CN112125926B - Novel chlorine-containing carboxylic acid metal complex, preparation method and application thereof - Google Patents

Abstract

The invention provides a novel chlorine-containing carboxylic acid metal complex, its preparation method and application, and belongs to the technical field of drug synthesis. The coordination structure diagram of the metal complex is shown in Fig. 1 . The preparation method of this novel chlorine-containing carboxylic acid metal complex comprises the following steps: dissolving 2-chloro-5-nitrobenzoic acid and 2-amino-6-methoxybenzothiazole in a solvent, stirring and dissolving, and preparing Mixed solution: dissolving copper nitrate in a solvent, stirring and dissolving to obtain a copper nitrate solution, adding the copper nitrate solution dropwise to the mixed solution, after the addition is complete, adjusting the pH between 5-7, stirring and reacting for a period of time at room temperature, sealing The film is placed in a cool place, and after standing for 12‑28d, brown blocky crystals are precipitated. The metal complex provided by the invention has good anticancer activity and the preparation method is simple.

Description

A novel chlorinated carboxylic acid metal complex, preparation method and use thereof

Technical Field

The invention relates to the technical field of drug synthesis, and in particular to a novel chlorine-containing carboxylic acid metal complex, a preparation method and application thereof.

Background Art

Deoxyribonucleic acid (DNA) is an important carrier of genetic information in life and is involved in the expression of genetic information in cells. DNA is the target molecule of many drugs. Studying the interaction between drugs and DNA helps to understand the medicinal mechanism of drug molecules and provide a theoretical basis for the design and development of new drugs. Metal complexes have potential applications in the biomedical field. Therefore, establishing an in vitro model of metal complexes binding to DNA and studying the binding mode, binding site and interaction between the two are of great significance for the design and in vitro screening of anti-tumor drugs.

HSA is an abundant carrier protein in the human circulatory system. It has multiple binding sites and can bind to many endogenous and exogenous substances, thus playing an important role in storage and transportation. Studying the interaction between HSA and metal complexes is of great significance to clarify the transport and pharmacological mechanism of metal complexes in the body.

In recent years, the medicinal value of metal complexes has gradually been discovered and applied in clinical practice. However, the clinical use of platinum metal drugs is hindered due to their large toxic side effects and strong drug resistance. Therefore, finding new metal complexes as anticancer drugs has become a hot research area.

Summary of the invention

The purpose of the present invention is to provide a novel chlorinated carboxylic acid metal complex, a preparation method and use thereof in view of the above-mentioned problems. The complex provided by the present invention has good anticancer activity and the preparation method is simple.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

A novel chlorinated carboxylic acid metal complex, the coordination structure of the complex is as follows:

Wherein, the unit structure diagram of the complex is as follows:

The invention also provides a preparation method of the novel chlorine-containing carboxylic acid metal complex, comprising the following steps: dissolving 2-chloro-5-nitrobenzoic acid and 2-amino-6-methoxybenzothiazole in a solvent, stirring and dissolving to prepare a mixed solution; dissolving copper nitrate in the solvent, stirring and dissolving to obtain a copper nitrate solution, dripping the copper nitrate solution into the mixed solution, adjusting the pH to between 5 and 7 after the dripping is complete, stirring and reacting at room temperature for a period of time, sealing the film and placing in a cool place, and precipitating brown block crystals after standing for 12 to 28 days.

In the present invention, preferably, the molar ratio of 2-chloro-5-nitrobenzoic acid, 2-amino-6-methoxybenzothiazole and copper nitrate is 9:3:1.5-2.

In the present invention, preferably, the solvent is methanol.

In the present invention, preferably, 1.0 mol·L ^-1 NaOH solution is used to adjust the pH value.

In the present invention, preferably, the copper nitrate solution is added dropwise for 20-40 min, and the stirring reaction is continued for 20-50 min at room temperature.

The complex obtained by the present invention has good anti-cancer activity after experimental verification. Therefore, the present invention also provides the use of the novel chlorinated carboxylic acid metal complex in the preparation of cancer therapeutic drugs. More specifically, the use of the novel chlorinated carboxylic acid metal complex in the preparation of cervical cancer, lung cancer and liver cancer therapeutic drugs.

