CN113788782A

CN113788782A - Terpyridine derivative and synthesis method and application thereof

Info

Publication number: CN113788782A
Application number: CN202111141769.1A
Authority: CN
Inventors: 钟玉君; 顾运琼; 杨啟源; 陈振锋; 梁宏
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2021-12-14
Anticipated expiration: 2041-09-28
Also published as: CN113788782B

Abstract

The invention discloses a series of terpyridine derivatives, and a synthesis method and application thereof. The terpyridine derivative is prepared by taking 2-acetylpyridine and 3-bromosalicylaldehyde or 5-bromosalicylaldehyde as raw materials, adding ammonia water into low-carbon alcohol, and reacting under the condition that the pH value of a system is adjusted to be more than or equal to 10. Furthermore, the invention also discloses two cases of copper terpyridine complexes obtained by taking the obtained terpyridine derivatives as ligands and coordinating with copper. The terpyridine derivative and the copper complex have good inhibition effect on various tumor cell strains, and are expected to be used as anti-cancer drugs.

Description

Terpyridine derivative and synthesis method and application thereof

Technical Field

The invention relates to a terpyridine derivative and a synthesis method and application thereof, belonging to the technical field of medicines.

Background

The terpyridine and the derivatives thereof have application potential in the fields of supramolecular chemistry, asymmetric catalysis, photosensitivity, tumor resistance and the like. Recently, there have been studies onThe terpyridine derivative is found to have good anti-tumor activity, and Katarzyna Mala and the like can detect that the terpyridine derivative has good anti-tumor activity on various tumor cell strains: has high inhibitory effect on breast cancer (MCF-7), colorectal cancer (HCT 116), lung cancer (A549), pancreatic cancer (PANC-1) and malignant glioma (U-251), and IC₅₀All values are below 1. mu.M, especially IC for MCF-7 cell line₅₀A value of about 0.04. mu.M (Malarz K, Zych D, Kuczak M, et al. anticancer activity of4 '-phenyl-2, 2':6', 2' -terpyridines-while the metal completion [ J ]]A terpyridine derivative (4' - (1-decyl-2,3-triazol-4-yl) phenyl-2,2':6',2 "-terpyridine) was synthesized in European Journal of Medicinal Chemistry,2020,189: 112039-112050.). However, the terpyridine derivative has a relatively complex structure and contains not only phenyl, but also triazole and decyl.

Terpyridine is a heterocyclic compound consisting of three pyridine rings, and a stable complex can be formed by the terpyridine and metal due to a special structure. In recent years, terpyridine complexes have been widely studied because of their excellent DNA binding ability and antitumor activity. Such as lanthanide complexes with photoactivation antitumor activity synthesized by taking terpyridine derivatives as ligands, monofunctional ruthenium (II) complexes synthesized by taking terpyridine derivatives as ligands, and the like. Copper (II) terpyridine complexes have also been designed and synthesized, and these copper (II) complexes have nuclease activity, cytotoxicity, high DNA affinity, and antitumor activity. Biljana

The two terpyridyl copper (II) complexes are synthesized, in vitro experiments prove that the complexes have good anti-proliferation performance on human non-small cell lung cancer (A549), and molecular docking research shows that the two complexes can interact with a DNA major groove and have stronger affinity (Biljana)

G,Jasmina N-R,Tatjana I-T,et al.Synthesis,cytotoxic activity and DNA-binding properties of copper(II)complexes with terpyridine[J]Polyhedron,2018,139: 313-322). Sofia Gama et al with terpyridine and bipyridineSeveral novel copper (II) complexes were synthesized for ligands, which could lead to double-stranded DNA breaks in the absence of exogenous oxidants or reductants, and were highly cytotoxic (four times as much as cisplatin) to ovarian cancer A2780 cells (Gama S, Rodrigues I, Marques F, et al.New tertiary bipyridine-recurring chip (II) complexes-selecting-activating chemical molecules [ J].RSC.Advances,2014,4(106):61363-61377.)。

Disclosure of Invention

The invention aims to solve the technical problems of providing two terpyridine derivatives with novel structures and synthesis methods thereof, and a copper complex taking the terpyridine derivative as a ligand and a synthesis method and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the terpyridine derivative disclosed by the invention is a compound shown as the following formula (I) or a pharmaceutically acceptable salt thereof:

where, when R1 ═ Br, R2 ═ H (also referred to herein as H-L)^cOr ligand H-L^c) (ii) a When R1 ═ H, R2 ═ Br (also referred to herein as H-L)^dOr ligand H-L^d)。

The synthesis method of the terpyridine derivative comprises the steps of putting 2-acetylpyridine and 3-bromosalicylaldehyde or 5-bromosalicylaldehyde into low-carbon alcohol, adding ammonia water, adjusting the pH value of a system to be more than or equal to 10, reacting under the heating condition or the non-heating condition, recovering a solvent from the obtained reactant, and collecting solids to obtain the target compound. When 2-acetylpyridine and 3-bromosalicylaldehyde are used as raw materials, ligand H-L is correspondingly obtained^c(ii) a When 2-acetylpyridine and 5-bromosalicylaldehyde are used as raw materials, ligand H-L is correspondingly obtained^d。

In the synthesis method of the terpyridine derivative, the lower alcohol is an alcohol containing 1-4 carbon atoms, specifically is one or a combination of more than two of methanol, ethanol, n-propanol and n-butanol, and preferably is methanol and/or ethanol. The lower alcohol is preferably used in an amount capable of dissolving the raw materials participating in the reaction. Specifically, the total amount of the lower alcohols used by all raw materials is generally 2-3 mL calculated by taking 1mmol of 3-bromosalicylaldehyde or 5-bromosalicylaldehyde as a reference.

In the method for synthesizing the terpyridine derivative, the reaction is followed and detected by Thin Layer Chromatography (TLC), and the reaction is preferably carried out under heating conditions, and more preferably at 35 to 50 ℃. When the reaction is carried out at 35-50 ℃, the reaction time is usually controlled to be 20-48 h. The solid collected after completion of the reaction may be further washed with a solvent at the time of synthesis to remove unreacted materials, preferably with methanol and/or ethanol.

In the synthesis method of the terpyridine derivative, the target product is generated only in the presence of ammonia water, and the adding amount of the ammonia water is usually more than 1 time, preferably more than 1 time of the amount of the 3-bromosalicylaldehyde or 5-bromosalicylaldehyde substance. The molar ratio of the 2-acetylpyridine to the 3-bromosalicylaldehyde or the 5-bromosalicylaldehyde is a stoichiometric ratio, and the 2-acetylpyridine can be used in a relatively excessive amount in an actual operation process.

In the synthesis method of the terpyridine derivative, the pH value of a conventional alkaline substance adjusting system which can enable the system to reach the condition that the pH value is more than or equal to 10 in the prior art is adopted, and preferably, alkaline substances such as sodium hydroxide, potassium hydroxide, barium hydroxide and the like are adopted to adjust the pH value of the system. The pH of the adjustment system is more preferably 12 or more, and the pH of the adjustment system is more preferably 12 to 13.

The applicant discovers that the terpyridine derivative has a remarkable cell proliferation inhibition effect on various tumor cells through an in vitro cell growth inhibition test, so that the invention also comprises the application of the terpyridine derivative or the pharmaceutically acceptable salt thereof in preparing the antitumor drugs. Further, the present invention also includes a pharmaceutical composition comprising a therapeutically effective amount of the above terpyridine derivative or a pharmaceutically acceptable salt thereof.

