A dihydroporphyrin compound, its preparation method and application

Abstract

This invention belongs to the field of organic synthesis technology, specifically relating to a dihydroporphyrin compound, its preparation method, and its applications. This invention provides a dihydroporphyrin compound having the structure shown in any of Formulas I to VII. This invention improves the physicochemical properties of the dihydroporphyrin core by structural modification and coupling it with amino acids, optimizing its distribution in tissues and its tumor-targeting ability. Simultaneously, it introduces p-toluenesulfonamide as an endoplasmic reticulum-oriented functional module, significantly enhancing its localization ability to the endoplasmic reticulum, thereby improving the compound's antitumor activity and immune response activation ability in vitro and in vivo, achieving precise localization and efficient enrichment in tumor tissues and cells.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a dihydroporphyrin compound, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are non-invasive targeted therapies. Their main principle is to deliver photosensitizing or sonodynamic drugs to the target site, using specific wavelengths of light or specific frequencies of ultrasound for targeted local irradiation. This causes the photosensitizing or sonodynamic drugs at the target site to generate cytotoxic reactive oxygen species (ROS), inducing apoptosis in target cells. PDT is widely used in the treatment of superficial tumors due to its high ROS yield and significant killing effect. However, its limited tissue penetration depth (usually <1 cm) poses a challenge in the treatment of deep solid tumors. SDT, developed from PDT, leverages the strong penetrating power of ultrasound (up to 7-10 cm) to overcome PDT's shortcomings in tissue penetration. It is particularly suitable for the treatment of deep tumors and possesses advantages such as high specificity and few side effects, showing promising clinical translation prospects. The efficacy of PDT and SDT is highly dependent on the performance of the photosensitizers and sonodynamic agents. Among these, tumor targeting capability, subcellular localization accuracy, and biosafety are key parameters determining their clinical effectiveness and translational prospects. Therefore, developing sensitizers that combine high targeting efficiency with good biocompatibility to achieve a highly efficient delivery system that targets tumors at the organelle level is a crucial strategy for improving the precision of treatment and promoting its clinical translation. In the field of photosensitizers or acoustic sensitizers, dihydroporphyrin compounds have attracted considerable attention due to their excellent photophysical properties. These compounds exhibit strong absorption in the 650-800 nm near-infrared region, significantly improving tissue penetration depth compared to first-generation photosensitizers, and possessing good structural modifiability, providing an important foundation for the development of highly efficient and low-toxicity photosensitizers and acoustic sensitizers. Currently, several dihydroporphyrin drugs, such as verteporfen, temoporfen, and taraporfen (NPe6), are widely used clinically. However, existing dihydroporphyrin sensitizers still have many limitations: poor water solubility, slow in vivo clearance, insufficient tumor targeting, and a tendency to cause adverse reactions such as skin phototoxicity; simultaneously, their immune activation capacity is weak, making it difficult to effectively induce anti-tumor immune responses, thus limiting their long-term efficacy and metastasis inhibition effects. Therefore, rationally modifying the structure of dihydroporphyrin molecules to improve their water solubility, enhance tumor targeting, optimize pharmacokinetic properties, and improve their anti-tumor immune activation has become a key research direction for further improving the therapeutic performance of this type of compound.

[0003] Improving tumor targeting not only enhances the generation of reactive oxygen species (ROS) at tumor sites, thereby increasing treatment efficiency, but also reduces off-target damage to normal tissues, lowers systemic toxicity, and broadens the therapeutic window. However, due to the extremely short lifespan of ROS (approximately 40 ns) and limited diffusion radius (approximately 20 nm), their biological effects are highly dependent on the precise localization of photosensitive or acoustic sensitizers in subcellular structures. The endoplasmic reticulum (ER), a key organelle regulating protein synthesis and folding, calcium homeostasis, and cellular stress responses, can experience ER stress due to dysfunction. This stress can activate unfolded protein responses, and sustained ER stress can further induce immunogenic cell death (ICD) and promote the release of damage-associated molecular patterns (DAMPs), ultimately initiating a systemic anti-tumor immune response and achieving effective control of distant tumors and metastatic lesions. Based on this mechanism, achieving the cascade enrichment of dihydroporphyrin-based photosensitive or acoustic sensitizers in tumor cells and the ER has become an effective strategy to improve treatment efficiency and synergize photodynamic or acoustic therapy with immunotherapy.

[0004] In summary, addressing the limitations of existing dihydroporphyrin-based photosensitizers in terms of targeting, water solubility, in vivo clearance rate, and immune activation, this invention optimizes their structure through rational molecular design: on one hand, it introduces a lipophilic module targeting the endoplasmic reticulum (ER) to enhance its affinity and localization ability to the ER and improve cell membrane penetration efficiency; on the other hand, it connects a hydrophilic fragment to improve the compound's water solubility and blood circulation stability. This strategy aims to synergistically optimize the drug's pharmacokinetic properties and tumor-targeting ability, achieving cascade targeting from tumor tissue to the ER while reducing systemic toxicity. These novel sensitizers not only enhance ROS-mediated direct killing effects but also effectively induce ICD and activate anti-tumor immune responses, thereby achieving synergistic effects of photodynamic or sonodynamic therapy and immunotherapy at the single-molecule level. This invention is expected to overcome the technical bottlenecks of existing dihydroporphyrin-based sensitizers, providing crucial support for their clinical translation. Summary of the Invention

[0005] The purpose of this invention is to provide a dihydroporphyrin compound, its preparation method, and its application. This compound can be used as a photosensitizer or sonosensitizer in the field of antitumor therapy, enabling cascade targeting from tumor tissue to the endoplasmic reticulum, achieving single-molecule-mediated PDT or SDT synergistic therapy with immunotherapy, thereby significantly enhancing the antitumor effect.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a dihydroporphyrin compound having the structure shown in any of formulas I to VII: ; R1, R8, and R9 are independently hydroxyl groups. Formula R1-1 , Formula R 1-2 or Formula R 1-3 ; In equations I and III-VII, R² is... Formula R 2-1 ; In Equation II, R2 is H or Formula R 2-1 ; n is any integer from 1 to 5.

[0007] The dihydroporphyrin compounds provided by this invention have the following advantages: (1) On the one hand, the endoplasmic reticulum targeting functional module is coupled with methylbenzenesulfonamide to improve its endoplasmic reticulum targeting ability and achieve cascade targeting of tumors. At the same time, the module has a certain lipophilicity to improve membrane permeability.

[0008] (2) On the other hand, the amino acids or coupled amino acids significantly change their physicochemical properties, optimize their tissue distribution and improve their tumor targeting ability.

[0009] (3) Light or sound activation: The local ROS (half-life < 50 ns) generated by light or sound sensitizers in the endoplasmic reticulum directly acts on the calcium ion channels, a key component of the endoplasmic reticulum, selectively disrupts the homeostasis of the endoplasmic reticulum, amplifies the stress level of the endoplasmic reticulum, strengthens the ICD effect, promotes the maturation of dendritic cells and the infiltration of cytotoxic T cells, and establishes long-term anti-tumor immune memory.

[0010] Data from the embodiments show that the dihydroporphyrin compounds provided by this invention exhibit significantly superior tumor targeting in vivo compared to clinical control drugs, enabling imaging of small tumor foci, and demonstrating accelerated in vivo metabolism and higher biosafety. In in vitro and in vivo sonodynamic antitumor activity evaluations, the compounds showed significantly higher sonodynamic activity against the 4T1 breast cancer cell line than the clinical positive control drug, and exhibited enhanced endoplasmic reticulum targeting ability, effectively inducing ICD, thereby enhancing the therapeutic effect on in situ tumors, metastatic tumors, and deep tumors. In in vitro photodynamic antitumor activity evaluations, the compounds showed significant inhibitory activity against both the B16 melanoma cell line and the MOC-1 oral squamous cell carcinoma cell line, with photodynamic antitumor effects significantly superior to existing clinical drugs.

[0011] The method for preparing the dihydroporphyrin compound provided by this invention is simple and suitable for industrial production. The dihydroporphyrin derivatives and their pharmaceutically acceptable salts of this invention, as novel photo / sound-sensitive drugs with excellent performance, can be used in photo / sound-dynamic therapy, immunotherapy, and combined therapy for tumors. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Fluorescence kinetic imaging of compound 2c in a 4T1 in situ breast cancer model; Figure 2 Image A shows solid tumor imaging of compound 2c in a 4T1 in situ breast cancer model; Image B shows microtumor imaging of compound 2c in a 4T1 in situ breast cancer model. Figure 3 Fluorescence kinetic imaging of compound 1b in a 4T1 in situ breast cancer model; Figure 4 This demonstrates the endoplasmic reticulum targeting ability of compound 1a. Figure 5 The ICD enhancement of compound 1a under ultrasound or without ultrasound is shown in Figure A, where A represents the immunofluorescence of calreticulin in each group; B represents the immunofluorescence of high-mobility group box B1 in each group; and C represents the bar graph of cellular ATP release. P <0.01~0.001); Figure 6 Compound 1a activates dendritic cells with or without ultrasound. Figure 7 A bar chart showing the effects of compounds 1a and 1b on the survival rate of 4T1 breast cancer cells. P <0.01~0.001); Figure 8 The bar graph shows the effect of compounds 1a and 1b on the survival rate of 4T1 breast cancer cells under ultrasound conditions. P <0.01~0.001); Figure 9 A bar graph showing the inhibitory effect of compound 1a on the weight of in situ breast cancer tumors under ultrasound or without ultrasound. P <0.01~0.001); Figure 10 The inhibitory effect of compound 1a on the number of lung metastatic nodules under ultrasound or without ultrasound ( P <0.01~0.001); Figure 11 The bar graph shows the inhibitory effect of compound 1a on tumor weight in an orthotopic pancreatic cancer model under ultrasound or without ultrasound. P <0.01~0.001); Figure 12 In the graph, A is a bar chart showing the effect of compounds 2a and 3a on the survival rate of B16 cells under light illumination; B is a bar chart showing the effect of compounds 2a and 3a on the survival rate of MOC-1 cells under light illumination. P <0.01~0.001); Figure 13 The bar graph shows the inhibitory effect of compound 3a on tumor weight in a B16 melanoma model under light conditions. P <0.01~0.001). Detailed Implementation

[0014] This invention provides a dihydroporphyrin compound having the structure shown in any of formulas I to VII: ; R1, R8, and R9 are independently hydroxyl groups. Formula R 1-1 , Formula R 1-2 or Formula R 1-3 ; In equations I and III-VII, R² is... ; In Equation II, R2 is H or ; n is any integer from 1 to 5.

[0015] This invention also provides a method for preparing the dihydroporphyrin compounds described in the above technical solution, including the following six cases: The first type: When the dihydroporphyrin compound has the structure shown in Formula I, Formula IV or Formula VI and R1 is a hydroxyl group: This invention involves methylating compound 1, compound 4, or compound 6 under acidic conditions to obtain compound 1a-1, compound 4a-1, or compound 6a-1. Specifically, the methylation is performed by dissolving compound 1, compound 4, or compound 6 in a mixed solution of CH3OH and acid, and then methylating the resulting solution.

[0016] In one embodiment of the present invention, the acid can be concentrated sulfuric acid; the concentration of the concentrated sulfuric acid can be 18.4 mol / L; the volume ratio of CH3OH to concentrated sulfuric acid is preferably 1:0.05~0.01; the concentration of compound 1, compound 4 or compound 6 in the solution can be 0.05~0.5 M; in one embodiment of the present invention, the methyl esterification temperature can be room temperature, and the time can be 2~6 h; in one embodiment of the present invention, after the methyl esterification reaction, the reaction solution is further subjected to solvent evaporation, ethyl acetate dilution, and washing in sequence, the resulting organic phase is dried and concentrated, and the concentrate is purified by silica gel column chromatography, wherein the eluent for the silica gel column chromatography is preferably a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone in the petroleum ether-acetone system is preferably 10:1.

[0017] The structural formulas of compounds 1, 4, and 6 are as follows: ; The structural formulas of compounds 1a-1, 4a-1, or 6a-1 are as follows: ; After obtaining compound 1a-1, compound 4a-1 or compound 6a-1, the present invention performs a first olefin metathesis reaction between compound 1a-1, compound 4a-1 or compound 6a-1 and a substituted terminal alkene under the condition of a catalyst to obtain compound 1a-2, compound 4a-2 or compound 6a-2.

[0018] The structural formula of the substituted terminal olefin is: ; The structural formulas of compounds 1a-2, 4a-2, or 6a-2 are as follows: ; In one embodiment of the present invention, the catalyst for the first olefin metathesis reaction is preferably a Grubbs' catalyst, and the solvent for the first olefin metathesis reaction is preferably one or more of dichloroethane, dichloromethane, and toluene; the molar ratio of compound 1a-1, compound 4a-1 or compound 6a-1, the substituted terminal olefin, and the catalyst is preferably 1:2~30:0.01~0.7, more preferably 1:4:0.4; the temperature of the first olefin metathesis reaction is preferably 40~70℃, more preferably 40~60℃; the holding time is preferably 6~36 h, more preferably 12~20 h. In another embodiment of the present invention, after the first olefin metathesis reaction, the reaction solution is washed, the resulting organic phase is dried and evaporated, and the residue is subjected to silica gel column chromatography, wherein the eluent for the silica gel column chromatography is preferably a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone in the petroleum ether-acetone system is preferably 6:1.

[0019] After obtaining compound 1a-2, 4a-2 or 6a-2, the present invention performs a first deprotection on compound 1a-2, compound 4a-2 or compound 6a-2 in an acid solution to obtain compound I, compound IV or compound VI when R1 is a hydroxyl group.

