A binuclear copper (II) complex and a preparation method and application thereof

Abstract

The application belongs to the field of metal complexes, and particularly relates to a binuclear copper (II) complex and a preparation method and application thereof. The complex structure formula is [Cu2(mu2-L)2(NO3)2Cl2], wherein L is 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride. The complex is prepared by using Cu(NO3)2.3H2O and 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride as raw materials through a heating reflux method. The complex can efficiently inhibit the activity of protein tyrosine phosphatase 1B (PTP1B), and can also effectively inhibit the proliferation of breast cancer (MCF7) cells and cervical cancer (Hela) cells. Therefore, the complex is not only a high-efficiency specific PTP1B inhibitor, but also a potential candidate drug for anti-breast cancer and anti-cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of metal complexes, specifically relating to a binuclear copper(II) complex, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a major threat to human health. Currently, the main chemotherapeutic drugs used clinically to treat malignant tumors are classic platinum-based drugs, cisplatin, which target DNA in the body. However, their anti-tumor specificity is poor, and they also have toxic side effects and drug resistance. Therefore, the continued development of highly effective and low-toxicity novel anti-tumor drugs is of great practical significance. Copper, as an essential metal element for the human body, is increasingly recognized by studies as participating in cell proliferation and apoptosis pathways. Therefore, copper-based inorganic metal drugs are ideal candidates for anti-tumor treatment. Once copper ions are properly complexed, their corresponding copper complexes hold promise as potential highly effective and low-toxicity anti-tumor drugs.

[0003] With the rapid development of tumor biology and related disciplines, the research and development philosophy of anti-tumor drugs has also undergone a significant transformation. The focus of research has gradually shifted from traditional DNA-targeted drug design to novel drugs targeting abnormal signaling systems within tumor cells. Protein tyrosine phosphatases (PTPs) are a class of important enzymes widely expressed in the human body and involved in cell signal transduction. Their overexpression or abnormal activity is associated with various human diseases. Recent studies have found that protein tyrosine phosphatase 1B (PTP1B) is overexpressed in some tumor cells, and this overexpression of PTP1B enzyme is related to tumor development and spread. Therefore, the development of antitumor drugs based on PTP1B target inhibition has important theoretical significance and potential application value. However, due to the influence of another enzyme with high homology to PTP1B (T-cell protein tyrosine phosphatase, abbreviated as TCPTP), the design and synthesis of inhibitors that can efficiently and specifically inhibit PTP1B activity still face great challenges. The development of antitumor drugs based on PTP1B target inhibition is still very limited. Therefore, selecting appropriate ligands, adopting convenient experimental methods, designing novel coordination structures, and obtaining antitumor metal complexes that can efficiently and specifically inhibit the PTP1B target is a new approach for the development of new antitumor drugs. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical situation by providing a 3-(2-pyridyl)pyrazole derivative binuclear copper(II) complex and its preparation method, as well as the application of this complex as a highly efficient and specific PTP1B inhibitor in the treatment of breast cancer and cervical cancer.

[0005] This invention provides a binuclear copper(II) complex with the structural formula: [Cu2(µ2-L)2(NO3)2Cl2], where L is 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride. The structural formula is:

[0006]

[0007] The binuclear copper(II) complex crystal provided by this invention belongs to the monoclinic crystal system, space group P21 / n, with the following cell parameters: a = 8.964(3) Å, b = 12.541(5) Å, c = 16.022(6) Å, α = 90º, β = 104.641(9)°, γ = 90º. The asymmetric structural unit of this complex contains one Cu 2+ 1 ligand L - And 1 NO3 - And 1 Cl - Each Cu(II) ion coordinates with five atoms to form a twisted planar square pyramidal configuration. From the coordination environment of this complex, it can be seen that the two central copper ions are bridged by two chloride ions in the ligand hydrochloride, forming a binuclear structure. The binuclear copper complex (II) provided by this invention has been confirmed by X-ray powder diffraction to be homogeneous and stable in crystalline samples.

