Macrocyclic peptide compounds having cell membrane permeability and metabolic stability and libraries containing the 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 is composed 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 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.

Description

Technical Field

[0001] This invention provides a macrocyclic peptide compound with cell membrane permeability and metabolic stability, as well as a library containing the macrocyclic peptide compound. Background Technology

[0002] Medium-molecular-weight compounds (molecular weight: 500 to 2000) have attracted attention as a means of drug development targeting challenging targets such as protein-protein interaction inhibition (Non-Patent Literature 1). Developing peptides as drugs is considered difficult because peptides generally 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 can be improved by cyclizing peptides or by using non-natural amino acids, such as N-methyl amino acids, in peptides (Non-Patent Literature 2 and 3). Cyclic peptides containing non-natural amino acids (especially those containing N-substituted amino acids) are known to have drug-like properties (Patent Literature 1). Library compounds of cyclic peptides containing non-natural amino acids have also been proposed for the development of protein-protein interaction inhibitors (Non-Patent Literature 4). Furthermore, cyclic peptide compounds with high membrane permeability, libraries containing such compounds, methods for their production, and screening methods have been reported in recent years (Patent Literature 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 Literature 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, and particularly preferably 9 to 11.

[0004] Patent Document 2 discloses a pre-incubation method using Caco-2 cells as a more accurate method for 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 sufficiently studied.

[0006] [List of Citations]

[0007] [Patent Literature]

[0008] [Patent Document 1]

[0009] International Publication Number WO 2013 / 100132

[0010] [Patent Document 2]

[0011] International Publication Number WO 2018 / 225864

[0012] [Patent Document 3]

[0013] International Publication Number WO 2020 / 122182

[0014] [Non-patent literature]

[0015] [Non-patent literature 1]

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

[0017] [Non-patent literature 2]

[0018] Acc. Chem. Res. 2008, 41, 1331-1342.

[0019] [Non-patent literature 3]

[0020] Angew. Chem. Int. (ed.), 2013, 52, 254-269.

[0021] [Non-patent literature 4]

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

[0023] [Technical Issues]

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

[0025] [Solution to the problem]

[0026] It is believed that as the size of a cyclic peptide increases, i.e., the number of amino acid residues constituting the cyclic moiety increases, the surface area available for contact with target molecules 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 pharmacomorphism 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 found 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 more efficiently obtain cyclic peptide compounds.

[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 portion consists 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.

[0032] (3) The ClogP / total amide bond (ClogP / total AB) ratio 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 as described in [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 as described in [A1] or [A2], wherein the number of amino acid residues constituting the cyclic peptide compound is 14.

[0039] [A3]

[0040] The 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] The 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 as described in [A1] or [A2], wherein the number of N-substituted amino acids constituting the cyclic moiety is eight or more.

[0045] [A6]

[0046] The cyclic peptide compound optionally linked to a nucleic acid according to claim [A5], wherein 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 number of N-substituted amino acid residues is 60% or greater in proportion to the total number of amino acid residues constituting the cyclic moiety.

[0051] [A8-1]

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

[0053] [A8-2]

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

[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] The 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] The 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] The 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]

[0066] The cyclic peptide compound optionally linked to a nucleic acid according to [A13], wherein the ClogP / total AB of the cyclic peptide compound is from 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], wherein 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 three 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 together form a ring.

[0073] [A18]

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

[0075] [A19]

[0076] Cyclic peptide compounds optionally linked to nucleic acids according to [A17] or [A18], 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.

[0077] [A20]

[0078] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A17] to [A19], wherein the aryl 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.

[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 optionally substituted C5-C. 10 aryl C1-C6 alkyl groups, wherein the C5-C 10 The aryl group is optionally substituted by 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.

[0081] [A22]

[0082] A cyclic peptide compound optionally linked to a nucleic acid according to any one of [A17] to [A21], 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.

[0083] [A23]

[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], wherein the amino acids constituting the cyclic portion 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 portion include α-amino acids, and the number of the 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 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).

[0095] [A29]

[0096] A cyclic peptide compound optionally linked to a nucleic acid according to [A27] or [A28], wherein, among 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 portion 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], wherein the amino acids constituting the cyclic portion 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.

[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 include 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 include gMeAbu.

[0107] [A35]

[0108] A cyclic peptide compound optionally linked to a nucleic acid according to [A33] or [A34], wherein, among the amino acids constituting the cyclic moiety, 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 portion 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 portion comprise natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0113] [A38]

[0114] The 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 the number of non-natural amino acids in the amino acids constituting the cyclic portion 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 moiety 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]

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

[0123] [A43]

[0124] The cyclic peptide compound optionally linked to a nucleic acid according to [A44], wherein Caco-2 P app The value (cm / sec) is calculated through membrane permeability assessment under the following conditions:

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

[0126] (b) After pre-incubation, the pre-incubation solution on the top and bottom sides was 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;

[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. The permeation of the compound was measured by liquid chromatography-mass spectrometry (LC / MS); and

[0128] (d) Calculate the permeability coefficient (Caco-2 P) from the measured permeability. app (cm / sec)).

[0129] [A44]

[0130] A cyclic peptide compound selected from 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] According to [A45], the cyclic peptide-nucleic acid complex has a cyclic portion and a linear portion, and the nucleic acid binds to the linear portion.

[0135] [A47]

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

[0137] The nucleic acid binds to the linear portion via an adapter.

[0138] [A48]

[0139] According to [A47], the cyclic peptide-nucleic acid complex contains puromycin.

[0140] [A49]

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

[0142] [A50]

[0143] The 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] The 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] The cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid contains a nucleotide sequence encoding a cyclic peptide portion forming the cyclic peptide-nucleic acid complex.

[0148] [B1]

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

[0150] [B2]

[0151] A library comprising cyclic peptide compounds optionally linked to nucleic acids.

[0152] The cyclic peptide compound contains a cyclic moiety, and

[0153] The number of cyclic peptide compounds comprising 13 or 14 amino acid residues constitutes the cyclic moiety is 10% or greater relative to the total number of cyclic peptide compounds contained in the library.

[0154] [B2-1]

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

[0156] [B2-2]

[0157] According to the library described in [B1] or [B2], the number of cyclic peptide compounds constituting the cyclic portion having 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 described in [B1] or [B2], the number of cyclic peptide compounds constituting the cyclic portion having 13 or 14 amino acid residues is 60% or greater in proportion to the total number of cyclic peptide compounds contained in the library.

[0160] [B2-4]

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

[0162] [B3]

[0163] According to the library described in [B1] or [B2], it is essentially composed of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion.

[0164] [B4]

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

[0166] [B4-1]

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

[0168] [B4-2]

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

[0170] [B4-3]

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

[0172] [B4-4]

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

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

[0178] [B7]

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

[0180] [B8]

[0181] According to the library described in [B7], 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 described in [B7], the number of N-substituted amino acids constituting the cyclic moiety in the cyclic peptide compound contained in the library is on average 8 to 13.

[0184] [B10]

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

[0186] [B11]

[0187] According to any one of [B1] to [B6], 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 10% or greater.

[0188] [B11-1]

[0189] According to any one of [B1] to [B6], 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 20% or greater.

[0190] [B11-2]

[0191] According to any one of [B1] to [B6], 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 40% or greater.

[0192] [B11-3]

[0193] According to any one of [B1] to [B6], 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], 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], 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] According to any one of [B1] to [B6], 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 20% or greater.

[0200] [B12-2]

[0201] According to any one of [B1] to [B6], 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 40% or greater.

[0202] [B12-3]

[0203] According to any one of [B1] to [B6], 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 60% or greater.

[0204] [B12-4]

[0205] According to any one of [B1] to [B6], the proportion of cyclic peptide compounds comprising 80% or more of the number of N-substituted amino acids constituting the cyclic moiety in the cyclic peptide compounds contained in the library is 80% to 13.

[0206] [B13]

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

[0208] [B13-1]

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

[0210] [B13-2]

[0211] According to any one of [B1] to [B6], the proportion of cyclic peptide compounds comprising 8 to 10 N-substituted amino acids in the cyclic moiety of the cyclic peptide compounds contained in the library is 40% or greater.

[0212] [B13-3]

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

[0214] [B13-4]

[0215] According to any one of [B1] to [B6], the proportion of cyclic peptide compounds comprising 80% or more of the number of N-substituted amino acids constituting the cyclic moiety in the cyclic peptide compounds contained in the library is 80% to 10%.

[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 described in [B14], the N-alkyl amino acid is an N-C1-C6 alkyl amino acid.

[0220] [B16]

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

[0222] [B17]

[0223] According to the library described in [B14], 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] 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 1.1 to 1.4.

[0230] [B19]

[0231] 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 1.0 to 1.8.

[0232] [B19-1]

[0233] 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 1.0 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]

[0237] The library according to any one of [B1] to [B19-2], wherein the average ClogP of each cyclic peptide compound contained 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 of 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] This cyclic peptide compound has a hydrogen bond donor, and

[0243] The library contains an average of eight or fewer hydrogen bond donors for each cyclic peptide compound.

[0244] [B22-1]

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

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

[0247] The library contains an average of eight or fewer hydrogen bond donors in the cyclic moiety of each cyclic peptide compound.

[0248] [B23]

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

[0250] [B23-1]

[0251] According to the library described in [B22], 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] The 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] According to any one of [B1] to [B24], in each cyclic peptide compound contained in the library, the average number of amino acids having the same side chain in the amino acids constituting the cyclic portion is 3 or less.

