Radiation-activatable compound and use thereof

Abstract

A radiation-activatable compound, a drug conjugate thereof and the use thereof, and the use of the compound as a drug conjugate linking group.

Description

Radiation-activatable compounds and their uses Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a radiation-activatable compound, a drug conjugate thereof, and applications thereof in treating or diagnosing diseases, such as treating or diagnosing cancer. Background Art

[0002] More than 50% of cancer cases require radiotherapy (or "radiation"), which uses ionizing radiation to kill cancer cells. Ionizing radiation, including X-rays and gamma rays, has high tissue penetration and low scattering properties. Modern radiotherapy techniques enable tumor targeting with high temporal and spatial resolution, delivering high radiation doses to tumors while minimizing damage to healthy tissue surrounding the tumor. The high clinical relevance, high tissue penetration, and high tumor targeting of radiotherapy make X-rays (or gamma rays) the optimal exogenous stimulus for controlled drug release strategies.

[0003] However, although strategies to activate prodrugs using radiotherapy have been reported in the 1990s, their application in tumors has been limited by efficiency or chemoselectivity. In tumors, radiotherapy primarily ionizes water rather than prodrug molecules because their concentrations are significantly different. The main products of water radiolysis are hydroxyl radicals (·OH), hydrogen radicals (·H), and hydrated electrons (e - aq ).

[0004] In recent years, a series of ·OH, ·H or e - aq Radiation-mediated removal of the group (Figure 1) can be used for prodrug activation in tumors. Notably, the strategy of radiotherapy-activated prodrugs has been rapidly applied to drug development in different fields, including PROTACs, drug conjugates, etc.

[0005] Radiation-induced prodrug activation is an ideal approach to reduce the systemic toxicity of chemotherapy in cancer treatment. Despite numerous efforts, maintaining the stability of prodrugs prior to irradiation while improving the efficiency and selectivity of radiation activation remains an unresolved challenge.

[0006] Summary of the Invention

[0007] The inventors of the present invention have creatively developed a radiation-removable protecting group (RPG) through unremitting efforts that is stable before radiation and has high radiation activation efficiency, and have provided a new strategy for the effective and rational design of radiation-activated prodrugs.

[0008] Specifically, the present invention provides a radiation-activated compound, a drug conjugate thereof, and uses thereof, wherein the compound or drug conjugate is activated by radiation to achieve high-efficiency and high-selectivity controlled release, thereby obtaining a new RPG category.

[0009] In one aspect, the present invention provides a radiation-activatable compound for use as a linker group for an ADC, and the corresponding ADC. The compound or ADC can be cleaved by radiation to release the drug or fluorescent molecule at the tumor site, thereby providing a possible path for the development of drugs that produce diagnostic or therapeutic effects through radiation activation.

[0010] Furthermore, the present invention provides a radiation-activatable compound, its use as a drug conjugate linker, and its corresponding drug conjugate. The compound or drug conjugate has good stability and high reactivity in biological systems, and its excellent performance in anti-tumor treatment also provides new insights into synchronous chemoradiotherapy.

[0011] Furthermore, the present invention provides a radiation-activatable compound and its corresponding drug conjugate, which exhibits strong responsiveness under physiological conditions.

[0012] In another aspect, the present invention also provides a method for activating the compound or drug conjugate using radiation.

[0013] In yet another aspect, the present invention provides a method for treating or diagnosing a disease, comprising administering to a subject a radiation-activatable compound or drug conjugate of the present invention.

[0014] In yet another aspect, the present invention also provides use of the compound or drug conjugate for the preparation of a medicament for treating or diagnosing a disease in a subject, wherein the subject is also irradiated.

[0015] In another aspect, the present invention also provides use of the compound or drug conjugate for preparing a combination drug for treating or diagnosing a disease in an individual, wherein the combination drug comprises the compound or drug conjugate and radiation.

[0016] In one aspect, the present invention provides a radiation-activatable compound, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound has a structure of Formula (I) or Formula (II):

[0017] Its characteristics are:

[0018] A is CR 1 , N or NR 2 , and only one A in formula (I) is N or NR 2, in formula (II), only one A is N or NR 2 The carbon atoms on the aromatic ring in formula (I) or formula (II) are optionally replaced by R 1 Substituted, where R 1 Selected from the group consisting of -H and electron-withdrawing groups, preferably selected from the group consisting of -H, -NO2, -CN, and halogen, more preferably selected from the group consisting of -H, -NO2, -CN, and -F;

[0019] When A is NR 2 When R 2 is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2 Optionally substituted with a linker group comprising one or more groups that are or are derived from: amino, amide, heterocyclyl, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid;

[0020] B is any therapeutic agent or chromogenic agent that can be coupled to the aryl ester moiety of the compound, as long as it can be separated from the aryl ester moiety under the action of radiation and release a therapeutically active or chromogenic molecule, for example, B is a fluorescent group, or a drug group, preferably a drug group for treating cancer, more preferably a chemotherapeutic drug group;

[0021] R 3 is selected from the group consisting of -H and electron-donating groups, preferably selected from the group consisting of -H, alkyl and aryl, more preferably selected from the group consisting of -H, C 1-6 Alkyl, C 6-10 The group consisting of an alkyl group and an aryl group, wherein the alkyl group and the aryl group are optionally substituted.

[0022] In the compound of formula (I) or (II), the other parts (aryl ester group part) except part B are radiation-responsive groups, that is, preferably in the presence of water, after being irradiated, the aryl ester group in the compound of formula (I) or (II) can be removed and B is released.

[0023] A preferred embodiment of the present invention relates to a radiation-activatable compound, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound has a structure of one of the following formulae:

[0024] wherein the carbon atoms on the aromatic ring are optionally replaced by R 1 Replacement, R 1 、R 2 、R 3 and B have the meanings as previously stated,

[0025] Preferably, in the presence of water, upon irradiation, the aryl ester group in the compound can be removed and B is released.

[0026] B in the compound of formula (I) or (II) can be any chromogenic agent or therapeutic agent that can be coupled to the aryl ester group portion as shown, as long as it can be separated from the aryl ester group portion under the action of radiation and release a therapeutically active or chromogenic molecule. In the present disclosure, the released therapeutically active or chromogenic molecule is generally the same structure as B in the compound of formula (I) or (II). In some embodiments, the released therapeutically active or chromogenic molecule has a slightly different structure from B in the compound of formula (I) or (II), that is, the therapeutic agent or chromogenic molecule undergoes a chemical reaction under the action of radiation but does not significantly affect its therapeutic activity or chromogenic properties.

[0027] Suitable molecules that can be used as part B include, but are not limited to, polypeptides, oligopeptides, peptidomimetics, amino acids, enzyme inhibitors, hormones, toxins, antibiotics, anti-inflammatory substances, fluorescent molecules, and the like.

[0028] In a preferred embodiment, the molecule corresponding to the B component is a therapeutic agent for treating cancer.

[0029] In one embodiment, the present disclosure uses a drug containing a primary amine or a secondary amine as a molecule corresponding to part B, wherein part B is connected to an aryl ester group portion (radiation response group) through an optionally substituted N atom. In a preferred embodiment, the present disclosure uses an anticancer drug containing a primary amine or a secondary amine as a molecule corresponding to part B. Drugs containing primary or secondary amines include, for example, ibrutinib, acalabrutinib, zanubrutinib, doxorubicin, mitomycin-C, mitomycin-A, daunorubicin, aminopterin, actinomycin, bleomycin, 9-aminocamptothecin, N8-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazide, yunnanmycin, gemcitabine, cytarabine, dolastatin, dacarbazine, 5-fluorouracil and derivatives thereof. Drugs containing primary or secondary amines also include amino derivatives of drugs that do not naturally contain amino groups. In other words, drugs that do not originally contain an amino group can be chemically modified to have an amino group, and then coupled to a radiation-responsive group using the primary or secondary amine coupling method described in the present disclosure.

[0030] In one embodiment, the present disclosure uses a hydroxyl-containing drug as the molecule corresponding to part B, wherein part B can be connected to the aryl ester group portion (radiation responsive group) through an O atom. In a preferred embodiment, the present disclosure uses a hydroxyl-containing anticancer drug as the molecule corresponding to part B. Hydroxyl-containing therapeutic agents include, for example, paclitaxel, docetaxel, gemcitabine, cytarabine, etc. Hydroxyl-containing molecules also include hydroxyl derivatives of drugs that do not naturally contain hydroxyl groups. In other words, drugs that originally do not contain hydroxyl groups can be chemically modified to have hydroxyl groups, and then coupled to the radiation responsive group through the hydroxyl coupling method described in the present disclosure.

[0031] In one embodiment, the present disclosure uses a thiol-containing drug as the molecule corresponding to part B, wherein part B can be connected to the aryl ester group portion (radiation responsive group) through an S atom. In a preferred embodiment, the present disclosure uses a thiol-containing anticancer drug as the molecule corresponding to part B. Examples of molecules corresponding to part B containing thiol groups include 6-mercaptopurine and the like. Thiol-containing molecules also include thiol derivatives of drugs that do not naturally contain thiol groups. In other words, drugs that originally do not contain thiol groups can be chemically modified to have thiol groups, and then coupled to the radiation responsive group through the thiol coupling method described in the present disclosure.

[0032] A preferred embodiment of the present invention relates to a radiation-activatable compound as described above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein:

[0033] R 1 All are -H,

[0034] R 2 If present, it is methyl, which is optionally substituted with a linking group, and / or

[0035] R 3 For -H,

[0036] The linking group has the meaning as described above.

[0037] A preferred embodiment of the present invention relates to a radiation-activatable compound as described above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein:

[0038] One or more R in formula (I) or formula (II) 1 Selected from the group consisting of -NO2, -CN, -F,

[0039] R 2 If present, it is methyl, and / or

[0040] R 3 is -H.

[0041] A preferred embodiment of the present invention relates to a radiation-activatable compound as described above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein

[0042] R 1 All are -H,

[0043] R 2 If present, it is methyl, and / or

[0044] R 3 C 1-3 Alkyl or phenyl.

[0045] A preferred embodiment of the present invention relates to the radiation-activatable compound as described above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein B is coumarin and its derivatives, rhodamine and its derivatives, or auristatin and its derivatives.

