Hypoxic microenvironment-responsive artificial deoxyribonucleic acid and applications thereof

Abstract

The present application provides a hypoxic microenvironment-responsive artificial deoxyribonucleic acid and application. Specifically, the present application provides a compound of formula (I), or an optical isomer, hydrate, solvate thereof, or a pharmaceutically acceptable salt thereof. The compound of the present application can be used as a molecular probe responding to hypoxic microenvironment stress, has superior microenvironment targeting and biological stability, and can be used for diagnosis and treatment of cancer or tumor (especially solid tumor) characterized by hypoxic microenvironment.

Description

Technical Field

[0001] This invention relates to the fields of compounds and biochemistry, specifically to artificial deoxyribose responsive to hypoxia microenvironment and its application in cancer diagnosis and treatment. Background Technology

[0002] The hypoxic microenvironment is the most prominent physiological characteristic of malignant solid tumors and one of the decisive factors in the treatment effect. As early as the 1960s, Tomlinson and Gray discovered the existence of hypoxia in human tumor tissues. Semenza, Ratcliffe, and Kaelin conducted in-depth research on this phenomenon, first elucidating the mechanism by which cells sense and adapt to changes in the oxygen supply environment, and discovered that hypoxia-inducible factors (HIF) are key proteins controlling cellular responses to changes in oxygen levels.

[0003] Numerous studies have demonstrated that the hypoxic microenvironment not only significantly impacts tumor cell proliferation, apoptosis, migration, invasion, and angiogenesis, but it is also a major cause of drug resistance during cancer treatment.

[0004] Developing new diagnostic and therapeutic technologies targeting the hypoxic microenvironment of malignant tumors has become a highly anticipated cutting-edge research area. Rajendran et al. utilized the responsiveness of the PET probe [F-18]methoxymethylnitroimidazole ethanol (FMISO) to the hypoxic microenvironment to effectively diagnose the prognosis of 58 head and neck cancer patients. Guo et al. developed a non-covalent azo molecular switch-fluorescent dye system to achieve conditional imaging of hypoxic tissues; Chan et al. developed a bioreducing probe for photoacoustic imaging of the hypoxic microenvironment.

[0005] PET technology maintains high specificity and sensitivity in complex physiological environments within the body, making it a preferred method for visualizing hypoxic microenvironments. The small molecule compound nitroimidazole exhibits good targeting properties for hypoxic microenvironments, and researchers have achieved widespread success in cancer diagnosis by designing a series of PET probes based on this compound. Currently, more than five PET probes have been approved by the FDA for clinical diagnosis of various tumors. However, the targeting performance and biostability of existing PET probes in hypoxic microenvironments remain unsatisfactory.

[0006] Therefore, there is an urgent need in this field to develop new molecular probes for hypoxic microenvironments that possess excellent targeting properties and superior biostability, in order to diagnose and treat malignant tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a novel molecular probe for hypoxic microenvironments that exhibits excellent targeting properties and superior biological stability.

[0008] In a first aspect, the present invention provides a compound of formula (I), or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof:

[0009]

[0010] In the formula,

[0011] R1 is H, -OH, or -ORa;

[0012] R2 is selected from the group consisting of: H, -P(Rc)Rd, wherein Rc is -N(Re)Rf, wherein Re and Rf are each independently H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; Rd is -OH, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 cycloalkoxy; wherein "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, C1-C3 alkyl, or combinations thereof;

[0013] R3 is selected from the following group: H, or -C(Rg)3, where Rg is a substituted or unsubstituted phenyl or C1-C8 alkyl group;

[0014] Ra is H, substituted or unsubstituted nitroimidazolyl or -(substituted or substituted C1-C6 alkylene)-(substituted or unsubstituted nitroimidazolyl); wherein, "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, C1-C3 alkyl, or combinations thereof.

[0015] Rb is free, H, substituted or unsubstituted nitroimidazolium, -CO-substituted or unsubstituted C6-C10 aryl, -CO-substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl;

[0016] Cyclic B represents a nucleotide base.

[0017] In another preferred embodiment, the additional condition is that Rb is absent when Ra is attached to the N atom on the main ring of the nucleotide base.

[0018] In another preferred embodiment, when the nucleotide base is an exocyclic NH2 type base, both Ra and Rb are attached to the N atom that is directly attached to the main ring of the nucleotide base.

[0019] In another preferred embodiment, when the nucleotide base is an exocyclic NH2 type base, Ra is a substituted or unsubstituted nitroimidazolium group or -(substituted or substituted C1-C6 alkylene group)-(substituted or unsubstituted nitroimidazolium group); while Rb is H, a substituted or unsubstituted nitroimidazolium group, -CO-substituted or unsubstituted C6-C10 aryl group, -CO-substituted or unsubstituted C1-C8 alkyl group, or a substituted or unsubstituted C3-C8 cycloalkyl group.

[0020] In another preferred embodiment, Ra is -(C1-C3 alkylene)-(substituted or unsubstituted nitroimidazolyl), wherein "substituted" means substituted by one or more substituents selected from the group consisting of halogens, C1-C3 alkyl groups, or combinations thereof.

[0021] In another preferred embodiment, Ra and Rb are both connected to N atoms that are directly connected to the nucleotide backbone (i.e., the N atoms are connected to the backbone via covalent bonds; preferably, the N atoms are connected to the ring C atoms on the backbone via covalent bonds).

[0022] In another preferred embodiment, Rb is absent, and Ra is attached to the N atom on the nucleotide backbone (i.e., the N atom located on the backbone).

[0023] Preferably, when the nucleotide base is T or U, Rb is absent, and Ra is attached to the N atom on the nucleotide base backbone.

[0024] In another preferred embodiment, the nucleotide bases are selected from the group consisting of A, T, C, G, I, U, or combinations thereof.

[0025] In another preferred embodiment, the ring B, Ra, and Rb constitute groups selected from the group consisting of:

[0026]

[0027] In the formula, Ra and Rb are defined as above.

[0028] In another preferred embodiment, R1, R2, R3, Ra, Rb, and ring B are each independently the corresponding groups in the compounds prepared in the examples.

[0029] In another preferred embodiment, the compound represented by formula (I), or a pharmaceutically acceptable salt thereof, has a structure selected from any of the compounds in Table A.

[0030] Table A

[0031] Example compound 1 Compounds 2-9 2 Compounds 11-18 3 Compounds 20-27 4 Compounds 29-36 5 Compounds 38-45 6 Compounds 47-54

[0032] A second aspect of the present invention provides a nucleic acid molecular pharmaceutical composition comprising the compound as claimed in claim 1 or an optical isomer thereof, a hydrate, a solvate thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, the nucleic acid molecular drug composition is selected from the group consisting of gene drugs composed of deoxyribose units, small interfering RNA drugs, or nucleic acid aptamer drugs.

[0034] In another preferred embodiment, the pharmaceutical composition is a gene therapy composed of deoxyribose units.

[0035] In another preferred embodiment, the pharmaceutical composition is a small nucleic acid interfering RNA drug.

[0036] In another preferred embodiment, the pharmaceutical composition is a nucleic acid aptamer drug.

[0037] A third aspect of the invention provides the use of a compound according to the first aspect of the invention or a pharmaceutical composition according to the third aspect of the invention in the treatment of cancer or tumor.

[0038] In another preferred embodiment, the pharmaceutical composition is a gene therapy composed of deoxyribose units.

[0039] In another preferred embodiment, the pharmaceutical composition is a small nucleic acid interfering RNA drug.

[0040] In another preferred embodiment, the pharmaceutical composition is a nucleic acid aptamer drug.

[0041] In another preferred embodiment, the cancer is selected from cancers including, but not limited to, those from the group consisting of: lung cancer, bladder cancer, breast cancer, stomach cancer, liver cancer, prostate cancer, kidney cancer, pancreatic cancer, or combinations thereof.

[0042] In a fourth aspect, the present invention provides an active-targeting contrast agent comprising: a compound of formula (I) as claimed in claim 1, or an optical isomer, hydrate, solvate, or a pharmaceutically acceptable salt thereof.

[0043] In another preferred embodiment, the contrast agent is a hypoxia-microenvironment-responsive contrast agent.

[0044] In another preferred embodiment, the contrast agent is used to create images of tumor tissue.

[0045] A fifth aspect of the present invention provides the use of a compound of formula (I) according to a first aspect of the present invention, or an optical isomer, hydrate, solvate, or pharmaceutically acceptable salt thereof, for the preparation of a contrast agent.

[0046] In another preferred embodiment, the contrast agent is a hypoxia-microenvironment-responsive contrast agent.

[0047] In another preferred embodiment, the contrast agent is used to create images of tumor tissue.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0049] Figure 1 The first line shows the chemical structures of deoxyribose dA, dT, dC, and dG; the second line shows the corresponding chemical structures of dA-module, dT-module, dC-module, and dG-module; the third line shows the chemical structure of nitroimidazole; and the fourth line shows the chemical structure of DMTr-.

[0050] Figure 2 The chemical structures of deoxyribose dAh, dTh, dCh, and dGh are shown.

[0051] Figure 3 Compounds 2-9, prepared from compound 1, are shown.

[0052] Figure 4 Compounds 11-18, prepared from compound 10, are shown.

[0053] Figure 5 Compounds 20-27, prepared from compound 19, are shown.

[0054] Figure 6 Compounds 29-36, prepared from compound 28, are shown.

[0055] Figure 7 Compounds 38-45, prepared from compound 37, are shown.

[0056] Figure 8 Compounds 47-54, prepared from compound 46, are shown. Detailed Implementation

[0057] Through long-term and in-depth research, the inventors have unexpectedly developed a novel molecular probe (hereinafter referred to as "the molecular probe of this invention") suitable for hypoxic microenvironments. Experiments show that the molecular probe of this invention is an artificial deoxyribose with the structure of Formula I. The series of molecular probes of this invention that respond to hypoxic microenvironment stress exhibit superior microenvironment targeting and biological stability, and can be used for the diagnosis and treatment of cancers or tumors (especially solid tumors) characterized by hypoxic microenvironments. This invention was completed based on this foundation.

[0058] the term

[0059] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0060] As used in this article, the term "hypoxic microenvironment" refers to a typical pathophysiological characteristic of tumor cells in malignant tumors, especially malignant solid tumors. The hypoxic microenvironment is caused by a variety of factors, such as rapid tumor cell growth leading to vascular disturbances, decreased blood supply, and increased oxygen consumption. It is also closely related to the involvement of various cytokines and signal transduction pathways. The hypoxic microenvironment significantly reduces the effectiveness of radiotherapy, chemotherapy, and surgery on tumor cells and is closely related to tumor invasion, distant metastasis, and resistance to radiotherapy and chemotherapy.

[0061] As used herein, the term "deoxyribose" includes deoxyribose dA, deoxyribose dT, deoxyribose dC, and deoxyribose dG. These are compounds formed by chemically bonding four different bases—adenine (A), guanine (G), thymine (T), and cytosine (C)—to the 1' carbon atom of the pentose sugar deoxyribose (chemical formula C4H9O3CHO). Figure 1 As shown in the first row).

[0062] As used herein, the terms “dA-module,” “dT-module,” “dC-module,” and “dG-module” refer to compounds obtained by substituting the hydroxyl hydrogen atom attached to the 2' and 3' carbon atoms of deoxyribose dA, deoxyribose dT, deoxyribose dC, and deoxyribose dG; wherein the substitution of the hydroxyl hydrogen atom attached to the 2' carbon atom is -P(Rc)Rd, where Rc is -N(Re)Rf, and Re and Rf are each independently H, substituted or unsubstituted C. 1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl; Rd is -OH, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 cycloalkoxy; wherein, "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, C1-C3 alkyl, or combinations thereof; the hydroxyl hydrogen attached to the 3' carbon atom is substituted with -C(Rg)3, where Rg is a substituted or unsubstituted phenyl or C1-C8 alkyl; in a preferred embodiment of the invention, a typical substitution is as follows Figure 1 As shown, but not limited to Figure 1 The second line shows the format.

[0063] As used herein, the term "nitroimidazole" refers to a class of organic compounds having a 5-nitroimidazole ring structure. In the examples herein, a series of hypoxia-responsive deoxyriboses containing a 5-nitroimidazole ring were prepared by substituting the hydroxyl group on N-1-methyl-2-nitro-5-hydroxymethylimidazole. The chemical structures of nitroimidazoles are shown below. Figure 1 As shown in the third row.

[0064] As used herein, the typical structure of "DMtr-" used in the embodiments is as follows: Figure 1 As shown in the fourth line.

[0065] As used in this article, the terms "deoxyribose dAh, dTh, dCh, dGh" specifically refer to compounds formed when the hydrogen atom on the N atom of the nucleotide backbone (when the base is T), or the N atom directly connected to the nucleotide backbone (when the base is A, C, or G), is replaced by a nitroimidazol group. Its typical chemical structure is as follows: Figure 2 As shown.

[0066] Unless otherwise specified, in all compounds of the present invention, each chiral carbon atom (chiral center) may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0067] As used herein, the term "alkyl" refers to a straight-chain (i.e., unbranched) or branched alkyl group, or a combination thereof, having 1 to 8 carbon atoms, whether alone or as part of other substituents. The alkyl group may be saturated, monounsaturated, or polyunsaturated, and may include divalent or polyvalent groups. When the alkyl group is preceded by a carbon number limitation (e.g., C...), the term is used to indicate the number of carbon atoms. 1-10 When ), it means that the alkyl group contains 1-10 carbon atoms, for example, C 1-8 Alkyl groups may include straight-chain or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0068] As used herein, the term "cycloalkyl" refers to a monocyclic, bicyclic, or tricyclic (including fused, bridged, or spirocyclic) ring system, whether alone or as part of other substituents. The cycloalkyl group may have 3-12 (e.g., 3-10, or 5-10) carbon atoms. When a cycloalkyl group is preceded by a carbon number limitation (e.g., C...), the term is used to indicate the number of carbon atoms in the cycloalkyl group. 3-10 When ), it refers to the cycloalkyl group containing 3-10 carbon atoms. In some preferred embodiments, the term "C" is used. 3-8 "Cycloalkyl" refers to a saturated or partially saturated monocyclic or bicyclic alkyl group having 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or similar groups.