In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

1. The present invention provides a chlorinated carboxylic acid metal complex with anticancer activity and a preparation method thereof, and pharmacological analysis is performed to prove that the complex has good anticancer activity. The IC ₅₀ values of the complex after acting on cervical cancer cells Hela, lung cancer cells A549 and liver cancer cells HepG2 for 48 hours are 25.17 μM, 20.88 μM and 20.53 μM, respectively; the preparation method is simple, has low requirements on equipment, and is suitable for promotion and application in the field of anticancer.

2. The present invention confirms that the synthesised novel chlorinated carboxylic acid metal complex binds to CT-DNA in an insertion manner through ultraviolet absorption spectrum, fluorescence spectrum and viscosity; the synthesised novel chlorinated carboxylic acid metal complex binds to HSA in an insertion manner through ultraviolet absorption spectrum and fluorescence spectrum, and has a strong binding with HSA, with a binding constant _Ka = 3.344 × ¹⁰⁵ L·mol ^-1 and a binding site number of 1.

3. After the novel chlorocarboxylic acid metal complex synthesized by the present invention acted on HepG2 cells for 24h and 48h, the ratio of the number of cells in the G0/G1 phase increased with the increase of drug concentration, indicating that the complex blocked the cycle of HepG2 cells in the G0/G1 phase.

4. The novel chlorocarboxylic acid metal complex synthesized by the present invention has an effect on the apoptosis of HepG2 cells and can induce apoptosis of HepG2 cells.

【Brief Description of the Drawings】

FIG1 is a coordination structure diagram of the novel chlorinated carboxylic acid metal complex of the present invention.

FIG. 2 is a unit structure diagram of the novel chlorinated carboxylic acid metal complex of the present invention.

FIG3 is a TG diagram of the novel chlorinated carboxylic acid metal complex synthesized in the present invention.

FIG. 4 is a UV absorption spectrum of the interaction between the novel chlorocarboxylic acid metal complex synthesized by the present invention and calf thymus DNA (CT-DNA).

FIG5 is a fluorescence spectrum diagram of the interaction between the novel chlorocarboxylic acid metal complex synthesized by the present invention and calf thymus DNA (CT-DNA).

FIG6 is a graph showing the change in viscosity of calf thymus DNA (CT-DNA) after the novel chlorocarboxylic acid metal complex synthesized by the present invention acts on CT-DNA.

FIG. 7 is a UV absorption spectrum of the interaction between the novel chlorinated carboxylic acid metal complex synthesized by the present invention and human serum albumin (HSA).

FIG8 is a fluorescence spectrum diagram of the interaction between the novel chlorinated carboxylic acid metal complex synthesized by the present invention and human serum albumin (HSA).

FIG. 9 is a graph showing the test results of the cytotoxicity of the novel chlorinated carboxylic acid metal complex synthesized by the present invention to cervical cancer cells Hela, lung cancer cells A549 and liver cancer cells HepG2.

FIG. 10 is a graph showing the test results of the effect of the novel chlorinated carboxylic acid metal complex synthesized by the present invention on the cell cycle of liver cancer cells HepG2.

FIG. 11 is a graph showing the test results of the effect of the novel chlorocarboxylic acid metal complex synthesized by the present invention on apoptosis of HepG2 liver cancer cells.

[Specific implementation method]

It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. It should also be understood that when the terms "comprise" and/or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and/or combinations thereof.

In order to obtain more complexes with higher anticancer activity, the present invention provides the following chlorinated carboxylic acid metal complexes after research and verification. In a typical embodiment of the present invention, a novel chlorinated carboxylic acid metal complex is provided, and the coordination structure diagram of the complex is shown in Figure 1. Among them, the unit structure diagram of the complex is shown in Figure 2.

In this embodiment, 2-chloro-5-nitrobenzoic acid and 2-amino-6-methoxybenzothiazole are used as ligands, and react with copper nitrate to generate a hexacoordinated complex centered on copper ions. This structure greatly improves the anticancer activity of the complex.

The novel chlorinated carboxylic acid metal complex with anticancer activity belongs to the triclinic system and the P-1 space group, and the unit cell parameters are a=7.7070(4)nm, b=9.2158(4)nm, c=12.2725(6)nm; α=103.303(4)°, β=104.468(4)°, γ=96.042(4)°; Z=1, Dc=1.694mg/m ³ , F(000)=419, and the final structure residual factor R ₁ =0.0266, wR ₂ =0.0727.

