Modified Amino Acids and Their Use

Abstract

The present disclosure relates to modified amino acids modified to change their physicochemical properties. The present disclosure further relates to the use of such modified amino acids to provide peptides having modified physicochemical properties, particularly cell membrane permeable peptides.

Description

Technical Field

[0001] The present disclosure relates to modified amino acids modified to change their physicochemical properties. The present disclosure further relates to the use of such modified amino acids to provide peptides having modified physicochemical properties, particularly cell-penetrating peptides.

Background Art

[0002] Cell membranes can be a troublesome obstacle to the development of small molecules and biological drug entities. Cell membranes, composed of phospholipids, membrane-bound glycoproteins, and fluid modifiers (e.g., cholesterol), are responsible for maintaining a non-equilibrium state within the cell compared to its extracellular environment. Recent progress in drug discovery has focused on understanding how the physicochemical properties of small molecules affect cell uptake, but equivalent design criteria for the development of larger molecular weight biopolymers (biological) drugs such as therapeutic proteins and oligonucleotides can be more difficult to achieve. As a result, the development of delivery vehicles, either conjugated to the drug of interest or used as part of a multi-component nanovector (e.g., nanoparticles, liposome formulations), can be used to expand the types of biomolecules that can be targeted by biopharmaceuticals and non-Lipinski compliant small molecules.

[0003] Cell-penetrating peptides (CPPs) are one example of a class of delivery vehicles that can be used to deliver drugs into cells. Cell-penetrating peptides have been used as biotechnology tools, however, the emergence of toxicity associated with CPPs has hindered their use in clinical applications (Guidotti et al, Cell-Penetrating Peptides: From Basic Research to Clinics. Trends Pharmacol. Sci. 2017, 38, 406-424). Thus, one challenge is the development of CPP scaffolds that maintain high levels of uptake and intracellular distribution but with reduced levels of toxicity. Most of the structural features of CPPs are such that their sequences typically contain multiple arginine (Arg) residues (see, for example, Fig. 1a). The positively charged guanidinium side chains of Arg residues form stable branched non-covalent ion-pair interactions with negatively charged phospholipids and sulfated glycoproteins. In combination with Arg residues, a balance of positive charge and hydrophobicity is typically required for efficient translocation across cell membranes and to avoid endosomal entrapment. However, the use of multiple positively charged Arg residues in CPPs can also be a source of toxicity. As a result, extensive efforts have been directed towards identifying novel CPPs that use primarily naturally occurring amino acids in order to reduce the toxicity associated with high Arg content while still maintaining the ability to enhance the uptake of therapeutic payloads. Some examples are shown in Fig. 1b (Dinca, A.; Chien, W.-M.; Chin, M. T. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease. Int.l J. Mol. Sci. 2016, 17, 263; Park, S. E.; Sajid, M. I.; Parang, K.; Tiwari, R. K. Cyclic Cell-Penetrating Peptides as Efficient Intracellular Drug Delivery Tools. Mol. Pharmaceutics 2019, 16, 3727-3743; and Silva, S.; Almeida, A. J.; Vale, N. Combination of Cell-Penetrating Peptides with Nanoparticles for Therapeutic Application: A Review. Biomolecules 2019, 9).

[0004] Examples of modified and / or unnatural amino acids are disclosed in Patent No. 06495714 B2 (Makoto et al.), International Publication No. 2020210916 (Guay et al.), and U.S. Patent No. 10253099 B2 (Strieker et al.). Further examples can be found in Behrouz et al, Tetrahedron Lett. Vol 81 2021, page 153342, Hosseini et al, Molecular Pharmacology, Vol. 80(4), 2011, pages 585-597, International Publication No. 2022014704 (Santen Pharmaceutical Co Ltd), International Publication No. 96 / 40743 (Cor Therapeutics), Zhang et al, Bioorganic & Medicinal Chemistry, Vol. 10(11), 2002, pages 3401-3413, International Publication No. 2022 / 129047 (H Lundbeck AS), International Publication No. 99 / 31061 (Merck&Co.Inc.), International Publication No. 2009 / 051397 (Choongwae Pharma Corp.), International Publication No. 2020 / 006315 (Pliant Therapeutics Inc.), European Patent Application Publication No. 2995612 (University of Strasbourg). However, there remains a need to identify further modified amino acids and cell membrane permeable peptides to address some of the challenges noted in the art.

Summary of the Invention

[0005] The present disclosure is based on the finding that modified amino acids can be used to provide peptides (e.g., cell membrane permeable peptides) with improved properties. In particular, targeted modifications to change the physicochemical properties of the side chains of some amino acids can result in improved cellular uptake, intracellular distribution, and / or reduced toxicity for cell membrane permeable peptides containing residues derived from these modified amino acids. In fact, as described above, a number of cell membrane permeable peptides typically contain multiple arginine residues. A novel class of modified amino acids has been identified for the purpose of addressing some of the issues associated with the use of multiple arginine residues. In particular, the inventors herein aim to mimic the naturally occurring guandinium group present in the arginine residue, but have identified a cohort of modified amino acids that are specifically modified to alter some of the physicochemical properties (e.g., basicity, hydrophobicity, amphiphilicity, pKa, lipophilicity, etc.). For example, the modified amino acids disclosed herein can include increased lipophilicity and / or hydrophobicity compared to arginine. These modified amino acids have been found to provide enhanced cellular uptake, intracellular distribution and / or reduced toxicity when used to replace arginine in a number of cell membrane permeable peptides.

[0006] Specifically, targeted modification of the side chain of arginine can alter the physicochemical properties and increase the "drug-like" properties of the cell membrane permeable peptide containing this amino acid and / or the amino acid residue derived from this amino acid. For example, it has been found that the biological equivalent substitution of the guandinium group of arginine by an amidine group (or an amidine mimetic group) changes the lipophilicity and / or basicity of the amino acid. Alternatively, other targeted modifications can be made to the side chain of arginine to change the lipophilicity and / or basicity of the guandinium group at the end of the side chain. The use of such modified amino acids as a substitution for arginine in a CPP can significantly enhance cellular uptake and intracellular distribution and may not show an adverse effect on toxicity.

[0007] As an example, the modified amino acid is specifically modified to have a lower pKa than arginine (which has a pKa of about 12.5 at 25 °C). Thus, the modified amino acids of the present disclosure can have a pKa of less than about 12.5 at 25 °C. As a further example, the modified amino acids of the present disclosure can have a pKa of from about 4 to about 12, or from about 5 to about 11 at 25 °C.

[0008] Additionally or alternatively, the modified amino acid is specifically modified to increase its hydrophobicity compared to arginine (which has a LogD or cLogD of about -3.5 at pH 7.4). Thus, the modified amino acids of the present disclosure can have a LogD or cLogD greater than about -3.5 at pH 7.4. As a further example, the modified amino acid can have a LogD or cLogD of about -3 to about 2, or about -2 to about 1 at pH 7.4. As used herein, the LogD value is a partition coefficient that can be used to provide an indication of the lipophilicity of an ionizable compound at a particular pH. As used herein, in the cLogD value, the "c" indicates that the value has been calculated.

[0009] As used herein, a cell-penetrating peptide (CPP) can be meant to be a peptide that can facilitate the cellular uptake and / or distribution of a drug of interest (sometimes referred to as a "payload" or "cargo"). Typically, a cell-penetrating peptide can comprise from about 2 to 100 amino acid residues, such as from about 5 to 50 or from about 7 to 20 amino acid residues. In some examples, a cell-penetrating peptide can comprise from about 2 to 30 amino acid residues. Cell-penetrating peptides can be broadly classified into several categories including cationic, amphiphilic, membrane-directed, and hydrophobic. Without wishing to be bound by theory, hydrophilicity and hydrophobicity are thought to correlate with their different modes of interacting with the membrane bilayer. Cationic CPPs can be rich in arginine, lysine, and histidine residues, particularly arginine residues. Thus, the modified amino acids as described herein may find particular use in cationic CPPs. As used herein, a cationic CPP has a net positive charge. Without wishing to be bound by theory, this net positive charge is understood to be important for its ability to cross the cell membrane. The amino acids disclosed herein can provide their charge while exhibiting altered basicity and / or pKa, and thus may find particular utility in cationic CPPs.

[0010] The agent of interest can associate with the peptide either through a chemical linkage (e.g., a covalent bond) and / or through non-covalent interactions using methods known in the art. One of ordinary skill in the art will understand that the linker functions to tether the agent of interest to the cell membrane permeable peptide while allowing both of their respective functions to be exerted and / or also enabling binding to its target. In particular, the linker functions to tether the agent of interest to the cell membrane permeable peptide while reducing the potential for the cell membrane permeable peptide to interfere with, disrupt, and / or inhibit (i) the binding of the agent of interest to any target; and / or (ii) the activity or desired function of the agent of interest. Additionally or alternatively, the linker functions to tether the agent of interest to the cell membrane permeable peptide while reducing the potential for the agent of interest to interfere with, disrupt, and / or inhibit the binding and / or interaction of the cell membrane permeable peptide (e.g., its function in altering, facilitating, and / or promoting the delivery of the agent of interest to a cell).

[0011] In some examples, the agent of interest can associate with the cell membrane permeable peptide by a covalent linkage that can include a moiety that contains an ester, amide, disulfide, or thioester group. The linker is an amino-substituted C1-C 20 alkyl carboxylic acid (e.g., an amino-substituted C1-C 10It can be derived from amino-substituted carboxylic acids such as (alkyl carboxylic acid). In some examples, the linker can be derived from 6-aminohexanoic acid. As an example, the agent of interest can associate with the peptide using a maleimide, succindyl ester or isothiocyanate linkage (e.g., such as shown in Figure 4B of Jones and Sayers, “Cell entry of cell penetrating peptides: tales of tails wagging dogs”, Journal of Controlled Release, 161(2012), 582-591, the content of which is incorporated herein by reference. In this figure (and with reference to the present disclosure), the term “Fluor” can represent any agent of interest).

[0012] As a further example, the agent of interest can associate with the cell-penetrating peptide by a common linker selected from one of the following structures: (i)

Chemical formula

[0013]

Chemical formula

Chemical formula

[0014]

Chemical formula

Chemical formula

[0015] In each of the above structures, the junctions where the wavy lines intersect indicate the points of attachment of the structure to the cell membrane-permeable peptide and the agent of interest. The agent of interest can be of a type that requires delivery to cells and can be, for example, a therapeutic agent, a diagnostic agent, or a contrast agent. The agent of interest can be a biological molecule (e.g., a nucleic acid-based molecule (e.g., siRNA, antisense oligonucleotide, DNA, plasmid, etc.), an antibody, a polysaccharide, a polypeptide, or a protein). In some examples, the agent of interest can be a particle (e.g., a nano-sized particle) or a compound.

[0016] Further information regarding cell membrane-permeable peptides and their use in the delivery of payloads to cells can be found in Jones and Sayers, “Cell entry of cell penetrating peptides: tales of tails wagging dogs”, Journal of Controlled Release, 161(2012), 582-591 and Falanga et al, “The world of cell penetrating: the future of medical applications”, Future Medicinal Chemistry, 2020, 12(15), 1431-1446, the contents of which are incorporated herein by reference.

[0017] Accordingly, modified amino acids according to formula (I) are described: [Chemical formula] (wherein, R 1 is H or a protecting group; X is absent or may be a C1-C6 alkyl which may be substituted, a C1-C6 alkyl-NR 2 (C=O) and a -C1-C6 alkyl-(C=O)NR 2selected from; R 2 is selected from H, optionally substituted C1-C6 alkyl, and a protecting group; wherein A is: (i)

[0018]

Chemical formula

Chemical formula

[0019] In some examples, at least one modified amino acid does not include the following structure:

Chemical formula

[0020] In the above formula (I), the stereochemistry at the position of the asterisk ( * ) can be (R) or (S). In some examples, the amino acid may be provided with a single chiral configuration at this position (e.g., as a single enantiomer or a substantially pure single enantiomer). In other examples, the amino acid may be provided as a racemic mixture (e.g., a mixture containing equimolar amounts of both enantiomers). The amino acid of formula (I) may be provided in a substantially single optical form (e.g., the (+) (dextrorotatory) or (-) (levorotatory) form). As already mentioned, the modified amino acid according to formula (I) may have a pKa of less than about 12.5 at 25°C. As a further example, the modified amino acid according to formula (I) may have a pKa of about 4 to about 12, or about 5 to about 11 at 25°C. Additionally or alternatively, the modified amino acid according to formula (I) may have a LogD or cLogD of greater than about -3.5 at a pH of 7.4. As a further example, the modified amino acid according to formula (I) may have a LogD or cLogD of about -3 to about 2, or about -2 to about 1 at a pH of 7.4.

[0021] Additionally or alternatively, the modified amino acid according to formula (I) can comprise chemical moieties of suitable length and / or shape in the X and A moieties to provide a vector projection of an amidino or guanidino functional group (or an amidine-like motif) similar to that of the guanidino group of an arginine amino acid. In particular, as used herein, a vector projection can mean the direction and distance in space of an amidino or guanidino functional group (or an amidine-like motif) of the modified amino acid relative to the central carbon atom on the amino acid basic structure (e.g., the backbone α-carbon of the amino acid). In other words, a vector projection can mean the direction and distance of these groups from the backbone of the amino acid when incorporated into a peptide. As described above, this vector projection may be similar to that of the guanidino group in arginine relative to the amino acid basic structure / the central carbon atom on the backbone of this amino acid when incorporated into a peptide. As will be understood by those skilled in the art, this vector projection can be controlled by appropriate selection and combination of chemical moieties (e.g., those with restricted degrees of freedom such as rings and / or unsaturated groups) and / or chain lengths (e.g., the number of atoms in the chain and / or between the amidino or guanidino group (or amidine-like motif) and the amino acid backbone).

[0022] In some examples, the vector projection of an amidino or guanidino functional group (or an amidine-like motif) can project at least about 7 or at least about 8 angstroms from the backbone α-carbon of the amino acid. In a preferred example, the vector projection of an amidino or guanidino functional group (or an amidine-like motif) can project about 9 angstroms from the backbone α-carbon of the amino acid. For example, the vector can project from about 9 angstroms to about 11 angstroms from the backbone α-carbon. In some examples, there can be about 6 to about 12 linking atoms, about 7 to about 11 linking atoms, or about 8 to about 10 linking atoms between the amidino or guanidino functional group (or amidine-like motif) and the backbone α-carbon atom of the amino acid functional group. When an amidino functional group or an amidine-like motif is present, such a structure can include a hydrogen bond donor group and a hydrogen bond acceptor group that are located about 2 angstroms to about 6 angstroms from each other.

