Aminopeptidase activated photodynamic photosensitizer as well as preparation method and application thereof

Abstract

The invention belongs to the field of fluorescent probes, and particularly relates to a photodynamic photosensitizer activated by aminopeptidase as well as a preparation method and application of the photodynamic photosensitizer. In order to increase the selectivity of the photosensitizer to tumor cells, an activated photodynamic photosensitizer SeR-APN-NO activated by aminopeptidase (APN) overexpressed on the surface of cancer cells is developed on the basis of a selenium rhodamine spiro ring off-on mechanism, and after the photosensitizer is activated by the APN overexpressed on the surface of the cancer cells, the activated photodynamic photosensitizer SeR-APN-NO is activated by the APN overexpressed on the surface of the cancer cells. The strong cell killing ability is shown only under illumination, so that no damage is caused to normal cells / tissues.

Description

Technical Field

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

[0002] Photodynamic therapy (PDT) shows great potential in tumor treatment due to its advantages such as non-invasiveness, low side effects, no drug resistance, and repeated treatment. 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] In order to increase the selectivity of the photosensitizer 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. The photosensitizer can kill tumor cells with high selectivity under light without any damage to normal cells, and has potential clinical application value.

[0004] To achieve the above object, 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 nitrogen protection, 2-iodobenzoic acid and anhydrous THF are mixed, and the resulting solution is cooled, and then an isopropylmagnesium chloride-lithium chloride solution is added dropwise to react, and 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 and distilled under reduced pressure in an ice-water bath, and 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;

[0010] Step 2: Under nitrogen protection, 1,3-dimethylbarbituric acid and Pd(PPh 3 ) 4 Add to the THF solution of compound 2, stir and react at room temperature, add NaOH aqueous solution after the reaction is completed, extract, and the combined organic phase is dried, concentrated and purified by silica gel column chromatography to obtain compound 3;

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

[0012] 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 to continue the stirring reaction. 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.

[0013] Furthermore, in step 1, the molar ratio of 2-iodobenzoic acid, isopropylmagnesium chloride-lithium chloride, and compound 1 is 10:20:1.

[0014] Furthermore, in the step 1, the obtained solution is cooled to -78°C, and isopropylmagnesium chloride-lithium chloride solution is added dropwise for 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 for further reaction for 30 minutes, and the temperature is gradually restored to room temperature for reaction 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.

[0015] Further, in step 2, 1,3-dimethylbarbituric acid, compound 2 and Pd(PPh 3 ) 4 The molar ratio is 12:6:1.

[0016] Furthermore, in step 2, the reaction was stirred at room temperature for 24 hours, the concentration of the NaOH aqueous solution was 2 mol / L, and the developing solvents used for silica gel column chromatography were EA and MeOH in a volume ratio of 10:1.

[0017] Furthermore, in the 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.

[0018] Furthermore, in step 3, the reaction is carried out at room temperature for 2 hours, and the developing solvents used for silica gel column chromatography are PE and EA, and the volume ratio thereof is 10:1.

[0019] Furthermore, 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, and the volume ratio thereof is 5:1.

[0020] The present invention also provides an application of an aminopeptidase-activated photodynamic photosensitizer for killing cancer cells / tissues under the irradiation of a 561nm LED light source.

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

[0022] Due to the lack of targeting to cancer cells / tissues, traditional photosensitizers may increase the risk of damage to normal cells / tissues in addition to killing cancer cells / tissues during photodynamic therapy. The present invention has developed an "activation-type" photodynamic photosensitizer based on the "off-on" mechanism of the selenorhodamine spirocycle. The photosensitizer itself exists in the form of a closed-ring lactone, has extremely low cytotoxicity and phototoxicity, and only exhibits strong phototoxicity after being activated by APN overexpressed on the surface of cancer cells. Therefore, during photodynamic therapy, the photosensitizer developed by the present invention can kill cancer cells with high selectivity without causing any damage to normal cells / tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 (A) and (B) are PBS solutions of SeR-APN-NO (2 μM) and SeR-APN-NO / ABDA under 565 nm LED light (10 mW / cm 2 ) UV-visible absorption spectrum changes under irradiation.

