CN108976277B

CN108976277B - 3R-indolylmethyl-6S-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof

Info

Publication number: CN108976277B
Application number: CN201710395110.6A
Authority: CN
Inventors: 赵明; 彭师奇; 王玉记; 吴建辉; 张可欣
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2017-05-30
Filing date: 2017-05-30
Publication date: 2021-07-02
Anticipated expiration: 2037-05-30
Also published as: CN108976277A

Abstract

3R-indolylmethyl-6S-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof. The invention discloses (3R,6S) -3- (AA-amino n-hexanoyl amino n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-diketone (wherein AA is L-Asn residue, L-Ser residue and L-Gln 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, and thus the invention discloses the application thereof in preparing antitumor drugs, anti-tumor metastasis drugs and anti-inflammatory drugs.

Description

3R-indolylmethyl-6S-polar amino acid modified piperazine-2, 5-dione, and synthesis, activity and application thereof

Technical Field

The invention relates to (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione. To their preparation, to their antitumor activity, to their tumor metastasis inhibiting activity and to their anti-inflammatory activity, and thus to their use in antitumor, antitumor metastasis inhibiting and anti-inflammatory agents. 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 amino n-butyl group of the diketopiperazine in the S, R-configuration. After 3 years of exploration, the amino n-butyl group of diketopiperazine acylated with amino n-hexanoic acid acylated with polar side chain amino acids (L-Asn, L-Ser and L-Gln) not only can reduce the minimum effective dose of anti-tumor metastasis to 0.5 mu mol/kg, but also can reduce the minimum effective dose of anti-tumor and anti-inflammatory 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,6S) -3- (AA-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione of the formula (wherein AA is a L-Asn residue, a L-Ser residue and a L-Gln residue).

The second aspect of the present invention provides a method for synthesizing (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue), comprising:

(1) D-Boc-Lys (Cbz) and L-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 a saturated ethyl acetate solution of a 5% sodium bicarbonate water solution to obtain (3R,6S) -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,6S) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2);

(5) condensing amino methyl hexanoate and Cbz-AA (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue) to obtain Cbz-AA-amino methyl hexanoate (3 a-c);

(6) saponifying and removing methyl ester from the compound 3a-c to obtain Cbz-AA-amino n-hexanoic acid (4a-c) (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue);

(7) condensing the compound 2 and a compound 4a-c to obtain (3R,6S) -3- (Cbz-AA-amino n-hexanoylamino n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-diketone (5a-c) (wherein AA is an L-Asn residue, an L-Ser residue and an L-Gln residue);

(8) the compound 5a-c is subjected to hydrogenolysis and debenzylation oxycarbonyl to obtain (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a-c) (wherein AA is an L-Asn residue, an L-Ser residue and an L-Gln residue).

The third aspect of the present invention is to evaluate the activity of (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue) 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,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue) on the inflammation of ICR mice.

A fifth aspect of the present invention is the evaluation of the use of (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (wherein AA is L-Asn residue, L-Ser residue and L-Gln residue) for inhibiting tumor growth in S180 mice.

Drawings

FIG. 1 is a scheme for the synthesis of (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a-g) AA is the residue L-Asn in 3a-6 a; AA in 3b-6b is L-Ser residue; 3c-6c, wherein AA is an L-Gln residue, i) Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM); ii) a solution of hydrogen chloride in ethyl acetate; iii) ethyl acetate, 5% aqueous sodium bicarbonate; iv) Pd/C, H₂(ii) a v) 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) -L-Trp-OBzl

