Triple negative breast cancer targeting compound and preparation method and application thereof

Abstract

The present application relates to the technical field of breast cancer targeting compounds, in particular to a triple negative breast cancer targeting compound and a preparation method and application thereof, comprising the following steps: preparation of arsine precursor; synthesis of triple negative breast cancer targeting compound; the present application discloses a triple negative breast cancer targeting compound and a preparation method and application thereof, a plurality of organic arsine compounds are prepared through innovative organic synthesis, and a series of high-efficiency low-toxicity targeted anti-tumor organic arsine compounds are obtained through screening and exploration at the levels of calculation simulation, macromolecules, cells and small animals, the single-component organic arsine of the series has higher thioredoxin reductase inhibition capacity and a longer in-vivo circulation half-life, under the action of lower dosing and lower dosing frequency, the targeted inhibition of tumors is realized, the inhibition effect on triple negative breast cancer is very remarkable, and the series has a certain degree of application prospect.

Description

Technical Field

[0001] This invention relates to the field of breast cancer targeting compound technology, specifically to a triple-negative breast cancer targeting compound, its preparation method, and its application. Background Technology

[0002] Based on the successful case of China's first original targeted drug, arsenic trioxide, used for acute promyelocytic leukemia, two organic arsenic compounds (GSAO and ZIO101) were developed internationally and approved by the U.S. Food and Drug Administration for cancer treatment. Subsequently, the development of organic arsenic compounds has mainly focused on expanding the indications of the above three arsenic agents, lacking original innovative drug research and development.

[0003] In addition, the three arsenic preparations already on the market still have the following drawbacks during treatment: low activity, rapid metabolism, and the need for higher doses and higher frequency of administration to achieve a more ideal therapeutic effect.

[0004] Breast cancer is the most common cancer among women worldwide and the second leading cause of cancer death, with triple-negative breast cancer accounting for approximately 20%. Triple-negative breast cancer (TNBC) is a highly malignant tumor, and currently there are no targeted therapies for TNBC. Clinical treatment for TNBC primarily involves chemotherapy, with commonly used drugs including doxorubicin (DOX) and paclitaxel. These chemotherapeutic drugs exhibit non-targeted distribution in TNBC treatment, which can cause severe side effects. Therefore, the development of novel targeted therapies for TNBC is an urgent problem to be solved.

[0005] Novel organic arsine compounds were synthesized by introducing microsomal enzyme metabolism inhibitory groups into organic arsine precursors. This series of single-component organic arsine compounds exhibits higher antitumor activity and longer in vivo circulation time, improving antitumor efficiency while reducing dosage and avoiding toxic side effects. This opens up new therapeutic possibilities for targeting triple-negative breast cancer, a disease with high incidence and mortality rates. Furthermore, by targeting thioredoxin reductase, a biomarker of triple-negative breast cancer, highly effective and low-toxicity targeted drugs can be prepared, offering new possibilities for solving the treatment challenges of this type of breast cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a targeted compound for triple-negative breast cancer, its preparation method, and its application. The invention discloses a targeted compound for triple-negative breast cancer and its preparation method. Through innovative organic synthesis, various organic arsine compounds are prepared. Through computational simulation, macromolecular, cellular, and small animal screening and exploration, a series of highly efficient and low-toxicity targeted anti-tumor organic arsine compounds are obtained. This series of single-component organic arsine compounds exhibits higher thioredoxin reductase inhibitory activity and a longer in vivo circulating half-life. It achieves targeted tumor inhibition at lower dosages and lower dosing frequencies, demonstrating a significant inhibitory effect on triple-negative breast cancer, which is of practical significance.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A triple-negative breast cancer targeting compound, wherein the molecular structural formula of the triple-negative breast cancer targeting compound is as follows:

[0009]

[0010] Wherein, R1 is one or more of alkyl, olefin, imino, alkylimino, -CO-NH-, -CO-O-, R3-CO-NH-, R3-CO-O-, aryl, heterocyclic substituent, alkoxy, and alkylamino;

[0011] R2 is one or more of a halogen atom, a nitro group, a hydroxyl group, or a hydrogen atom;

[0012] R3 is one or more of alkyl, olefin or aryl groups.

[0013] Preferably, the alkyl, olefin, alkoxy, heterocyclic substituent, alkylamino, and aminoalkyl groups have 1-12 carbon atoms.

[0014] Preferably, the aryl group is one or more selected from phenyl, alkylphenyl, nitrophenyl, hydroxyphenyl, halophenyl, naphthyl, anthracene, or biphenyl;

[0015] The heterocyclic substituent is one or more selected from cycloazoalkyl, cyclooxyethylene, cyclothioalkyl, acridine, pyrrolidine, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, furanyl, thiophenyl, piperidinyl, pyridinyl, morpholinyl, piperazine, azepine, oxazepine, or thiazolyl;

[0016] The halogen atom is one or more of fluorine, chlorine, bromine, and iodine.

[0017] A method for preparing a triple-negative breast cancer targeting compound, the method being as follows:

[0018] Preparation of arsine precursor: 8-12 mL of 70% ammonium thioglycolate was heated to 50 °C, and 3.5-4.5 g of benzoarsine 1a-1d was added. After 1 h, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant pressure dropping funnel. After reacting for 2-4 h, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursor 2a-2d.

