Star graft copolymer

Abstract

A novel star polymer comprising a graft copolymer backbone arm useful for delivering an active agent and / or a contrast agent to a target cell or tissue, for example.

Description

Technical Field

[0001] The present invention relates to novel star polymers comprising graft copolymer backbone arms useful for delivering, for example, active agents and / or contrast agents to target cells or tissues.

Background Art

[0002] Considerable efforts have been made to develop novel polymer structures with specificity for use as targeted drug delivery systems, including large molecules such as polypeptides and nucleic acids.

[0003] Delivery of an active ingredient to a target site located within a cell requires a suitable delivery carrier that provides appropriate protection and enables efficient delivery of the active ingredient to a specific tissue in the body.

[0004] Delivery carriers must overcome different extracellular and intracellular barriers to reach their target sites within the cell. Viral vectors may be more efficient in gene delivery compared to non-viral vectors, but their use involves several risks including toxicity, immunogenicity, and limitations on the size of the genetic cargo. Non-viral vectors are generally safer and more convenient for large-scale production.

[0005] EP 3331937 B1 discloses non-viral vectors made from a family of 3-arm star polypeptide derivatives consisting of a 1,3,5-benzenetricarboxamide-related central core and three polypeptide backbone arms used as initiators for the ring-opening polymerization of N-carboxy anhydride monomers. The novel graft copolymer compounds described herein are not directly and explicitly disclosed in EP 3331937 B1.

Summary of the Invention

[0006] The problem to be solved by the present invention can be considered to be to provide a novel graft copolymer having novel and improved properties and capable of delivering, for example, an active agent and / or a contrast agent to target cells or tissues.

[0007] According to the technical field, the term "graft copolymer" generally relates to a general class of segmented copolymers consisting of a linear backbone of one composition and randomly distributed branches of different compositions (see, for example, FIG. 1 in this specification for illustration).

[0008] As discussed above, European Patent No. 3331937B1 discloses a non-viral vector made from a family of three-arm star-shaped polypeptide derivatives. As is known in the technical field, star-shaped polypeptides are branched-chain polymers consisting of various linear chains linked to a central core.

[0009] Claim 4 of European Patent No. 3331937B1 discloses a compound structure including a (C 1 ~C 500 )-alkyl that can be substituted at different positions, for example, a "R 2··· " lysine (Lys) or glutamic acid (Glu) side-chain radical bonded to a so-called CL radical. The CL radical is not directly and clearly described in the relevant amino acid structure in this specification, that is, this specification does not directly and clearly describe the structure in which the "R 2··· " amino acid side chain of the R-arm structure of claim 4 is bonded to an amino acid-based structure.

[0010] In other words, European Patent No. 3331937B1 does not disclose a graft copolymer in which the amino acid side chain of the linear backbone is bonded to a branch having an amino acid structure.

[0011] Therefore, as a mere example, European Patent No. 3331937B1 discloses an R 2···is the Lys(PLys) side chain, and it is a graft copolymer structure (see, for example, the example of the "StPLys-g-PGA" structure in FIG. 2 herein) where it is bonded to, for example, a glutamate (PGA)-based branched structure, or R 2··· is the Glu(PGA) side chain, and it does not disclose a graft copolymer structure (see, for example, the example of the "PGA-g-PSar-g-PHis" structure in FIG. 2 herein) where it is bonded to a sarcosine (PSar)-based branched structure.

[0012] Although not limited to theory as a whole, the present invention is based on the inventors' discovery that by preparing a graft copolymer in which, for example, the linear backbone Lys / Orn (ornithine) and / or Glu / Asp (aspartic acid) amino acid side chains are bonded to branches having an amino acid structure, a graft copolymer having novel and improved properties compared to the star polypeptide structure in European Patent No. 3331937B1 can be obtained.

[0013] The novel graft copolymers considered herein may sometimes be generally referred to as star brushes herein.

[0014] As discussed in more detail below (see, for example, the examples), the novel graft copolymers considered herein can have many different useful advantages, such as not requiring a self-assembly process to form a structure in the nanometer range (e.g., a radius of 4 - 300 nm) that can be useful for delivering an active agent to a target cell, for example, because the molecular weight (MW) is very high.

[0015] As considered in

[0007] of European Patent No. 3331937B1, the star polypeptides considered there generally need to undergo a self-assembly process (which can become complex to control on an industrial scale) to form structures in the nanometer range. Therefore, the fact that a self-assembly process may not be required in the case of the novel star brush graft copolymers considered herein can be regarded as an advantage over the star polypeptides described in European Patent No. 3331937B1.

[0016] A further advantage of the novel graft copolymers considered herein is that by selecting different / suitable amino acid side chains having the desired properties (e.g., hydrophobic / hydrophilic or cationic / anionic), they have very high flexibility for preparing graft copolymer compounds for the targeted required purposes.

[0017] For example, Example 1 below describes an example of a graft copolymer designed to obtain a unimolecular aggregate having a hydrophobic pocket along with the ability to encapsulate the desired hydrophobic payload / drug (see, for example, Compounds 8-11 below).

[0018] For example, graft copolymers having both different degrees of hydrophobicity and hydrophilicity can also be prepared (see, for example, Compounds 8-12 of Example 1 below).

[0019] For example, a drug of interest (e.g., a small molecule or a cell targeting agent such as a therapeutic peptide, for example) can also be bound to the graft copolymers described herein (see, for example, Example Compounds 15, 18, 22 considered below).

[0020] Therefore, a first aspect of the present invention is of formula Ia comprising a homopolypeptide or a random or block or graft copolymer polypeptide:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0021] For the sake of mere illustration regarding the scope of the first aspect, the exemplary structure of the figures in this specification is considered below.

[0022] The exemplary "St-PLys-g-PGA" structure (also known as "St-PLys-g-PGlu") in Figure 2 of this specification may be regarded, in the language of the first aspect, as a structure in which grafted Lys / Orn-related units are present (i.e., the integer "o" is not 0), and grafted Glu / Asp-related units are not present (i.e., the integer "r" is 0).

[0023] The term "St" in the "St-PLys-g-PGA" nomenclature relates to a central core structure within the scope of formula (Ia) of the first aspect, such as the star-shaped 1,3,5-benzenetricarboxamide ring-related central core of the structure illustrated in Figure 2.

[0024] The exemplary "St-PLys-g-PHis-b-PGA" structure in Figure 2 also includes a PGA block (b), i.e., the integer "q" is not 0.

[0025] The exemplary "St-PGA-g-PSar-g-PHis" structure in Figure 2 may be regarded, in the language of the first aspect, as a structure in which grafted Glu / Asp-related units are present (i.e., the integer "r" is not 0), and grafted Lys / Orn-related units are not present (i.e., the integer "o" is 0).

[0026] The exemplary compound 10D below, St-PLys(18)-g-PHis(5)-b-PSar(20), may be regarded, in the language of the first aspect, as a structure in which the integer "o" is 18, the integer "p" is 5, and the integer "q" is 20.

[0027] As discussed above, the first aspect is interpreted as "each of the main repeating units defined by square brackets together with the numerical value 'r' may be the same or different from each other, and may include blocks of secondary monomer units defined by square brackets together with the numerical values's' and 't' which may be the same or different from each other according to the definition of the different substituents."

[0028] Accordingly, the exemplary "PGA-g-PSar-g-PHis" structure of FIG. 2 is an example of a situation within the scope of claim 1, where some contain PSar and some contain Phis substituents, so the "s" units are different.

[0029] According to the art, as shown in FIG. 1 herein · The term "Grafting from" relates to a method that includes an active site on a linear backbone capable of initiating polymerization and thus a functional group that functions as an initiator for grafting therefrom, and · The term "Grafting to" relates to a method that includes the use of a backbone chain having a functional group that is not used as such to initiate polymerization and can thus be said to be used as a "bonding point" for a pre-synthesized polymer.

[0030] As is known in the art, the "H 3 N + " (amino) group of the Lys side chain (see, for example, the "St-PLys-g-PGA" structure in FIG. 2) may be the backbone initiator active site in the "Grafting from" method being considered herein, i.e., the amine group functions as an initiator for grafting therefrom.

[0031] As is known in the art, the "COO -The (carboxyl) group (for example, refer to the "PGA-g-PSar-g-PHis" structure in Figure 2) may be the backbone "bonding point" functional group in the "Grafting to" method being considered in this specification, that is, the carboxyl group is used as a "bonding point" for a pre-synthesized polymer, for example, via their terminal amines.

[0032] In short, "Grafting from" and "Grafting to" are methods well-known to those skilled in the art. That is, based on the technical information and common general knowledge disclosed in this specification, those skilled in the art can routinely identify a process suitable for the synthesis of the graft copolymer compound of the first aspect, that is, those skilled in the art can routinely prepare the compound of the first aspect.

[0033] In the formula (I) of the first aspect, the main repeating unit defined by square brackets together with the numerical value "t" can be regarded as being related to the use of, for example, alanine / sarcosine / glycine (Ala / Sar / Gly).

[0034] Some of the examples in this specification describe structures where "t" is an integer of zero, and thus the integer "t" may preferably be 0.

[0035] As understood by those skilled in the art in this context, in the phrase of the first aspect interpreted as "a graft copolymer compound containing the backbone of the repeating structural unit of formula (I)", the term "containing" should be understood to mean that the graft copolymer compound of the present invention may contain other structures (such as repeating units) other than the structures explicitly shown for the formula (I) of the first aspect.

[0036] For example, the graft copolymer compound of the present invention may contain repeating units similar to the unit "t", and this unit contains an amino acid-related structure different from the Ala / Sar-related amino acids of the unit "t" (such as arginine (Arg), cysteine (Cys), etc.).

[0037] The second aspect of the present invention relates to a polymer complex comprising the graft copolymer of the first aspect and / or its embodiments and at least a pharmaceutical, veterinary or cosmetic active agent.

[0038] As considered herein, based on common general knowledge and the technical disclosure herein, it is routine work for a person skilled in the art to prepare the polymer complex of the second aspect of the present invention and / or its embodiments.

[0039] The third aspect of the present invention relates to a pharmaceutical, veterinary or cosmetic composition comprising at least one polymer complex of the second aspect and / or its embodiments together with one or more suitable excipients or carriers.

[0040] Based on common general knowledge and the technical disclosure herein, it is routine work for a person skilled in the art to prepare the pharmaceutical, veterinary or cosmetic composition of the third aspect of the present invention and / or its embodiments.

[0041] The fourth aspect of the present invention relates to the polymer complex of the second aspect and / or its embodiments for use as a drug or the pharmaceutical composition according to the third aspect and / or its embodiments.

[0042] The fifth aspect of the present invention relates to the polymer complex of the second aspect and / or its embodiments or the pharmaceutical composition according to the third aspect and / or its embodiments for use as (i) a transfection reagent for transfecting at least one active agent into cells, (ii) for in vivo or ex vivo production of a biologic encoding a recombinant protein, peptide or antibody or production of a recombinant virus, (iii) for use as a therapeutic or prophylactic vaccine against viral infection or a therapeutic vaccine against cancer, or (v) for use in genome engineering, cell reprogramming, cell differentiation or gene editing.

[0043] Based on common general knowledge and the technical disclosure in this specification, it is routine work for a person skilled in the art to identify the suitable medical uses of the polymer composites and / or pharmaceutical compositions described herein.

[0044] For example, many of the medical uses described in the art for related polymer compounds (see, for example, European Patent No. 3331937B1 discussed above) may also be relevant to the graft copolymer compounds of the present invention.

[0045] A sixth aspect of the present invention relates to a method for delivering a pharmaceutically active agent (e.g., a nucleic acid) into a target cell, comprising administering to an animal including a human a composition (e.g., a solution) containing the polymer composite of the second aspect and / or its embodiments so as to physically contact the target cell with the composite, thereby delivering the pharmaceutically active agent into the cell.

[0046] Based on common general knowledge and the technical disclosure in this specification, it is routine work for a person skilled in the art to identify a method suitable for delivering a desired active agent to a desired target cell.

[0047] The examples in this specification describe that the graft copolymer compounds of the present invention can physically contact a target cell of interest, thereby delivering the active agent of interest into the cell.

[0048] Furthermore, it is also described that related polymer compounds (see, for example, European Patent No. 3331937B1 discussed above) are useful for delivering an active agent into a target cell of interest.

[0049] A seventh aspect of the present invention is (i) a step of using the compound of the first aspect and / or its embodiments as a starting compound, (ii) a step of subjecting the compound of step (i) to a structural change to obtain a compound structurally different from the starting compound of (i) Relates to a process for preparing a compound structurally different from the graft copolymer starting compound of the first aspect and / or its embodiments, which comprises

[0050] Based on common general knowledge and the technical disclosure herein, it is routine work for those skilled in the art to prepare a structurally different compound using the graft copolymer compound described herein as a starting compound.

