Cyclic peptide compound having high membrane permeability, and library containing the same

Abstract

To provide, in a non-limiting aspect, a library of cyclic peptide compounds for screening cyclic peptide compounds with high cell membrane permeability (also called membrane permeability), and a library of cyclic peptide compounds for efficiently screening cyclic peptide compounds that can specifically bind to a target molecule in a non-limiting aspect.SOLUTION: There is provided a library of cyclic peptide compounds for use in the identification of compounds that can specifically bind to a target molecule, in which the library substantially consists of cyclic peptide compounds without (1) an indole skeleton and (2) a substituted or unsubstituted hydroxyphenyl group in a side chain of a ring part, the average number of aromatic rings contained in the side chain of the ring part of the cyclic peptide compounds contained in the library is 0-3 and the average number of amino acids comprising the ring part of the cyclic peptide compounds contained in the library is 7-14.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cyclic peptide compound having high membrane permeability, a library containing the same, and methods for producing and screening the same. [Background technology]

[0002] In recent years, medium-molecular-weight compounds (molecular weight: 500-2000) have attracted attention because they are superior to small-molecular-weight compounds in terms of their ability to access tough targets, and may be superior to antibodies in terms of their ability to be transported into cells. Cyclosporine A, a natural product, is a representative example, and is an orally administered peptide that inhibits the intracellular target cyclophilin.

[0003] Most of the medium-sized compounds that have been developed so far are natural products or their derivatives, and often require complex chemical synthesis. Therefore, the current situation is that the elucidation of the drug-likeness (excellent properties of metabolic stability and membrane permeability) of medium-sized compound drug development has not progressed as compared to drug development using low-molecular-weight compounds (molecular weight 500 or less). Peptides are representative molecular species of medium-sized compounds, but peptides have generally been considered to have low metabolic stability and membrane permeability. However, in recent years, conditions necessary for medium-sized peptides to satisfy drug-likeness (for example, having a cyclic portion, the number of N-substituted amino acids, the range of the number of amino acid residues, and lipophilicity) have been reported (Patent Document 1). In addition, there is a report that an N-methyl peptide library was produced by translational synthesis, focusing on N-methyl amino acids, which are unnatural amino acids (Patent Document 2).

[0004] Regarding library design, a guideline for designing a synthetic macrocyclic molecule library has been proposed based on the analysis results of known macrocyclic molecules (Non-Patent Document 1). However, since this guideline was derived from macrocyclic molecules including molecules other than peptides, it is unclear how this guideline can be applied to peptides. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 100132 [Patent Document 2] International Publication No. 2012 / 033154 [Non-patent literature]

[0006] [Non-Patent Document 1] Villar et al., Nat Chem Biol.2014 September; 10(9): 723-731 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of such circumstances, and in one non-limiting aspect, it is an objective of the present invention to provide a library of cyclic peptide compounds for screening cyclic peptide compounds with high cell membrane permeability (also referred to as "membrane permeability"). In another non-limiting aspect, it is an objective of the present invention to provide a library of cyclic peptide compounds for efficiently screening cyclic peptide compounds capable of specifically binding to a target molecule. In another aspect, it is an objective of the present invention to provide a cyclic peptide compound with high membrane permeability, or a production method or screening method thereof. [Means for solving the problem]

[0008] The inventors have found that, when screening for cyclic peptide compounds capable of specifically binding to a target molecule, the hit rate for cyclic peptide compounds capable of specifically binding to a target molecule can be improved, i.e., the efficiency of screening can be increased, by using a library containing cyclic peptide compounds having a long side chain in the cyclic portion.

[0009] On the other hand, they surprisingly discovered that tryptophan and tyrosine residues, which are amino acid residues having an indole skeleton or a hydroxyphenyl group that have traditionally been used in oral small molecule drugs, are actually not suitable for peptides aiming for high membrane permeability, meaning that peptides with the same structure have difficulty in exhibiting high membrane permeability.

[0010] In particular, the inventors have found that by repeatedly panning for compounds capable of binding to a target molecule using a library containing cyclic peptide compounds having amino acid residues with long side chains, such as tryptophan or tyrosine residues, cyclic peptide compounds with low membrane permeability are enriched as hit compounds.

[0011] Furthermore, without necessarily intending to be bound by any particular theory, even if an attempt is made to obtain a cyclic peptide compound having high membrane permeability by a hit-to-lead method from the cyclic peptide compounds concentrated as described above, the long side chains that are thought to contribute to binding to the target molecule themselves have a structure that makes it difficult to exhibit high membrane permeability, and therefore it is considered unlikely that both (i) binding affinity with the target molecule and (ii) high membrane permeability can be achieved at the same time.

[0012] Therefore, the inventors discovered a library of cyclic peptide compounds that have long side chains in the cyclic portion and do not have side chains that are unlikely to exhibit high membrane permeability, as one embodiment of a library that can efficiently screen cyclic peptide compounds that have high membrane permeability and can specifically bind to target molecules, and thus completed the present invention.

[0013] The present invention includes, in one non-limiting specific embodiment, the following. [A1] A cyclic peptide compound, (1) The side chain of the cyclic moiety does not have a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group; (2) when the cyclic moiety has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10; (3) when the cyclic moiety has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10; (4) having a long side chain in the cyclic portion, the side chain length being 6.0 to 13 angstroms; (5) the long side chain is R 1 where R 1 teeth, (a) a phenyl group, or (b) a 4- to 6-membered heterocyclic group having 1 to 3 heteroatoms independently selected from the group consisting of oxygen atoms and nitrogen atoms in the ring; Here, (a) and (b) may each have at least one substituent independently selected from the following Group A: Group A: oxo, a halogen atom, a C1-C4 alkyl group optionally substituted with a halogen atom, and a C1-C4 dialkylamino group (wherein one or two non-adjacent methylene groups in the C1-C4 alkyl group may be optionally substituted with an oxygen atom); Or, (c) a compound represented by the following general formula (II), (III), or (IV): [ka] [In the formula, X 1 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, X 2 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, Y 1 is a single bond, an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Y 2 is a single bond, a C1-C2 alkylene group optionally substituted with a C1-C4 alkyl group, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Z 1represents a single bond, a methylene group which may have at least one substituent independently selected from the following Group B, or an oxygen atom, R 2 and R 3 each independently represents a hydrogen atom, a C1-C6 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, wherein the C1-C6 alkyl group is optionally substituted with a halogen atom, and one or two non-adjacent methylene groups in the C1-C6 alkyl group are optionally substituted with an oxygen atom; R 2 and R 3 , or R 2 and X 1 may be bonded to form a 4- to 6-membered ring structure, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 4 represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C1-C6 alkanoyl group; R 5 represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group; R 4 and / or R 5 In the C1-C6 alkyl group, one or two non-adjacent methylene groups may be replaced by an oxygen atom; Or R 4 and R 5 , or R 5 and X 2 may form a 4- to 6-membered ring structure together with the atom to which they are attached, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 6 , R 7 , R 8 , R 9 , and R 10each independently represents a hydrogen atom, a halogen atom, a C1-C4 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, in which one or two non-adjacent methylene groups in the C1-C4 alkyl group may be replaced by an oxygen atom; [ka] represents a bonding point. Group B: halogen atoms, C1-C4 alkyl groups, and C1-C2 alkoxy groups. is a group represented by The cyclic peptide compound. [A2] The long side chain is represented by the following general formula (I-3): -(CH 2 ) n -R 1 (I-3) [In the formula, n is an integer from 1 to 5; one or two non-adjacent methylene groups of the alkylene group may be replaced by an oxygen atom; R 1 is the same as the description in [A1].] The cyclic peptide compound according to [A1], wherein the side chain is represented by the following formula: [A3] The cyclic peptide compound according to [A1] or [A2], wherein the heterocyclic group is a group selected from the group consisting of a pyridyl group, a piperidino group, a morpholino group, and an azetidinyl group, and these groups may have at least one substituent independently selected from the group A. [A4] The cyclic peptide compound according to any one of [A1] to [A3], wherein the side chain of the cyclic moiety does not have a condensed ring structure formed of two or more aromatic rings. [A5] The cyclic peptide compound according to any one of [A1] to [A4], wherein the pKa of the acidic side chain is 5.0 to 10. [A6] The cyclic peptide compound according to any one of [A1] to [A5], wherein the basic pKa of the basic side chain is 4.0 to 7.5. [A7] The cyclic peptide compound according to any one of [A1] to [A6], wherein the number of amino acids constituting the cyclic portion is 5 to 15. [A8] The cyclic peptide compound according to any one of [A1] to [A7], wherein the number of amino acids constituting the peptide portion other than the nucleic acid linkage is 5 to 20. [A9] The cyclic peptide compound according to any one of [A1] to [A8], wherein the number of N-substituted amino acids constituting the peptide portion other than the nucleic acid linkage is 3 or more. [A10] The cyclic peptide compound according to any one of [A1] to [A9], wherein the number of unnatural amino acids constituting the peptide portion other than the nucleic acid linkage is 4 or more. [A11] The cyclic peptide compound according to any one of [A1] to [A10], wherein ClogP / total aa is 1.0 to 1.8. [A12] The cyclic peptide compound according to any one of [A1] to [A11], which has an amide bond at the cyclization site. [A13] P app is 1.0×10 -6 The cyclic peptide compound according to any one of [A1] to [A12], wherein the viscosity is 100 MPa or more. [A14] The cyclic peptide compound according to any one of [A1] to [A13], wherein the number of aromatic rings contained in the side chain of the cyclic moiety is 0 to 3. [A15] The cyclic peptide compound according to any one of [A1] to [A13], wherein the number of aromatic rings contained in the side chain of the cyclic moiety is 1 to 3. [A16] The cyclic peptide compound according to any one of [A1] to [A15], wherein the long side chain contains 1 to 3 aromatic rings. [A17] The cyclic peptide compound according to any one of [A1] to [A15], wherein the long side chain contains 2 to 3 aromatic rings. [A18] The library according to any one of [A1] to [A17], wherein the long side chain does not contain an amide bond on the side chain or is a side chain containing one amide bond. [A19] The cyclic peptide compound according to any one of [A1] to [A18], wherein the long side chain is the longest side chain contained in at least one amino acid selected from the following Group C: Group C: amino acids listed in Tables 2-1 to 2-6. [A20] A cyclic peptide compound selected from pd100 to pd247 and pd300 to pd504 listed in Table 26.

[0014] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [B1] A library containing a cyclic peptide compound according to any one of [A1] to [A20]. [B2] A library of cyclic peptide compounds, the library essentially consisting of cyclic peptide compounds not having an indole skeleton in the side chain of the cyclic portion. [B3] A library of cyclic peptide compounds, the library being essentially composed of cyclic peptide compounds that do not have a fused ring structure formed by two or more aromatic rings in the side chain of the cyclic portion. [B4] The library according to any one of [B1] to [B3], wherein the library essentially consists of cyclic peptide compounds having no substituted or unsubstituted hydroxyphenyl group in the side chain of the cyclic portion. [B5] The library according to any one of [B1] to [B4], wherein when the library contains a cyclic peptide compound having an acidic side chain in the cyclic portion, the cyclic peptide compound essentially consists of a cyclic peptide compound whose acidic side chain has a pKa of 3.5 to 10. [B6] The library according to [B5], wherein the pKa is 4.5 to 10. [B7] The library according to [B5], wherein the pKa is 5.0 to 10. [B8] The library according to any one of [B1] to [B7], wherein when the library contains a cyclic peptide compound having a basic side chain in the cyclic moiety, the cyclic peptide compound essentially consists of a cyclic peptide compound having a basic pKa of 4.0 to 10 in the basic side chain. [B9] The library according to [B8], wherein the basic pKa is 4.0 to 9.5. [B10] The library described in [B8], wherein the basic pKa is 4.0 to 9.0. [B11] The library according to [B8], wherein the basic pKa is 4.0 to 8.5. [B12] The library according to [B8], wherein the basic pKa is 4.0 to 7.5. [B13] The library described in [B8], wherein the basic pKa is 4.0 to 7.2. [B14] The library according to any one of [B1] to [B13], wherein the library contains cyclic peptide compounds having a long side chain having a length of 5.4 to 13 angstroms in the cyclic portion. [B15] The library according to any one of [B1] to [B13], wherein the library contains cyclic peptide compounds having a long side chain having a length of 6.0 to 13 angstroms in the cyclic portion. [B16] The library according to any one of [B1] to [B13], wherein the library contains cyclic peptide compounds having a long side chain having a length of 6.0 to 10 angstroms in the cyclic portion. [B17] The library according to any one of [B14] to [B16], wherein the long side chain does not contain an amide bond on the side chain or is a side chain containing one amide bond. [B18] The library according to any one of [B1] to [B17], wherein the library contains a cyclic peptide compound having an aromatic ring in the side chain of the cyclic portion. [B19] The library according to any one of [B14] to [B18], wherein at least one of the long side chains is a side chain having an aromatic ring. [B20] The library according to any one of [B14] to [B18], wherein two or more of the long side chains are side chains having an aromatic ring. [B21] The library according to any one of [B1] to [B20], wherein the library essentially consists of cyclic peptide compounds having no methylthio group in the side chain of the cyclic portion. [B22] The library according to any one of [B1] to [B21], wherein the library essentially consists of cyclic peptide compounds having no thiol group in the side chain of the cyclic portion. [B23] The library according to any one of [B1] to [B22], wherein the average number of amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library is 5 to 15. [B24] The library according to any one of [B1] to [B22], wherein the average number of amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library is 8 to 13. [B25] The library according to any one of [B1] to [B22], wherein the average number of amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library is 8 to 11. [B26] The library according to any one of [B1] to [B25], wherein the average number of amino acids constituting the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compounds contained in the library is 5 to 20. [B27] The library according to any one of [B1] to [B25], wherein the average number of amino acids constituting the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compounds contained in the library is 8 to 13. [B28] The library according to any one of [B1] to [B27], wherein the average ClogP of the cyclic peptide compounds contained in the library is 4 to 18. [B29] The library according to any one of [B1] to [B28], wherein the average ClogP / total aa of the cyclic peptide compounds contained in the library is 1.0 to 1.8. [B30] The library according to any one of [B1] to [B29], wherein the average number of unnatural amino acids contained in peptide sites other than the nucleic acid linkage sites of cyclic peptide compounds contained in the library is 4 or more. [B31] The library according to any one of [B1] to [B30], wherein the average ratio of the number of N-substituted amino acids to the number of amino acids contained in the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compounds contained in the library is 30% or more. [B32] The library according to any one of [B1] to [B30], wherein the average number of N-substituted amino acids contained in peptide sites other than the nucleic acid linkage portion of cyclic peptide compounds contained in the library is 3 or more. [B33] The library according to any one of [B1] to [B32], wherein the cyclic peptide compounds contained in the library are cyclic peptide compounds having an amide bond at the cyclization site. [B34] The library according to any one of [B1] to [B33], wherein the library is used for identifying a compound capable of specifically binding to a target molecule. [B35] The library according to any one of [B1] to [B34], wherein the library is used for identifying an orally administrable compound or a precursor thereof. [B36] The library is P app is 1.0×10 -6 The library according to any one of [B1] to [B35], which is used to obtain the above cyclic peptide compound. [B37] The library is 1×10 4 The library according to any one of [B1] to [B36], having the above diversity. [B38] The library is 1×10 10 The library according to any one of [B1] to [B36], having the above diversity. [B39] The library according to any one of [B1] to [B38], wherein the average molecular weight of cyclic peptide compounds contained in the library is 500 to 2,000. [B40] The long side chain is R 1 where R 1 The library according to any one of [B1] and [B14] to [B39], wherein the above is synonymous with the description of [A1]. [B41] The long side chain is represented by the following general formula (I-3): -(CH 2 ) n -R 1 (I-3) [In the formula, n is an integer from 1 to 5; one or two non-adjacent methylene groups of the alkylene group may be replaced by an oxygen atom; R 1 is synonymous with [B40].] The library according to any one of [B1] and [B14] to [B40], wherein the side chain is represented by the following formula: [B42] The library according to [B40] or [B41], wherein the heterocyclic group is a group selected from the group consisting of a pyridyl group, a piperidino group, a morpholino group, and an azetidinyl group, and these groups may have at least one substituent independently selected from the group A. [B43] Said R 1 is at least one group selected from the group consisting of (a) and (b). [B44] The above R 1 is a group selected from the following (1) to (6): (1) In the above general formula (II), Y 1 is a single bond or an oxygen atom, and X 1 is a single bond or an optionally substituted C1-C2 alkylene group, R 2 and R 3 each independently represents a hydrogen atom or a C1-C6 alkyl group, wherein the C1-C6 alkyl group may be substituted with a halogen atom; (2) In the above general formula (III), R 5 is a hydrogen atom, and Y 2 is a single bond, a carbonyl group or a sulfonyl group, and X 2 is an optionally substituted C1-C2 alkylene group, and Z1 is an oxygen atom; (3) In the above general formula (III), R 5 is a C1-C6 alkyl group, does not form a ring structure, and Y 2 is a single bond, an optionally substituted C1-C2 alkylene group, a carbonyl group or a sulfonyl group, and X 2 is a single bond or an optionally substituted C1-C2 alkylene group, Z 1 is a single bond or an optionally substituted methylene group; (4) In the above general formula (III), R 4 and R 5form a 4- to 6-membered ring structure together with the atoms to which they are bonded, and the ring may contain an oxygen atom, and the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom; X 2 is a single bond or an optionally substituted C1-C2 alkylene group; Y 2 is a single bond or an optionally substituted C1-C2 alkylene group; Z 1 is a single bond, an optionally substituted methylene group, or an oxygen atom; (5) In the above general formula (III), R 5 and X 2 form a 4- to 6-membered ring structure together with the atoms to which they are bonded, and the ring may contain an oxygen atom, and the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom; Y 2 is a single bond or an optionally substituted C1-C2 alkylene group; Z 1 is an oxygen atom, and R 4 is a C1-C4 alkyl group; (6) In the above general formula (IV), R 6 , R 7 , R 8 , R 9 , and R 10 are each independently a hydrogen atom, a halogen atom, a methyl group, or an ethyl group. [B45] The library according to any one of [B1] and [B14] to [B44], wherein the long side chain is the longest side chain contained in at least one amino acid selected from group C described in [A19]. [B46] The library according to any one of [B1] and [B14] to [B44], wherein at least one of the long side chains is a side chain containing an aromatic ring. [B47] Said R 1 is a phenyl group which may have at least one substituent independently selected from the group A. [B48] The library described in any one of [B1], [B14] to [B47], wherein the long side chain is a side chain containing at least one group selected from the group consisting of a halophenyl group and a C1-C4 haloalkylphenyl group. [B49] The library according to any one of [B1] to [B48], wherein the average number of aromatic rings contained in the side chain of the cyclic portion of the cyclic peptide compounds contained in the library is 0 to 3. [B50] The library described in any of [B1] to [B49], wherein the average ratio of the number of aromatic rings to the number of amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library is 40% or less. [B51] The library according to any one of [B1] to [B50], wherein the average number of aromatic rings contained in the long side chain of the cyclic portion of the cyclic peptide compounds contained in the library is 1 to 3. [B52] The library according to any one of [B1] to [B51], wherein the library is a nucleic acid display library. [B53] A library of nucleic acids encoding the library of cyclic peptide compounds according to [B1] to [B52].

[0015] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [C1] A method for screening a cyclic peptide compound capable of specifically binding to a target molecule, comprising the steps of: (i) contacting a cyclic peptide compound contained in the library according to any one of [B1] to [B52] with the target molecule; and (ii) selecting a cyclic peptide compound capable of specifically binding to the target molecule. [C2] The method according to [C1], wherein the target molecule is a protein.

[0016] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [D1] A cell-free translation system for producing a peptide compound, the cell-free translation system substantially not including at least one selected from the group consisting of the following (a) to (c): (a) an amino acid having an indole skeleton in the side chain, or a nucleic acid encoding such an amino acid; (b) an amino acid having a fused ring structure with two or more aromatic rings in the side chain, or a nucleic acid encoding such an amino acid; and (c) An amino acid having a substituted or unsubstituted hydroxyphenyl group in the side chain, or a nucleic acid encoding such an amino acid. [D2] The cell-free translation system according to [D1], which is substantially free of (a), (b), and (c) according to [D1]. [D3] The cell-free translation system according to [D1] or [D2], wherein when the translation system comprises a tRNA in which an amino acid having an acidic side chain is acylated, and a nucleic acid encoding the amino acid, the pKa of the side chain of the amino acid is 3.5 to 10. [D4] The cell-free translation system according to any one of [D1] to [D3], wherein when the translation system comprises a tRNA in which an amino acid having a basic side chain is acylated, and a nucleic acid encoding the amino acid, the basic pKa of the side chain of the amino acid is 4.0 to 10. [D5] The cell-free translation system according to [D3] or [D4], wherein the pKa is 5.0 to 10. [D6] The cell-free translation system according to [D4] or [D5], wherein the basic pKa is 4.0 to 7.5. [D7] The cell-free translation system according to any one of [D1] to [D6], which contains an amino acid having a long side chain having a length of 5.4 to 13 angstroms. [D8] The cell-free translation system according to [D7], wherein the length of the long side chain is 6.0 to 10 angstroms. [D9] The cell-free translation system according to [D7] or [D8], wherein at least one of the long side chains is a side chain having an aromatic ring. [D10] The cell-free translation system described in [D7] or [D8], wherein two or more of the long side chains are side chains having an aromatic ring. [D11] The cell-free translation system according to any one of [D1] to [D10], wherein 40% or more of the types of amino acids contained in the translation system are N-substituted amino acids. [D12] The cell-free translation system according to any one of [D1] to [D11], wherein the peptide compound is a cyclic peptide compound. [D13] The cell-free translation system according to any one of [D1] to [D12], wherein the ratio of the number of types of amino acids having an aromatic ring to the total number of types of amino acids contained in the translation system is 40% or less. [D14] A cell-free translation system in which the average number of aromatic rings contained in the side chains of a cyclic portion of a cyclic peptide compound having a cyclic portion composed of 8 to 11 amino acids is adjusted to be 0 to 3.

[0017] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [E1] A method for producing a library of peptide compounds, comprising at least one step selected from the group consisting of the following (a) and (b): (a) preparing an amino acid pool substantially free of amino acids having an indole skeleton in the side chain, and synthesizing a peptide compound using some or all of the amino acids contained in the pool as constituent amino acids; and (b) preparing a template pool substantially free of nucleic acids encoding amino acids having an indole skeleton in the side chain, and synthesizing a peptide compound from the template pool. [E2] The method according to [E1], wherein the pools in steps (a) and (b) are pools that are substantially free of amino acids having a fused ring structure with two or more aromatic rings in the side chain, and nucleic acids encoding such amino acids. [E3] The method according to [E1] or [E2], wherein the pools in steps (a) and (b) are pools that are substantially free of amino acids having a substituted or unsubstituted hydroxyphenyl group in the side chain, and nucleic acids encoding such amino acids, respectively. [E4] The method according to any of [E1] to [E3], wherein when the amino acid pool used in the synthesis of a peptide compound contains an amino acid having an acidic side chain and the template pool contains a nucleic acid encoding the amino acid, the pKa of the side chain of the amino acid is 3.5 to 10. [E5] The method according to [E4], wherein the pKa is 5.0 to 10. [E6] The method according to any of [E1] to [E5], wherein when the amino acid pool used in the synthesis of a peptide compound contains an amino acid having a basic side chain and the template pool contains a nucleic acid encoding the amino acid, the basic pKa of the side chain of the amino acid is 4.0 to 10. [E7] The method according to [E6], wherein the basic pKa is 4.0 to 7.5. [E8] The method according to any one of [E1] to [E7], wherein the amino acid pool used in the synthesis of the peptide compound is a pool containing amino acids having a long side chain having a length of 5.4 to 13 angstroms. [E9] The method according to any one of [E1] to [E7], wherein the amino acid pool used in synthesizing the peptide compound is a pool containing amino acids having a long side chain having a length of 6.0 to 10 angstroms. [E10] The method according to [E8] or [E9], wherein at least one of the long side chains is a side chain having an aromatic ring. [E11] The method according to any one of [E1] to [E10], wherein 50% or more of the types of amino acids contained in an amino acid pool used in the synthesis of a peptide compound are unnatural amino acids. [E12] The method according to any one of [E1] to [E11], wherein 40% or more of the types of amino acids contained in an amino acid pool used in the synthesis of a peptide compound are N-substituted amino acids. [E13] The method according to any one of [E1] to [E12], wherein the peptide compound is a cyclic peptide compound. [E14] The method according to [E13], comprising at least one step selected from the group consisting of the following (c) and (d): (c) preparing an amino acid pool adjusted so that the average ratio of the number of amino acids having an aromatic ring to the number of amino acids constituting a cyclic portion in a cyclic peptide compound to be translationally synthesized is 40% or less, and synthesizing a peptide compound using some or all of the amino acids contained in the pool as constituent amino acids; (d) preparing a template pool adjusted so that the average ratio of the number of amino acids having an aromatic ring to the number of amino acids constituting the cyclic portion in the cyclic peptide compound to be translationally synthesized is 40% or less, and synthesizing a peptide compound from the template pool. [E15] A method for producing a library of peptide compounds, comprising a step of synthesizing a peptide compound using the cell-free translation system according to any one of [D1] to [D14]. [E16] The method according to any one of [E1] to [E13], comprising a step of synthesizing a peptide compound using the cell-free translation system according to any one of [D1] to [D14]. [E17] The library is 1 × 10 4 The method according to any one of [E1] to [E16], having the above diversity. [E18] The method described in any of [E1] to [E17], wherein the library is a nucleic acid display library. [E19] The method according to any one of [E1] to [E18] for producing a library according to any one of [B1] to [B44]. [E20] A library obtained by the method according to any one of [E1] to [E19]. [E21] A cyclic peptide compound constituting the library described in [E20].

[0018] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [F1] A method for producing a cyclic peptide compound, comprising the steps of: (i) contacting a cyclic peptide compound contained in the library according to any one of [B1] to [B52] with a target molecule; (ii) selecting a cyclic peptide compound capable of binding to the target molecule; and (iii) producing a cyclic peptide compound based on the amino acid sequence of the cyclic peptide compound selected in (ii); [F2] A cyclic peptide compound produced by the method according to [F1].

[0019] Furthermore, the present invention includes the following in one non-limiting specific embodiment. [G1] An amino acid having a long side chain of 5.4 to 13 angstroms in length, said long side chain containing R1, wherein R1 is as defined in [A1]. [G2] An amino acid having a long side chain of 5.4 to 13 angstroms in length. [G3] An amino acid selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6. [G4] An amino acid according to any one of [G1] to [G3], wherein the main chain amino group is protected by a protecting group. [G5] An amino acid according to any one of [G1] to [G3], in which the main chain amino group is protected with an Fmoc group. [G6] An amino acid according to any one of [G1] to [G5] for use in the synthesis of a cyclic peptide compound. [G7] A pdCpA amino acid, or a pCpA amino acid, in which pdCpA or pCpA is linked to the carboxy group of an amino acid described in [G1] to [G5] via an ester bond. [G8] An amino acid selected from any one of [G1] to [G5] and [G7] for use in the translation of a cyclic peptide compound. [H1] An amino acid having a group represented by the following general formula (II) or (III) in the side chain: [ka] [In the formula, X 1 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, X 2 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, Y 1 is a single bond, an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Y 2represents a single bond, a C1-C2 alkylene group optionally substituted with a C1-C4 alkyl group, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Z 1 represents an oxygen atom, R 2 and R 3 each independently represents a hydrogen atom, a C1-C6 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, wherein the C1-C6 alkyl group may be substituted with a halogen atom, and one or two non-adjacent methylene groups in the C1-C6 alkyl group may be substituted with an oxygen atom. R 2 and R 3 , or R 2 and X 1 may be bonded to form a 4- to 6-membered ring structure, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 4 represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C1-C6 alkanoyl group; R 5 represents a hydrogen atom, and R 4 In the C1-C6 alkyl group, one or two non-adjacent methylene groups may be replaced by an oxygen atom; [ka] represents a bonding point. Group B: halogen atoms, C1-C4 alkyl groups, and C1-C2 alkoxy groups. [H2] A peptide compound library containing peptide compounds containing at least one amino acid selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6. [H3] A method for producing a peptide compound library containing peptide compounds containing at least one amino acid selected from the group consisting of amino acids listed in Tables 2-1 to 2-6, the method comprising a step of translationally synthesizing a peptide compound in a cell-free translation system comprising the following (i) and (ii): (i) a tRNA having at least one amino acid selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6 bound thereto; (ii) a nucleic acid library encoding the peptide compound library; Here, the nucleic acid library contains a nucleic acid containing at least one codon corresponding to the anticodon of the tRNA. [H4] A cell-free translation system for producing a peptide compound containing at least one amino acid selected from the group consisting of amino acids listed in Tables 2-1 to 2-6, comprising the following (i) and (ii): (i) a tRNA having at least one amino acid selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6 bound thereto; (ii) a nucleic acid encoding said peptide compound; Here, the nucleic acid contains at least one codon that corresponds to the anticodon of the tRNA. [H5] A method for translational synthesis of a peptide compound containing at least one amino acid selected from the group consisting of amino acids listed in Tables 2-1 to 2-6, comprising the following (i) and (ii): (i) preparing a tRNA having at least one amino acid bound thereto selected from the group consisting of amino acids listed in Tables 2-1 to 2-6; (ii) 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. Effect of the Invention

[0020] According to one aspect of the present invention, it is possible to efficiently screen for a cyclic peptide compound capable of specifically binding to a target molecule. Also, according to one aspect of the present invention, it is possible to obtain a cyclic peptide compound having high cell membrane permeability. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a diagram showing an outline of one embodiment of a method for measuring membrane permeability. [Diagram 2] This is a conceptual diagram showing the premise and reality of cell membrane permeability measurement using Caco-2 cells. [Figure 3-1] FIG. 1 shows mass spectra of translationally synthesized peptide compounds containing unnatural amino acids. [Figure 3-2] This figure shows a continuation of Figure 3-1. [Figure 3-3] This figure shows the continuation of Figure 3-2. [Figure 4] FIG. 1 shows mass spectra of translationally synthesized peptide compounds containing unnatural amino acids. [Diagram 5] FIG. 1 shows mass spectra of translationally synthesized peptide compounds containing unnatural amino acids. [Figure 6] FIG. 1 shows mass spectra of translationally synthesized peptide compounds containing unnatural amino acids. [Figure 7] FIG. 1 shows the transition of TEER when Caco-2 cells were incubated for up to 3 hours according to a conventional method. [Figure 8] FIG. 1 shows the change in TEER over time when Caco-2 cells were incubated for up to 24 hours using the improved method. [Figure 9] FIG. 1 shows the correlation between Papp and Fa measured by a conventional method. [Figure 10] Graph showing the correlation between Papp and Fa measured by the improved method. The improved method showed a higher correlation with Fa than the conventional method. [Figure 11] 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 12] 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 13] FIG. 1 shows the relationship between ClogP / total AA and Papp when a population of cyclic peptide compounds having a side chain of Phe(4-CF3) is used. [Figure 14] FIG. 1 shows the relationship between the ARC of a cyclic peptide compound and cell membrane permeability. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Preferred, non-limiting embodiments of the present disclosure are described below.

