Method for modifying drug molecule through thioimine and application thereof

Abstract

The invention discloses a medicine molecule modified by thioimine and application of the medicine molecule. The pharmaceutical molecule has a structure as shown in formula (I) or a pharmaceutically acceptable salt thereof: A-B (I); wherein the structure of the group A is shown in the specification; the group B is an anti-cancer drug group, and S in the group A is connected with amino in the group B through a sulfur imine bond. According to the compound, the cytotoxicity of the anti-cancer drug is shielded through the A group, the cytotoxicity of the anti-cancer drug can be reduced, when the compound enters a tumor tissue, a sulfur imine bond is induced to break, activate and release the anti-cancer drug through radiation, the anti-tumor activity of the original anti-cancer drug is recovered, and particularly, the high radiolysis yield of 240 nM / Gy or above can be achieved. Therefore, the compound can be used as a radiotherapy response drug for treating cancers, can enhance the treatment effects of chemotherapy and radiotherapy, and has the advantages of improving the biological safety and the like.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to a drug molecule modified with thioimine and its use, and more specifically to the use of 10-methylphenothiazine as a toxicity-masking molecule for anticancer drugs, thioimine-protected compounds, their preparation methods and uses. Background Technology

[0002] Chemotherapy is a common treatment for cancer, effectively killing target cells through the cytotoxicity of the drug molecules themselves. However, due to the lack of selectivity of chemotherapy drugs, the toxic side effects on patients are quite significant during treatment. In recent years, experimental studies and clinical results have shown that using controlled delivery and release of chemotherapy drugs can effectively reduce the toxic side effects on normal cells and improve patients' quality of life.

[0003] Since its initial proposal in 1958, the prodrug strategy has been widely used to address drug delivery challenges in cancer treatment. Ideally, prodrugs can deliver more than 50 times the normal dose of drug to the desired site, effectively curing tumors that are typically resistant to chemotherapy after activation. Therefore, how to effectively activate prodrugs within the target tumor tissue is one of the most critical issues in prodrug strategies. However, due to the complex characteristics of the tumor microenvironment, efficient and controlled activation of prodrugs within tumor tissue remains a long-standing challenge in clinical practice. Therefore, finding suitable chemical tools for highly selective and efficient spatiotemporally precise prodrug activation within tumor tissue is an urgent need in current cancer treatment practice.

[0004] Inspired by the applications of photodynamic and photothermal therapy in clinical cancer treatment, clinically relevant ionizing radiation (X-rays, gamma rays, etc.) also has the potential to become a precise perturbation tool in vivo. This is because it possesses precise and deep tissue penetration (up to 15 cm), allowing energy to penetrate superficial tissues and focus deep into the cancerous tissue, thereby efficiently activating prodrugs. Furthermore, although over 50% of cancer patients receive radiotherapy, it may not achieve the desired effects due to total dose limitations (usually less than 60 Gy) and hypoxia resistance. Combined radiotherapy and chemotherapy is also a common treatment approach in clinical practice. Establishing a chemotherapy strategy that activates targeted radiotherapy holds promise for reducing the overall toxicity of dual therapy and achieving better treatment outcomes.

[0005] To design targeted radiotherapy activation chemotherapy strategies, it is necessary to recognize that the radiochemical effects of ionizing radiation on molecules include both direct and indirect effects. Among these, the indirect radiochemical effects of radiation on environmental components are dominant in biological systems. It primarily works through the radiolytic decomposition of water, as water is a major component of biological tissues, accounting for 70% of tissue weight. 80%. For example... Figure 1 As shown, various reactive particles are instantaneously formed through the radioactive decomposition of water, followed by a cascade reaction within 10... 4 Completed within seconds, the main product is hydrated electrons (e - aq. ·OH radicals and hydroxyl radicals (·OH) have been used in previous studies to report the cleavage chemistry of prodrug molecules induced by hydroxyl radicals and hydrated electrons. However, due to the limitations of redox reactions of chemical bonds themselves, these prodrug molecules can only utilize a single active species (e.g., utilizing only hydrated electrons or only hydroxyl radicals), and common bond breaking is mostly a two-electron process. This severely limits the efficiency of prodrug activation by radiation. Therefore, there is an urgent need to find a prodrug activation strategy that can simultaneously utilize hydrated electrons and hydroxyl radicals to efficiently achieve prodrug activation in tumor tissues. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a thioimine-protected compound.

[0007] This application is based on the following discoveries of the inventors: Radiotherapy-activated prodrugs (RAPs) are a class of low-toxicity prodrugs that can be activated by medical radiation (ionizing radiation) into a highly active form for use in cancer treatment. They utilize the targeting and tissue penetration (up to 15 cm or more) of radiation therapy rays and their high clinical relevance (more than 60% of cancer patients receive radiotherapy). By irradiating water molecules, they generate hydroxyl radicals and hydrated electrons, selectively breaking covalent bonds to release the therapeutic drug, thereby enhancing the effects of radiotherapy, chemotherapy, or immunotherapy while reducing systemic toxicity.

[0008] However, current radiotherapy-responsive drugs have drawbacks such as low radiation sensitivity, cumbersome preparation, and high production costs. Specifically: 1) Existing radiotherapy-responsive drugs often only utilize one active substance produced by water radiolysis, such as solely utilizing hydrated electrons or solely utilizing hydroxyl radicals, resulting in low efficiency in utilizing radiation energy. Therefore, a larger dose of radiation is required to ensure complete drug release; 2) Existing radiotherapy-responsive drugs often require multi-step synthesis and complex separation from expensive precursors to obtain the final drug, resulting in high production costs.

[0009] Based on this, the inventors of this application have developed a thioimine-modified compound as a radiotherapy-responsive drug. This compound exhibits high radiation sensitivity, effectively shielding the toxicity of anticancer drugs before irradiation. Furthermore, only a 1 Gy dose is required to fully activate the anticancer drug within the compound, achieving the same cytotoxicity as free anticancer drugs. In addition, the thioimine-modified compound is simple to synthesize. Compared to the complex synthetic steps of other radiation-activated drugs, the thioimine-modified compound utilizes an oxidative click reaction, yielding the target product in one step. Separation only requires sedimentation to obtain the pure product.

