Iron complex, preparation method and application thereof

Abstract

The application belongs to the technical field of antitumor drugs, and discloses an iron complex as well as a preparation method and application thereof.The preparation steps of the iron complex are as follows: 1, reacting 2-hydrazinylbenzothiazole, a ketone compound and a solvent to obtain a ligand; and 2, reacting the ligand, ferric chloride and a solvent, and then cooling and crystallizing to obtain the iron complex.The obtained iron complex can effectively induce cancer cell ferroptosis, can effectively inhibit the growth of breast cancer, lung cancer, liver cancer or glioma, has high selectivity, has small toxicity to normal cell strains, and has better treatment effect compared with existing clinical drugs, such as cisplatin.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and in particular to an iron complex, its preparation method and application. Background Technology

[0002] With the continuous rise in cancer incidence, research on anti-tumor drugs has become a hot topic in the global medical and pharmaceutical fields. Malignant tumors are characterized by high invasiveness, high recurrence rate, and high mortality rate, seriously threatening patients' lives and health. Although traditional treatments such as surgery, radiotherapy, and chemotherapy can control tumor growth to some extent, their efficacy is limited and they are highly toxic to normal tissues, resulting in low survival rates and quality of life for patients.

[0003] Ferroptosis, as a novel form of cell death, has attracted widespread attention due to its unique molecular mechanism and potential anti-tumor effects. Ferroptosis is a cell death process induced by iron-catalyzed lipid peroxidation, significantly different from traditional cell death mechanisms such as apoptosis, necrosis, and autophagy. Studies have shown that inducing ferroptosis in cancer cells can specifically inhibit the growth and proliferation of tumor cells without damaging normal cells, thus providing a new approach to cancer treatment. However, currently, there are relatively few drugs available clinically for inducing ferroptosis in cancer cells, and these drugs have some limitations. For example, some drugs have poor water solubility, resulting in low bioavailability; others, while inducing ferroptosis in cancer cells, also exhibit significant toxicity to normal cells, limiting their clinical application.

[0004] Therefore, the discovery of a novel, highly efficient iron complex with low toxicity to normal cells, its preparation method, and its application in the preparation of antitumor drugs are of great significance. Summary of the Invention

[0005] In view of this, the present invention provides an iron complex, its preparation method and application, the purpose of which is to solve the problem that existing drugs also produce significant toxicity to normal cells when inducing cancer cell death.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides an iron complex, wherein the iron complex has the structural formula C1 or C2:

[0008]

[0009] The present invention also provides a method for preparing an iron complex, comprising the following steps:

[0010] 1) The ligand is obtained by reacting 2-hydrazinobenzothiazole, ketone compounds and solvent;

[0011] 2) After reacting the ligand, ferric chloride and solvent, the mixture is cooled and crystallized to obtain the iron complex.

[0012] Preferably, in step 1), the ketone compound is 2-benzoylpyridine or 6,7-dihydro-5H-quinoline-8-one.

[0013] Preferably, in step 1), the ratio of 2-hydrazinobenzothiazole, ketone compound and solvent is 8-12 mmol: 8-12 mmol: 25-35 mL.

[0014] Preferably, in step 1), the reaction temperature is 55–65°C and the reaction time is 4–6 hours.

[0015] Preferably, in step 2), the ratio of ligand, ferric chloride and solvent is 0.2-0.4 mmol: 0.2-0.4 mmol: 15-25 mL.

[0016] Preferably, in step 2), the reaction temperature is 30–50°C and the reaction time is 1–3 hours.

[0017] Preferably, in step 2), the cooling crystallization temperature is 0–6°C, and the cooling crystallization time is 5–8 days.

[0018] Preferably, the solvent used in steps 1) and 2) is anhydrous ethanol.

[0019] This invention also provides the application of iron complexes in the preparation of antitumor drugs.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0021] The iron complex described in this invention can effectively induce ferroptosis in cancer cells, effectively inhibit the growth of breast cancer, lung cancer, liver cancer, or glioma, exhibits high selectivity, and has low toxicity to normal cell lines; compared with the existing clinical drug cisplatin, it has superior therapeutic effects.