[0023] All elements described in the following examples are intended to be considered as being equivalently described in this "Form for Carrying Out the Invention" without being bound by any patent practice, custom, laws, regulations, etc. that may attempt to limit the interpretation of the contents described in the examples in a country in which the patent application is intended to be granted.

[0024] Any combination of part or all of one or more elements described anywhere in the present disclosure is intended to be included in the present disclosure, unless there is a technical inconsistency based on the technical common sense of a person skilled in the art, and is described as being naturally understood by a person skilled in the art.

[0025] In the present specification, the following abbreviations are used: 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 Tables 2-1 to 2-6 and Tables 4-1 to 4-32 are used.

[0026] In the present specification, "alkyl" refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and 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 is an alkyl group having 1 to 20 carbon atoms (C1-C20, hereinafter "Cp-Cq" means that the number of carbon atoms is p to q), and preferably includes C1-C6 alkyl groups, C1-C5 alkyl groups, C1-C4 alkyl groups, C1-C3 alkyl groups, etc. Specific examples of the alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, tert-butyl, sec-butyl, 1-methylpropyl, 1,1-dimethylpropyl, 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.

[0027] As used herein, "alkenyl" refers to a monovalent group having at least one double bond (two adjacent SP2 carbon atoms). Depending on the arrangement of the double bond and the substituents (if any), the geometry of the double bond can be in an Entgegen (E) or Zusammen (Z), cis or trans configuration. Alkenyl includes linear or branched chains, including linear chains containing internal olefins. C2-C10 alkenyl is preferred, and C2-C6 alkenyl and C2-C4 alkenyl are more preferred. Specific examples of such alkenyl include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl.

[0028] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Examples of the alkynyl include linear or branched alkynyl, including internal alkylene. Preferred are C2-C10 alkynyl, more preferably C2-C6 alkynyl and C2-C4 alkynyl. Specific examples of the alkynyl include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2'-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.

[0029] In the present specification, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including a monocyclic ring, a bicyclic ring, and a spiro ring. A C3-C10 cycloalkyl is preferred. The cycloalkyl group may be partially unsaturated. Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[2.2.1]heptyl.

[0030] As used herein, the term "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably C6-C10 aryl. Specific examples of aryl include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

[0031] As used herein, "heteroaryl" refers to a monovalent aromatic ring group containing preferably 1 to 5 heteroatoms in the atoms constituting the ring (also referred to as "within the ring" in the present specification), which may be partially saturated. The ring may be a single ring or two condensed rings (for example, a bicyclic heteroaryl condensed with benzene or a single ring heteroaryl). The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl.

[0032] In the present specification, 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 aryl, preferably, "C5-C10 aryl C1-C6 alkyl", for example, benzyl.

[0033] In the present specification, "alkylene" refers to a divalent group derived by further removing one arbitrary hydrogen atom from the above-mentioned "alkyl", and 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 methylene, 1,2-ethylene, 1,1-ethylene, 1,3-propylene, tetramethylene, pentamethylene, and hexamethylene.

[0034] In the present specification, "arylene" means a divalent group derived by further removing one arbitrary hydrogen atom from the aryl. 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-C10 arylene). Specific examples of the arylene include phenylene and naphthylene.

[0035] In the present specification, the term "heteroarylene" refers to a divalent group derived by removing one arbitrary hydrogen atom from the heteroaryl. The heteroarylene may be a single ring or a condensed ring. The number of atoms constituting the ring is not particularly limited, but is preferably 5 to 10 (5-membered to 10-membered heteroarylene). Specific examples of the heteroarylene include pyrrolediyl, imidazolediyl, pyrazolediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazolediyl, triazinediyl, isoxazolediyl, oxazolediyl, oxadiazolediyl, isothiazolediyl, thiadiazolediyl, thiadiazolediyl, furandiyl, and thiophenediyl.

[0036] In the present specification, the term "alkylenearylene" refers to a divalent group in which the alkylene and the arylene are bonded at any position, and the alkylene is bonded to the basic skeleton side. Specific examples of the alkylenearylene include -C1-C6 alkylene-C6-C10 arylene.

[0037] In the present specification, the term "arylene alkylene" refers to a divalent group in which the arylene and the alkylene are bonded at any position, and the arylene is bonded to the basic skeleton side. Specific examples of the arylene alkylene include -C6-C10 arylene-C1-C6 alkylene.

[0038] In the present specification, the term "alkyleneheteroarylene" refers to a divalent group in which the alkylene and the heteroarylene are bonded at any position, and the alkylene is bonded to the basic skeleton side. Specific examples of the alkyleneheteroarylene include -C1-C6 alkylene-5-membered to 10-membered heteroarylene.

[0039] In the present specification, the term "heteroarylene alkylene" refers to a divalent group in which the heteroarylene and the alkylene are bonded at any position, and the heteroarylene is bonded to the basic skeleton side. Specific examples of the heteroarylene alkylene include -5-membered-10-membered heteroarylene-C1-C6 alkylene.

[0040] As used herein, the term "active ester" refers to a group containing a carbonyl that reacts with an amino group to form an amide bond, and is a group in which, for example, OBt, OAt, OSu, OPfp, SR1, or the like is bound to the carbonyl, and is a group that can promote the reaction with an amine.

[0041] In this specification, the term "reaction promoting group" refers to a group that is introduced near a functional group to be bonded in order to selectively cause a reaction at a desired position, and activates the functional group for the bonding reaction. For example, in order to react a carbonyl with an amine, a reaction promoting group can be introduced to either or both of the carbonyl and amine sides. Such a reaction promoting group can be removed during or after the bonding reaction.

[0042] As used herein, the term "amino acid" includes natural amino acids and non-natural amino acids. As used herein, the term "natural amino acid" refers to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. Non-natural amino acids are not particularly limited, but examples thereof include β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, amino acids whose side chains are different from those in nature, and hydroxycarboxylic acids. As used herein, amino acids may have any configuration. There are no particular limitations on the selection of the side chain of the amino acid, and the side chain may be freely selected from, in addition to a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group, and one or two non-adjacent methylene groups in these groups may be replaced by an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -). Each of them may be substituted with a substituent, and the substituents are not limited, and may be independently selected from any substituents including a halogen atom, an O atom, an S atom, an N atom, a B atom, an Si atom, or a P atom. That is, examples of the substituents include an optionally substituted alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and a cycloalkyl group. In addition, the amino acid in the present specification may have a "long side chain" as a side chain, which will be described later. In a non-limiting embodiment, the amino acid in the present specification may be a compound 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 acid).

[0043] The main chain amino group of the amino acid is unsubstituted (NH 2 R may be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group that may have a substituent, and one or two non-adjacent methylene groups in these groups may be an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2-), or the carbon chain bonded to the N atom and the carbon atom at the α position may form a ring, as in proline. The substituent of R is selected in the same manner as the substituent in the amino acid side chain described above. When the main chain amino group is substituted, R is included in the "amino acid side chain" in this specification. The R may be a "long side chain" as described later. Such an amino acid in which the main chain amino group is substituted is referred to as an "N-substituted amino acid" in this specification. As used herein, the "N-substituted amino acid" is preferably exemplified by N-alkyl amino acid, N-C1-C6 alkyl amino acid, N-C1-C4 alkyl amino acid, N-methyl amino acid, and N-substituted amino acid having a long side chain, but is not limited thereto.

[0044] In this specification, the "amino acids" constituting the peptide compounds 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 represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl etc. are included.

[0045] In the present specification, examples of halogen atoms include F, Cl, Br, and I, with F or Cl being preferred. Examples of substituents containing halogen atoms include, but are not limited to, fluoro (-F), chloro (-Cl), bromo (-Br), and iodine (-I). Examples of groups substituted with one or more of these and having halogen as a substituent include alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, and more specifically, examples of groups include fluoroalkyl, difluoroalkyl, and trifluoroalkyl.

[0046] Substituents containing O atoms include hydroxy (-OH), oxy (-OR), carbonyl (-C=OR), carboxy (-CO 2 H), oxycarbonyl (-C=O-OR), carbonyloxy (-OC=OR), thiocarbonyl (-C=O-SR), carbonylthio (-SC=OR), aminocarbonyl (-C=O-NHR), carbonylamino (-NH-C=OR), oxycarbonylamino (-NH-C=O-OR), sulfonylamino (-NH-SO 2 -R), aminosulfonyl (-SO 2 -NHR), sulfamoylamino (-NH-SO 2 -NHR), thiocarboxyl (-C(=O)-SH), carboxylcarbonyl (-C(=O)-CO 2 H).

[0047] Examples of oxy (-OR) include alkoxy, cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, aralkyloxy, etc. As the alkoxy, C1-C4 alkoxy and C1-C2 alkoxy are preferred, and among them, methoxy and ethoxy are preferred.

[0048] Examples of carbonyl (-C=OR) include formyl (-C=OH), alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, arylcarbonyl, heteroarylcarbonyl, aralkylcarbonyl, and the like.

[0049] Examples of oxycarbonyl (-C=O-OR) include alkyloxycarbonyl, cycloalkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl, and the like.

[0050] Examples of carbonyloxy (-OC=OR) include alkylcarbonyloxy, cycloalkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, arylcarbonyloxy, heteroarylcarbonyloxy, aralkylcarbonyloxy, and the like.

[0051] Examples of thiocarbonyl (-C=O-SR) include alkylthiocarbonyl, cycloalkylthiocarbonyl, alkenylthiocarbonyl, alkynylthiocarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, aralkylthiocarbonyl, and the like.

[0052] Examples of carbonylthio (-SC=OR) include alkylcarbonylthio, cycloalkylcarbonylthio, alkenylcarbonylthio, alkynylcarbonylthio, arylcarbonylthio, heteroarylcarbonylthio, aralkylcarbonylthio, and the like.

[0053] Examples of aminocarbonyl (-C=O-NHR) include alkylaminocarbonyl (e.g., C1-C6 or C1-C4 alkylaminocarbonyl, particularly ethylaminocarbonyl, methylaminocarbonyl, etc.), 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 included.

[0054] 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.

[0055] 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 an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl are also included.

[0056] Sulfonylamino (-NH-SO 2Examples of -R include alkylsulfonylamino, cycloalkylsulfonylamino, alkenylsulfonylamino, alkynylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, and the like. In addition, -NH-SO 2 Examples of the compound include compounds in which the H atom bonded to the N atom in -R is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0057] Aminosulfonyl (-SO 2 Examples of -NHR include alkylaminosulfonyl, cycloalkylaminosulfonyl, alkenylaminosulfonyl, alkynylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, aralkylaminosulfonyl, and the like. 2 Examples of the compound include compounds in which the H atom bonded to the N atom in -NHR is further substituted with an alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or aralkyl.

[0058] Sulfamoylamino (-NH-SO 2 Examples of -NHR include alkylsulfamoylamino, cycloalkylsulfamoylamino, alkenylsulfamoylamino, alkynylsulfamoylamino, arylsulfamoylamino, heteroarylsulfamoylamino, aralkylsulfamoylamino, and the like. 2 The two H atoms bonded to the N atom in -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.

[0059] Substituents containing S atoms include thiol (-SH), thio (-SR), sulfinyl (-S=OR), and sulfonyl (-SO 2 -R), sulfo (-SO 3H).

[0060] Examples of thio (-SR) are selected from among alkylthio, cycloalkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, aralkylthio, and the like.

[0061] Sulfonyl (-SO 2 Examples of -R include alkylsulfonyl, cycloalkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aralkylsulfonyl, and the like.

[0062] As a substituent containing a nitrogen atom, azide (-N 3 , also known as "azido group"), cyano (-CN), primary amino (-NH 2 ), secondary amino (-NH-R; also called monosubstituted amino), tertiary amino (-NR(R'); also called disubstituted amino), amidino (-C(=NH)-NH 2 ), substituted amidino (-C(=NR)-NR'R"), guanidino (-NH-C(=NH)-NH 2 ), substituted guanidino (-NR-C(=NR''')-NR'R"), aminocarbonylamino (-NR-CO-NR'R"), pyridyl, piperidino, morpholino, azetidinyl and the like groups.

[0063] Examples of secondary amino (-NH-R; monosubstituted amino) include alkylamino, cycloalkylamino, alkenylamino, alkynylamino, arylamino, heteroarylamino, aralkylamino, and the like.

[0064] Examples of tertiary amino (-NR(R'); disubstituted amino) include, for example, alkyl(aralkyl)amino and other amino groups having any two substituents independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, etc., and these two substituents may form a ring. Specific examples include dialkylamino, particularly C1-C6 dialkylamino, C1-C4 dialkylamino, dimethylamino, diethylamino, etc. In the present specification, "C p -C q "Dialkylamino group" means an amino group having C p -C q A group in which two alkyl groups are substituted, both C p -C q The alkyl groups can be the same or different.

[0065] 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.

[0066] 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 form a ring.

[0067] Examples of aminocarbonylamino (-NR-CO-NR'R") include those in which R, R', and R" are each independently selected from a hydrogen atom, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, and an aralkyl, or these groups form a ring.

[0068] In this specification, "having a heteroatom in the ring" means that a heteroatom is contained in an atom constituting the ring, and examples of such a group include heteroaryl groups such as a pyridyl group, a piperidino group, a morpholino group, an azetidinyl group, etc. When the heteroatom is an oxygen atom, it is expressed as "having an oxygen atom in the ring".

[0069] As used herein, a "translated amino acid" or a "translatable amino acid" refers to an "amino acid" that has a side chain that can be translated. In a non-limiting embodiment, an amino acid as used herein may be a translatable amino acid.

[0070] As used herein, "drug-likeness" or "drug-like" refers to the property of excellent metabolic stability and membrane permeability. As used herein, "drug-like amino acids" refer to α, β, and γ amino acids that have a main chain amino group (NH 2 One of the two hydrogen atoms of the main chain methylene group (-CH 2-group), one or two of the hydrogen atoms may be substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or the like. These substituents may be further substituted with a "substituent that contributes to drug-likeness". Preferred examples of drug-like amino acids include amino acids having a "long side chain" as described below. Drug-like amino acids may also be L-amino acids, D-amino acids, α,α-disubstituted amino acids, N-substituted amino acids, and the like. Drug-like amino acids do not necessarily need to be translatable. They include the side chain portion of a peptide obtained from a "translated amino acid" (for example, when a hit compound is obtained with D-tyrosine, a D-amino acid chemically modified therefrom, and when a hit compound is obtained with β-alanine, a β-amino acid chemically modified therefrom), or amino acids that can be chemically synthesized by structural optimization of the N-substituted portion by chemical conversion of an N-methyl amino acid. These amino acids function as components of a drug-like peptide compound, and are selected from a range that makes the peptide compound drug-like by chemical modification performed after translation. As described below, for example, lysine having an aminoalkyl group is not included in drug-like amino acids if the amino group is not involved in post-translational modification. However, when the amino group of lysine is utilized as a reactive functional group in post-translational modification (e.g., a crossover unit), the lysine unit is included as a drug-like amino acid unit. In this way, whether or not an amino acid is a "drug-like amino acid" is determined by the functional group after conversion by post-translational modification. Examples of such substituents that can be used include, from the substituents defined separately above, for example, ester groups (-CO-OR), thioester groups (-CO-SR), thiol groups (-SH), or protected thiol groups, amino groups (-NH 2 ), mono-substituted amino group (-NH-R) or di-substituted amino group (-NRR'), or a protected amino group, a substituted sulfonylamino group (-NH-SO 2 -R), alkylborane group (-BRR'), alkoxyborane group (-B(OR)(OR')), azide group (-N 3 ), keto acid group (-CO-CO2 Examples of such methylene groups include an indole skeleton and / or a substituted or unsubstituted hydroxyphenyl group. The details will be described later, but in one embodiment of the present disclosure, the drug-like amino acid does not include an indole skeleton and / or a substituted or unsubstituted hydroxyphenyl group. The non-adjacent 1 or 2 methylene groups in the side chain of the drug-like amino acid are replaced by an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) may be substituted.

[0071] In the present specification, examples of the "substituent that contributes to drug-likeness" include halogen (F, Cl, Br, I, etc.), hydroxy (-OH), alkoxy group (-OR), oxy (-OR), amide (-NR-COR' or -CO-NRR'), sulfonyl (-SO 2 -R), sulfinyl (-SOR), oxyamino (-NR-OR'), aminooxy (-O-NRR'), oxycarbonyl (-CO-OR), thiocarbonyl (-CO-SR), thiol (-SH), thio (-SR), primary amino (-NH 2 ), secondary amino (-NHR) or tertiary amino (-NRR'), sulfonylamino (-NH-SO 2 -R), boryl (-BRR'), dioxyboryl (-B(OR)(OR')), azide (-N 3 ), carboxycarbonyl (-CO-CO 2 Examples of the substituents include hydrogen, phosphorylcarbonyl (--CO--PO(R)(R')), carbonyloxyamino (--NH--O--CO--R), hydroxyamino (--NR--OR'), and aminohydroxy (--O--NRR').

[0072] As used herein, the term "fused ring structure" refers to a cyclic structure in which two or more rings share two or more atoms in a cyclic compound having two or more rings. The term "fused ring structure of two or more aromatic rings" refers to a cyclic structure in which two or more aromatic rings share two or more atoms in a cyclic compound having two or more aromatic rings. 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.

[0073] In the present specification, the term "substituted hydroxyphenyl group" refers to a group in which at least one hydrogen atom of the aromatic ring of the hydroxyphenyl group is substituted with a substituent. The substituent is selected in the same manner as the substituent in the amino acid side chain described above, and is preferably exemplified by halogen, among which fluorine is preferred. Although not intended to be limiting, an example of the substituted hydroxyphenyl group is 3-fluoro-4-hydroxyphenyl group. Note that the hydrogen atom of the aromatic ring in the present specification does not include H of the hydroxy group (-OH) in the hydroxyphenyl group. For example, a methoxyphenyl group is not included in either the "substituted hydroxyphenyl group" or the "unsubstituted hydroxyphenyl group" in the present specification.

[0074] In this specification, the term "unsubstituted hydroxyphenyl group" refers to a hydroxyphenyl group that has no substituent. In addition, a substituted hydroxyphenyl group and an unsubstituted hydroxyphenyl group may be collectively referred to as a "substituted or unsubstituted hydroxyphenyl group."

[0075] In this specification, the phrase "having no substituted or unsubstituted XX group" means having neither a substituted XX group nor an unsubstituted XX group.

[0076] In the present specification, the term "heterocyclic group" refers to a group having at least one heteroatom (e.g., N, O, S, etc.) in the ring. 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. Preferred examples of the heterocyclic group include a pyridyl group, a piperidino group, a morpholino group, and an azetidinyl group.

[0077] As used herein, the term "translational synthesis" refers to the synthesis of a peptide moiety by translation from a nucleic acid (e.g., DNA, RNA) that codes for the peptide moiety. Translation is a process in which a linear peptide is obtained by repeating amide bond and / or ester bond reactions using mRNA as a template by the action of ribosomes.

[0078] As used herein, the term "post-translational modification" refers to a chemical reaction that occurs automatically or by enzymes or reagents after translation other than by the action of ribosomes, and examples of such a reaction include, but are not limited to, a deamino acid reaction, a cyclization reaction, a desulfurization reaction, a deprotection reaction, and a reaction for forming a linear portion.

[0079] In this specification, "consisting essentially of" means that the components listed therein (examples include, but are not limited to, cyclic peptide compounds, amino acids, and nucleic acids) are the main components, and other components may be included as long as they do not negatively affect the effects of one aspect of the present invention, or in amounts or forms that do not negatively affect the effects of one aspect of the present invention. For example, components not listed therein (e.g., by-products, unreacted products, etc.) may be included as long as they do not negatively affect the effects of one aspect of the present invention, or in amounts or forms that do not negatively affect the effects of one aspect of the present invention.

[0080] As used herein, "substantially free of" means that it does not contain the components listed therein (examples include, but are not limited to, cyclic peptide compounds, amino acids, nucleic acids, etc.), or if it contains the components, the components do not have a negative effect on the effects of one aspect of the present invention, or the amount or aspect does not have a negative effect. For example, it may contain even the components listed therein, so long as they do not have a negative effect on the effects of one aspect of the present invention, or the amount or aspect does not have an effect.

[0081] In this specification, the term "negative impact" as used in the context of the effects of an invention refers to an impact that cancels out 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.

[0082] For example, when the hit rate that should be obtained by panning is 1%, a negative impact can be said to occur when the hit rate becomes 0.3% or less. In addition, when the rate of hit compounds not containing Trp that should be obtained by panning is 100%, a negative impact can be said to occur when the rate of hit compounds not containing Trp becomes 30% or less, but the negative impact is not limited to these examples.

[0083] Peptide Compounds Although not intended to be limiting, the "peptide compound" in the present disclosure includes linear peptide compounds and cyclic peptide compounds. Furthermore, the peptide compound in the present disclosure includes peptides, complexes of peptides and nucleic acids (peptide-nucleic acid complexes), complexes of peptides, ribosomes and nucleic acids, and the like. Although not intended to be limiting, the peptide compound in the present disclosure includes linear peptides, cyclic peptides, complexes of linear peptide moieties and nucleic acids (linear peptide-nucleic acid complexes), and complexes of cyclic peptide moieties and nucleic acids (cyclic peptide-nucleic acid complexes). In a non-limiting embodiment, the cyclic portion of the cyclic peptide compound is formed by subjecting a peptide compound synthesized by translation to a cyclization reaction. In the present specification, the peptide itself or the peptide portion in the peptide-nucleic acid complex may be referred to as the "peptide portion". Furthermore, the "nucleic acid" in the present disclosure includes DNA, mRNA, and tRNA. In the present specification, the "amino acids" constituting the peptide compound may be referred to as "amino acid residues". In the present specification, a peptide compound having a cyclic portion may be referred to as a "cyclic peptide compound". As used herein, the "cyclic portion" of a peptide compound refers to a cyclic portion formed by linking two or more amino acid residues. As used herein, the "straight chain portion" used to refer to a partial structure of a cyclic peptide compound refers to a portion not included in the main chain structure of the cyclic portion, which has at least one amide bond and / or ester bond on the chain of the portion. In addition, the "peptide compound" as used herein may include its pharma- ceutically acceptable salts.

[0084] The "peptide moiety" of the peptide compound in the present specification refers to a moiety in which 2 to 100, 3 to 50, or 4 to 30 amino acids are linked by amide bonds and / or ester bonds. However, when the peptide moiety includes a cyclic moiety, the bonding mode of the cyclization moiety is not particularly limited. The bonding mode of the cyclization moiety is preferably cyclized via a covalent bond 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, or a bond via a fluorophore structure, and among these, an amide bond is preferred because of its high metabolic stability. That is, in one embodiment, the cyclic peptide compound in the present disclosure preferably has an amide bond at the cyclization moiety. The "bonding mode of the cyclization moiety" refers to the bonding mode of the moiety in which the cyclization moiety is formed by a cyclization reaction.

[0085] As used herein, the term "cyclization reaction" refers to a reaction that forms a cyclic portion at the peptide portion of a peptide compound. In one embodiment, a peptide compound obtained by further chemically modifying or reacting a peptide compound in which a cyclic portion has been formed by a cyclization reaction is also included in the peptide compound of the present disclosure. In addition, chemical modification or the like may be performed before the cyclization reaction.

[0086] Scheme A is an example of a cyclization reaction of the peptide portion of a peptide compound according to the present disclosure. The open circle (○) unit (intersection unit), the black circle (●) unit (cyclic main chain unit), the square (■) unit (straight main chain unit), and the triangle (▲) unit (cyclized N-terminal unit) each represent an amino acid residue constituting the peptide portion. Each unit may be the same amino acid or different amino acids.

[0087] [ka]

[0088] As used herein, the term "unit" refers to any of the amino acids before cyclization after synthesis of the linear peptide compound of the present disclosure, the amino acids after cyclization, and the amino acids at the time point when chemical modification after cyclization is completed. The amino acid residues at the time point when chemical modification after cyclization is completed include, for example, amino acid residues that have been translated by one tRNA and have been chemically converted or backbone-converted by post-translational chemical modification.

[0089] In Scheme A, the cyclic portion is a portion consisting of one triangle unit, eight black circle units, and one white circle unit, and the linear portion is a portion consisting of six square units. The curved portion in Scheme A is the portion to be cyclized (also referred to as the "cyclization portion" or "post-translational cyclization portion" in this specification).

[0090] In one embodiment, the peptide moiety of the peptide compound of the present disclosure requires reactive functional groups in the triangle unit or intersection unit shown in Scheme A, and therefore, in the peptide compound before cyclization, drug-like amino acids are not necessarily selected for the triangle unit or intersection unit.

[0091] In one aspect, even if the triangle unit or intersection unit in a peptide compound before cyclization is not a drug-like amino acid or is an amino acid that is not originally preferred, the triangle unit or intersection unit can be made into a drug-like amino acid or a preferred amino acid by performing chemical modification or the like after cyclization.

[0092] In one embodiment, the triangle unit (pre-cyclization N-terminal unit) in the peptide portion of the peptide compound of the present disclosure before cyclization can be an amino acid residue having a thiol group adjacent to the amino group, both of which are protected by protecting groups.

[0093] In one embodiment, the intersection unit in the peptide portion before cyclization of the peptide compound of the present disclosure preferably uses an amino acid having a functional group (second reaction site) in its side chain that can be cyclized by reacting with a functional group (first reaction site) possessed by an amino acid in the triangle unit. Since the amino group and carboxy group in the main chain are used to form a covalent bond with other amino acid residues in translation synthesis or the like, and a third functional group is required for cyclization, the intersection unit preferably has a total of three or more functional groups. Among these, it is preferable to use a functional group in the side chain portion of the intersection unit for the cyclization reaction.

[0094] The intersection unit can be incorporated at any position in the peptide moiety before cyclization as long as it is a position that allows cyclization, but it is preferably incorporated at a position that results in a cyclic portion having 5 to 20 or 7 to 20 amino acids after cyclization or post-translational modification after cyclization having 5 to 13 or 8 to 13 amino acids, and more preferably at a position that results in 8 to 11 amino acids. That is, the intersection unit of the peptide compound of the present disclosure is preferably incorporated at a position at least 4 amino acid residues (e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues) C-terminal to the triangle unit.

[0095] The black circle units and square units are selected from amino acids, preferably drug-like amino acids.

[0096] In a non-limiting embodiment, the peptide moiety of the cyclic peptide compound of the present disclosure may have one or more, or two or more linear portions, and the number of linear portions is, for example, 0, 1, or 2. The cyclic peptide compound of the present disclosure is not particularly limited, and can be described, for example, as Scheme A-1 or A-2. [ka]

[0097] As used herein, the "number of amino acids" refers to the number of amino acid residues (amino acid units) constituting a peptide, and means the number of amino acid units generated when the amide bonds, ester bonds, and cyclized bonds that link the amino acids are cleaved. Therefore, the number of amino acids in a peptide compound or peptide moiety (also referred to as the number of amino acids constituting the peptide compound or peptide moiety) refers to the number of amino acid units generated when the amide bonds, ester bonds, and cyclized bonds contained in the entire peptide moiety, including the cyclic and linear portions, excluding the nucleic acid portion, are cleaved.

[0098] As used herein, "amino acids constituting the cyclic portion" (also referred to as "amino acids of the cyclic portion") refers to the amino acids present on the main chain of the cyclic portion among the amino acids generated when the amide bond, ester bond, and bond of the cyclized portion present in the peptide portion of a cyclic peptide compound are cleaved, and does not include the amino acids constituting the linear portion. As used herein, "the number of amino acids constituting the cyclic portion" refers to the number of amino acid units present on the main chain of the cyclic portion.

