CN112300245A - RGDS and theanine co-modified 5-fluorouracil, and synthesis, activity and application thereof - Google Patents

RGDS and theanine co-modified 5-fluorouracil, and synthesis, activity and application thereof Download PDF

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CN112300245A
CN112300245A CN201910695564.4A CN201910695564A CN112300245A CN 112300245 A CN112300245 A CN 112300245A CN 201910695564 A CN201910695564 A CN 201910695564A CN 112300245 A CN112300245 A CN 112300245A
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赵明
彭师奇
康贵峰
任智奇
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Abstract

The invention discloses RGDS and theanine co-modified 5-fluorouracil of the following formula. The invention discloses a preparation method thereof, and discloses anti-tumor and anti-tumor metastasis activities thereof, so that the invention discloses an application of the compound in preparing anti-tumor drugs, an application in preparing anti-tumor metastasis drugs and an application in preparing anti-tumor and anti-tumor metastasis dual-activity drugs.
Figure RE-DDA0002206102530000011

Description

RGDS and theanine co-modified 5-fluorouracil, and synthesis, activity and application thereof
Technical Field
The present invention relates to 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil, to a process for its preparation, to its antitumor activity andthe invention relates to the application of the compound in preparing anti-tumor drugs, the application of the compound in preparing anti-tumor metastasis drugs and the application of the compound in preparing drugs with dual functions of anti-tumor and anti-tumor metastasis. The invention belongs to the field of biological medicine.
Background
5-fluorouracil (5-FU) is a pyrimidine anti-metabolic anti-tumor drug and has a wide anti-tumor spectrum. It is used to treat digestive tract tumor, breast cancer, ovarian cancer, bladder cancer, and hepatocarcinoma. 5-FU has several drawbacks in clinical use. For example, the first-pass effect is obvious when the medicine is taken orally, and the clinical administration route is intravenous injection. The half-life of intravenous injection is no more than 20 min. For this reason, 5-FU is frequently administered by intravenous continuous infusion in clinical applications. Patients have poor compliance with continuous intravenous instillation. For example, 5-FU has a large therapeutic dose and poor tumor selectivity, and has obvious gastrointestinal reactions (nausea, vomiting and diarrhea) and bone marrow suppression (decrease in platelet and leukocyte counts) and other adverse reactions. These drawbacks limit the clinical application of 5-FU. To overcome the drawbacks of 5-FU, a number of structural modifications have been made. However, the desired effect is not achieved. The inventor discovers that the 1-position and the 3-position of 5-fluorouracil are respectively substituted by CH through years of exploration2CO-Arg-Gly-Asp-Ser and CH2The CO-The modification can be orally taken at an extremely low dose, can avoid bone marrow toxicity, can enhance The anti-tumor activity, and can obtain The anti-tumor metastasis activity. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
A first aspect of the present invention is to provide 1- (CH) of the formula2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil.
Figure BDA0002149252920000011
A second aspect of the present invention is to provide 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2A synthesis method of CO-The) -5-fluorouracil comprises The following steps:
(1) reacting 5-fluorouracil with bromoacetic acid in KOH aqueous solution at 60 ℃ for 8h, and then treating with concentrated hydrochloric acid at 0 ℃ to generate 1-carboxymethyl-5-fluorouracil;
(2) 1-carboxymethyl-5-fluorouracil is reacted with Arg (NO)2) Coupling of-Gly-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-5-fluorouracil;
(3)1-[CH2CO-Arg(NO2)-Gly-OBzl]reaction of-5-fluorouracil with tert-butyl bromoacetate under potassium carbonate condition to prepare 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2tBu) -5-fluorouracil;
(4)1-[CH2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2removing benzyl ester protecting group from tBu) -5-fluorouracil in 2N NaOH solution to prepare 1- [ CH2CO-Arg(NO2)-Gly]-3-(CH2CO2tBu) -5-fluorouracil;
(5)1-[CH2CO-Arg(NO2)-Gly]-3-(CH2CO2coupling of tBu-5-Fluorouracil with Asp (OBzl) -Ser-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2tBu) -5-fluorouracil;
(6)1-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2removing tert-butyl ester protecting group from tBu) -5-fluorouracil in 4N hydrogen chloride/ethyl acetate reagent to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2COOH) -5-fluorouracil;
(7)1-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2coupling of COOH) -5-fluorouracil with The-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO-The-OBzl) -5-fluorouracil;
(8) 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2Removing protective group by CO-The-OBzl) -5-fluorouracil through acid removal reaction to prepare 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil.