[0022] The method for preparing the iron complex described in this invention has mild reaction conditions, is simple to operate, has low cost, and is suitable for large-scale industrial production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The crystal structure of the iron complex C1 obtained in Example 1 is shown, wherein the large white spheres are carbon elements and the small white spheres are hydrogen elements;

[0025] Figure 2 The crystal structure of the iron complex C2 obtained in Example 2 is shown, wherein the large white spheres are carbon elements and the small white spheres are hydrogen elements;

[0026] Figure 3 The images show the results of Western blotting using different reagents.

[0027] Figure 4 This is a graph showing cell viability for different reagents. Detailed Implementation

[0028] This invention provides an iron complex, wherein the iron complex has the structural formula C1 or C2:

[0029]

[0030] The present invention also provides a method for preparing an iron complex, comprising the following steps:

[0031] 1) The ligand is obtained by reacting 2-hydrazinobenzothiazole, ketone compounds and solvent;

[0032] 2) After reacting the ligand, ferric chloride and solvent, the mixture is cooled and crystallized to obtain the iron complex.

[0033] In this invention, in step 1), the ketone compound is preferably 2-benzoylpyridine or 6,7-dihydro-5H-quinoline-8-one.

[0034] In this invention, in step 1), the preferred ratio of 2-hydrazinobenzothiazole, ketone compound and solvent is 8-12 mmol:8-12 mmol:25-35 mL, more preferably 9-11 mmol:9-11 mmol:26-33 mL, and even more preferably 10 mmol:10 mmol:28-30 mL.

[0035] In this invention, in step 1), the reaction temperature is preferably 55-65°C, more preferably 57-62°C, and even more preferably 58-60°C, and the reaction time is preferably 4-6 hours, more preferably 4.5-5.5 hours, and even more preferably 5 hours.

[0036] In this invention, in step 1), the reaction is preferably a reflux stirring reaction, and the stirring speed is preferably 80-120 rpm, more preferably 90-110 rpm, and even more preferably 100 rpm;

[0037] After the reaction is completed, solid-liquid separation, washing and drying are performed sequentially. The solid-liquid separation is preferably vacuum evaporation. The pressure of vacuum evaporation is preferably 15-25 mmHg, more preferably 18-23 mmHg, and more preferably 20-22 mmHg. The temperature of vacuum evaporation is preferably 50-100℃, more preferably 60-90℃, and more preferably 70-80℃. The purpose of vacuum evaporation is to remove excess solvent and obtain a light yellow powder.

[0038] The washing reagent is preferably anhydrous ethanol, and the number of washings is preferably 2 to 6 times, more preferably 3 to 5 times, and even more preferably 4 times.

[0039] The drying time is preferably 2 to 6 hours, more preferably 3 to 5 hours, and even more preferably 4 hours. The drying temperature is preferably 60 to 70°C, more preferably 62 to 68°C, and even more preferably 64 to 65°C.

[0040] In this invention, in step 2), the preferred ratio of the amount of ligand, ferric chloride and solvent is 0.2-0.4 mmol: 0.2-0.4 mmol: 15-25 mL, more preferably 0.25-0.35 mmol: 0.25-0.35 mmol: 17-23 mL, and even more preferably 0.3 mmol: 0.3 mmol: 18-20 mL.

[0041] In this invention, in step 2), the reaction temperature is preferably 30-50°C, more preferably 35-45°C, and even more preferably 38-40°C, and the reaction time is preferably 1-3 hours, more preferably 1.5-2.5 hours, and even more preferably 2 hours.

[0042] In this invention, in step 2), the reaction is preferably a reflux reaction.

[0043] In this invention, in step 2), the cooling crystallization temperature is preferably 0-6°C, more preferably 1-5°C, and even more preferably 3-4°C, and the cooling crystallization time is preferably 5-8 days, and even more preferably 6-7 days.

[0044] In this invention, the solvent used in steps 1) and 2) is preferably anhydrous ethanol.

[0045] This invention also provides the application of iron complexes in the preparation of antitumor drugs.

