1-Imidazole-β-carboline-3-carboxyl-LDV and its preparation, anticancer metastasis activity and application

Abstract

The present invention discloses 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val of the following formula, discloses its preparation method, discloses its activity of inhibiting tumor cell invasion, discloses its activity of inhibiting Lewis mouse tumor lung metastasis. Therefore, the present invention discloses its use in the preparation of anti-tumor metastasis drugs.

Description

Technical Field

[0001] The present invention relates to 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val, a preparation method thereof, its activity in inhibiting tumor cell invasion, and its activity in inhibiting Lewis mouse tumor lung metastasis. Therefore, the present invention relates to its application in the preparation of anti-tumor metastasis drugs. The present invention belongs to the field of biomedicine. Background Art

[0002] At present, cancer is the second leading cause of death in the world. According to statistics, there are 9.96 million cancer deaths in the world in 2020. Most of the cancer deaths are caused by tumor metastasis. In addition, there is still a lack of safe and effective anti-tumor metastasis drugs in the clinic. It can be seen that the development of new anti-tumor metastasis drugs has important practical significance.

[0003] β-Carbolines are a class of indole alkaloids with a pyrido[3,4-b]indole structure, which are widely found in animals and plants in nature. β-Carbolines can affect multiple tumor-related targets. For example, they can act on DNA, regulate the cell cycle, induce apoptosis, inhibit DNA topoisomerase and histone deacetylase, etc. Imidazole has the potential to produce antitumor effects by interacting with DNA covalently and non-covalently, and is an important building block for compounds with anticancer activity.

[0004] Macrophages are the most abundant immune cells that penetrate tumors. Tumor-associated macrophages (TAMs) can promote tumor cell migration and invasion, and thus have an important impact on cancer metastasis. Part of the mechanism by which tumor-associated macrophages promote tumor cell migration and invasion involves the ability of interleukin-1α (IL-1α) expressed by cancer cells to recruit cyclooxygenase-2 (COX2)-expressing macrophages. In turn, the recruited macrophages promote tumor cell migration and invasion, further advancing the process of tumor metastasis.

[0005] The above knowledge shows that compounds that can enter the active pockets of interleukin-1α and cyclooxygenase-2 will be able to inhibit macrophage recruitment, and thus have the ability to inhibit tumor cell migration and invasion, as well as the ability to inhibit cancer metastasis. After analyzing the morphology of the active pockets of interleukin-1α and cyclooxygenase-2, the inventors designed 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val. Using molecular docking technology, the inventors docked the compound with interleukin-1α and cyclooxygenase-2. It was found that 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val can well enter the active pockets of interleukin-1α and cyclooxygenase-2 (molecular docking diagram omitted here). These theoretical studies have enabled the inventors to realize that 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val can inhibit tumor cell migration and invasion, and thus inhibit cancer metastasis. Based on this understanding, the inventors completed the subsequent experimental studies. Summary of the invention

[0006] The first content of the present invention is to confirm that 1-(1H-imidazol-2-yl)-β-carboline-3-carbonyl-Leu-Asp-Val of the following formula is a completely new compound.

[0007]

[0008] The second content of the present invention is to prepare 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp-Val by the following steps:

[0009] 1) L-Trp-OBzl and imidazole-2-carboxaldehyde undergo Pictet-Spengler condensation under the catalysis of trifluoroacetic acid to obtain 3S-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester;

[0010] 2) 3S-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester is oxidized with 2,3-dichloro-5,6-dicyano-p-benzoquinone in tetrahydrofuran to obtain 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester;

[0011] 3) Debenzylating 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester with Pd / C catalytic hydrogenation in tetrahydrofuran to obtain 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid;

[0012] 4) preparing Leu-Asp(OBzl)-Val-OBzl by conventional peptide synthesis methods;

[0013] 5) 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid is coupled with Leu-Asp(OBzl)-Val-OBzl to prepare 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl;

[0014] 6) Removing the protecting group of 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl to prepare 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp-Val.

[0015] The third content of the present invention is to use the MTT method to confirm that 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp-Val is not a cytotoxic compound.

[0016] The fourth aspect of the present invention is to evaluate the inhibitory effect of 1-(1H-imidazol-2-yl)-β-carboline-3-carbonyl-Leu-Asp-Val on tumor cell metastasis and invasion using the Transwell chamber model.

