Macrocyclic peptide compound having cell membrane permeability and metabolic stability, and library containing same

Abstract

The present invention provides a cyclic peptide compound optionally linked to a nucleic acid, the cyclic peptide compound having a cyclic moiety wherein: (1) the cyclic moiety consists of 13 or 14 amino acid residues; (2) among the amino acid residues constituting the cyclic moiety, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues in a single side chain is 3 or less, and the number of natural amino acid residues is 4 or less; and (3) ClogP / total amide linkage (ClogP / total AB) is from 1.0 to 1.8.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480068603.7 (22) Application Date 2024.11.05 (30) Priority Data 2023-189465 2023.11.06 JP (85) PCT International Application Entering National Phase Date 2026.04.24 (86) PCT International Application Application Data PCT / JP2024 / 039298 2024.11.05 (87) PCT International Application Publication Data WO2025 / 100405 JA 2025.05.15 (71) Applicant Chugai Pharmaceutical Co., Ltd. Address Japan (72) Inventors Yuya Morita and Shota Tanaka (74) Patent Agency Zhongke Patent & Trademark Agency Co., Ltd. 11021 Patent Attorney Zhang Yiqun (51) Int.Cl. C07K 7 / 54 (2006.01) C40B 40 / 10 (2006.01) (54) Invention Title: Macrocyclic peptide compound with cell membrane permeability and metabolic stability and library containing said macrocyclic peptide compound (57) Abstract: This invention provides a cyclic peptide compound optionally linked to a nucleic acid, said cyclic peptide compound having a cyclic moiety, wherein: (1) said cyclic moiety is composed of 13 or 14 amino acid residues; (2) of said amino acid residues constituting said cyclic moiety, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues in a single side chain is 3 or less, and the number of native amino acid residues is 4 or less; and (3) ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8. Claims 2 pages, Description 100 pages, Drawings 1 page, CN 122094968 A 2026.05.26 CN 1 22 09 49 68 A 1. A cyclic peptide compound optionally linked to a nucleic acid, the compound comprising a cyclic moiety, wherein (1) the cyclic moiety is composed of 13 or 14 amino acid residues, (2) of the amino acid residues constituting the cyclic moiety, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues having the same side chain is 3 or fewer, and the number of native amino acid residues is 4 or fewer, and (3) the ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8. 2. The cyclic peptide compound optionally linked to a nucleic acid according to claim 1,3. The number of N-substituted amino acids constituting the cyclic moiety is 7 to 13. 4. The cyclic peptide compound optionally linked to a nucleic acid according to claim 1 or 2, wherein the N-substituted amino acid is an N-alkyl amino acid. 5. The cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 3, wherein the cyclic peptide compound has 3 or fewer hydrogen bond donors. 6. The cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 4, wherein the side chain of the amino acid constituting the cyclic moiety is composed of at least one of the following: alkyl group, cycloalkyl group, aralkyl group, and amide group, wherein any atom of the alkyl group and the nitrogen atom of the amino group of the amino acid can form a ring together. 7. The cyclic peptide compound optionally linked to a nucleic acid according to claim 5, wherein the alkyl group is a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of: halogen atom, hydroxyl group, C3-C8 cycloalkyl group, and C1-C6 alkoxy group. 7. The cyclic peptide compound optionally linked to a nucleic acid according to claim 5 or 6, wherein the cycloalkyl group is a C3-C10 cycloalkyl group optionally substituted with at least one group selected from the group consisting of: a halogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group. 8. The cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 5 to 7, wherein the aralkyl group is a C7-C14 aralkyl group optionally substituted with at least one group selected from the group consisting of: a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group. 9. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 5 to 8, wherein the amide group is represented by the formula: -CONR1R2, wherein R1 and R2 are each independently a hydrogen atom or a C1-C6 alkyl group, or R1 and R2 together with the nitrogen atom to which they are linked form a 4- to 8-membered saturated heterocycle, and said 4- to 8-membered saturated heterocycle is optionally substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups, and 4- to 7-membered heterocyclic groups. 10. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 9, wherein among the amino acids constituting said cyclic moiety,The number of natural amino acids is 1 to 4. 11. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 10, wherein the amino acids constituting the cyclic moiety comprise natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. 12. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 11, wherein the number of non-natural amino acids in the amino acids constituting the cyclic moiety is 9 or more. Claims 1 / 2 Page 2 CN 122094968 A 13. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 12, wherein the amino acids constituting the cyclic portion comprise non-natural amino acids selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). 14. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 13, wherein the value of Caco-2 Papp (cm / sec) of the cyclic peptide compound is 0.4 × 10⁻⁶ or greater. 15. A library comprising a cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 14. 16. A method for screening cyclic peptide compounds capable of specifically binding to a target molecule, the method comprising the steps of: (i) contacting the library according to claim 15 with the target molecule; and (ii) selecting cyclic peptide compounds capable of specifically binding to the target molecule. Claims 2 / 2 Page 3 CN 122094968 A Macrocyclic Peptide Compound with Cell Membrane Permeability and Metabolic Stability and a Library Containing the Macrocyclic Peptide Compound Technical Field

[0001] The present invention provides a macrocyclic peptide compound with cell membrane permeability and metabolic stability and a library containing the macrocyclic peptide compound. Background Art

[0002] Medium molecular weight compounds (molecular weight: 500 to 2000) have attracted attention as a means of drug development targeting intractable targets such as protein-protein interaction inhibition (Non-Patent Literature 1). Developing peptides as drugs is considered difficult because peptides typically have poor drug-like properties (e.g., metabolic stability and cell membrane permeability, hereinafter also referred to as "membrane permeability"). In recent years,It has been found that metabolic stability or membrane permeability is improved by cyclizing peptides or by using non-natural amino acids such as N-methyl amino acids in peptides (Non-Patent Documents 2 and 3). Cyclic peptides containing non-natural amino acids (especially cyclic peptides containing N-substituted amino acids) are known to have drug-like properties (Patent Document 1). It has also been proposed that library compounds of cyclic peptides containing non-natural amino acids can be used to develop protein-protein interaction inhibitors (Non-Patent Document 4). In addition, cyclic peptide compounds with high membrane permeability, libraries containing such compounds, methods for their production and screening have been reported in recent years (Patent Document 2), and amino acids that can improve the membrane permeability of peptide compounds, peptide compounds containing such amino acids, and methods for their production have also been reported (Patent Document 3).

[0003] Patent Document 1 describes that when considering the achievement of membrane permeability and metabolic stability, the number of amino acids and amino acid analogs constituting the cyclic moiety is preferably 5 to 12, particularly preferably 9 to 11.

[0004] Patent Document 2 discloses a pre-incubation method using Caco-2 cells as a method for more accurately measuring the membrane permeability of highly lipophilic drugs, etc., compared to conventional methods. This method is used to evaluate the membrane permeability of cyclic peptides mainly composed of 11 amino acid residues (Patent Documents 2 and 3).

[0005] However, for cyclic peptides containing 13 or more amino acid residues in the cyclic portion,The types of cyclic peptides with drug-like properties have not been fully studied.

[0006] [Citation List]

[0007] [Patent Documents]

[0008] [Patent Document 1]

[0009] International Publication No. WO 2013 / 100132

[0010] [Patent Document 2]

[0011] International Publication No. WO 2018 / 225864

[0012] [Patent Document 3]

[0013] International Publication No. WO 2020 / 122182

[0014] [Non-Patent Documents]

[0015] [Non-Patent Document 1]

[0016] Future Med. Chem., 2009, 1, 1289-1310.

[0017] [Non-Patent Document 2]

[0018] Acc. Chem. Res. 2008, 41, 1331-1342. Specification 1 / 100 pages 4 CN 122094968 A

[0019] [Non-Patent Literature 3]

[0020] Angew. Chem. Int. Edit, 2013, 52, 254-269.

[0021] [Non-Patent Literature 4]

[0022] Chem. Rev., 2019, 119, 10360-10391. Summary of the Invention

[0023] [Technical Problem]

[0024] The present invention was made under such circumstances, and the object of the present invention is to provide a library for screening cyclic peptides with excellent metabolic stability and membrane permeability. In another aspect, the object of the present invention is to provide cyclic peptide compounds with high membrane permeability, methods for their production or screening.

[0025] [Solution to the Problem]

[0026] It is believed that as the size of the cyclic peptide increases, i.e. the number of amino acid residues constituting the cyclic portion increases, the surface area available for contact with the target molecule increases. Therefore, from the perspective of achieving interaction with target molecules, peptides with a large number of amino acid residues constituting the cyclic moiety are considered advantageous. Consequently, the inventors conducted in-depth research to determine whether there exist regions in cyclic peptides with 13 or more amino acid residues constituting the cyclic moiety, which deviates from the conventionally considered advantageous range for achieving both membrane permeability and metabolic stability. Specifically, various cyclic peptides with different side chains and consisting of 13 or 14 amino acid residues have been synthesized, and the drug-likeness of cyclic peptide compounds with 13 or 14 amino acid residues constituting the cyclic moiety has been evaluated using a liver microsomal metabolic stability assay and a Caco-2 cell membrane permeability assay. Therefore,The inventors have discovered that peptide compounds consisting of 13 or 14 amino acid residues (where physical and chemical properties such as the number of N-substituted amino acids and lipophilicity are controlled) can exhibit excellent membrane permeability and metabolic stability, and have developed libraries to obtain cyclic peptide compounds more efficiently.

[0027] The present invention provides the following [A1] to [A52], [B1] to [B73] and [C1] to [C6].

[0028] [A1]

[0029] A cyclic peptide compound optionally linked to a nucleic acid, the compound comprising a cyclic moiety, wherein

[0030] (1) the cyclic moiety is composed of 13 or 14 amino acid residues,

[0031] (2) of the amino acid residues constituting the cyclic moiety, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues having the same side chain is 3 or fewer, and the number of native amino acid residues is 4 or fewer, and

[0032] (3) the ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8.

[0033] [A2]

[0034] The cyclic peptide compound optionally linked to a nucleic acid according to [A1], wherein the number of amino acid residues constituting the cyclic peptide compound is 13 or 14.

[0035] [A2-1]

[0036] A cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the number of amino acid residues constituting the cyclic peptide compound is 13.

[0037] [A2-2]

[0038] A cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the number of amino acid residues constituting the cyclic peptide compound is 14.

[0039] [A3] Specification 2 / 100 pages 5 CN 122094968 A

[0040] A cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the number of N-substituted amino acids constituting the cyclic moiety is 7 to 13.

[0041] [A4]

[0042] A cyclic peptide compound optionally linked to a nucleic acid according to claim [A3], wherein the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10.

[0043] [A5]

[0044] A cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the number of N-substituted amino acids constituting the cyclic moiety is 8 or more.

[0045] [A6]

[0046] A cyclic peptide compound optionally linked to a nucleic acid according to claim [A5],The number of N-substituted amino acids constituting the cyclic moiety is 8 to 13.

[0047] [A7]

[0048] The cyclic peptide compound optionally linked to a nucleic acid according to claim [A6], wherein the number of N-substituted amino acids constituting the cyclic moiety is 8 to 10.

[0049] [A8]

[0050] The cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic moiety is 60% or greater.

[0051] [A8-1]

[0052] The cyclic peptide compound optionally linked to a nucleic acid according to [A8], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic moiety is 65% or greater.

[0053] [A8-2]

[0054] A cyclic peptide compound optionally linked to a nucleic acid according to [A8], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic moiety is 70% or greater.

[0055] [A9]

[0056] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A8], wherein the N-substituted amino acid is an N-alkyl amino acid.

[0057] [A10]

[0058] A cyclic peptide compound optionally linked to a nucleic acid according to [A9], wherein the N-alkyl amino acid is an N-C1-C6 alkyl amino acid.

[0059] [A11]

[0060] A cyclic peptide compound optionally linked to a nucleic acid according to [A10], wherein the N-alkyl amino acid is an N-methyl amino acid or an N-ethyl amino acid.

[0061] [A12]

[0062] A cyclic peptide compound optionally linked to a nucleic acid according to [A11], wherein the N-alkyl amino acid is an N-methyl amino acid.

[0063] [A13]

[0064] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A12], wherein the ClogP / total AB of the cyclic peptide compound is 1.0 to 1.5.

[0065] [A14] Specification 3 / 100 pages 6 CN 122094968 A

[0066] A cyclic peptide compound optionally linked to a nucleic acid according to [A13], wherein the ClogP / total AB of the cyclic peptide compound is 1.1 to 1.4.

[0067] [A15]

[0068] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A14],The ClogP of the cyclic peptide compound is 13 to 20.

[0069] [A16]

[0070] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A15], wherein the cyclic peptide compound has 3 or fewer hydrogen bond donors.

[0071] [A17]

[0072] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A16], wherein the side chain of the amino acid constituting the cyclic portion is composed of at least one of the group consisting of alkyl groups, cycloalkyl groups, aralkyl groups and amide groups, wherein any atom of the alkyl group of the amino acid and the nitrogen atom of the amino group can form a ring together.

[0073] [A18]

[0074] A cyclic peptide compound optionally linked to a nucleic acid according to [A17], wherein the alkyl group is a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of: a halogen atom, a hydroxyl group, a C3-C8 cycloalkyl group, and a C1-C6 alkoxy group.

[0075] [A19]

[0076] A cyclic peptide compound optionally linked to a nucleic acid according to [A17] or [A18], wherein the cycloalkyl group is a C3-C10 cycloalkyl group optionally substituted with at least one group selected from the group consisting of: a halogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0077] [A20]

[0078] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A17] to [A19], wherein the aralkyl group is a C7-C14 aralkyl group optionally substituted by at least one group selected from the group consisting of: halogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0079] [A21]

[0080] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A17] to [A20], wherein the aryl group is an optionally substituted C5-C10 arylC1-C6 alkyl group, wherein the C5-C10 aryl group is optionally substituted by at least one group selected from the group consisting of: halogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0081] [A22]

[0082] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A17] to [A21],The amide group is represented by the formula: -CONR1R2, wherein R1 and R2 are each independently a hydrogen atom or a C1-C6 alkyl group, or R1 and R2 together with the nitrogen atom to which they are attached form a 4- to 8-membered saturated heterocycle, and the 4- to 8-membered saturated heterocycle is optionally substituted by at least one group independently selected from the group consisting of: halogen atom, oxo group, C1-C6 alkyl group and 4- to 7-membered heterocyclic group.

[0083] [A23] Specification 4 / 100 pages 7 CN 122094968 A

[0084] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A22], wherein the cyclic peptide compound contains an amide bond formed between at least one carboxyl group contained in the side chain of an amino acid and the nitrogen atom of the amino group of another amino acid.

[0085] [A24]

[0086] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A23], wherein the cyclic peptide compound contains amino acids selected from the group consisting of Asp-pip and MeAsp-pip.

[0087] [A25]

[0088] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A24], wherein the amino acids constituting the cyclic portion comprise amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0089] [A26]

[0090] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A25],The amino acids constituting the cyclic moiety are each independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0091] [A27]

[0092] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A26], wherein the amino acids constituting the cyclic moiety include α-amino acids, and the number of α-amino acids is 10 to 14.

[0093] [A28]

[0094] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A27], wherein the amino acids constituting the cyclic portion include α-amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0095] [A29]

[0096] A cyclic peptide compound optionally linked to a nucleic acid according to [A27] or [A28], wherein in the amino acids constituting the cyclic moiety, each α-amino acid is independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0097] [A30]

[0098] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A31], wherein the amino acids constituting the cyclic moiety include amino acids having a β-amino acid backbone, and the number of amino acids having a β-amino acid backbone is 0 to 2.

[0099] [A31]

[0100] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A30],The amino acids constituting the cyclic portion include amino acids having a β-amino acid backbone selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip. Specification 5 / 100 pages 8 CN 122094968 A

[0101] [A32]

[0102] A cyclic peptide compound optionally linked to a nucleic acid according to [A30] or [A31], wherein, among the amino acids constituting the cyclic portion, the amino acids having a β-amino acid backbone are each independently selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0103] [A33]

[0104] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A32], wherein the amino acids constituting the cyclic portion comprise amino acids having a γ-amino acid backbone, and the number of amino acids having a γ-amino acid backbone is 0 to 2.

[0105] [A34]

[0106] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A33], wherein the amino acids constituting the cyclic portion comprise gMeAbu.

[0107] [A35]

[0108] A cyclic peptide compound optionally linked to a nucleic acid according to [A33] or [A34], wherein in the amino acids constituting the cyclic portion, each γ-amino acid is independently gMeAbu.

[0109] [A36]

[0110] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A35], wherein the number of natural amino acids constituting the cyclic moiety is 1 to 4.

[0111] [A37]

[0112] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A36], wherein the amino acids constituting the cyclic moiety comprise natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0113] [A38]

[0114] A cyclic peptide compound optionally linked to a nucleic acid according to [A37], wherein the natural amino acids constituting the cyclic moiety are each independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0115] [A39]

[0116] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A38], wherein, among the amino acids constituting the cyclic moiety,The number of non-natural amino acids is nine or more.

[0117] [A40]

[0118] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A39], wherein the amino acids constituting the cyclic portion comprise non-natural amino acids selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0119] [A41]

[0120] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A36] to [A40], wherein, among the amino acids constituting the cyclic moiety, the non-natural amino acids are each independently selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0121] [A42] Specification 6 / 100 pages 9 CN 122094968 A

[0122] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A41], wherein the value of the Caco-2 Papp (cm / sec) of the cyclic peptide compound is 0.4 × 10-6 or greater.

[0123] [A43]

[0124] According to [A44], the cyclic peptide compound optionally linked to a nucleic acid, wherein the value of Caco-2 Papp (cm / sec) is calculated by membrane permeability assessment under the following conditions:

[0125] (a) Caco-2 cells are cultured in 96-well Transwells for 3 weeks, then the compound to be evaluated, DMEM medium and FaSSIF (containing 1% DMSO) are added to the top side, DMEM medium is added to the bottom side, and the cells are pre-incubated for 24 hours at 37°C with shaking at 80 rpm under a 5% CO2 atmosphere;

[0126] (b) After pre-incubation, the pre-incubation solutions on the top and bottom sides are aspirated and washed.And a FaSSIF / HBSS buffer solution containing the compound (containing 1% DMSO, pH 6.0) was added to the top side, and an HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the bottom side;

[0127] (c) Each well was shaken at 80 rpm at 37°C under a 5% CO2 atmosphere, and the sample on the bottom side was collected 180 minutes after the start of shaking, and the permeation amount of the compound was measured by liquid chromatography-mass spectrometry (LC / MS); and

[0128] (d) the permeation coefficient (Caco-2 Papp (cm / sec)) was calculated from the measured permeation amount.

[0129] [A44]

[0130] A cyclic peptide compound selected from the compounds pd001 to pd168 listed in Table 15.

[0131] [A45]

[0132] A cyclic peptide compound (cyclic peptide-nucleic acid complex) linked to a nucleic acid according to any one of [A1] to [A44].

[0133] [A46]

[0134] A cyclic peptide-nucleic acid complex according to [A45], wherein the cyclic peptide compound has a cyclic portion and a linear portion, and the nucleic acid binds to the linear portion.

[0135] [A47]

[0136] A cyclic peptide-nucleic acid complex according to [A46], wherein the cyclic peptide compound further comprises a linker, and

[0137] the nucleic acid binds to the linear portion via the linker.

[0138] [A48]

[0139] A cyclic peptide-nucleic acid complex according to [A47], wherein the linker contains puromycin.

[0140] [A49]

[0141] A cyclic peptide-nucleic acid complex according to any one of [A45] to [A48], wherein the nucleic acid is DNA or RNA.

[0142] [A50]

[0143] A cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid contains a nucleic acid encoding the cyclic peptide compound.

[0144] [A51]

[0145] A cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid is a nucleic acid encoding the cyclic peptide compound.

[0146] [A52]

[0147] A cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid contains a nucleotide sequence encoding the cyclic peptide portion forming the cyclic peptide-nucleic acid complex.

[0148] [B1]

[0149] A library,It comprises a cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A52].

[0150] [B2]

[0151] A library comprising a cyclic peptide compound optionally linked to a nucleic acid,

[0152] wherein the cyclic peptide compound comprises a cyclic moiety, and

[0153] wherein the number of cyclic peptide compounds constituting the cyclic moiety of 13 or 14 amino acid residues is 10% or more in proportion to the total number of cyclic peptide compounds contained in the library.

[0154] [B2-1]

[0155] According to the library of [B1] or [B2], wherein the number of cyclic peptide compounds constituting the cyclic moiety of 13 or 14 amino acid residues is 20% or more in proportion to the total number of cyclic peptide compounds contained in the library.

[0156] [B2-2]

[0157] According to the library of [B1] or [B2], the number of cyclic peptide compounds constituting the cyclic moiety of 13 or 14 amino acid residues is 40% or more of the total number of cyclic peptide compounds contained in the library.

[0158] [B2-3]

[0159] According to the library of [B1] or [B2], the number of cyclic peptide compounds constituting the cyclic moiety of 13 or 14 amino acid residues is 60% or more of the total number of cyclic peptide compounds contained in the library.

[0160] [B2-4]

[0161] According to the library of [B1] or [B2], the number of cyclic peptide compounds constituting the cyclic moiety of 13 or 14 amino acid residues is 80% or more of the total number of cyclic peptide compounds contained in the library.

[0162] [B3]

[0163] The library according to [B1] or [B2] is substantially composed of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion.

[0164] [B4]

[0165] The library according to any one of [B1] to [B3], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic peptide compound to the total number of cyclic peptide compounds contained in the library is 10% or greater.

[0166] [B4-1]

[0167] The library according to any one of [B1] to [B4], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic peptide compound to the total number of cyclic peptide compounds contained in the library is 20% or greater.

[0168] [B4-2]

[0169] The library according to any one of [B1] to [B4],The ratio of the number of cyclic peptide compounds constituting the cyclic peptide compound having 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 40% or greater.

[0170] [B4-3]

[0171] In the library according to any one of [B1] to [B4], the ratio of the number of cyclic peptide compounds constituting the cyclic peptide compound having 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 60% or greater.

[0172] [B4-4]

[0173] In the library according to any one of [B1] to [B4], the ratio of the number of cyclic peptide compounds constituting the cyclic peptide compound having 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 80% or greater.

[0174] [B5]

[0175] The library according to any one of [B1] to [B4] is substantially composed of a cyclic peptide compound having 13 or 14 amino acid residues constituting the cyclic peptide compound.

[0176] [B6]

[0177] The library according to any one of [B1] to [B5], wherein the ratio of the number of N-substituted amino acids to the total number of amino acids constituting the cyclic moiety in the cyclic peptide compound contained in the library is on average 50% or greater.

[0178] [B7]

[0179] The library according to any one of [B1] to [B6], wherein the number of N-substituted amino acids is on average 7 to 13.

[0180] [B8]

[0181] The library according to [B7], wherein the number of N-substituted amino acids constituting the cyclic moiety in the cyclic peptide compound contained in the library is on average 7 to 10.

[0182] [B9]

[0183] According to the library of [B7], wherein in the cyclic peptide compound contained in the library, the number of N-substituted amino acids constituting the cyclic moiety is on average 8 to 13.

[0184] [B10]

[0185] According to the library of [B7], wherein in the cyclic peptide compound contained in the library, the number of N-substituted amino acids constituting the cyclic moiety is on average 8 to 10.

[0186] [B11]

[0187] According to the library of any one of [B1] to [B6], wherein in the cyclic peptide compound contained in the library,The proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10% or greater.

