Artificial deoxyribose responding to low-oxygen microenvironment and application of artificial deoxyribose

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

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

[0084] As used herein, the term "pharmaceutically acceptable salts" refers to salts of compounds of the present invention with pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include (but are not limited to): hydrochloric acid, hydrogen Bromic acid, phosphoric acid, nitric acid, sulfuric acid; preferred organic acids include (but are not limited to): formic acid, acetic acid, propionic acid, succinic acid, naphthalenedisulfonic acid (1,5), succinic acid, oxalic acid, tartaric acid, lactic acid , salicylic acid, benzoic acid, valeric acid, diethyl acetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, Gluconic acid, ascorbic acid, niacin, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids.

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

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

[0087] Application method

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

[0089] Since the compounds of the present invention have excellent hypoxic microenvironmental response, the compounds of the present invention and their pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient are useful For the treatment, prevention and mitigation of diseases associated with the hypoxic microenvironment. According to the prior art, the compounds of the present invention can be used to treat (but not limited to) the following diseases: various cancers, such as lung cancer, bladder cancer, breast cancer, stomach cancer, liver cancer, prostate cancer, kidney cancer, pancreatic cancer and the like. The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0090] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gelling substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0091] The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical 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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0093] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

[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 employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.

[0095] Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

[0096] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

[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 for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

[0099] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0100] When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as people) in need of treatment, and the dose is a pharmaceutically considered effective dose when administered, and for a person with a body weight of 60kg, the daily dose is The administration dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

[0101] The main advantages of the present invention include:

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

[0103] (2) The nucleic acid probe library of the series of nitroimidazoles prepared by artificial deoxyribose responsive to the hypoxic microenvironment provided by the present invention is precisely and controllable in the number of artificial bases, so that the molecular weight of the probe and the number of nitroimidazoles change in a gradient, Thus, the targeting speed and enrichment degree of each molecule to the hypoxic microenvironment based on nitroimidazole can be adjusted.

[0104] (3) The artificial deoxyribose responsive to the hypoxic microenvironment provided by the present invention exhibits a correlation between the number of responsive functional groups, the molecular weight and the hypoxic concentration, and can be developed into a nucleic acid-based "nanobioscaler for accurately measuring hypoxia concentration" ".

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

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

[0109] The specific operation process is as follows:

[0110]

[0111] To a solution of N-1-methyl-2-nitro-5-hydroxymethylimidazole (10 mmol) and DIPEA (20 mmol) dissolved in ultra-dry dichloromethane at 0°C under nitrogen protection, PPhBr was slowly added 3 (19 mmol) and maintained at 0 °C for 5 hours, after monitoring the completion of the reaction by TLC, the solvent was removed by concentration under reduced pressure, and the target compound was isolated in high yield by column chromatography (81% yield).

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

[0113] 1 H NMR (500MHz, CDCl 3 ),δ(ppm)=7.21(s,1H),4.50(s,2H),4.07(s,3H); 13 C NMR (126MHz, CDCl 3 ),δ(ppm)=152.20,133.08,128.35,34.07,18.95.MS(ESI)m/z forC 5 H 6 BrN 3 O 2 : 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 20 mL of DMF were added to the reaction tube of sodium hydride (11 mmol) to the Schlenk tube with magnetron at 0 °C, and after stirring at 0 °C for 45 minutes, The temperature was raised to room temperature, and the reaction was continued for 8 hours. The reaction was monitored by TCL, and when all the starting materials were reacted, water was added to quench, 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 (yield 67%).

[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, CD 3 CN)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); 13 C NMR (126MHz, CD 3 CN),δ(ppm)=171.37,158.62,151.72,152.67,151.56,145.02,143.64,135.91,135.82,135.59,134.71,131.04,130.00,129.97,128.58,128.09,127.96,127.90,127.80,127.10,126.81, 113.01, 86.21, 86.02, 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 N 8 O 8 Na:[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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetron, and under nitrogen protection The reaction was stirred in ultra-dry DCM solvent for 3 hours under low temperature. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure, and separated by column chromatography to obtain the target compound (the yield was 63%).

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

[0123] 1 H NMR (500MHz, CD 3 CN)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 C NMR (126MHz, CD 3 CN),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 P NMR (202MHz, CD 3 CN), δ (ppm) = 148.11, 148.00; MS (ESI) m/z for C 52 H 58 N 10 O 9 P:[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 magnetron, 10 mL of DMF solvent was added, and the mixture was heated to 80° C. and stirred for 8 hours. The reaction was monitored by TCL and extracted with ethyl acetate (3x) when all the starting materials were reacted. 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 (86% yield).

