CN115397846A

CN115397846A - Hydrophobic modified albumin and preparation method and application thereof

Info

Publication number: CN115397846A
Application number: CN202180018719.6A
Authority: CN
Inventors: 柯天一; 檀琳; 劳芳; 孙楚楚; 于海勇; 欧阳芳幸
Original assignee: Kunshan Xinyunda Biotech Co ltd
Current assignee: Kunshan Xinyunda Biotech Co ltd
Priority date: 2020-03-06
Filing date: 2021-03-04
Publication date: 2022-11-25
Also published as: WO2021175275A1

Abstract

Hydrophobic modified albumin, methods of making and uses thereof are disclosed. The hydrophobic modified albumin is obtained by introducing a hydrophobic group at a thiol group of albumin. The hydrophobically modified albumin can be used as a carrier for encapsulating poorly water soluble pharmaceutical or cosmetic actives. Also disclosed are carrier compositions comprising the hydrophobically modified albumin, pharmaceutical or cosmetic compositions comprising the hydrophobically modified albumin or the carrier compositions, and modifiers for preparing the hydrophobically modified albumin.

Description

Hydrophobic modified albumin and preparation method and application thereof

Technical Field

The present application relates to hydrophobically modified albumins, in particular to modified albumins obtained by introducing hydrophobic groups at the free thiol groups of albumin, methods for their preparation and use, and compounds for the preparation of such hydrophobically modified albumins.

Background

The biggest obstacles to the administration (especially injection administration) of poorly water-soluble drugs (e.g., many small molecule anticancer agents such as paclitaxel) are the low solubility, difficulty in preparing suitable formulations, and the need for various methods to increase the solubility, such as salt formation, modification of the drug, addition of latent solvents, addition of co-solvents, and solubilization by addition of surfactants. These methods suffer from various drawbacks. Since salt formation often requires strong acid or strong base conditions, it is often not suitable for many drugs. Physiologically safe latent solvents are small and limited in amount. The safety of the co-solvent which increases the solubility of the drug by forming a complex with the drug is not easily ensured. Solubilization by surfactant micelles presents the following problems: the micelle is easy to dissociate during clinical dilution, so that the medicine is separated out; the application of the surfactant is easy to cause various toxic and side effects, such as drug-induced rash, tachypnea, bronchospasm, hypotension, renal toxicity, neurotoxicity, cardiotoxicity and the like.

Human Serum Albumin (HSA) is a naturally occurring water-soluble macromolecule. Mature HSA is a single polypeptide chain of 585 amino acid residues with a large number of free amino and carboxyl groups on the chain, but only 1 free thiol in the cysteine residue at position 34. One important application of HSA in the pharmaceutical field is as a carrier for pharmaceutically active ingredients. There have been various strategies to modify albumin with its free amino, carboxyl or thiol groups to use it as a carrier. One strategy is to modify hydrophobic groups such as fatty acids on the active pharmaceutical ingredients (e.g. antineoplastic agents) so that the drugs can bind to albumin in vivo to prolong the half-life of the drugs, such as CN101384623B, CN108187063A, CN105440112A, etc.; alternatively, groups such as PEG are modified on albumin free thiol groups to increase the water solubility of albumin, such as CN102378576A, CN101709085B, and the like. Another strategy is to make hydrophobic modifications to the free amino and carboxyl groups of albumin, which self-assembles into nanoparticles in aqueous media due to its amphiphilicity, which nanoparticles can encapsulate poorly water soluble compounds to increase their solubility, e.g., CN101220093A and CN102172404A, etc. In addition to such hydrophobic modifications, hydrophilic modifications of the free amino, carboxyl or thiol groups of albumin are also possible, for example CN101543630A. However, due to the large number of free amino and carboxyl groups present in albumin, the modified albumin obtained using this strategy carries a large number of hydrophobic modifying groups, which form nanoparticles having an average particle size of greater than 100 nm. Furthermore, the outer shell of the albumin nanoparticle has a significant number of hydrophobic groups, as shown in fig. 1A. The entry of these hydrophobic groups of non-natural origin into the body increases the risk of the body to develop an immune response, which may present a safety risk. In addition, the modification mode is multi-point modification of a plurality of groups on the surface of the albumin, and the modification degree and the specific modified sites are not controllable, so that the quality control and the patent medicine are not facilitated.

Therefore, there is still a need in the art to develop hydrophobically modified albumin with uniform quality and controllable modification sites to form nanoparticles with approximate natural particle size of albumin and no or low immunogenicity as carriers of poorly water soluble compounds.

Disclosure of Invention

According to the method, site-specific hydrophobic modification is carried out only on the free sulfydryl of the albumin, and a hydrophobic group is introduced, so that the novel hydrophobic modified albumin is obtained. Because one albumin molecule only has one free sulfydryl, the hydrophobic modification has high controllability, and the obtained product has small and uniform particle size and is beneficial to quality control and patent medicine. In addition, the obtained hydrophobic modified albumin spontaneously assembles in an aqueous medium (e.g., aqueous solution, blood, etc.) to form a structure in which the hydrophobic group faces inward (hydrophobic interface) and the natural albumin group faces outward (hydrophilic interface), as shown in fig. 1B. It is expected that this structure reduces the risk that the modified hydrophobic group may elicit an immune response, i.e. reduces the immunogenicity of the modified albumin molecule, improving safety.

Thus, in a first aspect, the present application provides a modified albumin consisting of albumin and a hydrophobicity-enhancing moiety attached to the S atom in the free thiol group of said albumin, said modified albumin being representable by the general formula (I):

wherein:

q is an S atom in a free thiol group of the albumin;

is the remainder of the albumin;

c is the hydrophobicity-enhancing moiety and is represented by the general formula (II):

wherein:

l is a connection unit connected to Q;

z is a spacer;

m is 1 or 0; and is

M is a hydrophobic group.

In a second aspect, the present application provides a compound of general formula (X):

R-(Z) _m -M

(X)

wherein:

r is a moiety containing a reactive group capable of linking a compound of formula (X) to S in a free thiol group of albumin by reaction with said free thiol group and providing a hydrophobicity-enhancing moiety of formula (II) as described above;

z is a spacer as defined above;

m is as described above; and is

M is a hydrophobic group as described above.

In a third aspect, the present application provides a method of preparing a modified albumin of the present application, said method comprising the step of reacting said albumin with a compound of general formula (X) as described above under conditions allowing the free thiol group of said albumin to react with R.

In a fourth aspect, the present application provides a composition comprising a modified albumin of the present application and optionally an unmodified albumin.

In a fifth aspect, the present application provides a pharmaceutical or cosmetic composition comprising:

a carrier encapsulating a poorly water soluble compound having pharmaceutical or cosmetic activity, said carrier being a modified albumin of the present application or a composition according to the fourth aspect above; and

optionally other pharmaceutically or cosmetically acceptable carriers.

In a sixth aspect, the present application provides a method of increasing the solubility of a poorly water soluble compound having pharmaceutical or cosmetic activity in an aqueous medium, the method comprising the steps of:

(1) Providing a solution of the modified albumin of the present application or the composition according to the fourth aspect above in an aqueous medium;

(2) Providing a solution of the poorly water soluble compound in an organic solvent;

(3) Mixing the two solutions in the step (1) and the step (2);

(4) The organic solvent is removed.

In a seventh aspect, the present application provides the use of a modified albumin of the present application or a composition according to the fourth aspect above as a carrier for poorly water soluble compounds with pharmaceutical or cosmetic activity.

In an eighth aspect, the present application provides the use of a modified albumin of the present application or a composition according to the fourth aspect above as a carrier in the preparation of a pharmaceutical or cosmetic composition comprising a poorly water soluble compound having pharmaceutical or cosmetic activity.

In a ninth aspect, the present application provides a kit comprising at least: i) Poorly water-soluble compounds with pharmaceutical or cosmetic activity, ii) unmodified albumin, and iii) compounds of the general formula (X) as described above.

Drawings

FIG. 1: fig. 1A is a schematic diagram showing nanoparticles formed in an aqueous medium of modified albumin resulting from hydrophobic modification of free amino and carboxyl groups of HSA according to the prior art, wherein a large number of hydrophobic modification groups face outward (hydrophilic surface) in addition to a portion of the hydrophobic modification groups facing inward (hydrophobic interface), exposed to the human environment. Fig. 1B is a schematic diagram showing nanoparticles formed in an aqueous medium of hydrophobically modified albumin according to the present invention, with the hydrophobic modification group facing inward (hydrophobic interface) and the native albumin group facing outward (hydrophilic interface).

FIG. 2 is a schematic diagram: LC-MS profile of rHA modified with SYN-LPD19002 (rHA-CA) prepared in example 7.

FIG. 3: deconvolution of LC-MS spectra of figure 2.

FIG. 4 is a schematic view of: rHA modified with SYN-LPD19005 (rHA-PEG) prepared in example 8 ₂ -PA).

FIG. 5: example 8 preparation of rHA modified with SYN-LPD19005 (rHA-PEG) ₂ -PA) LC-MS spectrum.

FIG. 6: deconvolution of the LC-MS spectra of figure 5.

FIG. 7: HPLC profile of rHA modified with SYN-LPD19001 (rHA-PA) prepared in example 9.

FIG. 8: LC-MS profile of rHA modified with SYN-LPD19001 (rHA-PA) prepared in example 9.

FIG. 9: deconvolution of the LC-MS spectra of figure 8.

FIG. 10: fig. 10A shows the particle size of the unmodified rHA sample (n = 3); fig. 10B shows the particle size of the product sample of example 7 (n = 3); fig. 10C shows the particle size of the product sample of example 8 (n = 3); fig. 10D shows the particle size (n = 3) of the product sample of example 9.

Detailed Description

Definition of

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to the techniques used herein is intended to refer to those techniques commonly understood in the art, including those variations of or alternatives to those techniques that would be apparent to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

Herein, the terms "hydrophobically modified albumin" and "modified albumin" are used interchangeably and mean modified albumin obtained by introducing hydrophobic groups at the free thiol groups of albumin. In some embodiments, the free thiol group of the albumin is the thiol group of the albumin at a cysteine residue corresponding to position 34 of SEQ ID NO: 1. Amino acid residue correspondence between different amino acid sequences can be determined by sequence alignment methods well known in the art.

The terms "modifier" or "conjugation modifier" herein include compounds of the general formulae (X), (XII) and (XIII) as defined herein, and may be used interchangeably.

The terms "comprising," "including," "having," "containing," or "involving," and other variants thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps, although not necessarily present (i.e., these terms also encompass the terms "consisting essentially of 8230; \8230; consisting of, and" consisting of \8230; \8230).

The term "alkyl" as used herein is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 30 (i.e., C) _1-30 Alkyl), e.g. C _1-12 Alkyl radical, C _1-10 Alkyl radical, C _1-6 Alkyl radical, C _1-4 Alkyl radical, C _4-8 Alkyl radical, C _5-25 Alkyl radical, C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl radical, C _7-15 An alkyl group. Examples that may be mentioned include, but are not limited to: ethyl (Et), n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, heptene, octyl, C ₁₁ Alkyl or C ₁₅ An alkyl group. The alkyl group may be optionally substituted.

As used herein, the term "alkenyl" means a linear or branched hydrocarbyl group, which may contain from 2 to 230 carbon atoms (i.e., C) _2-30 Alkenyl) such as C _2-12 Alkenyl radical, C _2-10 Alkenyl radical, C _2-6 Alkenyl radical, C _2-4 Alkenyl radical, C _4-8 Alkenyl radical, C _5-25 Alkenyl radical, C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl radical, C _7-15 Alkenyl, and may contain 1 or more double bonds, for example 1-8, or 1,2, 3,4, 5, or 6 double bonds. Examples that may be mentioned include, but are not limited to: vinyl, 1-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl, heptenyl, octenyl, C ₁₁ Alkenyl radical, C ₁₅ Alkenyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl or C ₂₁ Alkenyl groups, such as heptadec-8-enyl, nonadecadec-4, 7,10,13, 16-pentaenyl, nonadecadec-4, 7,10, 13-tetraenyl, heneicosyl-3, 6,9,12,15, 18-hexaenyl. The alkenyl group may be optionally substituted.

As used herein, the term "alkynyl" means a linear or branched hydrocarbyl group that may contain from 2 to 30 carbon atoms (i.e., C) _2-30 Alkynyl), for example C _2-12 Alkynyl, C _2-10 Alkynyl, C _2-6 Alkynyl, C _2-4 Alkynyl, C _4-8 Alkynyl, C _5-25 Alkynyl, C _7-23 Alkynyl, C _9-21 Alkynyl, C _11-19 Alkynyl, C _13-17 Alkynyl, C _7-15 Alkynyl and may contain 1 or more triple bonds, for example 1-8, or 1,2, 3,4, 5 or 6 triple bonds. Examples that may be mentioned include, but are not limited to: ethynyl, 1-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, octynyl, C ₁₁ Alkynyl, C ₁₅ Alkynyl, C ₁₇ Alkynyl, C ₁₉ Alkynyl or C ₂₁ Alkynyl and the like. Said alkynyl groupMay be optionally substituted.

As used herein, the term "alkoxy" means an alkyl group (as defined above) attached to the rest of the molecule through an oxygen atom, having the structure "-O-alkyl". Alkoxy is preferably C _1-8 Alkoxy radical, C _1-6 Alkoxy radical, C _1-4 Alkoxy or C _1-3 An alkoxy group. C _1-6 Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, pentyloxy, hexyloxy, and the like. The alkoxy group may be optionally substituted.

As used herein, the term "alkylthio" means an alkyl group (as defined above) attached to the rest of the molecule through a sulfur atom, having the structure "-O-alkyl". Alkylthio is preferably C _1-8 Alkylthio radical, C _1-6 Alkylthio radical, C _1-4 Alkylthio or C _1-3 Alkylthio, for example methylthio or ethylthio. The alkylthio group may be optionally substituted.

As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring. The cycloalkyl group has 3 to 15, such as 3 to 10 carbon atoms, 3 to 7 carbon atoms, 3 to 6 carbon atoms, 3 to 5 carbon atoms, 5 to 7 carbon atoms, 4 to 6 carbon atoms, or 5 to 6 carbon atoms, and the like. For example, as used herein, the term "C ₃ -C ₁₀ Cycloalkyl "refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring having 3 to 10 ring-forming carbon atoms. Examples that may be mentioned include, but are not limited to: monocyclic cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl; and bicyclic cycloalkyl groups, including spiro, fused or bridged systems, such as bicyclo [1.1.1]Pentyl, bicyclo [2.2.1]Heptyl, and the like. The cycloalkyl group may be optionally substituted.

As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system. As herein describedAs used, the term "C ₆ - ₁₀ Aryl "means an aryl group containing 6 to 10 carbon atoms, preferably phenyl or naphthyl. The aryl group may be optionally substituted.

The term "heteroaryl" as used herein refers to a monocyclic, bicyclic or tricyclic aromatic ring system having 5 to 14 ring atoms, in particular having 5, 6, 7,8,9,10,11,12,13 or 14 ring atoms, in particular 1,2, 3,4, 5, 6, 7,8,9 or 10 carbon atoms, and which comprises at least one heteroatom which may be the same or different (for example oxygen, nitrogen or sulfur), and which, in addition, may be benzo-fused in each case. For example, as used herein, the term "5-to 10-membered heteroaryl" means a heteroaryl group containing 5 to 10 ring atoms. Specific examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl and the like, or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like, or azaindole, and benzo derivatives thereof such as indole, benzimidazole, quinoline, isoquinoline and the like. The heteroaryl group may be optionally substituted.

As used herein, the term "halo" or "halogen" group is defined to include F, cl, br or I.

As used herein, the term "amino acid" includes natural amino acids and unnatural amino acids. In some embodiments, the amino acid has the following structure:

wherein R is ^3’ Selected from the group consisting of: h; c _1-6 An alkyl group; is selected from SH, SCH ₃ 、NH ₂ 、OH、COOH、-CONH ₂ 、-NHCOCH ₃ 、-NHCHO、-NHCONH ₂ Guanidino, optionally substituted C _6-10 Aryl (preferably phenyl, 4-OH-phenyl, biphenyl)Phenyl, naphthyl) and optionally substituted 5-to 10-membered heteroaryl (preferably imidazole, indole) C substituted with 1 or more substituents _1-6 An alkyl group; c _3-10 Cycloalkyl (preferably cyclohexyl); c _6-10 Aryl (preferably phenyl);

preferably, R ^3’ Selected from H, CH ₃ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ ) ₂ Phenyl, cyclohexyl, benzyl, p-hydroxybenzyl, phenethyl, p-hydroxyphenylethyl, pyridin-2-ylmethyl-, pyridin-3-ylmethyl-, pyridin-4-ylmethyl-, -CH ₂ OH、 -CH(OH)CH ₃ 、-CH ₂ CH ₂ SCH ₃ 、-CH ₂ CONH ₂ 、-CH ₂ COOH、-CH ₂ CH ₂ CONH ₂ 、-CH ₂ CH ₂ COOH、-(CH ₂ ) ₃ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₃ NH ₂ 、-(CH ₂ ) ₃ NHCOCH ₃ 、-(CH ₂ ) ₃ NHCHO、-(CH ₂ ) ₄ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₄ NHCOCH ₃ 、-(CH ₂ ) ₄ NHCHO、-(CH ₂ ) ₃ NHCONH ₂ 、-(CH ₂ ) ₄ NHCONH ₂ 、-CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ 、

As used herein, the term "peptide" refers to a compound formed from two or more amino acids as described above through a peptide bond. The amino acids may be the same or different. Preferably, the peptide is formed from 2 to 20 identical or different amino acids, for example 2 to 15, 2 to 10, 2 to 5 or 3 to 4.

