Cyclic peptides for the delivery of therapeutic agents - Patents.com

Abstract

The present disclosure relates to the synthesis of cyclic peptides that can effectively deliver cargo, such as therapeutic moieties (TM), inside cells to treat a variety of conditions and diseases.

Description

[Technical field]

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

[0002] Nucleic acids and their synthetic analogues hold enormous potential as therapeutic agents, especially against targets that are difficult to reach with traditional drug modalities (eg, defective / faulty proteins caused by genetic mutations).

[0003] However, one challenge in translating such therapeutic potential into the clinic is their limited ability to reach intracellular compartments when administered systemically. To facilitate intracellular delivery, carrier systems, such as polymers, cationic liposomes, or chemical modifications, such as covalently binding cholesterol molecules, have been used. Nevertheless, the intracellular delivery efficiency of these approaches is often low, and improved delivery systems to improve the efficacy of intracellular delivery have remained elusive.

[0004] There is a need for effective compositions for delivering therapeutic molecules to intracellular compartments to treat disease, and several have been recently reported, however, there remains a need for 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 cyclic peptide of formula (A): [ka] [During the ceremony, R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic 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; AA SC is an amino acid side chain; q is 1, 2, 3 or 4. or a protonated form thereof, which is any one of the methods described herein for a compound of formula (I).

[0007] The present disclosure relates to a cyclic peptide of formula (I): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: Compound of formula (1) [ka] with a compound of formula (II) [ka] to give a compound of formula (III) [ka] To form wherein X and X' are independently protecting groups, X" is H or a protecting group, X"' is H or an activating group (e.g., an NHS ester), and m is 0-3.

[0008] The present disclosure also provides a cyclic peptide of formula (I): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: Compound of formula (IX) [ka] [During the ceremony, [ka] is a solid support. with a compound of formula (X) [ka] wherein X and X' are independently protecting groups and Z is a radical of an amino acid side chain. to give a compound of formula (XI) [ka] [In the formula, p is an integer of 1 to 30] The present invention also relates to a method thereof, comprising forming

[0009] The present disclosure relates to a cyclic peptide of formula (I): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: Compound of formula (XIII) [ka] wherein X is a protecting group and Z is a radical of an amino acid side chain; [ka] is a solid support. with a compound of formula (XIV) [ka] to give a compound of formula (XV) [ka] The method includes obtaining a

[0010] The present disclosure relates to [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; Z is an amino acid side chain radical; 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; [ka] is a solid support. The present invention relates to a compound selected from the group consisting of

[0011] The present disclosure relates to [ka] [ka] [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; p is an integer from 1 to 30; 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; [ka] is a solid support. The present invention relates to a compound selected from the group consisting of

[0012] The present disclosure relates to [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; Z is an amino acid side chain radical; 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; [ka] is a solid support. The present invention relates to a compound selected from the group consisting of [Brief description of the drawings]

[0013] [Figure 1A] The synthetic pathway for producing EEV is shown. [Figure 1B-1] The synthetic pathway for producing EEV is shown. [Figure 1B-2] The synthetic pathway for producing EEV is shown. [Diagram 2] 1 shows a synthetic route for making a cyclic peptide. [Diagram 3] 1 shows a synthetic route for making a cyclic peptide. [Figure 4] 1 shows a synthetic route for making a cyclic peptide. [Diagram 5] The synthetic pathway for producing EEV is shown. [Figure 6] The synthetic pathway for producing EEV is shown. [Figure 7] 1 shows a synthetic route for making a cyclic peptide. [Figure 8] The synthetic pathway for producing EEV is shown. [Figure 9]1 shows the first generation EEV-PMO synthesis, where PMO is a phosphorodiamidate morpholino oligomer. [Figure 10] The synthesis of second generation EEV-PMO is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure relates to methods for making endosomal escape vehicles (EEVs).

[0015] Endosomal escape vehicles (EEVs) Provided herein is an endosomal escape vehicle (EEV) that can be used to transport cargo across a cell membrane, for example, to deliver cargo to the cytosol or nucleus of a cell. The cargo can include a macromolecule, for example, a peptide or oligonucleotide, or a small molecule. The EEV can include a cell penetrating peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP) conjugated to an exocyclic peptide (EP). The EP can include a sequence of a nuclear localization signal (NLS). The EP can be bound to the cargo. The EP can be bound to the cCPP. The EP can be bound to the cargo and the cCPP. The coupling between the EP, cargo, cCPP, or combinations thereof can be a non-covalent or covalent bond. The EP can be bound to the N-terminus of the cCPP via a peptide bond. The EP can be bound to the C-terminus of the cCPP via a peptide bond. The EP can be bound to the cCPP via a side chain of an amino acid in the cCPP. The EP can be attached to the cCPP via the side chain of the lysine, which can be conjugated to the side chain of the glutamine in the cCPP. The EP can be conjugated to the 5' or 3' end of the oligonucleotide cargo. The EP can be attached to the linker. The exocyclic peptide can be conjugated to the amino group of the linker. The EP can be coupled to the linker by the C-terminus of the EP and to the cCPP via the side chain on the cCPP and / or on the EP. For example, the EP can contain a terminal lysine, which can then be coupled to the cCPP containing glutamine via an amide bond. If the EP contains a terminal lysine and the side chain of the lysine can be used to attach the cCPP, the C-terminus or N-terminus can be attached to the linker on the cargo.

[0016] Exocyclic peptides The exocyclic peptide (EP) may contain from 2 to 10 amino acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues (including all ranges and values ​​therebetween). The EP may contain from 6 to 9 amino acid residues. The EP may contain from 4 to 8 amino acid residues.

[0017] Each amino acid in the exocyclic peptide may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homologue of a natural amino acid in that it has a structure similar to the natural amino acid so as to mimic the structure and reactivity of the natural amino acid. An unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. An unnatural amino acid may be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, 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, derivatives thereof, or combinations thereof. These and other amino acids, along with their abbreviations used herein, are listed in Table 1. For example, an amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.

[0018] The EP may contain at least one positively charged amino acid residue, for example, at least one lysine residue, and / or at least one amino acid residue containing a side chain containing a guanidine group or a protonated form thereof. The EP may contain one or two amino acid residues containing a side chain containing a guanidine group or a protonated form thereof. The amino acid residue containing a side chain containing a guanidine group may be an arginine residue. The protonated form may refer to a salt thereof throughout this disclosure.

[0019] The EP may include at least two, at least three, or at least four or more lysine residues. The EP may include two, three, or four lysine residues. The amino group on the side chain of each lysine residue may be substituted with a protecting group, such as, for example, a trifluoroacetyl (-COCF3) group, an allyloxycarbonyl (Alloc) group, a 4-methyltrityl (Mtt), a 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) group, or a (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-3)-methylbutyl (ivDde) ​​group. The amino group on the side chain of each lysine residue may be substituted with a trifluoroacetyl (-COCF3) group. The protecting group may be included to allow amide conjugation. The protecting group may be removed after the EP is conjugated to the cCPP.

[0020] The EP may comprise at least two amino acid residues having a hydrophobic side chain. The amino acid residues having a hydrophobic side chain may be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residue having a hydrophobic side chain may be valine or proline.

[0021] The EP may contain at least one positively charged amino acid residue, e.g., at least one lysine residue and / or at least one arginine residue. The EP may contain at least two, at least three, or at least four or more lysine and / or arginine residues.

[0022] EP is 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, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, K KKKR, 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, where B is beta alanine. The amino acids in EP may have D or L stereochemistry.

[0023] EP may include 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 include PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP may have D or L stereochemistry.

[0024] EP can be comprised 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 can be comprised of PKKKRKV, RR, RRR, RHR, RBR, RBRBR, RBHBR, or HBRBH, where B is beta-alanine. The amino acids in EP can have D or L stereochemistry.

[0025] The EP may comprise an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may comprise an NLS comprising the amino acid sequence PKKKRKV. The EP may consist of an NLS comprising the amino acid sequence PKKKRKV. The EP may comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK. The EP may consist of an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF, RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, and RKCLQAGMNLEARKTKK.

[0026] All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, the EP may have the structure: Ac-PKKKRKV.

[0027] Cell-penetrating peptides (CPPs) 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 may penetrate the cell membrane. An exocyclic peptide (EP) may be conjugated to the cCPP, and the resulting construct may be called an endosomal escape vehicle (EEV). The cCPP may guide a cargo (e.g., a therapeutic moiety (TM), e.g., an oligonucleotide, a peptide, or a small molecule) to penetrate the cell membrane. The cCPP may deliver the cargo to the cytosol of the cell. The cCPP may deliver the cargo to the cellular location where the target (e.g., pre-mRNA) is located. To conjugate the cCPP to a cargo (e.g., a peptide, an oligonucleotide, or a small molecule), at least one bond or lone pair on the cCPP may be replaced.

[0028] The total number of amino acid residues in a cCPP can range from 6 to 20 amino acid residues, e.g., 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. A cCPP can contain 6 to 13 amino acid residues. A cCPP disclosed herein can contain 6 to 10 amino acids. By way of example, a cCPP containing 6-10 amino acid residues can be represented by Formulas IA to IE:

[0029] [ka] wherein AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA 10 is an amino acid residue.

[0030] The cCPP may contain 6 to 8 amino acids. The cCPP may contain 8 amino acids.

[0031] Each amino acid in a cCPP may be a natural or unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a homologue of a natural amino acid in that it has a structure similar to the natural amino acid so as to mimic the structure and reactivity of the natural amino acid. An unnatural amino acid may be a modified amino acid and / or an amino acid analog that is not one of the 20 common natural amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. An unnatural amino acid may be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, 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, derivatives thereof, or combinations thereof. These and other amino acids, along with their abbreviations used herein, are listed in Table 1.

[0032] [Table 1-1] [Table 1-2]

[0033] A cCPP can contain from 4 to 20 amino acids, where (i) at least one amino acid has a side chain that includes a guanidine group or a protonated form thereof, and (ii) at least one amino acid has no side chain, or [ka] or a protonated form thereof, and (iii) at least two amino acids have side chains that, independently, contain an aromatic or heteroaromatic group.

[0034] At least two of the amino acids may have no side chains, or [ka] or a protonated form thereof. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on the carbon atom(s) connecting the amine and the carboxylic acid (e.g., -CH2-).

[0035] The amino acid having no side chain can be glycine or β-alanine.

[0036] The cCPP may comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least one amino acid may be a glycine, β-alanine, or 4-aminobutyric acid residue, (ii) at least one amino acid may have a side chain that includes an aryl or heteroaryl group, (iii) at least one amino acid may have a guanidine group, [ka] or having a side chain containing the protonated form thereof.

[0037] The cCPP can comprise 6 to 20 amino acid residues forming the cCPP, where (i) at least two amino acids can be independently glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid can have a side chain that includes an aryl or heteroaryl group, (iii) at least one amino acid can have a guanidine group, [ka] or having a side chain containing the protonated form thereof.

[0038] The cCPP may comprise from 6 to 20 amino acid residues forming the cCPP, where (i) at least three amino acids may be, independently, glycine, β-alanine, or 4-aminobutyric acid residues, (ii) at least one amino acid may have a side chain that includes an aromatic or heteroaromatic group, (iii) at least one amino acid may have a guanidine group, [ka] or a side chain containing the protonated form thereof.

[0039] Glycine and related amino acid residues The cCPP may include (i) 1, 2, 3, 4, 5 or 6 glycine β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 2 glycine β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may contain (i) three or four glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0040] The cCPP may include (i) 1, 2, 3, 4, 5, or 6 glycine residues. The cCPP may include (i) 2 glycine residues. The cCPP may include (i) 3 glycine residues. The cCPP may include (i) 4 glycine residues. The cCPP may include (i) 5 glycine residues. The cCPP may include (i) 6 glycine residues. The cCPP may include (i) 3, 4, or 5 glycine residues. The cCPP may include (i) 3 or 4 glycine residues. The cCPP may include (i) 2 or 3 glycine residues. The cCPP may include (i) 1 or 2 glycine residues.

[0041] The cCPP may include (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 6 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof. The cCPP may include (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues, or a combination thereof.

[0042] The cCPP may include at least three glycine residues. The cCPP may include (i) three, four, five, or six glycine residues. The cCPP may include (i) three glycine residues. The cCPP may include (i) four glycine residues. The cCPP may include (i) five glycine residues. The cCPP may include (i) six glycine residues. The cCPP may include (i) three, four, or five glycine residues. The cCPP may include (i) three or four glycine residues.

[0043] In embodiments, none of the glycine, β-alanine, or 4-aminobutyric acid residues in the cCPP are adjacent. Two or three glycine, β-alanine, 4- or aminobutyric acid residues may be adjacent. Two glycine, β-alanine, or 4-aminobutyric acid residues may be adjacent.

[0044] In embodiments, none of the glycine residues in the cCPP are adjacent. Each glycine residue in the cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be adjacent. Two glycine residues may be adjacent.

[0045] Amino acid side chains containing aromatic or heteroaromatic groups The cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 2 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 3 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 4 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 5 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 6 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. The cCPP may comprise (ii) 2, 3 or 4 amino acid residues that independently have a side chain that includes an aromatic or heteroaromatic group. A cCPP can contain (ii) two or three amino acid residues that independently have side chains that contain an aromatic or heteroaromatic group.

[0046] The cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 3 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 4 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 5 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 6 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2, 3 or 4 amino acid residues that independently have a side chain that comprises an aromatic group. The cCPP may comprise (ii) 2 or 3 amino acid residues that independently have a side chain that comprises an aromatic group.

[0047] The aromatic group can be 6- to 14-membered aryl. The aryl can be phenyl, naphthyl, or anthracenyl, each of which is optionally substituted. The aryl can be phenyl or naphthyl, each of which is optionally substituted. The heteroaromatic group can be 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. The heteroaryl can be pyridyl, quinolyl, or isoquinolyl.

