1-Imidazol-β-carboline-3-carboxyl-RGDV for inhibiting invasion and migration, its preparation and application

Abstract

This invention discloses 1-(1H-imidazol-2-yl)-β-carbamoline-3-formyl-Arg-Gly-Asp-Val, its preparation method, its activity in inhibiting tumor cell invasion and migration, and its activity in inhibiting tumor metastasis to the lungs in Lewis mice. Therefore, this invention discloses its application in the preparation of drugs for treating cancer metastasis.

Description

Technical Field

[0001] This invention relates to 1-(1H-imidazol-2-yl)-β-carbamo-3-formyl-Arg-Gly-Asp-Val, its preparation method, its activity in inhibiting tumor cell invasion and migration, and its activity in inhibiting tumor metastasis to the lungs in Lewis mice. Therefore, this invention discloses its application in the preparation of drugs for treating cancer metastasis. This invention belongs to the field of biomedicine. Background Technology

[0002] Currently, cancer is the second leading cause of death worldwide. Statistics show that 9.96 million people died from cancer globally in 2020. Of these, 3 million died in my country, accounting for 30% of the global total. The majority of these cancer deaths are due to tumor metastasis. Furthermore, safe and effective drugs for treating tumor metastasis are still lacking in clinical practice. Therefore, developing novel drugs for treating tumor metastasis is of significant practical importance.

[0003] β-Carboline compounds are a class of indole alkaloids with a pyrido[3,4-b]indole structure, widely found in plants and animals in nature. β-Carboline compounds can affect multiple tumor-related targets. For example, they can act on DNA, regulate the cell cycle, induce apoptosis, and inhibit DNA topoisomerases and histone deacetylases, etc. Imidazoles have the potential to exert antitumor effects through covalent and non-covalent interactions with DNA, serving as an important building block for compounds with anticancer activity.

[0004] Macrophages are the most abundant immune cells in penetrating tumors. Tumor-associated macrophages (TAMs) can promote tumor cell migration and invasion, thus having a significant impact on cancer metastasis. Part of the mechanism by which TAMs promote tumor cell migration and invasion involves the recruitment of cyclooxygenase-2 (COX2)-expressing macrophages by interleukin-1α (IL-1α) expressed by cancer cells. In turn, the recruited macrophages further promote tumor cell migration and invasion, thus advancing the tumor metastasis process.

[0005] The above knowledge indicates that compounds capable of entering the active pockets of interleukin-1α and cyclooxygenase-2 will be able to inhibit macrophage recruitment, thereby inhibiting tumor cell migration and invasion, and cancer metastasis. After analyzing the morphology of the active pockets of interleukin-1α and cyclooxygenase-2, the inventors designed 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Arg-Gly-Asp-Val. Using molecular docking technology, the inventors docked this compound with interleukin-1α and cyclooxygenase-2. It was found that 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Arg-Gly-Asp-Val can enter the active pockets of interleukin-1α and cyclooxygenase-2 very well (molecular docking diagram omitted here). These theoretical studies led the inventors to realize that 1-(1H-imidazol-2-yl)-β-carboline-3-carboxyl-Arg-Gly-Asp-Val can inhibit tumor cell migration and invasion, and thus inhibit cancer metastasis. Based on this understanding, the inventors completed the subsequent experimental research. Summary of the Invention

[0006] The first aspect of this invention is the confirmation that 1-(1H-imidazol-2-yl)-β-carbamo-3-formyl-Arg-Gly-Asp-Val is a novel compound.

[0007]

[0008] The second aspect of this invention is the preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Arg-Gly-Asp-Val using the following steps:

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

[0010] 2) 3S-1-(1H-imidazol-2-yl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was oxidized in tetrahydrofuran with 2,3-dichloro-5,6-dicyanobenzoquinone to give 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester.

[0011] 3) In tetrahydrofuran, 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester was hydrogenated and debenzyl ester was obtained by palladium-catalyzed hydrogenolysis to give 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid;

[0012] 4) Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl was prepared using conventional methods for peptide synthesis;

[0013] 5) 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid was coupled with Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl to prepare 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl;

[0014] 6) Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-carboxy ...

[0015] The third aspect of this invention is the use of the MTT assay to confirm that 1-(1H-imidazol-2-yl)-β-carbamo-3-formyl-Arg-Gly-Asp-Val is not a cytotoxic compound.