[0051] Hereinafter, embodiments of the present invention will be described merely as examples.

[0052] As will be apparent to those skilled in the art in this context, the preferred embodiments described herein (e.g., preferred radical structures) may preferably be combined with another described preferred embodiment (e.g., another preferred radical structure), i.e., a combination of two individual preferred embodiments described herein is understood to be a more preferred embodiment.

Brief Description of the Drawings

[0053]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0054] Graft copolymer - star core structure of the first aspect In many of the compounds in the examples herein (see, for example, Figure 2 and the examples below), the compound includes a star-related central core similar to the star core described in European Patent No. 3331937B1 discussed above.

[0055] Structure X may also be considered to provide the possibility of attaching additional branches to the star core structure.

[0056] There may be a case where the structure X does not exist, that is, the integers ω, ω’ and ω’’ are preferably 0 (for example, in the case of the graft copolymer compound in the examples in this specification).

[0057] Preferably, K, K’ and K’’ are each -NH-.

[0058] As considered in the examples above and in this specification, the novel graft copolymers considered in this specification can have a very high molecular weight (MW), and thus, for example, a self-assembly process may not be required to form a structure in the nanometer range (e.g., a radius of 4 to 300 nm) that may be useful for the delivery of an active agent to target cells.

[0059] Therefore, in a preferred embodiment, the graft copolymer of the first aspect and its related embodiments in this specification is a graft copolymer having a molecular weight (MW) of 3 to 500,000 kilodaltons (kDa), more preferably 10 to 100,000 kilodaltons (kDa), even more preferably 10 to 20,000 kilodaltons (kDa), and most preferably 20 to 10,000 kilodaltons (kDa).

[0060] As is known in the art, the molecular weight (MW) of a compound having a known structure may be calculated / determined from information from the periodic table.

[0061] For a suitable experimental MW determination method, refer to, for example, the "size exclusion chromatography (SEC)" analysis method in the examples of the present inventors in this specification.

[0062] The preferred embodiment relates to the graft copolymer of the first aspect and its related embodiments in this specification, which is a single-molecule structure having a radius of 4 to 300 nm.

[0063] For a suitable radius determination method, refer to, for example, the "size distribution" analysis method in the examples of the present inventors in this specification.

[0064] The graft copolymer of the first aspect - radical R 1 and R 2 As discussed above, R 2 is selected from -O-, -NH-. In some exemplary structures in this specification, R 2 = -NH-. Therefore, it is preferable that R 2 = -NH-.

[0065] As discussed above, R 1 is

Chemical formula

[0066] The radical R 1 may be regarded as an essentially covalent linker radical, i.e., the left - hand end " * " bonding point of formula (I) in the first aspect as a linker.

[0067] In the exemplary structure of the star - shaped polypeptide in Figure 2 in this specification, R 1 = ethyl, i.e., the "R 1 = ethyl" linker is used to attach / bond the illustrated star - related central core to the left - hand end " * " bonding point of the illustrated graft copolymer of formula (I). See also the star - shaped polypeptide of formula (Ia) in this specification.

[0068] R 1 The linker - related radical is, for example, similar to R of claim 1 of European Patent No. 3331937 B1 discussed above 1It may be regarded as corresponding to a linker-related radical.

[0069] As considered in this EPB1 patent (see, for example, claims 1 and examples - see Table 1 on page 29, for example), such an R 1 linker-related radical may be of many different suitable structures, such as a structure containing a disulfide bond cleavable in vivo in human cells.

[0070] In short, those skilled in the art know many different suitable linker-related structures of different molecular weight (MW) sizes from the art.

[0071] Y may preferably be a biradical having a molecular weight (MW) of 8 to 1000 g / mol or more preferably 2 to 500 g / mol.

[0072] Preferably, the Y biradical of R 1 is -NH-, -NH(C 1 ~C 6 )alkyl-, -O-, -(C 1 ~C 6 )alkyl-COO-, linear or branched -(C 1 ~C 30 )alkylene- as well as a biradical selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI):

Chemical formula

[0073] More preferably, R 1 is -CH 2 CH 2 -S-S-CH 2 CH 2 -, -CH 2 CH 2 CH 2 -S-S-CH 2 CH 2 CH 2 -, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 2 CH 3 )CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 COO-, -CH 2 CH 2 COO-, -CH 2 CHCH 3 COO-, -CH 2 CH 2 CH 3 CH 2A radical selected from the group consisting of COO- and a radical selected from the group consisting of (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) and (XI) as defined herein.

[0074] Most preferably, R 1 is ethylene.

[0075] Graft copolymer of the first aspect - Lys / Orn related units having the integers "o" and "u" In formula (I), the main repeating unit defined by square brackets together with the numerical values "o" and "u" may be regarded as being related to the use of, for example, lysine / ornithine (Lys / Orn).

[0076] In one embodiment of the present invention, the integer "o" is not 0.

[0077] L is preferably a single bond.

[0078] Orn may be a suitable amino acid, i.e., α may preferably be an integer of 2.

[0079] In some exemplary structures herein, Lys is used, and thus α is preferably an integer of 3.

[0080] It should be understood that when the integer "o" is not 0 and "u" is zero, all Lys / Orn related repeating units have the amino acid structure of the repeating unit "p" and are optionally also attached to a branch having the amino acid structure of "q".

[0081] The examples herein describe structures in which the majority of Lys / Orn related repeating units are attached to a branch having an amino acid structure.

[0082] Accordingly, in a preferred embodiment, the value of the integer "u" is less than 25% of the value of the integer "o". That is, when the value of the integer "o" is 100, the value of the integer "u" is less than 25.

[0083] More preferably, the value of the integer "u" is less than 15% of the value of the integer "o". Even more preferably, the value of the integer "u" is less than 5% of the value of the integer "o". Most preferably, the value of the integer "u" is less than 0.5% of the value of the integer "o".

[0084] In some exemplary structures in this specification, the integer "o" is not 0, and there is no Glu / Asp-related unit having the integers "r" and "v", that is, the integers "r" and "v" are zero.

[0085] Accordingly, in a preferred embodiment, the integer "o" is not 0, and the integers "r" and "v" are 0.

[0086] Preferably, the value of the integer "o" is 5 to 3000 (more preferably 10 to 2500, even more preferably 50 to 2000, most preferably 100 to 1500).

[0087] Preferably, the value of the integer "p" is 3 to 1000 (more preferably 5 to 750, even more preferably 8 to 250, most preferably 10 to 200).

[0088] As shown in the "St-PLys-g-PHis-b-PGA" structure in Figure 2, the graft copolymer compound may also include an amino acid-related block (b) structure, that is, the integer "q" is not 0.

[0089] Accordingly, there may be a case where it is preferable that the value of the integer "q" is 3 to 1000 (more preferably 5 to 750, even more preferably 8 to 250, most preferably 10 to 200).

[0090] Preferably, each R 11 and R 12is independently selected from H and the radicals of formulas (XII), (XIII), (XIV) and (XVI).

[0091] Preferably, R 11 is independently selected from H and the radicals of formulas (XII), (XIII), (XIV) and (XVI).

[0092] As considered in the examples above and herein, the novel graft copolymers considered herein can have very high molecular weights (MW), and thus for example, a self-assembly process may not be required to form structures in the nanometer range (e.g., a radius of 4 - 300 nm) that can be useful for the delivery of active agents to target cells.

[0093] A high MW may be obtained by having relatively high numerical values of the integer "o" and the integer "p", and thus in a preferred embodiment, the graft copolymer compound of the first aspect · the numerical value of the integer "o" is 25 - 5000 (more preferably 100 - 2500), and the numerical value of the integer "p" is 2 - 2000 (more preferably 10 - 500), and · the molecular weight (MW) is 3 - 500000 kilodaltons (kDa), more preferably 10 - 100000 kilodaltons (kDa), even more preferably 10 - 20000 kilodaltons (kDa), and most preferably 20 - 10000 kilodaltons (kDa) is a graft copolymer compound.

[0094] The preferred embodiment relates to the graft copolymers of the first aspect and related embodiments herein that are single - molecule structures having a radius of 4 - 300 nm.

[0095] Example 1 below describes examples of St - PLys - g - PGlu (e.g., see Compounds 7A - 7H) and provides data showing the advantageous properties of these St - PLys - g - PGlu structures.

[0096] Accordingly, preferred embodiments relate to such St-PLys-g-PGlu-related compounds, i.e., compounds in which the integer "o" is not 0, α is preferably an integer of 3, and R 11 is (XVI).

[0097] Example 1 below describes an example of a graft copolymer designed to obtain a unimolecular aggregate having a hydrophobic pocket along with the ability to encapsulate a hydrophobic payload (see, for example, Compounds 8-11).

[0098] Accordingly, preferred embodiments relate to such "hydrophobic pocket"-related compounds, i.e., compounds in which the integer "o" is not 0, α is preferably an integer of 3, and R 11 is (XII) or (XIII).

[0099] For example, since it also has block-P (hydrophilic aa) such as St-PLys-g-PBG-PGlu (Compound 9) or St-PLys-g-PHis-PSar (Compound 10), the integer "o" is not 0, α is preferably an integer of 3, R 11 is (XII) or (XIII), the integer "q" is not 0, and R 12 is preferably (XVI) or H in some cases.

[0100] Example 1 below describes an example of a graft copolymer designed to obtain a less dense structure such as St-(PLys-g-PGlu)-stat-PSar (Compound 13).

[0101] Accordingly, preferred embodiments relate to such "stat-PSar"-related compounds, i.e., compounds in which the integer "o" is not 0, α is preferably an integer of 3, R 11 is (XVI), the integer "t" is not 0, and R 15 is H.

[0102] Compound 14 of Example 1 is an example of the binding of the hydrophobic drug vitamin E (which may be regarded as a cell targeting agent herein, for example).

[0103] Accordingly, preferred embodiments relate to such "vitamin E" - related compounds, i.e., compounds in which "o" is not 0 and R 11 is vitamin E, preferably compounds in which α is preferably an integer of 3.

[0104] Compound 15 of Example 2 is an example of the binding of a small molecule, and compound 18 of Example 3 is an example of the binding of a therapeutic peptide.

[0105] Accordingly, preferred embodiments relate to such binding - related compounds, i.e., compounds in which the integer "o" is not 0, α is preferably an integer of 3, and R 11 and / or R 12 is (XIV), and compounds in which Q of (XIV) is a cell targeting agent such as a small molecule (e.g., the Toll - like receptor agonist (TLR7 / 8) imiquimod) or a therapeutic peptide (e.g., MHC - I and / or MHC - II, etc.).

[0106] Preferably, the graft copolymer of the first aspect is St - PLys - g - PGlu St - PLys - g - PHis St - PLys - g - PHis - b - PGlu St - PLys - g - PHis - b - PSar St - PLys - g - PBG St - PLys - g - PBG - b - PGlu St - PLys - g - PBG - b - PSar St - PLys - g - PSar St - PLys - g - PSar - g - VitE St - (PLys - g - PGlu) - stat - PSar, and St - PLys - b - PSar - g - PGA is a compound comprising a compound selected from the group consisting of Here, the term "St" is the star structure of formula (Ia) of the first aspect and / or its embodiments, the term "PBG" is poly(benzyl glutamate).

[0107] As will be understood by those skilled in the art in this context, compounds containing St-PLys-g-PHis include, for example, compounds containing St-PLys-g-PHis-b-PGlu, since the latter also contains St-PLys-g-PHis.

[0108] Similarly, compounds containing St-PLys-g-PHis include, for example, compounds containing St-PLys-g-PHis and a conjugated drug of interest, since the latter also contains St-PLys-g-PHis.

[0109] As considered in the following examples, for example, PBG is poly(benzyl glutamate).

[0110] Glu / Asp-related units having integers "r" and "v" of the graft copolymer of the first aspect In formula (I), the main repeating unit defined by square brackets together with the numerical values "r" and "v" may be regarded as being related to the use of, for example, glutamic acid / aspartic acid (Glu / Asp).

[0111] In one embodiment of the present invention, the integer "r" is not 0.

[0112] Asp may be a suitable amino acid, that is, β may preferably be an integer of 0.

[0113] In some exemplary structures herein, Glu is used, and thus β is preferably an integer of 1.

[0114] It should be understood that when the integer "r" is not zero and "v" is zero, all Glu / Asp-related repeating units are attached to the branches having the amino acid structure of the repeating unit "s".

[0115] Examples in this specification describe structures in which an appropriate amount of Glu / Asp-related repeating units are attached to the branches having an amino acid structure.

