CN108929313B - 3R-indolylmethyl-6R-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof - Google Patents
3R-indolylmethyl-6R-aliphatic amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses (3R,6R) -3- (AA-amino n-hexanoyl amino n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-diketone (in the formula, AA is L-Ala residue, Gly residue, L-Met residue and L-IIe residue). Discloses a preparation method thereof, discloses the antitumor activity thereof, discloses the anti-tumor metastasis activity thereof and discloses the anti-inflammatory activity thereof, so that the invention discloses the application thereof in preparing antitumor drugs, anti-tumor metastasis drugs and anti-inflammatory drugs.
Description
Technical Field
The invention relates to (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione. To their preparation, to their antitumor activity, to their antitumor metastatic activity, and to their anti-inflammatory activity, and thus to their use in the preparation of antitumor, antitumor metastatic and anti-inflammatory drugs. 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 inhibited tumor metastasis to the lung in C57BL/6 mice at a dose of 5. mu. mol/kg. However, the lowest effective dose is 5. mu. mol/kg. 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, it was found that acylation of n-butylamino group of diketopiperazine of R, R-configuration with aminocaproic acid acylated with aliphatic amino acids (L-Ala, Gly, L-Met and L-IIe) not only reduced the minimum effective anti-tumor metastasis dose to 0.5. mu. mol/kg, but also reduced the minimum effective anti-tumor and anti-inflammatory doses to 0.5. mu. mol/kg. Because the toxic and side effects of the medicine can disappear along with the reduction of the dosage, the reduction of the effective dosage by 10 times shows that the structure modification has outstanding technical effect. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention provides (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione of the formula (wherein AA is a L-Ala residue, a Gly residue, a L-Met residue and a L-IIe residue).
In a second aspect, the present invention provides a process for the synthesis of (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is a L-Ala residue, a Gly residue, a L-Met residue and a L-IIe residue), which comprises:
(1) D-Boc-Lys (Cbz) and D-Trp-OBzl are condensed to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) removing Boc from D-Boc-Lys (Cbz) -D-Trp-OBzl in ethyl acetate solution of hydrogen chloride to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) cyclizing D-Lys (Cbz) -D-Trp-OBzl in saturated ethyl acetate containing 5% sodium bicarbonate water solution to obtain (3R,6R) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione (1);
(4) the compound 1 is subjected to hydrogenolysis to remove benzyloxycarbonyl to obtain (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione (2);
(5) condensing amino methyl hexanoate and Boc-AA to obtain Boc-AA-amino methyl hexanoate;
(6) carrying out saponification on Boc-AA-amino-n-hexanoic acid methyl ester to remove methyl ester to obtain Boc-AA-amino-n-hexanoic acid;
(7) condensing the compound 2 and Boc-AA-amino-n-hexanoic acid to obtain (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) piperazine-2, 5-dione;
(8) boc removal of (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) piperazine-2, 5-dione in a solution of hydrogen chloride in ethyl acetate gives (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione.
In a third aspect of the present invention, (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Ala residue, Gly residue, L-Met residue and L-IIe residue) was evaluated for its activity in inhibiting the metastasis of lung cancer in C57BL/6 mice.
A fourth aspect of the present invention is to evaluate the inhibitory effect of (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Ala residue, Gly residue, L-Met residue and L-IIe residue) on the inflammation in ICR mice.
The fifth aspect of the present invention is to evaluate the use of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Ala residue, Gly residue, L-Met residue and L-IIe residue) for inhibiting tumor growth in S180 mice.
Drawings
FIG. 1 is a synthetic route for (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a-g) AA is the L-Ala residue in 3a-6 a; AA in 3b-6b is Gly residue; AA in 3c-6c is L-Met residue; AA in 3d-6d is L-Ile residue; i) dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), Tetrahydrofuran (THF); ii) a solution of hydrogen chloride in ethyl acetate; iii) Ethyl acetate, 5%Sodium bicarbonate; iv) Dimethylformamide (DMF), Pd/C, H2(ii) a v) Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), N, N-Dimethylformamide (DMF); vi) methanol, NaOH (2M).
