HER2 (human epidermal growth factor receptor 2) targeting antibody coupling gold (III) porphyrin medicine as well as synthesis and tumor treatment application of HER2 targeting antibody coupling gold (III) porphyrin medicine

Abstract

The invention relates to an HER2 (human epidermal growth factor receptor 2) targeting antibody coupling gold (III) porphyrin medicine as well as a synthesis method and tumor treatment application thereof. The antibody coupling gold (III) porphyrin drug targeting HER2 is formed by connecting an anti-HER-2 trastuzumab and an A3B type gold (III) porphyrin complex through a linker, and the linker comprises a maleimide joint connected with an antibody and a self-elimination joint connected with the A3B type gold (III) porphyrin complex. The A3B type gold (III) porphyrin complex with anti-tumor activity is combined with trastuzumab with a specific recognition function as a carrier, the antibody is utilized to accurately strike tumor cells, a medicine is delivered to a tumor environment, the tumor targeting property of the gold (III) porphyrin complex is improved, and accurate treatment is realized. Distribution of drugs in the body can be improved, accumulation of the drugs in non-tumor sites can be reduced, the dosage of metal complexes is reduced, and toxic and side effects are reduced; the efficient delivery of gold drugs to tumor tissues can also be realized, and the cancer treatment effect is improved.

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 for the extension of human life span. According to the International Agency for Research on Cancer (IARC) in 2022, there are nearly 20 million new cases of cancer and 9.7 million deaths worldwide. In view of this, the implementation of cancer control strategies and interventions is particularly urgent. Effective prevention and treatment methods can not only significantly improve the quality of life of patients, but also are essential to protecting people's health and well-being. At present, chemotherapy is still the main means of cancer treatment and plays an important role in the treatment of tumors. Among them, metal-based drugs occupy an important position in chemotherapeutic drugs. Gold (III) porphyrin compounds have a dominant position in the field of anticancer drug development due to their significant anti-proliferative properties, but their biocompatibility is poor and off-target toxicity is large. How to exert a stronger and more lasting anti-tumor effect is an urgent problem to be solved.

[0003] Antibody-Drug Conjugates (ADCs) couple monoclonal antibodies to small molecule cytotoxic drugs through 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. ADCs recognize and bind to antigens and then are internalized into cells. After being degraded by lysosomes, the payload is released in a biologically active form to kill 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 few types and single mechanism of action. At present, there is relatively little research on metal-based complexes as cytotoxin molecules for ADCs, and most of the research focuses on platinum drugs that have been used in clinical treatment, such as cisplatin and oxaliplatin. Although these platinum drugs show certain efficacy in cancer treatment, they are often accompanied by many side effects and drug resistance problems. Therefore, finding and developing 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, and 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, improve its distribution in the body, and 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 a physiologically stable A3B-type gold (III) porphyrin complex, which exhibits high-efficiency cytotoxicity at the micromolar to nanomolar level against a variety of tumor cell lines in vitro and effectively inhibits tumor growth in a number of cancer animal models.

[0010] The synthesis method of the above-mentioned A3B-type gold (III) porphyrin complex comprises: preparing an A3B-type porphyrin ring, introducing a gold metal center into the porphyrin ring, and finally forming an 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, thereby introducing a gold metal center into the porphyrin ring to obtain a gold porphyrin ring intermediate;

[0014] (3) The gold porphyrin ring intermediate obtained in step (2) is mixed with an alkali solution (the alkali solution can be potassium carbonate, potassium hydroxide, sodium hydroxide, sodium carbonate, etc., potassium carbonate is the best) and dissolved in MeOH and H 2 O mixed solution, hydrolyzed to form carboxyl sites;

[0015] (4) Add the product obtained in step (3) to DMF, then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and then 2 N-aminoethylpiperazine was added 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 an anti-HER2 monoclonal antibody, trastuzumab; the linker is a maleimide linker connected to the antibody and a self-eliminating linker connected to the A3B type gold (III) porphyrin complex; the dipeptide is valine-citrulline; and each antibody conjugate carries about 4 drug molecules.

