Iron complex as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antitumor drugs, and discloses an iron complex as well as a preparation method and application thereof. The preparation method of the iron complex comprises the following steps: 1, reacting 2-hydrazinobenzothiazole, a ketone compound and a solvent to obtain a ligand; and reacting the ligand, ferric chloride and a solvent, cooling and crystallizing to obtain the iron complex. The iron complex obtained by the invention can effectively induce ferroptosis of cancer cells, can effectively inhibit growth of breast cancer, lung cancer, liver cancer or gliocytoma, has relatively high selectivity, and has relatively low toxicity to normal cell strains; and compared with the existing clinical drug cis-platinum, the treatment effect is better.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and particularly relates to an iron complex, a preparation method thereof, and an application thereof. Background Art

[0002] With the continuous increase in the incidence of cancer, the 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, seriously threatening the life and health of patients. Although traditional treatment methods such as surgery, radiotherapy, and chemotherapy can control the growth of tumors to a certain extent, their curative effects are limited, and they have relatively high toxicity to normal tissues, resulting in low survival rates and quality of life of patients.

[0003] Ferroptosis, as a new type of cell death, has received extensive attention due to its unique molecular mechanism and potential anti-tumor effects. Ferroptosis is a process of cell death caused by iron-catalyzed lipid peroxidation reactions, which is significantly different from traditional cell death methods such as apoptosis, necrosis, and autophagy. Research has shown that by inducing ferroptosis in cancer cells, the growth and proliferation of tumor cells can be specifically inhibited without damaging normal cells, thus providing a new idea for cancer treatment. However, currently, there are relatively few types of drugs used clinically to induce ferroptosis in cancer cells, and there are some limitations. For example, some drugs have poor water solubility, resulting in low bioavailability; there are also some drugs that can cause relatively high toxicity to normal cells while inducing ferroptosis in cancer cells, limiting their clinical applications.

[0004] Therefore, it is of great significance to study a new type of highly efficient iron complex with low toxicity to normal cells, its preparation method, and its application in the preparation of anti-tumor drugs. Summary of the Invention

[0005] In view of this, the present invention provides an iron complex, a preparation method thereof, and an application thereof, aiming to solve the problem that existing drugs also cause relatively high toxicity to normal cells when inducing cancer cell death.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides an iron complex, and the structural formula of the iron complex is C1 or C2:

[0008]

[0009] The present invention also provides a preparation method of an iron complex, including the following steps:

[0010] 1) React 2-hydrazinobenzothiazole, a ketone compound, and a solvent to obtain a ligand;

[0011] 2) React the ligand, iron chloride and the solvent, and then cool and crystallize to obtain the iron complex.

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

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

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

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

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

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

[0018] Preferably, the solvent in the step 1) and the step 2) is absolute ethanol.

[0019] The present invention also provides an application of the iron complex in the preparation of anti-tumor drugs.

[0020] According to the above technical solutions, compared with the prior art, the present invention has the following beneficial effects:

[0021] The iron complex of the present invention can effectively induce ferroptosis of cancer cells, can effectively inhibit the growth of breast cancer, lung cancer, liver cancer or glioblastoma, has high selectivity, and has less toxicity to normal cell lines; compared with the existing clinical drug cisplatin, the treatment effect is better.

[0022] The preparation method of the iron complex of the present invention has mild reaction conditions, simple operation, low cost, and is suitable for large-scale industrial production. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0024] Figure 1 The crystal structure of the iron complex C1 obtained in Example 1, where the large white spheres are C elements and the small white spheres are H elements;

[0025] Figure 2 The crystal structure of the iron complex C2 obtained in Example 2, where the large white spheres are C elements and the small white spheres are H elements;

[0026] Figure 3 The result diagram of Western blotting of different reagents;

[0027] Figure 4 The cell survival rate diagram of different reagents. Detailed implementation manners

[0028] The present invention provides an iron complex, and the structural formula of the iron complex is C1 or C2:

[0029]

[0030] The present invention also provides a preparation method of an iron complex, including the following steps:

[0031] 1) React 2-hydrazinobenzothiazole, a ketone compound and a solvent to obtain a ligand;

[0032] 2) React the ligand, ferric chloride and a solvent, and then cool and crystallize to obtain the iron complex.

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

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

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

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

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

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

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

[0040] In the present invention, in step 2), the dosage ratio of the ligand, iron chloride, and the solvent is preferably 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 the present invention, in step 2), the reaction temperature is preferably 30-50 °C, more preferably 35-45 °C, and even more preferably 38-40 °C. The reaction time is preferably 1-3 h, more preferably 1.5-2.5 h, and even more preferably 2 h.

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

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

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

[0045] The present invention also provides an application of an iron complex in the preparation of an anti-tumor drug.

