CN110577570B - RGD tetrapeptide modified S, R-heptacyclic aldehyde, and synthesis, activity and application thereof - Google Patents

RGD tetrapeptide modified S, R-heptacyclic aldehyde, and synthesis, activity and application thereof Download PDF

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CN110577570B
CN110577570B CN201810595952.0A CN201810595952A CN110577570B CN 110577570 B CN110577570 B CN 110577570B CN 201810595952 A CN201810595952 A CN 201810595952A CN 110577570 B CN110577570 B CN 110577570B
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赵明
蒋雪云
桂琳
彭师奇
薛双悦
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Abstract

The invention discloses S, R-heptacyclaldehyde-Arg-Gly-Asp-AA, discloses a preparation method thereof, discloses anti-venous thrombosis activity thereof, discloses anti-arterial thrombosis activity thereof, discloses activity of inhibiting in vivo GPIIb/IIIa expression thereof, discloses activity of inhibiting in vivo P-selectin expression thereof, and discloses dose-effect relationship of anti-arterial and venous thrombosis activity of S, R-heptacyclaldehyde-Arg-Gly-Asp-Ser. Therefore, the invention discloses the application of the derivatives in preparing anti-arterial thrombosis medicaments, the application in preparing anti-venous thrombosis medicaments, the application in preparing GPIIb/IIIa antagonists and the application in preparing P-selectin antagonists.

Description

RGD tetrapeptide modified S, R-heptacyclic aldehyde, and synthesis, activity and application thereof
Technical Field
The present invention relates to S, R-heptacyclaldehyde-Arg-Gly-Asp-AA, to a process for their preparation, to their anti-thrombogenic activity, to their anti-arterial-thrombotic activity, to their activity in inhibiting the expression of glycoprotein IIb/IIIa (IIb/IIIa) in vivo, and to their activity in inhibiting the expression of P-selectin in vivo. Therefore, the invention relates to the application of the derivatives in preparing anti-arterial thrombosis medicaments, anti-venous thrombosis medicaments, GPIIb/IIIa antagonists and P-selectin antagonists. The invention belongs to the field of biological medicine.
Background
Thrombosis is a common pathology of ischemic heart disease, ischemic stroke and venous thrombosis. The number of deaths from ischemic heart disease and ischemic stroke is 1/4 out of all deaths from disease worldwide. Venous thrombosis is a major disease burden in less-developed, moderately-developed and highly-developed countries. Thrombosis can exacerbate a range of related diseases, for example, rarely occurring blocked thrombosis of a biomaterial tube can have unpredictable consequences for patients who undergo repeated tube changes, or undergo thrombolytic therapy or prolonged anticoagulant therapy, can cause re-embolization of patients who undergo percutaneous intracoronary intervention, can be associated with heparin-induced thrombocytopenia, and tortuous coronary thrombosis can cause acute coronary syndrome. In addition, thrombosis is a complication of related diseases, for example massive cerebral venous thrombosis is a complication in early pregnant women with epilepsy, and stent thrombosis is a serious complication in patients undergoing percutaneous intracoronary intervention. Therefore, the novel antithrombotic drug has clinical importance.
The beta-carboline is an important pharmacophore for inhibiting thrombus. However, the effective dose of beta-carboline, such as 3S-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid, is as high as 5. mu. mol/kg, and still remains to be reduced. The inventors hypothesized that two β -carboline pharmacophores fused, for example, 1 (S) -1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid and 1 (R) -1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid are intermolecularly condensed to (2S,5S) -tetrahydropyrazino [1,2:1,6] and bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] oxindole } -1, 4-dione, which is then acidolyzed to (2S,5S) -Tetrahydropyrazine [1,2:1,6] bis { (1S,1R) - [ 1-carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione (S, R-heptacyclal for short). The S, R-heptacyclic aldehyde should have strong antithrombotic activity. The inventors further hypothesized that the aldehyde group of this novel heptacyclic aldehyde should have greater antithrombotic activity linked to Arg-Gly-Asp-AA (where AA is a Phe residue, a Val residue, or a Ser residue). Based on this assumption, the inventors have proposed the present invention.
Disclosure of Invention
In a first aspect of the invention, there is provided S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is a Phe residue, a Val residue or a Ser residue) of the formula.