The invention further comprises a terpyridine copper complex taking the terpyridine derivative as a ligand, which is shown in the following formula (II) or formula (III):

the synthetic method of the terpyridine copper complex comprises the following steps: taking terpyridine derivative shown in the formula (I) and copper perchlorate (Cu (ClO)₄)₂·6H₂O) is dissolved in a mixed solvent, the reaction is carried out under the heating condition, the reactant is cooled, crystals are separated out, and the crystals are collected, thus obtaining the copper terpyridyl complex; wherein the content of the first and second substances,

when R1 ═ Br and R2 ═ H in the terpyridine derivative represented by formula (I), and the mixed solvent is prepared from chloroform or dichloromethane and methanol in a ratio of 1: 11.5-12.5, the copper terpyridine complex (also called as complex 5 in the application) shown in the formula (II) is obtained;

when R1 ═ H, R2 ═ Br in the terpyridine derivative represented by formula (I), and the mixed solvent is prepared from dimethyl sulfoxide and methanol according to the weight ratio of 1: 24.5-25.5 volume ratio, the obtained copper terpyridine complex (also called complex 7 in the application) is shown in formula (III).

In the synthesis method of the terpyridine copper complex, the molar ratio of the terpyridine derivative shown in the formula (I) to the copper perchlorate is a stoichiometric ratio, and the amount of the copper perchlorate can be relatively excessive in the actual operation process.

In the synthesis method of the terpyridine copper complex, the reaction is preferably carried out at a temperature of not less than 35 ℃, more preferably at a temperature of not less than 40 ℃, and even more preferably at a temperature of 50-65 ℃. When the reaction is carried out at 50-65 ℃, the reaction time is usually controlled to be 24-72 h. The reaction usually uses a thick-walled hard glass tube with one end closed to contain the feed liquid. The mixed solvent is preferably used in such an amount that the raw materials participating in the reaction can be dissolved. Specifically, the total amount of the mixed solvent used for all the raw materials is usually 20 to 30mL based on 1mmol of the terpyridine derivative represented by formula (I), wherein when R1 ═ Br and R2 ═ H in the terpyridine derivative represented by formula (I), the total amount of the mixed solvent used for all the raw materials is more preferably 25 to 30 mL; when R1 ═ H, R2 ═ Br in the terpyridine derivative represented by formula (I), the total amount of the mixed solvent used for all the raw materials is more preferably 20 to 22 mL.

The applicant discovers that the copper terpyridyl complex has extremely obvious cell proliferation inhibition effect on various tumor cells through in vitro cell growth inhibition tests, so that the invention also comprises the application of the copper terpyridyl complex or pharmaceutically acceptable salts thereof in preparing antitumor drugs. Further, the invention also includes a pharmaceutical composition containing a therapeutically effective amount of the copper terpyridyl complex or the pharmaceutically acceptable salt thereof.

Compared with the prior art, the invention provides two terpyridine derivatives with novel structures and synthesis methods thereof, and two terpyridine copper complexes taking the terpyridine derivatives as ligands and synthesis methods thereof; the test results of the applicant show that the compound of the invention has good inhibition effect on various tumor cell strains, and is expected to be used as an anticancer drug.

Drawings

FIG. 1 shows the crystal structure (ellipsoid fraction 50%) of complex 5.

FIG. 2 shows the crystal structure (ellipsoid fraction 50%) of complex 7.

FIG. 3 is a diagram of ligands H-L^cAnd complex 5, wherein (a) is ligand H-L^c0H, (b) is ligand H-L^c48h, (c) is complex 50 h, and (d) is complex 548 h.

FIG. 4 shows ligands H-L^dAnd complex 7, wherein (a) is ligand H-L^d0H, (b) is ligand H-L^d48h, 70 h for (c), and 748 h for (d).

FIG. 5 is a graph showing the effect of the blank control and different concentrations of complex 5 on the cell cycle 48h after BEL-7402.

FIG. 6 is a graph showing the effect of different concentrations of complex 7 on the cell cycle 48h after BEL-7402.

FIG. 7 is a graph showing the effect of different concentrations of complex 5 on the cyclin expression of BEL-7402, wherein (a) is a graph showing the protein expression of different proteins of the complex 5 on BEL-7402 cells, and (b) is a histogram showing the protein expression of different proteins of the complex 5 on BEL-7402 cells, and the histogram shows blank control, 0.45. mu.M, 0.9. mu.M and 1.8. mu.M from left to right.

FIG. 8 is a graph showing the effect of different concentrations of complex 7 on the cyclin expression of BEL-7402 cells, wherein (a) is a graph showing the protein expression of different proteins of BEL-7402 cells by different concentrations of complex 7, and (b) is a histogram showing the protein expression of different proteins of BEL-7402 cells by different concentrations of complex 7, and the histogram shows blank control, 0.45. mu.M, 0.9. mu.M and 1.8. mu.M from left to right.

FIG. 9 is a graph showing the effect of different concentrations of complex 5 on apoptosis of BEL-7402 cells.

FIG. 10 is a graph showing the effect of different concentrations of complex 7 on apoptosis of BEL-7402 cells.

FIG. 11 is a graph showing the effect of different concentrations of complex 5 on the mitochondrial membrane potential of BEL-7402 cells.

FIG. 12 is a graph showing the effect of different concentrations of complex 7 on the mitochondrial membrane potential of BEL-7402 cells.

FIG. 13 is a graph showing the effect of different concentrations of complex 5 on intracellular reactive oxygen species in BEL-7402.

FIG. 14 is a graph showing the effect of different concentrations of complex 7 on intracellular reactive oxygen species in BEL-7402.

FIG. 15 shows the different concentrations of complex 5 and 7 versus Ca in BEL-7402 cells²⁺Graph of the effect of concentration, where (a) is complex 5 (the curve not labeled is control) and (b) is complex 7 (the curve not labeled is control).

FIG. 16 is a graph showing the effect of different concentrations of Complex 5 on the expression level of Caspase-3/9 in BEL-7402 cells.

FIG. 17 shows the effect of different concentrations of Complex 7 on the expression level of Caspase-3/9 in BEL-7402 cells.

FIG. 18 is a graph showing the effect of complex 5 on the expression level of BEL-7402 apoptosis-related protein, wherein (a) is a graph showing the protein expression level of complex 5 on BEL-7402 apoptosis-related protein at different concentrations, and (b) is a histogram showing the expression level of complex 5 on BEL-7402 apoptosis-related protein at different concentrations, and blank control, 0.45. mu.M, 0.9. mu.M and 1.8. mu.M are sequentially arranged from left to right in each protein histogram.

FIG. 19 is a graph showing the effect of complex 7 on the expression level of BEL-7402 apoptosis-related protein, wherein (a) is a graph showing the protein expression level of complex 7 on BEL-7402 apoptosis-related protein at different concentrations, and (b) is a histogram showing the expression level of complex 7 on BEL-7402 apoptosis-related protein at different concentrations, and blank control, 0.45. mu.M, 0.9. mu.M and 1.8. mu.M are sequentially arranged from left to right in each protein histogram.

Detailed Description

In order to better explain the technical solution of the present invention, the following describes the present invention in further detail with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, technical features used in the embodiments may be replaced with other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept.