The term "substituted" means that one or more (e.g., 1,2, 3, or 4) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the present circumstances is not exceeded and the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

If a group is described as "optionally substituted" \8230; … substituted "or" optionally substituted ", that group may be (1) unsubstituted or (2) substituted. Unless otherwise indicated, where substituted, the groups are substituted with one or more substituents independently selected from the group consisting of: hydroxy, halogen, C ₁ -C ₆ Alkyl, halo C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₃ -C ₇ Cycloalkyl, cyano, nitro, -C (O) C ₁ -C ₆ Alkyl, -C (O) OH, -C (O) OC ₁ -C ₆ Alkyl, -C (O) NH ₂ 、-C(O)NH(C ₁ -C ₆ Alkyl), -C (O) N (C) ₁ -C ₆ Alkyl radical) ₂ 、NH ₂ -C ₁ -C ₆ Alkyl-, NH (C) ₁ -C ₆ Alkyl), -NH ₂ 、-N(C ₁ -C ₆ Alkyl radical) ₂ 、-NHC(O)(C ₁ -C ₆ Alkyl), -OC ₁ -C ₆ alkyl-OC ₁ -C ₆ Alkyl, hydroxy C ₁ -C ₆ Alkyl radical, C ₆ -C ₁₀ Aryl and 5-to 10-membered heteroaryl, preferably hydroxy、-NH ₂ Halogen, C ₁ -C ₆ Alkyl, NH ₂ -C ₁ -C ₆ Alkyl- (e.g., aminomethyl),.

If a substituent is described as being "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

If a variable or substituent can be selected from different variations and the variable or substituent occurs more than once, then the variations can be the same or different.

As used herein, the term "one or more" means 1 or more than 1, such as 2,3,4, 5 or 10, under reasonable conditions.

As used herein, unless otherwise indicated, the point of attachment of a substituent may be from any suitable position of the substituent.

When a bond of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

The term "poorly water soluble compound" refers to a compound having a water solubility of less than 0.01g at 20 ℃. Examples of poorly water soluble compounds include, but are not limited to, curcumin, paclitaxel, docetaxel, cisplatin, and the like.

The term "pharmaceutically or cosmetically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other components comprising the pharmaceutical or cosmetic composition and/or the mammal being treated therewith.

The term "about" means within. + -. 10%, preferably within. + -. 5%, more preferably within. + -. 2% of the stated value.

The numerical ranges set forth herein should be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" should be interpreted to include not only the explicitly recited values of 1 to 10, but also to include any individual value within the range of 1 to 10 (e.g., 2,3,4, 5, 6, 7,8, and 9) and the sub-ranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). The same principle applies to ranges using only one value as the minimum or maximum value.

All documents mentioned herein are incorporated by reference in their entirety.

In a first aspect, the present application provides a modified albumin consisting of albumin and a hydrophobicity-enhancing moiety attached to the S atom in a free thiol group (e.g., the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) of the albumin, the modified albumin being represented by the general formula (I):

wherein:

q is the S atom in the free thiol group (e.g., the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) of the albumin;

is the remainder of the albumin;

wherein:

l is a linker connected to Q;

z is a spacer;

m is 1 or 0; and is

M is a hydrophobic group.

Albumin

Human Serum Albumin (HSA) is a natural water-soluble macromolecule, and one important application in the pharmaceutical field is as a carrier for pharmaceutically active ingredients. Mature HSA is a single polypeptide chain of 585 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 1) with a large number of free amino and carboxyl groups on the chain, but only 1 free thiol in the cysteine residue at position 34. Additional exemplary amino acid sequences of human serum albumin are available through GenBank accession numbers such as AAN17825.1, CAA23754.1, or AAX 63425.1.

Any albumin known to those skilled in the art may be used to form the modified albumin of the present application. In some embodiments, the albumin may be serum albumin isolated and purified from a host. The serum albumin may be mammalian serum albumin known to those skilled in the art, including but not limited to mouse, rat, rabbit, guinea pig, dog, cat, sheep, cow, sheep, horse, or human albumin. In some preferred embodiments, the albumin is human serum albumin. Suitable human-isolated serum albumin is commercially available, for example, from shenzhen remarkable Biotechnology, inc. under the accession number 70024-90-7, or from ProSpec under the accession number PRO-354, or from Starfish Pond under the accession number EY-XQ 0489.

Any recombinant albumin known to those skilled in the art may be used to form the modified albumin of the present application. The recombinant albumin may be a mammalian albumin known to those skilled in the art, including but not limited to recombinant mouse, rat, rabbit, guinea pig, dog, cat, sheep, cow, sheep, horse, or human albumin. In some preferred embodiments, the recombinant albumin is recombinant human albumin (rHA). Suitable recombinant albumin is commercially available, for example from north china pharmaceuticals under the cat number 201801D2002, or Sigma-Aldrich under the cat number a9731, or from a cereal organism under the cat number HYC002M 01. In addition, provided by Albumedix corporation

A range of products may also be used in this application.

In some embodiments of the present application, the recombinant albumin is produced in a host cell. Any host cell capable of producing exogenous recombinant proteins can be used to produce recombinant albumin. In some embodiments, the host cell is a yeast, bacterial, plant, insect, animal, or human cell that is transformed to produce recombinant albumin. In some embodiments, the host is cultured in a liquid medium. In some embodiments, the host may be a bacterial strain, such as e.coli and b. In other embodiments, the host may be a yeast strain, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris (Pichia pastoris), kluyveromyces lactis (Kluyveromyces lactis), arxua adeninivorans, and Hansenula polymorpha (Hansenula polymorpha). In a preferred embodiment, the host is pichia pastoris. In some embodiments, the host may be a plant, such as rice (Oryza Sativa) or rice seed culture.

In certain embodiments, albumin useful herein includes molecular variants of albumin. Variants of albumin include natural variants produced by polymorphisms in human albumin. Genetic variants of over 30 distinctly different human serum albumins have been identified by electrophoretic analysis under different conditions. See, weitkamp, ann.hum.genet,36 (4): 381-92 (1973); weitkamp, isr.j.med.sci.,9 (9): 1238-48 (1973); fine et al, biomedicine,25 (8): 291-4 (1976); fine et al, rev. Fr. Trans/us. Immunohematol,25 (2): 149-63 (1982); rochu et al, rev.fr.transfus.immunohematol,31 (5): 725-33 (1988); arai et al, proc.natl.acad.sci.u.s.a 86 (2): 434-8 (1989), the contents of which are incorporated herein by reference in their entirety. Thus, modified albumins formed as variants of the albumin molecule are included within the scope of the present invention.

In some embodiments, derivatives of albumin that are substantially homologous to albumin may also be used herein. For example, the albumin derivative has at least 75%, at least 80%, at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence identity to albumin.

In some embodiments, the albumin, or a variant or homologue thereof has a free thiol group. In some embodiments, the albumin, or variant or homologue thereof, comprises a single free thiol group. In some embodiments, the single free thiol group of the albumin, or a variant or homologue thereof, is the thiol group corresponding to the cysteine residue at position 34 of SEQ ID No. 1.

In some embodiments, the albumin is selected from albumins having a cysteine residue corresponding to position 34 of SEQ ID No. 1; preferably selected from Human Serum Albumin (HSA), recombinant human albumin (rHA), bovine serum albumin and porcine serum albumin. For example, the albumin comprises the amino acid sequence shown in SEQ ID NO. 1.

Linking group

From the above, the modified albumin of the present application may be obtained by reacting albumin with a compound of general formula (X) as described above under conditions allowing the free thiol group of the albumin (e.g. the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) to react with R. The reaction may be selected from: michael addition reactions, thiol-disulfide exchange reactions, substitution reactions, and click chemistry. The skilled person will understand that the linker L in the modified albumin of the present application may be derived from R in the general formula (X) by said reaction.

In some embodiments, the present application provides the modified albumin described above, wherein L is represented by the general formula (III):

wherein:

the bond identified by a is bonded to Q, the bond identified by b is bonded to (Z) _m -M bonding;

R ¹ is S, or is selected from the group consisting of optionally substituted:

wherein:

the bond designated by f is bonded to Q, the bond designated by k is bonded to R ² Bonding;

R ¹¹ each independently selected from: H. optionally substituted C _1-12 Alkyl, -COOH, -COOC _1-6 Alkyl, -CH (R) ¹⁵ )NH-CO-C _1-6 Alkyl radical, C _6-10 Aryl and 5 to 10 membered heteroaryl;

R ¹² selected from H, optionally substituted C _1-12 Alkyl and optionally substituted C _2-12 An alkenyl group;

R ¹³ selected from: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl and

R ^13’ selected from: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 An alkenyl group,

Wherein:

u is 0,1, 2 or 3;

R ¹⁴ and R ¹⁵ Each independently is H or C _1-6 An alkyl group;

R ¹⁶ selected from H and optionally substituted C _1-12 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-4 Alkyl);

p is 0,1 or 2;

R ^a each independently selected from hydroxy, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkylthio and halogen;

R ² selected from: direct bond, O, optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) -, - (optionally substituted C _1-6 Alkyl) -C _3-6 Cycloalkyl-, -O- (optionally substituted C) _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -, optionally substituted with (R) ^b ) _q Substituted phenyl, optionally substituted (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -;

wherein:

j is an integer from 1 to 10, such as 2;

q is 0,1, 2 or 3;

R ^b each independently selected from H, hydroxyl, nitro, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkylthio and halogen;

or

R ¹ And R ² Together being optionally substituted C _1-6 An alkyl group; and is

X ¹ Selected from the group consisting of: o, CO, S (O) ₂ P (O) (OR') and NH; and is

Wherein R' is selected from H, optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl, preferably H.

In some preferred embodiments, R ¹¹ Each independently selected from: h; optionally substituted C _1-6 Alkyl, preferably optionally substituted C _1-4 An alkyl group; -COOH; -COOC _1-4 An alkyl group; -CH (R) ¹⁵ )NH-CO-C _1-4 An alkyl group; a phenyl group; and 5 to 7 membered heteroaryl.

In some preferred embodiments, R ¹² Selected from: h; optionally substituted C _1-10 Alkyl, more preferably optionally substituted C _4-8 An alkyl group; and optionally substituted C _2-10 Alkenyl, more preferably optionally substituted C _4-8 Alkenyl, more preferably-CH = CH (CH) ₂ ) _i CH ₃ Wherein i is 1,2, 3 or 4.

In some preferred embodiments, u is 1 or 2.

In some preferred embodiments, R ¹⁴ And R ¹⁵ Each independently is H or C _1-4 Alkyl, preferably H, methyl or ethyl; more preferably H or methyl.

In some preferred embodiments, R ¹³ Selected from: optionally substituted C _1-4 Alkyl, optionally substituted C _2-4 An alkenyl group,

In some preferred embodiments, R ^13’ Selected from: optionally substituted C _1-4 An alkyl group; optionally substituted C _2-4 An alkenyl group;

more preferably

Wherein R is ¹⁵ Is C _1-4 Alkyl, preferably methyl; or

In some preferred embodiments, R ¹⁶ Selected from H and optionally substituted C _1-6 Alkyl, more preferably optionally substituted C _1-4 An alkyl group.

In some preferred embodiments, R ² Selected from: direct bond, O, optionally substituted C _2-5 Alkyl (preferably-CH) ₂ -CH ₂ -or- (CH) ₂ ) ₅ -)、-CH ₂ -CH＝CH(CH ₂ ) ₃ -、-(CH ₂ CH ₂ O) ₂ -CH ₂ -、-CH ₂ Cyclohexane-, -O- (optionally substituted C _1-4 Alkyl) -, -NH- (optionally substituted C _1-4 Alkyl) -, -O- (optionally substituted C _2-4 Alkenyl) -, -NH- (optionally substituted C _2-4 Alkenyl) -, optionally substituted with (R) ^b ) _q Substituted phenyl, optionally substituted (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-4 Alkyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-4 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-4 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -; wherein, preferably, C is _1-4 Alkyl is-CH ₂ -CH ₂ -。

In some preferred embodiments, R ^b Each independently selected from H, hydroxyl, nitro, C _1-4 Alkyl radical, C _1-4 Alkoxy radical, C _1-4 Alkylthio and halogen, preferably H or nitro.

In some preferred embodiments, R ¹ And R ² Together being optionally substituted C _1-4 An alkyl group.

In some preferred embodiments, R' is selected from H, optionally substituted C _1-4 Alkyl and optionally substituted C _2-4 Alkenyl, preferably H.

In some embodiments, the present application provides the modified albumin described above, wherein X is ¹ And Z (when M = 1) or M (when M = 0) by one selected from: -O-; -CO-NH-; -NH-CO-; -CO-O-; -O-CO-; -S (O) ₂ -NH-；-NH-S(O) ₂ -；-S(O) ₂ -O-；-O-S(O) ₂ -; -P (O) (OR ") -NH-; -NH-P (O) (OR ") -; -P (O) (OR ") -O-; and-O-P (O) (OR') -.

In some embodiments, the present application provides a modified albumin as described above, wherein Z (when M = 1) is linked to M by one selected from the group consisting of: -O-; -CO-NH-; -NH-CO-; -CO-O-; -O-CO-; -S (O) ₂ -NH-；-NH-S(O) ₂ -；-S(O) ₂ -O-；-O-S(O) ₂ -; -P (O) (OR ") -NH-; -NH-P (O) (OR ") -; -P (O) (OR ") -O-; and-O-P (O) (OR') -.

In some embodiments, the present application provides a modified albumin as described above, wherein:

R ¹ is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) -, - (optionally substituted C _1-6 Alkyl) -C _3-6 Cycloalkyl-, -O- (optionally substituted C) _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; and is provided with

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH.

In some preferred embodiments, R ² Selected from: optionally substituted C _2-5 Alkyl, optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-4 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-4 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-4 Alkyl) -, -NH- (optionally substituted C _1-4 Alkyl) -, -O- (optionally substituted C _2-4 Alkenyl) -, -NH- (optionally substituted C _2-4 Alkenyl) -;

wherein, the C _1-4 Alkyl is preferably-CH ₂ -CH ₂ -。

More preferably, R ² Is selected from C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -。

In some preferred embodiments, X ¹ Is CO or NH.

In some embodiments, the present application provides the modified albumin described above, wherein

R ¹ Selected from the group consisting of optionally substituted:

R ¹¹ each independently selected from: H. optionally substituted C _1-6 Alkyl, -COOH, -COOC _1-6 Alkyl radical, C _6-10 Aryl and 5 to 10 membered heteroaryl;

R ² selected from the group consisting of: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl, -O- (optionally substituted C) _1-6 Alkyl) -and-NH- (optionally substituted C) _1-6 Alkyl) -;

X ¹ selected from: CO, S (O) ₂ P (O) (OR') and NH.

In some preferred embodiments, R ¹¹ Each independently selected from: H. optionally substituted C _1-4 Alkyl, -COOH, -COOC _1-4 Alkyl, phenyl and 5 to 7 membered heteroaryl.

In some preferred embodiments, R ² Selected from: optionally substituted C _1-4 Alkyl, optionally substituted C _2-4 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -, -O- (optionally substituted C) _1-4 Alkyl) -and-NH- (optionally substituted C _1-4 Alkyl) -.

In some preferred embodiments, X ¹ Is CO or NH.

R ¹ Is the following optionally substituted group:

R ¹² is optionally substituted C _1-12 Alkyl or optionally substituted C _2-12 An alkenyl group;

R ² selected from optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 An alkenyl group;

X ¹ selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO.

In some preferred embodiments, R ¹² Is optionally substituted C _1-10 Alkyl, more preferably C _4-8 An alkyl group; or optionally substituted C _2-10 Alkenyl, preferably C _6-8 Alkenyl, more preferably-CH = CH (CH) ₂ ) _i CH ₃ Wherein i is 1,2, 3 or 4.

In some preferred embodiments, R ² Selected from optionally substituted C _1-4 Alkyl and optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -)。

In some preferred embodiments, X ¹ Is CO.

R ¹ Selected from the group consisting of optionally substituted:

R ² selected from optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 An alkenyl group; and is provided with

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH.

In some preferred embodiments, R ² Selected from optionally substituted C _1-4 Alkyl and optionally substituted C _2-6 Alkenyl (for example)-CH ₂ -CH＝CH(CH ₂ ) ₃ -)。

In some preferred embodiments, X ¹ Is CO.

R ¹ Selected from the group consisting of optionally substituted:

R ¹³ selected from the group consisting of:

R ^13’ selected from:

R ² selected from the group consisting of: optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 An alkenyl group;

X ¹ selected from: o, CO, S (O) ₂ P (O) (OR') and NH; and is

u and R ¹⁵ Each as defined above.

In a preferred embodiment, R ¹³ Is composed of

In a preferred embodiment, R ^13’ Is composed of

In the preferred practiceIn the scheme, R ² Selected from the group consisting of: optionally substituted C _1-4 Alkyl and optionally substituted C _2-4 An alkenyl group.

In a preferred embodiment, X ¹ Is CO.

R ¹ In the form of S, the content of the S,

R ² selected from the group consisting of: optionally substituted (R) ^b ) _q Substituted phenyl, optionally substituted with (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -;

X ¹ selected from: o, CO, S (O) ₂ P (O) (OR ") and NH, preferably CO OR NH; and is

q and R ^b Each as defined above.

R ¹ and R ² Together being optionally substituted C _1-4 Alkyl, preferably methyl; and is provided with

X ¹ Selected from: CO, S (O) ₂ And P (O) (OR "), preferably CO.