[0188] [B11-1]

[0189] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10% is 20% or greater.

[0190] [B11-2]

[0191] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10% is 40% or greater.

[0192] [B11-3]

[0193] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising 7 to 10 N-substituted amino acids in the cyclic moiety of the cyclic peptide compounds contained in the library is 60% or greater.

[0194] [B11-4]

[0195] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising 7 to 10 N-substituted amino acids in the cyclic moiety of the cyclic peptide compounds contained in the library is 80% or greater.

[0196] [B12]

[0197] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising 8 to 13 N-substituted amino acids in the cyclic moiety of the cyclic peptide compounds contained in the library is 10% or greater.

[0198] [B12-1]

[0199] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 13 N-substituted amino acids is 20% or greater in the cyclic peptide compounds contained in the library.

[0200] [B12-2]

[0201] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 13 N-substituted amino acids is 40% or greater in the cyclic peptide compounds contained in the library.

[0202] [B12-3]

[0203] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 13 N-substituted amino acids is 40% or greater in the cyclic peptide compounds contained in the library.The proportion of cyclic peptide compounds comprising 8 to 13 N-substituted amino acids in the cyclic moiety is 60% or greater.

[0204] [B12-4]

[0205] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising 8 to 13 N-substituted amino acids in the cyclic moiety included in the library is 80% or greater.

[0206] [B13]

[0207] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising 8 to 10 N-substituted amino acids in the cyclic moiety included in the library is 10% or greater.

[0208] [B13-1]

[0209] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 10 N-substituted amino acids is 20% or greater in the cyclic peptide compounds included in the library.

[0210] [B13-2]

[0211] According to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 10 N-substituted amino acids is 40% or greater in the cyclic peptide compound specification 10 / 100 pages 13 CN 122094968 A included in the library.

[0212] [B13-3]

[0213] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 10 N-substituted amino acids is 60% or greater in the cyclic peptide compounds contained in the library.

[0214] [B13-4]

[0215] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds comprising the cyclic moiety of 8 to 10 N-substituted amino acids is 80% or greater in the cyclic peptide compounds contained in the library.

[0216] [B14]

[0217] The method according to any one of [B1] to [B10], wherein the N-substituted amino acid is an N-alkyl amino acid.

[0218] [B15]

[0219] According to the library of [B14], wherein the N-alkyl amino acid is an N-C1-C6 alkyl amino acid.

[0220] [B16]

[0221] According to the library of [B14],The N-alkyl amino acid is an N-methyl amino acid or an N-ethyl amino acid.

[0222] [B17]

[0223] The library according to [B14], wherein the N-alkyl amino acid is an N-methyl amino acid.

[0224] [B18]

[0225] The library according to any one of [B1] to [B17], wherein the average ClogP / total AB of each cyclic peptide compound contained in the library is 1.0 to 1.8.

[0226] [B18-1]

[0227] The library according to any one of [B1] to [B17], wherein the average ClogP / total AB of each cyclic peptide compound contained in the library is 1.0 to 1.5.

[0228] [B18-2]

[0229] A library according to any one of [B1] to [B17], wherein the average ClogP / total AB of each cyclic peptide compound contained in the library is 1.1 to 1.4.

[0230] [B19]

[0231] A library according to any one of [B1] to [B18-2], wherein the average ClogP / total AB of the peptide portion other than the nucleic acid linker in each cyclic peptide compound contained in the library is 1.0 to 1.8.

[0232] [B19-1]

[0233] A library according to any one of [B1] to [B18-2], wherein the average ClogP / total AB of the peptide portion other than the nucleic acid linker in each cyclic peptide compound contained in the library is 1.0 to 1.5.

[0234] [B19-2]

[0235] The library according to any one of [B1] to [B18-2], wherein the average ClogP / total AB of the peptide portion other than the nucleic acid linker in each cyclic peptide compound contained in the library is 1.1 to 1.4.

[0236] [B20] Specification 11 / 100 pages 14 CN 122094968 A

[0237] The library according to any one of [B1] to [B19-2], wherein the average ClogP of the peptide portion other than the nucleic acid linker in the library is 13 to 20.

[0238] [B21]

[0239] The library according to any one of [B1] to [B20], wherein the average ClogP of the peptide portion other than the nucleic acid linker in each cyclic peptide compound contained in the library is 13 to 20.

[0240] [B22]

[0241] The library according to any one of [B1] to [B21], wherein

[0242] the cyclic peptide compound has a hydrogen bond donor,

[0243] The average number of hydrogen bond donors in each cyclic peptide compound contained in the library is 8 or less.

[0244] [B22-1]

[0245] The library according to any one of [B1] to [B21], wherein

[0246] the cyclic peptide compound contains a hydrogen bond donor, and

[0247] the average number of hydrogen bond donors in the cyclic portion of each cyclic peptide compound contained in the library is 8 or less.

[0248] [B23]

[0249] The library according to [B22], wherein the average number of hydrogen bond donors in each cyclic peptide compound contained in the library is 3 to 8.

[0250] [B23-1]

[0251] The library according to [B22], wherein the average number of hydrogen bond donors in the cyclic portion of each cyclic peptide compound contained in the library is 3 to 8.

[0252] [B24]

[0253] A library according to any one of [B1] to [B23], wherein the cyclic peptide compound has a hydrogen bond donor in its cyclic moiety.

[0254] [B25]

[0255] A library according to any one of [B1] to [B24], wherein in each cyclic peptide compound included in the library, the average number of amino acids having the same side chain among the amino acids constituting the cyclic moiety is 3 or less.

[0256] [B26]

[0257] A library according to any one of [B1] to [B24], wherein in each cyclic peptide compound included in the library, the number of amino acids having the same side chain among the amino acids constituting the cyclic moiety is 3 or less.

[0258] [B27]

[0259] According to any one of [B1] to [B26], wherein in each cyclic peptide compound, the side chain of each amino acid constituting the cyclic moiety is composed of at least one of the following groups: alkyl group, cycloalkyl group, aralkyl group and amide group, wherein any atom of the alkyl group of the amino acid can form a ring together with the nitrogen atom of the amino group.

[0260] [B28]

[0261] According to the library of [B27], wherein the alkyl group is a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of: halogen atom, hydroxyl group, C3-C8 cycloalkyl group and C1-C6 alkoxy group.

[0262] [B29] Specification 12 / 100 pages 15 CN 122094968 A

[0263] According to the library of [B27] or [B28],The cycloalkyl group is a C3-C10 cycloalkyl group optionally substituted with at least one group selected from the group consisting of: halogen atom, hydroxyl group, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0264] [B30]

[0265] According to any one of [B27] to [B29], the aralkyl group is a C7-C14 aralkyl group optionally substituted with at least one group selected from the group consisting of: halogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0266] [B31]

[0267] The library according to any one of [B27] to [B30], wherein the aryl group is optionally substituted C5-C10 arylC1-C6 alkyl group, wherein the C5-C10 aryl group is optionally substituted by at least one group selected from the group consisting of: one or more halogen atoms, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0268] [B32]

[0269] The library according to any one of [B27] to [B31], wherein in each cyclic peptide compound, the amide group is represented by the formula: -CONR1R2, wherein R1 and R2 are each independently a hydrogen atom or a C1-C6 alkyl group, or R1 and R2 together with the nitrogen atom to which they are attached form a 4- to 8-membered saturated heterocycle, and the 4- to 8-membered saturated heterocycle is optionally substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups and 4- to 7-membered heterocyclic groups.

[0270] [B33]

[0271] The library according to any one of [B1] to [B32], wherein in each cyclic peptide compound,The amino acids constituting this cyclic moiety include those selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0272] [B34]

[0273] The library according to any one of [B1] to [B33], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety are each independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip and gMeAbu.

[0274] [B35]

[0275] The library according to any one of [B1] to [B34], wherein the amino acids constituting the cyclic moiety comprise α-amino acids, and the number of the α-amino acids is on average 10 to 14.

[0276] [B36]

[0277] The library according to any one of [B1] to [B35], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety comprise α-amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0278] [B37] Specification 13 / 100 pages 16 CN 122094968 A

[0279] The library according to any one of [B35] or [B36], wherein in each cyclic peptide compound,The α-amino acids constituting the cyclic moiety are each independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0280] [B38]

[0281] According to any one of [B1] to [B37], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety include amino acids having a β-amino acid backbone, and the number of amino acids having a β-amino acid backbone is on average 0 to 2.

[0282] [B39]

[0283] The library according to any one of [B1] to [B38], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety comprise amino acids having a β-amino acid backbone selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0284] [B40]

[0285] The library according to [B38] or [B39], wherein in each cyclic peptide compound, the amino acids having a β-amino acid backbone among the amino acids constituting the cyclic moiety are each independently selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0286] [B41]

[0287] According to any one of [B1] to [B40], wherein in each cyclic peptide compound, the amino acid constituting the cyclic moiety comprises an amino acid having a γ-amino acid backbone, and the number of amino acids having a γ-amino acid backbone is on average 0 to 2.

[0288] [B42]

[0289] According to any one of [B1] to [B41], wherein in each cyclic peptide compound, the amino acid constituting the cyclic moiety comprises gMeAbu.

[0290] [B43]

[0291] According to the library of [B40] or [B41], wherein in each cyclic peptide compound, the amino acid having a γ-amino acid backbone among the amino acids constituting the cyclic moiety is gMeAbu.

[0292] [B44]

[0293] The library according to any one of [B1] to [B43], wherein in each cyclic peptide compound, among the amino acids constituting the cyclic moiety,The number of natural amino acids is on average 1 or more.

[0294] [B45]

[0295] According to any one of [B1] to [B44], the number of natural amino acids constituting the cyclic portion in each cyclic peptide compound is on average 5 or less.

[0296] [B46]

[0297] According to any one of [B1] to [B45], the number of natural amino acids constituting the cyclic portion in each cyclic peptide compound is on average 1 to 5.

[0298] [B47]

[0299] According to the library of [B46], the number of natural amino acids constituting the cyclic portion in each cyclic peptide compound is on average 1 to 4.

[0300] [B48] Specification 14 / 100 pages 17 CN 122094968 A

[0301] According to any one of [B1] to [B47], wherein in each cyclic peptide compound, the ratio of the number of native amino acids to the number of amino acids constituting the cyclic portion is on average 10% or greater.

[0302] [B49]

[0303] According to any one of [B1] to [B48], wherein in each cyclic peptide compound, the ratio of the number of native amino acids to the number of amino acids constituting the cyclic portion is on average 40% or less.

[0304] [B50]

[0305] According to any one of [B1] to [B49], wherein in each cyclic peptide compound, the ratio of the number of native amino acids to the number of amino acids constituting the cyclic portion is on average from 10% to 40%.

[0306] [B51]

[0307] The library according to any one of [B1] to [B50], wherein the amino acids constituting the cyclic moiety in each cyclic peptide compound comprise naturally occurring amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0308] [B52]

[0309] The library according to any one of [B1] to [B51], wherein the amino acids constituting the cyclic moiety in each cyclic peptide compound independently comprise naturally occurring amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0310] [B53]

[0311] The library according to any one of [B1] to [B52], wherein in each cyclic peptide compound, among the amino acids constituting the cyclic moiety,The number of non-natural amino acids is on average 7 or more.

[0312] [B54]

[0313] According to any one of [B1] to [B53], in each cyclic peptide compound, the number of non-natural amino acids constituting the cyclic moiety is on average 8 or more.

[0314] [B55]

[0315] According to any one of [B1] to [B54], in each cyclic peptide compound, the ratio of the number of non-natural amino acids to the total number of amino acids constituting the cyclic moiety is on average 50% or more.

[0316] [B55-1]

[0317] According to the library of [B55], in each cyclic peptide compound, the ratio of the number of non-natural amino acids to the total number of amino acids constituting the cyclic moiety is on average 60% or more.

[0318] [B55-2]

[0319] According to the library of [B55], in each cyclic peptide compound, the ratio of the number of non-natural amino acids to the total number of amino acids constituting the cyclic moiety is on average 70% or greater.

[0320] [B55-3]

[0321] According to the library of [B55], in each cyclic peptide compound, the ratio of the number of non-natural amino acids to the total number of amino acids constituting the cyclic moiety is on average 80% or greater.

[0322] [B56]

[0323] The library according to any one of [B1] to [B55-3], wherein the cyclic peptide compound in each cyclic peptide compound comprises a non-natural amino acid selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly and Ser(tBu). Specification 15 / 100 pages 18 CN 122094968 A

[0324] [B57]

[0325] The library according to any one of [B1] to [B56], wherein in each cyclic peptide compound,Each of the amino acids constituting the cyclic moiety independently comprises a non-natural amino acid selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0326] [B58]

[0327] According to any one of [B1] to [B57], wherein in each cyclic peptide compound, the amino acid constituting the cyclic peptide compound does not have any of the following in its side chain: a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group.

[0328] [B59]

[0329] The library according to any one of [B1] to [B58], wherein the average molecular weight of each cyclic peptide compound is 500 to 2000.

[0330] [B60]

[0331] The library according to any one of [B1] to [B59], wherein the average molecular weight of the peptide portion, excluding the nucleic acid linker, of each cyclic peptide compound is 500 to 2000.

[0332] [B61]

[0333] The library according to any one of [B1] to [B60], wherein the library is used for the identification of orally administered compounds or their precursors.

[0334] [B62]

[0335] The library according to any one of [B1] to [B61], wherein the library is a library used for the identification of cyclic peptide compounds capable of specifically binding to a target molecule.

[0336] [B63]

[0337] A library according to any one of [B1] to [B62], wherein the library is used in the identification of compounds capable of specifically binding to a target molecule.

[0338] [B64]

[0339] The method according to any one of [B1] to [B63], wherein the library has a diversity of 1 × 10² or more.

[0340] [B64-1]

[0341] A library according to [B64], wherein the library has a diversity of 1 × 10³ or more.

[0342] [B64-2]

[0343] A library according to [B64], wherein the library has a diversity of 1 × 10⁴ or more.

[0344] [B64-3]

[0345] A library according to [B64],The library has a diversity of 1 × 10⁶ or more.

[0346] [B64-4]

[0347] The library according to [B64], wherein the library has a diversity of 1 × 10⁸ or more.

[0348] [B64-5]

[0349] The library according to [B64], wherein the library has a diversity of 1 × 10¹⁰ or more.

[0350] [B65]

[0351] The library according to any one of [B1] to [B63], wherein the library contains 1 × 10² or more of the type of cyclic peptide compounds optionally linked to nucleic acids.

[0352] [B65-1]

[0353] The library according to [B65], wherein the library contains 1 × 10³ or more of the type of cyclic peptide compounds optionally linked to nucleic acids.

[0354] [B65-2]

[0355] The library according to [B65], wherein the library contains 1 × 10⁴ or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0356] [B65-3]

[0357] The library according to [B65], wherein the library contains 1 × 10⁶ or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0358] [B65-4]

[0359] The library according to [B65], wherein the library contains 1 × 10⁸ or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0360] [B65-5]

[0361] The library according to [B65], wherein the library contains 1 × 10¹⁰ or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0362] [B66]

[0363] A library according to any one of [B1] to [B65-5], wherein the library contains at least one of the cyclic peptide compounds (i.e., pd001 to pd168) listed in Table 15.

[0364] [B67]

[0365] A library according to any one of [B1] to [B65-5], wherein the library contains all the cyclic peptide compounds (i.e., pd001 to pd168) listed in Table 15.

[0366] [B68]

[0367] A library according to any one of [B1] to [B67], wherein the cyclic peptide compounds contained in the library are cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes).

[0368] [B69]

[0369] A library according to [B68],The nucleic acid in the cyclic peptide-nucleic acid complex contains a nucleic acid encoding the cyclic peptide.

[0370] [B70]

[0371] According to the library of [B68], the nucleic acid in the cyclic peptide-nucleic acid complex is a nucleic acid encoding the cyclic peptide.

[0372] [B70-1]

[0373] According to any one of [B68] to [B69], the nucleic acid in the cyclic peptide-nucleic acid complex contains a nucleotide sequence encoding the cyclic peptide portion forming the cyclic peptide-nucleic acid complex.

[0374] [B71]

[0375] According to any one of [B1] to [B70], the library is a display library.

[0376] [B72]

[0377] According to the library of [B71], the display library is a nucleic acid display library.

[0378] [B73] Specification 17 / 100 pages 20 CN 122094968 A

[0379] The library according to [B72], wherein the nucleic acid display library is an mRNA display library.

[0380] [C1]

[0381] A method for screening cyclic peptide compounds that can specifically bind to a target molecule, the method comprising the steps of:

[0382] (i) contacting the library according to any one of [B1] to [B73] with the target molecule; and

[0383] (ii) selecting cyclic peptide compounds that can specifically bind to the target molecule.

[0384] [C2]

[0385] The method according to [C1], wherein the target molecule is a protein.

[0386] [C3]

[0387] The method according to [C1] or [C2], wherein in (ii), the cyclic peptide compound that can specifically bind to the target molecule is selected as a drug candidate compound.

[0388] [C4]

[0389] The method according to any one of [C1] to [C3] comprises the following steps:

[0390] chemically synthesizing a cyclic peptide compound that can be used as a candidate; and

[0391] measuring the membrane permeability of the chemically synthesized cyclic peptide compound using Caco-2 cells.

[0392] [C5]

[0393] The method according to any one of [C1] to [C4], wherein the membrane permeability of the chemically synthesized cyclic peptide compound is determined by a value of Papp (cm / sec).

[0394] [C6]

[0395] The method according to [C5], wherein the value of Papp (cm / sec) is calculated by membrane permeability assessment under the following conditions:

[0396] (a) culturing Caco-2 cells in 96-well Transwells for 3 weeks,Then, the compound to be evaluated, DMEM medium, and FaSSIF (containing 1% DMSO) were added to the top side, and DMEM medium was added to the bottom side. The cells were pre-incubated for 24 hours at 37°C with shaking at 80 rpm under a 5% CO2 atmosphere;

[0397] (b) After pre-incubation, the pre-incubation solutions on the top and bottom sides were aspirated and washed, and FaSSIF / HBSS buffer solution containing the compound (containing 1% DMSO, pH 6.0) was added to the top side, and HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the bottom side;

[0398] (c) Each well was shaken at 80 rpm at 37°C under a 5% CO2 atmosphere, and the sample on the bottom side was collected 180 minutes after the start of shaking. The permeation of the compound was measured by liquid chromatography-mass spectrometry (LC / MS); and

[0399] (d) The permeability coefficient (Caco-2 Papp (cm / sec)) is calculated from the measured permeate volume.

[0400] In the above numbering, unless otherwise stated, the number referenced in the dependent entry includes the branch number of the number. For example, [B5] referenced in the dependent entry indicates that not only [B5] is included, but also its branch numbers [B5-1], [B5-2], [B5-3] and [B5-4]. The same applies to other numbers.

[0401] [Beneficial Effects of the Invention]

[0402] In one aspect, the present invention provides guidance for inducing cyclic peptide compounds, which can serve as candidates for drugs with excellent membrane permeability and metabolic stability. In another aspect, the present invention enables the discovery of drugs composed of cyclic peptides formed of 13 or 14 amino acid residues, which, despite the benefit of interaction with the target, are difficult to achieve drug-likeness. On the other hand, a screening method for obtaining cyclic peptide lead compounds with excellent drug-like properties can be provided by designing a library of cyclic peptide compounds based on the present invention. (Page 18 / 100, CN 122094968 A)

[0403] [Figure 1] Figure 1 is a schematic diagram of an example of a method for generating peptide compounds using a solid-phase method.

[0404] In the following, non-limiting preferred embodiments of the present disclosure will be described.

[0405] Any elements described in the embodiments mentioned below are intended to be described in a manner naturally considered equivalent in the “Description of Embodiments”, without being limited by any patent practices, customs, laws, etc., in countries where this patent application is intended to be examined, which attempt to narrowly interpret the content described in the embodiments.

[0406] In this disclosure,Any combination of all or part of one or more elements described herein is also intended to be described as included in this disclosure and naturally understood by those skilled in the art, provided that it does not technically contradict the technical knowledge of those skilled in the art.

[0407] The following abbreviations are used herein: Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), and Val (valine). In addition to these, the abbreviations listed in Table 1 are also used.

[0408] As used herein, the term "alkyl" is a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon and having a hydrocarbon group structure or a subset thereof that does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds and contains hydrogen and carbon atoms in the main chain. Alkyl groups include not only straight-chain forms but also branched forms. Preferred examples of alkyl groups include alkyl groups having 1 to 20 carbon atoms (C1-C20; hereinafter "Cp-Cq" means the number of carbon atoms is p to q), preferably C1-C10 alkyl groups, and more preferably C1-C6 alkyl groups. Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl (also known as 2-methylpropyl), n-pentyl, sec-pentyl (also known as 1-methylbutyl), tert-pentyl (also known as 1,1-dimethylpropyl), neopentyl (also known as 2,2-dimethylpropyl), isopentyl (also known as 3-methylbutyl), 3-pentyl (also known as 1-ethylpropyl), 1,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, and 2-ethylbutyl.

[0409] As used herein, the term "alkoxy" means an oxygen group bonded to an "alkyl" as defined above. C1-C6 alkoxy groups are preferred, and C1-C4 alkoxy groups are more preferred. Specific examples of alkoxy groups include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and 3-methylbutoxy.

[0410] As used herein,"Haloalkoxy" means a group in which one or more hydrogen atoms of the "alkoxy" defined above are replaced by a halogen. Halo-C1-C6 alkoxy groups are preferred as haloalkoxy groups, and fluoro-C1-C6 haloalkoxy groups are more preferred. Specific examples of haloalkoxy groups include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 3-fluoropropoxy, and 2,2-difluoropropoxy.

[0411] As used herein, the term "alkenyl" is a monovalent group having at least one double bond (two adjacent sp2 carbon atoms). Depending on the conformation of the double bond and the substituent (if present), the geometry of the double bond can exhibit iso-side (E) or iso-side conformations, as well as cis or trans conformations. Alkenyl groups include not only straight-chain forms but also branched forms. Preferred examples of alkenyl groups include C2-C10 alkenyl groups, and more preferred examples include C2-C6 alkenyl groups. Specific examples of alkenyl groups include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, and hexenyl.

[0412] As used herein, the term "alkynyl" is a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alynyl groups include not only straight-chain forms but also branched forms. Preferred examples of alkynyl groups include C2-C10 alkynyl groups, and more preferably C2-C6 alkynyl groups. Specific examples of alkynyl groups include ethynyl, 1-propynyl, propynyl, 3-butynyl, penynyl, 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.

[0413] As used herein, the term "cycloalkyl" means a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group and includes monocyclic, bicyclic, and spirocyclic groups. Cycloalkyl groups are preferably C3-C8 or C3-C7 cycloalkyl groups, and more preferably C3-C6 cycloalkyl groups. Specific examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclic [2.2.1]heptyl, and spiro[3.3]heptyl.

[0414] As used herein, the term "aryl" means a monovalent aromatic hydrocarbon ring, i.e., an aromatic hydrocarbon cyclic group. Preferred examples of aryl groups include C6-C10 aryl groups. Specific examples of aryl groups include phenyl and naphthyl groups (e.g., 1-naphthyl and 2-naphthyl).

[0415] As used herein, the term "aryl group" may include the following "heteroaryl".