To a solution of 1.90g (5.0mmol) of D-Boc-Lys (Cbz) and 20mL of dried Tetrahydrofuran (THF) were added 0.68g (5.0mmol) of 1-hydroxybenzotriazole (HOBt) and 1.24g (6.0mmol) of Dicyclohexylcarbodiimide (DCC) under ice-cooling and stirred for 30 minutes to obtain reaction solution A. 1.47g (5.0mmol) of L-Trp-OBzl was dissolved in 20mL of dry THF, and N-methylmorpholine (NMM) was added to adjust the pH to 9 to obtain reaction solution B. The reaction solution B was added to the reaction solution A and reacted at room temperature for 12 hours, and TLC (methylene chloride/methanol, 40/1) showed completion of the reaction. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 50mL of ethyl acetate. The resulting solution was sequentially saturated NaHCO₃Aqueous solution (25 mL. times.3), saturated aqueous NaCl solution (25 mL. times.3), 5% KHSO₄Aqueous wash (25 mL. times.3), saturated aqueous NaCl wash (25 mL. times.3), saturated aqueous NaHCO₃An aqueous wash (25 mL. times.3) and a saturated aqueous NaCl wash (25 mL. times.3). Anhydrous Na for ethyl acetate layer₂SO₄Drying for 12 hours. Filtering off Na₂SO₄The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol, 40/1) to give 2.94g (90%) of the title compound as a colorless solid. ESI-MS (M/z):657[ M + H]⁺。

EXAMPLE 2 preparation of D-Lys (Cbz) -L-Trp-OBzl

To 2.62g (4.0mmol) of D-Boc-Lys (Cbz) -L-Trp-OBzl was slowly added 30mL of a solution of hydrogen chloride in ethyl acetate (4M) under ice-bath and stirred for 4 hours, TLC (dichloromethane/methanol, 40/1) showed the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 30mL of anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure, and the residue was dissolved in 30mL of anhydrous ethyl acetate. This operation was repeated three times. The residue was suspended with 30mL of anhydrous ether by means of a sonicator and after standing the ether was removed to give 2.07g (93%) of the title compound as a yellow powder. ESI-MS (M/z):557[ M + H ]]⁺。

EXAMPLE 3 preparation of (3R,6S) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (1)

A solution of 1.95g (3.5mmol) D-Lys (Cbz) -L-Trp-OBzl and 50mL ethyl acetate was treated with saturated NaHCO₃After the aqueous solution was sufficiently washed (25 mL. times.3), the ethyl acetate layer was stirred at 80 ℃ for 56 hours. TLC (dichloromethane/methanol, 20/1) showed the reaction was complete. The reaction mixture was allowed to stand well at room temperature and filtered to give 0.72g (46%) of the title compound as a colorless solid. ESI-MS (M/z):449[ M + H ]]⁺。

EXAMPLE 4 preparation of (3R,6S) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2)

To a solution of 0.67g (1.5mmol) of (3R,6S) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (1) in 10mL of Dimethylformamide (DMF) was added 0.07g of Pd/C (10%) and the mixture was taken up in H at room temperature₂TLC (dichloromethane/methanol, 20/1) showed the reaction was complete for 48 hours. Pd/C was filtered off from the reaction mixture, and the filtrate was concentrated under reduced pressure to give 0.48g (95%) of the title compound as a colorless solid. ESI-MS (M/z) 315[ M + H ]]⁺。

EXAMPLE 5 preparation of Boc-amino-n-hexanoic acid

To a solution of 0.26g (2.0mmol) of aminon-hexanoic acid in 5mL of distilled water was added 0.58g (2.6 mmol) (Boc) with stirring₂Solution of O with 5mL dioxane. The resulting solution was adjusted to pH 9 with an aqueous solution of NaOH (2M) in ice bath. After stirring for 30 minutes in an ice bath, the mixture was stirred at room temperature and pumped down by a water pump. The pH was monitored during stirring and kept at 9 all the time until TLC (dichloromethane/methanol, 3/1) indicated completion of the reaction. The reaction mixture was saturated with KHSO under ice-bath₄Adjusting pH of the aqueous solution to 7, and concentrating under reduced pressure. The aqueous phase is saturated KHSO₄Adjusting pH to 2 with water solution, washing with 10mL ethyl acetate for three times, washing with 10mL saturated NaCl water solution for three times to make solution pH 7, and adding anhydrous Na₂SO₄Drying for 12 hours. Filtering to remove Na₂SO₄The filtrate was concentrated under reduced pressure to give 0.41g (89%) of the title compound as a colorless solid. ESI-MS (M/e): 232[ M + H]⁺。

EXAMPLE 6 preparation of Cbz-Asn-amino-n-hexanoic acid methyl ester (3a)