Example 1: ligand H-L^cPreparation of

Adding 2-acetylpyridine (2.24mL, 20mmol) and 3-bromosalicylaldehyde (2.01g, 10mmol) into a 100mL round-bottom flask, measuring 50mL of ethanol, adding the ethanol into the round-bottom flask, shaking uniformly, measuring 0.75mL of 25% ammonia water, adding the ammonia water into the ammonia water, weighing 0.14g of potassium hydroxide, carefully adding the potassium hydroxide into the round-bottom flask (the pH value of the system is 12-13 after the potassium hydroxide is dissolved and stirred uniformly), placing a magneton, installing a reflux reaction device, and reacting for 24 hours in a water bath at 40 ℃ (the initial solution is light brown, and light yellow solid is generated along with the reaction). And after the reaction is finished, taking down the round-bottom flask, performing rotary evaporation, evaporating half of the solvent in the flask, performing suction filtration, collecting a filter cake, washing with ethanol, and drying to obtain a light yellow powdery solid. The yield was 92.3% (3.72g, based on 3-bromosalicylaldehyde).

The product obtained in this example was characterized:

(1) high resolution mass spectrometry: ESI-MS M/z 405.4[ M + H ]]⁺Wherein M is a ligand H-L^aMolecular weight of (2)。

(2) Nuclear magnetic hydrogen spectrum:¹H NMR(500MHz,DMSO-d₆):

δ15.71(s,1H),9.08(d,J＝1.3Hz,1H),8.90(d,J＝1.2Hz,1H),8.86–8.84(m,2H),8.49(d,J＝8.2Hz,1H),8.37(d,J＝6.9Hz,1H),8.23(d,J＝7.9Hz,1H),8.13(td,J＝7.7,1.7Hz,1H),8.09–8.05(m,1H),7.72–7.69(m,1H),7.61–7.57(m,2H),6.96(d,J＝7.9Hz,1H)。

(3) nuclear magnetic carbon spectrum:¹³C NMR(125MHz,DMSO-d₆):

δ156.98,156.41,153.60,153.52,153.14,150.52,150.50,149.71,138.49,138.20,135.27,127.50,125.49,125.47,122.52,121.12,120.42,120.33,117.75,117.57,111.92.

(4) elemental analysis (C)₂₁H₁₄BrN₃O):

Calculated value (%): c62.39, H3.49, N10.39.

Found (%): c62.36, H3.55, N10.37.

(5) Infrared spectrum: selected IR (KBr, cm)^–1):

3491,3054,3013,1563,1470,1392,1226,1177,1078,795。

From this, it was confirmed that the pale yellow powdery solid obtained in this example was the ligand H-L as the objective product^c。

Comparative examples 1 to 1

Example 1 was repeated except that no ammonia was added before the addition of potassium hydroxide.

As a result, a pale yellow powdery solid was not obtained.

Example 2: ligand H-L^cPreparation of

Example 1 was repeated, except that methanol was used instead of ethanol, sodium hydroxide was used instead of potassium hydroxide, and the amount of sodium hydroxide added was controlled so as to adjust the pH of the system to 12, and the reaction was carried out in a water bath at 50 ℃.

As a result, a pale yellow powdery solid was obtained. The yield was 90.6% (based on 3-bromosalicylaldehyde).

Performing high resolution mass spectrometry, nuclear magnetic characterization, elemental analysis and infrared analysis on the product obtained in the embodiment, and determining that the obtained pale yellow powdery solid is the target product ligand H-L^c。

Example 3: ligand H-L^dPreparation of

Example 1 was repeated, except that 5-bromosalicylaldehyde was used instead of 3-bromosalicylaldehyde.

As a result, 3.67g of a pale yellow powdery solid was obtained. The yield was 91.1% (based on 5-bromosalicylaldehyde).

The product obtained in this example was characterized:

(1) high resolution mass spectrometry: ESI-MS M/z 404.04[ M + H ]]⁺Wherein M is a ligand H-L^dMolecular weight of (2).

(2) Nuclear magnetic hydrogen spectrum:¹H NMR(400MHz,DMSO-d₆)：

δ10.31(s,1H),8.74(ddd,J＝4.8,1.8,0.9Hz,2H),8.68(t,J＝1.1Hz,1H),8.66(d,J＝1.1Hz,1H),8.65(s,2H),8.03(td,J＝7.7,1.8Hz,2H),7.65(d,J＝2.5Hz,1H),7.54–7.51(m,2H),7.50(dd,J＝3.3,1.8Hz,1H),7.03(dd,J＝8.7,3.0Hz,1H)。

(3) nuclear magnetic carbon spectrum:¹³C NMR(101MHz,DMSO-d₆)：

δ155.55,155.39,154.71,154.54,149.81,147.25,147.22,137.91,133.23,132.51,127.85,124.90,121.32,121.12,119.11,111.29,111.23。

(4) elemental analysis (C)₂₁H₁₄BrN₃O):

Calculated value (%): c62.39, H3.49, N10.39.

Found (%): c62.37, H3.57, N10.34.

(5) Infrared spectrum: SelectdIR (KBr, cm)^–1):

3545,3049,3012,1585,1567,1471,1385,1282,1256,1239,790。

From this, it was confirmed that the pale yellow powdery solid obtained in this example was the ligand H-L as the objective product^d。

Comparative example 3-1

Example 3 was repeated except that no ammonia was added before the addition of potassium hydroxide.

As a result, a pale yellow powdery solid was not obtained.

Example 4: ligand H-L^dPreparation of

Example 3 was repeated except that n-propanol was used instead of ethanol, the amount of ammonia water was changed to 2mL, the amount of potassium hydroxide was controlled so as to adjust the pH of the system to 13, the reaction was carried out in a water bath at 35 ℃ and the reaction time was changed to 48 hours.

As a result, a pale yellow powdery solid was obtained. The yield was 84.3% (based on 5-bromosalicylaldehyde).

Performing high resolution mass spectrometry, nuclear magnetic characterization, elemental analysis and infrared analysis on the product obtained in the embodiment, and determining that the obtained pale yellow powdery solid is the target product ligand H-L^d。

Example 5: ligand H-L^dPreparation of

Example 3 was repeated except that n-butanol was used instead of ethanol and the reaction was carried out at 25 ℃ at room temperature for 72 hours.

As a result, a pale yellow powdery solid was obtained. The yield was 80.2% (based on 5-bromosalicylaldehyde).

Example 6: copper terpyridyl complex [ Cu (H-L) shown as formula (II)^c)Cl]ClO₄(Compound 5) preparation

10mg (0.025mmol) of ligand H-L are weighed out^cAnd 10mg (0.027mmol) of Cu (ClO)₄)₂·6H₂O into a 25cm Pyrex thick-walled glass tube with one closed end, 0.05mL of chloroform and 0.6mL of methanol (the volume ratio of chloroform to methanol is 1: 12) are added dropwise,the open end of the glass tube was melt-sealed and placed in an oven at 50 ℃ for reaction for 72 hours. After the reaction is finished, the temperature is reduced to room temperature in a gradient way, blue blocky crystals can be observed to be generated in the tube, and the crystals are collected and dried. The yield was 65.9% (9.84mg, based on ligand H-L)^cCalculated as a benchmark).

Selecting single crystals with proper size and shape, determining the structure of the single crystals by adopting methods such as single crystal X-ray diffraction analysis and the like, wherein the specific characterization data is as follows:

(1) high resolution mass spectrometry: ESI-MS M/z 542.8[ M-CH ]₃CN-H₂O+H]⁺Wherein M is the molecular weight of complex 5.

(2) Elemental analysis (C)₂₁H₁₄BrCl₂CuN₃O₅):

Calculated value (%): c41.85, H2.34, N6.97;

found (%): c41.83, H2.39, N6.96.

(3) Infrared analysis of Selected IR (KBr, cm)^–1):

ν(O-H)3396(m)；ν(Ar-H)3081(m)；ν(C-O)1235(m)；ν(C-Br)737(m).

