Star graft copolymer

Abstract

Novel star polymers comprising graft copolymer backbone arms, for example, for the delivery of active agents and / or imaging agents to target cells or tissues.

Description

Technical Field

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

[0002] Considerable efforts have been made to develop novel polymer structures with specific properties for use as targeted drug delivery systems, including macromolecules such as peptides and nucleic acids.

[0003] The delivery of active ingredients to target sites located within cells requires appropriate delivery vehicles that provide adequate protection and allow the active ingredients to be effectively delivered to specific tissues in the body.

[0004] Delivery vehicles must overcome different extracellular and intracellular barriers to reach their target sites within the cell. Although viral vectors may be more effective for gene delivery than non-viral vectors, their use is associated with 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] EP3331937B1 discloses non-viral vectors made from a family of 3-arm star-shaped polypeptide derivatives, which consist of a 1,3,5-benzenetricarboxamide-related central core, which serves as an initiator for the ring-opening polymerization of N-carboxylic anhydride monomers, and three polypeptide backbone arms. EP3331937B1 does not directly and explicitly disclose the novel graft copolymer compounds described herein. Summary of the invention

[0006] The problem addressed by the present invention can be seen as providing novel graft copolymers having novel / improved properties and capable of delivering, for example, active agents and / or imaging agents to target cells or tissues.

[0007] According to the prior art - the term "graft copolymer" relates to the general class of segment-type copolymers and usually consists of a linear backbone of one composition and randomly distributed branches of a different composition (see for example herein Figure 1 for illustration purposes).

[0008] As discussed above - EP3331937B1 discloses non-viral vectors made from a family of 3-armed star polypeptide derivatives. As known in the art - star polypeptides are branched polymers consisting of various linear chains connected to a central core.

[0009] Claim 4 of EP3331937B1 discloses a compound structure which encompasses, for example, "R 2ˊˊˊLysine (Lys) or glutamic acid (Glu) side chain free radicals, CL free radicals are generalized (C 1 -C 500 )-alkyl. The CL radical does not directly and explicitly describe the relevant amino acid structure in this document - that is, this document does not directly and explicitly describe such a structure, in which the "R" of the R arm structure of claim 4 2ˊˊˊ “Amino acid side chains are bound to the amino acid base structure.

[0010] In other words, EP3331937B1 does not disclose a graft copolymer in which amino acid side chains of a linear main chain are bonded to branches having an amino acid structure.

[0011] Thus, and only as an illustrative example - EP 3331937 B1 does not disclose a graft copolymer structure in which R 2ˊˊˊ is a Lys (PLys) side chain and is connected to, for example, a glutamic acid (PGA)-based branched structure (see, for example, herein Figure 2 Illustrative example "StPLys-g-PGA" structure) combined with or R 2″′ It is a Glu (PGA) side chain and it is bound to a sarcosine (PSar) based branch structure (see, e.g., the illustrative example "PGA-g-PSar-g-PHis").

[0012] In general, and without being limited by theory - it is believed that the present invention is based on the inventors' identification that graft copolymers with novel / improved properties compared to the star-shaped polypeptide structure in EP3331937B1 can be obtained by preparing graft copolymers in which, for example, Lys / Orn (ornithine) and / or Glu / Asp (aspartic acid) amino acid side chains of a linear backbone are attached to branches having an amino acid structure.

[0013] The novel graft copolymers discussed herein may generally be referred to herein as Starbrushes.

[0014] As discussed in further detail below (see, e.g., the working examples), the novel graft copolymers discussed herein can have a number of different useful advantages - for example, molecular weights (MW) that are high enough to allow for the preparation of nanometer-scale (e.g., 4-300 nm in radius) structures that can be used to deliver, for example, active agents to target cells without the need for self-assembly processes.

[0015] For example, as discussed in

[0007] of EP3331937B1, the star-shaped polypeptides discussed therein typically need to undergo a self-assembly process to prepare nanoscale structures, which may be difficult to control on an industrially relevant scale - therefore, the fact that the new star-shaped brush graft copolymers discussed herein can be prepared without a self-assembly process can be regarded as an advantage over the star-shaped polypeptides described in EP3331937B1.

[0016] Another advantage of the novel graft copolymers discussed herein relates to the fact that by selecting different / appropriate amino acid side chains with interesting properties (e.g., hydrophobic / hydrophilic properties or cationic / anionic properties) - one has tremendous flexibility to prepare graft copolymer compounds of desired purpose of interest.

[0017] For example - Example 1 below describes examples of graft copolymers designed to obtain unimolecular aggregates with a hydrophobic pocket that has the ability to encapsulate a hydrophobic payload / agent of interest - (see, for example, compounds 8-11 below).

[0018] One can also, for example, prepare graft copolymers having varying degrees of both hydrophobicity and hydrophilicity (see, for example, compounds 8-12 of Example 1 below).

[0019] One can also, for example, conjugate an agent of interest, such as a cell targeting agent (such as, for example, a small molecule) or, for example, a therapeutic peptide, to the graft copolymers described herein - see, for example, exemplary compounds 15, 18, 22 discussed below.

[0020] Therefore, the first aspect of the present invention relates to a graft copolymer, wherein the graft copolymer is any stereoisomer or mixture of stereoisomers of any one of the compounds of formula Ia, their pharmaceutically acceptable salts or compounds of formula (Ia) or their pharmaceutically acceptable salts, including homopolymeric polypeptides or random or block or graft copolymeric polypeptides:

[0021]

[0022] wherein A, A' and A" are each independently selected from a polymer comprising a main chain of repeating structural units of formula (I); and each of the A, A' and A" subunits may be the same or different;

[0023] wherein K, K' and K" are each independently selected from -O-, -NH-;

[0024] X is selected from

[0025]

[0026] wherein ω, ω' and ω" are integers from 0 to 1; each wavy line represents a point of connection with A, A' or A"; and "*" represents a point of connection with K, K' or K";

[0027] and wherein formula (I) is a graft copolymer compound comprising a main chain of repeating structural units of formula (I)

[0028]

[0029] wherein although the main repeating units defined by square brackets with their values ​​o, r, t, u and v, respectively, are shown in a particular order for ease of description, the main repeating units may be present in any order and may be present in blocks or randomly;

[0030] wherein each of the primary repeating units defined by square brackets with the value "o" may be the same or different from each other, and may include blocks of secondary monomeric units defined by square brackets with the values ​​"p" and "q", which blocks may be the same or different from each other depending on the definitions of their different substituents;

[0031] wherein each of the primary repeating units defined by square brackets with the value "r" may be the same or different from each other, and may include blocks of secondary monomeric units defined by square brackets with the values ​​"s" and "t", which blocks may be the same or different from each other depending on the definitions of their different substituents;

[0032] Where "*" indicates a connection point;

[0033] Wherein " / " indicates that the order of the repeating units on both sides of the symbol is arbitrary;

[0034] R 1 is a diradical selected from the following

[0035]

[0036] The wavy lines indicate the connection points;

[0037] wherein y and z are independently integers ranging from 1 to 20; and

[0038] Where Y is a diradical with a molecular weight (MW) of 5 to 3000 g / mol

[0039] R 2 is selected from -O-, -NH-;

[0040] wherein o and r are each independently selected from integers from 0 to 5000; provided that at least one of o or r ≠ 0;

[0041] p and s are each independently selected from an integer from 1 to 1000;

[0042] q, t, u and v are each independently selected from integers from 0 to 1000;

[0043] α, α' are each independently an integer selected from 1 to 4;

[0044] β and β' are each independently an integer selected from 0 to 3;

[0045] Each R 3 are independently selected from H and -CH 3 ;

[0046] Each R 4 are independently selected from H and -CH 3 ;

[0047] Each R 5 are independently selected from H and -CH 3 ;

[0048] Each R 6 are independently selected from H and -CH 3 ;

[0049] Each R 7 are independently selected from H and -CH 3 ;

[0050] Each R 8 are independently selected from H and -CH 3 ;

[0051] Each R 9 are independently selected from H and -CH 3 ;

[0052] Each R 10 are independently selected from H and -CH 3 ;

[0053] Each R 15 are independently selected from H and -CH 3 ;

[0054] R 11 and R 12 Each independently selected from H, an imaging agent or a labeling agent, a cell targeting agent and a free radical of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII)

[0055]

[0056] wherein Q is selected from -OH, an imaging agent or a labeling agent, a cell targeting agent, and

[0057] a, b, c, d, e and f are each independently selected from an integer from 0 to 4;

[0058] Each R 13 are independently selected from H, -CH 3 , imaging or labeling agents, cell targeting agents and free radicals of formula (XII), (XIII), (XIX), (XX), (XXI), (XXII), (XVIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIII)

[0059]

[0060] wherein g, h, i and j are each independently selected from integers from 0 to 4;

[0061] T is selected from H and -OH;

[0062] Each R 14 independently selected from H, -OH, an imaging agent or labeling agent, and a cell targeting agent;

[0063] wherein L is selected from a single bond, and a diradical of formula (XXXIX), (XL), (XLI), (XLII), (XLIII) or (XLIV)

[0064]

[0065] wherein k, l, m, n, w and x are each independently selected from an integer from 0 to 4;

[0066] Wherein "*" indicates the connection point;

[0067] Each X 1 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 2 、-(C 1 -C 4 ) alkylNHCH 3 、-(C 1 -C 4 )alkylN(CH 3 ) 2 、-O-(C 1 -C 4 )alkyl-NH 2 、-O-(C1 -C 4 )alkyl-NHCH 3 、-O-(C 1 -C 4 )alkyl-N(CH 3 ) 2 , imaging or labeling agents, cell targeting agents, and free radicals of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII);

[0068] Each X 2 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 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 , an imaging or labeling agent, a cell targeting agent, and a free radical of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII); and

[0069] Each X 3 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 2 、-(C 1 -C 4 ) alkylNHCH 3 、-(C 1 -C 4 )alkylN(CH 3 ) 2 、-O-(C 1 -C4 )alkyl-NH 2 、-O-(C 1 -C 4 )alkyl-NHCH 3 、-O-(C 1 -C 4 )alkyl-N(CH 3 ) 2 , imaging or labeling agents, cell targeting agents, and free radicals of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII).

[0070] For purposes of illustration only regarding the scope of the first aspect, the illustrative example structures of the figures herein are discussed below.

[0071] In this article Figure 2 The example "St-PLys-g-PGA" structure (alternatively referred to as "St-PLys-g-PGlu") can be viewed as a structure in which there are grafted Lys / Orn related units (i.e., the integer "o" is ≠0) and there are no grafted Glu / Asp related units (i.e., the integer "r" is 0) in the terms of the first aspect.

[0072] The term "St" in the "St-PLys-g-PGA" nomenclature relates to the central core structure within the scope of formula (Ia) of the first aspect - such as, for example Figure 2 The structure shown has a star-shaped 1,3,5-benzenetricarboxamide ring associated with the central core.

[0073] Figure 2 The example "St-PLys-g-PHis-b-PGA" structure also includes a PGA block (b) - that is, the integer "q" is ≠0.

[0074] Figure 2 The example "St-PGA-g-PSar-g-PHis" structure can be regarded as a structure in which there are grafted Glu / Asp related units (i.e., the integer "r" is ≠0) and there are no grafted Lys / Orn related units (i.e., the integer "o" is 0) in the terms of the first aspect.

[0075] The example of compound 10D below, St-PLys(18)-g-PHis(5)-b-PSar(20)-, can be viewed in terms of the first aspect as a structure wherein the integer "o" is 18, the integer "p" is 5, and the integer "q" is 20.

[0076] As discussed above - the first aspect states that "each of the primary repeating units defined by square brackets with the value "r" may be the same or different from each other, and may include blocks of secondary monomer units defined by square brackets with the values ​​"s" and "t", which blocks may be the same or different from each other according to the definitions of their different substituents."

[0077] therefore, Figure 2 The illustrative "PGA-g-PSar-g-PHis" structure is an example of a situation within the scope of claim 1, where the "s" units differ in that some include PSar while others include PHis substituents.

[0078] According to the prior art and as herein Figure 1 As shown:

[0079] - the term "grafting from" relates to a process wherein the linear backbone comprises active sites capable of initiating polymerization, thus functional groups acting as initiators to be grafted therefrom; and

[0080] - The term "grafting to" relates to a process involving the use of a backbone having functional groups which are not used to initiate such polymerization and which can therefore be considered to serve as "attachment points" for the pre-synthesized polymer.

[0081] As is known in the art, the "H" of the -Lys side chain 3 N + "(amino) group (see e.g. Figure 2 The "St-PLys-g-PGA" structure) can be the main chain initiator active site in the "grafting from" method discussed in this article - that is, the amine group acts as an initiator to graft from it.