Figure GDA0002910106010000021
The second aspect of the present invention provides a method for synthesizing S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is Phe residue, Val residue or Ser residue), which comprises:
(1) carrying out Pictet-Spengler condensation on L-tryptophan benzyl ester and 1,1,3, 3-tetramethoxypropane under the catalysis of trifluoroacetic acid to obtain 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate (1);
(2) in methanol solution, 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate reacts with H under the catalysis of Pd/C2The benzyl ester is removed by reaction to obtain 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid (2);
(3) performing intermolecular condensation of 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid in anhydrous N, N-dimethylformamide in the presence of dicyclohexylcarbodiimide and N-hydroxybenzotriazole to obtain (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole } -1, 4-dione (3);
(4) adding (2S,5S) -tetrahydropyrazino [1,2:1,6] and bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione in the presence of glacial acetic acid, water and concentrated hydrochloric acid, and carrying out acidolysis reaction to obtain (2S,5S) -tetrahydropyrazino [1,2:1,6] and bis { (1S,1R) - [ 1-carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione, namely S, R-heptacyclal (4);
(5) uses dicyclohexylcarbodiimide and N-hydroxybenzotriazole as catalysts to synthesize HCl & Arg (NO) by adopting a liquid phase condensation method2) -Gly-asp (OBzl) -AA-OBzl (wherein AA is a Phe residue, a Val residue or a Ser residue);
(6) in the presence of sodium cyanoborohydride, HCl & Arg (NO)2) -Gly-Asp (OBzl) -AA-OBzl and (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-Carbonylmethyl group]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And then the indole } -1, 4-diketone is subjected to ammoniation reduction to obtain (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ N-ethyl ]-Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione (wherein AA is a Phe residue, a Val residue or a Ser residue);
(7) (2S,5S) -tetrahydropyrazines [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-AA-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione in the presence of trifluoroacetic acid and trifluoromethanesulfonic acid to obtain (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione, S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (where AA is a Phe residue, a Val residue or a Ser residue).
The third content of the invention is to evaluate the antithrombotic activity and dose-effect relationship of S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is Phe residue, Val residue or Ser residue).
The fourth aspect of the present invention is to evaluate the antithrombotic activity and dose-effect relationship of S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is Phe residue, Val residue or Ser residue).
The fifth aspect of the present invention is to evaluate the activity of S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is Phe residue, Val residue or Ser residue) for inhibiting GPIIb/IIIa expression in vivo.
The sixth aspect of the present invention is to evaluate the activity of S, R-heptacyclaldehyde-Arg-Gly-Asp-AA (wherein AA is Phe residue, Val residue or Ser residue) for inhibiting the expression of P-selectin in vivo.
Drawings
FIG. 1 is a synthetic route for (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (wherein AA is a Phe residue, a Val residue or a Ser residue) i) dichloromethane, trifluoroacetic acid, 1,1,3, 3-tetramethoxypropane; ii) palladium on carbon, hydrogen, methanol; iii) N-hydroxybenzotriazole, dicyclohexylcarbodiimide, N-methylmorpholine, N, N-dimethylformamide; iv) glacial acetic acid, concentrated hydrochloric acid, water and ice bath; v) dicyclohexylcarbodiimide, N-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; vi) ethyl hydrogen chloride acetate; vii)4N NaOH, methanol, ice bath; viii) sodium cyanoborohydride, pH 8; ix) trifluoroacetic acid, trifluoromethanesulfonic acid, ice bath,
5a (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione;
5b (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione;
5c (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Ser-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione;
6a (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-Phe ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione;
6b (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione;
6c (2S,5S) -Tetrahydropyrazine [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-Ser ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione.
