CN110551108B - 3R-indolylmethyl-6R-amino acid modified 2, 5-diketopiperazines, synthesis, activity and use thereof - Google Patents
3R-indolylmethyl-6R-amino acid modified 2, 5-diketopiperazines, synthesis, activity and use thereof Download PDFInfo
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Abstract
The present invention discloses 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino of the formulaN-butyl]-2, 5-diketopiperazine (AA is L-Ala residue, L-Ile residue, L-Leu residue, L-Met residue, L-Pro residue, L-Val residue), their preparation method, their anti-tumor metastasis activity, so the invention discloses their application in preparing anti-tumor metastasis medicine.
Description
Technical Field
The invention relates to 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino n-butyl ] -2, 5-diketopiperazine, a preparation method thereof and anti-tumor metastasis activity thereof, and thus the invention relates to application thereof in preparing anti-tumor metastasis medicaments. The invention belongs to the field of biological medicine.
Background
Tumors seriously threaten the health of human beings. In addition to the poor prognosis of patients with tumors by themselves, metastasis associated with tumors further worsens the prognosis of patients. For example, more than 90% of patients with tumors die from metastases. Because the existing antitumor drugs have no effect of resisting tumor metastasis, the clinical curative effect of tumor chemotherapy is not ideal. The invention relates to a medicament for resisting tumor metastasis, which is an urgent clinical need. The inventors have previously disclosed that diketopiperazines of the four S, S-, R, R-, R, S-and S, R-configurations inhibit migration and invasion of HCCLM3 (highly metastatic human liver cancer cells) at a concentration of 0.5. mu.M. Later, the inventors further disclosed that R, R-configured diketopiperazines inhibit tumor metastasis to the lung in C57BL/6 mice at a minimum effective dose of 5. mu. mol/kg/day. To reduce the minimum effective dose, the inventors have developed various modifications to the butylamino group of the diketopiperazine in the R, R-configuration. After 3 years of exploration, the 4-amino n-butyl group of diketopiperazine with R, R-configuration acylated by L-Lys in which side chain amino groups were acylated by L-Ala, L-Ile, L-Leu, L-Met, L-Pro and L-Val was found to reduce the minimum effective dose of anti-tumor metastasis to 0.5. mu. mol/kg/day. The 10-fold reduction of the lowest effective dose indicates that the structural modification has outstanding technical effects. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention provides 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine of the formula, AA being a L-Ala residue, a L-Ile residue, a L-Leu residue, a L-Met residue, a L-Pro residue, a L-Val residue.
The second aspect of the present invention provides a process for synthesizing 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine (wherein AA is a L-Ala residue, a L-Ile residue, a L-Leu residue, a L-Met residue, a L-Pro residue, a L-Val residue), which comprises:
(1) D-Boc-Lys (Cbz) is coupled with D-Trp-OBzl to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) D-Boc-Lys (Cbz) -D-Trp-OBzl removes Boc protecting group in 4N hydrogen chloride/ethyl acetate solution to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) cyclizing D-Lys (Cbz) -D-Trp-OBzl in ethyl acetate containing 5% sodium bicarbonate water solution to obtain 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine (1);
(4)1 catalyzing hydrogen to remove carbobenzoxy to obtain 3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine (2);
(5)2 is coupled with L-Boc-Lys (Cbz) to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino n-butyl ] -2, 5-diketopiperazine (3);
(6)3, removing benzyloxycarbonyl by catalytic hydrogenolysis to obtain 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino n-butyl) -2, 5-diketopiperazine (4);
(7)4 and Boc-AA (AA is L-Ala residue, L-Ile residue, L-Leu residue, L-Met residue, L-Pro residue, L-Val residue) to obtain a compound 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino n-butyl ] -2, 5-diketopiperazine (5 a-f);
(8)5a-f, removing Boc protecting group in 4N hydrogen chloride/ethyl acetate reagent to obtain 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino N-butyl ] -2, 5-diketopiperazine (6 a-f).