[0082] As is known in the art, the "COO-" (carboxyl) group of the -Glu side chain (see e.g. Figure 2 The "PGA-g-PSar-g-PHis" structure) can be the backbone "attachment point" functional group in the "grafting to" method discussed in this article - that is, the carboxyl group is used as the "attachment point" for example of a pre-synthesized polymer through its terminal amine.

[0083] In summary, "grafting from" and "grafting to" are methods well known to those skilled in the art - that is, those skilled in the art can determine a method suitable for synthesizing the graft copolymer compound of the first aspect based on the technical information disclosed herein and general common knowledge - that is, those skilled in the art can routinely prepare the compound of the first aspect.

[0084] In formula (I) of the first aspect - the main repeating units defined by square brackets with a value "t" may be considered to be related to the use of, for example, Ala / Sar / Gly.

[0085] Several of the working examples herein describe structures where the integer "t" is zero - therefore, it may be preferred that the integer "t" is zero.

[0086] As those skilled in the art understand in this context, the description of the first aspect “ include The term "comprising" in the phrase "graft copolymer compound having a main chain of repeating structural units of formula (I)" should be understood as meaning that the graft copolymer of the present invention may include other structures (e.g. repeating units) in addition to the structures explicitly shown in formula (I) with respect to the first aspect.

[0087] For example, the graft copolymer compounds of the present invention may include repeating units similar to unit "t", wherein the unit includes an amino acid related structure different from the Ala / Sar related amino acid of unit "t" (such as, for example, arginine (Arg), cysteine ​​(Cys), etc.).

[0088] A second aspect of the present invention relates to a polymer composite comprising the graft copolymer of the first aspect and / or embodiments thereof, and at least one pharmaceutical, veterinary or cosmetic active agent.

[0089] As discussed herein - based on common general knowledge and the technical disclosure herein - the preparation of the polymer composite of the second aspect of the present invention and / or embodiments thereof is a routine task for a person skilled in the art.

[0090] A third aspect of the present invention relates to a pharmaceutical, veterinary or cosmetic composition comprising at least one polymer complex according to the second aspect and / or embodiments thereof, and one or more suitable excipients or carriers.

[0091] Based on common general knowledge and the technical disclosure herein, it is a routine task 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.

[0092] The fourth aspect of the present invention relates to the polymer complex according to the second aspect and / or embodiments thereof, or the pharmaceutical composition according to the third aspect and / or embodiments thereof, for use as a medicament.

[0093] 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, which is (i) used as a transfection reagent for transfecting at least one active agent into a cell; (ii) used for in vivo or ex vivo production of biological preparations encoding recombinant proteins, peptides or antibodies, or for producing recombinant viruses; (iii) used as a therapeutic or prophylactic vaccine against viral infection or as a therapeutic vaccine against cancer; or (v) used for genome engineering, for cell reprogramming, for differentiating cells or for gene editing.

[0094] Based on common general knowledge and the technical disclosure herein, it is a routine task for a person skilled in the art to determine the suitable medical use of the polymer complex and / or pharmaceutical composition described herein.

[0095] For example, many of the medical uses described in the art for related polymer compounds (see, for example, EP 3331937 B1 discussed above) may also be relevant to the graft copolymer compounds of the present invention.

[0096] 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 a composition (e.g., a solution) comprising the polymer complex of the second aspect and / or its embodiments to an animal, including a human, so that the complex is in physical contact with the target cell, thereby delivering the pharmaceutically active agent into the cell.

[0097] Based on common general knowledge and the technical disclosure herein - it is a routine task for those skilled in the art to determine a suitable method to deliver an active agent of interest to a target cell of interest.

[0098] The working examples herein describe that the graft copolymer compounds of the present invention can be brought into physical contact with a target cell of interest, thereby delivering an active agent of interest to the cell.

[0099] Furthermore, related polymer compounds (see, for example, EP 3331937 B1 discussed above) have also been described for use in delivering active agents into target cells of interest.

[0100] A seventh aspect of the present invention relates to a method for preparing a compound that is structurally different from the starting compound of the graft copolymer of the first aspect and / or its embodiments, comprising the following steps:

[0101] (i): using the compound of the first aspect and / or its embodiments as a starting compound;

[0102] (ii) making structural changes to the compound of step (i) to obtain a compound that is structurally different from the starting compound of step (i).

[0103] Based on common general knowledge and the technical disclosure herein, it is a routine task for those skilled in the art to prepare structurally different compounds using the graft copolymer compounds described herein as starting compounds.

[0104] Various embodiments of the present invention are described below by way of example only.

[0105] It is obvious to a person skilled in the art that a preferred embodiment described herein (e.g. a preferred free radical structure) may preferably be combined with another described preferred embodiment (e.g. another preferred free 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

[0106] Figure 1 : Illustrative examples of graft-from / graft-to methods for preparing graft copolymers.

[0107] Figure 2 : Illustrative examples of graft copolymer compounds / structures within the scope of the first aspect (i.e., claim 1) herein. DETAILED DESCRIPTION

[0108] Graft copolymer of the first aspect - star-shaped core structure

[0109] Among the various compounds of the working examples herein (see e.g. Figure 2 and the working examples below) - these compounds include a stellate-related central core similar to the stellate core described in EP3331937B1 discussed above.

[0110] Structure X can be viewed as offering the possibility to connect even more branches to the star-shaped core structure.

[0111] It may be preferred (for example in the graft copolymer compounds of the working examples herein) that the structure X is absent - ie the ω, ω' and ω" integers are 0.

[0112] Preferably, K, K' and K" are each -NH-.

[0113] As discussed above and in the working examples herein, the molecular weight (MW) of the novel graft copolymers discussed herein can be high enough to allow the preparation of nanometer-range (e.g., 4-300 nm in radius) structures useful for delivering, for example, active agents to target cells without the need for self-assembly processes.

[0114] Therefore, in a preferred embodiment, the graft copolymer of the first aspect and related embodiments thereof herein is a graft copolymer wherein the molecular weight (MW) is from 3 to 500,000 kilodaltons (kDa) - more preferably from 10 to 100,000 kilodaltons (kDa), even more preferably from 10 to 20,000 kilodaltons (kDa), and most preferably from 20 to 10,000 kilodaltons (kDa).

[0115] As is known in the art - the molecular weight (MW) of a compound of known structure can be calculated / determined from information in the periodic table.

[0116] For suitable experimental MW determination methods - see, for example, the "Size Exclusion Chromatography (SEC)" analytical methods in our Examples text herein.

[0117] A preferred embodiment relates to the graft copolymer of the first aspect and related embodiments thereof herein, wherein the graft copolymer is a unimolecular structure with a radius of 4-300 nm.

[0118] For suitable radius determination methods - see for example the "Size Distribution" analysis method in our Examples text herein.

[0119] The graft copolymer of the first aspect - the free radical R 1 and R 2

[0120] As discussed above -R 2 Selected from -O-, -NH-.

[0121] In several structures exemplified in this article, R 2 =-NH-

[0122] Therefore, R is preferred 2 =-NH-

[0123] As discussed above -R 1 is a diradical selected from the following

[0124]

[0125] The wavy lines indicate the connection points;

[0126] wherein y and z are independently integers ranging from 1 to 20; and

[0127] wherein Y is a diradical having a molecular weight (MW) of 5 to 3000 g / mol.

[0128] Free Radical R 1 Essentially it can be considered as a covalent linker radical - ie a linker to the left end "*" of formula (I) of the first aspect.

[0129] In this article Figure 2 In the illustrative structure of a star-shaped peptide, R 1 = ethyl - that is, "R 1 =Ethyl" linker is used to connect / attach the star-related central core shown to the left end "*" connection point of the graft copolymer shown of formula (I) - see also star polypeptide formula (Ia) herein.

[0130] R 1 The linker-related radical may be considered to correspond to, for example, the analogous R 1 Linker-related free radicals.

[0131] As discussed in this EP 1 patent (see e.g. claim 1 and working examples - e.g. Table 1 on page 29) - this R 1 The linker-associated free radical can be of a variety of different suitable structures - including, for example, structures that can cleave disulfide bonds in human cells in vivo.

[0132] In summary - the skilled person knows from the art a number of different suitable linker-related structures of different molecular weight (MW) sizes.

[0133] It may be preferred that Y is a diradical having a molecular weight (MW) of 8 to 1000 g / mol or more preferably 2 to 500 g / mol.

[0134] Preferably, R 1 The Y diradical is selected from the following diradicals: -NH-, -NH(C 1 -C 6 )alkyl-, -O-, -(C 1 -C 6 )alkyl-COO-, straight or branched-(C 1 -C 30 )alkylene- and a diradical of formula (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI)

[0135]

[0136] Where "*" indicates a connection point;

[0137] Among them, Y-(C 1 -C 30 ) The alkylene diradical is selected from -OH, -NR a R b , -SH 2 、-NHNH 2 、-COOR c , -CF 3 、-OCF 3 and one or more free radicals of halogen, wherein R a , R b and R c is a free radical independently selected from the following: H, -phenyl, -(C 1 -C 30 )alkyl, -(C 2 -C 30 )alkenyl, -(C 1 -C 30 ) alkylphenyl and -phenyl (C 1 -C 30 )alkyl.

[0138] More preferably, R 1 is a diradical selected from the following: -CH 2 CH 2 -SS-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -SS-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 2 COO- and a diradical selected from (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) and (XI) as defined herein.

[0139] Most preferably, R 1 It's vinyl.

[0140] Graft copolymers of the first aspect - Lys / Orn related units with integers "o" and "u"

[0141] In formula (I) - the main repeating units defined by square brackets with the values ​​"o" and "u" can be considered to be related to the use of, for example, lysine / ornithine (Lys / Orn).

[0142] In one embodiment of the present invention - the integer "o" is ≠0.

[0143] L may preferably be a single bond.

[0144] Orn may be any suitable amino acid - ie it may be preferred that α is the integer 2.

[0145] Lys is used in several of the structures exemplified herein - therefore, it is preferred that α is an integer of 3.

[0146] If the integer "o" is ≠0 and "u" is zero, it is understood that all Lys / Orn related repeat units are bound to a branch having a repeat unit "p" and optionally a "q" amino acid structure.

[0147] The working examples herein describe the structures of most of the Lys / Orn related repeating units combined with branches having amino acid structures.

[0148] Thus, in a preferred embodiment, the value of the integer "u" is less than 25% of the value of the integer "o" - that is, if the value of the integer "o" is 100, then the value of the integer "u" is less than 25.

[0149] 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" - and most preferably, the value of the integer "u" is less than 0.5% of the value of the integer "o".

[0150] In several of the structures exemplified herein - the integer "o" is ≠0 and the integers "r" and "v" of the Glu / Asp related units are absent - ie, the integers "r" and "v" are zero.

[0151] Therefore, in a preferred embodiment - the integer "o" is ≠0 and the integers "r" and "v" are 0.

[0152] Preferably, the integer "o" has a value from 5 to 3000 (more preferably from 10 to 2500, even more preferably from 50 to 2000, and most preferably from 100 to 1500).

[0153] Preferably, the integer "p" has a value from 3 to 1000 (more preferably from 5 to 750, even more preferably from 8 to 250, and most preferably from 10 to 200).

[0154] like Figure 2 As shown in the structure of "St-PLys-g-PHis-b-PGA" - the graft copolymer compound may also include an amino acid related block (b) structure - that is, the integer "q" is ≠0.

[0155] Therefore, it may be preferred that the value of the integer "q" is from 3 to 1000 (more preferably from 5 to 750, even more preferably from 8 to 250, and most preferably from 10 to 200).

[0156] Preferably, R 11 and R 12 Each is independently selected from H and free radicals of formula (XII), (XIII), (XIV) and (XVI).

[0157] Preferably, R 11 is independently selected from H and free radicals of formula (XII), (XIII), (XIV) and (XVI).

[0158] As discussed above and in the working examples herein, the molecular weight (MW) of the novel graft copolymers discussed herein can be high enough to allow the preparation of nanometer-range (e.g., 4-300 nm in radius) structures useful for delivering, for example, active agents to target cells without the need for self-assembly processes.

[0159] High MW can be obtained by having relatively high values ​​of integer "o" and integer "p" - thus, in a preferred embodiment, the graft copolymer compound of the first aspect is a graft copolymer compound wherein

[0160] - the integer "o" has a value from 25 to 5000 (more preferably from 100 to 2500) and the integer "p" has a value from 2 to 2000 (more preferably from 10 to 500); and

[0161] - a molecular weight (MW) of 3 to 500,000 kDa - more preferably 10 to 100,000 kDa, even more preferably 10 to 20,000 kDa, and most preferably 20 to 10,000 kDa.

