Prodrug activation of N-oxides by radiotherapy

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a method for activating an N-oxide prodrug by utilizing radiation, and a method for treating or inhibiting cancer, reducing cancer severity, reducing cancer risk or inhibiting cancer metastasis in a subject. The method comprises administering to the subject a therapeutically effective amount of an N-oxide prodrug and radiating the subject with a therapeutically effective amount of radiotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for activating an N-oxide prodrug using radiation, and a method for treating or inhibiting cancer, reducing cancer severity, reducing cancer risk, or inhibiting cancer metastasis in a subject. The method comprises administering a therapeutically effective amount of an N-oxide prodrug to the subject, and irradiating the subject with a therapeutically effective amount of radiotherapy. Background Art

[0002] Since it was first proposed in 1958, the prodrug strategy has been widely used to address drug delivery issues in tumor therapy. Ideally, a prodrug can deliver 50 times more than the normal dose of drug to the desired location and cure tumors that are usually resistant to chemotherapy. Effective activation of the parent drug is perhaps the most critical step in the prodrug strategy. However, due to the lack of tumor-selective activation strategies, controlled activation of prodrugs remains a long-standing challenge in the clinic. Therefore, spatiotemporally precise prodrug activation in a tumor-selective manner is an urgent need and a challenging problem, prompting us to find suitable chemical tools.

[0003] Inspired by the application of light in phototherapy, clinically relevant ionizing radiation (X-rays, gamma rays, etc.) also has the potential to become a precise perturbation tool in living bodies due to its precise and deep tissue penetration (up to 15 cm). In addition, although more than 50% of cancer patients are treated with radiotherapy (RT), RT may fail due to total dose limitations (usually less than 60 Gy) and hypoxia resistance, as oxygen is involved in the fixation of radiation-induced DNA damage. Therefore, radiotherapy combined with chemotherapy has a better chance of curing cancer. Establishing a chemotherapy strategy activated by targeted radiotherapy is expected to reduce the comprehensive toxicity of dual treatment and achieve better therapeutic effects.

[0004] Radiation chemical changes to molecules include direct and indirect effects. Among them, the indirect radiation chemical effect of radiation on environmental components is dominant in biological systems. It mainly works through the radiolysis of water, because water accounts for 70%-80% of the weight of tissues. Figure 1 As shown, various reactive particles are formed instantly by the radiolysis of water, and the subsequent cascade reaction occurs within 10 -4 The cleavage chemistry of prodrug molecules induced by hydroxyl radicals (·OH, as one of the main products from water radiolysis) and hydrogen radicals (·H) has been previously reported. However, its application in living systems is hampered by the fact that ·OH is instantaneously quenched in reducing environments (e.g., hypoxic tumors). Interestingly, another major product, hydrated electrons (e- aq ), which increases in reducing environments, has been reported to be a "super reducing agent" for challenge reactions. Therefore, in the present invention, we sought to investigate the potential role of radiation-induced e - aq The mediated reaction serves as a chemical tool for prodrug activation in a tumor-selective manner. Summary of the invention

[0005] Precise activation of chemotherapeutic drugs in a tumor-selective manner is an ideal way to treat cancer without causing systemic toxicity. Despite many efforts, the development of spatiotemporally controllable activation methods remains an unsolved problem. Here, a new prodrug activation strategy using radiotherapy (X-rays) is presented. Due to their precision and deep tissue penetration, X-rays meet the requirements of modifying molecules in tumors by water radiolysis. The study demonstrates for the first time that radiation-generated hydrated electrons (e - aq ) can effectively reduce N-oxides. - aq The present invention then applies this strategy to activate N-oxide prodrugs. Camptothecin (CPT), an anticancer drug based on N-oxide prodrugs, shows significant anticancer effects when activated by radiotherapy. This radiation-induced in vivo chemical reaction can achieve the multifunctional design of radiotherapy-activated prodrugs, which has significant clinical significance because more than 50% of cancer patients need to receive radiotherapy. This radiation-induced in vivo chemical reaction can also greatly improve the selectivity of chemotherapeutic or fluorescent compounds, improve the efficacy of radiotherapy, and reduce the toxicity of the administered chemotherapeutic drugs or fluorescent compounds by controlling the release of the corresponding chemotherapeutic prodrugs or fluorescent prodrugs.

[0006] In one aspect, the present invention provides a method for activating an N-oxide compound using radiation, characterized in that the N-oxide compound contains at least one aromatic or heteroaromatic group, and the N-oxide compound can be reduced to the corresponding tertiary amine after irradiation with the radiation.

[0007] In another aspect, the present invention provides a method of treating or diagnosing a disease, the method comprising administering a therapeutically effective amount of an N-oxide prodrug to a subject, and irradiating the subject with a therapeutically effective amount of radiation.

[0008] In another aspect, the present invention provides the use of an N-oxide prodrug in the preparation of a medicament for treating or diagnosing a disease in a subject, wherein the subject is also exposed to radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1Radiation-induced prodrug activation in tumors was shown. Water radiolysis by ionizing radiation produces various reactive particles (G value represents the number of molecules formed by absorbing 100 eV of energy in the system). Among them, hydroxyl radical (·OH) has been reported to be used for controlled drug release. In this study, N-oxide was activated by hydrated electrons (e - aq ) reduction. Thanks to this reaction, nitrogen-containing drugs (mainly tertiary amines and N-heterocyclic compounds) can be “caged” in a highly atom-economical manner (via a single oxygen atom) and activated in a spatiotemporally dependent manner at the tumor site by radiotherapy.

[0010] Figure 2 The radiation-induced reduction of N-oxides is shown. (a) Schematic diagram of the radiation-induced reduction of a model N-oxide compound (NO-1) with water to produce e - aq Fluorescence reaction of abs =380 nm) and (c) fluorescence spectra (λ ex =380 nm and λ em =460 nm). Inset: Fluorescence images of the radiation dose dependence. (d) Fluorescence emission detection (λ ex =380 nm, λ em =460 nm). Compared with the instantaneous reduction induced by 60 Gy irradiation, NO-1 (10 µM) could be reduced by the bioreductant (1 mM) after 24 h in PBS. n = 3. (e) Representative confocal fluorescence images of living CT26 and HCT116 cell lines. Cells were pretreated with NO-1 (10 µM in RPMI-1640 medium) and then irradiated at different doses. Scale bar: 20 µm.

[0011] Figure 3 Structural screening of radiation-induced reduction of N-oxides is shown. (a) Chemical structures of N-oxides (NO-2 to NO-10) detected by radiation-induced reduction (10 µM in PBS, 60 Gy). The reduction products were detected by UPLC-MS. (b) G values ​​of the reduction products generated from NO-2 to NO-10. (c) Radiation dose dependence of the reduction product (A-8) (m / z=146) generated from the aromatic heterocycle NO-8 (10 µM in PBS) determined by UPLC-MS.

[0012] Figure 4 The e of N-oxide is shown - aqMechanistic study of induced reduction. (a) Proposed e - aq The mechanism of mediated reduction. (b) The molecular orbital of NO-1, which shows that e - aq Captured by the π system, not by N + -O - antibonding orbital capture of the bond. (c) Anionic radical intermediate and ground state of N-oxide | |with e - aq of | (d) shows the HOMO and LUMO energy levels and energy gaps (E) of representative N-oxides: NO-1 (aniline N-oxide), NO-7 (heterocyclic N-oxide), and NO-9 (alkyl N-oxide). gap ).

[0013] Figure 5 Radiation-induced activation of N-oxide prodrugs is shown. (a) Chemical structure of N-oxide drugs (10 µM in PBS) synthesized by 60 Gy radiation-induced reduction assay. (b) G-values ​​of the reduction products generated as shown in (a), n = 3. The dashed line is the G-value of DHBC (3,5-dihydroxybenzylcarbamate, which is the most efficient group that can be removed by hydroxyl radicals in water radiolysis). (c) Schematic diagram of the single oxygen atom "caging" and radiotherapy "decaging" prodrug strategies. NO-CPT was used as a model prodrug. Cell viability assay of radiation-activated NO-CPT in (d) HCT116 cells and (e) CT26 cells. (16 Gy X-ray [NO-CPT] = 0.5 µM, n = 6, two-tailed unpaired Student's t-test, ***P < 0.001).