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 benzyl 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylate (1)
5mL of 1,1,3, 3-tetramethoxypropane and 5mL of trifluoroacetic acid were sequentially added to 150mL of dichloromethane under ice bath conditions, and activated in an ice water bath for 40 min. 5.00g (17.00mmol) L-tryptophan benzyl ester were then added. The solution was reddish brown. The reaction mixture was stirred first for 1h on ice and then for 12h at room temperature. The reaction solution was washed with saturated aqueous sodium bicarbonate solution 6 times, and a large amount of bubbles were generated. And then washed with saturated aqueous sodium chloride solution for 3 times. The dichloromethane layer was collected, dried for 2 hours by adding anhydrous sodium sulfate. Filtering at normal pressure, concentrating the filtrate under reduced pressure, and purifying by silica gel column chromatographyThe reaction mixture was converted (v: v; petroleum ether: ethyl acetate ═ 1:1) to give 5.39g (13.68mmol) of the title compound as a yellow oil. The yield was 81%. ESI-MS (M/e)393[ M + H]-。
EXAMPLE 2 preparation of 1- (2, 2-Dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid (2)
To 5.39g (13.68mmol) of Compound 1 and 100mL of methanol solution were added 540mg of Pd/C, and H was purged2The reaction was stirred at room temperature for 4 h. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 3.34g (11.00mmol) of the title compound as a yellow oil in 80% yield. ESI-MS (m/e): 303[ M-H]-。
EXAMPLE 3 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (3)
3.34g (11.00mmol) of Compound 2 was suspended in 60mL of N-dimethylformamide, and 1.49g (11.00mmol) of N-hydroxybenzotriazole and 2.72g (13.20mmol) of dicyclohexylcarbodiimide were sequentially added to the suspension under ice bath, followed by adjusting the pH of the reaction solution to 8-9 with N-methylmorpholine. The reaction was carried out at room temperature for 8 hours. The reaction solution was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure. Then dissolved in 100mL of ethyl acetate, and a white solid was precipitated or not dissolved. Then, the mixture was filtered under reduced pressure, and the filtrate was collected. Washing the obtained ethyl acetate solution with saturated sodium bicarbonate water solution for 3 times, saturated sodium chloride water solution for 3 times, 5% potassium bisulfate water solution for 3 times, saturated sodium chloride water solution for 3 times, saturated sodium bicarbonate water solution for 3 times and saturated sodium chloride water solution for 3 times in sequence; the ethyl acetate layer was collected, dried over anhydrous sodium sulfate for 2 hours, and then filtered under reduced pressure, and the filtrate was collected. The solvent was removed under reduced pressure and the resulting yellow syrup was isolated by silica gel column chromatography (v: v; petroleum ether: ethyl acetate ═ 1:1) to give 670mg (1.17mmol) of the title compound as a yellow solid in 21% yield.
1H-NMR(300MHz,DMSO-d6)δ/ppm：11.00(d,J＝6.9Hz,2H),7.47(m,4H),7.11(m,4H),6.99(s,1H),5.23(s,1H),4.45(m,3H),4.19(s,1H),3.53(m,3H),4.19(s,1H),3.29(s,4H),3.21(s,3H),3.15(s,3H),3.10(s,3H),2.84(m,3H),2.19(s,2H)。
EXAMPLE 4 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-Carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione (4)
139mg (24.30mmol) of compound 3 was dispersed in 9ml of glacial acetic acid under ice-bath conditions, and 2ml of water and 1ml of concentrated hydrochloric acid were added in this order. The reaction was carried out for 1 hour under ice-bath conditions. Under the ice-water bath condition, the pH value of the reaction solution was adjusted to 7 with a 2N aqueous solution of sodium hydroxide. A large amount of yellow solid precipitated from the reaction solution. Extraction was carried out with 30ml of ethyl acetate three times, insoluble matter was present in the ethyl acetate layer, and the ethyl acetate phases were combined. Then carrying out extraction washing by using a saturated sodium bicarbonate solution, combining ethyl acetate phases after 8 times of extraction washing. A large amount of bubbles are generated in the process of extraction and washing. The ethyl acetate phase was then dried over anhydrous sodium sulfate for 2 hours, filtered under reduced pressure, and the filtrate was collected and spin-dried under reduced pressure to give 105mg (21.87mmol) of the title compound as a yellow solid in 90% yield. ESI-MS (M/e):479[ M-H]-。
