A hydroxyl radical-based ultrasound-responsive conjugate and applications thereof

Abstract

The application provides a conjugate based on hydroxyl radical ultrasonic response and application, having a structure shown in formula I or a salt thereof; the research is started from a reactive substance such as a hydroxyl radical generated by the cavitation effect of ultrasonic, and a plurality of meta-disubstituted benzyl alcohol ultrasonic response structures are designed and screened. After the conjugate is formed by covalent bond between the structure and a to-be-masked molecule such as a drug or a fluorescent contrast molecule, the active groups of the to-be-masked molecule can be masked, and the activity of the to-be-masked molecule can be inhibited, and a molecule obtained is called a prodrug, the prodrug can be a small molecule, and can also be used for a polymer drug loading system. The prodrug can capture the hydroxyl radical generated by the cavitation effect of ultrasonic under the condition of no ultrasound sensitizer, and release the target molecule by elimination reaction. Through experimental screening, it is found that the meta-benzene diether structure is the best ultrasonic response group.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, and particularly relates to an ultrasonically responsive conjugate based on hydroxyl radicals and its application. Background Technology

[0002] To combat cancer, a wide variety of small-molecule chemotherapy drugs have been developed. These drugs are widely applicable, highly toxic, and economical to use. However, small-molecule drugs also suffer from significant systemic toxicity and poor pharmacokinetics, causing unnecessary suffering for patients. To reduce these toxicities, improving drug selectivity is a common approach, making the development of spatiotemporally controllable prodrug release systems crucial.

[0003] Traditional methods involve selectively activating and releasing non-toxic prodrugs at the tumor site. Among these, exogenous stimulation therapies, such as radiotherapy and photodynamic therapy, offer advantages such as non-invasiveness and spatiotemporal control, showing great promise. Ultrasound, a novel exogenous stimulation therapy, enters the body non-invasively via mechanical sound waves. Through cavitation, it forms bubbles in the liquid, generating a large number of free radical reactive substances. Compared to the relatively mature radiotherapy, it offers advantages such as greater penetration depth and greater safety. However, current ultrasound response structures are relatively limited, and most rely on sound-sensitive agents and reducing substances such as riboflavin and NADH, restricting its application in vivo. Summary of the Invention

[0004] The purpose of this invention is to provide a hydroxyl radical-based ultrasonically responsive conjugate and its application. The conjugate in this invention can capture hydroxyl radicals generated by ultrasound without the need for an ultrasound sensitizer and release the target molecule through an elimination reaction. Experimental screening revealed that m-phenylene diether structures are the optimal ultrasonically responsive groups.

[0005] The present invention provides an ultrasonically responsive conjugate based on hydroxyl radicals, having the structure shown in Formula I or a salt thereof;

[0006] Formula I;

[0007] In Formula I, R1 and R2 are independently selected from substituted or unsubstituted C1-C10 alkyl groups, substituted or unsubstituted C1-C10 alkoxy groups, -NHCOR′ or -OCOR′′, and at least one of R1 and R2 is a substituted or unsubstituted C1-C10 alkoxy group.

[0008] Preferably, R1 and R2 are both independently selected from substituted or unsubstituted C1 to C10 alkoxy groups, such as substituted or unsubstituted C1 alkoxy groups, substituted or unsubstituted C2 alkoxy groups, substituted or unsubstituted C3 alkoxy groups, substituted or unsubstituted C4 alkoxy groups, substituted or unsubstituted C5 alkoxy groups, substituted or unsubstituted C6 alkoxy groups, substituted or unsubstituted C7 alkoxy groups, substituted or unsubstituted C8 alkoxy groups, substituted or unsubstituted C9 alkoxy groups, or substituted or unsubstituted C10 alkoxy groups.

[0009] The substituents in the substituted C1-C10 alkoxy groups and the substituents in the substituted C1-C10 alkyl groups are independently selected from amino, carboxyl, or hydroxyl groups;

[0010] R′ and R′′ are independently selected from C1 to C10 alkyl groups, such as C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl or C10 alkyl.

[0011] R3 is the residue of the molecule to be masked after losing a -H or -OH group.

[0012] Preferably, R1 and R2 are independently selected from substituted or unsubstituted C1-C5 alkyl groups, substituted or unsubstituted C1-C5 alkoxy groups, -NHCOR′ or -OCOR′′, and at least one of R1 and R2 is a substituted or unsubstituted C1-C5 alkoxy group.

[0013] The substituents in the substituted C1-C5 alkoxy groups and the substituents in the substituted C1-C5 alkyl groups are independently selected from amino, carboxyl, or hydroxyl groups;

[0014] R′ and R′′ are independently selected from C1 to C5 alkyl groups.

[0015] Preferably, R1 and R2 are independently selected from methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, -OCH2NH2, -OCH2CH2NH2, -OCH2CH2CH2NH2, -OCH2CH2CH2CH2NH2, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2CH2CH3, -OCOCH3, -OCOCH2CH3 or -OCOCH2CH2CH3, and at least one of R1 and R2 is methoxy, ethoxy, propoxy, isopropoxy, butoxy or tert-butoxy.

[0016] Preferably, the molecule to be masked includes a drug and / or a fluorescent reagent.

[0017] Preferably, the molecule to be masked includes one or more of fatty amine compounds, fatty alcohol compounds, carboxylic acid compounds, and aromatic amine compounds. Specifically, in some embodiments of the present invention, the molecule to be masked is eczema mesylate, p-aminobenzoic acid, camptothecin, or 4-methyl-7-aminocoumarin.

[0018] Preferably, the salt having the structure shown in Formula I includes one or more of carboxylates, hydrochlorides, p-benzenesulfonates, methanesulfonates, and phosphates. Specifically, in some embodiments of the present invention, the salt is a trifluoroacetate.

[0019] Preferably, the ultrasonically responsive conjugate based on hydroxyl radicals has the structures shown in Formulas I-1 to I-8:

[0020]

[0021] .

[0022] In this invention, the ultrasonically responsive conjugates based on hydroxyl radicals with the structure shown in Formula I can all be synthesized by conventional methods in the art.

[0023] The present invention provides an ultrasound-responsive polymeric drug delivery system based on hydroxyl radicals, comprising a polymeric compound and the aforementioned ultrasound-responsive conjugate based on hydroxyl radicals loaded onto the polymeric compound.

[0024] Preferably, the polymeric compound includes polyglutamic acid.

[0025] This invention provides the application of an ultrasound-responsive compound in the preparation of an antitumor drug, wherein the ultrasound-responsive compound is either the ultrasound-responsive conjugate based on hydroxyl radicals described above, or the ultrasound-responsive polymeric drug delivery system based on hydroxyl radicals described above.

[0026] In this invention, the ultrasonic-responsive compound can still exhibit significant ultrasonic responsiveness in a liquid environment without acoustic sensitizers or reducing agents, and can selectively activate the prodrug through the cavitation effect generated by ultrasound.

[0027] In this invention, the power of the ultrasound is preferably 1.5~2.5W / cm. 2 More preferably 1.5~2W / cm 2 For example, 1.5W / cm 2 1.6 W / cm 2 1.8 W / cm 2 1.9 W / cm 2 2.0 W / cm 2 2.1 W / cm2 2.2 W / cm 2 2.3 W / cm 2 2.4W / cm 2 2.5 W / cm 2 The preferred values ​​are those within the range of any of the above values ​​as the upper or lower limit; the preferred ultrasound duration is 8-10 min, such as 8 min, 8.5 min, 9 min, 9.5 min, 10 min, preferably within the range of any of the above values ​​as the upper or lower limit; the preferred ultrasound duty cycle is 40-60%, more preferably 45-55%, such as 40%, 45%, 50%, 55%, 60%, preferably within the range of any of the above values ​​as the upper or lower limit; the preferred ultrasound frequency is 0.5-2 MHz, more preferably 1-1.5 MHz, such as 0.5 MHz, 0.6 MHz, 0.7 MHz, 0.8 MHz, 0.9 MHz, 1.0 MHz, 1.1 MHz, 1.2 MHz, 1.3 MHz, 1.4 MHz, 1.5 MHz, 1.6 MHz, 1.7 MHz, 1.8 MHz, 1.9 MHz, 2 MHz, preferably within the range of any of the above values ​​as the upper or lower limit.

