Carboxyl fullerene derivative for inhibiting tumor growth

Abstract

The invention relates to the field of biological medicines, and provides a carboxyl fullerene derivative for inhibiting tumor growth. The carboxyl fullerene derivative disclosed by the invention plays an anti-tumor role by blocking a tumor cell cycle and reducing the expression quantity of STEAP1, and is relatively high in safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an anti-tumor carboxyl fullerene derivative. BACKGROUND

[0002] Cancer (malignant tumor) is a phenomenon of abnormal proliferation and metastasis caused by genetic mutation of cells, which has become one of the major causes of death of the world population. The existing tumor treatment methods mainly include surgical resection, chemotherapy, radiotherapy and targeted therapy, etc., among which the targeted therapy has strong specificity and small side effects, and the development of high-efficiency and safe anti-tumor drugs targeting specific targets has become one of the major problems to be solved in contemporary medicine.

[0003] Fullerenes are a class of closed cage-like molecules composed of an even number of carbon atoms. As a class of low-toxicity nanomaterials, fullerenes and their functional derivatives have been proven to play an important role in regulating various biochemical processes, and have been widely used in the treatment of diseases such as cancer, pulmonary fibrosis, radiation and chemotherapy damage, metabolic diseases, and neurodegenerative diseases. Among them, hydroxylated fullerene derivatives can inhibit tumor growth through blocking tumor blood vessels or inhibiting angiogenesis, enhancing immune infiltration, and reducing oxidative stress. Amino-functionalized fullerene derivatives with a clear molecular structure are easily taken up by cells due to their positive charge, can directly bind to proteins regulating cell movement and inhibit their function, and arrest the cell cycle at the G0 / G1 phase, directly inhibiting the growth and metastasis of tumor cells and reversing the epithelial-mesenchymal transition process. In contrast, carboxylated fullerene derivatives also have anti-tumor activity, but there is currently no basic and applied research on the biological molecular mechanisms of carboxylated fullerene derivatives in the field of tumor treatment. SUMMARY

[0004] The purpose of the present application is to provide a new water-soluble carboxylated fullerene derivative for inhibiting tumor growth. The carboxylated fullerene has amphiphilic properties and good solubility in water. The carboxylated fullerene derivative can specifically target and bind to STEAP1 protein, down-regulate the expression of STEAP1, and further arrest the cell cycle at the G0 / G1 phase and inhibit the migration of tumor cells. The carboxylated fullerene derivative has a significant inhibitory effect on malignant tumors with high expression of STEAP1.

[0005] In one aspect of the present application, a fullerene derivative of formula (I) or a salt thereof is provided for use in the preparation of a medicament for treating tumors with high expression of STEAP1.

[0006]

[0007] wherein,

[0008] F is selected from fullerenes, wherein the fullerene is selected from one or more of hollow fullerenes, metallofullerenes, heterocyclic fullerenes, and endogenous fullerenes;

[0009] R is a modifying group on a fullerene;

[0010] m is an integer selected from 1 to 12;

[0011] R is selected from -NR 1 R 2 C 1-10 Alkyl, -C 1-10 Alkyl-NR 1 R 2 -P(C=O)R 3 R 4 or

[0012] R1, R2, R3, and R4 are each independently selected from hydrogen, hydroxyl, halogen, and C. 1-10 Alkyl, C 1-10 Alkoxy, C 1-10 Alkyl-C1-10 alkoxy, C 1-10 Alkyl-C 1-10 Carboxyl group, C 6-10 Aryl, C 1-10 Alkyl-C 6-10 Aryl, C 1-10 Alkyl-5 to 20-membered heterocyclic aryl groups; the C 1-10 In the alkyl group, 1, 2, 3, 4, 5, 6, 7, 8, and 9 -CH2- can be replaced by -O-CH2CH2-; or R1 and R2 form a 5- to 20-membered heterocyclic alkyl group with their adjacent N atom; the C 1-10 Alkyl, C 1-10 Alkoxy, C 6-10 aryl, 5 to 20-membered heterocyclic aryl, optionally surrounded by one or more members selected from R 11 Substituents of the substituents;

[0013] Alternatively, when m is not 1, two arbitrarily connected Rs form C. 3-20 Cycloalkyl or 3 to 20-membered heterocycloalkyl; the C 3-20 Cycloalkyl or 3 to 20-membered heterocyclic alkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents;

[0014] n1 and n2 are each independently selected from 0, 1 or 2;

[0015] X is selected from CH or N;

[0016] R 11 Selected from amino, C 1-6 Alkyl, C 1-6 Carboxyl group, -C1-6 Alkyl-amino, C 1-10 Alkyl-C 1-10 Carboxyl group, C 1-10 Alkyl-C 6-10 Aryl, -(C=O)OC 1-10 Alkyl-5 to 10-membered heterocyclic aryl group, wherein the 5 to 10-membered heterocyclic aryl group is optionally surrounded by one or more amino groups, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-6 Alkyl-C 1-6 Carboxyl substitution.

[0017] In one aspect, the present invention provides a fullerene derivative of formula (I) or a salt thereof for treating tumors with high STEAP1 expression:

[0018]

[0019] in,

[0020] F is selected from fullerenes, wherein the fullerene is selected from one or more of hollow fullerenes, metallofullerenes, heterocyclic fullerenes, and endogenous fullerenes;

[0021] R is a modifying group on a fullerene;

[0022] m is an integer selected from 1 to 12;

[0023] R is selected from -NR 1 R 2 C 1-10 Alkyl, -C 1-10 Alkyl-NR 1 R 2 -P(C=O)R 3 R 4 or

[0024] R1, R2, R3, and R4 are each independently selected from hydrogen, hydroxyl, halogen, and C. 1-10 Alkyl, C 1-10 Alkoxy, C 1-10 Alkyl-C1-10 alkoxy, C 1-10 Alkyl-C 1-10 Carboxyl group, C 6-10 Aryl, C 1-10 Alkyl-C 6-10 Aryl, C 1-10 Alkyl-5 to 20-membered heterocyclic aryl groups; the C 1-10In the alkyl group, 1, 2, 3, 4, 5, 6, 7, 8, and 9 -CH2- can be replaced by -O-CH2CH2-; or R1 and R2 form a 5- to 20-membered heterocyclic alkyl group with their adjacent N atom; the C 1-10 Alkyl, C 1-10 Alkoxy, C 6-10 aryl, 5 to 20-membered heterocyclic aryl, optionally surrounded by one or more members selected from R 11 Substituents of the substituents;

[0025] Alternatively, when m is not 1, two arbitrarily connected Rs form C. 3-20 Cycloalkyl or 3 to 20-membered heterocycloalkyl; the C 3-20 Cycloalkyl or 3 to 20-membered heterocyclic alkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents;

[0026] n1 and n2 are each independently selected from 0, 1 or 2;

[0027] X is selected from CH or N;

[0028] R 11 Selected from amino, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-10 Alkyl-C 1-10 Carboxyl group, C 1-10 Alkyl-C 6-10 Aryl, -(C=O)OC 1-10 Alkyl-5 to 10-membered heterocyclic aryl group, wherein the 5 to 10-membered heterocyclic aryl group is optionally surrounded by one or more amino groups, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-6 Alkyl-C 1-6 Carboxyl substitution.

[0029] In one aspect, the present invention provides a method for treating tumors with high STEAP1 expression, wherein the method comprises administering a fullerene derivative of formula (I) or a salt thereof to a subject in need.

[0030]

[0031] in,

[0032] F is selected from fullerenes, wherein the fullerene is selected from one or more of hollow fullerenes, metallofullerenes, heterocyclic fullerenes, and endogenous fullerenes;

[0033] R is a modifying group on a fullerene;

[0034] m is an integer selected from 1 to 12;

[0035] R is selected from -NR 1 R 2 C 1-10 Alkyl, -C 1-10 Alkyl-NR 1 R 2 -P(C=O)R 3 R 4 or

[0036] R1, R2, R3, and R4 are each independently selected from hydrogen, hydroxyl, halogen, and C. 1-10 Alkyl, C 1-10 Alkoxy, C 1-10 Alkyl-C1-10 alkoxy, C 1-10 Alkyl-C 1-10 Carboxyl group, C 6-10 Aryl, C 1-10 Alkyl-C 6-10 Aryl, C 1-10 Alkyl-5 to 20-membered heterocyclic aryl groups; the C 1-10 In the alkyl group, 1, 2, 3, 4, 5, 6, 7, 8, and 9 -CH2- can be replaced by -O-CH2CH2-; or R1 and R2 form a 5- to 20-membered heterocyclic alkyl group with their adjacent N atom; the C 1-10 Alkyl, C 1-10 Alkoxy, C 6-10 aryl, 5 to 20-membered heterocyclic aryl, optionally surrounded by one or more members selected from R 11 Substituents of the substituents;

[0037] Alternatively, when m is not 1, two arbitrarily connected Rs form C. 3-20 Cycloalkyl or 3 to 20-membered heterocycloalkyl; the C 3-20 Cycloalkyl or 3 to 20-membered heterocyclic alkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents;

[0038] n1 and n2 are each independently selected from 0, 1 or 2;

[0039] X is selected from CH or N;

[0040] R 11 Selected from amino, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-10 Alkyl-C 1-10 Carboxyl group, C 1-10 Alkyl-C 6-10 Aryl, -(C=O)OC 1-10Alkyl-5 to 10-membered heterocyclic aryl group, wherein the 5 to 10-membered heterocyclic aryl group is optionally surrounded by one or more amino groups, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-6 Alkyl-C 1-6 Carboxyl substitution.

[0041] In one aspect, the present invention provides a fullerene derivative of formula (I) or a salt thereof for the treatment of tumors with high STEAP1 expression.

[0042]

[0043] in,

[0044] F is selected from fullerenes, wherein the fullerene is selected from one or more of hollow fullerenes, metallofullerenes, heterocyclic fullerenes, and endogenous fullerenes;

[0045] R is a modifying group on a fullerene;

[0046] m is an integer selected from 1 to 12;

[0047] R is selected from -NR 1 R 2 C 1-10 Alkyl, -C 1-10 Alkyl-NR 1 R 2 -P(C=O)R 3 R 4 or

[0048] R1, R2, R3, and R4 are each independently selected from hydrogen, hydroxyl, halogen, and C. 1-10 Alkyl, C 1-10 Alkoxy, C 1-10 Alkyl-C1-10 alkoxy, C 1-10 Alkyl-C 1-10 Carboxyl group, C 6-10 Aryl, C 1-10 Alkyl-C 6-10 Aryl, C 1-10 Alkyl-5 to 20-membered heterocyclic aryl groups; the C 1-10 In the alkyl group, 1, 2, 3, 4, 5, 6, 7, 8, and 9 -CH2- can be replaced by -O-CH2CH2-; or R1 and R2 form a 5- to 20-membered heterocyclic alkyl group with their adjacent N atom; the C 1-10 Alkyl, C 1-10 Alkoxy, C 6-10 aryl, 5 to 20-membered heterocyclic aryl, optionally surrounded by one or more members selected from R 11 Substituents of the substituents;

[0049] Alternatively, when m is not 1, two arbitrarily connected Rs form C. 3-20 Cycloalkyl or 3 to 20-membered heterocycloalkyl; the C 3-20 Cycloalkyl or 3 to 20-membered heterocyclic alkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents;

[0050] n1 and n2 are each independently selected from 0, 1 or 2;

[0051] X is selected from CH or N;

[0052] R 11 Selected from amino, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-10 Alkyl-C 1-10 Carboxyl group, C 1-10 Alkyl-C 6-10 Aryl, -(C=O)OC 1-10 Alkyl-5 to 10-membered heterocyclic aryl group, wherein the 5 to 10-membered heterocyclic aryl group is optionally surrounded by one or more amino groups, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-6 Alkyl-C 1-6 Carboxyl substitution.

