A HER2-targeting antibody-conjugated gold (III) porphyrin drug and its synthesis and tumor treatment application

Abstract

The present invention relates to a HER2-targeted antibody-coupled gold (III) porphyrin drug, a synthesis method thereof, and tumor treatment applications. The HER2-targeted antibody-coupled gold (III) porphyrin drug of the present invention is formed by connecting anti-HER‑2 trastuzumab and an A3B-type gold (III) porphyrin complex via a connector, wherein the connector includes a maleimide linker connected to the antibody and a self-eliminating linker connected to the A3B-type gold (III) porphyrin complex. The present invention combines an A3B-type gold (III) porphyrin complex with anti-tumor activity with trastuzumab with a specific recognition function as a carrier, utilizes antibodies to accurately strike tumor cells, delivers the drug to the tumor environment, improves the tumor targeting of the gold (III) porphyrin complex, and achieves precision treatment. Not only can the distribution of the drug in the body be improved, but also the accumulation in non-tumor sites can be reduced, the amount of metal complex used can be reduced, and toxic side effects can be reduced; it can also achieve efficient delivery of gold drugs to tumor tissues, thereby improving the effect of cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicines, and specifically relates to an antibody-coupled gold (III) porphyrin drug targeting HER2, and its synthesis and tumor treatment application. Background Art

[0002] Cancer is a global public health priority, causing profound harm to human survival and health, and is the main limiting factor in human life expectancy. According to data from the International Agency for Research on Cancer (IARC) in 2022, there were nearly 20 million new cases of cancer worldwide, with 9.7 million deaths. Given this, the implementation of cancer control strategies and interventions is particularly urgent. Effective prevention and treatment methods can not only significantly improve patients' quality of life, but are also crucial to protecting people's health and well-being. Currently, chemotherapy remains the mainstay of cancer treatment and plays an important role in the treatment of tumors. Metal-based drugs occupy a key position in chemotherapeutic drugs. Gold (III) porphyrin compounds have a dominant position in the field of anticancer drug development due to their significant antiproliferative properties. However, they have poor biocompatibility and high off-target toxicity. How to exert stronger and more sustained antitumor effects is an urgent problem to be solved.

[0003] Antibody-Drug Conjugates (ADCs) couple monoclonal antibodies to small molecule cytotoxic drugs via linkers, combining the high selectivity of monoclonal antibodies for specific tumor-associated antigens with the potent cytotoxicity of small molecule drugs to achieve specific effects on tumor cells while minimizing damage to normal tissues. After ADCs recognize and bind to antigens, they are internalized into cells and degraded by lysosomes. The payload is released in a biologically active form, killing tumor cells. Compared with traditional non-targeted and non-specific chemotherapy methods, ADCs technology provides an innovative anti-tumor mechanism of action that can effectively improve the therapeutic index of cytotoxic compounds. That is, by binding to large molecule antibodies, it limits the penetration of small molecule hydrophobic toxins into normal tissue cell membranes, reduces toxic side effects on healthy cells, and thus optimizes the safety and effectiveness of treatment.

[0004] At present, the cytotoxins used in ADCs still face the problem of limited types and single mechanisms of action. Currently, there is relatively little research on metal-based complexes as cytotoxic molecules for ADCs, and most research focuses on platinum drugs that have been used in clinical treatment, such as cisplatin and oxaliplatin. Although these platinum drugs have shown certain efficacy in cancer treatment, they are often accompanied by many side effects and drug resistance problems. Therefore, the search and development of new metal-based complexes as cytotoxins, with their potential high efficiency and low toxicity, has a very broad space in the research and application of ADCs.

[0005] Gold compounds are a class of metallodrugs with great potential in cancer therapy. Over the past two decades, a variety of gold(III) compounds have demonstrated relevant antiproliferative properties against selected human tumor cell lines in vitro, making them excellent candidates for further pharmacological evaluation. Summary of the Invention

[0006] The purpose of the present invention is to provide an A3B-type gold (III) porphyrin complex with anti-tumor activity, combine it with a trastuzumab carrier to prepare an antibody-coupled gold (III) porphyrin drug targeting HER2, so as to improve the tumor targeting of the A3B-type gold (III) porphyrin complex and its distribution in the body, so as to achieve the same or even better tumor treatment effect with a lower dosage of the metal complex, thereby achieving the purpose of high-efficiency and low-toxic tumor treatment.

[0007] An A3B-type gold (III) porphyrin complex, the structural formula of which is as follows:

[0008]

[0009] The present invention is based on physiologically stable A3B-type gold (III) porphyrin complexes, which exhibit high cytotoxicity at the micromolar to nanomolar level against various tumor cell lines in vitro and effectively inhibit tumor growth in multiple cancer animal models.

