CN108623638B - 12-chlorobenzimidazole-1, 8-naphthalimide-platinum complex and preparation method and application thereof - Google Patents

Abstract

The invention discloses a 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex and a preparation method and application thereof. The preparation method of the complex mainly comprises the following steps: taking the compound represented by the following formula (II) and the compound represented by the formula (III) in an organic solvent, and carrying out a coordination reaction under heating or non-heating conditions to obtain the target product. The inhibitory activity of the complex on some cancer cells is comparable to that of the commonly used antitumor drug cisplatin, but its toxicity to human liver normal cells HL-7702 is lower. The structure of the complex described in the present invention is shown in the following formula (I), and the structures of the compound represented by the formula (II) and the compound represented by the formula (III) involved in the preparation of the complex are as follows:

Description

12-chlorobenzimidazole-1,8-naphthalimide-platinum complex and its preparation method and application

technical field

The invention relates to the technical field of medicine, in particular to a 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex and a preparation method and application thereof.

Background technique

Platinum-based metal-based anti-tumor complexes are an important class of anti-tumor drug libraries. The design and screening of novel platinum-based metal-based anti-tumor complexes with high efficiency and low toxicity have attracted great attention from medicinal chemists.

Naphthalimide compounds are a class of classic small-molecule DNA intercalators, and their representative compounds, amonafide and mitonafide, have high anticancer activity against multiple cancer cells. But there is no report about the preparation method of 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex and its cytotoxicity.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex with novel structure, high toxicity to some tumor cells and low toxicity to normal liver cells, and Its preparation method and application.

The present invention relates to a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

The present invention also provides the preparation method of the above-mentioned compound, which mainly comprises the following steps: taking the compound represented by the following formula (II) and the compound represented by the formula (III) in an organic solvent, and carrying out a coordination reaction under heating or non-heating conditions, to obtain the target product;

In the above-mentioned preparation method, the compound shown in the formula (II) is 12-chlorobenzimidazole-1,8-naphthalimide, and this compound can refer to existing literature (Banerji K D, Sen K K, Mazumdar A K D, Synthesis of Somenaphthoylenebenzimidazole, Journal of the Indian Chemical Society, 197653(11), 1159-61) can be synthesized, and a synthetic route can also be designed for synthesis. The following methods are preferably used for synthesis: .

Take 1,8-naphthalene anhydride and p-chloro-o-phenylenediamine (4-chloro-o-phenylenediamine) in a stoichiometric ratio and place them in a polar solvent (selected from acetic acid, xylene, toluene, DMF, DMSO and ethylene glycol methyl ester) One or a combination of two or more ethers) in the reflux reaction (about 4-8h), after the reaction is completed, cooling, a solid is precipitated, the solid is collected, and dried to obtain the compound 12 shown in the formula (II) -Chlorobenzimidazole-1,8-naphthalimide (yellow crystal or powder), the specific synthetic route is as follows:

In the above-mentioned preparation method, the compound shown in the formula (III) is cis-dichloro-bis (dimethyl sulfoxide) platinum (II), and can refer to the existing literature (Al-Allaf TA K, et al.Transit. Met.Chem., 1998) prepared.

In the above preparation method, the mol ratio of the compound shown in the formula (II) and the compound shown in the formula (III) is a stoichiometric ratio. In actual operation, the compound shown in the formula (II) or the compound shown in the formula (III) The compound may also be present in relative excess, but the excess will result in an impure product.

In the above preparation method, the organic solvent can be one selected from methanol, ethanol, chloroform, dichloromethane, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF) or A combination of two or more. When the selection of the organic solvent is a mixture of two or more of the above selections, the ratio between them can be any ratio. The amount of the organic solvent can be determined as required. In the specific dissolving step, the compound represented by the formula (II) and the compound represented by the formula (III) can be respectively dissolved in an organic solvent, and then mixed together to react; the compound represented by the formula (II) and the formula (III) can also be mixed together. ) are mixed and then dissolved in an organic solvent.

