WO2023217093A1

WO2023217093A1 - Prodrug activation of n-oxides by radiotherapy

Info

Publication number: WO2023217093A1
Application number: PCT/CN2023/092817
Authority: WO
Inventors: Zhibo LIU; Zexuan DING
Original assignee: Peking University
Priority date: 2022-05-07
Filing date: 2023-05-08
Publication date: 2023-11-16

Abstract

Provided herein pertains to the biomedical field, and particularly relates to a method for activating N-oxide prodrugs with radiation, and a method of treating or suppressing a cancer, reducing its severity, lowering its risk or inhibiting its metastasis in an individual. The method comprises administering to the individual a therapeutically effective amount of an N-oxide prodrug, and radiating the individual with a therapeutically effective amount of radiotherapy.

Description

Prodrug activation of N-oxides by radiotherapy

Technical Field

The present invention pertains to the biomedical field, and particularly relates to a method for activating N-oxide prodrugs with radiation, and a method of treating or suppressing a cancer, reducing its severity, lowering its risk or inhibiting its metastasis in an individual. The method comprises administering to the individual a therapeutically effective amount of an N-oxide prodrug, and radiating the individual with a therapeutically effective amount of radiotherapy.

Background of the Invention

Since first introduced in 1958, prodrug strategies have been widely used to address drug delivery problems in tumor therapy. Ideally, prodrugs can deliver over 50-fold the normal dose to the desired location and cure tumors normally resistant to chemotherapy. Effectively activating the parent drugs is perhaps the most critical step of prodrug strategy. However, controlled activation of prodrug is still a long-standing challenge in clinics due to the lack of tumor-selective activating strategies. Therefore, spatiotemporally precise prodrug activation in a tumor-selective manner is a pressing need and full of challenges, prompting us to look for suitable chemical tools.

Inspired by the application of light in phototherapy, clinically relevant ionizing radiation (X-ray, γ-ray, etc. ) also has the potential to be a precise perturbing tool in living bodies as it features precision and deep tissue penetration (up to 15 cm) . Besides, though over 50%of cancer patients take radiotherapy, radiotherapy may fail because the total dose limitation (generally less than 60 Gy) and hypoxia resistance since oxygen is involved in the fixation of radiation-induced DNA damage. Consequently, radiotherapy combined with chemotherapy has better chances to treat cancers. Establishing site-directed radiotherapy-activated chemotherapy strategies are expected to reduce the combined toxicities of the dual treatments and achieve better therapeutic efficacy.

Radiochemical alterations towards molecules include direct and indirect effects. Among them, the indirect radiochemical effect that radiation deposits on environment components is dominant in living systems. It is mainly through water radiolysis as water accounts for 70-80%of tissue weight. As shown in Figure 1, various reactive particles instantly formed through the water radiolysis, followed by a cascade of reactions that complete within 10^-4 seconds. Cleavage chemistry on prodrug molecules induced by hydroxyl radical (·OH, as one of the major products from water radiolysis) and hydrogen radical (·H) has been reported previously. Nevertheless, due to the instant ·OH quenching in the reductive environments (e.g., hypoxia tumors) , the application in living systems is hampered. Interestingly, another major product, hydrated electron (e^- _aq) , which is reported as a “super reductant” for challenging reactions, increases in the reductive environment. Hence in the present invention, we seek to investigate whether an e^- _aq-mediated reaction induced by radiotherapy will function as a chemical tool applied for prodrug activation in a tumor-selective manner.

Summary of the Invention

Precise activating chemotherapeutic prodrugs in a tumor-selective manner is an ideal way to cure cancers without causing systemic toxicities. Though many efforts have been made, developing spatiotemporally controllable activation methods is still an unmet challenge. Here the present invention reports a novel prodrug activation strategy using radiotherapy (X-ray) . Due to its precision and deep tissue penetration, X-ray matches the need for altering molecules in tumors through water radiolysis. It is first demonstrated that N-oxides can be effectively reduced by hydrated electron (e^- _aq) generated from radiation both in tubes and living cells. A screening is performed to investigate the structure-reduction relationship and mechanism of the e^- _aq-mediated reductions. The present invention then applies the strategy to activate N-oxide prodrugs. The anticancer drug camptothecin (CPT) based N-oxide prodrug shows a remarkable anticancer effect upon activation by radiotherapy. This radiation-induced in vivo chemistry may enable versatile designs of radiotherapy-activated prodrugs, which are of remarkable clinical relevance, as over 50%of cancer patients take radiotherapy. This radiation-induced in vivo chemistry can also greatly increase the selectivity of chemotherapy or fluorescent compounds, increase the efficacy of the radiotherapy, and reduce the toxicity of administering chemotherapeutic drugs or fluorescent compounds through controlled release of the corresponding chemotherapeutic prodrugs or fluorescent prodrugs.

In one aspect, the invention provides a method for activating N-oxide compound with radiation, characterized in that the N-oxide compound comprises at least one aryl or heteroaryl group, and the N-oxide compound can be reduced to a corresponding tertiary amine after irradiated with the radiation.

In another aspect, the invention provides a method of treating or diagnosing a disease, the method comprises administering to an individual a therapeutically effective amount of an N-oxide prodrug, and radiating the individual with a therapeutically effective amount of radiation.

In another aspect, the invention provides use of an N-oxide prodrug for preparing a medicament for treating or diagnosing a disease in an individual, wherein the individual is also radiated with radiation.

Brief Description of the Drawings

Figure 1 shows radiation-induced prodrug activation in tumors. The water radiolysis by ionizing radiation generates various reactive particles (G-value means the number of molecules formed by absorbing 100 eV energy in the system) . Among them, hydroxyl radical (·OH) was previously reported for controlled drug release. In this work, N-oxides are found to be reduced by hydrated electrons (e^- _aq) generated from water radiolysis. Benefiting from this reaction, nitrogen-containing drugs (mainly tertiary amine and N-heterocyclic ones) can be “caged” in a high atom economic way (by single oxygen atom) and spatiotemporally activated at tumor sites by radiotherapy.

Figure 2 shows Radiation-induced reduction of N-oxides. (a) Schematic illustration: fluorogenic reaction of a model N-oxide compound (NO-1) with e^- _aq from water radiolysis. Radiation dosage–dependent generation of A-1 from NO-1 (10 μM in PBS) determined by (b) UPLC-UV (detected λ_abs= 380 nm) and (c) fluorescence spectrum (λ_ex= 380 nm and λ_em= 460 nm) . Inset: photography of radiation dosage-dependent fluorescence (d) Fluorescence emission assay (λ_ex= 380 nm and λ_em= 460 nm) of possible reduction of NO-1 (10 μM) by biologically reductants (1 mM) for 24 h in PBS compared with 60 Gy radiation-induced instant reduction, n = 3. (e) Representative confocal fluorescence images of living CT26 and HCT116 cell lines. The cells were pretreated with NO-1 (10 μM in RPMI-1640 medium) followed by different amounts of radiation. Scale bar: 20 μm.

