Salinomycin derivative as well as preparation method and application thereof

Abstract

The invention discloses salinomycin derivatives as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The salinomycin derivative comprises a general formula structure shown in a formula (1), and compared with salinomycin, the salinomycin derivative has the advantage that the activity is obviously improved; the C-20 site group of the salinomycin derivative contains a disulfide bond, so that the salinomycin derivative can be quickly and efficiently coupled with polypeptide or antibody through the displacement reaction of the disulfide bond to obtain the GSH-sensitive salinomycin derivative containing polypeptide or antibody or the salt of the salinomycin derivative. According to the salinomycin derivative containing the polypeptide or the antibody or the salt thereof, on one hand, the solubility is improved; on the other hand, the targeting property is enhanced, the drug selectivity and the biological activity are improved, the side killing effect is achieved, tumor cells with high expression antigens can be killed, and the obvious inhibiting effect on cells with low expression antigens is achieved. In addition, the compound has the effects of reversing drug resistance of tumor cells and enhancing drug sensitivity, and can be combined with other drugs for use to further improve the curative effect.

Description

Technical Field

[0001] This application belongs to the field of biological medicine technology, and specifically relates to salinomycin derivatives, their preparation methods and applications. Background Art

[0002] Tumors have always been one of the greatest threats to human health. Although there are various treatment methods, such as chemotherapy and immunotherapy, for treating cancer (malignant tumors), the mortality rate caused by tumors remains high. Existing studies have shown that only a small part of tumor cells with tumorigenic ability are the main factors in the occurrence and development of tumors, and these cells are called tumor stem cells. In addition, during the tumor treatment process, there is also a phenomenon of drug resistance, and there is still a lack of effective treatment methods for drug-resistant and relapsing patients. Finding effective tumor stem cell inhibitors is one of the main directions for treating tumors, and finding drugs that can reverse drug resistance and enhance drug sensitivity 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 anti-cancer activity, has obvious inhibitory effects on various tumor cells including tumor stem cells, and also has the ability to reverse drug-resistant cells and enhance drug sensitivity. As a new generation of anti-tumor candidate drug, its further application and development also face great challenges: such as the almost water-insoluble property of salinomycin, the biological activity needs to be further enhanced, the drug selectivity is relatively low, and it shows a certain degree of toxic and 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 related technologies, for example, the Chinese invention patent with the publication number CN105732655A discloses the preparation and application of a salinomycin derivative with a novel structure. Through a selective Mitsunobu reaction, nucleophilic substitution of the 20-position hydroxyl group is achieved while the configuration is inverted, obtaining a 20-position azide derivative of salinomycin. Then, through a selective click reaction of an alkyne and the 20-position azide derivative of salinomycin, a salinomycin derivative with a triazole structure is obtained, and its activity is higher than that of salinomycin. Another example is the Chinese invention patent with the publication number CN107428772A, which discloses a nitrogen-containing analogue of salinomycin, its synthesis, and its uses in anti-cancer stem cells and malaria, and its activity is also significantly improved. Another example is the Chinese invention patent with the publication number CN115052879A, which discloses a compound composed of C20-modified salinomycin derivatives, a method for obtaining the same, a composition containing the same, the uses of the compound, and a method for obtaining intermediate products, obtaining a C20-N-acyl derivative of salinomycin, and its anti-cancer activity is also significantly improved. Another example is that Huang et al. adopted a semi-synthetic method to dehydroxylate, acylate the C20 position of salinomycin or perform hydrogenation reduction on C18-C19, obtaining 5 derivatives, and their killing effects on breast cancer tumor stem cells are all better than that of salinomycin. However, due to reasons such as the modification site and 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 3 derivatives of salinomycin, but their anti-tumor ability and antibacterial activity are weaker than those of the original salinomycin. Another example is that connecting an L-amino acid methyl ester to the carboxyl group at the C1 position has good killing effects on the cell proliferation of leukemia and doxorubicin-resistant colon cancer cells, but when performing monosubstitution halogenation or nitration N-benzylamide amidation on the carboxyl group, the anti-tumor ability and antibacterial activity of the derived compounds are weaker than those of the original salinomycin.

[0005] The above chemical structure modifications mainly focus on modifying salinomycin through groups and using group modification to improve the biological activity (anti-cancer activity) of salinomycin derivatives. However, the above chemical structure modifications do not pay attention to the selectivity problem of drugs. Given that salinomycin or salinomycin derivatives may have a certain degree of toxic side effects, their drug selectivity is also worthy of attention.

[0006] Obtaining a salinomycin derivative with a targeting effect, enhancing the drug targeting, and then improving the biological activity of salinomycin, etc., is one of the solutions that is expected to solve the problem of its low selectivity. However, there has been no relevant report so far. Summary of the Invention

[0007] 1. Object of the Invention

[0008] One of the purposes of the present application is to provide a salinomycin derivative or its salt. The salinomycin derivative is a compound obtained by modifying the hydroxyl group at the C-20 position of salinomycin. The modified group at the C-20 position contains a disulfide bond. On the one hand, it has higher biological activity compared with salinomycin; on the other hand, the presence of the disulfide bond facilitates the conjugation of the salinomycin derivative with a polypeptide or an antibody to obtain a salinomycin derivative or its salt containing a polypeptide or an antibody.

[0009] Another purpose of the present application is to provide the above-mentioned salinomycin derivative or its salt containing a polypeptide or an antibody. On the one hand, the salinomycin derivative containing a polypeptide or an antibody can improve the physical and chemical properties of salinomycin by virtue of the physical and chemical properties of the polypeptide or the antibody, thereby enhancing its solubility; on the other hand, by virtue of the property of specific binding of the polypeptide or the antibody, the targeting property can be enhanced, and the drug selectivity and biological activity can be improved.

[0010] A third purpose of the present application is to provide the application of the above-mentioned salinomycin derivative or the above-mentioned salinomycin derivative containing a polypeptide or an antibody.

[0011] 2. Technical solutions

[0012] To solve the above problems, the technical solutions adopted in the present application are as follows:

[0013] In the first aspect of the present application, a salinomycin derivative or its salt is provided. The salinomycin derivative has the structure of formula (1) as follows:

[0014]

[0015] Wherein:

[0016] -X is H, Li, Na or K.

[0017] Further, for the above-mentioned salinomycin derivative or its salt, the salinomycin derivative has the structure of formula (2) as follows:

[0018] Further, for the above-mentioned salinomycin derivative or its salt, the salinomycin derivative has the structure of formula (3) as follows:

[0019] Further, for the above-mentioned salinomycin derivative or its salt, the salinomycin derivative has the structure of formula (4) as follows:

[0020] Further, for the above-mentioned salinomycin derivative or its salt, the salinomycin derivative has the structure of formula (5) as follows:

[0021]

[0022] The second aspect of the present application provides a method for preparing the above-mentioned salinomycin derivative or its salt, which includes oxidizing the C20-OH of salinomycin using an oxidizing agent, and then using a reductive amination reaction to reduce the C20-OH of salinomycin to C20-NH2, followed by an esterification reaction modification. Reducing the C20-OH to C20-NH2 can enhance the chemical reaction activity.

[0023] Further, the above-mentioned preparation method includes:

[0024] (1) Prepare compound A by reacting 3-mercaptopropionic acid with 2,2'-dithiopyridine, and then prepare compound B by reacting oxalyl chloride with compound A;

[0025] (2) Selectively oxidize the 20-OH of sodium salinomycin using MnO2 to obtain compound C; then prepare compound D using a reductive amination reaction;

[0026] (3) React compound B with D to obtain the salinomycin derivative of formula (1).

