Oleanolic acid-Leu-Asp-Val, its synthesis, activity and application

Abstract

The present invention discloses oleanolic acid-Leu-Asp-Val having the following structure and a preparation method thereof, discloses its anti-tumor activity and anti-tumor metastasis activity, and further discloses its inhibitory activity on MMP-2. In addition, the present invention discloses its use in preparing a drug having the dual effects of anti-tumor metastasis and inhibiting MMP-2 expression. #imgabs0#

Description

Technical Field

[0001] The present invention relates to oleanolic acid-Leu-Asp-Val and a preparation method thereof, and relates to its anti-tumor activity, anti-tumor metastasis activity and MMP-2 inhibition activity. It further relates to its application in the preparation of a drug having the dual effects of anti-tumor metastasis and inhibition of MMP-2 expression. The present invention belongs to the field of biomedicine. Background Art

[0002] Oleanolic acid is one of the active ingredients of the traditional Chinese medicine Ligustrum lucidum. It is a pentacyclic triterpene compound and is clinically used as an adjuvant treatment for acute and chronic hepatitis. However, low water solubility limits the clinical application of oleanolic acid. Based on the relationship between inflammation and tumor metastasis, especially the role of matrix metalloproteinase-2 (MMP-2) in inflammation and tumor metastasis, the inventors realized that oleanolic acid-Leu-Asp-Val obtained by modifying oleanolic acid with Leu-Asp-Val has potential anti-tumor metastasis activity. In order to theoretically support this understanding, the inventors docked oleanolic acid and oleanolic-Leu-Asp-Val with MMP-2 (PDB number: 1HOV) through the CDOCKING module of the Discovery Studio software, and completed the theoretical design based on virtual screening. Instructions attached Figure 2 The morphology and score of oleanolic acid-Leu-Asp-Val and oleanolic acid docked into the active pocket of MMP-2.

[0003] From the attached Figure 2 It can be seen that compared with oleanolic acid, oleanolic acid-Leu-Asp-Val is more suitable for entering the active pocket of MMP-2. In other words, compared with oleanolic acid, oleanolic acid-Leu-Asp-Val can inhibit MMP-2 more. Furthermore, oleanolic acid-Leu-Asp-Val is a potential anti-tumor metastasis compound, which is worth experimental study. So the inventor carried out experimental research on oleanolic acid-Leu-Asp-Val. The experimental study shows that the anti-tumor metastasis activity and inhibition of MMP-2 of oleanolic acid-Leu-Asp-Val are indeed better than oleanolic acid. Therefore, based on these experimental research results, the inventor proposed this application. Summary of the invention

[0004] The first object of the present invention is to provide a oleanolic acid-Leu-Asp-Val having the following structure:

[0005]

[0006] The second object of the present invention is to provide a method for preparing oleanolic acid-Leu-Asp-Val having the above structure, the method comprising:

[0007] 1) Synthesis of Boc-Leu-Asp(OBzl)-Val-OBzl;

[0008] 2) Synthesis of HCl·Leu-Asp(OBzl)-Val-OBzl;

[0009] 3) using 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensation agent, condensing oleanolic acid with HCl·Leu-Asp(OBzl)-Val-OBzl to prepare oleanolic acid-Leu-Asp(OBzl)-Val-OBzl;

[0010] 4) Using H2 / Pd to deprotect the OBzl in oleanolic acid-Leu-Asp(OBzl)-Val-OBzl to prepare oleanolic acid-Leu-Asp-Val.

[0011] The synthetic route of ursolic acid-Tyr-Gly-Phe-Gly-Gly of the present invention is shown in the attached figure. Figure 1 shown.

[0012] The third object of the present invention is to evaluate the effect of oleanolic acid-Leu-Asp-Val in the preparation of anti-tumor metastasis drugs.

[0013] The fourth objective of the present invention is to evaluate the effect of oleanolic acid-Leu-Asp-Val in preparing drugs for inhibiting tumor cell migration.

[0014] The fifth objective of the present invention is to evaluate the effect of oleanolic acid-Leu-Asp-Val in preparing drugs for inhibiting tumor cell invasion.

