CN115466315A - 1-imidazole-beta-carboline-3-formyl-RGDS for inhibiting invasion, preparation, anti-cancer metastasis effect and application - Google Patents
1-imidazole-beta-carboline-3-formyl-RGDS for inhibiting invasion, preparation, anti-cancer metastasis effect and application Download PDFInfo
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Abstract
The invention discloses 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser with the following formula, discloses a preparation method thereof, discloses the activity of inhibiting tumor cell invasion and discloses the activity of inhibiting tumor lung metastasis of Lewis mice. Therefore, the invention discloses the application of the compound in preparing the anti-tumor metastasis medicaments.
Description
Technical Field
The invention relates to 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser. Relates to its preparation and to its anti-tumor metastasis effect. 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
Cancer is currently the second leading cause of death worldwide. According to statistics, the number of cancer death cases in the world is 996 ten thousand in 2020. Wherein the number of cancer deaths in China is 300 ten thousand, accounting for 30 percent of the total number of cancer deaths in the world. The majority of the cancer deaths die from tumor metastases. In addition, safe and effective anti-tumor metastasis medicaments are still lacking in clinical application at present. Therefore, the development of novel anti-tumor metastasis medicaments has important practical significance.
The beta-carboline compounds are indole alkaloids with pyrido [3,4-b ] indole structure, and widely exist in animals and plants in nature. The beta-carboline compounds can affect a plurality of tumor-related targets. For example, it may act on DNA, regulate cell cycle, induce apoptosis, inhibit DNA topoisomerase and histone deacetylase, and the like. Imidazoles have the potential to produce anti-tumor effects by interacting covalently and non-covalently with DNA, as an important building block for compounds with anti-cancer activity.
Macrophages are the most abundant immune cells that penetrate tumors. Tumor-associated macrophages (TAMs) can promote tumor cell migration and invasion, and thus have a significant impact on cancer metastasis. Part of the mechanism by which tumor-associated macrophages promote tumor cell migration and invasion involves the recruitment of cyclooxygenase-2 (COX 2) expressing macrophages by interleukin-1 alpha (IL-1 alpha) expressed by cancer cells. In turn, recruited macrophages promote tumor cell migration and invasion, further advancing the tumor metastasis process.
The above knowledge suggests that compounds capable of entering the active pockets of interleukin-1 alpha and cyclooxygenase-2 would have the ability to inhibit macrophage recruitment, and thus tumor cell 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 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser. By utilizing a molecular docking technology, the inventor docks the compound with interleukin-1 alpha and cyclooxygenase-2. It was found that 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Ser could enter the active pockets of interleukin-1 α and cyclooxygenase-2 very well (the molecular docking scheme is omitted here). These theoretical studies led the inventors to recognize that 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Ser inhibits tumor cell migration and invasion and thus cancer metastasis. Based on this knowledge, the inventors have completed the following experimental studies.
Disclosure of Invention
The first aspect of the present invention is to confirm that 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Ser of the following formula is a novel compound.
The second content of the invention is that 1- (1H-imidazole-2-group) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser is prepared by the following steps:
1) Performing Pictet-Spengler condensation on L-Trp-OBzl and imidazole-2-formaldehyde under the catalysis of trifluoroacetic acid to obtain 3S-1- (1H-imidazole-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;
2) Oxidizing 3S-1- (1H-imidazol-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester with 2, 3-dichloro-5, 6-dicyan-p-benzoquinone in tetrahydrofuran to obtain 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid benzyl ester;
3) Carrying out catalytic hydrogen decomposition on 1- (1H-imidazole-2-yl) -beta-carboline-3-benzyl carboxylate in tetrahydrofuran by using Pd/C to obtain 1- (1H-imidazole-2-yl) -beta-carboline-3-carboxylic acid;
4) Preparation of Arg (NO) by conventional methods of polypeptide Synthesis 2 )-Gly-Asp(OBzl)-Ser-OBzl；
5) 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid with Arg (NO) 2 ) -Gly-Asp (OBzl) -Ser-OBzl coupling preparation of 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Ser-OBzl；
6) Removing 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 ) Preparation of protecting group of (E) -Gly-Asp (OBzl) -Ser-OBzl 1- (1H-imidazole-2-yl) -beta-carboline-3-methylacyl-Arg-Gly-Asp-Ser.