Hydrophobic groups

Albumin is a natural water-soluble macromolecule with a large number of free amino and carboxyl groups available for hydrophobic modifications. The application only selects the free sulfydryl of albumin as a modification site and introduces a hydrophobic group. The albumin modified in the way can be self-assembled into nanoparticles in an aqueous medium, the affinity of the albumin to water-insoluble compounds is increased, and the encapsulation effect is good.

The hydrophobic group M in the modified albumin of the present application may be derived from aliphatic hydrocarbons (e.g. octyl, dodecyl, hexadecyl, octadecyl), aliphatic alcohols (e.g. stearyl alcohol, sphingosine), aliphatic aldehydes (e.g. palmitaldehyde, hexadecenal), fatty acids (e.g. caprylic acid, stearic acid, dodecanoic acid, hexadecanoic acid, oleic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid), fatty acid esters (e.g. fatty acid glycerides), ceramides, phospholipids (e.g. 1, 2-heptacosanoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1, 2-phytanoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (sodium salt) and phosphatidylinositol), sterols (e.g. cholesterol, desmosterol, cholestanol, sitosterol, dihydrositostanol, glycosylated sterols, phytosterols, stigmasterol, campesterol), bile acid compounds (e.g. cholic acid, deoxycholic acid, hyocholic acid and lithocholic acid) and derivatives thereof.

In some embodiments, the present application provides a modified albumin as described above, wherein M is selected from the group consisting of:

(1) C terminal L- (Z) _m Linked, N-terminal by-CO-R ³ or-S (O) ₂ -R ³ A substituted amino acid;

(2) N terminal and L- (Z) _m To which is attached a-OH group of the C-terminal carboxyl group by-NH-R ³ or-O-R ³ A substituted amino acid;

(3) A group having a structure represented by general formula (IV):

-X ⁴ -R ⁴

(IV)

wherein

X ⁴ Selected from the group consisting of: direct bond, O, CO, S (O) ₂ P (O) (OR' ") and NH;

r' "is selected from: h and R ⁴¹ ；

R ³ And R ⁴ Each independently selected from:

1)R ⁴¹ and by R ^41’ -O-、R ^41’ -C (O) O-or R ^41’ -C (O) NH-substituted R ⁴¹ (ii) a And

2)

R ⁴¹ 、R ^41’ 、R ⁴² and R ⁴³ Each independently selected from:

C _1-30 an alkyl group;

C _2-30 an alkenyl group; and

C _2-30 an alkynyl group; and

3) Optionally substituted groups derived from sterols and bile acids.

In some preferred embodiments, R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from:

C _5-25 alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 Alkyl radicals, e.g. C ₅ Alkyl radical, C ₇ Alkyl radical, C ₁₁ Alkyl or C ₁₅ An alkyl group;

C _5-25 alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl radical, C _7-15 Alkenyl radicals, e.g. C ₅ Alkenyl radical, C ₇ Alkenyl radical, C ₁₁ Alkenyl radical, C ₁₅ Alkenyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl or C ₂₁ An alkenyl group; and

C _5-25 alkynyl, preferably C _7-23 Alkynyl, C _9-21 Alkynyl, C _11-19 Alkynyl, C _13-17 Alkynyl or C _7-15 Alkynyl, e.g. C ₅ Alkynyl, C ₇ Alkynyl, C ₁₁ Alkynyl, C ₁₅ Alkynyl, C ₁₇ Alkynyl, C ₁₉ Alkynyl or C ₂₁ Alkynyl.

In some preferred embodiments, the optionally substituted group derived from sterols and bile acids is:

more preferably

In some preferred embodiments, R ³ Each independently selected from: c ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ An alkenyl group;

preferably, the following components are used: n-octyl, n-pentadecyl,

In some preferred embodiments, X ⁴ Is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

R ⁴ selected from: r ⁴¹ And by R ^41’ -C (O) O-and R ^41’ -C (O) NH-substituted R ⁴¹ ；

R ⁴¹ And R ^41’ Each independently selected from: c ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ An alkenyl group;

preferably: n-octyl, n-pentadecyl,

In some preferred embodiments, X ⁴ Is O or NH;

R ⁴ is composed of

Preference is given to

In some preferred embodiments, R ⁴² And R ⁴³ Each independently selected from:

C ₇ alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ An alkenyl group;

preferably: n-octyl, n-pentadecyl,

In some preferred embodiments, X ⁴ Is CO, S (O) ₂ OR P (O) (OR' "), preferably CO; and is

R ⁴ Is optionally substituted

Preferably a

In some preferred embodiments, X ⁴ Is O; and is provided with

R ⁴ Is optionally substituted

Preferably a

wherein:

R ³ as defined above;

R ^3’ selected from: h; optionally substituted C _1-6 An alkyl group; is selected from SH, SCH ₃ 、NH ₂ 、OH、COOH、-CONH ₂ 、-NHCOCH ₃ 、-NHCHO、-NHCONH ₂ Guanidino, optionally substituted C _6-10 C substituted with 1 or more substituents of aryl and optionally substituted 5-to 10-membered heteroaryl _1-6 An alkyl group; c _3-10 A cycloalkyl group; and C _6-10 And (3) an aryl group.

In such embodiments, the C is _6-10 Aryl is selected from phenyl, 4-OH-phenyl, biphenyl and naphthyl; the 5-to 10-membered heteroaryl is selected from imidazole, indole.

In some preferred embodiments, R ^3’ Selected from H, C _1-4 Alkyl (e.g. CH) ₃ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ ) ₂ ) Phenyl, cyclohexyl, benzyl, p-hydroxybenzyl, phenethyl, p-hydroxyphenylethyl, -CH ₂ OH、-CH(OH)CH ₃ 、-CH ₂ CH ₂ SCH ₃ 、-CH ₂ CONH ₂ 、-CH ₂ COOH、-CH ₂ CH ₂ CONH ₂ 、-CH ₂ CH ₂ COOH、-(CH ₂ ) ₃ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₃ NH ₂ 、-(CH ₂ ) ₃ NHCOCH ₃ 、-(CH ₂ ) ₃ NHCHO、-(CH ₂ ) ₄ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₄ NHCOCH ₃ 、-(CH ₂ ) ₄ NHCHO、-(CH ₂ ) ₃ NHCONH ₂ 、-(CH ₂ ) ₄ NHCONH ₂ 、-CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ Pyridin-2-ylmethyl-, pyridin-3-ylmethyl-, pyridin-4-ylmethyl-, (I) and (II),

Spacer base

The modified albumin of the present application optionally comprises a divalent spacer Z linking the linker L to the hydrophobic group M.

In some embodiments, the present application provides the modified albumin described above, wherein Z may be represented by the general formula (V):

-{X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -[NH-X ⁵ -(CH ₂ ) _g ] _h -X ³ }-

(V)

wherein:

when m is 1, X ² With L in the general formula (II) or X in the general formula (III) ¹ Is connected to, and X ³ With M in the formula (II) or X in the formula (IV) ⁴ Connecting;

X ² selected from: direct bond, O, CO, S (O) ₂ P (O) (OR') and NH;

X ³ selected from: direct bond, O, CO, S (O) ₂ P (O) (OR' ") and NH;

X ⁵ selected from: CO and S (O) ₂ ；

g is 1,2, 3,4, 5 or 6;

h is 0 or 1;

r is 0,1, 2,3,4, 5 or 6;

t is 0,1, 2,3,4, 5 or 6;

s is an integer from 0 to 100;

r "and R'" are each as defined above.

Preferably, g is 1,2 or 3, more preferably 2; preferably, h is 0, or, h is preferably 1; preferably, r is 1,2 or 3, more preferably 2; preferably, t is 0,1, 2 or 3, more preferably 0; preferably, s is an integer from 0 to 10, preferably 0,1, 2,3,4, 5 or 6, more preferably 0,1 or 2.

In some preferred embodiments, the present application provides a modified albumin as described above, wherein Z may be represented by the general formula (V-i):

-[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ]-

(V-i)。

in some such embodiments, preferably, X ² Selected from: o, CO, S (O) ₂ P (O) (OR ") and NH, preferably CO OR NH; and r is 1,2 or 3, more preferably 2. In these embodiments, more preferably, X ¹ Is CO, S (O) ₂ OR P (O) (OR') and X ² Is O or NH; or X ¹ Is O or NH, and X ² Is CO, S (O) ₂ OR P (O) (OR ").

In other such embodiments, preferably, X ² Is a direct bond; and r is 0. In these embodiments, more preferably, X ¹ Is CO, S (O) ₂ OR P (O) (OR ").

In yet other such embodiments, preferably, X ³ Selected from: o, CO, S (O) ₂ P (O) (OR' "), and NH, preferably CO OR NH. In these embodiments, more preferably, X ³ Is O or NH, and X ⁴ Is CO, S (O) ₂ OR P (O) (OR' "); or X ³ Is CO, S (O) ₂ OR P (O) (OR' "), and X ⁴ Is a direct bond, O or NH.

In some embodiments described above, - [ X ] X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ]-preferably selected from: -O-CH ₂ CH ₂ -CO-、-O-(CH ₂ CH ₂ ) ₂ -CO-、-CO-CH ₂ CH ₂ -O-、-O-CH ₂ CH ₂ -O-、-CO-CH ₂ CH ₂ -CO-、-NH-CH ₂ CH ₂ -NH-、-CO-CH ₂ CH ₂ -NH-、-NH-CH ₂ CH ₂ -CO-、-O-CH ₂ CH ₂ -S(O) ₂ -、-S(O) ₂ -CH ₂ CH ₂ -O-、-NH-CH ₂ CH ₂ -O-、-O-CH ₂ CH ₂ -NH-、-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-、-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -CO-、-CO-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-、-O-CH ₂ CH ₂ -P(O)(OR”’)-、-O-(CH ₂ CH ₂ ) ₂ -P(O)(OR”’)-、 -CO-CH ₂ CH ₂ -P(O)(OR”’)-、-NH-CH ₂ CH ₂ -P (O) (OR' ") -and-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -P(O)(OR”’)-。

In some embodiments, the present application provides the modified albumin described above, wherein the modified albumin has a structure represented by the following general formula (VI):

wherein:

Q、M、m、R ¹ 、R ² 、X ¹ 、X ² 、X ³ r, s and t are each as defined above.

In some embodiments, the present application provides the modified albumin described above, wherein the modified albumin has a structure represented by the following general formula (VII):

wherein:

Q、m、R ¹ 、R ² 、X ¹ 、X ² 、X ³ 、r、s、t、R ⁴ and X ⁴ Each as defined above.

In some further embodiments, the present application provides the modified albumin described above, wherein

R ¹ Is composed of

R ² Selected from the group consisting of: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl or optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -；

X ¹ Is O, CO, S (O) ₂ P (O) (OR') OR NH;

m is 0;

or

m is 1, and

X ² is CO, S (O) ₂ P (O) (OR') OR NH, preferably CO OR NH, and

X ³ is CO, S (O) ₂ P (O) (OR' ") OR NH, preferably CO OR NH;

R ⁴ selected from:

1)R ⁴¹ and by R ^41’ -O-、R ^41’ -C (O) O-or R ^41’ -C (O) NH-substituted R ⁴¹ ；

2)

Preference is given to

Wherein R is ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from: c _5-25 Alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl radical, C _7-15 An alkyl group; and C _5-25 Alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl radical, C _7-15 An alkenyl group; and

3) Optionally substituted group selected from:

preferably, it is

In some of the above-described embodiments, R ² Preferably selected from: optionally substituted C _2-5 Alkyl and optionally substituted C _2-4 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-4 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-4 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-4 Alkyl) -, -NH- (optionally substituted C _1-4 Alkyl) -, -O- (optionally substituted C _2-4 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-4 Alkyl or optionally substituted C _2-4 An alkenyl group;

wherein, the C _1-4 The alkyl group is preferably-CH ₂ -CH ₂ -。

In some of the above-described embodiments, R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Preferably each independently selected from C ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

In some of the above-described embodiments, preferably, m is1，X ² Is CO or NH, and X ³ Is CO or NH.

In some of the above-described embodiments, R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Preferably each is independently selected from: c _7-23 Alkyl, preferably C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 An alkyl group; and C _7-23 Alkenyl, preferably C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl or C _7-15 An alkenyl group.

In some of the above-described embodiments, R ⁴ Preferably optionally substituted

In some embodiments, the present application provides the modified albumin described above, wherein m is 1. In some such embodiments, preferably r is 2; s is 2; and t is 0; or r is 2; s is 0; and t is 0. In some such embodiments, preferably, X ⁴ Is a direct bond, O, CO or NH.

In other preferred embodiments, the present application provides a modified albumin as described above, wherein Z is represented by the general formula (V-ii):

-[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ]-

(V-ii)。

in some such embodiments, preferably, X ² Selected from the group consisting of: o, CO, S (O) ₂ P (O) (OR') and NH,preferably NH; and r is 1,2 or 3, more preferably 2. In these embodiments, more preferably, X ¹ Is CO, S (O) ₂ OR P (O) (OR') and X ² Is NH.

In some of the above-described embodiments, s is preferably 1,2, 3,4, 5 or 6, more preferably 2.

In some of the above-described embodiments, X ³ Preferably selected from: CO, S (O) ₂ P (O) (OR' "), and NH, preferably CO. In these embodiments, more preferably, X ³ Is CO, and X ⁴ Is O or NH, preferably O.

In some of the above-described embodiments, X ⁵ Preferably CO.

In some of the embodiments described above, - [ X [ ] ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ]-preferably selected from:

-NH-CH ₂ CH ₂ -OCH ₂ CH ₂ -NH-CO-CH ₂ -CO-，

-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ -CO-，

-NH-CH ₂ CH ₂ -OCH ₂ CH ₂ -NH-CO-CH ₂ CH ₂ -CO-, and

-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ CH ₂ -CO-；

preferably-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ CH ₂ -CO-。

In some embodiments, the present application provides the modified albumin described above, wherein the modified albumin has a structure represented by the following general formula (VI'):

wherein:

Q、M、m、R ¹ 、R ² 、X ¹ 、X ² 、X ³ 、X ⁵ g, r and s are each as defined above.

In some embodiments, the present application provides the modified albumin described above, wherein the modified albumin has a structure represented by the following general formula (VII'):

wherein:

Q、m、R ¹ 、R ² 、R ⁴ 、X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ g, r and s are each as defined above.

In some preferred embodiments, m is 1.

R ¹ Is composed of

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ² is NH;

X ³ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

X ⁴ is O or NH;

R ⁴ selected from the group consisting of:

2)

Preference is given to

Wherein R is ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from: c _5-25 Alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 An alkyl group; and C _5-25 Alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl or C _7-15 An alkenyl group; and

3) Optionally substituted group selected from:

preferably, it is

In some preferred embodiments, R ² Selected from: optionally substituted C _2-5 Alkyl and optionally substituted C _2-4 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-4 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-4 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-4 Alkyl) -, -NH- (optionally substituted C _1-4 Alkyl) -, -O- (optionally substituted C _2-4 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-4 Alkyl or optionally substituted C _2-4 An alkenyl group;

wherein, the C _1-4 The alkyl group is preferably-CH ₂ -CH ₂ -。

In some preferred embodiments, R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from C ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

R ² Is optionally substituted C _1-6 Alkyl, preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -；

X ¹ Is CO;

X ² is NH;

X ³ is CO;

X ⁴ is O;

X ⁵ is CO;

R ⁴ is composed of

And is

R ⁴² And R ⁴³ Each independently selected from: c _5-25 Alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 An alkyl group.

One skilled in the art will appreciate that some linkers for antibody-drug conjugates (ADCs) may be used as spacers for this application. The ADC linker that may be used as a spacer in the present application may be selected from:

(1) A peptide, wherein when m is 1, the N-terminus of the peptide is linked to L in formula (II) or X in formula (III) ¹ And the C-terminus of the peptide is linked to M in formula (II) or X in formula (IV) ⁴ Connecting; and

(2) A peptide-p-aminobenzyloxycarbonyl (PAB) linker represented by general formula (VIII);

wherein: when m is 1, the N-terminus of the peptide is linked to L in formula (II) or X in formula (III) ¹ The peptide is linked to N (R) in the general formula (VIII) via the C-terminal CO ^Z1 ) Connecting;

preferably, the peptides described in items (1) and (2) above are formed from 2 to 20 (e.g. 2 to 15, 2 to 10, 2 to 5 or 3 to 4) identical or different amino acids;

more preferably, the peptide is a dipeptide represented by the general formula (IX):

(3) A moiety comprising the structure of item (1) or (2) above;

preferably, the moiety additionally comprises an oligosaccharide, - (CH) ₂ ) _n -and/or- (CH) ₂ CH ₂ O) _n -；

Preferably, the moiety may be selected from:

(4) A moiety selected from:

wherein: when m is 1, the bond designated by z is bonded to L in the formula (II) or X in the formula (III) ¹ Linking, the bond designated by v to M in formula (II) or X in formula (IV) ⁴ Connecting;

wherein:

each R ^Z1 Independently selected from: H. optionally substituted C _1-12 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-3 Alkyl groups such as methyl);

each R ^Z2 Independently selected from: h; c _1-6 Alkyl (preferably C) _1-4 Alkyl); is selected from SH, SCH ₃ 、NH ₂ 、OH、COOH、CONH ₂ Guanidino, optionally substituted C _6-10 Substituted by 1 or more substituents of aryl (preferably phenyl, 4-OH-phenyl, biphenyl) and optionally substituted 5-to 10-membered heteroaryl (preferably imidazole, indole)C _1-6 An alkyl group; c _3-10 Cycloalkyl (preferably cyclohexyl); and C _6-10 Aryl (preferably phenyl);

preferably, each R ^Z2 Independently selected from: H. CH (CH) ₃ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ ) ₂ Phenyl, cyclohexyl, benzyl, p-hydroxybenzyl, phenethyl, p-hydroxyphenylethyl, pyridin-2-ylmethyl-, pyridin-3-ylmethyl-, pyridin-4-ylmethyl-, -CH ₂ OH、-CH(OH)CH ₃ 、-CH ₂ CH ₂ SCH ₃ 、-CH ₂ CONH ₂ 、-CH ₂ COOH、-CH ₂ CH ₂ CONH ₂ 、-CH ₂ CH ₂ COOH、-(CH ₂ ) ₃ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₃ NH ₂ 、-(CH ₂ ) ₃ NHCOCH ₃ 、-(CH ₂ ) ₃ NHCHO、-(CH ₂ ) ₄ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₄ NHCOCH ₃ 、-(CH ₂ ) ₄ NHCHO、-(CH ₂ ) ₃ NHCONH ₂ 、-(CH ₂ ) ₄ NHCONH ₂ 、-CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ 、

n is an integer selected from 1 to 10, such as 1,2, 3,4, 5, 6 or 7.