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

[0017] The fifth aspect of this invention is to evaluate the inhibitory effect of 1-(1H-imidazol-2-yl)-β-carboxylate-3-carboxyformyl-Arg-Gly-Asp-Val on lung metastasis in Lewis lung cancer-bearing mice. Attached Figure Description

[0018] Figure 1 Synthetic route of 1-(1H-imidazol-2-yl)-β-carbamoline-3-carboxyformyl-Arg-Gly-Asp-Val: i) trifluoroacetic acid, dichloromethane; ii) dichlorodicyanobenzoquinone, tetrahydrofuran; iii) hydrogen, palladium on carbon, tetrahydrofuran; iv) 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N-methylmorpholine, N,N-dimethylformamide; v) trifluoroacetic acid, trifluoromethanesulfonic acid, diethyl ether; vi) dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; vii) CH3OH, NaOH (2N); viiii) ethyl acetate solution of hydrogen chloride (4M). Detailed Implementation

[0019] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be construed 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] 3 g (10.0 mmol) of L-tryptophan benzyl ester was dissolved in 100 mL of dichloromethane at 0 °C with stirring, followed by the addition of 2 mL of trifluoroacetic acid, and the reaction was allowed to proceed for 30 minutes. Then, 1.18 g (12.0 mmol) of imidazole-2-carboxaldehyde was added, and the reaction was allowed to proceed for 12 hours at room temperature. Subsequent TLC (dichloromethane:methanol = 20:1) showed the disappearance of L-tryptophan benzyl ester. The residue was concentrated under reduced pressure to remove the remaining trifluoroacetic acid, and the residue was dissolved in 100 mL of dichloromethane. The solution was washed successively with saturated NaHCO3 solution (20 mL × 3) and saturated NaCl solution (20 mL × 3). The dichloromethane layer was dried over anhydrous Na2SO4 for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to give 3 g of a colorless solid. This solid was purified by silica gel column chromatography (DCM:MeOH, 20:1) to give 3.5 g (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 under stirring at 0 °C. 5.7 g (25.0 mmol) of dichlorocyanobenzoquinone was added, and the mixture was reacted at room temperature for 48 h. 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. The residue was washed successively with saturated NaHCO3 aqueous solution (20 mL × 3) and saturated NaCl aqueous solution (20 mL × 3). The ethyl acetate layers were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to dryness under reduced pressure to give 4.2 g of colorless solid. The solid was purified by silica gel column chromatography (petroleum ether:ethyl acetate, 1.5:1) to give 2.4 g (53%) of the title compound as a colorless powder. ESI / MS (m / e): 391 [M+Na] + ; 1 H NMR(300MHz,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-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester (2) was dissolved in 50 mL of tetrahydrofuran under stirring. 200 mg of palladium on carbon was added, and hydrogen gas was bubbled through the solution to initiate the reaction. After reacting at room temperature for 24 hours, TLC (dichloromethane:methanol = 20:1) showed the disappearance of 1-(1H-imidazol-2-yl)-β-carboline-3-carboxylic acid benzyl ester (2). The palladium on carbon was filtered off, and the solvent was removed by concentration under reduced pressure to give 1.0 g (70%) of the title compound as a yellow powder. ESI / MS (m / e): 279 [M+H] + ; 1H 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-Arg(NO2)-Gly-OBzl

[0027] 3.19 g (10.0 mmol) of Boc-Arg(NO2) was dissolved in 100 mL of anhydrous tetrahydrofuran at 0 °C with stirring. Then, 1.49 g (11.0 mmol) of 1-hydroxybenzotriazole and 2.47 g (1.0 mmol) of dicyclohexylcarbodiimide were added, and the reaction was allowed to proceed for 30 minutes. Afterward, 2.22 g (11.0 mmol) of Gly-OBzl was added. Finally, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, and the resulting reaction solution was stirred at room temperature for 10 hours. The solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate, and the resulting solution was washed successively 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, allowed to stand for 6 hours to allow the solid to precipitate, and filtered to give 4.45 g (96%) of the title compound as a colorless powder. ESI-MS (m / e): 467 [M+H] + ; 1 H NMR(300MHz,DMSO-d6)δ8.49(s, 1H),8.29(t,J=5.7Hz,1H),7.83(s,2H),7.37(d,J=3.7Hz,4H),6.92(d,J=8.0Hz,1H),5.76 (s,1H),5.12(s,2H),3.89(tt,J=17.4,8.2Hz,3H),3.33(s,2H),1.64(s,1H),1.50(s,2H), 1.38(s,9H).

