CN115466316A - Migration-inhibiting 1-imidazole-beta-carboline-3-formyl-RGDF, preparation, anti-cancer metastasis effect and application - Google Patents
Migration-inhibiting 1-imidazole-beta-carboline-3-formyl-RGDF, preparation, anti-cancer metastasis effect and application Download PDFInfo
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Abstract
1-imidazole-beta-carboline-3-formyl-RGDF for inhibiting migration, preparation, anti-cancer metastasis and application thereof. The invention discloses 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the following formula, discloses a preparation method thereof, discloses the activity of inhibiting tumor cell migration, and discloses the activity of inhibiting Lewis mouse tumor lung metastasis. Therefore, the invention discloses the application of the compound in preparing the anti-tumor metastasis medicaments.
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Description
Technical Field
The invention relates to 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe, a preparation method thereof, activity for inhibiting tumor cell migration and activity for inhibiting Lewis mouse tumor metastasis to lung. Therefore, the invention relates to the application of the compound in preparing the anti-tumor metastasis medicaments. The invention belongs to the field of biological medicine
Background
Cancer is currently the second leading cause of death worldwide. The majority of the cancer deaths die from tumor metastases. In addition, at present, a safe and effective anti-tumor metastasis medicament is still lacking clinically. 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 will have the ability to inhibit macrophage recruitment and thus 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-1 alpha and cyclooxygenase-2, the inventors designed 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe. 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-Phe enters the active pockets of interleukin-1 α and cyclooxygenase-2 well (the molecular docking diagram is omitted here). These theoretical studies led the inventors to recognize that 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Phe 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 invention provides a brand new compound 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the following structure,
the second content of the invention provides a method for preparing 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the structure as shown in the formula:
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 in tetrahydrofuran with 2, 3-dichloro-5, 6-dicyan-p-benzoquinone 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)-Phe-OBzl；
5) 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid with Arg (NO) 2 ) -Gly-Asp (OBzl) -Phe-OBzl coupling preparation of 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl；
6) Removing 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl protecting group to prepare 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe.
The third content of the invention is that the MTT method is adopted to confirm that the 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the structure of the formula is not a cytotoxic compound.
The fourth content of the invention is that the Transwell chamber model is adopted to evaluate the inhibition effect of the 1- (1H-imidazole-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the structure on the migration and invasion of tumor cells.
The fifth aspect of the present invention is to evaluate the effect of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the structure in inhibiting tumor metastasis to lung.
Drawings
FIG. 1.1 is a synthetic route for 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Phe, i) trifluoroacetic acid, dichloromethane; ii) dichlorodicyanobenzoquinone, tetrahydrofuran; iii) Hydrogen, palladium on carbon, 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 and should not be taken as limiting the invention.
EXAMPLE 1 preparation of benzyl (S) -1- (1H-imidazol-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (1)