In some preferred embodiments, Z may be selected from:

Val-Leu-Lys、Gly-Gly-Arg、

Val-Lys(Ac)-PAB、Phe-Lys-PAB、Phe-Lys(Ac)-PAB、

thus, in some embodiments, the present application provides modified albumins wherein Z is as described above. In some preferred such embodiments, m is 1 and X ⁴ Is a direct bond, O or NH.

In some preferred such embodiments, the present application provides modified albumins, wherein

R ¹ Is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (C)CH ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl or optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -；

X ¹ Is O, NH, CO, S (O) ₂ OR P (O) (OR ");

R ⁴ selected from the group consisting of:

2)

Preference is given to

Wherein R is ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from: c _5-25 Alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl radical, C _7-15 An alkyl group; and C _5-25 Alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical、C _13-17 Alkenyl radical, C _7-15 An alkenyl group; and

3) Optionally substituted group selected from:

preferably, it is

In some further preferred embodiments, R ² Selected from: optionally substituted C _2-5 Alkyl and optionally substituted C _2-4 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-4 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-4 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-4 Alkyl) -, -NH- (optionally substituted C _1-4 Alkyl) -, -O- (optionally substituted C _2-4 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-4 Alkyl or optionally substituted C _2-4 An alkenyl group;

wherein, the C _1-4 The alkyl group is preferably-CH ₂ -CH ₂ -。

In some preferred embodiments, R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from C ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ An alkenyl group,C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

In some embodiments, the present application provides the modified albumin described above, wherein m is 0.

In some preferred such embodiments, X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO, and X ⁴ Is O or NH; or

X ¹ Is O or NH, preferably NH, and X ⁴ Is CO, S (O) ₂ OR P (O) (OR' "), preferably CO.

In some further preferred such embodiments, R ⁴ Selected from: r ⁴¹ And by R ^41’ -O-、R ^41’ -C (O) O-and R ^41’ -C (O) NH-substituted R ⁴¹ 。

In yet other further preferred such embodiments, X ¹ Is O or NH, preferably NH;

X ⁴ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO; and is

R ⁴ Selected from optionally substituted groups selected from:

preferably, it is

In yet further preferred such embodiments, X ¹ Is CO, S (O) ₂ Or P (O)) (OR "), preferably CO;

X ⁴ is O; and is

R ⁴ Selected from the group consisting of optionally substituted:

preferably, it is

In some embodiments, the present application provides a modified albumin represented by general formula (I):

wherein:

q is the S atom in the free thiol group (e.g., the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) of HSA or recombinant human albumin (rHA);

is the remainder of the HSA or rHA;

c is:

and

compounds as modifiers

In a second aspect, the present application provides a compound of general formula (X) for use as a modifying agent for the preparation of a modified albumin as described above:

R-(Z) _m -M

(X)

wherein:

r is a moiety containing a reactive group capable of linking a compound of formula (X) to S in a free thiol group of albumin by reaction with said free thiol group (e.g. the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) and providing a hydrophobicity-enhancing moiety of formula (II) as described above;

z is a spacer, as defined above;

m is as defined above; and is provided with

M is a hydrophobic group, as defined above.

It will be appreciated by those skilled in the art that the linker L in the hydrophobicity-enhancing moiety of formula (II) results from the reaction of the free thiol group of the albumin with the reactive group-containing moiety R of the conjugation modifier of formula (X).

In some embodiments, the reaction between the reactive group and the free thiol group is selected from: michael addition reactions, thiol-disulfide exchange reactions, substitution reactions, and click chemistry.

In some preferred embodiments, the reactive group is selected from michael reaction acceptors and groups containing disulfide bonds.

In some more preferred embodiments, R is represented by formula (XI):

R ^1’ -R ² -X ¹ -

(XI)

wherein

R ^1’ Is a reactive group selected from the group consisting of optionally substituted:

and

preference is given to

Wherein:

selected from the group consisting of: 6-to 10-membered aryl and 5-to 6-membered heteroaryl;

x is CH or N;

or alternatively

R ^1’ Is halogen, such as F, cl, br or I;

R ¹¹ 、R ¹² 、R ¹³ 、R ^13’ 、R ¹⁴ 、R ¹⁶ 、R ^a 、R ^b 、R ² 、X ¹ p and q are each as defined above.

Preferably, R ^1’ Is composed of

R ¹¹ Each independently selected from: H. optionally substituted C _1-6 Alkyl, -COOH, -COOC _1-6 Alkyl radical, C _6-10 Aryl (e.g., phenyl) and 5 to 10 membered heteroaryl;

R ² selected from: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -, -O- (optionally substituted C) _1-6 Alkyl) -and-NH- (optionally substituted C _1-6 Alkyl) -;

X ¹ selected from the group consisting of: CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH.

Preferably, R ^1’ Is composed of

And R is ¹² 、R ² And X ¹ Each as defined above.

Preferably, R ^1’ Selected from:

R ² selected from optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -) according to the formula (I); and is

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO.

Preferably, R ^1’ Is composed of

And R is ¹³ 、R ¹³ 、R ² And X ¹ Each as defined above.

Preferably, R ^1’ Is composed of

R ² Selected from: optionally substituted (R) ^b ) _q Substituted phenyl, optionally substituted (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -; x ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH.

Preferably, R ^1’ Is halogen, such as F, cl, br or I; r ² Is optionally substituted C _1-6 Alkyl, preferably optionally substituted C _1-4 Alkyl, more preferably methyl; and X ¹ Selected from CO, S (O) ₂ And P (O) (OR "), preferably CO.

More preferably, R ^1’ Is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -; and is provided with

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH.

In some preferred embodiments, the present application provides the modifier described above, wherein the modifier is represented by the general formula (XII):

R ^1’ -R ² -X ¹ -[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ] _m -M

(XII)；

wherein:

R ^1’ 、R ² 、X ¹ 、X ² 、X ³ r, s, t, M and M are each as defined above.

In such embodiments, more preferably, the modifying agent is represented by formula (XIII):

R ^1’ -R ² -X ¹ -[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ] _m -X ⁴ -R ⁴

(XIII)；

wherein:

R ^1’ 、R ² 、X ¹ 、X ² 、X ³ 、r、s、t、X ⁴ 、R ⁴ and m are each as defined above.

In such embodiments, the present application provides further preferred modifiers, wherein

R ^1’ Is composed of

And is provided with

R ² 、X ¹ 、X ² 、X ³ 、R ⁴ And m are each as defined above.

Preferably, m is 1. In some preferred such embodiments, r is 2; s is 2; and t is 0; or r is 2; s is 0; and t is 0. In some further preferred such embodiments, X is ⁴ Is a direct bond, O, CO or NH.

In other preferred embodiments, the present application provides the modifying agent described above, wherein the modifying agent is represented by formula (XIV):

R ^1’ -R ² -X ¹ -[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ] _m -M

(XIV)

wherein:

R ^1’ 、R ² 、X ¹ 、X ² 、X ³ 、X ⁵ g, r, s, M and M are each as defined above.

In such embodiments, more preferably, the modifying agent is represented by general formula (XV):

R ^1’ -R ² -X ¹ -[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ] _m -X ⁴ -R ⁴

(XV)

wherein:

R ^1’ 、R ² 、R ⁴ 、X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ g, r, s and m are each as defined above.

In some of the above-described embodiments, m is preferably 1.

Preferably, in some of the above-described embodiments,

R ^1’ is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably renOptionally substituted C _1-6 Alkyl or optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -；

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ² is NH;

X ³ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

X ⁴ is O or NH;

R ⁴ selected from:

2)

Preference is given to

3) Optionally substituted group selected from:

preferably, it is

More preferably, in some embodiments,

X ¹ Is CO;

X ² is NH;

X ³ is CO;

X ⁴ is O;

X ⁵ is CO;

R ⁴ is composed of

And is

In other preferred embodiments, the present application provides the modifying agent described above, wherein:

R ^1’ is composed of

Z is an ADC linker as defined above that can be used as a spacer in the present application; and is

R ² 、X ¹ 、X ⁴ 、R ⁴ Each as defined above;

m is as defined above, preferably 1.

In still other preferred embodiments, the present application provides the modifying agent described above, wherein m is 0.

X ⁴ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO; and is

R ⁴ Selected from optionally substituted groups selected from:

preferably a

In yet further preferred such embodiments, X is ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ⁴ is O; and is

R ⁴ Selected from the group consisting of optionally substitutedThe following groups of (a):

preferably a

In some embodiments, the present application provides the modifying agent described above selected from the following table:

preparation method

In a third aspect, the present application provides a method of preparing a modified albumin as described above, the method comprising contacting the albumin with a modifying agent as described above, at a position which allows for the free thiol group of the albumin (e.g. the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) to react with R or R ^1’ And (3) reacting under the reaction condition. Preferably, the reaction is selected from the group consisting of michael addition reactions, thiol-disulfide exchange reactions and substitution reactions.

In some embodiments, the method comprises reacting a free thiol group of the albumin (e.g., the thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1) with a thiol group of the albumin

(1) A compound of the formula (X)

R-(Z) _m -M

(X)

Wherein

R is represented by the general formula (XI):

R ^1’ -R ² -X ¹ -

(XI)

(2) A compound of the general formula (XII):

(XII); or

(3) A compound of formula (XIII):

(XIII)

the following reactions were performed to produce the modified albumins of the present application:

(a) Michael addition reaction, with the conditions: r ^1’ Is the following optionally substituted group:

preferably, it is

Wherein Z, M, R ² 、X ¹ 、X ² 、X ³ 、X ⁴ 、R ⁴ 、R ¹¹ 、R ¹² 、R ¹³ 、R ^13’ 、R ¹⁴ 、R ¹⁶ 、R ^a M, r, s, t and p are as defined above;

(b) Mercapto-disulfide bond exchange reaction with the proviso that R ^1’ Is composed of

Preference is given to

Wherein Z, M, R ² 、X ¹ 、X ² 、X ³ 、X ⁴ 、R ⁴ 、

X、R ^b M, r, s, t and q are as defined above;

or alternatively

(c) Substitution reaction with the proviso that R ^1’ Is halogen, such as F, cl, br or I; r ² Is optionally substituted C _1-6 Alkyl, preferably optionally substituted C _1-4 Alkyl, more preferably methyl; and X ¹ Selected from CO, S (O) ₂ And P (O) (OR "), preferably CO;

wherein Z, M, R ² 、X ¹ 、X ² 、X ³ 、X ⁴ 、R ⁴ M, r, s and t are asAs defined above.

For embodiments in which the modified albumin of the present application is prepared by a michael addition reaction, the method of the present application may comprise the steps of:

(1) Providing a solution of said albumin;

for example, HSA (or rHA) can be fed as a 20% aqueous solution diluted to a suitable concentration (e.g., about 31.25 mg/mL) with 10mM PB buffer (pH = 6.5). The resulting aqueous solution was used as the aqueous phase for the reaction.

(2) Providing a solution of the modifying agent;

for example, the modifier may be dissolved in a suitable solvent (e.g., ethanol, or a mixed solvent of ethanol and ethyl acetate) to prepare a solution having a suitable concentration (e.g., about 2.01 mg/mL) as an ethanol phase for the reaction.

(3) Adding the solution of the modifying agent of step (2) to the solution of albumin of step (1) to produce the modified albumin.

For example, a solution of the modifying agent can be added dropwise to a solution of albumin in a suitable volume ratio (e.g., albumin solution: modifying agent solution = about 1 to 12, preferably 3 to 5.

The present application also provides a process for the preparation of compounds of formula (X) for use as modifying agents.

In some embodiments, the compounds of formula (X) may be prepared by:

reaction scheme 1

Wherein:

r is R ^1’ -R ² -X ¹ -；

M is-X ⁴ -R ⁴ ；

m is 0 or 1;

R ^1’ as defined above, it is preferred

R ^a And p is as defined above, preferably p is 0;

R ² as defined above, preferably C _1-6 Alkyl, more preferably C _2-5 Alkyl radicals, e.g. -CH ₂ -CH ₂ -；

X ¹ Is NH;

X ² is CO;

X ³ as defined above, preferably CO;

r is as defined above, preferably 2;

t is as defined above, preferably 0;

s is as defined above, preferably 0;

R ⁴ as defined above, preferably:

X ⁴ is CO, O or NH;

preferably, X is 0 ⁴ Is CO and R ⁴ Comprises the following steps:

preferably, when m is 1, X ³ Is CO and X ⁴ Is O or NH:

in some preferred embodiments, the compound of formula In-2 is selected from:

the method comprises the step of subjecting an amine of formula In-1 to a condensation reaction with an acid of formula In-2 to provide a compound of general formula (X).

The condensation reaction may be carried out in a suitable organic solvent, which may be selected from: DMF, DMSO, acetonitrile, THF, NMP, and mixtures of two or more thereof. The condensation reaction may be carried out in the presence of a suitable condensing agent, which may be selected from: HBTU, HOBT/EDCI, DCC, and CDI. The condensation reaction may be carried out in the presence of a suitable organic base, which may be selected from: pyridine, triethylamine, diisopropylethylamine and the like. The condensation reaction may be carried out at a temperature of 20 to 40 ℃ for a suitable time, for example 4h or more. The resulting product may optionally be purified, for example by column chromatography or high performance liquid chromatography.

In some embodiments, the compound of formula (X) may be prepared by:

reaction scheme 2

Wherein:

r is R ^1’ -R ² -X ¹ -；

M is-X ⁴ -R ⁴ ；

m is 0;

R ^1’ as defined above, preferably

R ^a And p is as defined above, preferablyP is 0;

X ¹ Is CO;

R ⁴ as defined above, preferably is

More preferably

X ⁴ Is O;

the method comprises the following steps:

step 1: the acid of formula In-3 was reacted with oxalyl chloride of formula In-4 to provide the acid chloride of formula In-5.

The reaction may be carried out in a suitable organic solvent which may be selected from dichloromethane, chloroform, 1, 2-dichloroethane, THF, acetonitrile or mixtures of two or more thereof. The reaction may be carried out at a temperature of from 0 to 30 ℃ for a suitable time, for example 4h or more. In some embodiments, oxalyl chloride may be replaced with thionyl chloride.

Step 2: esterification of an acid chloride of formula In-5 with an alcohol of formula In-6 provides a conjugate of general formula (X).

The esterification reaction may be carried out in a suitable organic solvent which may be selected from DMF, dichloromethane, chloroform, 1, 2-dichloroethane, THF, DMSO, dioxane, NMP and mixtures of two or more thereof. The condensation reaction may be carried out at a temperature of 20 to 40 ℃ for a suitable time, for example for a period of 4 hours or more. The resulting product may optionally be purified, for example by column chromatography or high performance liquid chromatography.

In some embodiments, the compound of formula (X) may be prepared by:

reaction scheme 3

Wherein:

r is R ^1’ -R ² -X ¹ -；

M is-X ⁴ -R ⁴ ；

Z is- [ X ] ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ]-

m is 1;

R ^1’ as defined above, preferably

R ^a And p is as defined above, preferably p is 0;

X ¹ Is CO;

X ² is NH;

X ³ as defined above, preferably CO or NH;

r is as defined above, preferably 2;

t is as defined above, preferably 0;

s is as defined above, preferably 2;

R ⁴ as defined above, preferably:

X ⁴ as defined above, preferably CO, NH or O;

in some preferred embodiments, the compound of formula In-8 is selected from:

the method comprises the step of reacting an ester of formula In-7 with an amine of formula In-8 to provide a compound of general formula (X).

The reaction may be carried out in a suitable organic solvent which may be selected from dichloromethane, chloroform, 1, 2-dichloroethane, THF, DMF, DMSO, dioxane, NMP and mixtures of two or more thereof. The reaction may be carried out at a temperature of 20-40 ℃ for a suitable time, for example 4h or more. The resulting product may optionally be purified, for example by column chromatography or high performance liquid chromatography.

In some embodiments, the compounds of formula (X) may be prepared by:

reaction scheme 4

Wherein:

r is R ^1’ -R ² -X ¹ -；

M is-X ⁴ -R ⁴ ；

Z is- [ X ] ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ]-；

m is 1;

R ^1’ as defined above, preferably

R ^a And p is as defined above, preferably p is 0;

R ² as defined above, preferably is C _1-6 Alkyl, more preferably C _2-5 Alkyl radicals, e.g. -CH ₂ -CH ₂ -；

X ¹ As defined above, preferably CO;

X ² as defined above, preferably NH;

X ³ as defined above, preferably CO;

g is as defined above, preferably 2;

r is as defined above, preferably 2;

s is as defined above, preferably 2;

R ⁴ as defined above, preferably:

X ⁴ as defined above, preferably O or NH, more preferably O; and is

X ⁵ As defined above, preferably CO;

wherein the compound of formula In-9 may also be provided In the form of a salt, such as a hydrochloride salt;

in some preferred embodiments, the compound of formula In-9 is:

or a salt thereof, such as the hydrochloride;

in some preferred embodiments, the compound of formula In-10 is:

the method comprises the step of subjecting an amine of formula In-9 or a salt thereof to a condensation reaction with an acid of formula In-10 to provide a compound of general formula (X).