[0017] The fifth aspect of the present invention is to evaluate the inhibitory effect of 1-(1H-imidazol-2-yl)-β-carboline-3-carbonyl-Leu-Asp-Val on the lung metastasis of Lewis lung cancer-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The synthetic route of 1-(1H-imidazole-2-yl)-β-carboline-3-formyl-Leu-Asp-Val: i) trifluoroacetic acid, dichloromethane; ii) dichlorodicyanobenzoquinone, tetrahydrofuran; iii) hydrogen, palladium on carbon, tetrahydrofuran; iv) dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; v) ethyl acetate solution of hydrogen chloride (4M); vi) 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N-methylmorpholine, N,N-dimethylformamide; vii) trifluoroacetic acid, trifluoromethanesulfonic acid, ether. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 Preparation of (S)-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (1)

[0021] 3g (10.0mmol) of L-tryptophan benzyl ester was dissolved in 100mL of dichloromethane at 0℃ and stirred, and then 2mL of trifluoroacetic acid was added and reacted for 30 minutes. Then 1.18g (12.0mmol) of imidazole-2-carboxaldehyde was added and reacted at room temperature for 12 hours. After that, TLC (developing solution) showed that L-tryptophan benzyl ester disappeared. The remaining trifluoroacetic acid was removed by vacuum concentration, and the residue was dissolved in 100mL of dichloromethane, washed with saturated NaHCO3 solution (20mL×3) and saturated NaCl solution (20mL×3) in turn. The dichloromethane layer was dried with anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 3g of colorless solid. The solid was purified by silica gel column chromatography (DCM:MeOH, 20:1) to obtain 3.5g (94%) of the title compound as a colorless powder. 1 H NMR (800MHz, DMSO-d6) δ10.43 (s, 1H), 7.44 (d, J = 7.6Hz, 2H), 7.45–7.39 (m, 3H), 7.39–7.21(m,3H),7.01(t,J=7.5Hz,1H),6.99(s,2H),6.95(t,J=7.4Hz,1H),5.41(s,1H ),5.27–5.19(m,2H),3.96(dd,J=11.2,4.1Hz,1H),3.09–3.04(m,1H),2.82(ddd,J=13.9, 11.1,2.5Hz,1H).

[0022] Example 2 Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester (2)

[0023] 4.5 g (12.0 mmol) of (S)-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was dissolved in 150 mL of tetrahydrofuran at 0°C with stirring, and 5.7 g (25.0 mmol) of dichlorodicyanobenzoquinone was added. After reacting at room temperature for 48 hours, TLC (dichloromethane:methanol, 20:1) showed that (S)-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (1) disappeared. The mixture was concentrated under reduced pressure, and the residue was dissolved in 150 mL of ethyl acetate, and washed with saturated aqueous NaHCO3 solution (20 mL×3) and saturated aqueous NaCl solution (20 mL×3) in sequence. The ethyl acetate layers were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 4.2 g of colorless solid. The solid was purified by silica gel column chromatography (petroleum ether:ethyl acetate, 1.5:1) to obtain 2.4 g (53%) of the title compound as a colorless powder. ESI / MS (m / e): 391 [M+Na] + ; 1H NMR (300 MHz, DMSO-d6) δ12.89(s,1H),11.97(s,1H),8.94(s,1H),8.42(d,J=7.9Hz,1H),7.98(d, J=8.3Hz,1H),7.75–7.65(m,1H),7.60(dd,J=13.4,7.2Hz,3H),7.38(dp,J=19.9,7.1Hz,6H),5.50(s,2H).

[0024] Example 3 Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid (3)

[0025] 1.5 g (4.0 mmol) of 1-(1H-imidazole-2-yl)-β-carboline-3-carboxylic acid benzyl ester (2) was dissolved in 50 mL of tetrahydrofuran under stirring, 200 mg of palladium carbon was added and hydrogen was introduced to react. After 24 h of reaction at room temperature, TLC (dichloromethane: methanol, 20:1) showed that 1-(1H-imidazole-2-yl)-β-carboline-3-carboxylic acid benzyl ester (2) disappeared. The palladium carbon was filtered off, and the solvent was removed by concentration under reduced pressure to obtain 1.0 g (70%) of the title compound as a yellow powder. ESI / MS (m / e): 279 [M+H] + ; 1 H NMR(300MHz,DMSO-d6)δ13.28(s,1H),12.46(s,1H),11.98(s,1H),8.94(s,1H),8.43(d, J=7.9Hz, 1H), 7.98 (d, J=8.3Hz, 1H), 7.60 (d, J=8.1Hz, 2H), 7.34 (dd, J=12.8, 5.0Hz, 2H).