[0162] A preferred embodiment relates to the graft copolymer of the first aspect and related embodiments thereof herein, wherein the graft copolymer is a unimolecular structure with a radius of 4-300 nm.

[0163] Example 1 below describes examples of St-PLys-g-PGlu (see, e.g., Compounds 7A-7H) and provides data showing the advantageous properties of these St-PLys-g-PGlu structures.

[0164] Therefore, a preferred embodiment relates to St-PLys-g-PGlu related compounds - that is, wherein the integer "o" is ≠0, α is preferably an integer of 3 and R 11 Yes (XVI).

[0165] Example 1 below describes examples of graft copolymers designed to obtain unimolecular aggregates with a hydrophobic pocket that has the ability to encapsulate a hydrophobic payload - (see, eg, compounds 8-11).

[0166] Therefore, preferred embodiments relate to such "hydrophobic pocket" related compounds - i.e., where the integer "o" is ≠0, α is preferably the integer 3 and R 11 is (XII) or (XIII).

[0167] In order to also have a block -P (hydrophilic aa) as in, for example, St-PLys-g-PBG-PGlu (compound 9) or St-PLys-g-PHis-PSar (compound 10), the integer "o" is ≠0, α is preferably the integer 3, R 11 is (XII) or (XIII), the integer "q" is ≠ 0, and R 12 Is (XVI) or H.

[0168] Example 1 below describes an example of a graft copolymer designed to obtain a less dense structure, such as, for example, St-(PLys-g-PGlu)-stat-PSar (Compound 13).

[0169] Therefore, preferred embodiments relate to such "stat-PSar" related compounds - i.e., wherein the integer "o" is ≠0, α is preferably the integer 3, R 11 is (XVI), the integer "t" is ≠ 0 and R 15 It's H.

[0170] Compound 14 of Example 1 is an example of conjugation of a hydrophobic drug to vitamin E (which may be considered herein as, for example, a cell targeting agent).

[0171] Therefore, a preferred embodiment relates to such "vitamin E" related compounds - that is, wherein "o" is ≠0 and R 11 is vitamin E - preferably wherein α is preferably an integer of 3.

[0172] Compound 15 of Example 2 is an example of small molecule conjugation and Compound 18 of Example 3 is an example of therapeutic peptide conjugation.

[0173] Therefore, a preferred embodiment relates to such conjugated related compounds - that is, wherein the integer "o" is ≠ 0, α is preferably an integer of 3, R 11 and / or R 12 is (XIV), wherein Q of (XIV) is a cell targeting agent, such as, for example, a small molecule (eg, toll-like receptor agonist (TLR7 / 8) imiquimod) or a therapeutic peptide (eg, for example, MHC-I and / or MHC-II).

[0174] Preferably, the graft copolymer of the first aspect is a compound comprising compounds selected from the group consisting of:

[0175] St-PLys-g-PGlu

[0176] St-PLys-g-PHis

[0177] St-PLys-g-PHis-b-PGlu

[0178] St-PLys-g-PHis-b-PSar

[0179] St-PLys-g-PBG

[0180] St-PLys-g-PBG-b-PGlu

[0181] St-PLys-g-PBG-b-PSar

[0182] St-PLys-g-PSar

[0183] St-PLys-g-PSar-g-VitE

[0184] St-(PLys-g-PGlu)-stat-PSar; and

[0185] St-PLys-b-PSar-g-PGA, and

[0186] wherein the term "St" is a star structure of formula (Ia) of the first aspect and / or embodiments thereof; and

[0187] The term "PBG" herein refers to polybenzyl glutamate.

[0188] As will be appreciated by those skilled in the art - compounds comprising St-PLys-g-PHis will for example encompass compounds comprising St-PLys-g-PHis-b-PGlu, as the latter also comprises St-PLys-g-PHis.

[0189] Similarly - compounds comprising St-PLys-g-PHis would, for example, encompass compounds comprising St-PLys-g-PHis as well as the conjugates of interest, since the latter also comprise St-PLys-g-PHis.

[0190] As discussed in the working examples below -PBG is polybenzyl glutamate.

[0191] Graft copolymers of the first aspect - Glu / Asp related units with integers "r" and "v"

[0192] In formula (I) - the main repeating unit defined by square brackets with the values ​​"r" and "v" can be considered to be related to the use of, for example, glutamic acid / aspartic acid (Glu / Asp).

[0193] In an embodiment of the present invention: the integer "r" is ≠0.

[0194] Asp may be a suitable amino acid - ie it may be preferred that β is the integer 0.

[0195] Glu- is used in several of the structures exemplified herein, therefore, preferably β is an integer of 1.

[0196] If the integer "r" is ≠0 and "v" is zero, it is understood that all Glu / Asp related repeat units are bound to a branch having a repeat unit "s" amino acid structure.

[0197] The working examples herein describe structures in which relevant amounts of Glu / Asp related repeating units are attached to branches having amino acid structures.

[0198] Thus, it may be relevant that the value of the integer "r" is at least 2% of the value of the integer "v" - ie if the value of the integer "v" is 100, then the value of the integer "r" is at least 2.

[0199] Alternatively - it may be relevant that the value of the integer "r" is at least 10% of the value of the integer "v".

[0200] In several structures exemplified herein - the integer "r" is ≠0 and the integers "o" and "u" of the Lys / Orn related units are absent - ie the integers "o" and "u" are zero.

[0201] Therefore, in a preferred embodiment - the integer "r" is ≠0 and the integers "o" and "u" are 0.

[0202] Preferably, the integer "r" has a value from 3 to 1000 (more preferably from 5 to 750, even more preferably from 7 to 500, and most preferably from 8 to 200).

[0203] Preferably, the integer "s" has a value from 2 to 400 (more preferably from 3 to 350, even more preferably from 5 to 250, and most preferably from 10 to 200).

[0204] Preferably, R 13 is independently selected from H and free radicals of formula (XII), (XIII), (XXI) and (XXVIII).

[0205] It is preferred that each R 14 Independently selected from H, -OH.

[0206] As discussed above and in the working examples herein, the molecular weight (MW) of the novel graft copolymers discussed herein can be high enough to allow the preparation of nanometer-range (e.g., 4-300 nm in radius) structures useful for delivering, for example, active agents to target cells without the need for self-assembly processes.

[0207] High MW can be obtained by having relatively high values ​​of integer "r" and integer "s" - thus, in a preferred embodiment, the graft copolymer compound of the first aspect is a graft copolymer compound wherein

[0208] - the integer "r" has a value from 3 to 1000 (more preferably from 5 to 750) and the integer "s" has a value from 2 to 400 (more preferably from 3 to 350); and

[0209] - a molecular weight (MW) of 3 to 500,000 kDa - more preferably 10 to 100,000 kDa, even more preferably 10 to 20,000 kDa, and most preferably 20 to 10,000 kDa.

[0210] A preferred embodiment relates to the graft copolymer of the first aspect and related embodiments thereof herein, wherein the graft copolymer is a unimolecular structure with a radius of 4-300 nm.

[0211] Example 7 below describes an example of a graft copolymer having a St-PGlu-g-PSar structure (Compound 19) or a St-PGlu-g-PLysTG structure (Compound 20).

[0212] Therefore, preferred embodiments relate to compounds wherein the integer "r" is ≠0, β is preferably the integer 1, and R 13 It is H or (XXI).

[0213] Example 8 below describes an example of a graft copolymer having a structure related to St-PGA-g-PSar-g-PHis (alternatively referred to as St-PGlu-g-PSar-g-PHis) (Compound 21).

[0214] Therefore, preferred embodiments relate to compounds wherein the integer "r" is ≠0, β is preferably the integer 1, and R 13 is H and / or (XII).

[0215] Example 10 below describes an embodiment of conjugation of a hydrophobic drug to St-PGlu-g-Psar (Compound 22).

[0216] Therefore, preferred embodiments relate to compounds wherein the integer "r" is ≠0, "v" is ≠0, β is preferably the integer 1, R 13 Yes H, R 14 is a cell targeting agent (such as, for example, a hydrophobic drug, such as, for example, glycine-tocopherol).

[0217] Preferably, the graft copolymer of the first aspect is a compound comprising compounds selected from the group consisting of:

[0218] St-PGlu-g-PSar

[0219] St-PGlu-g-PHis

[0220] St-PGlu-g-PLys

[0221] St-PGlu-g-PLysTG; and

[0222] St-PGlu-g-PSar-g-PHis; and

[0223] wherein the term "St" is a star structure of formula (Ia) of the first aspect and / or embodiments thereof; and

[0224] PLysTG is poly-lysine-thioglycerated.

[0225] As will be appreciated by one skilled in the art - a compound comprising St-PGlu-g-PSar will for example encompass a compound comprising St-PGlu-g-PSar-g-PHis, as the latter also includes St-PGlu-g-PSar.

[0226] Similarly - compounds comprising St-PGlu-g-PSar would encompass, for example, compounds comprising St-PGlu-g-PSar as well as conjugates of interest since the latter also comprise St-PGlu-g-PSar.

[0227] As discussed in the working examples below -PLysTG is poly-lysine-thioglycerate.

[0228] Graft copolymers of the first aspect - Ala / Sar related units with integer "t"

[0229] In formula (I) - the main repeating unit defined by the square brackets with the value "t" can be considered to be associated with the use of, for example, Ala / Sar / Gly.

[0230] Several of the working examples herein describe structures where the integer "t" is zero - therefore, it may be preferred that the integer "t" is zero.

[0231] It may be preferred that the integer "t" has a value of 1 to 5000 (more preferably 3 to 4000).

[0232] Graft copolymer of the first aspect - other matters

[0233] It is preferred that:

[0234] Each R 3 are independently selected from -H;

[0235] Each R 4 are independently selected from -H;

[0236] Each R 5 are independently selected from -H;

[0237] Each R 6 Independently selected from -CH 3 ;

[0238] Each R 7 are independently selected from -H;

[0239] Each R 8 Independently selected from -CH 3 ;

[0240] Each R 9 are independently selected from -H;

[0241] Each R 10 Independently selected from CH 3 ;

[0242] Each R 15 are independently selected from -H;

[0243] It is preferred that each X 1 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 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 ;

[0244] It is preferred that each X 2 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 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 .

[0245] It is preferred that each X 3 independently selected from H, -C(O)H, -S(O)H, -NHCH(CH 3 ) 2 、-(C 1 -C 4 ) alkyl NH 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 .

[0246] The polymer composite of the second aspect

[0247] As discussed above, the second aspect of the present invention relates to a polymer composite comprising the graft copolymer of the first aspect and / or embodiments thereof, and at least one pharmaceutical, veterinary or cosmetic active agent.

[0248] Preferably, the 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.

[0249] The nucleic acid can, for example, be selected from plasmid DNA, clDNA, siRNA, mRNA, microRNA, donor DNA, sgDNA, crDNA, shRNA, antisense nucleic acid, decoy nucleic acid, aptamer and ribozyme.

[0250] As is known in the art, the active agent of interest can be covalently linked to the polypeptide backbone through amino acid side residues, via amide, ester, anhydride conjugation or through a linker comprising one or more functional groups, including but not limited to alkynes, azides, reactive disulfides, maleimides, hydrazides, hydrazones, Schiff bases, acetals, aldehydes, carbamates and reactive esters. In alternative embodiments, the covalent linkage is a bioreactive covalent linkage.

[0251] As known in the art, the active agent of interest can be linked to the polypeptide backbone by electrostatic interactions. Thus, when mixed in an aqueous medium, anionic compounds having more negative charges than positive charges can form polymer complexes with the graft copolymer of the first aspect by electrostatic interactions. Examples of anionic compounds include proteins, polysaccharides, lipids, and nucleic acids.

[0252] As is known in the art - the active agent of interest can be linked to the polypeptide backbone via, for example, hydrophobic and / or hydrophilic related interactions.

[0253] For example, Example 1 below describes examples of graft copolymers designed to achieve unimolecular aggregates with a hydrophobic pocket that has the ability to encapsulate a hydrophobic payload (eg, an active agent).

[0254] Therefore, based on common common knowledge and the technical disclosure herein, it is a routine task for those skilled in the art to prepare the polymer composite of the second aspect of the present invention and / or its embodiments.

[0255] Preferably, the polymer composite of the second aspect is a polymer composite wherein:

[0256] - the active agent is covalently linked to the polypeptide backbone of the polymer complex;

[0257] - the active agent is linked to the polypeptide backbone of the polymer complex via electrostatic interactions; or

[0258] - The active agent is linked to the polypeptide backbone of the polymer complex via hydrophobic and / or hydrophilic related interactions.

[0259] The pharmaceutical, veterinary or cosmetic composition of the third aspect

[0260] 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 embodiments thereof, and one or more suitable excipients or carriers.