Composition and pharmaceutical or cosmetic composition comprising same

In a fourth aspect, the present application provides a composition comprising a modified albumin as described above and optionally unmodified albumin. The composition can be used as a carrier, preferably a carrier for poorly water soluble compounds, in particular poorly water soluble compounds with pharmaceutical or cosmetic activity. In other words, the composition may be considered a carrier composition.

In some embodiments, the carrier composition comprises the modified albumin and an unmodified albumin. In such embodiments, the modified albumin comprises from about 1% to about 99%, for example from about 5% to about 70%, more preferably from about 5% to about 20%, by weight of the sum of the weight of the unmodified albumin and the modified albumin.

Preferably, the carrier composition is in solid (e.g. lyophilized powder) or liquid form (e.g. aqueous solution).

a carrier encapsulating a poorly water soluble compound having pharmaceutical or cosmetic activity, said carrier being a modified albumin as described above or a carrier composition according to the fourth aspect above; and

optionally other pharmaceutically or cosmetically acceptable carriers.

In the present application, the term "drug" or "pharmaceutical composition" includes agents for the prevention or treatment of a disease or disorder as well as diagnostic agents for the diagnosis of a disease or disorder.

The choice of the pharmaceutically or cosmetically acceptable carrier depends on the dosage form of the pharmaceutical or cosmetic composition, firstly on the route of administration of the dosage form (for example dosage forms for oral, nasal, intradermal, subcutaneous, topical, intramuscular or intravenous administration), and secondly on the formulation of the dosage form. For example, the pharmaceutically or cosmetically acceptable carrier may include water (e.g., water for injection), buffers, isotonic salt solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ascorbic acid, lactic acid, ethanol, polyalkylene glycols such as polyethylene glycol (e.g., polyethylene glycol 4000) or polypropylene glycol, triglycerides, and the like.

In addition, the pharmaceutical or cosmetic composition of the present application may further comprise various additives such as a wetting agent, an emulsifier or a buffer, and the like, as necessary.

Preferably, the pharmaceutical or cosmetic composition is in the form of a solid, semi-solid or liquid, preferably a solution, suspension, emulsion or lyophilized powder, more preferably injectable.

(1) Providing a solution of the modified albumin described above or the carrier composition according to the fourth aspect above in an aqueous medium;

(3) Mixing the two solutions in the step (1) and the step (2);

(4) Removing the organic solvent.

In a seventh aspect, the present application provides the use of a modified albumin as described above or a carrier composition according to the fourth aspect above as a carrier for poorly water soluble compounds having pharmaceutical or cosmetic activity.

In an eighth aspect, the present application provides the use of a modified albumin as described above or a carrier composition according to the fourth aspect above as a carrier in the preparation of a pharmaceutical or cosmetic composition comprising a poorly water soluble compound having pharmaceutical or cosmetic activity,

preferably, the pharmaceutical or cosmetic composition is in the form of a solid, semisolid or liquid, preferably a solution, suspension, emulsion or lyophilized powder, more preferably injectable.

In a ninth aspect, the present application provides a kit comprising at least: i) Poorly water soluble compounds having pharmaceutical or cosmetic activity, ii) unmodified albumin, and iii) the modifying agents described above.

The kit may be used to prepare a pharmaceutical or cosmetic composition as described herein for administration in accordance with the methods described in the third and sixth aspects above in the vicinity of administration of the poorly water soluble compound having pharmaceutical or cosmetic activity.

The poorly water soluble compounds having pharmaceutical or cosmetic activity include (but are not limited to): antioxidants such as, but not limited to, flavonoids and polyphenols; antineoplastic agents such as, but not limited to, docetaxel, taxanes, platins, camptothecins, vincas, anthraquinones, nucleosides, podophyllotoxins, DNA topoisomerase inhibiting compounds; immunomodulators, such as cyclosporines; steroidal or non-steroidal anti-inflammatory agents; a cardiovascular agent; (ii) an antibiotic; antifungal agents; and antiviral agents. Examples that may be mentioned include, but are not limited to: curcumin, paclitaxel, docetaxel, cisplatin, hydroxycamptothecin, camptothecin, vincamide, teniposide, etoposide, methotrexate, cyclosporin a, rapamycin, nimodipine, doxorubicin, breviscapine, bilobalide, silymarin, daunorubicin, mitomycin, indomethacin, ibuprofen, ketoprofen, naproxen, nitroerylate, fenofibrate, itraconazole, amphotericin B, biphenyldicarboxylate, progesterone, dihydrotestosterone, haloperidol, and risperidone.

Detailed description of the preferred embodiments

The following embodiments are within the scope of the invention:

1. a modified albumin consisting of albumin and a hydrophobicity-enhancing moiety attached to the S atom in the free thiol group of the albumin, the modified albumin being represented by the general formula (I):

wherein:

q is an S atom in a free thiol group of the albumin;

is the remainder of the albumin;

wherein:

l is a linker connected to Q;

z is a spacer;

m is 1 or 0; and is

M is a hydrophobic group.

2. The modified albumin of embodiment 1, wherein L is represented by the general formula (III):

wherein:

R ¹ is S, or is selected from the group consisting of optionally substituted:

wherein:

R ¹¹ each independently selected from: H. optionally substituted C _1-12 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-4 Alkyl), -COOH, -COOC _1-6 Alkyl, -CH (R) ¹⁵ )NH-CO-C _1-6 Alkyl radical, C _6-10 Aryl (e.g., phenyl) and 5 to 10 membered heteroaryl;

R ¹² selected from: H. optionally substituted C _1-12 Alkyl (preferably C) _1-10 Alkyl, more preferably C _4-8 Alkyl) and optionally substituted C _2-12 Alkenyl (preferably C) _2-10 Alkenyl, more preferably C _4-8 Alkenyl, more preferably-CH = CH (CH) ₂ ) _i CH ₃ Wherein i is 1,2, 3 or 4);

(preferred)

)；

R ^13’ Selected from the group consisting of: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 An alkenyl group,

(preferred)

)，

Wherein:

u is 0,1, 2 or 3, preferably 1 or 2;

R ¹⁴ and R ¹⁵ Each independently is H or C _1-6 Alkyl, preferably H or C _1-4 Alkyl, more preferably H, methyl or ethyl; more preferably H or methyl;

preferably, R ¹³ Is composed of

And wherein R ¹⁵ Preferably C _1-4 Alkyl, more preferably methyl;

p is 0,1 or 2;

R ² selected from the group consisting of: direct bond, O, optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl), optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -)、-(CH ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -, optionally substituted by (R) ^b ) _q Substituted phenyl, optionally substituted (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -;

wherein:

j is an integer from 1 to 10, such as 2;

q is 0,1, 2 or 3;

R ^b each independently selected from H, hydroxyl, nitro and C _1-6 Alkyl radical, C _1-6 Alkoxy radicalBase, C _1-6 Alkylthio and halogen;

or alternatively

R ¹ And R ² Together being optionally substituted C _1-6 Alkyl (e.g. C) _1-4 Alkyl groups); and is

X ¹ Selected from the group consisting of: o, CO, S (O) ₂ P (O) (OR') and NH; and is provided with

3. The modified albumin of embodiment 2 wherein X ¹ And Z (when M = 1) or M (when M = 0) by one selected from: -O-; -CO-NH-; -NH-CO-; -CO-O-; -O-CO-; -S (O) ₂ -NH-；-NH-S(O) ₂ -；-S(O) ₂ -O-；-O-S(O) ₂ -; -P (O) (OR ") -NH-; -NH-P (O) (OR ") -; -P (O) (OR ") -O-; and-O-P (O) (OR') -.

4. The modified albumin of

embodiment

2 or 3, wherein Z (when M = 1) is linked to M by one selected from: -O-; -CO-NH-; -NH-CO-; -CO-O-; -O-CO-; -S (O) ₂ -NH-；-NH-S(O) ₂ -；-S(O) ₂ -O-；-O-S(O) ₂ -; -P (O) (OR ") -NH-; -NH-P (O) (OR ") -; -P (O) (OR ") -O-; and-O-P (O) (OR') -.

5. The modified albumin of any one of embodiments 2-4, wherein:

R ¹ is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -; and is

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH.

6. The modified albumin of any one of embodiments 2-4, wherein

R ¹ Selected from the group consisting of optionally substituted:

R ² selected from: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -, -O- (optionally substituted C) _1-6 Alkyl) -and-NH- (optionally substituted C) _1-6 Alkyl) -;

X ¹ selected from: CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH.

7. The modified albumin of any one of embodiments 2-4, wherein

R ¹ Is the following optionally substituted group:

R ¹² is optionally substituted C _1-12 Alkyl (preferably C) _1-10 Alkyl, more preferably C _4-8 Alkyl) or optionally substituted C _2-12 Alkenyl (preferably C) _2-10 Alkenyl, more preferably C _6-8 Alkenyl, more preferably-CH = CH (CH) ₂ ) _i CH ₃ Wherein i is 1,2, 3 or 4);

R ² selected from optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -)；

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO.

8. The modified albumin of any one of embodiments 2-4, wherein

R ¹ Selected from the group consisting of optionally substituted:

R ² selected from optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -) according to the formula (I); and is provided with

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO.

9. The modified albumin of any one of embodiments 2-4, wherein

R ¹ Selected from the group consisting of optionally substituted:

R ¹³ selected from:

preferably, it is

R ^13’ Selected from the group consisting of:

preferably, it is

R ² Selected from: optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 An alkenyl group;

X ¹ selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO; and is provided with

u and R ¹⁵ Each as defined in any one of embodiments 2 to 4.

10. The modified albumin of any one of embodiments 2-4, wherein

R ¹ In the form of S, the content of the S,

R ² selected from: optionally substituted (R) ^b ) _q Substituted phenyl groups,Optionally substituted (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -;

q and R ^b Each as defined in any one of embodiments 2 to 4.

11. The modified albumin of any one of embodiments 2-4, wherein:

X ¹ Selected from: CO, S (O) ₂ And P (O) (OR "), preferably CO.

12. The modified albumin of any one of embodiments 1-11, wherein M is selected from the group consisting of:

(1) C terminal and L- (Z) _m Linked, N-terminal by-CO-R ³ or-S (O) ₂ -R ³ A substituted amino acid;

(2) N terminal and L- (Z) _m To which is attached the-OH of the C-terminal carboxyl group by-NH-R ³ or-O-R ³ A substituted amino acid;

(3) A group having a structure represented by general formula (IV):

-X ⁴ -R ⁴

(IV)

wherein

X ⁴ Selected from: direct bond, O, CO, S (O) ₂ P (O) (OR' ") and NH;

r' "is selected from: h and R ⁴¹ ；

R ³ And R ⁴ Each independently selected from:

2)

R ⁴¹ 、R ^41’ 、R ⁴² and R ⁴³ Each independently selected from:

C _1-30 alkyl, preferably C _5-25 Alkyl, preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 Alkyl radicals, e.g. C ₅ Alkyl radical, C ₇ Alkyl radical, C ₁₁ Alkyl or C ₁₅ An alkyl group;

C _2-30 alkenyl, preferably C _5-25 Alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl or C _7-15 Alkenyl radicals, e.g. C ₅ Alkenyl radical, C ₇ Alkenyl radical, C ₁₁ Alkenyl radical, C ₁₅ Alkenyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl or C ₂₁ An alkenyl group; and

C _2-30 alkynyl, preferably C _5-25 Alkynyl, preferably C _7-23 Alkynyl, C _9-21 Alkynyl, C _11-19 Alkynyl, C _13-17 Alkynyl or C _7-15 Alkynyl, e.g. C ₅ Alkynyl, C ₇ Alkynyl, C ₁₁ Alkynyl, C ₁₅ Alkynyl, C ₁₇ Alkynyl, C ₁₉ Alkynyl or C ₂₁ An alkynyl group; and

3) Optionally substituted groups derived from sterols and bile acids; preferably, the group is selected from:

more preferably

13. The modified albumin of embodiment 12, wherein R ³ Each independently selected from: c ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

14. The modified albumin of embodiment 12, wherein:

X ⁴ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

R ⁴ selected from the group consisting of: r is ⁴¹ And by R ^41’ -C (O) O-and R ^41’ -C (O) NH-substituted R ⁴¹ ；

R ⁴¹ And R ^41’ Each independently selected from: c ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl, preferably: n-octyl, n-pentadecyl,

15. The modified albumin of embodiment 12, wherein

X ⁴ Is O or NH;

R ⁴ is composed of

Preference is given to

16. The modified albumin of embodiment 15, wherein R ⁴² And R ⁴³ Each independently selected from:

C ₇ alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

17. The modified albumin of embodiment 12, wherein:

X ⁴ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO; and is

R ⁴ Is optionally substituted

More preferably

18. The modified albumin of embodiment 12, wherein:

X ⁴ is O; and is

R ⁴ Is optionally substituted

Preferably a

19. The modified albumin of any one of embodiments 1-12, wherein M is selected from the group consisting of:

wherein:

R ³ as defined in embodiment 12;

R ^3’ selected from the group consisting of: h; c _1-6 An alkyl group; is selected from SH, SCH ₃ 、NH ₂ 、OH、COOH、-CONH ₂ 、-NHCOCH ₃ 、-NHCHO、-NHCONH ₂ Guanidino, optionally substituted C _6-10 C substituted by 1 or more substituents of aryl (preferably phenyl, 4-OH-phenyl, biphenyl, naphthyl) and optionally substituted 5-to 10-membered heteroaryl (preferably imidazole, indole) _1-6 An alkyl group; c _3-10 Cycloalkyl (preferably cyclohexyl); and C _6-10 Aryl (preferably phenyl);

preferably, R ^3’ Selected from H, CH ₃ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ ) ₂ Phenyl, cyclohexyl, benzyl, p-hydroxybenzyl, phenethyl, p-hydroxyphenylethyl, pyridin-2-ylmethyl-, pyridin-3-ylmethyl-, pyridin-4-ylmethyl-、-CH ₂ OH、-CH(OH)CH ₃ 、-CH ₂ CH ₂ SCH ₃ 、-CH ₂ CONH ₂ 、-CH ₂ COOH、-CH ₂ CH ₂ CONH ₂ 、-CH ₂ CH ₂ COOH、-(CH ₂ ) ₃ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₃ NH ₂ 、-(CH ₂ ) ₃ NHCOCH ₃ 、-(CH ₂ ) ₃ NHCHO、-(CH ₂ ) ₄ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₄ NHCOCH ₃ 、-(CH ₂ ) ₄ NHCHO、-(CH ₂ ) ₃ NHCONH ₂ 、-(CH ₂ ) ₄ NHCONH ₂ 、 -CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ 、

20. The modified albumin of any one of embodiments 1-19, wherein Z is represented by the general formula (V):

(V)

wherein:

when m is 1, X ² With L in the formula (II) or X in the formula (III) ¹ Is connected to, and X ³ With M in the formula (II) or X in the formula (IV) ⁴ Connecting;

X ² selected from: direct bond, O, CO, S (O) ₂ P (O) (OR') and NH;

X ³ selected from: direct bond, O, CO, S (O) ₂ P (O) (OR' ") and NH;

X ⁵ selected from: CO and S (O) ₂ ；

g is 1,2, 3,4, 5 or 6, preferably 1,2 or 3, more preferably 2;

h is 0 or 1;

r is 0,1, 2,3,4, 5 or 6, preferably 1,2 or 3, more preferably 2;

t is 0,1, 2,3,4, 5 or 6, preferably 0,1, 2 or 3, more preferably 0;

s is an integer from 0 to 100, preferably an integer from 0 to 10, preferably 0,1, 2,3,4, 5 or 6, more preferably 0,1 or 2;

r "is as defined in embodiment 2; and is

R' "is as defined in embodiment 12.

21. The modified albumin of embodiment 20, wherein Z is represented by the general formula (V-i):

-[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ]-

(V-i)。

22. the modified albumin of embodiment 21 wherein

X ² Selected from: o, CO, S (O) ₂ P (O) (OR') and NH, preferably CO OR NH; and is

r is 1,2 or 3, more preferably 2.

23. The modified albumin of embodiment 22 wherein

X ¹ Is CO, S (O) ₂ OR P (O) (OR') and X ² Is O or NH; or alternatively

X ¹ Is O or NH, and X ² Is CO, S (O) ₂ OR P (O) (OR ").

24. The modified albumin of embodiment 21, wherein:

X ² is a direct bond; and is

r is 0.

25. The modified albumin of embodiment 24, wherein:

X ¹ is CO, S (O) ₂ OR P (O) (OR ").

26. The modified albumin of any one of embodiments 21-25, wherein:

t is 0,1, 2 or 3, more preferably 0.

27. The modified albumin of any one of embodiments 21-26, wherein:

s is 0,1, 2,3,4, 5 or 6, more preferably 0,1 or 2.

28. The modified albumin of any one of embodiments 21 to 27, wherein

X ³ Selected from: o, CO, S (O) ₂ P (O) (OR' ") and NH, preferably CO OR NH.

29. The modified albumin of embodiment 28 wherein

X ³ Is O or NH, and X ⁴ Is CO, S (O) ₂ OR P (O) (OR' "); or

X ³ Is CO, S (O) ₂ OR P (O) (OR' "), and X ⁴ Is a direct bond, O or NH.