[0026] Example 4 Preparation of Boc-Asp(OBzl)-Val-OBzl

[0027] 3.23 g (10.0 mmol) of Boc-Asp (OBzl) was dissolved in 100 mL of anhydrous tetrahydrofuran at 0°C with stirring, and then 1.49 g (11.0 mmol) of 1-hydroxybenzotriazole and 2.47 g (11.0 mmol) of dicyclohexylcarbodiimide were added and reacted for 30 minutes. After that, 2.67 g (11.0 mmol) of Val-OBzl was added. Finally, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, and the obtained reaction solution was stirred at room temperature for 10 hours. Filtered, the filtrate was concentrated under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate, and the obtained solution was washed with saturated NaHCO3 aqueous solution (30 mL × 3), saturated NaCl aqueous solution (20 mL × 3), 5% KHSO4 aqueous solution (20 mL × 3), saturated NaCl aqueous solution (20 mL × 3), saturated NaHCO3 aqueous solution (20 mL × 3) and saturated NaCl aqueous solution (20 mL × 3). The ethyl acetate layer was dried over anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 30 mL of dichloromethane and allowed to stand for 6 hours to allow the solid to fully precipitate. After filtration, 5.50 g (89.7%) of the title compound was obtained as a colorless solid powder. 1 HNMR(300MHz,DMSO-d6)δ7.97(d,J=8.3Hz,1H),7.43- 7.27(m,9H),7.22(d,J=8.2Hz,1H),5.22-5.00(m,4H),4.44(td,J=8.7,5.1Hz,1H),4.24 (dd,J=8.3,5.8Hz,1H),4.03(q,J=7.1Hz,1H),2.82-2.51(m,2H),2.08(h,J=6.7Hz,1H), 1.99(s,1H),1.38(s,8H),1.18(t,J=7.1Hz,1H),0.85(d,J=6.8Hz,6H).

[0028] Example 5 Preparation of Asp(OBzl)-Val-OBzl

[0029] 1.05 g (2.0 mmol) of Boc-Asp(OBzl)-Val-OBzl was dissolved in 20 mL of ethyl acetate solution (4 M) of hydrogen chloride at 0°C with stirring and stirred for 1 hour. TLC (dichloromethane: methanol = 20:1) showed that Boc-Asp(OBzl)-Val-OBzl disappeared. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 15 mL of anhydrous ethyl acetate. The solution was concentrated under reduced pressure again, and the residue was added with 10 mL of anhydrous ether and ultrasonically vibrated to completely suspend, and the supernatant was discarded to obtain 0.88 g (98%) of the title compound as a yellow oil. ESI-MS (m / e): 413 [M+H] + .

[0030] Example 6 Preparation of Boc-Leu-Asp(OBzl)-Val-OBzl

[0031] Using the method of Example 4, 1.05 g (60%) of the title compound was obtained as a colorless solid from 0.75 g (2.00 mmol) of Boc-Leu and 0.99 g (2.20 mmol) of HCl.Asp(OBzl)-Val-OBzl. 1 H NMR(300MHz, DMSO-d6)δ8.32(d,J=8.3Hz,1H),8.16(d,J=7.8Hz,1H),7.36(dd,J=8.7,4.9Hz,10H), 6.83(d,J=8.5Hz,1H),5.22-4.97(m,4H),4.66(q,J=6.7Hz,1H),4.25(dd,J=8.3,6.1Hz, 1H),3.99(td,J=8.9,5.8Hz,1H),2.80-2.60(m,2H),2.02(h,J=6.7Hz,1H),1.60(s,1H), 1.36(s,11H),0.82(dt,J=6.6,2.2Hz,12H).

[0032] Example 7 Preparation of Leu-Asp(OBzl)-Val-OBzl

[0033] 0.25 g (87.4%) of the title compound was obtained as a colorless solid from 0.32 g (0.50 mmol) of Boc-Leu-Asp(OBzl)-Val-OBzl using the method of Example 5. ESI-MS (m / e): 346 [M+H] + .