[0261] Based on common common knowledge and the technical disclosure herein, it is a routine task for those skilled in the art to prepare the pharmaceutical, veterinary or cosmetic composition of the third aspect of the present invention and / or its embodiments.

[0262] As known in the art - the pharmaceutical, veterinary or cosmetic composition further comprises an acceptable excipient - ie a pharmaceutically, veterinary or cosmetically acceptable excipient.

[0263] Medical Use - Fourth and Fifth Aspects

[0264] As discussed above, the fourth aspect of the present invention relates to the polymer complex of the second aspect and / or embodiments thereof, or the pharmaceutical composition of the third aspect and / or embodiments thereof, for use as a medicament.

[0265] As discussed above, the fifth aspect of the 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, which is (i) used as a transfection reagent for transfecting at least one active agent into a cell; (ii) used for in vivo or ex vivo production of biological preparations encoding recombinant proteins, peptides or antibodies, or for producing recombinant viruses; (iii) used as a therapeutic or prophylactic vaccine against viral infection or as a therapeutic vaccine against cancer; or (v) used for genome engineering, for cell reprogramming, for differentiating cells or for gene editing.

[0266] Based on common general knowledge and the technical disclosure herein, it is a routine task for a skilled person to determine suitable medical uses of the polymer complexes and / or pharmaceutical compositions described herein.

[0267] For example, many of the medical uses described in the art for related polymer compounds (see, for example, EP 3331937 B1 discussed above) may also be relevant to the graft copolymer compounds of the present invention.

[0268] Delivery of pharmaceutically active agents into target cells - Sixth Aspect

[0269] 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 a composition (e.g., a solution) comprising the polymer complex of the second aspect and / or embodiments thereof to an animal, including a human, such that the complex is in physical contact with the target cell, thereby delivering the pharmaceutically active agent into the cell.

[0270] Based on common general knowledge and the technical disclosure herein - it is a routine task for those skilled in the art to determine a suitable method to deliver an active agent of interest to a target cell of interest.

[0271] As will be understood by the skilled person - the term "the complex is brought into physical contact with the target cell" may for example relate to the complex being introduced into the target cell.

[0272] Alternatively, the complex may, for example, be bound to the surface of a target cell and the active agent delivered into the cell as a result of it, for example, crossing a surface cell membrane, thereby entering the cell.

[0273] The working examples herein describe that the graft copolymer compounds of the present invention can be brought into physical contact with a target cell of interest, thereby delivering an active agent of interest to the cell.

[0274] Furthermore, related polymer compounds (see, for example, EP 3331937 B1 discussed above) have also been described for use in delivering active agents into target cells of interest.

[0275] Use of the graft copolymer of the present invention for preparing different compounds - Seventh Aspect

[0276] As discussed above, the seventh aspect of the present invention relates to a method for preparing a compound that is structurally different from the graft copolymer starting compound of the first aspect and / or embodiments thereof, comprising the steps of:

[0277] (i): using the compound of the first aspect and / or its embodiments as a starting compound;

[0278] (ii) making structural changes to the compound of step (i) to obtain a compound that is structurally different from the starting compound of step (i).

[0279] Preferably, the structurally different compound obtained in step (ii) is a compound having similar functionality / capabilities as the starting compound of step (i) - i.e., preferably, the structurally different compound obtained in step (ii) is a compound capable of forming a polymer complex comprising the structurally different compound obtained in step (ii) and at least one pharmaceutical, veterinary or cosmetic active agent.

[0280] Preferably, the structurally different compound obtained in step (ii) is a compound capable of forming the following polymer complex: comprising the structurally different compound obtained in step (ii) and at least one pharmaceutical, veterinary or cosmetic active agent, and wherein the active agent is an active agent with which the starting compound of step (i) is also capable of forming a polymer complex.

[0281] The structurally different compound obtained in step (ii) may be, for example, a compound outside the protection scope of the graft copolymer compound of the first aspect and / or embodiments thereof.

[0282] Optionally, the structurally different compound obtained in step (ii) is a compound within the protection scope of the graft copolymer compound of the first aspect and / or embodiments thereof.

[0283] Based on common general knowledge and the technical disclosure herein, it is a routine task for a skilled person to prepare structurally different compounds using the graft copolymer compounds described herein as starting compounds.

[0284] As will be appreciated by those skilled in the art - the process of the seventh aspect is based on the technical disclosure herein - in that it uses the graft copolymer compound of the first aspect (ie the present invention) to prepare a structurally different compound.

[0285] Method for synthesizing the compound of the first aspect - separate aspect

[0286] A separate aspect of the present invention relates to a method for synthesizing the graft copolymer compounds of the first aspect and / or embodiments thereof, which method generally comprises polymerizing the N-carboxy anhydride (NCA) of protected or unprotected amino acids known per se with a 3-arm star initiator (St); followed by a deprotection reaction and then a second and optionally additional polymerization reaction.

[0287] According to this aspect, the 3-arm star initiator (St), namely N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1) (ie, St-initiator (1)), can be generally obtained by: reacting 1,3,5-benzenetricarbonyl chloride (1,3,5-benzenetricarbonyl trichloride) is reacted with N,N'-diisopropylethylenediamine (DIEA) to obtain the intermediate 1,3,5-((benzenetricarbonyltris-(azanediyl))tris(ethane-2,1-diyl))tricarbamate (2); the intermediate (2) is then reacted with TFA to obtain 1,3,5-(benzenetricarbonyltris(azanediyl))-triethanamonium TFA salt (3) (i.e., the star polymer TFA-protected intermediate (3)); and the TFA group is then removed by an alkaline deprotection cleavage reaction (e.g., by stirring in an appropriate ion exchange resin) to obtain the St-initiator (1).

[0288]

[0289] According to this aspect, a method for synthesizing a compound of formula (I) according to the first aspect and / or its embodiments, the method generally comprises:

[0290] i) polymerizing N-carboxylic anhydrides (NCA) of amino acids known per se, protected (e.g. in the form of tetrafluoroborate or ammonium trifluoroacetate salts) or unprotected, with a 3-armed star initiator, i.e. St-initiator (1) or St-protected initiator intermediate (3) in a sequential or statistical manner to obtain block copolymers or, alternatively, random copolymers;

[0291] ii) optionally, reacting the amine group at the N-terminal position with an amine-reactive group to introduce a capping group;

[0292] iii) optionally, orthogonally removing amino acid side chain protecting groups;

[0293] iv) optionally, reacting the amine group at the terminal position of the side chain with an amine-reactive group to introduce structural extension, conjugation, labeling or shielding;

[0294] v) optionally purifying the product obtained in step i), ii), iii) or iv) by fractionation, precipitation, ultrafiltration, dialysis, size exclusion chromatography, affinity chromatography or tangential flow filtration.

[0295] The above step i) may comprise: a) ring-opening polymerization of unprotected amino acid N-carboxy anhydride (NCA) monomers by reacting the tetrafluoroborate or ammonium trifluoroacetate form of the St-protected initiator intermediate (3) with the selected unprotected NCA-amino acid monomer; or alternatively ring-opening polymerization of protected amino acid N-carboxy anhydride (NCA) monomers (e.g. tetrafluoroborate or ammonium trifluoroacetate form) by reacting the St-initiator (1) with the selected protected NCA-amino acid monomer; wherein the monomer / initiator ratio allows control of the degree of polymerization (DP). The polymerization with different amino acid NCA monomers may be carried out in a sequential manner, i.e. wherein the block copolypeptide is prepared in a sequential manner after polymerization reaction a) by consuming the first NCA monomer and the resulting product may or may not be purified before adding the next NCA monomer to build the subsequent polypeptide block; or alternatively in a statistical manner, wherein the random copolypeptide is prepared in a statistical manner after polymerization reaction a) by mixing all NCA monomers before starting polymerization.

[0296] The above step ii) corresponds to capping, wherein the amine group at the N-terminal position is reacted with an amine-reactive group to introduce a capping functional group.

[0297] The above step iii) corresponds to a deprotection, wherein the amino acid side chains are removed orthogonally according to the protecting groups.

[0298] Step iv) corresponds to conjugation, wherein the amine group at the terminal position of the side chain of the shielding polymer, active small molecule, targeting agent or imaging agent is reacted with an amine-reactive group.

[0299] Example

[0300] Analytical methods

[0301] NMR spectroscopy: In the 300Ultrashield from Bruker (Billerica MA, USA) TM NMR spectra were recorded at 27°C (300 K). Data were processed using the software Topspin (Bruker GmbH, Karlsruhe, Germany). Samples were prepared at a concentration of approximately 20-10 mg / mL in the desired solvent.

[0302] 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 flow rate of 0.7 mL / min at 60°C using a TSKgel Alpha-4000 column from Tosoh Bioscience. A Viscotek TDA-302 was used as an integrated detection system. The system was calibrated with polymethyl methacrylate (PMMA) from PSS (Mw = 65 kDa; PDI = 1.05). For this Mw determination, an integrated triple detection system [refractive index, light scattering (two angles: 7° and 90°) and UV-visible detector] was used.

[0303] For SEC measurements in aqueous solutions containing 0.1M sodium nitrate and 0.005% sodium azide as additives, a GPC max (Malvern Instruments) pump and autosampler were used, with a flow rate of 0.7 mL / min, 25°C, using a TSKGel PWXL G5000 column from Tosoh Bioscience. Viscotek TDA-305 was used as an integrated detection system. The system was calibrated with 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 UV-visible detector] was used.

[0304] Size distribution.Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer NanoZS instrument equipped with a 532 nm laser and a fixed scattering angle of 173°. Polymer solutions were prepared under different conditions (MilliQ or PBS at different concentrations and temperatures), sonicated for 10 min and aged for the required time, filtered through a 1.20 μm cellulose membrane filter and measured. The size distribution (diameter, nm) of each polymer was measured in triplicate with n>3 measurements, automatically optimizing beam focus and attenuation for each sample.

[0305] Example 1. Preparation of compounds of formula (I) - Lys combined with branches having different amino acid structures

[0306]

[0307] Formula (Ia) - Formula (Ib1) above regarding the first aspect herein is an example where the integer "o" is ≠0 and α is the integer 3 - i.e., involves the use of Lys bound to a branch having a different amino acid structure.

[0308] In general, to synthesize the compound of formula (Ib1) according to the present disclosure, first, a 3-arm star initiator is obtained in 2-3 steps. Such an initiator is used to polymerize N-trifluoroacetyl-L-lysine N-carboxylic anhydride or lysine (TFA) NCA to obtain a TFA-protected star polymer (St-PLys (TFA)). The TFA group is removed by alkaline deprotection cleavage reaction to obtain the corresponding free NH 2 Star-PLys at the active site to initiate the next polymerization step.

[0309] Scheme 1 shows a specific example of the polymerization, deprotection steps and grafting process from the poly-L-lysine backbone.

[0310] Solution 1

[0311]

[0312] Example 1A: Synthesis of 3-arm star-shaped initiator

[0313] Scheme 2 discloses a method leading to a 3-arm star initiator, namely N 1 ,N 3 ,N 5 -Synthetic route of tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1).

[0314] Solution 2

[0315]

[0316] Step (a): Synthesis of tri-tert-butyl 1,3,5-((benzenetricarbonyltri(azanediyl))tri(ethane-2,1-diyl))tricarbamate (2):

[0317]

[0318] Equipped with a stirring rod and N 2 In a double-necked round-bottom flask with an inlet and an outlet, 500 mg of 1,3,5-benzenetricarboxylic acid chloride (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 eq.) was added to the reaction mixture, followed by dropwise addition of N-Boc-ethylenediamine (1.34 g, 6.22 mmol, 3.3 eq.) over a 10 min period. The reaction was then allowed to proceed for 2 hours. Afterwards, the solvent was completely removed under vacuum. The product was redissolved in chloroform and washed with deionized water (ddH 2 O) and washed 3 times with acidic water (pH ~ 3). Finally, the organic phase was separated in vacuo and the product was recrystallized 3 times in THF / methanol / hexane to give a white crystalline solid. The product was then dried under high vacuum and stored at -20 ° C.

[0319] Yield: 82%. 1 H NMR (300MHz, DMSO) δ8.68-8.65(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 C NMR (75MHz, CDCl 3 )δ166.80(C=O),156.84(C=O),134.58(CAr quaternary),128.47(CH Ar ),79.57 (C quartet),40.93 (CH 2 ),40.43(CH 2 ),28.45(CH 3 ).