[0023] In some examples, the modified amino acid can include (S) stereochemistry. Thus, in some examples, formula (I) can be represented by the following formula:

Chemical formula

[0024] As used herein, the term "alkyl" means a straight-chain or branched-chain hydrocarbyl group. The chain can be saturated or unsaturated, and for example, in some cases, the chain can include one or more double or triple bonds. As used herein, "C1-C 20 alkyl" can be selected from straight-chain or branched-chain hydrocarbyl groups containing 1 to 20 carbon atoms. As used herein, "C1-C 10"Alkyl" can be selected from straight-chain or branched-chain hydrocarbyl groups containing 1 to 10 carbon atoms. As used herein, "C1-C6 alkyl" can be selected from straight-chain or branched-chain hydrocarbyl groups containing 1 to 6 carbon atoms. Representative examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, neohexyl, etc. When a C1-C6 alkyl group is substituted, any hydrogen atom, CH3, CH2 or CH group can be replaced by a substituent, provided that the valence is satisfied.

[0025] As used herein, a protecting group can mean any type of group or chemical moiety that can help prevent an atom to which it is attached, typically nitrogen or oxygen, from participating in unwanted reactions. In particular, the use of protecting groups can be important for controlling the reactions of reactive groups on the side chains or termini of amino acids during peptide synthesis. Suitable protecting groups include side-chain protecting groups and amino or N-terminal protecting groups. In particular, when R 1 is a protecting group in the above formula (I), it can be considered an N-terminal protecting group. In examples where one or more of R 2 , R 3 and R 5 are protecting groups, they can be considered side-chain protecting groups.

[0026] Protecting groups can be removed under various conditions. For example, protecting groups can be removed by a base (e.g., base-labile), an acid (e.g., acid-labile), fluoride, light (photolabile), or hydrogenolysis. The type of protecting group can be selected according to the desired peptide synthesis strategy. Typically, a side chain protecting group having orthogonal reactivity to the N-terminal protecting group will be selected. In other words, the selection of orthogonal protecting groups for the N-terminal and side chain protecting groups can allow for the selective and / or specific deprotection of one of these types of groups without affecting the others.

[0027] Representative examples of suitable protecting groups include, but are not limited to, the following protecting groups: acyl type protecting groups (such as formyl, acrylyl (Acr), benzoyl (Bz), and acetyl (Ac)); aromatic urethane type protecting groups (such as benzyloxycarbonyl (Z) and substituted Z, for example, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl); aliphatic urethane protecting groups (such as t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl); cycloalkyl urethane type protecting groups (such as 9-fluorenyl-methyloxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl); thiourethane type protecting groups (such as phenylthiocarbonyl); 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); tert-butyl, triphenylmethyl (trityl); tetrahydropyranyl; benzyl ether (Bzl); 2,6-dichlorobenzyl (DCB); nitro, p-toluenesulfonyl (Tos); adamantyloxycarbonyl; xanthyl (Xan); benzyl; methyl; ethyl; -butyl ester, t-amyloxycarbonyl; photocleavable groups (such as nitro, veratryloxycarbonyl (NVOC)) and fluoride labile groups (such as tetramethylsilylethyloxycarbonyl (TEOC)). In some examples, the protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and t-butyloxycarbonyl (BOC).

[0028] In some examples, R 1 can be a labile protecting group under the first set of conditions, and R 2 , R 3 and R 5 any one or more of can each independently be selected from labile protecting groups under the second set of conditions. In other words, the protecting group of R 1 can have orthogonal reactivity with respect to the protecting groups of R 2 , R 3 and R 5 if there are protecting groups present at these positions. In some examples, R 1 can be a protecting group labile to base, and R 2 , R 3 and R 5 any one or more of can each independently be selected from protecting groups having orthogonal reactivity (such as protecting groups labile to acid). In some examples, R 1 can be Fmoc. In some examples, R 2 , R 3 and R 5 any one or more of can each independently be selected from Pbf and Boc. Modified amino acids of formula (I) containing protecting groups having these types of orthogonal reactivity can find particular uses in Fmoc / tBu peptide synthesis strategies.

[0029] As used herein, the term "aryl" can be a single or fused ring system having one or more aromatic rings. The term "aryl" can mean a monocyclic or polycyclic aromatic hydrocarbon system having 6 to 14 carbon ring atoms, particularly 6 to 10 carbon ring atoms. Representative examples of suitable "aryl" groups include, but are not limited to, phenyl, biphenyl, naphthyl, 1-naphthyl, 2-naphthyl, and anthracenyl. As used herein, "substituted aryl" means an aryl group as defined herein that contains one or more substituents on the aromatic ring. When the aryl group is substituted, any hydrogen atom can be replaced by a substituent, provided that the valence is satisfied.

[0030] As used herein, the term "heteroaryl" can be a single or fused ring system having one or more aromatic rings containing one or more O, N, and / or S heteroatoms. The term "heteroaryl" can mean a monocyclic or polycyclic heteroaromatic hydrocarbon system having 5 to 14 carbon ring atoms, particularly 5 to 10 carbon ring atoms. Representative examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyrimidyl, pyridazinyl, pyrazinyl, indolyl, benzofuranyl, benzothiazolyl, benzimidazolyl, indazolyl, benzoxazolyl, benzisoxazolyl, etc. As used herein, "substituted heteroaryl" means a heteroaryl group as defined herein that contains one or more substituents on the heteroaromatic ring. When the heteroaryl group is substituted, any hydrogen atom can be replaced by a substituent, provided that the valence is satisfied. As used herein, the term "optionally substituted" means that the moiety can contain one or more substituents.

[0031] As used herein, the "substituent" includes, but is not limited to, the following groups: hydroxyl, thiol, carboxyl, cyano (CN), nitro (NO2), halo, haloalkyl (e.g., C1-C6 haloalkyl), alkyl group (e.g., C1-C 10 or C1-C6), aryl (e.g., phenyl and substituted phenyl, such as benzyl or benzoyl), alkoxy group (e.g., C1-C6 alkoxy) or aryloxy (e.g., phenoxy and substituted phenoxy), thioether (e.g., C1-C6 alkyl or aryl), keto (e.g., C1-C6 keto), ester (e.g., C1-C6 alkyl or aryl which may be present as oxyester or carbonyl ester on the substituted moiety), thioester (e.g., C1-C6 alkyl or aryl), alkylene ester (rather than an ester functional group which may be substituted by a C1-C6 alkyl or aryl group, but rather such that the linkage is on an alkylene group), amine (including 5- or 6-membered cyclic alkyleneamine, further including C1-C6 alkylamine or C1-C6 dialkylamine, and the alkyl group can be substituted by one or two hydroxyl groups), amide (e.g., which can be substituted by one or two C1-C6 alkyl groups (including carboxamide which may be substituted by one or two C1-C6 alkyl groups)), alkanol (e.g., C1-C6 alkyl or aryl), or carboxylic acid (e.g., C1-C6 alkyl or aryl), sulfoxide, sulfone, sulfonamide, and urethane (-O-C(O)-NR2 or -N(R)-C(O)-O-R, etc., wherein each R in this context is independently selected from C1-C6 alkyl or aryl).

[0032] As used herein, "C1-C6 alkoxy" as used herein means a C1-C6 alkyl group as defined above attached to the parent molecular moiety through an oxy group -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, etc.

[0033] As used herein, "haloalkyl" can be an alkyl group in which one or more hydrogen atoms thereon are replaced by halogen atoms. As a representative example, C1-C6 haloalkyl can be a haloalkyl containing 1 to 6 carbon atoms. The haloalkyl can be a fluoroalkyl such as trifluoromethyl (-CF3) or 1,1-difluoroethyl (-CH2CHF2). As used herein, the "halo" group can be F, Cl, Br, or I, typically F.

[0034] As used herein, a bicyclic ring system can mean a chemical structure or moiety containing two rings joined together (e.g., covalently linked to each other). As used herein, a fused ring system can mean a chemical structure or moiety containing two rings sharing two adjacent atoms (or sharing a single covalent bond). The bicyclic ring system can contain 5 to 10 ring atoms. Representative examples include, but are not limited to, quinazolinyl, benzimidazolyl, tetrahydronaphthyridinyl (e.g., 1,2,3,4-tetrahydro-1,8-naphthyridinyl).

[0035] As used herein, the terms "aryl", "substituted aryl", "heteroaryl", "substituted heteroaryl", and "C1-C6 alkyl" can each mean either a monovalent radical species or a divalent radical species. For example, within the context of the various formulas described herein, R 3 is typically a monovalent group attached to the parent structure, and thus the term C1-C6 alkyl should be understood to represent a monovalent radical moiety. As a further example, X (as shown in formula (I)) is typically the asterisk ( *It is a divalent group that is commonly linked to both the carbon atom at the position of ()) and the A group. Therefore, in these examples, the term "C1-C6 alkyl" should be understood to represent a divalent radical moiety. Similar considerations apply to B (as shown in formula (I)), where again, B is typically a divalent group that is commonly linked to both X and Y. Therefore, in these examples, with reference to group B, the terms "aryl", "heteroaryl" and "C1-C6 alkyl" should each be understood to represent a divalent radical moiety.

[0036] As described above, R 1 can be selected from H and protecting groups. In some examples, R 1 can be a protecting group that is labile to a base. Representative examples include, but are not limited to, 9-fluorenylmethyloxycarbonyl (Fmoc), benzoyl (Bz), acetyl (Ac), etc. In some examples, R 1 can be H or Fmoc. As described above, X can be absent. In examples where X is absent, A is directly linked (e.g., using a covalent bond) to the starred carbon atom shown in formula (I).

[0037] In other examples, X can be selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkyl-NR 2 (C=O)-, and optionally substituted -C1-C6 alkyl-(C=O)NR 2 -. In other examples, X can be selected from optionally substituted C1-C3 alkyl, optionally substituted C1-C3 alkyl-NR 2 (C=O)-, and optionally substituted -C1-C3 alkyl-(C=O)NR 2 -. In some examples, X can be absent or selected from -CH2- and -CH2NR 2 (C=O)-(e.g., -CH2NH(C=O)-), and -CH2(C=O)NR 2 -(e.g., -CH2(C=O)NH-).

[0038] As described above, A can be the following formula:

Chemical formula

[0039] In some examples, B can be a 6- to 10-membered aryl ring optionally substituted by 1 to 3 substituents independently selected from halo, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. In some examples, B can be optionally substituted phenyl. For example, B can be phenyl. In other examples, B can be halo-substituted phenyl containing one or more halo substitutions (such as fluoro, chloro, bromo, iodo, etc.). In some examples, B can be a difluoro-substituted phenyl group. In some examples, B can be a 5- to 10-membered heteroaryl ring containing 1 to 3 heteroatoms selected from N, O, and S and optionally substituted by 1 to 3 substituents independently selected from halo, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. In some examples, B can be optionally substituted pyridyl. When B is an optionally substituted C1-C6 alkyl, Y is absent. In such examples, B can be an ethyl group.

[0040] As described above, R 3 can be selected from H, optionally substituted C1-C6 alkyl, and a protecting group. In some examples, R 3 can be C1-C6 alkyl (e.g., methyl). In other examples, R 3 can be a protecting group such as an acid-labile protecting group (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf)).

[0041] As already described, A can be a bicyclic fused ring system or can contain it. The bicyclic ring system can contain an amidine-like motif according to the following formula:

Chemical formula

[0042] In some examples, the bicyclic ring can be a heterocyclic ring containing one or more heteroatoms (e.g., N atoms). In some examples, the bicyclic ring can be a 9- or 10-membered ring system. In some examples, two N atoms of the amidine-like motif shown above also form part of the skeleton of the bicyclic ring system. In other examples, only the N atom directly linked to the R 7 group forms part of the skeleton of the bicyclic ring system (and in such examples, the NR 4 R 5 group is a substituent on the bicyclic ring system). In some examples, the bicyclic ring can include at least one aromatic or heteroaromatic ring. The bicyclic ring can be selected from quinazolinyl, benzimidazolyl, and tetrahydronaphthyridinyl (e.g., 1,2,3,4-tetrahydro-1,8-naphthyridinyl).

[0043] Representative examples of suitable Group A are shown below:

Chemical formula

[0044] In each of the structures shown above, R 3a can be selected from H, C1-C6 alkyl (e.g., methyl) and acid-labile protecting groups (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) or Boc); and / or R 5a can be selected from H, C1-C6 alkyl (e.g., methyl) and acid-labile protecting groups (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) or Boc).

[0045] In each of the above structures, the bond where the wavy lines intersect indicates the connection point of the structure to the group X shown in formula (I). In some examples, the modified amino acid can be represented by formula (Ia):

[0046]

Chemical formula

[0047] As a further example, R 1 , X, R 2 , B, and R 3 are further defined as described herein and above with respect to formula (I) (unless the context indicates otherwise).

[0048] Representative examples of modified amino acids according to the present disclosure are shown in the following formula (Ib):

Chemical formula

[0049] In this example, two groups can be attached to the aryl ring in any substitution pattern with respect to each other, e.g., ortho, meta, or para substitution pattern. In particular, the pendant group can covalently bond to a carbon atom on the aryl ring at any chemically suitable position (e.g., by replacing a hydrogen atom).

[0050] Further representative examples of modified amino acids according to the present disclosure are shown below:

Chemical formula

[0051] Further representative examples of modified amino acids according to the present disclosure are shown as formula (Ic) below:

Chem.

[0052] In this example, the group containing the amino acid moiety can be attached to the benzimidazole core at any suitable position, for example, at the 4, 5, 6, or 7 position, using a covalent bond to a carbon atom on the heteroaryl ring (e.g., by replacing a hydrogen atom at that position). In some examples, the modified amino acid can include one of the following structures:

Chem.

[0053] R 5a In some of the above examples where R 5a is H, the two structures shown above can exist as a mixture (since they represent tautomers of each other). In some cases where R 5a represents a protecting group, the synthesis of the modified amino acid can provide the two structures shown above as a mixture. The mixture can be used to synthesize cell membrane permeable peptides. After removal of the protecting group (e.g., after the cell membrane permeable peptide has been synthesized), R 5a is H, and the two structures can be readily converted via tautomerization.

