ALA derivative as well as preparation method and application thereof

Abstract

Aiming at the problems, the invention provides the ALA derivative and the preparation method and application thereof, the structural formula of the ALA derivative is as follows: # imgabs0, and the conception of the ALA derivative is that the physicochemical properties of ALA are improved through natural amino acid modification, the uptake and accumulation of ALA in tumor cells are improved, and the stability of the ALA derivative under acidic, neutral and alkaline conditions is improved. In-vitro experiments show that compared with a parent compound ALA or a derivative thereof (such as ALA-OMe), the lipophilicity and stability of the ALA derivative shown in the formula I are improved, so that the ALA derivative can more effectively generate PpIX in triple negative breast cancer cells, and meanwhile, the generation amount of the PpIX is in positive correlation with overexpression of the Cath B level in tumor cells. More importantly, the biotin part of the compound significantly improves the phototoxicity of the compound, which may be due to the targeting effect of biotin on tumors, improves the enrichment degree of ALA in tumor cells, and generates a stronger photodynamic effect. Therefore, the ALA derivative as shown in the formula I and the pharmaceutically acceptable acid or alkali addition salt and stereochemical isomer form thereof can be used for treating tumors with high Cath B expression level.

Description

Technical Field

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

[0002] Cancer is a major threat to human health. Traditional cancer treatments have the disadvantages of large side effects and high risks. Therefore, it is urgent to develop effective cancer treatments. Photodynamic therapy (PDT) is a commonly used treatment for human cancer, which has the advantages of minimal invasiveness and low side effects. Photosensitizers play an important role in PDT. When photosensitizers are exposed to light in an aerobic environment, they will produce reactive oxygen species (ROSs), which will cause oxidative damage to tumor cells or tissues. To date, researchers have designed and synthesized many photosensitizers for PDT. However, only a few photosensitizers have been approved for clinical use. Among them, 5-aminolevulinic acid (ALA) has received widespread attention. Unlike other photosensitizers, ALA itself has no photosensitivity, but can be converted into photosensitizer porphyrin (Pp IX) through the intracellular heme biosynthesis pathway. Compared with other photosensitizers, ALA has the advantages of rapid clearance, low skin photosensitivity, and low systemic toxicity. Under physiological conditions, ALA is a zwitterion with strong hydrophilicity and cannot effectively penetrate biological barriers such as cell membranes. Therefore, increasing the lipophilicity of ALA is the most effective way to improve its cell permeability, and esterification of ALA is a common method to increase lipophilicity, among which the most representative are ALA-OMe (Metvix) and ALA-OHex (Cysview). At present, Metvix has been widely used in the treatment of actinic keratosis and basal cell carcinoma, while Cysview is approved for photodetection of bladder cancer. However, ALA and its ester derivatives still have the disadvantage of instability under physiological conditions. This is mainly due to the high nucleophilicity of the 5-amino group. ALA and its ester derivatives are prone to form Schiff base dimers under physiological conditions. Therefore, modifying the amino terminus of ALA can prevent dimerization and improve the stability of ALA.

[0003] In addition, almost all photosensitizers, including ALA and its derivatives, lack tumor specificity. Researchers have proposed various approaches to address this problem, which can be summarized into two aspects: (1) using tumor-specific ligands to target photosensitizers; (2) using the tumor microenvironment to activate photosensitizers. In recent decades, a variety of ALA ester derivatives linked to tumor-specific ligands (such as vitamins, peptides, and nucleosides) have been designed to improve tumor selectivity. However, these tumor-targeted ALA ester derivatives are still unstable under physiological conditions. At the same time, researchers have developed ALA derivatives that can be activated by the tumor microenvironment. Unlike ALA ester derivatives, the synthesis of these stress-responsive ALA derivatives is mainly through the modification of the 5-amino group in ALA with a group that is easily activated by tumor-related stimulatory factors (such as cathepsin E, β-glucuronidase, and tumor-highly expressed aminopeptidase). Compared with ALA and its ester derivatives, these N-modified derivatives are very stable under physiological conditions. The effective release of ALA is essential for N-modified ALA prodrugs to achieve their pharmacological activity. Recently, researchers have also developed dual-targeting strategies that include both targeting ligands that enhance tumor-selective uptake and response factors that can be activated by the tumor microenvironment, further improving the tumor targeting of photosensitizers.

[0004] In summary, it is an important research direction to use various strategies to modify the structure of ALA to solve the problems of ALA's lipid solubility, stability and selectivity. Summary of the invention

[0005] In view of the above problems, the present invention proposes an ALA derivative, a preparation method and an application thereof, the idea of ​​which is to improve the physicochemical properties of ALA by modifying natural amino acids, increase the uptake and accumulation in tumor cells, and improve the stability of ALA derivatives under acidic, neutral and alkaline conditions.

[0006] In the first aspect, the present invention provides an ALA derivative, the structural formula of which is as follows:

[0007]

[0008] Where R 1 is one of hydroxyl, methoxy or n-hexyloxy;

[0009] R 2 is one of L-alanine, L-lysine or L-citrulline;

[0010] R 3 is L-valine, L-phenylalanine, L-lysine or One of;

[0011] R 4 is acetyl, benzyloxycarbonyl, ethylamino or One of the following. (preferably ethylamino)

[0012] More specifically, the structural formula of the above-mentioned ALA derivative is one of the following:

[0013]

[0014]

[0015]

[0016] In a second aspect, the present invention provides a method for preparing the above-mentioned ALA derivative, the steps of which include:

[0017] Step 1: dissolving the raw material represented by Formula 1 and N-hydroxysuccinimide (NHS) in an organic solvent, stirring at 0°C, then adding 1,3-dicyclohexylcarbodiimide (DCC), stirring evenly at 0°C (preferably for 1 hour), then moving to room temperature and continuing to stir for 16-24 hours, and monitoring the reaction progress by TLC; the molar ratio of the raw material represented by Formula 1, NHS and DCC is 1:1-2:1-2;

[0018] After the reaction is completed, the reaction is filtered to obtain a filtrate; the filtrate is concentrated under reduced pressure, the concentrated product is redissolved in DCM, placed at -20°C for 12 hours, filtered again to obtain a filtrate, and the filtrate is concentrated under reduced pressure to obtain a crude product of the compound represented by Formula 2;

[0019] Step 2: dissolving the crude product of the compound of formula 2 obtained in step 1 and the compound of formula 3 in an organic solvent, then adding an aqueous solution of a basic compound, stirring evenly at 0° C. (preferably for 1 hour), and then stirring at room temperature for 16-24 hours, and monitoring the reaction progress by TLC; the molar ratio of the raw material of formula 1, the compound of formula 3, and the basic compound is 1:1-1.2:1-2;

[0020] After the reaction is completed, a saturated sodium bicarbonate solution is added to quench the reaction, the reaction solution is washed with ethyl acetate, the pH of the aqueous phase is adjusted to 2-3 with 10% dilute hydrochloric acid at 0°C (preferably 10% dilute hydrochloric acid), the filter cake is filtered, the filter cake is washed with 10% dilute hydrochloric acid at 0°C, and the filter cake is vacuum dried to obtain a compound represented by formula 4;

[0021] Step 3: dissolving the compound represented by Formula 4 and 1-hydroxybenzotriazole (HOBT) in an organic solvent, adding a basic compound, stirring at 0°C, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), stirring evenly at 0°C (preferably for 1 hour), adding the compound represented by Formula 5, and reacting at room temperature for 2 to 4 hours, and monitoring the reaction progress by thin layer chromatography (TLC); the molar ratio of the compound represented by Formula 4, HOBT, the basic compound, EDCI, and the compound represented by Formula 5 is 1:1 to 1.2:1 to 2:1 to 1.5:1 to 2;

[0022] After the reaction is completed, the reaction solution obtained in step 3 is concentrated under reduced pressure, the concentrated product is redissolved in ethyl acetate, and washed with saturated sodium bicarbonate aqueous solution, saturated ammonium chloride aqueous solution, and saturated brine in sequence; the organic phase is dried over anhydrous sodium sulfate, concentrated under reduced pressure, separated and purified by column chromatography, and dried to obtain a compound represented by formula 6;

[0023] Step 4-1: dissolving the compound represented by Formula 6 in THF, adding 1 mol / L lithium hydroxide aqueous solution, stirring at room temperature for 2-4 hours, and monitoring the reaction progress by thin layer chromatography (TLC); the molar ratio of the compound represented by Formula 6 to lithium hydroxide is 1:1-5;

[0024] After the reaction is completed, a cation exchange resin is added to the reaction solution obtained in step 4-1, the pH of the reaction solution is adjusted to 6-7, and then the cation exchange resin is filtered out, the filtrate is concentrated under reduced pressure, anhydrous ethyl acetate is added for slurrying, and a filter cake is obtained by suction filtration, and the filter cake is vacuum dried to obtain a compound represented by formula 7;

[0025] Step 5-1: dissolve the compound represented by Formula 7 in an organic solvent, add an acidic solution, wherein the acidic solution is one or more of HCl-EA solution, TFA, MsOH, and TsOH, and the molar ratio of the compound represented by Formula 7 to the acidic component in the acidic solution is 1:1.2-10; stir the obtained mixed solution at 25-35°C for 2-5h, and monitor the reaction progress by TLC; after the reaction is completed, concentrate the reaction solution under reduced pressure, add ether, grind and stir thoroughly, centrifuge, remove the supernatant, add ether again, grind and stir thoroughly, centrifuge, remove the supernatant, collect the lower solid, and vacuum dry to obtain the finished product, i.e., the compound represented by Formula I.

[0026] The present invention further proposes a variant of the above preparation method, characterized in that after completing step 3, the operations of step 4-1 to step 5-1 are replaced by connecting step 4-2 and step 5-2:

[0027] The described operations are as follows:

[0028] Step 4-2: Dissolve the compound represented by Formula 6 in an organic solvent, add an acidic solution, wherein the acidic solution is one or more of HCl-EA solution, TFA, MsOH, and TsOH, and the molar ratio of the compound represented by Formula 6 to the acidic component in the acidic solution is 1:1.2 to 10; stir the obtained mixed solution at 25-35°C for 2-5h, and monitor the reaction progress by TLC; after the reaction is completed, concentrate the reaction solution under reduced pressure, add ether, grind and stir thoroughly, centrifuge, remove the supernatant, add ether again, grind and stir thoroughly, centrifuge, remove the supernatant, collect the lower solid, and vacuum dry to directly obtain the finished product, i.e., the compound represented by Formula I.

