CN112300245B - RGDS and theanine co-modified 5-fluorouracil, and synthesis, activity and application thereof - Google Patents

Abstract

The invention discloses 5-fluorouracil co-modified by RGDS of the following formula and theanine. Its preparation method is disclosed, its anti-tumor and anti-tumor metastasis activities are disclosed, so the present invention discloses its application in the preparation of anti-tumor drugs, its application in the preparation of anti-tumor metastasis drugs and its application in the preparation of anti-tumor and anti-tumor metastasis Application of Dual Active Drugs in Tumor Metastasis.

Description

5-fluorouracil co-modified by RGDS and theanine, its synthesis, activity and application

technical field

The present invention relates to 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil, to its preparation method, to its anti-tumor activity and anti-tumor metastasis activity , and thus the present invention relates to its application in the preparation of antitumor drugs, its application in the preparation of anti-tumor metastasis drugs, and its application in the preparation of anti-tumor and anti-tumor metastasis drugs. The invention belongs to the field of biomedicine.

Background technique

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite antineoplastic drug with a broad antitumor spectrum. It is mostly used in the treatment of digestive tract tumors, breast cancer, ovarian cancer, bladder cancer, and liver cancer. 5-FU has some defects in clinical application. For example, the oral first-pass effect is obvious, and the clinical route of administration is intravenous injection. The half-life of intravenous injection does not exceed 20 minutes. For this reason, 5-FU is often infused intravenously in clinical practice. Patients have poor compliance with continuous intravenous infusion. Another example is the large therapeutic dose of 5-FU, which has poor selectivity for tumors, and adverse reactions such as obvious gastrointestinal reactions (nausea, vomiting, diarrhea) and bone marrow suppression (decrease in the number of platelets and white blood cells) may occur. These defects limit the clinical application of 5-FU. In order to overcome the defects of 5-FU, a large number of structural modifications have been done. But it didn't achieve the desired effect. After several years of exploration, the inventors found that modifying the 1-position and 3-position of 5-fluorouracil with CH ₂ CO-Arg-Gly-Asp-Ser and CH ₂ CO-The can not only be administered orally at a very low dose, but also can avoid bone marrow toxicity, can enhance anti-tumor activity, and can obtain anti-tumor metastasis activity. Based on these findings, the inventors have made the present invention.

Contents of the invention

The first content of the present invention is to provide 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil of the following formula.

The second content of the present invention is to provide a synthesis method of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil, the method comprising:

(1) React 5-fluorouracil with bromoacetic acid at 60°C for 8 hours in KOH aqueous solution, then treat with concentrated hydrochloric acid at 0°C to generate 1-carboxymethyl-5-fluorouracil;

(2) Coupling 1-carboxymethyl-5-fluorouracil with Arg(NO ₂ )-Gly-OBzl to prepare 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-5-fluorouracil;

(3) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-5-fluorouracil reacts with tert-butyl bromoacetate under the condition of potassium carbonate to prepare 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-( _CH2CO2tBu )-5 _- fluorouracil;

(4) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil deprotected benzyl ester in 2N NaOH solution to prepare 1-[ CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil;

(5) 1-[CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil coupled with Asp(OBzl)-Ser-OBzl to prepare 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil;

(6) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil in 4N hydrogen chloride/ethyl acetate Remove the tert-butyl ester protecting group in the ester reagent to prepare 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ COOH)-5-fluorouracil;

(7) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ COOH)-5-fluorouracil is coupled with The-OBzl to prepare 1-[ _CH2CO -Arg(NO2)-Gly _- Asp(OBzl)-Ser-OBzl]-3-( _CH2CO -The-OBzl)-5-fluorouracil;

(8) Remove 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO-The-OBzl)-5-fluorouracil by acid dereaction Protecting group, preparation of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil.

The third content of the present invention is to evaluate the antitumor activity of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil.

The fourth content of the present invention is to evaluate the anti-tumor metastasis activity of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil.

