Azacyclo-ligand modified polyacid compound as well as synthesis method and application thereof

Abstract

The chemical formula of the polyacid compound is H14Na4 (CoC6H12N4) 3 (NaC6H12N4) (SbW9O33) 2.22 H2O, the crystal structure parameters of the polyacid compound are as follows: the molecular formula is C24Co3N16Na5O88Sb2W18, the molecular weight is 5764.94, the polyacid compound belongs to an orthogonal crystal system, the space group is P212121, the cell parameters are as follows: a / is 15.4423 (11), b / is 25.7133 (18), c / is 31.886 (2), alpha / degree, beta / degree and gamma / degree are all 90, and the cell volume / 3 is 12661.2 (16). The compound prepared by the invention is more prominent in antitumor activity, has a remarkable inhibition effect on hepatoma carcinoma cells, and provides an important basis for developing novel efficient antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of anti-tumor drugs and relates to a synthesis method and application of a polyacid compound modified by a nitrogen heterocyclic ligand. Background Art

[0002] Polyoxometalates (POMs) are a class of metal oxygen clusters with oxygen-rich surfaces, which have been widely used in biology, magnetism, catalysis and materials science. A series of physical and chemical properties of polyoxometalates, such as polarity, redox potential, surface charge distribution, shape and acidity, are the basis of their pharmaceutical activity and are conducive to the targeted recognition of biomacromolecules. The organic functionalization of POMs is a major research hotspot in the field of bioactive POMs, because pure inorganic POMs usually have toxic side effects and limited cell permeability due to their surface characteristics. The introduction of organic parts into the POM framework can change its surface, charge, polarity and redox properties, thereby forming a new compound with reduced toxicity and improved cell permeability. Organically modified POMs are generally more stable in aqueous solution, and their interactions with biological targets are enhanced and more specific. Therefore, by designing and modifying the structure of metal oxide cluster anions, their drug activity and selectivity can be further improved, which has important application research value in the field of new anticancer drug development.

[0003] For example, Joshi et al. synthesized a new Strandberg-type polyoxomolybdate-based organic-inorganic hybrid crystalline material [{4,4'-H2bpy}{4,4'-Hbpy}2{H2P2Mo5O 23 Compound (1) was tested for its in vitro antitumor activity against human breast cancer (MCF-7), human lung cancer (A549) and human liver cancer (HepG2) cells. The IC values ​​of HepG2, A549 and MCF-7 were 50 The values ​​were 33.79, 25.17 and 32.11 μmol L -1 In vitro cytotoxicity studies have shown that compound (1) is effective as an anti-tumor drug against MCF-7, A549 and HepG2 cancer cells, and has low toxicity to normal cell lines. The polyacid compound synthesized in the present invention has a better inhibitory effect on liver cancer cells than compound (1), IC 50 21.62±0.39μmol L -1 (DOI:10.1039 / d0dt01042a.) Summary of the invention

[0004] Technical problem to be solved: The present invention provides a polyacid compound modified with a nitrogen heterocyclic ligand having high anti-tumor activity and low toxicity, and a synthesis method and application thereof. 33 )9- Based on the introduction of transition metal Co and urotropine, the polyacid compound H modified by nitrogen heterocyclic ligands was synthesized by self-assembly. 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O.

[0005] Technical solution: a polyacid compound modified with a nitrogen heterocyclic ligand, wherein the chemical formula of the polyacid compound is H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O, its crystal structure parameters are as follows, molecular formula C 24 Co3N 16 Na5O 88 SbW 18 , molecular weight is 5764.94, belongs to the orthorhombic crystal system, space group is P212121, unit cell parameters are is 15.4423(11), is 25.7133(18), is 31.886(2), α / °, β / ° and γ / ° are all 90, and the unit cell volume / It is 12661.2(16).

[0006] The preparation method of the polyacid compound comprises the following steps: 21 O 86 ] 18- and cobalt chloride were mixed and dissolved in distilled water, and the [NaSb9W 21 O 86 ] 18- The molar ratio of cobalt chloride to 1: (2.5-10) is then adjusted to pH 7-7.5 by adding urotropine, and then adjusted to pH 8.5-9.5 with sodium hydroxide, heated at 90-110°C for 1-3 hours, filtered and collected, and evaporated at room temperature to obtain crystalline H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O.