The third content of the invention is that 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser is proved not to be a cytotoxic compound by adopting an MTT method.
The fourth content of the invention is that 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser is evaluated by adopting a Transwell chamber model to inhibit the metastasis and invasion of the anti-tumor cells.
The fifth content of the invention is to evaluate the lung metastasis effect of 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser in the tumor inhibition of the Lewis lung cancer-bearing mice.
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FIG. 1.1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Ser synthesis route i) CF 3 COOH,CH 2 Cl 2 (ii) a ii) dichlorodicyanobenzoquinone, tetrahydrofuran; iii) H 2 Pd/C, tetrahydrofuran; iv) 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N-methylmorpholine, N, N-dimethylformamide; v) trifluoroacetic acid, trifluoromethanesulfonic acid, diethyl ether; vi) dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; vii) CH 3 OH, naOH (2N); viii) hydrogen chloride in ethyl acetate (4M).
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 (S) -1- (1H-imidazol-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester (1)
3g (10.0 mmol) of L-tryptophan benzyl ester are dissolved in 100mL of dichloromethane at 0 ℃ with stirring, and 2mL of trifluoroacetic acid are added and reacted for 30 minutes. Then, 1.18g (12.0 mmol) of imidazole-2-carbaldehyde was added thereto, and the reaction was carried out at room temperature for 12 hours. After which TLC (giving developing solution) showed disappearance of L-tryptophan benzyl ester. The remaining trifluoroacetic acid is removed by concentration under reduced pressure, the residue is taken up in 100mL of dichloromethane and successively saturated NaHCO 3 The solution (20 mL. Times.3) and a saturated NaCl solution (20 mL. Times.3) were washed, and the dichloromethane layer was washed with anhydrous Na 2 SO 4 Drying for 12 hours, filtering, and concentrating the filtrate under reduced pressure to dryness to give 3g of a colorless solid. The solid was purified by silica gel column chromatography (DCM: meOH, 20. 1 H NMR(800MHz,DMSO-d 6 )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)。
EXAMPLE 2 preparation of benzyl 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylate (2)
4.5g (12.0 mmol) of benzyl (S) -1- (1H-imidazol-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate was dissolved in 150mL of tetrahydrofuran at 0 ℃ with stirring, 5.7g (25.0 mmol) of dichlorodicyanobenzoquinone was added, and TLC (dichloromethane: methanol, 20, 1) showed disappearance of benzyl (S) -1- (1H-imidazol-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (1) after 48 hours reaction at room temperature. Concentrated under reduced pressure, the residue is taken up in 150mL of ethyl acetate and, in turn, saturated NaHCO 3 An aqueous wash (20 mL. Times.3) and a saturated aqueous NaCl wash (20 mL. Times.3). Combining ethyl acetate layers, adding anhydrous Na 2 SO 4 Drying, filtering and concentrating the filtrate under reduced pressure to dryness gave 4.2g of a colourless solid. The solid was purified by silica gel column chromatography (petroleum ether: ethyl acetate, 1.5, 1) to give 2.4g (53%) of the title compound as a colorless powder. ESI/MS (m/e) 391[ 2 ], [ M ] +Na] + ； 1 H NMR(300MHz,DMSO-d 6 )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)。
EXAMPLE 3 preparation of 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid (3)