[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, CD 3 CN)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, CD 3 CN), δ(ppm)=163.00, 158.80, 150.64, 144.95, 135.84, 134.66, 134.41, 130.06, 128.81, 128.03, 127.93, 126.97, 113.12, 109.52, 80.38, 85.99, 45.1, 54.2, 70 34.27, 11.97; MS(ESI) m/z for C 36 H 37 N 5 O 9 Na:[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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetron at 0°C , and stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure and separated by column chromatography to obtain the target compound (the yield was 71%).

[0132] Compound 5: (2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-methyl-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, CD 3 CN)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); 13 C NMR (126MHz, CD 3CN),δ(ppm)=162.96,158.80,150.69,144.89,130.10,130.08,128.84,128.83,128.05,128.00,127.93,127.01,126.99,118.57,118.41,113.13,86.50,86.41,85.57,85.48,85.18, 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, CD 3 CN), δ (ppm) = 148.02, 147.99; MS (ESI) m/z for C 45 H 54 N 7 O 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 filled with magnetrons. 10 mL of DMF was added to the reaction tube, and stirred at room temperature for 2 hours. The reaction was monitored by TCL and extracted with ethyl acetate (3x) when all the starting materials were reacted. 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 (86% yield).

[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, CD 3 CN)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, CD 3 CN)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 N 8 O 9 : 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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetron at 0°C , and stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure, and separated by column chromatography to obtain the target compound (the yield was 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, CD 3 CN)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, CD 3 CN)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 for C 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 the Schlenk tube with magnetron at 0°C, and 20 mL of DMF was added to the reaction tube, stirred for 45 minutes, and then brought to room temperature The reaction was continued for 8 hours. The reaction was monitored by TCL, and when all the raw materials were reacted, water was added to quench and the aqueous layer was extracted with ethyl acetate (3×). 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 77%).

[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, CD 3 CN)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, 3H), 3.98–3.94 (m, 1H), 3.77 (s, 6H), 3.38–3.30 (m, 2H), 2.39–2.27 (m, 3H); 13 C NMR (126MHz, CD 3 CN),δ(ppm)=176.56,158.65,155.21,149.92,144.70,137.33,135.83,135.74,135.56,134.28,132.54,130.07,130.02,129.34,128.37,128.16,128.09,127.90,126.95,113.09,91.21, 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 N 6 O 9 Na:[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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetron at 0°C , and stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure and separated by column chromatography to obtain the target compound (the yield was 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] 1 H NMR (500MHz, CD 3 CN)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, CD 3 CN),δ(ppm)=176.56,170.68,158.75,155.03,149.96,144.62,137.29,137.24,135.80,135.79,135.62,135.54,135.49,135.45,134.24,132.55,130.08,129.34,128.36,128.19,128.12, 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, CD 3 CN), δ (ppm) = 148.26, 148.22; MS (ESI) m/z for C 51 H 57 N 8 O 10 PNa:[M+Na] + : 995.383(calcd.), 995.384(expt.).

[0154] The compounds prepared in this example are listed in image 3.

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

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

[0157]

[0158] To a solution of N-1-methyl-2-nitro-5-hydroxymethylimidazole (10 mmol) and DIPEA (20 mmol) dissolved in ultra-dry dichloromethane at 0°C under nitrogen protection, PPhBr was slowly added 3 (19 mmol), and maintained at 0 °C for 30 min, then slowly raised to room temperature and continued to react for 5 h. After TLC monitoring was completed, the solvent was concentrated under reduced pressure to remove the solvent, and the target compound was isolated in high yield by column chromatography (85% yield).

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

[0160] 1 H NMR (500MHz, CDCl 3 ),δ(ppm)=7.81(s,1H),4.50(s,2H),3.82(s,3H); 13 C NMR (126MHz, CDCl 3 ), δ(ppm)=162.08, 139.44, 136.35, 34.06, 28.85; MS(ESI) m/z forC 5 H 6 BrN 3 O 2 : 218.9643(calcd.), 218.9655(expt.).

[0161] 2.2 Preparation of compound 11

[0162]

[0163] Under nitrogen protection, superdry 20 mL of DMF-dissolved D1 (10 mmol) and compound 10 (10 mmol) were added to a Schlenk tube containing magnetron and sodium hydride (11 mmol) at 0°C, stirred for 45 minutes, and then brought to room temperature and continued to stir for 8 Hour. The reaction was monitored by TCL, and when all the raw materials were reacted, water was added to quench and the aqueous layer was extracted with ethyl acetate (3×). 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 (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 (500MHz, CD 3 CN)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 (126MHz, CD 3 CN),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 N 8 O 8 :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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetron at 0°C , and stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure, and the target compound was obtained by column chromatography (the yield was 73%).