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 solid. 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,6R) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (1)
3.45g (6.2mmol) of D-Boc-Lys (Cbz) -D-Trp-OBzl was dissolved in 150mL of ethyl acetate. After the resulting solution was washed three times with a 5% aqueous solution of sodium hydrogencarbonate, 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,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2)
Add 200mg Pd/C to a solution of 1.9g (4.2mmol) of (3R,6R) -3- (benzyloxycarbonylamino N-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (1) in 20mL of anhydrous N, N-Dimethylformamide (DMF) and let H in at room temperature2And (5) 48 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 amino-n-hexanoic acid methyl ester
5.6mL of thionyl chloride was added dropwise to 40mL of methanol, and after stirring for 30min, 2.62g (20mmol) of aminohexanoic acid was added and the mixture was stirred at room temperature for 12 hours. Pumping the reaction solution under reduced pressure by using a water pump under stirring of the reaction compound in a 37 ℃ warm water bath, adding dry methanol for dissolving, then pumping the reaction solution under reduced pressure by using a water pump, and repeating the steps for 3 times; the suspension was suspended by adding anhydrous ether, and the mixture was dried under reduced pressure with stirring in a warm water bath at 37 ℃ and repeated 3 times to obtain 2.72g (95%) of the title compound. ESI-MS (M/e) 145[ M + H]+
EXAMPLE 6 preparation of Boc-Ala-amino-n-hexanoic acid methyl ester (3a)
Using the method of example 1, from 945mg (5.0mmol) Boc-Ala and 720mg (5.0mmol) methyl aminohexanoate, 1480mg (94%) of the title compound is obtained as a colorless solid. ESI-MS (M/e) 317[ M + H ]]+。
EXAMPLE 7 preparation of Boc-Gly-amino-n-hexanoic acid methyl ester (3b)
Using the method of example 1, from 880mg (5.0mmol) Boc-Gly and 720mg (5.0mmol) methyl aminohexanoate, 1390mg (92%) of the title compound were obtained as colorless solid. ESI-MS (M/e):303[ M + H]+。
EXAMPLE 8 preparation of Boc-Met-amino-n-hexanoic acid methyl ester (3c)
Using the method of example 1, from 1245mg (5.0mmol) Boc-Met and 720mg (5.0mmol) methyl aminohexanoate, 1540mg (82%) of the title compound as a colorless solid. ESI-MS (M/e):377[ M + H]+。
EXAMPLE 9 preparation of Boc-Ile-amino-n-hexanoic acid methyl ester (3d)
Using the method of example 1, from 1160mg (5.0mmol) Boc-Ile and 720mg (5.0mmol) methyl aminohexanoate 1580mg (89%) of the title compound was obtained as a colorless solid. ESI-MS (M/e):359[ M + H]+。
EXAMPLE 10 preparation of Boc-Ala-amino-n-hexanoic acid (4a)
0.94g (3.0mmol) Boc-Ala-amino-n-hexanoic acid methyl ester (3a) in 20mL CH3OH is dissolved. And adding NaOH aqueous solution (2M) into the obtained solution under the stirring of ice bath to adjust the pH value to 12, and stirring and reacting for 6 hours in ice bath. The reaction solution was saturated KHSO with ice-bath stirring4The pH value of the solution is adjusted to 7, and the obtained solution is concentrated under reduced pressure. The residue was treated with 5% KHSO4The aqueous solution adjusted the pH to 2. The resulting solution was extracted 3 times with ethyl acetate, and the ethyl acetate solution was washed with saturated aqueous NaCl solution to neutrality. Anhydrous Na for ethyl acetate layer2SO4Drying for 12h, filtering, and concentrating the filtrate under reduced pressure to dryness. 1.12g of the title compound are obtained as a pale yellow syrup. ESI-MS (M/e):303[ M + H]+。