[0099] The number of amino acids constituting the peptide portion in the cyclic peptide compound of the present disclosure is not particularly limited, but is preferably 30 or less. In order to obtain high membrane permeability, the number of amino acids in the peptide portion other than the nucleic acid linking portion is preferably 20 or less, more preferably 18 or less, 16 or less, 15 or less, or 14 or less, particularly preferably 13 or less, specifically exemplified as 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. In order to obtain high metabolic stability, the number of amino acids in the peptide portion other than the nucleic acid linking portion is preferably 8 or more, more preferably 9 or more, even more preferably 10 or more, and particularly preferably 11 or more. In consideration of both membrane permeability and metabolic stability, the number of amino acids in the peptide portion other than the nucleic acid linker is preferably 5 to 20, or 7 to 20, more preferably 7 to 17, 8 to 16, 9 to 16, or 10 to 16, even more preferably 8 to 13, 10 to 15, 11 to 15, 10 to 14, 10 to 13, or 11 to 14, and particularly preferably 11 to 13.

[0100] In this specification, the term "peptide site other than the nucleic acid linking part" refers to a part of the cyclic peptide-nucleic acid complex excluding the linear part (also referred to as "nucleic acid linking part") to which the nucleic acid is linked. In addition, in a peptide compound that does not have a nucleic acid linking part, the entire peptide site is referred to as the "peptide site other than the nucleic acid linking part". For example, in the above scheme A-2, when a nucleic acid is linked to the end of the linear part 1, the part other than the linear part 1, i.e., the part consisting of the cyclic part and the linear part 2, becomes the "peptide site other than the nucleic acid linking part". Therefore, in the cyclic peptide-nucleic acid complex, the "number of amino acids constituting the peptide site other than the nucleic acid linking part" or the "number of amino acids of the peptide site other than the nucleic acid linking part" refers to the number of amino acid units of the part excluding the amino acids of the linear part to which the nucleic acid is linked from the amino acids constituting the peptide site. The "peptide site other than the nucleic acid linking part" is preferably a part excluding the nucleic acid linking part in the cyclic peptide-nucleic acid complex.

[0101] The number of amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure is not limited, and examples thereof include 4 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 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 5 to 15, more preferably 5 to 14, 7 to 14, or 8 to 14, further preferably 8 to 13, 9 to 13, 8 to 12, 8 to 11, or 9 to 12, and particularly preferably 9 to 11.

[0102] 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. In a non-limiting embodiment, the "linear portion" in this specification may contain natural amino acids and non-natural amino acids (including chemically modified or backbone-converted amino acids).

[0103] In a non-limiting embodiment, the number of unnatural amino acids contained in the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compound of the present disclosure is preferably 2 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 preferably 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 10 or less, or 9 or less. Examples of the number of unnatural amino acids contained in the cyclic peptide compound of the present disclosure are 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 or the number of amino acids contained in the peptide portion other than the nucleic acid linkage portion.

[0104] In a non-limiting embodiment, the number of N-substituted amino acids contained in the peptide site other than the nucleic acid linkage of the cyclic peptide compound of the present disclosure is preferably 3 or more, more preferably 4 or more, 5 or more, or 6 or more, even more preferably 7 or more, particularly preferably 8 or more, and also 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 the cyclic peptide compound of the present disclosure is exemplified by 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 or the number of amino acids contained in the peptide site other than the nucleic acid linkage. The N-substituted amino acid referred to here may be preferably an N-alkyl amino acid, more preferably an N-methyl amino acid. That is, in a non-limiting embodiment, the number of the N-substituted amino acids is exemplified as the number of N-alkyl amino acids or the number of N-methyl amino acids, but even in this case, it is not denied that other N-substituted amino acids are contained in the peptide site other than the nucleic acid linkage.

[0105] Although not intended to be limiting, as shown in the examples below, the number or ratio of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion can affect the membrane permeability of the cyclic peptide compound. Even in a cyclic peptide compound that does not contain tryptophan or tyrosine residues, if the number or ratio of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion exceeds a certain number, the probability of obtaining a compound with high membrane permeability may decrease.

[0106] In a non-limiting embodiment, for high membrane permeability, the number of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion of the cyclic peptide compound of the present disclosure is preferably 3 or less, preferably 0, 1, 2, or 3, and preferably 0 to 3, 1 to 3. Furthermore, the ratio of the number of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion to the number of amino acids constituting the peptide portion other than the nucleic acid linking portion, or the cyclic portion, is preferably 40% or less, preferably 35% or less, 30% or less, 27% or less, 25% or less, or 20% or less.

[0107] In a non-limiting embodiment, since the number of aromatic rings that may be contained in the cyclic peptide compound of the present disclosure is limited, it is preferable that the aromatic ring is present in the long side chain of the cyclic portion, which is a portion that can contribute to binding with a target molecule, in order to achieve high membrane permeability. That is, in one embodiment, the number of aromatic rings contained in the long side chain of the cyclic portion of the cyclic peptide compound of the present disclosure is 1, 2, or 3, and is exemplified in the range of 1 to 3 or 2 to 3. In addition, the ratio of the number of aromatic rings contained in the long side chain of the cyclic portion to the number of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion of the cyclic peptide compound of the present disclosure is 30% or more, 45% or more, 60% or more, 80% or more, or 100%.

[0108] As used herein, the "number of aromatic rings" (also referred to as "Aromatic Ring Count" (ARC)) refers to the number of aromatic rings contained in the peptide portion other than the nucleic acid linking portion of a cyclic peptide compound, the cyclic portion, or the side chain of the cyclic portion; 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.

[0109] In a non-limiting embodiment, the ClogP of the cyclic peptide compound of the present disclosure 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, and examples thereof include 4 to 18, 5 to 17, and 6 to 16. In the present specification, ClogP is a partition coefficient calculated by a computer, and can be calculated using Daylight Version 4.9 from Daylight Chemical Information Systems, Inc.

[0110] In a non-limiting embodiment, ClogP / total aa of the cyclic peptide compound of the present disclosure is preferably 1.0 or more, more preferably 1.1 or more, even more preferably 1.2 or more, and is preferably 1.8 or less, 1.7 or less, 1.6 or less, or 1.5 or less, and is exemplified by 1.0 to 1.8, 1.0 to 1.7, 1.1 to 1.6, and 1.1 to 1.5. In this specification, "total aa" (also written as "total AA") means the number of amino acids constituting the peptide portion other than the nucleic acid linkage portion of the peptide compound. For example, the total aa of a cyclic peptide compound in which the cyclic portion is composed of 10 amino acids and the linear portion to which the nucleic acid is not linked is composed of one amino acid is 11. In this specification, ClogP / total aa is calculated by dividing ClogP by total aa.

[0111] In a non-limiting embodiment, the molecular weight of the cyclic peptide compound of the present disclosure, or the peptide portion thereof other than the nucleic acid linking portion, may be 500-2,000.

[0112] 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 not included in the main chain structure of each. However, when a nucleic acid is bound to the peptide portion of a cyclic peptide compound, the nucleic acid portion and the nucleic acid linking portion are not included in the side chain of the cyclic portion.

[0113] In the present specification, the side chain of an amino acid includes a chain bound to a carbon atom (e.g., α, β, or γ-carbon atom) contained in the amino acid, and a chain bound to a nitrogen atom. In the present specification, the length of the side chain of an amino acid can be determined by the following method. That is, the N-terminus of the amino acid unit is capped with an acetyl group and the C-terminus with a methylamino group, a conformation is generated by LowModeMD of the molecular modeling software MOE (Chemical Computing Group, Inc.), and the distance from the atom to which the side chain moiety is bound (in the case of natural amino acids, the α-carbon atom (Cα carbon)) to the atom of the same side chain that is the furthest (excluding hydrogen atoms) can be determined. As an example, the method for determining the side chain length of Phe or nBuGly is shown below. [ka]

[0114] In addition, when an amino acid side chain forms a ring with part of the main chain structure, there are multiple atoms to which the side chain moiety is bonded. In that case, the length of the side chain is calculated as the longest distance between the atom to which each side chain moiety is bonded and the atom in the side chain that is the furthest away (excluding hydrogen atoms). For example, in the case of Hyp(Et), the distance of the side chain from the nitrogen atom is 6.04 Å, and the distance from the Cα carbon is 6.09 Å, so the distance of the side chain of Hyp(Et) is 6.09 Å. [ka]

[0115] The lengths of amino acid side chains calculated by the above method are shown in Tables 1-1 and 1-2. [Table 1-1] [Table 1-2]

[0116] In the present specification, the term "side chain of the cyclic part" refers to a part that is not included in the main chain structure of the cyclic part of the cyclic peptide compound. The starting point of the side chain of the cyclic part (the atom to which the side chain part is bonded) is not particularly limited, and is exemplified by a carbon atom and a nitrogen atom that constitute the cyclic part, among which the carbon atom is exemplified by the α-, β-, and γ-carbon atoms of the amino acid that constitutes the cyclic part. The length of the side chain of the cyclic part of the cyclic peptide compound can be calculated for each amino acid that constitutes the cyclic part by using the above-mentioned calculation method for the length of the side chain of the amino acid. In the present specification, the "side chain of the cyclic part" may have an amide bond and / or an ester bond on the side chain. For example, when the cyclic peptide compound has a linear part (for example, linear part 2 in Scheme A-2, which is composed of three or more amino acids) and the linear part is not linked to a nucleic acid, the linear part can also be the "side chain of the cyclic part". In the present specification, the term "side chain of the peptide part" refers to the side chain of the amino acid that constitutes the peptide part.

[0117] In a non-limiting embodiment, the amino acids constituting the cyclic portion or peptide portion of the cyclic peptide compound of the present disclosure may include at least one, two, or three amino acids having long side chains. In addition, the amino acids having long side chains may be consecutive, or may be sandwiched between other amino acids. The cyclic peptide compound of the present disclosure may include at least one, two, or three long side chains in its cyclic portion or peptide portion. In one embodiment, the cyclic peptide compound of the present disclosure may have a long side chain in its cyclic portion or peptide portion. This long side chain may provide high binding affinity with the target molecule. In one embodiment, by appropriately selecting the long side chain, a cyclic peptide compound having high membrane permeability can be obtained while maintaining binding affinity with the target molecule.

[0118] In this specification, "long side chain" refers to a side chain having a length of 5.4 angstroms or more. The length of the side chain is preferably 5.8 angstroms or more or 6.0 angstroms or more, more preferably 6.2 angstroms or more, and particularly preferably 6.4 angstroms or more. There is no particular upper limit to the length of the side chain, 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. The length of the side chain is exemplified as 5.4 to 20 angstroms, 5.4 to 15 angstroms, 5.4 to 13 angstroms, 5.4 to 10 angstroms, 5.4 to 9.0 angstroms, 6.0 to 20 angstroms, 6.0 to 15 angstroms, 6.0 to 13 angstroms, 6.0 to 10 angstroms, 6.0 to 9.0 angstroms, 6.0 to 8.5 angstroms, and 6.0 to 8.0 angstroms. In one embodiment, the "long side chain" may be (i) a side chain that does not contain an amide bond or contains one amide bond on the side chain, or (ii) a side chain that does not contain an amide bond on the side chain. That is, in a non-limiting embodiment, a preferred example is a cyclic peptide compound that does not have two or more amide bonds in the side chain and has a long side chain of 6.0 to 10 angstroms in length in the cyclic part. The long side chains in the present disclosure may or may not have an aromatic ring. In a non-limiting embodiment, the cyclic peptide compounds in the present disclosure may have an aromatic ring in at least one, or two or more, long side chains contained in the cyclic portion.

[0119] In the present specification, the "long side chain" may be a chain having 6 or more, 7 or more, or 8 or more atoms from the atom to which the side chain moiety is bonded (the α-carbon atom in the case of natural amino acids) to the atom in the same side chain that is the furthest (excluding hydrogen atoms) and may be 15 or less, 13 or less, 11 or less, 10 or less, 9 or less, or 8 or less. Specific examples of the number of atoms include 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 or less. When counting the "number of atoms in the chain from the atom to which the side chain moiety is bonded to the atom in the same side chain that is the furthest," the atom to which the side chain moiety is bonded (the α-carbon atom in the case of natural amino acids, but may be a nitrogen atom in the case of N-substituted amino acids, for example) is included. That is, for example, the number of atoms in Leu, Phe, and Tyr is 4, 6, and 7, respectively.

[0120] In one non-limiting embodiment, the long side chain herein is R 1 may include:

[0121] In a non-limiting embodiment, the long side chain in this specification may be a side chain represented by the following general formula (I-1) or (I-2).

[0122] [ka]

[0123] In this specification, [ka] represents the attachment point, [ka] represents the point of attachment to a nitrogen or carbon atom, [ka] represents the point of attachment to the carbon atom.

[0124] [ka] The carbon atom to which is bonded is [ka] may be the same as or different from the carbon atom to which it is attached.

[0125] B 1 is preferably a single bond, an optionally substituted C1-C5 alkylene group, or a C3-C8 cycloalkylene group, in which one or two non-adjacent methylene groups of the alkylene group or cycloalkylene group may be substituted with an oxygen atom. 1 When is an alkylene group, the long side chain in this specification is also represented by the following general formula (I-3).

[0126] -(CH 2 ) n -R 1 (I-3) [In the formula, n is an integer from 1 to 5; One or two non-adjacent methylene groups in the alkylene group may be replaced by an oxygen atom.]

[0127] B 2 As the group, a group represented by the following general formula (I-4) or (I-5) is preferable.

[0128] [ka] [In the formula, q is an integer from 1 to 3; M 1 represents an optionally substituted C1-C4 alkylene group, in which 1 or 2 non-adjacent methylene groups are optionally substituted with an oxygen atom; M 2 represents a hydrogen atom or an optionally substituted C1-C4 alkyl group; M 1 and M. 2may form a 3- to 6-membered ring structure together with the atom to which they are bonded, and the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, and may have an oxygen atom in the ring.

[0129] In the general formula (I-4), M 1 and M. 2 Examples of the partial structure of an amino acid having a side chain in which the ring structure is formed of 3 to 6 members together with the atom to which it is bonded include the following. [ka]

[0130] In one non-limiting embodiment, R 1 teeth, (a) a phenyl group, or (b) a 4- to 6-membered heterocyclic group having 1 to 3 heteroatoms independently selected from the group consisting of oxygen atoms and nitrogen atoms in the ring; Here, (a) and (b) may each have at least one substituent independently selected from the following Group A: Group A: oxo, a halogen atom, a C1-C4 alkyl group optionally substituted with a halogen atom, and a C1-C4 dialkylamino group (wherein one or two non-adjacent methylene groups in the C1-C4 alkyl group may be substituted with an oxygen atom). Or, (c) a compound represented by the following general formula (II), (III), or (IV): [ka] [In the formula, X 1 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, X 2 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, Y 1is a single bond, an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Y 2 is a single bond, a C1-C2 alkylene group optionally substituted with a C1-C4 alkyl group, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Z 1 represents a single bond, a methylene group which may have at least one substituent independently selected from the following Group B, or an oxygen atom, R 2 and R 3 each independently represents a hydrogen atom, a C1-C6 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, wherein the C1-C6 alkyl group is optionally substituted with a halogen atom, and one or two non-adjacent methylene groups in the C1-C6 alkyl group are optionally substituted with an oxygen atom; R 2 and R 3 , or R 2 and X 1 may form a 4- to 6-membered ring structure together with the atom to which they are attached, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 4 represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C1-C6 alkanoyl group; R 5 represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a C2-C6 alkynyl group; R 4 and / or R 5 In the C1-C6 alkyl group, one or two non-adjacent methylene groups may be replaced by an oxygen atom; R 4 and R 5 , or R 5 and X 2may form a 4- to 6-membered ring structure together with the atom to which they are attached, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 6 , R 7 , R 8 , R 9 , and R 10 each independently represents a hydrogen atom, a halogen atom, a C1-C4 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, and one or two non-adjacent methylene groups in the C1-C4 alkyl group may be replaced by an oxygen atom. Group B: halogen atoms, C1-C4 alkyl groups, and C1-C2 alkoxy groups. It may be.

[0131] In one embodiment, the M 1 and M. 2 R when they form a ring structure together with the atoms to which they are attached 1 In addition to the above, may be a group selected from the group consisting of a halogen atom, an optionally substituted C1-C4 alkyl group, and a C1-C4 alkoxy group.

[0132] In a non-limiting embodiment, Y in the general formula (II) 1 is preferably an oxygen atom, and X 1 is preferably an optionally substituted C1-C2 alkylene group, and more preferably an unsubstituted C1-C2 alkylene group; 2 and R 3 are each independently preferably a hydrogen atom or a C1-C6 alkyl group, and the C1-C6 alkyl group is preferably substituted with a halogen atom.

[0133] In one non-limiting embodiment, R 5 If is a hydrogen atom, Y 2 is a single bond, a carbonyl group or a sulfonyl group, of which a carbonyl group is preferred; X 2is an optionally substituted C1-C2 alkylene group, preferably an unsubstituted C1-C2 alkylene group; Z 1 is preferably an oxygen atom.

[0134] In a non-limiting embodiment, R in the general formula (III) 5 is a C1-C6 alkyl group that does not form a cyclic structure, Y 2 is a single bond, an optionally substituted C1-C2 alkylene group, a carbonyl group or a sulfonyl group, among which an unsubstituted C1-C2 alkylene group or a carbonyl group is preferable; X 2 is preferably a single bond, an optionally substituted C1-C2 alkylene group, or an unsubstituted C1-C2 alkylene group; Z 1 is preferably a single bond, an optionally substituted methylene group, or an unsubstituted methylene group.

[0135] In one non-limiting embodiment, R 4 and R 5 When they form a 4- to 6-membered ring structure together with the atom to which they are bonded, the ring may contain an oxygen atom, and the ring structure may be substituted with one or more C1-C4 alkyl groups and / or halogen atoms; X 2 is preferably a single bond, an optionally substituted C1-C2 alkylene group, or an unsubstituted C1-C2 alkylene group; Y 2 is a single bond or an optionally substituted C1-C2 alkylene group, preferably an unsubstituted C1-C2 alkylene group; Z 1 is preferably a single bond, an optionally substituted methylene group, an unsubstituted methylene group, or an oxygen atom.

[0136] In one non-limiting embodiment, R 5 and X 2 When Y is bonded to an atom that is a 4- to 6-membered ring, the ring may contain an oxygen atom, and the ring may be substituted with one or more C1-C4 alkyl groups and / or halogen atoms. 2is a single bond or an optionally substituted C1-C2 alkylene group, preferably an unsubstituted C1-C2 alkylene group; Z 1 is preferably an oxygen atom, R 4 is preferably a C1-C4 alkyl group.

[0137] In a non-limiting embodiment, R in general formula (IV) 6 , R 7 , R 8 , R 9 , and R 10 are each independently preferably a hydrogen atom, a halogen atom, a methyl group, or an ethyl group. As the halogen atom, F or Cl is preferable, and F is more preferable.

[0138] In one non-limiting embodiment, R 1 In the case where the phenyl group (a) has a substituent, the substituent is preferably a halogen atom, a C1-C4 haloalkyl group, or a C1-C4 dialkylamino group, and among these, F, Cl, a fluoromethyl group (such as a trifluoromethyl group or a difluoromethyl group), or a dimethylamino group. When multiple substituents are selected, they may be the same or different.

[0139] In one non-limiting embodiment, R 1 In the above formula (b), the 4- to 6-membered heterocyclic group having 1 to 3 heteroatoms independently selected from the group consisting of oxygen and nitrogen atoms in the ring is preferably a pyridyl group, a piperidino group, a morpholino group, or an azetidinyl group, and these groups may have at least one substituent independently selected from the following Group A: Group A: oxo, a halogen atom, a C1-C4 alkyl group optionally substituted with a halogen atom, and a C1-C4 dialkylamino group (wherein 1 or 2 non-adjacent methylene groups in the C1-C4 alkyl group may be optionally substituted with an oxygen atom). Preferred examples of halogen atoms include F and Cl, and preferred examples of C1-C4 alkyl groups optionally substituted with halogen atoms include fluoromethyl groups (trifluoromethyl groups, difluoromethyl groups, etc.), methyl groups, and ethyl groups, and preferred examples of C1-C4 dialkylamino groups include dimethylamino groups. When multiple substituents are selected, they may be the same or different.

[0140] In a non-limiting embodiment, the optionally substituted phenyl group in the present disclosure includes an unsubstituted phenyl group, a phenyl group substituted with 1 to 5, preferably 1 to 3, halogen atoms (halophenyl group), a phenyl group substituted with a C1-C4 alkyl group (C1-C4 alkyl group or C1-C4 haloalkyl group) optionally substituted with a halogen atom (C1-C4 alkyl group or C1-C4 haloalkylphenyl group), and a dialkylaminophenyl group. Specific examples include a chlorophenyl group (e.g., 2-, 3-, or 4-chlorophenyl group), a dichlorophenyl group, a trichlorophenyl group, a fluorophenyl group (e.g., 2-, 3-, or 4-fluorophenyl group), a difluorophenyl group, a trifluorophenyl group, a fluoromethylphenyl group (e.g., trifluoromethylphenyl group, difluoromethylphenyl group), and a dimethylaminophenyl group.

[0141] In a non-limiting aspect, examples of the optionally substituted pyridyl group in the present disclosure include an unsubstituted pyridyl group, a chloropyridyl group (e.g., a 2-, 3-, or 4-chloropyridyl group), a fluoropyridyl group (e.g., a 2-, 3-, or 4-fluoropyridyl group), a methylpyridyl group, an ethylpyridyl group, and a dimethylaminopyridyl group.

[0142] In a non-limiting embodiment, examples of the optionally substituted piperidino group in the present disclosure include an unsubstituted piperidino group, a chloropiperidino group (e.g., a 2-, 3-, or 4-chloropiperidino group), a dichloropiperidino group (e.g., a 2,2-, 3,3-, or 4,4-dichloropiperidino group), a fluoropiperidino group (e.g., a 2-, 3-, or 4-fluoropiperidino group), a difluoropiperidino group (e.g., a 2,2-, 3,3-, or 4,4-difluoropiperidino group), a methylpiperidino group, and an ethylpiperidino group.

[0143] In a non-limiting embodiment, examples of the optionally substituted morpholino group in the present disclosure include an unsubstituted morpholino group, a chloromorpholino group (e.g., a 2- or 3-chloromorpholino group), a fluoromorpholino group (e.g., a 2- or 3-fluoromorpholino group), a methylmorpholino group, and an ethylmorpholino group.

[0144] In a non-limiting aspect, examples of the optionally substituted azetidinyl group in the present disclosure include an unsubstituted azetidinyl group, a chloroazetidinyl group (e.g., a 1-, 2-, or 3-chloroazetidinyl group), a fluoroazetidinyl group (e.g., a 1-, 2-, or 3-fluoroazetidinyl group), a methylazetidinyl group (e.g., a 1-, 2-, or 3-methylazetidinyl group, an N-methylazetidinyl group), and an ethylazetidinyl group (e.g., a 1-, 2-, or 3-ethylazetidinyl group).

[0145] The "long side chain" in the present specification is not particularly limited, but examples of amino acids having such long side chains are shown in Tables 2-1 to 2-6. Although not intended to be limiting, the side chain portions of these amino acids (when one amino acid has two or more side chains, the longest side chain portion) are exemplified as preferred embodiments of the long side chain in the present specification.

[0146] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0147] In a non-limiting embodiment, in order for the cyclic peptide compound of the present disclosure to achieve high membrane permeability, it is preferable that it does not have a side chain that negatively affects membrane permeability. In one embodiment, even if the peptide compound before cyclization contains a side chain that negatively affects the membrane permeability, high membrane permeability can be achieved if the cyclic peptide compound after cyclization does not contain such a side chain. Therefore, in one embodiment, for example, by performing chemical modification after the cyclization reaction, a cyclic peptide compound that does not have a side chain that negatively affects membrane permeability can be obtained. In addition, in a non-limiting embodiment, in the optimization step after obtaining a cyclic peptide compound that can specifically bind to a target molecule, chemical modification or the like can be performed to increase membrane permeability, and at this time, a side chain that negatively affects membrane permeability may be changed.

[0148] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure may be a cyclic peptide compound that does not have at least one selected from the group consisting of the following (A) to (D) in the side chain of the peptide portion other than the cyclic portion or the nucleic acid linking portion: (A) an indole skeleton; (B) a fused ring structure of two or more aromatic rings; (C) an unsubstituted hydroxyphenyl group; and (D) a substituted hydroxyphenyl group. Among these, a cyclic peptide compound that does not have the above (A), (C) and (D) in the side chain of the cyclic portion is preferred, and a cyclic peptide compound that does not have the above (B), (C) and (D) in the side chain of the cyclic portion is more preferred. In a non-limiting embodiment, the side chain of the cyclic portion may be a long side chain of the cyclic portion. In a non-limiting embodiment, the "fused ring structure of two or more aromatic rings" may be a "fused ring structure".

[0149] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure may be a cyclic peptide compound that does not have at least one or both of the following selected from the group consisting of (A) and (B) in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion: (A) a methylthio group; and (B) a thiol group.

[0150] In a non-limiting embodiment, in order to achieve high membrane permeability, it is preferable that the cyclic peptide compound of the present disclosure does not have a functional group that is highly ionized at neutral (e.g., pH=7.0) in the peptide portion other than the nucleic acid linkage portion or in the cyclic portion. As used herein, pKa refers to the measured pKa unless otherwise specified. In addition, the pKa value calculated by ADMETPredictor described later is referred to as the calculated pKa. As used herein, Basic pKa refers to the measured Basic pKa unless otherwise specified. In addition, the Basic pKa value calculated by ADMETPredictor described later is referred to as the calculated Basic pKa.

[0151] The pKa and basic pKa can be measured by a conventional method. 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 the Examples. In addition, when the pKa value of the side chain of the amino acid to be measured and the basic pKa value are difficult to obtain 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.

[0152] In a non-limiting embodiment, when the cyclic peptide compound of the present disclosure has an acidic side chain at the cyclic portion, the pKa of the acidic side chain is preferably 3.5 or more, more preferably 3.9 or more, more preferably 4.5 or more, more preferably 5.0 or more, more preferably 5.5 or more, more preferably 5.7 or more, and preferably 10 or less. The pKa is preferably in the range of 3.5 to 10, 3.9 to 10, 4.5 to 10, 5.0 to 10, or 5.5 to 10, 5.7 to 10. When these pKa are expressed as calculated values, the calculated pKa is preferably 3.5 or more, more preferably 4.5 or more, preferably 5.0 or more, preferably 5.4 or more, preferably 8.0 or more, preferably 8.3 or more, and preferably 10 or less. The calculated pKa is preferably in the range of 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.

[0153] In a non-limiting embodiment, when the cyclic peptide compound of the present disclosure has a basic side chain at the cyclic portion, the basic pKa of the basic side chain is preferably 10 or less, more preferably 9.5 or less, more preferably 9.0 or less, more preferably 8.5 or less, more preferably 7.5 or less, more preferably 7.2 or less, particularly preferably 6.5 or less, and preferably 4.0 or more. The basic pKa is preferably exemplified in the range of 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 are expressed as calculated values, the basic calculated pKa is preferably 10 or less, more preferably 9.5 or less, preferably 9.0 or less, preferably 8.8 or less, preferably 8.6 or less, preferably 8.5 or less, preferably 7.5 or less, preferably 6.5 or less, and preferably 4.0 or more. Preferred examples of the basic calculated pKa are in the range of 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.

[0154] As used herein, an "acidic side chain" refers to a side chain having a pKa of 10 or less, and a "basic side chain" refers to a side chain having a basic pKa of 4 or more. As used herein, a side chain having a pKa of more than 10 and a side chain having a basic pKa of less than 4 are defined as neutral side chains.

[0155] In this specification, the calculated pKa and Basic calculated pKa of the side chain of an amino acid or the side chain of the cyclic portion of a cyclic peptide compound can be determined using ADMETPredictor (Simulations Plus Inc., ver. 8.0). The calculated pKa and Basic calculated pKa are calculated using a partial structure in which the side chain portion is extracted from the side chain β position (carbon directly connected to the main chain). As an example, the case of Lys is shown below. The Basic calculated pKa was calculated to be 10.5 using a partial structure including the side chain β position (carbon directly connected to the main chain). Similarly, as for acids, the calculated pKa of the side chain carboxyl group of Asp was 4.3, the calculated pKa of the side chain phenolic hydroxyl group of Tyr was 9.9, the calculated pKa of the side chain phenolic hydroxyl group of 3-fluorotyrosine (Tyr(3-F)) was 8.7, and the calculated pKa of tetrazole was 3.7. On the other hand, as bases, the calculated basic pKa of the side chain guanidino group of Arg is 12.7, the calculated basic pKa of the imidazolyl group of His is 7.6, and the calculated basic pKa of pyridine is 5.4. At least one side chain selected from the group consisting of the side chain of an amino acid in the present disclosure, the side chain of a cyclic portion of a cyclic peptide compound, a long side chain, and a basic side chain may be a side chain having no proton donor. [ka]

[0156] Some of the amino acids having basic side chains for which the Basic calculated pKa values ​​have been calculated using the method described herein are shown in the table below. [Table 33]

[0157] Some of the amino acids with acidic side chains for which pKa have been calculated using the methods described herein are listed in the table below. [Table 34]

[0158] In a non-limiting embodiment, the peptide compound of the present disclosure may be post-translationally modified. The post-translational modification may be performed before and / or after cyclization. Examples of the post-translational modification include, but are not limited to, a deamino acid reaction, a cyclization reaction, a desulfurization reaction, a deprotection reaction, and a reaction for forming a linear portion.