[0054] Further representative examples of modified amino acids according to the present disclosure are shown below:

Chem.

[0055] For each of the above compounds, R 1b is either Fmoc or H; and R 3b is a protecting group (e.g., Pbf or Boc), H, or methyl.

[0056] The modified amino acids as described above can be used to provide peptides and can be particularly useful in providing cell-permeable peptides for replacing arginine therewith. Accordingly, peptides containing residues derived from the modified amino acids as described herein, particularly cell-permeable peptides, are further provided. Accordingly, in the various formulas and structures described above, it will be understood that the amino acid residue can be present at R 1 (instead of H and the protecting group), and / or the amino acid residue can be present in place of H at the carboxylic acid group. In particular, when incorporated into a cell-permeable peptide, there will be at least one amino acid residue present at R 1 or in place of H at the carboxylic acid group. In some examples, there can be an amino acid residue present at R 1 and an amino acid residue present in place of H at the carboxylic acid group. Although the various formulas and structures for at least one modified amino acid are given in the context of the isolated amino acid, it will be understood that these are equally applicable to cell-permeable peptides containing residues derived from the modified amino acid. By way of example, the various options for (A) and (X) as given for the modified amino acid of formula (I) above are equally applicable to cell-permeable peptides containing residues derived from this modified amino acid.

[0057] By way of example, peptides containing at least one modified amino acid residue according to formula (II), particularly cell-permeable peptides, are provided:

Chemical formula

[0058] At least one modified amino acid residue can be derived from a modified amino acid not including the following structure:

Chemical formula

[0059] In such an example, the cell membrane permeable peptide can include at least one modified amino acid together with one additional amino acid residue at either R 1 or R 8 . In a further example, the cell membrane permeable peptide can include at least one modified amino acid together with two additional amino acid residues, one at each of R 1 and R 8 .

[0060] As a further example, the peptide can include a modified amino acid residue according to formula (IIa) or (IIb):

Chemical formula

[0061] A peptide (e.g., a cell-penetrating peptide) can contain any number of amino acid residues, e.g., about 2 to 100 amino acid residues, e.g., about 5 to 50 or about 7 to 20 amino acid residues. A cell-penetrating peptide can contain about 2 to 30 amino acid residues. A peptide (e.g., a cell-penetrating peptide) can fall within the scope of formula (II), (IIa), or (IIb), or can contain a plurality of modified amino acid residues derived from modified amino acids as defined or described in relation to any one of formula (I), (Ia), (Ib), or (Ic). By way of example, a peptide can fall within the scope of formula (II), (IIa), or (IIb), or can contain at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues derived from modified amino acids as defined or described in relation to any one of formula (I), (Ia), (Ib), or (Ic). In some cases, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the amino acid residues present in the peptide can be one or more amino acid residues derived from modified amino acids that follow formula (II) or are defined or described in relation to any one of formula (I), (Ia), (Ib), or (Ic).

[0062] The peptide can be a cell-permeable peptide such as a cationic cell-permeable peptide (a cell-permeable peptide having a net positive charge). As already described, cell-permeable peptides generally contain a relatively high number and / or proportion of arginine residues. In such peptides, one or more arginine residues can be replaced with amino acid residues derived from a modified amino acid as defined or described in accordance with formula (II), (IIa), or (IIb), or any one of formulas (I), (Ia), (Ib), or (Ic). Indeed, as already explained, the modified amino acids disclosed herein can be used to provide an overall positive charge while exhibiting a changed basicity and / or pKa, and thus can find particular utility in cationic CPPs. For example, when an amidino group replaces a guanidino group (as found in an arginine residue) (or a different crosslinking group is used between the α-carbon of the amino acid and the guanidino or amidino group of the modified amino acid), it can result in the same overall positive charge, but the distribution of that charge throughout the structure may be different, providing a changed basicity and / or pKa.

[0063] In some examples, the cell-permeable peptide can have a cLogP value of from about -42.12 to about 2.97 (see, for example, Oliveira et al, Nature Scientific Reports, 2021, 11, 7628). In some examples, the cell membrane permeable peptide can comprise an overall formal charge of from about +1 to about +4 (such as about +2 or +3). In some examples, the cell membrane permeable peptide can comprise an overall formal charge of about +2. In fact, it has been observed that in order to facilitate cell entry of the peptide, the peptide can preferably retain a positive charge, otherwise little or no cell uptake may be seen using fluorescence detection methods. This phenomenon has generally been investigated in the context of stapled peptides (see, for example, Verdine et al, Med. Chem. Commun., 2015, 6, 111-119, where a number of stapled peptides with various overall charges were tested for cell membrane permeability), and while not wishing to be bound by theory, the inventors hypothesize that a similar pattern may exist for cell membrane permeable peptides.

[0064] The cell membrane permeable peptide can comprise any suitable membrane diffusion rate and / or cell uptake rate that enables the cell membrane permeable peptide to pass through the membrane at a suitable rate (which may depend on the cargo and / or the purpose of the cargo) and / or transport the cargo into the cell. In some examples, the suitable membrane diffusion rate can be in the vicinity of 1×10 -6 cm / s or greater P app (apparent permeability) (for example, in a Caco permeability assay or a parallel artificial membrane permeability assay (PAMPA)) (see, for example, Pei et al, Chem. Rev. 2019, 119, 10241-10287).

[0065] In some examples, the cell membrane permeable peptide can include a hydrophobic moment of at least about 0.30, at least about 0.40, or at least about 0.50. In some examples, the cell membrane permeable peptide can include a hydrophobic moment of about 0.55 (see, for example, Bird et al, Nature Chemical Biology, Vol. 12, October 2016, 845, which describes the study of stapled peptides and shows a linear relationship between hydrophobic moment and cell access, and the data appears to show a linear regression without any specified top value. Without wishing to be bound by theory, the inventors hypothesize that a similar pattern may exist for cell membrane permeable peptides).

[0066] Examples of cell membrane permeable peptide sequences are shown in FIG. 1b. In particular, an example of a cell membrane permeable peptide is Tat (Green, M.; Ishino, M.; Loewenstein, P. M. Mutational analysis of HIV-1 Tat minimal domain peptides: Identification of <em>trans< / em>-dominant mutants that suppress HIV-LTR-driven gene expression. Cell 1989, 58, 215-223). The sequence of Tat is shown as SEQ ID NO: 1 below. Further examples include TP-1 (SEQ ID NO: 2), TP-2 (SEQ ID NO: 3), and PiP6 (SEQ ID NO: 4) (Guha et al, Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem. Rev. 2019, 119, 6040-6085; Marks et al, Spontaneous Membrane-Translocating Peptides by Orthogonal High-Throughput Screening. J. Am. Chem. Soc. 2011, 133, 8995-9004; and Betts et al, Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment. Mol. Ther. Nucleic Acids 2012, 1). RKKRRQRRR - SEQ ID NO: 1 PLILLRLLRG - SEQ ID NO: 2 PLIYLRLLRG - SEQ ID NO: 3 RXRRBRRYQFLIRBRXR - SEQ ID NO: 4 In SEQ ID NO: 4 above, X in the amino acid sequence represents 6-aminohexanoic acid (Ahx), and B in the amino acid sequence represents β-alanine.

[0067] By way of example only, an amino acid residue of formula (II) (e.g., formula (IIa) or (IIb)), or an amino acid residue derived from a modified amino acid as defined or described in connection with any one of formula (I), (Ia), (Ib), or (Ic) can be used in place of one or more arginine residues in any one or more of the above sequences. As a further example, a cell membrane-permeable peptide containing an amino acid residue according to formula (II) is shown below as SEQ ID NO: 5. PLIYLXLLXG - SEQ ID NO: 5 X in the amino acid sequence is an amino acid residue according to formula (II), or a residue derived from a modified amino acid as defined or described in connection with any one of formulas (I), (Ia), (Ib) or (Ic).

[0068] In some examples, X in the amino acid sequence can be an amino acid residue according to formula (IIa) or (IIb). Additional examples of cell membrane-permeable peptides in which one or more arginine residues can be substituted by an amino acid residue according to the amino acid residue of formula (II), or by an amino residue derived from a modified amino acid as defined or described in connection with any one of formulas (I), (Ia), (Ib) or (Ic), are shown below: RRRRRRRRR - SEQ ID NO: 6 ("R9") RQIKIWFQNRRMKWKK - SEQ ID NO: 7 (penetratin) MVRRFLVTLRIRRACGPPRVRV - SEQ ID NO: 8 (ARF(1-22)

[0069] In some examples, the cell membrane-permeable peptide can contain a sequence as shown in SEQ ID NO: 9: XPLIYLAmLLAmG - SEQ ID NO: 9 Here, X in the above sequence (SEQ ID NO: 9) is a residue derived from 6-aminohexanoic acid (Ahx), and each Am in the above sequence is a modified amino acid residue according to the following structure:

Chemical formula

[0070] In the above structure, the wavy line intersects the peptide bond formed between the Am residue and the adjacent amino acid. In some examples, the cell membrane-permeable peptide can contain a sequence as shown in SEQ ID NO: 10: XPLIYLBimLLBimG - Sequence number 10 Here, X in the above sequence (sequence number 10) is a residue derived from 6 - aminohexanoic acid (Ahx), and each Bim is a modified amino acid residue following the structure below:

Chemical formula

[0071] In the above structure, the wavy line intersects the peptide bond formed between the Bim residue and the adjacent amino acid. As already explained, the Bim - modified amino acids may exist in different tautomeric forms in the cell - membrane - permeable peptide structure, and they can readily interconvert:

Chemical formula

[0072] In some examples, the cell - membrane - permeable peptide can contain a sequence as shown in sequence number 11: XPLIYLmAmLLmAmG - Sequence number 11 Here, X in the above sequence (sequence number 11) is a residue derived from 6 - aminohexanoic acid (Ahx), and each mAm is a modified amino acid residue following the structure below:

Chemical formula

[0073] As previously described, the agent of interest can be any type of entity that requires delivery to cells and / or distribution throughout the cell. The agent of interest can be, for example, a therapeutic agent, a diagnostic agent, or a contrast agent. The agent of interest can be a biological molecule (e.g., a nucleic acid-based molecule (e.g., siRNA, antisense oligonucleotide, DNA, plasmid, etc.), a polypeptide, or a protein). In some examples, the agent of interest can be a particle (e.g., a nano-sized particle) or a compound.

[0074] In some examples, the agent of interest can be a fluorescent tag (such as a fluorescein derivative such as fluorescein or fluorescein isothiocyanate (FITC)). In some examples, the fluorescent tag can be covalently linked to a peptide at any chemically suitable position.

[0075] When the agent of interest is a therapeutic agent, the cell-penetrating peptide can be useful for promoting cellular uptake and / or distribution of the agent. Accordingly, there is further provided a cell-penetrating peptide as described herein for use in therapy and / or medicine. Indeed, the present disclosure also encompasses a method of treatment comprising administering to a subject in need thereof a cell-penetrating peptide as described herein (e.g., a cell-penetrating peptide comprising a therapeutic agent). The cell-penetrating peptide can be administered in a therapeutically effective amount. There is also provided the use of a cell-penetrating peptide as described herein (e.g., a cell-penetrating peptide comprising a therapeutic agent) in the manufacture of a medicament for use in therapy and / or medicine. When the cell-penetrating peptide is for use in therapy and / or medicine (e.g., a cell-penetrating peptide comprising a therapeutic agent), the peptide can be formulated into a pharmaceutical composition. For example, the cell-penetrating peptides of the present disclosure can be formulated as a sterile pharmaceutical composition suitable for administration to a subject.

[0076] Such formulations can include one or more pharmaceutically acceptable excipients, carriers, and / or diluents. Representative examples include, but are not limited to, water, physiological saline, phosphate buffered saline, dextrose, glycerol, ethanol, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, aqueous salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycon, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene - polypropylene block polymers, polyethylene glycol, and lanolin, etc., or combinations thereof.

[0077] The pharmaceutical composition can be formulated and / or prepared, for example, for oral, parenteral, topical, and / or mucosal / inhalation administration. According to a further aspect, there is provided the use of a modified amino acid as described herein (e.g., as per formula (I), (Ia), (Ib), or (Ic)) in the synthesis of a peptide. In particular, there is provided a method for preparing a peptide (e.g., a cell - permeable peptide) that includes the use of a modified amino acid according to formula (I), (Ia), (Ib), or (Ic). In particular, the method can include contacting a first amino acid (or a peptide fragment containing a residue of the first amino acid) with a second amino acid (or a peptide fragment containing a residue of the second amino acid) under conditions that promote and / or facilitate a condensation reaction between the first and second amino acids (or the peptide fragments containing their residues) to provide a new peptide linkage. At least one of the first and second amino acids can be a modified amino acid as described herein (e.g., a modified amino acid according to formula (I), (Ia), (Ib), or (Ic)). That is, the method provides a peptide comprising at least one modified amino acid residue according to formula (II).

[0078] As will be appreciated by those skilled in the art, the peptide can be prepared using a chemical synthesis approach, such as by solid-phase or liquid-phase peptide synthesis. Such a chemical synthesis approach for peptides generally involves a number of coupling (e.g., condensation) reactions between amino acids. The method can include a series of (a) deprotection and (b) coupling steps that are repeated until the desired or target peptide is obtained. After each series of deprotection and coupling steps, an amino acid can be added to the growing peptide fragment. In this way, the synthesis of the peptide can be controlled by the sequential addition of amino acids.

[0079] In particular, an orthogonal protecting group strategy can be used in such a synthesis (e.g., different classes of protecting groups can be used to protect the N-terminus of an amino acid relative to those used to protect the reactive groups on the side chain of the amino acid). Such a strategy can be used to ensure minimizing side reactions during peptide synthesis and is widely known in the art. Representative examples include, but are not limited to, the Boc / Bzl protecting group strategy (e.g., when the N-terminus of an amino acid is protected with an acid-labile Boc group and the side chain protecting group is benzyl or a benzyl-based group) and the Fmoc / tBu or Fmoc / Boc protecting group strategy (e.g., when the N-terminus of an amino acid is protected with a base-labile Fmoc group and the side chain protecting group is an acid-labile group (e.g., tBu or Boc)).