[0320] Step (b): Synthesis of 1,3,5-(benzenetricarbonyltri(azanediyl))triethylammonium TFA salt (3):

[0321]

[0322] In a round bottom flask equipped with a stirring bar and a stopper, 200 mg of tri-tert-butyl 1,3,5-((benzenetricarbonyltri(azanediyl))tri(ethane-2,1-diyl))tricarbamate (2) (0.33 mmol, 1 eq.) was dissolved in 5 mL of anhydrous dichlorometane and 2.5 mL of TFA was added. The reaction was stirred for 2 hours under a nitrogen atmosphere and the completion of the reaction was monitored by TLC. The solvent was evaporated in vacuo. The TFA salt of the initiator (220 mg) was obtained in quantitative yield and dried in vacuo.

[0323] Yield: 98%. 1 H NMR (300 MHz, D 2 O)δ8.36(s,3H),3.75(t,J=5,9Hz,6H)3.29(t,J=6,0Hz,6H). 19 F NMR (300MHz, D 2 O)δ-75.84.

[0324] Step (c): N 1 ,N 3 ,N 5 -Synthesis of tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (1):

[0325]

[0326] 220 mg of 1,3,5-(benzenetricarbonyltri(azanediyl))triethylammonium TFA salt (3) was dissolved in 22 mL of mixture H 2 O: MeOH (7: 3) and stirred with excess weakly basic Amberlyst ion exchange resin (1000 mol%) for 24 hours. Then, the mixture was filtered and the filtrate was concentrated by removing methanol. The aqueous solution was lyophilized to obtain high purity free base amine.

[0327] Yield: 98%. 1 H NMR (300 MHz, D 2 O) δ8.13(s,3H),3.45(t,J=6,3Hz,6H)2.86(t,J=6,3Hz,6H).

[0328] Example 1B: Polymerization of the backbone.

[0329] The synthesis of compound (5) was carried out according to the synthetic route of Scheme 3.

[0330] Solution 3

[0331]

[0332] Briefly, L-lysine (TFA)-NCA (2.0 g, 7.46 mmol) was added to a flask equipped with a stir bar, a stopper and 3 cycles of vacuum / N 2 In the Schlenk tube of circulation purge, and dissolved in anhydrous DMF (4mL). Then, the star initiator (St) (16.72mg, 0.05mmol) is dissolved in DMF (1mL) and added to the reaction mixture. The mixture is stirred at 10°C for 48 hours. After completion, the complete conversion of the monomer can be detected by IR. The reaction mixture is poured into diethyl ether (ratio of 1:10) to precipitate the product. The precipitate is separated by centrifugation (3750rpm, 4min) and dried under vacuum. White solid star-poly (L-Lys (TFA)) (St-PLys (TFA)) (4) is isolated. In the second step, the TFA protecting group from St-PLys (TFA) (4) is deprotected under alkaline conditions. In brief, polymer (4) (0.45g, 2.00mmol) is dissolved in MeOH (1.5mL) and NaOH 4.5M (aq) (0.67mL) is added to the reaction mixture. The mixture was stirred at 4 °C for 16 hours. Afterwards, TFA5M (aq) (0.55 mL) solution was poured into acetonitrile (ratio 1:10) to precipitate the product. The precipitate was separated by centrifugation (3750 rpm, 4 min) and dried under vacuum. White solid star-poly (L-Lys) (St-PLys) (5) was isolated.

[0333] Yield: 70-90% 1 H NMR (D 2 O):δ=1.46(m,2H,CH 2 ),1.64-1.90(width m,4H,CH 2, CH 2 ),3.03(t,2H,CH 2 -NH 2 ),3.34-3.66(width m,12H,CH 2 -CH 2 ),4.33(t,1H,quiral CH),8.31(s,3H,aryl CH).

[0334] Table 1 shows the different DP (degree of polymerization) obtained for St-PLy of different formulas (5), demonstrating the versatility and accuracy of the experimental procedure.

[0335] Table 1.

[0336]

[0337] a Confirmed by NMR. bDetermined by SEC. Mn and DP refer to the number average molar mass and degree of polymerization, respectively. represents the polydispersity determined by SEC-MALS software analysis.

[0338] Example 1C: Grafted from:

[0339] Synthesis of star-poly(L-Lys)-graft-poly(L-PGlu)(St-PLys-g-PGlu)(7). Scheme 4 shows the synthetic route to obtain compound 7.

[0340] Solution 4

[0341]

[0342] L-glutamic acid γ-benzyl ester N'carboxylic anhydride (99.1g, 0.38mol) was added to a Schlenk tube equipped with a stirring rod, a stopper and purged with 3 vacuum / Ar cycles, and dissolved in 490mL of anhydrous DMF. The required equivalent of the macroinitiator compound (5) (837mg, 3.45mmol) for grafting was dissolved in DMF (10mL), added, and the mixture was placed at rt and stirred for 2 days under an inert atmosphere. After completion, the complete conversion of the monomer can be detected by IR. Finally, the reaction mixture was poured into a large excess of cold diethyl ether (ratio of 1:10), and a white polymer star-poly (L-Lys)-grafted-poly (L-PBG), i.e. (St-PLys-g-PBG) (6) was obtained after separation by filtration or centrifugation (3750rpm, 4min), and dried under vacuum. According to this strategy, the degree of polymerization was controlled by the feed ratio [monomer] / [macroinitiator] to obtain a graft copolymer. In the second step, the benzyl protecting group of St-PLys-g-PBG (6) was deprotected under alkaline conditions. Briefly, polymer (6) (69.1 g, 0.32 mol) was dissolved in THF (2.8 L) and NaOH 0.7 M (aq) (0.7 L) was added to the reaction mixture. The mixture was stirred at 0° C. for 16 hours. Thereafter, the solution was poured into a mixture of diethyl ether / acetonitrile (3 / 1) to precipitate the product. The precipitate was separated by centrifugation (3750 rpm, 4 min) and dried under vacuum. White solid star-shaped-poly (L-Lys)-grafted-poly (L-PGlu), i.e. (St-PLys-g-PGlu) (7) was isolated.

[0343] Yield: 70-90% 1 H NMR (TFA): δ = 2.92 (m, 2H, CH 2 ),4.85(s,1H,CH),5.05(m,2H,benzylCH 2), 7.13 (s, 5H, aryl CH), 8.38 (s, aryl CH).

[0344] Table 2 shows the different DP (degree of polymerization) obtained for St-PLys-g-PGlu of different formula (7), demonstrating the versatility and accuracy of the experimental procedure.

[0345] Table 2

[0346]

[0347] a Determined by SEC. Mn and DP refer to the number average molar mass and degree of polymerization, respectively. represents the polydispersity determined by SEC-MALS software analysis. *GE: grafting efficiency

[0348] Example 1D: Star-poly(L-Lys)-graft-poly(L-hydrophobic aa)-block-P(hydrophilic aa), ie compounds 8-11.

[0349] This graft copolymer is designed to obtain unimolecular aggregates with hydrophobic pockets that can encapsulate hydrophobic payloads. Scheme 5 shows the general synthetic scheme to obtain compounds 8-11.

[0350] Solution 5

[0351]

[0352] Similar to the synthesis of compound 6, the first amino acid NCA is polymerized using a macroinitiator St-PLys (5), and then the second amino acid NCA is polymerized. In the specific case of the present embodiment, the amino acid NCA-1 is L-glutamic acid y-benzyl ester NCA or N (Im) -(2,4-dinitrophenyl) -L-histidine NCA (His (DNP) NCA). In short, the specific NCA-1 is added to a Schlenk tube equipped with a stirring rod, a stopper and purged with 3 vacuum / Ar cycles, and dissolved in anhydrous DMF. The required equivalent of the macroinitiator compound (5) for grafting is added and the mixture is stirred at 10 ° C for 3-4 hours under an inert atmosphere. After completion, the complete conversion of the monomer can be detected by IR. Next, the second monomer is added to polymerize the second block in a one-pot manner. In the specific case of this example, the amino acid NCA-2 is sarcosine NCA added to anhydrous DMF or glutamic acid 5-tert-butyl ester NCA (Glu(OtBu)NCA) added to anhydrous THF. After completion, the complete conversion of the second monomer can be detected by IR. Finally, the reaction mixture is poured into a large excess of cold diethyl ether or H 2O (depending on NCA-2), a white polymer was obtained after separation by filtration or centrifugation (3750 rpm, 4 min) and dried under vacuum.

[0353] For compound 8, St-PLys-g-PBG-PSar, this was the last step of the synthetic procedure.

[0354] Yield: 70-90% 1 H NMR (TFA): δ = 2.20 (broad m, 1H, CH 2 ),2.40(width m,1H,CH 2 ),2.71(width m,2H,CH 2 ),3.20-3.59(width s,30H,N-CH 3 ),4.49-5.01(broad m,1H,quiralCH(PBG),20H,CH 2 (PSar)), 5.32 (broad s, 2H, benzyl CH 2 ),7.47(s,5H,arylCH)

[0355] For compound 9, St-PLys-g-PBG-PGlu, it is necessary to carry out another deprotection step to the OtBu group. In brief, the polymer precursor of 9 (St-PLys-g-PBG-P (Otbu) Glu (38.3g, 0.207mol)) is dissolved in pure TFA (380mL). The mixture is stirred for 3.5 hours at rt. Afterwards, the solution is poured into diethyl ether to precipitate the product. Separate the precipitation by filtering or centrifuging (3750rpm, 4min) and dry it under vacuum to obtain an off-white solid.

[0356] 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, quiralCH), 6.97 (broad s, 5H, arylCH).

[0357] For compound 10, St-PLys-g-PHis-PSar, it is necessary to use mercaptoethanol to carry out additional steps to the DNP protecting group of histidine under reducing conditions. In brief, the polymer precursor of 10 is dissolved in DMF (100mg / mL) and excessive mercaptoethanol (10eq) is added to the reaction mixture. The mixture is stirred at rt for 16 hours. Afterwards, the solution is poured into diethyl ether to precipitate the product. Separate the precipitate by centrifugation (3750rpm, 4min) and dry it under vacuum. Isolate the yellow solid St-PLys-g-PHis-PSar (10).

[0358] 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).

[0359] For compound 11, St-PLys-g-PHis-PGlu, the deprotection of DNP with histidine and the deprotection of OtBu with glutamic acid must be carried out in two steps. First, the polymer precursor of 10 is dissolved in a mixture of DMF / THF 3 / 1 and an excess of thioethanol (10eq) is added to the reaction mixture. The mixture is stirred at rt for 16 hours. Afterwards, the solution is poured into diethyl ether to precipitate the product. The precipitate is separated by centrifugation (3750rpm, 4min) and dried under vacuum. A yellow solid St-Plys-g-PHis-PGlu (OtBu) is isolated. Furthermore, the polymer is dissolved in a pure TFA solution and the mixture is stirred at rt for 3.5 hours. Afterwards, the solution is poured into diethyl ether to precipitate the product. The precipitate is separated by filtration or centrifugation (3750rpm, 4min) and dried under vacuum to obtain a yellow solid.

[0360] Yield: 70-90% 1 H NMR (D2O-HFIP)

[0361] Similarly, and for comparison purposes, the linear analog of compound 10 (compound 12) was synthesized using the same procedure but using n-butylamine as initiator instead of the star-initiator compound 1.

[0362] Table 3 shows the different DPs obtained for different star-poly(L-Lys)-graft-poly(L-hydrophobic aa)-block-P(hydrophilic aa) (ie compounds 8-11). Various graft copolymers with hydrophobic pockets for encapsulation of hydrophobic molecules are shown.

[0363] Table 3

[0364]

[0365]

[0366] a Confirmed by NMR. b Determined by SEC. Mn and DP refer to the number average molar mass and degree of polymerization, respectively. represents the polydispersity determined by SEC-MALS software analysis. Nd: not determined and in the case of compounds 9C-9D due to solubility issues. *Note: Grafted DP is related to the degree of polymerization of each branch.

[0367] Example 1E: Star-shaped poly((L-Lys)-graft-(poly(L-Glu))-stat-poly(Sar), i.e. St-(PLys-g-PGlu)-stat-PSar (13).

[0368] The synthesis of compound 13 aims to obtain a graft copolymer with the same polymer backbone but a less dense structure. This is achieved by introducing non-reactive sites in the polymer backbone by statistical copolymerization of amino acids without introducing functional reactive sites that can initiate polymerization in a further step. In this specific embodiment, as depicted in Scheme 6, the amino acid NCA selected for copolymerization with Lys(TFA)NCA is sarcosine NCA.

[0369] Solution 6

[0370]

[0371] In brief, the synthesis of polymer backbone St-PLys-stat-PSar is similar to St-PLys, but monomer sarcosine NCA is introduced simultaneously with Lys (TFA) NCA. Afterwards, as described for the synthesis of compound 5 St-PLys, the TFA group is deprotected. Next, statistical copolymer St-PLys-stat-PSar is used as a macroinitiator for polymerizing monomer Glu (OBzl) NCA. The benzyl protecting group of the obtained polymer is removed according to the same procedure described for the synthesis of compound 7.