[0015] The sixth object of the present invention is to evaluate the effect of oleanolic acid-Leu-Asp-Val in the preparation of MMP-2 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the synthetic route of oleanolic acid-Leu-Asp-Val. In the figure: i) N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine, anhydrous tetrahydrofuran; ii) hydrogen chloride in ethyl acetate solution (4M); iii) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, anhydrous N,N-dimethylformamide, anhydrous N,N-diisopropylethylamine; iv) 10% palladium on carbon, hydrogen.

[0017] Figure 2 The morphology and score diagram of oleanolic acid-Leu-Asp-Val and oleanolic acid docked to the active pocket of MMP-2. Figure 2 On the left is the morphology of oleanolic acid-Leu-Asp-Val docked into the active pocket of MMP-2. In the figure -CDOCKERInteraction Energy 58.055 kcal / mol; Figure 2 The morphology of right oleanolic acid docked into the active pocket of MMP-2, in the figure -CDOCKER Interaction Energy 28.114 kcal / mol. DETAILED DESCRIPTION

[0018] In order to further illustrate the present invention, a series of examples are given below. These examples are completely illustrative, and they are only used to specifically describe the present invention and should not be understood as limiting the present invention.

[0019] Example 1 Preparation of Boc-Asp(OBzl)-Val-OBzl

[0020] Weigh 1.6 g (5.0 mmol) of Boc-L-Asp (OBzl), 0.7 g (5.5 mmol) of 1-hydroxybenzotriazole (HOBt), 50 mL of anhydrous tetrahydrofuran and 1.2 g (6 mmol) of N,N'-dicyclohexylcarbodiimide (DCC) and stir at 0°C for 0.5 hours to obtain reaction solution A. Add 1.3 g (5 mmol) of HCl·L-Val-OBzl to reaction solution A, and adjust the pH value to 9 with N-methylmorpholine (NMM) at 0°C. Then, stir the reaction mixture at room temperature for 4 hours. When TLC shows that HCl·L-Val-OBzl disappears, stirring is terminated. The reaction mixture is concentrated under reduced pressure, and the residue is dissolved in 100 mL of ethyl acetate. The obtained solution was filtered, and the filtrate was washed three times with 30 mL of saturated NaHCO3 solution, three times with 30 mL of saturated NaCl solution, three times with 30 mL of 5% KHSO4 solution, three times with 30 mL of saturated NaCl solution, and dried over anhydrous Na2SO4 for 12 hours. Na2SO4 was filtered off, and the filtrate was concentrated under reduced pressure to obtain 2.2 g (85%) of Boc-Asp(OBzl)-Val-OBzl as a colorless solid. TLC (petroleum ether / ethyl acetate, 4 / 1; UV color development; Rf=0.3), ESI-MS (m / e): 513 [M+H] + .

[0021] Example 2 Preparation of HCl·Asp(OBzl)-Val-OBzl

[0022] Dissolve 2.0 g (3.9 mmol) of Boc-Asp (OBzl) -Val-OBzl in 30 mL of ethyl acetate solution (4 M) of hydrogen chloride and stir at 0°C for 4 hours. TLC shows that Boc-Asp (OBzl) -Val-OBzl disappears and stirring is terminated. The reaction mixture is concentrated under reduced pressure, and the residue is dissolved in 10 mL of anhydrous ethyl acetate and concentrated under reduced pressure. This operation is repeated 3 times. The residue is suspended in 10 mL of petroleum ether, and the suspension is concentrated under reduced pressure. This operation is repeated 3 times. 1.5 g (93%) of HCl · Asp (OBzl) -Val-OBzl is obtained as a light yellow solid, which is directly used in the subsequent reaction. ESI-MS (m / e): 413 [M + H] + .

[0023] Example 3 Preparation of Boc-Leu-Asp(OBzl)-Val-OBzl

[0024] According to the scheme of Example 1, 1.0 g (4.0 mmol) of Boc-L-Leu and 1.7 g (4 mmol) of HCl·Asp(OBzl)-Val-OBzl were used to obtain 1.6 g (66%) of Boc-Leu-Asp(OBzl)-Val-OBzl as a colorless solid. TLC (petroleum ether / ethyl acetate, 1:1; UV color development; Rf=0.3), ESI-MS (m / e): 626 [M+H] + .