[0046] A preferred embodiment of the present invention relates to the radiation-activatable compound as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein B is selected from the group consisting of the following structures:

[0047] in represents the site where B is connected to the ester group in formula (I) or (II);

[0048] The ring carbon atoms in formula (B1), formula (B2) and formula (B3) may be optionally replaced by -R 5 、-NR 6 R 7 replace;

[0049] E is NR 4 Group or O, preferably NR 4 group;

[0050] R 8 NR 9 R 10 OR 9 ;

[0051] When B is auristatin and its derivatives, B is 4 The group is connected to the ester group in formula (I) or (II);

[0052] R 4 is -H or alkyl, preferably -H or C 1-6 Alkyl, more preferably -H or C 1-4 alkyl;

[0053] R 5 、R 6 、R7 、R 9 and R 10 The groups are each independently selected from H and alkyl, the alkyl being preferably C 1-6 Alkyl, more preferably C 1-4 alkyl;

[0054] In formula (B4), R 12 to R 23 Each independently selected from H and alkyl, the alkyl is preferably C 1-6 Alkyl, more preferably C 1-4 alkyl;

[0055] R 24 is selected from H, hydroxy, carboxyl, alkyl, alkoxy, halogen, aryl or heteroaryl, wherein the aryl is preferably C 6- 10 Aryl, the heteroaryl is preferably C 5-10 heteroaryl;

[0056] R 25 Selected from H, hydroxy, alkyl, alkoxy, halogen;

[0057] R 26 is selected from aryl or heteroaryl, wherein the aryl or heteroaryl is optionally further substituted by a substituent selected from H, halogen, hydroxyl, alkyl, alkoxy, and the aryl is preferably C 6-10 Aryl, the heteroaryl is preferably C 5-10 Heteroaryl.

[0058] A preferred embodiment of the present invention relates to the radiation-activatable compound as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein B is selected from the group consisting of the following structures:

[0059] where R 5 、R 6 、R 7 、R 9 、R 13 to R 17 、R 22 The groups are each independently C 1-6 Alkyl, more preferably C 1-4 Alkyl, more preferably R 6 and R 7 is ethyl, R 5 and R 9 is methyl;

[0060] R 24 Selected from H, hydroxyl, carboxyl, C 1-6 Alkyl, C 1-6 Alkoxy, halogen, C6-10 Aryl and C 5-10 Heteroaryl; R 25 Selected from H, hydroxyl, C 1-6 alkyl;

[0061] A preferred embodiment of the present invention relates to a radiation-activatable compound or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the radiation-activatable compound is selected from the following group:

[0062] where R 2 C 1-6 alkyl.

[0063] In another aspect, the present invention relates to the use of the radiation-activatable compound as described above or its pharmaceutically acceptable salt, solvate, or stereoisomer in the preparation of a drug conjugate, characterized in that only one A in formula (I) or formula (II) is NR 2 , R 2 is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2 Optionally substituted with a linker group comprising one or more groups that are or are derived from amino, amide, heterocyclyl, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid.

[0064] In a preferred embodiment, the present invention relates to the use as described above, wherein the drug moiety is via R 2 , or by R 2 The reaction is carried out to connect to formula (I) or formula (II), thereby preparing the drug conjugate.

[0065] In another aspect, the present invention relates to a drug conjugate, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein the drug conjugate comprises a targeting moiety, a linking moiety, and a drug moiety, wherein the linking moiety couples the targeting moiety and the drug moiety, and wherein the linking moiety comprises a structure of formula (I') or formula (II'):

[0066] Its characteristics are:

[0067] A is CR 1 or NR 2’ , and only one A in formula (I') is NR 2’ , in formula (II') only one A is NR 2’ The carbon atoms on the aromatic ring in formula (I') or formula (II') are optionally replaced by R 1 Substituted, where R1 Selected from the group consisting of -H and electron-withdrawing groups, preferably selected from the group consisting of -H, -NO2, -CN, and halogen, more preferably selected from the group consisting of -H, -NO2, -CN, and -F;

[0068] R 2’ is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2’ is substituted with a linking group that is attached to the targeting moiety;

[0069] B is a fluorescent group, or a drug group, preferably a chemotherapeutic drug group;

[0070] R 3 is selected from the group consisting of -H and electron-donating groups, preferably selected from the group consisting of -H, alkyl and aryl, more preferably selected from the group consisting of -H, C 1-6 Alkyl, C 6-10 The group consisting of an alkyl group and an aryl group, wherein the alkyl group and the aryl group are optionally substituted;

[0071] The targeting moiety is an antibody.

[0072] In a preferred embodiment, the present invention relates to the drug conjugate as described above, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the structure of formula (I') or formula (II') has one of the following structures:

[0073] wherein the carbon atoms on the aromatic ring are optionally replaced by R 1 Replacement, R 1 、R 2’ 、R 3 and B have the meanings as described above in 1.

[0074] In a preferred embodiment, the present invention relates to the drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein

[0075] R 1 Both -H, and / or

[0076] R 3 is -H.

[0077] In a preferred embodiment, the present invention relates to the drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein

[0078] One or more R in formula (I) or formula (II) 1 Selected from the group consisting of -NO2, -CN, -F, and / or

[0079] R3 is -H.

[0080] In a preferred embodiment, the present invention relates to the drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein

[0081] R 1 Both -H, and / or

[0082] R 3 C 1-3 Alkyl or phenyl.

[0083] In a preferred embodiment, the present invention relates to a drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein the linking group comprises one or more groups that are or are derived from the following: amino, amide, heterocyclic, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid.

[0084] In another aspect, the present invention relates to a method for activating a radiation-activatable compound or drug conjugate using radiation, characterized in that the radiation-activatable compound or drug conjugate is a compound or drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof.

[0085] In a preferred embodiment of the present invention, in the method, the radiation is high-energy radiation, preferably X-rays or gamma rays, more preferably the intensity of the X-rays or gamma rays is 60 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, 10 Gy or less, 8 Gy or less, 6 Gy or less and 4 Gy or less.

[0086] In another aspect, the present invention relates to a method for treating or diagnosing a disease, which comprises administering to an individual a radiation-activatable compound or drug conjugate, and irradiating the individual with a therapeutically effective amount, wherein the radiation-activatable compound or drug conjugate is a compound or drug conjugate as described above, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof.

[0087] In a preferred embodiment of the invention, the method is used to treat or inhibit, reduce the severity of, reduce the risk of, or inhibit the metastasis of cancer in an individual.

[0088] In a preferred embodiment of the present invention, the cancer is selected from breast cancer, colorectal cancer, lung cancer and gastric cancer.

[0089] In a preferred embodiment of the present invention, the subject is irradiated after administration of the compound or the drug conjugate, preferably 24 hours, 48 ​​hours, 96 hours, and / or 144 hours after administration of the compound or the drug conjugate.

[0090] In a preferred embodiment of the invention, the compound or the drug conjugate is administered in an amount of about 0.005 mg / kg to about 100 mg / kg, for example, a dose of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100 mg / kg.

[0091] In a preferred embodiment of the present invention, the compound or the drug conjugate and radiation are administered once a week, once every two weeks, once every three weeks, or once every four weeks, and the compound or the drug conjugate and radiation are administered continuously for at least 1 round, at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, or at least 10 rounds.

[0092] In a preferred embodiment of the present invention, the compound or the drug conjugate is administered parenterally, such as intravenously.

[0093] In a preferred embodiment of the present invention, the radiation is high-energy radiation, preferably X-rays or gamma rays, more preferably the intensity of the X-rays or gamma rays is 60 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, 10 Gy or less, 8 Gy or less, 6 Gy or less and 4 Gy or less.

[0094] Preferably, the radiation-activatable compound of the present invention has a structure selected from the group consisting of: BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The present invention will be further described below with reference to the accompanying drawings.

[0096] FIG1 shows the prior art of the method of using ·OH or e - aq Structures used in the radiation-induced prodrug activation strategy.

[0097] FIG2 shows the e-produced by water radiolysis of N-alkyl-pyridine (NAP) groups in the present invention. - aq Reduced, amino (or carboxyl)-containing fluorescent molecules or drugs can be activated in a spatiotemporal manner by radiotherapy in tumors.

[0098] Figure 3 shows that the pyridine group or NAP group was identified as a masking group that can be generated by water hydrolysis.- aq Easy to remove.

[0099] FIG4 shows the efficacy of compounds 1-9 in releasing coumarin: 1-9 (10 μM) were subjected to 60 Gy X-ray irradiation in PB (phosphate buffer, pH 7.4, 0.1% dimethyl sulfoxide as a cosolvent).

[0100] FIG5 shows the release efficiency of compound 7 after adding 10 mM HCOONa or KNO3 (10 μM, in PB, pH 7.4) to the irradiation system.

[0101] Figure 6 shows the energy released by the electrons captured by the NAP group during the reduction reaction (E C ) and the activation energy of free radical bond scission (E a ), when E C The smaller the size, the easier it is to bind hydrated electrons, and the smaller the activation energy, the easier it is for the CO bond to break. The calculation results also provide evidence for the efficiency of releasing reporter molecules after irradiation of different groups in the experiment.

[0102] FIG7 shows a schematic diagram of the radiation-induced reduction of N-alkyl-pyridine carbamate (NAPC) that can remotely release the "caged" amino molecule 7-amino-4-methylcoumarin (AMC).

[0103] Figure 8 shows the UPLC spectra of compound 10 (10 μM, 0.02 M PB, pH 7.4) subjected to a clinically relevant radiation gradient (0–60 Gy) at 25°C.

[0104] FIG9 shows the release of AMC versus radiation dose (0–60 Gy).

[0105] FIG10 shows the chemical structures of other compounds in the prior art.

[0106] Figure 11 shows the UPLC spectra of compound 10 mixed with compound 11 (10 μM 10 + 10 μM 11), compound 12 (10 μM 10 + 10 μM 12), and compound 13 (10 μM 10 + 10 μM 13), respectively: in PB (10 μM, 0.02 M PB, pH 7.4), with or without 60 Gy irradiation at 25°C.

[0107] FIG12 shows the percentage of competition reaction after compound 10 was mixed with compound 11 (10 μM 10+10 μM 11), compound 12 (10 μM 10+10 μM 12), and compound 13 (10 μM 10+10 μM 13).

[0108] FIG13 shows a schematic diagram of the radiation-induced reduction that releases the “fluorescent cage” MeRho from carbamate-containing NAPs.

[0109] FIG14 shows radiation dose dependence fluorescence imaging.

[0110] Figure 15 shows the fluorescence emission spectrum and UPLC spectrum of NAPC-MeRho (10 μM, 0.02 M PB, pH 7.4) after treatment with a radiation gradient, confirming that NAPC-MeRho produces MeRho in a radiation dose-dependent manner. The detector wavelength is 481 nm, which is the characteristic absorption wavelength of MeRho.

[0111] Figure 16 shows confocal fluorescence images. Cells were pretreated with NAPC-MeRho (10 μM, 0.1% DMSO) for 30 minutes under hypoxic conditions and then irradiated with different amounts of radiation. Scale bar, 50 μm.

[0112] FIG17 shows the fluorescence intensity of each single 4T1-FAP cell (left), HT1080-FAP cell (middle), and U87MG cell (right) in the confocal microscopy field: 10 cells were randomly selected for each experiment (two-tailed unpaired Student's t-test, ***P<0.001).

[0113] FIG18 shows representative confocal fluorescence images of tumor sections: scale bar, 100 μm.

[0114] FIG19 shows the mean fluorescence intensity of tumor sections: five randomly selected fields per experiment (two-tailed unpaired t-test, ***P<0.001) using BALB / c mice subcutaneously implanted with 4T1-FAP cells.

