CN108947979B - 3R-indolylmethyl-6R-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof - Google Patents
3R-indolylmethyl-6R-polar 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-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (AA is L-Lys residue, L-Ser residue and L-Thr residue) with the following formula. 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 the butylamino group of the R, R-configured diketopiperazine with aminocaproic acid acylated with polar amino acids (L-Lys, L-Ser and L-Thr) not only reduced the minimum effective dose against tumor metastasis to 0.5. mu. mol/kg, but also reduced the minimum effective dose against both tumor and inflammation 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 (AA is a L-Lys residue, a L-Ser residue and a L-Thr residue).
The second aspect of the present invention provides a method for synthesizing (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (AA is L-Lys residue, L-Ser residue and L-Thr residue), which comprises:
(1) D-Boc-Lys (Cbz) and D-Trp-OBzl are condensed to obtain Boc-Lys (Cbz) -Trp-OBzl;
(2) Boc-Lys (Cbz) -Trp-OBzl is subjected to Boc removal in an ethyl acetate solution of hydrogen chloride to obtain Lys (Cbz) -Trp-OBzl;
(3) lys (Cbz) -Trp-OBzl is cyclized in saturated ethyl acetate containing 5 percent of sodium bicarbonate water solution 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-n-hexanoic acid methyl ester with Boc-AA (AA is Lys (Boc) residue, Ser residue and Thr residue) to obtain Boc-AA-amino-n-hexanoic acid methyl ester (3 a-c);
(6) saponifying and removing methyl ester from the compound 3a-c to obtain Boc-AA-amino-n-hexanoic acid (4 a-c);
(7) condensing the compound 2 and a compound 4a-c to obtain (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-diketone (5 a-c);
(8) boc removal of the compounds 5a-c in hydrogen chloride in ethyl acetate gives (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (6 a-c).
The third aspect of the present invention is to evaluate the anti-lung cancer metastasis activity of (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (AA is L-Lys residue, L-Ser residue and L-Thr residue) 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 (AA is L-Lys residue, L-Ser residue and L-Thr residue) on ICR mouse inflammation.
A 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 (AA is the L-Lys residue, L-Ser residue and L-Thr residue) for inhibiting tumor growth in S180 mice.
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FIG. 1 is a scheme for the synthesis of (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a-e) AA in 3a-6a is the L-Lys residue; AA in 3b-6b is Ser residue; AA in 3c-6c is an L-Thr 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 Boc-Lys (Cbz) -Trp-OBzl
7.7g (20mmol) of 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. Thereafter, 8.0g (25mmol) of 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 Lys (Cbz) -Trp-OBzl
3.8g (5mmol) Boc-Lys (Cbz) -Trp-OBzl was slowly mixed with 52mL of an ethyl acetate solution of hydrogen chloride (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) Lys (Cbz) -Trp-OBzl were dissolved in 150mL ethyl acetate. The resulting solution was washed three times with 5% strength aqueous sodium bicarbonate solution, and acetic acidThe ethyl ester solution was stirred at room temperature for 12h to precipitate a colorless solid sufficiently. 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)
To a solution of 1.9g (4.2mmol) of (3R,6R) -3- (benzyloxycarbonyl butylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (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 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 activation for 30min, 2.62g (20mmol) of aminohexanoic acid was added thereto and 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-Lys (Boc) -amino-n-hexanoic acid methyl ester (3a)
From 1730mg (5.0mmol) Boc-Lys (Boc) and 720mg (5.0mmol) methyl aminohexanoate, 1970mg (83%) of the title compound was obtained as colorless solid by the method of example 1. ESI-MS (M/e):474[ M + H]+。
EXAMPLE 7 preparation of Boc-Ser-amino-n-hexanoic acid methyl ester (3b)
From 1030mg (5.0mmol) Boc-Ser and 720mg (5.0mmol) methyl aminohexanoate, 1410mg (85%) of the title compound were obtained as colorless solid by the method of example 1. ESI-MS (M/e):333[ M + H]+。