[0116] Therefore, the value of the integer "r" may suitably be at least 2% of the value of the integer "v", that is, when the value of the integer "v" is 100, the value of the integer "r" may suitably be at least 2.

[0117] Alternatively, the value of the integer "r" may suitably be at least 10% of the value of the integer "v".

[0118] In some exemplary structures in this specification, the integer "r" is not zero and there are no Lys / Orn-related units having the integers "o" and "u", that is, the integers "o" and "u" are zero.

[0119] Therefore, in a preferred embodiment, the integer "r" is not zero and the integers "o" and "u" are 0.

[0120] Preferably, the value of the integer "r" is from 3 to 1000 (more preferably from 5 to 750, even more preferably from 7 to 500, and most preferably from 8 to 200).

[0121] Preferably, the value of the integer "s" is from 2 to 400 (more preferably from 3 to 350, even more preferably from 5 to 250, and most preferably from 10 to 200).

[0122] Preferably, R 13 is independently selected from H and the radicals of formulas (XII), (XIII), (XXI) and (XXVIII).

[0123] Each R 14It may preferably be independently selected from H, -OH in some cases.

[0124] As considered in the examples above and in the present specification, the novel graft copolymers considered in the present specification can have a very high molecular weight (MW), and thus may not require a self-assembly process to form structures in the nanometer range (e.g., with a radius of 4 - 300 nm) that can be useful for, for example, the delivery of active agents to target cells.

[0125] The high MW may be obtained by having relatively high numerical values of the integer "r" and the integer "s", and thus in a preferred embodiment, the graft copolymer compound of the first aspect · the numerical value of the integer "r" is 3 - 1000 (more preferably 5 - 750), and the numerical value of the integer "s" is 2 - 400 (more preferably 3 - 350), and · the molecular weight (MW) is 3 - 500000 kilodaltons (kDa), more preferably 10 - 100000 kilodaltons (kDa), even more preferably 10 - 20000 kilodaltons (kDa), and most preferably 20 - 10000 kilodaltons (kDa) is a graft copolymer compound.

[0126] The preferred embodiments relate to graft copolymers of the first aspect and related embodiments herein that are single - molecule structures having a radius of 4 - 300 nm.

[0127] Example 7 below describes examples of graft copolymers having a St - PGlu - g - PSar (Compound 19) or a St - PGlu - g - PLysTG structure (Compound 20).

[0128] Thus, the preferred embodiments relate to such compounds, i.e., compounds in which the integer "r" is not 0, β is preferably an integer of 1, and R 13 is H or (XXI).

[0129] Example 8 below describes an example of a graft copolymer having a structure related to St-PGA-g-PSar-g-PHis (also known as St-PGlu-g-PSar-g-PHis) (Compound 21).

[0130] Accordingly, preferred embodiments relate to such compounds, i.e., compounds in which the integer "r" is not 0, β is preferably an integer of 1, and R 13 is H and / or (XII).

[0131] Example 10 below describes an example of the binding of a hydrophobic drug to St-PGlu-g-Psar (Compound 22).

[0132] Accordingly, preferred embodiments relate to such compounds, i.e., compounds in which the integer "r" is not 0, "v" is not 0, β is preferably an integer of 1, R 13 is H, R 14 is a cell targeting agent (such as a hydrophobic drug such as glycine-vitamin E).

[0133] Preferably, the graft copolymer of the first aspect is St-PGlu-g-PSar St-PGlu-g-PHis St-PGlu-g-PLys St-PGlu-g-PLysTG, and St-PGlu-g-PSar-g-Phis a compound comprising a compound selected from the group consisting of, where the term "St" is the star structure of formula (Ia) of the first aspect and / or its embodiments, PLysTG is polylysine thioglycerate.

[0134] As will be understood by those skilled in the art in this context, compounds containing St-PGlu-g-PSar include, for example, compounds containing St-PGlu-g-PSar-g-PHis, since the latter also contains St-PGlu-g-PSar.

[0135] Similarly, the compounds containing St-PGlu-g-PSar include, for example, compounds containing St-PGlu-g-PSar and the medicament of interest bound thereto, because the latter also contains St-PGlu-g-PSar.

[0136] As considered in the following examples, for instance, PLysTG is polylysine thioglycerate.

[0137] Graft copolymer of the first aspect - Ala / Sar-related unit having an integer "t" In formula (I), the main repeating unit defined by brackets together with the numerical value "t" may be regarded as being related to the use of, for example, alanine / sarcosine / glycine (Ala / Sar / Gly).

[0138] Some of the examples herein describe structures where the integer "t" is zero, and thus the integer "t" may preferably be 0.

[0139] The numerical value of the integer "t" may preferably be from 1 to 5000 (more preferably from 3 to 4000).

[0140] Graft copolymer of the first aspect - Other matters Each R 3 is independently selected from -H, Each R 4 is independently selected from -H, Each R 5 is independently selected from -H, Each R 6 is independently selected from -CH 3 selected from, Each R 7 is independently selected from -H, Each R 8 is independently selected from -CH 3 selected from, Each R 9 is independently selected from -H, Each R 10is independently CH 3 selected from each R 15 is preferably independently selected from H in some cases.

[0141] each X 1 is independently H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 , -(C 1 ~C 4 )alkylNH 2 , -(C 1 ~C 4 )alkylNHCH 3 , -(C 1 ~C 4 )alkylN(CH 3 ) 2 , -O-(C 1 ~C 4 )alkyl-NH 2 , -O-(C 1 ~C 4 )alkyl-NHCH 3 and -O-(C 1 ~C 4 )alkyl-N(CH 3 ) 2 and is preferably selected from in some cases.

[0142] each X 2 is independently H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 , -(C 1 ~C 4 )alkylNH 2 , -(C 1 ~C 4 )alkylNHCH 3 , -(C 1 ~C 4 )alkylN(CH 3 ) 2 , -O-(C 1 ~C 4 )alkyl-NH 2 , -O-(C 1 ~C 4 )alkyl-NHCH 3 and -O-(C 1~C 4 ) Alkyl-N(CH 3 ) 2 may preferably be selected from.

[0143] Each X 3 is independently H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 , -(C 1 ~C 4 ) alkylNH 2 , -(C 1 ~C 4 ) alkylNHCH 3 , -(C 1 ~C 4 ) alkylN(CH 3 ) 2 , -O-(C 1 ~C 4 ) alkyl-NH 2 , -O-(C 1 ~C 4 ) alkyl-NHCH 3 and -O-(C 1 ~C 4 ) alkyl-N(CH 3 ) 2 may preferably be selected from.

[0144] The polymer complex of the second aspect As discussed above, the second aspect of the present invention relates to a polymer complex comprising a graft copolymer of the first aspect and / or an embodiment thereof and at least a pharmaceutical, veterinary or cosmetic active agent.

[0145] Preferably, at least one pharmaceutical, veterinary or cosmetic active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers and combinations thereof.

[0146] The nucleic acid may be selected from the group consisting of, for example, plasmid DNA, clDNA, siRNA, mRNA, microRNA, donor DNA, sgDNA, crDNA, shRNA, antisense nucleic acid, decoy nucleic acid, aptamer and ribozyme.

[0147] As is known in the art, the active agent of interest may be covalently bound to the polypeptide backbone by, for example, amino acid side chain residues via amide, ester, anhydride linkages, or by a linker containing one or more functional groups including, but not limited to, alkyne, azide, reactive disulfide, maleimide, hydrazide, hydrazone, Schiff base, acetal, aldehyde, carbamate and reactive ester. In other embodiments, the covalent linkage is bioreactive.

[0148] As is known in the art, the active agent of interest may be bound to the polypeptide backbone by electrostatic interactions. Thus, an anionic compound having more negative charges than positive charges may form a polymer complex with the graft copolymer of the first aspect when mixed in an aqueous medium by electrostatic interactions. Examples of anionic compounds include proteins, polysaccharides, lipids and nucleic acids.

[0149] As is known in the art, the active agent of interest may be bound to the polypeptide backbone by, for example, hydrophobic and / or hydrophilic related interactions.

[0150] For example, Example 1 below describes an example of a graft copolymer designed to obtain a unimolecular aggregate having a hydrophobic pocket along with the ability to encapsulate a hydrophobic payload (e.g., an active agent).

[0151] Thus, based on common general knowledge and the technical disclosure herein, it is routine work for those skilled in the art to prepare the polymer complex of the second aspect of the present invention and / or its embodiments.

[0152] The polymer complex of the second aspect is · wherein the active agent is covalently bound to the polypeptide backbone of the present polymer complex · The active agent is bound to the polypeptide backbone of the present polymer complex by electrostatic interaction, or · The active agent is bound to the polypeptide backbone of the present polymer complex by hydrophobic and / or hydrophilic related interactions The polymer complex may preferably be one.

[0153] The pharmaceutical, veterinary or cosmetic composition of the third aspect As discussed above, the third aspect of the present invention relates to a pharmaceutical, veterinary or cosmetic composition comprising at least one polymer complex of the second aspect and / or an embodiment thereof together with one or more suitable excipients or carriers.

[0154] Based on common general knowledge and the technical disclosure in this specification, it is routine work for those skilled in the art to prepare the pharmaceutical, veterinary or cosmetic composition of the third aspect of the present invention and / or an embodiment thereof.

[0155] As is known in the art, pharmaceutical, veterinary or cosmetic compositions also include acceptable excipients, i.e., excipients acceptable in pharmaceutical, veterinary or cosmetic terms.

[0156] Medical use - Fourth and fifth aspects As discussed above, the fourth aspect of the present invention relates to the polymer complex of the second aspect and / or an embodiment thereof for use as a medicament or the pharmaceutical composition according to the third aspect and / or an embodiment thereof.

[0157] As discussed above, the fifth aspect of the present invention relates to a polymeric complex of the second aspect and / or an embodiment thereof or a pharmaceutical composition according to the third aspect and / or an embodiment thereof for use as (i) a transfection reagent for transfecting at least one active agent into cells, (ii) for the in vivo or ex vivo production of biopharmaceuticals encoding recombinant proteins, peptides or antibodies or the production of recombinant viruses, (iii) for use as a therapeutic or prophylactic vaccine against viral infections or a therapeutic vaccine against cancer, or (v) for use in genome engineering, cell reprogramming, cell differentiation or gene editing.

[0158] Based on common general knowledge and the technical disclosure herein, it is routine work for a person skilled in the art to identify medical uses suitable for the polymeric complexes and / or pharmaceutical compositions described herein.

[0159] For example, many of the medical uses described in the art for related polymeric compounds (see, for example, European Patent No. 3331937B1 discussed above) may also be relevant to the graft copolymer compounds of the present invention.

[0160] Delivery of a pharmaceutically active agent into a target cell - Sixth aspect As discussed above, the sixth aspect of the present invention relates to a method for delivering a pharmaceutically active agent (e.g., a nucleic acid) into a target cell, comprising administering to an animal, including a human, a composition (e.g., a solution) containing a polymeric complex of the second aspect and / or an embodiment thereof such that the complex is physically contacted with the target cell, thereby delivering the pharmaceutically active agent into the cell.

[0161] Based on common general knowledge and the technical disclosure herein, it is routine work for a person skilled in the art to identify a method suitable for delivering a desired active agent to a desired target cell.

[0162] As will be understood by those skilled in the art, the term "physically contacting the complex with the target cell" may relate to, for example, introducing the complex into the target cell.

[0163] Alternatively, the complex may be bound, for example, to the surface of the target cell, and the active agent is delivered into the cell, for example, by crossing the surface cell membrane and thereby entering the cell.

[0164] The examples in this specification describe that the graft copolymer compound of the present invention can be physically contacted with the target cell of interest, whereby the active agent of interest is delivered to the cell.

[0165] Furthermore, related polymer compounds (see, for example, European Patent No. 3331937B1 discussed above) are described as being useful for delivering an active agent into a target cell of interest.

[0166] Use of the graft copolymer of the present invention for preparing different compounds - Seventh aspect As discussed above, the seventh aspect of the present invention is a process for preparing a compound that is structurally different from the graft copolymer starting compound of the first aspect and / or its embodiments, (i) using the compound of the first aspect and / or its embodiments as a starting compound, (ii) subjecting the compound of step (i) to a structural change to obtain a compound that is structurally different from the starting compound of (i) relates to a process comprising.

[0167] Preferably, the structurally different compound obtained in step (ii) is a compound having the same function / capability as the starting compound of step (i), i.e., the structurally different compound obtained in step (ii) is preferably a compound capable of forming a polymer complex comprising at least the structurally different compound obtained in step (ii) and a pharmaceutical, veterinary or cosmetic active agent.