[0069] As used herein, the term "alkoxy" or "alkyloxy" refers to an alkyl group (e.g., -O-alkyl) linked by an oxygen atom, wherein the alkyl group is as described above. Examples of specific alkoxy groups include (but are not limited to) methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups. An alkoxy group may be substituted with one or more substituents, such as halogens, amino groups, cyano groups, or hydroxyl groups. Alkoxy groups may be straight-chain or branched. When an alkoxy group is preceded by a carbon number determination (e.g., C...), the alkoxy group is considered to have a higher carbon number. 1-8 When ), it means that the cycloalkyl group contains 1-8 carbon atoms.

[0070] As used herein, the term "halogen" refers to F, Cl, Br, and I, either alone or as part of other substituents.

[0071] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or fused-ring aromatic hydrocarbon group, whether alone or as part of another substituent. The aryl group may be substituted or unsubstituted. When an aryl group is preceded by a carbon number limit (e.g., C...), it is considered a substituted or unsubstituted group. 6-12 When ), it means that the aryl group contains 6-12 carbon atoms. Examples of aryl groups include (but are not limited to): phenyl, biphenyl, naphthyl, or similar groups (each carbon atom of which may be arbitrarily substituted).

[0072] As used herein, the term "heteroaryl" refers, alone or as part of other substituents, to a monocyclic, bicyclic, or fused-ring aromatic group having a specific number of cyclic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 4-10 This refers to a ring containing 4-10 cyclic carbon atoms, and including at least one identical or different heteroatom selected from N, O, or S. Atoms on each ring can be arbitrarily substituted. The heteroaryl group can be 5- to 15-membered aromatic cyclic group having 1-5 heteroatoms, each independently selected from N, O, or S. Examples of heteroaryl groups include (but are not limited to): pyridine, pyrimidine, pyrrole, indazole, indole, furan, benzofuran, thiophene, or similar groups.

[0073] As used herein, the term "heterocyclic group," whether alone or as part of other substituents, refers to a saturated or partially saturated monocyclic, bicyclic, or tricyclic (including fused, bridged, or spirocyclic) ring system. The group has a specific number of cyclic carbon atoms (e.g., C...). 3-11 This refers to a heterocyclic group having 3-11 cyclic carbon atoms, and including at least one identical or different heteroatom selected from N, O, or S. The heterocyclic group can be 3- to 15-membered, having 1-5 heteroatoms, each independently selected from N, O, or S. Examples of heterocyclic groups include (but are not limited to): nitrogen heterocyclic groups, oxygen heterocyclic groups, sulfur heterocyclic groups, nitrogen-oxygen heterocyclic groups, nitrogen-sulfur heterocyclic groups, oxygen-sulfur heterocyclic groups, etc., more preferably the heterocyclic groups appearing in the embodiments of this application. In this invention, the heterocyclic group can be a monocyclic, bicyclic, or tricyclic (including fused rings, bridged rings, or spirocyclic rings).

[0074] As used herein, the terms “arbitrary” or “optional” (e.g., “arbitrarily substituted”) mean that the said part is substituted or unsubstituted, and that the substitution occurs only at chemically realizable positions. For example, H, covalent bonds, or -C(=O)- groups cannot be substituted by substituents.

[0075] As used in this article, "oxygen" or "oxygen group" refers to =O.

[0076] As used herein, unless otherwise specified, the term "pharmaceutically acceptable salt" means a salt suitable for contact with the tissues of an object (e.g., a human) without producing undesirable side effects. In some embodiments, a pharmaceutically acceptable salt of a compound of the present invention includes salts of the compounds of the present invention having acidic groups (e.g., potassium, sodium, magnesium, calcium salts) or salts of the compounds of the present invention having basic groups (e.g., sulfates, hydrochlorides, phosphates, nitrates, carbonates).

[0077] As used herein, the term "substitution" (with or without "arbitrarily" modified) refers to the substitution of one or more hydrogen atoms on a particular group by a particular substituent. The particular substituent is the substituent described accordingly above, or the substituent appearing in the various examples. Unless otherwise specified, an arbitrarily substituted group may have a substituent selected from a particular group at any substituted site of that group, and the substituents may be the same or different at each position. Cyclic substituents, such as heterocyclic alkyl groups, may be attached to another ring, such as a cycloalkyl group, thereby forming a spirobicyclic system, for example, where the two rings share a common carbon atom. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible. The substituents include, but are not limited to, halogens, hydroxyl groups, carboxyl groups (-COOH), C1-C6 alkyl groups, C3-C8 cycloalkyl groups, 3- to 12-membered heterocyclic groups, aryl groups, heteroaryl groups, amino groups, C1-C6 alkoxy groups, etc.

[0078] For convenience and to conform to common understanding, the terms "arbitrary substitution" or "optional substitution" apply only to sites that can be substituted by substituents, and do not include chemically impossible substitutions.

[0079] Unless otherwise specified, the word “or” as used in this article has the same meaning as “and / or” (referring to both “or” and “and”).

[0080] General Synthesis Method

[0081] The compounds of the present invention can be prepared by the following method; however, the conditions of this method, such as reactants, solvent, base, amount of compound used, reaction temperature, and reaction time, are not limited to those explained below. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations being readily performed by those skilled in the art.

[0082] In the preparation method of the present invention, each reaction is typically carried out in an inert solvent at a reaction temperature of -78°C to 150°C (preferably 20°C to 120°C). The reaction time for each step is typically 0.5 to 48 h, preferably 2 to 12 h.

[0083] Pharmaceutically acceptable salts, solvates, stereoisomers, and tautomers

[0084] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include (but are not limited to): hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1,5), linalool, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids.

[0085] As used herein, the term "pharmaceuticalally acceptable solvate" refers to a compound of the present invention forming a solvate with a pharmaceutically acceptable solvent, wherein the pharmaceutically acceptable solvent includes (but is not limited to): water, ethanol, methanol, isopropanol, tetrahydrofuran, and dichloromethane.

[0086] As used herein, the term "pharmaceutically acceptable stereoisomer" means that the chiral carbon atom involved in the compounds of this invention can be in the R configuration, the S configuration, or a combination thereof.

[0087] Application method

[0088] The compounds of this invention can be used as contrast agents for the imaging of various cancers, such as solid tumors including lung cancer, bladder cancer, breast cancer, stomach cancer, liver cancer, prostate cancer, kidney cancer, and pancreatic cancer.

[0089] Because the compounds of this invention exhibit excellent response to hypoxic microenvironments, the compounds of this invention, their pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of this invention as the main active ingredient can be used to treat, prevent, and alleviate diseases related to hypoxic microenvironments. According to the prior art, the compounds of this invention can be used to treat (but are not limited to): various cancers, such as lung cancer, bladder cancer, breast cancer, gastric cancer, liver cancer, prostate cancer, kidney cancer, pancreatic cancer, etc. The pharmaceutical compositions of this invention contain the compounds of this invention or their pharmacologically acceptable salts and pharmacologically acceptable excipients or carriers within a safe and effective amount range. "Safe and effective amount" refers to an amount of compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of this invention per dose, more preferably, 5-200 mg of the compound of this invention per dose. Preferably, "one dose" is one capsule or tablet.

[0090] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0091] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0092] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0093] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0094] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0095] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0096] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0097] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0098] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0099] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0100] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0101] The main advantages of this invention include:

[0102] (1) The artificial deoxyribose that responds to the hypoxia microenvironment provided by the present invention has strong targeting and good biological stability.

[0103] (2) The nucleic acid probe library prepared by artificial deoxyribose in response to hypoxia microenvironment provided by the present invention has a series of nitroimidazoles with precisely controllable artificial base positions, so that the molecular weight of the probe and the number of nitroimidazoles change in a gradient, thereby regulating the targeting speed and enrichment degree of each molecule in hypoxia microenvironment based on nitroimidazoles.

[0104] (3) The artificial deoxyribose that responds to the hypoxia microenvironment provided by the present invention has a correlation between the number of responding functional groups, the molecular weight and the hypoxia concentration, and can be developed into a nucleic acid "nanobiometric ruler" for accurately measuring the hypoxia concentration.

[0105] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. All solvents, raw materials, and reagents, unless otherwise specified, are of analytical or chemical purity; the anhydrous treatment of solvents during the preparation process is carried out according to conventional methods. Unless otherwise specified, all methods are conventional methods. Unless otherwise specified, all raw materials are obtainable from publicly available commercial sources.

[0106] In the following examples, DCM represents dichloromethane; DMF represents N,N-dimethylformamide. All compounds were characterized by NMR, high-resolution mass spectrometry, and other methods.

[0107] Example 1: Preparation of a stress-responsive deoxyribose probe based on 5-(bromomethyl)-1-methyl-2-nitro-1H-imidazolium

[0108] 1.1 Preparation of Compound 1 (5-(bromomethyl)-1-methyl-2-nitro-1H-imidazolium)

[0109] The specific operating procedure is as follows:

[0110]

[0111] Under nitrogen protection, PPhBr3 (19 mmol) was slowly added to a solution of N-1-methyl-2-nitro-5-hydroxymethylimidazolium (10 mmol) and DIPEA (20 mmol) dissolved in ultra-dry dichloromethane at 0 °C, and the reaction was maintained at 0 °C for 5 hours. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (yield 81%).

[0112] Compound 1: (5-(Bromomethyl)-1-methyl-2-nitro-1H-imidazole):

[0113] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.21 (s, 1H), 4.50 (s, 2H), 4.07 (s, 3H); 13C NMR (126MHz, CDCl3), δ (ppm) = 152.20, 133.08, 128.35, 34.07, 18.95. MS (ESI) m / z for C5H6BrN3O2: 218.9643 (calcd.), 218.9651 (expt.).

[0114] 1.2 Preparation of Compound 2

[0115]

[0116] Under nitrogen protection, D1 (10 mmol), compound 1 (10 mmol), and sodium hydride (11 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. 20 mL of DMF was added to the reaction tube, and the mixture was stirred at 0 °C for 45 minutes. The temperature was then raised to room temperature, and the reaction continued for 8 hours. The reaction was monitored by TCL. Once all the reactants had reacted completely, water was added to quench the reaction, and the aqueous layer was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (67% yield).

[0117] Compound 2: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)benzamide:

[0118] 1 H NMR (500MHz, CD3CN) δ (ppm) = 8.48 (s, 1H), 8.18 (s, 1H), 7.39–7.37 (m, 4H), 7.27–7.23 (m, 9H), 7.10 (t, 2H), 7.01 (s, 1H), 6.82 (d, 4H) ,6.36(t,1H),5.58(s,2H),4.60(d,1H),4.06–4.03(m,1H),4.02(d,3H),3.77(s,6H),3.24(d,2H),2.84–2.76(m,1H),2.40(d,2H); 13C NMR (126MHz, CD3CN), δ (ppm) = 171.37, 158.62, 151.72, 152.67, 151.56, 14 5.02,143.64,135.91,135.82,135.59,134.71,131.04,130.00,129.97,1 28.58,128.09,127.96,127.90,127.80,127.10,126.81,113.01,86.21,8 6.02,84.29,70.93,63.49,60.00,54.93,41.50,38.93,34.18; MS(ESI)m / z for C 43 H 40 N8O8Na:[M+Na] + :819.286(calcd.),819.287(expt.).

[0119] 1.3 Preparation of Compound 3

[0120]

[0121] Under nitrogen protection, compound 2 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. The reaction was stirred in ultra-dry DCM solvent under nitrogen protection for 3 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 63%).

[0122] Compound 3:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-((1-methyl-2-nitro-1H-imidaz) ol-5-yl)methyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0123] 11H NMR (500 MHz, CD3CN) δ (ppm) = 8.49 (d, 1H), 8.19 (d, 1H), 7.42–7.34 (m, 4H), 7.29–7.19 (m, 9H), 7.09 (t, 2H), 7.01 (s, 1H), 6.81 (d, 4H), 6.37 (q, 1H), 5.58 (s, 2H), 4.91–4.78 (m, 1H), 4.18 (d, 1H), 4.02 (d, 3H), 3.88–3.79 (m, 1H), 3.78 (d, 7H), 3.75–3.66 (m, 1H), 3.61 (d, 2H), 3.39–3.30 (m, 1H), 3.23 (t, 1H), 3.00–2.91 (m, 1H), 2.62–2.50 (m, 2H), 1.21–1.15 (m, 10H), 1.10 (d, 3H); 13 13C NMR (126 MHz, CD3CN), δ (ppm) = 171.31, 158.64, 152.78, 152.54, 151.54, 144.97, 143.90, 143.86, 135.83, 135.79, 135.73, 135.58, 134.70, 131.05, 130.00, 129.97, 128.57, 128.10, 127,97, 127.92, 127.88, 127.79, 127.20, 127.16, 126.81, 126.80, 113.01, 86.02, 85.37, 85.14, 84.45, 73.13, 73.00, 72.57, 72.44, 63.14, 62.98, 59.99, 58.63, 58.52, 58.48, 58.37, 54.93, 43.10, 43.06, 43.00, 42.97, 41.49, 38.08, 38.05, 37.95, 37.91, 34.16, 23.97, 23.94, 23.91, 23.88, 23.83, 20.17, 20.11, 20.05, (20.03, 19.97); 31 31P NMR (202 MHz, CD3CN), δ (ppm) = 148.11, 148.00; MS (ESI) m / z for C 52 H 58 N 10 O9P: [M + H] + : 997.412 (calcd.), 997.411 (expt.).

