Cyclic peptides for delivery of therapeutic agents

Abstract

The present disclosure relates to the synthesis of cyclic peptides that are effective to deliver a cargo, such as a therapeutic moiety (TM), into a cell to treat a variety of conditions and diseases.

Description

[0001] This application claims the benefit of priority to the filing dates of U.S. Provisional Application Serial No. 63 / 363,450 filed on April 22, 2022, U.S. Provisional Application Serial No. 63 / 354,471 filed on June 22, 2022, and U.S. Provisional Application Serial No. 63 / 377,754 filed on September 30, 2022, the contents of which are specifically incorporated herein by reference in their entirety Background Art

[0002] Nucleic acids and their synthetic analogs have great potential as therapeutic agents, especially against targets that are challenging to treat with traditional drug approaches (e.g., missing / defective proteins resulting from genetic mutations).

[0003] However, a challenge in translating the potential of such therapies to the clinic is that nucleic acids and their synthetic analogs have limited ability to enter intracellular compartments when administered systemically. Carrier systems such as polymers, cationic liposomes or chemical modifications (e.g., covalent attachment of cholesterol molecules) have been used to facilitate intracellular delivery. However, the intracellular delivery efficiency of these methods is generally low, and it is still difficult to find improved delivery systems that improve the efficacy of intracellular delivery.

[0004] There is a need for effective compositions to deliver therapeutic molecules to intracellular compartments for the treatment of disease, and some such compositions have been reported recently. However, there remains a need for the efficient synthesis of such compounds.

[0005] The present disclosure addresses these and other problems. Summary of the invention

[0006] The present disclosure relates to a method for preparing a cyclic peptide of formula (A):

[0007] or its protonated form,

[0008] in:

[0009] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0010] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0011] R4, R5, R6, and R7 are independently H or an amino acid side chain;

[0012] At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;

[0013] AA SC is an amino acid side chain; and

[0014] q is 1, 2, 3, or 4;

[0015] Wherein the method is any one of the methods described herein for the compound of formula (I).

[0016] The present disclosure relates to a method for preparing a cyclic peptide of formula (I):

[0017] or its protonated form,

[0018] in:

[0019] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0020] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0021] R4 and R6 are independently H or an amino acid side chain;

[0022] AA SC is the amino acid side chain;

[0023] q is 1, 2, 3, or 4; and

[0024] Each m is independently an integer 0, 1, 2 or 3;

[0025] The method comprises:

[0026] The compound of formula (1) With the compound of formula (II) Reaction to form a compound of formula (III)

[0027] wherein X and X' are independently protecting groups, X" is H or a protecting group, and X'" is H or an activating group (eg, NHS ester) and m is 0-3.

[0028] The present disclosure also relates to a method for preparing a cyclic peptide of formula (I):

[0029] or its protonated form,

[0030] in:

[0031] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0032] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0033] R4 and R6 are independently H or an amino acid side chain;

[0034] AA SC is the amino acid side chain;

[0035] q is 1, 2, 3, or 4; and

[0036] Each m is independently an integer 0, 1, 2 or 3,

[0037] The method comprises:

[0038] The compound of formula (IX) in is a solid support, and a compound of formula (X) wherein X and X' are independently protecting groups and Z is a group of an amino acid side chain, reacted to form a compound of formula (XI) Where p is an integer from 1 to 30.

[0039] The present disclosure relates to a method for preparing a cyclic peptide of formula (I):

[0040] or its protonated form,

[0041] in:

[0042] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0043] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0044] R4 and R6 are independently H or an amino acid side chain;

[0045] AA SC is the amino acid side chain;

[0046] q is 1, 2, 3, or 4; and

[0047] Each m is independently an integer 0, 1, 2 or 3,

[0048] The method comprises: making a compound of formula (XIII) wherein X is a protecting group and Z is a group of an amino acid side chain and is a solid support, and a compound of formula (XIV) Reaction to obtain a compound of formula (XV)

[0049] The present disclosure relates to a compound selected from the group consisting of:

[0050]

[0051] in:

[0052] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0053] R4 is H or an amino acid side chain;

[0054] Z is a group on the amino acid side chain;

[0055] q is 1, 2, 3, or 4;

[0056] X, X' and X" are each independently a protecting group; each m is independently an integer from 0 to 3; and It is a solid support.

[0057] The present disclosure relates to compounds selected from the group consisting of:

[0058]

[0059]

[0060]

[0061] in:

[0062] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0063] R4 is H or an amino acid side chain;

[0064] p is an integer from 1 to 30;

[0065] q is 1, 2, 3, or 4

[0066] X and X" are each independently a protecting group;

[0067] Each m is independently an integer from 0 to 3; and It is a solid support.

[0068] The present disclosure relates to compounds selected from the group consisting of:

[0069]

[0070] in:

[0071] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0072] R4 is H or an amino acid side chain;

[0073] Z is a group on the amino acid side chain;

[0074] q is 1, 2, 3, or 4;

[0075] X' and X" are each independently a protecting group;

[0076] Each m is independently an integer from 0 to 3; and

[0077] It is a solid support. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1A and 1B A synthetic route for the preparation of EEV is shown.

[0079] Figure 2 A synthetic route for preparing cyclic peptides is shown.

[0080] Figure 3 A synthetic route for preparing cyclic peptides is shown.

[0081] Figure 4 A synthetic route for preparing cyclic peptides is shown.

[0082] Figure 5 A synthetic route for the preparation of EEV is shown.

[0083] Figure 6 A synthetic route for the preparation of EEV is shown.

[0084] Figure 7 A synthetic route for preparing cyclic peptides is shown.

[0085] Figure 8 A synthetic route for the preparation of EEV is shown.

[0086] Fig. 9 The synthesis of Generation 1 EEV-PMO is shown, where PMO is a phosphorodiamidate morpholino oligomer.

[0087] Fig.10 The 2nd generation EEV-PMO synthesis is shown. DETAILED DESCRIPTION

[0088] The present disclosure relates to a method for preparing an endosomal escape vector (EEV).

[0089] Endosomal escape vector (EEV)

[0090] Provided herein is an endosomal escape vector (EEV), which can be used to transport cargo across the cell membrane, for example, to deliver cargo to the cytoplasm or nucleus of a cell. Cargo can include macromolecules, such as peptides or oligonucleotides, or small molecules. EEV can include a cell penetrating peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP) conjugated to an extracyclic peptide (EP). EP can include a sequence of a nuclear localization signal (NLS). EP can be coupled to cargo. EP can be coupled to cCPP. EP can be coupled to cargo and cCPP. The coupling between EP, cargo, cCPP or a combination thereof can be non-covalent or covalent. EP can be attached to the N-terminus of cCPP by a peptide bond. EP can be attached to the C-terminus of cCPP by a peptide bond. EP can be attached to cCPP by the side chain of an amino acid in cCPP. EP can be attached to cCPP by the side chain of lysine, and lysine can be conjugated to the side chain of glutamine in cCPP. EP can be conjugated to the 5' or 3' end of an oligonucleotide cargo. The EP can be coupled to a linker. The exocyclic peptide can be conjugated to the amino group of the linker. The EP can be coupled to the linker via the C-terminus of the EP and the cCPP through the side chains on the cCPP and / or the EP. For example, the EP can contain a terminal lysine, which can then be coupled to a cCPP containing glutamine through an amide bond. When the EP contains a terminal lysine and the side chain of the lysine can be used to attach the cCPP, the C-terminus or the N-terminus can be attached to the linker on the cargo.

[0091] Exocyclic peptide

[0092] The exocyclic peptide (EP) may comprise 2 to 10 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, including all ranges and values ​​therebetween. The EP may comprise 6 to 9 amino acid residues. The EP may comprise 4 to 8 amino acid residues.

[0093] Each amino acid in the exocyclic peptide can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid because it has a structure similar to that of a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 common naturally occurring amino acids, nor are they rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives or combinations thereof. These and other amino acids are listed in Table 1 together with the abbreviations they use herein. For example, the amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.

[0094] The EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and / or at least one amino acid residue comprising a side chain containing a guanidine group or a protonated form thereof. The EP may comprise 1 or 2 amino acid residues comprising a side chain containing a guanidine group or a protonated form thereof. The amino acid residue comprising a side chain containing a guanidine group may be an arginine residue. The protonated form may refer to a salt thereof throughout the disclosure.

[0095] EP may comprise at least two, at least three or at least four or more lysine residues. EP may comprise 2, 3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted by a protecting group, and the protecting group includes, for example, trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 4-methyltrityl (Mtt), 1-(4,4-dimethyl-2,6-dioxocyclohexyl)ethyl (Dde) or (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene-3)-methylbutyl (ivDde) ​​groups. The amino group on the side chain of each lysine residue may be substituted by a trifluoroacetyl (-COCF3). Protective groups may be included to achieve amide conjugation. Protective groups may be removed after EP is conjugated to cCPP.

[0096] EP may contain at least 2 amino acid residues with hydrophobic side chains. The amino acid residue with a hydrophobic side chain may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue with a hydrophobic side chain may be valine or proline.

[0097] EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and / or at least one arginine residue. EP may comprise at least two, at least three or at least four or more lysine residues and / or arginine residues.

[0098] EP can contain KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, KHKK, K KHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, wherein B is beta-alanine. The amino acids in the EP can have D or L stereochemistry.

[0099] The EP may comprise KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. The EP may comprise PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β-alanine. The amino acids in the EP may have D or L stereochemistry.

[0100] EP may consist of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV, or PKKKRKG. EP may consist of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, wherein B is β-alanine. The amino acids in EP may have D or L stereochemistry.

[0101] The EP may comprise an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may consist of an amino acid sequence identified as a nuclear localization sequence (NLS) in the art. The EP may comprise an NLS comprising an amino acid sequence PKKKRKV. The EP may consist of an NLS comprising an amino acid sequence PKKK RKV. The EP may comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKK K, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRR RVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKI KKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK and RKCLQ AGMNLEARKTKK. The EP may consist of an NLS comprising an amino acid sequence selected from the group consisting of NLSKRPAA IKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKF KNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKR PRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PK QKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVA KKKSKK, and RKCLQAGMNLEARKTKK

[0102] All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, EP may have the following structure: Ac-PKKKRKV.

[0103] Cell Penetrating Peptides (CPP)

[0104] The cell penetrating peptide (CPP) may comprise 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). The cCPP is capable of penetrating the cell membrane. An exocyclic peptide (EP) may be conjugated to the cCPP, and the resulting construct may be referred to as an endosomal escape vector (EEV). The cCPP may guide cargo (e.g., a therapeutic moiety (TM), such as an oligonucleotide, a peptide, or a small molecule) to penetrate the cell membrane. The cCPP may deliver cargo to the cytoplasm of the cell. The cCPP may deliver cargo to the cellular location where the target (e.g., pre-mRNA) is located. In order to conjugate the cCPP to cargo (e.g., a peptide, an oligonucleotide, or a small molecule), at least one bond or lone pair of electrons on the cCPP may be replaced.

[0105] The total number of amino acid residues in the cCPP is in the range of 6 to 20 amino acid residues, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, including all ranges and subranges therebetween. The cCPP may comprise 6 to 13 amino acid residues. The cCPP disclosed herein may comprise 6 to 10 amino acids. By way of example, a cCPP comprising 6-10 amino acid residues may have a structure according to any one of Formulas IA to IE:

[0106]

[0107] Among them, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9 and AA 10 is an amino acid residue.

[0108] cCPP may comprise 6 to 8 amino acids. cCPP may comprise 8 amino acids.

[0109] Each amino acid in cCPP can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a homologue of a natural amino acid because it has a structure similar to that of a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 common naturally occurring amino acids, nor are they rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives or combinations thereof. These and other amino acids are listed in Table 1 together with the abbreviations they use herein.

[0110] Table 1. Amino acid abbreviations

[0111]

[0112]

[0113] *Single letter abbreviations: When shown herein with a capital letter, it refers to the L-amino acid form, and when shown herein with a lower case letter, it refers to the D-amino acid form.

[0114] The cCPP may comprise 4 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid has no side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acids independently have a side chain comprising an aromatic or heteroaromatic group.

[0115] At least two amino acids may have no side chains or have side chains comprising Or its protonated form of the side chain. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg, -CH2-).

[0116] The amino acid having no side chain may be glycine or β-alanine.

[0117] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least one amino acid may be a glycine, b-alanine or 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0118] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least two amino acids may independently be glycine, b-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0119] The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least three amino acids may independently be glycine, b-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.

[0120] Glycine and related amino acid residues

[0121] cCPP may contain (i) 1, 2, 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.

[0122] cCPP may comprise (i) 1, 2, 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.

[0123] cCPP may contain (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.

[0124] cCPP may comprise at least three glycine residues. cCPP may comprise (i) 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues.

[0125] In an embodiment, none of the glycine, β-alanine or 4-aminobutyric acid residues in the cCPP are contiguous. Two or three glycine, β-alanine or 4-aminobutyric acid residues may be contiguous. Two glycine, β-alanine or 4-aminobutyric acid residues may be contiguous.

[0126] In an embodiment, no glycine residues in a cCPP are contiguous. Each glycine residue in a cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be contiguous. Two glycine residues may be contiguous.

[0127] Amino acid side chains with aromatic or heteroaromatic groups

[0128] cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group.

[0129] cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues independently having a side chain comprising an aromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic group.

[0130] The aromatic group may be a 6 to 14 membered aryl group. The aryl group may be a phenyl, naphthyl or anthracenyl group, each of which may be optionally substituted. The aryl group may be a phenyl or naphthyl group, each of which may be optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S. The heteroaryl group may be a pyridyl, quinolyl or isoquinolyl group.

[0131] The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be bis(homonaphthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine or tyrosine, each of which is optionally substituted with one or more substituents. The amino acids having side chains containing aromatic or heteroaromatic groups may each independently be selected from:

[0132]

[0133] wherein the N-terminal H and / or the C-terminal H are replaced by a peptide bond.

[0134] The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis(homonaphthylalanine) or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine. At least two amino acid residues having a side chain containing an aromatic group may be a residue of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.

[0135] In an embodiment, none of the amino acids having side chains comprising aromatic or heteroaromatic groups are adjacent. Two amino acids having side chains comprising aromatic or heteroaromatic groups can be adjacent. Two adjacent amino acids can have opposite stereochemistry. Two adjacent amino acids can have the same stereochemistry. Three amino acids having side chains comprising aromatic or heteroaromatic groups can be adjacent. Three adjacent amino acids can have the same stereochemistry. Three adjacent amino acids can have alternating stereochemistry.

[0136] The amino acid residue containing an aromatic or heteroaromatic group can be an L-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a D-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a mixture of a D-amino acid and an L-amino acid.

[0137] The optional substituent may be any atom or group that does not significantly reduce (e.g., more than 50%) the cytoplasmic delivery efficiency of the cCPP, for example compared to an otherwise identical sequence without the substituent. The optional substituent may be a hydrophobic substituent or a hydrophilic substituent. The optional substituent may be a hydrophobic substituent. The substituent may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent may be a halogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, a cycloalkynyl, a heterocyclyl, an aryl, a heteroaryl, an alkoxy, an aryloxy, an acyl, an alkylcarbamoyl, an alkylcarboxamidyl, an alkoxycarbonyl, an alkylthio, or an arylthio. The substituent may be a halogen.