[0168] Preferably, the structurally different compounds obtained in step (ii) are compounds capable of forming a polymer complex comprising the structurally different compounds obtained in step (ii) and at least one pharmaceutical, veterinary or cosmetic active agent, and the active agent is an active agent with which the starting compound of step (i) can also form a polymer complex.

[0169] The structurally different compounds obtained in step (ii) may be, for example, compounds outside the scope of protection of the graft copolymer compounds of the first aspect and / or its embodiments.

[0170] Alternatively, the structurally different compounds obtained in step (ii) are compounds within the scope of protection of the graft copolymer compounds of the first aspect and / or its embodiments.

[0171] Based on common general knowledge and the technical disclosure herein, it is routine work for those skilled in the art to prepare structurally different compounds using the graft copolymer compounds described herein as starting compounds.

[0172] As will be understood by those skilled in the art, the process of the seventh aspect is based on the technical disclosure herein because the graft copolymer compounds of the first aspect (i.e., the present invention) are used to prepare structurally different compounds.

[0173] Process for the synthesis of the compounds of the first aspect - a separate aspect A separate aspect of the present invention relates to a process for the synthesis of the graft copolymer compounds of the first aspect and / or its embodiments, which generally comprises polymerizing N-carboxy anhydrides (NCAs) of protected or unprotected amino acids known per se using a 3-arm star initiator (St), followed by a deprotection reaction, and then performing second and optionally further polymerization reactions.

[0174] According to this aspect, the 3-arm star initiator (St), N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1) (i.e., St-initiator (1)) can generally be obtained by reacting 1,3,5-benzenetricarbonyl trichloride with N,N'-diisopropylethylenediamine (DIEA) to obtain the intermediate 1,3,5-tri-tert-butyl ((benzene-tricarbonyltris-(azanediyl))tris(ethane-2,1-diyl)) tricarbamate (2), and then reacting the intermediate (2) with TFA to obtain 1,3,5(benzene-tricarbonyltris(azanediyl))-triethaneammonium TFA salt (3) (i.e., the star polymer intermediate (3) protected by TFA), and then removing the TFA group by a cleavage reaction involving basic deprotection (e.g., by stirring in a suitable ion exchange resin) to obtain St-initiator (1).

Chemical formula

[0175] According to this aspect regarding the process for the synthesis of the compound of formula (I) of the first aspect and / or its embodiments, the process generally involves i) polymerizing a protected (e.g., in the form of ammonium tetrafluoroborate or ammonium trifluoroacetate salt) N-carboxy anhydride (NCA) or an amino acid known per se without protection continuously or statistically with a 3-arm star initiator, i.e., St-initiator (1) or a protected St-initiator intermediate (3) to obtain a block copolymer or alternatively a random copolymer, ii) optionally, reacting the amine group at the N-terminal position with an amine-reactive group to introduce an end-capping group, iii) optionally, orthogonally removing the amino acid side chain protecting groups, iv) optionally, reacting the amine group at the side chain terminal position with an amine-reactive group to introduce structural elongation, bonding, labeling or shielding, v) Purifying the product obtained in step i), ii), iii) or iv) optionally by fractionation, precipitation, ultrafiltration, dialysis, size exclusion chromatography, affinity chromatography or tangential flow filtration comprising.

[0176] Said step i) may include ring-opening polymerization of an unprotected amino acid N-carboxy anhydride (NCA) monomer by reacting a tetrafluoroboric acid or ammonium trifluoroacetate salt form of a protected St-initiator intermediate (3) with a selected unprotected NCA-amino acid monomer, or alternatively ring-opening polymerization of a protected amino acid N-carboxy anhydride (NCA) monomer (e.g., in tetrafluoroboric acid or ammonium trifluoroacetate salt form) by reacting an St-initiator (1) with a selected protected NCA-amino acid monomer (wherein the monomer / initiator ratio allows control of the degree of polymerization (DP)). Polymerization with different amino acid NCA monomers may be carried out continuously, i.e., here a block copolymer polypeptide is continuously prepared after polymerization reaction a), allowing consumption of the first NCA monomer, and the product obtained may or may not be purified before adding the next NCA-monomer to build subsequent polypeptide blocks, or alternatively may be carried out statistically, here a random copolymer polypeptide is prepared according to a statistical polymerization reaction a), and all NCA-monomers are mixed before starting the polymerization.

[0177] Said step ii) corresponds to end-capping, where the amine group at the N-terminal position is reacted with an amine-reactive group to introduce an end-capping functional group.

[0178] Said step iii) corresponds to deprotection, where the amino acid side chains are orthogonally removed depending on the protecting group.

[0179] Step iv) corresponds to conjugation, where the amine group at the side chain terminal position of a shielding polymer, active small molecule, targeting agent or contrast agent is reacted with an amine-reactive group.

Example

[0180] Analysis method NMR spectroscopy: The NMR spectra were recorded at 27 °C (300 K) using a 300 Ultrashield (trademark) from Bruker (Billerica, Massachusetts, USA). The data were processed using Topspin software (Bruker, Karlsruhe, Germany). Samples were prepared at a concentration of approximately 20 - 10 mg / mL in the required solvent.

[0181] Size exclusion chromatography (SEC): For SEC measurements in dimethylformamide (DMF) containing 0.1% (w / w) lithium bromide as an additive, a GPC max (Malvern Instruments) autosampler was used with a TSKgel Alpha - 4000 column from Tosoh Bioscience at a flow rate of 0.7 mL / min and 60 °C. A Viscotek TDA - 302 was used as an integrated detection system. This system was calibrated using polymethylmethacrylate (PMMA) (Mw = 65 kDa; PDI = 1.05) from PSS. For this Mw determination, an integrated triple - detection system [refractive index, light scattering (two angles: 7 and 90°), and ultraviolet - visible detector] was used.

[0182] For SEC measurements in an aqueous solution containing 0.1 M sodium nitrate and 0.005% sodium azide as an additive, a GPC max (Malvern Instruments) pump and autosampler were used with a TSKGel PWXL G5000 column from Tosoh Bioscience at a flow rate of 0.7 mL / min and 25 °C. A Viscotek TDA - 305 was used as an integrated detection system. This system was calibrated using polyethylene oxide (PEO) (Mw = 24 kDa; PDI = 1.01) from Malvern Panalytical. For this Mw determination, an integrated triple - detection system [refractive index, light scattering (two angles: 7 and 90°), and ultraviolet - visible detector] was used.

[0183] Size distribution. Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer NanoZS instrument equipped with a 532 nm laser at a fixed scattering angle of 173°. Polymer solutions were prepared under different conditions (MilliQ or PBS at different concentrations and temperatures), the solutions were sonicated for 10 minutes, left to stand for the required time, filtered through a 1.20 μm cellulose membrane filter, and measured. The size distribution (diameter: nm) was measured three times for each polymer at measurements with n > 3 for automatic optimization of beam focusing, and attenuation was applied for each sample.

[0184] Example 1: Preparation of the compound of formula (I) - Lys attached to a branch having different amino acid structures

Chem.

[0185] In connection with formula (Ia) of the first aspect herein, the above formula (Ib1) is an example regarding the use of Lys for attaching a branch having different amino acid structures, where the integer "o" is not 0 and α is an integer of 3.

[0186] Generally speaking, to synthesize the compound of formula (Ib1) according to the present disclosure, first a 3-arm star initiator was obtained within 2 to 3 steps. Using such an initiator, N-trifluoroacetyl-L-lysine N-carboxy anhydride, i.e., lysine (TFA) NCA, was polymerized to obtain a protected star polymer TFA (St-PLys(TFA)). The TFA groups were removed by a cleavage reaction involving basic deprotection to obtain the corresponding star-PLys having a free NH 2 active site and the next polymerization step was initiated.

[0187] Scheme 1 shows a specific example of the polymerization, deprotection process, and grafting from method from the poly-L-lysine backbone.

Chem.

[0188] Example 1A: Synthesis of a 3-arm star initiator The 3-arm star initiator, namely N 1 ,N 3 ,N 5 The synthetic route to -tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1) is disclosed in Scheme 2.

Chemical formula

[0189] Step (a): Synthesis of 1,3,5-tri-tert-butyl ((benzene tricarbonyltris-(azanediyl))tris(ethane-2,1-diyl)) tricarbamate (2)

Chemical formula

[0190] In a two-necked round-bottom flask equipped with a stir bar and an N 2 inlet and an outlet, 500 mg of 1,3,5-benzenetricarbonyl trichloride (1.88 mmol, 1 equivalent) was dissolved in 12 mL of anhydrous THF. N,N'-diisopropylethylenediamine (DIEA) (803.31 mg, 6.22 mmol, 3.3 equivalents) was added to the reaction mixture, and then N-Boc-ethylenediamine (1.34 g, 6.22 mmol, 3.3 equivalents) was added dropwise over 10 minutes. The reaction was then allowed to proceed for 2 hours. Thereafter, the solvent was completely removed under vacuum. The product was redissolved in chloroform and washed three times with deionized water (ddH 2 O) and then three times with acidic water (pH of about 3). Finally, the organic phase was isolated under vacuum, and the product was recrystallized three times from THF / methanol / hexane to obtain a white crystalline solid. The product was then dried under high vacuum and stored at -20 °C. Yield: 82%. 11H NMR (300 MHz, DMSO) δ 8.68 - 8.65 (m, 3H), 8.41 (s, 3H), 6.92 - 6.88 (m, 3H), 3.34 - 3.31 (m, 6H), 3.16 - 3.13 (m, 6H), 1.37 (s, 27H). 13 13C NMR (75 MHz, CDCl 3 ) δ 166.80 (C=O), 156.84 (C=O), 134.58 (quaternary CAr), 128.47 (CH Ar ), 79.57 (quaternary C), 40.93 (CH 2 ), 40.43 (CH 2 ), 28.45 (CH 3 ).

[0191] Step (b): Synthesis of 1,3,5-(benzenetricarbonyltris(azanediyl))-triethanammonium TFA salt (3)

Chemical Structure

[0192] In a round-bottom flask equipped with a stir bar and a stopper, 200 mg of 1,3,5-tri-tert-butyl ((benzenetricarbonyltris(azanediyl))tris(ethane-2,1-diyl)) tricarbamate (2) (0.33 mmol, 1 equivalent) was dissolved in 5 mL of anhydrous dichloromethane, and 2.5 mL of TFA was added. The reaction system was stirred under a nitrogen atmosphere for 2 hours, and the completion of the reaction was monitored by TLC. The solvent was evaporated in vacuo. The initiator TFA salt (220 mg) was obtained in quantitative yield and dried under vacuum. Yield: 98%. 1 1H NMR (300 MHz, D 2 MSO) δ 8.36 (s, 3H), 3.75 (t, J = 5.9 Hz, 6H) 3.29 (t, J = 6.0 Hz, 6H). 19 19F NMR (300 MHz, D 2 MSO) δ -75.84.

[0193] Step (c): N 1 ,N 3 ,N 5-Synthesis of tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1)

Chemical formula

[0194] Example 1B: Polymerization of the main chain Compound (5) was synthesized according to the synthetic route of Scheme 3.

Chemical formula

[0195] Briefly, L-lysine (TFA)-NCA (2.0 g, 7.46 mmol) was placed in a flask equipped with a stir bar and a stopper, and subjected to 3 cycles of vacuum / N 2It was added to the Schlenk tube purged with argon and dissolved in anhydrous DMF (4 mL). Subsequently, the star initiator (St) (16.72 mg, 0.05 mmol) was dissolved in DMF (1 mL) and added to the reaction mixture. The mixture was stirred at 10 °C for 48 h. Complete conversion of the monomer could be detected by IR upon completion. The reaction mixture was poured into diethyl ether (1:10 ratio), and the product was precipitated. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Star-shaped poly(L-Lys(TFA)) (St-PLys(TFA)) (4) was isolated as a white solid. In the second step, the TFA protecting groups were removed from St-PLys(TFA) (4) under basic conditions. Briefly, the polymer (4) (0.45 g, 2.00 mmol) was dissolved in MeOH (1.5 mL), and 4.5 M NaOH (aqueous solution) (0.67 mL) was added to the reaction mixture. The mixture was stirred at 4 °C for 16 h. Then, a 5 M TFA (aqueous solution) (0.55 mL) solution was poured into acetonitrile (1:10 ratio) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. Star-shaped poly(L-Lys) (St-PLys) (5) was isolated as a white solid. Yield: 70 - 90%. 1 H NMR (D 2 O): δ = 1.46 (m, 2H, CH 2 ), 1.64 - 1.90 (broad m, 4H, CH 2, CH 2 ), 3.03 (t, 2H, CH 2 -NH 2 ), 3.34 - 3.66 (broad m, 12H, CH 2 -CH 2 ), 4.33 (t, 1H, chiral CH), 8.31 (s, 3H, aryl CH).