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

[0020] The linker is used to achieve the connection between the antibody and the gold (III) porphyrin complex, and the gold (III) porphyrin complex is transformed into a linker-gold (III) porphyrin complex form, and then the antibody is coupled to the linker-gold (III) porphyrin complex by a chemical method of reducing the amide bond between the antibody chains. The linker-gold (III) porphyrin complex has a high reactivity with the thiol 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 conjugated 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 sites, so as to improve the tumor targeting and biodistribution characteristics of the gold (III) porphyrin complex and reduce toxic side effects; at the same time, it can effectively kill tumor cells and achieve the purpose of high-efficiency and low-toxicity treatment.

[0022] The present invention has previously conducted experiments to explore the possibility of combining gold (III) porphyrin complexes with trastuzumab. The experiments found that A3B-type gold (III) porphyrin complexes have efficient anti-tumor properties, can significantly increase the level of reactive oxygen in ovarian cancer cells SK-OV-3, lead to mitochondrial dysfunction and impaired cell energy metabolism, and ultimately induce tumor cell death by activating the apoptosis pathway. The present invention utilizes the respective advantages of gold (III) porphyrin complexes and trastuzumab, and uses the antibody-drug conjugate synthesized by gold (III) porphyrin complexes and trastuzumab to enhance the inhibitory effect of gold (III) porphyrin complexes on tumor cells, and improve the targeting of gold (III) porphyrin complexes, reduce the amount of metal complexes used, and further reduce the toxicity of gold (III) porphyrin complexes to surrounding healthy cells, and direct the cytotoxic drug to 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 tri(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 destroy 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 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 dropping 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 use of the above-mentioned A3B type gold (III) porphyrin complex in the preparation of anti-tumor drugs, wherein 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 antiproliferative 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 sites 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 poor stability and low drug-antibody ratio of current conjugates, thereby obtaining an antibody-drug conjugate 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 that is time-consuming and energy-saving, 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 may improve 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 its subsequent clinical application transformation. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0040] Figure 3 It is the connection between the linker and the A3B-type gold (III) porphyrin complex, i.e., the preparation route diagram of Linker-Au;

[0041] Figure 4 is 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 by the present invention;

[0044] Figure 7 It is a synthetic route diagram of the antibody-coupled gold (III) porphyrin drug targeting HER2, i.e., ADC-Au of the present invention;

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

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

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

[0048] Fig.11 This is an analysis diagram of the affinity of the antibody-coupled gold (III) porphyrin drug of the present invention to HER2 protein;

[0049] Fig.12 It is a diagram showing the binding of the antibody-coupled gold (III) porphyrin drug of the present invention to different tumor cell lines;

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

[0051] Fig.14 The in vitro antitumor activity of the antibody-coupled gold (III) porphyrin drug of the present invention;

[0052] Fig.15The present invention is an in vivo evaluation of the anti-tumor activity of the antibody-coupled gold (III) porphyrin drug, wherein (a) is a curve diagram of tumor volume changes during treatment, (b) is a curve diagram of mouse weight changes during treatment, (c) is the weight and inhibition rate of the tumor after the treatment, and (d) is a tumor anatomical 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 further described in 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, 4-formylbenzoic acid methyl ester, 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 protection for 30 minutes.

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

[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 hydrogen nuclear magnetic resonance spectrum: 1 H NMR (600MHz, Chloroform-d) δ8.90-8.73(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).

[0062] 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.

[0063] Intermediate MCTPP mass spectrum HR-ESI-MS: m / z Calcd.for C 46 H 32 N 4 O 2 ([M+H] + ):673.2559,found:673.2593.