[0048] The amino acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be bis(homonapthylalanine), 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-(naphthalen-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 acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be [ka] wherein H on the N-terminus and / or H on the C-terminus is replaced by a peptide bond.

[0049] Amino acid residues having a side chain containing an aromatic or heteroaromatic group may each independently be a residue 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 a side chain containing an aromatic group may each independently be a residue 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, or homophenylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue of phenylalanine, naphthylalanine, homophenylalanine, homophenylalanine, bis(homonaphthylalanine), or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having a side chain containing an aromatic group may each independently be a residue 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.

[0050] In embodiments, none of the amino acids having a side chain containing an aromatic or heteroaromatic group are adjacent. Two amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. Two adjacent amino acids may have opposite stereochemistry. Two adjacent amino acids may have the same stereochemistry. Three amino acids having a side chain containing an aromatic or heteroaromatic group may be adjacent. Three adjacent amino acids may have the same stereochemistry. Three adjacent amino acids may have alternate stereochemistry.

[0051] The amino acid residues containing aromatic or heteroaromatic groups may be L-amino acids. The amino acid residues containing aromatic or heteroaromatic groups may be D-amino acids. The amino acid residues containing aromatic or heteroaromatic groups may be a mixture of D- and L-amino acids.

[0052] An optional substituent may be, for example, any atom or group that does not significantly (e.g., more than 50%) reduce the cytoplasmic delivery efficiency of the cCPP compared to an otherwise identical sequence without the substituent. An optional substituent may be a hydrophobic or hydrophilic substituent. An optional substituent may be a hydrophobic substituent. A substituent may increase the solvent accessible surface area (as defined herein) of a hydrophobic amino acid. A substituent may be halogen, alkyl, alkenyl, alkynylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio. A substituent may be halogen.

[0053] Without wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups with higher hydrophobicity values ​​(i.e., amino acids with side chains containing aromatic or heteroaromatic groups) can improve the cytoplasmic delivery efficiency of cCPPs compared to amino acids with lower hydrophobicity values. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of glycine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than that of alanine. Each hydrophobic amino acid can independently have a hydrophobicity value equal to or greater than that of phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values ​​for 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), each of which is incorporated herein by reference in its entirety. Hydrophobicity can be measured using the hydrophobicity scale reported in Engleman et al.

[0054] [Table 2]

[0055] Amino acid residues having a side chain containing a guanidine group, a guanidine substituent, or the protonated form thereof As used herein, guanidine has the structure: [ka] Refers to...

[0056] As used herein, the protonated form of guanidine has the structure: [ka] Refers to...

[0057] A guanidine substituent refers to a functional group on the side chain of an amino acid that is positively charged at or above physiological pH, or a functional group that can reproduce the hydrogen bond donating and accepting activity of a guanidinium group.

[0058] The guanidine substituents facilitate cell penetration and delivery of therapeutic agents while reducing toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one amino acid having a side chain that includes a guanidine or guanidinium substituent. The cCPP may include at least two amino acids having side chains that include a guanidine or guanidinium substituent. The cCPP may include at least three amino acids having side chains that include a guanidine or guanidinium substituent.

[0059] The guanidine or guanidinium group may be an isostere of guanidine or guanidinium. The guanidine or guanidinium substituent may be less basic than guanidine.

[0060] As used herein, a guanidine substituent is: [ka] or its protonated form.

[0061] The present disclosure provides 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, and (ii) at least one amino acid residue has no side chain, or [ka] or a protonated form thereof, and (iii) at least two amino acid residues have side chains that, independently, contain an aromatic or heteroaromatic group.

[0062] At least two of the amino acid residues may have no side chains, or [ka] or a protonated form thereof. 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 carboxylic acid (e.g., -CH2-).

[0063] cCPP consists of the following parts: [ka] or at least one amino acid having a side chain that includes one of its protonated forms.

[0064] The cCPP can include at least two amino acids, each amino acid independently comprising the moiety: [ka] or one of its protonated forms. At least two of the amino acids are [ka] or its protonated form. At least one amino acid may have a side chain comprising the same moiety selected from [ka] or a protonated form thereof. At least two of the amino acids may have a side chain containing [ka] or its protonated form. [ka] or its protonated form. [ka] or its protonated form. [ka] or a side chain containing the protonated form thereof. [ka] or its protonated form can be attached to the terminus of an amino acid side chain. [ka] can be attached to the terminus of an amino acid side chain.

[0065] The cCPP may comprise 2, 3, 4, 5 or 6 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 2 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 3 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 4 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 5 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise 6 amino acid residues that independently have a side chain that comprises (iii) a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4, or 5 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2, 3, or 4 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues that independently have a side chain that includes a guanidine group, a guanidine substituent, or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue that has a side chain that includes a guanidine group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues that have a side chain that includes a guanidine group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues that have a side chain that includes a guanidine group or a protonated form thereof.

[0066] The amino acid residues may independently have side chains that contain non-adjacent guanidine groups, guanidine substituents, or their protonated forms. Two amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Three amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Four amino acid residues may independently have side chains that contain guanidine groups, guanidine substituents, or their protonated forms may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.

[0067] The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be L-amino acids. The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be D-amino acids. The amino acid residues that independently have side chains that include a guanidine group, a guanidine substituent, or a protonated form thereof can be a mixture of L or D amino acids.

[0068] Each amino acid residue having a side chain containing 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 containing a guanidine group or a protonated form thereof may independently be a residue of arginine or a protonated form thereof.

[0069] Each amino acid having a side chain containing a guanidine substituent or a protonated form thereof can independently be [ka] or a protonated form thereof.

[0070] Without wishing to be bound by theory, it is hypothesized that the guanidine substituents have reduced basicity compared to arginine and, in some cases, are uncharged (e.g., -N(H)C(O)) at physiological pH and can maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane that are believed to facilitate effective membrane binding and subsequent internalization. Removal of the positive charge is also believed to reduce the toxicity of cCPPs.

[0071] One of skill in the art will understand that the N-terminus and / or C-terminus of the above non-natural aromatic hydrophobic amino acids form an amide bond upon incorporation into the peptides disclosed herein.

[0072] A cCPP may include a first amino acid having a side chain that includes an aromatic or heteroaromatic group and a second amino acid having a side chain that includes an aromatic or heteroaromatic group, where the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain that includes 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 that includes an aromatic or heteroaromatic group. By convention, the term "first amino acid" often refers to the N-terminal amino acid of a peptide sequence, however, as used herein, "first amino acid" is used to distinguish a reference amino acid from another amino acid (e.g., a "second amino acid") in a cCPP, and thus the term "first amino acid" may refer to the amino acid located at the N-terminus of a peptide sequence.

[0073] The cCPP can include an N-terminus of a second glycine that forms a peptide bond with an amino acid having a side chain that includes an aromatic or heteroaromatic group, and a C-terminus of the second glycine that forms a peptide bond with an amino acid having a side chain that includes a guanidine group or a protonated form thereof.

[0074] The cCPP can include 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.

[0075] The cCPP may comprise asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine residues. The cCPP may comprise asparagine residues. The cCPP may comprise glutamine residues.

[0076] cCPPs may contain 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0077] Without wishing to be bound by theory, it is believed that the chirality of amino acids in a cCPP may affect cytoplasmic uptake efficiency. A cCPP may include at least one D amino acid. A cCPP may include 1 to 15 D amino acids. A cCPP may include 1 to 10 D amino acids. A cCPP may include 1, 2, 3, or 4 D amino acids. A cCPP may include 2, 3, 4, 5, 6, 7, or 8 adjacent amino acids with alternating D and L chirality. A cCPP may include three adjacent amino acids with the same chirality. A cCPP may include 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 alternate stereochemistry with respect to each other. At least three amino acids with alternate chirality with respect to each other may be adjacent to each other. At least four amino acids have alternate stereochemistry relative to each other. At least four amino acids with alternate chirality relative to each other can be adjacent to each other. At least two amino acids can have the same chirality. At least two amino acids with the same chirality can 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 can be adjacent to at least two amino acids with the same chirality. Thus, adjacent amino acids in a cCPP can have any of the following sequences: DL, LD, DLLD, LDDL, LDLLD, DLDDL, DLLDL, or LDDLD. Amino acid cCPPs can include the following sequences: D / LXD / L, D / LXD / LX, D / LXD / LXD / L, DXD, DXDX, DXDXD, LXL, LXLX, or LXLXL (where D / L means that the amino acid has D or L stereochemistry and X is an achiral amino acid). The achiral amino acid can be glycine.

[0078] [ka] or its protonated form can be adjacent to an amino acid having a side chain that includes an aromatic or heteroaromatic group. [ka] or its protonated form may be adjacent to at least one amino acid having a side chain comprising a guanidine or its protonated form. An amino acid having a side chain comprising a guanidine or its protonated form may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. [ka] or its protonated form may be adjacent to each other. Two amino acids having a side chain containing guanidine or its protonated form are adjacent to each other. A cCPP comprises at least two adjacent amino acids having side chains that may contain an aromatic or heteroaromatic group, [ka] or a protonated form thereof. A cCPP may have at least two contiguous amino acids having side chains that include an aromatic or heteroaromatic group, [ka] or at least two non-adjacent amino acids having side chains that include the protonated form thereof. The adjacent amino acids may have the same chirality. The adjacent amino acids may have opposite chiralities. Other combinations of amino acids may have any arrangement of D and L amino acids, such as any of the sequences described in the previous paragraph.

[0079] [ka] or its protonated form alternate with at least two amino acids having a side chain containing a guanidine group or its protonated form.

[0080] cCPP has the formula (A): [ka] or a protonated form thereof, During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidyl)alanine; AA SC is an amino acid side chain, q is 1, 2, 3 or 4.

[0081] In embodiments, at least one of R4, R5, R6, and R7 is independently an uncharged, non-aromatic side chain of an amino acid. In embodiments, at least one of R4, R5, R6, and R7 is independently H or the side chain of citrulline.

[0082] In an embodiment, a compound is provided, comprising a cyclic peptide having 6-12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, wherein at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids, and wherein at least two amino acids of the cyclic peptide are uncharged non-aromatic amino acids. In an embodiment, at least two amino acids of the cyclic peptide are arginine. In an embodiment, at least two aromatic hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthalanine (3-naphth-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 having 6-12 amino acids, wherein two amino acids of the cyclic peptide are arginine, wherein at least two amino acids are aromatic hydrophobic amino acids selected from phenylalanine, naphthalanine, and combinations thereof, and wherein at least two amino acids are uncharged non-aromatic amino acids selected from citrulline, glycine, and combinations thereof.

[0083] In embodiments, the cyclic peptide of formula (A) is not a cyclic peptide having the following sequence:

[0084] [Table 3]

[0085] A cCPP has the structure of formula (I): [ka] or a protonated form thereof, During the ceremony, R1, R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group; At least one of R1, R2, and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain, q is 1, 2, 3 or 4; Each m is independently an integer 0, 1, 2, or 3.

[0086] R1, R2, and R3 can each independently be H, -alkylene-aryl, or alkylene-heteroaryl. R1, R2, and R3 can each independently be H, -C 1~3 Alkylene-aryl, or C 1~3 R1, R2, and R3 may each independently be H or alkylene-aryl. R1, R2, and R3 may each independently be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R1, R2, and R3 are each independently selected from H, -C ... 1~3 Alkylene-Ph or C 1~3 alkylene-naphthyl. R1, R2, and R3 can each independently be H, -CH2Ph, or CH2naphthyl. R1, R2, and R3 can each independently be H or CH2Ph.

[0087] R1, R2, and R3 can each independently be the 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-pyridinylalanine, 3-pyridinylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.

[0088] R1 can be the side chain of tyrosine. R1 can be the side chain of phenylalanine. R1 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R1 can be the side chain of tryptophan. R1 can be the side chain of 3-benzothienylalanine. R1 can be the side chain of 4-phenylphenylalanine. R1 can be the side chain of 3,4-difluorophenylalanine. R1 can be the side chain of 4-trifluoromethylphenylalanine. R1 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 can be the side chain of homophenylalanine. R1 can be the side chain of β-homophenylalanine. R1 can be the side chain of 4-tert-butyl-phenylalanine. R1 can be the side chain of 4-pyridinylalanine. R1 can be the side chain of 3-pyridinylalanine. R1 can be the side chain of 4-methylphenylalanine. R1 can be the side chain of 4-fluorophenylalanine. R1 can be the side chain of 4-chlorophenylalanine. R1 can be the side chain of 3-(9-anthryl)-alanine.

[0089] R2 can be the side chain of tyrosine. R2 can be the side chain of phenylalanine. R2 can be the side chain of 1-naphthylalanine. R1 can be the side chain of 2-naphthylalanine. R2 can be the side chain of tryptophan. R2 can be the side chain of 3-benzothienylalanine. R2 can be the side chain of 4-phenylphenylalanine. R2 can be the side chain of 3,4-difluorophenylalanine. R2 can be the side chain of 4-trifluoromethylphenylalanine. R2 can be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R2 can be the side chain of homophenylalanine. R2 can be the side chain of β-homophenylalanine. R2 can be the side chain of 4-tert-butyl-phenylalanine. R2 can be the side chain of 4-pyridinylalanine. R2 can be the side chain of 3-pyridinylalanine. R2 can be the side chain of 4-methylphenylalanine. R2 can be the side chain of 4-fluorophenylalanine. R2 can be the side chain of 4-chlorophenylalanine. R2 can be the side chain of 3-(9-anthryl)-alanine.