[0053] In some implementations, F is selected from C. 2n M@C 2n M2@C 2n MA@C 2n M3N@C 2n M2C2@C 2n M2S@C 2n M2O@C 2n and M x A 3-x N@C 2n Any one or a mixture thereof, wherein M and A are both metallic elements, and M and A are selected from any one of Sc, Y and lanthanide elements, and 30≤n≤60.

[0054] In some embodiments, the fullerene is selected from C-containing... 2n One or more fullerene molecules, wherein 2n is the number of carbon atoms, 30 ≤ n ≤ 60; in some embodiments, the fullerene is selected from C 60 C 70 C 76 C 78 C 80 and C 84 One or more of the following.

[0055] In some embodiments, the fullerene is selected from C 60 and C 70 One or more of the following.

[0056] In some implementations, R is selected from -NR 1 R 2 C 1-6 alkyl, or

[0057] Alternatively, when m is not 1, two adjacent R are connected to form C. 3-10 Cycloalkyl or 3 to 10-membered heterocycloalkyl; the C 3-10 Cycloalkyl or 3 to 10-membered heterocycloalkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents;

[0058] Among them, R 1 R 2 n1, n2, X, R 11 Each custom definition is the same as before;

[0059] In some implementations, R is selected from -NR 1 R 2 C 1-3 alkyl, or

[0060] Alternatively, when m is not 1, two adjacent Rs are linked to form a 3- to 6-membered heterocyclic alkyl group; the 3- to 6-membered heterocyclic alkyl group is optionally connected by one or more elements selected from Rs. 11 Substituents of the substituents;

[0061] Among them, R 1 R 2 n1, n2, X, R 11 Each definition is the same as before.

[0062] In some implementations, R is selected from -NR 1 R 2 C 1-3 alkyl, or

[0063] Alternatively, when m is not 1, two adjacent Rs are connected to form an epoxy propylene alkyl group or a pyrrolidinyl group; the epoxy propylene alkyl group or the pyrrolidinyl group is optionally composed of one or more Rs selected from Rs. 11 Substituents of the substituents;

[0064] Among them, R 1 R 2 n1, n2, X, R 11 Each definition is the same as before.

[0065] In some implementation schemes, R 1 R 2 Each is independently selected from hydrogen, hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl-C 1-6 Alkoxy, C 1-6 Alkyl-C 1-6 Carboxyl, phenyl, C 1-6 Alkyl-phenyl, C 1-6 alkyl-5 or 6-membered heterocyclic aryl; the C 1-6 In alkyl groups, the 1st, 2nd, 3rd, 4th, and 5th -CH2- can be replaced by -O-CH2CH2-; or R 1 R 2 The adjacent N atom forms a 5- to 10-membered heterocyclic alkyl group; the 5- to 10-membered heterocyclic alkyl group is optionally surrounded by one or more atoms selected from R. 11 Substituents of R; wherein, R 11 The definition is the same as before.

[0066] In some embodiments, the salt is selected from ammonium salts and metal salts.

[0067] In some embodiments, the salt is selected from alkali metal salts and alkaline earth metal salts.

[0068] In some embodiments, the salt is an alkali metal salt.

[0069] In some embodiments, the salt is a sodium salt.

[0070] One aspect of the present invention provides the use of the fullerene derivative of formula (I) or a salt thereof in the preparation of a STEAP1 degrading agent.

[0071] In one aspect, the present invention provides the use of the fullerene derivative of formula (I) or a salt thereof in the preparation of a medicament for blocking the tumor cell cycle and inhibiting tumor migration.

[0072] Another aspect of the invention provides a protein degrading agent of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0073]

[0074] Among them, F, R, and m are each defined as before;

[0075] L is -L1-L2-;

[0076] L1 is -NR La R Lb ;

[0077] R La R LbEach is independently selected from: hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 Alkoxy; the C 1-10 Alkyl, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 The -CH2- in the alkoxy group is optionally replaced by an oxygen atom;

[0078] L2 is selected from: -(C=O)-NH-, -NH-;

[0079] E3 is a ubiquitin ligand.

[0080] In some implementation schemes, L is selected from:

[0081] m1, m2, and m3 are each independently selected from integers from 1 to 10.

[0082] In some implementations, E3 is Wherein, T1, T2, and T3 are each independently CH, C, or N; the group is optionally surrounded by one or more elements selected from hydrogen, =O, C. 1-6 Alkyl substituents.

[0083] In some implementations, E3 is selected from:

[0084] In some implementations, E3 is selected from:

[0085] In some implementations, E3 is selected from:

[0086] In some embodiments, the protein degrader of formula (II) is as shown in formula (III): In the formula, L is defined as before.

[0087] In some embodiments, the protein degrader of formula (II) is as shown in formula (IV):

[0088] In the formula, L is defined as before.

[0089] In some embodiments, the protein degrading agent or its stereoisomer, or a pharmaceutically acceptable salt, is selected from:

[0090]

[0091] In one aspect, the present invention provides the use of the aforementioned protein degrading agent or its stereoisomer, or a pharmaceutically acceptable salt, in the preparation of a medicament for treating tumors with high STEAP1 expression.

[0092] In one aspect of the invention, a formula (II) protein degrader or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is provided for treating tumors with high STEAP1 expression.

[0093] In another aspect of the present invention, a method for treating tumors with high STEAP1 expression is provided, wherein the method comprises administering to a subject in need a protein degrader of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0094] In some implementations, the STEAP1-highly expressed tumors are selected from STEAP1-highly expressed prostate cancer, renal cell carcinoma, bladder cancer, human Ewing's tumor, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, lung cancer, endometrial cancer, melanoma, glioma, kidney cancer, and / or liver cancer.

[0095] In some embodiments, the STEAP1-highly expressed tumors are selected from STEAP1-highly expressed breast cancer, lung cancer, melanoma, prostate cancer, glioma, liver cancer, and / or human Ewing's tumor.

[0096] In some implementations, the STEAP1-overexpressing tumor is STEAP1-overexpressing prostate cancer.

[0097] In some implementations, the STEAP1-overexpressing prostate cancer is metastatic castration-resistant prostate cancer.

[0098] In some implementations, the treatment of tumors with high STEAP1 expression involves inhibiting tumor proliferation, blocking the tumor cell cycle, and / or inhibiting tumor cell migration.

[0099] In some implementations, the treatment of tumors with high STEAP1 expression is a treatment for tumor proliferation.

[0100] In some embodiments, the medicament comprises a fullerene derivative of formula (I) or a salt thereof, or a protein degrader of formula (II) or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0101] In some embodiments, the formulation of the drug is selected from one or more of the following: solution, granules, lyophilized powder, emulsion, suspension, oil, and nanoformulation; in some embodiments, the solution is an injection.

[0102] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-1000 μM.

[0103] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-100 μM.

[0104] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-90 μM.

[0105] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-80 μM.

[0106] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-70 μM.

[0107] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-60 μM.

[0108] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-50 μM.

[0109] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-40 μM.

[0110] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-30 μM.

[0111] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-20 μM.

[0112] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-10 μM.

[0113] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from: 1 μM, 2 μM, 4 μM, 5 μM, 8 μM, 10 μM.

[0114] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-100 mM.

[0115] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1-10 mM.

[0116] In some embodiments, the concentration of the fullerene derivative of formula (I) or its salt in the drug is selected from 1 mM, 2 mM, or 4 mM.

[0117] In some embodiments, the concentration of the formula (II) protein degrader or its stereoisomer, or a pharmaceutically acceptable salt thereof, in the drug is selected from 1-1000 μM.

[0118] In some embodiments, the concentration of the formula (II) protein degrader or its stereoisomer, or a pharmaceutically acceptable salt thereof, in the drug is selected from 0.05, 0.1, 0.2, or 0.5 μM.

[0119] In some embodiments, the concentration of the formula (II) protein degrader or its stereoisomer, or a pharmaceutically acceptable salt of the drug is selected from 2 mM. Attached Figure Description

[0120] Figure 1 The molecular structure (left) and high-resolution mass spectrum (right) of carboxy-fullerene TCPC are shown.

[0121] Figure 2 The liquid chromatography of carboxylated fullerene TCPC is shown;

[0122] Figure 3 It shows the result of C 60 Reaction route diagram for the preparation of carboxy-fullerene derivative TCPC from Cl6 precursor;

[0123] Figure 4 The half-inhibitory concentration (WIC) fitting curves of carboxy-fullerene TCPC on A549, 4T1, U87, MCF-7, and B16 cells under serum-containing conditions are shown.

[0124] Figure 5 The half-inhibition concentration (WIC) fitting curves of carboxy-fullerene TCPC on A549, 4T1, U87, MCF-7, and B16 cells under serum-free conditions are shown.

[0125] Figure 6 The molecular structures of other carboxylated fullerenes BH, TH, PH, CPTA, CPBA, MCMA, MCBA, MCTA-3, and MCTA-4 are shown.

[0126] Figure 7 High-resolution mass spectra of other carboxylated fullerenes BH, TH, PH, CPTA, CPBA, MCMA, MCBA, MCTA-3, and MCTA-4 are shown.

[0127] Figure 8 It shows the result of C 60 Reaction route diagram for the preparation of carboxylated fullerene derivative BH;

[0128] Figure 9 It shows the result of C 60 Reaction route diagram for the preparation of carboxylated fullerene derivative TH from Cl6 precursor;

[0129] Figure 10 It shows the result of C 60 Reaction route diagram for preparing carboxy-fullerene derivative PH from Cl6 precursor;

[0130] Figure 11 It shows the result of C 60 Reaction route diagram for the preparation of carboxylated fullerene derivatives CPTA and CPBA;

[0131] Figure 12 It shows the result of C 60 Reaction route diagram for the preparation of carboxylated fullerene derivatives MCMA, MCBA, MCTA-3, and MCTA-4;

[0132] Figure 13 The half-inhibitory concentration (WIC) curves of other carboxylated fullerenes BH, TH, PH, CPTA, CPBA, MCMA, MCBA, MCTA-3, and MCTA-4 on 4T1 cells under serum-containing conditions are shown.

