A prodrug composition and a method of activating the prodrug composition with ultrasound

Abstract

The application relates to a prodrug composition and a method for activating the prodrug composition by ultrasound, relates to the field of medicinal chemistry, and solves the problems that the existing N-oxide prodrug activation process depends on the endogenous microenvironment of tumors, the activation efficiency and the stability of curative effect are poor, the existing invasive activation method lacks precise space-time control, normal tissue damage is easily caused, and the substrate application range of the existing N-oxide activation system is limited. The prodrug composition provided by the application comprises an N-oxide, an electron donor and a sonosensitizer; the prodrug composition is subjected to ultrasonic treatment, the N-oxide is reduced and deoxidized, is converted into a corresponding deoxidized product and an active form, and the activation of the prodrug composition is completed. The application can be applied to the preparation of tumor treatment drugs.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a prodrug composition and a method for activating the prodrug composition by ultrasound triggering. Background Technology

[0002] In the field of cancer treatment, solid tumors generally possess hypoxic microenvironments of varying degrees, and hypoxia is typically associated with increased tumor invasiveness, decreased sensitivity to radiotherapy and chemotherapy, and treatment tolerance. Therefore, hypoxia-activated prodrug strategies, which leverage the hypoxic microenvironment to achieve selective tumor activation, have gained increasing attention. These strategies typically involve converting bioactive drug structures into less active or temporarily inactivated prodrug forms, which are then bioreduced in the hypoxic tumor region and reverted to their active form, thereby improving tumor selectivity and reducing toxicity to normal tissues. Among these, N-oxides, due to their ability to reversibly shield amine or heteroaromatic nitrogen-containing centers through the N→O structure, have become one of the important structural types in hypoxia-activated prodrug systems.

[0003] Currently, the in vivo activation of existing N-oxide prodrugs mainly depends on the tumor's endogenous microenvironment, such as hypoxia, reductase expression, or abnormal redox states. However, solid tumors typically exhibit significant heterogeneity, with substantial differences in hypoxia and enzyme expression levels among different tumor types, patients, and even different regions within the same tumor. This results in N-oxide prodrugs not being fully, stably, and predictably activated within the tumor, leading to significant fluctuations in activation efficiency and hindering stable and predictable therapeutic effects, thus weakening efficacy and affecting reproducibility. For example, banoanthraquinone (AQ4N), as an N-oxide prodrug of AQ4, is highly dependent on a hypoxic-reduction environment. Only under extremely low oxygen conditions can its reduction rate effectively exceed the oxygen-mediated reoxidation process. When the local oxygen concentration slightly increases, some reduction intermediates are easily reoxidized, making it difficult for the active product AQ4 to accumulate sufficiently. Meanwhile, due to significant heterogeneity in the degree of hypoxia, distribution of reductase, and supply of reducing equivalent within the tumor, AQ4N typically exhibits slow activation rate, limited activation range, and insufficient exposure to effective drugs in vivo, resulting in poor stability of its therapeutic effect in vivo.

[0004] To improve the activation efficiency and local tumor response of N-oxide prodrugs, previous studies have attempted to introduce exogenous stimulation to induce reductive deoxygenation reactions in N-oxides. For example, ionizing radiation can be used to induce reductive deoxygenation reactions in N-oxides to achieve prodrug activation. This type of method typically involves applying ionizing radiation, such as X-rays, to an aqueous system, initiating a radiolysis process and generating hydrated electrons and other reactive species with strong reducing capabilities. This, in turn, promotes the reduction and transformation of the NO bond in the N-oxide structure, restoring it to the corresponding amine or active drug form, thereby achieving local tumor prodrug activation. However, this type of method usually requires ionizing radiation for prodrug activation, and its activation effect may be affected by factors such as local oxygen status, radiation dose distribution, and tissue conditions, thus limiting the stability and controllability of the prodrug activation process to some extent. Furthermore, the ability of existing activation methods to regulate the activation location and timing still needs improvement, especially in deep tumor environments. Achieving more regionally selective and spatiotemporally controllable prodrug activation remains a problem that needs further research in this field. Therefore, current technologies lack a non-invasive, deep-penetrating, spatiotemporally controllable, and broadly applicable reductive deoxygenation activation method. Furthermore, most existing N-oxide activation systems revolve around specific single prodrugs, and research on the applicability of extended π-conjugated N-oxide structures remains limited, significantly restricting the precise local activation of this type of N-oxide structure in tumors. Summary of the Invention

[0005] To address the problems of existing N-oxide prodrug activation processes relying on the tumor microenvironment, resulting in poor activation efficiency and therapeutic stability; existing invasive activation methods lacking precise spatiotemporal control and easily causing damage to normal tissues; and the limited substrate applicability of existing N-oxide activation systems, this invention proposes a prodrug composition and a method for ultrasound-triggered activation of the prodrug composition. The technical solution of this invention is as follows: A prodrug composition comprising an N-oxide, an electron donor, and a sound sensitizer; The N-oxide is selected from any one or at least two combinations of anthraquinone diamine N-oxides, nitrogen-containing aromatic heterocyclic N-oxides, and tertiary amine N-oxides; the electron donor is any one or at least two combinations of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADH), ascorbic acid, and glutathione; the sonication agent is any one or at least two combinations of riboflavin tetrabutyrate (TBR), riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD).

[0006] Furthermore, the molar ratio of the N-oxide, electron donor, and sound sensitizer is 1:(1-20):(0.5-10).

[0007] Further, the N-oxide is any one or at least a combination of two of the following: banoanthraquinone (AQ4N), quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide.

[0008] Furthermore, the prodrug composition also includes a pharmaceutically acceptable carrier / excipient and a buffer salt solution to ensure the dispersion and stability of the sonosensitive agent and the substrate in an aqueous environment.

[0009] Furthermore, the concentration of the N-oxide is 1~500 μM.