[0196] Table 1 shows the different DPs (degree of polymerization) obtained for different St-Plys of formula (5), demonstrating the generality and accuracy of the experimental procedure.

Table 1

[0197] Example 1C: Grafting from Synthesis of star poly(L-Lys) graft-poly(L-PGlu), i.e., (St-PLys-g-PGlu) (7). Scheme 4 shows the synthetic route for obtaining compound 7.

Chemical formula

[0198] L-Glutamic acid γ-benzyl N'-carboxy anhydride (99.1 g, 0.38 mol) was added to a Schlenk tube equipped with a stir bar, a stopper, and purged with 3 cycles of vacuum / Ar, and dissolved in 490 mL of anhydrous DMF. Then, the equivalent amount of compound (5) (837 mg, 3.45 mmol) required as a macroinitiator for grafting was dissolved in DMF (10 mL), added, and the mixture was stirred at room temperature under an inert atmosphere for 2 days. Upon completion, complete conversion of the monomer could be detected by IR. Finally, the reaction mixture was poured into a large excess of cold diethyl ether (1:10 ratio), and after isolation by filtration or centrifugation (3750 rpm, 4 min), the white polymer star poly(L-Lys)-graft-poly(L-PBG), i.e., (St-PLys-g-PBG) (6), was obtained and dried in vacuo. After this strategy, the degree of polymerization was controlled by the feed ratio [monomer] / [macroinitiator] to obtain the graft copolymer. In the second step, the benzyl protecting group was deprotected from St-PLys-g-PBG (6) under basic conditions. Briefly, the polymer (6) (69.1 g, 0.32 mol) was dissolved in THF (2.8 L), and 0.7 M NaOH (aqueous solution) (0.7 L) was added to the reaction mixture. The mixture was stirred at 0 °C for 16 h. Then, this solution was poured into a mixture of diethyl ether / acetonitrile (3 / 1), and the product was precipitated. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried in vacuo. Star poly(L-Lys)-graft-poly(L-PGlu), i.e., (St-PLys-g-PGlu) (7), was isolated as a white solid. Yield: 70 - 90%. 1 H NMR (TFA): δ = 2.92 (m, 2H, CH2 ), 4.85 (s, 1H, CH), 5.05 (m, 2H, benzyl CH 2 ), 7.13 (s, 5H, aryl CH), 8.38 (s, aryl CH).

[0199] Table 2 shows the different DPs (degree of polymerization) obtained for different St-PLys-g-Pglu of formula (7), demonstrating the generality and accuracy of the experimental procedure.

Table 2

[0200] Example 1D: Star-shaped poly(L-Lys)-graft-poly(L-hydrophobic aa)-block-P(hydrophilic aa), namely Compounds 8 - 11 These graft copolymers were designed to obtain unimolecular aggregates with hydrophobic pockets along with the ability to encapsulate hydrophobic payloads. Scheme 5 shows the general synthetic scheme for obtaining Compounds 8 - 11.

Chemical formula

[0201] Similar to the synthesis of compound 6, the first amino acid NCA and then the second amino acid NCA were polymerized using the macroinitiator St-PLys(5). In the specific case of this example, the amino acid NCA-1 was either L-glutamic acid γ-benzyl NCA or N(Im)-(2,4-dinitrophenyl)-L-histidine NCA (His(DNP)NCA). Briefly, a specific NCA-1 was added to a Schlenk tube equipped with a stir bar, stopper, and purged with 3 cycles of vacuum / Ar, and dissolved in anhydrous DMF. Then, the equivalent amount of compound (5) required as a macroinitiator for grafting was added, and the mixture was stirred at 10 °C for 3 - 4 hours under an inert atmosphere. Complete conversion of the monomer could be detected by IR upon completion. Next, the second monomer was added to polymerize the second block in one pot. In the specific case of this example, the amino acid NCA-2 was either sarcosine NCA added in anhydrous DMF or glutamic acid 5-tert-butyl ester NCA (Glu(OtBu)NCA) added in anhydrous THF. Complete conversion of the second monomer could be detected by IR upon completion. Finally, the reaction mixture was poured into a large excess of cold diethyl ether or H 2 O (determined by NCA-2), and a white polymer was obtained after isolation by filtration or centrifugation (3750 rpm, 4 min) and dried in vacuo.

[0202] For compound 8, i.e., St-PLys-g-PBG-PSar, this was the final step of the synthetic procedure. Yield: 70 - 90%. 1 1H NMR (TFA): δ = 2.20 (broad m, 1H, CH 2 ), 2.40 (broad m, 1H, CH 2 ), 2.71 (broad m, 2H, CH 2 ), 3.20 - 3.59 (broad s, 30H, N-CH 3 ), 4.49 - 5.01 (broad m, 1H, chiral CH (PBG), 20H, CH 2 (PSar)), 5.32 (broad s, 2H, benzyl CH 2 ), 7.47 (s, 5H, aryl CH).

[0203] For compound 9, namely St-PLys-g-PBG-PGlu, a further deprotection step of the OtBu group is required. Briefly, the polymer precursor of 9 (St-PLys-g-PBG-P(Otbu)Glu) (38.3 g, 0.207 mol) was dissolved in pure TFA (380 mL). The mixture was stirred at room temperature for 3.5 h. Then, this solution was poured into diethyl ether to precipitate the product. The precipitate was isolated by filtration or centrifugation (3750 rpm, 4 min) and dried in vacuo to obtain a grayish white solid. Yield: 70 - 90%. 1 H NMR (D 2 O-HFIP): δ = 1.90 (m, 26H, CH 2 ), 2.20 (m, 26H, CH 2 ), 4.23 (dd, 12H, chiral CH), 6.97 (broad s, 5H, aryl CH).

[0204] For compound 10, namely St-PLys-g-PHis-PSar, a further step for the DNP protecting group of histidine under reductive conditions using mercaptoethanol is required. Briefly, the polymer precursor of 10 was dissolved in DMF (100 mg / mL), and an excess amount of mercaptoethanol (10 equiv) was added to the reaction mixture. The mixture was stirred at room temperature for 16 h. Then, this solution was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried in vacuo. St-PLys-g-PHis-PSar (10) was isolated as a yellow solid. Yield: 70 - 90%. 1 H NMR (TFA): δ = 3.35 - 3.97 (m, 2H, CH 2 (PHis); 23H, CH 3 -NH), 4.95 (s, 15H, CH 2 ), 7.83 (s, 1H, imidazole), 9.00 (s, 1H, imidazole).

[0205] For compound 11, i.e., St-PLys-g-PHis-PGlu, the deprotection of DNP from histidine and the deprotection of OtBu from glutamic acid must be carried out in two additional steps. First, 10 of the polymer precursors were dissolved in a mixture of DMF / THF (3 / 1), and an excess amount of thioethanol (10 equivalents) was added to the reaction mixture. The mixture was stirred at room temperature for 16 h. Then, this solution was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. St-Plys-g-PHis-PGlu(OtBu) was isolated as a yellow solid. Next, the polymer was dissolved in pure TFA, and the mixture was stirred at room temperature for 3.5 h. Then, this solution was poured into diethyl ether to precipitate the product. The precipitate was isolated by filtration or centrifugation (3750 rpm, 4 min) and dried under vacuum to obtain a yellow solid. Yield: 70 - 90%. 1 H NMR (D 2 O-HFIP)

[0206] Similarly, for comparison, the same procedure was used, but n-butylamine was used as the initiator instead of the star initiator compound 1 to synthesize a linear analog (compound 12) with compound 10.

[0207] Table 3 shows the different DPs obtained for different star poly(L-Lys)-graft-poly(L-hydrophobic aa)-block-P(hydrophilic aa), i.e., compounds 8 - 11. Various graft copolymers with hydrophobic pockets for hydrophobic molecular encapsulation are exemplified.

Table 3

[0208] Example 1E: Star poly((L-Lys)-graft-(poly(L-Glu))-stat-poly(Sar), i.e., St-(PLys-g-PGlu)-stat-PSar(13) Compound 13 was synthesized with the aim of obtaining a graft copolymer having the same polymer backbone but a less dense structure. This is achieved by introducing unreacted sites into the polymer backbone by statistical copolymerization of amino acids that do not have functional reaction sites capable of initiating polymerization in further steps. In this particular example, the amino acid NCA selected for copolymerization with Lys(TFA)NCA is sarcosine NCA shown in Scheme 6.

Chemical formula

[0209] Briefly, the polymer backbone St-PLys-stat-PSar was synthesized in the same manner as St-PLys, except that sarcosine NCA monomer was introduced simultaneously with Lys(TFA)NCA. Subsequently, the TFA groups were deprotected as described for the synthesis of St-PLys, compound 5. Next, the statistical copolymer St-PLys-stat-PSar was used as a macroinitiator to polymerize the monomer Glu(OBzl)NCA. The benzyl protecting groups of the resulting polymer were removed according to the same procedure described for the synthesis of compound 7.

[0210] Table 4 shows the different DPs (degree of polymerization) obtained for St-St-(PLys-g-PGlu)-stat-PSar of formula (13).

Table 4

[0211] Example 1F: Star-shaped poly(L-Lys)-graft-poly(Sar)-poly(Lys-vitE) (14)

Chemical formula

[0212] Prior to the synthesis of the graft copolymer shown in Scheme 7, the hydrophobic drug vitamin E was conjugated. Briefly, St-PLys (Compound 5B) (30.0 mg, 0.12 mmol) was dissolved in 0.5 mL of anhydrous DMF. Next, DMTMMBF 4 (8.1 mg, 0.2 mmol) was added to 0.2 mL of anhydrous DMF, and the reaction mixture was allowed to proceed for 10 - 15 minutes. Then, succinate tocopherol (13.2 mg, 0.02 mmol) in anhydrous DMF was added to the reaction mixture, and the pH was adjusted to 8 - 9 with the aid of the addition of 50 μL of triethylamine (TEA). The reaction was allowed to proceed at room temperature under a nitrogen atmosphere for 24 hours. Then, Sar-NCA (228.0 mg, 2.0 mmol) was dissolved in anhydrous DMF (2 mL) and added to the reaction mixture under an inert atmosphere. This reaction was allowed to proceed for 16 hours. Upon completion, complete conversion of the monomer could be detected by IR. Finally, the mixture was poured into a large excess of diethyl ether, and the product was isolated as a white solid by filtration / centrifugation. The grafting ratio by succinate tocopherol was 1 calculated by 1H-NMR. Yield: 70 - 90%. 1 1H NMR (MeOD): δ = 0.87 (m, 12H, CH 3 ), 0.94 (t, 3H, CH 3 -), 1.02 - 1.60 (broad m, 32H, CH 2 &CH) , 1.98 - 2.09 (s, 12H, CH 3 -), 2.59 - 3.23 (broad m, 390H, terminal N-CH 3 ), 3.79 - 4.66 (broad m, 260H, -CH 2 -&-CH 2 -).

[0213] Example 1G: Formation of Unimolecular Aggregates from the Compounds in Example 1 One of the main characteristics of the graft copolymers of the present inventors is the possibility of forming single-molecule aggregates having high molecular weights and sizes suitable for their use in drug delivery, among others. To investigate this behavior, the inventors evaluated their hydrodynamic size in aqueous solution by using light scattering techniques. The results obtained for the graft copolymers tested are summarized in Table 5.

[0214] Table 5 shows the hydrodynamic size of the graft copolymer single-molecule aggregates formed in solution.

Table 5

[0215] Example 2: Binding of small molecules to the compound of formula Ib1 Proof-of-concept for the binding of small molecules was achieved. As a model molecule, a derivative of the Toll-like receptor agonist (TLR7 / 8) imiquimod was bound to compound 7B shown in Scheme 8 to obtain compound 15.

Chemical formula

[0216] Briefly, compound 7B was dissolved as the acid form at a concentration of 12 - 3 mg / mL in anhydrous DMSO in a round-bottom flask equipped with a stir bar, stopper, and previously purged with N 2 . Next, DMTMMBF 4 (1.5 equivalents of the desired modification rate) was added to anhydrous DMSO to activate the carboxylic acid. The reaction was allowed to proceed at room temperature for 10 - 15 minutes. Next, the imiquimod derivative (1.5 equivalents of the desired modification rate) and anhydrous DMSO were added, and the pH was adjusted to 8 by the addition of the DIPEA base. The final polymer concentration was 10 mg / mL. The reaction system was stirred at room temperature and under a nitrogen atmosphere for 20 hours. For purification, this reaction system was precipitated in THF, and the solid was washed 3 times with THF:Et 2 O (5:95). For conversion of the conjugate to the water-soluble sodium salt form, the product was suspended in water, which was 0.5 M NaHCO 3It dissolved upon addition. Next, the compound was desalted by either Sephadex G25 purification and / or dialysis using Vivaspin (MWCO: 30 kDa). A white solid was obtained after resuspension in water and lyophilization. The ratio of the bound TLR7 / 8 agonist was determined by NMR to be 3.8 mol% (87.4% yield).

