Salinomycin derivative, and preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of biological medicine, and discloses a salinomycin derivative, and a preparation method therefor and a use thereof. The salinomycin derivative comprises a general formula structure of formula (1), and compared with salinomycin, the activity of the salinomycin derivative is significantly improved. A group at the C-20 position contains a disulfide bond. By means of a replacement reaction of the disulfide bond, quick and efficient coupling with a polypeptide or an antibody can occur to obtain a GSH-sensitive salinomycin derivative containing the polypeptide or the antibody, or a salt thereof. The salinomycin derivative containing the polypeptide or the antibody, or the salt thereof has improved solubility, and also has enhanced targeting performance, has improved drug selectivity and biological activity, has a bystander effect, and in addition to being able to kill tumor cells highly expressing antigens, has a significant inhibitory effect on cells lowly expressing antigens. In addition, the salinomycin derivative or the salt thereof has the effects of reversing drug resistance in tumor cells and enhancing drug sensitization and can be used in combination with other drugs to further improve the efficacy.

Description

Salinomycin derivatives and their preparation methods and applications Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to salinomycin derivatives and preparation methods and applications thereof. Background Art

[0002] Tumors have always been one of the biggest threats to human health. Although a variety of treatments, such as chemotherapy and immunotherapy, are available to treat cancer (malignant tumors), the mortality rate from tumors remains high. Studies have shown that only a small number of tumor cells with tumorigenic ability are the main factors in the occurrence and development of tumors. These cells are called cancer stem cells. In addition, drug resistance also exists in the process of tumor treatment. There is also a lack of effective treatment for patients with drug resistance and high relapse rate. Finding effective tumor stem cell inhibitors is one of the main directions in tumor treatment, and finding drugs that can reverse drug resistance and enhance sensitization is also a meaningful direction.

[0003] Salinomycin (Sal) is a polyether ionophore antibiotic produced by Streptomyces albus and is a broad-spectrum antibacterial agent. Existing studies have shown that salinomycin also has anticancer activity, with significant inhibitory effects on various tumor cells, including cancer stem cells. It also has the ability to reverse drug-resistant cells and enhance drug sensitization. As a new generation of anti-tumor drug candidate, salinomycin faces significant challenges in its further application and development, including its near-insoluble nature in water, the need for further enhancement of its biological activity, its low drug selectivity, and the presence of certain toxic side effects.

[0004] Chemical structure modification to obtain salinomycin derivatives is one of the means to solve the problems existing in the application of salinomycin. In the related art, for example, Chinese invention patent publication number CN105732655A discloses the preparation and application of a novel structural salinomycin derivative, which achieves nucleophilic substitution of the 20-hydroxyl group and configuration inversion by selective Mitsunobu reaction to obtain a 20-azido derivative of salinomycin, and then obtains a salinomycin derivative with a triazole structure by selective click reaction of alkyne and the 20-azido derivative of salinomycin, which has higher activity than salinomycin; for example, Chinese invention patent publication number CN107428772A discloses a novel structural salinomycin derivative. A nitrogen-containing analogue of salinomycin, its synthesis and use against cancer stem cells and malaria, and its activity is also significantly improved; for example, the Chinese invention patent with publication number CN115052879A discloses a compound composed of C20-modified salinomycin derivatives, a method for obtaining the same, a composition containing the same, the use of the compound and a method for obtaining an intermediate product, and obtains a C20-N-acyl derivative of salinomycin, whose anti-cancer activity is also significantly improved; for example, Huang et al. used a semi-synthetic method to dehydroxylate, acylate or hydrogenate C18-C19 of salinomycin at the C20 position to obtain five derivatives, all of which have better killing effects on breast cancer stem cells than salinomycin. However, due to reasons such as the modification site and the modification group, the results of chemical structure modification are not completely controllable. For example, Jiang et al. blocked the slnM gene in S. albus and obtained three salinomycin derivatives, but their anti-tumor ability and antibacterial activity were weaker than those of the original salinomycin. For another example, the carboxyl group at the C1 position was connected with L-amino acid methyl ester, which had a good killing effect on leukemia cell proliferation and doxorubicin-resistant colon cancer cells. However, the anti-tumor ability and antibacterial activity of the derived compounds were weaker than those of the original salinomycin by single-substituted halogenation or nitration of N-benzylamine amidation at the carboxyl group.

[0005] The aforementioned chemical structure modifications all focus on modifying salinomycin via groups, thereby enhancing the biological activity (anticancer activity) of salinomycin derivatives. However, none of these chemical structure modifications address the issue of drug selectivity. Given that salinomycin or salinomycin derivatives may have certain toxic side effects, their drug selectivity also warrants attention.

[0006] Obtaining a targeted salinomycin derivative to enhance the drug's targeting and subsequently improve the biological activity of salinomycin is one of the promising solutions to its low selectivity. However, no relevant reports have been published so far. Summary of the Invention

[0007] 1. Purpose of the Invention

[0008] One of the purposes of the present application is to provide a salinomycin derivative or a salt thereof, wherein the salinomycin derivative is a compound obtained by modifying the C-20 hydroxyl group of salinomycin, and the modified C-20 group contains a disulfide bond. On the one hand, the derivative has higher biological activity than salinomycin; on the other hand, the presence of the disulfide bond facilitates the coupling of the salinomycin derivative with a polypeptide or antibody to obtain a salinomycin derivative or a salt thereof containing a polypeptide or antibody.

[0009] The second purpose of the present application is to provide the above-mentioned polypeptide- or antibody-containing salinomycin derivative or its salt. On the one hand, the polypeptide- or antibody-containing salinomycin derivative can improve the physicochemical properties of salinomycin by means of the physicochemical properties of the polypeptide or antibody, thereby improving its solubility; on the other hand, by means of the specific binding properties of the polypeptide or antibody, it can enhance targeting, improve drug selectivity and biological activity.

[0010] The third object of the present application is to provide the application of the above-mentioned salinomycin derivatives or the above-mentioned salinomycin derivatives containing polypeptides or antibodies.

[0011] 2. Technical solution

[0012] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0013] In a first aspect, the present application provides a salinomycin derivative or a salt thereof, wherein the salinomycin derivative has the structure of the following formula (1):

[0014] in:

[0015] -X is H, Li, Na or K.

[0016] Furthermore, the above-mentioned salinomycin derivative or its salt has a structure of the following formula (2):

[0017] Furthermore, the above-mentioned salinomycin derivative or its salt has a structure of the following formula (3):

[0018] Furthermore, the above-mentioned salinomycin derivative or its salt has a structure of the following formula (4):

[0019] Furthermore, the above-mentioned salinomycin derivative or its salt has a structure of the following formula (5):

[0020] The second aspect of the present application provides a method for preparing the above-mentioned salinomycin derivative or its salt, comprising oxidizing the C20-OH of salinomycin using an oxidant, then reducing the salinomycin C20-OH to C20-NH2 using a reductive amination reaction, and then performing an esterification reaction to modify it. Reducing C20-OH to C20-NH2 can enhance the chemical reaction activity.

[0021] Furthermore, the above preparation method comprises:

[0022] (1) preparing compound A by reacting 3-mercaptopropionic acid with 2,2-disulfide dipyridine, and then reacting compound A with oxalyl chloride to prepare compound B;

[0023] (2) selectively oxidizing the 20-OH group of sodium salinomycin using MnO2 to obtain compound C; then using reductive amination to prepare compound D;

[0024] (3) Compound B is reacted with D to obtain a salinomycin derivative of formula (1).

[0025] Furthermore, the above preparation method comprises:

[0026] Dissolve salinomycin sodium in DCM, add MnO2, and stir overnight at 25°C under N2 protection; filter through celite, combine the DCM phases, wash with hydrochloric acid, separate the layers, dry over anhydrous Na2SO4, and concentrate to obtain compound C as a white solid;

[0027] Compound C was dissolved in anhydrous methanol (MeOH), and NH3 / MeOH solution and 5 drops of acetic acid (HAC) were added, followed by the addition of 20 ml of DCM for complete dissolution, and the mixture was stirred at 25°C. Subsequently, cerium trichloride heptahydrate (CeCl3·7H2O) was added to the system, and the mixture was stirred at room temperature. Subsequently, sodium cyanoborohydride (NaBH3CN) / 50 ml of MeOH solution was added dropwise evenly using a constant pressure dropping funnel, and the mixture was stirred overnight at 25°C. After the reaction, the system was concentrated to dryness, dissolved in DCM, washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and subjected to column chromatography (eluent: MeOH / DCM = 1:50 - MeOH / DCM = 1:20) to obtain compound D.