[0159] Although not intended to be limiting, the deamination reaction in this specification includes a reaction to remove an N-terminal amino acid. The N-terminal amino acid may be a translation initiation amino acid, and specific examples thereof include fMet (formylmethionine), fNle (formylnorleucine), Met, and Nle. In one embodiment, the reaction to remove the N-terminal amino acid of a peptide compound includes a reaction to treat with peptide deformylase (PDF) and / or methionine aminopeptidase (MAP).

[0160] Peptide-Nucleic Acid Complexes In one embodiment, the peptide compound in the present disclosure may be a complex of a peptide and a nucleic acid (peptide-nucleic acid complex). The peptide-nucleic acid complex may be bound to a nucleic acid at the C-terminus side of the peptide site to form a complex. The binding method between the peptide and the nucleic acid is not particularly limited, but may be bound via a linker (spacer). In one embodiment, the nucleic acid forming a complex with the peptide site may be DNA or RNA, preferably RNA, and particularly preferably mRNA. In one embodiment, the nucleic acid forming a complex with the peptide may be a template used in the translational synthesis of the peptide site, and the template is preferably mRNA. As a more specific embodiment, a peptide compound in which the C-terminus of the peptide site is linked to the 3'-end of the template mRNA via a linker (spacer) containing an antibiotic puromycin, which is an analog of aminoacyl-tRNA, is preferably exemplified.

[0161] Cell membrane permeability of peptide compounds In this specification, "cell membrane permeability" may be referred to as "membrane permeability". A method using human colon cancer cell line Caco-2 cells has been reported as a method for measuring the membrane permeability of drugs and the like (also called "conventional method"; Artursson, P., 2001. Adv Drug Deliv Rev 46, 27-43.; Mason, AK, 2013. Drug Metab Dispos 41, 1347-1366.; Polli, JW, 2001. J Pharmacol Exp Ther 299, 620-628.; Sun, H. 2008. Expert Opin Drug Metab Toxicol 4, 395-411.). This method involves adding a substance to be measured (also called a "test substance") to the apical side of a cultured Caco-2 cell layer, and calculating the membrane permeability coefficient (P app ) is a method for measuring the membrane permeability of a test substance (see Figure 1). It has been revealed that the permeability of Caco-2 cells evaluated in such experiments correlates with the oral absorption rate in humans, and this correlation makes it possible to predict the oral absorption rate in humans. Caco-2 cells form a monolayer membrane when cultured under certain conditions, and the permeability of drugs through this monolayer membrane shows a good correlation with the oral absorption rate in humans, so this method is widely used as a method for evaluating oral absorption in vitro. However, as described below, it is difficult to accurately measure the membrane permeability of drugs with high lipid solubility with this conventional method.

[0162] In the membrane permeability test using Caco-2 cells, the membrane permeability coefficient (P appThe assumptions for calculating the drug concentration are that the intracellular distribution of the drug can be ignored, that the drug concentration on the basal side increases linearly, that the drug does not return to the cell once it has penetrated (i.e., sink condition), that the concentration change on the apical side is small, and that the accumulation of the drug in the cell is not taken into account (Bhoopathy, S., et al 2014. Methods Mol Biol 1113, 229-252.;Knipp, GT, et al 1997. J Pharm Sci 86, 1105-1110.;Korzekwa, KR, et al 2012. Drug Metab Dispos 40, 865-876.;Sun, H., et al 2008. Drug Metab Dispos 36, 102-123.).

[0163]

number

[0164] However, using the above formula 1, P app It has been reported that there are compounds for which the prerequisites for calculating the . For example, Ozeki et al. conducted a membrane permeability test using Caco-2 cells for drugs that permeate the membrane by passive diffusion (atenolol, metoprolol, propranolol) and substrates of P-glycoprotein (P-gp) (digoxin, cyclosporine, verapamil), and evaluated the concentration transitions on the apical side, intracellular side, and basal side. As a result, it was reported that the concentrations of metoprolol and propranolol on the apical side decreased by about 20% and about 40%, respectively, after 120 minutes of incubation (Ozeki K., et al 2015, Int J Pharm. 495, 963-971.). In other words, it is suggested that the concentration change on the apical side is large. In addition, for propranolol, cyclosporine, and verapamil, approximately 8% of the added drug was distributed inside the cells at the end of incubation, suggesting intracellular accumulation. Furthermore, there was a lag time (the intersection of the approximation line of the linear region and the x-axis) in the change in the basal concentration of cyclosporine (Ozeki K., et al 2015, Int JPharm. 495, 963-971.), and the time until the start of the linear portion (three times the lag time) exceeded 300 minutes in both the test in which the drug was added to the apical side and sampled from the basal side (AtoB) and the test in which the drug was added to the basal side and sampled from the apical side (BtoA), indicating that no linear region existed within the 120-minute incubation time. In other words, it is suggested that the drug concentration on the basal side does not increase linearly. P calculated using formula 1 only from the basal concentration after 120 minutes in the AtoB test was 0.01, and the linear region was 0.01, and the linear region was 0.01, and the linear region was 0.01, respectively. app is calculated using the optimal value of the model that takes into account the intracellular distribution. app This is thought to be because cyclosporine binds strongly to the intracellular matrix, slowing its appearance on the basal side (Ozeki K., et al 2015, Int J Pharm. 495, 963-971.). app The value is 2.5x10 -5 It has been reported that for compounds with a ClogP of 1.0 cm / sec, the linear region ends (also called "plateaus") within 120 minutes of incubation. Furthermore, drugs with a large ClogP bind strongly to the intracellular matrix, which delays the onset of the linear region, while the P of highly lipid-soluble drugs app This suggests that it is difficult to accurately evaluate this using conventional methods (Figure 2).

[0165] Therefore, in order to measure the membrane permeability of peptide compounds, particularly cyclic peptide compounds, an improved method of the conventional method has been developed. As shown in the examples, by using this improved method, i.e., the method for measuring membrane permeability in the present disclosure, it is possible to accurately measure the membrane permeability of peptide compounds. Note that the test substances that can be measured by this improved method are not limited to peptide compounds.

[0166] In the conventional method, the membrane permeability is measured without a pre-incubation step in a state where the test substance is mixed in. In the conventional method, a short incubation may be performed before the measurement of the membrane permeability in order to allow the buffer to blend in, but this is not an incubation in a state where the test substance is mixed in, and is therefore different from the pre-incubation in the present disclosure.

[0167] In a non-limiting embodiment, the method for measuring membrane permeability in the present disclosure (also referred to as the "measurement method in the present disclosure") includes a step of pre-incubating Caco-2 cells and a test substance mixed together. In the measurement method in the present disclosure, "pre-incubation" or "pre-incubation" refers to mixing Caco-2 cells and a test substance and incubating them before the membrane permeability measurement step. In the present specification, "mixing Caco-2 cells and a test substance and (pre-)incubating" is not particularly limited as long as there is a state in which Caco-2 cells and a test substance coexist during (pre-)incubation, and may be performed by, for example, mixing Caco-2 cells and a test substance in a medium and starting incubation. In addition, it may be arranged so that one side of a Caco-2 cell layer is in contact with a solution containing a test substance, and the other side of the Caco-2 cell layer is in contact with a solution not containing the test substance, and incubated for a predetermined time. In this specification, unless otherwise specified, a test substance is added to the solution on the apical side at the start of preincubation, but not to the solution on the basal side.

[0168] In a non-limiting embodiment of the measurement method of the present disclosure, a solution containing a test substance may be placed on the apical side of the Caco-2 cell layer during pre-incubation, or vice versa. The pre-incubation time in the measurement method of the present disclosure is not particularly limited, and examples thereof include 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 18 hours or more, 20 hours or more, and 24 hours or more. The upper limit of the pre-incubation time is not limited as long as it does not affect the measurement of membrane permeability, but a time that does not cause damage to Caco-2 cells is preferable, and examples thereof include 72 hours or less, 48 ​​hours or less, 36 hours or less, 30 hours or less, 26 hours, and 24 hours or less.

[0169] As used herein, the term "preincubation solution" refers to a solution that is brought into contact with Caco-2 cells during preincubation. The preincubation solution for the measurement method in the present disclosure is not particularly limited, but it is preferable to use a culture medium because preincubation does not damage Caco-2 cells. That is, preincubation in the present disclosure may be performed by contacting Caco-2 cells with a culture medium. As used herein, "no damage to Caco-2 cells" can be confirmed by measuring the transepithelial electrical resistance (TEER) of Caco-2 cells. As used herein, if the TEER value at the time of measurement is 70% or more, preferably 80% or more, compared to the initial value before preincubation, it is determined that no damage has occurred to Caco-2 cells at the time of measurement. TEER can be measured by a method known to those skilled in the art.

[0170] In a non-limiting embodiment, in the preincubation of the measurement method in the present disclosure, a medium may be used as a preincubation solution. In one embodiment, in the preincubation in the present disclosure, the preincubation solution on the apical side may be a medium. In one embodiment, as the preincubation solution, a medium may be used only on the apical side or only on the basal side, or a medium may be used on both sides, but it is preferable to use a medium at least on the apical side. In one embodiment, it is preferable to include a test substance in the preincubation solution on the apical side of Caco-2 cells. In the present specification, the medium is not particularly limited, but a medium that does not cause damage to Caco-2 cells is preferable, a cell culture medium is more preferable, and a medium containing non-essential amino acids is even more preferable. Specific examples of the medium include Dulbecco's modified Eagle's medium (DMEM), MEM, and RPMI 1640 medium, and among them, DMEM is preferable. In one embodiment, other components may be added to the medium. Examples of the components that can be added include, but are not limited to, organic solvents and solubilizers. Specific examples include fasted state simulated intestinal and stomach fluids (FaSSIF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), methanol, acetonitrile, surfactants, Tween, and the like. In this specification, a description such as "the medium is DMEM" does not exclude the inclusion of other components in the medium. The medium and / or added components on the apical side and the basal side may be the same or different. A preferred example of the medium on the apical side is a mixed medium of FaSSIF and DMEM, and a preferred example of the medium on the basal side is DMEM. In a non-limiting embodiment, the pH of the pre-incubation solution is not particularly limited, but is exemplified by pH 5.0 to 8.0. Examples of the temperature for pre-incubation include 5 to 45°C, preferably 20 to 40°C, and more preferably 37°C.

[0171] The measurement method of the present disclosure includes a step of measuring membrane permeability, in which a solution containing a test substance is placed in contact with one side of a Caco-2 cell layer, and a solution not containing the test substance is placed in contact with the other side of the Caco-2 cell layer, and after a predetermined time has elapsed, the amount of the test substance in the solution on the other side and / or the amount of the test substance in the solution on the one side is measured, thereby measuring the membrane permeability of the test substance.

[0172] The "membrane permeability measurement step" in the measurement method of the present disclosure can be performed according to a conventional method. In one embodiment, after the preincubation step, the preincubation solutions on the apical and basal sides are aspirated and removed, a solution containing the test substance is added to the apical side, and a solution not containing the test substance is added to the basal side, and the membrane permeability can be measured. In the membrane permeability measurement step, for example, a mixed solution (containing 1% DMSO) (pH 6.0) of FaSSIF and Hanks' Balanced Salt Solutions (HBSS) containing the test substance is added to the apical side, and HBSS (4% BSA) (pH 7.4) is added to the basal side, and the mixture is cultured under a condition of 37°C. After a predetermined time has elapsed, the amount of the test substance on the basal side is measured by LC / MS, and the membrane permeability coefficient is calculated from the amount of permeation, whereby the membrane permeability can be measured.

[0173] The compound (test substance) measured by the measurement method of the present disclosure is not particularly limited, but examples thereof include highly lipophilic compounds for which accurate measurement of membrane permeability is difficult by conventional methods. Examples of highly lipophilic compounds include compounds with ClogP of 3 or more, 4 or more, 5 or more, 6 or more, or 8 or more. Examples of highly lipophilic compounds include compounds with ClogP / total aa of 0.8 or more, 1.0 or more, 1.1 or more, 1.2 or more, or 1.3 or more. In a non-limiting embodiment, the test substance may be a peptide compound, preferably a cyclic peptide compound.

[0174] In a non-limiting embodiment, the membrane permeability of a plurality of test substances is measured by the measurement method of the present disclosure, and a test substance having a desired membrane permeability can be selected from the plurality of test substances based on the membrane permeability data obtained thereby. That is, the present specification discloses a method for screening test substances using the measurement method of the present disclosure.

[0175] In a non-limiting embodiment, a test substance having a level of membrane permeability required for development as a pharmaceutical can be selected by a screening method for a test substance using the measurement method of the present disclosure. The criteria used in this case can be selected according to the purpose of screening, such as the location of the target molecule in the body and the administration route, but the membrane permeability coefficient (P app ) is 5.0×10 -7 cm / sec or more, 8.0×10 -7 cm / sec or more, 9.0×10 -7 cm / sec or more, 1.0×10 -6 cm / sec or more, 3.0×10 -6 In this specification, the term "10 -n " is sometimes written as "En" (for example, 1.0 x 10 -6 is sometimes written as 1.0E-6 or 1.0E-06).

[0176] According to the measurement method (improved method) of the present disclosure, it is possible to appropriately evaluate the membrane permeability of a test substance with high lipid solubility, which was difficult to evaluate by conventional methods. Therefore, it is possible to screen test substances with high lipid solubility using the same criteria as in the past. For example, by using the measurement method of the present disclosure, it is possible to screen P app as 1.0 x 10 -6 A velocity of 0.1 cm / sec or higher can be set as the standard for development of an oral drug (P. Arturssonand J. et al 1991, Biochem Biophys Res Commun. 175,880-885.).

[0177] In a non-limiting embodiment, the test substance selected by the screening method of the present disclosure may be a peptide compound, and is particularly preferably a cyclic peptide compound. In a non-limiting embodiment, after selecting a peptide compound having a desired property by the screening method, a peptide compound having a desired property can be produced based on the amino acid sequence of the selected peptide compound.

[0178] The present disclosure also provides a pre-incubation method included in the above-mentioned measurement method of the present disclosure. That is, in one aspect, the present disclosure provides a pre-incubation method for measuring membrane permeability. The explanation, examples, preferred range, aspects, etc. of the pre-incubation method of the present disclosure are as described above.

[0179] Non-limiting aspects of methods for measuring membrane permeability in the present disclosure include the following: <1> A method for measuring membrane permeability of a test substance using Caco-2 cells, the method comprising a step of preincubating the Caco-2 cells and the test substance in a mixed state; <2> A method for measuring membrane permeability of a test substance using Caco-2 cells, the method comprising a step of pre-incubating Caco-2 cells and the test substance for 2 hours or more; <3> The method includes a step of pre-incubating Caco-2 cells and the test substance for 4 hours or more. <1> or <2> The method according to <4> The test involves mixing Caco-2 cells with the test substance and pre-incubating for at least 6 hours. <1> ~ <3> 2. A method according to any one of claims 1 to 11, <5> The test involves mixing Caco-2 cells with the test substance and pre-incubating for 8 hours or more. <1> ~ <4> 2. A method according to any one of claims 1 to 11, <6> The test involves mixing Caco-2 cells with the test substance and pre-incubating for 12 hours or more. <1> ~ <5> 2. A method according to any one of claims 1 to 11, <7> The test involves mixing Caco-2 cells with the test substance and pre-incubating for at least 24 hours. <1> ~ <6> 2. A method according to any one of claims 1 to 11, <8> The preincubation time in the preincubation step is 48 hours or less. <1> ~ <7> 2. A method according to any one of claims 1 to 11, <9> A pre-incubation method for measuring membrane permeability, which includes mixing Caco-2 cells with a test substance and incubating them for 4 hours or more; <10> This involves mixing Caco-2 cells with the test substance and incubating them for at least 24 hours. <9> The method according to <11> The preincubation is carried out by contacting Caco-2 cells with a medium; <1> ~ <9> 2. A method according to any one of claims 1 to 11, <12> The medium is a cell culture medium <11> The method according to <13> The medium is DMEM. <11> The method according to <14> The test substance has a ClogP of 3 or more. <1> ~ <13> 2. A method according to any one of claims 1 to 11, <15> The test substance is a peptide compound. <1> ~ <14> 2. A method according to any one of claims 1 to 11, <16> The test substance is a cyclic peptide compound. <1> ~ <15> 2. A method according to any one of claims 1 to 11, <17> The process further comprises the steps of: <1> ~ <16> Either way: a step of arranging a solution containing a test substance in contact with one side of a Caco-2 cell layer and a solution not containing the test substance in contact with the other side of the Caco-2 cell layer, and measuring the amount of the test substance in the solution on the other side and / or the amount of the test substance in the solution on the one side after a predetermined time has elapsed, thereby measuring membrane permeability of the test substance; <18> A method for screening a test substance, comprising the steps of: (a) <1> ~ <17> Measuring the membrane permeability of a plurality of test substances by any one of the methods described above; and (b) selecting a desired test substance from the plurality of test substances based on the membrane permeability data obtained in (a); <19> The step (b) is a step of determining the membrane permeability coefficient (P app ) is 1.0 × 10 -6Selecting a test substance that is faster than <18> The method according to <20> A method for producing a peptide compound, comprising the steps of: (i) <1> ~ <17> Measuring the membrane permeability of a plurality of peptide compounds by any one of the methods described above; and (ii) selecting a desired peptide compound from the plurality of peptide compounds based on the membrane permeability data obtained in (i); (iii) producing a peptide compound based on the amino acid sequence of the peptide compound selected in (ii); <21> <20> A peptide compound produced by the method described in claim 1.

[0180] The membrane permeability of the peptide compound of the present disclosure can be measured by the method for measuring membrane permeability of the present disclosure. That is, it can be measured by the following method. As the apical medium, a mixed medium of FaSSIF and DMEM containing 1% DMSO was used, and as the basal medium, DMEM was added, and the medium was incubated under 5% CO 2 Caco-2 cells and peptide compounds are pre-incubated for 20-24 hours under conditions of 37°C and 80 rpm. After that, the medium on the apical and basal sides is aspirated and washed, and the peptide compounds are added to FaSSIF / HBSS buffer (1% DMSO) (pH 6.0) containing 1% DMSO on the apical side, and HBSS buffer (pH 7.4) containing 4% BSA is added to the basal side to start the measurement of membrane permeability. Each well is shaken at 5% CO2, 37°C, and 80 rpm, and a sample from the basal side is taken approximately 180 minutes after the start. The amount of peptide compound in the sample is measured by LC / MS, and the membrane permeability coefficient is calculated from the amount of permeation. The membrane permeability coefficient (P app ) can be calculated using the above-mentioned formula 1.

[0181] In a non-limiting embodiment, when measuring the membrane permeability of the peptide compound of the present disclosure, it is preferable to use a peptide compound that does not have a nucleic acid portion serving as a template for the peptide site as a test substance. In one embodiment, as described below, after performing panning in the state of a peptide-nucleic acid complex, a peptide-nucleic acid complex that can specifically bind to a target molecule can be selected, and a peptide compound can be synthesized based on the base sequence of the nucleic acid portion. A preferred example is measuring the membrane permeability using the peptide compound synthesized in this way as a test substance. In one embodiment, a derivative of the peptide compound encoded by the base sequence may be synthesized and used as a test substance to measure the membrane permeability, or a derivative may be synthesized from the test substance based on the results of the membrane permeability, and the membrane permeability of the derivative may be measured.

[0182] In one non-limiting embodiment, P of the cyclic peptide compound of the present disclosure app is 5.0×10 -7 cm / sec or more, 8.0×10 -7 cm / sec or more is preferable, and 9.0×10 -7 More preferably, 1.0×10 cm / sec or more is used. -6 More preferably, 3.0×10 cm / sec or more. -6 Especially preferred is cm / sec or more.

[0183] In this specification, the "membrane permeability coefficient (P app )" means, unless otherwise specified, a value measured using the membrane permeability measurement method (improved method) in the present disclosure, and more specifically, a value measured using the improved method of the membrane permeability test described in the Examples.

[0184] Metabolic stability of peptide compounds In a non-limiting embodiment, the peptide compound of the present disclosure preferably has good metabolic stability. For 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 one embodiment, it is preferable that the peptide compound does not have a methylthio group, since it is easily oxidized and may hinder metabolic stability.

[0185] nucleic acid In a non-limiting embodiment, the nucleic acid in the present disclosure is preferably exemplified by a nucleic acid encoding a peptide compound in the present disclosure. The nucleic acid may or may not be linked to a peptide moiety. When linked, it may be linked via a spacer or linker. In one embodiment, the "nucleic acid encoding a peptide compound" refers to a template nucleic acid that is / was used in translational synthesis of the peptide compound.

[0186] Library The libraries in the present disclosure include libraries containing the peptide compounds in the present disclosure and libraries containing nucleic acids encoding the peptide compounds in the present disclosure. The libraries in the present disclosure include libraries of the peptide compounds in the present disclosure and libraries of peptide-nucleic acid complexes, among which libraries of cyclic peptide compounds or cyclic peptide-nucleic acid complexes are preferred, and libraries of cyclic peptide-mRNA complexes are particularly preferred. As the library, a display library is preferred. As the display library, a library using a display is exemplified, among which mRNA display library, DNA display library, and ribosome display library are preferred, and mRNA display library is more preferred.

[0187] In this specification, the term "(cyclic) peptide compound encoded by a nucleic acid" is not limited to a peptide compound directly synthesized by translating the nucleic acid as a template, but may include a peptide compound that can be synthesized by post-translational modification. For example, when a linear peptide compound is synthesized by translating a nucleic acid and a cyclic peptide compound is synthesized by a subsequent 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 translationally synthesized using a nucleic acid as a template, or a complex of a peptide and the nucleic acid that have subsequently been post-translationally modified (peptide-nucleic acid complex) are also included in the (cyclic) peptide compound encoded by a nucleic acid.

[0188] In a non-limiting embodiment, the average number of amino acids constituting the peptide site other than the nucleic acid linkage of the cyclic peptide compound contained in the library of the present disclosure or the cyclic peptide compound encoded by the nucleic acid contained in the library is not particularly limited, but is preferably 30 or less. In order to obtain high membrane permeability, the average number of amino acids in the peptide site other than the nucleic acid linkage is preferably 20 or less, more preferably 18 or less, 16 or less, 15 or less, or 14 or less, particularly preferably 13 or less, specifically exemplified as 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. In order to obtain high metabolic stability, the average number of amino acids in the peptide site other than the nucleic acid linkage is preferably 8 or more, more preferably 9 or more, even more preferably 10 or more, and particularly preferably 11 or more. In consideration of achieving both membrane permeability and metabolic stability, the average number of amino acids in the peptide portion other than the nucleic acid linking portion is preferably 5 to 20, 7 to 20, 7 to 17, 8 to 16, 9 to 16, 10 to 16, 8 to 13, 9 to 14, 9 to 14, 9 to 13, 10 to 15, 11 to 15, 10 to 14, 10 to 13, or 11 to 14, and more preferably 11 to 13. In one embodiment, the peptide portion other than the nucleic acid linking portion may have a linear portion, and the average number of the linear portions is not particularly limited, but is exemplified as 1 or more, or 2 or more, and specifically exemplified as 0, 1, or 2.

[0189] In a non-limiting embodiment, the average number of amino acids constituting the cyclic portion of the cyclic peptide compound contained in the library of the present disclosure or the cyclic peptide compound encoded by the nucleic acid contained in the library is not limited, and examples thereof include 4 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 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 average number of amino acids constituting the cyclic portion is preferably 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.

[0190] In a non-limiting aspect, the average number of amino acids (number of units) in the linear portion of a cyclic peptide compound contained in a library of the present disclosure, or a cyclic peptide compound encoded by a nucleic acid contained in the library, is preferably 0 to 8, more preferably 0 to 5, and more preferably 0 to 3.

[0191] In a non-limiting embodiment, the average number of unnatural amino acids contained in the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compound contained in the library of the present disclosure, or the cyclic peptide compound encoded by the nucleic acid contained in the library, is preferably 2 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. Examples of the cyclic peptide compound contained in the library of the present disclosure, or the cyclic peptide compound encoded by the nucleic acid contained in the library, are 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, and 80% or more in terms of the average number of unnatural amino acids among the number of amino acids constituting the cyclic portion, or the average ratio of the number of unnatural amino acids among the number of amino acids contained in the peptide portion other than the nucleic acid linkage portion of the cyclic peptide compound.

[0192] In a non-limiting embodiment, the average number of N-substituted amino acids contained in the peptide site other than the nucleic acid linkage of the cyclic peptide compound contained in the library of the present disclosure, or the cyclic peptide compound encoded by the nucleic acid contained in the library, is preferably 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. The cyclic peptide compound contained in the library of the present disclosure, or the cyclic peptide compound encoded by the nucleic acid contained in the library, is exemplified by 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more as the average number of N-substituted amino acids in the number of amino acids constituting the cyclic portion, or the average ratio of the number of N-substituted amino acids in the number of amino acids contained in the peptide site other than the nucleic acid linkage of the cyclic peptide compound. The N-substituted amino acid referred to here may be preferably an N-alkyl amino acid, more preferably an N-methyl amino acid. That is, in a non-limiting embodiment, the average number of the N-substituted amino acids is exemplified as the average number of N-alkyl amino acids or the average number of N-methyl amino acids.

[0193] In a non-limiting embodiment, for high membrane permeability, the average number of aromatic rings contained in the peptide portion other than the nucleic acid linkage portion, the cyclic portion, or the side chain of the cyclic portion of the cyclic peptide compound contained in the library of the present disclosure, or the cyclic peptide compound encoded by the nucleic acid contained in the library, is preferably 3 or less, and a preferred range is 0 to 3, or 1 to 3. Furthermore, the average ratio of the number of aromatic rings to the number of amino acids constituting the peptide portion other than the nucleic acid linkage portion, or the cyclic portion, 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.

[0194] In a non-limiting embodiment, since the number of aromatic rings that may be contained in the cyclic peptide compound of the present disclosure is limited, it is preferable that the long side chain of the cyclic portion, which is a portion that can contribute to binding with a target molecule, has an aromatic ring in order to achieve high membrane permeability. That is, in one embodiment, the average number of aromatic rings contained in the long side chain of the cyclic portion of the cyclic peptide compound of the present disclosure is exemplified as 0 to 3, 1 to 3, or 2 to 3. In addition, the average ratio of the number of aromatic rings contained in the long side chain of the cyclic portion to the number of aromatic rings contained in the peptide portion other than the nucleic acid linking portion, the cyclic portion, or the side chain of the cyclic portion of the cyclic peptide compound of the present disclosure is exemplified as 30% or more, 45% or more, 60% or more, 80% or more, or 100%.

[0195] In a non-limiting embodiment, the average number of unnatural amino acids, N-substituted amino acids, or amino acids having aromatic rings contained in the cyclic peptide compounds contained in the library of the present disclosure, or in the cyclic peptide compounds encoded by the nucleic acids contained in the library, can be adjusted by the frequency of occurrence of the codons assigned to the amino acids when synthesizing the DNA library. For example, in a peptide library containing 10 variable amino acid residues, assuming that 20 types of amino acids are selected, 10 types of N-substituted amino acids are selected from the 20 types, so that the codon units of the N-substituted amino acids occupy 10 / 20 (50%) per variable site. In this manner, the average ratio of the number of unnatural amino acids, the number of N-substituted amino acids, the number of aromatic rings, or the number of amino acids having aromatic rings in the present specification may be calculated.

[0196] In a non-limiting aspect, the average ClogP of the cyclic peptide compounds contained in the library of the present disclosure, or the cyclic peptide compounds encoded by the nucleic acids contained in the library, is preferably 4 or more, more preferably 5 or more, even more preferably 6 or more, and particularly preferably 8 or more; preferred examples include 18 or less, 17 or less, and 16 or less, with 4 to 18, 5 to 17, and 6 to 16 being examples.

[0197] In a non-limiting aspect, the average ClogP / total aa of the cyclic peptide compounds contained in the library of the present disclosure, or the cyclic peptide compounds encoded by the nucleic acids contained in the library, is preferably 1.0 or more, more preferably 1.1 or more, even more preferably 1.2 or more, and particularly preferably 1.3 or more, with preferred examples being 1.8 or less, 1.7 or less, 1.6 or less, and 1.5 or less, and examples of these being 1.0 to 1.8, 1.0 to 1.7, 1.1 to 1.6, and 1.1 to 1.5.

[0198] In a non-limiting embodiment, the library of the present disclosure may include a cyclic peptide compound having at least one or at least two long side chains in the cyclic portion, or a nucleic acid encoding the same. In one embodiment, the library of the present disclosure may include a cyclic peptide compound having at least one or at least two amino acids with long side chains as amino acids constituting the cyclic portion, or a nucleic acid encoding the same. The cyclic peptide compound included in the library of the present disclosure or encoded by the nucleic acid included in the library may have a high binding affinity with a target molecule by having a long side chain in the cyclic portion. In one embodiment, by appropriately selecting the long side chain, a cyclic peptide compound with high membrane permeability can be obtained while maintaining the binding affinity with the target molecule.

[0199] In a non-limiting embodiment, the library of the present disclosure may include a cyclic peptide compound having the above-mentioned long side chain in the cyclic portion, and / or a nucleic acid encoding the same. The above-mentioned examples, preferred ranges, and embodiments of the "long side chain" are applicable to the "long side chain".

