Copper (ii) complex with photodynamic anti-glioblastoma activity, preparation method and application thereof

Abstract

The present application relates to copper (II) complex with photodynamic anti-glioblastoma activity and its preparation method and application. The present application takes diiodofluoroborondipyrrin (I2BC) as ligand, reacts with copper perchlorate to generate copper (II) complex I2BC-Cu, and introduces the second ligand curcumin to generate copper (II) complex I2BC-Cu-Cur with Cu (II). The two copper (II) complexes designed and synthesized creatively for the first time have not been reported in the field and the existing literature, and have excellent photodynamic activity on GBM, and can be applied in the preparation of photodynamic anti-glioblastoma drugs. The two copper (II) complexes have the following structural formula:

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a copper (II) complex with photodynamic anti-glioblastoma activity and a preparation method and application thereof. BACKGROUND

[0002] Glioma is the most common primary brain malignancy of the central nervous system, which is composed of tumors of different phenotypes and molecular types. It is the tumor with the highest incidence among primary brain tumors, among which glioblastoma (GBM) is the most invasive. GBM is a highly invasive central nervous system malignancy, and the survival time of GBM patients is relatively low, about 10 to 15 months. Current treatment methods include tumor resection, chemotherapy, radiotherapy and anti-vascular targeting drugs, but its strong proliferative capacity and invasive progression still makes traditional methods unable to completely treat glioblastoma, prolong the survival period of patients and improve the quality of life.

[0003] Temozolomide (TMZ) is a first-line chemotherapy drug approved by the US Food and Drug Administration (FDA) for the treatment of GBM. However, the clinical application of temozolomide is accompanied by some side effects such as drug resistance and neurological dysfunction. SUMMARY

[0004] The application aims at the technical problems existing in the prior art, and provides a copper (II) complex with photodynamic anti-glioblastoma activity and a preparation method and application thereof. The copper (II) complex has very high toxic activity on glioblastoma under specific wavelength light, and has weak toxicity on normal glial cells under light-avoiding conditions.

[0005] In a first aspect, the application provides a copper (II) complex I2BC-Cu with photodynamic anti-glioblastoma activity, which adopts the following technical scheme:

[0006] The copper (II) complex I2BC-Cu with photodynamic anti-glioblastoma activity has the following structural formula:

[0007] .

[0008] In a second aspect, the application provides a copper (II) complex I2BC-Cu-Cur with photodynamic anti-glioblastoma activity, which adopts the following technical scheme:

[0009] The copper (II) complex I2BC-Cu-Cur with photodynamic anti-glioblastoma activity has the following structural formula:

[0010] .

[0011] In a third aspect, the present invention provides a method for preparing the copper (II) complex I2BC-Cu, using the following technical solution:

[0012] The synthesis route of the copper (II) complex I2BC-Cu is as follows:

[0013] .

[0014] On the basis of the above technical solution, the present invention can also be improved as follows.

[0015] Furthermore, the preparation method of the copper (II) complex I2BC-Cu includes the following steps: taking I2BC and copper perchlorate powder and dissolving them in methanol, heating them at 65°C under N2 protection for 12 hours, filtering and vacuum drying to obtain a purple-red I2BC-Cu complex.

[0016] Preferably, the ratio of I2BC to copper perchlorate is 1 mmol:1 mmol.

[0017] In a fourth aspect, the present invention provides a method for preparing the above-mentioned copper (II) complex I2BC-Cu-Cur, using the following technical solution:

[0018] The synthesis route of the copper (II) complex I2BC-Cu-Cur is as follows:

[0019] .

[0020] Furthermore, the preparation method of the copper (II) complex I2BC-Cu-Cur comprises the following steps:

[0021] 1) Dissolve I2BC and copper perchlorate powder in methanol and heat at 65°C under N2 protection for 12 h.

[0022] 2) Curcumin and triethylamine were weighed and added to the system in step 1), and the reaction was continued for 6 h. The mixture was filtered and vacuum dried to obtain the product I2BC-Cu-Cur.