The fifth content of the present invention is to evaluate the bone marrow toxicity of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil.

Description of drawings

Figure 1 Synthetic route of 1-(CH ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil. i) 60℃, bromoacetic acid, concentrated hydrochloric acid; ii) bicyclic Hexylcarbodiimide, 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

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1 Preparation of 1-(CH ₂ CO ₂ H)-5-fluorouracil (1)

2.6 g (20 mmol) of 5-fluorouracil (5FU) was dissolved in an aqueous potassium hydroxide solution at 0°C. Then activate at 60°C for 1 hour. Add 4.14g (30mmol) bromoacetic acid aqueous solution dropwise, and stir at 60°C for 8h. After the reaction was detected by TLC, the reaction mixture was cooled to room temperature. The reaction mixture was adjusted to pH 5 with concentrated hydrochloric acid at 0°C and stirred for 30 min. After filtration, the filtrate was added dropwise with concentrated hydrochloric acid to adjust the pH to 2. Stir at 0°C for 2.5h. After filtration, the filter residue was washed 3 times with distilled water, and dried to obtain 3.05 g (81%) of the title compound as a colorless solid. ESI-MS (m/e): 187 [MH] ^- . ¹ H NMR (300MHz, DMSO-d ₆ ): 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

Dissolve 3.19g (10mmol) Boc-Arg (NO ₂ ) in anhydrous tetrahydrofuran (THF) at 0°C, and add 1.35g (10mmol) 1-hydroxybenzotriazole and 2.68g (13mmol) bis Cyclohexylcarbodiimide. After stirring for 30 min, 3.71 g (1.1 mmol) of Gly-OBzl in tetrahydrofuran was added to the reaction solution, and N-methylmorpholine was added dropwise to the reaction solution at 0°C to adjust the pH to 8. The reaction solution was stirred at room temperature until TLC showed that the reaction was complete. 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 3.53 g (75%) of the title compound as a colorless solid. ESI-MS (m/e): 467 [M+H] ⁺ .

Example 3 Preparation of Arg(NO ₂ )-Gly-OBzl

466 mg (1 mmol) Boc-Arg(NO ₂ )-Gly-OBzl was dissolved in hydrogen chloride in ethyl acetate solution (4M) at 0°C, and stirred until TLC showed that the reaction was complete. Afterwards, the reaction solution was concentrated under reduced pressure at 37°C to completely remove free hydrogen chloride. The obtained solid was suspended in 5mL of anhydrous ether and washed thoroughly. The precipitate was collected to give 389 mg (96%) of the title compound as a colorless solid. ESI-MS (m/e): 365 [MH] ^- .

Example 4 Preparation of 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-5-fluorouracil (2)

Using the method of Example 2, 491 mg (46%) of the title compound were obtained from 376 mg (2 mmol) 1-(CH ₂ CO ₂ H)-5-fluorouracil (1) and 805 mg (2 mmol) Arg(NO ₂ )-Gly-OBzl, It is a colorless solid. ESI-MS (m/e): 535[MH] ^- ; ¹ HNMR (300MHz, DMSO-d ₆ ): 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-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (3)

Dissolve 536mg (1mmol) of 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-5-fluorouracil (2) in DMF, add 276mg of potassium carbonate at 0°C, and stir for 1h. After that, 390 mg (2 mmol) tert-butyl bromoacetate was slowly added dropwise. The reaction mixture was stirred until the reaction was complete as detected by TLC, the insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The obtained yellow oil was redissolved in dichloromethane and subjected to silica gel column chromatography to obtain 271 mg (41%) of the title compound as a colorless solid.