[0124] 1.4 Preparation of Compound 4

[0125]

[0126] Under nitrogen protection, compound 1 (5 mmol), D2 (5 mmol), and potassium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar, followed by 10 mL of DMF solvent. The mixture was heated to 80 °C and stirred for 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 86%).

[0127] Compound 4:1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)pyr-imidine-2,4(1H,3H)-dione:

[0128] 1 H NMR(500MHz,CD3CN)δ7.55(s,1H),7.46(d,2H),7.34(d,6H),7.26(t,1H),7.11(s,1H),6.89(d,4H),6.28(t,1H),5.10(s,2H),4 .49(d,1H),4.06(s,3H),3.96(d,1H),3.79(s,6H),3.35–3.24(m,2H),2.92(s,1H),2.80(s,1H),2.32–2.27(m,2H),1.55(s,3H); 13 CNMR (126MHz, CD3CN), δ (ppm) = 163.00, 158.80, 150.64, 144.95, 135.84, 134.66, 134.41, 130.06, 128.81, 128.0 3,127.93,126.97,113.12,109.52,86.38,85.99,85.42,70.91,63.51,54.94,40.04,34.27,11.97; MS(ESI)m / z for C 36 H 37 N5O9Na:[M+Na] + :706.2489(calcd.),706.2491(expt.).

[0129] 1.5 Preparation of Compound 5

[0130]

[0131] Under nitrogen protection, compound 4 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 71%).

[0132] Compound 5:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-met hyl-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)tetra hydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0133] 1 H NMR(500MHz,CD3CN)δ7.57(d,1H),7.47(d,2H),7.38–7.29(m,6H),7.29–7.23(m,1H), 7.11(d,1H),6.88(d,4H),6.30(d,1H),5.10(s,2H),4.70–4.61(m,1H),4.14(d,1H),4. 09(t,1H),4.06(s,3H),3.86–3.81(m,1H),3.78(s,6H),3.72–3.53(m,3H),3.42–3.28 (m,2H),2.66(t,1H),2.54(t,1H),2.41(d,1H),1.56(d,3H),1.18(d,9H),1.07(d,3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 162.96, 158.80, 150.69, 144.89, 130.10, 130.08, 128.84, 128.8 3,128.05,128.00,127.93,127.01,126.99,118.57,118.41,113.13,86.50,86.41,85.57,85. 48,85.18,85.14,84.92,84.87,73.17,73.04,72.72,63.15,62.99,58.47,58.32,54.95,54.94,43.08,42.98,39.28,39.26,39.10,34.27,23.93,23.87,23.81,20.04,13.33,11.97,11.96; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.02, 147.99; MS (ESI) m / z for C 45 H 54 N7O 10 PNa:[M+Na] + :906.3567(calcd.),906.3571(expt.).

[0134] 1.6 Preparation of Compound 6

[0135]

[0136] Under nitrogen protection, compound 1 (5 mmol), D3 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 86%).

[0137] Compound 6: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)isobutyramide:

[0138] 1 H NMR(500MHz,CD3CN)δ8.76(s,1H),8.03(s,1H),7.45(s,1H),7.34(d,2H),7. 24–7.21(m,4H),7.20–7.17(m,2H),6.80–6.70(m,4H),6.33(d,1H),5.77–5. 63(m,2H),4.75(d,1H),4.11–4.00(m,4H),3.75(d,6H),3.44–3.32(m,1H),3 .27–3.13(m,1H),2.94(d,1H),2.43(d,1H),2.03–1.91(m,2H),1.18(d,6H). 13 C NMR (126MHz, CD3CN) δ175.22,159.29,158.54,158.50,152.99,151.77,145.14,141.54,135.95,135.92,133.06,130.00,129.93,129 .91,128.00,127.66,126.70,86.51,85.86,84.40,71.11,64.24,57.08,54.88,54.88,38.95,35.36,34.27,18.74,18.73.MS(ESI)m / z for C 40 H 42 N8O9:778.3075(calcd.),778.3095(expt.).

[0139] 1.7 Preparation of Compound 7

[0140]

[0141] Under nitrogen protection, compound 6 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 74%).

[0142] Compound 7: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0143] 1 H NMR(500MHz,CD3CN)δ8.98(s,1H),8.12(s,1H),7.56(s,1H),7.45(d,2H),7.33–7.11(m,4H),7.31–7.27(m,2H),6.88–6.68(m,4H),6.43(d,1H),5.79–5.59(m,2H),4.85(d,1H),4.21–4.01(m,6H),3.85-3.50(m,8H),3.34–3.22(m,1H),3.17–3.03(m,1H),2.99-2.82(m,3H),2.43(d,1H),2.13–1.99(m,2H),1.42(d,6H),1.31(d,6H)1.18(d,6H). 13 C NMR(126MHz,CD3CN)δ176.20,159.52,158.84,158.70,153.09,151.97,145.24,141.44,135.99,135.98,133.26,130.01,129.13,129.91,128.08,127.68,126.90,117.90,86.71,85.96,84.50,71.51,64.34,62.6,56.08,54.98,54.88,47.20,39.95,36.36,34.67,23.50,23.46,19.12,18.79,18.93. 31 P NMR(202MHz,CD3CN)δ148.22,147.98.m / z forC 49 H 59 N 10 O 10 P:978.4153(calcd.),978.4173(expt.).

[0144] 1.8 Preparation of Compound 8

[0145]

[0146] Under nitrogen protection, D4 (10 mmol), compound 1 (10 mmol), and sodium hydride (11 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. 20 mL of DMF was added to the reaction tube, and the mixture was stirred for 45 minutes. The reaction was then brought to room temperature and continued for 8 hours. The reaction was monitored by a TCL (ThinPrep Chromatography-Concentration) until all reactants had reacted completely. The reaction was quenched with water, and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (77% yield).

[0147] Compound 8: N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)benzamide:

[0148] 1 H NMR (500MHz, CD3CN) δ (ppm) = 8.01–7.97 (m, 2H), 7.67 (d, 1H), 7.59 (t, 1H) ,7.49–7.41(m,4H),7.30(d,6H),7.25–7.20(m,1H),7.14(s,1H),6.87(d, 4H),6.21–6.17(m,1H),6.09(d,1H),5.38(s,2H),4.49(d,1H),4.05(s,3 H),3.98–3.94(m,1H),3.77(s,6H),3.38–3.30(m,2H),2.39–2.27(m,3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 176.56, 158.65, 155.21, 149.92, 144.70, 137.33, 135.83, 135.74, 135.56, 134.28, 132.54, 130.07, 130.02, 1 29.34,128.37,128.16,128.09,127.90,126.95,113.09,91.21,86.50 ,86.12,85.89,69.66,62.58,54.90,40.35,36.33,34.35; MS(ESI)m / z for C 42 H 40 N6O9Na:[M+Na] + :795.275(calcd.),795.275(expt.).

[0149] 1.9 Preparation of Compound 9

[0150]

[0151] Under nitrogen protection, compound 8 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 72%).

[0152] Compound 9:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)benzamido)-2-oxopyrimidi n-1(2H)-yl)tetrahydro-furan-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0153] 1H NMR(500MHz,CD3CN)δ7.99(d,2H),7.72–7.64(m,1H),7.59(d,1H),7.49–7.40(m,4H),7.30(d ,6H),7.23(d,1H),7.14(d,1H),6.86(dd,4H),6.21(d,1H),6.08(d,1H),5.37(s,2H),4.73–4 .61(m,1H),4.05(s,3H),3.87–3.80(m,1H),3.76(d,6H),3.74–3.53(m,3H),3.41–3.34(m,2H ),2.67(t,1H),2.56(t,1H),2.50(d,1H),2.43(d,1H),1.97(d,5H),1.19(d,9H),1.08(d,4H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 176.56, 170.68, 158.75, 155.03, 149.96, 144.62, 137.29, 137.24, 135.80, 135.79, 135.62, 135.54, 1 35.49,135.45,134.24,132.55,130.08,129.34,128.36,128.19,128.12,128.07,127.90,126.98,126.97,118.61,118.45,113.11, 97.32,86.58,86.18,86.15,85.17,85.13,84.94,84.89,72.14,72.00,71.49,71.36,62.31,62.10,59.99,58.56,58.41,58.25,43.12,43.08,43.02,42.98,39.59,39.58,39.33,39.29,36.34,34.34,23.95,23.89,23.87,23.80,20.17,20.11,20.05,20.01,13.53; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.26, 148.22; MS (ESI) m / z for C 51 H 57 N8O 10 PNa:[M+Na] + :995.383(calcd.),995.384(expt.).

[0154] The compounds prepared in this embodiment are listed below. Figure 3 .

[0155] Example 2: Preparation of a stress-responsive deoxyribose probe based on 2-(bromomethyl)-1-methyl-5-nitro-1H-imidazolium

[0156] 2.1 Preparation of compound 10 (2-(bromomethyl)-1-methyl-5-nitro-1H-imidazolium)

[0157]

[0158] Under nitrogen protection, PPhBr3 (19 mmol) was slowly added to a solution of N-1-methyl-2-nitro-5-hydroxymethylimidazolium (10 mmol) and DIPEA (20 mmol) dissolved in ultra-dry dichloromethane at 0 °C. The reaction was maintained at 0 °C for 30 min, then slowly raised to room temperature and continued for 5 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (85%).

[0159] Compound 10:2-(Bromomethyl)-1-methyl-5-nitro-1H-imidazole:

[0160] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.81 (s, 1H), 4.50 (s, 2H), 3.82 (s, 3H); 13 C NMR (126MHz, CDCl3), δ (ppm) = 162.08, 139.44, 136.35, 34.06, 28.85; MS (ESI) m / z for C5H6BrN3O2: 218.9643 (calcd.), 218.9655 (expt.).

[0161] 2.2 Preparation of Compound 11

[0162]

[0163] Under nitrogen protection, at 0°C, D1 (10 mmol) dissolved in ultra-dry 20 mL LMF and compound 10 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (11 mmol). The mixture was stirred for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (79% yield).

[0164] Compound 11: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)benzamide

[0165] 1 H NMR(500 MHz, CD3CN) δ(ppm) = 8.57(s, 1H), 8.23(s, 1H), 7.44–7.34(m, 4H), 7.29–7.25(m, 9H), 7.22(t, 2H), 7.12(s, 1H), 6.92(d, 4H), 6.56(t, 1H), 5.48(s, 2H), 4.82(d, 1H), 4.52(d, 3H), 4.11–4.00(m, 1H), 3.73(s, 6H), 3.45(d, 2H), 2.89–2.72(m, 1H), 2.30(d, 2H); 13 C NMR(126 MHz, CD3CN), δ(ppm) = 172.41, 161.57, 159.68, 152.72, 151.96, 146.04, 142.74, 136.81, 136.62, 135.59, 133.51, 130.94, 130.50, 128.97, 128.38, 128.19, 127.76, 127.55, 127.30, 127.07, 126.91, 112.11, 87.21, 86.089, 84.29, 75.93, 66.39, 59.80, 55.73, 40.90, 39.93, 32.09; MS(ESI) m / z for C 43 H 40 N8O8: 796.2969(calcd.), 796.2989(expt.).

[0166] 2.2 Preparation of Compound 12

[0167]

[0168] Under nitrogen protection, compound 11 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 73%).

[0169] Compound 12:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-((1-methyl-5-nitro-1H-imidaz) ol-2-yl)methyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0170] 1 H NMR(500MHz,CD3CN)δ(ppm)=8.89(d,1H),8.66(d,1H),7.86–7.63(m,4H),7.42–7 .21(m,9H),7.11(t,2H),7.00(s,1H),6.61(d,4H),6.05(q,1H),5.54(s,2H),4.87 –4.71(m,1H),4.08(d,1H),4.00(d,3H),3.78–3.59(m,3H),3.48(d,7H),3.35–3. 16(m,1H),3.00–2.91(m,3H),2.32–2.20(m,2H),1.41–1.25(m,10H),1.02(d,3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 170.29, 159.84, 153.88, 153.84, 152.64, 147.97, 144.65, 142.16, 136.13, 135.99, 135.73, 135.6 8,134.99,132.45,131.20,130.97,129.33,129.10,128,57,127.98,127.74,127.68,127.10,127.00,126.81,126.55,115.41,8 9.41,87.47,86.25,84.45,74.47,73.50,72.97,71.24,66.15,62.57,59.23,58.71,58.67,58.38,58.27,55.94,45.10,44.06,43.88,42.77,41.89,39.08,38.15,37.45,37.01,35.26,23.95,23.74,23.51,23.38,23.13,20.07,20.00,19.88,19.26,18.97; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.11, 148.70; MS (ESI) m / z for C 52 H 57 N 10 O9P:996.4048(calcd.),996.4066(expt.).

[0171] 2.3 Preparation of Compound 13

[0172]

[0173] Under nitrogen protection, compound 10 (5 mmol), D2 (5 mmol), and potassium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar, followed by 10 mL of DMF. The mixture was heated to 80 °C and stirred for 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 81%).

[0174] Compound 13:1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione:

[0175] 1 H NMR(500MHz,CD3CN)δ8.01(s,1H),7.86(d,2H),7.64(d,6H),7.46(t,1H),7.03(s,1H),6.66(d,4H),6.05(t,1H),4.99(s,2H),4 .39(d,1H),4.15(s,3H),4.01(d,1H),3.66(s,6H),3.39–3.24(m,2H),2.98(s,1H),2.86(s,1H),2.82–2.67(m,2H),1.79(s,3H); 13 CNMR (126MHz, CD3CN), δ (ppm) = 165.00, 159.80.152.77, 147.58, 137.25, 136.64, 135.21, 129.26, 128.91, 128.3 3,128.03,126.97,115.21,111.52,89.38,84.74,83.87,72.11,66.53,55.77,41.26,37.53,12.07; MS(ESI)m / z for C 36 H 37 N5O9:683.2591(calcd.),683.2598(expt.).

