阅读说明：本技术 一种含吡啶的化合物的抗肿瘤用途 (Anti-tumor application of pyridine-containing compound ) 是由 许兴智 程永现 彭斌 晏永明 楼慧强 黄丹灵 郑惜媛 王甫城 于 2021-08-05 设计创作，主要内容包括：本申请涉及一种含吡啶的化合物的抗肿瘤用途。所述含吡啶的化合物为化合物JKW-14,能够对多种肿瘤细胞具有抗增殖的作用。(The application relates to an anti-tumor application of a pyridine-containing compound. The pyridine-containing compound is a compound JKW-14, and can have an antiproliferative effect on various tumor cells.)

1. Compound JKW-14

Or a pharmaceutically acceptable derivative or salt thereof, or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating tumors.

2. The use according to claim 1, wherein the compound JKW-14 or a pharmaceutically acceptable derivative or salt thereof, or a pharmaceutical composition thereof, is administered orally, intranasally, intrarectally, sublingually, buccally or parenterally.

3. The use of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

4. The use according to claim 3, wherein the pharmaceutical composition is a tablet, capsule, dragee, lozenge, parenteral dosage form, liquid or powder.

5. The use according to claim 4, wherein the pharmaceutically acceptable excipient is one or more of talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, optionally further comprising a pigment or flavoring agent.

6. The use according to any one of claims 1 to 5, wherein the tumor is selected from nervous system cancer, brain cancer, thyroid cancer, head and neck cancer, melanoma, leukemia, multiple myeloma, Hodgkin's disease, breast cancer, bladder cancer, prostate cancer, colorectal cancer, colon cancer, kidney cancer, renal cell cancer, pancreatic cancer, esophageal cancer, lung cancer, mesothelioma, ovarian cancer, cervical cancer, endometrial cancer, uterine cancer, germ cell tumor, testicular cancer, stomach cancer, osteosarcoma, or other cancers.

7. Use according to claim 6, wherein the tumors are breast cancer and osteosarcoma, in particular human breast cancer and human osteosarcoma.

Technical Field

The invention relates to the field of medicinal chemistry, in particular to an anti-tumor application of a pyridine-containing compound.

Background

Non-steroidal anti-inflammatory drugs represented by aspirin have been used for centuries, and their action mechanism is mainly to inhibit synthesis of prostaglandins by cyclooxygenase (COX-1, COX-2, etc.) to exert anti-inflammatory and analgesic effects. Among them, COX-2 is expressed in a very low amount under normal conditions, and is expressed and promoted only under conditions such as inflammatory stimulation. Traditional non-steroidal anti-inflammatory drugs are still the most clinically-important drugs for treating inflammation, but most of them have no selectivity for COX-1 and COX-2, and are easy to cause serious gastrointestinal side effects and even are life-threatening. Therefore, finding a highly specific and active COX-2 inhibitor is the main direction in the study of nsaids.

The inventor separates and purifies a compound JKW-14(CN109810049A) with anti-inflammatory activity from insect traditional Chinese medicine lutea beetles with anti-inflammatory and fasciculate block

Disclosure of Invention

The content of the compound JKW-14 in the blaps beetles is low, and the subsequent experiment and production requirements are difficult to meet by extracting the compound with natural raw materials. The inventors completed the in vitro chemical synthesis of this compound using the Suzuki reaction.

Accordingly, in a first aspect of the present application, a method of synthesis of compound JKW-14 is provided. The synthetic route for compound JKW-14 is shown in scheme 1.

Scheme 1. reagents and conditions for the synthesis of compound JKW-14: (a) palladium triphenylphosphine, sodium bicarbonate and 1, 4-dioxane react for 8 hours at 90 ℃, and the yield is 89%; (b) boron tribromide and dichloromethane are reacted for 4 hours at room temperature, and the yield is 57%.

The inventor discovers through further research that the compound JKW-14 also has good anti-tumor activity and can play an anti-proliferation role on various tumor cells. Thus, in a second aspect, the present application provides the use of the compound JKW-14 or a pharmaceutically acceptable derivative thereof and a salt thereof for the prophylaxis or treatment of tumours.

Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.

Drawings

The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not to limit the disclosure.

FIG. 1 shows the effect of compound JKW-14 on MDA-MB-231 cell cycle.

FIG. 2 shows the effect of compound JKW-14 on the mitotic index of MDA-MB-231 cells.

