa03) (32 g, 48%). LCMS(ESI) m / z = 296 (M+Na)+ Retention time: 1.39 minutes (Analysis conditions SMD method7)

[0200] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-L-serine (compound aa04, Fmoc-Ser(iPen)-OH) [ka] N-(tert-butoxycarbonyl)-O-(3-methylbut-2-en-1-yl)-L-serine (compound aa03) (185 g, 676.85 mmol), ammonia methanol solution (2 M, 555 mL), Pd / C (18.5 g), and methanol (1500 mL) were mixed and stirred under a hydrogen atmosphere (5 atm) at room temperature for 16 hours. The solid was removed by filtration, and the solvent was removed by concentration under reduced pressure to give N-(tert-butoxycarbonyl)-O-isopentyl-L-serine (Boc-Ser(iPen)-OH) (183 g). The resulting N-(tert-butoxycarbonyl)-O-isopentyl-L-serine (Boc-Ser(iPen)-OH) (175 g) was dissolved in 1,4-dioxane (500 mL), concentrated hydrochloric acid (300 mL) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product (122 g) was dissolved in 1,4-dioxane / water (1000 mL / 1000 mL). Potassium carbonate (239 g, 1.73 mol) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (234 g, 694.36 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was then washed three times with hexane. After washing, the pH of the aqueous layer was adjusted to 2-3 with 6 M aqueous hydrochloric acid, and the mixture was extracted twice with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The resulting crude product was washed with ether, hexane, and ethyl acetate, and further purified by reverse-phase column chromatography (water / acetonitrile = 100 / 0 → 30 / 70) to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-L-serine (compound aa04, Fmoc-Ser(iPen)-OH) (95 g, 41%) as a white solid. LCMS(ESI) m / z = 398 (M+H)+ Retention time: 2.29 minutes (Analysis conditions SMD method 18)

[0201] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound aa05, Fmoc-MeHph-OH) [ka] Under a nitrogen atmosphere, tosylic acid (TsOH) (2.575 g, 14.95 mmol) and paraformaldehyde (15.96 g, 488.77 mmol) were added to a toluene solution (1.5 L) of commercially available (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-phenylbutanoic acid (100 g, 244.11 mmol) at room temperature, and the mixture was stirred at 110 °C for 16 hours. Water was added to the organic layer, and the mixture was washed three times with water. The organic layer and the aqueous layer were extracted with ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 100 g of (9H-fluoren-9-yl)methyl (S)-5-oxo-4-phenethyloxazolidine-3-carboxylate as a crude product. To a dichloromethane solution (700 mL) of (S)-5-oxo-4-phenethyloxazolidine-3-carboxylate (9H-fluoren-9-yl) (50 g, 118.51 mmol) obtained as described above, triethylsilane (EtSiH) and trifluoroacetic acid (TFA) (700 mL) were added and the mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated under reduced pressure, and aqueous potassium carbonate solution was added to the resulting residue, followed by washing with petroleum ether. The resulting aqueous layer was adjusted to pH 3 with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Methanol and hexane were added to the resulting residue, and the mixture was concentrated under reduced pressure again to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (Compound aa05, Fmoc-MeHph-OH) (29.5 g, 58%, 2 steps). LCMS(ESI)m / z=416(M+H)+ Retention time: 0.95 minutes (Analysis conditions SQDFA05)

[0202] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa12, Fmoc-Ser(3-F-5-Me-Pyr)-OH) N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa12, Fmoc-Ser(3-F-5-Me-Pyr)-OH) was synthesized via the following route. [ka]

[0203] Synthesis of (5-fluoropyridin-3-yl)methanol (compound aa07) [ka] Under a nitrogen atmosphere, commercially available 5-fluoronicotinic acid (compound aa06) (14.1 g, 100 mmol) was added to THF (200 mL) and stirred using a mechanical stirrer. Triethylamine (19.51 mL, 140 mmol) was then added at room temperature and stirred until the solid was completely dissolved. The reaction mixture was then cooled in an ice bath. Ethyl chloroformate (11.5 mL, 120 mmol) was then added dropwise and stirred in an ice bath for 30 minutes. The reaction mixture was then cooled to below -60°C using a dry ice bath, and a THF solution of LiAlH (lithium aluminum hydride) (2.5 M, 40 mL, 100 mmol) was added dropwise over 5 minutes, ensuring that the reaction temperature did not exceed -20°C. The reaction mixture was stirred at -78°C for 3 hours, after which ethyl acetate (69 mL) was added so that the reaction temperature did not exceed -20°C. After stirring for another hour in an ice bath, water (18 mL) was added and the mixture was stirred for 15 minutes at room temperature. The reaction mixture was filtered through NH silica gel and concentrated under reduced pressure to give (5-fluoropyridin-3-yl)methanol (compound aa07) (9.28 g, 73%) as a yellow oil. LCMS(ESI)m / z=128(M+H) + Retention time: 0.28 minutes (Analysis conditions SQDFA05)

[0204] Synthesis of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa08) [ka] A round-bottom flask was connected to an acetone / dry ice-cooled condenser, and under a nitrogen atmosphere, (5-fluoropyridin-3-yl)methanol (compound aa07) (8.21 g, 64.6 mmol) and 25% HBr in acetic acid (96 mL, 388 mmol) were added. The reaction was carried out in an open system, and 1 M aqueous sodium hydroxide was used to trap the resulting HBr. The reaction mixture was gradually heated from room temperature to 100 °C with stirring and stirred for an additional 3 h. The reaction mixture was then cooled to room temperature, and diisopropyl ether (48 mL) was slowly added three times. The resulting solid was filtered to give 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa08) (12.43 g, 71%) as a gray solid. 1 H NMR (Varian400-MR, 400MHz, d-DMSO) δ4.77 (2H, s), 7.85-7.88 (1H, m), 8.55-8.56 (2H, m)

[0205] Synthesis of O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa10, Trt-Ser(3-F-5-Me-Pyr)-OH) [ka] Under a nitrogen atmosphere, sodium t-pentoxide (NaOtPen) (13.7 g, 125 mmol) was added to THF (45.8 mL) and stirred. Then, commercially available triethylamine salt of tritylserine (Trt-Ser-OH triethylamine salt) (compound aa09) (11.24 g, 25 mmol) was added in three portions and stirred for 30 minutes at room temperature. The reaction mixture was stirred for 15 minutes in an ice bath, cooled, and then a DMF solution (16 mL) of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa08) (8.13 g, 30 mmol) was added dropwise, followed by the addition of DMF (14 mL). After stirring the reaction mixture in an ice bath for 45 minutes, a DMF solution (4.0 mL) of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa08) (2.03 g, 7.5 mmol) was added dropwise, followed by the addition of DMF (4.0 mL). The reaction mixture was then stirred at room temperature for an additional hour, and water (125 mL) was added. The resulting mixture was washed with t-butyl methyl ether (TBME), and the organic layer was extracted with water. The resulting aqueous layers were combined, adjusted to pH 7 with saturated aqueous sodium dihydrogen phosphate (15 mL), and then extracted twice with ethyl acetate. The resulting organic layers were combined and washed three times with a solution of saturated brine diluted two-fold, followed by two times with saturated brine. The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa10, Trt-Ser(3-F-5-Me-Pyr)-OH) (11.07 g, 97%) as a yellow amorphous substance. LCMS(ESI)m / z=455(M−H)- Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0206] Synthesis of O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa11, H-Ser(3-F-5-Me-Pyr)-OH) [ka] To a 1.4-dioxane solution (21.5 mL) of O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa10, Trt-Ser(3-F-5-Me-Pyr)-OH) (10.76 g, 23.57 mmol) was added 5-10% hydrochloric acid / methanol solution (64.2 mL) at room temperature. The reaction mixture was stirred at room temperature for 10 to 20 minutes, and then 1.4-dioxane (135 mL) was added. An additional 1.4-dioxane (90 mL) was added, followed by the addition of a small amount of seed crystals (5 mg) prepared in advance, as shown below, and the mixture was further stirred at room temperature for 30 minutes. The resulting solid was filtered, washed four times with diisopropyl ether (50 mL), and dried under reduced pressure to give O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa11, H-Ser(3-F-5-Me-Pyr)-OH)) (6.58 g, 95%) as the hydrochloride salt. LCMS(ESI)m / z=213(M−H)- Retention time: 0.24 minutes (Analysis conditions SQDAA05)

[0207] Preparation of seed crystals of O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa11, H-Ser(3-F-5-Me-Pyr)-OH) A 1.4-dioxane solution (819 μL) of O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa10, Trt-Ser(3-F-5-Me-Pyr)-OH) (98 mg, 2.69 mmol) was added to 5-10% hydrochloric acid / methanol solution (2.45 mL) at room temperature and stirred for 5 hours. 1.4-Dioxane (6.0 mL) was added to the reaction mixture, and the resulting solid was filtered, washed with diisopropyl ether, and dried under reduced pressure to give O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa11, H-Ser(3-F-5-Me-Pyr)-OH)) (222.5 mg, 86%) as the hydrochloride salt. LCMS(ESI)m / z=213(M−H)- Retention time: 0.24 minutes (Analysis conditions SQDAA05)

[0208] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa12, Fmoc-Ser(3-F-5-Me-Pyr)-OH) [ka] O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa11, H-Ser(3-F-5-Me-Pyr)-OH) hydrochloride (6.37 g, 22.19 mmol) was added to water (42 mL), 1,4-dioxane (115 mL), and diisopropylethylamine (DIPEA) (13.53 mL, 78 mmol) at room temperature and stirred. N-succinimidyl 9-fluorenylmethyl carbonate (Fmoc-OSu) (7.86 g, 23.3 mmol) was then added to the reaction mixture at room temperature and stirred. After confirming the disappearance of the starting material by LC-MS, water (56.2 mL) was added to the reaction mixture at room temperature and washed twice with 25% tert-butyl methyl ether (MTBE) / hexane solution. The resulting aqueous layer was adjusted to pH 6.1 using saturated aqueous sodium dihydrogen phosphate (NaH2PO4). The resulting organic layers were then extracted twice with ethyl acetate, washed with a solution of saturated saline diluted twice, and saturated saline. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa12, Fmoc-Ser(3-F-5-Me-Pyr)-OH) (9.47 g, 98%) as a yellow solid. LCMS(ESI)m / z=437(M+H)+ Retention time: 0.86 minutes (Analysis conditions SQDAA05)

[0209] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa16, Fmoc-Ser(Ph-3-Cl)-OH) (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa16, Fmoc-Ser(Ph-3-Cl)—OH) was synthesized via the following route. [ka]

[0210] Synthesis of (S)-methyl 3-(3-chlorophenoxy)-2-(tritylamino)propanoate (Trt-Ser(Ph-3-Cl)-OMe) (compound aa14) [ka] Under a nitrogen atmosphere, a 2.2 M toluene solution of diethyl azocarboxylate (DEAD) (15.06 mL, 33.2 mmol) was added to a toluene solution (35 mL) of commercially available (S)-methyl 3-hydroxy-2-(tritylamino)propanoate (compound aa13, Trt-Ser-OMe) (6.0 g, 16.6 mmol) and triphenylphosphine (PPh3) (8.71 g, 33.2 mmol) at room temperature, and the reaction mixture was stirred at 50 °C for 30 min. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase silica gel column chromatography (10 mM aqueous ammonium acetate / methanol) to give (S)-methyl 3-(3-chlorophenoxy)-2-(tritylamino)propanoate (Trt-Ser(Ph-3-Cl)-OMe) (compound aa14) (4.43 g, 57%). 1 H NMR (Varian400-MR, 400MHz, d-DMSO) δ7.42-7.44(6H, m), 7.28-7.30(7H, m), 7.20-7.21(3H, m), 6.99- 7.01 (2H, m), 6.83-6.85 (1H, m), 4.19-4.22 (1H, m), 4.06-4.08 (1H, m), 3.49-3.51 (1H, m), 3.17 (3H, s)

[0211] Synthesis of (S)-2-amino-3-(3-chlorophenoxy)propanoic acid (compound aa15, H-Ser(Ph-3-Cl)-OH) [ka] To a solution (80 mL) of (S)-methyl 3-(3-chlorophenoxy)-2-(tritylamino)propanoate (Trt-Ser(Ph-3-Cl)-OMe) (Compound aa14) (3.9 g, 8.26 mmol) in 1,4-dioxane, 1.0 M lithium hydroxide / methanol solution (83 mL) was added at room temperature, and the reaction mixture was stirred at 50°C for 3 hours. The reaction mixture was concentrated under reduced pressure, and trifluoroacetic acid (TFA) (30 mL) was added to the resulting residue, followed by stirring at 50°C for 10 minutes. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile) to give (S)-2-amino-3-(3-chlorophenoxy)propanoic acid (Compound aa15, H-Ser(Ph-3-Cl)-OH) (850 mg, 48%, 2 steps). LCMS(ESI)m / z=216(M+H)+ Retention time: 0.37 minutes (Analysis conditions SQDFA05)

[0212] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa16, Fmoc-Ser(Ph-3-Cl)-OH) [ka] (S)-2-Amino-3-(3-chlorophenoxy)propanoic acid (compound aa15, H-Ser(Ph-3-Cl)-OH) (850 mg, 3.94 mmol) and N-succinimidyl 9-fluorenylmethyl carbonate (Fmoc-OSu) (1.33 g, 3.94 mmol) were added to 1,4-dioxane (20 mL) and water (20 mL) at room temperature. Cesium carbonate (2.569 g, 7.88 mmol) was then added and the mixture was stirred at room temperature. After confirming the disappearance of the starting material by LC-MS, water (20 mL) was added and the mixture was washed twice with diethyl ether (40 mL). The resulting aqueous layer was adjusted to pH 2 with 5N aqueous hydrochloric acid and extracted twice with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile) to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (Compound aa16, Fmoc-Ser(Ph-3-Cl)—OH) (1.42 g, 82%). LCMS(ESI)m / z=438(M+H)+ Retention time: 0.91 minutes (Analysis conditions SQDFA05)

[0213] Synthesis of (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tetrahydro-2H-pyran-2-yl)oxy)hexanoic acid (compound aa17, Fmoc-Nle(6-OTHP)-OH) [ka] A solution (10 mL) of commercially available (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-hydroxyhexanoic acid (Fmoc-Nle(6-OH)-OH) (3 g, 8.12 mmol) and pyridinium p-toluenesulfonate (PPTS) (408 mg, 1.62 mmol) in toluene was concentrated under reduced pressure and subjected to azeotropic dehydration. The resulting residue was dissolved in tetrahydrofuran (15 mL), and dihydropyran (5.41 mL, 56.8 mmol) was added, followed by stirring at 50°C for 1 hour. Ethyl acetate was added to the reaction mixture at room temperature, and the organic layer was washed twice with saturated brine. The resulting organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (30 mL), and phosphate buffer (30 mL) adjusted to pH 8 was added, followed by stirring at 50°C for 3 hours. The reaction mixture was extracted twice with ethyl acetate at room temperature. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was dissolved in diethyl ether (50 mL), and heptane (50 mL) was added to precipitate the crude product. The diethyl ether was removed by concentration under reduced pressure, and the remaining supernatant solution was decanted. This process was repeated twice to obtain the crude product. The resulting crude product was dissolved in tert-butyl methyl ether (150 mL), and 0.05 M aqueous phosphoric acid (150 mL) was added. The mixture was stirred at room temperature for 1 hour. The organic layer was collected, and the aqueous layer was extracted with ethyl acetate. The obtained organic layers were combined and then washed twice with saturated brine. The obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tetrahydro-2H-pyran-2-yl)oxy)hexanoic acid (Compound aa17, Fmoc-Nle(6-OTHP)-OH) (2.925 g, 79%). LCMS(ESI)m / z=454 (M+H)+ Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0214] Synthesis of methyl N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serinate (compound aa18, Cbz-Ser(EtOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (Cbz-Azy-OMe) (25.0 g, 106 mmol) and 2-(benzyloxy)ethan-1-ol (23.8 g, 156 mmol) were dissolved in dichloromethane (100 mL) and cooled to 0°C. Boron trifluoride diethyl ether complex (2.00 mL, 15.9 mmol) was added and the mixture was stirred at 0°C for 1 hour. Water was added to the reaction mixture, which was then extracted twice with dichloromethane. The organic layer was then washed with aqueous sodium carbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to obtain methyl N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serinate (compound aa18, Cbz-Ser(EtOBn)-OMe) (30.0 g, 73%). LCMS(ESI) m / z = 388 (M+H)+ Retention time: 1.13 minutes (Analysis conditions SMD method 9)

[0215] Synthesis of N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serine (compound aa19, Cbz-Ser(EtOBn)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide monohydrate (13.9 g, 331 mmol) and calcium chloride (129 g, 1.24 mol) were dissolved in water (321 mL). A 2-propanol / tetrahydrofuran solution (1.28 L / 321 mL) of methyl N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serinate (Compound aa18, Cbz-Ser(EtOBn)-OMe) (30.0 g, 77.4 mmol) was added at room temperature and stirred for 3 hours. A 2M aqueous solution of hydrochloric acid was added until the pH reached 2. The organic layer was removed, and the aqueous layer was extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serine (Compound aa19, Cbz-Ser(EtOBn)-OH) (30.0 g) as a crude product, which was used in the next step without purification.