[0028] 3-Mercaptopropionic acid and 2,2-dithiodipyridine were dissolved in ethanol; after 15 minutes, 10 drops of acetic acid (HAC) were added to the system and stirred at room temperature; after the reaction, the system was concentrated, and the excess 2,2-dithiodipyridine was removed by column chromatography (EA / PE = 1:6), and purified using EA / PE = 1:5 + 1% HAC, and concentrated to dryness using a rotary evaporator to obtain compound A;

[0029] Compound A was dissolved in DCM, and oxalyl chloride (oxalyl chloride) was added to the system under N2 protection and ice-water bath conditions. After about 30 seconds of dropwise addition, the ice-water bath was removed, and 2 drops of N,N-dimethylformamide (DMF) were added to the system, and stirred at 25°C. After the reaction, the system was concentrated and drained, and DCM was added again and concentrated to dryness to obtain compound B.

[0030] Compound D was dissolved in DCM and triethylamine (C6H 15 N), N2 displacement protection, and ice-water bath conditions, compound B / 30 ml of ultra-dry DCM solution was added dropwise to the system. After 20 minutes, the addition was completed, and the mixture was naturally warmed to room temperature and stirred overnight. After the reaction, the reaction system was diluted with DCM, washed once with HCl aqueous solution and once with saturated NaCl, concentrated, and purified by column chromatography (eluent: first with EA / PE = 1:4 + 1% HAC until there were no impurities, and then purified with MeOH / DCM = 1:50) to obtain compound E, i.e., a compound with the structure of formula (2).

[0031] The third aspect of the present application provides the use of the above-mentioned salinomycin derivative or its salt in the preparation of a salinomycin derivative or its salt containing a polypeptide or an antibody.

[0032] Furthermore, the above application includes: coupling the salinomycin derivative or its salt with the polypeptide or antibody through a disulfide bond replacement reaction between the disulfide bond in the above salinomycin derivative or its salt and the sulfhydryl group in the polypeptide or antibody to obtain a salinomycin derivative or its salt containing the polypeptide or antibody.

[0033] Furthermore, the above-mentioned polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or functional antibody connected to a compound containing a thiol group.

[0034] Furthermore, the above-mentioned compound containing a thiol group includes: cysteine ​​(Cys), and the side chain group of cysteine ​​contains a thiol group (-SH).

[0035] Furthermore, the above-mentioned thiol-containing compounds include: compounds containing thiol (-SH) such as mercaptoethylamine, thioglycolic acid, and mercaptopropionic acid.

[0036] Furthermore, the above-mentioned polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or functional antibody and a compound containing a thiol group linked in sequence.

[0037] Furthermore, the above-mentioned polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or functional antibody and cysteine ​​linked in sequence.

[0038] Furthermore, the above-mentioned polypeptides or antibodies include: polypeptides or antibodies composed of functional polypeptides or functional antibodies linked in sequence with compounds such as mercaptoethylamine, thioglycolic acid, and mercaptopropionic acid.

[0039] Furthermore, the above functional polypeptides include: cell penetrating peptides, targeting peptides, etc.

[0040] Furthermore, the above-mentioned targeting peptides include: polypeptides targeting the tumor microenvironment, polypeptides targeting proteins highly expressed in tumor cells, etc.

[0041] Furthermore, one or more sarcosines (Sar) are connected between the functional polypeptide or functional antibody and cysteine, and the one or more sarcosines (Sar) can reduce the interaction between the functional polypeptide or functional antibody and the small molecule (the above-mentioned salinomycin derivative or its salt).

[0042] Furthermore, the number of the sarcosine is 1 to 12.

[0043] Furthermore, the number of the sarcosine is 6 to 10.

[0044] Furthermore, the number of the sarcosine is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Still further, the number of the sarcosine is 10.

[0045] Furthermore, the above-mentioned targeting peptide is polypeptide A6 (KPSSPPEE, SEQ ID NO.1), which targets CD44 protein. CD44 protein is a complex transmembrane adhesion glycoprotein expressed in a variety of human cells, including embryonic stem cells, differentiated cells and cancer cells. It is a recognized marker of tumor stem cells and a key regulator of epithelial-mesenchymal transition. It is involved in the occurrence, progression and metastasis of tumors. Targeting CD44 protein means being able to bind to CD44 protein in a targeted manner, thereby delivering salinomycin derivatives or salts containing polypeptides or antibodies to tumor stem cells, etc., thereby enhancing the targeting of drugs, improving the selectivity of drugs, increasing the activity of drugs, and reducing the toxicity of drugs.

[0046] Furthermore, the above polypeptide or antibody includes: polypeptide A6-Sar, whose structure is: Ac-KPSSPPEE(Sar) 10 C-NH2 (SEQ ID NO. 2).

[0047] Furthermore, the above application includes: coupling the salinomycin derivative or its salt with the polypeptide A6-Sar through a disulfide bond replacement reaction between the disulfide bond in the above-mentioned salinomycin derivative or its salt and the sulfhydryl group of cysteine ​​in the polypeptide A6-Sar, thereby obtaining a salinomycin derivative or its salt containing the polypeptide A6-Sar, wherein the A6 sequence (KPSSPPEE) contained therein can target the CD44 protein.

[0048] Furthermore, the above application includes: the disulfide bond replacement reaction is carried out in an organic solvent.

[0049] Furthermore, the organic solvent includes dimethyl sulfoxide (DMSO).

[0050] The fourth aspect of the present application provides a salinomycin derivative or a salt thereof containing a polypeptide or an antibody prepared in the above application.

[0051] Furthermore, the above-mentioned polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (6):

[0052] -X is H, Li, Na or K;

[0053] -Y is a functional polypeptide or a functional antibody;

[0054] n=1~12.

[0055] Furthermore, the above-mentioned polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (7):

[0056] -X is H, Li, Na or K.

[0057] Furthermore, the above-mentioned polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (8):

[0058] Furthermore, the polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (9):

[0059] Furthermore, the polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (10):

[0060] Furthermore, the above-mentioned polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (11):

[0061] The fifth aspect of the present application provides the use of the above-mentioned salinomycin derivative or its salt, or the above-mentioned salinomycin derivative or its salt containing a polypeptide or antibody in the preparation of a drug, which is used to treat tumors, or inhibit angiogenesis, etc.

[0062] Furthermore, the above-mentioned tumor treatment includes: inhibiting tumor cells / tissues, tumor stem cells, and / or reversing the drug resistance of tumor cells / tissues.

[0063] Furthermore, the above-mentioned tumors include:

[0064] Non-small cell lung cancer, including but not limited to lung epithelial cell carcinoma, lung adenocarcinoma, and human lung squamous cell carcinoma;

[0065] Ovarian cancer, including but not limited to epithelial ovarian cancer and ovarian cystadenocarcinoma;

[0066] Kidney cancer, including but not limited to renal cell carcinoma;

[0067] Prostate cancer, including but not limited to prostate adenocarcinoma;

[0068] stomach cancer;

[0069] Uterine corpus cancer;

[0070] Breast cancer, including but not limited to breast adenocarcinoma, inflammatory breast cancer, and metastatic adenocarcinoma;

[0071] pancreatic cancer;

[0072] Leukemia, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma;

[0073] Colorectal (colon) cancer, including but not limited to colorectal adenocarcinoma and colon epithelial cell carcinoma;

[0074] Central nervous system tumors, including but not limited to brain tumors such as gliomas;

[0075] Melanoma, including but not limited to malignant melanoma, epithelial melanoma, and non-epithelial melanoma;

[0076] or sarcoma.

[0077] Furthermore, the above-mentioned drug administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc., and can be administered in unit dosage form.

[0078] Furthermore, the above-mentioned drugs can be in liquid dosage form, solid dosage form or semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), emulsion (including o / w type, w / o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drops, nasal drops, lotion and liniment, etc.; the solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; the semi-solid dosage form can be an ointment, gel, paste, etc.

[0079] Furthermore, the above-mentioned drugs can be conventional preparations, sustained-release preparations, controlled-release preparations, targeted preparations or various microparticle delivery systems.

[0080] In a sixth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned salinomycin derivative or a salt thereof, or the above-mentioned salinomycin derivative containing a polypeptide or an antibody or a salt thereof.

[0081] Furthermore, the above-mentioned pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0082] Furthermore, the pharmaceutical composition further comprises a platinum-based therapeutic agent, also known as a platinum. Platinums cause DNA crosslinking, causing them to inhibit DNA repair and / or DNA synthesis, primarily in rapidly multiplying cells such as cancer cells. Furthermore, the platinum-based therapeutic agent is selected from the group consisting of cisplatin; carboplatin; oxaliplatin; nedaplatin, picoplatin; and satraplatin.

