Preparation method and application of a 2-(2-pyridyl)benzimidazole derivative curcumin copper complex

Abstract

This invention discloses a method for preparing and applying a 2-(2-pyridyl)benzimidazole derivative curcumin copper complex. The specific steps of the preparation method are as follows: curcumin, a 2-(2-pyridyl)benzimidazole derivative, and a copper salt are dissolved in a solvent, the pH is adjusted to 9-10 with an alkali, and after vacuum sealing, the reaction is carried out at 45-80℃ for 14-48 hours. The resulting crystals after cooling are the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex. In vitro cell experiments and in vivo mouse experiments using the prepared complex as a photodynamic anticancer drug showed that its antitumor activity was poor under dark conditions, but strong under light conditions, and far stronger than cisplatin. This offers hope for solving the problem of current anticancer drugs having good efficacy but high toxicity.

Description

Technical Field

[0001] This invention belongs to the field of photoactivated antitumor drug technology, specifically relating to a method for preparing and applying a 2-(2-pyridyl)benzimidazole derivative curcumin copper complex. Background Technology

[0002] Cancer, also known as malignant tumor, is a disease caused by uncontrolled cell proliferation and growth. Most types of cancer form visible growths, or tumors, within the body. Although platinum-based drugs developed from cisplatin are broad-spectrum drugs and highly effective against various cancer types, their application is limited due to drug resistance and serious side effects. Photodynamic therapy (PDT) is a medical technology that uses photosensitizers under aerobic conditions and specific wavelengths of light to trigger photochemical reactions, producing singlet oxygen and other substances that kill tumor cells, thereby treating tumors. Compared to other traditional treatments (such as surgery, chemotherapy, and radiotherapy), PDT is controllable in terms of space and time. Furthermore, PDT can utilize interventional techniques such as fiber optics and endoscopy to avoid open-chest and open-abdomen surgery, resulting in little or no trauma. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing and applying a 2-(2-pyridyl)benzimidazole derivative curcumin copper complex.

[0004] The structural formula of the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex is as follows:

[0005]

[0006] Where R 1 For H or CH3, R 2 It can be H, CH3, F, Cl, or Br.

[0007] The preparation method of the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex is as follows: curcumin, 2-(2-pyridyl)benzimidazole derivative and copper salt are dissolved in a solvent, the pH is adjusted to 9-10 with alkali, and after vacuum sealing, the reaction is carried out at 45-80℃ for 14-48h. The crystals obtained after cooling are the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex.

[0008] The 2-(2-pyridyl)benzimidazole derivative is one or more of 2-(2-pyridyl)benzimidazole, 5-methyl-2-(pyridin-2-yl)-1H-benzimidazole, 5,6-dimethyl-2-(pyridin-2-yl)-1H-benzimidazole, 5-fluoro-2-(pyridin-2-yl)-1H-benzimidazole, 5-chloro-2-(pyridin-2-yl)-1H-benzimidazole, and 5-bromo-2-(pyridin-2-yl)-1H-benzimidazole.

[0009] The copper salt is copper nitrate or copper nitrate containing water of crystallization.

[0010] The solvent is methanol and n-hexane in a volume ratio of 1:2 to 2:1.

[0011] The alkali mentioned is triethylamine or ammonia.

[0012] The molar ratio of curcumin, 2-(2-pyridyl)benzimidazole derivative and copper salt is 1:1:0.8-2.

[0013] The application of the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex prepared above in the preparation of photodynamic anticancer drugs.

[0014] This invention synthesizes a Cu(II) anticancer complex using curcumin as the main photosensitive ligand and 2-(2-pyridyl)benzimidazole derivative as the auxiliary ligand. In vitro cell experiments and in vivo mouse experiments show that its antitumor activity is poor under dark conditions, but strong under light conditions, and much stronger than cisplatin. This brings hope for solving the problem that current anticancer drugs are effective but highly toxic. Attached Figure Description

[0015] Figure 1 This is the chemical reaction formula for preparing the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex according to the present invention.