[0416] As used herein, the term "heteroaryl" means an aromatic cyclic monovalent group containing a carbon atom and 1 to 5 heteroatoms.That is, an aromatic heterocyclic group. The ring can be a monocyclic ring or fused with other rings, and can also be partially saturated. The number of atoms in the ring constituting the heteroaryl group is preferably 5 to 10 (5-membered to 10-membered heteroaryl), and more preferably 5 to 7 (5-membered to 7-membered heteroaryl).

[0417] Specific examples of heteroaryl groups include furanyl, thiophene, pyrrole, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiophene, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzooxadiazolyl, benzoimidazolyl, benzotriazolyl, indolyl, isindolyl, azaindolyl, quinolinyl, isoquinolinyl, cyclolinyl, quinazolinyl, quinoxolinyl, benzodioxacyclopentenyl, inazinyl, and imidazopyridyl, pyrazolpyridyl, imidazopyridyl, triazolpyridyl, pyrrolepyridyl, and fluoropyridyl.

[0418] As used herein, “aralkyl (arylalkyl)” means a group in which at least one hydrogen atom of an “alkyl” as defined herein is replaced by an “aryl” as defined herein. Aralkyl is preferably a C7-C14 aralkyl or a C5-C10 aryl-C1-C6 alkyl, more preferably a C7-C10 aralkyl. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0419] As used herein, the term “alkylene” means a divalent group derived from an “alkyl” as used herein by further removal of any hydrogen atom, and is preferably a C4-C8 alkylene. Examples of alkylene groups specifically include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)CH2-, -C(CH3)2-, -(CH2)4-, -CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, -CH2CH(CH2CH3)-, -(CH2)5-, -CH(CH3)CH(CH2CH3)-, -(CH2)6-, -(CH2)7-, and -(CH2)8-. Alkylene groups are preferably C1-C8 alkylene groups, and more preferably C4-C8 alkylene groups.

[0420] As used herein,"Arylidene" means a divalent group derived from "aryl" as defined herein by further removal of any hydrogen atom. Arylidene can be a monocyclic or condensed ring. The number of atoms in the ring constituting the arylidene is not particularly limited and is preferably 6 to 10 (C6-C10 arylidene). Specific examples of arylidene include 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene, 1,3-naphthylene, and 1,4-naphthylene.

[0421] As used herein, "heteroarylidene" means a divalent group derived from "heteroaryl" as defined herein by further removal of any hydrogen atom. Heteroarylidene can be a monocyclic or condensed ring. The number of atoms in the ring is not particularly limited and is preferably 5 to 10 (5-membered to 10-membered heteroarylidene). Specific examples of heteroarylene groups include pyrrolediyl, imidazolediyl, pyrazolediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, triazolediyl, triazinediyl, isoxazolediyl, oxazolediyl, oxadiazolediyl, isothiazazolediyl, thiazolediyl, thiadiazazolediyl, furandiyl, and thiophenediyl.

[0422] As used herein, the term "alkylarylene" means a divalent group in which an alkylene group and an arylene group are bonded at any position, and the alkylene group is attached to the base skeleton side. Specific examples of alkylarylene groups include -C1-C6 alkylarylene and -C6-C10 arylene groups.

[0423] As used herein, the term "alkylarylene" means a divalent group in which an arylene group and an alkylene group are bonded at any position, and the arylene group is attached to the base skeleton side. Specific examples of arylene alkylene include -C6-C10 arylene-C1-C6 alkylene.

[0424] As used herein, the term "alkylene heteroarylene" means a divalent group in which an alkylene and a heteroarylene are bonded at any position, and the alkylene is attached to the base skeleton side. Specific examples of alkylene heteroarylene include -C1-C6 alkylene-5- to 10-membered heteroarylene.

[0425] As used herein, the term "heteroarylene alkylene" means a divalent group in which a heteroarylene and an alkylene are bonded at any position, and the heteroarylene is attached to the base skeleton side. Specific examples of heteroarylene alkylene include 5- to 10-membered heteroarylene-C1-C6 alkylene.

[0426] As used herein, the term "active ester group" refers to a group containing a carbonyl group that reacts with an amino group to form an amide bond, wherein the carbonyl group is associated with, for example, OBt, OAT, OSu, OPfp, SRI, etc., and is capable of promoting the reaction with the amino group.

[0427] A "reaction auxiliary group" is a group introduced near the functional group to be bound to activate the binding reaction of the functional group and selectively induce the reaction at the desired position. For example, a reaction auxiliary group can be introduced onto a carbonyl group or an amino group.Alternatively, both can be introduced to promote the reaction between the carbonyl group and the amino group. Examples of such reaction auxiliary groups include SH groups. This type of reaction auxiliary group can be removed either during or after the binding reaction.

[0428] As used herein, the term "amino acid" refers to a carboxylic acid having an amino group and includes both natural and non-natural amino acids. As used herein, the term "amino acid" can mean an amino acid residue. As used herein, the term "natural amino acid" means any of the L-type amino acids selected from the following: Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. The term "non-natural amino acid" means an amino acid other than a natural amino acid. Examples of non-natural amino acids include β-amino acids, γ-amino acids, L-amino acids (other than the natural amino acids mentioned above), D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids with side chains different from those of natural amino acids. Modified amino acids in this article may be represented as "MeAsp-pip," etc. "MeAsp-pip" refers to an amino acid in which the N-terminal amino group of aspartic acid is replaced by a methyl group (i.e., N-methylated), and the C-terminal carboxyl group is piperidine-amidized. In similar notations, the three-letter abbreviation of the amino acid is shown in the center, with the letter on the left indicating the substituent of the N-terminal amino group and the letter on the right indicating the substituent of the C-terminal carboxyl group. Symbols without letters on the left and right represent the amino and carboxyl groups, respectively. "bAla" represents β-alanine, and "gMeAbu" represents 4-(methylamino)butyric acid. β-amino acids and γ-amino acids are represented by the letters "b" and "g" on the far left, respectively.

[0429] As used herein, any spatial configuration is acceptable for amino acids. The choice of amino acid side chains is not particularly limited as per specification 21 / 100 pages 24 CN 122094968 A, and, in addition to hydrogen atoms, side chains can be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, heteroarylalkyl groups, cycloalkyl groups, and cycloalkyl groups bonded in a spirocyclic manner. Substituents can be further added to each of these substituents. Such substituents are not limited, and can be one or two or more substituents, each independently and freely selected from any substituent including: halogen atoms, oxygen atoms, nitrogen atoms, sulfur atoms, boron atoms, silicon atoms, or phosphorus atoms. That is,Examples of side chains include optionally substituted alkyl groups, alkoxy groups, alkoxyalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, or cycloalkyl groups having optional added substituents. Examples of substituents that may be optionally added to the side chains of amino acids include deuterium, cyano, nitro, and aminocarbonyl. In a non-limiting embodiment, amino acids as used herein can be compounds having a carboxyl group and an amino group in the same molecule. Even in this case, compounds in which the nitrogen atom of the amino group and any atom of the side chain together form a ring (such as proline, hydroxyproline, and aziridine-2-carboxylic acid) are included in amino acids.

[0430] As used herein, the term "main chain of an amino acid" refers to the chain portion consisting of an amino group, an α-carbon, and a carboxyl group in the case of α-amino acids; in the case of β-amino acids, it refers to the chain portion consisting of an amino group, a β-carbon, an α-carbon, and a carboxyl group; and in the case of γ-amino acids, it refers to the chain portion consisting of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group. As used herein, in the case of an amino acid having two carboxyl groups (such as aspartic acid, which can be considered an α-amino acid or a β-amino acid), the amino acid is defined as having a shorter amino acid chain consisting of an amino group, a carbon, and a carboxyl group. That is, in the case of aspartic acid, it is defined as an "α-amino acid". Furthermore, the term "α-amino acid backbone" refers to the chain portion consisting of an amino group, an α-carbon, and a carboxyl group; the term "β-amino acid backbone" refers to the chain portion consisting of an amino group, a β-carbon, an α-carbon, and a carboxyl group; and the term "γ-amino acid backbone" refers to the chain portion consisting of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group. For example, aspartic acid also falls under the category of "amino acids having a β-amino acid backbone" because it has a β-amino acid backbone. Similarly, glutamic acid also falls under the category of "amino acids having a γ-amino acid backbone" because it has a γ-amino acid backbone.

[0431] The main chain amino group of an amino acid can be unsubstituted (NH2 group) or substituted (i.e., -NHR group, where R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group optionally having a substituent, and one or both non-adjacent methylene groups of any such group can be substituted with an oxygen atom, a carbonyl group (-C (=O)-) or a sulfonyl group (-SO2-); and the carbon chain bonded to the N atom and the carbon atom at position α can form a ring, as in proline). The substituents of R are selected in the same manner as the substituents of the amino acid side chains described above. In the case of a substituted main chain amino group, R is included in "the side chain of the amino acid" as used herein.Such amino acids having substituted main-chain amino groups are called “N-substituted amino acids”. As used herein, “N-substituted amino acids” are preferably N-alkyl amino acids, and may be N-C1-C6 alkyl amino acids or N-C1-C4 alkyl amino acids. Preferred N-substituted amino acids may be N-methyl amino acids or N-ethyl amino acids, and more preferably N-methyl amino acids.

[0432] As used herein, the term “side chain” refers to a portion other than the main chain structure and is used in the context of the side chain of an amino acid, the side chain of the cyclic portion of a cyclic peptide compound, etc. However, in the cyclic peptide-nucleic acid complexes mentioned below, the nucleic acid portion and the nucleic acid linking portion are not included in the side chain.

[0433] The “amino acid” constituting the peptide compound includes all isotopes corresponding to each amino acid. An isotope of an “amino acid” is a form in which at least one atom is replaced by atoms having the same atomic number (number of protons) and different mass numbers (the sum of the number of protons and neutrons). Examples of isotopes included 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, chlorine atoms, etc., including, for example, 2H, 3H; 13C, 14C; 15N; 17O, 18O; 31P, 32P; 35S; 18F; 36Cl.

[0434] Examples of halogen atoms include fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I). Examples of substituents derived from halogen atoms, in addition to halogen atoms, include alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups that can be substituted by one or more halogen atoms.

[0435] Examples of substituents include halogen atoms, hydroxyl groups (-OH), carboxyl groups, oxo groups (=O), mercapto groups (-SH, also known as thiols), thiocarboxyl groups, oxy groups (-O-), thioether groups (-S-), sulfoxide groups (-S(=O)-), sulfone groups (-S(=O)2-), sulfonyl groups (-SO3H), azide groups (-N3), and cyano groups (-CN). Substituents can be combinations of two or more substituents that form esters, thioesters, amides, carbamates, alkylthio groups (-SR), sulfonamides, thioamides, thiocarboxylic acid groups (-C(=O)-SH), keto acids (-C(=O)-CO2H), or urea groups.

[0436] The alkoxy group (-OR) is selected from alkylalkoxy groups, cycloalkylalkoxy groups, alkenylalkoxy groups, alkynylalkoxy groups, arylalkoxy groups, heteroarylalkoxy groups, aralkylalkoxy groups, etc. Examples of substituents derived from oxo groups include hydrocarbon groups (-CHO,Substituents that form esters include aldehydes, alkyl carbonyl groups (producing ketones as compounds), cycloalkyl carbonyl groups, alkenyl carbonyl groups, alkynyl carbonyl groups, aryl carbonyl groups, heteroaryl carbonyl groups, and aralkyl carbonyl groups. Examples of substituents forming esters include carbonyloxy groups (-O-C(=O)-) and alkoxycarbonyl groups (-C(=O)O-). Alkoxycarbonyl groups are selected from alkoxycarbonyl groups, cycloalkoxycarbonyl groups, alkenyloxycarbonyl groups, alkynyloxycarbonyl groups, aryloxycarbonyl groups, heteroaryloxycarbonyl groups, and arylalkoxycarbonyl groups. Carbonyloxy groups are selected from alkylcarbonyloxy groups, cycloalkylcarbonyloxy groups, alkenylcarbonyloxy groups, alkynylcarbonyloxy groups, arylcarbonyloxy groups, heteroarylcarbonyloxy groups, and aralkylcarbonyloxy groups.

[0437] Examples of substituents forming thioesters include carbonyl thio groups (-S-C(=O)-) and alkyl thiocarbonyl groups (-C(=O)-SR). The substituents forming thioesters are selected from, for example, alkyl carbonyl thio groups, cycloalkyl carbonyl thio groups, alkenyl carbonyl thio groups, alkynyl carbonyl thio groups, aryl carbonyl thio groups, heteroaryl carbonyl thio groups, aralkyl carbonyl thio groups, etc., or selected from alkyl thiocarbonyl groups, cycloalkyl thiocarbonyl groups, alkenyl thiocarbonyl groups, alkynyl thiocarbonyl groups, aryl thiocarbonyl groups, heteroaryl thiocarbonyl groups, and aralkyl thiocarbonyl groups.

[0438] Examples of substituents forming amides include alkyl carbonyl amino groups (-NH-C(=O)-R), cycloalkyl carbonyl amino groups, alkenyl carbonyl amino groups, alkynyl carbonyl amino groups, cycloalkyl carbonyl amino groups, aryl carbonyl amino groups, heteroaryl carbonyl amino groups, and aralkyl carbonyl amino groups.Or it may include alkylaminocarbonyl groups (-C(=O)-NHR), cycloalkylaminocarbonyl groups, alkenylaminocarbonyl groups, alkynylaminocarbonyl groups, arylaminocarbonyl groups, heteroarylaminocarbonyl groups, and aralkylaminocarbonyl groups. Further examples include compounds in which the H atom bonded to the N atom is replaced by an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0439] Examples of substituents forming urethane esters include alkoxycarbonylamino groups (-NH-CO-OR), cycloalkoxycarbonylamino groups, alkenyloxycarbonylamino groups, alkynyloxycarbonylamino groups, aryloxycarbonylamino groups, heteroaryloxycarbonylamino groups, and arylalkoxycarbonylamino groups. Further examples include compounds in which the H atom bonded to the N atom is replaced by an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0440] Examples of substituents forming sulfonamides include alkylsulfonylamino groups (-NH-SO2-R), cycloalkylsulfonylamino groups, alkenylsulfonylamino groups, alkynylsulfonylamino groups, cycloalkylsulfonylamino groups, arylsulfonylamino groups, heteroarylsulfonylamino groups, and aralkylsulfonylamino groups, or including alkylaminosulfonyl groups (-SO2-NHR), cycloalkylaminosulfonyl groups, alkenylaminosulfonyl groups, alkynylaminosulfonyl groups, arylaminosulfonyl groups, heteroarylaminosulfonyl groups, and aralkylaminosulfonyl groups. Further examples include compounds in which the H atom bonded to the N atom is replaced by an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0441] Examples of substituents forming thioamides include N-alkylaminosulfonylamino groups (-NH-SO2-NHR), N-cycloalkylaminosulfonylamino groups, N-alkenylaminosulfonylamino groups, N-alkynylaminosulfonylamino groups, N-cycloalkylaminosulfonylamino groups, N-arylaminosulfonylamino groups, N-heteroarylaminosulfonylamino groups, and N-aralkylaminosulfonylamino groups. Furthermore, compounds in which the hydrogen atom bonded to the nitrogen atom is replaced by two substituents selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, these two substituents may be the same or different.And optionally, a ring can be formed.

[0442] The substituents forming the thioester are selected from alkylthio groups, cycloalkylthio groups, olefin thio groups, alkynylthio groups, aryl thio groups, heteroaryl thio groups, arylalkylthio groups, etc. The substituents forming the sulfoxide group (-S(=O)-R) are selected from alkylsulfinyl groups, cycloalkylsulfinyl groups, alkenylsulfinyl groups, alkynylsulfinyl groups, arylsulfinyl groups, heteroarylsulfinyl groups, arylalkylsulfinyl groups, etc. The substituents forming the sulfone group (-S(=O)2-R) are selected from alkylsulfonyl groups, cycloalkylsulfonyl groups, alkenylsulfonyl groups, alkynylsulfonyl groups, arylsulfonyl groups, heteroarylsulfonyl groups, arylalkylsulfonyl groups, etc.

[0443] Examples of substituents forming the primary amine include amino groups (-NH2). Examples of substituents forming secondary amines (-NH-R) include alkylamino groups, cycloalkylamino groups, alkenylamino groups, alkynylamino groups, arylamino groups, heteroarylamino groups, and aralkylamino groups. Examples of substituents forming tertiary amines (-NR(R1)) (such as alkyl(aralkyl)amino groups) include any two substituents selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, which may be the same or different, and may optionally form a ring. Examples of substituents forming amidine groups (-C(=NR)-NR1R2) include amidine groups (-C(=NH)-NH2) and groups in which the three substituents on the nitrogen atom are replaced by three of the following: alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, which may be the same or different, such as alkyl(aralkyl)(aryl)amidinyl groups. Examples of substituents forming a guanidine group (-NR-C(=NR3)-NR1R2) include a guanidine group (-NH-C(=NH)-NH2) and a group in which R, R1, R2, and R3 are replaced by four substituents selected from: alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, which may be the same or different and may optionally form a ring.

[0444] Examples of substituents forming a urea group include an aminocarbamoyl group (-NR-CO-NR1R2). R, R1, and R2 are any three substituents, which may be the same or different, selected from hydrogen atoms, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, and may optionally form a ring together.

[0445] Examples of functional groups derived from the boron atom include alkylboranes (-BR(R1)) and alkoxyboranes (-B(OR)(OR1)). Any two substituents are acceptable.They can be the same or different, selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, and can optionally form a ring together.

[0446] In this way, various functional groups commonly used in low molecular weight compounds, containing O, N, S, B, P, Si, and halogen atoms, can be present in one, two, or more of them. In other words, one of these substituents (such as alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups) can be further substituted by one or more other substituents. The condition that the functional group satisfies all these requirements is defined as the free choice of substituents. In the case of β-amino acids or γ-amino acids, any stereochemical configuration is allowed, similar to α-amino acids, and the choice of side chains is not as particularly restricted as in the case of α-amino acids. Amino Acid Specification 24 / 100 Pages 27 CN 122094968 The amino group portion of the main chain of an amino acid can be free (-NH2 group) or N-alkylated, such as N-methylated (-NHR group, where R represents an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group or cycloalkyl group, and the carbon atom bonded to the N atom and the carbon atom from the side chain (e.g., at the α-position) can together form a ring such as in proline. Substituents can be freely chosen, and examples include halogen atoms, ether groups and hydroxyl groups.

[0447] As used herein, the term “translatable amino acid” or “translatable amino acid” refers to those “amino acids” having side chains that can be translated. In a non-limiting embodiment, the amino acid used herein can be a translateable amino acid.

[0448] As used herein, the term “drug-like” or “pharmaceutical-like” refers to properties of excellent metabolic stability and membrane permeability.

[0449] As used herein, the term "drug-like amino acid" refers to an amino acid having the same backbone as "amino acid," namely α, β, and γ amino acids, wherein one of the two hydrogen atoms of the main chain amino group (NH2 group) and one or both hydrogen atoms of the methylene group (-CH2- group) can be replaced by an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, etc. The group in which the hydrogen atom of the -CH2- group is replaced by the aforementioned group forms a side chain. These substituents include those that are further replaced by substituents used as components of the drug-like peptide compound. These are preferably selected from the substituents defined separately above. For example, it can be replaced by one or more substituents selected from: hydroxyl group, alkoxy group, amide group, sulfonyl group, sulfinyl group, halogen atom, hydroxyamino group, aminohydroxyl group, etc. Furthermore,It can correspond to L-amino acids or D-amino acids, or α,α-dialkyl amino acids. Drug-like amino acids are not necessarily translateable. Drug-like amino acids include the side chain portion of peptides obtained from “translated amino acids” (e.g., if a seed compound is obtained with D-tyrosine, the chemically modified D-type amino acid is derived from D-tyrosine, and if a seed compound is obtained with β-alanine, the chemically modified β-amino acid is derived from β-alanine) and all amino acids that can be chemically synthesized through structural optimization of the N-substitution moiety by chemical conversion of N-methyl amino acids. These amino acids serve as components of drug-like peptide compounds and are therefore selected from a range that ensures that the resulting peptide compounds obtained through post-translational chemical modification exhibit drug-like properties. For example, lysine with an aminoalkyl group is not included in drug-like amino acids when the amino group is not involved in post-translational modification, as described below. However, when the amino group of lysine is used as a reactive functional group for post-translational modification (such as a cross-unit), the lysine unit is included as a unit of the drug-like amino acid. In this manner, whether an amino acid corresponds to a "drug-like amino acid" is determined by the functional group transformed through post-translational modification. Examples of substituents that can be used as such substituents include, among those separately defined above, ester groups, thioester groups, mercapto groups or protected thiol groups, amino groups, monosubstituted or disubstituted amino groups, protected amino groups, substituted sulfonylamino groups, alkylborane groups, alkoxyborane groups, azide groups, keto acid groups, thiocarboxyl groups, phosphoryl ester groups, and acyloxyamino groups. Details will be described later, but in one embodiment of this disclosure, the indole skeleton and / or substituted or unsubstituted hydroxyphenyl groups are not included in the drug-like amino acids of this disclosure.

[0450] As used herein, the term "substituted hydroxyphenyl group" means a group in which at least one hydrogen atom of the aromatic ring of the hydroxyphenyl group is replaced by a substituent. Substituents are selected in the same manner as the substituents of the amino acid side chains described above. Examples of substituted hydroxyphenyl groups include, but are not limited to, 3-fluoro-4-hydroxyphenyl groups. As used herein, the hydrogen atom of the aromatic ring does not include the H of the hydroxyl group (-OH) in the hydroxyphenyl group. For example, the methoxyphenyl group is not included in "substituted hydroxyphenyl group" or "unsubstituted hydroxyphenyl group" as used herein.

[0451] As used herein, the term "unsubstituted hydroxyphenyl group" means an unsubstituted hydroxyphenyl group. Substituted hydroxyphenyl groups and unsubstituted hydroxyphenyl groups can be collectively referred to as "substituted or unsubstituted hydroxyphenyl groups". Specification 25 / 100 pages 28 CN 122094968 A

[0452] As used herein,The phrase “not having substituted or unsubstituted XX groups” means that there are no substituted or unsubstituted XX groups.

[0453] As used herein, the term “translation-based synthesis” means the translation and synthesis of a peptide compound from a nucleic acid (e.g., DNA, RNA) encoding the peptide compound. Translation is the process of obtaining a linear peptide by repeating an amide or ester bond reaction through the action of ribosomes using mRNA as a template.

[0454] The term “post-translational modification” refers to a chemical reaction that occurs spontaneously after translation, independently of the action of ribosomes, or after the addition of another reagent, such as cyclization and deprotection reactions.

[0455] As used herein, the term “consistently of” means that the ratio of the theoretical number of peptide compounds and / or nucleic acids encoding them contained in the library to the total number of theoretical variants of peptide compounds and / or nucleic acids encoding them contained in the library may be, but is not limited to, 90% or greater, 93% or greater, 95% or greater, 97% or greater, 98% or greater, or 99% or greater.

[0456] As used herein, the term “substantially free” means that the listed components (such as, but not limited to, cyclic peptide compounds, amino acids, or nucleic acids) are not included, or even if they are included, the components will not negatively affect the effect in one embodiment of the invention or in an amount or embodiment that does not have a negative effect. For example, the listed components may be included as long as they do not negatively affect the effect in one embodiment of the invention or in an amount or embodiment that does not have a negative effect.

