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

Abstract

The present invention provides a cyclic peptide compound optionally linked to a nucleic acid, the cyclic peptide compound having a cyclic moiety, wherein: (1) the cyclic moiety 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 less, and the number of natural amino acid residues is 4 or less; and (3) ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8.

Description

Macrocyclic peptide compounds having cell membrane permeability and metabolic stability, and libraries containing the same

[0001] The present invention relates to macrocyclic peptide compounds having cell membrane permeability and metabolic stability, and libraries containing the same.

[0002] Medium-sized molecules (molecular weight 500-2000) have attracted attention as a potential modality for drug discovery against tough targets, such as protein-protein interaction inhibitors (Non-Patent Document 1). Peptides generally have low drug-likeness (e.g., metabolic stability and cell membrane permeability (hereinafter also referred to as "membrane permeability")), making their development as pharmaceuticals difficult. Recently, it has been found that metabolic stability or membrane permeability can be improved by peptide cyclization or by using unnatural amino acids, such as N-methylamino acids, in peptides (Non-Patent Documents 2 and 3). It has become clear that cyclic peptides containing unnatural amino acids, particularly those containing N-substituted amino acids, can have drug-like properties (Patent Document 1). It has also been suggested that library compounds of cyclic peptides containing unnatural amino acids are useful for the creation of inhibitors of protein-protein interactions (Non-Patent Document 4). Furthermore, in recent years, cyclic peptide compounds with high membrane permeability, libraries containing such compounds, and methods for producing and screening them have been reported (Patent Document 2), and amino acids that can improve the membrane permeability of peptide compounds, peptide compounds containing such amino acids, and methods for producing them have been reported (Patent Document 3).

[0003] In Patent Document 1, it is stated that, in consideration of both 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 residues.

[0004] Patent Document 2 discloses a pre-incubation method using Caco2 cells as a method for measuring the membrane permeability of highly lipid-soluble drugs more accurately than conventional methods. This method has been used to evaluate the membrane permeability of cyclic peptides consisting mainly of 11 amino acid residues (Patent Documents 2 and 3).

[0005] However, for cyclic peptides having a cyclic structure and 13 or more amino acid residues, what kind of cyclic peptides have drug-like properties has not yet been fully verified.

[0006] International Publication No. WO 2013 / 100132 International Publication No. WO 2018 / 225864 International Publication No. WO 2020 / 122182

[0007] Future Med. Chem., 2009, 1,1289-1310.Acc. Chem. Res., 2008, 41,1331-1342.Angew. Chem. Int. Ed., 2013, 52,254-269.Chem. Rev., 2019, 119,10360-10391.

[0008] The present invention has been made in view of the above circumstances, and aims to provide a library for screening cyclic peptides having excellent metabolic stability and membrane permeability. In another aspect, the present invention aims to provide a cyclic peptide compound having high membrane permeability, or a method for producing or screening for the same.

[0009] It is believed that the larger the cyclic peptide, i.e., the greater the number of amino acid residues constituting the cyclic portion, the greater the surface area available for contact with the target molecule. Therefore, peptides with a greater number of amino acid residues constituting the cyclic portion are considered to be advantageous in terms of achieving interaction with the target molecule. Therefore, the present inventors conducted extensive research to determine whether there exists a region in cyclic peptides with 13 or more amino acid residues constituting the cyclic portion, which is outside the previously considered preferred range, that can achieve both membrane permeability and metabolic stability. Specifically, cyclic peptides having various side chains and composed of 13 or 14 amino acid residues were synthesized, and the drug-likeness of cyclic peptide compounds with 13 and 14 amino acid residues constituting the cyclic portion was evaluated by metabolic stability tests using liver microsomes and membrane permeability tests using Caco2 cells. As a result, it was found that peptide compounds with excellent membrane permeability and metabolic stability can be obtained even from cyclic peptides composed of 13 or 14 amino acid residues, with physicochemical properties such as the number of N-substituted amino acids and lipophilicity controlled, and a library for more efficiently obtaining cyclic peptide compounds was also discovered.

[0010] The present invention provides the following [A1] to [A52], [B1] to [B73], and [C1] to [C6]. [A1] A cyclic peptide compound optionally linked to a nucleic acid, wherein the cyclic peptide compound has a cyclic portion, and (1) the cyclic portion is composed of 13 or 14 amino acid residues, and (2) among the amino acid residues constituting the cyclic portion, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues with the same side chain is 3 or less, and the number of natural amino acid residues is 4 or less, and (3) ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8. [A2] 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. [A2-1] The cyclic peptide compound optionally linked to a nucleic acid according to [A1] or [A2], wherein the number of amino acid residues constituting the cyclic peptide compound is 13. [A2-2] A cyclic peptide compound optionally linked to the nucleic acid described in [A1] or [A2], wherein the number of amino acid residues constituting the cyclic peptide compound is 14. [A3] A cyclic peptide compound optionally linked to the nucleic acid described in [A1] or [A2], wherein the number of N-substituted amino acids constituting the cyclic portion is 7 to 13. [A4] A cyclic peptide compound optionally linked to the nucleic acid described in [A3], wherein the number of N-substituted amino acids constituting the cyclic portion is 7 to 10. [A5] A cyclic peptide compound optionally linked to the nucleic acid described in either [A1] or [A2], wherein the number of N-substituted amino acids constituting the cyclic portion is 8 or more. [A6] A cyclic peptide compound optionally linked to the nucleic acid described in [A5], wherein the number of N-substituted amino acids constituting the cyclic portion is 8 to 13. [A7] A cyclic peptide compound optionally linked to the nucleic acid described in [A6], wherein the number of N-substituted amino acids constituting the cyclic portion is 8 to 10. [A8] The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A1] or [A2], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic portion is 60% or more.[A8-1] The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A8], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic portion is 65% or more. [A8-2] The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A8], wherein the ratio of the number of N-substituted amino acid residues to the total number of amino acid residues constituting the cyclic portion is 70% or more. [A9] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any of [A1] to [A8], wherein the N-substituted amino acid is an N-alkyl amino acid. [A10] The N-alkyl amino acid is N-C. １ -C ６[A11] The cyclic peptide compound optionally linked to a nucleic acid according to [A10], wherein the N-alkylamino acid is an N-methyl amino acid or an N-ethyl amino acid. [A12] The cyclic peptide compound optionally linked to a nucleic acid according to [A11], wherein the N-alkylamino acid is an N-methyl amino acid. [A13] The cyclic peptide compound optionally linked to a nucleic acid according to any of [A1] to [A12], wherein the cyclic peptide compound has a ClogP / total AB of 1.0 to 1.5. [A14] The cyclic peptide compound optionally linked to a nucleic acid according to [A13], wherein the cyclic peptide compound has a ClogP / total AB of 1.1 to 1.4. [A15] The cyclic peptide compound optionally linked to a nucleic acid according to any of [A1] to [A14], wherein the cyclic peptide compound has a ClogP of 13 to 20. [A16] The 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 less hydrogen bond donors. [A17] The 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 moiety is composed of at least one selected from the group consisting of an alkyl group, a cycloalkyl group, an aralkyl group, and an amide group, and wherein any atom of the alkyl group and the nitrogen atom of the amino group of the amino acid may form a ring together. [A18] The cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A16], wherein the alkyl group is selected from the group consisting of a halogen atom, a hydroxy group, a C ３ -C ８ Cycloalkyl groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of alkoxy groups １ -C ６ [A19] The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A17], wherein the cycloalkyl group is an alkyl group. １ -C ６ Alkyl group, C １ -C ６Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ３ -C １０ [A20] A cyclic peptide compound optionally having a nucleic acid linked thereto according to [A17] or [A18], wherein the aralkyl group is a cycloalkyl group. １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ７ -C １４ [A21] A cyclic peptide compound to which the nucleic acid according to any one of [A17] to [A19] is optionally linked, wherein the aralkyl group is an optionally substituted C ５ -C １０ Aryl C １ -C ６ is an alkyl group, ５ -C １０ The aryl group may be a halogen atom, １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ [A22] A cyclic peptide compound to which the nucleic acid according to any one of [A17] to [A20] is optionally linked, wherein the amide group is a group represented by the formula: -CONR １ R ２ wherein R １ and R ２ are each independently a hydrogen atom or C １ -C ６alkyl group or R １ and R ２ form a 4- to 8-membered saturated heterocyclic ring together with the nitrogen atom to which they are attached, and the 4- to 8-membered saturated heterocyclic ring is not further substituted by a halogen atom, an oxo group, C １ -C ６[A23] The cyclic peptide compound to which the nucleic acid according to any one of [A1] to [A22] may be linked, wherein the cyclic peptide compound contains at least one amide bond formed between a carboxy group contained in a side chain of an amino acid and a nitrogen atom of an amino group of another amino acid. [A24] The cyclic peptide compound to which the nucleic acid according to any one of [A1] to [A23] may be linked, wherein the cyclic peptide compound contains an amino acid selected from the group consisting of Asp-pip and MeAsp-pip. [A25] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A1] to [A24], wherein the amino acids constituting the cyclic moiety include 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. [A26] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A1] to [A25], wherein 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. [A27] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A1] to [A26], wherein the amino acids constituting the cyclic moiety include α-amino acids, and the number of said α-amino acids is 10 to 14.[A28] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A1] to [A27], wherein the amino acids constituting the cyclic moiety include an α-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). [A29] The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A27] or [A28], wherein 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). [A30] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any of [A1] to [A31], wherein the amino acids constituting the cyclic moiety include an amino acid having a β-amino acid backbone, and the number of amino acids having the β-amino acid backbone is 0 to 2. [A31] The 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. [A32] The 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. [A33] The cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A32], wherein the amino acids constituting the cyclic portion comprise amino acids having a γ-amino acid backbone, and the number of amino acids having the γ-amino acid backbone is 0 to 2.[A34] A cyclic peptide compound optionally linked to the nucleic acid of any one of [A1] to [A33], wherein the amino acids constituting the cyclic portion include gMeAbu. [A35] A cyclic peptide compound optionally linked to the nucleic acid of [A33] or [A34], wherein the γ-amino acids constituting the cyclic portion are each independently gMeAbu. [A36] A cyclic peptide compound optionally linked to the nucleic acid of any one of [A1] to [A35], wherein the number of naturally occurring amino acids constituting the cyclic portion is 1 to 4. [A37] A cyclic peptide compound optionally linked to the nucleic acid of any one of [A1] to [A36], wherein the amino acids constituting the cyclic portion include naturally occurring amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. [A38] The cyclic peptide compound according to [A37], optionally linked to a nucleic acid, 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. [A39] The cyclic peptide compound according to any one of [A1] to [A38], optionally linked to a nucleic acid, wherein the number of unnatural amino acids constituting the cyclic moiety is 9 or more. [A40] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A1] to [A39], wherein the amino acids constituting the cyclic moiety comprise an unnatural 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).[A41] The cyclic peptide compound optionally having a nucleic acid linked thereto according to any one of [A36] to [A40], wherein, among the amino acids constituting the cyclic moiety, unnatural 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). [A42] The cyclic peptide compound Caco-2 P. ａｐｐ (cm / sec) is 0.4 × 10 －６ [A43] The cyclic peptide compound optionally linked to the nucleic acid according to any one of [A1] to [A41]. ａｐｐ The cyclic peptide compound optionally having a nucleic acid linked thereto according to [A44], wherein the value of (cm / sec) is calculated by membrane permeability evaluation under the following conditions: (a) Caco-2 cells are cultured on a 96-well transwell for 3 weeks, and then the compound to be evaluated, DMEM medium, and FaSSIF (containing 1% DMSO) are added to the apical side, and DMEM medium is added to the basal side, and the compound is cultured under 5% CO ２ (b) After the preincubation, the preincubation solution on the apical and basal sides was removed by suction and washed, and a FaSSIF / HBSS buffer solution (containing 1% DMSO, pH 6.0) containing the compound was added to the apical side, and an HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the basal side. (c) Each well was incubated at 5% CO ２ The mixture was shaken at 80 rpm at 37°C under atmospheric pressure, and a sample was taken from the basal side 180 minutes after the start of shaking. The amount of permeation of the compound was measured by liquid chromatography mass spectrometry (LC / MS). (d) The permeation coefficient (Caco-2 P ａｐｐ(cm / sec)) is calculated. [A44] A cyclic peptide compound selected from compounds pd001 to pd168 listed in Table 15. [A45] A cyclic peptide compound (cyclic peptide-nucleic acid complex) according to any one of [A1] to [A44], to which a nucleic acid is linked. [A46] The cyclic peptide-nucleic acid complex according to [A45], wherein the cyclic peptide compound has a cyclic portion and a linear portion, and the nucleic acid is bound to the linear portion. [A47] The cyclic peptide-nucleic acid complex according to [A46], wherein the cyclic peptide compound further comprises a linker, and the nucleic acid is bound to the linear portion via the linker. [A48] The cyclic peptide-nucleic acid complex according to [A47], wherein the linker comprises puromycin. [A49] The cyclic peptide-nucleic acid complex according to any one of [A45] to [A48], wherein the nucleic acid is DNA or RNA. [A50] The cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid comprises a nucleic acid encoding a cyclic peptide compound. [A51] The cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid is a nucleic acid encoding a cyclic peptide compound. [A52] The cyclic peptide-nucleic acid complex according to any one of [A45] to [A49], wherein the nucleic acid comprises a base sequence encoding a cyclic peptide moiety that forms a cyclic peptide-nucleic acid complex.

