CN111269433A - 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, preparation method and application - Google Patents
2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, preparation method and application Download PDFInfo
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Abstract
The invention discloses a 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, a preparation method and application thereof, and the technical scheme is that 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid is used as a ligand, and a coordination polymer is constructed with metal ion cobalt to obtain a brand-new 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex. The complex has the advantages of simple synthesis method, mild reaction conditions and high purity. Experimental results show that the complex has stronger binding capacity with BSA. And the compound has an inhibiting effect on the proliferation of esophageal cancer cells Eca109, is similar to cisplatin, and has an increased inhibiting rate of the compound on Eca109 compared with 24 hours in 48 hours. Therefore, the complex is expected to be developed into a novel complex anti-tumor drug for treating esophageal cancer caused by Eca109 cells.
Description
Technical Field
The invention relates to a 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex and a preparation method and application thereof, belonging to the field of coordination chemistry.
Background
Imidazole dicarboxylic acid is used as a polydentate chelating ligand, simultaneously contains heterocyclic imidazole rings and carboxyl, has coordination atoms N and O with different functions, and can be coordinated with various metals to form a complex. In addition, the structure of the target product can be regulated and controlled by adjusting the substituent at the 2-position of the imidazole dicarboxylic acid. The introduction of the 2-position substituent does not change the coordination site of the imidazole dicarboxylic acid, but can increase the steric effect to influence the structure and the performance of the generated complex. Researches show that the imidazole derivatives have various physiological effects of resisting bacteria, viruses and the like, and the tetrazole compounds have various biological activities of resisting bacteria, malaria, tuberculosis and the like.
Cobalt is a necessary trace element for the body and has important physiological action, the cobalt is a component of vitamin B12, and the cobalt element can stimulate the hematopoietic system of human bone marrow, promote the synthesis of hemoglobin and increase the number of red blood cells; the cobalt has a possible effect on the function of the thyroid gland, and animal experiment results show that the synthesis of thyroxine may need the cobalt which can resist the influence caused by iodine deficiency; the pancreas also contains a large amount of cobalt for the synthesis of insulin and some of the enzymes necessary for sugar and fat metabolism.
Serum Albumin (SA) is often used as a model protein to explore the interaction between small-molecule drugs and proteins, and SA is a soluble carrier protein rich in plasma and plays a role in transporting and storing drugs and other endogenous and exogenous substances. By analyzing the interaction of the two, the method has important guiding significance on the drug design and the action mechanism research.
The 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid ligand with bioactivity is coordinated with cobalt ions to obtain a complex, and the research on the interaction mechanism of the complex and Bovine Serum Albumin (BSA) is not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex and a preparation method and application thereof. The complex has the effect of inhibiting tumor cells, has strong interaction with BSA, and has the advantages of simple synthesis method, mild reaction conditions and high purity.
In order to achieve the above object, one of the technical solutions of the present invention is: 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex with molecular formula C7H8N6O6And Co, wherein the ligand is 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid.
One of the technical schemes of the invention is as follows: a preparation method of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex comprises the following steps:
(1) dissolving 0.05mmol of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid in 2ml of water to prepare a ligand solution;
0.1mmol of CoSO4·7H2Dissolving O in 4ml water to obtain CoSO4A solution;
(2) adding CoSO4And dropwise adding the solution into the ligand solution, then adding 2 drops of DMF, uniformly mixing, reacting, and cooling to room temperature after the reaction is finished to obtain the catalyst.
The reaction temperature was 120 ℃ and the reaction time was 72 h.
The cooling rate was 5 ℃/h.