[0064] Example 2

[0065] 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 metalation reaction of the porphyrin ring is realized, and the gold metal center is successfully introduced into the porphyrin ring to prepare the gold porphyrin intermediate, which mainly includes the following steps:

[0066] Weigh the intermediate MCTPP (0.1mmol, 1.0eq.), potassium tetrachloroaurate (0.2mmol, 2.0eq.) and sodium acetate (1mmol, 10.0eq.) and dissolve them in glacial acetic acid (5mL) and heat under reflux for 3 hours. After the reaction is completed, extract with dichloromethane and distilled water to remove excess acetic acid. Collect the organic phase, dry it with anhydrous sodium sulfate, concentrate it, purify the product by neutral alumina column chromatography, and elute with dichloromethane.

[0067] The purified product was dissolved in dichloromethane, and excess lithium chloride was added and stirred overnight. Filtered and the solvent was removed to obtain a red solid (intermediate Au-1). The yield was 70%. After the porphyrin ring (MCTPP) was introduced into the metal center, the δ-2.77ppm pyrrole N hydrogen peak in the hydrogen spectrum disappeared, and the m / z in the mass spectrum also increased.

[0068] The obtained intermediate Au-1 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).

[0069] Intermediate Au-1 carbon NMR spectrum: 13 C 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.

[0070] Mass spectrum of intermediate Au-1: HR-ESI-MS: m / z Calcd.for C 48 H 30 AHr 4 O 2 ([M-Cl] + ):867.2029,found:867.2018.

[0071] Example 3

[0072] 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:

[0073] The intermediate Au-1 (0.1 mmol, 1.0 eq.) was reacted with K 2 CO 3 (0.5 mmol, 5.0 eq.) was dissolved in MeOH (2 mL) and H 2 O (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.

[0074] The reaction mixture was acidified with 1M HCl solution, extracted with dichloromethane (50 mL x 3), the organic phases were combined, and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate under reduced pressure to obtain a red solid (intermediate Au-2). Yield: 98%.

[0075] The obtained intermediate Au-2 has hydrogen NMR 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).

[0076] Intermediate Au-2 carbon NMR spectrum: 13 C 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.

[0077] Intermediate Au-2 mass spectrum: HR-ESI-MS: m / z Calcd.for C 45 H 28 AHr 4 O 2 ([M-Cl] + ):853.1878,found:853.1860.

[0078] Example 4

[0079] 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:

[0080] To a solution of intermediate Au-3 (0.05 mmol, 1.0 eq.) in ultra-dry DMF (3 mL) was added HATU (0.15 mmol, 3.0 eq.) at room temperature. 2 N-aminoethylpiperazine (0.5 mmol, 10.0 eq) was added under reduced pressure. The reaction mixture was stirred for 24 hours. Then, the solution was concentrated under reduced pressure. It was then purified by neutral alumina column chromatography (DCM: MeOH = 95: 5) to obtain a red solid (complex Au-3). Yield: 62%.

[0081] 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).

[0082] 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..

[0083] Mass spectrum of Au-3 complex: HR-ESI-MS: m / z Calcd.for C 51 H 41 AHr 7 O([M-Cl] + ):964.3033,found:964.3020.

[0084] Example 5

[0085] 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:

[0086] (1) Preparation of the linker-Au by connecting the complex Au-3 to 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:

[0087] Weigh Linker (0.06 mmol, 1.2 eq.), dissolve in ultra-dry N,N-dimethylformamide (2 ml), stir at 0°C, add N,N-diisopropylethylamine (0.3 mmol, 6.0 eq.), and slowly drop Au-3 (0.05 mmol, 1.0 eq., dissolved in ultra-dry N,N-dimethylformamide). When TLC (CH 2 Cl 2 / CH 3 After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by reverse phase liquid preparation (C18, 10% acetonitrile / water to 100% acetonitrile, v / v) to obtain a red powder. The yield was 48%.