[0016] Figure 2 These are molecular structure diagrams of the six 2-(2-pyridyl)benzimidazole derivative curcumin copper complexes prepared in Examples 1-6 of this invention.

[0017] Figure 3 The six 2-(2-pyridyl)benzimidazole derivative curcumin copper complexes CH1 and CH6 (50 mg / kg) prepared in Examples 1 and 6 of this invention were subjected to light or dark conditions (450 nm, 50 mW / cm²). 2 The results of the in vivo antitumor effect evaluation of Hep-G2 xenograft tumors in mice (300s) are shown in the figure. (a) Changes in tumor volume in mice; (b) Changes in average body weight in mice; (c) Tumor weight in mice; (d) Tumor photographs of each group after drug administration. Detailed Implementation

[0018] Example 1: Synthesis and Characterization of Complex CH1

[0019] Accurately weigh HL 1 The ligand 2-(2-pyridyl)benzimidazole (0.02 mmol, 0.0039 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a circular, pressure-resistant glass tube with a length of 180 mm and an inner diameter of 10 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added, and the pH of the entire reaction system was adjusted by adding 20 μL of triethylamine. The mixture was thoroughly mixed and sealed under vacuum. The reaction was then carried out at 60 °C for 24 h. After slow cooling, brownish-yellow blocky crystals were obtained. Clean and suitable crystals were picked and separated by washing. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 34 H 32 CuN4O 10 Yield 42.8%. Experimental values ​​(%): C, 56.46; H, 4.30; N, 7.51; Cu, 8.48%. Theoretical values ​​(%): C, 56.71; H, 4.48; N, 7.78; Cu, 8.82%. IR (KBr, cm⁻¹) -1 ): 1622m, 1597m, 1511s, 1452w, 1384s, 1281s, 1169m, 988w, 963w, 849w, 821w, 745w. ESI-MS m / z:M + 625.1284 (calcd 625.1274).

[0020] Example 2: Synthesis and Characterization of Complex CH2

[0021] Accurately weigh three substances: HL 2 The ligands 5-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (0.02 mmol, 0.0042 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a circular pressure-resistant glass tube with an inner diameter of 10 mm and a length of 180 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added, along with 25 μL of triethylamine. The reaction system was thoroughly mixed and sealed under vacuum, and the reaction was carried out at 60 °C for 24 hours. After the reaction was completed, the mixture was slowly cooled to obtain brownish-yellow blocky crystals. Clean crystals were picked and washed. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 35 H 34 CuN4O10 Yield 37.6%. Experimental values ​​(%): C, 57.41; H, 4.90; N, 7.39; Cu, 8.90%. Theoretical values ​​(%): C, 57.26; H, 4.67; N, 7.63; Cu, 8.66%. IR (KBr, cm⁻¹) -1 ): 1623m, 1600m, 1590m, 1510s, 1466w, 1430w, 1384s, 1286s, 1172m, 1122m, 1032m, 991m, 963m, 849w, 821w, 805w. ESI-MS m / z:M + 639.1439 (calcd 639.1431).

[0022] Example 3: Synthesis and Characterization of Complex CH3

[0023] First, accurately weigh HL 3 The ligands 5,6-dimethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole (0.02 mmol, 0.0045 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a circular pressure-resistant glass tube with a length of 180 mm and an inner diameter of 10 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added, along with 10 μL of triethylamine, to adjust the pH of the reaction system. After thoroughly mixing the reaction system, it was sealed under vacuum and reacted at 60 °C for 24 hours. Subsequently, it was slowly cooled to obtain brownish-yellow blocky crystals. Clean and suitable crystals were picked and separated and washed. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 36 H 36 CuN4O 10 Yield 32.4%. Experimental values ​​(%): C, 57.91; H, 4.66; N, 7.21; Cu, 8.75%. Theoretical values ​​(%): C, 57.79; H, 4.85; N, 7.49; Cu, 8.49%. IR (KBr, cm⁻¹) -1 ): 1620m, 1589m, 1513s, 1453w, 1427w, 1384s, 1290s, 1161m, 1123m, 1028w, 988w, 963w, 850w, 812w. ESI-MS m / z:M + 653.1563 (calcd 653.1587).