[0115] FIG20 shows the design of the NAPC-ADC conjugate: the NAPC linker is reduced to release the carbamate, which undergoes decarboxylation and returns to the highly toxic MMAE.

[0116] FIG21 shows the purity of NAPC-ADC conjugates determined by HIC HPLC (left) and MALDI-TOF analysis (right), respectively.

[0117] FIG. 22 shows drug release in a radiation dose gradient: [NPC-ADC] = 100 nM.

[0118] FIG23 shows a cell viability assay of 4T1-FAP cells treated with NAPC-ADC alone or in addition to X-ray treatment under hypoxic conditions.

[0119] Figure 24 shows the detection of α-tubulin immunofluorescence by confocal microscopy to determine whether NAPC-ADC + X-ray-generated MMAE destabilizes tubulin in the cytoskeleton of 4T1-FAP cells. Scale bar = 25 μm.

[0120] Figure 25 shows that radiotherapy-induced controlled release of NAPC-ADC triggers tumor regression in mice, i.v. 89 Dynamic positron emission tomography-computed tomography images of 4T1-FAP tumor-bearing mice at the indicated time points after [Zr]NAPC-ADC.

[0121] Figure 26 shows the difference between blood, muscle and tumor [ 89 Time activity curve (TAC) of Zr]NAPC-ADC.

[0122] Figure 27 shows the treatment regimen for 4T1-FAP tumor-bearing mice: 20 BABL / c mice were subcutaneously implanted with 4T1-FAP cells and randomly divided into 4 groups (n=5) and treated with PBS; NAPC-ADC (5 mg / kg, intravenous injection); radiotherapy as indicated; NAPC-ADC (5 mg / kg) and radiotherapy (4 Gy each time, once every two days).

[0123] FIG28 shows the tumor concentration of MMAE detected after mice were injected with NAPC-ADC (5 mg / kg) alone or in addition to X-ray injection (4, 8, 12, and 16 Gy).

[0124] FIG29 shows the tumor growth curves of tumor-bearing mice after treatment.

[0125] FIG30 shows the average tumor weight of tumor-bearing mice on day 21 after treatment.

[0126] FIG31 shows the body weight change curve of mice (***P<0.001, **P=0.0018, two-tailed unpaired t-test).

[0127] FIG32 shows the radiation release efficiency of Compound 1: liquid chromatography characterization data of the release of coumarin model molecules from Compound 1 at a concentration of 10 μM under 60 Gy irradiation conditions.

[0128] Figure 33 shows the irradiation release efficiency test under different redox conditions: (A) Liquid chromatography profiles of 10 μM compound 1 releasing coumarin model molecules under 20 Gy irradiation, 20 Gy + 10 mM KNO3, and 20 Gy + 10 mM HCO2Na. (B) The release efficiency of coumarin was 16.7% under irradiation conditions, 1.3% under potassium nitrate + irradiation, and 27.3% under sodium formate + irradiation.

[0129] FIG34 shows the irradiation release efficiency of compounds 2 to 9 (10 μM) under 60 Gy X-ray conditions.

[0130] Figure 35 shows the release of compound 7 under different irradiation doses: (A) liquid chromatograms at different doses; (B) statistical data of coumarin release under irradiation at different doses, showing a linear relationship; (c) UV-visible absorption spectra of compound 7 and released carboxycoumarin.

[0131] Figure 36 shows the release efficiency of compound 7 under different redox conditions: (A) UPLC chromatograms of 10 μM compound 7, 20 Gy + 10 mM KNO3, and 20 Gy + 10 mM HCO2Na after 20 Gy irradiation; (B) Under the control conditions, the coumarin release rate was 27.6%, the oxidizing KNO3 was 0.0%, and the reducing HCO2Na was 53.8%.

[0132] Figure 37 shows the release efficiency of compound 10 under different radiation doses: (A) UPLC-MS chromatogram of compound 10 release as the radiation dose increases (324 nm, characteristic absorption wavelength of compound 10); (B) UPLC-MS chromatogram of compound 10 release as the radiation dose increases (342 nm, characteristic absorption wavelength of coumarin); (C) The released concentration of coumarin is linearly related to the radiation dose; (D) UV-visible absorption spectra of compound 10 (NAPC-AMC) and coumarin (AMC).

[0133] Figure 38 shows the characteristics of pyridinium irradiation products: (A) 4-[(acetyloxy)methyl]-1-methylpyridine (Intermediate 1) produces 1,4-dimethylpyridine (Intermediate 2) after irradiation; (B) UPLC-MS chromatogram of 1 mm Intermediate 1 after 5000 gy x-ray irradiation; (C) Mass spectrometry signal of the retained peak of Intermediate 1; (D) Mass spectrometry signal of the retained peak of Intermediate 2.

[0134] FIG39 shows the in vitro stability of NAPC-MeRho: (A) NAPC-MeRho (10 μM) was treated with 100 e.q VcNa, pH 7.4; (B) Fluorescence intensity of released MeRho under different reducing conditions.

[0135] Figure 40 (A) Cell viability of 4T1-FAP cells treated with 10 μM NAPC-MeRho; (B) Cell viability of HT1080-FAP cells treated with 10 μM NAPC-MeRho; (C) Cell viability of U87-MG cells treated with 10 μM NAPC-MeRho.

[0136] FIG41 shows the cell viability of 4T1-FAP treated with MMAE and NAPC-ADC: IC of MMAE on 4T1-FAP 50 The IC value of NAPC-ADC for 4T1-FAP is 1.694nM. 50 It is 547.1nM.

[0137] Figure 42 shows in vitro immunofluorescence staining of NAPC-ADC. α-Tubulin immunodetection by confocal microscopy revealed that NAPC-ADC + X-ray-generated MMAE destabilizes tubulin in the cytoskeleton of HT1080-FAP cells. Scale bar = 25 μm.

[0138] FIG43 shows photographs of representative tumors at day 21 after different treatments.

[0139] Figure 44 shows the hematoxylin and eosin stained tissue sections of the major organs (heart, liver, spleen, lung, and kidney) of the different treatment groups. No obvious abnormalities were found in these major organs of the mice after treatment. DETAILED DESCRIPTION

[0140] The present invention provides a radiation-activated compound, a drug conjugate thereof and use thereof.

[0141] Before further describing the present invention, the following sections collect certain terms used in the specification, examples, and appended claims. The definitions listed herein should be read and understood by those skilled in the art in light of the remainder of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0142] definition

[0143] Unless otherwise indicated, when any type of range is disclosed or claimed, it is intended to disclose or claim individually every possible value that the range may reasonably encompass, including any subranges encompassed therein. For example, a radical number of 1 to 6 indicates an integer within the range, where 1-6 is understood to include 1, 2, 3, 4, 5, 6, and also includes subranges of 1-5, 1-4, and 1-3.

[0144] The description of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonding.In some cases, it may be possible to remove a hydrogen atom in order to accommodate a substituent at a given position.

[0145] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0146] The term "pharmaceutically acceptable" in this disclosure means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or with the human or mammal for which it is used to prevent or treat the disease or condition.

[0147] "Stereoisomers" refer to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0148] "Radiation" in the context of this disclosure refers to radiation other than visible light or ultraviolet light, preferably high energy radiation, such as preferably X-rays or gamma rays, for example any radiation used in radiotherapy.

[0149] When "about" is followed by a value or ratio, it means such value or ratio ±10%, preferably ±5%, more preferably ±1%.

[0150] The term "group" refers to the portion of a molecule formed by removing one, two, or more hydrogen radicals. Specifically, a "fluorescent group" or "drug group" refers to a group formed by removing one, two, or more hydrogen radicals from a fluorescent molecule or drug molecule commonly used in the art.

[0151] The expression "optionally substituted" means that one, two, three or more hydrogen atoms in the group may be replaced independently of one another by respective substituents. The substituents may be selected from alkyl, alkenyl, alkoxy, halogen, cyano, amino, nitro, -OH.

[0152] The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably 1 to 6 carbon atoms, most preferably 1 to 3 carbon atoms. The alkyl group is, for example, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, amyl or octyl. The alkyl group is optionally substituted.

[0153] The term "alkoxy" includes -O-alkyl groups and alkyl groups wherein the O atom is within the alkyl chain, for example -CH2-O-CH3, which contain 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and most preferably 1 to 3 carbon atoms. The alkoxy group is optionally substituted.

[0154] The term "alkenyl" includes both straight-chain and branched-chain alkyl groups containing at least two carbon atoms and at least one carbon-carbon double bond, and comprising from 2 to 10 carbon atoms, preferably from 2 to 6 carbon atoms, and most preferably from 2 to 3 carbon atoms. The alkenyl group is optionally substituted.

[0155] The term "alkynyl" refers to a group having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, and containing 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and most preferably 2 to 3 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3), and the like. The alkynyl group is optionally substituted.

[0156] The term "aryl" preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthyl or anthracenyl. Aryl is optionally substituted.

[0157] The term "heteroaryl" preferably refers to a five- or six-membered aromatic monocyclic ring in which at least one carbon atom is replaced by 1, 2, 3 or 4 (for a five-membered ring) or by 1, 2, 3, 4 or 5 (for a six-membered ring) identical or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system in which 1, 2, 3, 4, 5 or 6 of the 8, 9, 10, 11 or 12 carbon atoms are replaced by identical or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system in which 1, 2, 3, 4, 5 or 6 of the 13, 14, 15 or 16 carbon atoms are replaced by identical or different heteroatoms, preferably selected from O, N and S. The N atom present in the heteroaryl group may be present in the form of an onium group, for example, an alkyl group or an oxygen group is attached to the trivalent aromatic N to form an onium group, i.e., "heteroaryl" in the context of the present application includes heteroaryl groups in free form (N atom not in salt form) and heteroaryl groups in the form of onium salts. Examples are pyridyl, N-oxypyridyl, N-alkylpyridyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-phenylenediazole ... benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoloxazinyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, quinolyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0158] The term "heterocyclyl" or "heterocycle" refers to a non-aromatic or aromatic 5-, 6- or 7-membered ring or polycyclic group, including but not limited to a bicyclic or tricyclic group comprising a fused 6- or 7-membered ring, wherein at least one carbon atom of one ring is replaced by a heteroatom. Each heteroatom is independently selected from an atom of oxygen, sulfur (including sulfoxides and sulfones) and / or nitrogen (which may be oxidized or quaternized). The term "heterocyclyl", "heterocycle" or "heterocycle" is intended to include groups in which the -CH2- in the ring is replaced by -C(=O)-, such as cyclic urea groups (e.g., 2-imidazolidinone) and cyclic amide groups (e.g., β-lactam, γ-lactam, δ-lactam and ε-lactam) and piperazin-2-one; wherein (i) each 5-membered ring has 0 to 2 double bonds and each 6-membered ring has 1 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, and (iii) the nitrogen heteroatom may be optionally quaternized. Representative heterocycles include, but are not limited to, pyrrolyl, pyrrolidinyl, dioxopyrrolyl, pyrazolinyl, imidazolyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolidinyl, furanyl, and tetrahydrofuranyl.