Figure 3 shows structure screening of N-oxides for radiation-induced reduction. (a) Chemical structures of N-oxides (NO-2 to NO-10) that have been assayed by radiation-induced reduction (10 μM in PBS, 60 Gy) . Reduction products are detected by UPLC-MS. (b) G-values of generated reduction products from NO-2 to NO-10. (c) Radiation dosage–dependent generation of reduction products (A-8) from aromatic heterocyclic NO-8 (10 μM in PBS) determined by UPLC-MS (m/z = 146) .

Figure 4 shows mechanism study of the e^- _aq-mediated reduction of N-oxides. (a) Proposed mechanism of the e^- _aq-mediated reduction. (b) Molecular orbitals of NO-1 which indicates e^- _aq is captured by π system instead of the antibonding orbital of the N⁺-O^- bond. (c) |ΔG| between the anion radical intermediate and ground state of N-oxides compared with |ΔG| of e^- _aq. (d) For representative N-oxides: NO-1 (aniline N-oxide) , NO-7 (heterocyclic N-oxide) and NO-9 (alkyl N-oxide) , the energy levels of HOMO and LUMO as well as the energy gap (E_gap) are indicated.

Figure 5 shows radiation-induced activation of N-oxide prodrug. (a) Chemical structures of synthesized N-oxide drugs (10 μM in PBS) that have been assayed by 60 Gy radiation-induced reduction. (b) G-value of generated reduction products shown in (a) , n = 3. The dash lines are G-value of DHBC (3, 5-dihydroxylbenzyl carbamate, which is the most efficient group that could be removed by hydroxyl radicals from water radiolysis) . (c) Schematic of single oxygen atom “caging” and radiotherapy “decaging” prodrug strategy. NO-CPT was used as a model prodrug. Cell viability assay of radiation-activated NO-CPT in (d) HCT116 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) .

Figure 6 shows radiotherapy activates a prodrug that rejects tumor growth. (a) Treatment scheme. Nu/Nu mice were implanted subcutaneously with HCT116 cells, followed by intravenous (iv) injection of NO-CPT (10 mg/kg) and radiotherapy (4 Gy each treatment) . (b) Time-dependent accumulation of NO-CPT in tumor tissue detected by UPLC-UV, n = 3. (c) Tumor growth curves of tumor-bearing mice after the indicated treatments. (d) Average tumor weight at day 27after the first treatment. (e) Weight change curves of mice subjected to different treatment. n = 6, two-tailed unpaired Student’s t-test, ***P < 0.001.

Figure 7 shows chemical structures of both synthetic raw materials and proposed reduction products of N-oxides (NO-1 to -NO-10, NO-IMQ, NO-APX, NO-PNP, NO-LRT, NO-CPT, respectively) .

Figure 8 shows, (a) UPLC-MS spectra (selected-ion monitoring signal of [A-1+H] ⁺, m/z = 232) of generated A-1 from NO-1 (10 μM in PBS) after 60 Gy radiation. (b) Positive ion mode mass spectrum is shown for reduction product A-1 (retention time = 4.08 min in (a) ) . (c) UPLC-UV spectra (λ_abs= 320 nm) and (d) UV-Vis absorbance spectra of NO-1 (10 μM in PBS) after radiation. (e) Radiation dosage–dependent generation of A-1 (10 μM NO-1 in PBS under X-ray radiation) determined by fluorescence emission spectrum (λ_ex= 380 nm) . (f) Fluorescence emission spectrum of NO-1 (10 μM in PBS with 10%FBS) before and after 60 Gy radiation (λ_ex= 380 nm) .

Figure 9 shows normalized amount of generated A-1 from NO-1 (100 μM in PBS, treated with e^- _aq quencher and hydroxyl radical quenchers, respectively) after 60 Gy radiation determined by UPLC-UV (λ_abs= 380 nm) .

Figure 10 shows cell viability assay of NO-1 incubated with (a) HCT116 and (b) CT26 cells for 12 h, n = 5.

Figure 11 shows radiation dosage–dependent reduction of NO-CPT (10 μM in PBS) determined by (a) UPLC-MS, m/z = 349, (b) positive ion mode mass spectrum is shown for CPT (retention time = 2.66 min) and (c) fluorescence emission spectrum (λ_ex= 380 nm) .

Figure 12 shows radiation-induced reduction of 10 μM NO-CPT in (a) PBS containing 10%FBS, (b) HCT116 cell lysate, determined by UPLC-MS (selected-ion monitoring signal of [CPT+H] ⁺, m/z = 349) and (c) PBS containing 10%FBS, (d) HCT116 cell lysate, determined by UPLC-UV (λ_abs= 380 nm) .

Figure 13 shows cell viability assays of (a) HCT116 and (b) CT26 cells incubated with CPT and NO-CPT, respectively, for48 h. n = 5.

Figure 14 shows photographs of representative tumors at day 27 after different treatments.

Figure 15 shows hematoxylin & eosin (H&E) stained tissue sections from the major organs (heart, liver, spleen, lung, kidney) . No noticeable abnormality can be observed in these major organs of mice after treatment.

Detailed Description of the Invention

Definitions

Unless otherwise defined below, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques that are generally understood in the art, including those obvious changes or equivalent replacements of the techniques for those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

As used herein, the terms "including" , "comprising" , "having" , "containing" or "comprising" , and other variants thereof, are inclusive or open, and do not exclude other unlisted elements or method steps.

As used herein, “chemotherapeutic agent” or “chemotherapeutic drug” refers to chemotherapeutic drugs that can kill tumor cells, and these drugs can act on different stages of tumor cell growth and reproduction, thereby inhibit or kill tumor cells.

The term "alkyl" as used herein, alone or as part of another group, refers to an unsubstituted straight or branched aliphatic hydrocarbon containing from 1 to 12 carbon atoms (ie, C_1-12 alkyl) or an indicated 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 such as methyl, ethyl, n-propyl or isopropyl, or the like. In one embodiment, the alkyl is C_1-4 alkyl. Non-limiting examples of C_1-12 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl and decyl. Examples of C_1-4 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl.

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, which comprises 1 or 2 rings having 3 to 12 carbon atoms or an indicated number of carbon atoms (i.e., C_3-12 cycloalkyl) . In one embodiment, the cycloalkyl has two rings. In one embodiment, the cycloalkyl has one ring. In another embodiment, the cycloalkyl group is selected from the group consisting of C_3-8 cycloalkyl groups. In another embodiment, the cycloalkyl group is selected from the group consisting of C_3-6 cycloalkyl groups. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decahydronaphthyl, adamantyl, cyclohexenyl, and cyclopentenyl.

The term "alkoxyl" or “alkoxy” as used herein, alone or as part of another group, refers to an oxygen linked to an alkyl group as defined above.

The term “heteroalkyl” as used herein, alone or as part of another group, refers to an alkyl group wherein one carbon atom is replaced with a heteroatom (such as N, O or S) or a carbonyl group (C=O) .