The third aspect of the present invention is to evaluate 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil has antitumor activity.
The fourth aspect of the present invention is evaluation 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2The anti-tumor metastasis activity of CO-The) -5-fluorouracil.
The fifth aspect of the present invention is to evaluate 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2Bone marrow toxicity of CO-The) -5-fluorouracil.
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FIG. 11- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2A synthetic route of CO-The) -5-fluorouracil, i)60 ℃, bromoacetic acid and concentrated hydrochloric acid; ii) dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine; iii) potassium carbonate, tert-butyl bromoacetate; iv) sodium hydroxide solution (2M); v) hydrogen chloride/ethyl acetate solution (4M); vi) trifluoroacetic acid, trifluoromethanesulfonic acid.
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 1- (CH)2CO2H) -5-Fluorouracil (1)
2.6g (20mmol) of 5-fluorouracil (5FU) were dissolved in aqueous potassium hydroxide at 0 ℃. Followed by activation at 60 ℃ for 1 hour. 4.14g (30mmol) of an aqueous bromoacetic acid solution was added dropwise thereto, and the mixture was stirred at 60 ℃ for 8 hours. After completion of the reaction, the reaction mixture was cooled to room temperature by TLC. The reaction mixture was adjusted to pH 5 with concentrated HCl at 0 ℃ and stirred for 30 min. Filtering, and dripping concentrated hydrochloric acid into the filtrate to adjust the pH value to 2. Stirring at 0 ℃ for 2.5 h. Filtration and washing of the filter residue with distilled water 3 times followed by air drying gave 3.05g (81%) of the title compound as a colorless solid. ESI-MS (m/e): 187[ M-H]-。1H NMR(300MHz,DMSO-d6):δ/ppm＝11.889(d,J＝2.7Hz,1H),8.069(d,J＝3.9Hz,1H),4.366(s,1H)。
EXAMPLE 2 preparation of Boc-Arg (NO)2)-Gly-OBzl
3.19g (10mmol) of Boc-Arg (NO) are added at 0 deg.C2) Dissolving (THF) in anhydrous tetrahydrofuran, and sequentially adding1.35g (10mmol) of 1-hydroxybenzotriazole and 2.68g (13mmol) of dicyclohexylcarbodiimide are added. After stirring for 30min, 3.71g (1.1mmol) of Gly-OBzl in tetrahydrofuran was added to the reaction mixture, and N-methylmorpholine was added dropwise to the reaction mixture at 0 ℃ to adjust pH 8. The reaction was stirred at room temperature until TLC showed completion of the reaction. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate solution. Purification by silica gel column chromatography gave 3.53g (75%) of the title compound as a colourless solid. ESI-MS (m/e): 467[ M + H ]]+。
EXAMPLE 3 preparation of Arg (NO)2)-Gly-OBzl
466mg (1mmol) of Boc-Arg (NO) at 0 deg.C2) -Gly-OBzl in hydrogen chloride in ethyl acetate (4M) and stirred until TLC indicated complete reaction. Thereafter, the reaction mixture was concentrated under reduced pressure at 37 ℃ to completely remove free hydrogen chloride. The resulting solid was suspended in 5mL of anhydrous ether and washed thoroughly. The precipitate was collected to yield 389mg (96%) of the title compound as a colorless solid. ESI-MS (m/e): 365[ M-H]-。
EXAMPLE 4 preparation of 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-5-Fluorouracil (2)
From 376mg (2mmol) of 1- (CH) by the method of example 22CO2H) -5-Fluorouracil (1) and 805mg (2mmol) of Arg (NO)2) -Gly-OBzl gave 491mg (46%) of the title compound as a colourless solid. ESI-MS (m/e): 535[ M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝11.833(s,1H),8.481(m,3H),7.989(d,J＝6.9Hz,1H),7.792(s,2H),7.365(s,5H),5.129(s,2H),4.346(m,3H),3.917(m,2H),3.128(m,2H),1.689(m,1H),1.512(m,3H)。
EXAMPLE 5 preparation of 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2tBu) -5-Fluorouracil (3)
536mg (1mmol) of 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-5-Fluorouracil (2) was dissolved in DMF, and 276mg of potassium carbonate was added at 0 ℃ and stirred for 1 hour. Thereafter, 390mg (2mmol) of tert-butyl bromoacetate are slowly added dropwise. The reaction mixture was stirred until the reaction was complete by TLC, insoluble material was filtered off and the filtrate was concentrated under reduced pressure. The resulting yellow oil was redissolved in dichloromethane and chromatographed on a silica gel column to give 271mg (41%) of the title compound,as a colorless solid.