[0216] Synthesis of O-(2-hydroxyethyl)-L-serine (compound aa20, H-Ser(EtOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serine (Compound aa19, Cbz-Ser(EtOBn)-OH) (34.0 g, 91.1 mmol) and palladium on carbon (6.80 g, 20% w / w) were dissolved in methanol (500 mL) and stirred at room temperature for 16 hours. After filtering the reaction solution, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain O-(2-hydroxyethyl)-L-serine (Compound aa20, H-Ser(EtOH)-OH) (10.7 g) as a crude product. This was used in the next step without further purification.

[0217] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-L-serine (compound aa21, Fmoc-Ser(EtOH)-OH) [ka] Under a nitrogen atmosphere, O-(2-hydroxyethyl)-L-serine (compound aa20, H-Ser(EtOH)-OH) (5.00 g, 33.5 mmol), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (12.4 g, 36.9 mmol), and sodium carbonate (10.6 g, 100 mmol) were dissolved in water / 1,4-dioxane (136 mL / 56.0 mL) and stirred at room temperature for 3 h. The reaction mixture was washed three times with t-butyl methyl ether. 2M aqueous hydrochloric acid was added to the aqueous layer until the pH reached 2, and the mixture was extracted three times with ethyl acetate. The resulting organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was then further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-L-serine (compound aa21, Fmoc-Ser(EtOH)-OH) (12.0 g, 96%). LCMS(ESI) m / z = 372 (M+H)+ Retention time: 1.31 minutes (Analysis conditions SMD method33)

[0218] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-L-serine (compound aa22, Fmoc-Ser(EtOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (PPTS) (54.1 mg, 0.215 mmol) was added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-L-serine (compound aa21, Fmoc-Ser(EtOH)-OH) (1.6 g, 4.31 mmol) and 3,4-dihydro-2H-pyran (2.728 mL, 30.3 mmol) in tetrahydrofuran (8.616 mL), and the mixture was stirred at 50°C for 2 hours. After cooling the mixture, ethyl acetate was added. The organic layer was then washed three times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (10 mL) and then 0.05 M phosphate buffer (100 mL) (prepared by mixing 1 M aqueous sodium dihydrogen phosphate (NaH2PO4) (94.3 mL) and 1 M aqueous sodium dihydrogen phosphate (Na2HPO4) (5.7 mL)) was added. The mixture was stirred at room temperature for 10 minutes, then extracted with t-butyl methyl ether. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-L-serine (compound aa22, Fmoc-Ser(EtOTHP)-OH) (1.75 g, 96%). LCMS(ESI)m / z=456 (M+H)+ Retention time: 0.81 minutes (Analysis conditions SQDFA05)

[0219] Synthesis of (S)-benzyl 4-((2-(benzyloxy)ethoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa23) [ka] N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-L-serine (Compound aa19, Cbz-Ser(EtOBn)-OH) (30.0 g, 80.3 mmol), paraformaldehyde (7.20 g), and paratoluenesulfonic acid (0.83 g, 4.82 mmol) were dissolved in toluene (300 mL) and stirred at 110 °C for 16 h. After cooling to room temperature and washing with aqueous sodium bicarbonate, the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain crude (S)-benzyl 4-((2-(benzyloxy)ethoxy)methyl)-5-oxooxazolidine-3-carboxylate (Compound aa23) (27.0 g). This was used in the next step without further purification.

[0220] Synthesis of N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-N-methyl-L-serine (compound aa24, Cbz-MeSer(EtOBn)-OH) [ka] Under a nitrogen atmosphere, triethylsilane (24.4 g, 210 mmol) and trifluoroacetic acid (450 mL) were added to a dichloromethane solution (450 mL) of (S)-4-((2-(benzyloxy)ethoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa23) (27.0 g, 70.1 mmol), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was evaporated under reduced pressure and further dried under reduced pressure using a vacuum pump. The residue was then redissolved in aqueous potassium carbonate, washed with diethyl ether, and concentrated hydrochloric acid was added until the pH reached 2, followed by extraction twice with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-N-methyl-L-serine (compound aa24, Cbz-MeSer(EtOBn)-OH) (24.0 g) as a crude product, which was used in the next step without further purification.

[0221] Synthesis of O-(2-hydroxyethyl)-N-methyl-L-serine (compound aa25, H-MeSer(EtOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-(2-(benzyloxy)ethyl)-N-methyl-L-serine (Compound aa24, Cbz-MeSer(EtOBn)-OH) (24.0 g, 62.0 mmol) and palladium on carbon (2.40 g, 10% w / w) were dissolved in methanol (400 mL) and stirred at room temperature for 16 hours. After filtering the reaction mixture, the solvent was evaporated under reduced pressure, and the residue was dried under reduced pressure using a vacuum pump to obtain crude O-(2-hydroxyethyl)-N-methyl-L-serine (Compound aa25, H-MeSer(EtOH)-OH) (12.0 g). This was used in the next step without further purification.

[0222] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-N-methyl-L-serine (compound aa26, Fmoc-MeSer(EtOH)-OH) [ka] Under a nitrogen atmosphere, O-(2-hydroxyethyl)-N-methyl-L-serine (compound aa25, H-MeSer(EtOH)-OH) (12 g, 73.54 mmol), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (27.3 g, 81.0 mmol), and sodium carbonate (27.3 g, 258 mmol) were dissolved in water / 1,4-dioxane (320 mL / 160 mL) and stirred at room temperature for 3 hours. The reaction mixture was washed three times with t-butyl methyl ether, and then 2M aqueous hydrochloric acid was added to the aqueous layer until the pH reached 2. The mixture was extracted three times with ethyl acetate. The resulting organic layers were mixed, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was further dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-N-methyl-L-serine (compound aa26, Fmoc-MeSer(EtOH)-OH) (20.0 g) as a crude product, which was used in the next step without purification.

[0223] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-L-serine (compound aa27, Fmoc-MeSer(EtOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (0.59 g, 2.34 mmol) and 3,4-dihydro-2H-pyran (27.5 g, 327 mmol) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxyethyl)-N-methyl-L-serine (compound aa26, Fmoc-MeSer(EtOH)-OH) (18.0 g, 46.7 mmol) in tetrahydrofuran (100 mL), and the mixture was stirred at 50 °C for 3 hours. The mixture was cooled to 25 °C, and ethyl acetate was added. The organic layer was then washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. 25.0 g of the resulting residue (31 g) was dissolved in tetrahydrofuran (200 mL), and then 1.0 M phosphate buffer (200 mL) adjusted to pH 6.8 was added. The mixture was stirred at 50°C for 3 hours. After cooling to 25°C, ethyl acetate was added, and the organic and aqueous layers were separated. Ethyl acetate was added to the aqueous layer for extraction, and then all the resulting organic layers were mixed and washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-L-serine (compound aa27, Fmoc-MeSer(EtOTHP)-OH) (20.0 g). LCMS(ESI)m / z = 487 (M+NH4)+ Retention time: 0.68 minutes (Analysis conditions SMD method11)

[0224] Synthesis of (4S)-4-methyl-2-phenyl-1,3-dioxolane (compound aa28) [ka] Under a nitrogen atmosphere, benzaldehyde (29.3 g, 276 mmol) and p-toluenesulfonic acid (0.45 g, 2.61 mmol) were added to a solution of (S)-propane-1,2-diol (20.0 g, 263 mmol) in toluene (200 mL), and the mixture was stirred at 130°C for 16 hours. The reaction mixture was concentrated, and the residue was purified by normal-phase column chromatography (hexane / ethyl acetate) to give (4S)-4-methyl-2-phenyl-1,3-dioxolane (compound aa28) (31.5 g, 73%). LCMS(ESI) m / z = 165 (M+H)+ Retention time: 0.89 minutes (Analysis conditions SMD method 12)

[0225] Synthesis of (2S)-2-(benzyloxy)propan-1-ol (compound aa29) [ka] Under a nitrogen atmosphere, a 1M hexane solution of diisobutylaluminum hydride (390 mL, 390 mmol) was added to a dichloromethane (1.00 L) solution of (4S)-4-methyl-2-phenyl-1,3-dioxolane (Compound aa28) (32 g, 194.88 mmol) at -50°C. The mixture was stirred at this temperature for 15 minutes and then at room temperature for 2 hours. The reaction was terminated by the addition of saturated aqueous ammonium chloride, followed by extraction twice with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was then further dried under reduced pressure using a vacuum pump to obtain (2S)-2-(benzyloxy)propan-1-ol (Compound aa29) (35.5 g) as a crude product. This was used in the next step without further purification.

[0226] Synthesis of methyl N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serinate (compound aa30, Cbz-Ser(S-2-PrOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (Cbz-Azy-OMe) (25.0 g, 106 mmol) and (2S)-2-(benzyloxy)propan-1-ol (compound aa29) (26.5 g, 159 mmol) were dissolved in dichloromethane (188 mL) and cooled to 0°C. Boron trifluoride diethyl ether complex (2.75 mL) was added and the mixture was stirred at 0°C for 1 hour and 30 minutes. Water was added to the reaction mixture, which was then extracted with dichloromethane. The organic layer was then washed with saturated aqueous sodium carbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain methyl N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serinate (compound aa30, Cbz-Ser(S-2-PrOBn)-OMe) (30.0 g) as a crude product, which was used in the next step without purification.

[0227] Synthesis of N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serine (compound aa31, Cbz-Ser(S-2-PrOBn)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide monohydrate (10.5 g) and calcium chloride (103 g) were dissolved in water (300 mL). A 2-propanol / tetrahydrofuran solution (1.20 L / 300 mL) of methyl N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serinate (compound aa30, Cbz-Ser(S-2-PrOBn)-OMe) (25 g, 62.3 mmol) was added at room temperature and stirred for 5 hours. 2M aqueous hydrochloric acid was added until the pH reached 2. The organic layer was removed, and the aqueous layer was extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serine (Compound aa31, Cbz-Ser(S-2-PrOBn)-OH) (25.0 g) as a crude product, which was used in the next step without further purification.

[0228] Synthesis of O-((S)-2-hydroxypropyl)-L-serine (compound aa32, H-Ser(S-2-PrOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serine (Compound aa31, Cbz-Ser(S-2-PrOBn)-OH) (3.00 g, 7.74 mmol) and palladium on carbon (0.30 g, 10% w / w) were dissolved in methanol (50.0 mL) and stirred at room temperature for 16 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump to give O-((S)-2-hydroxypropyl)-L-serine (Compound aa32, H-Ser(S-2-PrOH)-OH) (0.78 g) as a crude product. This was used in the next step without further purification.

[0229] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-L-serine (compound aa33, Fmoc-Ser(S-2-PrOH)-OH) [ka] Under a nitrogen atmosphere, O-((S)-2-hydroxypropyl)-L-serine (compound aa32, H-Ser(S-2-PrOH)-OH) (0.78 g, 4.78 mmol), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (1.61 g, 4.78 mmol), and N-ethyl-isopropylpropan-2-amine (DIPEA) (0.93 mg, 7.17 mmol) were dissolved in water / 1,4-dioxane (8.00 mL / 22.0 mL) and stirred at room temperature for 30 minutes. The reaction mixture was washed twice with hexane (18.0 mL) and t-butyl methyl ether (6.00 mL). The aqueous layer was then diluted with 2M hydrochloric acid until the pH reached 2, and extracted twice with ethyl acetate (20.0 mL). The resulting organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (0.1% formic acid in water / 0.1% formic acid in acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-L-serine (compound aa33, Fmoc-Ser(S-2-PrOH)-OH) (1.62 g, 88%). LCMS(ESI) m / z = 386 (M+H)+ Retention time: 0.68 minutes (Analysis conditions SQDFA05)

[0230] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2S)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa34, Fmoc-Ser(S-2-PrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (2.71 mg, 0.01 mmol) and 3,4-dihydro-2H-pyran (0.13 g, 1.51 mmol) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-L-serine (compound aa33, Fmoc-Ser(S-2-PrOH)-OH) (83 mg, 0.22 mmol) in tetrahydrofuran (1.00 mL), and the mixture was stirred at 50°C for 2 hours. The mixture was cooled to 25°C, and ethyl acetate was added. The organic layer was then washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (1.00 mL), and then 1.0 M phosphate buffer (1.00 mL) adjusted to pH 6.8 was added. This mixture was stirred at 50°C for 3 hours. After cooling to 25°C, ethyl acetate (175 mL) was added, and the organic and aqueous layers were separated. Ethyl acetate (175 mL) was added to the aqueous layer for extraction, and then all the resulting organic layers were combined and washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2S)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa34, Fmoc-Ser(S-2-PrOTHP)-OH) (88.0 mg, 87%). LCMS(ESI) m / z = 468 (M−H)- Retention time: 0.86 minutes, 0.87 minutes (Analysis conditions SQDFA05)

[0231] Synthesis of (S)-4-(((S)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate benzyl (compound aa35) [ka] N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-L-serine (compound aa31, Cbz-Ser(S-2-PrOBn)-OH) (25.0 g, 64.53 mmol), paraformaldehyde (5.80 g), and paratoluenesulfonic acid (0.67 g, 3.89 mmol) were dissolved in toluene (250 mL) and stirred at 110 °C for 16 h. After cooling to room temperature and washing with aqueous sodium bicarbonate, the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump to obtain crude (S)-4-(((S)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa35) (18.6 g). This was used in the next step without further purification.

[0232] Synthesis of N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-N-methyl-L-serine (compound aa36, Cbz-MeSer(S-2-PrOBn)-OH) [ka] Under a nitrogen atmosphere, triethylsilane (16.2 g, 139 mmol) and trifluoroacetic acid (296 mL) were added to a dichloromethane solution (296 mL) of benzyl (S)-4-(((S)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa35) (18.6 g, 46.6 mmol), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was evaporated under reduced pressure and further dried under reduced pressure using a vacuum pump. The residue was then redissolved in aqueous potassium carbonate, washed with diethyl ether, and concentrated hydrochloric acid was added until the pH reached 2. The mixture was then extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-N-methyl-L-serine (Compound aa36, Cbz-MeSer(S-2-PrOBn)-OH) (9.4 g) as a crude product. This was used in the next step without further purification.

[0233] Synthesis of O-((S)-2-hydroxypropyl)-N-methyl-L-serine (compound aa37, H-MeSer(S-2-PrOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-((S)-2-(benzyloxy)propyl)-N-methyl-L-serine (Compound aa36, Cbz-MeSer(S-2-PrOBn)-OH) (9.40 g, 23.4 mmol) and palladium on carbon (1.80 g, 10% w / w) were dissolved in methanol (185 mL) and stirred at room temperature for 16 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump to give O-((S)-2-hydroxypropyl)-N-methyl-L-serine (Compound aa37, H-MeSer(S-2-PrOH)-OH) (3.40 g) as a crude product. This was used in the next step without further purification.

[0234] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-N-methyl-L-serine (compound aa38, Fmoc-MeSer(S-2-PrOH)-OH) [ka] Under a nitrogen atmosphere, O-((S)-2-hydroxypropyl)-N-methyl-L-serine (Compound aa37, H-MeSer(S-2-PrOH)-OH) (3.40 g, 19.2 mmol), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (7.12 g, 22.1 mmol), and sodium carbonate (7.13 g, 67.3 mmol) were dissolved in water / 1,4-dioxane (80.0 mL / 40.0 mL) and stirred at room temperature for 3 hours. The reaction mixture was washed three times with t-butyl methyl ether, and then 2 M aqueous hydrochloric acid was added to the aqueous layer until the pH reached 2. The mixture was extracted three times with ethyl acetate. The resulting organic layers were combined and washed with saturated brine. The organic layers were then dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was then further dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-N-methyl-L-serine (compound aa38, Fmoc-MeSer(S-2-PrOH)-OH) (8.00 g) as a crude product, which was used in the next step without further purification.