Detailed Description

In a first aspect of the application, a method for synthesizing a compound JKW-14 is provided. The synthesis method comprises the steps of reacting 3, 4-dimethoxy phenyl boric acid and 3, 5-dibromo pyridine in 1, 4-dioxane at 90 ℃ for 8 hours in the presence of triphenylphosphine palladium and sodium bicarbonate to obtain an intermediate 1; and (3) taking dichloromethane as a solvent, and reacting the intermediate 1 with boron tribromide for 4 hours at room temperature to generate a target compound JKW-14. The compound JKW-14 may then be formed into pharmaceutically acceptable derivatives and salts by means commonly used in the art.

In a second aspect, the present application provides the use of the compound JKW-14 or a pharmaceutically acceptable derivative thereof and a salt thereof, or a pharmaceutical composition thereof, for the prophylaxis or treatment of tumours.

In some embodiments, the pharmaceutical composition comprises compound JKW-14 or a pharmaceutically acceptable derivative thereof and salts thereof, optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In some embodiments, the compound JKW-14 or a pharmaceutically acceptable derivative thereof and a salt thereof, or a pharmaceutical composition thereof may be adapted for oral, intranasal, intrarectal, sublingual, buccal or parenteral administration at a dose of 0.1mg to 5 g/time, once or more per day, or once every two or three days. In some embodiments, the pharmaceutical composition may be a tablet, capsule, dragee, lozenge, parenteral dosage form, liquid or powder, but is not limited to such dosage forms. Generally, oral dosage forms are contemplated to be most convenient. All dosage forms can be prepared using standard methods known in the art.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, and the like. Coloring or flavoring agents may also be added to the formulations designed for oral administration. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1-2 propylene glycol, polyethylene glycols, dimethyl sulfoxide, fatty alcohols, triglycerides, partial glycerides, and the like. The gastrointestinal compositions can be prepared using conventional techniques and include sterile isotonic saline, water, 1, 3-butanediol, ethanol, 1, 2-propanediol, polyethylene glycol mixed with water, and the like.

The inventor finds that the compound JKW-14 can inhibit the cell cycle in the G2/M phase. Thus, it is contemplated that compound JKW-14 is capable of inhibiting multiple types of cancers such as, but not limited to, nervous system cancer, brain cancer, thyroid cancer, head and neck cancer, melanoma, leukemia, multiple myeloma, hodgkin's disease, breast cancer, bladder cancer, prostate cancer, colorectal cancer, colon cancer, kidney cancer, renal cell cancer, pancreatic cancer, esophageal cancer, lung cancer, mesothelioma, ovarian cancer, cervical cancer, endometrial cancer, uterine cancer, germ cell tumor, testicular cancer, gastric cancer, osteosarcoma, or other cancers.

In some embodiments, compound JKW-14 is capable of inhibiting breast cancer and osteosarcoma, particularly human breast cancer MCF-7, T47D, MDA-MB-231, and human osteosarcoma U2OS cell lines.

Example 1: synthesis of Compound JKW-14

7.28g of 3, 4-dimethoxyphenylboronic acid and 4.68g of 3, 5-dibromopyridine were dissolved in 150mL of dioxane, stirred uniformly, and the air in the system was removed by bubbling argon. Then 1.15g of tetrakistriphenylphosphine palladium and 5mL of saturated sodium bicarbonate solution were added and bubbling with argon was continued for 5 minutes. Reacting at 90 ℃ for more than 8 hours under the protection of argon atmosphere, returning to room temperature when the thin layer chromatography detects that the raw materials disappear, filtering, washing filter residues twice by dioxane, combining filtrates, distilling under reduced pressure to remove the solvent, extracting the obtained residue by ethyl acetate and saturated saline solution, washing an organic phase twice by saturated saline solution, drying by anhydrous sodium sulfate, distilling under reduced pressure to remove the solvent to obtain 6.24g of light yellow solid, namely the compound 1, wherein the yield is 89%.

Dissolving 351mg of compound 1 in 25mL of anhydrous dichloromethane, bubbling argon to remove air in the system, cooling to-10 ℃ while stirring, slowly dropwise adding a dichloromethane solution of boron tribromide (1.1g of boron tribromide is dissolved in 25mL of dichloromethane), naturally heating to room temperature for reaction for more than 4 hours after dropwise addition, slowly adding a reaction solution into an ice-water mixture which is continuously stirred until the raw materials disappear after tracking of thin layer chromatography, slowly adding a saturated sodium bicarbonate solution until the solution is neutral, extracting dichloromethane (10mL multiplied by 3), combining organic phases, washing with saturated saline (100mL), and drying with anhydrous sodium sulfate. Vacuum concentration to obtain yellow solid, and column chromatography separation (DCM/MeOH,20: 1-10: 1) of the crude product to obtain 168.1mg of yellow solid, namely JKW-14, with the yield of 57%. ESIMS M/z 296[ M + H ]]⁺；HRESIMS m/z 296.0919[M+H]⁺(calcd for C₁₇H₁₃NO₄,296.0917)；¹H NMR(500MHz,MeOD)δ_H 8.58(d,J＝2.1Hz,1H),8.05(d,J＝2.1Hz,1H),7.12(d,J＝2.2Hz,2H),7.05(dd,J＝8.2,2.2Hz,2H),6.91(d,J＝8.2Hz,2H)；¹³C NMR(125MHz,MeOD)δ_C147.3,147.1,145.6,138.7,133.3,130.3,119.8,117.1,115.0.

