Use of conjugates having ultrasound radiation responsive groups

Abstract

The invention discloses application of a conjugate with an ultrasonic radiation response group, and belongs to the technical field of medicinal chemistry. The conjugate has a structure as shown in a formula I: PG-L-D, and is applied to preparation of ultrasonic responsive drugs or imaging agents. The embodiment of the invention can be used for preparing a medicine or an imaging agent with the general formula based on the chemical characteristics of a specific ultrasonic response group PG. PG is an ultrasonic radiation response group; l is a linking group; d is a residue of a masked active molecule. According to the embodiment of the invention, hydrogen free radicals generated by an ultrasonic cavitation effect are directly utilized to induce free radical cracking reduction of PG groups, so that chemical bonds are broken, and masked active molecules are released. According to the scheme, a specific PG group is introduced, dependence on a complex biological endogenous reducing agent is avoided, the advantages of deep tissue penetration, high biological safety and non-invasiveness of ultrasonic waves are fully utilized, and a new means is provided for realizing space-time controllable release of drugs in focus parts such as tumors and inflammations.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and specifically relates to the use of conjugates having ultrasonic radiation responsive groups. Background Technology

[0002] Highly active therapeutic drugs (such as chemotherapy drugs, anti-inflammatory drugs, and thrombolytics) are crucial in clinical treatment, and systemic administration is a common route of delivery. However, systemic administration lacks specificity in targeting lesions, leading to insufficient drug concentrations at the lesion site and easily causing severe systemic toxicity. Although toxicity can be reduced through prodrug modification, existing prodrugs mostly rely on endogenous enzymatic or hydrolytic processes, resulting in insufficient targeting. Therefore, utilizing exogenous stimuli to achieve spatiotemporally controlled drug release at the lesion site has become a research hotspot.

[0003] Existing external field activation methods mainly involve light and radiation activation. While light activation is controllable, it has shallow tissue penetration (<1 cm) and phototoxicity, making it difficult to apply to deep lesions. Radiation activation, although highly penetrating, is accompanied by severe ionizing radiation damage and systemic toxicity, and the equipment is expensive.

[0004] In contrast, ultrasound possesses both excellent tissue penetration (>10 cm) and high biocompatibility. It utilizes the extreme physical environment (transient high temperature, high pressure, and highly reactive free radicals, etc.) generated by cavitation to activate drugs, showing broad application prospects. However, most existing ultrasound response systems rely on acoustic sensitizers as mediators and consume endogenous biological reducing agents (such as glutathione). Due to the heterogeneity of the biological microenvironment, their activation efficiency is unstable. Furthermore, to achieve precision medicine, clinicians need a probe that can sensitively respond to the ultrasound cavitation environment for real-time monitoring or imaging of the treatment process. Therefore, developing an ultrasound response platform that does not rely on the endogenous biological environment and is versatile for drug delivery and imaging detection is one of the urgent technical problems to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, one objective of this invention is to provide an application of a conjugate with an ultrasonic radiation-responsive group. This invention can selectively remove the responsive group in the conjugate molecule through ultrasonic radiation, releasing the masked active molecule, and has broad application prospects.

[0006] One technical solution adopted in this invention is to provide the use of the conjugate in the preparation of ultrasound-responsive drugs or imaging agents, wherein the conjugate has the structure of Formula I: PG-LD Formula I; PG is an ultrasonic radiation-responsive group with a structure of formula II, wherein R is a C1 to C5 alkyl group. Indicates the connection site with L; Formula II; L represents a carbonyl group (-C=O-) or a carbon single bond, and D represents a residue of the active molecule to be masked that has lost a hydrogen (H) or hydroxyl (OH) group. The active molecule contains at least one of amino, hydroxyl, thiol, and carboxyl groups. Under ultrasonic radiation, the drug or imaging agent breaks bonds and releases the active molecule DH or D-OH.

[0007] In embodiments of the present invention, the conjugate has the general formula PG-LD (Formula I), where PG is an ultrasonic radiation-responsive group. The ultrasonic radiation-responsive group PG is the ultrasonically activatable group of the present invention, and is the structural group shown in Formula II; when water is the main medium, the hydrogen free radicals generated by ultrasonic cavitation can be used to achieve selective deprotection of the active molecule.

[0008] In Formula II, R is a C1 to C5 alkyl group; preferably, R is methyl (CH3) or ethyl (CH2CH3). In a preferred embodiment of the invention, R in PG shown in Formula II is selected from methyl.

[0009] In Formula I, L is a linking group; preferably, L is a carbonyl group.

[0010] The D is the active molecule to be masked that has lost a -H or -OH residue; the conjugate is broken under ultrasonic radiation, releasing the active molecule DH (when D is a residue that has lost a -H) or D-OH (when D is a residue that has lost a -OH).

[0011] In Formula I, D represents a residue of the active molecule to be masked. The active molecule contains at least one group, including but not limited to a therapeutic agent or imaging agent, such as an amino group (NH₂), a hydroxyl group (OH), a mercapto group (SH), and a carboxyl group (COOH). Preferably, the active molecule comprises a benzene ring structure, such as a benzene ring or a benzo[a]heterocyclic ring, and D is a residue formed by linking an amino or carboxyl group in the active molecule to L; more preferably, it is linked via an amino group.

[0012] In a specific embodiment of the present invention, the conjugate has any of the following structures: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5; Formula I-6; Formula I-7; Formula I-8; Formula I-9.

[0013] Another technical solution adopted in this embodiment of the invention is: providing a method for preparing an ultrasound-responsive formulation, using a conjugate and a pharmaceutically acceptable carrier or excipient; the formulation is in the form of a liquid injection or a lyophilized powder; the conjugate has the structure of Formula I: PG-LD Formula I; PG is an ultrasonic radiation-responsive group with a structure of formula II, wherein R is an alkyl group from C1 to C5; Formula II; L is a carbonyl group or a carbon single bond, and D is a residue of the active molecule to be masked that has lost a hydrogen or hydroxyl group. The active molecule includes at least one of amino, hydroxyl, thiol, and carboxyl groups.

[0014] In embodiments of the present invention, the conjugate may be commercially available or prepared by conventional methods. Furthermore, the formulation is prepared using methods well-known to those skilled in the art.

[0015] In a preferred embodiment, the formulation is a pharmaceutical composition comprising a conjugate, and is of the type of liquid, solid, or semi-solid formulation. More preferably, the pharmaceutical composition is an injection, a lyophilized powder for injection, a tablet, or a capsule. In a specific embodiment, the pharmaceutical composition is a formulation prepared by combining the conjugate with phosphate-buffered saline (PBS). Alternatively, it may be an imaging agent composition.

[0016] The present invention studies, starting from traditional chemical protecting groups, screened a large number of ultrasound-responsive, safe and universal protecting groups, which can be selectively removed under the action of ultrasound to achieve the release of active drugs or groups.

[0017] The core mechanism of this invention lies in the following: utilizing the specific response of the PG group to the hydrogen free radicals generated by ultrasonic cavitation, the pharmacophores of active molecules such as active drugs and imaging agents are coupled through a linker to block their activity and obtain an ultrasonically responsive conjugate; under ultrasonic radiation, the chemical bond between PG and the linker is broken (addition, cleavage, reduction or further decarboxylation), thereby releasing the active molecule DH or D-OH at a specific site.

[0018] In embodiments of the present invention, the ultrasonically responsive conjugate can achieve selective release / activation of active molecules under ultrasonic radiation in an aqueous medium free of acoustic sensitizers and reducing agents.