[0024] Figure 2(A) shows the UV-visible absorption spectrum of SeR-APN-NO (6 μM) PBS solution at 370°C for 1 hour, and (B) shows the UV-visible absorption spectrum of SeR-APN-NO / APN (50 ng / mL, 37°C, 1 h) / 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 images 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 490-560nm (λex=488nm) and 570-750nm (λex=561nm), respectively. Scale bar: 50μm.

[0027] Figure 5 In normoxia (21% O 2 ) and hypoxia (2% O 2 ) experimental results of phototoxicity and dark toxicity of SeR-APN-NO on HepG2 and LO2 cells under different conditions.

[0028] Figure 6 (A) shows 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 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 embodiments.

[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, and then 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 and then warmed to 0 ° C, and then a THF solution of compound 1 (200 mg, 0.5 mmol) was added dropwise, and the reaction solution was reacted at 0 ° C for 30 minutes and then gradually returned to room temperature, and 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, the mixture was distilled under reduced pressure to remove the solvent, the obtained solid was redissolved in DCM, NaOH (2.0 mol / L 10.0 mL) was added, and the mixture was stirred for 30 min, 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 (600 MHz, CDCl 3 )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 (150 MHz, CDCl 3 )δ170.24,153.27,150.03,148.39,134.00,133.04,130.53,130.51,129.26,128.02,127.93,125 .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, in N 2 Under protection, 1,3-dimethylbarbituric acid (129.5 mg, 0.8 mmol) and Pd(PPh 3 ) 4(34.6 mg, 0.07 mmol) was 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, an aqueous solution of NaOH (2 mol / L 20.0 mL) was added to the above solution, extracted with EA, and the combined organic phase was washed with anhydrous Na 2 SO 4 After drying, concentration and purification by silica gel column chromatography (EA / MeOH=10 / 1), compound 3 was obtained as a red solid (169.9 mg, 98%). 1 H NMR (600MHz, CD 3 CN)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),6.81(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, CD 3 CN)δ169.54,150.44,148.55,134.46,130.44,129.76,128.52,128.15,125.96,12 5.50,124.56,122.77,121.44,113.36,113.11,111.23,110.95,39.38; HR-MS[M+H] + :calculated for 423.0606,Found423.0606.

[0036] Step 3, in N 2 Under 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 above solution, and EA was used for extraction. The combined organic phase was purified by Na 2 SO 4 After drying, concentration and purification by silica gel column chromatography (PE / EA=10 / 1), compound 4 was obtained as a purple-red solid (283.3 mg, 60%). 1 H NMR (600MHz, CD 3CN)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),7.21-7.11(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, CD 3 CN)δ169.54,150.44,148.55,134.46,130.44,129.76,128.52,128.15,125.96,12 5.50,124.56,122.77,121.44,113.36,113.11,111.23,110.95,39.38; 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), and then n-butyl nitrite (23.7 mg, 0.23 mmol) was added to the above solution, and the mixture was stirred at 60°C for 4 hours. After the reaction was completed by TLC, the mixture was distilled under reduced pressure to remove the solvent. The obtained solid was redissolved in dichloromethane (4 mL), and after the solution temperature was lowered to -10°C, TFA (0.5 mL) was added dropwise, and the mixture was stirred at this temperature for 3 hours. The mixture was distilled under reduced pressure to remove the solvent, and the crude product was analyzed and purified by silica gel column chromatography (PE / EA=5 / 1) to obtain the photosensitizer Se-APN-NO, which was a white powder (20.2 mg, 17%). 1 H NMR (600MHz, CD 3 CN)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); 13 C NMR (150MHz, CD 3CN)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. Solution preparation

[0040] Chromatographically pure CH 3 CN was used to prepare the stock solution of SeR-APN-NO (2 mM), which was diluted to the required concentration with PBS (10 mM, pH = 7.4) when tested. 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA, 5 mM) was prepared with ultrapure water.

[0041] 2. Study on photosensitizer photostability

[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 PBS as a ring-closed lactone, 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 O 2 The capture agent, Rose Bengal (RB) was used as a reference to test the production of SeR-APN-NO in PBS 1 O 2 Such as 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 remained unchanged. The experimental results show that SeR-APN-NO does not produce 1 O 2 , no photodynamic activity.