EXAMPLE 5 preparation of Boc-Arg (NO)2)-Gly-OBzl
2.52g (7.89mmol) of Boc-Arg (NO)2) OH (E-OH) was suspended in 40mL of anhydrous tetrahydrofuran, and 1.19g (8.81mmol) of N-hydroxybenzotriazole and 1.82g (8.81mmol) of dicyclohexylcarbodiimide were added to the suspension in this order while stirring in an ice bath for 30 minutes. 2.18g (8.81mmol) of tos.Gly-OBzl was added, and the pH was adjusted to 8 to 9 with N-methylmorpholine. The reaction was carried out at room temperature for 8 hours, filtered and the insoluble matter was removed by filtration. The solvent was concentrated under reduced pressure. Then, the mixture was dissolved in 50mL of ethyl acetate to precipitate a white solid or to dissolve the white solid. And filtering again, and collecting the filtrate. Washing the obtained ethyl acetate solution with saturated sodium bicarbonate water solution for 3 times, saturated sodium chloride water solution for 3 times, 5% potassium bisulfate water solution for 3 times, saturated sodium chloride water solution for 3 times, saturated sodium bicarbonate water solution for 3 times and saturated sodium chloride water solution for 3 times in sequence; the ethyl acetate layer was collected, dried over anhydrous sodium sulfate for 2 hours, and then filtered under reduced pressure, and the filtrate was collected. The filtrate was concentrated under reduced pressure to give a yellow oily compound. Recrystallization from dichloromethane and filtration under reduced pressure gave the title compound as a colorless solid, 3.06g (6.57mmol), 83% yield. ESI-MS (M/e) 467[ M + H]+。
EXAMPLE 6 preparation of Boc-Arg (NO)2)-Gly-OH
5.00g (8.85mmol) of Boc-Arg (NO)2) -Gly-OBzl was dissolved in 60mL of methanol. The pH was adjusted to 13 with 4N NaOH in an ice bath. The reaction was carried out in ice bath for 3 h. The pH was adjusted to 7 with a saturated aqueous solution of potassium hydrogen sulfate in ice bath, and water was removed by evaporation under reduced pressure. In ice bath, regulating pH to 2 with saturated potassium hydrogen sulfate solution water solution, separating out colorless solid, and extracting with ethyl acetate for three times; washing with saturated aqueous sodium chloride solution (repeated three times); the ethyl acetate layer was collected, dried over anhydrous sodium sulfate for 2 hours, and then filtered under reduced pressure, and the filtrate was collected. After concentrating the filtrate under reduced pressure, 3.18g (8.46mmol) of the title compound were obtained as a colorless solid in a yield of 96%. ESI-MS (M/e):377[ M + H]+。
EXAMPLE 7 preparation of Boc-Asp (OBzl) -Phe-OBzl
From 2.00g (6.19mmol) of Boc-Asp (OBzl) -OH and 1.99g (6.81mmol) of HCl Phe-OBzl, 3.04g (5.44mmol) of the title compound were obtained in the form of a colorless solid with a yield of 88% by the method of example 5. ESI-MS (M/e):561[ M + H]+。
EXAMPLE 8 preparation of HCl Asp (OBzl) -Phe-OBzl
3.04g (5.44mmol) of Boc-Asp (OBzl) -Phe-OBzl are slowly mixed with stirring in 30mL of hydrogen chloride in ethyl acetate (4N). The resulting solution was stirred in an ice bath for 1 h. After that, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in 30mL of anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. This operation was repeated three times. The residue was sufficiently washed with dehydrated ether to give 2.64g (5.33mmol) of the title compound as a colorless solid in 98% yield.
ESI-MS(m/e):496[M+H]+。
EXAMPLE 9 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl
From 1.66g (4.43mmol) of Boc-Arg (NO) by the method of example 52) -Gly-OH and 2.45g (5.31mmol) HCl. Asp (OBzl) -Phe-OBzl, and separating by column chromatography on silica gel (v: v; dichloromethane: methanol ═ 20:1) yielded 2.77g (3.38mmol) of the title compound as a colorless solid in 76% yield. ESI-MS (M/e) 819[ M + H]+。