EXAMPLE 11 preparation of Boc-Gly-amino-n-hexanoic acid (4b)
From 0.90g (3.0mmol) Boc-Gly-amino-n-hexanoic acid methyl ester (3b) 1.10g of the title compound were obtained as light yellow syrup using the method of example 10. ESI-MS (M/e):289[ M + H]+。
EXAMPLE 12 preparation of Boc-Met-amino-n-hexanoic acid (4c)
From 1.12g (3.0mmol) Boc-Met-amino-n-hexanoic acid methyl ester (3c) 1.50g of the title compound was obtained as light yellow syrup using the procedure of example 10. ESI-MS (M/e) 363[ M + H]+。
EXAMPLE 13 preparation of Boc-Ile-amino-n-hexanoic acid (4d)
From 1.07g (3.0mmol) of Boc-Ile-amino-n-hexanoic acid methyl ester (3d) 1.35g of the title compound was obtained as light yellow syrup using the method of example 10. ESI-MS (m/e):345[M+H]+。
EXAMPLE 14 preparation of (3R,6R) -3- (Boc-Ala-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a)
From 1120mg of Boc-Ala-amino-n-hexanoic acid (4a) in pale yellow syrup and 940mg (3.0mmol) (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) by the method of example 1, 780mg (44%) of the title compound are obtained as colorless solid. ESI-MS (M/e):599[ M + H]+；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.896(m,1H),8.975(d,J＝1.5Hz,1H),7.966(d,J＝1.5Hz,1H),7.741(t,J＝5.4Hz,1H),7.581(m,2H),7.306(m,1H),7.048(m,2H),6.927(t,J＝7.2Hz,2H),6.820(d,J＝7.5Hz,1H),4.130(m,1H),3.919(m,1H),3.515(m,1H),3.269(dd,J1＝14.4Hz,J2＝4.2Hz,1H),3.012(m,3H),2.766(m,2H),2.014(t,J＝7.2Hz,2H),1.420(m,2H),1.367(m,11H),1.255(m,6H),0.952(m,3H),0.585(m,3H)。
EXAMPLE 15 preparation of (3R,6R) -3- (Boc-Gly-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b)
From 1100mg of Boc-Gly-amino-n-hexanoic acid (4b) and 940mg (3.0mmol) of (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) as pale yellow syrup using the method of example 1, 390mg (22%) of the title compound are obtained as colorless solid. ESI-MS (M/e):585[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.881(s,1H),8.044(d,J＝1.8Hz,1H),7.937(d,J＝1.8Hz,1H),7.721(t,J＝5.1Hz,1H),7.576(m,2H),7.298(d,J＝6.9Hz,1H),7.023(m,2H),6.912(m,2H),4.110(m,1H),3.482(m,2H),3.345(m,1H),3.247(dd,J1＝4.2Hz,J2＝14.4Hz,1H),3.022(m,3H),2.764(m,2H),2.006(t,J＝7.2Hz,2H),1.436(m,12H),1.224(m,2H),0.964(m,3H),0.554(m,3H)。
EXAMPLE 16 preparation of (3R,6R) -3- (Boc-Met-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c)
From 1500mg of Boc-Met-amino-n-hexanoic acid (4c) and 940mg (3.0mmol) of (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) as a pale yellow syrup, 310mg (16%) of the title compound were obtained using the method of example 1 as colorless solid. ESI-MS (M/e) 659[ M + H ] +; 1H NMR (300MHz, DMSO-d6): δ/ppm ═ 10.886(d, J ═ 1.5Hz,1H),8.059(d, J ═ 2.1Hz,1H),7.949(d, J ═ 2.1Hz,3H),7.787(t, J ═ 5.4Hz,1H),7.575(m,2H),7.298(d, J ═ 8.1Hz,1H),7.022(m,2H),6.926(m,2H),4.111(m,1H),3.931(m,1H),3.499(m,1H),3.248(dd, J1 ═ 14.4Hz, J2 ═ 4.2Hz,1H),2.989(m,3H),2.734(m,2H),2.416(m,1H),2.002(m,5H), 1.552 (m,1H), 1H, 200.960H, 13H, 1H, 200H, 1H, and m, 1H).