[0020] In one embodiment of the present invention, during the first deprotection, the acid solution comprises a strong acid and acetone, wherein the strong acid comprises sulfuric acid and / or hydrochloric acid; the hydrochloric acid is preferably concentrated hydrochloric acid; the volume concentration of the strong acid in the acid solution is preferably 10-60%, more preferably 33%; the concentration of compound 1a-2, 4a-2 or 6a-2 in the reaction solution is preferably 0.05-0.5 M, more preferably 0.1 M; the reaction temperature is preferably 0-30℃, more preferably 0℃; the reaction time is preferably 0.5-8 h, more preferably 4 h. In another embodiment of the present invention, after the first deprotection, the reaction further includes evaporating the solvent from the first deprotection reaction solution, diluting it with ethyl acetate, washing it, drying the resulting organic phase, concentrating it, and purifying the residue by silica gel column chromatography, wherein the eluent is preferably a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol is preferably 20:1.

[0021] Taking the preparation of compound I (denoted as 1a) with R1 as a hydroxyl group as an example, the chemical reaction formula is as follows: ; The second type: when the dihydroporphyrin compound has the structure shown in formula I, IV or VI and R1 is R 1-1 R 1-2 Or R 1-3 The preparation method includes the following steps: In this invention, compound 1, compound 4 or compound 6 are reacted with amino acid tert-butyl hydrochloride under alkaline conditions and condensing agent to undergo a first amide condensation reaction to obtain compound 1b-1, compound 4b-1 or compound 6b-1. The structural formulas of compounds 1b-1, 4b-1, or 6b-1 are as follows: ; R6 is , , ; The general structural formula of the amino acid tert-butyl hydrochloride is as follows: , or .

[0022] In one embodiment of the present invention, the condensing agent for the first amide condensation reaction preferably includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), dicyclohexylcarbodiimide (DCC), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride). One or more of the following are preferred: the alkaline substance preferably includes N,N-diisopropylethylamine (DIPEA) and / or triethylamine (TEA); the solvent for the first amide condensation reaction is preferably dichloromethane and / or DMF, more preferably dichloromethane; the molar ratio of compound 1, compound 4 or compound 6, condensing agent, alkaline substance and amino acid tert-butyl hydrochloride is preferably 1:1~10:1~10:1~10, more preferably 1:1~3:1~3:1~3, and even more preferably 1:3:3:1.2. As one embodiment of the present invention, the temperature of the first amide condensation reaction is preferably 30°C; the reaction time is preferably 4~20 h, more preferably 12 h. In one embodiment of the present invention, after the first amide condensation reaction, the first amide condensation reaction is further washed, the organic phase obtained by washing is dried and concentrated, and the residue is purified by silica gel column chromatography. The eluent is preferably a dichloromethane-ethyl acetate system; the volume ratio of dichloromethane to ethyl acetate is preferably 100~30:1; the elution is preferably gradient elution.

[0023] After obtaining compound 1b-1, compound 4b-1 or compound 6b-1, compound 1b-1, compound 4b-1 or compound 6b-1 are subjected to a second olefin metathesis reaction with a substituted terminal alkene under the condition of a catalyst to obtain compound 1b-2, compound 4b-2 or compound 6b-2.

[0024] The structures of compound 1b-2, compound 4b-2, or compound 6b-2 are respectively: ; In one embodiment of the present invention, the molar ratio of compound 1b-1, compound 4b-1 or compound 6b-1, the substituted terminal alkene, and the catalyst is 1:2~30:0.01~0.7, more preferably 1:4:0.4; the solvent, catalyst, temperature, and time of the second hydrocarbon metathesis reaction are preferably the same as those of the first olefin metathesis reaction, and will not be repeated here. In another embodiment of the present invention, after the second olefin metathesis reaction, it is preferable to further wash the second olefin metathesis reaction solution, dry and concentrate the obtained organic phase, and purify the residue by silica gel column chromatography, with the eluent preferably being a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone in the petroleum ether-acetone system is preferably 5:1.

[0025] After obtaining compound 1b-2, compound 4b-2, or compound 6b-2, the present invention performs a second deprotection on said compound 1b-2, compound 4b-2, or compound 6b-2 in an acid solution to obtain R1 as R 1-1 R 1-2 Or R 1-3 Compound I, compound IV, or compound VI at that time.

[0026] In one embodiment of the present invention, the acid in the acid solution preferably includes trifluoroacetic acid and / or hydrochloric acid; the solvent of the acid solution preferably includes acetone and / or tetrahydrofuran; the volume concentration of the acid in the acid solution is preferably 10-60%, more preferably 33%; the concentration of compound 1a-2, 4a-2 or 6a-2 in the reaction solution is preferably 0.05-0.5 M, more preferably 0.1 M. In one embodiment of the present invention, the reaction temperature for the second deprotection is preferably 25°C, and the reaction time is preferably 0.5-8 h, more preferably 4 h. In one embodiment of the present invention, after the second deprotection, the mixture is further washed after evaporation, the resulting organic layer is dried and concentrated, and the residue is purified by silica gel column chromatography; the eluent for the silica gel column chromatography purification is a dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is preferably 20:1.

[0027] Let R1 be R 1-1 Taking compound I (denoted as 1b) as an example, the chemical equation is: ; The third type: When the dihydroporphyrin compound has the structure shown in formula II, III, V or VII and R1, R8 and R9 are hydroxyl groups: In this invention, compound 2, compound 3, compound 5 or compound 7 are reacted with iodomethane under alkaline conditions to undergo a first methylation reaction to obtain compound 2a-1, compound 3a-1, compound 5a-1 or compound 7a-1; The structural formulas of compound 2, compound 3, compound 5, or compound 7 are as follows: ; The compounds 2a-1, 3a-1, 5a-1, or 7a-1 are respectively: ; In one embodiment of the present invention, the alkaline conditions for the first methylation reaction are provided by anhydrous potassium carbonate; the molar ratio of compound 2, compound 3, compound 5 or compound 7, iodomethane and anhydrous potassium carbonate is preferably 1:3~20:3~20, more preferably 1:3~10:3~20, and even more preferably 1:10:10~20. In another embodiment of the present invention, the temperature of the first methylation is preferably 20~40°C, more preferably 25°C, and the time is preferably 1~4 h.

[0028] In this invention, compound 2a-1, compound 3a-1, compound 5a-1 or compound 7a-1 are subjected to a third olefin metathesis reaction with a substituted terminal alkene under catalytic conditions to obtain compound 2a-2, compound 3a-2, compound 5a-2 or compound 7a-2.

[0029] The structural formulas of compounds 2a-2, 3a-2, 5a-2, or 7a-2 are as follows: ; As one embodiment of the present invention, the molar ratio of compound 2a-1, compound 3a-1, compound 5a-1 or compound 7a-1, the substituted terminal alkene and the catalyst is preferably 1:2~30:0.01~0.7, more preferably 1:4:0.4; the catalyst, solvent, temperature and time of the third olefin metathesis reaction are preferably the same as those of the second olefin metathesis reaction, and will not be repeated here.

[0030] In this invention, compound 2a-2, compound 3a-2, compound 5a-2 or compound 7a-2 are subjected to a third deprotection in an acid solution to obtain compound 2a-3, compound 3a-3, compound 5a-3 or compound 7a-3.

[0031] The compounds 2a-3, 3a-3, 5a-3, or 7a-3 are respectively: ; In one embodiment of the present invention, the acid in the acid solution is preferably trifluoroacetic acid; the volume concentration of the acid in the acid solution is preferably 25-60%, more preferably 33%; the temperature for the third deprotection is preferably 0-25°C, and the time is preferably 4 hours.

[0032] After obtaining compound 2a-3, compound 3a-3, compound 5a-3 or compound 7a-3, the present invention performs a first alkali deprotection on 2a-3, compound 3a-3, compound 5a-3 or compound 7a-3 in an alkaline solution to obtain compound II, compound III, compound V or compound VII with R1 being a hydroxyl group.

[0033] In one embodiment of the present invention, the concentration of compound 3a-3, compound 5a-3, or compound 7a-3 in the reaction solution is preferably 0.05~0.5 M; the alkaline solution is obtained by mixing a strong base solution and an organic solvent; the strong base in the strong base solution is preferably one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide; the concentration of the base in the alkaline solution is preferably 0.1~1 M, more preferably 1 M; the organic solvent is preferably tetrahydrofuran; the volume ratio of the strong base solution to the organic solvent is 1~3:1, more preferably 1:1. In one embodiment of the present invention, the deprotection temperature of the first base is preferably room temperature, and the reaction time is preferably 2~24 h, more preferably 12 h.

[0034] Taking the preparation of compound III (denoted as 3a) with R1 as a hydroxyl group as an example, the chemical equation is as follows: ; The fourth type: When the dihydroporphyrin compound has the structure shown in formula III or VII and R1 and R9 are hydroxyl groups, and R8 is a compound of formula R... 1-1 R 1-2 Or R 1-3 hour: The present invention involves reacting compound 3 or compound 7 with an amino acid methyl ester compound under alkaline conditions and a condensing agent to undergo a second amide condensation reaction, yielding compound 3b-1 or compound 7b-1.

[0035] The structures of compounds 3, 7, 3b-1, and 7b-1 are as follows: ; R7 is , , ; The structural formula of the amino acid methyl ester compound is: , , .

[0036] In one embodiment of the present invention, the solvent and condensing agent of the second amide condensation reaction are preferably the same as those of the first amide condensation reaction, and will not be described again; the molar ratio of compound 3 or compound 7, condensing agent, basic substance and amino acid methyl ester compound is preferably 1:1~1.2:1~2:1~1.2, more preferably 1:1.2:2:1.2; the temperature of the second amide condensation reaction is preferably room temperature, and the time is preferably 2~24 h, more preferably 12 h.

[0037] After compound 3b-1 or compound 7b-1, the present invention further involves reacting compound 3b-1 or compound 7b-1 with iodomethane under alkaline conditions to undergo a second methylation reaction, yielding compound 3b-2 or compound 7b-2.

[0038] The structural formulas of compound 3b-2 and compound 7b-2 are respectively; In one embodiment of the present invention, the alkaline conditions for the second methylation reaction are provided by anhydrous potassium carbonate; the molar ratio of compound 3b-1 or compound 7b-1, iodomethane and anhydrous potassium carbonate is preferably 1:2~10:2~20, more preferably 1:10:20; the temperature and time of the second methylation are preferably the same as those of the first methylation, and will not be repeated here.

[0039] After obtaining compound 3b-2 or compound 7b-2, the present invention reacts compound 3b-2 or compound 7b-2 with a substituted terminal alkene in the presence of a catalyst to undergo a fourth olefin metathesis reaction, yielding compound 3b-3 or compound 7b-3; the structural formulas of compound 3b-3 or compound 7b-3 are as follows: ; In one embodiment of the present invention, the molar ratio of compound 3b-2 or compound 7b-2, the substituted terminal alkene, and the catalyst is preferably 1:2~15:0.01~0.7, more preferably 1:4:0.4; the catalyst and solvent for the fourth olefin metathesis reaction are preferably the same as those for the second olefin metathesis reaction, and will not be described again here; the temperature of the fourth olefin metathesis reaction is 35~70℃, more preferably 40℃, and the holding time is preferably 12~36 h, more preferably 20 h.

[0040] After obtaining compound 3b-3 or compound 7b-3, the present invention performs a fourth deprotection on compound 3b-3 or compound 7b-3 in an acid solution to obtain compound 3b-4 or compound 7b-4.

[0041] The structures of compound 3b-4 or compound 7b-4 are as follows: ; In one embodiment of the present invention, during the fourth deprotection, the acid in the acid solution is preferably trifluoroacetic acid; the concentration and volume of the acid solution are preferably 25-60%, more preferably 30-33%. In another embodiment of the present invention, the reaction temperature for the fourth deprotection is preferably 0-25°C, more preferably 25°C; the reaction time is preferably 0.5-8 h, more preferably 4 h.

[0042] After obtaining compound 3b-4 or compound 7b-4, the present invention performs a second base deprotection on compound 3b-4 or compound 7b-4 in an alkaline solution to obtain R1 and R9 being hydroxyl groups and R8 being a compound of formula R. 1-1 R 1-2 Or R 1-3 Compound III or Compound VII. As one embodiment of the present invention, the conditions for the second base deprotection are preferably the same as those for the first base deprotection, and will not be repeated here.

[0043] To prepare R1 and R9 as hydroxyl groups, and R8 as R 1-1 Taking compound III(3b) as an example, the chemical equation is: Fifth type: When the dihydroporphyrin compound has the structure shown in Formula II or Formula V, R1 and R8 are R 1-1 R 1-2 Or R 1-3 R2 is R 2-1 The preparation method includes the following steps: Compound 2 or compound 5 is reacted with an amino acid methyl ester compound under alkaline conditions and a condensing agent to undergo a third amide condensation reaction to obtain compound 2b-1 or compound 5b-1. The structural formulas of compounds 2 and 5 are as follows: ; The structural formulas of compound 2b-1 and compound 5b-1 are as follows: In one embodiment of the present invention, the solvent and condensing agent of the third amide condensation reaction are preferably the same as those of the second amide condensation reaction, and will not be repeated here; the molar ratio of compound 2 or compound 5, condensing agent, basic substance and amino acid methyl ester compound is preferably 1:2~10:2~10:2~10, more preferably 1:2.5:2.5:2.5; the temperature and time of the third amide condensation reaction are preferably the same as those of the second amide condensation reaction, and will not be repeated here.

[0044] After obtaining compound 2b-1 or compound 5b-1, the present invention reacts compound 2b-1 or compound 5b-1 with a substituted terminal alkene under catalytic conditions to undergo a fourth olefin metathesis reaction to obtain compound 2b-2 or compound 5b-2. In one embodiment of the present invention, the catalyst and solvent for the fourth olefin metathesis reaction are preferably the same as those for the third olefin metathesis reaction, and will not be described again here.

[0045] After obtaining compound 2b-2 or compound 5b-2, the present invention performs a fifth deprotection on compound 2b-2 or compound 5b-2 in an acid solution to obtain compound 2b-3 or compound 5b-3; the structures of compound 2b-3 or compound 5b-3 are as follows: ; In this invention, the conditions for the fifth deprotection are preferably the same as those for the fourth deprotection, and will not be repeated here.