[0034] Example 8 Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl (4)

[0035] 200 mg (0.7 mmol) of 1-(1H-imidazole-2-yl)-β-carboline-3-carboxylic acid was dissolved in 20 mL of N,N-dimethylformamide at 0°C with stirring, and 450 mg (0.8 mmol) of Leu-Asp(OBzl)-Val-OBzl and 280 mg (0.8 mmol) of 2-(7-benzotriazole oxide)-, N,N',N'-tetramethyluronium hexafluorophosphate were added in sequence. The pH of the reaction solution was adjusted to 9 with N-methylmorpholine. After reacting at room temperature for 12 hours, the solution was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the obtained solution was washed with saturated NaHCO3 aqueous solution (20 mL×3) and saturated NaCl aqueous solution (20 mL×3) in sequence. The combined ethyl acetate layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain 413 mg of colorless solid. The solid was purified by silica gel column chromatography to obtain 270 mg (49%) of the title compound as a colorless powder. 1 H NMR(300MHz,DMSO-d6)δ9.12(d,J=8.5Hz, 1H),8.80(s,1H),8.57(d,J=7.7Hz,1H),8.34(d,J=7.9Hz,1H),7.97(t,J=7.8Hz,2H), 7.59(ddd,J=8.3,7.0,1.2Hz,1H),7.50(s,2H),7.43–7.17(m,11H),5.22–4.95(m,4H), 4.74(qd,J=9.9,9.1,4.4Hz,2H),4.22(dd,J=8.2,6.0Hz,1H),2.84(dd,J=16.3,5.4H z,1H),2.77–2.61(m,1H),2.17–1.95(m,1H),1.95–1.66(m,3H),1.01–0.77(m,12H).

[0036] Example 9 Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-carbonyl-Leu-Asp-Val (5)

[0037] 100 mg (0.11 mmol) of 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl (4) was dissolved in 1 mL of trifluoroacetic acid and 0.3 mL of trifluoromethanesulfonic acid at 0°C with stirring, and stirred for 0.5 hours. The reaction mixture was concentrated under reduced pressure. 10 mL of anhydrous ether was added to the residue and stirred for 10 minutes. Solid precipitated. After standing for 10 minutes, the supernatant was discarded. This operation was repeated 3 times. The collected solid was dissolved in 50% water and 50% methanol, and the pH was adjusted to 8 with 10% ammonia water. It was purified by C18 column with 40% water and 60% methanol as eluent. The eluate was lyophilized to obtain 29 mg (36.6%) of the title compound as a yellow solid. FT-ESI-MS (m / e): 606.27560 [M+H] + ;M P 201.3-202.6℃, 1 HNMR(300MHz,DMSO-d6)δ13.60(s,1H),11.82(s,1H), 9.17(d,J=8.7Hz,1H),8.80(s,1H),8.63(s,1H),8.38(d,J=7.9Hz,1H),7.96(d,J=8.3Hz ,1H),7.59(t,J=7.7Hz,3H),7.49(s,1H),7.32(dd,J=16.2,8.6Hz,2H),4.75(td,J=10.1, 9.6, 3.9Hz, 1H), 4.64 (d, J = 7.2Hz, 1H), 4.09 (dd, J = 8.5, 5.1Hz, 1H), 2.74 (dd, J = 16.3, 6. 2Hz,1H),2.58(d,J=7.3Hz,1H),2.05(dq,J=11.0,5.4,4.3Hz,1H),1.87(t,J=9.6Hz,1H), 1.77-1.61(m,2H),1.22-0.82(m,12H).