[0217] Example 3: Binding of Therapeutic Peptides to the Compound of Formula lb1 Proof-of-concept for the binding of therapeutic peptides was achieved.

[0218] Specifically, two peptide antigens were conjugated to compound 7B after prior 7B derivatization with pyridyldithioethylamine (PD). Next, two different peptide antigens of major histocompatibility complex class-I and -II (MHC-I and MHC-II), namely gp100 MHC class I and MHC class II peptides, were conjugated via reversible disulfide bonds (see Scheme 9).

Chemical Structure

[0219] A) Derivatization with Pyridyldithioethylamine (PD): Briefly, 900 mg of 7B (6.98 mmol, represented by the glutamic acid unit i.e., GAU) was added to a 250 mL round-bottom flask, and N 2It was purged. Next, 86 mL of anhydrous DMSO was added and the solid was dissolved with stirring at room temperature for 40 minutes. Then, DMTMM·BF4 (148 mg, 0.523 mmol for 5% GAU derivatization) was added to another 2 mL of anhydrous DMSO. After a 30-minute reaction, pyridyldithioethylamine (PD) (117 mg, 0.523 mmol for 5% GAU derivatization) dissolved in 2 mL of anhydrous DMSO was added. The pH was adjusted to 8 by adding DIEA, and the reaction was allowed to proceed with stirring at room temperature under an anhydrous atmosphere for 48 hours. Then, the reaction system was precipitated in 1 L of fresh THF with stirring and cooled to 4 °C for 16 hours to promote precipitation. Next, the solid was recovered by centrifugation and washed with THF and THF:Et 2 O (5:95), and freeze-dried to obtain a white solid of Compound 16. A small amount of sodium bicarbonate was added to D 2 in O 1 The identity, purity, and conjugation efficiency were determined by 1H-NMR. Yield: 90% (1050 mg), conjugation efficiency: 70% (3.5% mol GAU derivatization with PD).

[0220] B) Peptide conjugation to PD-modified graft copolymers (Compounds 17 and 18): Briefly, for MHC-I peptide conjugation, the PD-modified graft copolymer (350 mg, 2.57 mmol of GAU) was dissolved in 35 mL of anhydrous DMSO, and then 59.72 mg of MHC-I peptide (0.0495 mmol, 0.019 equivalent, 1207 g / mol for 1.9% modification) was dissolved in 5 mL of anhydrous DMSO, and then 0.15 equivalent of tris(2-carboxyethyl)phosphine hydrochloride, TCEP (0.722, 0.0029 mmol) was added. This reaction was allowed to proceed with stirring under an N 2 atmosphere at room temperature for 48 hours. Then, the compound was precipitated in cold THF at a ratio of 1:20. The precipitate was isolated by centrifugation and washed with THF. Finally, a white solid was obtained by freeze-drying. This solid was NaHCO 3It was converted to the sodium salt form of the product by addition of and subsequent desalting dialysis using 30 kDa Vivaspin. The peptide content was determined to be 6.07 wt% by amino acid analysis. Yield: 40%, binding efficiency: 49% (0.86% mol of bound MHC-1).

[0221] In another example, gp100 MHC-I and MHC-II peptides were linked in series in the same polymer backbone (Compound 18). Briefly, after reacting with the MHC-I peptide as described above for 48 h, instead of precipitating the gp100 MCH-I conjugate, after addition of gp100 MHC-II peptide (0.019 equivalents for 1.9% modification, 2208 g / mol) dissolved in 5 mL of anhydrous DMSO and 0.15 equivalents of TCEP (0.722, 0.0029 mmol), the reaction was allowed to proceed for an additional 48 h. The compound was then precipitated in cold THF at a ratio of 1:20. The precipitate was isolated by centrifugation and washed with THF. Finally, a white solid was obtained by lyophilization. This solid was treated with NaHCO 3 It was converted to the sodium salt form of the product by addition of and subsequent desalting dialysis using 30 kDa Vivaspin. The peptide content was determined to be 4.11 wt% for the MHC-I peptide and 4.99 wt% for the MHC-II peptide by amino acid analysis. Yield: 60%, binding efficiency: 36%.

[0222] Example 4: Encapsulation of a drug in a compound of formula Ib1 Compounds 8 - 12 shown in Examples 1D and 1E were designed to retain a hydrophobic pocket for drug encapsulation due to their amphiphilicity and the presence of highly hydrophobic blocks of polyamino acids (i.e., PBLG or PHis).

[0223] In a first attempt to characterize the system, Nile Red was selected as a hydrophobic drug model with logP: 2.98.

[0224] In the specific case of compound 9, i.e., St-PLys-g-PBG-PGlu, the presence of a hydrophobic pocket was demonstrated by the ability to incorporate the hydrophobic molecule Nile Red into the structure. Nile Red is commonly used as a lipophilic stain. In a hydrophilic environment, Nile Red emits little fluorescence, but when encapsulated / confined in a hydrophobic environment, it can emit strong fluorescence in various colors from strong red (in the case of an environment like a polar membrane) to strong golden yellow (in the neutral case). The presence of the hydrophobic pocket and the response of the polymer's hydrophobic / hydrophilic balance to pH changes were determined by a fluorescence Nile Red assay. This test is based on the significant solvatochromic effect and increased fluorescence emitted by this molecule in the presence of the aforementioned hydrophobic microenvironment. To do this, increasing amounts of 5 mM Nile Red in ethanol solution (final concentrations of 10 - 120 μM, see the following table) were added to a 10 mg / mL aqueous polymer solution. After mixing the samples at room temperature for 24 hours, the samples were centrifuged at 13300 for 2 minutes. Next, the absorbance at 560 nm was measured three times in a 96-well dark well plate using a plate reader.

[0225] Table 6 shows the maximum Nile Red encapsulated in compound 9 at different concentrations in ppm.

Table 6

[0226] In the specific cases of compounds 10 and 12, which have a pH-sensitive hydrophobic block of histidine, potentiometric titration was performed to find the hydrophobic range of drug encapsulation prior to the Nile Red assay.

[0227] Potentiometric titration: The pK of the cationic polymer is determined by acid-base titration that measures the pH of the solution throughout the process. a is then determined from the titration graph. aTo obtain. To perform the measurement, a 1 mg / mL cationic polymer solution was prepared with Milli-Q water, and a known amount of 0.1 M HCl was added until the pH of the solution reached approximately 2. At this point, titration was performed using 0.2 M NaOH with an automatic Methrom 916Ti touch potentiometer together with a Dosin 800 dispenser. The titration rate was set to 0.1 mL / min with a signal variation of 50 mV / min. The titration was completed when the pH reached 12. The relationship between pH and the degree of protonation of the polycation was calculated from the obtained titration curve. Further, the pK = pH + log[α / (1-α)] (where K is the effective dissociation constant) value was plotted against 1-α.

[0228] Table 7 shows the pK a values obtained for different compounds.

Table 7

[0229] Using these molecules, the Nile red test was modified. The test consists of preparing two types of Milli-Q aqueous solutions of Nile red ([Nile red] = 30 μM) at pH 2 with and without the polymer. Successive volumes of 1 M NaOH (10 μL) were added to these solutions until the final pH of 11 was reached. Since Nile red is insoluble in polar solvents such as water, it does not show fluorescence. After adding NaOH and raising the pH of the medium in the presence of a hydrophobic environment (sample containing the polymer), observe how the color of this solution turns a strong purple, and observe the fluorescence indicating that the polymer can encapsulate hydrophobic molecules and thereby make them soluble in an aqueous medium. No change was observed in the solution without the polymer, and no fluorescence signal was present.

[0230] Not only Nile red, but also other exemplary hydrophobic dyes such as DIL and molecules / drugs such as vitamin E were successfully encapsulated using the same procedure.

[0231] DIL, also known as DiIC18(3), is a fluorescent lipophilic cationic indocarbocyanine dye (cLogP: 22.539) with a maximum absorption at 550 nm and a maximum emission at 565 nm. DIL encapsulation was demonstrated by size exclusion chromatography (SEC) measurements and visual observation. For this purpose, a PBS buffer of DIL blank was prepared, and a colorless solution was observed after filtration of the sample, indicating that the DIL dye remained insoluble in PBS in the filter. However, when the DIL dye was encapsulated in the graft copolymer micelle core and this solution was filtered, the colorimetric quantification characteristics of DIL could be visually confirmed when this solution maintained a pink color. λ abs SEC measurements using a diode array detector (DAD) with λ = 550 nm confirmed the absorbance of only the encapsulated DIL when compared to the filtered blank and polymer alone.

[0232] Similarly, partial encapsulation of Vit E into these polymers was demonstrated. Partial encapsulation of vitamin E was evaluated by visual observation (before and after filtration of the aqueous solution, similar to the case of DIL) and a turbidity assay that monitors the absorbance at a high λ due only to the turbidity of the sample. The observed decrease in turbidity indicated Vit E encapsulation.

[0233] Example 5: Biological Evaluation of Compounds of Formula Ib1 - In Vivo Distribution Profile In vivo distribution experiment and fluorescence quantification. An in vivo distribution experiment was performed using 5- to 7-week-old male Balb / c mice from Envigo. The animals were housed on a 12-hour light and 12-hour dark cycle. In all cases, water and food were provided ad libitum throughout the experiment, and the general aspects, behavior, and body weight were evaluated daily to confirm that the animals were healthy. All animal protocols were approved by the Animal Experimentation Committee of the Centro de Investigacion Principe Felipe (CIPF, Valencia, Spain). For the in vivo distribution study, six mice with a body weight of 25 ± 4.5 were used at each time point. The labeled polymer was administered intravenously via the tail vein in serum saline at a dose of 1 mg / Kg equivalent of Cy5.5. The mice were euthanized at 0 hours, 1 hour, 4 hours, and 24 hours after administration. Similarly, Cy5.5 was injected into another three mice at 1 mg / Kg for 4 hours, and three mice were used as a control injected with PBS for basal fluorescence. Blood, kidneys, liver, spleen, heart, lungs, axillary lymph, and inguinal lymph nodes were collected after flushing with 10 mL of PBS, and their fluorescence was measured in an IVIS (registered) Spectrum (PerkinElmer, Waltham, Massachusetts, USA). The organ weights were then measured and stored at -80 °C for subsequent image acquisition by IVIS Spectrum and fluorescence quantification. For image acquisition by IVIS Spectrum, the settings used were "Exc640 / Em700, Bining Medium, F / Stop 2, Exp 0.5 s, FOV 13.2 (C)". Subsequently, the images were analyzed using Living Image 4.5.4 software. Regions of interest (ROIs) were automatically drawn in each organ and manually drawn as circular ROIs in the basal organs. The lymph nodes were analyzed separately. The total radiation efficiency (p / s) / (μW / cm 2 ) of the organs of interest was obtained, and the basal value was subtracted. Then, the total radiation efficiency was normalized according to the organ weight.

[0234] The results show the discriminative in vivo distribution profiles of two different graft copolymers according to their physicochemical properties. The graft copolymer compound 7B shows a renal excretion profile with accumulation in major organs such as the liver and spleen, and specific targeting of the inguinal and axillary lymph nodes (ILN and ALN, respectively). This selective accumulation makes this type of compound an excellent candidate for lymphatic targeting. In the case of compound 9A, which includes the incorporation of a hydrophobic pocket, the accumulation in major organs such as the liver and spleen is increased compared to that observed for 7B, without a distinct renal excretion profile. In this case, the specific targeting to the lymph nodes is completely altered by the introduction of the hydrophobic moiety and does not occur.

[0235] Example 6: Biological evaluation in a primary model of a B16F10 melanoma mouse model Immunomodulatory ability of the graft copolymer St-PLys-g-PGlu (compound 7B). High efficacy and high response rate + safety profile: Preliminary data were obtained.

[0236] The compounds obtained in Example 3 were evaluated for their antitumor effects in a primary model of B16F10 melanoma (the most aggressive type of skin cancer), where the gp100 MHC class I and MHC class II peptides are bound or combined as a single conjugate (St-Br-gp100 MHCI, St-Br-gp100 MHCII) to St-Plys-g-PGlu (alias: St-Br) (St-Br-gp100 MHCI_gp100 MHCII).