[0010] The synthesis method of the above-mentioned A3B-type gold (III) porphyrin complex includes: preparing an A3B-type porphyrin ring, introducing a gold metal center into the porphyrin ring, and finally forming the A3B-type gold (III) porphyrin complex Au-3 through acid-amine condensation.

[0011] The synthesis method of the above-mentioned A3B type gold (III) porphyrin complex comprises the following steps:

[0012] (1) preparing an A3B-type porphyrin ring; preferably using methyl 4-formylbenzoate, pyrrole, and benzaldehyde as raw materials, cyclizing under the catalysis of boron trifluoride etherate, and oxidizing with 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ) to obtain an A3B-type porphyrin ring;

[0013] (2) adding the A3B type porphyrin ring to a glacial acetic acid solution of potassium tetrachloroaurate and sodium acetate to cause a metallation reaction of the porphyrin ring, introducing a gold metal center into the porphyrin ring, and obtaining a gold porphyrin ring intermediate;

[0014] (3) mixing the gold porphyrin ring intermediate obtained in step (2) with an alkali solution (the alkali solution can be potassium carbonate, potassium hydroxide, sodium hydroxide, sodium carbonate, etc., with potassium carbonate being the best) and dissolving it in a mixed solution of MeOH and H2O to hydrolyze and form a carboxyl site;

[0015] (4) The product obtained in step (3) was added to DMF, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and then N-aminoethylpiperazine was added under N2, and the mixture was stirred for 24-48 hours.

[0016] Furthermore, in step (1), the molar ratio or mass ratio of the A3B type porphyrin ring, potassium tetrachloroaurate, and sodium acetate added is 1:2-3:10-15.

[0017] A HER2-targeted antibody-coupled gold (III) porphyrin drug is prepared by connecting a monoclonal antibody and the above-mentioned A3B-type gold (III) porphyrin complex via a linker.

[0018] Furthermore, the antibody is the anti-HER2 monoclonal antibody trastuzumab; the linker is a maleimide linker connecting the antibody and a self-immolative linker connecting the A3B-type gold (III) porphyrin complex; the dipeptide is valine-citrulline; and each antibody conjugate carries approximately four drug molecules.

[0019] Furthermore, the linker is maleimide-amide-polyethylene glycol-valine-citrulline-amino carbonate.

[0020] The linker is used to connect the antibody to the gold(III) porphyrin complex. The gold(III) porphyrin complex is converted into a linker-gold(III) porphyrin complex. The antibody is then conjugated to the linker-gold(III) porphyrin complex by chemically reducing the amide bonds between the antibody chains. The linker-gold(III) porphyrin complex has a high reactivity with the sulfhydryl group of the trastuzumab monoclonal antibody.

[0021] The present invention couples a monoclonal antibody with a specific recognition function with the anti-tumor active gold (III) porphyrin complex to form an antibody-gold (III) porphyrin conjugate drug, thereby achieving efficient delivery of gold drugs to tumor tissues through the targeting effect of the monoclonal antibody, thereby improving the therapeutic effect; it can also effectively reduce the accumulation of drugs in non-tumor areas, thereby improving the tumor targeting and biodistribution characteristics of the gold (III) porphyrin complex and reducing toxic side effects; at the same time, it can effectively kill tumor cells and achieve the goal of high-efficiency and low-toxicity treatment.

[0022] The present invention previously conducted experiments to explore the possibility of combining gold (III) porphyrin complexes with trastuzumab. The experiments found that the A3B-type gold (III) porphyrin complex has highly effective anti-tumor properties and can significantly increase the level of reactive oxygen species in ovarian cancer cells SK-OV-3, leading to mitochondrial dysfunction and impaired cellular energy metabolism, and ultimately inducing tumor cell death by activating the apoptosis pathway. The present invention utilizes the respective advantages of the gold (III) porphyrin complex and trastuzumab. By synthesizing the antibody-drug conjugate of the gold (III) porphyrin complex and trastuzumab, it can enhance the inhibitory effect of the gold (III) porphyrin complex on tumor cells and improve the targeting of the gold (III) porphyrin complex, thereby reducing the amount of metal complex used, thereby reducing the toxicity of the gold (III) porphyrin complex to surrounding healthy cells and directing the cytotoxic drug to the tumor cells.