[0090] R3 may be the side chain of tyrosine. R3 may be the side chain of phenylalanine. R3 may be the side chain of 1-naphthylalanine. R3 may be the side chain of 2-naphthylalanine. R3 may be the side chain of tryptophan. R3 may be the side chain of 3-benzothienylalanine. R3 may be the side chain of 4-phenylphenylalanine. R3 may be the side chain of 3,4-difluorophenylalanine. R3 may be the side chain of 4-trifluoromethylphenylalanine. R3 may be the side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be the side chain of homophenylalanine. R3 may be the side chain of β-homophenylalanine. R3 may be the side chain of 4-tert-butyl-phenylalanine. R3 may be the side chain of 4-pyridinylalanine. R3 may be the side chain of 3-pyridinylalanine. R3 can be the side chain of 4-methylphenylalanine. R3 can be the side chain of 4-fluorophenylalanine. R3 can be the side chain of 4-chlorophenylalanine. R3 can be the side chain of 3-(9-anthryl)-alanine.

[0091] R4 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R4 can be H or alkylene-aryl. R4 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R4 can be H, -C 1~3 Alkylene-Ph or C 1~3R4 can be H or the side chain of an amino acid of Table 1 or Table 3. 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.

[0092] R5 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R5 can be H or alkylene-aryl. R5 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R5 can be H, -C 1~3 Alkylene-Ph or C 1~3 R5 can be H or the side chain of an amino acid of Table 1 or Table 3. R4 can be H or an amino acid residue having a side chain containing an aromatic group. R5 can be H, -CH2Ph, or CH2 naphthyl. R4 can be H or CH2Ph.

[0093] R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R6 can be H or alkylene-aryl. R6 can be H or C 1~3 It can be an alkylene-aryl. 1~3The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl with one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R6 can be H, -C 1~3 Alkylene-Ph or C 1~3 R6 can be alkylene-naphthyl. R6 can be H or the side chain of an amino acid of Table 1 or Table 3. R6 can be H or an amino acid residue having a side chain containing an aromatic group. R6 can be H, -CH2Ph, or CH2 naphthyl. R6 can be H or CH2Ph.

[0094] R7 can be H, -alkylene-aryl, -alkylene-heteroaryl. 1~3 Alkylene-aryl, or C 1~3 R7 can be H or alkylene-aryl. R7 can be H or C 1~3 It can be an alkylene-aryl. 1~3 The alkylene can be methylene. The aryl can be 6- to 14-membered aryl. The heteroaryl can be 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. The aryl can be selected from phenyl, naphthyl, or anthracenyl. The aryl can be phenyl or naphthyl. The aryl can be phenyl. The heteroaryl can be pyridyl, quinolyl, and isoquinolyl. R7 can be H, -C 1~3 Alkylene-Ph or C 1~3 R7 can be alkylene-naphthyl. R7 can be H or the side chain of an amino acid of Table 1 or Table 3. R7 can be H or an amino acid residue having a side chain containing an aromatic group. R7 can be H, -CH2Ph, or CH2 naphthyl. R7 can be H or CH2Ph.

[0095] One, two, or three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. One of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Two of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. At least one of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph. Up to four of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0096] 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.

[0097] One, two or three of R1, R2, R3, R4, R5, R6, and R7 can be H. One of R1, R2, R3, R4, R5, R6, and R7 can be H. Two of R1, R2, R3, R4, R5, R6, and R7 can be H. Three of R1, R2, R3, R5, R6, and R7 can be H. At least one of R1, R2, R3, R4, R5, R6, and R7 can be H. Up to three of R1, R2, R3, R4, R5, R6, and R7 can be -CH2Ph.

[0098] 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.

[0099] At least one of R4, R5, R6, and R7 can be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4, R5, R6, and R7 can be a side chain of 4-guanidino-2-aminobutanoic acid. At least one of R4, R5, R6, and R7 can be a side chain of arginine. At least one of R4, R5, R6, and R7 can be a side chain of homoarginine. At least one of R4, R5, R6, and R7 can be a side chain of N-methylarginine. At least one of R4, R5, R6, and R7 can be a side chain of N,N-dimethylarginine. At least one of R4, R5, R6, and R7 can be a side chain of 2,3-diaminopropionic acid. At least one of R4, R5, R6, and R7 can be a side chain of 2,4-diaminobutanoic acid, lysine. At least one of R4, R5, R6, and R7 can be the side chain of N-methyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N-ethyllysine. At least one of R4, R5, R6, and R7 can be the side chain of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least one of R4, R5, R6, and R7 can be the side chain of citrulline. At least one of R4, R5, R6, and R7 can be the side chain of N,N-dimethyllysine, β-homoarginine. At least one of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0100] At least two of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutanoic acid. At least two of R4, R5, R6, and R7 can be the side chains of arginine. At least two of R4, R5, R6, and R7 can be the side chains of homoarginine. At least two of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least two of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least two of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutanoic acid, lysine. At least two of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least two of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least two of R4, R5, R6, and R7 can be the side chains of citrulline. At least two of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least two of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0101] At least three of R4, R5, R6, and R7 can be the side chains of 3-guanidino-2-aminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 4-guanidino-2-aminobutanoic acid. At least three of R4, R5, R6, and R7 can be the side chains of arginine. At least three of R4, R5, R6, and R7 can be the side chains of homoarginine. At least three of R4, R5, R6, and R7 can be the side chains of N-methylarginine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethylarginine. At least three of R4, R5, R6, and R7 can be the side chains of 2,3-diaminopropionic acid. At least three of R4, R5, R6, and R7 can be the side chains of 2,4-diaminobutanoic acid, lysine. At least three of R4, R5, R6, and R7 can be the side chains of N-methyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N-ethyllysine. At least three of R4, R5, R6, and R7 can be the side chains of N,N,N-trimethyllysine, 4-guanidinophenylalanine. At least three of R4, R5, R6, and R7 can be the side chains of citrulline. At least three of R4, R5, R6, and R7 can be the side chains of N,N-dimethyllysine, β-homoarginine. At least three of R4, R5, R6, and R7 can be the side chain of 3-(1-piperidinyl)alanine.

[0102] AA SC can be the side chain of an asparagine, glutamine, or homoglutamine residue. SC can be the side chain of a glutamine residue. SCFor example, a cCPP may further comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. Thus, a cCPP may further comprise a linker conjugated to an asparagine, glutamine, or homoglutamine residue. A cCPP may further comprise a linker attached to a glutamine residue.

[0103] 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.

[0104] m can be 1 to 3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m can be 3.

[0105] The cCPP of formula (A) has the structure of formula (I): [ka] or a protonated form thereof, wherein AA SC , R1, R2, R3, R4, R6, m and q are as defined herein.

[0106] The cCPP of formula (A) is represented by formula (Ia) or formula (Ib): [ka] or its protonated form, SC , R1, R2, R3, R4, and m are as defined herein.

[0107] The cCPP of formula (A) is represented by the formula (I-1), (I-2), (I-3) or (I-4): [ka] or a protonated form thereof, wherein AA SC and m is as defined herein.

[0108] The cCPP of formula (A) is represented by the formula (I-5) or (I-6): [ka] or a protonated form thereof, wherein AA SC is as defined herein.

[0109] The cCPP of formula (A) is represented by the formula (I-1): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0110] The cCPP of formula (A) is represented by the formula (I-2): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0111] The cCPP of formula (A) is represented by the formula (I-3): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0112] The cCPP of formula (A) is represented by the formula (I-4): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0113] The cCPP of formula (A) is represented by the formula (I-5): [ka] or a protonated form thereof, During the ceremony, A.A. SC and m is as defined herein.

[0114] The cCPP of formula (A) is represented by the formula (I-6): [ka] or a protonated form thereof, wherein AA SC and m is as defined herein.

[0115] The cCPP may include one of the following sequences: FGFGRGR, GfFGrGr, FfΦGRGR, FfFGRGR, or FfΦGrGr. The cCPP may have one of the following sequences: FGFGRGRQ, GfFGrGrQ, FfΦGRGRQ, FfFGRGRQ, or FfΦGrGrQ.

[0116] The present disclosure also provides a method for producing a compound having the structure of formula (II): [ka] or a protonated form thereof, During the ceremony, AA SC is an amino acid side chain, R 1a , R 1b , and R 1c are each independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl; R 2a , R 2b , R 2c and R 2d are independently amino acid side chains, R 2a , R 2b , R 2c and R 2d At least one of the [ka] or a protonated form thereof, R 2a , R 2b , R 2c and R 2d at least one of is guanidine or a protonated form thereof; each n" is independently an integer 0, 1, 2, 3, 4, or 5; each n' is independently an integer from 0, 1, 2, or 3; If n' is 0, R 2a , R 2b , R 2b or R 2d does not exist.

[0117] R 2a , R 2b , R 2c and R 2d At least two of the [ka] or its protonated form. 2a , R 2b , R 2c and R 2d Two or three of them are [ka] or its protonated form. 2a , R 2b , R 2c and R 2d One of them is [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of the [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder of R may be guanidine or its protonated form. 2a , R 2b , R 2c and R 2d At least two of the [ka] or its protonated form. 2a , R 2b , R 2c and R 2d The remainder may be guanidine or its protonated form.

[0118] R 2a , R 2b , R 2c and R 2d All of the above [ka] or its protonated form. 2a , R 2b , R 2c and R 2d At least one of the [ka] or its protonated form, R 2a , R 2b , R 2c and R 2d The remainder of R may be a guaninide or a protonated form thereof. 2a , R 2b , R 2c and R 2d The base is [ka] or its protonated form, R 2a , R 2b , R 2cand R 2d The remainder is guanidine or its protonated form.

[0119] R 2a , R 2b , R 2c and R 2d can each independently be 2,3-diaminopropionic acid, 2,4-diaminobutyric acid side chain, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homo-lysine, serine, homo-serine, threonine, allo-threonine, histidine, 1-methylhistidine, 2-aminobutanedioic acid, aspartic acid, glutamic acid, or homo-glutamic acid.

[0120] AA SC teeth, [ka] where t can be an integer from 0 to 5. SC teeth, [ka] where t can be an integer from 0 to 5. t can be 1 to 5. t is 2 or 3. t can be 2. t can be 3.

[0121] R 1a , R 1b , and R 1c Each R can independently be 6- to 14-membered aryl. 1a , R 1b , and R 1c R may each independently be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, or S. 1a , R 1b , and R 1c Each R may be independently selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl, or isoquinolyl. 1a , R 1b , and R 1cEach R may be independently selected from phenyl, naphthyl, or anthracenyl. 1a , R 1b , and R 1c Each R may independently be phenyl or naphthyl. 1a , R 1b , and R 1c may each independently be selected from pyridyl, quinolyl, or isoquinolyl.

[0122] Each n' can independently be 1 or 2. Each n' can be 1. Each n' can be 2. At least one n' can be 0. At least one n' can be 1. At least one n' can be 2. At least one n' can be 3. At least one n' can be 4. At least one n' can be 5.

[0123] Each n" can independently be an integer from 1 to 3. Each n" can independently be 2 or 3. Each n" can be 2. Each n" can be 3. At least one n" can be 0. At least one n" can be 1. At least one n" can be 2. At least one n" can be 3.

[0124] Each n" can be independently 1 or 2, and each n' can be independently 2 or 3. Each n" can be independently 1, and each n' can be independently 2 or 3. Each n" can be independently 1, and each n' can be 2. Each n" is 1, and each n' is 3.

[0125] The cCPP of formula (II) has the structure of formula (II-1): [ka] or a protonated form thereof, In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , A.A. SC, n′ and n″ are as defined herein.

[0126] The cCPP of formula (II) has the structure of formula (IIa): [ka] or a protonated form thereof, In the formula, R 1a , R 1b , R 1c , R 2a , R 2b , R 2c , R 2d , A.A. SC and n' are as defined herein.

[0127] The cCPP of formula (II) has the structure of formula (IIb): [ka] or a protonated form thereof, In the formula, R 2a , R 2b , A.A. SC and n' is as defined herein.

[0128] cCPP has the formula (IIb): [ka] or its protonated form, During the ceremony, AA SC and n' are as defined herein.

[0129] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0130] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0131] The cCPP of formula (IIa) has the following structure: [ka] wherein AA SC and n is as defined herein.

[0132] The cCPP of formula (II) has the structure: [ka] may have:

[0133] The cCPP of formula (II) has the structure: [ka] may have:

[0134] cCPP has the structure of formula (III): [ka] or a protonated form thereof, During the ceremony, AA SC is an amino acid side chain, R 1a , R 1b , and R 1c are each independently a 6- to 14-membered aryl or a 6- to 14-membered heteroaryl; R 2a and R 2c are each independently H, [ka] or its protonated form, R 2band R 2d are each independently guanidine or a protonated form thereof; each n″ is independently an integer from 1 to 3; each n' is independently an integer from 1 to 5; Each p' is independently an integer from 0 to 5.

[0135] The cCPP of formula (III) has the structure of formula (III-1): [ka] or a protonated form thereof, During the ceremony, AA SC , R 1a , R 1b , R 1c , R 2a , R 2c , R 2b , R 2d , n', n" and p' are as defined herein.

[0136] The cCPP of formula (III) has the structure of formula (IIIa): [ka] or a protonated form thereof, During the ceremony, AA SC , R 2a , R 2c , R 2b , R 2d , n', n'', and p' are as defined herein.

[0137] In formulae (III), (III-1), and (IIIa), R a and R c can be H. R a and R c can be H, R b and R d R may each independently be guanidine or a protonated form thereof. a can be H. R bcan be H. p' can be 0. R a and R c may be H, and each p' may be 0.

[0138] In formulae (III), (III-1) and (IIIa), R a and R c can be H, R b and R d can each independently be guanidine or a protonated form thereof; n″ can be 2 or 3; and each p′ can be 0.

[0139] p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.

[0140] cCPP has the structure: [ka] may have:

[0141] The cCPP of formula (A) can be selected from:

[0142] [Table 4]

[0143] The cCPP of formula (A) can be selected from:

[0144] [Table 5]

[0145] In embodiments, the cCPP is selected from the following:

[0146] [Table 6]

[0147] AASC can be conjugated to a linker.