[0133] Figure 14 The half-maximal inhibitory concentration (WMC) fitting curves of other carboxylated fullerenes BH, TH, PH, CPTA, CPBA, MCMA, MCBA, MCTA-3, and MCTA-4 on 4T1 cells under serum-free conditions are shown.

[0134] Figure 15 The study demonstrated that carboxylated fullerene TCPC inhibited the migration ability of 4T1 cells.

[0135] Figure 16 The molecular structure (left) and high-resolution mass spectra (right) of TCPC-FFF, a probe used to identify carboxyfullerene TCPC-binding proteins, are shown.

[0136] Figure 17 The liquid chromatography of the carboxylated fullerene probe TCPC-FFF is shown;

[0137] Figure 18 The reaction route for preparing the carboxy-fullerene probe TCPC-FFF from TCPC is shown.

[0138] Figure 19 The image shows the results of in-gel fluorescence imaging (left) and mass spectrometry identification (right) of carboxy-fullerene TCPC target proteins using the probe TCPC-FFF via active proteomics.

[0139] Figure 20 The molecular structure of TCPC-pom, a degrading agent for the efficient degradation of TCPC-binding proteins, is shown.

[0140] Figure 21The high-resolution mass spectra of TCPC-PEG4-pom are shown as representative.

[0141] Figure 22 The liquid chromatography of TCPC-PEG4-pom is shown as a representative example;

[0142] Figure 23 The reaction route diagram for preparing TCPC-pom from TCPC is shown;

[0143] Figure 24 The half-maximal inhibitory concentration (WMC) curve of TCPC-pom against 4T1 cells under serum-containing conditions is shown.

[0144] Figure 25 The half-maximal inhibitory concentration (WMC) curve of TCPC-pom against 4T1 cells under serum-free conditions is shown.

[0145] Figure 26 The results of identifying differentially expressed proteins using TCPC-C2-pom via proteomics are shown.

[0146] Figure 27 The MST method was used to determine the affinity of carboxy-fullerene TCPC for STEAP1 protein.

[0147] Figure 28 The half-inhibition concentration fitting curves of carboxyfullerene TCPC under serum (left) and serum-free (middle) conditions for LNCaP, A673, and DU145 cells with different STEAP1 expression levels are shown, as well as the relative content of STEAP1 in LNCaP, A673, and DU145 cells (right).

[0148] Figure 29 The image shows the expression levels of STEAP1 in wild-type and STEAP1-overexpressing DU145 cells detected by Western blot (left) and the half-inhibition concentration (WIC) curves of carboxyfullerene TCPC on wild-type and STEAP1-overexpressing DU145 cells (right).

[0149] Figure 30 The image shows the expression levels of STEAP1 in wild-type and STEAP1-knockdown LNCaP cells detected by Western blot (left) and the half-inhibition concentration fitting curves of carboxyfullerene TCPC on wild-type and STEAP1-knockdown LNCaP cells (right).

[0150] Figure 31 The effect of different concentrations of TCPC on STEAP1 expression levels in LNCaP cells was shown by Western blot analysis.

[0151] Figure 32This study demonstrates the effect of TCPC at different time points on STEAP1 expression levels in LNCaP cells, as detected by Western blot.

[0152] Figure 33 The image shows the expression level of STEAP1 in LNCaP cells at different time points after TCPC removal, as detected by Western blot.

[0153] Figure 34 The effect of different concentrations of TCPC-C2-pom on STEAP1 expression levels in LNCaP cells was shown by Western blot analysis.

[0154] Figure 35 This study demonstrates the cell cycle blockade of LNCaP cells by carboxylated fullerene TCPC.

[0155] Figure 36 The effects of different concentrations of TCPC on the expression levels of LNCaP-related proteins STEAP1, C-MYC, CCND1, pRB, PCNA, p-53, N-cadherin, MMP-9, and actin were shown by Western blot analysis.

[0156] Figure 37 A schematic diagram is shown of the animal tumor suppression experiment of carboxylated fullerene TCPC and TCPC-C2-pom in a CDX model of LNCaP cells;

[0157] Figure 38 Optical photographs (left) and relative tumor weight (right) of tumors after treatment with different concentrations of carboxy-fullerene TCPC and TCPC-C2-pom on a CDX model of LNCaP are shown.

[0158] Figure 39 The changes in body weight (left) and relative tumor volume (right) of mice treated with different concentrations of carboxy-fullerene TCPC and TCPC-C2-pom in the CDX model of LNCaP are shown.

[0159] Figure 40 The expression levels of STEAP1, CCND1, pRB, and N-cadherin in the tumor were detected by Western blot after treatment with different concentrations of carboxy-fullerene TCPC and TCPC-C2-pom in the CDX model of LNCaP. Detailed Implementation

[0160] I. Definition

[0161] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are all widely used terms and routine procedures in the respective fields. To better understand this invention, definitions and explanations of related terms are provided below.

[0162] Unless otherwise stated, all contents in this invention are by mass percentage, and % contents are by mass percentage.

[0163] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0164] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0165] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0166] In this description, "treatment" refers to a series of proactive interventions aimed at alleviating symptoms, controlling the condition, repairing damage, eliminating the cause, restoring physiological function, or improving quality of life through medical means, interventions, or methods targeting diseases, injuries, functional impairments, or abnormal physiological states. Its core objectives are to reduce suffering, halt disease progression, promote recovery, or prolong life.

[0167] As used herein, the term "fullerene" is a series of spherical cluster molecules consisting of an even number of carbon atoms, with 12 five-membered rings and the remainder being six-membered rings. Fullerenes include hollow fullerenes and endohedral fullerenes, wherein the endohedral fullerene is a fullerene whose carbon cage structure contains a metal or a cluster of metal atoms.

[0168] As used in this article, the terms "metal fullerene" and "endo-fullerene" refer to compounds with special structures and properties formed by incorporating various metals or metal atom clusters into the carbon cage structure of fullerenes. These compounds are usually called endohedral fullerenes and are generally represented in the form M@C2n, where M represents a metal element.

[0169] As used in this article, the term "topical preparation" refers to a class of drug preparations that are applied directly to the surface of the skin, mouth, nose, vagina, eyes, or other body cavities without passing through the human digestive tract, in order to exert a local therapeutic effect, or to exert a therapeutic effect through absorption through the skin or mucous membranes.

[0170] In this invention, the disclosure of all ranges should be regarded as the disclosure of all sub-ranges and all point values ​​within the range. For example, the disclosure of 1-1000 should be regarded as the disclosure of ranges such as 1-200 and 200-300, as well as point values ​​such as 200, 300, 400, 500, 600, 700, 800, 900, and 1000.

[0171] As used herein, the term "salt" refers to a compound formed by the ionic bonding of a cation (including metal cations or ammonium cations) and an acid radical anion; the ammonium salt refers to a compound whose cation is an ammonium ion (NH4+). + A quaternary ammonium salt is a salt formed by the combination of an ammonium ion and an acid radical anion (such as chloride ion, sulfate ion, nitrate ion, etc.). The quaternary ammonium salt refers to a salt whose cation is a quaternary ammonium ion (R4N). + The term "metal salt" refers to a salt formed when all four hydrogen atoms in an ammonium ion are replaced by an organic substituent (such as alkyl, aryl, etc., which may be the same or different), where R is an organic substituent (such as alkyl, aryl, etc., which may be the same or different). The term "metal salt" refers to a salt compound of a cationic metal ion, including cations of Group IA, Group IIA, transition metals, and other metal elements in the periodic table (such as Na+). + K + Mg 2+ Ca 2+ Cu 2+ Salts formed by combining with acid radical anions (such as halide ions, sulfate ions, nitrate ions, etc.). When the fullerene derivative of formula (I) of this application contains a -COOH group, it can optionally react with a base (such as ammonia, sodium hydroxide, potassium hydroxide, etc.) to form a stable salt, including but not limited to: reacting with ammonia to form an ammonium salt (the cation is NH4). + It reacts with alkali metal hydroxides (such as NaOH, KOH) to form alkali metal salts (with Na+ as the cation). + K + (etc.). When the fullerene derivative of formula (I) of this application contains a -NH2 group, it can optionally react with an inorganic acid or an organic acid to form a salt; when the fullerene derivative of formula (I) of this application contains a tertiary amine group, a fourth organic substituent can be introduced through an alkylation reaction to generate a quaternary ammonium salt.

[0172] As used in this article, the term "cell cycle" refers to the entire process a cell undergoes from the completion of one division to the end of the next, divided into two phases: interphase and mitotic phase. Interphase is further divided into three phases: pre-DNA synthesis (G1 phase), DNA synthesis (S phase), and post-DNA synthesis (G2 phase). Cell cycle regulation is primarily achieved through the arrest of the G1 phase. The G0 phase refers to a state of cell arrest, a temporary cessation of cell division outside the cell cycle, but under suitable stimuli, the cell can re-enter the cycle.

[0173] As used in this article, the term "degrading agent" refers to a class of compounds or biomolecules that can induce the specific degradation of target molecules (such as proteins) through a specific mechanism. Their core mechanism of action involves interacting with target molecules and natural intracellular degradation systems (such as the ubiquitin-proteasome system and autophagy) to label, recognize, and ultimately break down target molecules, thereby reducing their abundance within the cell and subsequently regulating related biological processes or exerting therapeutic effects.

[0174] II. Examples

[0175] The following detailed embodiments further illustrate the above-described content of this disclosure. However, this should not be construed as limiting the scope of the subject matter of this disclosure to the following embodiments. All technologies implemented based on the above-described content of this disclosure fall within the scope of this disclosure.

[0176] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below. The described embodiments should not be considered as limitations on the invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0177] Before providing a further detailed description of the embodiments of the present invention, the nouns and terms involved in the embodiments of the present invention will be explained, and the nouns and terms involved in the embodiments of the present invention shall be interpreted as follows.

[0178] Example 1: Preparation of carboxylated fullerene TCPC

[0179] Synthesis method: 1g of C 60 Dissolve Cl6 in 1 L of toluene. After complete dissolution, add 1.5 mL of tert-butyl 4-piperidinecarboxylate, a trace amount of cumene hydroperoxide (80%), and excess K2CO3. Stir at room temperature until C... 60After the Cl6 was exhausted and the reaction was complete, the solution changed from orange-red to brownish-red, accompanied by the precipitation of a white solid. The solution was washed successively with saturated NH4Cl solution and saturated NaHCO3 solution, and the organic phase was collected and filtered. The solvent was removed by vacuum distillation to obtain a reddish-brown crude product. After dissolving in a suitable amount of toluene, the product was separated on a silica gel column using ethyl acetate:toluene as the eluent to obtain the target product, an orange-red solid, namely the tert-butyl ester-protected carboxylated fullerene derivative.