3g (10.0 mmol) of L-tryptophan benzyl ester are dissolved in 100mL of dichloromethane at 0 ℃ with stirring, and then 2mL of trifluoroacetic acid are added thereto to react for 30 minutes. Then, 1.18g (12.0 mmol) of imidazole-2-carbaldehyde was added thereto, and the mixture was reacted 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 Solution (20 mL. Times.3) and saturated NaCl solution(20 mL. Times.3) 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 Aqueous wash (20 mL. Times.3) and 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). ESI/MS (m/e): 391[ M ] +Na] + ； 1 H NMR(300 MHz,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 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid benzyl ester (2) was dissolved in 50mL of tetrahydrofuran with stirring, 200mg of palladium on carbon was added and hydrogen was introduced to react. 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 )δ 13.28(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) was 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 (1.0 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 is adjusted to 9 by N-methylmorpholine, and the obtained reaction solution is stirred for 10 hours at room temperature. 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 (30 mL. Times.3) and saturated aqueous NaCl solution (20 mL. Times.3), 5% KHSO 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, allowed to stand for 6 hours to precipitate a solid sufficiently, and 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 methanol, the reaction solution pH was adjusted to 12 with aqueous sodium hydroxide (2M), stirred to TLC (dichloromethane: methanol =20 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 Adjusting pH to 2, extracting water layer with ethyl acetate (20 mL × 3), washing ester layer with saturated sodium chloride aqueous solution (20 mL × 3), and adding ethyl acetateAnhydrous Na for layer 2 SO 4 Drying for 12 hours, filtration and concentration of the filtrate under reduced pressure 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) -Phe-OBzl
6.1g (90.8%) of the title compound are obtained as a colorless solid by the method of example 4 from 3.23g (10.0 mmol) of Boc-Asp (OBzl) and 3.20g (11.0 mmol) of Phe-OBzl. ESI-MS (m/e): 561[ 2 ] M + H] + 。
EXAMPLE 7 preparation of Asp (OBzl) -Phe-OBzl
1.12g (2.0 mmol) of Boc-Asp (OBzl) -Phe-OBzl are dissolved with stirring in 20mL of hydrogen chloride in ethyl acetate (4M) and stirred for 1 hour at 0 ℃. TLC (dichloromethane: methanol = 20) showed disappearance of Boc-Asp (OBzl) -Phe-OBzl. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 15mL of anhydrous ethyl acetate. The solution was concentrated under reduced pressure, and the residue was suspended thoroughly by sonication in 10mL of anhydrous ether, and the supernatant was discarded to give 0.97g (97.5%) of the title compound as a colorless solid. ESI-MS (m/e) 461[ m ] +H ]] + 。
EXAMPLE 8 preparation of Boc-Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl
Using the method of example 4 from 0.75g (2.0 mmol) Boc-Arg (NO) 2 ) -Gly and 1.10g (2.2 mmol) Asp (OBzl) -Phe-OBzl gave 1.30g (80%) of the title compound as a colorless solid. ESI-MS (m/e): 819 [ M + H ]] + ； 1 H NMR(300MHz,DMSO-d 6 )δ8.48(s,1H),8.39(d,J＝7.5Hz,1H),8.21(d,J＝ 8.3Hz,1H),8.02(t,J＝5.3Hz,1H),7.45–7.27(m,7H),7.36–7.12(m,7H),6.99(d,J＝7.7 Hz,1H),5.17–4.96(m,4H),4.72(td,J＝8.5,5.1Hz,1H),4.50(td,J＝8.0,6.4Hz,1H),3.95 (d,J＝6.1Hz,1H),3.69(d,J＝5.3Hz,2H),3.18–2.88(m,4H),2.72(dd,J＝16.2,5.2Hz, 1H),2.51(d,J＝25.0Hz,0H),1.66(s,1H),1.56–1.45(m,4H),1.37(s,9H)。
EXAMPLE 9 preparation of Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl
Starting from 0.41g (0.5 mmol) of Boc-Arg (NO) by the method of example 7 2 ) -Gly-Asp (OBzl) -Phe-OBzl gave 0.34g (91%) of the title compound as a colorless solid. ESI-MS (m/e): 719[ 2 ] M + H] + ； 1 H NMR(300MHz, DMSO-d 6 )δ8.76(t,J＝5.6Hz,1H),8.51(d,J＝7.5Hz,1H),8.40(d,J＝8.2Hz,1H),8.26(s, 3H),7.98(s,2H),7.46–7.14(m,13H),5.17–4.96(m,4H),4.73(td,J＝8.6,5.0Hz,1H), 4.49(q,J＝7.6Hz,1H),3.90–3.67(m,3H),3.00(qd,J＝13.8,7.3Hz,2H),2.72(dd,J＝16.2, 5.0Hz,1H),2.56(d,J＝9.1Hz,1H),1.73(t,J＝7.9Hz,2H),1.57(s,2H)。
EXAMPLE 10 preparation of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl(4)
Dissolving 200mg (0.7 mmol) of 1- (1H-imidazol-2-yl) -beta-carboline-3-carboxylic acid in 20mL of N, N-dimethylformamide at 0 ℃ under stirring, and sequentially adding 600mg (0.8 mmol) of Arg (NO) 2 ) -Gly-Asp (OBzl) -Phe-OBzl and 280mg (0.8 mmol) of 2- (7-benzotriazole oxide) -, N, N ', N' -tetramethylurea hexafluorophosphate, the pH of the reaction solution was adjusted to 9 with nitromethylmorpholine, and the reaction solution was concentrated under reduced pressure after 12 hours at room temperature. 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 475mg (43%) of the title compound as a colorless powder. 1 H NMR(300MHz,DMSO-d 6 )δ13.39(s,1H), 11.83(s,1H),9.25(d,J＝8.1Hz,1H),8.82(s,1H),8.58(s,1H),8.37(dd,J＝11.2,7.5Hz, 3H),8.26(d,J＝9.1Hz,1H),7.97(d,J＝8.3Hz,2H),7.82(s,1H),7.61(d,J＝6.4Hz,2H), 7.43–7.09(m,18H),5.12–4.83(m,4H),4.73(ddd,J＝22.9,11.6,6.1Hz,2H),4.49(q,J＝ 7.3Hz,1H),3.77(qd,J＝16.9,5.4Hz,2H),3.33–3.19(m,2H),3.06–2.89(m,2H),2.76(dd, J＝16.2,5.1Hz,1H),2.02–1.83(m,2H),1.77–1.56(m,2H)。