[0014] Figure 6 Prodrugs showing radiotherapy-activated inhibition of tumor growth. (a) Treatment regimen. Nu / Nu mice were subcutaneously implanted with HCT116 cells, followed by intravenous (iv) injection of NO-CPT (10 mg / kg) and radiotherapy (4 Gy each time). (b) Time-dependent accumulation of NO-CPT in tumor tissues detected by UPLC-UV, n = 3. (c) Tumor growth curves of tumor-bearing mice after receiving the indicated treatments. (d) Average tumor weight on day 27 after the first treatment. (e) Body weight change curves of mice receiving different treatments. n = 6, two-tailed unpaired Student's t-test, ***P < 0.001.

[0015] Figure 7The chemical structures of the synthetic starting materials and the proposed N-oxide reduction products (NO-1 to NO-10, NO-IMQ, NO-APX, NO-PNP, NO-LRT, NO-CPT, respectively) are shown.

[0016] Figure 8 (a) UPLC-MS spectrum of A-1 generated from NO-1 (10 µM in PBS) after 60 Gy irradiation ([A-1+H] + (a) UPLC-UV spectrum of NO-1 (10 µM in PBS) after irradiation (λ abs =320 nm) and (d) UV-Vis absorption spectra. (e) Fluorescence emission spectra (λ ex =380 nm). (f) Fluorescence emission spectra (λ) of NO-1 (10 µM in PBS containing 10% FBS) before and after 60 Gy irradiation. ex =380 nm).

[0017] Fig. 9 Display UPLC-UV (λ abs =380 nm) after 60 Gy irradiation from NO-1 (10 µM in PBS, with e - aq The normalized amount of A-1 generated by the quencher and hydroxyl radical quencher, respectively.

[0018] Fig.10 Shown are cell viability assays of (a) HCT116 cells and (b) CT26 cells incubated with NO-1 for 12 h, n = 5.

[0019] Fig.11 Shown is the radiation dose-dependent reduction of NO-CPT (10 µM in PBS) as measured by: (a) UPLC-MS, m / z = 349, (b) positive ion mode mass spectrum of CPT (retention time = 2.66 min) and (c) fluorescence emission spectrum (λ ex =380 nm).

[0020] Fig.12 Shown is the radiation-induced reduction of 10 µM NO-CPT in (a) PBS containing 10% FBS and (b) HCT116 cell lysate as measured by UPLC-MS ([CPT+H] +Selected ion monitoring signals of (a) 50 μg / mL, m / z = 349) and (c) PBS containing 10% FBS, and (d) HCT116 cell lysate were analyzed by UPLC-UV (λ abs =380nm) measurement.

[0021] Fig.13 Shown are cell viability assays of (a) HCT116 cells and (b) CT26 cells incubated with CPT and NO-CPT for 48 h, respectively. n = 5.

[0022] Fig.14 Photographs of representative tumors at day 27 after different treatments are shown.

[0023] Fig.15 Tissue sections showing major organs (heart, liver, spleen, lung, and kidney) stained with hematoxylin and eosin (H&E). After treatment, no obvious abnormalities were observed in these major organs of mice. DETAILED DESCRIPTION

[0024] definition Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. The technology used herein refers to the technology generally understood in the art, including obvious changes or equivalent substitutions to these technologies by those skilled in the art. Although it is believed that the following terms are well known to those skilled in the art, in order to better illustrate the present invention, the following definitions are provided.

[0025] As used herein, the terms "comprises," "including," "having," "containing," or "having," and variations thereof, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0026] As used herein, "chemotherapeutic agents (chemotherapeutic agents)" or "chemotherapeutic drugs" refer to chemotherapeutic drugs that can kill tumor cells, and these drugs can act on different stages of tumor cell growth and proliferation, thereby inhibiting or killing tumor cells.

[0027] The term "alkyl" as used herein, alone or as part of another group, refers to a alkyl radical containing 1 to 12 carbon atoms (i.e., C 1-12 alkyl) or an unsubstituted straight or branched aliphatic hydrocarbon with a specified number of carbon atoms, for example, C1 alkyl such as methyl, C2 alkyl such as ethyl, C3 alkyl such as n-propyl or isopropyl, C 1-3 Alkyl groups are methyl, ethyl, n-propyl or isopropyl groups. 1-4 Alkyl. C 1-12Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1-4 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl.

[0028] The term "cycloalkyl" as used herein, alone or as part of another group, refers to a saturated or partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbon containing 1 or 2 rings having 3 to 12 carbon atoms or the specified number of carbon atoms (i.e., C 3-12 In one embodiment, the cycloalkyl group has two rings. In one embodiment, the cycloalkyl group has one ring. In another embodiment, the cycloalkyl group is selected from C 3-8 In another embodiment, the cycloalkyl group is selected from the group consisting of C 3-6 Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalinyl, adamantyl, cyclohexenyl, and cyclopentenyl.

[0029] The term "alkoxy" or "alkoxy" as used herein, alone or as part of another group, refers to an oxygen attached to an alkyl group as defined above.

[0030] The term "heteroalkyl" as used herein, alone or as part of another group, refers to an alkyl group in which one carbon atom is replaced by a heteroatom (eg, N, O, or S) or a carbonyl group (C=O).

[0031] The term "alkenyl" is intended to include hydrocarbon chains of straight or branched configurations having a specified number of carbon atoms and one or more carbon-carbon double bonds, preferably 1 to 2 carbon-carbon double bonds, which may occur at any stable point of the chain. For example, "C2-C6 alkenyl" is intended to include C2, C3, C4, C5 and C6 alkenyl. Examples of alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl and 4-methyl-3-pentenyl.

[0032] The terms "aryl", "heteroaryl" and "aromatic heterocycle" as used herein refer to preferably 3-14 carbon atoms having a stable monocyclic or polycyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated group, each of which may be substituted or unsubstituted. In certain embodiments of the present invention, "aryl" refers to a monocyclic or bicyclic carbocyclic system having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, etc. In certain embodiments of the present invention, as used herein, the term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic aromatic group having 5 to 15, preferably 5 to 10 ring atoms, wherein one ring atom is selected from S, O and N; 0, 1 or 2 ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the free radical of which is attached to the rest of the molecule via any ring atom, such as pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furanyl, quinolyl, isoquinolyl, etc.

[0033] As used herein, the term "heterocyclyl", "heterocyclic" or "heterocycle" refers to a non-aromatic 5-, 6- or 7-membered ring or polycyclic group, including but not limited to a bicyclic or tricyclic group containing a fused six-membered ring or seven-membered ring, wherein at least one carbon atom of one ring is replaced by a heteroatom. Each heteroatom is independently selected from the group consisting of oxygen, sulfur (including sulfoxides and sulfones) and / or nitrogen (which may be oxidized or quaternized). The terms "heterocyclyl", "heterocyclic" or "heterocycle" are intended to include groups in which -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, ε-lactam) and piperazin-2-one; wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may be optionally quaternized, and (iv) any of the above heterocycles may be fused to an aromatic or heteroaromatic ring, such as a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuranyl.

[0034] In certain embodiments, a "substituted aryl, heteroaryl, heterocyclyl or heterocyclic" group is used and, as used herein, refers to an aryl, heteroaryl, heterocyclyl or heterocyclic group as defined above, wherein one, two or three hydrogen atoms are independently replaced by substituents, which are not limited to alkyl; heteroalkyl; aryl; heteroaryl; aralkyl; heteroaralkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO2; -CN; -CF3; -CH2CF3; - CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)R x ; -CO2(R x );-CON(R x )2;-OC(O)R x ;-OCO2R x ;-OCON(R x )2;-N(R X )2; -S(O)2R x ;-NR x (CO)R x , each time R appears x Independently include but are not limited to alkyl, heteroalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, wherein any alkyl, heteroalkyl, aralkyl or heteroaralkyl substituent described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any aryl or heteroaryl substituent described above and herein may be substituted or unsubstituted. Other examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples described herein.