[0010] A method for ultrasound-triggered activation of the above-mentioned prodrug composition includes the following steps: A reaction solution containing N-oxide, a sonication agent, and an electron donor is prepared as a prodrug composition. The prodrug composition is then subjected to ultrasonic treatment. During this process, the sonication agent generates a reducing equivalent under ultrasonic excitation and mediates electron transfer or hydrogen atom transfer, thereby promoting the reduction and deoxygenation of N-oxide, converting it into the corresponding deoxygenation product and active form, thus completing the activation of the prodrug composition.

[0011] Furthermore, the ultrasonic processing frequency is 20 kHz to 10 MHz; the ultrasonic intensity is 0.1 to 5 W / cm². 2 The duty cycle is 1~100%; the ultrasonic treatment time is 1 min~60 min.

[0012] The above-mentioned method for activating the ultrasound-triggered prodrug composition is applied to the preparation of tumor therapeutic drugs.

[0013] Compared with existing technologies, this invention solves the problems of existing N-oxide prodrug activation processes relying on the tumor endogenous microenvironment, poor activation efficiency and efficacy stability, existing invasive activation methods lacking precise spatiotemporal control and easily causing damage to normal tissues, and the limited substrate applicability of existing N-oxide activation systems. Specifically, the beneficial effects are as follows: 1. This invention constructs an external activation system composed of ultrasound, TBR, and NADPH. These three components work synergistically under mild ultrasound conditions to promote the N–O bond breaking of AQ4N and its conversion into active AQ4, achieving efficient conversion to the active product AQ4. TBR acts as a redox relay, and NADPH acts as a reduction equivalent donor. Due to the advantages of ultrasound, such as deep penetration, focusability, adjustable parameters, and strong spatiotemporal control, in addition to mechanical and thermal effects, it can also generate special sonochemical effects through acoustic cavitation. The synergistic effect of these three components forms a high-energy transient microenvironment and induces highly reducing intermediates, thereby driving the reduction reaction. This externally controllable, non-invasive ultrasound triggering method can actively initiate the reductive deoxygenation activation of AQ4N at the target site. This transforms the activation of nitrogen oxides in existing technologies from being primarily controlled by the tumor's endogenous microenvironment into a local activation process that can be precisely controlled by external ultrasound.

[0014] 2. This invention constructs a non-invasive activation strategy using ultrasound as an exogenous trigger and TBR / NADPH as a reduction relay system. This strategy enables spatiotemporally controllable reduction and deoxygenation conversion of N-oxide prodrugs like AQ4N at tumor lesions. After specific ultrasound triggering, the active product AQ4 is efficiently generated, significantly enhancing the in vitro killing ability of hypoxic tumor cells and substantially reducing the IC50. 50 In this activation mode, TBR and ultrasound itself showed no significant non-specific cellular or in vivo toxicity, demonstrating good safety. It further drove the maturation and activation of dendritic cells, increasing CD4 levels in the tumor microenvironment. + CD8 + The invention enhances effector T cell infiltration levels and upregulates the secretion of immune factors such as TNF-α and IFN-γ, significantly inhibiting the growth of solid tumors in vivo while activating a systemic anti-tumor immune response. It combines the advantages of precise prodrug activation, highly efficient tumor suppression, and synergistic immunotherapy. Furthermore, the ultrasound-triggered AQ4N activation system constructed in this invention exhibits excellent tumor-selective activation capabilities, synergistic tumor suppression effects, and immune microenvironment regulation, demonstrating significant tumor suppression and immune activation effects in both the CT26 and MC38 tumor models.

[0015] 3. The ultrasound-responsive N-oxide prodrug composition reduction and activation system constructed in this invention has a clear electron transport pathway and good thermodynamic feasibility, effectively ensuring the activation efficiency and reaction stability of the prodrug composition. Furthermore, this invention is not only applicable to AQ4N, but can also effectively activate various N-oxide substrates with different structures, such as quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide, demonstrating excellent substrate compatibility and system universality. This overcomes the limitations of existing technologies that typically only construct activation systems for single prodrugs, providing a new technical pathway for precise release and controllable treatment of tumor prodrugs. Attached Figure Description