[0044] 3. Study on the photodynamic effect of photosensitizer 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 had good thermal stability at 37°C by UV-visible absorption spectroscopy ( Figure 2 A). Subsequently, APN (50 ng / mL) was added to the PBS solution of SeR-APN-NO (6 μM) and reacted at 37°C for 1 h, and then ABDA was added. 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 O 2 Subsequently, the enzymatic hydrolysis products of SeR-APN-NO and the products after illumination were detected by HPLC-MS ( 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, next, laser confocal microscopy (CLSM) was used to evaluate whether SeR-APN-NO can be specifically activated by cancer cells. HepG2 cells with high APN activity and LO2 cells with low APN activity were selected. The results of the live / dead cell staining experiment showed ( Figure 4 ), HepG2 cells loaded with SeR-APN-NO showed bright red fluorescence signals of PI after illumination, and the green fluorescence of Calcein-AM was negligible. HepG2 cells loaded with SeR-APN-NO / APN inhibitor (Ube, 100μM) only observed the green fluorescence signal of Calcein-AM after illumination; however, LO2 cells loaded with SeR-APN-NO only observed the green fluorescence signal of Calcein-AM after illumination. The results show that Se-APN-NO can selectively kill HepG2 cells under illumination without affecting 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, and the non-illumination group was placed in a cell culture incubator for 24 hours. 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 figure, Se-APN-NO had an effect on HepG2 cells and LO2 cells under normoxia (21% O 2 ) or hypoxia (2% O 2 ) conditions, they all showed low dark properties; 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% O 2 ) and 0.71 μM (2% O 2 ), while the survival rate of LO2 cells was not affected by light and Se-APN-NO concentration. The above experimental results show that Se-APN-NO has excellent photodynamic therapy effect on cancer cells and the therapeutic effect is not dependent on oxygen concentration. It is a photodynamic photosensitizer with potential clinical application value.

[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 illuminated 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 tumor 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 tumor was 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 mouse. However, the tumor volume of 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 process ( Figure 6These results indicate that SeR-APN-NO has good biocompatibility and can be activated by APN overexpressed in HepG2 cells, thus exhibiting excellent PDT effect in the HepG2 tumor-bearing mouse model with almost no effect on normal tissues.

[0051] In summary, the present invention has developed an APN-activated photodynamic photosensitizer SeR-APN-NO. Due to the double "locking" (electron-withdrawing effect) of NO and alanylamino substituents, Se-APN-NO itself exists in the form of a ring-closed lactone and does not show photodynamic effect under 565nm LED light irradiation; however, when the alanylamino part of Se-APN-NO is cleaved by APN overexpressed on the surface of HepG2 cells, the cleavage product can induce HepG2 cell death under 565nm LED light irradiation, and under normoxia (21% O 2 ) and hypoxia (2% O 2 ) were 0.65μM and 0.71μM respectively. In vivo experiments showed that the tumor sites of tumor-bearing nude mice injected with Se-APN-NO in situ were completely ablated 12 days after a single light exposure.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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, and then an isopropylmagnesium chloride-lithium chloride solution is added dropwise to react, and 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 and distilled under reduced pressure in an ice-water bath, and 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 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 are added to DMF, and reacted at room temperature. After the reaction is completed, water is added and extracted. The combined organic phase is dried, concentrated and purified by silica gel column chromatography to obtain compound 4; Step 4, dissolving compound 4 in ultra-dry THF, then adding n-butyl nitrite to react with stirring, after the reaction is detected by TLC, distilling under reduced pressure, re-dissolving the obtained solid in dichloromethane, lowering the solution temperature, adding TFA dropwise and continuing to react with stirring, after the reaction is completed, distilling under reduced pressure to remove, and purifying the crude product 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 isopropylmagnesium chloride-lithium chloride solution is added dropwise for 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 for further reaction for 30 minutes, and the temperature is gradually restored to room temperature for reaction 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 is stirred at room temperature for 24 hours, the concentration of the NaOH aqueous solution is 2 mol / L, and the developing solvents used for silica gel column chromatography are EA and MeOH, and the volume ratio thereof is 10:

1.

7. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In the 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 is carried out at room temperature for 2 hours, and the developing solvent used for silica gel column chromatography is PE and EA, and the volume ratio thereof is 10:

1.

9. The method for preparing an aminopeptidase-activated photodynamic photosensitizer according to claim 2, characterized in that: In the 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, and the volume ratio thereof is 5:

1.

10. A 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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