[0235] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-((2S)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa39, Fmoc-MeSer(S-2-PrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (0.26 g, 1.02 mmol) and 3,4-dihydro-2H-pyran (11.8 g, 140 mmol) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-2-hydroxypropyl)-N-methyl-L-serine (compound aa38, Fmoc-MeSer(S-2-PrOH)-OH) (80.0 g, 20.0 mmol) in tetrahydrofuran (40.0 mL), and the mixture was stirred at 50°C for 2 hours. The mixture was cooled to 25°C, and ethyl acetate was added. The organic layer was then washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue (9.30 g) was dissolved in tetrahydrofuran (50.0 mL), and then 1.0 M phosphate buffer (50.0 mL) adjusted to pH 6.8 was added. The mixture was stirred at 50°C for 3 hours. After cooling to 25°C, ethyl acetate was added, and the organic and aqueous layers were separated. Ethyl acetate was added to the aqueous layer for extraction, and then all the resulting organic layers were combined. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (0.5% aqueous ammonium bicarbonate / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-((2S)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa39, Fmoc-MeSer(S-2-PrOTHP)-OH) (7.00 g, 72%). LCMS(ESI) m / z = 501 (M+NH4)+ Retention time: 0.82 minutes (Analysis conditions SMD method 10)

[0236] Synthesis of (4R)-4-methyl-2-phenyl-1,3-dioxolane (compound aa40) [ka] Under a nitrogen atmosphere, benzaldehyde (29.3 g, 276 mmol) and p-toluenesulfonic acid (0.45 g, 2.61 mmol) were added to a solution of (R)-propane-1,2-diol (20 g, 262.83 mmol) in toluene (200 mL), and the mixture was stirred at 130°C for 16 hours. The reaction mixture was concentrated, and the residue was purified by normal-phase column chromatography (hexane / ethyl acetate) to give (4R)-4-methyl-2-phenyl-1,3-dioxolane (compound aa40) (31.5 g, 73%). LCMS(ESI) m / z = 163 (M−H)- Retention time: 7.21 minutes (Analysis conditions GC01)

[0237] Synthesis of (2R)-2-(benzyloxy)propan-1-ol (compound aa41) [ka] Under a nitrogen atmosphere, a 1M hexane solution of diisobutylaluminum hydride (390 mL, 390 mmol) was added to a dichloromethane (1.00 L) solution of (4R)-4-methyl-2-phenyl-1,3-dioxolane (Compound aa40) (32.0 g, 195 mmol) at -50°C. The mixture was stirred at this temperature for 15 minutes and then at room temperature for 2 hours. The reaction was terminated by the addition of saturated aqueous ammonium chloride, followed by extraction twice with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The mixture was then further dried under reduced pressure using a vacuum pump to obtain (2R)-2-(benzyloxy)propan-1-ol (Compound aa41) (35.5 g) as a crude product. This was used in the next step without further purification.

[0238] Synthesis of methyl N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serinate (compound aa42, Cbz-Ser(R-2-PrOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (Cbz-Azy-OMe) (25 g, 106.28 mmol) and (2R)-2-(benzyloxy)propan-1-ol (compound aa41) (26.5 g, 159 mmol) were dissolved in dichloromethane (188 mL) and cooled to 0°C. Then, boron trifluoride diethyl ether complex (2.75 mL) was added and the mixture was stirred at 0°C for 1 hour and 30 minutes. Water was added to the reaction mixture, which was then extracted with dichloromethane. The organic layer was then washed with saturated aqueous sodium carbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain methyl N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serinate (compound aa42, Cbz-Ser(R-2-PrOBn)-OMe) (50.0 g) as a crude product, which was used in the next step without purification.

[0239] Synthesis of N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serine (compound aa43, Cbz-Ser(R-2-PrOBn)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide monohydrate (10.5 g) and calcium chloride (103 g) were dissolved in water (300 mL). A 2-propanol / tetrahydrofuran solution (1.50 L / 150 mL) of methyl N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serinate (compound aa42, Cbz-Ser(R-2-PrOBn)-OMe) (25.0 g, 62.3 mmol) was added at room temperature and stirred for 5 hours. 2M aqueous hydrochloric acid was added until the pH reached 2. The organic layer was removed, and the aqueous layer was extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serine (Compound aa43, Cbz-Ser(R-2-PrOBn)-OH) (25.0 g) as a crude product, which was used in the next step without further purification.

[0240] Synthesis of O-((R)-2-hydroxypropyl)-L-serine (compound aa44, H-Ser(R-2-PrOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serine (compound aa43, Cbz-Ser(R-2-PrOBn)-OH) (2.87 g, 7.41 mmol) and palladium on carbon (570 mg, 20% w / w) were dissolved in methanol (50 mL) and stirred at room temperature for 16 hours. After filtering the reaction mixture, the solvent was evaporated under reduced pressure. The resulting residue was recrystallized from ethyl acetate at 60 °C to give O-((R)-2-hydroxypropyl)-L-serine (compound aa44, H-Ser(R-2-PrOH)-OH) (900 mg, 74%).

[0241] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-L-serine (compound aa45, Fmoc-Ser(R-2-PrOH)-OH) [ka] O-((R)-2-hydroxypropyl)-L-serine (compound aa44, H-Ser(R-2-PrOH)-OH) (5.0 g, 30.64 mmol), synthesized by the method described above, and sodium bicarbonate (7.71 g, 91.8 mmol) were dissolved in water / 1,4-dioxane (127 mL / 52.9 mL). N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (10.85 g, 32.2 mmol) was added at room temperature and stirred for 4 hours under a nitrogen atmosphere. Water was added to the reaction solution, and the mixture was washed with t-butyl methyl ether. After adding concentrated hydrochloric acid to the aqueous layer until the pH reached 3, the mixture was extracted twice with t-butyl methyl ether. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-L-serine (compound aa45, Fmoc-Ser(R-2-PrOH)-OH) (8 g, 68%). LCMS(ESI) m / z = 386 (M+H)+ Retention time: 2.08 minutes (Analysis conditions SMD method49)

[0242] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2R)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa46, Fmoc-Ser(R-2-PrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (143 mg, 5.7 mmol) and 3,4-dihydro-2H-pyran (6.7 g, 79.8 mmol) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-L-serine (compound aa45, Fmoc-Ser(R-2-PrOH)-OH) (4.4 g, 11.4 mmol) in tetrahydrofuran (50 mL), and the mixture was stirred at 50°C for 3 hours. The mixture was cooled to room temperature, and t-butyl methyl ether was added, followed by washing with saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered. The resulting solvent was evaporated under reduced pressure to give the crude product (8 g). A mixture of tetrahydrofuran (10 mL) and 1.0 M phosphate buffer (10 mL) adjusted to pH 6.8 was added to the obtained crude product (800 mg) and stirred at 50°C for 3 hours. The reaction mixture was cooled with room temperature salt, and then t-butyl methyl ether was added. The mixture was washed with saturated brine and filtered. The resulting solution was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (0.5% aqueous ammonium bicarbonate / acetonitrile). The resulting fractions were combined and the acetonitrile was evaporated under reduced pressure. To the resulting aqueous layer was added 1.0 M phosphate buffer adjusted to pH 6.8, followed by extraction three times with t-butyl methyl ether. The solvent was then evaporated under reduced pressure to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2R)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa46, Fmoc-Ser(R-2-PrOTHP)-OH) (400 mg, 65% yield over two steps). LCMS(ESI) m / z = 468 (M−H)- Retention time: 0.85 minutes, 0.87 minutes (Analysis conditions SQDFA05)

[0243] Synthesis of (S)-4-(((R)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate benzyl (compound aa47) [ka] N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-L-serine (compound aa43, Cbz-Ser(R-2-PrOBn)-OH) (25.0 g, 64.5 mmol), paraformaldehyde (5.80 g), and paratoluenesulfonic acid (0.67 g, 3.89 mmol) were dissolved in toluene (250 mL) and stirred at 110 °C for 16 h. After cooling to room temperature and washing with aqueous sodium bicarbonate, the organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump to give crude (S)-4-(((R)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa47) (18.6 g). This was used in the next step without further purification.

[0244] Synthesis of N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-N-methyl-L-serine (compound aa48, Cbz-MeSer(R-2-PrOBn)-OH) [ka] Under a nitrogen atmosphere, triethylsilane (16.2 g, 139 mmol) and trifluoroacetic acid (296 mL) were added to a dichloromethane solution (296 mL) of benzyl (S)-4-(((R)-2-(benzyloxy)propoxy)methyl)-5-oxooxazolidine-3-carboxylate (Compound aa47) (18.6 g, 46.6 mmol), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was evaporated under reduced pressure and further dried under reduced pressure using a vacuum pump. The residue was then redissolved in aqueous potassium carbonate, washed with diethyl ether, and concentrated hydrochloric acid was added until the pH reached 2. The mixture was then extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-N-methyl-L-serine (Compound aa48, Cbz-MeSer(R-2-PrOBn)-OH) (9.40 g) as a crude product, which was used in the next step without further purification.

[0245] Synthesis of O-((R)-2-hydroxypropyl)-N-methyl-L-serine (compound aa49, H-MeSer(R-2-PrOH)-OH) [ka] Under a hydrogen atmosphere, N-((benzyloxy)carbonyl)-O-((R)-2-(benzyloxy)propyl)-N-methyl-L-serine (compound aa48, Cbz-MeSer(R-2-PrOBn)-OH) (9.40 g, 23.4 mmol) and palladium on carbon (1.80 g, 10% w / w) were dissolved in methanol (185 mL) and stirred at room temperature for 16 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure. The residue was further dried under reduced pressure using a vacuum pump to obtain crude O-((R)-2-hydroxypropyl)-N-methyl-L-serine (compound aa49, H-MeSer(R-2-PrOH)-OH) (3.40 g). This was used in the next step without further purification.

[0246] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-N-methyl-L-serine (compound aa50, Fmoc-MeSer(R-2-PrOH)-OH) [ka] Under a nitrogen atmosphere, O-((R)-2-hydroxypropyl)-N-methyl-L-serine (Compound aa49, H-MeSer(R-2-PrOH)-OH) (3.40 g, 19.2 mmol), N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (7.12 g, 22.1 mmol), and sodium carbonate (7.13 g, 67.3 mmol) were dissolved in water / 1,4-dioxane (80.0 mL / 40.0 mL) and stirred at room temperature for 3 hours. The reaction mixture was washed three times with t-butyl methyl ether, and then 2 M aqueous hydrochloric acid was added to the aqueous layer until the pH reached 2. The mixture was extracted three times with ethyl acetate. The resulting organic layers were combined and washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was then further dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-N-methyl-L-serine (compound aa50, Fmoc-MeSer(R-2-PrOH)-OH) (8.00 g) as a crude product. This was used in the next step without further purification.

[0247] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-((2R)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa51, Fmoc-MeSer(R-2-PrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (0.26 g, 1.02 mmol) and 3,4-dihydro-2H-pyran (11.8 g, 139 mmol) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-2-hydroxypropyl)-N-methyl-L-serine (compound aa50, Fmoc-MeSer(R-2-PrOH)-OH) (8.00 g, 20.0 mmol) in tetrahydrofuran (40.0 mL), and the mixture was stirred at 50 °C for 2 hours. The mixture was cooled to 25 °C, and ethyl acetate was added. The organic layer was then washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue (9.3 g) was dissolved in tetrahydrofuran (100 mL), and then 1.0 M phosphate buffer (100 mL) adjusted to pH 6.8 was added. The mixture was stirred at 50°C for 3 hours. After cooling to 25°C, ethyl acetate was added, and the organic and aqueous layers were separated. Ethyl acetate was added to the aqueous layer for extraction, and then all the resulting organic layers were mixed and washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-((2R)-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa51, Fmoc-MeSer(R-2-PrOTHP)-OH) (8.50 g). LCMS(ESI) m / z = 501 (M+NH4)+ Retention time: 0.82 minutes (Analysis conditions SMD method 10)

[0248] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa54, Fmoc-Ser(tBuOTHP)-OH) was synthesized according to the following scheme. [ka]

[0249] Synthesis of methyl O-(2-(benzyloxy)-2-methylpropyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa52, Cbz-Ser(tBuOBn)-OMe) [ka] (S)-2-methyl-1-benzyl aziridine-1,2-dicarboxylate (Cbz-Azy-OMe) (2.0 g, 8.50 mmol) and 2-benzyloxy-2-methyl-propan-1-ol (2.30 g, 12.75 mmol) were dissolved in dichloromethane (7.5 mL), and boron trifluoride diethyl ether complex (BF3·OEt2) (0.160 mL, 1.28 mmol) was added dropwise over 5 minutes under ice cooling. After stirring for 30 minutes under ice cooling, water (2.0 mL) was added and the mixture was stirred for 10 minutes to quench the reaction. Saturated aqueous sodium bicarbonate solution was then added. The aqueous layer was extracted with dichloromethane, and the organic layer was dried over anhydrous magnesium sulfate. After filtration, the organic solvent was removed by concentration under reduced pressure, and the resulting residue was purified by normal phase column chromatography (hexane / ethyl acetate = 100 / 0 → 75 / 25) to obtain methyl O-(2-(benzyloxy)-2-methylpropyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa52, Cbz-Ser(tBuOBn)-OMe) (2.36 g, 67%). LCMS(ESI) m / z = 416 (M+H)+ Retention time: 0.93 minutes (Analysis conditions SQDFA05)

[0250] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-L-serine (compound aa53, Fmoc-Ser(tBuOH)-OH) [ka] Methyl O-(2-(benzyloxy)-2-methylpropyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa52, Cbz-Ser(tBuOBn)-OMe) (2.36 g, 5.68 mmol) was dissolved in methanol (8.0 mL). A solution of lithium hydroxide monohydrate (0.477 g, 11.36 mmol) in water (8.0 mL) was added and stirred at room temperature for 1 hour. Then, 2 M aqueous hydrochloric acid (8.52 mL, 17.04 mmol) was added. The aqueous layer was extracted three times with ethyl acetate, and the organic layer was dried over anhydrous magnesium sulfate. After filtration, the ethyl acetate was removed by concentration under reduced pressure to obtain O-(2-(benzyloxy)-2-methylpropyl)-N-((benzyloxy)carbonyl)-L-serine (Cbz-Ser(tBuOBn)-OH). The O-(2-(benzyloxy)-2-methylpropyl)-N-((benzyloxy)carbonyl)-L-serine (Cbz-Ser(tBuOBn)-OH) obtained above was dissolved in methanol (45 mL), Pd / C (456 mg) was added, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. After removing the Pd / C by filtration through Celite, the methanol was removed by concentration under reduced pressure to quantitatively obtain O-(2-hydroxy-2-methylpropyl)-L-serine (H-Ser(tBuOH)-OH) (1.05 g). The resulting O-(2-hydroxy-2-methylpropyl)-L-serine (H-Ser(tBuOH)-OH) (1.59 g, 8.97 mL) and sodium carbonate (2.85 g, 26.9 mmol) were dissolved in water (36 mL) and 1,4-dioxane (15 mL), and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (3.18 g, 9.42 mmol) was slowly added. After stirring at room temperature for 30 min, water (40 mL) was added to the reaction mixture, which was then washed three times with t-butyl methyl ether (60 mL). The aqueous layer was then adjusted to pH 1 with 2 M aqueous hydrochloric acid (26.9 mL, 53.8 mmol) and extracted three times with t-butyl methyl ether (60 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and then t-butyl methyl ether was concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-L-serine (Compound aa53, Fmoc-Ser(tBuOH)-OH) (2.62 g, 73%). LCMS(ESI) m / z = 400 (M+H)+ Retention time: 0.71 minutes (Analysis conditions SQDFA05)

[0251] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa54, Fmoc-Ser(tBuOTHP)-OH) [ka] N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-L-serine (compound aa53, Fmoc-Ser(tBuOH)-OH) (1.2 g, 3.00 mmol) and 3,4-dihydro-2H-pyran (1.90 mL, 21.03 mmol) were dissolved in tetrahydrofuran (6.0 mL), and pyridinium p-toluenesulfonate (PPTS) (0.038 g, 0.150 mmol) was added. The mixture was stirred at 50°C for 2 hours. The reaction mixture was then diluted with ethyl acetate (15 mL) and washed three times with saturated brine (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The resulting residue was dissolved in tetrahydrofuran (24.0 mL), and 1.0 M phosphate buffer (24.0 mL) adjusted to pH 6.8 was added. The mixture was stirred at 50°C for 3 hours. The reaction mixture was diluted with ethyl acetate (45 mL), washed three times with saturated brine (45 mL), and the organic layer was dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the residue was purified by reverse-phase column chromatography (10 mM aqueous ammonium acetate / methanol) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa54, Fmoc-Ser(tBuOTHP)-OH) (1.05 g, 72%). LCMS(ESI) m / z = 484(M+H)+ Retention time: 0.91 minutes (Analysis conditions SQDFA05)