The preparation method of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid comprises the following steps:
(1)2- (1H-tetrazole-1-methyl) -1H-benzimidazole
Mixing 40mmol of o-phenylenediamine and 44mmol of tetrazoleacetic acid, adding 50ml of 4mol/L hydrochloric acid, stirring for dissolving, heating to reflux, reacting for 4 hours, cooling to room temperature, adding the reaction solution into 100ml of ice water, uniformly mixing, adjusting the pH to 8.0, precipitating, carrying out suction filtration, washing to neutrality by using distilled water, drying to obtain a crude product, recrystallizing the crude product by using distilled water to obtain a silver gray long needle-shaped crystal, namely 2- (1H-tetrazol-1-methyl) -1H-benzimidazole;
(2)2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid
Adding 15ml of 98% concentrated sulfuric acid with mass fraction into 13mmol of 2- (1H-tetrazole-1-methyl) -1H-benzimidazole, heating to 110-115 ℃, dropwise adding 15ml of 30% hydrogen peroxide with volume fraction, continuing to react for 2H after 1H of dropwise addition, stopping heating, cooling to room temperature, adding the reaction solution into 80ml of ice water, refrigerating and standing at 4 ℃, precipitating a light yellow crystal, filtering, and drying to obtain light yellow crystal powder, namely 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid.
The reagent for adjusting the pH is a saturated sodium hydroxide solution.
In the step (2), the temperature is intermittently raised from room temperature to 110-115 ℃, the temperature per liter is 10 ℃, and the temperature is kept for 5 min.
One of the technical schemes of the invention is as follows: an application of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex in preparing antineoplastic drugs.
The tumor is esophageal cancer.
One of the technical schemes of the invention is as follows: the interaction of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex and bovine serum albumin.
The synthetic route of the ligand of the invention is as follows:
synthetic route of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid
The invention has the beneficial effects that:
1. the invention takes 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid as a ligand to construct a coordination polymer with metal ion cobalt, so as to obtain a brand new 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex. According to the invention, a tetrazole substituent is introduced to the 2-position of the imidazole dicarboxylic acid skeleton for the first time to obtain a hybrid, and a cobalt complex is constructed, so that the lipid solubility, antibacterial activity and drug-resistant activity of the medicine are expected to be improved, and the toxic and side effects of the medicine are reduced.
2. The invention adopts a hydrothermal method to prepare CoSO4·7H2And reacting O with 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid to coordinate the ligand with corresponding metal ions, thereby obtaining the novel 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex. The synthesis method is simple, convenient and safe, has mild reaction conditions and high purity, and is beneficial to subsequent activity research.
3. The invention adopts the fluorescence spectrometry to research the interaction between the complex and BSA. The result shows that the complex has stronger binding capacity with BSA.
4. The invention adopts an MTT method to detect the influence of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex on the proliferation of tumor cells. The result shows that the complex has an inhibiting effect on the proliferation of esophageal cancer cells Eca109, is similar to cisplatin, and the inhibiting rate of the complex on Eca109 is increased after 48 hours compared with 24 hours. Therefore, the complex is expected to be developed into a novel complex anti-tumor drug for treating esophageal cancer caused by Eca109 cells.
Drawings
FIG. 1 is the nuclear magnetic resonance hydrogen spectrum of 2- (1H-tetrazole-1-methyl) -1H-benzimidazole.
FIG. 2 shows 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid (H)6tmidc) nuclear magnetic resonance hydrogen spectrum.
FIG. 3 is a coordination environment diagram of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex.
FIG. 4 is a one-dimensional supermolecule chain structure diagram of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex.
FIG. 5 is a three-dimensional supermolecular net structure diagram of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex.
FIG. 6 shows the change of fluorescence spectrum of BSA solution with increasing concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex at different temperatures (the direction of the arrow indicates the direction of increasing concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex), (a: 298K, b: 308K, c: 313K).
FIG. 7 is a Stern-volmer plot of cobalt 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylate complexes versus BSA at different temperatures (a: 298K, b: 308K, c: 313K).
FIG. 8 is a double logarithmic graph of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex to BSA at different temperatures (a: 298K, b: 308K, c: 313K).
FIG. 9 shows the simultaneous fluorescence spectra of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex for BSA at different temperatures at. DELTA.λ.15 nm (the direction indicated by the arrow indicates the direction in which the concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex increases), (a: 298K, b: 308K, c: 313K).
FIG. 10 shows simultaneous fluorescence spectra of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex for BSA at different temperatures at. DELTA.λ.60 nm (the direction indicated by the arrow indicates the direction in which the concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex increases), (a: 298K, b: 308K, c: 313K).