[0027] Further, the above-mentioned preparation method includes:

[0028] Dissolve sodium salinomycin in DCM, add MnO2, stir overnight under N2 protection at 25 °C; filter through diatomaceous earth, combine the DCM phases, wash with hydrochloric acid, separate the layers, dry the DCM phase with anhydrous Na2SO4, concentrate, and obtain the white solid compound C;

[0029] Dissolve compound C in anhydrous methanol (MeOH), add NH3 / MeOH solution, 5 drops of acetic acid (HAC), then add 20 ml of DCM to completely dissolve, stir at 25 °C; then add cerium trichloride heptahydrate (CeCl3·7H2O) to the system and stir at room temperature; then use a constant pressure dropping funnel to dropwise add sodium cyanoborohydride (NaBH3CN) / 50 ml of MeOH solution, add dropwise evenly, and stir overnight at 25 °C; after the reaction is completed, concentrate the system to dryness, add DCM to dissolve, wash with saturated NaCl, dry with anhydrous Na2SO4, concentrate, and perform column chromatography (eluent: MeOH / DCM = 1:50 - MeOH / DCM = 1:20) to obtain compound D;

[0030] Dissolve 3-mercaptopropionic acid and 2,2'-dithiopyridine in ethanol; add 10 drops of acetic acid (HAC) to the system after 15 min and stir at room temperature; after the reaction is completed, concentrate the system, perform column chromatography (EA / PE = 1:6) to remove the excess 2,2'-dithiopyridine, purify using EA / PE = 1:5 + 1% HAC, and concentrate and dry by rotary evaporation to obtain compound A;

[0031] Dissolve compound A in DCM. Under N2 protection and in an ice-water bath, add oxalyl chloride (oxalic dichloride) to the system. After the addition is completed dropwise in about 30 seconds, remove the ice-water bath, add 2 drops of N,N-dimethylformamide (DMF) to the system, and stir at 25 °C. After the reaction is completed, concentrate and dry the system by evaporation, then add DCM again and concentrate and dry by evaporation to obtain compound B.

[0032] Dissolve compound D in DCM, add triethylamine (C6H 15 N). Under N2 replacement protection and in an ice-water bath, dropwise add a solution of compound B in 30 ml of ultradry DCM to the system. After the addition is completed in 20 minutes, let it rise to room temperature naturally and stir overnight. After the reaction is completed, dilute the reaction system with DCM, wash it once with an aqueous HCl solution, wash it once with saturated NaCl, concentrate it, and then purify it by column chromatography (eluent: first use EA / PE = 1:4 + 1% HAC until there are no impurities, and then use MeOH / DCM = 1:50 for purification) to obtain compound E, that is, the compound of formula (2).

[0033] 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.

[0034] Further, the above application includes: through a disulfide bond substitution reaction between the disulfide bond in the above-mentioned salinomycin derivative or its salt and the thiol group in the polypeptide or antibody, coupling the salinomycin derivative or its salt with the polypeptide or antibody to obtain a salinomycin derivative or its salt containing a polypeptide or an antibody.

[0035] Further, the above polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or a functional antibody linked to a compound containing a thiol group.

[0036] Further, the above compound containing a thiol group includes: cysteine (Cys), and the side chain group of cysteine contains a thiol group (-SH).

[0037] Further, the above compound containing a thiol group includes: compounds containing a thiol group (-SH) such as mercaptoethylamine, mercaptoacetic acid, and mercaptopropionic acid.

[0038] Further, the above polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or a functional antibody linked in sequence with a compound containing a thiol group.

[0039] Further, the above polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or a functional antibody linked in sequence with cysteine.

[0040] Further, the above polypeptide or antibody includes: a polypeptide or antibody composed of a functional polypeptide or a functional antibody linked in sequence with compounds such as mercaptoethylamine, mercaptoacetic acid, and mercaptopropionic acid.

[0041] Furthermore, the above-mentioned functional polypeptides include: cell-penetrating peptides, targeting peptides, etc.

[0042] Furthermore, the above-mentioned targeting peptides include: polypeptides targeting the tumor microenvironment, polypeptides targeting proteins highly expressed in tumor cells, etc.

[0043] Furthermore, one or more sarcosines (Sar) are also connected between the above-mentioned functional polypeptide or functional antibody and cysteine, and one or more sarcosines (Sar) can reduce the mutual influence between the functional polypeptide or functional antibody and the small molecule (the above-mentioned salinomycin derivative or its salt).

[0044] Furthermore, the number of the above-mentioned sarcosines is 1 to 12.

[0045] Furthermore, the number of the above-mentioned sarcosines is 6 to 10.

[0046] Furthermore, the number of the above-mentioned sarcosines is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Further, the number of the above-mentioned sarcosines is 10.

[0047] Furthermore, the above-mentioned targeting peptide is polypeptide A6 (KPSSPPEE, SEQ ID NO.1), which targets the CD44 protein. The CD44 protein is a complex transmembrane adhesion glycoprotein that is expressed in a variety of human cells, including embryonic stem cells, differentiated cells, and cancer cells. It is a recognized marker of cancer stem cells and a key regulator of epithelial-mesenchymal transition, and is involved in the occurrence, progression, and metastasis of tumors. Targeting the CD44 protein means being able to targetedly bind to the CD44 protein, thereby targeting and delivering the salinomycin derivative or its salt containing the polypeptide or antibody to cancer stem cells, etc., enhancing the targeting of the drug, improving the selectivity of the drug, increasing the activity of the drug, and reducing the toxicity of the drug.

[0048] Furthermore, the above-mentioned polypeptide or antibody includes: polypeptide A6-Sar, and its structure is: Ac-KPSSPPEE(Sar) 10 C-NH2 (SEQ ID NO.2).

[0049] Furthermore, the above-mentioned application includes: through the disulfide bond displacement reaction between the disulfide bond in the above-mentioned salinomycin derivative or its salt and the sulfhydryl group of cysteine in polypeptide A6-Sar, the salinomycin derivative or its salt is coupled with polypeptide A6-Sar to obtain the salinomycin derivative or its salt containing polypeptide A6-Sar, and the A6 sequence (KPSSPPEE) contained therein can target the CD44 protein.

[0050] Furthermore, the above-mentioned application includes: the disulfide bond displacement reaction is carried out in an organic solvent.

[0051] Further, the above-mentioned organic solvent includes dimethyl sulfoxide (DMSO).

[0052] The fourth aspect of the present application provides a salinomycin derivative or its salt containing a polypeptide or an antibody prepared in the above application.

[0053] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (6):

[0054]

[0055] -X is H, Li, Na or K;

[0056] -Y is a functional polypeptide or a functional antibody;

[0057] n = 1 to 12.

[0058] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (7):

[0059]

[0060] -X is H, Li, Na or K.

[0061] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (8):

[0062]

[0063] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (9):

[0064]

[0065] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (10):

[0066]

[0067] Further, the salinomycin derivative or its salt containing a polypeptide or an antibody has the structure of the following formula (11):

[0068]

[0069] 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 an antibody in the preparation of a drug for treating tumors or inhibiting angiogenesis, etc.

[0070] Further, the above-mentioned treatment of tumors includes: inhibiting tumor cells / tissues, tumor stem cells, and / or reversing the drug resistance of tumor cells / tissues.

[0071] Further, the above-mentioned tumors include:

[0072] Non-small cell lung cancer, including but not limited to lung epithelial cell carcinoma, lung adenocarcinoma, human lung squamous cell carcinoma;

[0073] Ovarian cancer, including but not limited to epithelial ovarian cancer, ovarian cystadenocarcinoma;

[0074] Renal cancer, including but not limited to renal cell carcinoma;

[0075] Prostate cancer, including but not limited to prostate adenocarcinoma;

[0076] Gastric cancer;

[0077] Corpus cancer of uterus;

[0078] Breast cancer, including but not limited to breast adenocarcinoma, inflammatory breast cancer, metastatic adenocarcinoma;

[0079] Pancreatic cancer;

[0080] Leukemia, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma;

[0081] Colorectal (colon) cancer, including but not limited to colorectal adenocarcinoma, colon epithelial cell carcinoma;

[0082] Central nervous system tumors, including but not limited to brain tumors, such as gliomas;

[0083] Melanoma, including but not limited to malignant melanoma, epithelial melanoma, non-epithelial melanoma;

[0084] Or sarcoma.

[0085] Further, the above-mentioned drug administration routes can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc., and can be administered in unit dose form.