[0019] Synthesis of the triple-negative breast cancer targeting compound: The triple-negative breast cancer targeting compound was prepared by mixing and reacting the arsine precursors 2a-2d prepared in S1 with the starting compound.

[0020] Preferably, when the raw material compound is piperic acid 3a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows:

[0021] S101. Dissolve 4.5-5.5 mM piperic acid 3a in 20 mL of dichloromethane, add 7-8 mM oxaloyl chloride and 30-50 μL of N,N-dimethylformamide, stir at room temperature for 40-50 min, and then dry the mixture to obtain a pale yellow solid piperic acid chloride 3b.

[0022] S102. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM pipericyl chloride 3b was dissolved in 8-15 mL of dichloromethane, 3-4 mM arsine precursors 2a-2d were added, and then pyridine was added as an acid-binding agent according to the acyl chloride:pyridine molar ratio of 1:1-1:2. The product was purified by silica gel column chromatography to obtain products 3c-3f.

[0023] The preparation route and structure of the triple-negative breast cancer targeting compound 3c-3f are as follows:

[0024]

[0025] Among them, in triple-negative breast cancer targeting compound 3c, X = 4-NH and R = H; in triple-negative breast cancer targeting compound 3d, X = 4-O and R = H; in triple-negative breast cancer targeting compound 3e, X = 4-O and R = 3-NO2; and in triple-negative breast cancer targeting compound 3f, X = 2-NH and R = H.

[0026] Preferably, when the raw material compound is piperine 4a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows:

[0027] S201. Dissolve 5 mg of piperine 4a in 15-25 mL of DCM, add 0.5%-5% of the reactant MnO2 and react for 8-12 h, then filter and dry to obtain piperine 4b;

[0028] S202. Dissolve 5-6 mM arsine precursors 2a-2d and 6 mM piperine 4b in 15-25 mL of anhydrous ethanol. Heat the solution under reflux at 75-85 °C and stir for 8-12 h. After cooling, crystals are produced to obtain products 4c-4d.

[0029] The preparation route and structure of the triple-negative breast cancer targeting compound 4c-4d are as follows:

[0030]

[0031] In the triple-negative breast cancer targeting compound 4c, -NH= is 4-NH=; in the triple-negative breast cancer targeting compound 4d, -NH= is 2-NH=.

[0032] Preferably, when the raw material compound is 5a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows:

[0033] S301. Dissolve 5 mM 5a in 15-25 mL of dichloromethane, add 7.5 mM oxalyl chloride and 0.5%-5% molar amount of N,N-dimethylformamide, stir at room temperature for 40 min, and then dry the mixture to obtain a pale yellow solid 5b;

[0034] S302. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM of 5b was dissolved in 8-12 mL of dichloromethane, 3-4 mM of arsine precursor 2a-2d was added, and then pyridine was added as an acid-binding agent according to the molar ratio of acyl chloride to pyridine of 1:1-1:2. The product was purified by silica gel column chromatography to obtain product 5c-5f.

[0035] The preparation route and structure of the triple-negative breast cancer targeting compound 5c-5f are as follows:

[0036]

[0037] Among them, in triple-negative breast cancer targeting compound 5c, X = 4-NH and R = H; in triple-negative breast cancer targeting compound 5d, X = 4-O and R = H; in triple-negative breast cancer targeting compound 5e, X = 4-O and R = 3-NO2; and in triple-negative breast cancer targeting compound 5f, X = 2-NH and R = H.

[0038] Preferably, when the raw material compound is 6a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows:

[0039] S401. Dissolve 30-35g KOH in 120-180mL of anhydrous ethanol, heat to 65-75℃, add 17.5mmol of piperine in portions, heat the solution under reflux at 75-85℃, stir for 8-12h, and filter to obtain brownish-yellow solid 6a.

[0040] S402. Dissolve the solid in 200-300 mL of water, adjust the pH to <1, cool until a yellow solid precipitates, filter to obtain a yellow solid, and recrystallize to obtain intermediate product 6b;

[0041] S403. Dissolve 5 mM 6b in 15-25 mL of dichloromethane, add 7.5 mM oxalyl chloride and 0.5%-5% molar amount of N,N-dimethylformamide, stir at room temperature for 40 min, and then dry the mixture to obtain a pale yellow solid 6c;

[0042] S404. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM of 6c was dissolved in 8-12 mL of dichloromethane, 3-4 mM of arsine precursor 2a-2d was added, and then pyridine was added as an acid-binding agent according to the molar ratio of acyl chloride to pyridine of 1:1-1:2. The product 6d-6e was obtained by silica gel column chromatography for separation and purification.

[0043] The preparation route and structure of the triple-negative breast cancer targeting compound 6d-6e are as follows:

[0044]

[0045] In the triple-negative breast cancer targeting compound 6d, -NH- is 4-NH-; in the triple-negative breast cancer targeting compound 6e, -NH- is 2-NH-.

[0046] Preferably, in the arsenic precursors 2a-2d, arsenic precursor 2a has a yield of 60% and the R group is 4-NH2; arsenic precursor 2b has a yield of 52% and the R group is 4-OH; arsenic precursor 2c has a yield of 45% and the R group is 3-NO2,4-OH; and arsenic precursor 2d has a yield of 41% and the R group is 2-NH2.