[0016] Figure 1 Bar chart comparing the AQ4 activation rates of Examples 1, 5, 6, 26 and Comparative Examples 1, 2, 4, 5 under different ultrasonic treatment times; Figure 2 The effect of different ultrasound parameters on the activation effect of ultrasound-triggered prodrug compositions; among which, Figure 2 (a) is a graph showing the detection results of AQ4 generation content under different ultrasonic intensity conditions in Examples 1, 7–9 and Comparative Example 4; Figure 2 (b) is a graph showing the detection results of AQ4 formation content under different ultrasonic frequency conditions in Examples 1, 10 and Comparative Example 4; Figure 2 (c) is a graph showing the detection results of AQ4 generation content under different ultrasonic duty cycles in Examples 1, 11, 12 and Comparative Example 4; Figure 2 (d) is a graph showing the detection results of AQ4 formation content under different ultrasonic time conditions in Examples 1–5 and Comparative Example 4; Figure 2 (e) is a graph showing the comparison results of AQ4 generation content between Example 1 and Comparative Examples 1, 3, 4, 6, and 7 under different system components and different triggering conditions; Figure 2 (f) is a representative LC-MS / MS extractive ion chromatogram and corresponding mass spectrum of the activated product of Example 1; Figure 3 This is the result of the synergistic effect of ultrasound and endogenous reductase; among which, Figure 3 (a) HPLC chromatograms of AQ4, AQ4N standard samples and samples prepared in Comparative Examples 8, 13 and 18; Figure 3 (b) A graph showing the quantitative statistical results of AQ4 generation in each group; Figure 4 Driven by ultrasound N -Substrate selection results for oxide deoxygenation reactions; among which... Figure 4 (a) is a schematic diagram of the reduction reaction of quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide as substrates in the system of the present invention; Figure 4 (b) and (c) are quantitative bar charts of reaction efficiency and load release rate for each substrate, respectively; Figure 5 The graph shows the results of the inhibition experiment; where, Figure 5 (a) is the HPLC elution spectrum of Example 1 and samples with different quenchers added; Figure 5 (b) A bar chart showing the quantitative inhibition analysis of AQ4 formation by different quenchers; such as Figure 5 (c) is a schematic diagram of the ultrasonic activation catalytic reaction mechanism; Figure 5 (d) and (e) are plots of Gibbs free energy changes calculated by DFT; Figure 6This is a diagram illustrating the in vitro tumor cell killing effect; among them, Figure 6 (a) and (b) are bar charts showing cell viability under different treatment conditions; Figure 6 (c) and (d) are dose-response curves under different treatment conditions; Figure 6 (e) is a fluorescence staining characterization diagram of Calcein-AM / PI live and dead cells; Figure 7 This image shows the therapeutic effect in a mouse CT26 subcutaneous tumor model; among them, Figure 7 (a) and (b) are the tumor growth curves and individual growth trajectories for each group; Figure 7 (c, d) are representative tumor photographs and weight statistics at the treatment endpoint; Figure 7 (e) is a graph showing the changes in body weight of mice in each group; Figure 7 (f) is a bar chart showing the detection of serum cytokine levels; Figure 8 Flow cytometry analysis results of key immune cell subsets in the subcutaneous tumor microenvironment of mouse CT26 tumors; among them, Figure 8 (a, b) are scatter plots and quantitative statistical results of flow cytometry analysis of tumor-infiltrating activated dendritic cells; Figure 8 (c, d) represent tumor-infiltrating CD4+. + With CD8 + Flow cytometry analysis scatter plot and quantitative statistical results of T cells; Figure 9 This image shows the therapeutic effect in a mouse MC38 subcutaneous tumor model; among them, Figure 9 (a) and (b) are the tumor growth curves and individual growth trajectories for each group; Figure 9 (c, d) are representative tumor photographs and weight statistics at the treatment endpoint; Figure 9 (e) is a graph showing the changes in body weight of mice in each group; Figure 9 (f) is a bar chart showing the detection of serum cytokine levels; Figure 10 Flow cytometry analysis results of key immune cell subsets in the subcutaneous tumor microenvironment of mouse MC38 cells; among them, Figure 10 (a, b) are scatter plots and quantitative statistical results of flow cytometry analysis of tumor-infiltrating activated dendritic cells; Figure 10 (c, d) represent tumor-infiltrating CD4+. + With CD8 + Flow cytometry scatter plot and quantitative statistical results of T cells. Detailed Implementation

[0017] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0018] Example 1. Weigh out AQ4N, NADPH, and TBR and mix them, wherein the molar ratio of AQ4N, NADPH, and TBR is 1:5:2. Add DMSO (volume fraction 1%), ethanol / polyoxyethylene castor oil mixed solution (volume ratio of ethanol to polyoxyethylene castor oil is 1:1, volume fraction 10%), and buffer salt solution to the mixture, stir and react to obtain a homogeneous AQ4N / NADPH / TBR prodrug composition reaction solution. The reaction solution was placed under conditions of oxygen concentration below 0.5 mg / L and subjected to ultrasonic treatment for 10 min. The ultrasonic frequency was set to 1 MHz and the ultrasonic intensity to 2.0 W / cm². 2 With a duty cycle of 50%, AQ4N activation is completed.

[0019] The amount of AQ4 generated was quantitatively detected and the activation rate was calculated using high-performance liquid chromatography (HPLC). The formula for calculating the activation rate is as follows: AQ4 activation rate (%) = ×100%.

[0020] Example 2. The difference between this embodiment and Embodiment 1 is that the ultrasonic treatment time is 1 min, while the remaining preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0021] Example 3. The difference between this embodiment and Embodiment 1 is that the ultrasonic treatment time is 3 minutes, while the remaining preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0022] Example 4. The difference between this embodiment and Embodiment 1 is that the ultrasonic treatment time is 5 minutes, while the remaining preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0023] Example 5. The difference between this embodiment and Embodiment 1 is that the ultrasonic treatment time is 20 min, while the remaining preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0024] Example 6. The difference between this embodiment and Example 1 is that the ultrasonic treatment time is 30 min, while the remaining preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0025] Example 7. The difference between this embodiment and Embodiment 1 is that the ultrasonic intensity is 0.5 W / cm². 2 The remaining preparation steps and conditions are the same as in Example 1, and the activation of AQ4N is completed.

[0026] Example 8. The difference between this embodiment and Embodiment 1 is that the ultrasonic intensity is 1 W / cm². 2 The remaining preparation steps and conditions are the same as in Example 1, and the activation of AQ4N is completed.

[0027] Example 9. The difference between this embodiment and Embodiment 1 is that the ultrasonic intensity is 1.5 W / cm². 2 The remaining preparation steps and conditions are the same as in Example 1, and the activation of AQ4N is completed.

[0028] Example 10. The difference between this embodiment and Embodiment 1 is that the ultrasonic frequency is 3 MHz, while the rest of the preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0029] Example 11. The difference between this embodiment and Example 1 is that the duty cycle is 10%, while the remaining preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0030] Example 12. The difference between this embodiment and Example 1 is that the duty cycle is 20%, while the remaining preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0031] Example 13. The difference between this embodiment and Example 1 is that the molar ratio of AQ4N, NADPH and TBR is 1:10:4. The remaining preparation steps and conditions are the same as in Example 1, and the activation of AQ4N is completed.