[0019] In embodiments of the present invention, the ultrasonic radiation is low-intensity ultrasound. The power of the ultrasonic radiation is selected from 0.8 to 2.8 W / cm². 2 Preferably, it is 1.0 to 2.4 W / cm². 2 More preferably, it is 1.6 to 2.0 W / cm².2 For example, 1.8 W / cm 2 1.9 W / cm 2 Or 2.0 W / cm 2 The duration of ultrasonic radiation is selected from 1 to 16 min, preferably 3 to 15 min, and more preferably 5 to 10 min; in addition, the duty cycle of the ultrasonic radiation is selected from 10% to 100%, such as 10%, 20%, 50% or 100%; the frequency of ultrasonic radiation is selected from 0.5 to 5 MHz, preferably 1 to 3 MHz, and more preferably 1 MHz.

[0020] In this invention, tert-butoxy groups were selected as the optimal PG, which has the advantages of high release efficiency, no toxic side effects, and good stability. After being coupled with active molecules (including but not limited to therapeutic agents / imaging agents) by a linker, their activity can be masked. The resulting conjugate can be re-cleaved under ultrasonic radiation to release the active molecules, thereby achieving selective treatment or imaging.

[0021] This invention, based on the chemical properties of a specific ultrasound-responsive group (PG), constructs a drug delivery system with the general formula PG-LD for pharmaceutical applications. Here, PG is an ultrasound-responsive group (selected from residues of tertiary alcohols, particularly tert-butoxy groups); L is a divalent linker; preferably, PG and L together form a tert-butoxycarbonyl structure; and D is a residue of the masked active molecule (therapeutic agent or imaging agent). This invention directly utilizes hydrogen free radicals generated by the ultrasonic cavitation effect to induce free radical cleavage and reduction of the PG group, leading to the breaking of chemical bonds and the release of the masked active molecule DH or D-OH. This technical solution, by introducing a specific PG group, does not rely on complex endogenous biological reducing agents and fully utilizes the advantages of deep tissue penetration, high biocompatibility, and non-invasiveness of ultrasound, providing a new means to achieve spatiotemporally controlled drug release at lesion sites (such as tumors, inflammatory sites, etc.). Attached Figure Description

[0022] Figure 1 This illustrates the effect of radiation time at different sound intensities on the normalized release rate of compound 14 in this embodiment of the invention. Figure 2 This describes the effect of radiated acoustic intensity on the normalized release rate of compound 14 in the embodiments of the present invention. Figure 3 This describes the effect of duty cycle on the normalized release rate of compound 14 in the embodiments of the present invention. Figure 4 This describes the effect of frequency at different sound intensities on the normalized release rate of compound 14 in this embodiment of the invention. Figure 5 This is the radiation emission curve of compound 5 in the embodiments of the present invention; Figure 6 The method used is high-performance liquid chromatography to test the radiation release of compound 5 in an air atmosphere; Figure 7 The method used was high-performance liquid chromatography to test the radiation release of compound 5 in a nitrogen atmosphere. Figure 8 This is the radiation emission curve of compound 10 in the embodiments of the present invention; Figure 9 The method used is high performance liquid chromatography to test the radiation release of compound 10 in an air atmosphere; Figure 10 The method used is high performance liquid chromatography to test the radiation release of compound 10 in a nitrogen atmosphere; Figure 11 This is the radiation emission curve of compound 12 in the embodiments of the present invention; Figure 12 The method used is high-performance liquid chromatography to test the radiation release of compound 12 in an air atmosphere; Figure 13 The method used is high-performance liquid chromatography to test the radiation release of compound 12 in a nitrogen atmosphere; Figure 14 This is the radiation emission curve of compound 13 in the embodiments of the present invention; Figure 15 The method used is high-performance liquid chromatography to test the radiation release of compound 13 in an air atmosphere; Figure 16 The method used was high-performance liquid chromatography to test the radiation release of compound 13 in a nitrogen atmosphere. Figure 17 This is the radiation emission curve of compound 16 in the embodiments of the present invention; Figure 18 The method used is high-performance liquid chromatography to test the radiation release of compound 16 in an air atmosphere; Figure 19 The method used was high-performance liquid chromatography to test the radiation release of compound 16 in a nitrogen atmosphere. Figure 20 Radiation emission curve of compound 17 in the embodiments of the present invention; Figure 21 The fluorescence spectrophotometric method was used to test the radiation release of compound 17 in a nitrogen atmosphere; Figure 22 This is a photograph showing the enhanced fluorescence of compound 17 due to ultrasonic triggering; Figure 23 The stability of compound 17 in the presence of common biological redox agents and ions was tested by fluorescence spectrophotometry. Figure 24 This is the curve showing the relationship between the release rate of compound 5 and the TEMPO equivalent in the embodiments of the present invention; Figure 25 The effect of Fenton's reagent on compound 5 was determined by high performance liquid chromatography. Figure 26 This describes the effect of isopropanol on the release rate of compound 5 in the embodiments of the present invention; Figure 27 It is the effect of oxygen on the ultrasonic radiation release of compound 5; Figure 28 The effect of CCl4 on the ultrasonic radiation release of compound 5 was determined by high performance liquid chromatography. Figure 29 This is a schematic diagram of the mechanism of bond breaking in the ultrasonic response of the conjugate in an embodiment of the present invention (taking R as a methyl group as an example). Figure 30 This is the LC-MS spectrum of 5-(tert-butyl)-2,2-dimethyl-3,4-dihydro-2H-pyrrole-1-oxide, the product of the addition of DMPO and tert-butyl radicals in the embodiments of the present invention. Figure 31 This is the LC-MS spectrum of 5-(tert-butyl)-2,2-dimethylpyrrolidine-1-ol, the product of the addition of DMPO and tert-butyl radicals in the embodiments of the present invention; Figure 32 This is the radiation emission curve of compound 14 in an embodiment of the present invention; Figure 33 The method used is high-performance liquid chromatography to test the radiation release of compound 14 in an air atmosphere; Figure 34 The method used was high-performance liquid chromatography to test the radiation release of compound 14 in a nitrogen atmosphere. Figure 35 This is the radiation emission curve of compound 15 from an embodiment of the present invention; Figure 36 The method used is high-performance liquid chromatography to test the radiation release of compound 15 in an air atmosphere; Figure 37 The method used was high-performance liquid chromatography to test the radiation release of compound 15 in a nitrogen atmosphere. Note: In the accompanying drawings of this application, all packing chromatograms involving High Performance Liquid Chromatography (HPLC) (specifically including...) Figure 6 , Figure 7 , Figure 9 , Figure 10 , Figure 12 , Figure 13 , Figure 15 , Figure 16 , Figure 18 , Figure 19 , Figure 33 , Figure 34 , Figure 36 , Figure 37The vertical fluctuations represent relative intensities (au). To avoid spectral overlap and facilitate visual comparison, the spectra of each sample have been offset along the vertical axis, so the vertical axis is omitted in the figure. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention.

[0024] To better understand the technical content of this application, specific embodiments are provided below to further illustrate this application. All raw materials involved are commercially available conventional products and are not subject to special restrictions. Specifically, compounds 1 to 5, 7, 8, 10, and 12 to 17 are obtained through synthesis, while the remaining compounds are obtained through purchase and have no special requirements.

[0025] .

[0026] Example 1: Synthesis of the compound

[0027] Compound 1: The synthesis reaction formula for compound 1.

[0028] Weigh p-aminobenzoic acid (2.0 g, 14.6 mmol, 1.00 eq) and place it in a 100 mL round-bottom flask equipped with a stirrer. Add 20 mL of methanol (MeOH), followed by dimethyl dicarbonate (2.05 g, 15.3 mmol, 1.05 eq). React at room temperature for 12 h. After the reaction is complete, monitor the reaction by thin-layer chromatography (TLC). Remove the solvent from the system by vacuum distillation. Grind the obtained solid three times with a solvent (ethyl acetate / n-hexane = 1 / 5), centrifuge, and remove the supernatant. Dry under vacuum to obtain 1.1 g of white solid powder, designated as compound 1. Analyze the compound using 1H NMR (1-H NMR) spectra. 1 ¹H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (… 13 C NMR (126 MHz, Bruker AVANCEIII HD) was used to characterize the successful synthesis of the compound.