[0028] Example 5: Preparation of Boc-Arg(NO2)-Gly

[0029] 2.33 g (5.00 mmol) of Boc-Arg(NO2)-Gly-OBzl was dissolved in 45 mL of methanol at 0 °C with stirring. The pH of the reaction solution was adjusted to 12 with 2 M sodium hydroxide aqueous solution. The mixture was stirred until TLC (dichloromethane:methanol = 20:1) showed complete disappearance of Boc-Arg(NO2)-Gly-OBzl. The reaction solution was adjusted to pH 7 at 0 °C with saturated KHSO4 aqueous solution, concentrated under reduced pressure, and the residue was added to 15 mL of distilled water. The pH was then adjusted to 2 with saturated KHSO4 aqueous solution. The aqueous layer was extracted with ethyl acetate (20 mL × 3), and the ester layer was washed with saturated sodium chloride aqueous solution (20 mL × 3). The ethyl acetate layer was dried over anhydrous Na2SO4 for 2 h, filtered, and the filtrate was concentrated under reduced pressure to give 1.7 g (90%) of the title compound as a colorless solid powder. ESI-MS (m / e): 377 [M+H] + .

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

[0031] Using the method of Example 4, 5.50 g (90%) of the title compound was obtained from 3.23 g (10.0 mmol) of Boc-Asp (OBzl) and 2.67 g (11.0 mmol) of Val-OBzl, as a yellow oil. 1H NMR(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,1 H), 1.99 (s, 1H), 1.38 (s, 8H), 1.18 (t, J = 7.1Hz, 1H), 0.85 (d, J = 6.8Hz, 6H).

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

[0033] 1.05 g (2.0 mmol) of Boc-Asp(OBzl)-Val-OBzl was dissolved in 20 mL of ethyl acetate solution of hydrogen chloride (4 M) at 0 °C with stirring, and stirred for 1 hour. TLC (dichloromethane:methanol = 20:1) showed the disappearance of Boc-Asp(OBzl)-Val-OBzl. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 15 mL of anhydrous ethyl acetate. The solution was concentrated again under reduced pressure, and the residue was sonicated with 10 mL of anhydrous diethyl ether to completely suspend it. The supernatant was discarded to give 0.88 g (98%) of the title compound as a yellow solid. ESI-MS (m / e): 413 [M+H] + .

[0034] Example 8: Preparation of Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl

[0035] 0.75 g (61%) of the title compound was obtained as a colorless solid from 0.75 g (2.0 mmol) of Boc-Arg(NO2)-Gly and 0.96 g (2.2 mmol) of Asp(OBzl)-Val-OBzl using the method of Example 4. ESI-MS (m / e): 771 [M+H] + ; 1HNMR(300MHz,DMSO-d6)δ8.48(s,1H),8.24(d,J=8.1Hz,1H),8.15(d,J=8.1Hz,1H ),8.05(d,J=5.8Hz,1H),7.45–7.25(m,10H),6.96(d,J=7.7Hz,1H),5.21–4.98(m, 4H),4.85–4.68(m,1H),4.18(dd,J=8.1,6.2Hz,1H),3.70(d,J=5.4Hz,2H),3.12(d,J= 5.9Hz,2H),2.76(dd,J=16.2,5.1Hz,1H),2.65–2.51(m,1H),2.07(h,J=6.8 Hz, 1H), 1.65 (s, 1H), 1.50 (s, 4H), 1.37 (s, 9H), 0.85 (dd, J = 6.8, 1.7Hz, 6H).

[0036] Example 9: Preparation of Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl

[0037] The title compound was obtained as a colorless solid from 0.4 g (0.5 mmol) of Boc-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl using the method of Example 7. ESI-MS (m / e): 671 [M+H] + ;1 H NMR (300MHz, DMSO-d6) δ8.77(t,J=5.6Hz,1H),8.57(s,1H),8.45(d,J=7.9Hz,1H),8.28(d,J=8.3Hz, 1H),8.27(s,2H),7.97(s,2H),7.46–7.23(m,9H),5.20–4.99(m,4H),4.79(td,J=8.7,4.9 Hz,1H),4.18(dd,J=8.0,6.2Hz,1H),3.83(t,J=8.1Hz,6H),2.74(dd,J=16.2,5.0Hz,1H), 2.58(dd,J=16.2,9.1Hz,1H),2.17–2.01(m,1H),1.80–1.65(m,2H),1.57(s,2H),0.85(dd, J=6.8,1.9Hz,6H).