[0029] The present invention further proposes a variant of the above preparation method, characterized in that after completing step 3, the operations of step 4-1 to step 5-1 are replaced by connecting step 4-3 and step 5-2:

[0030] Step 4-3: dissolving the compound represented by Formula 6 in ethyl acetate, adding HCl-EA solution, wherein the molar ratio of the compound represented by Formula 6 to the amount of HCl in the HCl-EA solution is 1:1.2-10; stirring the obtained mixed solution at 25-35°C for 2-5h, and monitoring the reaction progress by TLC; after the reaction is completed, concentrating the reaction solution under reduced pressure, adding ether, grinding and stirring thoroughly, centrifuging, removing the supernatant, adding ether again, grinding and stirring thoroughly, centrifuging, removing the supernatant, collecting the lower solid, and vacuum drying to obtain the compound represented by Formula 8;

[0031] Step 5-2: dissolving the compound represented by Formula 8 and biotin-N-succinimidyl ester in an organic solvent, slowly adding a basic compound, stirring at room temperature for 24 hours, and monitoring the reaction progress by TLC; the basic compound is one or more of DIPEA, triethylamine, and sodium bicarbonate; the molar ratio of the compound represented by Formula 8, biotin-N-succinimidyl ester and the basic substance is 1:1.2~1.5:1.2~1.5; after the reaction is completed, the reaction solution is concentrated under reduced pressure, water is added to precipitate the solid, filtered, the filter residue is redispersed in water, heated for recrystallization, filtered, and the filter cake is vacuum dried to obtain the compound represented by Formula I.

[0032] Preferably, the organic solvent in step 1 is one or more of DCM, THF, acetonitrile, methanol or DMF; the volume molar ratio of the organic solvent to the raw material represented by formula 1 is 4 to 10:1 mL / mmol.

[0033] Preferably, the organic solvent in step 2 is one or more of THF, DCM, acetonitrile, and ethylene glycol dimethyl ether (DME); the amount of the organic solvent in step 2 is 4 to 10:1 mL / mmol based on the amount of the compound represented by Formula 2; the alkaline compound is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and DIPEA; the volume ratio of the aqueous solution of the alkaline compound to the organic solvent is 1:1 to 1.5.

[0034] Preferably, the organic solvent in step 3 is one or more of THF, DCM, DMF, and acetonitrile; the amount of the organic solvent in step 3 is 4 to 10:1 mL / mmol based on the amount of the compound represented by formula 4; the basic compound is one or more of DIPEA, NMM, NMI, triethylamine, pyridine, and 2,6-lutidine.

[0035] Preferably, the volume ratio of the lithium hydroxide aqueous solution to THF in step 4-1 is 1:1 to 3; the organic solvent in step 5-1 is one or more of DCM, EA, THF, and hexafluoroisopropanol (HFIP); the amount of the organic solvent is 4 to 10:1 mL / mmol based on the amount of the compound represented by Formula 7.

[0036] In a third aspect, the present invention provides an ALA derivative and a pharmaceutically acceptable salt thereof for use in treating malignant tumors and skin diseases.

[0037] The malignant tumor or skin disease is basal cell carcinoma, squamous cell carcinoma, actinic keratosis, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, bladder cancer, cervical cancer, condyloma acuminatum, psoriasis and Bowen's disease.

[0038] The beneficial effects of the present invention are:

[0039] The ALA derivatives shown in Formula I of the present invention are photodynamic therapy compounds that have the ability to be selectively activated by the tumor microenvironment and / or selectively taken up by tumor cells. The structures of these compounds contain at least two of the following three parts, and must contain the ALA part: (1) ALA or its ester derivatives (such as ALA methyl ester and ALA hexyl ester), which can be converted into photosensitizer protoporphyrin (PpIX) through the intracellular heme biosynthesis pathway; (2) biotin, which is used to improve the targeting of the molecule to tumors and enhance the selective uptake of tumor cells or tissues; (3) a short peptide part, including natural amino acids and cathepsin B (Cathepsin B, Cath B) responsive dipeptide (pseudo-peptide) linkers, which modify the 5-amino terminus of ALA or its derivatives. The natural amino acid modification can improve the physicochemical properties of ALA and increase the uptake and accumulation in tumor cells. The Cath B responsive dipeptide (pseudo-peptide) linker can be recognized and selectively cleaved by Cath B overexpressed in tumor cells to release ALA or its ester derivatives. These compounds have high stability under acidic, neutral and alkaline conditions.

[0040] In vitro experiments showed that compared with the parent compound ALA or its derivatives (such as ALA-OMe), the improved lipophilicity and stability of the ALA derivatives shown in Formula I enabled them to more effectively produce PpIX in triple-negative breast cancer cells. At the same time, the amount of PpIX generated was positively correlated with the overexpression of Cath B levels in tumor cells. More importantly, the biotin part of the compound significantly increased the phototoxicity of the compound, which may be attributed to the targeting effect of biotin on tumors, increasing the enrichment of ALA in tumor cells and producing a stronger photodynamic effect. In short, the dual-targeting strategy of selective activation of the tumor microenvironment and selective uptake of biotin significantly improved the anti-tumor effect of ALA-based PDT.

[0041] Therefore, the ALA derivatives represented by formula I of the present invention, and pharmaceutically acceptable acid or base addition salts and stereochemical isomers thereof, can be used to treat tumors with high Cath B expression levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 These are the test results of the compounds A1-A21, MAL and HAL of the present invention inducing the production of PpIX in MCF-7 cells.

[0043] Figure 2 These are the test results of the compounds A1-A21, MAL and HAL of the present invention inducing the production of PpIX in MDA-MB-231 cells.

[0044] Figure 3 The graph shows the time curve of the compounds A1, A7 and A20 of the present invention inducing the production of PpIX in MCF-7 cells.

[0045] Figure 4 The graph shows the time curve of the compounds A1, A7 and A20 of the present invention inducing the production of PpIX in MDA-MB-231 cells.

[0046] Figure 5 This is the correlation curve between PpIX production and cathepsin B of compound A20 of the present invention in MDA-MB-231 cells.

[0047] Figure 6 These are the dark toxicity test results of compounds A1-A21, MAL and HAL of the present invention on MCF-7 cells.

[0048] Figure 7 The phototoxicity test results of the compounds A1-A21, MAL and HAL of the present invention on MCF-7 cells are shown in FIG. Figure 8 These are the dark toxicity test results of compounds A1-A21, MAL and HAL of the present invention on MDA-MB-231 cells.

[0049] Fig. 9 These are the phototoxicity test results of the compounds A1-A21, MAL and HAL of the present invention on MDA-MB-231 cells. DETAILED DESCRIPTION

[0050] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0051] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0052] Example 1

[0053] Synthesis method of compound (A1)

[0054] The raw material benzyloxycarbonyl-L-valine-L-citrulline (1.225 g, 3.00 mmol) and HOBT (0.418 g, 3.09 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.516 g, 5.10 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.690 g, 3.60 mmol), and continued stirring at 0°C for 0.5 h before adding methyl 5-aminolevulinate hydrochloride (MAL·HCl) The reaction mixture was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM: MeOH = 30: 1, v / v) to obtain 0.836 g of white solid A1 with a yield of 52%.

[0055] 1 H NMR (400MHz, DMSO-d6) δ8.22(t,J=5.6Hz,1H),7.95(d,J=7.8Hz,1H),7.40–7.25(m,6H),5.91(d,J=6.0Hz, 1H),5.36(s,2H),5.03(d,J=3.0Hz,2H),4.32(td,J=8.2,5.4Hz,1H),4.02–3.85(m,3H),3.57(s,3H),2.94 (dp,J=13.5,7.3,6.8Hz,2H),2.69(t,J=6.6Hz,2H),2.47(d,J=6.6Hz,2H),1.97(h,J=6.7Hz,1H),1.65(dq ,J=12.6,6.1Hz,1H),1.56–1.46(m,1H),1.36(ddt,J=17.3,12.9,8.1Hz,2H),0.83(dd,J=14.5,6.8Hz,6H). 13 CNMR(101MHz,DMSO-d6)δ205.19,172.61,171.85,170.98,158.73,156.12,137.06,128.33,127.75, 127.64,65.40,60.09,52.11,51.38,48.31,39.97,33.87,30.32,29.64,27.12,26.51,19.23,18.02.

[0056] Example 2

[0057] Synthesis method of compound (A2)

[0058] The raw material benzyloxycarbonyl-L-valyl-L-alanine (0.935 g, 2.90 mmol) and HOBT (0.404 g, 3.00 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.499 g, 4.93 mmol). The mixture was stirred at 0°C for 10 min, and then EDCI (0.667 g, 3.48 mmol) was added. After stirring at 0°C for 0.5 h, MAL·HCl (0.632 g , 3.48mmol), and then reacted at room temperature for 3h, and the reaction progress was monitored by TLC; after the reaction was completed, the reaction solution was concentrated under reduced pressure, redissolved with EA (30mL), washed once with saturated sodium bicarbonate aqueous solution (50mL), saturated ammonium chloride aqueous solution (50mL), and saturated brine (50mL) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=30:1, v / v) to obtain 0.782g of white solid A2 with a yield of 60%.

[0059] 1 H NMR(400MHz,Chloroform-d)δ7.41–7.28(m,5H),6.85(s,1H),6.57(d,J=7.5Hz,1H) ,5.38(d,J=8.3Hz,1H),5.10(s,2H),4.55(p,J=7.0Hz,1H),4.25–4.09(m,2H),4.01( t,J=7.2Hz,1H),3.65(s,3H),2.73(dd,J=9.4,4.2Hz,2H),2.68–2.60(m,2H),2.13( h,J=6.7Hz,1H),1.39(d,J=7.0Hz,3H),0.96(d,J=6.8Hz,3H),0.91(d,J=6.8Hz,3H). 13 C NMR (100MHz, CDCl3) δ203.71,172.97,172.28,171.34,156.71,136.34,128.67,128.33 ,128.21,67.25,60.53,52.06,49.26,48.80,34.60,31.29,27.65,19.36,18.60,17.90.

[0060] Example 3

[0061] Synthesis method of compound (A3)

[0062] The raw material 1-(ethylcarbamoyl)cyclobutane-1-carboxylic acid (0.856 g, 5.00 mmol) and NHS (0.633 g, 5.50 mmol) were placed in a 100 mL round-bottom flask, and solvent THF (50 mL) was added to dissolve, stirred at 0°C for 5 min, and then DCC (1.083 g, 5.25 mmol) was added, and stirring was continued at 0°C for 1 h, then moved to room temperature and continued stirring for 24 h, and the reaction progress was monitored by TLC; after the reaction was completed, the obtained reaction solution was filtered, the filtrate was concentrated under reduced pressure, and redissolved with dichloromethane (50 mL), and then placed at -20°C to cool for 12 h, filtered again, and the filtrate was concentrated under reduced pressure to obtain a crude product of 2,5-dioxopyrrolidin-1-yl-1-(ethylcarbamoyl)cyclobutane-1-carboxylate, which was directly used in the next step without further purification.

