CN115466314A - Anti-migration and anti-invasion 18 beta-glycyrrhetinic acid-RGDV, synthesis, anti-cancer metastasis activity and application thereof - Google Patents
Anti-migration and anti-invasion 18 beta-glycyrrhetinic acid-RGDV, synthesis, anti-cancer metastasis activity and application thereof Download PDFInfo
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Abstract
The invention discloses 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val with the following formula, discloses a preparation method thereof, discloses the activity of inhibiting the migration and invasion of tumor cells, and further discloses that the invention can inhibit the metastasis of cancer to lung. Therefore, the invention discloses the application of the compound in preparing the anti-tumor metastasis medicaments.
Description
Technical Field
The invention relates to 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val, relates to a preparation method thereof, and relates to the inhibition of tumor cell migration and invasion and metastasis, and further relates to the inhibition of cancer metastasis to lung. The invention thus relates to its use in the preparation of a medicament for the treatment of tumor metastasis. The invention belongs to the field of biological medicine.
Background
Glycyrrhetinic acid is the main metabolite of glycyrrhizic acid in human body. Glycyrrhizic acid is the main effective component of licorice. Glycyrrhetinic acid contains multiple chiral carbons. One of the 18-position carbon atoms is. If only the configuration of the 18-position chiral carbon atom is changed, then both α and β epimers occur. 97% of the natural glycyrrhetinic acid is 18 β -glycyrrhetinic acid. Although 18 beta-glycyrrhetinic acid has various pharmacological activities, the application of 18 beta-glycyrrhetinic acid is severely limited due to low activity. In order to overcome the defects of the 18 beta-glycyrrhetinic acid, various chemical modifications of the 18 beta-glycyrrhetinic acid are carried out by the technicians in the industry. But the effect is not significant.
Macrophages are the most abundant immune cells that penetrate tumors. Tumor-associated macrophages (TAMs) can promote tumor cell adhesion, migration and invasion, and thus have a significant impact on cancer metastasis. Part of the mechanism by which tumor-associated macrophages promote tumor cell adhesion, migration and invasion involves the ability of interleukin-1 alpha (IL-1 alpha) expressed by cancer cells to recruit cyclooxygenase-2 (COX 2) -expressing macrophages. In turn, recruited macrophages promote tumor cell adhesion, migration and invasion, further advancing the tumor metastasis process. These findings suggest that compounds capable of entering the activity pockets of interleukin-1 alpha and cyclooxygenase-2 should have the ability to inhibit macrophage recruitment, and thus the ability to inhibit tumor cell adhesion, migration and invasion and to inhibit cancer metastasis. After analyzing the morphology of the active pockets of interleukin-1 alpha and cyclooxygenase-2, the inventors designed 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val of the following formula. By utilizing a molecular docking technology, the inventor docks the compound with interleukin-1 alpha and cyclooxygenase-2. It was found that 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val could well enter the active pockets of interleukin-1 α and cyclooxygenase-2 (the molecular docking scheme is omitted here). These theoretical studies led the inventors to recognize that 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val inhibits tumor cell migration and invasion and thus metastasis of the planted tumor to lung cancer. Based on this knowledge, the inventors have completed the following experimental studies.
Disclosure of Invention
The invention provides a new compound 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val with the following structure,
the second aspect of the present invention is to provide a method for producing 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val having the above structure, the method comprising:
1) Adopting dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to synthesize Arg (NO) in a liquid phase 2 )-Gly-Asp(OBzl)-Val-OBzl；
2) Adopting dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to react Arg (NO) 2 ) condensing-Gly-Asp (OBzl) -Val-OBzl and 18 beta-glycyrrhetinic acid to prepare 18 beta-glycyrrhetinic acid-Arg (NO) 2 )-Gly-Asp(OBzl)-Val-OBzl；
3) 18 beta-glycyrrhetinic acid-Arg (NO) 2 ) And (4) removing a protecting group from-Gly-Asp (OBzl) -Val-OBzl to prepare 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val.
The third content of the invention is to evaluate the activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val with the structure of the formula for inhibiting the migration and invasion of tumor cells.