[0047] The application of a triple-negative breast cancer targeting compound prepared according to the aforementioned triple-negative breast cancer targeting compound or the preparation method of the aforementioned triple-negative breast cancer targeting compound in the course of treatment for triple-negative breast cancer.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] 1. The method for preparing triple-negative breast cancer targeted compounds provided by the present invention involves introducing microsomal enzyme metabolism inhibitory groups into organic arsine precursors to synthesize novel organic arsine compounds. This series of single-component organic arsine compounds have higher antitumor activity and longer in vivo circulation time, thereby improving antitumor efficiency and reducing the dosage and avoiding toxic side effects.

[0050] 2. The preparation method of the targeted compound for triple-negative breast cancer provided by this invention addresses the predicament of the lack of marketable targeted drugs for triple-negative breast cancer, which has a high incidence and high mortality rate. It targets thioredoxin reductase, a biomarker of triple-negative breast cancer, to prepare a highly effective and low-toxicity targeted drug. Compared with auronoxine and arsenic trioxide, which are already on the market and target the same disease, this series of organic arsine compounds has a more significant inhibitory effect on triple-negative breast cancer under conditions of lower dosage and lower dosing frequency, and is expected to achieve a breakthrough in targeted drugs for triple-negative breast cancer. Attached Figure Description

[0051] Figure 1 This is a multi-process flow diagram of the preparation method of the triple-negative breast cancer targeting compound of the present invention;

[0052] Figure 2 This is a fluorescence signal detection diagram of the triple-negative breast cancer targeting compound of the present invention;

[0053] Figure 3 This is a Western blot image of the triple-negative breast cancer targeting compound of the present invention;

[0054] Figure 4 This is an electron micrograph of the triple-negative breast cancer targeting compound of the present invention;

[0055] Figure 5 This is a dual-channel fluorescence signal detection diagram of the triple-negative breast cancer targeting compound of the present invention;

[0056] Figure 6 The following are ICP-MS quantitative detection curves of the triple-negative breast cancer targeting compounds of the present invention: Figure A shows the activity curves of CYP1A2 or CYP3A4 after incubation with different concentrations of recognized inhibitors; Figure B shows the activity curves of CYP1A2 after incubation with different concentrations of 2d, 5f, or 6e; Figure C shows the activity curves of CYP3A4 after incubation with different concentrations of 2d, 5f, or 6e; EC50 is the half-maximal inhibitory concentration.

[0057] Figure 7 The following are pharmacokinetic experimental results for the triple-negative breast cancer targeting compound of the present invention: Figure A is a flowchart of the pharmacokinetic experiment; Figure B is a graph showing the change of arsenic concentration in blood over time; Figure C is a graph showing the change of arsenic concentration in urine over time; Figure D is a graph showing the change of arsenic concentration in feces over time.

[0058] Figure 8The following diagrams illustrate the antitumor experimental detection of the triple-negative breast cancer targeting compound of the present invention: Figure A is a flowchart of the antitumor experimental operation; Figure B is a curve of tumor volume change over time; Figure C is a tumor anatomy diagram; Figure D is a comparison of tumor weight in each group; Figure E is a diagram of arsenic content in each group of tumors; Figure F is a diagram of ROS staining of tumor sections, in Figure F (1) solvent group / negative control group, (2) 3 mg / kg 2d, (3) 5 mg / kg 2d, (4) 0.5 mg / kg 5f, (5) 1 mg / kg 5f, (6) 1 mg / kg 6e;

[0059] Figure 9 The following are biometric data of the mice used in this invention: Figure A is a graph showing the change in mouse body weight over time; Figure B is a graph showing the organ coefficients of each group of mice, where liver and spleen are the liver and spleen, respectively; Figure C is a graph showing the liver and kidney function indicators of mice, where UA, CREA, UN, ALP, AST, and ALT are the urea, CREA, UN, ALP, AST, and ALT are the alanine aminotransferase, respectively; Figure D is a graph showing the detection of the content of thioredoxin reductase (TrxR) in mice, where Tumor, liver, spleen, actin, (1) solvent group / negative control group, (2) 3 mg / kg 2d, (3) 5 mg / kg 2d, (4) 0.5 mg / kg 5f, (5) 1 mg / kg 5f, (6) 1 mg / kg 6e; Figure E is a HE-stained section of tumor cells.

[0060] Figure 10 The following are the blood routine data of the experimental mice of this invention: Figure A is the white blood cell (WBC) data; Figure B is the neutrophil (NEUT) data; Figure C is the lymphocyte (LYMPH) data; Figure D is the monocyte (MON) data; In Figures A and D: (1) solvent group or negative control group, (3) 5mg / kg 2d, (5) 1mg / kg 5f, (6) 1mg / kg 6e, N normal mouse group. Detailed Implementation

[0061] The present invention will now be clearly and completely described in conjunction with embodiments thereof. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0062] Please see Figure 1-10 The present invention provides a technical solution:

[0063] A compound targeting triple-negative breast cancer, the molecular structural formula of which is:

[0064]

[0065] Wherein, R1 is one or more of alkyl, olefin, imino, alkylimino, -CO-NH-, -CO-O-, R3-CO-NH-, R3-CO-O-, aryl, heterocyclic substituent, alkoxy, and alkylamino;

[0066] R2 is one or more of a halogen atom, a nitro group, a hydroxyl group, or a hydrogen atom;

[0067] R3 is one or more of alkyl, olefin or aryl groups.