ESI-MS (m/e): 649[MH] ^- ; ¹ H NMR (300MHz, DMSO-d ₆ ): 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-[CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (4)

Dissolve 792 mg (1.2 mmol) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (3) in methanol at 0°C, and dissolve it with sodium hydroxide solution (2M) to adjust the pH value of the reaction solution to 13. The reaction was continued at 0°C until TLC showed that the reaction was complete. The pH of the reaction solution was adjusted to neutral with 2N hydrochloric acid solution. Concentrate under reduced pressure to remove methanol. Add absolute ethanol, ultrasonically shake for 10 min, filter, and wash the filter cake 3 times with absolute ethanol. Concentration under reduced pressure gave the title compound as a pale yellow oil, which was directly used in subsequent reactions. ESI-MS (m/e): 559 [MH] ^- .

Example 7 Preparation of Boc-Asp(OBzl)-Ser-OBzl

Using the method of Example 2, 3.86 g (77%) of the title compound was obtained from 3.32 g (10 mmol) Boc-Asp (OBzl) and 2.78 g (1.2 mmol) Ser-OBzl as a pale yellow solid. ESI-MS (m/e): 501 [M+H] ⁺ .

Example 8 Preparation of Asp(OBzl)-Ser-OBzl

Using the method of Example 3, 2.05 g (94%) of the title compound was obtained from 2.50 g (5 mmol) of Boc-Asp(OBzl)-Ser-OBzl as a pale yellow solid. ESI-MS (m/e): 399 [MH] ^- .

Example 9 Preparation of 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (5)

Using the method of Example 2, from 682mg (1.2mmol) 1-[CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (4) and 532mg (1.2mmol) Asp(OBzl)-Ser-OBzl afforded 509 mg (44%) of the title compound as a colorless solid. ESI-MS (m/e): 941 [MH] ^- . ¹ H NMR (300MHz, DMSO-d ₆ ): 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-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ COOH)-5-fluorouracil (6)

Using the method of Example 3, from 496mg (0.5mmol) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ tBu)-5-fluorouracil (5) Obtained 450 mg (98%) of the title compound as light yellow powder. ESI-MS (m/e): 885 [MH] ^- .

Example 11 Preparation of 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO-The-OBzl)-5-fluorouracil (7)

Using the method of Example 3, from 485 mg (0.5 mmol) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ COOH)-5-fluorouracil (6 ) and 206 mg (1.5 mmol) of The-OBzl yielded 106 mg (17%) of the title compound as a colorless solid. ESI-MS (m/e): 1131[MH] ^- ; ¹ H NMR (300MHz, DMSO-d ₆ ): 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); 75MHz, DMSO-d ₆ ): 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, 1628, 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 ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The)-5-fluorouracil (8)

(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。120 mg (0.1 mmol) 1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO-The-OBzl)-5-fluorouracil (7 ) was dissolved in 2 mL of trifluoroacetic acid, and 0.7 mL of trifluoromethanesulfonic acid was added to dissolve it. After the reaction was complete, pre-cooled anhydrous diethyl ether was added, and stirred for 40 min until a solid precipitated. Centrifuge, discard the supernatant, repeat three times, and dry the solid obtained by centrifugation. Add H ₂ O to dissolve, adjust the pH value to 7 with saturated sodium bicarbonate solution at 0°C, and filter to remove the filter residue. The filtrate was purified by Sephdax-G10 gel column chromatography and C18 column chromatography, and 47 mg (54%) of the title compound was obtained as a colorless solid after lyophilization. Mp 180～181℃,

(c=0.10, H ₂ O); ESI-MS (m/e): 816[MH] ^- ; ¹ H NMR (300MHz, DMSO-d ₆ ): 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). ¹³ C NMR (125MHz, DMSO-d ₆ ): 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 normal saline, and normal saline was used as a blank control.

2) Compound 8, 5-FU and normal saline were administered intragastrically, the dose of compound 8 was 1 nmol/kg/day, the dose of 5-FU was 150 μmol/kg/day, and the dose of normal saline was 0.1 mL/10 g/day. sky.

3) The experimental animals are clean grade ICR male mice with a body weight of 20±2g.