[0025] Example 4 Preparation of HCl·Leu-Asp(OBzl)-Val-OBzl

[0026] 0.9 g (100%) of HCl·Leu-Asp(OBzl)-Val-OBzl was obtained from 1.0 g (1.6 mmol) of Boc-Leu-Asp(OBzl)-Val-OBzl by the method of Example 2 as a light yellow solid, which was used directly in the subsequent reaction. ESI-MS (m / e): 526 [M+H] + .

[0027] Example 5 Preparation of oleanolic acid-Leu-Asp(OBzl)-Val-OBzl.

[0028] 2.06 g (46%) of oleanolic acid-Leu-Asp(OBzl)-Val-OBzl was prepared from 1.7 g (3.8 mmol) of oleanolic acid and 2.8 g (5.0 mmol) of HCl·Leu-Asp(OBzl)-Val-OBzl using the method of Example 1. It was a colorless solid. ESI-MS (m / e): 965 [M+H] + . 1H NMR (300MHz, CDCl3) δ=7.40-7.30(m,10H),7.27-7.28(m,2H),7.19-7.16(d,J=8.4,1H),5.42(m,1H),5.23-5.09(m,4H),4.83- 4.77(m,1H),4.53-4.46(m,2H),4.44-4.37(m,1H),3.06-2.99(m,1H),2.70-2.60(m,2H),2.02-1.97(m,1H),1.90-1.87(m,2H) ,1.80-1.76(m,1H),1.71-1.52(m,13H),1.48-1.44(m,21H),1.41-1.35(m,2H),1.24-1.18(m,3H),1.15(s,3H),1.10-1.02(m, 3H),0.98(s,2H),0.96-0.95(m,3H),0.93(s,3H),0.93-0.92(m,7H),0.89(s,2H),0.87(s,6H),0.82-0.79(m,4H),0.68(s,3H).

[0029] Example 6 Preparation of Oleanolic-Leu-Asp-Val

[0030] Dissolve 0.1 g (0.103 mmol) of oleanolic acid-Leu-Asp(OBzl)-Val-OBzl in 10 mL of methanol. Add 10 mg of palladium carbon to the obtained solution. After the air is removed from the obtained suspension, stir at room temperature and pass hydrogen gas for 12 hours. TLC shows that oleanolic acid-Leu-Asp(OBzl)-Val-OBzl disappears, and the hydrogenolysis is terminated. The suspension is removed of palladium carbon, and the filtrate is concentrated under reduced pressure and eluted with 90% methanol on an octadecyl reverse silica gel column to obtain 0.07 g (85%) of oleanolic acid-Leu-Asp-Val as a colorless solid. ESI-MS (m / e): 783 [MH] - ;Mp:186.2-186.5℃; IR / cm -1 =3379,2946,2873,1653,1476,1468,1384,1268,653 1H NMR (300MHz, DMSO-d6) δ / ppm=12.4(s,1H),8.1(s,1H),7.49-7.47(d,J=6Hz,1H),7.22-7.21(d,J=3Hz,1H),5.14 (s,1H)4.57-4.53(m,1H),4.25-4.21(m,2H),4.16-4.10(m,1H),2.98-2.95(m,1H),2.75-2.63(m,2H),2.45-2.44 (m,1H),2.0-1.99(m,1H),1.95-1.89(m,1H),1.78-1.76(m,2H),1.65-1.60(m,3H),1.57-1.51(m,4H),1.45-1.3 4(m,9H),1.31-1.25(m,2H),1.06-1.02(m,5H),0.93-0.90(m,2H),0.87-0.80(m,27H),0.65(s,3H),0.62(s,3H); 13 C NMR(75MHz,DMSO-d6)δ / ppm=176.81,173.16,173.01,172.15,170.80,144.32,121.82,77.27,57.43 ,55.28,51.54,49.54,47.59,46.46,45.72,41.79,41.34,40.97,40.75,40.48,40.20,39.92,39.64 ,39.36,39.20,39.09,38.83,38.54,37.00,36.05,34.07,33.38,33.11,32.49,30.78,30.60,28.70,27.39,25.99,24.65,23.94,23.76,23.40,22.78,21.93,19.43,18.52,18.17,17.37,16.47,15.56.