The third aspect of the present invention is to evaluate the activity of 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine (AA is L-Ala residue, L-Ile residue, L-Leu residue, L-Met residue, L-Pro residue, L-Val residue) for inhibiting the metastasis of lung cancer in C57BL/6 mice.
Drawings
FIG. 13R- (indol-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl]A synthetic route for 2, 5-diketopiperazines (in the formula AA is a L-Ala residue, a L-Ile residue, a L-Leu residue, a L-Met residue, a L-Pro residue, a L-Val residue) 6a-f, 5a,6a AA is a L-Ala residue; AA in 5b and 6b is L-Ile residue; AA in 5c and 6c is L-Leu residue; AA in 5d and 6d is L-Met residue; AA in 5e and 6e is L-Pro residue; AA in 5f and 6f is L-Val residue; i) dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), Tetrahydrofuran (THF); ii)4N hydrogen chloride-ethyl acetate reagent; iii) ethyl acetate, 5% sodium bicarbonate; iv) Dimethylformamide (DMF), Pd/C, H2。
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 D-Boc-Lys (Cbz) -D-Trp-OBzl
7.7g (20mmol) of D-Boc-Lys (Cbz) was suspended in 100mL of anhydrous Tetrahydrofuran (THF), and 2.7g (20mmol) of 1-hydroxybenzotriazole (HOBt) and 5.0g (25mmol) of Dicyclohexylcarbodiimide (DCC) were added to the suspension in this order under ice bath, followed by stirring for 30 min. Then, 8.0g (25mmol) of D-Trp-OBzl was added. The reaction mixture was adjusted to pH 9 by dropwise addition of N-methylmorpholine (NMM). The reaction mixture was stirred first for 1h on ice and then for 12h at room temperature. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 150mL of ethyl acetate solution. The ethyl acetate solution obtained was successively treated with 5% KHSO4The aqueous solution was washed 3 times, and the saturated NaCl aqueous solution was washed 3 times. Anhydrous Na for ethyl acetate layer2SO4Drying for 12h, filtering, and concentrating the filtrate under reduced pressure to dryness. The resulting yellow syrup was purified by silica gel column Chromatography (CH)2Cl2/CH3OH,100:1) 12.0g (88%) of the title compound are obtained as a colorless powder. ESI-MS (M/e):657[ M + H]+。
EXAMPLE 2 preparation of D-Lys (Cbz) -D-Trp-OBzl
3.8g (5mmol) D-Boc-Lys (Cbz) -D-Trp-OBzl were slowly mixed with 52mL of hydrogen chloride in ethyl acetate (4M) with stirring in an ice bath. The resulting solution was stirred in an ice bath for 5 h. After that, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 50mL of anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. This operation was repeated three times. The residue was washed thoroughly with anhydrous ether to give 3.41g (93%) of the title compound as a yellow powder. ESI-MS (M/e):557[ M + H ]]+。
EXAMPLE 3 preparation of 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino-n-butyl) -2, 5-diketopiperazine (1)
3.45g (6.2mmol) D-Lys (Cbz) -D-Trp-OBzl were dissolved in 150mL ethyl acetate. The resulting solution was washed three times with a 5% aqueous solution of sodium hydrogencarbonate, and then the ethyl acetate solution was stirred at room temperature for 12 hours to sufficiently precipitate a colorless solid. 1.8g (51%) of the title compound are filtered off. ESI-MS (M/e):449[ M + H ]]+。
EXAMPLE 4 preparation of 3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine (2)