[0200] In a non-limiting embodiment, a cyclic peptide compound included in a library of the present disclosure, or a cyclic peptide compound encoded by a nucleic acid included in a library, may have an aromatic ring in a side chain of its cyclic portion, and at least one, or two or more, long side chains included in the cyclic portion may be a side chain that includes an aromatic ring.

[0201] In a non-limiting embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially composed of cyclic peptide compounds that do not have an indole skeleton in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. Also, in one embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially free of cyclic peptide compounds that have an indole skeleton in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the side chain of the cyclic portion may be a long side chain of the cyclic portion.

[0202] In a non-limiting embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially composed of cyclic peptide compounds that do not have a condensed ring structure of two or more aromatic rings in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially free of cyclic peptide compounds that have a condensed ring structure of two or more aromatic rings in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the side chain of the cyclic portion may be a long side chain of the cyclic portion. In a non-limiting embodiment, the "condensed ring structure of two or more aromatic rings" may be a "condensed ring structure".

[0203] In a non-limiting embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially composed of cyclic peptide compounds that do not have a substituted or unsubstituted hydroxyphenyl group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. Also, in one embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially free of cyclic peptide compounds that have a substituted or unsubstituted hydroxyphenyl group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the side chain of the cyclic portion may be a long side chain of the cyclic portion.

[0204] In a non-limiting embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially composed of cyclic peptide compounds that do not have a methylthio group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. Also, in one embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially free of cyclic peptide compounds that have a substituted or unsubstituted methylthio group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the side chain of the cyclic portion may be a long side chain of the cyclic portion.

[0205] In a non-limiting embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially composed of cyclic peptide compounds that do not have a thiol group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the cyclic peptide compounds of the present disclosure or the library of nucleic acids encoding them may be libraries that are substantially free of cyclic peptide compounds that have a substituted or unsubstituted thiol group in the side chain of a peptide portion other than the cyclic portion or the nucleic acid linking portion, or nucleic acids encoding them. In one embodiment, the side chain of the cyclic portion may be the long side chain of the cyclic portion. In one embodiment, the side chain of the cyclic portion may be the long side chain of the cyclic portion.

[0206] In a non-limiting embodiment, the library of the present disclosure may be a library essentially consisting of cyclic peptide compounds that do not have functional groups that are highly ionized at neutral (e.g., pH=7.0) in order to achieve high membrane permeability, and / or nucleic acids encoding these.

[0207] In a non-limiting embodiment, when the library in the present disclosure includes a cyclic peptide compound having an acidic side chain in the cyclic portion and / or a nucleic acid encoding the same, the cyclic peptide compound may be a library substantially composed of cyclic peptide compounds having a pKa of the acidic side chain of 3.5 to 10. The pKa of the acidic side chain is preferably 3.5 or more, more preferably 3.9 or more, more preferably 4.5 or more, more preferably 5.0 or more, more preferably 5.5 or more, more preferably 5.7 or more, and preferably 10 or less. The pKa is preferably in the range of 3.5 to 10, 3.9 to 10, 4.5 to 10, 5.0 to 10, or 5.5 to 10, 5.7 to 10. When these pKa are expressed as calculated values, the calculated pKa is preferably 3.5 or more, more preferably 4.5 or more, preferably 5.0 or more, preferably 5.4 or more, preferably 8.0 or more, preferably 8.3 or more, and preferably 10 or less. The calculated pKa is preferably in the range of 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. In a non-limiting embodiment, the acidic side chain may be an acidic long side chain.

[0208] In a non-limiting embodiment, when the library of the present disclosure includes a cyclic peptide compound having a basic side chain at the cyclic portion and / or a nucleic acid encoding the same, the cyclic peptide compound may be a library substantially consisting of cyclic peptide compounds whose basic side chain has a basic pKa of 4.0 to 10. The basic pKa of the basic side chain is preferably 10 or less, more preferably 9.5 or less, more preferably 9.0 or less, more preferably 8.5 or less, more preferably 7.5 or less, more preferably 7.2 or less, particularly preferably 6.5 or less, and preferably 4.0 or more. Preferred examples of the basic pKa include ranges of 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 are expressed as calculated values, the basic calculated pKa is preferably 10 or less, more preferably 9.5 or less, preferably 9.0 or less, preferably 8.8 or less, preferably 8.6 or less, preferably 8.5 or less, preferably 7.5 or less, preferably 6.5 or less, and preferably 4.0 or more. The basic calculated pKa is preferably exemplified in the range of 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. In a non-limiting embodiment, the basic side chain may be a basic long side chain.

[0209] With respect to the libraries of cyclic peptide compounds and / or nucleic acids encoding them in the present disclosure, "consisting essentially of" may mean that the ratio of the theoretical number of cyclic peptide compounds and / or nucleic acid variations encoding them listed therein to the theoretical total number of cyclic 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 thereto.

[0210] With respect to the library of cyclic peptide compounds and / or the nucleic acids encoding them in the present disclosure, "substantially free of" may mean that the ratio of the theoretical number of cyclic peptide compounds and / or nucleic acid variations encoding them listed therein to the theoretical total number of cyclic peptide compounds and / or nucleic acid variations encoding them contained in the library is 10% or less, 7% or less, 5% or less, 3% or less, 2% or less, or 1% or less, but is not limited thereto.

[0211] In the library of the present disclosure, when determining the "theoretical (total) number of variations" of a cyclic peptide compound, those that cannot actually be produced as a cyclic 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 translated and synthesized, so that the peptide compound containing such an amino acid is 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 does not contain a nucleic acid encoding the same amino acid as a template; (2) when a translation solution is used in which the nucleic acid serving as the template for the peptide compound contains a base sequence but does not contain the corresponding amino acid. In addition, when there are side-reactants or unreacted substances in the process of producing a cyclic peptide compound, these are not included in the theoretical (total) number.

[0212] 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, if the translation initiation amino acid is fixed to methionine, the theoretical number of variations is 20 10 It becomes.

[0213] In a non-limiting embodiment, the library of the present disclosure may contain, in addition to the cyclic peptide compounds and / or the nucleic acids encoding them, other components necessary for screening peptide compounds capable of specifically binding to a target molecule. In addition, other components may be contained within a range that does not negatively affect the effect of the library of the present disclosure.

[0214] The library of the present disclosure includes a plurality of cyclic peptide compounds or nucleic acids encoding them. In this specification, the number of variations (types) of cyclic peptide compounds included in the library is referred to as "diversity." The diversity of cyclic peptide compounds included in the library of the present disclosure or nucleic acids encoding them is not particularly limited, but may be as high as 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 measured values, and may be theoretical values.

[0215] In a non-limiting aspect, the average ClogP of peptide compounds contained in a library of the present disclosure or encoded by a nucleic acid contained in the library is preferably 4 or more, more preferably 5 or more, even more preferably 6 or more, particularly preferably 8 or more, and preferably 15 or less.

[0216] In a non-limiting embodiment, the ClogP / total aa of a peptide compound contained in a library of the present disclosure or encoded by a nucleic acid contained in the library is preferably 0.8 or more, more preferably 1.0 or more, more preferably 1.1 or more, even more preferably 1.2 or more, and particularly preferably 1.3 or more.

[0217] In a non-limiting embodiment, the average molecular weight of the peptide portion other than the nucleic acid linkage portion of a cyclic peptide compound contained in a library of the present disclosure 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.

[0218] membrane permeability In a non-limiting embodiment, the library of the present disclosure may be a library in which cyclic peptide compounds contained in the library or encoded by nucleic acids contained in the library have high membrane permeability, or high membrane permeability can be easily obtained by derivatizing these. In a non-limiting embodiment, the library of the present disclosure may be a library in which cyclic peptide compounds found as compounds capable of specifically binding to a target molecule have high membrane permeability, or high membrane permeability can be easily obtained by derivatizing these. As used herein, "high membrane permeability" refers to a membrane permeability coefficient (P app ) is 5.0×10 -7 cm / sec or more, preferably 8.0×10 -7 cm / sec or greater, or 9.0 x 10 -7 cm / sec or more, more preferably 1.0×10 -6 cm / sec or more, more preferably 3.0×10 -6 cm / sec or more, more preferably 5.0×10 -6 In a non-limiting embodiment, the library in the present disclosure may be a library for identifying an orally administrable compound or a precursor thereof. As used herein, the term "orally administrable" refers to a compound having a membrane permeability coefficient (P app ) is 1.0×10 -6 cm / sec or more, preferably 3.0×10 -6 cm / sec or more, more preferably 5.0×10 -6 The term "precursor" as used herein means a compound prior to chemical modification or the like for optimization, such as a hit compound from panning prior to optimization.

[0219] Hit rate of peptide compounds that can specifically bind to target molecules In a non-limiting embodiment, the library in the present disclosure may be a library for identifying a compound capable of specifically binding to a target molecule. In one embodiment, the library in the present disclosure has a higher hit rate of peptide compounds capable of specifically binding to a target molecule compared to conventional libraries. That is, by using the library in the present disclosure, a large number of peptide compounds capable of specifically binding to a target molecule can be obtained. In addition, in one embodiment, a peptide compound having a high binding affinity with a target molecule can be obtained. Therefore, it is possible to select a desired compound from a large number of selected peptide compounds, taking into account other factors such as metabolic stability and membrane permeability. In this specification, the "hit rate" means the ratio of the number of variations of hit compounds obtained by panning to the number of variations of compounds contained in the library. The number of variations of compounds contained in the library may be a theoretical number.

[0220] In a non-limiting embodiment, a peptide compound that is not present in the library as a cyclic peptide compound for obtaining a hit compound (e.g., a peptide compound that is clearly not capable of binding to a target molecule even before panning) is not considered to be a peptide compound constituting a library in the present disclosure.

[0221] By analyzing the frequency of occurrence of amino acids using cyclic peptide compounds enriched by panning, it is possible to find groups of amino acids that can contribute to binding to a target molecule.

[0222] Display Library A display library is a library in which a peptide, which is a phenotype, is associated with the RNA or DNA that codes for that peptide, which is its genotype. This library can be used to identify peptide compounds that can specifically bind to a target molecule. For example, peptides that bind to the target can be enriched by contacting the library with a desired immobilized target and washing away molecules that do not bind to the target (panning method). The sequence of the peptide that bound to the target can be revealed by analyzing the genetic information associated with the peptides selected through this process. For example, a method utilizing the fact that the antibiotic puromycin, an analogue of aminoacyl-tRNA, is nonspecifically linked to a protein during mRNA translation elongation by the ribosome has been reported as mRNA display (Proc Natl Acad Sci USA. 1997; 94: 12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins. Roberts RW, Szostak JW.) or 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 proteinon the ribosome in vitro. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H.).

[0223] When a spacer such as puromycin is attached to the 3' end of an mRNA library obtained by transcription from a DNA library containing a promoter such as the T7 promoter, and the mRNA is translated into protein in a cell-free translation system, puromycin is mistaken for an amino acid and incorporated into the protein by the ribosome, and the mRNA and the protein encoded by it are linked, resulting in a library in which the mRNA and its product are associated. This process is highly efficient because it does not involve transformation of E. coli, and it is possible to construct a large-scale display library. The sequence of the bound protein can be revealed by synthesizing cDNA from the mRNA, which is a tag containing genetic information attached to the molecules enriched and selected by panning, amplifying it by PCR, and analyzing the base sequence.

[0224] In addition to mRNA display, cell-free translation systems have been used to display libraries such as 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 stabilizationofmRNA-protein fusions.Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M,Kubo T, Funatsu T, Husimi Y, Nemoto N.), ribosome display, which utilizes the relatively stable complex between ribosomes and translation products during mRNA translation (Proc Natl Acad Sci US 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.), and bacteriophage endonuclease Covalent display, which utilizes the covalent bond between P2A and DNA (Nucleic Acids Res. 2005;33:e10.Covalent antibody display--an in vitro antibody-DNA library selection system.Reiersen H, LobersliI, 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 replication initiator protein RepA of microbial plasmids to the replication origin ori (Proc Natl AcadSci US 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 addition, in vitro compartmentalization (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.) is also known, 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 a translation reaction is carried out. The above method can be carried out by using a known method as appropriate.

[0225] Nucleic Acid Libraries The "nucleic acid" of the present invention may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a nucleotide derivative having an artificial base. It may also include peptide nucleic acid (PNA). The nucleic acid of the present invention may be any of these nucleic acids or a mixture thereof, so 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.

[0226] Examples of nucleic acid libraries serving as templates for peptide compounds contained in a peptide compound library include mRNA libraries and DNA libraries. Nucleic acid libraries can be obtained by synthesizing sites on peptide sequences where amino acid residues are not fixed by mixing bases. For example, DNA libraries can be synthesized with A, T, G, and C, and RNA libraries can be synthesized with a mixture of four bases each, A, U, G, and C, repeated a multiple of three times, or with N as the first and second letters of the codon and a mixture of two bases, such as W, M, K, and S, as the third letter. Furthermore, if the number of types of amino acids to be introduced is limited to 16 or less, there is also a method in which the third letter is a single type of base. In addition, 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 use in synthesis.

[0227] 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 of RNA, DNA, a polymer of hexaethylene glycol (spc18) (e.g., a polymer of five), etc., between the nucleic acid library and a compound that is incorporated into a peptide during translation by the ribosome, such as puromycin or a derivative thereof.

[0228] Method for producing peptide compounds and libraries In one embodiment, the production methods of the present disclosure include methods for producing peptide compounds and libraries containing peptide compounds, including, but not limited to, chemical synthesis, translational synthesis, or a combination thereof.

[0229] Chemical synthesis method of peptide compounds The chemical synthesis method of the peptide compound in the present disclosure includes, for example, a liquid phase synthesis method, a solid phase synthesis method using Fmoc synthesis or Boc synthesis, and a combination thereof. In the Fmoc synthesis, the main chain amino group is protected by an Fmoc group, the side chain functional group is protected as necessary by a protecting group that is not cleaved by a base such as piperidine, and the main chain carboxylic acid is not protected, and the basic unit is an amino acid. The basic unit is not particularly limited as long as it has an Fmoc-protected amino group and a carboxylic acid group. For example, a dipeptide may be used as the basic unit. The basic 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 carboxylic acid group is supported on the solid phase by a chemical reaction with a functional group of the solid phase support. The Fmoc group is then deprotected with a base such as piperidine or DBU, and the newly generated amino group is condensed with the protected amino acid having a carboxylic acid of the basic unit to be added subsequently to generate a peptide bond. In the condensation reaction, various combinations are possible, such as a combination of DIC and HOBt, a combination of DIC and HOAt, or a combination of HATU and DIPEA. The desired peptide sequence can be generated by repeating the removal of the Fmoc group and the 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 of the side chain functional groups introduced 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 are performed under the same conditions, for example, 90:10 TFA / H 2O may be used, or deprotection may be performed under different conditions as necessary. Cleavage from the solid phase may be performed with a weak acid such as 1% TFA, or Pd may be used as a protecting group to utilize the orthogonality of the chemical reaction between the two. A step such as cyclization may be performed between or at the end of these steps. For example, a side chain carboxylic acid may be condensed with an amino group in the N-terminal main chain, or a side chain amino group may be condensed with a carboxylic acid in the C-terminal main chain. In this case, an orthogonal reaction is required between the carboxylic acid on the C-terminal side and the side chain carboxylic acid to be cyclized, or between the main chain amino group or hydroxy group on the N-terminal side and the side chain amino group to be cyclized, and as mentioned above, the protecting group is selected taking into consideration the orthogonality of the protecting group. The reaction product obtained in this way can be purified using a reverse phase column, a molecular sieve column, or the like. Details of these are described, for example, in the Solid Phase Synthesis Handbook published by Merck Ltd. on May 1, 2002.

[0230] Translational synthesis method for peptide compounds The translational synthesis method of the peptide compound in the present disclosure is exemplified by a synthesis method using a cell-free translation system, including a synthesis method using a reconstituted cell-free translation system. The use of a reconstituted cell-free translation system is preferable because it allows the removal of factors to be eliminated.

[0231] In one aspect, the present disclosure provides a method for translational synthesis of a peptide compound containing at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids set forth in Tables 2-1 to 2-6. Without being limited thereto, such a translational synthesis method may include the following (i) and (ii): (i) preparing a tRNA having at least one, two or more, or three or more amino acids bound thereto selected from the group consisting of the amino acids set forth in Tables 2-1 to 2-6; (ii) 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 one non-limiting embodiment, the translation synthesis method of the present disclosure may be a translation synthesis method for a cyclic peptide compound.

[0232] Cell-free translation system In the present specification, the term "cell-free translation system" refers to a combination of ribosomes extracted from cells, protein factors involved in translation, tRNA, amino acids, energy sources such as ATP, and a regeneration system thereof, and is not limited as long as it can translate mRNA into protein. In addition, a system in which a translation synthesis reaction is in progress may also be included in the "cell-free translation system" in the present specification. The cell-free translation system in the present specification may contain nucleic acids that serve as templates for translating peptides, and may also contain initiation factors, elongation factors, release factors, aminoacyl-tRNA synthetases, and the like. These factors can be obtained by purifying them from extracts of various cells. Examples of cells for purifying factors include prokaryotic cells or eukaryotic cells. Examples of prokaryotic cells include Escherichia coli cells, extreme thermophile cells, and Bacillus subtilis cells. Examples of eukaryotic cells include those using yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, or animal cells as materials. In addition to naturally occurring tRNA and aminoacyl-tRNA synthetases (ARS), artificial tRNA and artificial aminoacyl-tRNA synthetases that recognize unnatural amino acids can also be used. By using artificial tRNA and artificial aminoacyl-tRNA synthetases, peptides with unnatural amino acids introduced site-specifically can be synthesized. If necessary, transcription from template DNA can be performed by adding an RNA polymerase such as T7 RNA polymerase to the cell-free translation system.

[0233] As used herein, the phrase "cell-free translation system containing a certain substance" also includes an embodiment in which the substance is synthesized and contained in the system during the process of translation synthesis, even if the substance is not contained at the start of translation synthesis. For example, when tRNA in which an amino acid is acylated is synthesized during the process of translation synthesis, the cell-free translation system is understood to contain the aminoacyl-tRNA.

[0234] PURESYSTEM (registered trademark) (BioComber, Japan) is a reconstituted cell-free translation system in which protein factors, energy regeneration enzymes, and ribosomes required for translation in E. coli are extracted and purified, and then mixed with tRNA, amino acids, ATP, GTP, etc. Not only does it contain a small amount of 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;19:751-5. Cell-free translationreconstituted 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.).

[0235] For example, many methods have been reported that use stop codons to introduce unnatural amino acids, but by using the PURESYSTEM described above, a synthesis system can be constructed that excludes natural amino acids and ARS. This allows unnatural amino acids to be associated with codons that code for 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 degeneracy of codons, unnatural amino acids can be added without excluding natural amino acids (Kwon I,et al. Breaking the degeneracy of the genetic code. J Am ChemSoc. 2003, 125, 7512-3.). Peptides containing N-methyl amino acids can be synthesized in the ribosome using a cell-free translation system such as the PURESYSTEM.

[0236] More specifically, the translation synthesis may involve the use of, 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 the necessary one 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 done by adding mRNA to a known cell-free translation system such as PURESYSTEM, which contains a mixture of pH 7.6, magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, UTP, and the like, selected appropriately. Furthermore, by adding T7 RNA polymerase, transcription and translation from a template DNA containing a T7 promoter can be coupled. Furthermore, by adding a desired aminoacyl-tRNA group or a non-natural amino acid group (e.g., F-Tyr) that is acceptable to aminoacyl-tRNA synthetase (ARS) to the system, a peptide compound containing a non-natural amino acid can be translationally synthesized (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, by including a modified ARS in the system instead of or in addition to a natural ARS, and also including a group of unnatural amino acids in the system, it is possible to translationally synthesize a peptide compound containing unnatural amino acids.Alternatively, the efficiency of translational incorporation of unnatural amino acids can be increased by using 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 amino acid tolerance of protein biosynthesis system. J Am Chem Soc. 2007, 129, 14458-62., Park HS, et al. Expanding the genetic code of Escherichia coli withphosphoserine. Science. 2011, 333, 1151-4.).

[0237] In a non-limiting embodiment, the cell-free translation system in the present disclosure (also referred to as the "translation system in the present disclosure") may be a translation system for producing a peptide compound, and is preferably a translation system for producing a cyclic peptide compound.

[0238] In a non-limiting embodiment, the translation system of the present disclosure may be a cell-free translation system that is substantially free of at least one, two, or all selected from the group consisting of the following (a) to (c): (a) an amino acid having an indole skeleton in the side chain, or a nucleic acid encoding the amino acid; (b) an amino acid having a fused ring structure with two or more aromatic rings in the side chain, or a nucleic acid encoding the amino acid; and (c) an amino acid having a substituted or unsubstituted hydroxyphenyl group in the side chain, or a nucleic acid encoding the amino acid. In a non-limiting embodiment, the translation system of the present disclosure may be a cell-free translation system that is substantially free of at least one, two, or all selected from the group consisting of the following (d) to (f): (d) a nucleic acid encoding an amino acid having an indole skeleton in the side chain; (e) a nucleic acid encoding an amino acid having a fused ring structure with two or more aromatic rings in the side chain; and (f) a nucleic acid encoding an amino acid having a substituted or unsubstituted hydroxyphenyl group in the side chain. In a non-limiting embodiment, the translation system in the present disclosure may be a cell-free translation system that does not substantially contain at least one, two, or all of the following selected from the group consisting of (g) to (i): (g) an amino acid having an indole skeleton in the side chain; (h) an amino acid having a condensed ring structure with two or more aromatic rings in the side chain; and (i) an amino acid having a substituted or unsubstituted hydroxyphenyl group in the side chain. Even if a certain amino acid or nucleic acid is contained in the translation system, if it is contained in such a manner that it is not used in translation synthesis, it is understood that the translation system does not substantially contain the amino acid or nucleic acid. For example, there is a case where an amino acid is present but the corresponding ARS is not contained, so that the amino acid is not used in translation synthesis. In a non-limiting embodiment, the "condensed ring structure with two or more aromatic rings" may be a "condensed ring structure".

[0239] In a non-limiting embodiment, when the translation system of the present disclosure includes a tRNA in which an amino acid having an acidic side chain is acylated, and a nucleic acid encoding the amino acid, the pKa of the side chain of the amino acid may be 3.5 to 10. In one embodiment, the pKa is preferably in the range of 3.5 to 10, 3.9 to 10, 4.5 to 10, 5.0 to 10, 5.5 to 10, or 5.7 to 10. When these pKa are expressed as calculated pKa, the ranges of 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 are preferably exemplified.

[0240] In a non-limiting embodiment, when the translation system in the present disclosure includes a tRNA in which an amino acid having a basic side chain is acylated, and a nucleic acid encoding the amino acid, the basic pKa of the side chain of the amino acid may be 4.0 to 10. In one embodiment, the basic pKa is preferably in the range of 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, or 4.0 to 6.5. When these pKa are expressed as calculated pKa, the ranges of 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, or 4.0 to 6.5 are preferably exemplified.

[0241] In a non-limiting embodiment, the translation system of the present disclosure may include amino acids having long side chains, e.g., 5.4 to 13 or 5.4 to 10 angstroms in length. The "long side chains" in one embodiment may be as described in the "Peptide Compound" section. In one embodiment, at least one of the long side chains, or two or more of the long side chains, may be an aromatic ring-containing side chain.

[0242] In a non-limiting embodiment, the translation system of the present disclosure 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, of the types of amino acids contained in the translation system of the present disclosure, 50% or more, 60% or more, 70% or more, or 80% or more may be unnatural amino acids.

[0243] In a non-limiting embodiment, the translation system of the present disclosure may contain 5 to 28 or 5 to 20 types of N-substituted amino acids, preferably 5 to 18 or 5 to 15. In one embodiment, of the number of types of amino acids contained in the translation system of the present disclosure, 40% or more, 50% or more, 60% or more, or 70% or more may be N-substituted amino acids. Here, the N-substituted amino acids may refer to N-alkyl amino acids or N-methyl amino acids, but even in this case, it is not denied that the translation system of the present disclosure may contain other N-substituted amino acids.

[0244] In a non-limiting embodiment, the translation system of the present disclosure may be adjusted so that the average number of aromatic rings contained in the cyclic peptide compound produced using the translation system is within a certain range. For example, the translation system of the present disclosure may be adjusted so that the average ratio of the number of amino acids having aromatic rings to the number of amino acids constituting the cyclic portion in the cyclic peptide compound to be translationally synthesized is 40% or less, 35% or less, 30% or less, 27% or less, 25% or less, or 20% or less, or the average number of aromatic rings contained in the side chain of the cyclic portion of a cyclic peptide compound having a cyclic portion composed of 8 to 11 amino acids is 0 to 3. The adjustment method is not particularly limited, and examples thereof include adjusting the number of types of amino acids having aromatic rings to the number of types of amino acids contained in the translation system.

[0245] In a non-limiting aspect, the translation system of the present disclosure may contain amino acids having aromatic rings, and the proportion of the number of types of amino acids having aromatic rings in 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.

[0246] In a non-limiting embodiment, the cell-free translation system of the present disclosure may be a cell-free translation system for producing a peptide compound containing at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6. Without being limited thereto, such a cell-free translation system may include the following (i) and (ii): (i) a tRNA to which at least one, two or more, or three or more kinds of amino acids selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6 are bound; (ii) a nucleic acid encoding said peptide compound; Here, the nucleic acid may contain at least one codon that corresponds to the anticodon of the tRNA.

[0247] tRNA Translational incorporation of unnatural amino acids into peptides requires aminoacylation of tRNA, which is orthogonal and efficiently incorporated into ribosomes ((i)Biochemistry. 2003;42:9598-608.Adaptation of an orthogonalarchaeal leucyl-tRNA and synthetase pair for four-base, amber, and opalsuppression. Anderson JC, Schultz PG., (ii)Chem Biol. 2003;10:1077-84.Using asolid-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:

[0248] Within cells, aminoacyl-tRNA synthetases (ARSs) are prepared for each amino acid as enzymes for the aminoacylation of tRNA. Therefore, the first method is to utilize the fact that some ARSs tolerate unnatural amino acids such as N-Me His, or to prepare and use mutant aminoacyl-tRNA synthetases that tolerate unnatural amino acids. ((i)Proc NatlAcad Sci US A. 2002;99:9715-20. An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translationand its application in a wheat germ cell-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, YokoyamaS. (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) Proc NatlAcad Sci US A. 2006;103:4356-61.Enzymatic aminoacylation of tRNA with unnatural amino acids.Hartman MC, Josephson K, Szostak JW.) Secondly, a method of aminoacylation of tRNA in vitro followed by chemical modification of amino acids can also be used (J AmChem Soc. 2008;130:6131-6.Ribosomal synthesis of N-methyl peptides.Subtelny AO,Hartman MC, Szostak JW.).Thirdly, aminoacyl-tRNA can be obtained by removing CA from the CCA sequence at the 3' end of tRNA and ligating it with aminoacylated pdCpA prepared separately 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 performed using flexizyme, a ribozyme that carries active esters of various unnatural amino acids onto tRNA (J Am Chem Soc. 2002;124:6834-5.Aminoacyl-tRNA synthesis by aresin-immobilized ribozyme.Murakami H, Bonzagni NJ, Suga H.). Fourthly, a method of ultrasonically mixing tRNA and an amino acid activated ester in a cationic micelle 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.). Fifthly, aminoacylation can also be achieved by adding to tRNA a PNA that is complementary to the 3' end of tRNA and an amino acid activated ester bound thereto (J Am Chem Soc. 2004;126:15984-9. In situ chemical aminoacylation with amino acid thioesterslinked to a peptide nucleic acid. Ninomiya K, Minohata T, NishimuraM, Sisido M.).

[0249] More specifically, aminoacyl-tRNA can be prepared using the following method. A template DNA encoding a desired tRNA sequence and with a T7, T3 or SP6 promoter located upstream is prepared, and RNA can be synthesized by transcription using an RNA polymerase suitable for the promoter, such as T7 RNA polymerase, T3 or SP6 RNA polymerase. tRNA can be extracted and purified from cells, and the desired tRNA can be extracted using a probe with a complementary sequence to the tRNA sequence. In this case, cells transformed with an expression vector for the desired tRNA can also be used as the source. RNA of the desired sequence can also be synthesized by chemical synthesis. For example, aminoacyl-tRNA can be obtained by binding the thus obtained tRNA with CA removed from the 3'-terminal CCA sequence to a separately prepared aminoacylated pdCpA or pCpA using RNA ligase (pdCpA method, pCpA method). The tRNA is useful in the production of peptide compounds. Alternatively, aminoacylation can be performed by preparing a full-length tRNA and using flexizyme, a ribozyme that carries active esters of various non-natural amino acids on tRNA. In addition, although not intended to be limiting, aminoacyl-tRNA can be produced using natural ARS or a modified version thereof. When natural ARS or a modified version thereof is used, aminoacyl-tRNA once consumed in the translation system can be regenerated by the natural ARS or a modified version thereof, so there is no need to have a large amount of pre-produced aminoacyl-tRNA present in the translation system. Such modified ARS is described in WO2016 / 148044. These methods for producing aminoacyl-tRNA can also be combined as appropriate.