In some embodiments of the present invention, a preparation method of the novel chlorine-containing carboxylic acid metal complex is also provided, comprising the following steps: dissolving 2-chloro-5-nitrobenzoic acid and 2-amino-6-methoxybenzothiazole in a solvent, stirring and dissolving to prepare a mixed solution; dissolving copper nitrate in a solvent, stirring and dissolving to obtain a copper nitrate solution, and dripping the copper nitrate solution into the mixed solution. After the dripping is complete, adjusting the pH to between 5 and 7, stirring and reacting at room temperature for a period of time, sealing the film and placing in a cool place, and precipitating brown block crystals after standing for 12 to 28 days.

In order to make the ligand raw materials react fully and obtain the target complex, the molar ratio of 2-chloro-5-nitrobenzoic acid, 2-amino-6-methoxybenzothiazole and copper nitrate is 9:3:1.5-2.

In order to make the ligand raw materials dispersed evenly and react fully, and at the same time improve the separation and purification efficiency of the product, the preferred solvent is methanol.

In order to precipitate the ligand compound, the pH value was adjusted using a 1.0 mol·L ^-1 NaOH solution.

For better reaction, preferably, the stirring reaction time at room temperature is 20-50 minutes.

In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

1. Preparation Example

Example 1

Synthesis of the complex: 2-chloro-5-nitrobenzoic acid (0.9 mmol) and 2-amino-6-methoxybenzothiazole (0.3 mmol) were dissolved in 5 ml of methanol, stirred and dissolved to prepare a mixed solution; copper nitrate (0.15 mmol) was dissolved in 10 ml of methanol solvent, stirred and dissolved to obtain a copper nitrate solution, and the copper nitrate solution was added dropwise to the mixed solution within 20 minutes. The solution showed a yellow-green color. After the addition was complete, a 1.0 mol·L ^-1 NaOH solution was used to adjust the pH to 5. The reaction was stirred at room temperature for 20 minutes, and the film was sealed and placed in a cool place. After standing for 12 days, brown block crystals were precipitated.

Example 2

Synthesis of the complex: 2-chloro-5-nitrobenzoic acid (0.9 mmol) and 2-amino-6-methoxybenzothiazole (0.3 mmol) were dissolved in 5 ml of methanol, stirred and dissolved to prepare a mixed solution; copper nitrate (0.18 mmol) was dissolved in 10 ml of methanol solvent, stirred and dissolved to obtain a copper nitrate solution, and the copper nitrate solution was added dropwise to the mixed solution within 30 minutes. The solution showed a yellow-green color. After the addition was complete, a 1.0 mol·L ^-1 NaOH solution was used to adjust the pH to 6. The reaction was stirred at room temperature for 30 minutes, the film was sealed and placed in a cool place, and brown block crystals were precipitated after standing for 20 days.

Example 3

Synthesis of the complex: 2-chloro-5-nitrobenzoic acid (0.9 mmol) and 2-amino-6-methoxybenzothiazole (0.3 mmol) were dissolved in 5 ml of methanol, stirred and dissolved to prepare a mixed solution; copper nitrate (0.2 mmol) was dissolved in 10 ml of methanol solvent, stirred and dissolved to obtain a copper nitrate solution, and the copper nitrate solution was added dropwise to the mixed solution within 40 minutes. The solution showed a yellow-green color. After the addition was complete, a 1.0 mol·L ^-1 NaOH solution was used to adjust the pH to 7. The reaction was stirred at room temperature for 50 minutes, the film was sealed and placed in a cool place, and brown block crystals were precipitated after standing for 28 days.

2. Confirmation of the complex

The brown block crystals prepared in Example 1-3 were subjected to infrared testing, elemental analysis testing and crystallographic testing, and the results were as follows:

Infrared data: R(KBr)ν/cm ^-1 :3409,2918,2849,1612,1547,1473,1463,1343,1280,1212,1061,729,719.

Elemental analysis data: Elemental analysis according to [C ₃₀ H ₂₂ Cl ₂ CuN ₆ O ₁₀ S ₂ ], calculated value (%): C, 43.67; H, 2.69; N, 10.19. Found value (%): C, 43.42; H, 2.75; N, 10.60.

Crystallographic data: The crystallographic data, main bond lengths and bond angles, and main torsion angles of the complex [C ₃₀ H ₂₂ Cl ₂ CuN ₆ O ₁₀ S ₂ ] are listed in Tables 1, 2, and 3, respectively.