EXAMPLE 17 preparation of (3R,6R) -3- (Boc-Ile-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5d)
From 1350mg of Boc-Ile-amino-n-hexanoic acid (4d) and 940mg (3.0mmol) of (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) as a pale yellow syrup, 710mg (37%) of the title compound were obtained using the method of example 1 as a colorless solid. ESI-MS (M/e):641[ M + H]+；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.890(s,1H),8.070(d,J＝1.5Hz,1H),7.956(d,J＝1.5Hz,1H),7.845(t,J＝5.1Hz,1H),7.586(m,2H),7.295(m,1H),7.031(m,2H),6.923(t,J＝7.2Hz,1H),4.109(m,1H),3.732(t,J＝8.4Hz,2H),3.497(m,1H),3.248(dd,J1＝14.1Hz,J2＝3.9Hz,1H),3.033(m,3H),2.743(m,2H),1.997(t,J＝7.5Hz,2H),1.635(m,1H),1.444(m,15H),1.204(m,3H),0.976(m,3H),0.800(m,6H),0.556(m,3H)。
EXAMPLE 18 preparation of (3R,6R) -3- (Ala-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a)
From 300mg (0.5mmol) (3R,6R) -3- (Boc-Ala-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a) 230mg (92%) of the title compound were obtained as colorless solid according to the method of example 3. ESI-MS (M/e):499[ M + H]+；Mp148-150℃；
＝-19.6(C＝0.1,H2O)；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.901(d,J＝1.5Hz,1H),8.250(t,J＝5.5Hz,1H),8.020(d,J＝2Hz,1H),7.922(d,J＝2Hz,1H),7.601(t,J＝5.5Hz,1H),7.569(d,J＝8Hz,1H),7.302(d,J＝8Hz,2H),7.016(m,2H),6.915(m,1H),4.105(m,1H),3.637(m,1H),3.499(m,1H),3.236(dd,J1＝14.5Hz,J2＝4.5Hz,1H),3.059(m,3H),2.757(m,2H),2.016(m,2H),1.440(m,4H),1.255(m,5H),0.974(m,3H),0.612(m,3H)。
EXAMPLE 19 preparation of (3R,6R) -3- (Gly-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6b)
From 300mg (0.5mmol) (3R,6R) -3- (Boc-Gly-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b) 220mg (90%) of the title compound are obtained as colorless solid according to the method of example 3. ESI-MS (M/e):499[ M + H]+；Mp148–150℃；
＝-19.6(C＝0.1,H2O)；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.901(d,J＝1.5Hz,1H),8.250(t,J＝5.5Hz,1H),8.020(d,J＝2Hz,1H),7.922(d,J＝2Hz,1H),7.601(t,J＝5.5Hz,1H),7.569(d,J＝8Hz,1H),7.302(d,J＝8Hz,2H),7.016(m,2H),6.915(m,1H),4.105(m,1H),3.637(m,1H),3.499(m,1H),3.236(dd,J1＝14.5Hz,J2＝4.5Hz,1H),3.059(m,3H),2.757(m,2H),2.016(m,2H),1.440(m,4H),1.255(m,5H),0.974(m,3H),0.612(m,3H)。
EXAMPLE 20 preparation of (3R,6R) -3- (Met-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6c)
From 300mg (0.4mmol) (3R,6R) -3- (Boc-Met-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c) 120mg (47%) of the title compound were obtained as colorless solid according to the method of example 3. ESI-MS (M/e):559[ M + H]+；Mp137–138℃；
＝-9.8(C＝0.1,H2O)；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.922(s,1H),8.572(s,1H),8.294(s,3H),8.035(s,1H),7.936(s,1H),7.592(m,2H),7.308(d,J＝8.7Hz,1H),7.028(m,2H),6.931(m,1H),4.115(m,1H),3.808(m,1H),3.505(m,1H),3.147(m,4H),2.768(m,2H),2.019(m,5H),1.450(m,6H),1.258(m,3H),1.063(m,3H),0.618(m,3H)。
EXAMPLE 21 preparation of (3R,6R) -3- (Ile-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6d)
From 300mg (0.5mmol) (3R,6R) -3- (Boc-Ile-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5d) 230mg (92%) of the title compound were obtained as colorless solid according to the method of example 3. ESI-MS (M/e):541[ M + H]+；Mp162–164℃；
＝-11.2(C＝0.1,H2O)；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.914(d,J＝1.5Hz,1H),8.416(t,J＝5.0Hz,1H),8.021(d,J＝2.0Hz,1H),7.923(d,J＝2.0Hz,1H),7.877(m,2H),7.618(t,J＝5.5Hz,1H),7.569(d,J＝8.0Hz,1H),7.297(d,J＝8.0Hz,1H),7.035(m,2H),6.921(m,1H),4.103(m,1H),3.506(m,2H),3.234(dd,J1＝14.5Hz,J2＝4.0Hz,1H),3.181(m,1H),3.024(m,2H),2.757(m,2H),2.019(t,J＝7.5Hz,2H),1.784(m,1H),1.444(m,5H),1.246(m,2H),1.093(m,1H),0.975(m,1H),0.870(m,6H),0.593(m,3H)。
EXAMPLE 22 preparation of (3R,6R) -3- (Boc-aminocaproyl butylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (7)
From 0.97g (4.2mmol) Boc-aminocaproic acid and 1.9g (3.5mmol) (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) 0.641g (21%) of the title compound were obtained as colorless solid by the method of example 1. ESI-MS (M/e):528[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝10.872(s,1H),8.035(d,J＝1.8Hz,1H),7.928(d,J＝1.8Hz,1H),7.560(m,2H),7.303(d,J＝5.7Hz,1H),7.034(m,2H),6.925(t,J＝7.5Hz,1H),6.763(t,J＝5.1Hz,1H),4.108(m,1H),3.499(m,1H),3.246(dd,J1＝14.4Hz,J2＝4.2Hz,1H),3.009(dd,J1＝14.4Hz,J2＝4.2Hz,1H),2.890(q,J＝6.6Hz,2H),2.750(q,J＝6.6Hz,2H),2.002(t,J＝7.2Hz,2H),1.465(m,2H),1.350(m,12H),1.205(m,3H),0.951(m,3H),0.553(m,3H)。