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1: preparation method of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid
(1)2- (1H-tetrazole-1-methyl) -1H-benzimidazole (bimt)
Placing 40mmol of o-phenylenediamine and 44mmol of tetrazoleacetic acid in a round-bottom flask, adding 50ml of 4mol/L hydrochloric acid, stirring for dissolving, heating to reflux for reacting for 4 hours, cooling to room temperature, adding the reaction solution into 100ml of ice water, uniformly mixing, adding a saturated sodium hydroxide solution to adjust the pH value to 8.0, precipitating a large amount of precipitate, carrying out suction filtration, washing to be neutral by distilled water, drying to obtain a crude product, recrystallizing the crude product by distilled water to obtain 5.4614g of silver gray long needle-shaped crystals, namely 2- (1H-tetrazol-1-methyl) -1H-benzimidazole, wherein the yield is: 68.20 percent;
(2)2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid (H)6tmidc)
Adding 15ml of 98% concentrated sulfuric acid with mass fraction into 13mmol of 2- (1H-tetrazole-1-methyl) -1H-benzimidazole, heating to 110 ℃ (intermittently heating from room temperature to 110 ℃, keeping the temperature at 10 ℃ per liter for 5min), dropwise adding 15ml of 30% hydrogen peroxide with volume fraction, continuing to react for 2H after 1H of dropwise adding, stopping heating, cooling to room temperature, adding the reaction solution into 80ml of ice water, refrigerating and standing at 4 ℃, separating out a light yellow crystal, performing suction filtration and drying to obtain 3.7003g of light yellow crystal powder, namely 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid, wherein the yield is 76.61%.
Example 2: structural characterization of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid
The 2- (1H-tetrazole-1-methyl) -1H-benzimidazole (bimt) and 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid (H) -obtained in example 1 were mixed with tetramethylsilane as a standard by a Bruker ABANCE III 500MHz NMR spectrometer6tmidc) are respectively dissolved by MeOD and deuterated DMSO, hydrogen spectrum analysis is carried out, and the test result is as follows:
FIG. 1 shows the NMR spectrum of 2- (1H-tetrazole-1-methyl) -1H-benzimidazole (bimt),1HNMR (MeOD,500MHz), δ 9.37(s,1H, Tetra-H), hydrogen on tetrazole, δ 7.58(m,2H, Ar-H), δ 7.27-7.29(m,2H, Ar-H), δ 6.02(t,2H, CH), CH2)。
FIG. 2 shows 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid (H)6tmidc) of the nuclear magnetic resonance spectrum,1HNMR (DMSO,500MHz), δ ═ 9.50(s, 1H, Tetra-H), δ ═ 5.87(s, 2H, CH), due to hydrogen on tetrazole2). The shifts of H and the number of hydrogens given in the figure are in theoretical agreement with the shifts and numbers of H for the compounds synthesized, indicating that the compounds were synthesized successfully.
Example 3: preparation method of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex
(1) Dissolving 0.05mmol of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid in 2ml of water to prepare a ligand solution;
0.1mmol of CoSO4·7H2Dissolving O in 4ml water to obtain CoSO4A solution;
(2) adding CoSO4Dropwise adding the solution into a ligand solution, then adding 2 drops of DMF (dimethyl formamide), uniformly mixing, placing the mixture in a reaction kettle with a polytetrafluoroethylene lining, reacting for 72H in an oven at 120 ℃, and cooling to room temperature at the speed of 5 ℃/H after the reaction is finished to obtain a bulk pink transparent crystal, namely the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, wherein the yield is 78%.