From 0.40g (1.5mmol) Cbz-Asn and 0.22g (1.5mmol) methyl aminohexanoate, 0.52g (88%) of the title compound was obtained as a colorless solid by the method of example 1. ESI-MS (M/z):394[ M + H]⁺。

EXAMPLE 7 preparation of Cbz-Ser-amino-n-hexanoic acid methyl ester (3b)

From 0.36g (1.5mmol) Cbz-Ser and 0.22g (1.5mmol) methyl aminohexanoate, 0.45g (81%) was obtained as a colorless solid using the method of example 1. ESI-MS (M/z):367[ M + H]⁺。

EXAMPLE 8 preparation of Cbz-Gln-amino-n-hexanoic acid methyl ester (3c)

From 0.42g (1.5mmol) Cbz-Gln and 0.22g (1.5mmol) methyl aminohexanoate, 0.49g (81%) was obtained as a colorless solid using the method of example 1. ESI-MS (M/z) 408[ M + H ]]⁺。

EXAMPLE 9 preparation of Cbz-Asn-amino-n-hexanoic acid (4a)

0.47g (1.2mmol) Cbz-Asn-amino-n-hexanoic acid methyl ester (3a) was dissolved in 8mL methanol, the pH was adjusted to 12 with NaOH (2M) in water in ice bath, and then stirred at room temperature for 4 hours, TLC (dichloromethane/methanol, 20/1) showed the reaction was complete. The reaction mixture was saturated with KHSO in ice bath₄Adjusting pH of the aqueous solution to 7, concentrating under reduced pressure, and adding saturated KHSO to the aqueous phase₄The aqueous solution was adjusted to pH 2 and extracted thoroughly three times with 10mL of ethyl acetate. The ethyl acetate layer was washed three times with 10mL of a saturated aqueous NaCl solution to give a solution pH of 7, and then with anhydrous Na₂SO₄Drying for 12 hours. Filtering to remove Na₂SO₄The filtrate was concentrated under reduced pressure to give 0.39g (86%) of the title compound as a colorless solid. ESI-MS (M/z) 380[ M + H]⁺。

EXAMPLE 10 preparation of Cbz-Ser-amino-n-hexanoic acid (4b)

From 0.44g (1.2mmol) of Cbz-Ser-amino-n-hexanoic acid methyl ester (3b) 0.38g (89%) of the title compound are obtained as colorless solid by the method of example 9. ESI-MS (M/z):353[ M + H]⁺。

EXAMPLE 11 preparation of Cbz-Gln-amino-n-hexanoic acid (4c)

From 0.49g (1.2mmol) of Cbz-Gln-amino-n-hexyl there is employed the process of example 9Methyl ester (3c) gave 0.45g (96%) of the title compound as a colorless solid. ESI-MS (M/z):394[ M + H]⁺。

EXAMPLE 12 preparation of (3R,6S) -3- (Cbz-Asn-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a)

To a solution of 0.38g (1.0mmol) of Cbz-Asn-aminon-hexanoic acid (4a) and 5mL of anhydrous DMF was added 0.14g (1.0mmol) of HOBt and 0.25g (1.2mmol) of DCC under ice-bath, and the mixture was stirred for 30 minutes to obtain reaction solution A. 0.31g (1.0mmol) of (3R,6S) -3- (amino-N-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) was dissolved in 5mL of anhydrous DMF, and pH was adjusted to 9 with N-methylmorpholine to give reaction solution B. Reaction B was added to reaction A and stirred at room temperature for 12 hours, and TLC (dichloromethane/methanol, 10/1) showed completion of the reaction. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol, 50/1) to give 0.14g (21%) of the title compound as a colorless solid. ESI-MS (M/z):676 [ M + H]⁺；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝10.895(s,1H),8.038(s,1H),7.848(s,1H), 7.798(t,J＝5.4Hz,1H),7.680(t,J＝5.4Hz,1H),7.570(d,J＝7.8Hz,1H),7.333(m,5H), 7.266(s,1H),7.045(m,2H),6.948(t,J＝7.5Hz,1H),6.879(s,1H),5.020(s,2H),4.280(m,1 H),4.093(m,1H),3.239(dd,J₁＝14.4Hz,J₂＝4.2Hz,1H),2.995(m,6H),2.400(m,2H), 2.001(t,J＝7.2Hz,2H),1.429(m,6H),1.240(m,6H)。