[0046] After obtaining compound 2b-3 or compound 5b-3, the present invention performs a third alkali deprotection on compound 2b-3 or compound 5b-3 in an alkaline solution to obtain R1 and R8 as R 1-1 R 1-2 Or R 1-3 And R2 is R 2-1 Compound II or Compound V. As one embodiment of the present invention, the conditions for the third base deprotection are preferably the same as those for the second base deprotection, and will not be described again.

[0047] Using R1 and R8 as preparations 1-1、 R2 is R 2-1 Taking compound II (denoted as 2b) as an example, the chemical equation is: ; The sixth type: When the dihydroporphyrin compound has the structure shown in Formula II, R1 and R8 are R 1-1 R 1-2 Or R 1-3 When R2 is H, the preparation method includes the following steps: Compound 2b-1 was subjected to a fourth base deprotection in an alkaline solution to obtain R1 and R8. 1-1 R 1-2 Or R 1-3 R2 is compound II or compound V, which is H. As one embodiment of the present invention, the conditions for the fourth base deprotection are preferably the same as those for the third base deprotection, and will not be described again.

[0048] The present invention also provides metal complexes of dihydroporphyrin compounds as described in the above technical solutions, having the structure shown in any one of formulas Ia to VII-a: ; In formulas Ia to VII-a, M is a divalent, trivalent, or tetravalent metal ion.

[0049] In one embodiment of the present invention, the divalent metal ion preferably includes Cu. 2+ Fe 2+ Zn 2+ Mg 2+ Ni 2 + Co 2+ Pt 2+ or Rh 2+ Trivalent metal ions preferably include Ir 3+ Tetravalent metal ions include Sn 4+ Ti 4+ Or Ru 4+ .

[0050] In one embodiment of the present invention, when M is a trivalent metal ion or a tetravalent metal ion, the anion can be Cl. - .

[0051] This invention also provides a method for preparing metal complexes of dihydroporphyrin compounds as described in the above technical solution, comprising the following steps: The dihydroporphyrin compound described in the above technical solution is mixed with methanol, metal chloride or metal acetate complex, and heated under reflux to obtain a metal complex of the dihydroporphyrin compound.

[0052] In one embodiment of the present invention, the molar ratio of the dihydroporphyrin compound, metal chloride, or acetate complex is preferably 1:1 to 6, more preferably 1:3; the heating reflux time is preferably 2 to 8 hours.

[0053] This invention also provides metal salts of the dihydroporphyrin compounds described in the above technical solutions, having a structure shown in any of formulas a to g: ; R1, R8, and R9 are independent or , , R3 is either Na or K.

[0054] This invention also provides a method for preparing metal salts of dihydroporphyrin compounds as described in the above technical solution, comprising the following steps: A strong alkaline solution and a dihydroporphyrin compound are mixed and then subjected to salt formation to obtain a metal salt of the dihydroporphyrin compound.

[0055] Specifically, it is divided into (1) to (3): (1) When the dihydroporphyrin compound has the structure shown in Formula I, Formula IV or Formula VI and R1 is a hydroxyl group, the strong base solution is preferably an aqueous solution of NaOH or KOH; the concentration of the strong base solution is 0.001~0.5 M, more preferably 0.1 M; the molar ratio of the strong base to the dihydroporphyrin compound in the strong base solution is preferably 1:1; the hydrolysis temperature is preferably 4~45℃, more preferably 25℃, and the reaction time is preferably 1~4 h, more preferably 1 h.

[0056] (2) When the dihydroporphyrin compound has the structure shown in Formula I, Formula IV or Formula VI and R1 is R 1-1 R 1-2 R 1-3 hour: In one embodiment of the present invention, the strong alkali solution is preferably an aqueous solution of NaOH or KOH; the concentration of the strong alkali solution is 0.5~5 M, more preferably 1 M; the hydrolysis temperature is preferably 20~45℃, more preferably 35℃; and the reaction time is preferably 1~4 h, more preferably 1 h.

[0057] (3) When the dihydroporphyrin compound has the structure shown in formula II, III, V or VII and R1 is a hydroxyl group, or when the dihydroporphyrin compound has the structure shown in formula III or VII and R1 and R9 are hydroxyl groups, and R8 is a hydroxyl group of formula R 1-1 R 1-2 Or R 1-3 Alternatively, when the dihydroporphyrin compound has the structure shown in Formula II or Formula V, R1 and R8 are R 1-1 R 1-2 Or R 1-3 R2 is R 2-1 Alternatively, when the dihydroporphyrin compound has the structure shown in Formula II, R1 and R8 are R 1-1 R 1-2 Or R 1-3 When R2 is H: In one embodiment of the present invention, the strong alkali solution is preferably an aqueous solution of NaOH or KOH; the concentration of the strong alkali solution is 0.5~5 M, more preferably 1 M; the hydrolysis temperature is preferably 20~45℃, more preferably 35℃; and the reaction time is preferably 1~4 h, more preferably 1 h.

[0058] This invention improves the physicochemical properties of dihydroporphyrin by structurally modifying its core and coupling it with amino acids, thereby optimizing its distribution in tissues and its tumor-targeting ability. Simultaneously, it introduces p-toluenesulfonamide as an endoplasmic reticulum-targeting functional module, significantly enhancing its localization ability to the endoplasmic reticulum. This, in turn, enhances the compound's antitumor activity and ability to activate immune responses in vitro and in vivo, achieving precise localization and efficient enrichment in tumor tissues and cells.

[0059] This invention also provides metal complexes of the metal salts of dihydroporphyrin compounds described in the above technical solutions, having a structure shown in any one of the formulas am to gm: ; In the formula am~gm, M is a divalent, trivalent, or tetravalent metal ion.

[0060] The present invention also provides the application of the dihydroporphyrin compounds, metal complexes of dihydroporphyrin compounds, metal salts of dihydroporphyrin compounds, or metal complexes of metal salts of dihydroporphyrin compounds described in the above technical solutions in the preparation of photo / sound sensitive agents or antitumor drugs.

[0061] As one embodiment of the present invention, the application may further include pharmaceutically acceptable salts or pharmaceutically acceptable excipients of the above-mentioned drugs (dihydroporphyrin compounds, metal complexes of dihydroporphyrin compounds, metal salts of dihydroporphyrin compounds, or metal complexes of metal salts of dihydroporphyrin compounds).

[0062] In one embodiment of the present invention, when administered via photodynamic therapy as a photosensitizing drug, a light source with a wavelength of 600-800 nm can be used, more preferably 660-680 nm; when administered via sonodynamic therapy as a photosensitizing drug, the sonodynamic therapy can employ a wavelength of 1-1.5 W / cm². 2 Ultrasonic waves of high intensity.

[0063] In one embodiment of the present invention, the antitumor drug may be an injectable formulation.

[0064] In one embodiment of the present invention, the tumor includes benign tumors and / or malignant tumors; specifically, the tumor includes carcinoma in situ and / or metastatic carcinoma; the carcinoma in situ preferably includes one or more of breast tumors, liver tumors, lung tumors and colorectal tumors; the metastatic carcinoma includes one or more of liver metastatic tumors, lung metastatic tumors and colorectal metastatic tumors.

[0065] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1 0.9 mmol of compound 1 was dissolved in 20 mL of concentrated sulfuric acid / methanol (CH3OH to concentrated sulfuric acid, volume ratio 1:0.05). The mixture was stirred for 2 h at room temperature under N2 protection. The solvent was evaporated, and the solution was diluted with 200 mL of ethyl acetate. The solution was washed with saturated sodium bicarbonate and sodium chloride aqueous solutions, and the organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was subjected to silica gel column chromatography with petroleum ether-acetone system in a volume ratio of 10:1 to give a dark green solid compound 1a-1 in 91% yield.

[0067] The NMR and mass spectrometry data of compound 1a-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.26 (s,1H), 9.17 (s, 1H), 8.52 (s, 1H), 7.87 (dd, J = 17.8, 11.6 Hz, 1H), 6.21 (d, J =17.9 Hz, 1H), 6.10 (d, J = 11.5 Hz, 1H), 5.25 (d, J = 19.7 Hz, 1H), 5.10 (d, J =19.7 Hz, 1H), 4.47 (dd, J = 9.5, 3.1 Hz, 1H), 4.27 (d, J = 8.5 Hz, 1H), 3.65 (s,3H), 3.57 (s, 3H), 3.51 (m, 2H), 3.36 (s, 3H), 3.08 (s, 3H), 2.76 - 2.52 (m,3H), 2.35 - 2.24 (m, 2H), 1.83 (d, J = 7.2 Hz, 3H), 1.62 (t, J = 7.6 Hz, 3H), -1.83 (s, 1H). HRMS (ESI) m / z: C 34 H 37 N4O3 + [M+H] + Calculated value: 549.2866, Detected value: 549.2865.

[0068] A solution of compound 1a-1 (1.0 eq), a substituted terminal alkene (4.0 eq), and a Grubbs' II catalyst (0.4 eq) in anhydrous dichloroethane was stirred at 60 °C under nitrogen for 12 h. The resulting organic phase was washed successively with saturated ammonium chloride and sodium chloride aqueous solutions. The organic phase was dried over anhydrous sodium sulfate and evaporated. The residue was purified by silica gel column chromatography using a petroleum ether-acetone system with a volume ratio of 6:1 to give a dark green solid compound 1a-2 in 51% yield. In this embodiment, the substituted terminal olefin is... .

[0069] The NMR and mass spectrometry data of compound 1a-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.54 (s,1H), 9.39 (s, 1H), 8.60 (s, 1H), 8.02 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.22(d, J = 8.0 Hz, 2H), 6.88 (dt, J = 15.8, 6.1 Hz, 1H), 5.29 (d, J = 19.8 Hz, 1H), 5.13 (d, J = 19.8 Hz, 1H), 5.06 (d, J = 6.0 Hz, 2H), 4.51 (dd, J = 14.9, 7.9 Hz, 1H), 4.32 (d, J = 7.8 Hz, 1H), 3.70 (m, 2H), 3.68 (s, 3H), 3.62 (s, 3H), 3.41(s, 3H), 3.13 (s, 3H), 2.71 - 2.57 (m, 2H), 2.39 (m, 2H), 2.33 (s, 3H), 1.83(d, J = 7.2 Hz, 3H), 1.69 (t, J = 7.5 Hz, 3H), 1.47 (s, 9H), -1.71 (s, 1H). HRMS(ESI) m / z C 47 H 54 N5O7S + [M+H] + Calculated value: 832.3744, Detected value: 832.3736.

[0070] 0.09 mmol of compound 1a-2 was dissolved in 2 mL of concentrated hydrochloric acid / acetone solution (obtained by mixing concentrated hydrochloric acid and acetone, with a volume concentration of 33%), and stirred for 4 h under nitrogen protection at 0 °C. After evaporating the reaction solution, it was diluted with ethyl acetate, and then washed successively with saturated sodium bicarbonate and saturated sodium chloride aqueous solutions. The organic layer was dried with anhydrous sodium sulfate and evaporated. The residue was purified by silica gel column chromatography (using a dichloromethane-methanol system with a volume ratio of 20:1) to obtain a dark green solid compound 1a (compound I when R1 is a hydroxyl group), with a yield of 82%.

[0071] The NMR and mass spectrometry data of compound 1a are as follows: 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.44 (s,1H), 9.03 (s, 1H), 8.66 (s, 1H), 8.33 (s, 1H), 7.86 (d, J = 7.5 Hz, 2H), 7.64(d, J = 16.0 Hz, 1H), 7.40 (d, J = 7.4 Hz, 2H), 6.37 (dd, J = 25.9, 6.9 Hz, 1H), 5.16 (d, J = 22.9 Hz, 1H), 4.98 (d, J = 21.2 Hz, 1H), 4.48 (d, J = 7.3 Hz, 1H), 4.21 (d, J = 8.6 Hz, 1H), 3.93 (d, J = 2.6 Hz, 2H), 3.53 (m, 2H), 3.46 (s, 3H), 3.06 (s, 3H), 3.04 (s, 3H), 2.34 (m, 2H), 2.28 (s, 3H), 2.14 – 2.03 (m, 2H), 1.72 (d, J = 4.4 Hz, 3H), 1.55 (t, J = 7.4 Hz, 3H), -2.30 (s, 1H). HRMS (ESI) m / zC 41 H 44 N5O5S + [M+H] + Calculated value: 718.3063, Detected value: 718.3052.

[0072] Example 2 Compound 1 (1.0 eq), HATU (3.0 eq), and DIPEA (3.0 eq) were dissolved in dichloromethane and stirred at 30 °C under nitrogen protection for 1 h (monitored by TLC). Then, the mixture was added... L -Aspartic acid dimethyl ester hydrochloride L-Asp(t-Bu3)2 (1.2 eq) was stirred at 30 °C under nitrogen for 12 h. After the reaction was completed, the reaction solution was washed with 5 wt% citric acid, saturated NaHCO3 and NaCl aqueous solution, the organic layer was dried with anhydrous Na2SO4 and evaporated, and the residue was purified by silica gel column chromatography (the eluent was a dichloromethane-ethyl acetate system, the volume ratio of dichloromethane to ethyl acetate was 100:1→30:1, gradient elution) to give a dark green solid compound 1b-1, with a yield of 88%.

[0073] The NMR and mass spectrometry data of compound 1b-1 are as follows: 1 H-NMR (400 MHz, CDCl3): δ 9.32 (s,1H), 9.24 (s, 1H), 8.55 (s, 1H), 7.92 (dd, J = 17.8, 11.5 Hz, 1H), 6.31 (d, J =8.1 Hz, 1H), 6.23 (d, J = 17.9 Hz, 1H), 6.12 (d, J = 11.5 Hz, 1H), 5.33 - 5.02(m, 2H), 4.62 (dd, J = 8.0, 4.1 Hz, 1H), 4.50 (d, J = 6.7 Hz, 1H), 4.33 (d, J = 6.8Hz, 1H), 3.70 – 3.48 (m, 5H), 3.38 (s, 3H), 3.12 (s, 3H), 2.84 – 2.63 (m,3H), 2.53 (dd, J = 17.0, 4.1 Hz, 1H), 2.48 – 2.25 (m, 2H), 1.81 (d, J = 7.2 Hz, 3H), 1.61 (t, J = 7.5 Hz, 3H), 1.39 (s, 9H), 1.29 (s, 9H), -1.87 (s, 1H). HRMS(ESI) m / z C 45 H 56 N5O6 + [M+H] + Calculated value: 762.4231, Detected value: 762.4227.