(4) Single crystal X-ray diffraction analysis:

selecting a blue blocky crystal with moderate size, placing the blue blocky crystal on an Agilent Supernova X-ray single crystal diffractometer, and adopting Mo-K monochromatized by a graphite monochromator_α

The single crystal structure was measured using the radiation as a light source. The initial crystal structure of the product obtained in this example was solved by the SHELXS-97 direct method, the diffraction data was corrected by LP factors and empirical absorption, the full matrix least squares refinement was performed on all non-hydrogen atomic coordinates and their anisotropic thermal parameters, and all hydrogen atomic coordinates were determined by theoretical calculation. The obtained crystallographic and structural refinement data are shown in the following table 1, the partial bond length and bond angle data are shown in the following table 2, the crystal structure of the obtained blue bulk crystal is shown in fig. 1, and the planar structure is shown in the following formula (II). Therefore, the obtained blue bulk crystal is determined to be the target product of the copper terpyridyl complex shown in the formula (II)Substance [ Cu (H-L)^c)Cl]ClO₄I.e., Compound 5.

TABLE 1 crystallography and Structure correction data for

complexes

5 and 7

^aR₁＝Σ||F_o|–|F_c||/Σ|F_o|；^bωR₂＝[Σw(F_o ²–F_c ²)²/Σw(F_o ²)²]^0.5.

TABLE 2 partial bond lengths of

complexes

5 and 7

Angle of harmony key [ ° ]]

The single crystal X-ray diffraction analysis result shows that: the complex 5 belongs to a monoclinic system, P2₁The/n space group, complex 5 consists of a cation [ Cu (H-L)^c)(Cl)]⁺And an anion ClO₄ ^-And (4) forming. Cation [ Cu (H-L)^c)(Cl)]⁺With a chloride ion and from the ligand H-L^cThe three N atoms are coordinated to form a four-coordination plane quadrilateral geometrical configuration.

Example 6-1

Example 6 was repeated, except that 0.05mL of chloroform and 0.55mL of methanol (chloroform/methanol ratio by volume 1: 11) were added dropwise.

As a result, no crystals or precipitates of the desired product were formed.

Example 6 to 2

Example 6 was repeated, except that 0.05mL of chloroform and 0.65mL of methanol (chloroform/methanol ratio by volume 1: 11) were added dropwise.

As a result, no crystals or precipitates of the desired product were formed.

Examples 6 to 3

Example 6 was repeated except that the mixed solvent was changed to a single solvent such as methanol, acetone, acetonitrile, dichloromethane, chloroform, DMF or DMSO. As a result, no crystals or precipitates of the desired product were formed.

Comparative examples 6 to 4

Example 6 was repeated except that methanol in the mixed solvent was replaced with ethanol, acetonitrile, DMF or DMSO, or chloroform in the mixed solvent was replaced with acetone, DMF or DMSO, or the like. As a result, no crystals or precipitates of the objective product were formed.

Example 7: preparation of Complex 5

Example 6 was repeated, except that 0.05mL of methylene chloride and 0.575mL of methanol (the volume ratio of methylene chloride to methanol was 1: 11.5) were added dropwise, the reaction was carried out in a water bath at 40 ℃ and the reaction time was changed to 96 hours.

As a result, blue bulk crystals were obtained. Yield 58.7% (8.76mg, based on ligand H-L)^cCalculated as a benchmark).

The product obtained in the embodiment is subjected to high resolution mass spectrometry, elemental analysis, infrared analysis and single crystal diffraction analysis, and the obtained blue bulk crystal is determined to be the target product complex 5.

Example 8: preparation of Complex 5

Example 6 was repeated, except that 0.05mL of chloroform and 0.62mL of methanol (chloroform/methanol ratio by volume 1: 12.4) were added dropwise.

As a result, blue bulk crystals were obtained. Yield 63.7% (9.51mg based on ligand H-L)^cCalculated as a benchmark).

Example 9: preparation of Complex 5

Example 6 was repeated except that the reaction was carried out in a water bath at 60 ℃.

As a result, blue bulk crystals were obtained. Yield 71.8% (10.07mg, based on ligand H-L)^cCalculated as a benchmark).

Example 10: copper terpyridyl complex [ Cu (H-L) shown as formula (III)^d)(CH₃OH)(DMSO)](Cl O₄)₂(Compound 7) preparation

10mg (0.025mmol) of ligand H-L are weighed out^dAnd 10mg (0.027mmol) of Cu (ClO)₄)₂.6H₂O into a 25cm Pyrex thick-walled glass tube with one closed end, 0.02mL of DMSO and 0.5mL of methanol (volume ratio of DMSO to methanol is 1: 25) are added dropwise, and the open end of the glass tube is sealed by melting and placed in an oven at 65 ℃ for reaction for 72 hours. After the reaction is finished, the temperature is reduced to room temperature in a gradient way, the generation of blue strip crystals in the tube can be observed, and the crystals are collected and dried. The yield was 61.8% (11.9mg, based on ligand H-L)^dCalculated as a benchmark).

(1) high resolution mass spectrometry: ESI-MS M/z 502.3[ M-DMSO- (ClO)₄)₂+H]⁺Wherein M is the molecular weight of complex 7.

(2) Elemental analysis (C)₂₄H₂₃BrCl₂CuN₃O₁₁S):

Calculated value (%): C37.15, H2.99, N5.42.

Found (%): C37.12, H3.06, N5.40.

(3) Infrared analysis of Selected IR (KBr, cm)^–1):

ν(O-H)3481(m)；ν(Ar-H)3063(m)；ν(C-O)1239(s)；ν(C-Br)788(s)。

(4) Single crystal X-ray diffraction analysis:

selecting blue strip crystals with moderate size, placing the blue strip crystals on an Agilent Supernova X-ray single crystal diffractometer, and adopting Mo-K monochromatized by a graphite monochromator_α

The single crystal structure was measured using the radiation as a light source. The initial crystal structure of the product obtained in this example was solved by the SHELXS-97 direct method, the diffraction data was corrected by LP factors and empirical absorption, the full matrix least squares refinement was performed on all non-hydrogen atomic coordinates and their anisotropic thermal parameters, and all hydrogen atomic coordinates were determined by theoretical calculation. The obtained crystallographic and structural refinement data are shown in table 1 above, the partial bond length and bond angle data are shown in table 2 above, respectively, the crystal structure of the obtained blue stripe crystal is shown in fig. 2, and the planar structure is shown in formula (III) below. Therefore, the obtained blue strip-shaped crystal is determined to be the target product of the copper terpyridyl complex [ Cu (H-L)^d)(CH₃OH)(DMSO)](ClO₄)₂I.e., compound 7.

The single crystal X-ray diffraction analysis result shows that: the complex 7 belongs to a monoclinic system, P2₁The c space group, the central copper (II) ion with the O atom of one methanol molecule, the S atom of one DMSO molecule and from the ligand H-L^dThe three N atoms are coordinated to form a penta-coordinated distorted tetragonal pyramid geometry. The whole complex ion is +2 valence, and the outside has 2 ClO₄ ^-As a counter anion.

Comparative example 10-1

Example 10 was repeated, except that 0.02mL of DMSO and 0.48mL of methanol were added dropwise (DMSO to methanol ratio 1: 24 by volume).

As a result, no crystals or precipitates of the desired product were formed.

Comparative examples 10 to 2

Example 10 was repeated, except that 0.02mL of DMSO and 0.52mL of methanol were added dropwise (DMSO to methanol ratio 1: 26 by volume).

As a result, no crystals or precipitates of the desired product were formed.