[0031] Example 7 Evaluation of the anti-tumor cell proliferation effect of oleanolic acid-Leu-Asp-Val

[0032] The oleanolic acid-Leu-Asp-Val and oleanolic acid of the present invention are prepared into the required concentrations using a culture medium containing 0.1% DMSO. Oleanolic acid is purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0033] The tumor cells used in the experiment were A549 (human non-small cell lung cancer cells) and HCCLM3 (human highly metastatic hepatocarcinoma cells).

[0034] A549 cells were cultured in RPMI-1640 medium containing 10% inactivated fetal bovine serum and 1×10 5 U / L penicillin and 100 mg / L streptomycin. HCCLM3 cells were cultured in RPMI-DMEM medium containing 10% inactivated fetal bovine serum and 1×10 5 U / L penicillin and 100mg / L streptomycin.

[0035] The cells in good growth state and logarithmic growth phase were cultured at 3×10 4 The cells were inoculated at a density of 100 μL per well in a 96-well plate and cultured in a cell incubator at 37°C and 5% CO2 for 4 hours. Then, a solution prepared by sterilized oleanolic acid-Leu-Asp-Val, Arg-Gly-Asp-Ser and oleanolic acid and a culture medium containing 0.1% DMSO was added according to a preset concentration gradient, 25 μL per well, and an equal volume of solvent was added to the control group. After continued culture for 48 hours, 25 μL of 5 mg / mL MTT solution was added to each well and cultured in a cell incubator at 37°C and 5% CO2 for 4 hours. After carefully removing the supernatant, 100 μL of DMSO was added to each well, and the purple residue (formazan) was dissolved by shaking for about 10 minutes. The OD value (absorbance) was immediately detected on an ELISA reader at a wavelength of 490 nm. The activity of the compound in inhibiting tumor cell proliferation at each concentration was calculated as follows:

[0036] Inhibition rate = [(mean OD value of negative control group - mean OD value of test compound group) / mean OD value of negative control group] × 100%

[0037] The data in Table 1 show that the percentage of oleanolic acid and oleanolic acid-Leu-Asp-Val inhibiting tumor cell proliferation at 100 μM is less than 55%. It can be seen that the anti-tumor cell migration and invasion effects of oleanolic acid and oleanolic acid-Leu-Asp-Val have nothing to do with anti-tumor cell proliferation. The data in Table 1 further show that the percentage of oleanolic acid-Leu-Asp-Val inhibiting tumor cell proliferation at 100 μM is less than 37%. It can be seen that the anti-tumor cell migration and invasion effects of oleanolic acid-Leu-Asp-Val have nothing to do with anti-tumor cell proliferation.

[0038] Table 1 Inhibition rate of tumor cell proliferation by 100 μM oleanolic acid-Leu-Asp-Val and oleanolic acid

[0039]

[0040]

[0041] Example 8 Evaluation of the anti-tumor cell migration effect of oleanolic acid-Leu-Asp-Val

[0042] The oleanolic acid-Leu-Asp-Val and oleanolic acid of the present invention are prepared into 20 μM in a culture medium containing 0.1% DMSO.

[0043] Cells in good growth state and in logarithmic growth phase were cultured at 5×10 5 The density of cells / mL was inoculated in the upper chamber of the Transwell chamber, 100 μL per well, and 25 μL of culture medium containing oleanolic acid-Leu-Asp-Val and oleanolic acid and 0.1% DMSO was added; 600 μL of serum-containing culture medium (10%) was added to the lower chamber, and cultured in a 37°C, 5% CO2 incubator for 12 hours. The remaining liquid in the upper chamber was aspirated, and the unmigrated cells in the upper chamber were gently wiped with a cotton swab, and repeated 3 times. The remaining liquid in the lower chamber was aspirated, 600 μL of 4% paraformaldehyde was added to each well, and the cells were fixed for 30 minutes, the fixative was aspirated, and the cells were washed 3 times with PBS, and 600 μL of crystal violet staining solution was added to each well, and the staining was performed for 15 minutes. The staining solution was aspirated, and the chamber was washed 3 times with deionized water. Under a microscope, 5 fields of view with roughly the same position and uniform cell distribution were selected in each chamber for photographing and counting (avoiding the uneven distribution of cells at the edge of the chamber). The experimental data were statistically tested using a one-way analysis of variance, and the number of migrating cells was expressed as (mean ± SD). The results are shown in Table 2. Oleanolic acid-Leu-Asp-Val significantly inhibited the migration of A549 cells and HCCLM3 cells, significantly better than oleanolic acid and the positive control Arg-Gly-Asp-Ser. It can be seen that the present invention has an unexpected technical effect of inhibiting tumor cell migration.