Table 1 Crystallographic data of the complexes

Table 2 Main bond lengths and bond angles of complexes

#1-x+2,-y+1,-z+2; #1-x+2,-y+1,-z+2;

Table 3 Main torsion angles of the complexes

#1-x+2,-y+1,-z+2

3. Test of thermodynamic stability of complexes:

The thermal stability of the complex was determined using a HQT-4 fully automatic microcomputer differential thermal analyzer. 9.8 mg of the crystal was weighed and placed in a clean crucible, with nitrogen as the protective gas, a flow rate of 15 mL/min, and a heating rate of 10°C/min, and the thermal stability of the complex was tested within 25.00-800°C.

Fig. 3 is a TG diagram of a novel chlorinated carboxylic acid metal complex synthesized by the present invention. It can be seen that the complex can maintain a stable crystal form at room temperature, and with the increase of temperature, the complex has three weight loss stages. Before 216.43°C, the weight loss rate is 8.40%, which may be that the complex loses 1 chloride ion (theoretical value is 8.48%); at 216.43-390.16°C, the total weight loss is 44.28%, which may be that the complex loses 1 2-chloro-5-nitrophenyl and 1 methoxy group (theoretical value 45.46%); at 390.16-767.67°C, the complex is still decomposing, and at 767.67°C, the sample residue is 22.41%, and Cu may exist in the form of CuO ₂ or CuO ₃ (theoretical value of CuO ₂ is 23.16%, theoretical value of CuO ₃ is 27.03%).

IV. Ultraviolet absorption spectrum of the interaction between the complex of the present invention and DNA

Solution preparation:

Tris-HCl/NaCl buffer: Weigh 50 mmol (2.9008 g) of NaCl solid and 5 mmol (0.6057 g) of Tris (hydroxymethylaminomethane) respectively, dissolve in 1 L of double distilled water, and then adjust the pH value to 7.2 with dilute hydrochloric acid for later use.

CT-DNA solution: Weigh an appropriate amount of CT-DNA and dissolve it in Tris-HCl/NaCl buffer solution (pH=7.2). Measure its absorbance at 260nm and 280nm. If _A260 / _A280 =1.8-1.9, it means that it is basically protein-free and no further treatment is required. Measure the absorbance _A260 (ε=6600L·mol ^-1 ·cm ^-1 ) of CT-DNA at 260nm, and then calculate its concentration according to formula (1):

[DNA]＝K×A ₂₆₀ /6600(mol·L ^-1 ) (1)

K is the dilution factor. The prepared CT-DNA solution was placed in a 4°C refrigerator and used within 3 days.

Ultraviolet absorption spectrum of the novel chlorinated carboxylic acid metal complex synthesized by the present invention and CT-DNA: Scan the baseline with a Tris-HCl/NaCl buffer solution of pH=7.2, and subtract the blank background. Add 2.5mL of Tris-HCl/NaCl buffer solution and 5×10 ^-5 mol·L ^-1 complex solution to a blank cuvette (reference cell) and a sample cuvette (sample cell), respectively, and measure the ultraviolet absorption spectrum within the range of 200-450nm. Use a pipette to drop 20μL of 2mmol·L ^-1 CT-DNA solution into the reference cell and the sample cell, respectively, for a total of 15 times. After each drop, react for 5min, and then scan the complex solution.

FIG4 is a UV absorption spectrum of the interaction between the novel chlorocarboxylic acid metal complex synthesized by the present invention and calf thymus DNA (CT-DNA). It can be seen that as the concentration of CT-DNA increases, the complex has a weak hypochromic effect at 269nm, which indicates that the complex has an insertion effect on CT-DNA. The binding constant of the interaction between the complex and CT-DNA and its UV molar absorption coefficient conform to formula (2):

[DNA]/(ε _a -ε _f )=[DNA]/[(ε _b -ε _f )+1/K _b (ε _b -ε _f )] (2)

Where ε _a is the molar extinction coefficient of the coordination compound at different CT-DNA concentrations (L/(mol·cm)), ε _f is the molar extinction coefficient of the coordination compound when no CT-DNA is added (L/(mol·cm)), and ε _b is the molar extinction coefficient of the coordination compound when it is completely bonded to CT-DNA (L/(mol·cm)). The binding constant of the complex with DNA, K _b = 4.54×10 ² L·mol ^-1 , was calculated.