[0028] This invention provides an ultrasonically responsive conjugate based on hydroxyl radicals, having the structure shown in Formula I or a salt thereof; in Formula I, R1 and R2 are independently selected from substituted or unsubstituted C1-C10 alkyl groups, substituted or unsubstituted C1-C10 alkoxy groups, -NHCOR′ or -OCOR′′, and at least one of R1 and R2 is a substituted or unsubstituted C1-C10 alkoxy group; the substituent in the substituted C1-C10 alkoxy group and the substituent in the substituted C1-C10 alkyl group are independently selected from amino, carboxyl, or hydroxyl groups; R′ and R′′ are independently selected from C1-C10 alkyl groups; R3 is a residue of the molecule to be masked after losing a -H or -OH group. This study utilizes the property that ultrasound cavitation generates hydroxyl radicals to design and screen several meta-disubstituted benzyl alcohol-based ultrasonically responsive structures. When this type of structure forms a conjugate with the molecule to be masked, such as a drug or a fluorescent imaging molecule, through covalent bonds, it can mask their active groups and inhibit their activity. The resulting molecule is called a prodrug. This prodrug can be a small molecule or used in a polymeric drug delivery system. This prodrug can capture hydroxyl radicals generated by ultrasonic cavitation under conditions without a sonic sensitizer and release the target molecule through an elimination reaction. Experimental screening revealed that m-phenylenediether structures are the optimal ultrasonic responsive groups. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 The NMR spectrum of compound 19 in Example 1 of this invention;

[0031] Figure 2 The ultrasonic release rates of compounds 1-14 in Example 2 of this invention;

[0032] Figure 3 The ultrasonic reduction rate of compounds 1-14 in Example 2 of this invention;

[0033] Figure 4 This is a schematic diagram of the HPLC results of compound 12 after ultrasonication in Example 2 of the present invention;

[0034] Figure 5 The target molecule release rate of compound 12 under different power levels in Example 3 of this invention;

[0035] Figure 6 This illustrates the effect of radiation time at different sound intensities on the release rates of compounds 8, 11, and 12 in Example 3 of the present invention.

[0036] Figure 7 The release rate of the target molecule of compound 12 under different ultrasound times in Example 3 of the present invention;

[0037] Figure 8 The reduction rate of compound 12 under different ultrasound times in Example 3 of the present invention;

[0038] Figure 9 The ultrasonic release curve of compound 15 in Example 4 of this invention;

[0039] Figure 10 The ultrasonic release curve of compound 16 in Example 4 of this invention;

[0040] Figure 11 The ultrasonic release curve of compound 17 in Example 4 of this invention;

[0041] Figure 12 The ultrasonic release curve of compound 18 in Example 4 of this invention;

[0042] Figure 13 The ultrasonic release curve of compound 19 in Example 4 of this invention;

[0043] Figure 14This is the curve showing the relationship between the release rate of compound 12 and the volume fraction of isopropanol in Example 5 of the present invention;

[0044] Figure 15 This is the curve showing the relationship between the release rate of compound 12 and the Fenton reaction equivalent in Example 5 of the present invention;

[0045] Figure 16 The above are the HPLC results of compound 15 in Example 5 of this invention after ultrasonication and incubation for different times.

[0046] Figure 17 The results are high-resolution mass spectrometry results of compound 15 after sonication in Example 5 of this invention. Detailed Implementation

[0047] To further illustrate the present invention, the following detailed description of an ultrasonically responsive conjugate based on hydroxyl radicals and its application is provided in conjunction with embodiments, but it should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1: Synthesis of compounds 1-19

[0049]

[0050]

[0051] Compound 1:

[0052]

[0053] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3,5-dibromobenzyl alcohol (1030.8 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 608 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 401 mg of white powder, compound 3. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0054] 1H NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1H), 10.20 (s, 3H), 7.89 – 7.84(m, 6H), 7.80 (t, J = 1.8 Hz, 2H), 7.67 (d, J = 1.8 Hz, 5H), 7.60 – 7.54 (m,6H), 5.16 (s, 6H).

[0055] 13C NMR (126 MHz, DMSO-d6) δ 167.42, 153.32, 143.56, 141.61, 133.35,130.98, 130.19, 125.04, 122.95, 117.86, 64.80.

[0056] Compound 2:

[0057]

[0058] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.00 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3,5-dichlorobenzyl alcohol (687 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 592 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 350 mg of white powder, compound 3. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0059] 1 H NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1H), 10.20 (s, 1H), 7.90 – 7.84(m, 2H), 7.60 – 7.54 (m, 3H), 7.50 (d, J = 1.9 Hz, 2H), 5.17 (s, 2H).

[0060] 13C NMR (126 MHz, DMSO-d6) δ 167.42, 153.33, 143.56, 141.16, 134.57,130.98, 128.04, 126.94, 125.04, 117.86, 64.91.

[0061] Compound 3:

[0062]

[0063] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3,5-difluorobenzyl alcohol (560 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 568 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was then dried under vacuum to obtain 344 mg of white powder, compound 3. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0064] 1H NMR (500 MHz, DMSO-d6) δ 12.66 (s, 1H), 10.21 (s, 1H), 7.91 – 7.84(m, 2H), 7.61 – 7.54 (m, 2H), 7.23 – 7.12 (m, 3H), 5.18 (s, 2H).

[0065] 13C NMR (126 MHz, DMSO-d6) δ 167.43, 163.88, 163.77, 161.92, 161.81,153.35, 143.58, 141.54, 141.46, 141.39, 130.97, 125.04, 117.87, 111.31,111.26, 111.15, 111.10, 104.03, 103.83, 103.62, 65.07, 65.05.

[0066] Compound 4:

[0067]

[0068] Weigh 50 mg (0.26 mmol, 1.0 eq) of tert-butyl p-aminobenzoate and 38.4 mg (0.13 mmol, 0.5 eq) of triphosgene into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 10 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 10.4 mg, 0.78 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 87 mg (3.9 mmol, 1.5 eq) of methyl 3,5-dicarboxymethylbenzyl alcohol and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 74 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 48 mg of white powder, compound 4. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0069] 1H NMR (500 MHz, DMSO-d6) δ 12.67 (s, 1H), 10.23 (s, 1H), 8.26 (d, J= 1.7 Hz, 1H), 7.89 – 7.83 (m, 1H), 7.60 – 7.54 (m, 1H), 5.32 (s, 1H), 3.89(s, 3H).

[0070] 13 C NMR (126 MHz, DMSO-d6) δ 167.41, 165.65, 153.45, 143.59, 138.81,133.35, 130.96, 130.94, 129.63, 125.02, 117.87, 65.39, 53.05.

[0071] Compound 5:

[0072]

[0073] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.00 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add benzyl alcohol (419 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 623 mg of white powder was obtained as an intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 369 mg of white powder, compound 5. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0074] 1 H NMR (500 MHz, DMSO-d6) δ 12.66 (s, 1H), 10.15 (s, 1H), 7.90 – 7.84(m, 2H), 7.61 – 7.55 (m, 2H), 7.46 – 7.36 (m, 4H), 7.36 – 7.30 (m, 1H), 5.17(s, 2H).

[0075] 13 C NMR (126 MHz, DMSO-d6) δ 167.46, 153.67, 143.78, 136.81, 130.96,128.95, 128.70, 128.62, 124.88, 117.80, 66.53.

