A kind of aminopeptidase activated photodynamic photosensitizer and its preparation method and application

Abstract

The application belongs to the field of fluorescent probes, and particularly relates to an aminopeptidase-activated photodynamic photosensitizer as well as a preparation method and application thereof. In order to increase the selectivity of the photosensitizer to tumor cells, based on the selenium rhodamine spiro ring 'off-on' mechanism, an 'activated' photodynamic photosensitizer SeR-APN-NO activated by cancer cell surface overexpressed aminopeptidase (APN) is developed. The photosensitizer only exhibits strong cell killing ability under light irradiation after being 'activated' by the cancer cell surface overexpressed APN, and therefore does not cause any damage to normal cells / tissues.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes, and particularly relates to an aminopeptidase-activated photodynamic photosensitizer and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) has shown great potential in tumor treatment due to its advantages such as non-invasiveness, low side effects, no drug resistance, and repeatability. PDT mainly causes local oxidative stress in cancer cells through the combined action of photosensitizers (PS), light sources, and oxygen, leading to apoptosis or necrosis of tumor cells. In other words, PS is non-toxic when not irradiated with light, but after irradiation with specific light, it will produce free radicals such as singlet oxygen or superoxide at the tumor site, causing blood vessel damage or directly killing tumor cells. However, due to the lack of targeting to cancer cells / tissues, most photosensitizers may increase the risk of damage to normal cells / tissues in addition to killing cancer cells / tissues during photodynamic therapy. Therefore, how to increase the selectivity of photosensitizers for tumor cells still faces many challenges. Summary of the Invention

[0003] To increase the selectivity of photosensitizers for tumor cells, the present invention has developed an "activated" photodynamic photosensitizer SeR-APN-NO based on the "off-on" mechanism of selenorhodamine spirocycle, which is activated by aminopeptidase (APN) overexpressed on the surface of cancer cells. This photosensitizer can highly selectively kill tumor cells under light without any damage to normal cells, and has potential clinical application value.

[0004] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0005] The present invention provides an aminopeptidase-activated photodynamic photosensitizer, the structural formula of which is:

[0006]

[0007] The present invention also provides a method for preparing an aminopeptidase-activated photodynamic photosensitizer, comprising the following steps:

[0008]

[0009] Step 1, under the protection of nitrogen, 2-iodobenzoic acid and anhydrous THF were mixed, and the resulting solution was cooled, then isopropyl magnesium chloride-lithium chloride solution was added dropwise to react, after warming, THF solution of compound 1 was added dropwise to continue to react, then gradually returned to room temperature to react, after the reaction was completed, the reaction solution was quenched in an ice water bath, and then distilled under reduced pressure, the obtained solid was redissolved in DCM, NaOH aqueous solution was added, stirred, suction filtered, the filtrate was extracted, and the combined organic phase was concentrated and purified by neutral alumina column chromatography to obtain compound 2;

[0010] Step 2, under the protection of nitrogen, 1,3-dimethylbarbituric acid and Pd(PPh3)4 were added to THF solution of compound 2, and stirred at room temperature to react, after the reaction was completed, NaOH aqueous solution was added, extracted, the combined organic phase was dried, concentrated and purified by silica gel column chromatography to obtain compound 3;

[0011] Step 3, under the protection of nitrogen, compound 3, HATU, Boc-Glu-OtBu and DIEA were added to DMF, and reacted at room temperature, after the reaction was completed, water was added and extracted, the combined organic phase was dried, concentrated and purified by silica gel column chromatography to obtain compound 4;

[0012] Step 4, compound 4 was dissolved in super-dry THF, then n-butyl nitrite was added to stir, after the reaction was completed, detected by TLC, distilled under reduced pressure, the obtained solid was redissolved in dichloromethane, the solution was cooled, then TFA was added dropwise to continue to stir, after the reaction was completed, distilled under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the photosensitizer.

[0013] Further, in step 1, the molar ratio of 2-iodobenzoic acid, isopropyl magnesium chloride-lithium chloride and compound 1 was 10:20:1.

[0014] Further, in step 1, the resulting solution was cooled to-78℃, isopropyl magnesium chloride-lithium chloride solution was added dropwise to react at-78℃ for 30 minutes, after warming to 0℃, THF solution of compound 1 was added dropwise to continue to react for 30 minutes, then gradually returned to room temperature to react for 32 hours, the concentration of NaOH aqueous solution was 2 mol / L, stirred for 30 minutes, and the developing agent used in neutral alumina column chromatography was PE and EA, and the volume ratio was 2:1.

