Distearoylphosphatidylethanolamine-polyethylene glycol derivative and liposome containing distearoylphosphatidylethanolamine-polyethylene glycol derivative

Abstract

Disclosed are a substance represented by formula (I), and a preparation method therefor and a use thereof, wherein R is a protein containing an amino group, a polypeptide containing an amino group or a small molecule containing an amino group, M is a monovalent cation, m is 10 to 100, and hydrogen atoms in the substance represented by formula (I) are optionally substituted.

Description

Distearoylphosphatidylacetamide-polyethylene glycol derivative and liposome containing distearoylphosphatidylacetamide-polyethylene glycol derivative

[0001] Citation of Related Applications

[0002] This disclosure claims all rights and interests in the Chinese invention patent application with application number 202310744434.1 filed with the State Intellectual Property Office of the People's Republic of China on June 21, 2023, entitled Distearoylphosphatidylacetamide-polyethylene glycol derivatives and liposomes containing distearoylphosphatidylacetamide-polyethylene glycol derivatives, and incorporates the entire contents thereof into this disclosure by reference.

[0003] field

[0004] The present disclosure relates generally to the field of biomedicine, and more specifically to the field of protein degradation targeting chimeras (PROTACs).

[0005] background

[0006] The use of small molecule inhibitors or monoclonal antibodies to target non-specific or mutant expressed proteins to treat malignant tumors is widely used in cancer treatment. However, the vast majority of proteins in cells lack active sites that bind to small molecule inhibitors or monoclonal antibodies, and the chemotherapy drugs used in clinical practice so far are all non-targeted drugs. In 2001, PROTAC (targeted protein degradation chimera) was first proposed as a new technology for targeted cancer treatment. PROTAC contains heterobifunctional groups in its structural design, one end is a group that targets the target protein ligand, and the other end is a group that recruits the E3 ubiquitin ligase ligand. A linker is used in the middle to connect the target protein ligand group and the E3 ubiquitin ligase ligand group together through the linker, and the cell's intrinsic 26S proteasome degradation pathway is used to recognize and degrade the marked target protein, thereby triggering the ubiquitination process to induce target protein degradation, thereby achieving the effect of disease treatment.

[0007] The first generation of peptide PROTACs were based on peptide ligands that bind to β-TrCP and VHL as E3 ligases. However, their clinical application was limited by issues such as cell permeability and chemical stability. The second generation of small molecule-based PROTACs, which use MDM2, VHL, IAP, and Cereblon as ligands for E3 ligases, have been extensively studied.

[0008] Folate receptor α is highly expressed in many cancers, such as ovarian, lung, and breast cancer, while normal tissues or cells express only minimal or no folate receptor α. Currently, research on folate receptor α-targeted drugs is in Phase II / III clinical trials.

[0009] Overview

[0010] In one aspect, the present disclosure relates to a substance represented by formula (I),

[0011] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0012] In another aspect, the present disclosure relates to a method for preparing a substance represented by formula (I),

[0013] The method comprises: reacting a compound represented by formula (II) with R,

[0014] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0015] In another aspect, the present disclosure relates to a substance of formula (I) prepared by a method comprising the following steps:

[0016] The method comprises: reacting a compound represented by formula (II) with R,

[0017] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0018] In another aspect, the present disclosure relates to a liposome comprising a substance represented by formula (I) wherein R is a ligand R1 of an E3 ubiquitin ligase, and a substance represented by formula (I) wherein R is a ligand R2 of a target protein.

[0019] In another aspect, the present disclosure relates to a method for preparing a liposome comprising:

[0020] The liposome is obtained by self-assembling a substance represented by formula (I) in which R is a ligand R1 of an E3 ubiquitin ligase and a substance represented by formula (I) in which R is a ligand R2 of a target protein using a thin film dispersion method.

[0021] In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising the liposomes of the present disclosure and a pharmaceutically acceptable excipient.

[0022] In yet another aspect, the present disclosure relates to a method for treating or preventing tumors, comprising administering a therapeutically or prophylactically effective amount of the liposomes of the present disclosure to a subject in need thereof.

[0023] In another aspect, the present disclosure relates to a method for degrading a target protein (Protein of Interest, POI), comprising administering the liposome of the present disclosure and an E3 ubiquitin ligase to the target protein (Protein of Interest, POI).

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 shows a technical roadmap for modifying small molecules VHL, Tam, Mitb and Cer using DSPE-PEG2000-NHS in certain embodiments of the present disclosure;

[0026] Figure 2 shows (a) the small molecule high-resolution LC-MS spectrum of DSPE-PEG2000-NHS raw material (the compound has two charges); (b) the small molecule high-resolution LC-MS spectrum after DSPE-PEG2000-NHS modification of VHL (the compound has two charges); (c) the small molecule high-resolution LC-MS spectrum after DSPE-PEG2000-NHS modification of Tam small molecule (the compound has two charges); (d) the small molecule high-resolution LC-MS spectrum after DSPE-PEG2000-NHS modification of Mitb small molecule (the compound has one charge); and (e) the small molecule high-resolution LC-MS spectrum after DSPE-PEG2000-NHS modification of Cer small molecule (the compound has one charge).

[0027] Figure 3 shows the DSL hydrated particle size graphs of liposomes prepared according to certain embodiments of the present disclosure, including (a) the DSL hydrated particle size graph of FA-V-Tam-L liposomes; (b) the DSL hydrated particle size graph of V+Mitb-L liposomes; and (c) the DSL hydrated particle size graph of V-Cer-L liposomes.

[0028] Figure 4 shows the ZETA potential diagrams of liposomes prepared according to certain embodiments of the present disclosure, including (a) the ZETA potential diagram of FA-V-Tam-L liposomes; (b) the ZETA potential diagram of V+Mitb-L liposomes; and (c) the ZETA potential diagram of V-Cer-L liposomes.

[0029] Figure 5 shows graphs of the proliferation inhibition of cancer cells by liposomes prepared in certain embodiments of the present disclosure, including (a) a graph of CCK-8 cell proliferation inhibition of breast cancer cells MCF7; (b) a graph of CCK-8 cell proliferation inhibition of melanoma cells A375; and (c) a graph of CCK-8 cell proliferation inhibition of human non-small cell lung cancer NCI-H2228.

[0030] Figure 6 shows immunoblot gel electrophoresis patterns of different liposomes prepared in certain embodiments of the present disclosure on cancer cells overexpressing target proteins: immunoblot gel electrophoresis pattern of the degradation of estrogen receptor protein (MCF7) by FA-V-Tam-L liposomes, wherein (a) different concentration gradients and (b) different time gradients; immunoblot gel electrophoresis pattern of the degradation of mitogen-activated protein kinase (A375) by V-Mitb-L liposomes, wherein (c) different concentration gradients and (d) different time gradients; and immunoblot gel electrophoresis pattern of the degradation of anaplastic lymphoma kinase (NCI-H2228) by V-Cer-L liposomes, wherein (e) different concentration gradients and (f) different time gradients.

[0031] Figure 7 shows the cellular uptake trend of liposomes labeled with fluorescent substance FITC (with and without FA targeting) prepared in certain embodiments of the present disclosure after incubation with breast cancer cells MCF7 (folate receptor positive) and human monocytic leukemia cells THP-1 (folate receptor negative) for a certain period of time.

[0032] Details

[0033] In the following description, certain specific details are included to provide a comprehensive understanding of each disclosed embodiment. However, one skilled in the relevant art will recognize that the embodiments can still be implemented without one or more of these specific details and with other methods, components, materials, etc.

[0034] Unless otherwise required in this application, throughout the specification and the appended claims, the words "including," "comprising," "containing," and "having" should be interpreted in an open, inclusive sense, that is, "including but not limited to."

[0035] As used in this disclosure and the appended claims, singular references without indications of quantity include plural references unless the context clearly dictates otherwise.

