Light-activated chemotherapeutic drug as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly discloses a light-activated chemotherapy medicine as well as a preparation method and application thereof. The light-activated chemotherapeutic drug comprises a therapeutically effective amount of a compound as shown in a formula (I) or pharmaceutically acceptable salts, esters, isomers and solvates thereof, and pharmaceutically acceptable auxiliary materials. According to the compound, a nitrogen mustard-containing group is coordinated into a ruthenium (II) complex, so that the nitrogen mustard-containing group is caged by the ruthenium (II) complex, the biological activity of the nitrogen mustard group is masked in a dark environment, good biocompatibility is shown, and the nitrogen mustard-containing group has good biocompatibility under a low-power visible light condition. The nitrogen mustard group ligand can be quickly and effectively released, the light-controllable accurate release of the nitrogen mustard medicine is realized, the toxic and side effects of the nitrogen mustard medicine in the treatment process are effectively reduced, and the treatment effect of the compound on cancers is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a photoactivated chemotherapy drug and a preparation method and application thereof. Background Art

[0002] In clinical cancer treatment, chemotherapy is one of the main treatment methods. Nitrogen mustard drugs have been used in cancer chemotherapy for more than 70 years. As a biological alkylating agent, nitrogen mustard enters the body to form active electron-deficient intermediates or other compounds with electrophilic active groups. These active substances can undergo electrophilic reactions with electron-rich groups in substances such as proteins, DNA and glutathione (GSH) to form covalent bonds, thereby inhibiting the activity of biological molecules in the body. Among them, the binding and cross-linking of nitrogen mustard with DNA leads to DNA alkylation, inhibiting DNA replication and cell proliferation, and producing strong cytotoxicity. However, nitrogen mustard drugs have defects such as poor selectivity and serious adverse reactions during treatment.

[0003] Photoactivated chemotherapy is an emerging strategy for cancer treatment. Photoactivated chemotherapy involves the encapsulation of the active molecule within the body and tumors, resulting in low toxicity or bioactivity in the absence of light. Irradiation with light of an appropriate wavelength releases the active molecule, restoring its original bioactivity. Ruthenium(II) complexes possess a wealth of photophysical and photochemical properties, and their overall structure is easily tunable. Generally, photoactivated chemotherapeutic prodrugs based on ruthenium(II) complexes undergo ligand dissociation upon illumination, resulting in a ruthenium(II) hydrate that covalently binds to DNA in a manner similar to cisplatin. Simultaneously, the released free ligand also exhibits some bioactivity, resulting in a synergistic antitumor activity. Currently reported photoactivated chemotherapeutic prodrugs based on ruthenium(II) complexes generally require high-power, prolonged irradiation to achieve effective dissociation of the active ligand. Furthermore, their ability to induce non-apoptotic cell death and activate immunity has not been studied. Summary of the Invention

[0004] In order to overcome at least one technical problem existing in the above-mentioned prior art, one of the objects of the present invention is to provide a compound represented by formula (I),

[0005]

[0006] In this compound, the ruthenium-based complex cages the nitrogen mustard group ligand, making the compound low-toxic in the dark, but rapidly releasing the nitrogen mustard group under light. The released nitrogen mustard group acts on cancer cells, achieving the effect of effectively killing cancer cells, thereby achieving light-controlled precise release, effectively solving the defect of uncontrollable toxic side effects of nitrogen mustard drugs during treatment.

[0007] A second object of the present invention is to provide a method for preparing the above compound.

[0008] A third object of the present invention is to provide a photoactivated chemotherapy drug composition.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable salts, esters, isomers, and solvates in the preparation of drugs for photoactivated tumor treatment.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a compound represented by formula (I),

[0012]

[0013] or a pharmaceutically acceptable salt, ester, isomer, or solvate thereof, wherein

[0014] Each R1 is independently selected from C 1～10 Alkyl, C 1～10 Alkoxy, -OH;

[0015] Each x is independently selected from 0, 1, 2, 3, 4 or 5;

[0016] X is selected from F, Cl, Br or I;

[0017] m and n are independently selected from 1, 2, 3, 4 or 5.

[0018] In some embodiments of the present invention, each R1 is independently selected from C 1～5 In some embodiments of the present invention, each R1 is independently selected from C 1～3 In some embodiments of the present invention, each R1 is independently selected from methyl.