[0010] Therefore, in a first aspect of this application, the use of 10-methylphenothiazine as a toxicity-masking molecule for anticancer drugs is proposed, wherein the sulfur (S) of the 10-methylphenothiazine is linked to the amino group of the anticancer drug via a thioimine bond. This application discovers that by coupling 10-methylphenothiazine with an anticancer drug, the synthesized compound, as an anticancer prodrug, can reduce the cytotoxicity of the anticancer drug. When the anticancer prodrug enters tumor tissue, the thioimine bond is induced by radiation to break and activate the anticancer drug, releasing it and restoring the original antitumor activity of the anticancer drug.

[0011] In a second aspect, this application provides a compound. According to embodiments of this application, the compound has the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: AB(I); Wherein, group A has the structure shown in formula (Ia): (Ia); R1 is selected from C 1~6 Alkyl, C 1~6 Halogenated alkyl groups, or 5-8 membered aromatic groups; R2 is selected from H, halogens, and C. 1~6 Alkyl, C 1~6 Halogenated alkyl, or C 1~6 oxyalkyl; R a - is an anion; Group B is an anticancer drug group, and the S in group A is linked to the amino group in group B through a thioimine bond.

[0012] The compound described in this application masks the cytotoxicity of anticancer drugs through its A-group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drugs through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, especially achieving a high radiolysis yield of over 240 nM / Gy. Therefore, this compound can be used as a radiotherapy-responsive drug for cancer treatment, enhancing the efficacy of radiotherapy and chemotherapy while reducing cytotoxicity.

[0013] In a third aspect of this application, a method for preparing the compound described in the second aspect is provided. According to embodiments of this application, the method includes: subjecting 10-methylphenothiazine to an oxidative click reaction with an anticancer drug to obtain the compound; wherein the anticancer drug contains an amino group. The method of this application, by subjecting 10-methylphenothiazine to an oxidative click reaction with an anticancer drug, can obtain the aforementioned compound in one step, and its synthetic method is simple and has low production costs.

[0014] In a fourth aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises the compound described in the second aspect or a compound prepared according to the method described in the third aspect. As is known before, the aforementioned compound masks the cytotoxicity of anticancer drugs through its A group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drug through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, particularly achieving a high radiolysis yield of over 240 nM / Gy. Therefore, a drug containing the aforementioned compound can be used as a radiotherapy-responsive drug for treating cancer, enhancing the therapeutic effect of radiotherapy and chemotherapy, and possessing advantages such as reduced cytotoxicity.

[0015] In a fifth aspect of this application, the use of the compound described in the second aspect, the compound prepared according to the method described in the third aspect, or the pharmaceutical composition described in the fourth aspect as an anticancer prodrug is proposed. This application discovers that an anticancer prodrug can be synthesized by coupling 10-methylphenothiazine with an anticancer drug. This anticancer prodrug can reduce the cytotoxicity of the anticancer drug. When the anticancer prodrug enters tumor tissue, it releases the anticancer drug through radiation-induced cleavage and activation of the thioimine bond, restoring the antitumor activity of the original anticancer drug.

[0016] In a sixth aspect of this application, the use of the compound described in the second aspect, the compound prepared according to the method described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a drug for treating cancer is disclosed. The compound described in this application shields the cytotoxicity of anticancer drugs through its A group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drug through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, particularly achieving a high radiolysis yield of over 240 nM / Gy. Therefore, this compound can be used as a radiotherapy-responsive drug for treating cancer, enhancing the efficacy of radiotherapy and chemotherapy, and possessing advantages such as reduced cytotoxicity.

[0017] In a seventh aspect of this application, a method is provided for activating the compound described in the second aspect or the compound prepared according to the method described in the third aspect. According to an embodiment of this application, the method includes: irradiating the compound to release an anticancer drug from the compound. According to an embodiment of this application, the anticancer drug is released by radiation-induced thioimine bond cleavage activation, restoring the antitumor activity of the original anticancer drug, particularly achieving a high radiolysis yield of over 240 nM / Gy.

[0018] In its eighth aspect, this application discloses a method for treating cancer. According to embodiments of this application, the method comprises: administering to a subject a pharmaceutically acceptable dose of the compound described in the second aspect, a compound prepared according to the method described in the third aspect, or a pharmaceutical composition described in the fourth aspect, and irradiating the subject with an effective dose of radiation. The compound of this application's method exhibits low cytotoxicity prior to activation, and releases an anticancer drug through radiation-induced thioimine bond cleavage activation, restoring the original anticancer drug's antitumor activity, thereby effectively treating cancer.

[0019] Beneficial effects: 1. Thioimine-protected drug molecules exhibit excellent radioresponsiveness, with radiolysis yields generally exceeding 260 nM / Gy, enabling efficient release of the drug molecules upon radiation activation. Specifically, the results of Example 3 of this application demonstrate that thioimine protection can reduce the cytotoxicity of MMAE molecules by 770-fold, making them relatively cell-friendly under unactivated conditions; simultaneously, only a dose of 1 Gy is required to fully activate the drug molecules, achieving the same cytotoxicity as free drug molecules.

[0020] 2. Thioimine-protected drug molecules are characterized by their simple synthesis. Compared with the complex synthesis steps of other radiation-activated drugs, thioimine-protected drug molecules use an oxidative click reaction to obtain the target product in one step. The pure product can be obtained by precipitation only during separation.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The main products of water irradiated by gamma rays and their radiolysis yield.