4) The tumor source used for the modeling of transplanted mouse S180 ascites fibrosarcoma was S180 mouse ascites tumor cells, which were purchased from the Animal Experiment Center of Peking University Health Science Center. One week after subculture, ICR male mice (tumor-derived mice) in good growth state were taken and killed after anesthesia. Under sterile conditions, the S 180 tumor fluid in the peritoneal cavity was collected, centrifuged at 1000rpm for 10min, the supernatant was discarded, and the residue was used for Wash with a small amount of 4°C normal saline to remove floating blood, tissue cell debris and other non-cellular components. After trypan blue staining, the cells were counted and the concentration of viable cells and cell viability were calculated according to the formula.

The tumor fluid with cell viability greater than 90% was diluted with 4°C saline to make a cell suspension of 2×10 ⁷ cells/mL. Inoculate the right axilla of ICR mice with 0.2 mL/mouse tumor fluid (complete the inoculation as soon as possible). The tumor growth in the axilla of the mice was observed every day. According to tumor volume, they were evenly divided into groups, with 12 rats in each group. After grouping, the mice were orally administered compound 8 or 5-FU or normal saline every day according to the dose described above for 8 consecutive days. On the 9th day, the mice in each group were weighed, anesthetized, and the blood was collected from the eyeball in the blood vessel containing EDTA, and the blood routine count was performed to observe the effect of the test compound 8 on the blood routine cells. The mice were sacrificed, and the tumor tissues in the axilla of the mice were bluntly dissected, and the sarcoma was removed and weighed. The tumor weight was expressed as the mean ± SD g, and the experimental data were statistically analyzed by 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 can effectively inhibit the growth of tumors, and its activity has no significant difference from that of 5-fluorouracil at a dose of 150 μmol/kg/day (P>0.05). It can be seen that the antitumor activity of compound 8 is equivalent to 150,000 times that of 5-FU. The invention has remarkable technical effects.

Antitumor activity of compound 8 in table 1

a) P<0.01 compared with normal saline; b) P<0.01 compared with normal saline, P>0.05 compared with 5-FU; n=12.

Example 14 Determination of anti-tumor cell migration activity of compound 8

1) Compound 8 was prepared into the desired concentration with medium containing 0.1% DMSO.

2) The tumor cells were A549 (human non-small cell lung cancer cells) and 95D (human highly metastatic non-small cell lung cancer cells) were cultured in RPMI-1640 medium, and the medium contained 10% inactivated fetal bovine serum and 1×10 ⁵ U/L penicillin and 100 mg/L streptomycin.

3) A549 cells in good growth state and in the logarithmic growth phase were prepared at a density of 5×10 ⁵ cells/mL and 95D cells were prepared at a density of 1×10 ⁶ cells/mL. Inoculate the upper chamber of the Transwell with serum-free medium, add 100 μL to each chamber, and add compound 8 (final concentration: 20 μM). At the same time, add 600 μL of medium containing 10% FBS to the lower chamber, put the Transwell chamber into a 24-well culture plate, and culture it in a 5% carbon dioxide incubator at 37°C for 48 hours, wipe off the cells in the upper chamber with a cotton swab, and discard the lower chamber The cells were fixed with 4% paraformaldehyde fixative solution for 0.5h, discarded the fixative solution, washed twice with PBS, stained with crystal violet for 10min, washed with water to remove the floating color, and observed under a microscope at 400 times. Randomly select 9 different fields of view to observe cells and calculate the number of migration. The results are shown in Table 2. It can be confirmed that compound 8 effectively inhibits tumor cell migration at a concentration of 20 μM. In addition, their activity was not significantly different from that of Arg-Gly-Asp-Ser(RGDS) at the concentration of 20μM. This is an outstanding technical effect of the present invention.

Table 2 The effect of compound 8 on the migration of A549 and 95D cells

a) P<0.01 compared with blank control; b) P<0.01 compared with blank control, P>0.05 compared with RGDS; n=3 Example 15 Determination of the anti-tumor cell invasion activity of compound 8

1) Compound 8 was prepared into the desired concentration with medium containing 0.1% DMSO.