[0019] In some embodiments of the present invention, x is all 0.

[0020] In some embodiments of the present invention, X is Cl or Br.

[0021] In some embodiments of the present invention, m is selected from 1, 2 or 3.

[0022] In some embodiments of the present invention, n is selected from 1, 2 or 3.

[0023] In some embodiments of the present invention, the compound is selected from

[0024] The second aspect of the present invention provides a method for preparing the compound of the first aspect of the present invention, comprising the following steps: S1: and Perform reductive amination reaction to obtain a ligand; S2: Make the ligand react with reaction, and then SOX2 reaction, prepared;

[0025] The structural formula of the ligand is:

[0026] y is selected from 0, 1, 2, 3 or 4;

[0027] X, R1, x, m, and n are as defined above.

[0028] In some embodiments of the present invention, SOX2 is SOCl2.

[0029] In some embodiments of the present invention, The reaction is carried out in the presence of silver trifluoromethanesulfonate.

[0030] The third aspect of the present invention provides a photoactivated chemotherapy drug composition comprising a therapeutically effective amount of the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt, ester, isomer, or solvate thereof, and a pharmaceutically acceptable excipient.

[0031] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, oral solutions, injections, ointments, patches, aerosols, gels or drops.

[0032] The fourth aspect of the present invention provides use of the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt, ester, isomer, or solvate in the preparation of a drug for photoactivated cancer treatment.

[0033] In some embodiments of the present invention, the cancer comprises lung cancer, esophageal cancer, cervical cancer, breast cancer, prostate cancer, gastric cancer, leukemia, neuroblastoma, nasopharyngeal cancer, thyroid cancer, oral cancer, ovarian cancer, endometrial cancer, bladder cancer, kidney cancer, colorectal cancer, liver cancer or pancreatic cancer.

[0034] The beneficial effects of the present invention are as follows: the compound of the present invention is prepared by coordinating a nitrogen mustard group to a ruthenium (II) complex, thereby caged by the ruthenium (II) complex. In a dark environment, the biological activity of the nitrogen mustard group is masked, and the compound exhibits good biocompatibility. Under low-power visible light conditions, the nitrogen mustard group ligand can be quickly and effectively released, thereby achieving light-controlled and precise release of the nitrogen mustard drug, effectively reducing the uncontrollable toxic side effects of the nitrogen mustard drug during treatment, and improving the therapeutic effect of the compound on cancer.

[0035] In addition, the compounds of the present invention have excellent photoactivation properties. Under low-power visible light irradiation conditions, they rapidly release nitrogen mustard ligands and exhibit 30-fold enhanced cell killing activity in cisplatin-resistant lung cancer cells A549R, further demonstrating that the compounds of the present invention achieve light-controlled release of nitrogen mustard drugs and achieve an anti-tumor effect of "1+1>2" under the synergistic effect of ruthenium-based complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0037] Figure 2 This is the mass spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0038] Figure 3 This is the electrospray ionization mass spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 before and after irradiation in Example 1.

[0039] Figure 4 This is the UV-visible absorption spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1 under continuous light irradiation.

[0040] Figure 5 This is a nonlinear fitting curve of the absorption value of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 at 445nm in Example 1.

[0041] Figure 6 1 is a high performance liquid chromatogram of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1 under light and darkness.

[0042] Figure 7 This is a graph showing the cell viability test of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1 under the treatment of different death inhibitors.

[0043] Figure 8 This is a diagram of the cell DNA damage test of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1 under dark and light conditions.

[0044] Figure 9This is the confocal imaging of the GSH level of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0045] Figure 10 This is the confocal imaging image of lipid peroxides of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0046] Figure 11 This is a Western blot analysis of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0047] Figure 12 This is the CRT immunofluorescence confocal imaging of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0048] Figure 13 This is the HMGB1 immunofluorescence confocal imaging image of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1.

[0049] Figure 14 This is a graph showing the extracellular ATP release test of A549R cells using [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in Example 1. DETAILED DESCRIPTION

[0050] The specific implementation of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0051] The meanings of the abbreviations in the following embodiments are as follows:

[0052] DNA: deoxyribonucleic acid; GSH: glutathione; A549R: cisplatin-resistant human non-small cell lung cancer cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC 50 : half inhibitory concentration; PI: phototoxicity index; GSH: glutathione; CRT: calreticulin; HMGB1: high-mobility group box 1 protein; ATP: adenosine triphosphate.