[0036] Reference throughout this specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that at least one embodiment includes the specific referenced elements, structures, or features described in connection with that embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," "in another embodiment," or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the specific elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0037] It should be understood that the singular articles "a," "an," and "the" used in the specification and appended claims of this disclosure include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a liposome comprising "a substance represented by formula (I)" includes one substance represented by formula (I) or two or more substances represented by formula (I).

[0038] definition

[0039] Accordingly, unless otherwise indicated to the contrary, the following terms used in the specification and appended claims shall have the following meanings:

[0040] Certain chemical groups named in this disclosure are preceded by abbreviations indicating the total number of carbon atoms present in the indicated chemical group. For example, C1-C4 alkyl describes an alkyl group as defined below having a total of 1 to 4 carbon atoms, while C3-C4 alkyl describes an alkyl group ... 10 Cycloalkyl describes a cycloalkyl group as defined below having a total of 3 to 10 carbon atoms. The total number of carbons in the shorthand notation does not include carbons that may be present in substituents of the group being described.

[0041] In the present disclosure, the term "Ubiquitin-Proteasome System (UPS)" refers to a system composed of ubiquitin (Ub), ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3), proteasome and its substrates (proteins). It is the main pathway for intracellular protein degradation and is involved in the degradation of more than 80% of intracellular proteins.

[0042] In the present disclosure, the term "E3 ubiquitin ligase" refers to an enzyme that is capable of attaching a ubiquitin molecule to a lysine of a target protein.

[0043] In this disclosure, the term "target protein (Protein of Interest, POI)" refers to a protein in the body that has a pharmacological function and can be acted upon by a drug.

[0044] In this disclosure, the term "ubiquitin" refers to a small protein present in most eukaryotic cells. Its main function is to mark proteins that need to be broken down so that they can be hydrolyzed.

[0045] In this disclosure, the term "proteasome" refers to a multi-subunit complex widely distributed in the cytoplasm and nucleus. It is composed of approximately 50 protein subunits. It has multiple proteolytic enzyme activities and can degrade target proteins tagged with ubiquitin.

[0046] In this disclosure, the term "protein degradation targeting chimera (PROTAC)" refers to the regulation of post-translational protein levels by co-opting the ubiquitin-proteasome system.

[0047] In the present disclosure, the term "ligand" refers to a substance that has the ability to recognize and bind to a receptor.

[0048] In the present disclosure, the term "membrane lipid" refers to the skeleton components for preparing liposomes, such as cholesterol / phospholipids and other auxiliary components.

[0049] In the present disclosure, the term "estrogen receptor" refers to an important cellular protein receptor that regulates gene expression by binding to estrogen and plays a key role in multiple physiological processes and disease development.

[0050] In this disclosure, the term "pharmaceutically acceptable" refers to carriers, vehicles, diluents, excipients and / or salts that must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0051] In the present disclosure, the term "pharmaceutically acceptable excipients" includes but is not limited to any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers approved by the U.S. Food and Drug Administration for use in humans or animals, and any carriers in various forms that have no side effects on the composition of the pharmaceutical composition.

[0052] In this disclosure, the terms “optional” or “optionally” mean that the subsequently described event or circumstance can or cannot occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0053] Certain chemical groups named in this disclosure are preceded by abbreviations indicating the total number of carbon atoms present in the indicated chemical group. For example, C1-C4 alkyl describes an alkyl group as defined below having a total of 1 to 4 carbon atoms, while C3-C4 alkyl describes an alkyl group ... 10 Cycloalkyl describes a cycloalkyl group as defined below having a total of 3 to 10 carbon atoms. The total number of carbons in the shorthand notation does not include carbons that may be present in substituents of the group being described.

[0054] In this disclosure, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0055] In this disclosure, the term "hydroxyl" refers to an -OH group.

[0056] In this disclosure, the term "amino" refers to a -NH2 group.

[0057] In this disclosure, the term "cyano" refers to a -CN group.

[0058] In this disclosure, the term "alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing no unsaturated bonds, having 1 to 12 carbon atoms, and which is attached to the rest of the molecule by a single bond.

[0059] In certain embodiments, an alkyl group has 1 to 8 carbon atoms.

[0060] In certain embodiments, an alkyl group has 1 to 6 carbon atoms.

[0061] In certain embodiments, an alkyl group has 1 to 4 carbon atoms.

[0062] In certain embodiments, illustrative examples of alkyl groups that can be used in the present disclosure include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, and 2-methylhexyl.

[0063] An alkyl group can have from 1 to 8 carbon atoms (each occurrence in this disclosure of a numerical range such as "1 to 8" refers to each integer in the given range; e.g., "1 to 8" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 8 carbon atoms, although this definition also covers occurrences of the term "alkyl" without specifying a numerical range).

[0064] The alkyl group may be optionally substituted, i.e., substituted or unsubstituted. When substituted, the substituent groups are individually and independently selected from one or more of the following: cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiol, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxyl, O-carboxyl, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl as defined herein), or amino, including mono- and di-substituted amino groups, and protected derivatives thereof.

[0065] In certain embodiments, illustrative examples of alkyl groups that can be used in the present disclosure include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. Whenever a substituent is described as being "optionally substituted," the substituent may be substituted by one of the substituents described above.

[0066] In certain embodiments, "C1-C4 alkyl" refers to an alkyl group as defined above containing one to four carbon atoms. A C1-C4 alkyl group may be optionally substituted as defined for an alkyl group.

[0067] In certain embodiments, "C1-C6 alkyl" refers to an alkyl group as defined above containing one to six carbon atoms. A C1-C6 alkyl group may be optionally substituted as defined for an alkyl group.

[0068] In certain embodiments, "C1-C8 alkyl" refers to an alkyl group as defined above containing one to eight carbon atoms. A C1-C8 alkyl group may be optionally substituted as defined for an alkyl group.

[0069] In this disclosure, the term "alkenyl" refers to a straight or branched hydrocarbon chain group consisting of two to eight carbon atoms and at least one carbon-carbon double bond, and connected to the rest of the molecule by a single bond.

[0070] Alkenyl groups may be optionally substituted, i.e., substituted or unsubstituted. When substituted, the substituent groups are individually and independently selected from one or more of the following: cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxyl, O-carboxyl, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl as defined herein), or amino, including mono- and di-substituted amino groups, and protected derivatives thereof.

[0071] In certain embodiments, illustrative examples of alkenyl groups that can be used in the present disclosure include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, and penta-1,4-dienyl.

[0072] In certain embodiments, "C2-C4 alkenyl" refers to an alkenyl group as defined above containing two to four carbon atoms. A C2-C4 alkenyl group may be optionally substituted as defined for an alkenyl group.

[0073] In certain embodiments, "C2-C6 alkenyl" refers to an alkenyl group as defined above containing two to six carbon atoms. A C2-C6 alkenyl group may be optionally substituted as defined for an alkenyl group.

[0074] In certain embodiments, "C2-C8 alkenyl" refers to an alkenyl group as defined above containing two to eight carbon atoms. A C2-C8 alkenyl group may be optionally substituted as defined for an alkenyl group.

[0075] In this disclosure, the term "alkynyl" refers to a straight or branched hydrocarbon chain group consisting of two to eight carbon atoms and at least one carbon-carbon triple bond, and connected to the rest of the molecule by a single bond.

[0076] Alkynyl groups may be optionally substituted, i.e., substituted or unsubstituted. When substituted, the substituent groups are individually and independently selected from one or more of the following: cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxyl, O-carboxyl, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl as defined herein), or amino including mono- and di-substituted amino groups, and protected derivatives thereof.

[0077] In certain embodiments, illustrative examples of alkynyl groups that can be used in the present disclosure include, but are not limited to, ethynyl, propynyl, and butynyl.

[0078] In certain embodiments, "C2-C4 alkynyl" refers to an alkynyl group as defined above containing two to four carbon atoms. A C2-C4 alkynyl group may be optionally substituted as defined for an alkynyl group.