[0032] Example 14. The difference between this embodiment and Example 1 is that the N-oxide is quinoline N-oxide, while the rest of the preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0033] Example 15. The difference between this embodiment and Example 1 is that the N-oxide is 8-hydroxyquinoline N-oxide, while the rest of the preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0034] Example 16. The difference between this embodiment and Example 1 is that clozapine N-oxide is selected as the N-oxide, while the remaining preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0035] Example 17. The difference between this embodiment and Example 1 is that olanzapine N-oxide is selected as the N-oxide, while the rest of the preparation steps and conditions are the same as in Example 1, to complete the activation of AQ4N.

[0036] Example 18. The difference between this embodiment and Example 13 is that 0.1 mg of recombinant mouse CYP3A11 (0.1 mg / mL) was added. The remaining preparation steps and conditions were the same as in Example 13 to complete the activation of AQ4N.

[0037] Example 19. The difference between this embodiment and Embodiment 1 is that NADH is selected as the electron donor, while the rest of the preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0038] Example 20. The difference between this embodiment and Embodiment 1 is that ascorbic acid is selected as the electron donor, while the remaining preparation steps and conditions are the same as in Embodiment 1, thus completing the activation of AQ4N.

[0039] Example 21. The difference between this embodiment and Example 1 is that glutathione is selected as the electron donor, while the remaining preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0040] Example 22. The difference between this embodiment and Example 1 is that riboflavin is selected as the sound-sensing agent, while the rest of the preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0041] Example 23. The difference between this embodiment and Example 1 is that FMN is selected as the sound-sensing agent, while the rest of the preparation steps and conditions are the same as in Example 1, thus completing the activation of AQ4N.

[0042] Example 24. The difference between this embodiment and Example 1 is that FAD is selected as the sound-sensitive agent, while the rest of the preparation steps and conditions are the same as in Example 1, to complete the activation of AQ4N.

[0043] Example 25. The difference between this comparative example and Example 6 is that the ultrasonic treatment was carried out under normal oxygen conditions, while the remaining preparation steps and conditions were the same as in Example 6.

[0044] Comparative Example 1. Only AQ4N is provided, without ultrasonic treatment, and AQ4N is placed under normal oxygen conditions.

[0045] Comparative Example 2. The difference between this comparative example and Comparative Example 1 is that AQ4N was placed under conditions where the oxygen concentration was below 0.5 mg / L.

[0046] Comparative Example 3. The difference between this comparative example and Comparative Example 1 is that AQ4N was subjected to ultrasonic treatment under the same conditions as in Example 1.

[0047] Comparative Example 4. The difference between this comparative example and Example 1 is that the AQ4N / NADPH / TBR prodrug composition was not subjected to ultrasonic treatment; the remaining preparation steps and conditions were the same as in Example 1.

[0048] Comparative Example 5. The difference between this comparative example and comparative example 4 is that the AQ4N / NADPH / TBR prodrug composition was placed under conditions where the oxygen concentration was below 0.5 mg / L, while the remaining preparation steps and conditions were the same as those in comparative example 4.

[0049] Comparative Example 6. The difference between this comparative example and Example 1 is that TBR is not introduced, the molar ratio of AQ4N to NADPH is 1:5, and the rest of the preparation steps and conditions are the same as in Example 1.

[0050] Comparative Example 7. The difference between this comparative example and Example 1 is that NADPH is not introduced, the molar ratio of AQ4N to TBR is 1:2, and the rest of the preparation steps and conditions are the same as in Example 1.

[0051] Comparative Example 8. The difference between this comparative example and Example 18 is that TBR is not introduced and ultrasonic treatment is not performed, while the other preparation steps and conditions are the same as in Example 18.

[0052] (I) Activation effect analysis: To verify that the present invention can effectively achieve specific reduction and activation of AQ4N, high performance liquid chromatography (HPLC) was used to quantitatively detect the prodrug compositions prepared in Examples 1, 5, 6, 26 and Comparative Examples 1, 2, 4, 5, to analyze the generation of AQ4 after activation. The HPLC detection conditions were as follows: the mobile phase was a water-acetonitrile-trifluoroacetic acid mixture with a volume ratio of 76:24:0.05; the flow rate was set to 1.0 mL / min; and an ultraviolet detector was used with a detection wavelength of 254 nm. Figure 1 The figures show the detection results of AQ4 generation content after activation in each group of samples. As can be seen from the figures, single AQ4N exhibits extremely low spontaneous conversion ability in both air and oxygen-deficient (inert gas) environments, with AQ4 generation below 5%, indicating that no effective reduction conversion occurred. Even after ultrasonic treatment of the single AQ4N system, the AQ4 generation did not show a significant increase, remaining below 5%, indicating that ultrasound itself is insufficient to directly induce effective activation of AQ4N. However, in Examples 1, 5, and 6, after further introducing NADPH and TBR into the AQ4N system, AQ4N was significantly converted into AQ4 under ultrasonic treatment, and the AQ4 generation showed a continuous upward trend with increasing ultrasonic treatment time. Specifically, at 10 min, 20 min, and 30 min, the AQ4 generation content reached 14.8%, 25.2%, and 37.2%, respectively. This demonstrates that ultrasound is a necessary condition for initiating this reduction activation. In this system, TBR acts as a redox relay, accelerating the electron transfer process; NADPH acts as a reduction equivalent donor, continuously providing an electron source for the reductive deoxygenation reaction of AQ4N. The synergistic effect of these three components effectively promotes the N–O bond cleavage of AQ4N under mild conditions, achieving its efficient conversion into the active product AQ4, thus verifying that the system of this invention possesses good ultrasonic responsiveness and prodrug-specific activation capability.