[0083] Furthermore, the above-mentioned pharmaceutical composition further includes a nucleoside inhibitor, or a therapeutic agent that interferes with normal DNA synthesis, protein synthesis, cell replication or inhibits rapidly proliferating cells in other ways, and the nucleoside inhibitor is selected from trabectedin, nitrogen mustard; vincristine; temozolomide; cytarabine injection; lomustine; azacitidine; homoharringtonine; Erwinia chrysanthemi; eribulin mesylate; cabazitaxel; capecitabine; bendamustine; ixabepilone; nelarabine; clofarabine; trifluridine and / or tipiracil.

[0084] Furthermore, the above-mentioned pharmaceutical composition further comprises one or more kinase inhibitors or VEGF-R antagonists, including: anti-VEGF monoclonal antibody bevacizumab; anti-VEGFR-2 antibodies ramucirumab and aflibercept. VEGFR inhibitors such as regorafenib; vandetanib; axitinib; and lenvatinib; Raf inhibitors such as sorafenib; dabrafenib; and vemurafenib; MEK inhibitors such as cobimetinib; trametinib; Bcr-Abl tyrosine kinase inhibitors such as imatinib, nilotinib; dasatinib; bosutinib; and ponatinib; Her2 and EGFR inhibitors such as gefitinib; erlotinib; lapatinib; afatinib; osimertinib; and brigatinib; c-Met and VEGFR2 inhibitors such as cabozantinib; and multikinase inhibitors such as sunitinib; pazopanib; ALK inhibitors such as crizotinib; ceritinib; and alectinib; Bruton's tyrosine kinase inhibitors such as ibrutinib; and Flt3 receptor inhibitors such as midostaurin.

[0085] Furthermore, the above-mentioned pharmaceutical composition also includes other kinase inhibitors and VEGF-R antagonists that are being developed and can be used in the present application, including tivozanib; vatalanib; delitinib; duvitinib; sioronib; linifanib; neratinib; radotinib; ruxolitinib; foretinib; quizartinib and / or motesanib.

[0086] Furthermore, the above pharmaceutical composition further comprises one or more other therapeutic agents which are mTOR inhibitors that inhibit cell proliferation, angiogenesis and glucose uptake, and the mTOR inhibitor is everolimus; temsirolimus; and / or sirolimus.

[0087] Furthermore, the pharmaceutical composition further comprises one or more other therapeutic agents that are proteasome inhibitors, such as bortezomib, carfilzomib, and / or ixazomib, or taxane compounds that cause the destruction of microtubules essential for cell division, and the taxane compounds are selected from taxane drugs such as paclitaxel, docetaxel, albumin-bound paclitaxel, and cabazitaxel.

[0088] Furthermore, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents which are aromatase inhibitors, such as exemestane, anazol and / or letrozole.

[0089] Furthermore, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents that are antagonists of the hedgehog pathway. Approved hedgehog pathway inhibitors that can be used in this application include sonidegi and / or vismodegib.

[0090] Furthermore, the above-mentioned pharmaceutical composition also includes a folic acid inhibitor, such as pemetrexed.

[0091] Furthermore, the above-mentioned pharmaceutical composition also includes a CC chemokine receptor 4 (CCR4) inhibitor, such as moglizumab.

[0092] Furthermore, the above-mentioned pharmaceutical composition also includes an isocitrate dehydrogenase (IDH) inhibitor.

[0093] Furthermore, the above-mentioned pharmaceutical composition also includes an arginase inhibitor.

[0094] Furthermore, the above-mentioned pharmaceutical composition also includes a glutaminase inhibitor.

[0095] Furthermore, the above-mentioned pharmaceutical composition also includes an antibody that binds to a tumor antigen, i.e., a protein expressed on the cell surface of tumor cells. Approved antibodies that bind to tumor antigens that can be used in this application include rituximab; ofatumumab; obinutuzumab, ibritumomab tiuxetan; daratumumab, denosumab; trastuzumab; and pertuzumab; and / or venetoclax, etc. antibody drugs.

[0096] Furthermore, the pharmaceutical composition further comprises a topoisomerase inhibitor. Approved topoisomerase inhibitors that can be used in this application include irinotecan; topotecan; and / or pixantrone.

[0097] Furthermore, the above-mentioned pharmaceutical composition also includes an inhibitor of anti-apoptotic proteins such as BCL-2, such as venetoclax and belintomax.

[0098] Furthermore, the above-mentioned pharmaceutical composition further comprises an androgen / estrogen receptor inhibitor, such as enzalutamide, abiraterone, and / or raloxifene.

[0099] Furthermore, the above-mentioned pharmaceutical composition further comprises a bone resorption inhibitor, such as denosumab.

[0100] Furthermore, the above-mentioned pharmaceutical composition further comprises an inhibitor of the interaction between two primary p53 inhibitory proteins MDMX and MDM2.

[0101] Furthermore, the above-mentioned pharmaceutical composition also includes an inhibitor of transforming growth factor β (TGF-beta or TGFβ).

[0102] Furthermore, the above-mentioned pharmaceutical composition also includes an ADC drug selected from vegbatumumab-monomethyl auristatin E and the like.

[0103] Furthermore, the above-mentioned pharmaceutical composition further comprises an anti-proliferative compound, such anti-proliferative compounds including but not limited to aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; anti-tumor antimetabolites; platinum compounds; compounds that target / reduce protein or lipid kinase activity and additional anti-angiogenic compounds; compounds that target, reduce or inhibit protein or lipid phosphatase activity; gonadorelin agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; anti-proliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds for treating hematological malignancies; compounds that target, reduce or inhibit Flt-3 activity; Hsp90 inhibitors; and / or MEK inhibitors.

[0104] 3. Beneficial effects

[0105] Compared with the prior art, the present application has the following advantages:

[0106] (1) The salinomycin derivatives provided in this application, and their preparation methods and applications, include the general structure of formula (1). Compared with salinomycin, the activity of the salinomycin derivatives is significantly improved; and the C-20 group of the salinomycin derivatives contains a disulfide bond, which can be quickly and efficiently coupled with a polypeptide or antibody through a replacement reaction of the disulfide bond to obtain a GSH-sensitive salinomycin derivative containing a polypeptide or antibody or a salt thereof. The salinomycin derivatives containing a polypeptide or antibody or their salts can, on the one hand, improve the physicochemical properties of salinomycin by virtue of the characteristics of the polypeptide or antibody, such as improving its solubility; on the other hand, by virtue of the specific binding properties of the polypeptide or antibody, enhance targeting, improve drug selectivity and biological activity, and have a bystander effect. In addition to being able to kill tumor cells with high antigen expression, they also have a significant inhibitory effect on cells with low antigen expression.

[0107] (2) The salinomycin derivatives provided in this application, and their preparation methods and applications, including the general structure of formula (1) or formula (6), have significant inhibitory effects on tumor stem cells and drug-resistant cells, and have the effects of reversing tumor cell resistance and drug sensitization. They can be used in combination with other drugs to further improve the therapeutic effect.

[0108] (3) The salinomycin derivatives provided in the application, as well as their preparation methods and applications, can reduce the interaction between the polypeptide or antibody and small molecules by introducing a spacer region (i.e., Sar region) into the functional polypeptide or antibody when preparing the salinomycin derivatives or their salts. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 shows the MS identification results of compound A.

[0110] Figure 2 shows the MS identification results of compound C.

[0111] FIG3 is the MS identification result of compound D.

[0112] FIG4 is the MS identification result of compound E.

[0113] FIG5 shows the MS identification results of peptide A6-Sar.

[0114] FIG6 shows the MS identification results of the peptide-containing salinomycin derivative Sal-A6.

[0115] FIG7 shows the activity detection results of Sal and compound E at multiple tumor cell levels.

[0116] FIG8 shows the active toxicity effects of Sal and compound E on HUVEC cells at 24 h.

[0117] FIG9 shows the effects of Sal and compound E on the invasion ability of HUVEC cells in vitro at 24 h.

[0118] Figure 10 shows the effects of different concentrations of Sal and compound E on the invasion ability of HUVEC cells in vitro, #compared with Control, ###p<0.001, ####p<0.0001; *compared with Sal group; ****p<0.0001.

[0119] FIG11 shows the effects of Sal and compound E on the activity of HUVEC cells at 6 h.

[0120] FIG12 shows the effects of Sal and compound E on HUVEC cell tubule formation.

[0121] FIG13 is the statistical effect of Sal and compound E on the number of HUVEC cell tube formation; *compared with the Control group; **p<0.01.

[0122] FIG14 shows the solubility test results of Sal and Sal-A6 in water.

[0123] FIG15 shows the expression levels of CD44 protein in different cells.

[0124] FIG16 shows the activity of different cells detected by CCK8.