[0079] In certain embodiments, "C2-C6 alkynyl" refers to an alkynyl group as defined above containing two to six carbon atoms. A C2-C6 alkynyl group may be optionally substituted as defined for an alkynyl group.

[0080] In certain embodiments, "C2-C8 alkynyl" refers to an alkynyl group as defined above containing two to eight carbon atoms. A C2-C8 alkynyl group may be optionally substituted as defined for an alkynyl group.

[0081] In the present disclosure, the term "alkoxy" refers to the general formula -O-alkyl, wherein alkyl is as defined in the present disclosure. Illustrative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and tert-pentoxy.

[0082] In the present disclosure, the term "cycloalkyl" refers to a stable non-aromatic monocyclic or bicyclic alkyl group consisting solely of carbon and hydrogen atoms, having from three to fifteen carbon atoms, in certain embodiments from three to twelve carbon atoms, which is saturated and attached to the rest of the molecule by a single bond, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclodecyl, and the like. Unless otherwise specifically stated in the present disclosure, the term "cycloalkyl" is intended to include cycloalkyl groups as defined above optionally substituted by one or more substituents selected from the group consisting of cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfinylamino, N-sulfinylamino, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl groups as defined in the present disclosure), or amino including mono- and di-substituted amino groups, and protected derivatives thereof.

[0083] In certain embodiments, "C3-C6 cycloalkyl" refers to a cycloalkyl group as defined above having three to six carbon atoms. A C3-C6 cycloalkyl group may be optionally substituted as defined above for a cycloalkyl group.

[0084] In certain embodiments, "C3-C 10 "Cycloalkyl" refers to a cycloalkyl group as defined above having three to ten carbon atoms. 10 Cycloalkyl groups may be optionally substituted as defined above for cycloalkyl.

[0085] In certain embodiments, "C3-C 12 "Cycloalkyl" refers to a cycloalkyl group as defined above having three to twelve carbon atoms. 12 Cycloalkyl groups may be optionally substituted as defined above for cycloalkyl.

[0086] In the present disclosure, the term "aryl" refers to a carbocyclic ring (all carbon) or two or more fused rings (rings sharing two adjacent carbon atoms) having a completely delocalized pi electron system. Aryl groups include, but are not limited to, fluorenyl, phenyl, and naphthyl. Aryl groups can, for example, have five to twelve carbon atoms. The aryl groups of the present disclosure can be substituted or unsubstituted. When substituted, the hydrogen atoms are replaced by one or more groups independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, protected hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl as defined in the present disclosure), or protected amino. Whenever a substituent is described as being "optionally substituted," the substituent may be substituted by one of the substituents described above.

[0087] In the present disclosure, the term "heteroaryl" refers to a 5- to 18-membered aromatic ring group consisting of one to seventeen carbon atoms and one to ten heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, the heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl group may be optionally oxidized; the nitrogen atom may be optionally quaternized. Illustrative examples of heteroaryl groups include, but are not limited to, azepine, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepanyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophene yl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindololinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-phenyl-1H-pyrrolyl, phenanthrazinyl, phenanthiazinyl, phenanthroxazinyl, phthalazinyl, The heteroaryl groups of the present disclosure may be substituted or unsubstituted. When substituted, the hydrogen atoms are replaced by one or more groups independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, protected hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, -NR'R" (R' and R" are alkyl as defined in the present disclosure), or protected amino. Whenever a substituent is described as being "optionally substituted," the substituent may be substituted by one of the substituents described above.

[0088] In this disclosure, the term "mammal" refers to animals including, for example, dogs, cats, cows, sheep, horses, and humans, etc. In certain embodiments, the mammal includes humans.

[0089] In this disclosure, the term "patient" refers to animals (e.g., humans), companion animals (e.g., dogs, cats, or horses), and livestock (e.g., cattle, pigs, and sheep). In certain embodiments, the patient is a mammal, including males and females. In certain embodiments, the patient is a human.

[0090] As used herein, the term "pharmaceutical composition" refers to a formulation of a compound described herein and a medium generally accepted in the art for delivering the bioactive compound to mammals, such as humans. Such a medium includes any pharmaceutically acceptable carrier, diluent, or excipient.

[0091] In the present disclosure, the term "therapeutically effective amount" refers to an amount of a compound or combination of compounds that ameliorates, reduces, or eliminates a particular disease or condition and symptoms of a particular disease or condition, or prevents or delays the onset of a particular disease or condition or symptoms of a particular disease or condition. The amount of a compound described in the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, and the age, weight, etc. of the mammal to be treated, but the amount of a compound described in the present disclosure can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0092] As used herein, "treating" or "treatment" encompasses treating a relevant disease or condition in a mammal, such as a human, suffering from the relevant disease or condition, and includes:

[0093] (i) preventing a disease or disease state from occurring in a mammal, particularly where the mammal is susceptible to said disease state but has not yet been diagnosed with such disease state;

[0094] (ii) inhibiting the disease or disease state, i.e., preventing its occurrence; or

[0095] (iii) ameliorating the disease or condition, even if the disease or condition regresses or does not progress.

[0096] As used in this disclosure, the terms "disease" and "disease state" may be used interchangeably, or may be distinct in that a particular disease or disease state may have no known causative agent (and therefore cannot be explained etiologically) and therefore is not recognized as a disease, but rather is considered an undesirable disease state or condition in which clinicians have identified a more or less specific constellation of symptoms. DETAILED DESCRIPTION

[0097] In one aspect, the present disclosure relates to a substance represented by formula (I),

[0098] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0099] In certain embodiments, m is from 10 to 90.

[0100] In certain embodiments, m is from 10 to 80.

[0101] In certain embodiments, m is from 10 to 70.

[0102] In certain embodiments, m is from 10 to 60.

[0103] In certain embodiments, m is from 10 to 50.

[0104] In certain embodiments, m is from 10 to 40.

[0105] In certain embodiments, m is from 10 to 30.

[0106] In certain embodiments, m is from 10 to 20.

[0107] In certain embodiments, m is 40 to 50.

[0108] In certain embodiments, m is 45.

[0109] In certain embodiments, exemplary examples of M that can be used in the present disclosure include, but are not limited to, H + 、Li + 、Na + , K + , Rb + 、Cs + 、Fr + 、Ag + and NH4 + .

[0110] In certain embodiments, the hydrogen atoms in the species of formula (I) are unsubstituted.

[0111] In certain embodiments, one or more hydrogen atoms in the substance represented by formula (I) are substituted by halogen, hydroxy, amino, cyano, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, or heteroaryl.

[0112] In certain embodiments, illustrative examples of R that can be used in the present disclosure include, but are not limited to, a ligand R1 for an E3 ubiquitin ligase and a ligand R2 for a target protein.

[0113] In certain embodiments, illustrative examples of ligands R1 that can be used for the E3 ubiquitin ligases of the present disclosure include, but are not limited to, VHL ligands, MDM2 ligands, CRBN ligands, DCAF15 ligands, DCAF16 ligands, RNF4 ligands, RNF114 ligands, IAP ligands, and cIAP1 ligands.

[0114] In certain embodiments, illustrative examples of VHL ligands that can be used in the present disclosure include, but are not limited to

[0115] In certain embodiments, illustrative examples of MDM2 ligands that can be used in the present disclosure include, but are not limited to

[0116] In certain embodiments, illustrative examples of CRBN ligands that can be used in the present disclosure include, but are not limited to

[0117] In certain embodiments, illustrative examples of DCAF15 ligands that can be used in the present disclosure include, but are not limited to

[0118] In certain embodiments, illustrative examples of DCAF16 ligands that can be used in the present disclosure include, but are not limited to

[0119] In certain embodiments, illustrative examples of RNF4 ligands that can be used in the present disclosure include, but are not limited to

[0120] In certain embodiments, illustrative examples of RNF114 ligands that can be used in the present disclosure include, but are not limited to

[0121] In certain embodiments, illustrative examples of IAP ligands that can be used in the present disclosure include, but are not limited to

[0122] In certain embodiments, illustrative examples of cIAP1 ligands that can be used in the present disclosure include, but are not limited to

[0123] In certain embodiments, illustrative examples of ligands R2 that can be used for the target proteins of the present disclosure include, but are not limited to, estrogen receptor α ligands, Alk ligands, MEK ligands, BCL-XL ligands, BCR-ABL ligands, BRD2 / 3 / 4 ligands, BTK ligands, CDK6 / 9 ligands, CRABP-1 ligands, FLT-1 ligands, FAK ligands, FKBP12 ligands, FLT-3 ligands, PARP1 ligands, PCAF / GCN5 ligands, RIPK2 ligands, STAT3 ligands, TACC3 ligands, TBK1 ligands, TRIM24 ligands, and P38 ligands.