[0029] 1H NMR (500 MHz, DMSO-d6) δ (ppm) 12.65 (s, 1H), 10.03 (s, 1H), 7.87(d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 3.69 (s, 3H), . 13 C NMR (126MHz, DMSO) δ (ppm) 167.18, 154.02, 143.58, 130.66, 124.52, 117.47, 52.06.

[0030] Compound 2: The synthesis reaction formula for compound 2.

[0031] Weigh p-aminobenzoic acid (2.0 g, 14.6 mmol, 1.00 eq) and place it in a 100 mL round-bottom flask equipped with a stir bar. Add 20 mL of methanol, followed by diethyl dicarbonate (2.48 g, 15.3 mmol, 1.05 eq). React at room temperature for 12 h. After the reaction is complete, monitor the reaction by thin-layer chromatography (TLC). Remove the solvent from the system by vacuum distillation. Grind the obtained solid three times with a solvent (ethyl acetate / n-hexane = 1 / 5), centrifuge, and remove the supernatant. Dry under vacuum to obtain 1.2 g of white solid powder, designated as compound 2. Analyze the compound using 1H NMR spectroscopy (1H NMR spectroscopy). 1 ¹H NMR, 500 MHz, Bruker AVANCE III HD, and carbon NMR (1H NMR spectroscopy). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0032] 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.63 (s, 1H), 9.98 (s, 1H), 7.85 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 4.13 (q, J = 7.0 Hz, 2H), 1.23(t, J = 6.9 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ (ppm) 167.02, 153.39, 143.50,130.48, 124.27, 117.29, 60.52, 14.46.

[0033] Compound 3: The synthesis reaction formula for compound 3.

[0034] The reaction was carried out under anhydrous and oxygen-free conditions. 500.0 mg (2.59 mmol, 1.00 eq) of tert-butyl p-aminobenzoate and 384.3 mg (1.3 mmol, 0.5 eq) of triphosgene were weighed into a 50 mL round-bottom flask. After purging with an inert gas, the system was sealed, and 20 mL of anhydrous dichloromethane (DCM) was added. The reaction apparatus was placed in an ice bath, and after the system temperature dropped to 0 °C, N,N-diisopropylethylamine (DIPEA) diluted 2-fold with DCM (1004.1 mg, 7.8 mmol, 3.00 eq) was slowly added. The reaction was allowed to proceed at room temperature for 10 min. Then, isopropanol (310.8 mg, 5.2 mmol, 2.00 eq) was added, and the reaction was allowed to proceed at room temperature for 12 h. The crude product was purified and separated by rapid silica gel chromatography (ethyl acetate / n-hexane = 1 / 4), yielding 538.2 mg of a white solid powder. The intermediate product was dissolved in 5 mL of DCM, and 5 mL of trifluoroacetic acid (TFA) was slowly added. The reaction was carried out at room temperature for 4 h. Then, 20 mL of ultrapure water was added to quench the reaction, resulting in the precipitation of a large amount of white solid. The solid was filtered, and the filter cake was washed three times with ultrapure water. The solid was collected, dried under vacuum, and yielded 279.2 mg of the product, designated as compound 3. ¹H NMR spectroscopy was used to analyze the product. 1 H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0035] 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.64 (s, 1H), 9.94 (s, 1H), 8.02 –7.73 (m, 2H), 7.67 – 7.38 (m, 2H), 4.91 (d, J = 6.3 Hz, 1H), 1.26 (d, J = 6.3Hz, 6H). 13 C NMR (126 MHz, DMSO) δ (ppm) 167.03, 152.98, 143.59, 130.46,124.20, 117.27, 67.93, 21.93.

[0036] Compound 4: The synthesis reaction formula for compound 4.

[0037] Anhydrous and oxygen-free operation. Weigh p-aminobenzoic acid (2.0 g, 14.6 mmol, 1.00 eq) and sodium hydroxide (NaOH, 583.2 mg, 14.6 mmol, 1.00 eq) into a 100 mL round-bottom flask equipped with a stir bar. Add 20 mL of anhydrous tetrahydrofuran (THF) to dissolve the substrate. Place the reaction apparatus in an ice bath. After the system temperature drops to 0 °C, slowly add isobutyl chloroformate (2.2 g, 16.1 mmol, 1.10 eq), and then react at room temperature for 12 h. Remove the solvent by vacuum distillation; wash the obtained solid three times with ultrapure water and dry under vacuum. Grind the obtained solid three times with a solvent (ethyl acetate / n-hexane = 1 / 5), centrifuge, and remove the supernatant. Dry under vacuum to obtain 1.7 g of white solid powder, designated as compound 4. Analyze the compound using 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0038] 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.64 (s, 1H), 10.00 (s, 1H), 7.92 –7.83 (m, 2H), 7.64 – 7.54 (m, 2H), 3.89 (d, J = 6.7 Hz, 2H), 1.92 (dq, J =13.4, 6.7 Hz, 1H), 0.93 (d, J = 6.7 Hz, 6H). 13 C NMR (126 MHz, DMSO) δ (ppm)167.02, 153.51, 143.50, 130.47, 124.28, 117.32, 70.38, 27.55, 18.89.

[0039] Compound 5 corresponds to compound formula I-1: The synthesis reaction formula for compound 5.

[0040] Weigh p-aminobenzoic acid (2.0 g, 14.6 mmol, 1.00 eq), add 20 mL of water and 40 mL of 1,4-dioxane to a 100 mL round-bottom flask equipped with a stir bar. Add sodium hydroxide (613.2 mg, 15.33 mmol, 1.05 eq), then add di-tert-butyl dicarbonate (4.8 g, 21.9 mmol, 1.50 eq), and react at room temperature for 24 h. Remove excess solvent by vacuum distillation, adjust the pH to approximately 3 with 1 M hydrochloric acid solution, then filter, and wash the filter cake with water. Collect the filter cake and dry it. Further grind and wash the solid with solvent (ethyl acetate / n-hexane = 1 / 5). Collect the solid and dry it to obtain 1.5 g of white solid powder, designated as compound 5. Analyze the compound using 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, 500 MHz, Bruker AVANCE IIIHD), and carbon NMR ( 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0041] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 12.61 (s, 1H), 9.73 (s, 1H), 7.89 –7.72 (m, 2H), 7.66 – 7.43 (m, 2H), 1.48 (s, 9H). 13 C NMR (126 MHz, DMSO) δ (ppm) 167.05, 152.56, 143.81, 130.39, 123.97, 117.23, 79.66, 28.06.

[0042] Compound 7: The synthesis reaction formula for compound 7.

[0043] The reaction was carried out under anhydrous and oxygen-free conditions. Tert-butyl p-aminobenzoate (966.2 mg, 5.00 mmol, 1.00 eq) and triphosgene (741.9 mg, 2.5 mmol, 0.5 eq) were weighed into a 50 mL round-bottom flask. After purging with an inert gas, the system was sealed, and 20 mL of anhydrous dichloromethane (DCM) was added. The reaction apparatus was placed in an ice bath, and after the system temperature dropped to 0 °C, N,N-diisopropylethylamine (DIPEA) diluted 2-fold with DCM (1.94 g, 15.00 mmol, 3.00 eq) was slowly added, and the reaction was carried out at room temperature for 10 min. Then, p-nitrobenzyl alcohol (1.53 g, 10 mmol, 2.00 eq) was added, and the reaction was carried out at room temperature for 12 h. The crude product was purified and separated by rapid silica gel chromatography (ethyl acetate / n-hexane) to obtain 2.2 g of white solid powder. 2.0 g of the intermediate product was dissolved in 12 mL of DCM, and 4 mL of trifluoroacetic acid (TFA) was slowly added. The reaction was carried out at room temperature for 4 h. A large amount of white solid precipitated out. The product was stirred, washed, and centrifuged with DCM, and the supernatant was removed three times until the supernatant was colorless and transparent. The solid was collected, dried under vacuum, and 1.1 g of white solid powder was obtained, designated as compound 7. A proton nuclear magnetic resonance (NMR) spectrum was used to analyze the product. 1 H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0044] 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 12.66 (s, 1H), 10.28 (s, 1H), 8.35 – 8.20 (m, 2H), 7.91 – 7.85 (m, 2H), 7.73 – 7.66 (m, 2H), 7.63 – 7.54 (m, 2H),5.32 (s, 2H). 13 C NMR (126 MHz, DMSO) δ (ppm) 166.98, 152.96, 147.15, 144.31,143.13, 130.53, 128.59, 124.62, 123.67, 117.44, 64.80.