[0076] Compound 6:

[0077]

[0078] Weigh tert-butyl p-aminobenzoate (500.0 mg, 2.59 mmol, 1.00 eq) and triphosgene (384.3 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Under ice bath conditions, slowly add an anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.00 eq). After reacting at room temperature for 30 min, add 3,5-dimethylbenzyl alcohol (528 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 589 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 345 mg of white powder, compound 6. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0079] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.11 (s, 1H), 7.89 – 7.83(m, 2H), 7.60 – 7.54 (m, 2H), 7.01 (d, J = 1.6 Hz, 2H), 6.96 (s, 1H), 5.08(s, 2H), 2.26(s, 6H).

[0080] 13 C NMR (126 MHz, DMSO-d6) δ 167.44, 153.68, 143.80, 137.98, 136.59,130.95, 129.94, 126.38, 124.85, 117.77, 66.56, 21.31.

[0081] Compound 7:

[0082]

[0083] (1) Synthesis of m-acetaminobenzyl alcohol:

[0084] 1.0 g (8.1 mmol, 1.0 eq) of m-aminobenzyl alcohol was weighed into a flask, and 20 mL of anhydrous dichloromethane (DCM) was added. Acetic anhydride (0.87 g, 8.6 mmol, 1.05 eq) was then added dropwise under ice bath conditions. The reaction was allowed to proceed for 12 h. The product was purified by rapid silica gel chromatography (methanol:dichloromethane = 2:98). 823 mg of a white powder was obtained, yielding acetaminobenzyl alcohol. Characterization was performed using 1H NMR (300 MHz, Bruker AVANCE III HD).

[0085] 1 H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.46 (dt, J = 7.5, 1.3 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 6.96 (dt, J = 7.7,1.2 Hz, 1H), 5.20 (t, J = 5.7 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 2.03 (s,3H).

[0086] (2) Synthesis of compound 7:

[0087] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add m-acetaminophenethanol (641 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 607 mg of white powder was obtained as an intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 432 mg of white powder, compound 7. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0088] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.16 (s, 1H), 9.98 (s,1H), 7.90 – 7.83 (m, 2H), 7.64 (t, J = 1.9 Hz, 1H), 7.60 – 7.54 (m, 2H), 7.53(ddd, J = 8.2, 2.2, 1.1 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.08 (dt, J = 7.7,1.3 Hz, 1H), 5.13 (s, 2H), 2.02 (s, 3H).

[0089] 13C NMR (126 MHz, DMSO-d6) δ 168.85, 167.44, 153.64, 143.77, 139.97,137.39, 130.95, 129.31, 124.88, 123.21, 119.18, 119.02, 117.80, 66.46, 24.47.

[0090] Compound 8:

[0091]

[0092]

[0093] (1) Synthesis of 3-methoxy,5-hydroxymethylacetanilide:

[0094] Weigh 1.0 g (6.0 mmol, 1.0 eq) of 3-methoxy-5-aminobenzoic acid into a flask, add 20 mL of anhydrous dichloromethane (DCM), and then add acetic anhydride (0.61 g, 6.3 mmol, 1.05 eq) dropwise under ice bath conditions. After reacting for 12 h, a white slurry appears. Filter, wash the filter cake sequentially with dichloromethane and deionized water, and dry. 956 mg of white powder is obtained, and the crude product can be directly proceeded to the next step without purification. Dissolve the crude product completely in 20 mL of anhydrous tetrahydrofuran (THF), and add a BH3 THF solution (1 mol / L, 12 mL, 2.0 eq) dropwise under ice bath conditions. Purify the product using rapid silica gel chromatography (methanol:dichloromethane = 2:98) to obtain a white solid 3-methoxy,5-hydroxymethylacetanilide. Characterized by proton nuclear magnetic resonance spectroscopy (1H NMR, 300 MHz, Bruker AVANCE III HD).

[0095] 1 H NMR (300 MHz, DMSO-d6) δ 12.71 (s, 1H), 10.20 (s, 1H), 10.00 (s,1H), 7.87 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.26 (t, J = 2.2 Hz,1H), 7.17 (s, 1H), 6.69 (s, 1H), 5.10 (s, 2H), 3.73 (s, 3H), 2.02 (s, 3H).

[0096] (2) Synthesis of compound 8:

[0097] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3-methoxy,5-hydroxymethylacetanilide (758 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate:n-hexane = 1:9). 569 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 348 mg of white powder, compound 8. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0098] 1 H NMR (500 MHz, DMSO-d6) δ 12.66 (s, 1H), 10.16 (s, 1H), 9.96 (s,1H), 7.90 – 7.83 (m, 2H), 7.61 – 7.54 (m, 2H), 7.25 (t, J = 2.1 Hz, 1H), 7.17(t, J = 1.6 Hz, 1H), 6.67 (dd, J = 2.4, 1.4 Hz, 1H), 5.09 (s, 2H), 3.72 (s,3H), 2.01 (s, 3H).

[0099] 13C NMR (126 MHz, DMSO-d6) δ 168.91, 167.44, 160.01, 153.62, 143.76,141.05, 138.50, 130.95, 124.89, 117.81, 111.21, 108.53, 104.84, 67.49, 66.39,55.56, 24.53.

[0100] Compound 9:

[0101]

[0102]

[0103] (1) Synthesis of 3-methoxy,5-hydroxymethylacetylphenol:

[0104] Weigh 1.0 g (6.5 mmol, 1.0 eq) of 3-methoxy-5-hydroxybenzaldehyde into a flask, dissolve it in 5 mL of pyridine, and then add acetic anhydride (0.70 g, 6.9 mmol, 1.05 eq) dropwise under ice bath conditions. React for 16 h. Remove pyridine under reduced pressure, wash with ethyl acetate and saturated brine, combine the ethyl acetate phases, remove water by adding anhydrous sodium sulfate, and evaporate the ethyl acetate to dryness to obtain the crude product, which can be directly proceeded to the next step without purification. Dissolve the crude product in methanol, add a methanol solution of sodium borohydride (0.26 g, 19.5 mmol, 3.0 eq) under ice bath conditions, and react for 2 h. Remove methanol under reduced pressure, wash with ethyl acetate and saturated brine, and purify the product using rapid silica gel chromatography (methanol:dichloromethane = 2:98). Obtain the white solid 3-methoxy,5-hydroxymethylacetylphenol. Characterized by proton nuclear magnetic resonance spectroscopy (1H NMR, 300 MHz, Bruker AVANCE III HD).

[0105] 1 H NMR (300 MHz, DMSO-d6) δ 11.85 (s, 1H), 6.78 (dd, J = 2.4, 1.3 Hz, 2H), 6.65 (dd, J = 2.1, 1.2 Hz, 2H), 6.58 (t, J = 2.3 Hz, 2H), 4.47 (s, 4H), 3.74 (s, 6H), 2.25 (s, 6H), 1.99 (s, 1H), 1.91 (s, 4H).

[0106] (2) Synthesis of compound 9:

[0107] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3-methoxy,5-hydroxymethylacetylphenol (765 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate:n-hexane = 1:9). 423 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 301 mg of white powder, compound 9. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0108] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.18 (s, 1H), 7.90 – 7.84(m, 2H), 7.60 – 7.54 (m, 2H), 6.91 (dd, J = 2.3, 1.4 Hz, 1H), 6.77 (dd, J =2.0, 1.3 Hz, 1H), 6.71 (t, J = 2.2 Hz, 1H), 5.14 (s, 2H), 3.75 (s, 3H), 2.24(s, 3H).

[0109] 13 C NMR (126 MHz, DMSO-d6) δ 169.54, 167.43, 160.56, 153.52, 152.00,143.70, 139.24, 130.97, 124.94, 117.83, 113.81, 111.59, 107.93, 65.83, 56.00,21.30.

[0110] Compound 10:

[0111]

[0112] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3-methoxy,5-methylbenzyl alcohol (593 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate:n-hexane = 1:9). 399 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 259 mg of white powder, compound 5. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0113] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.13 (s, 1H), 7.89 – 7.83(m, 2H), 7.57 (d, J = 8.5 Hz, 2H), 6.83 – 6.77 (m, 2H), 6.74 – 6.70 (m, 1H), 5.09 (s, 2H), 3.73 (s, 3H), 2.27 (s, 3H).