[0180] 50 mg of the separated product was dissolved in dichloromethane, and trifluoroacetic acid was added. After stirring at room temperature until fully reacted, the solvent was removed by vacuum distillation to obtain the reddish-brown carboxylated fullerene derivative TCPC. Figure 3 The solution was dissolved in NaOH aqueous solution and dialyzed until the outer water layer was electrically neutral. The aqueous solution of TCPC was filtered through a 0.22 μm pore size filter membrane and then freeze-dried to obtain the sodium salt of the deep red carboxylated fullerene derivative TCPC.

[0181] Structural characterization: The molecular weight of TCPC was determined using electrospray ionization mass spectrometry (ESI-MS, positive ion mode). Figure 1 The purity of TCPC was determined using high-performance liquid chromatography (HPLC, Vanquish Core, Thermo Fisher), a C18 column (4.6*150mm, Zorbax Extend-C18, Agilent), with water and acetonitrile as the mobile phase, a UV detector (detection wavelength: 310nm), and a flow rate of 1mL / min. Figure 2 ).

[0182] Conclusion: The molecular weight detected by ESI-MS is completely consistent with the theoretical molecular weight of TCPC, and the purity detected by HPLC is above 95%, which can be used for further biological experiments.

[0183] Example 2: Carboxy-fullerene molecules (TCPC) inhibit tumor cell growth.

[0184] Cells: 4T1 (mouse breast cancer cell line), A549 (human non-small cell lung cancer cell line), B16F10 (mouse skin melanoma cells), MCF-7 (human breast cancer cell line), U87 (human brain astrocytoma cell line).

[0185] Grouping: The experimental groups were aqueous solutions of TCPC with concentration gradients of 0-100 μM.

[0186] Experimental Methods: CCK-8 assay – All cells were cultured in DMEM medium (Thermo Fisher, C11995500BT) containing 10% fetal bovine serum (Excell, FSP500) and 1% penicillin-streptomycin (Thermo Fisher, 15070063) at 37°C and 5% CO2. After normal culture for 24 hours, cells were digested and counted using 0.25% trypsin (Thermo Fisher, 25200072) and seeded at a density of 5000 cells / well in 96-well plates. After 24 hours of culture, cells were treated with different concentrations of TCPC (with and without serum for each cell type) for 24 hours. Cell Counting Kit-8 (CCK-8, Bairuiji) was then diluted 10-fold with fresh culture medium, and the drug-containing medium was replaced. After a period of culture, the absorbance at 450 nm per well was measured using a microplate reader (Tecan, Infifinite M1000) to assess cell viability. The half-inhibitory concentration (IC50) was calculated for each data point. 50 Fitting was performed to obtain the IC50 of TCPC for different tumor cells. 50 value.

[0187] Conclusion: Compared with serum-treated conditions, TCPC significantly reduced the activity of various tumor cell types, including 4T1, A549, B16, MCF-7, and U87, in a serum-free environment. Figures 4-5 TCPC affects the IC50 of the above five cell types under serum-containing conditions. 50 The values ​​were 88.48 μM, 720.5 μM, 63.63 μM, 102 μM, and 55.54 μM, respectively; the IC50 values ​​of TCPC for the above five cell types under serum-free conditions were... 50 The values ​​were 3.14 μM, 22.81 μM, 22.26 μM, 9.77 μM, and 10.3 μM, respectively. Lower concentrations of TCPC can effectively inhibit tumor cell activity.

[0188] Example 3: Preparation of other carboxylated fullerenes BH, TH, PH, CPTA, CPBA, MCMA, MCBA, MCTA-3, and MCTA-4

[0189] Synthesis method: BH: 1.76 g of malonic acid and 5.13 mL of 3-acetylen-1-butanol were dissolved in toluene, and p-toluenesulfonic acid was added as a catalyst at room temperature. The solution was heated and stirred for 6 h. During the reaction, the byproduct water was removed from the reaction system using a water separator. After the reaction, the mixture changed from colorless and transparent to yellow. The reaction solution was washed successively with NaOH aqueous solution and deionized water, dried with anhydrous Na2SO4, and filtered. The solvent was removed by vacuum distillation. Since the reactants and possible byproducts can be removed by washing with brine or vacuum distillation, the resulting liquid is diacetylenic malonate diester, and no further purification is required. C 60 CBr4, diacetylammonopropyl diester, and 1,8-diazocyclo[5.4.0]undecyl-7-ene (DBU) were dissolved in toluene and stirred at room temperature for 1 h. After the reaction was complete, the mixture was washed with water to quench the DBU, dried with anhydrous Na2SO4, and filtered. The solvent was removed by vacuum distillation. The target product CB1, a brownish-red solid, was obtained by separation on a silica gel column using toluene as the eluent. CB1 and azidoacetic acid were dissolved in 0.5 mL of dichloromethane. CuBr and DIPEA were added. The mixture was stirred until CB1 was exhausted. After the reaction was complete, trifluoroacetic acid was added and stirred for 5 min, followed by the addition of diethyl ether. The precipitated solid was washed successively with trichloromethane and acetonitrile. The solvent was removed by vacuum distillation to obtain a brown solid, the target product BH, which did not require further purification. Figure 8 ).

[0190] TH: C 60 Cl6, 80% CHP, and N-methylpropylamine were dissolved in toluene. The reaction was stirred at room temperature for 1 h, and the solvent was removed by vacuum distillation to obtain a brownish-red crude product. Using toluene as the eluent, the crude product was separated on a silica gel column, and the target product CB2 was an orange-red solid. CB2 and azidoacetic acid were dissolved in dichloromethane. CuBr and DIPEA were added. The mixture was stirred until CB2 was exhausted. After the reaction was complete, trifluoroacetic acid was added and stirred for 5 min, followed by the addition of diethyl ether. The precipitated solid was washed successively with trichloromethane and acetonitrile. The solvent was removed by vacuum distillation to obtain an orange-red solid, which was the target product TH, requiring no further purification. Figure 9 ).

[0191] PH: C 60Cl6 was dissolved in toluene, and 3-buten-1-amine hydrochloride was added. 5 mL of 10% NaOH aqueous solution was added to remove the hydrochloric acid. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the organic reaction solution changed from orange-red to dark red. After filtration, the solvent was removed by vacuum distillation. Using ethyl acetate / toluene as the eluent, the product was separated on a silica gel column; the target product CB3 was an orange-red solid. CB3 and azidoacetic acid were dissolved in 0.5 mL of dichloromethane. DIPEA was added. The mixture was stirred until CB3 was exhausted. After the reaction was complete, trifluoroacetic acid was added and stirred for 5 min, followed by the addition of diethyl ether. The precipitated solid was washed successively with trichloromethane and acetonitrile. The solvent was removed by vacuum distillation to obtain an orange-red solid, which was the target product pH and required no further purification. Figure 10 ).

[0192] The obtained fullerene derivatives BH, PH, and TH were dissolved in NaOH aqueous solution, and then the aqueous solution was dialyzed and lyophilized to obtain the sodium salts of water-soluble carboxylated fullerene derivatives BH, PH, and TH.

[0193] CPTA, CPBA: (This likely refers to a specific combination of terms, possibly related to C or CPBA.) 60 Dissolve in chlorobenzene, add diethyl iminodiacetic acid and ethyl glyoxylate. Stir at 140℃ for 2 h. Wash the mixture with water, dry with Na2SO4, remove the solvent by vacuum distillation, and separate the brown product with pure toluene on a silica gel column. C 60 - Pyrrolidine triethyl ester was dissolved in toluene, and then 20 mg of NaH was added under anhydrous and oxygen-free conditions. The mixture was stirred at room temperature for 1 h, and the solvent was removed by vacuum distillation. The product was washed successively with toluene and chloroform, acidified with 3M hydrochloric acid solution, filtered and dried to obtain CPTA. CPBA was synthesized in the same way as CPTA, except that the starting materials were replaced with diethyliminodiacetic acid ester and paraformaldehyde. Figure 11 ).

[0194] MCMA, MCBA, MCTA-3, MCTA-4: Change C 60 Dissolved in toluene, di-tert-butyl malonate, DBU, and CBr4 were added, and the reaction was carried out at room temperature for 8 hours. The mixture was then washed with water and dried over Na2SO4. The solvent was removed by vacuum distillation. The purple-red product, diethyl malonate C, was obtained by separation on a silica gel column using hexane-toluene. 60 The single adduct (DCMA) was synthesized by dissolving DCMA in dichloromethane, adding trifluoroacetic acid, stirring, and removing the solvent by vacuum distillation. The solution was washed with toluene and dried to obtain MCMA. MCBA was synthesized in the same way as MCMA, using di-tert-butyl malonate, DBU, and CBr4 instead of the starting materials. MCTA-3 was synthesized in the same way as MCMA, using di-tert-butyl malonate, DBU, and CBr4 instead of the starting materials. MCTA-4 was synthesized in the same way as MCMA, using di-tert-butyl malonate, DBU, and CBr4 instead of the starting materials. Figure 12 ).

[0195] Structural characterization: The molecular weight of the above compounds was determined using electrospray ionization mass spectrometry (ESI-MS, positive ion mode). Figures 6-7 ).

[0196] Conclusion: The molecular weights detected by ESI-MS are completely consistent with the theoretical molecular weights of the above compounds.

[0197] Example 4: Cytotoxicity of other carboxylated fullerene molecules on 4T1 cells

[0198] Cells: 4T1 cell line.

[0199] Grouping: The experimental groups consisted of aqueous solutions of various carboxylated fullerenes with concentration gradients of 0-100 μM.

[0200] Experimental Methods: CCK-8 assay – 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded at a density of 5000 cells / well in 96-well plates. After 24 hours of culture, cells were treated with different concentrations of carboxyfullerene (with and without serum for each derivative) for 24 hours. Cell Counting Kit-8 was then diluted 10-fold with fresh medium and the drug-containing medium was replaced. After a period of culture, the absorbance at 450 nm per well was measured using a microplate reader to assess cell viability. The half-inhibitory concentration (IC50) was calculated for each data point. 50 Fitting was performed to obtain the IC50 of various carboxylated fullerenes on 4T1 cells. 50 value.

[0201] Conclusion: Compared to TCPC, only BH, TH, and PH exhibit similar biological activities. BH, TH, and PH show positive effects on the IC50 of 4T1 in the presence of serum. 50 The values ​​were 125.5 μM, 313.9 μM, and 61.81 μM, respectively; the IC50 values ​​of BH, TH, and PH against 4T1 under serum-free conditions were... 50 The values ​​were 49.52 μM, 9.0 μM, and 9.26 μM, respectively. CPTA, CPBA, CPTA-3, and CPTA-4 showed no significant cytotoxicity against 4T1 cells. Figures 13-14 This indicates that the cytotoxicity of carboxy-fullerenes is highly correlated with their structure, and carboxy-fullerenes similar to TCPC have a good inhibitory effect on tumor cells.