[0063] The crude product of 2,5-dioxopyrrolidin-1-yl-1-(ethylcarbamoyl)cyclobutane-1-carboxylate and L-citrulline (0.920 g, 5.25 mmol) were placed in a 100 mL round-bottom flask, and solvent DME (25 mL) was added to dissolve. The mixture was stirred at 0°C for 5 min, and then an aqueous solution (25 mL) of sodium bicarbonate (0.630 g, 7.50 mmol) was slowly added. After stirring at 0°C for 1 h, the mixture was stirred at room temperature for 24 h, and the reaction progress was monitored by TLC. After the reaction, saturated aqueous sodium bicarbonate solution (10 mL) was added to the reaction solution to quench, and then the reaction solution was washed once with EA (20 mL), the organic phase was backwashed with saturated sodium bicarbonate solution (10 mL), the aqueous phases were combined, and the pH of the aqueous phase was adjusted to 2-3 with 10% dilute hydrochloric acid at 0°C, and the mixture was filtered, and the filter cake was washed with icy 10% dilute hydrochloric acid, and the filter cake was dried in vacuo to obtain 0.788 g of (S)-2-(1-(ethylcarbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanoic acid, with a two-step yield of 48%.

[0064] (S)-2-(1-(ethylcarbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanic acid (0.788 g, 2.40 mmol) and HOBT (0.334 g, 2.47 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (25 mL) was added, followed by NMM (0.413 g, 4.08 mmol), and the mixture was stirred at 0°C for 10 min, followed by EDCI (0.552 g, 2.88 mmol), and the mixture was stirred at 0°C for 0.5 h, and then MAL·H Cl (0.523 g, 2.88 mmol), and then react at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM: MeOH = 25: 1, v / v) to obtain 0.492 g of white solid A3 with a yield of 45%.

[0065] 1 H NMR (400MHz, DMSO-d6) δ8.30(t,J=5.6Hz,1H),7.77(t,J=5.6Hz,1H),7.65(d,J=8.0Hz,1H),5.95(t,J=5. 8Hz,1H),5.39(s,2H),4.31(td,J=8.7,4.7Hz,1H),4.04–3.90(m,2H),3.57(s,3H),3.09(dtd,J=12.3,7.1 ,5.3Hz,2H),2.93(q,J=6.7Hz,2H),2.69(t,J=6.5Hz,2H),2.49–2.45(m,2H),2.38(q,J=7.6,6.9Hz,4H), 1.71(ttd,J=10.6,7.0,3.2Hz,3H),1.54(tt,J=9.3,4.7Hz,1H),1.41–1.30(m,2H),0.99(t,J=7.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ205.24,172.70,172.27,171.80,171.76,158.85,53.98,52 .58,51.45,48.37,34.01,33.94,29.37,29.24,29.05,27.16,26.61,15.49,14.50.

[0066] Example 4

[0067] Synthesis method of compound (A4)

[0068] The raw material N-acetyl-L-phenylalanine (1.036 g, 5.00 mmol) and NHS (0.633 g, 5.50 mmol) were placed in a 100 mL round-bottom flask, and solvent THF (50 mL) was added to dissolve, and stirred at 0°C for 5 min, then DCC (1.083 g, 5.25 mmol) was added, and stirring was continued at 0°C for 1 h, and then moved to room temperature and continued stirring for 24 h. The reaction progress was monitored by TLC; after the reaction was completed, the obtained reaction solution was filtered, the filtrate was concentrated under reduced pressure, and it was redissolved with dichloromethane (50 mL), and then placed at -20°C to cool for 12 h, and filtered again, and the filtrate was concentrated under reduced pressure to obtain the crude product of N-acetyl-L-valine-N-hydroxysuccinimide ester, which was directly put into the next step without further purification.

[0069] The crude N-acetyl-L-phenylalanine-N-hydroxysuccinimide ester was mixed with N ε -(tert-Butoxycarbonyl)-L-lysine (1.293 g, 5.25 mmol) was placed in a 100 mL round-bottom flask, and solvent DME (25 mL) was added to dissolve, stirred at 0°C for 5 min, and then slowly added an aqueous solution (25 mL) of sodium bicarbonate (0.630 g, 7.50 mmol), continued to stir at 0°C for 1 h, and then stirred at room temperature for 24 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated sodium bicarbonate aqueous solution (10 mL) was added to the reaction solution to quench, and then the reaction solution was washed once with EA (20 mL), the organic phase was backwashed with saturated sodium bicarbonate solution (10 mL), the aqueous phase was combined, and the pH of the aqueous phase was adjusted to 2-3 with 10% dilute hydrochloric acid at 0°C, and the filter cake was washed with 10% dilute hydrochloric acid in ice, and the filter cake was vacuum dried to obtain N-acetyl-L-phenylalanine-N ε -(tert-Butyloxycarbonyl)-L-lysine 1.372 g, two-step yield 63%.

[0070] N-acetyl-L-phenylalanine-N ε-(tert-Butoxycarbonyl)-L-lysine (1.372 g, 3.15 mmol) and HOBT (0.439 g, 3.26 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.542 g, 5.36 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.725 g, 3.78 mmol). After stirring at 0°C for 0.5 h, MAL·HCl (0.686 g, 3.78 mmol) was added, and the mixture was reacted at room temperature for 3 h, and monitored by TLC. Reaction process: After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated sodium bicarbonate aqueous solution (50 mL), saturated ammonium chloride aqueous solution (50 mL), and saturated brine (50 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 1.241 g of methyl 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoate, with a yield of 70%.

[0071] Place methyl 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoate (1.241 g, 2.21 mmol) and p-toluenesulfonic acid (0.455 g, 2.65 mmol) in a 100 mL round-bottom flask, add solvent HFIP (10 mL) to dissolve, stir at room temperature for 2 h, and monitor the reaction progress by TLC. After the reaction is completed, concentrate the reaction solution under reduced pressure, add anhydrous ether, grind and stir thoroughly, centrifuge, remove the supernatant, add anhydrous ether again, repeat the operation, collect the lower solid, and dry in vacuo to obtain 1.206 g of white solid A4 with a yield of 86%.

[0072] 1H NMR (400MHz, DMSO-d6) δ8.19–8.02(m,3H),7.64(s,3H),7.49(d,J=7.8Hz,2H),7.33–7.15(m,5H ),7.12(d,J=7.8Hz,2H),4.50(ddd,J=10.4,8.1,4.4Hz,1H),4.28(td,J=8.5,4.9Hz,1H),3.95(t ,J=5.2Hz,2H),3.56(s,3H),3.01(dd,J=13.9,4.3Hz,1H),2.72(dt,J=22.4,6.8Hz,5H),2.48(t ,J=4.7Hz,2H),2.28(s,3H),1.75(s,4H),1.52(h,J=8.2,6.2Hz,3H),1.32(h,J=7.3,6.6Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ205.25,172.68,171.77,171.53,169.40,145.51,138.04,137.80,129.16,128.14,128. 04,126.24,125.51,54.06,52.19,51.43,48.33,38.77,37.33,33.92,31.39,27.13,26.58,22.46,22.18,20.81.

[0073] Example 5

[0074] Synthesis method of compound (A5)

[0075] The raw material N 2 -Acetyl-N 6 -(tert-Butoxycarbonyl)-L-lysine (1.142 g, 5.00 mmol) and NHS (0.633 g, 5.50 mmol) were placed in a 100 mL round-bottom flask, and solvent THF (50 mL) was added to dissolve, stirred at 0°C for 5 min, and then DCC (1.083 g, 5.25 mmol) was added, and stirring continued at 0°C for 1 h, then moved to room temperature and continued stirring for 24 h, and the reaction progress was monitored by TLC; after the reaction was completed, the obtained reaction solution was filtered, the filtrate was concentrated under reduced pressure, and it was redissolved with dichloromethane (50 mL), and then placed at -20°C to cool for 12 h, filtered again, and the filtrate was concentrated under reduced pressure to obtain N 2 -Acetyl-N 6 The crude product of -(tert-butoxycarbonyl)-L-lysine-N-hydroxysuccinimide ester was directly used in the next step without further purification.

[0076] N 2-Acetyl-N 6 -(tert-Butyloxycarbonyl)-L-lysine-N-hydroxysuccinimide ester crude product and N ε -(tert-Butyloxycarbonyl)-L-lysine (1.293 g, 5.25 mmol) was placed in a 100 mL round-bottom flask, and solvent DME (25 mL) was added to dissolve, stirred at 0°C for 5 min, and then slowly added an aqueous solution (25 mL) of sodium bicarbonate (0.630 g, 7.50 mmol), continued to stir at 0°C for 1 h, and then stirred at room temperature for 24 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated sodium bicarbonate aqueous solution (10 mL) was added to the reaction solution to quench, and then the reaction solution was washed once with EA (20 mL), the organic phase was backwashed with saturated sodium bicarbonate solution (10 mL), the aqueous phase was combined, and the pH of the aqueous phase was adjusted to 2-3 with 10% dilute hydrochloric acid at 0°C, and then filtered, and the filter cake was washed with 10% dilute hydrochloric acid in ice, and the filter cake was vacuum dried to obtain N 2 -(N 2 -Acetyl-N 6 -(tert-Butyloxycarbonyl)-L-lysine)-N 6 -(tert-Butyloxycarbonyl)-L-lysine 1.757 g, two-step yield 68%.

[0077] N 2 -(N 2 -Acetyl-N 6 -(tert-Butyloxycarbonyl)-L-lysine)-N 6 -(tert-Butoxycarbonyl)-L-lysine (1.757 g, 3.40 mmol) and HOBT (0.473 g, 3.50 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (35 mL) was added, followed by NMM (0.677 g, 5.78 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.782 g, 4.08 mmol), and stirred at 0°C for 0.5 h, followed by MAL·HCl (0.739 g, 4.08 mmol), and reacted at room temperature for 3 h. The reaction progress was monitored by TLC. After completion, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 1.270 g of methyl 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanoyl)hexanoyl)-4-oxopentanoate with a yield of 58%.

[0078] Place 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanamido)hexanamido)-4-oxopentanoic acid methyl ester (1.270 g, 1.97 mmol) and p-toluenesulfonic acid (0.814 g, 4.73 mmol) in a 100 mL round-bottom flask, add solvent HFIP (10 mL) to dissolve, stir at room temperature for 2 h, and monitor the reaction progress by TLC. After the reaction is completed, concentrate the reaction solution under reduced pressure, add anhydrous ether, grind and stir thoroughly, centrifuge, remove the supernatant, add anhydrous ether again, repeat the operation, collect the lower solid, and dry in vacuo to obtain 1.165 g of white solid A5 with a yield of 75%.