[0053] Example 1

[0054] This example provides a photoactivated chemotherapeutic prodrug, designated as [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2, with the following structural formula:

[0055]

[0056] The synthetic route of the photoactivated chemotherapy prodrug in this example is as follows:

[0057]

[0058] The synthesis steps of the photoactivated chemotherapeutic prodrug in this example are as follows:

[0059] (1) Synthesis of Compound 1

[0060] 4-(4-pyridyl)benzaldehyde (0.1832 g, 1 mmol) and N,N-bis(2-hydroxyethyl)ethylenediamine (0.1628 g, 1.1 mmol) were weighed into a 50 mL round-bottom flask, and 20 mL of anhydrous methanol was added. After stirring at room temperature for 1 hour, sodium borohydride (0.1135 g, 3 mmol) was added to the mixture in small amounts and then stirred at room temperature for 0.5 hours. After the reaction, the excess sodium borohydride was quenched with ice water, and the mixture was extracted three times with n-octanol. The organic phases were combined and evaporated under reduced pressure to remove the solvent to obtain the crude ligand product. The product was further purified by chromatography on a 100-200 mesh silica gel column with dichloromethane / methanol (volume ratio 5:1) as the mobile phase to obtain compound 1, which was used for the next step.

[0061] (2) Synthesis of intermediate a

[0062] To a 100mL round-bottom flask, [Ru(dip)2Cl2] 2H2O (i.e., compound 2, 125.40mg, 0.15mmol) and a mixture of the above-purified compound 1 (47.25mg, 0.15mmol) and 30mL of water / ethanol (volume ratio 1:3) were added, and the mixture was stirred under reflux in the dark for 12 hours under an argon atmosphere. The reaction solution was cooled to room temperature, vacuum rotary evaporation was performed to remove the solvent to obtain a crude intermediate a product. The product was further purified by column chromatography using 100-200 mesh alumina as the stationary phase and dichloromethane / methanol (volume ratio 15:1) as the mobile phase, vacuum rotary evaporation was performed to remove the eluent to obtain intermediate a for standby use.

[0063] (3) Synthesis of intermediate b

[0064] Silver trifluoromethanesulfonate (0.0771 g, 0.3 mmol), intermediate a (0.1674 g, 0.15 mmol) and 30 mL of ethanol were added to a 50 mL round-bottom flask, and the mixture was stirred under reflux in the dark for 8 hours under an argon atmosphere. The mixture was filtered while hot to collect an orange-red liquid. The orange-red liquid was transferred to a new 50 mL round-bottom flask, 4,4-bipyridine (0.0471 g, 0.3 mmol) was added, and the mixture was stirred under reflux in the dark for 12 hours under an argon atmosphere. After the reaction was completed, it was cooled to room temperature, evaporated under reduced pressure, and the solvent was removed to obtain a crude intermediate b product. The product was further purified by column chromatography using 100-200 mesh alumina as the stationary phase and dichloromethane / methanol (volume ratio 10: 1) as the mobile phase and set aside.

[0065] (4) Synthesis of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2

[0066] An appropriate amount of dichloromethane was added to dissolve intermediate b, followed by an excess of thionyl chloride. After stirring for 4 hours, the reaction was evaporated under reduced pressure to remove the liquid, yielding a crude Ru-nitrogen mustard complex [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2. This was purified by recrystallization from dichloromethane / toluene, washed with ether, and dried to afford a reddish-brown solid. Yield: 62 mg, 32% yield.

[0067] The H NMR spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 is as follows Figure 1 As shown, the mass spectrum is Figure 2 As shown, the nuclear magnetic hydrogen spectrum and mass spectrum data are as follows:

[0068] 1 H NMR (400MHz, MeOD) δ = 9.81 (s, 2H), 9.11 (d, J = 15.6Hz, 2H), 8.97 (d, J = 6.4Hz, 2H), 8.91 (s, 2H), 8.49–8.42(m,4H),8.35–8.26(m,4H),8.20(d,J=9.4Hz,2H),8.00(s,2H),7.89(d,J=8.0Hz,3H ),7.80(t,J=7.1Hz,6H),7.76–7.65(m,10H),7.59(s,10H),4.37(s,2H),3.71(q,J=6.8Hz,4H) ,3.55(d,J=6.8Hz,1H),3.51(d,J=6.9Hz,1H),3.26(t,J=5.6Hz,2H),3.04(t,J=6.4Hz,4H)ppm.