[0202] Example 5: Carboxy-fullerene molecules TCPC inhibit 4T1 cell migration

[0203] Cells: 4T1 (mouse breast cancer cell line).

[0204] Grouping: The experimental groups were TCPC solutions with concentrations of 0, 1, 5, and 10 μM, respectively.

[0205] Experimental Methods: Transwell assay – 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded at a density of 500,000 cells / well in 6-well plates. After overnight culture until cell attachment, cells were treated with different concentrations of TCPC (with and without serum) for 24 hours. After washing cells with PBS, cells were digested with 0.25% trypsin, centrifuged at 1000 rpm for 15 min to collect cells, and adjusted to a cell concentration of 1 × 10⁶ cells / well using serum-free medium. 5 Cells / mL were added to 200 μL of cell suspension in a transwell chamber, and 500 μL of culture medium containing 20% ​​serum was added to a 24-well plate. The transwell chambers were placed in the culture medium in the 24-well plate and cultured for 24 hours. The lower surface of the chamber was fixed by immersing it in 4% paraformaldehyde solution at room temperature for 15 min, washed twice with PBS, and the cells on the upper surface of the chamber were wiped clean with a cotton swab. The lower surface of the chamber was then stained with crystal violet solution at room temperature for 15 min, and washed twice with PBS. After drying, the cells were observed and photographed under a microscope, with 5 locations observed on the surface of each chamber. The number of stained cells was counted.

[0206] Conclusion: TCPC significantly inhibited the migration of 4T1 cells from the upper to the lower surface of the transwell chamber under both serum-free and serum-free conditions. Low concentrations of TCPC could also inhibit tumor cell migration. Figure 15 ).

[0207] Example 6: Preparation of the probe TCPC-FFF for carboxy-fullerene TCPC

[0208] Synthesis method: 50 mg of TCPC protected by tert-butyl ester was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 2 h, and the solvent was removed by vacuum distillation at 40 °C to obtain a reddish-brown carboxylated fullerene derivative TCPC. The solution was dried under vacuum at 40 °C overnight. TCPC was dissolved in N,N-dimethylacetamide (DMA) to obtain a 5 mM solution. A 5 mM solution of 3-aminoethyl-3-(but-3-ynyl)bisacrylamide (FFF) was prepared using DMA. Solutions of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N-methylmorpholine (NMM) were prepared using DMA. TCPC, FFF, HATU, and NMM were mixed in a molar ratio of 1:1:1.2:12 and stirred at room temperature in the dark for 2 h. Figure 18 The product was analyzed using a C18 column (10*250 mm, XBridgeBEH C18, Waters) on a semi-preparative high-performance liquid chromatography (LC-20AR) system. The mobile phase consisted of water containing 0.5% ammonia and acetonitrile containing 0.5% ammonia. Gradient elution was performed, increasing the acetonitrile concentration from 25% to 80%. The target product was collected as a yellow solution. The solvent was concentrated by vacuum distillation at 45°C, dialyzed for 1 h to remove ammonia and acetonitrile, and then further distilled under reduced pressure until the product concentration reached approximately 1 mM, resulting in a reddish-brown solution.

[0209] Structural characterization: The molecular weight of TCPC-FFF was determined using electrospray ionization mass spectrometry (ESI-MS, positive ion mode). Figure 16 The purity of TCPC-FFF was determined using high-performance liquid chromatography (HPLC, Vanquish Core, Thermo Fisher) with a C18 column (4.6*150mm, Zorbax Extend-C18, Agilent), water containing 0.5% ammonia and acetonitrile containing 0.5% ammonia as the mobile phase, and a UV detector (detection wavelength: 310nm) at a flow rate of 1mL / min. Figure 17 ).

[0210] Conclusion: The molecular weight detected by ESI-MS is completely consistent with the theoretical molecular weight of TCPC. Due to the symmetry of the TCPC structure, the probe structure has two isomers (close to the epoxy and far from the epoxy). HPLC detection shows that its purity is above 90%, which can be used for further biological experiments.

[0211] Example 7: Screening the appropriate labeling concentration of the probe TCPC-FFF for intracellular proteins in 4T1 cells

[0212] Cells: 4T1 (mouse breast cancer cell line).

[0213] Groups: control group, directly labeled experimental groups were TCPC-FFF solutions with concentrations of 6.25, 12.5, 25, 50, and 100 μM, respectively, and competitive experimental groups were 100 μM TCPC-FFF, 100 μM TCPC-FFF + 200 μM TCPC, 100 μM TCPC-FFF + 500 μM TCPC, and 100 μM TCPC-FFF + 1000 μM TCPC solutions, respectively.

[0214] Experimental Methods: 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded into 6cm culture dishes. When cell confluence reached 90%, 4T1 cells were treated with different concentrations of TCPC-FFF (700μL / dish, serum-free) for 0.5 hours. The culture medium was discarded, and the cells were gently washed twice with PBS. The cells were then irradiated under 365nm UV light for 15 minutes. All operations were performed on ice. Cells were lysed using lysis buffer supplemented with protease inhibitors and PMSF, and then sonicated. Centrifuge at 20000×g for 15 minutes, collect the supernatant, quantify the protein concentration using the BCA method, and adjust the protein concentration to 2 mg / mL using PBS. Take 50 μL of each protein sample and add 1 μL of 1.7 mM TBTA, 1 μL of 50 mM CuSO4, 3 μL of 1.25 mM TAMAR-azide, and 1 μL of 50 mM TCEP sequentially to achieve final concentrations of 0.1 mM, 1 mM, 50 μM, and 1 mM, respectively. Mix well and react at room temperature for 2 hours, then stop the reaction by adding 5X loading buffer. Separate the protein on a 4-20% sodium dodecyl sulfate-polyacrylamide gel, wash the gel twice with ultrapure water, and observe the fluorescence signal in the gel under the Cy3 channel of a fluorescence imaging system (SH-523, Shenhua Technology).

[0215] Similarly, for the competitive experimental group, cells were first incubated for 1 hour with the corresponding concentration of TCPC-containing medium (700 μL / plate, serum-free), then incubated for 0.5 hours with 100 μM TCPC-FFF, and in-gel fluorescence imaging was performed.

[0216] Conclusion: 100 μM TCPC-FFF showed the highest labeling effect on intracellular proteins. Under competition from TCPC, the protein signal labeled by TCPC-FFF was significantly weakened, and 500 μM TCPC almost completely eliminated the band. This demonstrates that TCPC-FFF can label proteins that specifically bind to TCPC and can be used for the identification of TCPC protein targets. Figure 19 Left).

[0217] Example 8: Identification of carboxy-fullerene TCPC target proteins using the probe TCPC-FFF via active proteomics

[0218] Cells: 4T1 (mouse breast cancer cell line).

[0219] Grouping: control group, direct labeling experimental group (100 μM TCPC-FFF solution), competition experimental group (100 μM TCPC-FFF + 500 μM TCPC solution).

[0220] Experimental Methods: 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded into 10cm culture dishes. When cell confluence reached 90%, the direct labeling group was treated with 100μM TCPC-FFF (1.5mL / dish, serum-free) for 0.5 hours, while the competitive labeling group was first treated with 500μM TCPC for 1 hour, followed by 100μM TCPC-FFF for 0.5 hours. The culture medium was discarded, and the cells were gently washed twice with PBS. The cells were then irradiated under 365nm UV light for 15 minutes. All operations were performed on ice. Cells were lysed using lysis buffer supplemented with protease inhibitors and PMSF, and then sonicated. Centrifuge at 20000×g for 15 minutes, collect the supernatant, quantify the protein concentration using the BCA method, and adjust the protein concentration to 2 mg / mL using PBS. Take 1 mL of each protein sample and add 60 μL of 1.7 mM TBTA, 20 μL of 50 mM CuSO4, 10 μL of 10 mM TAMAR-azide, and 20 μL of 50 mM TCEP sequentially to achieve final concentrations of 0.1 mM, 1 mM, 100 μM, and 1 mM, respectively. Mix well and react at room temperature for 2 hours. Then add 5 volumes of pre-cooled acetone / chloroform. Resuspend the precipitated protein in SDS-PBS. Biotin-labeled proteins were enriched using streptavidin-coated magnetic beads. The proteins were washed with SDS-PBS at room temperature for 10 minutes, followed by three 3-minute washes with PBS, and three 3-minute washes with TEBA. The beads were resuspended in urea / TEBA, and disulfide bonds were reduced using DTT at 37°C for 30 minutes. Alkylation was then performed using IAA at 35°C for 30 minutes. After cooling to room temperature, the proteins were diluted with TEBA, the supernatant was discarded, and the beads were washed three times with EPPS at pH 8.5. The beads were resuspended in EPPS, and the proteins were digested overnight at 37°C using trypsin and LysC. Digestion was terminated by adding 1% formic acid. The digested peptides were labeled using TMT 10plex and reacted at room temperature for 1 hour. The reaction was terminated by adding 5% hydroxylamine and incubated at room temperature for 15 minutes. Equal volumes of peptide samples labeled with different isotopes were then mixed, dried at 45°C, desalted using a desalting column, and divided into 12 aliquots. The samples were then dried.The obtained peptides were reconstituted with 0.1% formic acid solution and then analyzed by LC-MS / MS. LC separation was performed using a low-pH mobile phase (Easy-nLC 1200 HPLC-Q-Exactive HF-X Orbitrap mass spectrometer, Thermo Fisher Scientific). Mobile phase A consisted of 0.1% (v / v) formic acid aqueous solution, and mobile phase B consisted of 0.1% formic acid acetonitrile solution. The chromatographic column was a reversed-phase analytical column (25 cm, 75 μm id.). The gradient was set as follows: 0 min 3% B – 5 s 5% B – 24 min 15% B – 45 min 28% B – 52 min 30 s 38% B – 52 min 35 s 100% B – 65 min 100% B – stop. The constant flow rate was 300 nL / min. Mass spectrometry acquisition conditions were as follows: positive ion mode, Orbitrap mass analyzer, primary spectrum scan range 350 to 1800 Da, resolution 60000, profile format; secondary spectrum data acquisition mode data-dependent, centroid format, using the 15 highest intensity ion peaks for secondary HCD (high-energy collision-induced dissociation) fragmentation, secondary spectrum resolution 45000. Other parameters to be set included isolation window, 0.7 m / z units; default charge, 2+; normalized collision energy, 35%; maximum IT, 50 ms; dynamic exclusion, 30.0 s. Mass spectrometry data were used for library search and quantitative analysis using Thermo Proteome Discoverer 2.5. The above experiments were repeated three times under the same conditions, and p-values ​​were calculated.

[0221] Conclusion: Under the conditions of a competition threshold of 2 and a p-value less than 0.05, a total of 172 differentially expressed proteins were enriched, suggesting that these proteins may be TCPC-specific binding proteins. Figure 19 (Right). STEAP1 is a six-transmembrane protein specifically expressed in prostate cancer tissue. It promotes the growth and metastasis of tumor cells through multiple pathways, while TCPC can specifically bind to STEAP1.