The fourth content of the invention is to evaluate the activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val with the structure of the formula for inhibiting the metastasis of planted tumors to lung cancer.
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FIG. 1A scheme for the synthesis of beta-glycyrrhetinic acid-Arg-Gly-Asp-Val (i) (v) anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole; (ii) Anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole; (iii) aqueous sodium hydroxide (2N), methanol; (iv) hydrogen chloride in ethyl acetate (4N); (v) Anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole; (vi) hydrogen chloride in ethyl acetate (4N); (vii) Anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole; (viii) methanol, palladium on carbon, hydrogen.
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
EXAMPLE 1 preparation of Boc-Arg (NO) 2 )-Gly-OBzl
1.6g (5.0 mmol) of Boc-L-Arg (NO) 2 ) 0.6g (4.5 mmol) of N-hydroxybenzotriazole and 1.2g (6.0 mmol) of dicyclohexylcarbodiimide were dissolved in 50mL of anhydrous tetrahydrofuran and stirred for half an hour in ice bath. Then, 1.9g (5.5 mmol) of tos.Gly-OBzl was added to the reaction mixture, and the reaction mixture was adjusted to pH9 with N-methylmorpholine. The reaction was carried out at room temperature for 12 hours. TLC (dichloromethane: methanol, 20 2 ) And (4) disappearing. The reaction was stopped, dicyclohexylurea was filtered off and the filtrate was concentrated under reduced pressure. The residue was taken up in 60mL of ethyl acetate and the solution was filtered to remove dicyclohexylurea. The filtrate is sequentially processed with 5 percent NaHCO 3 Aqueous solution (30 mL. Times.3) and saturated aqueous NaCl solution (30 mL. Times.3), 5% KHSO 4 Aqueous solution (30 mL. Times.3), saturated aqueous NaCl solution (30 mL. Times.3), 5% NaHCO 3 Aqueous solution (30 mL. Times.3) and saturated aqueous NaCl solution (30 mL. Times.3). The ethyl acetate layer was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave a yellow powder which was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 10, 1) to give 1.9g (85%) of the title compound as a colorless solid. ESI-MS (m/e) 467[ 2 ], [ M ] +H] + 。
EXAMPLE 2 preparation of Boc-Asp (OBzl) -Val-OBzl
From 3.7g (11.5 mmol) Boc-L-Asp (OBzl) and 3.8g (10.0 mmol) Tos.L-Val-OBzl, 4.3g (84%) of the title compound were obtained as a colorless solid by the method of example 1. ESI-MS (m/e) 513[ 2 ], [ M ] +H] + 。
EXAMPLE 3 preparation of Boc-Arg (NO) 2 )-Gly
4.7g (10.2 mmol) of Boc-Arg (NO) 2 ) -Gly-OBzl in 15mL methanol. The reaction solution was adjusted to pH 12 with aqueous NaOH (2N) in ice bath and reacted for 6 hours. TLC (dichloromethane: methanol, 20 2 ) -Gly-OBzl disappeared. The reaction was terminated, the content of KHSO was 5% 4 The pH of the reaction solution was adjusted to 7 with an aqueous solution, and the reaction solution was concentrated under reduced pressure. 5% KHSO in ice bath 4 The pH value of the reaction solution is adjusted to 2 by the aqueous solution,extraction was performed with ethyl acetate (30 mL. Times.3), and the ethyl acetate layers were combined and washed with saturated aqueous NaCl (30 mL. Times.3). The ethyl acetate layer was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave 3.4g (91%) of the title compound as a colorless oil. ESI-MS (m/e) 375[ M-H ], [ M ] H] - 。
EXAMPLE 4 preparation of HCl Asp (OBzl) -Val-OBzl