[0044] Table 2 Effects of oleanolic acid-Leu-Asp-Val and oleanolic acid on tumor cell migration

[0045]

[0046] a) Compared with the blank control group, P<0.01; b) Compared with the blank control group, P<0.01, compared with the oleanolic acid group, P<0.05, compared with the Arg-Gly-Asp-Ser group, P<0.05; c) Compared with the blank control group, P<0.05, compared with the oleanolic acid group, P>0.05; n=2.

[0047] Example 9 Evaluation of the anti-tumor cell invasion effect of oleanolic acid-Leu-Asp-Val

[0048] Compounds and cells were prepared using the scheme of Example 8. Liquid matrix gel was diluted with serum-free medium at a ratio of 1:9, and 100 μL was added to the upper chamber of the Transwell. After incubation for 5 h, 50 μL of serum-free medium was added to each well to hydrate the basement membrane. Cells that were in good growth state and in the logarithmic growth phase were then cultured at a rate of 5×10 5 The density of 100 / mL was inoculated in the upper chamber of the Transwell chamber, and the rest of the operation was the same as Example 8. The results are shown in Table 3. Oleanolic acid-Leu-Asp-Val significantly inhibited the invasion of A549 cells and HCCLM3 cells, and the activity was better than oleanolic acid and the positive control Arg-Gly-Asp-Ser. It can be seen that the present invention has an unexpected technical effect of significantly inhibiting tumor cell invasion.

[0049] Table 3 Effects of oleanolic acid-Leu-Asp-Val and oleanolic acid on tumor cell invasion

[0050]

[0051] a) Compared with the blank control group, P<0.01; b) Compared with the blank control group, P<0.01, compared with the oleanolic acid group, P<0.05, compared with the Arg-Gly-Asp-Ser group, P<0.05; c) Compared with the blank control group, P<0.05, compared with the Arg-Gly-Asp-Ser group, P>0.05; n=2.

[0052] Example 10 Evaluation of the anti-tumor effect of oleanolic acid-Leu-Asp-Val

[0053] Oleanolic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., sodium carboxymethyl cellulose was purchased from Sinopharm Chemical Reagent Co., Ltd., and doxorubicin was purchased from Beijing Huafeng United Technology Co., Ltd. SPF-grade ICR strain male mice (20±2g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The experiment used a transplanted mouse S180 sarcoma model.

[0054] Because using S180 mouse sarcoma as a tumor model is a common practice in routine experiments, the inventors use the experimental data of S180 mouse sarcoma as a representative to evaluate the tumor inhibition activity of the present invention. The tumor source used for the transplanted mouse S180 sarcoma model is S180 mouse sarcoma cells, which were purchased from the Animal Experiment Center of Peking University Health Science Center and retained by self-passaging according to the suspension cell culture method. Take a SPF-grade male ICR mouse bearing S180 ascites tumor that has been passaged for one week, anesthetize with an appropriate amount of ether and kill by dislocating the neck, soak in 75% alcohol for disinfection for 1 minute, open the abdominal cavity, take the ascites S180 tumor fluid, centrifuge at 1000r / min×10min, discard the supernatant, wash the residue with a small amount of cooled saline to remove non-cellular debris, tissue and floating blood, and use trypan blue staining to calibrate cell activity. The results show that the cell activity reaches 95.0%. The living cells are counted and suspended in cooled saline to a cell density of 2×10 7 / mL, and use it for inoculation as soon as possible. During inoculation, fix the mouse with the left hand, hold a 1mL syringe in the right hand and pierce the right armpit of the mouse about 2mm to the subcutaneous, gently and bluntly separate a small cavity, and inject 0.2mL of live cell suspension. Observe the mice every day after inoculation. When most mice have mung bean-sized solid tumors in their armpits (5th day of inoculation), group them into groups, with 10 mice in each group, and give them oleanolic acid-Leu-Asp-Val (oral dose of 2μmol / kg / day) for 10 consecutive days, doxorubicin (intraperitoneal injection dose of 2μmol / kg / day) for 10 consecutive days, CMCNa for 10 consecutive days, and oleanolic acid (oral dose of 2μmol / kg / day and 20μmol / kg / day) for 10 consecutive days. On the 11th day, weigh the mice in each group, anesthetize with ether, and kill them by cervical dislocation. Fix the site of solid tumor growth in the armpit of the mouse, cut the skin with scissors to fully expose the tumor, and bluntly separate the sarcoma along the skin and upper limbs to weigh it. The in situ tumor weight was expressed as mean ± SD g, and the SD value was first analyzed by SPSS software for variance analysis to test the homogeneity of variance, and then one-way ANOVA was performed for statistical comparison among the groups. Specific data are shown in Table 4 below.