Example 11: preparation of Complex 7

Example 10 was repeated, except that 0.02mL of DMSO and 0.49mL of methanol (DMSO/methanol ratio 1: 24.5 by volume) were added dropwise and the reaction was carried out in a water bath at 65 ℃ for 24 hours.

As a result, blue stripe crystals were obtained. Yield 58.7% (11.3mg based on ligand H-L)^dCalculated as a benchmark).

The product obtained in the embodiment is subjected to high resolution mass spectrometry, elemental analysis, infrared analysis and single crystal diffraction analysis, and the obtained blue strip crystal is determined to be the target product complex 7.

Example 12: preparation of Complex 7

Example 10 was repeated, except that 0.02mL of DMSO and 0.51mL of methanol (DMSO/methanol ratio 1: 25.5 by volume) were added dropwise and the reaction was carried out in a water bath at 40 ℃.

As a result, blue stripe crystals were obtained. Yield 53.2% (10.2mg, based on ligand H-L)^dCalculated as a benchmark).

Experimental example 1: stability test of the ligand and the Complex

1.1 determination of Compound stability by UV-Vis Spectroscopy

The applicant uses UV-Vis spectroscopy to detect the stability of the synthesized compound in Tris-HCl buffer solution with pH 7.35, and provides experimental basis for subsequent cell level experiments.

The concentration is 2 x 10^-5The ligand and the complex in mol/L are placed in Tris-HCl buffer solution with the pH value of 7.35 and are placed at room temperature for 0 and 48 hoursThen, the absorbance was measured once by each UV spectrometer. The experimental result shows that no red shift or blue shift phenomenon occurs in each group of absorption peaks, and no new absorption peak occurs, which indicates that the ligand and the complex synthesized by the invention can stably exist in Tris-HCl buffer solution for at least 48 h.

1.2 High Performance Liquid Chromatography (HPLC) for determining stability and purity of Compound

FIGS. 3 and 4 are ligands H-L, respectively^cAnd complexes thereof 5, H-L^dHigh performance liquid chromatogram of the complex 7 (all high performance liquid chromatograms (LC-20AT, SPD-20A HPLCCOLUMN,150 mm. times.5.0 μm I.D., 40 ℃ C., mobile phase: metalol/H2O (90:10), flow rate: 1.0Ml/min, sample size: 1.50. times.10)^-4M)). Liquid chromatography experiments show that: no new peak appears in the ligand and the complex after 48 hours, and the peak shift is almost unchanged, which indicates that the ligand and the complex can stably exist in DMSO stock solution for at least 48 hours at room temperature, and the experimental result is consistent with the result measured by ultraviolet-visible spectroscopy, and indicates that the compound has sufficient stability. In addition, HPLC experiments also show that the purity of the compounds is more than 99%, and cell experiments can be carried out.

Experimental example 2: in-vitro anti-tumor activity research of ligand and complex

2.1 instruments and reagents as shown in Table 3 below.

TABLE 3 instruments and reagents used in the experiments

2.2 cell lines and culture

The cell lines used in this example were: MGC80-3 gastric cancer cell, T-24 human bladder cancer cell, NCI-H460 human lung cancer cell, HeLa human cervical cancer cell, BEL-7402 human liver cancer cell, Hep-G2 liver cancer cell, SK-OV-3 ovarian cancer cell and HL-7702 human normal liver cancer cellA cell. All cell lines were in 5% CO₂And cultured in an incubator at 37 ℃. And (3) observing the growth state of the cells by using an inverted microscope, washing the cells for 2 times by using a PBS (phosphate buffer solution) after the cells adhere to 80-90% of the wall, digesting the cells by using trypsin, and taking the cells in the logarithmic phase for experiment.

2.3 determination of IC at cellular level of Compound by MTT method₅₀Value of

Firstly, digesting cells, and putting the cells in the logarithmic growth phase in a 96-well culture plate, wherein 180 mu L (about 4500-;

after the cells grow to about 2/3 pore area, adding 20 microliter of sample into each pore, setting 5 multiple pores in parallel, and setting corresponding blank control groups;

③ after 48 hours of culture, 10 microliter of MTT reagent (with the concentration of 5mg/mLPBS) is added into each well;

fourthly, after incubation for 4-6 hours, the culture solution is removed by suction, 150 mu L of DMSO is added into each hole, and shaking is carried out for 5-8 min, so that formazan crystals are fully dissolved;

and measuring OD value (wavelength 570/630nm) by using an enzyme-labeling instrument.

For screening compounds, typically 5 concentration gradients (20,10,5,2.5,1.25 μ M) were set, but for the more active complexes, the concentration gradient was set to (6,3,1.5, 0.75, 0.375 μ M), and all experiments were repeated 3 more times to average. The compounds synthesized in accordance with the present invention were dissolved in analytically pure DMSO and cisplatin in physiological saline.

2.4 results and discussion

In the experiment, IC of the compound synthesized by the invention on 7 tumor cell strains and 1 normal cell strain is calculated by using a Bliss method₅₀Values (in. mu.M, as shown in Table 4).

TABLE 4 ligands H-L^cAnd H-L^dAnd IC of

complexes

5 and 7 on different cell lines₅₀Value (μ M)

As can be seen from Table 4, the ligands H-L^cAnd H-L^dAnd the inhibition effect of the

complexes

5 and 7 on the selected tumor cell strains is higher than that of cisplatin and H-L^cAnd H-L^dIC for selected tumor cell lines₅₀Values were within 7. mu.M, while IC of

complexes

5 and 7 for selected tumor cell lines₅₀The values were all within 3. mu.M. Wherein, the complex 5 and 7 have the highest inhibitory effect on BEL-7402 human hepatoma cells, and IC₅₀The values are 0.91 + -0.14 and 1.01 + -0.17, respectively, which are much lower than the IC of cisplatin₅₀Value (17.10. + -. 0.87. mu.M). Meanwhile, as shown in table 4, the selective inhibition indexes of

complexes

5 and 7 on BEL-7402 human hepatoma cells were 3.4 and 2.8, respectively (selective inhibition index ═ IC of normal cells)₅₀/7402 IC₅₀). Because the complex has selective inhibition effect on BEL-7402 human liver cancer cells to a certain extent, the preliminary anti-tumor mechanism research is carried out on the

complexes

5 and 7.

Experimental example 3: research on in-vitro anti-tumor action mechanism of complex

3.1 Experimental instruments and reagents, as shown in Table 5 below.

TABLE 5 instruments and reagents used in the experiments

3.2 Experimental methods

3.2.1 flow cytometry detection of the cell cycle

In tumor cells, the cell cycle is closely related to apoptosis. Propidium Iodide (PI) can permeate damaged cell membranes to stain cell nucleuses of apoptotic cells, and DNA content in the cells is analyzed according to the strength of fluorescence, so that the cell cycle retardation condition is detected.

First, logarithmic phase cells in good growth state were digested, and about 2X 106 cells were inoculated into a 70mm dish and placed in an incubator.

② after the cells adhere to about 2/3 dishes, adding the prepared sample and cells for co-culture for 48 hours, and setting a blank control group.

③ wash the cells 2-3 times with PBS, trypsinize and collect all cells in a 15mL centrifuge tube.

Fourthly, centrifuging for 10 minutes at 1000rpm/min, pouring out the supernatant, adding PBS for washing and then centrifuging.

Fifthly, absorbing PBS, adding 1mL PBS to blow the cells evenly, adding 9mL of frozen sterile alcohol while shaking, and placing at-20 ℃ overnight.

Sixthly, taking out, centrifuging at 1500rpm/min for 10 minutes, discarding supernatant fluid, and then washing with PBS.