[0069] ESI-MS m / z:[M]2+ Calculated as C 76 H 63 Cl2N9Ru, 637.2; a molecular weight peak of 637.7 was detected.

[0070] Electrospray ionization mass spectrometry was used for mass spectrometry. The mother solution of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 was diluted to 10 μmol / L with dichloromethane. The sample was analyzed by a light source (450 nm, 2.4 J / cm 2 , 10min) irradiate the complex solution, and perform mass spectrometry analysis on the material composition of the complex solution before and after irradiation. The specific test figure is as follows Figure 3 As shown. Figure 3 It can be seen that under the irradiation of visible light of a certain wavelength, the monodentate ligand (i.e., compound 1) is released from the ruthenium metal center. Due to the changes in the substance before and after the irradiation, the free ligand peak and the solvent-coordinated ruthenium complex peak will be characterized in the mass spectrometry.

[0071] The UV-visible absorption spectrum of the complex solution under continuous illumination was measured by a Perkin-Elmer Lambda spectrophotometer. Figure 4 As shown in a, Figure 4 b in Figure 4 A partial enlargement of a in FIG. In order to quantitatively evaluate the photodissociation reaction rate under irradiation with light sources of different powers by monitoring the changes in the intensity of the characteristic peaks of the complex absorption spectrum, the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 mother solution was diluted to the required 10μmol / L with phosphate buffer. The experiment was carried out in a quartz cuvette containing 3 ml of the complex solution. The light source (450nm, 2.4J / cm 2 The stirred sample solution was irradiated for 10 min. After each irradiation period, the UV-visible absorption spectrum of the sample solution was measured and analyzed using Microsoft Excel. The photolysis rate of the complex was calculated as follows:

[0072] y=(y0―y max )exp(-kt)+y max

[0073] y0: Absorption value at 445nm at the beginning; y max : absorbance at 445 nm at the end; t: time; k: reaction rate obtained by nonlinear fitting; Figure 4It can be seen that the UV-visible absorption spectrum of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 also changes significantly before and after light irradiation. The intensity of the metal-ligand charge transfer absorption band at 445nm is weakened and red-shifted, which is caused by the photoinduced ligand dissociation of the complex. Figure 4 The absorption value at 445nm in the spectrum is fitted nonlinearly using the above formula, as shown in the following example: Figure 5 As shown in the figure, the calculated complex still has a good photodissociation reaction rate (k = 1.157min) under low power activation. -1 ).

[0074] [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2(i.e. Figure 6 The Ru-nitrogen mustard stock solution was diluted to 10 μmol / L with chromatography grade acetonitrile, and 1 ml of the diluted solution was placed in a 2 ml microcentrifuge tube and placed under an excitation light source (450 nm, 4 mW / cm 2 ) for 300 seconds. Using high performance liquid chromatography, C18 reverse phase chromatography column, with A (ultrapure water) and B (chromatographic grade methanol) as linear gradient, flow rate of 0.5 ml / min, monitoring UV absorption at 285 nm, the specific test figure is as follows Figure 6 As shown, Figure 6 a in the figure is the ligand dissociation process of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 under light. Figure 6 b in the figure is the stability test diagram of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 under dark conditions. Figure 6 It can be seen that under the conditions of 150s and 300s of light irradiation, both 4-ppyNM and 4,4'-bpy ligands dissociate, and with the extension of light irradiation time, the amount of ligand dissociation increases. However, when placed in the dark for 24h, the ligands do not dissociate. Therefore, in the present invention, [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 has only one peak corresponding to the complex [Ru(dip)2(4,4'-bpy)(4-ppyNM )]Cl2, indicating that its structure is stable; after illumination, the peak intensity of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 decreased significantly, and two new peaks were also observed, corresponding to the nitrogen mustard group ligand (4-ppyNM) and 4,4'-bipyridine ligand (4,4-ppy), indicating that [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 can quickly dissociate from the ligand under illumination conditions, thereby achieving a photoactivation effect.