[0079] 1 H NMR (400MHz, DMSO-d6) δ8.24–7.95(m,3H),7.67(s,6H),7.50(d,J=7.0Hz,4H),7.13(d,J=7.9Hz,4H),4.29–4.17(m,2H),4.03–3.87(m,2H),3.57 (s,3H),2.80–2.60(m,6H),2.49–2.38(m,2H),2.29(s,6H),1.85(s,3H) ,1.74–1.60(m,2H),1.50(q,J=7.6Hz,6H),1.30(dh,J=14.8,6.9Hz,4H). 13 C NMR (100MHz, DMSO-d6) δ205.29,172.68,171.92,171.80,169.56,145.10,138.09,128.24,125.51,52. 49,52.16,51.45,48.33,38.79,33.93,31.36,31.25,27.15,26.66,26.55,22.53,22.37,22.21,20.82.

[0080] Example 6

[0081] Synthesis method of compound (A6)

[0082] Acetyl-L-valine-N-hydroxysuccinimide ester (1.281 g, 5.00 mmol) was mixed with N ε-(tert-Butoxycarbonyl)-L-lysine (1.293 g, 5.25 mmol) was placed in a 100 mL round-bottom flask, and solvent DME (25 mL) was added to dissolve, stirred at 0°C for 5 min, and then slowly added an aqueous solution (25 mL) of sodium bicarbonate (0.630 g, 7.50 mmol), continued to stir at 0°C for 1 h, and then stirred at room temperature for 24 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated sodium bicarbonate aqueous solution (10 mL) was added to the reaction solution to quench, and then the reaction solution was washed once with EA (20 mL), the organic phase was backwashed with saturated sodium bicarbonate solution (10 mL), the aqueous phase was combined, and the pH of the aqueous phase was adjusted to 2-3 with 10% dilute hydrochloric acid at 0°C, and the filter cake was washed with 10% dilute hydrochloric acid in ice, and the filter cake was vacuum dried to obtain acetyl-L-valyl-N ε -(tert-Butyloxycarbonyl)-L-lysine 1.356 g, yield 70%.

[0083] Acetyl-L-valyl-N ε -(tert-Butoxycarbonyl)-L-lysine (1.356 g, 3.50 mmol) and HOBT (0.485 g, 3.60 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (35 mL) was added, followed by NMM (0.602 g, 5.95 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.805 g, 4.20 mmol). After stirring at 0°C for 0.5 h, MAL·HCl (0.763 g, 4.20 mmol) was added, and the mixture was reacted at room temperature for 2 h, and monitored by TLC. Reaction process: After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated sodium bicarbonate aqueous solution (50 mL), saturated ammonium chloride aqueous solution (50 mL), and saturated brine (50 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 1.225 g of methyl 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoate, with a yield of 68%.

[0084] Place 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid methyl ester (1.225 g, 2.38 mmol) and p-toluenesulfonic acid (0.492 g, 2.85 mmol) in a 100 mL round-bottom flask, add solvent HFIP (10 mL) to dissolve, stir at room temperature for 2 h, and monitor the reaction progress by TLC. After the reaction is completed, concentrate the reaction solution under reduced pressure, add anhydrous ether, grind and stir thoroughly, centrifuge, remove the supernatant, add anhydrous ether again, repeat the operation, collect the lower solid, and dry in vacuo to obtain 1.257 g of white solid A6 with a yield of 90%.

[0085] 1 H NMR (400MHz, DMSO-d6) δ8.16(t,J=5.6Hz,1H),8.00(d,J=7.9Hz,1H),7.90(d,J=8.4Hz,1H),7.68(s,3H),7.49(d,J= 7.7Hz,2H),7.12(d,J=7.7Hz,2H),4.27(td,J=8.5,5.1Hz,1H),4.12(t,J=7.6Hz,1H),3.95(qd,J=18.3,5.5Hz,2H), 3.57(s,3H),2.74(s,2H),2.69(t,J=6.6Hz,2H),2.47(d,J=7.4Hz,2H),2.29(s,3H),1.95(h,J=7.2,6.7Hz,1H),1.8 6(s,3H),1.67(dq,J=13.8,6.6Hz,1H),1.52(h,J=7.3,6.2Hz,3H),1.32(h,J=7.0,6.6Hz,2H),0.83(t,J=7.0Hz,6H). 13 C NMR (100MHz, DMSO-d6) δ205.28,172.68,171.85,171.21,169.58,145.48,137.83,128.15,125.52,57. 93,52.12,51.45,48.35,38.74,33.90,31.29,30.24,27.14,26.56,22.52,22.21,20.82,19.30,18.23.

[0086] Example 7

[0087] Synthesis method of compound (A7)

[0088] The raw material benzyloxycarbonyl-L-valine-L-citrulline (1.225 g, 3.00 mmol) and HOBT (0.418 g, 3.09 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.516 g, 5.10 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.690 g, 3.60 mmol), and continued stirring at 0°C for 0.5 h before adding 5-aminolevulinic acid hexyl ester hydrochloride (HAL·HCl) The reaction mixture was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM: MeOH = 40: 1, v / v) to obtain 0.872 g of a white solid A7 with a yield of 48%.

[0089] 1 H NMR (400MHz, DMSO-d6) δ8.22(t,J=5.6Hz,1H),8.00(d,J=8.0Hz,1H),7.91(d,J=8.7Hz,1H),7.43–7.19(m,5H),6.18(t,J=6 .0Hz,1H),6.05(dt,J=11.9,5.7Hz,2H),5.07(m,2H),4.27(td,J=8.4,5.2Hz,1H),4.16(dd,J=8.7,6.6Hz,1H),3.95(dt,J=1 9.2,6.8Hz,4H),3.07–3.02(m,2H),2.67(t,J=6.6Hz,2H),2.46(t,J=6.5Hz,2H),1.95(h,J=6.7Hz,1H),1.78–1.69(m,2H),1 .69–1.60(m,1H),1.52(dq,J=11.9,5.4,4.6Hz,2H),1.43–1.20(m,7H),0.97(t,J=7.2Hz,3H),0.83(dd,J=14.3,7.0Hz,6H). 13C NMR (100MHz, DMSO-d6) δ204.98,173.24,172.43,172.05,162.29,156.13,136.58,128.61,128.50,128.36,66.37,64. 75,59.15,52.61,49.80,39.22,35.85,31.41,30.53,28.97,28.88,28.84,25.85,25.69,22.53,18.59,18.54,14.07.

[0090] Example 8

[0091] Synthesis method of compound (A8)

[0092] The raw material benzyloxycarbonyl-L-valyl-L-alanine (0.967 g, 3.00 mmol) and HOBT (0.418 g, 3.10 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.516 g, 5.10 mmol). The mixture was stirred at 0°C for 10 min, and then EDCI (0.690 g, 3.60 mmol) was added. After stirring at 0°C for 0.5 h, HAL·HCl (0.906 g) was added. , 3.60mmol), and then reacted at room temperature for 3h, and the reaction progress was monitored by TLC; after the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30mL), washed once with saturated sodium bicarbonate aqueous solution (50mL), saturated ammonium chloride aqueous solution (50mL), and saturated brine (50mL) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 0.827g of white solid A8 with a yield of 53%.

[0093] 1H NMR(400MHz,Chloroform-d)δ7.41–7.22(m,6H),7.08(dd,J=36.0,7.7Hz,1H),6.58(dd,J=19.1,8 .5Hz,1H),5.10(s,2H),4.65(dp,J=14.4,7.2Hz,1H),4.43–4.25(m,1H),4.17(ddd,J=19.5,9.8,4 .8Hz,2H),4.04(t,J=6.8Hz,2H),2.74(t,J=6.5Hz,2H),2.63(t,J=6.0Hz,2H),2.09–1.97(m,1H), 1.60(p,J=6.7Hz,2H), 1.38(dd,J=7.0,4.0Hz,3H), 1.30(qt,J=9.9,5.5Hz,6H), 1.00–0.82(m,9H). 13 C NMR(100MHz,Chloroform-d)δ204.67,172.53,172.05,171.97,156.13,136.58,128.61,128.50,128.36,66.3 7,64.75,59.12,50.01,48.84,35.85,31.41,30.53,28.97,28.84,25.69,22.53,18.59,18.54,17.94,14.07.

[0094] Example 9

[0095] Synthesis method of compound (A9)

[0096] (S)-2-(1-(ethylcarbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanic acid (0.788 g, 2.40 mmol) prepared according to Example 3 and HOBT (0.334 g, 2.47 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (25 mL) was added, followed by NMM (0.413 g, 4.08 mmol), and the mixture was stirred at 0°C for 10 min, followed by EDCI (0.552 g, 2.88 mmol), and the mixture was stirred at 0°C for 0.5 h before addition of 1% paraformaldehyde. HAL·HCl (0.523 g, 2.88 mmol) was added, and the mixture was reacted at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=25:1, v / v) to obtain 0.492 g of white solid A9 with a yield of 45%.

[0097] 1 H NMR(400MHz,Chloroform-d)δ7.80(s,1H),7.48(s,1H),6.80(d,J=5.9Hz,1H),5.94(s,1H),5.41–5.26(m,2H),4.52(td,J =8.5,4.6Hz,1H),4.13(qd,J=18.9,5.2Hz,2H),4.01(t,J=6.8Hz,2H),3.22(dq,J=15.0,7.6Hz,2H),3.13(d,J=21.3Hz,2H) ,2.72(t,J=6.5Hz,2H),2.58(t,J=6.6Hz,2H),2.55–2.41(m,4H),1.83(ttd,J=11.0,7.9,7.2,2.8Hz,3H),1.68(q,J=6.5,5 .7Hz,1H),1.61–1.54(m,2H),1.48(d,J=7.3Hz,2H),1.28(dt,J=9.2,6.0Hz,6H),1.07(t,J=7.2Hz,3H),0.91–0.82(m,3H). 13C NMR(100MHz,Chloroform-d)δ204.69,173.52,172.82,172.53,172.40,160.23,65.17,54.67,53.19,49 .13,34.95,34.60,31.48,29.72,29.59,29.41,28.57,27.80,26.33,25.61,22.59,15.94,14.62,14.08.

[0098] Example 10

[0099] Synthesis method of compound (A10)

[0100] Acetyl-L-phenylalanine-N ε -(tert-Butoxycarbonyl)-L-lysine (1.307 g, 3.00 mmol) and HOBT (0.418 g, 3.10 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.516 g, 5.10 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.690 g, 3.60 mmol), and stirred at 0°C for 0.5 h, and then HAL·HCl (0.906 g, 3.60 mmol) was added, and the mixture was reacted at room temperature for 3 h, and monitored by TLC. Reaction process: After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated sodium bicarbonate aqueous solution (40 mL), saturated ammonium chloride aqueous solution (40 mL), and saturated brine (40 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=50:1, v / v) to obtain 1.177 g of 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid hexyl ester, with a yield of 62%.