Example 4: structural representation of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex
1. Analysis of single crystal structure
The 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex obtained in example 3 was subjected to a crystal structure test using a Bruker APEX-II CCD type X-ray single crystal diffractometer, and the test results were:
the complex is a monoclinic system, and the space group is P2 (1)/c; cell parameters
α＝90°，β＝90.0230(10)°，γ＝90°；V＝1241.69(7)A3；Z＝4；Dc＝1.771Mg/m3；R1＝0.0384，ωR2＝0.0921。
Mo-K α ray monochromatized by graphite
X-ray diffraction experiments were performed in an omega scan fashion and diffraction data were collected. Data reduction is carried out on diffraction data by a Crystalclar program, and data correction is carried out by adopting an Lp factor and a semi-absorption experience. The structure analysis and refinement are carried out by using SHELXS-97 program, metal atoms are determined by a direct method, and then the correction is carried out according to anisotropy by using a least square method and Fourier transform. All non-hydrogen atoms are corrected by an anisotropic thermal parameter method, the hydrogen atoms are obtained by a difference Fourier synthesis method, and the structural parameters are optimized by a full matrix least square method. To obtain 2- (1H-tetrazole-1-methyl) -1H-imidazole-4 shown in figure 3The coordination environment diagram of the 5-dicarboxylic acid cobalt complex is shown in a one-dimensional supramolecular chain structure diagram of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex shown in fig. 4, and is shown in a three-dimensional supramolecular net structure diagram of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex shown in fig. 5.
As can be seen from FIG. 3, the composition of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex is { [ Co (H)4tmidc)(H2O)2]n(n is an integer greater than or equal to 1, and the parenthesis represents the composition of the minimum asymmetric unit), and the molecular formula is C7H8N6O6And (3) Co. The asymmetric unit of the complex comprises one half of Co (II) ion and one half of partially deprotonated H4tmidc2-An anion and 1 coordinated water molecule. Co1 is hexacoordinated in a Co (II) complex and is connected with four oxygen atoms (O1, O1# 1, O3, O3#1) and two nitrogen atoms (N1, N1#1), wherein the two oxygen atoms (O1, O1#1) are respectively from carboxyl on the ligand, and the two N atoms (N1, N1#1) are from two symmetrically related H6N on the imidazolyl group of the tmidc ligand, the other two O atoms (O3, O3#1) come from two coordinated water molecules, Co1 is octahedral CoN2O4Coordination configuration. The equatorial plane of the octahedron is composed of O3, O3# 1, N1 and N1# 1, and the bond lengths of Co1-N1 and Co1-N1# 1 are as follows:
co1-O3 and Co1-O3# 1:
o1 and O1# 1 occupy the apex position, and the bond lengths of Co1-O1 and Co1-O # 1 are:
the bond angle O1-Co1-O1# 1 was 180. The bond lengths of the axial Co1-O1 and Co1-O # 1 are longer than those of equatorial Co1-N1, Co1-N1# 1, Co1-O3 and Co1-O3# 1. These Co-O/N are all within the normal bond length range and are consistent with the values reported in the literature [ Jing Chi, Xiao Li Wei, Zhou Qiu Xiang, Dai Fucai ] structure and magnetic properties of pyridyl imidazole carboxylic acid cobalt complexes [ J]University of south Henan school newspaper(Nature science edition), 2017, (01):22-26.]It indicates that the ligand successfully coordinates with the metal and the coordination atom forms a coordination bond with the metal.
As shown in FIG. 3, each Htmidc2-The tetradentate ligand coordinates with Co (II) ions in an N, O-chelating coordination mode and the heteroleptic Co (II) ions form one-dimensional long chains infinitely extending along the direction b, and the distance between adjacent Co & C is
In the same ligand, imidazole ring and tetrazole ring are not coplanar, and the dihedral angle between them is 75.2(5) °. As shown in FIG. 4, along the a direction, adjacent chains are connected into a two-dimensional layered structure through O-H.N hydrogen bonds between coordinated water and tetrazole rings. In addition, O-H.O hydrogen bond between coordinated water and carboxyl group exists in the complex. The adjacent layers are further connected with each other through such hydrogen bonds to form a three-dimensional supramolecular network structure as shown in fig. 5.
2. Infrared spectroscopic analysis
Infrared spectrum test (KBr tablet method, scanning at room temperature, test range 400--1). Characteristic absorption peak (cm) in infrared spectrum-1)：3420，3129，3016，1633，1571，1490，1399，1349，1276，1170，983，871，782，680，518。
Example 5: interaction of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex and bovine serum albumin
The test instrument: the fluorescence spectrophotometer model is F7000, manufactured by Hitachi, Japan.