[0045] Compound 8: The synthesis reaction formula for compound 8.

[0046] Anhydrous and oxygen-free operation. Weigh tert-butyl p-aminobenzoate (590.0 mg, 3.05 mmol, 1.00 eq) and triphosgene (453.1 mg, 1.53 mmol, 0.5 eq) into a 50 mL round-bottom flask. After purging with an inert gas, seal the system and add 20 mL of anhydrous dichloromethane (DCM). Place the reaction apparatus in an ice bath and, after the system temperature drops to 0 °C, slowly add N,N-diisopropylethylamine (DIPEA) diluted 2-fold with DCM (1.18 g, 9.15 mmol, 3.00 eq), and react at room temperature for 10 min. Then, add methyl p-4-hydroxymethylbenzoate (1.01 g, 6.1 mmol, 2.00 eq), and react at room temperature for 12 h. A large amount of white solid precipitated. After centrifugation to remove the supernatant, the product was washed with DCM by stirring, centrifugation, and removal of the supernatant. This process was repeated three times until the supernatant was colorless and transparent. The solid was collected and dried under vacuum to obtain 784.0 mg of white powder. 330.0 mg of the intermediate product was dissolved in 2.5 mL of DCM, and 10.5 mL of TFA was slowly added. The mixture was reacted at room temperature for 4 h. A large amount of white solid precipitated. The residual solvent was removed by vacuum distillation, and ultrapure water was added. The mixture was stirred and filtered. The filter cake was washed with ultrapure water until the pH of the filtrate was neutral. The solid was collected and dried under vacuum to obtain 195.0 mg of white powder, designated as compound 8. A proton nuclear magnetic resonance (NMR) spectrum was used to analyze the product. 1 H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, BrukerAVANCE III HD) was used to characterize the successful synthesis of the compound.

[0047] 1 H NMR (500 MHz, DMSO-d 6 ) δ (ppm) 12.66 (s, 1H), 10.23 (s, 1H), 8.04 –7.95 (m, 2H), 7.92 – 7.83 (m, 2H), 7.65 – 7.53 (m, 4H), 5.26 (s, 2H), 3.85(s, 3H). 13C NMR (126 MHz, DMSO) δ (ppm) 166.98, 165.99, 153.07, 143.21,141.90, 130.51, 129.38, 129.20, 127.93, 124.53, 117.40, 65.30, 52.19.

[0048] Compound 10 corresponds to compound formula I-2: The synthesis reaction formula for compound 10.

[0049] p-Aminobenzoic acid (2.5 g, 18.23 mmol, 1.00 eq) was suspended in thionyl chloride (25 mL) and heated to gentle reflux for 2 h. After the solution clarified, it was cooled and distilled under reduced pressure, azeotropically with dichloromethane (DCM) to remove excess thionyl chloride. The resulting acidic chloride was dissolved in 20 mL of DCM in an ice bath, followed by the addition of 8 mL of tert-butanol and 8 mL of dichloromethane, forming a solid white precipitate. Excess solvent was removed by reduced pressure distillation, and the collected solid was washed with ethyl acetate and neutralized with 10% sodium bicarbonate aqueous solution. The solid was collected, washed with ultrapure water, and dried to obtain 1.22 g of white solid powder, designated as compound 10. ¹H NMR spectroscopy was used to analyze the compound. 1 H NMR, 500 MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0050] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 6.73 (d, J = 8.4 Hz, 2H), 5.69 (d, J = 8.4 Hz, 2H), 5.02 (s, 2H), 0.64 (s, 9H). 13 C NMR (126 MHz, DMSO) δ (ppm)165.30, 153.09, 130.90, 117.64, 112.50, 78.85, 28.01.

[0051] Compound 12 corresponds to compound formula I-3: The synthesis reaction formula for compound 12.

[0052] Weigh L-phenylalanine (2.0 g, 12.1 mmol, 1.00 eq) into a 100 mL round-bottom flask equipped with a stirrer. Then add 30 mL of 1,4-dioxane and 10 mL of water, and stir at room temperature for 5 min. Next, add 15 mL of 1 M NaOH solution to fully dissolve the reactants. Add di-tert-butyl dicarbonate (3.2 g, 14.52 mmol, 1.20 eq) to the reaction apparatus in an ice bath. React for 10 min in an ice bath, then remove the ice bath, restore the reaction temperature to room temperature, and react for 4 h. Remove excess di-tert-butyl dicarbonate by extraction with ethyl acetate, then adjust the pH of the aqueous phase to 2–3 with citric acid, extract with ethyl acetate, wash the organic phase with saturated brine, and dry with anhydrous sodium sulfate. Remove excess solvent by vacuum distillation to obtain 2.54 g of white solid powder, designated as compound 12. Analyze the compound using 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, 500 MHz, Bruker AVANCE IIIHD), and carbon NMR ( 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0053] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 12.61 (s, 1H), 7.25 (td, J = 8.1,7.6, 5.5 Hz, 4H), 7.22 – 7.16 (m, 1H), 7.09 (d, J = 8.4 Hz, 1H), 4.09 (ddd, J = 10.3, 8.4, 4.5 Hz, 1H), 3.01 (dd, J = 13.8, 4.5 Hz, 1H), 2.82 (dd, J =13.8, 10.3 Hz, 1H), 1.31 (s, 9H).

[0054] 13 C NMR (126 MHz, DMSO) δ (ppm) 173.64, 155.46, 138.08, 129.23,129.11, 128.15, 126.32, 78.04, 55.18, 36.45, 28.17.

[0055] Compound 13 corresponds to compound formula I-4: The synthesis reaction formula for compound 13.

[0056] Weigh 2.0 g (13.23 mmol, 1.00 eq) of p-methylaminobenzoic acid and add 20 mL of water and 40 mL of 1,4-dioxane to a 100 mL round-bottom flask equipped with a stir bar. Add sodium hydroxide (555.6 mg, 13.89 mmol, 1.05 eq), then add di-tert-butyl dicarbonate (4.3 g, 19.85 mmol, 1.50 eq), and react at room temperature for 24 h. Remove excess solvent by vacuum distillation, adjust the pH to approximately 3 with 1 M hydrochloric acid solution, then filter, and wash the filter cake with water. Collect the filter cake and dry it. Further grind and wash the solid with a hexane solution containing 10% ethyl acetate. Collect the solid and dry it to obtain 1.3 g of white solid powder, designated as compound 13. Analyze the compound using 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, 500 MHz, Bruker AVANCEIII HD), and carbon NMR ( 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0057] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 12.87 (s, 1H), 7.96 – 7.84 (m, 2H), 7.49 – 7.34 (m, 2H), 3.22 (s, 3H), 1.41 (s, 9H). 13 C NMR (126 MHz, DMSO) δ (ppm) 166.86, 153.30, 147.35, 129.66, 126.79, 124.42, 80.27, 36.55, 27.86.

[0058] Compound 14 corresponds to compound formula I-5: The synthetic reaction formula for compound 14.