EXAMPLE 13 preparation of (3R,6S) -3- (Cbz-Ser-amino-n-hexanoylamino) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b)

From 0.35g (1.0mmol) of Cbz-Ser-amino-n-hexanoic acid (4b) and 0.31g (1.0mmol) of (3R,6S) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) by the method of example 12, 0.09g (13%) of the title compound are obtained as a colorless solid. ESI-MS (M/z):649[ M + H]⁺；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝11.168(s,1H), 8.126(s,1H),7.996(s,1H),7.888(s,2H),7.539(d,J＝8.1Hz,1H),7.298(m,6H),7.041(m,2 H),6.933(t,J＝7.2Hz,1H),5.006(s,2H),4.503(m,1H),4.045(m,1H),3.966(m,1H),3.432 (m,1H),3.203(dd,J₁＝14.4Hz,J₂＝4.2Hz,1H),2.944(m,6H),1.996(t,J＝7.2Hz,2H), 1.377(m,7H),1.216(m,6H)。

EXAMPLE 14 preparation of (3R,6S) -3- (Cbz-Gln-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c)

From 0.39g (1.0mmol) of Cbz-Gln-aminon-hexanoic acid (4c) and 0.31g (1.0mmol) of (3R,6S) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) by the method of example 12, 0.13g (18%) of the title compound are obtained as a colorless solid. ESI-MS (M/z):690[ M + H]⁺；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝10.896(s,1H), 8.040(s,1H),8.035(s,1H),7.850(s,1H),7.686(t,J＝5.4Hz,1H),7.570(d,J＝7.8Hz,1H), 7.333(m,6H),7.019(m,2H),6.948(t,J＝7.2Hz,1H),6.760(s,1H),5.019(s,2H),4.080(m,1 H),3.961(m,1H),3.256(dd,J₁＝14.7Hz,J₂＝4.8Hz,1H),3.000(m,6H),2.034(m,5H),1.487 (m,6H),1.181(m,6H)。

EXAMPLE 15 preparation of (3R,6S) -3- (Asn-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6a)

From 0.07g (0.1mmol) of (3R,6S) -3- (Cbz-Asn-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5a) 0.05g (97%) of the title compound are obtained as colorless solid according to the method of example 4. ESI-MS (M/z):542[ M + H]+；Mp 164-167℃；

(c ═ 0.1, methanol); IR (cm-¹):3396,3305,3195, 3086,2932,2866,1671,1634,1537,1455,1417,1330,1256,1236,1096,1009,740,675；¹H NMR (300MHz,DMSO-d₆)δ/ppm＝10.898(d,J＝1.2Hz,1H),8.043(d,J＝2.1Hz,1H),7.840(m,2 H),7.687(t,J＝5.4Hz,1H),7.569(d,J＝8.1Hz,1H),7.380(s,1H),7.318(d,J＝8.1Hz,1H), 7.043(m,2H),6.948(td,J₁＝8.1Hz,J₂＝0.9Hz,1H),6.832(s,1H),4.073(m,1H),3.439(dd, J₁＝9.0Hz,J₂＝4.2Hz,1H),3.256(dd,J₁＝14.4Hz,J₂＝4.2Hz,1H),3.006(m,6H),2.395(dd, J₁＝15.0Hz,J₂＝3.9Hz,1H),2.151(dd,J₁＝15.0Hz,J₂＝3.9Hz,1H),2.007(t,J＝7.2Hz,2H), 1.432(m,6H),1.229(m,6H)。

EXAMPLE 16 preparation of (3R,6S) -3- (Ser-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6b)

From 0.07g (0.1mmol) of (3R,6S) -3- (Cbz-Ser-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5b) 0.04g (85%) of the title compound are obtained as colorless solid according to the method of example 4. ESI-MS (M/z) 515[ M + H]+；Mp 187-188℃；