[0075] (1) Complex (photo) cytotoxicity test:

[0076] The photocytotoxicity of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2, 4,4'-bipyridine, chlorambucil, and cisplatin on cells was studied using the classical MTT assay. Human alveolar basal epithelial cells (i.e., A549R cells) were seeded in 96-well plates (1×10 4 After 24 hours of attachment, the cells were replaced with a medium containing a concentration gradient of complex dilutions and incubated for 24 hours. Fresh medium containing 10% fetal bovine serum was then replaced. The dark toxicity group was incubated in the dark for another 24 hours. The phototoxicity group was exposed to a laser light source (450 nm, 2.4 J / cm 2 , 10min) and then incubated in the dark for 24 hours. After incubation, MTT solution (20 μl, 5 mg / ml) was added to each well and incubated for another 4 hours. The culture medium in each well was removed and 150 μl of dimethyl sulfoxide was added. The absorbance of each well at 595 nm was measured on a microplate reader to calculate the IC 50 The phototoxicity index PI was calculated, PI (%) = IC 50,Dark / IC 50,Light The experimental data are expressed as “mean ± standard deviation (n=3)”, and the specific test results are shown in Table 1 below.

[0077] Table 1 (Photo)cytotoxicity IC of different drugs on A549R cells 50 value

[0078]

[0079] As shown in Table 1, the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 prepared in the present invention has almost no cytotoxicity (IC 50 value greater than 100 μmol / L), and will be rapidly released under light conditions, with high cytotoxicity (IC 50 The value was 2.94±0.24μmol / L), and the phototoxicity index to A549R cells was >34.01%, which showed a good tumor treatment effect.

[0080] By studying the cell viability of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 before and after illumination and treatment, and comparing it with chlorambucil, it was found that [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 had good biocompatibility under the condition of no illumination and its cytotoxicity IC value on A549R cells was 0. 50 Value (IC 50,Dark) is greater than 100 μmol / L, indicating that the dark cytotoxicity of [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex to A549R cells is negligible. Under light conditions, [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 exhibits sharply enhanced photocytotoxicity, with an IC value of 50 Value (IC 50,Light ) was 2.94 μmol / L, which was significantly stronger than chlorambucil. The phototoxicity index (IC 50,Dark / IC 50,Light ) is 34.01, which further illustrates that [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 achieves the controlled release of nitrogen mustard group ligands and exhibits excellent photoselective cytotoxicity, providing a new and effective solution strategy for solving the defects of nitrogen mustard drugs during use.

[0081] (2) Research on the mechanism of photoactivation-induced cell death:

[0082] A549R cells were seeded in 96-well plates (1×10 4 cells) and cultured in a 37°C, 5% CO2 incubator for 24 h. Then, apoptosis (z-VAD-fmk, 10 μmol / L), necrosis (necrostatin-1, Nec-1, 50 μmol / L), autophagy (3-methyladenine, 3-MA, 100 μmol / L), Fe 2+ Ferroptosis (deferoxamine mesylate, DFO, 100 μmol / L), ferroptosis (cycloheximide, CHX, 0.1 μmol / L), and non-Fe 2+ Cells were pretreated with dilutions of ferroptosis-dependent ferroptosis (ferrostatin-1, Fer-1, 50 μmol / L) and pyroptosis (disulfiram, 4 μmol / L) inhibitors for 0.5 h, and then a drug-only group was set up. The drug-only group did not use any death inhibitors, and then [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (3 μmol / L) was added and incubated for 12 h. The culture medium was replaced with 10% fetal bovine serum, and then light treatment (450 nm, 2.4 J / cm 2, 10min) or continue incubation in the dark for 12h, then add 10μL of MTT solution to each well. After incubation for 4h, remove the culture medium and add 150μL of DMSO solution to each well. Measure the absorbance of each well at 595nm using a microplate reader, and then calculate the cell viability using the formula: average absorbance of each group / average absorbance of the blank control group. Data are expressed as "mean ± standard deviation (n = 3)". Specific test results are as follows Figure 7 The specific test data are shown in Table 2 below.