Preparation of reagents and setting of test conditions:
1. preparation of sample liquid
Cobalt complex solution: weighing 0.0025g of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, and diluting to 10mL with DMSO (dimethyl sulfoxide) to prepare the compoundThe concentration is 0.75 × 10-3And (3) a cobalt complex solution in mol/L.
Tris-HCl buffer solution: 3.0250g of Tris (Tris hydroxymethyl aminomethane) and 1.4625g of sodium chloride are taken, the volume is adjusted to 250mL by distilled water, the mixture is shaken up, and then the pH value is adjusted to 7.36 by hydrochloric acid.
Bovine Serum Albumin (BSA) solution: weighing bovine serum albumin 0.0333g, dissolving with Tris-HCl buffer solution to obtain a solution with a concentration of 5 × 10-5Bovine serum albumin solution of mol/L.
2. Setting of fluorescence spectrum test conditions
Setting an excitation wavelength to be 280nm, setting excitation and emission narrow peaks to be 5nm, setting a voltage to be 400V, and scanning fluorescence spectra in the range of 285-500 nm under the conditions of 298K, 308K and 313K respectively; and the simultaneous fluorescence spectra thereof were scanned at wavelength differences Δ λ of 15nm and Δ λ of 60nm, respectively.
3. Measurement of fluorescence Spectroscopy
1.5mL of bovine serum albumin solution and 13.5mL of Tris-HCl buffer solution were transferred to a 100mL round-bottomed flask. Adding complex solutions with different volumes, and measuring the change of fluorescence spectrum (final concentration range of complex: 0-4.245 × 10)- 5mol/L)。
And (3) determining the interaction of the complex and bovine serum albumin by using a fluorescence spectroscopy:
(1) quenching of fluorescence
The interaction of the complex synthesized in example 3 with BSA was investigated by fluorescence spectroscopy, as shown in FIG. 6. BSA concentration of 5X 10-6mol/L, the endogenous fluorescence of BSA gradually decreases with the increase of the concentration of the complex, when the concentration of the complex is 4.245X 10-5At mol/L, the interaction of the complex and BSA is basically balanced. Indicating that at the 298K, 308K and 313K temperature, the endogenous fluorescence of the BSA can be effectively quenched by the complex, and the position of the maximum emission peak and the shape of the emission spectrum are not basically changed, indicating that a complex with weak fluorescence or no fluorescence is formed between the complex and the BSA, so that the endogenous fluorescence of the BSA is quenched.
(2) Quenching mechanism
There are two types of fluorescence quenching, dynamic quenching and static quenching, which are the interactions between drug small molecules and proteins, the former is an electron transfer process and does not affect the conformation of proteins, and the latter is fluorescence quenching due to binding reactions. Dynamic and static quenching can be judged by measuring quenching constants of the ligand and the complex to BSA fluorescence at different temperatures. The dynamic quenching process follows the Stern-volmer equation:
F0/F＝1+Ksv[Q]＝1+Kqτ0[Q]formula (1)
F0And F is the fluorescence intensity of the protein without and with quencher added, respectively; kqIs the rate constant for the bimolecular quenching process; ksvIs the quenching constant of the Stern-volmer equation; tau is0The average lifetime of the biomacromolecule without the quencher is about 10-8s；[Q]Is the concentration of the drug small molecule.
The rate constant K of the BSA quenching process can be determined from equation (1)q(see Table 1).
TABLE 1 Rate constants, binding constants, number of binding sites for cobalt complexes to interact with BSA
Table 1 shows that the rate constants of the quenching processes at the three temperatures are all larger than the maximum dynamic quenching rate constant of 2.0X 10 of each type of quenching agent to the biomacromolecule10L·mol-1·s-1The 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex has a fluorescence quenching rate larger than the maximum diffusion collision quenching rate to BSA (bovine serum albumin), and the quenching rate constant is reduced along with the increase of the temperature, so that the interaction of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex to BSA (bovine serum albumin) is judged to be endogenous fluorescence quenching of BSA (bovine serum albumin) caused by complex formation, and the quenching rate belongs to static quenching.
FIG. 7 is a Stern-volmer linear fit of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex to BSA at different temperatures.