[0372] Table 4 shows the different DP (degree of polymerization) obtained for St-St-(PLys-g-PGlu)-stat-PSar of formula (13).

[0373] Table 4

[0374]

[0375] a Determined by the SEC.

[0376] Example 1F: Star-poly(L-Lys)-graft-poly(Sar)-poly(Lys-vitE), i.e. (14).

[0377] Solution 7

[0378]

[0379] As shown in Scheme 7, the conjugation of hydrophobic drug vitamin E was performed before the synthesis of graft copolymers. In brief, St-PLys (Compound 5B) (30.0 mg, 0.12 mmol) was dissolved in 0.5 mL of anhydrous DMF. Next, DMTMMBF4 (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 min. Afterwards, tocopherol succinate (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 help of adding 50 μL of triethylamine (TEA). The reaction was allowed to proceed for 24 h at room temperature and under a nitrogen atmosphere. Afterwards, 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. The reaction was allowed to proceed for 16 hours. After completion, the complete conversion of the monomers can be detected by IR. Finally, the mixture was poured into a large excess of diethyl ether and the white solid product was isolated by filtration / centrifugation. 1 The grafting % of tocopherol succinate was calculated by H-NMR.

[0380] Yield: 70-90% 1 H NMR(MeOD): δ=0.87(m,12H,CH 3 ),0.94(t,3H,CH 3 -),1.02-1.60(width m,32H,CH 2 &CH) , 1.98-2.09(s,12H,CH 3 -), 2.59-3.23 (width m, 390H, terminal N-CH 3 ),3.79-4.66(width m,260H,-CH 2-&-CH 2 -).

[0381] Example 1G: The compound of Example 1 formed unimolecular aggregates.

[0382] One of the main features of our graft copolymers is their high molecular weight and the possibility to form unimolecular aggregates of a size suitable for their applications, not limited to drug delivery. To study this behavior, we evaluated their hydrodynamic size in aqueous solution by using light scattering techniques. The results obtained for the tested graft copolymers are summarized in Table 5.

[0383] Table 5 shows the hydrodynamic sizes of the graft copolymer monomolecular aggregates formed in solution.

[0384] Table 5

[0385]

[0386] Example 2. Conjugation of small molecules with compounds of formula Ib1

[0387] Proof of concept for small molecule conjugation has been achieved. As a model molecule, a derivative of the toll-like receptor agonist (TLR7 / 8) imiquimod was conjugated with compound 7B as shown in Scheme 8 to give compound 15.

[0388] Solution 8

[0389]

[0390] Briefly, a stirring rod, a stopper and a 2 Compound 7B was dissolved in anhydrous DMSO in the acid form at a concentration of 12-3 mg / mL in a purged round-bottom flask. Next, DMTMMBF was added to the anhydrous DMSO. 4 (1.5 equivalents of desired modification %) 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 desired modification %) and anhydrous DMSO were added, and the pH was adjusted to 8 by adding DIPEA base. The final polymer concentration was 10 mg / mL. The reaction was stirred at room temperature under nitrogen atmosphere for 20 hours. For purification, the reaction was precipitated in THF and washed with THF:Et 2 The solid was washed three times with 0.5 M NaHCO 3Then it was dissolved. Next, the compound was desalted by Sephadex G25 purification and / or dialysis using Vivaspin MWCO: 30 kDa. A white solid was obtained after water resuspension and lyophilization. The % of conjugated TLR7 / 8 agonist was 3.8 mol % (yield 87.4%) as determined by NMR.

[0391] Example 3. Conjugation of therapeutic peptides to the compound of formula 1b1.

[0392] Proof of concept for therapeutic peptide conjugation has been achieved.

[0393] Specifically, two peptide antigens were conjugated to 7B after pre-derivatization with pyridyl dithiol ethylamine (PD). Next, two different peptide antigens of major histocompatibility complex class I and class II (MHC-I and MHC-II), i.e., gp100 MHC class I and MHC class II peptides, were conjugated via reversible disulfide bonds (see Scheme 9).

[0394] Solution 9

[0395]

[0396] A) Derivatization with pyridyldithiolethylamine (PD): Briefly, 900 mg of 7B (6.98 mmol, expressed as glutamic acid units (GAU)) was added to a 250 mL round-bottom flask and derivatized with N 2 Purge. Next, add 86mL of anhydrous DMSO and stir at RT for 40min to dissolve the solid. Then, add DMTMM.BF4 (148mg, 0.523mmol, 5% GAU derivatization) to another 2mL of anhydrous DMSO. After 30min of reaction, add pyridyl dithiol ethylamine (PD) (117mg, 0.523mmol, 5% GAU derivatization) dissolved in 2mL of anhydrous DMSO. Add DIEA to adjust the pH to 8 and place the reaction under stirring at RT for 48h under anhydrous atmosphere. Afterwards, precipitate the reaction in 1L of fresh THF under stirring and cool to 4°C for 16 hours to facilitate precipitation. Then, collect the solid by centrifugation and use THF and THF:Et 2 O (5:95) and lyophilized to obtain white solid compound 16. 2 O 1 H-NMR confirmed the identity, purity and conjugation efficiency. Yield: 90% (1050 mg), conjugation efficiency: 70% (3.5% mol GAU derivatized with PD).

[0397] B) Peptide conjugation with PD-modified graft copolymer (compounds 17 and 18): Briefly, for MHC-I peptide conjugation, PD-modified graft copolymer (350 mg, 2.57 mmol GAU) was dissolved in 35 mL anhydrous DMSO. Next, 59.72 mg MHC-I peptide (0.0495 mmol, 0.019 eq, 1.9% modification achieved, 1207 g / mol) was dissolved in 5 mL anhydrous DMSO, followed by the addition of 0.15 eq tris(2-carboxyethyl)phosphine hydrochloride, TCEP (0.722, 0.0029 mmol). The reaction was allowed to react at RT, stirring and N 2 atmosphere for 48 hours. Afterwards, the compound was precipitated in cold THF at a ratio of 1:20. The precipitate was separated by centrifugation and washed with THF. A white solid was finally obtained by freeze drying. By adding NaHCO 3 The solid was converted to the product in sodium salt form and subsequently dialyzed with a 30 kDa Vivaspin for desalting. The peptide content was determined to be 6.07% wt by amino acid analysis. Yield: 40%, conjugation efficiency: 49% (0.86% mol MHC-1 conjugated).

[0398] In another embodiment, gp100 MHC-I and MHC-II peptides were conjugated in series in the same polymer backbone (Compound 18). Briefly, after reacting with MHC-I peptide for 48 hours as described above, the gp100 MCH-I conjugate was not precipitated, but the reaction was allowed to proceed for another 48 hours after the addition of gp100 MHC-II peptide (0.019 eq, achieving 1.9% modification, 2208 g / mol) and 0.15 eq TCEP (0.722, 0.0029 mmol) dissolved in 5 mL of anhydrous DMSO. Afterwards, the compound was precipitated in cold THF at a ratio of 1:20. The precipitate was isolated by centrifugation and washed with THF. A white solid was finally obtained by lyophilization. The reaction was carried out by adding NaHCO 3 The solid was converted to the product in the form of a sodium salt and subsequently dialyzed with a 30 kDa Vivaspin for desalting. Peptide content determined by amino acid analysis: 4.11% wt for MHC-I peptide and 4.99% wt for MHC-II peptide. Yield: 60%, conjugation efficiency: 36%.

[0399] Example 4. Encapsulation of Drugs in Compounds of Formula Ib1

[0400] Compounds 8-12 described in Examples ID and IE were designed to hold a hydrophobic pocket for drug encapsulation due to their amphiphilicity and the presence of highly hydrophobic blocks of polyamino acids (ie, PBLG or PHis).

[0401] In a first attempt and to characterize the system, Nile Red with logP: 2,98 was chosen as a model hydrophobic drug.

[0402] In the specific case of compound 9 (i.e. St-PLys-g-PBG-PGlu), the ability to incorporate Nile Red hydrophobic molecules into the structure indicates the presence of a hydrophobic pocket. Nile Red is commonly used as a lipophilic stain. In a hydrophilic environment, Nile Red hardly fluoresces, but when encapsulated / constrained in a hydrophobic environment, it emits strong fluorescence, and the color changes from deep red (for polar membrane-like environments) to strong golden emission (for neutral environments). The presence of a hydrophobic pocket and the response of the hydrophobic / hydrophilic balance of the polymer to pH changes are determined by fluorescent Nile Red assay. This test is based on the significant solvatochromic effect and increased fluorescence emitted by this molecule in the presence of the mentioned hydrophobic microenvironment. For this reason, an incremental 5mM Nile Red ethanol solution (final concentration from 10 to 120 μM, see table below) is added to a 10 mg / mL aqueous polymer solution. After the sample is mixed at room temperature for 24 hours, the sample is centrifuged at 13300 for 2 minutes. Next, the absorbance at 560 nm was measured in triplicate using a plate reader in a 96-dark well plate.

[0403] Table 6. Shows the maximum value of Nile red encapsulated in compound 9 in ppm at different concentrations.

[0404] Table 6.

[0405]

[0406]

[0407] In the specific case of compounds 10 and 12, where a pH-sensitive histidine hydrophobic block was present, potentiometric titration was performed to find the hydrophobic extent of drug encapsulation prior to Nile Red assay.

[0408] Potentiometric titration: The pK of the cationic polymer is determined by acid-base titration, measuring the pH of the solution throughout the process. a . Then the pK is obtained from the titration diagram a. For the measurement, a 1 mg / mL solution of the cationic polymer was prepared in Milli-Q water and a known amount of 0.1 M HCl was added until the pH of the solution was approximately 2. At this point, titration was performed with 0.2 M NaOH using an automatic Methrom 916ti-touch potentiometer with a Dosin 800 dispenser. The titration rate was set to 0.1 mL / min with a signal drift 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 titration curve obtained. In addition, the pK=pH+log[α / (1-α)] values ​​were plotted against 1-α, where K is the effective dissociation constant.

[0409] Table 7 shows the pK values ​​obtained for different compounds a value.

[0410] Table 7

[0411]

[0412] Using these molecules, the Nile Red test was adapted. The test consisted of preparing two Nile Red solutions ([Nile Red] = 30 μM) in Mili-Q water at pH 2 - with and without polymer. To these solutions were added successive volumes of 1 M NaOH (10 μL) until a final pH of 11 was reached. Nile Red is insoluble in polar solvents such as water and therefore does not show fluorescence. After adding NaOH and raising the pH of the medium in the presence of a hydrophobic environment (samples containing polymers), the color of the solution was observed to be highly purple and fluorescence was observed, showing that the polymer is able to encapsulate the hydrophobic molecules, making them soluble in aqueous media. In solutions without polymer, no changes were observed and there was no fluorescent signal.

[0413] In addition to Nile IR, other exemplary hydrophobic dyes (eg, DIL) and molecules / drugs (eg, vitamin E) were also successfully encapsulated using the same procedure.

[0414] DIL, also known as DiIC18 (3), is a fluorescent lipophilic cationic indocarbocyanine dye (cLogP: 22.539) with an absorption maximum at 550 nm and an emission maximum at 565 nm. DIL encapsulation was demonstrated by size exclusion chromatography (SEC) measurements and visual observation. To this end, a blank of DIL in PBS buffer was prepared and after filtration of the sample, a colorless solution was observed, 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 out, the colorimetric properties of the DIL could be visually appreciated as the solution remained pink. A diode array detector (i.e., DAD detector) was used to measure the color of the DIL at λabs SEC measurements performed at =550 nm confirmed the absorbance of the encapsulated DIL alone when compared to the filtered blank and polymer alone.

[0415] Similarly, partial encapsulation of Vit E in these polymers was also demonstrated. Partial encapsulation of Vitamin E was assessed by visual observation (similar to DIL, before and after filtration of the aqueous solution) and by turbidimetry (monitoring the absorbance at high lambda attributed solely to the turbidity of the sample). The observed reduction in turbidity indicated vit E encapsulation.