The term "alkenyl" is intended to include hydrocarbon chains of either straight or branched configuration having the specified number of carbon atoms and one or more, preferably one to two, carbon-carbon double bonds that may occur in any stable point along the chain. For example, "C₂-C₆ alkenyl" is intended to include C₂, C₃, C₄, C₅, and C₆ alkenyl groups. Examples of alkenyl include, but are not limited to, ethenyl, 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.

The term "aryl" and "heteroaryl" and “aromatic heterocyclic” , as used herein, refer to stable mono-or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. In certain embodiments of the present invention, "aryl" refers to a mono-or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl" , as used herein, refers to a monocyclic, bicyclic or tricyclic aromatic radical having from five to fifteen, preferably five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

The term "heterocyclyl" , "heterocyclic" or "heterocycle" , as used herein, refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group, including, but not limited to a bi-or tri-cyclic group comprising fused six-membered rings or seven-membered rings, wherein at least one carbon atom of one of the rings is replaced by a heteroatom. Each heteroatom is independently selected from the group consisting of atoms of oxygen, sulfur (including sulfoxide and sulfone) and/or nitrogen (which may be oxidized or quaternized) . The term "heterocyclyl" , "heterocyclic" or "heterocycle" is intended to include a group wherein -CH₂-in the ring is replaced by -C (=O) -, for example, cyclic ureido (such as 2-imidazolidinone) and cyclic amido (such as β-lactam, γ-lactam, δ-lactam, ε-lactam) and piperazin-2-one; wherein (i) each 5-membered ring has 0 to 1 double bond and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl 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 tetrahydrofuryl.

In certain embodiments, a "substituted aryl, heteroaryl, heterocyclyl or heterocycle" group is utilized and as used herein, refers to an aryl, heteroaryl, heterocyclyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to alkyl; heteroalkyl; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F;-Cl;-Br;-I; -OH;-NO₂;-CN;-CF₃; -CH₂CF₃;-CHC1₂;-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, wherein each occurrence of R_x independently includes, but is not limited to, alkyl, heteroalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the alkyl, heteroalkyl, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein.

The term “coumarine” means the molecule having the following structure, and the term “coumarine or its derivatives” means the molecules having the following skeleton structure, wherein one or more hydrogen atoms, preferably one hydrogen atom in the skeleton structure can be replaced with a substituting group, such as alkyl, e.g. C₁ to C₄ alkyl, or halogen. Preferably, the 4-position hydrogen atom in the skeleton structure is replaced with an alkyl group, e.g. C₁ to C₄ alkyl. When mentioning “a coumarine group or its derivatives” , it means the group generated when a hydrogen radical is removed from a coumarine molecule or its derivatives, for example, a hydrogen radical on the aromatic ring, such as the hydrogen radical at the 7-position.

The term “1, 8-naphthalic imide” means the molecule having the following structure, and the term “1, 8-naphthalic imide” means the molecules having the following skeleton structure, wherein one or more hydrogen atoms (such as the hydrogen atom on the aromatic ring, or the hydrogen atom connected to the nitrogen atom) , preferably one hydrogen atom in the skeleton structure can be replaced with a substituting group, such as alkyl, e.g. C₁to C₄ alkyl, or halogen. Preferably, the hydrogen atom connected to the nitrogen atom is replaced with an alkyl group, e.g. C₁to C₄ alkyl. When mentioning “a 1, 8-naphthalic imide group or its derivatives” , it means the group generated when a hydrogen radical is removed from a 1, 8-naphthalic imide molecule or its derivatives, such as the hydrogen radical at the 4-position.

The term “halogen” , as used herein, comprises F, Cl, Br, I, etc.

The term "pharmaceutically acceptable salt", as used herein, includes both acid addition salts and base addition salts of a compound.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclohexylaminosulfonate, ethanedisulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, aldarate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate.

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

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 the pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The term "solvate" as used herein is a substance formed by combination, physical binding and/or solvation of a compound of the invention with a solvent molecule, such as a disolvate, a monosolvate or a hemisolvate, wherein the ratio of the solvent molecule to the compound of the invention is about 2: 1, about 1: 1 or about 1: 2, respectively. This kind of physical bonding involves ionization and covalent bonding (including hydrogen bonding) in different degrees. In some cases (e.g., when one or more solvent molecules are incorporated into crystal lattice of crystalline solid) , the solvate can be isolated. Thus, the solvate comprises both solution phase and isolatable solvates. The compounds of the invention may be in solvated forms with pharmaceutically acceptable solvents (such as water, methanol and ethanol) , and the present application is intended to encompass both solvated and unsolvated forms of the compounds of the invention.

One type of solvate is a hydrate. "Hydrate" relates to a specific subset of solvates wherein the solvent molecule is water. Solvates generally function in the form of pharmacological equivalents. The preparation of solvates is known in the art, see for example, M. Caira et al, J. Pharmaceut. Sci., 93 (3) : 601-611 (2004) , which describes the preparation of a solvate of fluconazole with ethyl acetate and water. Similar methods for the preparation of solvates, hemisolvates, hydrates and the like are described by van Tonder et al, AAPS Pharm. Sci. Tech., 5 (1) : Article 12 (2004) and A.L. Bingham et al, Chem. Commun. 603-604 (2001) . A representative and non-limiting method for the preparation of solvate involves dissolving a compound of the invention in a desired solvent (organic solvent, water or a mixture thereof) at a temperature above 20 ℃ to about 25 ℃, and then the solution is cooled at a rate sufficient to form a crystal, and the crystal is separated by a known method such as filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in the crystal of the solvate.

"Pharmaceutically acceptable carrier" in the context of the present invention refers to a diluent, adjuvant, excipient or vehicle together with which the therapeutic agent is administered, and which is suitable for contacting a tissue of human and/or other animals within the scope of reasonable medical judgment, and without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.

The pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the invention include, but are not limited to, sterile liquids such as water and oils, including those oils derived from petroleum, animals, vegetables or synthetic origins, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol and the like. The pharmaceutical composition may further contain a small amount of a wetting agent, an emulsifier or a pH buffering agent as needed. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington’s Pharmaceutical Sciences (1990) .

The pharmaceutical compositions of the invention may act systemically and/or locally. For this purpose, they may be administered via a suitable route, for example by injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular administration, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical administration, in form of ophthalmic preparation or by inhalation.

For these routes of administration, the pharmaceutical compositions of the invention may be administered in a suitable dosage form.

The dosage forms include, but are not limited to, tablets, capsules, troches, hard candy, pulvis, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

The term "effective amount" as used herein refers to an amount of active ingredient that, after administration, will relieve to some extent one or more symptoms of the condition being treated.

As used herein, "individual" includes a human or a non-human animal. Exemplary human individual includes a human individual (referred to as a patient) suffering from a disease (such as the disease described herein) or a normal individual. "Non-human animal" in the present invention includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc. ) .

Therapeutic methods and uses

In one embodiment, the invention provides a method for activating N-oxide compound with radiation, characterized in that the N-oxide compound comprises at least one aryl or heteroaryl group, and the N-oxide compound can be reduced to a corresponding tertiary amine after irradiated with the radiation.

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.