[0252] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa55, Fmoc-MeSer(tBuOTHP)-OH) was synthesized according to the following scheme. [ka]

[0253] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa55, Fmoc-MeSer(tBuOTHP)-OH) [ka] N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-L-serine (compound aa53, Fmoc-Ser(tBuOH)-OH) (12 g, 30.04 mmol) and paraformaldehyde (2.75 g, 91.67 mmol) were dissolved in trifluoroacetic acid (31.4 g, 277.78 mmol) and toluene (60 mL) and stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in dichloromethane, washed with saturated aqueous sodium bicarbonate and saturated brine, and then dried over anhydrous sodium sulfate. After filtration, the dichloromethane was removed by concentration under reduced pressure to give (S)-(9H-fluoren-9-yl)methyl 4-((2-hydroxy-2-methylpropoxy)methyl)-5-oxooxazolidine-3-carboxylate (12 g, 97%). The resulting (S)-4-((2-hydroxy-2-methylpropoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl ester (1 g, 2.43 mmol) was dissolved in trifluoroacetic acid / dichloromethane (13 mL / 13 mL) and triethylsilane (EtSiH) (832 mg, 7.16 mmol) was added dropwise. After stirring at room temperature for 16 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was dissolved in aqueous potassium carbonate and washed with t-butyl methyl ether. The pH of the aqueous layer was adjusted to 2 with 1M aqueous hydrochloric acid. The aqueous layer was extracted twice with t-butyl methyl ether, and the organic layer was washed twice with water and saturated brine and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile = 100 / 0 → 50 / 50) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serine (Fmoc-MeSer(tBuOH)-OH) (700 mg, 70%). The obtained N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-hydroxy-2-methylpropyl)-N-methyl-L-serine (Fmoc-MeSer(tBuOH)-OH) (6 g, 14.51 mmol) and pyridinium p-toluenesulfonate (PPTS) (182 mg, 0.73 mmol) were dissolved in tetrahydrofuran (48 mL), and 3,4-dihydro-2H-pyran (8.5 g, 101.05 mmol) was added dropwise at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at 50°C for 5 hours, and then ethyl acetate was added to the reaction mixture for extraction. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serinate. The resulting mixture (50 g) was dissolved in phosphate buffer (1 M, pH 6.8, 1000 mL) and tetrahydrofuran (1000 mL) and stirred at 50°C for 4 hours. The reaction mixture was then extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (0-40% 0.5% aqueous ammonium bicarbonate solution / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(2-methyl-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa55, Fmoc-MeSer(tBuOTHP)-OH) (20 g). LCMS(ESI) m / z = 498 (M+H)+ Retention time: 0.94 minutes (Analysis conditions SMD method20)

[0254] Synthesis of 4,4-dimethyl-2-phenyl-1,3-dioxane (compound aa56) [ka] Under a nitrogen atmosphere, dimethoxymethylbenzene (87.6 g, 576 mmol) and p-toluenesulfonic acid (3.59 g, 18.9 mmol) were added to a solution of 3-methylbutane-1,3-diol (40.0 g, 384 mmol) in chloroform (400 mL), and the mixture was stirred at 0°C for 1.5 hours. The reaction mixture was concentrated, and the residue was purified by column chromatography (hexane / ethyl acetate) to give 4,4-dimethyl-2-phenyl-1,3-dioxane (Compound aa56) (70.0 g, 95%).

[0255] Synthesis of 3-(benzyloxy)-3-methylbutan-1-ol (compound aa57) [ka] Under a nitrogen atmosphere, a 1M hexane solution of diisobutylaluminum hydride (156 mL, 156 mmol) was added to a dichloromethane (130 mL) solution of 4,4-dimethyl-2-phenyl-1,3-dioxane (compound aa56) (5.00 g, 26.0 mmol) obtained by the method described above at -50°C. The mixture was stirred at this temperature for 15 minutes and then at room temperature for 1 hour. Methanol was added to quench the reaction, followed by extraction twice with dichloromethane. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (hexane / ethyl acetate) to obtain 3-(benzyloxy)-3-methylbutan-1-ol (compound aa57) (2.00 g, 40%).

[0256] Synthesis of methyl O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa58, Cbz-Ser(2-Me-BuOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (25 g, 106 mmol) and 3-(benzyloxy)-3-methylbutan-1-ol (compound aa57) (31.0 g, 159 mmol) were dissolved in dichloromethane (113 mL) and cooled to 0°C. Boron trifluoride diethyl ether complex (2.26 g, 15.9 mmol) was added and the mixture was stirred at 0°C for 3 hours. Water was added to the reaction mixture, which was then extracted with dichloromethane. The organic layer was then washed with saturated aqueous sodium carbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (hexane / ethyl acetate) to obtain methyl O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa58, Cbz-Ser(2-Me-BuOBn)-OMe) (28.0 g, 55%). LCMS(ESI) m / z = 452 (M+Na)+ Retention time: 1.46 minutes (Analysis conditions SMD method13)

[0257] Synthesis of O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa59, Cbz-Ser(2-Me-BuOBn)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide monohydrate (11.2 g) and calcium chloride (110 g) were dissolved in water (278 mL). A 2-propanol / tetrahydrofuran solution (278 mL / 1115 mL) of methyl O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serinate (Compound aa58, Cbz-Ser(2-Me-BuOBn)-OMe) (28.7 g, 68.4 mmol) was added at room temperature and stirred for 5 hours. 2M aqueous hydrochloric acid was added until the pH reached 2. The organic layer was removed, and the aqueous layer was extracted three times with ethyl acetate. The resulting organic layers were combined and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serine (Compound aa59, Cbz-Ser(2-Me-BuOBn)-OH) (28.0 g) as a crude product. This was used in the next step without further purification.

[0258] Synthesis of O-(3-hydroxy-3-methylbutyl)-L-serine (compound aa60, H-Ser(2-Me-BuOH)-OH) [ka] Under a hydrogen atmosphere, O-(3-(benzyloxy)-3-methylbutyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa59, Cbz-Ser(2-Me-BuOBn)-OH) (28.0 g, 67.4 mmol) and palladium on carbon (6.00 g, 20% w / w) were dissolved in methanol (500 mL) and stirred at room temperature for 16 hours. After filtration, the solvent was evaporated under reduced pressure. The residue was dried under reduced pressure using a vacuum pump and then recrystallized from methanol / dichloromethane (1 / 5) to give O-(3-hydroxy-3-methylbutyl)-L-serine (compound aa60, H-Ser(2-Me-BuOH)-OH) (7.00 g, 54%).

[0259] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-L-serine (compound aa61, Fmoc-Ser(2-Me-BuOH)-OH) [ka] Under a nitrogen atmosphere, O-(3-hydroxy-3-methylbutyl)-L-serine (Compound aa60, H-Ser(2-Me-BuOH)-OH) (2.80 g, 14.6 mmol) and sodium carbonate (4.66 g, 44.0 mmol) were dissolved in 1,4-dioxane (24.5 mL) / water (58.8 mL), and then N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (5.18 g, 15.4 mmol) was added and the mixture was stirred at room temperature for 3 hours. The reaction mixture was washed three times with t-butyl methyl ether, and then 2M aqueous hydrochloric acid was added to the aqueous layer until the pH reached 2. The mixture was extracted three times with ethyl acetate. The resulting organic layers were combined and washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-L-serine (Compound aa61, Fmoc-Ser(2-Me-BuOH)-OH) (5.80 g) as a crude product, which was used in the next step without further purification.

[0260] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-methyl-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa62, Fmoc-Ser(2-Me-BuOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (0.20 g, 0.82 mmol) and 3,4-dihydro-2H-pyran (10.3 mL) were added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-L-serine (compound aa61, Fmoc-Ser(2-Me-BuOH)-OH) (6.80 g, 16.5 mmol) in tetrahydrofuran (35.0 mL), and the mixture was stirred at 50 °C for 5 hours. The mixture was cooled to 25 °C, and ethyl acetate was added. The organic layer was then washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. 0.75 g of the resulting residue (11.0 g) was dissolved in tetrahydrofuran (20.0 mL), and then 1.0 M phosphate buffer (20.0 mL) adjusted to pH 6.8 was added. The mixture was stirred at 50°C for 4 hours. After cooling to 25°C, ethyl acetate was added, and the organic and aqueous layers were separated. Ethyl acetate was added to the aqueous layer for extraction, and then all the resulting organic layers were combined. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (0.5% aqueous ammonium bicarbonate / acetonitrile) to yield N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-methyl-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa62, Fmoc-Ser(2-Me-BuOTHP)-OH) (0.60 g, 80%). LCMS(ESI) m / z = 498 (M+H)+ Retention time: 0.72 minutes (Analysis conditions SMD method 11)

[0261] Synthesis of (S)-4-((3-hydroxy-3-methylbutoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa63) [ka] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-L-serine (compound aa61, Fmoc-Ser(2-Me-BuOH)-OH) (0.20 g, 0.48 mmol), paraformaldehyde (0.06 g), and trifluoroacetic acid (0.64 g, 5.62 mmol) were dissolved in toluene (10.0 mL) and stirred at room temperature for 16 hours. The reaction mixture was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give (S)-4-((3-hydroxy-3-methylbutoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (S)-4-((3-hydroxy-3-methylbutoxy)methyl)-5-oxooxazolidine-3-carboxylate (compound aa63) (0.16 g, 80%). LCMS(ESI) m / z = 448 (M+Na)+ Retention time: 2.47 minutes (Analysis conditions SMD method 14)

[0262] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-N-methyl-L-serine (compound aa64, Fmoc-MeSer(2-Me-BuOH)-OH) [ka] Under a nitrogen atmosphere, triethylsilane (0.75 mL) was slowly added dropwise to a dichloromethane solution (50.0 mL) of (S)-4-((3-hydroxy-3-methylbutoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (Compound aa63) (1.00 g, 2.35 mmol) and aluminum chloride (0.63 g, 4.70 mmol) at 0°C, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane, washed with 2 M aqueous hydrochloric acid and saturated brine, and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-3-methylbutyl)-N-methyl-L-serine (compound aa64, Fmoc-MeSer(2-Me-BuOH)-OH) (0.30 g, 30%). LCMS(ESI) m / z = 450 (M+Na)+ Retention time: 2.12 minutes (Analysis conditions SMD method 15)

[0263] Synthesis of (S)-2-(1,3-dioxoisoindolin-2-yl)-6-oxohexanoic acid methyl ester (compound aa65) [ka] Under a nitrogen atmosphere, a solution of commercially available (S)-2-((tert-butoxycarbonyl)amino)-6-hydroxyhexanoic acid (Boc-Nle(6-OH)-OH) (10 g, 40.4 mmol) in toluene / methanol (90 mL / 60 mL) was added dropwise to 2M (trimethylsilyl)diazomethane / hexane solution (24.26 mL, 48.5 mmol) at room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure using a rotary evaporator, and the resulting residue was dissolved in toluene / methanol (90 mL / 60 mL). After dissolving, 2M (trimethylsilyl)diazomethane / hexane solution (24.26 mL, 48.5 mmol) was added dropwise to the residue at room temperature and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure using a rotary evaporator to obtain a crude product, methyl (S)-2-((tert-butoxycarbonyl)amino)-6-hydroxyhexanoate (Boc-Nle(6-OH)-OMe) (13 g). The resulting crude product, (S)-2-((tert-butoxycarbonyl)amino)-6-hydroxyhexanoic acid methyl ester (Boc-Nle(6-OH)-OMe) (13 g), was added to a 4N hydrochloric acid / 1.4-dioxane solution (50 mL, 200 mmol) at room temperature and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure using a rotary evaporator to obtain the crude product, (S)-2-amino-6-hydroxyhexanoic acid methyl ester (H-Nle(6-OH)-OMe) hydrochloride (9.2 g). The crude product, (S)-methyl 2-amino-6-hydroxyhexanoate (H-Nle(6-OH)-OMe) hydrochloride (9.2 g) in acetonitrile (100 mL) was added with ethyl 1,3-dioxoisoindoline-2-carboxylate (9.74 g, 44.4 mmol) and N,N-diisopropylethylamine (DIPEA) (15.52 mL, 89 mmol) at room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure using a rotary evaporator, and the resulting residue was purified by normal-phase column chromatography (dichloromethane / methanol) to give (S)-methyl 2-(1,3-dioxoisoindolin-2-yl)-6-hydroxyhexanoate (14.6 g). Dess-Martin periodinane (CAS#87413-09-0, 11.34 g, 26.7 mmol) was added to a dichloromethane solution (100 mL) of the above-mentioned (S)-methyl 2-(1,3-dioxoisoindolin-2-yl)-6-hydroxyhexanoate (7.08 g, 24.3 mmol) at 0°C and stirred at room temperature for 3 hours. The reaction mixture was diluted with dichloromethane and washed with a saturated aqueous sodium bicarbonate / water (1 / 1) solution, a saturated aqueous sodium thiosulfate solution, and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by normal-phase column chromatography (hexane / ethyl acetate) to yield (S)-methyl 2-(1,3-dioxoisoindolin-2-yl)-6-oxohexanoate (compound aa65) (3.6 g, 51% yield over four steps). LCMS(ESI)m / z=290 (M+H)+ Retention time: 0.70 minutes (Analysis conditions SQDAA05)

[0264] Synthesis of (2S)-2-(1,3-dioxoisoindolin-2-yl)-7,7,7-trifluoro-6-hydroxyheptanoic acid methyl ester (compound aa66) [ka] Under a nitrogen atmosphere, (S)-2-(1,3-dioxoisoindolin-2-yl)-6-oxohexanoic acid methyl ester (compound aa65) (3.62 g, 12.51 mmol) in tetrahydrofuran solution (50 mL) was added at 0°C (trifluoromethyl)trimethylsilane (1.390 mL, 9.39 mmol), 1M tetrabutylammonium fluoride (TBAF) / tetrahydrofuran (THF) solution (0.626 mL, 0.626 mmol) and stirred for 10 minutes at 0°C. (Trifluoromethyl)trimethylsilane (1.390 mL, 9.39 mmol), 1M tetrabutylammonium fluoride (TBAF) / tetrahydrofuran (THF) solution (0.626 mL, 0.626 mmol) were added to the reaction mixture at 0°C and stirred for 30 minutes. To the reaction mixture, (trifluoromethyl)trimethylsilane (1.390 mL, 9.39 mmol) and 1 M tetrabutylammonium fluoride (TBAF) / tetrahydrofuran (THF) solution (0.626 mL, 0.626 mmol) were added at 0°C and stirred for 2 hours and 30 minutes. 1N aqueous hydrochloric acid (37.5 mL) was added to the reaction mixture at 0°C and stirred at room temperature for 20 minutes, followed by extraction with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (10 mM aqueous ammonium acetate / methanol) to yield methyl (2S)-2-(1,3-dioxoisoindolin-2-yl)-7,7,7-trifluoro-6-hydroxyheptanoate (compound aa66) (1.8 g, 40%). LCMS(ESI)m / z = 358 (M−H)- Retention time: 0.81 minutes (Analysis conditions SQDAA05)