[0023] Preferably, the usage ratio of I2BC, copper perchlorate, curcumin and triethylamine is 1 mmol:1 mmol:1 mmol:1 mmol.

[0024] In a fifth aspect, the present invention provides the application of the above copper (II) complex, using the following technical solution:

[0025] Application of the copper (II) complex I2BC-Cu and / or I2BC-Cu-Cur in the preparation of photodynamic anti-glioblastoma drugs.

[0026] The beneficial effects of the present invention are:

[0027] 1. The present invention creatively designs and synthesizes two divalent copper complexes containing diiodo BODIPY, namely the binary complex I2BC-Cu and the ternary complex I2BC-Cu-Cur. These two complexes are new compounds and have not been reported in the art or existing literature.

[0028] 2. The present invention determined the structures of the above two copper (II) complexes by HRMS characterization and proved that the method described can successfully prepare the target products, namely the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur.

[0029] 3. Through cytotoxic activity experiments and cell ROS experiments, the present invention confirms that I2BC-Cu and I2BC-Cu-Cur have good photodynamic activity against GBM. It is expected that they can provide valuable reference for overcoming the drug resistance and neurotoxicity of the existing chemotherapy drug TMZ, as well as for the future design and synthesis of new photodynamic active drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 (ab) are HRMS spectra of the copper (II) complex I2BC-Cu prepared in Example 1 of the present invention;

[0031] Figure 2 (ab) are HRMS spectra of the copper (II) complex I2BC-Cu-Cur prepared in Example 2 of the present invention;

[0032] Figure 3 (ab) are graphs showing the inhibition of U87MG cells by the copper (II) complexes prepared in Examples 1 and 2 of the present invention;

[0033] Figure 4 (ab) are graphs showing the inhibition of T98G cells by the copper (II) complexes prepared in Examples 1 and 2 of the present invention;

[0034] Figure 5 (ab) are graphs showing the inhibition of HEB cells by the copper (II) complexes prepared in Examples 1 and 2 of the present invention;

[0035] Figure 6 Fluorescence microscope images of ROS generated by the copper (II) complexes prepared in Examples 1 and 2 of the present invention in U87MG cells;

[0036] Figure 7 The relative attenuation curves of the ultraviolet absorbance of the copper (II) complex and methylene blue (MB) prepared in Examples 1 and 2 of the present invention at 417 nm tested by the DPBF method are shown. DETAILED DESCRIPTION

[0037] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0038] Compared with chemotherapy (chemotherapy, such as TMZ), photodynamic therapy is a promising local therapy for treating tumors. It has high anti-cancer activity and can effectively reduce toxic side effects due to the regional selectivity of light. The three core elements of photodynamic therapy are photosensitizer, light and O2. When a laser of a specific wavelength is irradiated on the tumor site enriched with photosensitizer, a large amount of reactive oxygen species such as 1 O2, ˙OH, O2 ˙- These excessive highly oxidative species are the main killers of cancer cells or tissues.

[0039] Based on this, the inventors have conducted extensive research and experiments on the synthesis process and mechanism of action of photosensitizers in an attempt to find or design efficient photosensitizers with good photodynamic anti-glioblastoma activity, thereby improving the clinical application of TMZ, which is accompanied by some side effects, such as drug resistance and neurological dysfunction.

[0040] During their experiments, the inventors unexpectedly discovered that I2BC-Cu, generated by reacting diiodofluoroboron dipyrrole (I2BC) with copper perchlorate, and I2BC-Cu-Cur, generated by introducing a secondary ligand, curcumin, to coordinate with Cu(II), both achieved unexpected results, exhibiting excellent photodynamic activity against GBM. Further testing and analysis suggest that this may be due to the high singlet oxygen quantum yield of these two complexes due to the introduction of heavy atom iodine into the BODIPY complex, which generates a large amount of reactive oxygen species under visible light irradiation, resulting in excellent photodynamic activity against U87MG and T98G. Cytotoxicity and cellular ROS assays also confirm that I2BC-Cu and I2BC-Cu-C possess potential for the preparation of photodynamic anti-glioblastoma drugs. This invention is based on these findings, providing a data foundation and reference for the development of novel photodynamic anti-glioblastoma drugs.