[0083] Table 2 A549R cell viability under treatment with different death inhibitors

[0084] Test Group Cell viability (%) Test Group Cell viability (%) Only drug group 51.5±1.2 DFO 56.9±4.1 z-VAD-fmk 45.4±3.8 CHX 52.6±2.3 Nec-1 46.9±2.1 Fer-1 83.6±7.5 3-MA 48.2±3.6 Disulfiram 71.5±5.2

[0085] As shown in Table 2, ferroptosis and pyroptosis inhibitors significantly increased cell survival after light treatment with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex, indicating that photoactivation of the complex-induced ferroptosis and pyroptosis is the primary cause of cell death. Based on these preliminary screening results, we further explored the biological role of photoactivation of the complex in these two cell death mechanisms.

[0086] (3) Comet assay:

[0087] The comet assay was used to investigate cellular DNA damage. A549R cells were treated with [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (3 μmol / L) (i.e., Ru3) and cisplatin (i.e., CDDP, 75.0 μmol / L). A control group (no treatment) was also established. After 12 hours, fresh culture medium containing 10% fetal bovine serum was replaced and the cells were irradiated (450 nm, 2.4 J / cm 2 , 10 min) or incubated in the dark for 12 hours. Afterwards, cells were harvested and washed three times with cold PBS. The suspension was mixed with 0.8% by mass low-melting point agarose at a volume ratio of 1:9 and applied to a slide coated with 1% by mass normal agarose. The low-melting point agarose was then spread on the slide. The slide was placed at 4°C for 15 minutes, then immersed in cold dissolution solution for 70 minutes and washed. The slide was then electrophoresed in alkaline electrophoresis buffer (0.3 mol / L sodium hydroxide and 1 mol / L EDTA, pH = 13) on ice for 20 minutes (25 V / 300 mA). After electrophoresis, the slide was washed twice in deionized water and then immersed in 70% alcohol for 5 minutes. Finally, the treated slides were stained with GeneFinder™ nuclear stain for 20 minutes and analyzed using a confocal microscope (Carl Zeiss, LSM 800, Germany). The specific test figure is shown in the figure below. Figure 8As shown. Figure 8 A549R cells co-incubated with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex in the dark showed no tailing, indicating that the nitrogen mustard ligand was caged by the ruthenium(II) photocage and inactive. In contrast, A549R cells co-incubated with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and then exposed to light showed a distinct broom-like tail, and the extent of the tail was more pronounced than in the cisplatin group. This confirms that the nitrogen mustard ligand and ruthenium(II) hydrate released by photoactivation of the complex induce severe DNA damage in cisplatin-resistant cells.

[0088] (4) Determination of intracellular GSH levels:

[0089] Intracellular GSH levels were detected using the GSHtracer probe. A549R cells were seeded in a 35 mm confocal dish and allowed to adhere for 24 h. [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (i.e., Ru3, 3 μmol / L) was added and incubated for 12 h. The cells were then irradiated with light (450 nm, 2.4 J / cm 2 , 10min) or continue incubation in the dark for 6h, then remove the culture medium, add GSHtracer dilution (10μmol / L) and incubate for 1.5h, remove the staining solution, wash three times with PBS, and examine the treated cells using a confocal microscope. The GSHtracer probe is excited at 405nm and the emission is collected at 500-550nm. The specific test diagram is shown in the figure below. Figure 9 As shown by Figure 9 It can be seen that the green fluorescence intensity in A549R cells co-incubated with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and treated with light is the weakest. This indicates that the photoactivation of the complex releases the nitrogen mustard group ligand, which can induce a decrease in intracellular GSH levels.

[0090] (5) Intracellular lipid peroxide (LPO) detection:

[0091] The lipid peroxide (LPO) content in cells was detected using a specific lipid peroxide (LPO) probe C11-BODIPY. 2 The cells were cultured in confocal dish for 24 h until adhered to the wall. [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (i.e., Ru3, 3 μmol / L) and positive control drug Erastin (10 μmol / L) were added and incubated for 12 h, and then treated in the dark or with light (450 nm, 2.4 J / cm 2, 10min) and then continued to incubate for 6h. After the incubation, the cells were washed with PBS and stained in C11-BODIPY (10μmol / L) diluted in serum-free medium for 30min. The treated cells were examined by confocal microscopy. The specific test figure is shown in the figure below. Figure 10 As shown. The C11-BODIPY probe was excited at 488 nm and the emission was collected at 500-550 nm. Figure 10 It can be seen that the lipid peroxide fluorescence intensity of cells co-incubated with [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and treated with light is stronger than that in other treatment groups, indicating that the photoactivation of the complex leads to an increase in the lipid peroxide content in A549R cells.