[0457] As used herein in the context of the effect of the invention, “negative effect” means an effect that negates the effect of the invention. For example, assuming the original effectiveness of the invention is 100%, if its effectiveness drops to 30%, 20%, 10%, or 5% or lower, a “negative effect” can be considered. For example, if the hit rate that should initially be obtained by panning is 1%, then if the hit rate drops to 0.3% or lower, a negative effect can be considered. Furthermore, if the proportion of Trp-free lead compounds that should initially be obtained by panning is 100%, then if the proportion of Trp-free lead compounds drops to 30% or lower, a negative effect can be considered. However, the negative impacts are not limited to these situations.

[0458] Peptide Compounds

[0459] Although not limited thereto, the term "peptide compound" in this disclosure includes linear peptide compounds and cyclic peptide compounds. The peptide compounds of this disclosure also include peptides, peptide-nucleic acid complexes (peptide-nucleic acid complexes), peptide-ribosome-nucleic acid complexes, etc. Although not limited thereto, the peptide compounds of this disclosure include 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). As used herein,The peptide portion in a peptide-nucleic acid complex may be referred to as a "peptide portion". The peptide compounds of this disclosure may contain α-hydroxycarboxylic acids in place of some constitutive amino acids. When a peptide compound contains α-hydroxycarboxylic acids, the bond between each residue in the peptide compound may be a bond other than an amide bond, such as an ester bond. When counting the number of amino acid residues in a peptide compound containing α-hydroxycarboxylic acids, the number of amino acid residues is counted, including the number of α-hydroxycarboxylic acids. Examples of α-hydroxycarboxylic acids include, in addition to glycolic acid, D-lactic acid, and L-lactic acid, compounds in which the amino group of the aforementioned "α-amino acid" is replaced by a hydroxyl group.

[0460] "Nucleic acid" in this disclosure includes DNA, mRNA, and tRNA. As used herein, the "amino acid" constituting a peptide compound is sometimes simply referred to as an "amino acid residue". As used herein, a "peptide compound" may also include its pharmaceutically acceptable salts.

[0461] As used herein, the terms "peptide compound" or "peptide portion" refer to a peptide in which 2 to 100, 3 to 50, or 4 to 30 amino acids are linked by amide bonds and / or ester bonds. In such cyclic peptide compounds, the carboxyl group present in the main chain or side chain of the corresponding linear peptide compound is cyclized with an amino group or a hydroxyl group to form a cyclic moiety. There are no particular limitations on the bonding mode of cyclization, but covalent bonds, such as amide bonds, carbon-carbon bonds, disulfide bonds, ester bonds, thioester bonds, thioether bonds, lactam bonds, bonds via triazole structures, or bonds via fluorophore structures are preferred, for example. Cycling via amide bonds tends to provide cyclic peptide compounds with higher metabolic stability.

[0462] As used herein, "cyclization reaction" refers to a reaction in which a carboxyl group present in the main chain or side chain of a linear peptide compound is linked with an amino group or a hydroxyl group to form a cyclic moiety. When the cyclic peptide compounds of this disclosure are chemically modified, the linear peptide can be chemically modified before the cyclization reaction, or the cyclic peptide obtained after the cyclization reaction can be chemically modified.

[0463] Scheme A is an example of a cyclization reaction. The white circular ( ) unit (cross-linked unit), black circular ( ) unit (cyclic main chain unit), square ( ) unit (linear main chain unit), and triangular ( ) unit (cyclic N-terminal unit) each represent an amino acid residue that constitutes the peptide moiety. Each unit is classified according to its function.And these amino acids may be the same as or different from each other.

[0464] [Formula 1]

[0465]

[0466] As used herein, “unit” means one of the following: an amino acid before cyclization after synthesis of the linear peptide compound of this disclosure, an amino acid after cyclization, or an amino acid at the point of completion of post-cyclization chemical modification. Examples of amino acid residues at the point of completion of post-cyclization chemical modification include amino acid residues translated from a tRNA and subsequently chemically transformed or structurally modified by post-translational chemical modification.

[0467] In Scheme A, the cyclic portion is a portion consisting of a triangular unit, eight black circular units, and a white circular unit, while the linear portion is a portion consisting of six square units. The curved portion of Scheme A is the portion to be cyclized (this portion is also referred to herein as the “cyclized portion” or “post-translational cyclized portion”).

[0468] In one embodiment, the peptide portion of the peptide compound of this disclosure requires reactive functional groups on the triangular or cross-shaped units in Scheme A. Therefore, prior to cyclization, drug-like amino acids are not necessarily selected for the triangular or cross-shaped units in the peptide compound.

[0469] In one embodiment, even if the triangular or cross-unit in the peptide compound prior to cyclization is not a drug-like amino acid or is initially an unfavorable amino acid, the triangular or cross-unit can be made into a drug-like amino acid or a favorable amino acid by chemical modification after cyclization.

[0470] In one embodiment, for the triangular unit in the peptide moiety prior to cyclization (pre-cyclization of the N-terminal unit) of the peptide compound of this disclosure, an amino acid residue having a thiol group near the amino group can be used, wherein both the thiol group and the amino group are protected by a protecting group.

[0471] In one embodiment, for the cross-unit in the peptide moiety prior to cyclization of the peptide compound of this disclosure, an amino acid having a cyclizable functional group (second reaction site) is preferably used by reacting with the functional group possessed by the amino acid in the triangular unit (first reaction site) in the side chain. Since the amino and carboxyl groups of the main chain are used to form covalent bonds with other amino acid residues in translation-based synthesis, and a third functional group is required for cyclization in the cross-unit, it is preferred that the cross-unit has a total of three or more functional groups. Of these functional groups, for cyclization reactions, the functional group of the side chain portion of the cross-linked unit is preferred.

[0472] The cross-linked unit can be incorporated at any position on the peptide moiety before cyclization, as long as that position allows cyclization. However, it is preferred to incorporate the cross-linked unit at a position where the number of amino acids in the cyclic moiety after cyclization or post-translational modification is 13 or 14. In other words, it is preferred to incorporate the cross-linked unit at least 12 amino acid residues from the triangular unit toward the C-terminus (e.g.,The cross-units of the peptide compounds of this disclosure are incorporated at positions of 12 or 13 amino acid residues.

[0473] The black circular and square units are selected from amino acids, preferably from drug-like amino acids.

[0474] - Cyclic Peptide Compounds

[0475] The cyclic peptide compounds of this disclosure are compounds formed by amino acids linked by amide or ester bonds and have a cyclic moiety. As used herein, the term "cyclic moiety" of a cyclic peptide compound refers to a cyclic structure formed by linking 13 or 14 amino acid residues or α-hydroxycarboxylic acids by amide bonds, ester bonds or carbon-carbon covalent bonds. Preferably, the cyclic peptide compound consists of a cyclic moiety and a linear moiety of a total of 30 or fewer residues. In view of the requirements for achieving excellent membrane permeability and metabolic stability (drug-likeness), the number of amino acids constituting the cyclic moiety is preferably 13 or 14. In a non-limiting embodiment, the cyclic moiety of the cyclic peptide compound is formed by subjecting a peptide compound obtained by translation-based synthesis to a cyclization reaction. The cyclic peptide compounds of the present invention can be further chemically modified. Examples of chemically modified cyclic peptide compounds include cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes). The nucleic acid can be linked to the cyclic peptide compound via a linker.

[0476] The cyclic portion of the cyclic peptide compound may have side chains (also referred to as “linear portions”) branching from the main chain constituting the cyclic structure, and the linear portion has at least one amide bond and / or ester bond. When the cyclic peptide compound of the present invention has a linear portion, it can be described, for example, as scheme A-1 or A-2. For example, when the cyclic peptide compound has a linear portion, it can be a peptide compound in which the cyclic portion comprises an amino acid such as Asp or Glu, and the carboxyl group of the main chain or side chain of Asp or Glu is linked to the amino group of the linear portion via an amide bond. The term “linear portion” as used herein contains both natural and non-natural amino acids (including chemically modified or backbone-converted amino acids). When the total number of amino acids in the cyclic and linear portions is 13 or 14 residues, membrane permeability tends to be higher. When the total number of amino acids in the cyclic and linear portions is 13 residues or more, metabolic stability tends to be greater. The number of amino acids in the linear portion is preferably 0 to 8 residues, more preferably 0 to 3 residues, and most preferably 0 (i.e., no linear portion exists).

[0477] [Formula 2]

[0478]

[0479] As used herein, the term "number of amino acids" refers to the total number of amino acid residues or α-hydroxycarboxylic acids constituting a peptide, and means the number of amino acids or α-hydroxycarboxylic acids produced when the amide bond, ester bond, and cyclized portion of the amino acid are cleaved.

[0480] As used herein,The term "amino acid constituting the cyclic moiety" refers to an amino acid present on the backbone of the cyclic moiety, resulting from the cleavage of amide bonds, ester bonds, and bonds in the cyclic moiety present in the cyclic peptide compound. Amino acids or α-hydroxycarboxylic acids corresponding to the cross-units shown in Scheme A-1 or A-2 above are included in the amino acids constituting the cyclic moiety as described on page 28 / 100 of CN 122094968 A.

[0481] In the cyclic peptide-nucleic acid complex, the cyclic peptide compound (peptide moiety) and the nucleic acid are directly linked or linked via a linker. In the cyclic peptide-nucleic acid complex, the nucleic acid is linked to the linear moiety but not to the cyclic moiety. As used herein, the entire linear moiety linked by the nucleic acid is referred to as the "nucleic acid-linked moiety," and the portion of the peptide moiety excluding the nucleic acid-linked moiety is referred to as the "peptide moiety excluding the nucleic acid-linked moiety." In peptide compounds not linked to nucleic acids, there is no nucleic acid-linked moiety, and therefore the entire peptide moiety is referred to as the "peptide moiety excluding the nucleic acid-linked moiety." For example, in scheme A-2 above, when the nucleic acid is connected to the tip of linear portion 1, the portion other than linear portion 1 (i.e., the combination of the cyclic portion and linear portion 2) becomes the "peptide portion other than the nucleic acid linker portion". In the linear peptide-nucleic acid complex, the entire linear peptide is the nucleic acid linker portion, so there is no "peptide portion other than the nucleic acid linker portion".

[0482] In a non-limiting embodiment, the cyclic peptide compound has a cyclic portion,

[0483] (1) the cyclic portion is composed of 13 or 14 amino acid residues,

[0484] (2) of the amino acid residues constituting the cyclic portion, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues having the same side chain is 3 or less, and the number of native amino acid residues is 4 or less, and

[0485] (3) the ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8.

[0486] The number of amino acids constituting the cyclic peptide compound (referred to as the peptide moiety when the cyclic peptide compound is linked to a nucleic acid) is not particularly limited in this disclosure, but is preferably 30 or less. When the cyclic peptide compound is linked to a nucleic acid, the chemical structure of the cyclic peptide compound can be divided into a nucleic acid-linked moiety and a peptide moiety other than the nucleic acid-linked moiety. When the number of amino acids constituting the peptide moiety other than the nucleic acid-linked moiety is 13 or 14, or when the number of amino acids constituting the cyclic moiety is 13 or 14, excellent membrane permeability and metabolic stability are tended to be achieved simultaneously.

[0487] In a non-limiting embodiment, the number of amino acid residues (number of units) of the linear moiety is preferably 0 to 8, more preferably 0 to 5, and most preferably 0 to 3.

[0488] Among the amino acid residues constituting the cyclic moiety,The number of amino acid residues having the same side chain is, for example, but not limited to, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer, preferably 4 or fewer, and more preferably 3 or fewer. In other words, the amino acids constituting the cyclic moiety may include up to five amino acids having the same side chain.

[0489] The number of amino acids constituting the cyclic moiety is not limited, but examples include 13 or more, 14 or fewer, 13 to 14, 13 or 14. Considering the requirement of achieving excellent membrane permeability and metabolic stability, it is preferred that the number of amino acids constituting the cyclic moiety is 13 to 14, 13 or 14.

[0490] In a non-limiting embodiment, a cyclic peptide compound exhibiting greater membrane permeability can be obtained when the ratio of the number of N-substituted amino acids to the total number of amino acids constituting the cyclic moiety is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more.

[0491] In a non-limiting embodiment, the number of N-substituted amino acids constituting the cyclic portion includes, but is not limited to, 7 or more, 8 or more, 9 or more, 10 or more, 14 or less, 13 or less, 12 or less, 11 or less, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 14, 10 to 13, 10 to 12, or 10 to 11. Considering the requirements for achieving excellent membrane permeability and metabolic stability, it is preferred that the number of N-substituted amino acids constituting the cyclic moiety is 7 or more, or 7 to 13, more preferably 8 or more, 9 or more, 10 or more, 7 to 10 or 8 to 10. Specification 29 / 100 pages 32 CN 122094968 A

[0492] The cyclic peptide compound of this disclosure is a compound with a ClogP / total amide bond (ClogP / total AB) ratio of 1.0 to 1.8. As used herein, “total AB” is calculated as follows. When the cyclic peptide compound is not linked to a nucleic acid, the sum of all amide bonds constituting the entire cyclic peptide compound (the number of amide bonds in the main chain and side chain of each amino acid) is used as “total AB”. On the other hand, when the cyclic peptide compound is linked to a nucleic acid,The sum of the amide bonds in the main chain and side chains of the amino acids constituting the peptide moiety other than the nucleic acid linker of the cyclic peptide compound is used as "total AB". When the bond between the nucleic acid linker and the peptide moiety other than the nucleic acid linker is an amide bond, that amide bond is also included in the calculation. ClogP / total AB is a value calculated by dividing ClogP by total AB. Cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes) can be used as components of display libraries for screening, but drug candidates expected to be used in practice are only the unlinked nucleic acid portion. Therefore, the properties of the peptide moiety other than the nucleic acid linker may become important when considering drug-likeness (e.g., high membrane permeability).

[0493] In a non-limiting embodiment, the ClogP of the cyclic peptide compounds of this disclosure is preferably 4 or greater and 25 or less to have good membrane permeability, and more preferably 10 or greater, 11 or greater, 12 or greater, or 13 or greater, and 25 or greater, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less, and further examples include 10 to 25, 11 to 24, 12 to 23, 13 to 22, 13 to 21, 13 to 20, 14 to 20 and 15 to 20. ClogP is a computer-calculated allocation coefficient and can be determined according to the principles described below: “CLOGP Reference Manual Daylight Version 4.9 (Published August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )”. Examples of methods for calculating ClogP include calculating ClogP using Daylight version 4.95 of Daylight Chemical Information Systems, Inc. (released August 1, 2011, ClogP algorithm version 5.4, database version 28, https: / / www.daylight.com / dayhtml / doc / release_notes / index.html).

[0494] “CLOGP Reference Manual Daylight Version 4.9 (released August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )” is described in paragraph

[0101] of International Publication No. WO 2024 / 214825.

[0495] As used herein, when the cyclic peptide compound is not linked to a nucleic acid,Calculate the ClogP of the entire cyclic peptide compound. When the cyclic peptide compound is linked to a nucleic acid, the ClogP of the peptide portion excluding the nucleic acid linker is calculated as "ClogP of the cyclic peptide compound". "ClogP of the peptide portion excluding the nucleic acid linker" is the calculated ClogP value of the hypothetical peptide in which the nucleic acid linker of the cyclic peptide compound is replaced by piperidine (pip). That is, assuming the peptide is a cyclic peptide, where piperidine (pip) binds to replace the amino group of the nucleic acid linker in the amide bond between the carboxyl group of the main chain or side chain of the amino acid constituting the cyclic portion (e.g., amino acid residues with carboxyl groups on the side chain such as Asp or Glu) and the amino group of the nucleic acid linker. Cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes) can be used as components of display libraries for screening, but in practice, only the unlinked portion of the drug candidate compound is expected to be used. Therefore, the properties of the peptide portion excluding the nucleic acid linker may become important when considering drug-likeness (e.g., high membrane permeability).

[0496] In a non-limiting embodiment, the ClogP / total AB ratio of the cyclic peptide compound of this disclosure is preferably 1.0 or greater, more preferably 1.1 or greater, and most preferably 1.2 or greater. Furthermore, the ClogP / total AB ratio of the cyclic peptide compound of this disclosure is preferably 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, and 1.4 or less. Examples of preferred ClogP / total AB ratios include 1.0 to 1.8, 1.0 to 1.7, 1.0 to 1.6, 1.0 to 1.5, 1.0 to 1.4, 1.0 to 1.3, 1.0 to 1.2, 1.1 to 1.6, 1.1 to 1.5, 1.1 to 1.4, 1.1 to 1.3, or 1.1 to 1.2. Specification 30 / 100 pages 33 CN 122094968 A

[0497] In a non-limiting embodiment, when the ClogP / total AB of the cyclic peptide compound of this disclosure is 1.0 or greater and the number of N-substituted amino acids is 7 or more, the compound exhibits superior membrane permeability. When the ClogP / total AB of the cyclic peptide compound of this disclosure is 1.1 or greater and the number of N-substituted amino acids is 8 or more, the membrane permeability of the compound is further enhanced.

[0498] In a non-limiting embodiment, the cyclic peptide compound included in this disclosure may have a hydrogen bond donor (proton donor) in the cyclic portion. A hydrogen bond donor provides the necessary hydrogen atoms to form hydrogen bonds,A hydrogen bond donor is a group that forms a hydrogen bond with a hydrogen bond acceptor (proton acceptor) in the same or nearby compound (including water molecules). A hydrogen bond donor is a group containing a hydrogen atom covalently bonded to an atom with relatively high electronegativity, and examples include hydroxyl groups, carboxyl groups, amino groups, unsubstituted amide groups, and monosubstituted amide groups. A hydrogen bond acceptor is, for example, a lone pair on the carbonyl oxygen in a carboxyl group, ester, or amide or amine nitrogen. When intramolecular hydrogen bonds are formed, the conformation of the peptide compound is fixed, thus enhancing the affinity for the target molecule. When intermolecular hydrogen bonds are formed with the target molecule, the affinity for the target molecule is also enhanced. Examples of the number of hydrogen bond donors include 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer. When the number of hydrogen bond donors is 5 or fewer, 4 or fewer, or 3 or fewer, a better balance can be achieved between excellent membrane permeability and metabolic stability. There is no particular lower limit to the number of hydrogen bond donors, but examples include 0 or more, 1 or more, and 2 or more. Examples of the number of hydrogen bond donors include 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4.

[0499] To improve the hit rate of peptide compounds with high membrane permeability and specific binding to target molecules, it is preferable that the side chains of the amino acids constituting the cyclic moiety of the cyclic peptide compound include groups selected from drug-like substituents. Drug-like substituents may be selected from, for example, optionally substituted alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aralkyl groups, aryl groups, and heteroaryl groups, which may be further substituted as substituents, for example, halogen atoms, amide groups (-CO-NRR' or -NR-CO-R'), sulfonyl groups (-SO2-R), or alkoxy groups (-OR), wherein R and R' are each independently selected from optionally substituted alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or in the case of amide groups, R and R' may form a 4- to 8-membered saturated heterocycle together with the nitrogen atom to which they are attached.The 4- to 8-membered saturated heterocycles may be substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups, and 4- to 7-membered heterocyclic groups.

[0500] However, since biological membranes are composed of phospholipids, highly ionizable polar functional groups (such as alkylamino groups and alkylguanidine groups) under physiological conditions (approximately pH = 7) are not well-suited for achieving high membrane permeability. Preferably, the cyclic peptide compounds of this disclosure do not contain these functional groups.

[0501] In a non-limiting embodiment, the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of this disclosure is composed of at least one group selected from the group consisting of alkyl groups, cycloalkyl groups, aralkyl groups, and amide groups. Here, any atom of the alkyl group of the amino acid and the nitrogen atom of the amino group may together form a ring.

[0502] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an alkyl group, the alkyl group may be a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a hydroxyl group, a C3-C8 cycloalkyl group, and a C1-C6 alkoxy group.

[0503] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is a cycloalkyl group, the cycloalkyl group may be a C3-C10 cycloalkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0504] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an aralkyl group, the aralkyl group may be a C7-C14 aralkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0505] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an aralkyl group, the aralkyl group may be optionally substituted with a defined C5-C10 arylC1-C6 alkyl group, wherein the C5-C10 aryl group is optionally substituted with at least one group selected from the group consisting of a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0506] In a non-limiting embodiment,When the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an amide group, the amide group may be represented by the following formula: -CONR1R2, wherein R1 and R2 are each independently a hydrogen atom or a C1-C6 alkyl group, or R1 and R2 together with the nitrogen atom to which they are attached form a 4- to 8-membered saturated heterocycle, and the 4- to 8-membered saturated heterocycle is optionally substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups and 4- to 7-membered heterocyclic groups.

[0507] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure may be a cyclic peptide compound containing at least one amide bond formed between a carboxyl group contained in the side chain of an amino acid and the nitrogen atom of the amino group of another amino acid.

[0508] In a non-limiting embodiment, the cyclic peptide compound of this disclosure may contain amino acids selected from the group consisting of Asp-pip (see Formula (i)) and MeAsp-pip (see Formula (ii)).

[0509] [Formula 3]

[0510]

[0511] In a non-limiting embodiment, at least one amino acid constituting the cyclic portion of the cyclic peptide compound of this disclosure may be an amino acid selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu. At least one amino acid constituting the cyclic portion of the cyclic peptide compound of this disclosure may each be an amino acid selected independently from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser (tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0512] In a non-limiting embodiment,The amino acids constituting the cyclic moiety of the cyclic peptide compounds disclosed herein may be, but are not particularly limited to, α-amino acids or amino acids having a β-amino acid backbone or a γ-amino acid backbone. The number of α-amino acids constituting the cyclic moiety of the cyclic peptide compound may be, for example, but not limited to, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 10, 11, 12, 13, or 14. The number of amino acids constituting the cyclic moiety of the cyclic peptide compound having a β-amino acid backbone may be, but is not limited to, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, 3 to 4, 0, 1, 2, 3, or 4. The number of amino acids constituting the cyclic portion of the cyclic peptide compound having a γ-amino acid backbone can be, but is not limited to, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, 3 to 4, 0, 1, 2, 3, or 4.

[0513] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure may include, but are not particularly limited to, α-amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). Each α-amino acid is independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0514] In a non-limiting embodiment,The amino acids constituting the cyclic portion of the cyclic peptide compound of this disclosure may include, but are not particularly limited to, amino acids having a β-amino acid backbone selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip. The amino acids having a β-amino acid backbone may each independently be amino acids selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0515] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of this disclosure may include, or may be, but are not particularly limited to, gMeAbu.

[0516] In a non-limiting embodiment, the number of natural amino acids in the cyclic portion constituting the cyclic peptide compound of the present disclosure is, for example, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, and 5 to 6, and preferably 1 to 4.

[0517] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure may include natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. The natural amino acids may each be independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0518] In one non-limiting embodiment, the number of non-natural amino acids in the cyclic portion constituting the cyclic peptide compound of the present disclosure is preferably 7 or more, more preferably 8 or more, and most preferably 9 or more. Examples of preferred numbers of non-natural amino acids include 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, and 10 or fewer.