[0023] The method for synthesizing the gold (III) porphyrin antibody conjugate targeting HER2 comprises the following steps:

[0024] (S1) connecting the A3B-type gold (III) porphyrin complex with a linker to obtain Linker-Au;

[0025] (S2) dissolving Linker-Au in a solvent (preferably DMF);

[0026] (S3) dissolving tris(2-carbonylethyl)phosphine hydrochloride (TCEP) in PBS buffer, adding dropwise to the PBS solution containing trastuzumab, and stirring at room temperature for 2-4 hours to disrupt the interchain disulfide bonds;

[0027] (S4) adding the product obtained in step (S3) to the solution obtained in step (S2), and stirring at room temperature for 1-5 hours;

[0028] (S5) The mixture was diluted with PBS buffer and then TM Purification can be performed by desalting centrifugal columns, dialysis, and other methods;

[0029] (S6) The sample was then concentrated using an ultrafiltration centrifuge tube and washed with PBS buffer.

[0030] Furthermore, the connection method in step (S1) is: dissolving the linker in DMF, stirring at 0°C and adding N,N-diisopropylethylamine, then dropwise adding A3B type gold (III) porphyrin, reacting for 6-10 hours, removing the solvent under reduced pressure, and purifying.

[0031] Furthermore, the molar ratio of the added trastuzumab to Linker-Au was 1:10.

[0032] The above-mentioned HER2-targeting gold (III) porphyrin antibody conjugate is used in the preparation of anti-tumor drugs, and the tumors mainly refer to breast cancer, ovarian cancer, etc. with high HER2 expression.

[0033] The above-mentioned A3B type gold (III) porphyrin complex is used in the preparation of anti-tumor drugs, and the tumors include breast cancer and ovarian cancer.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] (1) The present invention combines an A3B-type gold (III) porphyrin complex with anti-proliferative activity with a monoclonal antibody with a specific recognition function as a carrier to prepare a trastuzumab-gold (III) porphyrin complex conjugate drug, thereby improving the tumor targeting of the gold (III) porphyrin complex, improving its distribution in the body, and achieving efficient delivery of gold drugs to tumor tissues, thereby improving the therapeutic effect; it can effectively reduce the accumulation of drugs in non-tumor areas and reduce toxic side effects; and it can also achieve efficient and low-toxic tumor treatment effects by effectively killing tumor cells.

[0036] (2) The method of the present invention uses a more toxic and stable gold (III) porphyrin complex to avoid the shortcomings of the current conjugates, such as poor stability and low drug-antibody ratio, and obtains antibody-drug conjugates with stronger anti-tumor activity and high uniformity.

[0037] (3) The method of the present invention adopts a simple synthesis and selects a gold (III) porphyrin complex with low time and energy consumption, which can avoid the disadvantages of complex toxin modification and high synthesis cost, and obtain an antibody-drug conjugate with stronger anti-tumor activity and high uniformity. The antibody conjugate construction of the present invention will limit the damage of the gold (III) porphyrin complex to normal tissue cells by binding to tumor-related proteins (human epidermal growth factor receptor 2, HER2), thereby reducing the potential toxic side effects on healthy cells and possibly improving the therapeutic efficacy index. It will provide important experimental data and theoretical support for the development of new anti-tumor drugs, promote the research progress of metal-based ADCs, and lay the foundation for their subsequent clinical application transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the structure of the HER2-targeting antibody-coupled gold (III) porphyrin drug of the present invention;

[0039] Figure 2 This is a synthetic route diagram of the A3B-type gold (III) porphyrin complex of Example 1;

[0040] Figure 3 This is a preparation diagram of the connection between the linker and the A3B-type gold (III) porphyrin complex, namely Linker-Au;

[0041] Figure 4is the hydrogen nuclear magnetic resonance spectrum of Linker-Au prepared by the present invention;

[0042] Figure 5 is the carbon nuclear magnetic resonance spectrum of Linker-Au prepared by the present invention;

[0043] Figure 6 is the high resolution mass spectrum HRMS (m / z) of Linker-Au prepared in the present invention;

[0044] Figure 7 This is a synthetic route for the HER2-targeting antibody-coupled gold (III) porphyrin drug, namely ADC-Au, of the present invention;

[0045] Figure 8 This is an SDS-PAGE analysis diagram of the antibody-coupled gold (III) porphyrin drug of the present invention;

[0046] Figure 9 The UV spectrum and drug-antibody ratio (DAR value) of the antibody-coupled gold (III) porphyrin drug of the present invention;

[0047] Figure 10 The stability analysis of the antibody-conjugated gold (III) porphyrin drug of the present invention is as follows: (a) is the stability of ADC-Au in PBS solution, and (b) is the stability in PBS solution containing 10% FBS;