[0074] Compound 1b-1 (1.0 eq), the substituted terminal alkene (4.0 eq), and Grubbs' catalyst (0.4 eq) were mixed in anhydrous dichloromethane solvent and stirred at 40 °C under nitrogen atmosphere for 16 hours. The mixture was then washed three times (50 mL) with saturated NH4Cl and NaCl aqueous solutions. The organic phase was dried over anhydrous Na2SO4 and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent was a petroleum ether-acetone system with a volume ratio of 5:1) to give compound 1b-2 as a dark green solid in 42% yield. In this embodiment, the substituted terminal olefin is... .

[0075] The NMR and mass spectrometry data of compound 1b-2 are as follows: 1 H-NMR (400 MHz, CDCl3): δ 9.40 (s,1H), 9.30 (s, 1H), 8.58 (s, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 12.9 Hz, 1H), 7.20 (d, J = 8.1 Hz, 2H), 6.85 (dt, J = 16.0, 6.2 Hz, 1H), 6.36 (d, J = 8.2Hz, 1H), 5.19 (dd, J = 65.9, 19.9 Hz, 2H), 5.04 (d, J = 6.1 Hz, 2H), 4.65 (dt, J =8.3, 4.3 Hz, 1H), 4.52 (m, 1H), 4.36 (m, 1H), 3.64 (m, 2H), 3.60 (s, 3H), 3.40 (s, 3H), 3.08 (s, 3H), 2.78 (m, 2H), 2.57 (m, 2H), 2.42 (m, 2H), 2.31(s, 3H), 1.82 (d, J = 7.2 Hz, 3H), 1.65 (t, J= 7.6 Hz, 3H), 1.47 (s, 9H), 1.40 (s, 9H), 1.30 (s, 9H), -1.75 (s, 1H). HRMS (ESI) m / z C 58 H 73 N6O 10 S + [M+H] + Calculated value: 1045.5109, Detected value: 1045.5098.

[0076] 0.09 mmol / L of compound 1b-2 was dissolved in 2 mL of hydrochloric acid / acetone solution (prepared by mixing concentrated hydrochloric acid and acetone, with a volume concentration of 33%) at 0 °C. The mixture was stirred at 25 °C for 4 hours under nitrogen atmosphere and monitored by TLC. The mixture was evaporated and then washed with saturated NaHCO3 and NaCl aqueous solutions. The organic layer was dried over anhydrous Na2SO4 and evaporated under pressure by rotary evaporation. The residue was purified by silica gel column chromatography using a 20:1 (v / v) dichloromethane-methanol system to give a dark green solid compound 1b (R1 is R). 1-1 Compound I was obtained at a yield of 82%.

[0077] The NMR and mass spectrometry data of compound 1b are as follows: 1 H-NMR (400 MHz, DMSO- d 6 ): δ 12.50 (m,2H), 9.45 (s, 1H), 9.11 (s, 1H), 8.73 (s, 1H), 8.23 ​​(d, J = 7.8 Hz, 1H), 8.10(t, J = 5.7 Hz, 1H), 7.80 (d, J = 7.9 Hz, 2H), 7.74 (d, J = 16.0 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 6.43 (dt, J = 12.1, 5.7 Hz, 1H), 5.04 (dd, J = 60.0, 19.8 Hz, 2H),4.49 (m, 2H), 4.21 (m, 1H), 3.93 (d, J= 4.7 Hz, 2H), 3.51 (m, 2H), 3.44 (s,3H), 3.14 (s, 3H), 3.04 (s, 3H), 2.55 (m, 2H), 2.42 (m, 4H), 2.10 (m, 2H),1.71 (d, J = 7.1 Hz, 3H), 1.49 (t, J = 7.4 Hz, 3H), -2.22 (s, 1H). HRMS (ESI) m / z C 45 H 48 N6O8S + [MH] + Calculated value: 831.3176, Detected value: 831.3186.

[0078] Example 3 Compound Ib (1 eq) was mixed with copper acetate (1 eq) and dissolved in 2 mL of a mixture of dichloromethane and methanol (volume ratio of dichloromethane to methanol: 1:1). The mixture was stirred at room temperature for 1 h to obtain compound 1b-Cu (R1 is R). 1-1 M is compound I of Cu.

[0079] The mass spectrometry data for compound 1b-Cu are as follows: HRMS (ESI) m / z C 45 H 48 N6O8SCu + [MH] + Calculated value: 892.2472, Detected value: 892.2466.

[0080] Example 4 Compound 2 (1 eq) was weighed and placed in a 50 mL round-bottom flask. 3 mL of DMF was added to dissolve it, followed by the addition of anhydrous potassium carbonate (10 eq) and iodomethane (10 eq). The mixture was stirred at room temperature (25 °C) for 4 h under nitrogen protection. The reaction solution was diluted with dichloromethane and transferred to a separatory funnel. The solution was washed successively with deionized water and saturated sodium chloride solution to separate the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Further purification was achieved by silica gel column chromatography (200–300 mesh). The eluent was a dichloromethane-ethyl acetate system with a volume ratio of 100:1. Optimized elution effectively separated impurities, ultimately yielding a dark green powder 2a-1 with a yield of 91%.

[0081] The NMR and mass spectrometry data of compound 2a-1 are as follows: 1H NMR (400 MHz, CDCl3) δ 9.84 (s,1H), 9.70 (s, 1H), 9.56 (s, 1H), 8.75 (s, 1H), 8.03 (dd, J = 17.8, 11.5 Hz,1H), 6.31 (dd, J = 17.8, 1.0 Hz, 1H), 6.13 (dd, J = 11.5, 1.1 Hz, 1H), 4.57-4.48(m, 2H), 4.38 (s, 3H), 3.79 (s, 3H), 3.73 (dd, J = 13.4, 5.7 Hz, 2H), 3.66 (s,3H), 3.46 (s, 3H), 3.26 (s, 3H), 2.78 (ddd, J = 18.5, 10.0, 4.9 Hz, 1H), 2.64(ddd, J = 15.1, 9.6, 5.6 Hz, 1H), 2.57 -2.48 (m, 1H), 2.42 (ddd, J = 15.0,10.4, 5.4 Hz, 1H), 1.92 (d, J = 7.1 Hz, 3H), 1.71 (t, J = 7.7 Hz, 3H), -1.70 (s, 2H). MS(ESI) m / z C 34 H 39 N4O4 + [M+H] + Calculated value: 567.2971, Detected value: 567.41.

[0082] The obtained compound 2a-1 (1 eq) was placed in a 50 mL round-bottom flask, dissolved in 3 mL of dichloromethane, and then a substituted terminal alkene (4 eq, structural formula same as in Example 1) and a Grubbs second-generation catalyst (0.4 eq) were added. The reaction was carried out under nitrogen protection and oxygen-free light-shielding conditions at 40 °C with stirring for 16 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: petroleum ether / acetone = 4:1). After the reaction was completed, the crude product was directly concentrated under reduced pressure and purified by silica gel column chromatography with a mesh size of 200-300 mesh. The crude product was dissolved in dichloromethane and loaded onto the column. The eluent was 5:1 (v / v) of petroleum ether and acetone. The main color band was collected, and the eluents were combined and concentrated to dryness under reduced pressure to obtain a dark green powder product 2a-2 with a yield of 42%. The NMR and mass spectrometry data of compound 2a-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.83 (s,1H), 9.79 (s, 1H), 9.59 (s, 1H), 8.77 (s, 1H), 8.07 (d, J = 16.0 Hz, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.93 (dt, J = 16.0, 6.2 Hz, 1H), 5.08 (d, J = 6.0 Hz, 2H), 4.58 - 4.46 (m, 2H), 4.36 (s, 3H), 3.83 (s, 3H), 3.81 - 3.75 (m, 2H), 3.62 (s, 3H), 3.47 (s, 3H), 3.19 (s, 3H), 2.80 - 2.67(m, 1H), 2.59 (ddd, J = 16.2, 10.7, 6.2 Hz, 1H), 2.54 – 2.43 (m, 1H), 2.43 –2.32 (m, 2H), 2.31 (d, J = 9.9 Hz, 3H), 1.90 (d, J = 7.0 Hz, 3H), 1.72 (t, J = 7.5Hz, 3H), 1.48 (s, 9H), -1.68 (s, 2H). MS(ESI) m / z C 47 H 56 N5O8S + [M+H] + Calculated value: 850.3850, Detected value: 850.28.

[0083] Compound 2a-2 (1 eq) was transferred to a 25 mL round-bottom flask and dissolved in dichloromethane with 33 vol.% trifluoroacetic acid. The reaction system was sealed and purged three times with nitrogen. The reaction was then stirred continuously at room temperature for 4 h. After the reaction was stopped, the reaction solution was washed three times with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by silica gel column chromatography (200-300 mesh) using dichloromethane / methanol (200:1, v / v) as the eluent to remove nonpolar impurities. The main colored band was collected, the eluents were combined, and the solvent was concentrated under reduced pressure to obtain a dark green powder product 2a-3 in 84% yield.

[0084] The NMR and mass spectrometry data of compound 2a-3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.81 (s,1H), 9.57 (s, 1H), 9.25 (s, 1H), 8.65 (s, 1H), 7.84 (d, J = 7.8 Hz, 2H), 7.53(d, J = 16.6 Hz, 1H), 7.25 (d, J = 8.8 Hz, 2H), 6.48 – 6.32 (m, 1H), 5.16 (s,1H), 4.48 (d, J = 5.1 Hz, 2H), 4.39 – 4.25 (m, 5H), 3.71 (s, 3H), 3.63 (m,5H), 3.14 (d, 6H), 2.75 (m, 1H), 2.68 – 2.54 (m, 1H), 2.45 (d, J = 16.3 Hz, 2H), 2.29 (s, 3H), 1.88 (d, J = 6.7 Hz, 3H), 1.63 (t, J = 7.4 Hz, 3H), -1.96 (s, 2H). MS(ESI) m / z C 42 H 48 N5O6S + [M+H] + Calculated value: 750.3325, Detected value: 750.46.

[0085] 0.1 mmol of compound 2a-3 was dissolved in 2 mL of a sodium hydroxide-tetrahydrofuran mixture (prepared by mixing 1M sodium hydroxide solution and tetrahydrofuran at a volume ratio of 1:1). The reaction system was sealed and purged three times with nitrogen gas. The reaction was stirred continuously at room temperature for 12 h. After the reaction was stopped, the organic solvent was removed by rotary evaporation. The pH was then adjusted to 3-4, and a dark green solid precipitated. After filtration, the solid was dried under vacuum to obtain compound 2a (R2 is R). 2-1 Compound II (where R1 and R8 are hydroxyl groups) had a yield of 89%.

[0086] The NMR and mass spectrometry data of compound 2a are as follows: 1 H NMR (400 MHz, DMSO) δ 13.22 – 12.04(s, 1H), 9.74 (s, 2H), 9.54 (s, 1H), 8.96 (s, 1H), 7.99 – 7.85 (m, 3H), 7.44(d, J = 7.1 Hz, 2H), 6.62 (d, J = 7.6, 1H), 4.58 (m, 1H), 4.44 (m, 1H), 4.25 (s,3H), 4.06 (m, 2H), 3.71 (s, 3H), 3.36 (m, 5H), 2.77 – 2.59 (m, 4H), 2.27 (s,3H), 1.85 (t, J = 7.2 Hz, 3H), 1.64 (t, J = 6.5 Hz, 3H), -1.93 (d, 2H). MS(ESI) m / z C 40 H 42 N5O6S + [MH] + Calculated value: 720.2850, Detected value: 720.45.

[0087] Example 5 Compound 2 (1 eq) was dissolved in anhydrous DMF in a round-bottom flask. The reaction was activated by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.5 eq) at 25°C for 1 h. After the solution changed from dark green to purple-red, triethylamine (2.5 eq) and... L -Aspartic acid dimethyl ester hydrochloride ( L-Asp(OMe)2, 2.5 eq). The reaction system was placed in a heating device, under nitrogen protection, oxygen-free and light-shielded conditions, and stirred at room temperature for 12 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: dichloromethane / methanol = 10:1). The reaction was terminated when the product spot no longer changed. The reaction solution was transferred to a separatory funnel and extracted twice with saturated sodium chloride solution and dichloromethane. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. Subsequently, it was eluted by 200-300 mesh silica gel column chromatography using a gradient elution system of dichloromethane-methanol with a volume ratio of 100:1 to 25:1 to obtain the dark green solid 2b-1, with a yield of 52%.

[0088] The NMR and mass spectrometry data of compound 2b-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.70 (s,2H), 9.45 (s, 1H), 8.84 (s, 1H), 8.10 (dd, J = 17.8, 11.6 Hz, 1H), 7.91 (d, J =7.8 Hz, 1H), 6.54 (d, J = 7.7 Hz, 1H), 6.34 (d, J = 17.8 Hz, 1H), 6.15 (d, J = 11.5Hz, 1H), 5.69 – 5.53 (m, 1H), 5.33 (dq, J = 8.1, 4.2 Hz, 1H), 4.76 – 4.66 (m,1H), 4.58 – 4.50 (m, 1H), 3.98 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H), 3.60 (s,3H), 3.50 (s, 3H), 3.40 (dd, J = 7.2, 5.2 Hz, 2H), 3.32 (s, 3H), 3.28 (s, 3H), 2.78 – 2.53 (m, 4H), 2.28 (ddd, J = 34.3, 15.9, 5.9 Hz, 4H), 1.89 (d, J = 6.6 Hz, 3H), 1.71 (t, J = 7.4 Hz, 3H), -2.05 (s, 2H). HRMS (ESI) m / z C 44 H 53 N6O10 + [M+H] + Calculated value: 825.3818, Detected value: 825.3828.