30. The modified albumin of any one of embodiments 21 to 29, wherein- [ X [ ] ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -(CH ₂ ) _t -X ³ ]-is selected from: -O-CH ₂ CH ₂ -CO-、-O-(CH ₂ CH ₂ ) ₂ -CO-、-CO-CH ₂ CH ₂ -O-、-O-CH ₂ CH ₂ -O-、-CO-CH ₂ CH ₂ -CO-、-NH-CH ₂ CH ₂ -NH-、-CO-CH ₂ CH ₂ -NH-、-NH-CH ₂ CH ₂ -CO-、-O-CH ₂ CH ₂ -S(O) ₂ -、-S(O) ₂ -CH ₂ CH ₂ -O-、-NH-CH ₂ CH ₂ -O-、-O-CH ₂ CH ₂ -NH-、-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-、-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -CO-、-CO-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-、-O-CH ₂ CH ₂ -P(O)(OR”’)-、-O-(CH ₂ CH ₂ ) ₂ -P(O)(OR”’)-、-CO-CH ₂ CH ₂ -P(O)(OR”’)-、-NH-CH ₂ CH ₂ -P (O) (OR' ") -and-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -P(O)(OR”’)-。

31. The modified albumin of any one of embodiments 1-30, wherein the modified albumin has a structure represented by the following general formula (VI):

wherein:

q, M and M are each as defined in any one of embodiments 1 to 30;

R ¹ 、R ² and X ¹ Each as defined in any one of embodiments 2 to 30; and is

X ² 、X ³ R, s and t are each as defined in any one of embodiments 20 to 30.

32. The modified albumin of any one of embodiments 1-30, wherein the modified albumin has a structure represented by the following general formula (VII):

wherein:

q and m are each as defined in any one of embodiments 1 to 30;

R ¹ 、R ² and X ¹ Each as defined in any one of embodiments 2 to 30;

X ² 、X ³ r, s and t are each as defined in any one of embodiments 20 to 30; and is

R ⁴ And X ⁴ Each as defined in any one of embodiments 12 to 30.

33. The modified albumin of embodiment 32 wherein

R ¹ Is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl radical)- (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl or optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -；

X ¹ Is O, CO, S (O) ₂ P (O) (OR') OR NH;

m is 0;

or

m is 1, and

X ² is CO, S (O) ₂ P (O) (OR') OR NH, preferably CO OR NH, and

X ³ is CO, S (O) ₂ P (O) (OR' ") OR NH, preferably CO OR NH;

R ⁴ selected from:

2)

Preference is given to

Wherein R is ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from: c _5-25 An alkyl group, a carboxyl group,preferably C _7-23 Alkyl radical, C _9-21 Alkyl radical, C _11-19 Alkyl radical, C _13-17 Alkyl or C _7-15 An alkyl group; and C _5-25 Alkenyl, preferably C _7-23 Alkenyl radical, C _9-21 Alkenyl radical, C _11-19 Alkenyl radical, C _13-17 Alkenyl or C _7-15 An alkenyl group; and

3) Optionally substituted group selected from:

preferably a

34. The modified albumin of any one of embodiments 1-33, wherein

m is 1.

35. The modified albumin of embodiment 34 wherein

r is 2; s is 2; and t is 0; or

r is 2; s is 0; and t is 0.

36. The modified albumin of embodiment 34 or 35, wherein: x ⁴ Is a direct bond, O, CO or NH.

37. The modified albumin of embodiment 20, wherein Z is represented by the general formula (V-ii):

-[X ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ]-

(V-ii)。

38. the modified albumin of embodiment 37 wherein

X ² Selected from the group consisting of: o, CO, S (O) ₂ P (O) (OR') and NH, preferably NH; and is

r is 1,2 or 3, more preferably 2.

39. The modified albumin of embodiment 38, wherein

X ¹ Is CO, S (O) ₂ OR P (O) (OR') and X ² Is NH.

40. The modified albumin of any one of embodiments 37-39, wherein:

s is 1,2, 3,4, 5 or 6, more preferably 2.

41. The modified albumin of any one of embodiments 37-40, wherein

X ³ Selected from: CO, S (O) ₂ P (O) (OR' "), and NH, preferably CO.

42. The modified albumin of embodiment 41, wherein

X ³ Is CO, and X ⁴ Is O or NH, preferably O.

43. The modified albumin of any one of embodiments 37-42, wherein:

X ⁵ is CO.

44. The modified albumin of any one of embodiments 37 to 43, wherein- [ X [ ] ² -(CH ₂ ) _r -(OCH ₂ CH ₂ ) _s -NH-X ⁵ -(CH ₂ ) _g -X ³ ]-is selected from:

-NH-CH ₂ CH ₂ -OCH ₂ CH ₂ -NH-CO-CH ₂ -CO-，

-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ -CO-，

-NH-CH ₂ CH ₂ -OCH ₂ CH ₂ -NH-CO-CH ₂ CH ₂ -CO-, and

-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ CH ₂ -CO-；

is preferably-NH-CH ₂ CH ₂ -(OCH ₂ CH ₂ ) ₂ -NH-CO-CH ₂ CH ₂ -CO-。

45. The modified albumin of any of embodiments 1-20 and 37-44, wherein the modified albumin has a structure represented by the following general formula (VI'):

wherein:

q, M and M are each as defined in any one of embodiments 1 to 20 and 37 to 44;

R ¹ 、R ² and X ¹ Each as defined in any one of embodiments 2 to 20 and 37 to 44; and is provided with

X ² 、X ³ 、X ⁵ G, r and s are each as defined in embodiments 20 and 37 to 44.

46. The modified albumin of any one of embodiments 1-20 and 37-44, wherein the modified albumin has a structure represented by the following general formula (VII'):

wherein:

q and m are each as defined in any one of embodiments 1 to 20 and 37 to 44;

R ¹ 、R ² and X ¹ Each as defined in any one of embodiments 2 to 20 and 37 to 44;

X ² 、X ³ 、X ⁵ g, r and s are each as defined in any one of embodiments 20 and 37 to 44; and is

R ⁴ And X ⁴ Each as defined in any one of embodiments 12 to 20 and 37 to 44.

47. The modified albumin of any one of embodiments 37-46, wherein

m is 1.

48. The modified albumin of embodiment 47, wherein

R ¹ Is composed of

R ² Selected from: optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl) and optionally substituted C _2-6 Alkenyl, - (CH) ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -; preferably optionally substituted C _1-6 Alkyl or optionally substituted C _2-6 An alkenyl group; more preferably C _2-5 Alkyl, more preferably-CH ₂ -CH ₂ -or- (CH) ₂ ) ₅ -；

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ² is NH;

X ³ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

X ⁴ is O or NH;

R ⁴ selected from:

2)

Preference is given to

3) Optionally substituted group selected from:

preferably a

49. The modified albumin of embodiment 48, wherein

X ¹ Is CO;

X ² is NH;

X ³ is CO;

X ⁴ is O;

X ⁵ is CO;

R ⁴ is composed of

And is

50. The modified albumin of any one of embodiments 1-19, wherein Z is selected from the group consisting of:

(1) A peptide, wherein when m is 1, the N-terminus of the peptide is linked to L in formula (II) or X in formula (III) ¹ And the C-terminus of the peptide is linked to M in formula (II) or X in formula (IV) ⁴ Connecting;

(2) A moiety represented by general formula (VIII):

wherein: when m is 1, the N-terminus of the peptide is linked to L in the general formula (II) or X in the general formula (III) ¹ The peptide is linked to the general formula (V) via the C-terminal COIII) N (R) ^Z1 ) Connecting;

preferably, the peptides described in the above items (1) and (2) are formed of 2 to 20 identical or different amino acids;

wherein:

a bond designated by c with L in the formula (II) or X in the formula (III) ¹ A bond designated by d is linked to M in the formula (II), X in the formula (IV) ⁴ Or N (R) in the general formula (VIII) ^Z1 ) Connecting;

(3) A moiety comprising the structure described in (1) or (2) above;

preferably, said moiety additionally comprises an oligosaccharide, - (CH) ₂ ) _n -and/or- (CH) ₂ CH ₂ O) _n -；

Preferably, the moiety may be selected from:

(4) A moiety selected from:

wherein: when m is 1, the bond designated by z is bonded to L in the formula (II) or X in the formula (III) ¹ Linking the bond designated by v with M in formula (II) or X in formula (IV) ⁴ Connecting;

wherein:

each R ^Z2 Independently selected from: h; c _1-6 Alkyl (preferably C) _1-4 Alkyl groups); is selected from SH, SCH ₃ 、NH ₂ 、OH、COOH、CONH ₂ Guanidino, optionally substituted C _6-10 C substituted by 1 or more substituents of aryl (preferably phenyl, 4-OH-phenyl, biphenyl) and optionally substituted 5-to 10-membered heteroaryl (preferably imidazole, indole) _1-6 An alkyl group; c _3-10 Cycloalkyl (preferably cyclohexyl); and C _6-10 Aryl (preferably phenyl);

preferably, each R ^Z2 Independently selected from: H. CH (CH) ₃ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ ) ₂ Phenyl, cyclohexyl, benzyl, p-hydroxybenzyl, phenethyl, p-hydroxyphenylethyl, pyridin-2-ylmethyl-, pyridin-3-ylmethyl-, pyridin-4-ylmethyl-, -CH ₂ OH、-CH(OH)CH ₃ 、-CH ₂ CH ₂ SCH ₃ 、-CH ₂ CONH ₂ 、-CH ₂ COOH、-CH ₂ CH ₂ CONH ₂ 、-CH ₂ CH ₂ COOH、-(CH ₂ ) ₃ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₃ NH ₂ 、-(CH ₂ ) ₃ NHCOCH ₃ 、-(CH ₂ ) ₃ NHCHO、-(CH ₂ ) ₄ NHC(＝NH)NH ₂ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₄ NHCOCH ₃ 、-(CH ₂ ) ₄ NHCHO、-(CH ₂ ) ₃ NHCONH ₂ 、 -(CH ₂ ) ₄ NHCONH ₂ 、-CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ 、

n is an integer selected from 1 to 10, such as 1,2, 3,4, 5, 6 or 7;

preferably, Z is selected from:

Val-Leu-Lys、Gly-Gly-Arg、

Val-Lys(Ac)-PAB、Phe-Lys-PAB、Phe-Lys(Ac)-PAB、

51. the modified albumin of embodiment 50 wherein

m is 1 and X ⁴ Is a direct bond, O or NH.

52. The modified albumin of embodiment 50 or 51, wherein

R ¹ Is composed of

X ¹ Is O, NH, CO, S (O) ₂ OR P (O) (OR ");

R ⁴ selected from:

2)

Preference is given to

3) Optionally substituted group selected from:

preferably a

53. The modified albumin of any one of embodiments 1-33 and 50, wherein

m is 0.

54. The modified albumin of embodiment 53, wherein

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO, and X ⁴ Is O or NH; or

55. The modified albumin of embodiment 53 or 54, wherein

R ⁴ Selected from: r is ⁴¹ And by R ^41’ -O-、R ^41’ -C (O) O-and R ^41’ -C (O) NH-substituted R ⁴¹ 。

56. The modified albumin of embodiment 53 or 54, wherein

X ¹ Is O or NH, preferably NH;

X ⁴ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO; and is

R ⁴ Selected from optionally substituted groups selected from:

57. the modified albumin of embodiment 53 or 54, wherein

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ⁴ is O; and is

R ⁴ Selected from the group consisting of optionally substituted:

58. the modified albumin of any one of embodiments 33 to 55, wherein R ⁴¹ 、R ^41’ 、R ⁴² And R ⁴³ Each independently selected from C ₇ Alkyl radical, C ₁₅ Alkyl radical, C ₁₇ Alkenyl radical, C ₁₉ Alkenyl radical, C ₂₁ Alkenyl and C ₂₃ Alkenyl groups, preferably: n-octyl, n-pentadecyl,

59. The modified albumin of any of embodiments 1-58, wherein said free thiol group is a thiol group corresponding to the cysteine residue at position 34 of SEQ ID NO 1.

60. The modified albumin of any one of embodiments 1-59, wherein said albumin is selected from the group consisting of Human Serum Albumin (HSA), recombinant human albumin (rHA), bovine serum albumin, and porcine serum albumin; for example, the albumin comprises the amino acid sequence shown in SEQ ID NO. 1.

61. The modified albumin of any one of embodiments 1-59, represented by the general formula (I):

wherein:

q is the S atom of HSA or rHA in the sulfhydryl group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1;

is the remainder of the HSA or rHA;

c is as follows:

and

62. a compound of the general formula (X):

R-(Z) _m -M

(X)

wherein:

r is a moiety containing a reactive group capable of linking the compound of formula (X) to S in the thiol group by reaction with the free thiol group of albumin and providing a hydrophobicity-enhancing moiety of formula (II) as defined in embodiment 1;

z is a spacer, as defined in any one of embodiments 1 to 61;

m is as defined in any one of embodiments 1 to 61; and is

M is a hydrophobic group, as defined in any one of embodiments 1 to 61.

63. A compound of embodiment 62, wherein the reaction between said reactive group and said free thiol group is selected from the group consisting of: michael addition reactions, thiol-disulfide exchange reactions, substitution reactions, and click chemistry.

64. A compound according to embodiment 62 or 63, wherein the reactive group is selected from michael reaction acceptors and groups containing a disulfide bond.

65. A compound of any one of embodiments 62 to 64 wherein R is represented by general formula (XI):

R ^1’ -R ² -X ¹ -

(XI)

wherein

and

preference is given to

Wherein:

selected from: 6-to 10-membered aryl and 5-to 6-membered heteroaryl;

x is CH or N;

or alternatively

R ^1’ Is halogen, such as F, cl, br or I;

R ¹¹ 、R ¹² 、R ¹³ 、R ^13’ 、R ¹⁴ 、R ¹⁶ 、R ^a 、R ^b 、R ² 、X ¹ p and q are each as in embodiments 2 to 61One is defined as;

preferably, R ^1’ Is composed of

And R is ¹¹ 、R ² And X ¹ Each as defined in embodiment 6;

preferably, R ^1’ Is composed of

And R is ¹² 、R ² And X ¹ Each as defined in embodiment 7;

preferably, R ^1’ Selected from:

and R is ² And X ¹ Each as defined in embodiment 8;

preferably, R ^1’ Is composed of

And R is ¹³ 、R ¹³ 、R ² And X ¹ Each as defined in embodiment 9;

preferably, R ^1’ Is composed of

And R is ² And X ¹ Each as defined in embodiment 10;

preferably, R ^1’ Is halogen, such as F, cl, br or I; r is ² Is optionally substituted C _1-6 Alkyl, preferably optionally substituted C _1-4 Alkyl, more preferably methyl; and X ¹ Selected from CO, S (O) ₂ And P (O) (OR "), preferably CO;

more preferably, R ^1’ Is composed of

R ² And X ¹ Each as defined in embodiment 5.

66. The compound of any one of embodiments 62 to 65 wherein the compound is represented by the general formula (XII):

(XII)；

wherein:

R ^1’ as defined in embodiment 65;

R ² and X ¹ Each as defined in any one of embodiments 2 to 36;

X ² 、X ³ r, s and t are each as defined in any one of embodiments 20 to 36;

m and M are each as defined in any one of embodiments 1 to 36.

67. The compound of any one of embodiments 62 to 66, wherein said compound is represented by general formula (XIII):

(XIII)；

wherein:

R ^1’ as defined in embodiment 65;

R ² and X ¹ Each as defined in any one of embodiments 2 to 36;

X ² 、X ³ r, s and t are each as defined in any one of embodiments 20 to 36;

X ⁴ and R ⁴ Each as defined in any one of embodiments 12 to 36;

m is as defined in any one of embodiments 1 to 36.

68. A compound of embodiment 67 wherein

R ^1’ Is composed of

And is

R ² 、X ¹ 、X ² 、X ³ 、R ⁴ And m are each as defined in any one of embodiments 33 to 36.

69. A compound according to any one of embodiments 62 to 68, wherein

m is 1.

70. A compound of embodiment 69 wherein

r is 2; s is 2; and t is 0; or

r is 2; s is 0; and t is 0.

71. A compound of embodiment 69 or 70 wherein: x ⁴ Is a direct bond, O, CO or NH.

72. The compound of any one of embodiments 62 to 65, wherein the compound is represented by general formula (XIV):

(XIV)

wherein:

R ^1’ as defined in embodiment 65;

m and M are each as defined in any one of embodiments 1 to 20 and 37 to 44;

R ² and X ¹ Each as defined in any one of embodiments 2 to 20 and 37 to 44; and is provided with

X ² 、X ³ 、X ⁵ G, r and s are each as defined in any one of embodiments 20 and 37 to 44.

73. A compound according to any one of embodiments 62 to 65 and 72 wherein said compound is represented by general formula (XV):

(XV)

wherein:

R ^1’ as defined in embodiment 65;

each m is as defined in any one of embodiments 1 to 20 and 37 to 44;

R ² and X ¹ Each as defined in any one of embodiments 2 to 20 and 37 to 44;

74. A compound of embodiment 72 or 73 wherein

m is 1.

75. A compound of embodiment 74 wherein

R ^1’ Is composed of

X ¹ Is CO, S (O) ₂ OR P (O) (OR "), preferably CO;

X ² is NH;

X ³ is CO, S (O) ₂ OR P (O) (OR' "), preferably CO;

X ⁴ is O or NH;

R ⁴ selected from:

2)

Preference is given to

3) Optionally substituted group selected from:

preferably, it is

76. A compound of embodiment 75 wherein

X ¹ Is CO;

X ² is NH;

X ³ is CO;

X ⁴ is O;

X ⁵ is CO；

R ⁴ Is composed of

And is provided with

77. A compound of any one of embodiments 62 to 65 wherein:

R ^1’ is composed of

Z is as defined in embodiment 50; and is

R ² 、X ¹ 、X ⁴ 、R ⁴ And m are each as defined in any one of embodiments 51 to 58.