[0237] Furthermore, ligands for Toll-like receptor 7 / 8 also bind to St-Br ([St-Br-TLR7 / 8], i.e., the compound described in Example 2), delivering an adjuvant-like signal to antigen-presenting cells (APCs), thereby improving antigen presentation for broad T cell activation and proliferation (see Frega, G. et al., "Trial Watch: experimental TLR7 / TLR8 agonists for oncological indications". Oncoimmunology 9, 1796002 (2020)). Thus, the anti-tumor immune-mediated effects induced by St-Br conjugated to a TLR7 / 8 agonist were evaluated alone and in combination with a St-Br conjugate that delivers both gp100 MHCI and MHCII peptides, and compared to the responses induced by this double conjugate mixed with Toll-like receptor (TLR) 9 (CpG) and TLR3 agonists. The mean tumor volumes of mice treated with [St-Br-gp100 MHCI-gp100 MHCII] and TLR inducers, CpG and Poly I:C, were significantly lower compared to the PBS-treated group at 21 days post-tumor inoculation (P = 0.0335). None of the St-Br conjugates significantly changed the body weight of the mice, indicating the tolerance and safety of these carriers. Mice were sacrificed on day 22 post-tumor inoculation, tumors were harvested and later processed for FACS analysis.

[0238] The double nano-conjugate [St-Br-gp100 MHCI-gp100 MHCII] mixed with CpG and Poly I:C significantly induced infiltration of CD3 + and CD4 + T cells and decreased the proportion of CD3 + cells expressing PD-1. Significantly higher levels of antigen-specific intracellular IFN-γ + were observed in tumor-infiltrating cytotoxic CD8 + T lymphocytes, as well as TNF-α + and +It was also observed. Higher infiltration of T lymphocytes correlates with more potent tumor growth inhibition induced by [St-Br-gp100 MHCI_St-Br-gp100 MHCII] containing CpG and Poly I:C.

[0239] [St-Br-gp100MHCI-gp100MHCII] combined with CpG and Poly I:C promoted the infiltration of bone marrow DC2, but bone marrow DC1 cells, which correlate with a more potent cytotoxic immune response in the TME, were observed at higher levels in the TME of animals treated with [St-Br-TLR7 / 8] alone or in combination with [St-Br-gp100MHCI] or [St-Br-gp100MHCI-gp100MHCII].

[0240] The immune-mediated effects of these multivalent nanovaccines were also evaluated in a humanized patient-derived orthotopic melanoma model. To confirm the humanization process, the presence of human lymphocytes (human CD45 + cells) in the blood of mice was evaluated on days 7, 14, and 21 after tumor cell inoculation. Human CD45 + cells in the total lymphocyte population increased over time from 13.3% on day 7 to 71.6% on day 21 after tumor cell inoculation. 97.1% and 79.1% of CD45 + cells in the blood and spleen, respectively, on day 17 (endpoint day) were human, confirming the successful development of this melanoma patient-derived mouse model. Mice were sacrificed on day 17 after the start of treatment, and tumors and spleens were harvested and later processed for FACS analysis.

[0241] Humanized mice were treated with the conjugated branched-chain polypeptide [St-Br-gp100MHCI-gp100MHCII] alone or in combination with the PD-1 antibody (αPD-1) nivolumab. From day 15 after treatment, tumor growth was significantly reduced by both treatments compared to the PBS treatment group (P = 0.0081 and P = 0.0414 for [St-Br-gp100MHCI-gp100MHCII]+TLRl alone or in combination with αPD-1, respectively). However, no significant difference was observed between them. The individual tumor weights of the mice on day 17 after the start of treatment were significantly reduced in animals immunized with [St-Br-gp100MHCI-gp100 MHCII]+TLRl alone (P = 0.0070) or in combination with αPD-1 (P = 0.0381). No significant difference was observed in the body weights of the mice over time, indicating that the systemic toxicity was negligibly small.

[0242] [St-Br-gp100MHCI-gp100MHCII] in combination with αPD-1 resulted in extensive tumor infiltration of T lymphocytes (P<0.0001). Furthermore, CD8 + T cells were significantly increased in the spleen of [St-Br-gp100MHCI-gp100MHCII] in combination with αPD-1 (P<0.0001). Furthermore, PD-1 expression was quantified in the spleen and was significantly decreased in CD3 + and CD8 + T cells compared to that shown by animals treated with [St-Br-gp100MHCI-gp100MHCII] alone (P<0.0001 for both treatments).

[0243] Overall, these findings demonstrated the potential of MHCI and MHCII restricted antigens and / or St-Br conjugated to TLR ligands as cancer vaccines to modulate melanoma-immune cell crosstalk in the microenvironment. Furthermore, the results obtained after administration of this dual nanoconjugate to a xenograft (PDX) model derived from melanoma patients demonstrated the potential clinical utility of these St-Br conjugates as multivalent cancer vaccines.

[0244] Example 7: Preparation of Compounds of Formula (I) - Glu (Ib2) Linked to Branches with Different Amino Acid Structures

Chemical Structure

[0245] In connection with formula (Ia) of the first aspect herein, the above formula (Ib2) is an example regarding the use of Glu for binding to a branch having a different amino acid structure, where the integer "r" is not 0 and β is an integer of 1.

[0246] Compounds 19 - 20 of formula (Ib2) were synthesized according to the following synthetic scheme 10.

Chemical Structure

[0247] Briefly, the precursor St - PGlu was synthesized as described above for Compound 4, except that L - glutamic acid γ - benzyl NCA was used. In the second step, the benzyl protecting group was deprotected under acidic conditions. Briefly, the polymer was dissolved in pure TFA and the mixture was reacted at room temperature for 3 hours. Then, this solution was poured into diethyl ether to precipitate the product. The product St - PGlu was isolated by centrifugation (3750 rpm, 4 minutes) and vacuum - dried as a white solid.

[0248] In the next step, polymer St-PGlu (20.0 mg, 0.16 mmol of PGlu units) was dissolved in anhydrous DMF (2 mL). Next, 0.4 equivalents of the activator DMTMM was added to the reaction mixture and it was allowed to proceed for 10 - 15 minutes at room temperature under a nitrogen atmosphere. Then, the desired equivalents of the pre-synthesized polyamino acid chain (i.e., polysarcosine (352.0 mg, 0.06 mmol), polylysine thioglycerate dissolved in 4 mL of anhydrous DMF for a specific grafting rate) was added to the reaction mixture. The pH was adjusted to 8 - 9 with TEA (150 μL) and this reaction was allowed to proceed for 48 hours at room temperature under a nitrogen atmosphere. Then, this solution was poured into diethyl ether to precipitate the product. The product was isolated by centrifugation (3750 rpm, 4 minutes) and vacuum dried as a white solid.

[0249] The degree of grafting and grafting efficiency were estimated by NMR. Yield: 70 - 90%. 1 H NMR(D 2 O): δ = 1.21(m, 6H, CH 3 ), 2.05(m, 5H, CH 2 ), 2.35(m, 5H, CH 2 ), 2.83 - 3.30(m, 290H, N-CH 3 ), 4.06 - 4.64(m, 190H, chiral CH, CH 2 ). Grafting rate = 27.66%. Grafting efficiency = 69.15%.

[0250] Table 8 shows two examples of R13 and R6.

Table 8

[0251] As an example, a general procedure for obtaining a pre-polymerized graft chain for PSar is described: Sarcosine-NCA (1675 mg, 14.59 mmol) was placed in a flask equipped with a stir bar and a stopper and then subjected to 3 cycles of vacuum / N 2It was added to the Schlenk tube purged with argon and dissolved in 6 mL of anhydrous DMF. Then, isopropylamine was dissolved in DMF (2 mL) and added to the reaction mixture. The mixture was stirred at 10 °C for 16 h. When the reaction was complete, the reaction mixture became transparent. The complete conversion of the monomer could be detected by IR. Then, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried in vacuo. The final product was isolated as a white solid. Yield: 80%. 1 H NMR (D 2 O): δ = 1.08 (d, J = 6.7 Hz, CH 3 ), 2.73 - 3.12 (m, CH 3 ), 3.78 - 4.56 (m, CH 2 ). MW by SEC: 1222 Da (1.104).

[0252] Table 9 shows the main property evaluations of Compounds 19 and 20.

Table 9

[0253] Example 8: Synthesis of the compound of formula (lb3)

Chemical formula

[0254] Similar to the compound in Example 7, Compound 21 was synthesized according to the following Synthetic Scheme 11.

Chemical formula

[0255] Briefly, a star-shaped -PGlu precursor was synthesized using L - glutamic acid γ - benzyl NCA as described above. In the second step, the benzyl protecting group was deprotected under basic conditions. Briefly, the polymer was dissolved in THF (100 mg / mL), and NaOH (1.5 equivalents) was added. The mixture was stirred at 4 °C for 16 h. Upon completion, the reaction mixture was poured into tert - butyl methyl ether:acetonitrile (3:1) to precipitate the product. The precipitate was isolated by filtration and dried in vacuo. The homopolymer was isolated as a white solid. Yield: 70 - 90%. 1 H NMR(D 2 O):δ = 1.69 - 2.60(m, 2H, CH 2 ), 3.57(m, 2H, CH 2 ); 3.97 - 4.39(m, 1H, CH), 8.32(s, 1H, aryl CH).

[0256] The syntheses of iPrPSar(16) and nBuPHis(DNP)(5) were carried out in a similar manner to the polymerization of PBG. As an example, the synthesis of nBuPHis(DNP)(5) is described.

[0257] Scheme 12 shows a general procedure for the polymerization of PHis(DNP).

Chemical Structure

[0258] Briefly, L - His(DNP)NCA (2000 mg, 5.76 mmol) was added to a Schlenk tube equipped with a stir bar, a stopper, and then purged with 3 cycles of vacuum / N 2 and dissolved in 8 mL of anhydrous DMF. Then, butylamine was dissolved in DMF (2 mL) and added to the reaction mixture. The mixture was stirred at 10 °C for 16 h. Upon completion, the reaction mixture became clear. Complete conversion of the monomer could be detected by IR. Then, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried in vacuo. The final product was isolated as a brown solid. Yield: 80%. 1 H NMR (DMSO): δ = 0.81 (t, J = 7.1 Hz, CH3), 1.06 - 1.40 (m, CH2), 3.37 (m, CH2), 4.48 (m, CH), 7.20 (m, CH), 7.82 - 8.08 (m, CH), 8.22 - 8.46 (m, NH amide), 8.64 (brs, CH), 8.90 (brs, CH).

[0259] Next, both polymers (iPrPSar and nBuHis(DNP)) were continuously coupled to the main PGlu chain by activating them with DMTMMBF as follows. 4 St-PGlu (0.1 g) was added to a two-necked round-bottom flask equipped with a stir bar and a stopper and then purged with 3 cycles of vacuum / N 2 and dissolved in 1 mL of DMF. Then, DMTMMBF 4 (4 equivalents, 0.366 g) was added to the reaction mixture and stirred at room temperature for 30 minutes. After this time, the polymer iPrPSar dissolved in 0.5 mL of DMF was added. The mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 minutes) and dried in vacuo. The final product was isolated as a white solid. Yield: 80%.

[0260] The above procedure was followed for the binding of the polyhistidine polymer. The final product was isolated as a yellowish solid. Yield: 80%.

[0261] A final step consisting of the DNP protecting group of histidine under reducing conditions is required. Briefly, 10 polymer precursors were dissolved in DMF (100 mg / mL) and mercaptoethanol (10 equivalents) was added to the reaction mixture. The mixture was stirred at room temperature for 16 hours. Then, this solution was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 minutes) and dried in vacuo. St-PGlu-graft-into-PSar-graft-into-PHis was isolated as a yellowish solid. Yield: 70 - 90%

[0262] Table 10 shows the compositions and main property evaluations of these graft copolymers. [Table 10]

[0263] Example 9: Formation of unimolecular aggregates from the compound of formula lb2 One of the main properties of our graft copolymers is their high molecular weight and the ability to form unimolecular aggregates with a size suitable, among other things, for their use in drug delivery. To investigate this behavior, we evaluated their hydrodynamic size in aqueous solution by using light scattering techniques. The results obtained for the graft copolymers tested are summarized in Table 11.