EXAMPLE 10 preparation of HCl.Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl
From 2.77g (3.38mmol) of Boc-Arg (NO) using the method of example 82) -Gly-Asp (OBzl) -Phe-OBzl gives 2.46g (3.26mmol) of the title compound as a colorless solid in 97% yield. ESI-MS (M/e):754[ M + H]+。
EXAMPLE 11 preparation of Boc-Asp (OBzl) -Val-OBzl
From 2.00g (6.19mmol) of Boc-Asp (OBzl) -OH and 2.58g (6.81mmol) of Tos. Val-OBzl, 3.10g (6.04mmol) of the title compound were obtained as a colorless solid in 98% yield by the method of example 5. ESI-MS (M/e):513[ M + H]+。
EXAMPLE 12 preparation of HCl Asp (OBzl) -Val-OBzl
From 4.68g (9.14mmol) of Boc-Asp (OBzl) -Val-OBzl, 3.92g (5.94mmol) of the title compound were obtained as a white solid in 98% yield by the method of example 8. ESI-MS (M/e):448[ M + H]+。
EXAMPLE 13 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
From 1.66g (4.43mmol) of Boc-Arg (NO) by the method of example 52) -Gly-OH and 2.19g (5.31mmol) HCl. Asp (OBzl) -Val-OBzl. Separation by silica gel column chromatography (v: v; dichloromethane: methanol ═ 30:1) gave 2.67g (3.47mmol) of the title compound as a colorless solid in 78% yield. ESI-MS (M/e):771[ M + H]+。
EXAMPLE 14 preparation of HCl Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
From 2.67g (3.47mmol) of Boc-Arg (NO) using the method of example 82) -Gly-Asp (OBzl) -Val-OBzl gives 2.41g (3.42mmol) of the title compound as a colourless solid in 99% yield. ESI-MS (M/e):706[ M + H]+。
EXAMPLE 15 preparation of Boc-Asp (OBzl) -Ser-OBzl
From 2.00g (6.19mmol) of Boc-Asp (OBzl) -OH and 1.58g (6.81mmol) of HCl.Ser-OBzl, 1.72g (3.44mmol) of the title compound were obtained as colorless solid in 56% yield by the method of example 5. ESI-MS (M/e):501[ M + H]+。
EXAMPLE 16 preparation of HCl. Asp (OBzl) -Ser-OBzl
By using a blockThe procedure of example 8 gave 1.45g (3.32mmol) of the title compound as a colorless solid in 97% yield from 1.72g (3.44mmol) of Boc-Asp (OBzl) -Ser-OBzl. ESI-MS (M/e):436[ M + H]+。
EXAMPLE 17 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Ser-OBzl
From 1.66g (4.43mmol) of Boc-Arg (NO) by the method of example 52) -Gly-OH and 1.93g (5.31mmol) HCl. Asp (OBzl) -Ser-OBzl, silica gel column chromatography (v: v; dichloromethane: methanol ═ 20:1) yielded 2.12g (4.12mmol) of the title compound as a colorless solid in 48% yield. ESI-MS (M/e):759[ M + H]+。
EXAMPLE 18 preparation of HCl Arg (NO)2)-Gly-Asp(OBzl)-Ser-OBzl
2.12g (4.12mmol) of Boc-Arg (NO) was recovered by the method of example 82) -Gly-Asp (OBzl) -Ser-OBzl to give 2.58g (3.79mmol) of the title compound as a colourless solid in 92% yield. ESI-MS (M/e):694[ M + H ]]+。
EXAMPLE 19 preparation of (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione (5a)
100mg (0.21mmol) of Compound 4 was dispersed in 3mL of dichloromethane. Adding 2 drops of triethylamine to adjust the pH value to 8 under ice bath to obtain a solution A; 471mg (0.63mmol) of HCl Arg (NO)2) -Gly-asp (OBzl) -Phe-OBzl dispersed in 5mL of dichloromethane, and 2 drops of triethylamine added to adjust pH 8 in ice bath to obtain solution B; combine solutions a and B. And a drying agent magnesium sulfate is added to activate for 1 hour at normal temperature. Every 1 hour, 13mg (0.21mmol) of sodium cyanoborohydride as a reducing agent was added, 4 times in total, and 53mg (0.84mmol) of sodium cyanoborohydride in total was added. The reaction was terminated after 8 hours at room temperature, and the solvent was concentrated under reduced pressure. The residue was dissolved by adding 10mL of ethyl acetate. The obtained ethyl acetate solution is extracted and washed for 3 times by using a saturated sodium bicarbonate aqueous solution, a saturated sodium chloride aqueous solution is washed to be neutral, an ethyl acetate layer is collected, dried for 2 hours by using anhydrous sodium sulfate, filtered, and the filtrate is concentrated to be dry under reduced pressure. The resulting yellow solid was separated by silica gel column chromatography (v: v; dichloromethane: methanol ═ v)15:1) 64mg (0.04mmol) of the title compound are obtained as a yellow solid in 16% yield.