[0416] Example 5. Biological Evaluation: Biodistribution Profiles of Compounds of Formula Ib1

[0417] Biodistribution experiments and fluorescence quantification. Biodistribution experiments were performed using male Balb / c mice from Envigo at 5-7 weeks. Animals were housed in a 12-hour light and 12-hour dark cycle. Water and food were provided ad libitum throughout the experiment in all cases, and general aspects, behavior, and body weight were assessed daily to ensure animal health. All animal protocols were approved by the Institutional Animal Care and Use Committee of Centro de Investigación Príncipe Felipe (CIPF, Valencia, Spain). For biodistribution studies, 6 mice weighing 25 ± 4.5 g were used at each time point). The labeled polymer was administered intravenously through the tail vein at a dose of 1 mg / Kg Cy5.5 eq. in serum saline. At 0h, 1h, 4h, and 24h after administration, mice were euthanized. Cy5.5 was injected into 3 other mice at 1 mg / Kg for 4 hours in the same manner, and 3 mice were used as controls-basal fluorescence was achieved using PBS injection. After washing with 10 mL of PBS, blood, kidney, liver, spleen, heart, lung, axillary and inguinal lymph nodes were collected and Spectrum (PerkinElmer, Waltham, MA, USA) was used to measure its fluorescence. Then, the organs were weighed and stored at -80C for subsequent image acquisition by IVIS spectroscopy and fluorescence quantification. For IVIS spectral image acquisition, the settings used were (Exc 640 / Em 700, Bining Medium, F / Stop 2, Exp 0,5sec, FOV 13,2(C)). Afterwards, the images were analyzed using Living Image 4.5.4 software. The region of interest (ROI) of each organ was automatically drawn, and the circular ROI of the underlying organ was manually drawn. Lymph nodes were analyzed separately. The total radiant efficiency ((p / s) / (μW / cm 2) and subtract the basal value. The total radiation efficiency was then normalized according to the organ weight.

[0418] The results show that the biodistribution characteristics of two different graft copolymers are different due to their physicochemical properties. Graft copolymer compound 7B shows renal excretion characteristics, accumulates in major organs such as liver and spleen, and specifically targets inguinal and axillary lymph nodes (ILN and ALN, respectively). This selective accumulation makes this class of compounds an excellent candidate for lymphocyte delivery. In the case of compound 9A, which includes the incorporation of a hydrophobic pocket, the accumulation in major organs such as liver and spleen increases relative to the accumulation found in 7B, without clear renal excretion characteristics. In this case, the introduction of the hydrophobic part completely changes the specific targeting to lymph nodes, and it does not occur.

[0419] Example 6. Biological evaluation of the primary model of B16F10 melanoma mouse model.

[0420] Immunomodulatory potential of the graft copolymer St-PLys-g-PGlu (Compound 7B). High efficacy and high response rate, while being safe: preliminary data obtained.

[0421] The compounds obtained in Example 3, in which the gp100 MHC class I and MHC class II peptides had been conjugated to St-Plys-g-PGlu (also known as St-Br) as single conjugates (St-Br-gp100 MHCI, St-Br-gp100 MHCII) or in combined form (St-Br-gp100MHCI_gp100 MHCII), were evaluated for their antitumor effects in the primary model of B16F10 melanoma, the most aggressive type of skin cancer.

[0422] In addition, ligands of Toll-like receptor 7 / 8 have also been conjugated to St-Br ([St-Br-TLR7 / 8], a compound described in Example 2) to deliver adjuvant-like signals to antigen presenting cells (APCs), thereby improving antigen presentation and achieving extensive T cell activation and expansion (reference: Frega, G. et al. Trial Watch: experimental TLR7 / TLR8 agonists foroncological indications. Oncoimmunology 9, 1796002 (2020)). Therefore, the anti-tumor immune-mediated effects induced by St-Br conjugated to TLR7 / 8 agonists were evaluated alone and in combination with St-Br conjugates that delivered both gp100 MHC I and MHC II peptides, and compared with the responses induced by this dual conjugate mixed with Toll-like receptor (TLR) 9 (CpG) and TLR3 agonists. Compared with the PBS-treated group, the mean tumor volume of mice treated with [St-Br-gp100 MHCI-gp100 MHCII] and TLR inducers (i.e., CpG and Poly I:C) was significantly reduced 21 days after tumor inoculation (P=0.0335). None of these St-Br conjugates caused significant changes in mouse body weight, indicating the tolerability and safety of these carriers. Mice were sacrificed on day 22 after tumor inoculation and tumors were collected and then processed for FACS analysis.

[0423] The dual nanoconjugate [St-Br-gp100 MHCI-gp100 MHCII] mixed with CpG and Poly I:C significantly induced CD3 + and CD4 + T cell infiltration and decreased expression of PD-1 + CD3 + It was also observed that tumor-infiltrating cytotoxic CD8 + Antigen-specific intracellular IFN-γ in T lymphocytes + and TNF-α + The increased infiltration of T lymphocytes was associated with enhanced tumor growth inhibition induced by [St-Br-gp100 MHCI_St-Br-gp100 MHCII], CpG and Poly I:C.

[0424] [St-Br-gp100MHCI-gp100MHCII] combined with CpG and Poly I:C promoted the infiltration of myeloid DC2, while myeloid DC1 cells associated with a stronger cytotoxic immune response within the TME were found to be 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].

[0425] 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 number of human lymphocytes (human CD45 + The presence of human CD45 in the total lymphocyte population + The percentage of cells increased over time, from 13.3% on day 7 after tumor cell inoculation to 71.6% on day 21. On day 17 (end point), 97.1% and 79.1% of CD45 cells were expressed in the blood and spleen, respectively. + The cells 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 collected and subsequently processed for FACS analysis.

[0426] Humanized mice were treated with conjugated branched polypeptides [St-Br-gp100MHCI-gp100MHCII] alone or in combination with PD-1 antibody (αPD-1), namely Nivolumab. From the 15th day after treatment, both treatments significantly reduced tumor growth compared with the PBS-treated group (P=0.0081 and P=0.0414 for [St-Br-gp100MHCI-gp100MHCII]+TLR1 alone or in combination with αPD-1, respectively). However, no significant difference was observed between them. On the 17th day after the start of treatment, the individual tumor weights of mice were significantly reduced in animals immunized with [St-Br-gp100MHCI-gp100 MHCII]+TLR1 alone (P=0.0070) or in combination with αPD-1 (P=0.0381). No significant differences in mouse body weight were observed over time, indicating that systemic toxicity was negligible.

[0427] The combination of [St-Br-gp100MHCI-gp100MHCII] and αPD-1 resulted in extensive tumor infiltration of T lymphocytes (P<0.0001). +T cells were highly increased (P < 0.0001). In addition, compared with animals treated with [St-Br-gp100MHCI-gp100MHCII] alone, quantified CD3 + and CD8 + PD-1 expression in T cells was significantly decreased (P < 0.0001 for both treatments).

[0428] In summary, these findings demonstrate the potential of St-Br conjugated to MHCI and MHCII restricted antigens and / or TLR ligands as cancer vaccines to modulate melanoma-immune cell crosstalk within the microenvironment. In addition, the results obtained after administration of this dual nanoconjugate in a melanoma patient-derived xenograft (PDX) model validate the potential clinical application of these St-Br conjugates as multivalent cancer vaccines.

[0429] Example 7. Preparation of Compound (I) -Glu Combined with Branches Having Different Amino Acid Structures (Ib2)

[0430]

[0431] Formula (Ia) - Formula (Ib2) above regarding the first aspect of the present invention is an example where the integer "r" is ≠0 and β is the integer 1 - i.e., involves the use of Glu bound to branches having different amino acid structures.

[0432] Compounds 19-20 of formula (Ib2) were synthesized according to the next synthetic scheme 10.

[0433] Solution 10

[0434]

[0435] In brief, the synthesis of precursor St-PGlu is identical with the previous description of compound 4, but uses L-glutamic acid γ-benzyl ester NCA.In the second step, the benzyl protecting group is deprotected under acidic conditions.In brief, polymer is dissolved in pure TFA and the mixture is placed in rt and reacted 3 hours.Afterwards, solution is poured in diethyl ether so that product is precipitated.By centrifugal (3750rpm, 4min) separation product St-PGlu and under vacuum, it is dried to white solid.

[0436] In the next step, polymer St-PGlu (20.0mg, 0.16mmol PGlu unit) is dissolved in anhydrous DMF (2mL). Next, 0.4 equivalent of activator DMTMM is added to the reaction mixture and allowed to proceed for 10-15min at room temperature under a nitrogen atmosphere. Afterwards, for a certain grafting percentage, the required equivalent of pre-synthesized polyamino acid chain (i.e. polysarcosine (352.0mg, 0.06mmol), polylysine-thioglycerate) is added to the reaction mixture dissolved in 4mL anhydrous DMF. With TEA (150 μL), pH is adjusted to 8-9 and the reaction is allowed to proceed for 48 hours at room temperature and a nitrogen atmosphere. Afterwards, the solution is poured into diethyl ether to precipitate the product. The product is separated by centrifugation (3750rpm, 4min) and dried to a white solid under vacuum.

[0437] The grafting degree 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,quiralCH,CH 2 ). Grafting % = 27.66%. Grafting efficiency % = 69.15%.

[0438] Table 8 shows two examples of R13 and R6.

[0439] Table 8

[0440]

[0441] As an example, the general procedure for obtaining previously polymerized grafted chains is described for PSar: Sarcosine-NCA (1675 mg, 14.59 mmol) was added to a Schlenk tube equipped with a stir bar, a stopper, and then precipitated with 3 cycles of vacuum / N 2 Circulate and purge, and be dissolved in 6mL anhydrous DMF.Then, isopropylamine is dissolved in DMF (2mL) and added in the reaction mixture.The mixture is stirred at 10 ℃ for 16 hours.After completing, the reaction mixture becomes clear.The complete conversion of the monomer can be detected by IR.Then, the reaction mixture is poured into diethyl ether to precipitate the product.Separate the precipitate by centrifugation (3750rpm, 4min) and dry it under vacuum.Isolate the white solid final product.

[0442] Yield: 80%. 1H NMR (D 2 O):δ=1.08(d,J=6.7Hz,CH 3 ),2.73-3.12(m,CH 3 ),3.78 -4.56(m,CH 2 ).MW by SEC:1222Da(1.104)

[0443] Table 9 shows the main characterizations of compounds 19 and 20.

[0444] Table 9.

[0445]

[0446]

[0447] a In D 2 O 1 Data obtained by H-NMR. b Data obtained by SEC in MeOH.GuHCl.

[0448] Example 8. Synthesis of compound of formula (lb3).

[0449]

[0450] Analogously to the compound in Example 7, compound 21 was synthesized according to the next synthetic scheme 11.

[0451] Solution 11

[0452]

[0453] Briefly, the precursor star-PGlu was synthesized as previously described using L-glutamic acid γ-benzyl ester NCA. In the second step, the benzyl protecting group was deprotected under alkaline conditions. Briefly, the polymer was dissolved in THF (100 mg / mL) and NaOH (1.5 eq) was added. The mixture was stirred at 4 °C for 16 h. After 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 under vacuum. A white solid homopolymer was isolated. 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).

[0454] The synthesis of iPrPSar (16) and nBuPHis (DNP) (5) was carried out in a manner similar to the PBG polymerization. As an example, the synthesis of nBuPHis (DNP) (5) is described:

[0455] Scheme 12 shows the general procedure for the polymerization of PHis(DNP).

[0456] Solution 12

[0457]

[0458] 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 heated with 3 cycles of vacuum / N 2 Circulate and purge, and be dissolved in 8mL anhydrous DMF.Then, butylamine is dissolved in DMF (2mL) and added to the reaction mixture.The mixture is stirred at 10 ℃ for 16 hours.After completion, the reaction mixture becomes clear.The complete conversion of the monomer can be detected by IR.Then, the reaction mixture is poured into diethyl ether to precipitate the product.Separate the precipitate by centrifugation (3750rpm, 4min) and dry it under vacuum.Isolate the brown solid final product.

[0459] Yield: 80%. 1 H NMR(DMSO): δ=0.81(t,J=7.1Hz,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).

[0460] Next, use 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 vacuum / N was applied 3 times. 2 Circulate the purge and dissolve in 1 mL of DMF. Then, add DMTMMBF to the reaction mixture. 4 (4eq, 0.366g) and stirred at room temperature for 30 minutes. Afterwards, the polymer iPrPSar dissolved in 0.5mL 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 separated by centrifugation (3750rpm, 4min) and dried under vacuum. The white solid final product was isolated. Yield: 80%.

[0461] For coupling of polyhistidine polymer, the above procedure was followed. The final product was isolated as a pale yellow solid. Yield: 80%.

[0462] The last step is composed of the DNP protecting group of histidine and needs to be carried out under reducing conditions. In brief, 10 of the polymer precursor is dissolved in DMF (100mg / ml) and mercaptoethanol (10eq) is added to the reaction mixture. The mixture is stirred at room temperature for 16 hours. Afterwards, the solution is poured into diethyl ether to precipitate the product. Separate the precipitate by centrifugation (3750rpm, 4min) and dry it under vacuum. Isolate the pale yellow solid St-PGlu-grafted into-PSar-grafted into-PHis.

[0463] Yield: 70-90%

[0464] Table 10 shows the composition and main characteristics of these graft copolymers.