[0059] Weigh 2.0 g (16.24 mmol, 1.00 eq) of p-aminobenzyl alcohol into a 100 mL round-bottom flask, then add 40 mL of methanol to dissolve the substrate. Next, add 3.9 g (17.8 mmol, 1.10 eq) of ditert-butyl dicarbonate and react at room temperature for 12 h. Remove excess solvent by vacuum distillation, collect the solid, wash with n-hexane to remove excess ditert-butyl dicarbonate, and dry under vacuum to obtain 1.8 g of white solid powder, designated as compound 14. ¹H NMR spectroscopy was used to analyze the compound. 1 ¹H NMR, 500 MHz, Bruker AVANCE III HD, and carbon NMR (1H NMR spectroscopy). 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0060] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 9.27 (s, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 5.04 (t, J = 5.7 Hz, 1H), 4.40 (d, J = 5.6 Hz, 2H), 1.47 (s, 9H). 13 C NMR (126 MHz, DMSO) δ (ppm) 152.81, 138.13, 136.09,126.94, 117.84, 78.86, 62.64, 28.16.

[0061] Compound 15 corresponds to compound formula I-6: The synthetic reaction formula for compound 15.

[0062] Weigh p-aminobenzyl alcohol (123.0 mg, 1 mmol, 1.00 eq) into a 25 mL round-bottom flask equipped with a stirrer. Add 3 mL of methanol to dissolve the substrate, then add di-tert-amyl dicarbonate (258.0 mg, 1.05 mmol, 1.05 eq). React at room temperature for 12 h. The crude product was purified and separated by rapid silica gel chromatography (ethyl acetate / n-hexane = 1 / 4), yielding 112.3 mg of a pale yellow transparent solid, designated as compound 15. ¹H NMR spectroscopy was used to further analyze the compound. 1H NMR, 500 MHz, Bruker AVANCE IIIHD), and carbon NMR ( 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0063] 1 H NMR (500 MHz, DMSO-d6) δ (ppm) 9.25 (s, 1H), 7.39 (d, J = 8.1 Hz,2H), 7.26 – 7.15 (m, 2H), 5.04 (t, J = 5.6 Hz, 1H), 4.40 (d, J = 5.5 Hz, 2H), 1.79 (q, J = 7.5 Hz, 2H), 1.43 (s, 6H), 0.89 (t, J = 7.5 Hz, 3H). 13 C NMR (126MHz, DMSO) δ (ppm) 152.76, 138.16, 136.08, 126.94, 117.84, 81.09, 62.65, 33.01, 25.70, 8.19.

[0064] Compound 16 corresponds to compound formula I-8: The synthetic reaction formula for compound 16.

[0065] Lenalidomide (500.0 mg, 1.93 mmol, 1.00 eq) was weighed and added to a 50 mL round-bottom flask. 30 mL of 1,4-dioxane and 15 mL of ultrapure water were added to dissolve it. Then, di-tert-butyl dicarbonate (731.8 mg, 3.35 mmol, 1.73 eq) was added, and the mixture was reacted at room temperature for 48 h. The 1,4-dioxane was removed from the system by vacuum distillation, resulting in a solid precipitate. The precipitate was filtered. The filter cake was washed three times with ultrapure water and dried to obtain the crude product. The crude product was then purified and separated using a rapid silica gel column (dichloromethane / methanol = 20 / 1), yielding a white solid of 231.2 mg, designated as compound 16. The crude product was further purified using 1H NMR spectroscopy (1H NMR spectroscopy). 1 1H NMR, 500MHz, Bruker AVANCE III HD), and carbon NMR (1H NMR). 13 C NMR (126 MHz, Bruker AVANCE IIIHD) was used to characterize the successful synthesis of the compound.

[0066] 1H NMR (500 MHz, DMSO-d6) δ (ppm) 11.01 (s, 1H), 9.22 (s, 1H), 7.76 (dd, J = 7.1, 2.0 Hz, 1H), 7.53 7.37 (m, 2H), 5.12 (dd, J = 13.3, 5.1 Hz,1H), 4.48 – 4.28 (m, 2H), 2.91 (ddd, J = 17.3, 13.6, 5.4 Hz, 1H), 2.71 – 2.55(m, 1H), 2.35 (qd, J = 13.2, 4.4 Hz, 1H), 2.02 (dtd, J = 12.6, 5.2, 2.3 Hz, 1H), 1.48 (s, 9H). 13 C NMR (126 MHz, DMSO) δ (ppm) 172.91, 171.06, 167.88,152.95, 134.18, 133.00, 132.68, 128.64, 124.33, 118.15, 79.53, 51.59, 46.27,31.23, 28.07, 22.61.

[0067] Compound 17 corresponds to formula I-9: The synthetic reaction formula for compound 17.

[0068] 2-Amino-4-methoxycarbonylbenzoic acid (500.0 mg, 2.56 mmol, 1.00 eq) was weighed and added to a 50 mL round-bottom flask. 10 mL of 1,4-dioxane and 5 mL of ultrapure water were added, followed by di-tert-butyl dicarbonate (1117.0 mg, 5.12 mmol, 2.00 eq). Finally, triethylamine (520.0 mg, 5.12 mmol, 2.0 eq) was added to dissolve the solid. The reaction was allowed to proceed at room temperature for 24 h. 1,4-dioxane was removed from the system by vacuum distillation. The pH was adjusted to 3–4 with 1 M hydrochloric acid, resulting in the precipitation of a solid, which was then filtered. The filter cake was washed three times successively with ultrapure water and a 20% ethyl acetate solution in n-hexane, and dried to obtain 247.6 mg of a brown crystalline solid, designated as compound 17. ¹H NMR spectroscopy was used to analyze the compound. 1 H NMR, 500 MHz, Bruker AVANCE IIIHD), and carbon NMR ( 13 C NMR (126 MHz, Bruker AVANCE III HD) was used to characterize the successful synthesis of the compound.

[0069] 1 H NMR (500 MHz, DMSO- d 6) δ (ppm) 14.00 (s, 1H), 10.51 (s, 1H), 8.89(d, J = 1.7 Hz, 1H), 1.49 (s, 9H), 8.06 (d, J = 8.3 Hz, 1H), 7.60 (dd, J =8.2, 1.7 Hz, 1H), 3.88 (s, 3H). 13 C NMR (126 MHz, DMSO) δ (ppm) 168.96, 165.50,151.94, 141.48, 134.15, 131.66, 121.69, 118.89, 118.64, 80.59, 52.56, 27.89.

[0070] Example 2: Screening for ultrasonic protecting groups

[0071] Weigh out a certain amount of model compounds 1-11 and compound 14, and dissolve them in 300 μL DMSO to prepare a 100 mM stock solution. Dissolve an appropriate amount of the stock solution in phosphate buffer (10 mM, pH = 7.4), and filter through a 0.22 μm filter to prepare a 100 μM sample solution. Place 0.5 mL or 1 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap. Then, place the sample vial in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. The ultrasound radiation parameters are: 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The ultrasonically treated sample was immediately analyzed by High Performance Liquid Chromatography (HPLC). Specific test parameters are as follows: For compounds 1-11, 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL; For compound 14, 1. Detection wavelength: 235 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water:acetonitrile (90:10, v / v) to Water:acetonitrile (30:90, v / v), 20 min; Water:acetonitrile (30:90, v / v) to Water:acetonitrile (90:10, v / v), 5 min; Water:acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 30 min; 6. Injection volume: 20 μL.

[0072] Based on the above scheme, the ultrasonic radiation response capabilities of compounds 1-11 and 14 with different protecting group structures were tested, and their ability to release the corresponding active molecules was measured. The results are shown in Table 1. Among them, compounds 5, 10, and 14 showed higher release rates. Compounds 5, 10, and 14 all have a tert-butoxycarbonyl structure, and compounds with this structure have higher ultrasonic radiation response capabilities.