[0053] The AQ4 generation was analyzed using the ultrasound-triggered prodrug composition activation methods provided in Examples 1–5, 7–12, and Comparative Example 4 to evaluate the effects of different ultrasound parameters and system components on the AQ4N activation efficiency. The amount of AQ4 generated after activation in each group of samples was quantitatively detected using HPLC under the same HPLC conditions as described above. Figure 1 The testing conditions were consistent, and the results were as follows: Figure 2 As shown. Among them, Figure 2 (a) The results of AQ4 generation under different ultrasonic intensities for Examples 1, 7–9, and Comparative Example 4 are shown. As can be seen from the figure, the amount of AQ4 generated increases significantly with increasing ultrasonic intensity, indicating that increasing the ultrasonic energy input is beneficial to promoting the reduction and activation process of AQ4N. When the ultrasonic intensity reaches 2 W / cm², the AQ4 generation increases significantly. 2At this point, the AQ4 formation reached its highest level of 21%, significantly higher than that of control group 4 (1.8%) without ultrasonic triggering. This result indicates that ultrasonic intensity has a significant impact on the initiation of redox reactions and electron transfer efficiency in the system; higher intensity ultrasound can effectively enhance the conversion efficiency of AQ4N to AQ4.

[0054] Figure 2 (b) The results of AQ4 generation in Examples 1, 10, and Comparative Example 4 under different ultrasonic frequencies are shown. As can be seen from the figure, as the ultrasonic frequency increases from 1 MHz to 3 MHz, the AQ4 generation decreases slightly to 7.7%, but is still significantly higher than that of Comparative Example 4 without ultrasonic treatment. This indicates that lower frequency ultrasound is more conducive to the formation of cavitation effects in the system, thereby enhancing local energy release and free radical / electron transfer processes.

[0055] Figure 2 (c) The results of AQ4 generation under different ultrasonic duty cycles for Examples 1, 11, 12 and Comparative Example 4 are shown. As can be seen from the figure, the amount of AQ4 generated generally shows a continuous upward trend with the increase of ultrasonic duty cycle, indicating that ultrasonic duty cycle is a key process parameter for regulating the activation and conversion efficiency of AQ4N. Appropriately increasing the ultrasonic duty cycle can enhance the energy input of the system, further drive the reduction and breaking process of N-O bonds, and significantly improve the conversion efficiency of AQ4N to active AQ4.

[0056] Figure 2 (d) The results of AQ4 generation in Examples 1–5 and Comparative Example 4 under different ultrasonic time conditions are shown. The results show that the amount of AQ4 generated increases continuously with the extension of ultrasonic treatment time, indicating that the reduction and activation process of AQ4N has a significant time dependence. Even when the ultrasonic treatment is only 1 min, the amount of AQ4 generated has already shown an upward trend compared with Comparative Example 4 without ultrasonic triggering. Although the increase is relatively limited, it can prove that ultrasonic triggering can effectively start the activation process of AQ4N in a short time. With further extension of ultrasonic time, the amount of AQ4 generated increases significantly, indicating that the system can stably carry out the reduction and activation reaction under continuous ultrasonic treatment.

[0057] Figure 2(e) Comparison results of AQ4 generation content in Example 1 and Comparative Examples 1, 3, 4, 6, and 7 under different system components and triggering conditions. As shown in the figure, the AQ4 generation in Comparative Examples 1, 3, and 4 was less than 5%, indicating that no effective conversion occurred, fully demonstrating that ultrasound is the key triggering condition for initiating the reduction system. Comparative Examples 6 and 7 lacked TBR or NADPH, respectively. Although the AQ4 generation was increased under ultrasound, it was still less than 16%, significantly lower than in Example 1, indicating that relying solely on ultrasound or certain components is insufficient to achieve efficient activation. Specifically, the absence of TBR restricts the electron relay process, while the absence of NADPH leads to a lack of a stable source of reducing equivalence, thus significantly weakening the reduction and deoxygenation efficiency of AQ4N.

[0058] also, Figure 2 (f) shows the representative LC-MS / MS extractive ion chromatogram and corresponding mass spectrum of the activated product from Example 1, used to confirm the formation of AQ4. The LC-MS / MS detection conditions were as follows: mobile phase was a water-acetonitrile-formic acid mixture with a volume ratio of 76:24:0.1; flow rate was set to 0.8 mL / min; and ionization mode was electrospray ionization (ESI) positive ionization mode. + The figure clearly demonstrates that AQ4N can be effectively converted into the target product AQ4, with a reaction efficiency of 44.1% and a release load of 40.3%. This result further verifies at the molecular structure level that the ultrasound-responsive redox activation system constructed in this invention can achieve efficient, stable, and controllable release of AQ4N, providing a reliable basis for its application in precise local activation of tumors.

[0059] Ultrasound can not only directly promote the reduction and activation of AQ4N, but also significantly enhance the conversion efficiency of AQ4N with the participation of reductase. For example... Figure 3 (a) is the HPLC chromatogram of AQ4, AQ4N standard samples and samples prepared in Comparative Examples 8, 13, and 18, under the same test conditions as... Figure 1 The test conditions were consistent. As shown in the figure, the AQ4 and AQ4N standards exhibited characteristic retention peaks at 6.8 min and 9.4 min, respectively. Both Examples 13 and 18 showed obvious product peaks at the characteristic retention time of AQ4, indicating that AQ4N underwent effective reduction and transformation. The AQ4 product peak area in Example 18 was significantly higher than that in Example 13, indicating that the introduction of CYP3A11 on top of ultrasonic treatment further enhanced the activation efficiency of AQ4N. In contrast, Comparative Example 8, although CYP3A11 was introduced, did not undergo ultrasonic treatment, resulting in a weaker AQ4 product peak and only a small amount of AQ4 produced, indicating that relying solely on enzymatic action is insufficient to achieve efficient transformation of AQ4N. Figure 3(b) Quantitative statistical results of AQ4 production in each group. As can be seen from the figure, the average AQ4 yield of Example 18 reached 64.2%, which was significantly higher than 39.1% of Example 13 and 11.5% of Comparative Example 8, and the difference between groups was statistically significant (**). p <0.01, **** p <0.0001). The above results confirm that ultrasound can significantly enhance the CYP3A11-mediated enzymatic reduction process of AQ4N, indicating that in the tumor microenvironment, the synergistic effect of ultrasound and endogenous reductase can further improve the conversion efficiency of AQ4N to active AQ4, providing experimental evidence for improving the efficacy of prodrug therapy.