[0125] FIG. 17 shows the effects of Sal and Sal-A6 on the proliferation activity of SKOV3 cells at different time points.

[0126] FIG18 shows the effects of Sal and compound E on the proliferation activity of SKOV3 cells at different time points.

[0127] FIG19 shows the effects of Sal, Sal-A6, compound E and A6 on the migration of SKOV3 cells at 24 h.

[0128] Figure 20 shows the effects of Sal, Sal-A6, compound E (10 μM) and A6 (10 μM) on the migration rate of SKOV3 cells at 24 h; compared with Control, **p<0.01, ***p<0.001, ****p<0.0001; compared with Sal, ###p<0.001.

[0129] FIG21 shows the effects of Sal, Sal-A6 and compound E on the migration ability of SKOV3 cells in vitro at 48 h.

[0130] Figure 22 shows the inhibition rate of SKOV3 cell migration by Sal, Sal-A6 and Compound E at 48 h; *compared with the Sal group; *p<0.1, **p<0.01, ***p<0.001.

[0131] FIG23 shows the invasion rate statistics of Sal, Sal-A6 and compound E on SKOV3 cells at 24 h; *compared with Sal group; ***p<0.001, ***p<0.001.

[0132] FIG24 shows the effects of Sal, Sal-A6, and compound E on the cell cycle of SKOV3 cells.

[0133] FIG. 25 shows the effects of different concentrations of cisplatin on the proliferation activity of SKOV3 or SKOV3 / DDP at 48 h; ***p<0.001.

[0134] FIG26 shows the effects of Sal, Sal-A6, and compound E on the activities of different SKOV3 / DDPs.

[0135] FIG27 shows the effects of 1 μM Sal, compound E, and Sal-A6 on the activity of cisplatin at different concentrations.

[0136] FIG28 shows the effects of Sal, Sal-A6, and compound E on the spheroid formation of tumor stem cells.

[0137] FIG. 29 shows the effects of Sal, A6, and Sal-A6 on the activity of acclimated SKOV3 cells.

[0138] FIG30 shows the effects of Sal, A6 and Sal-A6 on the activity of acclimated A2780 cells.

[0139] FIG31 shows the effects of different concentrations of Sal-A6 on the activity of mixed cells, *compared with Control, ****p<0.0001.

[0140] FIG32 shows the effect of different concentrations of Sal-A6 on the fluorescence intensity of mixed cells.

[0141] FIG33 shows the inhibitory effects of Sal and Sal-A6 on SKOV3 subcutaneous transplanted tumors and their effects on the body weight of nude mice, *p<0.05, **p<0.01.

[0142] FIG34 shows the effects of Cetuximab and Sal-A6 on the body weight and tumor volume of nude mice in the Cal-27 nude mouse xenograft tumor model, *p<0.05, **p<0.001. DETAILED DESCRIPTION

[0143] The present application is further described below with reference to specific embodiments.

[0144] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0145] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0146] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0147] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0148] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0149] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0150] For all of the following examples, standard procedures and purification methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in ° C. The structures of the compounds were determined by H NMR spectroscopy ( 1 The purity was determined by H NMR) and mass spectrometry (MS) or by ultra-high performance liquid chromatography.

[0151] Example 1

[0152] This embodiment provides a salinomycin derivative or a salt thereof and a preparation method thereof. The salinomycin derivative has the structure of the following formula (1):

[0153] Wherein: -X is H, Li, Na or K.

[0154] In this embodiment, -X is H, that is, a salinomycin derivative having the following structure (2):

[0155] This embodiment also provides a method for preparing a salinomycin derivative of formula (2), which comprises oxidizing the C20-OH group of salinomycin with an oxidant, then reducing the C20-OH group of salinomycin to C20-NH2 using a reductive amination reaction, and then performing an esterification reaction to obtain the derivative. The method specifically comprises the following steps:

[0156] (1) Preparation of Compound A

[0157] 2.02g of 3-mercaptopropionic acid, 60ml of ethanol, and 8.35g of 2,2-disulfide dipyridine were added to a 250ml reaction flask, and the system changed from colorless and turbid to a yellow solution. After 15min, 10 drops of acetic acid (HAC) were added to the system and stirred at room temperature. The reaction progress was detected by thin layer chromatography (TLC). After the reaction was completed, the system was concentrated, and the excess 2,2-disulfide dipyridine was removed by column chromatography (EA / PE=1:6). EA / PE=1:5+1% HAC was used for purification, and compound A was obtained after concentration and drying using a rotary evaporator. The MS identification results of compound A are shown in Figure 1, and its reaction mechanism is as follows:

[0158] (2) Preparation of Compound B

[0159] In a 100ml reaction flask, 152mg of A and 30ml of ultra-dry dichloromethane (DCM) were added. Under N2 protection, 267mg of oxalyl chloride (oxalyl chloride) was added to the system under ice-water bath conditions. Bubbles were produced during the addition, and the color of the system changed from colorless to yellow. After about 30 seconds of dropwise addition, the ice-water bath was removed, and 2 drops of N,N-dimethylformamide (DMF) were added to the system. Stirring at 25°C, the system color changed to colorless, and a sample was taken for TLC (MeOH:DCM=1:20) detection. After the reaction was completed, the system was concentrated and drained, and DCM was added again to concentrate and drain to obtain compound B. The reaction mechanism is as follows:

[0160] (3) Preparation of Compound C

[0161] In a 250 ml reaction flask, 1.02 g of salinomycin sodium (Sal-Na, CAS No: 55721-31-8), 100 ml of ultra-dry DCM, and 3.36 g of MnO2 were added. Under N2 protection, the mixture was stirred overnight at 25°C to obtain a black turbid system. Samples were taken and detected by TLC (MeOH:DCM = 1:25). Filtered through celite, the DCM phases were combined, washed with 0.1 N hydrochloric acid (HCl), and after separation, the DCM phases were dried over anhydrous Na2SO4 and concentrated to obtain compound C as a white solid. The MS identification results of compound C are shown in Figure 2. The reaction mechanism is as follows:

[0162] (4) Preparation of Compound D

[0163] To a 250ml reaction flask, 810mg of C, 30ml of anhydrous methanol (MeOH), 1ml of NH3 / MeOH solution, and 5 drops of HAC were added. The yellow turbid solution was then completely dissolved in 20ml of ultra-dry DCM and stirred at 25°C. 400mg of cerium trichloride heptahydrate (CeCl3·7H2O) was then added to the system and stirred at room temperature until the system turned brown. A 133mg sodium cyanoborohydride (NaBH3CN) / 50ml MeOH solution was then added dropwise using a constant pressure dropping funnel. The mixture was stirred overnight at 25°C and the reaction was monitored by TLC (MeOH / DCM = 1:25) the next day. After the reaction, the system was concentrated to dryness, and 100 ml of DCM was added for dissolution. The product was washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and subjected to column chromatography (eluent: MeOH / DCM = 1:50-MeOH / DCM = 1:20) to obtain product D. The MS identification results of compound D are shown in FIG3 . The reaction mechanism is shown below:

[0164] (5) Preparation of Compound E

[0165] In a 100 ml reaction bottle, 102 mg of D, 20 ml of ultra-dry DCM, 30 mg of triethylamine (C6H 15N), N2 displacement protection, under ice-water bath conditions, to the system was added dropwise 50mg of B / 30ml of ultra-dry DCM solution. During the addition, the color of the system changed from colorless to light yellow. After 20 minutes of complete addition, the mixture was naturally warmed to room temperature and stirred overnight. TLC (MeOH / DCM = 1:20) was used to monitor the reaction. After the reaction, the reaction system was diluted with 50ml of DCM, washed once with 50ml of 1N HCl aqueous solution and once with 50ml of saturated NaCl. After concentration, it was purified by column chromatography (eluent: first EA / PE = 1:4 + 1% HAC until no impurities were present, then purified with MeOH / DCM = 1:50) to obtain product E. The MS identification results of compound E are shown in Figure 4, which is a compound of formula (2). The MS identification results of compound E are shown in Figure 4. Its reaction mechanism is as follows:

[0166] Example 2

[0167] This example provides the use of a salinomycin derivative (Compound E) in the preparation of a polypeptide-containing salinomycin derivative and the prepared polypeptide-containing salinomycin derivative.