[0176] 2.4 Preparation of Compound 14

[0177]

[0178] Under nitrogen protection, compound 13 (2 mmol), DIPEA (10 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (6 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 77%).

[0179] Compound 14: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-3-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0180] 1 H NMR (500 MHz, CD3CN) δ 8.17 (d, 1H), 7.89 (d, 2H), 7.84–7.68 (m, 6H), 7.59–7.43 (m, 1H), 7.23 (d, 1H), 6.93 (d, 4H), 6.56 (d, 1H), 5.27 (s, 2H), 4.88–4.73 (m, 1H), 4.36 (d, 1H), 4.29 (t, 1H), 4.18 (s, 3H), 3.89–3.84 (m, 1H), 3.78 (s, 6H), 3.72–3.53 (m, 3H), 3.39–3.25 (m, 2H), 2.87 (t, 1H), 2.75 (t, 1H), 2.56 (d, 1H), 1.68 (d, 3H), 1.09 (d, 9H), 1.11 (d, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 165.12, 159.66, 152.79, 146.52, 131.26, 130.85, 129.14, 128.53, 12 eighty-two point six, one hundred and twenty-eight point zero one, one hundred and twenty-seven point five six, one hundred and twenty-seven point one two, one hundred and twenty-four point nine seven, one hundred and nineteen point one seven, one hundred and eighteen point seven six, one hundred and fifteen point zero four, eighty-nine point two four, eighty-seven point three eight, eighty-six point eight two, eighty-five point eight five, eighty-five point two six, eighty-five point zero one, eighty-four point eight two, eighty-three point zero five, seventy-five point six four, seventy-four point three seven, seventy-three point six one, sixty-six point three eight, sixty-one point zero five, fifty-nine point four two, fifty-eight point eight six, fifty-six point nine five, fifty-five point nine zero, forty-five point four six, forty-one point seven four, thirty-nine point nine nine, thirty-nine point two two, thirty-nine point zero zero, thirty-five point three eight, twenty-seven point nine seven, twenty-three point four three, twenty-two point zero one, nineteen point zero eight, fourteen point three three, twelve point seven seven, eleven point eight nine; 31 P NMR (202 MHz, CD3CN), δ (ppm) = 149.15, 149.11; MS (ESI) m / z for C 45 H 54 N7O 10 It should be noted that in the translation of the C NMR data in item , there may be some inaccuracies in the original Chinese description. The correct chemical shift values are provided in the translation as accurately as possible based on the context.P:883.3670(calcd.),883.3677(expt.).

[0181] 2.5 Preparation of Compound 15

[0182]

[0183] Under nitrogen protection, compound 10 (5 mmol), D3 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 83%).

[0184] Compound 15:N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2 -yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)isobutyramide:

[0185] 1 H NMR(500MHz,CD3CN)δ8.88(s,1H),8.56(s,1H),7.89(s,1H),7.55(d,2H),7. 44–7.33(m,4H),7.19–7.08(m,2H),6.86–6.75(m,4H),6.39(d,1H),5.82–5. 74(m,2H),4.88(d,1H),4.33–4.21(m,4H),3.88(d,6H),3.61–3.48(m,1H),3 .33–3.20(m,1H),2.86(d,1H),2.55(d,1H),2.36–2.10(m,2H),1.38(d,6H). 13C NMR (126MHz, CD3CN) δ176.21,158.56,158.33,158.21,152.99,151.83,147.28,143.39,136.11,134.92,133.63,131.07,129.89,129 .56,128.71,127.83,126.11,87.57,85.96,84.79,73.57,63.11,59.62,55.71,53.82,39.17,36.56,35.63,19.15,17.01.MS(ESI)m / z for C 40 H 42 N8O9:778.3075(calcd.),778.3079(expt.).

[0186] 2.6 Preparation of Compound 16

[0187]

[0188] Under nitrogen protection, compound 15 (2 mmol), DIPEA (10 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (6 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 87%).

[0189] Compound 16:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-((1-methyl-5-nitro-1H-imidazol-2-yl)met hyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0190] 1H NMR(500MHz,CD3CN)δ8.73(s,1H),8.41(s,1H),7.91(s,1H),7.82(d,2H),7.58–7.45 (m,4H),7.28–7.07(m,2H),6.84–6.66(m,4H),6.36(d,1H),5.89–5.75(m,2H),4.43(d ,1H),4.32–4.20(m,6H),3.96-3.82(m,8H),3.66–3.55(m,1H),3.23–3.12(m,1H),2.8 3-2.72(m,3H),2.53(d,1H),2.32–2.21(m,2H),1.59(d,6H),1.46(d,6H)1.15(d,6H). 13 C NMR(126MHz,CD3CN)δ178.01,162.53,160.24,159.23,155.62,153.15,146 .39,143.36,139.56,136.12,132.58,131.01,130.13,129.91,128.85,127 .63,126.31,119.92,87.78,84.96,84.36,73.51,63.54,62.87,58.08,55.98,54.98,49.20,39.25,36.78,34.34,26.47,23.85,21.25,19.23,17.99. 31 P NMR(202MHz,CD3CN)δ148.82,147.68.MS(ESI)m / zfor C 49 H 59 N 10 O 10 P:978.4153(calcd.),978.4155(expt.).

[0191] 2.7 Preparation of Compound 17

[0192]

[0193] Under nitrogen protection, 20 mL of ultra-dry DMF-dissolved D4 (10 mmol) and compound 10 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred at low temperature for 45 minutes, then brought to room temperature and reacted for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 81%).

[0194] Compound 17: N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)benzamide:

[0195] 1 H NMR (500 MHz, CD3CN) δ (ppm) = 8.88–8.80 (m, 2H), 7.92 (d, 1H), 7.67 (t, 1H), 7.58–7.45 (m, 4H), 7.35 (d, 6H), 7.27–7.18 (m, 1H), 7.05 (s, 1H), 6.82 (d, 4H), 6.42–6.35 (m, 1H), 6.22 (d, 1H), 5.55 (s, 2H), 4.63 (d, 1H), 4.21 (s, 3H), 3.89–3.79 (m, 1H), 3.55 (s, 6H), 3.41–3.30 (m, 2H), 2.73–2.61 (m, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 175.26, 159.62, 156.36, 150.92, 146.56, 138.42, 136.52, 135.99, 134.56, 134.18, 133.56, 131.56, 130.73, 128.94, 128.89, 128.58, 128.23, 127.53, 126.85, 114.11, 9三、20, 87.47, 86.72, 85.53, 69.42, 63.14, 56.03, 41.12, 35.83, 33.25; MS (ESI) m / z for C 42 H 40 N6O9: 772.2857 (calcd.), 772.2863 (expt.).

[0196] 2.8 Preparation of Compound 18

[0197]

[0198] Under nitrogen protection, compound 17 (5 mmol), DIPEA (50 mmol), and 3-(chloro(diisopropylamino)phosphine oxide)propionitrile (7.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 83%).

[0199] Compound 18:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)benzamido)-2-oxopyrimidin-1(2H)-yl)tetrahydrofu ran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0200] 1 H NMR(500MHz,CD3CN)δ7.86(d,2H),7.73–7.65(m,1H),7.59(d,1H),7.45–7.37(m,4H),7.29(d ,6H),7.21(d,1H),7.15(d,1H),6.90(dd,4H),6.46(d,1H),6.14(d,1H),5.58(s,2H),4.88–4 .77(m,1H),4.23(s,3H),3.92–3.84(m,1H),3.72(d,6H),3.66–3.52(m,3H),3.42–3.31(m,2H ),2.79(t,1H),2.61(t,1H),2.49(d,1H),2.38(d,1H),2.01(d,5H),1.23(d,9H),1.05(d,4H); 13C NMR (126MHz, CD3CN), δ (ppm) = 178.01, 172.99, 159.70, 156.75, 147.56, 144.96, 139.64, 138.53, 137.88, 136.56, 135.98, 135.66, 1 35.34,135.28,134.19,131.55,130.28,129.82,128.96,128.20,128.15,128.00,127.70,126.85,126.30,119.35,118.05,114.11, 98.38,89.61,87.19,86.95,85.28,85.03,84.88,84.51,74.14,73.09,71.55,71.26,63.63,62.11,59.08,58.52,58.21,58.04,46.18,44.13,43.99,42.56,39.83,39.62,39.43,39.34,37.38,35.26,25.95,24.80,23.99,23.87,21.19,21.01,20.56,20.33,14.23; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.52, 148.42; MS (ESI) m / z for C 51 H 57 N8O 10 P:972.3935(calcd.),972.3938(expt.).

[0201] The compounds prepared in this embodiment are listed below. Figure 4 .

[0202] Example 3: Preparation of a stress-responsive deoxyribose probe based on 1-(bromomethyl)-2-nitro-1H-imidazolium

[0203] 3.1 Preparation of compound 19 (1-(bromomethyl)-2-nitro-1H-imidazolium)

[0204]

[0205] Under nitrogen protection, PPhBr3 (20 mmol) was slowly added to a solution of N-1-hydroxymethyl-2-nitroimidazole (10 mmol) and DIPEA (20 mmol) dissolved in ultra-dry dichloromethane at 0 °C. The reaction was maintained at 0 °C for 30 min, then slowly raised to room temperature and continued for 5 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (85%).

[0206] Compound 19:1-(Bromomethyl)-2-nitro-1H-imidazole:

[0207] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.86 (s, 1H), 7.21 (d, 1H), 5.84 (s, 2H); 13 C NMR (126MHz, CDCl3), δ (ppm) = 156.53, 130.52, 127.53, 50.91; MS (ESI) m / z for C4H4BrN3O2: 204.9487 (calcd.), 204.9498 (expt.).

[0208] 3.2 Preparation of Compound 20

[0209]

[0210] Under nitrogen protection, at a low temperature of 0°C, 20 mL of ultra-dry DMF dissolved in D1 (10 mmol) and compound 19 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (77% yield).

[0211] Compound 20: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-((2-nitro-1H-imidazol-1-yl)methyl)benz-amide:

[0212] 1H NMR (500MHz, CD3CN) δ (ppm) = 8.78 (s, 1H), 8.34 (s, 1H), 7.86 (d, 1H), 7.71 (d, 1H), 7.58–7.45 (m, 4H), 7.39–7.30 (m, 9H), 7.19 (t, 2H) ,6.98(d,4H),6.78(t,1H),5.74(s,2H),4.63(d,1H),4.46–4.28(m,1H),3.82(s,6H),3.33(d,2H),2.97–2.83(m,1H),2.63(d,2H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 171.80, 159.85, 152.93, 152.76, 152.06, 14 6.81,144.88,137.01,136.52,136.09,135.83,132.23,131.80,130.64,1 29.58,128.79,128.26,127.90,127.56,127.05,126.93,114.26,87.33,8 6.73,85.49,71.15,64.55,60.89,55.97,50.72,42.50,33.98; MS(ESI)m / z for C 42 H 38 N8O8:782.2813(calcd.),782.2817(expt.).

[0213] 3.3 Preparation of Compound 21

[0214]

[0215] Under nitrogen protection, compound 20 (5 mmol), DIPEA (10 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (15 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. The mixture was stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by vacuum concentration, and the target compound was obtained by column chromatography (yield 88%).

[0216] Compound 21: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-((2-nitro-1H-imidazol-1-yl)methyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0217] 1 H NMR (500 MHz, CD3CN) δ (ppm) = 8.56 (d, 1H), 8.39 (d, 1H), 7.96 (d, 1H), 7.86 (d, 1H), 7.53–7.44 (m, 4H), 7.31–7.23 (m, 9H), 7.13 (t, 2H), 6.89 (d, 4H), 6.57 (q, 1H), 5.68 (s, 2H), 4.98–4.83 (m, 1H), 4.27 (d, 1H), 3.93–3.83 (m, 1H), 3.78 (d, 7H), 3.65–3.52 (m, 1H), 3.41 (d, 2H), 3.26–3.13 (m, 1H), 3.03 (t, 1H), 2.83–2.71 (m, 1H), 2.61–2.49 (m, 2H), 1.42–1.28 (m, 10H), 1.11 (d, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 171.69, 159.86, 154.61, 153.57, 152.04, 148.97, 144.95, 143.75, 138.63, 136.18, 135.88, 135.74, 135.55, 131 P NMR (202MHz, CD3CN), δ (ppm) = 148.61, 148.49; MS (ESI) m / z forC 51 H 55 N 10 O9P:982.3891(calcd.),982.3894(expt.).

[0218] 3.4 Preparation of Compound 22

[0219]

[0220] Under nitrogen protection, compound 19 (5 mmol), D2 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was heated to 80 °C and stirred for 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 82%).

[0221] Compound 22: 1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-((2-nitro-1H-imidazol-1-yl)methyl)pyrimidine-2,4(1H,3H)-dione:

[0222] 1 H NMR(500MHz,CD3CN)δ8.56(d,1H),8.36(d,1H),7.89(s,1H),7.76(d,2H),7.55(d,6H),7.28(t,1H),6.94(d,4H),6.44(t,1H),5 .37(s,2H),4.85(d,1H),3.98(d,1H),3.83(s,6H),3.75–3.64(m,2H),2.98(s,1H),2.83(s,1H),2.62–2.47(m,2H),1.63(s,3H); 13CNMR (126MHz, CD3CN), δ (ppm) = 164.93, 159.89, 151.94, 146.05, 136.84, 134.96, 134.66, 131.06, 129.31, 128.7 3,127.73,126.56,115.12,110.76,87.13,86.13,85.22,69.91,64.51,53.76,51.71,41.46,11.53; MS(ESI)m / z for C 35 H 35 N5O9P:669.2435(calcd.),669.2445(expt.).