[0256] [B26]

[0257] According to any one of [B1] to [B24], in each cyclic peptide compound contained in the library, the number of amino acids having the same side chain among the amino acids constituting the cyclic portion is 3 or less.

[0258] [B27]

[0259] According to any one of [B1] to [B26], 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: 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 described in [B27], 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.

[0262] [B29]

[0263] According to the library described in [B27] or [B28], 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.

[0264] [B30]

[0265] The library according to any one of [B27] to [B29], 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.

[0266] [B31]

[0267] The library according to any one of [B27] to [B30], wherein the aralkyl group is optionally substituted C5-C 10 aryl C1-C6 alkyl groups, wherein the C5-C 10 The aryl group is optionally substituted by at least one group selected from the group consisting of one or more halogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, and C1-C6 haloalkoxy groups.

[0268] [B32]

[0269] According to any one of [B27] to [B31], wherein in each cyclic peptide compound, 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.

[0270] [B33]

[0271] According to any one of [B1] to [B32], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety 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.

[0272] [B34]

[0273] 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] According to any one of [B1] to [B34], the amino acids constituting the cyclic portion include α-amino acids, and the number of the α-amino acids is on average 10 to 14.

[0276] [B36]

[0277] 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]

[0279] 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], in each cyclic peptide compound, the amino acids constituting the cyclic portion 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] According to any one of [B1] to [B38], wherein in each cyclic peptide compound, the amino acids constituting the cyclic portion 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] According to the library described in [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], in each cyclic peptide compound, the amino acids constituting the cyclic portion include amino acids 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] The library according to any one of [B1] to [B41], wherein in each cyclic peptide compound, the amino acid constituting the cyclic moiety includes gMeAbu.

[0290] [B43]

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

[0292] [B44]

[0293] According to any one of [B1] to [B43], the number of natural amino acids constituting the cyclic portion in each cyclic peptide compound is on average one 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 fewer.

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

[0300] [B48]

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

[0302] [B49]

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

[0304] [B50]

[0305] According to any one of [B1] to [B49], in each cyclic peptide compound, the ratio of the number of natural amino acids to the number of amino acids in the cyclic moiety is on average 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 include natural 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 include naturally occurring amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0310] [B53]

[0311] According to any one of [B1] to [B52], in each cyclic peptide compound, the number of non-natural amino acids in the amino acids constituting the cyclic moiety is on average seven or more.

[0312] [B54]

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

[0316] [B55-1]

[0317] According to the library described in [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 greater.

[0318] [B55-2]

[0319] According to the library described in [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 described in [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 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).

[0324] [B57]

[0325] According to any one of [B1] to [B56], wherein in each cyclic peptide compound, the amino acids constituting the cyclic moiety each independently include 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).

[0326] [B58]

[0327] According to any one of [B1] to [B57], 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 molecular weight of each cyclic peptide compound is on average from 500 to 2000.

[0330] [B60]

[0331] According to any one of [B1] to [B59], the average molecular weight of the peptide portion, excluding the nucleic acid linker, in 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 in the identification of an orally administered compound or its precursor.

[0334] [B62]

[0335] The library according to any one of [B1] to [B61], wherein the library is a library for use in identifying cyclic peptide compounds that can specifically bind to a target molecule.

[0336] [B63]

[0337] The 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 1 × 10 2 or more diversity.

[0340] [B64-1]

[0341] According to the library described in [B64], wherein the library has 1 × 10 3 or more diversity.

[0342] [B64-2]

[0343] According to the library described in [B64], wherein the library has 1 × 10 4 or more diversity.

[0344] [B64-3]

[0345] According to the library described in [B64], wherein the library has 1 × 10 6 or more diversity.

[0346] [B64-4]

[0347] According to the library described in [B64], wherein the library has 1 × 10 8 or more diversity.

[0348] [B64-5]

[0349] According to the library described in [B64], wherein the library has 1 × 10 10 or more diversity.

[0350] [B65]

[0351] The library according to any one of [B1] to [B63], wherein the library contains 1 × 10 2 One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0352] [B65-1]

[0353] According to the library described in [B65], wherein the library contains 1 × 10 3 One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0354] [B65-2]

[0355] According to the library described in [B65], wherein the library contains 1 × 10 4One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0356] [B65-3]

[0357] According to the library described in [B65], wherein the library contains 1 × 10 6 One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0358] [B65-4]

[0359] According to the library described in [B65], wherein the library contains 1 × 10 8 One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0360] [B65-5]

[0361] According to the library described in [B65], wherein the library contains 1 × 10 10 One or more types of cyclic peptide compounds optionally linked to nucleic acids.

[0362] [B66]

[0363] The 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] The 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] The library according to any one of [B1] to [B67], wherein the cyclic peptide compound contained in the library is a cyclic peptide compound linked to a nucleic acid (cyclic peptide-nucleic acid complex).

[0368] [B69]

[0369] According to the library described in [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 described in [B68], the nucleic acid in the cyclic peptide-nucleic acid complex is a nucleic acid encoding the cyclic peptide.

[0372] [B70-1]

[0373] The cyclic peptide-nucleic acid complex according to any one of [B68] to [B69], wherein 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] The method according to any one of [B1] to [B70], wherein the library is a display library.

[0376] [B72]

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

[0378] [B73]

[0379] The library described in [B72] is an mRNA display library.

[0380] [C1]

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

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

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

[0384] [C2]

[0385] According to the method described in [C1], the target molecule is a protein.

[0386] [C3]

[0387] According to the method of [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] includes the following steps:

[0390] Chemically synthesized cyclic peptide compounds that can serve as candidates; and

[0391] The membrane permeability of this chemically synthesized cyclic peptide compound was measured using Caco-2 cells.

[0392] [C5]

[0393] According to any one of [C1] to [C4], the membrane permeability of the chemically synthesized cyclic peptide compound is determined by P app The value of (cm / sec) is determined.

[0394] [C6]

[0395] According to the method described in [C5], where P app The value (cm / sec) is calculated through membrane permeability assessment under the following conditions:

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

[0397] (b) After pre-incubation, the pre-incubation solution on the top and bottom sides was 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) Calculate the permeability coefficient (Caco-2 P) from the measured permeability. app (cm / sec)).

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

[0401] [Beneficial effects of the invention]

[0402] In one aspect, the present invention provides guidelines 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 from 13 or 14 amino acid residues, which, despite the benefits of interaction with a target, are difficult to achieve drug-likeness. Still in another aspect, a screening method for obtaining cyclic peptide lead compounds with excellent drug-likeness can be provided by designing libraries of cyclic peptide compounds based on the present invention. Attached Figure Description

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

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

[0405] Any element described in the embodiments mentioned below is intended to be described in a manner that is naturally considered equivalent in the “Description of Embodiments”, and is not subject to any patent practices, customs, laws, etc., in the countries where this patent application is to be examined that 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 to be 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 in this article: 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" refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon and having a hydrocarbon group structure free of heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds, and containing 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 those having 1 to 20 carbon atoms (C1-C2). 20 The following text "C"p -C q "Refers to alkyl groups with a carbon number of p to q, preferably C1-C2." 10 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" refers to 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 substituted with a halogen. Halogenated C1-C6 alkoxy groups are preferred, and fluoroC1-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" refers to an alkenyl group having at least one double bond (two adjacent sp bonds). 2 Alkenes are monovalent groups consisting of carbon atoms. Depending on the conformation of the double bond and substituents (if present), the geometry of the double bond can be either opposite-side (E) or same-side (Z), and cis or trans conformations. Alkenes include not only straight-chain forms but also branched forms. Preferred examples of alkenes include C2-C... 10 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 both straight-chain and branched forms. Preferred examples of alkynyl groups include C2-C... 10 The alkynyl group, and more preferably C2-C6 alkynyl. 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" refers to a monovalent aromatic hydrocarbon ring, i.e., an aromatic hydrocarbon ring group. Preferred examples of aryl groups include C6-C... 10 Aryl. Specific examples of aryl groups include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

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

[0416] As used herein, the term "heteroaryl" refers to an aromatic cyclic monovalent group containing a carbon atom and 1 to 5 heteroatoms, i.e., an aromatic heterocyclic group. The ring may be monocyclic or fused with other rings, and may also be partially saturated. The number of atoms in the ring constituting the heteroaryl 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, thiopheneyl, pyrroloyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiopheneyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzooxadiazolyl, benzoimidazolyl, benzotriazolyl, indoleyl, isoindoleyl, azaindoleyl, quinolinyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxolinyl, benzodioxacyclopentenyl, inazinyl, and imidazopyridyl, pyrazolopyridyl, imidazopyridyl, triazolopyridyl, pyrrolopyridyl, 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 C7-C. 14 Aryl or C5-C 10 aryl-C1-C6 alkyl, more preferably C7-C 10 Araneyl groups. Specific examples of araneyl groups 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. Specific examples of alkylene include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)CH2-, -C(CH3)2-, -(CH2)4-, -CH(CH3)CH2CH2-, -C(CH3)2CH2-, -CH2CH(CH3)CH2-, -CH2C(CH3)2-, -CH2CH2CH(CH3)-, -CH2CH(CH2CH3)-, -(CH2)5-, -CH(CH3)CH(CH2CH3)-, -(CH2)6-, -(CH2)7-, and -(CH2)8-. The alkylene group is preferably a C1-C8 alkylene group, and more preferably a C4-C8 alkylene group.