[0086] Furthermore, the above-mentioned drug can be in liquid dosage form, solid dosage form or semi-solid dosage form. The liquid dosage form can be solution (including true solution and colloidal solution), emulsion (including o / w type, w / o type and multiple emulsion), suspension, injection (including aqueous injection, powder for injection and infusion), eye drops, nasal drops, lotion and liniment, etc.; the solid dosage form can be tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, enteric-coated capsule), granule, powder, pellet, dropping pill, suppository, film, patch, aerosol (powder) and spray, etc.; the semi-solid dosage form can be ointment, gel, paste, etc.

[0087] Furthermore, the above-mentioned drug can be an ordinary preparation, sustained-release preparation, controlled-release preparation, targeted preparation or various particulate drug delivery systems.

[0088] The sixth aspect of the present application provides a pharmaceutical composition, comprising the above-mentioned salinomycin derivative or its salt, or the above-mentioned salinomycin derivative containing polypeptide or antibody or its salt.

[0089] Furthermore, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0090] Furthermore, the above-mentioned pharmaceutical composition further comprises a platinum-based therapeutic agent, also known as platinum drugs. Platinum drugs cause DNA cross-linking, so that they inhibit DNA repair and / or DNA synthesis, mainly in rapidly proliferating cells such as cancer cells. Further, the platinum-based therapeutic agent is selected from cisplatin; carboplatin; oxaliplatin; nedaplatin, picoplatin; and satraplatin and other platinum drugs.

[0091] Furthermore, the above-mentioned pharmaceutical composition further comprises a nucleoside inhibitor, or a therapeutic agent that interferes with normal DNA synthesis, protein synthesis, cell replication or otherwise inhibits rapidly proliferating cells. 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.

[0092] Further, 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-Ab1 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 multi-kinase inhibitors such as sunitinib; pazopanib; ALK inhibitors such as crizotinib; ceritinib; and alectinib; Bruton's tyrosine kinase inhibitor such as ibrutinib; and Flt3 receptor inhibitor such as midostaurin.

[0093] Further, the above-mentioned pharmaceutical composition further comprises other kinase inhibitors and VEGF-R antagonists that are under development and can be used in this application, including tivozanib; vatalanib; deritinib; dovitinib; sirolimus; linifanib; neratinib; radotinib; ruxolitinib; fruquintinib; quizartinib and / or motesanib.

[0094] Further, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents that are mTOR inhibitors, which inhibit cell proliferation, angiogenesis and glucose uptake, and the mTOR inhibitors are everolimus; temsirolimus; and / or sirolimus.

[0095] Further, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents that are proteasome inhibitors, such as: bortezomib; carfilzomib; and / or ixazomib, taxane compounds, which cause the disruption of microtubules essential for cell division, and the taxane compounds are selected from taxane drugs such as paclitaxel, docetaxel, albumin-bound paclitaxel, cabazitaxel, etc.

[0096] Further, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents that are aromatase inhibitors, such as: exemestane; anastrozole and / or letrozole.

[0097] Further, the above-mentioned pharmaceutical composition further comprises one or more other therapeutic agents that are antagonists of the hedgehog pathway, and the approved hedgehog pathway inhibitors that can be used in this application include sonidegib; and / or vismodegib.

[0098] Further, the above-mentioned pharmaceutical composition further comprises folic acid inhibitors, such as: pemetrexed.

[0099] Furthermore, the above-mentioned pharmaceutical composition further comprises a CC chemokine receptor 4 (CCR4) inhibitor, such as mogamulizumab.

[0100] Furthermore, the above-mentioned pharmaceutical composition further comprises an isocitrate dehydrogenase (IDH) inhibitor.

[0101] Furthermore, the above-mentioned pharmaceutical composition further comprises an arginase inhibitor.

[0102] Furthermore, the above-mentioned pharmaceutical composition further comprises a glutaminase inhibitor.

[0103] Furthermore, the above-mentioned pharmaceutical composition further comprises an antibody that binds to a tumor antigen, i.e., a protein expressed on the cell surface of tumor cells. Approved antibodies for binding to tumor antigens in the present application include rituximab; ofatumumab; obinutuzumab, ibritumomab tiuxetan; daratumumab, dinutuximab; trastuzumab; and pertuzumab; and / or antibody drugs such as brentuximab vedotin.

[0104] Furthermore, the above-mentioned pharmaceutical composition further comprises a topoisomerase inhibitor. Approved topoisomerase inhibitors for use in the present application include irinotecan; topotecan; and / or pixantrone.

[0105] Furthermore, the above-mentioned pharmaceutical composition further comprises an inhibitor of anti-apoptotic proteins such as BCL-2, such as venetoclax; and blinatumomab.

[0106] Furthermore, the above-mentioned pharmaceutical composition further comprises an androgen / estrogen receptor inhibitor. Such as enzalutamide; abiraterone; and / or raloxifene.

[0107] Furthermore, the above-mentioned pharmaceutical composition further comprises an osteoclast inhibitor. Such as denosumab.

[0108] Furthermore, the above-mentioned pharmaceutical composition further comprises an inhibitor of the interaction between two primary p53 inhibitory proteins, MDMX and MDM2.

[0109] Furthermore, the above-mentioned pharmaceutical composition further comprises an inhibitor of transforming growth factor β (TGF-beta or TGFβ).

[0110] Furthermore, the above-mentioned pharmaceutical composition further comprises an ADC drug selected from vedotin-monomethyl auristatin E.

[0111] Further, the above-mentioned pharmaceutical composition further comprises an anti-proliferative compound, and such anti-proliferative compounds include, but are not limited to, aromatase inhibitors; anti-estrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule-active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce the process of cell differentiation; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; anti-tumor antimetabolites; platinum compounds; compounds that target / reduce the activity of protein or lipid kinases and additional anti-angiogenic compounds; compounds that target, reduce or inhibit the activity of protein or lipid phosphatases; 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 the treatment of hematological malignancies; compounds that target, reduce or inhibit Flt-3 activity; Hsp90 inhibitors; and / or MEK inhibitors.

[0112] 3. Beneficial effects

[0113] Compared with the prior art, the beneficial effects of the present application are as follows:

[0114] (1) The salinomycin derivative, its preparation method and application provided by the present application include the general formula structure of formula (1). Compared with salinomycin, the activity of this salinomycin derivative is significantly improved; and the C-20 group of this salinomycin derivative contains a disulfide bond, and through the substitution reaction of the disulfide bond, it can be quickly and efficiently coupled with a polypeptide or an antibody to obtain a GSH-sensitive salinomycin derivative or its salt containing a polypeptide or an antibody. The salinomycin derivative or its salt containing a polypeptide or an antibody can, on the one hand, improve the physical and chemical properties of salinomycin by virtue of the characteristics of the polypeptide or the antibody, such as improving its solubility; on the other hand, by virtue of the property of specific binding of the polypeptide or the antibody, it enhances the targeting property, improves the drug selectivity and biological activity, and has a bystander effect. In addition to being able to kill tumor cells with high antigen expression, it has an obvious inhibitory effect on cells with low antigen expression.

[0115] (2) The salinomycin derivative, its preparation method and application provided by the present application include the general formula structure of formula (1) or formula (6), and both have significant inhibitory effects on tumor stem cells and drug-resistant cells, and have the effects of reversing tumor cell drug resistance and drug sensitization, and can be used in combination with other drugs to further improve the curative effect.

[0116] (3) When preparing the salinomycin derivative or its salt containing a polypeptide or an antibody in the salinomycin derivative, its preparation method and application provided by the application, by introducing a spacer region (i.e., the Sar region) into the functional polypeptide or antibody, the interaction between the polypeptide or antibody and the small molecule can be reduced. Brief description of the drawings

[0117] Figure 1 It is the MS identification result of compound A.

[0118] Figure 2 It is the MS identification result of compound C.

[0119] Figure 3 It is the MS identification result of compound D.

[0120] Figure 4 It is the MS identification result of compound E.

[0121] Figure 5 It is the MS identification result of polypeptide A6-Sar.

[0122] Figure 6 It is the MS identification result of the polypeptide-containing salinomycin derivative Sal-A6.

[0123] Figure 7 It is the activity detection result of Sal and compound E at multiple tumor cell levels.