[0223] 3.5 Preparation of Compound 23

[0224]

[0225] Under nitrogen protection, compound 22 (4.5 mmol), DIPEA (9 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (13.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar at 0 °C. Under nitrogen protection, 20 mL of ultra-dry DCM solvent was added. The mixture was slowly heated to room temperature and stirred for 3 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the mixture was separated by column chromatography to obtain the target compound (yield 81%).

[0226] Compound 23:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-3-((2-nitro-1H-imidazol-1-yl)m ethyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0227] 11H NMR (500 MHz, CD3CN) δ 8.23 (d, 1H), 7.98 (d, 1H), 7.78 (d, 1H), 7.69 (d, 2H), 7.56–7.43 (m, 6H), 7.25 (d, 1H), 6.92 (d, 4H), 6.56 (d, 1H), 5.23 (s, 2H), 4.84–4.72 (m, 1H), 4.46 (d, 1H), 4.23 (t, 1H), 3.93–3.86 (m, 1H), 3.78 (s, 6H), 3.63–3.50 (m, 3H), 3.41–3.29 (m, 2H), 2.84 (t, 1H), 2.63 (t, 1H), 2.36 (d, 1H), 1.98 (d, 3H), 1.33 (d, 9H), 1.21 (d, 3H); 13 13C NMR (126 MHz, CD3CN), δ (ppm) = 165.25, 161.83, 152.69, 146.23, 132.19, 131.08, 130.96, 129.56, 128.85, 128.63, 127.93, 127.74, 126.85, 120.25, 119.41, 113.59, 89.83, 88.41, 87.42, 86.66, 85.92, 85.78, 84.56, 76.17, 75.04, 73.72, 66.52, 64.15, 59.11, 58.31, 56.15, 55.38, 52.77, 47.78, 44.54, 41.82, 38.86, 38.25, 25.93, 24.66, 23.41, 22.18, 16.33, 13.96, 12.05; 31 31P NMR (202 MHz, CD3CN), δ (ppm) = 148.43, 148.15; MS (ESI) m / z for C 44 H 52 N7O 10 P: 869.3513 (calcd.), 869.3525 (expt.).

[0228] 3.6 Preparation of Compound 24

[0229]

[0230] Under nitrogen protection, compound 19 (3 ​​mmol), D3 (3 mmol), and cesium carbonate (4.5 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 86%).

[0231] Compound 24: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-((2-nitro-1H-imidazol-1-yl)methyl)isobutyramide:

[0232] 1 H NMR(500MHz,CD3CN)δ8.88(s,1H),8.45(s,1H),8.03(d,1H),7.86(d,1H),7.6 6(d,2H),7.45–7.33(m,4H),7.28–7.18(m,2H),6.95–6.82(m,4H),6.75(d,1H) ,5.85(s,2H),4.91(d,1H),4.63–4.51(m,1H),3.78(d,6H),3.55–3.39(m,1H) ,3.28–3.16(m,1H),2.98(d,1H),2.49(d,1H),2.10–1.99(m,2H),1.25(d,6H). 13 C NMR (126MHz, CD3CN) δ177.82,162.31,159.54,158.74,154.58,153.87,147.28,142.33,137.63,134.92,133.75,132.32,129.83,129 .52,128.23,127.69,126.52,86.85,85.71,84.31,72.63,65.33,56.99,55.52,55.06,52.28,39.86,37.28,19.29,18.27; MS(ESI)m / z for C 39 H 40 N8O9:764.2918(calcd.),764.2923(expt.).

[0233] 3.7 Preparation of Compound 25

[0234]

[0235] Under nitrogen protection, compound 24 (5 mmol), DIPEA (10 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (15 mmol) were dissolved in ultradry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 82%).

[0236] Compound 25:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-((2-nitro-1H-imidazol-1-yl)methyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydro furan-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0237] 1 H NMR(500MHz,CD3CN)δ8.92(s,1H),8.52(s,1H),8.05(d,1H),7.98(d,1H),7.77(d,2H), 7.63–7.52(m,4H),7.43–7.33(m,2H),6.92–6.82(m,4H),6.66(d,1H),5.85(s,2H),4.8 3(d,1H),4.32–4.18(m,3H),3.93-3.81(m,8H),3.58–3.41(m,1H),3.31–3.18(m,1H),2 .95-2.82(m,3H),2.56(d,1H),2.21–2.12(m,2H),1.56(d,6H),1.42(d,6H)1.07(d,6H). 13CNMR(126MHz,CD3CN)δ178.18,162.56,160.84,159.70,155.09,153.35,14 7.26,143.32,138.23,137.98,135.19,132.01,130.13,129.88,128.56,12 7.10, 126.92, 119.05, 88.71, 85.92, 83.50, 72.51, 65.34, 63.60, 57.11, 55.98, 54.56, 50.27, 48.23, 40.95, 38.36, 24.23, 23.78, 19.63, 18.18, 18.03. 31 P NMR(202MHz,CD3CN)δ148.33,147.12; MS(ESI)m / zfor C 48 H 57 N 10 O 10 P:964.3997(calcd.),964.3999(expt.).

[0238] 3.8 Preparation of Compound 26

[0239]

[0240] Under nitrogen protection, D4 (10 mmol) dissolved in 20 mL of ultra-dry DMF and compound 19 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol) at 0 °C. The mixture was stirred at 0 °C for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (70% yield).

[0241] Compound 26: N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-((2-nitro-1H-imidazol-1-yl)methyl)benzamide:

[0242] 1H NMR (500MHz, CD3CN) δ (ppm) = 8.32–8.24 (m, 2H), 8.02 (d, 1H), 7.94 (d, 1H) ,7.82(d,1H),7.68(t,1H),7.60–7.52(m,4H),7.42(d,6H),7.34–7.25(m, 1H),6.93(d,4H),6.76–6.63(m,1H),6.33(d,1H),5.77(s,2H),4.75(d,1 H),4.05–3.94(m,1H),3.66(s,6H),3.48–3.36(m,2H),2.56–2.39(m,3H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 177.86, 159.78, 157.36, 151.03, 146.18, 138.15, 136.98, 135.94, 135.77, 135.28, 134.41, 132.80, 131.56, 1 30.18,129.18,128.99,128.78,127.56,126.52,115.09,93.72,88.31 ,87.12,85.92,71.73,65.56,58.83,51.78,44.02,38.56; MS(ESI)m / z for C 41 H 38 N6O9:758.2700(calcd.),758.2721(expt.).

[0243] 3.9 Preparation of Compound 27

[0244]

[0245] Under nitrogen protection, compound 26 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 83%).

[0246] Compound 27: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-((2-nitro-1H-imidazol-1-yl)methyl)benzamido)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0247] 1 H NMR (500 MHz, CD3CN) δ 8.31 (d, 2H), 8.06 (d, 1H), 7.98 (d, 1H), 7.82–7.74 (m, 1H), 7.68 (d, 1H), 7.56–7.45 (m, 4H), 7.33 (d, 6H), 7.21 (d, 1H), 6.83 (d, 4H), 6.23 (d, 1H), 6.13 (d, 1H), 5.48 (s, 2H), 4.86–4.75 (m, 1H), 3.93–3.85 (m, 1H), 3.68 (d, 6H), 3.54–3.43 (m, 3H), 3.31–3.24 (m, 2H), 2.85 (t, 1H), 2.74 (t, 1H), 2.63 (d, 1H), 2.45 (d, 1H), 1.98 (d, 5H), 1.25 (d, 9H), 1.06 (d, 4H); 13C NMR (126MHz, CD3CN), δ (ppm) = 176.01, 171.38, 159.78, 156.21, 149.99, 145.82, 138.70, 137.514, 136.79, 136.52, 135.99, 135.74, 1 35.59,135.43,134.85,131.75,131.08,129.84,128.86,128.25,128.18,128.01,127.85,126.85,126.20,119.73,119.45,114.12, 98.12,87.68,86.73,86.30,85.55,85.23,84.99,84.79,73.14,72.56,71.33,71.26,63.81,62.77,59.90,58.65,58.38,58.05,50.70,44.18,43.25,43.18,42.88,39.48,39.85,39.35,39.21,37.24,24.75,24.59,23.98,23.74,21.20,20.85,20.18,20.01,13.15; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.72, 148.68; MS (ESI) m / z for C 50 H 55 N8O 10 P:958.3779(calcd.),958.3783(expt.).

[0248] The compounds prepared in this embodiment are listed below. Figure 5 .

[0249] Example 4: Preparation of a stress-responsive deoxyribose probe based on 2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl methanesulfonate

[0250] 4.1 Preparation of Compound 28 (2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl methanesulfonic acid)

[0251]

[0252] Under nitrogen protection, N-1-methyl-2-nitro-5-hydroxyethylimidazolium (10 mmol) and triethylamine (30 mmol) were dissolved in 20 mL of ultradry dichloromethane in a reaction tube. The system was maintained at 0 °C and methanesulfonyl chloride (15 mmol) was slowly added and reacted for 1 hour. The temperature was then slowly raised to room temperature and the reaction was continued for 5 hours. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (79%).

[0253] Compound 28:2-(1-Methyl-2-nitro-1H-imidazol-5-yl)ethylmethanesulfonate:

[0254] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.46 (s, 1H), 3.95 (t, 2H), 3.26 (s, 3H), 3.15 (s, 3H), 2.66 (t, 2H); 13 C NMR (126MHz, CDCl3), δ (ppm) = 156.26, 142.63, 122.62, 70.33, 38.87, 31.19, 21.82; MS (ESI) m / z for C7H 11 N3O5S:249.0419(calcd.),249.0552(expt.).

[0255] 4.2 Preparation of Compound 29

[0256]

[0257] Under nitrogen protection, at 0°C, D1 (10 mmol) dissolved in ultra-dry 20 mL LMF and compound 28 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (67% yield).

[0258] Compound 29: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)benzamide:

[0259] 1 H NMR (500 MHz, CD3CN) δ (ppm) = 8.66 (s, 1H), 8.34 (s, 1H), 7.88–7.74 (m, 4H), 7.48–7.35 (m, 9H), 7.21 (t, 2H), 7.08 (s, 1H), 6.76 (d, 4H), 6.22 (t, 1H), 5.42 (t, 2H), 4.85 (d, 1H), 4.21–4.14 (m, 1H), 4.05 (d, 3H), 3.83 (s, 6H), 3.37 (d, 2H), 2.85 (t, 2H), 2.77–2.63 (m, 1H), 2.50 (d, 2H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 171.59, 159.80, 152.83, 152.02, 151.89, 147.56, 144.84, 137.41, 136.82, 135.35, 134.88, 132.04, 131.09, 129.92, 128.82, 128.53, 127.99, 127.85, 127.63, 127.25, 126.84, 115.56, 87.21, 86.58, 84.11, 68.93, 66.32, 64.56, 61.02, 56.03, 43.11, 37.93, 33.24; MS (ESI) m / z for C 44 H 42 N8O8: 810.3126 (calcd.), 810.3134 (expt.).

[0260] 4.3 Preparation of Compound 30

[0261]

[0262] Under nitrogen protection, compound 29 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 79%).

[0263] Compound 30: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)di isopropylphosphoramidite:

[0264] 1 H NMR (500MHz, CD3CN) δ (ppm) = 8.56 (d, 1H), 8.28 (d, 1H), 7.74–7.62 (m, 4H), 7.38–7.27 (m, 9H), 7. 15(t,2H),7.02(s,1H),6.98(d,4H),6.56(q,1H),5.33(t,2H),4.85–4.74(m,1H),4.36(d,1H),4 .12(d,3H),3.94–3.82(m,1H),3.62(d,7H),3.54–3.42(m,1H),3.35(d,2H),3.28–3.21(m,1H),3 .11(t,1H),3.00–2.98(m,1H),2.71(t,2H),2.56–2.42(m,2H),1.45–1.32(m,10H),1.19(d,3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 172.01, 157.24, 153.41, 153.01, 152.24, 146.98, 144.90, 143.41, 137.83, 1 36.79,135.83,135.28,134.66,132.27,131.07,129.88,128.52,128.21,127,86,127.77,127.56,127.13 ,127.01,126.90,126.77,126.45,114.01,87.42,86.77,85.42,84.78,74.23,73.52,72.89,72.36,70.11,65.18,62.32,60.21,59.61,59.20,58.88,58.23,55.93,45.12,43.18,43.01,42.88,41.14,38.54,38.21 37.95, 37.88, 35.17, 24.91, 23.76, 23.55, 23.21, 23.12, 21.19, 20.81, 20.66, 20.18, 19.91; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.33, 148.21; MS (ESI) m / z for C 53 H 59 N 10 O9P:1010.4204(calcd.),1010.4210(expt.).

[0265] 4.4 Preparation of Compound 31

[0266]

[0267] Under nitrogen protection, compound 28 (5 mmol), D2 (5 mmol), and potassium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at 80 °C for 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 83%).

[0268] Compound 31:1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)pyr imidine-2,4(1H,3H)-dione:

[0269] 1 H NMR(500MHz,CD3CN)δ8.02(s,1H),7.92(d,2H),7.56(d,6H),7.34(t,1H),7.20(s,1H),6.72(d,4H),6.33(t,1H),5.52(t,2H),4.78(d, 1H),3.99(s,3H),3.81(d,1H),3.62(s,6H),3.52–3.44(m,2H),2.86(s,1H),2.72(s,1H),2.65(t,2H),2.44–2.12(m,2H),1.67(s,3H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 164.10, 159.76, 152.13, 146.94, 137.81, 135.12, 134.88, 131.14, 129.13, 129.03, 1 28.53,127.56,112.54,108.14,85.31,84.92,85.56,70.42,70.02,62.62,53.14,40.52,34.32,11.85; MS(ESI)m / z for C 37 H 39 N5O9:697.2748(calcd.),697.2753(expt.).