[0011] [B1] A library comprising a cyclic peptide compound optionally linked to a nucleic acid according to any one of [A1] to [A52]. [B2] A library comprising cyclic peptide compounds optionally linked to a nucleic acid, wherein the cyclic peptide compounds have a cyclic portion, and the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library is 10% or more. [B2-1] The library according to [B1] or [B2], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library is 20% or more. [B2-2] The library according to [B1] or [B2], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library is 40% or more. [B2-3] The library according to [B1] or [B2], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library is 60% or more. [B2-4] The library according to [B1] or [B2], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library is 80% or more. [B3] The library according to [B1] or [B2], wherein the library consists essentially of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion. [B4] The library according to any of [B1] to [B3], wherein the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic peptide compound to the total number of cyclic peptide compounds contained in the library is 10% or more. [B4-1] The library according to any one of [B1] to [B4], wherein the ratio of the number of cyclic peptide compounds each comprising 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 20% or more.[B4-2] The library according to any one of [B1] to [B4], wherein the ratio of the number of cyclic peptide compounds comprising 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 40% or more. [B4-3] The library according to any one of [B1] to [B4], wherein the ratio of the number of cyclic peptide compounds comprising 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 60% or more. [B4-4] The library according to any one of [B1] to [B4], wherein the ratio of the number of cyclic peptide compounds comprising 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library is 80% or more. [B5] The library according to any one of [B1] to [B4], wherein the library consists essentially of cyclic peptide compounds comprising 13 or 14 amino acid residues. [B6] The library according to any one of [B1] to [B5], wherein the ratio of the number of N-substituted amino acids to the total number of amino acids constituting the cyclic portion in the cyclic peptide compounds contained in the library is 50% or more on average. [B7] The library according to any one of [B1] to [B6], wherein the number of N-substituted amino acids is 7 to 13 on average. [B8] The library according to [B7], wherein the number of N-substituted amino acids constituting the cyclic portion in the cyclic peptide compounds contained in the library is 7 to 10 on average. [B9] The library according to [B7], wherein the number of N-substituted amino acids constituting the cyclic portion in the cyclic peptide compounds contained in the library is 8 to 13 on average. [B10] The library according to [B7], wherein the number of N-substituted amino acids constituting the cyclic portion in the cyclic peptide compounds contained in the library is 8 to 10 on average. [B11] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds containing 7 to 10 N-substituted amino acids constituting the cyclic portion is 10% or more among the cyclic peptide compounds contained in the library.[B11-1] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 7 to 10 N-substituted amino acids constituting the cyclic portion is 20% or more. [B11-2] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 7 to 10 N-substituted amino acids constituting the cyclic portion is 40% or more. [B11-3] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 7 to 10 N-substituted amino acids constituting the cyclic portion is 60% or more. [B11-4] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 7 to 10 N-substituted amino acids constituting the cyclic portion is 80% or more. [B12] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 13 N-substituted amino acids constituting the cyclic portion is 10% or more. [B12-1] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 13 N-substituted amino acids constituting the cyclic portion is 20% or more. [B12-2] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 13 N-substituted amino acids constituting the cyclic portion is 40% or more. [B12-3] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 13 N-substituted amino acids constituting the cyclic portion is 60% or more. [B12-4] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic portion is 8 to 13 is 80% or more among the cyclic peptide compounds contained in the library.[B13] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 10 N-substituted amino acids constituting the cyclic portion is 10% or more. [B13-1] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 10 N-substituted amino acids constituting the cyclic portion is 20% or more. [B13-2] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 10 N-substituted amino acids constituting the cyclic portion is 40% or more. [B13-3] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds having 8 to 10 N-substituted amino acids constituting the cyclic portion is 60% or more. [B13-4] The library according to any one of [B1] to [B6], wherein the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic portion is 8 to 10 is 80% or more among the cyclic peptide compounds contained in the library. [B14] The library according to any one of [B1] to [B10], wherein the N-substituted amino acids are N-alkyl amino acids. [B15] The N-alkyl amino acid is N-C. １ -C ６[B16] The library according to [B14], wherein the N-alkylamino acid is an N-methyl amino acid or an N-ethyl amino acid. [B17] The library according to [B14], wherein the N-alkylamino acid is an N-methyl amino acid. [B18] 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. [B18-1] 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. [B18-2] 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. [B19] The library according to any one of [B1] to [B18-2], wherein the average ClogP / totalAB of the peptide portion other than the nucleic acid linkage portion in each cyclic peptide compound contained in the library is 1.0 to 1.8. [B19-1] The library according to any one of [B1] to [B18-2], wherein the average ClogP / totalAB of the peptide portion other than the nucleic acid linkage portion in each cyclic peptide compound contained in the library is 1.0 to 1.5. [B19-2] The library according to any one of [B1] to [B18-2], wherein the average ClogP / totalAB of the peptide portion other than the nucleic acid linkage portion in each cyclic peptide compound contained in the library is 1.1 to 1.4. [B20] The library according to any one of [B1] to [B19-2], wherein the average ClogP of the cyclic peptide compound contained in the library is 13 to 20. [B21] The library according to any one of [B1] to [B20], wherein the average ClogP of the peptide portion other than the nucleic acid linkage portion of each cyclic peptide compound contained in the library is 13 to 20. [B22] The library according to any one of [B1] to [B21], wherein the cyclic peptide compound has a hydrogen bond donor, and the average number of the hydrogen bond donors in each cyclic peptide compound contained in the library is 8 or less.[B22-1] The library according to any one of [B1] to [B21], wherein the cyclic peptide compound has a hydrogen bond donor, and the average number of the hydrogen bond donors in the cyclic portion of each cyclic peptide compound included in the library is 8 or less. [B23] The library according to [B22], wherein the average number of the hydrogen bond donors in the cyclic portion of each cyclic peptide compound included in the library is 3 to 8. [B23-1] The library according to [B22], wherein the average number of the hydrogen bond donors in the cyclic portion of each cyclic peptide compound included in the library is 3 to 8. [B24] The library according to any one of [B1] to [B23], wherein the cyclic peptide compound has a hydrogen bond donor in its cyclic portion. [B25] The library according to any one of [B1] to [B24], wherein the average number of amino acids with the same side chain among the amino acids constituting the cyclic portion of each cyclic peptide compound included in the library is 3 or less. [B26] The library according to any of [B1] to [B24], wherein, in each cyclic peptide compound included in the library, the number of amino acids with the same side chain among the amino acids constituting the cyclic portion is 3 or less. [B27] The library according to any of [B1] to [B26], wherein, in each cyclic peptide compound, the side chain of each amino acid constituting the cyclic portion is composed of at least one selected from the group consisting of an alkyl group, a cycloalkyl group, an aralkyl group, and an amide group, and wherein the alkyl group may optionally form a ring together with any atom of the alkyl group and the nitrogen atom of the amino group of the amino acid. [B28] The library according to any of [B1] to [B26], wherein the alkyl group is selected from the group consisting of a halogen atom, a hydroxy group, C. ３ -C ８ Cycloalkyl groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of alkoxy groups １ -C ６ [B29] The library according to [B27], wherein the cycloalkyl group is an alkyl group. [B29] The library according to [B27], wherein the cycloalkyl group is a halogen atom, a hydroxy group, a C １ -C ６ Alkyl group, C １ -C ６Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ３ -C １０ [B30] The library according to [B27] or [B28], wherein the aralkyl group is a cycloalkyl group. １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ７ -C １４ [B31] The library according to any one of [B27] to [B29], wherein the aralkyl group is an optionally substituted C ５ -C １０ Aryl C １ -C ６ is an alkyl group, ５ -C １０ The aryl group may contain one or more halogen atoms, C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ [B32] The library according to any one of [B27] to [B30], wherein the amide group in each cyclic peptide compound is represented by the formula: -CONR １ R ２ wherein R １ and R ２ are each independently a hydrogen atom or C １ -C ６ alkyl group or R １ and R ２form a 4- to 8-membered saturated heterocyclic ring together with the nitrogen atom to which they are attached, and the 4- to 8-membered saturated heterocyclic ring is not further substituted by a halogen atom, an oxo group, C １ -C ６The library according to any one of [B27] to [B31], which is optionally substituted with at least one group independently selected from the group consisting of an alkyl group and a 4- to 7-membered heterocyclyl group. [B33] The library according to any of [B1] to [B32], wherein, in each cyclic peptide compound, the amino acids constituting the cyclic portion include 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. [B34] The library according to any one of [B1] to [B33], wherein, in each cyclic peptide compound, the amino acid constituting the cyclic portion is independently at least one 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. [B35] The library according to any one of [B1] to [B34], wherein 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. [B36] The library according to any one of [B1] to [B35], wherein, in each cyclic peptide compound, the amino acids constituting the cyclic portion include α-amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu).[B37] The library according to [B35] or [B36], wherein, in each cyclic peptide compound, the α-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, and Ser(tBu). [B38] The library according to any of [B1] to [B37], wherein, 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 the β-amino acid backbone is 0 to 2 on average. [B39] The library 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. [B40] The library according to [B38] or [B39], wherein, in each cyclic peptide compound, the amino acids having a β-amino acid backbone among the amino acids constituting the cyclic portion are each independently selected from the group consisting of Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip. [B41] The library according to any one of [B1] to [B40], wherein, in each cyclic peptide compound, the amino acids constituting the cyclic portion comprise amino acids having a γ-amino acid backbone, and the number of amino acids having the γ-amino acid backbone is 0 to 2 on average. [B42] The library according to any one of [B1] to [B41], wherein, in each cyclic peptide compound, the amino acids constituting the cyclic portion include gMeAbu. [B43] The library according to [B40] or [B41], wherein, in each cyclic peptide compound, an amino acid having a γ-amino acid backbone among the amino acids constituting the cyclic portion is gMeAbu. [B44] The library according to any one of [B1] to [B43], wherein, in each cyclic peptide compound, the number of natural amino acids among the amino acids constituting the cyclic portion is 1 or more on average.[B45] The library according to any one of [B1] to [B44], wherein the number of natural amino acids among the amino acids constituting the cyclic portion in each cyclic peptide compound is 5 or less on average. [B46] The library according to any one of [B1] to [B45], wherein the number of natural amino acids among the amino acids constituting the cyclic portion in each cyclic peptide compound is 1 to 5 on average. [B47] The library according to [B46], wherein the number of natural amino acids among the amino acids constituting the cyclic portion in each cyclic peptide compound is 1 to 4 on average. [B48] The library according to any one of [B1] to [B47], wherein the ratio of the number of natural amino acids to the total number of amino acids among the amino acids constituting the cyclic portion in each cyclic peptide compound is 10% or more on average. [B49] The library according to any one of [B1] to [B48], wherein the ratio of the number of natural amino acids to the total number of amino acids among the amino acids constituting the cyclic portion in each cyclic peptide compound is 40% or less on average. [B50] The library according to any one of [B1] to [B49], wherein the ratio of the number of natural amino acids to the total number of amino acids among the amino acids constituting the cyclic portion of each cyclic peptide compound is 10 to 40% on average. [B51] The library according to any one of [B1] to [B50], wherein the amino acids constituting the cyclic portion of each cyclic peptide compound comprise natural amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. [B52] The library according to any one of [B1] to [B51], wherein the amino acids constituting the cyclic portion of each cyclic peptide compound each independently comprise natural amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. [B53] The library according to any one of [B1] to [B52], wherein the number of unnatural amino acids among the amino acids constituting the cyclic portion of each cyclic peptide compound is 7 or more on average. [B54] The library according to any one of [B1] to [B53], wherein the number of unnatural amino acids among the amino acids constituting the cyclic portion of each cyclic peptide compound is 8 or more on average.[B55] The library according to any one of [B1] to [B54], wherein, in each cyclic peptide compound, the ratio of the number of unnatural amino acids to the total number of amino acids constituting the cyclic portion is 50% or more on average. [B55-1] The library according to [B55], wherein, in each cyclic peptide compound, the ratio of the number of unnatural amino acids to the total number of amino acids constituting the cyclic portion is 60% or more on average. [B55-2] The library according to [B55], wherein, in each cyclic peptide compound, the ratio of the number of unnatural amino acids to the total number of amino acids constituting the cyclic portion is 70% or more on average. [B55-3] The library according to [B55], wherein, in each cyclic peptide compound, the ratio of the number of unnatural amino acids to the total number of amino acids constituting the cyclic portion is 80% or more on average. [B56] The library according to any one of [B1] to [B55-3], wherein each cyclic peptide compound comprises an unnatural 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). [B57] The library according to any of [B1] to [B56], wherein, in each cyclic peptide compound, the amino acids constituting the cyclic moiety each independently comprise an unnatural 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). [B58] The library according to any one of [B1] to [B57], wherein, in each cyclic peptide compound, no amino acid constituting the cyclic peptide compound has a methylthio group, a thiol group, an indole skeleton, or a substituted or unsubstituted hydroxyphenyl group in its side chain.[B59] The library according to any one of [B1] to [B58], wherein the molecular weight of each cyclic peptide compound is 500 to 2000 on average. [B60] The library according to any one of [B1] to [B59], wherein the molecular weight of the peptide portion other than the nucleic acid linking portion of each cyclic peptide compound is 500 to 2000 on average. [B61] The library according to any one of [B1] to [B60], wherein the library is for use in identifying an orally administrable compound or a precursor thereof. [B62] The library according to any one of [B1] to [B61], wherein the library is for use in identifying a cyclic peptide compound capable of specifically binding to a target molecule. [B63] The library according to any one of [B1] to [B62], wherein the library is for use in identifying a compound capable of specifically binding to a target molecule. [B64] The library is 1 x 10. ２ [B64-1] The library according to any one of [B1] to [B63], having a diversity of 1 x 10 or more. ３ [B64-2] The library according to [B64], having a diversity of 1 x 10 or more. ４ [B64-3] The library according to [B64], having a diversity of 1 x 10 or more. ６ [B64-4] The library according to [B64], having a diversity of 1 x 10 or more. ８ [B64-5] The library according to [B64], having a diversity of 1 x 10 or more. １０ [B65] The library according to [B64], having a diversity of 1 x 10 or more. ２ [B65-1] The library according to any one of [B1] to [B63], comprising cyclic peptide compounds to which one or more types of nucleic acids may be linked. ３ [B65-2] The library according to [B65], comprising cyclic peptide compounds to which one or more types of nucleic acids may be linked. ４[B65-3] The library according to [B65], comprising cyclic peptide compounds to which one or more types of nucleic acids may be linked. ６ [B65-4] The library according to [B65], comprising cyclic peptide compounds to which one or more types of nucleic acids may be linked. ８ [B65-5] The library according to [B65], comprising cyclic peptide compounds to which one or more types of nucleic acids may be linked. １０ The library according to [B65], comprising a cyclic peptide compound to which one or more types of nucleic acids may be linked. [B66] The library according to any of [B1] to [B65-5], comprising at least one cyclic peptide compound selected from the cyclic peptide compounds listed in Table 15 (i.e., pd001 to pd168). [B67] The library according to any of [B1] to [B65-5], comprising all of the cyclic peptide compounds listed in Table 15 (i.e., pd001 to pd168). [B68] The library according to any of [B1] to [B67], wherein the cyclic peptide compounds included in the library are cyclic peptide compounds to which a nucleic acid is linked (cyclic peptide-nucleic acid complex). [B69] The library according to [B68], wherein the nucleic acid in the cyclic peptide-nucleic acid complex comprises a nucleic acid encoding the cyclic peptide. [B70] The library according to [B68], wherein the nucleic acid in the cyclic peptide-nucleic acid complex is a nucleic acid encoding the cyclic peptide. [B70-1] 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 comprises a base sequence encoding a cyclic peptide moiety that forms the cyclic peptide-nucleic acid complex. [B71] The library according to any one of [B1] to [B70], wherein the library is a display library. [B72] The library according to [B71], wherein the display library is a nucleic acid display library. [B73] The library according to [B72], wherein the nucleic acid display library is an mRNA display library.

[0012] [C1] A method for screening for a cyclic peptide compound capable of specifically binding to a target molecule, comprising the following steps: (i) contacting the target molecule with the library according to any one of [B1] to [B73]; and (ii) selecting a cyclic peptide compound capable of specifically binding to the target molecule. [C2] The method according to [C1], wherein the target molecule is a protein. [C3] The method according to [C1] or [C2], wherein in (ii), the cyclic peptide compound capable of specifically binding to the target molecule is selected as a candidate compound for a pharmaceutical. [C4] The method according to any one of [C1] to [C3], comprising chemically synthesizing a candidate cyclic peptide compound; and measuring the membrane permeability of the chemically synthesized cyclic peptide compound using Caco-2 cells. [C5] A method according to any one of [C1] to [C3], comprising the steps of: (i) contacting the target molecule with the library according to any one of [B1] to [B73]; and (ii) selecting a cyclic peptide compound capable of specifically binding to the target molecule. [C6] The method according to [C7], wherein the cyclic peptide compound is selected as a candidate compound for a pharmaceutical product. [C7] The method according to [C8], wherein the cyclic peptide compound is selected as a candidate compound for a pharmaceutical product. [C8] The method according to [C9], wherein the cyclic peptide compound is selected as a candidate compound for a pharmaceutical product. ａｐｐ [C6] The method according to any one of [C1] to [C4], wherein the value of P is determined by the value of (cm / sec). ａｐｐ The method according to [C5], wherein the value of (cm / sec) is calculated by membrane permeability evaluation according to the following steps: (a) after culturing Caco-2 cells on a 96-well transwell for 3 weeks, a compound to be evaluated, DMEM medium, and FaSSIF (containing 1% DMSO) are added to the apical side, and DMEM medium is added to the basal side, and the cells are incubated in a 5% CO atmosphere. ２ (b) After the preincubation, the preincubation solution on the apical and basal sides was removed by suction and washed, and a FaSSIF / HBSS buffer solution (containing 1% DMSO, pH 6.0) containing the compound was added to the apical side, and an HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the basal side. (c) Each well was incubated at 5% CO ２ The mixture was shaken at 80 rpm at 37°C under atmospheric pressure, and a sample was taken from the basal side 180 minutes after the start of shaking. The amount of permeation of the compound was measured by liquid chromatography mass spectrometry (LC / MS). (d) The permeation coefficient (Caco-2 P ａｐｐ (cm / sec) is calculated.

[0013] In the above numbering scheme, the numbers referred to in dependent claims include their sub-numbers unless otherwise specified. For example, [B5] referred to in a dependent claim means that it includes not only [B5] but also its sub-numbers [B5-1], [B5-2], [B5-3], and [B5-4]. The same applies to other numbering schemes.

[0014] In one aspect, the present invention provides guidelines for derivatizing cyclic peptide compounds that are potential drug candidates into compounds with excellent membrane permeability and metabolic stability. In another aspect, the present invention enables the development of cyclic peptides formed from 13 or 14 amino acid residues, which have previously been difficult to achieve drug-likeness despite their merit of interacting with a target. In yet another aspect, the present invention provides a screening method for obtaining hit cyclic peptide compounds with excellent drug-likeness by designing a library of cyclic peptide compounds based on the present invention.

[0015] FIG. 1 is a schematic diagram showing an example of a method for producing a peptide compound using a solid phase method.

[0016] Preferred, non-limiting embodiments of the present disclosure are described below.

[0017] All elements described in the following examples are intended to be considered to be equivalently described in this "Form for Carrying Out the Invention" without being bound by any patent practice, custom, laws, regulations, etc. in any country in which the patent application is intended to be granted that may attempt to restrictively interpret the contents described in the examples.

[0018] Any combination of part or all of one or more elements described anywhere in this disclosure is intended to be included in this disclosure, unless there is a technical contradiction based on the common technical knowledge of a person skilled in the art, and is described as being naturally understood by a person skilled in the art.

[0019] The following abbreviations are used herein: Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), Gln (glutamine), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), and Val (valine). In addition to these, the abbreviations listed in Table 1 are also used.

[0020] As used herein, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any one hydrogen atom, and does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, but has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. Alkyl includes not only linear but also branched chain alkyls. Alkyl preferably has 1 to 20 carbon atoms (C １ -C ２０ , hereinafter referred to as “C ｐ -C ｑ " means that the number of carbon atoms is p to q), and preferably C １ -C １０ Alkyl, more preferably C １ -C ６Specific examples of the alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, isobutyl (also referred to as 2-methylpropyl), n-pentyl, s-pentyl (also referred to as 1-methylbutyl), t-pentyl (1,1-dimethylpropyl), neopentyl (also referred to as 2,2-dimethylpropyl), isopentyl (also referred to as 3-methylbutyl), 3-pentyl (also referred to 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.

[0021] As used herein, "alkoxy" refers to an oxy group to which the above-defined "alkyl" is bonded. １ -C ６ Alkoxy is preferred, C １ -C ４ Alkoxy is more preferred. Specific examples of alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, pentyloxy, and 3-methylbutoxy.

[0022] As used herein, "haloalkoxy" refers to a group in which one or more hydrogen atoms of the "alkoxy" defined above are substituted with halogen. １ -C ６ Alkoxy is preferred, and fluoro C １ -C ６ Alkoxy is more preferred. Specific examples of haloalkoxy include difluoromethoxy, trifluoromethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 3-fluoropropoxy, and 2,2-difluoropropoxy.

[0023] As used herein, "alkenyl" refers to an alkyl group having at least one double bond (two adjacent SP２ It is a monovalent group having 1 carbon atom. Depending on the configuration of the double bond and the substituents (if any), the geometry of the double bond can be in the Entgegen (E) or Zusammen (Z), cis or trans configuration. Alkenyl includes not only straight chains but also branched chains. C is preferred as alkenyl. ２ -C １０ alkenyl, more preferably C ２ -C ６ Specific examples of alkenyl include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, 3-methyl-2-butenyl, and hexenyl.

[0024] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Alkynyl includes not only straight chain but also branched chain. C is preferred as alkynyl. ２ -C １０ Alkynyl, more preferably C ２ -C ６ Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, hexynyl, 3-phenyl-2-propynyl, 3-(2-fluorophenyl)-2-propynyl, 2-hydroxy-2-propynyl, 3-(3-fluorophenyl)-2-propynyl, and 3-methyl-(5-phenyl)-4-pentynyl.

[0025] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including monocyclic, bicyclic, and spirocyclic rings. ３ -C ８ Cycloalkyl, C ３ -C ７ Cycloalkyl, more preferably C ３ -C ６Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, spiro[3.3]heptyl, and the like.

[0026] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, i.e., an aromatic hydrocarbon ring group. Preferred examples of aryl include C ６ -C １０ Specific examples of the aryl include phenyl and naphthyl (for example, 1-naphthyl and 2-naphthyl).

[0027] In this specification, the above-mentioned "aryl group" may include the below-mentioned "heteroaryl".

[0028] As used herein, the term "heteroaryl" refers to an aromatic monovalent cyclic group containing 1 to 5 heteroatoms in addition to carbon atoms, i.e., an aromatic heterocyclic group. The ring may be a single ring or a condensed ring with another ring, and may be partially saturated. The number of atoms constituting the heteroaryl ring is preferably 5 to 10 (5- to 10-membered heteroaryl), and more preferably 5 to 7 (5- to 7-membered heteroaryl).

[0029] Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, benzotriazolyl, indolyl, isoindolyl, indazolyl, azaindolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, imidazopyridyl, pyrazolopyridyl, imidazopyridyl, triazolopyridyl, pyrrolopyrazinyl, and furopyridyl.

[0030] As used herein, "aralkyl (arylalkyl)" refers to a group in which at least one hydrogen atom of an "alkyl" as defined herein is substituted with an "aryl" as defined herein. ７ -C １４ Aralkyl, C ５ -C １０ Aryl C １ -C ６ Alkyl is preferred, C ７ -C １０ Aralkyl is more preferred. Specific examples of aralkyl include benzyl, phenethyl, and 3-phenylpropyl.

[0031] As used herein, "alkylene" refers to a divalent group derived from an "alkyl" as defined herein by further removing one optional hydrogen atom, and C ４ -C ８ Alkylene is preferred. Specific examples of alkylene include —CH ２ -, -(CH ２ ) ２ -, -(CH ２ ) ３ -, -CH(CH ３ ) CH ２ -, -C(CH ３ ) ２ -, -(CH ２ ) ４ -, -CH(CH ３ ) CH ２ CH ２ -, -C(CH ３ ) ２ CH ２ -, -CH ２ CH (CH ３ ) CH ２ -, -CH ２ C(CH ３ ) ２ -, -CH ２ CH ２ CH (CH ３ ) -, -CH ２ CH (CH ２ CH ３ ) -, -(CH ２ ) ５ -, -CH(CH ３ ) CH(CH ２ CH３ ) -, -(CH ２ ) ６ -, -(CH ２ ) ７ -, -(CH ２ ) ８ Examples of alkylene include C １ -C ８ Alkylene is preferred, C ４ -C ８ Alkylene is more preferred.

[0032] As used herein, "arylene" refers to a divalent group derived from "aryl" as defined herein by further removing one optional hydrogen atom. The arylene may be a single ring or a condensed ring. The number of atoms constituting the arylene ring is not particularly limited, but is preferably 6 to 10 (C ６ -C １０ Arylene. Specific examples of arylene include 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 1,2-naphthylene, 1,3-naphthylene, and 1,4-naphthylene.

[0033] As used herein, the term "heteroarylene" refers to a divalent group derived by further removing one optional hydrogen atom from a "heteroaryl" as defined herein. A heteroarylene may be a single ring or a fused ring. The number of atoms constituting the ring is not particularly limited, but is preferably 5 to 10 (5- to 10-membered heteroarylene). Specific examples of heteroarylene include pyrrolediyl, imidazolediyl, pyrazolediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazolediyl, triazinediyl, isoxazolediyl, oxazolediyl, oxadiazolediyl, isothiazolediyl, thiazolidinyl, thiadiazolediyl, furandiyl, and thiophenediyl.

[0034] In the present specification, the term "alkylenearylene" refers to a divalent group in which the alkylene and the arylene are bonded at any position, and the alkylene is bonded to the basic skeleton side. Specific examples of alkylenearylene include -C １ -C ６Alkylene-C ６ -C １０ Examples include arylene.

[0035] In the present specification, the term "arylene alkylene" refers to a divalent group in which the arylene and the alkylene are bonded at any position, and the arylene is bonded to the basic skeleton side. Specific examples of the arylene alkylene include -C ６ -C １０ Arylene-C １ -C ６ Alkylene and the like.

[0036] As used herein, the term "alkyleneheteroarylene" refers to a divalent group in which the alkylene and the heteroarylene are bonded at any position, and the alkylene is bonded to the basic skeleton side. Specific examples of alkyleneheteroarylene include -C １ -C ６ alkylene-5- to 10-membered heteroarylene.

[0037] In the present specification, the term "heteroarylene alkylene" refers to a divalent group in which the heteroarylene and the alkylene are bonded at any position, and the heteroarylene is bonded to the basic skeleton side. Specific examples of heteroarylene alkylene include -5-membered-10-membered heteroarylene-C １ -C ６ Alkylene and the like.

[0038] 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, and is a group in which, for example, OBt, OAt, OSu, OPfp, SR1, or the like is bound to the carbonyl group, and is a group that can promote the reaction with an amino group.

[0039] A "reaction promoting group" is a group that is introduced near a functional group to be bonded in order to selectively cause a reaction at a desired position, and that activates the functional group for the bonding reaction. For example, to react a carbonyl group with an amino group, a reaction promoting group can be introduced into either or both of the carbonyl group and the amino group. Examples of such reaction promoting groups include SH. Such reaction promoting groups can be removed during or after the bonding reaction.

[0040] As used herein, "amino acid" refers to a carboxylic acid having an amino group, and includes natural amino acids and unnatural amino acids. Furthermore, as used herein, "amino acid" may refer to an amino acid residue. As used herein, "natural amino acid" refers to any L-amino acid selected from Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro. As used herein, "unnatural amino acid" refers to an amino acid other than a natural amino acid. Examples of unnatural amino acids include β-amino acids, γ-amino acids, L-amino acids (excluding the natural amino acids described above), D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, and amino acids whose side chains differ from those of natural amino acids. Furthermore, as used herein, modified amino acids may be expressed as "MeAsp-pip" or the like. "MeAsp-pip" indicates an amino acid in which the N-terminal amino group of aspartic acid is substituted with a methyl group (i.e., N-methylated) and the C-terminal carboxy group is amidated with piperidine. 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 carboxy group. Notations with no letters on the left or right indicate an amino group or a carboxy group, respectively. Furthermore, "bAla" indicates β-alanine, and "gMeAbu" indicates 4-(methylamino)butanoic acid. β-amino acids and γ-amino acids are represented by the letters b and g, respectively, on the far left.