[0048] Figure 11 This is an analysis chart of the affinity of the antibody-conjugated gold (III) porphyrin drug of the present invention to the HER2 protein;

[0049] Figure 12 This is a diagram showing the binding of the antibody-conjugated gold (III) porphyrin drug of the present invention to different tumor cell lines;

[0050] Figure 13 This is a diagram showing the endocytosis of the antibody-coupled gold (III) porphyrin drug of the present invention;

[0051] Figure 14 The in vitro anti-tumor activity of the antibody-coupled gold (III) porphyrin drug of the present invention;

[0052] Figure 15 The present invention is an antibody-coupled gold (III) porphyrin drug for in vivo anti-tumor activity evaluation, wherein (a) is a graph showing changes in tumor volume during treatment, (b) is a graph showing changes in mouse body weight during treatment, (c) is a graph showing tumor weight and inhibition rate after treatment, and (d) is a tumor anatomy diagram; compared with the control group, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0054] Example 1

[0055] A synthetic method of porphyrin ring (intermediate MCTPP), such as Figure 2 As shown, methyl 4-formylbenzoate, pyrrole and benzaldehyde are used as raw materials, cyclized under the catalysis of boron trifluoride etherate, and oxidized by 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ) to prepare a porphyrin ring; specifically, the following steps are included:

[0056] (1) Under anhydrous conditions, methyl 4-formylbenzoate (5 mmol, 1.0 eq.), pyrrole (20 mmol, 4.0 eq.), benzaldehyde (15 mmol, 3.0 eq.), and dichloromethane (200 mL) were added, and the reaction system was deoxygenated under nitrogen for 30 min.

[0057] (2) Boron trifluoride etherate (333 μL) was added dropwise and reacted for 3 hours in the dark.

[0058] (3) Then, 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ, 15 mmol, 3.0 eq.) was added and stirring was continued for 3 hours to complete the reaction.

[0059] (4) The dark green precipitate generated by the reaction was separated by filtration, and the solvent was removed from the filtrate under reduced pressure.

[0060] (5) The crude product was purified by silica gel column chromatography to obtain a purple solid powder with a yield of 26%.

[0061] The obtained intermediate MCTPP has a hydrogen nuclear magnetic resonance spectrum: 1 H NMR (600 MHz, Chloroform-d) δ

[0062] 8.90-8.73(m,8H),8.45-8.28(m,4H),8.24-8.17(m,6H),7.79-7.72(m,9H),4.10(s,3H),2.77(s,2H).

[0063] The intermediate MCTPP carbon NMR spectrum: (151MHz, Chloroform-d)δ167.34,147.07,142.08,142.05,134.58,134.54,129.57,129.54,127.91,127.78,126.72,120.58,120.38,118.52,77.22,77.01,76.80,52.41.

[0064] Intermediate MCTPP mass spectrum HR-ESI-MS: m / z Calcd.for C 46 H 32 N4O2([M+H] + ):673.2559,found:673.2593.

[0065] Example 2

[0066] A method for synthesizing 5-(4-benzoic acid methyl ester)-10,15,20-triphenylporphyrin gold (III) complex, such as Figure 2 As shown, under the action of potassium tetrachloroaurate and sodium acetate, the metallation reaction of the porphyrin ring is realized, and a gold metal center is successfully introduced into the porphyrin ring to prepare a gold porphyrin intermediate. The specific steps mainly include the following steps:

[0067] The intermediate MCTPP (0.1 mmol, 1.0 eq.), potassium tetrachloroaurate (0.2 mmol, 2.0 eq.), and sodium acetate (1 mmol, 10.0 eq.) were weighed and dissolved in glacial acetic acid (5 mL) and heated under reflux for 3 hours. After the reaction, the product was extracted with dichloromethane and distilled water to remove excess acetic acid. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated. The product was purified by neutral alumina column chromatography, eluting with dichloromethane.

[0068] The purified product was dissolved in dichloromethane, excess lithium chloride was added, and the mixture was stirred overnight. Filtering and removal of the solvent yielded a red solid (intermediate Au-1). The yield was 70%. After the porphyrin ring (MCTPP) was introduced into the metal center, the pyrrole N hydrogen peak at δ-2.77 ppm disappeared in the proton spectrum, and the m / z in the mass spectrum also increased.

[0069] The obtained intermediate Au-1 has a hydrogen nuclear magnetic resonance spectrum: 1 H NMR (600MHz, Chloroform-d) δ9.32-9.11(m,8H),8.57-8.34(m,4H),8.32-8.18(m,6H),7.90-7.78(m,9H),4.12(s,3H).