[0270] 4.5 Preparation of Compound 32

[0271]

[0272] Under nitrogen protection, compound 31 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (70% yield).

[0273] Compound 32: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-3-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0274] 1 H NMR (500 MHz, CD3CN) δ 7.88 (d, 1H), 7.63 (d, 2H), 7.47–7.32 (m, 6H), 7.29–7.12 (m, 1H), 7.06 (d, 1H), 6.92 (d, 4H), 6.55 (d, 1H), 5.23 (t, 2H), 4.82–4.73 (m, 1H), 4.36 (d, 1H), 4.22 (t, 1H), 4.11 (s, 3H), 3.89–3.78 (m, 1H), 3.65 (s, 6H), 3.51–3.42 (m, 3H), 3.33–3.25 (m, 2H), 2.84 (t, 2H), 2.71 (t, 1H), 2.59 (t, 1H), 2.44 (d, 1H), 1.79 (d, 3H), 1.20 (d, 9H), 1.10 (d, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 163.88, 159.51, 149.32, 144.23, 132.25, 130.96, 129.84, 129.72, 128.62, 128.13, 127.93, 127.55, 126.21, 120.57, 119.41, 114.23, 87.50, 86.81, 85.67, 85.28, 85.08, 84.92, 84.66, 84.14, 74.32, 73.21, 72.52, 70.20, 64.15, 63.92, 59.27, 59.02, 56.95, 55.24, 44.25, 43.88, 40.65, 39.26, 38.10, 33.27, 25.14, 24.87, 23.66, 21.15, 15.43, 12.1, 11.57; 31 P NMR (202 MHz, CD3CN), δ (ppm) = 147.82, 147.75; MS (ESI) m / z for C46 H 56 N7O 10 P:897.3826(calcd.),897.3829(expt.).

[0275] 4.6 Preparation of Compound 33

[0276]

[0277] Under nitrogen protection, compound 28 (5 mmol), D3 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 87%).

[0278] Compound 33:N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2 -yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)isobutyramide:

[0279] 1 H NMR(500MHz,CD3CN)δ8.52(s,1H),8.25(s,1H),7.56(s,1H),7.42(d,2H),7.3 6–7.28(m,4H),7.18–7.11(m,2H),6.88–6.79(m,4H),6.53(d,1H),5.44(t,2H) ,4.82(d,1H),4.32–4.12(m,4H),3.82(d,6H),3.65–3.59(m,1H),3.44–3.29( m,1H),2.85(d,1H),2.72(t,2H),2.46(d,1H),2.10–1.99(m,2H),1.13(d,6H). 13C NMR (126MHz, CD3CN) δ175.56,159.23,158.77,158.63,154.90,152.77,145.45,141.22,137.95,136.92,133.23,131.00,130.91,129.52 ,128.22,127.82,126.42,86.31,85.86,82.40,73.11,71.32,65.24,58.08,56.88,55.88,38.55,35.32,34.17,18.84,18.21.MS(ESI)m / z for C 41 H 44 N8O9:792.3231(calcd.),792.3233(expt.).

[0280] 4.7 Preparation of Compound 34

[0281]

[0282] Under nitrogen protection, 3 mmol of DIPEA (6 mmol) and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 88%).

[0283] Compound 34:(2S,3R,5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)e thyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0284] 1H NMR(500MHz,CD3CN)δ8.78(s,1H),8.34(s,1H),7.71(s,1H),7.60(d,2H),7.42–7.32(m ,4H),7.24–7.15(m,2H),6.82–6.74(m,4H),6.51(d,1H),5.32(t,2H),4.74(d,1H),4.4 2–4.29(m,6H),3.79-3.65(m,8H),3.31–3.24(m,1H),3.19–3.12(m,1H),2.98-2.85(m, 3H),2.71(t,2H),2.52(d,1H),2.21–1.12(m,2H),1.44(d,6H),1.35(d,6H)1.20(d,6H). 13 CNMR(126MHz,CD3CN)δ177.21,160.51,159.44,159.12,154.18,153.97,146 .14,142.23,136.41,135.98,132.18,131.01,129.77,129.18,128.22,127.6 8,126.12,118.23,88.71,86.76,85.40,70.53,70.71,66.34,61.6,58.74,55.78,54.98,48.31,39.85,36.64,34.43,23.87,23.56,19.38,18.97,18.16. 31 P NMR(202MHz,CD3CN)δ148.13,147.92.MS(ESI)m / zfor C 50 H 61 N 10 O 10 P:992.4310(calcd.),992.4313(expt.).

[0285] 4.8 Preparation of Compound 35

[0286]

[0287] Under nitrogen protection, at 0°C, D4 (10 mmol) dissolved in 20 mL of ultra-dry LMF and compound 28 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (79% yield).

[0288] Compound 35: N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-(2-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)benzamide

[0289] 1 H NMR(500 MHz, CD3CN) δ(ppm) = 8.32–8.21 (m, 2H), 7.88 (d, 1H), 7.71 (t, 1H), 7.58–7.51 (m, 4H), 7.38 (d, 6H), 7.29–7.23 (m, 1H), 7.15 (s, 1H), 6.79 (d, 4H), 6.44–6.36 (m, 1H), 6.21 (d, 1H), 5.29 (t, 2H), 4.58 (d, 1H), 4.21 (s, 3H), 3.91–3.84 (m, 1H), 3.73 (s, 6H), 3.42–3.35 (m, 2H), 2.88 (t, 2H), 2.47–2.38 (m, 3H); 13 C NMR(126 MHz, CD3CN), δ(ppm) = 176.45, 159.13, 156.63, 150.74, 146.71, 139.13, 137.83, 136.74, 135.33, 134.52, 133.87, 132.20, 131.14, 130.39, 129.35, 128.82, 128.63, 127.92, 126.95, 114.04, 93.21, 87.50, 86.12, 85.74 70.14, 69.87, 62.74, 55.76, 40.41, 37.42, 33.87; MS(ESI) m / z for C 43 H 42 N6O9: 786.3013 (calcd.), 786.3018 (expt.).

[0290] 4.9 Preparation of Compound 36

[0291] [[ID=​​Under nitrogen protection, compound 35 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 79%).

[0293] Compound 36:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-(2-(1-methyl-2-nitro-1H-imidazol- 5-yl)ethyl)benzamido)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0294] 1 H NMR(500MHz,CD3CN)δ8.32(d,2H),8.01–7.94(m,1H),7.82(d,1H),7.66–7.56(m,4H),7.26(d,6H) ),7.14(d,1H),7.03(d,1H),6.74(dd,4H),6.33(d,1H),6.13(d,1H),5.52(t,2H),4.87–4.76(m, 1H),4.18(s,3H),3.95–3.87(m,1H),3.54(d,6H),3.76–3.65(m,3H),3.48–3.39(m,2H),2.88(t, 2H),2.59(t,1H),2.42(t,1H),2.33(d,1H),2.24(d,1H),1.89(d,5H),1.21(d,9H),1.13(d,4H); 13C NMR (126MHz, CD3CN), δ (ppm) = 176.71, 172.96, 159.84, 156.18, 151.93, 146.62, 141.29, 138.23, 137.80, 136.79, 135.98, 135.74, 1 35.52,135.25,134.26,131.45,131.13,130.14,129.88,129.12,128.78,128.56,127.85,126.41,126.13,119.61,118.85,113.74, 99.33,88.54,87.18,86.98,85.93,85.56,84.78,84.18,73.45,72.40,71.87,71.41,64.54,63.65,59.85,58.13,58.01,56.18,45.65,44.87,43.31,42.98,41.89,40.63,39.24,38.78,36.86,34.67,26.12,25.98,24.38,23.87,21.87,21.52,20.38,20.11,12.97; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.32, 148.24; MS (ESI) m / z for C 52 H 59 N8O 10 P:986.4092(calcd.),986.4095(expt.).

[0295] The compounds prepared in this embodiment are listed below. Figure 6 .

[0296] Example 5: Preparation of a stress-responsive deoxyribose probe based on 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl 4-methylbenzenesulfonic acid.

[0297] 5.1 Preparation of compound 37 (2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl 4-methylbenzenesulfonic acid)

[0298]

[0299] Under nitrogen protection, N-1-methyl-5-nitro-2-hydroxyethylimidazolium (10 mmol) and triethylamine (30 mmol) were dissolved in 20 mL of ultradry dichloromethane in a reaction tube. The system was maintained at 0 °C and p-toluenesulfonyl chloride (15 mmol) was slowly added and reacted for 1 hour. The temperature was then slowly raised to room temperature and the reaction was continued for 5 hours. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (87%).

[0300] Compound 37:2-(1-Methyl-5-nitro-1H-imidazol-2-yl)ethyl4-methylbenzenesulfonate:

[0301] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.86 (s, 1H), 7.78 (d, 2H), 7.48 (d, 2H), 3.85 (t, 2H), 3.76 (s, 3H), 2.75 (t, 2H), 2.46 (s, 3H); 13 C NMR (126MHz, CDCl3), δ (ppm) = 158.36, 144.43, 140.56, 139.72, 132.44, 130.51, 130.47, 128.49, 128.30, 70.42, 35.74, 21.41, 19.92.MS (ESI) m / z for C 13 H 15 N3O5S:325.0732(calcd.),325.0762(expt.).

[0302] 5.2 Preparation of Compound 38

[0303]

[0304] Under nitrogen protection, at a low temperature of 0°C, 20 mL of ultra-dry DMF dissolved in D1 (10 mmol) and compound 37 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred for 45 minutes and then brought to room temperature for 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 82%).

[0305] Compound 38: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)benzamide:

[0306] 1 H NMR(500MHz, CD3CN)δ(ppm) = 8.26(s, 1H), 8.12(s, 1H), 7.46–7.36(m, 4H), 7.31–7.27(m, 9H), 7.21(t, 2H), 7.10(s, 1H), 6.99(d, 4H), 6.71(t, 1H), 5.15(t, 2H), 4.73(d, 1H), 4.31(d, 3H), 4.20–4.16(m, 1H), 3.59(s, 6H), 3.39(d, 2H), 2.88(t, 2H), 2.78–2.69(m, 1H), 2.21(d, 2H); 13 C NMR(126MHz, CD3CN), δ(ppm) = 171.59, 161.14, 159.36, 152.15, 151.886, 145.04, 141.74, 135.65, 135.31, 135.11, 134.51, 132.18, 130.64, 129.95, 129.74, 128.75, 127.76, 127.24, 127.01, 126.91, 126. = 171.59, 161.14, 159.36, 152.15, 151.886, 145.04, 141.74, 135.65, 135.31, 135.11, 134.51, 132.18, 130.64, 129.95, 129.74, 128.75, 127.76, 127.24, 127.01, 126.91, 126.07, 111.85, 86.85, 85.89, 83.36, 44 H 42 N8O8: 810.3126(calcd.), 810. = 171.59, 161.14, 159.36, 152.15, 151.886, 145.04, 141.74, 135.65, 135.31, 135.11, 134.51, 132.18, 130.64, 129.95, 129.74, 128.75, 127.76, 127.24, 127.01, 126.91, 126.07, 111.85, 86.85, 85.89, 83.36, 74.71, 65.52, 58.73, 54.91, 40.74, 38.263, 31.75, 29.14; MS(ESI)m / z for C

[0307] 5.3 Preparation of Compound 39

[0308]

[0309] Under nitrogen protection, compound 38 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 85%).

[0310] Compound 39: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)di isopropylphosphoramidite:

[0311] 1 H NMR (500MHz, CD3CN) δ (ppm) = 8.36 (d, 1H), 8.15 (d, 1H), 7.92–7.85 (m, 4H), 7.65–7.51 (m,9H),7.32(t,2H),7.04(s,1H),6.88(d,4H),6.26(q,1H),5.13(t,2H),4.82–4.73 (m,1H),4.32(d,1H),4.14(d,3H),3.88–3.79(m,3H),3.53(d,7H),3.22–3.17(m,1H) ,3.07–2.99(m,3H),2.82(t,2H),2.54–2.42(m,2H),1.36–1.19(m,10H),1.04(d,3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 169.52, 158.76, 152.98, 152.74, 151.15, 146.75, 143.96,141.35,135.34,134.87,134.56,134.33,133.47,131.75,130.85,129. 32,128.87,128.54,127.74,126.67,126.45,126.25,126.01,125.94,125.81,125.55,114.75,88.841,86.35,85.62,83.74,73.485,72.21,71.74,7021,65.7,4 61.57.85,58.23,57.87,57.63,56.374,55.25,54.36,44.54,43.02,42.47,41.85,40.23,38.74,37.25,36.65,36.231,34.85,27.92,25.85,24.14,23.32,22.85,22.65,21.85,20.31,19.84,18.35,17.78; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.41, 148.32; MS (ESI) m / z for C 53 H 59 N 10 O9P:1010.4204(calcd.),1010.4220(expt.).

[0312] 5.4 Preparation of Compound 40

[0313]

[0314] Under nitrogen protection, compound 37 (5 mmol), D2 (5 mmol), and potassium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred for 45 minutes. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 82%).

[0315] Compound 40:1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)pyrimidine-2,4(1H,3H)-dione:

[0316] 1 H NMR(500MHz,CD3CN)δ8.23(s,1H),7.98(d,2H),7.74(d,6H),7.65(t,1H),7.32(s,1H),6.85(d,4H),6.32(t,1H),5.15(t,2H),4.85(d, 1H),4.22(s,3H),4.14(d,1H),3.87(s,6H),3.65–3.53(m,2H),2.99(s,1H),2.85(t,2H),2.71(s,1H),2.63–2.54(m,2H),1.85(s,3H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 164.85, 158.74.151.32, 146.65, 136.74, 135.68, 134.21, 128.12, 127.85, 127.62, 1 27.03,125.85,114.74,110.12,88.74,83.35,82.23,71.37,65.74,54.25,40.12,36.45,27.74,11.12; MS(ESI)m / z for C 37 H 39 N5O9:697.2748(calcd.),697.2755(expt.).