[0138] Although not wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups (i.e., amino acids with side chains comprising aromatic or heteroaromatic groups) having higher hydrophobicity values ​​relative to amino acids with lower hydrophobicity values ​​can improve the cytoplasmic delivery efficiency of cCPPs. Each hydrophobic amino acid can independently have a hydrophobicity value greater than glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value greater than alanine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values ​​of various amino acids reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. USA 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. USA 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492), the entire contents of each of which are incorporated herein by reference. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.

[0139] Table 2. Amino Acid Hydrophobicity

[0140]

[0141]

[0142] Amino acid residues having a side chain containing a guanidine group, a guanidine replacement group or a protonated form thereof

[0143] As used herein, guanidine refers to the following structure:

[0144]

[0145] As used herein, the protonated form of guanidine refers to the following structure:

[0146]

[0147] The guanidine replacement group refers to a functional group on the amino acid side chain that will be positively charged at or above physiological pH, or can reproduce the hydrogen bond donating and accepting activity of the guanidinium group.

[0148] The guanidine replacement group promotes cell penetration and delivery of therapeutic agents while reducing the toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group. The cCPP may include at least two amino acids having side chains comprising a guanidine or guanidinium replacement group. The cCPP may include at least three amino acids having side chains comprising a guanidine or guanidinium replacement group.

[0149] The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium displacement group may be less basic than guanidine.

[0150] As used herein, a guanidine replacement group refers to or its protonated form.

[0151] The present disclosure relates to cCPPs comprising 4 to 20 amino acid residues, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid residue does not have a side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.

[0152] At least two amino acid residues may have no side chains or have Or its protonated form of the side chain. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg, -CH2-).

[0153] The cCPP may comprise at least one amino acid having a side chain comprising one of the following moieties: or its protonated form.

[0154] The cCPP may comprise at least two amino acids, each of which independently has one of the following moieties: or a protonated form thereof. At least two amino acids may have a side chain comprising the same moiety selected from: or its protonated form. At least one amino acid may have or their protonated side chains. At least two amino acids may have or their protonated forms. One, two, three or four amino acids may have a or its protonated form. An amino acid may have a side chain comprising or their protonated side chains. The two amino acids may have or its protonated form. Or its protonated form may be attached to the terminus of the amino acid side chain. Can be attached to the terminal end of an amino acid side chain.

[0155] The cCPP may comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4 or 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3 or 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidine group or a protonated form thereof.

[0156] The amino acid residues may independently have side chains comprising non-adjacent guanidine groups, guanidine replacement groups or their protonated forms. Two amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Three amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.

[0157] The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof can be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a D-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a mixture of L-amino acids or D-amino acids.

[0158] Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine residue or a protonated form thereof.

[0159] Each amino acid having a side chain containing a guanidine replacement group or a protonated form thereof may independently be or its protonated form.

[0160] Without being bound by theory, it is hypothesized that the guanidine replacement group has reduced basicity relative to arginine and, in some cases, is uncharged (e.g., -N(H)C(O)) at physiological pH and is able to maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which is thought to promote efficient membrane binding and subsequent internalization. The removal of the positive charge is also thought to reduce the toxicity of cCPP.

[0161] It will be understood by those skilled in the art that the N-terminus and / or C-terminus of the above-mentioned non-natural aromatic hydrophobic amino acids form an amide bond after incorporation into the peptides disclosed herein.

[0162] The cCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although by convention, the term "first amino acid" generally refers to the N-terminal amino acid of a peptide sequence, as used herein, "first amino acid" is used to distinguish the referred amino acid from another amino acid (e.g., "second amino acid") in the cCPP, such that the term "first amino acid" may be or may refer to an amino acid located at the N-terminus of the peptide sequence.

[0163] The cCPP may comprise: the N-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising a guanidino group or a protonated form thereof.

[0164] The cCPP may comprise a first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidine group or a protonated form thereof.

[0165] The cCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. The cCPP may comprise residues of asparagine. The cCPP may comprise residues of glutamine.

[0166] cCPP may comprise residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0167] Although not wishing to be bound by theory, it is believed that the chirality of the amino acids in the cCPP can affect the efficiency of cytoplasmic uptake. The cCPP may comprise at least one D amino acid. The cCPP may comprise one to fifteen D amino acids. The cCPP may comprise one to ten D amino acids. The cCPP may comprise 1, 2, 3 or 4 D amino acids. The cCPP may comprise 2, 3, 4, 5, 6, 7 or 8 adjacent amino acids with alternating D and L chirality. The cCPP may comprise three adjacent amino acids with the same chirality. The cCPP may comprise two adjacent amino acids with the same chirality. At least two amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids may have alternating stereochemistry relative to each other. At least three amino acids with alternating chirality relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids with alternating chirality relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids with the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Thus, the adjacent amino acids in a cCPP may have any of the following sequences: DL; LD; DLLD; LDDL; LDLLD; DLDDL; DLLDL; or LDDLD. An amino acid cCPP may comprise the following sequence: D / LXD / L; D / LXD / LX; D / LXD / LXD / L; DXD; DXDX; DXDXD; LXL; LXLX; or LXLXL, wherein D / L means that the amino acid may have a D or L stereochemistry and X is an achiral amino acid. The achiral amino acid may be glycine.

[0168] Amino acids having side chains comprising:

[0169]

[0170] or its protonated form, adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. An amino acid having a side chain comprising: or a protonated form thereof, may be adjacent to at least one amino acid having a side chain comprising guanidine or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising: or a protonated form thereof, may be adjacent to each other. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. A cCPP may comprise at least two adjacent amino acids having side chains that may comprise an aromatic or heteroaromatic group, and at least two non-adjacent amino acids having side chains comprising: or a protonated form thereof. The cCPP may comprise at least two contiguous amino acids having side chains comprising an aromatic or heteroaromatic group and at least two amino acids having side chains comprising or non-adjacent amino acids with side chains in their protonated form. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the preceding paragraphs.

[0171] At least two amino acids having side chains comprising:

[0172]

[0173] or a protonated form thereof, alternating with at least two amino acids having a side chain comprising a guanidine group or a protonated form thereof.

[0174] The cCPP may comprise a structure of formula (A):

[0175] or its protonated form,

[0176] in:

[0177] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0178] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0179] R4, R5, R6, and R7 are independently H or an amino acid side chain;

[0180] At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;

[0181] AA SC is an amino acid side chain; and

[0182] q is 1, 2, 3, or 4.

[0183] In an embodiment, at least one of R4, R5, R6, R7 is independently an uncharged non-aromatic side chain of an amino acid. In an embodiment, at least one of R4, R5, R6, R7 is independently H or a side chain of citrulline.

[0184] In an embodiment, a compound comprising a cyclic peptide with 6 to 12 amino acids is provided, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In an embodiment, at least two charged amino acids of the cyclic peptide are arginine. In an embodiment, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphthyl-2-yl-alanine) or a combination thereof. In an embodiment, at least two uncharged non-aromatic amino acids of the cyclic peptide are citrulline, glycine or a combination thereof. In an embodiment, the compound is a cyclic peptide with 6 to 12 amino acids, wherein two amino acids of the cyclic peptide are arginine, at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthylalanine and a combination thereof, and at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine and a combination thereof.

[0185] In an embodiment, the cyclic peptide of formula (A) is not a cyclic peptide having the sequence:

[0186]

[0187] wherein F is L-phenylalanine, f is D-phenylalanine, Φ is L-3-(2-naphthyl)-alanine, Φ is D-3-(2-naphthyl)-alanine, R is L-arginine, r is D-arginine, Q is L-glutamine, q is D-glutamine, C is L-cysteine, U is L-selenocysteine, W is L-tryptophan, K is L-lysine, D is L-aspartic acid, and Ω is L-norleucine.

[0188] The cCPP may comprise the structure of formula (I):

[0189]

[0190] or its protonated form,

[0191] in:

[0192] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0193] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0194] R4 and R6 are independently H or an amino acid side chain;

[0195] AA SC is the amino acid side chain;

[0196] q is 1, 2, 3, or 4; and

[0197] Each m is independently an integer of 0, 1, 2 or 3.

[0198] R1, R2 and R3 may each independently be H, -alkylene-aryl or -alkylene-heteroaryl. R1, R2 and R3 may each independently be H, -C 1-3 Alkylene-aryl or -C 1-3 R1, R2 and R3 can each independently be H or -alkylene-aryl. R1, R2 and R3 can each independently be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R1, R2 and R3 may each independently be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R1, R2 and R3 may each independently be H, -CH2Ph ​​or -CH2Naphthyl. R1, R2 and R3 may each independently be H or -CH2Ph.

[0199] R1, R2 and R3 can each independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthryl)-alanine.

[0200] R1 may be a side chain of tyrosine. R1 may be a side chain of phenylalanine. R1 may be a side chain of 1-naphthylalanine. R1 may be a side chain of 2-naphthylalanine. R1 may be a side chain of tryptophan. R1 may be a side chain of 3-benzothienylalanine. R1 may be a side chain of 4-phenylphenylalanine. R1 may be a side chain of 3,4-difluorophenylalanine. R1 may be a side chain of 4-trifluoromethylphenylalanine. R1 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 may be a side chain of homophenylalanine. R1 may be a side chain of β-homophenylalanine. R1 may be a side chain of 4-tert-butyl-phenylalanine. R1 may be a side chain of 4-pyridylalanine. R1 may be a side chain of 3-pyridylalanine. R1 may be a side chain of 4-methylphenylalanine. R1 may be a side chain of 4-fluorophenylalanine. R1 may be a side chain of 4-chlorophenylalanine. R1 may be the side chain of 3-(9-anthryl)-alanine.

[0201] R2 may be a side chain of tyrosine. R2 may be a side chain of phenylalanine. R2 may be a side chain of 1-naphthylalanine. R1 may be a side chain of 2-naphthylalanine. R2 may be a side chain of tryptophan. R2 may be a side chain of 3-benzothienylalanine. R2 may be a side chain of 4-phenylphenylalanine. R2 may be a side chain of 3,4-difluorophenylalanine. R2 may be a side chain of 4-trifluoromethylphenylalanine. R2 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R2 may be a side chain of homophenylalanine. R2 may be a side chain of β-homophenylalanine. R2 may be a side chain of 4-tert-butyl-phenylalanine. R2 may be a side chain of 4-pyridylalanine. R2 may be a side chain of 3-pyridylalanine. R2 may be a side chain of 4-methylphenylalanine. R2 may be a side chain of 4-fluorophenylalanine. R2 may be a side chain of 4-chlorophenylalanine. R2 may be the side chain of 3-(9-anthryl)-alanine.

[0202] R3 may be a side chain of tyrosine. R3 may be a side chain of phenylalanine. R3 may be a side chain of 1-naphthylalanine. R3 may be a side chain of 2-naphthylalanine. R3 may be a side chain of tryptophan. R3 may be a side chain of 3-benzothienylalanine. R3 may be a side chain of 4-phenylphenylalanine. R3 may be a side chain of 3,4-difluorophenylalanine. R3 may be a side chain of 4-trifluoromethylphenylalanine. R3 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be a side chain of homophenylalanine. R3 may be a side chain of β-homophenylalanine. R3 may be a side chain of 4-tert-butyl-phenylalanine. R3 may be a side chain of 4-pyridylalanine. R3 may be a side chain of 3-pyridylalanine. R3 may be a side chain of 4-methylphenylalanine. R3 may be a side chain of 4-fluorophenylalanine. R3 may be a side chain of 4-chlorophenylalanine. R3 may be the side chain of 3-(9-anthryl)-alanine.

[0203] R4 can be H, -alkylene-aryl, -alkylene-heteroaryl. R4 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R4 can be H or -alkylene-aryl. R4 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R4 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R4 can be H or a side chain of an amino acid in Table 1. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R4 can be H, -CH2Ph ​​or -CH2naphthyl. R4 can be H or -CH2Ph.

[0204] R5 can be H, -alkylene-aryl, -alkylene-heteroaryl. R5 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R5 can be H or -alkylene-aryl. R5 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R5 may be H, -C1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R5 can be H or a side chain of an amino acid in Table 1. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R5 can be H, -CH2Ph ​​or -CH2naphthyl. R4 can be H or -CH2Ph.

[0205] R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R6 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R6 can be H or -alkylene-aryl. R6 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R6 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R6 can be H or a side chain of an amino acid in Table 1. R6 can be H or an amino acid residue having a side chain containing an aromatic group. R6 can be H, -CH2Ph ​​or -CH2naphthyl. R6 can be H or -CH2Ph.

[0206] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. R7 can be H, -C 1-3 Alkylene-aryl or -C 1-3 Alkylene-heteroaryl. R7 can be H or -alkylene-aryl. R7 can be H or -C 1-3 Alkylene-aryl. C 1-3 The alkylene group may be a methylene group. The aryl group may be a 6- to 14-membered aryl group. The heteroaryl group may be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R7 may be H, -C 1-3 Alkylene -Ph or -C 1-3 Alkylene-naphthyl. R7 can be H or a side chain of an amino acid in Table 1. R7 can be H or an amino acid residue having a side chain containing an aromatic group. R7 can be H, -CH2Ph ​​or -CH2naphthyl. R7 can be H or -CH2Ph.

[0207] One, two or three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. One of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. Two of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. Three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph. No more than four of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph.

[0208] One, two or three of R1, R2, R3 and R4 are -CH2Ph. One of R1, R2, R3 and R4 is -CH2Ph. Two of R1, R2, R3 and R4 are -CH2Ph. Three of R1, R2, R3 and R4 are -CH2Ph. At least one of R1, R2, R3 and R4 is -CH2Ph.

[0209] One, two or three of R1, R2, R3, R4, R5, R6 and R7 may be H. One of R1, R2, R3, R4, R5, R6 and R7 may be H. Two of R1, R2, R3, R4, R5, R6 and R7 may be H. Three of R1, R2, R3, R4, R5, R6 and R7 may be H. At least one of R1, R2, R3, R4, R5, R6 and R7 may be H. No more than three of R1, R2, R3, R4, R5, R6 and R7 may be -CH2Ph.

[0210] One, two or three of R1, R2, R3 and R4 are H. One of R1, R2, R3 and R4 is H. Two of R1, R2, R3 and R4 are H. Three of R1, R2, R3 and R4 are H. At least one of R1, R2, R3 and R4 is H.

[0211] At least one of R4, R5, R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6 and R7 may be a side chain of 4-guanidino-2-aminopropionic acid. At least one of R4, R5, R6 and R7 may be a side chain of arginine. At least one of R4, R5, R6 and R7 may be a side chain of homoarginine. At least one of R4, R5, R6 and R7 may be a side chain of N-methylarginine. At least one of R4, R5, R6 and R7 may be a side chain of N,N-dimethylarginine. At least one of R4, R5, R6 and R7 may be a side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6 and R7 may be a side chain of 2,4-diaminobutyric acid, lysine. At least one of R4, R5, R6 and R7 may be a side chain of N-methyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N, N-dimethyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N-ethyllysine. At least one of R4, R5, R6 and R7 may be a side chain of N, N, N-trimethyllysine or 4-guanidinophenylalanine. At least one of R4, R5, R6 and R7 may be a side chain of citrulline. At least one of R4, R5, R6 and R7 may be a side chain of N, N-dimethyllysine or β-homoarginine. At least one of R4, R5, R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.