[0124] Figure 8 It is the activity toxicity effect of Sal and compound E on HUVEC cells at 24 h.

[0125] Figure 9 It is the effect of Sal and compound E on the in vitro invasion ability of HUVEC cells at 24 h.

[0126] Figure 10 It is the effect of different concentrations of Sal and compound E on the in vitro invasion ability of HUVEC cells. # Compared with Control, p < 0.001, # p < 0.0001; * compared with the Sal group; **** p < 0.0001.

[0127] Figure 11 It is the activity effect of Sal and compound E on HUVEC cells at 6 h.

[0128] Figure 12 It is the effect of Sal and compound E on the tube formation of HUVEC cells.

[0129] Figure 13 It is the statistical count of the number of tube formations of HUVEC cells by Sal and compound E; * compared with the Control group; ** p < 0.01.

[0130] Figure 14 It is the solubility detection result of Sal and Sal-A6 in water.

[0131] Figure 15 It is the expression level of CD44 protein in different cells.

[0132] Figure 16 It is the detection of the activity of different cells by CCK8.

[0133] Figure 17 It is the effect of Sal and Sal-A6 on the proliferation activity of SKOV3 cells at different time points.

[0134] Figure 18 It is the effect of Sal and compound E on the proliferation activity of SKOV3 cells at different time points.

[0135] Figure 19 It is the effect of Sal, Sal-A6, compound E and A6 on the migration of SKOV3 cells at 24 h.

[0136] Figure 20 It is the effect of Sal, Sal-A6, compound E (10 μM) and A6 (10 μM) on the migration rate of SKOV3 cells at 2--h; compared with Control, **p < 0.01, ***p < 0.001, ****p < 0.0001; compared with Sal, p < 0.001.

[0137] Figure 21 It is the effect of Sal, Sal-A6 and compound E on the in vitro migration ability of SKOV3 cells at 48 h.

[0138] Figure 22 It is the statistics of the migration inhibition rate of Sal, Sal-A6 and compound E on SKOV3 cells at 48 h; * compared with the Sal group; *p < 0.1, **p < 0.01, ***p < 0.001.

[0139] Figure 23 It is the statistics of the invasion rate of Sal, Sal-A6 and compound E on SKOV3 cells at 24 h; * compared with the Sal group; ***p < 0.001, ***p < 0.001.

[0140] Figure 24 It is the effect of Sal, Sal-A6 and compound E on the cell cycle of SKOV3 cells.

[0141] Figure 25 It is the effect of different concentrations of cisplatin on the proliferation activity of SKOV3 or SKOV3 / DDP at 48 h; ***p < 0.001.

[0142] Figure 26 It is the effect of Sal, Sal-A6 and compound E on the activity of different SKOV3 / DDP.

[0143] Figure 27The effects of 1 μM Sal, Compound E, and Sal-A6 on the activity of cisplatin at different concentrations.

[0144] Figure 28 The effects of Sal, Sal-A6, and Compound E on the spheroid formation of tumor stem cells.

[0145] Figure 29 The effects of Sal, A6, and Sal-A6 on the activity of domesticated SKOV3 cells.

[0146] Figure 30 The effects of Sal, A6, and Sal-A6 on the activity of domesticated A2780 cells.

[0147] Figure 31 The effects of different concentrations of Sal-A6 on the activity of mixed cells, * compared with Control, **** p < 0.0001.

[0148] Figure 32 The effects of different concentrations of Sal-A6 on the fluorescence intensity of mixed cells.

[0149] Figure 33 The inhibitory effects of Sal and Sal-A6 on SKOV3 subcutaneous xenograft tumors and their effects on the body weight of nude mice, * p < 0.05, ** p < 0.01.

[0150] Figure 34 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 implementation manners

[0151] The following further describes the present application in combination with specific embodiments.

[0152] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope of implementation of the present application.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0154] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0155] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. A person skilled in the art can readily determine the degree of flexibility for a particular variable.

[0156] 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" expressly includes only A, only B, only C, and their respective combinations.

[0157] Concentrations, amounts, and other numerical data may be presented herein in a range format. It should be understood that such range formats are used merely for convenience and brevity and should be interpreted flexibly as including not only the numerical values explicitly recited as the limits of the range, but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted as including not only the explicitly recited limits of 1 to about 4.5, but also the individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that recite only one numerical value, such as "less than about 4.5", which should be interpreted as including all of the above values and ranges. In addition, this interpretation should apply regardless of the breadth of the range or feature being described.

[0158] For all of the following examples, standard operating and purification methods known to those skilled in the art may be used. Unless otherwise stated, all temperatures are in °C. The structure of the compound is determined by nuclear magnetic resonance hydrogen spectroscopy ( 1 1H NMR) and mass spectrometry (MS), or the purity is determined using ultra-high performance liquid chromatography.

[0159] Example 1

[0160] This example provides a salinomycin derivative or a salt thereof and a preparation method thereof. The salinomycin derivative has the structure of the following formula (1),

[0161]

[0162] wherein: -X is H, Li, Na, or K.

[0163] In this example, -X is H, that is, a salinomycin derivative having the structure of the following formula (2),

[0164]

[0165] This example also provides a method for preparing a salinomycin derivative of the structure of formula (2). The C20-OH of salinomycin is oxidized using an oxidizing agent, and then the C20-OH of salinomycin is reduced to C20-NH2 using reductive amination reaction, and then esterification reaction modification is carried out to obtain it, which specifically includes the following steps:

[0166] (1) Preparation of compound A

[0167] Add 2.02 g of 3-mercaptopropionic acid, 60 ml of ethanol, and 8.35 g of 2,2-dithiodipyridine into a 250 ml reaction flask. The system changes from colorless and turbid to a yellow solution. After 15 min, add 10 drops of acetic acid (HAC) to the system and stir at room temperature. Thin layer chromatography (TLC) is used to detect the reaction progress. After the reaction is completed, the system is concentrated, and excess 2,2-dithiodipyridine is removed by column chromatography (EA / PE = 1:6), and purification is carried out using EA / PE = 1:5 + 1% HAC. After concentration and drying by rotary evaporator, compound A is obtained. The MS identification result of compound A is as Figure 1 shown, and its reaction mechanism is as follows:

[0168]

[0169] (2) Preparation of compound B

[0170] In a 100 ml reaction flask, add 152 mg of A, 30 ml of ultra-dry dichloromethane (DCM), under N2 protection, add 267 mg of oxalyl chloride (oxalyl chloride) to the system under ice-water bath conditions. There are bubbles during the dropping process, and the color of the system changes from colorless to yellow. After about 30 seconds of dropping, remove the ice-water bath, add 2 drops of N,N-dimethylformamide (DMF) to the system, and stir at 25 °C. The color of the system changes to colorless, and a sample is taken for TLC (MeOH:DCM = 1:20) detection. After the reaction is completed, the system is concentrated and dried, and then DCM is added again for concentration and drying to obtain compound B. Its reaction mechanism is as follows:

[0171]

[0172] (3) Preparation of compound C

[0173] Add 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 into a 250 ml reaction flask. Protect with N₂ and stir overnight at 25 °C to obtain a black turbid system. Take a sample and detect it by TLC (MeOH:DCM = 1:25). Filter through diatomaceous earth, combine the DCM phases, wash with 0.1 N hydrochloric acid (HCl), separate the layers, dry the DCM phase with anhydrous Na₂SO₄, and concentrate to obtain white solid compound C. The MS identification result of compound C is as Figure 2 shown, and its reaction mechanism is as follows:

[0174]

[0175] (4) Preparation of compound D

[0176] Add 810 mg of C, 30 ml of anhydrous methanol (MeOH), 1 ml of NH₃ / MeOH solution, and 5 drops of HAC into a 250 ml reaction flask to obtain a yellow turbid solution. Then add 20 ml of ultra-dry DCM and it completely dissolves. Stir at 25 °C. Subsequently, add 400 mg of cerium(III) chloride heptahydrate (CeCl₃·7H₂O) to the system and stir at room temperature. The system turns coffee-colored. Then use a constant pressure dropping funnel to dropwise add 133 mg of sodium cyanoborohydride (NaBH₃CN) / 50 ml of MeOH solution into the system dropwise evenly and stir overnight at 25 °C. The next day, detect the reaction by TLC (MeOH / DCM = 1:25). After the reaction is completed, concentrate the system to dryness, add 100 ml of DCM to dissolve, wash with saturated NaCl, dry with anhydrous Na₂SO₄, concentrate, and perform column chromatography (eluent: MeOH / DCM = 1:50 - MeOH / DCM = 1:20) to obtain product D. The MS identification result of compound D is as Figure 3 shown, and its reaction mechanism is as follows:

[0177]

[0178] (5) Preparation of compound E

[0179] Add 102 mg of D, 20 ml of ultra-dry DCM, and 30 mg of triethylamine (C₆H 15(N), protected by N2 substitution. Under ice - bath conditions, 50 mg of B in 30 ml of ultra - dry DCM solution was added dropwise to the system. During the addition process, the color of the system changed from colorless to light yellow. After 20 min, the addition was completed, and it was allowed to rise to room temperature naturally and stirred overnight. The reaction was monitored by TLC (MeOH / DCM = 1:20). After the reaction ended, the reaction system was diluted with 50 ml of DCM, washed once with 50 ml of 1N HCl aqueous solution, and once with 50 ml of saturated NaCl, concentrated, and purified by column chromatography (eluent: first use EA / PE = 1:4 + 1% HAC until there are no impurities, and then use MeOH / DCM = 1:50 for purification) to obtain product E. The MS identification result of compound E is as Figure 4 shown, that is, the compound with the structure of formula (2). The MS identification result of compound E is as Figure 4 shown, and its reaction mechanism is as follows:

[0180]

[0181] Example 2

[0182] This example provides the application of salinomycin derivative (compound E) in the preparation of salinomycin derivative containing polypeptide and the prepared salinomycin derivative containing polypeptide.

[0183] This application includes: through the disulfide bond substitution reaction between the disulfide bond in compound E and the thiol group in the polypeptide, enabling the rapid coupling of compound E and the polypeptide to obtain a salinomycin derivative containing polypeptide. The polypeptide is: polypeptide A6 - Sar, and its structure is: Ac - KPSSPPEE(Sar) 10 C - NH2 (SEQ ID NO.2), which is a polypeptide formed by sequentially connecting functional polypeptide A6 (KPSSPPEE, SEQ ID NO.1), 10 sarcosines (Sar), and cysteine (Cys). The side - chain group of cysteine contains a thiol group (-SH), which undergoes a disulfide bond substitution reaction with the disulfide bond in compound E. The specific steps are as follows:

[0184] (1) Preparation of polypeptide A6 - Sar

[0185] This polypeptide is a polypeptide formed by sequentially connecting functional polypeptide A6 (KPSSPPEE, SEQ ID NO.1), 10 sarcosines (Sar), and cysteine (Cys). Introducing 10 sarcosines as a spacer can reduce the interaction between the targeting peptide (functional polypeptide A6) and the small molecule (compound E). Polypeptide A6 - Sar is synthesized by solid - phase synthesis method, and the specific steps are as follows:

[0186] 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.

[0187] 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.

[0188] 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;

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Repeat the above operation and add each amino acid to the peptide sequence in turn. Couple 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 in turn according to the peptide sequence, and then remove the Fmoc protecting group with a 20% piperidine / DMF solution;

[0193] Use acetic anhydride to cap the end. Specifically, use 5 ml of DCM as the solvent, add 0.29 ml of DIPEA and 0.2 ml of acetic anhydride, and bubble nitrogen for 30 min at room temperature; after the reaction is completed, wash with DMF four times;

[0194] Use 9.5 ml of TFA, 0.1 ml of H2O, 0.2 ml of EDT and 0.2 ml of Tis as the cleavage solution. Place the A6-Sar10-Cys protected peptide resin and the cleavage solution in a centrifuge tube. After reacting for 2 h, precipitate with ice ether and centrifuge three times to obtain a white solid. Dry it in a vacuum drying oven to obtain the crude product. Purify it by HPLC and freeze-dry it to obtain the white powder polypeptide A6-Sar. The MS identification result of polypeptide A6-Sar is as Figure 5 shown, and its structure is shown in formula (12):

[0195]

[0196] (2) Coupling of compound E and polypeptide A6-Sar

[0197] Weigh 200 mg of compound E, 800 mg of A6-Sar, and use 100 ml of DMSO as the solvent. React for 72 h under N2 protection at room temperature. After the reaction is completed, purify it by HPLC (mobile phase A: ACN + 0.1% formic acid, mobile phase B: water + 0.1% formic acid) to obtain the white powder Sal-A6, that is, the salinomycin derivative containing polypeptide with the structure of formula (8). The MS identification result is as Figure 6 shown.

[0198] Example 3

[0199] This example provides an in vitro anti-tumor activity test of the salinomycin derivative (compound E) prepared in Example 1.

[0200] Take cells in the logarithmic growth phase, digest, count, and prepare a cell suspension of 1×10 5 cells / mL, inoculate into a 96-well plate (100 μL / well), and culture in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, add salinomycin (Sal) or compound E at corresponding concentrations according to the dosages 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. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plate in the incubator and culture for 72 h. After completion, add 10 μL of CCK8 solution to each well, continue to incubate in the cell incubator for 1 hour, measure the absorbance value at 450 nm, and calculate the proliferation inhibition rate.

[0201] The results are as Figure 7 shown in Figure 7 and Table 1, which are the results of the activity detection of Sal and compound E at the cell levels of MKN45, HCCLM3, A431, A549, ACHN, Du145, and HeLa. The results show that at the cell levels of MKN45, HCCLM3, A431, A549, ACHN, Du145, and HeLa, the inhibitory effect of compound E on tumor cells is better than that of Sal.

[0202] Table 1 IC50 of Sal and compound E at multiple tumor cell levels

[0203] Cell Sal (IC50 μM) Compound E (IC50 μM) MKN-45 (Human gastric cancer cells) 6.59 1.91 HCCLM3 (Human liver cancer cells) 8.07 1.59 A431 (Human skin squamous carcinoma cells) 5.47 3.20 A549 (Human lung cancer cells) 25.62 13.96 ACHN (Human renal cancer cells) 62.35 6.73 Du145 (Human prostate cancer cells) 47.47 30.93 HeLa (Human cervical cancer cells) 24.60 3.76

[0204] Example 4

[0205] This example provides the effect of the salinomycin derivative (compound E) prepared in Example 1 on inhibiting angiogenesis.

[0206] Invasion experiment of HUVEC (human umbilical vein endothelial cells):

[0207] First, determine the toxicity of compound E and Sal to the cell HUVEC at a concentration of 1.25 μM. After co-incubating the above two drugs with the cells for 24 h, detect the cell toxicity. The results are as Figure 8 shown. At a concentration of 1.25 μM, Sal and compound E have almost no significant toxicity to the cell HUVEC.

[0208] Thaw Matrigel matrix glue in a 4°C refrigerator. Pre-chill the yellow pipette tip box and 96-well plate at -20°C. Take 600 μl of Matrigel matrix glue and add it to 900 μl of serum-free ECM medium containing 1% ECGS. Pipette and mix well, then add 50 μl to the chambers and place them in the 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. After termination, repeatedly pipette the bottom of the cell flask with a pipette gun to fully resuspend the cells, and transfer them to a 15 ml centrifuge tube. Centrifuge at 12,000 rpm for 5 min, resuspend the cells, and count. After mixing the prepared Sal, compound E, and cell suspension in a 1:1 ratio, seed HUVECs at a density of 4×10 5 cells / well into the chambers and culture them in a CO₂ constant temperature incubator for 24 h. Wash with PBS, fix with methanol, stain with crystal violet, and take pictures. The results are as shown in Figure 9 and Figure 10 , showing that both Sal and E can inhibit the invasion ability of HUVECs cells, and the efficacy of compound E is far better than that of Sal.