5. Fluorescence spectrum of the complex synthesized by the present invention interacting with CT-DNA

Mix 8μmol·L ^-1 ethidium bromide (EB) and 10μmol·L ^-1 CT-DNA solution in equal volumes (2.5mL) and react for 12h. Add 2.5mL of the EB-CT-DNA mixed solution into the sample pool and measure the emission spectrum in the wavelength range of 540-700nm at an excitation wavelength of 525nm and a scanning speed of 240nm·s ^-1 . Add 20μL of the complex solution with a concentration of 1mmol·L ^-1 to the EB-CT-DNA system, and add it 10 times in total. After each reaction for 5min, measure its emission spectrum.

FIG5 is a fluorescence spectrum of the interaction between the novel chlorocarboxylic acid metal complex synthesized by the present invention and calf thymus DNA (CT-DNA). It can be seen that the maximum emission wavelength of EB-CT-DNA is at 585nm, and the fluorescence intensity gradually decreases as the concentration of the complex increases. Under different r=[Complex]/[DNA] values, the initial fluorescence intensity drops from 97.63% to 69.56%. This indicates that the complex replaces a considerable number of EB molecules in the EB-CT-DNA system, freeing EB from the CT-DNA molecules, resulting in a decrease in the fluorescence intensity of the EB-CT-DNA system. It is inferred that the interaction mode between the complex and CT-DNA is similar to that of EB, which is a classic insertion mode.

The quenching of fluorescent molecules by fluorescence quenchers can be divided into dynamic quenching and static quenching. Dynamic quenching is the fluorescence quenching caused by the collision between the quencher and the excited state molecules of the fluorescent molecules. If the collision of the complex with the EB-CT-DNA molecule is regarded as dynamic quenching, according to the Stern-Volmer equation:

I ₀ /I＝1+K _q τ ₀ [C]＝1+K _sv [C] (3)

Where I and I ₀ are the fluorescence intensities of EB-CT-DNA when the complex is added and not added, respectively, K _q is the rate constant of the molecular quenching process, K _sv is the dynamic quenching constant, τ ₀ is the average lifetime of the fluorescent molecule when the quencher is not present, [C] is the concentration of the quencher, and the lifetime of the fluorescent molecule is about 10 ^-8 s. The quenching rate constant K _q = 1.994×10 ¹¹ L·mol ^-1 ·s ^-1 is obtained. Obviously, the quenching constant of the complex on EB-CT-DNA is much larger than the collision quenching constant 2.0×10 ¹⁰ L·mol ^-1 ·s ^-1 controlled by the maximum diffusion between the drug small molecule and the biological macromolecule. Therefore, the fluorescence quenching of the complex on EB-DNA is not due to dynamic quenching caused by intermolecular collision, but static quenching. The binding constant of the interaction between the complex and DNA is _Ka = 5.845×10 ⁴ L·mol ^-1 , and the binding site n = 1.

VI. Effect of the novel chlorocarboxylic acid metal complex synthesized by the present invention on the viscosity of CT-DNA

The reaction temperature was controlled at 29.0±0.1℃ by a thermostatic water bath, and 20mL of 200μmol·L ^-1 CT-DNA solution was added to the Ubbelohde viscometer. The concentration of the complex solution was gradually increased to make the concentration ratio of the complex to CT-DNA [Complex]/[DNA]=0, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, and the time (s) for the CT-DNA solution to flow through the effective scale of the capillary was recorded in sequence. Each group of experiments was repeated 3 times and the average value was taken. The relative viscosity of the measured solution was calculated according to the formula η=(tt ₀ )/t ₀ , where t ₀ is the time taken for the buffer solution to flow through the effective scale of the capillary, and t is the time taken for the complex of different concentrations to flow through the effective scale of the capillary when acting on the CT-DNA solution. By plotting [Complex]/[DNA] with (η/η ₀ ) ^1/3 (η ₀ is the relative viscosity of the CT-DNA solution without complex), we can obtain a graph showing the effect of the complex on DNA viscosity. Ethidium bromide (EB) was used as a positive control.

Figure 6 is a graph showing the viscosity change of the novel chlorocarboxylic acid metal complex synthesized by the present invention and calf thymus DNA (CT-DNA). It can be seen that, similar to EB, as the concentration of the complex increases, the viscosity of DNA increases, indicating that the complex binds to DNA in an intercalating manner.