[0159] The term "chemotherapeutic agent" or "chemotherapeutic drug" refers to chemotherapy drugs that can kill tumor cells. These drugs can act on different stages of tumor cell growth and proliferation, thereby inhibiting or killing tumor cells.

[0160] The term "amino acid" refers to any compound containing both an amino group and a carboxylic acid group. Although the amino group is most commonly found adjacent to a carboxyl functional group, the amino group can be located anywhere within the molecule. Amino acids can also contain additional functional groups such as amino, thio, carboxyl, carboxamide, imidazole, and the like. Amino acids can be synthetic or naturally occurring and can be used in their racemic or optically active (D- or L-) form.

[0161] The term "polyethylene glycol" refers to one or more repeating units of -CH2CH2O-.

[0162] The term "linking group" refers to any chemically suitable linking group. Preferably, the linking group does not break or only breaks slowly under physiological conditions.

[0163] The term "antibody" refers to an immunoglobulin molecule or its immunologically active portion that is bound to a specific antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen). In those embodiments where the targeting moiety is an antibody and the antibody is a full-length immunoglobulin molecule, the antibody includes two heavy chains and two light chains, each heavy chain and light chain comprising three complementary determining regions (CDRs). In those embodiments where the targeting moiety is an antibody and the antibody is an immunologically active portion of an immunoglobulin molecule, the antibody can be a single domain antibody (dAb), a double antibody, a three-antibody, a four-antibody, and the antibody used as the targeting moiety can be, for example, a natural antibody, a synthetic antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a multispecific antibody, a bispecific antibody, a dual-specific antibody, an anti-idiotypic antibody, or a fragment thereof that retains the ability to bind to a specific antigen.

[0164] The term "derivative" means that the skeletal structure is the same as the parent core structure, and the substituents on the skeleton may vary, for example, the ring atoms are replaced by alkyl, alkenyl, alkoxy, halogen, cyano, amino, alkylamino, nitro, or -OH, or the substituents on the skeleton can be alkyl, alkenyl, alkoxy, halogen, cyano, amino, alkylamino, nitro, or -OH.

[0165] The term "rhodamine" refers to a dye molecule having two backbone structures, wherein the ring atoms are optionally substituted.

[0166] The term "coumarin" refers to a dye molecule having the following backbone structure, wherein the ring atoms are optionally substituted.

[0167] The term "Auristatin" refers to a drug molecule having the following backbone structure, wherein the substituents R 11 to R 26 Each of them can independently be hydrogen, alkyl, alkenyl, alkoxy, halogen, hydroxy (—OH), carboxyl (—COOH), aryl, heteroaryl, cyano, amino, alkylamino or nitro.

[0168] The term "pharmaceutically acceptable salts" refers to relatively non-toxic addition salts of the compounds of the present disclosure. See, for example, SM Berge et al. "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.

[0169] Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be acid addition salts of compounds of the present disclosure having a nitrogen atom in a chain or ring with sufficient basicity, such as acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or acid addition salts formed with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-Hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

[0170] In addition, another suitable pharmaceutically acceptable salt of the compound of the present invention having sufficient acidity is an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt or a magnesium salt, an ammonium salt, a triethylamine salt, or a salt formed with an organic base which provides a physiologically acceptable cation, for example, a salt formed with the following substances: N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternized using reagents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides, such as benzyl and phenethyl bromides, etc.

[0171] Those skilled in the art will also recognize that acid addition salts of the claimed compounds can be prepared by reacting the compounds with a suitable inorganic or organic acid by any of a variety of known methods. Alternatively, alkali metal and alkaline earth metal salts of the acidic compounds of the present disclosure can be prepared by reacting them with a suitable base by various known methods.

[0172] The present invention includes all possible salts of the disclosed compounds, either as a single salt or as any mixture of such salts in any ratio.

[0173] The term "solvate" is a substance formed by combining, physically combining and / or solvating the compounds of the present invention with solvent molecules, such as a disolvate, a monosolvate or a hemisolvate, wherein the ratio of solvent molecules to the ratio of the compounds of the present invention is about 2:1, about 1:1 or about 1:2, respectively. This physical combination involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate can be separated. Therefore, solvates include solution phases and separable solvates. The compounds of the present invention can be in solvated form with pharmaceutically acceptable solvents (such as water, methanol and ethanol), and the application is intended to cover solvated and non-solvated forms of the compounds of the present invention. A solvate is a hydrate.

[0174] The term "halogen" refers to fluorine, chlorine, bromine, iodine and astatine.

[0175] The term "optionally" means that the situation may or may not occur.

[0176] The term "effective amount" refers to that amount of active ingredient which, upon administration, will relieve to some extent one or more of the symptoms of the disease being treated.

[0177] The term "subject" includes humans and non-human animals. Exemplary human subjects include human subjects suffering from diseases (e.g., the diseases described herein) (referred to as patients) or normal subjects. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0178] Example

[0179] Reagents and models used

[0180] The starting materials of the embodiments are commercially available and / or can be prepared by a variety of methods known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of experiment and aftertreatment) in the following synthetic methods. Those skilled in the art of organic synthesis will appreciate that the functional groups present in each part of the molecule should be compatible with the proposed reagents and reactions.

[0181] All reagents and compounds synthesized can be purchased through general commercial channels in China (excluding Hong Kong, Macao and Taiwan). Suppliers include J&K (China), Bidex Pharmaceuticals, Sinopharm Group, Beijing Chemical Plant, Yisheng Bio, and Abcam (ab64503).

[0182] Cell model: Mouse breast cancer 4T1 cell line transfected with fibroblast activation protein (4T1-FAP, WuXi AppTec, China) was grown in RPMI-1640 (Roswell Park Memorial Institute-1640) medium containing 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 2 μg / mL blasticidin S.

[0183] Human fibrosarcoma HT1080 cell line transfected with fibroblast activation protein (HT1080-FAP, WuXi AppTec, China) was grown in EME (Eagle's minimum essential medium) containing 10% FBS, 1% penicillin / streptomycin and 4 μg / mL blasticidin S.

[0184] The U87MG cell line was obtained from the American Type Culture Collection (ATCC) and was grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin.

[0185] All cell cultures were maintained at 37°C and 5% CO2.

[0186] Instruments used:

[0187] Nuclear magnetic resonance spectra were acquired using a Bruker AVANCE 400 MHz spectrometer and a Bruker AVANCE 500 MHz spectrometer.

[0188] Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using an ACQUITY UPLC H-Class PLUS instrument equipped with a Waters PDA eλ detector and a Waters ACQUITY QDA mass spectrometer.

[0189] High-resolution mass spectrometry was performed on a Bruker Fourier transform ion cyclotron resonance mass spectrometer.

[0190] The absorption spectrum was measured using a UV-1100 spectrophotometer.

[0191] Confocal fluorescence images were recorded on an A1R-si laser scanning confocal microscope (Nikon, Japan).

[0192] PET images were taken on a microPET (Mediso Medical Imaging Systems).

[0193] In order to make the purpose and technical scheme of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments.It should be understood that these examples are not intended to limit the scope of the present invention.In addition, the specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0194] As comparative examples, compounds 11, 12 and 13 were synthesized according to the following prior art documents:

[0195] [1] Sundararajan, C. & Falvey, DEC-O Bond Fragmentation of 4-Picolyl-and N-Methyl-4-picolinium Esters Triggered by Photochemical Electron Transfer.J.Org.Chem.69,5547-5554,doi:10.1021 / jo049501j(2004).

[0196] [2]Fu,Q.et al.External-Radiation-Induced Local Hydroxylation Enables Remote Release of Functional Molecules in Tumors.Angew.Chem.Int.Ed.59,21546-21552, doi:https: / / doi.org / 10.1002 / anie.202005612(2020).

[0197] Example 1: Synthesis of Compound 1

[0198] Synthetic routes of compounds 1 to 6

[0199] Pyridin-4-ylmethanol (109 mg, 1.0 mmol, 1.0 eq.), DCC (247 mg, 1.2 mmol, 1.2 eq.), and DMAP (12 mg, 0.1 mmol, 0.1 eq.) were dissolved in DCM (5 mL, anhydrous). 7-(diethylamino)-2-oxo-2H-1-chromene-3-carboxylic acid was added. The reaction mixture was stirred at room temperature for 4 hours and monitored by thin-layer chromatography (TLC). The reaction mixture was then washed with water and saturated sodium chloride solution and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by flash chromatography using DCM / MeOH (10:1) as the eluent. Compound 1, pyridin-4-ylmethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate, was obtained as a yellow powder in a yield of 63%.

[0200] 1 H NMR (400MHz, Methanol-d4) δ8.68–8.59(m,3H),7.75–7.69(m,2H),7.55(d,J=9.0Hz,1H),6.81(dd, J=9.1,2.5Hz,1H),6.55(d,J=2.4Hz,1H),5.46(s,2H),3.54(q,J=7.1Hz,4H),1.24(t,J=7.1Hz,6H);

[0201] 13 C NMR(151MHz,Methanol-d4)δ163.68,159.14,158.66,153.86,150.27,149.19,147.51 ,131.66,122.55,122.52,110.18,107.69,106.05,95.89,63.96,44.67,11.28,-1.44.

[0202] Example 2: Synthesis of Compound 2

[0203] Based on a synthetic method similar to Example 1, compound 2: pyridin-3-ylmethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate was obtained as a yellow powder in a yield of 75%.

[0204] 1H NMR (400MHz, DMSO-d6) δ8.99(s,1H),8.84(d,J=5.5Hz,1H),8.67(s,1H),8.54–8.51(m,1H),7.99–7.96(m,1H),7.66(d,J= 9.1Hz, 1H), 6.80 (dd, J = 9.1, 2.4Hz, 1H), 6.56 (d, J = 2.4Hz, 1H), 5.46 (s, 2H), 3.48 (q, J = 7.0Hz, 4H), 1.14 (t, J = 7.0Hz, 6H);

[0205] 13 C NMR(151MHz,DMSO-d6)δ163.63,158.75,157.60,153.62,150.56,149.95,143.71,143.17,143.00,135.92, 132.49,132.34,126.68,110.54,110.47,107.59,106.65,96.39,96.31,79.68,63.00,44.90,12.82,12.80

[0206] Example 3: Synthesis of Compound 3

[0207] Based on a synthetic method similar to Example 1, compound 3: pyridin-2-ylmethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate was obtained as a yellow powder in a yield of 61%.

[0208] 1 H NMR(400MHz, Methanol-d4)δ8.63(s,1H),8.54–8.52(m,1H),7.90–7.86(m,1H),7.65(d,J=7.8Hz,1H),7.51(d,J=9.0Hz,1H),7 .39–7.36(m,1H),6.78(dd,J=9.1,2.5Hz,1H),6.52(d,J=2.4Hz,1H),5.38(s,2H),3.52(q,J=7.1Hz,4H),1.23(t,J=7.1Hz,6H);

[0209] 13 C NMR (151MHz, Methanol-d4) δ164.59,160.88,159.27,158.42,153.62,149.83,147. 91,137.48,131.43,122.32,120.74,110.06,107.57,106.69,95.86,64.06,11.29.