1H-NMR(300MHz,DMSO-d6)δ/ppm：11.06(d,J＝29.7Hz,2H),8.20(m,13H),7.45(m,3H),7.30(m,16H),7.18(m,22H),7.01(m,5H),5.84(s,1H),5.25(s,1H),5.01(m,9H),4.70(m,2H),4.46(m,4H),4.30(m,2H),3.63(m,7H),3.00(m,14H),2.72(m,8H)。
EXAMPLE 20 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-Ethyl-Arg-Gly-Asp-Phe-OBzl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (6a)
30mg (0.02mmol) of 5a was dissolved in 1mL of trifluoroacetic acid while cooling on ice, and 333. mu.L of trifluoromethanesulfonic acid was added thereto, whereby the solution turned from yellow to dark brown. The reaction was carried out in ice bath for 0.5 hour, 15mL of precooled ether was added, a yellow solid precipitated, and the mixture was stirred well. Standing and discarding the supernatant. Additional pre-chilled ether was added and the procedure was repeated three times. 2mL of water was added, and the pH was adjusted to 7 with a 10-fold dilution of aqueous ammonia (the solution gradually changed from cloudy to clear as the pH was adjusted). Filtering under reduced pressure, and collecting filtrate. After passage through a Sephadex-G25 column, 16mg (0.01mmol) of the title compound are obtained as a yellow solid in 56% yield.
Melting range: 195.8-197.4 ℃; ESI-MS (m/e): 1436[ M + H]+IR(cm-1):3194.04,3089.37,2889.65,1657.64,1433.08,1226.04,1164.06,1028.24,841.64,764.57,724.89,704.83,628.43；
1H-NMR(300MHz,DMSO-d6)δ/ppm：11.40(s,1H),11.20(s,1H),10.05(s,1H),8.89(m,2H),8.50(s,1H),8.35(s,1H),7.42(m,12H),7.23(m,4H),7.08(m,10H),7.00(m,4H),5.81(s,1H),5.25(s,1H),4.36(m,4H),4.01(s,2H),3.72(d,J＝20.1Hz,4H),3.40(m,2H),2.95(m,14H),2.45(s,3H),2.30(m,5H),1.65(s,1H),1.46(m,9H)。
EXAMPLE 21 preparation of (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione (5b)
From 100mg (0.21mmol) of Compound 4 and 441mg (0.63mmol) of HCl Arg (NO) by the method of example 192)-Gly-Asp(OBzl) Val-OBzl gave 62mg (0.03mmol) of the title compound as a yellow solid in a yield of 17%1H-NMR(300MHz,DMSO-d6)δ/ppm：11.07(s,1H),11.00(s,1H),8.51(m,3H),8.21(m,5H),8.05(m,2H),7.95(s,1H),7.01(m,5H),7.45(m,2H),7.32(m,26H),7.09(m,6H),5.84(m,1H),5.35(s,1H),5.08(m,9H),4.75(m,2H),4.42(m,2H),4.31(m,1H),4.13(m,4H)。
EXAMPLE 22 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-Ethyl-Arg-Gly-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (6b)
From 30mg (0.02mmol) of the compound 5b, 15mg (0.01mmol) of the title compound are obtained as a yellow solid in 57% yield by the method of example 20. Melting range: 211.3-212.0 ℃; ESI-MS (m/e): 1340[ M + H]+IR(cm-1):3212.23,3069.01,2971.41,2837.83,1651.03,1543.25,1417.36,1330.21,1242.06,1225.18,1202.04,1168.10,1027.42,838.64,799.67,745.54,722.59,637.90；1H-NMR(300MHz,D2O)δ/ppm：7.47(t,J＝7.2Hz,2H),7.37(d,J＝8.1Hz,2H),7.10(m,4H),5.75(s,1H),5.20(s,1H),4.39(m,3H),3.87(m,7H),3.62(m,2H),3.52(m,4H),3.42(m,4H),3.17(m,2H),3.05(m,2H),2.57(s,4H),3.94(m,4H),1.90(s,4H),1.55(m,4H),1.35(s,4H),0.69(m,12H)。
EXAMPLE 23 preparation of (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-Ser-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione (5c)
From 100mg (0.21mmol) of Compound 4 and 438mg (0.63mmol) of HCl Arg (NO) by the method of example 192) -Gly-Asp (OBzl) -Ser-OBzl to give 52mg (0.03mmol) of the title compound as a yellow solid in 14% yield, ESI-MS (m/e): 1767[ M + H]+1H-NMR(300MHz,DMSO-d6)δ/ppm：11.04(s,1H),10.94(s,1H),8.05(m,11H),7.47(m,2H),7.32(s,24H),7.05(m,6H),5.86(s,1H),5.25(s,1H),5.07(m,10H),4.79(m,2H),4.45(m,5H),4.11(m,1H),4.75(m,2H),3.70(m,4H),3.50(m,2H),3.18(m,4H),3.04(m,4H),2.87(m,2H),2.74(m,2H),2.19(m,4H),2.02(m,4H),1.50(m,8H)。
EXAMPLE 23 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-Ethyl-Arg-Gly-Asp-Ser ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (6c)
From 30mg (0.02mmol) of the compound 5c, 10mg (0.01mmol) of the title compound, i.e., S, R-heptacyclaldehyde-Arg-Gly-Asp-Ser, was obtained as a yellow solid in a yield of 38% by using the method of example 20.