The 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex described in the present invention can be prepared by a solution method or a solvothermal method during specific preparation.

When the solution method is used to prepare, it mainly includes: dissolving the compound shown in formula (II) and the compound shown in formula (III) in an organic solvent, carrying out a coordination reaction under heating or without heating, and filtering while hot after the reaction is completed , the filtrate is cooled, a solid is precipitated, and the solid is collected, which is the target product.

In the above solution method, whether the reaction is complete can be detected by tracking by thin layer chromatography. In order to improve the yield of the reaction, the reaction is preferably carried out under heating conditions, more preferably carried out under the condition of ≥40 °C, more preferably carried out under the condition of 60-80 °C, when the reaction is carried out within this preferred temperature range, the reaction The time is usually 24-48h. In this method, 1 mmol of the compound represented by the formula (II) and 1 mmol of the compound represented by the formula (III) are usually dissolved in 6-25 mL of an organic solvent.

When prepared by solvothermal method, it mainly includes: dissolving the compound represented by formula (II) and the compound represented by formula (III) in an organic solvent, and then placing them in a container (usually a thick-walled glass tube with one end open or a pressure-resistant glass tube). bottle), evacuated to vacuum after being frozen in liquid nitrogen, melt-sealed, and then carry out a coordination reaction under heating conditions, cool, crystals are precipitated, and the solid is collected, which is the target product. In this method, the reaction is preferably carried out under the condition of ≥40°C, more preferably under the condition of 60-80°C. When the reaction is carried out within this preferred temperature range, the reaction time is usually 24-48h. When using this method, 1 mmol of the compound represented by the formula (II) and 1 mmol of the compound represented by the formula (III) are usually dissolved in 5-20 mL of an organic solvent.

The target product separated out by the above-mentioned solvothermal method is a crystal, and the product separated out by the solution method is usually powdery. In a mixed solvent consisting of one of ethanol and one of chloroform and dichloromethane, the reaction is carried out at 60-80° C. for 24-48 hours, and the crystalline target product can be obtained after cooling.

The present invention also includes the application of the above-mentioned 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs.

The present invention further includes a pharmaceutical composition comprising a therapeutically effective dose of the above-mentioned 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex or a pharmaceutically acceptable salt thereof.

Compared with the prior art, the present invention provides a 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex with novel structure, which has short preparation period, high yield and stable quality; the applicant The experimental results show that the inhibitory activity of the complex on some cell lines is much higher than that of its parent nucleus (such as Sk-ov-3) and cisplatin (such as SMMC-7721), and its toxicity to normal cells is much lower. It is used in common anticancer drugs (such as cisplatin and 5-fluorouracil), and is expected to be used in the preparation of antitumor drugs.

Description of drawings

Fig. 1 is the crystal structure diagram of the final product obtained in Example 1 of the present invention;

Figure 2 is the agarose gel electrophoresis image of the effect of compound I and Topo I at different concentrations.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments to better understand the content of the present invention, but the present invention is not limited to the following embodiments.

The compound represented by the formula (II) involved in the following examples (that is, 12-chlorobenzimidazole-1,8-naphthalimide) is prepared by the following method:

Take 1,8-naphthalene anhydride (5g, 25.2mmol, 1.0equiv) and 4-chloro-o-phenylenediamine (3.59g, 25.2mmol, 1.0equiv) in a 100ml round-bottomed flask, add 50mL of acetic acid, and heat the reaction to 118 ℃ stirring and refluxing for 6h. The reaction was stopped, cooled to room temperature naturally, a yellow solid was precipitated, and filtered to obtain 6.7 g of the compound represented by formula (II) (yellow powdery solid), yield 87.46% 1H NMR (400MHz, DMSO) δ8.71 (dd, J =14.3,7.4Hz,2H),8.54(d,J=8.1Hz,1H),8.48-8.29(m,2H),7.98-7.49(m,4H).13C NMR(101MHz,DMSO)δ160.58,151.25, 144.93,136.28,133.11,132.77,132.44,132.38,131.97,131.86,128.72,128.05,127.80,127.74,127.58,127.08,126.93,123.05,121.38,121.28,120.51,120.32,116.65,116.55.MS m/z:305.04 [M+H]+.