[0222] Example 9: Preparation of TCPC-pom, a degrader for TCPC-binding proteins

[0223] Synthesis method: 50 mg of TCPC protected by tert-butyl ester was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 2 h, and the solvent was removed by vacuum distillation at 40 °C to obtain a reddish-brown carboxylated fullerene derivative, TCPC. The solution was dried under vacuum at 40 °C overnight. TCPC was dissolved in N,N-dimethylacetamide (DMA) to obtain a 5 mM solution. A 5 mM solution of pomalidomide (pom) derivative was prepared using DMA. Solutions of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N-methylmorpholine (NMM) were prepared using DMA. TCPC, pomalidomide, HATU, and NMM were mixed in a molar ratio of 1:1:1.2:12 and stirred at room temperature in the dark for 2 h. Figure 23 The product was analyzed using a C18 column (10*250 mm, XBridgeBEH C18, Waters) on a semi-preparative high-performance liquid chromatography (LC-20AR) system. The mobile phase consisted of water containing 0.5% ammonia and acetonitrile containing 0.5% ammonia. Gradient elution was performed, increasing the acetonitrile concentration from 25% to 80%. The target product was collected as a yellow solution. The solvent was concentrated by vacuum distillation at 45°C, dialyzed for 1 h to remove ammonia and acetonitrile, and then further distilled under reduced pressure until the product concentration reached approximately 1 mM, resulting in a reddish-brown solution.

[0224] Structural characterization: The molecular weight of TCPC-pom was determined using electrospray ionization mass spectrometry (ESI-MS, positive ion mode). Figure 21 The purity of TCPC-PEG4-pom was determined using high-performance liquid chromatography (HPLC, Vanquish Core, Thermo Fisher) with a C18 column (4.6*150mm, Zorbax Extend-C18, Agilent), water containing 0.5% ammonia and acetonitrile containing 0.5% ammonia as the mobile phase, and a UV detector (detection wavelength: 310nm) at a flow rate of 1mL / min. Figure 22 ).

[0225] Conclusion: The molecular weight detected by ESI-MS is completely consistent with the theoretical molecular weight of TCPC-pom. Due to the symmetry of the TCPC structure, the degrading agent has two isomers (close to the epoxy and far from the epoxy). HPLC analysis shows that its purity is above 90%, which is sufficient for further biological experiments.

[0226] Example 10: Cytotoxicity of TCPC-pom on 4T1 cells

[0227] Cells: 4T1 cell line.

[0228] Grouping: The experimental groups consisted of aqueous solutions of various TCPC-pom solutions with concentration gradients of 0-100 μM.

[0229] Experimental Methods: CCK-8 assay – 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded at a density of 5000 cells / well in 96-well plates. After 24 hours of culture, cells were treated with different concentrations of carboxyfullerene (with and without serum for each derivative) for 24 hours. Cell Counting Kit-8 was then diluted 10-fold with fresh medium and the drug-containing medium was replaced. After a period of culture, the absorbance at 450 nm per well was measured using a microplate reader to assess cell viability. The half-inhibitory concentration (IC50) was calculated for each data point. 50 Fitting was performed to obtain the IC50 of various carboxylated fullerenes on 4T1 cells. 50 value.

[0230] Conclusion: TCPC-PEG2-CONH-pom, TCPC-PEG3-CONH-pom, TCPC-PEG4-CONH-pom, TCPC-PEG1-amine-pom, TCPC-PEG2-amine-pom, TCPC-PEG3-amine-pom, TCPC-PEG4-amine-pom, TCPC-C2-pom, TCPC-C3-pom, TCPC-C4-pom, and TCPC-C5-pom, under serum-containing conditions, showed an effect on the IC50 of 4T1. 50 The values ​​were 11313 μM, 908261 μM, 228855 μM, 274.2 μM, 301.2 μM, 224.0 μM, 361.4 μM, 174.7 μM, 359.1 μM, 192.4 μM, and 200.6 μM, respectively. The IC50 values ​​of TCPC-PEG2-CONH-pom, TCPC-PEG3-CONH-pom, TCPC-PEG4-CONH-pom, TCPC-PEG1-amine-pom, TCPC-PEG2-amine-pom, TCPC-PEG3-amine-pom, TCPC-PEG4-amine-pom, TCPC-C2-pom, TCPC-C3-pom, TCPC-C4-pom, and TCPC-C5-pom against 4T1 under serum-free conditions were... 50The values ​​were 34.24 μM, 49.34 μM, 551.1 μM, 7.99 μM, 17.17 μM, 25.22 μM, 205.2 μM, 23.01 μM, 16.11 μM, 9.04 μM, and 16.02 μM, respectively. Figures 24-25 Considering that TCPC-C2-pom has the best inhibitory effect, it was selected as a representative for subsequent biological experiments.

[0231] Example 11: Identification of differentially expressed proteins using TCPC-C2-pom via proteomics

[0232] Cells: 4T1 (mouse breast cancer cell line).

[0233] Grouping: control group, experimental group was 100 μM TCPC-C2-POM solution.

[0234] Experimental Methods: 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded in 10cm culture dishes. When cell confluence reached 90%, the directly labeled group was treated with 100μM TCPC-C2-pom for 24 hours. The culture medium was discarded, and the cells were gently washed twice with PBS. Sample lysis and protein extraction were performed using SDT (4% SDS, 100mM Tris-HCl, 1mM DTT, pH 7.6) buffer. Protein levels were determined using a BCA protein assay kit (Bio-Rad, USA). Protein digestion was performed using trypsin according to the Filter-Assisted Sample Preparation (FASP) procedure described by Matthias Mann. Digested peptides from each sample were desalted on a C18 column (Empore). TMSPE column C18 (standard density), 7 mm diameter, 3 mL volume (Sigma), concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v / v) formic acid. 100 μg peptide mixtures for each sample were labeled with TMT reagent according to the manufacturer's instructions (ThermoScientific). The labeled peptides were separated using a high-pH reversed-phase peptide separation kit (Thermo Scientific). The dried peptide mixture was reconstituted and acidified with 0.1% TFA solution and packed into an equilibrated high-pH reversed-phase spin fractionation column. The peptides bound to the hydrophobic resin under aqueous conditions and were desalted by washing the column with water at low speed. The bound peptides were then eluted into 10 distinct fractions by increasing the acetonitrile concentration gradient in a volatile high-pH elution solution. LC-MS / MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) coupled with an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) for 60 / 90 min. Peptides were loaded onto a reversed-phase capture column (Thermo Scientific Acclaim PepMap100, 100 μm * 2 cm, nanoViper C18), which was then separated from a C18 reversed-phase analysis column (Thermo Scientific Easy column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% formic acid) using a linear gradient in buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL / min. The mass spectrometer was operated in positive ion mode. Mass spectrometry data were acquired using a data-dependent top 10 method, dynamically selecting the most abundant precursor ions from the HCD fragmentation survey scans (300–1800 m / z). Automatic gain control (AGC) was set to a target of 3e6, with a maximum injection time of 10 ms. The dynamic exclusion time was 40.0 s. The measurement scan resolution was 70,000 m / z 200, the HCD spectral resolution was 17,500 m / z 200, and the isolation width was 2 m / z. The normalized collision energy was 30 eV, and the lower fill rate (i.e., the minimum percentage of the target value that can be reached within the maximum fill time) was defined as 0.1%. Peptide recognition mode was enabled during instrument operation. The above experiments were repeated three times under the same conditions, and the p-value was calculated.

[0235] Conclusion: Under the conditions of change thresholds of 0.75 and 1.25 and p-values ​​less than 0.05, differentially expressed proteins induced by TCPC-C2-pom treatment in 4T1 cells were identified. Figure 26 STEAP1 was identified as being significantly downregulated.

[0236] Example 12: Determination of the affinity between TCPC and STEAP1

[0237] Experimental Methods: Micro-thermophoresis (MST) – Tests were performed according to the instrument's instructions. Using the Momolith™ RED-NHS kit, the labeling efficiency of fluorescence was first measured to determine the appropriate protein and fluorescence concentration. Fluorescent molecules were labeled onto STEAP1 via an amino-coupled reaction, followed by the addition of TCPC solutions of varying concentrations. After capillary absorption, fluorescence changes were analyzed on a Monolith™ NT.115 (NanoTemper Technologies) instrument to detect and calculate the affinity constant between TCPC and STEAP1.

[0238] Conclusion: The affinity constant K between TCPC and STEAP1 d The value is approximately 270 nM, indicating a strong interaction between TCPC and STEAP1. Figure 27 ).

[0239] Example 13: Carboxy-fullerene molecule TCPC inhibits the growth of tumor cells with high STEAP1 expression.

[0240] Cells: LNCaP (human prostate cancer cell line with high STEAP1 expression), A673 (human Ewing's tumor cell line with high STEAP1 expression), DU145 (human prostate cancer cell line with low STEAP1 expression).

[0241] Grouping: The experimental groups were aqueous solutions of TCPC with concentration gradients of 0-100 μM.

[0242] Experimental Methods: CCK-8 assay – LNCaP cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. A673 and DU145 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded at a density of 5000 cells / well in 96-well plates. After 24 hours of culture, cells were treated with different concentrations of TCPC (with and without serum for each cell type) for 24 hours. Cell Counting Kit-8 was then diluted 10-fold with fresh medium and the drug-containing medium was replaced. After a period of culture, the absorbance at 450 nm per well was measured using a microplate reader to assess cell viability. The half-inhibitory concentration (IC50) was calculated for each data point. 50 Fitting was performed to obtain the IC50 of TCPC for different tumor cells. 50 value.

[0243] Conclusion: The sensitivity of tumor cells to TCPC is positively correlated with the expression level of STEAP1. Figure 28 TCPC affects the IC50 of the above three cell types under serum-containing conditions. 50 The values ​​were 7.46 μM, 27.47 μM, and 78.08 μM, respectively; the IC50 values ​​of TCPC for the above five cell types under serum-free conditions were... 50 The concentrations were 0.46 μM, 3.48 μM, and 5.19 μM, respectively, indicating that TCPC exhibited good inhibitory activity against the LNCaP prostate cancer cell line, which highly expresses STEAP1.

[0244] Example 14: Carboxy-fullerene molecules exert tumor-suppressive biological activity via TCPC in dependence on STEAP1.

[0245] Cells: DU145 cells, LNCaP cells.

[0246] Grouping: wild-type cells and cells overexpressing or knocking down STEAP1; drug treatment was with aqueous solutions of TCPC at concentration gradients of 0-100 μM.

[0247] Experimental Methods: DU145 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% pancreatic acid. Approximately 250,000 DU145 cells were seeded in T25 cell culture flasks and cultured for 24 hours. Lentiviral virus, mixed with infection enhancement solution, was added according to the cell MOI and viral titer. After 16 hours of culture at 37°C, the medium was replaced with fresh complete medium for further culture. Approximately 72 hours post-infection, medium containing an appropriate concentration of puromycin was added for at least 48 hours to screen for stable transfected cell lines.