[0092] (6) Western blot analysis:

[0093] A549R cells were seeded in 10 cm 2 [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (i.e., Ru3, 3μmol / L) was added and incubated for 12h. The cells were then irradiated with light (450nm, 2.4J / cm 2 After incubation for 10 minutes (10 minutes) or for 12 hours in the dark, the cells were washed three times with pre-chilled PBS, digested with trypsin, collected in a centrifuge tube, and centrifuged at 4°C. The supernatant was discarded. The cells were lysed with RIPA solution containing protease and phosphatase inhibitors at 4°C for 30 minutes. After lysis, the cells were centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was collected. After quantification of the protein concentration of each histone using a BCA protein assay kit, loading buffer was added to each histone solution in proportion. After thorough mixing, the solution was heated to 100°C for 5 minutes and stored at -20°C until use. An equal amount of protein sample was added to each lane of an SDS-PAGE gel for electrophoresis. The membrane was then transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk solution for 1 hour, washed three times with TBST buffer, incubated with a specific primary antibody overnight at 4°C, and then incubated with an HRP-conjugated secondary antibody for 1 hour at room temperature. The Omega Lum C imaging system (Aplegen, USA) was used to visualize and analyze the levels of GPX4, GSDMD-N, C-CASP1, and β-Actin in A549R cells under different treatments. Figure 11 As shown. Figure 11It can be seen that GPX4 expression was significantly downregulated in cells co-incubated with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and exposed to light, while the expression levels of cleaved Caspase-1 and GSDMD-N were significantly increased. Overall, these results suggest that upon light activation, the complex releases the nitrogen mustard group ligand, which depletes intracellular glutathione, leading to increased cellular lipid peroxide levels, downregulating GPX4 expression, and causing ferroptosis. This also leads to severe cellular DNA damage, further activating Caspase-1 to cleave GSDMD, and thus causing pyroptosis.

[0094] (7) Testing of damage molecule-related patterns:

[0095] CRT immunofluorescence staining experiment: A549R cells were seeded on 35 mm 2 The cells were cultured in a confocal dish for 24 h until they adhered to the wall. [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (i.e., Ru3, μmol / L) was added and incubated for 12 h. The cells were then irradiated with light (450 nm, 2.4 J / cm 2 , 10 min) or incubate in the dark for 12 h, then remove the culture medium, fix the cells with 4% paraformaldehyde for 10 min, permeabilize with cold methanol at -20°C for 15 min, wash three times with pre-cooled PBS, and add Calreticulin (D3E6) XP@Rabbit mAb (Alexa Fluor 500). 488Conjugate) dilution at 4 ° C overnight, washed three times with pre-cooled PBS, and then diluted with Hoechst (λ ex =405nm,λ em =430-460nm) staining for 15min, and finally, after washing three times with PBS, imaging was performed using a confocal microscope, with the fluorescent secondary antibody excited at 563nm and the emission collected at 600-640nm. Figure 12 As shown by Figure 12 It can be seen that after cells were co-incubated with [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and treated with light, the transfer of CRT from the endoplasmic reticulum to the cell membrane was clearly observed by immunofluorescence confocal imaging.

[0096] HMGB1 immunofluorescence staining experiment: A549R cells were seeded on 35 mm 2 The cells were cultured in a confocal dish for 24 h until adhered to the wall. [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (i.e., Ru3, 3 μmol / L) was added and incubated for 12 h. The cells were then irradiated with light (450 nm, 2.4 J / cm 2, 10 min) or continue incubation in the dark for 12 h, then remove the culture medium, fix the cells with 4% paraformaldehyde for 10 min, wash three times with pre-cooled PBS, incubate with HMGM1 antibody dilution at 4 ° C overnight, wash three times with PBS, add Anti-rabbit IgG (H + L), F (ab') 2 Fragment (Alexa 555Conjugate) fluorescent secondary antibody dilution solution was incubated at room temperature for 2 h, washed three times with PBS, and diluted with Hoechst (λ ex =405nm,λ em =430-460nm) staining for 15min, and finally, after washing three times with PBS, imaging was performed using a confocal microscope. The fluorescent secondary antibody was excited at 563nm and the emission was collected at 600-640nm. The specific test results are shown in Figure 2. Figure 13 As shown. Figure 13 It was found that HMGB1 protein also migrated from the nucleus to the cytoplasm in cells co-incubated with the [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and then irradiated with light. However, this phenomenon was not observed in the other groups.