[0264] Table 11 shows the hydrodynamic size of the graft copolymer unimolecular aggregates formed in solution. [Table 11]

[0265] Example 10: Binding of a drug to the compound of formula (lb2) We succeeded in binding a hydrophobic drug to Compound 19, i.e., St-PGlu-g-Psar, to obtain the following Compound 22. In this particular example, glycine-vitamin E was bound to St-PGlu-g-Psar (10%) according to the conditions described in Scheme 13. [Chemical formula]

[0266] Briefly, 50 mg of St-PGlu-g-PSar (10%) in sodium salt form (0.058 mmol, 1 equivalent) was dissolved in anhydrous DMSO (2 mL) in a 250 mL round-bottom flask equipped with a stir bar and a septum cap. Then, 3.92 mg (0.012 mmol, 0.2 equivalent) of DMTMMBF4 was added to another 1 mL of DMSO. In this particular case, the added DMTMMBF 4 amount corresponded to a 20% glutamic acid unit modification rate. This reaction was allowed to proceed for 30 minutes under an inert atmosphere with vigorous stirring. Next, 7.15 mg (0.015 mmol, 0.25 eq) of glycine-modified vitamin E was added to the reaction mixture in 1 mL of DMSO. This reaction was stirred for an additional 48 hours. Thereafter, the product was poured into a large excess of diethyl ether. The white solid was isolated by centrifugation and lyophilization. Yield: 36 mg, binding efficiency: 65%, proportion of incorporated Vit E: 13% ( 1 determined by 1H-NMR).

[0267] Example 11: Encapsulation of Hydrophobic Molecules into Compounds of Formula lb3 Due to their amphiphilicity and pH sensitivity of PHis and the presence of highly hydrophobic blocks, compound 21 shown in Example 8 was designed to hold a hydrophobic pocket for drug encapsulation.

[0268] In a first attempt, Nile Red was selected as a hydrophobic drug model with logP: 2.98 to characterize the system. A pH titration was also performed to find the hydrophobic range for drug encapsulation. The procedure followed was as described for Compounds 10 - 12. It was found that the pka for Compound 21A was 6.45, and the Nile Red assay showed a transition from a hydrophilic environment to a hydrophobic environment upon increasing pH.

[0269] Example 12: Preparation of Novel Compounds of Formula (I) - Lys Linked to Branches with Different Amino Acid Structures Compounds 22A - G are novel examples based on the previous compounds described in the section of Example 1. That is, Compounds 22A, 22C, and 22D are synthesized according to the procedure described in Example 1C, and Compounds 22B, 22E - G are synthesized according to the procedure described in Example 1E by changing the degree of polymerization and structure. The design of these polymers aims to vary structural parameters such as morphology, density, and lower ionic charge. [Table 12]

[0270] Minor note Merely as a note, according to the art, the term "pharmaceutically acceptable" is useful for preparing pharmaceutical compositions and is preferably non - toxic, that is, a "pharmaceutically acceptable salt" is preferably a non - toxic salt. Numerous different types of pharmaceutically acceptable salts are known to those skilled in the art.

[0271] List of references 1: European Patent No. 3331937B1

Claims

1. Formula Ia, including homopolypeptides or random, block, or graft copolymers: 【Chemistry 1】 A graft copolymer which is any stereoisomer or mixture of stereoisomers of any compound, a pharmaceutically acceptable salt thereof, or a compound of formula (Ia), or any pharmaceutically acceptable salt thereof, During the ceremony, A, A', and A'' are each independently selected from polymers containing a repeating structural unit skeleton of formula (I), and each of the A, A', and A'' subunits may be the same or different. K, K', and K'' are each independently selected from -O- and -NH-, X is, 【Chemistry 2】 Selected from, ω, ω', and ω'' are integers between 0 and 1, and each wavy line indicates a connection point to A, A', or A'', and " ＊ " indicates a connection point to K, K', or K''. Here, equation (I) is: 【Transformation 3】 It is a graft copolymer compound containing a backbone of repeating structural units, The main repeating units, each defined by square brackets along with the numerical values ​​o, r, t, u, and v, are shown in a specific order for the sake of explanation, but these main repeating units may exist in any order and may exist in blocks or randomly. Each of the main repeating units defined by square brackets with the numerical value "o" may be the same or different from each other, and may include a block of secondary monomer units defined by square brackets with the numerical values ​​"p" and "q", which may be the same or different from each other according to the definitions of their different substituents. Each of the main repeating units defined by square brackets with the numerical value "r" may be the same or different from each other, and may include a block of secondary monomer units defined by square brackets with the numerical values ​​"s" and "t", which may be the same or different from each other according to the definitions of their different substituents. " ＊ " indicates a connection point, The symbol " / " indicates that the order of the repeating units on either side of the symbol is arbitrary. R １ teeth, 【Chemistry 4】 A biradical selected from the group consisting of, The wavy line indicates the connection point. y and z are independent integers in the range of 1 to 20, and Y is a biradical having a molecular weight (MW) of 5 to 3000 g / mol. R ２ The is selected from -O- and -NH-. Given that at least one of o or r is not 0, o and r are integers independently selected from 0 to 5000. p and s are integers independently selected from 1 to 1000. q, t, u, and v are each integers independently selected from 0 to 1000. α and α' are integers independently selected from 1 to 4. β and β' are integers independently selected from 0 to 3. Each R ３ These are independently H and -CH ３ Selected from, Each R ４ These are independently H and -CH ３ Selected from, Each R ５ is independently selected from H and -CH ３ and Each R ６ These are independently H and -CH ３ Selected from, Each R ７ These are independently H and -CH ３ Selected from, Each R ８ These are independently H and -CH ３ Selected from, Each R ９ These are independently H and -CH ３ Selected from, Each R １０ These are independently H and -CH ３ Selected from, Each R １５ These are independently H and -CH ３ Selected from, Each R １１ and R １２ These are independently H, contrast agents, labeling agents, cell targeting agents, and formulas (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII): 【Transformation 5】 Selected from the radicals, Q is selected from -OH, contrast agents, labeling agents, and cell targeting agents, and a, b, c, d, e, and f are each integers independently selected from 0 to 4. Each R １３ H and -CH are independent of each other. ３ , contrast agents, labeling agents, cell targeting agents and formulas (XII), (XIII), (XIX), (XX), (XXI), (XXII), (XVIII), (XXIV), (XXV), (XXVII), (XXVII), (XXVII), (XXVII), (XXIX), (XXXX), (XXXXI), (XXXXII), (XXXIII), (XXXVIV), (XXXV), (XXXVII), (XXXVII), or (XXXVIIII): 【Transformation 6】 Selected from the radicals, g, h, i, and j are each integers independently selected from 0 to 4. T is selected from H and -OH, Each R １４ These are independently selected from H, -OH, contrast agents, labeling agents, and cell targeting agents. L is a single bond and formula: 【Transformation 7】 Selected from the biradicals, k, l, m, n, w, and x are each integers independently selected from 0 to 4. "*" indicates a connection point. Each X １ These are independently H, -C(O)H, -S(O)H, and -NHCH(CH ３ ) ２ , - (C １ ~C ４ ) alkyl NH ２ , - (C １ ~C ４ ) alkylNHCH ３ , - (C １ ~C ４ ) Alkyl N (CH ３ ) ２ , -O-(C １ ~C ４ ) Alkyl-NH ２ , -O-(C １ ~C ４ ) Alkyl-NHCH ３ , -O-(C １ ~C ４ ) Alkyl-N (CH ３ ) ２ , selected from contrast agents, labeling agents, cell targeting agents and radicals of formulas (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII), Each X ２ These are independently H, -C(O)H, -S(O)H, and -NHCH(CH ３ ) ２ , - (C １ ~C ４ ) alkyl NH ２ , - (C １ ~C ４ ) alkylNHCH ３ , - (C １ ~C ４ ) Alkyl N (CH ３ ) ２ , -O-(C １ ~C ４ ) Alkyl-NH ２ , -O-(C １ ~C ４ ) Alkyl-NHCH ３ , -O-(C １ ~C ４ ) Alkyl-N (CH ３ ) ２ , selected from contrast agents, labeling agents, cell targeting agents and radicals of formulas (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII), and Each X ３ is independently H, -C(O)H, -S(O)H, -NHCH(CH ３ ) ２ , -(C １ ~C ４ )alkylNH ２ , -(C １ ~C ４ )alkylNHCH ３ , -(C １ ~C ４ )alkylN(CH ３ ) ２ , -O-(C １ ~C ４ )alkyl-NH ２ , -O-(C １ ~C ４ )alkyl-NHCH ３ , -O-(C １ ~C ４ )alkyl-N(CH ３ ) ２ , a contrast agent, a labeling agent, a cell targeting agent, and selected from the radicals of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII) Graft copolymer.

2. R １ The Y radical of is -NH-, -NH(C １ ~C ６ )alkyl-, -O-, -(C １ ~C ６ )alkyl-COO-, linear or branched -(C １ ~C ３０ )alkylene and formula (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI): 【Transformation 8】 A biradical selected from the group consisting of the following biradicals: " ＊ " indicates a connection point, Y - (C １ ~C ３０ ) Alkylene biradicals are -OH, -NR ａ R ｂ , -SH ２ , -NHNH ２ , -COOR ｃ , -CF ３ , -OCF ３ (In the formula, R ａ , R ｂ and R ｃ These are independently H, -phenyl, -(C １ ~C ３０ ) alkyl, -(C ２ ~C ３０ ) Alkenil, - (C １ ~C ３０ ) alkylphenyl and -phenyl (C １ ~C ３０ (A radical selected from the group consisting of alkyls) and optionally substituted with one or more radicals selected from the group consisting of halogens, The graft copolymer according to claim 1.

3. The graft copolymer according to claim 1, wherein the graft copolymer has a molecular weight (MW) of 3 to 500,000 kilodaltons (kDa), and the graft copolymer is a monomolecule structure having a radius of 4 to 300 nm.

4. The integers ω, ω', and ω'' are 0, and R １ is ethylene, and R ２ The graft copolymer according to claim 1, wherein the coefficient is -NH-.

5. The graft copolymer according to claim 1, wherein the integer "o" is not 0, L is a single bond, α is an integer of 3, and the integers "r" and "v" are 0.

6. The graft copolymer according to claim 5, wherein the value of the integer "u" is less than 25% of the numerical value of the integer "o".

7. The graft copolymer according to claim 5, wherein the value of the integer "o" is between 5 and 3000, and the value of the integer "p" is between 3 and 1000.

8. Each R １１ and R １２ The graft copolymer according to claim 5, wherein is independently selected from H and radicals of formulas (XII), (XIII), (XIV), and (XVI).

9. The aforementioned graft copolymer is St-PLys-g-PGlu St-PLys-g-PHis St-PLys-g-PHis-b-PGlu St-PLys-g-PHis-b-PSar St-PLys-g-PBG St-PLys-g-PBG-b-PGlu St-PLys-g-PBG-b-PSar St-PLys-g-PSar St-Plys-g-PSar-g-VitE, and St-(PLys-g-PGlu)-stat-PSar A compound comprising a compound selected from the group consisting of, The term "St" refers to the star-shaped structure of formula (Ia) described in claim 1, The term "PBG" refers to poly(benzyl glutamate). The graft copolymer according to claim 5.

10. The graft copolymer according to claim 1, wherein the integer "r" is not 0, β is an integer of 1, and the integers "o" and "u" are 0.

11. The graft copolymer according to claim 10, wherein the value of the integer "r" is between 3 and 1000, and the value of the integer "s" is between 2 and 400.

12. R １３ The graft copolymer according to claim 10, wherein is independently selected from H and radicals of formulas (XII), (XIII), (XXI), and (XXVIII).

13. The aforementioned graft copolymer is St-PGlu-g-PSar St-PGlu-g-PHis St-PGlu-g-PLys St-PGlu-g-PlysTG, and St-PGlu-g-PSar-g-Phys A compound comprising a compound selected from the group consisting of, The term "St" refers to the star-shaped structure of formula (Ia) described in claim 1, PlysTG is polylysine thioglycerate. The graft copolymer according to claim 10.

14. A polymer complex comprising the graft copolymer described in Claim 1 and at least a pharmaceutical, veterinary, or cosmetic active agent.

15. A pharmaceutical, veterinary, or cosmetic composition comprising at least one polymer complex according to claim 14 together with one or more suitable excipients or carriers.

16. A polymer complex according to claim 14 or a pharmaceutical composition according to claim 15 for use as a medicine.

17. A composition for use in a method for delivering a pharmaceutically active drug into target cells, wherein the composition contains the polymer complex described in claim 14, and the method comprises administering the composition to an animal, including a human, to physically contact the complex with target cells, thereby delivering the pharmaceutically active drug into the target cells.

18. A process for preparing a compound structurally different from the graft copolymer starting compound described in claim 1, (i) A step of using the compound described in claim 1 as a starting compound, and (ii) A step of adding a structural change to the compound of step (i) to obtain a compound that is structurally different from the starting compound of step (i). Includes, A process wherein the structurally different compound obtained in step (ii) is a compound that can form a polymer complex comprising the structurally different compound obtained in step (ii) and at least one pharmaceutical, veterinary, or cosmetic active agent, and the active agent is an active agent that can form both the starting compound of step (i) and the polymer complex together.

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