[0124] In certain embodiments, illustrative examples of estrogen receptor alpha ligands that can be used in the present disclosure include, but are not limited to

[0125] In certain embodiments, illustrative examples of MEK ligands that can be used in the present disclosure include, but are not limited to

[0126] In certain embodiments, illustrative examples of Alk ligands that can be used in the present disclosure include, but are not limited to

[0127] In certain embodiments, illustrative examples of AR ligands that can be used in the present disclosure include, but are not limited to

[0128] In certain embodiments, illustrative examples of BRD2 / 3 / 4 ligands that can be used in the present disclosure include, but are not limited to

[0129] In certain embodiments, illustrative examples of BCL-XL ligands that can be used in the present disclosure include, but are not limited to

[0130] In certain embodiments, illustrative examples of BCR-ABL ligands that can be used in the present disclosure include, but are not limited to

[0131] In certain embodiments, illustrative examples of BTK ligands that can be used in the present disclosure include, but are not limited to

[0132] In certain embodiments, illustrative examples of CDK6 ligands that can be used in the present disclosure include, but are not limited to

[0133] In certain embodiments, illustrative examples of CDK9 ligands that can be used in the present disclosure include, but are not limited to

[0134] In certain embodiments, illustrative examples of CRABP-1 ligands that can be used in the present disclosure include, but are not limited to

[0135] In certain embodiments, illustrative examples of FAK ligands that can be used in the present disclosure include, but are not limited to

[0136] In certain embodiments, illustrative examples of FLT-3 ligands that can be used in the present disclosure include, but are not limited to

[0137] In certain embodiments, illustrative examples of p38 ligands that can be used in the present disclosure include, but are not limited to

[0138] In certain embodiments, illustrative examples of FKBP12 ligands that can be used in the present disclosure include, but are not limited to

[0139] In certain embodiments, illustrative examples of FLT-1 ligands that can be used in the present disclosure include, but are not limited to

[0140] In certain embodiments, exemplary PARP1 ligands that can be used in the present disclosure include, but are not limited to

[0141] In certain embodiments, illustrative examples of PCAF / GCN5 ligands that can be used in the present disclosure include, but are not limited to

[0142] In certain embodiments, illustrative examples of TBK1 ligands that can be used in the present disclosure include, but are not limited to

[0143] In certain embodiments, exemplary RIPK2 ligands that can be used in the present disclosure include, but are not limited to

[0144] In certain embodiments, illustrative examples of STAT3 ligands that can be used in the present disclosure include, but are not limited to, STAT3

[0145] In certain embodiments, illustrative examples of TACC3 ligands that can be used in the present disclosure include, but are not limited to

[0146] In certain embodiments, exemplary TRIM24 ligands that can be used in the present disclosure include, but are not limited to

[0147] In certain embodiments, illustrative examples of MEK ligands that can be used in the present disclosure include, but are not limited to

[0148] In certain embodiments, the substance represented by formula (I) is selected from:

[0149] In another aspect, the present disclosure relates to a method for preparing a substance represented by formula (I),

[0150] The method comprises: reacting a compound represented by formula (II) with R,

[0151] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0152] In certain embodiments, the molar ratio of the compound represented by formula (II) to R is about 1-5:1.

[0153] In certain embodiments, the molar ratio of the compound represented by formula (II) to R is about 1:1.

[0154] In certain embodiments, a basic reagent is added to react the compound represented by formula (II) with R.

[0155] In certain embodiments, illustrative examples of alkaline reagents that can be used in the present disclosure include, but are not limited to, N,N-diisopropylethylamine (DIPEA), triethylamine, and phosphate buffer.

[0156] In certain embodiments, the reaction of the compound of formula (II) with R is carried out at a pH of about 8.0.

[0157] In certain embodiments, the basic agent is added in an amount of about 1 to about 5 equivalents based on R.

[0158] In certain embodiments, the reaction of the compound represented by formula (II) and R is carried out in a polar solvent.

[0159] In certain embodiments, illustrative examples of polar solvents that can be used in the present disclosure include, but are not limited to, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, chloroform, and dichloromethane.

[0160] In certain embodiments, the reaction of the compound represented by formula (II) and R is carried out for about 12 to 48 hours.

[0161] In certain embodiments, the reaction of the compound of formula (II) with R is carried out at room temperature.

[0162] In certain embodiments, the reaction of the compound represented by formula (II) and R is carried out under stirring conditions.

[0163] In certain embodiments, stirring that can be used in the present disclosure includes, but is not limited to, mechanical stirring, electromagnetic stirring, or manual glass rod stirring.

[0164] In certain embodiments, the reaction of the compound represented by formula (II) and R is carried out under conditions of uniform stirring.

[0165] In certain embodiments, the reaction of the compound represented by formula (II) and R is carried out at a stirring rate of 200 to 600 rpm.

[0166] In certain embodiments, the product obtained by reacting the compound represented by formula (II) with R is dialyzed.

[0167] In certain embodiments, the product obtained by reacting the compound represented by formula (II) with R is dialyzed in an aqueous medium.

[0168] In certain embodiments, the product obtained by reacting the compound represented by formula (II) with R is dialyzed in an aqueous medium for about 24 to 72 hours.

[0169] In certain embodiments, illustrative examples of dialysis methods that can be used in the present disclosure include, but are not limited to, semipermeable membrane methods, reverse osmosis methods, or electrodialysis methods.

[0170] In certain embodiments, dialysis is performed using a semipermeable membrane using a dialysis bag.

[0171] In certain embodiments, the molecular weight of the dialysis tubing useful in the present disclosure is between about 1000 and 8000 Da.

[0172] In certain embodiments, the dialyzed product is lyophilized.

[0173] In another aspect, the present disclosure relates to a substance of formula (I) prepared by a method comprising the following steps:

[0174] The method comprises: reacting a compound represented by formula (II) with R,

[0175] Wherein, R is a protein containing an amino group, a polypeptide containing an amino group or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

[0176] In another aspect, the present disclosure relates to a liposome comprising a substance represented by formula (I) wherein R is a ligand R1 of an E3 ubiquitin ligase, and a substance represented by formula (I) wherein R is a ligand R2 of a target protein.

[0177] In certain embodiments, the substance represented by formula (I) wherein R is a ligand R1 of an E3 ubiquitin ligase is

[0178] In certain embodiments, R is a ligand R2 of a target protein, wherein the substance represented by formula (I) is

[0179] In certain embodiments, the liposomes of the present disclosure have a particle size of about 130 to 180 nm.

[0180] In certain embodiments, the thin film dispersion method is used to obtain the substance represented by formula (I) by self-assembly, wherein R is a ligand R1 of an E3 ubiquitin ligase, and R is a ligand R2 of a target protein.

[0181] In certain embodiments, the liposomes of the present disclosure have good stability at 4°C.

[0182] In certain embodiments, the liposomes of the present disclosure are spherical nanoparticles with uniform particle size.

[0183] In another aspect, the present disclosure relates to a method for preparing a liposome comprising:

[0184] The liposome is obtained by self-assembling a substance represented by formula (I) in which R is a ligand R1 of an E3 ubiquitin ligase and a substance represented by formula (I) in which R is a ligand R2 of a target protein using a thin film dispersion method.