[0148] Linker The cCPP of the present disclosure can be conjugated to a linker. The linker can link a cargo to the cCPP. The linker can be attached to the side chain of an amino acid of the cCPP, and the cargo can be attached to a suitable position on the linker.

[0149] The linker may be any suitable moiety that can conjugate the cCPP to one or more further moieties, such as an exocyclic peptide (EP) and / or a cargo. Prior to binding to the cCPP and one or more further moieties, the linker has two or more functional groups, each of which can independently form a covalent bond to the cCPP and one or more further moieties. When the cargo is an oligonucleotide, the linker can be covalently attached to the 5' end of the cargo or the 3' end of the cargo. The linker can be covalently attached to the 5' end of the cargo. The linker can be covalently attached to the 3' end of the cargo. When the cargo is a peptide, the linker can be covalently attached to the N-terminus or C-terminus of the cargo. The linker can be covalently attached to the backbone of an oligonucleotide or peptide cargo. The linker may be any suitable moiety that conjugates the cCPP described herein to a cargo, such as an oligonucleotide, peptide, or small molecule.

[0150] The linker may comprise a hydrocarbon linker.

[0151] The linker may comprise a cleavage site, which may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).

[0152] The linker may be (i) one or more D or L amino acids, each of which is optionally substituted; (ii) an optionally substituted alkylene; (iii) an optionally substituted alkenylene; (iv) an optionally substituted alkynylene; (v) an optionally substituted carbocyclyl; (vi) an optionally substituted heterocyclyl; (vii) one or more -(R 1- JR 2 )z″-subunits, wherein R 1 and R 2 each, at each occurrence, is independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl; and each J is independently selected from C, NR 3 , -NR 3 C(O)-, S, and O, where 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; 1- J)z”- or (JR 1 )z″-, wherein each R 1 is, at each occurrence, independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl; 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 include one or more of (i)-(x).

[0153] The linker may be one or more D or L amino acids and / or (R 1- JR 2 )z″-, wherein R 1 and R 2 each, at each occurrence, is independently alkylene; and each J is independently C, NR 3, -NR 3 C(O)-, S, and O, where R 4 is independently selected from H and alkyl, and z″ is an integer from 1 to 50, or a combination thereof.

[0154] The linker is -(OCH2CH2) z’ - (e.g., as a spacer), where z' is an integer from 1 to 23, e.g., 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'" can also be referred to as polyethylene glycol (PEG).

[0155] The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2CH2) z’ -, where z' is an integer from 1 to 23, and a peptide. The peptide may comprise 2 to 10 amino acids. The linker may further comprise a functional group (FG) that can react via click chemistry. FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.

[0156] The linker is (i) a β-alanine residue and a lysine residue, (ii) -(JR 1 )z″-, or (iii) combinations thereof. Each R 1 can be independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, and each J can be 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″ can be an integer from 1 to 50. Each R 1 can be alkylene and each J can be O.

[0157] The linker may be (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, carbocyclyl, or heterocyclyl, and each J can be 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″ can be an integer from 1 to 50. Each R 1 can be alkylene and each J can be O. The linker can include glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0158] The linker can be a trivalent linker. The linker has the structure: [ka] wherein A1, B1 and C1 are independently a hydrocarbon linker (e.g., NRH-(CH2) n -COOH), PEG linker (e.g., NRH-(CHO) n -COOH, where R is H, methyl or ethyl), or one or more amino acid residues, and Z is independently a protecting group. Linkers also include disulfides [NH2-(CHO) n -SS-(CH2O) n -COOH], or a cleavage site such as a caspase cleavage site (Val-Cit-PABC) can be incorporated.

[0159] The carbohydrate may be a residue of glycine or beta-alanine.

[0160] The linker is bivalent and can link a cCPP to a cargo. The linker is bivalent and can link a cCPP to an exocyclic peptide (EP).

[0161] The linker may be trivalent and may link the cCPP to the cargo and the EP.

[0162] The linker is a divalent or trivalent C1-C 50 It can be alkylene, where 1 to 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 It can be alkylene, where 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or combinations thereof.

[0163] The linker has the structure: [ka] wherein each AA is independently an amino acid residue; * AA SC AA SCis a side chain of an amino acid residue of cCPP, where 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 can be an integer from 1 to 5. x can be an integer from 1 to 3. x can be 1. y can be an integer from 2 to 4. y can be 4. z can be an integer from 1 to 5. z can be an integer from 1 to 3. z can be 1. Each AA can be independently selected from glycine, β-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.

[0164] The cCPP can be linked to the cargo via a linker ("L"). The linker can be conjugated to the cargo via a linking group ("M").

[0165] The linker has the structure: [ka] 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, and each AA is independently an amino acid residue; * AA SC AA SC is the side chain of an amino acid residue of a cCPP, and M is a linking group as defined herein.

[0166] The linker has the structure: [ka] It may have In the formula, 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. * AA SC AA SC is the side chain of an amino acid residue of a cCPP, and M is a linking group as defined herein.

[0167] The linker has the structure: [ka] It may have In the formula, 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. * AA SC AA SC are the side chains of amino acid residues of cCPP.

[0168] x can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

[0169] x' can be 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 (including all ranges and subranges therebetween). x' can be an integer from 5 to 15. x' can be an integer from 9 to 13. x' can be an integer from 1 to 5. x' can be 1.

[0170] y can be an integer from 1 to 5, for example, 1, 2, 3, 4, or 5 (including all ranges and subranges therebetween), and y can be an integer from 2 to 5. y can be an integer from 3 to 5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.

[0171] z can be an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges and subranges therebetween.

[0172] z' can be 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, including all ranges and subranges therebetween. z' can be an integer from 5 to 15. z' can be an integer from 9 to 13. z' can be 11.

[0173] As described above, the linker or M (wherein M is a part of the linker) can be covalently attached to the cargo at any suitable position on the cargo. The linker or M (wherein M is a part of the linker) can be covalently attached to the 3'-end of the oligonucleotide cargo or the 5'-end of the oligonucleotide cargo. The linker or M (wherein M is a part of the linker) can be covalently attached to the N-terminus or C-terminus of the peptide cargo. The linker or M (wherein M is a part of the linker) can be covalently attached to the backbone of the oligonucleotide or peptide cargo.

[0174] The linker can be attached to the side chain of an aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of a glutamine or asparagine (e.g., a reduced side chain bearing an amino group).The linker can be attached to the side chain of a lysine on the cCPP.

[0175] The linker may be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the peptide cargo, or to a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group).The linker may be attached to the side chain of lysine on the peptide cargo.

[0176] The linker has the structure: [ka] It may have During the ceremony, M is a group that conjugates L to a cargo, e.g. an oligonucleotide; AA s is the side chain or terminus of an amino acid on the cCPP, Each AA x are independently amino acid residues, o is an integer from 0 to 10; p is an integer from 0 to 5.

[0177] The linker has the structure: [ka] It may have During the ceremony, M is a group that conjugates L to a cargo, e.g. an oligonucleotide; AA s is the side chain or terminus of an amino acid on the cCPP, Each AA x are independently amino acid residues, o is an integer from 0 to 10; p is an integer from 0 to 5.

[0178] M can include alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M can be [ka] wherein R is alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl.

[0179] M is [ka] You can choose from R 10 is alkylene, cycloalkyl, or [ka] where a is 0 to 10.

[0180] M is [ka] R 10 teeth, [ka] where a is from 0 to 10. M can be [ka] It could be.

[0181] M is a heterobifunctional crosslinker, e.g., [ka] which 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.

[0182] M can be -C(O)-.

[0183] AA s may be the side chain or terminus of an amino acid on the cCPP. s Non-limiting examples of AA include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain bearing an amino group). s is AA as defined herein SC It could be.

[0184] Each AA x are independently natural or unnatural amino acids. x may be a natural amino acid. x may be a non-natural amino acid. x may be a β-amino acid. The β-amino acid may be β-alanine.

[0185] o can be an integer from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o can be 0, 1, 2, or 3. o can be 0. o can be 1. o can be 2. o can be 3.

[0186] p can be 0 to 5, e.g., 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p can be 2. p can be 3. p can be 4. p can be 5.

[0187] The linker has the structure: [ka] It may have In the formula, M, AA s , each -(R 1- JR 2 ) z″-, o, and z″ are defined herein. r can be 0 or 1.

[0188] r can be 0. r can be 1.

[0189] The linker has the structure: [ka] It may have In the formula, M, AA s , o, p, q, r, and z″ may be as defined herein.

[0190] z" can 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" can be an integer from 5 to 20. z" can be an integer from 10 to 15.

[0191] The linker has the structure: [ka] It may have During the ceremony, M, A.A. sand o are as defined herein.

[0192] Other non-limiting examples of suitable linkers include: [ka] [ka] In the formula, M and AA s is as defined herein.

[0193] Provided herein is a compound comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, further comprising L, a linker conjugated to the AC via a linking group (M), wherein M is [ka] The compound is provided,

[0194] Provided herein is a compound comprising a cCPP and a cargo comprising an antisense compound (AC), e.g., an antisense oligonucleotide, that is complementary to a target in a pre-mRNA sequence, further comprising L, wherein a linker is conjugated to the AC via a linking group (M), wherein M is [ka] Selected from R 1 is alkylene, cycloalkyl, or [ka] where t' is 0 to 10, each R is independently alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, and R 1 but, [ka] and t' is 2.

[0195] The linker has the structure: [ka] It may have During the ceremony, A.A. s is as defined herein, and m' is 0-10.

[0196] The linker has the formula: [ka] It may be of the following type.

[0197] The linker has the formula: [ka] where "base" corresponds to the 3'-terminal nucleobase of the cargo phosphorodiamidate morpholino oligomer.

[0198] The linker has the formula: [ka] where "base" corresponds to the 3'-terminal nucleobase of the cargo phosphorodiamidate morpholino oligomer.

[0199] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0200] The linker has the formula: [ka] where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.

[0201] The linker has the formula: [ka] It may be of the following type.

[0202] The linker can be covalently attached to the cargo at any suitable position on the cargo. The linker is covalently attached to the 3' end of the cargo or the 5' end of the oligonucleotide cargo. The linker can be covalently attached to the backbone of the cargo.

[0203] The linker can be attached to the side chain of an aspartic acid, glutamic acid, glutamine, asparagine, or lysine on the cCPP, or a modified side chain of a glutamine or asparagine (e.g., a reduced side chain bearing an amino group).The linker can be attached to the side chain of a lysine on the cCPP.

[0204] cCPP-linker conjugates The cCPP may be conjugated to a linker as defined herein. The linker may be any combination of the AA SC can be conjugated to

[0205] The linker is -(OCH2CH2) z’ subunits (e.g., as spacers), where 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 referred to as PEG. The cCPP-linker conjugate may have a structure selected from Table 3.

[0206] [Table 7]

[0207] The linker is -(OCH2CH2) z’-subunits, where z' is an integer from 1 to 23, and peptide subunits. The peptide subunits may comprise 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 4.

[0208] [Table 8]

[0209] An EEV is provided that includes a cyclic cell penetrating peptide (cCPP), a linker, and an exocyclic peptide (EP). The EEV has the structure of formula (B): [ka] or a protonated form thereof, During the ceremony, R1, R2, and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; EP is an exocyclic peptide as defined herein; each m is independently an integer from 0 to 3; n is an integer from 0 to 2, x' is an integer from 1 to 20; y is an integer from 1 to 5; q is 1 to 4; z' is an integer from 1 to 23.

[0210] R1, R2, R3, R4, R7, EP, m, q, y, x', z' are as described herein.

[0211] n can be 0. n can be 1. n can be 2.

[0212] EEV has the formula (Ba) or (Bb): [ka] or a protonated form thereof, 1 , R 2 , R 3 , R 4 , m and z' are as defined above in formula (B).

[0213] EEV is calculated by the formula (Bc): [ka] or a protonated form thereof, 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.

[0214] The EEV is represented by the formula (B-1), (B-2), (B-3), or (B-4): [ka] [ka] or a protonated form thereof, where EP is as defined above in formula (B).

[0215] The EEV may comprise the formula (B) and the structure: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG 12 -OH, or Ac-PKKKRKVAEEA-K(cyclo[GfFGrGrQ])-PEG 12 It may have -OH.

[0216] EEV has the formula: [ka] The cCPP may include

[0217] EEV may include the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3).