To a solution of 1.9g (4.2mmol) of 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino N-butyl) -2, 5-diketopiperazine (1) in 20mL of anhydrous N, N-Dimethylformamide (DMF) was added 200mg of Pd/C, and H was bubbled through2The reaction was stirred at room temperature for 12 h. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 1.2g (92%) of the title compound as a colorless powder. ESI-MS (M/e) 315[ M + H ]]+。
EXAMPLE 5 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl ] -2, 5-diketopiperazine (3)
Using the method of example 1, from 4.18g (11mmol) Boc-Lys (Cbz) and 3.14g (10.0mmol)3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine (2) 5.17g (76%) of the title compound were obtained as colorless powder. ESI-MS (M/e):677[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.852(s,1H),8.045(d,J＝1.8Hz,1H),7.916(d,J＝1.8Hz,1H),7.581(m,2H),7.345(m,6H),7.223(t,J＝5.4Hz1H),7.025(m,2H),6.930(t,J＝6.9Hz,1H),6.714(d,J＝8.4Hz,1H),4.115(m,1H),3.812(m,1H),3.499(m,1H),3.206(dd,J1＝16.5Hz,J2＝3.9Hz,1H),3.172(d,J＝3.9Hz,1H),2.993(m,3H),2.764(m,2H),1.478(m,2H),1.381(m,11H),1.426(m,2H),0.971(m,3H),0.600(m,3H)。
EXAMPLE 6 preparation of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4)
From 1.65g (2.44mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl using the method of example 4]-2, 5-diketopiperazine (3) gave 1.49g (90%) of the title compound as a colorless powder. ESI-MS (M/e):543[ M + H]+。
EXAMPLE 7 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Ala) -amino-n-butyl ] -2, 5-diketopiperazine (5a)
From 680mg (3.6mmol) Boc-Ala and 1.62g (3mmol)3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) 1.58g (74%) of the title compound was obtained as colorless powder using the method of example 1. ESI-MS (M/e):714[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.855(s,1H),8.038(s,1H),7.926(s,1H),7.593(s,1H),7.569(d,J＝4.8Hz 1H),7.302(d,J＝4.8Hz 1H),7.048(s,1H),7.027(t,J＝8.1Hz 1H),6.930(t,J＝Hz 1H),6.791(d,J＝3.9Hz 1H),6.708(d,J＝3.9Hz 1H),4.110(m,1H),3.909(m,1H),3.815(m,1H),3.499(m,1H),3.275(m,1H),3.043(m,3H),2.760(m,2H),1.480(m,2H),1.375(m,20H),1.239(m,2H),1.157(d,J＝6.9Hz 1H),0.969(m,3H),0.586(m,3H)。
EXAMPLE 8 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Ile) -amino-n-butyl ] -2, 5-diketopiperazine (5b)
From 832mg (3.6mmol) of Boc-Ile and 1.62g (3mmol) of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) 1.81g (80%) of the title compound were obtained as colorless powder by the method of example 1. ESI-MS (M/e):756[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.851(s,1H),8.025(d,J＝2.1Hz 1H),7.922(d,J＝2.1Hz 1H),7.830(m,1H),7.581(m,2H),7.300(d,J＝7.8Hz1H),7.048(s,1H),7.023(t,J＝9.0Hz 1H),6.928(t,J＝9.0Hz 1H),6.694(d,J＝7.8Hz 1H),6.580(d,J＝7.8Hz 1H),4.115(m,1H),3.773(m,1H),3.746(m,1H),3.495(m,1H),3.255(dd,J1＝14.4Hz,J2＝3.9Hz,1H),3.031(m,3H),2.754(m,2H),1.637(m,2H),1.376(m,20H),1.245(m,2H),0.989(m,3H),0.803(m,6H),0.591(m,3H)。
EXAMPLE 9 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Leu) -amino-n-butyl ] -2, 5-diketopiperazine (5c)
From 832mg (3.6mmol) of Boc-Leu and 1.62g (3mmol) of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) by the method of example 1, 1.81g (81%) of the title compound are obtained as colorless powder. ESI-MS (M/e):756[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.855(s,1H),8.036(d,J＝1.8Hz 1H),7.925(d,J＝1.8Hz 1H),7.769(m,1H),7.589(m,2H),7.300(d,J＝8.1Hz1H),7.047(s,1H),7.022(t,J＝8.1Hz 1H),6.928(t,J＝8.1Hz 1H),6.730(m,2H),4.116(m,1H),4.116(m,1H),3.927(m,1H),3.495(m,1H),3.255(dd,J1＝14.1Hz,J2＝3.9Hz,1H),3.029(m,3H),2.735(m,2H),1.433(m,2H),1.378(m,21H),1.247(m,2H),0.949(m,3H),0.858(m,6H),0.582(m,3H)。