[0023] Figure 2The radiation-induced thioimine protection SFI-1 in Example 1 of this application includes (a) the ¹H NMR spectra of the reactants and products of SFI-1 after dose gradient irradiation, (b) the radiation dose-dependent release of 10-methylphenothiazine (PTZ) is linearly correlated with a radiation dose of 270.9 nM / Gy (0-500 Gy) (R²=0.99), and (c) a comparison of radiolysis yields of various radiation-induced release systems.

[0024] Figure 3 Model molecules of thioimines protected by different substrates in Example 2 of this application and their radiolytic yields; Figure 4a This is a structural diagram of the MMAE molecule protected by thioimine in Example 3 of this application; Figure 4b The NMR spectrum of the MMAE molecule protected by thioimine in Example 3 of this application; Figure 4c The NMR C-spectrum of the MMAE molecule protected by thioimine in Example 3 of this application; Figure 5a This is a structural diagram of the nitrogen mustard molecule protected by thioimine in Example 3 of this application; Figure 5b The NMR spectrum of the nitrogen mustard molecule protected by thioimine in Example 3 of this application; Figure 5c The NMR C-spectrum of the nitrogen mustard molecule protected by thioimine in Example 3 of this application; Figure 6a This is a structural diagram of the thioimine-protected ubenimex molecule in Example 3 of this application; Figure 6b The NMR spectrum of the thioimine-protected ubenimex molecule in Example 3 of this application; Figure 6c Mass spectrometry of the thioimine-protected ubenimex molecule in Example 3 of this application; Figure 7a This is a high-resolution mass spectrometry image of the MMAE molecule protected by thioimine in Example 3 of this application; Figure 7b The high-resolution mass spectra of the thioimine-protected nitrogen mustard molecule of the thioimine-protected MMAE molecule in Example 3 of this application are shown. Figure 8 This is a verification result of the activation of the thioimine-protected MMAE molecule in Example 3 of this application, utilizing both hydrated electrons and hydroxyl radicals. Figure 9 This refers to the radiation-induced activation results of the thioimine-protected MMAE molecules in Example 3 of this application; wherein, Figure 9 b- Figure 9 e-use 60Co was used as the radiation source, with a radiation dose rate of 4.0 Gy / min. -1 All cell experiments were incubated for 36 hours after irradiation; Figure 9 c and Figure 9 In e, the scale bar is 25 μm; the fluorescent dyes used, and the fluorescence excitation wavelength (Ex) and emission wavelength (Em) are as follows: Hoechst 33342: Ex: 408 nm, Em: 460 nm; DCFH-DA: Ex: 488 nm, Em: 525 nm; Tubulin-tracker green: Ex: 495 nm, Em: 519 nm. Detailed Implementation

[0025] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0027] It should be noted that the structural and chemical formula descriptions in the embodiments or implementations of this application are intended to cover all alternatives, modifications, and equivalent technical solutions, all of which are within the scope of this application as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice this application. This application is by no means limited to the methods and materials described herein. In the event that one or more of the cited documents, patents, and similar materials differ from or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0028] It should be further appreciated that some features of this application, for clarity, have been described in multiple independent embodiments or implementations, but may also be provided in combination in a single embodiment or implementation. Conversely, various features of this application, for the sake of brevity, have been described in a single embodiment or implementation, but may also be provided individually or in any suitable sub-combination.

[0029] Unless otherwise stated, the technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and unless otherwise stated, all patent publications cited in the entirety of this application are incorporated herein by reference.

[0030] This application will apply the following definitions unless otherwise indicated. For the purposes of this application, chemical elements are defined according to the periodic table, CAS version, and Chemical Handbook, 75, 1994. Furthermore, general principles of organic chemistry are found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007; therefore, all contents of this application incorporate the references.

[0031] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0032] In this document, the compounds of this application also include isotopically labeled compounds of this application that are identical to those compounds described herein except that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those of naturally common atomic masses or mass numbers. Exemplary isotopes that may also be introduced into the compounds of this application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as... 2 H, 3 H, 13 C 14 C 15 N、 16 O、 17 O、 31 P, 32 P, 36 S, 18 F and 37 Cl.

[0033] Compounds of this application containing other isotopes of the aforementioned isotopes and / or other atoms, as well as pharmaceutically acceptable salts of said compounds, are included within the scope of this application. Isotope-labeled compounds of this application, such as radioactive isotopes, are also included. 3 H and 14 The incorporation of tritium into the compounds of this application can be used for drug and / or substrate tissue distribution analysis. Due to its ease of preparation and detection, tritium-substituted compounds... 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred. In addition, heavier isotopes, such as deuterium, are used. 2H substitution can offer therapeutic advantages stemming from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements. Therefore, it may be preferred in some cases.

[0034] In this document, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated.

[0035] In this document, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the compounds of this application. Pharmaceutically acceptable salts are well known in the art. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts and organic acid salts formed by reaction with amino groups, or obtained by other methods described in the literature, such as ion exchange.

[0036] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0037] In this article, the term "halogen" refers to a fluorine, chlorine, bromine, or iodine atom.

[0038] In this paper, the minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes, for example, prefix C. a~b This refers to a carbon atom, which is "a" to "b". For example, "C 1~n "C" refers to a saturated / unsaturated carbon chain, either straight or branched, containing 1, 2, 3, 4, 5, ..., or n carbon atoms; further understanding, "C" 1~n "Should be interpreted as any subranges included, such as C" 1~6 In, containing C 1~5 C 1~4 C 1~3 C 1~2 C 2~6 C 2~5 C 2~4 C 2~3 C 3~6 C 3~5 C 3~4 C 4~6 C 4~5 C 1~3 In, containing C 1~3 C 1~2 C 2~3 .

[0039] In this document, the term "alkyl" refers to a straight-chain or branched saturated monovalent hydrocarbon group having a carbon atom, such as C1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, C 1~3 Alkyl, C 2~5 Alkyl, C 2~4 Alkyl, C 2~3 Alkyl groups. These include, but are not limited to, methyl, ethyl, n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), and n-pentyl (-CH2CH2CH2C). H2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl ( -CH2CH2CH2CH2CH2CH3), 2-hexyl(-CH(CH3)CH2CH2CH2CH3), 3-hexyl(-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl(-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl(-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl(-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl(-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl(-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl(-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl(-CH(CH3)C(CH3)3), wherein the alkyl groups may be independently unsubstituted or substituted by one or more substituents described in this application.