[0519] In one non-limiting embodiment,The amino acids constituting the cyclic moiety of the cyclic peptide compounds of this disclosure may include, but are not particularly limited to, non-natural amino acids selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). Non-natural amino acids can be individually selected from the following groups: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0520] In a non-limiting embodiment, the Caco-2 Papp (cm / sec) value of the cyclic peptide compound of this disclosure may be, for example, 1.0 × 10⁻⁴ or lower, 9.0 × 10⁻⁵ or lower, 8.0 × 10⁻⁵ or lower, 7.0 × 10⁻⁵ or lower, 6.0 × 10⁻⁵ or lower, 5.0 × 10⁻⁵ or lower, 4.0 × 10⁻⁵ or lower, 3.0 × 10⁻⁵ or lower, 2.0 × 10⁻⁵ or lower, 1.0 × 10⁻⁵ or lower, 9.0 × 10⁻⁶ or lower, 8.0 × 10⁻⁶ or lower, 7.0 × 10⁻⁶ or lower, 6.0 × 10⁻⁶ or lower, or 5.99 × 10⁻⁶ or lower. In a non-limiting embodiment, the Caco-2 Papp (cm / sec) value of the cyclic peptide compound of this disclosure can be, for example, 1.0 × 10⁻⁹ or greater, 1.0 × 10⁻⁸ or greater, 1.0 × 10⁻⁷ or greater, 0.4 × 10⁻⁶ or greater, 1.0 × 10⁻⁶ or greater, or 1.0 × 10⁻⁵ or greater, preferably 0.4 × 10⁻⁶ or greater, and more preferably 1.0 × 10⁻⁶ or greater. In a non-limiting embodiment,The Caco-2 Papp (cm / sec) values ​​of the cyclic peptide compounds disclosed herein can range, for example, 1.0 × 10⁻⁹ or greater and 1.0 × 10⁻⁴ or less, 1.0 × 10⁻⁸ or greater and 1.0 × 10⁻⁴ or less, 1.0 × 10⁻⁷ or greater and 1.0 × 10⁻⁴ or less, 0.4 × 10⁻⁶ or greater and 1.0 × 10⁻⁴ or less, and 0.4 × 10⁻⁶ or greater and 1.0 × 10⁻⁵ or less.

[0521] - Membrane permeability of peptide compounds

[0522] The Caco-2 Papp (cm / sec) value serves as an indicator of membrane permeability in the cell membrane and can be calculated by membrane permeability assessment under the following conditions.

[0523] (a) Caco-2 cells were cultured in 96-well Transwells for 3 weeks. The compound to be evaluated, DMEM medium, and FaSSIF (containing 1% DMSO) were added to the top side, and DMEM medium was added to the bottom side. The cells were pre-incubated for 24 hours at 37°C with shaking at 80 rpm under a 5% CO2 atmosphere.

[0524] (b) After pre-incubation, the pre-incubation solutions on the top and bottom sides were aspirated and washed. FaSSIF / HBSS buffer solution containing the compound (containing 1% DMSO, pH 6.0) was added to the top side, and HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the bottom side.

[0525] (c) Each well was shaken at 37°C under a 5% CO2 atmosphere at 80 rpm, and 180 rpm was added after the shaking started. Minutes were taken to collect the sample on the bottom side and the permeation amount of the compound was measured by liquid chromatography-mass spectrometry (LC / MS); and

[0526] (d) the permeation coefficient (Caco-2 Papp (cm / sec)) was calculated from the measured permeation amount.

[0527] The permeation coefficient (Caco-2 Papp (cm / sec)) was calculated using numerical expression 1.

[0528] [Expression 1]

[0529]

[0530] dQ / dt represents the permeation rate of the drug (the amount of drug appearing on the bottom side per unit time), S represents the total surface area of ​​the cells, and C1(0) represents the concentration of the drug added on the top side.

[0531] Note that in the above measurements, a Pgp inhibitor may be added to each of the FaSSIF / HBSS buffer solution (1% DMSO) (pH 6.0) and the HBSS buffer solution (4% BSA) (pH 7.4).Such as Zosquidar. Additionally, the concentration of the compound to be evaluated on the donor side used to calculate the permeability coefficient in step (d) above can also be the concentration of the compound at the initial addition, or the concentration of the compound measured by collecting the solution on the tip side before starting pre-incubation or oscillation in step (c) above. In particular, when performing pre-incubation, it is preferable to use the concentration of the compound collected on the tip side before pre-incubation.

[0532] In a non-limiting embodiment, when measuring the membrane permeability of the peptide compounds of this disclosure, it is preferable to use a peptide compound that does not have a nucleic acid moiety serving as a template for the peptide moiety as the test substance. In a non-limiting embodiment, as described later, panning is performed in the state of a peptide-nucleic acid complex, and peptide-nucleic acid complexes that can specifically bind to the target molecule are selected, and peptide compounds can be synthesized based on the nucleotide sequence of the nucleic acid moiety of the selected peptide-nucleic acid complex. Preferred examples include using such synthesized peptide compounds as test substances to measure membrane permeability. In a non-limiting embodiment, derivatives of peptide compounds encoded by nucleotide sequences can be synthesized, and the derivatives can be used as test substances to measure membrane permeability, or derivatives can be synthesized from test substances based on membrane permeability results, and the membrane permeability of the derivatives can be measured. Specification 34 / 100 pages 37 CN 122094968 A

[0533] In a non-limiting embodiment, the cyclic peptide compounds of this disclosure can be selected from compounds pd001 to pd168 listed in Table 15.

[0534] - Peptide-Nucleic Acid Complex

[0535] In one embodiment, the peptide compounds of this disclosure can be complexes of peptides and nucleic acids (peptide-nucleic acid complexes). Peptide-nucleic acid complexes can be formed by binding nucleic acids to the C-terminus of the peptide moiety. The peptide-nucleic acid complexes of this disclosure are divided into a linear portion of nucleic acid linkage, i.e., a nucleic acid linkage portion, and a peptide moiety other than the nucleic acid linkage portion. In cyclic peptide-nucleic acid complexes, the nucleic acid is linked to the linear portion but not to the cyclic portion. The method of bonding peptides and nucleic acids is not particularly limited, but they can be bonded via linkers. When a peptide compound and a nucleic acid are bonded via a linker, the linker is considered part of the nucleic acid linker moiety. In one embodiment, the nucleic acid forming the complex with the peptide moiety can be DNA or RNA, preferably RNA, and more preferably mRNA. In one embodiment, the nucleic acid forming the complex with the peptide can be a template for translation-based synthesis of the peptide moiety, wherein mRNA is preferably used as the template. In a more specific embodiment, preferred examples include peptide compounds.The C-terminus of the peptide is linked to the 3' end of the mRNA serving as a template via a linker containing an antibiotic such as puromycin, which is an analog of an aminoacyl tRNA.

[0536] The peptide-nucleic acid complex can be generated, for example, by the method described in International Publication No. WO 2013 / 100132. Specifically, the peptide-nucleic acid complex can be generated by a method comprising the following steps.

[0537] A) Acylate dinucleotide pdCpA or pCpA with the desired amino acid or amino acid analog to provide aminoacyl-pdCpA or aminoacyl-pCpA,

[0538] B) Provide tRNA lacking the 3' terminal CA,

[0539] C) Link aminoacyl-pdCpA or aminoacyl-pCpA from step A to the tRNA lacking CA from step B to provide initiator tRNA acylated with the desired amino acid or amino acid analog,

[0540] D) Provide a cell-free translation system containing the initiator tRNA from step C) and excluding at least one of methionine, methionyl-tRNA synthetase (MetRS), initiator tRNA for methionine translation, a formyl donor, and methionyl-tRNA transferase,

[0541] E) Provide template DNA encoding an ATG downstream of a promoter. The peptide sequence of the start codon, the subsequent cysteine ​​codon UGU or UGC, and the codon downstream of the anticodon of the tRNA in step C) is translated,

[0542] F) mRNA is provided from the template DNA in step E),

[0543] G) the adapter is attached to the 3' end of the mRNA in step F), and

[0544] H) the mRNA attached to the adapter in step G) is added to the cell-free translation system in step D) and translated to provide a precyclic peptide-mRNA complex.

[0545] When the peptide is a cyclic peptide, the step of cyclizing the precyclic peptide-mRNA complex in step H) may be further included. In step H), a desulfurization reaction may be performed as needed. In addition, after step G), the step of synthesizing cDNA using primers annealed to the 3' region of the mRNA may be included.

[0546] In steps A to C,RNA can be synthesized by preparing template DNA encoding the desired tRNA sequence and setting a T7, T3, or SP6 promoter upstream and transcribing it with an RNA polymerase suitable for the promoter (such as T7 RNA polymerase and T3) and SP6 RNA polymerase. Alternatively, tRNA can be extracted and purified from cells, and the resulting tRNA of interest can be extracted using a probe with a complementary sequence to the tRNA sequence. Cells transformed with an expression vector of the tRNA of interest can then be used as the source. RNA containing the sequence of interest can also be synthesized chemically. For example, aminoacyl tRNA can be obtained by binding tRNA in which CA is removed from the 3' end of the CCA sequence obtained in this manner to an aminoacylated pdCpA (pdCpA method) prepared separately using RNA ligase. Alternatively, full-length tRNA can be prepared and aminoacylated using flexizyme, a ribozyme that charges tRNA with an active ester of various non-natural amino acids. Acylated tRNA can also be obtained using the methods described later.

[0547] In steps E to H, DNA in which the desired sequence is placed downstream of a promoter (such as the T7 promoter) is first chemically synthesized and then used as a template to obtain double-stranded DNA via a primer extension reaction. The obtained double-stranded DNA is transcribed into mRNA using an RNA polymerase such as T7 RNA polymerase. Puromycin is incorporated into the protein by linking a linker containing puromycin to the 3' end of the mRNA obtained by transcription and then translating the mRNA into a protein using a known cell-free translation system (such as the PURE system), thereby linking the mRNA and its encoded protein via puromycin. In this way, a peptide-mRNA complex associated with the mRNA and its encoded product (peptide) can be constructed.

[0548] - Metabolic stability of the peptide compound

[0549] In a non-limiting embodiment, the peptide compound of this disclosure preferably has good metabolic stability. For good metabolic stability, the peptide compound preferably contains 13 to 14 amino acids. When the peptide compound contains 13 to 14 amino acids, the membrane permeability conditions described above do not affect the metabolic stability of the peptide compound. In one embodiment, the peptide compound preferably does not have easily oxidized thioether bonds. In one embodiment, the peptide compound preferably does not have a methylthio group.Because the methylthio group is readily oxidized and potentially hinders metabolic stability.

[0550] The metabolic stability of the peptide compounds of the present invention can be confirmed using known methods, such as using hepatocytes, intestinal cells, liver microsomes, intestinal microsomes, liver S9 mixtures, etc. Specifically, for example, according to the description by LL von Moltke et al. in the following literature, the stability of the peptide compounds can be confirmed by measuring the peptide compounds in liver microsomes (Midazolam hydroxylation by human liver microsomes in vitro: inhibition by fluoxetine, norfluoxetine, and by azole antifungal agents. J Clin Pharmacol, 1996, 36(9), 783-791). More specifically, metabolic stability can be confirmed according to the method described in Example 3.

[0551] Regarding metabolic stability, for example, if the intrinsic hepatic clearance (CLh int (μL / min / mg protein)) value when measuring stability in liver microsomes according to the above method is 150 or lower, it can be considered that sufficient metabolic stability has been achieved for use as an oral medicine, preferably 100 or lower. In the case of a peptide compound that is a CYP3A4 metabolite, a preferred value of 78 or lower is preferred to avoid intestinal metabolism in humans (Non-patent literature: M. Kato et al. The intestinal first-pass metabolism of substances of CYP3A4 and P-glycoprotein-quantitative analysis based on information from the literature. Drug Metab. Pharmacokinet. 2003, 18(6), 365-372.), and a more preferred value of 35 or lower (assuming FaFg = 1 and protein binding = 0%) to indicate a bioavailability of about 30% or greater in humans.

[0552] - Nucleic Acids

[0553] In a non-limiting embodiment, the peptide compound of this disclosure may be linked to a nucleic acid, preferably via a linker. Preferred examples of nucleic acids of this disclosure include nucleic acids encoding the peptide compound of this disclosure. In one embodiment,The term "nucleic acid encoding peptide compound" refers to a nucleic acid used or already used as a template in the translation-based synthesis of the peptide compound.

[0554] - Library

[0555] The libraries of this disclosure include libraries of peptide compounds and libraries of peptide-nucleic acid complexes. The libraries of this disclosure may further include linear peptide compounds and linear peptide-nucleic acid complexes. For the libraries of this disclosure, libraries of cyclic peptide compounds or libraries of cyclic peptide-nucleic acid complexes are preferred, and libraries of cyclic peptide-mRNA complexes are particularly preferred. According to the library 39 CN 122094968 A on page 36 / 100 of this disclosure, compounds with excellent drug-like properties and high affinity for target molecules can be efficiently found in cyclic peptide compounds having a cyclic moiety formed by 13 or 14 amino acid residues. As a library, a display library is preferred. Examples of display libraries include display utilization libraries. Among them, mRNA display libraries, DNA display libraries, and ribosome display libraries are preferred, and mRNA display libraries are more preferred. When the library disclosed herein is a display library, the nucleic acids linked to the peptide compounds by panning and enrichment can be amplified by PCR, etc., and then transcribed and translated, thereby making the peptide compounds easy to synthesize (Yanagawa, H. et al. FEBS Lett., 1997, 414, 405-408. and Szostak J.W. et al. Proc. Natl. Acd. Sci. USA, 1997, 94, 12297-12302.).

[0556] The peptide compounds disclosed herein may be peptide compounds encoded by nucleic acids. When the peptide compounds of the disclosed present invention are linked to nucleic acids, the peptide compounds may be peptide compounds encoded by the linked nucleic acids or peptide compounds obtained by further modification of the peptide compounds. In other words, the term "(cyclic) peptide compounds encoded by nucleic acids" includes not only peptide compounds synthesized directly by translating nucleic acids as templates, but also peptides synthesized by further modification after translation. For example, when a linear peptide is synthesized by translating a nucleic acid and then a cyclic peptide is synthesized by a subsequent cyclization step, the cyclic peptide may be referred to as a cyclic peptide compound encoded by a preferred nucleic acid.

[0557] For a library of peptide compounds that can be linked to nucleic acids and / or the nucleic acids encoding them, the term “consistently of” as used herein means that the ratio of the theoretical number of said peptide compounds and / or the nucleic acids encoding them contained in the library to the total number of theoretical variants of said peptide compounds and / or the nucleic acids encoding them contained in the library may be, but is not limited to, 90% or greater, 93% or greater, 95% or greater, 97% or greater, 98% or greater, or 99% or greater.

[0558] In a non-limiting embodiment,The proportion of cyclic peptide compounds comprising 13 or 14 amino acid residues in the cyclic moiety to the total number of cyclic peptide compounds contained in the library of this disclosure may be, but is not limited to, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0559] In a non-limiting embodiment, the library of this disclosure may be a library consisting essentially of cyclic peptide compounds, wherein the number of amino acid residues constituting the cyclic moiety of the cyclic peptide compounds is 13 or 14.

[0560] In a non-limiting embodiment, the proportion of cyclic peptide compounds constituting 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library of this disclosure may be, but is not limited to, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0561] In a non-limiting embodiment, the library of this disclosure may be a library consisting essentially of cyclic peptide compounds, wherein the number of amino acid residues constituting the cyclic peptide compounds is 13 or 14.

[0562] In a non-limiting embodiment, the average number of amino acids (units) in the linear portion other than the nucleic acid linker in each cyclic peptide compound included in the library of this disclosure is preferably 0 to 8, more preferably 0 to 5, and most preferably 0.

[0563] In a non-limiting embodiment, in each cyclic peptide compound included in the library of this disclosure, the number of amino acids constituting the peptide portion other than the nucleic acid linker is, for example, but not limited to, 13 or more, 14 or less, 13 to 14, 13 or 14. Considering the requirement of achieving excellent membrane permeability and metabolic stability, it is preferred that the number of amino acids constituting the cyclic portion is 13, 14, or 13 to 14.

[0564] In a non-limiting embodiment, in each cyclic peptide compound included in the library of this disclosure,The average number of amino acids constituting the peptide portion, excluding the nucleic acid linker, is, for example, but not limited to, 13 or more, 14 or fewer, or 13 or 14. Considering the requirements for achieving excellent membrane permeability and metabolic stability, the average number of amino acids constituting the cyclic portion is preferably 13 to 14.

[0565] In a non-limiting embodiment, in each cyclic peptide compound included in the library of this disclosure, the average number of N-substituted amino acids constituting the cyclic moiety is, for example, 7 or more, 8 or more, 9 or more, 10 or more, 14 or less, 13 or less, 12 or less, 11 or less, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 14, 10 to 13, 10 to 12 or 10 to 11, preferably 7 or more, 8 or more, 7 to 13 The number of N-substituted amino acids constituting the cyclic moiety is 7 to 10 or 8 to 13, more preferably 7 to 10 or 8 to 10.

[0566] In a non-limiting embodiment, the proportion of cyclic peptide compounds containing 7 to 10 N-substituted amino acids constituting the cyclic moiety in the cyclic peptide compounds included in this disclosure is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, preferably 50% or more or 60% or more, more preferably 70% or more, and most preferably 80% or more.

[0567] In one non-limiting embodiment, among the cyclic peptide compounds included in this disclosure, the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 8 to 13 is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, preferably 50% or more or 60% or more, more preferably 70% or more, and most preferably 80% or more.

[0568] In one non-limiting embodiment, among the cyclic peptide compounds included in this disclosure,The proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 8 to 10 is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, preferably 50% or more or 60% or more, more preferably 70% or more, and most preferably 80% or more.

[0569] In the cyclic peptide compounds included in the library of this disclosure, the average proportion of the number of N-substituted amino acids in the number of amino acids constituting the cyclic moiety is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, preferably 50% or more, and more preferably 60% or more. The N-substituted amino acids described herein are preferably N-alkyl amino acids, more preferably N-C1-C6 alkyl amino acids, further preferably N-methyl amino acids or N-ethyl amino acids, and most preferably N-methyl amino acids. That is, in a non-limiting embodiment, the number of N-substituted amino acids is shown as the average of the number of N-alkyl amino acids, the number of N-C1-C6 alkyl amino acids, the number of N-ethyl amino acids, or the number of N-methyl amino acids.

[0570] In a non-limiting embodiment, the average ClogP / total AB of the cyclic peptide compounds contained in the library of this disclosure is preferably 1.0 or greater, more preferably 1.1 or greater, more preferably 1.2 or greater, and preferably 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or 1.4 or less. The average ClogP / total AB of the cyclic peptide compounds included in the library of this disclosure can be 1.0 to 1.8, 1.0 to 1.7, 1.0 to 1.6, 1.0 to 1.5, 1.0 to 1.4, 1.0 to 1.3, 1.0 to 1.2, 1.1 to 1.6, 1.1 to 1.5, 1.1 to 1.4, 1.1 to 1.3, or 1.1 to 1.2. "ClogP / total AB of the cyclic peptide compound" is the value calculated as described above. If the ClogP / total AB of each cyclic peptide compound included in the library of this disclosure is calculated, and the average value is within the above range, the probability of the selected compound being a compound with excellent permeability increases. (See page 41 of CN 122094968 A in the membrane specification 38 / 100).

[0571] In a non-limiting embodiment, the average ClogP of the cyclic peptide compounds included in the library of this disclosure is preferably 10 or greater, 11 or greater, 12 or greater, or 13 or greater.And preferably 25 or lower, 24 or lower, 23 or lower, 22 or lower, 21 or lower, or 20 or lower. The average ClogP of the cyclic peptide compounds included in the library of this disclosure may be 10 to 25, 11 to 24, 12 to 23, 13 to 22, 13 to 21, 13 to 20, 14 to 20, or 15 to 20. “ClogP of the cyclic peptide compound” is the value calculated as described above. If the ClogP of each cyclic peptide compound included in the library of this disclosure is calculated and the average value is within the above range, the probability of the selected compound being a compound with excellent membrane permeability increases.

[0572] In a non-limiting embodiment, the cyclic peptide compounds included in the library of this disclosure may have a hydrogen bond donor (proton donor). The average number of hydrogen bond donors is, for example, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, preferably 5 or less, more preferably 4 or less, and most preferably 3 or less. There is no particular limitation on the lower limit of the average number of hydrogen bond donors, but examples include 0 or more, 1 or more, and 2 or more. Examples of average hydrogen bond donor numbers include 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4. If the number of hydrogen bond donors for each cyclic peptide compound contained in the library of this disclosure is calculated and the average number is within the above range, the probability of the selected compound being a compound with excellent membrane permeability increases.

[0573] In one embodiment, the cyclic peptide compounds contained in the library of this disclosure may have hydrogen bond donors (proton donors) in the cyclic portion. A hydrogen bond donor is a group that provides the necessary hydrogen atoms to form a hydrogen bond and forms a hydrogen bond with a hydrogen bond acceptor (proton acceptor) in the same or nearby compounds (including water molecules). Hydrogen bond donors are groups containing hydrogen atoms covalently bonded to atoms with relatively high electronegativity, and examples include hydroxyl groups, carboxyl groups, amino groups, unsubstituted amide groups, and monosubstituted amide groups. Hydrogen bond acceptors are, for example, lone pairs on the carbonyl oxygen in carboxyl, ester, and amide or amine nitrogen groups. When intramolecular hydrogen bonds are formed, the conformation of the peptide compound is fixed, thus enhancing its affinity for the target molecule. When intermolecular hydrogen bonds are formed with the target molecule,This can enhance affinity for target molecules. The average number of hydrogen bond donors in the cyclic moiety is, for example, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer, preferably 5 or fewer, more preferably 4 or fewer, and most preferably 3 or fewer. There is no particular lower limit to the average number of hydrogen bond donors, but examples include 0 or more, 1 or more, and 2 or more. Examples of average hydrogen bond donor numbers include 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 8, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4.

[0574] In a non-limiting embodiment, the cyclic peptide compounds included in the library of this disclosure may include a plurality of amino acids having the same side chain as the amino acids constituting the cyclic portion. Here, the term “including amino acids having the same side chain” means that the amino acids constituting the whole cyclic peptide compound or the cyclic portion of the cyclic peptide compound include a plurality of amino acids having the same side chain. "Amino acids having the same side chain" are determined based on the similarity or dissimilarity of the side chains, without considering substituents on the nitrogen atom of the amino group in the main chain. For example, alanine and N-methylalanine are considered "amino acids having the same side chain". Specifically, when the cyclic portion of a cyclic peptide compound contains two Leu amino acids, the cyclic peptide compound includes a plurality of amino acids having the same side chain as the amino acids constituting the cyclic portion. Among the amino acids constituting the cyclic portion, the average number of amino acids having the same side chain is, for example, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, preferably 4 or less, and more preferably 3 or less. Specification 39 / 100 pages 42 CN 122094968 A

[0575] In a non-limiting embodiment, the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is composed of at least one selected from the group consisting of alkyl groups, cycloalkyl groups, aralkyl groups and amide groups. Here, any atom of the alkyl group of the amino acid and the nitrogen atom of the amino group can form a ring together.

[0576] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is an alkyl group,The alkyl group may be a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a hydroxyl group, a C3-C8 cycloalkyl group, and a C1-C6 alkoxy group.

[0577] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is a cycloalkyl group, the cycloalkyl group may be a C3-C10 cycloalkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a hydroxyl group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0578] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is an aralkyl group, the aralkyl group may be a C7-C14 aralkyl group optionally substituted with at least one group selected from the group consisting of a halogen atom, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkoxy group.