[0070] Intermediate Au-1 carbon NMR spectrum: 13C NMR(151MHz,Chloroform-d)δ166.89,143.46,138.80,137.12,137.02,136.46,134.36,132.51,132.31 ,132.27,131.70,131.07,129.30,128.69,127.58,123.87,123.79,122.10,77.25,77.04,76.83,52.62.

[0071] Intermediate Au-1 mass spectrum: HR-ESI-MS: m / z Calcd.for C 46 H 30 AuN4O2([M-Cl] + ):867.2029,found:867.2018.

[0072] Example 3

[0073] A method for synthesizing a 5-(4-benzoic acid)-10,15,20-triphenylporphyrin gold (III) complex, such as Figure 2 As shown, the intermediate Au-1 is hydrolyzed under the action of potassium carbonate to form a carboxyl site to obtain the intermediate Au-2, which mainly includes the following steps:

[0074] Intermediate Au-1 (0.1 mmol, 1.0 eq.) and K2CO3 (0.5 mmol, 5.0 eq.) were dissolved in MeOH (2 mL) and H2O (2 mL). The reaction mixture was stirred at 90°C for 12 h, and then the product was cooled to room temperature in the solvent.

[0075] The reaction mixture was acidified with 1M HCl solution and extracted with dichloromethane (50 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a red solid (Intermediate Au-2). Yield: 98%.

[0076] The obtained intermediate Au-2 has a hydrogen nuclear magnetic resonance spectrum: 1 H NMR (500MHz, Chloroform-d) δ9.37-9.24(m,8H),8.52-8.36(m,2H),8.32-8.21(m,8H),7.97-7.87(m,9H).

[0077] Intermediate Au-2 C NMR spectrum: 13C NMR (101MHz, Chloroform-d) δ138.44,136.94,136.71,134.27,133.57,132.44,129.58,128.74,127.85,123.73,77.52,77.20,76.88.

[0078] Intermediate Au-2 mass spectrum: HR-ESI-MS: m / z Calcd.for C 45 H 28 AuN4O2([M-Cl] + ):853.1878,found:853.1860.

[0079] Example 4

[0080] A method for synthesizing an A3B-type gold (III) porphyrin complex, such as Figure 2 As shown, the intermediate Au-2 undergoes a condensation reaction under the action of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) to obtain the complex Au-3, which specifically includes the following steps:

[0081] To a solution of intermediate Au-3 (0.05 mmol, 1.0 eq) in ultra-dry DMF (3 mL) at room temperature was added HATU (0.15 mmol, 3.0 eq.). N-aminoethylpiperazine (0.5 mmol, 10.0 eq.) was added under N₂. The reaction mixture was stirred for 24 hours. The solution was then concentrated under reduced pressure. Purification by neutral alumina column chromatography (DCM:MeOH = 95:5) afforded a red solid (complex Au-3). Yield: 62%.

[0082] The obtained complex Au-3 hydrogen nuclear magnetic resonance spectrum: 1 H NMR(400MHz,Chloroform-d)δ9.33-9.21(m,8H),8.33-8.28(m,4H),8.25-8.18(m,6H),7.95-7.81(m,10H),7 .62(t,J=4.8Hz,1H),3.71(q,J=5.8Hz,2H),2.98(t,J=4.7Hz,4H),2.74(t,J=6.1Hz,2H),2.62-2.55(m,4H).

[0083] Au-3 complex carbon NMR spectrum: 13C NMR(126MHz,Chloroform-d)δ167.09,140.90,138.39,138.30,136.98,136.96,136.86,136.73,135.70,134.32,134.19,134.16,132.74,132. 51,132.43,129.63,127.86,127.84,126.79,126.67,123.80,123.72,1 22.97,77.31,77.05,76.80,65.88,56.85,51.97,44.88,36.57,29.71..

[0084] Mass spectrum of Au-3 complex: HR-ESI-MS: m / z Calcd.for C 51 H 41 AuN7O([M-Cl] + ):964.3033,found:964.3020.

[0085] Example 5

[0086] A method for coupling a HER2-targeting antibody to a gold (III) porphyrin drug, such as Figure 3 and Figure 7 As shown, the following steps are included:

[0087] (1) Preparation of the linker-Au by connecting the complex Au-3 with the linker. Figure 3 As shown, the complex Au-3 and the linker (maleimide-amide-polyethylene glycol-valine-citrulline-amino carbonate) are used as raw materials to react in a DMF solution to generate Linker-Au. Specifically, the following steps are included:

[0088] Linker (0.06 mmol, 1.2 eq.) was weighed and dissolved in ultra-dry N,N-dimethylformamide (2 ml). The mixture was stirred at 0°C, and N,N-diisopropylethylamine (0.3 mmol, 6.0 eq.) was added. Au-3 (0.05 mmol, 1.0 eq., dissolved in ultra-dry N,N-dimethylformamide) was slowly added dropwise. When TLC (CH2Cl2 / CH3OH = 10:1, v:v) indicated the reaction was complete, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by reverse-phase preparative chromatography (C18, 10% acetonitrile / water to 100% acetonitrile, v / v) to obtain a red powder. The yield was 48%.