[0041] Unless otherwise noted, the raw materials used in this invention are conventional raw materials (commonly available products) in the art and are commercially available. The experimental methods and detection methods in the following examples are all conventional methods unless otherwise noted, and the instruments and equipment used in the experiments are all commercially available.

[0042] The I2BC used in the present invention was synthesized by the inventors in the laboratory. The specific synthesis method has been disclosed in the invention patent application with application number CN202111238299.0 and is not described here in detail. The specific structural formula of I2BC is as follows:

[0043] .

[0044] Example 1

[0045] Preparation of copper (II) complex I2BC-Cu having the following structural formula:

[0046] .

[0047] The synthetic route of copper (II) complex I2BC-Cu is as follows:

[0048] .

[0049] Specifically, the preparation method of the copper (II) complex I2BC-Cu comprises the following steps:

[0050] I2BC (55 mg, 0.074 mM) and copper perchlorate (27.5 mg, 0.074 mM) were dissolved in 30 mL of methanol. The mixture was heated at 65 °C under N2 protection for 12 h. After filtration, the mixture was vacuum-dried to obtain a purple-red I2BC-Cu complex (37 mg, yield: 62.1%).

[0051] See also Figure 1 , HRMS (CH3CN): m / z found: 801.9970, 929.9019 and 740.0751, C 28 H 30 BCuF2I2N5, [M-2ClO4-H] + ,[M-2ClO4+CH3CN+CH3OH+3H2O-H] + and [M-2ClO4-Cu+H] + The theoretical values ​​are: 801.9937, 928.0781 and 740.0730 respectively.

[0052] Example 2

[0053] Preparation of copper (II) complex I2BC-Cu-Cur having the following structural formula:

[0054] .

[0055] The synthetic route of copper (II) complex I2BC-Cu-Cur is as follows:

[0056] .

[0057] Specifically, the preparation method of the copper (II) complex I2BC-Cu-Cur comprises the following steps:

[0058] 1) Dissolve I2BC (46 mg, 0.062 mmol) and copper perchlorate powder (23 mg, 0.062 mmol) in 30 mL of methanol and heat at 65 °C under N2 for 12 h.

[0059] 2) Curcumin (23 mg, 0.062 mmol) was weighed and dissolved in 5 mL of methanol. Triethylamine (6.2 mg, 0.062 mmol) was added to the system in step 1). The reaction was continued for 6 h. After filtration, vacuum drying was performed to obtain the product I2BC-Cu-Cur (20.3 mg, yield: 27.9%).

[0060] See also Figure 2 , HRMS (CH3CN): m / z found: 1169.1113, 1078.0695 and 929.8984, C 49 H 49 O6BCuF2I2N5, [MH] + , [M-Cur+3CH3CN+3H2O-H] + and [M-Cur+CH3CN+ CH3OH+3H2O-H] + The theoretical values ​​are: 1169.1118, 1078.0541 and 928.0781 respectively.

[0061] Example 3

[0062] Cytotoxicity experiments were performed on the I2BC-Cu and I2BC-Cu-Cur prepared in Examples 1 and 2 above:

[0063] 1. Cell lines and cell culture

[0064] This experiment used human glioblastoma U87MG, T98G and human brain astrocyte normal cells HEB.

[0065] The cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin in a 5% CO2, 37°C incubator. When the cells reached 80% confluency, they were passaged or plated.

[0066] 2. Preparation of test compounds

[0067] The two copper (II) complexes in the experimental group were dissolved in DMSO to prepare a stock solution with a concentration of 25 mM, and then diluted with culture medium to a final solution of 0-100 μM. In the control group, curcumin was prepared into a stock solution with a concentration of 100 mM, and the TMZ concentration was 200 mM (DMSO concentration ≤ 1%).