[0008] The present invention provides a method for preparing a binuclear copper(II) complex, comprising the following steps:

[0009] (1) Cu(NO3)2·3H2O and 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride were added to a preheated alcohol solvent, a weak base was added to adjust the pH of the reaction system, and the reaction was carried out under reflux.

[0010] (2) After the reaction was completed, the mixture was cooled to room temperature to obtain a large amount of blue-green strip-shaped crystals. After washing with ethanol and diethyl ether, the mixture was dried under vacuum to obtain the binuclear copper(II) complex.

[0011] Further, the molar ratio of Cu(NO3)2·3H2O to 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride in step (1) is 1:1 to 1:2.

[0012] Further, the alcohol solvent mentioned in step (1) is any one of methanol, ethanol, a mixed solvent of methanol and water, or a mixed solvent of ethanol and water.

[0013] Furthermore, the preheating temperature in step (1) is 50~80 ℃, the reflux temperature is 65~85 ℃, and the time is 2~4 h.

[0014] Furthermore, the weak base mentioned in step (1) is triethylamine, and the reaction system is weakly acidic, neutral, or weakly alkaline with pH > 6.

[0015] The binuclear copper(II) complex provided by this invention can efficiently inhibit PTP1B activity, and its IC 50 The value was 0.06 μM, and the complex inhibited the IC50 of PTP1B. 50 The value is its inhibition of TCPTP activity (IC). 50 The concentration (value 1.15 μM) is about 1 / 19 of that of PTP1B, showing that the complex selectively inhibits PTP1B activity and can be used as a highly efficient and specific inhibitor of PTP1B activity.

[0016] The binuclear copper(II) complex provided by this invention inhibited tumor cell proliferation by IC50 after 48 hours of treatment with MCF7 and HeLa cells. 50 The values ​​were 6.78 and 2.10 μM, respectively. This inhibitory ability was stronger than that of cisplatin against the two cell types reported in the literature, showing highly effective anti-breast cancer and anti-cervical cancer activity, and can be used as a potential anti-breast cancer drug and anti-cervical cancer drug.

[0017] Advantages and effects of the present invention:

[0018] The binuclear copper complex of this invention is obtained under conventional reflux reaction conditions, with a simple preparation process, high yield and purity, and novel structure with excellent biological properties. The complex can efficiently and specifically inhibit PTP1B activity, and can serve as a PTP1B target inhibitor. Furthermore, the complex exhibits superior anti-breast cancer and anti-cervical cancer cell proliferation activity compared to cisplatin, making it a novel multifunctional binuclear copper(II) complex. Attached Figure Description

[0019] Figure 1. Crystal structure diagram of the binuclear copper(II) complex of the present invention.

[0020] Figure 2 The π-π interaction diagram of the binuclear copper(II) complex of the present invention (symmetry code: i = -1+x, y, z; ii = -1+x, 1+y, z).

[0021] Figure 3. X-ray powder diffraction pattern of the binuclear copper(II) complex of the present invention at 298 K (experimental and simulation diagram).

[0022] Figure 4. IC50 of the present invention’s binuclear copper(II) complex inhibiting PTP1B and TCPTP activity. 50 Value determination chart.

[0023] Figure 5. MTT assay showing the inhibition of MCF7 cell proliferation by the binuclear copper(II) complex of this invention.

[0024] Figure 6. MTT assay of the inhibition of HeLa cell proliferation by the binuclear copper(II) complex of the present invention. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Example 1. Preparation method of the complex of the present invention.

[0027] Weigh 0.0520 g of ligand L (0.2 mmol) and add it to a round-bottom flask containing 12 mL of preheated methanol. Heat and reflux at 75 °C with stirring until the ligand is completely dissolved. Then weigh 0.0500 g of Cu(NO3)2·3H2O (0.2 mmol) and dissolve it in 2 mL of methanol. Add the dissolved copper nitrate solution to the ligand solution. Adjust the pH to neutral with triethylamine. Continue heating and reflux for 3 h to stop the reaction. A large number of blue-green strip-shaped crystals appear in the flask. After rapidly cooling the reaction solution with running ice water, filter it. The filtrate can continue to precipitate the above-mentioned blue-green strip-shaped crystals after being left at room temperature for about three to four days to volatilize. The yield is about 87.9%.