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

[0421] As used herein, “heteroaryl” means a divalent group derived from a “heteroaryl” as defined herein by further removal of any hydrogen atom. Heteroaryl groups can be monocyclic or condensed rings. There is no particular limitation on the number of atoms constituting the ring, but it is preferably 5 to 10 (5-membered to 10-membered heteroaryl groups). Specific examples of heteroaryl groups include pyrrolediyl, imidazolediyl, pyrazolediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazolediyl, triazinediyl, isoxazolediyl, oxazolediyl, oxadiazolediyl, isothiazazolediyl, thiazolediyl, thiadiazazolediyl, furandiyl, and thiophenediyl.

[0422] As used herein, the term "alkylene arylene" refers to a divalent group in which an alkylene group and an arylene group are bonded at any position, with the alkylene group attached to the base skeleton side. Specific examples of alkylene arylene groups include -C1-C6 alkylene groups and -C6-C6 alkylene groups. 10 Alpha-aryl.

[0423] As used herein, the term "arylene alkylene" refers to a divalent group in which an arylene and an alkylene group are bonded at any position, with the arylene group attached to the base skeleton side. Specific examples of arylene alkylenes include -C6-C... 10 arylene-C1-C6 alkylene.

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

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

[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, SR1, etc., and is capable of promoting the reaction with the amino group.

[0427] A "reaction accessory group" is a group introduced near the functional group to be bound to activate the binding reaction and selectively induce a reaction at the desired site. For example, a reaction accessory group can be introduced onto a carbonyl group or an amino group, or both, to promote a reaction between the carbonyl and amino groups. Examples of such reaction accessory groups include SH groups. This type of reaction accessory group can be removed 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" may refer to an amino acid residue. As used herein, the term "natural amino acid" refers to any L-type amino acid 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" refers to an amino acid other than a natural amino acid. Examples of non-natural amino acids include β-amino acids, γ-amino acids, L-type amino acids (other than the natural amino acids mentioned above), D-type amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids having side chains different from those of natural amino acids. Modified amino acids in this document 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. The unlettered symbols on the left and right represent the amino group and carboxyl group, 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. There are no particular restrictions on the choice of amino acid side chains, and in addition to hydrogen atoms, the 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 may be further added to each of these substituents. Such substituents are not limited and can be freely selected independently from one, two, or more substituents including any substituents comprising: 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 further having optionally added substituents. Examples of substituents that may be optionally added to the amino acid side chains include deuterium, cyano, nitro, and aminocarbonyl. In a non-limiting embodiment, as used herein, an amino acid can be a compound 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 atoms of the side chain together form a ring (such as proline, hydroxyproline, and aziridine-2-carboxylic acid) are also included in the amino acid.

[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 amino acids having two carboxyl groups (such as aspartic acid, which can be considered either 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 by 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 substituent of R is selected in the same manner as the substituents of the side chains of the amino acids described above. In the case of a substituted main chain amino group, R is included in "side chain of amino acid" as used herein. As used herein, such amino acids having a substituted main chain amino group are referred to as "N-substituted amino acids". As used herein, "N-substituted amino acids" are preferably N-alkyl amino acids, and can be N-C1-C6 alkyl amino acids or N-C1-C4 alkyl amino acids. The preferred N-substituted amino acid can be an N-methyl amino acid or an N-ethyl amino acid, and more preferably an N-methyl amino acid.

[0432] As used herein, the term "side chain" refers to the portion other than the main chain structure and is used in the context of side chains of amino acids, side chains of cyclic moieties in cyclic peptide compounds, etc. However, in the cyclic peptide-nucleic acid complexes mentioned below, the nucleic acid moieties and nucleic acid linker moieties are not included in the side chain.

[0433] The "amino acids" constituting the peptide compounds include 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) but 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... 2 H, 3 H; 13 C 14 C; 15 N; 17 O、 18 O; 31 P, 32 P; 35 S; 18 F; 36 Cl.

[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 forming 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, producing aldehydes as compounds), alkylcarbonyl groups (producing ketones as compounds), cycloalkylcarbonyl groups, alkenylcarbonyl groups, alkynylcarbonyl groups, arylcarbonyl groups, heteroarylcarbonyl groups, and aralkylcarbonyl groups. Examples of substituents forming esters include carbonyloxy groups (-OC(=O)-) and alkoxycarbonyl groups (-C(=O)O-). The alkoxycarbonyl group is selected from alkoxycarbonyl groups, cycloalkoxycarbonyl groups, alkenyloxycarbonyl groups, alkynyloxycarbonyl groups, aryloxycarbonyl groups, heteroaryloxycarbonyl groups, aralkyloxycarbonyl groups, etc. The carbonyloxy group is selected from alkyl carbonyloxy groups, cycloalkyl carbonyloxy groups, alkenyl carbonyloxy groups, alkynyl carbonyloxy groups, aryl carbonyloxy groups, heteroaryl carbonyloxy groups, aralkyl carbonyloxy groups, etc.

[0437] Examples of substituents forming thioesters include carbonyl thio groups (-SC(=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 carbonylamino groups (-NH-C(=O)-R), cycloalkyl carbonylamino groups, alkenyl carbonylamino groups, alkynyl carbonylamino groups, cycloalkyl carbonylamino groups, aryl carbonylamino groups, heteroaryl carbonylamino groups, and aralkyl carbonylamino groups, or 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, alkenoxycarbonylamino groups, alkynoxycarbonylamino 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 may optionally form a ring.

[0442] The substituents forming thioesters are selected from alkylthio groups, cycloalkylthio groups, olefin thio groups, alkynylthio groups, aryl thio groups, heteroaryl thio groups, arylalkylthio groups, etc. The substituents forming sulfoxide groups (-S(=O)-R) are selected from alkylsulfinyl groups, cycloalkylsulfinyl groups, alkenylsulfinyl groups, alkynylsulfinyl groups, arylsulfinyl groups, heteroarylsulfinyl groups, arylalkylsulfinyl groups, etc. The substituents forming sulfone groups (-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 primary amines 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(R)) include... 1 Examples of substituents (such as alkyl (aralkyl)amino groups) include any two 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. An amidine group (-C(=NR)-NR) is formed. 1 R 2 Examples of substituents 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 groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, which may be the same or different, such as alkyl(aralkyl)(aryl)amidinyl groups. Guanidinyl groups are formed (-NR-C(=NR)-NH2). 3 )-NR 1 R 2Examples of substituents include guanidine groups (-NH-C(=NH)-NH2) and those wherein R, R 1 R 2 and R 3 The group replaced by four substituents selected from the following: alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group and aralkyl group, which may be the same or different and may optionally form a ring.

[0444] Examples of substituents that form urea groups include aminocarbamoyl groups (-NR-CO-NR). 1 R 2 R, R 1 and R 2 The three substituents can 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 can optionally form a ring together.

[0445] Examples of functional groups derived from boron atoms include alkylboranes (-BR(R)). 1 )) and alkoxyborane (-B(OR)(OR) 1 These two substituents can be any two substituents, which may be the same or different, selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and aralkyl groups, and may optionally form a ring together.

[0446] In this way, various functional groups commonly found 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, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups) can be further substituted by one or more additional substituents. The condition for a functional group to satisfy all these requirements is defined as the free choice of substituents. In the case of β- or γ-amino acids, any stereochemical configuration is permitted, similar to α-amino acids, and the choice of side chains is not as particularly restricted as in the case of α-amino acids. The amino group portion of the amino acid backbone 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, 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" that have side chains that are capable of being translated. In a non-limiting embodiment, the amino acid used herein may be a translatable amino acid.

[0448] As used herein, the terms “drug-like” or “pharmaceutical-like” refer 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 groups forms a side chain. These substituents include those that are further replaced by substituents used as components of drug-like peptide compounds. These are preferably selected from the substituents defined separately above. For example, it can be replaced by one or more substituents selected from: hydroxyl groups, alkoxy groups, amide groups, sulfonyl groups, sulfinyl groups, halogen atoms, hydroxyamino groups, aminohydroxy groups, 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 translatable. 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 via the chemical transformation 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 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 (such as a cross-unit) for post-translational modification, the lysine unit is included as a unit of the drug-like amino acid. In this way, 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 thiols, 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 pharmaceutical-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 those for the substituents in 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 atom of the hydroxyl group (-OH) in the hydroxyphenyl group. For example, methoxyphenyl groups are not included in "substituted hydroxyphenyl groups" or "unsubstituted hydroxyphenyl groups" as used herein.

[0451] As used herein, the term "unsubstituted hydroxyphenyl group" means an unsubstituted hydroxyphenyl group. Substituted and unsubstituted hydroxyphenyl groups can be collectively referred to as "substituted or unsubstituted hydroxyphenyl groups".

[0452] As used in this article, 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" refers to the translation and synthesis of peptide compounds from nucleic acids (e.g., DNA, RNA) that encode peptide compounds. Translation is the process of obtaining linear peptides through repeated amide or ester bond reactions via the action of ribosomes using mRNA as a template.

[0454] The term "post-translational modification" refers to chemical reactions that occur spontaneously after translation, independent of the action of the ribosome, 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 the peptide compounds and / or nucleic acids encoding them contained in the library to the total number of the theoretical variants of the 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 of" 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 impact. 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 impact.