ESI-MS(m/e)：649[M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝8.527(m,3H),8.138(d,J＝6.6Hz,1H),7.885(m,3H),7.369(s,5H),5.134(s,2H),4.452(m,4H),4.394(m,1H),3.924(m,2H),3.141(m,2H),1.691(m,1H),1.518(m,3H),1.398(s,9H)。
EXAMPLE 6 preparation of 1- [ CH2CO-Arg(NO2)-Gly]-3-(CH2CO2tBu) -5-Fluorouracil (4)
792mg (1.2mmol) of 1- [ CH at 0 DEG C2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2tBu) -5-Fluorouracil (3) was dissolved in methanol, and the pH of the reaction solution was adjusted to 13 with sodium hydroxide solution (2M). The reaction was continued at 0 ℃ until TLC showed completion of the reaction. The reaction solution was adjusted to neutral pH with 2N hydrochloric acid solution. The mixture was concentrated under reduced pressure to remove methanol. Adding absolute ethyl alcohol, ultrasonically shaking for 10min, filtering, and washing the filter cake with absolute ethyl alcohol for 3 times. Concentrated under reduced pressure to give the title compound as a pale yellow oil which was used directly in the subsequent reaction. ESI-MS (m/e): 559[ M-H ]]-。
EXAMPLE 7 preparation of Boc-Asp (OBzl) -Ser-OBzl
Using the method of example 2, 3.86g (77%) of the title compound were obtained as a pale yellow solid from 3.32g (10mmol) of Boc-Asp (OBzl) and 2.78g (1.2mmol) of Ser-OBzl. ESI-MS (m/e): 501[ M + H [ ]]+。
EXAMPLE 8 preparation of Asp (OBzl) -Ser-OBzl
Using the method of example 3, 2.05g (94%) of the title compound was obtained as a pale yellow solid from 2.50g (5mmol) of Boc-Asp (OBzl) -Ser-OBzl. ESI-MS (m/e): 399[ M-H ]]-。
EXAMPLE 9 preparation of 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2tBu) -5-Fluorouracil (5)
From 682mg (1.2mmol) of 1- [ CH using the method of example 22CO-Arg(NO2)-Gly]-3-(CH2CO2tBu) -5-Fluorouracil (4) and 532mg (1.2mmol) Asp (OBzl) -Ser-OBzl gave 509mg (44%) of the title compound as a colorless solid. ESI-MS (m/e): 941[ M-H ]]-。1H NMR(300MHz,DMSO-d6):δ/ppm＝8.503(d,J＝7.8Hz,1H),8.294(m,2H),8.160(d,J＝6.6Hz,1H),7.357(m,10H),5.190(m,5H),4.758(m,1H),4.446(m,3H),4.378(m,2H),4.028(m,1H),3.718(m,4H),3.138(m,2H),2.729(m,1H),2.601(m,1H),1.687(m,2H),1.519(m,2H),1.392(m,9H)。
EXAMPLE 10 preparation of 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2COOH) -5-fluorouracil (6)
From 496mg (0.5mmol) of 1- [ CH using the method of example 32CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2tBu) -5-Fluorouracil (5) gave 450mg (98%) of the title compound as a pale yellow powder. ESI-MS (m/e): 885[ M-H ]]-。
EXAMPLE 11 preparation of 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO-The-OBzl) -5-fluorouracil (7)
From 485mg (0.5mmol) of 1- [ CH ] using the method of example 32CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2COOH) -5-fluorouracil (6) and 206mg (1.5mmol) of The-OBzl gave 106mg (17%) of The title compound as a colorless solid. ESI-MS (m/e): 1131[ M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝8.678(d,J＝6.6Hz,1H),8.493(m,2H),8.276(m,3H),8.130(d,J＝6.3Hz,1H),7.757(s,1H),7.361(s,15H),5.098(m,7H),4.792(m,1H),4.412(m,7H),3.745(m,4H),3.142(m,1H),3.306(t,J＝7.2Hz,2H),2.738(m,1H),2.600(m,1H),2.137(t,J＝7.2Hz,2H),1.914(m,1H),1.843(m,1H),1.693(m,1H),1.572(m,3H),0.983(t,J＝7.2Hz,3H)；13C NMR(75MHz,DMSO-d6):δ/ppm＝171.9,1701.1,170.9,170.5,170.2,169.0,166.8,166.6,159.7,157.1,150.1,136.4,136.3,128.8,128.4,128.3,128.2,128.0,66.4,66.1,61.5,56.4,55.4,52.7,52.3,50.6,49.5,42.2,33.7,31.8,30.0,27.4,21.5,19.0,15.1。