Example 1

Measure 12-chlorobenzimidazole-1,8-naphthalimide (90.14 mg, 0.2958 mmol), dichlorobis(dimethylsulfoxide)platinum( _II )(Pt(DMSO)2Cl2 ₎ (124.53 mg, 0.2958 mmol), 5 ml of methanol and 5 ml of chloroform, placed in a round-bottomed flask, and stirred for 48 h at a temperature of 60 ° C. After the reaction was completed, the unreacted raw materials were removed by hot filtration, and the filtrate was cooled to room temperature, and a yellow solid was precipitated. , the solid was collected and dried to obtain 118.07 mg of yellow powder product with a yield of 55.00%.

The products obtained in this implementation are characterized by mass spectrometry, elemental analysis and X-single crystal diffraction, as follows:

(1) MS m/z: 690.9980[M-Cl+DMSO] ⁺ .

( _{2 )} Anal.Calc. (for _{C20H15Cl3N2O2PtS} ) C 37.02; H 2.33; N 4.32%, Found. _C37.04 ; H _2.30 ; N 4.32%.

(3) get 10mg of this implementation gained product and 5ml methanol/chloroform mixed solution (the volume ratio of methanol and chloroform is 1:1) and place in sealed tube, be warming up to 90 ℃, react 12h, cool, have crystals to separate out, collect crystals , dried to obtain yellow crystals. The obtained yellow crystal was analyzed by X-Ray single crystal diffraction, its crystallographic data are shown in the following Table 1, some bond lengths and bond angles are shown in the following Table 2, and the crystal structure of the obtained product is shown in Figure 1.

Table 1: Crystallographic and structural correction data for the product

和键角[°]Table 2: Partial health of the product

and bond angle [°]

Therefore, it can be determined that the yellow powder product obtained in this example is the target product 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex, and its structural formula is shown in the following formula (I):

Example 2

Take 12-chlorobenzimidazole-1,8-naphthalimide (304.73 mg, 1 mmol), Pt(DMSO) ₂ Cl ₂ (422.9 mg, 1 mmol) and 12 ml of methanol, put it in a thick-walled pressure bottle, dissolve After the reaction was stirred for 36 hours at a temperature of 70° C., and cooled to room temperature naturally, a yellow solid was precipitated, which was separated and dried to obtain 371.09 mg of yellow powder with a yield of 51.00%.

Mass spectrometry, elemental analysis and further X-ray single crystal diffraction analysis were performed on the product obtained in this example, and it was determined that the product obtained in this example was the target compound.

Example 3

Take 12-chlorobenzimidazole-1,8-naphthalimide (225.62 mg, 0.7404 mmol), Pt(DMSO) ₂ Cl ₂ (311.64 mg, 0.7404 mmol) and 6 ml DMSO, put it in a round bottom flask, dissolve After the reaction was stirred at 80° C. for 24 hours, and cooled to room temperature naturally, a yellow solid was precipitated, which was separated and dried to obtain 285.28 mg of yellow powder with a yield of 53.10%.

Mass spectrometry, elemental analysis and further X-ray single crystal diffraction analysis were performed on the product obtained in this example, and it was determined that the product obtained in this example was the target compound.

Example 4

Take 12-chlorobenzimidazole-1,8-naphthalimide (112.81 mg, 0.3702 mmol), Pt(DMSO) ₂ Cl ₂ (155.82 mg, 0.3702 mmol) and 10 ml of chloroform, put it in a round-bottomed flask, dissolve After the reaction was carried out at a temperature of 80°C, the reaction was stirred for 40 hours. After the reaction, the unreacted raw materials were removed by filtration while hot, and cooled to room temperature naturally. A yellow solid was precipitated, which was separated and dried to obtain 120.88 mg of yellow powder with a yield of 45.00%.