[0457] In this document, the term "negative impact" as used in the context of the effects of the invention refers to an impact that negates the effects of the invention. For example, assuming the original effectiveness of the invention is 100%, a "negative impact" can be considered if its effectiveness decreases to 30%, 20%, 10%, or 5% or lower. For instance, if the initial hit rate to be obtained through panning is 1%, a negative impact can be considered if the hit rate decreases to 0.3% or lower. Furthermore, if the initial proportion of Trp-free lead compounds to be obtained through panning is 100%, a negative impact can be considered if the proportion of Trp-free lead compounds decreases to 30% or lower. However, negative impacts are not limited to these cases.

[0458] peptide compounds

[0459] While 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. While 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 moieties in a peptide-nucleic acid complex may be referred to as "peptide moieties." The peptide compounds of this disclosure may contain α-hydroxycarboxylic acids to replace some constitutive amino acids. When a peptide compound contains α-hydroxycarboxylic acids, the bonds between each residue in the peptide compound can be bonds other than amide bonds, such as ester bonds. 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, in addition to glycolic acid, D-lactic acid, and L-lactic acid, include compounds in which the amino groups of the aforementioned "α-amino acids" are replaced by hydroxyl groups.

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

[0461] As used herein, the terms "peptide compound" or "peptide moiety" 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 or 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. Cyclation via amide bonds tends to provide cyclic peptide compounds with higher metabolic stability.

[0462] As used herein, "cyclization" refers to a reaction in which a carboxyl group present in the main chain or side chain of a linear peptide compound is linked to an amino or hydroxyl group to form a cyclic moiety. When chemically modifying the cyclic peptide compound of this disclosure, 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. (White circular...) ) unit (cross unit), black circle ( ) unit (ring-shaped main chain unit), square ( ) unit (linear part main chain unit) and triangle ( Each unit (cyclic N-terminal unit) represents an amino acid residue that makes up 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” refers to one of the following: an amino acid before cyclization, an amino acid after cyclization, or an amino acid at the point of completion of post-cyclization chemical modification of the linear peptide compound of this disclosure. 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 ring-shaped portion consists of one triangular unit, eight black circular units, and one white circular unit, while the linear portion consists of six square units. The curved portion of Scheme A is the portion to be ringized (this portion is also referred to herein as the "ringed portion" or "post-translational ringized portion").

[0468] In one embodiment, the peptide moiety of the peptide compound of this disclosure requires a reactive functional group on the triangular or cross-shaped unit of scheme A. Therefore, prior to cyclization, it is not necessarily required to select a drug-like amino acid targeting the triangular or cross-shaped unit in the peptide compound.

[0469] In one embodiment, even if the triangular or cross-shaped unit in the peptide compound is not a drug-like amino acid or is initially an unfavorable amino acid before cyclization, it can be chemically modified after cyclization to make the triangular or cross-shaped unit a drug-like amino acid or a favorable amino acid.

[0470] In one embodiment, for the triangular unit in the peptide portion prior to the 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 at the peptide moiety prior to cyclization of the peptide compound of this disclosure, it is preferable to use an amino acid having a cyclizable functional group (second reaction site) by reacting with the functional group possessed by the amino acid of the triangular unit in the side chain (first reaction site). 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 essential for cyclization in the cross-unit, it is preferred that the cross-unit have a total of three or more functional groups. Of these functional groups, for the cyclization reaction, the functional groups of the side chain portion of the cross-unit are preferably used.

[0472] Cross-linking units can be incorporated at any position on the peptide moiety prior to cyclization, as long as that position allows cyclization. However, it is preferred to incorporate cross-linking units at positions 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 cross-linking units of the disclosed peptide compounds at positions where at least 12 amino acid residues (e.g., 12 or 13 amino acid residues) are directed from the triangular unit toward the C-terminus.

[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 disclosed herein 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 acid residues via amide, ester, 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. To achieve 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 through 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 moiety of a cyclic peptide compound may have side chains (also referred to as "linear moiety") branching from the main chain constituting the cyclic structure, and the linear moiety has at least one amide bond and / or ester bond. When the cyclic peptide compound of the present invention has a linear moiety, it may be described, for example, scheme A-1 or A-2. For example, when the cyclic peptide compound has a linear moiety, it may be a peptide compound in which the cyclic moiety 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 moiety via an amide bond. The term "linear moiety" 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 moiety and the linear moiety is 13 or 14 residues, membrane permeability tends to be higher. When the total number of amino acids in the cyclic moiety and the linear moiety is 13 residues or more, metabolic stability tends to be greater. The number of amino acids in the linear moiety is preferably 0 to 8 residues, more preferably 0 to 3 residues, and most preferably 0 (i.e., no linear moiety).

[0477] [Equation 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 that make up a peptide, and means the number of amino acids or α-hydroxycarboxylic acids produced when the bonds of amide bonds, ester bonds, and cyclized moieties connecting amino acids 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 Schemes A-1 or A-2 above are included in the amino acids constituting the cyclic moiety.

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

[0482] In one non-limiting embodiment, the cyclic peptide compound has a cyclic moiety.

[0483] (1) The cyclic portion consists of 13 or 14 amino acid residues.

[0484] (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.

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

[0486] The number of amino acids constituting the cyclic peptide compound in this disclosure (referred to as the peptide moiety when the cyclic peptide compound is linked to a nucleic acid) is not particularly limited, 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 linker moiety and a peptide moiety excluding the nucleic acid linker moiety. When the number of amino acids constituting the peptide moiety excluding the nucleic acid linker moiety is 13 or 14, or when the number of amino acids constituting the cyclic moiety is 13 or 14, it is preferable to simultaneously achieve excellent membrane permeability and metabolic stability.

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

[0488] The number of amino acid residues having the same side chain in the cyclic moiety 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 requirements for 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 greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, or 75% or greater.

[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.

[0492] The cyclic peptide compounds disclosed herein are compounds with a ClogP / total amide bond ratio (ClogP / total AB) 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 chains of each amino acid) is used as “total AB”. Alternatively, 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 portion other than the nucleic acid linker portion of the cyclic peptide compound is used as “total AB”. When the bond between the nucleic acid linker portion and the peptide portion other than the nucleic acid linker portion 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 constituting display libraries for screening; however, in practice, only the unlinked portion of the drug candidate compound is expected to be used. Therefore, when considering drug-like properties (e.g., high membrane permeability), the properties of the peptide portion other than the nucleic acid linker may become important.

[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 partition 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 (Published 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, the ClogP of the entire cyclic peptide compound is calculated. 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 for a hypothetical peptide in which the nucleic acid linker of the cyclic peptide compound is replaced by piperidine (pip). That is, the hypothetical peptide is a cyclic peptide in which 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, when considering drug-like properties (e.g., high membrane permeability), the properties of the peptide portion other than the nucleic acid linker may become important.

[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.

[0497] In a non-limiting embodiment, when the ClogP / total AB ratio 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 ratio 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 compounds included in 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 compound or a nearby compound (including water molecules). A hydrogen bond donor is a group containing a hydrogen atom covalently bonded to an atom having a 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 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. A better balance between excellent membrane permeability and metabolic stability can be achieved when the number of hydrogen bond donors is 5 or fewer, 4 or fewer, or 3 or fewer. There is no particular limitation on the lower limit of 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 portion of the cyclic peptide compound include groups selected from drug-like substituents. The 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 by, 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, wherein the 4- to 8-membered saturated heterocycle 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 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 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 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 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.

[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 this disclosure is a cycloalkyl group, the cycloalkyl group may be a C3-C 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. 10 Cycloalkyl groups.

[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 this disclosure is an aralkyl group, the aralkyl group may be a C7-C1 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. 14 Aryl 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 this disclosure is an aralkyl group, the aralkyl group may optionally be a C5-C group as determined. 10 Aryl C1-C6 alkyl group, of which C5-C 10 The aryl group is optionally substituted by 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 this disclosure is an amide group, the amide group can be 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.

[0507] In a non-limiting embodiment, the cyclic peptide compound of this 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 a 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 the present 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 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 portion of the cyclic peptide compound of this disclosure 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 portion 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 portion of the cyclic peptide compound having a β-amino acid backbone may be, but 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 with a γ-amino acid backbone constituting the cyclic portion of a cyclic peptide compound 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 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). Each α-amino acid is independently 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, 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 one non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of this disclosure may include natural amino acids selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. Each natural amino acid may be independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0518] In a non-limiting embodiment, the number of non-natural amino acids in the cyclic portion constituting the cyclic peptide compound of this 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 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, 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 group consisting of the following: 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 one non-limiting embodiment, the Caco-2 P of the cyclic peptide compound of this disclosure app The value (cm / sec) can be, for example, 1.0 × 10⁻⁶. -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 -5Or 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 one non-limiting embodiment, the Caco-2 P of the cyclic peptide compound of this disclosure app The value (cm / sec) can be, for example, 1.0 × 10⁻⁶. -9 Or larger, 1.0 × 10 -8 Or larger, 1.0 × 10 -7 Or larger, 0.4 × 10 -6 Or larger, 1.0 × 10 -6 Or larger, or 1.0 × 10 -5 Or larger, preferably 0.4 × 10 -6 Or larger, and more preferably 1.0 × 10 -6 Or larger. In one non-limiting embodiment, the Caco-2 P of the cyclic peptide compound of this disclosure app The value range for (cm / sec) can be, for example, 1.0 × 10⁻⁶. -9 Or larger and 1.0 × 10 -4 Or lower, 1.0 × 10 -8 Or larger and 1.0 × 10 -4 Or lower, 1.0 × 10 -7 Or larger and 1.0 × 10 -4 Or lower, 0.4 × 10 -6 Or larger and 1.0 × 10 -4 Or lower and 0.4 × 10 -6 Or larger and 1.0 × 10 -5 Or lower.