EXAMPLE 8 preparation of Boc-Thr-amino-n-hexanoic acid methyl ester (3c)
From 1100mg (5.0mmol) Boc-Thr and 720mg (5.0mmol) methyl aminohexanoate 1440mg (83%) of the title compound were obtained as colorless solid by the method of example 1. ESI-MS (M/e):347[ M + H]+。
EXAMPLE 9 preparation of Boc-Lys (Boc) -amino-n-hexanoic acid (4a)
1.41g (3.0mmol) Boc-Lys (Boc) -amino-n-hexanoic acid methyl ester (3a) was reacted with 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. Stirring in ice bath, and reacting with saturated KHSO4The 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. 1.58g of the title compound are obtained in the form of a pale yellow syrup. ESI-MS (M/e):460[ M + H]+。
EXAMPLE 10 preparation of Boc-Ser-amino-n-hexanoic acid (4b)
0.99g (3.0mmol) of Boc-Ser-amino-n-hexanoic acid methyl ester (3b) was saponified as in example 9 to give 1.16g of a pale yellow syrup as the title compound. ESI-MS (M/e):319[ M + H]+。
EXAMPLE 11 preparation of Boc-Thr-amino-n-hexanoic acid (4c)
1.03g (3.0mmol) Boc-Thr-amino-n-hexanoic acid methyl ester (3c) was saponified as in example 9 to give 1.20g pale yellow syrup as the title compound. ESI-MS (M/e):333[ M + H]+。
EXAMPLE 12 preparation of (3R,6R) -3- (Boc-Lys (Boc) -amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a)
From 1580mg of Boc-Lys (Boc) -amino-n-hexanoic acid (4a) and 940mg (3.0mmol) of (3R,6R) -3-butylamino-6- (indole-3-methyl) -piperazine-2, 5-dione (2) as pale yellow syrup, 1060mg (47%) of the title compound was obtained using the method of example 1 as colorless solid. ESI-MS (M/e):756[ M + H]+；1H NMR(300MHz,DMSO-d6):/ppm＝10.871(s,1H),8.026(d,J＝1.8Hz,1H),7.929(d,J＝1.8Hz,1H),7.725(t,J＝5.7Hz,1H),7.557(m,2H),7.301(d,J＝8.1Hz,1H),7.040(m,2H),6.929(m,1H),6.720(m,1H),4.112(m,1H),3.811(m,1H),3.504(m,1H),3.249(dd,J1＝14.4Hz,J2＝4.2Hz,1H),3.071(m,3H),2.874(m,2H),2.760(m,2H),2.001(m,2H),1.389(m,25H),1.193(m,6H),0.975(m,3H),0.608(m,3H)。
EXAMPLE 13 preparation of (3R,6R) -3- (Boc-Ser-amino-n-butylaminon-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b)
From 1160mg of Boc-Ser-amino-n-hexanoic acid (4b) and 940mg (3.0mmol) of (3R,6R) -3-butylamino-6- (indole-3-methyl) -piperazine-2, 5-dione (2) as pale yellow syrup, 480mg (26%) of the title compound were obtained as colorless solid by the method of example 1. ESI-MS (M/e) 615[ M + H]+；1H NMR(300MHz,DMSO-d6):/ppm＝10.882(s,1H),8.046(s,1H),7.940(s,1H),7.753(s,1H),7.578(m,2H),7.306(d,J＝7.8Hz,1H),7.044(m,2H),6.930(t,J＝7.2Hz,1H),6.555(d,J＝8.1Hz,2H),4.793(t,J＝5.1Hz,1H),4.113(m,1H),3.925(m,1H),3.507(m,3H),3.270(m,1H),3.012(m,3H),2.755(m,2H),2.012(m,3H),1.386(m,13H),1.192(m,3H),0.969(m,3H),0.594(m,3H)。
EXAMPLE 14 preparation of (3R,6R) -3- (Boc-Thr-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c)
From 1200mg of Boc-Thr-amino-n-hexanoic acid (4c) and 940mg (3.0mmol) of (3R,6R) -3-butylamino-6- (indol-3-methyl) -piperazine-2, 5-dione (2) as pale yellow syrup using the method of example 1, 830mg (44%) of the title compound are obtained as colorless solid. ESI-MS (M/e):629[ M + H]+；1H NMR(500MHz,DMSO-d6):/ppm＝10.873(s,1H),8.035(s,1H),7.932(s,1H),7.739(m,1H),7.578(m,1H),7.303(m,1H),7.047(m,2H),6.931(t,J＝7.2Hz,1H),6.259(m,1H),4.721(m,1H),4.114(m,1H),3.875(m,1H),3.791(m,1H),3.504(m,1H),3.269(m,1H),3.031(m,3H),2.758(m,2H),2.007(t,J＝7.2Hz,2H),1.393(m,13H),1.227(m,3H),1.014(m,6H),0.603(m,3H)。
EXAMPLE 15 preparation of (3R,6R) -3- (Lys-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a)
From 300mg (0.4mmol) (3R,6R) -3- (Boc-Lys (Boc) -amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a) 190mg (89%) of the title compound was obtained as colorless solid according to the method of example 3. ESI-MS (M/e):556[ M + H]+；Mp:114–115℃；
(C＝0.1,H2O)；1H NMR(500MHz,DMSO-d6):/ppm＝10.901(s,1H),8.035(m,1H),7.932(m,1H),7.783(t,J＝5.5Hz,1H),7.570(d,J＝7.5Hz,2H),7.295(d,J＝8Hz,1H),7.017(m,2H),6.923(m,1H),4.105(m,1H),3.498(m,1H),3.225(m,3H),3.030(m,4H),2.748(m,2H),2.005(t,J＝7.5Hz,2H),1.462(m,3H),1.342(m,6H),1.217(m,3H),0.972(m,3H),0.594(m,3H)。
EXAMPLE 16 preparation of (3R,6R) -3- (Ser-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6b)
From 300mg (0.5mmol) (3R,6R) -3- (Boc-Ser-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b) 210mg (87%) of the title compound were obtained as colorless solid according to the method of example 3. ESI-MS (M/e) 515[ M + H]+；Mp:147–148℃；