[0068] Alkyl, olefin, alkoxy, heterocyclic substituent, alkylamino, and aminoalkyl groups have 1-12 carbon atoms.

[0069] The aryl group is one or more of the following: phenyl, alkylphenyl, nitrophenyl, hydroxyphenyl, halophenyl, naphthyl, anthracene, or biphenyl.

[0070] The heterocyclic substituent is one or more of cycloazoalkyl, cyclooxyethylene, cyclothioalkyl, acridine, pyrrolidine, pyrrolidinyl, pyrrolinyl, imidazolyl, pyrazolyl, furanyl, thiophene, piperidinyl, pyridinyl, morpholinyl, piperazine, azepine, oxazepine, or thiazolyl; the halogen atom is one or more of fluorine, chlorine, bromine, and iodine.

[0071] In this invention, the molecular structural formulas of triple-negative breast cancer targeting compounds are specifically disclosed, but not limited to, the molecular structural formulas of the following compounds, according to the following specific embodiments:

[0072]

[0073] Regarding the preparation method of the above-mentioned triple-negative breast cancer targeting compound, the present invention further discloses the following specific embodiments.

[0074] Example 1

[0075] A method for preparing a synthetic triple-negative breast cancer targeting compound includes the following steps:

[0076] 10 mL (13 g, 120 mM) of 70% ammonium thioglycolate was heated to 50 °C, and 4 g of benzoarsine was added in four portions. After about 1 hour, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant-pressure dropping funnel. After 2 hours of reaction, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursors 2a-2d.

[0077] 5 mM piperic acid was dissolved in 20 mL of dichloromethane, and 7.5 mM oxaloyl chloride and 50 μL of N,N-dimethylformamide were added. The mixture was stirred at room temperature for 40 min. The mixture was then dried to obtain a pale yellow solid pipericyl chloride. Under nitrogen protection in an ice bath at 1 °C, 4.5 mM pipericyl chloride was dissolved in 10 mL of dichloromethane, and 3 mM arsine precursor was added. Pyridine was then added as an acid-binding agent at a molar ratio of acyl chloride to pyridine of 1:1 to 1:2. The product was purified by silica gel column chromatography to obtain product 3C-3F.

[0078] The molecular structure of product 3c is as follows:

[0079] The molecular structure of product 3d is as follows:

[0080] The molecular structure of product 3e is:

[0081] The molecular structure of product 3f is as follows:

[0082] Example 2

[0083] A method for preparing a synthetic triple-negative breast cancer targeting compound includes the following steps:

[0084] 10 mL (13 g, 120 mM) of 70% ammonium thioglycolate was heated to 50 °C, and 4 g of benzoarsine was added in four portions. After about 1 hour, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant-pressure dropping funnel. After 2 hours of reaction, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursors 2a-2d.

[0085] 5 mg of piperine was dissolved in 20 mL of DCM, and 0.5% molar amount of MnO2 was added to react the mixture for 8 h. The mixture was filtered and dried to obtain piperine. 5 mM of the arsine precursor and 6 mM of piperine were dissolved in 20 mL of anhydrous ethanol. The solution was heated to reflux at 75 °C and stirred for 8 h. After cooling, crystals formed, yielding product 4c-4d.

[0086] The molecular structure of product 4c is as follows:

[0087] The molecular structure of product 4d is as follows:

[0088] Example 3

[0089] A method for preparing a synthetic triple-negative breast cancer targeting compound includes the following steps:

[0090] 10 mL (13 g, 120 mM) of 70% ammonium thioglycolate was heated to 50 °C, and 4 g of benzoarsine was added in four portions. After about 1 hour, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant-pressure dropping funnel. After 2 hours of reaction, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursors 2a-2d.

[0091] 5 mM 5a was dissolved in 20 mL of dichloromethane, and 7.5 mM oxalyl chloride and 4% molar amount of N,N-dimethylformamide were added. The mixture was stirred at room temperature for 40 min. The mixture was then dried to obtain a pale yellow solid 5b. Under a nitrogen atmosphere in an ice-water bath at 5 °C, 4.5 mM 5b was dissolved in 8 mL of dichloromethane, and 3 mM arsine precursor was added. Pyridine was then added as an acid-binding agent at a acyl chloride:pyridine molar ratio of 1:1 to 1:2. The mixture was purified by silica gel column chromatography to obtain products 5c-5f.

[0092] The molecular structure of product 5c is as follows:

[0093] The molecular structure of product 5d is as follows:

[0094] The molecular structure of product 5e is:

[0095] The molecular structure of product 5f is as follows:

[0096] Example 4

[0097] A method for preparing a synthetic triple-negative breast cancer targeting compound includes the following steps:

[0098] 10 mL (13 g, 120 mM) of 70% ammonium thioglycolate was heated to 50 °C, and 4 g of benzoarsine was added in four portions. After about 1 hour, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant-pressure dropping funnel. After 2 hours of reaction, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursors 2a-2d.