[0015] Further, in step 2, the molar ratio of 1,3-dimethylbarbituric acid, compound 2 and Pd(PPh3)4 was 12:6:1.

[0016] Further, the stirring reaction in step 2 is carried out at room temperature for 24 hours, the concentration of the aqueous NaOH solution is 2 mol / L, and the developing agent used in the silica gel column chromatography is EA and MeOH with a volume ratio of 10:1.

[0017] Further, the molar ratio of compound 3, HATU and Boc-Glu-OtBu in step 3 is 4:9:8, and the amount of DIEA used is 421.1 μL of DIEA per 0.4 mmol of compound 3.

[0018] Further, the stirring reaction in step 3 is carried out at room temperature for 2 hours, and the developing agent used in the silica gel column chromatography is PE and EA with a volume ratio of 10:1.

[0019] Further, the molar ratio of compound 4 and n-butyl nitrite in step 4 is 1:1.1, the stirring reaction is carried out at a temperature of 60°C for 4 hours, the solution temperature is then lowered to -10°C, TFA is added dropwise, and the stirring reaction is continued for 3 hours, and the developing agent used in the silica gel column chromatography is PE and EA with a volume ratio of 5:1.

[0020] The application also provides an application of the amino peptidase-activated photodynamic photosensitizer in killing cancer cells / tissues under the irradiation of an LED light source at 561 nm.

[0021] Compared with the prior art, the application has the following advantages:

[0022] Due to the lack of targeting to cancer cells / tissues, in the process of photodynamic therapy, the conventional photosensitizer may increase the risk of damage to normal cells / tissues in addition to killing cancer cells / tissues. The application develops an "activated" photodynamic photosensitizer based on the selenium rhodamine spiro "on-off" mechanism. The photosensitizer itself exists in the form of a lactone with a closed ring, has extremely small cell dark toxicity and phototoxicity, and only after being activated by the APN overexpressed on the surface of cancer cells, the photosensitizer exhibits strong phototoxicity. Therefore, in the process of photodynamic therapy, the photosensitizer developed by the application can kill cancer cells with high selectivity without causing any damage to normal cells / tissues. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 In the figure, (A) and (B) are the ultraviolet-visible absorption spectrum change diagrams of the PBS solutions of SeR-APN-NO (2 μM) and SeR-APN-NO / ABDA under the irradiation of an LED light source (10 mW / cm 2 ) at 565 nm.

[0024] Figure 2(A) is the UV-visible absorption spectrum of SeR-APN-NO (6 μM) PBS solution at 370°C for 1 hour, (B) is the UV-visible absorption spectrum of SeR-APN-NO / APN (50 ng / mL, 37°C, 1 hour) / ABDA PBS solution at 565 nm LED light source (10 mW / cm 2 ) UV-visible absorption spectrum changes under irradiation.

[0025] Figure 3 These are the HPLC-MS pictures of SeR-APN-NO (6 μM), SeR-APN-NO / APN and SeR-APN-NO / APN+hv.

[0026] Figure 4 These are the results of live / dead cell staining experiments after treatment with SeR-APN-NO, SeR-APN-NO+hν, and SeR-APN-NO+Ube+hν. The collection ranges of Calcein-AM and PI are 490-560 nm (λex=488 nm) and 570-750 nm (λex=561 nm), respectively. Scale bar: 50 μm.

[0027] Figure 5 The graph shows the experimental results of phototoxicity and dark toxicity of SeR-APN-NO on HepG2 and LO2 cells under normoxic (21% O2) and hypoxic (2% O2) conditions.

[0028] Figure 6 In the middle, (A) is a nude mouse bearing a tumor that was injected with SeR-APN-NO (20 μM, 125 μL) in situ and then illuminated with a 565 nm LED light source (100 mW / cm 2 (B) and (C) are the curves showing the changes in tumor volume and body weight of tumor-bearing mice, respectively. DETAILED DESCRIPTION

[0029] In order to further illustrate the technical solution of the present invention, the present invention is further described below through examples.