1.5g (4.0 mmol) of benzyl 1- (1H-imidazol-2-yl) - β -carboline-3-carboxylate (2) was dissolved in 50mL of tetrahydrofuran with stirring, 200mg of palladium on carbon was added and hydrogen was introduced to carry out a reaction. TLC (dichloromethane: methanol, 20: 1) after 24H reaction at room temperature showed 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 1.0g (70%) of the title compound as a yellow powder. ESI/MS (m/e): 279[ M ] +H] + ； 1 H NMR(300MHz,DMSO-d 6 )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)。
EXAMPLE 4 preparation of Boc-Arg (NO) 2 )-Gly-OBzl
3.19g (10.0 mmol) of Boc-Arg (NO) were added at 0 ℃ with stirring 2 ) Dissolved in 100mL of anhydrous tetrahydrofuran, and then added with 1.49g (11.0 mmol) of 1-hydroxybenzotriazole and 2.47g (11.0 mmol) of dicyclohexylcarbodiimide to react for 30 minutes. Thereafter, 2.22g (11.0 mmol) Gly-OBzl were 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. Filtering, and concentrating the filtrate under reduced pressure. The residue was dissolved in 50mL ethyl acetate and the resulting solution was sequentially saturated NaHCO 3 Aqueous solution washing (30 mL. Times.3), saturated aqueous NaCl washing (20 mL. Times.3), 5% 4 Aqueous solution (20 mL. Times.3), saturated aqueous NaCl solution (20 mL. Times.3), saturated aqueous NaHCO solution 3 Aqueous wash (20 mL. Times.3) and saturated aqueous NaCl wash (20 mL. Times.3). Anhydrous Na for ethyl acetate layer 2 SO 4 After drying for 12 hours, filtration and concentration of the filtrate under reduced pressure, the residue was dissolved in 30mL of dichloromethane and left to stand for 6 hours to precipitate a solid sufficiently, which was filtered to give 4.45g (96%) of the title compound as a colorless powder. ESI-MS (m/e) 467[ sic ] M + H] + ； 1 H NMR(300MHz,DMSO-d 6 )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)。
EXAMPLE 5 preparation of Boc-Arg (NO) 2 )-Gly
2.33g (5.00 mmol) of Boc-Arg (NO) was added at 0 ℃ with stirring 2 ) -Gly-OBzl was dissolved in 45mL of methanol, the reaction solution was adjusted to pH 12 with aqueous sodium hydroxide (2M), and stirred until TLC (dichloromethane: methanol, 20) showed Boc-Arg (NO 2 ) -Gly-OBzl disappeared completely. The reaction solution is saturated KHSO at 0 deg.C 4 Adjusting pH of the aqueous solution to 7, concentrating under reduced pressure, adding 15mL distilled water to the residue, and adding saturated KHSO 4 The pH of the aqueous solution was adjusted to 2, the aqueous layer was extracted with ethyl acetate (20 mL. Times.3), the ester layer was washed with saturated aqueous sodium chloride (20 mL. Times.3), and the ethyl acetate layer was washed with anhydrous Na 2 SO 4 Drying for 12 hr, filtering, and vacuum concentrating the filtrateConcentration gave 1.7g (90%) of the title compound as a colorless solid powder. ESI-MS (m/e) 377[ 2 ], [ M ] +H] + 。
EXAMPLE 6 preparation of Boc-Asp (OBzl) -Ser-OBzl
5.61g (93%) of the title compound were obtained as a yellow oil from 3.23g (10.0 mmol) of Boc-Asp (OBzl) and 2.54g (11.0 mmol) of Ser-OBzl by the method of example 4. ESI-MS (m/e): 518[ M ] +H] + 。
EXAMPLE 7 preparation of Asp (OBzl) -Ser-OBzl
1.00g (2.0 mmol) of Boc-Asp (OBzl) -Ser-OBzl are dissolved in 20mL of a solution of hydrogen chloride in ethyl acetate (4M) at 0 ℃ with stirring and stirred for 1 hour. TLC (dichloromethane: methanol, 20. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 15mL of anhydrous ethyl acetate. The solution was again concentrated under reduced pressure, and the residue was sonicated with 10mL of dry ether to thoroughly suspend, and the supernatant was discarded to give 0.85g (97%) of the title compound as a pale yellow solid. ESI-MS (m/e): 418[ M ] +H] + 。
EXAMPLE 8 preparation of Boc-Arg (NO) 2 )-Gly-Asp(OBzl)-Ser-OBzl
Starting from 0.75g (2.0 mmol) of Boc-Arg (NO) using the method of example 4 2 ) Gly and 0.96g (2.2 mmol) Asp (OBzl) -Ser-OBzl gave 0.75g (61%) of the title compound as a colorless solid. ESI-MS (m/e) 759[ m ] +H] + ； 1 HNMR(300MHz,DMSO-d 6 )δ8.47(s,1H),8.26(s,2H),8.04(s,2H),7.34(d,J＝11.5Hz,8H),6.97(s,1H),5.08(s,4H),5.04(s,1H),4.77(s,1H),4.42–4.29(m,1H),3.93(s,1H),3.70(s,4H),3.10(s,2H),2.75(s,1H),1.50(s,2H),1.36(d,J＝9.2Hz,9H)。