[0041] The amino acids herein may have any steric configuration. The side chain of an amino acid is not particularly limited, and may be freely selected from, in addition to a hydrogen atom, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a heteroaralkyl group, a cycloalkyl group, or a spiro-linked cycloalkyl group. Each of these groups may be substituted, and the substituents are not limited thereto. For example, one or more groups may be independently selected from any substituent containing a halogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a boron atom, a silicon atom, or a phosphorus atom. Specifically, examples of the amino acid include optionally substituted alkyl groups, alkoxy groups, alkoxyalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups, each of which may further optionally have an additional substituent. Furthermore, examples of substituents that may be optionally added to the side chain of an amino acid include deuterium, cyano, nitro, and aminocarbonyl. In a non-limiting embodiment, the amino acid herein may be a compound having a carboxyl group and an amino group in the same molecule. Even in this case, proline, hydroxyproline, azetidine-2-carboxylic acid, and the like, in which the nitrogen atom of the amino group and any atom of the side chain form a ring, are also included in the amino acids.

[0042] As used herein, the term "amino acid main chain" refers to the chain portion composed of an amino group, an α-carbon, and a carboxyl group in the case of an α-amino acid; the chain portion composed of an amino group, a β-carbon, an α-carbon, and a carboxyl group in the case of a β-amino acid; and the chain portion composed of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group in the case of a γ-amino acid. In the present specification, when an amino acid has two carboxyl groups, such as aspartic acid, and can be considered both an α-amino acid and a β-amino acid, the amino acid is defined as the amino acid with the shorter chain portion composed of an amino group, a carbon, and a carboxyl group. In other words, aspartic acid is defined as an "α-amino acid." Furthermore, the term "α-amino acid backbone" refers to the chain portion composed of an amino group, an α-carbon, and a carboxyl group; the term "β-amino acid backbone" refers to the chain portion composed 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 composed of an amino group, a γ-carbon, a β-carbon, an α-carbon, and a carboxyl group. For example, aspartic acid has a β-amino acid skeleton, and therefore also falls under the category of "amino acids having a β-amino acid skeleton." Furthermore, glutamic acid has a γ-amino acid skeleton, and therefore also falls under the category of "amino acids having a γ-amino acid skeleton."

[0043] The main chain amino group of the amino acid is unsubstituted (NH ２ group), and may be substituted (i.e., an —NHR group: R represents an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group which may have a substituent, and one or two non-adjacent methylene groups in these groups are an oxygen atom, a carbonyl group (—C(═O)—), or a sulfonyl group (—SO ２-), or the carbon chain bonded to the N atom and the carbon atom at the α-position may form a ring, as in proline. The substituent for R is selected in the same manner as the substituents in the amino acid side chains described above. When the main chain amino group is substituted, R is included in the "side chain of amino acid" in this specification. In this specification, an amino acid in which such a main chain amino group is substituted is referred to as an "N-substituted amino acid." As the "N-substituted amino acid" in this specification, an N-alkyl amino acid is preferred, and an N-C １ -C ６ Alkyl amino acids, N-C １ -C ４ The N-substituted amino acid may be an alkyl amino acid. The N-substituted amino acid may be a N-methyl amino acid or an N-ethyl amino acid, and more preferably an N-methyl amino acid.

[0044] As used herein, the term "side chain" refers to a portion other than the main chain structure, and is used in the context of the side chain of an amino acid, the side chain of the cyclic portion of a cyclic peptide compound, etc. However, in the cyclic peptide-nucleic acid complex described below, the nucleic acid portion and the nucleic acid linking portion are not included in the side chain.

[0045] The "amino acids" constituting the peptide compound include all corresponding isotopes. An isotope of an "amino acid" is one in which at least one atom has been replaced with an atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes contained in the "amino acids" constituting the peptide compound of the present invention include isotopes of hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., 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 and the like are included.

[0046] Examples of halogen atoms include fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), etc. Examples of substituents derived from halogen atoms include, in addition to halogen atoms, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, each of which may be substituted with one or more halogen atoms.

[0047] Examples of the substituent include a halogen atom, a hydroxy group (—OH), a carboxy group, an oxo group (═O), a sulfhydryl group (—SH, also called a thiol group), a thiocarboxy group, an oxy group (—O—), a thioether group (—S—), a sulfoxide group (—S(═O)—), a sulfone group (—S(═O) ２ -), sulfo group (-SO ３ H), azide group (-N ３ and a cyano group (—CN). The substituent may be a combination of two or more of these to form an ester, a thioester, an amide, a carbamate, an alkylthio group (—SR), a sulfonamide, a sulfamide, a thiocarboxylic acid group (—C(═O)—SH), a keto acid (—C(═O)—CO ２ H), or a urea group may be formed.

[0048] The alkoxy group (-OR) is selected from alkylalkoxy groups, cycloalkylalkoxy groups, alkenylalkoxy groups, alkynylalkoxy groups, arylalkoxy groups, heteroarylalkoxy groups, aralkylalkoxy groups, etc. Substituents derived from an oxo group include a hydrocarbonyl group (-CHO, which yields an aldehyde as a compound), an alkylcarbonyl group (which yields a ketone as a compound), a cycloalkylcarbonyl group, an alkenylcarbonyl group, an alkynylcarbonyl group, an arylcarbonyl group, a heteroarylcarbonyl group, and an aralkylcarbonyl group. Substituents that form esters include a carbonyloxy group (-O-C(=O)-) and an alkoxycarbonyl group (-C(=O)O-). The alkoxycarbonyl group is selected from an alkyloxycarbonyl group, a cycloalkyloxycarbonyl group, an alkenyloxycarbonyl group, an alkynyloxycarbonyl group, an aryloxycarbonyl group, a heteroaryloxycarbonyl group, an aralkyloxycarbonyl group, and the like; and the carbonyloxy group is selected from an alkylcarbonyloxy group, a cycloalkylcarbonyloxy group, an alkenylcarbonyloxy group, an alkynylcarbonyloxy group, an arylcarbonyloxy group, a heteroarylcarbonyloxy group, an aralkylcarbonyloxy group, and the like.

[0049] Examples of the substituent that forms a thioester include a carbonylthio group (-S-C(=O)-), an alkylthiocarbonyl group (-C(=O)-SR), etc. The substituent that forms a thioester is selected from, for example, an alkylcarbonylthio group, a cycloalkylcarbonylthio group, an alkenylcarbonylthio group, an alkynylcarbonylthio group, an arylcarbonylthio group, a heteroarylcarbonylthio group, an aralkylcarbonylthio group, etc., or an alkylthiocarbonyl group, a cycloalkylthiocarbonyl group, an alkenylthiocarbonyl group, an alkynylthiocarbonyl group, an arylthiocarbonyl group, a heteroarylthiocarbonyl group, or an aralkylthiocarbonyl group.

[0050] Examples of the substituent that forms an amide include an alkylcarbonylamino group (-NH-C(=O)-R), a cycloalkylcarbonylamino group, an alkenylcarbonylamino group, an alkynylcarbonylamino group, a cycloalkylcarbonylamino group, an arylcarbonylamino group, a heteroarylcarbonylamino group, and an aralkylcarbonylamino group; or an alkylaminocarbonyl group (-C(=O)-NHR), a cycloalkylaminocarbonyl group, an alkenylaminocarbonyl group, an alkynylaminocarbonyl group, an arylaminocarbonyl group, a heteroarylaminocarbonyl group, and an aralkylaminocarbonyl group. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0051] Examples of the substituent that forms a carbamate include an alkyloxycarbonylamino group (—NH—CO—OR), a cycloalkyloxycarbonylamino group, an alkenyloxycarbonylamino group, an alkynyloxycarbonylamino group, a cycloalkyloxycarbonylamino group, an aryloxycarbonylamino group, a heteroaryloxycarbonylamino group, an aralkyloxycarbonylamino group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or an aralkyl group.

[0052] The substituents that form sulfonamide include alkylsulfonylamino groups (—NH—SO ２ -R), a cycloalkylsulfonylamino group, an alkenylsulfonylamino group, an alkynylsulfonylamino group, a cycloalkylsulfonylamino group, an arylsulfonylamino group, a heteroarylsulfonylamino group, an aralkylsulfonylamino group, or an alkylaminosulfonyl group (-SO ２—NHR), cycloalkylaminosulfonyl group, alkenylaminosulfonyl group, alkynylaminosulfonyl group, arylaminosulfonyl group, heteroarylaminosulfonyl group, aralkylaminosulfonyl group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced with an alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or aralkyl group.

[0053] The substituent for forming a sulfamide is an N-alkylsulfamoylamino group (—NH—SO ２ —NHR), N-cycloalkylsulfamoylamino group, N-alkenylsulfamoylamino group, N-alkynylsulfamoylamino group, N-cycloalkylsulfamoylamino group, N-arylsulfamoylamino group, N-heteroarylsulfamoylamino group, N-aralkylsulfamoylamino group, etc. Further examples include compounds in which the H atom bonded to the N atom is replaced by any two of the same or different alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or by substituents selected from substituents in which these groups may form a ring.

[0054] Substituents forming a thioether are selected from alkylthio groups, cycloalkylthio groups, alkenylthio groups, alkynylthio groups, arylthio groups, heteroarylthio groups, aralkylthio groups, etc. Substituents forming a sulfoxide group (-S(=O)-R) are selected from alkylsulfinyl groups, cycloalkylsulfinyl groups, alkenylsulfinyl groups, alkynylsulfinyl groups, arylsulfinyl groups, heteroarylsulfinyl groups, aralkylsulfinyl groups, etc. Substituents forming a sulfone group (-S(=O)-R) are selected from alkylsulfinyl groups, cycloalkylsulfinyl groups, alkenylsulfinyl groups, alkynylsulfinyl groups, arylsulfinyl groups, heteroarylsulfinyl groups, aralkylsulfinyl groups, etc. ２ The substituent forming —R) is selected from an alkylsulfonyl group, a cycloalkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, a heteroarylsulfonyl group, an aralkylsulfonyl group, and the like.

[0055] The amino group (-NH ２ ) and examples of the substituent that forms a secondary amine (—NH—R) include an alkylamino group, a cycloalkylamino group, an alkenylamino group, an alkynylamino group, an arylamino group, a heteroarylamino group, and an aralkylamino group. １ Examples of the substituents forming the amidino group (-C(=NR)-NR) include an alkyl (aralkyl) amino group, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, and the like, and two or two of the same substituents, or substituents which may form a ring, are selected from these groups. １ R ２ ) as a substituent for forming an amidino group (—C(═NH)—NH ２ ), or three substituents on the N atom are substituted with any three of the following groups, which may be the same or different: alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and aralkyl groups, such as an alkyl (aralkyl) (aryl) amidino group; a guanidino group (—NR—C(═NR ３ )-NR １ R ２ As a substituent for forming a guanidine group (—NH—C(═NH)—NH ２ ) or R, R １ , R ２ , R ３ are any four of the same or different alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents selected from these groups which may form a ring.

[0056] The substituent that forms a urea group is an aminocarbamoyl group (—NR—CO—NR １ R ２ ) can be mentioned. １ , R ２are each a hydrogen atom, or any three of the same or different alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and aralkyl groups, or substituents selected from substituents which may form a ring.

[0057] In addition, alkylborane (-BR(R １ )) and alkoxyborane (-B(OR)(OR １ These two substituents may be any two or two identical substituents selected from alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, etc., or substituents selected from substituents which may form a ring.

[0058] In this way, one or more of various functional groups including O atom, N atom, S atom, B atom, P atom, Si atom, and halogen atom, which are usually used in low molecular weight compounds, may be added. In other words, one or more other substituents may be added to the alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and aralkyl group shown as one of these substituents. A functional group that satisfies all of these conditions is defined as freely selecting the substituent. In the case of β- or γ-amino acids, any steric configuration is allowed, just as in the case of α-amino acids, and the selection of the side chain is also not particularly limited, just like in the case of α-amino acids. In addition, the amino group site in the main chain of the amino acid may be a free (-NH ２ group), or may be N-alkylated such as N-methylated (-NHR group: R represents an alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, or cycloalkyl group which may be freely substituted, and the carbon atom bonded to the N atom and a carbon atom from the side chain (for example, the α-position) may form a ring, as in proline. The substituent can be freely selected, and examples thereof include a halogen atom, an ether group, and a hydroxy group).

[0059] As used herein, the term "translated amino acid" or "translatable amino acid" refers to an "amino acid" having a side chain that can be translated. In a non-limiting embodiment, the amino acid used herein may be a translatable amino acid.

[0060] As used herein, the term "drug-likeness" or "drug-like" refers to the properties of excellent metabolic stability and membrane permeability.

[0061] As used herein, "drug-like amino acids" refer to amino acids that have the same backbone as "amino acids," i.e., α, β, and γ amino acids, and have a main chain amino group (NH ２ one of the two hydrogen atoms of the methylene group (-CH ２ One or two of the hydrogen atoms of the - group may be substituted with an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, or the like. ２A group in which a hydrogen atom of a group is substituted with the above group is defined as a side chain. These substituents also include those further substituted with substituents that function as building blocks of drug-like peptide compounds. These are preferably selected from the substituents defined separately above, and may be substituted with one or more substituents selected from, for example, a hydroxy group, an alkoxy group, an amide group, a sulfonyl group, a sulfinyl group, a halogen atom, a hydroxyamino group, an aminohydroxy group, etc. Furthermore, they may correspond to L-amino acids, D-amino acids, and α,α-dialkylamino acids. Drug-like amino acids do not necessarily need to be translatable. They include the side chain portion of a peptide obtained from a "translated amino acid" (e.g., if a hit compound is obtained with D-tyrosine, a D-amino acid chemically modified from D-tyrosine; if a hit compound is obtained with β-alanine, a β-amino acid chemically modified from D-alanine), or all amino acids that can be chemically synthesized by structural optimization of the N-substituted portion through chemical conversion of an N-methyl amino acid. Because these amino acids function as building blocks of drug-like peptide compounds, they are selected from a range that will result in drug-like peptide compounds obtained by post-translational chemical modification. As described below, for example, lysine having an aminoalkyl group is not included in the drug-like amino acids if the amino group is not involved in post-translational modification. However, when the amino group of lysine is utilized as a reactive functional group in post-translational modification (e.g., a crossover unit), the lysine unit is included as a drug-like amino acid unit. Thus, whether an amino acid is a "drug-like amino acid" is determined by the functional group after conversion by post-translational modification. Examples of such substituents include, from the substituents defined separately above, an ester group, a thioester group, a sulfhydryl group, a protected thiol group, an amino group, a mono-substituted amino group, a di-substituted amino group, a protected amino group, a substituted sulfonylamino group, an alkylborane group, an alkoxyborane group, an azide group, a keto acid group, a thiocarboxy group, a phosphoryl ester group, and an acyloxyamino group.Although details will be described later, in one aspect of the present disclosure, an indole skeleton and / or a substituted or unsubstituted hydroxyphenyl group are not included in the drug-like amino acids of the present disclosure.

[0062] As used herein, the term "substituted hydroxyphenyl group" refers to a group in which at least one hydrogen atom in the aromatic ring of a hydroxyphenyl group is substituted with a substituent. The substituent is selected in the same manner as the substituents in the amino acid side chains described above. Although not intended to be limiting, an example of a substituted hydroxyphenyl group is a 3-fluoro-4-hydroxyphenyl group. Note that the hydrogen atoms in the aromatic ring in this specification do not include the H of the hydroxy group (—OH) in the hydroxyphenyl group. For example, a methoxyphenyl group is not included in either the "substituted hydroxyphenyl group" or the "unsubstituted hydroxyphenyl group" in this specification.

[0063] In this specification, the term "unsubstituted hydroxyphenyl group" refers to a hydroxyphenyl group that has no substituent. In addition, substituted hydroxyphenyl groups and unsubstituted hydroxyphenyl groups may be collectively referred to as "substituted or unsubstituted hydroxyphenyl groups."

[0064] In this specification, "having no substituted or unsubstituted XX group" means having neither a substituted XX group nor an unsubstituted XX group.

[0065] As used herein, "translational synthesis" refers to the synthesis of a peptide compound by translation from a nucleic acid (e.g., DNA, RNA) that encodes the peptide compound. Translation is a process in which a linear peptide is obtained by repeating amide bond and ester bond reactions using mRNA as a template through the action of ribosomes.

[0066] The term "post-translational modification" refers to a chemical reaction that occurs after translation automatically or by adding another reagent other than through the action of ribosomes, and examples thereof include cyclization and deprotection reactions.

[0067] In this specification, the term "substantially consisting of" may mean that the ratio of the theoretical number of peptide compounds and / or nucleic acid variations encoding them listed therein to the theoretical total number of peptide compounds and / or nucleic acid variations encoding them contained in the library is 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, but is not limited thereto.

[0068] As used herein, the phrase "substantially free of" means that the component listed therein (examples include, but are not limited to, cyclic peptide compounds, amino acids, nucleic acids, etc.) is not included, or if the component is included, the component does not have a negative effect on the effects of one aspect of the present invention, or the component is included in an amount or in a form that does not have a negative effect. For example, the component listed therein may be included as long as it does not have a negative effect on the effects of one aspect of the present invention, or the component is included in an amount or in a form that does not have a negative effect.

[0069] As used herein in the context of the effects of the invention, the term "negative effect" refers to an effect that negates the effects of the invention. For example, a "negative effect" can be defined as a case where the effect of the invention that would have been achieved is 30%, 20%, 10%, or 5% or less, where the effect is 100%. For example, a negative effect can be defined as a case where the hit rate that would have been achieved by panning is 1%, but the hit rate is 0.3% or less. Furthermore, a negative effect can be defined as a case where the percentage of hit compounds that do not contain Trp that would have been achieved by panning is 100%, but the percentage of hit compounds that do not contain Trp is 30% or less, but this is not a limiting example.

[0070] Peptide Compounds. While not intended to be limiting, the term "peptide compound" in the present disclosure includes linear peptide compounds and cyclic peptide compounds. Furthermore, the term "peptide compound" in the present disclosure also includes peptides, peptide-nucleic acid complexes (peptide-nucleic acid complexes), peptide-ribosome-nucleic acid complexes, and the like. While not intended to be limiting, the term "peptide compound" in the present disclosure also includes linear peptides, cyclic peptides, complexes of linear peptide moieties and nucleic acids (linear peptide-nucleic acid complexes), and complexes of cyclic peptide moieties and nucleic acids (cyclic peptide-nucleic acid complexes). In this specification, the peptide portion of a peptide-nucleic acid complex may be referred to as the "peptide moiety." The peptide compound in the present disclosure may contain α-hydroxycarboxylic acid in place of some of the constituent amino acids. When the peptide compound contains α-hydroxycarboxylic acid, the bonds between the residues in the peptide compound may 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 acid, the number of α-hydroxycarboxylic acid is counted as part of the amino acid residues. Examples of α-hydroxycarboxylic acids include glycolic acid, D-lactic acid, L-lactic acid, and compounds in which the amino group of the above-mentioned "α-amino acid" is replaced with a hydroxy group.

[0071] Furthermore, "nucleic acids" in the present disclosure include DNA, mRNA, and tRNA. In this specification, "amino acids" constituting peptide compounds may be referred to as "amino acid residues." In addition, "peptide compounds" in this specification may also include pharmaceutically acceptable salts thereof.

[0072] As used herein, a "peptide compound" or a "peptide moiety" refers 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. Such cyclic peptide compounds are formed by cyclization at a carboxy group and an amino or hydroxy group present in the main chain or side chain of the corresponding linear peptide compound to form a cyclic moiety. The type of cyclization bond is not particularly limited, but preferred are, for example, covalent bonds such as an amide bond, a carbon-carbon bond, a disulfide bond, an ester bond, a thioester bond, a thioether bond, a lactam bond, a bond via a triazole structure, or a bond via a fluorophore structure. In particular, cyclization via an amide bond tends to result in a cyclic peptide compound with higher metabolic stability.

[0073] As used herein, the term "cyclization reaction" refers to a reaction in which a cyclic moiety is formed by bonding a carboxy group present in the main chain or side chain of a linear peptide compound with an amino group or a hydroxy group. When the cyclic peptide compound of the present disclosure is chemically modified, the chemical modification may be carried out on the linear peptide before the cyclization reaction, or on the cyclized peptide obtained after the cyclization reaction.

[0074] Scheme A is an example of a cyclization reaction. The open circle (○) unit (intersection unit), black circle (●) unit (cyclic main chain unit), square (■) unit (linear main chain unit), and triangle (▲) unit (cyclization N-terminal unit) represent amino acid residues that make up the peptide moiety. Each unit is classified based on its role, and may be the same or different amino acids.