Seventhly, 0.5mL of RNase A (50 mu g/mL) is added, cells are evenly blown, and incubation is carried out for 20-30 min at 37 ℃.

And adding 20 mu L of PI (50 mu g/mL), dyeing in a dark place, passing through a membrane, and detecting the cell cycle by using a flow cytometer.

3.2.2 Western Blot method for detecting the influence of the Complex on the expression of the cycle-related protein

Western blotting is often used to detect protein expression levels, and the basic experimental steps are as follows:

(1) extraction of proteins

The method of culturing, adding medicine and collecting cell refers to the steps of 3.2.1.

② adding a proper amount of lysate (containing PMSF and with the concentration of 1mM), transferring the lysate to a 1.5mL centrifuge tube, and placing the centrifuge tube on ice for cracking for 30 min.

③ centrifuging at 4 ℃ for 15min (13000rpm), taking the supernatant and subpackaging in a small amount of new centrifuge tubes for determining the protein concentration.

Fourthly, according to the volume ratio of 3: 1 (supernatant: 4 × SDS-PAGE protein loading buffer), adding the loading buffer, mixing uniformly and boiling.

Naturally cooling and storing at-20 deg.c.

(2) Protein quantification

Protein samples were processed according to the BCA protein quantification kit, various reagents (shown in Table 6) were added to 96-well plates, and after incubation at 37 ℃ for 30 minutes, absorbance at 562nm was measured using a microplate reader, a standard curve was drawn, and the relative concentration of total protein in the samples was calculated.

TABLE 6 BCA Standard Curve preparation Table

(3) SDS-PAGE electrophoresis

TABLE 7 separation gel and concentrated gel preparation methods

Preparing glue: the separation gel and the concentrated gel were prepared according to table 7, and an appropriate volume of the separation gel was pipetted into the clamped glass plate and covered with secondary water. After solidification, the water in the upper layer is poured off. And filling the glass plate with the concentrated glue, inserting the comb, and pulling out the comb after solidification.

Sample loading: the prepared gel is put into an electrophoresis tank, electrophoresis solution is added, and samples (20 mu g/lane, 4 mu L Marker) are loaded in sequence.

③ electrophoresis: 80V and 150min, and stopping when bromophenol blue is about to run out.

Fourthly, transferring the film: the PVDF membrane was cut and activated in anhydrous methanol for 2 min. Placing the spongy cushion, the filter paper, the gel, the PVDF membrane, the filter paper and the spongy cushion in sequence, placing the spongy cushion into a membrane transferring groove, and then adding the precooled membrane transferring liquid. PVDF membrane is arranged on the positive pole, gel is arranged on the negative pole, the current is 250A, and the time is adjusted according to the molecular weight of the target protein.

Immune reaction: the PVDF membrane was washed twice with TBST for 5min each and transferred to the blocking solution for 2 h. The membrane after three times of blocking was washed with TBST, the TBST was blotted with filter paper, and the membrane was placed in the diluted primary antibody and incubated overnight at 4 ℃. After washing three times with TBST, the membrane was incubated in diluted secondary antibody at room temperature for 1 h. The membrane was washed three times in TBST for 10min each time.

Sixthly, chemiluminescence: and mixing the chemiluminescence liquid reagent A and the reagent B in equal volume, sucking a proper amount of mixed liquid to fully contact with the membrane, and exposing for 1-10 min.

And seventhly, performing gel imaging and image analysis.

3.2.3 flow cytometry detection of apoptosis

In the experiment, cells are doubly stained by Annexin-V-FITC and PI, and the percentage of apoptosis of the cells is quantitatively detected by flow cytometry. The experimental procedure was as follows:

first, the log phase cells were digested and seeded in six well plates (2 mL per well, approximately 1X 106 cells);

and secondly, when the cells grow to 80-90% of the area of the holes in an adherent manner, adding the compound and the cells for co-culture for 48 hours.

③ collect the cells, wash with PBS (2 times), centrifuge for 5min (1200rpm/min), and discard the supernatant.

And fourthly, staining the cells according to the apoptosis kit instruction, and detecting the apoptosis by using a flow cytometer.

3.2.4 detection of mitochondrial Membrane potential (. DELTA.. psi.m)

Flow cytometry can detect changes in Δ ψ m to analyze the apoptosis. The experimental process comprises the following steps:

culturing cells, adding medicine, incubating and collecting cells, and the third, fourth and third steps are 3.2.1.

② staining the cells according to the instructions of a mitochondrial membrane potential detection kit (JC-1), and detecting the change of delta ψ m by using a flow cytometer.

3.2.5 intracellular Reactive Oxygen Species (ROS) detection

The DCFH-DA fluorescent probe can detect the change of the intracellular reactive oxygen species level, is not fluorescent, but can be oxidized into the DCF with fluorescence by ROS, and the change of the intracellular ROS level can be known by detecting the DCF in the cells. The specific experimental steps are as follows:

firstly, the method for culturing cells and adding medicine refers to the steps of 3.2.1.

② washing the cells for 2-3 times by serum-free culture medium, adding diluted DCFH-DA into a six-hole plate, 1mL per hole, and dyeing for 20min in a dark place.

And thirdly, washing the cells for 2-3 times by using PBS, adding 1mL of serum-free culture medium, and detecting the change of the reactive oxygen species level in the cells by using a fluorescence inverted microscope.

3.2.6 intracellular Ca²⁺Concentration detection

Changes in intracellular calcium levels can be detected using flow cytometry with Ex/Em ═ 506nm/526 nm. The operation is as follows:

Secondly, staining the cells according to the instruction of the calcium ion detection kit, and detecting the concentration of Ca2+ by using a flow cytometer.

3.2.7 Activity assays of Caspase-3/9

Caspase-3/9 was detected using the CaspGLOWTM fluoroescein Active Caspase-3/9Staining Kit. The simple operation process is as follows:

culturing cells, adding medicine, incubating and collecting cells, and the first, second and third steps are 4.2.1.

② resuspending the cells with 300. mu.L PBS, adding 1. mu.L Caspase-3/9staining solution, mixing uniformly, and incubating for 30min at 37 ℃ in dark.

And thirdly, centrifuging for 3min (1200rpm/min), removing the working solution, and washing for 2-3 times by using corresponding Caspase-3/9 Buffer.

Fourthly, adding 0.5mL of Buffer again after centrifugation, and detecting by a flow cytometer after membrane filtration.

3.2.8Western Blot to detect the influence of the complex on the expression of apoptosis-related protein

The experimental procedure is referred to 4.2.2.

3.3 results and discussion

3.3.1 flow cytometry detection of the cell cycle

The cell cycle is a highly ordered process involved in events such as cell growth, tissue regeneration, DNA repair, and apoptosis. The cell cycle of eukaryotic cells is divided into two major phases: interphase and interphase, wherein the interphase consists essentially of three phases: the G1 phase (prophase of DNA synthesis), the S phase (DNA synthesis phase), and the G2 phase (mitosis preparatory phase). Cell cycle regulation requires the participation of a large number of signals, and the absence of signals can lead to cell cycle arrest. When cancer cells undergo cycle arrest under the influence of external conditions, cell proliferation is inhibited and it is also possible to induce apoptosis.

FIG. 5 is a graph of the cell cycle distribution of BEL-7402 cells after 48h incubation with Complex 5(0.45, 0.9 and 1.8. mu.M). Compared with the control group, after the complex 5 acts on BEL-7402 cells, the cells in the G1 stage are increased from 51.31% to 70.48%, 72.82% and 76.25%, and the relative content of the cells is respectively increased: 19.17%, 21.51% and 24.94%.