In a preferred embodiment, the N-oxide compound has the general formula I,

ArR₂N⁺-O^-Formula I,

wherein R is independently selected from the group consisting of alkyl, such as C_1-4 alkyl, and cycloalkyl, such as C_3-6 cycloalkyl,

Ar is C₆-C₂₀ aryl group, preferably C₆-C₁₄ aryl group, most preferably C₆-C₁₀ aryl group, wherein the aryl group is optionally substituted with alkyl, hydroxyl, amino, aminosulfonyl (NH₂-SO₂-) , -C (O) HR’, -OC (O) HR’, -C (O) -NR¹, wherein optionally two substituting groups on the aryl group can form heterocycle, preferably 6-membered unsaturated heterocycle, preferably 6-membered heterocycle comprising O, NR¹, CR¹, C=O as the ring member,

R¹ and R’ are independently selected from the group consisting of hydrogen and alkyl, such as C_1-4 alkyl.

In a preferred embodiment of Formula I, R is independently selected from the group consisting of C₁-C₄ alkyl, and is preferably selected from the group consisting of methyl and ethyl, and/or

Ar is phenyl or naphthyl, wherein the phenyl or naphthyl is unsubstituted or substituted with alkyl, alkenyl, hydroxyl, amino, aminosulfonyl (NH₂-SO₂-) , -C (O) HR’, -OC (O) HR’, -C (O) -NR¹, wherein optionally two substituting groups on the phenyl or naphthyl group can form heterocycle, preferably 6-membered unsaturated heterocycle, preferably6-membered heterocycle comprising O, NR¹, CR¹, C=O as the ring member,

R¹ and R’ are independently selected from the group consisting of hydrogen and alkyl, such as C_1-4 alkyl,

preferably, Ar is naphythyl optionally substituted with alkyl, or aminosulfonyl (NH₂-SO₂-) , or Ar with two substituting groups thereon form heterocycle is a coumarine group or its derivatives, or a 1, 8-naphthalic imide group or its derivatives.

In another preferred embodiment, the N-oxide compound has the general formula II,

wherein R², R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, amido, which can be in turn substituted with heterocycle, the heterocycle is optionally substituted with alkylcarbonatealkoxyl group; and optionally the substituting groups on the above illustrated ring can form one or two aryl or heteroaryl groups or heterocycles condensed on the above illustrated ring, said one or two aryl or heteroaryl groups or heterocycle can in turn be substituted by amino, halogen, alkyl, hydroxyl, carboxyl alkyl or heterocycle, which heterocycle is optionally substituted with alkoxylcarbonyl group;

with the proviso that R², R³, R⁴, R⁵ and R⁶ are not simultaneously hydrogen.

In a preferred embodiment of Formula II, R², R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C₁-C₄ alkyl, amino, hydroxyl, amido, which can be in turn substituted with5-, 6-, or 7-membered heterocycle, the heterocycle is optionally substituted with (C₁-C₄) alkylcarbonate (C₁-C₄) alkoxyl group; and optionally the substituting groups on the above illustrated ring can form one or two phenylene or5-, 6-, or 7-membered heteroaryl groups or heterocycles condensed on the above illustrated ring, said one or two aryl or heteroaryl groups or heterocycle can in turn be substituted by amino, halogen, C₁-C₄ alkyl, hydroxyl, carboxyl (C₁-C₄) alkyl or 5-, 6-, or 7-membered heterocycle, which heterocycle is optionally substituted with (C₁-C₄) alkoxylcarbonyl group;

In a preferred embodiment of Formula II, the N-oxide compound has the general formula IIa, IIb, IIc, IId, IIe or IIf:

wherein, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, hydroxyl, amino, halogen, C₁-C₅ alkyl, (C₁-C₄) alkyl-O (CO) - (C₁-C₄) alkyl, and (C₁-C₄) alkyl-O (CO) H, and

optionally one or more hydrogen atoms on the ring in the general formula IIa, IIb, IIc, IId, IIe or IIf can be replaced with hydroxyl, amino, halogen, or C₁-C₅ alkyl, preferably halogen or C₁-C₄ alkyl;

preferably, R⁷, R⁹, and R¹³ are independently selected from the group consisting of hydrogen, hydroxyl, amino, and halogen,

R⁸, R¹⁰, R¹¹, R¹², and R¹⁴ are 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

optionally one or more hydrogen atoms on the aromatic ring in the general formula IIa, IIb, IIc, IId, IIe or IIf can be replaced with halogen or C₁-C₄ alkyl.

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.

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

In an embodiment, the invention provides a method of treating or diagnosing a disease, the method comprises administering to an individual a therapeutically effective amount of an N-oxide prodrug, and radiating the individual with a therapeutically effective amount of radiation.

In a preferred embodiment, the N-oxide prodrug is the one as defined above.

In a preferred embodiment, said method is for treating or suppressing a cancer, reducing its severity, lowering its risk or inhibiting its metastasis in an individual.

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.

In a preferred embodiment, the individual is radiated after administering the N-oxide prodrug, preferably 20 mins, 1 hour, 2 hours, 4 hours after administering the N-oxide prodrug.

In a preferred embodiment, the N-oxide prodrug is administrated in an amount of from about 0.005 mg/kg to about 100 mg/kg, such as an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100mg/kg.

In a preferred embodiment, 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..

In a preferred embodiment, the N-oxide prodrug is administrated orally or parenterally.

In a preferred embodiment, the radiation is X-ray or gamma ray, and the intensity of the X-ray or gamma ray is 60 Gy or lower, 50 Gy or lower, 40 Gy or lower, 30 Gy or lower, 20 Gy or lower, 10 Gy or lower, 8 Gy or lower, 6 Gy or lower, 4 Gy or lower.

Examples

In order to make the objects and technical solutions of the present invention clearer, the present invention will be further described below in conjunction with specific example. It should be understood that the examples are not intended to limit the scope of the invention. Further, specific experimental methods not mentioned in the following examples were carried out in accordance with a conventional experimental method.

Example 1: Study of radiation induced reduction of NO-1 in aqueous solution and in biological environment

To pursue the present invention, it is important to design molecules that are not only stable in the biological environment, but also selectively reactive to e^- _aq. It is noticed that N-oxide, an oxidation state of tertiary amine, was reported as a bio-orthogonal reagent, and is biocompatible and stable in the blood circulation. Therefore, remotely reducing N-oxides with e^- _aq in the living system may provide an opportunity for developing in vivo chemical tools, yet has not been established.

Of note, e^- _aq is one of the major products (G-value～280 nM/Gy, the number of molecules formed by absorbing 100 eV energy in the system) of water radiolysis, and also one of the strongest reductants. It is speculated that radiation may decompose water to locally generate e^- _aq that would efficiently reduce N-oxides (Figure 2a) . To test this idea, a coumarin-based N-oxide NO-1 (an oxidation product of A-1) was synthesized as a model compound. Firstly, a deoxygenated solution of NO-1 (10 μM in PBS) was irradiated by X-ray (radiotherapy-dose, 10-60 Gy) from a small animal irradiator. Interestingly, radiation dosage–dependent reduction instantly occurred and generation of the product A-1 was detected by UPLC (Figure 2b) . As fluorophore always loses its fluoresce emission when its amine is blocked by N-oxide group, the reduction of NO-1 can be readily indicated by the released fluorescence from coumarin (A-1) . The fluorescence data and corresponding photography suggested that the amount of released coumarin is linearly correlated to the given radiation dose (Figure 2c) .