[0035] The term "coumarin" refers to a molecule having the following structure, and the term "coumarin or a derivative thereof" refers to a molecule having the following backbone structure, wherein one or more hydrogen atoms, preferably one hydrogen atom in the backbone structure may be substituted by a substituent, for example an alkyl group such as a C1 to C4 alkyl group, or a halogen. Preferably, the 4-position hydrogen atom in the backbone structure is substituted by an alkyl group, for example a C1 to C4 alkyl group. When referring to a "coumarin group or a derivative thereof", it means a group generated when a hydrogen radical is removed from a coumarin molecule or a derivative thereof, for example a hydrogen radical on an aromatic ring, for example a hydrogen radical at position 7.

[0036]

[0037] The term "1,8-naphthalimide" refers to a molecule having the following structure, and the term "1,8-naphthalimide" refers to a molecule having the following backbone structure, wherein one or more hydrogen atoms (e.g., hydrogen atoms on an aromatic ring or hydrogen atoms attached to a nitrogen atom), preferably one hydrogen atom in the backbone structure, may be substituted by a substituent, for example, an alkyl group such as a C1 to C4 alkyl group, or a halogen. Preferably, the hydrogen atom attached to the nitrogen atom is substituted by an alkyl group, for example, a C1 to C4 alkyl group. When referring to "1,8-naphthalimide group or a derivative thereof", it means a group generated when a hydrogen radical is removed from a 1,8-naphthalimide molecule or a derivative thereof, for example, a hydrogen radical at the 4-position.

[0038]

[0039] As used herein, the term "halogen" includes F, Cl, Br, I, and the like.

[0040] As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts of the compound.

[0041] Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, e), lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, aldarate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and xinofoate.

[0042] Suitable base addition salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts.

[0043] For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0044] The term "solvate" as used herein refers to a substance formed by the combination, physical association and / or dissolution of a compound of the present invention with a solvent molecule, such as a disolvate, a monosolvate or a hemisolvate, wherein the ratio of solvent molecules to the compound of the present invention is about 2:1, about 1:1 or about 1:2, respectively. This physical association involves varying degrees of ionization and covalent bonding (including hydrogen bonding). 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, the solvate includes a solution phase and a separable solvate. The compounds of the present invention can form a solvated form with a pharmaceutically acceptable solvent (such as water, methanol and ethanol), and the present application is intended to cover the solvated and non-solvated forms of the compounds of the present invention.

[0045] A solvate is a hydrate. "Hydrate" refers to a specific subset of solvates in which the solvent molecule is water. Solvates generally function as pharmacological equivalents. The preparation of solvates is known in the art, for example, see M. Caira et al., J. Pharmaceut. Sci., 93(3): 601-611 (2004), which describes the preparation of solvates of fluconazole using ethyl acetate and water. Similar methods for preparing solvates, hemisolvates, hydrates, etc. are described in van Tonder et al., AAPS Pharm. Sci. Tech., 5(1): Article 12 (2004) and AL Bingham et al., Chem. Commun. 603-604 (2001). A representative and non-limiting method for preparing solvates is to dissolve the compound of the present invention in a desired solvent (organic solvent, water or a mixture thereof) at a temperature of greater than 20°C to about 25°C, then cool the solution at a rate sufficient to form crystals, and separate the crystals by known methods such as filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of solvent in solvate crystals.

[0046] "Pharmaceutically acceptable carrier" in the context of the present invention refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered and is suitable for contact with the tissues of humans and / or other animals within the scope of sound medical judgment and without excessive toxicity, irritation, allergic response, or other problems or complications corresponding to a reasonable benefit / risk ratio.

[0047] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including oils derived from petroleum, animals, vegetables or synthetics, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. As needed, the pharmaceutical composition may also contain a small amount of a wetting agent, emulsifier or pH buffer. Oral preparations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

[0048] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered via a suitable route, such as by injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or orally, buccal, nasal, transmucosal, topical, in the form of an ophthalmic preparation or by inhalation.

[0049] For these administration routes, the pharmaceutical composition of the present invention can be administered in appropriate dosage forms.

[0050] Dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0051] As used herein, the term "effective amount" refers to that amount of the active ingredient which, after administration, will relieve to some extent one or more of the symptoms of the condition being treated.

[0052] As used herein, "subject (individual)" includes human or non-human animals. Exemplary human subjects include human subjects (referred to as patients) suffering from diseases (e.g., diseases described herein) or normal human 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, cattle, pigs, etc.).

[0053] Treatments and uses In one embodiment, the present invention provides a method for activating an N-oxide compound using radiation, characterized in that the N-oxide compound contains at least one aromatic or heteroaromatic group and the N-oxide compound can be reduced to the corresponding tertiary amine after irradiation with the radiation.

[0054] In a preferred embodiment, the nitrogen atom in the N-oxide group is directly bonded to the at least one aryl or heteroaryl group, or the nitrogen atom in the N-oxide group is an aromatic atom constituting the at least one aryl or heteroaryl group.

[0055] In a preferred embodiment, the N-oxide compound has the structure of Formula I, , Wherein R is independently selected from alkyl groups (e.g., C 1-4 alkyl) and cycloalkyl (e.g. C 3-6 cycloalkyl), Ar is C6-C 20 Aryl, preferably C6-C 14 Aryl, most preferably C6-C 10 Aryl, wherein the aryl group is optionally substituted by alkyl, hydroxy, amino, aminosulfonyl (NH2-SO2-), -C(O)HR', -OC(O)HR', -C(O)-NR 1 Substituted, wherein optionally, the two substituents on the aryl group can form a heterocyclic ring, preferably a 6-membered unsaturated heterocyclic ring, preferably containing O, NR 1 , CR 1 , a 6-membered heterocyclic ring having C=O as a ring member, R 1 and R' are independently selected from hydrogen and alkyl (e.g. C 1-4 alkyl).

[0056] In a preferred embodiment of formula I, R is independently selected from the group consisting of C1-C4 alkyl, and preferably selected from the group consisting of methyl and ethyl, and / or Ar is phenyl or naphthyl, wherein phenyl or naphthyl is unsubstituted or substituted by alkyl, alkenyl, hydroxyl, amino, aminosulfonyl (NH2-SO2-), -C(O)HR', -OC(O)HR', -C(O)-NR 1 substituted, wherein optionally, the two substituents on the phenyl or naphthyl group may form a heterocyclic ring, preferably a 6-membered unsaturated heterocyclic ring, preferably containing O, NR 1 , CR 1 , a 6-membered heterocyclic ring with C=O as a ring member, R 1 and R' are independently selected from hydrogen and alkyl (e.g. C 1-4 alkyl), Preferably, Ar is a naphthyl group optionally substituted by an alkyl group or an aminosulfonyl group (NH2-SO2-), or the heterocyclic ring formed by Ar and two substituents thereon is a coumarin group or a derivative thereof, or a 1,8-naphthalimide group or a derivative thereof.

[0057] In another preferred embodiment, the N-oxide compound has the structure of Formula II,

[0058] Where R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, acylamino, which may be substituted by heterocycle, which may be optionally substituted by alkylcarbonatealkoxyl; and the substituents on the above-mentioned rings may optionally form one or two aryl or heteroaryl groups or heterocycles condensed on the above-mentioned rings, which may be substituted by amino, halogen, alkyl, hydroxyl, carboxyalkyl or heterocycle, which may be optionally substituted by alkoxycarbonyl; The premise is R 2 , R 3 , R 4 , R 5 and R 6 Not hydrogen at the same time.

[0059] In a preferred embodiment of Formula II, R 2 , R 3 , R 4 , R 5 and R 6Independently selected from the group consisting of hydrogen, C1-C4 alkyl, amino, hydroxyl, acylamino, which may be substituted by a 5-, 6- or 7-membered heterocycle, which may be optionally substituted by a (C1-C4) alkyl carbonate (C1-C4) alkoxy group; and the substituents on the ring shown above may optionally form one or two phenyl groups or 5-, 6- or 7-membered heteroaryl or heterocycle condensed on the ring shown above, and the one or two aryl or heteroaryl or heterocycle may be substituted by amino, halogen, C1-C4 alkyl, hydroxyl, carboxyl (C1-C4) alkyl or a 5-, 6- or 7-membered heterocycle, which may be optionally substituted by a (C1-C4) alkoxycarbonyl group; The premise is R 2 , R 3 , R 4 , R 5 and R 6 Not at the same time as hydrogen.