[0038] Example 10 Evaluation of the cytotoxic effect of compound 5

[0039] Compound 5 was prepared into the required concentration using 1640 medium containing 0.5% DMSO. HCT-116, A549, S180, LLC, HL60, U2OS, and L02 cells in good growth state and logarithmic growth phase were cultured at 4×10 4The cells were inoculated with a density of 100 μL / mL in a 96-well plate. The cells were cultured in a 37°C, 5% CO2 incubator for 4 hours. The sterilized compound 5 solution was added according to the preset concentration gradient of 100 μM, 50 μM, 25 μM, 10 μM and 1 μM. The control group was added with an equal volume of 1640 medium containing 0.5% dimethyl sulfoxide to dissolve the sample. After 48 hours of continuous culture, 25 μL of 5 mg / mL MTT solution was added to each well and incubated at 37°C for 4 hours. After carefully removing the supernatant, 100 μL of dimethyl sulfoxide was added to each well and the precipitate was dissolved by shaking for about 15 minutes. The OD (absorbance) value was immediately measured at a wavelength of 570 nm on an ELISA reader. The inhibition rate was calculated as follows: inhibition rate = [(OD average value of the 1640 medium group containing 0.5% dimethyl sulfoxide – OD average value of the compound) / OD value of the 1640 medium group containing 0.5% dimethyl sulfoxide] × 100%. The experiment was repeated 6 times in parallel, and the inhibition rate was plotted against the compound concentration to calculate the IC of the compound of the present invention. 50 (half effective inhibitory concentration) value.

[0040] The results are listed in Table 1. From the results, it can be seen that compound 5 of the present invention has no inhibitory effect on the proliferation of six tumor cells and normal liver cells L02. It can be seen that compound 5 of the present invention is not a cytotoxic compound.

[0041] Table 1 IC of compound 5 for inhibiting tumor cell proliferation 50

[0042]

[0043] n=6

[0044] Example 11 Evaluation of the inhibitory activity of compound 5 on tumor cell migration

[0045] LLC and 95D are both anchorage-dependent cells and are cultured in a monolayer format. Remove the cell culture flask from the incubator and observe the cell status under a microscope to see if it meets the requirements. Wash, digest, disperse and count the cells. Then, dilute the cell concentration to 5×10 5 / mL. Serum-free medium (1640) was used for dispersion and dilution of 95D cells. Serum-free medium (DMEM) was used for dispersion and dilution of LLC cells. 100 μL of cell suspension was added to the upper chamber of the chamber first, and then 25 μL of compound 5 aqueous solution was added to the concentration of 20 μM. Each plate was set up with positive control RGDS and negative control serum-free medium, and two secondary wells were set up for the control and compounds 3 and 5. After adding the test compound and the control solution, gently tap the four walls of the 24-well plate to mix the solution in the upper chamber of the chamber evenly, and add 600 μL of medium rich in 10% fetal bovine serum to the lower chamber as soon as possible and remove the bubbles under the membrane. After culturing each cell line at 37°C and 5% CO2 for the required time, post-treatment was performed. Post-processing method: Use a pipette to carefully remove the residual liquid in the upper chamber, add 100 μL PBS buffer to each upper chamber, carefully wipe the cells in the upper chamber with a cotton swab, and repeat this operation twice to make sure that there is no cell residue on the upper chamber membrane (to prevent damage to the Transwell chamber membrane). Remove the culture medium in the lower chamber, add 600 μL 4% tissue fixative, fix the cells on the lower side of the upper chamber membrane in a 4°C refrigerator for 1 hour, remove the residual liquid in the lower chamber, add 600 μL crystal violet dye to each lower chamber, and dye for 30 minutes. Remove the recovered dye solution, rinse the remaining crystal violet dye on the polycarbonate membrane with triple distilled water, and take pictures under an inverted microscope. Select nine different fields of view for each chamber, fix the angle and magnification, and the cells in the field of view should be evenly distributed. Avoid selecting the field of view of cells at the edge of the chamber for counting. Use image J to count the number of cells in the photos, expressed as mean ± SD, and t-test p < 0.05 indicates statistical difference. The results are listed in Tables 2 and 3. The results showed that compounds 3 and 5 of the present invention had no effect on the migration of 95D and LLC cells at a concentration of 20 μM.

[0046] Table 2 Effects of compound 5 on 95D cell migration

[0047] Compound Concentration (μM) The number of migrated cells is the mean ± SD 1640 - 164±37 RGDS 20 <![CDATA[114±12 a ]]> Compound 3 20 <![CDATA[181±39 b ]]> Compound 5 20 <![CDATA[157±23 b ]]>

[0048] a) p<0.01 compared with 1640; b) p>0.05 compared with 1640; n=9.