FIG. 6 is a graph of the cell cycle distribution of BEL-7402 cells after 48h incubation with complex 7(0.5, 1.0 and 2.0. mu.M). Compared with a control group, after the complex 7 acts on BEL-7402 cells, the cells in the G1 stage are increased from 51.52% to 67.38%, 72.90% and 72.21%, and the relative content of the cells in the G1 stage is respectively increased: 15.86%, 21.38% and 20.69%.

3.3.2Western Blot to examine the Effect of the Complex on tumor cell cycle-associated proteins

In order to ensure the orderly progress of the cell cycle, a large amount of protein participates in the regulation and control of the cell cycle, and a cell cycle check point is a check mechanism for ensuring the accurate and error-free progress of the cell cycle. Checkpoint sensors Chk1(checkpoint kinase 1) and Chk2(checkpoint kinase 2) belong to the serine/threonine protein kinase family and are also substrates for the apical checkpoint kinases ATM and ATR. The primary checkpoint response of mammalian cells to DNA damage is the ATM (ATP)/Chk2(Chk1) -p53-p21 pathway, which induces long-term and even permanent G1 phase arrest. The DNA replication checkpoint (i.e. the phase-within-S checkpoint) is activated when DNA replication is abnormal, when the effector kinase Chk1 phosphorylates downstream Cdc25A, Cdc25A activates mainly the cyclin E-CDK2 and cyclin a-CDK2 complexes during the G1-S phase transition. Therefore, cells can prevent DNA replication through Chk1/Cdc25A-CDK2 pathway and arrest cells in S phase, and when cyclin E/A-CDK2 complex is inactivated, cells are also arrested in G1 phase.

FIGS. 7 and 8 show the expression of complexes 5(0.45, 0.9 and 1.8. mu.M) and 7(0.5, 1.0 and 2.0. mu.M) on the protein Chk1, Chk2, Cdc25A, CyclinA, CyclinE, CDK2, P53 and P21 of BEL-7402 cells by Western Blot, respectively. Compared with a control group, the expression level of the P53 and P21 proteins is increased along with the addition of the complex; while the expression levels of Chk1, Chk2, Cdc25A, CyclinA, CyclinE and CDK2 proteins were inhibited. The experimental results show that the

complexes

5 and 7 can block BEL-7402 cells in the G1 phase by inhibiting the activity of the cyclin E/A-CDK2 complex, thereby inhibiting the cell proliferation. The results are consistent with the cell cycle experiments examined by flow cytometry.

3.3.3 flow cytometry detection of Complex-induced apoptosis in tumor cells

Annexin-V-FITC is a fluorescent probe which can be combined with Phosphatidylserine (PS) in the presence of calcium ions. When the cell undergoes early apoptosis, PS begins to transfer from the inside of the cell to the outside of the cell membrane, and at the moment, Annexin-V can be combined with the everted PS, and the early apoptosis of the cell can be detected. When the cell undergoes late apoptosis, PI (pyridine iodide) can stain DNA through a damaged cell membrane, and the late apoptosis of the cell can be detected. Thus, this section examines the percentage of apoptosis induced by the complex by flow cytometry.

FIG. 9 shows the apoptosis of BEL-7402 cells after 48h of complex 5(0.45, 0.9 and 1.8. mu.M) action. After the complex 5 is added, the apoptosis percentages are respectively as follows: 17.74%, 28.4% and 56.4%, respectively, the percentage of apoptosis was increased compared to the control group (0.487%): 17.253%, 27.91% and 55.91%, the percentage of apoptosis increased with increasing dosing. Indicating that the complex 5 can induce BEL-7402 cells to generate apoptosis.

FIG. 10 shows the apoptosis of BEL-7402 cells after 48h of complex 7(0.5, 1.0 and 2.0. mu.M). After the complex 7 is added, the apoptosis percentages are respectively as follows: 14.66%, 30.6% and 39.9%, respectively, the percentage of apoptosis increased compared to the control group (0.487%): 14.173%, 30.11% and 39.41%, the percentage of apoptosis increased with increasing dosing. Experiments show that the complex 7 can induce BEL-7402 cells to generate apoptosis.

3.3.4 detection of mitochondrial Membrane potential (. DELTA.. psi.m)

Mitochondria play a key role in the apoptotic process (e.g., affecting intracellular ATP levels, alterations in Δ ψ m, alterations in mitochondrial membrane permeability, production of reactive oxygen species, etc.). Mitochondrial outer membrane permeabilization is an important event in mitochondrial-mediated apoptosis pathway, and releases apoptosis-promoting proteins such as cytochrome c (CYT-c) into cytoplasm through mitochondria, and the CYT-c can start caspase-dependent pathways, so that PARP (poly ADP-ribose polymerase, a DNA repair enzyme, which is a cleavage substrate of caspases) is cleaved, and finally apoptosis is caused. While disruption of mitochondrial membrane potential (Δ ψ m) plays a key role in the increase of mitochondrial membrane permeability, it is considered to be a prerequisite for mitochondrial-mediated apoptosis.

FIGS. 11 and 12 show the changes in the mitochondrial membrane potential of cells after 48h exposure of complexes 5(0.45, 0.9 and 1.8. mu.M) and 7(0.5, 1.0 and 2.0. mu.M) to BEL-7402 cells, respectively. Δ ψ m of BEL-7402 cells was decreased compared to the control (1.88%): after the complex 5 acts, the percentage of reduction is as follows: 23.9%, 32.0%, 54.9%; after the complex 7 acts, the percentage of reduction is as follows: 17.9%, 27.9%, 38.5%; the experimental results show that both

complexes

5 and 7 can induce the decrease of delta psi m of BEL-7402 cells, and the apoptosis of BEL-7402 cells is possibly induced by mitochondria.

3.3.5 Effect of complexes on Reactive Oxygen Species (ROS) in cancer cells

Elevated intracellular Reactive Oxygen Species (ROS) levels serve as a signal for oxidative stress and may also activate apoptotic signaling pathways. Mitochondria are both the target cells for ROS and the source of more ROS produced. Several studies have shown that ROS production exacerbates the breakdown of mitochondrial membrane potential and then releases more cytochrome c (cyt c), leading to caspase-3 lysis and ultimately apoptosis. In addition, reactive oxygen species can interact with purine bases, pyrimidine bases and ribose, can break single or double strands of DNA, activate DNA-dependent protein kinases and P53, and lead to apoptosis.

FIGS. 13 and 14 are the intracellular ROS release profile of complexes 5(0.45, 0.9 and 1.8. mu.M) and 7(0.5, 1.0 and 2.0. mu.M) on BEL-7402 cells after 48 h. Observed by a fluorescence inverted microscope to find: compared with a control group, the green fluorescence emitted by cells is enhanced after the coordination compound acts, and the coordination compound 5 and the coordination compound 7 can cause the ROS level of BEL-7402 cells to be increased, so that the apoptosis is induced.

3.3.6 calcium ion (Ca)²⁺) Detection of Release

The intrinsic mechanism of mitochondrially mediated apoptosis involves mitochondria and related mitochondrial proteins, DNA damage or oncogene overexpression in cells can activate this apoptotic pathway. At the same time, other factors (e.g. growth factor deprivation, Ca)²⁺Up-regulation of levels, oxidizing agents and targeted drugs, etc.) will also cause the pathway to be activated. We have already known that Ca²⁺The signal controls many important cellular functions, the homeostasis of which within the cell is of paramount importance for both cell survival and cell death. Research shows that when cells receive an apoptosis signal, calcium ions are transferred from endoplasmic reticulum to mitochondria, so that mitochondrial membranes are damaged, membrane permeability is increased, cytochrome c (Cyt c) and apoptosis protease activating factor 1(Aapaf-1) are released, a series of key events causing apoptosis are caused, and finally the apoptosis is caused.