[0265] Synthesis of methyl (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-7,7,7-trifluoro-6-hydroxyheptanoate (compound aa67, Fmoc-Hnl(7-F3-6-OH)-OMe) [ka] To a solution (30 mL) of (2S)-2-(1,3-dioxoisoindolin-2-yl)-7,7,7-trifluoro-6-hydroxyheptanoate (compound aa66) (3.1 g, 8.63 mmol) obtained by the method described above, hydrazine monohydrate (1.258 mL, 25.9 mmol) and acetic acid (1.482 mL, 25.9 mmol) were added at room temperature and stirred overnight. To remove the methanol, the reaction mixture was concentrated under reduced pressure using a rotary evaporator. The resulting solution was diluted with dimethyl sulfoxide and purified by reverse-phase column chromatography (10 mM aqueous ammonium acetate / methanol) to give (2S)-2-amino-7,7,7-trifluoro-6-hydroxyheptanoate (H-Hnl(7-F3-6-OH)-OMe) (2 g). To the (2S)-2-amino-7,7,7-trifluoro-6-hydroxyheptanoate methyl ester (H-Hnl(7-F3-6-OH)-OMe) (2 g, 8.73 mmol), water (25 mL), sodium carbonate (2.93 g, 34.9 mmol), tetrahydrofuran (50 mL), and N-succinimidyl 9-fluorenylmethyl carbonate (Fmoc-OSu) (3.53 g, 10.47 mmol) were added at room temperature, and the reaction mixture was stirred for 2 hours. To remove the tetrahydrofuran, the reaction mixture was concentrated under reduced pressure using a rotary evaporator. Ethyl acetate and 1N aqueous hydrochloric acid were added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by reverse-phase column chromatography (0.1% formic acid in water / 0.1% formic acid in acetonitrile) and normal-phase column chromatography (dichloromethane / methanol), followed by further purification by reverse-phase column chromatography (10 mM ammonium acetate in water / methanol). The collected fractions were evaporated under reduced pressure, extracted twice with ethyl acetate, and washed with saturated potassium hydrogen sulfate and saturated brine. The organic solvent was evaporated under reduced pressure to give methyl (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-7,7,7-trifluoro-6-hydroxyheptanoate (compound aa67, Fmoc-Hnl(7-F3-6-OH)-OMe) (1.4 g, 36% yield over two steps). LCMS(ESI) m / z = 452 (M+H)+ Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0266] Synthesis of (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-7,7,7-trifluoro-6-hydroxyheptanoic acid (compound aa68, Fmoc-Hnl(7-F3-6-OH)-OH) [ka] Lithium hydroxide monohydrate (0.521 g, 12.40 mmol) was added to a solution of calcium chloride (5.16 g, 46.5 mmol) in water (7.00 mL) at room temperature and stirred for 5 minutes. Methyl (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-7,7,7-trifluoro-6-hydroxyheptanoate (Compound aa67, Fmoc-Hnl(7-F3-6-OH)-OMe) (1.4 g, 3.10 mmol) in an isopropanol / tetrahydrofuran solution (28 mL / 7 mL) was added dropwise at room temperature, and the reaction mixture was stirred overnight. A 1N aqueous solution of hydrochloric acid was added to the reaction mixture, which was then extracted twice with t-butyl methyl ether, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (0.1% aqueous formic acid / 0.1% formic acid acetonitrile solution) to give (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-7,7,7-trifluoro-6-hydroxyheptanoic acid (compound aa68, Fmoc-Hnl(7-F3-6-OH)-OH) (950 mg, 70%). LCMS(ESI) m / z = 438.6 (M+H)+ Retention time: 0.76 minutes (Analysis conditions SQDFA05)

[0267] Synthesis of methyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serinate (compound aa69, Fmoc-Ser(1-CF3-EtOH)-OMe) [ka] Under a nitrogen atmosphere, boron trifluoride diethyl ether complex (BF3·OEt2) (7.8 μL, 0.062 mmol) was added to a dichloromethane solution (3.0 mL) of separately synthesized (S)-aziridine-1,2-dicarboxylate 2-methyl 1-((9H-fluoren-9-yl)methyl) (compound aa73, Fmoc-Azy-OMe) (0.20 g, 0.62 mmol) and 3,3,3-trifluoropropane-1,2-diol (0.24 g, 1.9 mmol) at 0°C, and the mixture was stirred for 30 minutes. In addition, under a nitrogen atmosphere, boron trifluoride diethyl ether complex (BF3·OEt2) (0.19 mL, 1.55 mmol) was added to a dichloromethane solution (77.0 mL) of (S)-aziridine-1,2-dicarboxylate 2-methyl 1-((9H-fluoren-9-yl)methyl) (compound aa73, Fmoc-Azy-OMe) (5.0 g, 15.46 mmol) and 3,3,3-trifluoropropane-1,2-diol (6.03 g, 46.4 mmol) at 0°C, and the mixture was stirred for 30 minutes. Furthermore, under a nitrogen atmosphere, boron trifluoride diethyl ether complex (BF3·OEt2) (0.14 mL, 1.11 mmol) was added to a dichloromethane solution (55.7 mL) of (S)-aziridine-1,2-dicarboxylate 2-methyl 1-((9H-fluoren-9-yl)methyl) (compound aa73, Fmoc-Azy-OMe) (3.6 g, 11.13 mmol) and 3,3,3-trifluoropropane-1,2-diol (4.34 g, 33.4 mmol) at 0°C, and the mixture was stirred for 30 minutes. Saturated aqueous sodium bicarbonate was added to each of the three reaction mixtures. All of the reaction mixtures were combined, and most of the organic solvent was removed under reduced pressure. Ethyl acetate (300 mL) was added to the remaining mixture for extraction. The organic layer was washed with saturated brine, and the organic solvent was removed under reduced pressure. The resulting residue was purified by normal-phase silica gel column chromatography (hexane / ethyl acetate) to give methyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serinate (compound aa69, Fmoc-Ser(1-CF3-EtOH)-OMe) (6.57 g, 53%, purity 95%) and the same compound (3.1 g, 25%, purity 87%). LCMS(ESI)m / z=454 (M+H)+ Retention time: 1.18 minutes (Analysis conditions SMD method45)

[0268] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa70, Fmoc-Ser(1-CF3-EtOH)-OH) [ka] Calcium chloride (2.06 g, 18.5 mmol) was dissolved in water (5.2 mL), and lithium hydroxide monohydrate (207 mg, 4.94 mmol) was added. The mixture was stirred at room temperature for 10 minutes. To the solution was added a solution of methyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serinate (compound aa69, Fmoc-Ser(1-CF3-EtOH)-OMe) (560 mg, 1.24 mmol) in tetrahydrofuran (THF) (5.15 mL) and isopropanol (20.6 mL), and the mixture was stirred at room temperature for 2 hours. 1N aqueous hydrochloric acid was added to terminate the reaction. Further, similar reactions were carried out using 0.15 g, 1.11 g, 0.48 g, and 0.96 g of Fmoc-Ser(1-CF3-EtOH)-OMe, respectively. All reaction solutions were pooled, and most of the solvent was evaporated under reduced pressure. Ethyl acetate and water were added to the resulting solution, followed by extraction. After evaporation of the organic layer under reduced pressure, the resulting residue was purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to yield N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa70, Fmoc-Ser(1-CF3-EtOH)-OH) (3.10 g, 98%). LCMS(ESI)m / z=462 (M+Na)+ Retention time: 2.24 minutes (Analysis conditions SMD method46)

[0269] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa71, Fmoc-MeSer(1-CF3-EtOH)-OH) [ka] Under a nitrogen atmosphere, a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa70, Fmoc-Ser(1-CF3-EtOH)-OH) (1.84 g, 4.19 mmol) in dichloromethane (20.9 mL) was added with paraformaldehyde (151 mg, 5.03 mmol), anhydrous magnesium sulfate (1.26 g, 10.5 mmol), and boron trifluoride diethyl ether complex (BF3·OEt2) (526 μL, 4.19 mmol) and stirred at room temperature for 3 hours. The reaction mixture was filtered to remove insoluble material and then washed with dichloromethane (10 mL). The filtered reaction mixture was added with triethylsilane (2.0 mL, 12.6 mmol) and boron trifluoride diethyl ether complex (BF3·OEt2) (1.58 mL, 12.6 mmol) and stirred at room temperature for 30 minutes. The reaction mixture was diluted with saturated brine and extracted with dichloromethane. The resulting organic layer was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa71, Fmoc-MeSer(1-CF3-EtOH)-OH) (640 mg, 34%). LCMS(ESI) m / z = 452 (M−H) Retention time: 2.33 minutes (Analysis conditions SMD method47)

[0270] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa72, Fmoc-Ser(1-CF3-EtOTHP)-OH) [ka] To a dichloromethane solution (19.12 mL) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-hydroxypropyl)-L-serine (compound aa70, Fmoc-Ser(1-CF3-EtOH)-OH) (2.52 g, 5.74 mmol), 3,4-dihydro-2H-pyran (3.92 mL, 43.0 mmol) and pyridinium p-toluenesulfonate (0.144 g, 0.574 mmol) were added, and the mixture was stirred at 40°C overnight. Water was then added to the reaction mixture, which was then extracted with dichloromethane. The organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by concentration under reduced pressure. The resulting residue was dissolved in tetrahydrofuran (29 mL), and 1 M aqueous phosphoric acid solution (pH = 8, 29 mL) was added, followed by stirring at 50°C for 3 hours. The reaction mixture was diluted with water and extracted twice with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The resulting residue was dissolved in dichloromethane (60 mL) and heptane (60 mL), and the dichloromethane was concentrated under reduced pressure to precipitate an oily crude product. The heptane solution was removed by decantation. This procedure was repeated twice. The resulting crude product was dissolved in ethyl acetate and washed twice with 0.05 M aqueous phosphoric acid and once with saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3,3,3-trifluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa72, Fmoc-Ser(1-CF3-EtOTHP)-OH) (2.89 g, 96%). LCMS(ESI)m / z=522(M−H)- Retention time: 1.00 minutes (Analysis conditions SQDAA05-2)

[0271] Synthesis of (S)-aziridine-1,2-dicarboxylate 2-methyl 1-((9H-fluoren-9-yl)methyl) (compound aa73, Fmoc-Azy-OMe) [ka] Under a nitrogen atmosphere, trifluoroacetic acid (33 mL) was added dropwise to a chloroform / methanol solution (145 mL / 145 mL) of commercially available (S)-1-tritylaziridine-2-methyl carboxylate (Trt-Azy-OMe) (50 g, 145.60 mmol) at 0°C, and the reaction mixture was stirred for 7 hours. N,N-Diisopropylethylamine (DIPEA) (127 mL) and a 1,4-dioxane solution (145 mL) of (9H-fluoren-9-yl)methyl carbonochloridate (Fmoc-Cl) (36 g, 139.16 mmol) were added dropwise to the reaction mixture at 0°C, and the reaction mixture was stirred at 0°C for 1 hour and 30 minutes. The solvent was evaporated under reduced pressure, the mixture was diluted with ethyl acetate, and the mixture was washed twice with water, saturated aqueous ammonium chloride, and twice with saturated brine. The obtained organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was then distilled off under reduced pressure to obtain a residue. The residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to obtain (S)-aziridine-1,2-dicarboxylate 2-methyl 1-((9H-fluoren-9-yl)methyl) (Compound aa73, Fmoc-Azy-OMe) (40 g, 85% yield over two steps). LCMS(ESI)m / z=324(M+H)+ Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0272] Synthesis of tert-butyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-methyl-L-glutamate (compound aa74, Fmoc-Gln(Me)-OtBu) [ka] To a solution of (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (Fmoc-Glu-OtBu) (20.0 g, 47.0 mmol) and 1-hydroxy-7-azabenzotriazole (HOAt) (8.8 g) in dimethylformamide (DMF) (300 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC·HCl) (13.5 g) under ice cooling and stirred for 10 minutes. While maintaining the temperature of the reaction solution below 5°C, a 2 mol / L solution of methylamine in tetrahydrofuran (29.5 mL) was added and stirred at room temperature for 16 hours. The reaction solution was diluted with hexane / ethyl acetate (1 / 1, 1400 mL) and aqueous ammonium chloride solution (500 mL), and the organic layer was separated. The organic layer was washed with aqueous sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain tert-butyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-methyl-L-glutamate (compound aa74, Fmoc-Gln(Me)-OtBu) (36.2 g) as a crude product. LCMS(ESI) m / z = 439 (M+H)+ Retention time: 1.05 minutes (Analysis conditions SMD method23)

[0273] Synthesis of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-methyl-L-glutamine (compound aa75, Fmoc-Gln(Me)-OH) [ka] To a solution of tert-butyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-methyl-L-glutamate (compound aa74, Fmoc-Gln(Me)-OtBu) (43.6 g) in dichloromethane (DCM) (300 mL), trifluoroacetic acid (TFA) (300 mL) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated under reduced pressure, and diethyl ether and aqueous sodium bicarbonate solution were added to the resulting residue, and the organic phase was separated. The pH of the resulting aqueous layer was adjusted to between 1 and 2 with 5 mol / L aqueous hydrochloric acid, and the precipitated solid was collected by filtration to give N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N5-methyl-L-glutamine (compound aa75, Fmoc-Gln(Me)-OH) (26.7 g). LCMS(ESI) m / z = 383 (M+H)+ Retention time: 1.75 minutes (Analysis conditions SMD method5)

[0274] Synthesis of 2-bromo-N-tert-butylacetamide (compound aa76) [ka] Under a nitrogen atmosphere, a solution of 2-bromoacetic acid (50.0 g, 360 mmol) in N,N-dimethylformamide (80.0 mL) was mixed with 2-methylpropan-2-amine (26.7 g, 365 mmol), N-ethyl-isopropylpropan-2-amine (DIPEA) (139 g, 1.08 mol), and propylphosphonic anhydride (T3P) (229 g, 720 mmol) and stirred at room temperature for 2 hours. Water was added to the reaction mixture, which was then extracted twice with ethyl acetate. The resulting organic layers were mixed, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was washed with hexane to give 2-bromo-N-tert-butylacetamide (compound aa76, 36.5 g) as a crude product. This was used in the next step without further purification.

[0275] Synthesis of O-(2-(tert-butylamino)-2-oxoethyl)-N-trityl-L-serine (compound aa77, Trt-Ser(NtBu-Aca)-OH) [ka] Under a nitrogen atmosphere, sodium hydride (0.52 g, 13.0 mmol, 60% oil dispersion) was added to a solution of triethylamine salt of trityl-L-serine (Trt-Ser-OH) (1.50 g, 4.32 mmol) in dimethylformamide (DMF) (2.00 mL) and stirred at room temperature for 2 hours. To this solution, a solution of 2-bromo-N-tert-butylacetamide (Compound aa76) (0.96 g) in DMF (1.00 mL) was added and stirred at room temperature for 1 hour. Water was added to the reaction mixture, which was then extracted twice with ethyl acetate. The resulting organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was then further dried under reduced pressure using a vacuum pump to obtain O-(2-(tert-butylamino)-2-oxoethyl)-N-trityl-L-serine (compound aa77, Trt-Ser(NtBu-Aca)-OH) (1.60 g) as a crude product, which was used in the next step without further purification.

[0276] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(tert-butylamino)-2-oxoethyl)-L-serine (compound aa78, Fmoc-Ser(NtBu-Aca)-OH) [ka] Under a nitrogen atmosphere, trifluoroacetic acid (11.2 g, 99.1 mmol) was added to a solution of O-(2-(tert-butylamino)-2-oxoethyl)-N-trityl-L-serine (compound aa77, Trt-Ser(NtBu-Aca)-OH) (15.3 g, 33.2 mmol) in dichloromethane (150 mL) / water (150 mL) at 4°C, and the mixture was stirred at the same temperature for 2 hours. The aqueous layer was separated, and N-ethyl-isopropylpropan-2-amine (DIPEA) was added until the pH reached 7. 1,4-Dioxane (150 mL) was added, followed by the addition of N-ethyl-isopropylpropan-2-amine (DIPEA) (14.9 g, 115 mmol) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (22.3 g, 231 mmol), and the mixture was stirred at 25°C for 16 hours. Water was added to the reaction mixture, which was then washed twice with hexane. Concentrated hydrochloric acid was added to the aqueous layer until the pH reached 2, and the mixture was extracted twice with ethyl acetate. The resulting organic layers were mixed, washed with saturated brine, and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(tert-butylamino)-2-oxoethyl)-L-serine (compound aa78, Fmoc-Ser(NtBu-Aca)-OH) (14.2 g, 98%). LCMS(ESI) m / z = 441 (M+H)+ Retention time: 1.08 minutes (Analysis conditions SMD method 9)

[0277] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(tert-butylamino)-2-oxoethyl)-N-methyl-L-serine (compound aa79, Fmoc-MeSer(NtBu-Aca)-OH) [ka] Under a nitrogen atmosphere, a toluene solution (1.2 mL) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(tert-butylamino)-2-oxoethyl)-L-serine (compound aa78, Fmoc-Ser(NtBu-Aca)-OH) (1.0 g, 2.270 mmol), paraformaldehyde (203 mg, 6.81 mmol), and ((1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonic acid (26 mg, 0.114 mmol) was stirred at 80°C for 1 hour. Saturated aqueous sodium bicarbonate was added to the reaction mixture, which was then extracted three times with ethyl acetate and washed with saturated brine. The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (S)-4-((2-(tert-butylamino)-2-oxoethoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (S)-(2-tert-butylamino)-2-oxoethoxy ... Under a nitrogen atmosphere, triethylsilane (EtSiH) (1.096 mL, 6.86 mmol) and water (40 μL) were added to a dichloromethane (DCM) solution (2.86 mL) of the crude product (S)-4-((2-(tert-butylamino)-2-oxoethoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (1.035 g). The mixture was stirred at room temperature for 2 minutes. Then, boron trifluoride diethyl etherate (BFOEt) (0.580 mL, 4.57 mmol) was added and the mixture was stirred at room temperature for 24 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture, which was stirred at room temperature for 40 minutes. The mixture was then extracted twice with dichloromethane (DCM). The resulting organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was added with acetonitrile and washed with hexane. Acetonitrile was evaporated under reduced pressure to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(tert-butylamino)-2-oxoethyl)-N-methyl-L-serine (compound aa79, Fmoc-MeSer(NtBu-Aca)-OH) (0.9449 g, 92% over two steps). LCMS(ESI)m / z=455 (M+H)+ Retention time: 0.79 minutes (Analysis conditions SQDFA05)

[0278] Synthesis of 2-bromo-N-methylacetamide (compound aa80) [ka] Under a nitrogen atmosphere, dichloromethane (100 mL) was added to potassium carbonate (11.61 g, 84 mmol), followed by the addition of methylamine hydrochloride (2.84 g, 42.0 mmol) at room temperature. 2-Bromoacetyl bromide (3.47 mL, 40 mmol) was added dropwise to the reaction mixture at 0°C, and the mixture was stirred at room temperature for 1 hour. Water (20 mL) was added to the reaction mixture, which was then extracted with dichloromethane. The mixture was then concentrated under reduced pressure and dried on an oil pump to obtain 2-bromo-N-methylacetamide (compound aa80) (5.2 g) as a crude product. This was used in the next step without further purification.