[0099] Dissolve 30 g KOH in 150 mL of anhydrous ethanol, heat to 70 °C, add 17.5 mmol piperine in portions, and reflux at 80 °C for 10 h. Filter to obtain a brownish-yellow solid. Dissolve the solid in 250 mL of water, adjust the pH to <1, cool until a yellow solid precipitates, filter to obtain a yellow solid, and recrystallize to obtain intermediate 6b.

[0100] 5 mM 6b was dissolved in 15-25 mL of dichloromethane, and 7.5 mM oxalyl chloride and 3% molar amount of N,N-dimethylformamide were added. The mixture was stirred at room temperature for 40 min, and then dried to obtain a pale yellow solid 6c. In a nitrogen atmosphere at 2 °C, 4.5 mM 6c was dissolved in 9 mL of dichloromethane, and 3 mM arsine precursor was added. Pyridine was then added as an acid-binding agent at a molar ratio of acyl chloride to pyridine of 1:1-1:2. The mixture was purified by silica gel column chromatography to obtain products 6d-6e.

[0101] The molecular structure of product 6d is as follows:

[0102] The molecular structure of product 6e is as follows:

[0103] Comparative example:

[0104] Comparative Example 1

[0105] 10 mL (13 g, 120 mM) of 70% ammonium thioglycolate was heated to 50 °C, and 4 g of benzoarsine was added in four portions. After about 1 hour, 2 mL of 1,3-propanedithiol was slowly added dropwise through a constant-pressure dropping funnel. After 2 hours of reaction, the resulting suspension was extracted with dichloromethane in small batches, and the organic phases were combined and concentrated by rotary evaporation. The product was purified by silica gel column chromatography and collected to obtain arsine precursors 2a-2d.

[0106] Cellular experiments were conducted on Examples 1-4 and Comparative Example 1, and relevant tests were performed:

[0107] 1. MTT assay for the antiproliferative capacity of organic arsine

[0108] Experimental protocol: 4T1 and COS-7 cells were seeded into 96-well plates and cultured overnight to recover growth. Different concentrations of organic arsine were added, and the cells were cultured for 24 or 48 hours. After incubation with MTT, the cells were lysed, and the absorbance at 490 nm was measured to calculate the IC50. 50 The values ​​and calculation results are shown in Table 1:

[0109]

[0110]

[0111] Table 1

[0112] Data Analysis:

[0113] Among all organic arsines, organic arsine 6e exhibited the lowest half-maximal inhibitory concentration (WMC) against triple-negative breast cancer 4T1 cell proliferation, a relatively high WMC against normal cell COS-7 proliferation, and the highest IC50 between normal and cancer cells. 50The ratio (last two columns in the table) indicates that organic arsine 6e has high antitumor activity, low ability to interfere with normal cell proliferation, and high selectivity between cancer cells and normal cells.

[0114] 2. The targeted inhibitory ability of organoarsine on the target TrxR

[0115] Experimental procedure: The structure of the organoarsine was optimized using Gaussian or ORCA; molecular docking of the organoarsine with TrxR was performed using ledock, and the binding energy ΔG was calculated.

[0116] 4T1 cells were seeded in 10cm culture dishes and cultured overnight. After incubation for 24 hours with 0.3μM organic arsine, the cells were collected, homogenized and lysed, and centrifuged (8000g, 4℃, 10min) to remove insoluble matter. The activity of TrxR in the supernatant was detected using a kit.

[0117] NADPH-reduced TrxR recombinase (80 nM) was incubated with different concentrations of organic arsine in 96-well plates for 10 min. TE buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 50 μL) of DTNB (2 mM) and NADPH (200 μM) was added. The rate of change in absorbance at 412 nm was detected using a microplate reader, and the half-maximal inhibitory concentration (HMC) was calculated. The results are shown in Table 2.

[0118] Table 2. Interactions between organic arsine and TrxR

[0119]

[0120] Data Analysis:

[0121] Molecular docking experiments showed that the series of organic arsines had a higher binding capacity to TrxR than arsine precursors 2a-2d. The organic arsine 6e exhibited the largest absolute value of the Gibbs free energy change ΔG when binding to TrxR, indicating that organic arsine 6e possessed the strongest TrxR binding capacity. In vitro TrxR holoenzyme activity assays revealed that organic arsine 6e had the lowest half-maximal inhibitory concentration (WMC) for TrxR, indicating that organic arsine 6e had the highest inhibitory capacity for TrxR. When all organic arsines were used at the same concentration, the organic arsine 6e group showed the lowest TrxR activity, indicating that at the cellular level, organic arsine 6e had the strongest inhibitory capacity for TrxR activity.

[0122] 3. Oxidative stress and endoplasmic reticulum stress induce apoptosis.

[0123] Experimental protocol: 4T1 cells were seeded into 6-well plates and cultured overnight. After incubation with organic arsine for 24 hours, reactive oxygen species (ROS) detection reagent DCFH-DA was added and incubated for half an hour. Cells were then collected, and fluorescence signals were detected by flow cytometry. Results are shown below. Figure 2 As shown.

[0124] 4T1 cells were seeded into 10cm dishes and cultured overnight. After incubation with organic arsine for 24 hours, cells were collected, lysed with RIPA strong lysis buffer, and analyzed by Western blot. Results are shown below. Figure 3 As shown.