[0218] EEV can be Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-AEEA-K-(cyclo[FGFGRGRQ])-PEG12-OH. EEV is

Chemical formula

[0219] EEV can be Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH. EEV is

Chemical formula

[0220] EEV is Ac-rr-miniPEG2-Dap[cyclo(FfΦ-Cit-r-Cit-rQ)]-PEG12-OH, Ac-frr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rfr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rbfbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rrr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-rbrbr-PEG2-Dap(cyclo(FfΦ-Cit-r-Cit-rQ))-PEG12-OH, Ac-hh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rbhbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-hbrbh-PEG2-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-PEG12-OH、 Ac-rr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-frr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rfr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbfbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rrr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbrbr-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-hh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-hbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-hbhbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-rbhbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-hbrbh-Dap(シクロ(FfΦ-Cit-r-Cit-rQ))-b-OH、 Ac-KKKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2、 Ac-KGKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KKGK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KKK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KGK-miniPEG2-Lys(シロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBKBK-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-KR-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-KBR-miniPEG2-Lys(シロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2; Ac-PKKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-PKKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-PGKKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2. Ac-PKGKRKV-miniPEG2-Lys(シクロ(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 Ac-PKKGRKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKGKV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKRGV-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-PKKKRKG-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-KKKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, Ac-KKRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2, and Ac-KRK-miniPEG2-Lys(cyclo(Ff-Nal-GrGrQ))-miniPEG2-K(N3)-NH2 may be selected from:

[0221] EEV is Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG 12 -K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦGrGrQ])-miniPEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2, Ac-KR-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKGKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKG-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FFΦGRGRQ])-miniPEG2-K(N3)-NH2, Ac-PKKKRKV-miniPEG2-Lys(cyclo[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2, and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FfΦSrSrQ])-miniPEG2-K(N3)-NH2 may be selected from:

[0222] EEV is Ac-PKKKRKV-miniPEG2-Lys(cyclo(GfFGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFKRKRQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRGRGQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRrRQ])-PEG 12 -OH, Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFGRRRQ])-PEG 12 -OH, and Ac-PKKKRKV-miniPEG2-Lys(cyclo[FGFRRRRQ])-PEG 12 -OH may be selected from:

[0223] EEV is Ac-KKKRKG-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKKRK-miniPEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKRKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KRKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-KKKKR-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-RKKKK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH, and Ac-KKKRK-PEG4-K(cyclo[FGFGRGRQ])-PEG 12 -OH may be selected from:

[0224] EEV is Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG2-K(N3)-NH2, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2, and Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH may be selected from:

[0225] The cargo may be an AC and the EEV may be Ac-PKKKRKV-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-PKKKRKV-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12-OH, Ac-rrr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rrr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FfΦGrGrQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG12 -OH、 Ac-rbrbr-PEG2-K(シ[FfΦGrGrQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(シ[FfΦCit-r-Cit-rQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(シ[FfFGRGRQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(FGFGRGRQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(FGFGRRRQ])-PEG 12 -OH、 Ac-rbrbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(シ[FfΦGrGrQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(。[FfΦCit-r-Cit-rQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(シ[FfFGRGRQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(FGFGRGRQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(FGFGRRRQ])-PEG 12 -OH、 Ac-rbhbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH、 Ac-hbrbh-PEG2-K(シ[FfΦGrGrQ])-PEG 12 -OH、 Ac-hbrbh-PEG2-K(cyclo[FfΦCit-r-Cit-rQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FfFGRGRQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH, Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH, and Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH where b is beta-alanine and the exocyclic sequence may be of D or L stereochemistry.

[0226] In an embodiment, the cCPP is

[0227] [Table 9] It could be.

[0228] The cargo may be a protein and the EEV may be Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(FGFGRRRQ])-PEG 12 -OH Ac-PKKKRKV-PEG2-K(シ[FGFRRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(。[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(。[FGFGRGRQ])-PEG 12 -OH Ac-rr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH Ac-rr-PEG2-K(。[FGFGRRRQ])-PEG 12 -OH Ac-rr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(。[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rrr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(FGFGRGRQ])-PEG 12 -OH Ac-rrr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH Ac-rrr-PEG2-K(FGFGRRRQ])-PEG 12 -OH Ac-rrr-PEG2-K(。[FGFRRRRQ])-PEG12 -OH Ac-rhr-PEG 2- K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rhr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-rbr-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(FGFGRGRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(FGFGRRRQ])-PEG 12 -OH Ac-rbrbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(。[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(シ[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(シ[FfF-GRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(FGFGRGRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(シ[GfFGrGrQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(FGFGRRRQ])-PEG 12 -OH Ac-rbhbr-PEG2-K(。[FGFRRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(。[Ff-Nal-GrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(。[Ff-Nal-Cit-r-Cit-rQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FfF-GRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRGRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[GfFGrGrQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFGRRRQ])-PEG 12 -OH Ac-hbrbh-PEG2-K(cyclo[FGFRRRRQ])-PEG 12 -OH where b is beta-alanine and the exocyclic sequence may be of D or L stereochemistry.

[0229] cargo A cell penetrating peptide (CPP), such as a cyclic cell penetrating peptide (cCPP), can be conjugated to a cargo. The cargo can be a therapeutic drug 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 to the cargo. The cargo can be conjugated to the cCPP by a linker. The cargo can be linked to the AA SC At least one atom of the cCPP can be replaced by a therapeutic drug moiety, or at least one lone pair of the cCPP forms a bond to a therapeutic drug moiety. A hydroxyl group on an amino acid side chain of the cCPP can be replaced by a bond to a cargo. A hydroxyl group on a glutamine side chain of the cCPP can be replaced by a bond to a cargo. The cargo can be conjugated to the cCPP by a linker. The cargo can be linked to an AA SC can be conjugated to

[0230] 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-peptidyl therapeutic agent. The cargo may be an antibody or an antigen-binding fragment thereof, such as, but not limited to, an scFv or a nanobody.

[0231] Cyclic cell-penetrating peptides (cCPPs) conjugated to cargo moieties A cyclic cell penetrating peptide (cCPP) can be conjugated to a cargo moiety.

[0232] The cargo moiety may be conjugated to the cCPP via a linker. The cargo moiety may comprise a therapeutic drug moiety. The therapeutic drug moiety may comprise an oligonucleotide, a peptide, or a small molecule. The oligonucleotide may comprise an antisense oligonucleotide. The cargo moiety is conjugated to the linker at the terminal carbonyl group to have the following structure: [ka] [wherein EP is an exocyclic peptide; M, AA SC , cargo, x', y, and z' are as defined above; * AA SC is the attachment point to x' can be 1. y can be 4. z' can be 11. -(OCH2CH2) x’ -and / or-(OCH2CH2) z’ The - may be independently replaced by one or more amino acids, such as glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or combinations thereof.

[0233] An endosomal escape vehicle (EEV) can comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, conjugated to a cargo to have the structure of formula (C): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; R4 is H or an amino acid side chain; EP is an exocyclic peptide as defined herein; Cargo is a moiety as defined herein; each m is independently an integer from 0 to 3; n is an integer from 0 to 2; x' is an integer from 2 to 20; y is an integer from 1 to 5; q is an integer from 1 to 4; z' is an integer from 2 to 20. or a protonated form thereof.

[0234] R1, R2, R3, R4, EP, cargo, m, n, x', y, q and z' are as defined herein.

[0235] The EEV can be conjugated to a cargo, the EEV conjugate having the structure of formula (Ca) or (Cb): [ka] wherein EP, m, and z are as defined above in formula (C). or a protonated form thereof.

[0236] The EEV can be conjugated to a cargo, the EEV conjugate having the structure of formula (Cc): [ka] or a protonated form thereof, 1 , R 2 , R 3 , R 4and m are as defined above in formula (III), AA can be an amino acid as defined herein, n can be an integer from 0 to 2, x can be an integer from 1 to 10, y can be an integer from 1 to 5, and z can be an integer from 1 to 10.

[0237] The EEV may be conjugated to an oligonucleotide cargo, the EEV-oligonucleotide conjugate having the structure of formula (C-1), (C-2), (C-3) or (C-4): [ka] [ka] or a protonated form thereof.

[0238] How to Make Cyclic Peptides The present disclosure relates to a cyclic peptide of formula (A): [ka] [During the ceremony, R1, R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4, R5, R6, R7 are independently H or an amino acid side chain; At least one of R4, R5, R6, and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic 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; AA SC is an amino acid side chain; q is 1, 2, 3 or 4. or a protonated form thereof, which is any one of the methods described herein for a compound of formula (I).

[0239] The present disclosure relates to a cyclic peptide of formula (I): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: Compound of formula (1) [ka] with a compound of formula (II) [ka] to give a compound of formula (III) [ka] To form wherein X and X' are independently protecting groups, X" is H or a protecting group, X"' is H or an activating group (e.g., an NHS ester), and m is 0-3. In some embodiments, X" is methyl or t-butyl.

[0240] 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, for example, N,N'-dicyclohexylcarbodiimide (DCC). The method may further include treating with an activating agent, such as N-hydroxysuccinimide. The method may further include treating with a base. The base may be NMM. In embodiments, the combination of reagent(s) and / or solvent(s) may be DCC / N-hydroxysuccinimide / THF. In embodiments, the combination of reagent(s) and / or solvent(s) may be NMM / DMF.

[0241] The method comprises reacting a compound of formula (III) with a compound of formula (IV) [ka] The method may further include converting the

[0242] The process may include deprotecting or converting a compound of formula (III) to a compound of formula (IV) in the presence of a base or a weak acid.

[0243] The method also includes reacting a compound of formula (IV) with a compound of formula (V) [ka] to give a compound of formula (VI) [ka] where Z is a radical of an amino acid side chain; [ka] is a solid support.

[0244] 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, e.g., DIC, HATU, DEPBT, an additive, e.g., HOAt / Oxyma / K-Oxyma, Oxyma-B, and a base, e.g., DIPEA / NMM. In an embodiment, the combination of reagent(s) and / or solvent(s) may be DIC / Oxyma. In an embodiment, the combination of reagent(s) and / or solvent(s) may be DIC / HOAt. In an embodiment, the combination of reagent(s) and / or solvent(s) may be DEPBT / DIPEA. In an embodiment, the combination of reagent(s) and / or solvent(s) may be DEPBT / NMM. In an embodiment, the combination of reagent(s) and / or solvent(s) may be HATU / NMM. In embodiments, the combination of reagent(s) and / or solvent(s) may be DIC / K-Oxyma. In embodiments, the combination of reagent(s) and / or solvent(s) may be DIC / Oxyma-B. In some embodiments, the solvent comprises DMF.

[0245] The compound of formula (IV) [ka] and the compound of formula (VI) is [ka] It could be.

[0246] The method comprises reacting a compound of formula (VII) [ka] with a coupling agent, an additive and a base to obtain a compound of formula (VIII): [ka] The method may further include obtaining:

[0247] 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.

[0248] In embodiments, the combination of reagent(s) and / or solvent(s) may be HATU / HOAt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyAOP / HOAt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyAOP / HOAt / NMM. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyBOP / HOBt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyBrop / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyOxim / Oxyma / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be DIC / HOBt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be HBTU / HOBt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be TBTU / HOBt / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be COMU / Oxyma / DIPEA. In embodiments, the combination of reagent(s) and / or solvent(s) may be DEPBT / DIPEA. In some embodiments, the solvent comprises DMF.

[0249] The compound of formula (VII) [ka] and the compound of formula (VIII) is [ka] It could be.

[0250] The present disclosure relates to a cyclic peptide of formula (Ia): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; Z is an amino acid side chain radical; P1 is [ka] and; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: Compound of formula (IX) [ka] wherein X is a protecting group; [ka] is a solid support. with a compound of formula (X) [ka] wherein each X' is independently a protecting group and Z is a radical of an amino acid side chain. to give a compound of formula (XI) [ka] 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, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000].

[0251] The compound of formula (X) [ka] and the compound of formula (XI) [ka] It could be.

[0252] The method comprises treating a compound of formula (XI) to form a compound of formula (XII) [ka] The method may further include forming

[0253] The method may include treating a compound of formula (XI) with a compound of formula (XII) in the presence of a base, for example, piperidine / hydrazine / DBU / sodium hydroxide / pyrrolidine / morpholine / diethylamine / tert-butylamine. The method may further include adding Pd(PPh3)4 / PhSiH3 / DCM. The method may further include adding a coupling reagent, such as PyOxim, an additive, such as Oxyma, and a base, such as DIPEA.

[0254] In embodiments, the combination of reagent(s) and / or solvent(s) may be piperidine. In embodiments, the combination of reagent(s) and / or solvent(s) may be piperidine / formic acid. In embodiments, the combination of reagent(s) and / or solvent(s) may be piperidine / Oxyma. In embodiments, the combination of reagent(s) and / or solvent(s) may be DBU. In embodiments, the combination of reagent(s) and / or solvent(s) may be DBU / piperidine. In embodiments, the combination of reagent(s) and / or solvent(s) may be DBU / piperidine / Oxyma. In embodiments, the combination of reagent(s) and / or solvent(s) may be DBU / piperidine / HOBt. In embodiments, the combination of reagent(s) and / or solvent(s) may be DBU / piperidine / formic acid. In embodiments, the combination of reagent(s) and / or solvent(s) may be tert-butylamine, pyrrolidine. In embodiments, the combination of reagent(s) and / or solvent(s) may be morpholine. In embodiments, the combination of reagent(s) and / or solvent(s) may be diethylamine. In embodiments, the combination of reagent(s) and / or solvent(s) may be sodium hydroxide. In embodiments, the combination of reagent(s) and / or solvent(s) may be Pd(PPh3)4 / PhSiH3 / DCM for allyl ester removal. In embodiments, the combination of reagent(s) and / or solvent(s) may be PyOxim / Oxyma / DIPEA / DMF / DCM for cyclization.

[0255] The compound of formula (XII) [ka] It could be.

[0256] The present disclosure relates to a cyclic peptide of formula (I): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; or a protonated form thereof, comprising the steps of: [ka] wherein X' is a protecting group and Z is a radical of an amino acid side chain; [ka] is a solid support. with a compound of formula (XIV) [ka] to give a compound of formula (XV) [ka] The method includes obtaining a

[0257] 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, followed by assembly according to 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].

[0258] The compound of formula (XIII) [ka] and the compound of formula (XIV) [ka] and the compound of formula (XV) [ka] How to be.

[0259] The method comprises treating a compound of formula (XV) to produce a compound of formula (XVI): [ka] The method may further include obtaining:

[0260] The method may include treating a compound of formula (XV) in the presence of standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids.

[0261] The compound of formula (XVI) [ka] It could be.

[0262] Alternative cyclic peptide formation The present disclosure also relates to a compound of formula (D) [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; Y is [ka] It is. q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; Each n is independently an integer of 0, 1, 2, or 3. or a protonated form thereof.

[0263] Macrolactamization The present disclosure relates to a cyclic peptide of formula (DI): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; Y is [ka] is or a protonated form thereof, comprising the steps of: Compound of formula (XVII) [ka] wherein Z is a radical of an amino acid side chain; [ka] is a solid support. The present invention also relates to a method of the present invention, comprising cyclizing

[0264] An example of a synthesis scheme is shown in Scheme 1. [ka]

[0265] Methods may include standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids, e.g., using coupling reagents such as PyOxim, additives such as Oxyma, and bases such as DIPEA for cyclization, and treatment with, e.g., HFIP or TFA for cleavage from the solid.

