阅读说明：本技术 一类小分子化合物在制备抗肿瘤转移药物中的应用 (Application of micromolecular compound in preparation of anti-tumor metastasis medicine ) 是由 钱朝南 李长志 陈金东 周红娟 黄婕 于 2020-06-04 设计创作，主要内容包括：本发明属于医药技术领域,具体涉及一类小分子化合物在制备抗肿瘤转移药物中的应用。本发明的通过邻甲基苯甲酸出发,制备得到了小分子化合物,小分子化合物影响肿瘤细胞转移时骨架蛋白的重组,对肿瘤细胞转移具有显著的抑制作用。(The invention belongs to the technical field of medicines, and particularly relates to an application of a small molecular compound in preparation of an anti-tumor metastasis medicine. The small molecular compound is prepared by starting from o-methylbenzoic acid, influences the recombination of the skeleton protein during tumor cell metastasis, and has a remarkable inhibiting effect on tumor cell metastasis.)

1. A micromolecular compound is characterized in that a compound with a structural formula (I),

wherein R1, R2, R3, R4, R5, R6, R7 and R8 are respectively selected from H, F, Cl, Br, I and NH₂、NO₂、D、CH₃、

2. The small molecule compound according to claim 1, wherein said small molecule compound comprises

3. The method for preparing a small molecule compound according to claim 2, comprising the steps of:

s1) adding o-methylbenzoic acid and N, N-dimethylformamide into a round-bottom flask, plugging a bottle stopper, adding anhydrous dichloromethane by using an injector, dropwise adding oxalyl chloride under the protection of argon, and reacting at room temperature; after the reaction, spin-drying the reaction liquid to obtain o-methylbenzoyl chloride;

s2) adding a compound S2 into the o-methylbenzoyl chloride obtained in the step S1, plugging a bottle stopper, adding anhydrous dichloromethane and triethylamine, and reacting at room temperature; monitoring the reaction by using a TLC point plate to obtain a product;

s3) purifying the product of step S2 with silica gel column to obtain small molecule compound.

4. The method for preparing a small molecule compound according to claim 3, wherein the silica gel column purification process uses ethyl acetate/petroleum ether as an eluent.

5. The use of the small molecule compound according to claim 1 or 2 for the preparation of a medicament against tumor metastasis.

6. The use according to claim 5, wherein said tumors comprise breast, lung, stomach, nasopharynx, pancreas and bladder cancer.

7. A preparation of a small molecule compound comprising the small molecule compound of claim 1 or 2 and a pharmaceutically acceptable excipient.

8. The preparation of small molecule compound of claim 6, wherein the preparation is in the form of oral preparation or injection.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a small molecular compound in preparation of an anti-tumor metastasis medicine.

Background

In addition to infiltrating and growing at the primary site and affecting adjacent organs, malignant tumors can also spread to other parts of the body by means of lymphatic vessels, blood vessels, etc., and this process is called tumor metastasis. In recent years, the prevalence and mortality of malignant tumors have been increasing year by year. Moreover, the malignant tumor is easy to metastasize, so that clinical medication, surgical treatment and the like are very difficult, and more than 90% of malignant tumor patients die of tumor metastasis. At the same time, tumor metastasis can occur in an "early" state where the primary tumor is still small. More and more evidence shows that: in the state where the primary tumor is still microscopic, distant dissemination of tumor cells has already occurred. Therefore, prevention of tumor metastasis becomes a key point for the treatment of malignant tumors.

Currently, over 200 over anticancer drugs have been approved by FDA, and cytotoxic drugs remain the basic and major approaches to anti-tumor, but there is still a lack of drugs in clinical use that can achieve the goal of high efficacy and low toxicity, with less inhibition of tumor metastasis. Cytotoxic drugs often injure normal cells while killing tumor cells, and cannot effectively inhibit tumor cell metastasis, so that clinical common tumor primary lesions are relieved, but a plurality of metastasis lesions appear, and finally the disease condition is worsened. Therefore, for modern tumor treatment, it is very important to provide a drug capable of inhibiting tumor cell metastasis.

Disclosure of Invention

The invention aims to provide application of a small molecular compound in preparing a medicine for resisting tumor metastasis.

In order to achieve the purpose, the invention adopts the following technical scheme:

a micromolecular compound, a compound with a structural formula (I),

wherein R1, R2, R3, R4, R5, R6, R7 and R8 are respectively selected from H, F, Cl, Br, I and NH₂、NO₂、D、CH₃、

One kind of (1).