[0080] The deprotection step (a) can include removing a protecting group from the terminus of the amino acid (typically the N-terminus). By way of example only, the deprotection step includes removing a protecting group from the N-terminus of an amino acid (or a peptide fragment containing at least one amino acid residue). In some examples, the deprotection step can include removing a base-labile protecting group (such as Fmoc) from the N-terminus of an amino acid (or a peptide fragment containing at least one amino acid residue). The coupling step (b) can include contacting a first amino acid (or a peptide fragment) with a second amino acid under conditions that promote and / or facilitate a condensation reaction between the N-terminus of the first amino acid (or peptide fragment) and the C-terminus of the second amino acid to provide a peptide or a fragment thereof. The second amino acid can include a protecting group on the N-terminus that can be stable under the coupling conditions (and thus can prevent or reduce unwanted condensation reactions). To promote and / or facilitate the coupling step, an activator and / or a catalyst can be added to increase the reactivity of the N-terminus acid and / or the C-terminus of the first amino acid and / or the second amino acid.

[0081] Suitable activating agents are known to those skilled in the art. Representative examples include, but are not limited to, carbodiimide-based reagents (e.g., dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC)), 1-hydroxy-benzotriazole (HOBt), and 1-hydroxy-7-azabenzotriazole (HOAt), ammonium, uronium, and / or phosphonium salts (e.g., HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate), HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), etc.). In some examples, the amino acid can be converted to an acid halide (e.g., acyl fluoride or acyl chloride) to drive the condensation step.

[0082] In some examples, the method can further include the step of deprotecting one or more or all of the side chain protecting groups (if present). The step of deprotecting one or more or all of the side chain protecting groups can be carried out after the desired peptide length or sequence has been obtained and / or after cleavage of the peptide from the solid support. When using a solid support, the method can include the step of providing a first amino acid that can be optionally linked (e.g., covalently linked) to the solid support. In such examples, the peptide can be cleaved from the support when the desired peptide length or sequence has been obtained.

[0083] When solid-phase synthesis is utilized, any type of support suitable for performing solid-phase peptide synthesis (SPPS) can be used. By way of example, the support can include a resin that can be manufactured from one or more polymers, copolymers, or combinations of polymers such as polyamides, polysulfamides, substituted polyethylene, polyethylene glycol, phenolic resins, polysaccharides, or polystyrene. The solid support typically includes a linking moiety to which the growing peptide can be coupled during synthesis and that can be cleaved under desired conditions to release the peptide from the support.

[0084] Suitable solid supports can include linkers that are photocleavable, trifluoroacetic acid cleavable (TFA cleavable), HF cleavable, fluoride ion cleavable, reductively cleavable, Pd(0) cleavable, nucleophilically cleavable, or radically cleavable. In some examples, the linking moiety can be cleavable under conditions where any side-chain protecting groups are stable and / or are removed under the conditions used to cleave the linker. In some examples, solid supports include acid-sensitive solid supports such as Rink amide resin, hydroxymethyl-polystyrene-divinylbenzene polymer resin (see “Wang” resin, Wang, S. S. 1973, J. Am. Chem. Soc., 95:1328-33), 2-chlorotrityl chloride resin (see Barlos et al. (1989) Tetrahedron Letters 30(30):3943-3946), and 4-hydroxymethyl-3-methoxyphenoxybutyric acid resin (see Richter et al. (1994), Tetrahedron Letters 35(27):4705-4706), as well as functionalized crosslinked poly-N-acryloxypyrrolidone resin, and chloromethylpolystyrene divinylbenzene polymer resin.

[0085] A further aspect of the disclosure is directed to a method for screening cell membrane-permeable peptides. The method can include providing a candidate peptide that contains at least one modified amino acid residue according to formula (II) or that contains at least one modified amino acid residue derived from a modified amino acid of formula (I), (Ia), (Ib), or (Ic). The method can include contacting the candidate peptide with cells. The method can include determining the effect of the candidate peptide on the cells. The method can be an in vitro method.

[0086] By way of example only, the method can (i) cellular uptake of the candidate peptide; (ii) intracellular distribution of the candidate peptide; and / or (iii) toxicity (to the cells) of the candidate peptide include determining one or more of these. If it is determined that the candidate peptide is taken up by the cells and / or is distributed throughout the cells, it can be determined that the candidate peptide is suitable for use as a cell-permeable peptide. Additionally or alternatively, if the candidate peptide exhibits minimal or low levels of toxicity, it can be determined that the candidate peptide is suitable for use as a cell-permeable peptide. In some examples, the candidate peptide can be labeled (e.g., with a fluorescent label). Such labeling can assist in determining and / or detecting the effect of the candidate peptide on the cells.

[0087] In some examples, the method can include identifying a peptide sequence that can function as a cell-permeable peptide (e.g., a peptide sequence known to act as a cell-permeable peptide). Such a peptide can be designated as a parent peptide and can contain at least one arginine residue. The method can include replacing one or more arginine residues in the parent peptide with a modified amino acid residue according to formula (II). Such a modified peptide may be referred to as a candidate peptide. The method can include a step of comparing the effect of a candidate peptide on cells with a reference level. The reference level can be obtained by determining the effect of a parent peptide on cells.

[0088] By way of example only, the method can include, in order to provide a reference level, (i) cellular uptake of the parent peptide; (ii) intracellular distribution of the parent peptide; and / or (iii) toxicity (to cells) of the parent peptide in one or more of the steps of determining. If the effect of the candidate peptide is more favorable than the reference level observed using the parent peptide (e.g., to facilitate use as a cell membrane permeable peptide), the candidate peptide can provide a useful and / or improved cell membrane permeable peptide, and / or the modified amino acid can find particular utility in cell membrane permeable peptides.

[0089] By way of example only, if the candidate peptide exhibits increased cellular uptake and / or intracellular distribution compared to the parent peptide, the effect of the candidate peptide can be considered more favorable. In some examples, if the candidate peptide exhibits a reduced level of toxicity compared to the parent peptide, the effect of the candidate peptide can be considered more favorable.

[0090] The inventors have further identified a series of novel compounds that can find particular use, for example, in the synthesis of cell membrane permeable peptides. In particular, according to a further aspect of the present disclosure, there is provided a modified amino acid according to formula (I''):

Chemical formula

Chemical formula

[0091]

Chemical formula

[0092] In some examples of formula (I''), R 1can be H or 9-fluorenylmethyloxycarbonyl (Fmoc). In some examples of formula (I''), X is -CH2NH(C=O)-. In some examples of formula (I''), A is: [Chemical formula] and; B is optionally substituted phenyl or optionally substituted pyridyl; Y is absent; and R 3 is optionally substituted C1-C6 alkyl or a protecting group labile to acids.

[0093] In some examples, the modified amino acid can be represented by formula (Ia''): [Chemical formula] (wherein, R 1 is H or a protecting group; R 8 is H or a protecting group; X is selected from optionally substituted C2-C4 alkyl, optionally substituted -C1-C3 alkyl-NR 2 (C=O)- and optionally substituted C1-C3 alkyl-(C=O)NR 2 -; R 2 is selected from H, optionally substituted C1-C6 alkyl and a protecting group; B is selected from optionally substituted aryl and optionally substituted heteroaryl; and R 3 is selected from H, optionally substituted C1-C6 alkyl and a protecting group). In preferred examples, R 1 is a protecting group and R 8 is H.

[0094] In some examples of formula (I''), A is: [Chemical formula] selected from wherein R 3a is selected from H, C1-C6 alkyl (e.g., methyl) and an acid-labile protecting group (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) or Boc); and R 5a is selected from H, C1-C6 alkyl (e.g., methyl) and an acid-labile protecting group (e.g., Pbf or Boc).

[0095] In some examples, the modified amino acid can comprise a structure according to formula (Ib''):

Chemical formula

[0096] Definition In the foregoing discussion, a number of terms are referenced and, unless the context indicates otherwise, should be understood to have the meanings provided below. The nomenclature used herein to define compounds, particularly the compounds described herein, is intended to follow the rules of the International Union of Pure and Applied Chemistry (IUPAC) regarding compounds, specifically the "IUPAC Compendium of Chemical Terminology (Gold Book)" (see A. D. Jenkins et al., Pure & Appl. Chem., 68, 2287-2311 (1996)). To avoid doubt, where the IUPAC rules differ from the definitions provided herein, the definitions in this specification should be given precedence.

[0097] The present disclosure also includes any compound of a formula disclosed herein, or of any of the formulas disclosed herein including formulas (I), (Ia) and (II) (including the corresponding subgeneric formulas defined herein), and also includes various deuterated forms of the example compounds (1) to (16) of the present disclosure, respectively, or pharmaceutically acceptable salts and / or their corresponding tautomeric forms (including subgeneric formulas as defined above). Each available hydrogen atom attached to a carbon atom can independently exist as a deuterium atom. One of ordinary skill in the art will know how to synthesize the deuterated forms of any compound of a formula disclosed herein, or of any of the formulas disclosed herein including formulas (I), (Ia) and (II) (including the corresponding subgeneric formulas defined herein), and also of the example compounds (1) to (16) of the present disclosure, respectively, or pharmaceutically acceptable salts and / or their corresponding tautomeric forms (including subgeneric formulas as defined above). For example, deuterated materials such as alkyl groups can be prepared by conventional techniques (see, for example, methyl-d3-amine, catalog number 489,689-2, available from Aldrich Chemical Co., Milwaukee, WI).

[0098] Apart from the fact that one or more atoms are present as atoms having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, the compounds shown in any of the formulas disclosed herein, including formulas (I), (Ia) and (II) (including the corresponding subgeneric formulas defined herein), and the isotopically labeled compounds identical to the example compounds (1) to (16), or pharmaceutically acceptable salts and / or their corresponding tautomeric forms (including subgeneric formulas as defined above) of the present disclosure are also included. Examples of isotopes that can be incorporated into the compounds of the present disclosure include 3 H, 11 C, 14 C, 18 F, 123 I or 125 I and the like, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine and chlorine. The compounds of the present disclosure containing the above-mentioned isotopes and / or other isotopes of other atoms and the pharmaceutically acceptable salts of said compounds are within the scope of the present disclosure.

[0099] It should be noted that the terms "comprise", "comprising" and / or "comprises" are used to indicate that aspects and embodiments of the present invention "comprise" certain features or a plurality of features. These terms can also encompass aspects and / or embodiments "consisting essentially of" or "consisting of" the relevant features or a plurality of features. Detailed Description This application is now further described by way of example only with reference to the following drawings.

Brief Description of the Drawings

[0100]

Figure 1a

Figure 1b

Figure 1c

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0101] [Examples] General Information All reagents and solvents were obtained from commercial suppliers and used without further purification. All reactions were carried out under air unless otherwise specified. Reactions were monitored by thin layer chromatography (TLC) using Merck silica plates coated with a fluorescent indicator UV254. TLC plates were analyzed using 254 / 365 nm UV light or developed using potassium permanganate solution.

[0102] Peptide Synthesis Peptide synthesis was completed on an automated Tribute® peptide synthesizer with an IntelliSynth UV monitoring system and a feedback control system. Rink amide resin (100 - 200 mesh, 0.65 mmol / g), Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Tyr(O t Bu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Cit-OH, Fmoc-Lys(Boc)-OH and Fmoc-εAhx-OH were purchased from Merck Millipore or Fluorochem and used without further purification. Fluorescein 5-isothiocyanate (FITC) was purchased from Sigma-Aldrich and used without further purification.

[0103] HPLC for analysis RP-HPLC was performed using an Aeris 3.6 μm, 250 × 4.6 mm widepore XB C18 column with a DIONEX 3000 series HPLC equipped with a VWD3400 photodiode array detector. Samples were eluted using water (0.1% TFA) as solvent A and acetonitrile (0.1% TFA) as solvent B and analyzed at a flow rate of 1.0 mL / min. RP-HPLC method A for analysis: Absorbance detection was set at 220 nm.

[0104] [Table 1] Purification of the product Normal-phase flash chromatography was performed using ZEOprep 60 HYD 40 - 63μm silica gel. Semi-preparative reverse-phase HPLC purification was carried out on a Kinetex 5μm, 150×21.2mm XB C18 column using a DIONEX 3000 series HPLC equipped with a VWD3400 variable wavelength detector. Purification was performed using water (0.1% TFA) as solvent A and acetonitrile (0.1% TFA) as solvent B, and analyzed at a flow rate of 12.0 mL / min. RP-HPLC method A: Absorbance detection was set at 220 nm.

[0105]

Table 2

[0106]

Chem.

[0107]

Chem.

[0108] [Chemical formula] Scheme 3: Synthetic route used to synthesize the modified amino acid S2b.

[0109] Synthesis procedure Methyl 4-(N-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzoate (S2) [Chemical formula]

[0110] Methyl 4-carbamimidoylbenzoate hydrochloride (2.00 g, 9.32 mmol) was suspended in acetone (40.0 mL) at 0 °C, and then 3 M NaOH solution (6.00 mL, 18.0 mmol) was added to form a clear solution. Separately, Pbf chloride (3.23 g, 11.2 mmol) was dissolved in acetone (13.5 mL) and then added dropwise to the reaction mixture. The resulting pale yellow solution was stirred at 0 °C for 2 hours until a white suspension formed, and then the reaction was stirred at room temperature (rt) for an additional 2 hours. The reaction was then acidified to pH 6 using 3 M HCl solution, and the suspension was filtered. The solid was washed with water (15 mL) and then with acetone (15 mL). The filtrate was then extracted with ethyl acetate (EtOAc) (3 × 50 mL), and the combined organic layers were washed with brine (100 mL) and then dried over MgSO4. The organic solution was then concentrated under vacuum, and the resulting residue was purified by silica gel chromatography in a gradient of 0% - 40% EtOAc in petroleum ether (40 - 60) to give a colorless solid (3.03 g, 75%). mp 400 °C with decomposition. 11H NMR (500 MHz, CDCl3): δ / ppm: 8.21 (br s, 1H, NH), 8.05 (d, 2H, J = 8.5 Hz, H3CO-C(O)-C q -CH×2), 7.82 (d, 2H, J = 8.5 Hz, H3CO-C(O)-C q -CH-CH×2), 6.22 (br s, 1H, NH), 3.92 (s, 3H, C q -C(O)-OCH3), 2.96 (s, 2H, O-C q (CH3)2-CH2), 2.62 (s, 3H, S(O)2-C q -C q -(CH3)-C q -(CH3)), 2.55 (s, 3H, S(O)2-C q -C q -(CH3)-C q ), 2.10 (s, 3H, S(O)2-C q -C q (CH3)-C q -(CH3)), 1.46 (s, 6H, C q -CH3×2). 13 C{ 1 H} NMR (125 MHz, CDCl3): δ / ppm: 166.0 (C q ), 160.2 (C q ), 159.6 (C q ), 139.3 (C q ), 137.9 (C q ), 133.5 (C q ), 133.2 (C q ), 130.9 (C q ), 129.9 (2×C-H), 127.3 (2×C-H), 124.9 (C q ), 117.8 (C q ), 86.7 (C q ), 52.4 (CH3), 43.1 (CH2), 28.6 (2×CH3), 19.2 (CH3), 17.9 (CH3), 12.4 (CH3). IR (neat): νmax / cm -1 : 3450 (N-H stretching), 3334 (N-H stretching), 2973 (C-H stretching), 2929 (C-H stretching), 1703 (C=O stretching), 1628 (C=N stretching), 1578 (C=C stretching), 1533 (C=C stretching). HRMS (ESI): C 22 H 27 N2O5S + Calculated value. Theoretical value: 431.1635 Measured value: 431.1637.