(3) Binding constant and number of binding sites
The binding constant and the number of binding sites of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex and BSA (bovine serum albumin) interaction can be calculated by using a double logarithmic equation:
log[(F0-F)/F]＝logKb+nlog[Q]formula (2)
KbAs the binding constant, n is the number of binding sites, the calculation results (Table 1) show that the binding constant K of the cobalt complexes with BSAbAre respectively 7.345X 104L·mol-1、2.428×105L·mol-1、4.562×104L·mol-1The number n of binding sites is 1.166, 1.285 and 0.909 respectively, which indicates that the complex has stronger interaction with BSA.
FIG. 8 is a double logarithmic graph of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex to BSA at different temperatures.
(4) Synchronous fluorescence spectroscopy
The BSA molecule emits strong endogenous fluorescence due to the fact that the BSA molecule contains amino acid residues such as tyrosine and tryptophan, wherein the fluorescence intensity of the tyrosine and the tryptophan is large, the excitation spectra of the tyrosine and the tryptophan are similar, and the emission spectra are overlapped, so that the synchronous fluorescence spectrum is used for selecting proper delta lambda, and the purposes of simplifying the spectrum, narrowing the band and reducing light scattering interference can be achieved. In general, a synchronous fluorescence spectrum with Δ λ of 15nm may express a characteristic spectrum of tyrosine, and a synchronous fluorescence spectrum with Δ λ of 60nm may express a characteristic spectrum of tryptophan. The synchronous fluorescence spectrum can also provide important information for the conformational change of the protein, and the influence of the interaction of the drug and BSA on tryptophan or tyrosine residues can be presumed according to the change of the emission wavelength of the synchronous fluorescence spectrum, so that the change of the protein conformation caused by the action of the drug and the protein can be judged.
Fig. 9 shows the change of BSA with increasing concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex in the simultaneous fluorescence spectrum at 298K, 308K and 313K, respectively, and Δ λ of 15nm, which shows that the intensity of the maximum absorption peak of tyrosine residues gradually decreases, and the simultaneous fluorescence spectrum undergoes a slight blue shift with increasing temperature, indicating that the hydrophobicity of the microenvironment in which the tyrosine residues are located increases and the hydrophilicity decreases.
Fig. 10 shows the change of the fluorescence spectrum of BSA with increasing concentration of 2- (1H-tetrazol-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex at 298K, 308K and 313K, respectively, and Δ λ 60nm, which shows that the intensity of the maximum absorption peak gradually decreases, indicating that the complex forms a complex with BSA, which is weakly fluorescent or non-fluorescent. In addition, the peak position of the characteristic fluorescence spectrum of the tryptophan residue generates red shift, which shows that the polarity of the microenvironment where the tryptophan residue is positioned is increased, the hydrophilicity is enhanced, the hydrophobic cavity of the protein is loosened, and the change of the fluorescence spectrum of the tryptophan residue is more obvious than that of the tyrosine residue, thereby proving that the combination site of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-cobalt dicarboxylate complex and BSA (bovine serum albumin) is closer to the tryptophan residue.
In summary, the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex has a binding effect with tyrosine and tryptophan residues in BSA, but mainly binds with tryptophan residues in BSA.