[0024] Example 4: Synthesis and Characterization of Complex CH4

[0025] Accurately weigh HL 4The ligands 5-fluoro-2-(pyridin-2-yl)-1H-benzo[d]imidazole (0.02 mmol, 0.0043 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a pressure-resistant circular glass tube with an inner diameter of 10 mm and a length of 180 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added, along with 30 μL of triethylamine to adjust the pH of the reaction system. After thorough mixing, the mixture was sealed under vacuum and reacted at 60 °C for 24 hours. Subsequently, the temperature was gradually reduced to room temperature, yielding brownish-yellow blocky crystals. Clean and suitable crystals were selected for separation and washing. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 34 H 31 CuFN4O 10 Yield 35.5%. Experimental values ​​(%): C, 55.49; H, 4.46; N, 7.87; Cu, 8.86%. Theoretical values ​​(%): C, 55.32; H, 4.23; N, 7.59; Cu, 8.61%. IR (KBr, cm⁻¹) -1 ): 1621m, 1600m, 1511s, 1466w, 1430w, 1385s, 1284s, 1160s, 1124m, 1026w, 987w, 962w, 848w, 821w, 695w, 610w. ESI-MS m / z:M + 643.1152 (calcd 643.1180).

[0026] Example 5: Synthesis and Characterization of Complex CH5

[0027] Accurate weighing of HL 5 The ligands 5-chloro-2-(pyridin-2-yl)-1H-benzo[d]imidazole (0.02 mmol, 0.0046 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a pressure-resistant circular glass tube with an inner diameter of 10 mm and a length of 180 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added dropwise, followed by 15 μL of triethylamine to adjust the pH of the reaction system. After thorough mixing, the mixture was sealed under vacuum and reacted at 60 °C for 24 hours. Subsequently, the temperature was gradually reduced to room temperature, yielding yellow flaky crystals. Clean and suitable crystals were selected for separation and washing. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 34 H 31 ClCuN4O 10Yield 38.1%. Experimental values ​​(%): C, 54.35; H, 4.37; N, 7.65; Cu, 8.65%. Theoretical values ​​(%): C, 54.11; H, 4.14; N, 7.42; Cu, 8.42%. IR (KBr, cm⁻¹) -1 ): 1621m, 1589m, 1511s, 1466w, 1430w, 1385s, 1283s, 1161m, 1124m, 1052w, 1026w, 931w, 849w, 821w, 695w. ESI-MS m / z:M + ,659.0839(calcd 659.0884).

[0028] Example 6: Synthesis and Characterization of Complex CH6

[0029] Accurate weighing of HL 6 The ligands 5-bromo-2-(pyridin-2-yl)-1H-benzo[d]imidazole (0.02 mmol, 0.0055 g), curcumin (0.02 mmol, 0.0074 g), and Cu(NO3)2·3H2O (0.02 mmol, 0.005 g) were placed in a pressure-resistant circular glass tube with an inner diameter of 10 mm and a length of 180 mm. Then, 1 mL of methanol and 1 mL of n-hexane were added, and 10 μL of triethylamine was added dropwise to adjust the pH of the reaction system. After thorough mixing, the mixture was sealed under vacuum and reacted at 60 °C for 24 hours. Subsequently, the temperature was gradually reduced to room temperature, and the mixture was removed to obtain yellow needle-like crystals. Clean and suitable crystals were picked and separated by washing. The crystals obtained by this synthetic method are suitable for single-crystal testing, yielding the molecular formula C. 34 H 31 BrCuN4O 10 Yield 46.4%. Experimental values ​​(%): C, 51.34; H, 3.77; N, 7.23; Cu, 7.78%. Theoretical values ​​(%): C, 51.10; H, 3.91; N, 7.01; Cu, 7.95%. IR (KBr, cm⁻¹) -1 ):1621m,1588m,1511s,1467w,1428w,1385s,1288s,1161m,1123m,1026w,987w,921w,849w,816w. ESI-MS m / z:M + 705.0311 (calcd 705.0360).