[0073] Table 1. Ultrasonic release rates of compounds 1-11 and 14

[0074] Example 3: Screening for optimal ultrasound conditions: Compound 14 was selected to screen for optimal ultrasound conditions. A certain amount of model compound 14 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. An appropriate amount of the stock solution was dissolved in phosphate buffer (10 mM, pH = 7.4) and filtered through a 0.22 μm filter to prepare a 100 μM sample solution. 0.5 mL or 1 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The vial was then placed in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. The sample treated with ultrasound was immediately analyzed by HPLC. The specific test parameters are as follows: 1. Detection wavelength: 235 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water:acetonitrile (90:10, v / v) to Water:acetonitrile (10:90, v / v), 10 min; Water:acetonitrile (10:90, v / v) to Water:acetonitrile (90:10, v / v), 5 min; Water:acetonitrile (90:10, v / v), 5 min; 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0075] Based on the above scheme, taking therapeutic ultrasound as an example, the ultrasonic release of compound 14 under different technical conditions was screened, and the specific classification is as follows: (a) Screening based on radiation time and sound intensity Taking therapeutic ultrasound as an example, the specific parameters include a duty cycle of 50%, an ultrasound frequency of 1 MHz, a radiation time of 2–16 min, a 1-min interval between each sample group, and at least 3 parallel samples per group for testing. The sound intensity is grouped from 0.8 to 2.8 W / cm². 2 The interval between each sample group was 0.4 W / cm. 2 At least three parallel samples were used for testing in each group. Results are as follows: Figure 1 , Figure 2 As shown, the normalized release rate of compound 14 gradually increased with increasing ultrasound time and acoustic intensity, and then showed a certain decreasing trend. Therefore, the final ultrasound time was selected as 10 min and the acoustic intensity as 2.0 W / cm². 2 As the optimal ultrasound conditions.

[0076] (ii) Duty cycle screening

[0077] Taking therapeutic ultrasound as an example, specifically including setting the ultrasound intensity to 2.0 W / cm². 2 The ultrasonic time was set to 10 minutes, and the ultrasonic frequency was set to 1 MHz. Duty cycles were grouped at 10%, 20%, 50%, and 100%, with at least three parallel samples per group for testing. Results are as follows: Figure 3 As shown, the higher the ultrasound duty cycle, the higher the release rate of compound 14.

[0078] (III) Frequency Filtering

[0079] Taking therapeutic ultrasound as an example, specifically including setting the ultrasound intensity to 1.0 W / cm². 2 1.5 W / cm 2 2.0 W / cm 2 The duty cycle was set to 50%, and the ultrasonic time was set to 10 minutes. Frequency groups were set at 1 MHz and 3 MHz, with at least three parallel samples in each group for testing. Results are as follows: Figure 4As shown, this demonstrates that the lower the ultrasonic frequency, the higher the release rate of compound 14.

[0080] Example 4: Verification of the ultrasonic release capacity of different model compounds in simulated normoxic or hypoxic (nitrogen) environments.

[0081] Compounds 5, 10, 12, 13, and 16 were selected to verify the ultrasonic release capabilities of tert-butyloxycarbonyl-protected aromatic amines, carboxylic acids, fatty amines, secondary amines, and marketed drug models, respectively.

[0082] (a) Aromatic amine model: Weigh a certain amount of model compound 5 and dissolve it in 300 μL DMSO to prepare a 100 mM stock solution. Take an appropriate amount of the stock solution and dissolve it in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4; simulating the hypoxic environment inside a tumor) under normal oxygen or after three freeze-thaw cycles to prepare a 100 μM sample solution. Take 1 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap, and then place the sample vial in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. The ultrasound intensity is set to 2.0 W / cm². 2 The duty cycle was set to 50%, and the ultrasonic time was set to 0–10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). The samples treated with ultrasonic radiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0083] like Figure 5 , Figure 6 and Figure 7 As shown, under ultrasonic irradiation, compound 5 undergoes responsive bond cleavage and releases p-aminobenzoic acid (…). p-Aminobenzoic acid (PABA). Quantitative analysis results showed that after 10 min of ultrasonic irradiation, the highest release rate was 9.86% in air; while in an oxygen-deficient (nitrogen) atmosphere, the release rate significantly increased, reaching 15.7%.

[0084] (ii) Carboxylic acid model: Weigh a certain amount of model compound 10 and dissolve it in 300 μL DMSO to prepare a 100 mM stock solution. Take an appropriate amount of the stock solution and dissolve it in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) under normal oxygen or after three freeze-thaw cycles to prepare a 100 μM sample solution. Filter the sample solution through a 0.22 μm filter to prepare a 100 μM sample solution. Place 0.5 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap. Then, place the sample vial in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. Set the ultrasound intensity to 2.0 W / cm². 2 The duty cycle was set to 50%, and the ultrasonic time was set to 0-10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). The samples treated with ultrasonic radiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0085] like Figure 8 , Figure 9 and Figure 10 As shown, under ultrasonic irradiation, compound 10 undergoes responsive bond cleavage and releases p-aminobenzoic acid (PABA). Quantitative analysis results indicate that after 10 min of ultrasonic irradiation, the highest release rate is 14.7% in air; while in an oxygen-deficient (nitrogen) atmosphere, the release rate significantly increases, reaching 44.3%.

[0086] (III) Fatty Amine Model: Weigh a certain amount of model compound 12 and dissolve it in 300 μL DMSO to prepare a 100 mM stock solution. Take an appropriate amount of the stock solution and dissolve it in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) under normal oxygen or after three freeze-thaw cycles to prepare a 100 μM sample solution. Filter the sample solution through a 0.22 μm filter to prepare a 100 μM sample solution. Place 0.5 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap. Then, place the sample vial in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. Set the ultrasound intensity to 2.0 W / cm². 2 The duty cycle was set to 50%, and the ultrasonic time was set to 0–10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). The samples treated with ultrasonic radiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 210 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.1% TFA): Methanol (90:10, v / v) to Water (0.1% TFA): Methanol (10:90, v / v), 12 min; Water (0.1% TFA): Methanol (10:90, v / v) to Water (0.1% TFA): Methanol (90:10, v / v), 3 min; Water (0.1% TFA): Methanol (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0087] like Figure 11 , Figure 12 and Figure 13 As shown, under ultrasonic irradiation, compound 12 undergoes responsive bond cleavage and releases L-Phenylalanine (Phe). Quantitative analysis results indicate that after 10 min of ultrasonic irradiation, the highest release rate is 10.6% in air; while in an oxygen-deficient (nitrogen) atmosphere, the release rate significantly increases, reaching 19.0%.

[0088] (iv) Secondary amine model: Weigh a certain amount of model compound 13 and dissolve it in 300 μL DMSO to prepare a 100 mM stock solution. Take an appropriate amount of the stock solution and dissolve it in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) under normal oxygen or after three freeze-thaw cycles to prepare a 100 μM sample solution. Filter the sample solution through a 0.22 μm filter to prepare a 100 μM sample solution. Place 1.0 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap. Then, place the sample vial in the center of the metal probe of the physiotherapy device coated with ultrasound coupling agent. Set the ultrasound intensity to 2.0 W / cm². 2 The duty cycle was set to 50%, and the ultrasonic time was set to 0–10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). The samples treated with ultrasonic radiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 300 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0089] like Figure 14 , Figure 15 and Figure 16 As shown, under ultrasonic irradiation, compound 13 undergoes responsive bond cleavage and releases p-methylaminobenzoic acid (m-methylaminobenzoic acid). p -Methylaminobenzoic acid, p -MABA). Quantitative analysis results showed that after 10 minutes of ultrasonic radiation, the highest release rate was 12.75% in an air atmosphere; while in an oxygen-deficient (nitrogen) atmosphere, the release rate significantly increased to 23.23%.