VII. Interaction between the novel chlorinated carboxylic acid metal complex synthesized by the present invention and human serum albumin (HSA)

1. Ultraviolet absorption spectrum of the complex synthesized by the present invention and HSA:

Solution preparation:

Tris-HCl/NaCl buffer (pH=7.4): Weigh 0.05 mol of tris(hydroxymethyl)aminomethane and 0.15 mol of NaCl solid respectively and dissolve them in 1 L of double distilled water, and adjust the pH value to 7.4 with dilute hydrochloric acid solution for later use.

Preparation of human serum albumin (HSA) solution: Weigh an appropriate amount of HSA and dissolve it in a Tris-HCl-NaCl (pH=7.4) buffer solution. After it is completely dissolved, measure its concentration. The concentration of HSA solution can be measured by using a UV-visible spectrophotometer to measure its absorbance at 280nm, and then calculate its concentration according to the following formula (4):

[HSA]＝K×A ₂₈₀ /35700mol·L ^-1 (4)

Where K is the dilution factor. The prepared HSA stock solution is generally stored at 4°C and used within 4 days.

The ultraviolet absorption spectrum of the novel chlorinated carboxylic acid metal complex synthesized by the present invention and HSA is as follows: 2.5 mL of 200 μmol·L ^-1 HSA solution is added to the sample cell, and the same amount of Tris-HCl/NaCl buffer solution is added to the reference cell, and the ultraviolet absorption spectrum thereof in the wavelength range of 190 to 400 nm is measured. 2.5 μL of 0.25 μmol·L ^-1 complex solution is added to the reference cell and the sample cell respectively by a pipette, for a total of 9 times, and each time the addition is completed, the solution is placed at room temperature for 5 minutes before scanning and measuring.

Figure 7 is a UV absorption spectrum of the interaction between the novel chlorinated carboxylic acid metal complex synthesized by the present invention and human serum albumin (HSA). The results show that as the concentration of the complex increases, the absorption peak at 218nm undergoes a hypochromic effect, and the absorption peak position has a slight red shift phenomenon, indicating that the complex interacts with HSA.

2. Fluorescence spectrum of the complex synthesized by the present invention and HSA:

2.5mL of 5μmol·L ^-1 HSA solution was added to the sample pool, and the fluorescence emission spectrum of the complex was measured at an excitation wavelength of 280nm and a wavelength range of 300-500nm. Then 1μL of 1mmol·L ^-1 complex solution was added to the sample pool with a pipette, for a total of 11 times. After each addition, it was placed at room temperature for 5min before scanning and measurement. FIG8 is a fluorescence spectrum of the interaction between the novel chlorocarboxylic acid metal complex synthesized by the present invention and human serum albumin (HSA). It can be seen that as the concentration of the complex increases, the fluorescence intensity of HSA decreases. At the same time, a slight red shift occurs at the maximum emission wavelength (312nm). At different r=[Complex]/[HSA] values, the initial fluorescence intensity drops from 91.27% to 28.59%. It shows that the complex has a quenching effect on the fluorescence of HSA.

The quenching intensity can be calculated using the Stern-Volmer equation (5):

F ₀ /F＝1+K _sv [Q]＝1+K _q τ ₀ [M] (5)

Where F ₀ and F represent the fluorescence intensity of the fluorescent molecule in the absence and presence of the quencher, respectively, K _sv is the quenching constant, K _q is the bimolecular quenching reaction rate constant, τ ₀ is the average lifetime of the fluorescent molecule in the absence of the quencher (τ ₀ = 10 ^-8 s), and [M] is the concentration of the complex. The quenching rate constant K _q = 6.516×10 ¹² L·mol ^-1 ·s ^-1 is obtained, which is greater than the collision quenching constant value of 2.0×10 ¹⁰ L·mol ^-1 ·s ^-1 for the maximum diffusion control of various quenchers on biomacromolecules, indicating that the fluorescence quenching of HSA by the complex is a static quenching process.

The binding constant and number of binding sites of the complex and human serum protein HSA can be obtained by formula (6):

log[F ₀ -F]＝logK _a +nlog[M] (6)

In the formula, _Ka value is the binding constant, n is the number of binding sites, and the linear fitting calculation of the binding constant _Ka = 3.344 × 10 ⁵ L·mol ^-1 and the number of binding sites n = 1 indicates that the binding interaction between the two is strong.