[0212] At least two of R4, R5, R6 and R7 may be the side chain of 3-guanidino-2-aminopropionic acid. At least two of R4, R5, R6 and R7 may be the side chain of 4-guanidino-2-aminobutyric acid. At least two of R4, R5, R6 and R7 may be the side chain of arginine. At least two of R4, R5, R6 and R7 may be the side chain of homoarginine. At least two of R4, R5, R6 and R7 may be the side chain of N-methylarginine. At least two of R4, R5, R6 and R7 may be the side chain of N,N-dimethylarginine. At least two of R4, R5, R6 and R7 may be the side chain of 2,3-diaminopropionic acid. At least two of R4, R5, R6 and R7 may be the side chain of 2,4-diaminobutyric acid, lysine. At least two of R4, R5, R6 and R7 may be the side chain of N-methyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N, N-dimethyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N-ethyllysine. At least two of R4, R5, R6 and R7 may be the side chain of N, N, N-trimethyllysine, 4-guanidinophenylalanine. At least two of R4, R5, R6 and R7 may be the side chain of citrulline. At least two of R4, R5, R6 and R7 may be the side chain of N, N-dimethyllysine, β-homoarginine. At least two of R4, R5, R6 and R7 may be the side chain of 3-(1-piperidinyl)alanine.

[0213] At least three of R4, R5, R6 and R7 may be the side chain of 3-guanidino-2-aminopropionic acid. At least three of R4, R5, R6 and R7 may be the side chain of 4-guanidino-2-aminobutyric acid. At least three of R4, R5, R6 and R7 may be the side chain of arginine. At least three of R4, R5, R6 and R7 may be the side chain of homoarginine. At least three of R4, R5, R6 and R7 may be the side chain of N-methylarginine. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethylarginine. At least three of R4, R5, R6 and R7 may be the side chain of 2,3-diaminopropionic acid. At least three of R4, R5, R6 and R7 may be the side chain of 2,4-diaminobutyric acid, lysine. At least three of R4, R5, R6 and R7 may be the side chain of N-methyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N-ethyllysine. At least three of R4, R5, R6 and R7 may be the side chain of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least three of R4, R5, R6 and R7 may be the side chain of citrulline. At least three of R4, R5, R6 and R7 may be the side chain of N,N-dimethyllysine, β-homoarginine. At least three of R4, R5, R6 and R7 may be the side chain of 3-(1-piperidinyl)alanine.

[0214] AA SC Can be the side chain of an asparagine, glutamine or homoglutamine residue. SC It can be a side chain of a glutamine residue. cCPP can also contain SC (e.g., residues of asparagine, glutamine, or homoglutamine). Thus, the cCPP may also comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. The cCPP may also comprise a linker conjugated to a glutamine residue.

[0215] q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.

[0216] m can be 1-3. m can be 1 or 2. m can be 0, m can be 1. m can be 2. m can be 3.

[0217] The cCPP of formula (A) may comprise the structure of formula (I):

[0218] or its protonated form, wherein AA SC , R1, R2, R3, R4, R6, m and q are as defined herein.

[0219] The cCPP of formula (A) may comprise the structure of formula (Ia) or formula (Ib):

[0220]

[0221] or its protonated form, wherein AA SC , R1, R2, R3, R4 and m are as defined herein.

[0222] The cCPP of formula (A) may comprise the structure of formula (I-1), (I-2), (I-3) or (I-4):

[0223]

[0224]

[0225] or its protonated form, wherein AA SC and m are as defined herein. The cCPP of formula (A) may comprise a structure of formula (I-5) or (I-6):

[0226]

[0227] or its protonated form, wherein AA SC As defined herein.

[0228] The cCPP of formula (A) may comprise the structure of formula (I-1):

[0229] or its protonated form, wherein AA SC and m are as defined herein.

[0230] The cCPP of formula (A) may comprise the structure of formula (I-2):

[0231] or its protonated form, wherein AA SC and m are as defined herein.

[0232] The cCPP of formula (A) may comprise the structure of formula (I-3):

[0233] or its protonated form, wherein AA SC and m are as defined herein.

[0234] The cCPP of formula (A) may comprise the structure of formula (I-4):

[0235] or its protonated form, wherein AA SC and m are as defined herein.

[0236] The cCPP of formula (A) may comprise a structure of formula (I-5):

[0237] or its protonated form, wherein AA SC and m are as defined herein.

[0238] The cCPP of formula (A) may comprise the structure of formula (I-6):

[0239] or its protonated form, wherein AA SC and m are as defined herein.

[0240] cCPP may comprise one of the following sequences: FGFGRGR; GfFGrGr; FfΦGRGR; FfFGRGR; or FfΦGrGr. cCPP may have one of the following sequences: FGFGRGRQ; GfFGrGrQ; FfΦGRGRQ; FfFGRGRQ; or FfΦGrGrQ.

[0241] The present disclosure also relates to a cCPP having the structure of formula (II):

[0242] or its protonated form,

[0243] in:

[0244] AA SC is the amino acid side chain;

[0245] R 1a , R 1b and R 1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;

[0246] R 2a , R 2b , R 2c and R 2d independently, an amino acid side chain;

[0247] R 2a , R 2b , R 2c and R 2d At least one of or its protonated form;

[0248] R 2a , R 2b , R 2c and R 2d At least one of is guanidine or a protonated form thereof;

[0249] Each n" is independently an integer 0, 1, 2, 3, 4 or 5;

[0250] Each n' is independently an integer of 0, 1, 2 or 3; and

[0251] If n' is 0, then R 2a , R 2b , R 2b or R 2d Does not exist.

[0252] R 2a , R 2b , R 2c and R 2d At least two of them can be or its protonated form. 2a , R 2b , R 2c and R 2d Two or three of the or its protonated form. 2a , R 2b , R 2c and R 2d One of them can be or its protonated form. At least one of R2a, R2b, R2c and R2d may be or a protonated form thereof, and the remainder of R2a, R2b, R2c and R2d may be guanidine or a protonated form thereof. At least two of R2a, R2b, R2c and R2d may be or a protonated form thereof, and the remainder of R2a, R2b, R2c and R2d may be guanidine or a protonated form thereof.

[0253] All R 2a , R 2b , R 2c and R 2d Can be or its protonated form. At least one of R2a, R2b, R2c and R2d may be or a protonated form thereof, and the remainder of R2a, R2b, R2c and R2d may be guanidine or a protonated form thereof. At least two of R2a, R2b, R2c and R2d may be or a protonated form thereof, and the remainder of R2a, R2b, R2c and R2d may be guanidine or a protonated form thereof.

[0254] Each of R2a, R2b, R2c and R2d can independently be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, the side chain of ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminobutyric acid, aspartic acid, glutamic acid or homoglutamic acid.

[0255] AA SC Can be Wherein t can be an integer from 0 to 5. SC Can be Wherein t may be an integer from 0 to 5. t may be from 1 to 5. t is 2 or 3. t may be 2. t may be 3.

[0256] R 1a , R 1b and R 1c R1a, R1b and R1c may each independently be a 6- to 14-membered aryl group. R1a, R1b and R1c may each independently be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O or S. R1a, R1b and R1c may each independently be selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl or isoquinolyl. 1a , R 1b and R 1c R may be independently selected from phenyl, naphthyl or anthracenyl. 1a , R 1b and R 1c R may be independently phenyl or naphthyl. 1a , R 1b and R 1c It may be pyridyl, quinolyl or isoquinolyl, each independently selected.

[0257] Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.

[0258] Each n" may independently be an integer from 1 to 3. Each n" may independently be 2 or 3. Each n" may be 2. Each n" may be 3. At least one n" may be 0. At least one n" may be 1. At least one n" may be 2. At least one n" may be 3.

[0259] Each n" may independently be 1 or 2, and each n' may independently be 2 or 3. Each n" may be 1 and each n' may independently be 2 or 3. Each n" may be 1 and each n' may be 2. Each n" is 1 and each n' is 3.

[0260] The cCPP of formula (II) may have the structure of formula (II-1):

[0261] or its protonated form,

[0262] Where R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d AA SC , n' and n" are as defined herein.

[0263] The cCPP of formula (II) may have the structure of formula (IIa):

[0264] or its protonated form,

[0265] Where R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d AA SC and n' are as defined herein.

[0266] The cCPP of formula (II) may have the structure of formula (IIb):

[0267] or its protonated form,

[0268] Where R 2a , R 2b AA SC and n' are as defined herein.

[0269] The cCPP may have the structure of formula (IIb):

[0270] or a protonated form thereof, wherein:

[0271] AA SC and n' are as defined herein.

[0272] The cCPP of formula (IIa) has one of the following structures:

[0273]

[0274]

[0275] Among them, AA SCand n are as defined herein. The cCPP of formula (IIa) has one of the following structures:

[0276]

[0277]

[0278] Among them, AA SC and n are as defined herein. The cCPP of formula (IIa) has one of the following structures:

[0279]

[0280] Among them, AA SC and n are as defined herein. The cCPP of formula (II) may have the following structure:

[0281]

[0282] The cCPP of formula (II) may have the following structure:

[0283]

[0284]

[0285] The cCPP may have the structure of formula (III):

[0286] or a protonated form thereof, wherein:

[0287] AA SC is the amino acid side chain;

[0288] R 1a , R 1b and R 1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;

[0289] R 2a and R 2c Each independently is H, or its protonated form;

[0290] R 2b and R 2d each independently is guanidine or a protonated form thereof;

[0291] Each n" is independently an integer from 1 to 3;

[0292] Each n' is independently an integer from 1 to 5; and

[0293] Each p' is independently an integer from 0 to 5.

[0294] The cCPP of formula (III) may have the structure of formula (III-1):

[0295] or its protonated form,

[0296] in:

[0297] AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d , n', n" and p' are as defined herein.

[0298] The cCPP of formula (III) may have the structure of formula (IIIa):

[0299] or its protonated form,

[0300] in:

[0301] AA SC , R 2a , R 2c , R 2b , R 2d , n', n" and p' are as defined herein.

[0302] In formula (III), (III-1) and (IIIa), R a and R c It can be H. R a and R c Can be H and R b and R d R and R are independently guanidine or a protonated form thereof. a It can be H. R b It can be H. p' can be 0. R a and R c can be H and each p' can be 0.

[0303] In formula (III), (III-1) and (IIIa), Ra and Rc may be H, Rb and Rd may each independently be guanidine or a protonated form thereof, n" may be 2 or 3, and each p' may be 0.

[0304] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5

[0305] cCPP can have the following structure:

[0306]

[0307] The cCPP of formula (A) may be selected from:

[0308]

[0309] The cCPP of formula (A) may be selected from:

[0310]

[0311] In an embodiment, the cCPP is selected from:

[0312]

[0313] Φ = L-naphthylalanine; -naphthylalanine; Ω = L-norleucine

[0314] AA SC Can be conjugated with a linker.

[0315] Connectors

[0316] The cCPP of the present disclosure can be conjugated with a linker. The linker can connect the cargo to the cCPP. The linker can be attached to the side chain of the amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker.

[0317] The linker can be any suitable part that can conjugate the cCPP to one or more additional parts (e.g., an exocyclic peptide (EP) and / or a cargo). Prior to conjugation with the cCPP and the one or more additional parts, the linker has two or more functional groups, each of which is capable of independently forming a covalent bond with the cCPP and the one or more additional parts. If the cargo is an oligonucleotide, the linker can be covalently bound to the 5' end of the cargo or the 3' end of the cargo. The linker can be covalently bound to the 5' end of the cargo. The linker can be covalently bound to the 3' end of the cargo. If the cargo is a peptide, the linker can be covalently bound to the N-terminus or C-terminus of the cargo. The linker can be covalently bound to the backbone of the oligonucleotide or peptide cargo. The linker can be any suitable part for conjugating the cCPP described herein to a cargo (such as an oligonucleotide, a peptide, or a small molecule).

[0318] The connector may comprise a hydrocarbon connector.

[0319] The linker may comprise a cleavage site. The cleavage site may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).

[0320] The linker may comprise: (i) one or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R 1- JR 2 )z"-subunit, where R 1 and R 2 Each occurrence is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl and heterocyclyl, and each J is independently C, NR 3 、-NR 3 C(O)-, S and O, among which R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; (viii)-(R 1- J)z"-or-(JR 1 )z”-, where R 1 Each occurrence is independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, and each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; or (ix) the linker may comprise one or more of (i) to (x).

[0321] The linker may comprise one or more D or L amino acids and / or -(R 1- JR 2 )z”-, where R 1 and R 2 is independently alkylene at each occurrence, and each J is independently C, NR 3 、-NR 3 C(O)-, S and O, among which R 4 is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.

[0322] The linker may contain -(OCH2CH2) z’ -(OCH2CH2)z') (e.g., as a spacer), wherein z' is an integer from 1 to 23, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. "-(OCH2CH2)z'" may also be referred to as polyethylene glycol (PEG).

[0323] The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2CH2) z’ - and a peptide, wherein z' is an integer from 1 to 23. The peptide may contain 2 to 10 amino acids. The linker may also contain a functional group (FG) that can react by click chemistry. FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

[0324] The linker may comprise (i) a β-alanine residue and a lysine residue; (ii) -(JR 1 )z”; or (iii) any combination thereof. Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic, each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R 1 may be alkylene and each J may be O.

[0325] The linker may comprise (i) a residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof; and (ii) -(R 1- J)z"-or-(JR 1 )z". Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic, each J is independently C, NR 3 、-NR 3 C(O)-, S or O, where R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R 1 may be an alkylene group and each J may be O. The linker may comprise glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof.

[0326] The linker can be a trivalent linker. The linker can have the following structure: Wherein A1, B1 and C1 can independently be a hydrocarbon linker (e.g., NRH-(CH2) n -COOH), PEG linkers (e.g., NRH-(CH2O) n -COOH, wherein R is H, methyl or ethyl) or one or more amino acid residues, and Z is independently a protecting group. The linker may also incorporate a cleavage site, including a disulfide [NH2-(CH2O) n-SS-(CH2O) n -COOH] or a caspase cleavage site (Val-Cit-PABC).

[0327] The hydrocarbon may be the residue of glycine or beta-alanine.

[0328] The linker may be bivalent and connect the cCPP to the cargo.The linker may be bivalent and connect the cCPP to the exocyclic peptide (EP).

[0329] The linker can be trivalent and connects the cCPP to the cargo and the EP.

[0330] The linker can be a divalent or trivalent C1-C 50 Alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker can be a divalent or trivalent C1-C 50 Alkylene wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.

[0331] The joint can have the following structures:

[0332] Where: Each AA is independently an amino acid residue; * is SC The attachment point, and AA SC is a side chain of an amino acid residue of a cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10. x may be an integer from 1 to 5. x may be an integer from 1 to 3. x may be 1. y may be an integer from 2 to 4. y may be 4. z may be an integer from 1 to 5. z may be an integer from 1 to 3. z may be 1. Each AA may be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, and 6-aminohexanoic acid.