4.3g (8.3 mmol) of Boc-Asp (OBzl) -Val-OBzl are dissolved in 40mL of a solution of hydrogen chloride in ethyl acetate (4M) and reacted for 4 hours in ice bath. TLC (dichloromethane: methanol, 20. After the reaction, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 40mL of anhydrous ethyl acetate, and the solution was concentrated under reduced pressure. This operation was repeated 3 times to completely remove free hydrogen chloride. The resulting residue was suspended in 15mL of petroleum ether, and the suspension was sonicated and allowed to stand. The petroleum ether was decanted, the resulting residue was resuspended in 15mL of petroleum ether and the suspension was sonicated. This operation was repeated 3 times to give 4.0g (93%) of the title compound as a pale yellow oil. ESI-MS (m/e): 413[ m ] +H] + 。
EXAMPLE 5 preparation of Boc-Arg (NO) 2 )-Gly-Asp(OBzl)-Val-OBzl
From 4.3g (11.5 mmol) Boc-Arg (NO) by the method of example 1 2 ) -Gly and 4.5g (10.0 mmol) HCl. Asp (OBzl) -Val-OBzl gave 5.2g (67%) of the title compound as a colorless solid. ESI-MS (m/e) 7712 [ M + H ]] + 。
EXAMPLE 6 preparation of HCl.Arg (NO) 2 )-Gly-Asp(OBzl)-Val-OBzl
Starting from 5.2g (6.7 mmol) of Boc-Arg (NO) by the method of example 4 2 ) -Gly-Asp (OBzl) -Val-OBzl gives 5.8g (87%) of the title compound as a yellow oil. ESI-MS (m/e): 671[ M ] +H] + 。
EXAMPLE 7 preparation of 18 beta-Glycyrrhetinic acid-Arg (NO) 2 )-Gly-Asp(OBzl)-Val-OBzl
From 0.06g (0.12 mmol) of glycyrrhetinic acid and 0.06g (0.10 mmol) of HCl.Arg (NO) by the method of example 1 2 ) -Gly-Asp (OBzl) -Val-OBzl gives 0.07g (62%) of the title compound as a colorless solid. ESI-MS (m/e): 1124[ m ] +H] + 。
EXAMPLE 8 preparation of 18 beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val
0.2g (0.2 mmol) of 18 beta-glycyrrhetinic acid-Arg (NO) 2 ) -Gly-Asp (OBzl) -Val-OBzl was dissolved in 6mL of methanol, 20mg of palladium on carbon was added, air was evacuated, hydrogen was introduced with stirring, and the reaction was carried out at room temperature for 12 hours, TLC (ethyl acetate: water: glacial acetic acid, 4: 1) showed 18 β -glycyrrhetinic acid-Arg (NO 2 ) -Gly-Asp (OBzl) -Val-OBzl disappeared. The palladium-carbon is filtered off, and the filtrate is concentrated under reduced pressure. The residue was triturated with ether to give 0.1g (80%) of the title compound as a colourless solid. FT-MS (m/e) 898.5747[ 2 ] M + H] + (theoretical value: 898.5758); 1 H-NMR(300MHz,DMSO-d 6 ):δ/ppm＝10.33(s, 1H),9.02(d,J＝9.0Hz,1H),8.57(s,1H),7.53(d,J＝6.0Hz,1H),7.00(d,J＝9.0Hz,2H),5.50(s, 1H),4.32(s,2H),3.95(m,2H),3.47(m,5H),3.17(s,1H),3.01(d,J＝6.0Hz,2H),2.55(m,5H), 2.28(d,J＝12.0Hz,2H),1.99(m,4H),1.5(dd,J 1 ＝12.0Hz,J 2 ＝16.0Hz,5H),1.34(m,7H), 1.10(m,7H),0.90(s,3H),0.80(d,J＝6.0Hz,4H),0.70(d,J＝9.0Hz,5H).
example 9 evaluation of 18 beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val anti-cell adhesion Activity
1) Preparation of samples
Preparing 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val into a solution with the concentration of 10mM by using DMSO, diluting the solution into a solution with the final concentration of 2.5 mu M by using a DMEM medium, and simultaneously controlling the content of the DMSO to be 0.5%. Arg-Gly-Asp-Val (RGDV tetrapeptide) was prepared at a concentration of 25. Mu.M in DMEM medium containing 0.5% DMSO. 18 β -glycyrrhetinic acid was diluted to a concentration of 25 μ M with a DMEM medium containing 0.5% DMSO. DMEM medium containing 0.5% DMSO was prepared.