[0111] 4-(N-((2,2,4,6,7-Pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzoic acid (S3)

Chem.

[0112] Amidine S2 (2.50 g, 5.81 mmol) was dissolved in tetrahydrofuran (THF) (29.1 mL), and then 1 M NaOH solution (29.1 mL, 29.1 mmol) was added dropwise. The resulting solution was stirred at rt for 2 h. THF was removed under vacuum, and the reaction was diluted with water (25 mL). Subsequently, the aqueous solution was acidified to pH 5 using 1 M HCl solution to form an off-white gum. The suspension was filtered, and the solid was washed with water (25 mL). Subsequently, the colorless solid was dried under high vacuum and purified by SiO2 chromatography in a gradient of 0% - 50% EtOAc in petroleum ether (40 - 60) containing 1% acetic acid (AcOH) modifier to obtain a colorless solid (1.90 g, 78%). mp 400 °C with decomposition. 1 H NMR (500 MHz, DMSO-d6): δ / ppm: 13.23 (br s, 1H, CO2H), 8.98 (br s, 1H, NH), 8.06 (br s, 1H, NH), 8.00 (d, 2H, J = 8.7 Hz, HO2C-C q-CH×2), 7.91 (d, 2H, J = 8.7 Hz, HO2C-C q -CH-CH×2), 3.00 (s, 2H, O-C q (CH3)2-CH2), 2.52 (s, 3H, S(O)2-C q -C q -(CH3)-C q -(CH3)), 2.46 (s, 3H, S(O)2-C q -C q -(CH3)-C q ), 2.04 (s, 3H, S(O)2-C q -C q (CH3)-C q -(CH3)), 1.43 (s, 6H, C q -CH3×2). 13 C{ 1 H} NMR (125 MHz, DMSO-d6): δ / ppm: 167.1 (C q ), 160.9 (C q ), 158.8 (C q ), 138.5 (C q ), 138.0 (C q ), 134.2 (C q ), 132.7 (C q ), 132.5 (C q ), 129.8 (2×C-H), 128.4 (2×C-H), 125.3 (C q ), 117.2 (C q ), 87.2 (C q ), 42.7 (CH2), 28.7 (2×CH3), 19.3 (CH3), 18.1 (CH3), 12.7 (CH3). IR (neat): ν max / cm -1 : 3358 (N-H stretching), 3223 (N-H stretching), 3091 (CO2H stretching), 2972 (C-H stretching), 2937 (C-H stretching), 1710 (C=O stretching), 1670 (C=N stretching), 1589 (C=C stretching), 1533 (C=C stretching). HRMS (ESI): C 21 H 25 N2O5S + Calculated value. Theoretical value: 417.1479 Measured value: 417.1474.

[0113] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(N-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzamide)propanoic acid (S4) [Chemical formula]

[0114] Amidine S3 (1.10 g, 2.64 mmol) and HATU (1.00 g, 2.64 mmol) were dissolved in DMF (26.4 mL), and then N,N-diisopropylethylamine (DIPEA) (920 μL, 5.28 mmol) was added dropwise. The resulting yellow solution was stirred at room temperature (rt) for 30 minutes. The reaction mixture turned dark orange, and Fmoc-Dap-OH (1.15 g, 3.17 mmol) was added portionwise thereto, and the reaction mixture was stirred at rt for 4.5 hours. EtOAc (80 mL) and water (80 mL) were added, and the organic layer was separated. Subsequently, the aqueous layer was extracted with EtOAc (2 × 80 mL). Subsequently, the combined organic layers were washed with brine (150 mL), dried over MgSO4, then filtered, and then concentrated under vacuum. The obtained residue was purified by SiO2 chromatography in a gradient of 0% - 4% MeOH in DCM containing 1% AcOH modifier to obtain a colorless solid (905 mg, 51%).

[0115] mp 113 - 117 °C. [α] D = -16.86 (c = 0.011 g mL -1 , MeOH) 1 1H NMR (500 MHz, CD3OD): δ / ppm: 7.76 (d, 2H, J = 8.6 Hz, HN=C q -Cq -CH-CH×2), 7.72 (d, 2H, J = 8.6 Hz, HN=C q -C q -CH×2), 7.62 (dd, 2H, J = 7.5, 3.3 Hz, CO2-CH2-CH-C q -CH-CH-CH-CH×2), 7.50 (d, 2H, J = 7.5 Hz, CO2-CH2-CH-C q -CH×2), 7.22 (2H, m, CO2-CH2-CH-C q -CH-CH-CH×2), 7.12 (2H, m, CO2-CH2-CH-C q -CH-CH×2), 4.41 (1H, m, HO2C-CH), 4.19 (2H, m, CO2-CH2), 4.06 (1H, t, J = 7.1 Hz, CO2-CH2-CH) 3.74 (1H, m, HO2C-CH-CH A H B ), 3.63 (1H, m, HO2C-CH-CH A H B ), 2.91 (s, 2H, O-C q (CH3)2-CH2), 2.49 (s, 3H, S(O)2-C q -C q -(CH3)- C q -(CH3)), 2.44 (s, 3H, S(O)2-C q -C q- (CH3)-C q ), 1.99 (s, 3H, S(O)2-C q -C q (CH3)-C q -CH3), 1.35 (s, 6H, C q -CH3×2). 13 C{ 1 H} NMR (125 MHz, CD3OD): δ / ppm: 172.1 (C q ), 168.3 (C q ), 161.6 (C q ), 159.2 (C q ), 157.2 (C q), 143.8 (C q ), 143.7 (C q ), 141.1 (2×C q ), 138.7 (C q ), 137.4 (C q ), 136.6 (C q ), 132.9 (C q ), 131.3 (C q ), 127.6 (2×C-H), 127.4 (2×C-H), 127.2 (2×C-H), 126.7 (2×C-H), 125.0 (C q ), 124.8 (2×C-H), 119.5 (2×C-H), 117.3 (C q ), 86.6 (C q ), 66.7 (CH2), 53.8 (CH), 46.8 (CH), 42.4 (CH2), 40.9 (CH2), 27.3 (2×CH3), 18.0 (CH3), 16.9 (CH3), 11.0 (CH3). IR (neat): ν max / cm -1 : 3394 (N-H stretch), 3311 (N-H stretch), 3115 (CO2H stretch), 2972 (C-H stretch), 2927 (C-H stretch), 1716 (C=O stretch), 1705 (C=O stretch), 1627 (C=N stretch), 1575 (C=C stretch), 1527 (C=C stretch). HRMS (ESI): C 39 H 41 N4O8S + calculated value. Theoretical value: 725.2640 Measured value: 725.2624.

[0116] Methyl 3-(N-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzoate (S3a)

Chemical Structure

[0117] Methyl 3-carbamimidoylbenzoate hydrochloride (200 mg, 932 μmol) was suspended in acetone (4.0 mL) at 0 °C, and then 3 M NaOH solution (621 μL, 18.0 mmol) was added to form a clear solution. Separately, Pbf chloride (323 mg, 1.12 mmol) was dissolved in acetone (1.35 mL) and then added dropwise to the reaction mixture. The resulting solution was stirred at 0 °C for 2 h and then at rt for an additional 2 h. The reaction was then acidified to pH 6 using HCl (3 M aqueous), followed by extraction with EtOAc (5 × 10 mL), and the combined organic layers were dried (MgSO4). The crude mixture was then purified by column chromatography (3:2, Hex:EtOAc) to give the title compound as a colorless solid (260 mg, 65%). mp 400 °C decomp. 1 1H NMR (500 MHz, d6-DMSO): δ / ppm: 9.05 (s, 1H, NH), 8.38 (s, 1H, NH), 8.12 (d, 1H, J = 7.8 Hz, ArH), 8.10 - 8.02 (m, 2H, 2×ArH), 7.63 (t, 1H, J = 7.8 Hz, ArH), 3.88 (s, 3H, OCH3), 2.99 (s, 2H, CH2), 2.50 (s, 3H, CH3 - hidden by DMSO), 2.45 (s, 3H, CH3), 2.03 (s, 3H, CH3), 1.42 (s, 6H, 2×CH3). 13 13C{ 1 1H} NMR (125 MHz, d6-DMSO): δ / ppm: 166.0 (C q ), 160.9 (C q ), 158.8 (C q ), 138.47 (C q ), 134.6 (C q ), 132.9 (C q ), 132.7 (CH), 132.7 (C q ), 132.5 (C q), 130.4 (CH), 129.7 (CH), 128.9 (CH), 125.38 (C q ), 117.2 (C q ), 87.27 (C q ), 52.9 (CH3), 42.7 (CH2), 28.7 (2×CH3), 19.3 (CH3), 18.0 (CH3), 12.7 (CH3). m / z: ESI+: 453.5 ([M+Na] + , 18%), 431.3 ([M+H] + , 100).

[0118] 3-(N-((2,2,4,6,7-Pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzoic acid (S4a)

Chem.

[0119] S3a (200 mg, 465 μmol) was dissolved in THF (2.3 mL), and then 1 M LiOH solution (2.3 mL, 9.20 mmol) was added dropwise. The resulting solution was stirred at rt for 2 h. THF was removed under vacuum, and the reaction mixture was diluted with water (5 mL). Subsequently, the aqueous solution was acidified to pH 5 with HCl (1 M aqueous), and extracted with EtOAc (5 × 10 mL) and DCM (5 × 5 mL). The organic layer was dried (MgSO4) and concentrated under vacuum. Subsequently, the crude mixture was purified by column chromatography (4:1, chloroform:EtOH) to give the title compound as a colorless solid (176 mg, 91%). 11H NMR (500 MHz, d6-DMSO): δ / ppm: 13.22 (broad singlet, 1H, COOH), 9.03 (singlet, 1H, NH), 8.37 (singlet, 1H, NH), 8.10 (doublet, 1H, J = 7.8 Hz, ArH), 8.07 - 7.98 (multiplet, 2H, 2×ArH), 7.60 (triplet, 1H, J = 7.8 Hz, ArH), 2.99 (singlet, 2H, CH2), 2.50 (singlet, 3H, CH3 - hidden by DMSO), 2.46 (singlet, 3H, CH3), 2.03 (singlet, 3H, CH3), 1.42 (singlet, 6H, 2×CH3). 13 C{ 1 H} NMR (125 MHz, d6-DMSO): δ / ppm: 166.6 (C q ), 160.5 (C q ), 158.3 (C q ), 138.0 (C q ), 134.0 (C q ), 132.6 (C q ), 132.2 (CH), 132.1 (C q ), 131.8 (CH), 131.1 (C q ), 129.0 (CH), 128.6 (CH), 124.9 (C q ), 116.7 (C q ), 86.8 (C q ), 42.3 (CH2), 28.3 (2×CH3), 18.8 (CH3), 17.5 (CH3), 12.2 (CH3). m / z: ESI+: 439.2 ([M+Na] + , 30%), 417.2 ([M+H] + , 100).

[0120] (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(N-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)carbamimidoyl)benzamide)propanoic acid (S5a) [Chem.]

[0121] S4a (150 mg, 360 μmol) and HATU (137 mg, 360 μmol) were placed under an inert atmosphere and dry DCM (1.50 mL) was added. Subsequently, DIPEA (125 μL, 720 μmol) was added dropwise, and immediately afterwards the mixture was stirred for 30 minutes to dissolve the initial suspension. Subsequently, solid Fmoc-Dap-OH (141 mg, 432 μmol) was added in small portions with stirring to maintain solubility. Subsequently, the reaction was stirred for about 18 hours, volatiles were removed, H2O (5 mL) was added, and the pH was adjusted to 5 using HCl (1 M aqueous). Subsequently, the aqueous mixture was extracted with EtOAc (5 × 20 mL) and DCM (5 × 10 mL), the combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum. Subsequently, the crude mixture was purified by column chromatography (7:3, chloroform:EtOH), taken up in H2O:ACN, and lyophilized to give the title compound as a colorless solid (142 mg, 54%). 11H NMR (500 MHz, d6-DMSO): δ / ppm: 8.92 (s, 1H, NH), 8.67 (s, 1H, CONH), 8.25 (s, 1H, ArH), 8.03 (s, 1H, NH), 7.94 (dd, 2H, J = 13.2, 7.9 Hz, 2×ArH), 7.87 (d, 2H, J = 7.5 Hz, 2×Fmoc-ArH), 7.67 (d, 2H, J = 7.5 Hz, 2×Fmoc-ArH), 7.56 (t, 1H, J = 7.8 Hz, ArH), 7.42 - 7.36 (m, 2H, 2×Fmoc-ArH), 7.32 - 7.24 (m, 2H, 2×Fmoc-ArH), 4.30 - 4.25 (m, 2H, Fmoc-CH2), 4.22 - 4.18 (m, 1H, Fmoc-CH), 4.15 (br s, 1H, αCH), 3.62 - 3.57 (m, 2H, βCH2), 2.98 (s, 2H, Pbf-CH2), 2.50 (s, 3H, CH3 - hidden by DMSO), 2.45 (s, 3H, CH3), 2.02 (s, 3H, CH3), 1.41 (s, 6H, 2×CH3). 13 C{ 1 H} NMR (125 MHz, d6-DMSO): δ / ppm: 165.7 (C q ), 160.8 (C q ), 158.3 (2×C q ), 143.8 (2×C q ), 140.7 (2×C q ), 138.0 (C q ), 134.7 (C q ), 133.9 (C q ), 132.2 (C q ), 132.1 (C q ), 130.4 (CH), 130.3 (CH), 128.6 (CH), 127.6 (2×CH), 127.1 (2×CH), 126.8 (CH), 125.2 (2×CH), 124.9 (C q ), 120.0 (2×CH), 116.7 (Cq ), 86.8 (C q ), 65.6 (CH2), 49.4 (CH), 46.6 (CH), 42.2 (CH2), 41.5 (CH2), 28.3 (2×CH3), 18.8 (CH3), 17.6 (CH3), 12.2 (CH3). m / z: ESI+: 747.0 ([M+Na] + , 14%), 449.3 (20), 324.3 (70), 282.2 (100).