[0521] - Membrane permeability of peptide compounds

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

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

[0524] (b) After pre-incubation, the pre-incubation solution on the top and bottom sides was 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;

[0525] (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

[0526] (d) Calculate the permeability coefficient (Caco-2 P) from the measured permeability. app (cm / sec)).

[0527] Permeability coefficient (Caco-2 P) app (cm / sec) is calculated using numerical expression 1.

[0528] [Expression 1]

[0529]

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

[0531] Note that in the above measurements, a Pgp inhibitor, such as Zosquidar, can 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). Additionally, the concentration of the compound to be evaluated on the donor side used to calculate the permeability coefficient in step (d) above can be the initial concentration of the compound, or the concentration of the compound measured by collecting the solution on the top side before starting pre-incubation or shaking in step (c) above. In particular, when performing pre-incubation, it is preferable to use the concentration of the compound collected on the top side before pre-incubation.

[0532] In one 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 one 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 a 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 one 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 the test substance based on the results of membrane permeability, and the membrane permeability of the derivatives can be measured.

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

[0534] - Peptide-nucleic acid complex

[0535] In one embodiment, the peptide compound of this disclosure may be a complex of a peptide and a nucleic acid (peptide-nucleic acid complex). The peptide-nucleic acid complex can be formed by binding a nucleic acid to the C-terminus of the peptide moiety. The peptide-nucleic acid complex of this disclosure is 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 a cyclic peptide-nucleic acid complex, the nucleic acid is linked to the linear portion but not to the cyclic portion. The method of bonding the peptide and nucleic acid is not particularly limited, but they can be bonded via a linker. When the peptide compound and nucleic acid are bonded via a linker, the linker is considered part of the nucleic acid linkage portion. In one embodiment, the nucleic acid forming the complex with the peptide moiety may be DNA or RNA, preferably RNA, and more preferably mRNA. In one embodiment, the nucleic acid forming the complex with the peptide may 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 in which the C-terminus of the peptide moiety is linked to the 3' end of the mRNA used as a template via a linker containing an antibiotic such as puromycin, which is an analog of aminoacyl-tRNA.

[0536] Peptide-nucleic acid complexes can be generated, for example, by the method described in International Publication No. WO 2013 / 100132. Specifically, peptide-nucleic acid complexes 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 the aminoacyl pdCpA or aminoacyl pCpA from step A to the tRNA lacking CA from step B to provide an initiator tRNA acylated with the desired amino acid or an 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, formyl donor, and methionyl-tRNA transferase.

[0541] E) Provide template DNA encoding a translation start codon downstream of the promoter, followed by the cysteine ​​codon UGU or UGC, and a peptide sequence downstream of the codon corresponding to the anticodon of the tRNA in step C).

[0542] F) Provide mRNA from the template DNA in step E).

[0543] G) Attach the adapter to the 3' end of the mRNA from step F), and

[0544] H) The mRNA ligated to the adapter in step G) is added to the cell-free translation system in step D) and translated to provide a procyclic 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. Additionally, after step G), a step of synthesizing cDNA using primers annealed to the 3' region of the mRNA may be included.

[0546] In steps A through 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, the 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 a source. The RNA 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 CCA sequence obtained in this manner to an aminoacylated pdCpA (the pdCpA method) prepared separately using an 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 through H, DNA with the desired sequence positioned 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 integrated into the protein by attaching a linker containing puromycin to the 3' end of the transcribed mRNA, and then translating the mRNA into a protein using a known cell-free translation system (like the PURE system), thereby linking the mRNA and its encoded protein via puromycin. In this way, a peptide-mRNA complex associated with the encoded product (peptide) can be constructed.

[0548] - Metabolic stability of peptide compounds

[0549] In a non-limiting embodiment, the peptide compound of this disclosure preferably exhibits 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 aforementioned membrane permeability conditions 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 methylthio groups are easily oxidized and potentially hinder 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 (CL) in liver microsomes is measured according to the method described above, stability can be assessed. h intA value of 150 (μL / min / mg protein) or lower is considered sufficient for metabolic stability for oral administration, and this value is preferably 100 or lower. In the case of a peptide compound that is a CYP3A4 metabolite, a 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 is 35 or lower (assuming FaFg = 1 and protein binding = 0%) to indicate a bioavailability of approximately 30% or greater in humans.

[0552] - Nucleic acid

[0553] In one 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 the 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 disclosed herein 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 libraries of this disclosure, compounds with excellent drug-like properties and high affinity for target molecules can be efficiently found among cyclic peptide compounds having a cyclic moiety formed by 13 or 14 amino acid residues. Display libraries are preferred. Examples of display libraries include display utilization libraries. Among these, 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, nucleic acids linked to the peptide compounds by panning and enrichment can be amplified by PCR or the like, 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 JW et al. Proc. Natl. Acd. Sci. USA, 1997, 94, 12297-12302.).

[0556] The peptide compounds disclosed herein can be peptide compounds encoded by nucleic acids. When the peptide compounds of this disclosure are linked to nucleic acids, the peptide compounds can 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 compound encoded by nucleic acid" includes not only peptide compounds synthesized directly by translating a nucleic acid as a template, but also peptides synthesized post-translationally by further modification. 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 can be referred to as a cyclic peptide compound encoded by a superior nucleic acid.

[0557] For a library of peptide compounds that can be linked to nucleic acids and / or the nucleic acids that encode them, the term “substantially composed of” as used herein means that the ratio of the theoretical number of said peptide compounds and / or the nucleic acids that encode them contained in the library to the total number of theoretical variants of said peptide compounds and / or the nucleic acids that encode 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 constituting the cyclic portion having 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.

[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 portion of the cyclic peptide compound 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 of each cyclic peptide compound contained in the library of this disclosure, excluding the nucleic acid linker, 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 moiety other than the nucleic acid linker is, for example, but not limited to, 13 or more, 14 or fewer, 13 to 14, 13 or 14. Preferably, the number of amino acids constituting the cyclic moiety is 13, 14, or 13 to 14, taking into account the requirements for achieving excellent membrane permeability and metabolic stability.

[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 moiety other than 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 moiety 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, or 8 to 13, more preferably 7. Up to 10 or 8 to 10.

[0566] In a 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 7 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.

[0567] In a 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 a 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 this disclosure library, the average proportion 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 included 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 may 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 a value calculated as described above. If the ClogP / total AB of each cyclic peptide compound contained in the library of this disclosure is calculated and the average value is within the range described above, the probability of the selected compound being a compound with excellent membrane permeability increases.

[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 less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less. 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 finding a compound with excellent membrane permeability during screening increases.

[0572] In a non-limiting embodiment, the cyclic peptide compounds included in the library of this disclosure may have hydrogen bond donors (proton donors). 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 counts 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 count falls within the above ranges, the probability of finding a compound with excellent membrane permeability during screening increases.

[0573] In one embodiment, the cyclic peptide compounds included in the library of this disclosure may have a hydrogen bond donor (proton donor) in the cyclic moiety. 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 compound or a nearby compound (including water molecules). A hydrogen bond donor is a group containing a hydrogen atom covalently bonded to an atom having 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. The average number of hydrogen bond donors in the cyclic portion 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.

[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 chains as the amino acids constituting the cyclic moiety. Here, the term "including amino acids having the same side chains" means that the amino acids constituting the whole cyclic peptide compound or the cyclic moiety of the cyclic peptide compound include a plurality of amino acids having the same side chains. "Amino acids having the same side chains" is determined based on the identity or non-identity of the side chains, and substituents on the nitrogen atom of the amino group in the main chain are not considered. For example, alanine and N-methylalanine are considered "amino acids having the same side chains". Specifically, when the cyclic moiety of the cyclic peptide compound contains two Leu amino acids, the cyclic peptide compound includes a plurality of amino acids having the same side chains as the amino acids constituting the cyclic moiety. The average number of amino acids having the same side chains among the amino acids constituting the cyclic moiety 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.

[0575] In one non-limiting embodiment, the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound included 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 together form a ring.

[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 included in the library of this disclosure is a cycloalkyl group, the cycloalkyl group may be a C3-C 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. 10 Cycloalkyl groups.

[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 included in the library of this disclosure is an aralkyl group, the aralkyl group may be a C7-C1 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. 14 Aryl 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 included in the library of this disclosure is an aralkyl group, the aralkyl group may optionally be a C5-C group as determined. 10 Aryl C1-C6 alkyl group, of which C5-C 10 The aryl group is optionally substituted by 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.

[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 included in the library of this disclosure is an amide group, the amide group may be represented by the following formula: -CONR 1 R2 , 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.

[0581] In a non-limiting embodiment, at least one amino acid of the cyclic portion of each of the cyclic peptide compounds comprising 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 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.