[0333] The cCPP can be attached to the cargo via a linker ("L"). The linker can be conjugated to the cargo via a binding group ("M").

[0334] The joint can have the following structures:

[0335] Wherein: x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10; each AA is independently an amino acid residue; * is a residue with AA SC The attachment point, and AA SC is the side chain of an amino acid residue of cCPP; and M is a binding group as defined herein.

[0336] The joint can have the following structures:

[0337]

[0338] Where: x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * is the same as AA SC The attachment point, and AA SC is the side chain of an amino acid residue of cCPP; and M is a binding group as defined herein.

[0339] The joint can have the following structures:

[0340]

[0341] Wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; and z' is an integer from 1 to 23; * is the same as AA SC The attachment point, and AA SC It is the side chain of the amino acid residue of cCPP.

[0342] x can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.

[0343] x' may be an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. x' may be an integer from 5 to 15. x' may be an integer from 9 to 13. x' may be an integer from 1 to 5. x' may be 1.

[0344] y may be an integer from 1 to 5, such as 1, 2, 3, 4, or 5, including all ranges and subranges therebetween. y may be an integer from 2 to 5. y may be an integer from 3 to 5. y may be 3 or 4. y may be 4 or 5. y may be 3. y may be 4. y may be 5.

[0345] z can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.

[0346] z' may be an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. z' may be an integer from 5 to 15. z' may be an integer from 9 to 13. z' may be 11.

[0347] As discussed above, the linker or M (wherein M is part of a linker) can be covalently bound to a cargo (any suitable position on the cargo). The linker or M (wherein M is part of a linker) can be covalently bound to the 3' end of an oligonucleotide cargo or the 5' end of an oligonucleotide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the N-terminus or C-terminus of a peptide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the backbone of an oligonucleotide or peptide cargo.

[0348] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.

[0349] The linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the peptide cargo, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be bound to the side chain of lysine on the peptide cargo.

[0350] The joint can have the following structures:

[0351]

[0352] in

[0353] M is a group that conjugates L to a cargo, such as an oligonucleotide;

[0354] AA s It is the side chain or end of the amino acid on the cCPP;

[0355] Each AA x are independently amino acid residues;

[0356] o is an integer from 0 to 10; and

[0357] p is an integer from 0 to 5.

[0358] The joint can have the following structures:

[0359]

[0360] in

[0361] M is a group that conjugates L to a cargo, such as an oligonucleotide;

[0362] AA s It is the side chain or end of the amino acid on the cCPP;

[0363] Each AA x are independently amino acid residues;

[0364] o is an integer from 0 to 10; and

[0365] p is an integer from 0 to 5.

[0366] M may include alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic groups, each of which is optionally substituted. M may be selected from:

[0367]

[0368] wherein R is an alkyl, alkenyl, alkynyl, carbocyclic or heterocyclic group.

[0369] M can be selected from:

[0370]

[0371]

[0372] Where: R 10 is an alkylene group, a cycloalkyl group or Where a is 0 to 10.

[0373] M can be R 10 Can be and a is 0 to 10. M can be

[0374] M may be a heterobifunctional cross-linker, such as It is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, which is incorporated herein by reference in its entirety.

[0375] M may be -C(O)-.

[0376] AA s It can be the side chain or the end of an amino acid on cCPP. s Non-limiting examples include aspartic acid, glutamic acid, glutamine, asparagine or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). s May be AA as defined hereinSC .

[0377] Each AA X is independently a natural or non-natural amino acid. One or more AAX may be a natural amino acid. One or more AAX may be a non-natural amino acid. One or more AAX may be a β-amino acid. The β-amino acid may be β-alanine.

[0378] o may be an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o may be 0, 1, 2, or 3. o may be 0. o may be 1. o may be 2. o may be 3.

[0379] p may be 0 to 5, for example 0, 1, 2, 3, 4 or 5. p may be 0. p may be 1. p may be 2. p may be 3. p may be 4. p may be 5.

[0380] The joint can have the following structures:

[0381]

[0382] Among them, M, AA s , each-(R 1- JR 2 )z"-, o and z" are as defined herein; r can be 0 or 1.

[0383] r can be 0. r can be 1.

[0384] The joint can have the following structures:

[0385]

[0386] Among them, M, AA s , o, p, q, r and z" may each be as defined herein.

[0387] z" may be an integer from 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, including all ranges and values ​​therebetween. z" may be an integer from 5-20. z" may be an integer from 10-15.

[0388] The joint can have the following structures:

[0389]

[0390] in:

[0391] M, AA s and o are as defined herein.

[0392] Other non-limiting examples of suitable linkers include:

[0393]

[0394]

[0395]

[0396] Among them, M and AA s As defined herein.

[0397] Provided herein are compounds comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, the compound further comprising L, wherein a linker is conjugated to AC via a binding group (M), wherein M is

[0398] Provided herein are compounds comprising a cCPP and a cargo, the cargo comprising an antisense compound (AC), such as an antisense oligonucleotide, which is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein a linker is conjugated to AC via a binding group (M), wherein M is selected from:

[0399]

[0400] Where: R 1 is an alkylene group, a cycloalkyl group or wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, wherein R 1 yes And t' is 2.

[0401] The joint can have the following structures:

[0402]

[0403] Among them, AA s As defined herein, and m' is 0-10.

[0404] The linker may have the formula:

[0405]

[0406] The linker may have the formula: where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0407] The linker may have the formula:

[0408] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0409] The linker may have the formula:

[0410] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0411] The linker may have the formula: where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0412] The linker may have the formula:

[0413]

[0414] The linker may be covalently bound to the cargo at any suitable position on the cargo. The linker may be covalently bound to the 3' end of the cargo or the 5' end of the oligonucleotide cargo. The linker may be covalently bound to the backbone of the cargo.

[0415] The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.

[0416] cCPP-linker conjugate

[0417] The cCPP may be conjugated to a linker as defined herein. The linker may be conjugated to the AA of the cCPP as defined herein. SC Conjugation.

[0418] The linker may contain -(OCH2CH2) z’ -subunit (e.g. as a spacer), wherein z' is an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. "-(OCH2CH2) z’ " is also called PEG. The cCPP-linker conjugate may have a structure selected from Table 3:

[0419] Table 3: cCPP-linker conjugates

[0420] <![CDATA[Cyclo(FfФ-4gp-r-4gp-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Cit-r-Cit-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Pia-r-Pia-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Dml-r-Dml-rQ)-PEG4-K-NH2]]> <![CDATA[Cyclo(FfФ-Cit-r-Cit-rQ)-PEG 12 -OH]]> <![CDATA[Cyclo(fФR-Cit-R-Cit-Q)-PEG 12 -OH]]>

[0421] The linker may contain -(OCH2CH2) z’-subunit and a peptide subunit, wherein z' is an integer from 1 to 23. The peptide subunit may contain 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 4:

[0422] Table 4: cCPP-linker conjugates

[0423] <![CDATA[Ac-PKKKRKV-Lys(cyclo[FfФ-R-r-Cit-rQ])-PEG 12 -K(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-Lys(cyclo[FfФ-Cit-r-R-rQ])-PEG 12 -K(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-K(cyclo(FfФR-cit-R-cit-Q))-PEG 12 -K(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-B-k(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG2-k(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-PEG2-Lys(cyclo[Phe-Cit-r-Cit-rGln])-PEG4-k(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N3)-NH2]]> <![CDATA[Ac-pkkkrkv-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-k(N3)-NH2]]> Ac-rrv-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-rQ])-PEG12-OH <![CDATA[Ac-PKKKRKV-PEG2-Lys(cyclo[Phe-Cit-r-Cit-r-Q])-PEG12-k(N3)-NH2]]> <![CDATA[Ac-PKKK-Cit-KV-PEG2-Lys(Ring[FfФ-Cit-r-Cit-r-Q])-PEG12-k(N3)-NH2]]> <![CDATA[Ac-PKKKRKV-PEG2-Lys(cyclo[FfФ-Cit-r-Cit-r-Q]-PEG12-K(N3)-NH2]]>

[0424] Provided are EEVs comprising a cyclic cell penetrating peptide (cCPP), a linker and an exocyclic peptide (EP). The EEV may comprise a structure of formula (B):

[0425] or its protonated form,

[0426] in:

[0427] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0428] R4 and R6 are independently H or an amino acid side chain;

[0429] EP is an exocyclic peptide as defined herein;

[0430] Each m is independently an integer from 0 to 3;

[0431] n is an integer from 0 to 2;

[0432] x' is an integer from 1 to 20;

[0433] y is an integer from 1 to 5;

[0434] q is 1-4; and

[0435] z' is an integer from 1 to 23.

[0436] R1, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.

[0437] n can be 0. n can be 1. n can be 2.

[0438] The EEV may comprise a structure of formula (Ba) or (Bb):

[0439]

[0440] or its protonated form, wherein EP, R 1 , R 2 , R 3 , R 4 , m and z' are as defined above in formula (B).

[0441] The EEV may comprise a structure of formula (Bc):

[0442]

[0443] or its protonated form, wherein EP, R 1 , R 2 , R 3 , R 4 and m are as defined above in formula (B); AA is an amino acid as defined herein; M is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10.

[0444] The EEV may have a structure of formula (B-1), (B-2), (B-3) or (B-4):

[0445]

[0446]

[0447]

[0448] or a protonated form thereof, wherein EP is as defined above in formula (B).

[0449] The EEV may comprise formula (B) and may have the following structure: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH or Ac-PKKKRKVAEEA-K(cyclo[GfFGrGr Q])-PEG 12 -OH.

[0450] The EEV may comprise a cCPP of the formula:

[0451]

[0452] The EEV may comprise the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0453] The EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-AEEA-K-(cyclo[FGFGRGRQ])-PEG12-OH. The EEV can be:

[0454]

[0455] The EEV may be Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH. The EEV may be:

[0456]

[0457] EEV can be selected from

[0458] Ac-rr-miniPEG2-Dap[cyclo(FfΦ-Cit-r-Cit-rQ)]-PEG 12 -OH

[0459] Ac-frr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0460] Ac-rfr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0461] Ac-rbfbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0462] Ac-rrr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0463] Ac-rbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0464] Ac-rbrbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0465] Ac-hh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0466] Ac-hbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0467] Ac-hbhbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0468] Ac-rbhbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0469] Ac-hbrbh-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG 12 -OH

[0470] Ac-rr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0471] Ac-frr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0472] Ac-rfr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0473] Ac-rbfbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0474] Ac-rrr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0475] Ac-rbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0476] Ac-rbrbr-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0477] Ac-hh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0478] Ac-hbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0479] Ac-hbhbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0480] Ac-rbhbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0481] Ac-hbrbh-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-b-OH

[0482] Ac-KKKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0483] Ac-KGKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0484] Ac-KKGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0485] Ac-KKK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0486] Ac-KK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0487] Ac-KGK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0488] Ac-KBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0489] Ac-KBKBK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0490] Ac-KR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0491] Ac-KBR-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0492] Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0493] Ac-PKKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0494] Ac-PGKKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2Ac-PKGKRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0495] Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0496] Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0497] Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0498] Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0499] Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2

[0500] Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 and

[0501] Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2.

[0502] EEV can be selected from:

[0503] Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG 12 -K(N3)-NH2

[0504] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦGrGrQ])-miniPEG2-K(N3)-NH2

[0505] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2

[0506] Ac-KR-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0507] Ac-PKKKGKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0508] Ac-PKKKRKG-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0509] Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0510] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FFΦGRGRQ])-miniPEG2-K(N3)-NH2

[0511] Ac-PKKKRKV-miniPEG2-Lys(cyclo[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2 and

[0512] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦSrSrQ])-miniPEG2-K(N3)-NH2.

[0513] EEV can be selected from:

[0514] Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQ])-PEG 12 -OH

[0515] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ])-PEG 12 -OH

[0516] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ])-PEG 12 -OH

[0517] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG 12 -OHAc-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRrRQ])-PEG 12 -OH

[0518] Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ])-PEG 12 -OH and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ])-PEG 12 -OH.

[0519] EEV can be selected from:

[0520] Ac-KKKRKG-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0521] Ac-KKKRK-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0522] Ac-KKRKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0523] Ac-KRKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0524] Ac-KKKKR-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0525] Ac-RKKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH and

[0526] Ac-KKKRK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH.

[0527] EEV can be selected from:

[0528] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2

[0529] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0530] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2 and

[0531] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH.

[0532] Cargo can be AC ​​and EEV can be selected from:

[0533] Ac-PKKKRKV-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0534] Ac-PKKKRKV-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0535] Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0536] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0537] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0538] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0539] Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0540] Ac-rr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0541] Ac-rr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0542] Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0543] Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0544] Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0545] Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0546] Ac-rr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0547] Ac-rrr-PEG2-K(cyclo[FfΦGrGrQ])-PEG12 -OH

[0548] Ac-rrr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0549] Ac-rrr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0550] Ac-rrr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0551] Ac-rrr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0552] Ac-rrr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0553] Ac-rrr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0554] Ac-rhr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0555] Ac-rhr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0556] Ac-rhr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0557] Ac-rhr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0558] Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0559] Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0560] Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0561] Ac-rbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0562] Ac-rbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0563] Ac-rbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0564] Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0565] Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0566] Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0567] Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0568] Ac-rbrbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0569] Ac-rbrbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0570] Ac-rbrbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0571] Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0572] Ac-rbrbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0573] Ac-rbrbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0574] Ac-rbrbr-PEG2-K(cyclo[FGFRRRRQ])-PEG12 -OH

[0575] Ac-rbhbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0576] Ac-rbhbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0577] Ac-rbhbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0578] Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0579] Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0580] Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0581] Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0582] Ac-hbrbh-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH

[0583] Ac-hbrbh-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH

[0584] Ac-hbrbh-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH

[0585] Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0586] Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0587] Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH and

[0588] Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH,

[0589] wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.

[0590] In an embodiment, the cCPP may be:

[0591]

[0592] The cargo may be a protein and the EEV may be selected from:

[0593] Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0594] Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0595] Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0596] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0597] Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0598] Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0599] Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0600] Ac-rr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0601] Ac-rr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0602] Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0603] Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0604] Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0605] Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0606] Ac-rr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0607] Ac-rrr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0608] Ac-rrr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0609] Ac-rrr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0610] Ac-rrr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0611] Ac-rrr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0612] Ac-rrr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0613] Ac-rrr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0614] Ac-rhr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0615] Ac-rhr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG12 -OH

[0616] Ac-rhr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0617] Ac-rhr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0618] Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0619] Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0620] Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0621] Ac-rbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0622] Ac-rbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0623] Ac-rbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0624] Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0625] Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0626] Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0627] Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0628] Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0629] Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0630] Ac-rbrbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0631] Ac-rbrbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0632] Ac-rbrbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0633] Ac-rbrbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0634] Ac-rbrbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0635] Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0636] Ac-rbhbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0637] Ac-rbhbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0638] Ac-rbhbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0639] Ac-rbhbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0640] Ac-rbhbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0641] Ac-rbhbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0642] Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH

[0643] Ac-hbrbh-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH

[0644] Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH

[0645] Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH

[0646] Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH

[0647] Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH

[0648] Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH

[0649] wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.