[0250] Methods for Cyclizing Peptide Moieties In a non-limiting embodiment, the peptide moiety of the peptide compound of the present disclosure preferably has a cyclic portion. The method of cyclization of the peptide compound is not particularly limited, but for example, the peptide compound can be synthesized by chemical synthesis or translation synthesis, and then the cyclization reaction can be performed. For example, cyclization using an amide bond, a carbon-carbon bond, a thioether bond, a disulfide bond, an ester bond, a thioester bond, a lactam bond, a bond via a triazole structure, a bond via a fluorophore structure, etc. can be mentioned. The synthesis step of the peptide compound and the cyclization reaction step may be separated or may proceed continuously. The cyclization can be performed by a method known to those skilled in the art, for example, as described in WO2013 / 100132, WO2008 / 117833, WO2012 / 074129, etc.

[0251] The cyclization portion is not limited, and may be any of a bond between the N-terminus and C-terminus of a peptide, a bond between the N-terminus of a peptide and a side chain of another amino acid residue, a bond between the C-terminus of a peptide and a side chain of another amino acid residue, or a bond between side chains of amino acid residues, or may be a combination of two or more of these. When a peptide-nucleic acid complex having a nucleic acid linked to the C-terminus is cyclized, the bond may be a bond between the N-terminus of a peptide and a side chain of another amino acid residue, or a bond between side chains of amino acid residues.

[0252] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure can be produced by the following method. For example, the production method can include the following steps: (1) synthesizing an acyclic peptide compound composed of amino acid residues or amino acid residues and an N-terminal carboxylic acid analog by translating a nucleic acid encoding the peptide compound, the acyclic peptide compound including an amino acid residue having a reactive site at one side chain on the C-terminus side and an amino acid residue or an N-terminal carboxylic acid analog having another reactive site on the N-terminus side; and (2) A step of bonding the reactive site of an amino acid residue on the N-terminus or an N-terminal carboxylic acid analog with the reactive site of an amino acid residue in the side chain on the C-terminus to form an amide bond, a carbon-carbon bond or a thioether bond. The steps (1) and (2) may be carried out separately or consecutively.

[0253] A non-limiting embodiment of the method for cyclizing a peptide compound by an amide bond is a method in which a peptide compound having cysteine ​​or a cysteine ​​analog at the N-terminus and an active ester at the side chain of the amino acid on the C-terminus is translated and cyclized using native chemical ligation. There is no particular limitation on the method for translationally synthesizing a peptide compound having cysteine ​​or a cysteine ​​analog at the N-terminus, which is a precursor of this cyclic peptide compound.

[0254] In one embodiment, for example, the following three methods can be used to synthesize a peptide compound having an amino acid other than Met or Nle at the N-terminus of the peptide compound.

[0255] The first method is to cleave the N-terminal amino acid or the N-terminal peptide of a peptide initiated with formylmethionine or Met using an enzyme, such as peptide deformylase (PDF) and / or methionine aminopeptidase (MAP).

[0256] The second method is to use a reconstituted cell-free translation system, remove Met from the system in advance, place the amino acid to be placed at the N-terminus at the second residue from the N-terminus, and perform translational synthesis by skipping the start codon Met (defined as the initiation readthrough (iRT) method).

[0257] The third method is to perform translation synthesis by adding aminoacyl-tRNA, which is prepared by aminoacylation of the amino acid to be placed at the N-terminus, as an initiator tRNA to a reconstituted cell-free translation system that does not contain Met or translation initiation methionine tRNA (defined as the initiation suppression (iSP) method).

[0258] In a non-limiting embodiment, the C-terminal moiety of the cyclic peptide compound of the present disclosure may be chemically modified rather than remaining a carboxylic acid. For example, the carboxylic acid moiety may be reacted with piperidine or the like to convert it to a piperidine amide or the like.

[0259] Library manufacturing method In a non-limiting embodiment, when producing a library in the present disclosure, it is preferable to include constituent amino acids that contribute to desired properties and not include constituent amino acids that have undesirable properties. In addition, in one embodiment, it is desirable to expand the diversity of the library as much as possible. Specifically, it is preferable to include, as constituent amino acids, amino acids that can contribute to at least one selected from the group consisting of the following (1) to (4): (1) improving the hit rate of peptide compounds that can specifically bind to target molecules; (2) increasing the binding affinity with target molecules; (3) increasing metabolic stability; and (4) increasing membrane permeability. On the other hand, it is preferable not to include, as constituent amino acids, amino acids that can have a negative effect on at least one selected from the group consisting of (1) to (4).

[0260] In a non-limiting embodiment, the production of the library in the present disclosure can be performed according to the method for producing a peptide compound in the present disclosure described above, and can be combined with a known method as appropriate. In one embodiment, the library of peptide compounds in the present disclosure can be produced using the cell-free translation system in the present disclosure described above. That is, the method for producing a library in the present disclosure may include a step of synthesizing a peptide compound using the cell-free translation system in the present disclosure. In one embodiment, the examples, preferred ranges, and aspects described in the cell-free translation system in the present disclosure can be directly applied to the method for producing a library in the present disclosure.

[0261] In one non-limiting embodiment, the method for producing a library of peptide compounds according to the present disclosure may include the following step (a): (a) preparing an amino acid pool substantially free of amino acids having an indole skeleton in their side chains, and synthesizing a peptide compound using some or all of the amino acids contained in the pool as constituent amino acids. Also, in one embodiment, the method for producing a library of peptide compounds according to the present disclosure may include the following step (b): (b) preparing a template pool substantially free of nucleic acids encoding amino acids having an indole skeleton in their side chains, and synthesizing a peptide compound from the template pool.

[0262] In this specification, even if an amino acid pool contains an amino acid, if the amino acid is contained in a form that is not used in translation synthesis or is contained in a very small amount, it is understood that the amino acid pool does not substantially contain the amino acid.For example, even if an amino acid pool contains an amino acid, if the amino acid is not used in translation synthesis because it does not contain the ARS corresponding to the amino acid, it is understood that the amino acid pool does not substantially contain the amino acid.In addition, even if it does not contain an ARS, if the corresponding amino acid is contained in a form that is used in translation synthesis, it is understood that the amino acid pool contains the amino acid.

[0263] As used herein, even if the template pool contains a nucleic acid encoding a certain amino acid, if the nucleic acid is contained in such a manner that the amino acid is not utilized in translational synthesis or if the nucleic acid is contained in only a very small amount, it is understood that the template pool is substantially free of the nucleic acid.

[0264] In a non-limiting embodiment, the pools in steps (a) and (b) may each be pools that are substantially free of amino acids having a fused ring structure with two or more aromatic rings in the side chain, and nucleic acids encoding said amino acids. In one embodiment, the pools in steps (a) and (b) may each be pools that are substantially free of amino acids having a substituted or unsubstituted hydroxyphenyl group in the side chain, and nucleic acids encoding said amino acids. In a non-limiting embodiment, the "fused ring structure with two or more aromatic rings" may be a "fused ring structure".

[0265] In a non-limiting embodiment, the amino acid pool used in the synthesis of the peptide compound may contain an amino acid having an acidic side chain, and the template pool may contain a nucleic acid encoding the amino acid, in which case the pKa of the side chain of the amino acid is preferably in the range of 3.5 to 10, 3.9 to 10, 4.5 to 10, 5.0 to 10, 5.5 to 10, or 5.7 to 10. When these pKa are expressed as calculated pKa, preferably in the range of 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.

[0266] In a non-limiting embodiment, the amino acid pool used in the synthesis of the peptide compound may contain an amino acid having a basic side chain, and the template pool may contain a nucleic acid encoding the amino acid, in which case the basic pKa of the side chain of the amino acid is preferably in the range of 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, or 4.0 to 6.5. When these pKa are expressed as calculated pKa, the ranges of 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, or 4.0 to 6.5 are preferably exemplified.

[0267] In a non-limiting embodiment, the amino acid pool used in the synthesis of the peptide compound may contain amino acids having the above-mentioned long side chains. The above-mentioned examples, preferred ranges, and embodiments are applicable to the "long side chains". In one embodiment, at least one, two, or three or more of the long side chains contained in the amino acid pool may be side chains having an aromatic ring.

[0268] In a non-limiting embodiment, the amino acid pool used in the synthesis of the peptide compound may contain the above-mentioned unnatural amino acids, N-substituted amino acids, and amino acids having an aromatic ring, and the examples, preferred ranges, and embodiments described in the section "Cell-free translation system" can be applied as they are. The proportion of the number of types of unnatural amino acids, N-substituted amino acids, or amino acids having an aromatic ring to the number of types of amino acids contained in the amino acid pool may be selected similarly to the proportions thereof in the translation system of the present disclosure.

[0269] In a non-limiting embodiment, the mRNA display library can be produced as follows. First, a DNA library in which a desired sequence is placed downstream of a promoter such as a T7 promoter is chemically synthesized, and this is used as a template to form double-stranded DNA by a primer extension reaction. This is used as a template to transcribe into mRNA using an RNA polymerase such as T7 RNA polymerase. A linker (spacer) to which an antibiotic such as puromycin, which is an analog of aminoacyl tRNA, is linked is attached to the 3' end of this RNA. This is added to a known cell-free translation system such as the above-mentioned PURESYSTEM, and incubated to translate the mRNA, and the mRNA and the peptide encoded thereby are linked via a linker containing puromycin. In this way, a display library consisting of a complex 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. In addition, post-translational modifications such as cyclization can be performed by the above-mentioned method or a known method, if necessary.

[0270] In a non-limiting embodiment, the library in 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 the amino acids listed in Tables 2-1 to 2-6. A method for producing such a library may include a step of translationally synthesizing a peptide compound in a cell-free translation system comprising the following (i) and (ii): (i) a tRNA to which at least one, two or more, or three or more kinds of amino acids selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6 are bound; (ii) a nucleic acid library encoding the peptide compound library; Here, the nucleic acid library may contain a nucleic acid containing at least one codon corresponding to the anticodon of the tRNA.

[0271] Screening Methods In a non-limiting embodiment, a peptide compound capable of specifically binding to a target molecule can be selected by screening using the library in the present disclosure. The screening method in the present disclosure is not particularly limited, but for example, an mRNA display method can be used.

[0272] In a non-limiting embodiment, the screening method of the present disclosure can enrich peptide compounds that bind to the target molecule by contacting the library of cyclic peptide compounds of the present disclosure with the target molecule and washing away peptide compounds that do not bind to the target molecule (panning). In one embodiment, the amino acid sequence of the bound peptide can be clarified by synthesizing cDNA from mRNA, which is a tag containing base sequence information contained in the peptide compound selected in this manner, amplifying it by PCR, and analyzing the base sequence. In one embodiment, an mRNA library may be obtained by transcribing the amplified cDNA as described above, and a library of peptide compounds may be produced again using these as templates. Since this library is enriched with peptide compounds that can bind to the target molecule, it is possible to further enrich peptide compounds that can specifically bind to the target molecule by performing panning again using this library. By repeating this process multiple times, the target peptide compound can be further enriched. In one embodiment, it is possible to identify the amino acid sequence based on the base sequence information contained in the peptide compound enriched in this manner, and produce a peptide compound that can specifically bind to the target molecule. In one embodiment, the screening method of the present disclosure can be carried out in vitro.

[0273] In a non-limiting embodiment, the peptide compound obtained by the screening method of the present disclosure may be optimized by chemically modifying it by a known method, etc. In this specification, "optimization" means chemically modifying the peptide compound to become more drug-like by converting the structure of each amino acid in the translationally synthesized compound, chemically modifying the peptide compound to become a peptide compound having stronger activity against a pharmacological target, and / or chemically modifying the peptide compound to become a peptide compound with less toxicity.

[0274] In one non-limiting embodiment, the screening method of the present disclosure comprises the steps of: (a) contacting a target molecule with a peptide compound included in a library of the present disclosure; (b) selecting a peptide compound capable of binding to said target molecule.

[0275] In one embodiment, the screening method of the present disclosure may enrich for peptide compounds capable of specifically binding to a target molecule by repeating the above steps (a) and (b) two or more times.

[0276] target molecule The target molecule used in the screening method of the present disclosure is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, sugars, lipids, etc., among which proteins are preferably targeted. In one embodiment, the screening method of the present disclosure can obtain a peptide compound that inhibits protein-protein interactions (PPI). Although it is not intended to be bound by a particular theory, it is believed that, in particular, in terms of the action of PPI, there is a site called a hot spot, which is said to enhance the binding strength of PPI, and therefore, PPI can be inhibited by a long side chain entering this site. In other words, it is believed that the library and screening method of the present disclosure can obtain a peptide compound that inhibits PPI regardless of the type of target molecule.

[0277] The location of the target molecule in the living body is not particularly limited. In one aspect, the library and screening method of the present disclosure can obtain a cyclic peptide compound with high membrane permeability or a cyclic peptide compound whose membrane permeability can be easily increased by derivatization, and therefore, it is also possible to target a protein in a cell.

[0278] In a non-limiting embodiment, the target molecule used in the screening method of the present disclosure is immobilized on a carrier. The carrier is not particularly limited as long as it can immobilize the target molecule, and examples thereof include beads and resins. The target molecule can be immobilized on the carrier by a known method.

[0279] As described above, the target molecules of the library and screening method of the present disclosure are not particularly limited, but examples thereof include GTPase KRas (KRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), mitogen-sctivated protein kinase 3 (ERK1), and interleukin 6 receptor (IL-6R). As described in the Examples, the library of the present disclosure contains peptide compounds capable of specifically binding to various target molecules, and therefore, in one embodiment, may enable drug discovery for tough targets that have been considered difficult to discover in the past.

[0280] In one non-limiting embodiment, a method for producing a cyclic peptide compound of the present disclosure may include the steps of: (i) contacting a target molecule with a cyclic peptide compound included in the library of cyclic peptide compounds of the present disclosure; (ii) selecting a cyclic peptide compound capable of binding to the target molecule; and (iii) producing a cyclic peptide compound based on the amino acid sequence of the cyclic peptide compound selected in (ii);

[0281] In one non-limiting embodiment, the methods for producing libraries, producing peptide compounds, and / or screening methods of the present disclosure may be performed in vitro.

[0282] In one aspect, the present disclosure provides an amino acid having a "long side chain" as defined herein in its side chain. Examples of such amino acids include amino acids selected from the group consisting of the amino acids listed in Tables 2-1 to 2-6.

[0283] In one aspect, the present disclosure provides an amino acid having a group represented by the following general formula (II) or (III) at its side chain: [ka] [In the formula, X 1 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, X 2 represents a single bond or a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, Y 1 is a single bond, an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Y 2 is a single bond, a C1-C2 alkylene group optionally substituted with a C1-C4 alkyl group, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Z 1 represents an oxygen atom, R 2 and R 3 each independently represents a hydrogen atom, a C1-C6 alkyl group, a C2-C4 alkenyl group, or a C2-C4 alkynyl group, wherein the C1-C6 alkyl group may be substituted with a halogen atom, and one or two non-adjacent methylene groups in the C1-C6 alkyl group may be substituted with an oxygen atom. R 2 and R 3 , or R 2 and X 1 may be bonded to form a 3- to 6-membered ring structure, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 4 represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C1-C6 alkanoyl group; R 5 represents a hydrogen atom, and R 4 In the C1-C6 alkyl group, one or two non-adjacent methylene groups may be replaced by an oxygen atom; [ka] represents a bonding point. Group B: halogen atoms, C1-C4 alkyl groups, and C1-C2 alkoxy groups.

[0284] In one aspect, the present disclosure provides an amino acid having a group represented by the following general formula (II) or (III) at its side chain: [ka] [In the formula, X 1 represents a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, X 2 represents a C1-C2 alkylene group which may have at least one substituent independently selected from the following Group B, Y 1 is an oxygen atom, a carbonyl group (-CO-), or a sulfonyl group (-SO 2 -) and Y 2 is a carbonyl group (-CO-) or a sulfonyl group (-SO 2 -) and Z 1 represents an oxygen atom, R 2 and R 3 each independently represents a hydrogen atom or a C1-C6 alkyl group, wherein the C1-C6 alkyl group may be substituted with a halogen atom, and one or two non-adjacent methylene groups in the C1-C6 alkyl group may be substituted with an oxygen atom. R 2 and R 3 , or R 2 and X 1 may be bonded to form a 3- to 6-membered ring structure, in which case the ring structure may be substituted with a C1-C4 alkyl group or a halogen atom, may have an oxygen atom in the ring, and may be partially unsaturated; R 4 represents a C1-C6 alkyl group, R5 represents a hydrogen atom, and R 4 In the C1-C6 alkyl group, one or two non-adjacent methylene groups may be replaced by an oxygen atom; [ka] represents a bonding point. Group B: halogen atoms, C1-C4 alkyl groups, and C1-C2 alkoxy groups.

[0285] the above X 1 is preferably a C1-C2 alkylene group which may have at least one substituent independently selected from the group B, X 2 is preferably a C1-C2 alkylene group which may have at least one substituent independently selected from the group B, Y 1 is preferably an oxygen atom, Y 2 is preferably a carbonyl group (-CO-), R 2 and R 3 are each independently preferably a hydrogen atom or a C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally substituted with a halogen atom, wherein the halogen atom is preferably fluorine; R 4 is preferably a C1-C6 alkyl group. The halogen atom in the group B is preferably a fluorine atom.

[0286] In a non-limiting embodiment, an amino acid in the present disclosure may be a pdCpA amino acid or a pCpA amino acid in which pdCpA or pCpA is linked to the carboxy group via an ester bond. The structures of pdCpA and pCpA and the linkage point via the ester bond to the carboxy group of the amino acid are shown below: [ka] [In the formula, [ka] represents the point of attachment via an ester bond to the carboxy group of the amino acid. Such amino acids can be prepared, for example, using the methods described in the Examples.

[0287] In a non-limiting embodiment, the main chain amino group of the amino acid, pdCpA amino acid, and / or pCpA amino acid in the present disclosure may be protected by a protecting group. Examples of the protecting group for the amino group include, but are not limited to, Fmoc, Boc, and Cbz groups. Methods for protecting the amino group may be known in the art, and may be methods described in the Examples.

[0288] In a non-limiting embodiment, the amino acids in the present disclosure may be used in chemical synthesis and / or translational synthesis of peptide compounds. For example, but not limited to, pdCpA amino acids and / or pCpA amino acids may be used to perform translational synthesis of peptide compounds, and amino acids protected with Fmoc groups may be used to perform chemical synthesis of peptide compounds. The peptide compounds herein are preferably, but are not limited to, cyclic peptide compounds.

[0289] In one aspect, the present disclosure provides amino acids having "long side chains" in their side chains for use in the synthesis of peptide compounds. In another aspect, the present disclosure provides amino acids having "long side chains" in their side chains for use in the translation of peptide compounds. The peptide compounds herein are preferably, but not limited to, cyclic peptide compounds.

[0290] The chemical synthesis method and translational synthesis method of the peptide compound using the amino acids in the present disclosure can be carried out in accordance with techniques known in the art or methods described in the Examples.

[0291] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0292] The present invention is further illustrated by, but not limited to, the following examples.

[0293] The following abbreviations are used in the examples: AA Ammonium Acetate Acbz 4-azidobenzyloxycarbonyl group [ka] AcONH 4 Ammonium acetate CH 2 CN Cyanomethyl group DBU 1,8-diazabicyclo[5.4.0]-7-undecene DCM Dichloromethane DCE 1,2-dichloroethane DMF N,N-Dimethylformamide DIC N,N'-Diisopropylcarbodiimide DIPEA N-ethyl-isopropylpropan-2-amine or N,N-diisopropylethylamine MeCN Acetonitrile MeOH Methanol NMP N-Methyl-2-pyrrolidone FA Formic Acid TFA Trifluoroacetic acid TFE 2,2,2-trifluoroethanol HFIP 1,1,1,3,3,3-Hexafluoroisopropyl alcohol HOAt 1-Hydroxy-7-azabenzotriazole HOBt 1-Hydroxybenzotriazole WSCI·HCl 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride TBME t-Butyl methyl ether TIPS Triisopropylsilane HATU O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate Allyl dba Dibenzylideneacetone DEAD Diethyl azodicarboxylate DIAD Diisopropyl azodicarboxylate DAST Dimethylaminosulfur trifluoride DMAP 4-Dimethylaminopyridine DMSO Dimethyl sulfoxide Fmoc-Cl carbonochloridic acid (9H-fluoren-9-yl)methyl Fmoc-OSu (2,5-dioxopyrrolidin-1-yl)(9H-fluoren-9-yl)methyl carbonate or N-(9-fluorenylmethoxycarbonyloxy)succinimide or N-succinimidyl 9-fluorenylmethyl carbonate F-Pnaz 4-(2-(4-fluorophenyl)acetamido)benzyloxycarbonyl group [ka] GC Gas Chromatography MeAbu (S)-2-(methylamino)butanoic acid Ns Nosyl group [ka] pCpA dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl [ka] pCpA(THF) dihydrogen phosphate ((2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)-4-((tetrahydrofuran-2-yl)oxy)tetrahydrofuran-2-yl)methyl [ka] PPTS Pyridinium p-Toluenesulfonate TBAF Tetrabutylammonium fluoride Tf 2 O Trifluoromethanesulfonic anhydride THF Tetrahydrofuran TMSCl Chlorotrimethylsilane T3P Propylphosphonic anhydride Trt Trityl group

[0294] The reaction solvents used in peptide synthesis and solid-phase synthesis were those for peptide synthesis (purchased from Watanabe Chemical and Wako Pure Chemical). For example, DCM, DMF, NMP, 2% DBU in DMF, 20% piperidine in DMF, etc. In addition, for reactions in which water was not added as a solvent, dehydrated solvents, ultra-dehydrated solvents, and anhydrous solvents (purchased from Kanto Chemical and Wako Pure Chemical, etc.) were used.

[0295] The LC / MS analysis conditions are as follows. [Table 3] TIFF2025038213000042.tif231170 TIFF2025038213000043.tif231170 TIFF2025038213000044.tif231170 TIFF2025038213000045.tif231170 TIFF2025038213000046.tif231170 TIFF2025038213000047.tif229170

[0296] Example 1 Chemical synthesis of peptide compounds According to the peptide synthesis method by the Fmoc method described in WO2013 / 100132, peptide elongation was performed by the following basic route. That is, the process is five steps: 1) peptide elongation reaction by the Fmoc method from the N-terminus of Asp, in which the carboxylic acid of the Asp side chain was supported on the 2-chlorotrityl resin, 2) peptide excision process from the 2-chlorotrityl resin, 3) amide cyclization by condensation of the carboxylic acid of the Asp side chain generated by being removed from the 2-chlorotrityl resin in the excision process with the amino group of the N-terminus (triangle unit) of the peptide chain, 4) deprotection of the protecting group of the side chain functional group contained in the peptide chain, and 5) purification of the compound by preparative HPLC. In this example, unless otherwise stated, the peptide compound was synthesized based on this basic route. [ka]

[0297] 1-1. Fmoc-amino acids for peptide synthesis using a peptide synthesizer In the peptide synthesis described herein, the following Fmoc-amino acids were used in the synthesis by a peptide synthesizer. The abbreviations for amino acids are shown in Tables 4-1 to 4-32. Fmoc-MeLeu-OH, Fmoc-Leu-OH, Fmoc-MeTrp-OH, Fmoc-MePhe-OH, Fmoc-MeIle-OH, Fmoc-Thr(Trt)-OH, Fmoc-MeGly-OH, Fmoc-g-MeAbu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc -Trp-OH, Fmoc-MeAla-OH, Fmoc-Val-OH, Fmoc-D-Ala-OH, Fmoc-MeVal-OH, Fmoc-Ala-OH, Fmoc-D-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(Clt)-OH, Fmoc-D-Leu-OH, Fmoc-Phe(4-CF3 )-OH, Fmoc-D-MeAla-OH, Fmoc-Pro-OH, Fmoc-Pic(2)-OH, Fmoc-EtGly-OH, Fmoc-D-Pro-OH, Fmoc-A bu-OH, Fmoc-Ala(3-Pyr)-OH, FmocAla(4-Pyr)-OH, Fmoc-Phe(3-Cl)-OH, Fmoc-Phg-OH, Fmoc-Met(O 2 )-OH, Fmoc-Ser(Bn)-OH, Fmoc-Hph-OH, Fmoc-Phe{#(CH 2 Fmoc-nPrGly-OH, Fmoc-MePhe(3-Cl)-OH, Fmoc-MeAla(4-Thz)-OH, Fmoc-MeSer(DMT)-OH, and the like were purchased from Watanabe Chemical, Chempep, Chem-Impex, or Bachem. Fmoc-nPrGly-OH, Fmoc-MePhe(3-Cl)-OH, Fmoc-MeAla(4-Thz)-OH, Fmoc-MeSer(DMT)-OH, and the like were synthesized by the method described in WO2013 / 100132. In addition, Fmoc-Thr(THP)-OH (compound aa01), Fmoc-Tyr(3-F,tBu)-OH (compound aa02), Fmoc-Tyr(3-F,Pis)-OH (compound aa03), Fmoc-MePhe(4-Cl)-OH (compound aa04), Fmoc-MeHis(Trt)-OH (compound aa05), Fmoc-MeSer(THP)-OH (compound aa06), Fmoc-Ser(3-F-5-Me-Pyr)-OH (compound aa10), Fmoc-MeHph-OH (compound aa11), Fmoc-Ser(Ph-3-Cl)-OH (compound aa12), etc. were synthesized as follows. Fmoc-MeAbu-OH, Fmoc-D-Abu-OH, Fmoc-D-MeLeu-OH, Fmoc-MeNva-OH, Fmoc-(PhEt)NGly-OH, Fmoc-Hse(Et)-OH, Fmoc-Lys(Ac)-OH, Fmoc-Phe(4-CHF2)-OH, and Fmoc-Phe(4-OCHF2)-OH were purchased from Amatek, Watanabe Chemical Industry, ChemBioBank, etc. Fmoc-Ser(nPr)-OH (compound aa51), Fmoc-MeSer(nPr)-OH (compound aa52), Fmoc-Ser(iPen)-OH (compound aa54), Fmoc-MeSer(iPen)-OH (compound aa55), Fmoc-Hnl(7-F2)-OH (compound aa58), Fmoc-MeHnl(7-F2)-OH (compound aa59), Fmoc-Ser(Ph-2-Cl)-OH (compound aa60), Fmoc-MeSer(Ph-2-Cl)-OH (compound aa65), Fmoc-MeSer(3-F-5 -Me-Pyr)-OH (compound aa67), Fmoc-Ser(F(4)nPr)-OH (compound aa72), Fmoc-Hph(2-Cl)-OH (compound aa73), Fmoc-MeHph(2-Cl)-OH (compound aa75), Fmoc-Hph(3-Cl)-OH (compound aa76), Fmoc-MeHph(3-Cl)-OH (compound aa78), Fmoc-Hph(4-Cl)-OH (compound aa79), Fmoc-MeHph(4-Cl)-OH (compound aa81), Fmoc-MePhe{#(CH2)2}-OH (compound aa8 2), Fmoc-MeSer(Bn)-OH (compound aa83), Fmoc-Hyp(Et)-OH (compound aa84), Fmoc-Ala(3-Pyr-4-NMe2)-OH (compound aa88), Fmoc-nPenGly-OH (compound aa89), Fmoc-nHexGly (compound aa90), Fmoc-(EtOEt)NGly-OH (compound aa91), Fmoc-(PhOEt)NGly-OH (compound aa92), Fmoc-Gln(Me2)-OH (compound aa94), Fmoc-MeGln(Me2)-OH (compound aa95), 6), Fmoc-Gln(Me)-OH (compound aa98), Fmoc-MeGln(Me)-OH (compound aa101), Fmoc-Ser(NtBu-Aca)-OH (compound aa104), Fmoc-MeSer(NtBu-Aca)-OH (compound aa105), Fmoc-MeHse(Et)-OH (compound aa106), Fmoc-Nle(6-OTHP)-OH (compound aa107), Fmoc-Abu(pip-4-F2)-OH (compound aa109), Fmoc-MeAbu(pip-4-F2)-OH (compound aa110),Fmoc-MeAbu(pip-3-F2)-OH (compound aa116), Fmoc-Abu(Mor)-OH (compound aa123), Fmoc-MeAbu(Mor)-OH (compound aa125), Fmoc-(2-(pip-4-F2)-Et)Gly-OH (compound aa126), Fmoc-Pro(pip-4-F2)-OH (compound aa130), Fmoc-cisPro(pip-4-F2)-OH (compound aa133), Fmoc-Ahp(2)(3-R-OTHP)-OH (compound aa137), Fmoc-Ser(EtOTHP)-OH (compound aa142), Fmoc-MeSer(EtOTHP)-OH (compound aa147), Fmoc-Ser(S-2-PrOTHP)-OH (compound aa154), Fmoc-MeSer(S-2-PrOTHP)-OH (compound aa159), Fmoc-Ser(R-2-PrOTHP)-OH (compound aa166), Fmoc-MeSer(R-2-PrOTHP)-OH (compound aa171), Fmoc-Ser(tBuOH)-OH (compound aa173), F moc-Ser(tBuOTHP)-OH (compound aa174), Fmoc-MeSer(tBuOTHP)-OH (compound aa175), Fmoc-Ser(2-Me-BuOH)-OH (compound aa181), Fmoc-Ser(2-Me-BuOTHP)-OH (compound aa182), Fmoc-MeSer(2-Me-BuOH)-OH (compound aa184), Fmoc- MeAla(3-Pyr-4-NMe2)-OH (compound aa186), Fmoc-MePhe(4-CF3)-OH (compound aa187), Fmoc-MePhe(4-CHF2)-OH (compound aa188), Fmoc-MePhe(4-OCHF2)-OH (compound aa189), Fmoc-Ser(Ph-4-Cl)-OH (compound aa190), Fmoc-Ser(Et -2-Mor)-OH (compound aa191), Fmoc-nBuGly-OH (compound aa192), Fmoc-iPenGly-OH (compound aa193), Fmoc-Abu(5-Oxo-Odz)-OH (compound aa194), Fmoc-MeAbu(5-Oxo-Odz)-OH (compound aa196), Fmoc-MeAla(3-Pyr)-OH (compound aa197),Fmoc-Ser(Et-2-NMe2)-OH(compound aa198), Fmoc-Gln(Ms)-OH(compound aa199), Fmoc-Ser(1-CF3-EtOH)-OH(compound aa201), Fmoc-MeSer( 1-CF3-EtOH)-OH (compound aa202), Fmoc-Ser (1-CF3-EtOTHP)-OH (compound aa203), Fmoc-Hnl (7-F3-6-OH)-OH (compound aa208) were synthesized as follows. ,

[0298]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

Table 4-29

Table 4-30

Table 4-31

Table 4-32

[0299] 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)

change

[0300] Toluene (50mL) was added to a mixture of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid monohydrate (monohydrate of Fmoc-Thr-OH, purchased from Tokyo Kasei, 5.0g, 13.9mmol) and pyridinium p-toluenesulfonate (PPTS, 0.175g, 0.70mmol), and the water contained therein was removed by azeotropy by distilling off the toluene under reduced pressure. Super-dehydrated tetrahydrofuran (THF, 28mL) and 3,4-dihydro-2H-pyran (8.8mL, 97mmol) were added to the resulting residue, and the mixture was stirred at 50°C for 4 hours under a nitrogen atmosphere. After confirming the disappearance of the raw materials by LCMS (SQDFA05), the mixture was cooled to 25°C, and ethyl acetate (30mL) was added. Saturated aqueous sodium chloride solution (30 mL) was then added to wash the organic layer, and the aqueous layer was extracted with ethyl acetate (30 mL). All the organic layers obtained were mixed and washed twice with saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (9.3 g).