[0210] Example 4: Synthesis of Compound 4

[0211] Based on a synthetic method similar to that of Example 1, compound 4: quinolin-4-ylmethyl-7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate was obtained as a yellow powder with a yield of 56%.

[0212] 1 H NMR (400MHz, DMSO-d6) δ8.93(d,J=4.4Hz,1H),8.68(s,1H),8.15(d,J=8.4Hz,1H),8.09(d,J=8.4Hz,1H),7.82(t,J=7.7Hz,1H), 7.74–7.64(m,3H),6.79(dd,J=9.0,2.4Hz,1H),6.57(d,J=2.3Hz,1H),5.84(s,2H),3.49(q,J=7.1Hz,4H),1.14(t,J=7.0Hz,6H);

[0213] 13 C NMR(151MHz,DMSO-d6)δ163.71,158.77,157.59,157.09,153.59,150.93,150.47,148.01,142.15,132.53,130.11,130 .01,127.44,125.76,124.20,119.74,110.42,107.63,106.89,96.32,63.00,47.97,44.89,33.83,25.80,24.94,12.82.

[0214] Example 5: Synthesis of Compound 5

[0215] Based on a synthetic method similar to that of Example 1, compound 5: 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid quinolin-4-ylmethyl ester was obtained as a yellow powder in a yield of 58%.

[0216] 1H NMR(400MHz,Chloroform-d)δ9.06(d,J=2.1Hz,1H),8.54(s,1H),8.46(s,1H),8.32(d,J=8.6Hz,1H),7.95(d,J=8.2Hz,1H),7.82(t,J=7.7Hz,1H),7.6 6(t,J=7.6Hz,1H),7.36(d,J=9.0Hz,1H),6.61(dd,J=9.1,2.4Hz,1H),6.46 (d,J=2.4Hz,1H),5.58(s,2H),3.45(q,J=7.1Hz,4H),1.24(t,J=7.1Hz,6H)

[0217] Example 6: Synthesis of Compound 6

[0218] Based on a synthetic method similar to that of Example 1, compound 6: 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid quinolin-2-ylmethyl was obtained as a yellow powder in a yield of 54%.

[0219] 1 H NMR(400MHz,Chloroform-d)δ8.55(s,1H),8.28(d,J=8.5Hz,1H),8.18(d,J=8.5Hz,1H),7.89–7.72(m,3H),7.58(t,J=7.5Hz,1H),7 .37(d,J=9.0Hz,1H),6.61(dd,J=9.0,2.5Hz,1H),6.48(d,J=2.4Hz,1H),5.70(s,2H),3.46(q,J=7.1Hz,4H),1.24(t,J=7.1Hz,6H).

[0220] Example 7: Synthesis of Compound 7

[0221] Compound 1 (100 mg, 0.284 mmol, 1.0 eq.) was dissolved in a 1:1 methanol-iodomethane mixture (5 mL). The reaction mixture was stirred at 80°C for 2 hours and then evaporated under reduced pressure. The crude product was purified by high-performance liquid chromatography using water / MeCN (70:30) as the eluent. Compound 7: 4-((7-(diethylamino)-2-oxo-2h-chromene-3-carbonyl)oxy)methyl-1-methylpyridin-1-ium was obtained as a yellow powder in a 90% yield.

[0222] 1H NMR (400MHz, DMSO-d6) δ9.03(d,J=6.4Hz,2H),8.75(s,1H),8.20(d,J=6.2Hz,2H),7.69(d,J=9.0Hz,1H),6.83(dd ,J=9.0,2.4Hz,1H),6.58(d,J=2.3Hz,1H),5.62(s,2H),4.36(s,3H),3.51(q,J=7.0Hz,4H),1.15(t,J=7.0Hz,6H).

[0223] Example 8: Synthesis of Compound 8

[0224] Based on a synthetic method similar to Example 7, compound 8: 3-((7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)oxy)methyl-1-methylpyridin-1-ium was obtained as a yellow powder in a yield of 93%.

[0225] 1 H NMR (400MHz, Methanol-d4) δ9.15 (s, 1H), 8.88 (d, J = 6.1Hz, 1H), 8.70 (d, J = 8.2Hz, 1H), 8.66 (s, 1H), 8.54 (s, 1H), 8.15-8.07 (m, 1H), 7.54 ( d, J=9.0Hz, 1H), 6.82 (dd, J=9.1, 2.5Hz, 1H), 6.55 (d, J=2.4Hz, 1H), 5.54 (s, 2H), 4.46 (s, 3H), 3.55 (q, J=7.1Hz, 4H), 1.24 (t, J=7.1Hz, 6H).

[0226] 13 C NMR (151MHz, DMSO-d6) δ163.44, 158.79, 157.57, 153.71, 150.70, 145.44, 144.66, 144.24, 137.55, 132.51, 127.96, 110.55, 107.57, 106.38, 96.32, 62.35, 48.64, 44.91, 44.44, 12.84, 12.82.

[0227] Example 9: Synthesis of Compound 9

[0228] Based on a synthetic method similar to Example 7, compound 9: (2-((7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)oxy)methyl-1-methylpyridin-1-ium) was obtained as a yellow powder in 89% yield.

[0229] Example 10: Synthesis of Compound 10

[0230] 7-Dimethylamino-4-methylcoumarin (1.75 g, 10 mmol, 1.0 eq.) and triphosgene (1.48 g, 5 mmol, 0.5 eq.) were dissolved in DCM (35 mL, anhydrous). DIPEA (3.87 g, 30 mmol, 3.0 eq.) was added under a N2 atmosphere. The reaction mixture was stirred for 30 minutes and became transparent. The mixture was used in the next step without purification. Pyridin-4-ylmethanol (10a) (1.20 g, 11 mmol, 1.1 eq.) was added. The reaction mixture was stirred at room temperature for 4 hours and then evaporated under reduced pressure. The crude product was purified by flash chromatography using DCM / MeOH (10:1) as the eluent to obtain compound 10b as a pink powder in a yield of 72%.

[0231] Compound 10b (100 mg, 0.322 mmol, 1.0 eq.) was dissolved in a 1:1 methanol-iodoethane mixture (5 mL). The reaction mixture was stirred at 65°C for 2 hours and then evaporated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) using water / MeCN (70:30) as the eluent to afford compound 10 as a pink powder in an 83% yield.

[0232] Example 11: Synthesis of Compound 11 (NAPC-MeRho)

[0233] Synthesis of NAPC-MeRho: MeRho (17) (1.75 g, 10 mmol, 1.0 eq.) and triphosgene (1.48 g, 5 mmol, 0.5 eq.) were dissolved in DCM (35 mL, anhydrous). DIPEA (3.87 g, 30 mmol, 3.0 eq.) was added under N2 atmosphere. The reaction mixture was stirred for 30 minutes and became transparent. The mixture was used in the next step without purification. 14 (1.20 g, 11 mmol, 1.1 eq.) The reaction mixture was stirred at room temperature for 4 hours and then evaporated under reduced pressure. The crude product was purified by column chromatography using DCM / MeOH (10:1) as the eluent. 15 was obtained as an orange powder. 18 (100 mg, 0.322 mmol, 1.0 eq.) was dissolved in a 1:1 mixture of methanol and iodoethane (5 mL). The reaction mixture was stirred at 65°C for 2 hours, evaporated under reduced pressure, and the crude product was purified by HPLC using water / MeCN (70:30) as eluent. NAPC-MeRho was an orange powder (yield: 69%).

[0234] Example 12: Synthesis of drug conjugate (ADC)

[0235] (1) Synthesis of Intermediate I: Dissolve 6-maleimidocaproic acid (840 mg, 4.0 mmol, 1.0 equivalent) and sodium acetate (720 mg, 8.8 mmol, 2.2 equivalent) in acetic acid (8 mL) in a tube at 0°C; add bromine (450 μL, 8.8 mmol, 2.2 equivalent) to the mixture and seal the tube. Stir at 135°C for 4 hours; cool to 0°C, add 10 mL of water to the mixture; extract the solution with ethyl acetate (3×15 mL); combine the organic phases and wash with sodium thiosulfate (2×15 mL); dry the reaction mixture with magnesium sulfate; concentrate the reaction mixture under reduced pressure; co-evaporate the residual acetic acid with toluene under reduced pressure; and purify the crude residue by flash chromatography to obtain Intermediate I.

[0236] (2) Synthesis of intermediate II: Pyridin-4-ylmethanol (2 g, 18.32 mmol, 1.0 equiv) and tert-butyl (3-bromopropyl)carbamate (5.24 g, 22.00 mmol, 1.2 equiv) were dissolved in 10 mL of MeCN; the reaction was monitored by thin layer chromatography (TLC); after complete consumption of the alcohol, the solid residue was discarded and the liquid residue was purified by flash chromatography to obtain intermediate II.

[0237] (3) Synthesis of Intermediate III: 5 mL of concentrated HCl was added to Intermediate II (1066 mg, 3.1 mmol) and stirred for 5 minutes; then, the reaction mixture was concentrated under reduced pressure and lyophilized.

[0238] (4) Synthesis of Intermediate IV: Intermediate III (800 mg, 2.2 mmol, 1.0 equiv), NaHCO₃ (370 mg, 4.4 mmol, 2.0 equiv), and Intermediate I (1000 mg, 2.6 mmol, 1.2 equiv) were dissolved in 20 mL of H₂O:DMSO = 1:1; the reaction was monitored by thin layer chromatography (TLC). After completion of the reaction, water and ethyl acetate were added to the reaction mixture. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Purification was performed using a reverse phase C-18 column.

[0239] (5) Synthesis of intermediate V: Intermediate IV (300 mg, 0.56 mmol, 1.0 equiv), DCM (20 mL) and DIPEA (0.30 mL, 1.68 mmol, 3.0 equiv) were prepared in a round bottom container equipped with a stirring bar; DSC (430 mg, 1.68 mmol, 3.0 equiv) was added to the reaction mixture, stirred at room temperature for 16 h, and the solvent was evaporated. The raw material was purified by reverse phase C-18 column chromatography to obtain intermediate V.

[0240] (6) Synthesis of compound VI: Intermediate product V (30 mg, 0.044 mmol, 1.0 equiv), DCM (2 mL) and DIPEA (7.7 μL, 0.044 mmol, 1.0 equiv) and MMAE (57 mg, 0.044 mmol, 1.0 equiv) were mixed in a round bottom container equipped with a stirring bar and stirred at room temperature for 16 h. The solvent was evaporated and the raw material was purified by reverse phase C-18 column chromatography to obtain product VI.

[0241] (7) The antibody (150 kDa) was dissolved in ADC buffer (pH 7.4) to obtain a 10 mg / mL (66 μM) protein solution, which was then reduced with 4.8 equivalents of TCEP at 37°C for 1 hour. Five equivalents of compound VI were then added, and the reaction was maintained at 37°C for 1 hour. Maleimide was quantitatively inserted into the disulfide bond to obtain the antibody-drug conjugate.