[0075] As used herein, the term "unit" refers to any of the amino acids before cyclization after synthesis of the linear peptide compound of the present disclosure, the amino acids after cyclization, and the amino acids at the time when chemical modification after cyclization is completed. The amino acid residues at the time when chemical modification after cyclization is completed include, for example, amino acid residues that have been translated by one tRNA and then chemically converted or backbone-converted by post-translational chemical modification.

[0076] In Scheme A, the cyclic portion is a portion consisting of one triangle unit, eight black circle units, and one white circle unit, and the linear portion is a portion consisting of six square units. The curved portion in Scheme A is the portion to be cyclized (also referred to herein as the "cyclization portion" or "post-translational cyclization portion").

[0077] In one aspect, the peptide moiety of the peptide compound of the present disclosure requires a reactive functional group in the triangle unit or intersection unit shown in Scheme A. Therefore, in the peptide compound before cyclization, drug-like amino acids are not necessarily selected for the triangle unit or intersection unit.

[0078] In one aspect, even if the triangle unit or intersection unit in a peptide compound before cyclization is not a drug-like amino acid or is an amino acid that is not originally preferred, the triangle unit or intersection unit can be converted into a drug-like amino acid or a preferred amino acid by performing chemical modification or the like after cyclization.

[0079] In one aspect, the triangle unit (pre-cyclization N-terminal unit) in the peptide moiety of the peptide compound of the present disclosure can be an amino acid residue having a thiol group near the amino group, in which both the thiol group and the amino group are protected by protecting groups.

[0080] In one aspect, the intersection unit in the peptide portion of the peptide compound of the present disclosure before cyclization preferably uses an amino acid having a functional group (second reactive site) in its side chain that can be cyclized by reacting with a functional group (first reactive site) possessed by an amino acid in the triangle unit. The intersection unit preferably has a total of three or more functional groups, since the amino group and carboxyl group in the main chain are used to form covalent bonds with other amino acid residues in translational synthesis, etc., and the third functional group is required for cyclization. Among these, it is preferred to use a functional group in the side chain portion of the intersection unit for the cyclization reaction.

[0081] The intersection unit can be incorporated at any position in the peptide moiety before cyclization, as long as it is a position that allows cyclization, but it is preferably incorporated at a position that results in a cyclic portion having 13 to 14 amino acids after cyclization or post-translational modification after cyclization. That is, the intersection unit of the peptide compound of the present disclosure is preferably incorporated at a position at least 12 amino acid residues (e.g., 12 or 13 amino acid residues) C-terminal to the triangle unit.

[0082] The black circle units and square units are selected from amino acids, preferably drug-like amino acids.

[0083] Cyclic Peptide Compounds The cyclic peptide compounds of the present invention are compounds formed by amide or ester bonds between amino acids and have a cyclic portion. As used herein, the "cyclic portion" of a cyclic peptide compound refers to a cyclic structure formed by 13 or 14 amino acid residues or α-hydroxycarboxylic acids linked together via amide bonds, ester bonds, or carbon-carbon covalent bonds. The cyclic peptide compound preferably comprises 30 or fewer residues, including the cyclic portion and linear portion. Considering both excellent membrane permeability and metabolic stability (drug-likeness), the number of amino acids constituting the cyclic portion is preferably 13 or 14. In a non-limiting embodiment, the cyclic portion of the cyclic peptide compound is formed by subjecting a translationally synthesized peptide compound to a cyclization reaction. The cyclic peptide compounds of the present invention may be further chemically modified. Examples of chemically modified cyclic peptide compounds include cyclic peptide compounds linked to a nucleic acid (cyclic peptide-nucleic acid complexes). The nucleic acid may be linked to the cyclic peptide compound via a linker.

[0084] The cyclic portion of a cyclic peptide compound may have a side chain (also referred to as a "linear portion") branching from the main chain constituting the cyclic structure, and the linear portion has at least one amide bond and / or ester bond. When the cyclic peptide compound of the present invention has a linear portion, it can be depicted, for example, as shown in Scheme A-1 or A-2. For example, when the cyclic peptide compound has a linear portion, the amino acids constituting the cyclic portion may include Asp or Glu, and the carboxy group of the main chain or side chain of the Asp or Glu may be linked to the amino group of the linear portion via an amide bond. As used herein, the term "linear portion" includes natural amino acids and unnatural amino acids (including chemically modified or backbone-converted amino acids). When the total number of amino acids in the cyclic portion and the linear portion is 13 or 14, membrane permeability tends to be higher. When the total number of amino acids in the cyclic portion and the linear portion is 13 or more, metabolic stability tends to be higher. The number of amino acid residues in the linear portion is preferably 0 to 8, more preferably 0 to 3, and most preferably 0 (ie, no linear portion).

[0085] As used herein, the term "number of amino acids" refers to the total number of amino acid residues or α-hydroxycarboxylic acids constituting a peptide, and means the number of amino acids or α-hydroxycarboxylic acids generated upon cleavage of the amide bonds, ester bonds, and bonds at the cyclized portion that link the amino acids.

[0086] As used herein, the term "amino acids constituting the cyclic moiety" refers to amino acids present on the main chain of the cyclic moiety, among amino acids generated upon cleavage of an amide bond, an ester bond, or a bond in the cyclized portion present in a cyclic peptide compound. The amino acids or α-hydroxycarboxylic acids corresponding to the intersection units shown in Scheme A-1 or A-2 above are included in the amino acids constituting the cyclic moiety.

[0087] In a cyclic peptide-nucleic acid complex, a cyclic peptide compound (peptide portion) and a nucleic acid are linked directly or via a linker. In the cyclic peptide-nucleic acid complex, the nucleic acid is linked to the linear portion, but not to the cyclic portion. In this specification, the entire linear portion to which a nucleic acid is linked is referred to as the "nucleic acid linkage portion," and the portion of the peptide portion other than the nucleic acid linkage portion is referred to as the "peptide portion other than the nucleic acid linkage portion." Furthermore, in a peptide compound to which no nucleic acid is linked, there is no nucleic acid linkage portion, and therefore the entire peptide portion is referred to as the "peptide portion other than the nucleic acid linkage portion." For example, in Scheme A-2, when a nucleic acid is linked to the end of linear portion 1, the portion other than linear portion 1, i.e., the combined portion of the cyclic portion and linear portion 2, is the "peptide portion other than the nucleic acid linkage portion." In a linear peptide-nucleic acid complex, the entire linear peptide is the nucleic acid linkage portion, and therefore there is no "peptide portion other than the nucleic acid linkage portion."

[0088] In a non-limiting embodiment, the cyclic peptide compound has a cyclic portion, (1) the cyclic portion is composed of 13 or 14 amino acid residues, (2) among the amino acid residues constituting the cyclic portion, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues having the same side chain is 3 or less, and the number of natural amino acid residues is 4 or less, and (3) ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.8.

[0089] The number of amino acids constituting the cyclic peptide compound (referred to as the peptide portion when the cyclic peptide compound is linked to a nucleic acid) in the present disclosure 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 classified into the nucleic acid linkage portion and the other peptide portion. When the number of amino acids constituting the peptide portion other than the nucleic acid linkage portion is 13 or 14, or when the number of amino acids constituting the cyclic portion is 13 or 14, excellent membrane permeability and metabolic stability tend to be compatible.

[0090] In a non-limiting embodiment, the number of amino acids (number of units) in the linear portion is preferably 0 to 8, more preferably 0 to 5, and most preferably 0 to 3.

[0091] Among the amino acid residues constituting the cyclic portion, the number of amino acid residues with the same side chain is not limited, and examples include 5 or less, 4 or less, 3 or less, or 2 or less, preferably 4 or less, and more preferably 3 or less. In other words, the amino acids constituting the cyclic portion may include up to 5 amino acids with the same side chain.

[0092] The number of amino acids constituting the cyclic portion is not limited, and may be, for example, 13 or more, 14 or less, 13 to 14, 13, or 14. In consideration of achieving both excellent membrane permeability and metabolic stability, the number of amino acids constituting the cyclic portion is preferably 13 to 14, 13, or 14.

[0093] In a non-limiting embodiment, when the ratio of the number of N-substituted amino acids to the total number of amino acids constituting the cyclic portion is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, the cyclic peptide compound can exhibit higher membrane permeability.

[0094] In a non-limiting embodiment, the number of N-substituted amino acids constituting the cyclic portion is not limited, and examples thereof include 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. In consideration of achieving both excellent membrane permeability and metabolic stability, 7 or more or 7 to 13 are preferred, and 8 or more, 9 or more, 10 or more, 7 to 10, or 8 to 10 are more preferred examples.

[0095] A cyclic peptide compound in the present disclosure is a compound having a ClogP / total amide bond (ClogP / total AB) of 1.0 to 1.8. In this specification, "total AB" is calculated as follows: When a cyclic peptide compound is not linked to a nucleic acid, the sum of the number of all amide bonds constituting the entire cyclic peptide compound (the number of amide bonds in the main chain and side chain of each amino acid) is used as "total AB." On the other hand, when a cyclic peptide compound is linked to a nucleic acid, the sum of the number of amide bonds in the main chain and side chain of the amino acids constituting the peptide portion other than the nucleic acid linking portion of the cyclic peptide compound is used as "total AB." When the bond between the nucleic acid linking portion and the peptide portion other than the nucleic acid linking portion is an amide bond, this amide bond is also included in the calculation. ClogP / total AB is a value calculated by dividing ClogP by total AB. Nucleic acid-linked cyclic peptide compounds (cyclic peptide-nucleic acid complexes) can be used to constitute display libraries used in screening, and the only drug candidate compounds that are actually envisioned are the portions not linked to nucleic acids. Therefore, when considering drug-likeness (for example, high membrane permeability), the properties of the peptide portion other than the nucleic acid linker can be important.

[0096] In a non-limiting embodiment, in order to have good membrane permeability, the ClogP of the cyclic peptide compound of the present disclosure is preferably 4 or more and 25 or less, more preferably 10 or more, 11 or more, 12 or more, or 13 or more, and more preferably 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less, and examples thereof 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 partition coefficient calculated by computer and can be determined in accordance with the principles described in the "CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / )." An example of a method for calculating ClogP is to use Daylight Version 4.95 from Daylight Chemical Information Systems, Inc. (release date: August 1, 2011, ClogP algorithm version 5.4, database version 28, https: / / www.daylight.com / dayhtml / doc / release_notes / index.html).

[0097] The principles described in "CLOGP Reference Manual Daylight Version 4.9 (Release Date: August 1, 2011, https: / / www.daylight.com / dayhtml / doc / clogp / ") are as described in paragraph

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

[0098] In this specification, when a cyclic peptide compound is not linked to a nucleic acid, the ClogP of the entire cyclic peptide compound is calculated. When a cyclic peptide compound is linked to a nucleic acid, the ClogP of the peptide portion other than the nucleic acid linker is calculated as the "ClogP of the cyclic peptide compound." The "ClogP of the peptide portion other than the nucleic acid linker" is the ClogP calculated for a hypothetical peptide in which the nucleic acid linker of a cyclic peptide compound is replaced with piperidine (pip). That is, the hypothetical peptide is a cyclic peptide in which piperidine (pip) is bonded to the amide bond between the main chain or side chain carboxy group of the amino acid constituting the cyclic portion (e.g., an amino acid residue having a carboxy group in its side chain, such as Asp or Glu) and the amino group of the nucleic acid linker, replacing the amino group of the nucleic acid linker. Nucleic acid-linked cyclic peptide compounds (cyclic peptide-nucleic acid complexes) can be used to construct display libraries used in screening, and only the portion not linked to the nucleic acid constitutes a potential drug candidate. Therefore, when considering drug-likeness (e.g., high membrane permeability), the properties of the peptide portion other than the nucleic acid linker can be important.

[0099] In a non-limiting embodiment, the ClogP / total AB of the cyclic peptide compound of the present 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 of the cyclic peptide compound of the present disclosure is preferably 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, or 1.4 or less. Preferred ClogP / total AB values ​​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, and 1.1 to 1.2.

[0100] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure has better membrane permeability when ClogP / totalAB is 1.0 or more and the number of N-substituted amino acids is 7 or more. Furthermore, the cyclic peptide compound has even better membrane permeability when ClogP / totalAB is 1.1 or more and the number of N-substituted amino acids is 8 or more.

[0101] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure may have a hydrogen bond donor (proton donor) in its cyclic portion. The hydrogen bond donor is a group that presents a hydrogen atom necessary for forming a hydrogen bond and forms a hydrogen bond with a hydrogen bond acceptor (proton acceptor) in the same compound or another compound (including water molecules) present in the vicinity. The hydrogen bond donor is a group having a hydrogen atom covalently bonded to an atom with a relatively high electronegativity, such as a hydroxyl group, a carboxy group, an amino group, an unsubstituted amide group, or a monosubstituted amide group. The hydrogen bond acceptor is, for example, a lone pair such as a carbonyl oxygen of a carboxy group, an ester, or an amide, or an amine nitrogen. Forming an intramolecular hydrogen bond can fix the conformation of the peptide compound and increase its affinity with a target molecule. Forming an intermolecular hydrogen bond with a target molecule can increase its affinity with the target molecule. The number of hydrogen bond donors can be, 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. When the number of hydrogen bond donors is 5 or less, 4 or less, or 3 or less, both of better membrane permeability and metabolic stability can be achieved. 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.

[0102] In order for peptide compounds to exhibit higher membrane permeability and increase the hit rate of peptide compounds capable of specifically binding to target molecules, the side chains of the amino acids constituting the cyclic portion of the cyclic peptide compound preferably contain a group selected from among drug-like substituents. The drug-like substituent can be selected from, for example, optionally substituted alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, aralkyl groups, aryl groups, and heteroaryl groups, and examples of the substituent include halogen atoms, amide groups (-CO-NRR' or -NR-CO-R'), sulfonyl groups (-SO ２ and an alkoxy group (—OR) (R and R′ are each independently selected from an optionally substituted alkyl group, cycloalkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, and aralkyl group, or in the case of an amide group, R and R′ together with the nitrogen atom to which they are bonded form a 4- to 8-membered saturated heterocycle, and the 4- to 8-membered saturated heterocycle is not further substituted with a halogen atom, an oxo group, C １ -C ６ It may be substituted with at least one group independently selected from the group consisting of alkyl groups and 4- to 7-membered heterocyclyl groups.

[0103] On the other hand, since biological membranes are composed of phospholipids, polar functional groups (e.g., alkylamino groups, alkylguanidine groups, etc.) that are highly ionized in vivo (around pH = 7) are not particularly suitable for achieving high membrane permeability. The cyclic peptide compounds of the present disclosure preferably do not contain these functional groups.

[0104] In a non-limiting embodiment, the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is composed of at least one selected from the group consisting of an alkyl group, a cycloalkyl group, an aralkyl group, and an amide group, wherein any atom of the alkyl group may form a ring together with the nitrogen atom of the amino group of the amino acid.

[0105] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an alkyl group, a halogen atom, a hydroxy group, C ３ -C ８ Cycloalkyl groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of alkoxy groups １ -C ６ It may be an alkyl group.

[0106] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is a cycloalkyl group, a halogen atom, a hydroxy group, C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ３ -C １０ It may also be a cycloalkyl group.

[0107] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an aralkyl group, a halogen atom, C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ７ -C １４ It may also be an aralkyl group.

[0108] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an aralkyl group, optionally substituted C ５-C １０ Aryl C １ -C ６ may be an alkyl group, ５ -C １０ The aryl group may be a halogen atom, １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ It may be substituted with at least one group selected from the group consisting of haloalkoxy groups.

[0109] In one non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound of the present disclosure is an amide group, the amide group has the formula: -CONR １ R ２ wherein R １ and R ２ are each independently a hydrogen atom or C １ -C ６ alkyl group or R １ and R ２ form a 4- to 8-membered saturated heterocyclic ring together with the nitrogen atom to which they are attached, and the 4- to 8-membered saturated heterocyclic ring is not further substituted by a halogen atom, an oxo group, C １ -C ６ It may be substituted with at least one group independently selected from the group consisting of alkyl groups and 4- to 7-membered heterocyclyl groups.

[0110] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure may be a cyclic peptide compound that contains at least one amide bond formed between a carboxy group contained in the side chain of an amino acid and the nitrogen atom of the amino group of another amino acid.

[0111] In one non-limiting embodiment, the cyclic peptide compounds of the present disclosure may comprise an amino acid selected from the group consisting of Asp-pip (see formula (i)) and MeAsp-pip (see formula (ii)).

[0112] 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 portion of the cyclic peptide compound of the present disclosure 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, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0113] In a non-limiting embodiment, the amino acids constituting the cyclic portion of a cyclic peptide compound of the present disclosure are not particularly limited, but may all be 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 a cyclic peptide compound is not limited, but may be, for example, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 10, 11, 12, 13, or 14. The number of amino acids having a β-amino acid backbone constituting the cyclic portion of a cyclic peptide compound is not limited, but may be 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 having a γ-amino acid backbone constituting the cyclic portion of the cyclic peptide compound is not limited, and may be 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.

[0114] In one non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds of the present disclosure may include, but are not limited to, α-amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). The α-amino acids may each independently 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, and Ser(tBu).

[0115] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure may include, but are not 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.

[0116] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure are not particularly limited, but may include gMeAbu or may be gMeAbu.

[0117] In a non-limiting aspect, the number of natural amino acids among the amino acids constituting the cyclic portion of 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, or 5 to 6, with 1 to 4 being preferred.

[0118] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure may comprise naturally occurring amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. The naturally occurring amino acids may each independently be an amino acid selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0119] In a non-limiting embodiment, the number of unnatural amino acids among the amino acids constituting the cyclic portion of the cyclic peptide compound of the present disclosure is preferably 7 or more, more preferably 8 or more, and most preferably 9 or more. Preferred examples of the number of unnatural amino acids include 14 or less, 13 or less, 12 or less, 11 or less, and 10 or less.

[0120] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds of the present disclosure may include, but are not limited to, unnatural 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). The unnatural amino acids can each independently be an 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).

[0121] In one non-limiting embodiment, the cyclic peptide compound of the present disclosure is Caco-2 P ａｐｐ The value of (cm / sec) is, for example, 1.0×10 －４ Below, 9.0 x 10 －５ Below, 8.0 x 10 －５ Below, 7.0 x 10 －５ Below, 6.0 x 10－５ Below, 5.0 x 10 －５ Below, 4.0 x 10 －５ Below, 3.0 x 10 －５ Below, 2.0 x 10 －５ Below, 1.0 x 10 －５ Below, 9.0 x 10 －６ Below, 8.0 x 10 －６ Below, 7.0 x 10 －６ Below, 6.0 x 10 －６ or less, or 5.99 x 10 －６ In one non-limiting embodiment, the cyclic peptide compound of the present disclosure may be Caco-2 P ａｐｐ The value of (cm / sec) is, for example, 1.0×10 －９ That's it, 1.0 x 10 －８ That's it, 1.0 x 10 －７ That's it, 0.4 x 10 －６ That's it, 1.0 x 10 －６ or more, or 1.0 x 10 －５ or more, preferably 0.4×10 －６ More preferably, 1.0 × 10 －６ In one non-limiting embodiment, the cyclic peptide compound of the present disclosure may be Caco-2 P ａｐｐ The range of the value of (cm / sec) is, for example, 1.0×10 －９ Above 1.0 x 10 －４ Below, 1.0 x 10 －８ Above 1.0 x 10 －４ Below, 1.0 x 10 －７ Above 1.0 x 10 －４ Below, 0.4 x 10 －６ Above 1.0 x 10 －４ Below, 0.4 x 10 －６ Above 1.0 x 10 －５ The following is an example:

[0122] ・Membrane permeability of peptide compounds Caco-2 P ａｐｐ The value of (cm / sec) is an index of membrane permeability in the cell membrane, and can be calculated by membrane permeability evaluation under the following conditions.

[0123] (a) Caco-2 cells were cultured on a 96-well transwell for 3 weeks, and then the compound to be evaluated, DMEM medium, and FaSSIF (containing 1% DMSO) were added to the apical side, and DMEM medium was added to the basal side. ２ (b) After the preincubation, the preincubation solution on the apical and basal sides was removed by suction and washed, and a FaSSIF / HBSS buffer solution (containing 1% DMSO, pH 6.0) containing the compound was added to the apical side, and an HBSS buffer solution (containing 4% BSA, pH 7.4) was added to the basal side. (c) Each well was incubated at 5% CO ２ The mixture was shaken at 80 rpm at 37°C under atmospheric pressure, and a sample was taken from the basal side 180 minutes after the start of shaking. The amount of permeation of the compound was measured by liquid chromatography mass spectrometry (LC / MS). (d) The permeation coefficient (Caco-2 P ａｐｐ (cm / sec) is calculated.

[0124] Permeability coefficient (Caco-2 P ａｐｐ (cm / sec) is calculated using Equation 1. In the above measurement, a Pgp inhibitor (e.g., Zosquidar) can be added to FaSSIF / HBSS buffer (1% DMSO) (pH 6.0) and HBSS buffer (4% BSA) (pH 7.4), respectively. The concentration of the donor-side target compound used to calculate the permeability coefficient in step (d) above can be the concentration of the compound initially added, or the compound concentration measured by sampling the apical solution before starting preincubation or before starting shaking in step (c) above can also be used. In particular, when preincubation is performed, it is preferable to use the concentration of the compound sampled from the apical side before preincubation.