[0030] Example 1

[0031] An aminopeptidase-activated photodynamic photosensitizer, the structural formula of which is:

[0032]

[0033] A method for preparing an aminopeptidase-activated photodynamic photosensitizer comprises the following steps:

[0034] Step 1: 2-iodobenzoic acid (1.3 g, 5.0 mmol) was added to a dry round-bottom flask. Anhydrous THF (10.0 mL) was added under nitrogen protection, and the resulting solution was cooled to -78°C. Subsequently, isopropylmagnesium chloride-lithium chloride solution (1.3 M, 7.7 mL, 10.0 mmol) was added dropwise. The reaction solution was reacted at -78°C for 30 minutes, then the temperature was raised to 0°C, and a THF solution of compound 1 (200 mg, 0.5 mmol) was then added dropwise. The reaction solution was reacted at 0°C for 30 minutes, then gradually returned to room temperature. The reaction solution was reacted at room temperature for 32 hours. After the reaction, the reaction solution was quenched with methanol (5.0 mL) in an ice-water bath, and the mixture was distilled under reduced pressure to remove the solvent. The obtained solid was redissolved in DCM, and NaOH (2.0 mol / L 10.0 mL) was added. After stirring for 30 min, the mixture was filtered and the filtrate was extracted with EA. The combined organic phases were concentrated and purified by neutral alumina column chromatography (PE / EA = 2 / 1, V / V, the same below) to obtain compound 2 as a bluish-white solid (164 mg, 65%). 1 H NMR (600MHz, CDCl3) δ7.92(d,J=7.5Hz,1H),7.77(d,J=7.6Hz,1H),7.61(t,J=7.5Hz,1H),7.53(t,J=7.4Hz,1H ),6.96-6.85(m,4H),6.43(t,J=11.1Hz,2H),5.83-5.76(m,2H),5.16-5.11(m,4H),3.89(s,4H),2.93(s,6H); 13 C NMR (150MHz, CDCl3) δ170.24,153.27,150.03,148.39,134.00,133.04,130.53,130.51,129.26,128.02,12 7.93,125.90,125.82,124.37,122.32,116.29,111.63,111.55,110.86,110.79,52.57,40.23; HR-MS[M+H] + :calculated for 503.1232,Found 503.1229.

[0035] Step 2: Under N2 protection, 1,3-dimethylbarbituric acid (129.5 mg, 0.8 mmol) and Pd(PPh3)4 (34.6 mg, 0.07 mmol) were added to a solution of compound 2 (205.0 mg, 0.4 mmol) in THF (10.0 mL), and the mixture was stirred at room temperature for 24 h. After the reaction, aqueous NaOH solution (2 mol / L 20.0 mL) was added to the solution, and the mixture was extracted with EA. The combined organic phases were dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography (EA / MeOH = 10 / 1) to afford compound 3 as a red solid (169.9 mg, 98%). 1 H NMR(600MHz,CD3CN)δ7.90(d,J=7.6Hz,1H),7.76-7.62(m,3H),6.95(d,J=2.5Hz,1H),6.90(d,J=2.2Hz,1H),6.8 1(d,J=9.0Hz,1H),6.73(d,J=8.6Hz,1H),6.50(dd,J=9.0,2.6Hz,1H),6.41(dd,J=8.6,2.3Hz,1H),2.91(s,6H); 13 C NMR(150MHz,CD3CN)δ169.54,150.44,148.55,134.46,130.44,129.76,128.52,128.15,12 5.96,125.50,124.56,122.77,121.44,113.36,113.11,111.23,110.95,39.38; HR-MS[M+H] + :calculated for 423.0606,Found423.0606.

[0036] Step 3: Under N₂ protection, compound 3 (153.3 mg, 0.4 mmol), HATU (332.2 mg, 0.9 mmol), Boc-Glu-OtBu (241.0 mg, 0.8 mmol), and 0.4 (421.1 μL) were added to DMF (10.0 mL), and the mixture was reacted at room temperature for 2 h. After the reaction, water was added to the solution, and the mixture was extracted with EA. The combined organic phases were dried over Na₂SO₄, concentrated, and purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound 4 as a purple-red solid (283.3 mg, 60%). 1H NMR (600MHz, CD3CN) δ8.09-8.03(m,1H),7.91(d,J=5.4Hz,2H),7.57(d,J=4.7Hz,1H),7.54-7.50(m,1H),7.21-7.1 1(m,3H),6.91(s,1H),6.53(s,1H),4.98(s,1H),4.30(s,1H),2.96(s,6H),2.82(s,3H),1.46(s,9H).,2.91(s,6H); 13 C NMR(150MHz,CD3CN)δ169.54,150.44,148.55,134.46,130.44,129.76,128.52,128.15,12 5.96,125.50,124.56,122.77,121.44,113.36,113.11,111.23,110.95,39.38; HR-MS[M+H] + :calculated for 594.1502,Found594.1505.