[0088] (2) Preparation of antibody-conjugated gold (III) porphyrin drugs. Figure 7 As shown, TCEP (0.22 μmol, 3.0 eq.) was dissolved in PBS buffer, added dropwise to a PBS solution containing trastuzumab (0.07 μmol, 1.0 eq.), and stirred at room temperature for 2 hours to destroy the interchain disulfide bonds; then Linker-Au (0.7 μmol, 10.0 eq.) dissolved in DMF was added and stirred at room temperature for 1 hour;

[0089] After the reaction was complete, the mixture was diluted with 5 mL of PBS buffer and then lysed using Zeba TM The sample was purified using a desalting centrifugal column (Thermo, 40K MWCO, 2 mL) to remove uncoupled Linker-Au. The sample was then concentrated using an ultrafiltration centrifugal 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.

[0090] Linker-Au H NMR spectrum Figure 4 As shown: 1 H NMR (500MHz, Methanol-d 4 )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).

[0091] Linker-Au carbon NMR spectrum Figure 5 As shown: 13 C NMR (126MHz, Methanol-d 4)δ173.00,172.40,171.55,170.79,170.66,168.06,160.85,155.37,141.80,138.69,138.10,137.11,137.04,137.01,136. 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.

[0092] Linker-Au mass spectrometry Figure 6 Shown: HR-ESI-MS: m / z Calcd.for C 88 H 94 AHr 14 O 14 ([M-Cl] + ):1767.6734,found:1767.6716.

[0093] Performance Testing

[0094] 1. SDS-PAGE and UV spectrophotometry analysis of HER2-targeted antibody-conjugated gold (III) porphyrin drug

[0095] The successful coupling of the gold (III) porphyrin complex to the antibody was demonstrated by UV spectrophotometry, gel electrophoresis, and bicinchoninic acid method. Figure 8 , Fig. 9 As shown. The conjugate has an antibody characteristic peak of 280 nanometers and a complex characteristic peak of 528 nanometers. By observing and comparing the positions of the light and heavy chain bands of the antibody conjugate and trastuzumab under white light, it was observed that the migration positions of the light chain and heavy chain bands of the conjugate were both shifted upward, which is consistent with the increase in molecular weight and the slowing of migration speed after coupling. This shows that the gold (III) porphyrin complex was successfully coupled to the light chain and heavy chain of trastuzumab.

[0096] 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 Fig. 9 As shown, the concentration of the complex in the conjugate is calculated, and combined with the antibody concentration determined by the BCA method, the DAR value of the antibody conjugate can be calculated to be approximately 4.

[0097] 2. Stability of HER2-targeted antibody-conjugated gold (III) porphyrin drugs

[0098] 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. Fig.10 The absorption peak of ADC-Au showed no significant change within 48h or 72h. The absorption peaks of the electron transfer between the metal and the ligand of the gold (III) porphyrin complex Au-3 were around 410nm and 528nm. Fig.10 (a)) and in PBS solution containing 10% FBS for 72 hours ( Fig.10 In (b), the contents 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.

[0099] 3. Analysis of the affinity of HER2-targeted antibody-conjugated gold (III) porphyrin drug to HER2 protein

[0100] First, we need to understand the affinity between ADCs and target proteins, and compare the affinity between ADCs and naked monoclonal antibodies, which is crucial for their targeting and efficacy in vivo, to ensure that the drugs can accurately target tumor cells and exert the best therapeutic effect. Enzyme-linked immunosorbent assay (ELISA) can be used to evaluate the affinity of both for antigens. Fig.11 As shown, the EC of ADC-Au affinity for HER2 protein 50 (half effective concentration) is 0.0203nM, which is comparable to trastuzumab (EC 50 =0.0147 nM), these experimental results show that in terms of affinity, the binding ability of the antibody part of the conjugated drug to the HER2 protein is not significantly affected.