[0125] 4T1 cells were seeded into 6-well plates and cultured overnight. After incubation with organic arsine for 24 hours, cells were collected, fixed, sectioned, and examined under a transmission electron microscope. Results are shown below. Figure 4 As shown.

[0126] 4T1 cells were seeded into 6-well plates and cultured overnight. After 24 hours of co-incubation with organic arsine, apoptosis detection reagent was added, and cells were collected. Dual-channel fluorescence signals were detected by flow cytometry. Results are shown below. Figure 5 As shown.

[0127] Data Analysis:

[0128] like Figure 2 As shown, flow cytometry analysis results indicated that both organic arsine 5f and 6e could induce an increase in reactive oxygen species (ROS) levels in 4T1 cells. At the same dosage (0.3 or 0.45 μM), the ROS level in the 6e group was higher than that in the 5f group.

[0129] like Figure 3 As shown, Western blot results indicate that organic arsine 6e induces increased cellular ubiquitination levels (left figure), causing endoplasmic reticulum stress, and upregulating the expression of related proteins such as GRP78, ATF4, and CHOP (right figure). This suggests that organic arsine 6e induces cellular oxidative stress and endoplasmic reticulum stress by inhibiting TrxR activity, and further induces apoptosis.

[0130] like Figure 4 As shown, electron microscopy results after cell sectioning revealed that incubation with organic arsine 6e disrupted the mitochondrial structure of the cells, resulting in phenomena such as reduced contents and damage to the inner membrane.

[0131] like Figure 5 As shown, organic arsine 6e induces cellular oxidative stress and endoplasmic reticulum stress, disrupting the structure and function of organelles such as mitochondria, and further inducing apoptosis. Compared with the control group (C), only 30.8% of the cells incubated with 6e remained normal, with the rest undergoing apoptosis. The apoptosis inhibitor Z-VAD-fmk (Z) had no apoptosis-inducing function and significantly inhibited the apoptosis-inducing ability of 6e, restoring the proportion of remaining normal cells to 84.1%.

[0132] 4.6e enhances antitumor activity by inhibiting the activity of microsomal enzymes, thus prolonging its half-life in vivo.

[0133] Experimental protocol: CYP1A2 and CYP3A4 were co-incubated with recognized inhibitors (1A2-naphthoflavone, 3A4-ketoconazole) or organoarsine, and the substrates phenacetin or midazolam were added. The content of the reaction product acetaminophen or 1-hydroxymidazolam was detected, and the half-maximal inhibitory concentration (IC50) of the organoarsine for CYP1A2 and CYP3A4 was calculated.

[0134] Adult healthy male SD rats were selected and injected with 3 mg / kg organic arsine via the tail vein. At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 14 h, 19 h, 24 h, 28 h, 36 h, 44 h, and 48 h post-administration, 60 μL of blood, urine, and feces were collected from the orbital cavity. All samples were hydrolyzed with concentrated nitric acid and then quantitatively analyzed by ICP-MS. The results are shown below. Figure 6 , Figure 7 As shown.

[0135] Data Analysis:

[0136] like Figure 6 As shown, the half-maximal inhibitory concentration (C50) of naphthol, a recognized inhibitor of microsomal CYP1A2, for CYP1A2 is... 50 The half-maximal inhibitory concentration (WMC) of ketoconazole for CYP3A4 was 0.00815 μM, and the WMC for CYP3A4 was 0.0242 μM. Compared to naphthaleneflavonoids, organoarsine precursors 2d, 5f, or 6e showed weaker inhibitory activity against CYP1A2 and higher WMCs. Compared to ketoconazole, organoarsine precursor 2d showed very weak inhibitory activity against CYP3A4, while organoarsine 5f or 6e showed moderate inhibitory activity.

[0137] like Figure 7 As shown, compared to the arsine precursor 2d, organic arsine 5f and 6e have significantly longer half-lives in the blood and are excreted relatively slowly through urine or feces.

[0138] Consistent with the experiments on microsomal enzymes mentioned above, the benzo[a]cephalotaxene structure in organic arsenic inhibits the activity of microsomal enzyme 3A4, slows down its metabolism in vivo, prolongs its half-life in vivo, and enhances the antitumor activity of organic arsenic.

[0139] 5. Organic arsine inhibits tumor growth and restores vital signs in mice by targeting and accumulating in tumors.

[0140] Experimental protocol: Female BALB / c mice around 4 weeks old were divided into two groups: a normal mouse group (normal control group) and a tumor-bearing mouse group. The tumor-bearing mouse group included solvent group (1), 3 mg / kg 2d (2), 5 mg / kg 2d (3), 0.5 mg / kg 5f (4), 1 mg / kg 5f (5), and 1 mg / kg 6e (6), with 4 mice in each group. 106 4T1 cells in 0.1 mL of whole DMEM medium were subcutaneously injected into the hind leg of the mice. After 7-10 days, the tumors grew to an average size of 100-150 mm. 3 (Tumor volume = 1 / 2 × length × width) 2 Based on the mouse's body weight, the total injection volume was controlled at 100 μL, and organic arsine was injected via the tail vein every 4 days. On day 14, blood samples were collected from the tail vein for routine blood tests. Mice were euthanized by cervical dislocation, and their organs were dissected for subsequent testing. The test results are as follows: Figure 8 , Figure 9 , Figure 10 As shown.