EXAMPLE 9 preparation of Arg (NO) 2 )-Gly-Asp(OBzl)-Ser-OBzl
Using the method of example 7 from 0.4g (0.5 mmol) Boc-Arg (NO) 2 ) -Gly-Asp (OBzl) -Ser-OBzl gives 0.33g (89%) of the title compound as a colorless solid. ESI-MS (m/e): 659[ 2 ] M + H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ8.79(t,J＝5.6Hz,1H),8.57(s,1H),8.42(dd,J＝19.0,7.9Hz,2H),8.25(s,2H),7.96(s,2H),7.45–7.22(m,8H),5.20–4.99(m,4H),4.81(td,J＝8.5,5.1Hz,1H),4.37(dt,J＝7.5,4.7Hz,1H),3.96–3.60(m,4H),2.78(dd,J＝16.3,5.1Hz,1H),2.58(dd,J＝16.2,8.8Hz,1H),1.74(d,J＝9.1Hz,2H),1.57(s,2H)。
EXAMPLE 10 preparation of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Ser-OBzl(4)
200mg (0.7 mmol) of 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid was dissolved in N, N-dimethylformamide (20mL) at 0 deg.C under stirring, and 550mg (0.8 mmol) of Arg (NO) was sequentially added 2 ) -Gly-Asp (OBzl) -Ser-OBzl and 280mg (0.8 mmol) of 2- (7-benzotriazole oxide) -, N, N ', N' -tetramethylurea hexafluorophosphate, adjusting the pH of the reaction solution to 9 by using nitromethylmorpholine, reacting at room temperature for 12 hours, and concentrating under reduced pressure. The residue was dissolved in ethyl acetate and the resulting solution was successively saturated with NaHCO 3 An aqueous wash (20 mL. Times.3) and a saturated aqueous NaCl wash (20 mL. Times.3). The combined ethyl acetate layers were washed with anhydrous Na 2 SO 4 Drying, filtration and concentration of the filtrate under reduced pressure gave 400mg of a colourless solid. The solid was purified by silica gel column chromatography to give 276mg (43%) of the title compound as a colorless powder. 1 H NMR(300MHz,DMSO-d 6 )δ13.40(s,1H),11.83(s,1H),9.24(d,J＝8.2Hz,1H),8.82(d,J＝8.4Hz,1H),8.59(s,1H),8.43–8.23(m,4H),8.05(s,2H),7.96(d,J＝8.5Hz,1H),7.60(s,2H),7.42–7.23(m,11H),5.18–4.91(m,4H),4.82(t,J＝7.1Hz,1H),4.67(q,J＝7.8Hz,1H),4.38(q,J＝5.5Hz,1H),3.91–3.59(m,3H),3.25(s,2H),2.88–2.62(dd,J＝16.5,8.7Hz,2H),2.08–1.88(d,J＝11.9Hz,2H),1.65(s,2H)。
EXAMPLE 11 preparation of 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid-formyl-Arg-Gly-Asp-Ser (5)
100mg (0.11 mmol) of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg (NO) is added under stirring at 0 DEG C 2 ) -Gly-Asp (OBzl) -Ser-OBzl (4) was dissolved in 1mL trifluoroacetic acid and 0.3mL trifluoromethanesulfonic acid, stirred for 0.5 h, and the reaction mixture was concentrated under reduced pressure. 10mL of anhydrous ether was added to the residue, and the mixture was stirred for 10 minutes to precipitate a solid, and the mixture was allowed to stand for 10 minutes, and then the supernatant was discarded. This operation was repeated 3 times. The collected solid was dissolved in 50% water and 50% methanol, adjusted to pH 8 with 10% ammonia water, purified on a C18 column eluting with 40% water and 60% methanol and the eluent lyophilized to give 23mg (30%) of the title compound as a yellow solid. FT-ESI-MS (m/e): 694.27880[ M ] +H] + ； 1 H NMR(300MHz,DMSO-d 6 )δ13.61(s,1H),11.83(s,1H),9.74(s,1H),9.26(d,J＝8.6Hz,1H),8.81(s,1H),8.63(dd,J＝25.5,7.0Hz,2H),8.39(d,J＝7.9Hz,1H),7.96(d,J＝8.3Hz,1H),7.58(q,J＝7.9Hz,2H),7.46(d,J＝6.9Hz,1H),7.32(dd,J＝15.4,7.8Hz,2H),7.11(s,2H),4.78–4.63(m,1H),4.55–4.42(m,1H),4.10–3.77(m,2H),3.77–3.53(m,2H),3.27(dd,J＝12.9,6.2Hz,1H),3.16–3.04(m,2H),2.66–2.44(dd,J＝16.1,5.2Hz,2H),2.26(s,2H),1.90(d,J＝10.6Hz,2H),1.63(s,2H)。
EXAMPLE 12 evaluation of the cytotoxic Effect of Compound 5
The results are shown in Table 1. The results show that the compound 5 in the compound has no proliferation inhibiting effect on six tumor cells and normal liver cells L02. It can be seen that compound 5 of the present invention is not a cytotoxic compound.