[0242] Example 13: Radiation Experiment

[0243] The compounds were irradiated using the following procedure:

[0244] Deoxygenation step

[0245] The pre-treated solution (about 1-2 ml) was sealed in a glass bottle with a rubber stopper and a metal cap. All tube radiation experiments were pre-treated by freeze-thaw method to remove dissolved oxygen from the solution. Freeze the solution and then vacuum for a few minutes until it thawed to room temperature.

[0246] Irradiation step

[0247] X-ray irradiation was performed using an X-ray generator (RS2000 Pro 225, 225 kV, 17.7 mA; Rad Source Technologies, Inc).

[0248] X-rays were used to irradiate the test tubes with a total dose of 0 to 60 Gy at a rate of 2 Gy / min. The cells were irradiated with different doses of X-rays at a rate of 2 Gy / min.

[0249] The local irradiation dose to the tumor was 4 Gy at a dose rate of 2 Gy / min, and the rest of the body was shielded with 5 mm thick lead.

[0250] Gamma ray irradiation by 60 Co source provided.

[0251] Use the following steps to perform the analysis:

[0252] Standard solutions and calibration curves

[0253] 1. Standard Preparation and Calibration Curve. Dissolve the standard (coumarin, MeRho, or MMAE) in DMSO to prepare a 10 mM stock solution. Dilute the stock solution with deionized water to obtain a solution with an appropriate concentration gradient. Analyze these standard solutions by UPLC-MS to generate calibration curves for each standard compound. The peak area of ​​the target compound should be within the linear range of the curve. The relative standard deviation of two injections of each standard solution should be less than 5.0%.

[0254] 2. Analysis and Calculation. After the solutions were treated differently, the reaction products were analyzed by ultra-high performance liquid chromatography-mass spectrometry. The quantity of the product was calculated based on the equivalent of its respective external standard.

[0255] Release of detection compounds

[0256] 1) Radiation

[0257] As a representative example, the detailed procedure for X-ray-induced reduction of compound 1 is described below. A stock solution of compound 1 (10 mM in DMSO) was diluted in PB (0.02 M, pH 7.4) to a final concentration of 10 μM. The solution was deoxygenated using the previously described deoxygenation procedure before X-ray irradiation of 0 to 60 Gy. The reaction mixture was analyzed by UPLC and fluorescence spectrometry, and the amount of product released was quantified using a standard curve.

[0258] 2) Use reducing solution

[0259] 20 μM NAPC-MeRho was mixed with equal volumes of 2 mM sodium ascorbate solution, incubated and detected at different time points using fluorescence spectrometry and UPLC-MS. Stability experiments in cysteine ​​and γ-glutathione solutions yielded similar results.

[0260] UPLC-MS method

[0261] The elution procedure for compounds 1-9 and NAPC-MeRho was as follows: 95% H2O + 5% ACN (0 min) → 95% H2O + 5% ACN (0.2 min) → 5% H2O + 95% ACN (2.8 min) → 5% H2O + 95% ACN (2.9 min) → 95% H2O + 5% ACN (3.0 min) → 95% H2O + 5% ACN (4.0 min). Injection volume: 10 μL.

[0262] Compound 10, a mixture of compound 7 and other RPGs, and a mixture of compound 10 and other RPGs were eluted using Method B: 95% H2O + 5% ACN (0 min) → 95% H2O + 5% ACN (0.2 min) → 70% H2O + 30% ACN (3.0 min) → 70% H2O + 30% ACN (3.6 min) → 95% H2O + 5% ACN (3.7 min) → 95% H2O + 5% ACN (5.0 min). Injection volume: 10 μL.

[0263] Example 13a: Product analysis confirmed that the CO bond of 4-pyridyl ester breaks after single-electron reduction

[0264] In compound 1, the marker molecule 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (7-Deacca) is blocked by a 4-pyridyl group.

[0265] 10 mM KNO3 (10 μM in PB, pH 7.4) was added to the irradiation system of Compound 1.

[0266] Reactivity (10 μM, 0.1% DMSO) test: The release of 7-Deacca in PB (20 mM, pH 7.4) after deoxygenation and 60 Gy X-ray irradiation was measured. Encouragingly, UPLC-MS detected the release of coumarin (Figure 32), with a release efficiency of 33.5 ± 9.1% (Figure 4), resulting in a value of 55.8 nM / Gy.

[0267] Compared with the control group, the release efficiency in the KNO3 solution of this embodiment is significantly reduced (Figures 5 and 33), indicating that when KNO3 quenches e - aq When, e - aq Plays a key role in the reaction.

[0268] Example 13b: Radiation reduction efficiency of RPG

[0269] 7-Deacca was designed to react with a series of molecules protected by pyridyl, quinolyl, and N-methylpyridyl groups, compounds 2-9. Stock solutions of compounds 2-9 (10 mM in DMSO) were diluted in PB (10 μM, 0.1% DMSO) and then irradiated with 60 Gy X-rays and analyzed by UPLC-MS to determine the yield of free 7-Deacca ( FIG. 35 ).

[0270] The inventors discovered that the amount of 7-Deacca released from compounds 1-9 is determined by the substitution position and electron density on the pyridine moiety. More specifically, ortho- and para-substituted esters (compounds 1, 3, 4, 6, 7, and 9) are more reactive to radiation reduction than meta-substituted esters (compounds 2, 5, and 8). The NAP esters of compounds 7 and 9 have particularly high release efficiency. When compound 7 was irradiated with X-rays in a dose gradient from 0 to 60 Gy, the release of the marker molecule was shown to be dose-dependent as detected by UPLC ( FIG. 37 ), with a G value of 140 nM / Gy.

[0271] Example 13c: Compound 7 is reduced via a radiation-mediated hydrated electron reduction mechanism

[0272] 10 mM HCOONa or KNO3 (10 μM, in PB, pH 7.4) was added to the radiation system of compound 7. The results of compound 7 and compound 1 in KNO3 solution were the same, and the release efficiency was significantly reduced (Figure 5 and Figure 36). Among them, the addition of HCOONa increased the reduction yield of compound 7 by about two times. This can be explained by the fact that HCOONa in the radicalized water reacts with ·OH and hydrogen radicals (·H) to generate carboxyl radicals (·CO 2- ), which is another reducing agent with a standard electrode potential of -1.90 V, which may also be a possible explanation for the difference in their radiation reduction efficiency. Compared with the quinolinyl moiety with a higher electron density (lower reduction potential), the positive charge on the pyridyl moiety with a lower electron density (higher reduction potential) has a negative effect on the electronegativity of the quinolinyl moiety. - aq It has a higher affinity, resulting in the highest radiation reduction efficiency of the pyridyl moiety.

[0273] Example 13d: RPG Adaptability

[0274] In addition to the carboxyl group, the amide group is another important moiety in many bioactive molecules because it can form hydrogen bonds with residues in the target binding pocket. Using an N-alkyl-4-pyridylcarbamate (NAPC) system (in which the carbamate carbon is linked to a labeling molecule), the inventors demonstrated that RPG is adaptable and releases functional molecules in their native and unmodified active form.

[0275] The inventors synthesized compound 10 (NAPC-AMC), containing NAP-protected 7-amino-4-methylcoumarin (AMC). Upon irradiation, this compound released AMC through C-O bond cleavage and decarboxylation in a dose-dependent manner (Figure 7) (Figure 8). The amount of AMC released was proportional to the radiation dose (Figure 9), indicating that the radiation reduction reaction occurred stoichiometrically (G = 145 nM / Gy).

[0276] Recently, there has been a boom in the development of radiotherapy-induced controlled release systems, such as o-carbamoylmethylene quaternary ammonium (Compound 11), azide (Compound 12), and N-oxide (Compound 13) systems (Figure 10). Although they are all capable of - aq receptors, but they are the same under the same conditions - aq The reaction capacity is different. Biological systems are complex and unpredictable, with numerous redox processes, and the radiation reduction reaction is very likely to be interfered with by oxidizing substances. A higher cross section of the radiation reduction reaction means a better ability to react. - aq , resulting in higher release efficiency in biological environments.

[0277] The inventors mixed the reported RPG with compound 10 to provide solutions of [10+11], [10+12], and [10+13], respectively (each component: 10 μM PB, pH 7.4). The solutions were deoxygenated and irradiated with 60 Gy of X-rays. The release efficiency was analyzed by UPLC-MS (Figure 11) and summarized in Figure 12. The lower and upper columns represent the activation ratios of compound 10 and the competing component in each irradiated system, respectively. Competitive inhibition experiments demonstrated that the NMP group is a more reactive single-electron reduction substrate than previously reported structures, generating 20-40% more at the same concentration.

[0278] Example 14: POC in cells and tumors

[0279] The inventors have demonstrated the proof-of-concept of a radiation-responsive fluorescent probe, NAPC-MeRho ( FIG. 13 ), designed for the quantitative analysis of radiation-induced release. The NAPC linker selectively responds to the e- - aq , where the CO bond of the carbamate is broken and then decarboxylated to release fluorescent MeRho (Figure 13). Under physiological conditions, 10μM NAPC MeRho was irradiated with a gradient dose ranging from 0 to 60Gy, resulting in a linear increase in fluorescence intensity (Figures 14 and 15), and the G value of MeRho release was determined to be 140nM / Gy by UPLC spectroscopy (Figure 15). To test whether the radiation reduction reaction can occur in living cells without interference, the stability to bioreductants and the cytotoxicity to different cell lines were evaluated. When treated with 100 equivalents of VcNa at pH 7.4, NAPC-MeRho (10μM) was highly stable, and the cell viability of 10μM NAPC-MeRho was greater than 95%.

[0280] Cells were typically seeded at a density of 2 × 10 cells / mL in a 35-mm confocal dish and cultured in an anoxic environment at 37°C for 24 h in an incubator containing 5% CO2. A 10 mM stock solution of NAPC-MeRho was prepared in DMSO. Adherent cells were washed twice with PBS before adding the fluorescent probe to a final concentration of 10 μM in 1 mL of deoxygenated HBSS. After incubation at 37°C for 30 min, cells were irradiated with X-rays for varying durations (0, 4, 8, and 16 Gy), and fluorescence images were captured using a laser scanning confocal microscope (Nikon, Japan). The excitation wavelength was 488 nm, and the fluorescence emission window was 500–530 nm. For image quantification, all cells in the image were designated as regions of interest, and fluorescence intensity was measured using Nikon Image software (version 4.0).

[0281] 4T1-FAP, HT1080-FAP and U87MG cell lines were incubated with 10 μM NAPC-MeRho for 30 minutes under hypoxia, showing that MeRho did not spontaneously release (Figure 16, left lane). Subsequently, 4, 8 and 16 Gy were irradiated, showing the appearance of green fluorescence released by MeRho (Figure 16, right lane), and the fluorescence intensity was positively correlated with the radiation dose (Figure 17). Under 4 Gy, 8 Gy and 16 Gy radiation, the fluorescence intensity of 4T1-FAP cells increased by 14.4-, 27.9 and 54.5 times, respectively (Figure 17, left panel). Similar experimental results were obtained in HT1080-FAP cells (Figure 17, middle panel) and U87MG cells (Figure 17, right panel), indicating that NAPC-MeRho is stable in mammalian cells and ready for radiation reduction reaction.