[0579] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is an aralkyl group, the aralkyl group may be optionally a defined C5-C10 arylC1-C6 alkyl group, wherein the C5-C10 aryl group is optionally substituted by at least one group selected from the group consisting of: halogen atom, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group and C1-C6 haloalkoxy group.

[0580] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound contained in the library of this disclosure is an amide group, the amide group may be represented by the following formula: -CONR1R2, wherein R1 and R2 are each independently a hydrogen atom or a C1-C6 alkyl group, or R1 and R2 together with the nitrogen atom to which they are attached form a 4- to 8-membered saturated heterocycle, and the 4- to 8-membered saturated heterocycle is optionally substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups, and 4- to 7-membered heterocyclic groups.

[0581] In a non-limiting embodiment,At least one amino acid of the cyclic portion of each of the cyclic peptide compounds contained in the library of this disclosure may be an amino acid selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu. At least one amino acid constituting the cyclic moiety may be an amino acid selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser (tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0582] In a non-limiting embodiment, the amino acids constituting the cyclic moiety of the cyclic peptide compounds contained in the library of this disclosure are not particularly limited and may be selected from α-amino acids, amino acids having a β-amino acid backbone, and amino acids having a γ-amino acid backbone. The average number of α-amino acids in the cyclic moiety of the cyclic peptide compounds contained in the library is, for example, but not limited to, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 10, 11, 12, 13, or 14. The average number of amino acids in the β-amino acid backbone of the cyclic moiety of the cyclic peptide compounds contained in the library is, for example, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, 3 to 4, 0, 1, 2, 3, or 4. The average number of γ-amino acids constituting the cyclic moiety of the cyclic peptide compounds contained in the library is, but is not limited to, for example, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, 3 to 4, 0, 1, 2, 3, or 4. To achieve high membrane permeability,Preferably, the average number of α-amino acids constituting the cyclic moiety is 10 to 14, the average number of amino acids having the β-amino acid backbone is 0 to 2, and the average number of γ-amino acids is 0 to 2.

[0583] In a non-limiting embodiment, the amino acids constituting the cyclic moiety of the cyclic peptide compounds contained in the library of this disclosure may include, but are not particularly limited to, α-amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). The α-amino acids may be independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0584] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds included in the library of this disclosure may include, but are not particularly limited to, amino acids having a β-amino acid backbone selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip. The amino acids having a β-amino acid backbone can each be independently selected from the group consisting of: Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0585] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library of this disclosure may include or may be, but are not particularly limited to, gMeAbu.

[0586] In a non-limiting embodiment, the average number of natural amino acids in the number of amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library of this disclosure is, for example, 1 or more, 2 or more, 3 or more, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 7, 3 to 6, 3 to 5, or 3 to 4, preferably 1 or more, 7 or fewer, or 1 to 7.More preferably, one or more, six or fewer, five or fewer, one to six or one to five, more preferably one or more, four or fewer or one to four. In the cyclic peptide compounds included in the library of this disclosure, the average proportion of the number of naturally occurring amino acids constituting the cyclic moiety can be 10% or more or 20% or more, and can be 50% or less, 40% or less, or 30% or less. The average proportion of the number of naturally occurring amino acids constituting the cyclic moiety can be 10% to 50%, 15% to 50%, 20% to 50%, 25% to 50%, 30% to 50%, 40% to 50%, 10% to 40%, 15% to 40%, 20% to 40%, 25% to 40%, 30% to 40%, 10% to 30%, 15% to 30%, or 20% to 30%.

[0587] In one non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds included in the library of this disclosure may include natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. The natural amino acids may each be independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0588] In one non-limiting embodiment, the average number of non-natural amino acids constituting the cyclic portion of the cyclic peptide compounds included in the library of this disclosure is preferably 7 or more, more preferably 8 or more, 9 or more, or 10 or more. In the cyclic peptide compounds included in this disclosed library, the average percentage of non-natural amino acids constituting the cyclic moiety may be 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, or 80% or greater.And it can be 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less. The average percentage of non-natural amino acids in the amino acid composition of the cyclic moiety can be 50% to 100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 55% to 100%, 55% to 95%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 60% to 100%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 70% to 100%, 70% to 95%, 70% to 90%, 70% to 85%, or 70% to 80%.

[0589] In a non-limiting embodiment, the amino acids contained in the cyclic peptide compound or constituting the cyclic portion of the cyclic peptide compound of the present disclosure may include, but are not particularly limited to, non-natural amino acids selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). Non-natural amino acids can be independently selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

[0590] In a non-limiting embodiment, each cyclic peptide compound included in the library of this disclosure may be a cyclic peptide compound in which the amino acids contained in the peptide moiety, excluding the nucleic acid linker, do not have substituted or unsubstituted hydroxyphenyl groups on the side chains. In one embodiment, the library of this disclosure may be a library of cyclic peptide compounds that substantially do not contain substituted or unsubstituted hydroxyphenyl groups on the side chains of the peptide moiety, excluding the nucleic acid linker. Because the substituted or unsubstituted hydroxyphenyl groups on the side chains of the peptide moiety other than the nucleic acid linker in cyclic peptide compounds can hinder membrane permeability,Therefore, it is preferred that the cyclic peptide compounds do not have substituted or unsubstituted hydroxyphenyl groups.

[0591] In a non-limiting embodiment, each cyclic peptide compound included in the library of this disclosure may be a cyclic peptide compound that does not have an indole skeleton on the side chain of the peptide portion other than the nucleic acid linker. In one embodiment, the library of this disclosure may be a library of cyclic peptide compounds that substantially do not have an indole skeleton on the side chain of the peptide portion other than the nucleic acid linker. Since the presence of an indole skeleton on the side chain of the peptide portion other than the nucleic acid linker of the cyclic peptide compound would impede membrane permeability, it is preferred that the cyclic peptide compound does not have an indole skeleton.

[0592] In a non-limiting embodiment, the library of this disclosure may be a library consisting substantially of cyclic peptide compounds that do not have methylthio groups on the side chain of the peptide portion other than the nucleic acid linker. In one embodiment, the library of this disclosure may be a library of cyclic peptide compounds that substantially do not have substituted or unsubstituted methylthio groups on the side chain of the peptide portion other than the nucleic acid linker. In one embodiment, it is preferred that the cyclic peptide compound does not have a methylthio group, because methylthio groups are readily oxidized and potentially impede metabolic stability.

[0593] In a non-limiting embodiment, the library of this disclosure may be a library consisting essentially of cyclic peptide compounds that do not have thiol groups on the side chains of the peptide moiety other than the nucleic acid linker. In one embodiment, the library of this disclosure may be a library consisting essentially of cyclic peptide compounds that do not have substituted or unsubstituted thiol groups on the side chains of the peptide moiety other than the nucleic acid linker. In one embodiment, it is preferred that the cyclic peptide compound does not have a thiol group, because thiol groups are readily oxidized and potentially impede metabolic stability.

[0594] In a non-limiting embodiment, the average molecular weight of the cyclic peptide compounds included in the library of this disclosure may be from 500 to 2,000. In one embodiment, the average molecular weight of the cyclic peptide compounds included in the library of this disclosure may be from 500 to 2,000. As used herein, when the cyclic peptide compound is not linked to a nucleic acid, the molecular weight of the entire cyclic peptide compound specification (page 42 / 100, CN 122094968 A) is calculated. When the cyclic peptide compound is linked to a nucleic acid, the molecular weight of the peptide portion other than the nucleic acid linker is calculated as the "molecular weight of the cyclic peptide compound." The "molecular weight of the peptide portion other than the nucleic acid linker" is the calculated molecular weight of the hypothetical peptide in which the nucleic acid linker of the cyclic peptide compound is replaced by piperidine. That is, assuming the peptide is a cyclic peptide, wherein the amide bond between the carboxyl group of the main chain or side chain of the amino acid constituting the cyclic portion (e.g., amino acid residues with carboxyl groups on the side chain such as Asp or Glu) and the amino group of the nucleic acid linker...Piperidine binds to the amino group that replaces the nucleic acid linker. Cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes) can be used as components of display libraries for screening, but drug candidates intended for use in practice are only the unlinked portions of the nucleic acids. Therefore, the properties of the peptide portion other than the nucleic acid linker may become important when considering drug-likeness (e.g., high membrane permeability).

[0595] In the libraries of this disclosure, when determining the “theoretical (total) number of variants” of cyclic peptide compounds, those that cannot actually be generated as cyclic peptide compounds are not included in the theoretical (total) number of variants. For example, in the following cases (1) and (2), the relevant amino acids are not synthesized via translation, and therefore peptide compounds containing such amino acids are not included in the theoretical (total) number: (1) even if the amino acid is added as material to the translation solution as a peptide compound, it does not contain a nucleic acid encoding the same amino acid as a template; (2) when a translation mixture containing a nucleic acid nucleotide sequence as a template for a peptide compound but not containing the corresponding amino acid is used. Furthermore, if byproducts or unreacted substances are present during the production of cyclic peptide compounds, they are not included in the theoretical (total) quantity.

[0596] For example, using a total of 20 natural amino acids and a codon table that assigns a set of codon units to one type of amino acid, when an 11-residue peptide is synthesized by translation from a nucleotide sequence randomly arranged from codon units, if the initiator amino acid is fixed to methionine, the theoretical number of variations is 2010.

[0597] As used herein, whether the average value of the cyclic peptide compounds contained in the library is within a specific range can be determined by summing the values ​​of each cyclic peptide compound actually contained in the library and then dividing by the total number of all cyclic peptide compounds contained in the library (the measured number mean). However, if the number of cyclic peptide compounds in the library is large, it is difficult to calculate the measured number mean using the above method, so the theoretical number mean is used instead of the measured number mean. The theoretical mean is calculated by assuming all types of cyclic peptide compounds that could theoretically be included in the library, summing their individual values, and then dividing the total by the theoretical number of types of cyclic peptide compounds that could theoretically be included in the library.

[0598] The library of this disclosure contains multiple cyclic peptide compounds or nucleic acids encoding these compounds. The number of variations (types) of the cyclic peptide compounds included in this library is referred to as “diversity”. The diversity of the cyclic peptide compounds or nucleic acids encoding them included in the library of this disclosure is not particularly limited, and examples include 10² or greater, 10³ or greater, 10⁴ or greater, 10⁵ or greater, 10⁶ or greater, 10⁷ or greater, 10⁸ or greater, 10⁹ or greater, 10¹⁰ or greater, 10¹¹ or greater, and 10¹² or greater. These diversity values ​​are not limited to the measured values.This can be a theoretical value.

[0599] The cyclic peptide compounds included in the library of this disclosure may include at least one selected from the cyclic peptide compounds pd001 to pd168 listed in Table 15, or may include all of them.

[0600] While the characteristics of the cyclic peptide compounds included in the library of this disclosure have been described to date, these descriptions may also be applied to the characteristics of cyclic peptide compounds included in the library of this disclosure that have 13 or 14 amino acid residues constituting the cyclic moiety. For example, the description in the paragraph regarding the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10 in the cyclic peptide compounds included in the library herein may also be used to explain the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic moiety is 7 to 10 in the cyclic peptide compounds included in the library that have 13 or 14 amino acid residues.

[0601] - Membrane Permeability Specification 43 / 100 pages 46 CN 122094968 A

[0602] In a non-limiting embodiment, the library of this disclosure contains cyclic peptide compounds with relatively high membrane permeability, and peptide compounds with even higher membrane permeability can be readily obtained by further derivatizing cyclic peptide compounds. Furthermore, when screening using the library of this disclosure, cyclic peptide compounds that are hit as compounds capable of specifically binding to target molecules may have relatively high membrane permeability. As used herein, the term "high membrane permeability" means a membrane permeability coefficient (Papp) of 4.0 × 10⁻⁷ cm / s or greater, preferably 8.0 × 10⁻⁷ cm / s or greater, or 9.0 × 10⁻⁷ cm / s or greater, more preferably 1.0 × 10⁻⁶ cm / s or greater, more preferably 3.0 × 10⁻⁶ cm / s or greater, and most preferably 5.0 × 10⁻⁶ cm / s or greater. In a non-limiting embodiment, the library of this disclosure may be a library for identifying orally administered compounds or their precursors. "Orally administered" herein means a membrane permeability coefficient (Papp) of 0.4 × 10⁻⁶ cm / s or greater, preferably 1.0 × 10⁻⁶ cm / s or greater, and more preferably 5.0 × 10⁻⁶ cm / s or greater. As used herein, the term "precursor" refers to a compound prior to chemical modifications for optimization, etc., and examples include hit compounds from panning prior to any optimization.

[0603] - Hit rate of peptide compounds capable of specifically binding to target molecules

[0604] In a non-limiting embodiment, the library of this disclosure can be used to identify compounds capable of specifically binding to target molecules. In one embodiment,Compared to conventional libraries, the libraries of this disclosure contain a greater number of amino acids constituting peptide compounds. This increases the diversity of amino acids and results in a higher hit rate of peptide compounds capable of specifically binding to target molecules. That is, by using the libraries of this disclosure, not only can peptide compounds with high binding affinity to target molecules be obtained, but also a large number of peptide compounds capable of specifically binding to target molecules can be obtained. Therefore, desired compounds can be selected from a large number of selected peptide compounds, taking into account other factors such as metabolic stability and membrane permeability. As used herein, the term "hit rate" refers to the proportion of the number of variations of the number of compounds obtained by panning the number of variations of compounds contained in the library. The number of variations of compounds contained in the library may be a theoretical figure.

[0605] In a non-limiting embodiment, peptide compounds that are not present in the library as cyclic peptide compounds for obtaining hit compounds (e.g., peptide compounds that obviously do not have the ability to bind to target molecules before panning) are not considered as peptide compounds constituting the library of this disclosure.

[0606] By using the cyclic peptide compounds enriched by panning, amino acid groups that may contribute to binding to target molecules can be identified by analyzing the frequency of occurrence of amino acids.

[0607] - Display Library

[0608] A display library is a library in which peptides (which are phenotypes) and RNA or DNA (which are genotypes) encoding the peptides are associated. This library can be used to identify peptide compounds that can specifically bind to target molecules. For example, peptides that bind to a desired target can be enriched by contacting the library with the desired immobilized target and washing away molecules that do not bind to the target (panning method). By analyzing the genetic information associated with the peptides selected through this process, the peptide sequences that bind to the target can be clarified. For example, using the method of the antibiotic puromycin, an analog of aminoacyl-tRNA, which nonspecifically binds to proteins during ribosomal mRNA translation expansion,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 protein on the ribosome in vitro. Nemoto N, Miyamoto‑Sato E, Husimi Y, Yanagawa H.).

[0609] When a linker 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 this mRNA is translated into a protein in a cell-free translation system, puromycin is mistakenly incorporated into the protein as an amino acid by ribosomes, and the mRNA and protein encoded thereby are linked to produce a library in which the mRNA and its product are associated. High efficiency can be achieved in this process because transformation (such as with E. coli) is not involved, and large display libraries can be constructed. The sequence of the bound protein can be identified by synthesizing cDNA from the mRNA, the mRNA being labeled to enriched and selected molecules containing genetic information, the cDNA being subjected to PCR amplification, and the nucleotide sequence being analyzed.

[0610] Examples of known display libraries utilizing cell-free translation systems include, in addition to mRNA display,cDNA display is a method of displaying cDNA libraries encoding peptides and puromycin complexes (Nucleic Acids Res. 2009;37(16):e108. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N.), utilizing ribosome display of relatively stable ribosome complexes 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.), which is a covalent display.Among them, the phage endonuclease P2A forms a covalent bond with DNA (Nucleic Acids Res. 2005;33:e10. Covalent antibody display--an in vitro antibody-DNA library selection system. Reiersen H, Lobersli I, Loset GA, Hvattum E, Simonsen B, Stacy JE, McGregor D, Fitzgerald K, Welschof M, Brekke OH, Marvik OJ.) and the CIS display, which utilizes the binding of the replication initiation protein RepA of microbial plasmids to the replication origin ori (Proc Natl Acad Sci USA. 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.). In vitro compartmentalization is also known, in which the transcription-translation system is encapsulated in an oil-in-water emulsion or liposome for each DNA molecule constituting the DNA library and the translation reaction is carried out (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.). Where appropriate, known methods can be used to apply the above methods.

[0611] - Nucleic acid molecules

[0612] The term "nucleic acid" in this invention may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotide derivatives having artificial bases, and peptide nucleic acids (PNA). The nucleic acid in this invention may be any of these nucleic acids or mixtures thereof, as long as the genetic information of interest is preserved. That is, the nucleic acids of this invention also include DNA-RNA hybrid nucleotides or chimeric nucleic acids,Different nucleic acids, such as DNA and RNA, are linked in a single strand.

[0613] The peptide compounds contained in the library can be synthesized from the template nucleic acid through translation. One non-limiting embodiment of this disclosure is a nucleic acid library used as a template. Examples of nucleic acid libraries include mRNA libraries and DNA libraries. Nucleic acid libraries can be obtained by adding bases to positions where amino acid residues are not fixed on the peptide sequence and synthesizing them. For example, nucleic acid libraries can be synthesized as a mixture of A, T, G, C in a DNA library and A, U, G, C in an RNA library (the number of which is 3 times that of 4 bases (N)) or a mixture of two bases, such as the first and second letters of a codon being N and the third letter being W, M, K, and S. When the types of amino acids introduced are reduced to 16 or less, there is a method to set the third letter base to one type of base. Furthermore, codon units corresponding to the three letters of a codon are prepared. The frequency of occurrence of amino acid residues can be freely adjusted by mixing in any proportion and used in synthesis. Each nucleic acid in the nucleic acid library can be linked to puromycin at its 3' end. By translating the nucleic acid library with puromycin linked to its 3' end, a peptide compound encoded by the nucleic acid is synthesized, and the resulting peptide compound is linked to the corresponding nucleic acid to synthesize a display library.

[0614] Libraries of corresponding cyclic peptides can be synthesized by translating these nucleic acid libraries using a cell-free translation system. When using a cell-free translation system, it is preferable to include a spacer sequence encoding the downstream of the nucleic acid of interest. Examples of spacer sequences include, but are not limited to, sequences containing glycine and serine. It is also preferred that the linker formed by polymers of RNA, DNA, hexaethylene glycol (spc18), etc. (e.g., five polymers) includes a compound incorporated into the peptide during ribosomal translation between the nucleic acid library and the nucleic acid library, such as puromycin or a derivative thereof.

[0615] - Screening Method

[0616] In a non-limiting embodiment, peptide compounds capable of specifically binding to a target molecule can be selected by screening using a library of the present disclosure. The screening method of the present disclosure is not particularly limited, and, for example, an mRNA display method can be used.

[0617] In a non-limiting embodiment, the screening method of the present disclosure involves contacting a library of cyclic peptide compounds of the present disclosure with a target molecule and washing away peptide compounds that do not bind to the target molecule, thereby enriching peptide compounds that bind to the target molecule (panning). In one embodiment, the amino acid sequence of the bound peptide can be identified by synthesizing cDNA from mRNA, which is a tag containing nucleotide sequence information of the peptide compounds selected in this manner, subjecting it to PCR amplification and nucleotide sequence analysis. In one embodiment,An mRNA library can be obtained by transcribing the amplified cDNA described above. Using this mRNA library as a template, a peptide compound library can be generated again. This library is rich in peptide compounds that can bind to target molecules, allowing for further enrichment of peptide compounds that can specifically bind to target molecules by panning again using this library. By repeating this process multiple times, the desired peptide compounds can be further enriched. In one embodiment, amino acid sequences can be identified based on the nucleotide sequence information contained in the peptide compounds enriched in this manner, and peptide compounds that can specifically bind to target molecules can be generated. In one embodiment, the screening method according to this disclosure can be performed in vitro.

[0618] In a non-limiting embodiment, the peptide compounds obtained by the screening method according to this disclosure can be optimized by known methods such as chemical modification. As used herein, the term "optimization" means chemically modifying the structure of each amino acid in the compounds obtained by screening, etc., to produce more drug-like peptide compounds, peptide compounds with stronger activity against pharmacological targets, and / or peptide compounds with reduced toxicity.

[0619] In a non-limiting embodiment, the screening method of this disclosure includes the following steps:

[0620] (a) contacting a peptide compound contained in the library of this disclosure with a target molecule; and

[0621] (b) selecting peptide compounds capable of binding to the target molecule.

[0622] In one embodiment, the screening method of this disclosure can enrich peptide compounds that specifically bind to the target molecule by repeating steps (a) and (b) above two or more times (cycles). In this case, in step (a), starting from the second time, a library of peptide compounds selected in the previous cycle can be used. This allows for an increase in the enrichment rate of the target peptide compound in each additional cycle. The number of repetitions can be, for example, 2 or more cycles, 3 or more cycles, 4 or more cycles, 5 or more cycles, 6 or more cycles, 7 or more cycles, 8 or more cycles, 9 or more cycles, or 10 or more cycles. By repeating the cycles, peptide compounds that bind to the target can be enriched.

[0623] In a non-limiting embodiment, the screening method of this disclosure may select a peptide compound that binds to the target molecule as the drug candidate compound in step (b) above.

[0624] In a non-limiting embodiment,The screening method of this disclosure further includes the following steps:

[0625] (c) chemically synthesizing a peptide compound that can be used as a candidate; and

[0626] (d) using Caco-2 cells to measure the membrane permeability of the chemically synthesized peptide compound.

[0627] - Target Molecule

[0628] There are no particular limitations on the target molecules used in the screening method of this disclosure, and examples include proteins, peptides, nucleic acids, sugars, lipids, etc., but proteins are preferred as target molecules. In one embodiment, according to the screening method of this disclosure, peptide compounds that inhibit protein-protein interactions (PPIs) can be obtained. While not intended to be bound by any specific theory, it is believed that sites called “hot spots” exist on the surfaces of the two proteins involved in a PPI, which enhance the binding strength of the PPI. Examples of PPIs include hydrophobic interactions, electrostatic interactions, van der Waals forces, hydrogen bonds, photocrosslinking, and salt bridges. Through such PPIs, protein phosphorylation, conformational changes, or localization, as well as the formation of protein-protein complexes (e.g., multi-subunit formation), can occur, which may involve protein modification, transport, folding alteration, signal transduction, etc. While not intended to be bound by any specific theory, it is believed that the side chains of the amino acids constituting the peptides contained in the library of this disclosure can inhibit PPIs by entering this site. In other words, it is believed that peptide compounds that inhibit PPIs can be obtained regardless of the type of target molecule, according to the library and screening method of this disclosure.

[0629] In one embodiment, when the library used in the screening method of this disclosure contains a cyclic peptide compound linked to a nucleic acid (cyclic peptide-nucleic acid complex), the actual expected drug candidate compound may consist only of the portion other than the nucleic acid and the nucleic acid linker (e.g., the cyclic portion). Target molecules can be selected based on the presence or absence or strength of the interaction with the cyclic peptide portion. In the cyclic peptide-nucleic acid complex, when the nucleic acid portion contains a nucleotide sequence encoding the cyclic peptide portion, the nucleic acid portion can facilitate the understanding of the chemical structure and synthesis of the cyclic peptide portion and help improve screening efficiency.

[0630] In vivo target molecules are not particularly limited to their location. In one embodiment, according to the library and screening method of this disclosure, cyclic peptide compounds with high membrane permeability, or cyclic peptide compounds whose membrane permeability is easily enhanced by derivatization, can be obtained, thereby allowing targeting of intracellular proteins.

[0631] In a non-limiting embodiment, the target molecules used in the screening method of this disclosure are used by immobilization on a support. The support is not particularly limited as long as the target molecule can be immobilized, but examples include beads or resins. Target molecules can be immobilized on a support by known methods.