[0122] 1-(tert-Butoxycarbonyl)-1H-benzo[d]imidazole-(6 / 5)-carboxylic acid (S1b)

Chemical formula

[0123] 1H-Benzimidazole-6-carboxylic acid (100 mg, 614 μmol) was dissolved in 1,4-dioxane (1 mL) and cooled to 0 °C. Subsequently, Na2CO3 (1.63 mL, 10% aqueous) was added, and Boc2O (162 mg, 740 μmol) was added portionwise. Subsequently, the reaction mixture was warmed to rt and stirred overnight. Dioxane was removed under reduced pressure, H2O (5 mL) was added, and the pH was adjusted to 5. Subsequently, the aqueous mixture was extracted with EtOAc (5 × 10 mL), the organic layer was dried (MgSO4), filtered, and concentrated under vacuum. Subsequently, the crude mixture was purified by column chromatography (9:1, chloroform:EtOH) to give the title compound as a colorless solid (162 mg, 45%). mp 400 °C with decomposition. 1 1H NMR (500 MHz, d6-DMSO): δ / ppm: 13.01 (br s, 1H, COOH), 8.76 (s, 1H, NCHN), 8.27 (s, 1H, ArH), 8.03 (s, 2H, 2×ArH), 1.66 (s, 9H, 3×CH3). 13 C{ 11H NMR (125 MHz, d6-DMSO): δ / ppm: 167.2 (C q ), 147.3 (C q ), 144.4 (CH), 143.5 (C q ), 134.1 (C q ), 126.9 (C q ), 126.2 (CH), 121.5 (CH), 114.1 (CH), 86.0 (C q ), 27.5 (3×CH3). m / z: ESI+: 263.1 ([M+H] + , 30%), 207.0 (40), 163.0 ([M-Boc+H] + , 100).

[0124] (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-benzo[d]imidazole-(5 / 6)-carboxamide)propanoic acid (S2b)

Chemical Structure

[0125] S1b (50 mg, 191 μmol) and HATU (72 mg, 191 μmol) were placed under an inert atmosphere, and dry DCM (600 μL) was added. Subsequently, DIPEA (53.0 μL, 381 μmol) was added dropwise, and immediately afterwards the mixture was stirred for 30 minutes to dissolve the initial suspension. Subsequently, solid Fmoc-Dap-OH (75.0 mg, 229 μmol) was added in small portions with stirring to maintain solubility. Subsequently, the reaction was stirred for approximately 18 hours, the volatiles were removed, H2O (5 mL) was added, and the pH was adjusted to 5 using HCl (1 M aqueous). Subsequently, the aqueous mixture was extracted with EtOAc (5 × 10 mL) and DCM (5 × 5 mL), the combined organic layers were dried (MgSO4), filtered, and concentrated under vacuum. Subsequently, the crude mixture was purified by column chromatography (7:3, chloroform:EtOH), taken up in H2O:ACN, and lyophilized to give the title compound as a colorless solid and a mixture of isomers (85.0 mg, 78%). 1 H NMR (500 MHz, d6-DMSO): δ / ppm: 8.77 - 8.62 (m, 2H, NCHN & CONH), 8.56 - 8.44 (m, 0.5H, Iso1 ArH), 8.34 - 8.22 (m, 0.5H, Iso1 ArH), 8.00 - 7.79 (m, 4H, 2×FmocCH & 2×Iso2 ArH & Iso1 / 2 ArH), 7.72 - 7.66 (m, 2H, 2×FmocCH), 7.65 - 7.58 (m, 1H, OCONH), 7.45 - 7.33 (m, 2H, 2×FmocCH), 7.33 - 7.23 (m, 2H, 2×FmocCH), 4.34 - 4.25 (m, 3H, FmocCH2& αCH), 4.24 - 4.13 (m, 1H, FmocCH), 3.68 (br s, 2H, βCH2), 1.71 - 1.60 (m, 9H, 3×CH3). 13 C{ 1 H} NMR (125 MHz, d6-DMSO): δ / ppm: 172.2 (C q ), 166.6 (Iso1C q), 166.4 (Iso2C q ), 156.0 (C q ), 147.4 (Iso1C q ), 147.3 (Iso2C q ), 145.7 (C q ), 144.8 (C q ), 144.1 (CH), 143.8 (Iso1C q ), 143.4 (Iso2C q ), 140.7 (C q ), 133.0 (C q ), 131.3 (C q ), 130.9 (Iso1C q ), 130.5 (Iso2C q ), 127.6 (2×CH), 127.1 (2×CH), 125.2 (Iso1CH), 125.2 (Iso2CH), 124.6 (CH), 123.3 (CH), 120.1 (2×CH),119.7 (Iso1CH), 119.2 (Iso2CH), 113.8 (CH), 85.8 (Iso1C q ), 85.7 (Iso2C q ), 65.8 (CH2), 53.9 (CH), 46.6 (CH), 40.7 (CH2), 27.5 (Iso1 3×CH3), 27.5 (Iso2 3×CH3). m / z: ESI-: 569.1 ([M-H] - , 100%), 424.8 (8)

[0126] General peptide synthesis protocol On a Tribute (registered trademark) solid-phase peptide synthesizer, Rink amide resin (231 mg, 0.15 mmol, 0.65 mmol g -1) was swollen in dichloromethane (DCM) (5.00 mL) for 30 minutes. The terminal Fmoc group was deprotected using two 10-minute stirrings with a 20% (v / v) solution of piperidine in dimethylformamide (DMF) (5.00 mL). Activation of the Fmoc-protected amino acid (0.45 mmol, 3.00 equivalents) was achieved using HATU (0.38 mmol, 2.50 equivalents) and 0.5 M DIPEA in DMF (5.00 mL). Subsequently, the reaction mixture of the activated ester of the Fmoc amino acid was sequentially added to the resin and stirred at room temperature for 20 minutes. As soon as the final coupling of the appropriate residue was successful, the resin was washed with DCM (5.00 mL) and dried under N2. Subsequently, the resin was removed from the automated synthesizer and manually swollen in DCM (5.00 mL) for 30 minutes. After swelling, the resin was washed with DMF (4 × 2.00 mL), and the terminal Fmoc group was deprotected using two 10-minute stirrings with a 20% (v / v) solution of piperidine in DMF (5.00 mL). Separately, FITC (117 mg, 0.30 mmol) was dissolved in DMF (1.50 mL) in a 5.00 mL glass vial, and DIPEA (183 μL, 1.05 mmol) was added to form a bright red solution. Subsequently, the red FITC solution was added to the resin, protected from light, and stirred at room temperature for 16 hours. After 16 hours of stirring, the resin was washed with DMF (4 × 2.00 mL), then with MeOH (4 × 2.00 mL), and finally with DCM (4 × 2.00 mL). Subsequently, the peptide was cleaved from the resin using a mixture of TFA:phenol:water:TIPS (90:5:5:2, 3.00 mL) and stirred for 4 hours. Subsequently, the cleavage solution was filtered from the resin, and the peptide was precipitated using cold Et2O (30 mL, -20 °C) to obtain an orange precipitate. The solid was washed with Et2O (3 × 10 mL) to remove excess TFA, and the resulting orange solid was purified by RP-HPLC method A.

[0127] Peptide Characterization FITC-XRKKRRQRRR (Tat) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0128] [Chemical formula]

[0129] Using general peptide synthesis protocol A, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (22 mg, 6%). HRMS: C 80 H 133 N 33 O 16 S 4+ Theory: 658.3235 Observed: 658.3228. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 17.4 minutes, 100%.

[0130] FITC-XRLLRLLR (Pep-1) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)). [Chemical formula] Using general peptide synthesis protocol A, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (68 mg, 25%). HRMS: C 69 H 107 N 19 O 13 S 2+ Theory: 720.9003 Observed: 720.9060. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 24.6 minutes, 99%.

[0131] FITC-XRLLRRLLR (Pep-2) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0132] [Chemical formula]

[0133] Using general peptide synthesis protocol A, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (70 mg, 23%). HRMS: C 75 H 119 N 23 O 14 S 2+ Theory: 798.9508 Observed: 798.9549. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 24.3 minutes, 98%.

[0134] FITC-XRXRRBRRYQFLIRBRXR(PiP6) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0135]

Chemical formula

[0136] Using general peptide synthesis protocol A on a 0.20 mmol scale resin, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (20 mg, 3%).

[0137] HRMS: C 128 H 206 N 45 O 25 S.CF3CO2H 5+ Theory: 584.1176 Observed: 584.1211.

[0138] RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 19.7 minutes, 99%.

[0139] FITC-XPLILLRLLRG(TP-1) (Here, X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx).)

[0140]

Chem.

[0141] FITC-XPLIYLRLLRG (TP-2) (Here, X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx).)

[0142]

Chem.

[0143] FITC-XPLIYLKLLKG (TP-3) (Here, X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx).)

[0144]

Chem.

[0145] Using the general peptide synthesis protocol A, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (17 mg, 6%). HRMS: C 85 H 125 N 15 O 17 S 2+ Theoretical: 829.9544 Observed: 829.9544. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 29.0 minutes, 97%.

[0146] FITC-XPLIYLCitLLCitG(TP-4) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

Chem.

[0147] FITC-XPLIYLrLLrG(TP-5) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0148] [Chemical formula]

[0149] Using general peptide synthesis protocol A on a resin scale of 0.10 mmol. The appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (12 mg, 6%). HRMS: C 85 H 125 N 19 O 17 S 2+ Theory: 857.9605 Observed: 857.9600. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 28.6 minutes, 97%.

[0150] FITC-XPLIYLELLEG(TP-6) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0151] [Chemical formula]

[0152] Using general peptide synthesis protocol A on a resin scale of 0.75 mmol. The appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (8 mg, 6%). HRMS: C 83 H 115 N 13 O 21 S 2+ Theory: 830.9020 Observed: 830.9004. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 31.1 minutes, 98%.

[0153] FITC-XPLIYLAmLLAmG(Am-TP-2) (Here, X in the above array is a residue derived from 6-aminohexanoic acid (Ahx).)

[0154]

Chemical formula

[0155] On a Tribute (registered trademark) solid-phase peptide synthesizer, Rink amide chem matrix resin (50 mg, 0.026 mmol, 0.52 mmol g -1It was swollen in DCM (2.00 mL) for 30 minutes. The terminal Fmoc group was deprotected using two 10-minute stirrings with a 20% (v / v) solution of piperidine in DMF (4.00 mL). Activation of the Fmoc-protected amino acid (78.0 μmol, 3.00 equivalents) was achieved using HATU (65 μmol, 2.50 equivalents) and 0.5 M DIPEA in DMF (2.00 mL). Activation of the amidine S4 and the 6-position of Fmoc-Leu-OH was achieved using DIC (11.0 μL, 70.0 μmol, 2.70 equivalents) and HOAt (10.0 mg, 74.0 μmol, 2.85 equivalents) in DMF (2.00 mL). Subsequently, the reaction mixture of the activated ester of the Fmoc amino acid except for the amidine S4 and the 6-position of Fmoc-Leu-OH was sequentially added to the resin and stirred at room temperature for 20 minutes. The amidine S4 and the 6-position of Fmoc-Leu-OH activation solution was added to the resin and heated to 75 °C for 30 minutes. Once the final coupling of the appropriate residues was achieved, the resin was washed with DCM (2.00 mL) and dried under N2. Subsequently, the resin was removed from the automated synthesizer and manually swollen in DCM (2.00 mL) for 30 minutes. After swelling, the resin was washed with DMF (4 × 2.00 mL), and the terminal Fmoc group was deprotected using two 10-minute stirrings with a 20% (v / v) solution of piperidine in DMF (2.00 mL). Separately, FITC (28 mg, 0.078 mmol) was dissolved in DMF (1.00 mL) in a 5.00 mL glass vial, and DIPEA (32 μL, 0.182 mmol) was added to form a bright red solution. Subsequently, the red FITC solution was added to the resin, protected from light, and stirred at room temperature for 16 hours. After 16 hours of stirring, the resin was washed with DMF (4 × 2.00 mL), then with MeOH (4 × 2.00 mL), and finally with DCM (4 × 2.00 mL). Subsequently, the peptide was cleaved from the resin using a mixture of TFA:phenol:water:TIPS (90:5:5:2, 3.00 mL) and stirred at 60 °C for 4 hours. Subsequently, the cleavage solution was filtered from the resin, and the peptide was precipitated using cold Et2O (30 mL, -20 °C) to obtain an orange precipitate.The solid was washed with Et2O (3 × 10 mL) to remove excess TFA, and the resulting orange solid was purified by RP-HPLC method A. The appropriate HPLC fractions were lyophilized to give a powdery orange solid (2 mg, 4%).

[0156] HRMS: C 95 H 125 N 19 O 19 S 2+ . Theory: 933.9554 Observed: 933.9534. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 28.2 minutes, 98%.

[0157] FITC-XPLILLrLLrG(TP-1-D-Arg) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0158]

Chemical formula

[0159] FITC-XPLILLKLLKG(TP-1-Lys) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

Chemical formula

[0160] FITC-XPLILLCitLLCitG(TP-1-Cit) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0161]

Chemical formula

[0162] Using the general peptide synthesis protocol A, the appropriate HPLC fraction was lyophilized to obtain a powdery orange solid (7 mg, 3%). HRMS: C 82 H 125 N 17 O 18 S 2+ Theory: 833.9549 Observed: 833.9548. RP-HPLC (Analytical RP-HPLC method A, Kinetex 5μm 150×21.2mm XB C18 column) R t = 33.7 minutes, 98%.