[0037] Step 4: Compound 4 (136.3 mg, 0.2 mmol) was dissolved in ultra-dry THF (15.0 mL). Then, n-butyl nitrite (23.7 mg, 0.23 mmol) was added to the solution. The mixture was stirred at 60° C. for 4 hours. After the reaction was completed by TLC, the solvent was removed from the mixture by distillation under reduced pressure. The resulting solid was redissolved in dichloromethane (4 mL). After the solution temperature was lowered to −10° C., TFA (0.5 mL) was added dropwise. The mixture was stirred at this temperature for 3 hours. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA=5 / 1) to obtain the photosensitizer Se-APN-NO as a white powder (20.2 mg, 17%). 1 H NMR(600MHz,CD3CN)δ9.59(s,1H),8.16(s,1H),8.01-7.94(m,3H),7.75-7.65(m,2H),7.50 -7.36(m,3H),7.27(d,J=8.7Hz,1H),3.55-3.48(m,1H),3.40(s,3H),1.32(d,J=6.7Hz,3H); 13C NMR (150MHz, CD3CN) δ174.84,169.40,153.14,142.47,139.21,135.26,133.05,130.45,129.55,129.53,128.90,128.23,128. 21,127.59,127.28,125.97,123.91,123.88,123.53,123.51,119.21,118.96,87.06,51.12,30.55,20.46,13.41; HR-MS[M+H] + :calculated for 509.0723,Found 509.0726.

[0038] Example 2

[0039] 1. Preparation of solution

[0040] A stock solution of SeR-APN-NO (2 mM) was prepared with chromatographically pure CH3CN and diluted to the desired concentration with PBS (10 mM, pH = 7.4). 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 5 mM) was prepared with ultrapure water.

[0041] 2. Photostability study of photosensitizers

[0042] The photostability of the photosensitizer SeR-APN-NO (6 μM) was tested using a UV-visible absorption spectrometer. Figure 1 As shown in A, SeR-APN-NO exists in the form of a closed-ring lactone in PBS, that is, the photosensitizer is in a "locked" state. When illuminated by a 565 nm LED light source (10 mW / cm 2 ) After continuous irradiation for 2 minutes, the absorption peak hardly changed, indicating that SeR-APN-NO has good photostability in PBS.

[0043] Taking ABDA as 1 O2 capture agent, using Rose Bengal (RB) as reference, to test the production of SeR-APN-NO in PBS 1 O2's capabilities. Figure 1 As shown in B, a 565nm LED light source (10mW / cm 2 When the ABDA / SeR-APN-NO solution was continuously irradiated for 2 min, the absorption peak at 565 nm remained unchanged, and the absorbance value of ABDA at 380 nm also remained unchanged. The experimental results showed that SeR-APN-NO would not produce 1 O2, no photodynamic activity.

[0044] 3. Study on the photodynamic effect of photosensitizers in vitro

[0045] We investigated whether SeR-APN-NO could be activated by CD13 / aminopeptidase N (APN, EC3.4.11.2) overexpressed on cancer cell membranes. Considering that enzymatic reactions usually need to be carried out at 37°C, we first confirmed that SeR-APN-NO has good thermal stability at 37°C by UV-visible absorption spectroscopy ( Figure 2 APN (50 ng / mL) was then added to the PBS solution of SeR-APN-NO (6 μM) and reacted at 37°C for 1 h before adding ABDA. Figure 2 As shown in B, a 565nm LED light source (10mW / cm 2 When the ABDA / SeR-APN-NO / APN solution was continuously irradiated for 2 min, the absorbance of ABDA at 380 nm decreased rapidly. The experimental results showed that after SeR-APN-NO reacted with APN and then irradiated with light, NO and photosensitizer SeR were released, and a large amount of 1 O2. Subsequently, the enzymatic hydrolysis products of SeR-APN-NO and the products after illumination ( Figure 3 ).