TABLE 1 IC for Compound 5 inhibition of tumor cell proliferation 50
n＝6
EXAMPLE 13 evaluation of the Activity of Compound 5 to inhibit tumor cell migration
LLC and 95D belong to anchorage dependent cells and are in monolayer culture. And taking out the cell culture bottle from the incubator, and observing the cell state by using a microscope to meet the conditions. Cells were washed, digested, dispersed and counted. Thereafter, the cell concentration was diluted to 5X 10 5 one/mL. The 95D cells were dispersed and diluted by pipetting in serum-free medium 1640. LLC cells were dispersed and diluted in serum-free medium (DMEM). In the chamber first 100 u L cell suspension and then 25 u L compound 5 aqueous solution to a concentration of 20M. Each plate was equipped with a positive control RGDS and a negative control serum-free medium, and the control and compounds 3 and 5 were equipped with two secondary wells. After the addition of the test compound and the control solution, the solution in the upper chamber of the chamber was mixed by gently tapping the walls of the 24-well plate, and 600. Mu.L of a medium rich in 10% fetal calf serum was added to the lower chamber as soon as possible to remove the air bubbles under the membrane. Standing at 37 deg.C, 5% CO 2 After culturing each cell line under the conditions of (1) for a desired time, the post-treatment is carried out. The post-treatment method was carried out by carefully aspirating the supernatant in the upper chamber with a pipette, adding 100. Mu.L of PBS buffer to each upper chamber, carefully wiping the cells in the upper chamber with a cotton swab, and repeating this operation twice so that no cells remained on the upper membrane side (preventing the membrane of the Transwell chamber from being damaged). The lower chamber medium was aspirated, 600. Mu.L of 4% tissue fixative was added, cells on the lower side of the upper chamber membrane were fixed in a refrigerator at 4 ℃ for 1 hour, the residual liquid in the lower chamber was aspirated, 600. Mu.L of crystal violet stain was added to each lower chamber, and staining was carried out for 30 minutes. The staining solution was recovered by blotting, and the residual crystal violet staining solution on the polycarbonate film was rinsed with triple distilled water and photographed under an inverted microscope. Nine different visual fields are selected for each chamber, the angle and the magnification are fixed, cells in the visual fields are uniformly distributed, and the cell visual fields at the edges of the chambers are prevented from being selected for counting. The number of cells in the image is counted by image J, and is expressed as mean + -SD, and the statistical difference is found when P < 0.05 is tested by t. The results are shown in tables 2 and 3. The results show that compounds 3 and 5 of the invention do not affect 95D and LLC cell migration at a concentration of 20 μ M.
TABLE 2 Effect of Compound 5 on 95D cell migration
| Compound (I) | Concentration (μ M) | Mean value of + -SD |
| 1640 | - | 164±37 |
| RGDS | 20 | 114±12 a |
| |
20 | 181±39 b |
| |
20 | 170±16 b |
a) P is less than 0.01 compared with serum-free medium; b) P is greater than 0.05 compared to serum-free medium; n =9.