[0163] FITC-XPLILLELLEG(TP-1-Glu) (wherein X in the above sequence is a residue derived from 6-aminohexanoic acid (Ahx)).

[0164]

Chemical formula

[0165] PepS1 FITC-XPLIYLBimLLBimG (Bim-TP-2) (where X is a residue derived from 6-aminohexanoic acid (Ahx); Bim is a residue derived from the modified amino acid S2b).

[0166]

Chemical Structure

[0167] On a Biotage Alstra® solid-phase peptide synthesizer, Tentagel S RAM resin (100 mg, 0.025 mmol, 0.25 mmol g -1) was swollen in DCM (2.00 mL) for 30 minutes. The N-Fmoc group was removed using two 10-minute stirrings with a 20% (v / v) solution of piperidine in DMF. All amino acids were coupled using DIC (1 equivalent to the amino acid) and oxyma pure (1 equivalent to the amino acid) in DMF at 75 °C for 30 minutes using microwave irradiation. Amino acids were used in a 3-fold excess during the coupling step. The coupling of the terminal FITC group was performed manually using FITC (7 equivalents) and DIPEA (14 equivalents) in DMF (1 mL) and stirred at room temperature for 16 hours. After 16 hours of stirring, the resin was washed with DMF (4 × 2.00 mL), then with MeOH (4 × 2.00 mL), and finally with DCM (4 × 2.00 mL). Subsequently, the peptide was cleaved from the resin using a mixture of TFA:phenol:water:TIPS (90:5:5:2, 3.00 mL) and stirred at 60 °C for 4 hours. Subsequently, the cleavage solution was filtered from the resin, and the peptide was precipitated using cold Et2O (30 mL, -20 °C) to obtain an orange precipitate. The solid was washed with Et2O (3 × 10 mL) to remove excess TFA, and the resulting orange solid was purified by RP-HPLC method A. The appropriate HPLC fractions were lyophilized to obtain a powdery orange solid (15 mg, 15%).

[0168] RP-HPLC (Analytical RP-HPLC method A, Aeris 2.6 μm 250×2.1 mm Peptide-XB C18 column) R t = 17.4 minutes, 98%. m / z: ESI+: 1863.0 ([M+H] + , 16%), 931.9 ([M+2H] 2+ , 100)

[0169] PepS2 FITC-XPLIYLmAmLLmAmG (mAm-TP-2) (where X is a residue derived from 6-aminohexanoic acid (Ahx); mAm is a residue derived from the modified amino acid 5a).

[0170]

Chemical Structure

[0171] On a Biotage Alstra (registered trademark) solid-phase peptide synthesizer, Tentagel S RAM resin (100 mg, 0.025 mmol, 0.25 mmol g -1 ) was swollen in DCM (2.00 mL) for 30 minutes. The N-Fmoc group was removed using two 10-minute stirrings with a 20% (v / v) solution of piperidine in DMF. All amino acids were coupled using DIC (1 equivalent) and oxyma pure (1 equivalent) in DMF at 75 °C for 30 minutes using microwave irradiation. The coupling of the terminal FITC group was performed manually using FITC (7 equivalents) and DIPEA (14 equivalents) in DMF (1 mL) and stirred at room temperature for 16 hours. After stirring for 16 hours, the resin was washed with DMF (4 × 2.00 mL), then with MeOH (4 × 2.00 mL), and finally with DCM (4 × 2.00 mL). Subsequently, the peptide was cleaved from the resin using a mixture of TFA:phenol:water:TIPS (90:5:5:2, 3.00 mL) and stirred at 60 °C for 4 hours. Subsequently, the cleavage solution was filtered from the resin, and the peptide was precipitated using cold Et2O (30 mL, -20 °C) to obtain an orange precipitate. The solid was washed with Et2O (3 × 10 mL) to remove excess TFA, and the resulting orange solid was purified by RP-HPLC method A. The appropriate HPLC fractions were lyophilized to obtain a powdery orange solid (7 mg, 15%).

[0172] RP-HPLC (analytical RP-HPLC method A, Aeris 2.6 μm 250 × 2.1 mm Peptide-XB C18 column) R t = 17.7 minutes, 95%. m / z: ESI+: 1866.7 ([M + H] + , 4%), 934.0 ([M + 2H] 2+ , 92), 623.1 ([M + 3H] 3+ , 100)

[0173] Cell culture HeLa cells and U2OS cells were maintained in a medium consisting of Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin at pH 7.4. HepG2 cells were maintained in a medium consisting of Dulbecco's Modified Eagle Medium (DMEM) without glucose and phenol red but containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin at pH 7.4. Cells were cultured in a humidified incubator at 37 °C with a 5% CO2 atmosphere. The PBS used was 1× PBS consisting of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 adjusted to pH 7.4. BSA, FBS, L-glutamine, penicillin / streptomycin, and TrypLE Express were purchased aseptically from sigma-aldrich or Thermo Fisher scientific and used without further treatment.

[0174] Flow cytometry HeLa cells or U2OS cells were cultured in a 24-well plate (4.5×10 5 cells per well) for 24 hours. On the day of the experiment, the cells were incubated with 5 μM FITC-peptide in DMEM containing 10% FBS at 37 °C or 4 °C for the required time. Subsequently, the medium was removed, the wells were washed with PBS (2×500 μL per well), and then the cells were detached from the plate using TrypLE Express (500 μL per well). PBS (500 μL) containing 2% bovine serum albumin was added to each well, and the cells were transferred to 5 mL polystyrene FACS tubes. The cells were centrifuged (5 minutes, 5000 rpm), washed with PBS (2×500 μL) containing 2% bovine serum albumin, and then resuspended in PBS (1.00 mL per FACS tube) containing 2% bovine serum albumin.

[0175] Finally, laser excitation / emission set at 490 nm / 519 nm and 200 μL aliquots -1Using the flow rate, flow cytometry data obtained by analyzing cells on a Thermo fisher Attune NxT flow cytometer were analyzed using FlowJo software with gating set in the range of 51K to 285K side scatter and 251K to 815K forward scatter dispersion for each sample. The median fluorescence intensity value was calculated from the gating range using FlowJo, The reported median fluorescence values were standardized by subtracting the mean background fluorescence at 490 nm / 519 nm of a set of untreated cells (analyzed with each experiment) from the values of each FITC-labeled peptide.

[0176] Cell viability AlamarBlue assay HeLa cells, U2OS cells or HepG2 cells were cultured in a 96-well plate (2.6×10 3 cells per well) for 24 hours. On the day of the experiment, the cells were incubated for 24 hours at 37°C in DMEM containing 10% FBS (total well volume of 100 μL) with 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM FITC-peptide in a 5% CO2 atmosphere. The experiment was performed in triplicate for each concentration. Subsequently, AlamarBlue® was added to each well (0.5 mM, 10 μL, 10% v / v) and incubated for an additional 6 hours for HeLa cells and HepG2 cells and 24 hours for U2OS cells. Subsequently, the cells were analyzed using an Hldex plate reader by laser excitation / emission set at 560 nm / 595 nm. The fluorescence of untreated cells was used as a 100% survival control, 1% Triton X was used as a negative control, and the cell viability was calculated as a percentage of the control fluorescence value using OriginPro 2019b software.

[0177] Confocal microscopy HeLa cells were seeded in an 8-well plate (Ibidi plate) (3.5×10 4They were cultured in (cells) for 24 hours. On the day of the experiment, the cells were incubated in DMEM containing 10% FBS (total well volume of 200 μL) with 5 μM FITC-peptide at 37 °C for 4 hours in a 5% CO2 atmosphere. After 3.5 hours of incubation, Hoechst 33258 was added at a concentration of 5.6 μM, and the cells were incubated at 37 °C for the last 0.5 hour. The cells were removed from the incubator and the medium was removed. Subsequently, each well was washed with serum- and phenol red-free medium (2 × 200 μL), and then incubated with 4% formaldehyde in PBS (200 μL) at room temperature for 20 minutes. After incubation, the 4% formaldehyde solution was removed and PBS (200 μL) was added to each well. Subsequently, the cells were imaged using a Leica S8 confocal microscope. The excitation / emission was set at 352 nm / 461 nm for Hoechst 33258 imaging and 495 nm / 519 nm for FITC-labeled peptide. The images were processed using ImageJ software. The images were averaged overall for each channel set with "rolling ball" to remove background fluorescence. Additional synthetic routes for further exemplary modified amino acids are shown below.

[0178]

Chemical formula

[0179]

Chemical formula

[0180]

Chemical formula

[0181] [Chemistry] Scheme 7. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure.

[0182] [Chemistry] Scheme 8. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure.

[0183] [Chemistry] Scheme 9. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure.

[0184] [Chemistry] Scheme 10. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure.

[0185] [Chemistry] Scheme 11. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure. The route is shown in the context of a compound having a para-substitution pattern with respect to the phenyl ring. Similar synthetic routes are used for analogs having ortho and meta substitution patterns with respect to the phenyl ring.

[0186] [Chemistry] Scheme 12. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure. The route is shown in the context of a compound having a para-substitution pattern with respect to the phenyl ring. Similar synthetic routes are used for analogs having ortho and meta substitution patterns with respect to the phenyl ring.

[0187] [Chemistry] Scheme 13. Synthetic route for synthesizing a modified amino acid according to an embodiment of the present disclosure.

[0188]

Chemical formula

[0189] Results and Discussion In the following studies, the use of amidine groups as potential arginine mimetics was investigated. The approach was to first identify an existing cell-penetrating peptide (CPP) sequence that exhibits high levels of cell uptake and distribution but minimal levels of cytotoxicity. This provided a robust basis for investigating the effect of replacing Arg residues with Am building blocks (such as those shown in Fig. 1c).

[0190] Three CPP prototypes selected for comparative studies are shown in Fig. 1b. Tat is a prototypical CPP that has been extensively investigated as a delivery tool (Green, M.; Ishino, M.; Loewenstein, P. M. Mutational analysis of HIV-1 Tat minimal domain peptides: Identification of <em>trans< / em>-dominant mutants that suppress HIV-LTR-driven gene expression. Cell 1989, 58, 215-223). TP-1 and TP-2 are synthetic CPPs identified by high-throughput screening (Guha et al, Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem. Rev. 2019, 119, 6040-6085; and Marks et al, Spontaneous Membrane-Translocating Peptides by Orthogonal High-Throughput Screening. J. Am. Chem. Soc. 2011, 133, 8995-9004), while PiP6 was developed to enhance the uptake of a therapeutic oligonucleotide (PMO) sequence for the treatment of Duchenne muscular dystrophy (Betts et al, Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment. Mol. Ther. Nucleic Acids 2012, 1).

[0191] Cell viability studies revealed that the lowest cytotoxicity across all three classes of CPPs investigated was for TP-1 and TP-2, both of which were well tolerated by HeLa cells up to 100 μM (Figure 2a). Tat showed a moderate level of toxicity at 100 μM, while cells incubated with PiP6 showed only 15% cell viability at 30 μM and 5% at 100 μM. This toxicity trend was also consistent in the HepG2 and U2OS cell lines. Previous studies on the TP series emphasized the importance of the Arg placement with respect to the hydrophobic core in the central Arg-Leu-Leu-Arg motif (Marks, J. R.; Placone, J.; Hristova, K.; Wimley, W. C. Spontaneous Membrane-Translocating Peptides by Orthogonal High-Throughput Screening. J. Am. Chem. Soc. 2011, 133, 8995-9004). To further investigate this, repeats of this core motif were evaluated (Pep-1, Pep-2). Cell viability studies revealed that the placement of Arg and Leu residues and the nature of the repeat unit played a significant role in toxicity, with Pep-2 showing cytotoxicity at 100 μM compared to Pep-1.

[0192] Cell uptake / retainment studies in the HeLa cell line revealed the time-dependent profiles for Tat and PiP6 showing maximal uptake at 37 °C for 1 h (Figure 2b). This suggests an energy-dependent uptake mechanism for the TP series (Madani, F.; Lindberg, S.; Langel, U.; Futaki, S.; Graslund, A. Mechanisms of Cellular Uptake of Cell-Penetrating Peptides. J. Biophys. 2011, 2011, 414729), which is different from the active endocytosis mechanism of uptake for Tat and PiP6 (Guha, S.; Ghimire, J.; Wu, E.; Wimley, W. C. Mechanistic Landscape of Membrane-Permeabilizing Peptides. Chem. Rev. 2019, 119, 6040-6085; Boisguerin, P.; Deshayes, S.; Gait, M. J.; O'Donovan, L.; Godfrey, C.; Betts, C. A.; Wood, M. J. A.; Lebleu, B. Delivery of therapeutic oligonucleotides with cell penetrating peptides. Adv. Drug Deliv. Rev. 2015, 87, 52-67; Kauffman, W. B.; Guha, S.; Wimley, W. C. Synthetic molecular evolution of hybrid cell penetrating peptides. Nat. Commun. 2018, 9). This is in contrast to the enhanced uptake levels for the TP series at 4 h; this trend was also observed in the U2OS cell line. Due to enhanced intracellular retention and reduced toxicity, the peptides of the TP series were advanced for further evaluation.

[0193] Subsequently, the effects of both Arg residues in the TP series on toxicity, cellular uptake, and distribution were investigated. Cellular viability studies of the TP peptides revealed very small changes in toxicity when both Arg residues were replaced by Lys, Glu, Cit, or D-Arg, but analysis of cellular uptake of TP-1 and TP-2 (Figure 3a) revealed the importance of the positive charges present at these sites. For example, TP-3 (L-Lys) showed no difference in cellular uptake compared to the TP-2 prototype. Subsequently, a reduction in cellular uptake was observed in TP-4 incorporating two L-Cit residues, followed by a further reduction in uptake in TP-5 (L-Glu). Inversion of the stereochemistry to D-Arg (TP-5) enhanced uptake compared to TP peptides containing L-Arg at both positions (e.g., TP-1 and TP-2). A related observation is the enhanced level of uptake observed for the TP peptides compared to Pip6 and Tat at 4 hours. Thus, the inventors conclude that the cellular uptake characteristics of the TP series are associated with positively charged residues at both Arg positions in TP-1 and TP-2.