[0101] 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid hexyl ester (1.177 g, 1.86 mmol) and p-toluenesulfonic acid (0.384 g, 2.23 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, and the lower layer of solid was collected and dried in vacuo to obtain 1.075 g of white solid A10 with a yield of 82%.

[0102] 1 H NMR (400MHz, DMSO-d6) δ8.21–8.02(m,3H),7.64(s,3H),7.49(d,J=8.1Hz,2H),7.27–7.15(m,5H ),7.12(d,J=7.8Hz,2H),4.50(ddd,J=10.2,8.1,4.3Hz,1H),4.28(td,J=8.7,5.0Hz,1H),4.02–3 .89(m,4H),3.01(dd,J=13.9,4.3Hz,1H),2.82–2.63(m,5H),2.49–2.45(m,2H),2.29(s,3H),1.7 5(s,3H),1.73–1.63(m,1H),1.53(tt,J=13.6,6.4Hz,5H),1.37–1.21(m,8H),0.90–0.81(m,3H). 13 C NMR(100MHz,DMSO-d6)δ205.23,172.22,171.75,171.53,169.40,145.51,138.04,137.79,129.16,128.14,128.03,126.23,125.5 1,64.00,54.07,52.19,48.34,38.77,37.32,33.92,31.38,30.87,28.06,27.32,26.57,25.01,22.46,22.19,22.01,20.81,13.91.

[0103] Embodiment 11

[0104] Synthesis method of compound (A11)

[0105] The N prepared according to Example 5 was 2 -(N 2 -Acetyl-N 6 -(tert-Butyloxycarbonyl)-L-lysine)-N 6-(tert-Butoxycarbonyl)-L-lysine (1.550 g, 3.00 mmol) and HOBT (0.426 g, 3.15 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.455 g, 4.50 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.690 g, 3.60 mmol), and continued stirring at 0°C for 0.5 h, and then HAL·HCl (0.906 g, 3.60 mmol) was added, and the mixture was reacted at room temperature for 3 h, and the reaction progress was monitored by TLC; After completion, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 1.306 g of 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanoyl)hexanoyl)-4-oxopentanoic acid hexyl ester with a yield of 61%.

[0106] 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanamido)hexanamido)-4-oxopentanoic acid hexyl ester (1.306 g, 1.83 mmol) and p-toluenesulfonic acid (0.788 g, 4.58 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, and the lower layer of solid was collected and dried in vacuo to obtain 11.162 g of white solid A1 with a yield of 74%.

[0107] 1H NMR(400MHz,Chloroform-d)δ8.28–7.97(m,3H),7.67(s,6H),7.50(d,J=7.9Hz,4H),7.13(d ,J=7.8Hz,4H),4.31–4.16(m,2H),3.95(dt,J=16.2,5.5Hz,4H),2.74(q,J=6.6Hz,4H),2.67 (td,J=6.8,2.4Hz,2H),2.46(dd,J=6.7,2.9Hz,2H),2.29(s,6H),1.85(s,3H),1.67(dt,J=1 4.1,8.0Hz,2H),1.59–1.44(m,8H),1.27(td,J=12.4,11.5,7.2Hz,10H),0.90–0.81(m,3H). 13 C NMR (101MHz, DMSO-d6) δ205.72,172.67,172.38,172.30,170.24,145.54,138.52,128.67,125.95,64.43,53.34,52.96,52. 62,48.79,39.23,34.34,31.61,31.49,31.32,28.51,27.75,27.08,26.95,25.46,22.97,22.90,22.82,22.45,21.25,14.34.

[0108] Example 12

[0109] Synthesis method of compound (A12)

[0110] Acetyl-L-valyl-N ε-(tert-Butoxycarbonyl)-L-lysine (1.162 g, 3.00 mmol) and HOBT (0.418 g, 3.10 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.516 g, 5.10 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.690 g, 3.60 mmol), and continued stirring at 0°C for 0.5 h, and then HAL·HCl (0.906 g, 3.60 mmol) was added, and the mixture was reacted at room temperature for 3 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated sodium bicarbonate aqueous solution (50 mL), saturated ammonium chloride aqueous solution (50 mL), and saturated brine (50 mL) in sequence, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=40:1, v / v) to obtain 1.070 g of 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid hexyl ester, with a yield of 61%.

[0111] 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid hexyl ester (1.070 g, 1.83 mmol) and p-toluenesulfonic acid (0.378 g, 2.19 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, the lower layer of solid was collected, and vacuum dried to obtain 1.022 g of white solid A12 with a yield of 85%.

[0112] 1H NMR (400MHz, DMSO-d6) δ8.29–7.88(m,3H),7.67(s,3H),7.49(d,J=7.8Hz,2H),7.12(d,J=7.8Hz,2H) ,4.25(dtd,J=17.1,8.7,4.9Hz,1H),4.09(dt,J=24.4,7.3Hz,1H),4.03–3.84(m,4H),2.74(t,J=6.3 Hz,2H),2.67(q,J=5.6,4.8Hz,2H),2.46(t,J=6.7Hz,2H),2.29(s,3H),2.00–1.90(m,1H),1.86(d,J =3.3Hz,3H),1.75–1.62(m,1H),1.60–1.45(m,5H),1.38–1.21(m,8H),0.85(dt,J=10.0,6.6Hz,9H). 13 C NMR(101MHz,DMSO-d6)δ205.68,172.65,172.26,171.65,170.00,145.92,138.25,128.58,125.95,64.43,59.15,58.37, 52.55,48.80,34.33,31.71,31.32,30.67,30.38,28.51,27.77,27.00,25.46,22.95,22.45,21.25,19.73,18.66,14.35.

[0113] Example 13

[0114] Synthesis method of compound (A13)

[0115] The methyl 5-((S)-2-((S)-2-benzyloxycarbonyl-3-methylbutyramido)-5-ureidopentanamido)-4-oxopentanoate (1.607 g, 3.00 mmol) prepared according to Example 1 was placed in a 100 mL round-bottom flask, and solvent THF (15 mL) was added to dissolve it. Subsequently, lithium hydroxide aqueous solution (10 mL, 1 M) was added during stirring, and stirring was continued at room temperature for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, a cation exchange resin was added to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The cation exchange resin was then filtered off, and the filtrate was concentrated under reduced pressure. Anhydrous EA was added for pulping, suction filtered, and the filter cake was dried in vacuo to obtain 1.220 g of white solid A13 with a yield of 78%.

[0116] 1H NMR(400MHz, DMSO-d6)δ8.29–8.16(m,1H),7.99–7.92(d,J=8Hz,1H),7.42–7.22(m,6H),6.41–6.25( d,J=1.2Hz,1H),6.06(s,2H),5.10–4.93(m,2H),4.36–4.12(m,2H),3.98–3.85(m,2H),3.00–2.90(m ,2H),2.63(td,J=7.7,6.9,3.1Hz,2H),2.40(dt,J=7.4,3.7Hz,2H),1.98–1.87(m,1H),1.65(dt,J=1 9.8,6.0Hz,1H),1.49(qt,J=9.9,4.7Hz,1H),1.37(dtt,J=22.4,11.1,5.6Hz,2H),0.89–0.78(m,6H). 13 C NMR(100MHz,DMSO-d6)δ204.74,176.35,173.24,172.43,162.29,156.13,136.58,129.41,128.5 0,128.13,66.37,59.15,52.61,49.80,39.22,36.31,30.53,28.99,28.88,25.85,18.59,18.54.

[0117] Embodiment 14

[0118] Synthesis method of compound (A14)

[0119] The methyl 5-((S)-2-((S)-2-acetylamino-3-methylbutyramido)propionamido)-4-oxopentanoate (1.349 g, 3.00 mmol) prepared according to Example 2 was placed in a 100 mL round-bottom flask, and solvent THF (15 mL) was added to dissolve it. Subsequently, an aqueous lithium hydroxide solution (10 mL, 1 M) was added during stirring, and stirring was continued at room temperature for 4 h. After the reaction was completed, a cation exchange resin was added to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The cation exchange resin was then filtered out, and the filtrate was concentrated under reduced pressure. Anhydrous EA was added for pulping, suction filtered, and the filter cake was dried in vacuo to obtain 1.071 g of white solid A14 with a yield of 78%.

[0120] 1H NMR (400MHz, DMSO-d6) δ12.14(s,1H),8.17(t,J=5.6Hz,1H),8.01(d,J=7.4Hz ,1H),7.46–7.22(m,6H),5.03(d,J=2.1Hz,2H),4.34(p,J=7.0Hz,1H),4.00–3 .82(m,3H),2.63(t,J=6.5Hz,2H),2.40(t,J=6.5Hz,2H),1.95(dd,J=13.5,6. 8Hz, 1H), 1.23 (d, J = 7.0Hz, 3H), 0.86 (d, J = 6.8Hz, 3H), 0.81 (d, J = 6.6Hz, 3H). 3 C NMR(100MHz,DMSO-d6)δ205.42,173.62,172.45,170.78,156.16,137.09,128.36,12 7.78,127.65,65.40,59.98,48.37,47.93,33.96,30.33,27.41,19.23,18.39,18.00.

[0121] Embodiment 15

[0122] Synthesis method of compound (A15)

[0123] The (S)-5-(2-(1-(ethylcarbamoyl)cyclobutane-1-carboxamido)-5-ureidopentanamido)-4-oxopentanoic acid methyl ester (1.367 g, 3.00 mmol) prepared according to Example 3 was placed in a 100 mL round-bottom flask, and solvent THF (15 mL) was added to dissolve it. Subsequently, lithium hydroxide aqueous solution (10 mL, 1 M) was added during stirring, and stirring was continued at room temperature for 4 h. After the reaction was completed, a cation exchange resin was added to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The cation exchange resin was then filtered out, and the filtrate was concentrated under reduced pressure. Anhydrous EA was added for pulping, suction filtered, and the filter cake was vacuum dried to obtain 0.821 g of white solid A15 with a yield of 62%.

[0124] 1H NMR (400MHz, DMSO-d6) δ8.30(t,J=5.6Hz,1H),7.77(t,J=5.6Hz,1H),7.65(d,J=8.0Hz,1H),5.95(t,J= 5.8Hz,1H),5.39(s,2H),4.31(td,J=8.7,4.7Hz,1H),4.04–3.90(m,2H),3.09(dtd,J=12.3,7.1,5.3Hz, 2H),2.93(q,J=6.7Hz,2H),2.69(t,J=6.5Hz,2H),2.49–2.45(m,2H),2.38(q,J=7.7,7.1Hz,4H),1.71(d tp,J=10.7,6.8,4.2,3.3Hz,3H),1.54(tt,J=9.3,4.7Hz,1H),1.41–1.30(m,2H),0.99(t,J=7.2Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ204.67,173.18,173.15,172.83,172.51,162.29,53.91 ,51.89,49.80,39.22,35.89,35.78,31.12,28.87,28.23,25.85,15.31,14.83.