[0060] To verify the ultrasonic drive provided by the present invention N - The substrates applicable to oxide deoxygenation reactions are broad. The reaction efficiency and loading release rate of the prodrug compositions prepared in Examples 14-17 were tested. The formulas for calculating the reaction efficiency and loading release rate are as follows: Reaction efficiency (%) = 1 - ×100%; Load release rate (%) = ×100%.

[0061] like Figure 4 (a) is a schematic diagram of the reduction reaction of quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide as substrates in the system of the present invention, showing that different types of N-oxides can undergo deoxygenation reaction in the ultrasound / NADPH / TBR system to generate the corresponding free load. Figure 4 (b) and (c) are quantitative bar charts showing the reaction efficiency and load release rate of each substrate. The charts show that the reaction efficiencies of quinoline N-oxide and 8-hydroxyquinoline N-oxide are 61.1% and 61.5%, respectively, with release loads of 57.8% and 55.8%. The reaction efficiencies of clozapine N-oxide and olanzapine N-oxide are 34.5% and 43.7%, respectively, with release loads of 31.6% and 37.6%. This demonstrates that the ultrasonic activation and reduction system provided by this invention has good universality for N-oxide prodrugs with different structures and can effectively achieve load release. Furthermore, the reaction efficiency and release rate of different substrates exhibit a certain structure dependence, which is related to the electronic and steric effects of the substrate's own N-oxide.

[0062] (ii) Inhibition test: To demonstrate that the reaction described in this invention is essentially a stepwise reductive deoxygenation process mediated by free radicals / electron transfer, Example 1 was used as a control group. Free radical / electron quenchers TEMPO (5.0 eq), BMPO (5.0 eq), CCl4 (5.0 eq), KNO3 (10.0 eq), and a combination of CCl4 / KNO3 were added to the mixture for treatment, and the formation of AQ4 was analyzed by HPLC. Figure 5 (a) is the HPLC elution spectrum of Example 1 and samples with different quenchers added. Figure 5 (b) is a bar chart showing the quantitative inhibition analysis of AQ4 formation by different quenchers. The chart shows that TEMPO significantly inhibits AQ4 formation (yield decreased from 34.3% to 9.8%), CCl4 and KNO3 reduced the conversion to 21.6% and 22.4% respectively, with the inhibitory effect further enhanced when used in combination (yield decreased to 9.6%), while BMPO had a weak inhibitory effect on the yield (only decreasing to 29.2%). These results indicate that the activation process is related to radical-mediated electron / hydrogen atom transfer, and the key active intermediate in the system is not the oxygen-center free radical that BMPO can capture, but a highly reducing transient species that mediates the stepwise reduction and cleavage of the N-oxide of AQ4N. Further, combined with... Figure 5 (c) shows a schematic diagram of the ultrasonic (US) activated catalytic reaction mechanism and Figure 5 The DFT calculations of Gibbs free energy changes shown in (d) and (e), combined with the mechanism in the figures, prove that the reaction pathway for ultrasonic-induced AQ4N activation proposed in this invention is as follows: after ultrasonic-induced TBR excitation, NADPH provides electrons, and through single-electron transfer and subsequent hydrogen atom / proton-electron transfer processes, the N–O bonds are gradually broken, first forming a partially deoxygenated intermediate AQ4M, and then further generating AQ4. Thermodynamic calculation results show that this stepwise deoxygenation pathway has good thermodynamic feasibility, with ΔG of -49.8 kcal / mol for AQ4N→AQ4M and -45.4 kcal / mol for AQ4M→AQ4.

[0063] (III) In vitro tumor cell killing effect: Mouse colon cancer CT26 cells were used as a model. CT26 cells were cultured under normoxic or hypoxic conditions (1% O2) (hypoxic cells were pre-cultured in a 1% O2 hypoxic incubator for 24 h to establish a stable hypoxic cell model). Experimental setups included a PBS (phosphate-buffered saline) control group, a TBR + ultrasound (US) group, different concentrations of AQ4N treatment groups, and an AQ4N + TBR + ultrasound group. Ultrasound parameters were set at a frequency of 1 MHz and a power density of 2 W / cm². 2The cell viability was assessed using a 50% duty cycle and pulsed treatment for 2 minutes. Cell viability was then determined using the CCK-8 assay to obtain bar charts of cell viability under different treatment conditions. Figure 6 (a), (b)) and dose-response curves ( Figure 6 (c) and (d) show that under normoxic conditions, cell viability remained at a high level in both the AQ4N-treated groups and the AQ4N+TBR+ultrasound group, without significant cytotoxicity. However, under hypoxic conditions, cell viability decreased in a concentration-dependent manner in both the AQ4N-treated group and the AQ4N+TBR+ultrasound group with increasing AQ4N concentration, and the cell-killing effect of the AQ4N+TBR+ultrasound group was significantly better than that of the AQ4N-treated group alone. Quantitative analysis showed that cell viability in the TBR+ultrasound group was not significantly different from that in the PBS control group, indicating that this treatment itself had almost no significant cytotoxicity. Under hypoxic conditions, the IC50 of the AQ4N+TBR+ultrasound group was significantly higher than that of the control group. 50 Treatment with AQ4N alone reduced the concentration from 36.7 mg / L to 2.8 mg / L, resulting in a 13-fold increase in antitumor activity. Figure 6 (e) is a fluorescence staining characterization diagram of Calcein-AM / PI live and dead cells. Green fluorescence marks live cells and red fluorescence marks apoptotic and necrotic cells. The staining results of each group show that the PBS group and the TBR+ultrasound group are dominated by green live cell fluorescence, indicating that the cells are in good condition. The AQ4N alone treatment group shows only a slight increase in red dead cell fluorescence, while the AQ4N+TBR+ultrasound group shows a significant increase in the range and intensity of red fluorescence and a significant increase in the proportion of dead cells. From the perspective of cell morphology and fluorescence distribution, this further confirms that ultrasound combined with TBR can efficiently activate AQ4N under hypoxic conditions and significantly enhance the killing ability of CT26 tumor cells.