[0028] Example 2. X-ray diffraction experimental conditions and crystal structure analysis of single crystals of the complexes of the present invention.

[0029] High-quality single crystals of various complexes were selected and mounted on a Bruker Smart CCD single-crystal diffractometer. Using a graphite monochromator Mo-Ka (λ = 0.71073 Å) target as the radiation source, diffraction data of the complex crystals were collected using a ω-2θ scanning method. The data were then refined and analyzed using SHELXS-97 software to obtain the crystal structure and π-π interaction diagram of the complexes, as shown below. Figure 1 and Figure 2 As shown, detailed crystallographic information of the coordination compounds is shown in Table 1.

[0030] Table 1 Crystallographic data of the coordination compounds

[0031]

[0032] Example 3. X-ray powder diffraction experimental conditions and results analysis of the complexes of the present invention.

[0033] X-ray powder diffraction experiments were conducted using a Bruker D8 instrument from Germany. The test conditions were as follows: Cu-Kα radiation source, scanning rate of 2º / min, and scanning range of 5~50º.

[0034] X-ray powder diffraction results showed that the crystalline sample had a homogeneous phase, and the experimental diffraction pattern was consistent with the powder diffraction pattern simulated based on the crystal structure. Figure 3 .

[0035] Example 4. IC50 of the complex of the present invention inhibiting the activity of PTP1B and TCPTP 50 Value determination.

[0036] IC 50 Measurement principle:

[0037] IC 50 IC50 refers to the concentration of an inhibitor that reduces enzyme activity to half of its original activity. It is one of the standards for evaluating the inhibitory effect of an inhibitor. 50 The smaller the value, the stronger the inhibitory effect. Protein tyrosine phosphatases (PTPs) can decompose their substrate, disodium p-nitrophenyl phosphate (pNPP), into yellow p-nitrophenol (pNP). pNP has strong ultraviolet absorption at 405 nm, and its molar extinction coefficient ε = 1.78 × 10⁻⁶. 4 (mol -1 ·L·cm) -1 In the PTPs inhibition experiment, the change in enzyme activity can be indirectly detected by detecting the change in absorbance at 405 nm.

[0038] IC 50 Experimental steps for IC50 determination: First, add 83 µL of enzyme-containing MOPS buffer solution (pH = 7.2) of appropriate concentration to each of the first three rows of the microplate. Add the same volume of MOPS solution (without enzyme) to the fourth row as a control. Then, add 10 µL of DMSO solution of the complex at different concentration gradients in each column, with the concentration increasing from left to right. Incubate in a 37°C water bath for 30 min. Then, add 2 µL of substrate pNPP to each well, gently shake to start the reaction, and stop the reaction by adding 5 µL of NaOH solution (2 M) after the solution turns yellow. Measure the absorbance of each well at 405 nm using a microplate reader. Use Origin to process the data and fit the curve to obtain the IC50 value. 50 Value. Repeat the experiment three times to reduce experimental error.

[0039] Experimental results showed that the binuclear copper complex could inhibit the activity of PTP1B and TCPTP, such as Figure 4 As shown, its IC 50 The values ​​were 0.06 and 1.15 μM, respectively. The IC50 values ​​were compared. 50 The magnitude of the values ​​indicates that the complex can efficiently and selectively inhibit PTP1B activity and can serve as a target inhibitor of PTP1B.

[0040] Example 5. IC50 of the complex of the present invention on the inhibition of MCF7 and HeLa cell proliferation 50 Value determination.