2) Experimental procedures
Fibronectin was prepared into 10. Mu.g/mL solution with PBS buffer, 100. Mu.L was added to each well, and after overnight at 4 ℃, the wells were washed 3 times with PBS buffer, then blocked with 1% bovine serum albumin in an incubator at 37 ℃ for 1 hour, then washed 3 times with PBS buffer, and pre-plated 96-well plates were used for cell anti-adhesion experiments.
And (3) taking LLC cells in a good growth state in an exponential growth phase, pouring out a cell culture solution, adding 2ml PBS buffer solution for washing, and pouring out the PBS buffer solution. This operation was repeated 2 times. 1mL of trypsin-EDTA digest was added and digested in an incubator. On a microscopeThe state of cell digestion was observed, and if most of the cells changed from irregular shape to regular round particles and fell off the bottle wall, the digestion was terminated by adding 2mL of mem medium, the dropper repeatedly and forcefully blows along the bottle wall to disperse in the liquid and transfers the liquid to a sterilized 15mL centrifuge tube, and centrifuge at 1368g for 3 minutes. Discarding supernatant, adding DMEM culture medium, blowing to disperse cells uniformly, counting under microscope with cell counting plate, diluting cells with DMEM culture medium to final concentration of 5 × 10 5 Cell suspension per mL. Then the cell suspension was inoculated uniformly into a 96-well plate, and 100. Mu.L of the cell suspension was added to each well of the 96-well plate while 25. Mu.L of a solution of 18. Beta. -glycyrrhetinic acid or a solution of 18. Beta. -glycyrrhetinic acid-Arg-Gly-Asp-Val or a solution of RGDV was added to each well, and the reaction was allowed to proceed at 37 ℃ in 5% CO 2 The cell incubator (2) was incubated for 1 hour, and the unadhered cells were washed away with PBS buffer. The area where 9 cells were evenly distributed was selected under an inverted microscope for image counting.
3) Results of the experiment
The number of microscopic cells is expressed as the mean ± SD, and statistical comparisons between groups were made by one-way anova, and the results are shown in table 1. The data show that neither 18 β -glycyrrhetinic acid nor 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val is able to inhibit LLC cell adhesion. As can be seen, the molecular docking has deviation when predicting the anti-LLC cell adhesion activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val.
TABLE 1 Activity of beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val against LLC cell adhesion
a) P <0.05 to DMEM; b) P >0.05 to DMEM; n =8.
EXAMPLE 10 evaluation of 18 beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val anti-cell migration Activity
1) Preparation of samples
Samples were prepared using the method of example 9.
2) Experimental procedures
Taking LLC cells with good growth state in logarithmic growth phase, and pouring out cells for cultureAfter washing with 2ml PBS buffer, the PBS buffer was removed. This operation was repeated 2 times. 1mL of trypsin-EDTA digest was added and digested in an incubator. The state of cell digestion was observed under a microscope, and if most of the cells changed from irregular shape to regular round particles and fell off the bottle wall, 2ml of MEM was added to terminate the digestion. The dropper repeatedly blows forcefully along the vial wall to disperse the cells in the liquid and transfers the liquid to a sterilized 15mL centrifuge tube, which is centrifuged at 1368g for 3 minutes. Discarding supernatant, adding DMEM without fetal calf serum to blow and beat cells to disperse uniformly, counting under microscope with cell counting plate, diluting cells with DMEM without fetal calf serum to final concentration of 5 × 10 5 Each well of the cell suspension was filled with 100. Mu.L of the cell suspension per well in the upper chamber of the Transwell chamber, and each well was filled with 25. Mu.L of a solution of 18. Beta. -glycyrrhetinic acid, or a solution of 18. Beta. -glycyrrhetinic acid-Arg-Gly-Asp-Val, or a solution of RGDV. Adding 600 μ L of DMEM containing 10% fetal bovine serum into the lower chamber, placing at 37 deg.C and 5% 2 Is cultured in an incubator for 7 hours.