78. A compound according to any one of embodiments 62 to 77, wherein the free thiol group is the thiol group corresponding to the cysteine residue at position 34 of SEQ ID No. 1.

79. The compound of any one of embodiments 62 to 78, wherein said albumin is selected from the group consisting of Human Serum Albumin (HSA), recombinant human albumin (rHA), bovine serum albumin, and porcine serum albumin; for example, the albumin comprises the amino acid sequence shown in SEQ ID NO. 1.

80. A compound of embodiment 62 selected from:

and

81. a method of preparing the modified albumin of any one of embodiments 1 to 61, the method comprising contacting the albumin with the compound of any one of embodiments 62 to 80 while allowing for free thiol groups of the albumin to react with R or R ^1’ A step of reacting under the reaction conditions;

preferably, the reaction is selected from the group consisting of michael addition reactions, thiol-disulfide exchange reactions and substitution reactions.

82. The method of embodiment 81, wherein the free thiol group is a thiol group corresponding to a cysteine residue at position 34 of SEQ ID NO. 1.

83. The method of embodiment 81 or 82, wherein said albumin is selected from the group consisting of Human Serum Albumin (HSA), recombinant human albumin (rHA), bovine serum albumin, and porcine serum albumin; for example, the albumin comprises the amino acid sequence set forth in SEQ ID NO 1.

84. A composition comprising a modified albumin of any one of embodiments 1 to 61 and optionally unmodified albumin;

preferably, the composition is in solid (e.g., lyophilized powder) or liquid form (e.g., aqueous solution);

preferably, the composition comprises the modified albumin as well as unmodified albumin; more preferably, the modified albumin comprises from about 1% to about 99%, preferably from about 5% to about 70%, more preferably from about 5% to about 20%, by weight of the sum of the weight of the unmodified albumin and the modified albumin.

85. A pharmaceutical or cosmetic composition comprising:

a carrier encapsulating a poorly water soluble compound having pharmaceutical or cosmetic activity, the carrier being the modified albumin of any one of embodiments 1 to 61 or the composition of embodiment 84; and

optionally a pharmaceutically or cosmetically acceptable other carrier;

86. A method of increasing the solubility of a poorly water soluble compound having pharmaceutical or cosmetic activity in an aqueous medium, the method comprising the steps of:

(1) Providing a solution of the modified albumin of any one of embodiments 1 to 61 or the composition of embodiment 84 in an aqueous medium;

(3) Mixing the two solutions in the step (1) and the step (2);

(4) Removing the organic solvent.

87. Use of the modified albumin of any one of embodiments 1 to 61 or the composition of embodiment 84 as a carrier, in particular as a carrier for poorly water soluble compounds having pharmaceutical or cosmetic activity.

88. Use of the modified albumin of any one of embodiments 1 to 47 or the composition of embodiment 84 as a carrier in the preparation of a pharmaceutical or cosmetic composition comprising a poorly water soluble compound having pharmaceutical or cosmetic activity,

preferably, the pharmaceutical or cosmetic composition is in solid, semi-solid or liquid form, preferably as a solution, suspension, emulsion or lyophilized powder, more preferably injectable.

89. A kit comprising at least: i) A poorly water soluble compound having pharmaceutical or cosmetic activity, ii) an unmodified albumin, and iii) a compound of any one of embodiments 62 to 80.

90. The pharmaceutical or cosmetic composition of embodiment 85, the method of embodiment 86, the use of embodiment 87 or 88, or the kit of embodiment 89, wherein the poorly water soluble compound having pharmaceutical or cosmetic activity is selected from the group consisting of:

antioxidants such as, but not limited to, flavonoids and polyphenols; antineoplastic agents, such as, but not limited to, docetaxel, taxanes, platins, camptothecins, vinblastines, anthraquinones, nucleosides, podophyllotoxins, DNA topoisomerase inhibitors; immunomodulators, such as cyclosporins; steroidal or non-steroidal anti-inflammatory agents; a cardiovascular agent; (ii) an antibiotic; antifungal agents; and an antiviral agent;

such as but not limited to: curcumin, paclitaxel, docetaxel, cisplatin, hydroxycamptothecin, camptothecin, vincamide, teniposide, etoposide, methotrexate, cyclosporin a, rapamycin, nimodipine, doxorubicin, breviscapine, bilobalide, silymarin, daunorubicin, mitomycin, indomethacin, ibuprofen, ketoprofen, naproxen, nitroerylate, fenofibrate, itraconazole, amphotericin B, biphenyldicarboxylate, progesterone, dihydrotestosterone, haloperidol, and risperidone.

Advantageous effects

The modified albumin of the present application can serve as a carrier platform for a variety of pharmaceutically active ingredients, particularly poorly water soluble compounds. As the modified albumin is introduced with hydrophobic groups only at the free sulfydryl, the controllability of the hydrophobic modification is high, the product is more uniform, and the product is beneficial to product control and patent medicine. In addition, when the hydrophobic modified albumin obtained in the aqueous medium self-assembles in vivo, the introduced hydrophobic groups are positioned in the cavities of the formed nanoparticles, and the natural albumin structure is exposed to the environment, so that the risk of in vivo immune reaction caused by exposure of the hydrophobic groups to the environment is reduced, namely the immunogenicity of the modified albumin molecule is reduced, and the safety is improved. Moreover, the modified albumin of the present application also has the following advantages: (1) The nanoparticles formed in the aqueous medium have good stability; (2) Has excellent solubilizing effect on the compound which is difficult to dissolve in water; (3) Long cycles of the encapsulated active ingredient are facilitated; (4) When the antitumor agent is encapsulated, tumor targeted delivery can be realized by utilizing the EPR effect of tumor tissues, so that the antitumor effect is improved.

Examples

The invention will be further illustrated in the following examples. These examples are intended to illustrate the invention, but not to limit it in any way.

In the conventional syntheses as well as in the examples and intermediate syntheses, the abbreviations have the following meanings.

Abbreviations	Means of
DMSO	Dimethyl sulfoxide
DIEA	N, N-diisopropylethylamine
HATU	O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
DMF	N, N-dimethylformamide
TLC	Thin layer chromatography
HPLC	High performance liquid chromatography
DCM	Methylene dichloride
DCC	N, N-dicyclohexylcarbodiimide
TEA	Triethylamine
EA	Ethyl acetate
FA	Formic acid
ACN	Acetonitrile (ACN)
TFA	Trifluoroacetic acid
Boc	Tert-butyloxycarbonyl radical
LCMS	Liquid phase mass spectrum combined instrument
MS	Mass spectrometry

Example 1: preparation of N- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) palmitamide (SYN-LPD 19001)

Palmitic acid (256mg, 1.0mmol), N- (2-aminoethyl) maleimide hydrochloride (175mg, 1.0mmol), DIEA (480. Mu.l, 3.0 mmol) and HATU (570mg, 1.5 mmol) were dissolved in DMF (3 mL), and stirred at room temperature overnight. After TLC detection indicated complete reaction, 5mL H was added ₂ O, solid is separated out, filtered and filtered by H ₂ O (10 mL. Multidot.2) was washed twice and dried to obtain the title compound (250 mg, yield: 66%).

¹ H NMR(400MHz,CDCl ₃ )δ:7.85(s,1H),3.47-3.44(t,2H),3.23-3.19(t,2H),1.99-1.95(t,2H),1.46-1.42(t,2H),1.26(s,26H),0.90-0.86(t,3H).

LC-MS:m/z＝379.3[M+H] ⁺ .

Example 2: preparation of N- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) cholamide (SYN-LPD 19002)

Cholic acid (408mg, 1.0mmol), N- (2-aminoethyl) maleimide hydrochloride (175mg, 1.0mmol), DIEA (570. Mu.l) and HATU (570mg, 1.5mmol) were dissolved in DMF (4 mL), and the reaction was stirred at room temperature overnight. After TLC detection showed complete reaction of the starting material, H was added ₂ O (20 mL), extracted with DCM (20ml × 2). The organic phase was dried over anhydrous sodium sulfate and spin-dried to obtain the title compound (400 mg, yield: 75.4%).

¹ HNMR(400MHz,CDCl ₃ )δ8.01(s,1H),7.86(s,2H),4.77(s,1H),4.49(s,1H)，4.14(S,1H),3.60-3.34(m,7H),3.21-3.19(m,23H),0.88-0.86(s,6H),0.81(s,3H).

LC-MS：m/z＝531.3[M+H] ⁺ .

Example 3: preparation of (R) -3- ((4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutanoyl) oxy) propan-1, 2-diyl dipalmitate (SYN-LPD 19003)

Step 1: preparation of (R) -1, 2-dipalmitoyl-3-succinin glyceride

1, 2-Dipalmitin (382mg, 0.68mmol) and succinic anhydride (134mg, 1.34mmol) were dissolved in a mixture of pyridine (4 mL) and dichloromethane (2 mL) and stirred overnight at 60 ℃. TLC showed the starting material had reacted completely, the reaction was spun dry and then dissolved in dichloromethane (15 mL), the solid was filtered, and the dichloromethane layer was washed with H ₂ After washing 4 times with O (20mL. Multidot.4), the organic phase was washed with 10% citric acid and 20mL of water. The organic phase was then spin dried to give the title compound of this step (300 mg), which was used directly in the next reaction.

Step 2: preparation of (R) -3- ((4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutanoyl) oxy) propan-1, 2-diyl dipalmitate

1, 2-Dipalmitoyl-3-succinin glyceride (100mg, 0.15mmol), N- (2-aminoethyl) maleimide hydrochloride (25mg, 0.15mmol), DIEA (78. Mu.L), and HATU (70mg, 0.22mmol) were dissolved in DMF (2 mL) and stirred at room temperature overnight. TLC showed the starting material had reacted completely, after adding an appropriate amount of water under stirring, followed by suction filtration and drying, the title compound of this step (66 mg, yield: 54.8%) was obtained.

¹ H NMR(400MHz,DMSO-d6)δ7.99(s,1H),7.02(s,2H),5.20(s,1H),4.32-4.14(m,4H)，3.46-3.45(d,2H),3.21-3.19(d,2H),2.48(s,2H),2.30-2.28(m,6H),1.53(s,4H),1.26(s,52H),0.88-0.86(s,6H).

LC-MS：m/z＝792.8[M+H] ⁺ .

Example 4: preparation of (3S, 8S,9S,10R,13R,14S, 17R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-decatetrahydro-1H-cyclopenta [ a ] phenanthren-3-yl 3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionate (SYN-LPD 19004)

3-Maleimidopropionic acid (0.50g, 2.96mmol), cholesterol (1.26g, 3.25mmol), DCC (0.73g, 3.55mmol) were stirred in dichloromethane (100.0 mL) at room temperature for 24 h. Quench with water (20.0 mL) and separate the dichloromethane layer. With anhydrous Na ₂ SO ₄ Drying, filtering and concentrating. After purification by column chromatography (EA/PE = 1/10), the title compound (0.20g, 12.6%) was obtained as a white solid.

¹ H NMR(500MHz,CDCl ₃ ):δ6.70(s,2H),5.37(d,J＝5.0Hz,1H),4.63-4.59(m,1H),3.82(t,J＝7.0Hz,2H),2.61(t,J＝7.5Hz,2H),2.29-2.28(m,2H),2.05-1.98(m,2H),2.00-1.80(m,3H),1.56-0.96(m,38H),0.63(s,3H).

LC-MS m/z:538.8[M+H] ⁺ .

Example 5: preparation of N- (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) palmitamide (SYN-LPD 19005)

Step 1: preparation of tert-butyl (2- (2- (2-palmitoylethoxy) ethoxy) ethyl) carbamate

Palmitic acid (256mg, 1.0mmol), (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamic acid tert-butyl ester (248mg, 1.0mmol), DIEA (480. Mu.I, 3.0 mmoL) and HATU (570mg, 1.5mmol) were dissolved in DMF (3 mL) and reacted at room temperature for 4 hours. TLC assay showed the starting material was reacted completely, then 15mL water was added followed by extraction with ethyl acetate (20ml × 2). The organic phase was dried over anhydrous sodium sulfate and then spin dried to give the crude title compound of this step (ca 480 mg), which was used directly in the next step.

And 2, step: preparation of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) palmitamide

The crude product from step 1 (480 mg) was dissolved in TFA/DCM (1 mL/1 mL) and stirred at room temperature for 2 h. After completion of the reaction as checked by TLC, the solvent was distilled off under reduced pressure to obtain the crude title compound (about 580 mg) of this step.

And 3, step 3: preparation of N- (2- (2- (2- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propionamido) ethoxy) ethyl) palmitamide

The crude product (102 mg) obtained in step 2 was dissolved in DMF (2 mL) together with N-hydroxysuccinimide 3-maleimidopropionate (70 mg) and DIEA (100 mg), and reacted at room temperature overnight. TLC assay showed the starting material had reacted completely, 10mL of water was added and extracted with DCM (20ml × 2). The organic phase was dried over anhydrous sodium sulfate, followed by distillation under the reduced pressure to give the title compound of this step (about 52mg, yield: 55.0%).

¹ H NMR(400MHz,CDCl ₃ )δ[ppm]8.03(s,1H),7.82(s,1H),3.64-3.60(t,2H),3.52(s,4H),3.43-3.35(m,8H),3.21-3.17(m,4H),2.38-2.34(t,2H),2.09-2.05(t,2H),1.49(s,2H),1.26(s,24H),0.90-0.86(t,3H).

LC-MS:m/z＝538.2[M+H] ⁺ .

Example 6: preparation of Cholesterol-3- [2- (2-Maleimidopropionamidoethoxy) ethoxy ] propionate (SYN-LPD 19008)

Step 1: preparation of cholesterol-3- [2- (2-tert-butoxycarbonyl-aminoethoxy) ethoxy ] propionate

3- [2- (2-t-Butoxycarbonylaminoethoxy) ethoxy ] propionic acid (1.0 g,3.6 mmol) and cholesterol (2.09g, 5.4 mmol) were dissolved in methylene chloride (50 mL) and DCC (890 mg, 4.32mmol) was slowly added. The reaction was stirred at room temperature for 16 hours. The reaction solution was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (PE/EA = 3/1) to obtain the title compound of the present step (2.3 g, yield: 95%) as an oil.

And 2, step: preparation of cholesterol-3- [2- (2-aminoethoxy) ethoxy ] propionate

The compound prepared in step 1 (2.3 g, 3.56mmol) was dissolved in HCl in dioxane (20.0 mL) and stirred at room temperature for 2 hours. The reaction solution was concentrated to obtain the title compound of this step (1.8 g, yield: 92.7%) as a white oil.

And 3, step 3: preparation of cholesterol-3- [2- (2-maleimidopropionamidoethoxy) ethoxy ] propionate

The compound prepared in step 2 (840mg, 1.54mmol), 3-maleimidopropionic acid (200mg, 1.18mmol), HATU (493mg, 1.30mmol) and TEA (155mg, 1.53mmol) were dissolved in DCM (20 mL) and stirred at room temperature for 2 hours. Water (20 mL) was added to the reaction and extracted with DCM (50mL × 2). The organic layers were combined, washed once with brine (50mL. Times.1), and the organic phase was washed with Na ₂ SO ₄ Drying, filtration, concentration and purification by column chromatography (DCM: methanol = 9) gave the title compound SYN-LPD19008 of this step (340 mg, yield: 41.4%) as a yellow solid.

¹ H NMR(500MHz,CDCl ₃ ):δ6.69(s,2H),6.37(s,1H),5.36(d,J＝3.5Hz,1H),4.65-4.59(m,1H),3.85(t,J＝7.0Hz,2H),3.77(t,J＝6.5Hz,2H),3.63-3.58(m,4H),3.53-3.52(m,2H),3.44-3.41(m,2H),2.59-2.52(m,4H),2.31(d,J＝7.5Hz,2H),2.02-1.94(m,2H),1.87-1.79(m,2H),1.62-1.40(m,7H),1.39-1.10(m,12H),1.08-0.85(m,15H),0.67(s,3H).

LC-MS m/z:697.2[M+H] ⁺ .

Example 7: rHA modified with SYN-LPD19002

As shown in scheme 5, albumin is hydrophobically modified by the Michael addition reaction between the free thiol group of albumin (e.g., the thiol group corresponding to cysteine residue 34 (Cys-34) in SEQ ID NO: 1) and the maleimide group of the reactive group:

reaction scheme 5

Wherein formula (a) represents albumin (e.g., rHA), and

wherein-SH represents a free thiol group of the albumin (e.g. rHA),

representing the remainder of the albumin (e.g. rHA).

Taking rHA as an example, the preparation method comprises the following steps:

a. taking 20% rHA (North China pharmaceutical) raw material, diluting with 10mM PB buffer (pH = 6.5) to a concentration of about 31.25mg/mL to be used as an aqueous phase;

b. the product of example 2, SYN-LPD19002, was dissolved in ethanol and formulated into an ethanol phase at a concentration of about 2.01 mg/mL;

c. slowly dropwise adding the ethanol phase into the water phase according to the volume ratio of the water phase to the ethanol phase of about 8;

d. after the dropwise addition is finished, stirring at room temperature for reaction for 1h;

e. extracting unreacted SYN-LPD19002 in the system with equal volume of ethyl acetate, and collecting a water phase;

f. adding arginine into the water phase to ensure that the concentration of the arginine is 5mg/mL, continuously stirring at room temperature for 30min, and then removing the organic solvent in the system by rotary evaporation;

g. the product obtained was analyzed after filtration through a 0.22 μm filter. The resulting hydrophobically modified albumin (rHA-CA) was determined by LC-MS using HPLC-Q-TOF-MS (Agilent, USA).