[0089] The prepared 2b-1 (1 eq) was placed in a 50 mL round-bottom flask, dissolved in dichloromethane, and then a substituted terminal alkene (4 eq, structural formula same as in Example 1) and a Grubbs second-generation catalyst (0.4 eq) were added. The reaction was carried out under nitrogen protection at 40 °C with stirring for 14 h, and the reaction progress was monitored by thin-layer chromatography (developing solvent: petroleum ether / acetone = 3:1). After the reaction was complete, the product was directly concentrated under reduced pressure and purified by silica gel column chromatography with a mesh size of 200-300. The crude product was dissolved in dichloromethane and loaded onto the flask, eluted with a petroleum ether-acetone system with a volume ratio of 5:1. The main color band was collected, and the eluents were combined and concentrated to dryness under reduced pressure to obtain a dark green powder product 2b-2, with a yield of 39%.

[0090] The NMR and mass spectrometry data of compound 2b-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.75 (s,1H), 9.67 (s, 1H), 9.46 (s, 1H), 8.84 (s, 1H), 8.11 (d, J = 16.0 Hz, 1H), 8.00(d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.94 (dt, J = 12.7, 5.9 Hz, 1H), 5.61 (t, J = 4.4 Hz, 1H), 5.29 (s, 1H), 5.08 (d, J = 5.8 Hz, 2H), 4.72 (d, J = 5.1Hz, 1H), 4.53 (dd, J = 18.7, 7.1 Hz, 2H), 3.98 (s, 3H), 3.80 (s, 3H), 3.77 (s,3H), 3.62 (s, 3H), 3.50 (s, 3H), 3.40 (dd, J = 8.7, 4.5 Hz, 2H), 3.30 (s, 3H), 3.22 (s, 3H), 2.99 (m, 4H), 2.73 (m, 2H), 2.57 (m, 2H), 2.30 (s, 3H), 1.89(d, J= 7.0 Hz, 3H), 1.73 (t, J = 7.5 Hz, 3H), 1.48 (s, 9H), -2.00 (s, 1H). HRMS(ESI) m / z : C 57 H 70 N7O 14 S + [M+H] + Calculated value: 1108.4696, Detected value: 1108.4708.

[0091] Compound 2b-2 (1 eq) was accurately weighed and transferred to a pear-shaped flask. A solution of 30 vol% trifluoroacetic acid in dichloromethane was added to the flask. The reaction system was sealed and purged three times with nitrogen to ensure an inert atmosphere. The reaction was stirred continuously for 4 h at room temperature (25°C). After the reaction was stopped, the reaction solution was washed three times sequentially with saturated sodium bicarbonate, water, and saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and purified directly by silica gel column chromatography (200–300 mesh). Nonpolar impurities were eluted using dichloromethane / methanol (200:1, v / v). The main band fraction was collected, and the eluents were combined and concentrated under reduced pressure to obtain a dark green powder, 2b-3, in 90% yield.

[0092] The NMR and mass spectrometry data of compound 2b-3 are as follows: 1 H NMR (500 MHz, CDCl3) 1H NMR (500MHz, CDCl3) δ 9.70 (s, 1H), 9.47 (s, 2H), 8.78 (s, 1H), 7.91 (d, J = 7.9 Hz, 2H), 7.81 – 7.66 (m, 1H), 7.34 (d, J = 7.9 Hz, 2H), 6.62 – 6.44 (m, 2H), 5.70 – 5.52 (m, 1H), 5.01 – 4.84 (m, 1H), 4.80 – 4.67 (m, 1H), 4.50 (m, 2H), 4.12(m, 2H), 3.98 (s, 3H), 3.81 (s, 3H), 3.60 (s, 3H), 3.51 (s, 3H), 3.40 (dd, J= 16.0, 4.2 Hz, 2H), 3.31 (s, 3H), 3.28 (s, 3H), 3.27 (s, 3H), 2.86 – 2.56(m, 4H), 2.38 (s, 3H), 2.28 – 2.14 (m, 4H), 1.89 (d, J = 6.3 Hz, 3H), 1.70 (t, J = 7.5 Hz, 3H), -1.92 – -2.38 (m, 2H). HRMS (ESI) m / z : C 52 H 62 N7O 12 S + [M+H] + Calculated value: 1008.4172, Detected value: 1008.4180.

[0093] 0.1 mmol of compound 2b-1 was dissolved in 2 mL of a sodium hydroxide-tetrahydrofuran mixture (obtained by mixing 1M sodium hydroxide solution and tetrahydrofuran at a volume ratio of 1:1). The reaction system was sealed and purged three times with nitrogen to ensure an inert atmosphere. The reaction was carried out with continuous stirring at room temperature for 12 h. After the reaction was stopped, the organic solvent was removed by rotary evaporation, and the pH was adjusted to 3-4. A dark green solid precipitated out, which was then filtered and dried under vacuum to obtain compound 2c (R2 is H and R1 and R8 are R). 1-1 Compound II), with a yield of 92%.

[0094] The NMR and mass spectrometry data of compound 2c are as follows: 1H NMR (500 MHz, DMSO) δ 13.61 – 11.86 (s, 3H), 9.88 (s, 2H), 9.34 (s, 2H), 9.28 (s, 1H), 9.16 (s, 1H), 8.23 ​​(dd, J =21.1, 8.0 Hz, 1H), 6.46 (d, J = 17.8 Hz, 1H), 6.22 (d, J = 11.3 Hz, 1H), 5.28 –5.18 (m, 1H), 4.69 (dd, J = 14.7, 7.4 Hz, 1H), 4.53 (dd, J = 13.4, 7.7 Hz, 1H), 4.47 (dd, J = 9.0, 5.6 Hz, 1H), 3.86 (dd, J = 15.4, 8.2 Hz, 3H), 3.68 (s, 3H), 3.56 (s, 3H), 3.32(q, 2H), 3.16 – 2.93 (m, 2H), 2.73 – 2.57 (m, 2H), 2.31 –2.11 (m, 2H), 1.93 – 1.84 (m, 3H), 1.68 (t, J = 7.4 Hz, 3H), -2.27 – -2.49 (s, 2H). MS (ESI) m / z: C 40 H 43 N6O 10 + [MH] + Calculated value: 767.3035, Detected value: 767.38.

[0095] ② Compound 2b-3 was dissolved in a sodium hydroxide-tetrahydrofuran mixture (obtained by mixing 1M sodium hydroxide solution and tetrahydrofuran at a volume ratio of 1:1). After sealing the reaction system, nitrogen gas was purged three times. Then, the reaction was stirred continuously for 12 h at room temperature (25℃). After the reaction was stopped, the organic solvent was removed by rotary evaporation. The pH was then adjusted to 3-4, and a dark green solid precipitated. After filtration, the solid was dried under vacuum to obtain compound 2b (R2 is R). 2-1 And R1 and R8 are R 1-1 Compound II), with a yield of 91%.

[0096] The NMR and mass spectrometry data of compound 2b are as follows: 1H NMR (400 MHz, DMSO) δ 12.77 – 12.54(s, 4H), 9.84 (s, 2H), 9.78 (s, 1H), 9.25 (s, 1H), 9.09 (s, 1H), 8.14 (d, J =15.6 Hz, 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 6.70 (dt, J =13.7, 6.4 Hz, 1H), 5.22 (t, J = 8.4 Hz, 1H), 5.07 – 5.01 (m, 1H), 4.66 (dd, J =11.8, 3.8 Hz, 1H), 4.53 (d, J = 9.4 Hz, 1H), 4.12 (m, 2H), 3.88 – 3.83 (m, 2H), 3.76 – 3.73 (m, 2H), 3.69 – 3.67 (s, 3H), 3.42 (s, 3H), 3.36 (s, 3H), 3.13 –2.97 (m, 4H), 2.67 – 2.60 (m, 4H), 2.30 (s, 3H), 1.87 (d, J = 6.6 Hz, 3H), 1.70(m, 3H), -2.32 – -2.41 (s, 2H). HRMS (ESI) m / z : C 48 H 52 N7O 12 S + [MH] + Calculated value: 950.3389, Detected value: 950.3394.

[0097] Example 6 0.1 mmol of compound 3 (1 eq) was placed in a 50 mL round-bottom flask and dissolved in 3 mL of DMF. Anhydrous potassium carbonate (20 eq) and iodomethane (10 eq) were added sequentially, and the mixture was stirred at room temperature (25 °C) for 4 h under nitrogen protection. The reaction solution was diluted with dichloromethane, transferred to a separatory funnel, and washed three times with deionized water and saturated sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was further purified by silica gel column chromatography using a 200–300 mesh silica gel column, eluted with a dichloromethane-ethyl acetate system at a volume ratio of 100:1, to obtain a dark green powder 3a-1 in 88% yield.

[0098] The NMR and mass spectrometry data of compound 3a-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.71 (s,1H), 9.57 (s, 1H), 8.76 (s, 1H), 8.05 (dd, J = 18.3, 11.0 Hz, 1H), 6.35 (d, J =17.9 Hz, 1H), 6.14 (d, J = 11.5 Hz, 1H), 5.48 – 5.13 (m, 2H), 4.45 (dd, J = 12.4,5.1 Hz, 2H), 4.26 (s, 3H), 3.83 – 3.73 (m, 5H), 3.65 (s, 3H), 3.58 (s, 3H), 3.47 (s, 3H), 3.30 (s, 3H), 2.64 – 2.50 (m, 1H), 2.21 (td, J = 14.6, 3.6 Hz,2H), 1.76 (m, 4H), 1.71 (t, J = 7.5 Hz, 3H), -1.31 (s, 1H), -1.44 (s, 1H).

[0099] The obtained compound 3a-1 (1 eq) was placed in a 50 mL round-bottom flask, dissolved in 3 mL of dichloromethane, and then a substituted terminal alkene (4 eq, structural formula same as in Example 1) and a Grubbs second-generation catalyst (0.4 eq) were added. The reaction was carried out under nitrogen protection at 40 °C in an oxygen-free, light-shielded environment, with stirring for 16 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: petroleum ether / acetone = 3:1, v:v). After the reaction was complete, the product was directly concentrated under reduced pressure and purified by silica gel column chromatography (200-300 mesh). The crude product was dissolved in dichloromethane and loaded onto the flask. Elution was performed using a petroleum ether-acetone system with a volume ratio of 5:1. The main color band was collected, and the eluents were combined and concentrated to dryness under reduced pressure to obtain a dark green powder product 3a-2, with a yield of 40%.

[0100] The NMR and mass spectrometry data of compound 3a-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.74 (s,1H), 9.53 (s, 1H), 8.81 (s, 1H), 8.07 (s, 1H), 8.04 (d, J = 7.9 Hz, 2H), 7.22(d, J = 8.1 Hz, 2H), 6.97 (dt, J = 16.0, 6.2 Hz, 1H), 5.38 (t, J = 22.5 Hz, 2H),5.29 (s, 1H), 5.10 (d, J = 6.2 Hz, 2H), 4.58 - 4.38 (m, 2H), 4.31 (m, 5H), 3.82(s, 3H), 3.68 (s, 3H), 3.63 (s, 3H), 3.50 (s, 3H), 3.19 (d, J = 9.1 Hz, 3H),2.70 - 2.50 (m, 1H), 2.35 - 2.15 (m, 5H), 1.81 (d, J = 7.1 Hz, 3H), 1.74 (t, J =7.5 Hz, 3H), 1.52 (s, 9H), -1.34 (d, 2H). MS(ESI) m / z : C 50 H 60 N5O 10 S + [M+H] + Calculated value: 922.4055, Detected value: 922.45.

[0101] Compound 3a-2 was transferred to a 25 mL round-bottom flask and dissolved in a 33 vol% trifluoroacetic acid solution in dichloromethane. The reaction system was sealed and purged with nitrogen three times. The mixture was then stirred continuously at 0 °C for 4 h. After the reaction was stopped, the reaction solution was washed three times sequentially with saturated sodium bicarbonate, water, and saturated brine. It was dried over anhydrous sodium sulfate, filtered, and purified directly by silica gel column chromatography (200–300 mesh). Nonpolar impurities were washed away using a dichloromethane-methanol system (200:1 v / v). The main colored band was collected, the eluents were combined, and the solvent was concentrated under reduced pressure and evaporated to obtain a dark green powder product 3a-3 in 84% yield.

[0102] The NMR and mass spectrometry data of compound 3a-3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.61 (s,1H), 9.11 (s, 1H), 8.62 (s, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 16.1 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 6.22 (dt, J = 15.9, 6.1 Hz, 1H), 5.26 (dd, J = 33.8, 19.0 Hz, 2H), 5.10 (t, J = 6.0 Hz, 1H), 4.39 (d, J = 7.3 Hz, 2H), 4.22 (s, 3H), 3.84 (q, J = 5.6 Hz, 2H), 3.78 (d, J = 38.3 Hz, 3H), 3.68 - 3.62 (m, 5H), 3.53(s, 3H), 3.12 (s, 3H), 3.03 (s, 3H), 2.52 (dd, J = 16.6, 9.4 Hz, 2H), 2.22 (s,3H), 2.15 (m, 2H), 1.71 (d, J = 7.1 Hz, 3H), 1.63 (t, J = 7.6 Hz, 3H), -1.60 (s, 1H). HRMS (ESI) m / z: C 45 H 52 N5O8S + [M+H]+ Calculated value: 822.3531, Detected value: 822.3530.

[0103] Compound 3a-3 was dissolved in a sodium hydroxide-tetrahydrofuran mixture (obtained by mixing 1M sodium hydroxide solution and tetrahydrofuran at a volume ratio of 1:1). After sealing the reaction system, nitrogen gas was purged three times, and the reaction was carried out under continuous stirring at room temperature for 12 h. After the reaction was stopped, the organic solvent was removed by rotary evaporation, and the pH was adjusted to 3-4. A dark green solid precipitated out, which was then filtered and dried under vacuum to obtain compound 3a (compound III when R1, R8, and R9 are hydroxyl groups), with a yield of 89%.