1H NMR(300MHz,DMSO-d6):/ppm＝10.935(s,1H),8.458(s,1H),8.157(s,3H),8.059(s,1H),7.947(s,1H),7.648(m,1H),7.547(d,J＝8.1Hz,1H),7.307(d,J＝7.8Hz,1H),7.045(m,2H),6.942(m,1H),5.490(s,1H),4.115(s,1H),3.753(m,3H),3.503(m,1H),3.398(m,4H),3.111(m,5H),2.762(m,2H),2.028(m,2H),1.453(m,4H),1.241(m,2H),1.097(t,J＝6.6Hz,1H),0.976(m,3H),0.602(m,3H)。
EXAMPLE 17 preparation of (3R,6R) -3- (Thr-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6c)
From 300mg (0.4mmol) of (3R,6R) -3- (Boc-Thr-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c) 230mg (94%) of the title compound are obtained as colorless solid according to the method of example 3. ESI-MS (M/e):529[ M + H]+；Mp:147–148℃；
1H NMR(500MHz,DMSO-d6):/ppm＝10.903(s,1H),8.468(t,J＝5.5Hz,1H),8.020(d,J＝2Hz,1H),7.921(d,J＝2Hz,1H),7.853(s,2H),7.611(t,J＝5.5Hz,1H),7.568(d,J＝8Hz,1H),7.297(d,J＝8.0Hz,1H),7.016(m,1H),6.923(m,1H),5.484(s,1H),4.104(m,1H),3.448(d,J＝7.0Hz,1H),3.235(m,1H),3.145(m,1H),3.048(m,2H),2.757(m,2H),2.019(t,J＝7.5Hz,2H),1.435(m,4H),1.243(m,2H),1.120(d,J＝6.5Hz,3H),0.989(m,3H),0.626(m,3H)。
EXAMPLE 18 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-butylamino-6- (indole-3-methyl) -piperazine-2, 5-dione (2) 0.641g (21%) of the title compound was obtained as a colorless solid using the method of example 1. ESI-MS (M/e):528[ M + H]+；1H NMR(300MHz,DMSO-d6):/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 19 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]+；1HNMR(300MHz,DMSO-d6):/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 20 determination of the anti-tumor metastasis Activity of Compounds 6a-c
Lewis mouse lung carcinoma cells (LLC, purchased from ATCC) used for the determination model) Inoculating with DMEM medium (containing 10% inactivated fetal calf serum, 1 × 10)5U/L penicillin and 100mg/L streptomycin), passaging every two days according to an adherent cell culture method, enriching cells, digesting the cells when the cells are in a good growth state and in a logarithmic growth phase, and adjusting the cell density to 1 × 10 by using physiological saline7one/mL. Staining with placental blue to count viable cells>95 percent, inbred line C57BL/6 male mice (SPF grade, weight 20 +/-2 g) are taken, mice are fixed by the left hand, the right forelimb armpit skin of the mice is disinfected by 75 percent ethanol, LLC tumor cell suspension is injected to the armpit part of the mice by holding 1mL sterile injector in the right hand, each mouse is injected with 0.2mL, after the mice are inoculated for 10 days, tumors with the diameter of about 4-5mm are grown out, namely tumor sources, the Lewis lung cancer tumor-bearing mice inoculated for 10 days are anesthetized by ethyl ether, cervical vertebra is removed and killed, the mice are soaked for 10min by 75 percent ethanol, disinfected and tumor bodies are stripped on an ultra-clean workbench, well-grown tumor tissues are selected to be cut into pieces in an aseptic flat dish, the pieces are placed in a tissue homogenizer made of glass, physiological saline with the temperature of 4 ℃ is added according to the ratio of the weight of the tumor mass to the volume of 1 to 3(g to mL), the weight of the physiological saline is lightly ground to prepare cell suspension, the cell suspension passes through a 200-mesh cell sieve to prepare the single cell suspension, and the cell density is adjusted to 17one/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 recognized physiological saline solution of the anti-tumor metastasis peptide RGDS (dose of 20. mu. mol/kg/day) or compounds 6a-c (dose of 0.5. mu. mol/kg/day) or compound 8 (dose of 5. mu. mol/kg/day) or compound 8 (dose of 10 mL/kg/day), 1 dose per day, for 12 consecutive days, and tumor volumes were measured and recorded every two days. Measuring tumor volume the next day of last administration, killing by removing cervical vertebra under ether anesthesia, weighing tumor of mouse, collecting lung of mouse, and calculating tumor node transferred from lungAnd (4) counting. Statistical analysis of the data was performed using the t-test. The results are shown in Table 1. Compounds 6a-c 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-c
a) P <0.01 to saline, p >0.05 to RGDS and compound 8; n is 11
EXAMPLE 21 determination of the anti-tumor growth Activity of Compounds 6a-c