[0168] The application includes: a disulfide bond replacement reaction between the disulfide bond in compound E and the sulfhydryl group in the polypeptide is carried out, so that compound E and the polypeptide are quickly coupled to obtain a salinomycin derivative containing a polypeptide, the polypeptide being: polypeptide A6-Sar, and having the structure: Ac-KPSSPPEE(Sar) 10 C-NH2 (SEQ ID NO. 2) is a polypeptide formed by sequentially linking functional polypeptide A6 (KPSSPPEE, SEQ ID NO. 1), 10 sarcosines (Sar), and cysteine ​​(Cys). The side chain of cysteine ​​contains a sulfhydryl group (-SH), which undergoes a disulfide bond replacement reaction with the disulfide bond in compound E. Specifically, the steps include:

[0169] (1) Preparation of polypeptide A6-Sar

[0170] The polypeptide is a polypeptide formed by sequentially linking a functional polypeptide A6 (KPSSPPEE, SEQ ID NO. 1), 10 sarcosines (Sar), and cysteine ​​(Cys). The introduction of the 10 sarcosines as a spacer can reduce the interaction between the targeting peptide (functional polypeptide A6) and the small molecule (compound E). The polypeptide A6-Sar is synthesized using a solid-phase synthesis method. The specific steps are as follows:

[0171] Weigh 1 g of RinkAM resin with a degree of substitution of 0.5 mmol / g and add it to a peptide solid phase reactor. Add 5 ml of DCM and swell with nitrogen bubbling for 10 minutes. Drain and remove Fmoc twice with 20% piperidine / DMF solution. At room temperature, use nitrogen bubbling for 10 minutes and 5 minutes respectively. After the reaction, wash with DMF 5 times. Use ninhydrin:phenol:piperidin (2:1:1) to detect for 30 seconds. If the resin turns dark blue, it proves that Fmoc has been successfully removed.

[0172] Accurately weigh 0.878 g of Fmoc-Cys(trt)-OH and 0.57 g of HBTU, use DMF as solvent, add 0.29 ml of DIEA, and react at room temperature with nitrogen for 1 h. Then drain and rinse twice with DMF.

[0173] Use acetic anhydride to seal the head. Specifically, use 5 ml of DCM as solvent, add 0.29 ml of DIPEA and 0.2 ml of acetic anhydride, and react with nitrogen at room temperature for 30 minutes. After the reaction is completed, use DMF to wash 4 times;

[0174] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0175] Weigh 0.47 g of Fmoc-Sar-OH and 0.57 g of HBTU into a reactor, use DMF as solvent, and add 0.29 ml of DIPEA. React at room temperature with nitrogen bubbling for 60 min, then drain and rinse four times with DMF. Detect with ninhydrin:phenol:piperidin (2:1:1) for 3 min. If the resin is colorless, the coupling is successful.

[0176] Remove Fmoc twice using 20% ​​piperidine / DMF solution at room temperature with nitrogen bubbling for 10 min and 5 min respectively. Wash with DMF five times after the reaction. Detect with ninhydrin:phenol:piperidin (2:1:1) for 30 s. If the resin turns dark blue, it indicates that Fmoc has been successfully removed.

[0177] Repeat the above operation to add each amino acid in the peptide sequence. Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Glu(otBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ser(tbu)-OH, Fmoc-Ser(tbu)-OH, Fmoc-Pro-OH and Fmoc-Lys(Boc)-OH were coupled in sequence according to the peptide sequence, and then the Fmoc protecting group was removed with 20% piperidine / DMF solution;

[0178] Use acetic anhydride to seal the head. Specifically, use 5 ml of DCM as solvent, add 0.29 ml of DIPEA and 0.2 ml of acetic anhydride, and react with nitrogen at room temperature for 30 minutes. After the reaction is completed, use DMF to wash 4 times;

[0179] 9.5 ml of TFA, 0.1 ml of H2O, 0.2 ml of EDT and 0.2 ml of Tis were used as lysis solution. The A6-Sar10-Cys protected peptide resin and the lysis solution were placed in a centrifuge tube together. After reacting for 2 h, the resin was precipitated with icy ether and centrifuged three times to obtain a white solid. The solid was dried in a vacuum drying oven to obtain a crude product. The crude product was purified by HPLC and lyophilized to obtain a white powder of the polypeptide A6-Sar. The MS identification results of the polypeptide A6-Sar are shown in FIG5 . Its structure is shown in formula (12):

[0180] (2) Conjugation of compound E with peptide A6-Sar

[0181] 200 mg of compound E, 800 mg of A6-Sar, and 100 ml of DMSO were weighed as solvent, and the reaction was carried out at room temperature under N2 protection for 72 h. After the reaction, purification was performed using HPLC (mobile phase A: ACN + 0.1% formic acid, mobile phase B: water + 0.1% formic acid) to obtain white powder Sal-A6, a polypeptide-containing salinomycin derivative with the structure of formula (8). The MS identification results are shown in Figure 6.

[0182] Example 3

[0183] This example provides an in vitro antitumor activity test of the salinomycin derivative (Compound E) prepared in Example 1.

[0184] Cells in logarithmic growth phase were taken, digested, counted, and prepared into 1×10 5Cell suspensions of 100 μL / well were plated in 96-well plates (100 μL / well) and cultured in a 37 ° C, 5% CO2 incubator for 24 hours. Subsequently, salinomycin (Sal) or compound E containing the corresponding concentrations was added according to 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM. A negative control group and a blank group were also set up, with 5 replicates per group, and the plate was placed in an incubator and cultured for 72 hours. After the end, 10 μL of CCK8 solution was added to each well, and the cells were incubated in a cell culture incubator for another 1 hour. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated.

[0185] The results are shown in Figure 7 and Table 1. Figure 7 shows the activity test results of Sal and Compound E in MKN45, HCCLM3, A431, A549, ACHN, Du145, and HeLa cells. The results showed that Compound E had a better inhibitory effect on tumor cells than Sal in MKN45, HCCLM3, A431, A549, ACHN, Du145, and HeLa cells.

[0186] Table 1 IC50 of Sal and compound E at multiple tumor cell levels

[0187] Example 4

[0188] This example provides the angiogenesis-inhibiting effect of the salinomycin derivative (Compound E) prepared in Example 1.

[0189] HUVEC (human umbilical vein endothelial cell) invasion assay:

[0190] First, the cytotoxicity of Compound E and Sal at a concentration of 1.25 μM on HUVEC cells was determined. After incubating the cells with the two drugs for 24 hours, the cytotoxicity was tested. As shown in Figure 8, at a concentration of 1.25 μM, Sal and Compound E had almost no significant toxicity to HUVEC cells.

[0191] Thaw the Matrigel matrix gel in a 4°C refrigerator, and pre-cool the yellow pipette tip box and 96-well plate at -20°C. Take 600μl of Matrigel matrix gel and add 900μl of serum-free ECM culture medium containing 1% ECGS, mix well by pipetting, add to a 50μl chamber, and place in a cell culture incubator overnight. When the cells grow to a density of 80-90%, discard the supernatant, add PBS to wash the culture flask, add trypsin solution to digest the cells, and after termination, use a pipette to repeatedly blow the bottom of the cell flask to fully resuspend the cells and transfer them to a 15ml centrifuge tube. Centrifuge at 12000rpm for 5min, resuspend the cells, and count. After mixing the prepared Sal, compound E and cell suspension in a 1:1 ratio, HUVEC were cultured at a rate of 4×10 5 Cells were seeded at a density of 1000 cells / well in a small chamber and cultured in a CO2 incubator for 24 hours. The cells were then washed with PBS, fixed with methanol, stained with crystal violet, and photographed. The results, shown in Figures 9 and 10, demonstrate that both Sal and E can inhibit the invasive ability of HUVECs, with compound E being far more effective than Sal.

[0192] HUVEC angiogenesis assay:

[0193] First, the cytotoxicity of Compound E and Sal at concentrations of 1.25 μM and 2.5 μM on HUVEC cells was determined. After incubating the cells with the two drugs for 6 hours, the cytotoxicity was measured. As shown in Figure 11, Sal and Compound E showed almost no significant cytotoxicity to HUVEC cells at concentrations of 1.25 μM and 2.5 μM.

[0194] Thaw the Matrigel matrix gel in a 4°C refrigerator, and pre-cool the yellow pipette tip box and 96-well plate at -20°C. Take 600μl of Matrigel matrix gel and add 900μl of serum-free ECM culture medium containing 1% ECGS, mix well by pipetting, add to a 50μl chamber, and place in a cell culture incubator overnight. When the cells grow to a density of 80-90%, discard the supernatant, add PBS to wash the culture flask, add trypsin solution to digest the cells, and after termination, use a pipette to repeatedly blow the bottom of the cell flask to fully resuspend the cells and transfer them to a 15ml centrifuge tube. Centrifuge at 12000rpm for 5min, resuspend the cells, and count. After mixing the prepared Sal, compound E and cell suspension in a 1:1 ratio, HUVEC were cultured at a rate of 4×10 4 The cells were seeded at a density of 100 μg / well in a small chamber and incubated in a CO2 incubator for 6 hours. The cells were then observed and photographed under a microscope. The results, shown in Figures 12 and 13, show that both Sal and E inhibited the tubule formation ability of HUVECs. At a concentration of 2.5 μM, compound E was more potent than Sal, and the difference in efficacy was statistically significant at this concentration.