[0064] (iv) Therapeutic effects and immune enhancement in mouse tumor models: Establish a subcutaneous CT26 tumor model in immune-intelligent BALB / c mice, and wait for the tumor volume to grow to 200 mm. 3 Mice with tumors were randomly divided into four groups: a PBS control group (G1), a TBR + ultrasound (US) group (G2), an AQ4N monotherapy group (G3), and an AQ4N + TBR + US synergistic therapy group (G4), with n=5 in each group. Drug administration was performed on days 0, 2, 4, and 6. AQ4N was administered intravenously at 10 mg / kg, and TBR was administered intraperitoneally at 59 mg / kg. T30 min after administration, ultrasound treatment (1 MHz, 2 W / cm²) was applied to the tumor site. 2(50% duty cycle, effective duration of action 5 min). Tumor volume and mouse weight were monitored regularly during treatment. At the treatment endpoint (D14), tumor tissue was removed and weighed, and the AQ4 level in the tumor was quantitatively detected by HPLC / LC-MS. At the same time, the proportion of tumor-infiltrating immune cells was analyzed by flow cytometry, and the serum cytokine level was detected by ELISA.

[0065] like Figure 7 (a) and (b) show the tumor growth curves and individual growth trajectories for each group. The change in tumor volume over the number of days after drug administration characterizes the treatment effect. The results show that the tumor in group G1 showed a continuous and rapid growth trend, the tumor growth in group G2 was not significantly different from that in group G1, group G3 had a certain inhibitory effect on tumor growth, and the tumor growth in group G4 was significantly inhibited, with the final tumor volume being only 75.3 ± 33.3 mm. 3 This is significantly lower than that of Group G1 (972.4±87.1mm). 3 (G3 group (207.2±69.8mm)) 3 The tumor suppression rate (TSR) reached 113.1%, and the synergistic effect Q value was 1.35, demonstrating that the synergistic treatment of AQ4N+TBR+US has a clear anti-tumor effect; Figure 7 (c, d) show representative tumor photographs and weight statistics at the treatment endpoint. The average tumor weight in group G4 was 50.4 mg, only 1 / 5.5 of that in group G3 (279.2 mg), further validating the potent tumor-suppressing effect of the synergistic treatment of AQ4N+TBR+US; Figure 7 (e) shows the weight change curves of mice in each group. No significant decrease in weight was observed in any group, indicating good in vivo safety of the treatment regimen. Quantitative results of AQ4 in tumor tissue showed that the AQ4 content in the tumor tissue of group G4 reached 604.3 μg / g, a 25.8-fold increase compared to group G3 (23.4 μg / g), confirming that the ultrasound-driven system can efficiently promote the conversion of AQ4N to the active product AQ4 at the tumor site in vivo. Figure 7 (f) is a bar chart showing serum cytokine levels. In the G4 group of mice, serum TNF-α and IFN-γ levels reached 70.5 pg / mL and 67.5 pg / mL, respectively, significantly higher than in other groups, demonstrating that the synergistic treatment of AQ4N+TBR+US can effectively induce a systemic anti-tumor immune response. Figure 8 (a, b) represent tumor-infiltrating activated dendritic cells (CD86). + MHCII + Flow cytometry scatter plot and quantitative statistical results of DCs, CD86 in tumors of group G4 + MHCII + The proportions of DCs were 35.8% and 34.1%, respectively, significantly higher than those in the G1 group (10.2% and 8.0%), demonstrating enhanced DC maturation and antigen presentation function; Figure 8 (c, d) represent tumor-infiltrating CD4+. + With CD8 + Flow cytometry scatter plot and quantitative statistical results of T cells, and CD4 infiltration of tumor cells in group G4. + With CD8 + The proportions of T cells were 1.7% and 2.1%, respectively, significantly higher than those in the G3 group (0.8% and 0.9%), indicating that the synergistic treatment of AQ4N+TBR+US can effectively promote tumor infiltration of effector T cells. In summary, the ultrasound-driven AQ4N activation system can significantly inhibit CT26 tumor growth in vivo by efficiently generating active AQ4, while inducing immune activation in the tumor microenvironment and activating a systemic anti-tumor immune response, without significant toxic side effects, and has good potential for in vivo application.

[0066] Furthermore, this invention established a mouse MC38 subcutaneous tumor model under the same conditions as establishing an immune-healthy BALB / c mouse CT26 subcutaneous tumor model. The tumor-bearing mice were randomly divided into four groups: a PBS control group (G1), a TBR + ultrasound (US) group (G2), an AQ4N monotherapy group (G3), and an AQ4N + TBR + US synergistic therapy group (G4), with n=5 in each group.

[0067] like Figure 9 (a) and (b) show the tumor growth curves and individual growth trajectories of each group in the MC38 subcutaneous tumor model. The results showed that the tumor volume in group G1 increased rapidly and continuously with the number of days after drug administration. In group G2, TBR+US treatment alone had a limited inhibitory effect on tumor growth. In group G3, AQ4N monotherapy could delay tumor growth to some extent. However, in group G4, tumor growth was significantly inhibited after synergistic treatment with AQ4N+TBR+US, and the final tumor volume was significantly lower than that in groups G1, G2, and G3. The tumor inhibition rate (TSR) reached 120.5%, indicating that the ultrasound triggering mechanism provided by this invention also has a significant synergistic anti-tumor effect in the MC38 tumor model, and the synergistic effect is strong.