LC-MS detection:

(1) Instrument system and parameter setting

(2) A detection step:

mu.l of the sample +40ul of 0.1% aqueous FA solution were mixed, 2. Mu.l of the mixture was applied to an autosampler, subjected to HPLC separation, and subjected to mass spectrometry, wherein a profile spectrum of 600-2000M/Z was collected, deconvoluted using Deconfuel (MS): protein software, deconfuel algorithm: maximum Entrophy, mass range: 10000-100000Da.

(3) Results of LC-MS measurement

The results are shown in FIGS. 2 and 3.

The results show that the most abundant molecular ion peaks are: m/z =67014.07[ m ] +H] ⁺ Indicating that SYN-LPD19002 was successfully bound to rHA.

Example 8: rHA modified with SYN-LPD19005

Albumin comprising a hydrophobic modification (rHA-PEG) was prepared according to the method described in example 7, replacing the product SYN-LPD19002 of example 2 with the product SYN-LPD19005 of example 5 ₂ -PA) and subjecting the resulting product to HPLC analysis and LC-MS determination.

HPLC detection method:

(1) The HPLC chromatographic conditions were as follows:

(2) Results of HPLC measurement

The results are shown in FIG. 4. As shown in FIG. 4, the resulting product was separated into unmodified rHA and modified rHA (rHA-PEG) ₂ -PA), rHA-PEG ₂ The retention time of-PA was longer than that of rHA (16.098min vs 15.055min), indicating rHA-PEG ₂ Increased hydrophobicity of PA. Calculation according to peak area ratio, with unmodified rHA and rHA-PEG ₂ Total of-PA, rHA-PEG ₂ -PA accounts for more than 70%.

LC-MS detection

The results of the measurements are shown in FIGS. 5 and 6.

As shown in fig. 5 and 6, the most abundant molecular ion peaks are: m/z =67001.15[M+H] ⁺ Indicating successful binding of SYN-LPD19005 to albumin.

Example 9: rHA modified with SYN-LPD19001

In analogy to the procedure described in example 7, a product comprising hydrophobically modified albumin (rHA-PA) was prepared with SYN-LPD19001 and rHA prepared in example 1 and the resulting product was subjected to HPLC analysis and LC-MS assay.

The preparation method comprises the following steps:

a. raw material rHA (north China pharmaceutical) was mixed with 10mM PB buffer (pH = 6.5) containing 5% (v/v) ethyl acetate at 30 ℃ so that the final concentration of rHA in the resulting system was 25mg/ml as phase a;

b. the product SYN-LPD19001 of example 1 was dissolved in a mixed solvent of 95% ethanol and 5% (v/v) ethyl acetate as phase B;

c. slowly (1 ml/min) dropwise adding phase B to phase a under magnetic stirring according to a volume ratio of phase a to phase B of about 8;

d. after the dropwise addition is finished, continuously reacting for 1h under the condition of magnetic stirring;

e. extracting unreacted SYN-LPD19001 in the system by ethyl acetate;

f. removing the organic solvent in the system by rotary evaporation;

g. after filtration through a 0.22 μm filter, the product, which contained SYN-LPD19001 modified rHA (rHA-PA), was obtained.

The HPLC profile and LC-MS measurement results of the obtained product are shown in FIGS. 7 to 9.

As shown in FIG. 7, the resulting product separated into two major peaks for unmodified rHA and modified rHA (rHA-PA), with longer retention time for rHA-PA than rHA (16.190min vs 15.039min), indicating increased hydrophobicity of rHA-PA. Calculated according to the peak area ratio, the rHA-PA accounts for more than 70 percent of the total sum of the unmodified rHA and the rHA-PA.

As shown in fig. 9, the most abundant molecular ion peaks are: m/z =66840.31[ 2 ], [ M ] +H] ⁺ Indicating successful binding of SYN-LPD19001 to rHA.

EXAMPLE 10 particle size determination of the products prepared in examples 7-9

rHA was diluted with 10mM PB Buffer (pH = 6.5) to a concentration of 25mg/mL for use.

The particle size of samples of the products prepared in examples 7-9 was determined using an instrumental Nano ZSE Nanosizer (Malvern, UK) with parameter settings: material is Protern and Dispersant is Water, an automatic mode scan is selected, each sample is scanned three times, and the results are averaged. Samples of rHA, prepared as described above and prepared in examples 7-9, were scanned in a cuvette at approximately 200. Mu.L each.

The results of the particle size measurement are shown in table 1 and fig. 10.

TABLE 1 results of particle size measurement of products prepared in examples 7 to 9

Sample (I)	Average particle diameter (d, nm)	Average PDI
rHA	5.230	0.028
rHA-CA (example 7)	5.236	0.049
rHA-PEG 2-PA (example 8)	5.673	0.067
rHA-PA (example 9)	5.347	0.081

The results in Table 1 and FIG. 10 show that rHA-CA and rHA-PEG ₂ -PA and rHA-PA make it possible to obtain monodisperse protein solutions.

Example 11 preparation of nanoparticles encapsulating paclitaxel Using modified Albumin as Carrier and testing of the stability

1. Preparation of nanoparticles

Nanoparticles encapsulating Paclitaxel (PTX) (rHA/PTX NPs) were prepared with rHA according to the method described in example 1 of patent EP1023050B 1. The specific experimental steps are as follows:

a. diluting rHA with deionized water to a 1% (w/v) rHA solution;

b. dissolving 30mg of PTX in 3mL of dichloromethane to prepare an organic phase;

c. dropping the organic phase into 27mL of 1% (w/v) rHA solution, and dispersing at low shear (IKA T25 easy clean, IKA) for 5min;

d. high pressure homogenization at 20000psi for 10 cycles;

e. the mixture was transferred to a low pressure (30 mm Hg) and rotary evaporated (rotary evaporator, RE-52AA, shanghai Yarong) at 40 ℃ for 20-30 minutes to rapidly spin off the organic solvent, resulting in a dispersion containing PTX-encapsulated nanoparticles (rHA/PTX NPs).

Nanoparticles containing encapsulated PTX (rHA-CA) were prepared as described above except that in step a the 1% (w/v) rHA solution was replaced with a solution in which the total protein content of rHA and rHA-CA was 1% (w/v) and rHA-CA accounted for 65% of the total protein ₆₅ PTX NPs).

Nanoparticles containing encapsulated PTX (rHA-CA) were prepared as described above except that in step a the 1% (w/v) rHA solution was replaced with a solution in which the total protein content of rHA and rHA-CA was 1% (w/v) and rHA-CA accounted for 7.25% of the total protein _7.25 PTX NPs).

Except that in step a 1% (w/v) rHA solution was treated with rHA and rHA-PEG ₂ -PA Total protein content 1% (w/v) and rHA-CA 10% of Total protein instead of the solution, nanoparticles containing encapsulated PTX (rHA-PEG) were prepared as described above ₂ -PA ₁₀ PTX NPs).

The particle size of each dispersion sample containing nanoparticles prepared was determined as described in example 10, except that each dispersion sample was diluted with deionized water to a PTX concentration of about 1 mg/mL. The results are shown in Table 2 below.

TABLE 2 particle size measurement results for each sample of the dispersion containing nanoparticles

Sample(s)	Average particle diameter (nm)	PDI
rHA/PTX NPs	189.2	0.138
rHA-CA ₆₅ /PTX NPs	193.2	0.144
rHA-CA _7.25 /PTX NPs	172.6	0.135
rHA-PEG ₂ -PA ₁₀ /PTX NPs	168.1	0.129

The results show that the nanoparticles obtained by encapsulation of PTX with hydrophobically modified albumin are comparable to unmodified rHA (rHA-CA) ₆₅ PTX NPs) or less (rHA-CA) _7.25 PTX NPs and rHA-PEG ₂ -PA ₁₀ PTX NPs).

2. Nanoparticle stability assay

(1) Determination of disintegration stability

After diluting the sample with deionized water to a PTX concentration of about 1mg/mL, the sample was diluted with deionized water in a gradient of 5, 10, 15, 20, 30, 40, 50 or more times to study the variation in the size of Nanoparticles (NPs) in the sample. When multiple peaks are detected, micron-sized particles and particles smaller than 10nm exist, indicating that the Nano Particles (NPs) in the sample are disintegrated. The lower the disintegration concentration of the sample, the more stable the nanoparticles formed by the binding of proteins (including unmodified albumin and modified albumin) in the sample to PTX.

TABLE 3 results of measurement of disintegration stability

Sample (I)	Disintegration test
rHA/PTX NPs	Dilution to PTX concentration of about 0.025mg/mL, i.e. 40-fold gradient dilution
rHA-CA ₆₅ /PTX NPs	Diluting to PTX concentration of about 0.025mg/mL, i.e. gradient dilution 40 times rapid disintegration
rHA-CA _7.25 /PTX NPs	Dilution to a PTX concentration of about 0.02mg/mL, i.e., a 50-fold gradient dilution
rHA-PEG ₂ -PA ₁₀ /PTX NPs	Dilution to PTX concentration of about 0.025mg/mL, i.e. 40-fold gradient dilution

The results show that nanoparticles prepared using hydrophobically modified albumin have comparable or higher disintegration stability compared to unmodified rHA, where rHA-CA _7.25 The best stability of the/PTX NPs.

(2) Long term stability assay

TABLE 4 Long-term stability assay results

The results show that the long-term stability of the four samples is ranked as: rHA-CA _7.25 /PTX NPs>rHA-PEG ₂ -PA ₁₀ /PTX NPs>rHA/PTX NPs>rHA-CA ₆₅ PTX NPs, wherein rHA-CA _7.25 the/PTX NPs showed the most excellent long-term stability.

Equivalents and incorporation by reference

The modified albumins, conjugate modifiers, compositions and methods of the present application have been described herein with reference to certain preferred embodiments. However, since specific variations thereof will be apparent to those skilled in the art based on the disclosure set forth herein, the application should not be construed as limited thereto.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification and claims, the singular also includes the plural unless the context clearly dictates otherwise.

Furthermore, to the extent that the methods of the present application do not rely on the particular order of steps set forth herein, the particular order of steps recited in the claims should not be construed as limitations on such claims.

All patents, patent applications, references, and publications cited herein are incorporated by reference in their entirety.

Claims

A modified albumin consisting of albumin and a hydrophobicity-enhancing moiety attached to the S atom in the free thiol group of the albumin, the modified albumin being represented by the general formula (I):

wherein:

q is an S atom in the free thiol group of the albumin;

is the remainder of the albumin;

c is the hydrophobicity-enhancing moiety and is represented by the general formula (II):

wherein:

l is a linker connected to Q;

z is a spacer;

m is 1 or 0; and is

M is a hydrophobic group.
The modified albumin of claim 1, wherein L is represented by the general formula (III):

wherein:

the bond identified by a is bonded to Q, the bond identified by b is bonded to (Z) _m -M bonding;

R ¹ is S, or is selected from the group consisting of optionally substituted:

wherein:

the bond designated by f is bonded to Q, the bond designated by k is bonded to R ² Bonding;

R ¹¹ each independently selected from: H. optionally substituted C _1-12 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-4 Alkyl), -COOH, -COOC _1-6 Alkyl, -CH (R) ¹⁵ )NH-CO-C _1-6 Alkyl radical, C _6-10 Aryl (e.g., phenyl) and 5 to 10 membered heteroaryl;

R ¹² selected from the group consisting of: H. optionally substituted C _1-12 Alkyl (preferably C) _1-10 Alkyl, more preferably C _4-8 Alkyl) and optionally substituted C _2-12 Alkenyl (preferably C) _2-10 Alkenyl, more preferably C _4-8 Alkenyl, more preferably-CH = CH (CH) ₂ ) _i CH ₃ Wherein i is 1,2, 3 or 4);

R ¹³ selected from the group consisting of: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 Alkenyl and
(preferred is
)；

R ^13’ Selected from the group consisting of: optionally substituted C _1-6 Alkyl, optionally substituted C _2-6 An alkenyl group,
(preferred)
)，

Wherein:

u is 0,1, 2 or 3, preferably 1 or 2;

R ¹⁴ and R ¹⁵ Each independently is H or C _1-6 Alkyl, preferably H or C _1-4 Alkyl, more preferably H, methyl or ethyl; more preferably H or methyl;

preferably, R ¹³ Is composed of
And wherein R ¹⁵ Preferably C _1-4 Alkyl, more preferably methyl;

R ¹⁶ selected from H and optionally substituted C _1-12 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-4 Alkyl groups);

p is 0,1 or 2;

R ^a each independently selected from hydroxy, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkylthio and halogen;

R ² selected from: direct bond, O, optionally substituted C _1-6 Alkyl (e.g. C) _2-5 Alkyl), optionally substituted C _2-6 Alkenyl (e.g. -CH) ₂ -CH＝CH(CH ₂ ) ₃ -)、-(CH ₂ CH ₂ O) _j - (optionally substituted C) _1-6 Alkyl) - (preferably- (CH) ₂ CH ₂ O) ₂ -CH ₂ -, - (optionally substituted C) _1-6 Alkyl) -C _3-6 Cycloalkyl- (preferably-CH) ₂ -cyclohexane-), -O- (optionally substituted C _1-6 Alkyl) -, -NH- (optionally substituted C _1-6 Alkyl) -, -O- (optionally substituted C _2-6 Alkenyl) -, -NH- (optionally substituted C _2-6 Alkenyl) -, optionally substituted by (R) ^b ) _q Substituted phenyl, optionally substituted with (R) ^b ) _q Substituted pyridyl, - (optionally substituted (R) ^b ) _q Substituted phenyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _1-6 Alkyl) -, - (optionally substituted by (R) ^b ) _q Substituted phenyl) - (optionally substituted C _2-6 Alkenyl) -and- (optionally substituted by (R) ^b ) _q Substituted pyridyl) - (optionally substituted C _2-6 Alkenyl) -;

wherein:

j is an integer from 1 to 10, such as 2;

q is 0,1, 2 or 3;

R ^b each independently selected from H, hydroxyl, nitro, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkylthio and halogen;

or

R ¹ And R ² Together being optionally substituted C _1-6 Alkyl (e.g. C) _1-4 Alkyl); and is

X ¹ Selected from: o, CO, S (O) ₂ P (O) (OR') and NH; and is

Wherein R' is selected from H, optionally substituted C _1-6 Alkyl and optionally substituted C _2-6 Alkenyl, preferably H.
The modified albumin of claim 1 or 2, represented by the general formula (I):

wherein:

q is the S atom of HSA or rHA in the sulfhydryl group corresponding to the cysteine residue at position 34 of SEQ ID NO: 1;

is the remainder of the HSA or rHA;

c is as follows:

and
a compound of the general formula (X):

R-(Z) _m -M

(X)

wherein:

r is a moiety containing a reactive group capable of linking a compound of formula (X) to S in a free thiol group of albumin by reaction with said thiol group and providing a hydrophobicity-enhancing moiety of formula (II) as defined in claim 1;

z is a spacer group as defined in any one of claims 1 to 3;

m is as defined in any one of claims 1 to 3; and is

M is a hydrophobic group, as defined in any one of claims 1 to 3.
A method of preparing the modified albumin of any one of claims 1 to 3, the method comprising contacting the albumin with the compound of claim 4 while allowing for free sulfhydryl groups of the albumin to react with R or R ^1’ A step of reacting under the reaction conditions;

preferably, the reaction is selected from the group consisting of michael addition reactions, thiol-disulfide exchange reactions, and substitution reactions.
A composition comprising the modified albumin of any one of claims 1 to 3 and optionally unmodified albumin;

preferably, the composition is in solid (e.g., lyophilized powder) or liquid form (e.g., aqueous solution);

preferably, the composition comprises the modified albumin as well as unmodified albumin; more preferably, the modified albumin comprises from about 1% to about 99%, preferably from about 5% to about 70%, more preferably from about 5% to about 20%, by weight of the sum of the weight of the unmodified albumin and the modified albumin.
A pharmaceutical or cosmetic composition comprising:

a carrier encapsulating a poorly water soluble compound having pharmaceutical or cosmetic activity, the carrier being the modified albumin of any one of claims 1 to 3 or the composition of claim 6; and

optionally a pharmaceutically or cosmetically acceptable other carrier;

preferably, the pharmaceutical or cosmetic composition is in the form of a solid, semi-solid or liquid, preferably a solution, suspension, emulsion or lyophilized powder, more preferably injectable.
A method of increasing the solubility of a poorly water soluble compound having pharmaceutical or cosmetic activity in an aqueous medium, the method comprising the steps of:

(1) Providing a solution of the modified albumin of any one of claims 1 to 3 or the composition of claim 6 in an aqueous medium;

(2) Providing a solution of the poorly water soluble compound in an organic solvent;

(3) Mixing the two solutions in the step (1) and the step (2);

(4) The organic solvent is removed.
Use of the modified albumin of any one of claims 1 to 3 or the composition of claim 6 as a carrier, in particular as a carrier for poorly water soluble compounds with pharmaceutical or cosmetic activity.
Use of the modified albumin of any one of claims 1 to 3 or the composition of claim 6 as a carrier in the preparation of a pharmaceutical or cosmetic composition comprising a poorly water soluble compound with pharmaceutical or cosmetic activity,

preferably, the pharmaceutical or cosmetic composition is in solid, semi-solid or liquid form, preferably as a solution, suspension, emulsion or lyophilized powder, more preferably injectable.
A kit comprising at least: i) A poorly water soluble compound having pharmaceutical or cosmetic activity, ii) an unmodified albumin, and iii) a compound of any one of claim 4.