[0195] Example 5

[0196] This example provides a solubility test of the polypeptide-containing salinomycin derivative Sal-A6 prepared in Example 2.

[0197] 4 mM Sal and Sal-A6 were prepared in physiological saline and their solubility was tested.

[0198] The results are shown in FIG14 . Sal-A6 can be significantly dissolved in water, while salinomycin is almost insoluble in water.

[0199] Example 6

[0200] This example provides flow cytometry screening of cells that highly express CD44.

[0201] Cells HT-29 (human colorectal cancer cells), MKN-45 (human gastric cancer cells), SKOV3 (human ovarian cancer cells), Cal-27 (human tongue squamous cell carcinoma cells), HepG2 (human liver cancer cells), A431 (human skin squamous cell carcinoma cells), A549 (human lung cancer cells), and A2780 (human ovarian cancer cells) were revived and cultured for more than three passages. After three passages of cell culture, the cells were washed several times with 4°C pre-cooled commercial PBS at an appropriate density to remove poorly functioning cells and floating objects, and then digested with trypsin solution. After the cells became round, an equal volume of culture medium was added. Terminate the digestion and centrifuge at 1000 rpm for 5 min; discard the supernatant and block with 2% commercially available BSA blocking solution at 4°C for 1 h; take 100 μl of the above-mentioned cells in 1.5 ml centrifuge tubes, then add anti-human PE-CD44 antibody (purchased from Biolegend) and PE-CD44 isotype control IgG (purchased from Biolegend) at 4°C and incubate in the dark for 30 min; the above cells are centrifuged at 4°C and 2000 rpm for 5 min, washed 3 times with PBS, and then detected using a flow cytometer (CytoFLEX).

[0202] The results are shown in FIG15 , and all cells tested except A2780 cells highly expressed CD44.

[0203] Table 2 Detection of CD44 expression on different cells

[0204] Example 7

[0205] This example provides an in vitro antitumor activity test of the polypeptide-containing salinomycin derivative Sal-A6:

[0206] Experimental methods:

[0207] SKOV3 cells in logarithmic growth phase were digested, counted and prepared into 1×10 5Cell suspensions of 100 μL / well were plated in 96-well plates (100 μL / well) and cultured in a 37°C, 5% CO2 incubator for 24 hours. Sal, compound E, Sal-A6, and peptide drug A6 containing the corresponding concentrations were then added according to the dosage of 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM. A negative control group and a blank group were also set up, with 5 replicates per group. The plates were placed in an incubator and cultured for 0 h, 24 h, 48 h, and 72 h. After the incubation, 10 μL of CCK8 solution was added to each well and incubated in a cell culture incubator for another 1 hour. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated.

[0208] The results are shown in Figure 16. The activities of compound E and Sal-A6 were improved compared to Sal, while peptide A6 had almost no inhibitory effect on tumor cells.

[0209] Example 8

[0210] Detection of onset time of compound E and Sal-A6:

[0211] Experimental methods:

[0212] Cells in logarithmic growth phase (SKOV3 cells) were digested, counted, and prepared into 1×10 5 A cell suspension of 100 μL / well was plated in a 96-well plate (100 μL / well) and incubated at 37°C in a 5% CO2 incubator for 24 hours. Sal, compound E, and Sal-A6 were then added at 20 μM, 10 μM, and 5 μM, respectively. A negative control group and a blank group were also established, with 5 replicates per group. The plates were then incubated in an incubator for 72 hours. Afterwards, 10 μL of CCK8 solution was added to each well, and the cells were incubated in a cell culture incubator for another hour. The absorbance was measured at 450 nm, and the proliferation inhibition rate was calculated.

[0213] The results are shown in Figures 17-18. Sal-A6 has a slower onset of action than Sal, but compound E significantly increases the onset of action compared to Sal, which may be related to fat solubility.

[0214] Example 9

[0215] This example provides an experiment on the effects of compound E and Sal-A6 on SKOV3 cell scratches:

[0216] Experimental methods:

[0217] (1) Resuscitating and culturing the CD44-high-expressing cell line SKOV3 obtained in the early screening, and stably passaged for more than three generations for future use;

[0218] (2) When the cells grow to a density of approximately 85%, discard the supernatant, add PBS solution to rinse the culture flask, and then add trypsin solution to digest the cells. After complete digestion, use a pipette to repeatedly blow the bottom of the culture flask to fully resuspend the cells. Then transfer them to a 10 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, resuspend the cells, and count them;

[0219] (3) Add complete culture medium containing 10% serum until the cell concentration is about 4×10 5 / mL, plated, and three replicate wells were inoculated in each group;

[0220] (4) When the cell density reaches 90%, use a 100 μL sterile pipette tip to draw a 2 mm wide line, discard the culture medium in the 24-well plate, add blank culture medium, and administer different doses of SAL, SAL-Na, SAL-SS, polypeptide A6, and SAL-A6; culture in a CO2 constant temperature incubator;

[0221] (5) The cells were removed after 0 and 24 h, and the healing status was observed under a microscope. Photos were taken, ensuring that the same field of view and the same objective lens and eyepiece were selected for each photo. Photoshop software was used for analysis.

[0222] The results are shown in Figures 19-20. 24 hours after administration of Sal, compound E, polypeptide A6, and Sal-A6, the migration of ovarian cancer SKOV3 cells was significantly inhibited compared with the blank control group, and the inhibitory effect was compound E>Sal-A6>SAL≈A6.

[0223] Example 10

[0224] This example provides an experiment on the effects of compound E and Sal-A6 on cell migration:

[0225] Experimental methods:

[0226] SKOV3 cells were cultured. When the cells grew to a density of 80-90%, the supernatant was discarded, PBS was added to wash the culture flask, and trypsin solution was added to digest the cells. After termination, the bottom of the cell flask was repeatedly blown up and down with a pipette to fully resuspend the cells. The cells were transferred to a 15 ml centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The cells were resuspended and counted. After the prepared Sal, compound E and cell suspension were mixed in a ratio of 1:1, SKOV3 was plated at 4×10 5 The cells were inoculated at a density of 1000 / well in a small chamber and cultured in a carbon dioxide constant temperature incubator for 48 h. The cells were observed and photographed under a microscope.

[0227] The results are shown in Figures 21-22. Sal, Sal-A6, and E were all able to inhibit the in vitro migration ability of SKOV3 cells in a dose-dependent manner, and the inhibitory ability of compound E>Sal-A6>Sal.

[0228] Example 11

[0229] This example provides an experiment on the effects of compound E and Sal-A6 on cell invasion:

[0230] Experimental methods:

[0231] Thaw Matrigel in a 4°C refrigerator, take 600 μl of Matrigel and add 900 μl of serum-free ECM culture medium containing 1% 1640, mix well by pipetting, add to a 50 μl chamber, and place in a cell culture incubator overnight.

[0232] When the cells (SKOV3) grew to a density of 80-90%, the supernatant was discarded, PBS was added to wash the culture flask, and trypsin solution was added to digest the cells. After termination, the bottom of the cell flask was repeatedly blown by a pipette to fully resuspend the cells and transferred to a 15ml centrifuge tube. Centrifuged at 12000rpm for 5min, the cells were resuspended and counted. After the prepared Sal, compound E and cell suspension were mixed in a ratio of 1:1, HUVEC were plated at 6×10 4 The cells were inoculated at a density of 1000 / well in a small chamber and cultured in a carbon dioxide constant temperature incubator for 12 h. The cells were observed and photographed under a microscope.

[0233] The results are shown in FIG23 . Sal, Sal-A6, and compound E were all able to inhibit the in vitro invasion ability of SKOV3 cells in a dose-dependent manner, and the inhibitory ability was compound E>Sal-A6>Sal.

[0234] Example 12

[0235] This example provides an experiment on the effects of compound E and Sal-A6 on cell cycle:

[0236] Experimental methods:

[0237] (1) Resuscitate and culture SKOV3 cells, and passage them for more than three generations;

[0238] (2) When the cell fusion rate reaches 90%, discard the culture medium, wash the cells 2-3 times with PBS, and then add trypsin solution to digest the cells;

[0239] (3) When the cells become round, the digestion process is terminated with complete medium and centrifuged at 1000 rpm for 5 min.