EXAMPLE 10 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Met) -amino-n-butyl ] -2, 5-diketopiperazine (5d)
From 896mg (3.6mmol) of Boc-Met and 1.62g (3mmol) of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) by the method of example 1, 1.81g (65%) of the title compound was obtained as colorless powder. ESI-MS (M/e):774[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.861(s,1H),8.052(m,1H),7.931(m,1H),7.801(m,1H),7.587(m,2H),7.296(m,1H),7.019(m,2H),6.923(m,2H),6.728(m,1H),4.111(m,1H),3.947(m,1H),3.806(m,1H),3.489(m,1H),3.252(dd,J1＝14.1Hz,J2＝3.9Hz,1H),3.034(m,3H),2.742(m,2H),2.047(m,2H),1.765(m,2H),1.456(m,2H),1.374(m,20H),1.235(m,2H),0.957(m,3H),0.568(m,3H)。
EXAMPLE 11 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Pro) -amino-n-butyl ] -2, 5-diketopiperazine (5e)
Starting from 774mg (3.6mmol) Boc-Pro and 1.62g (3mmol)3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2 using the method of example 15-Diketopiperazine (4) gives 950.4mg (45%) of the title compound as a colorless powder. ESI-MS (M/e):740[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.899(s,1H),8.056(m,1H),7.936(m,1H),7.778(m,1H),7.587(m,2H),7.281(m,2H),7.039(s,1H),7.020(t,J＝7.2Hz 1H),6.923(t,J＝7.2Hz 1H),6.767(m,2H),4.120(m,1H),3.812(m,1H),3.491(m,1H),3.458(m,1H),3.249(dd,J1＝14.1Hz,J2＝3.6Hz,1H),3.002(m,3H),2.732(m,2H),1.978(m,2H),1.799(m,3H),1.476(m,4H),1.373(m,20H),1.235(m,2H),0.955(m,3H),0.562(m,3H)。
EXAMPLE 12 preparation of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Val) -amino-n-butyl ] -2, 5-diketopiperazine (5f)
From 781mg (3.6mmol) of Boc-Val and 1.62g (3mmol) of 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine (4) by the method of example 1, 1.49g (67%) of the title compound were obtained as colorless powder. ESI-MS (M/e):742[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.852(s,1H),8.029(d,J＝2.1Hz 1H),7.922(d,J＝2.1Hz 1H),7.825(m,1H),7.581(d,J＝4.8Hz 1H),7.302(d,J＝4.8Hz 1H),7.047(s,1H),7.022(t,J＝8.1Hz 1H),6.928(t,J＝8.1Hz 1H),6.698(d,J＝8.4Hz 1H),6.548(d,J＝8.4Hz 1H),4.112(m,1H),3.801(m,1H),3.721(m,1H),3.495(m,1H),3.277(dd,J1＝14.1Hz,J2＝3.9Hz,1H),3.176(m,3H),2.750(m,2H),1.889(m,1H),1.481(m,2H),1.379(m,18H),1.243(m,2H),1.015(m,3H),0.817(m,3H),0.587(m,3H)。
EXAMPLE 13 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Ala) -amino-n-butyl ] -2, 5-diketopiperazine (6a)
From 100mg (0.14mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Ala) -amino-n-butyl using the method of example 2]-2, 5-diketopiperazine gave 58.5mg (80%) of the title compound as a colorless powder. ESI-MS (M/e):514[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.918(s,1H),8.058(m,1H),7.945(m,1H),7.767(m,1H),7.643(m,1H),7.576(d,J＝4.8Hz,1H),7.301(d,J＝4.8Hz,1H),7.041(s,1H),7.035(t,J＝7.5Hz 1H),6.927(t,J＝7.5Hz 1H),4.113(m,1H),3.505(m,1H),3.021(m,3H),2.980(m,4H),2.772(m,2H),1.999(m,3H),1.538(m,1H),1.379(m,5H),1.108(m,3H),0.955(m,3H),0.595(m,3H)。
EXAMPLE 14 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Ile) -amino-n-butyl ] -2, 5-diketopiperazine (6b)