EXAMPLE 23 preparation of (3R,6R) -3- (aminocaproyl-butylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (8)
From 2.21g (4mmol) (3R,6R) -3- (Boc-aminocaproyl butylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (7) 1.45g (82%) of the title compound was obtained as colorless powder using the method of example 3. ESI-MS (M/e):428[ M + H]+；1H NMR(300MHz,DMSO-d6):δ/ppm＝11.003(s,1H),8.086(s,1H),7.999(s,1H),7.748(s,1H),7.578(s,J＝8.1Hz,1H),7.314(m,J＝8.1Hz,1H),7.023(m,2H),6.926(m,1H),4.117(m,1H),3.502(m,1H),3.269(m,1H),3.041(m,1H),2.736(m,4H),2.030(m,2H),1.542(m,4H),1.260(m,2H),0.981(m,3H),0.590(m,3H)。
EXAMPLE 24 determination of the anti-tumor metastasis Activity of Compounds 6a-d
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 fixed inbred C57BL/6 male mice,the right anterior limb axillary skin of the mouse 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 dosed 1 time daily for 12 consecutive days with tumor volumes measured and recorded every two days, with an oral administration of either the putative anti-tumor metastasis peptides Arg-Gly-Asp-Ser (RGDS) in saline (dose of 20. mu. mol/kg/day) or compounds 6a-d in saline (dose of 0.5. mu. mol/kg/day) or compound 8 in saline (dose of 5. mu. mol/kg/day) or 10 mL/kg/day. 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. Compounds 6a-d were not only effective in inhibiting tumor lung metastasis at 0.5 μmol/kg dose, but also had no significant difference in activity from RGDS, which was 40-fold higher in dose and compound 8, which was 10-fold higher in dose. These data indicate that the present invention has significant technical effects.
TABLE 1 antitumor metastatic Activity of Compounds 6a-d
a) P <0.01 to saline; b) p <0.01 to saline, p >0.05 to RGDS compound 8; n is 11
EXAMPLE 25 determination of the anti-tumor growth Activity of Compounds 6a-d
Doxorubicin, compound 8 and compounds 6a-d were all dissolved in saline prior to assay for administration to S180 mice. Taking S180 ascites tumor liquid which is inoculated in a male ICR mouse and grows vigorously for 10 days in a sterile environment, diluting the S180 ascites tumor liquid into liquid (1:2) by using normal saline, fully mixing the liquid, dyeing the tumor cell suspension by using freshly prepared 0.2% trypan blue, uniformly mixing the liquid and the liquid, counting the liquid according to a white cell counting method, wherein the blue-dyed cell is a dead cell, and the non-dyed cell is a live cell. Press thinCell concentration is 4 check viable cell number/4 × 104The cell density was calculated as x dilution factor ═ cell number/mL, and the cell survival rate was calculated as live cell number/(live cell number + dead cell number) × 100%. Homogenizing tumor solution with survival rate of more than 90% to density of 2.0 × 107Cell suspension per mL. This cell suspension was inoculated subcutaneously (0.2 mL/mouse) in the right axilla of a mouse to prepare S180 tumor-bearing mice. 24h after inoculation, S180 tumor-bearing mice were intraperitoneally injected daily with a physiological saline solution of doxorubicin (at a dose of 2. mu. mol/kg/day g), or daily orally administered with a physiological saline solution of Compound 8 (at a dose of 5. mu. mol/kg/day), or daily orally administered with a physiological saline solution of Compounds 6a-d (at a dose of 0.5. mu. mol/kg/day). The administration is once daily for 12 days. The day after the last dose, tumor volume was measured, cervical spine was removed under ether anesthesia and sacrificed, then the right axillary tumor growth site of the mouse was fixed with forceps, and the skin was excised and the tumor was blunt-stripped and weighed. Efficacy was expressed as tumor weight (mean ± SD g), and data were analyzed by t-test and variance. The results are shown in Table 2. The data that compounds 6a-d are not only effective in inhibiting tumor growth at 0.5 μmol/kg dose, but also that there is no significant difference in activity from compound 8, which is 10 times higher than them at the dose, indicate that the present invention has significant technical effects.