[0007] Preferably, the above [NaSb9W 21 O 86 ] 18- The molar ratio of tantalum to cobalt chloride is 1:5.

[0008] Preferably, the concentration of the sodium hydroxide is 1M.

[0009] Preferably, the pH is adjusted to 7.4 with the above-mentioned urotropine, and the pH is adjusted to 9 with sodium hydroxide.

[0010] Application of the polyacid compound modified by the nitrogen heterocyclic ligand in the preparation of anti-tumor drugs.

[0011] The above tumors are liver cancer cells.

[0012] An antitumor drug preparation comprising the polyacid compound modified with the nitrogen heterocyclic ligand as an active ingredient.

[0013] The preparation is in the form of oral tablets, capsules, injections or external preparations.

[0014] Beneficial effects: The present invention not only fully utilizes the structural characteristics and potential anti-tumor biological effects of POM by cleverly introducing organic groups into the polyoxometalate (POM) framework, but also significantly changes the surface charge, polarity and redox properties of POM through modification, successfully creating a functionalized POM with lower toxicity and significantly enhanced cell penetration ability. This nitrogen heterocyclic ligand-modified polyacid compound can bind tightly to DNA, effectively causing the inactivation of DNA active sites, and thus exhibiting a powerful ability to inhibit tumor cell proliferation. More importantly, compared with unmodified polyacids, the compounds prepared by the present invention are more outstanding in anti-tumor activity and have a significant inhibitory effect on liver cancer cells, providing an important basis for the development of new and highly effective anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )Experimental and theoretical values ​​of X-ray powder diffraction data of 2·22H2O.

[0016] Figure 2 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )Infrared spectrum of 2·22H2O.

[0017] Figure 3 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )Thermogravimetric diagram of 2·22H2O.

[0018] Figure 4 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )Raman spectrum of 2·22H2O.

[0019] Figure 5 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )The UV absorption spectrum of 2·22H2O changes with the increase of CT-DNA concentration.

[0020] Figure 6 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O cytotoxicity results on HepG2 and HEK293T cells.

[0021] Figure 7 H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O polyhedral structure diagram. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0023] [NaSb 21 O 86 ] 18- Preparation reference (Gilbert Hervéa. Inorganic Syntheses. 1990, 27.)

[0024] Example 1

[0025] [NaSb9W 21 O 86 ] 18- (0.35 g, 0.05 mmol) was dissolved in 10 mL of distilled water, cobalt chloride (0.0584 g, 0.25 mmol) was added, and the mixture was heated at 100 °C to dissolve. The pH was adjusted to 7.4 with urotropine and to 9 with sodium hydroxide. After reacting for 2 hours, the filtrate was collected by filtration and evaporated at room temperature. 21 O86 ] 18- Decomposed into (SbW90 33 ) 9- , get crystal H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O.

[0026] Example 2

[0027] This embodiment is basically the same as embodiment 1, except that [NaSb9W 21 O 86 ] 18- The molar ratio of tantalum to cobalt chloride is 1:2.5.

[0028] Example 3

[0029] This embodiment is basically the same as embodiment 1, except that [NaSb9W 21 O 86 ] 18- The molar ratio of tantalum to cobalt chloride is 1:10.

[0030] Comparative Example 1

[0031] [NaSb 21 O 86 ] 18- When the molar ratio of tantalum to cobalt chloride is 1:5, the pH of the solution is adjusted to above 9 and no crystals grow.

[0032] Comparative Example 2

[0033] [NaSb 21 O 86 ] 18- The molar ratio of cobalt chloride to water was 1:2.5, and the pH value was adjusted according to the method of Example 1, and no crystals grew.