[0209] Angiogenesis experiment of HUVEC:

[0210] First, determine the toxicity of compound E and Sal to HUVEC cells at concentrations of 1.25 μM and 2.5 μM. After co-incubating the above two drugs with the cells for 6 h, detect the cell toxicity. The results are as shown in Figure 11 , showing that at concentrations of 1.25 μM and 2.5 μM, Sal and compound E have almost no significant toxicity to HUVEC cells.

[0211] Thaw Matrigel matrix glue in a 4°C refrigerator. Pre-chill the yellow pipette tip box and 96-well plate at -20°C. Take 600 μl of Matrigel matrix glue and add it to 900 μl of serum-free ECM medium containing 1% ECGS. Pipette and mix well, then add 50 μl to the chambers and place them in the 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. After termination, repeatedly pipette the bottom of the cell flask with a pipette gun to fully resuspend the cells, and transfer them to a 15 ml centrifuge tube. Centrifuge at 12,000 rpm for 5 min, resuspend the cells, and count. After mixing the prepared Sal, compound E, and cell suspension in a 1:1 ratio, seed HUVECs at a density of 4×10 4 cells / well into the chambers and culture them in a CO₂ constant temperature incubator for 6 h. Observe and take pictures under the microscope. The results are as shown in Figure 12 and Figure 13As shown, it is shown that both Sal and E can inhibit the tubule formation ability of HUVECs cells. At a concentration of 2.5 μM, the efficacy of compound E is better than that of Sal, and there is a statistical difference in efficacy at a concentration of 2.5 μM.

[0212] Example 5

[0213] This example provides a solubility test of the salinomycin derivative Sal-A6 containing a polypeptide prepared in Example 2.

[0214] Prepare 4 mM Sal and Sal-A6 using physiological saline and detect their solubility.

[0215] The results are as Figure 14 shown, Sal-A6 can be significantly dissolved in water, while salinomycin is almost insoluble in water.

[0216] Example 6

[0217] This example provides a flow cytometry screening of cells with high CD44 expression.

[0218] Resuscitate and culture the cells HT-29 (human colorectal cancer cells), MKN-45 (human gastric cancer cells), SKOV3 (human ovarian cancer cells), Cal-27 (human tongue squamous carcinoma cells), HepG2 (human liver cancer cells), A431 (human skin squamous carcinoma cells), A549 (human lung cancer cells) and A2780 (human ovarian cancer cells), and passage them more than three times for standby; after culturing the cells for three generations, under appropriate density conditions, wash them several times with commercially available PBS pre-cooled at 4 °C to remove cells in poor condition and floating substances, and then digest them with trypsin solution; when the cells become round, add an equal volume of medium to terminate digestion, centrifuge at 1000 rpm for 5 min; discard the supernatant, and block them with 2% commercially available BSA blocking solution at 4 °C for 1 h; respectively take 100 μl of the above-mentioned concentration of cells into a 1.5 ml centrifuge tube, and 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-mentioned cells are centrifuged at 4 °C and 2000 rpm for 5 min, washed 3 times with PBS, and then detected on a flow cytometer (CytoFLEX).

[0219] The results are as Figure 15 shown, among the cells tested, all except A2780 cells are highly expressing CD44.

[0220] Table 2 Detection of CD44 expression in different cells

[0221]

[0222] Example 7

[0223] This example provides an in vitro anti-tumor activity test of the salinomycin derivative Sal-A6 containing a polypeptide:

[0224] Experimental method:

[0225] Take the SKOV3 cells in the logarithmic growth phase, digest, count, and prepare a cell suspension of 1×10 5 cells / mL, inoculate into a 96-well plate (100 μL / well), and culture in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, add Sal, compound E, Sal-A6, and polypeptide drug A6 containing the corresponding concentrations according to the dosages 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. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plate in the incubator and culture for 0 h, 24 h, 48 h, and 72 h. After the end, add 10 μL of CCK8 solution to each well, continue to incubate in the cell incubator for 1 hour, measure the absorbance at 450 nm, and calculate the proliferation inhibition rate.

[0226] The results are as Figure 1 shown. The activities of compound E and Sal-A6 are both improved compared with Sal, while polypeptide A6 has almost no inhibitory effect on tumor cells.

[0227] Example 8

[0228] Detection of the onset time of compound E and Sal-A6:

[0229] Experimental method:

[0230] Take the cells in the logarithmic growth phase (SKOV3 cells), digest, count, and prepare a cell suspension of 1×10 5 cells / mL, inoculate into a 96-well plate (100 μL / well), and culture in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, add Sal, compound E, and Sal-A6 containing the corresponding concentrations according to the dosages of 20 μM, 10 μM, and 5 μM. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plate in the incubator and culture for 72 h. After the end, add 10 μL of CCK8 solution to each well, continue to incubate in the cell incubator for 1 hour, measure the absorbance at 450 nm, and calculate the proliferation inhibition rate.

[0231] The results are as ​ shown. Compared with Sal, the onset speed of Sal-A6 is slower, but the onset speed of compound E compared with Sal is significantly increased, which may be related to liposolubility.

[0232] Example 9

[0233] This example provides an experiment on the effect of compound E and Sal-A6 on the scratch of SKOV3 cells:

[0234] Experimental method:

[0235] (1) Resuscitate and culture the CD44 highly expressed cell line SKOV3 obtained from the previous screening, and reserve it for stable passage for more than three generations;

[0236] (2) When the cell density grows to about 85%, first discard the supernatant, then add PBS solution to rinse the culture flask, and then add trypsin solution to digest the cells. After complete digestion, repeatedly pipette the bottom of the culture flask with a pipette to fully resuspend the cells, and then transfer them to a 10 mL sterile centrifuge tube. After centrifugation at 1000 rpm for 5 min, resuspend the cells and perform cell counting;

[0237] (3) Add complete medium containing 10% serum to a cell concentration of about 4×10 5 cells / mL, plate, and inoculate 3 replicate wells for each group;

[0238] (4) When the cell density increases to 90%, use a 100 μL sterile pipette tip to draw a 2 mm wide straight line, discard the medium in the 24-well plate, add blank medium, and perform drug administration treatments with different doses of SAL, SAL-Na, SAL-S-S, polypeptide A6, and SAL-A6; culture in a CO2 constant temperature incubator;

[0239] (5) Take out the cells at 0 and 24 h respectively, observe the healing condition under the microscope, take pictures, ensure that the same field of view is selected for each picture and the same objective lens and eyepiece are used, and analyze with Photoshop software.

[0240] The results are as ​ shown. After 24 h of drug administration, the cell migration of ovarian cancer SKOV3 is significantly inhibited compared with the blank control group, and the inhibitory effect is compound E > Sal-A6 > SAL ≈ A6.

[0241] Example 10

[0242] This example provides an experiment on the effect of compound E and Sal-A6 on cell migration:

[0243] Experimental method:

[0244] Culture SKOV3 cells. When the cell density reaches 80 - 90%, discard the supernatant, add PBS to wash the culture flask, add trypsin solution to digest the cells. After termination, repeatedly pipette the bottom of the cell flask with a pipette gun to fully resuspend the cells, and transfer them to a 15 ml centrifuge tube. Centrifuge at 12,000 rpm for 5 min, resuspend the cells, and count. After mixing the prepared Sal, compound E, and cell suspension in a 1:1 ratio, seed SKOV3 at a density of 4×10 5 cells / well into the chamber, and culture in a carbon dioxide incubator at a constant temperature for 48 h. Observe and take pictures under the microscope.

[0245] The results are as ​ shown. Both Sal, Sal - A6, and E can inhibit the in vitro migration ability of SKOV3 cells, and show a dose - dependence. Moreover, the inhibitory ability is compound E > Sal - A6 > Sal.

[0246] Example 11

[0247] This example provides an experiment on the effect of compound E and Sal - A6 on cell invasion:

[0248] Experimental method:

[0249] Thaw Matrigel matrix glue in a 4°C refrigerator. Take 600 μl of Matrigel matrix glue and add it to 900 μl of serum - free ECM medium containing 1% 1640. Pipette and mix well, then add 50 μl to the chamber and place it in the cell culture incubator overnight.