Example 2: in vitro antitumor Activity test

Cells and culture conditions thereof

DMEM medium, fetal bovine serum and 0.25% Trypsin (Trypsin-EDTA) were purchased from Hyclone. Diabesin (penicillin-streptomycin) was purchased from Gemini. CellTiter used for in vitro antitumor activity experimentMTS cell proliferation kit was purchased from Promega.

All tumor cells were 5% CO at 37 deg.C₂Culturing in a cell culture box. After the cells have spread to about 80-90% of the bottom of the dish, the original medium is aspirated off, and about 5mL of 1 XPBS buffer is slowly added along the sides of the dish to rinse the cells. PBS was aspirated, 1mL of 0.25% trypsin was added, the dish was gently shaken to fill the entire bottom of the dish and make full contact with the cells, and trypsin was aspirated. Placing the culture dish in incubator, and digesting for 2-3 min. The cell digestion was stopped by adding a suitable volume of fresh medium and the cells were gently pipetted to disperse the cells in a single suspension in the medium. Aspirate 1mL of cell suspension and transfer to a new dish and replenish the dish to 10mL of medium. The culture dish is slightly shaken to uniformly disperse the cells, and the cells are placed into an incubator for culture.

Cytotoxic Activity assay

CellTiter was used for this experimentThe AQueous One Solution Cell Proliferation Assay kit (Promega). Test Compound Pre-preparation 10⁴mu.M DMSO solution, stored at-20 ℃, thawed and used, diluted to the desired concentration with culture medium. The experimental data are calculated by three independent parallel experiments.

Cell suspension, 5X 10, was prepared according to the cell passaging method described above³The density of the/well was seeded in 96-well plates leaving two wells as blanks and adding 90. mu.L of cell suspension per remaining well. After culturing for 24h in the incubator, the old culture medium is aspirated, 100 μ L of new culture medium is added to the blank and background wells, the prepared culture medium solution of the compound to be tested is added to the other wells, and the culture is continued for 72h in the incubator. Preserved by freezing in advanceThe MTS solution was thawed at room temperature, 20. mu.L of the MTS solution was added to each well of a 96-well plate, which was placed in an incubator for 2 hours, and the absorbance (OD value) of each well at a wavelength of 492nm was measured using a microplate reader (Molecular Devices SpectraMax M5). The Cell proliferation inhibition rate (Cell proliferation inhibition) was calculated as follows:

Cell proliferation inhibition(％)＝[1-(OD_{cell+test compound}-OD_blank)/(OD_cell+CM-OD_blank)]×100％

wherein OD_{cell+test compound}、OD_cell+CM、OD_blankValues represent absorbance at 492nm wavelength for the test, blank, and background groups, respectively. The half Inhibitory Concentration (IC) of the compound was calculated by plotting an S-shaped curve of the inhibition ratio using SPSS 20for Windows software₅₀)。

The cytotoxic activity of the target compound against tumor cells was measured by the MTS method, and the results are shown in table 1. The anti-tumor drug 5-fluorouracil (5-FU) in the market is used as a positive control, and the semi-Inhibitory Concentration (IC) of the compound JKW-14 on cell lines of human breast cancer MCF-7, T47D, MDA-MB-231, osteosarcoma U2OS and the like is determined₅₀). The results show that JKW-14 has good antiproliferative activity on the tumor cell lines and is obviously superior to the positive control.

TABLE 1 IC of Compound JKW-14 in MCF-7, T47D, MDA-MB-231, and U2OS cells₅₀The value is obtained.