[0114] 13C NMR (126 MHz, DMSO-d6) δ 167.44, 159.83, 153.64, 143.78, 139.65,138.04, 130.95, 124.87, 121.39, 117.79, 114.61, 111.25, 66.41, 55.51, 21.56.

[0115] Compound 11:

[0116]

[0117]

[0118] (1) Synthesis of 3,5-di-tert-butyldimethylsiloxane benzyl alcohol:

[0119] Weigh 1.0 g (7.2 mmol, 1.0 eq) of m-dihydroxybenzaldehyde and 2.95 g (43.2 mmol, 6.0 eq) of imidazole into a flask, dissolve in 40 mL of anhydrous tetrahydrofuran, and then add dimethyl tert-butylchlorosilane (2.3 g, 15.1 mmol, 2.1 eq) dropwise under ice bath conditions. React for 16 h. Remove the solvent under reduced pressure, wash with ethyl acetate and saturated brine, combine the ethyl acetate phases, remove water with anhydrous sodium sulfate, and remove ethyl acetate under reduced pressure to obtain the crude product, which can be directly proceeded to the next step without purification. Dissolve the crude product in methanol, add a methanol solution of sodium borohydride (0.26 g, 19.5 mmol, 3.0 eq) under ice bath conditions, and react for 2 h. Remove methanol under reduced pressure, wash with ethyl acetate and saturated brine, and purify the product using rapid silica gel chromatography (methanol:dichloromethane = 2:98). A colorless oily substance, 3,5-di-tert-butyldimethylsiloxane benzyl alcohol, was obtained. It was characterized by 1H NMR (300 MHz, Bruker AVANCE III HD).

[0120] 1 H NMR (300 MHz, DMSO-d6) δ 6.43 (s, 2H), 6.13 (s, 1H), 5.15 (q, J =5.0, 3.8 Hz, 1H), 4.38 (s, 2H), 1.07 (s, 1H), 0.93 (t, J = 3.6 Hz, 17H), 0.84(d, J = 4.2 Hz, 1H), 0.77 (s, 2H), 0.21 – 0.12 (m, 11H), 0.09 (s, 3H).

[0121] (2) Synthesis of compound 11:

[0122] Weigh 500 mg (2.6 mmol, 1.0 eq) of tert-butyl p-aminobenzoate and 384 mg (1.3 mmol, 0.5 eq) of triphosgene into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 1.4 g (3.9 mmol, 1.5 eq) of 3,5-di-tert-butyldimethylsiloxane benzyl alcohol and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 878 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 10 mL of THF, and tetrabutylammonium fluoride (1 mol / L, 8 mL, 3.0 eq) was added to remove the silicon protecting group. Then, THF was removed under reduced pressure, and 10 mL of DCM was added to dissolve the residue. 2 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, and a white solid precipitated. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 105 mg of white powder, compound 11. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0123] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.13 (s, 1H), 9.28 (s,2H), 7.89 – 7.83 (m, 2H), 7.60 – 7.54 (m, 2H), 6.24 (d, J = 2.2 Hz, 2H), 6.15(t, J = 2.2 Hz, 1H), 4.98 (s, 2H), 1.26 (s, 0H).

[0124] 13C NMR (126 MHz, DMSO-d6) δ 167.45, 158.95, 153.69, 143.81, 138.81, 130.96, 124.84, 117.78, 106.23, 102.52, 66.39, 40.86.

[0125] Compound 12:

[0126]

[0127] Weigh tert-butyl p-aminobenzoate (500 mg, 2.6 mmol, 1.0 eq) and triphosgene (384 mg, 1.3 mmol, 0.5 eq) into a dehydrated single-necked round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Slowly add anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1010 mg, 7.8 mmol, 3.0 eq) under ice bath conditions. After reacting at room temperature for 30 min, add 3,5-dimethoxybenzyl alcohol (653 mg, 3.9 mmol, 1.5 eq) and react at room temperature for 16 h. Remove dichloromethane by vacuum distillation, and purify the product using rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). 625 mg of white powder was obtained as the intermediate. The intermediate product was dissolved in 5 mL of DCM, and 1 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. 20 mL of ultrapure water was added to the system, resulting in the precipitation of a large amount of white solid. The mixture was filtered under reduced pressure, and the filter cake was repeatedly washed with DCM. The filter cake was dried under vacuum to obtain 399 mg of white powder, compound 5. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0128] 1 H NMR (500 MHz, DMSO-d6) δ 12.65 (s, 1H), 10.15 (s, 1H), 7.89 – 7.83(m, 2H), 7.60 – 7.54 (m, 2H), 6.58 (d, J = 2.3 Hz, 2H), 6.45 (t, J = 2.3 Hz,1H), 5.09 (s, 2H), 3.73 (s, 6H).

[0129] 13 C NMR (126 MHz, DMSO-d6) δ 167.44, 161.01, 153.61, 143.75, 139.09, 130.96, 124.90, 117.82, 106.30, 100.16, 66.35, 55.70.

[0130] Compound 13:

[0131]

[0132] Weigh 1.0 g (7.3 mmol, 1.00 eq) of p-aminobenzoic acid into a single-necked round-bottom flask, add 20 mL of methanol, and dropwise add a methanol solution of dimethyl dicarbonate (1.02 g, 7.6 mmol, 1.05 eq) under ice bath conditions. Stir the reaction mixture at room temperature for 16 h. Monitor the reaction using thin-layer chromatography (TLC). Remove the solvent methanol by vacuum distillation. Pulverize the concentrate with a 1:5 mixture of ethyl acetate and n-hexane, and centrifuge to remove the supernatant. Dry under vacuum at 40°C for 2 h to obtain 0.5 g of a white solid powder, compound 13. Characterize the compound using 1H NMR (300 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0133] 1 H NMR (300 MHz, DMSO-d6) δ 12.69 (s, 1H), 10.06 (s, 1H), 7.87 (d, J =8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 3.69 (s, 3H).

[0134] 13 C NMR (126 MHz, DMSO-d6) δ 167.45, 154.28, 143.84, 130.93, 124.78,117.74, 56.50, 55.11, 52.33.

[0135] Compound 14:

[0136]

[0137] Weigh 1.0 g (7.3 mmol, 1.00 eq) of p-aminobenzoic acid into a single-necked round-bottom flask, add 20 mL of methanol, and dropwise add a methanol solution of diethyl dicarbonate (1.24 g, 7.6 mmol, 1.05 eq) under ice bath conditions. Stir the reaction mixture at room temperature for 16 h. Monitor the reaction using thin-layer chromatography (TLC). Remove the methanol solvent by vacuum distillation. Pulverize the concentrate with a 1:5 mixture of ethyl acetate and n-hexane, and centrifuge to remove the supernatant. Dry under vacuum at 40°C for 2 h to obtain 0.5 g of a white solid powder, compound 14. Characterize the compound using 1H NMR (300 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0138] 1 H NMR (300 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.03 (s, 1H), 7.86 (d, J =8.3 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 4.15 (q, J = 7.1 Hz, 2H), 1.25 (t, J =7.1 Hz, 3H).

[0139] 13 C NMR (126 MHz, DMSO-d6) δ 167.45, 153.83, 143.93, 130.91, 124.71,117.73, 60.96, 14.90.

[0140] Compound 15:

[0141]

[0142] (1) Synthesis of 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde:

[0143] Weigh 1.0 g (6.6 mmol, 1.0 eq) of 3-methoxy-5-hydroxybenzaldehyde, 2.95 g (13.2 mmol, 2.0 eq) of tert-butoxycarbonyl ethyl bromide, 1.65 mg (9.9 mmol, 1.5 eq) of potassium iodide, and 1.82 g (13.2 mmol, 2.0 eq) of potassium carbonate into a flask, and dissolve in 40 mL of N,N-dimethylformamide (DMF). React at 70 °C for 24 h. Remove DMF under reduced pressure, and wash with ethyl acetate and saturated brine. Purify by rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9) to obtain the product 3-(N-tert-butoxycarbonylaminoethoxy)5-methoxybenzaldehyde, a yellow oily liquid. Characterize by 1H NMR (500 MHz, Bruker AVANCE III HD).