Example 12 preparation of 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil (8)
120mg (0.1mmol) of 1- [ CH at 0 DEG C2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO-The-OBzl) -5-fluorouracil (7) is dissolved in 2mL of trifluoroacetic acid and addedDissolving 0.7mL of trifluoromethanesulfonic acid, adding precooled anhydrous ether after the reaction is completed, and stirring for 40min until solid is separated out. Centrifuging, discarding supernatant, repeating for three times, and air drying the solid obtained by centrifuging. Addition of H2Dissolving O, adjusting pH to 7 with saturated sodium bicarbonate solution at 0 deg.C, and filtering to remove residue. The filtrate was purified by Sephadax-G10 gel column chromatography and C18 column chromatography to give 47mg (54%) of the title compound as a colorless solid after lyophilization. The temperature of Mp is 180-181 ℃,
Figure BDA0002149252920000051
(c＝0.10,H2O)；ESI-MS(m/e)：816[M-H]-；1H NMR(300MHz,DMSO-d6):δ/ppm＝9.384(s,1H),8.569(d,J＝5.7Hz,1H),8.476(d,J＝8.7Hz,1H),8.155(d,J＝6.6Hz,1H),7.877(d,J＝7.8Hz,1H),7.763(d,J＝5.4Hz,1H),7.284(s,1H),4.441(m,5H),3.873(m,3H),3.634(s,4H),3.029(m,5H),2.052(m,2H),1.901(m,1H),1.725(m,2H),1.513(m,3H),0.984(t,J＝7.2Hz,3H)。13C NMR(125MHz,DMSO-d6):δ/ppm＝175.5,172.1,170.5,166.9,165.7,157.6,157.3,156.9,150.1,140.7,137.7,130.8,130.3,54.4,52.2,52.1,51.5,44.3,33.7,32.9,30.4,29.1,25.6,15.1。
EXAMPLE 13 determination of the antitumor Activity of Compound 8
1) Compound 8 and the positive control 5-FU were dissolved in physiological saline, which served as a blank control.
2) The compound 8,5-FU and physiological saline were both gavaged, the dose of the compound 8 was 1 nmol/kg/day, the dose of the 5-FU was 150. mu. mol/kg/day, and the dose of the physiological saline was 0.1mL/10 g/day.
3) The experimental animals were male mice of clean grade ICR, and the body weight was 20 + -2 g.
4) The tumor source for modeling the transplanted mouse S180 ascites type fibrosarcoma is S180 mouse ascites tumor cells, and is purchased from animal experiment center of department of medicine of Beijing university. Taking ICR male mice (tumor source mice) with good growth state after one week of passage, killing after anesthesia, taking S180 tumor liquid in the abdominal cavity under aseptic condition, centrifuging for 10min at 1000rpm, discarding supernatant, washing residues with a small amount of 4 ℃ physiological saline, and removing floating blood, histiocyte fragments and other non-cellular components. Then, trypan blue staining is carried out, cells are counted, and the concentration of the viable cells and the cell viability are calculated according to the formula.