[0248] LNCaP cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, the cells were digested and counted using 0.25% trypsin. Approximately 500,000 DU145 cells were seeded in 6-well plates and cultured for 24 hours. Lipofectamine was added according to the instructions. TM 3000 nmol of siRNA was mixed with 100 nmol of siRNA and incubated with LNCaP cells for 24 hours. Then the culture medium was replaced with fresh complete medium and cultured for another 48 hours.

[0249] Western blot analysis of target protein expression efficiency: Cells were lysed in RIPA lysis buffer (Beyotime, P0013) containing a mixture of protease phosphatase inhibitors (Beyotime, P1048), and proteins were collected by centrifugation at 12000×g at 4°C for 15 minutes. Protein concentrations were determined using the BCA method, and the concentrations of various cell proteins were adjusted to 2 mg / mL. Subsequently, the protein extracts were boiled in 5x loading buffer (Beyotime, P0015) for 10 minutes, separated by SDS-polyacrylamide gel electrophoresis (Genscript, M42012), and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with primary antibody and then incubated for 1 hour at room temperature with secondary antibody (Cell Signaling Technology, 7074 / 7076). All antibodies were diluted in blocking buffer. Finally, the antigen-antibody reaction was visualized using an enhanced chemiluminescence kit (Absin, abs920). The primary antibodies used in the experiment were anti-β-Actin (CST, #4970), anti-STEAP1 antibody (Abcam, ab290723), and DYKDDDDK tag monoclonal antibody (Proteintech, 66008-4-lg).

[0250] The CCK-8 assay was used to detect the toxicity of TCPC to wild-type and STEAP1-overexpressing DU145 cells and wild-type and STEAP1-knockdown LNCaP cells. LNCaP cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. DU145 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. After 24 hours of normal culture, cells were digested and counted using 0.25% trypsin and seeded at a density of 5000 cells / well in 96-well plates. After 24 hours of culture, cells were treated with different concentrations of TCPC for 24 hours. Cell Counting Kit-8 was then diluted 10-fold with fresh medium and the drug-containing medium was replaced. After a period of culture, the absorbance at 450 nm per well was measured using a microplate reader to assess cell viability. The half-inhibitory concentration (IC50) was calculated for each data point. 50 Fitting was performed to obtain the IC50 of TCPC for different tumor cells. 50 value.

[0251] Conclusion: The expression level of STEAP1 in DU145 cells was increased by 2-3 times. Figure 29 TCPC's IC50 values ​​for wild-type DU145 and DU145 overexpressing STEAP1 50The concentrations were 51.96 μM and 37.45 μM, respectively; the expression level of STEAP1 in DU145 cells was knocked down to 30% of its original level. Figure 30 TCPC transfection of wild-type LNCaP cells with siRNA vectors resulted in an IC50 of LNCaP cells transfected with two siRNAs. 50 The concentrations were 10.22 μM, 11.00 μM, 18.85 μM, and 21.71 μM, respectively. This indicates that STEAP1 plays an important role in the antitumor activity of TCPC.

[0252] Example 15: Carboxy-fullerene molecules (TCPC) downregulate STEAP1 expression.

[0253] Cells: LNCaP cells

[0254] Grouping: The concentration-dependent experimental groups were treated with TCPC solutions at concentrations of 0, 1, 2, 5, and 10 μM, respectively; the time-dependent experimental groups were treated for 0, 2, 4, 8, 12, 24, and 48 hours, respectively; and the rescue experimental groups were treated with 10 μM TCPC for 24 hours followed by normal incubation for 2, 4, 8, 12, 24, and 48 hours.

[0255] Experimental Methods: Western blot detection of STEAP1 protein levels in cells: After normal culture for 24 hours, cells were treated with different concentrations of TCPC for 24 hours; then treated with 10 μM TCPC for different times; after 24 hours of treatment with 10 μM TCPC, the drug-containing medium was replaced with complete medium and cultured for different times. The treated cells were lysed in RIPA lysis buffer containing a mixture of protease phosphatase inhibitors, and proteins were collected by centrifugation at 12000g at 4°C for 10 minutes. Protein concentrations were determined using the BCA method, and various cellular proteins were adjusted to 2 mg / mL. Subsequently, the protein extracts were boiled in 5x loading buffer for 10 minutes, then separated by SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with STEAP1 and actin primary antibodies, and then incubated with secondary antibodies at room temperature for 1 hour. All antibodies were diluted in blocking buffer. Finally, the antigen-antibody reaction was visualized using an enhanced chemiluminescence kit.

[0256] Conclusion: TCPC can significantly downregulate STEAP1 expression in a concentration- and time-dependent manner. Figures 31-32 After removal of TCPC, the expression level of STEAP1 in LNCaP cells remained at a low level, indicating that TCPC has a long-lasting antitumor effect. Figure 33 ).

[0257] Example 16: TCPC-C2-pom downregulates STEAP1 expression

[0258] Cells: LNCaP cells

[0259] Grouping: The experimental groups were TCPC-C2-pom solutions with concentrations of 0, 0.05, 0.1, 0.2, and 0.5 μM, respectively.

[0260] Experimental Methods: Western blot detection of STEAP1 protein levels in cells: After culturing normally for 24 hours, the cells were treated with different concentrations of TCPC-C2-pom for 24 hours. The treated cells were lysed in RIPA lysis buffer containing a mixture of protease phosphatase inhibitors and centrifuged at 12000g at 4°C for 10 minutes to collect proteins. Protein concentrations were determined using the BCA method, and various cellular proteins were adjusted to 2 mg / mL. Subsequently, the protein extracts were boiled in 5x loading buffer for 10 minutes, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with STEAP1 and actin primary antibodies, and then incubated with secondary antibodies at room temperature for 1 hour. All antibodies were diluted in blocking buffer. Finally, the antigen-antibody reaction was visualized using an enhanced chemiluminescence assay kit.

[0261] Conclusion: TCPC-C2-pom can significantly downregulate STEAP1 expression in a concentration-dependent manner. Figure 34 ).

[0262] Example 17: Carboxyfullerene TCPC blocks cell cycle

[0263] Cells: LNCaP cells

[0264] Grouping: Control group, 5μM and 10μM TCPC groups

[0265] Experimental methods: Cell cycle determination – at a density of 1×10⁻⁶ 6 Cells were seeded into 6-well plates at a 1 / well ratio, treated with TCPC for 24 hours, and then digested into single cells with trypsin. Cells were fixed overnight at 4°C with pre-chilled ethanol. The fixed cells were stained in the dark for 30 minutes using a cell cycle kit containing PI and RNase A (DOJINDO), then resuspended in 500 μL PBS and analyzed by flow cytometry. (NxT, Thermo Fisher). The relative proportions of G1 / G0, S, and G2 / M cell cycle stages were calculated using Modfit.

[0266] Conclusion: After TCPC treatment, the proportion of cells in G0 / G1 phase increased compared to the control group. Figure 35 Therefore, the inhibitory effect of TCPC on cancer cell proliferation is mainly attributed to cell cycle arrest in the G0 / G1 phase.

[0267] Example 18: Carboxy-fullerene molecules downregulate the expression of downstream proteins related to cell growth and metastasis associated with STEAP1 via TCPC.

[0268] Cells: LNCaP cells

[0269] Grouping: Control group, 4 and 8 μM TCPC solution

[0270] Experimental Methods: Western blot was used to detect the expression levels of c-myc, CCND1, pRb, PCNA, p-53, N-cadherin, and MMP-9 proteins in cells. After 24 hours of normal culture, cells were treated with different concentrations of TCPC for 24 hours. The treated cells were lysed in RIPA lysis buffer containing a mixture of protease phosphatase inhibitors and centrifuged at 12000g at 4°C for 10 minutes to collect proteins. Protein concentrations were determined using the BCA method, and the concentrations of various cellular proteins were adjusted to 2 mg / mL. Subsequently, the protein extracts were boiled in 5x loading buffer for 10 minutes, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with primary antibody and then incubated with secondary antibody at room temperature for 1 hour. All antibodies were diluted in blocking buffer. Finally, the antigen-antibody reaction was visualized using an enhanced chemiluminescence assay kit. The primary antibodies used in the experiment included anti-β-Actin (CST, #4970), anti-STEAP1 antibody (Abcam, ab290723), DYKDDDDKtag monoclonal antibody (Proteintech, 66008-4-lg), C-MYC monoclonal antibody (Proteintech, 67447-1-Ig), Cyclin D1 polyclonal antibody (Proteintech, 26939-1-AP), and Phospho-Rb (Ser807 / 811)(D20B12). Rabbit mAb (CST, 8516S), PCNA Polyclonalantibody (Proteintech, 10205-2-AP), P53 Polyclonal antibody (Proteintech, 21891-1-AP), Anti-N Cadherin antibody (Abcam, ab18203), MMP-9 (N-terminal) Polyclonalantibody (Proteintech, 10375-2-AP).

[0271] Conclusion: TCPC significantly downregulated STEAP1 expression, and also downregulated PCNA, pRb, N-cadherin, and MMP-9 in a concentration-dependent manner, while having no effect on CCND1 expression. Further investigation was conducted on c-Myc protein, an upstream protein of CCND1. Myc is an essential early response gene in the cell cycle transition from G1 to S phase, also known as the "progression factor" of G1 phase. TCPC treatment significantly reduced c-Myc protein expression in LNCaP. This indicates that TCPC inhibited STEAP1 activity, thereby suppressing the expression of the G1-S phase response gene c-Myc, which in turn inhibited the activity of the CCND1 / CDK4 complex, reduced Rb phosphorylation levels, and prevented E2F release, thus arresting the cell cycle in the G0 / G1 phase. Simultaneously, the expression levels of N-cadherin and MMP-9, proteins related to cell migration, were also downregulated, thereby inhibiting cell migration. On the other hand, the upregulation of the tumor suppressor gene p-53 also participates in cell cycle arrest. Figure 36 ).

[0272] Example 19: In vivo antitumor therapy using carboxy-fullerene molecules TCPC

[0273] Animal strain: NCG male rat, 4 weeks old, weighing between 16-20g.

[0274] Tumor model: Human prostate cancer LNCaP cells with high STEAP1 expression

[0275] Experimental groups: Experimental groups: 1mM TCPC group, 2mM TCPC group, 4mM TCPC group, 2mM TCPC-C2-pom group; Control group: saline group

[0276] Administration method: Tail vein injection

[0277] Dosage: 0.1 mL / animal by injection.