EXAMPLE 11 preparation of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg-Gly-Asp-Phe (5)
100mg (0.11 mmol) of 1- (1H-imidazol-2-yl) -beta-carboline-3-formyl-Arg (NO) at 0 ℃ with stirring 2 ) -Gly-Asp (OBzl) -Phe-OBzl (4) was dissolved with 1mL trifluoroacetic acid and 0.3mL trifluoromethanesulfonic acid, stirred for 0.5 h, and the reaction mixture was concentrated under reduced pressure. Adding 10mL of anhydrous ether into the residue, stirring for 10 min, separating out a solid, standing for 10 minThe 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, purified on a C18 column eluting with 40% water and 60% methanol and the eluent was lyophilized to give 34mg (41%) of the title compound as a yellow solid. FT-ESI-MS (m/e) 754.31692[ 2 ] M + H] + ； 1 H NMR(300MHz, DMSO-d 6 )δ14.27(s,1H),11.82(s,1H),10.22(s,1H),9.44(d,J＝9.0Hz,1H),8.89(d,J＝ 8.2Hz,1H),8.80(s,1H),8.65(s,1H),8.38(d,J＝7.9Hz,1H),7.96(d,J＝8.3Hz,1H),7.59 (ddd,J＝8.3,7.0,1.2Hz,1H),7.46(s,1H),7.38–7.23(m,3H),7.16(q,J＝7.3Hz,4H),7.08 (td,J＝6.4,5.7,2.4Hz,1H),4.75(td,J＝9.2,4.3Hz,1H),4.33(dt,J＝9.1,4.9Hz,1H),4.09 (d,J＝6.4Hz,1H),3.86(d,J＝15.9Hz,1H),3.64(dd,J＝16.3,5.3Hz,1H),3.22(s,1H),3.01 (dd,J＝13.5,6.5Hz,2H),2.88(dd,J＝13.2,5.1Hz,1H),2.60(dd,J＝16.0,4.6Hz,1H),2.34 (s,2H),2.00(d,J＝8.2Hz,1H),1.62(s,2H)。
EXAMPLE 12 evaluation of the cytotoxic Effect of Compound 5
1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Phe (Compound 5) was prepared to the desired concentration in 1640 medium containing 0.5% DMSO. Respectively subjecting the HCT-116, the A549, the S180, the LLC, the HL60 and the U with good growth state in logarithmic growth phase to 2 OS,L0 2 Cells were as per 4X 10 4 The density of individual/mL was seeded in 96-well plates at 100. Mu.L per well. At 37 ℃,5% CO 2 After 4 hours of incubation in an incubator, a sterilized compound 5 solution was added at a predetermined concentration gradient of 100. Mu.M, 50. Mu.M, 25. Mu.M, 10. Mu.M and 1. Mu.M, and an equal volume of a sample-dissolved 1640 medium containing 0.5% dimethyl sulfoxide was added to the control group. After an additional 48 hours of incubation, 25. Mu.L of 5mg/mL MTT solution was added to each well, incubated at 37 ℃ for 4 hours, carefully removed the supernatant and 100. Mu.L DMSO was added to each well and the pellet was dissolved by shaking for about 15 min. OD (absorbance) values were immediately measured at a wavelength of 570nm on a microplate reader. In terms of inhibition = [ (OD mean value of 1640 medium group containing 0.5% dimethyl sulfoxide-OD mean value of compound)/OD value of 1640 medium group containing 0.5% dimethyl sulfoxide]X 100% the inhibition was calculated. The experiment was repeated 6 times in parallel and the inhibition was plotted against compound concentration to calculate the IC of the compounds of the invention 50 (median effective inhibitory concentration) value。
The results are shown in Table 1. From the results, it can be seen that compound 5 in the compound of the present invention has no proliferation inhibiting effect on six tumor cell lines and normal liver cell line 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 tumor cell migration inhibitory Activity of Compound 5
LLC and 95D belong to anchorage dependent cells and are 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 95D cells were dispersed and diluted by pipetting in serum-free medium 1640. The LLC cells are dispersed and diluted by pipetting 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. Absorbing and recovering the dyeing liquid, rinsing the polycarbonate with triple distilled waterThe crystal violet dye solution remained on the ester film and was 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 is less than 0.05 by t test. The results are shown in tables 2 and 3. The results show that compound 3 of the present invention does not affect 95D and LLC cell migration at a concentration of 20 μ M, and that compound 5 of the present invention effectively inhibits 95D and LLC cell migration at a concentration of 20 μ M.
TABLE 2 Effect of Compound 5 on 95D cell migration
a) P is less than 0.01 compared with 1640; b) P >0.05 to 1640; c) P is less than 0.01 compared with 1640, and p is more than 0.05 compared with RGDS; n =9.