[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] In this invention, RIPA lysis buffer was purchased from Beyotime Biotechnology Co., Ltd.; 5× Loading buffer was purchased from Beyotime Biotechnology Co., Ltd.; primary antibody GPX4 was purchased from Wuhan Sanying Biotechnology Co., Ltd.; SLC7A11 was purchased from Wuhan Sanying Biotechnology Co., Ltd.; secondary antibody GAPDH was purchased from Wuhan Sanying Biotechnology Co., Ltd.; and MDA-MB-231, A549, HepG2, U87MG and HL-7702 cell lines were all purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0048] Example 1

[0049] Preparation of iron complex C1:

[0050] 10 mmol of 2-hydrazinobenzothiazole, 10 mmol of 2-benzoylpyridine and 30 mL of anhydrous ethanol were mixed and refluxed at 60 °C for 5 h at 100 rpm. After the reaction was completed, the mixture was evaporated under reduced pressure (20 mmHg) at 80 °C to remove excess solvent, and a pale yellow powder was obtained. The obtained pale yellow powder was washed three times with anhydrous ethanol and dried at 60 °C for 5 h to obtain the ligand with a yield of 90.3%.

[0051] 0.3 mmol of ligand, 0.3 mmol of ferric chloride and 20 mL of anhydrous ethanol were refluxed at 40 °C for 2 h. After the reaction was completed, the resulting solution was filtered and the filtrate was placed in an environment of 4 °C to volatilize and crystallize for 7 days to obtain dark green crystals, which are the iron complex C1, with a yield of 87%.

[0052] Elemental analysis shows that the molecular formula of the iron complex C1 is C 38 H 26 Cl4Fe2N8S2, theoretical values: C: 50.03; H: 2.87; N: 12.28; S: 7.03. Measured values: C: 50.05; H: 2.86; N: 12.27; S: 7.04.

[0053] Example 2

[0054] Preparation of iron complex C2:

[0055] 10 mmol of 2-hydrazinobenzothiazole, 10 mmol of 6,7-dihydro-5H-quinoline-8-one and 30 mL of anhydrous ethanol were mixed and stirred under reflux at 60 °C for 5 h at 100 rpm. After the reaction was completed, the mixture was evaporated under reduced pressure (20 mmHg) at 70 °C to remove excess solvent, and a pale yellow powder was obtained. The obtained pale yellow powder was washed three times with anhydrous ethanol and dried at 70 °C for 4 h to obtain the ligand with a yield of 92.8%.

[0056] 0.3 mmol of ligand, 0.3 mmol of ferric chloride and 20 mL of anhydrous ethanol were refluxed at 50 °C for 2 h. After the reaction was completed, the resulting solution was filtered and the filtrate was placed in an environment of 4 °C to volatilize and crystallize for 7 days to obtain dark green crystals, which are the iron complex C2, with a yield of 87%.

[0057] Elemental analysis shows that the molecular formula of the iron complex C2 is C 32 H 26 Cl4Fe2N8S2, theoretical values: C: 45.74; H: 3.12; N: 13.34; S: 7.63. Measured values: C: 45.78; H: 3.11; N: 13.33; S: 7.62.

[0058] Comparative Example 1

[0059] Cisplatin, purchased from Beijing Innocare Technology Co., Ltd., model number A82733.

[0060] The iron complex C1 obtained in Example 1 and the iron complex C2 obtained in Example 2 were subjected to crystal diffraction to obtain diffraction data, and the results are shown in Table 1. The crystal structures of the iron complexes C1 and C2 obtained in Example 1 were analyzed using OLEX 2 software. The crystal structure of the iron complex C1 obtained in Example 1 is shown in Table 1. Figure 1 As shown, the crystal structure of the iron complex C2 obtained in Example 2 is as follows. Figure 2 As shown.

[0061] Table 1. Crystal diffraction data of iron complex C1 and iron complex C2

[0062]

[0063]

[0064] Cancer cell inhibition performance test: The iron complexes obtained in Examples 1 and 2 and the cisplatin described in Comparative Example 1 were subjected to tumor cell proliferation inhibition test using the thiazolyl blue method.

[0065] Cancer cell lines selected were MDA-MB-231, A549, HepG2, and U87MG, while normal cell line selected was HL-7702.

[0066] Thiazol blue method: The above cell lines were cultured to the logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells / mL. 4Cells / mL; 180 μL of the above logarithmic phase cells were taken and placed in 96-well plates, and cultured in DMEM medium at 5% CO2 and 37°C for 18 h. MDA-MB-231, A549, HepG2, U87MG and HL-7702 cells were divided into three groups, which were designated as iron complex C1 group, iron complex C2 group and cisplatin group, respectively. Each group was further divided into 5 subgroups.