To confirm that radiation-induced reduction is mediated by e^- _aq from water radiolysis, NO-1 (100 μM in PBS) was irradiated along with 10 mM of isopropyl alcohol (i-PrOH) , sodium formate (CH₃OONa) , and KNO₃, respectively. The amount of generated A-1 from each group was determined by UPLC. As expected, compared with the control group, the amount of generated A-1 with i-PrOH and CH₃OONa increased, as they are known to produce extra e^- _aq under irradiation. In contrast, the addition of KNO₃, a quencher of e^- _aq, significantly reduced the amount of generated A-1 (Figure 9) .

It was further investigated whether such radiation-induced reduction could work in the biological environment, which is often a balance between biological stability and reactivity. To determine the stability of the N-oxide in a reducing environment, NO-1 (10 μM in PBS) was treated with biological reductants including glutathione (GSH, 1mM) , cysteine (Cys, 1 mM) and ascorbate (Vc, 1 mM) , respectively, for 24 h. The result was determined by the fluorescence emission spectrum, indicating the reductions of NO-1 by these reductants are almost negligible compared with the instant reduction after radiation (Figure 2d) .

Next, it was tested the biocompatibility of radiation-induced reduction. NO-1 (10 μM) in PBS containing 10%fetal bovine serum (FBS) was subjected to 60 Gy radiation and analyzed by fluorescence emission spectrum (Figure 8f) . The generated fluorescence shows that the reduction works even in serum environment, which is promising to be operated in the living systems. Furthermore, NO-1 was also used as a fluorogenic probe to confirm that the reduction worked in living cells. CT26 and HCT116 cell lines were pretreated with NO-1 (10 μM) , followed by low-dose radiation (0, 4, 8, 16 Gy) in hypoxia milieu. Representative confocal fluorescence images of living cells are shown in Figure 2e, indicating that after radiation the fluorescent A-1 molecules were released and the intensity is positively correlated to the radiation dose. The aforementioned data indicate that radiation-induced reduction of N-oxides does have the potential to be a chemical tool for radiotherapy-activated prodrug strategy.

Example 2: Screening N-oxides that can undergo the radiation induced reduction

A screening of N-oxides was performed to investigate the structure-reduction relationship for the purpose of developing N-oxides based prodrug systems. As shown in Figure 3a, N-oxides including aniline types (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 (10 μM in PBS) via 60 Gy radiation. The G-value of generated reduction products from NO-2 to NO-10 were quantified by UPLC and shown in Figure 3b. No reduction products from NO-6, NO-9, and NO-10 were detected. Interestingly, aromatic heterocyclic N-oxides such as NO-7 and NO-8 were efficiently reduced by radiation (Figure 3b, c) , which expanded the scope of radiation-responsive substrates.

It is worth noting that G-values of generated reduction products are almost half that of e^- _aq (280 nM/Gy) , suggesting the two-electron reduction mechanism. Accordingly, for e^- _aq-mediated N-oxides reduction we propose a hypothesis that the reaction progresses mainly in two steps (Figure 4a) . Firstly, N-oxide captures e^- _aq from water radiolysis to become an N-oxide anion radical intermediate. Then the intermediate abstracts a proton from the surrounding environment, deoxidizes via electron transfer to release a tertiary amine and an·OH which continues capturing another e^- _aq to produce a water molecule.

To further test this hypothesis, the B3LYP method was used for density functional theory (DFT) calculation. According to the LUMO orbital distribution of the model compound NO-1 (Figure 4b) , it is suggested that the e^- _aq should fill in the π system of N-oxide instead of directly in the antibonding orbital of the N⁺-O^-bond. Due to the tunnel effect of e^- _aq in the reaction process, the first step is diffusion controlled. Hence only thermodynamic factors need to be considered in the first step reaction: the reaction occurs when the absolute value of Gibbs free energy change (|ΔG|) between N-oxide and its anion radical intermediate in the aqueous phase, is greater than that of e^- _aq. The |ΔG| of different N-oxides compared with that of e^- _aq are shown in Figure 4c, among them, NO-1, NO-2, NO-3, NO-4, NO-5, NO-7, and NO-8with larger π system have better reactivity, which is consistent with the experimental data (Figure 3b, Table 1) . In consideration of that e^- _aqfills in the π system of N-oxide, the conjugation degree of π system will also affect the accessing of e^- _aq to LUMO. Since the energy difference (E_gap) between HOMO and LUMO can also reflect the conjugation degree of molecules, the energy levels of HOMO and LUMO as well as the E_gap of representative N-oxides including NO-1 (aniline N-oxide) , NO-7 (aromatic heterocyclic N-oxide) and NO-9 (alkylated N-oxide) are calculated and shown in Figure 4d.

Example 3: Study of radiation induced reduction of aromatic heterocyclic N-oxide drug molecules

Having found that aromatic heterocyclic NO-7 and NO-8 can be efficiently reduced by X-ray (Figure 3b-c) , it was noted that the percentage of aromatic N-heterocycles in FDA-approved small molecule pharmacophores continue to rise for the last five years. The nitrogen atoms of many biologically active N-heterocycles are readily interacting with DNA or proteins through hydrogen bonding, showing the structural significance of N-heterocycles for drug design and discovery. Thus, we synthesized a series of aromatic heterocyclic N-oxide drug molecules through one-spot oxidation (Figure 5a) . According to the parent drugs, these N-oxide drugs were termed as NO-Imiquimod (NO-IMQ) , NO-Ampiroxicam (NO-APX) , NO-Pranoprofen (NO-PNP) , NO-Loratadine (NO-LRT) , and NO-Camptothecin (NO-CPT) . Next, we tested their radiation-responsive reactivities. After being treated with 60 Gy radiation, the reduction of these N-oxide drugs was analyzed by UPLC. Compared with DHBCs (3, 5-dihydroxylbenzyl carbamate, the most efficient group that could be removed by hydroxyl radicals from water radiolysis) , the reduction G-value of the N-oxide drugs are much higher, indicating that e^- _aq mediated reaction is much more efficient than that mediated by ·OH (Figure 5b) .

Encouraged by the reactivity of these N-oxide drugs with relatively complex chemical structures, towards radiation-induced reduction, we took into account establishing a strategy that radiotherapy activates chemotherapeutic prodrugs of which activity are “caging” by a single oxygen atom (Figure 5c) . Anticancer drug CPT, known as a topoisomerase inhibitor containing a pyrrolo [3, 4-β] -quinoline moiety, was selected to prepare a model prodrug NO-CPT (Scheme S2) . After oxidation by hydrogen peroxide (H₂O₂) , the chemical structure of NO-CPT is similar to that of CPT besides an extra oxygen atom, which is highly atom economic. As expected, in vitro reduction (10 μM in PBS) took place in a radiation-dose-dependent manner, yielding CPT as a reduction product (Figure 11) . Moreover, activation of NO-CPT (10 μM) in PBS containing 10%FBS and cell lysate also have been tested and analyzed by UPLC (Figure 12) , indicating the NO-CPT can be activated in the biological environment by radiation.