[0266] Ring-closing metathesis (RCM) The present disclosure also relates to a cyclic peptide of formula (D-II): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; each n is independently an integer of 0, 1, 2, or 3; Y is [ka] is A method for preparing a compound of formula (XVIII): [ka] wherein Z is a radical of an amino acid side chain; [ka] is a solid support).

[0267] An example of a synthesis scheme is shown in Scheme 2. [ka]

[0268] Methods may include standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids, e.g., using coupling reagents such as PyOxim, additives such as Oxyma, and bases such as DIPEA for cyclization, and treatment with, e.g., HFIP or TFA for cleavage from the solid.

[0269] Thioester stapling agents The present disclosure also relates to a cyclic peptide of formula (D-III): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AASC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; each n is independently an integer of 0, 1, 2, or 3; Y is [ka] is or a protonated form thereof, comprising reacting a compound of formula (XIX): [ka] wherein Z is a radical of an amino acid side chain; [ka] is a solid support).

[0270] An example of a synthesis scheme is shown in Scheme 3. [ka]

[0271] Methods may include standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids, e.g., using coupling reagents such as PyOxim, additives such as Oxyma, and bases such as DIPEA for cyclization, and treatment with, e.g., HFIP or TFA for cleavage from the solid. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)

[0272] The present disclosure also relates to a cyclic peptide of formula (D-IV): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; Y is [ka] is or a protonated form thereof, comprising reacting a compound of formula (XX): [ka] wherein Z is a radical of an amino acid side chain; [ka] is a solid support.

[0273] An example of a synthesis scheme is shown in Scheme 4. [ka]

[0274] Methods may include standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids, e.g., using coupling reagents such as PyOxim, additives such as Oxyma, and bases such as DIPEA for cyclization, and treatment with, e.g., HFIP or TFA for cleavage from the solid.

[0275] Thioether Cyclization The present disclosure also provides a cyclic peptide of formula (DV): [ka] [During the ceremony, R1, R2, and R3 can each independently be H, or an amino acid residue having a side chain that includes an aromatic group; At least one of R1, R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid; R4 and R6 are independently H or an amino acid side chain; AA SC is an amino acid side chain; q is 1, 2, 3 or 4; each m is independently an integer of 0, 1, 2, or 3; Y is [ka] is or a protonated form thereof, comprising reacting a compound of formula (XXI): [ka] wherein Z is a radical of an amino acid side chain; [ka] is a solid support.

[0276] An example of a synthesis scheme is shown in Scheme 5. [ka]

[0277] Methods may include standard solid phase peptide synthesis conditions [Chan, WC, White PD, ed. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, 2000] for deprotection and coupling of amino acids, e.g., using coupling reagents such as PyOxim, additives such as Oxyma, and bases such as DIPEA for cyclization, and treatment with, e.g., HFIP or TFA for cleavage from the solid. [ka]

[0278] How to Make Phosphorodiamidate Morpholino Oligomers (PMOs) PMOは, when the technology is divided into arbitrary methods, examples, Summerton et al. U.S. Patent No. 5,166,315 (November 24, 1992), Summerton et al. U.S. Patent No. 5,185,444 (February 9, 1993), Summerton et al. al. U.S. Patent No. 5,217,866 (June 8, 1993), Summerton et al. U.S. Patent No. 5,235,033 (August 10, 1993), Summerton et al. U.S. Patent No. 5,506,337 (April 9, 1996), Summerton et al. al. U.S. Patent No. 5,521,063 (May 28, 1996), Summerton et al. Antisense Nucleic Acid Drug Dev. 1997, 7: 187-195, Iversen, P. International Patent WO02 / 092617A1 (November 21, 2002), Stein et al. U.S. Patent No. 6,828,105 B2 (December 7, 2004), Ivesen et al. U.S. Patent Application 2005 / 0261249 A1 (November 24, 2005), Moich et al. U.S. Patent Application 2006 / 0276425 A1 (December 7, 2006), Stein et al. U.S. Patent Application 2007 / 0004661A1 (January 4, 2007), Stein et al. U.S. Patent Application 2007 / 0129323 A1 (June 7, 2007), Multon et al. International Patent Application WO2009 / 005793 A2 (January 8, 2009), Multon et al. United States Patent Application 2010 / 0016215 A1 (January 21, 2010), Sazani et al. United States Patent Application 2010 / 0130591 (May 27, 2010), Weller et al. al. U.S. Patent No. 2010 / 0234281 A1 (September 16, 2010), Weller et al. U.S. Patent No. 7,943,762 B2 (May 17, 2011), Weller et al. U.S. Patent No. 8,067,571 B2 (November 29, 2011), Reves et al. al. United States Patent No. 8,076,476 B2 (December 13, 2011), Fox et al. U.S. Patent No. 8,299,206B2 (October 30, 2012), Linsley et al.U.S. Patent Application 2014 / 030238 (October 9, 2014), Linsley et al. US Patent Application 2014 / 0329772 (November 6, 2014), Ueda, T. US Patent 8,969,551B2 (March 3, 2015), Hanson, G. US Patent No. 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 WO2019 / 0-60862A1 (March 28, 2019), Torii et al. US Patent No. 10,415,036 B2 (September 17, 2019), Cai et al. al. U.S. Patent Application 2019 / 0292208 A1 (September 26, 2019), Bestwick et al. U.S. Patent Application 2019 / 0365918 A1 (December 5, 2019), Passini et al. U.S. Patent Application 2020 / 0377886 A1 (December 3, 2020), Sinha et al. U.S. Patent Application 2021 / 0130379 (May 6, 2021), Fang et al. International Patent WO2022 / 125987A1, and Scheme 6. [ka]

[0279] The method may include, for example, treatment with 4-cyanopyridine and TFA for detritylation, treatment with DIPEA for neutralization, addition of a PMO monomer in the presence of a base such as NEM for coupling, the method may include 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.

[0280] Peptide + PMO Conjugation Method Cyclic peptides can be conjugated to PMOs according to any method known in the art, such as those described in [Hanson, G. Peptide Oligonucleotide Conjugates. U.S. Patent No. 9,161,948 B2 (October 20, 2015)] and Scheme 7. Various reaction conditions demonstrating activation of N3-terminal peptides by treatment with base and a "coupling reagent" followed by addition of a PMO are also shown in Example 4.

[0281] [ka] The method may include treatment with a coupling reagent such as DIC / HATU / PyAOP, an additive such as Oxyma, and a base such as DIPEA. The method may include treatment with a base such as sodium hydroxide / lithium hydroxide / potassium hydroxide / potassium carbonate / potassium chloride for deprotection.

[0282] compound The present disclosure relates to [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; AA SC is an 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; [ka] is a solid support. The present invention also relates to a compound selected from:

[0283] The present disclosure relates to [ka] [ka] [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; AA SC is an 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; [ka] is a solid support. The present invention also relates to a compound selected from:

[0284] The present disclosure relates to [ka] [During the ceremony, R1, R2, and R3 are each independently H or the side chain of tyrosine, phenylalanine, or tryptophan; R4 is H or an amino acid side chain; AA SC is an 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; [ka] is a solid support.

[0285] The synthesis can be performed manually or automatically, or a combination of both.

[0286] The resin loading level may be about 0.1-1.0 mmol / g. The resin loading level may be about 0.30-0.50 mmol / g. The resin loading level may be about 0.20-0.30 mmol / g. The resin loading level may be about 0.20-0.25 mmol / g. The resin loading level may be about 0.2-0.50 mmol / g. The resin loading level may be about 0.2-0.60 mmol / g. The resin loading level may be about 0.2-0.70 mmol / g. The resin loading level may be about 0.2-0.80 mmol / g. The resin loading level may be about 0.22-0.90 mmol / g. The resin loading level may be about 0.22-0.92 mmol / g. The resin loading level may be about 0.22 mmol / g. The resin loading level may be about 0.39 mmol / g. The resin loading level may be about 0.46 mmol / g. The resin loading level may be about 0.64 mmol / g. The resin loading level may be about 0.77 mmol / g. The resin loading level may be about 0.92 mmol / g.

[0287] Specific Definitions As used in this 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.

[0288] "about"(" ~The term "about" (also written "about 50"), when preceding a numerical value, means a range (e.g., plus or minus 10% of the value). For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless the context of this disclosure indicates otherwise or is inconsistent with such an interpretation. For example, in a list of numerical values ​​such as "about 49, about 50, about 55, ...", "about 50" means a range that spans less than half the interval between the preceding value and the following value, e.g., greater than 49.5 to less than 52.5. Furthermore, phrases less than "about" a value or greater than "about" a value should be understood in light of the definition of the term "about" provided herein. Similarly, the term "about" when preceding a series of numerical values ​​or a range of values ​​(e.g., "about 10, 20, 30" or "about 10 to 30") refer to all of the values ​​in the series or to the end points of the range, respectively.

[0289] "2-[2-[2-aminoethoxy]ethoxy]acetic acid" is also called AEEA or miniPEG.

[0290] As used herein, the term "cyclic cell-penetrating peptide" or "CPP" refers to a peptide that facilitates delivery of a cargo, such as a therapeutic drug moiety.

[0291] As used herein, the term "endosomal escape vehicle" (EEV) refers to a CPP conjugated by a chemical bond (i.e., a covalent bond or a non-covalent interaction) to a linker, as defined herein, and / or an exocyclic peptide, as defined herein. An EEV of the present disclosure is an EEV of formula (B):

[0292] As used herein, the term "EEV conjugate" refers to an endosomal escape vehicle as defined herein conjugated to a cargo by chemical bond (i.e., covalent bond or non-covalent interaction). The cargo can be a therapeutic moiety (e.g., an oligonucleotide) that can be delivered intracellularly by the EEV. The EEV conjugate of the present disclosure can be an EEV conjugate of formula (C).

[0293] As used herein, the terms "exocyclic peptide" (EP) and "modulatory peptide" (MP) may be used interchangeably to refer to two or more amino acid residues linked by a peptide bond that may be conjugated to the cyclic peptides disclosed herein. The EP, when conjugated to the cyclic peptides disclosed herein, alters 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 large T antigen of the SV40 virus, the minimal functional unit of which is the seven amino acid sequence PKKKRKV, the nucleoplasmin bipartite NLS having the sequence NLSKRPAAIKKAGQAKKKK, the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD or RQRRNELKRSF, the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha, the sequences VSRKRPRP and PPKKARED of the sarcoma T protein, the sequence PQPKKKPL of human p53, the sequence PQPKKKPL of mouse c-abl Examples of NLS include the sequence SALIKKKKKMAP of IV, the sequences DRLRR and PKQKKRK of influenza virus NS1, the sequence RKLKKKIKKL of hepatitis virus delta antigen and the sequence REKKKFLKRR of mouse MxI protein, the sequence KRKGDEVDGVDEVAKKKSKK of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK of steroid hormone receptor (human). Additional examples of NLSs are described in WO 2001 / 038547, which is incorporated herein by reference in its entirety.

[0294] As used herein, "linker" or "L" refers to a moiety that covalently attaches one or more moieties (e.g., an exocyclic peptide (EP), as well as a cargo, e.g., an oligonucleotide, peptide, or small molecule) to a cyclic peptide. The linker may comprise a natural or unnatural amino acid or polypeptide. The linker may be a synthetic compound that contains two or more suitable functional groups suitable for attaching the cyclic peptide to a cargo moiety, thereby forming a compound disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. For example, a cyclic peptide may be covalently attached to an AC via a linker.

[0295] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of an oligonucleotide can be modified. An oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). An oligonucleotide can be composed of natural and / or modified nucleobases, sugars and covalent internucleoside linkages, and can further comprise non-nucleic acid conjugates.

[0296] The terms "peptide", "protein" and "polypeptide" are used interchangeably to refer to natural or synthetic molecules that contain two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of the other amino acid. Two or more amino acid residues may be linked by the carboxyl group of one amino acid to the alpha amino group. Two or more amino acids of a polypeptide may be linked by peptide bonds. A polypeptide may contain peptide backbone modifications in which two or more amino acids are covalently linked by bonds other than peptide bonds. A polypeptide may contain 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 amino acids and artificially occurring amino acids. The term polypeptide includes peptides that contain, for example, about 2 to about 100 amino acid residues, as well as proteins, such as therapeutic proteins, including, for example, but not limited to, antibodies, enzymes, receptors, soluble proteins, and the like.

[0297] The term "therapeutic polypeptide" refers to a polypeptide having therapeutic, prophylactic or other biological activity. A therapeutic polypeptide can be produced in any suitable manner. For example, a therapeutic polypeptide can be isolated or purified from a naturally occurring environment, can be chemically synthesized, can be recombinantly produced, or a combination thereof.

[0298] The term "small molecule" refers to an organic compound having pharmacological activity and a molecular weight of less than about 2000 daltons, or less than about 1000 daltons, or less than about 500 daltons. Small molecule therapeutic agents are typically produced by chemical synthesis.

[0299] As used herein, the term "adjacent" refers to two amino acids that are covalently connected. For example, [ka] In the context of a representative cyclic peptide such as, AA1 / AA2, AA2 / AA3, AA3 / AA4, and AA5 / AA1 exemplify pairs of adjacent amino acids.

[0300] A residue of a chemical species, as used herein, refers to a derivative of a chemical species present in a particular product. To form a product, at least one atom of the chemical species is replaced by a bond to another moiety such that the product contains a derivative or residue of the chemical species. For example, the cyclic peptides described herein have an amino acid (e.g., arginine) incorporated therein through the formation of one or more peptide bonds. An amino acid incorporated into a cyclic peptide may be referred to as a residue, or simply an amino acid. Thus, an arginine or arginine residue may be: [ka] Refers to...