[0632] The target molecules of the screening method of this disclosure are not particularly limited. The library of this disclosure includes peptide compounds capable of specifically binding to a variety of target molecules, in one embodiment,This peptide compound enables drug discovery for thorny targets considered difficult to drug develop.

[0633] - Production method for peptide compound and library

[0634] In one embodiment, the cyclic peptide compound of the present invention and the library containing such cyclic peptide compound can be produced, for example, by chemical synthesis, translation-based synthesis or a combination thereof.

[0635] - Chemical synthesis method for peptide compound

[0636] Examples of chemical synthesis methods for peptide compounds of the present disclosure include liquid-phase synthesis, solid-phase synthesis using Fmoc synthesis or Boc synthesis, and combinations thereof. In Fmoc synthesis, an amino acid whose main chain amino group is protected with an Fmoc group, an amino acid whose side chain functional group is protected by a protecting group (not cleaved by a base such as piperidine if needed), and an amino acid whose main chain carboxylic acid is not protected are used as the base unit. As for the base unit, there are no specific limitations as long as they are combinations of amino and carboxylic acid groups with Fmoc protection. For example, a dipeptide can be used as the base unit. The base unit placed at the N-terminus can be a unit other than an Fmoc amino acid. For example, it can be a Boc amino acid, or it can be a carboxylic acid analog without an amino group, as described on page 47 / 100 of CN 122094968 A. The main-chain carboxylic acid group is fixed to the solid phase through a chemical reaction with the functional groups of the solid-phase support. Next, the Fmoc group is deprotected using a base such as piperidine or DBU, and the newly formed amino group is condensed with the protected amino acid of the carboxylic acid having the subsequently added base unit to form a peptide bond. Various combinations can be used in the condensation reaction, such as the combination of DIC and HOBt, the combination of DIC and HOAt, and the combination of HATU and DIPEA. By repeating the deprotection of the Fmoc group followed by the peptide bond formation reaction, the desired peptide sequence can be generated. Once the desired sequence is obtained, cleavage from the solid phase and deprotection of any side chain functional groups introduced as needed are performed. Structural transformation or cyclization of the peptide can also be performed before cleavage from the solid phase. Solid-phase cleavage and deprotection can be carried out under the same conditions (e.g., 90:10 TFA / H2O) or under separate conditions, as needed. In some cases, solid-phase cleavage can be performed with a weak acid such as 1% TFA, while in others, Pd can be used as the protecting group, taking advantage of the orthogonality of the two chemical reactions. During or at the end of these processes, steps (such as cyclization) can also be performed. For example, a side-chain carboxylic acid can be condensed with an N-terminal main-chain amino group, or a side-chain amino group can be condensed with a C-terminal main-chain carboxylic acid. In this case, a cyclization step is required between the C-terminal carboxylic acid and the side-chain carboxylic acid to be cyclized.Or the orthogonality of the reaction between the N-terminal main chain amino group or hydroxyl group and the side chain amino group to be cyclized, and as described above, the protecting group is selected with consideration of the orthogonality of the protecting group. The reaction product obtained in this way can be purified using a reverse phase column or a molecular sieve column. Details of these are described in sources such as the “Solid Phase Synthesis Handbook” published by Merck KGaA on May 1, 2002.

[0637] - Translation-based synthetic methods for peptide compounds

[0638] The peptide compounds of this disclosure can be synthesized by translation-based synthetic methods. In this disclosure, “translation-based synthetic methods” refers to methods for synthesizing peptide compounds by recognizing the nucleotide sequence of mRNA with tRNA, rather than methods for synthesizing peptide compounds solely by organic synthesis techniques. Examples of translation-based synthetic methods for peptide compounds include methods using cell-free translation systems, which include synthetic methods using reconstructed cell-free translation systems. Reconstructed cell-free translation systems are preferred because they allow the exclusion of undesirable factors.

[0639] In one aspect, this disclosure provides a translation-based synthesis method for peptide compounds containing at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids described in (A).

[0640] (A) Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0641] This translation-based synthesis method may include, but is not limited to, the following (i) and (ii):

[0642] (i) the step of preparing tRNA that binds to at least one, two or more, or three or more amino acids selected from the group consisting of amino acids described in (A); and

[0643] (ii) the step of translating a nucleic acid containing at least one anticodon corresponding to the tRNA in a cell-free translation system to obtain a peptide compound.

[0644] In a non-limiting embodiment, the translation-based synthesis method of this disclosure may be a translation-based synthesis method for cyclic peptide compounds.

[0645] - Cell-free translation system

[0646] As used herein, the term "cell-free translation system" is not limited,As long as it contains ribosomes extracted from the west, a group of protein factors involved in translation, tRNA, amino acids, an energy source (such as ATP), and its regeneration system, it can be combined to translate mRNA into proteins. Systems in which translation-based synthesis is carried out can be included in the “cell-free translation system” described herein. The cell-free translation system described herein may contain nucleic acids used as templates for translated peptides, and may include initiation factors, elongation factors, dissociation factors, aminoacyl-tRNA synthetases, etc. These factors can be obtained by purification from extracts from various cells. Examples of cells used for purifying factors can include prokaryotic and eukaryotic cells. Examples of prokaryotic cells can include Escherichia coli cells, extreme thermophilic cells, or Bacillus subtilis cells. As eukaryotic cells, cells made from yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, or animal cells are known. As tRNA and aminoacyl-tRNA synthetases (ARS), not only naturally occurring tRNAs can be used, but also artificial tRNAs and artificial aminoacyl-tRNA synthetases that recognize non-natural amino acids can be used. By using artificial tRNAs and artificial aminoacyl-tRNA synthetases, it is possible to synthesize peptides in which non-natural amino acids are introduced at specific sites. If desired, RNA polymerases such as T7 RNA polymerase can be added to the cell-free translation system for transcription from template DNA.

[0647] PURESYSTEM® (manufactured by BioComber Co., Ltd., Japan) is a reconstructed cell-free translation system in which protein factors, energy regeneration system enzymes and ribosomes required for translation in E. coli are extracted and purified and mixed with tRNA, amino acids, ATP, GTP, etc. PURESYSTEM(R) not only has a low impurity content, but also allows for the easy preparation of systems that do not contain protein factors or amino acids that need to be eliminated, because it is a reconstituted system ((i) Nat Biotechnol. 2001; 19: 751-5. Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. (ii) Methods Mol Biol. 2010; 607: 11-21. PURE technology. Shimizu Y, Ueda T.).

[0648] For example,Numerous methods have been reported for using stop codons as codons to introduce non-natural amino acids. However, by using the aforementioned PURESYSTEM, a synthetic system excluding natural amino acids and ARS can be constructed. This allows non-natural amino acids to be associated with codons encoding the natural amino acid to be removed (J Am Chem Soc. 2005; 127: 11727–35. Ribosomal synthesis of non-natural peptides. Josephson K, Hartman MC, Szostak JW.). Furthermore, by decoding the degeneracy of the codons, non-natural amino acids can be added without excluding natural amino acids (Kwon I et al. Breaking the degeneracy of the genetic code. J Am Chem Soc. 2003, 125, 7512–3.). Peptides containing N-methyl amino acids can be synthesized in ribosomes using cell-free translation systems such as PURESYSTEM.

[0649] More specifically, translation-based synthesis can be performed by adding mRNA to a known cell-free translation system like PURESYSTEM, which is a protein factor appropriately selected from those necessary for E. coli translation (selected from methionyl-tRNA convertase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (selected from AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS, GluRS, GlyRS, HisRS, IleRS, LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, ValRS, if desired)), ribosomes, amino acids, creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase, E. coli-derived tRNA, creatine phosphate, potassium glutamate, HEPES-KOH A mixture of components including pH 7.6, magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, and UTP. When T7 RNA polymerase is added, transcription and translation from template DNA containing a T7 promoter can also be performed in a coupled manner. Furthermore, peptide compounds containing non-natural amino acids can be added to the system by adding a desired set of aminoacyl-tRNAs or a set of non-natural amino acids permitted by aminoacyl-tRNA synthetase (ARS) (e.g., ...).F-Tyr can be obtained through translation-based synthesis (Kawakami T et al., Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J Am Chem Soc. 2008, 130, 16861-3.; Kawakami T et al., Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol. 2009, 5, 888-90.). Furthermore, peptide compounds encoding non-natural amino acids can also be obtained through translation-based synthesis by adding a modified ARS (instead of a natural ARS or other than a natural ARS) to the system, and by adding a set of non-natural amino acids to the system. Alternatively, mutants of ribosomes and EF-Tu, etc., can be used to increase the efficiency of translational introduction of non-natural amino acids (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 with phosphoserine. Science 2011, 333, 1151-4.).

[0650] In a non-limiting embodiment, the cell-free translation system of this disclosure can be a translation system for producing peptide compounds, preferably a translation system for producing cyclic peptide compounds.

[0651] - tRNA

[0652] In the process of incorporating non-natural amino acids into peptides through translation,Aminoacylation of orthogonal and ribosome-integrated tRNA ((i) Biochemistry 2003; 42: 9598-608. Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. Anderson JC, Schultz PG.; (ii) Chem Biol. 2003; 10: 1077-84. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code. Murakami H, Kourouklis D, Suga H.). The following five methods can be used as methods for aminoacylation of tRNA.

[0653] In the cell, aminoacyl-tRNA synthetase (ARS) is provided individually for each amino acid as the enzyme for aminoacylation of tRNA. Therefore, as a first method, a method that allows certain ARS such as N-MeHis can be used.And methods for preparing and utilizing modified aminoacyl-tRNA synthetases that allow non-natural amino acids (i) Proc. Natl. Acad . Sci. US A. 2002; 99: 9715-20. An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of a non-natural amino acid into proteins in eukaryotic translation and 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, Yokoyama S. (ii) Science 2003; 301: 964-7. An expanded eukaryotic genetic code. Chin JW , Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG.Chin, JW. (iii) Proc.Natl.Acad .Sci. US A. 2006; 103: 4356-61. Enzymatic aminoacylation of tRNA with non-natural amino acids. Hartman MC, Josephson K, Szostak JW.). Secondly, a method can be used to aminoacylate tRNA and then chemically modify the amino acids in the test tube (J Am Chem Soc. 2008; 130: 6131-6. Ribosomal synthesis of N-methyl peptides. Subtelny AO , Hartman MC, Szostak JW.). Thirdly, tRNA can be bound to obtain aminoacyl-tRNA.The CA is removed from the CCA sequence at the 3' end to form 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 produces active esters of various non-natural amino acids carried on tRNA (J Am Chem Soc. 2002; 124: 6834-5. Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme. Murakami H, Bonzagni NJ, Suga H.). Fourth, the method of ultrasonically mixing tRNA and amino acid active esters in cationic micelles can also be used (Chem Commun (Camb). 2005; (34): 4321-3. Simple and quick chemical aminoacylation of tRNA in cationic micelle solution under ultrasonic agitation. Hashimoto N, Ninomiya K, Endo T, Sisido M.). Fifth, it is also possible to perform aminoacylation by adding an amino acid active ester that is complementary to the 3' end of tRNA and binds to PNA to tRNA (J Am Chem Soc. 2004; 126: 15984-9. In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid. Ninomiya K, Minohata T, Nishimura M, Sisido M.).

[0654] More specifically,Aminoacyl-tRNAs can be produced using the following methods. RNA can be synthesized by preparing template DNA encoding the desired tRNA sequence and setting a T7, T3, or SP6 promoter upstream, followed by transcription using an RNA polymerase suitable for the promoter (such as T7 RNA polymerase and T3) or SP6 RNA polymerase. Alternatively, tRNA can be extracted and purified from cells, and the resulting tRNA of interest can be extracted using a probe with a complementary sequence to the tRNA sequence. Cells transformed with an expression vector of the tRNA of interest can then be used as the source. RNA with the sequence of interest can also be synthesized chemically. For example, aminoacyl-tRNAs can be obtained by binding tRNA in which CA is removed from the 3' end of the CCA sequence obtained in this manner to an aminoacylated pdCpA or pCpA (pdCpA method, pCpA method) prepared separately using RNA ligase. tRNAs can be used to produce peptide compounds. Alternatively, full-length tRNA is prepared and aminoacylated by a flexizyme, a ribozyme that produces an active ester of the various non-natural amino acids carried on the tRNA. Aminoacyl tRNA can also be produced using natural ARS or modified ARS thereof, although this is not intended to be limiting. When using natural ARS or modified ARS thereof, it is not necessary to have a large amount of previously prepared aminoacyl tRNA in the translation system, as aminoacyl tRNA previously consumed in the translation system can be regenerated from natural ARS or modified ARS thereof. Such modified ARS are described in WO 2016 / 148044. These methods for producing aminoacyl tRNA can also be used in appropriate combinations.

[0655] - Methods for Cycling Peptide Moieties

[0656] The cyclic peptide compounds of this disclosure have cyclic moieties. There are no particular limitations on the cyclization methods of the peptide compounds, but for example, the cyclization reaction can be carried out after the peptide compound is synthesized by chemical synthesis or translation-based synthesis. Examples of cyclization include cyclization using amide bonds, carbon-carbon bonds, thioether bonds, disulfide bonds, ester bonds, thioester bonds, lactam bonds, bonds via triazole structures, or bonds via fluorophore structures. The synthetic steps of peptide compounds and the cyclization reaction steps can be performed individually or sequentially. Cyclization can be carried out using methods known to those skilled in the art.Methods such as those described in WO 2013 / 100132, WO 2008 / 117833, or WO 2012 / 074129.

[0657] The cyclization moiety may be, but is not limited to, 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, and two or more of these bonds may be used in combination. When cyclizing a peptide-nucleic acid complex in which a nucleic acid is attached to the C-terminus, it may involve a bond between the N-terminus of the peptide and a side chain of another amino acid residue or between side chains of amino acid residues.

[0658] In a non-limiting embodiment, the cyclic peptide compound of this disclosure may be prepared by the following method. Examples include a production method comprising the following steps:

[0659] (1) a step of synthesizing a noncyclic peptide compound consisting of amino acid residues or amino acid residues and an N-terminal carboxylic acid analog by translating from a nucleic acid encoding a peptide compound, wherein the noncyclic peptide compound comprises an amino acid residue having a reactive site on one side chain at the C-terminus and an amino acid residue or an N-terminal carboxylic acid analog having another reactive site at the N-terminus; and specification 51 / 100 pages 54 CN 122094968 A

[0660] (2) combining the reactive site of the amino acid residue or N-terminal carboxylic acid analog at the N-terminus with the reactive site of the amino acid residue at the side chain at the C-terminus to form an amide bond, a carbon-carbon bond, or a thioether bond.

[0661] These steps (1) and (2) may be performed independently or sequentially.

[0662] A non-limiting embodiment of the method of cyclizing peptide compounds via amide bonds is a method of translating a peptide compound having a cysteine ​​or cysteine ​​analog at the N-terminus and an active ester on an amino acid side chain at the C-terminus, and cyclizing it using natural chemical linkages. Translation-based synthetic methods for peptide compounds having a cysteine ​​or cysteine ​​analog at the N-terminus as a precursor of such cyclic peptide compounds are not particularly limited.

[0663] In one embodiment, to synthesize a peptide compound having an amino acid other than Met or Nle at the N-terminus, three methods can be used, for example.

[0664] The first method is a method of using an enzyme to cleave the N-terminal amino acid or N-terminal peptide of a peptide initiated by formylmethionine or Met. Examples of enzymes include peptide protease (PDF) and / or methionine aminopeptidase (MAP).

[0665] The second method is a method using a reconstructed cell-free translation system, removing Met from the system beforehand, and placing the amino acid desired to be located at the N-terminus from the second residue at the N-terminus.Translation-based synthesis is performed by reading the start codon Met. This method is defined as the initiation readthrough (iRT) method.

[0666] A third method is a translation-based synthesis method by adding aminoacyl tRNA to a recombinant cell-free translation system that does not contain Met or initiator methionine tRNA, the aminoacyl tRNA being prepared by aminoacylation of an amino acid that is desired to act as an initiator tRNA located at the N-terminus. This method is defined as the initiation inhibition (iSP) method.

[0667] In a non-limiting embodiment, the C-terminal portion of the cyclic peptide compound of this disclosure may be chemically modified instead of being retained as a carboxylic acid. For example, the carboxylic acid portion may be reacted with piperidine, etc., to convert to piperidine amide, etc.

[0668] [Examples]

[0669] The invention will be described in more detail based on the following examples, which should not be construed as limiting the invention. All starting materials and reagents were obtained from commercial suppliers or synthesized according to known methods.

[0670] In the examples, the following abbreviations are used.

[0671] AA: Ammonium acetate

[0672] DBU: 1,8-diazabicyclo[5.4.0]-7-undecene

[0673] DCE: 1,2-dichloroethane

[0674] DCM: Dichloromethane

[0675] DIC: N,N'-diisopropylcarbodiimide

[0676] DIPEA: N,N-diisopropylethylamine

[0677] DMF: N,N-dimethylformamide

[0678] DMSO: Dimethyl sulfoxide

[0679] Fmoc: 9-fluorenylmethyloxycarbonyl

[0680] HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate

[0681] HFIP: 1,1,1,3,3,3-Hexafluoro-2-propanol

[0682] HOAt: 1-Hydroxy-7-azabenzotriazole

[0683] NMP: N-Methyl-2-pyrrolidone

[0684] TBME: tert-butyl methyl ether

[0685] TFE: 2,2,2-trifluoroethanol

[0686] TIPS: Triisopropylsilane (Instruction manual 52 / 100 pages 55 CN 122094968 A

[0687] Trt: Triphenylmethyl

[0688] oxyma: Ethyl cyano(hydroxyimino)cyanoacetate

[0689] The reaction solvents used for peptide synthesis and solid-phase synthesis are reaction solvents used for peptide synthesis (purchased from KANTO CHEMICAL CO., INC., FUJIFILM Wako Pure Chemical Corporation or WATANABE CHEMICAL INDUSTRIES, LTD.). For example,DCM, DMF, DMSO, and DBU (2% DMF solution) were used. In reactions where water was not used as a solvent, dehydrated or anhydrous solvents (purchased from KANTO CHEMICAL CO., INC., FUJIFILM Wako Pure Chemical Corporation, etc.) were used.

[0690] Example 1. Synthesis of compounds such as amino acids for peptide synthesis

[0691] For the peptide synthesis described herein, the amino acids and amino acid supporting resins shown in Tables 1, 2, and 3 were used.

[0692] Example 1-1. Purchase and use of Fmoc-amino acids as is

[0693] The Fmoc-amino acids shown in Table 1 were purchased from commercial suppliers. As used herein, when -OH is described other than the abbreviations for amino acids in Table 1, it means that the C-terminus of the amino acid is a carboxyl group. For example, Gly-OH means that the C-terminus of glycine is a carboxyl group, and Fmoc-Gly-OH means that the N-terminal amino group of glycine is protected by the Fmoc group.And the C-terminus is a carboxyl group.

[0694] [Table 1]

[0695] Specification 53 / 100 pages 56 CN 122094968 A Specification 54 / 100 pages 57 CN 122094968 A

[0696] Examples 1-2. Synthesis of Fmoc-amino acids

[0697] The Fmoc-amino acids shown in Table 2 were synthesized according to the scheme shown below.

[0698] [Table 2]

[0699]

[0700] Compound aa001

[0701] Synthesis of (3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-piperidin-1-ylbutyric acid (Fmoc-Asp-pip)

[0702] [Formula 4]

[0703]

[0704] Compound aa001 was synthesized according to the method described in WO 2013 / 100132.

[0705] Compound aa002

[0706] Synthesis of (3S)-3-[9H-fluorene-9-ylmethoxycarbonyl(methyl)amino]-4-oxo-4-piperidin-1-ylbutyric acid (Fmoc-MeAsp-pip)

[0707] [Formula 5] Specification 55 / 100 pages 58 CN 122094968 A

[0708]

[0709] Compound aa002 was synthesized according to the method described in WO 2018 / 225864.

[0710] Compound aa003

[0711] Synthesis of (2S,3R)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-(oxane-2-yloxy)butyric acid (Fmoc-Thr (THP)-OH)

[0712] [Formula 6]

[0713]

[0714] Compound aa003 was synthesized according to the method described in WO 2018 / 225864.

[0715] Examples 1-3. Synthesis of amino acid-supporting resin

[0716] [Formula 7]

[0717]

[0718] As used herein, when a compound is combined with a polymer or resin, the polymer or resin moiety can be represented by [formula]. To clarify the reaction sites of the resin moiety, the chemical structure of the reaction sites can be shown as linked to [the resin moiety]. For example, in the structure Fmoc-Asp(O-Trt (2-Cl)-resin)-pip, the 2-chlorotriphenylmethyl group of the resin is bonded to the carboxyl group on the side chain of Asp via an ester bond. The term "pip" refers to piperidine, and in the above structure, the C-terminal carboxyl group forms an amide bond with piperidine. 2-chlorotriphenylmethyl chloride resin (1.25-1.60 mmol / g, 100 to 200 mesh),1% DVB was purchased from WATANABE CHEMICAL INDUSTRIES, LTD. and SUNRESIN Co. Ltd.

[0719] The Fmoc-amino acid-supporting resin in Table 3 was synthesized according to the scheme shown below.

[0720] [Table 3] Specification 56 / 100 pages 59 CN 122094968 A

[0721]

[0722] Compound rs001:

[0723] Synthesis of (3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-piperidin-1-ylbutyric acid-2-chlorotriphenylmethyl resin (Fmoc-Asp(O-Trt(2-Cl)-resin)-pip)

[0724] [Formula 8]

[0725]

[0726] 2-chlorotriphenylmethyl chloride resin (1.60 mmol / g, 50.0 g, 80.0 mmol) and DCM (676 mL) were added to a reaction vessel equipped with a filter, and the mixture was shaken at room temperature for 10 minutes. Nitrogen pressure was applied to remove DCM, and then a solution obtained by mixing compound aa001 (16.9 g, 40.0 mmol), methanol (12.9 mL, 320 mmol), DCM (676 mL), and DIPEA (33.4 mL, 192 mmol) was added to the reaction vessel and shaken at room temperature for 60 min. Nitrogen pressure was applied to remove the reaction solution, and then a mixture of methanol (99.7 mL, 2.46 mmol), DCM (676 mL), and DIPEA (33.4 mL, 192 mmol) was added to the reaction vessel and shaken at room temperature for 90 min. Nitrogen pressure was applied to remove the reaction solution, and then DCM (676 mL) was added. The reaction vessel was shaken for 10 min, and then nitrogen pressure was applied to remove the reaction solution. The resin washing operation with DCM was repeated twice, and the resulting resin was dried overnight under reduced pressure to obtain 61.8 g of compound rs001 (support mass 0.452 mmol / g).

[0727] Synthesis of compound rs002, (3R)-3-(9H-fluorene-9-ylmethoxycarbonylamino)butyric acid-2-chlorotriphenylmethyl resin (Fmoc-D-3-Abu-O-Trt (2-Cl) resin)

[0728] [Formula 9]

[0729]

[0730] 2-chlorotriphenylmethyl chloride resin (1.44 mmol / g, 200 g, 289 mmol) and Fmoc-D-3-Abu-OH (47.0 g, 144 mmol) were used as starting materials.230 g of compound rs002 (support amount 0.494 mmol / g) was obtained in the same manner as the synthesis of compound rs001.