[0194] Uptake was further investigated using unnatural amino acid building blocks (L-Am, Figure 1c). The underlying hypothesis for this study was that the combination of increased hydrophobicity and reduced basicity of the benzamidine building block compared to the guanidinium group would enhance uptake. Direct substitution of the Arg residue with two L-Am building blocks (Am-TP-2) resulted in a marked four-fold increase in uptake compared to TP-2, thereby confirming this hypothesis (Figure 3a). The time and temperature dependence of Am-TP-2 uptake followed the same trend as that observed for TP-1 and TP-2 (Figure 2b - c), with enhanced uptake observed at 4 hours of incubation compared to 1 hour (Figure 3b). Furthermore, the cellular uptake of Am-TP-2 appears to follow a similar energy-dependent pathway as observed by a 30-fold decrease at 4 °C compared to 37 °C (Figure 3c). Consistent with what was observed for TP-1 and TP-2, the trends in enhanced uptake of Am-TP-2 were also observed in the U2OS cell line.

[0195] Finally, the intracellular distribution profiles of fluorescein-tagged CPPs were investigated in the HeLa cell line. As previously confirmed in various studies, the distribution profile of Tat proceeds through an endocytosis mechanism that includes encapsulation within endosomes, which is reflected by the presence of fluorescent-tagged Tat accumulating in puncta (Figure 4a). The distribution profile of PiP6 is very different and includes high-level accumulation in the nucleus (Figure 4b). TP-1 (Figure 4c) and TP-2 (Figure 4d) are widely distributed throughout the cytoplasm, and while this distribution spreads into the nucleus, it is not at the same concentration as in the cytoplasm (Figure 4e). The cytoplasmic distribution profiles of the TP series also match those observed for Am-TP-2, suggesting that Arg residues within the TP series affect uptake but need to be considered in the context of the full-length sequence.

[0196] To date, the 4-fold increase in cellular uptake of Am-TP-2 compared to TP-2 emphasizes the relative flexibility of the cellular uptake profiles of the TP series. Initial reports of the TP series reported that the mechanism of uptake was via passive diffusion, but the results herein suggest that other energy-dependent pathways, including supramolecular interactions with phospholipid membranes and specific interactions with cell surface glycoproteins, can influence uptake. These studies demonstrate the potential to alter the cell membrane permeation properties of CPPs. In particular, these studies highlight the potential of the TP series as a CPP scaffold suitable for further evaluation as a delivery vector for large molecular cargos that may be through bioconjugate formation, or as part of a delivery vector such as a liposome formulation or multivalent nanoparticles. These studies also show that replacement of the guanidinium group of Arg by benzamidine is not only tolerated, but actually improves the cellular uptake and distribution properties when compared to TP-2.

[0197] References: TIFF2025520703000076.tif196153 TIFF2025520703000077.tif205153Although the present invention has been described in some detail for purposes of clarity of understanding by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. A cell membrane-permeable peptide containing at least one modified amino acid residue according to formula (II): 【Chemistry 1】 (In the formula, R 1 is selected from H, protecting groups, and amino acid residues; R 8 It is selected from H and amino acid residues; X is absent or may be substituted C 1 -C 6 alkyl, optionally substituted -C 1 -C 6 alkyl-NR 2 (C=O)-, and optionally substituted -C 1 -C 6 alkyl-(C=O)NR 2 -selected from; R 2 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; and In the formula, A is: (i) 【Chemistry 2】 (wherein B is an optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C) 1 -C 6 Selected from alkyl groups; Y is either absent or NH; In the formula, R 3 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; However, C may be substituted for B. 1 -C 6 If it is alkyl, Y is absent); or (ii) A bicyclic fused ring system containing an amidine-like motif that follows the following formula: 【Transformation 3】 (In the formula, R 4 and R 6 Each is independently selected from H and carbon atoms that form part of the bicyclic ring system skeleton; R 5 is selected from H and protecting groups; and R 7 (A carbon atom is a carbon atom that forms part of the framework of a bicyclic ring system.) (Selected from) And at least one modified amino acid residue has the following structure: 【Chemistry 4】 A cell membrane-permeable peptide that is not derived from modified amino acids, including those mentioned above.

2. The cell membrane permeable peptide according to claim 1, wherein Y is absent.

3. The cell membrane permeable peptide according to claim 1, wherein at least one arginine residue is replaced by at least one modified amino acid according to formula (II).

4. R 1 The cell membrane permeable peptide according to claim 1, wherein the peptide is selected from H, an amino acid residue, and 9-fluorenylmethyloxycarbonyl (Fmoc).

5. X is absent or -CH 2 -ien-CH 2 NH(C=O)- and -CH 2 A cell membrane-permeable peptide according to claim 1, selected from (C=O)NH-.

6. X is CH 2 The cell membrane permeable peptide according to claim 1, wherein it is NH(C=O)-.

7. A: 【Transformation 5】 and B is an optionally substituted phenyl or optionally substituted pyridyl; Y is either absent or NH; and R 3 C may be substituted. 1 -C 6 A protecting group that is unstable to alkyl or acid, The cell membrane-permeable peptide according to claim 1.

8. The cell membrane permeable peptide according to claim 1, wherein at least one modified amino acid is represented by formula (Ia'). 【Transformation 6】 (In the formula, R 1 is selected from H, protecting groups, and amino acid residues; X may be replaced by C. 1 -C 6 Alkyl, may be substituted -C 1 -C 6 Alkyl-NR 2 (C=O)-, and possibly substituted-C 1 -C 6 Alkyl-(C=O)NR 2 - Selected from; R 2 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; B is selected from optionally substituted aryls and optionally substituted heteroaryls; R 3 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; and R 8 (Selected from H and amino acid residues)

9. The cell membrane permeable peptide according to claim 1, wherein A is a bicyclic condensed ring system selected from the group consisting of quinazolinyl, benzimidazolyl, and tetrahydronaphthilidinyl (e.g., 1,2,3,4-tetrahydro-1,8-naphthilidinyl).

10. A: 【Transformation 7】 (In the formula, R 3a H, C 1 -C 6 Selected from alkyl groups (e.g., methyl) and acid-unstable protecting groups (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) or Boc); and R 5a H, C 1 -C 6 (Selected from alkyl groups (e.g., methyl) and acid-unstable protecting groups (e.g., Pbf or Boc)) A cell membrane-permeable peptide according to claim 1, selected from the above.

11. At least one modified amino acid is of formula (IIa) or formula (IIb): 【Transformation 8】 (In the formula, R 1b is selected from H, a protecting group (e.g., Fmoc), or an amino acid residue; R 3b H, C 1 -C 6 Selected from alkyl and protecting groups (e.g., Pbf); R 5a H, C 1 -C 6 Selected from alkyl and protecting groups (e.g., Pbf or Boc); and R 8 (Selected from H and amino acid residues) Includes a structure that follows; The group containing the amino acid moiety is added to the carbon atom on the aryl ring at any chemically suitable position. The cell membrane-permeable peptide according to claim 1.

12. The following array: (i) RKKRRQRRR - Sequence ID 1 (ii) PLILLRRLLRG - Sequence ID 2 (iii) PLIYLRLLRG - Sequence ID 3 (iv)RXRRBRRYQFLIRBRXR-Sequence No. 4 Select one or more of the following: X is 6-aminohexanoic acid (Ahx) and B is β-alanine; In each of sequence numbers 1-4, one or more arginine (R) residues are replaced by at least one modified amino acid residue. The cell membrane-permeable peptide according to claim 1.

13. Sequence ID 5, Sequence ID 9, Sequence ID 10, or Sequence ID 11: (i) PLIYLXLLLG-SEQ ID NO: 5 Each X in Sequence ID No. 5 is as defined in formula (II), and each X may be the same or a different modified amino acid; (ii)XPLIYLAmLLAmG-SEQ ID NO: 9 In Sequence ID No. 9, X is a residue derived from 6-aminohexanoic acid (Ahx), and each Am is a modified amino acid residue following the structure below: 【Chemistry 9】 (iii)XPLIYLBimMLLBimG-Sequence No. 10 In Sequence ID No. 10, X is a residue derived from 6-aminohexanoic acid (Ahx), and each Bim is a modified amino acid residue following the structure below: 【Chemistry 10】 (iv)XPLYLmAmLLmAmG-SEQ ID NO: 11 In Sequence ID No. 11, X is a residue derived from 6-aminohexanoic acid (Ahx), and each mAm is a modified amino acid residue following the structure below: 【Chemistry 11】 A cell membrane-permeable peptide according to claim 1, comprising a sequence that follows.

14. A cell membrane-permeable peptide according to claim 1, which is associated with and / or further comprises the target drug.

15. The cell membrane-permeable peptide according to claim 14, wherein the target drug is selected from the group consisting of therapeutic agents, diagnostic agents, and contrast agents, and optionally the target drug associates with the peptide via chemical linkage or non-covalent interactions.

16. A pharmaceutical composition comprising a cell membrane-permeable peptide and a therapeutic agent according to any one of claims 1 to 15, together with one or more pharmaceutically acceptable excipients, carriers and / or diluents.

17. A pharmaceutical composition according to claim 16 for use in therapy.

18. A therapeutic method comprising the step of administering to a non-human subject requiring such treatment a therapeutic dose of a cell membrane permeable peptide according to any one of claims 1 to 15, or a pharmaceutical composition comprising the cell membrane permeable peptide and a therapeutic agent together with one or more pharmaceutically acceptable excipients, carriers and / or diluents.

19. Use of a cell membrane-permeable peptide according to any one of claims 1 to 15 in the manufacture of a pharmaceutical product for therapeutic and / or medical use.

20. A method for preparing a cell membrane-permeable peptide comprising at least one modified amino acid as described in any one of claims 1 to 15.

21. The step of providing a novel peptide linkage by contacting a first amino acid (or a peptide fragment containing a residue of the first amino acid) with a second amino acid (or a peptide fragment containing a residue of the second amino acid) under conditions that promote and / or accelerate the condensation reaction between the first and second amino acids (or peptide fragments containing their residues). Includes, At least one of the first and second amino acids is at least one modified amino acid as defined in claim 1. The method according to claim 20.

22. A step of providing a candidate peptide comprising at least one modified amino acid residue as defined in any one of claims 1 to 11 (for example, as defined in formula (II), (IIa), or (IIb); Steps include contacting the candidate peptide with cells; and Steps to determine the effect of candidate peptides on cells. A method for screening cell membrane-permeable peptides, including [specific peptides].

23. (i) Cellular uptake of candidate peptides; (ii) Intracellular distribution of candidate peptides; and / or (iii) Toxicity of candidate peptides (against cells) One or more of the steps to determine The method according to claim 22, further comprising:

24. A step of identifying peptide sequences that can function as cell membrane-permeable peptides; and Steps to specify the aforementioned sequence as the parent peptide. The method according to claim 22, further comprising:

25. A step of providing a candidate peptide by replacing one or more arginine residues in the parent peptide with modified amino acid residues as defined in claim 1. The method according to claim 24, further comprising:

26. A step to compare the effects of candidate peptides on cells against a reference level. The method according to claim 25, further comprising, wherein the reference level is obtained by determining the action of the parent peptide on cells.

27. A modified amino acid following formula (I''). 【Chemistry 12】 (In the formula, R 1 is H or a protecting group; R 8 is H or a protecting group; and (i) X may be replaced by C 2 -C 4 Alkyl, may be substituted -C 1 -C 3 Alkyl-NR 2 (C=O)- and possibly substituted C 1 -C 3 Alkyl-(C=O)NR 2 - Selected from; R 2 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; And A is: 【Chemistry 13】 And, In the formula, B is an optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted C. 1 -C 6 Alkyl (C 1 -C 3 Selected from alkyl groups, etc.; Y is absent; R 3 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; or (ii) X is absent or may be substituted C 1 -C 6 alkyl (e.g., C 1 -C 3 alkyl), optionally substituted -C 1 -C 6 alkyl-NR 2 (C=O)-(e.g., -C 1 -C 3 alkyl-NR 2 (C=O)-), and optionally substituted C 1 -C 6 alkyl-(C=O)NR 2 -(e.g., C 1 -C 3 alkyl-(C=O)NR 2 -); is selected from R 2 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; and A is: 【Chemistry 14】 Selected from, In the formula, R 5a (Selected from H and protecting groups)

28. R 1 is H or 9-fluorenylmethyloxycarbonyl (Fmoc), and / or R 8 The modified amino acid according to claim 27, wherein is H.

29. X is CH 2 The modified amino acid according to claim 27, wherein it is NH(C=O)-.

30. A: 【Chemistry 15】 And; B is an optionally substituted phenyl or optionally substituted pyridyl; Y is absent; and R 3 C may be substituted. 1 -C 6 A protecting group that is unstable to alkyl or acid, The modified amino acid according to claim 27.

31. A modified amino acid according to claim 27, represented by formula (Ia''). 【Chemistry 16】 (In the formula, R 1 is H or a protecting group; R 8 is H or a protecting group; X may be replaced by C. 2 -C 4 Alkyl, may be substituted -C 1 -C 3 Alkyl-NR 2 (C=O)- and possibly substituted C 1 -C 3 Alkyl-(C=O)NR 2 - Selected from; R 2 H, and C which may be substituted. 1 -C 6 Selected from alkyl and protecting groups; B is selected from optionally substituted aryls and optionally substituted heteroaryls; and R 3 H, and C which may be substituted. 1 -C 6 (Selected from alkyl and protecting groups)

32. A: 【Chemistry 17】 Selected from, In the formula, R 3a H, C 1 -C 6 Selected from alkyl groups (e.g., methyl) and acid-unstable protecting groups (e.g., 2,2,4,6,7-pentamethyl-dihydro-benzofuran-5-sulfonyl (Pbf) or Boc); and R 5a H, C 1 -C 6 Selected from alkyl groups (e.g., methyl) and acid-unstable protecting groups (e.g., Pbf or Boc), The modified amino acid according to claim 27.

33. Structure following formula (Ib''): Structure following formula (Ib''): [Chemistry 18] (In the formula, R 1b is selected from H and a protecting group (e.g., Fmoc); and R 3b H, C 1 -C 6 Selected from alkyl and protecting groups (e.g., Pbf); R 8b H and protecting groups (e.g., C 1 -C 6 (Selected from alkyl) Including; The group containing the amino acid moiety is added to the carbon atom on the aryl ring at any chemically suitable position. The modified amino acid according to claim 27.

34. A peptide comprising a residue derived from a modified amino acid as described in any one of claims 27 to 33.

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