[0125] Example 16

[0126] Synthesis method of compound (A16)

[0127] The methyl 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoate (1.688 g, 3.00 mmol) prepared according to Example 4 was placed in a 100 mL round-bottom flask, and a solvent THF (15 mL) was added to dissolve it. Subsequently, an aqueous lithium hydroxide solution (10 mL, 1 M) was added during stirring, and stirring was continued at room temperature for 4 h. After the reaction was completed, a cation exchange resin was added to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The cation exchange resin was then filtered out, and the filtrate was concentrated under reduced pressure. Anhydrous EA was added for pulping, suction filtered, and the filter cake was vacuum dried to obtain 1.136 g of 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid, with a yield of 69%.

[0128] 5-((S)-2-((S)-2-acetamido-3-phenylpropionamido)-6-((tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid (1.097 g, 2.00 mmol) and p-toluenesulfonic acid (0.412 g, 2.40 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, and the lower layer of solid was collected and dried in vacuo to obtain 0.943 g of white solid A16 with a yield of 76%.

[0129] 1 H NMR(400MHz,DMSO-d6)δ8.19–8.02(m,3H),7.65(s,3H),7.49(dd,J=8.0,1.9Hz,2H),7.28 –7.15(m,5H),7.12(d,J=7.8Hz,2H),4.50(ddd,J=10.2,8.1,4.3Hz,1H),4.28(td,J=8.6, 5.0Hz,1H),3.95(t,J=5.7Hz,2H),3.01(dd,J=13.9,4.3Hz,1H),2.81–2.60(m,5H),2.41( t,J=6.5Hz,2H),2.29(s,3H),1.75(s,4H),1.53(h,J=7.8,6.7Hz,3H),1.41–1.26(m,2H). 13 C NMR(100MHz,DMSO-d6)δ205.37,173.65,171.75,171.52,169.41,138.05,129.17,128.17,128.15,128.0 5,126.25,125.52,54.07,52.20,48.37,38.78,37.33,34.02,31.41,27.44,26.59,22.47,22.18,20.82.

[0130] Embodiment 17

[0131] Synthesis method of compound (A17)

[0132] Methyl 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanamido)hexanamido)-4-oxopentanoate (1.932 g, 3.00 mmol) prepared according to Example 5 was placed in a 100 mL round-bottom flask, and THF (15 mL) was added to dissolve the mixture. Then, an aqueous lithium hydroxide solution (10 mL, 1 M) was added while stirring. The mixture was stirred at room temperature. The mixture was stirred for 4 h. After the reaction, a cation exchange resin was added to the reaction solution to adjust the pH of the reaction solution to 6-7, and then the cation exchange resin was filtered out, the filtrate was concentrated under reduced pressure, anhydrous EA was added for slurrying, suction filtered, and the filter cake was dried under vacuum to obtain 1.391 g of 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanoyl)hexanoyl)-4-oxopentanoic acid with a yield of 72%.

[0133] 5-((2S)-6-((tert-butoxycarbonyl)amino)-2-((2S)-6-((tert-butoxycarbonyl)amino)-2-acetamidohexanamido)hexanamido)-4-oxopentanoic acid (1.288 g, 2.00 mmol) and p-toluenesulfonic acid (0.412 g, 2.40 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, the lower layer of solid was collected, and vacuum dried to obtain 1.024 g of white solid A17 with a yield of 76%.

[0134] 1 H NMR (400MHz, DMSO-d6) δ8.24–7.97(m,3H),7.65(s,6H),7.49(d,J=8.0Hz,4H),7.13(d ,J=7.9Hz,4H),4.24(dtt,J=16.3,8.0,4.0Hz,2H),4.00–3.86(m,2H),2.74(q,J=6.8H z,4H),2.63(dt,J=7.6,3.7Hz,2H),2.41(td,J=6.5,3.1Hz,2H),2.29(s,6H),1.84(s, 3H), 1.66 (dq, J=19.8, 7.6Hz, 2H), 1.51 (p, J=8.5Hz, 6H), 1.30 (dq, J=15.9, 7.1Hz, 4H). 13C NMR (100MHz, DMSO-d6) δ205.82,174.10,172.33,172.24,169.94,145.84,138.32,128.61,125.95,52. 84,52.54,48.79,39.21,34.48,31.85,31.75,28.18,27.88,27.12,27.00,22.98,22.81,22.64,21.26.

[0135] Embodiment 18

[0136] Synthesis method of compound (A18)

[0137] The methyl 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoate (1.544 g, 3.00 mmol) prepared according to Example 6 was placed in a 100 mL round-bottom flask, and a solvent THF (15 mL) was added to dissolve it. Subsequently, an aqueous lithium hydroxide solution (10 mL, 1 M) was added during stirring, and stirring was continued at room temperature for 4 h. After the reaction was completed, a cation exchange resin was added to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The cation exchange resin was then filtered out, and the filtrate was concentrated under reduced pressure. Anhydrous EA was added for pulping, suction filtered, and the filter cake was vacuum dried to obtain 1.231 g of 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid, with a yield of 82%.

[0138] 5-((S)-2-((S)-2-acetamido-3-methylbutanamido)-6-(tert-butoxycarbonyl)amino)hexanamido)-4-oxopentanoic acid (1.001 g, 2.00 mmol) and p-toluenesulfonic acid (0.412 g, 2.40 mmol) were placed in a 100 mL round-bottom flask, and solvent HFIP (10 mL) was added to dissolve, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, and the mixture was thoroughly ground and stirred. The mixture was centrifuged to remove the supernatant, and anhydrous ether was added again. The operation was repeated, the lower layer of solid was collected, and vacuum dried to obtain 0.882 g of white solid A18 with a yield of 77%.

[0139] 1H NMR (400MHz, DMSO-d6) δ8.28–7.87(m,3H),7.64(s,3H),7.48(d,J=8.1Hz,2H),7.12(d,J=7. 8Hz,2H),4.31–4.20(m,1H),4.15–4.05(m,1H),3.98–3.89(m,2H),2.74(p,J=6.4,5.5Hz,2H) ,2.62(dt,J=6.9,4.5Hz,2H),2.40(t,J=6.8Hz,2H),2.29(s,3H),1.99–1.89(m,1H),1.86(s, 3H),1.76–1.62(m,1H),1.52(h,J=6.2,5.7Hz,3H),1.39–1.26(m,2H),0.84(q,J=7.4Hz,6H). 13 C NMR(100MHz,DMSO-d6)δ205.82,174.07,172.24,171.64,169.99,145.96,138.23,128.58,125.96, 58.36,52.54,48.82,39.19,34.43,31.76,30.68,27.87,27.01,22.95,22.64,21.25,19.74,18.67.

[0140] Embodiment 19

[0141] Synthesis method of compound (A19)

[0142] The raw material N-tert-butoxycarbonyl-L-valyl-L-alanine (0.836 g, 2.90 mmol) and HOBT (0.404 g, 3.00 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.499 g, 4.93 mmol), and stirred at 0°C for 10 min. Subsequently, EDCI (0.667 g, 3.48 mmol) was added, and stirring was continued at 0°C for 0.5 h. MAL·HCl (0.632 g, 3.48 mmol) was added, and the mixture was reacted at room temperature for 3 h. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=30:1, v / v) to obtain 0.939 g of methyl 5-((2S)-2-((2S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)-4-oxopentanoate in a yield of 78%.

[0143] 5-((2S)-2-((2S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)-4-oxopentanoic acid methyl ester (0.831 g, 2.00 mmol) was placed in a 100 mL round-bottom flask, anhydrous EA (10 mL) was added to dissolve the mixture, HCl-EA solution (6 mL) was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, the mixture was thoroughly ground and stirred, and the mixture was centrifuged to remove the supernatant. Anhydrous ether was added again, the operation was repeated, the lower solid was collected, and the mixture was dried in vacuo to obtain 0.577 g of 5-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)-4-oxopentanoic acid methyl ester hydrochloride in a yield of 82%.

[0144] 5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-4-oxopentanoic acid methyl ester hydrochloride (0.528 g, 1.50 mmol) and biotin-N-succinimidyl ester (0.615 g, 1.80 mmol) were placed in a 100 mL round-bottom flask, DMF (20 mL) was added to dissolve, triethylamine (0.182 g, 1.80 mmol) was slowly added dropwise, and the mixture was stirred at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, water was added to precipitate the solid, and the solid was filtered off by suction. The filter residue was redispersed in water, heated for recrystallization, filtered off by suction, and the filter cake was dried under vacuum to obtain 0.187 g of white solid A19 with a yield of 23%.

[0145] 1H NMR (400MHz, DMSO-d6) δ8.29–7.77(m,3H),6.40(d,J=22.0Hz,2H),4.31(td,J=7.6,7.1,4.2Hz,2H),4.20–4.04(m,2H),4.0 4–3.84(m,2H),3.57(s,3H),3.09(ddd,J=8.3,6.2,4.1Hz,1H),2.82(dd,J=12.4,5.0Hz,1H),2.69(t,J=6.5Hz,2H),2.57(d ,J=12.4Hz,1H),2.47(t,J=6.6Hz,2H),2.17(hept,J=7.1Hz,2H),1.93(dq,J=17.9,6.9Hz,1H),1.61(ddd,J=19.9,9.4,4.6 Hz,1H),1.48(tt,J=15.5,4.7Hz,3H),1.30(q,J=8.1,7.7Hz,2H),1.23(d,J=7.1Hz,3H),0.83(ddd,J=11.7,7.8,5.4Hz,6H). 13 C NMR(100MHz,DMSO-d6)δ205.36,172.68,172.54,172.33,170.90,162.78,61.07,59.24,57.45,55.45 ,51.44,48.37,48.00,39.89,34.93,33.86,30.39,28.16,28.03,27.14,25.43,19.31,18.25,18.13.

[0146] Embodiment 20

[0147] Synthesis method of compound (A20)

[0148] The raw material N-tert-butoxycarbonyl-L-valyl-L-citrulline (1.086 g, 2.90 mmol) and HOBT (0.404 g, 3.00 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.499 g, 4.93 mmol), and stirred at 0°C for 10 min. Subsequently, EDCI (0.667 g, 3.48 mmol) was added, and stirring was continued at 0°C for 0.5 h. HAL·HCl (0.873 g, 3.48 mmol) was added, and the mixture was reacted at room temperature for 3 h. The mixture was stirred at 4 °C for 10 min. The reaction progress was monitored by LC. After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=30:1, v / v) to obtain 1.210 g of 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-4-oxopentanoic acid hexyl ester with a yield of 73%.