[0125] In a non-limiting embodiment, when measuring the membrane permeability of a peptide compound of the present disclosure, it is preferable to use a peptide compound that does not have a nucleic acid moiety that serves as a template for the peptide site as a test substance. In one embodiment, as described below, panning is performed in the form of a peptide-nucleic acid complex, and then a peptide-nucleic acid complex that can specifically bind to a target molecule is selected, and a peptide compound can be synthesized based on the base sequence of the nucleic acid moiety. A preferred example is measuring membrane permeability using the peptide compound synthesized in this manner as a test substance. In one embodiment, a derivative of the peptide compound encoded by the base sequence may be synthesized and used as a test substance to measure membrane permeability. Alternatively, a derivative may be synthesized from the test substance based on the results of membrane permeability, and the membrane permeability of the derivative may be measured.

[0126] In one non-limiting aspect, the cyclic peptide compounds of the present disclosure may be selected from compounds pd001 to pd168 set forth in Table 15.

[0127] Peptide-Nucleic Acid Complex In one embodiment, the peptide compound of the present disclosure may be a complex of a peptide and a nucleic acid (peptide-nucleic acid complex). The peptide-nucleic acid complex may bind to a nucleic acid at the C-terminus of the peptide moiety to form a complex. The peptide-nucleic acid complex of the present disclosure is classified into a linear portion to which the nucleic acid is linked (i.e., the 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, not to the cyclic portion. The binding method between the peptide and the nucleic acid is not particularly limited, but may be via a linker. When the peptide compound and the nucleic acid are bound via a linker, the linker is considered to be part of the nucleic acid linkage portion. In one embodiment, the nucleic acid forming a complex with the peptide moiety may be DNA or RNA, with RNA being preferred and mRNA being more preferred. In one embodiment, the nucleic acid forming a complex with the peptide may be a template used in the translational synthesis of the peptide moiety, with mRNA being preferred as the template. A more specific preferred embodiment is a peptide compound in which the C-terminus of the peptide moiety is linked to the 3'-terminus of the template mRNA via a linker containing the antibiotic puromycin, which is an aminoacyl-tRNA analog, or the like.

[0128] The peptide-nucleic acid complex can be produced, for example, by the method described in WO 2013 / 100132. Specifically, the peptide-nucleic acid complex can be produced by a method including the following steps: A) acylating the dinucleotide pdCpA or pCpA with a desired amino acid or amino acid analog to provide aminoacyl-pdCpA or aminoacyl-pCpA; B) providing a tRNA lacking a CA at the 3' end; C) linking the aminoacyl-pdCpA or aminoacyl-pCpA of step A) with the CA-lacking tRNA of step B) to provide an initiator tRNA acylated with a desired amino acid or amino acid analog; D) providing a cell-free translation system comprising the initiator tRNA of step C) but not containing at least one of methionine, methionyl-tRNA synthetase (MetRS), a translation initiation tRNA for methionine, a formyl donor, and methionyl-tRNA transferase; E) providing template DNA encoding a peptide sequence comprising, downstream of a promoter, a translation initiation ATG followed by a cysteine ​​codon UGU or UGC, and downstream of that, a codon corresponding to the anticodon of the tRNA of step C); F) Step E) providing mRNA from template DNA; Step G) attaching a linker to the 3' end of the mRNA; and Step H) adding the linker-attached mRNA of Step G) to the cell-free translation system of Step D) and translating it to provide a pre-cyclized peptide-mRNA complex.

[0129] When the peptide is a cyclic peptide, a step of cyclizing the pre-cyclized peptide-mRNA complex in step H) may be further included. In step H), a desulfurization reaction can be carried out as necessary. Furthermore, after step G), a step of synthesizing cDNA using a primer that anneals to the 3' region of mRNA can be included.

[0130] In steps A to C, template DNA encoding the desired tRNA sequence and containing a T7, T3, or SP6 promoter upstream is prepared, and RNA can be synthesized by transcription using an RNA polymerase compatible with the promoter, such as T7 RNA polymerase, T3, or SP6 RNA polymerase. Alternatively, tRNA can be extracted and purified from cells, and the desired tRNA can be extracted using a probe with a sequence complementary to the tRNA sequence. Cells transformed with an expression vector for the desired tRNA can also be used as the source. RNA of the desired sequence can also be synthesized by chemical synthesis. For example, aminoacyl-tRNA can be obtained by ligating the thus-obtained tRNA, in which the CA has been removed from the CCA sequence at the 3' end, with separately prepared aminoacylated pdCpA using RNA ligase (pdCpA method). Aminoacylation can also be performed using full-length tRNA with Flexizyme, a ribozyme that attaches activated esters of various unnatural amino acids to tRNA. Acylated tRNA can also be obtained using the methods described below.

[0131] In steps E to H, DNA containing the desired sequence downstream of a promoter such as a T7 promoter is first chemically synthesized, and then double-stranded DNA is generated using this as a template through a primer extension reaction. The resulting double-stranded DNA is then transcribed into mRNA using an RNA polymerase such as T7 RNA polymerase. A linker containing puromycin or similar is attached to the 3' end of the mRNA obtained by transcription, and the mRNA is translated into protein using a known cell-free translation system such as the PURE system. Puromycin is then incorporated into the protein, linking the mRNA and the protein encoded thereby via puromycin. In this way, a peptide-mRNA complex can be constructed in which the mRNA and the product (peptide) encoded by it are associated.

[0132] Metabolic Stability of Peptide Compounds In a non-limiting aspect, the peptide compounds of the present disclosure preferably have good metabolic stability. For good metabolic stability, the number of amino acids contained in the peptide compound is preferably 13 to 14. If the number of amino acids contained in the peptide compound is 13 to 14, the above-mentioned conditions for membrane permeability do not affect the metabolic stability of the peptide compound. In one aspect, it is preferable that the peptide compound does not have a thioether bond that can be easily oxidized. In another aspect, it is preferable that the peptide compound does not have a methylthio group, as this is easily oxidized and can interfere with metabolic stability.

[0133] The metabolic stability of the peptide compounds of the present invention can be confirmed by known methods, for example, using hepatocytes, small intestinal cells, liver microsomes, small intestinal microsomes, liver S9 mix, etc. Specifically, for example, the stability of the peptide compounds in liver microsomes can be confirmed by measuring the stability according to the description of LL von Moltke et al. (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, the stability can be confirmed according to the method described in Example 3.

[0134] The metabolic stability can be measured, for example, by measuring the liver intrinsic clearance (CL) of the compound in liver microsomes according to the method described above. ｈ　ｉｎｔA metabolic stability sufficient for oral administration can be considered to be achieved when the value of (μL / min / mg protein) is 150 or less, with a value of 100 or less being preferable. For drugs metabolized by CYP3A4, a value of 78 or less is preferable to avoid small intestinal metabolism in humans (Non-Patent Document: 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 value of 35 or less (assuming FaFg = 1 and protein binding rate of 0%) is even more preferable to demonstrate a bioavailability of approximately 30% or more in humans.

[0135] Nucleic Acid In a non-limiting embodiment, the peptide compound of the present disclosure may be linked to a nucleic acid, preferably linked via a linker. A preferred example of the nucleic acid of the present disclosure is a nucleic acid that encodes the peptide compound of the present disclosure. In one embodiment, the "nucleic acid encoding the peptide compound" refers to a nucleic acid that is used or has been used as a template in the translational synthesis of the peptide compound.

[0136] Libraries in the present disclosure include libraries of peptide compounds and libraries of peptide-nucleic acid complexes in the present disclosure. The libraries in the present disclosure may further include linear peptide compounds and linear peptide-nucleic acid complexes. As the libraries in the present 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. With the libraries in the present disclosure, compounds that have excellent drug-likeness and high affinity for target molecules can be efficiently discovered from among cyclic peptide compounds having a cyclic portion formed from 13 or 14 amino acid residues. As the libraries, display libraries are preferred. Examples of display libraries include libraries that utilize display, and among these, mRNA display libraries, DNA display libraries, and ribosome display libraries are preferred, with mRNA display libraries being more preferred. When the library in the present disclosure is a display library, the peptide compounds can be easily synthesized by amplifying, by PCR or the like, nucleic acids linked to the peptide compounds enriched by panning, followed by transcription and translation (Yanagawa, H. et al. FEBS Lett., 1997, 414, 405-408. and Szostak JW et al. Proc. Natl. Acad. Sci. USA, 1997, 94, 12297-12302.).

[0137] The peptide compound of the present disclosure may be a peptide compound encoded by a nucleic acid. When the peptide compound of the present disclosure is linked to a nucleic acid, the peptide compound may be a peptide compound encoded by the linked nucleic acid, or a peptide compound obtained by further modifying the peptide compound. That is, as used herein, the term "(cyclic) peptide compound encoded by a nucleic acid" includes not only peptide compounds synthesized directly by translating the nucleic acid as a template, but also peptides synthesized by further post-translational modification. For example, when a linear peptide is synthesized by translating a nucleic acid and a cyclic peptide is subsequently synthesized by a cyclization step, the cyclic peptide may be referred to as a cyclic peptide compound encoded by the nucleic acid.

[0138] As used herein, with respect to a library of peptide compounds to which nucleic acids may be linked and / or nucleic acids encoding them, the term "consisting essentially of" may mean that the ratio of the theoretical number of peptide compounds and / or nucleic acid variations encoding them listed therein to the theoretical total number of peptide compounds and / or nucleic acid variations encoding them contained in the library is 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, but is not limited thereto.

[0139] In one non-limiting aspect, the ratio of the number of cyclic peptide compounds having 13 or 14 amino acid residues constituting the cyclic portion to the total number of cyclic peptide compounds contained in the library of the present disclosure may be 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, but is not limited to these.

[0140] In a non-limiting embodiment, the library of the present disclosure may be a library consisting essentially of cyclic peptide compounds in which the cyclic portion of the cyclic peptide compound comprises 13 or 14 amino acid residues.

[0141] In one non-limiting aspect, the ratio of the number of cyclic peptide compounds comprising 13 or 14 amino acid residues to the total number of cyclic peptide compounds contained in the library of the present disclosure may be 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, but is not limited to these.

[0142] In a non-limiting embodiment, the library of the present disclosure may be a library consisting essentially of cyclic peptide compounds each consisting of 13 or 14 amino acid residues.

[0143] In a non-limiting embodiment, the average number of amino acids (number of units) in the linear portion other than the nucleic acid linking portion of each cyclic peptide compound contained in the library of the present disclosure is preferably 0 to 8, more preferably 0 to 5, and most preferably 0.

[0144] In a non-limiting aspect, the number of amino acids constituting the peptide portion other than the nucleic acid linking portion in each cyclic peptide compound included in the library of the present disclosure is not limited, and may be, for example, 13 or more, 14 or less, 13 to 14, 13, or 14. In consideration of achieving both excellent membrane permeability and metabolic stability, the number of amino acids constituting the cyclic portion is preferably 13, 14, or 13 to 14.

[0145] In a non-limiting aspect, the average number of amino acids constituting the peptide portion other than the nucleic acid linking portion in each cyclic peptide compound included in the library of the present disclosure is not limited, and examples include 13 or more, 14 or less, 13, and 14. In consideration of achieving both excellent membrane permeability and metabolic stability, the average number of amino acids constituting the cyclic portion is preferably 13 to 14.

[0146] In a non-limiting aspect, each cyclic peptide compound included in the library of the present disclosure has an average number of N-substituted amino acids relative to the number of amino acids constituting the cyclic portion of 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, with 7 or more, 8 or more, 7 to 13, or 8 to 13 being preferred, and 7 to 10 or 8 to 10 being more preferred.

[0147] In a non-limiting embodiment, the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic portion is 7 to 10 among the cyclic peptide compounds contained in the library of the present disclosure is 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.

[0148] In a non-limiting aspect, the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic portion is 8 to 13 among the cyclic peptide compounds contained in the library of the present disclosure is 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.

[0149] In a non-limiting embodiment, the proportion of cyclic peptide compounds in which the number of N-substituted amino acids constituting the cyclic portion is 8 to 10 among the cyclic peptide compounds contained in the library of the present disclosure is 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.

[0150] In each cyclic peptide compound contained in the library of the present disclosure, the average percentage of N-substituted amino acids relative to the total number of amino acids constituting the cyclic portion 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, more preferably 60% or more. The N-substituted amino acids referred to here are preferably N-alkyl amino acids, more preferably N-C １ -C ６ In other words, in a non-limiting embodiment, the number of N-substituted amino acids is the number of N-alkylamino acids, N-C １ -C ６ It is exemplified as the average number of alkyl amino acids, the average number of N-ethyl amino acids or the average number of N-methyl amino acids.

[0151] In a non-limiting embodiment, the average ClogP / totalAB of the cyclic peptide compounds contained in the library of the present disclosure is preferably 1.0 or more, more preferably 1.1 or more, more preferably 1.2 or more, and is preferably 1.8 or less, 1.7 or less, 1.6 or less, 1.5, or 1.4 or less. The average ClogP / totalAB of the cyclic peptide compounds contained in the library of the present 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 a cyclic peptide compound" is a value calculated as described above. When the ClogP / total AB of each cyclic peptide compound contained in the library of the present disclosure is calculated and the average value is within the above range, the probability that the compound hit in screening will be a compound with superior membrane permeability increases.

[0152] In a non-limiting embodiment, the average ClogP of the cyclic peptide compounds contained in the library of the present disclosure is preferably 10 or more, 11 or more, 12 or more, or 13 or more, 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 contained in the library of the present 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. The "ClogP of a cyclic peptide compound" is a value calculated as described above. When the ClogP of each cyclic peptide compound contained in the library of the present disclosure is calculated and the average value is within the above range, the probability that a compound hit in screening will be a compound with superior membrane permeability increases.

[0153] In a non-limiting embodiment, the cyclic peptide compounds contained in the library of the present disclosure may have a hydrogen bond donor (proton donor). The average number of hydrogen bond donors is, for example, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, with 5 or less being preferred, 4 or less being more preferred, and 3 or less being most preferred. The lower limit of the average number of hydrogen bond donors is not particularly limited, but examples include 0 or more, 1 or more, or 2 or more. Examples of the average 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. When the number of hydrogen donors of each cyclic peptide compound contained in the library of the present disclosure is calculated and the average value is within the above range, the probability that the compound hit in screening will be a compound with better membrane permeability increases.

[0154] In one embodiment, the cyclic peptide compounds included in the library of the present disclosure may have a hydrogen bond donor (proton donor) in their cyclic portion. The hydrogen bond donor is a group that presents a hydrogen atom necessary for forming a hydrogen bond and forms a hydrogen bond with a hydrogen bond acceptor (proton acceptor) in the same compound or in another compound (including a water molecule) present in the vicinity. The hydrogen bond donor is a group having a hydrogen atom covalently bonded to an atom with a relatively high electronegativity, such as a hydroxyl group, a carboxyl group, an amino group, an unsubstituted amide group, or a monosubstituted amide group. The hydrogen bond acceptor is, for example, a lone pair such as a carbonyl oxygen of a carboxyl group, an ester, or an amide, or an amine nitrogen. Forming an intramolecular hydrogen bond fixes the conformation of the peptide compound, potentially increasing its affinity with a target molecule. Forming an intermolecular hydrogen bond with a target molecule may increase its affinity with the target molecule. 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, or 2 or more. Examples of the average number of hydrogen bond donors are 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.

[0155] In a non-limiting embodiment, the cyclic peptide compounds included in the library of the present disclosure may contain multiple amino acids with the same side chain as the amino acids constituting the cyclic portion. Here, "containing amino acids with the same side chain" means that multiple amino acids with the same side chain are contained in the entire cyclic peptide compound or among the amino acids constituting the cyclic portion of the cyclic peptide compound. "Amino acids with the same side chain" is determined based on whether or not the side chains are the same, without taking into account the substituents on the nitrogen atoms of the amino groups in the main chain. For example, alanine and N-methylalanine are considered to be "amino acids with the same side chain." Specifically, when the amino acids constituting the cyclic portion of a cyclic peptide compound include two Leu, the cyclic peptide compound contains multiple amino acids with the same side chain as the amino acids constituting the cyclic portion. The average number of amino acids with the same side chain among the amino acids constituting the cyclic portion may be 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, with 4 or less being preferred and 3 or less being more preferred.

[0156] In a non-limiting embodiment, the side chain of at least one amino acid constituting the cyclic portion of a cyclic peptide compound included in a library of the present disclosure is composed of at least one selected from the group consisting of an alkyl group, a cycloalkyl group, an aralkyl group, and an amide group, wherein any atom of the alkyl group may form a ring together with the nitrogen atom of the amino group of the amino acid.

[0157] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound included in the library of the present disclosure is an alkyl group, the side chain may be a halogen atom, a hydroxy group, C ３ -C ８ Cycloalkyl groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of alkoxy groups １ -C ６ It may be an alkyl group.

[0158] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound included in the library of the present disclosure is a cycloalkyl group, the side chain may be a halogen atom, a hydroxy group, a C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ３ -C １０ It may also be a cycloalkyl group.

[0159] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound included in the library of the present disclosure is an aralkyl group, a halogen atom, C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C optionally substituted with at least one group selected from the group consisting of haloalkoxy groups ７ -C １４ It may also be an aralkyl group.

[0160] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound included in the library of the present disclosure is an aralkyl group, an optionally substituted C ５ -C １０ Aryl C １ -C ６ may be an alkyl group, ５ -C １０ The aryl group may be a halogen atom, １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １-C ６ It may be substituted with at least one group selected from the group consisting of haloalkoxy groups.

[0161] In a non-limiting embodiment, when the side chain of at least one amino acid constituting the cyclic portion of the cyclic peptide compound included in the library of the present disclosure is an amide group, the amide group is represented by the formula: -CONR １ R ２ wherein R １ and R ２ are each independently a hydrogen atom or C １ -C ６ alkyl group or R １ and R ２ form a 4- to 8-membered saturated heterocyclic ring together with the nitrogen atom to which they are attached, and the 4- to 8-membered saturated heterocyclic ring is not further substituted by a halogen atom, an oxo group, C １ -C ６ It may be substituted with at least one group independently selected from the group consisting of alkyl groups and 4- to 7-membered heterocyclyl groups.

[0162] In a non-limiting embodiment, in each cyclic peptide compound included in the library of the present disclosure, at least one amino acid constituting the cyclic portion may be an amino acid selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu. At least one amino acid constituting the cyclic portion 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, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0163] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library of the present disclosure are not particularly limited, and may all be selected from the group consisting of 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 contained in the library is not limited, and examples thereof include 10 to 14, 10 to 13, 10 to 12, 10 to 11, 10, 11, 12, 13, or 14. The average number of amino acids having a β-amino acid backbone constituting the cyclic portion of the cyclic peptide compounds contained in the library is not limited, and examples thereof include 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 portion of the cyclic peptide compounds contained in the library is not limited, and examples include 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. In order to achieve high membrane permeability, it is preferable that the average number of α-amino acids constituting the cyclic portion is 10 to 14, the number of amino acids having a β-amino acid backbone is 0 to 2, and the number of γ amino acids is 0 to 2.

[0164] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the libraries of the present disclosure may include, but are not limited to, α-amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, and Ser(tBu). The α-amino acids may each independently be 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).

[0165] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds included in the library of the present disclosure may include, but are not 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 selected from the group consisting of Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, and MeAsp-pip.

[0166] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds contained in the library of the present disclosure are not particularly limited, but may include gMeAbu or may be gMeAbu.

[0167] In a non-limiting aspect, among the cyclic peptide compounds contained in the library of the present disclosure, the average number of natural amino acids in the total number of amino acids constituting the cyclic portion can be 1 or more, 2 or more, 3 or more, 7 or less, 6 or less, 5 or less, 4 or less, 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, with 1 or more, 7 or less, or 1 to 7 being preferred, 1 or more, 6 or less, 5 or less, 1 to 6, or 1 to 5 being more preferred, and 1 or more, 4 or less, or 1 to 4 being most preferred. Among the cyclic peptide compounds contained in the library of the present disclosure, the average proportion of the number of natural amino acids in the total number of amino acids constituting the cyclic portion can be 10% or more, or 20% or more, or 50% or less, 40% or less, or 30% or less. The average ratio of the number of natural amino acids to the number of amino acids constituting the cyclic portion may 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%.

[0168] In a non-limiting embodiment, the amino acids constituting the cyclic portion of the cyclic peptide compounds included in the library of the present disclosure may include natural amino acids selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro. The natural amino acids may each be independently selected from the group consisting of Gly, Ala, Val, Leu, Ile, Thr, Phe, and Pro.