[0240] (4) Discard the supernatant, add serum-free medium to resuspend the cells, and use a cell counter to measure the cell concentration to 4×10 5 / mL;

[0241] (5) Pipette 2 mL of the above cells into 6-well plates, mix well, and incubate in a cell culture incubator overnight;

[0242] (6) Dilute the cells with serum-free medium to a final concentration of 10 μM and 5 μM for standby use. Wash the cells three times with PBS solution, remove cells with poor growth status and floating objects, and then add the diluted Sal and Sal-A6 solutions to the corresponding sample wells, respectively. Inject serum-free medium into the blank wells and culture in a cell culture incubator for 72 hours.

[0243] (7) Wash cells three times with PBS, 2000 rpm, 5 min, and collect cells from each well by centrifugation;

[0244] (8) Use a cell counter to count the number of cells and adjust the density to 1×10 6 When the number of cells / mL is reached, mix with a pipette to obtain a single-cell suspension;

[0245] (9) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and then add 500 μL of the pre-chilled cell fixative prepared above for fixation at 4°C overnight;

[0246] (10) Centrifuge at 1000 rpm for 5 min, wash three times with PBS, and remove the fixative;

[0247] (11) Add 500 μL of RNase A / PI staining working solution prepared with this formula to each well and incubate at room temperature for 60 minutes.

[0248] Incubate in the dark;

[0249] (12) Flow cytometry testing.

[0250] The results are shown in Figure 24 and Table 3.

[0251] Table 3 Statistical table of the effects of Sal, Sal-A6 and compound E on the cell cycle of SKOV3 cells

[0252] SKOV3 cells were treated with various concentrations of Sal and Sal-A6, and their effects on cell cycle distribution were determined by flow cytometry. As shown in Table 3, the number of cells in the G0 / G1 phase decreased, while the number of cells in the S phase and G2 / M phase increased, indicating that both Sal-A6 and Compound E induced S-phase and G2 / M phase arrest in SKOV3 cells. These results demonstrate that Sal-A6 can induce S-phase and G2 / M phase arrest in SKOV3 cells. Furthermore, the effects of Sal and Sal-A6 on the cell cycle were largely consistent, indicating that the effects of Sal introduced into the A6 peptide on the cell cycle were largely consistent.

[0253] Example 13

[0254] This example provides an experiment on the effect of cisplatin on SKOV3 cells and SKOV3 / CDDP cells:

[0255] Experimental methods:

[0256] SKOV3 and SKOV3-DDP cells in logarithmic growth phase were digested, counted, and prepared into 1×10 4 The cell suspension of 100 μL / mL was inoculated into a 96-well plate (100 μL / well) and cultured in a 37° C., 5% CO 2 incubator for 24 hours.

[0257] Different concentrations of cisplatin were added to 96-well plates, and a negative control group and a blank group were set up, with 5 replicates in each group. The plates were placed in an incubator and cultured for 72 hours.

[0258] 10 μL of CCK8 solution was added to each well and the cells were incubated in a cell culture incubator for 1 hour. The absorbance was measured at 450 nm and the proliferation inhibition rate was calculated.

[0259] The results are shown in FIG25 . The inhibitory activity of cisplatin on SKOV3 cells was better than that on SKOV3 / CDDP cells, indicating that SKOV3 / CDDP cells have developed obvious resistance to cisplatin.

[0260] Example 14

[0261] This example provides the detection of CD44 expression in drug-resistant cells and the activity experiment of drug-resistant cells.

[0262] Detection of CD44 expression in drug-resistant cells

[0263] After three generations of culture, human ovarian cancer cell SKOV3-DDP cells were revived and cultured. They were washed several times with 4°C pre-cooled commercial PBS at an appropriate density to remove cells in poor condition and floating objects, and then digested with trypsin solution. After the cells became round, an equal volume of culture medium was added to terminate the digestion, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were blocked with 2% commercially available BSA blocking solution at 4°C for 1 hour. 100 μl of the cells at the above concentration were respectively taken into 1.5 ml centrifuge tubes, and then anti-human PE-CD44 antibody (purchased from Biolegend) and PE-CD44 isotype control IgG (purchased from Biolegend) were added at 4°C and incubated in the dark for 30 minutes. The above cells were centrifuged at 4°C and 2000 rpm for 5 minutes, washed 3 times with PBS, and then detected using a flow cytometer (CytoFLEX).

[0264] Drug-resistant cell activity assay

[0265] SKOV3-DDP cells in logarithmic growth phase were digested, counted, and prepared into 1×10 4 The cell suspension of 100 μL / mL was inoculated into a 96-well plate (100 μL / well) and cultured in a 37° C., 5% CO 2 incubator for 24 hours.

[0266] 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM of polypeptide A6, Sal, compound E, and Sal-A6 were respectively prepared. A negative control group and a blank group were also set up, with 5 replicates in each group. The plates were placed in an incubator and cultured for 72 h.

[0267] 10 μL of CCK8 solution was added to each well and the cells were incubated in a cell culture incubator for 1 hour. The absorbance was measured at 450 nm and the proliferation inhibition rate was calculated.

[0268] The results are shown in FIG26 . Compared with SKOV3 cells (see FIG16 ), the activities of Sal, Sal-A6, and Compound E on SKOV3 / DDP were enhanced, which also indicates that the stemness of drug-resistant cells SKOV3 / DDP was enhanced compared with SKOV3.

[0269] Example 15

[0270] This example provides a sensitization test of compound E and Sal-A6 on drug-resistant cells:

[0271] Experimental methods:

[0272] SKOV3-DDP cells in logarithmic growth phase were digested, counted, and prepared into 1×10 4The cell suspension of 100 μL / mL was inoculated into a 96-well plate (100 μL / well) and cultured in a 37° C., 5% CO 2 incubator for 24 hours.

[0273] 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM of polypeptide A6, Sal, compound E, and Sal-A6 were respectively prepared. A negative control group and a blank group were also set up, with 5 replicates in each group. The plates were placed in an incubator and cultured for 72 h.

[0274] The results are shown in FIG27 . Sal, compound E, or Sal-A6 all enhance the activity of cisplatin and increase its sensitivity to cells.

[0275] Example 16

[0276] This example provides an experiment on the effects of Compound E and Sal-A6 on tumor stem cells:

[0277] Tumor cell spheroidization is one of the main indicators for detecting tumor stem cells. SKOV3 cells were cultured in agarose with low serum and co-incubated with drugs to observe the effects of Sal, Sal-A6, and compound E on spheroidization ability.

[0278] The results are shown in FIG28 , showing that Sal, E or Sal-A6 can inhibit the activity of cancer stem cells.

[0279] Example 17

[0280] This example provides a test for the bystander effect of Sal-A6 on tumor cells:

[0281] Experimental methods:

[0282] Cell acclimation

[0283] (1) Remove the cell culture flask from the cell culture incubator and observe under a microscope. If the cells have covered the bottom surface, discard the culture medium in a clean bench and rinse the cells with 2 mL of PBS and discard. Repeat twice. Add 2 mL of 0.25% trypsin to digest the cells. End with the corresponding volume of serum-containing culture medium and transfer the cells to a 15 mL conical-bottom centrifuge tube.

[0284] (2) Centrifugation: 1000 rpm, 5 min, discard the supernatant, resuspend the cells in 2 mL of RPMI-1640 complete medium containing serum, add 1 mL of cell suspension to each cell bottle, and then replenish the medium in each bottle. Observe under a microscope and place in the incubator for further culture;

[0285] (3) When the cell fusion rate reaches 70%, discard the culture medium, wash with PBS 2-3 times, and then add a complete culture medium mixed with DMEM and RPMI-1640 (DMEM:RPMI-1640=1:4). Observe under a microscope and place in an incubator for further culture.

[0286] (4) When the cell fusion rate reaches 90%, discard the culture medium, wash with PBS 2-3 times, and then add trypsin solution to digest the cells;

[0287] (5) Repeat steps (2) to (4), and the ratio of the mixed complete culture medium is DMEM:RPMI-1640=1:4, DMEM:RPMI-1640=2:3, DMEM:RPMI-1640=1:1, DMEM:RPMI-1640=3:2, DMEM:RPMI-1640=4:1, and DMEM complete culture medium in sequence;

[0288] (6) The cells are stably propagated for more than three generations for future use.

[0289] Detection of Sal-A6 activity against SKOV3 after acclimation

[0290] SKOV3 cells in logarithmic growth phase were taken and digested, counted and prepared into 1×10 4 The cell suspension of 100 μL / mL was inoculated into a 96-well plate (100 μL / well) and cultured in a 37° C., 5% CO 2 incubator for 24 hours.

[0291] Sal-A6 at concentrations of 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM were prepared respectively. A negative control group and a blank group were set up with 5 replicates in each group. The plates were placed in an incubator and cultured for 72 h.

[0292] 10 μL of CCK8 solution was added to each well and the cells were incubated in a cell culture incubator for 1 hour. The absorbance was measured at 450 nm and the proliferation inhibition rate was calculated.