[0141] Data Analysis:

[0142] like Figure 8 As shown, the tumors in tumor-bearing mice gradually grew, with the tumors in the treatment group growing more slowly (Figure B above). The tumors in the group treated with organic arsine 6e gradually shrank, eventually reaching approximately 10% of the size of the control group (C and D). Arsenic content detection in the tumors revealed the highest arsenic accumulation in the 6e-treated group. 6e induces oxidative stress (F) in tumor cells, leading to tumor cell death and slowing tumor growth by targeting and accumulating in the tumor.

[0143] like Figure 9 As shown, the weight of mice did not change significantly during the treatment (Figure A above). Only the weight of mice in the untreated control group (1) showed a slight decreasing trend, possibly due to the large size of the tumor severely affecting the health of the mice. Organ coefficient data (B) showed that organic arsine alleviated hepatosplenomegaly in tumor-bearing mice by targeting and inhibiting tumor growth, and maintained the activity of liver and kidney-related enzymes (C). Moreover, organic arsine only significantly inhibited the target TrxR in the tumor, and had no significant inhibition on TrxR in the liver or spleen (D). HE staining also showed that organic arsine could significantly alleviate the damage of tumor to the liver of mice and restore the mice's physical condition to a normal state (E).

[0144] like Figure 10As shown, compared with normal mice (N in the figure above), tumor-bearing mice (1) showed a significant increase in white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), and monocytes (MON). Organic arsine can significantly reduce the number of the above cells in the blood, with organic arsine 6e showing the best performance, restoring the mouse blood routine to a level that is not significantly different from that of normal mice (ns in the figure: no significant difference).

[0145] Conclusion: Organic arsine 6e has highly efficient and low-toxicity antitumor activity.

[0146] The antitumor effects of the arsenic precursor 2d and organic arsenic 6e prepared in this invention were compared with those of inorganic arsenic trioxide and auronoxine, which are already used clinically. The relevant results are shown in Table 3.

[0147] Table 3. Comparison of anti-tumor effects

[0148] Dosage (mg / kg) 5 1 5 1 Frequency (per day) 4 4 4 4 Tumor inhibition rate 20％ 30％ 50％ 90％

[0149] Data Analysis:

[0150] Compared with clinically used inorganic arsenic trioxide, under the same dosage and frequency conditions, the organic arsenic prodrug 2d showed superior antitumor efficacy. Compared with the clinically used TrxR-targeting inhibitor aurinophene, under the same dosage and frequency conditions, the organic arsenic 6e showed significantly better antitumor efficacy. Compared to the arsenic prodrug, the organic arsenic 6e exhibited superior antitumor efficacy at lower dosages.

[0151] The above comparative experiments on anti-tumor effects demonstrate that organic arsine 6e has great potential for clinical application.

[0152] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A triple negative breast cancer targeting compound, characterized in that, The molecular structural formula of the triple-negative breast cancer targeting compound is as follows: ； Wherein, R1 is one or more of alkyl, olefin, imino, alkylimino, -CO-NH-, -CO-O-, R3-CO-NH-, R3-CO-O-, aryl, heterocyclic substituent, alkoxy, and alkylamino; R2 is one or more of a halogen atom, a nitro group, a hydroxyl group, or a hydrogen atom; R3 is one or more of alkyl, olefin or aryl groups; The alkyl, olefin, alkoxy, heterocyclic substituent, alkylamino, and aminoalkyl groups have 1-12 carbon atoms. The aryl group is one or more selected from phenyl, alkylphenyl, nitrophenyl, hydroxyphenyl, halophenyl, naphthyl, anthracene, or biphenyl. The heterocyclic substituent is one or more selected from cycloazoalkyl, cyclooxyethylene, cyclothioalkyl, acridine, pyrrolidine, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, furanyl, thiophenyl, piperidinyl, pyridinyl, morpholinyl, piperazine, azepine, oxazepine, or thiazolyl; The halogen atom is one or more of fluorine, chlorine, bromine, and iodine.

2. A method for preparing a triple negative breast cancer targeting compound according to claim 1, characterized in that, The preparation method includes: Preparation of arsine precursor: After heating 8-12 mL of 70% ammonium thioglycolate to 50 °C, add 3.5-4.5 g of benzoarsine 1a-1d. After 1 h, slowly add 2 mL of 1,3-propanedithiol dropwise through a constant pressure dropping funnel. After reacting for 2-4 h, extract the suspension with dichloromethane in small amounts several times, combine the organic phases, and concentrate by rotary evaporation. Separate and purify with silica gel column chromatography, collect the product, and obtain arsine precursor 2a-2d. Synthesis of the triple-negative breast cancer targeting compound: The triple-negative breast cancer targeting compound was prepared by reacting the arsine precursors 2a-2d with the starting compound.