From 789mg (1mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Ile) -amino n-butyl using the method of example 2]-2, 5-diketopiperazine (5b) gives 455mg (82%) of the title compound as a colorless powder. ESI-MS (M/e):556[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.931(s,1H),8.061(m,1H),7.947(m,1H),7.915(m,1H),7.783(m,1H),7.582(d,J＝7.8Hz 1H),7.308(d,J＝7.8Hz 1H),7.048(s,1H),7.028(t,J＝7.5Hz 1H),6.932(t,J＝7.5Hz 1H),4.118(m,1H),3.508(m,1H),3.255(dd,J1＝14.4Hz,J2＝3.3Hz,1H),3.101(m,3H),2.793(m,2H),1.676(m,1H),1.540(m,1H),1.371(m,4H),1.279(m,2H),1.062(m,3H),0.987(m,3H),0.863(m,6H),0.584(m,3H)。
EXAMPLE 15 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Leu) -amino-n-butyl ] -2, 5-diketopiperazine (6c)
From 789mg (1mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Leu) -amino-n-butyl using the method of example 2]-2, 5-diketopiperazine (5c) gave 421.8mg (76%) of the title compound as a colorless powder. ESI-MS (M/e):556[ M + H]+；1H NMR(300MHz,DMSO-d6):1H NMR(300MHz,DMSO-d6):δ/ppm＝10.922(s,1H),8.566(m,1H),8.341(m,1H),8.165(m,4H),8.079(m,1H),7.955(m,1H),7.583(d,J＝7.8Hz,1H),7.305(d,J＝7.8Hz,1H),7.051(s,1H),7.026(t,J＝7.8Hz,1H),6.093(t,J＝7.8Hz,1H),4.126(m,1H),3.681(m,2H),3.511(m,1H),3.259(dd,J1＝12.0Hz,J2＝3.6Hz,1H),3.137(m,1H),3.043(m,2H),2.835(m,2H),1.617(m,2H),1.554(m,3H),1.467(m,2H),1.437(m,2H),0.997(m,3H),0.891(m,6H),0.615(m,3H)。
EXAMPLE 16 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Met) -amino-n-butyl ] -2, 5-diketopiperazine (6d)
From 616mg (0.8mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Met) -amino-n-butyl using the method of example 2]-2, 5-Diketopiperazine (5d) to give 293mg (64%) of the title compound asA colorless powder. ESI-MS (M/e):574[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.995(s,1H),8.747(m,1H),8.508(m,1H),8.097(m,1H),8.038(m,1H),7.961(m,1H),7.578(d,J＝7.8Hz,1H),7.314(d,J＝7.8Hz,1H),7.045(s,1H),7.023(t,J＝7.5Hz 1H),6.926(t,J＝7.5Hz 1H),4.121(m,1H),3.841(m,1H),3.707(m,1H),3.505(m,1H),3.256(dd,J1＝14.4Hz,J2＝3.6Hz,1H),3.110(m,2H),3.038(dd,J1＝14.4Hz,J2＝3.6Hz,1H),2.828(m,2H),2.085(m,2H),1.770(m,2H),1.440(m,2H),1.330(m,2H),1.021(m,3H),0.595(m,3H)。
EXAMPLE 17 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Pro) -amino-n-butyl ] -2, 5-diketopiperazine (6e)
From 520mg (0.7mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Pro) -amino-n-butyl using the method of example 2]-2, 5-diketopiperazine (5e) gave 242.0mg (64%) of the title compound as a colorless powder. ESI-MS (M/e):540[ M + H [)]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.930(s,1H),8.039(s,1H),7.977(m,1H),7.937(s,1H),7.838(m,1H),7.778(d,J＝8.1Hz,1H),7.305(d,J＝8.1Hz,1H),7.047(s,1H),7.026(t,J＝7.8Hz,1H),6.928(t,J＝7.8Hz,1H),4.115(m,1H),3.549(m,1H),3.507(m,1H),3.257(dd,J1＝15.0Hz,J2＝3.6Hz,1H),3.062(m,3H),2.830(m,4H),1.621(m,4H),1.412(m,3H),1.249(m,2H),0.983(m,3H),0.609(m,3H)。
EXAMPLE 18 preparation of 3R- (indole-3-methyl) -6R- [4-Lys (Val) -amino-n-butyl ] -2, 5-diketopiperazine (6f)
From 1.48g (2mmol) of 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-Val) -amino-n-butyl using the method of example 2]-2, 5-diketopiperazine (5f) gave 973mg (90%) of the title compound as a colorless powder. ESI-MS (M/e) 542[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.953(s,1H),8.058(m,2H),7.984(m,1H),7.944(m,1H),7.578(d,J＝7.8Hz,1H),7.307(d,J＝7.8Hz,1H),7.047(s,1H),7.042(t,J＝7.2Hz,1H),6.927(t,J＝7.2Hz,1H),4.116(m,1H),3.505(m,1H),3.321(m,1H),3.257(dd,J1＝14.4Hz,J2＝4.8Hz,1H),3.070(m,4H),2.791(m,2H),1.589(m,1H),1.490(m,1H),1.408(m,2H),1.280(m,2H),0.997(m,2H),0.850(d,J＝6.9Hz,1H),0.825(d,J＝6.9Hz,3H),0.607(m,3H)。