[0060] In preferred embodiments of Formula II, the N-oxide compound has the structure of Formula IIa, IIb, IIc, IId, IIe or IIf:

[0061] Among them, R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 independently selected from hydrogen, hydroxy, amino, halogen, C1-C5 alkyl, (C1-C4)alkyl-O(CO)-(C1-C4)alkyl and (C1-C4)alkyl-O(CO)H, and One or more hydrogen atoms on the ring in the general formula IIa, IIb, IIc, IId, IIe or IIf are optionally substituted by hydroxyl, amino, halogen or C1-C5 alkyl, preferably halogen or C1-C4 alkyl; Preferably, R 7 , R 9 and R 13 are independently selected from the group consisting of hydrogen, hydroxy, amino and halogen, R 8 , R 10 , R 11 , R 12 and R 14 independently selected from the group consisting of C1-C5 alkyl, (C1-C4)alkyl-O(CO)-(C1-C4)alkyl, (C1-C4)alkyl-O(CO)H, and One or more hydrogen atoms on the aromatic ring in Formula IIa, IIb, IIc, IId, IIe or IIf are optionally substituted by halogen or C1-C4 alkyl.

[0062] In a preferred embodiment, the tertiary amine is a biologically active compound, such as a fluorescent compound, preferably a coumarin derivative or a chemotherapeutic agent.

[0063] In a preferred embodiment, the N-oxide compound is an aniline N-oxide or an aromatic heterocyclic N-oxide, such as a compound in the following table or a pharmaceutically acceptable salt or solvate thereof:

[0064] In one embodiment, the invention provides a method of treating or diagnosing a disease, the method comprising administering to a subject a therapeutically effective amount of an N-oxide prodrug, and irradiating the subject with a therapeutically effective amount of radiation.

[0065] In a preferred embodiment, the N-oxide prodrug is as defined above.

[0066] In a preferred embodiment, the method is used to treat or inhibit cancer, reduce the severity of cancer, reduce the risk of cancer, or inhibit cancer metastasis in a subject.

[0067] In a preferred embodiment, the cancer is selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma and malignant lymphoma.

[0068] In a preferred embodiment, the subject is irradiated after administration of the N-oxide prodrug, preferably 20 minutes, 1 hour, 2 hours, 4 hours after administration of the N-oxide prodrug.

[0069] In a preferred embodiment, the N-oxide prodrug is administered in an amount of about 0.005 mg / kg to about 100 mg / kg, for example, about 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 100 mg / kg.

[0070] In preferred embodiments, the N-oxide prodrug and radiation are administered once every two days, once every three days, once every four days, once every five days, once every seven days, once every ten days, once every two weeks, once every three weeks, once every four weeks, and the N-oxide prodrug and radiation are administered continuously for 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.

[0071] In a preferred embodiment, the N-oxide prodrug is administered orally or parenterally.

[0072] In a preferred embodiment, the radiation is X-rays or gamma rays, and 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, or 4 Gy or less.

[0073] Example In order to make the purpose and technical scheme of the present invention clearer, the present invention is further described below in conjunction with specific embodiments.It should be understood that these embodiments 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.

[0074] Example 1: Studies on radiation-induced NO-1 reduction in aqueous solutions and biological environments In order to realize the present invention, it is important to design a system that is not only stable in biological environments but also resistant to e - aq Molecules with selective reactions. N-oxides (oxidized states of tertiary amines) are reported to be bioorthogonal reagents, biocompatible, and stable in blood circulation. Therefore, in living systems, e - aq Remote reduction of N-oxides may offer opportunities to develop in vivo chemical tools, but this has not yet been established.

[0075] It is worth noting that e - aq It is one of the main products of water radiolysis (G value is about 280 nM / Gy, i.e. the number of molecules formed by absorbing 100 eV of energy in the system) and one of the strongest reducing agents. It is speculated that radiation can decompose water to produce e locally. - aq , thereby effectively reducing N-oxides ( Figure 2 a). To verify this idea, a coumarin-based N-oxide, NO-1 (oxidation product of A-1), was synthesized as a model compound. First, a deoxygenated solution of NO-1 (10 µM in PBS) was subjected to X-rays (radiotherapy dose, 10-60 Gy) from a small animal irradiator. Interestingly, radiation dose-dependent reduction occurred immediately and the product A-1 ( Figure 2 b). Fluorophores always lose their fluorescence emission when their amine is blocked by an N-oxide group, so the reduction of NO-1 can be easily indicated by the fluorescence released by coumarin (A-1). The fluorescence data and corresponding photographs show that the amount of fluorescence released by coumarin is linearly related to a given radiation dose ( Figure 2c).

[0076] To confirm that the radiation-induced reduction is caused by e from water radiolysis - aq In order to investigate the effect of irradiation on the A-1, NO-1 (10 µM in PBS) was irradiated with 10 mM isopropyl alcohol (i-PrOH), sodium formate (CH3OONa), and KNO3, respectively. The amount of A-1 generated in each group was determined by UPLC. As expected, the amount of A-1 generated by i-PrOH and CH3OONa increased compared with the control group because they produced additional e under irradiation. - aq Instead, adding KNO3 (an e - aq After quenching agent), the amount of A-1 generated was significantly reduced ( Fig. 9 ).

[0077] We further investigated whether this radiation-induced reduction could function in biological environments, where a balance between biological stability and reactivity is often required. To determine the stability of N-oxides in a reducing environment, NO-1 (10 µM in PBS) was treated with biological reductants including glutathione (GSH, 1 mM), cysteine ​​(Cys, 1 mM), and ascorbate (Vc, 1 mM) for 24 h. The results, as determined by fluorescence emission spectroscopy, showed that the reduction of NO-1 by these reductants was almost negligible compared to the immediate reduction after irradiation ( Figure 2 d).

[0078] Next, the biocompatibility of radiation-induced reduction was tested. NO-1 (10 µM) in PBS containing 10% fetal bovine serum (FBS) was irradiated with 60 Gy and analyzed by fluorescence emission spectroscopy ( Figure 8 f). The fluorescence produced indicates that reduction can occur even in a serum environment, and thus has the potential for use in living systems. In addition, NO-1 was used as a fluorescent probe to confirm that reduction works in living cells. CT26 and HCT116 cell lines were pretreated with NO-1 (10 µM) and subsequently subjected to low-dose irradiation (0 Gy, 4 Gy, 8 Gy, 16 Gy) in a hypoxic environment. Representative confocal fluorescence images of living cells are shown in Figure 2 e shows that fluorescent A-1 molecules are released after irradiation, and their intensity is positively correlated with the radiation dose. The above data indicate that radiation-induced N-oxide reduction does have the potential to become a chemical tool for radiotherapy-activated prodrug strategies.

[0079] Example 2: Screening for N-oxides that can undergo radiation-induced reduction In order to develop N-oxide-based prodrug systems, N-oxides were screened to study structure-reduction relationships. Figure 3 As shown in a, N-oxides (10 µM in PBS) including anilines (NO-2, NO-3, NO-4, NO-5, NO-6), aromatic heterocyclic types (NO-7, NO-8), and alkyl types (NO-9, NO-10) were synthesized and screened by 60 Gy irradiation. The G values ​​of the reduction products from NO-2 to NO-10 were quantified by UPLC and Figure 3 b. No reduction products of NO-6, NO-9, and NO-10 were detected. Interestingly, aromatic heterocyclic N-oxides such as NO-7 and NO-8 were also effectively reduced by radiation ( Figure 3 b, 3c), thereby expanding the range of radiation-responsive substrates.

[0080] It is worth noting that the G value of the resulting reduction product is almost e - aq (280 nM / Gy), indicating the existence of a two-electron reduction mechanism. - aq mediated N-oxide reduction, we propose the hypothesis that the reaction proceeds primarily in two steps ( Figure 4 a). First, N-oxides capture e from the radiolysis of water - aq The intermediate then extracts a proton from the surrounding environment, deoxygenates via electron transfer to release a tertiary amine and ·OH, and continues to capture another electron. - aq to produce water molecules.