[0097] ATP extracellular release assay: ATP bioluminescence assay kit (Promega) was used to detect extracellular ATP. A549R cells were seeded in 96-well plates (1 × 10 cells per well). 4 cells) and cultured for 24 h until adhered. [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 (3 μmol / L) was added and incubated for 12 h, followed by light treatment (450 nm, 2.4 J / cm 2 , 10min) or continue incubation in the dark for 12h. 100μL of supernatant from each well was taken out and placed in a 96-well white plate. An equal volume of ATP detection reagent was added. After thorough mixing, the chemiluminescent signal of ATP in each group was measured using a TECAN Infinite M200 PRO multifunctional microplate reader. The specific test results are shown in the figure. Figure 14 As shown. Figure 14 It can be seen that significant extracellular ATP release was detected only in cells co-incubated with [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 complex and treated with light.

[0098] In summary, the ruthenium-based photoactivated chemotherapy prodrug [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 in the present invention innovatively designs a new nitrogen mustard group ligand and combines it with a ruthenium-based photocage molecule to design and synthesize a new type of ruthenium-based photoactivated chemotherapy prodrug, which can realize the light-controlled and precise release of nitrogen mustard drugs, effectively solving the defect of uncontrollable toxic side effects of nitrogen mustard drugs during treatment, and exhibiting a stronger anti-tumor effect than nitrogen mustard under light, effectively overcoming tumor resistance; and [Ru(dip)2(4,4'-bpy)(4-ppyNM)]Cl2 releases the nitrogen mustard ligand through light, causing severe DNA damage to tumor cells, and also binds to intracellular GSH, downregulating GPX4 protein expression, leading to lipid peroxidation (LPO) accumulation, inducing A549R cell pyroptosis and ferroptosis, and further triggering immunogenic cell death. In general, the present invention utilizes a novel ruthenium (II) complex photocage molecule to cage a ligand containing a nitrogen mustard group, achieving light-controlled and precise release of the nitrogen mustard drug under low-power visible light irradiation, effectively solving the defect of uncontrollable toxic side effects of nitrogen mustard drugs during treatment, and providing new ideas for the development of novel low-power ruthenium-based photoactivated chemotherapy drugs to induce specific death modes of tumor cells and activate immunity.

[0099] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt, ester, isomer, or solvate thereof, wherein Each R1 is independently selected from C 1～10 Alkyl, C 1～10 Alkoxy, -OH; Each x is independently selected from 0, 1, 2, 3, 4 or 5; X is selected from F, Cl, Br or I; m and n are independently selected from 1, 2, 3, 4 or 5.

2. The compound according to claim 1, characterized in that: Each R1 is independently selected from C 1～5 of alkyl.

3. The compound according to claim 1, characterized in that: x is 0.

4. The compound according to claim 1, characterized in that: The compound is selected from 5. The method for preparing the compound according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Make and Perform reductive amination reaction to obtain a ligand; S2: Make the ligand reaction, and then SOX2 reaction, prepared; The structural formula of the ligand is: y is selected from 0, 1, 2, 3 or 4; X, R1, x, m, and n are as defined in any one of claims 1 to 4.

6. The method for preparing the compound according to claim 5, wherein: Said The reaction is carried out in the presence of silver trifluoromethanesulfonate.

7. A photoactivated chemotherapy drug composition, characterized in that: The invention comprises a therapeutically effective amount of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, ester, isomer or solvate thereof, and a pharmaceutically acceptable excipient.

8. The photoactivated chemotherapy drug composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is selected from tablets, capsules, granules, oral liquids, injections, ointments, patches, aerosols, gels or drops.

9. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, ester, isomer or solvate thereof in the preparation of a drug for photoactivated cancer treatment.

10. The use according to claim 9, characterized in that: The cancer includes lung cancer, esophageal cancer, cervical cancer, breast cancer, prostate cancer, gastric cancer, leukemia, neuroblastoma, nasopharyngeal cancer, thyroid cancer, oral cancer, ovarian cancer, endometrial cancer, bladder cancer, kidney cancer, colorectal cancer, liver cancer or pancreatic cancer.

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