[0293] To verify the bystander-killing effect of Sal-A6 on tumor cells, we co-cultured CD44+ SKOV3 cells with CD44- A2780 cells to verify its bystander-killing effect. Prior to this, we first adapted SKOV3 cells from 1640 culture medium to DMEM culture medium and measured CD44 expression and the effects of Sal-A6 and Sal on its activity. The results showed that SKOV3 / DMEM cells exhibited high CD44 expression, and the activity of Sal-A6 and Sal on CD44 was not significantly different from that of SKOV3 / 1640 cells.

[0294] A concentration that had no obvious killing effect on A2780 but had a significant killing effect on SKOV3 was selected to detect the bystander effect. SKOV3 and A2780 at different ratios were co-cultured in DMEM medium, and then 20μM, 10μM, 5μM, and 2.5μM Sal-A6 were given for incubation for 72 hours. The lethality of Sal-A6 on cells at different cell ratios was detected using a microplate reader. The results showed that when the ratio of SKOV3 to A2780 cells was 6:4, 5:5, and 4:6, Sal-A6 was able to effectively kill the mixed cells in a dose-dependent manner. When the drug concentration was 20μM, it could achieve an inhibitory effect of more than 80% on tumor cells, indicating that Sal-A6 should have a certain bystander effect.

[0295] To further verify the bystander killing ability of Sal-A6, we constructed a cell line A2780 / GFP stably transfected with GFP green fluorescent protein for CD44- tumor cells A2780, and co-incubated SKOV3 with A2780 / GFP cells at a ratio of 6:4, 5:5, and 4:6 and administered different concentrations of Sal-A6. The results showed that after administration, the fluorescence brightness of A2780 / GFP was significantly reduced in a dose-dependent manner. In summary, Sal-A6 is a PDC drug with a bystander killing effect. It has the ability to kill CD44- tumor cells in the presence of CD44+ tumor cells.

[0296] The results are shown in Figures 29-32. Figure 29 shows the effects of Sal, A6 and Sal-A6 on the activity of acclimated SKOV3 cells; Figure 30 shows the effects of Sal, A6 and Sal-A6 on the activity of acclimated A2780 cells; Figure 31 shows the effects of different concentrations of Sal-A6 on the activity of mixed cells; and Figure 32 shows the effects of different concentrations of Sal-A6 on the fluorescence intensity of mixed cells.

[0297] Example 18

[0298] This example provides an in vivo antitumor efficacy study of Compound E and Sal-A6:

[0299] Experimental methods:

[0300] Validation of Sal-A6 on SKOV3 nude mouse subcutaneous transplanted tumors

[0301] Experimental animals: balb / c female nude mice, cultured human ovarian cancer cells SKOV3 were collected, and the concentration was 1×10 7 Each 0.1 ml was inoculated subcutaneously in the right axilla of nude mice.

[0302] The calculation formula for tumor volume (TV) is:

[0303] TV=1 / 2×a×b 2 , where a and b represent the length and width respectively.

[0304] When the average tumor volume is 100 mm 3 Drug administration began around 5:00 a.m. and was observed every two days. Tumor volume was measured and body weight changes were recorded. The dosage was calculated based on the salinomycin dosage, and the groups were: saline group (Saline), salinomycin group (Sal 20 μg / kg), Sal-A6-1 group (equivalent to 15 μg / kg salinomycin), and Sal-A6-2 group (equivalent to 30 μg / kg salinomycin).

[0305] The results are shown in FIG33 . Sal-A6 can inhibit tumor growth even at low doses, and its efficacy is better than Sal itself, and it has no significant effect on the body weight of nude mice.

[0306] Example 19

[0307] This example provides a validation of Sal-A6 against Cal-27 nude mouse subcutaneous transplanted tumors.

[0308] Experimental animals: BALB / c female nude mice, cultured human head and neck cancer cell SKOV3, the concentration was 1×10 7 Each 0.1 ml was inoculated subcutaneously in the right axilla of nude mice.

[0309] The calculation formula for tumor volume (TV) is:

[0310] TV=1 / 2×a×b 2 , where a and b represent the length and width respectively.

[0311] When the average tumor volume is 90 mm 3 Drug administration began around 5:00 p.m. and was observed every two days. Tumor volume and body weight changes were measured. The dosage was calculated based on the salinomycin dose, and the following groups were used: saline group (Saline), cetuximab (ERBITUX 20 mg / kg), and Sal-A6 group (equivalent to 300 μg / kg of salinomycin).

[0312] In the Cal model, the in vivo efficacy of Sal-A6 was basically the same as that of cetuximab, and there was no significant difference in the body weight of nude mice compared with the blank group.

[0313] The results are shown in FIG34 . Sal-A6 can also inhibit tumor growth at low doses, with an efficacy comparable to that of the positive drug cetuximab, and has no significant effect on the body weight of nude mice.

Claims

1. A salinomycin derivative or a salt thereof, characterized in that: The salinomycin derivative has the structure of the following formula (1): in: -X is H, Li, Na or K.

2. The method for preparing a salinomycin derivative or a salt thereof according to claim 1, characterized in that: The method comprises: The C20-OH of salinomycin is oxidized by an oxidant, and then the C20-OH of salinomycin is reduced to C20-NH2 by a reductive amination reaction, and then modified by an esterification reaction.

3. Use of a salinomycin derivative or a salt thereof according to claim 1 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The applications include: The disulfide bond in the above-mentioned salinomycin derivative or its salt undergoes a disulfide bond replacement reaction with the sulfhydryl group in the polypeptide or antibody, so that the salinomycin derivative or its salt is coupled with the polypeptide or antibody to obtain the salinomycin derivative or its salt containing the polypeptide or antibody.

4. Use of a salinomycin derivative or a salt thereof according to claim 3 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The polypeptide or antibody includes: a polypeptide or antibody formed by connecting a functional polypeptide or functional antibody with a compound containing a thiol group.

5. Use of a salinomycin derivative or a salt thereof according to claim 4 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The polypeptide or antibody comprises a polypeptide or antibody composed of a functional polypeptide or a functional antibody and a compound containing a thiol group connected in sequence.

6. Use of a salinomycin derivative or a salt thereof according to claim 5 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: One or more sarcosines are further connected between the functional polypeptide or functional antibody and the compound containing sulfhydryl.

7. Use of a salinomycin derivative or a salt thereof according to claim 4-6 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The thiol-containing compound includes: cysteine, mercaptoethylamine, thioglycolic acid and / or mercaptopropionic acid.

8. Use of a salinomycin derivative or a salt thereof according to claim 7 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The functional polypeptides include cell penetrating peptides or targeting peptides.

9. Use of a salinomycin derivative or a salt thereof according to claim 8 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that: The polypeptide includes: polypeptide A6-Sar, whose structure is: Ac-KPSSPPEE (Sar) 10 C-NH2.

10. A salinomycin derivative containing a polypeptide or an antibody or a salt thereof, characterized in that: The method is obtained by using a salinomycin derivative or a salt thereof according to any one of claims 3 to 9 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody.

11. A salinomycin derivative or a salt thereof containing a polypeptide or an antibody according to claim 10, characterized in that: The polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (6): -X is H, Li, Na or K; -Y is a functional polypeptide or a functional antibody; n＝1～12。 12. A salinomycin derivative or a salt thereof containing a polypeptide or an antibody according to claim 11, characterized in that: The polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (7): -X is H, Li, Na or K.

13. A salinomycin derivative containing a polypeptide or an antibody or a salt thereof, characterized in that: The polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (6): -X is H, Li, Na or K; -Y is a functional polypeptide or a functional antibody; n＝1～12。 14. A salinomycin derivative containing a polypeptide or an antibody or a salt thereof, characterized in that: The polypeptide or antibody-containing salinomycin derivative or its salt has the structure of the following formula (7): -X is H, Li, Na or K.

15. Use of a salinomycin derivative or a salt thereof according to claim 1, or a salinomycin derivative or a salt thereof containing a polypeptide or an antibody according to any one of claims 10 to 14 in the preparation of a medicament for treating tumors and / or inhibiting angiogenesis.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a salinomycin derivative or a salt thereof according to claim 1, or a salinomycin derivative or a salt thereof containing a polypeptide or an antibody according to any one of claims 10-14.

17. A method for treating tumors and / or inhibiting angiogenesis, characterized in that: A therapeutically effective amount of the salinomycin derivative or a salt thereof according to claim 1 is administered to a subject.

18. A method for treating tumors and / or inhibiting angiogenesis, characterized in that: A therapeutically effective amount of the salinomycin derivative or salt thereof containing the polypeptide or antibody according to any one of claims 10 to 14 is administered to a subject.

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