[0185] In certain embodiments, a thin film dispersion method is used to self-assemble a substance represented by formula (I) in which R is a ligand R1 of an E3 ubiquitin ligase, a substance represented by formula (I) in which R is a ligand R2 of a target protein, and a lipid membrane excipient to obtain the liposome.

[0186] In certain embodiments, illustrative examples of lipid membrane excipients that can be used in the present disclosure include, but are not limited to, phospholipids, glycolipids, and cholesterol.

[0187] In certain embodiments, hydrogenated soybean lecithin, cholesterol, and DSPE-mPEG2000 (distearoylphosphatidyl acetamide-polyethylene glycol 2000) are used as lipid membranes, and a thin film dispersion method is used to self-assemble a substance represented by formula (I) in which R is a ligand R1 for an E3 ubiquitin ligase and a substance represented by formula (I) in which R is a ligand R2 for a target protein, thereby obtaining the liposome.

[0188] In certain embodiments, in a polar solvent, a substance represented by formula (I) in which R is a ligand R1 of an E3 ubiquitin ligase and a substance represented by formula (I) in which R is a ligand R2 of a target protein undergo a self-assembly reaction.

[0189] In certain embodiments, illustrative examples of polar solvents that can be used in the self-assembly reactions of the present disclosure include, but are not limited to, organic solvents such as chloroform, dichloromethane, ethanol, and methanol.

[0190] In certain embodiments, the molar ratio of hydrogenated soybean lecithin, cholesterol, DSPE-mPEG2000 (distearoylphosphatidyl acetamide-polyethylene glycol 2000), the substance represented by formula (I) in which R is a ligand R1 for E3 ubiquitin ligase, and the substance represented by formula (I) in which R is a ligand R2 for a target protein is approximately 45-55:35-45:1-5:1-5:1-10.

[0191] In certain embodiments, in a polar solvent, a substance represented by formula (I) in which R is a ligand R1 of an E3 ubiquitin ligase and a substance represented by formula (I) in which R is a ligand R2 of a target protein undergo a self-assembly reaction, and then the polar solvent is removed to obtain a thin film.

[0192] In certain embodiments, the film is immersed in deionized water or phosphate buffer and subjected to a hydration reaction at 55 to 65° C. to obtain a lipid dispersion.

[0193] In certain embodiments, the hydration reaction is carried out in a water bath or a metal bath.

[0194] In certain embodiments, the hydration reaction is carried out for about 0.5 to 5 hours.

[0195] In certain embodiments, the lipid dispersion solution is repeatedly extruded through an extruder having a pore size of about 100 to 400 nm to obtain preliminary liposomes.

[0196] In certain embodiments, illustrative examples of extruders that can be used in the present disclosure include, but are not limited to, polycarbonate membrane extruders, pneumatic liposome extruders, and multi-extruders.

[0197] In certain embodiments, the primary liposomes are washed with deionized water or phosphate buffered saline.

[0198] In certain embodiments, illustrative examples of washing methods that can be used in the present disclosure include, but are not limited to, ultrafiltration washing, semipermeable membrane dialysis, and high-speed centrifugation.

[0199] In certain embodiments, the ultrafiltration tubing used in the ultrafiltration wash has a specification of 100 kd.

[0200] In certain embodiments, the centrifuge used in the ultrafiltration wash rotates at a speed of about 1000 to 2500 g.

[0201] In certain embodiments, the centrifugation time in the ultrafiltration wash is about 5 to 30 minutes.

[0202] In certain embodiments, the centrifugation temperature during the ultrafiltration wash is about 2 to 10°C.

[0203] In yet another aspect, the present disclosure relates to a pharmaceutical composition comprising the liposomes of the present disclosure and a pharmaceutically acceptable excipient.

[0204] In yet another aspect, the present disclosure relates to a method for treating or preventing tumors, comprising administering a therapeutically or prophylactically effective amount of the liposomes of the present disclosure to a subject in need thereof.

[0205] In certain embodiments, illustrative examples of tumors that can be used in the present disclosure include, but are not limited to, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), low-grade serous ovarian cancer, neurofibroma, liver cancer, melanoma, non-small cell lung cancer, and esophageal cancer.

[0206] In certain embodiments, illustrative examples of individuals that can be used with the present disclosure include, but are not limited to, mammals.

[0207] In certain embodiments, illustrative examples of mammals that can be used in the present disclosure include, but are not limited to, humans.

[0208] In another aspect, the present disclosure relates to a method for degrading a target protein (Protein of Interest, POI), comprising administering the liposome of the present disclosure and an E3 ubiquitin ligase to the target protein (Protein of Interest, POI).

[0209] In certain embodiments, the method is performed in vitro.

[0210] Hereinafter, the present disclosure will be explained in detail through the following examples in order to better understand the various aspects and advantages of the present application. However, it should be understood that the following examples are non-limiting and are only used to illustrate certain embodiments of the present disclosure.

[0211] Example

[0212] The reagents and equipment used in the examples of this disclosure are conventional and commercially available. For example:

[0213] Example 1

[0214] (1) Modification of small molecule VHL compound: DSPE-PEG2000-NHS and VHL small molecule (1:1, molar ratio) were weighed in a 25 mL round-bottom flask, and dichloromethane (DCM) was added to fully dissolve them. Then, N,N-diisopropylethylamine (DIPEA) basic compound (1:2, DIPEA:VHL, molar ratio) was added to maintain the reaction system in an alkaline environment. The reaction was carried out at room temperature with uniform stirring for 24 h, and then the product was separated and purified.

[0215] (2) Modification of small molecule Tam compound: DSPE-PEG2000-NHS and Tam small molecule (1:1, molar ratio) were weighed, and after adding dichloromethane (DCM) to fully dissolve, N,N-diisopropylethylamine (DIPEA) basic compound (1:2, DIPEA:VHL, molar ratio) was added to maintain the reaction system in an alkaline environment. The reaction was carried out at room temperature with uniform stirring for 24 hours, and then the product was separated and purified.

[0216] (3) Modification of small molecule Mitb compound: DSPE-PEG2000-NHS and Mitb small molecule (1:1, molar ratio) were weighed, and after adding dichloromethane (DCM) to fully dissolve, N,N-diisopropylethylamine (DIPEA) basic compound (1:2, DIPEA:VHL, molar ratio) was added to maintain the reaction system in an alkaline environment. The reaction was carried out at room temperature with uniform stirring for 24 h, and then the product was separated and purified.

[0217] (4) Modification of small molecule Cer compounds: DSPE-PEG2000-NHS and Cer small molecules (1:1, molar ratio) were weighed and fully dissolved in dichloromethane (DCM). Then, N,N-diisopropylethylamine (DIPEA) alkaline compound (1:2, DIPEA:VHL, molar ratio) was added to maintain the reaction system in an alkaline environment. The reaction was carried out at room temperature with uniform stirring for 24 h, and then the product was separated and purified.

[0218] (5) Isolation and purification of compounds DSPE-PEG2000-VHL, DSPE-PEG2000-Tam, DSPE-PEG2000-Mitb, and DSPE-PEG2000-Cer: The reaction products were obtained according to the above method. First, the reaction solvent was dried using a rotary evaporator, and then the resolubilizing organic solvent DMF was added to dissolve the reaction product into a liquid state. Then, the dialysis bag (1000 Da) was cleaned with deionized water. The liquid product was then placed in the dialysis bag, sealed, and dialyzed with deionized water for 72 hours, with the water changed continuously during the dialysis. Finally, the purified product in the dialysis bag was collected and freeze-dried to obtain the modified product. A small amount of the product was dissolved and identified by small molecule high-resolution mass spectrometry (as shown in Figure 2).

[0219] DSPE-PEG2000-Tam, DSPE-PEG2000-VHL, DSPE-PEG2000-Mitb and DSPE-PEG2000-Cer compounds were obtained by the above method.