Preferably, the small molecule compound comprises

The invention provides a preparation method of the small molecule compound, which is characterized by comprising the following steps:

preferably, the method specifically comprises the following steps:

s1) adding o-methylbenzoic acid and N, N-dimethylformamide into a round-bottom flask, plugging a bottle stopper, adding anhydrous dichloromethane by using an injector, dropwise adding oxalyl chloride under the protection of argon, and reacting at room temperature; after the reaction, spin-drying the reaction liquid to obtain o-methylbenzoyl chloride;

s2) adding a compound S2 into the o-methylbenzoyl chloride obtained in the step S1, plugging a bottle stopper, adding anhydrous dichloromethane and triethylamine, and reacting at room temperature; monitoring the reaction by using a TLC point plate to obtain a product;

s3) purifying the product of step S2 with silica gel column to obtain small molecule compound.

Preferably, ethyl acetate/petroleum ether is used as eluent in the purification process of the silica gel column.

Preferably, the volume ratio of the ethyl acetate to the petroleum ether is 1: 10.

the invention provides application of the small molecular compound in preparing a medicine for resisting tumor metastasis.

Preferably, the tumor comprises breast cancer, lung cancer, stomach cancer, nasopharyngeal cancer, pancreatic cancer, and bladder cancer.

The tumor comprises nasopharyngeal carcinoma caused by nasopharyngeal carcinoma cells S18 and 5-8F, breast cancer caused by MDA-MB-231, lung cancer caused by non-small cell lung cancer cells A549 and non-small cell lung cancer H1299, gastric cancer caused by gastric cancer cells MKN45, gastric cancer cells MGC803 and gastric cancer cells MGC803, pancreatic cancer caused by pancreatic cancer cells PANC-1, and bladder cancer caused by bladder cancer cells UM-UC-3, bladder cancer cells J82, bladder cancer cells T24, bladder cancer cells SW780 and bladder cancer cells BIU 87.

The invention also provides a small molecule compound preparation, which comprises the small molecule compound as claimed in claim 1 and pharmaceutically acceptable auxiliary materials.

Preferably, the excipients include, but are not limited to, fillers, binders, lubricants, dispersants, glidants, wetting agents, disintegrants, flavors, or colors.

Preferably, the bulking agent includes, but is not limited to, natural sources of high molecular polymers, sugars, cellulose and its derivatives, starch and its derivatives, cyclodextrins and its derivatives, polymers, inorganic salts, PH modifiers.

Preferably, the high molecular polymers of natural origin include, but are not limited to: shellac, peach gum, gum arabic, xanthan gum, locust bean gum, guaiac gum, furcellaran gum, pectin, carrageenan, gelatin, karaya gum, ghatti gum, bletilla gum, sesbania gum, furcellaran gum, tragacanth gum, carrageenan, tara gum, locust bean gum, guar gum, locust bean gum, tara gum, karaya gum, carageenan, alginic acid and its salts, apricot gum, agar, dextran, chitin and its derivatives, zein, casein, and the like.

Preferably, the saccharides include, but are not limited to: glucose, fructose, sucrose, brown sugar, white sugar, crystal sugar, starch sugar, maltose, verbascose, lactose, dextrin, erythritol, xylitol, fructose, mannitol, sorbitol, lactitol, galactitol, maltitol, isomalt, palatinol, isomalt, arabitol, isomaltooligosaccharide, soy oligosaccharides, galacto-oligosaccharides, oligomannose, fructo-oligosaccharides, malto-oligosaccharides, xylo-oligosaccharides, gluco-oligosaccharides, lacto-oligosaccharides, fructo-oligosaccharides, branched fructo-oligosaccharides, milk oligosaccharides, trehalose raffinose, panose, glucose-based sucrose, conjugated sugars, lactulose, lacto-oligosaccharides, lactulose, stachyose, isomerolactose, sucrose, cello-oligosaccharides, lacto-sucrose oligosaccharides, hydrogenated starch hydrolysates, Arabinose, xylose, pluronic, etc.

Preferably, cellulose and its derivatives include, but are not limited to: low-substituted hydroxypropyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, cellulose acetate phthalate, microcrystalline cellulose, ethyl methyl cellulose, croscarmellose sodium, cellulose acetate, and the like.

Preferably, starches and their derivatives include, but are not limited to: modified starch, carboxymethyl starch, hydroxymethyl starch, hydroxy starch propionate, pregelatinized starch, dextrin, etc.

Preferably, cyclodextrins and derivatives thereof include, but are not limited to: cyclodextrins, methyl cyclodextrins, hydroxypropyl cyclodextrins, hydroxyethyl cyclodextrins, polymeric cyclodextrins, ethyl cyclodextrins, branched cyclodextrins, and the like.

Preferably, the polymers include, but are not limited to: polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl butyral, styrene-vinylpyridine copolymers, acrylic resins, crosslinked polyacrylic resins (carbomers), crospovidone, povidone, polyvinyl acetate, methyl acrylate copolymers, polylactic acid, polyamino acids, polycarboxyacetic acid, polyethylene glycol, polyethers, ion exchange resins, and the like.