[0149] 5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-4-oxopentanic acid hexyl ester (1.143 g, 2.00 mmol) was placed in a 100 mL round-bottom flask, anhydrous EA (10 mL) was added to dissolve the solvent, HCl-EA solution (6 mL) was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, the mixture was thoroughly ground and stirred, centrifuged, the supernatant was removed, anhydrous ether was added again, the operation was repeated, the lower solid was collected, and vacuum dried to obtain 0.619 g of 5-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-4-oxopentanic acid hexyl ester hydrochloride with a yield of 61%.

[0150] 5-((S)-2-((S)-2-amino-3-methylbutyramido)-5-ureidopentanamido)-4-oxopentanoic acid hexyl ester hydrochloride (0.508 g, 1.00 mmol) and biotin-N-succinimidyl ester (0.410 g, 1.20 mmol) were placed in a 100 mL round-bottom flask, DMF (10 mL) was added to dissolve, triethylamine (0.121 g, 1.20 mmol) was slowly added dropwise, and the mixture was stirred at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, water was added to precipitate the solid, and the solid was filtered off by suction. The filter residue was redispersed in water, heated for recrystallization, filtered off by suction, and the filter cake was dried under vacuum to obtain 0.140 g of white solid A20 with a yield of 20%.

[0151] 1 H NMR (400MHz, DMSO-d6) δ8.18(t,J=5.5Hz,1H),7.95(d,J=7.6Hz,1H),7.81(d,J=8.8Hz,1H),6.41(d,J=30.7Hz,2H),5. 92(t,J=5.8Hz,1H),5.38(s,2H),4.30(d,J=7.3Hz,2H),4.15(p,J=6.2,5.1Hz,2H),4.06–3.82(m,4H),3.09(s,1H),2. 94(s,2H),2.82(dd,J=12.4,4.9Hz,1H),2.66(d,J=6.5Hz,2H),2.57(d,J=12.5Hz,1H),2.47(s,2H),2.16(q,J=6.6Hz, 2H),2.01–1.89(m,1H),1.62(s,2H),1.51(dq,J=17.7,9.3,8.0Hz,6H),1.42–1.18(m,10H),0.83(q,J=8.0,7.6Hz,9H). 13 C NMR(100MHz,DMSO-d6)δ205.25,172.35,172.22,171.88,171.11,162.77,158.78,63.99,61.04,59.22,57.55,55.42,52.17,48.3 8,39.88,38.78,34.93,33.88,30.88,30.29,29.53,28.11,28.07,28.00,27.33,26.59,25.44,25.02,22.01,19.33,18.16,13.92.

[0152] Embodiment 21

[0153] Synthesis method of compound (A21)

[0154] The raw material tert-butoxycarbonyl-L-valyl-L-alanine (0.836 g, 2.90 mmol) and HOBT (0.404 g, 3.00 mmol) were placed in a 100 mL round-bottom flask, and solvent DCM (30 mL) was added, followed by NMM (0.499 g, 4.93 mmol), and stirred at 0°C for 10 min, followed by EDCI (0.667 g, 3.48 mmol), and continued stirring at 0°C for 0.5 h, and then HAL·HCl (0.873 g, 3.48 mmol) was added, and the mixture was reacted at room temperature for 3 h. The reaction progress was monitored by TLC. After the reaction, the reaction solution was concentrated under reduced pressure, redissolved with EA (30 mL), and washed once with saturated aqueous sodium bicarbonate solution (50 mL), saturated aqueous ammonium chloride solution (50 mL), and saturated brine (50 mL) in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by column chromatography (DCM:MeOH=30:1, v / v) to obtain 1.154 g of 5-((2S)-2-((2S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)-4-oxopentanoic acid hexyl ester with a yield of 82%.

[0155] 5-((2S)-2-((2S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)-4-oxopentanoic acid hexyl ester (0.971 g, 2.00 mmol) was placed in a 100 mL round-bottom flask, anhydrous EA (10 mL) was added to dissolve the solvent, HCl-EA solution (6 mL) was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, anhydrous ether was added, the mixture was fully ground and stirred, and the mixture was centrifuged to remove the supernatant, anhydrous ether was added again, the operation was repeated, the lower solid was collected, and the mixture was dried in vacuo to obtain 0.590 g of 5-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)-4-oxopentanoic acid hexyl ester hydrochloride with a yield of 70%.

[0156] 5-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-4-oxopentanoic acid hexyl ester hydrochloride (0.633 g, 1.50 mmol) and biotin-N-succinimidyl ester (0.615 g, 1.80 mmol) were placed in a 100 mL round-bottom flask, DMF (20 mL) was added to dissolve, triethylamine (0.182 g, 1.80 mmol) was slowly added dropwise, and the mixture was stirred at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, water was added to precipitate the solid, and the solid was filtered off by suction. The filter residue was redispersed in water, heated for recrystallization, filtered off by suction, and the filter cake was dried under vacuum to obtain 0.321 g of light yellow solid A2 with a yield of 35%.

[0157] 1 H NMR(400MHz, DMSO-d6)δ8.27–7.75(m,3H),6.39(d,J=23.1Hz,2H),4.30(qd,J=7.2,4.5Hz,2H),4 .19–4.05(m,2H),4.02–3.85(m,4H),3.13–3.04(m,1H),2.82(dd,J=12.5,4.9Hz,1H),2.72–2.63 (m,2H),2.57(d,J=12.4Hz,1H),2.46(t,J=6.4Hz,2H),2.16(q,J=7.3Hz,2H),1.93(dh,J=20.2,6 .8Hz,1H),1.66–1.58(m,1H),1.57–1.41(m,5H),1.35–1.19(m,11H),0.84(dq,J=9.4,6.5Hz,9H). 13 C NMR (101 MHz, DMSO) δ 205.77, 172.95, 172.74, 172.65, 171.32, 163.18, 64.43, 61.49, 59.65, 57.86, 55.89, 48.81, 48.41, 40.32, 35.35, 34.30, 31.33, 30.83, 28.59, 28.52, 28.46, 27.77, 25.86, 25.47, 22.46, 19.74, 18.67, 18.56, 14.36. Example 22

[0158] The following are the pharmacological experimental data of some compounds of the present invention:

[0159] 1. Detection of compound's ability to produce Pp IX

[0160] (1) Tumor cell culture

[0161] Human breast cancer cells MCF-7 and MDA-MB-231 were cultured in DMEM (dulbecco's modified eagle medium) containing 10% fetal bovine serum (FBS).

[0162] (2) Pp IX generation detection

[0163] Compound A1-21, ALA methyl ester and ALA hexyl ester were dissolved in DMSO to obtain a mother solution with a concentration of 10 mM; the mother solution was dissolved in culture medium (containing 1% Penicillin-streptomycin) to obtain a secondary mother solution with a concentration of 100 μM; the secondary mother solution was diluted with culture medium to the required concentration according to experimental requirements.

[0164] Tumor cells were counted at 8×103 The cells were seeded into a 96-well plate (701001, NEST) at 1000 μg / well and grown in an incubator for 12 h, after which the culture medium was discarded. Culture medium containing different concentrations of compounds A1-A21, ALA methyl ester and ALA hexyl ester was added, respectively, and the culture was continued for 8 h. After the culture was completed, the fluorescence intensity of Pp IX in the cells was measured using an enzyme-linked immunosorbent assay (Bio-Rad microplate reader) (excitation wavelength: 405 nm, emission wavelength: 635 nm).

[0165] The results are as follows Figure 1 and Figure 2 As shown, the PpIX production efficiency of compounds A1-A21 is affected by the modified part of the ALA amino terminus and increases with the increase of the lipophilicity of the compound. Among the derivatives of ALA ester amino terminus modified by Cath B selectively cleaving the dipeptide, compounds A1, A7 and A20 respectively show higher PpIX production efficiency than their parent compounds (ALA methyl ester or hexyl ester) at low concentrations. Compound A7 has a higher PpIX production efficiency than compound A1 due to the hexyl ester part increasing the lipophilicity of the molecule; compound A20 has a much higher targeting in tumor cells due to the introduction of the biotin part, which increases the uptake of the compound by tumor cells and enhances the enrichment of the photosensitizer in tumor cells, thus having a much higher PpIX production efficiency than ALA methyl ester or hexyl ester. However, at all concentrations, compounds A4-A6 and compounds A10-A18 all show weak PpIX fluorescence, which may be due to the presence of their p-toluenesulfonate form and the unmodified carboxyl terminus of ALA, which greatly increases the water solubility of the compound. As the lipophilicity of ALA derivatives increases, their membrane permeability increases. Therefore, it is reasonable to conclude from this experiment that the low lipophilicity of compounds A4-A6 and compounds A10-A18 limits their membrane permeability, resulting in low cellular uptake.

[0166] (3) Pp IX generation and time relationship curve

[0167] Tumor cells were counted at 8×10 3 The cells were seeded into a 96-well plate (701001, NEST) at 1000 cells / well, grown in an incubator for 12 h, and then the culture medium was discarded; culture medium containing 25 μM of compounds A1, A7 and A20 was added respectively, and the culture was continued for 2-12 h. After the culture was completed, the fluorescence intensity of Pp Ⅸ in the cells was measured using an enzyme-linked immunosorbent assay (Bio-Rad microplate reader) (excitation wavelength: 405 nm, emission wavelength: 635 nm).

[0168] like Figure 3 and Figure 4As shown, by detecting PpIX fluorescence at different time points, the PpIX fluorescence level produced by compound A20 gradually increased over time; in MCF-7 cells, the fluorescence level of compound A20 reached a peak after 8 hours, while in MDA-MB-231 cells, the fluorescence level was still on an upward trend at 12 hours.

[0169] (4) Correlation curve between Pp IX production and cathepsin B

[0170] Tumor cells were counted at 8×10 3 Cells were seeded into 96-well plates (701001, NEST) at 1000 cells / well and grown in an incubator for 12 h. The culture medium was then discarded. The culture medium containing or not containing compound A20 with CA-074Me (cathepsin B inhibitor) was added, and the fluorescence intensity of Pp Ⅸ in the cells was determined using an enzyme-linked immunosorbent assay (Bio-Rad microplate reader) (excitation wavelength: 405 nm, emission wavelength: 635 nm). The detection was performed every 5 min for a total of 3 h at 37 °C.

[0171] like Figure 5 As shown, the group with the addition of cathepsin B inhibitor did not produce Pp IX, indicating that compound A20 needs to be specifically cleaved by cathepsin B to release free ALA and further generate Pp IX.