(c ═ 0.1, methanol); IR (cm-¹):3396,3307,3207, 3094,2930,2865,1671,1635,1532,1455,1329,1256,1047,739；¹H NMR(500MHz,DMSO-d₆) δ/ppm＝10.882(d,J＝1.5Hz,1H),8.025(d,J＝2.0Hz,1H),7.837(s,1H),7.787(t,J＝5.5Hz, 1H),7.669(t,J＝5.5Hz,1H),7.563(d,J＝8.0Hz,1H),7.311(d,J＝8.0Hz,1H),7.060(d,J＝ 2.0Hz,1H),7.037(td,J₁＝8.0Hz,J₂＝1.0Hz,1H),6.942(dt,J₁＝8.0Hz,J₂＝1.0Hz,1H), 4.683(t,J＝5.5Hz,1H),4.065(m,1H),3.496(m,1H),3.455(m,1H),3.246(dd,J₁＝14.5Hz, J₂＝4.5Hz,1H),3.017(m,4H),2.934(m,2H),1.988(t,J＝7.5Hz,2H),1.503(m,2H),1.406 (m,5H),1.206(m,5H),1.095(m,1H)。

EXAMPLE 17 preparation of (3R,6S) -3- (Gln-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6c)

From 0.07g (0.1mmol) of (3R,6S) -3- (Cbz-Gln-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (5c) 0.05g (91%) of the title compound are obtained as colorless solid according to the method of example 4. ESI-MS (M/z):556[ M + H]+；Mp 102-103℃；

(c ═ 0.1, methanol); IR (cm-¹):3212,3079,2929, 2860,1659,1556,1454,1324,1259,1099,744；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝10.907(s, 1H),8.602(s,1H),7.865(s,1H),7.813(t,J＝5.4Hz,1H),7.704(t,J＝5.4Hz,1H),7.569(d,J ＝8.1Hz,1H),7.305(m,2H),7.046(m,2H),6.949(t,J＝7.2Hz,1H),6.716(s,1H),4.074(m, 1H),3.256(dd,J₁＝14.7Hz,J₂＝4.8Hz,1H),2.993(m,6H),2.033(m,5H),1.767(m,1H), 1.438(m,6H),1.229(m,6H)。

EXAMPLE 18 preparation of (3R,6S) -3- (Boc-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (7)

From 0.28g (1.2mmol) Boc-amino-n-hexanoic acid and 0.38g (1.2mmol) (3R,6S) -3- (amino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (2) 0.12g (19%) of the title compound was obtained as a colorless solid using the method of example 1. ESI-MS (M/z):528[ M + H]⁺；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝10.903(s,1H),8.046(d,J ＝1.8Hz,1H),7.851(s,1H),7.686(t,J＝5.4Hz,1H),7.556(d,J＝7.8Hz,1H),7.314(d,J＝ 7.8Hz,1H),7.039(m,2H),6.943(td,J₁＝7.8Hz,J₂＝0.6Hz,1H),6.758(t,J＝5.4Hz,1H), 4.068(m,1H),3.252(dd,J₁＝14.4Hz,J₂＝4.2Hz,1H),2.924(m,6H),1.995(t,J＝7.2Hz,2H), 1.292(m,21H)。

EXAMPLE 19 preparation of (3R,6S) -3- (amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (8)

From 0.11g (0.2mmol) (3R,6S) -3- (Boc-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (7) 0.08g (96%) of the title compound was obtained as colorless solid by the method of example 2. ESI-MS (M/z):428 [ M + H]⁺；¹H NMR(300MHz,DMSO-d₆)δ/ppm＝10.924(s,1H),8.071(s,1H),7.866(s,1H), 7.729(t,J＝5.4Hz,1H),7.568(d,J＝7.5Hz,1H),7.321(d,J＝7.5Hz,1H),7.044(m,2H), 6.949(t,J＝7.5Hz,1H),4.070(m,1H),3.239(dd,J₁＝14.4Hz,J₂＝3.6Hz,1H),2.991(m,4H), 2.694(m,2H),2.021(t,J＝7.5Hz,2H),1.467(m,6H),1.240(m,6H)。