Example 4: In vitro study of radiation induced reduction of NO-CPT

The cytotoxicity of NO-CPT and CPT was tested by incubating with HCT116 and CT26 cells, respectively, for 48 h (Figure 13) . The half-maximal inhibitory concentration (IC₅₀) of NO-CPT against CT26 cells is7.71 ±0.56 μM, which is 22-fold over the parent drug. IC₅₀of NO-CPT against HCT116 cells is 2.18 ±0.13 μM, which is 42-fold over the parent drug. In hypoxia conditions, HCT116 and CT26 cells were treated with NO-CPT, 16 Gy X-ray, CPT, X-ray treated NO-CPT (activated NO-CPT) , and NO-CPT + 16 Gy X-ray (NO-CPT and CPT are both 0.5 μM for each group) . After 48 h incubation, cell viability of group treated with activated NO-CPT and NO-CPT + 16 Gy X-ray were close to that of CPT treated group (Figure 5d, e) . As a contrast, NO-CPT only group showed almost no treatment efficacy in hypoxia conditions. The results suggest that NO-CPT has good stability in living cells and efficient reactivity toward radiation.

Example 5: In vivo study of radiation induced reduction of NO-CPT

The anti-tumor efficacy of the radiotherapy-induced prodrug activation was further investigated (Figure 6a) . To achieve the best therapeutic efficacy, it is crucial to determine the right time point for radiotherapy. Time-dependent accumulation of NO-CPT in tumor tissue shows that about 1 h post-injection is the best time point for irradiation (Figure 6b) . HCT116 tumor–bearing mice were randomly divided into 4 groups including the control group (PBS) , then were treated by NO-CPT, X-ray, and NO-CPT + X-ray (Figure 6 a) via intravenous injection. After four rounds of NO-CPT + X-ray treatments (the NO-CPT + X-ray treatment is administrated once every three days, which is called “a round” ) , the growth of tumors was inhibited (Figure 6c) . As for comparisons, treatments with PBS, NO-CPT, and X-ray could not prevent the growth of tumors. Tumor weight measurements and photography of harvested tumors confirmed the antitumor effect caused by radiotherapy combined with activated chemotherapy (Figure 6d, Figure 14) . Importantly, evaluations of body weight changes showed that the radiotherapy-activated prodrug displayed no gross toxicities (Figure 6e) . The biosafety has been further evaluated via histology analysis after NO-CPT + X-ray treatment, the results shown almost no tissue damage in the primary organs was observed (Figure 15) .

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) was purchased from Sigma-Aldrich and used as received. Dansylamide (A-5) , dimethyl (2-phenoxy ethyl) amine were purchased from Macklin and used as received. Quinoline N-oxide (A-7) , 8-hydroxyquinoline N-oxide (A-8) , 4-methyl-morpholin N-oxide (NO-9) , dimethyl (2-phenoxyethyl) amine (A-10) was purchased from Energy Chemical and used as received. Imiquimod (IMQ) , Ampiroxicam (APX) , Pranoprofen (PNP) , Loratadine (LRT) , Camptothecin (CPT) were purchased from J&K and used as received. KNO₃ (AR) , CH₃OONa (AR) , methanol (MeOH) (AR) , isopropyl alcohol (i-PrOH) (AR) , and dichloromethane (DCM) (AR) , H₂O₂, acetic acid (AR) were purchased from Beijing Chemical Works, and used as received. Glutathione (GSH) , tryptophan (Try) , phenylalanine (Phe) , cysteine (Cys) , Vitamin C (Vc) were purchased from Sigma-Aldrich and used as received. UPLC Solvents were of HPLC quality and were purchased from Sigma-Aldrich. 1x 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 with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ cm. 4-Dimethylamino-N-butyl-1, 8-naphthalic imide (A-4) , ¹ N, N-dimethylaniline N-oxide (NO-6) ²were synthesized according to previously reported literature procedures. All of the chemicals were used as received without further purification.

Characterization. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE 400 MHz spectrometer. Ultra-performance liquid chromatography (UPLC) was performed on ACQUITY UPLC H-Class PLUS instrument equipped with Waters PDA eλ Detector and a Waters Acquity QDA mass spectrometer. High-resolution mass spectroscopy was 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 on an A1R-si Laser Scanning Confocal Microscope (Nikon, Japan) .

Synthesis.

Scheme S1. Chemical synthesis of NO-1.

General synthetic route of N-oxides: A-1 (1 mmol, 1.0 eq. ) was dissolved in DCM (5 mL, anhydrous) and cooled in the ice bath to 0℃. m-CPBA (1.5 mmol, 1.5 eq. ) was added. The reaction mixture was warmed 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 obtained crude product was purified by flash chromatography with DCM/MeOH (10: 1) as eluents successively. NO-1was afforded as a pink powder (yield: 58%) : ¹H NMR (DMSO-d₆, 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) . ¹³C NMR (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/z calculated for [M + H] ⁺, 248.1280; found, 248.1281.

NO-2, NO-3, NO-4, NO-5, NO-10, NO-IMQ, NO-APX, NO-PNP, NO-LRT were synthesized according to similar procedures.

NO-2, yellow powder (yield: 54%) : ¹H NMR (DMSO-d₆, 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= 9.6 Hz, 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) . ¹³C 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/z calculated for [M + H] ⁺, 234.1124; found, 234.1125.

NO-3, yellow powder (yield: 55%) : ¹H NMR (DMSO-d₆, 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) . ¹³C NMR (DMSO, δ, ppm, 126 MHz) : 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/z calculated for [M + H] ⁺, 220.0968; found, 220.0968.

NO-4, yellow powder (yield: 49%) : ¹H NMR (DMSO-d₆, 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= 7.4 Hz, 3H) . ¹³C 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/z calculated for [M + H] ⁺, 313.1545; found, 313.1547.

NO-5, pale yellow powder (yield: 51%) : ¹H 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) . ¹³C 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/z calculated for [M +H]⁺, 267.0798; found, 267.0798.

NO-10, yellow solid (yield: 57%) : ¹H NMR (DMSO-d₆, 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) . ¹³C NMR (DMSO, δ, ppm, 126 MHz) : 158.41, 130.05, 121.34, 115.02, 68.80, 62.67, 60.29. High-resolution mass: m/z calculated for [M + H] ⁺, 182.1175; found, 182.1176.

NO-IMQ, pale grey powder (yield: 60%) : ¹H 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) . ¹³C 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/z calculated for [M + H] ⁺, 257.1397; found, 257.1397.

NO-APX, white powder (yield: 62%) : ¹H 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= 5.3 Hz, 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/z calculated for [M + H] ⁺, 257.1397; found, 257.1397.