[0040] In this document, the term "oxoalkyl" refers to an alkyl group containing the formula "-O-alkyl", where the term "alkyl" is as defined above. Examples include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentoxy, isopentoxy, and n-hexyloxy, or isomers of the above groups. In particular, the "C1-10 alkoxy" may contain 1, 2, 3, 4, 5, or 6 carbon atoms ("C1-10 alkoxy"). 1~6 Alkoxy group), preferably, may contain 1, 2, 3 or 4 carbon atoms ("C"). 1~4(alkoxy group).

[0041] In this document, the term "halogenated alkyl" refers to an alkyl group containing halogen, and the term "alkyl" is as defined above.

[0042] In this document, the term "aromatic" or "aryl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic system containing 5-8 ring atoms, wherein at least one ring system is aromatic. The aryl group is usually, but not necessarily, linked to the parent molecule through the aromatic ring of the aryl group. The term "aromatic" or "aryl" may be used interchangeably with the term "aromatic ring" or "aromatic ring". Examples of aryl groups may include, but are not limited to, phenyl.

[0043] In this paper, the thioimine bond refers to -S-NR1R2-.

[0044] The "group description in this application" "" is used to describe the position of the substituent group.

[0045] In this application, the chemical structure of the compound contains the bond " "" indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the bond " "can be " "", ", or both contain " "and" "Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as: tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers."

[0046] In the chemical structure of the ligands or compounds described in this disclosure, the bond " "" indicates that the configuration is not specified. If cis-trans isomerism exists in the chemical structure, the bond " The configuration of “” can be E type, Z type, or both E and Z types.

[0047] In this document, the term "pharmaceuticalally acceptable excipient" includes any solvent, salt, pharmaceutical stabilizer, or combination thereof, all of which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where any conventional excipient is incompatible with the active ingredient, it covers its use in therapeutic or pharmaceutical compositions.

[0048] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The compounds or pharmaceutical compositions of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the compositions of this application are administered via intravenous or subcutaneous injection.

[0049] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0050] In this document, the terms "cancer" or "tumor" can refer to any unregulated cell growth. Examples include, but are not limited to, non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer, etc.

[0051] This application discloses the use of 10-methylphenothiazine as a toxicity-masking molecule for anticancer drugs, a thioimine-protected compound, its preparation method, and its use, which will be described in detail below.

[0052] Uses of 10-methylphenthiazide In a first aspect of this application, the use of 10-methylphenothiazine as a toxicity-masking molecule for anticancer drugs is proposed, wherein the sulfur (S) of the 10-methylphenothiazine is linked to the nitrogen (N) of the anticancer drug via a thioimine bond. This application discovers that by coupling 10-methylphenothiazine with an anticancer drug, the synthesized compound, as an anticancer prodrug, can reduce the cytotoxicity of the anticancer drug. When the anticancer prodrug enters tumor tissue, the thioimine bond is induced by radiation to break and activate the anticancer drug, releasing it and restoring the original antitumor activity of the anticancer drug.

[0053] According to an optional embodiment of this application, the anticancer drug is selected from at least one of MMAE, MMAF, nitrogen mustard, and bestatin.

[0054] According to an optional embodiment of this application, the nitrogen mustard drug is selected from di(2-chloroethyl)amine.

[0055] In this paper, the structural formula of ubenmethoxam is: . compound In a second aspect, this application provides a compound. According to embodiments of this application, the compound has the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: AB(I); Wherein, group A has the structure shown in formula (Ia): (Ia); R1 is selected from C 1~6 Alkyl, C 1~6 Halogenated alkyl groups, or 5-8 membered aromatic groups; R2 is selected from H, halogens, and C. 1~6 Alkyl, C 1~6 Halogenated alkyl, or C 1~6 oxyalkyl; R a - is an anion; Group B is an anticancer drug group, and the S in group A is linked to the amino group in group B through a thioimine bond.

[0056] The compound described in this application masks the cytotoxicity of anticancer drugs through its A-group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drugs through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, especially achieving a high radiolysis yield of over 240 nM / Gy. Therefore, this compound can be used as a radiotherapy-responsive drug for cancer treatment, enhancing the efficacy of radiotherapy and chemotherapy while reducing cytotoxicity.

[0057] In this article, "anticancer drug group" refers to the group formed after the primary or secondary amine in the anticancer drug loses H after coupling with 10-methylphenothiazine.

[0058] According to embodiments of this application, the above-mentioned compound may further include at least one of the following technical features: According to an optional embodiment of this application, R1 is selected from C. 1~3 Alkyl or phenyl.

[0059] According to an optional embodiment of this application, R1 is selected from methyl.

[0060] According to an optional embodiment of this application, R2 is selected from H, Cl, and C. 1~3 Alkyl, C 1~3 Halogenated alkyl, or C 1~3 Oxyalkyl groups.

[0061] According to an optional embodiment of this application, R2 is selected from H.

[0062] According to an optional embodiment of this application, R1 is selected from C. 1~3 Alkyl groups and R2 are selected from H, Cl, and C. 1~3 Alkyl, C 1~3 Halogenated alkyl, or C 1~3 Oxyalkyl groups.

[0063] According to an optional embodiment of this application, R1 is selected from C. 1~3 Alkyl groups and R2 are selected from H.

[0064] According to an optional embodiment of this application, R1 is selected from methyl and R2 is selected from H.

[0065] According to an optional embodiment of this application, R1 is selected from phenyl, and R2 is selected from H, Cl, and C. 1~3 Alkyl, C 1~3 Halogenated alkyl, or C 1~3 Oxyalkyl groups.