[0067] 20 μL of aqueous solution of iron complex C1 was added to each of the five subgroups in iron complex C1, with concentrations of 1 μM, 5 μM, 10 μM, 20 μM, and 30 μM, respectively. 20 μL of aqueous solution of iron complex C2 was added to each of the five subgroups in iron complex C2, with concentrations of 1 μM, 5 μM, 10 μM, 20 μM, and 30 μM, respectively. 20 μL of aqueous solution of cisplatin was added to each of the five subgroups in cisplatin group, with concentrations of 5 μM, 10 μM, 20 μM, 30 μM, and 40 μM, respectively.

[0068] Continue culturing for 48 hours, then add 10 μL of thiazolyl blue aqueous solution (5 mg / mL) and incubate for 4 hours. Slowly remove all liquid and add 100 μL of dimethyl sulfoxide to dissolve.

[0069] The absorbance was measured at a wavelength of 570 nm using an ELISA reader, and the IC was calculated. 50 The values ​​are shown in Table 2.

[0070] Table 2 shows the IC50 of the iron complexes obtained in Examples 1-2 and the cisplatin in Comparative Example 1 for different cell lines. 50 value

[0071]

[0072] IC 50 The smaller the value, the better the inhibitory activity on cells. As shown in Table 2, the iron complexes obtained in Examples 1 and 2 all showed high toxicity to various tumor cell lines, and the effect was significantly better than that of cisplatin described in the comparative example. For human glioblastoma cells U87MG, which are less affected by cisplatin, iron complexes C1 and C2 still showed high toxicity, about 10 times and 13 times that of cisplatin, respectively. This indicates that iron complexes C1 and C2 have the potential to treat tumors. Furthermore, iron complexes C1 and C2 have a certain degree of selectivity and are less toxic to normal cells than cisplatin.

[0073] Ferropyrexia Study: The iron complexes prepared in Examples 1-2 of this invention were used to treat tumor cells, and the ferroptosis of these cells was studied using Western blot and thiazolyl blue assays. The cell line used was U87MG. The results are as follows: Figure 3 As shown.

[0074] Western blot method:

[0075] (1) 5 mL of U87MG cells (1×10⁻⁶) 6 The culture medium (cells / well) was seeded into three 10cm diameter culture dishes. After the cells adhered, 10μL of physiological saline (control group), 10μL of aqueous solution of iron complex C1 (concentration of 1.5mM) and 10μL of aqueous solution of iron complex C2 (concentration of 1.5mM) were added to each dish. The dishes were then cultured at 37℃ and 5% CO2 for 48h.

[0076] (2) Protein extraction: Collect cells, add RIPA lysis buffer to extract total protein, lyse on ice for 30 min, centrifuge at 12000 rpm for 15 min, and take the supernatant to determine the protein concentration by BCA method.

[0077] (3) Dilute the protein concentration of each sample to 1 mg / mL by adding water and 5× Loading buffer;

[0078] (4) Electrophoresis was performed using 10% SDS-polyacrylamide gel electrophoresis buffer. 10 μL of each protein sample was added to each well. The electrophoresis conditions were: 80 V for 30 min during the concentration phase, 120 V for 90 min during the separation phase, and PVDF membrane transfer for 120 min after electrophoresis.

[0079] (5) After adding blocking buffer (5% bovine serum albumin) and blocking at room temperature for 1 h, the PVDF bands were incubated with primary antibodies GAPDH, GPX4 and SLC7A11 at 4°C overnight, and washed three times with 1×TBST buffer for 5 min each time.

[0080] (6) Incubate with secondary antibody at room temperature for 2 hours, wash three times with 1×TBST buffer, and expose using a chemiluminescence analyzer. The results are as follows: Figure 3 As shown.

[0081] The results of Western blotting with different reagents are shown in the figure below. Figure 3 As shown. By Figure 3 As can be seen, compared with the control group, the expression levels of GPX4 (ferroptosis regulator) and SLC7A11 were significantly downregulated in the iron complex C1 and iron complex C2 groups, while the expression level of the internal reference protein GAPDH remained unchanged. This indicates that iron complexes C1 and C2 have the ability to induce ferroptosis in cancer cells.

[0082] Thiazole blue method:

[0083] The U87MG cell line was cultured to the logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells / year. 4Cells / mL; 180 μL of the above logarithmic phase cells were taken, placed in a 96-well plate, cultured in DMEM medium, and cultured for 18 h in an environment of 5% CO2 and 37°C as one group, and a total of ten groups were set up;

[0084] The first group was supplemented with physiological saline and served as a control group.