[0582] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds included 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 constituting the cyclic portion of the cyclic peptide compounds included 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 constituting the β-amino acid backbone of the cyclic portion of the cyclic peptide compounds included 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 compound 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, it is preferable that 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 portion of the cyclic peptide compounds included 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). Each α-amino acid 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 may 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 included 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 cyclic portion of the cyclic peptide compound 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 1 or more, 6 or fewer, 5 or fewer, 1 to 6, or 1 to 5, and even more preferably 1 or more, 4 or fewer, or 1 to 4. In the cyclic peptide compounds included in this disclosed library, the average proportion of the number of naturally occurring amino acids in the number of 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 in the number of 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. Each natural amino acid may be independently selected from the group consisting of: Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0588] In a 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 the library of this disclosure, the average proportion of non-natural amino acids in the number of amino acids constituting the cyclic portion may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, and may 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 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 independently 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).

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

[0591] In one 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 moiety 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 moiety other than the nucleic acid linker. Since the presence of an indole skeleton on the side chain of the peptide moiety other than the nucleic acid linker of the cyclic peptide compound can impede membrane permeability, it is preferred that the cyclic peptide compound does not have an indole skeleton.

[0592] In one non-limiting embodiment, the library of this disclosure may be a library consisting essentially of cyclic peptide compounds that do not have methylthio 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 of cyclic peptide compounds that do not have substituted or unsubstituted methylthio 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 compounds do not have methylthio groups because methylthio groups are readily oxidized and potentially impair metabolic stability.

[0593] In one 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 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 compounds do not have thiol groups because thiol groups are readily oxidized and potentially impair metabolic stability.

[0594] In one 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 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 a hypothetical peptide in which the nucleic acid linker of the cyclic peptide compound is replaced by piperidine. That is, the hypothetical peptide is a cyclic peptide in which piperidine 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 having 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; however, in practice, drug candidates are expected to be used only for the unlinked nucleic acid portion. Therefore, the properties of the peptide portion other than the nucleic acid-linked portion may become important when considering drug-likeness (e.g., high membrane permeability).

[0595] In this disclosed library, when determining the "theoretical (total) variation" of cyclic peptide compounds, those that cannot actually be produced as cyclic peptide compounds are not included in the theoretical (total) variation. 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) quantity: (1) even if the amino acid is added as a material to the translation solution for the 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 the 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 maps a set of codon units to one type of amino acid, when a nucleotide sequence randomly arranged from codon units is translated to synthesize an 11-residue peptide, the theoretical number of variants is 20 if the initiator amino acid is fixed to methionine. 10 .

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

[0598] This disclosed library contains multiple cyclic peptide compounds or nucleic acids encoding these compounds. The number of variations (types) of the cyclic peptide compounds contained in this library is referred to as "diversity". The diversity of the cyclic peptide compounds or nucleic acids encoding them contained in this disclosed library is not particularly limited, and examples include 10. 2 or larger, 10 3 Or larger, 10 4 or larger, 10 5 Or larger, 10 6 Or larger, 10 7 Or larger, 10 8 or larger, 10 9 or larger, 10 10 Or larger, 10 11 or larger and 10 12Or even greater. These variations are not limited to measured values; they can also be theoretical values.

[0599] The cyclic peptide compounds included in the library disclosed herein may include at least one, or all of the cyclic peptide compounds pd001 to pd168 listed in Table 15.

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

[0601] - Membrane permeability

[0602] In one 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 (P0) as measured by the method of this disclosure for measuring membrane permeability. app ) is 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, preferably 1.0 × 10 -6 cm / s or greater, preferably 3.0 × 10 -6 cm / s or greater, with 5.0 × 10⁻⁶ being the most preferred. -6 cm / s or greater. In one non-limiting embodiment, the library of this disclosure may be a library for identifying orally administerable compounds or their precursors. "Orally administerable" herein refers to membrane permeability coefficient (P0). app The value is 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 used for optimization, etc., and examples include hit compounds from panning prior to any optimization.

[0603] - Hit rate of peptide compounds that can specifically bind to target molecules

[0604] In one non-limiting embodiment, the library of this disclosure can be used to identify compounds capable of specifically binding to target molecules. In one embodiment, the library of this disclosure has a greater number of amino acids constituting peptide compounds compared to conventional libraries. This increases amino acid diversity and results in a higher hit rate for peptide compounds capable of specifically binding to target molecules. That is, by using the library 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 selection of 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 compounds obtained by panning the number of variations of compounds included in the library. The number of variations of compounds included 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 cyclic peptide compounds enriched through panning, amino acid groups that may facilitate binding to target molecules can be identified by analyzing the frequency of amino acid occurrence.

[0607] - Showcase Library

[0608] A display library is a library in which peptides (as phenotypes) are associated with RNA or DNA encoding the peptides (as genotypes). This library can be used to identify peptide compounds that can specifically bind to target molecules. For example, peptides binding to a desired target can be enriched by contacting the library with the desired 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 target-binding peptide sequences can be clarified. For example, the method using the antibiotic puromycin, an analog of aminoacyl-tRNA, which non-specifically binds to proteins during ribosome mRNA translational expansion, has been reported as mRNA display (Proc Natl Acad SciUSA. 1997;94:12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins. Roberts RW, Szostak JW.) 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 invitro. 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 including 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. This process is highly efficient because it does not involve transformation (such as with E. coli), 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 cDNA display, in addition to mRNA display, of cDNA libraries encoding peptides and puromycin complex-attached peptides (Nucleic Acids Res. 2009;37(16):e108. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N.), and ribosome display using 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 This is a covalent display, in which the bacteriophage 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 a CIS display that utilizes the binding of the replication initiation protein RepA of microbial plasmids to the replication origin ori (Proc Natl Acad Sci US A.2004;101:2806-10. CIS display: In vitro selection of peptides from libraries of protein-DNA complexes).Odegrip R, Coomber D, Eldridge B, Hederer R, Kuhlman PA, Ullman C, FitzGerald K, McGregor D.). 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 takes place (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments formolecular 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 can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotide derivatives with artificial bases, and peptide nucleic acids (PNA). The nucleic acid in this invention can be any of these nucleic acids or a mixture thereof, as long as the genetic information of interest is preserved. That is, the nucleic acids in this invention also include DNA-RNA hybrid nucleotides or chimeric nucleic acids, wherein 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 to the peptide sequence and synthesizing them. For example, nucleic acid libraries can be synthesized as a mixture of A, T, G, C from a DNA library and A, U, G, C from an RNA library (in three times the number of four bases (N)) or a mixture of two types of bases, such as N for the first and second letters of a codon and W, M, K, and S for the third letter. When the number of introduced amino acid types is reduced to 16 or less, there is a method to set the third-letter base to a single type of base. Furthermore, codon units corresponding to the three letters of a codon are prepared, and the frequency of occurrence of amino acid residues can be freely adjusted by mixing them in any proportion and for use in synthesis. Each nucleic acid in the nucleic acid library can be linked to puromycin at its 3' end. By translating a nucleic acid library linked to puromycin at the 3' end, peptide compounds encoded by nucleic acids are synthesized, and the resulting peptide compounds are linked to the corresponding nucleic acids 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 a polymer (e.g., five polymers) of RNA, DNA, hexadecimaltol (spc18), etc., 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] - Filtering methods

[0616] In a non-limiting embodiment, peptide compounds capable of specifically binding to the target molecule can be selected by screening using the libraries disclosed herein. The screening methods of this disclosure are not particularly limited, and, for example, mRNA display methods can be used.

[0617] In one non-limiting embodiment, the screening method of this disclosure involves contacting a cyclic peptide compound library of this 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, the mRNA being a tag containing nucleotide sequence information of the peptide compounds selected in this manner, subjected to PCR amplification, and the nucleotide sequence analyzed. 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 the target molecule, allowing for further enrichment of peptide compounds that specifically bind to the target molecule by panning again using this library. By repeating this process multiple times, the desired peptide compounds can be further enriched. In one embodiment, the amino acid sequence can be identified based on the nucleotide sequence information contained in the peptide compounds enriched in this manner, and peptide compounds that specifically bind to the target molecule 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 one non-limiting embodiment, the screening method of this disclosure includes the following steps:

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

[0621] (b) Select peptide compounds that can bind to target molecules.

[0622] In one embodiment, the screening method of this disclosure can enrich peptide compounds that specifically bind to a target molecule by repeating steps (a) and (b) above two or more times (cycles). In this case, in step (a), starting from the second cycle, 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 peptide compounds that bind to target molecules as drug candidate compounds in step (b) above.

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

[0625] (c) Chemical synthesis of peptide compounds that can serve as candidates; and

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

[0627] - Target molecules

[0628] The target molecules used in the screening method of this disclosure are not particularly limited, and examples include proteins, peptides, nucleic acids, sugars, lipids, etc., but proteins are preferred. In one embodiment, peptide compounds that inhibit protein-protein interactions (PPIs) can be obtained according to the screening method of this disclosure. 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 the 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 changes, 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 these sites. In other words, it is believed that peptide compounds that inhibit PPIs can be obtained according to the library and screening method of this disclosure, regardless of the type of target molecule.

[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 a portion other than the nucleic acid and the nucleic acid linker (e.g., a cyclic moiety). Target molecules can be selected based on the presence, absence, or strength of the interaction with the cyclic peptide moiety. In the cyclic peptide-nucleic acid complex, when the nucleic acid moiety contains a nucleotide sequence encoding the cyclic peptide moiety, the nucleic acid moiety can facilitate the understanding of the chemical structure and synthesis of the cyclic peptide moiety and contribute to improved screening efficiency.

[0630] The target molecule in vivo is not particularly limited to its 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 can be easily enhanced by derivatization, can be obtained, thereby allowing targeting of intracellular proteins.