Dissolving adriamycin, compound 8 and compounds 6a-c in normal saline before measurement, using the solution for administration to S180 mice, taking S180 ascites tumor fluid which is inoculated to male ICR mice and grows vigorously for 10 days in a sterile environment, diluting the S180 ascites tumor fluid with normal saline to obtain a liquid (1:2), fully mixing, staining tumor cell suspension with freshly prepared 0.2% trypan blue, counting the cells by a white cell counting method after mixing uniformly, wherein the cells stained blue are dead cells, the cells not stained are live cells, and the cell concentration is 4-square live cell number/4 × 104× the cell density is calculated by diluting times of cell number/mL, the cell survival rate is calculated by the cell survival rate of × 100% live cell number/(live cell number + dead cell number), the tumor liquid with the survival rate of more than 90% is prepared into the density of 2.0 × 10 by a homogenization method7Cell 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-c (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. Tumor weight (mean. + -. SD g) tableEfficacy was shown, and data were analyzed using t-test and variance. The results are shown in Table 2. Compounds 6a-e were not only effective at 0.5 μmol/kg dose in inhibiting tumor growth, 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 2 Effect of Compounds 6a-c on tumor growth in S180 mice
a) P <0.01 to saline, p >0.05 to compound 8; n is 12.
EXAMPLE 22 determination of the anti-inflammatory Activity of Compounds 6a-c
Since xylene-induced ear swelling in mice is recognized as an acute inflammation model, the present invention measures the therapeutic effect of compound 6b 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-c 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-c, 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 2. Compounds 6a-c 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-c 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,
2. a process for the preparation of (3R,6R) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione according to claim 1, which comprises:
(1) D-Boc-Lys (Cbz) and D-Trp-OBzl are condensed to obtain Boc-Lys (Cbz) -Trp-OBzl;
(2) Boc-Lys (Cbz) -Trp-OBzl is subjected to Boc removal in an ethyl acetate solution of hydrogen chloride to obtain Lys (Cbz) -Trp-OBzl;
(3) lys (cbz) -Trp-OBzl was dissolved in ethyl acetate, and the resulting solution was washed with 5% aqueous sodium bicarbonate solution, followed by cyclization to give (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 (3 a-c);
(6) saponifying and removing methyl ester from the compound 3a-c to obtain Boc-AA-amino-n-hexanoic acid (4 a-c);
(7) condensing the compound 2 and a compound 4a-c to obtain (3R,6R) -3- (Boc-AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-diketone (5 a-c);
(8) boc removal of the compounds 5a-c in hydrogen chloride in ethyl acetate gives (3R,6R) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (6 a-c).
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 anti-tumor medicament.
5. 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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| EP0181152A1 (en) * | 1984-11-02 | 1986-05-14 | Fujisawa Pharmaceutical Co., Ltd. | Piperazine compound as PAF-antagonist |
| JPS61112060A (en) * | 1984-11-02 | 1986-05-30 | Fujisawa Pharmaceut Co Ltd | Piperazine compound |
| WO2006132594A1 (en) * | 2005-06-10 | 2006-12-14 | Viogard Ab | Novel dipodazine compounds and applications |
| CN104098549A (en) * | 2014-08-01 | 2014-10-15 | 华东理工大学 | Piperazinedione derivative and preparation and application thereof |
| CN105503833A (en) * | 2014-10-20 | 2016-04-20 | 首都医科大学 | Indole piperazine dione compound, and preparation and treatment effect thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0181152A1 (en) * | 1984-11-02 | 1986-05-14 | Fujisawa Pharmaceutical Co., Ltd. | Piperazine compound as PAF-antagonist |
| JPS61112060A (en) * | 1984-11-02 | 1986-05-30 | Fujisawa Pharmaceut Co Ltd | Piperazine compound |
| WO2006132594A1 (en) * | 2005-06-10 | 2006-12-14 | Viogard Ab | Novel dipodazine compounds and applications |
| CN104098549A (en) * | 2014-08-01 | 2014-10-15 | 华东理工大学 | Piperazinedione derivative and preparation and application thereof |
| CN105503833A (en) * | 2014-10-20 | 2016-04-20 | 首都医科大学 | Indole piperazine dione compound, and preparation and treatment effect thereof |
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