[0030] The structural formulas of the six copper complexes prepared in Examples 1-6 above, with curcumin as the main ligand and 2-(2-pyridyl)benzimidazole derivatives as auxiliary ligands, are as follows: Figure 2As shown. The structures of the obtained complexes were characterized by single-crystal X-ray diffraction. All complexes CH1-CH6 have a mononuclear structure, monoclinic crystal system, and space group P21 / n. Crystallographic parameters are listed in Tables 1 and 2. The metal ions in the six complexes all have similar distorted planar square configurations, and each compound contains one NO3- ion for charge balance. - Counterion and a Cu(II) curcumin ligand (Cur - [Cu(Cur)(NN)] is composed of 1 / N and a pyridylbenzimidazole derivative ligand (NN). + A mononuclear cation and a free MeOH molecule.

[0031] Table 1. Data on the crystals of the complex CH1-CH3

[0032]

[0033]

[0034] Table 2. Data on the crystals of the complex CH4-CH6

[0035]

[0036]

[0037] Application Example 1: Optical Antitumor Activity Test

[0038] 1. Cell seeding and culture

[0039] The selected tumor cell lines for the experiments were: human cervical cancer HeLa cells, human bladder cancer T-24 cells, non-small cell lung cancer A549 cells, human liver cancer Hep-G2 cells, and normal hepatocytes HL-7702. The selected tumor cell lines were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and cultured under constant conditions of 37°C and 5% CO2. When the cells reached 80%–90% of the culture flask area, they were digested with trypsin, passaged 3–5 times, and cells in good growth condition and in the logarithmic growth phase were used for the experiments.

[0040] 2. Cell growth inhibition assay (MTT method)

[0041] MTT is a yellow powdery chemical reagent, its full name being 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide, also known as thiazolium blue. Its working principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to purple formazan crystals, while dead cells do not have this ability. Within a certain cell count range, the amount of formazan crystals produced is directly proportional to the number of living cells. Therefore, the number of living cells can be determined based on the measured absorbance value; the higher the absorbance value, the stronger the cell activity. Half-inhibitory concentration (IC50) 50 ( ) is the concentration required for a drug to inhibit cell growth and viral replication by 50%, and can be used as an indicator to measure the anti-tumor activity of a drug.

[0042] Experimental steps:

[0043] (1) Digesting cells: When the cells have grown to 80-90% in the culture medium bottle, discard the culture medium, wash twice with PBS buffer solution, then add 0.5-1 mL of trypsin to digest until the cells become round, immediately add about 5 mL of culture medium to stop digestion, and use a disposable sterile pipette to blow off the adherent cells and mix them evenly.

[0044] (2) Cell seeding: Seed cells in a 96-well plate at a ratio of 1.5 mL of cell stock solution to 12 mL of culture medium. Seed 180 μL of cell solution in each well, and finally add 200 μL of PBS solution around the outside of the 96-well plate to prevent evaporation. Place the seeded 96-well plate in an incubator for incubation.

[0045] (3) Drug incubation: After the cells in the well plate have grown to 70-80%, add 20 μL of the prepared test drug to each well. Set up 5 parallel experimental groups for each drug, and set up a blank control group without drug. Continue incubation for 48 h after drug addition.

[0046] (4) Activity assay: After 48 hours of drug treatment, 10 μL of MTT was added to each well and incubated for another 4 hours. Then, the culture medium was discarded and 100 μL of DMSO was added, followed by appropriate shaking to fully dissolve the formed formazan. The absorbance value of each well was measured using a microplate reader, and the IC50 of the test drug was calculated. 50 Values. Each group needs to be measured three times, and the average value is calculated.

[0047] Table 3. Half-maximal inhibitory concentrations (IC50) of the complexes against different tumor cell lines. 50 (μM)

[0048]

[0049]

[0050] aPre-incubate in the dark for 4 hours, then continue incubation in the dark for 20 hours after drug treatment.

[0051] b Pre-incubate in the dark for 4 hours, then treat with the drug and expose to visible light (400-700 nm, 10 mW / cm²). 2 Expose to light for 1 hour, then incubate in the dark for 19 hours.