[0046] 4. Study on the effect of photosensitizer on photodynamic therapy at the cellular level

[0047] Considering that APN is overexpressed in many cancer cells, we then used confocal laser scanning microscopy (CLSM) to evaluate whether SeR-APN-NO could be specifically activated by cancer cells. We selected HepG2 cells with high APN activity and LO2 cells with low APN activity. The results of the live / dead cell staining experiment showed that ( Figure 4 ), HepG2 cells loaded with SeR-APN-NO showed bright red fluorescence signals from PI after illumination, while the green fluorescence from Calcein-AM was negligible. HepG2 cells loaded with SeR-APN-NO / APN inhibitor (Ube, 100 μM) showed only green fluorescence signals from Calcein-AM after illumination; however, LO2 cells loaded with SeR-APN-NO showed only green fluorescence signals from Calcein-AM after illumination. These results indicate that Se-APN-NO can selectively kill HepG2 cells under illumination while having no effect on normal LO2 cells.

[0048] Subsequently, the phototoxicity and dark toxicity of Se-APN-NO to HepG2 and LO2 cells under normoxia or hypoxia were evaluated by CCK8 assay. HepG2 / LO2 cells were incubated with different concentrations of Se-APN-NO (0, 0.2, 0.5, 1, 2, 5 μM) for 4 hours. The non-illumination group was placed in a cell culture incubator for 24 hours, and the illumination group was illuminated with a 565 nm LED light source (30 mW / cm 2 , 30min) and then placed in a cell culture incubator for 24 hours. Figure 5 As shown in the results, Se-APN-NO showed low dark sensitivity to HepG2 cells and LO2 cells under both normoxic (21% O2) and hypoxic (2% O2) conditions. Under light conditions, the survival rate of HepG2 cells decreased rapidly with the increase of Se-APN-NO concentration, and the half-lethal concentration (IC 50 ) were 0.65 μM (21% O2) and 0.71 μM (2% O2), respectively. The survival rate of LO2 cells was unaffected by light exposure and Se-APN-NO concentration. These experimental results demonstrate that Se-APN-NO has excellent photodynamic therapy efficacy against cancer cells, and its therapeutic effect is independent of oxygen concentration, making it a photodynamic photosensitizer with potential clinical application.

[0049] 5. Study on the effect of photosensitizer on photodynamic therapy at the in vivo level

[0050] The PDT efficacy of Se-APN-NO in tumor-bearing nude mice was evaluated. The tumor-bearing nude mice were divided into PBS group, PBS+hv group, Se-APN-NO group, Se-APN-NO+hv group and Se-APN-NO+Ube+hv group. The tumor sites of the tumor-bearing nude mice were injected with PBS (125 μL) and Se-APN-NO (20 μM, 125 μL) in situ, and then irradiated with 565 nm LED light (100 mW / cm 2 ) for 30 minutes, and the tumor volume and weight of tumor-bearing mice were recorded every other day. Figure 6 As shown in A, the tumors in the Se-APN-NO+hv group gradually became smaller, and scabs were observed at the tumor site on the 6th day after illumination. The tumors were completely cleared on the 12th day after illumination. In addition, no obvious damage was observed after subcutaneous injection of SeR-APN-NO and illumination at the normal site on the other side of the nude mice. However, the tumors in the other groups continued to grow throughout the treatment process ( Figure 6 Middle B). The body weight of each group of tumor-bearing mice did not show abnormalities during the treatment ( Figure 6The results show that SeR-APN-NO has good biocompatibility, can be activated by APN overexpressed in HepG2 cells, and thus exhibits excellent PDT effect in the HepG2 tumor-bearing mouse model, with little effect on normal tissues.

[0051] In summary, the present application develops an APN-activated photodynamic photosensitizer SeR-APN-NO. Due to the double "locking" (electron-withdrawing effect) of NO and alanyl amine substituents, Se-APN-NO itself exists in the form of a ring-closed lactone and does not show photodynamic effect under LED light irradiation at 565 nm; however, after the alanyl amine moiety of Se-APN-NO is cleaved by APN overexpressed on the surface of HepG2 cells, the cleavage product can induce HepG2 cell death under LED light irradiation at 565 nm, with IC50 values of 0.65 μM and 0.71 μM under normoxia (21% O2) and hypoxia (2% O2), respectively. In vivo experiments show that the tumor site of a tumor-bearing nude mouse injected with Se-APN-NO in situ is completely ablated after a single light irradiation for 12 days.