[0038] Example 10 Preparation of 1-(1H-imidazol-2-yl)-β-carbline-3-formyl-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(4)

[0039] 200 mg (0.7 mmol) of 1-(1H-imidazol-2-yl)-β-carboxylin-3-carboxylic acid was dissolved in 20 mL of N,N-dimethylformamide under stirring at 0 °C. Then, 560 mg (0.8 mmol) of Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl and 280 mg (0.8 mmol) of 2-(7-benzotriazole oxide)-,N,N',N'-tetramethylurea hexafluorophosphate were added sequentially. The pH of the reaction solution was adjusted to 9 with N-methylmorpholine, and the mixture was reacted at room temperature for 12 hours. The solution was then concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the resulting solution was washed sequentially with saturated NaHCO3 aqueous solution (20 mL × 3) and saturated NaCl aqueous solution (20 mL × 3). The combined ethyl acetate layers were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give 400 mg of a colorless solid. This solid was purified by silica gel column chromatography to give 296 mg (46%) of the title compound as a colorless powder. 1H NMR (300MHz, DMSO-d6) δ13.40 (s, 1H), 11.83 (s, 1H), 9.24 (d, J = 8.0Hz, 1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.41–8.23 (m, 3H), 8.16(d,J=8.2Hz,1H),7.96(d,J=8.4Hz,1H),7.66–7.52(m,2H),7.42–7.24(m,13H),5 .05(dq,J=25.0,12.4Hz,4H),4.84–4.73(m,1H),4.65(d,J=7.0Hz,1H),4.16(t,J=7.2 Hz,1H),3.75(d,J=6.9Hz,2H),3.23(s,2H),2.78(dd,J=17.0,5.9Hz,1H),2.62(dd,J=16 .1,8.6Hz,1H),2.10–1.96(m,1H),1.84(s,1H),1.63(s,2H),1.23(s,2H),0.87–0.75(m, 6H).

[0040] Example 11 Preparation of 1-(1H-imidazol-2-yl)-β-carboline-3-formyl-Arg-Gly-Asp-Val(5)

[0041] 100 mg (0.11 mmol) of 1-(1H-imidazol-2-yl)-β-carbamo-3-carboxymethyl-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(4) was dissolved in 1 mL of trifluoroacetic acid and 0.3 mL of trifluoromethanesulfonic acid under stirring at 0 °C. The mixture was stirred for 0.5 hours, and the reaction mixture was concentrated under reduced pressure. 10 mL of anhydrous diethyl ether was added to the residue, and the mixture was stirred for 10 minutes. A solid precipitated out. The supernatant was discarded after standing for 10 minutes. 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. The solid was purified by C18 column using 40% water and 60% methanol as eluent. The eluent was lyophilized to give 27 mg (35%) of the title compound as a yellow solid. FT-ESI-MS (m / e): 706.31653 [M+H] + ; 1H NMR(300MHz, DMSO-d6)δ14.47(s,1H),11.83(s,1H),10.38(s,1H),9.71(s,1H),9.36(s,1H),8.95(s,1H), 8.82(s,1H),8.38(d,J=7.9Hz,1H),7.96(d,J=8.3Hz,1H),7.59(t,J=7.8Hz,1H),7.49(s,1 H),7.32(s,2H),7.29(d,J=7.2Hz,0H),7.09(s,3H),4.80(s,1H),4.22(s,1H),3.91(s,1H), 3.80(s,2H),3.21(d,J=23.8Hz,1H),3.03(s,1H),2.26(d,J=13.0Hz,1H),2.01(s,2H),1.65(s,2H),0.81(t,J=6.5Hz,6H).