[0081] To further test this hypothesis, density functional theory (DFT) calculations were performed using the B3LYP method. According to the LUMO orbital distribution of the model compound NO-1 ( Figure 4 b), e - aq should be filled in the π system of the N-oxide rather than directly in the N + -O - bond in the antibonding orbital. - aq The first step is diffusion controlled due to the tunneling effect. Therefore, only thermodynamic factors need to be considered in the first step: when the absolute value of the Gibbs free energy change (|ΔG|) of the N-oxide and its anion radical intermediate in the aqueous phase is greater than e - aq When the absolute value of , the reaction occurs. Figure 4 c shows the difference between different N-oxides and e -aq Compared with the |ΔG|, NO-1, NO-2, NO-3, NO-4, NO-5, NO-7 and NO-8 with larger π systems have better reactivity, which is consistent with the experimental data ( Figure 3 b, Table 1). Considering e - aq Filling in the N-oxide π system, the degree of conjugation of the π system also affects the e - aq Enter LUMO. Due to the energy difference between HOMO and LUMO (E gap ) can also reflect the degree of conjugation of the molecule, so the HOMO and LUMO energy levels of representative N-oxides including NO-1 (aniline N-oxide), NO-7 (aromatic heterocyclic N-oxide) and NO-9 (alkylated N-oxide) as well as E gap ,like Figure 4 As shown in d.

[0082] Example 3: Radiation-induced reduction study of aromatic heterocyclic N-oxide drug molecules After it was discovered that aromatic heterocycles NO-7 and NO-8 could be effectively reduced by X-rays ( Figure 3 bc), we noticed that the proportion of aromatic N-heterocycles in FDA-approved small molecule drugs has continued to rise over the past five years. The nitrogen atoms of many biologically active N-heterocycles readily interact with DNA or proteins through hydrogen bonds, suggesting that N-heterocycles have important structural significance in drug design and development. Therefore, we synthesized a series of aromatic heterocyclic N-oxide drug molecules ( Figure 5 a). Based on the parent drug, these N-oxide drugs are called NO-Imiquimod (NO-IMQ), NO-Ampicroxiam (NO-APX), NO-Pranoprofen (NO-PNP), NO-Loratadine (NO-LRT), and NO-Camptothecin (NO-CPT). Next, we tested their radiation-responsive reactivity. After treatment with 60 Gy irradiation, the reduction of these N-oxide drugs was analyzed by UPLC. Compared with DHBC (3,5-dihydroxybenzylcarbamate, the most effective radical that can be removed by hydroxyl radicals in water radiolysis), the reduction G values ​​of N-oxide drugs were much higher, indicating that e - aq The reaction mediated by ·OH is much more efficient than that mediated by ·OH ( Figure 5 b).

[0083] Inspired by the reactivity of these relatively complex N-oxide drugs that undergo radiation-induced reduction reactions, we considered developing a strategy to activate chemotherapeutic prodrugs whose activity is “caged” by a single oxygen atom ( Figure 5 c). The anticancer drug CPT is a topoisomerase inhibitor containing a pyrrolo[3,4-β]-quinoline moiety, which we chose to prepare the model prodrug NO-CPT (Scheme S2). After oxidation by hydrogen peroxide (H2O2), the chemical structure of NO-CPT is similar to that of CPT, except for an additional oxygen atom, which is highly atom-economical. As expected, in vitro reduction (10 µM in PBS) occurred in a radiation dose-dependent manner, and the resulting reduction product was CPT ( Fig.11 In addition, activation of NO-CPT (10 µM) in PBS and cell lysate containing 10% FBS has also been investigated by UPLC ( Fig.12 ) tests and analyses showed that NO-CPT can be activated by radiation in a biological environment.

[0084] Example 4: In vitro study of radiation-induced reduction of NO-CPT The cytotoxicity of NO-CPT and CPT was tested by incubating HCT116 cells and CT26 cells with NO-CPT and CPT, respectively, for 48 h. Fig.13 The half-maximal inhibitory concentration (IC) of NO-CPT on CT26 cells 50 ) was 7.71±0.56 µM, which was 22 times that of the parent drug. The IC of NO-CPT against HCT116 cells 50 It is 2.18±0.13 µM, which is 42 times that of the parent drug. Under hypoxic conditions, HCT116 cells and CT26 cells were treated with NO-CPT, 16 Gy X-ray, CPT, X-ray-treated NO-CPT (activated NO-CPT) and NO-CPT+16GyX-ray (NO-CPT and CPT were 0.5 µM in each group). After 48 hours of incubation, the cell viability of the activated NO-CPT and NO-CPT+16Gy X-ray-treated groups was close to that of the CPT-treated group ( Figure 5 d, 5e). In contrast, the group treated with NO-CPT alone had almost no therapeutic efficacy under hypoxic conditions. The results showed that NO-CPT has good stability in living cells and effective response activity to radiation.

[0085] Example 5: In vivo study of radiation-induced reduction of NO-CPT The antitumor efficacy of radiotherapy-induced prodrug activation was further investigated ( Figure 6a). In order to achieve the best therapeutic effect, it is crucial to determine the correct time point of radiotherapy. The accumulation of NO-CPT in tumor tissue changes over time and shows that about 1 h after injection is the best time point for irradiation ( Figure 6 b). HCT116 tumor-bearing mice were randomly divided into 4 groups, including a control group (PBS), and then injected intravenously with NO-CPT, X-rays, and NO-CPT+X-rays ( Figure 6 a) Treatment. After four rounds of NO-CPT+X-ray treatment (NO-CPT+X-ray treatment was administered every three days, referred to as a “round”), tumor growth was inhibited ( Figure 6 c). In contrast, treatment with PBS, NO-CPT, and X-rays failed to prevent tumor growth. Tumor weight measurements and photos of harvested tumors confirmed the antitumor effect of radiotherapy combined with activated chemotherapy ( Figure 6 d, Fig.14 Importantly, evaluation of body weight changes indicated that the radio-activated prodrug did not display significant toxicity ( Figure 6 e). Biosafety was further evaluated by histological analysis after NO-CPT+X-ray treatment, showing that little tissue damage was observed in major organs ( Fig.15 ).

[0086] Materials and synthesis Materials. m-Chloroperbenzoic acid (m-CPBA), 7-(diethylamino)-4-methylcoumarin (A-1), 7-(diethylamino)coumarin (A-2), 7-dimethylamino-4-methylcoumarin (A-3) were purchased from Sigma-Aldrich and used as received. Dansylamide (A-5) and dimethyl(2-phenoxyethyl)amine were purchased from Macklin and used as received. Quinoline N-oxide (A-7), 8-hydroxyquinoline N-oxide (A-8), 4-methylmorpholine N-oxide (NO-9), and dimethyl(2-phenoxyethyl)amine (A-10) were purchased from Energy Chemical and used as received. Imiquimod (IMQ), ampiroxicam (APX), pranoprofen (PNP), loratadine (LRT), and camptothecin (CPT) were purchased from J&K and used as received. KNO3 (AR), CH3OONa (AR), methanol (MeOH) (AR), isopropanol (i-PrOH) (AR), and dichloromethane (DCM) (AR), H2O2, and acetic acid (AR) were purchased from Beijing Chemical Works and used as received. Glutathione (GSH), tryptophan (Try), phenylalanine (Phe), cysteine ​​(Cys), and vitamin C (Vc) were purchased from Sigma-Aldrich and used as received. UPLC solvents were HPLC quality grade and purchased from Sigma-Aldrich. 1× phosphate buffered saline (PBS) was purchased from Meryer. Fetal bovine serum (FBS), penicillin, streptomycin, and RPMI-1640 medium were purchased from GIBCO and used as received. Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Institute. Ultrapure water was deionized using a Millio-Q SP reagent water system (Millipore) with a specific resistivity of 18.4 MΩ cm. 4-Dimethylamino-N-butyl-1,8-naphthalimide (A-4), 1 N,N-Dimethylaniline N-oxide (NO-6) 2 Synthesized according to previously reported literature methods. All chemicals were used as received without further purification.