Diluting tumor solution with cell activity greater than 90% with 4 deg.C physiological saline to obtain 2 × 107Cell suspension per mL. The right axilla of ICR mice were inoculated with 0.2 mL/tumor solution (inoculation was completed as soon as possible). Mice were observed daily for axillary tumor growth. The groups were evenly divided into 12 per group according to tumor volume. After grouping, mice were orally administered compound 8 or 5-FU or normal saline daily for 8 consecutive days according to the above-described dose. On day 9, each group of mice was weighed, anesthetized, and the eyeballs were bled in blood-drawing tubes containing EDTA, and blood-routine counting was performed to observe the effect of the test compound 8 on blood-routine cells. Mice were sacrificed, mouse axillary tumor tissue was isolated blunt, and sarcomas were removed and weighed. Tumor weights are expressed as mean ± SD g, and statistical analysis of experimental data was performed using SPSS statistical analysis software. The results are shown in Table 1. It can be seen that compound 8 at an oral dose of 1 nmol/kg/day is effective in inhibiting tumor growth, with no significant difference in activity from 5-fluorouracil at a dose of 150 μmol/kg/day (P)>0.05). As can be seen, the antitumor activity of Compound 8 is 150000 times as high as that of 5-FU. The invention has obvious technical effect.
TABLE 1 antitumor Activity of Compound 8
Figure BDA0002149252920000061
a) P <0.01 to saline; b) p <0.01 to normal saline, P >0.05 to 5-FU; n is 12.
EXAMPLE 14 determination of the antitumor cell migration Activity of Compound 8
1) Compound 8 was formulated to the desired concentration in media containing 0.1% DMSO.
2) The tumor cells are A549 (human non-small cell lung cancer cell) and 95D (human high-metastasis non-small cell lung cancer cell) which are cultured in RPMI-1640 culture medium containing 10% inactivated fetal calf serum and 1 × 105U/L penicillin and 100mg/L streptomycin.
3) Good growth state in logarithmic growth phaseA549 cells at 5X 105Cell density of 1X 10 cells/mL and 95D cells6Cell suspensions were prepared at a density of one/mL. Serum-free medium was used to inoculate the upper chamber of a Transwell, 100. mu.L of each was added, and Compound 8 (20. mu.M final concentration) was added. Meanwhile, 600. mu.L of a culture medium containing 10% FBS was added to the lower chamber, the Transwell chamber was placed in a 24-well culture plate, cultured in a 5% carbon dioxide incubator at 37 ℃ for 48 hours, the cells in the upper chamber were wiped off with a cotton swab, the culture medium in the lower chamber was aspirated, the cells were fixed with 4% paraformaldehyde fixing solution for 0.5 hour, the fixing solution was discarded, washed with PBS for 2 times, stained with crystal violet for 10 minutes, washed off by water to remove floating color, and observed with a 400-fold microscope. Randomly select 9 different visual fields to observe the cells and calculate the migration number. The results are shown in Table 2. It was confirmed that compound 8 effectively inhibited tumor cell migration at a concentration of 20 μ M. Furthermore, their activity was not significantly different from that of Arg-Gly-Asp-Ser (RGDS) at a concentration of 20. mu.M. This is a prominent technical effect of the present invention.
Table 2 effect of compound 8 on a549 and 95D cell migration
Figure BDA0002149252920000071
a) A blank-to-control ratio P < 0.01; b) p <0.01 to blank control, P >0.05 to RGDS; n-3 example 15 determination of antitumor cell invasion Activity of Compound 8
1) Compound 8 was formulated to the desired concentration in media containing 0.1% DMSO.
2) The tumor cells are A549 (human non-small cell lung cancer cell) and 95D (human high-metastasis non-small cell lung cancer cell) which are cultured in RPMI-1640 culture medium containing 10% inactivated fetal calf serum and 1 × 105U/L penicillin and 100mg/L streptomycin.