[0042] Example 12: Evaluation of the cytotoxic effects of compound 5

[0043] Compound 5 was prepared to the desired concentration using 1640 medium containing 0.5% DMSO. HCT-116, A549, S180, LLC, HL60, U2OS, and L02 cells in good growth condition and in logarithmic growth phase were respectively cultured at 4 × 10⁻⁶. 4 The sample was seeded at a density of 100 μL / mL in a 96-well plate. After incubation at 37°C in a 5% CO2 incubator for 4 hours, sterilized compound 5 solution was added at preset concentration gradients of 100 μM, 50 μM, 25 μM, 10 μM, and 1 μM. The control group was treated with an equal volume of 1640 medium containing 0.5% dimethyl sulfoxide (DMSO). After further incubation for 48 hours, 25 μL of 5 mg / mL MTT solution was added to each well, and the plate was incubated at 37°C for 4 hours. After carefully removing the supernatant, 100 μL of DMSO was added to each well, and the plate was shaken for approximately 15 min to dissolve the precipitate. The OD (absorbance) value was immediately measured at 570 nm using a microplate reader. The inhibition rate was calculated as: [(average OD value of the 1640 medium group containing 0.5% DMSO – average OD value of the compound) / OD value of the 1640 medium group containing 0.5% DMSO] × 100%. The experiment was repeated 6 times in parallel. The inhibition rate was plotted against the compound concentration to calculate the IC50 of the compound of this invention. 50 (Half-maximal inhibitory concentration) value.

[0044] The results are listed in Table 1. The results show that compound 5 of this invention had no inhibitory effect on the proliferation of the six tumor cell lines and normal L02 hepatocytes. Therefore, compound 5 of this invention is not a cytotoxic compound.

[0045] Table 1. IC50 of compound 5 in inhibiting tumor cell proliferation 50

[0046]

[0047] n = 6

[0048] Example 13: Evaluation of Compound 5's activity in inhibiting tumor cell migration

[0049] Both LLC and 95D are adherent cell lines, cultured in a monolayer. The cell culture flasks were removed from the incubator, and microscopic observation confirmed that the cells met the required conditions. The cells were then washed, digested, dispersed, and counted. The cell concentration was subsequently diluted to 5 × 10⁻⁶. 5 Cells / mL. 95D cells were dispersed and diluted using serum-free medium (1640) by pipetting. LLC cells were dispersed and diluted using serum-free medium (DMEM) by pipetting. In the upper chamber, 100 μL of cell suspension was added first, followed by 25 μL of compound 5 aqueous solution to a concentration of 20 μM. Each plate included a positive control (RGDS) and a negative control (serum-free medium). Two auxiliary wells were provided for the controls and compounds 3 and 5. After adding the test compounds and control solutions, the walls of the 24-well plate were gently tapped to mix the solution in the upper chamber. 600 μL of medium rich in 10% fetal bovine serum was added to the lower chamber as soon as possible, and air bubbles were removed from the membrane. The cells were cultured at 37°C and 5% CO2 for the required time before post-treatment. Post-processing: Carefully aspirate residual fluid from the upper chamber using a pipette. Add 100 μL of PBS buffer to each upper chamber and carefully wipe away cells with a cotton swab. Repeat this process twice to ensure no cells remain on one side of the upper chamber membrane (to prevent damage to the Transwell chamber membrane). Aspirate the culture medium from the lower chamber and add 600 μL of 4% tissue fixative. Fix cells on the underside of the upper chamber membrane at 4°C for 1 hour. Aspirate residual fluid from the lower chamber and add 600 μL of crystal violet staining solution to each lower chamber, staining for 30 minutes. Aspirate the recovered staining solution and rinse the polycarbonate membrane with triple-distilled water to remove any remaining crystal violet staining solution. Photograph the cells under an inverted microscope. Nine different fields of view were selected from each chamber, with fixed angles and magnifications. Cells should be evenly distributed within the field of view, avoiding selection of cells at the chamber edges. Cell counts were performed. The number of cells in the photographs was statistically analyzed using ImageJ, expressed as mean ± SD. A t-test was performed, with p < 0.05 considered statistically significant. Results are listed in Tables 2 and 3. The results showed that compound 5 of the present invention effectively inhibited the migration of 95D and LLC cells at a concentration of 20 μM.

[0050] Table 2 Effects of compound 5 on 95D cell migration

[0051] compound Concentration (μM) Number of migrating cells, mean ± SD 1640 - 164±37 RGDS 20 <![CDATA[114±12 a ]]> Compound 3 20 <![CDATA[181±39 b ]]> Compound 5 20 <![CDATA[66±13 c ]]>

[0052] a) Compared with 1640, p < 0.01; b) Compared with 1640, p > 0.05; c) Compared with 1640, p < 0.01; Compared with RGDS, p < 0.01; n = 9.