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

[0632] The target molecules of the screening methods disclosed herein are not particularly limited. The library disclosed includes peptide compounds capable of specifically binding to a variety of target molecules, and in one embodiment, these peptide compounds are capable of drug discovery for intractable targets considered difficult to drug develop.

[0633] - Production methods for peptide compounds and libraries

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

[0635] - Chemical synthesis methods for peptide compounds

[0636] Examples of chemical synthesis methods for the peptide compounds disclosed herein 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 groups are protected by protecting groups (which are not cleaved by bases such as piperidine if needed), and an amino acid whose main-chain carboxylic acid is not protected are used as the base unit. There are no specific limitations on the base units, as long as they are combinations of Fmoc-protected amino and carboxylic acid groups. For example, a dipeptide can be used as the base unit. The base unit located 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. The main-chain carboxylic acid group is fixed to the solid phase by 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. In condensation reactions, various combinations are possible, such as the combination of DIC and HOBt, DIC and HOAt, and HATU and DIPEA. The desired peptide sequence can be generated by repeatedly deprotecting the Fmoc group followed by a peptide bond formation reaction. 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 prior to cleavage from the solid phase. Cleavage from the solid phase 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 a protecting group, taking advantage of the orthogonality of the two chemical reactions. Steps such as cyclization can also be performed during or at the end of these processes. 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, orthogonality is required between the C-terminal carboxylic acid and the side-chain carboxylic acid to be cyclized, or between the N-terminal main-chain amino group or hydroxyl group and the side-chain amino group to be cyclized, and the protecting group is selected considering the orthogonality of the protecting group, as mentioned above. 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 disclosed herein can be synthesized using 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 using tRNA, rather than methods for synthesizing peptide compounds solely through organic synthesis techniques. Examples of translation-based peptide compound synthesis methods include methods using cell-free translation systems, including synthetic methods using reconstructed cell-free translation systems. The use of reconstructed cell-free translation systems is preferred because it allows for the elimination of undesirable factors.

[0639] In one aspect, this disclosure provides a translation-based method for synthesizing 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, MeI le, 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 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, which 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" of this document. The cell-free translation system of this document may contain nucleic acids used as templates for translated peptides, and may additionally contain 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, 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 synthases (ARS), not only naturally occurring tRNAs can be used, but also artificial tRNAs and artificial aminoacyl-tRNA synthases that recognize non-natural amino acids. By using artificial tRNAs and artificial aminoacyl-tRNA synthases, it is possible to synthesize peptides in which site-specific introduction of non-natural amino acids is achieved. If desired, RNA polymerases such as T7 RNA polymerase can be added to cell-free translation systems to transcribe from template DNA.

[0647] PURESYSTEM® (manufactured by BioComber Co., Ltd., Japan) is a reconstituted 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® 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, as it is a reconstituted system ((i) NatBiotechnol. 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, many methods have been reported to use stop codons as codons for introducing non-natural amino acids. However, by using the PURESYSTEM described above, synthetic systems that exclude 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 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 AmChem 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 contains a suitable selection of protein factors essential 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, and HEPES-KOH. A mixture of components including pH 7.6, magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, and UTP. Transcription and translation from template DNA containing a T7 promoter can also be performed in a coupled manner upon the addition of T7 RNA polymerase. Furthermore, peptide compounds containing non-natural amino acids can be obtained through translation-based synthesis by adding a desired set of aminoacyl-tRNAs or a set of non-natural amino acids (e.g., F-Tyr) permitted by aminoacyl-tRNA synthetase (ARS) to the system (Kawakami T et al., Ribosomal synthesis of polypeptoids and peptide-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 modified ARS (instead of natural ARS or other than 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, orthogonal tRNA effectively incorporated by ribosomes undergoes aminoacylation ((i) Biochemistry 2003; 42: 9598-608. Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opalsuppression. Anderson JC, Schultz PG.; (ii) Chem Biol. 2003; 10: 1077-84. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code. Murakami H, Kourouklis D, Suga H.). The following five methods can be used to aminoacylate tRNA.

[0653] Intracellularly, each aminoacyl-tRNA synthetase (ARS) is individually provided as an enzyme for the aminoacylation of tRNA. Therefore, as a first approach, methods that allow certain ARSs such as N-MeHis, and methods that prepare and utilize modified aminoacyl-tRNA synthetases that allow non-natural amino acids can be used (i) Proc. Natl. Acad. Sci. US A. 2002; 99: 9715-20. An engineered Escherichiacoli tyrosyl-tRNA synthetase for site-specific incorporation of a non-natural amino acid into proteins in eukaryotic translation and its application in awheat 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, SchultzPG.Chin, JW. (iii) Proc.Natl.Acad.Sci. US A. 2006; 103: 4356-61. Enzymaticaminoacylation 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, where CA is removed from the CCA sequence at the 3' end to 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, tRNA and amino acid active esters can be ultrasonically mixed in cationic micelles (Chem Commun (Camb. 2005; (34): 4321-3. Simple and quick chemicalaminoacylation of tRNA in cationic micellar solution under ultrasonic agitation. Hashimoto N, Ninomiya K, Endo T, Sisido M.).Fifth, aminoacylation may also be achieved by adding an active amino acid ester that is complementary to the 3' end of the tRNA and binds to PNA to the tRNA (JAm Chem Soc. 2004; 126: 15984-9. In situ chemical aminoacylation with aminoacid thioesters linked to a peptide nucleic acid. Ninomiya K, Minohata T, Nishimura M, Sisido M.).

[0654] More specifically, aminoacyl-tRNA 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) 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 a source. The RNA 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 or pCpA (pdCpA method, pCpA method) prepared separately using RNA ligase. tRNA can be used to produce peptide compounds. Alternatively, full-length tRNA is prepared and aminoacylated via 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, although this is not intended to be limiting. When using natural ARS or modified ARS, it is not necessary to have a large quantity of previously prepared aminoacyl-tRNA in the translation system, as aminoacyl-tRNA previously consumed in the translation system can be regenerated from the natural ARS or modified ARS. 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 cyclized peptide moieties

[0656] The cyclic peptide compounds disclosed herein have a cyclic moiety. There are no particular limitations on the cyclization method of the peptide compounds, but for example, the cyclization reaction can be carried out after the peptide compounds are 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 and cyclization reaction steps of the peptide compounds can be carried out individually or sequentially. Cyclization can be carried out by methods known to those skilled in the art, such as those described in WO 2013 / 100132, WO 2008 / 117833, or WO 2012 / 074129.

[0657] The cyclization moiety can 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 the side chain of another amino acid residue, a bond between the C-terminus of a peptide and the side chain of another amino acid residue, or a bond between the side chains of amino acid residues, and two or more of these bonds can 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 the side chain of another amino acid residue, or a bond between the side chains of amino acid residues.

[0658] In one non-limiting embodiment, the cyclic peptide compound of this disclosure can 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 an amino acid residue or an amino acid residue and an N-terminal carboxylic acid analog by translation 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

[0660] (2) To combine the reaction sites of amino acid residues at the N-terminus or N-terminal carboxylic acid analogs with the reaction sites of amino acid residues at the C-terminus of the side chain to form amide bonds, carbon-carbon bonds or thioether bonds.

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

[0662] A non-limiting example of a method for 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 precursors to 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 of the peptide compound, the following three methods can be used, for example.

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

[0665] The second approach uses a reconstructed cell-free translation system, in which Met is removed from the system beforehand, the desired amino acid positioned at the N-terminus is placed from the second residue at the N-terminus, and translation-based synthesis is performed by reading the start codon Met. This method is defined as the start-readthrough (iRT) method.

[0666] The third method is a translation-based synthesis approach that involves adding aminoacyl-tRNA to a recombinant cell-free translation system that does not contain Met or the initiator methionine tRNA. The aminoacyl-tRNA is prepared by aminoacylation of the amino acid at the N-terminus of the desired initiator tRNA. 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 moiety may be reacted with piperidine, etc., to convert it into piperidine amide, etc.

[0668] [Example]

[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 this example, 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

[0687] Trt: Triphenylmethyl

[0688] oxyma: Ethyl cyano(hydroxyimino)

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

[0690] Example 1. Compounds used in peptide synthesis, such as the synthesis of amino acids.

[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 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 an Fmoc group, and the C-terminus is a carboxyl group.

[0694] [Table 1]

[0695]

[0696] Example 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] (3S)-3-(9H-fluorene-9-ylmethoxycarbonylamino)-4-oxo-4-piperidin-1-ylbutyric acid (Fmoc-Asp- Synthesis of pip

[0702] [Formula 4]

[0703]

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

[0705] Compound aa002

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

[0707] [Formula 5]

[0708]

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

[0710] compound aa003

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

[0716] [Formula 7]

[0717]

[0718] As used herein, when a compound is combined with a polymer or resin, the polymer or resin portion can be... To clarify the reaction sites of the resin portion, the chemical structure of the reaction sites can be shown to connect to... For example, in the structure Fmoc-Asp(O-Trt (2-Cl)-resin)-pip described above, 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 chlororesin (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 following scheme.