[0085] The second group was supplemented with 20 μL of cisplatin aqueous solution (concentration of 350 μM);

[0086] The third group contains 10 μL of cisplatin aqueous solution (700 μM) and 10 μL of Ferrostatin-1 aqueous solution (200 μM).

[0087] In the fourth group, 10 μL of cisplatin aqueous solution (700 μM) and 10 μL of Ferrostatin-1 aqueous solution (400 μM) were added.

[0088] In the fifth group, 20 μL of an aqueous solution of iron complex C1 (concentration of 30 μM) was added;

[0089] In the sixth group, 10 μL of an aqueous solution of iron complex C1 (concentration of 60 μM) and 10 μL of an aqueous solution of Ferrostatin-1 (concentration of 200 μM) were added.

[0090] In the seventh group, 10 μL of an aqueous solution of iron complex C1 (concentration of 60 μM) and 10 μL of an aqueous solution of Ferrostatin-1 (concentration of 400 μM) were added.

[0091] In group 8, 20 μL of an aqueous solution of iron complex C2 (concentration of 30 μM) was added;

[0092] In the ninth group, 10 μL of an aqueous solution of iron complex C2 (concentration of 60 μM) and 10 μL of an aqueous solution of Ferrostatin-1 (concentration of 200 μM) were added.

[0093] In group 10, 10 μL of an aqueous solution of iron complex C2 (concentration of 60 μM) and 10 μL of an aqueous solution of Ferrostatin-1 (concentration of 400 μM) were added.

[0094] The above ten groups were cultured for another 48 hours. After incubation for 4 hours, 10 μL of thiazolyl blue aqueous solution (5 mg / mL) was added to each group. Then, all liquid was slowly removed and 100 μL of dimethyl sulfoxide was added to each group to dissolve them.

[0095] The absorbance was measured using a microplate reader at a wavelength of 570 nm, and the cell viability was calculated. The results are as follows: Figure 4 As shown.

[0096] Cell viability graphs of different cell iron inhibitors are shown below Figure 4 As shown. By Figure 4 It is evident that the ferroptosis inhibitor Ferrostatin-1 has a low effect on the activity of cisplatin, but it can effectively reduce the activity of iron complexes C1 and C2. This indicates that iron complexes C1 and C2 can inhibit ferroptosis in cancer cells and are potential ferroptosis inducers.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An iron complex, characterized in that, The iron complex has the structural formula C1 or C2: 、 C1 C2.

2. The method for preparing an iron complex according to claim 1, characterized in that, Includes the following steps: 1) The ligand is obtained by reacting 2-hydrazinobenzothiazole, ketone compounds, and solvents; 2) The ligand, ferric chloride, and solvent were reacted and then cooled to crystallize, yielding an iron complex; In step 1), the ketone compound is 2-benzoylpyridine or 6,7-dihydro-5H-quinoline-8-one.

3. The method for preparing an iron complex according to claim 2, characterized in that, In step 1), the ratio of 2-hydrazinobenzothiazole, ketone compounds and solvent is 8~12 mmol:8~12 mmol:25~35 mL.

4. The method for preparing an iron complex according to claim 3, characterized in that, In step 1), the reaction temperature is 55~65℃ and the reaction time is 4~6h.

5. The method for preparing an iron complex according to claim 4, characterized in that, In step 2), the ratio of ligand, ferric chloride and solvent is 0.2~0.4 mmol:0.2~0.4 mmol:15~25 mL.

6. The method for preparing an iron complex according to claim 5, characterized in that, In step 2), the reaction temperature is 30~50℃ and the reaction time is 1~3h.

7. A method for preparing an iron complex according to claim 5 or 6, characterized in that, In step 2), the cooling crystallization temperature is 0~6℃, and the cooling crystallization time is 5~8 days.

8. The method for preparing an iron complex according to claim 7, characterized in that, The solvent used in steps 1) and 2) is anhydrous ethanol.

9. The use of the iron complex according to claim 1 in the preparation of antitumor drugs, characterized in that, The anti-tumor drug is a drug that inhibits the growth of breast cancer, lung cancer, liver cancer, or glioma.

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