[0282] Then, when examining the reduction of NAPC-MeRho in tumor-bearing mice, 20 microliters of 100 μM NAPC-MeRho in PBS were locally injected into 4T1-FAP tumors and treated with 4 Gy of X-rays 30 minutes after injection. Due to the limited penetration of green light, the tumors were collected and quickly frozen, and the fluorescence intensity was quantified by confocal imaging. When treated with NAPC-MeRho alone, only a limited fluorescent signal was detected in the tumor sections (Figure 18, middle lane), which increased significantly when additional radiation was delivered (Figure 18, bottom lane). In addition, the MeRho signal was statistically greater than that in the non-irradiated group (Figure 19), providing conclusive evidence that the radiation-reduced response in vivo is feasible. The inventors have successfully demonstrated that radiation activation of fluorescent probes with NAPC structures is feasible in both cells and tumors, and has suitable stability and reactivity in complex biological environments.

[0283] Cell viability was assessed using the CCK-8 assay. Each experiment was repeated three times. To test the cytotoxicity of MMAE and NAPC-ADC, 4T1-FAP cells were seeded at a concentration of 5 × 10⁴ / mL in 96-well plates in 200 μL of RMI-1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. The cells were incubated at 37°C in a 5% CO₂ incubator for 24 hours. The cells were then incubated with various concentrations of MMAE and NAPC-ADC for 72 hours. A blank culture medium containing a final concentration of 0.5 mg / mL CCK-8 was then added. The cells were incubated at 37°C in 5% CO₂ for 2 hours, and the absorbance was measured at 450 nm. The absorbance of the treated cells was compared with that of the control group, with the viability of the control group set as 100%.

[0284] To evaluate the cytotoxicity of 4T1-FAP cells under different treatment conditions, 4T1-FAP cells were seeded at a concentration of 5 × 10⁴ / mL in 96-well plates in 200 μL of RMI-1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. The cells were cultured at 37°C in a 5% CO₂ incubator for 12 hours. They were then treated with 100 nM NAPC-ADC in hypoxia for 24 hours. The cells were irradiated with 4 Gy of X-rays and incubated for 48 hours. Afterward, blank medium containing CCK-8 at a final concentration of 0.5 mg / mL was added. The cells were incubated at 37°C in a 5% CO₂ incubator for 2 hours, and the absorbance was measured at 450 nm. The absorbance of the treated cells was compared with that of the control, with the viability of the control set as 100%.

[0285] 4T1-FAP and HT1080-FAP cells were seeded in 8-well plates (5 × 10 cells / well) and allowed to adhere overnight under hypoxic conditions. Cells were then treated with NAPC-ADC (100 nM), X-rays (4 Gy), or a combination for 12 h, fixed in cold methanol (4°C) for 30 min, and then incubated with FITC-conjugated anti-α-tubulin (Abbkine) at room temperature (1:100 V:V; λex = 488; λem = 570 nm). Nuclei were stained with DAPI. All samples were imaged using a Nikon confocal laser scanning microscope.

[0286] Example 15: ADC activation for in vivo anticancer activity

[0287] The following steps were used to measure the MMAE concentration after in vivo ADC activation:

[0288] Six-week-old Balb / c female mice were purchased from Beijing Weitong Lihua Laboratory and housed under specific pathogen-free conditions with free access to standard chow and water. Approximately 2 × 106 4T1-FAP cells suspended in 100 μL of PBS were subcutaneously implanted into the right shoulder of the mice for PET imaging, fluorescence imaging, and treatment. Tumor volume was calculated using the formula: volume = 1 / 2 × length × width 2 Fluorescence imaging and PET imaging were performed when the tumor volume reached approximately 100-200 mm³ (10-15 days after inoculation). The optimal timing for treatment was when the tumor volume reached 40-100 mm³ (7 days after inoculation). If the tumor volume exceeded 1500 mm³, the mice were sacrificed, and the treatment duration was 21 days.

[0289] Four groups of mice bearing BGC823 xenografts were injected with either NC-ADC or oxaliPt(IV)-ADC (5 mg / kg). ADC administration was followed by X-ray irradiation 48 hours after administration. Mice were then sacrificed, and tumor samples were collected from each group and weighed. Tumors (0.1-0.2 g) were transferred to glass tubes along with 1 mL of methanol. The tissues were homogenized and centrifuged (13,000 rpm, 5 minutes) to remove debris. The methanol was evaporated, and the residue was reconstituted in 10% acetonitrile in water (500 μL) and filtered through a 0.22 μm filter.

[0290] All samples were analyzed by LC-QTOF-MS, and the free MMAE content was quantified based on a standard curve. The detection limit of MMAE in this method was 5 nM.

[0291] Antibody drug conjugates (ADCs) are a perfect example of prodrugs that have extremely high tumor selectivity in clinical development, but their release is uncontrolled and inefficient, so third-generation ADCs with excellent stability and reactivity are needed. Radiotherapy-induced reduction reactions can be applied to ADCs to provide a new platform to solve this problem and improve the efficiency of drug release in tumors. Based on the model reaction, MMAE was conjugated to a humanized monoclonal antibody derived from sibutrazole using a pyridine linker through conventional chemical operations (Figure 20). This antibody has been developed for fibroblast activation protein (FAP) targeted radionuclide therapy with a mass of 149.8KDa and a binding affinity of 0.608nM. In this case, the N-alkyl-4-pyridylcarbamate (NAPC) linker is irradiated with e - aqSelective reduction cleaves the CO bond, followed by decarboxylation and release of the highly toxic free MMAE ( FIG. 20 ). By hydrophobic interaction high performance liquid chromatography (HIC HPLC) analysis, the sibotuzumab-(N-alkyl-4-picolinate)-MMAE conjugate (NAPC-ADC) is a homogeneous ADC, as evidenced by a single main peak ( FIG. 21 ). By MALDI-TOF analysis, the drug-to-antibody ratio (DAR) of the modified ADC is 4 ( FIG. 21 ), indicating that the main peak contains 4 payloads per antibody ( FIG. 21 ), which is optimal for in vivo tumor-specific drug delivery.

[0292] It has been shown that 4Gy of irradiation can cause 100nM ADC in PB (pH 7.4) to release more than 220nM of MMAE (Figure 22), which is sufficient for tumor treatment, and the IC50 of free MMAE was measured to be less than 1.694nM. The inventors also observed that ADC (100nM) was activated in a radiation dose-dependent manner, reaching a conversion rate close to 100% under 8Gy of radiation. To test whether radiation-induced release can achieve MMAE release from ADC, NAPC-ADC+X-ray cell viability assays were performed using 4T1-FAP and HT1080-FAP cell lines (Figure 23). Cells were treated with X-rays, NAPC-ADC alone, and NAPC-ADC+4Gy X-rays. As expected, 100 nM ADC or 4 Gy irradiation showed little toxicity to 4T1-FAP and HT1080-FAP cell lines, and cell viability after ADC treatment decreased significantly after irradiation due to the transient release of active MMAE, indicating that the MMAE released by NAPC-ADC is effective. In addition, FITC anti-α-tubulin antibody was applied to characterize tubulin damage under different conditions. Confocal imaging showed that tubulin damage only occurred when NAPC-ADC was incubated with X-rays after treatment (Figure 24). The ADC or X-ray treated groups showed clear tubulin structures, while the cells in the X-ray + ADC group shrank rapidly due to the loss of tubulin function. Therefore, NAPC appears to be a promising third-generation ADC linker with good stability and high release efficiency upon X-ray irradiation in a biological environment.

[0293] The above findings prompted the inventors to study the anti-cancer effects of radiation-activated ADCs in animals. To achieve the best therapeutic effect, the correct timing of radiotherapy activation is crucial. Therefore, the inventors first studied the pharmacokinetics of NAPC-ADCs by positron emission tomography (PET) to understand the dynamic distribution of ADCs in mice bearing 4T1-FAP xenografts. NAPC-ADCs were used according to the established protocol. 89 Zr radionuclide labeling. Intravenous injection [ 894T1-FAP tumor-bearing mice were scanned at 2, 6, 12, 24, 48, 72, 96, 120, and 144 hours after Zr]NAPC-ADC. Multiple time-point positron emission tomography-computed tomography (Figure 25) showed [ 89 Zr]NAPC-ADC showed good blood circulation and high tumor uptake. Tumor uptake began to be seen 2 hours after injection and gradually increased until it reached a plateau at 48 hours after injection (~4g / mL, Figure 26). 48 hours after injection, the radioactivity uptake in the tumor was significantly higher than that in the blood. As blood retention continued to decrease, the tumor uptake into the blood continued to increase (Figure 26). 89 The time-activity curve (TAC) of Zr]NAPC-ADC ( FIG. 26 ) showed that ADC mainly accumulated in the tumor site in a time-dependent manner and reached a plateau after 48 hours, with little enrichment in the blood and muscle.

[0294] As shown in Figure 28, 4T1-FAP tumor-bearing mice were randomly divided into five groups. All mice were administered 5 mg / kg ADCs on day 0. Following the previous in vivo biodistribution, X-rays were delivered at doses of 0 Gy, 4 Gy, 8 Gy, 12 Gy, and 16 Gy, and xenograft tumors were harvested and analyzed for intratumoral MMAE concentrations by LC-QTOF-MS. As expected, the results showed that 4T1-FAP tumors treated with NAPC-ADCs released MMAE in a dose-dependent manner after radiotherapy, indicating that MMAE release was dependent on radiation-induced reduction reactions. When irradiated with 12 Gy of radiation, 100 nM NAPC-ADCs completely released MMAE, with a G value of 35 nM / Gy for MMAE in the tumor. The success of the radiation-induced controlled release strategy using antibody drug delivery systems is expected to enhance therapeutic efficacy.

[0295] The effect of radiation-induced payload release was subsequently evaluated. Mice bearing tumors measuring 40-70 mm³ were randomly divided into four groups and treated with PBS, NAPC-ADC, X-rays, and NAPC-ADC + X-rays. All mice were administered 5 mg / kg of ADCs on day 0. For the treatment groups, 4 Gy of X-rays were administered on days 2, 4, and 6 after ADC injection, based on the fact that 12 Gy of X-rays can trigger complete MMAE release (Figure 27). NAPC-MMAE combined with 3 × 4 Gy of X-rays demonstrated significant therapeutic effects against 4T1-FAP tumors. As shown in Figure 29, by only day 21, the average tumor size was approximately 100 mm³, while tumors in the control group (PBS alone) reached 1500 mm³ (Figure 29). Administration of NAPC-ADC alone at a concentration of 5 mg / kg had no significant effect on tumor growth, indicating that the observed therapeutic response was specific to radiation-induced payload release in vivo. Therefore, the combined treatment of X-rays and NAPC-ADC significantly inhibited tumor growth (Figure 29), while NAPC-ADC treatment barely stopped tumor growth, and X-rays inhibited its growth in a limited way. Tumor-bearing mice were sacrificed on day 21, and tumors were harvested for tumor weight measurement and photography (Figure 30), confirming the therapeutic effect of NAPC-ADC + radiotherapy. There was a significant difference between the treatment group and the control group (P < 0.0001). Body weight assessments on day 21 (Figure 31) and histological analysis of key organs showed that the prodrug did not show overall toxicity, indicating that the biosafety of the inventor's strategy is satisfactory.