[0087] Characterization. Nuclear magnetic resonance (NMR) spectra were recorded by a Bruker AVANCE 400 MHz spectrometer. Ultra-performance liquid chromatography (UPLC) was performed on an ACQUITY UPLC H-Class PLUS instrument equipped with a Waters PDA eλ detector and a Waters Acquity QDA mass spectrometer. High-resolution mass spectra were performed on a Bruker Fourier transform ion cyclotron resonance mass spectrometer. Fluorescence spectra were measured on an F-7000 spectrophotometer (Hitachi, Japan). Confocal fluorescence images were recorded by an A1R-si laser scanning confocal microscope (Nikon, Japan).

[0088] synthesis.

[0089]

[0090] Scheme S1. Chemical synthesis of NO-1.

[0091] General synthetic route of N-oxides: A-1 (1 mmol, 1.0 eq.) was dissolved in DCM (5 mL, anhydrous) and cooled to 0 °C in an ice bath. m-CPBA (1.5 mmol, 1.5 eq.) was added. The reaction mixture was heated to room temperature and stirred for 2 h, monitored by thin layer chromatography (TLC). The reaction mixture was then evaporated under reduced pressure, and the crude product was purified by flash chromatography using DCM / MeOH (10:1) as the eluent. NO-1 was obtained as a pink powder (yield: 58%): 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 8.11 (s, 1H), 7.97 (dd, J = 8.7,2.1 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 6.49 (s, 1H), 3.88 (dt, J = 13.6, 6.8 Hz,2H), 3.46 (dq, J = 13.9, 7.1 Hz, 2H), 2.53 (s, 3H), 0.93 (t, J = 6.9 Hz, 6H). 13CNMR (DMSO, δ, ppm, 126 MHz): 159.62, 153.88, 152.73, 125.58, 119.27, 118.16,114.88, 111.26, 66.26, 40.16, 38.90, 18.19, 7.90. High resolution mass: [M + H] + Calculated m / z, 248.1280; found, 248.1281.

[0092] NO-2, NO-3, NO-4, NO-5, NO-10, NO-IMQ, NO-APX, NO-PNP, and NO-LRT were synthesized according to a similar method.

[0093]

[0094] NO-2, yellow powder (yield: 54%) 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 8.13-8.02(m, 2H), 7.90 (dd, J = 8.5, 2.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 6.54 (d, J = 9.6Hz, 1H), 3.84 (dq, J = 11.7, 6.9 Hz, 2H), 3.42 (dq, J = 11.7, 7.1 Hz, 2H), 0.89(t, J = 7.0 Hz, 6H). 13 C NMR (DMSO, δ, ppm, 126 MHz): 160.64, 155.43, 144.35,128.79, 123.49, 110.69, 99.92, 66.21, 49.48, 40.03, 10.58. High resolution mass: [M +H] + Calculated m / z, 234.1124; found: 234.1125.

[0095]

[0096] NO-3, yellow powder (yield: 55%): 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 8.24 (d,J =2.2 Hz, 1H), 8.13 (dd, J = 8.7, 2.3 Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 6.50 (d, J =1.4 Hz, 1H), 3.41 (s, 3H), 2.49 (d, J = 1.3 Hz, 6H). 13 C NMR (DMSO, δ, ppm, 126MHz): 159.59, 158.61, 152.59, 125.79, 119.47, 116.71, 114.92, 109.31, 63.26,39.36, 18.16. High resolution mass: [M + H] + Calculated m / z, 220.0968; found: 220.0968.

[0097]

[0098] NO-4, yellow powder (yield: 49%): 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 8.51-8.36(m, 3H), 7.78 (dd, J = 8.4, 7.3 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 4.07-3.94 (m,2H), 2.90 (s, 6H), 1.66-1.53 ​​(m, 2H), 1.33 (h, J = 7.4 Hz, 2H), 0.91 (t, J = 7.4Hz, 3H). 13 C NMR (DMSO, δ, ppm, 126 MHz): 163.53, 162.88, 159.24, 133.51,131.03, 128.64, 127.74, 126.29, 121.92, 120.92, 114.39, 107.80, 47.76, 44.38,41.87, 39.36, 29.71, 19.79, 13.71. High resolution mass: [M + H] + Calculated m / z, 313.1545; found: 313.1547.

[0099]

[0100] NO-5, light yellow powder (yield: 51%): 1 H NMR (MeOD, δ, ppm, 400 MHz): 9.64 (dt, J =8.9, 1.1 Hz, 1H), 9.01-8.88 (m, 1H), 8.36 (dd, J = 7.4, 1.1 Hz, 1H), 8.26-8.16(m, 1H), 8.09 (dd, J = 8.0, 0.9 Hz, 1H), 7.72 (ddd, J = 20.4, 8.8, 7.6 Hz, 3H), 3.94 (s, 6H). 13 C NMR (MeOH, δ, ppm, 126 MHz): 148.02, 141.72, 131.94, 131.65,129.67, 128.76, 127.92, 126.95, 126.49, 119.59, 62.47. High resolution mass: [M + H] + Calculated m / z, 267.0798; found: 267.0798.

[0101]

[0102] NO-10, yellow solid (yield: 57%): 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 7.37-7.22(m, 2H), 7.01-6.87 (m, 3H), 4.55-4.40 (m, 2H), 3.50 (d, J = 9.6 Hz, 2H), 3.06(s, 6H). 13 C NMR (DMSO, δ, ppm, 126 MHz): 158.41, 130.05, 121.34, 115.02,68.80, 62.67, 60.29. High resolution mass: [M + H] + Calculated m / z, 182.1175; found: 182.1176.

[0103]

[0104] NO-IMQ, light grey powder (yield: 60%):1 H NMR (MeOD, δ, ppm, 400 MHz): 8.52 (d, J = 8.7 Hz, 1H), 8.28-8.10 (m, 2H), 7.77 (t, J = 7.9 Hz, 1H), 7.60 (t, J = 7.7 Hz,1H), 4.47 (d, J = 7.5 Hz, 2H), 3.35 (s, 2H), 2.28 (dq, J = 14.0, 6.9 Hz, 1H),1.02 (d, J = 6.7 Hz, 6H). 13 C NMR (MeOH, δ, ppm, 126 MHz): 145.96, 145.56,137.38, 129.49, 129.01, 127.48, 125.40, 121.99, 118.17, 113.38, 55.03, 29.33,19.14. High resolution mass: [M + H] + Calculated m / z, 257.1397; found: 257.1397.

[0105]

[0106] NO-APX, white powder (yield: 62%): 1 H NMR (MeOD, δ, ppm, 400 MHz): 8.65 (dd, J =8.6, 1.8 Hz, 1H), 8.01-7.91 (m, 6H), 7.47 (t, J = 7.9 Hz, 2H), 6.36 (q, J = 5.3Hz, 1H), 4.03 (qq, J = 10.6, 7.1 Hz, 2H), 3.02 (s, 3H), 1.88 (d, J = 5.3 Hz, 3H),1.13 (t, J = 7.1 Hz, 3H). 13C NMR (MeOH, δ, ppm, 126 MHz): 168.52, 139.05,136.88, 135.71, 134.33, 134.05, 133.72, 131.69, 131.35, 130.74, 129.23,127.74, 124.80, 121.29, 116.84, 100.76, 65.93, 37.29, 20.65, 14.43. High resolution mass: [M + H] + Calculated m / z, 257.1397; found: 257.1397.

[0107]

[0108] NO-PNP, white powder (yield: 55%): 1 H NMR (DMSO- d 6, δ, ppm, 400 MHz): 12.38 (s,1H), 8.17 (dd, J = 6.5, 1.5 Hz, 1H), 7.34-6.98 (m, 5H), 4.16 (s, 2H), 3.68 (q, J = 7.1 Hz, 1H), 1.36 (d, J = 7.1 Hz, 3H). 13 C NMR (DMSO, δ, ppm, 126 MHz): 175.03,151.54, 148.59, 137.72, 127.77, 127.20, 125.86, 119.42, 119.26, 118.64,116.42, 43.76, 26.38, 18.32. High resolution mass: [M + H] + Calculated m / z, 272.0915; found: 272.0917.