TABLE 3 Effect of Compound 5 on LLC cell migration
| Compound (I) | Concentration (μ M) | Mean value of + -SD |
| DMEM | - | 242±63 |
| RGDS | 20 | 125±44 a |
| |
20 | 245±66 b |
| |
20 | 248±27 b |
a) P is less than 0.01 compared with serum-free medium; b) P is greater than 0.05 compared to serum-free medium; n =9.
EXAMPLE 14 evaluation of the Activity of Compound 5 for inhibiting tumor cell invasion
The matrigel was taken out of the-20 ℃ freezer and placed in the 4 ℃ freezer overnight to completely convert it from a solid state to a liquid state, and then diluted five times with serum-free medium and mixed well. 100. Mu.L of the prepared matrigel dilution was pipetted into the upper chamber of each well of the Transwell chamber. Placing the Transwell cell at 37 ℃,5% CO 2 Is incubated for 5 hours.
Taking out the incubated Transwell chamber, aspirating the liquid portion of the Transwell chamber, adding 50. Mu.L of serum-free medium to the upper chamber of each well of the Transwell chamber, adding the medium at 37 ℃, and 5% 2 After 30 minutes of incubation in the incubator, the Transwell chamber is sucked outOf (2) is used.
LLC is anchorage-dependent cell, and is in monolayer culture. And taking out the cell culture flask from the incubator, and observing the cell state by using a microscope to meet the conditions. Cells were washed, digested, dispersed and counted. Thereafter, the cell concentration was diluted to 5X 10 5 one/mL. The dispersion and dilution were performed by pipetting using a serum-free medium (DMEM). In the chamber first 100 u L cell suspension and then 25 u L compound 3 or 5 aqueous solution to the concentration of 20M. Each plate was equipped with positive and negative controls, and both controls and compound 3 or 5 were equipped with two secondary wells. After adding compound 3 or 5 and the control solution, gently patting the walls of the 24-well plate to mix the solution in the upper chamber, adding 600. Mu.L of 10% fetal bovine serum-rich medium to the lower chamber as soon as possible, and removing the air bubbles under the membrane. Standing at 37 deg.C, 5% CO 2 After culturing each cell line under the conditions of (1) for a desired time, post-treatment is carried out.
The post-treatment was carried out by carefully aspirating the supernatant from the upper chamber with a pipette, adding 100. Mu.L of PBS buffer to each upper chamber, carefully wiping the cells from the upper chamber with a cotton swab, and repeating this operation twice to leave no cells on the upper membrane side (to prevent damage to the Transwell chamber membrane). The lower chamber culture medium was aspirated, 600. Mu.L of 4% tissue fixative was added, cells on the lower side of the upper chamber membrane were fixed in a refrigerator at 4 ℃ for 1 hour, the residual liquid in the lower chamber was aspirated, 600. Mu.L of crystal violet stain was added to each lower chamber, and staining was performed for 30 minutes. Absorbing and recovering the staining solution, rinsing the residual crystal violet staining solution on the polycarbonate membrane by using triple distilled water, taking a picture under an inverted microscope, selecting nine different visual fields for each cell, fixing the angle and the magnification, uniformly distributing cells in the visual fields, and avoiding counting the visual fields of the cells at the edges of the selected cells. The number of cells in the photographs was counted using image J, and expressed as the mean. + -. SD, and there was a statistical difference when P < 0.05 by t-test. The results are shown in Table 4. From the results, it can be seen that compound 5 of the present invention is effective in inhibiting tumor cell invasion.
TABLE 4 Effect of Compound 5 on LLC cell invasion
| Compound (I) | Concentration (μ M) | Mean number of. + -. SD |
| DMEM | - | 410±89 |
| RGDS | 20 | 152±46 |
| |
20 | 287±76 a |
| |
20 | 183±33 b |
a) P <0.01 to DMEM; b) P is less than 0.01 to DMEM and compound 3 and p is more than 0.05 to RGDS; n =9
EXAMPLE 15 evaluation of the Activity of Compound 5 to inhibit tumor Lung metastasis
1) The positive control is RGDS tetrapeptide, the intraperitoneal injection dose is 20 mu mol/kg/day, and the administration is continuously carried out for 10 days; the blank control group is normal saline, and the oral dosage is 0.1mL/10 g/day; the oral dose of compound 3 was 1 μmol/kg/day; the oral dose of compound 5 was 1,0.1 and 0.01. Mu. Mol/kg/day for 10 consecutive days.