[0296] The results demonstrate that the radiation-induced controlled release strategy has the potential to treat tumors with limited toxicity. It also reveals another advantage of the radiation-reducing response of controlled release in vivo, namely, the double tumor targeting of prodrug activation. On the one hand, radiation therapy has been precisely applied to the spatial localization of tumors. On the other hand, e - aq It is quenched by high levels of oxygen in normal tissues to deny drug release, providing additional selectivity for hypoxic tumors to enhance the efficiency of radiotherapy-induced prodrug activation.

Claims

1. A radiation-activatable compound, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein the compound has a structure of formula (I) or formula (II): Features: A is CR 1 , N or NR 2 , and only one A in formula (I) is N or NR 2 , in formula (II), only one A is N or NR 2 The carbon atoms on the aromatic ring in formula (I) or formula (II) are optionally replaced by R 1 Substitution, where R 1 Selected from the group consisting of -H and electron withdrawing groups, preferably selected from the group consisting of -H, -NO2, -CN, and halogen, more preferably selected from the group consisting of -H, -NO2, -CN, and -F; When A is NR 2 When R 2 is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2 Optionally substituted with a linker group comprising one or more groups that are or are derived from: amino, amide, heterocyclyl, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid; B is any therapeutic agent or color developing agent that can be coupled to the aryl ester group of the compound, as long as it can be separated from the aryl ester group under radiation and release a molecule with therapeutic activity or color development, for example, B is a fluorescent group, or a drug group, preferably a drug group for treating cancer, more preferably a chemotherapeutic drug group; R 3 is selected from the group consisting of -H and an electron donating group, preferably selected from the group consisting of -H, an alkyl group and an aryl group, more preferably selected from the group consisting of -H, C 1-6 Alkyl, C 6-10 The group consisting of an aryl group, the alkyl group and the aryl group are optionally substituted.

2. The radiation-activatable compound according to claim 1, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound has a structure of one of the following formulae: The carbon atoms on the aromatic ring are optionally replaced by R 1 Replacement, R 1 , R 2 , R 3 and B has the meaning as defined in claim 1, Preferably, in the presence of water, after irradiation, the aryl ester group in formula (I) or formula (II) can be removed and B is released.

3. The radiation-activatable compound according to claim 1 or 2, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein R 1 All are -H, R 2 If present, it is methyl, which is optionally substituted by a linking group, and / or R 3 For -H, The linking group has the meaning as defined in claim 1.

4. The radiation-activatable compound according to claim 1 or 2, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein One or more R in formula (I) or formula (II) 1 Selected from the group consisting of -NO2, -CN, -F, R 2 If present, it is methyl, and / or R 3 is -H.

5. The radiation-activatable compound according to claim 1 or 2, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein R 1 All are -H, R 2 If present, it is methyl, and / or R 3 C 1-3 Alkyl or phenyl.

6. The radiation-activatable compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof, wherein B is coumarin and its derivatives, rhodamine and its derivatives, or auristatin and its derivatives.

7. The radiation-activatable compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein B is selected from the group consisting of the following structures: in represents the site where B is connected to the ester group in formula (I) or (II); The ring carbon atoms in formula (B1), formula (B2) and formula (B3) may be optionally replaced by -R 5 、-NR 6 R 7 replace; E is NR 4 Group or O, preferably NR 4 Group; R 8 NR 9 R 10 OR 9 ; When B is auristatin and its derivatives, B is 4 The group is connected to the ester group in formula (I) or (II); R 4 is -H or alkyl, preferably -H or C 1-6 Alkyl, more preferably -H or C 1-4 alkyl; R 5 , R 6 , R 7 , R 9 and R 10 The groups are each independently selected from H and alkyl, the alkyl being preferably C 1-6 Alkyl, more preferably C 1-4 alkyl; In formula (B4), R 12 To R 23 Each independently selected from H and alkyl, the alkyl is preferably C 1-6 Alkyl, more preferably C 1-4 alkyl; R 24 is selected from H, hydroxyl, carboxyl, alkyl, alkoxy, halogen, aryl or heteroaryl, wherein the aryl is preferably C 6- 10 Aryl, the heteroaryl is preferably C 5-10 heteroaryl; R 25 Selected from H, hydroxy, alkyl, alkoxy, halogen; R 26 is selected from aryl or heteroaryl, the aryl or heteroaryl is optionally further substituted by a substituent selected from H, halogen, hydroxyl, alkyl, alkoxy, the aryl is preferably C 6-10 Aryl, the heteroaryl is preferably C 5-10 Heteroaryl.

8. The radiation-activatable compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein B is selected from the group consisting of the following structures: Where R 5 , R 6 , R 7 , R 9 , R 13 To R 17 , R 22 The groups are each independently C 1-6 Alkyl, more preferably C 1-4 Alkyl, more preferably R 6 and R 7 Ethyl, R 5 and R 9 is methyl; R 24 Selected from H, hydroxyl, carboxyl, C 1-6 Alkyl, C 1-6 Alkoxy, halogen, C 6-10 Aryl and C 5-10 heteroaryl; R 25 Selected from H, hydroxyl, C 1-6 alkyl; The radiation-activatable compound is preferably selected from the following group: Where R 2 C 1-6 alkyl.

9. Use of a radiation-activatable compound or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof according to any one of claims 1 to 8 in the preparation of a drug conjugate, characterized in that: In formula (I) or formula (II), only one A is NR 2 , R 2 is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2 Optionally substituted with a linker group comprising one or more groups that are or are derived from amino, amide, heterocyclyl, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid.

10. The use according to claim 9, wherein the drug is partially expressed by R 2 , or by using R 2 Reaction to connect to formula (I) or formula (II), thereby preparing the drug conjugate.

11. A drug conjugate, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein the drug conjugate comprises a targeting moiety, a linking moiety, and a drug moiety, wherein the linking moiety couples the targeting moiety and the drug moiety, and wherein the linking moiety comprises a structure of formula (I') or formula (II'): Features: A is CR 1 or NR 2’ , and only one A in formula (I') is NR 2’ In formula (II'), only one A is NR 2’ The carbon atoms on the aromatic ring in formula (I') or formula (II') are optionally replaced by R 1 Substitution, where R 1 Selected from the group consisting of -H and electron withdrawing groups, preferably selected from the group consisting of -H, -NO2, -CN, and halogen, more preferably selected from the group consisting of -H, -NO2, -CN, and -F; R 2’ is an alkyl group, preferably C 1-6 Alkyl, more preferably C 1-3 Alkyl; R 2’ is substituted with a linking group, said linking group being attached to said targeting moiety; B is a fluorescent group, or a drug group, preferably a chemotherapeutic drug group; R 3 is selected from the group consisting of -H and an electron donating group, preferably selected from the group consisting of -H, an alkyl group and an aryl group, more preferably selected from the group consisting of -H, C 1-6 Alkyl, C 6-10 The group consisting of an alkyl group and an aryl group, wherein the alkyl group and the aryl group are optionally substituted; The targeting moiety is an antibody.

12. The drug conjugate according to claim 11, or a pharmaceutically acceptable salt, or solvate, or a stereoisomer thereof, wherein the structure of formula (I') or formula (II') has one of the following structures: The carbon atoms on the aromatic ring are optionally replaced by R 1 Replacement, R 1 , R 2’ , R 3 and B have the meanings as defined in claim 1.

13. The drug conjugate according to claim 11 or 12, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein R 1 Both -H, and / or R 3 is -H.

14. The drug conjugate according to claim 11 or 12, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein One or more R in formula (I) or formula (II) 1 Selected from the group consisting of -NO2, -CN, -F, and / or R 3 is -H.

15. The drug conjugate according to claim 11 or 12, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein R 1 Both -H, and / or R 3 C 1-3 Alkyl or phenyl.

16. The drug conjugate according to any one of claims 11 to 15, or a pharmaceutically acceptable salt, or a solvate, or a stereoisomer thereof, wherein the linking group comprises one or more groups that are or are derived from the following: amino, amide, heterocyclic, alkyl, alkenyl, alkynyl, polyethylene glycol (PEG), amino acid.

17. A method for activating a radiation-activatable compound or drug conjugate using radiation, characterized in that: The radiation-activatable compound or drug conjugate is a compound as claimed in any one of claims 1 to 8 or a drug conjugate as claimed in any one of claims 11 to 16, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof.

18. The method according to claim 17, wherein the radiation is high-energy radiation rays, preferably X-rays or gamma rays, more preferably the intensity of the X-rays or gamma rays is 60 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, 10 Gy or less, 8 Gy or less, 6 Gy or less and 4 Gy or less.

19. A method for treating or diagnosing a disease, the method comprising administering a radiation-activatable compound or drug conjugate to an individual and irradiating the individual with a therapeutically effective amount, the radiation-activatable compound or drug conjugate being a compound as described in any one of 1 to 8 or a drug conjugate as described in any one of claims 11 to 16, or a pharmaceutically acceptable salt, or solvate, or stereoisomer thereof.

20. The method of claim 19, which is used to treat or inhibit, reduce the severity of, reduce the risk of, or inhibit the metastasis of cancer in an individual.

21. The method of claim 19 or 20, wherein the cancer is selected from breast cancer, colorectal cancer, lung cancer and gastric cancer.

22. The method according to any one of claims 19 to 21, wherein the subject is irradiated after administration of the compound or the drug conjugate, preferably 24 hours, 48 ​​hours, 96 hours, and / or 144 hours after administration of the compound or the drug conjugate.

23. The method of any one of claims 19 to 22, wherein the compound or the drug conjugate is administered in an amount of about 0.005 mg / kg to about 100 mg / kg, for example, a dose of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100 mg / kg.

24. The method of any one of claims 19 to 23, wherein the compound or the drug conjugate and radiation are administered once a week, once every two weeks, once every three weeks, or once every four weeks, and the compound or the drug conjugate and radiation are administered continuously for at least 1 round, at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9 rounds, at least 10 rounds.

25. The method according to any one of claims 19 to 24, wherein the compound or the drug conjugate is administered parenterally, such as intravenously.

26. The method according to any one of claims 19 to 25, wherein: The radiation is high energy radiation, preferably X-rays or gamma rays, more preferably the intensity of the X-rays or gamma rays is 60 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, 10 Gy or less, 8 Gy or less, 6 Gy or less and 4 Gy or less.

Images