[0279] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(methylamino)-2-oxoethyl)-L-serine (compound aa81, Fmoc-Ser(NMe-Aca)-OH) [ka] Under a nitrogen atmosphere, a 1.9 M solution of sodium bis(trimethylsilyl)amide (NaHMDS) in THF (4.86 mL, 9.23 mmol) was added to a solution of triethylamine salt of trityl-L-serine (Trt-Ser-OH) (1.80 g) in tetrahydrofuran (THF) (2.00 mL) at 0°C and stirred for 15 minutes. A solution of 2-bromo-N-methylacetamide (compound aa80) (1.22 g) in THF (2.00 mL) was added to the reaction mixture at 0°C and stirred at room temperature for 15 minutes. The reaction mixture was added to a mixture of ethyl acetate (300 mL), water (60 mL), and 1N aqueous hydrochloric acid (4.8 mL). The resulting organic layer was evaporated under reduced pressure to give the crude product. Separately, under a nitrogen atmosphere, a 1.9 M solution of sodium bis(trimethylsilyl)amide (NaHMDS) in THF (4.65 mL, 8.83 mmol) was added to a solution of triethylamine salt of trityl-L-serine (Trt-Ser-OH) (1.80 g) in tetrahydrofuran (THF) (2.00 mL) at 0°C and stirred for 15 minutes. A solution of 2-bromo-N-methylacetamide (compound aa80) (1.22 g) in THF (2.00 mL) was added to the reaction mixture at 0°C and stirred at room temperature for 15 minutes. The reaction mixture was added to a mixture of ethyl acetate (300 mL), water (60 mL), and 1N aqueous hydrochloric acid (4.8 mL), and the resulting organic layer was evaporated under reduced pressure to obtain the crude product. The two crude products were combined and purified by reverse-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to give O-(2-(methylamino)-2-oxoethyl)-N-trityl-L-serine (Trt-Ser(NMe-Aca)-OH) (2.5 g). To a dichloromethane (DCM) solution (2.87 mL) of the obtained O-(2-(methylamino)-2-oxoethyl)-N-trityl-L-serine (Trt-Ser(NMe-Aca)-OH) (2.4 g, 5.73 mmol), trifluoroacetic acid (TFA) (1.325 mL, 17.20 mmol) was added at 0°C and stirred for 30 minutes. Water (2.87 mL) was added to the reaction mixture, and the resulting aqueous layer was purified by reverse-phase column chromatography (water / acetonitrile) to give O-(2-(methylamino)-2-oxoethyl)-L-serine (H-Ser(NtBu-Aca)-OH) (900 mg) as a crude product. This was used in the next step without further purification. The resulting crude product, O-(2-(methylamino)-2-oxoethyl)-L-serine (H-Ser(NtBu-Aca)-OH) (900 mg), was added with water (4.2 mL) and a 1,4-dioxane solution (14.0 mL) of N-ethyl-N-isopropylpropan-2-amine (DIPEA) (2.231 mL, 12.77 mmol) at room temperature. N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (1.723 g, 5.11 mmol) was then added at room temperature and stirred for 30 minutes. The reaction mixture was added to a mixture of ethyl acetate (150 mL), water (30 mL), and 1N aqueous hydrochloric acid (3.0 mL), and the resulting organic layer was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-(methylamino)-2-oxoethyl)-L-serine (compound aa81, Fmoc-Ser(NMe-Aca)-OH) (1.8 g). LCMS(ESI) m / z = 399 (M+H)+ Retention time: 0.63 minutes (Analysis conditions SQDFA05)

[0280] Synthesis of methyl N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serinate (compound aa82, Cbz-Ser(nPrOH)-OMe) [ka] Under a nitrogen atmosphere, commercially available (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (Cbz-Azy-OMe) (500 mg, 2.125 mmol) and propane-1,3-diol (810 mg, 10.645 mmol) were dissolved in dichloromethane (10 mL) and cooled to 0°C. Boron trifluoride diethyl etherate (BF3·Et2O) (46 mg, 0.324 mmol) was added and the mixture was stirred at 0°C for 4 hours. Dichloromethane was added to the reaction mixture, which was then washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give methyl N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serinate (compound aa82, Cbz-Ser(nPrOH)-OMe) (390 mg, 59%). LCMS(ESI) m / z = 334 (M+Na)+ Retention time: 1.09 minutes (Analysis conditions SMD method 13)

[0281] Synthesis of N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (compound aa83, Cbz-Ser(nPrOH)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide (3.14 g, 131.049 mmol) and calcium chloride (54.54 g, 491.434 mmol) were added to water (134 mL) and 2-propanol (538 mL) at room temperature. After stirring, a tetrahydrofuran solution (134 mL) of methyl N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serinate (Compound aa82, Cbz-Ser(nPrOH)-OMe) (10.2 g, 32.762 mmol) was added at room temperature and stirred for 5 hours. 6N aqueous hydrochloric acid was added to the reaction mixture until the pH reached 3. The organic solvent was removed under reduced pressure, and the aqueous layer was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was then dried under reduced pressure using a vacuum pump to obtain N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (Compound aa83, Cbz-Ser(nPrOH)-OH) (10.6 g) as a crude product, which was used in the next step without further purification.

[0282] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (compound aa84, Fmoc-Ser(nPrOH)-OH) [ka] Under a hydrogen atmosphere (~3 atm), a mixture of the crude product N-((benzyloxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (compound aa83, Cbz-Ser(nPrOH)-OH) (13 g), 10% palladium on carbon (4.65 g), and methanol (130 mL) was stirred at room temperature for 16 hours. After filtration, the solvent was evaporated under reduced pressure. The resulting residue was recrystallized from ethyl acetate to give O-(3-hydroxypropyl)-L-serine (H-Ser(nPrOH)-OH) (5.5 g, 78% yield over two steps). Under a nitrogen atmosphere, N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (11.94 g, 35.396 mmol) was added to a solution of O-(3-hydroxypropyl)-L-serine (H-Ser(nPrOH)-OH) (5.5 g, 33.707 mmol) and sodium bicarbonate (8.6 g, 102 mmol) in water / 1,4-dioxane (50 mL / 130 mL) at room temperature and stirred for 4 hours. Water was added to the reaction solution, which was then washed twice with t-butyl methyl ether. After adding 6N aqueous hydrochloric acid until the aqueous layer reached pH 3, the mixture was extracted with t-butyl methyl ether. The resulting organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (compound aa84, Fmoc-Ser(nPrOH)-OH) (8 g, 67%). LCMS(ESI) m / z = 386 (M+H)+ Retention time: 0.61 minutes (Analysis conditions SMD method50)

[0283] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa85, Fmoc-Ser(nPrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (PPTS) (32.6 mg, 0.130 mmol) was added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxypropyl)-L-serine (compound aa84, Fmoc-Ser(nPrOH)-OH) (1 g, 2.595 mmol) and 3,4-dihydro-2H-pyran (1.5 g, 17.83 mmol) in tetrahydrofuran (5 mL), and the mixture was stirred at 50°C for 4 hours. The reaction mixture was cooled, t-butyl methyl ether was added, and the mixture was washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under reduced pressure to give a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa85, Fmoc-Ser(nPrOTHP)-OH) and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serinate (Fmoc-Ser(nPrOTHP)-OTHP) (1.5 g). To a mixture (1.5 g) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa85, Fmoc-Ser(nPrOTHP)-OH) and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serinate (Fmoc-Ser(nPrOTHP)-OTHP), tetrahydrofuran (8 mL) and 1 M phosphate buffer (8 mL, pH 8.0) were added and stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated under reduced pressure. The resulting residue was recrystallized from diethyl ether / heptane to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa85, Fmoc-Ser(nPrOTHP)-OH) as a sodium salt (1 g). To the sodium salt (5 g) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa85, Fmoc-Ser(nPrOTHP)-OH) obtained as described above, t-butyl methyl ether (100 mL) and 0.05 M aqueous phosphoric acid solution (250 mL) were added and stirred at room temperature for 5 minutes. The reaction solution was extracted twice with t-butyl methyl ether, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa85, Fmoc-Ser(nPrOTHP)-OH) (4.9 g) as a crude product. This was used in peptide synthesis without further purification. LCMS(ESI)m / z = 468 (M-H)- Retention time: 0.84 minutes (Analysis conditions SQDFA05)

[0284] Synthesis of methyl N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serinate (compound aa86, Cbz-Ser(2-Me2-PrOH)-OMe) [ka] Under a nitrogen atmosphere, commercially available (S)-aziridine-1,2-dicarboxylate 2-methyl-1-benzyl (Cbz-Azy-OMe) (30 g, 127.53 mmol) and 2,2-dimethylpropane-1,3-diol (26.56 g, 255.06 mmol) were dissolved in dichloromethane (400 mL) and cooled to 0°C. Boron trifluoride diethyl etherate (BF3·Et2O) (2.72 g, 19.129 mmol) was added dropwise and stirred for 1 hour. The reaction mixture was washed with saturated aqueous sodium bicarbonate and saturated brine, and the resulting organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was then evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to obtain methyl N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serinate (compound aa86, Cbz-Ser(2-Me2-PrOH)-OMe) (26.7 g, 62%). LCMS(ESI) m / z = 326 (M+H)+ Retention time: 1.12 minutes (Analysis conditions SMD method 13)

[0285] Synthesis of N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa87, Cbz-Ser(2-Me2-PrOH)-OH) [ka] Under a nitrogen atmosphere, lithium hydroxide monohydrate (13.16 g, 313.505 mmol), calcium chloride (130.48 g, 1175.646 mmol), water (260 mL), and 2-propanol (1050 mL) were added at room temperature. After stirring, a tetrahydrofuran solution (260 mL) of methyl N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serinate (compound aa86, Cbz-Ser(2-Me2-PrOH)-OMe) (26.6 g, 78.376 mmol) was added at room temperature and stirred for 2 hours. 6N aqueous hydrochloric acid was added to the reaction mixture until the pH reached 3. The organic solvent was removed under reduced pressure, and the aqueous layer was extracted with ethyl acetate. The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and the resulting solvent was evaporated under reduced pressure to give N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa87, Cbz-Ser(2-Me2-PrOH)-OH) (25.0 g) as a crude product, which was used in the next step without purification.

[0286] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa88, Fmoc-Ser(2-Me2-PrOH)-OH) [ka] Under a hydrogen atmosphere (~3 atm), a mixture of the crude product N-((benzyloxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa87, Cbz-Ser(2-Me2-PrOH)-OH) (25.0 g), 10% palladium on carbon (5 g, 20% w / w), and methanol (250 mL) was stirred at room temperature for 16 hours. After filtration, the solvent was evaporated under reduced pressure. The resulting solid was washed three times with ethyl acetate to give O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (H-Ser(2-Me2-PrOH)-OH) (13.1 g, 87% yield over two steps). Under a nitrogen atmosphere, N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (24.08 g, 71.381 mmol) was added to a solution of O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (H-Ser(2-Me2-PrOH)-OH) (13 g, 67.982 mmol) and sodium bicarbonate (17.13 g, 203.946 mmol) in water / 1,4-dioxane (260 mL / 104 mL) at room temperature and stirred for 2 hours. Water was added to the reaction mixture, which was then washed twice with t-butyl methyl ether. 6N aqueous hydrochloric acid was added to the aqueous layer until the pH reached 3, and the mixture was extracted with t-butyl methyl ether. The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The mixture was then further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa88, Fmoc-Ser(2-Me2-PrOH)-OH) (26.5 g, 94%). LCMS(ESI) m / z = 414 (M+H)+ Retention time: 1.44 minutes (Analysis conditions SMD method51)

[0287] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (PPTS) (607.79 mg, 2.419 mmol) was added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(3-hydroxy-2,2-dimethylpropyl)-L-serine (compound aa88, Fmoc-Ser(2-Me2-PrOH)-OH) (20 g, 48.371 mmol) and 3,4-dihydro-2H-pyran (28.48 g, 338.598 mmol) in tetrahydrofuran (161 mL), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, and the mixture was washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under reduced pressure to give a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serinate (Fmoc-Ser(2-Me2-PrOTHP)-OTHP). Tetrahydrofuran (161 mL) and 1 M phosphate buffer (161 mL, pH 8.0) were added to a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) obtained as described above and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serinate (Fmoc-Ser(2-Me2-PrOTHP)-OTHP), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure. The resulting residue was recrystallized from diethyl ether / heptane to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) as a sodium salt (17.1 g). To the sodium salt (8 g) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) obtained as described above, t-butyl methyl ether (120 mL) and 0.05 M aqueous phosphoric acid solution (350 mL) were added and stirred at room temperature for 5 minutes. The reaction solution was extracted with t-butyl methyl ether, washed twice with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give the crude product N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2,2-dimethyl-3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-L-serine (Compound aa89, Fmoc-Ser(2-Me2-PrOTHP)-OH) (7 g). This was used in peptide synthesis without further purification. LCMS(ESI)m / z = 496 (M-H)- Retention time: 0.97 minutes (Analysis conditions SQDFA05)

[0288] Synthesis of (4S)-4-methyl-2-phenyl-1,3-dioxane (compound aa90) [ka] Under a nitrogen atmosphere, p-toluenesulfonic acid (TsOH) (2.64 g, 13.880 mmol) was added to a chloroform (CHCl3) solution (210 mL) of (S)-butane-1,3-diol (25 g, 277.407 mmol, CAS#24621-61-2) and (dimethoxymethyl)benzene (63.37 g, 416.688 mmol) at 0°C, and the mixture was stirred at room temperature for 16 hours. Dichloromethane was added to the reaction mixture, which was then washed with aqueous sodium carbonate and water, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in ethanol (EtOH) (135 mL), sodium borohydride (NaBH4) (5.25 g, 138.778 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Saturated aqueous sodium bicarbonate (210 mL) was added to the reaction mixture, which was stirred at room temperature for 2 hours, and then extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to give (4S)-4-methyl-2-phenyl-1,3-dioxane (Compound aa90) (42.7 g, 86%). LCMS(ESI) m / z = 179 (M+H)+ Retention time: 1.26 minutes (Analysis conditions SMD method13)

[0289] Synthesis of (S)-3-(benzyloxy)butan-1-ol (compound aa91) [ka] Under a nitrogen atmosphere, a 1M hexane solution (390 mL, 390 mmol) of diisobutylaluminum hydride (DIBAL) was added dropwise to a dichloromethane solution (960 mL) of (4S)-4-methyl-2-phenyl-1,3-dioxane (compound aa90) (35 g, 196.629 mmol) obtained as described above at -50°C. The reaction mixture was stirred at -50°C for 30 minutes and then at room temperature for an additional 2 hours. Methanol (MeOH) was added to the reaction mixture at -30°C to quench the reaction, followed by the addition of water. A 6N aqueous solution of hydrochloric acid was added until the reaction mixture reached pH 2, followed by extraction with dichloromethane. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to obtain (S)-3-(benzyloxy)butan-1-ol (compound aa91) (21.6 g, 61%). LCMS(ESI) m / z = 181 (M+H)+ Retention time: 1.16 minutes (Analysis conditions SMD method 13)

[0290] Synthesis of methyl O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa92, Cbz-Ser(S-2-BuOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-2-methyl-1-benzyl aziridine-1,2-dicarboxylate (Cbz-Azy-OMe) (18.8 g, 80.0 mmol) and (S)-3-(benzyloxy)butan-1-ol (compound aa91) (21.6 g, 120 mmol) obtained as described above were dissolved in dichloromethane (190 mL). Boron trifluoride diethyl etherate (BF3·Et2O) (1.51 mL, 11.976 mmol) was added at 0°C and the mixture was stirred at room temperature for 2 hours. Dichloromethane was added to the reaction mixture, which was then washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give methyl O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa92, Cbz-Ser(S-2-BuOBn)-OMe) (15 g, 30%). LCMS(ESI) m / z = 416 (M+H)+ Retention time: 1.39 minutes (Analysis conditions SMD method 13)

[0291] Synthesis of O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa93, Cbz-Ser(S-2-BuOBn)-OH) [ka] Under a nitrogen atmosphere, water (155 mL) and 2-propanol (615 mL) were added to lithium hydroxide monohydrate (6.1 g, 145.238 mmol) and calcium chloride (60.2 g, 542.342 mmol) at room temperature. After stirring, a tetrahydrofuran solution (155 mL) of methyl O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (Compound aa92, Cbz-Ser(S-2-BuOBn)-OMe) (15 g, 36.144 mmol) obtained as described above was added at room temperature. The reaction mixture was stirred at room temperature for 5 hours, and then 6N aqueous hydrochloric acid was added until the pH of the reaction mixture reached 3. The organic solvent was removed under reduced pressure, and the aqueous layer was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (Compound aa93, Cbz-Ser(S-2-BuOBn)-OH) (16.5 g) as a crude product, which was used in the next step without further purification.