Mass spectrometry, elemental analysis and further X-ray single crystal diffraction analysis were performed on the product obtained in this example, and it was determined that the product obtained in this example was the target compound.

Example 5

Take 12-chlorobenzimidazole-1,8-naphthalimide (112.81 mg, 0.3702 mmol), Pt(DMSO) ₂ Cl ₂ (155.82 mg, 0.3702 mmol), 5 ml of ethanol and 5 ml of dichloromethane, placed in a circle In the bottom flask, at a temperature of 40°C, the reaction was stirred for 48 hours. After the reaction was completed, the unreacted raw materials were removed by filtration while hot, and cooled to room temperature naturally. A yellow solid was precipitated, which was separated and dried to obtain 51.04 mg of yellow powder with a yield of 19.00%. .

Mass spectrometry, elemental analysis and further X-ray single crystal diffraction analysis were performed on the product obtained in this example, and it was determined that the product obtained in this example was the target compound.

Example 6

Example 5 was repeated, except that the reaction temperature was normal temperature.

Finally, 9.75 mg of yellow powder was obtained with a yield of 3.63%.

Mass spectrometry, elemental analysis and further X-ray single crystal diffraction analysis were performed on the product obtained in this example, and it was determined that the product obtained in this example was the target compound.

Experimental Example 1: In vitro antitumor activity test of the target compound of the present invention on various human tumor cell lines:

In order to illustrate the anti-tumor effect of the 12-chlorobenzimidazole-1,8-naphthalimide-platinum complexes of the present invention, the applicant conducted an anti-tumor activity experiment on the complexes (using common anti-tumor drugs 5). - Fluorouracil (5-FU) and cisplatin (Cis-platin) as reference), and the toxicity test on normal cells was carried out on the compounds prepared by the method described in Example 1 above.

1. Cell Seeding and Culture

The selected cell lines were placed in an incubator at 37°C and 5% CO ₂ fully humidified, and inoculated in PPMI1640 medium containing 10% inactivated newborn bovine serum. Observe the cell growth with an inverted microscope, change the medium 2-3 times a week, and pass it once every 6-7 days. When inoculating, it is digested with 0.25% trypsin for passage, usually 3-4 times of passage, and cells in the logarithmic growth phase are used. in the experiment.

2. Primary screening of compound activity at the cellular level

The compounds used in this experiment (wherein the complex of the present invention is prepared according to the method described in Example 1 above), the purity is ≥95%, all compounds are formulated into 100 μg/mL, and the final concentration of co-solvent DMSO does not exceed 1 %, test the inhibition rate of each compound on cancer cells at this concentration, where the inhibition rate is greater than 50% and conforms to the morphological changes of the cells that are inhibited (or damaged) under the light microscope (such as cell shrinkage, fragmentation, floating, etc.), and If the compound is not very toxic to normal cells, it is preliminarily determined that the compound is effective in the preliminary screening, that is, the next step is to obtain the IC ₅₀ stage.

3. Cell growth inhibition assay (MTT method)

MTT colorimetry, is a method to detect cell growth and survival. Detection principle: Different from dead cells, exogenous MTT can be reduced to water-insoluble blue-violet crystalline formazan (Formazan) by succinate dehydrogenase in the mitochondria of living cells and deposited in cells. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and its light absorption value can be measured by enzyme-linked immunosorbent assay at 490nm wavelength, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. This method has been widely used in the activity detection of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity test and tumor radiosensitivity measurement, etc. It has the characteristics of high sensitivity and economy.