[0068] 3. Cell growth inhibition assay (MTT assay)

[0069] Digest the cells grown on the culture dish with trypsin, remove the supernatant after centrifugation, count the cells with a hemocytometer, and adjust the cell suspension concentration to approximately 1×10 5 / mL, add 100 μL of cell suspension to each well of a 96-well plate, and maintain the cell density in each well at 8×10 3 -1×10 4 Cells were cultured overnight in an incubator. The original culture medium was removed after the cells grew to 80% adherence. Five concentrations of each drug were set, and five replicates were set for each concentration. 100 μL of culture medium containing different drug concentrations was added to each well. The two copper (II) complexes in the experimental group were exposed to cells in the dark for 24 h or in the dark for 3 h + light for 1 h (520 nm). The control group was incubated with cells in a dark environment for 48 h. After the exposure time, the well plate was removed and 10 μL of MTT reagent (5 mg / mL) was added to each well. The cells were incubated in the incubator for another 4 h and then removed. 150 μL of DMSO was added to each well. The cells were shaken on a shaker to fully dissolve the formazan crystals. The absorbance OD value of each well was measured using a microplate reader (490 nm).

[0070] 4. IC 50 calculate

[0071] See also Figures 3 to 5 , cell inhibition rate = (1-OD value of experimental group / OD value of blank group) × 100%, with inhibition rate as the ordinate and drug concentration as the abscissa, and the IC was calculated by curve fitting using Prism 8.0 software. 50 , the results are shown in Table 1 below.

[0072] 5. Calculation of Photodynamic Index (PI)

[0073] PI value is IC under dark conditions 50 / IC under light conditions 50 , the calculation results are shown in Table 2 below.

[0074] Table 1: IC values ​​of ligands and complexes for different cell lines 50 The lower the value, the better the inhibitory effect of the compound.

[0075]

[0076] Table 2: PI values ​​of complexes for U87MG, T98G and HEB

[0077]

[0078] 6. Data Analysis

[0079] From Table 1 and Table 2, we can see that:

[0080] (1) IC values ​​of the two copper (II) complexes under light conditions were 50 The values ​​were significantly lower than IC under dark conditions. 50 The results showed that the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur both had photosensitization effects, produced ROS after illumination, interacted with the DNA in the cells, and played a role in killing glioma cells.

[0081] (2) The copper (II) complexes I2BC-Cu-Cur and I2BC-Cu were more active against U87MG than T98G. For U87MG, the IC 50 The value was 0.808 μM, the PI value was 12.13; the IC value of I2BC-Cu after illumination was 0.808 μM, the PI value was 12.13, and the IC value of I2BC-Cu after illumination was 0.808 μM, the PI value was 12.13. 50 The value reached 0.147 μM and the PI value reached 20.54. This showed that both I2BC-Cu and I2BC-Cu-Cur had good anti-glioblastoma activity, and I2BC-Cu was slightly better than I2BC-Cu-C.

[0082] (3) IC of copper (II) complexes I2BC-Cu and I2BC-Cu-Cur on HEB cells 50 Both were higher than those of U87MG and T98G, indicating that the two copper (Ⅱ) complexes had less toxic activity on normal glial cells.

[0083] (4) The anti-glioblastoma activity of the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur is significantly better than that of the first-line glioblastoma chemotherapy drug TMZ. I2BC-Cu and I2BC-Cu-C are expected to be used as new photosensitizers in photodynamic therapy for glioblastoma and further used in subsequent studies of photodynamic anti-glioblastoma.

[0084] Example 4

[0085] On the basis of Example 3, in order to further confirm that the copper (II) complexes of the present invention have photodynamic anti-glioblastoma activity, the inventors measured the ability of the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur prepared in Examples 1 and 2 above to generate ROS in U87MG cells by fluorescence microscopy imaging.

[0086] The experimental results are shown in Figure 6 .

[0087] Depend on Figure 6 It can be seen that:

[0088] The copper (II) complexes I2BC-Cu and I2BC-Cu-Cur produce almost no reactive oxygen species in a dark environment, but can generate a large amount of reactive oxygen species in U87MG cells under light conditions of a certain wavelength (520 nm, 5 mW), and are expected to be used in the preparation of alternative drugs for photodynamic therapy of glioblastoma.