Cell cycle experiments

Cell cycle distribution was determined using propidium iodide staining and fluorescence activated sorting. Firstly, compounds to be detected with different concentrations are arranged according to requirements to prepare a culture medium solution. The cells were then aligned to 6X 10⁵The density of the well is inoculated in a six-hole plate and put into an incubator for cultivationAnd (5) cultivating for 24 hours. The original medium was aspirated off, the prepared medium solution was added, and 1.5-2mL of fresh medium was replenished per well. After adding medicine for 24h, collecting culture medium, adding 200 μ L of 0.25% trypsin per well, and digesting for 2-3 min. Adding the culture medium of the previous mobile phone to terminate cell digestion, gently blowing and beating the cells, collecting all the cells in a centrifuge tube, and centrifuging at 4 ℃ for 2min at 1500 g. The supernatant was discarded, 1mL of cold PBS was added to resuspend the cells, centrifuged at 1500g at 4 ℃ for 2min and aspirated. Add 200. mu.L of cold PBS to resuspend the cells, slowly add 200. mu.L of cold absolute ethanol, blow and mix well, add ethanol twice repeatedly to make the final concentration of ethanol 75%. After being fixed in a chromatography cabinet at 4 ℃ for 12 hours, the sample can be tested on a machine.

The fixed cells were centrifuged (4 ℃, 1500g, 2min) before testing, the supernatant was discarded and left open to evaporate the ethanol as much as possible. The cells were resuspended in 1mL PBS, centrifuged at 1500g for 2min at 4 deg.C, the supernatant aspirated, and the cells were washed twice. RNase A (10mg/mL, L:100) and PI (2mg/mL,1:200) were diluted with PBS, 400. mu.L of RNase A and PI stain dilutions were added to each sample tube, and staining was carried out in a water bath at 37 ℃ in the dark for 30 min.

After staining was complete, cells were dispersed evenly by gentle pipetting. Filtering the cells with a 500 mesh screen to prevent un-blown cell clumps from clogging the instrument tubing, and then using a flow cytometer (BD)C6) And (5) detecting the sample.

The results of the experiment are shown in figure 1. MDA-MB-231 cells are treated for 24h by JKW-14 with different concentrations, the distribution of different cell phases is analyzed by flow cytometry, and a DMSO solvent group without drug treatment is arranged. As a result, JKW-14 was found to significantly arrest the cell cycle in the G2/M phase, even at the minimum concentration (1. mu.M), with a proportion of cells in the G2/M phase as high as 90%.

Mitotic index

Cells were aligned at 2X 10⁵The density of the cells is inoculated in a six-hole plate, and after the cells grow adherent to the wall, the cells are treated by adding drugs for different time lengths. Cells were collected and fixed according to the cell cycle method. The fixed cells were centrifuged (4 ℃, 1500g, 2min), the supernatant discarded, and 1mL of 0.1% PBST was added for washingThe cells were centrifuged twice and the supernatant discarded. Add 500. mu.L blocking buffer (2% BSA, 0.1% PBST) and block for 30min at room temperature. Care was taken to gently shake the tube every 10min to resuspend the pelleted cells for adequate contact with BSA. Centrifugation (4 ℃, 1500g, 2min) and aspiration of the supernatant. mu.L of diluted primary anti-H3 pS10 was added and incubated in a water bath at 37 ℃ for 45 min. Centrifugation (4 ℃, 1500g, 2min) was carried out and the supernatant discarded. Cells were washed twice with 1mL of 0.1% PBST, 150. mu.L of diluted secondary antibody FITC was added, and incubated in 37 ℃ water bath in the dark for 30 min. Centrifugation (4 ℃, 1500g, 2min) was carried out and the supernatant discarded. The cells were washed twice with 1mL PBST, 400. mu.L of RNase A and PI stain in PBS diluted solution was added to each sample tube, and the cells were stained in a water bath at 37 ℃ for 30min in the dark. Flow cytometer (BD)C6) And (3) detecting a sample, and filtering cells by using a 500-mesh sieve before a machine is used for testing, so that the cell mass which is not blown away is prevented from blocking an instrument pipeline.

The effect of compound JKW-14 on the mitotic index of MDA-MB-231 cells was tested using flow cytometry. MDA-MB-231 cells were treated with JKW-14 at a concentration of 10. mu.M for different times, and as can be seen from FIG. 2, cells in M phase increased significantly and mitotic index increased significantly as treatment time was extended from 3h to 16 h. The results suggest that JKW-14 can promote HeLa cells to cross the G2/M checkpoint, blocking the cell cycle in M phase by activating the spindle assembly checkpoint.

The present application describes embodiments, but the description is illustrative rather than limiting and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature or element of any embodiment may be used in combination with or instead of any other feature or element in any other embodiment, unless expressly limited otherwise.

The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements that have been disclosed in this application may also be combined with any conventional features or elements to form unique inventive aspects as defined by the claims. Any feature or element of any embodiment may also be combined with features or elements from other inventive aspects to form yet another unique inventive aspect, as defined by the claims. Thus, it should be understood that any of the features shown and/or discussed in this application may be implemented alone or in any suitable combination. Accordingly, the embodiments are not limited except as by the appended claims and their equivalents. Furthermore, various modifications and changes may be made within the scope of the appended claims.