2) The tumor cells were A549 (human non-small cell lung cancer cells) and 95D (human highly metastatic non-small cell lung cancer cells) were cultured in RPMI-1640 medium, and the medium contained 10% inactivated fetal bovine serum and 1×10 ⁵ U/L penicillin and 100mg/L streptomycin.

3) A549 cells in good growth state and in the logarithmic growth phase were inoculated in the upper chamber of the Transwell at a density of 5×10 ⁵ cells/mL and 95D cells at a density of 1×10 ⁶ cells/mL with serum-free medium. , add 100 μL to each chamber, add compound 8 (final concentration 20 μM) and add 600 μL medium containing 10% FBS to the lower chamber, put the Transwell chamber into a 24-well culture plate, and incubate in a 5% carbon dioxide incubator at 37 °C for 48 h , wipe off the cells in the upper chamber with a cotton swab, discard the medium in the lower chamber, fix the cells with 4% paraformaldehyde fixative solution for 0.5h, discard the fixative solution, wash twice with PBS, stain with crystal violet for 10min, wash with water to remove the floating cells. Color, observed with a 400X microscope. Randomly select 9 different fields of view to observe cells and calculate the number of invasion. The results are shown in Table 3. It can be seen that compound 8 effectively inhibited tumor cell invasion at a concentration of 20 μM. In addition, their activity was not significantly different from that of Arg-Gly-Asp-Ser(RGDS) at the concentration of 20μM. This is an outstanding technical effect of the present invention.

Table 3 The effect of compound 8 on the invasion of A549 and 95D cells

a) P<0.01 compared with blank control; b) P<0.01 compared with blank control, P>0.05 compared with RGDS; n=3.

Example 16 Determination of the anti-tumor metastasis activity of compound 8

This compound 8 was dissolved in physiological saline. Lewis mouse lung cancer cells (LLC, purchased from ATCC) were cultured in DMEM medium containing 10% FBS, 1×10 ⁵ U·L ^-1 penicillin and 100 mg·L ^-1 streptomycin. Passage once a day to enrich cells. Digest the cells when they are in logarithmic growth phase and in good growth condition. Adjust the cell density to 2× ¹⁰⁷ /mL with saline.

Inbred C57BL/6 male mice with a body weight of 20±2 g were fixed with the left hand, and 75% ethanol was applied to the skin of the axilla of the right forelimb of the mouse for disinfection, and the right hand was injected with a 1 mL sterile syringe into the subcutaneous place of the tumor cell suspension. Each mouse was injected with 0.2 mL, and Lewis lung cancer tumor-bearing mice with good growth status were taken 10 days after inoculation, and sacrificed by cervical dislocation after ether anesthesia. Soak and sterilize with 75% ethanol for 10min, operate and strip the tumor body on an ultra-clean bench, select a well-growing tumor tissue, cut it into pieces in a sterile petri dish, and place it in a glass tissue homogenizer for grinding. When grinding, add 4°C pre-cooled physiological saline according to the ratio of tumor mass (g)/normal saline volume (mL) to 1/3. The cell suspension prepared by grinding was filtered with a 200-mesh nylon mesh, and the collected cells were adjusted to a concentration of 2×10 ⁷ cells/mL with normal saline. Take an inbred C57BL/6 male mouse with a body weight of 20±2 g, fix the mouse with the left hand, apply 75% ethanol to the skin of the right forelimb armpit of the mouse for disinfection, and inject tumor cells subcutaneously with a 1 mL sterile syringe in the right hand Suspension, each injected 0.2mL, 10 days after inoculation can grow into mung bean-sized tumors. Tumor volume was measured, and mice with tumor diameters of 4-6 mm were randomly divided into groups. Mice in compound 8 group were orally administered once a day at a dose of 0.1 nmol/kg/day for 10 consecutive days. Arg-Gly-Asp-Ser (RGDS, administered intraperitoneally at a dose of 20 μmol/kg/day for 10 consecutive days) was used as a positive control. Mice in the blank group were given normal saline orally every day at a dose of 0.2 mL/mouse/day for 10 consecutive days. On the 11th day after the administration, the mice were weighed, anesthetized with ether, and the lungs of the mice in each group were dissected to calculate the number of metastatic tumor nodes, and the tumors of the mice in each group 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. There is no significant difference in the activity of inhibiting tumor metastasis to the lung at the dose of 0.1nmol/kg/day and the activity of RGDS at the dose of 20μmol/kg/day. Visible, the present invention has outstanding technical effect.