NO-PNP, white powder (yield: 55%) : ¹H NMR (DMSO-d₆, 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) . ¹³C 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/z calculated for [M + H] ⁺, 272.0915; found, 272.0917.

NO-LRT, white powder (yield: 48%) : ¹H 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) . ¹³C 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/z calculated for [M + H] ⁺, 399.1470; found, 399.1470.

Scheme S2. Chemical synthesis of NO-CPT.

NO-CPT was synthesized according to previously reported literature procedures. ³To CPT (1 mmol, 1 eq. ) suspended in acetic acid (20 mL) was added 30%hydrogen peroxide (5 mL) . The reaction mixture was stirred overnight at 80 ℃, then poured into ice water (100 mL) . The precipitated needle crystals are collected by filtration and dried under reduced pressure (yield: 83%) . ¹H NMR (DMSO-d₆, 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= 7.3 Hz, 3H) . ¹³C 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/z calculated for [M + H] ⁺, 365.1131; found, 365.1132.

Test assays

Reduction of N-oxides under X-ray radiation. As a representative example, detailed procedures employed for the X-ray-induced reduction of NO-1 are described below. 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 radiation from 0 to 60 Gy (RS2000 PRO, 225 kV, 17.7 mA, Radsource Corporation) . The reaction mixtures were analyzed by UPLC and fluorescence spectra.

Computational studies with DFT calculation.

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

Calculation on comparing the reactivity of N-oxides with hydrated electrons It has been known that the reaction of hydrated electrons with N-oxides is diffusion-controlled, it is reliable to consider only thermodynamics. The electron affinity N-oxides (ΔG) refers to the free energy change of N-oxide to its anion (1 Hartree = 2625.5kJ/mol) :
ΔG= [NO] ^--NO

Comparing the ΔG with the enthalpy of formation of hydrated electrons, the reactivity of N-oxides can be judged. Electron affinity can be obtained by calculating the Gibbs free energies of N-oxides and N-oxides anion, respectively. Comparing ΔG with the absolute free energy of solvation of electrons (ΔG_e=35.5 kcal/mol) in water, it is possible to judge whether the reaction can occur. Using electrode potentials to represent the reactivity of different nitrogen oxides:

Table 1. Calculation Data of Representative N-oxides.

^a Free energy of N-oxides calculated by B3-LYP in water solvent; ^b free energy of N-oxides anion calculated by B3-LYP in water solvent; ^c the electron affinity of N-oxides (ΔG) ; ^d The electrode potentials of N-oxides

Cell Culture. CT26 and HCT116 cells were cultured in RPMI-1640 medium. The media were 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℃ and the medium was replaced every 2-3 days. After growing to 80%confluence, the cells were treated with trypsin and then seeded on dishes or 96-well plates overnight for further experiments.

Confocal imaging. Cells were typically seeded at a density of 2×10⁵ cells/mL in a 35-mm confocal dish and incubated in an incubator containing 5%CO₂ at 37 ℃ for 24 h. Stock solutions of NO-1 (10 mM) were prepared in DMSO. The adherent cells were washed twice with PBS before the addition of NO-1 (10 μM final concentration) in 1 mL in RPMI-1640 medium. The cells were then incubated with AnaeroPack (Mitsubishi Gas Chemical Company, Inc. ) at 37 ℃ for another 30 min to construct a hypoxic environment, followed by exposure to X-ray radiation. The fluorescent images were taken by a laser scanning confocal microscope, the excitation wavelength is 405 nm.

Cell viability assays. Cell viability was assessed with the CCK-8 assay following the protocol and treatment groups were normalized to controls.

To assay the cytotoxicity of NO-1, HCT116 and CT26 were seeded in a 96-well plate at a concentration of 1×10⁴/mL in 100 μL of RPMI-1640 medium with 10%FBS and 1%penicillin/streptomycin and maintained at 37 ℃ for 24 h. Then the cells were incubated with different concentrations of NO-1 for 12 h. Then the medium of each well was replaced by a blank medium containing a final concentration of 0.5 mg/mL CCK-8. The cells were incubated at 37 ℃ for 2 h and the absorbance was measured at 450 nm. The absorbance of treated cells was compared with the absorbance of the control group, of which the viability was set as 100%.

To assay the cytotoxicity of CPT and NO-CPT, HCT116 and CT26 were seeded in a 96-well plate at a concentration of 1×10⁴/mL in 100 μL of RPMI-1640 medium with 10%FBS and 1%penicillin/streptomycin and maintained at 37 ℃ for 24 h. Then the cells were incubated with different concentrations of CPT and NO-CPT, respectively, for 48 h. Then the medium of each well was replaced by a blank medium containing a final concentration of 0.5 mg/mL CCK-8. The cells were incubated at 37 ℃ for 2 h and the absorbance was measured at 450 nm. The absorbance of treated cells was compared with the absorbance of the control group, of which the viability was set as 100%.

To assay the cytotoxicity of NO-CPT activated by X-ray, HCT116, and CT26 were seeded in a 96-well plate at a concentration of 1×10⁴/mL in 100 μL of RPMI-1640 medium with 10%FBS and 1%penicillin/streptomycin, and maintained at 37 ℃ for 24 h and then treated with different ways:

1. Cells were incubated with NO-CPT (0.5 μM) for 48 h.

2. Cells were incubated with AnaeroPack for 30 min and exposed to 16 Gy X-ray, then incubated in the normoxic environment for 48 h.

3. Cells were incubated with CPT (0.5 μM) for 48 h.

4. Cells were incubated with 16 Gy X-ray irradiated NO-CPT (0.5 μM) for 48 h.

5. Cells were incubated with NO-CPT (0.5 μM) in an AnaeroPack for 30 min and exposed to 16 Gy X-ray, then incubated in the normoxic environment for 48 h.

Then the medium of each well was replaced by a blank medium containing a final concentration of 0.5 mg/mL CCK-8. The cells were incubated at 37℃ for 2 h and the absorbance was measured at 450 nm. The absorbance of treated cells was compared with the absorbance of the control group, of which the viability was set as 100%.

Animal Model. 6～8-week-old female BALB/c nude mice were ordered from Vital River Laboratories (Beijing, China) and kept under Specific Pathogen Free (SPF) condition with free access to standard food and water. Approximately 2×10⁶HCT116 cells suspended in 100 μL of PBS were implanted subcutaneously into the right thigh of BALB/c nude mice.

Biodistribution analysis. HCT116 tumor-bearing mice were injected with NO-CPT (10 mg/kg) via the intravenous route. The mice were then sacrificed at preset time points post-injection and tumor tissues were collected, wet weighed, and the concentration of NO-CPT was measured by UPLC-UV (detected λ_abs= 380 nm) .

Radiation-induced activation of NO-CPT in vivo. When the tumor size reached approximately 100 mm³, the mice were randomly divided into 4 groups (n = 6) for the treatment with PBS, X-ray, NO-CPT, NO-CPT+ X-ray (intravenous injection, NO-CPT = 10 mg/kg) . X-ray radiation was conducted at the time point of 1 h after injection (4Gy) . Tumor sizes were measured every other day using a caliper. Tumor volumes were calculated with the formula: the volume = length × width²/2. The body weights were measured every 2 days. After the mice were sacrificed, the solid tumors were taken out, weighed, and photographed.