FIG. 15 shows intracellular Ca after 48h of complexes 5(0.45, 0.9 and 1.8. mu.M) and 7(0.5, 1.0 and 2.0. mu.M) on BEL-7402 cells²⁺The influence of (c). The flow cytometry experiment result shows that: under the action of the complex, Ca in cancer cells²⁺The concentration is increased, and compared with a control group (black peak), the fluorescence is enhanced (peak is shifted to the right) after the corresponding complex is added, and Ca is added²⁺The increased release levels indicated that

complexes

5 and 7 were likely to induce apoptosis of BEL-7402 cells via the mitochondrial pathway.

3.3.7 Complex activates expression of Caspase-3/9 in tumor cells

Apoptotic cells have morphological characteristics different from pathological necrotic cells, and most of morphological changes in apoptosis are caused by a group of caspase-specific proteases (Caspases) which are specifically activated in apoptotic cells. The caspase family is divided into two groups: initiation factors (Caspase-2,8,9 and 10) and execution factors (Caspase-3,6 and 7). Wherein, Caspase-9 is a key initiation factor for triggering apoptosis, after cytochrome c (Cyt-c) is released from a mitochondrial membrane gap to cytoplasm, the cytochrome c combines with an apoptotic protease activation factor-1 (Apaf-1) and procaspase-9 to form an apoptotic body, so as to promote the activation of Caspase-9, the activated Caspase-9 is cracked and activates downstream caspases (such as Caspase-3, Caspase-6, Caspase-7 and the like), thereby forming a Caspase cascade reaction to integrate a pro-apoptotic signal, and finally leading to apoptosis. Caspase-3 is the key performer of apoptosis, and after caspase-9 is cleaved and activated, caspase-3 cleaves and cleaves hundreds of target proteins in cells, eventually leading to cell death. In this section, flow cytometry was used to detect the effect of

complexes

5 and 7 on Caspase-3/9 expression in cancer cells.

The activation of Caspase-3/9 in BEL-7402 cells was examined 48h after complexes 5(0.45, 0.9 and 1.8. mu.M) and 7(0.5, 1.0 and 2.0. mu.M) were applied to the cells by flow cytometry.

As shown in FIG. 16, the percentage of Caspase-3 activation in cells increased by 9.08%, 17.54%, and 46.77% after complex 5 acted on the cells, respectively. The percentage of Caspase-9 activation increased by 17.23%, 22.5%, 58.99%, respectively.

As shown in FIG. 17, the percentage of Caspase-3 activation in cells increased by 21.01%, 36.14%, and 50.55% after complex 7 acted on the cells, respectively. The percentage of Caspase-9 activation increased by 33.28%, 44.24%, 73.62%, respectively.

The experimental result shows that both the

complexes

5 and 7 can activate Caspase-3/9 in BEL-7402 cells. Caspase-3 and Caspase-9 are key factors for apoptosis in mitochondrial pathways, indicating that

complexes

5 and 7 may induce apoptosis via mitochondrial pathways.

3.3.8 Western Blot to detect the influence of the complex on the expression of apoptosis-related protein

Mitochondrial dysfunction has been shown to contribute to the development of apoptosis and is central to the apoptotic pathway. This pathway involves three key proteins: b cell lymphoma-2 (BCL-2) family proteins, Caspases and mitochondrial pro-apoptotic proteins. The BCL-2 family proteins play a key role in mitochondrial-mediated apoptosis by regulating the permeability of the mitochondrial membrane. The Bcl-2 protein in the BCL-2 family protein is a mitochondrial outer membrane protein and is used as an anti-apoptosis factor, and the over-expression of the Bcl-2 protein can block the apoptosis process of cells. Molecular therapy targeting anti-apoptotic proteins such as Bcl-2 can initiate cancer cell apoptosis, for example, Bcl-2 antagonist ABT-737 is known to effectively kill leukemia and lymphoma cells. Bax is a pro-apoptotic protein that can exist as a monomer in the cytosol or loosely associated with the mitochondrial membrane when not activated, but which is displaced toward and inserted into the outer mitochondrial membrane during activation. Activated Bax can form homooligomers with Bak, which participate in the formation of mitochondrial membrane pores and lead to increased mitochondrial membrane permeability, releasing cytochrome c (cyt c) into the cytosol. Cyt c, together with the apoptotic protease activator 1(Apaf-1) and dATP, causes the promoter procaspase-9 to accumulate and activate in apoptotic bodies. Subsequently, apoptotic bodies drive the activation of caspases, which lyse throughout the cell and inactivate numerous key proteins, ultimately leading to apoptosis.

As shown in FIGS. 18 and 19, after complexes 5(0.45, 0.9 and 1.8 μ M) and 7(0.5, 1.0 and 2.0 μ M) act on BEL-7402 cells for 48 hours, the expression levels of Apaf-1, Bax, Bak and Cytochrome-c protein can be obviously increased, and the expression level of Bcl-2 protein can be reduced, and the experimental results show that:

complexes

5 and 7 are likely to induce apoptosis in cells via the mitochondrial pathway.

Claims

1. A terpyridine derivative represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

wherein, when R1 ═ Br, R2 ═ H; when R1 ═ H, R2 ═ Br.

2. The method for synthesizing terpyridine derivatives as claimed in claim 1, wherein the target compound is obtained by adding 2-acetylpyridine and 3-bromosalicylaldehyde or 5-bromosalicylaldehyde into lower alcohol, adding ammonia water, adjusting pH of the system to be not less than 10, reacting under heating or non-heating conditions, recovering solvent from the obtained reactant, and collecting the solid.

3. The method according to claim 2, wherein the lower alcohol is an alcohol having 1 to 4 carbon atoms.

4. The method according to claim 2, wherein the reaction is carried out at 35 to 50 ℃.

5. The method according to claim 2, wherein the amount of the aqueous ammonia added is 1 time or more of the amount of the 3-bromosalicylaldehyde or 5-bromosalicylaldehyde substance.

6. The terpyridine derivative or pharmaceutically acceptable salt thereof as set forth in claim 1 for use in preparing antitumor medicine.

7. A copper terpyridine complex with the terpyridine derivative as the ligand in claim 1, which is represented by the following formula (II) or formula (III):

8. the synthesis method of the terpyridine copper complex as claimed in claim 7, characterized by dissolving the terpyridine derivative represented by formula (I) in claim 1 and copper perchlorate in a mixed solvent, reacting under heating conditions, cooling the reactant, precipitating crystals, and collecting the crystals to obtain the terpyridine copper complex; wherein the content of the first and second substances,

when R1 ═ Br and R2 ═ H in the terpyridine derivative represented by formula (I), and the mixed solvent is prepared from chloroform or dichloromethane and methanol in a ratio of 1: 11.5-12.5, the copper terpyridine complex shown in the formula (II) is obtained;

when R1 ═ H, R2 ═ Br in the terpyridine derivative represented by formula (I), and the mixed solvent is prepared from dimethyl sulfoxide and methanol according to the weight ratio of 1: 24.5-25.5 volume ratio, the obtained copper terpyridine complex is shown in the formula (III).

9. The method according to claim 8, wherein the reaction is carried out at a temperature of 40 ℃ or higher.

10. The use of the copper terpyridine complex or a pharmaceutically acceptable salt thereof as described in claim 7 in the preparation of an antitumor medicament.