EXAMPLE 19 determination of the anti-tumor metastasis Activity of Compounds 6a-f
The assay model was inoculated with Lewis mouse lung carcinoma cells (LLC, purchased from ATCC) in DMEM medium (containing 10% inactivated fetal bovine serum, 1X 10)5U/L penicillin and 100mg/L streptomycin), and the cells are enriched by passage every two days according to an adherent cell culture method. Digesting the cells when the cells are in good growth state and in logarithmic growth phase, and adjusting the cell density to 1 × 10 with physiological saline7one/mL. Staining with placental blue to count viable cells>95 percent. Inbred C57BL/6 male mice (SPF grade, body weight 20. + -.2 g) were taken and left-handed mice fixed. The right anterior limb axillary skin of the mouse was disinfected with 75% ethanol. The LLC tumor cell suspension is injected subcutaneously into the axilla of a mouse with a 1mL sterile syringe held in the right hand, and 0.2mL is injected into each mouse. After the mice are inoculated for 10 days, tumors with the diameter of about 4-5mm grow out, namely the tumor source. The Lewis lung cancer tumor-bearing mice are inoculated for 10 days and anesthetized by ether, and then the cervical vertebrae are removed for killing. Soaking in 75% ethanol for 10min, sterilizing, and removing tumor on clean bench. Well-grown tumor tissue was selected, minced in a sterile plate, and placed in a tissue homogenizer made of glass. Adding physiological saline with the temperature of 4 ℃ according to the ratio of the tumor mass to the volume of the physiological saline of 1 to 3(g to mL), and lightly grinding to prepare the cell suspension. The cell suspension is screened by 200-mesh cells to prepare single cell suspension. Adjusting the cell density of the single cell suspension to 1.5X 10 with physiological saline7one/mL. Staining with placental blue to count viable cells>95 percent. Left-handed inbred C57BL/6 male mice were fixed and their right anterior limb axillary skin was disinfected with 75% ethanol. The tumor cell suspension was injected subcutaneously into the mouse axilla with a 1mL sterile syringe in the right hand, 0.2mL each. 10 days after inoculation, the mice developed tumors of 4-5mm in diameter, and the inoculated mice were randomly grouped by the measured tumor volume. Each group had 12 mice. Mice on day 11 of tumor inoculation were orally administered either a normal saline solution of the putative antitumor metastatic peptide RGDS (at a dose of 20. mu. mol/kg/day) or compounds 6a-f (at a dose of 0.5. mu. mol/kg/day) or normal saline (at a dose of 10 mL/kg/day) 1 time a day for 12 consecutive daysTumor volumes were measured and recorded every two days. The next day of the last administration, tumor volume was measured, cervical spine was removed by ether anesthesia and sacrificed, tumor of the mice was weighed, lung of the mice was taken and tumor nodules transferred from the lung of the tumor were counted. Statistical analysis of the data was performed using the t-test. The results are shown in Table 1. At 0.5 μmol/kg/day dose (10-fold reduction of the least effective dose) 6a-f was not only effective in inhibiting tumor lung metastasis, but also had no significant difference in activity from RGDS at the 20 μmol/kg/day dose. These data show that this structural modification has a remarkable technical effect.