[0053] Table 3 Effects of compound 5 on LLC cell migration

[0054] compound Concentration (μM) Number of migrating cells, mean ± SD DMEM - 242±63 RGDS 20 <![CDATA[125±44 a ]]> Compound 3 20 <![CDATA[245±66 b ]]> Compound 5 20 <![CDATA[172±44 a ]]>

[0055] a) Compared with DMEM, p < 0.01; b) Compared with DMEM, p > 0.05; n = 9.

[0056] Example 14: Evaluation of Compound 5's activity in inhibiting tumor cell invasion

[0057] Remove the matrix gel from the -20°C freezer and place it in a 4°C freezer overnight to allow it to completely transform from a solid to a liquid state. Then, dilute it five-fold with serum-free medium and mix thoroughly. Add 100 μL of the prepared matrix gel dilution to the upper chamber of each well of the Transwell chamber. Incubate the Transwell chambers in a 37°C, 5% CO2 incubator for 5 hours.

[0058] Remove the incubated Transwell chambers, aspirate the liquid portion from the Transwell chambers, add 50 μL of serum-free culture medium to the upper chamber of each well of the Transwell chamber, incubate in a 37°C, 5% CO2 incubator for 30 minutes, and then aspirate the liquid from the Transwell chambers.

[0059] LLC cells are adherent cells and were cultured in a monolayer. The cell culture flasks were removed from the incubator, and microscopic observation confirmed that the cells met the required conditions. The cells were then washed, digested, dispersed, and counted. The cell concentration was subsequently diluted to 5 × 10⁻⁶. 5 Cells / mL. Dispersion and dilution were performed using serum-free medium (DMEM) via pipetting. In the upper chamber, 100 μL of cell suspension was added first, followed by 25 μL of aqueous solution of compound 3 or 5 to a concentration of 20 μM. Each plate included positive and negative control groups, and two auxiliary wells for both the control and compound 3 or 5 solutions. After adding compound 3 or 5 and the control solution, the walls of the 24-well plate were gently tapped to ensure thorough mixing in the upper chamber. 600 μL of medium rich in 10% fetal bovine serum was added to the lower chamber as soon as possible, and air bubbles were removed from the membrane. The cells were cultured at 37°C and 5% CO2 for the required time before post-treatment.

[0060] The post-processing method involved carefully aspirating residual fluid from the upper chamber using a pipette, adding 100 μL of PBS buffer to each upper chamber, and carefully wiping away cells with a cotton swab. This process was repeated twice to ensure no cells remained on one side of the upper chamber membrane (to prevent damage to the Transwell chamber membrane). The lower chamber culture medium was aspirated, and 600 μL of 4% tissue fixative was added. Cells on the underside of the upper chamber membrane were fixed at 4°C for 1 hour. Residual fluid in the lower chamber was aspirated, and 600 μL of crystal violet staining solution was added to each lower chamber for staining for 30 minutes. The staining solution was then removed and the remaining crystal violet staining solution on the polycarbonate membrane was rinsed with triple-distilled water. Images were taken under an inverted microscope, selecting nine different fields of view for each chamber, with fixed angles and magnifications. Cells should be evenly distributed within the field of view, avoiding counting cells at the chamber edges. The number of cells in the images was counted using ImageJ, expressed as mean ± SD. A t-test was performed, and P < 0.05 was considered statistically significant. The results are listed in Table 4. The results show that compound 5 of the present invention effectively inhibits tumor cell invasion.

[0061] Table 4. Effects of compound 5 on LLC cell invasion.

[0062] compound Concentration (μM) Number of invasive cells, mean ± SD DMEM - 410±89 RGDS 20 152±46 Compound 3 20 <![CDATA[287±76 a ]]> Compound 5 20 <![CDATA[182±41 b ]]>

[0063] a) p < 0.01 compared to DMEM; b) p < 0.01 compared to DMEM and compound 3, p > 0.05 compared to RGDS; n = 9

[0064] Example 15 evaluates the activity of compound 5 in inhibiting tumor lung metastasis.

[0065] 1) The positive control was RGDS tetrapeptide, administered intraperitoneally at a dose of 20 μmol / kg / day for 10 consecutive days; the blank control group was physiological saline, administered orally at a dose of 0.1 mL / 10 g / day; the oral dose of compound 3 was 20 μmol / kg / day; and the oral dose of compound 5 was 1 μmol / kg / day for 10 consecutive days.