[0144] 1 H NMR (500 MHz, DMSO-d6) δ 9.92 (s, 2H), 7.07 (d, J = 2.3 Hz, 4H), 7.03 (t, J = 5.8 Hz, 2H), 6.91 (ddd, J = 15.0, 2.3, 1.2 Hz, 1H), 6.83 (t, J =2.4 Hz, 2H), 4.03 (t, J = 5.8 Hz, 4H), 3.82 (s, 6H), 3.78 (s, 1H), 3.31 (q, J= 5.8 Hz, 4H), 1.38 (s, 19H), 1.25 (d, J = 8.6 Hz, 1H).

[0145] (2) Synthesis of 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol p-nitrophenol carbonate:

[0146] First, 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde was reduced to 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde. 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde (800 mg, 5.2 mmol, 1.0 eq) was dissolved in 10 mL of methanol. A methanol solution of sodium borohydride (0.58 g, 15.6 mmol, 3.0 eq) was added under ice bath conditions, and the reaction was allowed to proceed for 2 h. Methanol was removed under reduced pressure, and the mixture was washed with ethyl acetate and brine. Removal of ethyl acetate yielded an oily crude product of 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde, which was directly used in the next step. The crude product was dissolved in 20 mL of anhydrous DCM. A DCM solution of p-nitrophenol chloroformate (1.56 g, 7.8 mmol, 1.5 eq) was added under ice bath conditions, followed by dropwise addition of triethylamine (0.79 g, 7.8 mmol, 1.5 eq). The reaction was allowed to proceed at room temperature for 16 h. Rapid silica gel chromatography (ethyl acetate: n-hexane = 5: 95) was used to purify the product into a white solid, 3-(N-tert-butoxycarbonylaminoethoxy)5-methoxybenzyl alcohol p-nitrophenol carbonate. Characterization was performed using 1H NMR (300 MHz, Bruker AVANCE III HD).

[0147] 1 H NMR (300 MHz, DMSO-d6) δ 8.37 – 8.27 (m, 2H), 7.63 – 7.53 (m, 2H), 7.04 (t, J = 5.7 Hz, 1H), 6.62 (d, J = 2.2 Hz, 2H), 6.51 (t, J = 2.3 Hz, 1H), 5.23 (s, 2H), 3.96 (t, J = 5.8 Hz, 2H), 3.75 (s, 3H), 3.36 (s, 1H), 3.33 –3.23 (m, 2H), 1.38 (s, 9H).

[0148] (3) Synthetic compound 15

[0149] Weigh 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol p-nitrophenol carbonate (800 mg, 1.7 mmol, 1.0 eq) and eczemanetide (918 mg, 1.7 mmol, 1.0 eq) into a flask, add 20 mL of anhydrous DMF, and add triethylamine (0.79 g, 7.8 mmol, 1.5 eq) dropwise under ice bath. React at room temperature for 16 h. Remove DMF under reduced pressure and purify by rapid silica gel chromatography (methanol:dichloromethane = 3:97). Deprotect the crude product directly, dissolve it in 5 mL of DCM, add 1 mL of TFA under ice bath, and react for 2 h. Remove the solvent under reduced pressure, dissolve in deionized water, and lyophilize the resulting solution to obtain a yellow powder product, compound 15. The proton nuclear magnetic resonance (1H NMR, 500 MHz, Bruker AVANCE III HD) and carbon nuclear magnetic resonance (13C NMR, 126 MHz, Bruker AVANCE III HD) spectra were used for characterization.

[0150] 1 H NMR (500 MHz, DMSO-d6) δ 8.09 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 5.5Hz, 3H), 7.73 (d, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.63 – 6.58 (m, 2H), 6.46(d, J = 2.3 Hz, 1H), 5.41 (s, 2H), 5.26 (td, J = 7.8, 4.5 Hz, 1H), 5.20 (s,2H), 5.07 (s, 2H), 4.15 (t, J = 5.0 Hz, 2H), 3.70 (s, 3H), 3.21 (dq, J = 8.9,5.0 Hz, 3H), 3.09 (dd, J = 15.3, 8.6 Hz, 1H), 2.88 (s, 1H), 2.72 (s, 1H),2.34 (d, J = 1.9 Hz, 3H), 2.20 (s, 1H), 2.18 (dq, J = 21.1, 7.2, 6.1 Hz, 1H), 1.94 – 1.78 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H).

[0151] 13C NMR (126 MHz, DMSO-d6) δ 172.90, 163.06, 162.78, 161.08, 160.91,159.50, 158.86, 158.59, 157.17, 156.45, 152.87, 150.49, 148.42, 148.31,145.58, 141.20, 139.86, 136.81, 125.74, 124.23, 124.08, 121.93, 119.55,117.84, 115.49, 110.39, 110.20, 106.50, 106.40, 100.93, 97.19, 72.83, 66.18, 65.67, 64.94, 55.69, 50.19, 47.75, 38.87, 36.24, 31.23, 30.79, 28.64, 24.15, 11.42, 11.39, 8.22.

[0152] Compound 16:

[0153]

[0154] (1) Synthesis of 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde:

[0155] The method is the same as that for compound 15.

[0156] (2) Synthesis of compound 16:

[0157] 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde was reduced to 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol, using the same method as compound 15. The crude product was used for the next reaction. Camptothecin (500 mg, 1.44 mmol, 1.0 eq) and triphosgene (156 mg, 0.53 mmol, 1.1 eq) were weighed into a flask and dissolved in 20 mL of anhydrous DCM. A solution of N,N-dimethylaminopyridine (DMAP, 387 mg, 3.17 mmol, 2.2 eq) in DCM was added dropwise under ice bath conditions. The reaction was allowed to proceed for 1 h. After confirming complete dissolution of camptothecin, 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol was added and the reaction was allowed to proceed for 16 h. Rapid silica gel chromatography purification (methanol:dichloromethane = 3:97) yielded a product that was directly dissolved in 5 mL DCM. 1 mL TFA was added under ice bath conditions, and the reaction proceeded for 2 h. The solvent was removed under reduced pressure, and the product was reconstituted with deionized water and lyophilized to obtain a yellow powder, compound 16. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0158] 1 H NMR (500 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.18 – 8.09 (m, 2H), 8.05 –7.99 (m, 3H), 7.87 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.71 (ddd, J = 8.1, 6.8,1.2 Hz, 1H), 7.04 (s, 1H), 6.54 (dt, J = 6.8, 1.5 Hz, 2H), 6.43 (t, J = 2.3Hz, 1H), 5.52 (d, J = 2.8 Hz, 2H), 5.49 (s, 3H), 5.28 (d, J = 2.0 Hz, 2H),5.21 (s, 5H), 5.14 (d, J = 12.5 Hz, 1H), 5.07 (d, J = 12.5 Hz, 1H), 4.08 (t,J = 5.1 Hz, 2H), 3.64 (s, 3H), 3.14 (h, J = 5.9 Hz, 2H), 2.25 – 2.11 (m, 2H),0.91 (t, J = 7.4 Hz, 3H).

[0159] 13C NMR (126 MHz, DMSO-d6) δ 167.63, 160.95, 160.75, 159.46, 159.20,158.92, 158.64, 158.37, 156.94, 153.37, 152.63, 148.34, 146.72, 145.19,137.69, 132.11, 130.94, 130.23, 129.42, 129.03, 128.49, 128.26, 119.70,117.74, 115.41, 106.93, 106.60, 105.09, 101.49, 99.75, 94.75, 78.44, 70.08, 66.98, 64.90, 64.77, 63.18, 55.65, 55.56, 50.79, 38.86, 38.74, 30.86, 8.00.