[0292] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-3-hydroxybutyl)-L-serine (compound aa94, Fmoc-Ser(S-2-BuOH)-OH) [ka] Under a hydrogen atmosphere (~3 atm), a mixture of the crude product obtained above, O-((S)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa93, Cbz-Ser(S-2-BuOBn)-OH) (16.5 g), 10% palladium on carbon (8.25 g, 50% w / w), and methanol (280 mL) was stirred at room temperature for 16 hours. After filtering the reaction mixture, the solvent was evaporated under reduced pressure. The resulting solid was washed three times with ethyl acetate to give O-((S)-3-hydroxybutyl)-L-serine (H-Ser(S-2-BuOH)-OH) (5.85 g). N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (11.7 g, 34.6815 mmol) was added to a solution of O-((S)-3-hydroxybutyl)-L-serine (H-Ser(S-2-BuOH)-OH) (5.85 g, 33.03 mmol) and sodium bicarbonate (8.30 g, 98.81 mmol) in water / 1,4-dioxane (135 mL / 55 mL) under a nitrogen atmosphere at room temperature and stirred for 2 hours. Water was added to the reaction mixture, which was then washed twice with t-butyl methyl ether. After adding 6N aqueous hydrochloric acid until the aqueous layer reached pH 3, the mixture was extracted with t-butyl methyl ether. The resulting organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-3-hydroxybutyl)-L-serine (compound aa94, Fmoc-Ser(S-2-BuOH)-OH) (8.1 g, 61%). LCMS(ESI) m / z = 400 (M+H)+ Retention time: 1.21 minutes (Analysis conditions SMD method13)

[0293] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (PPTS) (255 mg, 1.014 mmol) was added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((S)-3-hydroxybutyl)-L-serine (compound aa94, Fmoc-Ser(S-2-BuOH)-OH) (8.10 g, 20.292 mmol) obtained as described above and 3,4-dihydro-2H-pyran (11.94 g, 141.94 mmol) in tetrahydrofuran (90 mL), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, and the mixture was washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under reduced pressure to give a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (Compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serinate (Fmoc-Ser(S-2-BuOTHP)-OTHP). Tetrahydrofuran (90 mL) and 1 M phosphate buffer (90 mL, pH 8.0) were added to a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) obtained as described above and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serinate (Fmoc-Ser(S-2-BuOTHP)-OTHP), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure. The resulting residue was recrystallized from diethyl ether / heptane to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (Compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) as a sodium salt (7.4 g). To the sodium salt (7.4 g) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) obtained as described above, t-butyl methyl ether (130 mL) and 0.05 M aqueous phosphoric acid solution (360 mL) were added and stirred at room temperature for 5 minutes. The reaction solution was extracted with t-butyl methyl ether, washed twice with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give a crude product, N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3S)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (Compound aa95, Fmoc-Ser(S-2-BuOTHP)-OH) (6.9 g). This was used in peptide synthesis without further purification. LCMS(ESI)m / z = 482 (M-H)- Retention time: 0.87 minutes, 0.88 minutes (Analysis conditions SQDFA05)

[0294] Synthesis of (4R)-4-methyl-2-phenyl-1,3-dioxane (compound aa96) [ka] Under a nitrogen atmosphere, p-toluenesulfonic acid (TsOH) (1.91 g, 11.096 mmol) was added to a chloroform (CHCl3) solution (200 mL) of (R)-butane-1,3-diol (20 g, 221.921 mmol, CAS#6290-03-5) and (dimethoxymethyl)benzene (50.66 g, 332.867 mmol) at 0°C, and the mixture was stirred at room temperature for 16 hours. Dichloromethane was added to the reaction mixture, which was then washed with aqueous sodium carbonate and water, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in ethanol (EtOH) (13 mL), sodium borohydride (NaBH4) (4.20 g, 110.961 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Saturated aqueous sodium bicarbonate (13 mL) was added to the reaction mixture, which was stirred at room temperature for 2 hours, and then extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to give (4R)-4-methyl-2-phenyl-1,3-dioxane (Compound aa96) (25 g, 63%). LCMS(ESI) m / z = 179 (M+H)+ Retention time: 1.29 minutes (Analysis conditions SMD method 13)

[0295] Synthesis of (R)-3-(benzyloxy)butan-1-ol (compound aa97) [ka] Under a nitrogen atmosphere, a 1M hexane solution (225 mL, 225 mmol) of diisobutylaluminum hydride (DIBAL) was added dropwise to a dichloromethane solution (561 mL) of (4R)-4-methyl-2-phenyl-1,3-dioxane (compound aa96) (20 g, 112.36 mmol) obtained as described above at -50°C. The reaction mixture was stirred at -50°C for 30 minutes and then at room temperature for an additional 2 hours. Methanol (MeOH) was added to the reaction mixture at -30°C to quench the reaction, followed by the addition of water. A 6N aqueous solution of hydrochloric acid was added until the reaction mixture reached pH 2, followed by extraction with dichloromethane. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to obtain (R)-3-(benzyloxy)butan-1-ol (compound aa97) (16.6 g, 82%). LCMS(ESI) m / z = 181 (M+H)+ Retention time: 1.11 minutes (Analysis conditions SMD method 13)

[0296] Synthesis of methyl O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa98, Cbz-Ser(R-2-BuOBn)-OMe) [ka] Under a nitrogen atmosphere, (S)-2-methyl-1-benzyl aziridine-1,2-dicarboxylate (Cbz-Azy-OMe) (14.4 g, 61.481 mmol) and (R)-3-(benzyloxy)butan-1-ol (compound aa97) (16.6 g, 92.2 mmol) obtained as described above were dissolved in dichloromethane (145 mL). Boron trifluoride diethyl etherate (BF3·Et2O) (1.31 g, 9.222 mmol) was added dropwise at 0°C and the mixture was stirred at room temperature for 2 hours. Dichloromethane was added to the reaction mixture, which was then washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give methyl O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (compound aa98, Cbz-Ser(R-2-BuOBn)-OMe) (13.4 g, 52%). LCMS(ESI) m / z = 416 (M+H)+ Retention time: 1.42 minutes (Analysis conditions SMD method 13)

[0297] Synthesis of O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa99, Cbz-Ser(R-2-BuOBn)-OH) [ka] Under a nitrogen atmosphere, water (134 mL) and 2-propanol (538 mL) were added to lithium hydroxide monohydrate (5.41 g, 128.92 mmol) and calcium chloride (53.69 g, 483.771 mmol) at room temperature. After stirring, a tetrahydrofuran solution (134 mL) of methyl O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serinate (Compound aa98, Cbz-Ser(R-2-BuOBn)-OMe) (13.4 g, 32.251 mmol) obtained as described above was added at room temperature. The reaction mixture was stirred at room temperature for 5 hours, and then 6N aqueous hydrochloric acid was added until the pH of the reaction mixture reached 3. The organic solvent was removed under reduced pressure, and the aqueous layer was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated under reduced pressure, and the residue was further dried under reduced pressure using a vacuum pump to obtain O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (Compound aa99, Cbz-Ser(R-2-BuOBn)-OH) (13.6 g) as a crude product, which was used in the next step without further purification.

[0298] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-3-hydroxybutyl)-L-serine (compound aa100, Fmoc-Ser(R-2-BuOH)-OH) [ka] Under a hydrogen atmosphere (~3 atm), a mixture of the crude product obtained above, O-((R)-3-(benzyloxy)butyl)-N-((benzyloxy)carbonyl)-L-serine (compound aa99, Cbz-Ser(R-2-BuOBn)-OH) (13.6 g), 10% palladium on carbon (5.44 g), and methanol (270 mL) was stirred at room temperature for 16 hours. After filtering the reaction mixture, the solvent was evaporated under reduced pressure. The resulting solid was washed three times with ethyl acetate to give O-((R)-3-hydroxybutyl)-L-serine (H-Ser(R-2-BuOH)-OH) (5.3 g, 93% yield over two steps). N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (10.58 g, 31.405 mmol) was added to a solution of O-((R)-3-hydroxybutyl)-L-serine (H-Ser(R-2-BuOH)-OH) (5.3 g, 29.91 mmol) and sodium bicarbonate (7.54 g, 89.73 mmol) in water / 1,4-dioxane (134 mL / 57 mL) under a nitrogen atmosphere at room temperature and stirred for 2 hours. Water was added to the reaction mixture, which was then washed twice with t-butyl methyl ether. After adding 6N aqueous hydrochloric acid until the aqueous layer reached pH 3, the mixture was extracted with t-butyl methyl ether. The resulting organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure, and the mixture was further dried under reduced pressure using a vacuum pump. The resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-3-hydroxybutyl)-L-serine (compound aa100, Fmoc-Ser(R-2-BuOH)-OH) (7.4 g, 62%). LCMS(ESI) m / z = 400 (M+H)+ Retention time: 1.20 minutes (Analysis conditions SMD method 13)

[0299] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) [ka] Under a nitrogen atmosphere, pyridinium p-toluenesulfonate (PPTS) (280 mg, 1.114 mmol) was added to a solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((R)-3-hydroxybutyl)-L-serine (compound aa100, Fmoc-Ser(R-2-BuOH)-OH) (8.90 g, 22.281 mmol) obtained as described above and 3,4-dihydro-2H-pyran (13.12 g, 155.971 mmol) in tetrahydrofuran (74 mL), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, and the mixture was washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under reduced pressure to give a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serinate (Fmoc-Ser(R-2-BuOTHP)-OTHP). Tetrahydrofuran (74 mL) and 1 M phosphate buffer (74 mL, pH 8.0) were added to a mixture of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) obtained as described above and tetrahydro-2H-pyran-2-yl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serinate (Fmoc-Ser(R-2-BuOTHP)-OTHP), and the mixture was stirred at 50°C for 3 hours. The reaction mixture was cooled, t-butyl methyl ether was added, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure. The resulting residue was recrystallized from diethyl ether / heptane to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (Compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) as a sodium salt (10.2 g). To the sodium salt (10 g) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) obtained as described above, t-butyl methyl ether (160 mL) and 0.05 M aqueous phosphoric acid solution (440 mL) were added and stirred at room temperature for 5 minutes. The reaction solution was extracted with t-butyl methyl ether, washed twice with saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give a crude product, N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((3R)-3-((tetrahydro-2H-pyran-2-yl)oxy)butyl)-L-serine (Compound aa101, Fmoc-Ser(R-2-BuOTHP)-OH) (8.8 g). This was used in peptide synthesis without further purification. LCMS(ESI)m / z = 482 (M-H)- Retention time: 0.86 minutes, 0.88 minutes (Analysis conditions SQDFA05)

[0300] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoic acid (compound aa102, Fmoc-Tyr(3-OMe)-OH) [ka] Commercially available (S)-2-amino-3-(4-hydroxy-3-methoxyphenyl)propanoic acid (H-Tyr(3-OMe)-OH) (2.5 g, 11.48 mmol) was added to water (49.9 mL), sodium carbonate (3.65 g, 34.4 mmol), and 1,4-dioxane (14.9 mL) at room temperature. A 1,4-dioxane solution (50 mL) of N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (3.10 g, 9.18 mmol) was added dropwise to the reaction mixture at 0°C. After stirring at 0°C for 20 minutes, N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (388 mg, 1.15 mmol) was added to the reaction mixture and stirred for 20 minutes. N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (388 mg, 1.15 mmol) was added to the reaction mixture at 0°C and stirred for 40 minutes. Water and t-butyl methyl ether (TBME) were added to the reaction mixture, and the resulting aqueous layer was acidified with 1N aqueous hydrochloric acid and then extracted three times with ethyl acetate. The resulting organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The solvent was then evaporated under reduced pressure. The residue was purified by reverse-phase column chromatography (0.1% aqueous formic acid / 0.1% formic acid acetonitrile solution) to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoic acid (compound aa102, Fmoc-Tyr(3-OMe)-OH) (3.53 g, 93%). LCMS(ESI) m / z = 434 (M+H)+ Retention time: 0.73 minutes (Analysis conditions SQD2FA05)

[0301] Synthesis of (S)-methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoate (compound aa103, Fmoc-Tyr(3-OMe)-OMe) [ka] Under a nitrogen atmosphere, methanol (20.75 mL) and acetonitrile (5.40 mL) were added to (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoic acid (compound aa102, Fmoc-Tyr(3-OMe)-OH) (2.29 g, 5.23 mmol) at room temperature, and the mixture was stirred at 0°C for 10 minutes. Then, a 2.0 M hexane solution of trimethylsilyldiazomethane (TMS diazomethane) (2.62 mL, 5.23 mmol) was added dropwise at 0°C. A 2.0 M solution of trimethylsilyldiazomethane (TMS diazomethane) in hexane (2.62 mL, 5.23 mmol) was added dropwise to the reaction mixture at 0°C, followed by the dropwise addition of a 2.0 M solution of trimethylsilyldiazomethane (TMS diazomethane) in hexane (3.93 mL, 7.85 mmol) at 0°C. The reaction was quenched by the addition of acetic acid (2.0 mL), followed by the addition of 50% saturated aqueous sodium bicarbonate and ethyl acetate, followed by washing with ethyl acetate. The organic layer was extracted with 50% saturated aqueous sodium bicarbonate, and the resulting aqueous layers were combined and washed three times with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product (S)-methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoate (Compound aa103, Fmoc-Tyr(3-OMe)-OMe) (2.39 g) was used in the next step without further purification.