Preferably, the inorganic materials include, but are not limited to: talc, calcium carbonate, magnesium carbonate, calcium sulfate, calcium hydrogen phosphate, calcium phosphate, magnesium oxide, aluminum hydroxide, silica, activated carbon, activated clay, alumina, sodium chloride, titanium dioxide, and the like.

Preferably, PH adjusting agents include, but are not limited to: sodium bicarbonate, potassium bicarbonate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, citric acid, tartaric acid, succinic acid, fumaric acid, adipic acid, malic acid, etc.

Preferably, the binder includes, but is not limited to: distilled water and ethanol.

Preferably, lubricants include, but are not limited to: magnesium stearate, stearic acid.

Preferably, dispersants include, but are not limited to: water-soluble dispersants and oily dispersants.

Preferably, glidants include, but are not limited to: silica or talc.

Preferably, disintegrants include, but are not limited to: low-substituted hydroxypropyl cellulose, croscarmellose sodium, sodium carboxymethyl starch, and crospovidone.

Preferably, the dosage form is an oral dosage form.

Preferably, the oral dosage is in the form of: tablets, hard or soft capsules, lozenges, dripping pills, pellets, aqueous or oily suspensions, emulsions, dispersible powders or granules, oral liquids, syrups or elixirs.

Preferably, the dosage form is an injection.

Preferably, the injection is in the form of: sterile aqueous or oily solutions, sterile powders, liposomes, emulsions or microcapsules.

The invention prepares a small molecular compound, and simultaneously researches whether the small molecular compound plays a role in controlling tumor metastasis on tumor cells. When tumor cells with metastatic properties undergo cell migration, the recombination of the skeleton protein is necessary to realize the spatial metastasis of the tumor cells. In the experiment of the invention, the influence of the medicine on the cell invasion capacity is determined by an experiment 'Transwell invasion experiment', and then the small molecular compound is found to be capable of obviously inhibiting the migration and invasion of tumor cells represented by nasopharyngeal carcinoma cells S18, breast cancer cells MDA-MB-231, non-small cell lung cancer cells A549, non-small cell lung cancer cells H1299, non-small cell lung cancer cells H1975 and gastric cancer cells MGC 803. Thus, it is assumed that the small molecule compound exerts an anti-tumor metastasis effect by disrupting the recombination of tumor cytoskeletal proteins.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention prepares a small molecular compound from o-methylbenzoic acid, thereby providing a new anti-tumor metastasis medicament.

(2) Experiments show that the small molecular compound has a remarkable inhibition effect on tumor cell metastasis, and provides a brand new direction for anti-tumor metastasis medicaments.

Drawings

FIG. 1 is a nuclear magnetic spectrum of a small molecule compound of the present invention.

FIG. 2 shows the growth of different tumor cells by the small molecule compounds of the present invention.

FIG. 3 shows the inhibitory effect of the small molecule compounds of the present invention on the invasion of nasopharyngeal carcinoma cells S18 in vitro.

FIG. 4 shows the effect of the small molecule compounds of the present invention on the inhibition of in vitro invasion of breast cancer cells MDA-MB-231.

FIG. 5 shows the inhibitory effect of the small molecule compounds of the present invention on the invasion of non-small cell lung cancer cell A549 in vitro.

FIG. 6 shows the inhibitory effect of the small molecule compounds of the present invention on the invasion of non-small cell lung cancer cell H1299 in vitro.

FIG. 7 shows the inhibitory effect of small molecule compounds of the present invention on the invasion of non-small cell lung cancer cells H1975 in vitro.

Fig. 8 shows the inhibitory effect of small molecule compounds of the present invention on the invasion of gastric cancer cells MGC803 in vitro.

In the figure, inhibition rate is inhibition rate, concentration is concentration, invaded cell shaped is invaded cell, Negative is Negative control, Positive is Positive control, and H03 is small molecule compound.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

Preparation of small molecule compounds

The method comprises the following steps:

the method specifically comprises the following steps:

s1) adding o-methylbenzoic acid (950mg) and one drop of N, N-dimethylformamide into a round-bottom flask, plugging a bottle stopper, adding anhydrous dichloromethane (10mL) by using a syringe, dropwise adding oxalyl chloride (710 mu L) under the protection of argon, and reacting for 1h at room temperature; after the reaction, spin-drying the reaction liquid to obtain o-methylbenzoyl chloride;

s2) adding the compound S2 to the o-methylbenzoyl chloride of step S1, wherein the molar ratio of the o-methylbenzoyl chloride to the compound S2 is 1:1, plugging a bottle stopper, adding two drops of anhydrous dichloromethane and triethylamine respectively, and reacting for 3 hours at room temperature; monitoring the reaction by using a TLC point plate to obtain a product;

s3) the product of step S2 was purified on a silica gel column with ethyl acetate/petroleum ether at 1:10 as eluent to give a white solid, i.e. a small molecule compound.