[0104] The NMR and mass spectrometry data of compound 3a are as follows: 1 H NMR (400 MHz, DMSO) δ 12.93 – 12.10(s, 3H), 9.76 (s, 1H), 9.64 (s, 1H), 9.06 (s, 1H), 8.07 (d, J = 9.8 Hz, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 6.67 (dt, J = 15.7, 6.0 Hz,1H), 5.34 (m, 2H), 4.59 (m, 2H), 4.22 (s, 1H), 4.11 (d, J = 5.7 Hz, 2H), 3.86 –3.76 (m, 2H), 3.57 (s, 3H), 3.37 (s, 3H), 3.31 (s, 3H), 2.71 – 2.55 (m, 2H),2.28 (s, 3H), 2.12 (m, 2H), 1.68 (m, 6H), -1.71 (s, 1H), -1.99 (s, 1H). MS(ESI) m / z: C 42 H 44 N5O8S + [MH] + Calculated value: 778.2905, Detected value: 778.41.

[0105] Example 7 Compound 3 (1 eq) was dissolved in anhydrous DMF in a flask, and the reaction was activated by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 eq) at 25°C for 1 h. After the solution changed from dark green to purple-red, triethylamine (2 eq) and L - Dimethyl aspartate hydrochloride (1.2 eq). The reaction system was placed in a heating apparatus and continuously stirred for 12 h under nitrogen protection, oxygen-free and light-proof conditions at room temperature. The reaction progress was monitored by thin-layer chromatography (developing solvent: dichloromethane / methanol = 10:1). The reaction was terminated when the product spot no longer changed. The reaction solution was transferred to a separatory funnel and extracted twice with dichloromethane using saturated sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. Subsequently, it was analyzed by silica gel column chromatography (200-300 mesh) (eluent: dichloromethane-methanol system with a volume ratio of 100:1 to 25:1) to obtain a dark green solid 3b-1, with a yield of 77%.

[0106] The NMR and mass spectrometry data of compound 3b-1 are as follows: 1 H NMR (500 MHz, DMSO- d 6 ) δ 9.77 (s,1H), 9.68 (s, 1H), 9.10 (s, 1H), 8.26 (dd, J = 17.9, 11.7 Hz, 1H), 6.42 (d, J =17.9 Hz, 1H), 6.14 (d, J = 11.6 Hz, 1H), 5.29 (dd, J = 79.1, 17.4 Hz, 2H), 4.73 -4.53 (m, 2H), 4.42 (d, J = 9.2 Hz, 1H), 3.88 - 3.69 (m, 2H), 3.60 (s, 3H), 3.59(s, 3H), 3.56 (s, 3H), 3.50 (s, 3H), 3.37 (s, 3H), 3.28 (s, 3H), 2.90 (dd, J =16.5, 7.0 Hz, 1H), 2.74 (dd, J = 16.7, 5.3 Hz, 1H), 2.68 - 2.57 (m, 1H), 2.39 -2.27 (m, 2H), 2.17 (d, J = 6.9 Hz, 1H), 1.67 (d, J= 9.8 Hz, 3H), 1.65 (s, 3H), -1.65 (s, 1H), -1.93 (s, 1H). HRMS (ESI) m / z: C 40 H 44 N5O9 + [MH] + Calculated value: 738.3134, Detected value: 738.3137.

[0107] The prepared compound 3b-1 (1 eq) was dissolved in a gaiwan-shaped flask containing dimethylformamide, followed by the sequential addition of anhydrous potassium carbonate (20 eq) and iodomethane (10 eq). After sealing the system, nitrogen gas was purged, and the reaction flask was fixed on a heating device and stirred continuously at 30°C for 3 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: dichloromethane / methanol = 20:1). After the reaction was completed, the reaction solution was diluted with dichloromethane and washed three times sequentially with water and saturated sodium chloride aqueous solution. The organic layer was collected, dried with anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated to obtain the crude product. Then, it was eluted using a 200-300 mesh silica gel column with a dichloromethane-methanol system at a volume ratio of 200:1. The main color band was collected, the eluents were combined, and the solvent was evaporated under reduced pressure to finally obtain the dark green powder product 3b-2, with a yield of 99%.

[0108] The NMR and mass spectrometry data of compound 3b-2 are as follows: 1 H NMR (500 MHz, CDCl3) δ 9.69 (s,1H), 9.56 (s, 1H), 8.93 (s, 1H), 8.04 (dd, J = 17.8, 11.5 Hz, 1H), 7.99 (s,1H), 6.34 (d, J = 17.9 Hz, 1H), 6.14 (d, J = 11.5 Hz, 1H), 5.24 (t, J = 19.5 Hz,2H), 4.87 - 4.78 (m, 1H), 4.56 - 4.40 (m, 2H), 4.26 (s, 3H), 3.77 (q, J = 7.6Hz, 2H), 3.55 (s, 6H), 3.46 (s, 3H), 3.38 (s, 3H), 3.28 (s, 3H), 3.08 (s,2H), 2.91 (s, 1H), 2.85 (d, J= 6.7 Hz, 2H), 2.65 - 2.51 (m, 1H), 2.40 - 2.16(m, 2H), 1.82 (dd, J = 23.4, 14.7 Hz, 1H), 1.79 - 1.63 (m, 6H), -1.36 (s, 2H). HRMS (ESI) m / z : C 42 H 50 N5O9 + [M+H] + Calculated value: 768.3603, Detected value: 768.3605.

[0109] The prepared 3b-2 (1 eq) was placed in a 50 mL round-bottom flask, dissolved in dichloromethane, and then a substituted terminal alkene (4 eq, structural formula same as in Example 1) and a Grubbs second-generation catalyst (0.4 eq) were added. The reaction was carried out under nitrogen protection at 40 °C with stirring for 14 h. The reaction progress was monitored by thin-layer chromatography (developing solvent: petroleum ether / acetone = 3:1). After the reaction was complete, the product was directly concentrated under reduced pressure and purified by silica gel column chromatography with a mesh size of 200-300. The crude product was dissolved in dichloromethane and loaded onto the flask. Elution was performed using a petroleum ether-acetone system with a volume ratio of 5:1. The main color band was collected, and the eluents were combined and concentrated to dryness under reduced pressure to obtain a dark green powder product 3b-3, with a yield of 42%.

[0110] The NMR and mass spectrometry data of compound 3b-3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.71 (s,1H), 9.53 (s, 1H), 8.78 (s, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.21(d, J = 7.9 Hz, 2H), 7.01 - 6.88 (m, 1H), 6.48 - 6.23 (m, 1H), 5.27 (dd, J =31.3, 21.9 Hz, 2H), 5.07 (d, J = 6.0 Hz, 2H), 4.82 (d, J = 7.0 Hz, 1H), 4.49 (dd, J = 12.2, 8.0 Hz, 2H), 4.26 (s, 3H), 3.77 (d, J = 7.2 Hz, 2H), 3.56 (d, J= 5.7Hz, 6H), 3.46 (s, 3H), 3.40 (s, 3H), 3.14 (s, 3H), 2.84 (s, 3H), 2.59 (dd, J =15.3, 7.4 Hz, 2H), 2.31 (s, 3H), 2.16 -2.04 (m, 4H), 1.71 (dd, J = 16.4, 8.2Hz, 6H), 1.48 (s, 9H), -1.44 (s, 2H). HRMS (ESI) m / z : C 55 H 67 N6O 13 S + [M+H] + Calculated value: 1051.4481, Detected value: 1051.4484.

[0111] 0.1 mmol of compound 3b-3 (1 eq) was transferred to a pear-shaped flask, and 2 mL of 30 vol.% trifluoroacetic acid in dichloromethane was added. The reaction system was sealed and purged with nitrogen three times. The reaction was stirred continuously at room temperature (25 °C) for 4 h. The reaction solution was washed three times successively with saturated sodium bicarbonate, water, and saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered and purified by direct silica gel column chromatography (200-300 mesh). Nonpolar impurities were washed away by elution with a dichloromethane-methanol system (200:1 v / v). The main band fraction was collected, the eluents were combined, and the solvent was concentrated under reduced pressure and evaporated to obtain a dark green powder product 3b-4 with a yield of 81%.

[0112] The NMR and mass spectrometry data of compound 3b-4 are as follows: 1 H NMR (400 MHz, CDCl3) δ 9.69 (s,1H), 9.36 (s, 1H), 8.73 (s, 1H), 8.10 (s, 1H), 7.89 (d, J = 8.1 Hz, 2H), 7.32(d, J = 7.9 Hz, 2H), 6.53 (dt, J = 15.9, 6.2 Hz, 1H), 5.24 (dd, J = 28.1, 11.6 Hz, 2H), 4.91 (d, J = 6.0 Hz, 1H), 4.86 - 4.76 (m, 1H), 4.47 (dd, J= 16.2, 8.6 Hz,2H), 4.27 (s, 3H), 4.22 (s, 3H), 4.20 (s, 2H), 3.76 (q, J = 7.4 Hz, 2H), 3.58(s, 3H), 3.55 (s, 3H), 3.41 (s, 3H), 3.28 (s, 3H), 3.25 (s, 3H), 3.14 (s,3H), 2.85 (t, J = 3.9 Hz, 2H), 2.65 - 2.55 (m, 1H), 2.35 (s, 3H), 1.70 (m, 6H), -1.52 (s, 2H). HRMS (ESI) m / z : C 50 H 59 N6O 11 S + [M+H] + Calculated value: 951.3957, Detected value: 951.3958.

[0113] Compound 3b-4 was dissolved in a sodium hydroxide-tetrahydrofuran mixture (obtained by mixing 1M sodium hydroxide solution and tetrahydrofuran at a volume ratio of 1:1). The reaction system was sealed and purged with nitrogen three times. The reaction was carried out under continuous stirring at room temperature for 12 h. After the reaction was stopped, the organic solvent was removed by rotary evaporation. The pH was then adjusted to 3-4, and a dark green solid precipitated out. After filtration and vacuum drying, R1 was obtained. 1-1 Compound III was obtained in 92% yield.

[0114] The NMR and mass spectrometry data of compound 3b are as follows: 1 H NMR (400 MHz, DMSO) δ 13.21 – 12.08(s, 2H), 9.78 (s, 1H), 9.69 (s, 1H), 9.09 (s, 1H), 8.27 – 8.17 (m, 1H), 8.09(dd, J = 15.3, 7.9 Hz, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 7.7 Hz, 2H), 6.78 – 6.63 (m, 1H), 5.11 (m, 2H), 4.61 (d, J = 6.0 Hz, 1H), 4.37 (d, J= 10.0Hz, 1H), 4.16 – 4.07 (m, 2H), 3.95 – 3.74 (m, 4H), 3.56 (s, 3H), 3.38 (s,3H), 3.30 (s, 3H), 2.99 (m, 2H), 2.29 (s, 3H), 2.14 (m, 2H), 1.97 (m, 2H),1.69 (t, J = 7.4 Hz, 3H), 1.65 – 1.58 (m, 3H), -1.82 – -1.90 (s, 1H), -2.08 – -2.22 (s, 1H). HRMS (ESI) m / z : C 46 H 49 N6O 11 S + [MH] + Calculated value: 893.3175, Detected value: 893.52.

[0115] Application Example 1: The good tumor targeting properties of compound 2c in vivo Small animal fluorescence imaging was used to assess the tumor enrichment of the compound in mice. First, 4T1 cells (3 × 10⁻⁶) were seeded into the second pair of mammary glands of mice. 5 An orthotopic 4T1 breast cancer model was constructed using cells (per cell). The tumor volume was allowed to grow to approximately 200 mm. 3 The volume was approximately [size missing], and compounds 2 and 2c, as well as the positive control drug NPe6 (photosensitizer), were injected via the tail vein for in vivo imaging. The in vivo imaging results are shown in [image missing]. Figure 1 Compound 2c showed peak fluorescence intensity at the tumor site 1 hour after administration, significantly higher than other tissue regions at the same time. Simultaneously, the tumor fluorescence intensity of 2c at 1 hour after administration was even significantly higher than the positive control NPe6 at 2 hours (its peak time), indicating that 2c has superior tumor targeting. Furthermore, compound 2c was rapidly metabolized, with a significantly weakened fluorescence signal at 12 hours; while NPe6 retained strong fluorescence at the same time point, indicating relatively slow clearance. These results suggest that compound 2c is metabolized more quickly and has better biocompatibility, highlighting its superiority over the existing clinical photosensitizer NPe6 in both tumor-selective enrichment and rapid in vivo clearance, demonstrating excellent tumor targeting and biocompatibility.

[0116] Application Example 2: Targeting ability of compound 2c for small tumor foci Compound 2c was further evaluated using small animal fluorescence imaging technology in a microtumor model (approximately 50 mm in volume). 3 The imaging performance in ) (the model construction method is the same as in application example 1). Figure 2 The tumor in image A has a volume of 200 mm. 3 Solid tumor models, Figure 2 The tumor in the B-mode image is 60 mm in size. 3 Small volume tumor models, such as Figure 2 As shown in Figures A and B, compound 2c not only exhibits significant targeting enrichment in solid tumors but also demonstrates clear and specific fluorescent recognition of small tumor lesions. These results confirm that compound 2c can be used not only as a photosensitizer / sound sensitizer for anti-tumor therapy but also has the potential to serve as a highly efficient tumor-targeting fluorescent probe, suitable for early tumor diagnosis and imaging. This reflects the dual application value and promising translational prospects of 2c in the field of tumor diagnosis and treatment.