Table 4 compound 8 inhibits tumor lung metastasis activity

a) P<0.01 compared with blank control; b) P<0.01 compared with blank control, P>0.05 compared with RGDS; n=10.

Example 17 Determination of the bone marrow toxicity of compound 8 to S180 mice

The bone marrow suppression toxicity of 5-FU is mainly manifested in the reduction of white blood cell and platelet counts in the blood. In order to investigate the potential bone marrow toxicity of compound 8 treatment, the present invention uses Mindray automatic three-differential hematology analyzer BC3000 to measure the white blood cell and platelet counts in the blood of S180 mice treated with compound 8. See Table 5 for the data. The data show that the effect of compound 8 on leukocyte and platelet counts in the blood of S180 mice at a dose of 1 nmol/kg/day is not different from that of normal saline. It can be seen that the treatment of compound 8 has no bone marrow toxicity to S180 mice. On the contrary, at the dose of 150μmol/kg/day, the effect of 5-FU on leukocyte and platelet count in the blood of S180 mice was significantly different from that of normal saline. Visible, 5-FU has myelotoxicity to S180 mice. On the premise that the antitumor activity is the same as that of 5-FU, compound 8 has no bone marrow toxicity, reflecting the outstanding technical effect of the present invention.

The influence of table 5 compound 8 on S180 mouse white blood cell and platelet count

a) P<0.05 compared with normal saline; b) P>0.05 compared with normal saline, n=8.

Claims (5)

1. 1- (CH) of the following Structure ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The) -5-fluorouracil,

2. the 1- (CH) of claim 1 ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ A 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- [ CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-5-fluorouracil;

(3)1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]reaction of-5-fluorouracil with tert-butyl bromoacetate under potassium carbonate condition to prepare 1- [ CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-(CH ₂ CO ₂ tBu) -5-fluorouracil;

(4)1-[CH ₂ CO-Arg(NO ₂ )-Gly-OBzl]-3-(CH ₂ CO ₂ removing benzyl ester protecting group from tBu) -5-fluorouracil in 2N NaOH solution to prepare 1- [ CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ tBu) -5-fluorouracil;

(5)1-[CH ₂ CO-Arg(NO ₂ )-Gly]-3-(CH ₂ CO ₂ coupling of tBu) -5-Fluorouracil with Asp (OBzl) -Ser-OBzl to prepare 1- [ CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ tBu) -5-fluorouracil;

(6)1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO ₂ removing tert-butyl ester protecting group from tBu) -5-fluorouracil in 4N hydrogen chloride/ethyl acetate reagent to prepare 1- [ CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ COOH) -5-fluorouracil;

(7)1-[CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ coupling of COOH) -5-fluorouracil with The-OBzl to prepare 1- [ CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ CO-The-OBzl) -5-fluorouracil;

(8) 1- [ CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl]-3-(CH ₂ Removing protective group by CO-The-OBzl) -5-fluorouracil through acid removal reaction to prepare 1- (CH) ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ CO-The) -5-fluorouracil.

3. The 1- (CH) of claim 1 ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ Application of CO-The) -5-fluorouracil in preparing antitumor drugs.

4. The 1- (CH) of claim 1 ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ Application of CO-The) -5-fluorouracil in preparing anti-tumor metastasis medicaments.

5. The 1- (CH) of claim 1 ₂ CO-Arg-Gly-Asp-Ser)-3-(CH ₂ Application of CO-The) -5-fluorouracil in preparing medicine with dual effects of resisting tumor and tumor metastasis.

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