[0169] In a non-limiting aspect, the average number of unnatural amino acids contained in the amino acids constituting the cyclic portion of cyclic peptide compounds contained in the library of the present disclosure is preferably 7 or more, and more preferably 8 or more, 9 or more, or 10 or more. The average proportion of the number of unnatural amino acids to the number of amino acids constituting the cyclic portion of cyclic peptide compounds contained in the library of the present disclosure may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more, or 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 the number of unnatural amino acids in the number of amino acids comprising the cyclic portion may be 50-100%, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 55-100%, 55-95%, 55-90%, 55-85%, 55-80%, 55-75%, 55-70%, 60-100%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 70-100%, 70-95%, 70-90%, 70-85%, or 70-80%.

[0170] In a non-limiting embodiment, the cyclic peptide compounds included in the libraries of the present disclosure, or the amino acids constituting the cyclic portion of the cyclic peptide compounds, may include, but are not limited to, unnatural 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). The unnatural amino acids may each independently be 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).

[0171] In a non-limiting embodiment, each cyclic peptide compound contained in the library of the present disclosure may be a cyclic peptide compound that does not have a substituted or unsubstituted hydroxyphenyl group in the side chain of an amino acid contained in a peptide portion other than the nucleic acid linkage portion. Also, in one embodiment, the library of the present disclosure may be a library that is substantially free of cyclic peptide compounds that have a substituted or unsubstituted hydroxyphenyl group in the side chain of a peptide portion other than the nucleic acid linkage portion. Since having a substituted or unsubstituted hydroxyphenyl group in the side chain of a peptide portion other than the nucleic acid linkage portion of a cyclic peptide compound can hinder membrane permeation, it is preferable that the cyclic peptide compound does not have a substituted or unsubstituted hydroxyphenyl group.

[0172] In a non-limiting embodiment, each cyclic peptide compound included in the library of the present disclosure may be a cyclic peptide compound that does not have an indole skeleton in the side chain of a peptide portion other than the nucleic acid linkage portion. Also, in one embodiment, the library of the present disclosure may be a library that substantially does not contain cyclic peptide compounds that have an indole skeleton in the side chain of a peptide portion other than the nucleic acid linkage portion. If a cyclic peptide compound has an indole skeleton in the side chain of a peptide portion other than the nucleic acid linkage portion, it may hinder membrane permeation, so it is preferable that the cyclic peptide compound does not have an indole skeleton.

[0173] In a non-limiting embodiment, the library of the present disclosure may be a library substantially consisting of cyclic peptide compounds that do not have a methylthio group in the side chain of a peptide portion other than the nucleic acid linkage. Furthermore, in another embodiment, the library of the present disclosure may be a library that does not substantially contain cyclic peptide compounds that have a substituted or unsubstituted methylthio group in the side chain of a peptide portion other than the nucleic acid linkage. In another embodiment, the cyclic peptide compounds preferably do not have a methylthio group, since methylthio groups are easily oxidized and can interfere with metabolic stability.

[0174] In a non-limiting embodiment, the library of the present disclosure may be a library substantially consisting of cyclic peptide compounds that do not have a thiol group in the side chain of a peptide portion other than the nucleic acid linkage portion. Furthermore, in another embodiment, the library of the present disclosure may be a library that does not substantially contain cyclic peptide compounds that have a substituted or unsubstituted thiol group in the side chain of a peptide portion other than the nucleic acid linkage portion. In another embodiment, it is preferable that the cyclic peptide compound does not have a thiol group, because thiol groups are easily oxidized and can interfere with metabolic stability.

[0175] In a non-limiting embodiment, the average molecular weight of the cyclic peptide compounds contained in the library of the present disclosure may be 500 to 2000. Also, in one embodiment, the average molecular weight of each cyclic peptide compound contained in the library of the present disclosure may be 500 to 2000. Herein, when a cyclic peptide compound is not linked to a nucleic acid, the molecular weight of the entire cyclic peptide compound is calculated. When a cyclic peptide compound is linked to a nucleic acid, the molecular weight of the peptide portion other than the nucleic acid linkage is calculated as the "molecular weight of the cyclic peptide compound." The "molecular weight of the peptide portion other than the nucleic acid linkage" is the calculated molecular weight of a hypothetical peptide in which the nucleic acid linkage of a cyclic peptide compound is replaced with piperidine (pip). That is, the hypothetical peptide is a cyclic peptide in which piperidine (pip) is bonded to the amide bond between the carboxy group of the main chain or side chain of the amino acid constituting the cyclic portion (e.g., an amino acid residue having a carboxy group in the side chain, such as Asp or Glu) and the amino group of the nucleic acid linkage. Nucleic acid-linked cyclic peptide compounds (cyclic peptide-nucleic acid complexes) can be used as components of display libraries used in screening, and the actual drug candidate compounds are only those that are not linked to nucleic acids. Therefore, when considering drug-likeness (e.g., high membrane permeability), the properties of the peptide moiety other than the nucleic acid linker may be important.

[0176] In determining the "theoretical (total) number" of variations of cyclic peptide compounds in the library disclosed herein, those that cannot actually be produced as cyclic peptide compounds are not included in the theoretical (total) number. For example, in the following cases (1) and (2), the corresponding amino acid is not synthesized by translation, and therefore peptide compounds containing such amino acids are not included in the theoretical (total) number: (1) when an amino acid is added to the translation solution as a material for the peptide compound, but a nucleic acid encoding the amino acid is not included as a template; or (2) when a translation solution is used in which the nucleic acid serving as a template for the peptide compound contains a base sequence but does not contain the corresponding amino acid. Furthermore, if there are by-products or unreacted materials in the process of producing a cyclic peptide compound, these are not included in the theoretical (total) number.

[0177] For example, when a peptide of 11 residues is translated and synthesized from a base sequence in which the codon units are randomly arranged using a codon table in which a set of codon units corresponds to one type of amino acid, using a total of 20 types of natural amino acids, and the translation initiation amino acid is fixed to methionine, the theoretical number of variations is 20. １０ This becomes:

[0178] In the present specification, whether or not the average value of the cyclic peptide compounds contained in the library is within a specific range can be determined by adding up the values ​​of all cyclic peptide compounds actually contained in the library and dividing the total by the total number of cyclic peptide compounds contained in the library (actually measured number average value). However, if the number of cyclic peptide compounds contained in the library is large and it is difficult to calculate the actual measured number average value by the method described above, the actual measured number average value is substituted for the theoretically measured number average value, which is calculated by assuming all types of cyclic peptide compounds that can theoretically be contained in the library, adding up the values ​​of each of them, and dividing the total by the theoretical number of types of cyclic peptide compounds that can theoretically be contained in the library.

[0179] The library of the present disclosure includes a plurality of cyclic peptide compounds or nucleic acids encoding them. In this specification, the number of variations (types) of cyclic peptide compounds included in the library is referred to as "diversity." The diversity of the cyclic peptide compounds included in the library of the present disclosure or the nucleic acids encoding them is not particularly limited, but may be 10 ２ That's it, 10 ３ That's it, 10 ４ That's it, 10 ５ That's it, 10 ６ That's it, 10 ７ That's it, 10 ８ That's it, 10 ９ That's it, 10 １０ That's it, 10 １１ The above and 10 １２ The above are examples. These variations are not limited to actual measurements, but may also be theoretical values.

[0180] The cyclic peptide compounds contained in the library of the present disclosure may include at least one selected from cyclic peptide compounds pd001 to pd168 listed in Table 15, or may include all of them.

[0181] While the characteristics of the cyclic peptide compounds contained in the library of the present disclosure have been described above, these descriptions can also be applied to describing the characteristics of the cyclic peptide compounds contained in the library of the present disclosure, whose cyclic moiety has 13 or 14 amino acid residues. For example, the description in the paragraph describing the proportion of the number of cyclic peptide compounds whose cyclic moiety has 7 to 10 N-substituted amino acids among the cyclic peptide compounds contained in the library of the present disclosure can also be applied to describing the proportion of the number of cyclic peptide compounds whose cyclic moiety has 7 to 10 N-substituted amino acids among the cyclic peptide compounds whose cyclic moiety has 13 or 14 amino acid residues among the cyclic peptide compounds contained in the library of the present disclosure.

[0182] Membrane permeability In a non-limiting embodiment, the cyclic peptide compounds contained in the library of the present disclosure have relatively high membrane permeability, and by further derivatizing these, peptide compounds with even higher membrane permeability can be easily obtained. Furthermore, when screening is performed using the library of the present disclosure, cyclic peptide compounds that are found as compounds capable of specifically binding to a target molecule may have relatively high membrane permeability. As used herein, "high membrane permeability" refers to a membrane permeability coefficient (P ａｐｐ ) is 4.0 × 10 －７ cm / sec or more, preferably 8.0 × 10 －７ cm / sec or more, or 9.0 x 10 －７ cm / sec or more, more preferably 1.0 × 10 －６ cm / sec or more, more preferably 3.0 × 10 －６ cm / sec or more, most preferably 5.0 x 10 －６ In a non-limiting embodiment, the library of the present disclosure may be a library for identifying orally administrable compounds or precursors thereof. As used herein, "orally administrable" means that the membrane permeability coefficient (P ａｐｐ ) is 0.4 × 10 －６ cm / sec or more, preferably 1.0 × 10 －６ cm / sec or more, more preferably 5.0 × 10 －６ As used herein, the term "precursor" refers to a compound prior to chemical modification or the like for optimization, such as a hit compound obtained by panning prior to optimization.

[0183] Hit Rate of Peptide Compounds Capable of Specific Binding to Target Molecules In a non-limiting embodiment, the library of the present disclosure can be used to identify compounds capable of specifically binding to target molecules. In one embodiment, the library of the present disclosure has a larger number of amino acids constituting peptide compounds than conventional libraries, thereby increasing the diversity of those amino acids and resulting in a higher hit rate of peptide compounds capable of specifically binding to target molecules. In other words, by using the library of the present 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, it is possible to select desired compounds from a large number of selected peptide compounds, taking into account other factors such as metabolic stability and membrane permeability. As used herein, the term "hit rate" refers to the ratio of the number of variations of hit compounds obtained by panning to the number of variations of compounds contained in the library. The number of variations of compounds contained in the library may be a theoretical number.

[0184] 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 are clearly not capable of binding to a target molecule even before panning) are not considered to be peptide compounds that constitute the library in the present disclosure.

[0185] By analyzing the frequency of occurrence of amino acids using cyclic peptide compounds enriched by panning, it is possible to find groups of amino acids that can contribute to binding to a target molecule.

[0186] Display libraries: A display library is a library in which a peptide (phenotype) corresponds to the RNA or DNA encoding the peptide (genotype). This library can be used to identify peptide compounds that can specifically bind to a target molecule. For example, peptides that bind to the target can be enriched by contacting the library with a desired immobilized target and washing away molecules that do not bind to the target (panning method). By analyzing the genetic information associated with peptides selected through this process, the sequences of peptides that bind to the target can be determined. For example, methods utilizing the nonspecific binding of the antibiotic puromycin, an aminoacyl-tRNA analog, to proteins during mRNA translation elongation by the ribosome have been reported as mRNA display (Proc Natl Acad Sci USA. 1997; 94: 12297-302. RNA-peptide fusions for the in vitro selection of peptides and proteins. Roberts RW, Szostak JW.) or in vitro virus (FEBS Lett. 1997; 414: 405-8. In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H.).

[0187] A linker such as puromycin is attached to the 3' end of an mRNA library transcribed from a DNA library containing a promoter such as the T7 promoter. When the mRNA is translated into protein in a cell-free translation system, the puromycin is mistaken for an amino acid and incorporated into the protein by the ribosome, linking the mRNA and the protein it encodes, resulting in a library in which mRNA and its product are associated. This process is highly efficient because it does not require transformation of E. coli or other organisms, making it possible to construct large-scale display libraries. The sequences of the bound proteins can be determined by synthesizing cDNA from mRNA, which is a tag containing genetic information attached to molecules enriched by panning, amplifying it via PCR, and analyzing the base sequence.

[0188] In addition to mRNA display, cell-free translation system-based display libraries include cDNA display, which is a library consisting of cDNAs encoding peptides bound to a peptide-puromycin complex (Nucleic Acids Res. 2009; 37(16): e108. cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N.), ribosome display, which utilizes the relatively stable complex between ribosomes and translation products during mRNA translation (Proc Natl Acad Sci U S A. 1994; 91: 9022-6. An in vitro polysome display system for identifying ligands from very large peptide libraries. Mattheakis LC, Bhatt RR, Dower WJ), and bacteriophage endonuclease (BHAT) display. Covalent display utilizes the covalent bond between P2A and DNA (Nucleic Acids Res. 2005;33:e10. Covalent antibody display--an in vitro antibody-DNA library selection system. Reiersen H, Lobersli I, Loset GA, Hvattum E, Simonsen B, Stacy JE, McGregor D, Fitzgerald K, Welschof M, Brekke OH, Marvik OJ.), and CIS display utilizes the binding of the microbial plasmid replication initiator protein RepA to the origin of replication, ori (Proc Natl Acad Sci U S A. 2004;101:2806-10).CIS display: In vitro selection of peptides from libraries of protein-DNA complexes. Odegrip R, Coomber D, Eldridge B, Hederer R, Kuhlman PA, Ullman C, FitzGerald K, McGregor D.) is known. In vitro compartmentalization (Nat Biotechnol. 1998;16:652-6. Man-made cell-like compartments for molecular evolution. Tawfik DS, Griffiths AD.) is also known, in which a transcription-translation system is encapsulated in a water-in-oil emulsion or liposome for each DNA molecule constituting a DNA library, and the translation reaction is carried out. The above methods can be carried out using known methods as appropriate.

[0189] Nucleic acid library In the present invention, "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and nucleotide derivatives having artificial bases, as well as peptide nucleic acid (PNA). The nucleic acid in the present invention can be any one of these nucleic acids or a mixture thereof, as long as the desired genetic information is retained. In other words, the nucleic acid in the present invention also includes DNA-RNA hybrid nucleotides and chimeric nucleic acids in which different nucleic acids, such as DNA and RNA, are linked in a single strand.

[0190] The peptide compounds contained in the library can be synthesized by translation from a template nucleic acid. A non-limiting embodiment of the present disclosure is a library of template nucleic acids. Examples of nucleic acid libraries include mRNA libraries and DNA libraries. Nucleic acid libraries can be obtained by synthesizing sites in peptide sequences where amino acid residues are not fixed by mixing bases. For example, DNA libraries can be synthesized using A, T, G, and C, while RNA libraries can be synthesized using a mixture of four bases (A, U, G, and C) repeated in multiples of three (N), or by synthesizing a codon in which the first and second letters are N and the third letter is a mixture of two bases such as W, M, K, or S. Furthermore, if the number of amino acids to be introduced is limited to 16 or less, a method can be used in which the third letter is a single base. Furthermore, the frequency of occurrence of amino acid residues can be freely adjusted by preparing codon units corresponding to the three letters of a codon and mixing them in any ratio for synthesis. Puromycin may be linked to the 3' end of each nucleic acid contained in the nucleic acid library. By translating a library of nucleic acids with puromycin linked to the 3' end, peptide compounds encoded by the nucleic acids are synthesized, and corresponding nucleic acids are linked to the resulting peptide compounds, thereby synthesizing a display library.

[0191] By translating these nucleic acid libraries using a cell-free translation system, a library of corresponding cyclic peptides can be synthesized. When using a cell-free translation system, it is preferable to include a sequence encoding a spacer downstream of the target nucleic acid. Examples of spacer sequences include, but are not limited to, sequences containing glycine or serine. In addition, it is preferable to include a linker formed from RNA, DNA, a polymer of hexaethylene glycol (spc18) (e.g., a five-polymer) between the nucleic acid library and a compound, such as puromycin or its derivative, that is incorporated into the peptide during ribosomal translation.

[0192] Screening Method In a non-limiting embodiment, a peptide compound capable of specifically binding to a target molecule can be selected by screening using the library of the present disclosure. The screening method of the present disclosure is not particularly limited, and for example, an mRNA display method can be used.

[0193] In one non-limiting embodiment, the screening method of the present disclosure can enrich for peptide compounds that bind to a target molecule by contacting a library of cyclic peptide compounds of the present disclosure with a target molecule and washing away peptide compounds that do not bind to the target molecule (panning). In one embodiment, cDNA is synthesized from mRNA, which is a tag containing nucleotide sequence information contained in the peptide compounds selected in this manner, and PCR amplification is performed. The nucleotide sequence can then be analyzed to determine the amino acid sequence of the bound peptide. In one embodiment, an mRNA library can be obtained by transcribing the amplified cDNA, and these can be used as templates to produce another library of peptide compounds. Since this library is enriched for peptide compounds that can bind to the target molecule, further panning using this library can further enrich for peptide compounds that can specifically bind to the target molecule. This process can be repeated multiple times to further enrich the desired peptide compounds. In one embodiment, amino acid sequences can be identified based on the nucleotide sequence information contained in the enriched peptide compounds, and peptide compounds that can specifically bind to the target molecule can be produced. In one embodiment, the screening method of the present disclosure can be performed in vitro.

[0194] In a non-limiting embodiment, the peptide compound obtained by the screening method of the present disclosure may be optimized by chemically modifying it using a known method, etc. As used herein, "optimization" refers to chemically modifying a compound obtained by screening, etc., to make it more drug-like, to make it have stronger activity against a pharmacological target, and / or to make it less toxic.

[0195] In one non-limiting embodiment, the screening method of the present disclosure comprises the following steps: (a) contacting a target molecule with a peptide compound contained in a library of the present disclosure; and (b) selecting a peptide compound capable of binding to the target molecule.

[0196] In one aspect, the screening method of the present disclosure may enrich peptide compounds capable of specifically binding to a target molecule by repeating the steps (a) and (b) two or more times (cycles). In this case, in step (a) from the second time onwards, the library of peptide compounds selected in the previous cycle can be used. This allows the enrichment rate of the target peptide compound to be increased with each increase in the number of cycles. The number of repetitions may be, for example, two or more cycles, three or more cycles, four or more cycles, five or more cycles, six or more cycles, seven or more cycles, eight or more cycles, nine or more cycles, or ten or more cycles. By repeating the cycles, peptide compounds that bind to the target can be enriched.

[0197] In a non-limiting embodiment, in the step (b) of the screening method of the present disclosure, a peptide compound that binds to a target molecule can be selected as a candidate compound for a pharmaceutical.

[0198] In a non-limiting embodiment, the screening method of the present disclosure further comprises the following steps: (c) chemically synthesizing a candidate peptide compound; and (d) measuring the membrane permeability of the chemically synthesized peptide compound using Caco-2 cells.

[0199] Target Molecules: The target molecules used in the screening methods of the present disclosure are not particularly limited, and examples include proteins, peptides, nucleic acids, sugars, and lipids. Proteins are particularly preferred as targets. In one aspect, the screening methods of the present disclosure can yield peptide compounds that inhibit protein-protein interactions (PPI). While not intending to be bound by any particular theory, it is believed that hotspots exist on the surfaces of both proteins involved in PPI, enhancing the binding strength of PPI. Examples of PPI include hydrophobic interactions, electrostatic interactions, van der Waals forces, hydrogen bonds, photocrosslinking, and salt bridges. Such PPIs can cause protein phosphorylation, conformational changes or localization, and protein-protein complex formation (e.g., multisubunit formation), which may contribute to protein modification, transport, folding changes, signal transduction, and the like. While not intending to be bound by any particular theory, it is believed that the side chains of the amino acids constituting the peptides included in the library of the present disclosure can enter these hotspots, thereby inhibiting PPI. That is, it is believed that the library and screening method of the present disclosure will enable the production of peptide compounds that inhibit PPI, regardless of the type of target molecule.

[0200] In one aspect, when the library used in the screening method of the present disclosure contains cyclic peptide compounds linked to nucleic acids (cyclic peptide-nucleic acid complexes), the actual anticipated drug candidate compound may consist only of the portion other than the nucleic acid and the nucleic acid linker (e.g., the cyclic portion). The target molecule may be selected based on the presence or absence, and the strength of, an interaction with the cyclic peptide portion. When the nucleic acid portion of the cyclic peptide-nucleic acid complex contains a base sequence encoding the cyclic peptide portion, the nucleic acid portion makes it easier to understand and synthesize the chemical structure of the cyclic peptide portion, which may contribute to more efficient screening.

[0201] The location of the target molecule in the body is not particularly limited. In one aspect, the library and screening method of the present disclosure can obtain a cyclic peptide compound with high membrane permeability or a cyclic peptide compound whose membrane permeability can be easily increased by derivatization, and therefore, can target intracellular proteins.

[0202] In a non-limiting embodiment, the target molecule used in the screening method of the present disclosure is immobilized on a carrier. The carrier is not particularly limited as long as it can immobilize the target molecule, and examples thereof include beads and resins. The target molecule can be immobilized on the carrier by a known method.

[0203] The target molecules of the screening method of the present disclosure are not particularly limited. The library of the present disclosure includes peptide compounds that can specifically bind to various target molecules, and therefore, in one embodiment, may enable drug discovery for tough targets that have been considered difficult to discover drugs for until now.