[0117] Application Example 3: Evaluation of the tumor targeting properties of compound 1b in vivo Small animal in vivo fluorescence imaging technology was used, following the method described in Application Example 1, to evaluate the tumor accumulation behavior of compound 1b in mice. The experiment included four groups: compound 1 (pyromethesthetized chlorophyll a, a commonly used photosensitizer), compound 1b, control drug Ppa-Asp (a monomodified aspartic acid derivative of compound 1), and positive control drug NPe6. Imaging results are shown in [Figure number missing]. Figure 3 The fluorescence intensity of compound 1b at the tumor site gradually increased over time, reaching a peak at 8 hours post-injection. Compared to the positive control NPe6, whose signal gradually weakened in the tumor region, compound 1b exhibited superior tumor accumulation capacity. Simultaneously, compound 1b showed limited distribution throughout the body, indicating a low level of systemic exposure, a characteristic that helps reduce systemic risks such as skin toxicity. Furthermore, compared to the parent compound 1 and the single-modification control Ppa-Asp, 1b showed significantly enhanced tumor accumulation capacity. This confirms the synergistic effect of the endoplasmic reticulum targeting group and amino acid modification in its structure. The results of this embodiment demonstrate that by jointly modifying the parent nucleus with the endoplasmic reticulum targeting group and amino acids, compound 1b not only significantly improves tumor targeting and accumulation capacity, but its limited systemic distribution also suggests better potential safety. Compared to current positive control drugs, 1b exhibits comprehensive advantages in both targeted accumulation and safety.

[0118] Application Example 4: The targeting ability of compounds to the endoplasmic reticulum The endoplasmic reticulum (ER) targeting ability of the compound was evaluated using a co-localization method with an ER fluorescent probe. Cells were evenly seeded in confocal culture dishes, and after adhesion, the compound was applied. After 4 h of incubation, the culture medium containing the compound was removed, and the ER probe was added for further incubation for 30 min. Images were then acquired and observed under a confocal microscope. The experimental results are as follows: Figure 4The results showed that compound 1a, modified with an endoplasmic reticulum (ER) targeting group, exhibited significant ER co-localization, with a Pearson correlation coefficient (PCC) as high as 0.95, while the PCC values ​​of compound 1 and the positive control HPPH (a phase II clinical photosensitizer) were both 0.89. Therefore, the degree of ER co-localization of compound 1a was significantly higher than that of the unmodified parent compound 1 and HPPH. These examples demonstrate that the introduction of the ER targeting group effectively enhanced the ER targeting ability of compound 1a, thereby achieving cascade targeting from tumor tissue to organelle level.

[0119] Application Example 5: Enhancing the ICD Effect Endoplasmic reticulum stress (ERS) is a core node in the signaling cascade leading to intracellular tumor cell death (ICD). Extensive evidence suggests that ICD triggered by ROS-directed ERS stress is significantly more immunogenic than that triggered by secondary or alternative ERS pathways. A key characteristic of ICD is the release of damage-associated molecular patterns (DAMPs) from tumor cells, such as calreticulin (CRT) exposure, high-mobility group box 1 (HMGB1) release, and ATP secretion, thereby activating an anti-tumor immune response. To assess the effects of ICD, this study used immunofluorescence to detect calreticulin CRT and HMGB1 release, and chemiluminescence to detect ATP secretion levels. 1 × 10⁻⁶ cells were used per well. 4 Cells were seeded in 96-well plates. After cell attachment, a gradient concentration of drug was added and the cells were cultured for 4 hours. The drug was washed off with pre-cooled PBS, and complete culture medium was added again. The cells were divided into sonication and non-sonication groups. The sonication group was sonicated (1.0 MHz, 1 W / cm²). 2 (10 min), continue culturing for 12 h, and then detect the corresponding indicators. Figure 5 As shown, the cell surface exposure CRT was significantly upregulated in the compound 1a + ultrasound group, far exceeding that in the compound 1a + ultrasound group. Simultaneously, HMGB1, a non-histone chromatin-binding protein, is primarily nuclearly localized. After treatment with compound 1a + ultrasound, the intranuclear HMGB1 level significantly decreased, indicating its active transport from the nucleus to the extranuclear space surrounding stressed cells. Consistent with this, ATP secretion also reached its highest level in the compound 1a + ultrasound group. Therefore, this example demonstrates that, compared to compound 1, the endoplasmic reticulum-targeting compound 1a exhibits stronger ICD-inducing ability under ultrasound, effectively promoting DAMPs release, indicating its promising application in activating anti-tumor immune responses.

[0120] Application Example 6: Dendritic Cell Activation This embodiment describes the use of immature mouse bone marrow-derived dendritic cells (iBMDCs) with compound 1 or compound 1a under ultrasound irradiation conditions (1.0 MHz, 1.5 W / cm²). 24T1 tumor cells treated with the compound for 10 min were co-cultured, and the activation levels of dendritic cells (DCs) were compared to assess the compound's ability to induce ICD. Experimental results are as follows: Figure 6 As shown, compared with the unirradiated group, ultrasound treatment alone had a limited effect on the activation of dendritic cells (DCs). However, compound 1a, when combined with ultrasound treatment, significantly promoted DC maturation compared with the compound 1 + ultrasound group, specifically by a significant upregulation of the expression of co-stimulatory molecules CD80 and CD86. This example demonstrates that compound 1a can more effectively enhance the immunogenicity of tumor cells under ultrasound activation conditions and drive DC activation by inducing high expression of co-stimulatory molecules, showing superior potential in stimulating anti-tumor immune responses.

[0121] Application Example 7: Evaluation of Dark Cytotoxicity Compounds 1a and 1b were evaluated for dark cytotoxicity using the MTT assay. An ideal sonosensitive agent should possess low dark toxicity and biocompatibility, while exhibiting tumor cell killing activity upon targeted SDT or PDT irradiation. 1 × 10⁻⁶ cells per well were used. 4 Cells were seeded in 96-well plates. After cell attachment, graded concentrations of the drug were added and cultured for 4 h. After drug withdrawal, the cells were cultured for another 12 h. Then, 20 μL of MTT solution (5 mg / ml) was added to each well, and the cells were incubated for 4 h before termination of culture. The culture medium was discarded, and 150 μL of DMSO was added to each well. The mixture was shaken for 2 min, and the absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated. The results showed that, without sonication, at a concentration of 40 µM, the viability of 4T1 cells in the HPPH and Compound 1 treatment groups decreased to 76.85 ± 1.66% and 45.36 ± 3.51%, respectively. In contrast, Compound 1a and Compound 1b did not show significant cytotoxicity at any of the tested concentrations. Figure 7 The differences were statistically significant. This example illustrates that both the single modification strategy of introducing an endoplasmic reticulum targeting group and the dual modification strategy of combining endoplasmic reticulum targeting and aspartic acid modification can significantly reduce the dark toxicity of the compound, thereby improving its biosafety.

[0122] Application Example 8: Evaluation of Cellular Sonotoxicity The acoustic dynamic activity of compounds 1a and 1b was also evaluated using the MTT assay. An ideal acoustic sensitizer should possess low dark toxicity and high acoustic toxicity. 1 × 10⁻⁶ ppm was applied to each well. 4 Cells were seeded in 96-well plates. After cell attachment, a gradient concentration of drug was added and the cells were cultured for 4 h. The drug was washed off with pre-cooled PBS, and complete culture medium was added again. The cells were divided into sonication and non-sonication groups. The sonication group was sonicated (1.0 MHz, 1 W / cm²). 2After incubation for 10 min, continue culturing for 12 h. Add 20 μl of MTT solution (5 mg / ml) to each well and incubate for 4 h, then terminate the culture. Discard the culture medium, add 150 μL of DMSO to each well, shake for 2 min to mix, and measure the absorbance at 570 nm using a microplate reader to calculate cell viability. Under ultrasonic irradiation, compound 1a can induce significant sonodynamic cytotoxicity at concentrations as low as 10 µM. Figure 8 This indicates that it possesses excellent acoustic activity. This effect may be attributed to its effective endoplasmic reticulum targeting capability. This example demonstrates that, compared to the positive control, compound 1a not only exhibits stronger sonotoxicity under ultrasound conditions but also lower dark toxicity, showing good therapeutic potential and application prospects.

[0123] Application Example 9: Evaluating Treatment Efficacy in Lung Metastases of In Situ Breast Cancer The in vivo antitumor activity of compound 1a was evaluated using an orthotopic 4T1 mouse breast cancer model with lung metastasis. 4T1 mouse breast cancer cells (3 × 10⁻⁶) were used. 5 Cells / mouse were inoculated into Balb / c (female, 5-6 weeks old, 18-22 g) mice directly below the fourth pair of nipples on the left side to establish a mouse model of orthotopic breast cancer with lung metastasis. After tumor growth, mice were randomly divided into 5 groups: control group, ultrasound group, compound 1a alone group (denoted as group 1a), compound 1a + ultrasound group (denoted as group 1 + ultrasound group), and compound 1a + ultrasound group (denoted as group 1a + ultrasound group), and tumor volume changes were monitored daily. Treatment began on day 14 after tumor inoculation. The treatment regimen was: tail vein injection of a sonosensitive agent (dose 16 mg / kg), followed by ultrasound-dynamic therapy on the tumor site 4 hours after administration (parameters: 1.0 MHz, 1.5 W / cm²). 2 (50% duty, 5 min). Treatment was administered every 2 days for a total of 10 sessions. Figure 9 It was found that the tumor mass of the 1a+ultrasound group was significantly reduced compared with the control group, ultrasound group, 1a group, and 1+ultrasound group. Further statistical analysis of the tumor inhibition rate was performed, as shown in Table 1. The tumor inhibition rate of each group was calculated based on the tumor weight data of its comparison with the control group. The tumor inhibition rate of the 1a+ultrasound group was 76.06±3.28%, significantly higher than the other groups. The tumor inhibition rate of the ultrasound group was 8.74±6.98%, the tumor inhibition rate of the 1a group was -17.18±26.42%, and the tumor inhibition rate of the 1+ultrasound group was -19.89±25.35%. Therefore, the examples demonstrate that compound 1a exhibits excellent antitumor activity under ultrasound, thanks to its excellent targeting ability to tumors and endoplasmic reticulum.

[0124] Table 1. Inhibition rate of in situ breast cancer tumors in each group with or without ultrasound.

[0125] Application Example 10: Number of lung metastatic nodules To evaluate the antitumor metastasis effect of compound 1a, a further counting analysis was performed on metastatic nodules in the lungs of mice. The results are as follows: Figure 10 The results showed that the number of lung metastatic nodules was significantly reduced in the 1a+ ultrasound group compared to the control group, ultrasound group, 1a group, and 1+ ultrasound group. Further statistical analysis of the lung nodule inhibition rate was performed, as shown in Table 2. The lung nodule inhibition rate of each group was calculated based on its lung nodule data compared to the control group. The lung nodule inhibition rate of the 1a+ ultrasound group was 89.55±2.88%, significantly higher than the other groups. The lung nodule inhibition rate of the ultrasound group was 48.26±4.19%, the 1a group was 40.3±5.73%, and the 1+ ultrasound group was 49.75±6.01%. This indicates that compound 1a not only effectively inhibits in situ tumor growth but also exhibits a strong anti-metastatic effect. Its mechanism of inhibiting tumor metastasis mainly lies in the fact that compound 1a can specifically accumulate in the endoplasmic reticulum of tumor cells, effectively inducing immunogenic cell death (ICD) under ultrasound stimulation, thereby activating a strong anti-tumor immune response and ultimately achieving effective inhibition of distant metastatic lesions.

[0126] Table 2. Inhibition rate of lung metastasis in each group under ultrasound or without ultrasound.

[0127] Application Example 11: Evaluating Treatment Efficacy in an Orthotopic Pancreatic Cancer Model To further evaluate the efficacy of compound 1a against deep tumors, this study employed a potent immunosuppressive in situ pancreatic cancer model. Pan02 tumor cells (2.5 × 10⁻⁶) were orally inoculated into the pancreas of C57BL / 6J (female, 5–6 weeks old, 18–22 g) mice. 5 Mice were observed daily and randomly divided into a control group, an ultrasound group, a 1a group, a 1a+ ultrasound group, and a 1a+ ultrasound group. Starting from day 9 of tumor implantation, treatment was administered every two days via tail vein injection of 16 mg / kg of a sonosensitive agent. Four hours after administration, sonic therapy (1.0 MHz, 1.5 W / cm²) was performed. 2 (50% duty, 5 min), for a total of 10 treatments. Figure 11 The graph presents the statistical results of tumor tissue weight in the orthotopic pancreatic cancer model for each treatment group at the experimental treatment endpoint. Figure 11 It was found that, compared with the control group, ultrasound group, 1a group, and 1+ ultrasound group, the tumor volume growth in the 1a+ ultrasound group was significantly inhibited. Statistical analysis showed that there was a significant difference in tumor weight after treatment between the 1a+ ultrasound group and other groups.

[0128] Further statistical analysis of the tumor inhibition rate was performed, as shown in Table 3. The tumor inhibition rate of each group was calculated based on the tumor weight data of the group and the control group. Among them, the tumor inhibition rate of the 1a + ultrasound group was 67.25±1.37%, which was much higher than that of the other groups. The tumor inhibition rate of the ultrasound group was 25.82±12.81%, the tumor inhibition rate of the 1a group was 24.12±11.19%, and the tumor inhibition rate of the 1 + ultrasound group was 25.56±3.26%. Therefore, compound 1a + ultrasound showed a significant inhibitory effect on deep tumors, which is attributed to both the penetration depth of ultrasound and the excellent tumor accumulation of compound 1a.

[0129] Table 3. Inhibition rate of tumors in the orthotopic pancreatic cancer model under ultrasound or without ultrasound.

[0130] Application Example 12: Evaluation of the photodynamic therapy effects of compounds 2a and 3a in cells The cellular-level PDT activity of compounds 2a and 3a was evaluated using B16 melanoma cells and MOC-1 head and neck squamous cell carcinoma cells. Logarithmically growing cells were seeded in 96-well plates (1 × 10⁻⁶). 4 Cell count / well), after cell adhesion, incubate with different concentrations of drug, wash off the drug with PBS after 4 h of incubation, replace with fresh culture medium, and light (2 min, 22 mW / cm²). 2 Alternatively, continue culturing. After 24 h of culture, add 20 μL of 5 mg / mL MTT solution to each well and continue culturing for another 4 h. Discard the supernatant and add 150 μL of DMSO. Measure the absorbance of each well at 570 nm using a microplate reader and calculate the cell viability.