The Transwell cell was subjected to CO reduction from 37 ℃ to 5% 2 The incubator was removed, the upper chamber liquid was aspirated, 100. Mu.L PBS buffer was added to the upper chamber, and the upper chamber cells and residual medium were gently wiped off with a loose cotton swab. This operation was repeated 3 times to leave the upper chamber free of cells. Add 600. Mu.L of 4% paraformaldehyde and fix at 4 ℃ for 30 min. The residual liquid in the lower chamber was aspirated, and 600. Mu.L of a crystal violet staining solution having a concentration of 0.1% was added thereto, followed by staining at room temperature for 15 minutes. And (3) absorbing the staining solution, washing away the residual staining solution on the small chamber, recovering the staining solution, airing the small chamber, and selecting 9 areas with uniformly distributed cells under an inverted microscope for photographing and counting.
3) Results of the experiment
Microscopic cell counts are expressed as mean ± SD, and statistical comparisons between groups were made by one-way anova, with the results shown in table 2. The result shows that the activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val for inhibiting LLC cell migration is obviously stronger than that of 18 beta-glycyrrhetinic acid and DMEM under the concentration of 2.5 mu M. Therefore, the invention has outstanding technical effects.
TABLE 2 Activity of beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val against LLC cell migration
a) P <0.05 to DMEM; b) The ratio P to glycyrrhetinic acid is less than 0.001, and the ratio P to RGDV is more than 0.05; n =9.
Example 11 evaluation of the Activity of 18 beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val against cell invasion
1) Preparation of samples
Samples were prepared using the method of example 9.
2) Experimental procedures
Coating matrigel: the yellow solid Matrigel preserved in a refrigerator at-20 ℃ was placed in the refrigerator at 4 ℃ overnight in advance, and the Matrigel became pink liquid. A matrigel solution was prepared according to the ratio of matrigel to DMEM1:9 without fetal bovine serum, and 100. Mu.L of the matrigel solution was added to the upper chamber of the Transwell cell. Put at 37 ℃ and 5% CO 2 Incubate for 5 hours.
Hydrated basement membrane: removing the residual liquid from the upper chamber of the Transwell chamber by aspiration, adding 50. Mu.L of DMEM without fetal bovine serum, setting the temperature at 37 ℃ and the content at 5% CO 2 Incubate 30 minutes, and then suck the chamber upper chamber residual liquid.
And (3) taking LLC cells in a good growth state in an exponential growth phase, pouring out a cell culture solution, adding 2ml PBS buffer solution for washing, and pouring out the PBS buffer solution. This operation was repeated 2 times. 1mL of trypsin-EDTA digestion solution was added and digested in an incubator. Observing the digestion state of the cells under a microscope, if most of the cells change from irregular shapes to regular round particles and fall off and float from the bottle wall, adding 2mL of MEM to terminate the digestion, repeatedly blowing the dropper along the bottle wall with force to disperse the cells in the liquid and transferring the liquid to a sterilized 15mL centrifuge tube, and centrifuging for 3 minutes at 1368 g. Discarding supernatant, adding DMEM medium without fetal calf serum to blow and beat cells to disperse uniformly, counting under microscope with cell counting plate, diluting cells with DMEM without fetal calf serum to final concentration of 5 × 10 5 100. Mu.L of cell suspension, and 25. Mu.L of 18. Beta. -glycyrrhetinic acid solution or 18. Beta. -glycyrrhetinic acid-Arg-Gly-Asp-Val solution or RGDV solution were added to the upper chamber of a Transwell chamber. Adding 600 μ L DMEM containing 10% fetal calf serum into lower chamber, standing at 37 deg.C and5％CO 2 is cultured for 7 hours in the cell incubator.