[0650] goods

[0651] A cell penetrating peptide (CPP), such as a cyclic cell penetrating peptide (e.g., cCPP), can be conjugated to a cargo. The cargo can be a therapeutic moiety. The cargo can be conjugated to the terminal carbonyl group of a linker. At least one atom of the cyclic peptide can be replaced by the cargo, or at least one lone pair can form a bond with the cargo. The cargo can be conjugated to the cCPP via a linker. The cargo can be conjugated to the AA via a linker. SC Conjugation. At least one atom in the cCPP can be replaced by a therapeutic moiety, or at least one lone pair in the cCPP forms a bond with the therapeutic moiety. The hydroxyl group on the amino acid side chain of the cCPP can be replaced by a bond with the cargo. The hydroxyl group on the glutamine side chain of the cCPP can be replaced by a bond with the cargo. The cargo can be conjugated to the cCPP via a linker. The cargo can be conjugated to the AA via a linker. SC Conjugation.

[0652] The cargo may comprise one or more detectable moieties, one or more therapeutic moieties, one or more targeting moieties, or any combination thereof. The cargo may be a peptide, an oligonucleotide, or a small molecule. The cargo may be a peptide sequence or a non-peptide-based therapeutic agent. The cargo may be an antibody or an antigen-binding fragment thereof, including but not limited to a scFv or a nanobody.

[0653] Cyclic cell penetrating peptide (cCPP) conjugated to cargo moiety

[0654] Cyclic cell penetrating peptides (cCPPs) can be conjugated to cargo moieties.

[0655] The cargo moiety can be conjugated to the cCPP via a linker. The cargo moiety can comprise a therapeutic moiety. The therapeutic moiety can comprise an oligonucleotide, a peptide, or a small molecule. The oligonucleotide can comprise an antisense oligonucleotide. The cargo moiety can be conjugated to the linker at the terminal carbonyl to provide the following structure:

[0656] in:

[0657] EP is an exocyclic peptide, and M, AA SC , Goods, x', y and z' are as defined above, * is related to AA SC Attachment point. x' can be 1. y can be 4. z' can be 11. -(OCH2CH2) x’ -and / or-(OCH2CH2) z’ - may be independently replaced by one or more amino acids, including, for example, glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminocaproic acid, or a combination thereof.

[0658] An endosomal escape vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, and may be conjugated to a cargo to form an EEV-conjugate comprising a structure of formula (C):

[0659] or its protonated form,

[0660] in:

[0661] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0662] R4 is H or an amino acid side chain;

[0663] EP is an exocyclic peptide as defined herein;

[0664] Goods are Parts as defined herein;

[0665] Each m is independently an integer from 0 to 3;

[0666] n is an integer from 0 to 2;

[0667] x' is an integer from 2 to 20;

[0668] y is an integer from 1 to 5;

[0669] q is an integer from 1 to 4; and

[0670] z' is an integer from 2 to 20.

[0671] R1, R2, R3, R4, EP, cargo, m, n, x', y, q and z' are as defined herein.

[0672] The EEV may be conjugated to a cargo, and the EEV-conjugate may comprise a structure of formula (Ca) or (Cb):

[0673]

[0674] or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).

[0675] The EEV may be conjugated to a cargo, and the EEV-conjugate may comprise a structure of formula (Cc):

[0676]

[0677] or its protonated form, wherein EP, R 1 , R 2 , R 3 , R 4 and m are as defined above in formula (III); AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; and z may be an integer from 1 to 10.

[0678] The EEV may be conjugated to an oligonucleotide cargo, and the EEV-oligonucleotide conjugate may comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):

[0679]

[0680]

[0681] Method for preparing cyclic peptides The present disclosure relates to a method for preparing a cyclic peptide of formula (A):

[0682] or its protonated form,

[0683] in:

[0684] R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;

[0685] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0686] R4, R5, R6, and R7 are independently H or an amino acid side chain;

[0687] At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;

[0688] AA SC is an amino acid side chain; and

[0689] q is 1, 2, 3, or 4;

[0690] Wherein the method is any one of the methods described herein for the compound of formula (I).

[0691] The present disclosure relates to a method for preparing a cyclic peptide of formula (I):

[0692] or its protonated form,

[0693] in:

[0694] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0695] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0696] R4 and R6 are independently H or an amino acid side chain;

[0697] AA SC is the amino acid side chain;

[0698] q is 1, 2, 3, or 4; and

[0699] Each m is independently an integer 0, 1, 2 or 3;

[0700] The method comprises:

[0701] The compound of formula (1) With the compound of formula (II) Reaction to form a compound of formula (III)

[0702] wherein X and X' are independently protecting groups, X" is H or a protecting group, X'" is H or an activating group (eg, NHS ester), and m is 0-3. In some embodiments, X" is methyl or tert-butyl.

[0703] The method may include coupling or reacting a compound of formula (I) with a compound of formula (II) in the presence of a coupling reagent such as N,N'-dicyclohexylcarbodiimide (DCC). The method may also include treating with an activator such as N-hydroxysuccinimide. The method may also include treating with a base. The base may be NMM. In an embodiment, the combination of reagents and / or solvents may be DCC / N-hydroxysuccinimide / THF. In an embodiment, the combination of reagents and / or solvents may be NMM / DMF.

[0704] The method may also include converting the compound of formula (III) into a compound of formula (IV)

[0705] The method may comprise deprotecting or converting the compound of formula (III) to a compound of formula (IV) in the presence of a base or a weak acid.

[0706] The method may further comprise reacting a compound of formula (IV) with a compound of formula (V) To form a compound of formula (VI) Wherein Z is a radical of an amino acid side chain and It is a solid support.

[0707] The method may include coupling or reacting a compound of formula (VI) with a compound of formula (IV) in the presence of a coupling reagent such as DIC, HATU, DEPBT, an additive such as HOAt / Oxyma / K-Oxyma, Oxyma-B, and a base such as DIPEA / NMM. In an embodiment, the combination of reagents and / or solvents may be DIC / Oxyma. In an embodiment, the combination of reagents and / or solvents may be DIC / HOAt. In an embodiment, the combination of reagents and / or solvents may be DEPBT / DIPEA. In an embodiment, the combination of reagents and / or solvents may be DEPBT / NMM. In an embodiment, the combination of reagents and / or solvents may be HATU / NMM. In an embodiment, the combination of reagents and / or solvents may be DIC / K-Oxyma. In an embodiment, the combination of reagents and / or solvents may be DIC / Oxyma-B. In an embodiment, the solvent comprises DMF.

[0708] The compound of formula (IV) can be And the compound of formula (VI) can be

[0709] The method may also include treating the compound of formula (VII) with a coupling agent, an additive and a base

[0710] To obtain a compound of formula (VIII):

[0711] In some embodiments, the coupling agent can be PyOxim, PyAOP, PyBOP, PyBrOP, HATU, DIC, HBTU, TBTU, COMU or DEPBT. In some embodiments, the additive can be Oxyma, HOAt or HOBt. In some embodiments, the base can be DIPEA or NMM.

[0712] In an embodiment, the combination of reagents and / or solvents may be HATU / HOAt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be PyAOP / HOAt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be PyAOP / HOAt / NMM. In an embodiment, the combination of reagents and / or solvents may be PyBOP / HOBt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be PyBrop / DIPEA. In an embodiment, the combination of reagents and / or solvents may be PyOxim / Oxyma / DIPEA. In an embodiment, the combination of reagents and / or solvents may be DIC / HOBt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be HBTU / HOBt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be TBTU / HOBt / DIPEA. In an embodiment, the combination of reagents and / or solvents may be COMU / Oxyma / DIPEA. In an embodiment, the combination of reagents and / or solvents may be DEPBT / DIPEA. In an embodiment, the solvent comprises DMF.

[0713] The compound of formula (VII) can be

[0714] And the compound of formula (VIII) can be

[0715] The present disclosure also relates to a method for preparing a cyclic peptide of formula (Ia):

[0716] or its protonated form,

[0717] in:

[0718] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0719] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0720] R4 and R6 are independently H or an amino acid side chain;

[0721] Z is a group on the amino acid side chain;

[0722] P1 is

[0723] q is 1, 2, 3, or 4; and

[0724] Each m is independently an integer 0, 1, 2 or 3,

[0725] The method comprises:

[0726] The compound of formula (IX) wherein X is a protecting group and is a solid support, and a compound of formula (X) wherein X' is independently a protecting group and Z is a group of an amino acid side chain, reacted to form a compound of formula (XI)

[0727]

[0728] The method may comprise coupling or reacting a compound of formula (IX) with a compound of formula (X) in the presence of standard solid phase peptide conditions [Chan, WC, White PD, eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000].

[0729] The compound of formula (X) can be And the compound of formula (XI) can be

[0730]

[0731] The method may also include treating the compound of formula (XI) to form a compound of formula (XII)

[0732]

[0733] The method may include treating a compound of formula (XI) with a compound of formula (XII) in the presence of a base such as piperidine / hydrazine / DBU / sodium hydroxide / pyrrolidine / morpholine / diethylamine / tert-butylamine. The method may also include adding Pd(PPh3)4 / PhSiH3 / DCM. The method may also include adding a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA).

[0734] In an embodiment, the combination of reagents and / or solvents may be piperidine. In an embodiment, the combination of reagents and / or solvents may be piperidine / formic acid. In an embodiment, the combination of reagents and / or solvents may be piperidine / Oxyma. In an embodiment, the combination of reagents and / or solvents may be DBU. In an embodiment, the combination of reagents and / or solvents may be DBU / piperidine. In an embodiment, the combination of reagents and / or solvents may be DBU / piperidine / Oxyma. In an embodiment, the combination of reagents and / or solvents may be DBU / piperidine / HOBt. In an embodiment, the combination of reagents and / or solvents may be DBU / piperazine / formic acid. In an embodiment, the combination of reagents and / or solvents may be tert-butylamine, pyrrolidine. In an embodiment, the combination of reagents and / or solvents may be morpholine. In an embodiment, the combination of reagents and / or solvents may be diethylamine. In an embodiment, the combination of reagents and / or solvents may be sodium hydroxide. In an embodiment, the combination of reagents and / or solvents may be Pd(PPh3)4 / PhSiH3 / DCM, which is used to remove allyl esters. In an embodiment, the combination of reagents and / or solvents used for cyclization can be PyOxim / Oxyma / DIPEA / DMF / DCM.

[0735] The compound of formula (XII) can be

[0736]

[0737] The present disclosure relates to a method for preparing a cyclic peptide of formula (I):

[0738] or its protonated form,

[0739] in:

[0740] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0741] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0742] R4 and R6 are independently H or an amino acid side chain;

[0743] AA SCis the amino acid side chain;

[0744] q is 1, 2, 3, or 4; and

[0745] Each m is independently an integer 0, 1, 2 or 3;

[0746] The method comprises: making a compound of formula (XIII) Where X' is a protecting group, Z is a group of the amino acid side chain and is a solid support, and a compound of formula (XIV) Reaction to obtain a compound of formula (XV)

[0747] The method may include coupling or reacting a compound of formula (XIII) with a compound of formula (XIV) in the presence of Pd(PPh3)4 / PhSiH3 / DCM to remove the allyl ester, and then assembling according to standard solid phase peptide synthesis conditions for deprotection and coupling of the amino acid [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000].

[0748] The method, wherein the compound of formula (XIII) is The compound of formula (XIV) is And the compound of formula (XV) is

[0749] The method may also comprise treating the compound of formula (XV) to obtain a compound of formula (XVI):

[0750]

[0751] The method may comprise treating the compound of formula (XV) in the presence of standard solid phase peptide synthesis conditions to effect deprotection and coupling of the amino acid [Chan, WC, White PD, eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000].

[0752] The compound of formula (XVI) can be

[0753]

[0754] Alternative cyclic peptide formation

[0755] The present disclosure also relates to the preparation of compounds of formula (D)

[0756] or its protonated form,

[0757] in:

[0758] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0759] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0760] R4 and R6 are independently H or an amino acid side chain;

[0761] AA SC is the amino acid side chain;

[0762] Y is

[0763] q is 1, 2, 3, or 4;

[0764] Each m is independently an integer 0, 1, 2 or 3, and

[0765] Each n is independently an integer of 0, 1, 2 or 3.

[0766] Macrolactamization

[0767] The present disclosure also relates to a method for preparing a cyclic peptide of formula (DI): or a protonated form thereof, wherein:

[0768] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0769] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0770] R4 and R6 are independently H or an amino acid side chain;

[0771] AA SC is the amino acid side chain;

[0772] q is 1, 2, 3, or 4;

[0773] Each m is independently an integer 0, 1, 2 or 3, and

[0774] Y is

[0775] The method comprises: cyclizing a compound of formula (XVII)

[0776]

[0777] Wherein Z is a radical of an amino acid side chain and It is a solid support.

[0778] An example of a synthetic scheme is illustrated in Scheme 1.

[0779] Solution 1

[0780]

[0781] The method may include standard solid phase peptide synthesis conditions for deprotection and coupling of amino acids [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000]. For example, cyclization using a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA), and treatment with, for example, HFIP or TFA to cleave from the solid.

[0782] Ring Closing Metathesis (RCM)

[0783] The present disclosure also relates to a method for preparing a cyclic peptide of formula (D-II):

[0784] or its protonated form,

[0785] in:

[0786] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0787] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0788] R4 and R6 are independently H or an amino acid side chain;

[0789] AA SC is the amino acid side chain;

[0790] q is 1, 2, 3, or 4;

[0791] Each m is independently an integer 0, 1, 2 or 3,

[0792] Each n is independently an integer 0, 1, 2 or 3, and

[0793]

[0794] The method comprises: making a compound of formula (XVIII): Cyclization, wherein Z is a radical of an amino acid side chain and It is a solid support.

[0795] An example of a synthetic scheme is illustrated in Scheme 2.

[0796] Solution 2

[0797]

[0798] The method may include standard solid phase peptide synthesis conditions for deprotection and coupling of amino acids [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000]. For example, cyclization using a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA), and treatment with, for example, HFIP or TFA to cleave from the solid.

[0799] Thioester stapling

[0800] The present disclosure also relates to a method for preparing a cyclic peptide of formula (D-III):

[0801] or its protonated form,

[0802] in:

[0803] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0804] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0805] R4 and R6 are independently H or an amino acid side chain;

[0806] AA SC is the amino acid side chain;

[0807] q is 1, 2, 3, or 4;

[0808] Each m is independently an integer 0, 1, 2 or 3,

[0809] Each n is independently an integer 0, 1, 2 or 3, and

[0810] Y is

[0811] The method comprises: making a compound of formula (XIX): Cyclization, wherein Z is a radical of an amino acid side chain and It is a solid support.

[0812] An example of a synthetic scheme is illustrated in Scheme 3.

[0813] Solution 3

[0814]

[0815] The method may include standard solid phase peptide synthesis conditions for deprotection and coupling of amino acids [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000]. For example, cyclization using a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA), and treatment with, for example, HFIP or TFA to cleave from the solid.