TABLE 1 antitumor metastatic Activity of Compounds 6a-f
a) P <0.01 to saline, p >0.05 to RGDS; b) p <0.05 to saline; n is 10.
Claims (3)
1. 3R- (indol-3-methyl) -6R- [4-Lys (AA) -amino-n-butyl ] -2, 5-diketopiperazine of the formula,
wherein AA is a L-Ala residue, a L-Ile residue, a L-Leu residue, a L-Met residue, a L-Pro residue or a L-Val residue.
2. A process for the preparation of 3R- (indole-3-methyl) -6R- [4-lys (aa) -amino-n-butyl ] -2, 5-diketopiperazine according to claim 1, which comprises:
(1) D-Boc-Lys (Cbz) is coupled with D-Trp-OBzl to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) D-Boc-Lys (Cbz) -D-Trp-OBzl removes Boc protecting group in 4N hydrogen chloride or ethyl acetate solution to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) D-Lys (Cbz) -D-Trp-OBzl in ethyl acetate is washed for 3 times by 5 percent sodium bicarbonate water solution and cyclized to obtain 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine;
(4) the 3R- (indole-3-methyl) -6R- (4-benzyloxycarbonylamino n-butyl) -2, 5-diketopiperazine catalyzes and hydrolyzes benzyloxycarbonyl to obtain 3R- (indole-3-methyl) -6R- (4-amino n-butyl) -2, 5-diketopiperazine;
(5)3R- (indole-3-methyl) -6R- (4-amino-n-butyl) -2, 5-diketopiperazine is coupled with L-Boc-Lys (Cbz) to obtain 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino-n-butyl ] -2, 5-diketopiperazine;
(6)3R- (indole-3-methyl) -6R- [4-Boc-Lys (Cbz) -amino n-butyl ] -2, 5-diketopiperazine is subjected to catalytic hydrogenolysis to remove benzyloxycarbonyl to obtain 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino n-butyl) -2, 5-diketopiperazine;
(7) coupling 3R- (indole-3-methyl) -6R- (4-Boc-Lys-amino-n-butyl) -2, 5-diketopiperazine with Boc-AA to obtain a compound 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino-n-butyl ] -2, 5-diketopiperazine;
(8) and removing the Boc protecting group of the 3R- (indole-3-methyl) -6R- [4-Boc-Lys (Boc-AA) -amino N-butyl ] -2, 5-diketopiperazine in a hydrogen chloride or ethyl acetate reagent with the concentration of 4N to obtain the 3R- (indole-3-methyl) -6R- [4-Lys (AA) -amino N-butyl ] -2, 5-diketopiperazine.
3. The use of 3R- (indole-3-methyl) -6R- [4-lys (aa) -amino-n-butyl ] -2, 5-diketopiperazine according to claim 1 for the preparation of a medicament for the treatment of tumor metastasis.
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