[0041] The principle of MTT assay for determining the inhibitory effect of complexes on cell proliferation:

[0042] MTT is a yellow thiazole salt that is readily soluble in water and slightly soluble in organic solvents. It is commonly used to detect cell viability. The principle is that MTT can be catalyzed and oxidized by succinate dehydrogenase in the mitochondria of all living cells to produce formazan crystals. Dissolving the formazan in DMSO yields a blue-purple solution. Measuring the absorbance of this solution at 490 nm using a microplate reader allows for the determination of the number of viable cells.

[0043] MTT assay procedure: Tumor cells grown to the logarithmic growth phase were digested in a clean bench, centrifuged to remove supernatant, resuspended, and counted. Appropriate densities were seeded into 96-well plates according to different culture times and incubated overnight. After cell adhesion, DMSO solutions with final concentrations of 0, 1, 5, 10, 25, and 50 µM complex, along with corresponding maximum concentrations of CuCl2·2H2O and ligand L in DMSO solutions, were added to the prepared 96-well plates, with 6 replicates per group. After incubation for 24 h, 48 h, and 72 h, 20 µL of LMTT solution was added to each well, and incubation continued for 4 h. The liquid in the wells was aspirated, and 150 µL of DMSO solution was added to each well. The plates were shaken for 10 min to completely dissolve the formazan crystals at the bottom. The absorbance of the solution at 490 nm was measured using a microplate reader. Data were processed using Origin to obtain cell viability at each concentration, and the IC50 inhibitory effect was obtained through fitting. 50 The value is used to assess the inhibitory effect of the complex on cell proliferation.

[0044] Experimental results show that, see Figure 5 and Figure 6 The binuclear copper complex effectively inhibited the proliferation of MCF7 and HeLa cells, and its IC50 value for inhibiting tumor cell proliferation was significantly reduced after 48 hours of treatment with MCF7 and HeLa cells. 50 The values ​​were 6.78 and 2.10 μM, respectively. This inhibitory ability was stronger than that of cisplatin against the two cell types reported in the literature, demonstrating highly efficient anti-breast cancer and anti-cervical cancer cell proliferation activity.

[0045] The embodiments described above are merely specific examples of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A binuclear copper(II) complex, characterized in that, The structural formula is: ； The crystal of this complex belongs to the monoclinic crystal system. P twenty one / n Space group, cell parameters are: a = 8.964(3) Å, b =12.541(5) Å, c = 16.022(6) Å, α = 90º, β = 104.641(9)°, γ = 90º.

2. A method for preparing the binuclear copper(II) complex according to claim 1, characterized in that, Includes the following steps: (1) Cu(NO3)2·3H2O and 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride were added to a preheated alcohol solvent, a weak base was added to adjust the pH of the reaction system, and the reaction was carried out under reflux. (2) After the reaction was completed, the mixture was cooled to room temperature to obtain a large amount of blue-green strip-shaped crystals. After washing with ethanol and diethyl ether, the mixture was dried under vacuum to obtain the binuclear copper(II) complex. The alcohol solvent mentioned in step (1) is any one of methanol, ethanol, a mixture of methanol and water, or a mixture of ethanol and water.

3. The method for preparing the binuclear copper(II) complex as described in claim 2, characterized in that, The molar ratio of Cu(NO3)2·3H2O to 4-[(3-(pyridin-2-yl)-1H-pyrazol-1-yl)methyl]benzonitrile hydrochloride in step (1) is 1:1 to 1:

2.

4. The method for preparing the binuclear copper(II) complex as described in claim 2, characterized in that, The preheating temperature in step (1) is 50~80℃, the reflux temperature is 65~85℃, and the time is 2~4 h.

5. The method for preparing the binuclear copper(II) complex as described in claim 2, characterized in that, The weak base mentioned in step (1) is triethylamine, and the reaction system is weakly acidic, neutral or weakly alkaline with pH > 6.

6. The use of the binuclear copper(II) complex of claim 1 in the preparation of highly efficient and specific PTP1B inhibitors.

7. The use of the binuclear copper(II) complex according to claim 1 in the preparation of an anti-breast cancer drug.

8. The use of the binuclear copper(II) complex according to claim 1 in the preparation of an anti-cervical cancer drug.

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