[0301] Of the obtained crude product, 4.65 g was dissolved in tetrahydrofuran (THF, 30 mL), and then 1.0 M phosphate buffer (30 mL) adjusted to pH 8.0 was added. This mixture was stirred at 50 degrees for 4 hours. After cooling to 25 degrees, ethyl acetate (30 mL) was added and the organic layer and aqueous layer were separated. After adding ethyl acetate (30 mL) to the aqueous layer and performing extraction, all the obtained organic layers were mixed and washed twice with saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and further dried at 25 degrees for 30 minutes under reduced pressure with a pump.

[0302] The resulting residue was dissolved in diethyl ether (50 mL), and then heptane (50 mL) was added. Under controlled reduced pressure (~100 hPa), only diethyl ether was distilled off, and the resulting mixture was filtered to obtain a solid. This washing operation with heptane was repeated twice. The obtained solid was dried under reduced pressure on a pump at 25°C for 2 hours to obtain the sodium salt of Fmoc-Thr(THP)-OH (2.80 g, 6.26 mmol).

[0303] Ethyl acetate (50mL) and 0.05M phosphoric acid aqueous solution (140mL) of pH 2.1 were added to the total amount of sodium salt of Fmoc-Thr(THP)-OH obtained, and the mixture was stirred at 25 degrees for 5 minutes, and then the organic layer and the aqueous layer were separated. After adding ethyl acetate (50mL) to the aqueous layer and extracting it, all the obtained organic layers were mixed and washed twice with saturated aqueous sodium chloride solution (50mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure at 25 degrees for 2 hours with a pump, and the obtained solid was dissolved in t-butyl methyl ether (TBME, 50mL), and the solvent was distilled off under reduced pressure. Further, by drying at 25 degrees under reduced pressure with a pump for 1 hour, (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) was obtained as a diastereomer derived from the asymmetric carbon on the THP protection. The obtained Fmoc-Thr(THP)-OH was stored in a freezer at -25 degrees. LCMS(ESI)m / z=424.2(MH) - Retention time: 0.84 minutes, 0.85 minutes (Analysis conditions SQDFA05)

[0304] Synthesis of (S)-methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoate (compound aa07, Fmoc-Tyr(3-F)-OMe) [ka]

[0305] (S)-2-Amino-3-(3-fluoro-4-hydroxyphenyl)propanoic acid (H 2N-Tyr(3-F)-OH (purchased from Astatech, 2.0 g, 10.0 mmol) was dissolved in 10% aqueous sodium carbonate, and then a solution of (2,5-dioxopyrrolidin-1-yl)(9H-fluoren-9-yl)methyl carbonate (Fmoc-OSu, 3.39 g, 10.0 mmol) in 1.4-dioxane (35 mL) was added at 0°C using a dropping funnel. The reaction solution was stirred at 25°C for 40 minutes, after which water (35 mL) and diethyl ether (70 mL) were added and washed three times with diethyl ether. The pH of the aqueous layer was adjusted to 2-3 with a 5N aqueous hydrochloric acid solution, and then extracted three times with ethyl acetate (100 mL x 3). The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the mixture was further dried by pumping. The resulting residue (4.08 g) was used as it was in the next reaction without further purification. The above residue (1.04 g) was dissolved in methanol (10 mL) and thionyl chloride (SOCl 2 , 539μL, 7.38mmol) was added dropwise at 0°C. The reaction solution was stirred at 60°C for 1 hour, cooled to room temperature, and the solvent was distilled off using an evaporator. Ethyl acetate and water were added to the resulting residue, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with saturated saline, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure, and further dried by pump. The resulting residue was purified by flash column chromatography (purif pack (registered trademark) SIZE200, hexane / ethyl acetate), and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoate (S)-methyl (compound aa07, Fmoc-Tyr(3-F)-OMe, 900mg, 2.07mmol) was obtained in a two-step yield of 84%. LCMS(ESI)m / z=436.4(M+H) + Retention time: 0.82 minutes (Analysis conditions SQDFA05)

[0306] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid (compound aa02, Fmoc-Tyr(3-F,tBu)-OH) [ka]

[0307] A solution (690 μL) of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoate (S)-methyl (compound aa07, Fmoc-Tyr(3-F)-OMe, 300 mg, 0.689 mmol) in tetrahydrofuran (THF) was added to tert-butyl 2,2,2-trichloroacetimidate (308 μL, 1.72 mmol) and a catalytic amount of boron trifluoride-ethyl ether complex (BF 3 -OEt 2 The reaction mixture was stirred at 25°C for 1 hour, and then the same amount of tert-butyl 2,2,2-trichloroacetimidate (308 μL, 1.72 mmol) and boron trifluoride-ethyl ether complex (BF 3 -OEt 2 , 13.1μL, 0.103mmol) was added again, and the reaction solution was stirred at 25°C for another hour. The reaction solution was diluted with dichloromethane (DCM), and saturated aqueous sodium bicarbonate was added. After extraction with dichloromethane, the organic layer was washed with saturated aqueous sodium bicarbonate and saturated saline. The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the mixture was dried by pump. The resulting residue was purified by flash column chromatography (purif pack (registered trademark) SIZE60, hexane / ethyl acetate) to obtain 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid (S)-methyl (Fmoc-Tyr(3-F,tBu)-OMe) as a mixture.

[0308] The mixture obtained above (40 mg) was dissolved in dichloroethane (DCE) (810 μL) and trimethyltin(IV) hydroxide (Me 3SnOH, 29.4 mg, 0.163 mmol) was added and stirred at 60 degrees for 1 hour. After adding formic acid (15.35 μL, 0.407 mmol) to the reaction solution, the mixture was purified by reverse phase chromatography (Wakosil 25C18 10 g, 0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(tert-butoxy)-3-fluorophenyl)propanoic acid (compound aa02, Fmoc-Tyr(3-F,tBu)-OH, 27 mg, 56.5 μmol) in a two-step yield of 93%. LCMS(ESI)m / z=478.3(M+H) + Retention time: 0.94 minutes (Analysis conditions SQDFA05)

[0309] Synthesis of (S)-methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoate (compound aa08, Fmoc-Tyr(3-F,Pis)-OMe) [ka]

[0310] 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-hydroxyphenyl)propanoate (S)-methyl (compound aa07, Fmoc-Tyr(3-F)-OMe, 200 mg, 0.459 mmol) was dissolved in THF (460 μL), and then 2,2,2-trichloroacetimidate 2-phenylpropan-2-yl (compound aa09, 322 mg, 1.15 mmol) and a catalytic amount of boron trifluoride-ethyl ether complex (BF 3 -OEt 2 The reaction mixture was stirred at room temperature for 30 minutes, and then the same amount of 2,2,2-trichloroacetimidate 2-phenylpropan-2-yl (322 mg, 1.15 mmol) and a catalytic amount of boron trifluoride-ethyl ether complex (BF 3 -OEt 2A 100% aqueous solution of 1,2-dichloromethane (8.73 μL, 0.069 mmol) was added dropwise at 0°C. The reaction solution was further stirred at room temperature for 30 minutes, then diluted with dichloromethane, and saturated aqueous sodium bicarbonate solution was added under ice-cooling. After extraction with dichloromethane, the organic layer was washed with saturated saline. The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure, and further dried with a pump. The obtained residue was washed twice with dichloromethane / hexane = 1 / 1 (20 mL, 10 mL), and the white solid was removed by filtration. The obtained filtrate was concentrated, and the residue was purified by flash column chromatography (purif pack SIZE 20, hexane / ethyl acetate, 0.1% diisopropylethylamine (DIPEA)), and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (S)-methyl (compound aa08, Fmoc-Tyr(3-F,Pis)-OMe, 210 mg, 0.379 mmol) was obtained in a yield of 83%. LCMS(ESI)m / z=554.4(M+H) + Retention time: 1.09 minutes (Analysis conditions SQDFA05)

[0311] Preparation of 2-phenylpropan-2-yl 2,2,2-trichloroacetimidate (compound aa09) [ka]

[0312] 2-Phenylpropan-2-ol (purchased from Wako, 2.0 g, 14.7 mmol) in diethyl ether (Et 2A 1.9M NaHMDS tetrahydrofuran (THF) solution (850μL, 1.62mmol) was added dropwise to a 1.9M NaHMDS (4.8mL) solution at 22°C. The reaction solution was stirred at the same temperature for 20 minutes, then cooled to 0°C, and 2,2,2-trichloroacetonitrile (1.47mL, 14.7mmol) was added dropwise. The reaction solution was stirred at 0°C for 10 minutes, then heated to 15°C and stirred for another hour. The reaction solution was concentrated using an evaporator, and hexane (1.8mL) and methanol (65μL) were added to the resulting residue and stirred at 15°C for 15 minutes. The resulting solid was filtered and washed three times with hexane (2.0mL), yielding 4.19g of 2,2,2-trichloroacetimidate 2-phenylpropan-2-yl (compound aa09). This was used in the reaction without further purification.

[0313] 1 H NMR (Varian400-MR, 400MHz, CDCl 3 )δ1.89(6H, s), 7.28(1H, m), 7.36(2H, m), 7.43(2H, m), 8.20(1H, brs)

[0314] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (compound aa03, Fmoc-Tyr(3-F,Pis)-OH) [ka]

[0315] (S)-Methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoate (compound aa08, Fmoc-Tyr(3-F,Pis)-OMe, 210 mg, 0.379 mmol) was dissolved in dichloroethane (DCE) (1.26 mL) and trimethyltin(IV) hydroxide (Me 3SnOH (137 mg, 0.379 mmol) was added and stirred at 60°C for 3 hours. The reaction solution was concentrated using an evaporator, and t-butyl methyl ether (TBME, 2.0 mL) and 0.05 M aqueous phosphoric acid solution (pH 2.1, 4.0 mL) were added and stirred at 25°C for 15 minutes. After separating the organic layer, the aqueous layer was extracted twice with t-butyl methyl ether (TBME, 1 mL). The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the mixture was further dried by pumping. The resulting residue was dissolved in a 0.1% formic acid acetonitrile solution and stirred for 15 minutes. The resulting solution was then purified by reverse phase chromatography (Wakosil 25C18 30 g, 0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-fluoro-4-((2-phenylpropan-2-yl)oxy)phenyl)propanoic acid (compound aa03, Fmoc-Tyr(3-F,Pis)-OH, 190 mg, 0.352 mmol) in a yield of 93%. LCMS(ESI)m / z=538.2(MH) - Retention time: 1.00 minutes (Analysis conditions SQDFA05)

[0316] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid (compound aa04, Fmoc-MePhe(4-Cl)-OH) [ka]

[0317] Paraformaldehyde (48 g, 1.60 mol) and 10-camphorsulfonic acid (CSA, 4.6 g, 19.83 mmol) were added to a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-chlorophenyl)propanoic acid (Fmoc-Phe(4-Cl)-OH, 170 g, 402.96 mmol) in toluene (2.5 L), and the mixture was stirred at 110 degrees for 16 hours. The reaction solution was then washed twice with a saturated aqueous sodium bicarbonate solution (1.0 L) and twice with a saturated aqueous sodium chloride solution (1.0 L). The organic layer was dried over sodium sulfate, the solid was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 160 g of 4-(4-chlorobenzyl)-5-oxooxazolidine-3-carboxylate (S)-(9H-fluoren-9-yl)methyl.

[0318] A solution of 4-(4-chlorobenzyl)-5-oxooxazolidine-3-carboxylate (S)-(9H-fluoren-9-yl)methyl (230 g, 530.10 mmol) mixed with another lot prepared by the same operation in dichloromethane (2.5 L) was mixed with triethylsilane (881 g, 7.58 mol) and trifluoroacetic acid (TFA, 2518 g, 22.28 mol) and stirred at 30 degrees for 12 hours. The solvent was then distilled off under reduced pressure, and the resulting residue was recrystallized with dichloromethane / hexane (1 / 10, v / v) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-chlorophenyl)propanoic acid (compound aa04, Fmoc-MePhe(4-Cl)-OH, 205 g). LCMS(ESI)m / z=436.3(M+H) + Retention time: 0.99 minutes (Analysis conditions SQDFA05)

[0319] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazol-4-yl)propanoic acid (compound aa05, Fmoc-MeHis(Trt)-OH) [ka]

[0320] A 3L flask was charged with a dichloromethane (1.0L) solution of (S)-3-(1H-imidazol-4-yl)-2-(methylamino)propanoic acid hydrochloride (75g, 364.71mmol), dichlorodimethylsilane (51g, 395.16mmol), and triethylamine (40g, 395.30mmol). Then, a dichloromethane (500mL) solution of (chloromethanetriyl)tribenzene (Trt-Cl, 111g, 398.17mmol) and triethylamine (40g, 395.30mmol) were added. The resulting reaction solution was stirred under heating and reflux for 4 hours, and further stirred at 20 degrees for 2 hours. Methanol was added to the reaction solution to stop the reaction, and the solvent was then distilled off under reduced pressure. The pH was adjusted to 8-8.5 with triethylamine, and 125g of a solid was obtained.

[0321] 1,4-dioxane (1.0L), potassium carbonate (84g, 603.39mmol), and water (1.0L) were added to the obtained solid. Furthermore, (2,5-dioxopyrrolidin-1-yl)(9H-fluoren-9-yl)methyl carbonate (Fmoc-OSu, 102g, 302.38mmol) was added and stirred at 0 degrees for 2 hours. After washing the obtained reaction solution with diethyl ether (2.0L), the pH of the solution was adjusted to 6-7 using acetic acid. The obtained solid was filtered to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(1-trityl-1H-imidazol-4-yl)propanoic acid (compound aa05, Fmoc-MeHis(Trt)-OH, 155g). LCMS(ESI)m / z=634.4(M+H) + Retention time: 1.07 minutes (Analysis conditions SQDAA05)

[0322] Synthesis of (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (compound aa06, Fmoc-MeSer(THP)-OH) [ka]

[0323] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-hydroxypropanoic acid (Fmoc-MeSer-OH) was synthesized by the method described in WO2013 / 100132. Pyridinium p-toluenesulfonate (PPTS, 0.552 g, 2.197 mmol) and 3,4-dihydro-2H-pyran (23.85 mL) were added to a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-hydroxypropanoic acid (Fmoc-MeSer-OH, 15 g, 43.9 mmol) in tetrahydrofuran (88 mL), and the mixture was stirred at 50° C. for 4 hours. The mixture was cooled to 25° C., and ethyl acetate (90 mL) was added. The organic layer was then washed with saturated aqueous sodium chloride solution (90 mL), and the aqueous layer was extracted with ethyl acetate (90 mL). All the organic layers obtained were mixed and washed twice with saturated aqueous sodium chloride solution (90 mL). The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure.

[0324] Of the resulting residue, 15.0 g was dissolved in tetrahydrofuran (175 mL), and then 1.0 M phosphate buffer (175 mL) adjusted to pH 8.0 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 layer and the aqueous layer were separated. After extraction by adding ethyl acetate (175 mL) to the aqueous layer, all the resulting organic layers were mixed and washed twice with saturated aqueous sodium chloride solution (175 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was dissolved in dichloromethane (100 mL), and then heptane (250 mL) was added. Under controlled reduced pressure (~100 hPa), only dichloromethane was distilled off, and the resulting mixture was filtered to obtain a solid. This washing operation with heptane was repeated twice. The obtained solid was dried at 25°C for 2 hours under reduced pressure by pumping.

[0325] To the residue obtained, t-butyl methyl ether (TBME, 250 mL) and 0.05 M aqueous phosphoric acid solution (700 mL) of pH 2.1 were added, and the mixture was stirred at 25 degrees for 5 minutes, after which the organic layer and the aqueous layer were separated. After the aqueous layer was extracted with t-butyl methyl ether (TBME, 250 mL), all the organic layers obtained were mixed and washed twice with saturated aqueous sodium chloride solution (250 mL). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dried under reduced pressure at 25 degrees for 2 hours with a pump to obtain (2S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-((tetrahydro-2H-pyran-2-yl)oxy)propanoic acid (compound aa06, Fmoc-MeSer(THP)-OH, 9.0 g, 30 mol% of t-butyl methyl ether (TBME) remained). The resulting Fmoc-MeSer(THP)-OH was stored in a freezer at -25°C. LCMS(ESI)m / z=426.4(M+H) + Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0326] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa10, Fmoc-Ser(3-F-5-Me-Pyr)-OH) N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa10, Fmoc-Ser(3-F-5-Me-Pyr)-OH) was synthesized according to the following route. [ka]

[0327] Synthesis of (5-fluoropyridin-3-yl)methanol (compound aa14) [ka] Under a nitrogen atmosphere, THF (200 mL) was added to commercially available 5-fluoronicotinic acid (compound aa13) (14.1 g, 100 mmol), and the mixture was stirred using a mechanical stirrer. Next, triethylamine (19.51 mL, 140 mmol) was added at room temperature, and the mixture was stirred until the solid was completely dissolved, and the reaction solution was cooled using an ice bath. Then, ethyl chloroformate (11.5 mL, 120 mmol) was added dropwise, and the mixture was stirred for 30 minutes in an ice bath. Then, the reaction solution was cooled to -60°C or lower using a dry ice bath, and LiAlH 4 A THF solution (2.5 M, 40 mL, 100 mmol) of (lithium aluminum hydride) was added dropwise over 5 minutes so that the reaction temperature did not exceed -20°C. The reaction solution was stirred at -78°C for 3 hours, and then ethyl acetate (69 mL) was added so that the reaction temperature did not exceed -20°C. The mixture was then stirred for another hour using an ice bath, water (18 mL) was added, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was filtered using NH silica gel and concentrated under reduced pressure to obtain (5-fluoropyridin-3-yl)methanol (compound aa14) (9.28 g, 73%) as a yellow oily component. LCMS(ESI)m / z=128(M+H) + Retention time: 0.28 minutes (Analysis conditions SQDFA05)

[0328] Synthesis of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa15) [ka] A condenser cooled with acetone / dry ice was connected to the round-bottom flask, and (5-fluoropyridin-3-yl)methanol (compound aa14) (8.21 g, 64.6 mmol) and 25% HBr in acetic acid (96 mL, 388 mmol) were added under a nitrogen atmosphere. The reaction was carried out in an open system, and 1 M aqueous sodium hydroxide was used to trap the HBr generated. The reaction solution was gradually heated from room temperature to 100°C while stirring, and further stirred for 3 hours. The reaction solution was then cooled to room temperature, and diisopropyl ether (48 mL) was slowly added three times, and the resulting solid was filtered to obtain 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa15) (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)

[0329] Synthesis of O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa17, Trt-Ser(3-F-5-Me-Pyr)-OH) [ka] Under a nitrogen atmosphere, THF (45.8 mL) was added to sodium t-pentoxide (NaOtPen) (13.7 g, 125 mmol) and stirred, then commercially available triethylamine salt of tritylserine (Trt-Ser-OH triethylamine salt) (11.24 g, 25 mmol) was added in three portions and stirred at room temperature for 30 minutes. The reaction solution was stirred for 15 minutes using an ice bath, cooled, and then a DMF solution (16 mL) of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa15, 8.13 g, 30 mmol) was added dropwise, followed by the addition of DMF (14 mL). After stirring the reaction solution for 45 minutes in an ice bath, a DMF solution (4.0 mL) of 3-(bromomethyl)-5-fluoropyridine hydrobromide (compound aa15, 2.03 g, 7.5 mmol) was added dropwise, and then DMF (4.0 mL) was added. The reaction solution was then stirred at room temperature for another 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 and adjusted to pH=7 using a saturated aqueous solution of 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 saline diluted twice, and then washed twice with saturated saline. 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 aa17, Trt-Ser(3-F-5-Me-Pyr)-OH) (11.07 g, 97%) as a yellow amorphous substance. LCMS(ESI)m / z=455(MH) - Retention time: 0.86 minutes (Analysis conditions SQDFA05)

[0330] Synthesis of O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa18, H-Ser(3-F-5-Me-Pyr)-OH) [ka] O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa17, Trt-Ser(3-F-5-Me-Pyr)-OH) (10.76 g, 23.57 mmol) in 1.4-dioxane solution (21.5 mL) was added at room temperature to 5-10% hydrochloric acid / methanol solution (64.2 mL). After stirring the reaction solution at room temperature for 10 to 20 minutes, 1.4-dioxane (135 mL) was added. After adding additional 1.4-dioxane (90 mL), a small amount of seed crystals (5 mg) prepared in advance was added 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 obtain O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa18, H-Ser(3-F-5-Me-Pyr)-OH)) (6.58 g, 95%) as the hydrochloride salt. LCMS(ESI)m / z=213(MH) - Retention time: 0.24 minutes (Analysis conditions SQDAA05)

[0331] Preparation of seed crystals of O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa18, H-Ser(3-F-5-Me-Pyr)-OH) O-((5-fluoropyridin-3-yl)methyl)-N-trityl-L-serine (compound aa17, Trt-Ser(3-F-5-Me-Pyr)-OH) (98 mg, 2.69 mmol) in 1.4-dioxane solution (819 μL) was added with 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 solution, and the obtained solid was filtered, washed with diisopropyl ether, and dried under reduced pressure to obtain O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa18, H-Ser(3-F-5-Me-Pyr)-OH)) (222.5 mg, 86%) as a hydrochloride salt. LCMS(ESI)m / z=213(MH) - Retention time: 0.24 minutes (Analysis conditions SQDAA05)

[0332] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa10, Fmoc-Ser(3-F-5-Me-Pyr)-OH) [ka] Water (42 mL), 1.4-dioxane (115 mL), and diisopropylethylamine (DIPEA) (13.53 mL, 78 mmol) were added to the hydrochloride salt (6.37 g, 22.19 mmol) of O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa18, H-Ser(3-F-5-Me-Pyr)-OH) at room temperature and stirred. Then, N-succinimidyl 9-fluorenylmethyl carbonate (Fmoc-OSu) (7.86 g, 23.3 mmol) was added to the reaction solution at room temperature and stirred at room temperature. After confirming the disappearance of the raw material by LC-MS, water (56.2 mL) was added to the reaction solution at room temperature and washed twice with 25% t-butyl methyl ether (MTBE) / hexane solution. The obtained aqueous layer was dissolved in saturated sodium dihydrogen phosphate aqueous solution (NaH 2 PO 4 ) was used to adjust the pH to 6.1. It was then extracted twice with ethyl acetate, and the resulting organic layers were combined and washed with a solution of saturated saline diluted twice and saturated saline. The resulting organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (compound aa10, 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)

[0333] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound aa11, 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 organic layer was combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 100 g of (S)-5-oxo-4-phenethyloxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl as a crude product. The above-obtained (S)-5-oxo-4-phenethyloxazolidine-3-carboxylate (9H-fluoren-9-yl) (50 g, 118.51 mmol) was dissolved in dichloromethane (700 mL) and triethylsilane (Et 3 SiH) and trifluoroacetic acid (TFA) (700 mL) were added and stirred at 25°C for 16 hours. The reaction solution was concentrated under reduced pressure, and an aqueous potassium carbonate solution was added to the resulting residue, which was then washed with petroleum ether. The resulting aqueous layer was adjusted to pH = 3 using 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 obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-4-phenylbutanoic acid (compound aa11, Fmoc-MeHph-OH) (29.5 g, 58%, 2 steps). LCMS(ESI)m / z=416(M+H) + Retention time: 0.95 minutes (Analysis conditions SQDFA05)

[0334] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa12, Fmoc-Ser(Ph-3-Cl)-OH) (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa12, Fmoc-Ser(Ph-3-Cl)-OH) was synthesized via the following route. [ka]

[0335] Synthesis of (S)-methyl 3-(3-chlorophenoxy)-2-(tritylamino)propanoate (Trt-Ser(Ph-3-Cl)-OMe) (compound aa20) [ka] Under a nitrogen atmosphere, commercially available (S)-methyl 3-hydroxy-2-(tritylamino)propanoate (compound aa19, Trt-Ser-OMe) (6.0 g, 16.6 mmol) and triphenylphosphine (PPh 3 A toluene solution (35 mL) of 2.2 M diethyl azocarboxylate (DEAD) (15.06 mL, 33.2 mmol) was added to the toluene solution (35 mL) of 2.2 M diethyl azocarboxylate (DEAD) (15.06 mL, 33.2 mmol) at room temperature, and the reaction solution was stirred at 50° C. for 30 minutes. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reversed-phase silica gel column chromatography (10 mM aqueous ammonium acetate solution / methanol) to obtain (S)-methyl 3-(3-chlorophenoxy)-2-(tritylamino)propanoate (Trt-Ser(Ph-3-Cl)-OMe) (compound aa20) (4.43 g, 57%). 1 H NMR (Varian400-MR, 400MHz, d-DMSO) δ3.17(3H, s), 3.49-3.51(1H, m), 4.06-4.08(1H, m), 4.19-4.22( 1H, m), 6.83-6.85 (1H, m), 6.99-7.01 (2H, m), 7.20-7.21 (3H, m), 7.28-7.30 (7H, m), 7.42-7.44 (6H, m)

[0336] Synthesis of (S)-2-amino-3-(3-chlorophenoxy)propanoic acid (compound aa21, H-Ser(Ph-3-Cl)-OH) [ka] 1.0M lithium hydroxide / methanol solution (83mL) was added to 1.4-dioxane solution (80mL) of 3-(3-chlorophenoxy)-2-(tritylamino)propanoic acid (S)-methyl (Trt-Ser(Ph-3-Cl)-OMe) (compound aa20) (3.9g, 8.26mmol), and the reaction solution was stirred at 50°C for 3 hours. After concentrating the reaction solution under reduced pressure, trifluoroacetic acid (TFA) (30mL) was added to the resulting residue and stirred at 50°C for 10 minutes. Thereafter, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reversed-phase silica gel column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile) to obtain (S)-2-amino-3-(3-chlorophenoxy)propanoic acid (compound aa21, H-Ser(Ph-3-Cl)-OH) (850mg, 48%, 2 steps). LCMS(ESI)m / z=216(M+H) + Retention time: 0.37 minutes (Analysis conditions SQDFA05)

[0337] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa12, Fmoc-Ser(Ph-3-Cl)-OH) [ka] (S)-2-amino-3-(3-chlorophenoxy)propanoic acid (compound aa21, 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, and then cesium carbonate (2.569 g, 7.88 mmol) was added and stirred at room temperature. After confirming the disappearance of the raw material by LC-MS, water (20 mL) was added and washed twice with diethyl ether (40 mL). The obtained aqueous layer was adjusted to pH = 2 using a 5N aqueous hydrochloric acid solution 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 obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-chlorophenoxy)propanoic acid (compound aa12, 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)

[0338] Fmoc-Ser(nPr)-OH (compound aa51) was synthesized according to the following scheme. [ka]

[0339] Synthesis of O-allyl-N-(tert-butoxycarbonyl)-L-serine (compound aa50, Boc-Ser(Allyl)-OH) [ka] (tert-Butoxycarbonyl)-L-serine (Boc-Ser-OH) (50g, 234.65mmol) was dissolved in dehydrated dimethylformamide (DMF) (250mL), and sodium hydride (22g, 916.67mmol, 60% oil dispersion) was added under ice cooling (below 10 degrees). After stirring for 30 minutes under ice cooling, 3-bromoprop-1-ene (36.7g, 303.37mmol) was added dropwise under ice cooling (below 10 degrees). After the addition, the reaction solution was stirred at 25 degrees for 16 hours, and then ice water (500mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (500mLx3 times). The organic layer was washed three times with saturated saline and dried over anhydrous sodium sulfate. After filtration, the ethyl acetate was removed by vacuum concentration to obtain a crude product containing the target product. The same reaction was carried out three times, and the crude products from the four reactions were combined and purified by column chromatography (petroleum ether / ethyl acetate) to obtain O-allyl-N-(tert-butoxycarbonyl)-L-serine (compound aa50, Boc-Ser(Allyl)-OH) (196.7 g, 82%). LCMS(ESI) m / z = 268 (M+Na)+ Retention time: 0.90 minutes (Analysis conditions SMD method17)