[0301] The term "protonated form thereof" refers to the protonated form of an amino acid. For example, the guanidine group on the side chain of arginine can be protonated to form a guanidinium group. The structure of the protonated form of arginine is: [ka] It is.

[0302] As used herein, the term "chirality" refers to "D" and "L" isomers of an amino acid or amino acid residue.

[0303] As used herein, the term "hydrophobic" refers to a moiety that does not dissolve in water or has minimal solubility in water. In general, neutral and / or non-polar moieties, or moieties that are predominantly neutral and / or non-polar, are hydrophobic. Hydrophobicity can be measured using one of the methods disclosed herein below.

[0304] As used herein, "aromatic" refers to an unsaturated cyclic molecule having 4n+2 pi-electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated cyclic molecule not included in the definition of aromatic.

[0305] "Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated, straight or branched hydrocarbon chain radical having from 1 to 40 carbon atoms and attached to the remainder of the molecule by a single bond. Alkyl containing any number of carbon atoms from 1 to 40 is included. Alkyl containing up to 40 carbon atoms is C1-C 40 Alkyl, alkyl containing up to 10 carbon atoms is C1-C 10 Alkyl, where an alkyl containing up to 6 carbon atoms is C1-C6 alkyl, and an alkyl containing up to 5 carbon atoms is 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 listed above for C1-C5 alkyl, but also includes C6 alkyl. C1-C 10 Alkyl includes all of the moieties listed above for C1-C5 alkyl and C1-C6 alkyl, but also includes C7, C8, C9 and C 10 Also includes alkyl. Similarly, C1-C 12 Alkyl includes all of the moieties described above, except that C 11 and C 12 Includes alkyl. C1~C 12 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0306] "Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated, straight or branched divalent hydrocarbon chain radical having from 1 to 40 carbon atoms.40 Non-limiting examples of alkylene include butylene ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted.

[0307] "Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 40 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, containing up to 10 carbon atoms, is C2-C 10 An alkenyl group containing up to 6 carbon atoms is C2-C6 alkenyl, and an 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 of the moieties listed above for C2-C5 alkenyl, but also includes C6 alkenyl. C2-C 10 Alkenyl includes all of the moieties listed above for C2-C5 alkenyl and C2-C6 alkenyl, but also includes C7, C8, C9 and C 10 Alkenyl is also included. Similarly, C2-C 12 Alkenyl includes all of the above moieties, except that C 11 and C 12 Includes alkenyl. C2~C 12Non-limiting examples of alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 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, nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-decenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.

[0308] "Alkenylene", "alkenylene chain" or "alkenylene group" refers to a linear or branched divalent hydrocarbon chain radical having from 2 to 40 carbon atoms and having one or more carbon-carbon double bonds. 40 Non-limiting examples of alkenylene include ethene, propene, butene, etc. Unless stated otherwise specifically in the specification, the alkenylene chain can be optional.

[0309] "Alkoxy" or "alkoxy group" refers to the -OR group, where R is alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl, as defined herein. Unless otherwise specifically stated in the specification, an alkoxy group can be optionally substituted.

[0310] "Acyl" or "acyl group" refers to the group -C(O)R, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, as defined herein. Unless otherwise specifically stated in the specification, acyl can be optionally substituted.

[0311] An "alkylcarbamoyl" or "alkylcarbamoyl group" is -OC(O)-NR a R b R a and R b are the same or different and are independently alkyl, alkenyl, aryl, heteroaryl, or R a R b may be taken together to form a cycloalkyl or heterocyclyl group, as defined herein. Unless otherwise specifically stated in the specification, an alkylcarbamoyl group may be optionally substituted.

[0312] An "alkylcarboxamidyl" or "alkylcarboxamidyl group" is -C(O)-NR a R b R a and R b are the same or different and are independently an alkyl group, an alkenyl group, an aryl group, a heteroaryl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, or a heterocyclyl group as defined herein, or R a R b can be taken together to form a cycloalkyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarboxamidyl group can be optionally substituted.

[0313] "Aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. In the present invention, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals 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 herein, the term "aryl" is meant to include aryl radicals that are optionally substituted.

[0314] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. In the present invention, the heteroaryl radical 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 radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, Examples of phenyl include isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 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.

[0315] The term "substituted" as used herein refers to any of the above groups (i.e., alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom of any of the groups has been replaced by a non-hydrogen atom, for example, but not limited to, a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl, alkoxy, ester groups; a sulfur atom in groups such as thiol, thioalkyl, sulfone, sulfonyl, sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, 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 bond (e.g., a double or triple bond) to a heteroatom, such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in groups such as imine, oxime, hydrazone, and nitrile. For example, "substituted" means that 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 SO2R h , -OC(=O)NR g R h , -OR g , -SR g -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2Rg , and -SO2NR g R h "Substituted" also includes any of the above groups in which one or more hydrogen atoms have been replaced with -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h In the above, R g and R h are the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further refers to any of the above groups where one or more atoms are replaced by amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl groups. "Substituted" can also refer to an amino acid in which one or more atoms on the side chain are substituted with an alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl. Additionally, each of the aforementioned substituents can also be optionally substituted with one or more of the above-mentioned substituents.

[0316] As used herein, "activating group" refers to an electron donating group that enhances the stability and overall reactivity of a compound / intermediate. The activating group can be, for example, an NHS ester, or PhSiH3.

[0317] As used herein, "subject" refers to an individual. Thus, "subject" can include domestic animals (e.g., cats, dogs, etc.), livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" can also include mammals (e.g., primates or humans). Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0318] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0319] "Reduce" or other forms of this word, such as "reducing" or "reduction", refers to the reduction of an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, although it is understood that a standard or relative value is not necessarily referenced. For example, "reducing tumor growth" refers to reducing the growth rate of a tumor compared to a standard or control (e.g., an untreated tumor).

[0320] The term "treatment" refers to the medical management of a patient with the intent of curing, ameliorating, stabilizing, or preventing a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of an associated disease, pathological condition, or disorder. In addition, the term includes symptomatic treatment, i.e., treatment designed for the relief of symptoms rather than a cure of a disease, pathological condition, or disorder, preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of an associated disease, pathological condition, or disorder, and adjunctive treatment, i.e., treatment used to supplement another specific therapy aimed at ameliorating an associated disease, pathological condition, or disorder.

[0321] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such an amelioration requires only a reduction or alteration, not necessarily elimination.

[0322] The term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0323] The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

[0324] As used herein, the term "pharmaceutical acceptable carrier" refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to 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.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Suitable inert carriers can include sugars, such as lactose.

[0325] [Table 10-1] [Table 10-2] EXAMPLES

[0326] Manufacturing method The compounds described herein can be prepared by various methods known to those skilled in the art of organic synthesis, or variations thereof as will be understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art. All reactions were carried out in solution (using a solvent or mixture of solvents) or neat (no solvent required at all).

[0327] Modification of the compounds described herein includes the addition, removal, or movement of various components as described for each compound. Similarly, the chirality of the molecule can be changed if one or more chiral centers are present in the molecule. Furthermore, synthesis of the compounds can include protection and deprotection of various chemical groups. Those skilled in the art can determine the use of protection and deprotection, and the selection of appropriate protecting groups. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0328] Starting materials and reagents used in preparing the compounds and compositions of the disclosure are available from Aldrich Chemical Corporation (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), Abercrombie & Fitch (New York, NY), and other companies. These compounds are available from commercial suppliers such as Eppendorf Ingelheim (Ingelheim, Germany), AstraZeneca (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 may be purchased from 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 Supplementals (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) according to procedures known to those skilled in the art.Other materials disclosed herein, such as the pharmaceutical carriers, can be obtained from commercial sources.

[0329] The reactions to 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, at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of two or more solvents. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) It can be monitored by infrared spectroscopy, spectrophotometry (eg, UV-visible), or mass spectrometry, or by chromatography, for example, high performance liquid chromatography (HPLC) or thin layer chromatography.

[0330] Example 1: Linear peptide synthesis (linker synthesis) Linear peptide synthesis - Fmoc-PEG on Wang resin 12 -CH2CH2COOH loading Add DCM to the glass peptide synthesis vessel containing Wang resin, allow the resin to swell, and drain the solvent. Add DCM to the resin. Add the desired amount of Fmoc-PEG. 12 -CH2CH2COOH is weighed into a vessel and dissolved in a minimum amount of DMF, the resulting solution is transferred to the resin vessel and pyridine is added. 2,6-Dichlorobenzoyl chloride is added and the resin suspension is stirred until the reaction is judged to be complete.

[0331] Fmoc deprotection Add DMF to the glass peptide synthesis vessel containing the resin-bound peptide, allow to swell, and drain. Add 20% piperidine in DMF to completely cover the resin and allow to react. Drain and wash sequentially with DMF, DCM, and more DMF.

[0332] Coupling of each amino acid (AA) Weigh out Fmoc-AA into a container and add an additive (e.g., Oxyma). Dissolve Fmoc-AA and additive in DMF until completely dissolved. Add DIC, mix, and allow to stand at room temperature. Add preactivated Fmoc-AA solution onto the resin and mix.

[0333] Example 2: Cyclic Peptide Synthesis Loading of the first Fmoc-AA onto 2-chlorotrityl chloride (CTC) resin Add DCM to the glass peptide synthesis vessel containing the CTC resin to swell the resin and drain the solvent. Weigh out the desired Fmoc-AA and dissolve in DMF. Add the AA solution to the resin. Add DIPEA and mix. When the reaction is judged complete, quench with methanol and wash the resin. Fmoc deprotection: as described in Example 1.

[0334] Methyltrityl (Mtt) deprotection of exocyclic peptides 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. Coupling of each AA: as described in Example 1.

[0335] Allyl ester deprotection for cyclic peptides Add a resin bed volume of DCM to a glass peptide synthesis vessel containing the linear precursor of an N-terminal Fmoc-protected and C-terminal allyl-protected cyclic peptide. Allow the resin to swell at room temperature. Weigh out 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 sequentially with DCM and DMF. Weigh out SDDC·(H2O)3 (sodium diethyldithiocarbamate trihydrate) and dissolve in DMF. Add the mixture to the resin and mix. Once complete, wash the resin with DCM and DMF.

[0336] Cleavage from the solid support - Cyclic peptides A mixture of HFIP:DCM is prepared and added to the peptide synthesis vessel containing the resin-bound cyclic peptide and mixed. The resin is washed with a minimum amount of DCM. The filtrate is evaporated to a minimum volume and triturated with cold MTBE. The resulting suspension is filtered and the cake is washed with MTBE.

[0337] Example 3: Coupling of cyclic and linear peptides Dissolve the crude cyclic peptide and HOAt in DMF. Add DIC, mix and let stand at room temperature. Add the preactivated cyclic peptide solution onto the resin and mix. Shake the reaction mixture at room temperature.

[0338] Cleavage from solid support A mixture of TFA:DCM 95:5 (v / v) is prepared and added to a glass peptide synthesis vessel containing the resin-bound cyclic peptide. The contents are mixed and filtered. The resin is washed with a minimum amount of DCM. The filtrate is evaporated to a minimum volume and triturated with cold MTBE. The resulting suspension is filtered and the cake is washed with MTBE. The resulting crude peptide can be purified by preparative RP-HPLC and further lyophilized.

[0339] Example 4: Synthesis of phosphorodiamidate morpholino oligomers (PMOs) Loading of PMO monomers onto solid supports: Aminomethyl polystyrene resin is suspended in NMP and the resin is allowed to swell. The resin is filtered to remove the NMP and washed with DCM, 5% DIPEA in DCM, and DCM. The PMO monomer functionalized as an activated succinate ester is dissolved in anhydrous NMP and added to the resin. The reaction mixture is stirred at room temperature. The resin is filtered and washed with NMP and DCM. The resin is dried and the loading is tested by trityl quantification. A 0.4 M solution of benzoic anhydride and NEM in NMP is prepared. The benzoic anhydride / NEM solution is added to the resin and stirred at room temperature. The resin is filtered and washed twice with NMP, twice with IPA, and twice with DCM. The resin is dried.

[0340] Determination of resin loading by trityl quantification Place the dry resin (50-100 mg) in a fritted syringe and swell it with DCM. Remove the DCM and add the 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 the filtrates, dilute the solution with water and measure the absorbance value 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: weight of resin, g; ε: molar extinction coefficient = 32500).

[0341] Resin swelling for PMO synthesis Before starting the PMO synthesis, the resin functionalized with the PMO monomer was suspended in NMP and allowed to swell for 2 h.

[0342] Trityl deprotection The resin is washed with DCM. The resin is treated with a solution of 4-cyanopyridine, TFA (CYTFA) in 80:20:1 DCM / TFE / EtOH.

[0343] Neutralization Treat the resin with a neutralization solution containing 5% DIPEA in 1:3 IPA / DCM. Wash the resin with DCM and anhydrous (1,3-dimethyl-2-imidazolidinone) DMI. Coupling Prepare a solution containing 0.2M PMO monomer and 0.4M NEM in anhydrous DMI. For the first coupling, use 5 equivalents of PMO monomer and NEM. For the next coupling, use 3 equivalents of PMO monomer and 6 equivalents of NEM up to the 10th position of the sequence. For couplings from the 10th position to the 20th position, use 4 equivalents of PMO monomer and 8 equivalents of NEM. For couplings at positions 20 and above, use 5 equivalents of PMO monomer and 10 equivalents of NEM. Add the coupling solution to the resin and react at room temperature up to 45°C. Coupling reactions performed at 45°C have been found to be more efficient and are completed in a shorter time frame (2-4 hours). Follow the coupling reaction by chloranil test (Pept Res., 8(4):236-7.1995). Wash the resin with DCM and 30% TFE in DCM. The resin can be stored overnight in a solution of 30% TFE in DCM. When the PMO sequence is complete, wash the resin four times with IPA and dry the resin.