3) A549 cells which grow well and are in logarithmic growth phase are treated according to the standard of 5 multiplied by 105Cell density of 1X 10 cells/mL and 95D cells6Per mL density Using serum-free medium seeded in the upper chamber of a Transwell, 100. mu.L of each chamber, Compound 8 (20. mu.M final concentration) was added while 600. mu.L of medium containing 10% FBS was added in the lower chamber, and Transw was addedell the chamber is put into a 24-hole culture plate, cultured in a 5% carbon dioxide incubator at 37 ℃ for 48h, the cells in the upper chamber are wiped off by a cotton swab, the culture medium in the lower chamber is discarded, the cells are fixed by 4% paraformaldehyde fixing solution for 0.5h, the fixing solution is discarded, the cells are washed by PBS for 2 times, the cells are dyed by crystal violet for 10min, the floating color is washed by clear water, and the cells are observed by a 400-fold microscope. Randomly selecting 9 different visual fields to observe cells and calculating the invasion number. The results are shown in table 3, and it can be seen that compound 8 effectively inhibits tumor cell invasion at a concentration of 20 μ M. Furthermore, their activity was not significantly different from that of Arg-Gly-Asp-Ser (RGDS) at a concentration of 20. mu.M. This is a prominent technical effect of the present invention.
Table 3 effect of compound 8 on a549 and 95D cell invasion
Figure BDA0002149252920000081
a) A blank-to-control ratio P < 0.01; b) p <0.01 to blank control, P >0.05 to RGDS; n is 3.
EXAMPLE 16 determination of the anti-metastatic Activity of Compound 8
This compound 8 was dissolved in physiological saline. Lewis mouse Lung cancer cells (LLC, available from ATCC) with 10% FBS and 1X 105U·L-1Penicillin and 100 mg.L-1Culturing streptomycin in DMEM medium. Passage is carried out once a day, and cells are enriched. The cells were digested while they were in the logarithmic growth phase and in good growth conditions. Adjusting cell density to 2X 10 with physiological saline7one/mL.
An inbred line C57BL/6 male mouse with the weight of 20 +/-2 g is fixed by the left hand, the right front limb armpit skin of the mouse is coated with 75% ethanol for sterilization, a 1mL sterile syringe is used for injecting tumor cell suspension into the sterilized subcutaneous part of the right hand, 0.2mL is injected into each male mouse, the Lewis lung cancer tumor-bearing mouse with good growth state for 10 days is inoculated, and the cervical vertebra dislocation is killed after ether anesthesia. Soaking in 75% ethanol for 10min, sterilizing, removing tumor on a clean bench, selecting well-grown tumor tissue, cutting in a sterile culture dish, and grinding in a glass tissue homogenizer. Grinding at 1/3 tumor mass/normal saline volume (mL) with 4 deg.C precooled raw materialAnd (6) treating the saline water. Filtering the cell suspension obtained by grinding with 200 mesh nylon net, and adjusting the concentration of the collected cells to 2 × 10 with physiological saline7one/mL. A male mouse of an inbred line C57BL/6 with the weight of 20 +/-2 g is taken, the mouse is fixed by the left hand, the right front limb armpit skin of the mouse is smeared with 75% ethanol for sterilization, the right hand is injected with 0.2mL of tumor cell suspension into a sterilized subcutaneous part by a 1mL sterile syringe, and the tumor cells can grow into the tumor with the size of mung bean 10 days after inoculation. Tumor volumes were measured and mice with tumor diameters of 4-6mm were randomly grouped. Compound 8 mice were orally administered once daily at a dose of 0.1 nmol/kg/day for 10 consecutive days. Arg-Gly-Asp-Ser (RGDS, intraperitoneal injection dose of 20. mu. mol/kg/day, continuous administration for 10 days) was used as a positive control. The mice in the blank group were orally administered with physiological saline daily at a dose of 0.2 mL/mouse/day for 10 consecutive days. Mice were weighed on day 11 of dosing, anesthetized with ether, lungs from each group of mice were dissected to count metastatic tumor nodules, and tumors from each group of mice were dissected and weighed. The results are shown in Table 4. It can be seen that compound 8 effectively inhibits tumor metastasis to the lung. The activity of the compound for inhibiting tumor metastasis to lung at the dose of 0.1 nmol/kg/day is not significantly different from the activity of RGDS at the dose of 20 mu mol/kg/day. Therefore, the invention has outstanding technical effects.