TABLE 2 Effect of Compounds 6a-d on tumor growth in S180 mice
a) P <0.01 to saline, p <0.05 to compound 8; n is 12.
EXAMPLE 26 determination of the anti-inflammatory Activity of Compounds 6a-d
Since xylene-induced ear swelling in mice is recognized as an acute inflammation model, the present invention measures the therapeutic effect of compounds 6b-d on a xylene-induced ear swelling model in mice. Because aspirin is a positive drug for treating acute inflammation, aspirin is selected as a positive control in the present invention. ICR male mice (body weight 20 ± 2g) were allowed to rest for 2 days at 22 ℃ with free access to water and food. Thereafter, the mice were randomly divided into a saline group (dose of 0.2 mL/mouse), an aspirin group (dose of 1.11mmol/kg), a compound 8 group (dose of 5. mu. mol/kg) and compound 6a-d groups (dose of 0.5. mu. mol/kg), and 12 mice were each group. Mice were tested either orally with normal saline, orally with aspirin, orally with compound 8, or orally with compounds 6a-d, as indicated. After 30min of administration, the left auricle of the mouse was evenly smeared with 30 μ L of xylene, and after 2h, the mouse was subjected to ether anesthesia, the neck was cut off, the left and right ears were cut off, round ears were taken at the same positions of the two ears by a 7mm punch, and the difference in swelling between the two ears was weighed and found to be the swelling degree. Namely the swelling degree is equal to the weight of the left ear disk to the weight of the right ear disk. The results are shown in Table 3. Compounds 6a-d were not only effective at 0.5 μmol/kg dose in inhibiting xylene-induced ear swelling in mice, but also had no significant difference in activity from compound 8, which was 10-fold higher than them at the dose. These data indicate that the present invention has significant technical effects.
TABLE 3 Effect of Compounds 6a-d on xylene-induced ear swelling in mice
a) P <0.01 to saline, p >0.05 to compound 8; n is 12.
Claims (5)
1. (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione of the formula,
wherein AA is a L-Ala residue, a Gly residue, a L-Met residue and a L-IIe residue.
2. A process for the preparation of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione as claimed in claim 1, which comprises:
(1) D-Boc-Lys (Cbz) and D-Trp-OBzl are condensed to obtain D-Boc-Lys (Cbz) -D-Trp-OBzl;
(2) removing Boc from D-Boc-Lys (Cbz) -D-Trp-OBzl in ethyl acetate solution of hydrogen chloride to obtain D-Lys (Cbz) -D-Trp-OBzl;
(3) D-Lys (Cbz) -D-Trp-OBzl is dissolved in ethyl acetate, and the obtained solution is washed by 5 percent sodium bicarbonate water solution, and then cyclization is carried out to generate (3R,6R) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione (1);
(4) the compound 1 is subjected to hydrogenolysis to remove benzyloxycarbonyl to obtain (3R,6R) -3- (amino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione (2);
(5) condensing amino methyl hexanoate and Boc-AA to obtain Boc-AA-amino methyl hexanoate;
(6) carrying out saponification on Boc-AA-amino-n-hexanoic acid methyl ester to remove methyl ester to obtain Boc-AA-amino-n-hexanoic acid;
(7) condensing the compound 2 and Boc-AA-amino-n-hexanoic acid to obtain (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) piperazine-2, 5-dione;
(8) boc removal of (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) piperazine-2, 5-dione in a solution of hydrogen chloride in ethyl acetate gives (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione.
3. Use of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione according to claim 1 for the preparation of a medicament against tumor metastases.
4. The use of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione according to claim 1 for the preparation of an antitumor medicament.
5. The use of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) piperazine-2, 5-dione according to claim 1 for the preparation of an anti-inflammatory medicament.
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