[0089] (2) Preparation of antibody-conjugated gold (III) porphyrin drugs. Figure 7As shown, TCEP (0.22 μmol, 3.0 eq.) was dissolved in PBS buffer and added dropwise to a PBS solution containing trastuzumab (0.07 μmol, 1.0 eq.), and stirred at room temperature for 2 hours to break the interchain disulfide bond; then, Linker-Au (0.7 μmol, 10.0 eq.) dissolved in DMF was added and stirred at room temperature for 1 hour;

[0090] After the reaction, the mixture was diluted with 5 mL of PBS buffer and then TM Purification was performed using a desalting centrifugal column (Thermo, 40K MWCO, 2 mL) to remove unconjugated Linker-Au; the sample was then concentrated using an ultrafiltration centrifuge tube (Amicon Ultra-15 10K NMWL), washed with PBS buffer (pH = 7.2), and diluted to a volume of 2 mL to obtain a trastuzumab-gold (III) porphyrin complex conjugate of appropriate concentration.

[0091] Linker-Au H NMR spectrum Figure 4 As shown: 1 H NMR(500MHz,Methanol-d4)δ9.39(s,8H),8.39(s,4H),8.32-8.23(m,6H),7.99-7.88(m,9H),7.61(d,J=8 .4Hz,2H),7.33(d,J=8.3Hz,2H),6.77(s,2H),5.49(s,1H),5.09(s,2H),4.48(dd,J=9.2,5.0Hz,1H),4.23 -4.18(m,1H),3.83-3.62(m,10H),3.62-3.53(m,12H),3.51-3.43(m, 4H),3.28-3.24(m,2H),3.20-3.05(m,2H),2.93(s,2H),2.79(s,4H),2 .54(t,J=6.1Hz,2H),2.41(t,J=6.9Hz,2H),2.16-2.02(m,1H),1.97-1.83(m,1H),1.81-1.67(m,1H),1.63-1.47(m,2H),1.01-0.90(m,6H).

[0092] Linker-Au carbon NMR spectrum Figure 5 As shown: 13C NMR(126MHz,Methanol-d4)δ173.00,172.40,171.55,170.79,170.66,168.06,160.85,155.37,141.80,138.69,138.10,137.11,1 37.04,137.01,136.63,135.23,134.12,134.03,132.22,132.12,132.07,131.73,129.26,128.20,127.53,126.33,123.70,123.59 ,122.20,119.57,70.12,70.09,70.07,70.00,69.90,69.78,68.99,66.84,66.50,65.51,59.20,56.82,53.50,53.43,52.52,48.46,48.12,47.95,47.78,47.61,47.44,47.27,47.10,38.94,36.77,35.93,34.29,33.98,30.31,28.91,26.50,18.40,17.38,14.05.

[0093] Linker-Au mass spectrometry Figure 6 Shown: HR-ESI-MS: m / z Calcd. for C 88 H 94 AU 14 O 14 ([M-Cl] + ):1767.6734,found:1767.6716.

[0094] Performance testing

[0095] 1. SDS-PAGE and UV spectrophotometric analysis of HER2-targeting antibody-conjugated gold (III) porphyrin drug

[0096] The successful coupling of gold (III) porphyrin complex to antibody was demonstrated by UV spectrophotometry, gel electrophoresis and bicinchoninic acid method. Figure 8 、 Figure 9 As shown. The conjugate exhibits a characteristic peak for the antibody at 280 nm and a characteristic peak for the complex at 528 nm. Observation and comparison of the positions of the light and heavy chain bands of the antibody conjugate and trastuzumab under white light revealed an upward shift in the migration of both the light and heavy chain bands of the conjugate, consistent with an increase in molecular weight and a decrease in migration speed after conjugation. This indicates that the gold(III) porphyrin complex was successfully conjugated to both the light and heavy chains of trastuzumab.

[0097] In addition, a standard curve was prepared based on the absorbance of the conjugate at the characteristic absorption wavelength (528 nm) of the complex, such as Figure 9 As shown, the concentration of the complex in the conjugate is calculated. Combined with the antibody concentration determined by the BCA method, the DAR value of the antibody conjugate can be calculated to be approximately 4.