[0278] Experimental method: Subcutaneous inoculation at a rate of 5 × 10 6 One LNCaP prostate cancer cell was inoculated, and seven days later, when the tumor volume reached 100 mm... 3 Mice were injected with 100 μL each of 1 mM TCPC, 2 mM TAPC, and 4 mM TCPC via the tail vein, while the control group received an equal volume of physiological saline via the tail vein. The drugs were administered every other day for a total of 12 doses. Mice were weighed and tumor size was measured at each administration to observe the tumor inhibition in the treatment group. When the tumor volume in the control group reached 500 mm², the tumor was considered inhibited. 3 Treatment was discontinued, and the mice were dissected. Major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were harvested. Some parts were cryopreserved in liquid nitrogen, and the rest were fixed in 4% paraformaldehyde fixative.Figure 37 ).

[0279] Experimental results: A significant difference was observed between the treatment group and the control group after treatment with TCPC and TCPC-C2-pom, indicating a decrease in tumor weight. Figure 38 ) and volume ( Figure 39 The right mouse weight was significantly smaller than the control group, indicating that TCPC and TCPC-C2-pom had significant tumor-inhibiting effects, and these effects were concentration-dependent. Meanwhile, there was no significant change in mouse body weight. Figure 39 (Left) This demonstrates that TCPC and TCPC-C2-pom have high security.

[0280] Example 20: Carboxy-fullerene molecule TCPC downregulates STEAP1 expression in tumors.

[0281] Sample: The aforementioned cryopreserved tumor in mice.

[0282] Experimental Methods: Western blot detection of STEAP1 protein levels in tumor tissues: 40 mg samples of tumor tissue from each group were cut and lysed in RIPA lysis buffer containing a mixture of protease phosphatase inhibitors. The tissues were then homogenized until no obvious lumps remained, followed by sonication. Proteins were collected by centrifugation at 12000g at 4°C for 10 minutes. Protein concentrations were determined using the BCA method, and various cellular proteins were adjusted to 2 mg / mL. Subsequently, the protein extracts were boiled in 5x loading buffer for 10 minutes, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with primary antibody and then incubated with secondary antibody at room temperature for 1 hour. All antibodies were diluted in blocking buffer. Finally, the antigen-antibody reaction was visualized using an enhanced chemiluminescence assay kit.

[0283] Conclusion: TCPC and TCPC-C2-pom can significantly downregulate the expression of STEAP1 in tumor tissues, and also downregulate proteins related to cell growth and metastasis such as CCND1, pRb, and N-cadherin. Figure 40 This is consistent with the findings at the cellular level, where TCPC inhibits tumor growth by degrading STEAP1.

Claims

1. Use of a fullerene derivative of formula (I) or a salt thereof in the preparation of a medicament for treating tumors with high STEAP1 expression; in, F is selected from fullerenes, wherein the fullerene is selected from one or more of hollow fullerenes, metallofullerenes, heterocyclic fullerenes, and endogenous fullerenes; R is a modifying group on a fullerene; m is an integer selected from 1 to 12; R is selected from -NR 1 R 2 C 1-10 Alkyl, -C 1-10 Alkyl-NR 1 R 2 -P(C=O)R 3 R 4 or R1, R2, R3, and R4 are each independently selected from hydrogen, hydroxyl, halogen, and C. 1-10 Alkyl, C 1-10 Alkoxy, C 1-10 Alkyl-C1-10 alkoxy, C 1-10 Alkyl-C 1-10 Carboxyl group, C 6-10 Aryl, C 1-10 Alkyl-C 6-10 Aryl, C 1-10 alkyl-5 to 20-membered heterocyclic aryl groups; the C 1-10 In the alkyl group, 1, 2, 3, 4, 5, 6, 7, 8, and 9 -CH2- can be replaced by -O-CH2CH2-; or R1 and R2 form a 5- to 20-membered heterocyclic alkyl group with their adjacent N atom; the C 1-10 Alkyl, C 1-10 Alkoxy, C 6-10 aryl, 5 to 20-membered heterocyclic aryl, optionally surrounded by one or more members selected from R 11 Substituents of the substituents; Alternatively, when m is not 1, two arbitrarily connected Rs form C. 3-20 Cycloalkyl or 3 to 20-membered heterocycloalkyl; the C 3-20 Cycloalkyl or 3 to 20-membered heterocyclic alkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents; n1 and n2 are each independently selected from 0, 1 or 2; X is selected from CH or N; R 11 Selected from amino, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-10 Alkyl-C 1-10 Carboxyl group, C 1-10 Alkyl-C 6-10 Aryl, -(C=O)OC 1-10 Alkyl-5 to 10-membered heterocyclic aryl group, wherein the 5 to 10-membered heterocyclic aryl group is optionally surrounded by one or more amino groups, C 1-6 Alkyl, C 1-6 Carboxyl group, -C 1-6 Alkyl-amino, C 1-6 Alkyl-C 1-6 Carboxyl substitution.

2. The use according to claim 1, wherein, F is selected from C 2n M@C 2n M2@C 2n MA@C 2n M3N@C 2n M2C2@C 2n M2S@C 2n M2O@C 2n and M x A 3-x N@C 2n Any one or a mixture thereof, wherein M and A are both metallic elements, and M and A are selected from any one of Sc, Y and lanthanide elements, and 30≤n≤60; Preferably, the fullerene is selected from C-containing... 2n One or more fullerene molecules, wherein 2n is the number of carbon atoms, 30 ≤ n ≤ 60; preferably, the fullerene is selected from C 60 C 70 C 76 C 78 C 80 and C 84 One or more; Preferably, the fullerene is selected from C 60 and C 70 One or more of the following.

3. The use according to claim 1 or 2, wherein, R is selected from -NR 1 R 2 C 1-6 alkyl, or Alternatively, when m is not 1, two adjacent R are connected to form C. 3-10 Cycloalkyl or 3- to 10-membered heterocycloalkyl; the C 3-10 Cycloalkyl or 3 to 10-membered heterocycloalkyl groups optionally composed of one or more selected from R 11 Substituents of the substituents; Among them, R 1 R 2 n1, n2, X, R 11 Each custom definition is the same as claim 1; Preferably, R is selected from -NR 1 R 2 C 1-3 alkyl, or Alternatively, when m is not 1, two adjacent Rs are linked to form a 3- to 6-membered heterocyclic alkyl group; the 3- to 6-membered heterocyclic alkyl group is optionally connected by one or more elements selected from Rs. 11 Substituents of the substituents; Among them, R 1 R 2 n1, n2, X, R 11 Each custom definition is the same as claim 1; Preferably, R is selected from -NR 1 R 2 C 1-3 alkyl, or Alternatively, when m is not 1, two adjacent Rs are connected to form an epoxy propylene alkyl group or a pyrrolidinyl group; the epoxy propylene alkyl group or the pyrrolidinyl group is optionally composed of one or more Rs selected from Rs. 11 Substituents of the substituents; Among them, R 1 R 2 n1, n2, X, R 11 Each custom definition is the same as claim 1; Preferably, R 1 R 2 Each is independently selected from hydrogen, hydroxyl, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl-C 1-6 Alkoxy, C 1-6 Alkyl-C 1-6 Carboxyl, phenyl, C 1-6 Alkyl-phenyl, C 1-6 alkyl-5 or 6-membered heterocyclic aryl; the C 1-6 In alkyl groups, the 1st, 2nd, 3rd, 4th, and 5th -CH2- can be replaced by -O-CH2CH2-; or R 1 R 2 The adjacent N atom forms a 5- to 10-membered heterocyclic alkyl group; the 5- to 10-membered heterocyclic alkyl group is optionally surrounded by one or more atoms selected from R. 11 Substituents of the substituents; Among them, R 11 The definition is the same as in claim 1; Preferably, the salt is selected from ammonium salts and metal salts; Preferably, the salt is selected from alkali metal salts and alkaline earth metal salts; Preferably, the salt is an alkali metal salt; Preferably, the salt is a sodium salt.

4. Use of the fullerene derivative of formula (I) or its salt according to any one of claims 1-3 in the preparation of STEAP1 degrading agent.

5. Use of the fullerene derivative of formula (I) or its salt according to any one of claims 1-3 in the preparation of a drug for blocking the tumor cell cycle and inhibiting tumor migration.

6. Formula (II) protein degrading agent or its stereoisomer, pharmaceutically acceptable salt: in, F, R, and m are each defined as in claim 1; L is -L1-L2-; L1 is -NR La R Lb ; R La R Lb Each is independently selected from: hydrogen, C 1-10 Alkyl, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 Alkoxy; the C 1-10 Alkyl, C 1-10 Alkoxy, halogenated C 1-10 Alkyl, Halogenated C 1-10 The -CH2- in the alkoxy group is optionally replaced by an oxygen atom; L2 is selected from: -(C=O)-NH-, -NH-; E3 is a ubiquitin ligand.

7. The protein degrading agent of formula (II) according to claim 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein, L is selected from: m1, m2, and m3 are each independently selected from integers from 1 to 10; Preferably, E3 is Wherein, T1, T2, and T3 are each independently CH, C, or N; the group is optionally surrounded by one or more elements selected from hydrogen, =O, C. 1-6 Alkyl substituents; Preferably, E3 is selected from: Preferably, E3 is selected from: Preferably, E3 is selected from: Preferably, the protein degrading agent of formula (II) is as shown in formula (III): In the formula, L and E3 are defined as before; Preferably, the protein degrading agent of formula (II) is as shown in formula (IV): In the formula, L is defined as before; Preferably, the protein degrading agent or its stereoisomer, or a pharmaceutically acceptable salt, is selected from:

8. Use of the protein degrader of formula (II) according to any one of claims 6-7 or its stereoisomers, or pharmaceutically acceptable salts, in the preparation of a medicament for treating tumors with high STEAP1 expression.

9. The use according to any one of claims 1-5, 8, wherein, The tumors that highly express STEAP1 are selected from STEAP1-highly expressing prostate cancer, renal cell carcinoma, bladder cancer, human Ewing's tumor, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, lung cancer, endometrial cancer, melanoma, glioma, kidney cancer and / or liver cancer. Preferably, the tumors that highly express STEAP1 are selected from breast cancer, lung cancer, melanoma, prostate cancer, glioma, liver cancer and / or human Ewing's tumor that highly express STEAP1. Preferably, the tumor with high STEAP1 expression is prostate cancer with high STEAP1 expression; Preferably, the STEAP1-overexpressing prostate cancer is metastatic castration-resistant prostate cancer; Preferably, the treatment of tumors with high STEAP1 expression involves inhibiting tumor proliferation, blocking the tumor cell cycle, and / or inhibiting tumor cell migration. Preferably, the treatment of tumors with high STEAP1 expression is for the treatment of tumor proliferation.

10. The use according to any one of claims 1-5, 8, wherein, The drug comprises a fullerene derivative of formula (I) or a salt thereof according to any one of claims 1-3, or a protein degrader of formula (II) or a stereoisomer thereof, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier according to any one of claims 6-7. Preferably, the formulation of the drug is selected from one or more of the following: solution, granules, lyophilized powder, emulsion, suspension, oil, and nano-formulation; preferably, the solution is an injection.

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