[0090] (v) Drug Models: Compound 16 was selected as the model drug for marketing. A certain amount of model compound 16 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. An appropriate amount of the stock solution was dissolved in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) under normal oxygen or after three freeze-thaw cycles to prepare a 100 μM sample solution. 0.5 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The sample vial was then placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound intensity was set to 2.0 W / cm². 2 The duty cycle was set to 50%, and the ultrasonic time was set to 0–10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). The samples treated with ultrasonic radiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 246 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water:acetonitrile (90:10, v / v) to Water:acetonitrile (10:90, v / v), 10 min; Water:acetonitrile (10:90, v / v) to Water:acetonitrile (90:10, v / v), 5 min; Water:acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0091] like Figure 17 , Figure 18 and Figure 19 As shown, under ultrasonic irradiation, compound 16 undergoes responsive bond cleavage and releases lenalidomide (LEN). Quantitative analysis results indicate that after 10 min of ultrasonic irradiation, the highest release rate is 10.9% in air; while in an oxygen-deficient (nitrogen) atmosphere, the release rate significantly increases to 22.2%.

[0092] (vi) Imaging agent model

[0093] (1). Ultrasonic release of compound 17

[0094] To verify the application of the conjugate in the preparation of ultrasound-responsive imaging agents, compound 17 was selected as the imaging agent model. Compound 17 is non-fluorescent under normal conditions; ultrasound-generated hydrogen radicals can release the caged fluorescent molecules, thus serving as an imaging agent for ultrasound action or hydrogen radical activation. A certain amount of model compound 17 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. An appropriate amount of the stock solution was dissolved in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) after three freeze-thaw cycles to remove oxygen, and filtered to prepare a 100 μM sample solution. 1.0 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The sample vial was then placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound intensity was set to 2.0 W / cm². 2 The frequency was set to 1 MHz, the duty cycle to 50%, and the ultrasonic time to 0–10 min (with a 2-min interval between each group, and at least 3 parallel samples per group for testing). After ultrasonic irradiation, the samples were diluted 10-fold with phosphate buffer (10 mM, pH = 7.4) and immediately analyzed using a fluorescence spectrophotometer. Specific test parameters are as follows: 1. Excitation wavelength λ ex = 360 nm; emission wavelength λ em = 465 nm; 2. Excitation slit 5 nm; emission slit 5 nm.

[0095] like Figure 20 , Figure 21 and Figure 22 As shown, under ultrasonic irradiation, compound 17 undergoes responsive bond cleavage and releases a fluorescently active substance. The fluorescence intensity of the aqueous solution of compound 17 significantly increases with increasing ultrasonic time. Quantitative analysis results indicate that after 10 min of ultrasonic irradiation in an oxygen-deficient (nitrogen) atmosphere, the release rate of compound 17 is 31.1%.

[0096] (2). Stability of compound 17 in the presence of common biological redox agents and ions

[0097] To verify that compound 17 remains stable in the presence of common biological redox agents and ions, a certain amount of model compound 17 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. An appropriate amount of the stock solution was dissolved in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) after three freeze-thaw cycles to prepare a 10 μM sample solution. Except for the blank group, the other groups were treated with potassium chloride, magnesium sulfate, calcium chloride, potassium nitrate, sodium nitrite, hydrogen peroxide, reduced glutathione, oxidized glutathione, cysteine, and sodium ascorbate, respectively, at a final concentration of 1 mM. After incubation at 25 ℃ for 2 h, the samples were detected using a fluorescence spectrophotometer. Specific test parameters are as follows: 1. Excitation wavelength λ ex = 360 nm; emission wavelength λ em = 465 nm; 2. Excitation slit 5 nm; emission slit 5 nm.

[0098] Compound 17 is a common biological redox agent and is almost not released into ions, exhibiting good stability, as shown in the results. Figure 23 As shown.

[0099] Example 5: Verification of the free radical release mechanism by ultrasound

[0100] (a) Using compound 5 as a model structure, the mechanism of tert-butyloxycarbonyl was verified.

[0101] The release of tert-butyloxycarbonyl groups was verified by quenching ultrasound-generated free radicals using the broad-spectrum free radical quencher 2,2,6,6-tetramethylpiperidine oxide (TEMPO) to verify whether the release process was mediated by ultrasound-generated free radicals. A certain amount of model compound 5 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. A certain amount of TEMPO was weighed and dissolved in a certain volume of DMSO to prepare 1 M and 10 M stock solutions, respectively. Appropriate amounts of the stock solutions of model compound 5 and TEMPO were dissolved in phosphate buffer (10 mM, pH = 7.4) and filtered through a 0.22 μm filter to prepare 100 μM sample solutions of compound 5, 10 equiv., and 100 equiv. TEMPO. 1 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample bottle with a plastic cap. The sample bottle was then placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound radiation parameters were set to 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The ultrasonically treated sample was immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0102] See results Figure 24 As the TEMPO equivalent increased, the release of compound 5 was completely suppressed, proving that its release was mediated by free radicals.

[0103] (II) Verification of the hydroxyl radical mechanism

[0104] The release of tert-butyloxycarbonyl groups was verified using hydroxyl radicals generated by Fenton's reagent to determine whether it was mediated by hydroxyl radicals generated by ultrasound. A certain amount of model compound 5 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. A certain amount of the stock solution was dissolved in phosphate buffer (10 mM, pH = 7.4) to prepare a 100 μM sample solution. Then, ferrous sulfate and disodium ethylenediaminetetraacetic acid (EDTA) were added in a 1:1 ratio to achieve a concentration of 1 mM (10 equiv.) of ferrous sulfate and disodium EDTA. The experiment was divided into two groups: one group received 10 mM hydrogen peroxide, and the other group did not. After incubation at room temperature overnight, the results were analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL; The results are as follows Figure 25 10 equiv. Hydroxyl radicals cannot release compound 5; indicating that the release process of tert-butyloxycarbonyl is not mediated by hydroxyl radicals.

[0105] (iii) Using isopropanol as a hydroxyl radical scavenger, we will verify whether the release of tert-butyloxycarbonyl is mediated by hydroxyl radicals generated by ultrasound.

[0106] Weigh a certain amount of model compound 5 and dissolve it in 300 μL DMSO to prepare a 100 mM stock solution. Dissolve a certain amount of the stock solution in phosphate buffer (10 mM, pH = 7.4) to prepare a 100 μM sample solution. The experiment was divided into two groups: one group with 100 equiv. isopropanol added, and the other group without. Take 1 mL of the sample solution into a 1.5 mL flat-bottomed glass sample vial with a plastic cap. Then, place the sample vial in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. Set the ultrasound radiation parameters to 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The samples treated with ultrasonic irradiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0107] Test results are as follows Figure 26 The addition of isopropanol does not stop the release of compound 5, indicating that the release of tert-butyloxycarbonyl is not mediated by hydroxyl radicals.

[0108] (iv) Verification of oxygen-related free radicals (Ultrasound mainly generates three types of highly active free radicals, including hydroxyl radicals, hydrogen radicals and superoxide anions).

[0109] The release of tert-butyloxycarbonyl groups was verified to be related to oxygen by deoxygenation. A certain amount of model compound 5 was weighed and dissolved in 300 μL DMSO to prepare a 100 mM stock solution. A certain amount of the stock solution was dissolved in nitrogen-saturated phosphate buffer solution after three freeze-thaw cycles to prepare a 100 μM sample solution. 1 mL of each sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The sample vial was then placed in the center of the metal probe of a physiotherapy device coated with an ultrasound coupling agent. The ultrasound radiation parameters were set to 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The samples treated with ultrasonic irradiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 275 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water (0.01% TFA): Acetonitrile (90:10, v / v) to Water (0.01% TFA): Acetonitrile (10:90, v / v), 10 min; Water (0.01% TFA): Acetonitrile (10:90, v / v) to Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min; Water (0.01% TFA): Acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0110] Test results are as follows Figure 27 The increased release rate of compound 5 in an oxygen-deficient (nitrogen) atmosphere indicates that the release of tert-butyloxycarbonyl is not mediated by oxygen-related free radicals.