[0034] Figure 1 Polyacid compound H modified with nitrogen heterocyclic ligand 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O X-ray powder diffraction data experimental values ​​and theoretical values. Figure 1 It can be seen that the peak positions of the experimental and theoretical X-ray powder diffraction values ​​of the crystals are consistent, indicating that the crystal structure analysis is accurate and the synthesized crystals have high purity. Figure 2 Polyacid compound H modified with nitrogen heterocyclic ligand 14 Na4(CoC6H 12N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O infrared spectrum. Figure 2 Low wave number area 400~1000cm -1 It can be seen in the range of 688, 933 and 856 cm -1 Stretching vibration peaks of ν(Sb-Oa), terminal ν(W-Ot) and shared angle ν(W-Ob) were observed at 2920, 1640, 1240 cm -1 The characteristic peaks of CH, NH, and CN of urotropine are shown in the infrared spectrum of the compound, which shows that the compound contains characteristic vibration absorption peaks of the ligand urotropine, indicating that the polyacid compound modified with nitrogen heterocyclic ligand does contain urotropine. Figure 3 Polyacid compound H modified with nitrogen heterocyclic ligand 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O thermogravimetric diagram, it can be seen that the compound loses 6.03% of its weight in the range of 60-275℃, corresponding to the loss of solvent water molecules, which is within the error range compared with the theoretical value of 6.74%; the second stage of weight loss is in the range of 275-580℃, and the loss of 9.63% corresponds to the loss of four urotropine ligands, and the theoretical value is 9.55%. This result is consistent with the single crystal structure analysis. Figure 4 Polyacid compound H modified with nitrogen heterocyclic ligand 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O Raman spectrum, it can be seen that the compound contains the characteristic vibration absorption peak of the ligand urotropine, which indicates that the compound does contain urotropine.

[0035] Test Example 1 DNA Binding Experiment

[0036] 1. Preparation of solution:

[0037] (1) Preparation of buffer solution

[0038] Weigh 0.6057 g of Tris (tris(hydroxymethyl)aminomethane)) and 0.2922 g of sodium chloride into a 100 mL volumetric flask, and adjust the pH to 7.2 with 1 M HCl to obtain 5 mM Tris buffer.

[0039] (2) Preparation of calf thymus DNA (CT-DNA)

[0040] Weigh 0.0044 g of CT-DNA, dissolve it in 10 mL of the above buffer, and store it in a 4°C refrigerator overnight.

[0041] (3) Preparation of compounds

[0042] 0.00058 g of the nitrogen heterocyclic ligand-modified polyacid compound (Example 1) was weighed and dissolved in 10 mL of the above buffer.

[0043] 2. Experimental steps:

[0044] The concentration was determined by ultraviolet absorption spectroscopy, and the ratio of the absorbance values ​​at 260nm and 280nm was calculated to be between 1.8 and 1.9, indicating that the solution did not contain protein and could be used for the experiment. It is known that the molar absorption coefficient at 260nm is 6600M -1 cm -1 According to the Lambert-Beer law, the concentration of the prepared solution can be calculated from the absorbance value of the solution at 260 nm. The CT-DNA solution was added dropwise to 1.0×10 -5 mol / L heteropolyacid salt-organic ligand compound solution. The intrinsic binding constant Kb is calculated by the relationship between [DNA] / (εa-εf) and [DNA] using formula (1):

[0045]

[0046] [DNA] indicates the concentration of DNA (mol / L);

[0047] εa represents the molar absorption coefficient of the compound (at different concentrations);

[0048] εb represents the molar absorption coefficient of the compound after complete binding with CT-DNA;

[0049] εf represents the molar absorption coefficient of the compound when not bound to CT-DNA;

[0050] like Figure 5 As shown in the figure, DNA was gradually added while keeping the compound concentration constant, and the UV absorption spectra at each corresponding DNA concentration were recorded. The occurrence of hyperchromic blue shift indicates that the polyacid compound modified with nitrogen heterocyclic ligand: H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O may have electrostatic interaction with DNA.

[0051] Test Example 2 Pharmacodynamics Experiment

[0052] In vitro experiment: MTT method to detect the inhibitory effect of drugs on tumor cells

[0053] 1. Experimental Materials

[0054] Tumor cells HepG2;

[0055] Normal cells HEK293T;

[0056] The culture medium contained 10% fetal bovine serum 1640 culture medium.