[0049] Table 3 Effect of compound 5 on LLC cell migration

[0050] Compound Concentration (μM) The number of migrated cells is the mean ± SD DMEM - 242±63 RGDS 20 <![CDATA[125±44 a ]]> Compound 3 20 <![CDATA[245±66 b ]]> Compound 5 20 <![CDATA[231±77 b ]]>

[0051] a) p<0.01 compared with DMEM; b) p>0.05 compared with DMEM; n=9.

[0052] Example 12 Evaluation of the inhibitory activity of compound 5 on tumor cell invasion

[0053] Take out the matrix gel from the -20℃ refrigerator and place it in a 4℃ refrigerator overnight to completely convert it from solid to liquid. Then dilute it five times with serum-free medium and mix it evenly. Pipette 100μL of the prepared matrix gel dilution and add it to the upper chamber of each well of the Transwell chamber. Place the Transwell chamber in an incubator at 37℃ and 5% CO2 for 5 hours.

[0054] Take out the incubated Transwell chamber, aspirate the liquid in the Transwell chamber, add 50 μL of serum-free culture medium to the upper chamber of each well of the Transwell chamber, place it in an incubator at 37°C and 5% CO2, incubate for 30 minutes, and then aspirate the liquid in the Transwell chamber.

[0055] LLC is an anchorage-dependent cell and is cultured in a monolayer format. Take out the cell culture flask from the incubator and observe the cell status under a microscope to see if it meets the requirements. Wash, digest, disperse and count the cells. Then, dilute the cell concentration to 5×10 5 / mL. Serum-free medium (DMEM) was used for dispersion and dilution. First, 100μL of cell suspension was added to the upper chamber of the chamber, and then 25μL of compound 3 or 5 aqueous solution was added to the concentration of 20μM. Positive and negative control groups were set up on each plate, and two secondary wells were set up for the control and compound 3 or 5. After adding compound 3 or 5 and the control solution, the four walls of the 24-well plate were gently tapped to mix the solution in the upper chamber of the chamber evenly. 600μL of culture medium rich in 10% fetal bovine serum was added to the lower chamber as soon as possible and the bubbles under the membrane were removed. After culturing each cell line at 37°C and 5% CO2 for the required time, post-treatment was performed.

[0056] The post-treatment method is to carefully remove the residual liquid in the upper chamber with a pipette, add 100 μL PBS buffer to each upper chamber, carefully wipe the cells in the upper chamber with a cotton swab, and repeat this operation twice so that there is no cell residue on the upper chamber membrane (to prevent damage to the Transwell chamber membrane). Remove the culture medium in the lower chamber, add 600 μL 4% tissue fixative, fix the cells on the lower side of the upper chamber membrane in a 4°C refrigerator for 1 hour, remove the residual liquid in the lower chamber, add 600 μL crystal violet dye to each lower chamber, and dye for 30 minutes. Remove the recovered dye solution, rinse the remaining crystal violet dye on the polycarbonate membrane with triple distilled water, and take pictures under an inverted microscope. Select nine different fields of view for each chamber, fix the angle and magnification, and the cells in the field of view should be evenly distributed, avoiding the selection of the cell field of view at the edge of the chamber for counting. Image J was used to count the number of cells in the photo, expressed as mean ± SD, and statistically significant when P < 0.05 was tested by t. The results are listed in Table 4. It can be seen from the results that compound 5 in the compounds of the present invention can effectively inhibit tumor cell invasion.

[0057] Table 4 Effect of compound 5 on LLC cell invasion

[0058] Compound Concentration (μM) The number of invasive cells, mean ± SD DMEM - 410±89 RGDS 20 152±46 Compound 3 20 <![CDATA[287±76 a ]]> Compound 5 20 <![CDATA[170±41 b ]]>

[0059] a) p<0.01 compared with DMEM; b) p<0.01 compared with DMEM and compound 3, p>0.05 compared with RGDS; n=9 Example 13 Evaluation of the inhibitory activity of compound 5 on tumor lung metastasis

[0060] 1) The positive control was RGDS tetrapeptide, with an intraperitoneal injection dose of 20 μmol / kg / day for 10 consecutive days; the blank control group was normal saline, with an oral dose of 0.1 mL / 10 g / day; the oral dose of compound 3 was 20 μmol / kg / day; the oral dose of compound 5 was 1 μmol / kg / day for 10 consecutive days.