2) Lewis lung carcinoma cells (LLC) were purchased from ATCC. The LLC is subcultured in DMEM complete medium containing 10% fetal calf serum to obtain cells in logarithmic growth phase. After washing, digesting, dispersing and counting the cells, the cells were washed with PBSDiluting to 2 × 10 7 Cell suspension per mL. The prepared cell suspension was inoculated into the axilla of C57BL/6N male mice, each at 0.2mL. When the solid tumor of the tumor-bearing mouse grows to the diameter of 1.5cm-2cm, the solid tumor is taken as a tumor source for standby.
3) Tumor-bearing mice as a tumor source were anesthetized with ether and sacrificed rapidly by dislocation of the cervical vertebrae. Grinding the solid tumor into cell suspension by using a tissue homogenizer, and sieving the cell suspension by using a 200-mesh cell sieve to obtain the single cell suspension. The single cell suspensions were pooled, washed and counted and diluted to 2X 10 with PBS buffer 7 Cell suspension per mL. The prepared cell suspension was inoculated into the axilla of C57BL/6N male mice, each at 0.2mL. Observing and measuring solid tumors of tumor-bearing mice every day from inoculation on the day, and administrating when the solid tumors grow to the size of soybean grains, and regrouping according to a completely random principle before administration. The body weight of the mice was monitored before daily administration and the tumor volume was measured using a vernier caliper, and the corresponding dose of compound 3 or 5 was administered according to the body weight for 10 consecutive days. On day 11 the mice were anesthetized with ether, decapped, sacrificed, implanted tumors were blunt dissected with surgical instruments, rapidly weighed and statistically analyzed for tumor weight in each group of mice. The results showed no significant difference in the weight of implanted tumors in saline, RGDS, compound 3 and compound 5 treated mice (data omitted here). RGDS, compound 3 and compound 5 were found to have no inhibitory effect on in situ tumor growth. The lungs were removed blunt with surgical instruments, photographed rapidly and the metastatic nodules counted. The results are shown in Table 5. From the results, it can be seen that compound 5 of the present invention is effective in inhibiting tumor metastasis to the lung.
TABLE 5 Compound 5 inhibitory Activity on tumor Lung metastasis
a) P <0.01 to saline and compound 3; b) P is less than 0.01 to normal saline and compound 3, and p is more than 0.05 to RGDS; n =10.
Claims (3)
1. 1-imidazole-beta-carboline-3-formyl-Arg-Gly-Asp-Ser with the following structure,
2. the method of claim 1, wherein the step of preparing 1-imidazole- β -carboline-3-formyl-Arg-Gly-Asp-Ser comprises the steps of:
2.1. performing Pictet-Spengler condensation on L-Trp-OBzl and imidazole-2-formaldehyde under the catalysis of trifluoroacetic acid to obtain 3S-1-imidazole-2, 3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;
2.2.3S-1-imidazole-2, 3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester is oxidized by 2, 3-dichloro-5, 6-dicyan p-benzoquinone in tetrahydrofuran to obtain 1-imidazole-beta-carboline-3-carboxylic acid benzyl ester;
2.3. carrying out Pd/C catalytic hydrogen decomposition on 1-imidazole-beta-carboline-3-carboxylic acid benzyl ester in tetrahydrofuran to obtain 1-imidazole-beta-carboline-3-carboxylic acid;
2.4. preparation of Arg (NO) by conventional methods of polypeptide Synthesis 2 )-Gly-Asp(OBzl)-Ser-OBzl；
2.5.1-imidazole-beta-carboline-3-carboxylic acid with Arg (NO) 2 ) Coupling of-Gly-Asp (OBzl) -Ser-OBzl to prepare 1-imidazole-beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Ser-OBzl；
2.6. Removing 1-imidazole-beta-carboline-3-formyl-Arg (NO) 2 ) Preparing 1-imidazole-beta-carboline-3-formyl-Arg-Gly-Asp-Ser from a protecting group of-Gly-Asp (OBzl) -Ser-OBzl.
3. The use of the 1-imidazole-2- β -carboline-3-formyl-Arg-Gly-Asp-Ser of claim 1 for the preparation of a medicament for the treatment of tumor metastasis.
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