[0250] When the cells (SKOV3) grow to a density of 80 - 90%, discard the supernatant, add PBS to wash the culture flask, add trypsin solution to digest the cells. After termination, repeatedly pipette the bottom of the cell flask with a pipette gun to fully resuspend the cells, and transfer them to a 15 ml centrifuge tube. Centrifuge at 12,000 rpm for 5 min, resuspend the cells, and count. After mixing the prepared Sal, compound E, and cell suspension in a 1:1 ratio, seed HUVEC at a density of 6×10 4 cells / well into the chamber, and culture in a carbon dioxide incubator at a constant temperature for 12 h. Observe and take pictures under the microscope.

[0251] The results are as ​ shown. Both Sal, Sal - A6, and compound E can inhibit the in vitro invasion ability of SKOV3 cells, and show a dose - dependence. Moreover, the inhibitory ability is compound E > Sal - A6 > Sal.

[0252] Example 12

[0253] This example provides an experiment on the effect of compound E and Sal - A6 on the cell cycle:

[0254] Experimental method:

[0255] (1) Resuscitate and culture SKOV3 cells, and passage them for more than three generations for standby;

[0256] (2) When the cell fusion rate of the above reaches 90%, discard the culture medium, wash it 2 - 3 times with PBS, and then add trypsin solution to digest the cells;

[0257] (3) When the cells become round, terminate the digestion process with complete culture medium, centrifuge at 1000 rpm for 5 min;

[0258] (4) Discard the supernatant, resuspend the cells by adding serum - free medium, and measure them with a cell counter, adjusting the cell concentration to 4×10 5 cells / mL;

[0259] (5) Pipette 2 mL of the above cells into a 6 - well plate respectively, mix evenly, and incubate overnight in a cell culture incubator;

[0260] (6) Dilute with serum - free medium according to the dilution ratio to a final concentration of 10 μM and 5 μM for standby. Wash 3 times with PBS solution, and remove the cells with poor growth status and floating substances in the cells themselves. Then add the above - diluted Sal and Sal - A6 solutions to the corresponding sample wells respectively, inject serum - free medium into the blank wells, and continuously culture in a cell culture incubator for 72 h;

[0261] (7) Wash the cells 3 times with PBS, centrifuge at 2000 rpm for 5 min, and collect the cells in each well;

[0262] (8) Use a cell counter to count the number of cells and adjust the density to 1×10 6 cells / mL, then mix well with a pipette to obtain a single - cell suspension;

[0263] (9) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and then add 500 μL of the pre - cooled cell fixative prepared above for fixation, and incubate overnight at 4℃;

[0264] (10) Centrifuge at 1000 rpm for 5 min, wash 3 times with PBS to remove the fixative;

[0265] (11) Add 500 μL of RnaseA / PI staining working solution prepared according to this formula to each well, incubate at room temperature for 60 min in the dark;

[0266] (12) Detect by flow cytometry.

[0267] The results are as ​ shown in and Table 3.

[0268] Table 3 Statistical table of the effects of Sal, Sal-A6, and Compound E on the cell cycle of SKOV3 cells

[0269] G1 S G2 ​ 60.0％ 36.8％ 0％ ​ 77.0％ 21.0％ 0％ ​ 45.8％ 32.3％ 20.3％ ​ 37.3％ 38.0％ 26.3％ ​ 37.3％ 45.6％ 17.5％ ​ 42.2％ 38.5％ 21.1％ ​ 26.4％ 44.4％ 28.1％

[0270] SKOV3 cells were treated with different concentrations of Sal and Sal-A6, and flow cytometry was used to determine the effect on cell cycle distribution. As shown in Table 3, in SKOV3 cells, 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 can induce SKOV3 cells to arrest in the S phase and G2 / M phase. The above results show that Sal-A6 can induce SKOV3 cells to arrest in the S phase and G2 / M phase. At the same time, the effects of Sal and Sal-A6 on the cell cycle are basically the same, indicating that the trend of the effect of Sal after introducing polypeptide A6 on the cell cycle is basically the same.

[0271] Example 13

[0272] This example provides an experiment on the effects of cisplatin on SKOV3 cells and SKOV3 / CDDP cells:

[0273] Experimental method:

[0274] Take logarithmically growing cells SKOV3 and SKOV3-DDP, digest, count, and make a cell suspension of 1×10 4 cells / mL, inoculate into 96-well plates (100 μL / well), and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0275] Prepare cisplatin with different concentrations and add it to the 96-well plates. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plates in the incubator and culture for 72 h.

[0276] Add 10 μL of CCK8 solution to each well, continue to incubate in the cell culture incubator for 1 hour, measure the absorbance value at 450 nm, and calculate the proliferation inhibition rate.

[0277] The results are as ​ shown. The inhibitory activity of cisplatin on SKOV3 cells is better than that on SKOV3 / CDDP cells, indicating that SKOV3 / CDDP cells have developed obvious drug resistance to cisplatin.

[0278] Example 14

[0279] This example provides the detection of CD44 expression in drug-resistant cells and the experiment on the activity of drug-resistant cells.

[0280] Detection of CD44 expression in drug-resistant cells

[0281] After resuscitation and culturing, the human ovarian cancer cell line SKOV3-DDP was cultured for three generations. Under appropriate density conditions, it was washed several times with commercially available PBS pre-cooled to 4°C to remove poorly conditioned cells and floating matter, and then digested with trypsin solution. After the cells became round, an equal volume of medium was added to terminate 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 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 the cells were 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 on a flow cytometer (CytoFLEX).

[0282] Drug-resistant cell activity experiment

[0283] Cells in the logarithmic growth phase of SKOV3-DDP were digested, counted, and made into a cell suspension of 1×10 4 cells / mL, and inoculated into 96-well plates (100 μL / well), and cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0284] Peptide A6, Sal, compound E, and Sal-A6 with 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 respectively prepared. At the same time, a negative control group and a blank group were set up, with 5 replicates in each group. The plate was placed in the incubator and cultured for 72 hours.

[0285] 10 μL of CCK8 solution was added to each well, and the plate was further incubated in the cell culture incubator for 1 hour. The absorbance value was measured at 450 nm, and the proliferation inhibition rate was calculated.

[0286] The results are as ​ shown. Compared with SKOV3 cells (reference ​ ), the activities of Sal, Sal-A6, and compound E against SKOV3 / DDP were all enhanced, which also indicates that the stemness of drug-resistant cells SKOV3 / DDP is enhanced compared to SKOV3.

[0287] Example 15

[0288] This example provides a sensitization test of compound E and Sal-A6 on drug-resistant cells:

[0289] Experimental method:

[0290] Cells in the logarithmic growth phase of SKOV3-DDP were digested, counted, and made into a cell suspension of 1×104 A cell suspension of

[0291] Prepare polypeptides A6, Sal, compound E, and 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 respectively. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plate in an incubator and culture for 72 h.

[0292] The results are as ​ shown. The use of Sal, compound E, or Sal-A6 can enhance the activity of cisplatin and improve its sensitivity to cells.

[0293] Example 16

[0294] This example provides an experiment on the effect of compound E and Sal-A6 on tumor stem cells:

[0295] The tumor cell sphere formation assay is one of the main indicators for detecting tumor stem cells. Using SKOV3 cells under the condition of low-serum agarose culture, and co-incubating with drugs, observe the effect of Sal, Sal-A6, and compound E on the sphere formation ability.

[0296] The results are as ​ shown. Sal, E, or Sal-A6 can all inhibit the activity of tumor stem cells.

[0297] Example 17

[0298] This example provides a detection of the bystander effect of Sal-A6 on tumor cells:

[0299] Experimental method:

[0300] Cell acclimation

[0301] (1) Take out the cell culture flask from the cell incubator and observe under the microscope. When the cells cover the bottom surface, in a clean workbench, discard the culture medium, rinse the cells with 2 mL of PBS and then discard, repeat 2 times, add 2 mL of 0.25% trypsin to digest, and terminate with the corresponding volume of serum-containing medium. Transfer the cells to a 15 mL conical bottom centrifuge tube.

[0302] (2) Centrifuge: 1000 rpm, 5 min, discard the supernatant, resuspend the cells with 2 mL of serum-containing RPMI-1640 complete medium, add 1 mL of cell suspension to each cell flask, and then supplement the medium in each flask. Observe under the microscope and place it back in the incubator for continued culture.