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

[0047] In the present invention, RIPA lysis buffer was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; 5×Loading buffer was purchased from Beyotime Biotechnology Co., Ltd. in Shanghai, China; 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.; MDA-MB-231, A549, HepG2, U87MG and HL-7702 cell lines were all purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection.

[0048] Example 1

[0049] Preparation of iron complex C1:

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

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

[0052] Elemental analysis showed that the molecular formula of iron complex C1 was 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-quinolin-8-one and 30 mL of absolute ethanol were mixed and refluxed with stirring at 60 °C at a speed of 100 rpm for 5 h. After the reaction was completed, vacuum evaporation was carried out at 70 °C (pressure was 20 mmHg) to remove the excess solvent, and a pale yellow powder was obtained. The obtained pale yellow powder was washed three times with absolute 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 absolute ethanol were refluxed at 50 °C for 2 h. After the reaction, the resulting solution was filtered. The filtrate was placed in an environment at 4 °C to volatilize and crystallize for 7 d, and a dark green crystal, namely iron complex C2, was obtained with a yield of 87%.

[0057] Elemental analysis showed that the molecular formula of iron complex C2 was 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 InnoChem Co., Ltd., with the 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. The results are shown in Table 1; The OLEX 2 software was used to analyze the iron complex C1 obtained in Example 1 and the iron complex C2 obtained in Example 2 to obtain the crystal structures of the iron complexes. The crystal structure of the iron complex C1 obtained in Example 1 is as Figure 1 shown, and the crystal structure of the iron complex C2 obtained in Example 2 is as Figure 2 shown.

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

[0062]

[0063]

[0064] Detection of cancer cell inhibitory performance: The iron complexes obtained in Examples 1-2 and the cisplatin described in Comparative Example 1 were subjected to a tumor cell inhibitory proliferation test, and the test method was the thiazolyl blue method.

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

[0066] Thiazolyl blue method: The above cell lines were respectively cultured to logarithmic-phase cells with a concentration of 5×10 4cells / mL; Take 180 μL of the above-mentioned logarithmic-phase cells, and place them in 96-well plates respectively. Using DMEM medium, continue to culture them in an environment of 5% CO2 and 37 °C for 18 h. Take MDA-MB-231, A549, HepG2, U87MG, and HL-7702 cells as a group, and set three groups in total, which are respectively recorded as iron complex C1 group, iron complex C2 group, and cisplatin group. Set 5 subgroups in each group;

[0067] Add 20 μL of the aqueous solution of iron complex C1 to 5 subgroups in the iron complex C1 group, and the concentrations are 1 μM, 5 μM, 10 μM, 20 μM, and 30 μM in sequence. Add 20 μL of the aqueous solution of iron complex C2 to 5 subgroups in the iron complex C2 group, and the concentrations are 1 μM, 5 μM, 10 μM, 20 μM, and 30 μM in sequence. Add 20 μL of the aqueous solution of cisplatin to 5 subgroups in the cisplatin group, and the concentrations are 5 μM, 10 μM, 20 μM, 30 μM, and 40 μM in sequence;

[0068] Continue to culture for 48 h. After adding 10 μL of the aqueous solution of thiazolyl blue (5 mg / mL) and incubating for 4 h, slowly remove all the liquid, and then continue to add 100 μL of dimethyl sulfoxide to dissolve;

[0069] Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 570 nm, and calculate the IC 50 value, and the results are shown in Table 2.

[0070] Table 2 IC 50 values of the iron complexes obtained in Examples 1-2 and the cisplatin described in Comparative Example 1 against different cell lines

[0071]

[0072] IC 50 The smaller the IC 50 value, the better the inhibitory activity on cells. As can be seen from Table 2, the iron complexes obtained in Examples 1-2 showed high toxicity to various tumor cell lines, and the effect was significantly better than that of the cisplatin described in the comparative example; for the human brain astrocytoma cell line U87MG, which is less affected by cisplatin activity, iron complex C1 and iron complex C2 still showed high toxicity, which were about 10 times and 13 times that of cisplatin respectively. This indicates that iron complex C1 and iron complex C2 have the potential to treat tumors; and iron complex C1 and iron complex C2 have a certain selectivity, and the toxicity to normal cells is relatively small compared to cisplatin.

[0073] Study on ferroptosis of cells: After treating tumor cells with the iron complexes prepared in Examples 1-2 of the present invention, the research method for ferroptosis of tumor cells adopts Western blot method and thiazolyl blue method. The cell line selected is U87MG. The detection results are as Figure 3 shown.