[0098] 2. Stability of HER2-targeting antibody-conjugated gold (III) porphyrin drugs

[0099] The UV-visible spectra of ADC-Au at different time periods of 0-48h and 0-72h were used to evaluate the stability of ADC-Au in PBS solution and PBS solution containing 10% FBS. Figure 10 The absorption peaks of ADC-Au showed no significant changes within 48h or 72h. The electron transfer absorption peaks of the gold (III) porphyrin complex Au-3 between the metal and the ligand were located at around 410nm and 528nm. Figure 10 (a)) and in PBS solution containing 10% FBS for 72 hours ( Figure 10 The contents in (b) are basically unchanged, indicating that ADC-Au is stable in these two solvents, the complex Au-3 will not fall off ADC-Au, and the coupling strategy is successful.

[0100] 3. Analysis of the affinity of HER2-targeted antibody-conjugated gold (III) porphyrin drug for HER2 protein

[0101] First, we need to understand the affinity between ADCs drugs and target proteins, and compare the affinity differences between ADCs and naked monoclonal antibodies. This is crucial for their targeting and efficacy in vivo, to ensure that the drugs can accurately target tumor cells and achieve the best therapeutic effect. Enzyme-linked immunosorbent assay (ELISA) can be used to evaluate the affinity of both for antigens. Figure 11 As shown, the EC of ADC-Au affinity for HER2 protein 50 (half-maximal effective concentration) is 0.0203nM, which is comparable to trastuzumab (EC 50 =0.0147 nM). These experimental results indicate that the affinity of the antibody portion of the conjugated drug to the HER2 protein is not significantly affected.

[0102] 4. Binding of HER2-targeted antibody-conjugated gold (III) porphyrin drugs to different tumor cell lines

[0103] Flow cytometry was used to evaluate the targeting properties of antibody-conjugated gold (III) porphyrin drugs and trastuzumab in HER2 high-expressing and HER2 low-expressing cells. Figure 12The results showed that the conjugate drug had similar HER2 antigen recognition as trastuzumab in HER2-high-expressing cells SK-OV-3, BT-474, and Calu-3, but had almost no binding in HER2-low-expressing cells MDA-MB-231, NCI-H460, and A2780. This indicates that the antibody portion of the conjugate drug retains a high degree of antigen recognition specificity, can specifically bind to HER2 antigens on the cell surface, and selectively binds to the surface of HER2-high-expressing cells, while not binding to HER2-low-expressing cells.

[0104] 4. Internalization of HER2-targeted antibody-conjugated gold (III) porphyrin drugs

[0105] In addition, under 37℃, the fluorescence intensity of antibody-conjugated gold (III) porphyrin drug and trastuzumab on the surface of BT-474 cells decreased by about 18.9% and 30.2%, respectively, indicating that they can be effectively internalized. In SK-OV-3 cells, the internalization efficiency of ADC-Au is high ( Figure 13 This suggests that drug conjugates have advantages in endocytosis efficiency compared to naked mAbs, particularly in SK-OV-3 cells. These findings are important for gaining a deeper understanding of the intracellular trafficking and therapeutic effects of antibody-drug conjugates.

[0106] 5. In vitro antitumor activity of HER2-targeting antibody-conjugated gold (III) porphyrin drugs

[0107] The in vitro antiproliferative activity of the antibody conjugates against different cell lines was determined using the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. The in vitro cytotoxicity of ADC-Au was evaluated using HER2-positive (SK-OV-3, BT-474, and Calu-3) and negative (MDA-MB-231, A2780, and NCI-H460) cancer cell lines. Figure 14 After 72 hours of continuous culture, cytotoxicity was assessed using the MTT assay. The gold(III) porphyrin complex significantly enhanced cytotoxicity upon binding to trastuzumab, with ADC-Au exhibiting more potent cytotoxic activity than trastuzumab. ADC-Au induced cell death in SK-OV-3, BT-474, and Calu-3 cells in a dose-dependent manner, whereas trastuzumab exhibited less inhibitory activity against HER2-positive cells. Furthermore, ADC-Au exhibited some in vitro cytotoxicity against cells with low HER2 expression.