[0816] Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)

[0817] The present disclosure also relates to a method for preparing a cyclic peptide of formula (D-IV):

[0818] or its protonated form,

[0819] in:

[0820] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0821] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0822] R4 and R6 are independently H or an amino acid side chain;

[0823] AA SC is the amino acid side chain;

[0824] q is 1, 2, 3, or 4;

[0825] Each m is independently an integer 0, 1, 2 or 3, and

[0826] Y is

[0827] The method comprises: making a compound of formula (XX): Cyclization, wherein Z is a radical of an amino acid side chain and It is a solid support.

[0828] An example of a synthetic scheme is illustrated in Scheme 4.

[0829] Solution 4

[0830]

[0831] The method may include standard solid phase peptide synthesis conditions for deprotection and coupling of amino acids [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000]. For example, cyclization using a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA), and treatment with, for example, HFIP or TFA to cleave from the solid.

[0832] Thioether cyclization

[0833] The present disclosure also relates to a method for preparing a cyclic peptide of formula (DV):

[0834] or its protonated form,

[0835] in:

[0836] R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;

[0837] At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;

[0838] R4 and R6 are independently H or an amino acid side chain;

[0839] AA SC is the amino acid side chain;

[0840] q is 1, 2, 3, or 4;

[0841] Each m is independently an integer 0, 1, 2 or 3, and

[0842] Y is

[0843] The method comprises: making a compound of formula (XXI) Cyclization, wherein Z is a radical of an amino acid side chain and It is a solid support.

[0844] An example of a synthetic scheme is illustrated in Scheme 5.

[0845] Solution 5

[0846]

[0847] The method may include standard solid phase peptide synthesis conditions for deprotection and coupling of amino acids [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000]. For example, cyclization using a coupling reagent (such as PyOxim), an additive (such as Oxyma) and a base (such as DIPEA), and treatment with, for example, HFIP or TFA to cleave from the solid.

[0848] Conjugation of cyclic peptides to linear peptides

[0849]

[0850] Method for preparing phosphorodiamidate morpholino oligomers (PMO)

[0851] PMO can be prepared according to any method known in the art, such as exemplified in: Summerton et al. US Patent 5,166,315. November 24, 1992; Summerton et al. US Patent 5,185,444, February 9, 1993; Summerton et al. US Patent 5,217,866, June 8, 1993; Summerton et al. US Patent 5,235,033. August 10, 1993; Summerton et al. US Patent 5,506,337, April 9, 1996; Summerton et al. US Patent 5,521,063, May 28, 1996; Summerton et al. Antisense Nucleic Acid Drug Dev. 1997, 7:187-195; Iversen, P.International Patent WO 02 / 092617 A1, November 21, 2002; Stein et al. US Patent 6,828,105 B2, December 7, 2004; Iversen et al. US Patent Application 2005 / 0261249 A1, November 24, 2005; Mourich et al. US Patent Application 2006 / 0276425 A1, December 7, 2006; Stein et al. US Patent Application 2007 / 0004661 A1, January 4, 2007; Stein et al. US Patent Application 2007 / 0129323 A1, June 7, 2007; Moulton et al. International Patent WO 2009 / 005793 A2, January 8, 2009; Moulton et al. US Patent Application 2010 / 0016215 A1, January 21, 2010; Sazani et al. US Patent Application, 2010 / 0130591, May 27, 2010; Weller et al. US Patent Application 2010 / 0234281 A1, September 16, 2010; Weller et al. US Patent 7,943,762, B2, May 17, 2011; Weller et al. US Patent 8,067,571 B2, November 29, 2011; Reeves et al. United States Patent 8,076,476 B2, December 13, 2011; Fox et al. United States Patent 8,299,206 B2, October 30, 2012; Linsley et al. United States Patent Application 2014 / 030238, October 9, 2014; Linsley et al. United States Patent Application 2014 / 0329772, November 6, 2014; Ueda, T. US Patent 8,969,551 B2, March 3, 2015; Hanson, G. US Patent 9,161,948, October 20, 2015; Bhadra et al. Nucleic Acid Chem. 2015, 62: 4.65.1-6.65.26; Totaro et al. International Patent WO 2019 / 0-60862A1, March 28, 2019; Torii et al. US Patent 10,415,036B2, September 17, 2019; Cai et al. US Patent Application 2019 / 0292208A1, September 26, 2019; Bestwick et al. US Patent Application 2019 / 0365918A1, December 5, 2019; Passini et al. US Patent Application 2020 / 0377886A1, December 3, 2020; Sinha et al. US Patent Application 2021 / 0130379, May 6, 2021; Fang et al. International Patent WO 2022 / 125987 A1, and Scheme 6. .

[0852] Solution 6

[0853]

[0854] The method may include, for example, treatment with 4-cyanopyridine and TFA for detritylation, treatment with DIPEA for neutralization, and addition of PMO monomer in the presence of a base such as NEM for coupling. The method may include, for example, further treatment with DTT in the presence of a base such as DBU for cleavage, and further treatment with a base such as ammonium hydroxide for deprotection.

[0855] Method of conjugating peptide + PMO

[0856] The cyclic peptide can be conjugated to the PMO according to any method known in the art, such as exemplified in: [Hanson, G. Peptide Oligonucleotide Conjugates. US Patent 9,161,948 B2, October 20, 2015] and Scheme 7. Example 4 also exemplifies various reaction conditions showing activation of the N3 terminal peptide by treatment with a base and a "coupling reagent" followed by addition of a PMO.

[0857]

[0858] Compound

[0859] The present disclosure also relates to a compound selected from the group consisting of:

[0860] in:

[0861] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0862] R4 is H or an amino acid side chain;

[0863] AA SC is the amino acid side chain;

[0864] q is 1, 2, 3, or 4;

[0865] X, X' and X" are each independently a protecting group;

[0866] Each m is independently an integer from 0 to 3; and It is a solid support.

[0867] The present disclosure also relates to a compound selected from the group consisting of:

[0868]

[0869]

[0870]

[0871] in:

[0872] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0873] R4 is H or an amino acid side chain;

[0874] AA SC is the amino acid side chain;

[0875] q is 1, 2, 3, or 4

[0876] X and X' are each independently a protecting group;

[0877] Each m is independently an integer from 0 to 3; and It is a solid support.

[0878] The present disclosure also relates to a compound selected from the group consisting of:

[0879]

[0880] in:

[0881] R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan;

[0882] R4 is H or an amino acid side chain;

[0883] AA SC is the amino acid side chain;

[0884] q is 1, 2, 3, or 4

[0885] X and X' are each independently a protecting group;

[0886] Each m is independently an integer from 0 to 3; and It is a solid support.

[0887] Synthesis can be done manually, automatically, or a combination of both.

[0888] The resin loading level may be ~0.1-1.0 mmol / g. The resin loading level may be ~0.30-0.50 mmol / g. The resin loading level may be ~0.20-0.30 mmol / g. The resin loading level may be ~0.20-0.25 mmol / g. The resin loading level may be ~0.2-0.50 mmol / g. The resin loading level may be ~0.2-0.60 mmol / g. The resin loading level may be ~0.2-0.70 mmol / g. The resin loading level may be ~0.2-0.80 mmol / g. The resin loading level may be ~0.2-0.90 mmol / g. The resin loading level may be ~0.22-0.92 mmol / g. The resin loading level may be ~0.22 mmol / g. The resin loading level may be ~0.39 mmol / g. The resin loading level may be ~0.46 mmol / g. The resin loading level may be ~0.64 mmol / g. The resin loading level may be -0.77 mmol / g. The resin loading level may be -0.92 mmol / g.

[0889] Some definitions

[0890] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, etc.

[0891] The term "about" (also written as "~") means a range (e.g., plus or minus 10% of the value) when it is immediately before a numerical value. For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless otherwise indicated by the context of the present disclosure, or inconsistent with such an explanation. For example, in a numerical list such as "about 49, about 50, about 55, ...", "about 50" means a range extending to less than half the interval between the preceding and following values, for example, greater than 49.5 to less than 52.5. In addition, the phrase "less than about" value or "greater than about" value should be understood according to the definition of the term "about" provided herein. Similarly, the term "about" when before a series of numerical values ​​or numerical ranges (e.g., "about 10, 20, 30" or "about 10-30") refers to all values ​​in the series or the endpoints of the range, respectively.

[0892] "2-[2-[2-aminoethoxy]ethoxy]acetic acid" is also known as AEEA or miniPEG.

[0893] As used herein, the term "cyclic cell penetrating peptide" or "CPP" refers to a peptide that facilitates the delivery of a cargo, such as a therapeutic moiety, into a cell.

[0894] As used herein, the term "endosomal escape vector" (EEV) refers to a CPP conjugated to a linker as defined herein and / or an exocyclic peptide as defined herein by chemical bonding (ie, covalent bond or non-covalent interaction). The EEV of the present disclosure is an EEV of formula (B).

[0895] As used herein, the term "EEV-conjugate" refers to an endosomal escape vector as defined herein that is conjugated to a cargo by chemical bonding (i.e., covalent bonding or non-covalent interaction). The cargo may be a therapeutic moiety (e.g., an oligonucleotide) that can be delivered into a cell by an EEV. The EEV-conjugate of the present disclosure may be an EEV-conjugate of formula (C).

[0896] As used herein, the terms "exocyclic peptide" (EP) and "modulatory peptide" (MP) are used interchangeably and refer to two or more amino acid residues connected by a peptide bond, which can be conjugated to the cyclic peptides disclosed herein. When conjugated to the cyclic peptides disclosed herein, the EP changes the tissue distribution and / or retention of the compound. Typically, the EP may contain at least one positively charged amino acid residue, such as at least one lysine residue and / or at least one arginine residue. Non-limiting examples of EPs are described herein. The EP may be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the SV40 virus large T antigen, whose minimum functional unit is the seven amino acid sequence PKKKRKV, the bipartite nucleoplasmic protein NLS having the sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of importin-α, the sequences VSRKRPRP and PPKKARE D of myoma T protein, the sequence PQPKKKPL of human p53, the sequence SALIKKKKK of mouse c-abl IV. MAP, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human) glucocorticoid. Additional examples of NLSs are described in International Publication No. 2001 / 038547 and are incorporated herein by reference in their entirety.

[0897] As used herein, "linker" or "L" refers to a moiety that covalently bonds one or more moieties (e.g., an exocyclic peptide (EP) and a cargo such as an oligonucleotide, peptide, or small molecule) to a cyclic peptide. The linker may comprise a natural or non-natural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding the cyclic peptide to the cargo moiety to form a compound disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. For example, the cyclic peptide may be covalently bound to the cargo via a linker.

[0898] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of connected nucleotides or nucleosides. One or more nucleotides of an oligonucleotide may be modified. An oligonucleotide may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). An oligonucleotide may be composed of natural and / or modified nucleobases, sugars, and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

[0899] The terms "peptide", "protein" and "polypeptide" are used interchangeably and refer to natural or synthetic molecules comprising two or more amino acids connected by the carboxyl group of one amino acid to the alpha amino group of another amino acid. Two or more amino acid residues can be connected to the alpha amino group through the carboxyl group of one amino acid. Two or more amino acids of a polypeptide can be joined by peptide bonds. A polypeptide may include a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond. A polypeptide may include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides comprising about 2 to about 100 amino acid residues and proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins, such as antibodies, enzymes, receptors, soluble proteins, etc.

[0900] The term "therapeutic polypeptide" refers to a polypeptide having therapeutic, preventive or other biological activity. Therapeutic polypeptides can be produced in any suitable manner. For example, therapeutic polypeptides can be isolated or purified from naturally occurring environments, can be chemically synthesized, can be recombinantly produced, or a combination thereof.

[0901] The term "small molecule" refers to an organic compound that has pharmacological activity and has a molecular weight of less than about 2000 Daltons, or less than about 1000 Daltons, or less than about 500 Daltons. Small molecule therapeutics are typically made by chemical synthesis.

[0902] As used herein, the term "adjacent" refers to two amino acids linked by a covalent bond. For example, in representative cyclic peptides such as Contiguous amino acid pairs are exemplified in the cases of AA1 / AA2, AA2 / AA3, AA3 / AA4, and AA5 / AA1.

[0903] As used herein, a residue of a chemical substance refers to a derivative of the chemical substance present in a particular product. To form the product, at least one atom of the substance is replaced by a bond to another moiety, such that the product contains a derivative or residue of the chemical substance. For example, the cyclic peptides described herein have an amino acid (e.g., arginine) incorporated therein by forming one or more peptide bonds. The amino acid incorporated into the cyclic peptide can be referred to as a residue, or simply an amino acid. Thus, arginine or an arginine residue refers to

[0904] The term "a protonated form thereof" refers to the protonated form of an amino acid. For example, the guanidinium group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is

[0905] As used herein, the term "chirality" refers to the "D" and "L" isomers of an amino acid or amino acid residue.

[0906] As used herein, the term "hydrophobic" refers to a portion that is insoluble or has minimal solubility in water. Typically, a neutral portion and / or a non-polar portion, or a predominantly neutral and / or non-polar portion is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein below.

[0907] As used herein, "aromatic" refers to an unsaturated ring molecule having 4n+2 π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated ring molecule that does not fall within the definition of aromatic.

[0908] "Alkyl," "alkyl chain," or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain radical having from one to forty carbon atoms and attached to the rest of the molecule by a single bond. Alkyl groups containing any number of carbon atoms from 1 to 40 are included. Alkyl groups containing up to 40 carbon atoms are C1-C 40 Alkyl groups, containing up to 10 carbon atoms, are C1-C 10 Alkyl, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl, and an alkyl containing up to 5 carbon atoms is a C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl and C1 alkyl (i.e., methyl). C1-C6 alkyl includes all of the moieties described above for C1-C5 alkyl, but also includes C6 alkyl. C1-C 10 Alkyl includes all of the moieties described above for C1-C5 alkyl and C1-C6 alkyl, but also includes C7, C8, C9 and C 10 Similarly, C1-C 12 Alkyl includes all of the aforementioned moieties, but also includes C 11 and C 12 Alkyl. C1-C 12 The non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless specifically stated otherwise in the specification, the alkyl group may be optionally substituted.

[0909] "Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated straight or branched divalent hydrocarbon chain radical having from one to forty carbon atoms. 40 Non-limiting examples of alkylene include ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0910] "Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 40 are included. Alkenyl groups containing up to 40 carbon atoms are C2-C 40 Alkenyl, alkenyl containing up to 10 carbon atoms is C2-C 10 Alkenyl, alkenyl containing up to 6 carbon atoms is C2-C6 alkenyl, and alkenyl containing up to 5 carbon atoms is C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl and C2 alkenyl. C2-C6 alkenyl includes all the parts described above for C2-C5 alkenyl, but also includes C6 alkenyl. C2-C 10 Alkenyl includes all of the moieties described above for C2-C5 alkenyl and C2-C6 alkenyl, but also includes C7, C8, C9 and C 10 Similarly, C2-C 12 Alkenyl includes all of the aforementioned moieties, but also includes C 11 and C 12 Alkenyl. C2-C 12 Non-limiting examples of alkenyl groups include ethenyl / vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4- The alkyl groups may be optionally substituted, unless otherwise specifically stated in the specification, alkyl groups may be optionally substituted, or substituted.