[0066] 2) Lewis lung cancer cells (LLC) were purchased from ATCC. LLC cells were passaged in DMEM complete medium containing 10% fetal bovine serum to obtain cells in the logarithmic growth phase. After washing, digestion, dispersion, and counting, the cell concentration was diluted to 2 × 10⁻⁶ cells / mL using PBS. 7 Cell suspension of 0.2 mL / mL was prepared and inoculated into the axillae of male C57BL / 6N mice. Solid tumors in the tumor-bearing mice were used as tumor sources when they grew to a diameter of 1.5 cm-2 cm.

[0067] 3) Tumor-bearing mice, used as the tumor source, were anesthetized with ether and euthanized by rapid cervical dislocation. The solid tumors were homogenized 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 suspensions were combined, washed, and counted, then diluted with PBS buffer to 2 × 10⁻⁶ cells / mL. 7 Cell suspensions of 0.2 mL / mL were prepared and inoculated into the axillae of male C57BL / 6N mice. From the day of inoculation, the solid tumors of the tumor-bearing mice were observed and measured daily. When the solid tumors grew to the size of a soybean, the mice were regrouped according to a completely randomized principle before administration. The weight of the mice was monitored daily before administration, and the tumor volume was measured using calipers. The appropriate dose of compound 3 or 5 was administered according to the weight for 10 consecutive days. On day 11, the mice were anesthetized with ether, euthanized by cervical dislocation, and the implanted tumors were bluntly dissected using surgical instruments. The tumor weights of the mice in each group were quickly weighed and statistically analyzed. The results showed no significant difference in the weight of the implanted tumors in mice treated with saline, RGDS, compound 3, and compound 5 (data omitted here). This indicates that RGDS, compound 3, and compound 5 did not inhibit the growth of in situ tumors. The lungs were bluntly dissected using surgical instruments, and rapid photographs were taken to count the metastatic nodules. The results are listed in Table 5. The results show that compound 5 of the present invention effectively inhibits the metastasis of tumors to the lungs in Lewis mice.

[0068] Table 5. Compound 5's inhibitory activity against tumor lung metastasis.

[0069] compound Dosage (μmol / kg / day) Number of lung metastatic nodules, mean ± SD physiological saline - 37.2±14.0 RGDS 20 <![CDATA[9.4±2.0 a ]]> Compound 3 20 35.0±12.0 Compound 5 1 <![CDATA[7.2±4.0 b ]]>

[0070] a) p < 0.01 compared with physiological saline and compound 3; b) p < 0.01 compared with physiological saline and compound 3, p > 0.05 compared with RGDS; n = 10.

Claims

1. The following structure: 1-imidazol-β-carboline-3-formyl-Arg-Gly-Asp-Val, 2. The method for preparing 1-imidazolium-β-carbline-3-formyl-Arg-Gly-Asp-Val according to claim 1, wherein the method... Includes the following steps: 2.

1. L-Trp-OBzl and imidazole-2-carboxaldehyde under the catalysis of trifluoroacetic acid undergo Pictet-Spengler condensation to obtain 3S-1-imidazole-2,3,4,9-tetrahydro-β-carbamoline-3-carboxylic acid benzyl ester. 2.2.3S-1-imidazol-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was oxidized in tetrahydrofuran with 2,3-dichloro-5,6-dicyanobenzoquinone to give 1-imidazol-β-carboline-3-carboxylic acid benzyl ester; 2.

3. In tetrahydrofuran, 1-imidazolium-β-carboline-3-carboxylic acid benzyl ester was hydrogenated and debenzyl ester was obtained by Pd / C catalysis to yield 1-imidazolium-β-carboline-3-carboxylic acid; 2.

4. Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl was prepared using conventional methods for peptide synthesis; 2.5.1-Imidazole-β-carboline-3-carboxylic acid was coupled with Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl to prepare 1-imidazazole-β-carboline-3-carboxylic acid-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl; 2.

6. Preparation of 1-imidazol-β-carboline-3-carboxyl-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl by removing the protecting group of 1-imidazol-β-carboline-3-carboxyl-Arg-Gly-Asp-Val.

3. The use of 1-imidazol-β-carbamoline-3-carboxyl-Arg-Gly-Asp-Val as described in claim 1 in the preparation of an anti-lung cancer metastasis drug.

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