The Transwell cell was subjected to a weight reduction of 37 ℃ and 5% CO 2 The incubator was removed, the upper chamber liquid was aspirated, 100. Mu.L PBS buffer was added to the upper chamber, and the upper chamber cells and residual medium were gently wiped off with a loose cotton swab. This operation was repeated 3 times to leave the upper chamber free of cell residue. 600. Mu.L of 4% paraformaldehyde was added and fixed at 4 ℃ for 30 minutes. The residual liquid in the lower chamber was aspirated off, and 60. Mu.L of a crystal violet staining solution having a concentration of 0.1% was added thereto, followed by staining at room temperature for 15 minutes. And (3) absorbing the staining solution, washing away the residual staining solution on the small chamber, recovering the staining solution, airing the small chamber, and selecting 9 areas with uniformly distributed cells under an inverted microscope for photographing and counting.
3) Results of the experiment
Microscopic cell counts are expressed as mean ± SD, and statistical comparisons between groups were made by one-way anova, with the results shown in table 3. The results show that the activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val for inhibiting LLC cell invasion at the concentration of 2.5 mu M is obviously stronger than that of 18 beta-glycyrrhetinic acid and DMEM. Therefore, the invention has outstanding technical effects.
TABLE 3 anti-LLC cell invasion Activity of 18 β -Glycyrrhetinic acid-Arg-Gly-Asp-Val
a) P <0.05 to DMEM; b) The ratio P to glycyrrhetinic acid is less than 0.01, and the ratio P to RGDV is more than 0.05; n =9.
Example 12 evaluation of 18. Beta. -Glycyrrhetinic acid-Arg-Gly-Asp-Val inhibitory Activity on cancer metastasis
1) Laboratory animal
The mice were used as C57BL/6 mice, male, 20. + -.2 g, purchased from laboratory animals technology, inc. of Tokyo Wintolite, beijing.
Dosage and administration mode: the dose of 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val was 0.5 μmol/kg/day, the dose of 18 β -glycyrrhetinic acid was 5 μmol/kg/day, and the negative control was sodium carboxymethylcellulose solution (0.5% CMCNa); the positive control was RGDV tetrapeptide at a dose of 20. Mu. Mol/kg/day. They are all administered by intraperitoneal injection.
2) Experimental procedures
Lewis mouse lung carcinoma cells (LLC), purchased from ATCC, were self-cultured by monolayer cell culture. DMEM (containing penicillin and streptomycin) medium containing 10% inactivated fetal bovine serum is selected. According to the adherent cell culture method, the cells are enriched to the required number after passage once a day. PBS buffer solution, trypsin-EDTA digestive juice, corresponding culture medium required by cells and preheating for 30 minutes in a water bath at 37 ℃. When the cells are in good growth state, high transparency, few inner particles, no vacuole, clear cell membrane, clear and transparent culture solution supernatant, no suspended cells and no debris and are in logarithmic growth phase, when the cells grow to 80% of the area of the bottom of the bottle, removing the original cell culture solution, adding 1mL of PBS buffer solution to wash the residual culture medium for 1-3 times, discarding the buffer solution, adding 1mL of trypsin-EDTA digestive juice, placing the digested solution in an incubator for a certain time, observing the cell morphology under a microscope, adding 1mL of the culture medium to stop the digestion when most of the cells are changed from irregular shapes to regular circular particles and a small part of the particles fall off and float from the bottle wall, repeatedly blowing the cells along the bottle wall by a dropper to remove the walls and disperse in the liquid, transferring the liquid to a sterilized 15mL centrifuge tube, centrifuging for 3 minutes at 1368g, discarding the supernatant, and adjusting the cell concentration to 2 × 10 by using physiological saline at 4 DEG C 7 Counts per mL, trypan blue staining indicate viable cell number>95 percent. Inbred line C57BL/6 male mice were taken, left-handed mice were fixed, the right anterior limb axillary skin of the mice was disinfected with 75% ethanol, and Lewis mouse lung cancer cell suspension (0.2 mL/mouse) was injected subcutaneously into the axillary region of the mice with a 1mL sterile syringe held in the right hand. Solid tumor tissue formation was seen 10 days after inoculation. Solid tumor tissues with the diameter of about 2-3mm grow out on days 17-20 and are used as tumor sources for standby.