[0066] According to an optional embodiment of this application, R1 is selected from phenyl and R2 is selected from H.

[0067] According to an optional embodiment of this application, R a - Selected from Br-, Cl-, or PF6-.

[0068] According to an optional embodiment of this application, R a - Selected from Br-.

[0069] According to an optional embodiment of this application, group A has the following structure: , , , , .

[0070] According to an optional embodiment of this application, the anticancer drug group is derived from MMAE, MMAF, nitrogen mustard, and ubenmethoxazole.

[0071] According to an optional embodiment of this application, the anticancer drug group is selected from... , , ,or .

[0072] According to an optional embodiment of this application, the compound has the following structure: , , , , , , , , , , , , , , , , , , , .

[0073] Preparation methods of compounds In a third aspect of this application, a method for preparing the compound described in the second aspect is provided. According to embodiments of this application, the method includes: subjecting 10-methylphenothiazine to an oxidative click reaction with an anticancer drug to obtain the compound; wherein the anticancer drug contains an amino group. Compared to the complex synthetic steps of other radiation-activated drugs, the method of this application, by subjecting 10-methylphenothiazine to an oxidative click reaction with an anticancer drug, can obtain the aforementioned compound in one step, and its synthetic method is simple and has low production costs.

[0074] According to embodiments of this application, the above-mentioned compound may further include at least one of the following technical features: According to an optional embodiment of this application, the oxidant for the oxidative click reaction is selected from at least one of N-bromosuccinimide, N-chlorosuccinimide, N-chlorophthalimide, dibromohydantoin, N-iodosuccinimide, diiodohydantoin, and selective fluorine reagents.

[0075] According to an optional embodiment of this application, the solvent for the oxidative click reaction is selected from at least one of acetonitrile, water, dichloromethane, tetrahydrofuran, isopropanol, n-propanol, hexafluoroisopropanol, ethyl acetate, dioxane, N,N-dimethylacetamide, N,N-dimethylformamide, carbon tetrachloride, and chloroform.

[0076] According to an optional embodiment of this application, the solvent for the oxidative click reaction is selected from acetonitrile, water, dichloromethane, tetrahydrofuran, isopropanol, n-propanol, hexafluoroisopropanol, ethyl acetate, dioxane, N,N-dimethylacetamide, N,N-dimethylformamide, carbon tetrachloride, chloroform, or a mixture of at least two of these.

[0077] According to an optional embodiment of this application, the temperature of the oxidative click reaction is 0°C to 80°C (e.g., 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or any two of these values), and the time is 0.5h to 10h (e.g., 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h or any two of these values).

[0078] According to an optional embodiment of this application, the method further includes: extracting the oxidative click reaction product to obtain a crude product of the compound.

[0079] According to an optional embodiment of this application, the extractant for the extraction process is ethyl acetate.

[0080] According to an optional embodiment of this application, the method further includes: purifying the crude product of the compound to obtain the compound. Thus, only precipitation is required to obtain the pure product, making the operation simple.

[0081] Pharmaceutical Composition In a fourth aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises the compound described in the second aspect or a compound prepared according to the method described in the third aspect. As is known before, the aforementioned compound masks the cytotoxicity of anticancer drugs through its A group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drug through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, particularly achieving a high radiolysis yield of over 240 nM / Gy. Therefore, a drug containing the aforementioned compound can be used as a radiotherapy-responsive drug for treating cancer, enhancing the therapeutic effect of radiotherapy and chemotherapy, and possessing advantages such as reduced cytotoxicity.

[0082] According to embodiments of this application, the above-described pharmaceutical composition may further include at least one of the following technical features: According to embodiments of this application, the pharmaceutical composition further includes pharmaceutically acceptable excipients, carriers, and mediators.

[0083] In one optional embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients that are conventional in the pharmaceutical field, such as absorption enhancers, isotonic agents, stabilizers, regulators, etc.

[0084] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field.

[0085] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water).

[0086] In one alternative embodiment of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0087] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0088] The pharmaceutical composition of this application can be administered via various routes (e.g., orally or intravenously). Preferably, the pharmaceutical composition of this application is in solution form. Clinical dosing regimens are determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including patient size, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered medications. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously. The pharmaceutical composition of this application can also be administered directly to the target site, for example, by targeted delivery to internal or external target sites.

[0089] Uses of compounds In a fifth aspect of this application, the use of the compound described in the second aspect, the compound prepared according to the method described in the third aspect, or the pharmaceutical composition described in the fourth aspect as an anticancer prodrug is proposed. This application discovers that an anticancer prodrug can be synthesized by coupling 10-methylphenothiazine with an anticancer drug. This anticancer prodrug can reduce the cytotoxicity of the anticancer drug. When the anticancer prodrug enters tumor tissue, it releases the anticancer drug through radiation-induced cleavage and activation of the thioimine bond, restoring the antitumor activity of the original anticancer drug.

[0090] According to an alternative embodiment of this application, the compound masks the toxicity of the anticancer drug B group through the A group in formula (I).

[0091] In a sixth aspect of this application, the use of the compound described in the second aspect, the compound prepared according to the method described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a drug for treating cancer is disclosed. The compound described in this application shields the cytotoxicity of anticancer drugs through its A group, thereby reducing the cytotoxicity of the anticancer drugs. When the compound enters tumor tissue, it releases the anticancer drug through radiation-induced thioimine bond cleavage and activation, restoring the original anticancer drug's antitumor activity, particularly achieving a high radiolysis yield of over 240 nM / Gy. Therefore, this compound can be used as a radiotherapy-responsive drug for treating cancer, enhancing the efficacy of radiotherapy and chemotherapy, and possessing advantages such as reduced cytotoxicity.

[0092] According to an optional embodiment of this application, the drug activates its anticancer activity through radiation.

[0093] According to an optional embodiment of this application, the radiation yield of the drug is greater than 240 nM / Gy.