[0204] - Methods for Producing Peptide Compounds and Libraries In one embodiment, the cyclic peptide compounds of the present disclosure and libraries containing the cyclic peptide compounds can be produced, for example, by chemical synthesis, translational synthesis, or a combination thereof.

[0205] Chemical Synthesis Methods of Peptide Compounds: Examples of chemical synthesis methods for peptide compounds in the present disclosure include liquid-phase synthesis, solid-phase synthesis using Fmoc synthesis or Boc synthesis, and combinations thereof. In Fmoc synthesis, the base unit is an amino acid in which the main chain amino group is protected with an Fmoc group, and side chain functional groups are protected as needed with a protecting group that is not cleaved by a base, such as piperidine, without protecting the main chain carboxylic acid. The base unit is not particularly limited as long as it has an Fmoc-protected amino group and a carboxylic acid group. For example, a dipeptide may be used as the base unit. The base unit placed at the N-terminus may be other than an Fmoc amino acid. For example, it may be a Boc amino acid or a carboxylic acid analog without an amino group. The main chain carboxylic acid group is supported on the solid phase by chemical reaction with a functional group on the solid support. Subsequently, the Fmoc group is deprotected with a base such as piperidine or DBU, and the newly generated amino group is condensed with the subsequently added base unit, a protected amino acid with a carboxylic acid, to form a peptide bond. In the condensation reaction, various combinations are possible, such as the combination of DIC and HOBt, the combination of DIC and HOAt, or the combination of HATU and DIPEA. The desired peptide sequence can be produced by repeating the Fmoc group removal and subsequent peptide bond formation reaction. After the desired sequence is obtained, the peptide is cleaved from the solid phase and, if necessary, the protective groups of the introduced side chain functional groups are deprotected. It is also possible to perform structural transformation or cyclization of the peptide before cleavage from the solid phase. Cleavage from the solid phase and deprotection can be performed under the same conditions, for example, a 90:10 TFA / H ２Deprotection can be performed with 0 or under separate conditions as needed. Cleavage from the solid phase can be performed with a weak acid such as 1% TFA, or by using a protecting group such as Pd to take advantage of the orthogonal nature of the chemical reaction. Cyclization or other steps can be performed between or at the end of these steps. For example, a side-chain carboxylic acid can be condensed with an amino group in the N-terminal main chain, or a side-chain amino group can be condensed with a carboxylic acid in the C-terminal main chain. In this case, orthogonal reaction is required between the C-terminal carboxylic acid and the side-chain carboxylic acid to be cyclized, or between the N-terminal main-chain amino group or hydroxy group and the side-chain amino group to be cyclized. As mentioned above, the protecting groups are selected with consideration for their orthogonality. The resulting reaction product can be purified using a reverse-phase column or molecular sieve column. Details of these steps are described, for example, in the Solid-Phase Synthesis Handbook published by Merck on May 1, 2002.

[0206] - Translational synthesis method of peptide compounds Peptide compounds in the present disclosure may be synthesized by a translational synthesis method. In the present disclosure, a "translational synthesis method" refers to a method of synthesizing a peptide compound by recognizing the base sequence of mRNA with tRNA, rather than synthesis solely by organic synthesis technology. An example of a translational synthesis method of a peptide compound is a synthesis method using a cell-free translation system, which includes a synthesis method using a reconstituted cell-free translation system. The use of a reconstituted cell-free translation system is preferable because it allows for the removal of factors that are desired to be eliminated.

[0207] In one aspect, the present disclosure provides a method for translational synthesis of a peptide compound containing at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids set forth in (A) below: (A) Gly, Ala, Val, Leu, Ile, Thr, Phe, Pro, Abu, D-Ala, D-MeAla, D-MeVal, D-Val, Hph, MeAbu, MeAla, MeGly, MeIle, MeLeu, MePhe, MeThr, MeVal, nBuGly, Ser(tBu), Asp-pip, bAla, bMeAla, D-3-Abu, D-3-MeAbu, MeAsp-pip, and gMeAbu.

[0208] Such a translational synthesis method may include, but is not limited to, the following (i) and (ii): (i) a step of preparing a tRNA to which at least one, two or more, or three or more amino acids selected from the group consisting of the amino acids described in (A) are bound; and (ii) a step of translating, in a cell-free translation system, a nucleic acid containing at least one codon corresponding to the anticodon of the tRNA to obtain the peptide compound.

[0209] In a non-limiting embodiment, the translational synthesis method of the present disclosure may be a translational synthesis method for a cyclic peptide compound.

[0210] Cell-Free Translation System As used herein, the term "cell-free translation system" refers to a combination of ribosomes extracted from cells, protein factors involved in translation, tRNA, amino acids, an energy source such as ATP, and a regeneration system for these. It is not limited to any system capable of translating mRNA into protein. The term "cell-free translation system" as used herein also includes a system in which a translation synthesis reaction is in progress. The cell-free translation system used herein may contain nucleic acids that serve as templates for peptide translation, as well as initiation factors, elongation factors, release factors, aminoacyl-tRNA synthetases, and other factors. These factors can be obtained by purification from extracts of various cells. Examples of cells used to purify factors include prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include Escherichia coli cells, extreme thermophilic bacteria cells, and Bacillus subtilis cells. Examples of eukaryotic cells known include yeast cells, wheat germ, rabbit reticulocytes, plant cells, insect cells, and animal cells. In addition to naturally occurring tRNAs and aminoacyl-tRNA synthetases (ARSs), artificial tRNAs and artificial aminoacyl-tRNA synthetases that recognize unnatural amino acids can also be used. Using artificial tRNAs and artificial aminoacyl-tRNA synthetases allows for the site-specific synthesis of peptides incorporating unnatural amino acids. If necessary, transcription from template DNA can also be performed by adding an RNA polymerase such as T7 RNA polymerase to the cell-free translation system.

[0211] PURESYSTEM® (BioComber, Japan) is a reconstituted cell-free translation system in which protein factors, energy regeneration enzymes, and ribosomes required for translation in Escherichia coli are extracted and purified, and then mixed with tRNA, amino acids, ATP, GTP, and other components. Not only does it contain few impurities, but because it is a reconstituted system, it is easy to create a system that does not contain protein factors or amino acids that you want to eliminate. ((i) Nat Biotechnol. 2001;19:751-5. 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. PUREtechnology. Shimizu Y, Ueda T.)

[0212] For example, while many methods have been reported that use stop codons to introduce unnatural amino acids, the aforementioned PURESYSTEM can be used to construct a synthesis system that excludes natural amino acids and ARSs. This allows unnatural amino acids to be linked to the codons encoding the natural amino acids to be excluded (J. Am. Chem. Soc. 2005;127:11727-35. Ribosomal synthesis of unnatural peptides. Josephson K, Hartman MC, Szostak JW.). Furthermore, by breaking the codon degeneracy, unnatural amino acids can be added without excluding natural amino acids (Kwon I, et al. Breaking the degeneracy of the genetic code. J Am Chem Soc. 2003, 125, 7512-3.). Peptides containing N-methylamino acids can be synthesized in the ribosome using cell-free translation systems such as the PURESYSTEM.

[0213] More specifically, the translation synthesis may involve, for example, protein factors necessary for translation in E. coli (methionyl-tRNA transformylase, EF-G, RF1, RF2, RF3, RRF, IF1, IF2, IF3, EF-Tu, EF-Ts, ARS (select from AlaRS, ArgRS, AsnRS, AspRS, CysRS, GlnRS, GluRS, GlyRS, HisRS, IleRS, LeuRS, LysRS, MetRS, PheRS, ProRS, SerRS, ThrRS, TrpRS, TyrRS, and ValRS)), ribosomes, amino acids, creatine kinase, myokinase, inorganic pyrophosphatase, nucleoside diphosphate kinase, E. coli-derived tRNA, creatine phosphate, potassium glutamate, HEPES-KOH This can be done by adding mRNA to a known cell-free translation system such as PURESYSTEM, which contains a pH 7.6 mixture of magnesium acetate, spermidine, dithiothreitol, GTP, ATP, CTP, UTP, and other components. Furthermore, by adding T7 RNA polymerase, transcription and translation from a template DNA containing a T7 promoter can be coupled. Furthermore, peptide compounds containing unnatural amino acids can be translationally synthesized by adding desired aminoacyl-tRNAs or unnatural amino acids (e.g., F-Tyr) tolerated by aminoacyl-tRNA synthetase (ARS) to the system (Kawakami T, et al., Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids. J Am Chem Soc. 2008, 130, 16861-3; Kawakami T, et al., Diverse backbone-cyclized peptides via codon reprogramming. Nat. Chem. Biol. 2009, 5, 888-90). Furthermore, peptide compounds containing unnatural amino acids can be translationally synthesized by including a modified ARS in the system in place of or in addition to a natural ARS, as well as a group of unnatural amino acids.Alternatively, the efficiency of translational incorporation of unnatural amino acids can be increased by using mutant ribosomes or EF-Tu (Dedkova LM, et al. Construction of modified ribosomes for incorporation of D-amino acids into proteins. Biochemistry. 2006, 45, 15541-51; Doi Y, et al. Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system. J. Am. Chem. Soc. 2007, 129, 14458-62; Park HS, et al. Expanding the genetic code of Escherichia coli withphosphoserine. Science 2011, 333, 1151-4).

[0214] In a non-limiting embodiment, the cell-free translation system of the present disclosure may be a translation system for producing a peptide compound, preferably a translation system for producing a cyclic peptide compound.

[0215] - tRNA Translational incorporation of unnatural amino acids into peptides requires aminoacylation of orthogonal tRNA that is efficiently incorporated into ribosomes ((i) Biochemistry. 2003;42:9598-608. Adaptation of an orthogonalar leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. Anderson JC, Schultz PG., (ii) Chem. Biol. 2003;10:1077-84. Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code. Murakami H, Kourouklis D, Suga H.). The following five methods can be used to aminoacylate tRNA.

[0216] Within cells, aminoacyl-tRNA synthetases (ARSs) are prepared for each amino acid as enzymes for aminoacylation of tRNA. Therefore, the first method is to utilize the fact that certain ARSs can tolerate unnatural amino acids such as N-Me His, or to prepare and use mutant aminoacyl-tRNA synthetases that can tolerate unnatural amino acids ((i) Proc. Natl. Acad. Sci. USA. 2002;99:9715-20. An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system. Kiga D, Sakamoto K, Kodama K, Kigawa T, Matsuda T, Yabuki T, ShirouzuM, Harada Y, Nakayama H, Takio K, Hasegawa Y, Endo Y, Hirao I, Yokoyama S. (ii) Science2003;301:964-7. An expanded eukaryotic genetic code. Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG. Chin, JW. (iii) Proc. Natl. Acad.Sci. US A. 2006;103:4356-61. Enzymatic aminoacylation of tRNA with unnatural amino acids.Hartman MC, Josephson K, Szostak JW.).Second, aminoacylation of tRNA in vitro followed by chemical modification of the amino acid can be used (J. Am. Chem. Soc. 2008;130:6131-6. Ribosomal synthesis of N-methylpeptides. Subtelny AO, Hartman MC, Szostak JW.). Third, aminoacyl-tRNA can be obtained by removing the CA from the CCA sequence at the 3' end of tRNA and ligating this with separately prepared aminoacylated pdCpA using RNA ligase (Biochemistry 1984;23:1468-73. T4 RNA ligase-mediated preparation of novel "chemically misacylated" tRNAPheS. Heckler TG, Chang LH, Zama Y, Naka T, Chorghade MS, Hecht SM.). Aminoacylation can also be performed using flexizyme, a ribozyme that can attach activated esters of various unnatural amino acids to tRNA (J. Am. Chem. Soc. 2002;124:6834-5. Aminoacyl-tRNA synthesis by a resin-immobilized ribozyme. Murakami H, Bonzagni NJ, Suga H.). Fourth, ultrasonic mixing of tRNA and activated amino acid esters in cationic micelles can also be used (Chem. Commun. (Camb). 2005;(34):4321-3. Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation. Hashimoto N, Ninomiya K, Endo T, Sisido M.).Fifth, aminoacylation can also be achieved by adding a PNA complementary to the 3' end of tRNA, to which an amino acid activated ester has been attached, to the tRNA (J. Am. Chem. Soc. 2004;126:15984-9. In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid. Ninomiya K, Minohata T, Nishimura M, Sisido M.).

[0217] More specifically, aminoacyl-tRNA can be prepared using the following method. A template DNA encoding the desired tRNA sequence, with a T7, T3, or SP6 promoter upstream, is prepared, and RNA can be synthesized by transcription using an RNA polymerase compatible with the promoter, such as T7 RNA polymerase, T3, or SP6 RNA polymerase. The target tRNA can also be extracted and purified from cells, and then extracted using a probe with a sequence complementary to the tRNA sequence. Cells transformed with an expression vector for the target tRNA can also be used as the source. RNA of the target sequence can also be synthesized by chemical synthesis. For example, aminoacyl-tRNA can be obtained by ligating the thus-obtained tRNA, in which the CA has been removed from the CCA sequence at the 3' end, with separately prepared aminoacylated pdCpA or pCpA using RNA ligase (pdCpA method, pCpA method). This tRNA is useful in the production of peptide compounds. Alternatively, aminoacylation can also be carried out by preparing full-length tRNA and using flexizyme, which is a ribozyme that carries the active ester of various unnatural amino acids on tRNA.In addition, although not intended to be limited, natural ARS or its modified form can also be used to prepare aminoacyl-tRNA.When natural ARS or its modified form is used, the aminoacyl-tRNA once consumed in the translation system can be regenerated by natural ARS or its modified form, so there is no need to make a large amount of pre-prepared aminoacyl-tRNA in the translation system.Such ARS modified form is described in WO2016 / 148044.These methods for preparing aminoacyl-tRNA can also be combined appropriately.

[0218] Cyclization Method of Peptide Moiety The cyclic peptide compounds of the present disclosure have a cyclic moiety. The method for cyclizing a peptide compound is not particularly limited. For example, a cyclization reaction can be carried out after synthesizing the peptide compound by chemical synthesis, translational synthesis, or the like. Examples of cyclization methods include amide bonds, carbon-carbon bonds, thioether bonds, disulfide bonds, ester bonds, thioester bonds, lactam bonds, bonds via triazole structures, and bonds via fluorophore structures. The peptide compound synthesis process and the cyclization reaction process may be carried out separately or sequentially. Cyclization can be carried out by methods known to those skilled in the art, such as those described in WO2013 / 100132, WO2008 / 117833, WO2012 / 074129, etc.

[0219] The cyclization moiety is not limited, but may be a bond between the N-terminus and C-terminus of the peptide, a bond between the N-terminus of the peptide and a side chain of another amino acid residue, a bond between the C-terminus of the peptide and a side chain of another amino acid residue, or a bond between the side chains of amino acid residues, or a combination of two or more of these. When a peptide-nucleic acid complex having a nucleic acid linked to the C-terminus is cyclized, the bond may be a bond between the N-terminus of the peptide and a side chain of another amino acid residue, or a bond between the side chains of amino acid residues.

[0220] In a non-limiting embodiment, the cyclic peptide compound of the present disclosure can be produced by the following method. For example, the production method can include the following steps: (1) synthesizing an acyclic peptide compound composed of amino acid residues, or amino acid residues and an N-terminal carboxylic acid analog, by translating a nucleic acid encoding the peptide compound, wherein the acyclic peptide compound contains an amino acid residue having one reactive site on the C-terminal side chain and an amino acid residue or N-terminal carboxylic acid analog having another reactive site on the N-terminal side; and (2) bonding the reactive site of the N-terminal amino acid residue or N-terminal carboxylic acid analog with the reactive site of the amino acid residue in the C-terminal side chain to form an amide bond, a carbon-carbon bond, or a thioether bond.

[0221] The steps (1) and (2) may be carried out separately or consecutively.

[0222] A non-limiting embodiment of a method for cyclizing a peptide compound through an amide bond is a method in which a peptide compound having cysteine ​​or a cysteine ​​analog at the N-terminus and an activated ester in the side chain of the amino acid on the C-terminus is translated and cyclized using native chemical ligation. There are no particular limitations on the method for translationally synthesizing a peptide compound having cysteine ​​or a cysteine ​​analog at the N-terminus, which is a precursor of this cyclic peptide compound.

[0223] In one embodiment, for example, the following three methods can be used to synthesize a peptide compound having an amino acid other than Met or Nle at the N-terminus of the peptide compound.

[0224] The first method involves cleaving the N-terminal amino acid or N-terminal peptide of a peptide initiated with formylmethionine or Met using an enzyme, such as peptide deformylase (PDF) and / or methionine aminopeptidase (MAP).

[0225] The second method is to use a reconstructed cell-free translation system, remove Met from the system in advance, place the amino acid to be placed at the N-terminus at the second residue from the N-terminus, and perform translational synthesis by skipping the start codon Met (defined as the initiation readthrough (iRT) method).

[0226] The third method is a method of translation synthesis in which an aminoacyl-tRNA, prepared in advance by aminoacylation of an amino acid to be placed at the N-terminus, is added as an initiation tRNA to a reconstructed cell-free translation system that does not contain Met or translation initiation methionine-tRNA (defined as the initiation suppression (iSP) method).

[0227] In a non-limiting embodiment, the C-terminal moiety of the cyclic peptide compound of the present disclosure may be chemically modified rather than remaining as a carboxylic acid. For example, the carboxylic acid moiety may be converted to a piperidine amide or the like by reacting with piperidine or the like.

[0228] The present invention is further illustrated, but not limited, in the following examples. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods.

[0229] The following abbreviations are used in the examples: AA: ammonium acetate, DBU: 1,8-diazabicyclo[5.4.0]-7-undecene, DCE: 1,2-dichloroethane, DCM: dichloromethane, DIC: N,N'-diisopropylcarbodiimide, DIPEA: N,N-diisopropylethylamine, DMF: N,N-dimethylformamide, DMSO: dimethyl sulfoxide, Fmoc: 9-fluorenylmethyloxycarbonyl, HATU: O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol, HOAt: 1-hydroxy-7-azabenzotriazole, NMP: N-methyl-2-pyrrolidone, TBME: t-butyl methyl ether TFE: 2,2,2-trifluoroethanol, TIPS: triisopropylsilane, Trt: triphenylmethyl, oxyma: ethyl cyano(hydroxyimino)acetate. The reaction solvents used for peptide synthesis and solid-phase synthesis were those for peptide synthesis (purchased from Kanto Chemical, Wako Pure Chemical, or Watanabe Chemical). Examples include DCM, DMF, DMSO, and DBU (2% DMF solution). For reactions that did not use water as a solvent, dehydrated or anhydrous solvents (purchased from Kanto Chemical, Wako Pure Chemical, or other companies) were used.

[0230] Example 1. Synthesis of compounds such as amino acids used in peptide synthesis The amino acids and amino acid-supported resins listed in Tables 1, 2, and 3 were used in the peptide synthesis described herein.

[0231] Example 1-1. Fmoc-amino acids used as purchased products The Fmoc-amino acids listed in Table 1 were purchased from commercial suppliers. In this specification, when an amino acid abbreviation in Table 1 is followed by -OH, it means that the C-terminus of that 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 with an Fmoc group and the C-terminus is a carboxyl group.

[0232]

[0233] Example 1-2. Synthesis of Fmoc-amino acids The Fmoc-amino acids listed in Table 2 were synthesized according to the scheme shown below.

[0234] Synthesis of compound aa001 (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-piperidin-1-ylbutanoic acid (Fmoc-Asp-pip)

[0235] Compound aa001 was synthesized according to the method described in WO2013 / 100132.

[0236] Synthesis of compound aa002 (3S)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-4-oxo-4-piperidin-1-ylbutanoic acid (Fmoc-MeAsp-pip)

[0237] Compound aa002 was synthesized according to the method described in WO2018 / 225864.

[0238] Synthesis of compound aa003 (2S,3R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(oxan-2-yloxy)butanoic acid (Fmoc-Thr(THP)-OH)

[0239] Compound aa003 was synthesized according to the method described in WO2018 / 225864.

[0240] Examples 1-3. Synthesis of amino acid-supported resin

[0241] In this specification, when a polymer or resin is bonded to a compound, the polymer or resin site may be represented by a circle. Furthermore, to clarify the reactive site of the resin site, the chemical structure of the reactive site may be represented by connecting it to a circle. For example, in the above structure, Fmoc-Asp(O-Trt(2-Cl)resin)-pip, the 2-chlorotrityl group of the resin is bonded to the side chain carboxyl group of Asp via an ester bond. Note that pip refers to piperidine, and in the above structure, the C-terminal carboxyl group forms an amide bond with piperidine. 2-Chlorotrityl chloride resin (1.25-1.60 mmol / g, 100-200 mesh, 1% DVB) was purchased from Watanabe Chemical Industry Co., Ltd. and SUNRESIN.

[0242] The Fmoc-amino acid-supported resins shown in Table 3 were synthesized according to the scheme shown below.