[0160] Compound 17:

[0161]

[0162] (1) Synthesis of 3,5-dimethoxybenzyl alcohol (N-tert-butoxycarbonyl) p-aminomethylbenzoate:

[0163] 3,5-Dimethoxybenzyl alcohol (500 mg, 3.0 mmol, 1.0 eq), N-tert-butoxycarbonyl-p-aminomethylbenzoic acid (754 mg, 3.0 mmol, 1.0 eq), and dicyclohexylcarbodiimide (DCC, 742 mg, 3.6 mmol, 1.2 eq) were weighed into a flask, and anhydrous DCM was added. The reaction was carried out for 16 h. The resulting white solid was removed by filtration, and the filtrate was purified by rapid silica gel chromatography (ethyl acetate: dichloromethane = 10: 90) to obtain a white solid, 3,5-dimethoxybenzyl alcohol (N-tert-butoxycarbonyl)-p-aminomethylbenzoate. The sample was characterized by 1H NMR (300 MHz, Bruker AVANCE III HD).

[0164] 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.90 (dt, J = 6.1, 2.9 Hz, 2H), 7.60 (dt, J = 6.0, 2.8 Hz, 2H), 6.60 (d, J = 3.2 Hz, 2H), 6.47 (d, J =4.1 Hz, 1H), 5.23 (s, 2H), 3.74 (t, J = 2.9 Hz, 6H), 1.48 (s, 9H).

[0165] (2) Synthesis of compound 17:

[0166] 400 mg (1.0 mmol, 1.0 eq) of 3,5-dimethoxybenzyl alcohol (N-tert-butoxycarbonyl) p-aminomethylbenzoate was weighed into a flask, dissolved in 5 mL of anhydrous ethyl acetate, and ethyl hydrochloride solution (4 mol / L, 2.5 mL, 10 eq) was added dropwise. The reaction was allowed to proceed for 16 h. A large amount of white solid precipitated in the system. The solid was filtered, washed with ethyl acetate, and dried to obtain a white powder, compound 17. The compound was characterized by 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0167] 1 H NMR (500 MHz, DMSO-d6) δ 8.66 (s, 2H), 8.04 – 7.98 (m, 1H), 7.69 –7.63 (m, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.47 (t, J = 2.3 Hz, 0H), 5.27 (s,1H), 4.10 (d, J = 5.7 Hz, 1H), 3.73 (s, 3H).

[0168] 13 C NMR (126 MHz, DMSO-d6) δ 165.70, 161.05, 140.09, 138.76, 129.89,129.85, 129.75, 106.24, 100.14, 66.63, 55.71, 42.18.

[0169] Compound 18:

[0170]

[0171] (1) Synthesis of 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde:

[0172] The method is the same as that for compound 15.

[0173] (2) Synthesis of compound 18:

[0174] 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzaldehyde was reduced to 3-(N-tert-Butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol, using the same method as compound 15. The crude product was directly used for the next reaction. 4-Methyl-7-aminocoumarin (500 mg, 2.85 mmol, 1.0 eq) and triphosgene (423 mg, 1.43 mmol, 0.5 eq) were weighed into a dehydrated single-necked round-bottom flask. After purging with an inert gas, the system was sealed, and 20 mL of anhydrous dichloromethane (DCM) was added. An anhydrous dichloromethane solution of N,N-diisopropylethylamine (DIPEA, 1224 mg, 8.56 mmol, 3.0 eq) was slowly added dropwise under ice bath conditions. After reacting at room temperature for 30 min, 3-(N-tert-butoxycarbonylaminoethoxy)-5-methoxybenzyl alcohol (1273 mg, 4.28 mmol, 1.5 eq) was added, and the reaction was allowed to proceed for 16 h at room temperature. Dichloromethane was removed by vacuum distillation, and the product was purified by rapid silica gel chromatography (ethyl acetate: n-hexane = 1:9). A white powder, the intermediate, was obtained. The intermediate product was dissolved in 5 mL of ethyl acetate, and ethyl acetate hydrochloride solution (4 mol / L, 7.1 mL, 10 eq) was added dropwise under ice bath, and the reaction was allowed to proceed for 16 h. A large amount of white solid precipitated in the system. The solid was filtered, washed with ethyl acetate, and dried to give 198 mg of white powder, compound 18. The compound was characterized by 1H NMR (500 MHz, Bruker AVANCE III HD) and 13C NMR (126 MHz, Bruker AVANCE III HD).

[0175] 1H NMR (500 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.36 (t, J = 5.8 Hz, 3H), 7.67 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.42 (dd, J = 8.7, 2.1Hz, 1H), 6.64 (d, J = 2.2 Hz, 2H), 6.51 (t, J = 2.3 Hz, 1H), 6.21 (d, J = 1.3Hz, 1H), 5.12 (s, 2H), 4.19 (t, J = 5.1 Hz, 2H), 3.74 (s, 3H), 3.64 – 3.60(m, 2H), 3.16 (p, J = 5.6 Hz, 2H), 2.36 (d, J = 1.3 Hz, 3H).

[0176] 13 C NMR (126 MHz, DMSO-d6) δ 170.82, 160.95, 160.51, 159.56, 154.27,153.66, 153.59, 143.17, 139.18, 126.49, 114.86, 114.76, 112.39, 106.90,106.88, 104.93, 101.00, 66.37, 64.85, 60.23, 55.80, 38.65, 21.24, 18.47,14.55.

[0177] Compound 19:

[0178]

[0179] Polyglutamic acid (133 mg, 20 eq), compound 15 (40 mg, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (12 mg, 1.2 eq), and N-hydroxysuccinimide (NHS) (9 mg, 1.5 eq) were weighed into a dehydrated single-necked round-bottom flask. The system was sealed after purging with an inert gas, and 5 mL of DMF was added. After reacting at room temperature for 16 h, precipitation with ice-cold diethyl ether resulted in the precipitation of a large amount of white crude product. The crude product was dialyzed against pure water for three days and then lyophilized. 102 mg of yellow powder, compound 19, was obtained. Characterization was performed using 1H NMR (500 MHz, Bruker AVANCEIII HD). Figure 1 As shown.

[0180] Example 2: Screening for the optimal ultrasonic structure

[0181] Weigh out model compounds 1-14 and dissolve them in 500 μL of DMSO to prepare a 100 mmol / L stock solution. Add 10 μL of the corresponding DMSO stock solution to 10 mL of phosphate-buffered saline (PBS) solution (pH 7.4). Filter through a 0.22 μm filter to prepare a 100 μM test sample solution. Transfer 600 μL of the sample solution to a 1.5 mL headspace cap vial. Place the vial in the center of the physiotherapy probe, apply ultrasound coupling gel around the vial, and perform ultrasound. The ultrasound parameters are 1.5 W / cm². 2 1 MHz, 50% Duty, 10 min. The ultrasonically treated samples were incubated at 37℃ for 2 h and then analyzed by High Performance Liquid Chromatography (HPLC). The HPLC test parameters are as follows, and are the same for compounds 1–14:

[0182] 1. Detection wavelength: 275 nm

[0183] 2. Test temperature: 40˚C

[0184] 3. Elution gradient: Water (0.1‰ TFA): Acetonitrile (90:10, v / v) to Water (0.1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.1‰ TFA): Acetonitrile (10:90, v / v) to Water (0.1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0185] 4. Flow rate: 1 mL / min

[0186] 5. Data collection time: 20 min

[0187] 6. Injection volume: 20 μL

[0188] The ultrasonic response of different synthesized compounds was tested using the above method. Their ability to release target molecules was confirmed by the release amount of para-aminobenzoic acid, and their sensitivity to ultrasound itself was assessed by the substrate reduction amount. Results are as follows: Figure 2 , 3 As shown. The high-performance liquid chromatography characterization results of compound 12 are as follows: Figure 4 As shown.

[0189] Example 3: Screening for optimal ultrasound conditions

[0190] Compound 12 was used to screen for optimal ultrasonic conditions because it has good release and response rates.