[0109]

[0110] NO-LRT, white powder (yield: 48%): 1 H NMR (MeOD, δ, ppm, 400 MHz): 8.18 (dd, J =6.5, 1.2 Hz, 1H), 7.48 (dd, J = 7.7, 1.2 Hz, 1H), 7.41-7.26 (m, 2H), 7.18 (dq,J = 5.2, 2.3 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.75 (dq, J = 13.1, 7.2, 6.5 Hz,2H), 3.50-3.32 (m, 4H), 3.01-2.82 (m, 2H), 2.51 (ddd, J = 13.4, 8.4, 4.5 Hz,1H), 2.35 (ddd, J = 14.2, 6.5, 4.1 Hz, 1H), 2.25 (ddd, J = 13.4, 8.4, 4.4 Hz,1H), 1.97 (ddd, J = 14.1, 6.5, 4.0 Hz, 1H), 1.26 (t, J = 7.1 Hz, 3H). 13 C NMR (MeOH, δ, ppm, 126 MHz): 157.04, 150.59, 142.65, 140.20, 138.36, 134.72,133.42, 131.13, 130.01, 126.89, 126.03, 124.39, 62.65, 45.35, 44.93, 32.68,30.96, 14.80. High resolution mass: [M + H] + Calculated m / z, 399.1470; found: 399.1470.

[0111]

[0112] Scheme S2. Chemical synthesis of NO-CPT.

[0113] NO-CPT was synthesized according to the method previously reported in the literature. 3 30% hydrogen peroxide (5 mL) was added to CPT (1 mmol, 1 eq.) suspended in acetic acid (20 mL). The reaction mixture was stirred at 80 °C overnight and then poured into ice water (100 mL). The precipitated needle-like crystals were collected by filtration and dried under reduced pressure (yield: 83%). 1 H NMR (DMSO- d 6, δ, ppm,400 MHz): 8.61 (d, J = 8.6 Hz, 1H), 8.18 (d, J= 8.5 Hz, 2H), 8.08 (s, 1H), 7.89(ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 7.80 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H), 6.52 (s,1H), 5.41 (s, 2H), 5.26 (s, 2H), 1.98-1.71 (m, J = 7.1 Hz, 2H), 0.88 (t, J = 7.3Hz, 3H). 13 C NMR (DMSO, δ, ppm, 126 MHz): 172.57, 156.50, 150.00, 141.41,140.61, 137.34, 132.01, 130.65, 130.01, 129.66, 129.18, 119.97, 119.56,118.46, 101.67, 72.47, 65.39, 50.94, 30.71, 7.92. High resolution mass: [M + H] + Calculated m / z, 365.1131; found: 365.1132.

[0114] Test Method Reduction of N-oxides under X-ray irradiation. The detailed procedure is described below using X-ray-induced reduction of NO-1 as an example. A stock solution of NO-1 (10 mM in DMSO) was diluted in PBS to a final concentration of 10 µM. The solution was carefully degassed by three freeze-pump-thaw cycles and bubbling N2 before X-ray irradiation from 0 to 60 Gy (RS2000 PRO, 225 kV, 17.7 mA, Radsource). The reaction mixture was analyzed by UPLC and fluorescence spectroscopy.

[0115] Computational studies with DFT calculations.

[0116] Computational methods. All density functional theory (DFT) calculations were performed with Gaussian 09 series programs. The B3LYP function with the 6-311+G (d, p) basis set was used to calculate the single-point energy in water solvent to provide more accurate energy information. The solvent effect was considered by single-point calculations based on gas-phase N-oxides and the SMD continuum dissolution model. The Gibbs free energy of N-oxides calculated using the B3LYP function was used to discuss the energy. The molecular orbital energy and distribution of N-oxides calculated using the B3LYP function, especially the HOMO and LUMO, were studied.

[0117] Compare the calculations of the reactivity of N-oxides with that of hydrated electrons. It is known that the reaction of hydrated electrons with N-oxides is diffusion controlled, so it is feasible to consider only thermodynamics. The electron affinity (ΔG) of an N-oxide refers to the free energy change of the conversion of an N-oxide to its anion (1 Hartree = 2625.5 kJ / mol):

[0118] Will The reactivity of the N-oxide can be determined by comparing it with the enthalpy of formation of the hydrated electrons. The electron affinity can be obtained by calculating the Gibbs free energy of the N-oxide and the N-oxide anion separately. The absolute free energy of electron solvation in water ( e =35.5 kcal / mol) to determine whether the reaction has occurred. ) to indicate the reactivity of different nitrogen oxides:

[0119] Table 1. Calculation data of representative N-oxides

[0120] a Free energy of N-oxide in water solvent calculated by B3-LYP; b Free energy of N-oxide anion in water solvent calculated by B3-LYP; c The electron affinity of N-oxides ( ); d The electrode potential of N-oxide ( ).

[0121] Cell culture. CT26 cells and HCT116 cells were cultured in RPMI-1640 medium. The medium was supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin (100 units / mL), and streptomycin (100 µg / mL). All these cells were cultured in a 5% CO2 incubator at 37 °C, and the medium was changed every 2-3 days. After growing to 80% confluence, the cells were trypsinized and then seeded in culture dishes or 96-well plates overnight for further experiments.

[0122] Confocal imaging. Cells were usually imaged at 2 × 10 5 Cells / mL were seeded in 35 mm confocal dishes and cultured for 24 h at 37 °C in an incubator containing 5% CO2. A stock solution of NO-1 (10 mM) was prepared in DMSO. Adherent cells were rinsed twice with PBS before adding NO-1 (final concentration of 10 µM) to 1 mL of RPMI-1640 medium. Cells were then incubated with AnaeroPack (Mitsubishi Gas Chemical Company, Inc.) for 30 min at 37 °C to create a hypoxic environment and subsequently exposed to X-ray radiation. Fluorescence images were taken using a laser scanning confocal microscope with an excitation wavelength of 405 nm.

[0123] Cell viability assay. Cell viability was assessed using CCK-8 assay, and the treated groups were normalized to the control group.

[0124] To determine the cytotoxicity of NO-1, HCT116 and CT26 cells were cultured at 1 × 10 4 / mL concentration was inoculated into a 96-well plate, and 100 µL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin was used in each well. After culturing at 37 °C for 24 hours, the cells were incubated with different concentrations of NO-1 for 12 hours. The medium in each well was then replaced with blank medium containing CCK-8 at a final concentration of 0.5 mg / mL. The cells were incubated at 37 °C for 2 hours, and the absorbance was measured at a wavelength of 450 nm. The absorbance of the treated cells was compared with that of the control group, and the viability of the control group was set as 100%.

[0125] To determine the cytotoxicity of CPT and NO-CPT, HCT116 cells and CT26 cells were seeded in 96-well plates at a concentration of 1 × 10 4 / mL, 100 µL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin was used per well and cultured at 37°C for 24 hours. The cells were then incubated with different concentrations of CPT and NO-CPT for 48 hours. The medium in each well was then replaced with blank medium containing a final concentration of 0.5 mg / mL CCK-8. The cells were incubated at 37°C 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, and the viability of the control group was set as 100%.

[0126] To determine the cytotoxicity of NO-CPT activated by X-rays, HCT116 cells and CT26 cells were seeded in 96-well plates at a concentration of 1 × 10 4 / mL, 100 µL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin was used per well, kept at 37°C for 24 hours, and then treated in different ways: 1. Incubate cells with NO-CPT (0.5 µM) for 48 hours.

[0127] 2. Incubate cells with AnaeroPack for 30 minutes and expose to 16 Gy X-rays. Then incubate in normoxia for 48 hours.

[0128] 3. Incubate cells with CPT (0.5 µM) for 48 hours.

[0129] 4. Incubate cells with 16 Gy X-ray irradiated NO-CPT (0.5 µM) for 48 hours.

[0130] 5. Cells were incubated with NO-CPT (0.5 µM) for 30 minutes in the AneroPack and exposed to 16 Gy X-rays followed by incubation in normoxia for 48 hours.

[0131] The medium in each well was then replaced with blank medium containing CCK-8 at a final concentration of 0.5 mg / mL. The cells were incubated at 37 °C for 2 hours, and the absorbance was measured at a wavelength of 450 nm. The absorbance of the treated cells was compared with that of the control group, and the viability of the control group was set as 100%.