[0220] Example 2

[0221] (1) Hydrogenated soybean lecithin (HSPC), cholesterol (CHO), distearoylphosphatidyl acetamide-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-VHL, DSPE-PEG2000-Tam, and DSPE-PEG2000-FA were weighed in a molar ratio of 45:35:5:1:9:5 and dissolved in chloroform. The mixed solution was sonicated for 10 min at an input power of 50% and thoroughly mixed. The organic solvent was evaporated and removed at room temperature under ventilation to form a transparent film.

[0222] (2) Add 2 mL of deionized water to cover the liposome film in step (1), place in a constant temperature metal bath reactor, heat at 65 ° C, 650 r·min -1 Under the conditions of , micro-oscillation and incubation for 2 h to form a preliminary lipid mixed solution.

[0223] (3) The preliminary liposome solution prepared in step (2) is repeatedly extruded through a polycarbonate membrane extruder with a pore size of 100 to 400 nm to obtain preliminary liposomes.

[0224] (4) The preliminary liposomes obtained in step (3) were replaced with deionized water or phosphate buffer as the replacement liquid, and ultrafiltration was performed for 10 min at 4°C and 2000 rpm using a 100 kD ultrafiltration tube. The ultrafiltration was repeated to concentrate the liposome concentrate, which was stored in a refrigerator at 4°C until use (FA-V-Tam-L is the abbreviation of the assembled liposomes, which mainly contains DSPE-PEG2000-FA, DSPE-PEG2000-VHL and DSPE-PEG2000-Tam).

[0225] Example 3

[0226] (1) Hydrogenated soybean lecithin (HSPC), cholesterol (CHO), distearoylphosphatidyl acetamide-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-VHL and DSPE-PEG2000-Mitb were weighed in a molar ratio of 50:35:5:1:9 and dissolved in chloroform. The mixed solution was ultrasonicated at an input power of 50% for 10 min and mixed thoroughly. The organic solvent was evaporated and removed under ventilation at room temperature to form a transparent film.

[0227] (2) Add 2 mL of deionized water to cover the liposome film in step (1), place in a constant temperature metal bath reactor, heat at 65 ° C, 650 r·min -1 Under the conditions of , micro-oscillation and incubation for 2 h to form a preliminary lipid mixed solution.

[0228] (3) The preliminary liposome solution prepared in step (2) is repeatedly extruded through a polycarbonate membrane extruder with a pore size of 100 to 400 nm to obtain preliminary liposomes.

[0229] (4) The preliminary liposomes obtained in step (3) were replaced with deionized water or phosphate buffer as the replacement liquid, and ultrafiltration was performed for 10 min at 4°C and 2000 rpm using a 100 kD ultrafiltration tube. The ultrafiltration was repeated to concentrate the liposome concentrate, which was stored in a refrigerator at 4°C until use (V-Mitb-L is the abbreviation of the assembled liposomes, which mainly contains DSPE-PEG2000-VHL and DSPE-PEG2000-Mitb).

[0230] Example 4

[0231] (1) Hydrogenated soybean lecithin (HSPC), cholesterol (CHO), distearoylphosphatidyl acetamide-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-VHL, and DSPE-PEG2000-Cer were weighed in a molar ratio of 50:35:5:1:9 and dissolved in chloroform. The mixed solution was sonicated for 10 min at an input power of 50% and thoroughly mixed. The organic solvent was evaporated and removed under ventilation at room temperature to form a transparent film.

[0232] (2) Add 2 mL of deionized water to cover the liposome film in step (1), place in a constant temperature metal bath reactor, heat at 65 ° C, 650 r·min -1 Under the conditions of , micro-oscillation and incubation for 2 h to form a preliminary lipid mixed solution.

[0233] (3) The preliminary liposome solution prepared in step (2) is repeatedly extruded through a polycarbonate membrane extruder with a pore size of 100 to 400 nm to obtain preliminary liposomes.

[0234] (4) The preliminary liposomes obtained in step (3) were replaced with deionized water or phosphate buffer as the replacement liquid, and a 100 kD ultrafiltration tube was selected. Ultrafiltration was performed at 4°C and 2000 rpm for 10 min. The ultrafiltration was repeated to concentrate the liposome concentrate, which was stored in a refrigerator at 4°C until use (V-Cer-L is the abbreviation of the assembled liposomes, which mainly contains DSPE-PEG2000-VHL and DSPE-PEG2000-Cer).

[0235] Example 5

[0236] (1) Hydrogenated soybean lecithin (HSPC), cholesterol (CHO), distearoylphosphatidyl acetamide-polyethylene glycol 2000 (DSPE-mPEG2000), DSPE-PEG2000-FA, and DSPE-PEG2000-FITC were weighed at a molar ratio of 50:35:5:5:5 and dissolved in chloroform. The mixed solution was sonicated at an input power of 50% for 10 min and thoroughly mixed. The organic solvent was evaporated and removed under ventilation at room temperature to form a transparent film.

[0237] (2) Add 2 mL of deionized water to cover the liposome film in step (1) and place in a constant temperature oscillating reactor at 65°C and 650 r·min -1 Under the conditions of , micro-oscillation and incubation for 2 h to form a preliminary lipid mixed solution.

[0238] (3) repeatedly extruding the preliminary liposome solution prepared in step (2) through a polycarbonate membrane extruder having a pore size of 100 to 400 nm to obtain liposomes;

[0239] (4) The liposomes obtained in step (3) were replaced with deionized water or phosphate buffer solution, and ultrafiltration was performed for 10 min at 4°C and 2000 rpm using a 100 kD ultrafiltration tube. The ultrafiltration was repeated three times to obtain the liposomes, which were then placed in a refrigerator at 4°C for use.

[0240] The purpose of preparing fluorescein-containing liposomes is to detect whether breast cancer cells can better absorb the prepared liposomes by flow cytometry.

[0241] Example 6

[0242] Using the liposomes prepared in Example 2 of the present disclosure, a solution with a liposome concentration of 0.5 mg / mL (deionized water or phosphate buffer) was taken, and the particle size of the prepared liposomes was measured using a dynamic light scattering instrument DLS, which was less than 200 nm, and the dispersion index was less than 0.3 (as shown in Figure 3). A solution with a liposome concentration of 0.5 mg / mL (deionized water or phosphate buffer) was taken, and the absolute value of the surface Zeta potential of the prepared liposomes was measured using a dynamic light scattering instrument DLS, which was less than 30 mV (as shown in Figure 4).

[0243] It can be seen that the polypeptide liposomes prepared by this system are negatively charged, small-sized, and uniformly dispersed nanoparticles.

[0244] Example 7

[0245] In this example, breast cancer cell line MCF7 (folate receptor positive, estrogen receptor α positive) was selected as a representative to evaluate whether the prepared liposomes can affect the proliferation of cancer cells such as breast cancer cells.

[0246] (1) Cell culture: The breast cancer cells used in the experiment were adherent cells and were cultured according to the adherent cell culture method. MCF7 cells were cultured in MEM medium containing 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 0.001% human insulin at 37°C in a CO2 incubator with 5% humidity.

[0247] (2) Cell plating: Take cells in the logarithmic growth phase, digest them with trypsin, collect the cells by centrifugation, count them using a hemocytometer, and add 5,000 cells per well to inoculate them into a 96-well plate, where the outer surface is a pH 7.4 phosphate buffer. After the cells grow and adhere to the wall overnight, add the drug that needs to be stimulated.

[0248] (3) Drug dilution: Use pure MEM culture medium to dilute the drug. If it contains organic solvents such as DMSO, the content of DMSO solvent should be less than one thousandth, and the drug must be prepared before use.