[0055] Table 4 Antitumor activity of oleanolic acid and oleanolic acid-Leu-Asp-Val

[0056]

[0057] a) Compared with the blank control group, P<0.05; b) Compared with the blank control group, P>0.05, compared with the oleanolic acid group, P<0.05, compared with the Arg-Gly-Asp-Ser group, P<0.05; c) Compared with the blank control group, P>0.05; n=10.

[0058] The data in Table 4 show that continuous treatment with oleanolic acid-Leu-Asp-Val (oral dose of 2 μmol / kg / day) for 10 days has no anti-tumor growth effect, thus indicating that the anti-tumor metastasis effect of oleanolic acid-Leu-Asp-Val is unrelated to the inhibition of tumor growth.

[0059] Example 11 Evaluation of the anti-tumor metastasis effect of oleanolic acid-Leu-Asp-Val

[0060] Oleanolic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., sodium carboxymethyl cellulose was purchased from Sinopharm Chemical Reagent Co., Ltd., and doxorubicin was purchased from Beijing Huafeng United Technology Co., Ltd. SPF-grade C57BL / 6 strain male mice (20±2g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The experiment used the Lewis lung cancer spontaneous metastasis model.

[0061] Lewis lung cancer cell metastasis belongs to tumor cells that are difficult to inhibit, so it is generally used as a representative model in conventional experiments. Therefore, the inventors used the data of Lewis lung cancer cell experiments to evaluate the activity of the present invention in inhibiting tumor metastasis. The tumor source used in the Lewis lung cancer spontaneous metastasis model is LLC mouse lung cancer cells, which were purchased from ATCC. Cells with good growth status were selected, and the supernatant was discarded in a sterilized biosafety cabinet. The culture dish wall was washed 3 times with PBS buffer, 1 mL each time; 1 mL of trypsin-EDTA digestion solution was added, and the cells were placed in an incubator (37°C, 5% CO2) for digestion. When the cells were round in shape, the culture medium was aspirated with a sterile dropper to blow the cells; the cell solution was transferred to a 15 mL EP tube and centrifuged at 3000 rpm for 3 minutes. The supernatant was discarded in the clean bench, and the cell concentration was adjusted to 2×10 7 Cells / mL, counted by trypan blue staining. Inbred C57BL / 6 male mice were sterilized with 75% ethanol, and the cell suspension (0.2 mL / mouse) was injected subcutaneously into the right anterior armpit of the mice. Solid tumor tissue formation was observed 7 days after inoculation. On the 15th day, solid tumor tissue with a diameter of about 2-3 cm grew and was used as a tumor source.

[0062] The tumor-bearing mice were anesthetized with ether and then killed by dislocating the cervical vertebra. The tumors were sterilized with 75% ethanol, bluntly dissected, cut into pieces, ground with a glass tissue homogenizer, and passed through a 200-mesh cell sieve to prepare a single-cell suspension. The cells were resuspended with physiological saline and counted with trypan blue staining. The density was 1.9×10 7 Pieces / mL.

[0063] Take inbred C57BL / 6 male mice, fix the mice with the left hand, disinfect the right anterior armpit of the mice with 75% ethanol, and hold a 1mL sterile syringe in the right hand to inject the prepared cell suspension (0.2mL / mouse) into the right anterior armpit. Ten days after inoculation, the tumor diameter was about 1-2mm, the model was established, and the mice were randomly divided into groups. Then, oleanolic acid-Leu-Asp-Val (oral dose of 20μmol / kg / day), CMCNa, oleanolic acid (oral dose of 20μmol / kg / day), Arg-Gly-Asp-Ser (intraperitoneal injection dose of 20μmol / kg / day), and Leu-Asp-Val (intraperitoneal injection dose of 20μmol / kg / day) were given once a day for a total of 10 days. The tumor volume was measured and recorded every two days. On the 25th day, the mice were anesthetized with ether, killed by cervical dislocation, lung tissue was separated, and the number of tumor nodes metastasized to the lungs was counted. The number of tumor nodules was expressed as mean ± SD, and statistical comparison among groups was performed by one-way analysis of variance. The data are shown in Table 5.