[0052] The above merely provides the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An aminopeptidase-activated photodynamic photosensitizer, characterized in that: The structural formula of the photosensitizer is:

2. A method for preparing the aminopeptidase-activated photodynamic photosensitizer according to claim 1, characterized in that: The following steps are involved: Step 1: Under nitrogen protection, 2-iodobenzoic acid and anhydrous THF are mixed, and the resulting solution is cooled. An isopropylmagnesium chloride-lithium chloride solution is then added dropwise to react. After heating, a THF solution of compound 1 is added dropwise to continue the reaction, and then the temperature is gradually restored to room temperature for reaction. After the reaction is completed, the reaction solution is quenched in an ice-water bath and distilled under reduced pressure. The obtained solid is redissolved in DCM, an aqueous NaOH solution is added, stirred, and filtered. The filtrate is extracted, and the combined organic phase is concentrated and purified by neutral alumina column chromatography to obtain compound 2; Step 2: Under nitrogen protection, 1,3-dimethylbarbituric acid and Pd(PPh3)4 are added to a THF solution of compound 2, and the reaction is stirred at room temperature. After the reaction is completed, a NaOH aqueous solution is added, and the resulting mixture is extracted. The combined organic phase is dried, concentrated, and purified by silica gel column chromatography to obtain compound 3. Step 3, under nitrogen protection, compound 3, HATU, Boc-Glu-OtBu and DIEA were added to DMF and reacted at room temperature. After the reaction was completed, water was added and extracted. The combined organic phase was dried, concentrated and purified by silica gel column chromatography to obtain compound 4; Step 4, compound 4 is dissolved in ultra-dry THF, and then n-butyl nitrite is added to react with stirring. After the reaction is completed by TLC detection, the solid is redissolved in dichloromethane by vacuum distillation. After the solution temperature is lowered, TFA is added dropwise and continued to react with stirring. After the reaction is completed, it is removed by vacuum distillation, and the crude product is purified by silica gel column chromatography to obtain the photosensitizer; The structural formula of the compound 1 is: The structural formula of the compound 2 is: The structural formula of the compound 3 is: The structural formula of the compound 4 is:

3. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In step 1, the molar ratio of 2-iodobenzoic acid, isopropylmagnesium chloride-lithium chloride, and compound 1 is 10:20:

1.

4. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In the step 1, the obtained solution is cooled to -78°C, and an isopropylmagnesium chloride-lithium chloride solution is added dropwise for the reaction at a temperature of -78°C for 30 minutes. After the temperature is raised to 0°C, a THF solution of compound 1 is added dropwise and the reaction is continued for 30 minutes. The temperature is gradually restored to room temperature and the reaction is carried out for 32 hours. The concentration of the NaOH aqueous solution is 2 mol / L, and the mixture is stirred for 30 minutes. The developing solvents used for neutral alumina column chromatography are PE and EA in a volume ratio of 2:

1.

5. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In step 2, the molar ratio of 1,3-dimethylbarbituric acid, compound 2 and Pd(PPh3)4 is 12:6:

1.

6. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In the step 2, the reaction was stirred at room temperature for 24 hours. The concentration of the NaOH aqueous solution was 2 mol / L. The developing solvents used for silica gel column chromatography were EA and MeOH in a volume ratio of 10:

1.

7. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In step 3, the molar ratio of compound 3, HATU, and Boc-Glu-OtBu is 4:9:8, and the amount of DIEA used is 421.1 μL DIEA per 0.4 mmol of compound 3.

8. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In the step 3, the reaction was carried out at room temperature for 2 hours, and the developing solvents used for silica gel column chromatography were PE and EA in a volume ratio of 10:

1.

9. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In step 4, the molar ratio of compound 4 to n-butyl nitrite is 1:1.1, the stirring reaction temperature is 60°C, the time is 4 hours, the solution temperature is lowered to -10°C, TFA is added dropwise and the stirring reaction is continued for 3 hours, and the developing solvents used for silica gel column chromatography are PE and EA in a volume ratio of 5:

1.

10. Use of the aminopeptidase-activated photodynamic photosensitizer according to claim 1, characterized in that: Used to prepare reagents for killing cancer cells / tissues.

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