值)，计算细胞增殖抑制率，对初筛抗肿瘤效果好的受试化合物，继续用5个浓度梯度继续做相应细胞株的IC₅₀值，所有实验均重复3次后取平均值。实验结果详见下述表3。The cells in the logarithmic growth phase were taken, and 180 μL (about 4500-5000 cells) of cell-containing medium per well was inoculated into a 96-well culture plate, and cultured at 37° C. and 5% CO ₂ fully humidified for 24 h. After the cells adhered, samples were added in an amount of 20 μL per well, 6 duplicate wells were set for each sample, and a corresponding blank control was set at the same time. After culturing for 48 hours, add 10 μL MTT reagent (concentration 5 mg/mL) to each well, and after 4 hours of incubation, aspirate the supernatant, add 150 μL DMSO to each well, and react with slight shaking for 5-8 minutes to fully dissolve the crystal particles. The blank control group was adjusted to zero, and the absorbance value after removing the background light absorbance value was measured with a microplate reader at a wavelength of 490nm (

value), calculate the cell proliferation inhibition rate, and continue to use 5 concentration gradients to determine the IC ₅₀ value of the corresponding cell line for the test compound with good antitumor effect in the initial screening. All experiments are repeated 3 times and the average value is taken. The experimental results are shown in Table 3 below.

It can be seen from the data in Table 3 that the inhibitory activity of the complexes of the present invention on human hepatoma cell SMMC-7721 and human glioma cell line U251 is significantly better than that of the commonly used antitumor drug cisplatin, and the ligands and complexes are effective on human beings. The toxicity of HL-7702 in normal liver cells was significantly less than that of cisplatin. The above results show that a new type of 12-chlorobenzimidazole-1,8-naphthalimide-platinum was prepared by introducing 12-chlorobenzimidazole-1,8-naphthalimide nuclei into the platinum metal structure. Anti-tumor complexes are feasible, and novel anti-tumor complexes with high efficiency and low toxicity can be screened. However, the inhibitory activity of the complexes of the present invention against all human cancer cells is not all better than that of their ligands, that is, the introduction of 12-chlorobenzimidazole-1,8-naphthoimide nuclei into platinum metalloids The structure does not necessarily enhance cytotoxicity to tumor cells.

Table 3: Half-inhibitory concentration (C ₅₀ , μM) of compounds on different tumor cell lines.

nd ^a means no test.

Experimental Example 2: Anti-tumor mechanism of the complex of the present invention

In order to illustrate the anti-tumor mechanism of the 12-chlorobenzimidazole-1,8-naphthalimide-platinum complex of the present invention, the applicant studies the anti-tumor mechanism of the complex based on the topoisomerase I target .

1. Interaction study with topoisomerase I

Based on the fact that topoisomerase is a common target of naphthalimide and cisplatin, we used agarose gel electrophoresis and Western blotting to study the interaction of the complex with topoisomerase.

Fig. 2 is the agarose gel electrophoresis image of adding different concentrations of compound I and Topo I. Electrophoresis results showed that compound I had a strong inhibitory effect on TopoI in vitro. It could completely inhibit the catalytic function of 0.1U/L TopoI at a concentration of 20μM, and the compound could completely inhibit the activity of topoisomerase I at 80μM. The half-inhibitory concentration of topoisomerase I in vitro by the classical Topo I agent camptothecin (T) was about 17 μM, and the inhibitory activity of compound I on topoisomerase I was comparable to that.

The experiment shown in Figure 2 shows that Compound I has strong topoisomerase inhibitory activity, so we speculate that its anticancer activity may be through the inhibition of intracellular topoisomerase activity.

Claims (7)

1. A compound of the following formula (I) or a pharmaceutically acceptable salt thereof:

2. a process for the preparation of a compound according to claim 1, characterized in that: the method mainly comprises the following steps: putting a compound shown in a formula (II) and a compound shown in a formula (III) into an organic solvent, and carrying out a coordination reaction under the heating condition or the non-heating condition to obtain a target product;

Pt(DMSO)₂Cl₂(III)。

3. a process for the preparation of a compound according to claim 2, characterized in that: the organic solvent is one or the combination of more than two of methanol, ethanol, chloroform, dichloromethane, dimethyl sulfoxide and N, N-dimethylformamide.

4. A process for the preparation of a compound according to claim 2, characterized in that: the reaction is carried out at a temperature of more than or equal to 40 ℃.

5. A process for the preparation of a compound according to claim 2, characterized in that: the reaction is carried out at 60-80 ℃.

6. The use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-neoplastic drug.

7. A pharmaceutical composition characterized by: comprising a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

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