[0302] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-chlorophenyl)diphenylmethoxy)-3-methoxyphenyl)propanoic acid (compound aa104, Fmoc-Phe(4-OClt-3-OMe)-OH) [ka] Under a nitrogen atmosphere, acetonitrile (10.58 mL), 2-chlorotrityl chloride (1.656 g, 5.29 mmol), and N,N-diisopropylethylamine (0.906 mL, 5.29 mmol) were added to the crude product, methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3-methoxyphenyl)propanoate (Compound aa103, Fmoc-Tyr(3-OMe)-OMe) (2.39 g), and the mixture was stirred at room temperature for 1 hour. Water and t-butyl methyl ether were added to the reaction mixture, and the organic layer was concentrated under reduced pressure. The resulting residue was purified by reverse-phase column chromatography (10 mM aqueous ammonium acetate solution / methanol) to give methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-chlorophenyl)diphenylmethoxy)-3-methoxyphenyl)propanoate (Fmoc-Phe(4-OClt-3-OMe)-OMe) (2.6 g, 68%). Water (14.96 mL) and lithium hydroxide monohydrate (0.603 g, 14.36 mmol) were added to calcium chloride (5.98 g, 53.8 mmol) and stirred at room temperature for 5 minutes. To the reaction mixture, isopropanol (59.8 mL) and a tetrahydrofuran (THF) solution (14.96 mL) of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-chlorophenyl)diphenylmethoxy)-3-methoxyphenyl)methylpropanoate (Fmoc-Phe(4-OClt-3-OMe)-OMe) (2.6 g, 3.59 mmol) were added. After stirring for 4 hours, 0.05 M aqueous phosphoric acid and t-butyl methyl ether were added. The aqueous layer was extracted three times with t-butyl methyl ether. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((2-chlorophenyl)diphenylmethoxy)-3-methoxyphenyl)propanoic acid (Compound aa104, Fmoc-Phe(4-OClt-3-OMe)-OH) (2.8 g), was used in peptide synthesis without further purification. LCMS(ESI) m / z = 708 (M―H)― Retention time: 0.74 minutes (Analysis conditions SQD2AA50)

[0303] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (compound aa109, Fmoc-Ser(3-Me-5-Oxo-Odz)-OH) N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (compound aa109, Fmoc-Ser(3-Me-5-Oxo-Odz)-OH) was synthesized according to the following scheme. [ka] Under a nitrogen atmosphere, sodium hydride (34 g, 1420 mmol, 60% oil dispersion) was added to a dimethylformamide (DMF) solution (1000 mL) of commercially available (tert-butoxycarbonyl)-L-serine (Boc-Ser-OH) (50 g, 243.65 mmol) at 0°C and stirred for 1 hour. A dimethylformamide (DMF) solution (50 mL) of 2-bromoacetonitrile (37.2 mL, 536.03 mmol) was added dropwise to the reaction mixture at 0°C and stirred for an additional 1 hour. Water was added to the reaction mixture, which was then extracted three times with ethyl acetate. The resulting organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by normal phase column chromatography (petroleum ether / ethyl acetate) to give N-(tert-butoxycarbonyl)-O-(cyanomethyl)-L-serine (compound aa105, Boc-Ser(CHCN)-OH) (29 g, 49%). Hydroxylamine hydrochloride (22.4 g, 315.26 mmol) and triethylamine (58.8 mL, 425.60 mmol) were added to a solution of N-(tert-butoxycarbonyl)-O-(cyanomethyl)-L-serine (compound aa105, Boc-Ser(CHCN)-OH) (38.5 g, 157.63 mmol) obtained as described above in ethanol (EtOH) (400 mL) at room temperature, and the mixture was stirred at 85°C for 2 hours. The solvent was evaporated under reduced pressure to give the crude product, N-(tert-butoxycarbonyl)-O-(2-(hydroxyamino)-2-iminoethyl)-L-serine (compound aa106) (107 g). Under a nitrogen atmosphere, carbonyldiimidazole (CDI) (3.96 g, 24.42 mmol) and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (4.92 g, 32.32 mmol) were added to a 1,4-dioxane solution (120 mL) of the resulting crude product, N-(tert-butoxycarbonyl)-O-(2-(hydroxyamino)-2-iminoethyl)-L-serine (compound aa106) (12 g) at room temperature, and the mixture was stirred at 110°C for 2 hours. The reaction solution was adjusted to pH 2 with concentrated hydrochloric acid and then extracted twice with dichloromethane (DCM). The obtained organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give N-(tert-butoxycarbonyl)-O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (compound aa107, Boc-Ser(3-Me-5-Oxo-Odz)-OH) (1.2 g) as a crude product. Under a nitrogen atmosphere, a 4N hydrochloric acid / 1,4-dioxane solution (15 mL) was added to a 1,4-dioxane solution (10 mL) of the crude product, N-(tert-butoxycarbonyl)-O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (Compound aa107, Boc-Ser(3-Me-5-Oxo-Odz)-OH) (258 mg) at room temperature, followed by stirring for 16 hours. The solvent was evaporated under reduced pressure to give the crude product, O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (Compound aa108, H-Ser(3-Me-5-Oxo-Odz)-OH) (160 mg). Under a nitrogen atmosphere, N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (23 g, 68.2 mmol) was added to a solution of the crude product, O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (compound aa108, H-Ser(3-Me-5-Oxo-Odz)-OH) (22.4 g) and potassium carbonate (18.8 g, 135.04 mmol) in 1,4-dioxane / water (165 mL / 110 mL) at room temperature, followed by stirring for 3 hours. The reaction mixture was washed with t-butyl methyl ether / hexane (1 / 3). The resulting aqueous layer was adjusted to pH 2 with concentrated hydrochloric acid and then extracted twice with dichloromethane (DCM). The resulting organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)methyl)-L-serine (compound aa109, Fmoc-Ser(3-Me-5-Oxo-Odz)-OH) (5.69 g). LCMS(ESI)m / z=426 (M+H)+ Retention time: 0.72 minutes (Analysis conditions SQD2FA05)

[0304] Synthesis of ((2S)-4-diazo-3-oxo-1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)butan-2-yl)carbamate (9H-fluoren-9-yl)methyl (compound aa110) [ka] Under a nitrogen atmosphere, 4-methylmorpholine (0.355 mL, 3.23 mmol) and ethyl chloroformate (0.310 mL, 3.23 mmol) were added to a tetrahydrofuran solution (5.0 mL) of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-L-serine (Compound aa22, Fmoc-Ser(EtOTHP)-OH) (1.4 g, 3.07 mmol) while cooling with an ice bath prepared with ice and sodium chloride, and the mixture was stirred for 1 hour and 10 minutes. Subsequently, a separately prepared diazomethane solution in diethyl ether was added dropwise to the reaction mixture while cooling with an ice bath prepared with ice and sodium chloride, and the mixture was stirred for 30 minutes. Acetic acid (0.88 mL, 15.37 mmol) was added to the reaction mixture, which was stirred for 10 minutes. After adding ethyl acetate and water, the mixture was extracted with ethyl acetate. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated under reduced pressure, and the resulting residue was purified by normal-phase column chromatography (hexane / ethyl acetate) to give (9H-fluoren-9-yl)methyl ((2S)-4-diazo-3-oxo-1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)butan-2-yl)carbamate (Compound aa110) (1.13 g, 77%). LCMS (ESI) m / z = 502 (M + Na) + Retention time: 0.87 minutes (Analysis conditions SQDFA05)

[0305] Preparation of diazomethane in diethyl ether Diethyl ether (17.0 mL) was added to 1-methyl-3-nitro-1-nitrosoguanidine (5.0 g, purchased from Tokyo Chemical Industry or Fujifilm Wako Pure Chemical Industries), which contained approximately 50% water, and 50% aqueous potassium hydroxide solution (9.5 mL) was added dropwise at 0°C. After stirring the reaction solution for 1 hour, the yellow diethyl ether layer was separated using a Pasteur tube with a blunt tip, and the resulting solution was used directly in the above reaction.

[0306] Synthesis of (3R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)butanoic acid (compound aa111, Fmoc-bAla(3R-MeOEtOTHP)-OH) [ka] Under a nitrogen atmosphere, 4-methylmorpholine (0.974 mL, 8.86 mmol) and silver trifluoroacetate (AgOCF3) (0.078 mL, 0.355 mmol) were added to a solution of (9H-fluoren-9-yl)methyl ((2S)-4-diazo-3-oxo-1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)butan-2-yl)carbamate (Compound aa110) (1.7 g, 3.55 mmol) obtained as described above in tetrahydrofuran / water (18 mL / 1.8 mL) at 0°C, followed by stirring at room temperature for 2 hours and 30 minutes. Dimethyl sulfoxide (DMSO) was added to the reaction mixture, and the mixture was purified by reverse-phase column chromatography (water / acetonitrile) to give (3R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)butanoic acid (compound aa111, Fmoc-bAla(3R-MeOEtOTHP)-OH) (1.4 g, 84%). LCMS (ESI) m / z = 468 (M-H) Retention time: 0.78 minutes (Analysis conditions SQDFA05)

[0307] (2S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)methyl)propanoic acid (compound aa117) was synthesized according to the following scheme. [ka]

[0308] Synthesis of (2S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)methyl)propanoic acid (compound aa117, Fmoc-bAla(2S-MeOEtOTHP)―OH) Under a nitrogen atmosphere, sodium hydride (1.16 g, 28.9 mmol, 60% oil dispersion) was added to a dimethylformamide (DMF) solution (100 mL) of 2-(trityloxy)ethan-1-ol (CAS. No. 18325-45-6) (8.8 g, 28.9 mmol), synthesized by a method described in a paper (Beilstein Journal of Organic Chemistry, 2017, 13, 2428-2441.), and commercially available (R)-oxiran-2-ylmethyl 4-methylbenzenesulfonate (6.0 g, 26.3 mmol) at 0°C. The mixture was stirred at room temperature for 12 hours. Water was added to the reaction mixture, which was then extracted twice with ethyl acetate. The resulting organic layer was concentrated under reduced pressure, and the resulting residue was purified by normal phase column chromatography (hexane / ethyl acetate=5 / 1) to give (R)-2-((2-(trityloxy)ethoxy)methyl)oxirane (compound aa112) (7.32 g, 77%). LCMS(ESI)m / z=383 (M+Na)+ Retention time: 1.40 minutes (Analysis conditions SMD method45)

[0309] To the (R)-2-((2-(trityloxy)ethoxy)methyl)oxirane (compound aa112) (38.5 g, 157.63 mmol) obtained as described above, a 7 M solution of ammonia in methanol (196 mL) was added at room temperature, and the mixture was stirred at room temperature for 15 hours. The solvent was evaporated under reduced pressure, and water was added to the resulting residue, followed by extraction with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile). The fractions were collected, and saturated sodium bicarbonate solution (21.4 mL) and di-tert-butyl dicarbonate (BocO) (5.55 g) were added to the solution, followed by stirring at room temperature for 10 minutes. The solvent was evaporated under reduced pressure, and water was added to the resulting residue, followed by extraction with ethyl acetate. The obtained organic layer was concentrated under reduced pressure, and the obtained residue was purified by normal phase column chromatography (hexane / ethyl acetate = 4 / 1) to obtain tert-butyl (R)-(2-hydroxy-3-(2-(trityloxy)ethoxy)propyl)carbamate (compound aa113) (4.25 g, 70%). LCMS(ESI)m / z=500 (M+Na)+ Retention time: 1.40 minutes (Analysis conditions SMD method45)

[0310] Under a nitrogen atmosphere, a solution of the obtained (R)-(2-hydroxy-3-(2-(trityloxy)ethoxy)propyl)carbamate tert-butyl (Compound aa113) (4.53 g, 9.48 mmol) in dichloromethane (47.4 mL) was added with triethylamine (3.97 mL, 28.5 mmol) and methanesulfonic anhydride (2.48 g, 14.2 mmol) at room temperature and stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give (R)-12,12-dimethyl-10-oxo-1,1,1-triphenyl-2,5,11-trioxa-9-azatridecan-7-yl methanesulfonate (Compound aa114) (4.62 g, 88%). LCMS(ESI)m / z = 578 (M+Na)+ Retention time: 1.47 minutes (Analysis conditions SMD method45)

[0311] Under a nitrogen atmosphere, sodium cyanide (1.95 g, 39.7 mmol) was added to a dimethyl sulfoxide (DMSO) solution (39.6 mL) of the resulting (R)-12,12-dimethyl-10-oxo-1,1,1-triphenyl-2,5,11-trioxa-9-azatridecan-7-yl methanesulfonate (compound aa114) (4.41 g, 7.93 mmol) at room temperature, and the mixture was stirred at 80°C for 10 hours. Acetonitrile and water were added to the reaction mixture, and the mixture was purified by reverse-phase column chromatography (water / acetonitrile) to obtain tert-butyl (R)-(2-cyano-3-(2-(trityloxy)ethoxy)propyl)carbamate (compound aa115) (2.16 g, 53%). LCMS(ESI)m / z = 509 (M+Na)+ Retention time: 1.49 minutes (Analysis conditions SMD method45)

[0312] Under a nitrogen atmosphere, trifluoromethanesulfonic acid (3.5 mL, 39.5 mmol) was added to a chlorobenzene solution (9.3 mL) of the obtained (R)-(2-cyano-3-(2-(trityloxy)ethoxy)propyl)carbamate (compound aa115) (3.63 g, 7.46 mmol) at 0°C, and the mixture was stirred at room temperature for 1 hour. Subsequently, water (3.6 mL, 201 mmol) was added to the reaction solution at room temperature, and the mixture was stirred at 110°C for 3 hours. Water (20 mL), hexane (7 mL), and toluene (20 mL) were added to the reaction solution, and the aqueous layer was removed. Water (12 mL) was added to the obtained organic layer, and the mixture was washed. The resulting aqueous layer was collected, and acetonitrile (37.3 mL) and sodium bicarbonate (6.27 g, 74.6 mmol) were added. (1-Fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (3.77 g, 11.2 mmol) was added at room temperature and stirred for 30 minutes. Fmoc-OSu (1.26 g, 3.73 mmol) was then added at room temperature and stirred overnight. The reaction mixture was filtered and extracted with t-butyl methyl ether. 6N concentrated hydrochloric acid was added to the resulting aqueous layer until the pH reached 1. Purification by reverse-phase column chromatography (water / acetonitrile) afforded (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((2-hydroxyethoxy)methyl)propanoic acid (Compound aa116) (1.31 g, 46%). LCMS(ESI)m / z=386 (M+H)+ Retention time: 1.78 minutes (Analysis conditions SMD method47)

[0313] Under a nitrogen atmosphere, pyridinium paratoluenesulfonate (PPTS) (67 mg, 0.27 mmol) was added to a solution of the (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((2-hydroxyethoxy)methyl)propanoic acid (compound aa116) (1.03 g, 2.67 mmol) obtained above and 3,4-dihydro-2H-pyran (0.73 mL, 8.02 mmol) in dichloromethane (13.4 mL) at room temperature, and the mixture was stirred at 40°C for 10 hours. Water was added to the reaction solution, and the mixture was extracted with dichloromethane. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. A separately prepared phosphate buffer solution (pH = 8, 13.4 mL) was added to a solution of the obtained crude product in tetrahydrofuran (13.4 mL), and the mixture was stirred at 50°C for 20 hours. Water (50 mL) was added to the reaction solution, followed by extraction with t-butyl methyl ether / hexane (40 mL / 10 mL). The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The resulting organic layer was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (water / acetonitrile) to give (2S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)methyl)propanoic acid (Compound aa117) (1.04 g, 83%). LCMS(ESI)m / z=492 (M+Na)+ Retention time: 0.95 minutes (Analysis conditions SQDAA05-2)

[0314] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((R)-1-((allyloxy)carbonyl)-4,4-difluoropyrrolidin-2-yl)pentanoic acid (compound aa122, Fmoc-Nva(2-R-4-F2-Pyrro(N-Alloc))—OH) was synthesized according to the following scheme. [ka]

[0315] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((R)-1-((allyloxy)carbonyl)-4,4-difluoropyrrolidin-2-yl)pentanoic...

Claims

1. A protected amino acid in which the amino group and / or carboxyl group contained in the amino acid represented by the following formula is protected with a protecting group: 【Chemistry 1】 During the ceremony, R １ is hydrogen or C １ -C ６ is alkyl, L １ is fluorine, C １ -C ２ Alkyl groups, and C １ -C ２ a straight-chain C substituted with one or more substituents selected from the group consisting of fluoroalkyl ２ alkylene, fluorine, C １ -C ２ Alkyl group, C １ -C ２ a straight-chain C alkyl group optionally substituted with one or more substituents selected from the group consisting of fluoroalkyl, and oxo (=O); ３ It is alkylene.

2. The group consisting of: 【Chemistry 2】 The protected amino acid according to claim 1, wherein an amino group and / or a carboxyl group contained in the amino acid selected from the following is protected with a protecting group.

3. the amino-protecting group is selected from the group consisting of Fmoc, Boc, Cbz, Alloc, nosyl, dinitronosyl, t-Bu, trityl, and cumyl; and / or 3. The protected amino acid according to claim 1, wherein the carboxyl-protecting group is selected from the group consisting of a methyl group, an allyl group, a t-Bu group, a trityl group, a cumyl group, a methoxytrityl group, and a benzyl group.

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