[0172] 2. Compound cytotoxicity test

[0173] (1) Cellular light and dark toxicity experiment

[0174] Tumor cells were counted at 8×10 3 100 cells / well were seeded into 96-well plates (701001, NEST), placed in an incubator for 12 hours, and then the culture medium was discarded; culture medium containing different concentrations of compounds was added, and the culture was continued for 8 hours. The cells were irradiated with LED light (405nm) for 20 minutes, and the dark contrast experiment was carried out at the same time. After the irradiation, the culture medium was discarded, PBS was washed twice, and the culture medium containing 10% FBS was added again. The culture was continued for 16 hours, the culture medium was discarded, 0.5mg / mL of thiazolyl blue (MTT) was added, and the culture was continued for 3 hours. The culture medium was discarded and DMSO was added. The wavelength of 490nm was selected and the enzyme-linked immunosorbent monitor (Bio-Rad microplate reader) was used for reading.

[0175] (2) Cytotoxicity assay

[0176] Tumor cells were counted at 8×10 31000 cells / well were seeded into a 96-well plate (701001, NEST), placed in an incubator for 12 hours, and then the culture medium was discarded; culture medium containing different concentrations of compounds A1-A21 was added, and after 24 hours of culture, the culture medium was discarded, 0.5 mg / mL of thiazolyl blue (MTT) was added, and the culture was continued for 3 hours. The culture medium was discarded and DMSO was added. The wavelength of 490 nm was selected and the enzyme-linked immunosorbent monitor (Bio-Rad microplate reader) was used for reading. Figure 6-9 As shown, ALA methyl ester and ALA hexyl ester had almost no dark toxicity in the two cell lines, but showed certain phototoxicity under light of 425nm wavelength, but its phototoxicity was weak; compounds A1-A21 had no obvious dark toxicity; among all the compounds, A20 showed the best tumor activity, and at a concentration of 10μM, the survival rate of MDA-MB-231 cells could be reduced to 5%. Compared with compounds A1 and A7 without biotin moiety, the phototoxicity of compound A20 containing biotin moiety was greatly improved. This experiment believes that this may be due to the enhanced targeted anti-tumor effect of biotin.

Claims

1. An ALA derivative, the structural formula of which is as follows: Where R 1 is hydroxy, methoxy or n-hexyloxy; R 2 is L-alanine, L-lysine or L-citrulline; R 3 is L-valine, L-phenylalanine, L-lysine or R 4 is acetyl, benzyloxycarbonyl, ethylamino or 2. The ALA derivative according to claim 1, wherein the structural formula is one of the following:

3. The method for preparing an ALA derivative according to claim 1, comprising the steps of: Step 1: dissolving the raw material represented by Formula 1 and N-hydroxysuccinimide in an organic solvent, stirring at 0°C, then adding 1,3-dicyclohexylcarbodiimide, stirring evenly at 0°C, then moving to room temperature and continuing to stir for 16-24 hours, and monitoring the reaction progress by TLC; the molar ratio of the raw material represented by Formula 1, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide is 1:1-2:1-2; After the reaction is completed, the reaction is filtered to obtain a filtrate; the filtrate is concentrated under reduced pressure, the concentrated product is redissolved in DCM, and placed at -20°C for 12 hours, and the filtrate is filtered again to obtain a filtrate; the filtrate is concentrated under reduced pressure and dried to obtain a crude product of the compound shown in Formula 2; Step 2: dissolving the crude product of the compound of formula 2 obtained in step 1 and the compound of formula 3 in an organic solvent, then adding an aqueous solution of the basic compound (2), stirring evenly at 0°C, and then stirring at room temperature for 16-24 hours, and monitoring the reaction progress by TLC; the molar ratio of the raw material of formula 1, the compound of formula 3, and the basic compound is 1:1-1.2:1-2; After the reaction is completed, a saturated sodium bicarbonate solution is added to quench the reaction, the reaction solution is washed with ethyl acetate, the pH of the aqueous phase is adjusted to 2-3 at 0°C, the filter cake is filtered out, the filter cake is washed with dilute hydrochloric acid at 0°C, and the filter cake is vacuum dried to obtain a compound represented by formula 4; Step 3: dissolving the compound represented by Formula 4 and 1-hydroxybenzotriazole (HOBT) in an organic solvent, adding the basic compound (3), stirring at 0°C, then adding EDCI, stirring evenly at 0°C, adding the compound represented by Formula 5, and reacting at room temperature for 2 to 4 hours, and monitoring the reaction progress by thin layer chromatography (TLC); the molar ratio of the compound represented by Formula 4, HOBT, the basic compound, EDCI, and the compound represented by Formula 5 is 1:1 to 1.2:1 to 2:1 to 1.5:1 to 2; After the reaction is completed, the reaction solution obtained in step 3 is concentrated under reduced pressure, the concentrated product is redissolved in ethyl acetate, and washed with saturated sodium bicarbonate aqueous solution, saturated ammonium chloride aqueous solution, and saturated brine in sequence; the organic phase is dried over anhydrous sodium sulfate, concentrated under reduced pressure, separated and purified by column chromatography, and dried to obtain a compound represented by formula 6; Step 4-1: dissolving the compound represented by Formula 6 in THF, adding 1 mol / L lithium hydroxide aqueous solution, stirring at room temperature for 2-4 hours, and monitoring the reaction progress by thin layer chromatography (TLC); the molar ratio of the compound represented by Formula 6 to lithium hydroxide is 1:1-5; After the reaction is completed, a cation exchange resin is added to the reaction solution obtained in step 4-1, the pH is adjusted to 6-7, and then the cation exchange resin is filtered out, the filtrate is concentrated under reduced pressure, anhydrous ethyl acetate is added to slurry, and a filter cake is obtained by suction filtration, and the filter cake is vacuum dried to obtain a compound represented by formula 7; Step 5-1: dissolve the compound represented by Formula 7 in an organic solvent, add an acidic solution, wherein the acidic solution is one or more of HCl-EA solution, TFA, MsOH, and TsOH, and the molar ratio of the compound represented by Formula 7 to the acidic component in the acidic solution is 1:1.2-10; stir the obtained mixed solution at 25-35°C for 2-5h, and monitor the reaction progress by TLC; after the reaction is completed, concentrate the reaction solution under reduced pressure, add ether, grind and stir thoroughly, centrifuge, remove the supernatant, add ether again, grind and stir thoroughly, centrifuge, remove the supernatant, collect the lower solid, and vacuum dry to obtain the finished product, i.e., the compound represented by Formula I.

4. The method for preparing an ALA derivative according to claim 3, characterized in that: After completing step 3, the operations from step 4-1 to step 5-1 are replaced by connecting step 4-2 and step 5-2: The described operations are as follows: Step 4-2: Dissolve the compound represented by Formula 6 in an organic solvent, add an acidic solution, wherein the acidic solution is one or more of HCl-EA solution, TFA, MsOH, and TsOH, and the molar ratio of the compound represented by Formula 6 to the acidic component in the acidic solution is 1:1.2 to 10; stir the obtained mixed solution at 25-35°C for 2-5h, and monitor the reaction progress by TLC; after the reaction is completed, concentrate the reaction solution under reduced pressure, add ether, grind and stir thoroughly, centrifuge, remove the supernatant, add ether again, grind and stir thoroughly, centrifuge, remove the supernatant, collect the lower solid, and vacuum dry to directly obtain the finished product, i.e., the compound represented by Formula I.

5. The method for preparing an ALA derivative according to claim 3, characterized in that: After completing step 3, the operations from step 4-1 to step 5-1 are replaced by connecting step 4-3 and step 5-2: The step 4-3 is as follows: dissolving the compound represented by Formula 6 in ethyl acetate, adding HCl-EA solution, wherein the molar ratio of the compound represented by Formula 6 to HCl in the HCl-EA solution is 1:1.2-10; stirring the obtained mixed solution at 25-35°C for 2-5h, and monitoring the reaction progress by TLC; after the reaction is completed, concentrating the reaction solution under reduced pressure, adding ether, grinding and stirring thoroughly, centrifuging, removing the supernatant, adding ether again, grinding and stirring thoroughly, centrifuging, removing the supernatant, collecting the lower solid, and vacuum drying to obtain the compound represented by Formula 8; Step 5-2: dissolving the compound represented by Formula 8 and biotin-N-succinimidyl ester in an organic solvent, slowly adding a basic compound, stirring at room temperature for 24 hours, and monitoring the reaction progress by TLC; the basic compound is one or more of DIPEA, triethylamine, and sodium bicarbonate; the molar ratio of the compound represented by Formula 8, biotin-N-succinimidyl ester and the basic substance is 1:1.2~1.5:1.2~1.5; after the reaction is completed, the reaction solution is concentrated under reduced pressure, water is added to precipitate the solid, filtered, the filter residue is redispersed in water, heated for recrystallization, filtered, and the filter cake is vacuum dried to obtain the compound represented by Formula I.

6. The method for preparing an ALA derivative according to claim 3, characterized in that: The organic solvent in step 1 is one or more of DCM, THF, acetonitrile, methanol or DMF; the volume molar ratio of the organic solvent to the raw material represented by formula 1 is 4 to 10:1 mL / mmol.

7. The method for preparing an ALA derivative according to claim 3, characterized in that: The organic solvent in step 2 is one or more of THF, DCM, acetonitrile, and ethylene glycol dimethyl ether (DME); the amount of the organic solvent in step 2 is 4 to 10:1 mL / mmol based on the amount of the compound represented by formula 2; The alkaline compound (2) is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and DIPEA; the volume ratio of the aqueous solution of the alkaline compound to the organic solvent is 1:1 to 1.

5.

8. The method for preparing an ALA derivative according to claim 3, characterized in that: The organic solvent in step 3 is one or more of THF, DCM, DMF, and acetonitrile; the amount of the organic solvent in step 3 is 4 to 10:1 mL / mmol based on the amount of the compound represented by formula 4; The basic compound (3) is one or more of DIPEA, NMM, NMI, triethylamine, pyridine, and 2,6-lutidine.

9. The method for preparing an ALA derivative according to claim 3, characterized in that: The volume ratio of the lithium hydroxide aqueous solution to THF in step 4-1 is 1:1 to 3; the organic solvent in step 5-1 is one or more of DCM, EA, THF, and hexafluoroisopropanol (HFIP); the amount of the organic solvent is 4 to 10:1 mL / mmol based on the amount of the compound represented by Formula 7.

10. Use of an ALA derivative and a pharmaceutically acceptable salt thereof according to claim 1 in the treatment of malignant tumors and skin diseases; the malignant tumors or skin diseases are basal cell carcinoma, squamous cell carcinoma, actinic keratosis, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, bladder cancer, cervical cancer, condyloma acuminatum, psoriasis and Bowen's disease.

Images