Example 6: influence of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex on tumor cell proliferation
The MTT method is adopted to detect the influence of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex on the proliferation of tumor cells:
planting tumor cell suspension (esophageal cancer cell Eca109) of logarithmic growth phase in 96-well culture plate, adding 200 μ l per well, with cell density of 8000 per well, setting 3 multiple wells, placing at 37 deg.C and 5% CO2Incubate for 24h, discard the supernatant and add 200 μ l RPMI1640 medium containing drugs (cisplatin, ligand, cobalt complex) and 10% FBS at final concentrations: 55 mu mol/L, 50 mu mol/L, 45 mu mol/L, 40 mu mol/L, 35 mu mol/L, 25 mu mol/L, 20 mu mol/L and 10 mu mol/L, taking an equal volume of RPMI1640 culture medium containing 10% FBS and without drugs as a blank control group, continuing to incubate for 24 hours and 48 hours respectively, then discarding the supernatant, adding 100 mu L of MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide) with the concentration of 5mg/ml into each well, continuing to incubate for 4 hours under the same conditions, and terminating the culture. After the completion of the culture, the supernatant was discarded every timeAdding 150 μ l dimethyl sulfoxide into the hole, shaking thoroughly, mixing well to dissolve purple crystal, and measuring absorbance (OD) at 570nm with microplate reader. Adopting SPSS 18.0 software to calculate experimental data to obtain IC50Value, IC for inhibition of esophageal carcinoma cell Eca109 proliferation by cisplatin, ligands and cobalt complexes50The results are shown in Table 2. As shown in the table, the IC of the 24h and 48h complexes on esophageal cancer cell Eca10950The values are respectively: 77.17 +/-0.008 mu mol/L and 73.91 +/-0.014 mu mol/L, show that the complex has an inhibiting effect on the proliferation of esophageal cancer cells Eca109, and the inhibition rate of the complex on the Eca109 is increased compared with 24h in 48 h. The inhibition effect of the cobalt complex on the proliferation of esophageal cancer cells Eca109 is similar to that of cisplatin, and the effect of the cobalt complex is stronger than that of a ligand, which is a result of the synergistic effect between the ligand and metal ions. Therefore, the complex is expected to be developed into a novel complex anti-tumor drug for treating esophageal cancer caused by Eca109 cells.
TABLE 2 proliferation inhibition of esophageal carcinoma cells Eca109 by cisplatin, ligands and cobalt complexes
Claims (10)
1.2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex, which is characterized in that the molecular formula is C7H8N6O6And Co, wherein the ligand is 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid.
2. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 1, characterized by comprising the following steps:
(1) dissolving 0.05mmol of 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid in 2ml of water to prepare a ligand solution;
0.1mmol of CoSO4·7H2Dissolving O in 4ml water to obtain CoSO4A solution;
(2) adding CoSO4The solution is dripped into the ligand solution,and adding 2 drops of DMF, uniformly mixing, reacting, and cooling to room temperature after the reaction is finished to obtain the finished product.
3. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 2, wherein the reaction temperature is 120 ℃ and the reaction time is 72 hours.
4. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 2, wherein the cooling speed is5 ℃/H.
5. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex according to any one of claims 2 to 4, which is characterized in that the preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid comprises the following steps:
(1)2- (1H-tetrazole-1-methyl) -1H-benzimidazole
Mixing 40mmol of o-phenylenediamine and 44mmol of tetrazoleacetic acid, adding 50ml of 4mol/L hydrochloric acid, stirring for dissolving, heating to reflux, reacting for 4 hours, cooling to room temperature, adding the reaction solution into 100ml of ice water, uniformly mixing, adjusting the pH to 8.0, precipitating, carrying out suction filtration, washing to neutrality by using distilled water, drying to obtain a crude product, recrystallizing the crude product by using distilled water to obtain a silver gray long needle-shaped crystal, namely 2- (1H-tetrazol-1-methyl) -1H-benzimidazole;
(2)2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid
Adding 15ml of 98% concentrated sulfuric acid with mass fraction into 13mmol of 2- (1H-tetrazole-1-methyl) -1H-benzimidazole, heating to 110-115 ℃, dropwise adding 15ml of 30% hydrogen peroxide with volume fraction, continuing to react for 2H after 1H of dropwise addition, stopping heating, cooling to room temperature, adding the reaction solution into 80ml of ice water, refrigerating and standing at 4 ℃, precipitating a light yellow crystal, filtering, and drying to obtain light yellow crystal powder, namely 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid.
6. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 5, wherein the reagent for adjusting the pH is a saturated sodium hydroxide solution.
7. The preparation method of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 5, wherein the temperature rise in step (2) is intermittent temperature rise, the temperature is raised from room temperature to 110 ℃ -115 ℃, the temperature is raised to 10 ℃ per liter, and the temperature is kept for 5 min.
8. An application of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as claimed in claim 1 in preparing antitumor drugs.
9. The use of claim 8, wherein the tumor is esophageal cancer.
10. The interaction of the 2- (1H-tetrazole-1-methyl) -1H-imidazole-4, 5-dicarboxylic acid cobalt complex as defined in claim 1 and bovine serum albumin.
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