[0089] Example 5

[0090] The ability of the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur prepared in Examples 1 and 2 above to generate active oxygen under light of a specific wavelength was determined using the DPBF fluorescence probe method:

[0091] In this experiment, the DPBF stock solution was diluted 200 times with DMSO to form 5×10 -5 To prepare a DPBF / complex solution, add 4 μL of the complex stock solution to a 3 mL sample cell and mix thoroughly. This creates a mixed solution with a DPBF / complex concentration ratio of 25:1. UV absorption spectra of I2BC-Cu, I2BC-Cu-Cur, and methylene blue (MB) were scanned every 15 seconds. MB was used as a reference reagent (its ROS yield in DMSO solution is 0.52). The ROS yield of the complex was calculated according to the following formula.

[0092] The calculation formula of active oxygen production rate is:

[0093] Among them, Φ ∆ represents the ROS yield, K is the slope of the linear fit with the irradiation time as the x-axis and the UV absorbance of DPBF at 417 nm after the irradiation time as the y-axis, and F represents the absorption correction factor of the complex and the reference reagent, F = 1-10 -OD , OD represents the UV absorption value of the complex or MB at the corresponding maximum absorption wavelength.

[0094] Table 3 Summary of relevant parameters of the interaction between DPBF and copper (II) complexes

[0095]

[0096] The experimental results are shown in Figure 7 and Table 3.

[0097] Depend on Figure 7 From Table 3, we can see that:

[0098] Under the same conditions, the decrease rate of absorbance of I2BC-Cu-Cur and I2BC-Cu was significantly higher than that of MB. After calculation, the active oxygen production rate of I2BC-Cu-Cur was higher, which was 0.508.

[0099] In summary, the copper (II) complexes I2BC-Cu and I2BC-Cu-Cur described herein generate large amounts of reactive oxygen species under illumination with specific wavelengths and exhibit excellent anti-glioblastoma activity in vitro. The design concepts and synthetic routes for the copper (II) complexes synthesized herein with photodynamic anti-glioblastoma activity are both feasible. These two copper (II) complexes, I2BC-Cu and I2BC-Cu-Cur, can be used in the preparation of photodynamic anti-glioblastoma drugs.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A copper (II) complex I2BC-Cu with photodynamic anti-glioblastoma activity, characterized in that: Its structural formula is as follows: 。 2. A copper (II) complex I2BC-Cu-Cur with photodynamic anti-glioblastoma activity, characterized in that: Its structural formula is as follows: 。 3. The method for preparing the copper (II) complex I2BC-Cu having photodynamic anti-glioblastoma activity according to claim 1, characterized in that: The following steps are involved: I2BC and copper perchlorate powder were dissolved in methanol and heated at 65°C under N2 protection for 12 h. The mixture was filtered and dried under vacuum to obtain a purple-red I2BC-Cu complex. The specific structural formula of I2BC is as follows: 。 4. The method for preparing the copper (II) complex I2BC-Cu having photodynamic anti-glioblastoma activity according to claim 3, characterized in that: The usage ratio of I2BC and copper perchlorate is 1 mmol:1 mmol.

5. The method for preparing the copper (II) complex I2BC-Cu-Cur having photodynamic anti-glioblastoma activity according to claim 2, characterized in that: The following steps are involved: 1) Dissolve I2BC and copper perchlorate powder in methanol and heat at 65°C under N2 protection for 12 h. 2) Weigh curcumin and triethylamine and add them to the system in step 1), continue the reaction for 6 hours, filter, and vacuum dry to obtain the product I2BC-Cu-Cur; Among them, the specific structural formula of I2BC is as follows: 。 6. The method for preparing the copper (II) complex I2BC-Cu-Cur having photodynamic anti-glioblastoma activity according to claim 5, characterized in that: The usage ratio of the I2BC, copper perchlorate, curcumin and triethylamine is 1 mmol:1 mmol:1 mmol:1 mmol.

7. Use of the copper (II) complex according to claim 1 or 2 in the preparation of photodynamic anti-glioblastoma drugs.

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