[0191] (a) Screening the optimal power

[0192] The DMSO stock solution of compound 12 was dissolved in PBS and filtered through a 0.22 μm filter to prepare a 100 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap, and another 600 μL was placed in a 1.5 mL headspace cap vial. The sample vials were placed in the center of the physiotherapy device probe, and ultrasonic coupling agent was applied around the vials before ultrasonication. The ultrasonic duty cycle was set to 50%, the ultrasonic frequency to 1 MHz, and the time to 10 min. Three replicates were prepared for each group. The ultrasonically treated samples were incubated at 37℃ for 2 h and then analyzed by HPLC. The specific test conditions and parameters were the same as in Example 2. The results are as follows. Figure 5 As shown, by Figure 5 It can be seen that 1.5 W / cm 2 ~2.5 W / cm 2 Both have good effects. Choose 1.5 W / cm. 2 For optimal power.

[0193] (ii) Screening of ultrasonic concentrations

[0194] DMSO stock solutions of compounds 8, 11, and 12 from (I) were dissolved in PBS and filtered through a 0.22 μm filter to prepare 100 μM sample solutions. These solutions were then diluted with PBS to prepare 80 μM, 60 μM, 40 μM, 20 μM, and 10 μM sample solutions. 600 μL of each sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy device probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency to 1 MHz, and the treatment time to 10 min. Three replicates were prepared for each group. The ultrasound-treated samples were incubated at 37℃ for 2 h and then analyzed by HPLC. Specific test conditions and parameters were the same as in Example 2. The results are as follows: Figure 6 As shown, by Figure 6 It is evident that a higher yield is achieved around 20 μM.

[0195] (III) Screening ultrasound time

[0196] The DMSO stock solution of compound 12, which showed the highest response rate in (I), was dissolved in PBS and filtered through a 0.22 μm filter to prepare a 100 μM sample solution. 600 μL of this sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound administration. The ultrasound duty cycle was set to 50%, the ultrasound frequency to 1 MHz, and the power to 1.5 W / cm². 2 To investigate the release of target molecules under different sonication times, three replicates were set up for each group. The samples treated with ultrasound were incubated at 37℃ for 2 h and then analyzed by HPLC. Specific test conditions and parameters were the same as above. The results are as follows: Figures 7-8 As shown, the results indicate that compound 12 exhibits a rapid initial release and subsequent slowdown in both the release rate and reduction rate of the target product, with the inflection point occurring at approximately 8-10 minutes.

[0197] Example 4: Investigating the reducing power of different model compounds

[0198] Compounds 15, 16, 17, 18, and 19 were selected to investigate the ability of 3,5-diphenyl ether structures to protect aliphatic amines, aliphatic alcohols, carboxylic acids, and aromatic amines, respectively, and to release them via sonication. For solubility considerations, compounds 15, 16, and 18 were modified from compound 12 by changing the structure of 3,5-dimethoxybenzyl alcohol to 3-(2-aminoethoxy)-5-methoxybenzyl alcohol, utilizing the exposed primary amine to provide water solubility.

[0199]

[0200] Fatty amine model:

[0201] A certain amount of model compound 15 was weighed and dissolved in 500 μL DMSO to prepare a 100 mmol / L stock solution. The stock solution was dissolved in PBS, filtered through a 0.22 μm filter, and diluted with PBS to prepare an 80 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency was set to 1 MHz, and three replicates were prepared for each group. After ultrasound treatment, the samples were incubated at 37℃ for 2 h and then diluted with 600 μL acetonitrile for HPLC analysis. Specific test conditions and parameters are as follows:

[0202] 1. Detection wavelength: 254nm

[0203] 2. Test temperature: 40˚C

[0204] 3. Elution gradient: Water (1‰ TFA): Acetonitrile (90:10, v / v) to Water (1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (1‰ TFA): Acetonitrile (10:90, v / v) to Water (1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0205] 4. Flow rate: 1 mL / min

[0206] 5. Data collection time: 20 min

[0207] 6. Injection volume: 20 μL

[0208] The yield results as a function of ultrasound time are as follows: Figure 9 As shown, compound 15 had the highest yield of approximately 13% at 8 min.

[0209] Fatty alcohol model:

[0210] A certain amount of model compound 16 was weighed and dissolved in 500 μL DMSO to prepare a 100 mmol / L stock solution. The stock solution was dissolved in PBS, filtered through a 0.22 μm filter, and diluted with PBS to prepare an 80 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency was set to 1 MHz, and three replicates were prepared for each group. After ultrasound treatment, the samples were incubated at 37℃ for 2 h and then diluted with 600 μL DMF for HPLC analysis. Specific test conditions and parameters are as follows:

[0211] 1. Detection wavelength: 254nm

[0212] 2. Test temperature: 40˚C

[0213] 3. Elution gradient: Water (1‰ TFA): Acetonitrile (90:10, v / v) to Water (1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (1‰ TFA): Acetonitrile (10:90, v / v) to Water (1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0214] 4. Flow rate: 1 mL / min

[0215] 5. Data collection time: 20 min

[0216] 6. Injection volume: 20 μL

[0217] The yield results as a function of ultrasound time are as follows: Figure 10 As shown: Compound 16 had the highest yield of approximately 12.5% ​​at 10 min.

[0218] Carboxylic acid model:

[0219] A certain amount of model compound 17 was weighed and dissolved in 500 μL DMSO to prepare a 100 mmol / L stock solution. The stock solution was dissolved in PBS, filtered through a 0.22 μm filter, and diluted with PBS to prepare an 80 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency was set to 1 MHz, and three replicates were prepared for each group. After ultrasound treatment, the samples were incubated at 37℃ for 2 h and then diluted with 600 μL methanol for HPLC analysis. Specific test conditions and parameters are as follows:

[0220] 1. Detection wavelength: 225 nm

[0221] 2. Test temperature: 40˚C

[0222] 3. Elution gradient: Water (1‰ TFA): Methanol (90:10, v / v) for 5 min; Water (1‰ TFA): Methanol (90:10, v / v) to Water (1‰ TFA): Methanol (10:90, v / v) for 10 min; Water (1‰ TFA): Methanol (10:90, v / v) to Water (1‰% TFA): Methanol (90:10, v / v) for 5 min; Water (1‰ TFA): Methanol (90:10, v / v) for 5 min.

[0223] 4. Flow rate: 1 mL / min

[0224] 5. Data collection time: 25 min

[0225] 6. Injection volume: 20 μL

[0226] The yield results as a function of ultrasound time are as follows: Figure 11 As shown: Compound 17 had the highest yield of approximately 20% at 10 min.

[0227] Aromatic amine model:

[0228] A certain amount of model compound 18 was weighed and dissolved in 500 μL DMSO to prepare a 100 mmol / L stock solution. The stock solution was dissolved in PBS, filtered through a 0.22 μm filter, and diluted with PBS to prepare a 100 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency was set to 1 MHz, and three replicates were prepared for each group. After ultrasound treatment, the samples were incubated at 37℃ for 2 h and then diluted with 600 μL acetonitrile for detection using a fluorescence spectrophotometer. Specific test conditions and parameters are as follows:

[0229] 1. Excitation wavelength: 363 nm.

[0230] 2. Test temperature: 25˚C.

[0231] 3. Excitation slit: 5 nm; Emission slit: 5 nm; Gain: 1; PMT voltage: 550V.

[0232] 4. Maximum excitation wavelength: 442 nm.

[0233] The results are as follows Figure 12 As shown, compound 18 has a maximum yield of about 12% after sonication for 10 min.