[0108] 6. In vivo evaluation of the antitumor activity of HER2-targeting antibody-conjugated gold (III) porphyrin drugs

[0109] Tumor-bearing mice were purchased from Shanghai Model Organisms Co., Ltd. and implanted with HER2-high-expressing BT-474 tumor cells in the right groin. When the tumor grew to approximately 50-100 mm 3 At the same time, tumor-bearing mice were randomly divided into three different treatment groups (3 mice in each group). Antibody-conjugated gold (III) porphyrin drug ADC-Au (5 mg / kg, diluted in PBS) and trastuzumab (5 mg / kg, diluted in PBS) were injected into the tail vein once every 7 days. The control group was injected with an equal amount of PBS. The tumor volume and weight of the mice were measured every 2 days. The tumor size was measured with a digital caliper and the tumor volume (mm 3 )=1 / 2×L×W 2 (L represents length, W represents width) Calculate the tumor size. On day 28, the tumor volume of mice in the blank group reached 1000 mm 3 All mice were killed according to the animal treatment guidelines, and the tumors were dissected and weighed.

[0110] The inhibition rate of tumor growth was calculated using the following formula:

[0111] Tumor growth inhibition rate (%) = (1-average tumor weight of the treatment group / average tumor weight of the control group) × 100%.

[0112] All measurements are expressed as mean ± standard deviation; statistical analysis was performed using one-way analysis of variance and least significant difference (LSD) test. All data were analyzed by Graphpad Prism8. Figure 15 As shown, compared to the blank control group, the 5 mg / kg ADC-Au treatment group achieved a tumor inhibition rate of 73.99%, superior to the trastuzumab group (54.31%). Furthermore, nude mice in the ADC-Au treatment group showed stable growth and no weight loss during treatment. This demonstrates that ADC-Au has excellent in vivo anti-tumor activity.

Claims

1. An A3B-type gold (III) porphyrin complex, characterized in that: The structural formula is as follows:

2. The method for synthesizing A3B-type gold (III) porphyrin according to claim 1, characterized in that: The following steps are involved: (1) Preparation of A3B-type porphyrin ring; (2) adding the A3B type porphyrin ring to a glacial acetic acid solution of potassium tetrachloroaurate and sodium acetate to cause a metallation reaction of the porphyrin ring, introducing a gold metal center into the porphyrin ring, and obtaining a gold porphyrin ring intermediate; (3) mixing the gold porphyrin ring intermediate obtained in step (2) with alkali solution and dissolving it in a mixed solution of MeOH and H2O to hydrolyze it to form a carboxyl site; (4) adding the product obtained in step (3) to DMF, then adding HATU, and then adding N-aminoethylpiperazine under N2, and mixing and stirring for 24-48 hours; The structural formula of the A3B type porphyrin ring is as follows 3. The synthesis method according to claim 2, characterized in that In step (2), the molar ratio of the A3B type porphyrin ring, potassium tetrachloroaurate and sodium acetate is 1:2-3:10-15.

4. An antibody-coupled gold (III) porphyrin drug targeting HER2, characterized in that: The antibody is formed by connecting the A3B-type gold (III) porphyrin complex according to claim 1 via a linker.

5. The HER2-targeting antibody-conjugated gold (III) porphyrin drug according to claim 4, characterized in that: The antibody is trastuzumab; the linker comprises a maleimide linker and a self-eliminating linker connected to the A3B type gold (III) porphyrin complex.

6. The HER2-targeting antibody-conjugated gold (III) porphyrin drug according to claim 4, characterized in that: The linker is maleimide-amide-polyethylene glycol-valine-citrulline-amino carbonate.

7. The method for synthesizing the HER2-targeting antibody-coupled gold (III) porphyrin drug according to claim 4, characterized in that: The following steps are involved: (S1) connecting the A3B-type gold (III) porphyrin complex with a linker to obtain Linker-Au; (S2) dissolving Linker-Au in a solvent; (S3) dissolving tris(2-carbonylethyl)phosphine hydrochloride in PBS buffer, adding the solution dropwise to the PBS solution containing trastuzumab, and stirring at room temperature for 2-4 hours to disrupt the interchain disulfide bonds; (S4) adding the product obtained in step (S3) to the solution obtained in step (S2), and stirring at room temperature for 1-5 hours; (S5) diluting the mixture with PBS buffer and then purifying; (S6) The sample was concentrated again and washed with PBS buffer.

8. The synthesis method according to claim 7, characterized in that The connection method in step (S1) is as follows: dissolving the linker in DMF, stirring at 0°C and adding N,N-diisopropylethylamine, then dropwise adding A3B type gold (III) porphyrin, reacting for 6-10 hours, removing DMF under reduced pressure, and purifying.

9. The synthesis method according to claim 7, characterized in that The molar ratio of PBS solution containing trastuzumab to Linker-Au was 1:

10.

10. Use of the A3B-type gold (III) porphyrin complex according to claim 1 in the preparation of anti-tumor drugs, wherein the tumors include breast cancer and ovarian cancer.

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