Anesthetizing Lewis lung cancer tumor-bearing mice in a tumor source by using ether, removing cervical vertebrae, killing the tumor-bearing mice, soaking and sterilizing the tumor-bearing mice by using 75% ethanol for 10 minutes, stripping tumor bodies on a super clean workbench, shearing the tumor bodies in a sterile flat dish, placing the tumor bodies in a glass tissue homogenizer, slightly grinding the tumor bodies by using physiological saline which is precooled to 4 ℃ according to the proportion that the tumor mass (g) and the volume (mL) of the physiological saline is 1 to prepare cell suspension, and sieving the cell suspension by using 200-mesh cells to prepare single-cell suspensionAdjusting the cell concentration to 2X 10 with physiological saline 7 Counts per mL, trypan blue staining indicated viable cell number>95 percent. Inbred line C57BL/6 male mice were taken, left-handed mice were fixed, the right anterior limb axillary skin of the mice was disinfected with 75% ethanol, and Lewis mouse lung cancer cell suspension (0.2 mL/mouse) was injected subcutaneously into the axillary region of the mice with a 1mL sterile syringe held in the right hand. From the day after tumor inoculation, tumor-bearing mice are observed every day, and are regrouped when the mice axillary solid tumors grow to soybean size, the administration is started after the tumor state of each group of mice is basically consistent, the administration is performed for 1 time (0.1 ml/10 g) every day for 10 days continuously, and the mouse axillary tumor size is measured by a vernier caliper every 1 day and recorded. On day 10, mice were anesthetized with ether and sacrificed by cervical dislocation. Lungs were taken and the number of nodules that had metastasized above was quickly counted. Metastatic nodules were expressed as mean ± SDg, and were statistically compared between groups by one-way anova.
3) Results of the experiment
The activity of 18 beta-glycyrrhetinic acid for inhibiting tumor lung metastasis on a mouse Lewis lung cancer metastasis model is obviously stronger than that of CMCNa, and the activity of 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val for inhibiting tumor lung metastasis is obviously stronger than that of 18 beta-glycyrrhetinic acid. Therefore, the invention has outstanding technical effects.
TABLE 4 Activity of beta-Glycyrrhetinic acid-Arg-Gly-Asp-Val for inhibiting tumor metastasis to the lung
a) P <0.05 compared to CMCNa; p is greater than 0.05 compared with RGDS; b) And 0.5%; n =11.
Claims (4)
1. 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val with the structure as shown in the following formula,
2. a method for preparing 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val form according to claim 1, said method comprising the steps of:
2.1. liquid-phase synthesis of Arg (NO) by using dicyclohexylcarbodiimide as condensing agent and 1-hydroxybenzotriazole as catalyst 2 )-Gly-Asp(OBzl)-Val-OBzl；
2.2. Adopting dicyclohexylcarbodiimide as condensing agent and 1-hydroxybenzotriazole as catalyst to react Arg (NO) 2 ) condensing-Gly-Asp (OBzl) -Val-OBzl and 18 beta-glycyrrhetinic acid to prepare 18 beta-glycyrrhetinic acid-Arg (NO) 2 )-Gly-Asp(OBzl)-Val-OBzl；
2.3. 18 beta-glycyrrhetinic acid-Arg (NO) 2 ) And (3) removing a protecting group from-Gly-Asp (OBzl) -Val-OBzl to prepare 18 beta-glycyrrhetinic acid-Arg-Gly-Asp-Val.
3. Use of 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val of the structure according to claim 1 for the manufacture of a medicament for the treatment of tumor metastasis.
4. Use of 18 β -glycyrrhetinic acid-Arg-Gly-Asp-Val of the structure according to claim 1 for the manufacture of a medicament for inhibiting cell migration and cell invasion.
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| CN202110649859.5A CN115466314A (en) | 2021-06-10 | 2021-06-10 | Anti-migration and anti-invasion 18 beta-glycyrrhetinic acid-RGDV, synthesis, anti-cancer metastasis activity and application thereof |
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