[0911] "Alkenylene", "alkenylene chain" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain radical having two to forty carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene groups include ethylene, propylene, butylene, etc. Unless stated otherwise specifically in the specification, the alkenylene chain may be optional.

[0912] "Alkoxy" or "alkoxy group" refers to a group -OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl as defined herein. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.

[0913] "Acyl" or "acyl group" refers to the group -C(O)R, wherein R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, as defined herein. Unless stated otherwise specifically in the specification, an acyl group may be optionally substituted.

[0914] "Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group -OC(O)-NR a R b , where R a and R b are the same or different and are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, or R as defined herein. a R b Together they may form a cycloalkyl group or a heterocyclyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylcarbamoyl group may be optionally substituted.

[0915] "Alkylcarboxamido" or "alkylcarboxamido group" refers to the group -C(O)-NR a R b , wherein Ra and Rb are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl group as defined herein, or R a R b Together they may form a cycloalkyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylcarboxamido group may be optionally substituted.

[0916] "Aryl" refers to a hydrocarbon ring system group comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For the purposes of the present invention, an aryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a condensed ring or a bridged ring system. Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" is intended to include optionally substituted aryl groups.

[0917] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepanyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzophenylthio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, oxazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxypyridinyl, 1-oxypyrimidinyl, 1-oxypyrazinyl, 1-oxypyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group may be optionally substituted.

[0918] The term "substituted" as used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamido, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is replaced by a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more atoms are replaced by a higher order bond (e.g., double or triple bond) to a heteroatom (such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles). For example, "substituted" includes groups in which one or more atoms are replaced by -NR g R h 、-NR g C(=O)R h 、-NR g C(=O)NR g R h 、-NR g C(=O)OR h 、-NR g S02R h 、-OC(=O)NR g R h 、-OR g 、-SR g 、-SOR g 、-SO2R g 、-OSO2R g 、-SO2OR g , =NSO2R g and -SO2NR g R h "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by -C(=O)R g 、-C(=O)OR g 、-C(=O)NR g R h 、-CH2SO2R g 、-CH2SO2NR g R hIn the above, R g and R h The same or different, and independently are hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" also means any of the above groups in which one or more atoms are replaced by amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" may also mean an amino acid in which one or more atoms on the side chain are replaced by an alkyl, alkenyl, alkynyl, acyl, alkylcarboxamide, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In addition, each of the aforementioned substituents may also be optionally substituted by one or more of the above substituents.

[0919] As used herein, "activating group" means an electron donating group that increases the stability and overall reactivity of a compound / intermediate. The activating group can be, for example, an NHS ester or PhSiH3.

[0920] As used herein, "subject" means an individual. Thus, "subject" may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., physician).

[0921] The term "inhibit" refers to a reduction in an activity, reaction, disorder, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, reaction, disorder or disease. This may also include, for example, a 10% reduction in an activity, reaction, disorder or disease compared to a natural or control level. Thus, a reduction may be a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount therebetween compared to a natural or control level.

[0922] "Reduce" or other forms of the word, such as "reducing" or "reduction", means a decrease in an event or characteristic (e.g., tumor growth). It will be understood that this is typically relative to some standard or expected value, in other words, it is relative, but does not always require reference to a standard or relative value. For example, "reduce tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., an untreated tumor).

[0923] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to the amelioration of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed to the elimination of the cause of the disease, pathological condition or disorder in question. In addition, the term includes palliative treatment, i.e., treatment directed to the relief of symptoms rather than the cure of a disease, pathological condition or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the development of the disease, pathological condition or disorder in question; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the amelioration of the disease, pathological condition or disorder in question.

[0924] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only needs to be reduced or altered, not eliminated.

[0925] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0926] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in a subject.

[0927] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It can be ensured that the action of microorganisms is prevented by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Suitable inert carriers may include sugars such as lactose.

[0928]

[0929]

[0930]

[0931] Example

[0932] Preparation method

[0933] The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art. All reactions can be carried out in solution (using a solvent or a mixture of solvents) or neat (no solvent is required).

[0934] The variation of compounds described herein includes the addition, subtraction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in a molecule, the chirality of the molecule can be changed. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, Wuts and Greene, Protective Groups in Organic Synthesis, 4th edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0935] The starting materials and reagents used to prepare the disclosed compounds and compositions can be purchased from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or prepared by methods known to those skilled in the art according to the procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementary Editions (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.

[0936] The reactions that produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reactions are carried out. The reactions can be carried out in one solvent or in a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0937] Example 1: Linear peptide synthesis (linker synthesis)

[0938] Linear peptide synthesis - Fmoc-PEG 12 -CH2CH2COOH loaded onto Wang resin

[0939] Add DCM to the glass peptide synthesis vessel containing Wang resin, swell the resin and drain the solvent. Add DCM to the resin. Weigh the required amount of Fmoc-PEG 12 -CH2CH2COOH was placed in a container and dissolved in a minimum amount of DMF, the resulting solution was transferred to the resin container and pyridine was added. 2,6-Dichlorobenzoyl chloride was added and the resin suspension was stirred until the reaction was determined to be complete.

[0940] Fmoc deprotection

[0941] DMF was added to the glass peptide synthesis vessel containing the resin-bound peptide, allowed to swell and drained. A 20% piperidine solution in DMF was added to completely cover the resin and react. Drained and washed sequentially with DMF, DCM and then DMF again.

[0942] Conjugation of individual amino acids (AA)

[0943] Weigh Fmoc-AA into a container and add additives (e.g., Oxyma. Dissolve Fmoc-AA and additives in DMF until completely dissolved. Add DIC, mix, and let stand at room temperature. Add pre-activated Fmoc-AA solution onto the resin and mix.

[0944] Example 2: Cyclic Peptide Synthesis

[0945] Loading the first Fmoc-AA onto 2-chlorotrisphenyl chloride (CTC) resin

[0946] Add DMF to the glass peptide synthesis vessel containing CTC resin, swell the resin and drain the solvent. Weigh the desired Fmoc-AA and dissolve in DMF. Add the AA solution to the resin. Add DIPEA and mix. Once the reaction is determined to be complete, quench with methanol and wash the resin.

[0947] Fmoc deprotection: as described in Example 1

[0948] Deprotection of Methyltrityl (Mtt) Groups of Exocyclic Peptides

[0949] Prepare a solution of hexafluoroisopropanol (HFIP) in DCM. Add it to the vessel containing the resin-bound peptide. Mix the contents at room temperature. Drain and wash the resin with DCM. Repeat until deprotection is complete.

[0950] Coupling of each AA: as described in Example 1

[0951] Deprotection of allyl esters of cyclic peptides

[0952] Add a resin bed volume of DCM to a glass peptide synthesis vessel containing a cyclopeptide linear precursor with Fmoc protection at the N-terminus and allyl protection at the C-terminus. Swell the resin at room temperature. Weigh Pd(PPh3)4 and dissolve the solid in DCM. Drain the resin and add fresh DCM. Add PhSiH3 to the resin suspended in DCM. Add the Pd solution to the resin, protect from light, and mix at room temperature. Drain the resin and wash with DCM and DMF in turn. Weigh SDDC·(H2O)3 (sodium diethyldithiocarbamate trihydrate) and dissolve in DMF. Add the mixture to the resin and mix. After completion, wash the resin with DCM and DMF.

[0953] Cleavage from solid supports - cyclic peptides

[0954] Prepare HFIP:DCM mixture, add to peptide synthesis vessel containing resin-bound cyclic peptide and mix. Wash resin with minimal DCM. Evaporate filtrate to minimum volume and grind with cold MTBE. Filter resulting suspension and wash filter cake with MTBE.

[0955] Example 3: Coupling of cyclic peptides to linear peptides

[0956] Dissolve the crude cyclic peptide and HOAt in DMF. Add DIC, mix, and let stand at RT. Add the preactivated cyclic peptide solution onto the resin and mix. Shake the reaction mixture at room temperature.

[0957] Cleavage from solid support

[0958] Prepare a mixture of TFA: DCM 95: 5 (volume / volume) and add it to a glass peptide synthesis vessel containing a resin-bound cyclic peptide. Mix the contents and filter. Wash the resin with a minimum amount of DCM. Evaporate the filtrate to a minimum volume and grind with cold MTBE. Filter the resulting suspension and wash the filter cake with MTBE. The resulting crude peptide can be purified by preparative RP-HPLC and further lyophilized.

[0959] Example 4: Phosphorodiamidate Morpholino Oligomer (PMO) Synthesis

[0960] Loading of PMO monomers onto solid support:

[0961] Aminomethyl polystyrene resin is suspended in NMP and the resin is swollen. Filter the resin to remove NMP and wash with 5% DIPEA solution in DCM, DCM and DCM. PMO monomer is dissolved, functionalized to active succinate in dry NMP, and added to the resin. Stir the reaction mixture at RT. Filter the resin and wash with NMP and DCM. Dry the resin and quantitatively test the load by trityl. Prepare a solution of 0.4M benzoic anhydride and NEM in NMP solution. Add benzoic anhydride / NEM solution to the resin and stir at RT. Filter the resin and wash twice with NMP, twice with IPA and twice with DCM. Dry the resin.

[0962] Resin loading determined by trityl quantification

[0963] Place dry resin (50-100 mg) in a fritted syringe and swell with DCM. Remove DCM and add deprotection solution (3% TFA in DCM) to the resin. Stir the solution for 5 min and collect the filtrate. Repeat this process four more times until the solution is colorless. Combine all filtrates, dilute the solution with water, and measure the absorbance of the sample solution at 404 nm. Use 3% TFA in DCM as a blank during the test. Determine the resin loading using L=OD*N*12 / ε / W (L: resin loading, mmol / g; OD: OD value of the sample solution; N: dilution factor; W: resin weight, g; ε: molar absorptivity = 32500)

[0964] PMO Synthetic Resin Swelling

[0965] Before starting the PMO synthesis, the resin functionalized with the PMO monomer was suspended in NMP and swelled for 2 h.

[0966] Trityl deprotection

[0967] Wash the resin with DCM. Treat the resin with 4-cyanopyridine, TFA in 80:20:1 DCM / TFE / EtOH (CYTFA)

[0968] Neutralization

[0969] The resin was treated with a neutralizing solution of 1:3 IPA / DCM solution containing 5% DIPEA. The resin was washed with DCM and anhydrous (1,3-dimethyl-2-imidazolidinone) DMI.

[0970] Coupling

[0971] Prepare a solution containing an anhydrous DMI solution of 0.2M PMO monomer and 0.4M NEM. For the first coupling, 5 equivalents of PMO monomer and NEM are used. For the next coupling, until the 10th position of the sequence, 3 equivalents of PMO monomer and 6 equivalents of NEM are used. For couplings from the 10th position to the 20th position, 4 equivalents of PMO monomer and 8 equivalents of NEM are used. For couplings starting from the 20th position, 5 equivalents of PMO monomer and 10 equivalents of NEM are used. The coupling solution is added to the resin and reacted at RT to 45°C. The coupling reaction carried out at 45°C is more efficient and can be completed in a shorter timeframe (2-4h). The coupling reaction is monitored by the chloranil test (Pept Res., 8(4): 236-7.1995). The resin is washed with DCM and 30% TFE in DCM. The resin can be stored overnight in a 30% TFE DCM solution. If the PMO sequence has been completed, wash the resin four times with IPA and dry the resin.

[0972] Cleavage and deprotection

[0973] Treat the dried detritylated PMO bound resin with NMP and allow it to swell at 30°C for 3h. Drain the resin and add a cleavage mixture containing 1.0-1.4M DTT, 2.0-2.8M DBU in NMP to the resin and react at 40°C for 2h. Collect the filtrate into a clean filter bottle. Dilute the cleaved PMO solution with cold ammonium hydroxide to form a uniform mixture. Place the PMO solution in a pressure flask and incubate at 50°C for 18h at 120rpm. Repeat this process as detailed above for a second round of cleavage. Collect two cleavage solutions and dilute with water.

[0974] PMO Purification

[0975] The diluted PMO lysate solution was concentrated by tangential flow filtration (TFF). Diafiltration was performed with water until the conductivity was <300 μS / cm. The desalted PMO was concentrated to a sufficient volume for anion exchange (AEX) purification. The crude PMO was loaded and purified using an AEX column (TOYOPEARL SuperQ-650S) with the following gradient buffer: buffer A = 10-25 mM NaOH; buffer B = 10-25 mM NaOH + 0.5-1 M NaCl. Pure fractions were identified by ion pair reverse phase chromatography (IP-RP), combined, and desalted by TFF. The IP-RP method was performed using a C18 column and the following gradient buffers: Buffer A = 10 mM triethylamine (TEA), 4.3-8.6 mM Na3PO4, 10 mM dodecyltrimethylammonium bromide (DTMA) in 55% methanol; Buffer B = 10 mM TEA, 4.3 Na3PO4, 10 mM DTMA in 60% acetonitrile. Diafiltration was performed until the conductivity of the pure PMO solution was <350 μS / cm. The desalted pure PMO was concentrated and lyophilized to obtain a white powder. The purity of the pure PMO was determined by the above IP-RP method.

[0976] Example 5: EEV-PMO Synthesis - Method 1

[0977] EEV conjugation with PMO

[0978] Dissolve PMO in DMSO. Separately prepare DMSO solutions of EEV, DMSO solutions of HATU, and DMSO solutions of DIPEA. Add DIPEA, HATU, and EEV solutions to the dissolved PMO solution. Analyze the progress of the reaction by CEX or RP-HPLC.

[0979] TFA deprotection

[0980] For the 1st generation conditions, the conjugation reaction was diluted with water and mixed. The mixture was diluted with a solution containing 25 mM NaOH and 0.2 M KCl and mixed. The progress of the reaction was analyzed by CEX or RP-HPLC. After the deprotection was completed, the reaction was diluted with 0.5 M NaH2PO4 buffer and then purified.

[0981] For the second generation conditions, the mixture was diluted with a solution containing 320 mM NaOH and mixed. The reaction progress was analyzed by CEX or RP-HPLC. After the deprotection was completed, the reaction was diluted with 0.5 M NaH2PO4 buffer and then purified.

[0982] EEV-PMO purification

[0983] The diluted PMO lysate solution was concentrated by tangential flow filtration (TFF). The desalted PMO was concentrated to a sufficient volume for cation exchange (CEX) purification. The crude PMO was loaded and purified using a CEX column with the following gradient buffer: buffer A = water, or 10-25 mM NaH2PO4 in 15-20% acetonitrile; buffer B = 0.5-1 M NaCl, or 10-25 mM NaH2PO4, 0.5-1 M NaCl in 15-20% acetonitrile. The pure fractions were identified by ion pair reverse phase chromatography (IP-RP), combined, and desalted by TFF. The desalted pure PMO was concentrated and lyophilized to obtain a white powder. The purity of pure PMO was determined by CEX method using a strong cation exchange (SCX) column and the following gradient buffers: buffer A = 24 mM H3PO4 in 80% water and 20% acetonitrile; buffer B = 24 mM H, 2.0 M LiCl in 75% water and 25% acetonitrile.