[0132] Animal Model. Female BALB / c nude mice aged 6 to 8 weeks were ordered from Vital River Laboratories (Beijing, China) and housed under specific pathogen-free (SPF) conditions with free access to standard chow and water. Approximately 2 × 10 6HCT116 cells were implanted subcutaneously into the right thigh of BALB / c nude mice.

[0133] Biodistribution analysis. HCT116 tumor-bearing mice were injected with NO-CPT (10 mg / kg) by intravenous route. The mice were then sacrificed at the pre-set time points after injection, and tumor tissues were collected, weighed wet weight, and analyzed by UPLC-UV (λ abs =380 nm) to measure the concentration of NO-CPT.

[0134] In vivo radiation induces activation of NO-CPT. When tumor size reaches approximately 100 mm 3 At 1 hr post-injection, mice were randomly divided into 4 groups (n = 6) and treated with PBS, X-ray, NO-CPT, and NO-CPT + X-ray (intravenous injection, NO-CPT = 10 mg / kg). X-ray irradiation was performed at 1 hour post-injection (4 Gy). Tumor size was measured every other day using a caliper. Tumor volume was calculated as follows: Volume = Length × Width 2 / 2. Body weight was measured every 2 days. After killing the mice, solid tumors were removed, weighed, and photographed.

[0135] Statistical analysis. Statistical analysis was performed using GraphPad Prism 6. Statistical comparisons were performed using two-way analysis of variance.

[0136] Abbreviations and technical terms

Claims

1. A method for activating an N-oxide compound using radiation, characterized in that The N-oxide compound includes at least one aryl or heteroaryl group, and the N-oxide compound can be reduced to the corresponding tertiary amine after irradiation with radiation.

2. The method according to claim 1, characterized in that The nitrogen atom in the N-oxide group is directly bonded to the at least one aryl group or heteroaryl group, or the nitrogen atom in the N-oxide group is an aromatic atom constituting the at least one aryl group or heteroaryl group.

3. The method according to claim 1 or 2, wherein the N-oxide compound has a structure of general formula I, ， Wherein R is independently selected from alkyl groups (e.g., C 1-4 alkyl) and cycloalkyl (e.g. C 3-6 cycloalkyl), Ar is C6-C 20 Aryl, preferably C6-C 14 Aryl, most preferably C6-C 10 Aryl, wherein the aryl group is optionally substituted by alkyl, hydroxy, amino, aminosulfonyl (NH2-SO2-), -C(O)HR', -OC(O)HR', -C(O)-NR 1 Substituted, wherein optionally, the two substituents on the aryl group can form a heterocyclic ring, preferably a 6-membered unsaturated heterocyclic ring, preferably containing O, NR 1 , CR 1 , a 6-membered heterocyclic ring having C=O as a ring member, R 1 and R' are independently selected from hydrogen and alkyl (e.g. C 1-4 alkyl).

4. The method according to claim 3, wherein R is independently selected from the group consisting of C1-C4 alkyl, and preferably selected from the group consisting of methyl and ethyl, and / or Ar is phenyl or naphthyl, wherein phenyl or naphthyl is unsubstituted or substituted by alkyl, alkenyl, hydroxy, amino, aminosulfonyl (NH2-SO2-), -C(O)HR', -OC(O)HR', -C(O)-NR 1 substituted, wherein optionally, the two substituents on the phenyl or naphthyl group may form a heterocyclic ring, preferably a 6-membered unsaturated heterocyclic ring, preferably containing O, NR 1 , CR 1 , a 6-membered heterocyclic ring with C=O as a ring member, R 1 and R' are independently selected from hydrogen and alkyl (e.g. C 1-4 alkyl), Preferably, Ar is a naphthyl group optionally substituted by an alkyl group or an aminosulfonyl group (NH2-SO2-), or the heterocyclic ring formed by Ar and two substituents thereon is a coumarin group or a derivative thereof, or a 1,8-naphthalimide group or a derivative thereof.

5. The method according to claim 1 or 2, wherein the N-oxide compound has a structure of general formula II, ， Where R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, acylamino, which may be substituted by a heterocycle, which may be optionally substituted by an alkyl carbonate alkoxy group; and the substituents on the ring shown above may optionally form one or two aryl or heteroaryl or heterocycle condensed on the ring shown above, which may be substituted by amino, halogen, alkyl, hydroxyl, carboxyalkyl or heterocycle, which may be optionally substituted by an alkoxycarbonyl group; The premise is R 2 , R 3 , R 4 , R 5 and R 6 Not at the same time as hydrogen.

6. The method according to claim 5, wherein R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from the group consisting of hydrogen, C1-C4 alkyl, amino, hydroxyl, acylamino, which may be substituted by a 5-, 6- or 7-membered heterocycle, which may be optionally substituted by a (C1-C4) alkyl carbonate (C1-C4) alkoxy group; and the substituents on the ring shown above may optionally form one or two phenylene groups or 5-, 6- or 7-membered heteroaryl or heterocycle condensed on the ring shown above, and the one or two aryl or heteroaryl or heterocycle may be substituted by amino, halogen, C1-C4 alkyl, hydroxyl, carboxyl (C1-C4) alkyl or a 5-, 6- or 7-membered heterocycle, which may be optionally substituted by a (C1-C4) alkoxycarbonyl group; The premise is R 2 , R 3 , R 4 , R 5 and R 6 Not at the same time as hydrogen.

7. The method according to claim 1 or 2, wherein the N-oxide compound has the structure of Formula IIa, IIb, IIc, IId, IIe or IIf: in, R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 independently selected from hydrogen, hydroxy, amino, halogen, C1-C5 alkyl, (C1-C4)alkyl-O(CO)-(C1-C4)alkyl and (C1-C4)alkyl-O(CO)H, and One or more hydrogen atoms on the ring in the general formula IIa, IIb, IIc, IId, IIe or IIf are optionally substituted by hydroxyl, amino, halogen or C1-C5 alkyl, preferably halogen or C1-C4 alkyl; Preferably, R 7 , R 9 and R 13 are independently selected from the group consisting of hydrogen, hydroxyl, amino and halogen, R 8 , R 10 , R 11 , R 12 and R 14 independently selected from the group consisting of C1-C5 alkyl, (C1-C4)alkyl-O(CO)-(C1-C4)alkyl, (C1-C4)alkyl-O(CO)H, and One or more hydrogen atoms on the aromatic ring in Formula IIa, IIb, IIc, IId, IIe or IIf are optionally substituted by halogen or C1-C4 alkyl.

8. The method according to claim 1 or 2, wherein the tertiary amine is a biologically active compound, such as a fluorescent compound, preferably a coumarin derivative or a chemotherapeutic agent.

9. The method according to claim 1 or 2, wherein the N-oxide compound is an aniline N-oxide or an aromatic heterocyclic N-oxide, such as a compound in the following table or a pharmaceutically acceptable salt or solvate thereof: 。 10. A method of treating or diagnosing a disease, the method comprising administering to a subject a therapeutically effective amount of an N-oxide prodrug and irradiating the subject with a therapeutically effective amount of radiation, Preferably, the N-oxide prodrug is as defined in any one of claims 1-9.

11. The method of claim 10, which is used to treat or inhibit cancer, reduce cancer severity, reduce cancer risk, or inhibit cancer metastasis in a subject.

12. The method according to claim 10 or 11, wherein the cancer is selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma and malignant lymphoma.

13. The method according to any one of claims 10-12, wherein the subject is irradiated after administration of the N-oxide prodrug, preferably 20 minutes after, 1 hour after, 2 hours after, 4 hours after administration of the N-oxide prodrug.

14. The method of any one of claims 10-13, wherein the N-oxide prodrug is administered in an amount of about 0.005 mg / kg to about 100 mg / kg, for example, about 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 100 mg / kg.

15. The method of any one of claims 10-14, wherein the N-oxide prodrug and radiation are administered once every two days, once every three days, once every four days, once every five days, once every seven days, once every ten days, once every two weeks, once every three weeks, once every four weeks, and the N-oxide prodrug and radiation are administered continuously for 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.

16. The method of any one of claims 10-15, wherein the N-oxide prodrug is administered orally or parenterally.

17. The method of any one of claims 10-16, wherein the radiation is X-rays or gamma rays, and 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, or 4 Gy or less.

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