[0131] To evaluate the photodynamic therapy (PDT) activity of compounds 2a and 3a, their in vitro killing effects on B16 melanoma cells and MOC-1 head and neck squamous cell carcinoma cells were detected using the MTT assay. Cells in logarithmic growth phase were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and, after adhesion, incubated for 4 hours with different concentrations of the drug. Subsequently, the drug-containing medium was removed, the cells were washed three times with PBS, and replaced with fresh complete culture medium. Cells were divided into a light group and a dark control group: the light group received red light irradiation (wavelength 660 nm, power density 22 mW / cm²). 2 The cells were cultured for 2 hours (2 min), while the dark control group was not exposed to light. After culturing for another 24 hours, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 hours. The supernatant was then carefully discarded, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader, and the cell viability was calculated.

[0132] Figure 12 Figure A shows the cell viability test results of the B16 melanoma cell line after PDT treatment, and Figure B is a bar graph showing the effect of compounds 2a and 3a on the viability of MOC-1 cells under light irradiation. P <0.01~0.001); such as Figure 12 As shown in Figures A and B, compounds 2a and 3a showed better performance in the B16 cell line compared to compound 3 (positive control Ce6, clinical photosensitizer). Figure 12 (A) and MOC-1 cell line ( Figure 12 Compound B) showed stronger photodynamic antitumor activity than the positive control drug compound 3 (Ce6). Therefore, the series of endoplasmic reticulum-targeting compounds involved in this invention show promising application prospects as photosensitizers for antitumor therapy.

[0133] Application Example 13: Evaluation of the photodynamic therapy effect of compound 3a in vivo To further evaluate the antitumor effect of compound 3a in vivo, a melanoma model was used. B16 melanoma cells (4 × 10⁻⁶) were subcutaneously inoculated into the thigh of female C57BL / 6J mice (5–6 weeks old, 18–22 g). 5 Cells / animal) were used to establish a xenograft tumor model. The tumor was allowed to grow to 200 mm. 3 Subsequently, mice were randomly divided into three groups: a light irradiation group, a 3a+ light irradiation group, and a 3+ light irradiation group (positive control group). Photodynamic therapy was initiated on day 7 post-inoculation. The treatment regimen was as follows: a photosensitizer (5 mg / kg) was injected via the tail vein, followed immediately by light irradiation of the tumor site (power density 250 mW / cm²). 2 (5 min). Treatment was administered every 3 days for a total of 4 times. Figure 13 The results presented are the cell viability assay results of the MOC-1 cell line after PDT treatment, such as... Figure 13 As shown, while the 3+ light irradiation group exhibited some tumor inhibition effect, there were significant individual differences. In contrast, the 3a+ light irradiation group showed more significant tumor growth inhibition, and the efficacy remained consistent across individuals. Further statistical analysis of the tumor inhibition rate was performed, as shown in Table 4. The tumor inhibition rate for each group was calculated based on the tumor weight data of its group and the control group. The tumor inhibition rate of the 3a+ light irradiation group was 70.78±1.98%, higher than that of the positive control group. These results indicate that under the experimental conditions, the photodynamic antitumor efficacy of compound 3a is superior to that of the clinical photosensitizer Ce6 (compound 3), and the consistency of the treatment response is better.

[0134] Table 4. Inhibition rates of compounds 3a and 3 on tumors in a B16 melanoma model under light irradiation.

[0135] In vivo experimental results show that the compounds provided by this invention exhibit excellent tumor targeting, outperforming the clinical positive control drug tarapofen sodium (NPe6), and possessing a faster in vivo clearance rate and higher biocompatibility. Compared with the positive control drug photoclopramide (HPPH), they can precisely target the endoplasmic reticulum, enhancing the immunogenic cell death (ICD) effect, thereby initiating an anti-tumor immune response and synergistically inhibiting the growth of in situ and metastatic tumors. Compared with the clinically commonly used photosensitizer dihydroporphyrin e6 (Ce6), this series of compounds shows significant advantages in photodynamic therapy efficacy. In summary, this series of compounds, through structural design, achieves cascade targeting from tumor tissue to endoplasmic reticulum organelles. This precise targeting not only enhances the direct efficacy of PDT or SDT, but also, through efficient ICD induction, synergizes local treatment with a systemic anti-tumor immune response, achieving an upgrade in treatment modality. Therefore, this series of compounds demonstrates outstanding potential in terms of biocompatibility, therapeutic synergy, and ultimate efficacy, and has broad translational and application prospects in the field of integrated anti-tumor drug development and precision medicine.

[0136] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A dihydroporphyrin compound having the structure shown in any one of Formula II, Formula III, Formula V, or Formula VII: ； In formulas III, V, or VII, R1, R8, and R9 are independently hydroxyl groups. Formula R 1-1 , Formula R 1-2 or Formula R 1-3 R2 is Formula R 2-1 ; In Formula II, R1 and R8 are independently hydroxyl groups. Formula R 1-1 , Formula R 1-2 or Formula R 1-3 R2 is H or Formula R 2-1 ; When R1 and R8 are both hydroxyl groups, R2 is not H; n is any integer from 1 to 5.

2. The metal complex of the dihydroporphyrin compound according to claim 1, characterized in that, It has any of the structures shown in Formula II-a, Formula III-a, Formula Va, or Formula VII-a: ； M is a divalent, trivalent, or tetravalent metal ion; the divalent metal ion includes Cu. 2+ Fe 2+ Zn 2+ Mg 2+ Ni 2+ Co 2+ Pt 2+ or Rh 2+ Trivalent metal ions include Ir 3+ Tetravalent metal ions include Sn 4+ Ti 4+ Or Ru 4+ .

3. A metal salt of a dihydroporphyrin compound having the structure shown in any of formulas b, c, e, or g: ； In equations c, e, or g, R1, R8, and R9 are independently... , , or R2 is ; In equation b, R1 and R8 are independent. , , or R2 is H or When R1 and R8 are both When R2 is not H, R3 is Na or K.

4. The metal complex of the metal salt of the dihydroporphyrin compound according to claim 3, characterized in that, It has any of the structures shown in formula bm, formula cm, formula em, or formula gm: ； M can be a divalent, trivalent, or tetravalent metal ion.

5. The method for preparing the dihydroporphyrin compound according to claim 1, characterized in that, This includes the following four situations: The first method, when the dihydroporphyrin compound has the structure shown in formula II, III, V, or VII and R1, R8, and R9 are hydroxyl groups, includes the following steps: Compound 2, compound 3, compound 5 or compound 7 are subjected to a first methylation reaction with iodomethane under basic conditions to obtain compound 2a-1, compound 3a-1, compound 5a-1 or compound 7a-1; The structural formulas of compound 2, compound 3, compound 5, or compound 7 are as follows: ； The compounds 2a-1, 3a-1, 5a-1, or 7a-1 are respectively: ； Compound 2a-1, compound 3a-1, compound 5a-1 or compound 7a-1 are subjected to a third olefin metathesis reaction with a substituted terminal alkene under catalytic conditions to obtain compound 2a-2, compound 3a-2, compound 5a-2 or compound 7a-2. The structural formula of the substituted terminal olefin is: ; The structural formulas of compounds 2a-2, 3a-2, 5a-2, or 7a-2 are respectively; ; Compound 2a-2, compound 3a-2, compound 5a-2 or compound 7a-2 were subjected to a third deprotection in an acidic solution to obtain compound 2a-3, compound 3a-3, compound 5a-3 or compound 7a-3; The structures of compounds 2a-3, 3a-3, 5a-3, or 7a-3 are as follows: ； Compound 2a-3, compound 3a-3, compound 5a-3 or compound 7a-3 are subjected to a first base deprotection in an alkaline solution to obtain compound II, compound III, compound V or compound VII in which R1, R8 and R9 are hydroxyl groups; The second type: When the dihydroporphyrin compound has the structure shown in formula III or VII and R1 and R9 are hydroxyl groups, and R8 is a compound of formula R... 1-1 R 1-2 Or R 1-3 The preparation method includes the following steps: Compound 3 or compound 7 is reacted with an amino acid methyl ester compound under alkaline conditions and a condensing agent to undergo a second amide condensation reaction to obtain compound 3b-1 or compound 7b-1. The structures of compounds 3, 7, 3b-1, and 7b-1 are as follows: ； R7 is , , ; The structural formula of the amino acid methyl ester compound is as follows: , , ; Compound 3b-1 or compound 7b-1 is subjected to a second methylation reaction with iodomethane under basic conditions to obtain compound 3b-2 or compound 7b-2; The structural formulas of compound 3b-2 and compound 7b-2 are as follows: ； Compound 3b-2 or compound 7b-2 is reacted with a substituted terminal alkene in the presence of a catalyst to undergo a fourth olefin metathesis reaction, yielding compound 3b-3 or compound 7b-3; the structural formulas of compound 3b-3 or compound 7b-3 are as follows: ； The fourth deprotection of compound 3b-3 or compound 7b-3 was performed in an acid solution to obtain compound 3b-4 or compound 7b-4; the structures of compound 3b-4 or compound 7b-4 are as follows: ； Compound 3b-4 or compound 7b-4 is subjected to a second base deprotection in an alkaline solution to obtain R1 and R9, which are hydroxyl groups, and R8, which is of formula R. 1-1 R 1-2 Or R 1-3 Compound III or compound VII at that time; The third type: When the dihydroporphyrin compound has the structure shown in Formula II or Formula V, R1 and R8 are R 1-1 R 1-2 Or R 1-3 R2 is R 2-1 The preparation method includes the following steps: Compound 2 or compound 5 is reacted with an amino acid methyl ester compound under alkaline conditions and a condensing agent to undergo a third amide condensation reaction to obtain compound 2b-1 or compound 5b-1. The structural formulas of compounds 2 and 5 are as follows: ； The structural formulas of compound 2b-1 and compound 5b-1 are as follows: ； Compound 2b-1 or compound 5b-1 is reacted with a substituted terminal alkene in the presence of a catalyst to undergo a fifth olefin metathesis reaction, yielding compound 2b-2 or compound 5b-2. ； The fifth deprotection of compound 2b-2 or compound 5b-2 was performed in an acid solution to obtain compound 2b-3 or compound 5b-3; the structures of compound 2b-3 or compound 5b-3 are as follows: ； Compound 2b-3 or compound 5b-3 was subjected to a third alkali deprotection in an alkaline solution to obtain R1 and R8, which are R1 and R8, respectively. 1-1 R 1-2 Or R 1-3 R2 is R 2-1 Compound II or compound V; The fourth type: When the dihydroporphyrin compound has the structure shown in Formula II, R1 and R8 are R 1-1 R 1-2 Or R 1-3 When R2 is H, the preparation method includes the following steps: Compound 2b-1 was subjected to a fourth base deprotection in an alkaline solution to obtain R1 and R8. 1-1 R 1-2 Or R 1-3 Compound II, where R2 is H.

6. The preparation method according to claim 5, characterized in that, The catalyst for the third, fourth, or fifth olefin metathesis reaction is independently a Grubbs catalyst, the reaction solvent is independently one or more of dichloroethane, dichloromethane, and toluene, and the reaction time is independently 6 to 36 h; the molar ratio of compound 2a-1, compound 3a-1, compound 5a-1, or compound 7a-1, the substituted terminal olefin, and the catalyst is 1:2 to 30:0.01 to 0.7; The molar ratio of compound 3b-2 or compound 7b-2, the substituted terminal alkene, and the catalyst is 1:2~15:0.01~0.7; the molar ratio of compound 2b-1 or compound 5b-1, the substituted terminal alkene, and the catalyst is 1:2~15:0.01~0.

7.

7. The preparation method according to claim 5, characterized in that, The condensing agents for the second and third amide condensation reactions independently include one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; The basic substances in the second and third amide condensation reactions independently include N,N-diisopropylethylamine and / or triethylamine; the solvents in the first, second, and third amide condensation reactions are independently dichloromethane and / or DMF. The molar ratio of compound 3 or compound 7, condensing agent, basic substance and amino acid methyl ester compound is 1:1~1.2:1~2:1~1.2; the molar ratio of compound 2 or compound 5, condensing agent, basic substance and amino acid methyl ester compound is 1:2~10:2~10:2~10. The basic conditions for both the first and second methylation are provided by alkali metal carbonates; the alkali metal carbonates independently include anhydrous sodium carbonate and / or anhydrous potassium carbonate; the solvent for the second methylation is DMF; the molar ratio of compound 2, compound 3, compound 5 or compound 7, iodomethane and alkali metal carbonate is 1:3~20:3~20; the molar ratio of compound 3b-1 or compound 7b-1, iodomethane and alkali metal carbonate is 1:2~10:2~10; The concentration of compound 3b-1 or compound 7b-1 in the reaction solution is 0.05~0.5 M; the temperature of the first methylation and the second methylation are independently 20~40 °C; and the reaction time is independently 1~4 h.

8. The method for preparing the metal complex of dihydroporphyrin compounds according to claim 2, characterized in that, Includes the following steps: The dihydroporphyrin compound of claim 1, a metal chloride or a metal acetate complex are mixed and heated under reflux to obtain a metal complex of the dihydroporphyrin compound.

9. A method for preparing the metal salt of the dihydroporphyrin compound according to claim 3, comprising the following steps: The dihydroporphyrin compounds are salted in an alkaline solution of sodium hydroxide or potassium hydroxide to obtain the metal salts of the dihydroporphyrin compounds. The dihydroporphyrin compound is the dihydroporphyrin compound as described in claim 1.

10. The use of the dihydroporphyrin compounds II and III as described in claim 1 in the preparation of photosensitizers or antitumor drugs.

Images