8. Cytotoxicity of the novel chlorinated carboxylic acid metal complex synthesized by the present invention to tumor cells:

The cytotoxicity of the complex to cervical cancer cells Hela, lung cancer cells A549 and liver cancer cells HepG2 was determined by using an enzyme marker. Figure 9 is a cytotoxicity diagram of the novel chlorinated carboxylic acid metal complex synthesized by the present invention on lung cancer cells A549, cervical cancer cells Hela and liver cancer cells HepG2. The IC ₅₀ values of the novel chlorinated carboxylic acid complex and lung cancer cells A549, cervical cancer cells Hela and liver cancer cells HepG2 after 48 hours of action were calculated to be 25.17 μM, 20.88 μM and 20.53 μM, respectively. This shows that the chlorinated carboxylic acid metal complex synthesized by the present invention has good anticancer activity against tumor cells, so it can be used in the preparation of cancer therapeutic drugs, especially in the application of cervical cancer, lung cancer and liver cancer therapeutic drugs.

IX. Effect of the novel chlorinated carboxylic acid metal complex synthesized by the present invention on the cell cycle of HepG2 liver cancer cells:

The effect of the metal complex on the cell cycle of liver cancer cells HepG2 was determined by flow cytometry, and the effect of the novel chlorocarboxylic acid metal complex synthesized by the present invention on the cell cycle of liver cancer cells HepG2 was obtained. FIG10 is a graph showing the effect of the novel chlorocarboxylic acid metal complex synthesized by the present invention on the cell cycle of liver cancer cells HepG2.

It can be seen that after the new copper metal complex acts on HepG2 cells for 24h and 48h, the ratio of the number of cells in the G0/G1 phase gradually increases with the increase of drug concentration, indicating that the complex blocks the cycle of HepG2 cells in the G0/G1 phase.

10. Effect of the chlorocarboxylic acid metal complex synthesized by the present invention on apoptosis of HepG2 liver cancer cells

The effect of the novel metal complex on apoptosis of HepG2 liver cancer cells was measured by flow cytometry, and the results are shown in Figure 11. The effect of the novel chlorocarboxylic acid metal complex synthesized by the present invention on apoptosis of HepG2 liver cancer cells. The results show that the metal complex has an effect on apoptosis of HepG2 cells and can induce apoptosis of HepG2 cells.

The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims (8)

1. A novel chlorinated carboxylic acid metal complex, characterized in that the crystal structure parameters, main bond lengths and bond angles, and main torsion angle data of the complex are shown in Tables 1, 2, and 3 below: Table 1 Crystallographic data of the complexes

Table 2 Main bond lengths and bond angles of complexes

#1-x+2,-y+1,-z+2; #1-x+2,-y+1,-z+2; Table 3 Main torsion angles of the complexes

#1-x+2,-y+1,-z+2. 2. The method for preparing a novel chlorine-containing carboxylic acid metal complex according to claim 1, characterized in that it comprises the following steps: dissolving 2-chloro-5-nitrobenzoic acid and 2-amino-6-methoxybenzothiazole in a solvent, stirring and dissolving to prepare a mixed solution; dissolving copper nitrate in a solvent, stirring and dissolving to obtain a copper nitrate solution, and dropping the copper nitrate solution into the mixed solution. After the dropping is complete, adjusting the pH to between 5 and 7, stirring and reacting at room temperature for a period of time, sealing the film and placing it in a cool place, and precipitating brown block crystals after standing for 12 to 28 days. 3. The method for preparing a novel chlorinated carboxylic acid metal complex according to claim 2, characterized in that the molar ratio of the 2-chloro-5-nitrobenzoic acid, 2-amino-6-methoxybenzothiazole and copper nitrate is 9:3:1.5-2. 4. The method for preparing a novel chlorinated carboxylic acid metal complex according to claim 2, wherein the solvent is methanol. 5. The method for preparing a novel chlorinated carboxylic acid metal complex according to claim 2, characterized in that a 1.0 mol·L ^-1 NaOH solution is used to adjust the pH value. 6. The method for preparing a novel chlorinated carboxylic acid metal complex according to claim 2, characterized in that the copper nitrate solution is added dropwise for 20-40 min, and the stirring reaction is continued for 20-50 min at room temperature. 7. Use of the novel chlorinated carboxylic acid metal complex according to claim 1 in the preparation of cancer therapeutic drugs. 8. Use of the novel chlorinated carboxylic acid metal complex according to claim 7 in the preparation of therapeutic drugs for cervical cancer, lung cancer and liver cancer.

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