[0249] (4) Addition and testing of drugs: After the cells adhere to the wall, the culture medium is discarded and the diluted drug is slowly added along the wall of the well plate. After incubation for 24 hours, the commercial CCK-8 reagent is directly added, 10 μL per well, and incubated for 1 to 4 hours. During this period, the OD value of the 96-well plate is tested using an enzyme reader at a wavelength of 450 nm. The test results of the enzyme reader are normalized to obtain the data results of the cell proliferation experiment (as shown in Figure 5(a)).

[0250] Example 8

[0251] In this example, the melanoma A375 cell line was selected as a representative to evaluate whether the prepared liposomes can affect the proliferation of cancer cells such as melanoma cells.

[0252] (1) Cell culture: The melanoma cells used in the experiment were adherent cells and were cultured according to the adherent cell culture method. A375 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin in a 37°C, 5% humidity, carbon dioxide incubator.

[0253] (2) Cell plating: Take cells in the logarithmic growth phase, digest them with trypsin, collect the cells by centrifugation, count them using a hemocytometer, and add 5,000 cells per well to inoculate them into a 96-well plate, where the outer surface is a pH 7.4 phosphate buffer. After the cells grow and adhere to the wall overnight, add the drug that needs to be stimulated.

[0254] (3) Drug dilution: Use pure DMEM culture medium to dilute the drug. If it contains organic solvents such as DMSO, the content of DMSO solvent should be less than one thousandth, and the drug must be prepared before use.

[0255] (4) Drug addition and testing: After the cells adhere to the wall, the culture medium is discarded and the diluted drug is slowly added along the wall of the well plate. After incubation for 48 hours, the commercial CCK-8 reagent is directly added, 10 μL per well, and incubated for 1 to 4 hours. During this period, the OD value of the 96-well plate is tested using an enzyme reader at a wavelength of 450 nm. The test results of the enzyme reader are normalized to obtain the data results of the cell proliferation experiment (as shown in Figure 5(b)).

[0256] Example 9

[0257] In this example, the non-small cell lung cancer NCI-H2228 cell line was selected as a representative to evaluate whether the prepared liposomes can affect the proliferation of cancer cells such as non-small cell lung cancer.

[0258] (1) Cell culture: The non-small cell lung cancer cells used in the experiment were adherent cells and were cultured according to the adherent cell culture method. NCI-H2228 cells were cultured in 1640 medium containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin in a 37°C, 5% humidity, carbon dioxide incubator.

[0259] (2) Cell plating: Take cells in the logarithmic growth phase, digest them with trypsin, collect the cells by centrifugation, count them using a hemocytometer, and add 5,000 cells per well to inoculate them into a 96-well plate, where the outer surface is a pH 7.4 phosphate buffer. After the cells grow and adhere to the wall overnight, add the drug that needs to be stimulated.

[0260] (3) Drug dilution: Use pure 1640 culture medium to dilute the drug. If it contains organic solvents such as DMSO, the content of DMSO solvent must be less than one thousandth, and the drug must be prepared before use.

[0261] (4) Drug addition and testing: After the cells adhere to the wall, the culture medium is discarded and the diluted drug is slowly added along the wall of the well plate. After incubation for 24 hours, the commercial CCK-8 reagent is directly added, 10 μL per well, and incubated for 1 to 4 hours. During this period, the OD value of the 96-well plate is tested using an enzyme reader at a wavelength of 450 nm. The test results of the enzyme reader are normalized to obtain the data results of the cell proliferation experiment (as shown in Figure 5(c)).

[0262] Example 10

[0263] In order to evaluate the degradation of estrogen receptor α protein in breast cancer cells by the liposomes prepared in the present invention, this example selected the breast cancer MCF7 cell line (folate receptor positive, estrogen receptor α positive) as a representative to evaluate whether the prepared liposomes can degrade estrogen receptor α at the protein level and further affect the apoptosis of breast cancer cells.

[0264] (1) Liposomes were prepared according to the thin film dispersion method described in the present disclosure.

[0265] (2) The breast cancer MCF7 cell line in the logarithmic growth phase was selected, digested with trypsin, and the cells were collected by centrifugation. The cells were seeded into a 12-well plate. After the cells grew and adhered overnight, the prepared liposome drug was added and incubated with the cells for a certain period of time.

[0266] (3) Discard the co-incubation culture medium, wash with PBS 1 to 3 times, add commercial RIPA rapid lysis buffer containing protease inhibitors, lyse at 4°C for 30 minutes, then centrifuge at high speed for 5 to 10 minutes, and determine the protein concentration using a BCA kit.

[0267] (4) Use protein loading solution to dilute the extracted protein, and the protein loading amount per well is 5-20 μg.

[0268] (5) Western Blot (WB) was used to verify the degradation of estrogen receptor α protein in breast cancer cells by the prepared liposomes.

[0269] As shown in Figures 6a and 6b, after the liposomes prepared by the method were incubated with MCF7 cells for 24 hours, the cells were collected and lysed, and then detected by protein immunoblotting (Western Blot, WB) and incubated with anti-estrogen receptor α and anti-GAPDH antibodies, it can be seen that FA-V-Tam-L showed significant concentration-dependency and time-dependency in the degradation of 7 estrogen receptor α protein, indicating that the FA-V-Tam-L liposomes prepared by the method can degrade estrogen receptor α and promote the apoptosis of breast cancer cells MCF7.

[0270] Example 11

[0271] In order to evaluate the degradation of MEK1 / MEK2 overexpressed proteins in melanoma cells by the liposomes prepared in the present disclosure, this example selected the melanoma A375 cell line as a representative to evaluate whether the prepared liposomes can degrade MEK1 / MEK2 at the protein level and further affect the apoptosis of melanoma cells.

[0272] (1) Liposomes were prepared according to the thin film dispersion method described in the present disclosure.

[0273] (2) Melanoma A375 cell lines in the logarithmic growth phase were selected, digested with trypsin, and collected by centrifugation. The cells were seeded into a 12-well plate. After the cells grew and adhered overnight, the prepared liposome drug was added and incubated with the cells for a certain period of time.

[0274] (3) Discard the co-incubation culture medium, wash with PBS 1 to 3 times, add commercial RIPA rapid lysis buffer containing protease inhibitors, lyse at 4°C for 30 minutes, then centrifuge at high speed for 5 to 10 minutes, and determine the protein concentration using a BCA kit.

[0275] (4) Use protein loading solution to dilute the extracted protein, and the protein loading amount per well is 5-20 μg.

[0276] (5) Western Blot (WB) was used to verify the degradation of melanoma MEK1 / MEK2 protein by the prepared liposomes.

[0277] As shown in Figures 6c and 6d, after the liposomes prepared by the method were incubated with A375 cells for a certain period of time, the cells were collected and lysed, and then detected by protein immunoblotting (Western Blot, WB) using anti-estrogen receptor α and anti-GAPDH antibodies. It can be seen that V-Mitb-L showed significant concentration-dependency and time-dependency in the degradation of MKE1 / MEK2 proteins, indicating that the V-Mitb-L liposomes prepared by the method can degrade MEK1 / MEK2 and promote the apoptosis of melanoma cells A375.

[0278] Example 12

[0279] In order to evaluate the degradation of the human non-small cell lung cancer overexpressed protein Alk by the liposomes prepared in the present invention, this example selected the human non-small cell lung NCI-H2228 cell line as a representative to evaluate whether the prepared liposomes can degrade Alk at the protein level and further affect the apoptosis of human non-small cell lung cancer cells.

[0280] (1) Liposomes were prepared according to the thin film dispersion method described in the present disclosure.

[0281] (2) Human non-small cell lung cancer NCI-H2228 cell line in the logarithmic growth phase was selected, digested with trypsin, and the cells were collected by centrifugation. The cells were seeded into a 12-well plate. After the cells grew and adhered overnight, the prepared liposome drug was added and incubated with the cells for a certain period of time.

[0282] (3) Discard the co-incubation culture medium, wash with PBS 1 to 3 times, add commercial RIPA rapid lysis buffer containing protease inhibitors, lyse at 4°C for 30 minutes, then centrifuge at high speed for 5 to 10 minutes, and determine the protein concentration using a BCA kit.