[0061] 2) Lewis lung cancer cells (LLC) were purchased from ATCC. LLC was subcultured in DMEM complete medium containing 10% fetal bovine serum to obtain cells in the logarithmic growth phase. The cells were washed, digested, dispersed, counted, and diluted to a concentration of 2×10 7 The prepared cell suspension was inoculated into the armpits of C57BL / 6N male mice, with 0.2 mL inoculated per mouse. When the solid tumor of the tumor-bearing mouse grows to a diameter of 1.5 cm-2 cm, it is used as a tumor source for later use.

[0062] 3) The tumor-bearing mice were anesthetized with ether and quickly killed by cervical dislocation. The solid tumor was ground into a cell suspension using a tissue homogenizer and passed through a 200-mesh cell sieve to obtain a single-cell suspension. The single-cell suspension was combined, washed, counted, and diluted with PBS buffer to 2×10 7 / mL cell suspension. The prepared cell suspension was inoculated in the armpits of C57BL / 6N male mice, with 0.2mL inoculated per mouse. Since the day of inoculation, the solid tumors of tumor-bearing mice were observed and measured every day. When the solid tumors grew to the size of soybeans, the drugs were administered. Before administration, the groups were regrouped according to the principle of complete randomization. The weight of the mice was monitored and the tumor volume was measured using a vernier caliper before daily administration. The corresponding dose of compound 3 or 5 was administered according to the body weight for 10 consecutive days. On the 11th day, the mice were anesthetized with ether, killed by dislocation of the neck, and the implanted tumors were bluntly dissected with surgical instruments. The tumor weights of mice in each group were quickly weighed and statistically analyzed. The results showed that there was no significant difference in the weight of the implanted tumors of mice treated with normal saline, RGDS, compound 3 and compound 5 (data omitted here). It can be seen that RGDS, compound 3 and compound 5 have no inhibitory effect on the growth of in situ tumors. The lungs were bluntly dissected with surgical instruments, and the metastatic nodules were quickly photographed and counted. The results are listed in Table 5. From the results, it can be seen that compound 5 among the compounds of the present invention can effectively inhibit the lung metastasis of tumor.

[0063] Table 5 Inhibitory activity of compound 5 on tumor lung metastasis

[0064] Compound Dosage (μmol / kg / day) Number of lung metastatic tumor nodes, mean ± SD Normal saline - 37.2±14.0 RGDS 20 <![CDATA[9.4±2.0 a ]]> Compound 3 20 35.0±12.0 Compound 5 1 <![CDATA[7.3±3.1 b ]]>

[0065] a) p<0.01 compared with saline and compound 3; b) p<0.01 compared with saline and compound 3, p>0.05 compared with RGDS; n=10.

Claims

1. 1-imidazole-β-carboline-3-formyl-Leu-Asp-Val having the following structure, 2. A method for preparing 1-imidazole-β-carboline-3-formyl-Leu-Asp-Val of the structure of claim 1, characterized in that the method comprises the following steps: 2.

1. Under the catalysis of trifluoroacetic acid, L-Trp-OBzl and imidazole-2-carboxaldehyde underwent Pictet-Spengler condensation to obtain 3S-1-imidazole-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester; 2.2.3S-1-imidazole-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester is oxidized with 2,3-dichloro-5,6-dicyano-p-benzoquinone in tetrahydrofuran to give 1-imidazole-β-carboline-3-carboxylic acid benzyl ester; 2.

3. 1-imidazole-β-carboline-3-carboxylic acid benzyl ester was debenzylated with Pd / C catalytic hydrogenation in tetrahydrofuran to give 1-imidazole-β-carboline-3-carboxylic acid; 2.

4. Prepare Leu-Asp(OBzl)-Val-OBzl by conventional peptide synthesis; 2.5.1-imidazole-β-carboline-3-carboxylic acid is coupled with Leu-Asp(OBzl)-Val-OBzl to prepare 1-imidazole-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl; 2.

6. Removal of the protecting group of 1-imidazole-β-carboline-3-formyl-Leu-Asp(OBzl)-Val-OBzl to prepare 1-imidazole-β-carboline-3-formyl-Leu-Asp-Val.

3. Use of 1-imidazole-β-carboline-3-formyl-Leu-Asp-Val of the structure described in claim 1 in the preparation of drugs for inhibiting lung cancer metastasis.

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