[0052] Experimental results show that the IC of the complex CH1-CH6 under visible light (400-700nm) irradiation... 50 The value range is 6.23–15.77 μM, while the IC50 under dark conditions is... 50 The values ​​were all greater than 60 μM, indicating that the complex CH1-CH6 exhibited significant phototoxicity to all five cell lines. However, they were almost non-toxic in the dark, suggesting that the complex CH1-CH6 possesses excellent biocompatibility. (Ligand HL) 1 -HL 6 It showed almost no cytotoxicity to the tested cell lines (IC50). 50 >200μM), while curcumin has certain activity under both dark and light conditions (IC50). 50 The concentration of curcumin (<50 μM) also demonstrates its importance as a photosensitizer in such complexes. This provides an interesting example of the anticancer applications of curcumin and its role in combating malignant tumors.

[0053] The tumor inhibition experiment results of compounds CH1 and CH6 are as follows: Figure 3 As shown, on day 15 after drug administration, the tumor volume in the laser group was not significantly different from that in the model group (P>0.05). However, on day 15 after drug administration, the tumor volumes in the CH1+ and CH6+ laser groups were significantly smaller than those in the model group, much smaller than those in the cisplatin group, almost disappearing. On day 15 after drug administration, the relative tumor proliferation rates (T / C%) of the laser group, CH1 group, CH1+ laser group, CH6 group, and CH6+ laser group against Hep-G2 human liver cancer xenografts in nude mice were 88.0%, 83.0%, 10.1%, 80.4%, and 5.2%, respectively, with tumor inhibition rates of 12.2%, 19.6%, 91.4%, 26.8%, 94.8%, and 53.4%, respectively. Furthermore, on day 15 after drug administration, the relative tumor proliferation rate (T / C%) of the cisplatin group against Hep-G2 human liver cancer xenografts in nude mice was 45.8%, with a tumor inhibition rate of 53.4%. This indicates that Cu(II) complexes have excellent photo-controlled antitumor activity because they can precisely irradiate tumor tissue to stimulate the antitumor activity of the drug, while having extremely low cytotoxicity in other parts of the body when there is no light irradiation. Therefore, they will not cause damage to other organs and normal cells in the body, thus achieving highly effective, low-toxicity, and precise treatment.

Claims

1. A 2-(2-pyridyl)benzimidazole derivative curcumin copper complex, characterized in that, The structural formula of the complex is as follows: ， in R 1 It is H or CH3. R 2 It can be H, CH3, F, Cl, or Br.

2. A method for preparing a 2-(2-pyridyl)benzimidazole derivative curcumin copper complex, characterized in that, The specific operation of the preparation method is as follows: curcumin, 2-(2-pyridyl)benzimidazole derivative and copper salt are dissolved in solvent, the pH is adjusted to 9-10 with alkali, and after vacuum sealing, the reaction is carried out at 45-80 °C for 14-48 h. The crystals obtained after cooling are the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex. The 2-(2-pyridyl)benzimidazole derivative is one or more of 2-(2-pyridyl)benzimidazole, 5-methyl-2-(pyridin-2-yl)-1H-benzimidazole, 5,6-dimethyl-2-(pyridin-2-yl)-1H-benzimidazole, 5-fluoro-2-(pyridin-2-yl)-1H-benzimidazole, 5-chloro-2-(pyridin-2-yl)-1H-benzimidazole, and 5-bromo-2-(pyridin-2-yl)-1H-benzimidazole. The copper salt is copper nitrate or copper nitrate containing water of crystallization.

3. The preparation method according to claim 2, characterized in that, The solvent is methanol and n-hexane in a volume ratio of 1:2 to 2:

1.

4. The preparation method according to claim 2, characterized in that, The alkali mentioned is triethylamine or ammonia.

5. The preparation method according to claim 2, characterized in that, The molar ratio of curcumin, 2-(2-pyridyl)benzimidazole derivative and copper salt is 1:1:0.8-2.

6. The application of the 2-(2-pyridyl)benzimidazole derivative curcumin copper complex prepared by the method according to any one of claims 2-5 in the preparation of photodynamic anticancer drugs.

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