TABLE 3 Effect of Compound 5 on LLC cell migration
| Compound (I) | Concentration (μ M) | Number of migrating cells, mean. + -. SD |
| DMEM | - | 242±63 |
| RGDS | 20 | 125±44 a |
| |
20 | 245±66 b |
| |
20 | 102±36 c |
a) P <0.01 to DMEM; b) P >0.05 to DMEM; c) The ratio p to DMEM is less than 0.01, and the ratio p to RGDS is more than 0.05; n =9.
EXAMPLE 14 evaluation of the Activity of Compound 5 in inhibiting tumor cell invasion
The matrigel was removed from the-20 ℃ freezer and placed in a 4 ℃ freezer overnight to allow complete transition from the solid to the liquid state, 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.
Removing 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, and incubating at 37 ℃ for 5% CO 2 After 30 minutes incubation, the liquid in the Transwell chamber was aspirated.
LLC is anchorage-dependent cell, and is 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 dispersion and dilution were carried out by pipetting using 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. Positive and negative controls were set for each plateTwo secondary wells were provided for group, control and compound 3 or 5. After adding compound 3 or 5 and control, gently tap the walls of the 24-well plate to mix the solutions in the upper chamber of the chamber, add 600 μ L of medium enriched with 10% fetal calf serum to the lower chamber as soon as possible and 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 was carried out by carefully aspirating the upper chamber residue with a pipette, adding 100. Mu.L of PBS buffer to each upper chamber, carefully wiping the upper chamber cells with a cotton swab, and repeating this operation twice to leave no cells on the upper chamber membrane (to prevent damage to the Transwell chamber membrane). 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. 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 selecting the cell visual field at the edge of the cell 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 Table 4. From the results, it can be seen that compound 5 in the compound of the present invention has no inhibitory effect on tumor cell invasion.
TABLE 4 Effect of Compound 5 on LLC cell invasion
| Compound (I) | Concentration (μ M) | Number of invading cells, mean. + -. SD |
| DMEM | - | 410±89 |
| RGDS | 20 | 152±46 a |
| |
20 | 387±76 b |
| |
20 | 372±117 b |
a) P <0.01 to DMEM; b) P >0.05 to DMEM; 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 dosage is 20 mu mol/kg/day, and the administration is continuously carried out for 10 days; the blank control group is physiological saline, and the oral dosage is 0.1mL/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.
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. The cells were washed, digested, dispersed, counted and then diluted to a concentration of 2X 10 using PBS 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 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 combined, washed and counted before usePBS buffer diluted to 2X 10 7 Cell suspension per mL. The prepared cell suspension was inoculated into the axilla of C57BL/6N male mice, each 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 days. On day 11 the mice were anesthetized with ether, decapped, implanted tumors were blunt dissected with surgical instruments, rapidly weighed and the tumor weight of each group of mice was statistically analyzed. 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). It can be seen that RGDS, compound 3 and compound 5 have no inhibitory effect on the growth of in situ tumor. The lungs were blunt dissected with surgical instruments, and the metastases were rapidly photographed and 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
| Compound (I) | Dosage (mu mol/kg/day) | The mean value of the number of pulmonary metastasis nodules is +/-SD |
| Physiological saline | - | 37.2±14.0 |
| RGDS | 20 | 9.4±2.0 a |
| |
20 | 35.0±12.0 |
| |
1 | 9.0±5.7 b |
a) P <0.01 to saline and compound 3; b) P <0.01 to saline and compound 3, p >0.05 to RGDS; n =10.
Claims (4)
1. 1-imidazole-beta-carboline-3-formyl-Arg-Gly-Asp-Phe with the following structural formula,
2. a process for the preparation of 1-imidazole- β -carboline-3-formyl-Arg-Gly-Asp-Phe of the formula as claimed in claim 1, comprising 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 catalytic hydrogen decomposition on 1-imidazole-beta-carboline-3-carboxylic acid benzyl ester in tetrahydrofuran by using Pd/C 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)-Phe-OBzl；
2.5.1-imidazole-beta-carboline-3-carboxylic acid with Arg (NO) 2 ) -Gly-Asp (OBzl) -Phe-OBzl coupling preparation of 1-imidazole-beta-carboline-3-formyl-Arg (NO) 2 )-Gly-Asp(OBzl)-Phe-OBzl；
2.6. Removing 1-imidazole-beta-carboline-3-formyl-Arg (NO) 2 ) And (3) preparing 1-imidazole-beta-carboline-3-formyl-Arg-Gly-Asp-Phe from a protecting group of-Gly-Asp (OBzl) -Phe-OBzl.
3. The use of 1-imidazole- β -carboline-3-formyl-Arg-Gly-Asp-Phe of the structural formula of claim 1 in the preparation of a medicament for the treatment of tumor metastasis.
4. Use of 1- (1H-imidazol-2-yl) - β -carboline-3-formyl-Arg-Gly-Asp-Phe of the structural formula of claim 1 for the preparation of a medicament against invasion of tumor cells.
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