[0068] like Figure 9 (c, d) show representative tumor photographs and tumor weight statistics for each group at the treatment endpoint. The tumor volume of the excised tumors in groups G1 and G2 was larger, the tumor weight of group G3 was reduced, and the tumor in group G4 was the smallest. The tumor weight at the endpoint was significantly lower than that of other groups, indicating that the synergistic treatment of AQ4N+TBR+US can effectively inhibit the growth of MC38 tumors. Figure 9 (e) shows that the weight of mice in each group remained relatively stable during the treatment period, with no significant downward trend, indicating that the treatment regimen did not cause significant systemic toxicity in the MC38 model and had good in vivo safety. Figure 9(f) shows the results of serum cytokine level detection. The levels of TNF-α and IFN-γ in group G4 were significantly higher than those in groups G1, G2 and G3. Among them, TNF-α increased to above 74.7 pg / mL and IFN-γ increased to above 66.0 pg / mL. This indicates that the synergistic treatment of AQ4N+TBR+US can not only directly inhibit tumor growth, but also effectively activate the systemic anti-tumor immune response. This result is consistent with the immune activation trend in the CT26 subcutaneous tumor model.

[0069] like Figure 10 (a, b) show the flow cytometry analysis and quantitative statistical results of MHCII and CD86, dendritic cell maturation-related markers, in MC38 tumor tissue. Representative flow cytometry plots show that the positive rate of MHCII in group G4 increased from 0.18% in group G1 to 1.33%, and the positive rate of CD86 increased from 0.27% in group G1 to 2.27%. Quantitative results also show that MHCII in group G4... + DCs and CD86 + The proportion of dendritic cells (DCs) was significantly higher in the G1, G2, and G3 groups, indicating that the synergistic treatment of AQ4N+TBR+US can significantly promote the maturation of intratumoral DCs and enhance antigen presentation function. The proportion of DCs in the G3 group was also higher than that in the G1 group, but was lower than that in the G4 group overall, indicating that AQ4N treatment alone is insufficient to induce sufficient immune activation.

[0070] like Figure 10 (c, d) show the flow cytometry analysis and quantitative statistical results of tumor-infiltrating T cells. Representative flow cytometry plots show that CD8+ in the G4 group... + The proportion of T cells increased from 0.39% in the G1 group to 2.18%, CD4 + The proportion of T cells increased from 0.41% in group G1 to 1.67%; statistical results showed that CD8+ cells were present in the tumors of group G4. + T cells and CD4 + The T cell infiltration levels were significantly higher in all groups than in other groups. These results indicate that in the MC38 subcutaneous tumor model, the synergistic treatment of AQ4N+TBR+US can also promote DC maturation, enhance effector T cell infiltration, and induce an anti-tumor immune response. In summary, combined with... Figure 9 and Figure 10 The results showed that the ultrasound-driven AQ4N activation system not only exhibited good tumor suppression and immune activation effects in the CT26 model, but also demonstrated stable and significant antitumor activity and good in vivo safety in the MC38 model.

[0071] In summary, this invention constructs an external activation system composed of ultrasound, TBR, and NADPH. These three components work synergistically to promote the N–O bond cleavage of AQ4N under mild ultrasound conditions, converting it into active AQ4, thus achieving efficient conversion to the active product AQ4. Simultaneously, it enables the spatiotemporally controllable reduction and deoxygenation conversion of N-oxide prodrugs like AQ4N at tumor lesions, significantly enhancing the in vitro killing ability of hypoxic tumor cells and substantially reducing the IC50. 50 Furthermore, it exhibits no significant non-specific cellular or in vivo toxicity, demonstrating good safety and combining multiple advantages such as precise prodrug activation, efficient tumor suppression, and synergistic immunotherapy. In addition, this invention is not only applicable to AQ4N but can also effectively activate various N-oxide substrates with different structures, including quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide, demonstrating excellent substrate compatibility and system universality. This overcomes the limitations of existing technologies that typically only construct activation systems for single prodrugs, providing a new technical pathway for precise release and controllable treatment of tumor prodrugs.

[0072] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0073] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A prodrug composition, characterized in that, Including N-oxides, electron donors, and sound-sensitive agents; The N-oxide is selected from any one or at least two combinations of anthraquinone diamine N-oxides, nitrogen-containing aromatic heterocyclic N-oxides, and tertiary amine N-oxides; the electron donor is any one or at least two combinations of nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide phosphate, ascorbic acid, and glutathione; the sonication agent is any one or at least two combinations of riboflavin tetrabutyrate, riboflavin, flavin mononucleotide, and flavin adenine dinucleotide.

2. The prodrug composition according to claim 1, characterized in that, The molar ratio of the N-oxide, electron donor, and sonosensitive agent is 1:(1-20):(0.5-10).

3. The prodrug composition according to claim 1, characterized in that, The N-oxide is any one or a combination of at least two of the following: banoanthraquinone, quinoline N-oxide, 8-hydroxyquinoline N-oxide, clozapine N-oxide, and olanzapine N-oxide.

4. The prodrug composition according to claim 1, characterized in that, The prodrug composition also includes a pharmaceutically acceptable carrier / excipient and a buffer salt solution.

5. The prodrug composition according to claim 4, characterized in that, The concentration of the N-oxide is 1~500 μM.

6. A method for activating a prodrug composition by ultrasonic triggering as described in any one of claims 1-5, characterized in that, Includes the following steps: The prodrug composition is subjected to ultrasonic treatment to reduce and deoxygenate the N-oxide, converting it into the corresponding deoxygenated product and active form, thus completing the activation of the prodrug composition.

7. The method for activating the prodrug composition by ultrasound triggering according to claim 6, characterized in that, The frequency of the ultrasonic treatment is 20 kHz to 10 MHz; the ultrasonic intensity is 0.1 to 5 W / cm. 2 The duty cycle is 1-100%; the ultrasonic treatment time is 1 min-60 min.

8. The application of a method for activating a prodrug composition by ultrasound triggering as described in claim 6 or 7, characterized in that, It is used in the preparation of drugs for cancer treatment.

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