Performing nuclear magnetic identification on the obtained small molecular compound, as shown in figure 1,¹H NMR(400MHz, CDCl₃)8.59(d,J＝4.8Hz,1H)，8.28(d,J＝8.0Hz,1H)，7.90(d,J＝7.8Hz, 1H)，7.78(td,J＝7.8,1.7Hz,1H)，7.47-7.37(m,2H)，7.32-7.26(m,2H)，6.42 (br,1H)，5.55(br,1H)，2.65(s,3H)。

experiment I, MTT measures the influence of drugs on cell growth

1.1 subjects: nasopharyngeal carcinoma cells S18; breast cancer cells MDA-MB-231; non-small cell lung cancer cell a 549; non-small cell lung cancer cell H1299; non-small cell lung cancer cells H1975; gastric cancer cell MGC 803.

1.2 Experimental drugs: a small molecule compound.

1.3 Experimental methods: plate paving: cells in log phase were collected, cell suspension concentration was adjusted, 90ul was added to each well, and plating was performed to achieve a cell density of 10000 per well. Adding medicine: 5% CO₂Incubate at 37 ℃ until the cell monolayer is confluent at the bottom of the well (96-well flat bottom plate) and add the drug in a concentration gradient. 5% CO₂Incubation was carried out at 37 ℃ for 72 hours and observed under an inverted microscope. 20ul of MTT solution (5mg/ml, i.e.0.5% MTT) was added to each well and incubation was continued for 4 h. The culture was terminated and the culture medium in the wells was carefully aspirated. 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at the ELISA OD490nm to calculate the relative viability of the cells. In the experiment, a culture medium without cells is used as a blank control, and 20ul of MTT solution is added into each well to be used as an experiment control group and an experiment group containing samples.

1.4 relative survival rate ═ 100% (experimental OD-blank OD)/(control OD-blank OD) ×

The results show that: as can be seen from FIG. 2, the IC50 of the small molecule compound is 26.12ug/ml for nasopharyngeal carcinoma cell S18, 25.12ug/ml for breast carcinoma cell MDA-MB-231, 37.58ug/ml for non-small cell lung cancer cell A549, 64.2ug/ml for non-small cell lung cancer cell H1299, 76.28ug/ml for non-small cell lung cancer cell H1975, and 20.17ug/ml for gastric carcinoma cell MGC 803.

Second, Transwell invasion experiment is used for determining influence of drug on cell invasion capacity

2.1 subjects: nasopharyngeal carcinoma cells S18; breast cancer cells MDA-MB-231; non-small cell lung cancer a 549; non-small cell lung cancer H1299; non-small cell lung cancer H1975; gastric cancer MGC 803.

2.2 Experimental drugs: a small molecule compound.

2.3 Experimental methods: coating basement membrane Matrigel (4 ℃ procedure) Matrigel (10mg/ml to 250. mu.g/ml) was diluted with serum-free cell culture medium DMEM at 4 ℃. When in use, 50. mu.l of Matrigel was added to 1950. mu.l of DMEM in an EP tube. 100 μ l of the diluted gel was slowly added dropwise to the upper chamber of a 24-well transwell and incubated in an incubator for 1 hour. Preparing a cell suspension; digesting the cells, centrifuging after terminating digestion, discarding the culture solution, washing with PBS for 1 time, resuspending in a serum-free culture medium, and adjusting the cell density to a proper concentration. Inoculating cells; 200. mu.l of the cell suspension was added to the upper chamber of a Transwell and 2. mu.l of 10% BSA was added. Immediately below the 24-well plate, 800. mu.l of medium containing 10% FBS was typically added. And culturing for 24h conventionally. Staining cells; the Transwell chamber was removed, the medium in the well was discarded, the cells in the upper chamber were wiped off with a cotton swab and placed in a clean 24-well plate. Wash 1 time with calcium-free 1 × PBS, fix with methanol for 30 min, and air dry the chamber in a fume hood. 0.1% crystal violet stain for 20min, gently wipe off the upper non-migrated cells with a cotton swab, wash 1 time with PBS. The results were counted, observed under a 5X microscope, and cells were observed under a 10X microscope at random in five visual fields.

The results show that: as can be seen from FIGS. 3 to 8, the small molecule compound may affect the occurrence and development of nasopharyngeal carcinoma cells S18, breast cancer cells MDA-MB-231, non-small cell lung cancer cells A549, non-small cell lung cancer cells H1299, non-small cell lung cancer cells H1975 and gastric cancer cells MGC803 by affecting the invasion ability of the cells.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.