[0283] (4) Use protein loading solution to dilute the extracted protein, and the protein loading amount per well is 5-20 μg.

[0284] (5) Western Blot (WB) was used to verify the degradation of melanoma MEK1 / MEK2 protein by the prepared liposomes.

[0285] As shown in Figures 6e and 6f, after the liposomes prepared by the method were incubated with NCI-H2228 cells for a certain period of time, the cells were collected and lysed, and then detected by protein immunoblotting (Western Blot, WB) and incubated with anti-estrogen receptor α and anti-GAPDH antibodies, it can be seen that V-Cer-L showed significant concentration-dependency and time-dependency in the degradation of Alk protein, indicating that the V-Cer-L liposomes prepared by the method can degrade Alk protein and promote the apoptosis of human non-small cell lung cancer cells NCI-H2228.

[0286] Example 13

[0287] In order to evaluate the effect of the liposomes disclosed herein on the uptake of folate receptor-negative and -positive cells, this example selected folate receptor-negative cells (THP-1) and folate receptor-positive cells (MCF7) as representatives to evaluate whether the prepared folate-containing (FA-FITC) and folate-free fluorescent (FITC) liposomes affect the uptake of liposomes at the cellular level.

[0288] (1) Liposomes were prepared according to the thin film dispersion method described in the present disclosure.

[0289] (2) THP-1 and MCF7 cells in the logarithmic growth phase were selected, digested with trypsin, and collected by centrifugation. The cells were seeded into 12-well plates. After the cells grew overnight, the prepared liposome drugs were added at different time points and incubated with the cells.

[0290] (3) In the dark, MCF7 cells were collected by trypsin digestion, THP-1 cells were directly collected, and washed 2 to 3 times by repeated centrifugation (1000 rpm) with phosphate buffer. Finally, they were dispersed in 500 μL of phosphate buffer and tested on a BD flow cytometer.

[0291] Figure 7 shows the trend of FA-FITC and FITC liposomes after incubation with MCF7 and THP-1 cells for 1, 3, and 6 hours, respectively, followed by flow cytometry analysis. As shown in Figure 7, the uptake of FA-FITC liposomes by folate receptor-positive MCF7 cells was significantly time-dependent, as was the uptake of FITC liposomes. However, the uptake in the FA-FITC liposome group was significantly greater than that in the FITC liposome group. For folate receptor-negative THP-1 cells, both FA-FITC and FITC liposomes showed a weaker time-dependency, and there was essentially no difference in the uptake of liposomes containing or without folic acid by THP-1 cells.

[0292] Therefore, the liposomes disclosed herein that target estrogen receptor degradation with folate receptors have a strong folate targeting function.

[0293] In this disclosure, relational terms such as first and second, etc. are used merely to distinguish one entity or operation from another entity or operation, but do not necessarily require or imply any actual relationship or order between these entities or operations.

[0294] It will be appreciated from the foregoing that, although specific embodiments of the present disclosure have been described for illustrative purposes, various modifications or variations may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. Such modifications or variations are intended to fall within the scope of the appended claims of the present disclosure.

Claims

1. A substance represented by formula (I), in, R is a protein, a polypeptide or a compound containing an amino group, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

2. The substance represented by formula (I) according to claim 1, wherein R is a ligand R1 of E3 ubiquitin ligase.

3. The substance represented by formula (I) according to claim 2, wherein the ligand R1 of the E3 ubiquitin ligase is selected from VHL ligand, MDM2 ligand, CRBN ligand, DCAF15 ligand, DCAF16 ligand, RNF4 ligand, RNF114 ligand, IAP ligand or cIAP1 ligand.

4. The substance represented by formula (I) as claimed in claim 1, wherein R is a ligand R2 of a target protein.

5. The substance as described in claim 4, wherein the ligand R2 of the target protein is an estrogen receptor α ligand, an Alk ligand, a MEK ligand, a BCL-XL ligand, a BCR-ABL ligand, a BRD2 / 3 / 4 ligand, a BTK ligand, a CDK6 / 9 ligand, a CRABP-1 ligand, a FLT-1 ligand, a FAK ligand, a FKBP12 ligand, a FLT-3 ligand, a PARP1 ligand, a PCAF / GCN5 ligand, a RIPK2 ligand, a STAT3 ligand, a TACC3 ligand, a TBK1 ligand, a TRIM24 ligand and a P38 ligand, preferably selected from 4-hydroxytamoxifen (Tam), toremifene, tamoxifen, endoxifen, Ceritinib (Cer) or Mirdametinib (Mitb).

6. A substance as represented by formula (I) according to any one of claims 1 to 5, which is selected from:

7. A method for preparing a substance represented by formula (I), It includes: The compound represented by formula (II) is reacted with R, Wherein, R is a protein containing an amino group, a polypeptide containing an amino group, or a chemical containing an amino group. The compound is a monovalent cation, M is a monovalent cation, m is 10 to 100, and the hydrogen atoms in the substance represented by formula (I) are arbitrarily substituted.

8. The method according to claim 7, wherein the molar ratio of the compound represented by formula (II) to R is 1-5:1, preferably 1:

1.

9. The method according to claim 7 or 8, wherein an alkaline reagent is added to react the compound represented by formula (II) with R, and the alkaline reagent is preferably selected from N,N-diisopropylethylamine (DIPEA), triethylamine, phosphate buffer and any mixture thereof.

10. The method of claim 9, wherein the amount of the alkaline agent added is 1 to 5 equivalents based on the R.

11. The method of any one of claims 7 to 10, wherein the reaction of the compound of formula (II) with R is carried out in a polar solvent, the polar solvent being preferably selected from dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, chloroform, dichloromethane and any mixture thereof.

12. The substance represented by formula (I) prepared by the method according to any one of claims 7 to 11.

13. A liposome comprising the substance represented by the formula (I) according to claim 2 or 3 and the substance represented by the formula (I) according to claim 4 or 5.

14. The liposome according to claim 13, wherein the substance represented by formula (I) according to claim 2 or 3 is 15. The liposome according to claim 13 or 14, wherein the substance represented by formula (I) according to claim 4 or 5 is 16. The liposome according to any one of claims 13 to 15, wherein the particle size of the liposome is 130 to 180 nm.

17. The liposome according to any one of claims 13 to 16, wherein the liposome is obtained by self-assembly of the substance represented by formula (I) according to claim 2 or 3 and the substance represented by formula (I) according to claim 4 or 5 using a thin film dispersion method.

18. A method for preparing liposomes, comprising: The liposome is obtained by self-assembling the substance represented by formula (I) according to claim 2 or 3 and the substance represented by formula (I) according to claim 4 or 5 using a thin film dispersion method.

19. The method according to claim 18, wherein the thin film dispersion method is used to self-assemble the substance represented by formula (I) according to claim 102 or 103 and the substance represented by formula (I) according to claim 104 or 105 with a lipid membrane excipient to obtain the liposome.

20. The method of claim 19, wherein the lipid membrane is selected from the group consisting of phospholipids, glycolipids, cholesterol, and any mixture thereof.

21. A pharmaceutical composition comprising the liposome according to any one of claims 13 to 16, and a pharmaceutically acceptable excipient.

22. A method for treating or preventing tumors, comprising administering a therapeutically or preventively effective amount of the liposome according to any one of claims 13 to 16, or a therapeutically or preventively effective amount of the pharmaceutical composition according to claim 21, to an individual in need thereof.

23. The method of claim 22, wherein the individual is a mammal, preferably a human.

24. The method of claim 22 or 23, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), low-grade serous ovarian cancer, neurofibroma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, and esophageal cancer.

25. A method for degrading a target protein (Protein of Interest, POI), comprising administering the liposome according to any one of claims 13 to 16 and an E3 ubiquitin ligase to the target protein (Protein of Interest, POI).

Images