[0094] Methods of activating compounds In a seventh aspect of this application, a method is provided for activating the compound described in the second aspect or the compound prepared according to the method described in the third aspect. According to an embodiment of this application, the method includes: irradiating the compound to release an anticancer drug from the compound. According to an embodiment of this application, the anticancer drug is released by radiation-induced thioimine bond cleavage activation, restoring the antitumor activity of the original anticancer drug, particularly achieving a high radiolysis yield of over 240 nM / Gy.

[0095] According to an optional embodiment of this application, the radiation dose of the radiation irradiation treatment is 1 Gy to 60 Gy, for example, 1 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy.

[0096] Methods of treating cancer In its eighth aspect, this application discloses a method for treating cancer. According to embodiments of this application, the method comprises: administering to a subject a pharmaceutically acceptable dose of the compound described in the second aspect, a compound prepared according to the method described in the third aspect, or a pharmaceutical composition described in the fourth aspect, and irradiating the subject with an effective dose of radiation. The compound of this application's method exhibits low cellular activity prior to activation; by inducing thioimine bond cleavage and activation through radiation, it releases an anticancer drug, restoring the original antitumor activity of the anticancer drug, thereby effectively treating cancer.

[0097] According to an optional embodiment of this application, the radiation dose of the radiation irradiation treatment is 1 Gy to 60 Gy.

[0098] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0099] The effective amount of the compound described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0100] The compounds or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in various forms, such as liquids. Various routes of administration of the compounds, pharmaceutical compositions, or pharmaceuticals of this application are contemplated, including intravenous, intramuscular, and subcutaneous injection, but this application is not limited to these exemplified routes of administration.

[0101] According to embodiments of this application, the disease includes tumors.

[0102] According to embodiments of this application, the disease includes solid tumors or hematologic malignancies.

[0103] According to embodiments of this application, the diseases include breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, or leukemia.

[0104] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0105] Example 1: Preparation and detection of a model molecule protected by thioimine 1. Preparation of model molecules protected by thioimine The model molecule SFI-1 was prepared based on n-butylamine, 10-methylphenthiazide and N-bromosuccinimide (NBS).

[0106] 10-Methylphenothiazine (213.28 mg, 1 mmol) and n-butylamine (73.14 mg, 1 mmol) were dissolved in 10 mL of acetonitrile. NBS (177.99 mg, 1 mmol) was added at 60 °C, and the mixture was stirred for 4 hours while maintaining the temperature. The mixture was then evaporated to dryness to obtain the crude product. The crude product was separated by column chromatography to obtain SFI-1.

[0107] 1 H NMR (500 MHz, Acetonitrile-d3) δ 8.05 (dd, J = 7.9, 1.6 Hz, 2H), 7.87 (ddd, J = 8.8, 7.2, 1.6 Hz, 2H), 7.62 (dd, J = 8.8, 1.0 Hz, 2H), 7.44(ddd, J = 8.2, 7.2, 1.0 Hz, 2H), 6.73 (t, J = 5.7 Hz, 1H), 3.79 (s, 3H), 2.45(td, J = 6.8, 5.7 Hz, 2H), 1.27 – 1.15 (m, 2H), 1.13 – 1.01 (m, 2H), 0.67 (t,J = 7.3 Hz, 3H). 2. Determination of the radiolysis yield of the model molecule The radiation-induced controlled release of a model molecule protected by thioimine was verified using quantitative NMR spectroscopy. Specifically: A certain concentration of thioimine-protected drug molecules was dissolved in heavy water. At a radiation dose rate of 4.0 Gy / min and a total dose of 600 Gy, parallel experiments were conducted at 100 Gy intervals. After irradiation, a certain amount of dibromomethane was added to the system as an internal standard. The amount of thioimine-protected drug molecules released under radiation-induced radiation was calculated by the ratio of the NMR integrals of the product and the internal standard. Furthermore, the radiolysis yield was calculated using linear fitting. The results are as follows: Figure 2 As shown.

[0108] like Figure 2As shown in Figure a, with increasing irradiation dose, the peaks of SFI-1 in the 7.3–8.0 ppm and 0.56 ppm regions gradually disappeared. Simultaneously, peaks attributed to 10-methylphenthiazide (PTZ) gradually appeared in the 6.8–7.3 ppm region, and peaks attributed to n-BuNH2 methyl groups appeared in the 0.84 ppm region. After irradiation at 600 Gy, the peaks of SFI-1 completely disappeared, and only peaks attributed to PTZ and amines were produced. This indicates that SFI-1 is completely selectively converted to PTZ and nBuNH2. Figure 2 b shows that the released products were found to be dose-dependent, proportional to the radiation dose, and could produce a radiolytic yield of 270.9 nM / Gy. Figure 2 c shows that the radiolysis yield is the highest among direct radiation-induced bond-breaking systems, at 270.9 nM / Gy. Notably, the radiolysis yield of hydrated electrons is 272.6 nM / Gy, and the radiolysis yield of hydroxyl radicals is 281.9 nM / Gy. The radiolysis yield of SFI-1 is close to that of these primary reactive species.

[0109] Compared to conventional radiation-induced release systems that rely on multi-step elimination pathways (such as 1,4 or 1,6 elimination), the thioimine-modified drug in this application exhibits a real-time response without the need for post-irradiation incubation. These results demonstrate that SFI-1 undergoes efficient and selective bond breaking under ionizing radiation with a real-time response and clean decomposition in PTZ and n-BuNH2.