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

[0170] 1 H NMR (500MHz, CD 3 CN)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); 13 C NMR (126MHz, CD 3 CN),δ(ppm)=170.29,159.84,153.88,153.84,152.64,147.97,144.65,142.16,136.13,135.99,135.73,135.68,134.99,132.45,131.20,130.97,129.33,129.10,128,57,127.98,127.74, 127.68,127.10,127.00,126.81,126.55,115.41,89.41,87.47,86.25,84.45,74.47,73.50,72.97,71.24,66.15,62.57,59.23,58.71,58.67,58.38,58.27,55.94,45.10,44.06,43.88, 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, CD 3CN), δ (ppm) = 148.11, 148.70; MS (ESI) m/z for C 52 H 57 N 10 O 9 P: 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 with magnetron, and then 10 mL of DMF was added, and the mixture was heated to 80°C and stirred for 8 hours. The reaction was monitored by TCL and extracted with ethyl acetate (3x) when all the starting materials were reacted. 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 (81% yield).

[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, CD 3 CN)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, CD 3 CN), δ(ppm)=165.00, 159.80.152.77, 147.58, 137.25, 136.64, 135.21, 129.26, 128.91, 128.33, 128.03, 126.97, 115.21, 111.52, 89.38, 84.74, 83.8557, 72 37.53, 12.07; MS(ESI) m/z for C 36 H 37 N 5 O 9 : 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), 3-(chloro(diisopropylamino)phosphino)propionitrile (6 mmol) were added to a Schlenk tube with magneton at 0°C, Stir in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure and separated by column chromatography to obtain the target compound (the yield was 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 (500MHz, CD 3 CN)δ8.17(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 (126MHz, CD 3 CN),δ(ppm)=165.12,159.66,152.79,146.52,131.26,130.85,129.14,128.53,128.26,128.01,127.56,127.12,124.97,119.17,118.76,115.04,89.24,87.38,86.82,85.85,85.26, 85.01,84.82,83.05,75.64,74.73.73.61,66.38,61.05,59.42,56.95,5.90,41.74,39.22,38.38,23.7.01,19.01,19.01,19.01,19.08,3.3.01,19.08,3.3.3.01,19.08,3.3.01,19.08,3.3.01,19.08,3.3.01,19.01,3.3.01,19.01,3.3.3.01,19.01,3.3.01,19.01,19.01,19.01,3.3.01,19.01,19.01,19.01,19.01,19. 31 P NMR (202MHz, CD 3 CN), δ (ppm) = 149.15, 149.11; MS (ESI) m/z for C 45 H 54 N 7 O 10 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 containing a magnetron, and 10 mL of DMF was added to the reaction tube, and stirred at room temperature for 2 hours. The reaction was monitored by TCL and extracted with ethyl acetate (3x) when all the starting materials were reacted. 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 (83% yield).

[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, CD 3 CN)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). 13 C NMR (126MHz, CD 3 CN)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 N 8 O 9 : 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), 3-(chloro(diisopropylamino)phosphino)propionitrile (6 mmol) were added to a Schlenk tube with magnetron at 0°C, Stir in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were completely reacted, the solvent was removed by concentration under reduced pressure and separated by column chromatography to obtain the target compound (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)methyl)isobutyramido)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0190] 1 H NMR (500MHz, CD 3CN)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), 2.83–2.72 (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, CD 3 CN)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, superdry 20 mL DMF-dissolved D4 (10 mmol) and compound 10 (10 mmol) were added to a Schlenk tube filled with magnetron and sodium hydride (12 mmol), stirred at low temperature for 45 minutes, and then brought to room temperature to continue the reaction 8 Hour. The reaction was monitored by TCL, and when all the raw materials were reacted, water was added to quench and the aqueous layer was extracted with ethyl acetate (3×). 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 (81% yield).

[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 (500MHz, CD 3 CN)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 (126MHz, CD 3 CN),δ(ppm)=175.26,159.62,156.36,150.92,146.56,138.42,136.52,135.99,134.56,134.18,133.56,131.56,130.73,128.94,128.89,128.58,128.23,127.53,126.85,114.11,93.20, 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 N 6 O 9 :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), 3-(chloro(diisopropylamino)phosphino)propionitrile (7.5 mmol) were added to a Schlenk tube with magnetrons at 0°C , and stirred in ultra-dry DCM solvent for 3 hours under nitrogen protection. The reaction was monitored by TCL. When all the raw materials were reacted completely, the solvent was removed by concentration under reduced pressure and separated by column chromatography to obtain the target compound (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-oxopyrimdin-1(2H)-yl)tetrahydrofu ran-3-yl(2-cyanoethyl)diisopropylphosphoramidite:

[0200] 1 H NMR (500MHz, CD 3 CN)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); 13 C NMR (126MHz, CD 3 CN),δ(ppm)=178.01,172.99,159.70,156.75,147.56,144.96,139.64,138.53,137.88,136.56,135.98,135.66,135.34,135.28,134.19,131.55,130.28,129.82,128.96,128.20,128.15, 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, CD 3 CN), δ (ppm) = 148.52, 148.42; MS (ESI) m/z for C 51 H 57 N 8 O 10 P: 972.3935 (calcd.), 972.3938 (expt.).

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