[0340] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-propyl-L-serine (compound aa51, Fmoc-Ser(nPr)-OH) [ka] O-allyl-N-(tert-butoxycarbonyl)-L-serine (compound aa50, Boc-Ser(Allyl)-OH) (148 g, 603.41 mmol) was dissolved in methanol (1000 mL), and ammonia in methanol solution (2 M, 453 mL) was added and stirred at room temperature for 20 minutes. Then, Pd / C (29.6 g) was added and stirred at 25 degrees under a hydrogen atmosphere (5 atm) for 16 hours. Then, the solid was removed by filtration through Celite, and the solvent was removed by vacuum concentration to obtain N-(tert-butoxycarbonyl)-O-propyl-L-serine (Boc-Ser(nPr)-OH) (146.7 g, 98%). The obtained N-(tert-butoxycarbonyl)-O-propyl-L-serine (Boc-Ser(nPr)-OH) (146.7 g) was dissolved in 1,4-dioxane (500 mL), and concentrated hydrochloric acid (297 mL) was added dropwise, followed by stirring at room temperature for 16 hours. The reaction solution was adjusted to pH 7 to 8 by adding an aqueous potassium carbonate solution, and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (240.17 g) and potassium carbonate (165.1 g, 1.19 mol) were added and stirred at room temperature for 16 hours. The reaction solution was then washed three times with hexane, and the pH of the aqueous layer was adjusted to 1 to 2 by adding an aqueous hydrochloric acid solution (6 M), followed by extraction three times with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to remove the solvent. The resulting crude product was washed with hexane and ether and purified by reverse phase column chromatography to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-propyl-L-serine (compound aa51, Fmoc-Ser(nPr)-OH) (103.2 g, 24%) as a white solid. LCMS(ESI) m / z = 370 (M+H)+ Retention time: 2.11 minutes (Analysis conditions SMD method 18)

[0341] Compound aa52 was synthesized according to the following scheme. [ka]

[0342] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-propyl-L-serine (compound aa52, Fmoc-MeSer(nPr)-OH) [ka] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-propyl-L-serine (compound aa51, Fmoc-Ser(nPr)-OH) (67 g, 181.37 mmol), p-toluenesulfonic acid (TsOH) (1.86 g, 10.80 mmol) and paraformaldehyde ((CH 2(O)n) (10.86 g) was suspended in toluene (670 mL) and stirred at 110°C for 16 hours under a nitrogen atmosphere. The reaction solution was then diluted with ethyl acetate and washed twice with a saturated aqueous solution of sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding (S)-5-oxo-4-(propoxymethyl)oxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl as a crude product. The resulting crude product, (S)-(9H-fluoren-9-yl)methyl 5-oxo-4-(propoxymethyl)oxazolidine-3-carboxylate (30 g, 78.65 mmol), was dissolved in dichloromethane (DCM) (480 mL) and triethylsilane (Et 3 SiH) (28 g, 240.80 mmol) and trifluoroacetic acid (TFA) (480 mL) were added at room temperature. After stirring the reaction solution at room temperature for 2 days, the reaction solution was concentrated under reduced pressure. The resulting residue was dissolved in an aqueous potassium carbonate solution, which was washed twice with hexane. Then, concentrated hydrochloric acid was added to the aqueous layer to adjust the pH from 2 to 3, and the aqueous layer was extracted twice with ethyl acetate. The organic layer was washed three times with saturated saline, dried over anhydrous sodium sulfate, and filtered. The solvent was removed by concentration under reduced pressure to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-O-propyl-L-serine (compound aa52, Fmoc-MeSer(nPr)-OH) (24 g, 80%). LCMS(ESI) m / z = 384 (M+H)+ Retention time: 1.68 minutes (Analysis conditions SMD method 19)

[0343] Compound aa54 (Fmoc-Ser(iPen)-OH) was synthesized according to the following scheme. [ka]

[0344] Synthesis of N-(tert-butoxycarbonyl)-O-(3-methylbut-2-en-1-yl)-L-serine (compound aa53) [ka] (tert-Butoxycarbonyl)-L-serine (Boc-Ser-OH) (50g, 234.65mmol) was dissolved in dimethylformamide (DMF) (250mL), and sodium hydride (22g, 916.67mmol, 60% oil dispersion) was added under ice cooling (0 degrees). After stirring for 30 minutes under ice cooling, 1-bromo-3-methylbut-2-ene (44g, 295.24mmol) was added dropwise. After the addition, the reaction solution was stirred at 25 degrees for 16 hours, and then ice water was added to quench the reaction, and an aqueous hydrochloric acid solution (5M) was added to adjust the pH from 2 to 3. Then, it was extracted twice with ethyl acetate, and the organic layer was washed with saturated saline and dried over anhydrous sodium sulfate. After filtration, the ethyl acetate was removed by vacuum concentration to obtain a crude product containing the target product. 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 aa53) (32 g, 48%). LCMS(ESI) m / z = 296 (M+Na)+ Retention time: 1.39 minutes (Analysis conditions SMD method7)

[0345] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-L-serine (compound aa54, Fmoc-Ser(iPen)-OH) [ka] N-(tert-butoxycarbonyl)-O-(3-methylbut-2-en-1-yl)-L-serine (compound aa53) (185 g, 676.85 mmol), ammonia in methanol (2 M, 555 mL), Pd / C (18.5 g) and methanol (1500 mL) were mixed and stirred at room temperature under a hydrogen atmosphere (5 atm) for 16 hours. After removing the solid by filtration, the solvent was removed by vacuum concentration to obtain N-(tert-butoxycarbonyl)-O-isopentyl-L-serine (Boc-Ser(iPen)-OH) (183 g). The obtained 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 solution was concentrated under reduced pressure, and the obtained 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 solution was then washed three times with hexane. After washing, an aqueous hydrochloric acid solution (6 M) was added to adjust the pH of the aqueous layer from 2 to 3, 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 obtained 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 aa54, 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)

[0346] Compound aa55 (Fmoc-MeSer(iPen)-OH) was synthesized according to the following scheme. [ka]

[0347] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-N-methyl-L-serine (Fmoc-MeSer(iPen)-OH, compound aa55) [ka] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-L-serine (compound aa54, Fmoc-Ser(iPen)-OH) (75 g, 188.70 mmol), p-toluenesulfonic acid (TsOH) (1.95 g, 11.32 mmol) and paraformaldehyde (11.4 g) were suspended in toluene (750 mL) and stirred at 110 degrees for 16 hours under a nitrogen atmosphere. The reaction solution was then washed twice with a saturated aqueous solution of sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to remove the solvent, and (S)-4-((isopentyloxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl was obtained as a crude product. The obtained (S)-4-((isopentyloxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (40 g, 97.69 mmol) was dissolved in dichloromethane (670 mL) and triethylsilane (Et 3 SiH) (34 g, 292.41 mmol) and trifluoroacetic acid (TFA) (670 mL) were added at room temperature. After stirring the reaction solution at room temperature for 2 days, the reaction solution was concentrated under reduced pressure. The resulting residue was dissolved in an aqueous potassium carbonate solution, which was washed three times with petroleum ether. Then, concentrated hydrochloric acid was added to the aqueous layer to adjust the pH from 2 to 3, and the solution was extracted three times with ethyl acetate. The organic layer was washed three times with saturated saline, dried over sodium sulfate, and filtered. The solvent was removed by concentration under reduced pressure, and the resulting residue was dissolved in methanol, washed three times with hexane, and then the methanol was removed by concentration under reduced pressure to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-isopentyl-N-methyl-L-serine (Fmoc-MeSer(iPen)-OH, compound aa55) (35 g, 87%). LCMS(ESI) m / z = 412 (M+H)+ Retention time: 0.97 minutes (Analysis conditions SQDFA05)

[0348] Synthesis of 1,1-difluoro-4-iodobutane (compound aa56) [ka] Under a nitrogen atmosphere, 5-bromo-1,1-difluoropentane (5 g, 26.73 mmol) was dissolved in acetone (20 ml) and sodium iodide (6 g) was added at room temperature. The reaction solution was heated to 45°C and stirred for 1 hour. The reaction solution was filtered, and the obtained filtrate was concentrated under reduced pressure, hexane was added, and the mixture was washed with an aqueous sodium thiosulfate solution and saturated saline. The obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was purified by normal phase silica gel chromatography (hexane) to obtain 1,1-difluoro-4-iodobutane (compound aa56) (4.2 g, 67%). 1 H NMR (300 MHz, CDCl 3 -d):δ5.87(t,J=27.0Hz,1H),3.26(t,J=6.0Hz,2H),2.06-1.99(m,4H)

[0349] Synthesis of (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-7,7-difluoroheptanoic acid (compound aa58, Fmoc-Hnl(7-F2)-OH) [ka] Under a nitrogen atmosphere, (R)-2-(2,6-dichlorophenyl)-5-oxo-3-((R)-1-phenylethyl)imidazolidine-1-carboxylate tert-butyl (50 g, 114.85 mmol) was dissolved in tetrahydrofuran (300 ml) and cooled to -20 degrees. Sodium bis(trimethylsilyl)amide (1.9 M tetrahydrofuran solution, 64 ml, 121.6 mmol) was added dropwise and stirred for 10 minutes, and then 1,1-difluoro-4-iodobutane (aa56, 35 g, 149.56 mmol) diluted in 40 ml of tetrahydrofuran was added dropwise and stirred at -20 degrees for 30 minutes. 20% aqueous ammonium chloride solution was added to the reaction solution, and then extraction was performed twice with ethyl acetate. The organic layer was washed with a 20% aqueous ammonium acetate solution (twice) and 15% saline, and then the obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product (2R,4S)-2-(2,6-dichlorophenyl)-4-(5,5-difluoropentyl)-5-oxo-3-((R)-1-phenylethyl)imidazolidine-1-carboxylate tert-butyl (compound aa57, 74 g). The obtained (2R,4S)-2-(2,6-dichlorophenyl)-4-(5,5-difluoropentyl)-5-oxo-3-((R)-1-phenylethyl)imidazolidine-1-carboxylate tert-butyl (compound aa57, 74 g) was dissolved in chlorobenzene (200 ml). Trifluoromethanesulfonic acid (45 ml) was added dropwise over 30 minutes while keeping the internal temperature of the reaction below 10 degrees. The reaction solution was stirred at room temperature for 2 hours, and then 100 ml of water was added, heated to 110 degrees, and stirred for 4 hours. The reaction solution was returned to room temperature, and the aqueous layer was washed three times with 1:1 cyclopentyl methyl ether and hexane. The aqueous layer was then neutralized (pH = 7) with a 40% aqueous potassium phosphate solution. N,N-diisopropylethylamine (25.7 ml), acetonitrile (50 ml), and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (44.8 g) were added to the reaction mixture under a nitrogen atmosphere and stirred at room temperature for 16 hours. N,N-diisopropylethylamine (19 ml) was added to maintain the pH of the reaction mixture at 8-9. N,N-diisopropylethylamine (9 ml) was added to further advance the reaction. The reaction mixture was then filtered, and hexane:ethyl acetate (2:1) (150 ml) was added to the filtrate and stirred vigorously for 40 minutes. The middle layer of the three-layered reaction mixture was collected and washed nine times with a 2:1 hexane:ethyl acetate solution (150 ml). Concentrated hydrochloric acid was added to the washed aqueous layer until the pH was 1, and the mixture was extracted twice with ethyl acetate. The obtained organic layer was washed twice with water and with 10% saline, and then the obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-7,7-difluoroheptanoic acid (compound aa58, Fmoc-Hnl(7-F2)-OH) (33 g). LCMS(ESI)m / z=404 (M+H)+ Retention time: 3.29 minutes (Analysis conditions SMD method27)

[0350] Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-7,7-difluoroheptanoic acid (compound aa59, Fmoc-MeHnl(7-F2)-OH) [ka] Under a nitrogen atmosphere, a toluene solution (525 mL) of (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-7,7-difluoroheptanoic acid (compound aa58, Fmoc-Hnl(7-F2)-OH) (35 g, 86.76 mmol), paraformaldehyde (8.05 g, 268.33 mmol), and 10-camphorsulfonic acid (CSA) (1.01 g, 4.35 mmol) was heated to 95°C and stirred for 2 hours. After the reaction solution was cooled to room temperature, it was washed three times with saturated aqueous sodium bicarbonate solution. The resulting organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain 9H-fluoren-9-ylmethyl (4S)-4-(5,5-difluoropentyl)-5-oxo-1,3-oxazolidine-3-carboxylate as a crude product (33 g, 79.44 mmol). The crude product (4S)-4-(5,5-difluoropentyl)-5-oxo-1,3-oxazolidine-3-carboxylate 9H-fluoren-9-ylmethyl ester (33 g, 79.44 mmol) was dissolved in dichloromethane (DCM) (330 mL) and diluted with triethylsilane (25.4 mL, 12.42 mmol), water (1.43 mL), and boron trifluoride diethyl etherate (BF 3 ·OEt 2 ) (20.1 mL) was added at 0°C and stirred at room temperature for 16 hours. A 5% aqueous solution of ammonium chloride was added to the reaction solution, the organic layer was washed with saturated saline, and the obtained organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by reverse phase column chromatography (0.1% aqueous formic acid / 0.1% formic acid acetonitrile solution) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-7,7-difluoroheptanoic acid (compound aa59, Fmoc-MeHnl(7-F2)-OH) (27 g, 80% for two steps). LCMS(ESI)m / z=440 (M+Na)+ Retention time: 2.20 minutes (Analysis conditions SMD method 18)

[0351] The synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-L-serine (compound aa60, Fmoc-Ser(Ph-2-Cl)-OH) was carried out according to the following scheme. [ka]

[0352] Synthesis of methyl O-(2-chlorophenyl)-N-trityl-L-serinate (compound aa61, Trt-Ser(Ph-2-Cl)-OMe) [ka] Methyltrityl-L-serinate (Trt-Ser-OMe) (25.0 g, 69.2 mmol), triphenylphosphine (PPh 3 ) (20.0 g, 76.1 mmol), 2-chlorophenol (10.5 mL, 103.7 mmol) and toluene (57.2 mL) were mixed, and diisopropyl azodicarboxylate (DIAD) (40% toluene solution, 1.9 mol / l, 40.3 mL, 76.1 mmol) was added dropwise over 40 minutes under ice cooling. After the dropwise addition was completed, the reaction solution was warmed to room temperature and stirred for another 90 minutes. The reaction solution was then concentrated under reduced pressure, and ethanol / water (8 / 2, 180 mL) was added to the resulting residue and stirred at room temperature for 5 minutes. The resulting white solid was filtered, washed three times with ethanol / water (8 / 2, 100 mL), and dried under reduced pressure to obtain methyl O-(2-chlorophenyl)-N-trityl-L-serinate (compound aa61, Trt-Ser(Ph-2-Cl)-OMe) (24.3 g, 75%). LCMS(ESI) m / z = 494 (M+Na)+ Retention time: 1.16 minutes (Analysis conditions SQDFA05)

[0353] Synthesis of methyl O-(2-chlorophenyl)-L-serinate (compound aa62) [ka] Methyl O-(2-chlorophenyl)-N-trityl-L-serinate (compound aa61, Trt-Ser(Ph-2-Cl)-OMe) (24.3 g, 51.5 mmol) was dissolved in 4N hydrochloric acid / 1,4-dioxane solution (38.7 mL, 154.5 mmol) and stirred at room temperature for 15 minutes. The reaction solution was concentrated under reduced pressure, hexane (100 mL) was added to the obtained solid, and the mixture was stirred at room temperature for 10 minutes, filtered, and the solid was washed three times with hexane (100 mL), and dried under reduced pressure to obtain methyl O-(2-chlorophenyl)-L-serinate (compound aa62, H-Ser(Ph-2-Cl)-OMe) (13.5 g, 98%). LCMS(ESI) m / z = 230 (M+H)+ Retention time: 0.40 minutes (Analysis conditions SQDFA05)

[0354] Synthesis of O-(2-chlorophenyl)-L-serine (compound aa63, H-Ser(Ph-2-Cl)-OH) [ka] Methyl O-(2-chlorophenyl)-L-serinate (compound aa62, H-Ser(Ph-2-Cl)-OMe) (13.5 g, 50.7 mmol) was dissolved in water (75 mL), and a solution of lithium hydroxide monohydrate (4.68 g, 111.5 mmol) in water / methanol (30 mL / 30 mL) was added dropwise over 20 minutes under ice cooling. After the dropwise addition, the mixture was stirred for 60 minutes under ice cooling. Then, acetonitrile (400 mL) cooled in a refrigerator was added to the reaction solution, and a solid precipitated. The mixture was further stirred for 20 minutes under ice cooling, and then allowed to stand in a refrigerator for 5 hours. The solid was collected by filtration, and the solid precipitated in the mother liquor was also collected. The collected solids were combined, washed three times with acetonitrile (150 mL), and dried under reduced pressure to obtain O-(2-chlorophenyl)-L-serine (compound aa63, H-Ser(Ph-2-Cl)-OH) (compound aa63, H-Ser(Ph-2-Cl)-OH) (10.3 g, 91%). LCMS(ESI) m / z = 216 (M+H)+ Retention time: 0.34 minutes (Analysis conditions SQDFA05)

[0355] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-L-serine (compound aa60, Fmoc-Ser(Ph-2-Cl)-OH) [ka] O-(2-chlorophenyl)-L-serine (compound aa63, H-Ser(Ph-2-Cl)-OH) (1.0 g, 4.49 mmol) was added to an aqueous solution (3.8 mL) of sodium carbonate (0.952 g, 8.99 mmol) and 1,4-dioxane (2.0 mL), and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (1.59 g, 4.72 mmol) was slowly added under ice cooling. After the addition, the mixture was stirred at room temperature for 10 minutes, and water (12 mL) was added to dissolve the solid. After washing three times with t-butyl methyl ether (15 mL), 5M aqueous hydrochloric acid (4.49 mL, 22.46 mmol) was slowly added to adjust the pH of the aqueous layer to 1, and the aqueous layer was extracted three times with t-butyl methyl ether (15 mL). The organic layer was dried over anhydrous magnesium sulfate and then filtered. The solvent was removed by concentration in vacuo to give a white solid. The same procedure was carried out using O-(2-chlorophenyl)-L-serine (compound aa63, H-Ser(Ph-2-Cl)-OH) (9.28 g, 41.7 mmol), and the resulting solids were collected and dried under reduced pressure to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-L-serine (compound aa60, Fmoc-Ser(Ph-2-Cl)-OH) (19.6 g, 97%). LCMS(ESI) m / z = 438(M+H)+ Retention time: 0.90 minutes (Analysis conditions SQDFA05)

[0356] Synthesis of (S)-4-((2-chlorophenoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa64) [ka] A suspension of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-L-serine (compound aa60, Fmoc-Ser(Ph-2-Cl)-OH) (3.00 g, 6.85 mmol), paraformaldehyde (0.823 g, 27.4 mmol) and (+)-10-camphorsulfonic acid (80.0 mg, 0.343 mmol) in toluene (23 mL) was stirred at 80 degrees for 3 hours. After cooling, the reaction solution was diluted with ethyl acetate (15 mL) and filtered through Celite. A saturated aqueous solution of sodium bicarbonate / water (1 / 1, 15 mL) was added to the filtrate, and the mixture was extracted with ethyl acetate. The organic layer was washed twice with a saturated aqueous solution of sodium bicarbonate / water (1 / 1, 15 mL) and once with saturated saline / water (1 / 1, 15 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain (S)-4-((2-chlorophenoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa64) (3.23 g) as a crude product. LCMS(ESI) m / z = 450 (M+H)+ Retention time: 0.99 minutes (Analysis conditions SQDFA05)

[0357] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-N-methyl-L-serine (compound aa65, Fmoc-MeSer(Ph-2-Cl)-OH) [ka] Triethylsilane (4.79mL, 30.0mmol) and trifluoroacetic acid (TFA) (6.94mL, 90.0mmol) were added to a dichloroethane (DCE) (11mL) solution of (S)-4-((2-chlorophenoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa64, 1.50g, 3.33mmol) and the mixture was stirred at 70°C for 2.5 hours. The solvent was removed under reduced pressure, and the residue was dissolved in acetonitrile (25mL) and washed four times with hexane (25mL). The acetonitrile layer was evaporated under reduced pressure, and the residue was purified by reverse phase column chromatography to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(2-chlorophenyl)-N-methyl-L-serine (compound aa65, Fmoc-MeSer(Ph-2-Cl)-OH) (1.1 g, 83%). LCMS(ESI) m / z = 452 (M+H)+ Retention time: 0.93 minutes (Analysis conditions SQDFA05)

[0358] Synthesis of (S)-4-(((5-fluoropyridin-3-yl)methoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa66) [ka] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-L-serine (Fmoc-Ser(3-F-5-Me-Pyr)-OH) (compound aa10, 3.00 g, 6.87 mmol) in toluene (13.7 mL) was added with paraformaldehyde (0.619 g, 20.6 mmol) and trifluoroacetic acid (TFA) (4.77 mL, 61.9 mmol) and stirred at 70 degrees for 3 hours. The solvent was removed from the reaction solution under reduced pressure, and the residue was dissolved in dichloromethane (30 ml) and washed three times with saturated aqueous sodium bicarbonate solution / water (1:1, 15 mL). The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain (S)-4-(((5-fluoropyridin-3-yl)methoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa66) (3.08 g) as a crude product. LCMS(ESI) m / z = 449 (M+H)+ Retention time: 0.84 minutes (Analysis conditions SQDFA05)

[0359] Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-N-methyl-L-serine (compound aa67, Fmoc-MeSer(3-F-5-Me-Pyr)-OH) [ka] (S)-4-(((5-fluoropyridin-3-yl)methoxy)methyl)-5-oxooxazolidine-3-carboxylate (9H-fluoren-9-yl)methyl (compound aa66) (1.75 g, 3.90 mmol) in dichloroethane (DCE) (13 mL) was added with triethylsilane (5.61 mL, 35.1 mmol) and trifluoroacetic acid (8.12 mL, 105 mmol) and stirred at 70 degrees for 2 hours and 30 minutes. The solvent was distilled off under reduced pressure, and the residue was dissolved in acetonitrile (25 mL) and washed with hexane (25 mL). The acetonitrile layer was distilled under reduced pressure to remove the solvent, and the resulting crude product was purified by reverse phase column chromatography to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((5-fluoropyridin-3-yl)methyl)-N-methyl-L-serine (compound aa67, Fmoc-MeSer(3-F-5-Me-Pyr)-OH) (1.63 g, 93%). LCMS(ESI) m / z = 451 (M+H)+ Retention time: 0.77 minutes (Analysis conditions SQDFA05)

[0360] Synthesis of methyl((2-nitrophenyl)sulfonyl)-L-serinate (compound aa68, Ns-Ser-OMe) [ka] At room temperature, a solution obtained by dissolving sodium carbonate (7.76 g, 73.2 mmol) in water (75 mL) was added to commercially available methyl L-serinate hydrochloride (H-Ser-OMe·HCl) (5.0 g, 32.1 mmol), and then a solution of 2-nitrobenzenesulfonyl chloride (NsCl) (7.05 g, 31.8 mmol) in 1,4-dioxane (75 mL) was added and stirred at room temperature for 1 hour and 30 minutes. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate, 1N aqueous hydrochloric acid, and saturated saline, and the resulting organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain methyl ((2-nitrophenyl)sulfonyl)-L-serinate (compound aa68, Ns-Ser-OMe) (7.9 g, 82%). LCMS(ESI)m / z=305 (M+H)+ Retention time: 0.50 minutes (Analysis conditions SQDFA05)

[0361] Synthesis of (S)-1-((2-nitrophenyl)sulfonyl)aziridine-2-carboxylate methyl (compound aa69, Ns-Azy-OMe) [ka] Under a nitrogen atmosphere, dichloromethane (DCM) (69 mL) was added to methyl((2-nitrophenyl)sulfonyl)-L-serinate (compound aa68, Ns-Ser-OMe) (2.49 g, 8.18 mmol), and the solution was stirred at room temperature until it became transparent. Then, triphenylphosphine (PPh 3) (2.545 g, 9.82 mmol) was added at -10°C using an ice bath made of salt and ice, and the solution was stirred at -10°C until it became transparent. 40% diethyl azodicarboxylate (DEAD) toluene solution (4.46 mL, purchased from Tokyo Chemical Industry Co., Ltd.) was added at -10°C, and the reaction solution was stirred for 30 minutes, after which toluene (40 mL) was added and the dichloromethane (DCM) was removed by concentrating under reduced pressure using a rotary evaporator while keeping the temperature of the water bath below 15°C. Filtration was performed to remove solids, and the obtained filtrate was purified by normal phase silica gel chromatography (toluene / acetone) to obtain (S)-1-((2-nitrophenyl)sulfonyl)aziridine-2-carboxylate methyl (compound aa69, Ns-Azy-OMe) (1.9 g, 81%). LCMS(ESI)m / z=287 (M+H)+ Retention time: 0.64 minutes (Analysis conditions SQDFA05)

[0362] Synthesis of methyl N-((2-nitrophenyl)sulfonyl)-O-(2,2,3,3-tetrafluoropropyl)-L-serinate (compound aa70, Ns-Ser(F(4)nPr)-OMe) [ka] Under a nitrogen atmosphere, a mixture of (S)-1-((2-nitrophenyl)sulfonyl)aziridine-2-carboxylate methyl (compound aa69, Ns-Azy-OMe) (1.9 g, 6.64 mmol) and 2,2,3,3-tetrafluoropropan-1-ol (8.82 mL, 100 mmol) was treated with boron trifluoride diethyl etherate (BF 3 ·OEt 2 ) (2.52 mL, 19.91 mmol) was added at room temperature, and the mixture was stirred at 70° C. for 40 minutes. After cooling the reaction mixture to room temperature, it was diluted with dimethyl sulfoxide (DMSO) and purified by reversed-phase column chromatography (0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile solution) to obtain methyl N-((2-nitrophenyl)sulfonyl)-O-(2,2,3,3-tetrafluoropropyl)-L-serinate (compound aa70, Ns-Ser(F(4)nPr)-OMe) (1.3 g, 47%). LCMS(ESI)m / z=419 (M+H)+ Retention time: 0.77 minutes (Analysis conditions SQDFA05)

[0363] Synthesis of N-((2-nitrophenyl)sulfonyl)-O-(2,2,3,3-tetrafluoropropyl)-L-serine (compound aa71, Ns-Ser(F(4)nPr)-OH) [ka] Methyl N-((2-nitrophenyl)sulfonyl)-O-(2,2,3,3-tetrafluoropropyl)-L-serinate (compound aa70, Ns-Ser(F(4)nPr)-OMe) (142 mg, 0.339 mmol) was ...

Claims

1. A cyclic peptide, (1) the number of amino acids constituting the cyclic peptide is 5 to 20; (2) The amino acids constituting the cyclic portion of the cyclic peptide include at least one amino acid having a long side chain, and the amino acid having a long side chain is selected from the group consisting of: 【Chemistry 1】 【Chemistry 2】 A cyclic peptide selected from the group consisting of

2. The cyclic peptide according to claim 1 , wherein the side chain of the cyclic portion does not have an indole skeleton and a substituted or unsubstituted hydroxyphenyl group.

3. The cyclic peptide according to claim 1 or 2, which does not contain a methylthio group or a thiol group in the side chain of the cyclic portion.

4. The cyclic peptide according to any one of claims 1 to 3, wherein, when the cyclic portion has an acidic side chain, the pKa of the acidic side chain is 3.5 to 10.

5. The cyclic peptide according to any one of claims 1 to 4, wherein, when the cyclic portion has a basic side chain, the basic pKa of the basic side chain is 4.0 to 10.

6. The cyclic peptide according to any one of claims 1 to 5, wherein the number of amino acids constituting the cyclic portion is 5 to 15.

7. The cyclic peptide according to any one of claims 1 to 6, wherein the number of N-substituted amino acids constituting the cyclic peptide is 3 or more.

8. The cyclic peptide of claim 7, wherein the N-substituted amino acid is an N-alkyl amino acid.

9. The cyclic peptide of claim 8, wherein the N-alkyl amino acid is an N-methyl amino acid.

10. The cyclic peptide according to any one of claims 1 to 9, wherein the number of unnatural amino acids constituting the cyclic peptide is 4 or more.

11. The cyclic peptide according to any one of claims 1 to 10, wherein the number of aromatic rings contained in the side chain of the cyclic portion is 0 to 3.

12. The cyclic peptide according to any one of claims 1 to 11, wherein the side chain of the cyclic portion does not have a condensed ring structure formed by two or more aromatic rings.

13. The cyclic peptide according to any one of claims 1 to 12, wherein the ClogP / total aa of the cyclic peptide is 1.0 to 1.

8.

14. P of the cyclic peptide ａｐｐ is 1.0 x 10 －６ The cyclic peptide according to any one of claims 1 to 13, wherein the cyclic peptide has a viscosity of at least 1.0 cm / sec.

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