3. The method for preparing a triple-negative breast cancer targeting compound according to claim 2, characterized in that, When the raw material compound is piperic acid 3a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows: S101. Dissolve 4.5-5.5 mM piperic acid 3a in 20 mL of dichloromethane, add 7-8 mM oxaloyl chloride and 30-50 μL of N,N-dimethylformamide, stir at room temperature for 40-50 min, and then dry the mixture to obtain a pale yellow solid piperic acid chloride 3b; S102. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM pipericyl chloride 3b was dissolved in 8-15 mL of dichloromethane, 3-4 mM arsine precursors 2a-2d were added, and then pyridine was added as an acid-binding agent at a molar ratio of acyl chloride to pyridine of 1:1-1:

2. The product was purified by silica gel column chromatography to obtain products 3c-3f. The preparation route and structure of the triple-negative breast cancer targeting compound 3c-3f are as follows: ； Among them, in triple-negative breast cancer targeting compound 3c, X=4-NH and R=H; in triple-negative breast cancer targeting compound 3d, X=4-O and R=H; in triple-negative breast cancer targeting compound 3e, X=4-O and R=3-NO2; and in triple-negative breast cancer targeting compound 3f, X=2-NH and R=H.

4. The method of claim 2, wherein the compound is represented by the following formula: ###0002### 4 When the raw material compound is piperine 4a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows: S201. Dissolve 5 mg of piperine 4a in 15-25 mL of DCM, add 0.5%-5% of the reactant MnO2 and react for 8-12 h, then filter and dry to obtain piperine 4b; S202. Dissolve 5-6 mM arsine precursors 2a-2d and 6 mM piperine 4b in 15-25 mL of anhydrous ethanol. Heat the solution under reflux at 75-85 °C and stir for 8-12 h. After cooling, crystals are produced to obtain products 4c-4d. The preparation route and structure of the triple-negative breast cancer targeting compound 4c-4d are as follows: ； In the triple-negative breast cancer targeting compound 4c, -NH= is 4-NH=; in the triple-negative breast cancer targeting compound 4d, -NH= is 2-NH=.

5. The method for preparing a triple-negative breast cancer targeting compound according to claim 2, characterized in that, When the raw material compound is 5a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows: S301. Dissolve 5 mM 5a in 15-25 mL of dichloromethane, add 7.5 mM oxalyl chloride and 0.5%-5% molar amount of N,N-dimethylformamide, stir at room temperature for 40 min, and then dry the mixture to obtain a pale yellow solid 5b; S302. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM of 5b was dissolved in 8-12 mL of dichloromethane, 3-4 mM of arsine precursor 2a-2d was added, and then pyridine was added as an acid-binding agent according to the molar ratio of acyl chloride to pyridine of 1:1-1:

2. The product 5c-5f was obtained by silica gel column chromatography for separation and purification. The preparation route and structure of the triple-negative breast cancer targeting compound 5c-5f are as follows: ； Among them, in triple-negative breast cancer targeting compound 5c, X=4-NH and R=H; in triple-negative breast cancer targeting compound 5d, X=4-O and R=H; in triple-negative breast cancer targeting compound 5e, X=4-O and R=3-NO2; and in triple-negative breast cancer targeting compound 5f, X=2-NH and R=H.

6. The method for preparing a triple-negative breast cancer targeting compound according to claim 2, characterized in that, When the raw material compound is 6a, the synthesis steps of the triple-negative breast cancer targeting compound are as follows: S401. Dissolve 30-35g KOH in 120-180mL of anhydrous ethanol, heat to 65-75℃, add 17.5mmol of piperine in portions, heat the solution under reflux at 75-85℃, stir for 8-12h, and filter to obtain brownish-yellow solid 6a. S402. Dissolve the solid in 200-300 mL of water, adjust the pH to <1, cool until a yellow solid precipitates, filter to obtain a yellow solid, and recrystallize to obtain intermediate product 6b; S403. Dissolve 5 mM 6b in 15-25 mL of dichloromethane, add 7.5 mM oxalyl chloride and 0.5%-5% molar amount of N,N-dimethylformamide, stir at room temperature for 40 min, and then dry the mixture to obtain a pale yellow solid 6c; S404. In an ice-water bath at 1-5℃ under a nitrogen atmosphere, 4.5 mM of 6c was dissolved in 8-12 mL of dichloromethane, 3-4 mM of arsine precursor 2a-2d was added, and then pyridine was added as an acid-binding agent at a molar ratio of acyl chloride to pyridine. The product 6d-6e was obtained by silica gel column chromatography for separation and purification. The preparation route and structure of the triple-negative breast cancer targeting compound 6d-6e are as follows: ； In the triple-negative breast cancer targeting compound 6d, -NH- is 4-NH-; in the triple-negative breast cancer targeting compound 6e, -NH- is 2-NH-.

7. A method for preparing a synthetic triple-negative breast cancer targeting compound according to any one of claims 2-6, characterized in that, Of the arsenic precursors 2a-2d, arsenic precursor 2a has a yield of 60% and an R group of 4-NH2; arsenic precursor 2b has a yield of 52% and an R group of 4-OH; arsenic precursor 2c has a yield of 45% and an R group of 3-NO2,4-OH; and arsenic precursor 2d has a yield of 41% and an R group of 2-NH2.

8. The use of a triple-negative breast cancer targeting compound prepared by the method of preparation of the triple-negative breast cancer targeting compound according to claim 1 or any one of claims 2-7 in the preparation of a medicament for treating triple-negative breast cancer.

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