[0064] Table 5 Anti-tumor metastasis activity of oleanolic acid and oleanolic acid-Leu-Asp-Val

[0065]

[0066] a) Compared with CMCNa, P<0.05; b) Compared with CMCNa, P<0.01, compared with oleanolic acid (20μmol / kg), P<0.05, compared with Leu-Asp-Val, P<0.05, compared with Arg-Gly-Asp-Ser, P>0.05; n=10.

[0067] The data in Table 5 show that oleanolic acid-Leu-Asp-Val significantly inhibits tumor metastasis at an oral dose of 20 μmol / kg / day. In terms of inhibiting tumor metastasis, the activity of oleanolic acid-Leu-Asp-Val is significantly stronger than that of oleanolic acid and Leu-Asp-Val. It can be seen that the present invention has an unexpected technical effect of inhibiting tumor metastasis.

[0068] Example 12 Evaluation of the Effect of Oleanolic-Leu-Asp-Val on Matrix Metalloproteinase-2

[0069] The mouse matrix metalloproteinase-2 (MMP-2) enzyme-linked immunosorbent assay kit for this experiment was provided by Shanghai ELISA Biotechnology Co., Ltd. After ether anesthesia, blood was collected by removing the eyeballs of C57BL / 6 mice for evaluating mouse lung cancer metastasis. The blood was centrifuged (3000rpm, 15min) and the supernatant was taken. The assay was performed according to the kit operation. The obtained data were statistically analyzed. The standard wells (n=3) were averaged and calculated. The sample groups (n=6) were assayed to calculate the blood matrix metalloproteinase-2 content. The data are shown in Table 6.

[0070] Table 6 Effect of oleanolic acid-Leu-Asp-Val on the content of MMP-2 in mice

[0071]

[0072] a) P<0.05 compared with healthy mice; b) P>0.05 compared with healthy mice, P<0.05 compared with CMCNa, P<0.05 compared with oleanolic acid; c) P>0.05 compared with CMCNa; n=6

[0073] The data in Table 6 show that oleanolic acid-Leu-Asp-Val significantly inhibits the content of matrix metalloproteinase-2 in blood at an oral dose of 20 μmol / kg / day. In terms of inhibiting the expression of matrix metalloproteinase-2, the activity of oleanolic acid-Leu-Asp-Val is significantly stronger than that of oleanolic acid. It can be seen that the present invention has an unexpected technical effect of inhibiting matrix metalloproteinase-2.

Claims

1. A compound having the general formula I, oleanolic acid-Leu-Asp-Val, 2. The method for preparing oleanolic acid-Leu-Asp-Val having the general formula I according to claim 1, characterized in that: The method comprises the following steps: 1) Synthesis of Boc-Leu-Asp(OBzl)-Val-OBzl; 2) Synthesis of HCl·Leu-Asp(OBzl)-Val-OBzl; 3) using 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate as a condensation agent, condensing oleanolic acid with HCl·Leu-Asp(OBzl)-Val-OBzl to prepare oleanolic acid-Leu-Asp(OBzl)-Val-OBzl; 4) Using H2 / Pd to deprotect the OBzl in oleanolic acid-Leu-Asp(OBzl)-Val-OBzl to prepare oleanolic acid-Leu-Asp-Val.

3. Use of oleanolic acid-Leu-Asp-Val having the general formula I according to claim 1 in the preparation of drugs for preventing lung cancer and / or liver cancer metastasis.

4. Use of oleanolic acid-Leu-Asp-Val having the general formula I according to claim 1 in preparing a drug for inhibiting the migration of lung cancer and / or liver cancer cells.

5. Use of oleanolic acid-Leu-Asp-Val having the general formula I according to claim 1 in the preparation of drugs for inhibiting the invasion of lung cancer and / or liver cancer cells.

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