[0720] [Table 3]

[0721]

[0722] Compound rs001:

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

[0724] [Formula 8]

[0725]

[0726] Add 2-chlorotriphenylmethyl chloride resin (1.60 mmol / g, 50.0 g, 80.0 mmol) and DCM (676 mL) to a reaction vessel equipped with a filter, and shake the mixture at room temperature for 10 min. Apply nitrogen pressure to remove DCM, then add 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) to the reaction vessel and shake at room temperature for 60 min. Apply nitrogen pressure to remove the reaction solution, then add a mixture of methanol (99.7 mL, 2.46 mmol), DCM (676 mL), and DIPEA (33.4 mL, 192 mmol) to the reaction vessel and shake at room temperature for 90 min. Apply nitrogen pressure to remove the reaction solution, then add DCM (676 mL). The reaction vessel was shaken for 10 minutes, 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] Compound rs002, (3R)-3-(9H-fluorene-9-ylmethoxycarbonylamino)butyric acid-2-chlorotriphenylmethyl resin Synthesis of (Fmoc-D-3-Abu-O-Trt (2-Cl) resin)

[0728] [Formula 9]

[0729]

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

[0731] Compound rs003, (3R)-3-(9H-fluorene-9-ylmethoxycarbonyl(methyl)amino)butyric acid-2-chlorotriphenylmethyl Synthesis of 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] Compound rs004, (3S)-3-(9H-fluorene-9-ylmethoxycarbonyl(methyl)amino)-4-oxo-4-piperidine-1- Synthesis of 2-chlorotriphenylmethyl butyric acid resin (Fmoc-MeAsp(O-Trt(2-Cl)-resin)-pip)

[0736] [Equation 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 are synthesized by peptide elongation through solid-phase reaction, cleavage, cyclization, side chain deprotection and purification.

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

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

[0743] Peptide elongation through solid-phase reaction

[0744] Solution 1 was prepared by dissolving the Fmoc-protected amino acid (0.6 mol / L) constituting the peptide of interest and HOAt or oxyma (0.375 mol / L) as a carboxyl group activator in NMP. Solution 2 was prepared by mixing DIC with DMF to a concentration of 10% (v / v). A solid-phase reaction vessel with a filter (glass frit) was loaded with the resin (100 mg) supporting the Fmoc amino acid prepared in Examples 1 to 3 and placed in a 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 set in the peptide synthesizer, and automated 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 min in deprotection at the first residue and for 10 min in deprotection at the second and subsequent residues, and then the solution was drained from the glass frit. DMF (0.7 mL) was added, the mixture was allowed to stand for 5 min, and then the solution was drained from the glass frit. This numerical washing step was repeated at least 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 the mixture was allowed to react for 2.5 h 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 a 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, and then the solution was drained from the glass frit. The resulting resin was washed four times with DMF (0.7 mL) and then four times with DCM (0.7 mL).

[0746] Cutting from resin

[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 process 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 added to them, and then the solvent was removed by distillation under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12).

[0748] cyclization

[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 the 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 Genevac high-throughput centrifugal evaporator (HT-12). 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 is performed only on peptides containing Thr. For peptides that do not contain Thr, deprotection is omitted.

[0752] The deprotection of the Thr side chain was carried out by dissolving the residue obtained by the above method in a solution of HFIP / TIPS / DCE (117 / 2.4 / 1 (v / v / v)) in 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 under the synthetic conditions described above. 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 spectrum (observed mass; m / z), ionic form, and retention time (Rt; min) for 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]

[0758] [Table 5]

[0759]

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

[0761] The metabolic stability of the cyclic peptide compounds consisting 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, mouse liver microsomes (male, Corning Incorporated, USA) and 1 μM of the compound were prepared into microsomal solutions at a concentration of 0.5 mg protein / mL and subjected to metabolic reactions for 22, 44, or 66 minutes using 100 mM phosphate-buffered saline (pH 7.4). Protein removal was performed over time using acetonitrile, and the remaining amount of the compound without alteration was analyzed using LCMS to calculate intrinsic hepatic clearance (CLh, intrinsic) (Table 6).

[0763] [Table 6]

[0764]

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

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

[0767] The membrane permeability of the cyclic peptide compound composed 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 FaSSIF medium containing 1% DMSO and DMEM as the top-side medium and DMEM as the bottom-side medium, under 5% CO2, 37°C, and 80 rpm. Then, the top-side and bottom-side media were 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 under 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 (Table 7). When calculating the membrane permeability coefficient (P) from the permeate... app When ), the following expression can be used.

[0769] [Expression 2]

[0770]

[0771] dQ / dt represents the drug penetration 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]

[0773]

[0774] It has been demonstrated that the tested cyclic peptide compounds consisting of 13 or 14 amino acid residues include a wide 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 cyclic peptide compounds.

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

[0778] [Table 8]

[0779]

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

[0781] cLogP (the partition coefficient calculated by computer) was calculated using Daylight version 4.95 from Daylight Chemical Information Systems, Inc. Total AB (total amide bonds) refers to the number of amide bonds in the main chain and side chains present 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 in this example, it is used as an indicator of the lipophilicity of the peptide compound.

[0785] Example 5-1. Analysis of N-alkyl number and membrane permeability

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

[0787] [Table 10]

[0788]

[0789] In the case of compounds with 7 N-alkyl groups, 4 out of the 36 compounds tested showed P app ≥ 0.4 × 10 -6 cm / s, but no compound showed P app ≥ 1.0 × 10 -6 cm / s. In the case of compounds with 8 N-alkyl groups, 19 out of the 48 compounds tested showed P. app ≥ 0.4 × 10 -6 cm / s, and in addition, 7 of these compounds showed P app ≥ 1.0 × 10 -6 cm / s. In the case of compounds with 9 N-alkyl groups, 17 out of the 48 compounds tested showed P. app ≥ 0.4 × 10 -6 cm / s, and in addition, four of these compounds showed P app ≥ 1.0 × 10 -6 cm / s. In the case of compounds with 10 N-alkyl groups, 13 out of the 36 compounds tested showed P. app ≥ 0.4 × 10 -6 cm / s, and in addition, four of these compounds showed P app ≥ 1.0 × 10 -6 cm / s.

[0790] As can be seen from the above, compounds with 7 N-alkyl groups can achieve P app ≥ 0.4 × 10-6 Compounds with 8 or more N-alkyl groups can reach P cm / s. app ≥ 1.0 × 10 -6 cm / s. Furthermore, it was shown that compounds with 8 or more N-alkyl groups achieved P compared to compounds with 7 N-alkyl groups. app ≥ 0.4 × 10 -6 The proportion of compounds with a speed of cm / s is higher.

[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 a cLogP / total AB ratio < 1.0, 1 out of 20 compounds tested showed P app ≥ 0.4 × 10 -6 cm / s, but no compound showed P app ≥ 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 the 52 compounds tested showed P app ≥ 0.4 × 10 -6 cm / s, and in addition, one of these compounds showed P app ≥ 1.0 × 10 -6 cm / s. In the case of compounds with cLogP / total AB ≤ 1.1, 39 out of the 96 compounds tested showed P app ≥ 0.4 × 10 -6 cm / s, and in addition, 14 of these compounds showed P app ≥ 1.0 × 10 -6 cm / s.

[0796] As can be seen from the above, compounds with cLogP / totalAB < 1.0 are unlikely to achieve P.app ≥ 0.4 × 10 -6 Compounds with a velocity of cm / s and a coefficient of 1.0 ≤ cLogP / total AB < 1.1 can achieve P app ≥ 1.0 × 10 -6 Compounds with a velocity of cm / s and a value ≤ cLogP / totalAB can achieve P app ≥ 1.0 × 10 -6 cm / s. It also shows that, compared to compounds with cLogP / total AB < 1.1, for compounds with 1.1 ≤ cLogP / total AB, achieving P... app ≥ 0.4 × 10 -6 The proportion of compounds with a speed of cm / s is higher.

[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 / total AB < 1.0 were less likely to achieve P. app ≥ 0.4 × 10 -6 Therefore, the analysis was only performed on compounds with cLogP / totalAB ≥ 1.0.

[0799] [Table 12]

[0800]

[0801] In the case of compounds with a MeLeu ion, 5 out of 8 compounds tested showed P. app ≥ 0.4 × 10 -6 cm / s, in the case of compounds with two MeLeu, 20 out of the 75 compounds tested showed P app ≥ 0.4 × 10 -6 cm / s, and in the case of compounds with three MeLeu, 27 out of the 63 compounds tested showed P app ≥ 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 P. app ≥ 1.0 × 10 -6 cm / s.

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

[0803] Examples 3 through 5 reveal that having 8 or more N-alkyl groups and a cLogP / total AB ratio 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 changes in plasma concentrations of four types of peptides meeting these conditions after intravenous or oral administration to mice.

[0804] Male mice (C57BL / 6J, 10 weeks old, purchased from Charles River Laboratories Japan, Inc.: 3 mice / group) were administered the compound intravenously at a dose of 1 mg / kg or orally at a dose of 50 mg / kg, and blood was collected from the jugular vein over time over 24 hours after administration. The collected blood was partitioned 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 by non-compartmental analysis using Phoenix WinNonlin 6.1 analysis software (manufactured by Phasight Corporation, USA).

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

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

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

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

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

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

[0811] F: Bioavailability (%)

[0812] [Table 13]

[0813] Pharmacokinetic parameters during intravenous administration

[0814]

[0815] [Table 14]

[0816] Pharmacokinetic parameters during oral administration

[0817]

[0818] All tested compounds exhibited low systemic clearance (1.7 to 14 mL / min / kg) after intravenous administration, indicating high metabolic stability. The compounds showed bioavailability up to 40%, and the four peptides tested demonstrated oral absorption suitable for development as oral administration agents, as expected.

[0819] [Table 15]

[0820]

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.

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