[0317] 5.5 Preparation of Compound 41

[0318]

[0319] Under nitrogen protection, compound 40 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 83%).

[0320] Compound 41: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-3-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0321] 1 H NMR(500MHz,CD3CN)δ8.33(d,1H),7.95(d,2H),7.81–7.71(m,6H),7.66–7.56(m,1H),7.34(d,1H),6.82(d,4H),6.61(d,1H),5.38(t,2H),4.85–4.71(m,1H),4.42(d,1H),4.31(t,1H),4.21(s,3H),3.95–3.88(m,1H),3.72(s,6H),3.65–3.52(m,3H),3.41–3.32(m,2H),2.85(t,2H),2.72(t,1H),2.63(t,1H),2.51(d,1H),1.55(d,3H),1.21(d,9H),1.06(d,3H); 13 C NMR(126MHz,CD3CN),δ(ppm)=166.01,158.58,151.47,145.08,132.39,131.74,129.​​46 H 56 N7O 10 P:897.3826(calcd.),897.3839(expt.).

[0322] 5.6 Preparation of Compound 42

[0323]

[0324] Under nitrogen protection, compound 37 (5 mmol), D3 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred for 45 minutes. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (77% yield).

[0325] Compound 42:N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2 -yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)isobutyramide:

[0326] 1 H NMR(500MHz,CD3CN)δ8.62(s,1H),8.44(s,1H),7.83(s,1H),7.67(d,2H),7.53 –7.41(m,4H),7.27–7.17(m,2H),6.88–6.75(m,4H),6.51(d,1H),5.75–5.69(t, 2H),4.83(d,1H),4.52–4.38(m,4H),3.82(d,6H),3.72–3.65(m,1H),3.42–3.28 (m,1H),2.93(t,2H),2.77(d,1H),2.42(d,1H),2.29–2.13(m,2H),1.45(d,6H). 13C NMR (126MHz, CD3CN) δ175.74,160.35,159.36,157.47,151.58,150.69,148.14,142.25,135.36,133.01,132.24,131.35,130.68,129.57 ,127.84,126.95,125.20,86.28,84.39,83.17,72.67,62.27,58.19,54.27,52.55,38.38,35.41,34.14,27.25,18.57,16.68.MS(ESI)m / z for C 41 H 44 N8O9:792.3231(calcd.),792.3236(expt.).

[0327] 5.7 Preparation of Compound 43

[0328]

[0329] Under nitrogen protection, compound 42 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 85%).

[0330] Compound 43:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)e thyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0331] 1H NMR(500MHz,CD3CN)δ8.49(s,1H),8.26(s,1H),7.85(s,1H),7.69(d,2H),7.53–7.41(m ,4H),7.32–7.21(m,2H),6.78–6.63(m,4H),6.21(d,1H),5.19(t,2H),4.52(d,1H),4.4 1–4.33(m,6H),3.88-3.68(m,8H),3.52–3.44(m,1H),3.32–3.27(m,1H),2.86(t,2H),2 .75-2.61(m,3H),2.52(d,1H),2.45–2.36(m,2H),1.68(d,6H),1.59(d,6H)1.21(d,6H). 13 CNMR(126MHz,CD3CN)δ177.74,163.85,161.96,158.63,156.52,152.41,145 .10,144.02,139.23,138.35,132.57,131.24,130.48,129.59,128.26,127.0 3,126.08,119.71,86.28,85.49,84.39,73.74,62.05,61.28,57.25,54.19,53.27,48.50,38.08,35.81,33.72,27.85,25.82,22.17,20.75,19.28,18.17. 31 P NMR(202MHz,CD3CN)δ148.56,147.45.MS(ESI)m / z for C 50 H 61 N 10 O 10 P:992.4310(calcd.),992.4312(expt.).

[0332] 5.8 Preparation of Compound 44

[0333]

[0334] Under nitrogen protection, at a low temperature of 0°C, 20 mL of ultra-dry DMF dissolved in D4 (10 mmol) and compound 37 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). The mixture was stirred for 45 minutes, then brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 80%).

[0335] Compound 44:N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-(2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethyl)benzamide:

[0336] 1 H NMR (500MHz, CD3CN) δ (ppm) = 8.56–8.42 (m, 2H), 7.91 (d, 1H), 7.74 (t, 1H), 7. 62–7.51(m,4H),7.40(d,6H),7.32–7.23(m,1H),7.11(s,1H),6.66(d,4H),6 .39–6.28(m,1H),6.15(d,1H),5.22(t,2H),4.85(d,1H),4.32(s,3H),3.92– 3.81(m,1H),3.65(s,6H),3.52–3.41(m,2H),2.89(t,2H),2.69–2.55(m,3H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 174.24, 158.57, 157.24, 149.35, 145.84, 137.95, 135.30, 134.94, 133.75, 133.20, 132.74, 130.63, 129.85, 127. 94,127.89,127.54,127.32,126.21,125.65,113.54,92.47,86.58,85. 92,84.36,68.32,62.21,55.41,40.74,34.85,32.65,27.12; MS(ESI)m / z forC 43 H 42N6O9:786.3013(calcd.),786.3019(expt.).

[0337] 5.9 Preparation of Compound 45

[0338]

[0339] Under nitrogen protection, compound 44 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 85%).

[0340] Compound 45:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-(2-(1-methyl-5-nitro-1H-imidazol- 2-yl)ethyl)benzamido)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0341] 1 H NMR(500MHz,CD3CN)δ7.98(d,2H),7.85–7.78(m,1H),7.66(d,1H),7.54–7.42(m,4H),7.31(d,6H) ),7.22(d,1H),7.12(d,1H),6.67(dd,4H),6.32(d,1H),6.01(d,1H),5.19(t,2H),4.98–4.92(m, 1H),4.64(s,3H),3.90–3.82(m,1H),3.69(d,6H),3.52–3.33(m,3H),3.25–3.14(m,2H),2.88(t, 2H),2.67(t,1H),2.42(t,1H),2.31(d,1H),2.17(d,1H),2.04(d,5H),1.45(d,9H),1.19(d,4H); 13C NMR (126MHz, CD3CN), δ (ppm) = 177.74, 171.85, 158.96, 157.41, 146.52, 143.63, 140.30, 137.02, 136.21, 135.87, 135.42, 134.74, 134.23, 133 .96,132.57,130.68,130.21,129.87,129.25,128.35,127.85,127.45 ,126.59,126.15,125.48,118.78,117.35,113.65,97.87,88.72,86.36 85.75,85.14,84.78,83.85,82.32,73.85,72.63,71.25,70.14,62.53,61.68,60.14,59.75,58.86,57.42,45.10,43.68,42.35,41.74,40.68,39.24,38.36,37.74,37.24,36.26,27.75,24.40,23.61,22.89,21.32,20.36,20.12,19.90,18.84,13.63; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.31, 148.25; MS (ESI) m / z for C 52 H 59 N8O 10 P:986.4092(calcd.),986.4097(expt.).

[0342] The compounds prepared in this embodiment are listed below. Figure 7 .

[0343] Example 6: Preparation of a stress-responsive deoxyribose probe based on 2-(2-nitro-1H-imidazol-1-yl)ethyl methanesulfonate

[0344] 6.1 Preparation of Compound 46 (2-(2-nitro-1H-imidazol-1-yl)ethyl methanesulfonate)

[0345]

[0346] Under nitrogen protection, 2-nitro-1-hydroxyethylimidazolium (10 mmol) and triethylamine (30 mmol) were dissolved in 25 mL of ultradry dichloromethane in a reaction tube. The system was maintained at 0 °C and p-toluenesulfonyl chloride (15 mmol) was slowly added. After reacting for 1 hour, the temperature was slowly raised to room temperature and the reaction was continued for 5 hours. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography in high yield (85%).

[0347] Compound 46:2-(2-Nitro-1H-imidazol-1-yl)ethyl methanesulfonate:

[0348] 1 H NMR (500MHz, CDCl3), δ (ppm) = 7.66 (d, 1H), 7.18 (d, 1H), 4.85 (t, 2H), 4.05 (t, 2H), 3.26 (s, 3H); 13 C NMR (126MHz, CDCl3), δ (ppm) = 156.65, 131.93, 128.82, 60.96, 43.28, 38.32. MS (ESI) m / z for C6H9N3O5S: 235.0263 (calcd.), 235.0283 (expt.).

[0349] 6.2 Preparation of Compound 47

[0350]

[0351] Under nitrogen protection, at a low temperature of 0°C, 20 mL of ultra-dry DMF dissolved in D1 (10 mmol) and compound 46 (10 mmol) was added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol). After stirring for 45 minutes, the mixture was brought to room temperature and stirred for another 8 hours. The reaction was monitored by TCL. Once the reactants had completely reacted, the reaction was quenched with water, and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (73% yield).

[0352] Compound 47: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)benzam-ide:

[0353] 1H NMR (500MHz, CD3CN) δ (ppm) = 8.33 (s, 1H), 8.21 (s, 1H), 7.76 (d, 1H), 7.65 (d, 1H), 7.55–7.46 (m, 4H), 7.37–7.25 (m, 9H), 7.12 (t, 2H), 6.87 (d,4H),6.66(t,1H),5.34(t,2H),4.52(d,1H),4.49–4.32(m,1H),4.01(t,2H),3.79(s,6H),3.56(d,2H),2.88–2.81(m,1H),2.54(d,2H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 170.41, 158.75, 154.20, 153.74, 152.85, 147 .96,143.52,138.05,137.84,136.95,135.62,133.76,132.35,131.24,130. 35,129.57,128.98,127.08,127.37,126.84,125.95,114.30,88.87,87.35, 86.28,72.19,65.29,61.38,56.76,51.95,43.28,41.35,33.67; MS(ESI)m / z for C 43 H 40 N8O8:796.2969(calcd.),796.2975(expt.).

[0354] 6.3 Preparation of Compound 48

[0355]

[0356] Under nitrogen protection, compound 47 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 87%).

[0357] Compound 48: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)benzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0358] 1 H NMR (500 MHz, CD3CN) δ (ppm) = 8.45 (d, 1H), 8.32 (d, 1H), 7.85 (d, 1H), 7.69 (d, 1H), 7.51–7.40 (m, 4H), 7.32–7.24 (m, 9H), 7.14 (t, 2H), 6.87 (d, 4H), 6.41 (q, 1H), 5.52 (t, 2H), 4.82–4.77 (m, 1H), 4.21 (d, 1H), 4.12 (t, 2H), 3.94–3.85 (m, 1H), 3.77 (d, 7H), 3.62–3.50 (m, 1H), 3.40 (d, 2H), 3.22–3.11 (m, 1H), 3.01 (t, 1H), 2.87–2.74 (m, 1H), 2.62–2.54 (m, 2H), 1.45–1.31 (m, 10H), 1.17 (d, 3H); 13C NMR (126MHz, CD3CN), δ (ppm) = 170.74, 158.21, 155.42, 154.86, 151.32, 149.05, 145.72, 144.03,139.96,137.35,136.42,135.75,134.35,133.82,132.73,131.02,130.75,129. 85,129,56,128.65,127.84,127.63,127.11,127.03,125.32,124.86,114.02,89.75,88.32,87.76,85.14,76.75,75.74,74.85,73.74,65.25,64.36,60.85,59.75,58.32,57.74 56.71,55.21,51.43,46.74,45.78,44.82,43.82,42.32,41.36,40.05,39.70,38.32,37.96,26.74,24.86,23.57,22.75,21.12,20.99,20.86,20.79 20.65,19.23; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.49, 148.32; MS (ESI) m / z for C 52 H 57 N 10 O9P:996.4048(calcd.),996.4057(expt.).

[0359] 6.4 Preparation of Compound 49

[0360]

[0361] Under nitrogen protection, compound 46 (5 mmol), D2 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at 80 °C for 8 hours. The reaction was monitored by a TCL. After the reactants had completely reacted and the mixture was cooled to room temperature, it was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 83%).

[0362] Compound 49:1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methyl-3-(2-(2-nitro-1H-imidazol-1-yl)ethyl)pyrimidine-2,4(1H,3H)-dione:

[0363] 1 H NMR(500MHz,CD3CN)δ8.62(d,1H),8.45(d,1H),7.93(s,1H),7.79(d,2H),7.62(d,6H),7.35(t,1H),6.82(d,4H),6.53(t,1H),5.10(t, 2H),4.94(d,1H),4.35(t,2H),3.78(d,1H),3.66(s,6H),3.52–3.42(m,2H),2.85(s,1H),2.71(s,1H),2.58–2.43(m,2H),1.61(s,3H); 13 C NMR (126MHz, CD3CN), δ (ppm) = 165.14, 158.25, 150.85, 147.35, 137.86, 135.04, 134.75, 130.86, 130.32, 129.05, 1 27.36,125.75,114.86,111.34,88.71,87.37,84.76,68.91,63.73,52.38,50.29,48.76,40.09,11.98; MS(ESI)m / z for C 36 H 37 N5O9:683.2591(calcd.),683.2601(expt.).

[0364] 6.5 Preparation of Compound 50

[0365]

[0366] Under nitrogen protection, compound 49 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 86%).