[0111] (v) Verification of hydrogen radicals

[0112] To verify whether the release of tert-butyloxycarbonyl is mediated by hydrogen radicals, carbon tetrachloride was used as a hydrogen radical quencher to verify the mechanism. A certain amount of carbon tetrachloride was thoroughly mixed with phosphate buffer (10 mM, pH = 7.4) to obtain carbon tetrachloride-saturated phosphate buffer (10 mM, pH = 7.4). A certain amount of model compound 14 was weighed and dissolved in 300 μL LDMSO to prepare a 100 mM stock solution. An appropriate amount of the stock solution was dissolved in carbon tetrachloride-saturated phosphate buffer (10 mM, pH = 7.4) and filtered through a 0.22 μm filter to prepare a 100 μM sample solution; no carbon tetrachloride was added to the control group. 1 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample bottle with a plastic cap. Then, the sample bottle was placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound radiation parameters were set to 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The samples treated with ultrasonic irradiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 235 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water:acetonitrile (90:10, v / v) to Water:acetonitrile (10:90, v / v), 20 min; Water:acetonitrile (10:90, v / v) to Water:acetonitrile (90:10, v / v), 5 min; Water:acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 30 min; 6. Injection volume: 20 μL.

[0113] The results are as follows Figure 28 As shown, carbon tetrachloride does not inhibit the reduction of compound 14, but completely blocks its release, indicating that the release of tert-butyloxycarbonyl is likely mediated by hydrogen radicals.

[0114] (vi) Radical mechanism and verification of tert-butyloxycarbonyl response

[0115] This invention proposes an ultrasonic response bond breaking mechanism ( Figure 29 Ultrasound cleaves water molecules through cavitation, generating hydrogen radicals. These hydrogen radicals attack the carbon-oxygen double bond of the addition conjugate, forming a carbon radical reactive intermediate. This intermediate then undergoes β-cleavage, generating an unstable acidic intermediate, which spontaneously decarboxylates and releases the reactive molecule DH. When the reactive molecule loses its -H group through its amino, hydroxyl, or thiol group and connects to a linking group (such as tert-butyloxycarbonyl), the unstable intermediates formed by β-cleavage are carbamic acid, monocarbonate, or thiocarbonate, respectively. In this field, it is common knowledge that these unstable intermediates readily undergo spontaneous decarboxylation. Therefore, the mechanism described in this invention can be reasonably extended to cases involving hydroxyl and thiol groups.

[0116] Specifically, when the active molecule loses its -OH group and couples with a tert-butoxy group, the resulting tert-butyl ester structure is directly converted to D-OH via β-cleavage. Those skilled in the art will understand that the same chemical logic applies when R is an alkyl group of other definitions or when D is a residue of other active molecules.

[0117] Mechanism Validation: The intermediate free radical was captured using the free radical scavenger 5,5-dimethyl-1-pyrrolino-N-oxide (DMPO). A certain amount of compound 5 was dissolved in DMF to prepare a stock solution with a final concentration of 500 mM. An appropriate amount of the stock solution was mixed with pure water to prepare a 0.5 mM sample solution. DMPO with a final concentration of 1 M was added. The suspension was not filtered, and nitrogen was bubbled for 30 min to remove oxygen. 1 mL of the sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The sample vial was then placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound radiation parameters were set to 2.0 W / cm². 2 1 MHz, 50% Duty, 15 min. The ultrasonically treated samples were analyzed by liquid chromatography-mass spectrometry (LC-MS); results are as follows. Figure 30 , 31As shown, the tert-butyl radical was captured, indicating that the ultrasonic release of the tert-butyloxycarbonyl group indeed proceeds through hydrogen radical addition-induced β-cleavage. Based on this principle, during β-cleavage, when PG is a tert-butoxy group, it provides a more stable radical product, which is beneficial to the reaction thermodynamics, while a stable positive charge provides a more stable polarized intermediate state, which is beneficial to the reaction kinetics.

[0118] Since the tert-amyl group has a more stable positive charge center than the tert-butyl group, we selected compounds 14 and 15 for further comparative verification of the mechanism. A certain amount of model compounds 14 and 15 were weighed and dissolved in 300 μL DMSO to prepare 100 mM stock solutions. Appropriate amounts of each stock solution were dissolved in nitrogen-saturated phosphate buffer (10 mM, pH = 7.4) under normal oxygen or after three freeze-thaw cycles to prepare 100 μM sample solutions. 1 mL of each sample solution was placed in a 1.5 mL flat-bottomed glass sample vial with a plastic cap. The vial was then placed in the center of the metal probe of a physiotherapy device coated with ultrasound coupling agent. The ultrasound radiation parameters were: 2.0 W / cm². 2 The ultrasonic treatment was performed at 1 MHz, 50% Duty, with an ultrasonic time of 0–10 min (2 min intervals between each group, and at least 3 parallel samples per group). The samples treated with ultrasonic irradiation were immediately analyzed by HPLC. Specific test parameters are as follows: 1. Detection wavelength: 235 nm; 2. Test temperature: 40 ℃; 3. Elution gradient: Water:acetonitrile (90:10, v / v) to Water:acetonitrile (10:90, v / v), 10 min; Water:acetonitrile (10:90, v / v) to Water:acetonitrile (90:10, v / v), 5 min; Water:acetonitrile (90:10, v / v), 5 min. 4. Flow rate: 1 mL / min; 5. Data collection time: 20 min; 6. Injection volume: 20 μL.

[0119] Test results are as follows Figures 32 to 37 As shown, under ultrasonic irradiation, compounds 14 and 15 undergo responsive bond cleavage, releasing p-aminobenzyl alcohol (…). p -Aminobenzyl alcohol, p-ABOH). After 10 min of ultrasonic irradiation, the release rate of compound 14 was 13.39% in air and 38.33% in a nitrogen-deficient atmosphere. Correspondingly, the release rate of compound 15 was 17.27% in air and 37.22% in a nitrogen-deficient atmosphere. In air, the release rate of compound 15 was greater than that of compound 14, while in a nitrogen atmosphere, the release rates of the two were comparable, which is consistent with the ultrasonic response mechanism described above in this invention, further confirming the responsiveness of the structure described in this invention.

[0120] As can be seen from the above, the conjugates described in the embodiments of the present invention can utilize the hydrogen free radicals generated by ultrasonic cavitation to achieve selective deprotection of active molecules / groups. The embodiments of the present invention also describe the process and principle of hydrogen free radical-mediated reductive cleavage of tert-butyloxycarbonyl groups. Tert-butyloxycarbonyl groups can be selectively deprotected under the action of hydrogen free radicals generated by ultrasound, thereby releasing the protected drug molecules, imaging agents, etc. In the embodiments of the present invention, the active molecules protected by tert-butyloxycarbonyl groups can be selectively released at specific spatiotemporal locations in the organism under ultrasonic radiation, thereby achieving functions such as reducing systemic toxicity of drugs and precise imaging.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of the conjugate in the preparation of ultrasound-responsive drugs or imaging agents, characterized in that, The conjugate has the structure of Formula I: PG-LD Formula I; PG is an ultrasonic radiation-responsive group with a structure of formula II, wherein R is a C1 to C5 alkyl group. Indicates the connection site with L; Formula II; L is a carbonyl group or a carbon single bond, and D is a residue of the active molecule to be masked that has lost a hydrogen or hydroxyl group. The active molecule includes at least one of amino, hydroxyl, thiol, and carboxyl groups.

2. The use according to claim 1, characterized in that, R is methyl or ethyl.

3. The use according to claim 1, characterized in that, L stands for carbonyl group.

4. The use according to claim 1, characterized in that, The active molecule contains a benzene ring structure, and D is a residue formed by linking an amino or carboxyl group in the active molecule containing a benzene ring structure to L.

5. The use according to any one of claims 1 to 4, characterized in that, The conjugate has any of the following structures: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5; Formula I-6; Formula I-7; Formula I-8; Formula I-9.

6. The use according to any one of claims 1 to 4, characterized in that, The ultrasound-responsive drug or imaging agent is in the form of a liquid injection or a lyophilized powder.

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