[0731] Synthesis of compound rs003, (3R)-3-(9H-fluorene-9-ylmethoxycarbonyl(methyl)amino)butyric acid-2-chlorotriphenylmethyl resin (Fmoc-D-3-MeAbu-O-Trt (2-Cl) resin)

[0732] [Formula 10]

[0733] ​​

[0734] Using 2-chlorotriphenylmethyl chloride resin (1.25 mmol / g, 200 g, 250 mmol) and Fmoc-D-3-MeAbu-OH (42.5 g, 125 mmol) as starting materials, 228 g of compound rs003 (supporting mass 0.495 mmol / g) was obtained in the same manner as the synthesis of compound rs001.

[0735] Synthesis of compound rs004, (3S)-3-(9H-fluorene-9-ylmethoxycarbonyl(methyl)amino)-4-oxo-4-piperidin-1-ylbutyric acid-2-chlorotriphenylmethyl resin (Fmoc-MeAsp(O-Trt(2-Cl)-resin)-pip)

[0736] [Formula 11]

[0737]

[0738] Using 2-chlorotriphenylmethyl chloride resin (1.44 mmol / g, 200 g, 289 mmol) and compound aa002 (82.2 g, containing 23 w% TBME, 145 mmol) as starting materials, 239 g of compound rs004 (supporting mass 0.509 mmol / g) was obtained in the same manner as the synthesis of compound rs001.

[0739] Example 2. Synthesis of Cyclic Peptide Compounds

[0740] According to the scheme shown in Figure 1, cyclic peptide compounds consisting of 13 or 14 amino acid residues were synthesized by peptide extension through solid-phase reaction, cleavage, cyclization, side chain deprotection, and purification.

[0741] Using a peptide synthesizer (Multipep RS,Peptide extension of the solid-phase reaction described in this example was performed by the Fmoc method (manufactured by CEM Corporation (formerly Intavis AG)). Detailed operating procedures were performed according to the manual attached to the synthesizer.

[0742] The detailed synthesis conditions in this example are shown below.

[0743] Peptide extension by solid-phase reaction

[0744] Fmoc-protected amino acids constituting the peptide of interest (0.6 mol / L) and HOAt or oxyma (0.375 mol / L) as carboxyl group activators were dissolved in NMP to prepare solution 1. Additionally, DIC was mixed with DMF to a concentration of 10% (v / v) to prepare solution 2. A solid-phase reaction vessel with a filter (glass frit) was loaded with the resin (100 mg) supporting the Fmoc amino acids prepared in Examples 1 to 3 and placed in the peptide synthesizer. DCM (1.2 mL) was added, the mixture was allowed to stand for 30 minutes to allow the resin to swell, and then the solution was drained from the glass frit. Solutions 1 and 2 were placed in a peptide synthesizer, and the automatic synthesis was started using the peptide synthesizer.

[0745] A solution of DBU in DMF (2% v / v, 0.7 mL / column) was added to a solid-phase reaction vessel containing resin, and the Fmoc group removal reaction was carried out at room temperature. The mixture was allowed to react for 4.5 minutes in deprotection at the first residue and for 10 minutes in deprotection at the second and subsequent residues, and then the solution was drained from the glass frit. DMF (0.7 mL) was added to it, the mixture was allowed to stand for 5 minutes, and then the solution was drained from the glass frit. This numerical washing step was repeated more than three times. Subsequently, solution 1 (0.3 mL) was mixed with solution 2 (0.36 mL) in a mixing vial of the synthesizer, and then the mixture was added to the resin. The solid-phase reaction vessel was heated to 40°C and allowed to react for 2.5 hours to allow the amino groups on the resin to condense with the Fmoc-protected amino acids, and then the solution was drained from the glass frit. The resin was then washed three times with DMF (0.7 mL). The Fmoc group removal reaction, followed by the condensation reaction of the Fmoc amino acids, was performed as a cycle, and this cycle was repeated to extend the peptide on the resin surface. After peptide extension was complete, a DMF solution of DBU (2% v / v, 0.7 mL) was added to the solid-phase reaction vessel containing the resin, and the mixture was reacted at room temperature for 15 minutes to carry out the Fmoc group removal reaction.The solution was then drained from the glass frit. The obtained resin was washed four times with DMF (0.7 mL) and then four times with DCM (0.7 mL).

[0746] Resin cleavage

[0747] TFE / DCM (1 / 1, 2.0 mL) containing 0.75% (v / v) DIPEA was added to the obtained resin, and the mixture was reacted at room temperature for 2 hours to cleave the peptide chain with the resin. After the reaction, the solution in the tube was recovered from the glass frit. The operation of adding TFE / DCM (1 / 1 (v / v), 1.0 mL) to the remaining resin and recovering the solution from the glass frit was repeated twice. All the obtained cleavage solutions were mixed, DMF (4.0 mL) was mixed with it, and then the solvent was removed by distillation under reduced pressure using a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac.

[0748] Cycloning

[0749] The residue obtained by the above method was dissolved in a mixture of DMF (4.0 mL) and DCM (4.0 mL), and a solution of HATU in DMF (0.5 mol / L, 1.5 equivalents) and DIPEA (1.8 equivalents) was added. The mixture was stirred at room temperature for 2 hours to carry out a condensation cyclization reaction of the amino group of the N-terminal amino group and the carboxyl group as the part bound to the resin. The solvent was then removed by distillation under reduced pressure using a high-throughput centrifugal evaporator (HT-12) manufactured by Genevac. The equivalent number was calculated by multiplying the amount of peptide support on the resin used as a raw material (mmol / g) by the amount of resin used.

[0750] Deprotection

[0751] Deprotection was performed only on peptides containing Thr. For peptides without Thr, deprotection was omitted.

[0752] The Thr side chain was deprotected by dissolving the residue obtained by the above method in a solution of HFIP / TIPS / DCE (117 / 2.4 / 1 (v / v / v)) with tetramethylammonium hydrogen sulfate (0.10 mol / L, 4.0 mL) and stirring at room temperature for 1 hour. DIPEA (0.070 mL) was added, and the solvent was then removed by distillation under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12).

[0753] Purification

[0754] DMSO was added to the residue obtained by the above method, insoluble substances were removed by filtration, and then purified by preparative HPLC to obtain the desired cyclic peptide. A Waters automated purification system was used as the purification device.A YMC-Actus Triart C18 column (inner diameter: 20 mm and length: 100 mm) was used as the column, and an aqueous methanol-ammonium acetate solution (50 mmol / L) was used as the mobile phase.

[0755] Cyclic peptides consisting of 13 or 14 amino acid residues listed in Table 4 were synthesized according to the above synthesis conditions. In Table 4, the amino acid sequences are shown in order from N-terminus 1 to 14, and the precise mass, LC-MS analytical conditions (LCMS conditions), observed mass spectra (observed mass; m / z), ionic form, and retention time (Rt; min) of each compound are described. All peptides are cyclic peptides in which an amide bond is formed between the amino group at the N-terminus and the carboxyl group at the C-terminus (or the carboxyl group of the side chain when the C-terminus is Asp or MeAsp). The LCMS analytical conditions are shown in Table 5. The structural formula of each compound is shown in Table 15.

[0756] [Table 4]

[0757] Specification 60 / 100 pages 63 CN 122094968 A Specification 61 / 100 pages 64 CN 122094968 A Specification 62 / 100 pages 65 CN 122094968 A

[0758] [Table 5] Specification 63 / 100 pages 66 CN 122094968 A

[0759]

[0760] Example 3. Metabolic stability of cyclic peptide compounds

[0761] The metabolic stability of the cyclic peptide compounds composed of 13 or 14 amino acid residues synthesized in Example 2 was evaluated using a liver microsomal stability assay.

[0762] In the presence and absence of NADPH, mice liver microsomes (male, Corning Incorporated, USA) prepared to a concentration of 0.5 mg protein / mL using 100 mM phosphate buffer (pH 7.4) and 1 μM of the compound were subjected to metabolic reactions for 22, 44, or 66 minutes. Protein removal was performed using acetonitrile over time, and the remaining amount of the compound without alteration was analyzed using LCMS to calculate intrinsic hepatic clearance (CLh).(Inherent) (Table 6).

[0763] [Table 6] Specification 64 / 100 pages 67 CN 122094968 A

[0764] Specification 65 / 100 pages 68 CN 122094968 A Specification 66 / 100 pages 69 CN 122094968 A

[0765] This indicates that all tested cyclic peptide compounds consisting of 13 or 14 amino acid residues have high metabolic stability.

[0766] Example 4. Membrane permeability test of cyclic peptide compounds

[0767] The membrane permeability of the cyclic peptide compounds consisting of 13 or 14 amino acid residues synthesized in Example 2 was evaluated according to the method for measuring membrane permeability using Caco-2 cells disclosed in WO 2018 / 225864 as shown below.

[0768] Caco-2 cells and peptide compounds were pre-incubated for 20 to 24 hours at 37°C and 80 rpm using a mixed medium of FaSSIF containing 1% DMSO and DMEM as the top-side medium and DMEM as the bottom-side medium, with the culture medium at 5% CO2. The top-side and bottom-side mediums were then aspirated and washed. Peptide compounds were added to the top-side FaSSIF / HBSS buffer solution (1% DMSO) (pH 6.0) containing 1% DMSO, and to the bottom-side HBSS buffer solution (pH 7.4) containing 4% BSA. Membrane permeability measurements were then initiated. Each well was shaken at 37°C and 80 rpm at 5% CO2, and samples were collected from the bottom side 180 minutes after the start of shaking. The amount of peptide compounds in the samples was measured using LCMS, and the membrane permeability coefficient was calculated from the permeate volume (Table 7). When calculating the membrane permeability coefficient (Papp) from the permeate volume, the following expression can be used.

[0769] [Expression 2]

[0770]

[0771] dQ / dt represents the drug permeation rate (amount of drug appearing on the bottom side / unit time), C1(0) represents the concentration of drug added on the top side, and S represents the total surface area of ​​the cell.

[0772] [Table 7] Specification 67 / 100 pages 70 CN 122094968 A

[0773] Specification 68 / 100 pages 71 CN 122094968 A

[0774] It has been shown that the tested cyclic peptide compounds consisting of 13 or 14 amino acid residues include a range of compounds from those with high membrane permeability to those with low permeability.

[0775] Example 5. Analysis of various properties and membrane permeability of cyclic peptide compounds

[0776] As shown in Example 3,Since all the tested cyclic peptide compounds consisting of 13 or 14 amino acid residues were found to have high metabolic stability, we focused on membrane permeability as an indicator of drug-likeness, as shown in Example 4, and analyzed its relationship with various properties of the cyclic peptide compounds.

[0777] The membrane permeability and various properties values ​​for each cyclic peptide used in the analysis are shown in Table 8.

[0778] [Table 8] Specification 69 / 100 pages 72 CN 122094968 A

[0779] Specification 70 / 100 pages 73 CN 122094968 A Specification 71 / 100 pages 74 CN 122094968 A Specification 72 / 100 pages 75 CN 122094968 A Specification 73 / 100 pages 76 CN 122094968 A

[0780] In Table 8, total AA (total amino acids) represents the number of amino acid residues constituting each cyclic peptide, and the number of N-alkyl groups represents the number of N-alkylated amides in the main chain amide.

[0781] cLogP (partition coefficient calculated by computer) was calculated using Daylight version 4.95 of Daylight Chemical Information Systems, Inc. Total AB (total amide bonds) refers to the number of amide bonds in the main chain and side chains contained in each compound. For example, in the cyclic peptide shown in this example, it corresponds to one of the patterns listed in Table 9.

[0782] [Table 9]

[0783]

[0784] cLogP / total AB represents the value obtained by dividing cLogP by total AB, and it is used in this example as an indicator of the lipophilicity of the peptide compound.

[0785] Example 5-1. Analysis of N-alkyl number and membrane permeability 74 / 100 pages 77 CN 122094968 A

[0786] The relationship between the N-alkyl number of cyclic peptide compounds and membrane permeability was analyzed (Table 10).

[0787] [Table 10]

[0788]

[0789] In the case of compounds with an N-alkyl number of 7, 4 out of 36 compounds tested showed Papp ≥ 0.4 × 10⁻⁶ cm / s, but no compound showed Papp ≥ 1.0 × 10⁻⁶ cm / s. In the case of compounds with 8 N-alkyl groups, 19 out of 48 compounds tested showed a Papp ≥ 0.4 × 10⁻⁶ cm / s, and 7 of these compounds showed a Papp ≥ 1.0 × 10⁻⁶ cm / s. In the case of compounds with 9 N-alkyl groups,Of the 48 compounds tested, 17 showed a Papp ≥ 0.4 × 10⁻⁶ cm / s, and 4 of these compounds showed a Papp ≥ 1.0 × 10⁻⁶ cm / s. In the case of compounds with 10 N-alkyl groups, 13 of the 36 compounds tested showed a Papp ≥ 0.4 × 10⁻⁶ cm / s, and 4 of these compounds showed a Papp ≥ 1.0 × 10⁻⁶ cm / s.

[0790] Therefore, it can be seen that compounds with 7 N-alkyl groups can achieve a Papp ≥ 0.4 × 10⁻⁶ cm / s, and compounds with 8 or more N-alkyl groups can achieve a Papp ≥ 1.0 × 10⁻⁶ cm / s. Furthermore, it was found that a higher proportion of compounds with 8 or more N-alkyl groups achieved Papp ≥ 0.4 × 10⁻⁶ cm / s compared to compounds with 7 N-alkyl groups.

[0791] Example 5-2. Analysis of lipophilicity and membrane permeability

[0792] The relationship between the lipophilicity and membrane permeability of cyclic peptide compounds was analyzed (Table 11). In this example, cLogP / total AB was used as an indicator of lipophilicity.

[0793] [Table 11]

[0794]

[0795] In the case of compounds with cLogP / total AB < 1.0, 1 out of 20 compounds tested showed Papp ≥ 0.4 × 10⁻⁶ cm / s, but no compound showed Papp ≥ 1.0 × 10⁻⁶ cm / s. In the case of compounds with cLogP / total AB of 1.0 ≤ cLogP / total AB < 1.1, 14 out of 52 tested compounds showed Papp ≥ 0.4 × 10⁻⁶ cm / s, and one of these compounds showed Papp ≥ 1.0 × 10⁻⁶ cm / s. In the case of compounds with cLogP / total AB of 1.1 ≤ cLogP / total AB, 39 out of 96 tested compounds showed Papp ≥ 0.4 × 10⁻⁶ cm / s, and 14 of these compounds showed Papp ≥ 1.0 × 10⁻⁶ cm / s.

[0796] As can be seen from the above, it is difficult for compounds with cLogP / total AB < 1.0 to achieve Papp ≥ 0.4 × 10⁻⁶ cm / s.Compounds with 1.0 ≤ cLogP / total AB < 1.1 can achieve Papp ≥ 1.0 × 10⁻⁶ cm / s, and compounds with 1.1 ≤ cLogP / total AB can achieve Papp ≥ 1.0 × 10⁻⁶ cm / s. It is also shown that for compounds with 1.1 ≤ cLogP / total AB, the proportion of compounds achieving Papp ≥ 0.4 × 10⁻⁶ cm / s is higher than that for compounds with cLogP / total AB < 1.1.

[0797] Example 5-3. Analysis of MeLeu and Membrane Permeability

[0798] For each cyclic peptide compound, the relationship between the amount of MeLeu and membrane permeability was analyzed (Table 12). From Examples 5 to 2, it was found that compounds with cLogP / totalAB < 1.0 were unlikely to achieve Papp ≥ 0.4 × 10⁻⁶ cm / s, therefore the analysis was only performed on compounds with cLogP / totalAB ≥ 1.0.

[0799] [Table 12]

[0800]

[0801] In the case of compounds with one MeLeu, 5 out of 8 tested compounds showed Papp ≥ 0.4 × 10⁻⁶ cm / s; in the case of compounds with two MeLeu, 20 out of 75 tested compounds showed Papp ≥ 0.4 × 10⁻⁶ cm / s; and in the case of compounds with three MeLeu, 27 out of 63 tested compounds showed Papp ≥ 0.4 × 10⁻⁶ cm / s. On the other hand, in the case of compounds with four MeLeu, none of the four compounds tested showed Papp ≥ 1.0 × 10⁻⁶ cm / s.

[0802] Example 6. Mouse PK test of cyclic peptide compounds

[0803] Examples 3 to 5 revealed that having 8 or more N-alkyl groups and a cLogP / total AB of 1.1 or greater are conditions for cyclic peptide compounds consisting of 13 or 14 amino acid residues to exhibit particularly good drug-like properties. We evaluated the changes in plasma concentrations of four types of peptides that met these conditions after intravenous or oral administration to mice.

[0804] The compounds were administered intravenously to male mice (C57BL / 6J, 10 weeks old, purchased from Charles River Laboratories Japan, Inc.: 3 mice / group) at a dose of 1 mg / kg, or orally to male mice at a dose of 50 mg / kg.Blood was collected from the jugular vein over time within 24 hours after administration. The collected blood was dispensed into tubes pretreated with heparin as an anticoagulant. Plasma was separated from the blood by centrifugation, and plasma concentrations of the compound were measured using an LC / MS / MS apparatus (XEVO TQ-XS, manufactured by Waters Corporation) after deproteinization with acetonitrile. Based on the obtained plasma concentration-time curves, the following pharmacokinetic parameters (Tables 13 and 14) were calculated using non-compartmental analysis with Phoenix WinNonlin 6.1 analytical software (manufactured by Phalsight Corporation, USA).

[0805] AUCinf: Area under the plasma concentration-time curve (ng‧h / mL)

[0806] CL: Systemic clearance (mL / min / kg)

[0807] Vss: Steady-state volume of distribution (L / kg)

[0808] t1 / 2: Elimination half-life (hours)

[0809] Cmax: Maximum plasma concentration (ng / mL)

[0810] Tmax: Time to reach maximum plasma concentration (hours)

[0811] F: Bioavailability (%)

[0812] [Table 13]

[0813] Pharmacokinetic parameters for intravenous administration 76 / 100 pages 79 CN 122094968 A

[0814]

[0815] [Table 14]

[0816] Pharmacokinetic parameters for oral administration

[0817]

[0818] All test compounds showed low systemic clearance after intravenous administration (1.7 to 14). The concentration of the compound (mL / min / kg) indicates high metabolic stability. The bioavailability of the compound is as high as 40%, and the four peptides tested exhibit oral absorption suitable for development as oral administration agents.As expected.

[0819] [Table 15] Specification 77 / 100 pages 80 CN 122094968 A

[0820] Specification 78 / 100 pages 81 CN 122094968 A Specification 79 / 100 pages 82 CN 122094968 A Specification 80 / 100 pages 83 CN 122094968 A Specification 81 / 100 pages 84 CN 122094968 A Specification 82 / 100 pages 85 CN 122094968 A Specification 83 / 100 pages 86 CN 122094968 A Specification 84 / 100 pages 87 CN 122094968 A Specification 85 / 100 pages 88 CN 122094968 A Specification 86 / 100 pages 89 CN 122094968 Instruction Manual A, page 87 / 100, 90 CN 122094968; Instruction Manual A, page 88 / 100, 91 CN 122094968; Instruction Manual A, page 89 / 100, 92 CN 122094968; Instruction Manual A, page 90 / 100, 93 CN 122094968; Instruction Manual A, page 91 / 100, 94 CN 122094968; Instruction Manual A, page 92 / 100, 95 CN 122094968; Instruction Manual A, page 93 / 100, 96 CN 122094968; Instruction Manual A, page 94 / 100, 97 CN 122094968; Instruction Manual A, page 95 / 100, 98 CN 122094968; Instruction Manual A, page 96 / 100, 99 CN 122094968; Instruction Manual A, page 97 / 100, 100 CN 122094968 A Instruction Manual 98 / 100 pages 101 CN 122094968 A Instruction Manual 99 / 100 pages 102 CN 122094968 A Instruction Manual 100 / 100 pages 103 CN 122094968 A Figure 1 Instruction Manual Drawing 1 / 1 page 104 CN 122094968 A,

Claims

1. A cyclic peptide compound optionally linked to a nucleic acid, said compound comprising a cyclic moiety, wherein... (1) The cyclic portion consists of 13 or 14 amino acid residues. (2) Of the amino acid residues constituting the cyclic moiety, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues having the same side chain is 3 or fewer, and the number of native amino acid residues is 4 or fewer. (3) The ClogP / total amide bond (ClogP / total AB) ratio is 1.0 to 1.

8.

2. The cyclic peptide compound optionally linked to a nucleic acid according to claim 1, wherein the number of N-substituted amino acids constituting the cyclic moiety is 7 to 13.

3. The cyclic peptide compound optionally linked to a nucleic acid according to claim 1 or 2, wherein the N-substituted amino acid is an N-alkyl amino acid.

4. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 3, wherein the cyclic peptide compound has three or fewer hydrogen bond donors.

5. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 4, wherein the side chains of the amino acids constituting the cyclic portion are composed of at least one of the group consisting of alkyl groups, cycloalkyl groups, aralkyl groups, and amide groups, wherein any atom of the alkyl group and the nitrogen atom of the amino group of the amino acid can together form a ring.

6. The cyclic peptide compound optionally linked to a nucleic acid according to claim 5, wherein the alkyl group is a C1-C6 alkyl group optionally substituted with at least one group selected from the group consisting of: halogen atoms, hydroxyl groups, C3-C8 cycloalkyl groups, and C1-C6 alkoxy groups.

7. The cyclic peptide compound optionally linked to a nucleic acid according to claim 5 or 6, wherein the cycloalkyl group is a C3-C group optionally substituted with at least one group selected from the group consisting of the following. 10 Cycloalkyl groups: halogen atoms, hydroxyl groups, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, and C1-C6 haloalkoxy groups.

8. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 5 to 7, wherein the aralkyl group is a C7-C group optionally substituted with at least one group selected from the group consisting of the following. 14 Aryl groups: halogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups and C1-C6 haloalkoxy groups.

9. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 5 to 8, wherein the amide group is represented by the following formula: -CONR 1 R 2 , where R 1 and R 2 Each is independently a hydrogen atom or a C1-C6 alkyl group, or R 1 and R 2 Together with the nitrogen atoms to which they are attached, they form 4- to 8-membered saturated heterocycles, which are optionally substituted by at least one group independently selected from the group consisting of: halogen atoms, oxo groups, C1-C6 alkyl groups, and 4- to 7-membered heterocyclic groups.

10. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 9, wherein the number of natural amino acids constituting the cyclic portion is 1 to 4.

11. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 10, wherein the amino acids constituting the cyclic portion comprise natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

12. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 11, wherein the number of non-natural amino acids in the amino acids constituting the cyclic portion is nine or more.

13. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 12, wherein the amino acids constituting the cyclic portion comprise non-natural amino acids selected from the group consisting of: Abu, Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, D-Ala, D-MeAla, D-MeVal, D-Val, gMeAbu, Hph, MeAbu, MeAla, MeAsp-pip, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).

14. A cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 13, wherein the Caco-2 P of the cyclic peptide compound... app The value (cm / sec) is 0.4 × 10 -6 Or larger.

15. A library comprising a cyclic peptide compound optionally linked to a nucleic acid according to any one of claims 1 to 14.

16. A method for screening cyclic peptide compounds capable of specifically binding to a target molecule, the method comprising the following steps: (i) Contact the library according to claim 15 with the target molecule; as well as (ii) Select a cyclic peptide compound that can specifically bind to the target molecule.

Images