[0465] Table 10

[0466]

[0467] a pass 1 Data obtained by H-NMR. b Data obtained by GPC in MeOH / guanidine.

[0468] Example 9. Formation of single-molecule aggregates from compounds of formula lb2

[0469] One of the main features of our graft copolymers is their high molecular weight and the possibility of forming unimolecular aggregates of a size suitable for their application, not limited to drug delivery. In order to study this behavior, we evaluated their hydrodynamic size in aqueous solution by using light scattering techniques. The results obtained for the tested graft copolymers are summarized in Table 11.

[0470] Table 11 shows the hydrodynamic sizes of the graft copolymer monomolecular aggregates formed in solution.

[0471] Table 11

[0472]

[0473] Example 10. Conjugation of drugs with compounds of formula (lb2)

[0474] Hydrophobic drugs have been successfully conjugated to compound 19 (i.e., St-PGlu-g-Psar) to afford the following compound 22. In this specific example, glycine-tocopherol was conjugated to St-PGlu-g-Psar (10%) according to the conditions described in Scheme 13.

[0475] Solution 13

[0476]

[0477] Briefly, 50 mg of St-PGlu-g-PSar (10%) (0.058 mmol, 1 eq) in the form of sodium salt was dissolved in anhydrous DMSO (2 mL) in a 250 mL round-bottom flask equipped with a stirring bar and a septum stopper. Then, 3.92 mg (0.012 mmol, 0.2 eq) of DMTMMBF was added to another 1 mL of DMSO. 4 In this specific case, the added DMTMMBF 4 The amount corresponds to 20% modification of glutamic acid units. The reaction was allowed to proceed for 30 minutes under vigorous stirring and an inert atmosphere. 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. The reaction was allowed to stir for another 48 hours. Afterwards, the product was poured into a large excess of diethyl ether. A white solid was isolated by centrifugation and freeze drying. Yield: 36 mg; conjugation efficiency, 65%; % of incorporated Vit E: 13%, by 1 Confirmed by H-NMR.

[0478] Example 11. Encapsulation of hydrophobic molecules in the compound of formula 1b3.

[0479] Compound 21 described in Example 8 was designed to hold a hydrophobic pocket for drug encapsulation due to its amphiphilicity and the presence of a pH-sensitive and highly hydrophobic block of pHis.

[0480] In a first attempt and to characterize the system, Nile Red with logP: 2,98 was selected as a model hydrophobic drug. pH titration was also performed to find the hydrophobic range of drug encapsulation. The procedure followed was the same as described for compounds 10-12. The pka of compound 21A was found to be 6.45, and the Nile Red assay showed a transition from a hydrophilic environment to a hydrophobic environment with increasing pH.

[0481] Example 12. Preparation of novel compounds of formula (I) - Lys conjugated to branches with different amino acid structures.

[0482] Compounds 22A-22G are new examples based on the previous compounds described in the Example 1 section. That is, by changing the degree of polymerization and structure, compounds 22A, 22C and 22D were synthesized according to the procedure described in Example 1C, and compounds 22B, 22E-22G were synthesized according to the procedure described in Example 1E. These polymers were designed to change structural parameters such as morphology, compactness and lower ionic charge.

[0483] Table 12.

[0484]

[0485] a In NaNO 3 / NaN 3 Data obtained by SEC-RI-MALS in. Abbreviations: g = grafted; b = block; stat = statistical.

[0486] Tips

[0487] Just as a reminder, and according to the prior art - the term "pharmaceutically acceptable" means that it is used to prepare pharmaceutical compositions and is preferably non-toxic - that is, "pharmaceutically acceptable salts" are preferably non-toxic salts. Many different types of pharmaceutically acceptable salts are known to those skilled in the art.

[0488] Citation List

[0489] 1:EP3331937B1.

Claims

1. A graft copolymer, wherein the graft copolymer is any stereoisomer or mixture of stereoisomers of any one of the compounds of formula Ia, their pharmaceutically acceptable salts, or the compounds of formula (Ia) or their pharmaceutically acceptable salts, including homopolymeric polypeptides or random or block or graft copolymeric polypeptides: wherein A, A' and A" are each independently selected from a polymer comprising a main chain of repeating structural units of formula (I); and each of the A, A' and A" subunits may be the same or different; wherein K, K' and K" are each independently selected from -O-, -NH-; X is selected from wherein ω, ω' and ω" are integers from 0 to 1; each wavy line represents a point of connection with A, A' or A"; and "*" represents a point of connection with K, K' or K"; and wherein formula (I) is a graft copolymer compound comprising a main chain of repeating structural units of formula (I) wherein although the main repeating units defined by square brackets with their values ​​o, r, t, u and v, respectively, are shown in a specific order for ease of description, the main repeating units may be present in any order and may be present in blocks or randomly; wherein each of the primary repeating units defined by square brackets with the value "o" may be the same or different from each other, and may include blocks of secondary monomeric units defined by square brackets with the values ​​"p" and "q", which blocks may be the same or different from each other according to the definitions of their different substituents; wherein each of the primary repeating units defined by square brackets with the value "r" may be the same or different from each other, and may include blocks of secondary monomeric units defined by square brackets with the values ​​"s" and "t", which blocks may be the same or different from each other depending on the definitions of their different substituents; Where "*" indicates a connection point; Wherein " / " indicates that the sequential order of the repeating units on both sides of the symbol is arbitrary; R1 is a diradical selected from the following wherein the wavy line represents the connection point; wherein y and z are independently integers ranging from 1 to 20; and Where Y is a diradical with a molecular weight (MW) of 5 to 3000 g / mol R2 is selected from -O-, -NH-; wherein o and r are each independently selected from integers from 0 to 5000; provided that at least one of o or r ≠ 0; p and s are each independently selected from an integer from 1 to 1000; q, t, u and v are each independently selected from integers from 0 to 1000; α, α' are each independently an integer selected from 1 to 4; β and β' are each independently an integer selected from 0 to 3; Each R3 is independently selected from H and -CH3; Each R4 is independently selected from H and -CH3; Each R5 is independently selected from H and -CH3; Each R6 is independently selected from H and -CH3; Each R7 is independently selected from H and -CH3; Each R8 is independently selected from H and -CH3; Each R9 is independently selected from H and -CH3; Each R 10 Independently selected from H and -CH3; Each R 15 Independently selected from H and -CH3; R 11 and R 12 Each independently selected from H, an imaging agent or a labeling agent, a cell targeting agent and a free radical of formula (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII) wherein Q is selected from -OH, an imaging agent or a labeling agent, a cell targeting agent, and a, b, c, d, e and f are each independently selected from an integer from 0 to 4; Each R 13 independently selected from H, -CH3, an imaging agent or labeling agent, a cell targeting agent, and a free radical of formula (XII), (XIII), (XIX), (XX), (XXI), (XXII), (XVIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII) or (XXXVIII) wherein g, h, i and j are each independently selected from integers from 0 to 4; T is selected from H and -OH; Each R 14 independently selected from H, -OH, an imaging agent or labeling agent, and a cell targeting agent; Wherein L is selected from a single bond, and a diradical of the formula wherein k, l, m, n, w and x are each independently selected from an integer from 0 to 4; Wherein "*" indicates the connection point; Each X1 is independently selected from H, -C(O)H, -S(O)H, -NHCH(CH3)2, -(C1-C4)alkylNH2, -(C1-C4)alkylNHCH3, -(C1-C4)alkylN(CH3)2, -O-(C1-C4)alkyl-NH2, -O-(C1-C4)alkyl-NHCH3, -O-(C1-C4)alkyl-N(CH3)2, imaging or labeling agents, cell targeting agents and free radicals of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII); Each X2 is independently selected from H, -C(O)H, -S(O)H, -NHCH(CH3)2, -(C1-C4)alkylNH2, -(C1-C4)alkylNHCH3, -(C1-C4)alkylN(CH3)2, -O-(C1-C4)alkyl-NH2, -O-(C1-C4)alkyl-NHCH3, -O-(C1-C4)alkyl-N(CH3)2, an imaging or labeling agent, a cell targeting agent, and a free radical of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or (XXIII); and Each X3 is independently selected from H, -C(O)H, -S(O)H, -NHCH(CH3)2, -(C1-C4)alkylNH2, -(C1-C4)alkylNHCH3, -(C1-C4)alkylN(CH3)2, -O-(C1-C4)alkyl-NH2, -O-(C1-C4)alkyl-NHCH3, -O-(C1-C4)alkyl-N(CH3)2, imaging or labeling agents, cell targeting agents and free radicals of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII) or (XXIII).

2. The graft copolymer according to claim 1, wherein the Y diradical of R1 is a diradical selected from the following: -NH-, -NH(C1-C6)alkyl-, -O-, -(C1-C6)alkyl-COO-, straight or branched -(C1-C6)alkyl- 30 )alkylene- and a diradical of formula (IV), (V), (VI), (VII), (VIII), (IX), (X) or (XI) Wherein "*" indicates the connection point; Among them, Y-(C1-C 30 ) The alkylene diradical is selected from -OH, -NR a R b , -SH2, -NHNH2, -COOR c , -CF3, -OCF3 and one or more free radicals of halogen are optionally substituted, wherein R a , R b and R c is a free radical independently selected from the following: H, -phenyl, -(C1-C 30 )alkyl, -(C2-C 30 )alkenyl, -(C1-C 30 ) alkylphenyl and -phenyl (C1-C 30 )alkyl.

3. The graft copolymer according to any one of the preceding claims, wherein the graft copolymer is a graft copolymer, wherein the molecular weight (MW) is 3 to 500000 kilodaltons (kDa) and wherein the graft copolymer is a unimolecular structure with a radius of 4-300 nm.

4. A graft copolymer according to any one of the preceding claims, wherein the integers ω, ω' and ω" are 0, R1 is ethylene, and R2 = -NH-.

5. The graft copolymer according to any one of the preceding claims, wherein the integer "o" is ≠0; wherein L is a single bond; wherein α is the integer 3; and wherein the integers "r" and "v" are 0.

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

7. The graft copolymer according to any one of claims 5-6, wherein the value of integer "o" is 5 to 3000 and wherein the value of integer "p" is 3 to 1000.

8. The graft copolymer according to any one of claims 5 to 7, wherein R 11 and R 12 Each is independently selected from H and free radicals of formula (XII), (XIII), (XIV) and (XVI).

9. The graft copolymer according to any one of claims 5 to 8, wherein the graft copolymer is a compound comprising a compound selected from the group consisting of: 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, and wherein the term "St" is the star structure of formula (Ia) of claim 1; and Wherein the term "PBG" is polybenzyl glutamate.

10. The graft copolymer of any one of claims 1-4, wherein the integer "r" is ≠0; wherein β is the integer 1; and the integers "o" and "u" are 0.

11. The graft copolymer of claim 10, wherein the integer "r" has a value from 3 to 1000; and wherein the integer "s" has a value from 2 to 400.

12. The graft copolymer according to any one of claims 10 to 11, wherein R 13 is independently selected from H and free radicals of formula (XII), (XIII), (XXI) and (XXVIII).

13. The graft copolymer according to any one of claims 10 to 12, wherein the graft copolymer is a compound comprising a compound selected from the group consisting of: St-PGlu-g-PSar St-PGlu-g-PHis St-PGlu-g-PLys St-PGlu-g-PLysTG; and St-PGlu-g-PSar-g-PHis; and wherein the term "St" is the star structure of formula (Ia) of claim 1; and PLysTG is poly-lysine-thioglycerate.

14. A polymer composite comprising a graft copolymer according to any one of the preceding claims and at least one pharmaceutical, veterinary or cosmetic active agent; or A pharmaceutical, veterinary or cosmetic composition comprising at least one polymer complex according to claim 14, and one or more suitable excipients or carriers.

15. The polymer complex of claim 14 or the pharmaceutical composition of claim 14 for use as a medicament; or A method for delivering a pharmaceutically active agent (e.g., a nucleic acid) into a target cell, the method comprising administering a composition (e.g., a solution) comprising the polymer complex of claim 14 to an animal, including a human, such that the complex is in physical contact with the target cell, thereby delivering the pharmaceutically active agent into the cell.

16. A process for preparing a compound which is structurally different from the starting compound of a graft copolymer according to any one of claims 1 to 13, said process comprising the steps of: (i): using a compound according to any one of claims 1 to 13 as a starting compound; and (ii) making structural changes to the compound of step (i) to obtain a compound that is structurally different from the starting compound of step (i); and wherein the structurally different compound of step (ii) obtained is a compound capable of forming a polymer complex, the polymer complex comprising the structurally different compound of step (ii) obtained, and at least one pharmaceutical, veterinary or cosmetic active agent, and wherein the active agent is an active agent with which the starting compound of step (i) is also capable of forming a polymer complex.

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