TABLE 4 Compound 8 inhibits tumor Lung metastasis Activity
Figure BDA0002149252920000091
a) A blank-to-control ratio P < 0.01; b) p <0.01 to blank control, P >0.05 to RGDS; n is 10.
Example 17 determination of bone marrow toxicity of Compound 8 to S180 mice
The myelosuppressive toxicity of 5-FU is mainly manifested by a decrease in the white blood cell and platelet counts in the blood. To investigate the potential bone marrow toxicity of compound 8 treatment, the present invention measured leukocyte and platelet counts in the blood of compound 8 treated S180 mice using a michael fully automated three-classification hematology analyzer BC 3000. The data are shown in Table 5. The data show that compound 8 has no difference in its effect on white blood cell and platelet counts in the blood of S180 mice at the 1 nmol/kg/day dose compared to saline. As can be seen, compound 8 treatment was not myelotoxic to S180 mice. In contrast, the effect of 5-FU at the dose of 150. mu. mol/kg/day on the white blood cell and platelet counts in the blood of S180 mice was significantly different from that of normal saline. As can be seen, 5-FU has bone marrow toxicity to S180 mice. On the premise that the antitumor activity is the same as that of 5-FU, the compound 8 has no bone marrow toxicity, and the outstanding technical effect of the invention is reflected.
TABLE 5 Effect of Compound 8 on leukocyte and platelet counts in S180 mice
Figure BDA0002149252920000092
a) P <0.05 to saline; b) the ratio of P to physiological saline is greater than 0.05, and n is 8.

Claims (5)

1. 1- (CH) of the following Structure2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil,
Figure FDA0002149252910000011
2. the 1- (CH) of claim 12CO-Arg-Gly-Asp-Ser)-3-(CH2A method for preparing CO-The) -5-fluorouracil, comprising:
(1) reacting 5-fluorouracil with bromoacetic acid in KOH aqueous solution at 60 ℃ for 8h, and then treating with concentrated hydrochloric acid at 0 ℃ to generate 1-carboxymethyl-5-fluorouracil;
(2) 1-carboxymethyl-5-fluorouracil is reacted with Arg (NO)2) Coupling of-Gly-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-5-fluorouracil;
(3)1-[CH2CO-Arg(NO2)-Gly-OBzl]reaction of-5-fluorouracil with tert-butyl bromoacetate under potassium carbonate condition to prepare 1- [ CH2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2tBu) -5-fluorouracil;
(4)1-[CH2CO-Arg(NO2)-Gly-OBzl]-3-(CH2CO2removing benzyl ester protecting group from tBu) -5-fluorouracil in 2N NaOH solution to prepare 1- [ CH2CO-Arg(NO2)-Gly]-3-(CH2CO2tBu) -5-fluorouracil;
(5)1-[CH2CO-Arg(NO2)-Gly]-3-(CH2CO2coupling of tBu-5-Fluorouracil with Asp (OBzl) -Ser-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2tBu) -5-fluorouracil;
(6)1-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO2removing tert-butyl ester protecting group from tBu) -5-fluorouracil in 4N hydrogen chloride/ethyl acetate reagent to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2COOH) -5-fluorouracil;
(7)1-[CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2coupling of COOH) -5-fluorouracil with The-OBzl to prepare 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2CO-The-OBzl) -5-fluorouracil;
(8) 1- [ CH2CO-Arg(NO2)-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH2Removing protective group by CO-The-OBzl) -5-fluorouracil through acid removal reaction to prepare 1- (CH)2CO-Arg-Gly-Asp-Ser)-3-(CH2CO-The) -5-fluorouracil.
3. The 1- (CH) of claim 12CO-Arg-Gly-Asp-Ser)-3-(CH2Application of CO-The) -5-fluorouracil in preparing antitumor drugs.
4. The 1- (CH) of claim 12CO-Arg-Gly-Asp-Ser)-3-(CH2Application of CO-The) -5-fluorouracil in preparing anti-tumor metastasis medicaments.
5. The 1- (CH) of claim 12CO-Arg-Gly-Asp-Ser)-3-(CH2Application of CO-The) -5-fluorouracil in preparing medicine with dual effects of resisting tumor and tumor metastasis.
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