EXAMPLE 20 determination of the anti-tumor metastasis Activity of Compounds 6a-c

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), passaging once every two days according to an adherent cell culture method,and (4) enriching cells. 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 20 μmol/kg/day normal saline solution of the putative anti-tumor metastasis peptide Arg-Gly-Asp-Ser (RGDS) or a 5 μmol/kg/day normal saline solution of Compound 8 or a 0.5 μmol/kg/day normal saline solution of Compounds 6a-c or 10 mL/kg/day normal saline 1 time a day for 12 consecutive days. The next day of the last administration, cervical spine was removed by ether anesthesia, and the lungs of the mice were taken and the number of tumor nodules that metastasized in the tumor lungs was calculated. 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. mu. mol/kg dose, but there was no significant difference in activity versus RGDS at 40-fold higher doses and 8 at 10-fold higher doses. 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 RGDS and p >0.05 to compound 8 to saline; n is 12.

EXAMPLE 21 determination of the anti-tumor growth Activity of Compounds 6a-c

Doxorubicin, compound 8 and compounds 6a-c 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. The cell concentration is 4-large-grid 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 injected intraperitoneally with a physiological saline solution of doxorubicin (at a dose of 2. mu. mol/kg/day), or orally with a physiological saline solution of Compound 8 (at a dose of 5. mu. mol/kg/day), or orally with a physiological saline solution of Compounds 6a-c (at a dose of 0.5. mu. mol/kg/day), or orally with a physiological saline solution (at a dose of 10 mL/kg/day). The administration is once daily for 12 days. The next day of the last administration, cervical spine was removed under ether anesthesia, and 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. Not only was compound 6a-c effective at a dose of 0.5 μmol/kg in inhibiting tumor growth, but there was no significant difference in activity from 8, which was 10-fold higher than the dose. TheseThe data show that the invention has significant technical effect.

TABLE 2 Effect of Compounds 6a-g on tumor growth in S180 mice

a) P <0.01 to saline; b) 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 compounds 6a-c 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 (SPF grade, body weight 20 ± 2g) were allowed to rest for 2 days at 22 ℃ with free access to water and food. Thereafter, 12 mice were randomly divided into a saline group (dose of 0.2 mL/mouse), an aspirin group (dose of 1110. mu. mol/kg), a Compound 4 group (dose of 5. mu. mol/kg) and Compound 6a-c groups (dose of 0.5. mu. mol/kg), each group. The mice were tested either orally with normal saline, orally with aspirin, orally with Compound 8, or orally with Compound 6a-g, 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. Not only can compounds 6a-c effectively inhibit xylene-induced ear swelling in mice at a dose of 0.5 μmol/kg, but there was no significant difference in activity from aspirin at a dose 2220-fold higher than them and compound 8 at a dose 10-fold higher than them. 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 aspirin and compound 8; n is 12.

Claims

1. (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione of the formula,

wherein AA is L-Asn residue, L-Ser residue or L-Gln residue.

2. A process for the preparation of (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione as claimed in claim 1, which comprises:

(3) dissolving D-Lys (Cbz) -D-Trp-OBzl in ethyl acetate, washing the obtained solution with 5% sodium bicarbonate aqueous solution, and cyclizing to obtain (3R,6S) -3- (benzyloxycarbonylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (1);

(5) condensing amino methyl hexanoate and Cbz-AA to obtain Cbz-AA-amino methyl hexanoate (3a-c), wherein AA is an L-Asn residue, an L-Ser residue or an L-Gln residue;

(6) saponifying and removing methyl ester from the compound 3a-c to obtain Cbz-AA-amino n-hexanoic acid (4 a-c);

(7) condensing the compound 2 and a compound 4a-c to obtain (3R,6S) -3- (Cbz-AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-diketone (5 a-c);

(8) the compound 5a-c is subjected to hydrogenolysis to remove the benzyloxycarbonyl to obtain (3R,6S) -3- (AA-amino-n-hexanoylamino-n-butyl) -6- (indole-3-methyl) -piperazine-2, 5-dione (6 a-c).

3. The use of (3R,6S) -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 lung cancer metastasis.

4. The use of (3R,6S) -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 S180 ascites tumor.

5. Use of (3R,6S) -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 acute inflammation.