[0344] Cleavage and deprotection The dried detritylated PMO-bound resin is treated with NMP and allowed to swell for 3 hours at 30 °C. The resin is drained and a cleavage cocktail containing 1.0-1.4 M DTT, 2.0-2.8 M DBU in NMP is added to the resin and allowed to react for 2 hours at 40 °C. The filtrate is collected in a clean filter flask. The cleaved PMO solution is diluted with chilled ammonium hydroxide to form a homogenous mixture. The PMO solution is placed in a pressure flask and incubated at 50 °C for 18 hours at 120 rpm. This process is repeated for the second round of cleavage as detailed above. Both cleavage solutions are collected and diluted with water.

[0345] PMO purification The diluted PMO cleavage solution is concentrated by tangential flow filtration (TFF). Diafiltration is performed with water until the conductivity is less than 300 μS / cm. The desalted PMO is concentrated to an appropriate volume for anion exchange (AEX) purification. The crude PMO is loaded and purified using an AEX column (TOYOPEARL SuperQ-650S) with the following gradient buffers: Buffer A = 10-25 mM NaOH; Buffer B = 10-25 mM NaOH + 0.5-1 M NaCl. Pure fractions were identified by ion-pair reversed-phase chromatography (IP-RP) and pooled for desalting by TFF. IP-RP method was performed using a C18 column and the following gradient buffers: Buffer A = 55% methanol containing 10 mM triethylamine (TEA), 4.3-8.6 mM Na3PO4, 10 mM dodecyltrimethylammonium bromide (DTMA), Buffer B = 60% acetonitrile containing 10 mM TEA, 4.3 mM Na3PO4, 10 mM DTMA. Diafiltration is performed until the conductivity of the pure PMO solution is less than 350 μS / cm. The desalted pure PMO is concentrated and lyophilized to obtain a white powder. The purity of the pure PMO is determined by the above IP-RP method.

[0346] Example 5: EEV-PMO synthesis - Method 1 Conjugation of EEV to PMO PMO is dissolved in DMSO. Separate solutions of EEV in DMSO, HATU in DMSO, and DIPEA in DMSO are prepared. To the solution of dissolved PMO, DIPEA, HATU, and EEV solutions are added. Reaction progress is analyzed by CEX or RP-HPLC.

[0347] TFA deprotection In the first generation conditions, the conjugation reaction is diluted with water and mixed. The mixture is diluted with a solution containing 25 mM NaOH and 0.2 M KCl and mixed. The progress of the reaction is analyzed by CEX or RP-HPLC. Once deprotection is complete, the reaction is diluted with 0.5 M NaH2PO4 buffer and then purified.

[0348] In the second generation conditions, the mixture is diluted with a solution containing 320 mM NaOH and mixed. The progress of the reaction is analyzed by CEX or RP-HPLC. Once the deprotection is complete, the reaction is diluted with 0.5 M NaH2PO4 buffer and then purified.

[0349] Purification of EEV-PMO The diluted PMO cleavage solution is concentrated by tangential flow filtration (TFF). The desalted PMO is concentrated to an appropriate volume for cation exchange (CEX) purification. The crude PMO is loaded and purified using a CEX column with the following gradient buffers: Buffer A = water, or 10-25 mM NaH2PO4 in 15-20% acetonitrile; Buffer B = 0.5-1 M NaCl, or 10-25 mM NaH2PO4, 15-20% acetonitrile containing 0.5-1 M NaCl. Pure fractions were identified by ion-pair reversed-phase chromatography (IP-RP) and pooled for desalting by TFF. The desalted pure PMO is concentrated and lyophilized to obtain a white powder. The purity of the pure PMOs is determined by the CEX method performed using a strong cation exchange (SCX) column and the following gradient buffers: Buffer A = 80% water and 20% acetonitrile containing 24 mM H3PO4; Buffer B = 75% water and 25% acetonitrile containing 24 mM H, 2.0 M LiCl.

[0350] Example 6: Optimization of reaction conditions Optimization of linear peptide fragment synthesis. Fmoc-proline is replaced by Ac-proline to reduce the number of synthetic steps to prepare linear peptide fragments. In the first generation protocol, Fmoc-proline is coupled as the last amino acid of the linear peptide sequence. This is followed by Fmoc deprotection, after which the free amine of the proline residue is protected with an acetyl group (by reaction with acetic anhydride). This protocol can be performed manually for larger scale reactions (>1 g) and automatically using smaller scale (approximately 600 mg of 0.4 mmol / g input material per 40 mL reactor).

[0351] Suppression of epimerization during dipeptide coupling for the synthesis of cyclic peptides: Alternative protecting groups investigated as replacements for Fmoc for the dipeptide Gly-Arg(Pbf). Pht, Dde, and Fmoc / Dmb were investigated for use in cyclic peptide synthesis to assess epimerization throughout the synthetic process and in the final product.

[0352] [Table 11]

[0353] [Table 12]

[0354] [Table 13]

[0355] Dipeptide coupling screening to suppress epimerization during the synthesis of cyclic peptides. After adjusting the protocol to include coupling of pre-made dipeptides (initially intended to limit DKP by-products), alternative dipeptide coupling reagents / reaction conditions were evaluated. Epimerization was evaluated after dipeptide coupling, and optimized conditions were carried forward.

[0356] [Table 14] Optimization of deprotection of allyl esters (Figure 1A)

[0357] [Table 15]

[0358] Fmoc deprotection screening for linear peptide synthesis Alternative deprotection conditions were found that could replace the 20% piperidine / DMF deprotection without affecting the integrity / purity of the product. The milder conditions resulted in complete Fmoc removal, but impurities such as desPreg that typically result from harsh deprotection were not observed. 12 and desPreg 12 Lys(Mtt) was recognized.

[0359] [Table 16]

[0360] The above table lists the alternative Fmoc deprotection conditions in detail. The deprotection filtrate was analyzed by UV to quantify the removed Fmoc. In addition, LCMS was performed to assess the completeness / quality of the deprotected linear peptide product. 20%, 10%, 5% and 2% piperidine in DMF each gave similar results, indicating that reduction of piperidine can still completely deprotect Fmoc-protected peptides. The use of an organic base (DBU) and addition of acid were also performed to adjust the basicity of piperidine. Based on UV and LCMS analysis, the completeness of the linear peptide product was comparable to the control, so these alternative methods can also be used for Fmoc deprotection. No deletion products were detected by LCMS. Optimization of cyclization (Figure 1A)

[0361] [Table 17]

[0362] During the synthesis of the cyclic peptide fragments, additional cyclization reagents were screened. Cyclization screening was performed at two resin loading levels (high / low) to evaluate differences in product, epimer, and dimer formation.

[0363] [Table 18]

[0364] [Table 19]

[0365] In most conditions, the desired product formation and cyclization was completed with various amounts of epimer and dimer by-products. Unexpectedly, lower resin loadings resulted in more dimer formation in most cases, but less epimer.

[0366] Optimization to reduce dimerization during cyclic peptide synthesis To assess and minimize dimer formation during cyclization of linear precursors and its reproducibility on a large scale, a resin loading screen was performed.

[0367] [Table 20]

[0368] Screening of amide conjugation conditions All conditions were evaluated at room temperature with 2 mM PMO (50 mg / mL in DMSO). All reagents were prepared in DMSO: 1. DIC (300 mM DMSO) 2. Oxyma (300 mM DMSO) 3. PyAOP (300 mM DMSO) 4. HATU (300 mM DMSO) 5. DIPEA (300 mM DMSO) 6. EEV (100 mM DMSO) Prior to injection, a dilution / quenching treatment with 1:1 ACN / H2O + 0.1% TFA was performed.

[0369] [Table 21] Screening of Tfa deprotection conditions

[0370] [Table 22]

[0371] [Table 23]

[0372] [Table 24]

[0373] Screening of Tfa deprotection of EEV-PMO Alternative EEV-PMO deprotection conditions were evaluated using a variety of aqueous, organic, and amphipathic bases; amphipathic bases that result in fewer PMO regenerations and higher product purity, as well as more stringent conditions for faster deprotection without sacrificing purity, have also been screened.

[0374] [Table 25-1] [Table 25-2] [Table 25-3]

[0375] [Table 26]

[0376] Screening of Linear Cyclic Peptide Coupling Reactions A variety of conditions were used for the linear cyclic coupling to be screened. Experiments were performed at 0.6 mmol / g support material and at 0.4 mmol / g.

[0377] [Table 27]

[0378] [Table 28]

[0379] [Table 29]

[0380] [Table 30]

[0381] Mechanistic studies of EEV-PMO conjugation and deprotection reactions A time course evaluation of EEV-PMO conjugation / deprotection reactions was performed comparing the second and first generation protocols. Conjugation / deprotection reactions using PMOs of different purity, as well as deprotection of purified and protected EEV-PMO were also tested.

[0382] [Table 31]

[0383] [Table 32]

[0384] [Table 33]

[0385] [Table 34]

[0386] [Table 35]

[0387] [Table 36]

[0388] [Table 37]

[0389] [Table 38]

[0390] Experiments were performed to study the second generation conjugation / deprotection protocol (40 equivalents of NaOH), the first generation conjugation / deprotection protocol (conjugation stoichiometry is slightly different, deprotection uses 12.5 mM NaOH, 1 M KCl), deprotection of purified and protected EEV-PMO by the second generation method, and second generation conjugation / deprotection with PMO of various purity (four purity levels). In all of these experiments, both the conjugation and deprotection reactions were tracked and compared using UPLC integration and purity by LCMS mass spectrometry.

Claims

1. A method for preparing linear peptides, the following: (i) Fmoc-PEG １２ -CH ２ CH ２ COOH is coupled to Wang resin, as follows: 【Chemistry 1】 (In the formula, 【Chemistry 2】 (represents the aforementioned resin) The process of obtaining: (ii) A step of removing the Fmoc protecting group to obtain the reaction product; and (iii) A step of sequentially coupling the reaction product with Fmoc-protected amino acids (Fmoc-AA) to obtain a linear peptide. The method, including the method described above.

2. The method according to claim 1, wherein step (i) is carried out in the presence of 2,6-dichlorobenzoyl chloride and pyridine.

3. Step (i) involves adding 1 to 2 equivalents of Fmoc-PEG in the presence of 2 to 4 equivalents of 2,6-dichlorobenzoyl chloride and 4 to 8 equivalents of pyridine. １２ -CH ２ CH ２ The method according to claim 1, comprising coupling COOH to Wang resin.

4. The method according to claim 1, further comprising capping after step (i).

5. The method according to claim 4, wherein the capping is carried out using acetic anhydride in the presence of diisopropylethylamine (DIPEA).

6. The method according to claim 1, wherein step (ii) is carried out in the presence of 20% piperidine in DMF.

7. The method according to claim 1, wherein step (iii) is carried out in the presence of oxima, DMF and DIC.

8. The method according to claim 1, wherein step (iii) comprises deprotecting each sequentially bound Fmoc-protected amino acid.

9. The method according to claim 8, wherein the deprotection is carried out in the presence of piperidine.

10. The linear peptide is as follows: 【Transformation 3】 (In the formula, Mtt represents a methyltrityl protecting group.) The method according to claim 1, including the method described in claim 1.

11. The method according to claim 10, further comprising the step of acetylating the terminal pyrrolidinyl nitrogen.

12. The method according to claim 11, wherein acetylation is carried out in the presence of acetic anhydride.

13. Acetylated linear peptides are as follows: 【Chemistry 4】 The method according to claim 11 or 12, including the method described in claim 11 or 12.

14. A method for producing a cyclic peptide, the following: (a) Load Fmoc-Glu-OAll into 2-chlorotrityl chloride (CTC) resin and perform the following: 【Transformation 5】 (In the formula, 【Transformation 6】 (This represents resin.) The process of obtaining: (b) a step of removing the Fmoc protecting group to obtain the first reaction product; and (c) The first reaction product is coupled with Fmoc-Gly-Arg(Pbf)-OH to obtain the following: 【Transformation 7】 The method, which includes the step of obtaining [something].

15. Furthermore, the following: 【Transformation 8】 A step of deprotecting to obtain a second reaction product, and The second reaction product is sequentially coupled with an Fmoc-protected amino acid (Fmoc-AA) to obtain the following: 【Chemistry 9】 process to obtain The method, including the method described above.

16. Furthermore, the following formula: 【Chemistry 10】 By removing the allyl protecting group from the compound represented by, below: 【Chemistry 11】 Obtaining this, remove Fmoc, below: 【Chemistry 12】 The process of obtaining; and The following formula: 【Chemistry 13】 The compound represented by the following is cyclized: 【Chemistry 14】 The method according to claim 15, further comprising the step of obtaining

17. The method according to claim 16, wherein the cyclization is carried out in the presence of PyOxim, Oxyma, and DIPEA.

18. Furthermore, the following formula: 【Chemistry 15】 The resin is cut from the compound represented by the following: 【Chemistry 16】 The method according to claim 16, further comprising the step of obtaining a compound represented by [the specified compound].

19. A method for coupling a linear peptide with a cyclic peptide, formula: 【Chemistry 17】 A cyclic peptide represented by formula: [Chemistry 18] Coupling to a linear peptide represented by the following formula: 【Chemistry 19】 The method for obtaining a compound represented by the above.

20. The method according to claim 19, wherein the coupling is carried out in the presence of 3 equivalents of 3H-[1,2,3]triazolo[4,5-b]pyridine-3-ol (HOAt) and 3.3 equivalents of N,N'-diisopropylcarbodiimide (DIC).

21. The method according to claim 20, further comprising adding 3 equivalents of HOAt and 3.3 equivalents of DIC.

22. Furthermore, the following formula: 【Chemistry 20】 The resin is cut from the compound represented by the following formula: 【Chemistry 21】 The method according to claim 19, which includes the step of obtaining

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