[0984] Example 6: Optimization of reaction conditions

[0985] Optimization of linear peptide fragment synthesis.

[0986] The replacement of Fmoc-Proline by Ac-Proline reduces the number of synthetic steps for preparing linear peptide fragments. In the first generation protocol, Fmoc-Proline is coupled to the last amino acid in the linear peptide sequence. Fmoc deprotection is then performed, and the free amine of the proline residue is then protected by an acetyl group (via reaction with acetic anhydride). For larger scale reactions (>1 g), this protocol can be performed manually, while using a smaller scale (approximately 600 mg of 0.4 mmol / g load material per 40 mL reactor), this protocol can be performed in an automated manner.

[0987] Inhibition of epimerization during dipeptide coupling in cyclic peptide synthesis:

[0988] Alternative non-Fmoc protecting groups for the dipeptide Gly-Arg(Pbf) were investigated. The use of Pht, Dde, and Fmoc / Dmb in the synthesis of cyclopeptides was investigated and epimerization was evaluated throughout the synthesis and in the final product.

[0989] Table 5. Summary of Pht deprotection screens using hydrazine with Oxyma and hydrazine alone.

[0990]

[0991]

[0992] Table 6. Summary of Pht deprotection screening using hydrazine with allyl alcohol and methylhydrazine.

[0993]

[0994]

[0995] Table 7. Summary of the purity of cyclic peptide fragments synthesized with various Gly protecting groups. (Pure obtained by UPLC integration Spend)

[0996]

[0997] Dipeptide conjugation screening to inhibit epimerization during cyclopeptide synthesis

[0998] After adjusting the protocol to include coupling of preformed dipeptides (initially intended to limit DKP byproducts), alternative dipeptide coupling reagents / reaction conditions were evaluated. Epimerization was evaluated after dipeptide coupling and conditions optimized.

[0999] Table 8. Dipeptide coupling screening during cyclopeptide synthesis (purity obtained by UPLC integration)

[1000]

[1001]

[1002] Optimization of allyl ester deprotection ( Figure 1A )

[1003] Table 9. Deprotection of allyl esters

[1004]

[1005] Fmoc Deprotection Screening for Linear Peptide Synthesis

[1006] Alternative deprotection conditions were found that could replace deprotection with 20% piperidine / DMF without affecting product integrity / purity. Milder conditions were observed to result in complete Fmoc removal, as well as impurities such as desPeg that are typically caused by harsh deprotection. 12 and desPeg 12 Lys(Mtt).

[1007] Table 10. Fmoc deprotection

[1008]

[1009] *Average UV-Vis absorbance measurements at 304 nm under alternative deprotection conditions

[1010] The table above details alternative Fmoc deprotection conditions. The removed Fmoc was quantified by ultraviolet UV analysis of the deprotection filtrate. In addition, LCMS was performed to assess the integrity / quality of the deprotected linear peptide product. 20%, 10%, 5% and 2% piperidine in DMF all produced similar results, indicating that reducing piperidine can still completely deprotect the Fmoc-protected peptide. An organic base (DBU) was also used as well as adding acid to adjust the basicity of the piperidine. Based on UV and LCMS analysis, the integrity of the linear peptide product was comparable to the control, so these alternative methods can also be used for Fmoc deprotection. No missing product was detected by LCMS.

[1011] Cyclization optimization ( Figure 1A )

[1012] Table 11. Cyclization conditions

[1013]

[1014]

[1015] Additional cyclization agents were screened during the synthesis of the cyclic peptide fragment. The cyclization screen was performed at two resin loading levels (high / low) to evaluate differences in product, epimer, and dimer formation.

[1016] Table 12. Cyclization conditions

[1017]

[1018] *Comparison

[1019] Table 13. Cyclization conditions

[1020]

[1021]

[1022] *Comparison

[1023] Under most conditions, the formation of the desired product and completion of cyclization were accompanied by varying amounts of epimer and dimer byproducts. Unexpectedly, in most cases, lower resin loading levels resulted in higher amounts of dimer formation, but lower amounts of epimers.

[1024] Optimization to reduce dimerization during cyclic peptide synthesis

[1025] Resin loading screening was performed to evaluate and minimize dimer formation during cyclization of linear precursors and to assess its reproducibility at large scale.

[1026] Table 14. Resin loading levels, target products, diastereomers, and dimers detected after cyclization of linear precursors. Summary table.

[1027]

[1028] *1st generation resin loading is 0.48-0.51mmol / g

[1029] Screening of amide conjugation conditions

[1030] All conditions were evaluated at room temperature with 2 mM PMO (50 mg / mL in DMSO). All reagents were prepared in DMSO:

[1031] 1.DIC (300mM DMSO)

[1032] 2. Oxyma (300mM DMSO)

[1033] 3. PyAOP (300 mM DMSO)

[1034] 4.HATU (300mM DMSO)

[1035] 5.DIPEA (300mM DMSO)

[1036] 6. EEV (100 mM DMSO)

[1037] Dilute / quench with 1:1 ACN / H2O+0.1% TFA before injection

[1038] Table 15. Amide conjugation conditions

[1039]

[1040] Screening of Tfa deprotection conditions

[1041] Table 16.EEV-PMO Tfa deprotection

[1042]

[1043]

[1044] *(+) indicates complete deprotection, (-) indicates partial deprotection or no deprotection

[1045] Table 17. EEV-PMO conjugation

[1046]

[1047] Table 18. EEV-PMO conjugation

[1048]

[1049]

[1050] EEV-PMO Tfa deprotection screening

[1051] Alternative EEV-PMO deprotection conditions were evaluated using a variety of aqueous, organic, and amphiphilic bases, and milder deprotection conditions with less PMO regeneration and higher product purity were found, and more aggressive conditions were also screened for faster deprotection without sacrificing purity.

[1052] Table 19. Tfa deprotection screening conditions

[1053]

[1054]

[1055]

[1056] Table 20. Addition deprotection conditions

[1057]

[1058] Linear-Cyclic Peptide Coupling Reaction Screening

[1059] Linear-cyclic couplings were screened using various conditions. Experiments were performed at 0.6 mmol / g load material and 0.4 mmol / g load material.

[1060] Table 21. Summary of coupling reaction conditions for 0.6 mmol / g loaded linear peptide fragments

[1061]

[1062] Table 22. Summary of coupling reaction conditions for 0.4 mmol / g loading of linear peptide fragments

[1063]

[1064] Table 23. Analysis of 2-day conjugation with lower weight equivalent cyclic peptides.

[1065]

[1066] Table 24. Analysis of 2-day conjugation with lower purity equivalent cyclic peptides

[1067]

[1068] Study on the mechanism of EEV-PMO conjugation and deprotection reaction

[1069] The EEV-PMO conjugation / deprotection reaction was evaluated by comparing the time course of the Generation 2 and Generation 1 protocols. Conjugation / deprotection reactions using PMOs of varying purity as well as deprotection of purified and protected EEV-PMO were also investigated.

[1070] Table 25. EEV-PMO product formation based on UPLC integration data

[1071]

[1072] Table 26. PMO consumption during conjugation based on UPLC integration data (same time course as Table 25)

[1073]

[1074] Table 27. Time course of product formation in the deprotection reaction using PMO of different purities

[1075]

[1076]

[1077] Table 28. PMO regeneration time course for deprotection reactions using PMO of different purities

[1078]

[1079] Table 29. Time course of conjugation reaction product formation under generation 1 and generation 2 conditions

[1080]

[1081] Table 30. PMO consumption time course of conjugation reaction under 1st and 2nd generation conditions

[1082]

[1083] Table 31. Time course of deprotection product formation under 1st and 2nd generation conditions

[1084]

[1085] Table 32. PMO regeneration time course for deprotection reactions under 1st and 2nd generation conditions

[1086]

[1087] Experiments were performed to investigate the 2nd generation conjugation / deprotection protocol (40 eq NaOH), the 1st generation conjugation / deprotection protocol (slightly different conjugation stoichiometry, while deprotection used 12.5 mM NaOH, 1 M KCl), deprotection of purified and protected EEV-PMO using the 2nd generation method, and 2nd generation conjugation / deprotection using PMOs of varying purity (4 purity levels). In all of these experiments, both conjugation and deprotection reactions were monitored and compared using purity obtained by UPLC integration and LCMS mass spectrometry analysis.

Claims

1. A method for preparing a cyclic peptide of formula (I) or its protonated form, in: R1, R2 and R3 are each independently H or a residue of tyrosine, phenylalanine or tryptophan; R4 and R6 are independently H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4; and Each m is independently an integer from 0 to 3, The method comprises: The compound of formula (1) With the compound of formula (II) Reaction to form a compound of formula (III) wherein X and X' are independently protecting groups, X" is H or a protecting group, X'" is H or an activating group and m is 0-3.

2. The method of claim 1, further comprising converting the compound of formula (III) into a compound of formula (IV) 3. The method of claim 2, further comprising reacting a compound of formula (IV) with a compound of formula (V) To form a compound of formula (VI) Wherein Z is a radical of an amino acid side chain and It is a solid support.

4. The method of claim 3, wherein the compound of formula (IV) is And the compound of formula (VI) is 5. The method of claim 1, further comprising treating the compound of formula (VII) with PyOxim / Oxyma and a base To obtain a compound of formula (VIII):

6. The method of claim 5, wherein the compound of formula (VII) is And the compound of formula (VIII) is 7. The method of claim 5 or 6, wherein the base is DIPEA.

8. A method for preparing cyclic peptide or its protonated form, in: R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan; R4 and R6 are independently H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4; and Each m is independently an integer from 0 to 3, The method comprises: The compound of formula (IX) wherein X is a protecting group and is a solid support, and a compound of formula (X) wherein X' is a protecting group and Z is a group of an amino acid side chain, reacted to form a compound of formula (XI) 9. The method of claim 8, wherein the compound of formula (X) is And the compound of formula (XI) is 10. The method of claim 8, further comprising treating the compound of formula (XI) to form a compound of formula (XII) 11. The method of claim 10, wherein the compound of formula (XII) is 12. A method for preparing cyclic peptide or its protonated form, in: R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan; R4 and R6 are independently H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4; and Each m is independently an integer from 0 to 3, The method comprises: making a compound of formula (XIII) in is a solid support, and a compound of formula (XIV) Reaction to obtain a compound of formula (XV) 13. The method of claim 12, wherein the compound of formula (XIII) is The compound of formula (XIV) is And the compound of formula (XV) is 14. The method of claim 12, further comprising treating the compound of formula (XV) to obtain a compound of formula (XVI):

15. The method of claim 14, wherein the compound of formula (XVI) is 16. The method of any one of claims 1-15, wherein R4 is H or a side chain of tyrosine, phenylalanine or tryptophan.

17. The method of any one of claims 1-16, wherein R4 is the side chain of phenylalanine.

18. The method of any one of claims 1-17, wherein two of R1, R2, R3 and R4 are phenylalanine side chains.

19. The method of any one of claims 1-18, wherein two of R1, R2, R3 and R4 are H.

20. The method of any one of claims 1 to 19, comprising a structure of formula (I-1) or (I-2): or its protonated form, in: AA SC is an amino acid side chain; and Each m is independently an integer from 0 to 3.

21. The method of any one of claims 1 to 20, wherein the compound of formula (A) has the following structure: or its protonated form, in: AA SC is an amino acid side chain; and Each m is independently an integer from 0 to 3.

22. The method of any one of claims 1 to 21, wherein AA SC It is the side chain of an asparagine side chain, an aspartic acid side chain, a glutamine side chain, a glutamic acid side chain, a homoglutamine residue or a homoglutamic acid side chain.

23. The method of any one of claims 1-21, wherein AA SC It is the side chain of glutamine side chain.

24. The method of any one of claims 1-23, wherein the AA SC Conjugation to a linker.

25. The method of claim 24, wherein the linker comprises: (i)-(OCH2CH2) z -subunit, wherein z is an integer from 2 to 20 (ii) one or more amino acid side chains, such as residues of glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid or 6-aminovaleric acid, or combinations thereof; or (iii) A combination of (i) and (ii).

26. The method of claim 24 or 25, wherein the linker comprises -(OCH2CH2) x -subunit, wherein x is an integer from 2 to 20.

27. The method of any one of claims 24-26, wherein the linker has the following structure: in: x is an integer from 2 to 20; y is an integer from 1 to 5; and z is an integer from 2 to 20.

28. The method of claim 25 or 27, wherein z is 11.

29. The method of any one of claims 26-28, wherein x is 1.

30. The method of any one of claims 24-29, comprising an exocyclic peptide conjugated to the linker at an amino group.

31. The method of claim 30, wherein the exocyclic peptide comprises 2 to 10 amino acid side chains.

32. The method of claim 30 or 31, wherein the exocyclic peptide comprises 4 to 8 amino acid side chains.

33. The method of any one of claims 30-32, wherein the exocyclic peptide comprises 1 or 2 amino acid side chains comprising a side chain comprising a guanidino group or a protonated form thereof.

34. The method of any one of claims 30-33, wherein the exocyclic peptide comprises 2, 3 or 4 lysine side chains.

35. The method of claim 34, wherein the amino group on the side chain of each lysine side chain is substituted by a trifluoroacetyl (-COCF3), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) or (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) ​​group.

36. The method of any one of claims 30-35, wherein the exocyclic peptide comprises at least 2 amino acid side chains having hydrophobic side chains.

37. The method of claim 36, wherein the amino acid side chain having a hydrophobic side chain is selected from the group consisting of valine, proline, alanine, leucine, isoleucine and methionine.

38. The method of any one of claims 30-37, wherein the exocyclic peptide comprises one of the following sequences: PKKKRKV; KR; RR, KKK; KGK; KBK; KBR; KRK; KRR; RKK; RRR; KKKK; KKRK; KRKK; KRRK; RKKR; RRRR; KGKK ;KKGK; KKKKK; KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV; PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG.

39. The method of any one of claims 30-38, wherein the exocyclic peptide has the following structure: Ac-PKKKRKV-.

40. The method of any one of claims 24-39, conjugated to a cargo moiety, wherein the -OH of the terminal carboxylic acid group is replaced by the cargo moiety.

41. The method of claim 40, wherein the cargo moiety comprises a therapeutic molecule comprising an oligonucleotide, a peptide, or a small molecule.

42. The method of any one of claims 24-41, conjugated to a linker, wherein the linker has the structure: in: EP is an exocyclic peptide; AA SC is the amino acid side chain; x is an integer from 2 to 20; y is an integer from 1 to 5; and z is an integer from 2 to 20.

43. A compound selected from the group consisting of: in: R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan; R4 is H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4; X, X' and X" are each independently a protecting group; Each m is independently an integer from 0 to 3; and It is a solid support.

44. A compound selected from the group consisting of: in: R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan; R4 is H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4 X and X' are each independently a protecting group; Each m is independently an integer from 0 to 3; and It is a solid support.

45. A compound selected from the group consisting of: in: R1, R2 and R3 are each independently H or a side chain of tyrosine, phenylalanine or tryptophan; R4 is H or an amino acid side chain; AA SC is the amino acid side chain; q is 1, 2, 3, or 4 X and X' are each independently a protecting group; Each m is independently an integer from 0 to 3; and It is a solid support.

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