[0074] Western blot method:

[0075] (1) Inoculate 5 mL of medium containing U87MG cells (1×10 6 cells / well) onto 3 culture dishes with a diameter of 10 cm respectively. After the cells adhere to the wall, add 10 μL of normal saline (control group), 10 μL of aqueous solution of iron complex C1 (concentration 1.5 mM), and 10 μL of aqueous solution of iron complex C2 (concentration 1.5 mM) to them respectively, and continue to culture for 48 h in an environment of 37 °C and 5% CO2;

[0076] (2) Protein extraction: Collect the cells, add RIPA lysis buffer to extract total protein, lyse on ice for 30 min, centrifuge at a high speed of 12000 rpm for 15 min, and take the supernatant to measure 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) Perform electrophoresis using 10% SDS-polyacrylamide gel electrophoresis buffer. Add 10 μL of each protein sample to each well. The electrophoresis conditions are 80 V for 30 min in the concentration stage and 120 V for 90 min in the separation stage. After electrophoresis, transfer the membrane to a PVDF membrane for 120 min;

[0079] (5) After adding the blocking solution (5% bovine serum albumin) and blocking at room temperature for 1 h, incubate the PVDF strips with primary antibodies GAPDH, GPX4, and SLC7A11 respectively overnight at 4 °C, wash three times with 1×TBST buffer, 5 min each time;

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

[0081] The results of Western blot of different reagents are as Figure 3 shown. As Figure 3 can be seen, compared with the control group, GPX4 (a ferroptosis regulator) and SLC7A11 in the iron complex C1 and iron complex C2 groups are significantly down-regulated, while the expression level of the internal reference protein GAPDH has no obvious change. This indicates that iron complexes C1 and C2 have the ability to induce ferroptosis in cancer cells.

[0082] MTT method:

[0083] Culture the U87MG cell line until the cells are in the logarithmic phase, with a concentration of 5×10 4cells / mL; Take 180 μL of the above-mentioned logarithmic-phase cells, place them in a 96-well plate, use DMEM medium, and continue culturing for 18 h in an environment of 5% CO2 and 37 °C as a group, with a total of ten groups set up;

[0084] Add normal saline to the first group as the control group;

[0085] Add 20 μL of cisplatin aqueous solution (concentration: 350 μM) to the second group;

[0086] Add 10 μL of cisplatin aqueous solution (concentration: 700 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 200 μM) to the third group;

[0087] Add 10 μL of cisplatin aqueous solution (concentration: 700 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 400 μM) to the fourth group;

[0088] Add 20 μL of iron complex C1 aqueous solution (concentration: 30 μM) to the fifth group;

[0089] Add 10 μL of iron complex C1 aqueous solution (concentration: 60 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 200 μM) to the sixth group;

[0090] Add 10 μL of iron complex C1 aqueous solution (concentration: 60 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 400 μM) to the seventh group;

[0091] Add 20 μL of iron complex C2 aqueous solution (concentration: 30 μM) to the eighth group;

[0092] Add 10 μL of iron complex C2 aqueous solution (concentration: 60 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 200 μM) to the ninth group;

[0093] Add 10 μL of iron complex C2 aqueous solution (concentration: 60 μM) and 10 μL of Ferrostatin-1 aqueous solution (concentration: 400 μM) to the tenth group;

[0094] Continue culturing the above ten groups for 48 h, add 10 μL of thiazolyl blue aqueous solution (5 mg / mL) respectively, incubate for 4 h, then slowly remove all the liquid, and continue to add 100 μL of dimethyl sulfoxide to dissolve them respectively;

[0095] Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 570 nm and calculate the cell survival rate. The results are as Figure 4 shown.

[0096] The cell survival rate graphs of different cell iron inhibitors are as follows Figure 4 shown. It can be seen from Figure 4 that the ferroptosis inhibitor Ferrostatin-1 has a relatively low effect on the activity of cisplatin, but can effectively reduce the activities of iron complexes C1 and C2. This indicates that iron complexes C1 and C2 can inhibit ferroptosis of cancer cells and are potential ferroptosis inducers.

[0097] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

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

2. The method for preparing an iron complex according to claim 1, characterized in that: The steps include: 1) reacting 2-hydrazinobenzothiazole, a ketone compound and a solvent to obtain a ligand; 2) The ligand, ferric chloride and solvent are reacted and then cooled and crystallized to obtain an iron complex.

3. The method for preparing an iron complex according to claim 2, characterized in that: In the step 1), the ketone compound is 2-benzoylpyridine or 6,7-dihydro-5H-quinoline-8-one.

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

5. The method for preparing an iron complex according to claim 4, characterized in that: In the step 1), the reaction temperature is 55-65° C. and the reaction time is 4-6 hours.

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

7. The method for preparing an iron complex according to claim 6, characterized in that: In the step 2), the reaction temperature is 30-50° C. and the reaction time is 1-3 hours.

8. The method for preparing an iron complex according to claim 6 or 7, characterized in that: In the step 2), the temperature of cooling crystallization is 0-6°C, and the time of cooling crystallization is 5-8 days.

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

10. Use of the iron complex according to claim 1 in the preparation of anti-tumor drugs.

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