[0101] 4. Binding of HER2-targeted antibody-conjugated gold (III) porphyrin drugs to different tumor cell lines

[0102] Flow cytometry was used to evaluate the targeting binding characteristics of antibody-conjugated gold (III) porphyrin drugs and trastuzumab in HER2 high-expressing and HER2 low-expressing cells. Fig.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 almost no binding in HER2-low-expressing cells MDA-MB-231, NCI-H460, and A2780. This indicates that the antibody part of the conjugate drug retains a high degree of antigen recognition specificity, can specifically bind to the HER2 antigen on the cell surface, and selectively binds to the surface of HER2-high-expressing cells, but not to HER2-low-expressing cells.

[0103] 4. Internalization of HER2-targeted antibody-conjugated gold (III) porphyrin drugs

[0104] In addition, at 37°C, 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 was high ( Fig.13 ). This suggests that the conjugate drug has an advantage over naked monoclonal antibodies in terms of endocytosis efficiency, especially in SK-OV-3 cells. These findings are of great significance for a deeper understanding of the intracellular transport and therapeutic effects of antibody-drug conjugates.

[0105] 5. In vitro antitumor activity of HER2-targeted antibody-conjugated gold (III) porphyrin drugs

[0106] The in vitro antiproliferative activity of the antibody conjugates against different cell lines was detected 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. Fig.14 ). After continuous culture for 72 h, the cytotoxicity was evaluated by MTT assay. The gold (III) porphyrin complex significantly enhanced the cytotoxicity after binding to trastuzumab, and ADC-Au showed more effective cytotoxic activity than trastuzumab. ADC-Au induced cell death in SK-OV-3, BT-474, and Calu-3 cells in a dose-related manner, while trastuzumab had a lower inhibitory effect on HER2-positive cells. In addition, ADC-Au also showed certain in vitro cytotoxicity against HER2-low-expressing cells.

[0107] 6. Evaluation of the in vivo antitumor activity of HER2-targeted antibody-conjugated gold (III) porphyrin drugs

[0108] Tumor-bearing mice purchased from Shanghai Model Organisms Co., Ltd. were implanted with HER2-high-expressing BT-474 tumor cells in the right groin. When the tumor size grew to about 50-100 mm 3 At the same time, the tumor-bearing mice were randomly divided into 3 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, and 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 1000mm 3 All mice were killed according to the animal treatment guidelines, and the tumors were dissected and weighed.

[0109] The inhibition rate of tumor growth was calculated using the following formula:

[0110] Tumor growth inhibition rate (%) = (1-average tumor weight of the drug administration group / average tumor weight of the control group) × 100%.

[0111] All measurements were expressed as mean ± standard deviation; statistical analysis was performed using one-way ANOVA and least significant difference (LSD) test. All data were analyzed by Graphpad prism8. Fig.15 As shown in the figure, compared with the blank control group, the tumor inhibition rate of the 5 mg / kg ADC-Au treatment group reached 73.99%, which was better than the trastuzumab group (54.31%). The nude mice in the ADC-Au treatment group grew steadily during the treatment period, and their weight did not decrease. This shows that ADC-Au has good anti-tumor activity in vivo.

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) preparing 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, thereby introducing a gold metal center into the porphyrin ring to obtain a gold porphyrin ring intermediate; (3) mixing the gold porphyrin ring intermediate obtained in step (2) with an alkaline solution and dissolving it in a mixed solution of MeOH and H2O to hydrolyze it to form a carboxyl site; (4) Add the product obtained in step (3) to DMF, then add HATU, and then add N-aminoethylpiperazine under N2, and mix and stir for 24-48 hours.

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 and the A3B-type gold (III) porphyrin complex according to claim 1 are connected 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-aminocarbonate.

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 tri(2-carbonylethyl)phosphine hydrochloride in PBS buffer, adding dropwise to the PBS solution containing trastuzumab, and stirring at room temperature for 2-4 hours to destroy 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 dropping 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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