[0057] 2. Experimental Procedure

[0058] Routinely resuscitate cells, and when the cells are growing well, adjust the cell concentration to 5×10 5 3×10 cells / mL were added to each well. 3 200 μL of the suspension was placed in a 96-well plate and cultured at 37°C, 5% CO2 for 24 hours. The experimental group was divided into 6 concentrations, with five replicate wells for each concentration. 10 μL of the drug solution was added to each well. A positive control and a negative control (untreated cancer cell group) were also set up. The culture was continued in a 37°C, 5% CO2 incubator for 48 hours. 4 hours before the end of the experiment, 20 μL of 0.5 mg / mL MTT was added to each well and cultured for another 4 hours. The supernatant was discarded, and the MTT precipitate was dissolved with DMSO. After oscillation and mixing, the OD value was measured at 490 nm on a microplate reader. The inhibition rate was calculated according to the measured OD value using the following formula. The different inhibition rates obtained from different concentrations of the same sample were statistically processed to obtain the half inhibition concentration IC 50 ,

[0059] Inhibition rate = (1-OD value of experimental group / OD value of control group) × 100%.

[0060] The inhibitory activity of the polyacid compound modified with nitrogen heterocyclic ligand (Example 1) on the above two cells was studied at 0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively. 50 The value was 21.62±0.39μM, see Table 1. At the same time, a control group (SbW9O 33 ) 9- , cobalt chloride and methenamine on liver cancer cells HepG2 and normal cells HEK293T inhibitory activity experiment.

[0061] Table 1 Polyacid compounds modified with nitrogen heterocyclic ligands, (SbW9O 33 ) 9- IC values ​​of cytotoxicity of cobalt chloride and methenamine on HepG2 and HEK293T cells 50 :

[0062] Table 1

[0063] <![CDATA[IC 50 ]]> HepG2 cells HEK293T Example 1 Compound 21.62±0.39 ＞200 <![CDATA[(SbW9O 33 ) 9- ]]> 169.8±0.91 Cobalt chloride ＞200 Hexamethylenetetramine ＞200

[0064] Polyacid compounds modified with nitrogen heterocyclic ligands, (SbW9O 33 ) 9- The cytotoxic effects of cobalt chloride and methenamine on HepG2 and HEK293T cells are shown in Figure 6 , we can see that the simple building block (SbW9O 33 ) 9- The toxicity of cobalt chloride and urotropine to HepG2 and HEK293T cells is significantly lower than that of the compound. The inhibitory effect of the compound of the present invention on HepG2 is significantly improved, indicating that the polyacid compound modified with nitrogen heterocyclic ligands enhances the activity of inhibiting tumor cell growth, can induce apoptosis of liver cancer cells and thus inhibit their growth, showing good anti-tumor activity.

[0065] The above examples are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A polyacid compound modified with a nitrogen heterocyclic ligand, characterized in that: The chemical formula of the polyacid compound is H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O, its crystal structure parameters are as follows, molecular formula C 24 Co3N 16 Na5O 88 SbW 18 , molecular weight is 5764.94, belongs to the orthorhombic crystal system, space group is P212121, unit cell parameters are a / Å is 15.4423(11), b / Å is 25.7133(18), c / Å is 31.886(2), α / °, β / ° and γ / ° are all 90, unit cell volume / Å 3 It is 12661.2(16).

2. The method for preparing the polyacid compound according to claim 1, characterized in that: The steps include: 21 O 86 ] 18- and cobalt chloride were mixed and dissolved in distilled water, and the [NaSb9W 21 O 86 ] 18- The molar ratio of cobalt chloride to 1: (2.5-10) is then adjusted to pH 7-7.5 by adding urotropine, and then adjusted to pH 8.5-9.5 with sodium hydroxide, heated at 90-110°C for 1-3 hours, filtered and collected, and evaporated at room temperature to obtain crystalline H 14 Na4(CoC6H 12 N4)3(NaC6H 12 N4)(SbW9O 33 )2·22H2O.

3. The preparation method according to claim 2, characterized in that: [NaSb 21 O 86 ] 18- The molar ratio of tantalum to cobalt chloride is 1:

5.

4. The preparation method according to claim 2, characterized in that: The concentration of sodium hydroxide was 1M.

5. The preparation method according to claim 2, characterized in that: Adjust the pH to 7.4 with hexamethylenetetramine and adjust the pH to 9 with sodium hydroxide.

6. Use of the polyacid compound modified with a nitrogen heterocyclic ligand according to claim 1 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: The tumor is a liver cancer cell.

8. An antitumor pharmaceutical preparation comprising the polyacid compound modified with the nitrogen heterocyclic ligand according to claim 1 as an active ingredient.

9. The anti-tumor drug preparation according to claim 7, characterized in that: The preparation is in the form of oral tablets, capsules, injections or external preparations.

Images