[0243] Synthesis of compound rs001: (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-4-piperidin-1-ylbutanoic acid-2-chlorotrityl resin (Fmoc-Asp(O-Trt(2-Cl)resin)-pip)

[0244] 2-Chlorotrityl chloride resin (1.60 mmol / g, 50.0 g, 80.0 mmol) and DCM (676 mL) were placed in a filter-equipped reaction vessel and shaken at room temperature for 10 minutes. After removing the DCM using nitrogen pressure, a mixed solution of compound aa001 (16.9 g, 40.0 mmol), methanol (12.9 mL, 320 mmol), DCM (676 mL), and DIPEA (33.4 mL, 192 mmol) was added to the reaction vessel and shaken at room temperature for 60 minutes. After removing the reaction solution using nitrogen pressure, a mixed solution of methanol (99.7 mL, 2.46 mmol), DCM (676 mL), and DIPEA (33.4 mL, 192 mmol) was added to the reaction vessel and shaken at room temperature for 90 minutes. After removing the reaction solution by applying nitrogen pressure, DCM (676 mL) was added and the mixture was shaken for 10 minutes, after which the reaction solution was removed by applying nitrogen pressure. This resin washing procedure with DCM was repeated twice, and the resulting resin was dried overnight under reduced pressure to obtain 61.8 g of compound rs001 (loading amount 0.452 mmol / g).

[0245] Synthesis of compound rs002, (3R)-3-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid-2-chlorotrityl resin (Fmoc-D-3-Abu-O-Trt(2-Cl) resin)

[0246] Using 2-chlorotrityl 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 (loading amount 0.494 mmol / g) was obtained in the same manner as in the synthesis of compound rs001.

[0247] Synthesis of compound rs003, (3R)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]butanoic acid-2-chlorotrityl resin (Fmoc-D-3-MeAbu-O-Trt(2-Cl) resin)

[0248] Using 2-chlorotrityl 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 (loading amount 0.495 mmol / g) was obtained in the same manner as in the synthesis of compound rs001.

[0249] Synthesis of compound rs004, (3S)-3-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-4-oxo-4-piperidin-1-ylbutanoate-2-chlorotrityl resin (Fmoc-MeAsp(O-Trt(2-Cl)resin)-pip)

[0250] Using 2-chlorotrityl chloride resin (1.44 mmol / g, 200 g, 289 mmol) and compound aa002 (82.2 g, 23 wt % of TBME included, 145 mmol) as starting materials, 239 g of compound rs004 (supported amount 0.509 mmol / g) was obtained by the same method as in the synthesis of compound rs001.

[0251] Example 2 Synthesis of Cyclic Peptide Compounds According to the scheme shown in Figure 1, cyclic peptide compounds consisting of 13 or 14 amino acid residues were synthesized through peptide elongation by solid-phase reaction, excision, cyclization, side chain deprotection, and purification.

[0252] The peptide elongation by solid-phase reaction described in this example was carried out by the Fmoc method using a peptide synthesizer (Multipep RS; manufactured by CEM (formerly Intavis)). Detailed operating procedures were performed in accordance with the manual attached to the synthesizer.

[0253] Detailed synthesis conditions in this example are shown below.

[0254] Peptide elongation by solid-phase reaction. Solution 1 was prepared by dissolving the Fmoc-protected amino acids (0.6 mol / L) constituting the target peptide and HOAt or oxyma (0.375 mol / L) as a carboxyl group activator in NMP. Furthermore, this was mixed with DMF to obtain a DIC of 10% (v / v), to prepare Solution 2. The resin (100 mg) carrying the Fmoc amino acids prepared in Examples 1-3 was placed in a solid-phase reaction vessel equipped with a filter (frit) and set in a peptide synthesizer. After adding DCM (1.2 mL) and allowing to stand for 30 minutes to allow the resin to swell, the solution was discharged from the frit. Solutions 1 and 2 were set in the peptide synthesizer, and automated synthesis using the peptide synthesizer was initiated.

[0255] A DMF solution of DBU (2% v / v, 0.7 mL) was added to the solid-phase reaction vessel containing the resin, and the Fmoc group removal reaction was carried out at room temperature. The reaction was allowed to proceed for 4.5 minutes for deprotection of the first residue, and for 10 minutes for deprotection of the second and subsequent residues, after which the solution was drained through the frit. DMF (0.7 mL) was added, and after 5 minutes of standing, the solution was drained through the frit. This resin washing process was repeated three more times. Next, Solution 1 (0.3 mL) and Solution 2 (0.36 mL) were mixed in the synthesizer's mixing vial and added to the resin. The solid-phase reaction vessel was heated to 40°C and allowed to react for 2.5 hours, allowing the condensation reaction between the amino groups on the resin and the Fmoc-protected amino acid to proceed, after which the solution was drained through the frit. The resin was then washed three times with DMF (0.7 mL). This Fmoc group removal reaction followed by Fmoc amino acid condensation reaction constituted one cycle, and this cycle was repeated to elongate the peptide on the resin surface. After peptide elongation was completed, a DBU solution in DMF (2% v / v, 0.7 mL) was added to the solid-phase reaction vessel containing the resin and reacted at room temperature for 15 minutes to remove the Fmoc group. The solution was then drained through the frit. The resulting resin was washed four times with DMF (0.7 mL) and then four times with DCM (0.7 mL).

[0256] Cleavage from resin TFE / DCM (1 / 1, 2.0 mL) containing 0.75% (v / v) DIPEA was added to the obtained resin, and the reaction was carried out at room temperature for 2 hours to perform the cleavage reaction of the peptide chain from the resin. After the reaction, the solution in the tube was recovered through the frit. TFE / DCM (1 / 1 (v / v), 1.0 mL) was added to the remaining resin, and the solution was recovered through the frit. This operation was repeated twice. All the obtained cleavage solutions were mixed, and then DMF (4.0 mL) was added, and the solvent was evaporated under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12).

[0257] The residue obtained by the above method was dissolved in a mixture of DMF (4.0 mL) and DCM (4.0 mL), to which a DMF solution of HATU (0.5 mol / L, 1.5 equivalents) and DIPEA (1.8 equivalents) were added, and the mixture was stirred at room temperature for 2 hours to carry out a cyclocondensation reaction between the N-terminal amino group and the carboxyl group that had been the resin-binding site. The solvent was then distilled off under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12). The number of equivalents was calculated based on the peptide loading amount (mmol / g) of the resin used as the raw material multiplied by the amount of resin used.

[0258] Deprotection is performed only for Thr-containing peptides. For Thr-free peptides, the deprotection step is omitted.

[0259] The residue obtained by the above method was dissolved in a solution (0.10 mol / L, 4.0 mL) of tetramethylammonium hydrogen sulfate in HFIP / TIPS / DCE (117 / 2.4 / 1 (v / v / v)) and stirred at room temperature for 1 hour to deprotect the Thr side chain. After adding DIPEA (0.070 mL), the solvent was evaporated under reduced pressure using a Genevac high-throughput centrifugal evaporator (HT-12).

[0260] DMSO was added to the residue obtained by the above method, and insoluble matter was removed by filtration. The target cyclic peptide was obtained by preparative HPLC using a Waters Auto Purification System, a YMC-Actus Triart C18 column (inner diameter 20 mm, length 100 mm), and a methanol-ammonium acetate aqueous solution (50 mmol / L) as the mobile phase.

[0261] According to the above synthesis conditions, cyclic peptides consisting of 13 or 14 amino acid residues as shown in Table 4 were synthesized. Table 4 shows the amino acid sequences in columns 1 to 14, starting from the N-terminus, and lists the exact mass, LCMS analysis conditions (LCMS cond.), observed mass spectrum (obs. mass; m / z), ion form, and retention time (Rt; min) for each compound. All peptides are cyclic peptides in which an amide bond is formed between the N-terminal amino group and the C-terminal carboxyl group (or the side chain carboxyl group when the C-terminus is Asp or MeAsp). Table 5 lists the LCMS analysis conditions. Table 15 shows the structural formula of each compound.

[0262]

[0263]

[0264] Example 3. Metabolic stability test of cyclic peptide compounds The metabolic stability of the cyclic peptide compounds composed of 13 or 14 amino acid residues synthesized in Example 2 was evaluated by a stability test in liver microsomes.

[0265] Mouse liver microsomes (male, Corning, USA) were prepared in 100 mM phosphate buffer (pH 7.4) to a concentration of 0.5 mg protein / mL, and the compound was metabolically reacted with the compound at a concentration of 1 μM for 22, 44, and 66 minutes in the presence and absence of NADPH. The compound was then deproteinized with acetonitrile over time, and the amount of the unchanged compound was analyzed using LCMS to determine the intrinsic hepatic clearance (CL). ｈ，ｉｎｔ) was calculated (Table 6).

[0266]

[0267] All of the tested cyclic peptide compounds consisting of 13 or 14 amino acid residues were shown to have high metabolic stability.

[0268] Example 4. Membrane permeability test of cyclic peptide compounds The membrane permeability of the cyclic peptide compounds consisting of 13 or 14 amino acid residues synthesized in Example 2 was evaluated according to the method for measuring membrane permeability using Caco-2 cells disclosed in WO2018 / 225864 shown below.

[0269] The apical medium was a mixture of FaSSIF and DMEM containing 1% DMSO, and the basal medium was DMEM. ２ Caco-2 cells and peptide compounds are pre-incubated for 20 to 24 hours under conditions of 37°C and 80 rpm. Then, the medium on the apical and basal sides is aspirated and washed, and the peptide compound is added to FaSSIF / HBSS buffer (1% DMSO) (pH 6.0) containing 1% DMSO on the apical side. HBSS buffer (pH 7.4) containing 4% BSA is added to the basal side, and measurement of membrane permeability is initiated. Each well is incubated at 5% CO ２ The mixture was shaken at 37°C and 80 rpm, and a sample was taken from the basal side about 180 minutes after the start of shaking. The amount of peptide compound in the sample was measured by LCMS, and the membrane permeability coefficient was calculated from the amount of permeation (Table 7). ａｐｐ ) the following formula can be used to calculate

[0270] dQ / dt represents the drug permeation rate (the amount of drug appearing on the basal side per unit time), C1(0) represents the drug concentration added on the apical side, and S represents the surface area of ​​the entire cell.

[0271]

[0272] It was shown that the cyclic peptide compounds consisting of 13 or 14 amino acid residues that were tested include compounds with various membrane permeabilities, ranging from high to low.

[0273] Example 5 Analysis of Various Properties and Membrane Permeability of Cyclic Peptide Compounds As shown in Example 3, all of the tested cyclic peptide compounds consisting of 13 or 14 amino acid residues were found to have high metabolic stability. Therefore, focusing on the membrane permeability shown in Example 4 as an index of drug-likeness, the relationship between the various properties of the cyclic peptide compounds was analyzed.

[0274] Table 8 shows the membrane permeability values ​​and various properties of each cyclic peptide used in the analysis.

[0275] In Table 8, total AA (total amino acid) represents the number of amino acid residues constituting each cyclic peptide, and N-alkyl number represents the number of N-alkylated amides among the main chain amides.

[0276] cLogP is a computer-calculated partition coefficient, calculated using Daylight Version 4.95 from Daylight Chemical Information Systems, Inc. Total AB (total amide bond) is the number of amide bonds in the main chain and side chain contained in each compound, and for example, the cyclic peptide shown in this example corresponds to one of the patterns shown in Table 9.

[0277] cLogP / total AB represents the value obtained by dividing cLogP by total AB, and was used in this example as an index of the lipid solubility of the peptide compound.

[0278] Example 5-1. Analysis of N-alkyl number and membrane permeability The relationship between the N-alkyl number and membrane permeability of cyclic peptide compounds was analyzed (Table 10).

[0279] In the case of compounds with an N-alkyl number of 7, 4 out of 36 compounds tested were P ａｐｐ ≧0.4×10 －６ cm / sec., but P ａｐｐ ≧1.0×10 －６ In the case of compounds with an N-alkyl number of 8, 19 out of 48 compounds tested showed a P ａｐｐ ≧0.4×10 －６ cm / sec, and seven of these compounds were P ａｐｐ ≧1.0×10 －６ In the case of compounds with an N-alkyl number of 9, 17 out of 48 compounds tested showed P ａｐｐ ≧0.4×10 －６ cm / sec, and four of these compounds are P ａｐｐ ≧1.0×10 －６ In the case of compounds with an N-alkyl number of 10, 13 out of 36 compounds tested showed P ａｐｐ ≧0.4×10 －６ cm / sec, and four of these compounds are P ａｐｐ ≧1.0×10 －６ cm / sec.

[0280] From the above, the compound with an N-alkyl number of 7 is P ａｐｐ ≧0.4×10 －６ cm / sec can be achieved, and compounds having an N-alkyl number of 8 or more are P ａｐｐ ≧1.0×10 －６ It has been shown that it is possible to achieve a P ａｐｐ ≧0.4×10 －６ It was shown that the proportion of compounds achieving cm / sec was higher for compounds with an N-alkyl number of 8 or more than for compounds with an N-alkyl number of 7.

[0281] Example 5-2 Analysis of Lipid Solubility and Membrane Permeability The relationship between lipid solubility and membrane permeability of cyclic peptide compounds was analyzed (Table 11). In this example, cLogP / totalAB was used as an index of lipid solubility.

[0282]

[0283] When the cLogP / totalAB of a compound is cLogP / totalAB<1.0, one compound out of 20 compounds tested is P ａｐｐ ≧0.4×10 －６ cm / sec., but P ａｐｐ ≧1.0×10 －６ When the cLogP / totalAB of the compound was 1.0≦cLogP / totalAB<1.1, 14 out of 52 compounds tested showed P ａｐｐ ≧0.4×10 －６ cm / sec, and one of the compounds is P ａｐｐ ≧1.0×10 －６ When the cLogP / totalAB of the compound was 1.1 or less, 39 of the 96 compounds tested showed P ａｐｐ ≧0.4×10 －６ cm / sec, and 14 of these compounds were P ａｐｐ ≧1.0×10 －６ cm / sec.

[0284] From the above, compounds with cLogP / totalAB<1.0 are P ａｐｐ ≧0.4×10 －６ cm / sec., and compounds with 1.0≦cLogP / totalAB<1.1 are P ａｐｐ ≧1.0×10 －６ cm / sec and compounds with 1.1≦cLogP / totalAB are P ａｐｐ ≧1.0×10 －６ It has been shown that it is possible to achieve a P ａｐｐ ≧0.4×10 －６ It was shown that the proportion of compounds achieving cm / sec was higher for compounds with 1.1≦cLogP / totalAB than for compounds with cLogP / totalAB<1.1.

[0285] Example 5-3. Analysis of MeLeu and membrane permeability The relationship between the number of MeLeu and membrane permeability was analyzed for each cyclic peptide compound (Table 12). From Example 5-2, for compounds with cLogP / totalAB<1.0, P ａｐｐ ≧0.4×10－６ Because it is difficult to achieve cm / sec, only compounds with cLogP / totalAB≧1.0 were analyzed.

[0286]

[0287] In the case of compounds with one MeLeu, five of the eight compounds tested were P ａｐｐ ≧0.4×10 －６ In the case of compounds with two MeLeu units, 20 out of 75 compounds tested showed P ａｐｐ ≧0.4×10 －６ In the case of compounds with three MeLeu units, 27 out of 63 compounds tested showed P ａｐｐ ≧0.4×10 －６ On the other hand, in the case of a compound with four MeLeu units, the P ａｐｐ ≧1.0×10 －６ No compound exhibited cm / sec.

[0288] Example 6. Mouse PK test of cyclic peptide compounds Examples 3 to 5 revealed that the conditions for a cyclic peptide compound consisting of 13 or 14 amino acid residues to have particularly good drug-likeness are that the number of N-alkyl groups is 8 or more and cLogP / totalAB is 1.1 or more. Four types of peptides that fulfill these conditions were administered intravenously or orally to mice, and the time course of their plasma concentrations was evaluated.

[0289] Male mice (C57BL / 6J, 10 weeks old, manufactured by Charles River Japan, Inc.; 3 mice per 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 up to 24 hours after administration. The collected blood was dispensed into tubes treated with heparin as an anticoagulant. Plasma was separated from the blood by centrifugation, and after deproteinization with acetonitrile, plasma concentrations were measured using an LC / MS / MS system (XEVO TQ-XS, manufactured by Waters). The following pharmacokinetic parameters were calculated from the obtained plasma concentration profiles by non-compartmental analysis using the analysis software Phoenix WinNonlin 6.1 (manufactured by Pharsight Corporation, USA) (Tables 13 and 14).

[0290] AUC ｉｎｆ : Area under the plasma concentration-time curve (ng·hours / mL) CL: Total body clearance (mL / min / kg) Vss: Steady-state distribution volume (L / kg) t1 / 2: Elimination half-life (hours) Cmax: Maximum plasma concentration (ng / mL) Tmax: Time to reach maximum plasma concentration (hours) F: Bioavailability (%)

[0291]

[0292]

[0293] The total body clearance after intravenous administration was small (1.7-14 mL / min / kg), suggesting high metabolic stability. The bioavailability of the compounds was up to 40%, and the four peptides evaluated had oral absorption that would allow development as oral preparations, as expected.

[0294]

Claims

1. A cyclic peptide compound to which a nucleic acid may be linked, the cyclic peptide compound having a cyclic portion, (1) the cyclic portion being composed of 13 or 14 amino acid residues, (2) among the amino acid residues constituting the cyclic portion, the number of N-substituted amino acid residues is 7 or more, the number of amino acid residues in the same side chain is 3 or less, and the number of natural amino acid residues is 4 or less, and (3) the ClogP / total amide bond (ClogP / total AB) is 1.0 to 1.

8.

2. A cyclic peptide compound to which the nucleic acid according to claim 1 may be linked, wherein the number of N-substituted amino acids constituting the cyclic portion is 7 to 13.

3. A cyclic peptide compound to which the nucleic acid according to claim 1 or 2 may be linked, wherein the N-substituted amino acid is an N-alkyl amino acid.

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

5. A cyclic peptide compound to which the nucleic acid according to any one of claims 1 to 4 may be linked, wherein the side chain of the amino acid constituting the cyclic portion is composed of at least one selected from the group consisting of an alkyl group, a cycloalkyl group, an aralkyl group, and an amide group, and wherein the alkyl group may form a ring together with any atom of the alkyl group and the nitrogen atom of the amino acid.

6. The alkyl group is not a halogen atom, a hydroxyl group, or a C ３ -C ８ Cycloalkyl groups, and C １ -C ６ C which may be substituted with at least one group selected from the group consisting of alkoxy groups １ -C ６ A cyclic peptide compound to which the nucleic acid according to claim 5 may be linked, the nucleic acid being an alkyl group.

7. The cycloalkyl group is not a halogen atom, a hydroxyl group, or a C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C which may be substituted with at least one group selected from the group consisting of haloalkoxy groups ３ -C １０ A cyclic peptide compound to which the nucleic acid according to claim 5 or 6, which is a cycloalkyl group, may be linked.

8. The aralkyl group is selected from the group consisting of halogen atoms, C １ -C ６ Alkyl group, C １ -C ６ Haloalkyl group, C １ -C ６ Alkoxy groups, and C １ -C ６ C which may be substituted with at least one group selected from the group consisting of haloalkoxy groups ７ -C １４ A cyclic peptide compound to which the nucleic acid according to any one of claims 5 to 7, which is an aralkyl group, is optionally linked.

9. The amide group has the formula: -CONR １ R ２ In the formula, R １ and R ２ are each independently a hydrogen atom or C １ -C ６ is an alkyl group or R １ and R ２ form a 4- to 8-membered saturated heterocyclic ring together with the nitrogen atom to which they are attached, and the 4- to 8-membered saturated heterocyclic ring is not further substituted by a halogen atom, an oxo group, C １ -C ６ A cyclic peptide compound to which the nucleic acid according to any one of claims 5 to 8 may be linked, which may be substituted with at least one group independently selected from the group consisting of an alkyl group and a 4- to 7-membered heterocyclyl group.

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

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

12. A cyclic peptide compound to which the nucleic acid according to any one of claims 1 to 11 may be linked, wherein the number of unnatural amino acids among the amino acids constituting the cyclic portion is 9 or more.

13. The cyclic peptide compound optionally to which the nucleic acid according to any one of claims 1 to 12 is linked, wherein the amino acids constituting the cyclic moiety include an unnatural 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).

14. The cyclic peptide compound Caco-2 P ａｐｐ (cm / sec) is 0.4×10 －６ The cyclic peptide compound optionally linked to the nucleic acid according to any one of claims 1 to 13, wherein 15. A library comprising a cyclic peptide compound to which the nucleic acid according to any one of claims 1 to 14 may be linked.

16. A method for screening a cyclic peptide compound capable of specifically binding to a target molecule, comprising the steps of: (i) contacting the target molecule with the library described in claim 15; and (ii) selecting a cyclic peptide compound capable of specifically binding to the target molecule.