[0110] Example 2: Expansion of substrates in thioimine-protected model molecules To explore the scope of radiation-induced thioimine bond-modified model molecules, thioimine bond-modified model molecules with different substrates were synthesized. The specific synthesis methods are detailed in Examples 1-1, differing only in the substrate molecules. Following the methods in Examples 1-2, dose-dependent irradiation was applied to the thioimine bond-modified model molecules with different substrates to determine their radiolytic yields. Specifically, the thioimine bond-modified model molecules with different substrates were dissolved in 150 μM D₂O at room temperature and under nitrogen atmosphere, respectively. 60 The Co source produced gamma rays at a dose rate of 4.0 Gy / min and a total dose of 600 Gy (see [link to source]). Figure 3 a), the results show (see Figure 3 (b) At a concentration of 150 μM, all thioimine bond-modified model molecules could be quantitatively converted (>99%) after 600 Gy irradiation. Specifically, such as... Figure 3As shown in b, model molecules modified with thioimine bonds derived from 10-methylphenothiazine and various amines (including primary and secondary amines) exhibit high radiolytic yields of nearly 270 nM / Gy; model molecules modified with thioimine bonds containing aromatic rings such as benzene, substituted benzene, and thiophene also exhibit high radiolytic yields of 260.2 to 268.2 nM / Gy.

[0111] Example 3: Preparation and detection of thioimine-protected drug molecules 1. Preparation of drug molecules protected by thioimine 1.1 Preparation of MMAE molecules protected by MMAE, 10-methylphenthiazide, N-bromosuccinimide (NBS), and ammonium hexafluorophosphate with thioimine. 10-Methylphenothiazine (213.0 mg, 1.0 mmol) and MMAE molecules (717.98 mg, 1.0 mmol) were dissolved in 10 mL of acetonitrile. NBS (178.0 mg, 1.0 mmol) was added at 60 °C. After stirring for 4 hours while maintaining the temperature, 40 mL of ethyl acetate was added to the mixed solution. The mixture was then centrifuged to obtain the crude product containing thioimine-protected MMAE molecules.

[0112] The crude product was dissolved in deionized water, and the supernatant was collected after centrifugation. A 1 mM deionized aqueous solution of ammonium hexafluorophosphate was added to the supernatant, resulting in a large precipitate. The precipitate was separated by centrifugation, yielding pure thioimine-protected MMAE molecules (structure shown in Figure 1). Figure 3 (As shown).

[0113] The structure of the drug molecule protected by the thioimine bond was characterized as follows: the prepared complex was dissolved in a deuterated NMR solvent, and its structure was characterized by 1H NMR and 1C NMR spectroscopy, followed by electrospray ionization mass spectrometry. The characterization results are shown in Figure 4. Figure 7a As shown, in the 1H NMR spectrum, the hydrogen atoms in the MMAE region shift, and characteristic peaks of thioimine molecules appear; high-resolution mass spectrometry shows the formation of molecules with the mass number of thioimine. The results are: HRMS m / z (ESI): calculated for (C 52 H 77 N6O7S)+: 929.5569; found:929.5561. 1.2 Preparation of MMAE molecules protected by nitrogen mustard, 10-methylphenthiazide, N-bromosuccinimide (NBS), and ammonium hexafluorophosphate with thioimine. The specific preparation method is described in section 1.1 above, with the only difference being that MMAE is replaced with nitrogen mustard. The structure of the drug molecule protected by the thioimine bond was characterized using the methods described in section 1.1 above, and the characterization results are shown in Figure 5. Figure 7bThe result is: 1 H NMR (600 MHz, DMSO-d6) δ 8.33 (dd, J = 8.0, 1.6 Hz, 2H), 7.96 (ddd, J = 8.7, 7.1, 1.6 Hz, 2H), 7.83 (dd, J = 8.8, 1.0 Hz, 2H), 7.53 (ddd, J= 8.1, 7.1, 1.0 Hz, 2H), 3.84 (s, 3H), 3.54 (t, J = 6.0 Hz, 4H), 3.36 (t, J =6.0 Hz, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 143.16, 136.68, 133.24, 124.28, 118.08,107.40, 52.75, 41.94, 36.51. HRMS m / z (ESI): calculated for (C 17 H 19 N2SCl2) + : 353.0641; found:353.0641. 1.3 Preparation of MMAE molecules protected by ubenimex, 10-methylphenthiazide, N-bromosuccinimide (NBS), and ammonium hexafluorophosphate with thioimine. The specific preparation method is described in section 1.1 above, with the only difference being the replacement of MMAE with ubenimex. The structure of the drug molecule protected by the thioimine bond was characterized using the same method described in section 1.1 above, and the characterization results are shown in Figure 6. The results are as follows: HRMS m / z (ESI): calculated for (C29H34N3O4S)+: 520.2270; found:520.2268. 2. Verification of the simultaneous utilization of hydrated electrons and hydroxyl radicals by drug molecules protected by thioimine. The protection of thioimine-protected drug molecules by quencher experiments was verified using both hydrated electrons and hydroxyl radicals. Specifically, a certain concentration of thioimine-protected drug molecules was dissolved in D₂O, and a corresponding active species quencher (sodium nitrate as the hydrated electron quencher) was added. - aq DMSO (a hydroxyl radical quencher) was used to characterize the release of the prodrug after the addition of the quencher using quantitative NMR 1H2S spectroscopy. The results are as follows: Figure 8 As shown.

[0114] The results showed that the addition of sodium nitrate completely inhibited the reaction (radiolysis yield = 0), while the addition of dimethyl sulfoxide reduced the radiolysis yield by about half, reaching 144.3 nM / Gy. These results indicate that hydrated electrons are indispensable for the activation of thioimine-protected drug molecules, and that this activation reaction also utilizes hydroxyl radicals.

[0115] 3. Apoptosis assay of drug molecules protected by thioimine To achieve controlled activation of MMAE in living cells, SFI-MMAE was synthesized; see details below. Figure 9 a.