[0303] (3) When the cell fusion rate reaches 70%, discard the culture medium, wash with PBS 2-3 times, and then add the complete culture medium mixed with DMEM and RPMI-1640 (DMEM:RPMI-1640 = 1:4). Observe under the microscope and place it in the incubator for continued culture;

[0304] (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;

[0305] (5) Repeat the operations in (2)-(4), and gradually transition the ratio of the mixed complete culture medium from 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 to DMEM complete culture medium;

[0306] (6) Reserve the above-mentioned cells after stable passage for more than three generations.

[0307] Detection of the activity of Sal-A6 against domesticated SKOV3

[0308] Take the domesticated cells of SKOV3 in the logarithmic growth phase, digest, count, and prepare a cell suspension of 1×10 4 cells / mL, inoculate into a 96-well plate (100 μL / well), and culture in an incubator at 37 °C and 5% CO2 for 24 hours.

[0309] Prepare 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 respectively. At the same time, set up a negative control group and a blank group, with 5 replicates in each group. Place the plate in the incubator and culture for 72 h.

[0310] Add 10 μL of CCK8 solution to each well, continue to incubate in the cell incubator for 1 hour, measure the absorbance at 450 nm, and calculate the proliferation inhibition rate.

[0311] To verify the bystander killing effect of Sal-A6 on tumor cells, we co-incubated CD44+ SKOV3 cells and CD44- A2780 cells to verify whether Sal-A6 has a bystander killing effect. Before that, we first domesticated SKOV3 from 1640 medium to DMEM medium, and detected the CD44 expression level and the effects of Sal-A6 and Sal on its activity. The results showed that SKOV3 / DMEM presented a state of high CD44 expression, and there was no significant difference in the activity of Sal-A6 and Sal compared with SKOV3 / 1640.

[0312] Select the concentration that has no obvious killing effect on A2780 and has a significant killing effect on SKOV3 to detect the bystander effect. Co-culture different ratios of SKOV3 and A2780 in DMEM medium, and then incubate with 20 μM, 10 μM, 5 μM, and 2.5 μM of Sal-A6 for 72 h. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the killing effect of Sal-A6 on cells with different cell ratios. The results show that when the ratio of SKOV3 to A2780 cells is 6:4, 5:5, or 4:6, Sal-A6 can effectively kill the mixed cells and shows a dose-dependent relationship. When the drug concentration is 20 μM, it can achieve more than 80% inhibition of tumor cells, indicating that Sal-A6 should have a certain bystander drug effect.

[0313] To further verify the bystander ability of Sal-A6, we constructed a cell line A2780 / GFP stably transfected with GFP green fluorescent protein for the CD44-negative tumor cell A2780, and co-incubated with SKOV3 and A2780 / GFP cells at a ratio of 6:4, 5:5, or 4:6 and gave different concentrations of Sal-A6. The results show that after drug administration, the fluorescence intensity of A2780 / GFP can be significantly reduced and shows a dose-dependent relationship. In summary, Sal-A6 is a PDC drug with a bystander effect and has the ability to kill CD44-negative tumor cells in the presence of CD44+ tumor cells.

[0314] The results are as ​ shown, ​ the effects of Sal, A6, and Sal-A6 on the activity of domesticated SKOV3 cells; ​ the effects of Sal, A6, and Sal-A6 on the activity of domesticated A2780 cells; ​ the effects of different concentrations of Sal-A6 on the activity of mixed cells; ​ the effects of different concentrations of Sal-A6 on the fluorescence intensity of mixed cells.

[0315] Example 18

[0316] This example provides a study on the in vivo anti-tumor efficacy of compound E and Sal-A6:

[0317] Experimental method:

[0318] Verification of Sal-A6 on subcutaneous xenograft tumors of SKOV3 in nude mice

[0319] Experimental animals: Female balb / c nude mice. Collect the cultured human ovarian cancer cells SKOV3 at a concentration of 1×10 7 cells / ml and inoculate 0.1 ml per mouse subcutaneously into the right axilla of the nude mice.

[0320] The calculation formula for tumor volume (TV) is as follows:

[0321] TV = 1 / 2 × a × b 2 , where a and b represent the length and width respectively.

[0322] When the average tumor volume is about 100 mm 3 , administration starts. Administer the drug once every two days, observe, measure the tumor volume, and record the change in body weight. The dosing doses are calculated according to the dose of salinomycin, and are respectively: Saline group, Salinomycin group (Sal 20 μg / kg), Sal-A6-1 group (equivalent to 15 μg / kg of salinomycin), and Sal-A6-2 group (equivalent to 30 μg / kg of salinomycin).

[0323] The results are as ​ shown. Even at low doses, Sal-A6 can inhibit tumor growth, and its drug effect is better than that of Sal itself, and it has no significant effect on the body weight of nude mice.

[0324] Example 19

[0325] This example provides the verification of Sal-A6 on subcutaneous xenografts in Cal-27 nude mice

[0326] Experimental animals: balb / c female nude mice. Collect the cultured human head and neck cancer cells SKOV3, with a concentration of 1 × 10 7 cells / ml, and inoculate 0.1 ml per mouse subcutaneously into the right axilla of the nude mice.

[0327] The calculation formula for tumor volume (TV) is as follows:

[0328] TV = 1 / 2 × a × b 2 , where a and b represent the length and width respectively.

[0329] When the average tumor volume is about 90 mm 3 , administration starts. Administer the drug once every two days, observe, measure the tumor volume, and record the change in body weight. The dosing doses are calculated according to the dose of salinomycin, and are respectively: Saline group, Cetuximab (ERBITUX 20 mg / kg), and Sal-A6 group (equivalent to 300 μg / kg of salinomycin).

[0330] In the Cal model, the in vivo drug effect of Sal-A6 is basically the same as that of Cetuximab, and there is no significant difference in the body weight of nude mice compared with the blank group.

[0331] The results are as ​As shown, Sal-A6 can also inhibit tumor growth at low doses, with its efficacy being comparable to that of the positive drug cetuximab and having no significant effect on the body weight of nude mice.

Claims

1. A salinomycin derivative or its salt, characterized in that, The salinomycin derivative has the structure of the following formula (1): Wherein: - 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 includes: Oxidizing the C20-OH of salinomycin with an oxidant, then reducing the C20-OH of salinomycin to C20-NH2 using reductive amination reaction, and then obtaining it through esterification reaction modification.

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 application includes: Through the disulfide bond substitution reaction between the disulfide bond in the above salinomycin derivative or its salt and the sulfhydryl group in the polypeptide or antibody, the salinomycin derivative or its salt is coupled with the polypeptide or antibody to obtain a 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 composed of a functional polypeptide or a functional antibody linked to a compound containing a sulfhydryl 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 includes: a polypeptide or antibody composed of a functional polypeptide or a functional antibody linked to a compound containing a sulfhydryl group 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 also linked between the functional polypeptide or functional antibody and the compound containing a sulfhydryl group.

7. Use of a salinomycin derivative or a salt thereof according to claims 4-6 in the preparation of a salinomycin derivative or a salt thereof containing a polypeptide or an antibody, characterized in that, The compound containing a sulfhydryl group includes: cysteine, mercaptoethylamine, mercaptoacetic 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 polypeptide includes: a cell-penetrating peptide or a targeting peptide.

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, and its structure is: Ac-KPSSPPEE(Sar) 10 C-NH2.

10. A salinomycin derivative or its salt containing a polypeptide or an antibody, characterized in that, Obtained through the application of a salinomycin derivative or its salt according to any one of claims 3-9 in the preparation of a salinomycin derivative or its salt containing a polypeptide or antibody.

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

13. The application of a salinomycin derivative or its salt according to claim 1, or a salinomycin derivative or its salt containing a polypeptide or antibody according to any one of claims 11-12 in the preparation of a drug for treating tumors and / or inhibiting angiogenesis.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition includes a salinomycin derivative or its salt according to claim 1, or a salinomycin derivative or its salt containing a polypeptide or antibody according to any one of claims 11-12.

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