Statistical Analysis. Statistical analyses were performed using GraphPad Prism 6. Statistical comparisons were analyzed using two-way ANOVA.

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ABBREVIATIONS AND SPECIALIST TERMS

Claims

A method for activating N-oxide compound with radiation, characterized in that the N-oxide compound comprises at least one aryl or heteroaryl group, and the N-oxide compound can be reduced to a corresponding tertiary amine after irradiated with the radiation.
The method according to claim 1, wherein 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.
The method according to claim 1 or 2, wherein the N-oxide compound has the general formula I,

ArR₂N⁺-O^-Formula I,

wherein R is independently selected from the group consisting of alkyl, such as C_1-4 alkyl, and cycloalkyl, such as C_3-6 cycloalkyl,

Ar is C₆-C₂₀ aryl group, preferably C₆-C₁₄ aryl group, most preferably C₆-C₁₀ aryl group, wherein the aryl group is optionally substituted with alkyl, hydroxyl, amino, aminosulfonyl (NH₂-SO₂-) , -C (O) HR’, -OC (O) HR’, -C (O) -NR¹, wherein optionally two substituting groups on the aryl group can form heterocycle, preferably 6-membered unsaturated heterocycle, preferably 6-membered heterocycle comprising O, NR¹, CR¹, C=O as the ring member, R¹ and R’ are independently selected from the group consisting of hydrogen and alkyl, such as C_1-4 alkyl.
The method according to claim 3, wherein R is independently selected from the group consisting of C₁-C₄ alkyl, and is preferably selected from the group consisting of methyl and ethyl, and/or

Ar is phenyl or naphthyl, wherein the phenyl or naphthyl is unsubstituted or substituted with alkyl, alkenyl, hydroxyl, amino, aminosulfonyl (NH₂-SO₂-) , -C (O) HR’, -OC (O) HR’, -C (O) -NR¹, wherein optionally two substituting groups on the phenyl or naphthyl group can form heterocycle, preferably 6-membered unsaturated heterocycle, preferably 6-membered heterocycle comprising O, NR¹, CR¹, C=O as the ring member,

R¹ and R’ are independently selected from the group consisting of hydrogen and alkyl, such as C_1-4 alkyl,

preferably, Ar is naphythyl optionally substituted with alkyl, or aminosulfonyl (NH₂-SO₂-) , or Ar with two substituting groups thereon form heterocycle is a coumarine group or its derivatives, or a 1, 8-naphthalic imide group or its derivatives.
The method according to claim 1 or 2, wherein the N-oxide compound has the general formula II,

wherein R², R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, amido, which can be in turn substituted with heterocycle, the heterocycle is optionally substituted with alkylcarbonatealkoxyl group; and optionally the substituting groups on the above illustrated ring can form one or two aryl or heteroaryl groups or heterocycles condensed on the above illustrated ring, said one or two aryl or heteroaryl groups or heterocycle can in turn be substituted by amino, halogen, alkyl, hydroxyl, carboxyl alkyl or heterocycle, which heterocycle is optionally substituted with alkoxylcarbonyl group;

with the proviso that R², R³, R⁴, R⁵ and R⁶ are not simultaneously hydrogen.
The method according to claim 5, wherein R², R³, R⁴, R⁵ and R⁶ are independently selected from the group consisting of hydrogen, C₁-C₄ alkyl, amino, hydroxyl, amido, which can be in turn substituted with 5-, 6-, or 7-membered heterocycle, the heterocycle is optionally substituted with (C₁-C₄) alkylcarbonate (C₁-C₄) alkoxyl group; and optionally the substituting groups on the above illustrated ring can form one or two phenylene or 5-, 6-, or 7-membered heteroaryl groups or heterocycles condensed on the above illustrated ring, said one or two aryl or heteroaryl groups or heterocycle can in turn be substituted by amino, halogen, C₁-C₄ alkyl, hydroxyl, carboxyl (C₁-C₄) alkyl or 5-, 6-, or 7-membered heterocycle, which heterocycle is optionally substituted with (C₁-C₄) alkoxylcarbonyl group;

with the proviso that R², R³, R⁴, R⁵ and R⁶ are not simultaneously hydrogen.
The method according to claim 1 or 2, wherein the N-oxide compound has the general formula IIa, IIb, IIc, IId, IIe or IIf:

wherein, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, hydroxyl, amino, halogen, C₁-C₅ alkyl, (C₁-C₄) alkyl-O (CO) - (C₁-C₄) alkyl, and (C₁-C₄) alkyl-O (CO) H, and

optionally one or more hydrogen atoms on the ring in the general formula IIa, IIb, IIc, IId, IIe or IIf can be replaced with hydroxyl, amino, halogen, or C₁-C₅ alkyl, preferably halogen or C₁-C₄ alkyl;

preferably, R⁷, R⁹, and R¹³ are independently selected from the group consisting of hydrogen, hydroxyl, amino, and halogen,

R⁸, R¹⁰, R¹¹, R¹², and R¹⁴ are 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

optionally one or more hydrogen atoms on the aromatic ring in the general formula IIa, IIb, IIc, IId, IIe or IIf can be replaced with halogen or C₁-C₄ alkyl.
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.
The method according to claim 1 or 2, wherein the N-oxide compound is aniline N-oxide or aromatic heterocyclic N-oxide, such as a compound in the following table or a pharmaceutically acceptable salt or solvate thereof:
A method of treating or diagnosing a disease, the method comprises administering to an individual a therapeutically effective amount of an N-oxide prodrug, and radiating the individual with a therapeutically effective amount of radiation,

preferably the N-oxide prodrug is the one as defined in claims 1-9.
The method according to claim 10, said method is for treating or suppressing a cancer, reducing its severity, lowering its risk or inhibiting its metastasis in an individual.
The method according to claim 10 or 11, the cancer is selected from the group consisting of colon cancer, small cell lung cancer, Hodgkin's lymphoma, and malignant lymphoma.
The method according to any one of claims 10 to 12, wherein the individual is radiated after administering the N-oxide prodrug, preferably 20 mins, 1 hour, 2 hours, 4 hours after administering the N-oxide prodrug.
The method according to any one of claims 10 to 13, wherein the N-oxide prodrug is administrated in an amount of from about 0.005 mg/kg to about 100 mg/kg, such as an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100 mg/kg.
The method according to any one of claims 10 to 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..
The method according to any one of claims 10 to 15, wherein the N-oxide prodrug is administrated orally or parenterally.
The method according to any one of claims 10 to 16, wherein the radiation is X-ray or gamma ray, and the intensity of the X-ray or gamma ray is 60 Gy or lower, 50 Gy or lower, 40 Gy or lower, 30 Gy or lower, 20 Gy or lower, 10 Gy or lower, 8 Gy or lower, 6 Gy or lower, 4 Gy or lower.