[0116] To evaluate the cytotoxicity of free MMAE, SFI-MMAE, and irradiated SFI-MMAE, in vitro cell viability was assessed in 4T1 cells using the MTT assay. Specifically: Cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and incubated at 37 °C with 5% CO₂ for 24 h. Cells were then treated with different concentrations of SFI-MMAE and free MMAE for 12 h each. Cells were then irradiated with 0–20 Gy and incubated for another 36 h. Afterward, 10 μL of MTT solution (0.5 mg / mL) was added to each well and the cells were treated for another 4 h, followed by dissolution with 100 μL of DMSO for 10 min. Finally, the absorbance of each well was measured at 570 nm using a microplate reader (BioTek Epoch, USA). The results are shown below. Figure 9 As shown in b. The results showed that the toxicity of thioimine-masked MMAE molecules was reduced by 770-fold, from 7.1 nM to 5.5 uM. After receiving the usual single dose of radiotherapy (1gy), the MMAE prodrug was activated to release MMAE, and the toxicity increased to 9.5 nM.

[0117] Furthermore, the effect of this prodrug release on the cell cycle was analyzed by flow cytometry, and the results were as follows: Figure 9 As shown in Figure c. The results indicate that radiation-induced SFI-MMAE and free MMAE induced similar cell cycle effects, with more cells arresting in the G2 / M phase. In the untreated group, the 1 Gy γ-ray group and the unirradiated SFI-MMAE group showed that more cells arrested in the G1 phase. These results further support the feasibility of in situ activation of SFI-MMAE in living cells.

[0118] Furthermore, to verify the activation of SFI-MMAE in living cells, the production of reactive oxygen species (ROS) in the aforementioned 4T1 cells was monitored as an indicator of DNA damage. The results are as follows: Figure 9As shown in d. The results showed that both free MMAE and 1 Gy irradiated SFI-MMAE triggered strong ROS fluorescence, while the control groups (untreated control group, 1 Gy γ-ray group only, and SFI-MMAE group only) showed minimal ROS signal, indicating that the activation process was functioning well in the cell.

[0119] Furthermore, to demonstrate the damage of released MMAE to tubulin, the damage to tubulin in the aforementioned 4T1 cells under different treatment conditions was evaluated, and the results are as follows: Figure 9 As shown in Figure e. The results indicated that microtubule damage was observed only in the MMAE treatment group and the 1 Gy SFI-MMAE irradiation group, where the cytoskeleton shape changed from filamentous to a circular damage-associated shape. In contrast, no significant changes in cytoskeleton shape were observed in the control group, the unirradiated SFI-MMAE group, and the group receiving only 1 Gy irradiation.

[0120] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0121] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Use of 10-methylphenothiazine as a toxicity-masking molecule for anticancer drugs, wherein the S of the 10-methylphenothiazine is linked to the amino group of the anticancer drug by forming a thioimine bond.

2. The use according to claim 1, characterized in that, The anticancer drug is selected from at least one of MMAE, MMAF, nitrogen mustard, and ubenmexico.

3. A compound, characterized in that, The compound has the structure shown in formula (I) or a pharmaceutically acceptable salt thereof: AB(I); Wherein, group A has the structure shown in formula (Ia): (It); R1 is selected from C 1~6 Alkyl, C 1~6 Halogenated alkyl groups, or 5-8 membered aromatic groups; R2 is selected from H, halogens, and C. 1~6 Alkyl, C 1~6 Halogenated alkyl, or C 1~6 oxyalkyl; R a - is an anion; Group B is an anticancer drug group, and the S in group A is linked to the amino group in group B through a thioimine bond.

4. The compound according to claim 3, characterized in that, The compound satisfies one or more of the following conditions: 1) R1 is selected from C 1~3 Alkyl or phenyl; 2) R2 is selected from H, Cl, C 1~3 Alkyl, C 1~3 Halogenated alkyl, or C 1~3 oxyalkyl; 3) R a - Selected from Br-, Cl-, or PF6-; Optional, R a -Selected from Br-; 4) Group A has the following structure: 、 、 、 、 ； 5) The anticancer drug group is derived from MMAE, MMAF, nitrogen mustard, or ubenimex; 6) The anticancer drug group is selected from... , , ,or .

5. The compound according to claim 3, characterized in that, The compound has the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 6. A method for preparing the compound according to any one of claims 3 to 5, characterized in that, include: The compound was obtained by oxidative click reaction of 10-methylphenothiazine with an anticancer drug. The anticancer drug contains amino groups.

7. The method according to claim 6, characterized in that, The method satisfies one or more of the following conditions: 1) The oxidant in the oxidative click reaction is selected from at least one of N-bromosuccinimide, N-chlorosuccinimide, N-chlorophthalimide, dibromohydantoin, N-iodosuccinimide, diiodohydantoin, and selective fluorine reagents; 2) The solvent for the oxidative click reaction is selected from at least one of acetonitrile, water, dichloromethane, tetrahydrofuran, isopropanol, n-propanol, hexafluoroisopropanol, trifluoroethanol, ethyl acetate, dioxane, N,N-dimethylacetamide, N,N-dimethylformamide, carbon tetrachloride, and chloroform. 3) The temperature of the oxidation click reaction is 0℃~80℃ and the time is 0.5h~10h; 4) Further includes: extracting the product of the oxidative click reaction to obtain a crude product of the compound.

8. The method according to claim 7, characterized in that, The method satisfies one or more of the following conditions: 1) The extractant used in the extraction process is ethyl acetate; 2) Further includes: purifying the crude product of the compound to obtain the compound.

9. A pharmaceutical composition, characterized in that, include: The compound according to any one of claims 3 to 5 or the compound prepared by the method according to any one of claims 6 to 8, and optionally pharmaceutically acceptable excipients, carriers, or mediators.

10. Use of the compound according to any one of claims 3 to 5, the compound prepared by the method according to any one of claims 6 to 8, or the pharmaceutical composition according to claim 9 as an anticancer prodrug; Optionally, the compound masks the toxicity of the anticancer drug B group through the A group in formula (I).

11. Use of the compound according to any one of claims 3 to 5, the compound prepared by the method according to any one of claims 6 to 8, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for the treatment of cancer; Optionally, the drug's anticancer activity is activated by radiation; Optionally, the radiation yield of the drug is greater than 240 nM / Gy.

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