[0234] Drug-loaded polymer model:

[0235] A certain amount of model compound 19 was dissolved in PBS to prepare a 0.5 g / mL solution. The solution was filtered through a 0.22 μm filter, and 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy device probe, and ultrasonic coupling agent was applied around the vial before ultrasonication. The ultrasonic duty cycle was set to 50%, and the ultrasonic frequency was set to 1 MHz. Three replicates were prepared for each group. After ultrasonic irradiation treatment, the samples were incubated at 37℃ for 2 h and then diluted with 600 μL of acetonitrile for detection using high-performance liquid chromatography (HPLC). Specific test conditions and parameters are as follows:

[0236] 1. Detection wavelength: 225 nm

[0237] 2. Test temperature: 40˚C

[0238] 3. Elution gradient: Water (1‰ TFA): Acetonitrile (90:10, v / v) to Water (1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (1‰ TFA): Acetonitrile (10:90, v / v) to Water (1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0239] 4. Flow rate: 1 mL / min

[0240] 5. Data collection time: 20 min

[0241] 6. Injection volume: 20 μL

[0242] The results are as follows Figure 13 As shown, compound 19 has a yield of 6% after 10 min of sonication and can exhibit an ultrasonic response.

[0243] Example 5: Verification of the Free Radical Mechanism

[0244] Compound 12 was selected as a model structure to verify the mechanism of ultrasonic response of 3,5-dimethoxybenzyl alcohol structures.

[0245] (a) Isopropanol capture experiment

[0246] Isopropanol, a hydroxyl radical scavenger, was used to quench hydroxyl radicals generated by ultrasound. This verified that the ultrasonic response of the 3,5-dimethoxybenzyl alcohol structure is mediated via hydroxyl radicals. A stock solution of model compound 12 was dissolved in PBS and filtered through a 0.22 μm filter to prepare a 100 μM sample solution. Different amounts of isopropanol were added to obtain sample solutions with isopropanol concentrations of 0‰, 1‰, 3‰, and 5‰. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency to 1 MHz, and the treatment time to 10 min. Three replicates were prepared for each group. The ultrasound-treated samples were incubated at 37℃ for 2 h and then analyzed by HPLC. Specific test conditions and parameters are as follows:

[0247] 1. Detection wavelength: 275 nm

[0248] 2. Test temperature: 40˚C

[0249] 3. Elution gradient: Water (0.1‰ TFA): Acetonitrile (90:10, v / v) to Water (0.1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.1‰ TFA): Acetonitrile (10:90, v / v) to Water (0.1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0250] 4. Flow rate: 1 mL / min

[0251] 5. Data collection time: 20 min

[0252] 6. Injection volume: 20 μL

[0253] The results are as follows Figure 14 As shown, the release of compound 12 was gradually and completely suppressed as the isopropanol equivalent increased, indicating that the ultrasonic response was suppressed as the hydroxyl radicals were suppressed.

[0254] (ii) Fenton reaction

[0255] The use of hydroxyl radicals generated by Fenton's reagent further verified that the ultrasonic response of the 3,5-dimethoxybenzyl alcohol structure is caused by hydroxyl radicals. A certain amount of stock solution of model compound 12 (denoted as 2Ome-ABA) was dissolved in PBS and diluted with PBS to prepare a 20 μM sample solution. Different amounts of ferrous sulfate, disodium ethylenediaminetetraacetate, and hydrogen peroxide were added to the solution, with the molar ratio of the three fixed at 1:1:10. According to the ratio of hydrogen peroxide to substrate compound 12, the samples were divided into four groups: 0.2 eq, 1 eq, 2 eq, and 5 eq, denoted as groups 3-6. A group containing 1 eq of ferrous sulfate and disodium ethylenediaminetetraacetate without hydrogen peroxide was designated as group 1, and a group containing 1 eq of hydrogen peroxide without ferrous sulfate or disodium ethylenediaminetetraacetate was designated as control group 2. The samples were filtered through a 0.22 μm filter to obtain test samples of different groups. 600 μL of each sample was placed in a headspace vial and incubated at 37°C for 24 h. Three replicates were set up for each group. The incubated samples were analyzed by HPLC. The specific test conditions and parameters are as follows:

[0256] 1. Detection wavelength: 275 nm

[0257] 2. Test temperature: 40˚C

[0258] 3. Elution gradient: Water (0.1‰ TFA): Acetonitrile (90:10, v / v) to Water (0.1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.1‰ TFA): Acetonitrile (10:90, v / v) to Water (0.1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0259] 4. Flow rate: 1 mL / min

[0260] 5. Data collection time: 20 min

[0261] 6. Injection volume: 20 μL

[0262] The results are as follows Figure 15 As shown, the target product was gradually released as the equivalent of Fenton's reagent increased, while the control group did not release any product, indicating that hydroxyl radicals can trigger a response.

[0263] (III) Ultrasonic Intermediate Capture

[0264] An attempt was made to capture the intermediate product of hydroxyl radical addition to the aromatic ring. Compound 15 was used in the experiment. A certain amount of stock solution of model compound 15 was dissolved in PBS, filtered through a 0.22 μm filter to prepare a 100 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy instrument probe, and ultrasonic coupling agent was applied around the vial before ultrasonication. The ultrasonic duty cycle was set to 50%, the ultrasonic frequency was set to 1 MHz, and the time was 10 min. The ultrasonically treated sample was incubated at 37℃ for 0 h, 1 h, and 2 h, respectively, and then analyzed by HPLC. The specific test conditions and parameters are as follows:

[0265] 1. Detection wavelength: 254 nm

[0266] 2. Test temperature: 40˚C

[0267] 3. Elution gradient: Water (1‰ TFA): Acetonitrile (90:10, v / v) to Water (1‰ TFA): Acetonitrile (10:90, v / v), 10 min; Water (1‰ TFA): Acetonitrile (10:90, v / v) to Water (1‰% TFA): Acetonitrile (90:10, v / v), 5 min; Water (1‰ TFA): Acetonitrile (90:10, v / v), 5 min.

[0268] 4. Flow rate: 1 mL / min

[0269] 5. Data collection time: 20 min

[0270] 6. Injection volume: 20 μL

[0271] The results are as follows Figure 16 As shown, with increasing incubation time, the peak of the intermediate gradually disappears, while the target product gradually increases.

[0272] Further testing was performed using high-resolution mass spectrometry. A certain amount of stock solution of model compound 15 was dissolved in PBS, filtered through a 0.22 μm filter to prepare a 100 μM sample solution. 600 μL of the sample solution was placed in a 1.5 mL headspace cap vial. The vial was placed in the center of the physiotherapy device probe, and ultrasound coupling agent was applied around the vial before ultrasound treatment. The ultrasound duty cycle was set to 50%, the ultrasound frequency to 1 MHz, and the treatment time to 10 min. The ultrasound-treated sample was characterized by high-resolution mass spectrometry. The results are as follows: Figure 17 As shown, the intermediate resulting from the addition of hydroxyl radicals to the benzene ring was captured, confirming that the mechanism is indeed a hydroxyl radical process.

[0273] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hydroxyl radical-based ultrasonically responsive conjugate having the structure shown in Formula I-1, Formula I-2, Formula I-3, Formula I-5, Formula I-6, Formula I-7 or Formula I-8; 。 2. A hydroxyl radical-based ultrasonic-responsive polymeric drug delivery system, characterized in that, Including polymeric compounds and hydroxyl radical-based ultrasonically responsive conjugates supported on the structures shown in Formulas I-1 to I-8 of the polymeric compounds; 。 3. The ultrasonically responsive polymeric drug delivery system based on hydroxyl radicals according to claim 2, characterized in that, The polymer compound is polyglutamic acid.

4. The application of ultrasound-responsive compounds in the preparation of ultrasound-responsive antitumor drugs, characterized in that, The ultrasonically responsive compound is a hydroxyl radical-based ultrasonically responsive conjugate with the structure shown in Formula I-5 or Formula I-6 of claim 1, or is a hydroxyl radical-based ultrasonically responsive polymeric drug delivery system as described in any one of claims 2 to 3. The hydroxyl radical-based ultrasonic-responsive conjugate in the ultrasonic-responsive polymer drug delivery system is a hydroxyl radical-based ultrasonic-responsive conjugate with the structure shown in Formula I-5 or Formula I-6.

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