[0367] Compound 50: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-3-(2-(2-nitro-1H-imidazol-1-yl)ethyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0368] 1 H NMR (500 MHz, CD3CN) δ 8.39 (d, 1H), 8.02 (d, 1H), 7.85 (d, 1H), 7.77 (d, 2H), 7.59–7.45 (m, 6H), 7.26 (d, 1H), 6.86 (d, 4H), 6.58 (d, 1H), 5.01 (t, 2H), 4.82–4.75 (m, 1H), 4.57 (d, 1H), 4.32 (t, 1H), 4.15 (t, 2H), 3.98–3.88 (m, 1H), 3.72 (s, 6H), 3.65–3.52 (m, 3H), 3.45–3.33 (m, 2H), 2.89 (t, 1H), 2.65 (t, 1H), 2.32 (d, 1H), 1.85 (d, 3H), 1.38 (d, 9H), 1.25 (d, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 166.05, 160.52, 151.93, 147.84, 133.75, 132.09, 131.42, 130.93, 129.81, 127.92, 128.60, 127.14, 126.24, 121.37, 120.14, 114.3, 90.93, 89.74, 87.39, 86.91, 85.94, 85.88, 84.56, 76.37, 75.19, 73.37, 66.39, 64.74, 59.73, 58.74, 56.61, 55.83, 52.27, 47.91, 44.37, 43.2, 41.96, 39.64, 38.67, 25.34, 24.96, 23.67, 22.67, 16.96, 13.17, 12.28; 31 P NMR (202 MHz, CD3CN), δ (ppm) = 148.43, 148.15; MS (ESI) m / z for C 45 H 54 N7O10 P:883.3670(calcd.),883.3681(expt.).

[0369] 6.6 Preparation of Compound 51

[0370]

[0371] Under nitrogen protection, compound 46 (5 mmol), D3 (5 mmol), and cesium carbonate (10 mmol) were added to a Schlenk tube equipped with a magnetic stir bar. 10 mL of DMF was added to the reaction tube, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TCL. After the reactants had completely reacted, the mixture was extracted with ethyl acetate (3x). The collected organic phase was washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (yield 81%).

[0372] Compound 51: N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)isobutyramide:

[0373] 1 H NMR(500MHz,CD3CN)δ8.64(s,1H),8.33(s,1H),8.12(d,1H),7.88(d,1H),7.63(d ,2H),7.49–7.36(m,4H),7.29–7.15(m,2H),6.87–6.69(m,4H),6.54(d,1H),5.62 (t,2H),4.94(d,1H),4.74–4.66(m,1H),4.12(t,2H),3.84(d,6H),3.65–3.49(m, 1H),3.33–3.19(m,1H),2.87(d,1H),2.58(d,1H),2.22–2.01(m,2H),1.59(d,6H). 13C NMR (126MHz, CD3CN) δ178.25,161.35,158.68,157.84,155.40,154.13,147.42,143.81,136.93,135.76,134.16,133.37,130.76,129.67 ,128.84,127.37,126.64,87.87,86.34,85.87,73.64,65.52,57.83,56.05,55.46,53.27,44.74,38.25,36.37,18.56,17.19; MS (ESI) m / z forC 40 H 42 N8O9:778.3075(calcd.),778.3083(expt.).

[0374] 6.7 Preparation of Compound 52

[0375]

[0376] Under nitrogen protection, compound 51 (imidazolium-modified base G) (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 80%).

[0377] Compound 52:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(N-(2-(2-nitro-1H-imidazol-1-yl)ethyl) isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0378] 1H NMR(500MHz,CD3CN)δ8.52(s,1H),8.36(s,1H),8.14(d,1H),7.86(d,1H),7.72(d,2H),7.6 1–7.53(m,4H),7.46–7.34(m,2H),6.84–6.82(m,4H),6.61(d,1H),5.88(t,2H),4.89(d,1H ),4.33–4.19(m,3H),4.05(t,2H),3.92-3.80(m,8H),3.52–3.39(m,1H),3.30–3.13(m,1H) ,2.96-2.85(m,3H),2.51(d,1H),2.23–2.16(m,2H),1.59(d,6H),1.47(d,6H)1.13(d,6H). 13 C NMR(126MHz,CD3CN)δ=177.17,163.75,161.36,160.70,156.09,152.35,149 .22,141.52,139.74,136.32,135.74,131.32,130.42,129.41,128.75,127.3 2,126.01,118.75,87.78,86.32,81.74,73.32,67.79,62.82,58.11,56.37,55.28,51.76,48.64,43.75,41.6,36.37,25.77,21.15,20.63,17.74,16.283. 31 P NMR(202MHz,CD3CN)δ148.21,147.65; MS(ESI)m / z for C 49 H 59 N 10 O 10 P:978.4153(calcd.),978.4162(expt.).

[0379] 6.8 Preparation of Compound 53

[0380]

[0381] Under nitrogen protection, 20 mL of ultra-dry DMF-dissolved D4 (10 mmol) and compound 46 (10 mmol) were added to a Schlenk tube containing a magnetic stir bar and sodium hydride (12 mmol), and the mixture was stirred for 45 minutes. The reaction was monitored by TCL. After the reactants had completely reacted, the reaction was quenched with water, and the aqueous layer was extracted with ethyl acetate (3x). The organic phase was collected, washed with brine, dried over magnesium sulfate, concentrated under reduced pressure to remove the solvent, and separated by column chromatography to obtain the target compound (63% yield).

[0382] Compound 53: N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)benzamide

[0383] 1 H NMR (500 MHz, CD3CN) δ (ppm) = 8.41–8.38 (m, 2H), 8.12 (d, 1H), 7.99 (d, 1H), 7.79 (d, 1H), 7.56 (t, 1H), 7.47–7.36 (m, 4H), 7.25 (d, 6H), 7.19–7.11 (m, 1H), 6.85 (d, 4H), 6.69–6.53 (m, 1H), 6.42 (d, 1H), 5.57 (t, 2H), 4.81 (d, 1H), 4.33 (t, 2H), 4.21–4.14 (m, 1H), 3.77 (s, 6H), 3.42–3.36 (m, 2H), 2.67–2.53 (m, 3H); 13 C NMR (126 MHz, CD3CN), δ (ppm) = 178.10, 158.75, 157.65, 150.46, 144.87, 139.34, 137.39, 136.82, 135.17, 134.35, 133.76, 132.36, 131.76, 130.25, 129.13, 128.78, 127.05, 126.15, 125.722, 114.34, 92.76, 87.76, 86.25, 85.36, 70.64, 66.76, 57.32, 50.02, 44.74, 43.34, 37.86; MS (ESI) m / z for C 42 H 40 N6O9: 772.2857 (calcd.), 772.2863 (expt.).

[0384] 6.9 Preparation of Compound 54

[0385]

[0386] Under nitrogen protection, compound 53 (3 mmol), DIPEA (6 mmol), and 3-(chloro(diisopropylamino)phosphono)propionitrile (9 mmol) were dissolved in ultra-dry DCM (20 mL) at 0 °C and added to a Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 0 °C for 30 min, then slowly raised to room temperature and stirred for another 3 h. The reaction was monitored by TCL. After the reactants had completely reacted, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (yield 86%).

[0387] Compound 54:(2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(4-(N-(2-(2-nitro-1H-imidazol-1-yl) ethyl)benzamido)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0388] 1 H NMR(500MHz,CD3CN)δ8.76(d,2H),8.54(d,1H),8.02(d,1H),7.89–7.78(m,1H),7.69(d,1H),7. 51–7.42(m,4H),7.33(d,6H),7.24(d,1H),6.88(d,4H),6.46(d,1H),6.10(d,1H),5.56(t,2H),4 .88–4.73(m,1H),4.24(t,2H),3.98–3.82(m,1H),3.74(d,6H),3.53–3.43(m,3H),3.32–3.25(m, 2H),2.88(t,1H),2.78(t,1H),2.66(d,1H),2.48(d,1H),1.85(d,5H),1.35(d,9H),1.09(d,4H); 13C NMR (126MHz, CD3CN), δ (ppm) = 178.85, 172.23, 158.65, 155.89, 148.75, 146.82, 139.01, 138.36, 137.96, 136.32, 135.98, 135.77, 135 .56,135.35,133.20,132.85,131.14,129.75,128.87,128.44,128.36,128.07,127.82,126.74,125.36,119.85,118.23,114.52,98. 63,88.74,87.63,86.21,85.85,85.38,84.75,83.36,73.62,72.28,71.36,70.86,63.46,62.45,59.23,58.85,58.65,58.23,50.52,52.32,43.74,43.22,43.75,42.36,39.63,39.82,39.32,38.23,36.14,25.25,24.36,23.85,23.52,21.75,20.75,20.23,20.02,12.21; 31 P NMR (202MHz, CD3CN), δ (ppm) = 148.63, 148.42; MS (ESI) m / z for C 51 H 57 N8O 10 P:972.3935(calcd.),972.3949(expt.).

[0389] The compounds prepared in this embodiment are listed below. Figure 8 .

[0390] discuss

[0391] This invention links nitroimidazoles to DNA bases, developing a series of stress-responsive deoxyribose dTh, dAh, dCh, and dGh synthesized with corresponding solid-phase modules. A series of nucleic acid probe libraries with precisely controllable nitroimidazole quantities and artificial base positions are prepared using a DNA solid-phase synthesizer. This allows for gradient changes in probe molecular weight and nitroimidazole quantity, thereby regulating the targeting speed and enrichment degree of each molecule in the hypoxic microenvironment based on nitroimidazoles. PET imaging of tumor-bearing mice with malignant solid tumors at different hypoxic concentrations using labeled probes reveals a correlation between the number and molecular weight of responsive functional groups and hypoxic concentration, leading to the development of nucleic acid-based "nanobiometer scales" for accurately measuring hypoxic concentrations.

[0392] Stress-responsive deoxyribose dTh, dAh, dCh, and dGh solid-phase modules can also be used to construct nucleic acid drugs such as antisense nucleic acid drugs, giving them better biological stability and microenvironment targeting, thereby improving efficacy.

[0393] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (I) or an optical isomer thereof, or a pharmaceutically acceptable salt thereof: (I) In the formula, R1 is H or -OH; R2 is selected from the following group: H, -P(Rc)Rd, where, Rc is -N(Re)Rf, wherein Re and Rf are each independently H, substituted or unsubstituted C1-C8 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; Rd is -OH, substituted or unsubstituted C1-C8 alkoxy, or substituted or unsubstituted C3-C8 cycloalkoxy; wherein "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, and C1-C3 alkyl. R3 is selected from the group consisting of H or -C(Rg)3, where Rg is independently a substituted or unsubstituted phenyl or C1-C8 alkyl group, wherein "substituted" means substituted by one or more substituents selected from the group consisting of C1 alkoxy or halogen. R a It is (C1-C6 alkylene)-(substituted or unsubstituted nitroimidazolium); wherein, "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, C1-C3 alkyl; R b It can be H, -CO-C6-C10 aryl, or -CO-C1-C8 alkyl; The rings B and R mentioned above a and R b Groups selected from the following group: 、 、 、 。 2. The compound or its optical isomer, or a pharmaceutically acceptable salt, as described in claim 1, characterized in that... R1 is H; R2 is H; R3 is -C(Rg)3, and Rg is a substituted or unsubstituted phenyl or C1-C8 alkyl group.

3. The compound or its optical isomer, or a pharmaceutically acceptable salt, as described in claim 1, characterized in that... R1 is H; R2 is -P(Rc)Rd, where Rc is -N(Re)Rf, where Re and Rf are each independently H, substituted or unsubstituted C1-C8 alkyl, or substituted or unsubstituted C3-C8 cycloalkyl; Rd is -OH, substituted or unsubstituted C1-C8 alkoxy, or substituted or unsubstituted C3-C8 cycloalkoxy; wherein "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, and C1-C3 alkyl. R3 is -C(Rg)3, and Rg is a substituted or unsubstituted phenyl or C1-C8 alkyl group.

4. The compound of claim 1 or its optical isomer, or a pharmaceutically acceptable salt thereof, characterized in that, The rings B and R mentioned above a and R b Groups selected from the following group: , , ;in, R a It is -(C1-C6 alkylene)-(substituted or unsubstituted nitroimidazolium); wherein, "substituted" means substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, CN, nitro, C1-C3 alkyl; R b It is -CO-C6-C10 aryl or -CO-C1-C8 alkyl.

5. The compound of claim 1 or its optical isomer, or a pharmaceutically acceptable salt thereof, characterized in that, The rings B and R mentioned above a and R b It consists of the following groups: .

6. The compound of claim 1 or its optical isomer, or a pharmaceutically acceptable salt thereof, characterized in that, The rings B and R mentioned above a and R b It consists of the following groups: 。 7. The compound or its optical isomer, or a pharmaceutically acceptable salt, as described in any one of claims 4-6, characterized in that, R a It is -(C1-C3 alkylene)-(substituted or unsubstituted nitroimidazolyl), wherein "substituted" means substituted by one or more substituents selected from the group consisting of halogens and C1-C3 alkyl groups.

8. A compound, characterized in that, The structure of the compound is as described in compound 3. or compound 5 As shown.

9. A nucleic acid molecular pharmaceutical composition comprising a compound or an optical isomer thereof as described in any one of claims 1-7, a pharmaceutically acceptable salt or a compound as described in claim 8, and a pharmaceutically acceptable carrier.

10. The nucleic acid molecular pharmaceutical composition according to claim 9, characterized in that, The nucleic acid molecular drug composition is selected from the group consisting of: gene drugs composed of deoxyribose units, small nucleic acid interfering RNA drugs, or nucleic acid aptamer drugs.

11. An active targeted contrast agent, characterized in that, The contrast agent comprises: the compound or optical isomer of any one of claims 1-7, a pharmaceutically acceptable salt, or the compound of claim 8, and the contrast agent is a hypoxia microenvironment responsive contrast agent.

12. The active targeted contrast agent as described in claim 11, characterized in that, The contrast agent is used to create images of tumor tissue.

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