Melting range: 232.0-233.4 ℃; ESI-MS (m/e): 1316[ M + H]+IR(cm-1):3217.06,3078.47,1662.80,1424.40,1240.25,1185.66,1164.24,1025.91,841.08,766.70,724.16,668.88,636.26；
1H-NMR(300MHz,DMSO-d6)δ/ppm：11.15(s,2H),9.98(s,2H),8.51(m,12H),7.97(m,8H),7.39(m,4H),7.05(m,4H),5.82(s,1H),5.28(s,1H),4.44(m,8H),4.06(m,2H),3.89(m,6H),3.02(m,12H),2.14(m,8H),1.57(m,8H)。
Experimental example 24 evaluation of anti-venous thrombosis Activity of Compounds 6a to 6c
1) Experimental methods
SD rats (200 + -20 g) were acclimatized and fasted for one day before surgery, and the rats were gavaged with a dose of 0.3mL/100g body weight and anesthetized with a intraperitoneal administration of 20% urethane solution 2min before surgery 30min after administration. Preparing skin of abdomen of rat, sterilizing, opening abdominal cavity along the leucorrhea line, descending to coagulated gland, and ascending to expose one corner of liver. The organs such as small intestine in abdominal cavity are pulled out to expose inferior vena cava, and the pulled-out organs are wrapped with gauze soaked with normal saline. Blunt-separating connective tissue around blood vessel, exposing inferior vena cava and its branch, peeling off abdominal aorta and inferior vena cava below renal vein, ligating inferior vena cava at junction of inferior vena cava and left renal vein with suture soaked with physiological saline, placing organs such as intestine back into abdominal cavity according to anatomical position, and suturing abdominal cavity layer by layer with suture.
After operation, the rat is placed in an environment with the temperature of 25-28 ℃ for circulation for 4 hours, the abdominal cavity is opened, the branches of the rat are tied one by one, the 2 cm inferior vena cava is taken out from the tying position of the junction of the inferior vena cava and the left renal vein, and the thrombus is taken out from the inferior vena cava.
2) Methods of administration and dosages
The administration mode is intragastric administration. Blank control: physiological saline with the administration dosage of 0.3mL/100 g; positive control: warfarin (dose 4.87. mu. mol/kg); compound 4 (administered at a dose of 1. mu. mol/kg); compounds 6a to c of the present invention were administered at a dose of 0.1. mu. mol/kg.
3) Statistical method
The statistics of the experimental data are all performed by adopting a t test, and the suppository weight is expressed by (mean value +/-SD mg).
4) Results of the experiment
As can be seen from the results in Table 1, the ratio P of 6a to c to normal saline is less than 0.01, which indicates that 6a to c have good anti-venous thrombosis activity; 0.1 mu mol/kg6 a-c is equivalent to 4.87 mu mol/kg warfarin anti-thrombus activity (also equivalent to compound 4 anti-thrombus activity under the lavage dose of 1 mu mol/kg). Under the same activity condition, the compounds 6 a-c of the invention are reduced by 48.7 times compared with warfarin dosage and 10 times compared with compound 4 dosage. The present invention has an unexpected technical effect.
TABLE 1 anti-venous thrombosis Activity of Compounds 6 a-c
Figure GDA0002910106010000101
a) P <0.01 to normal saline, P >0.05 to 4.87 μmol/kg warfarin and 1 μmol/kg 4; n-12
Experimental example 25 dose-effect relationship for evaluating anti-thrombotic activity of Compound 6c
1) The experimental method comprises the following steps: same as Experimental example 24
2) Methods of administration and dosages
The administration mode is intragastric administration. Blank control: physiological saline with the administration dosage of 0.3mL/100 g; positive control: warfarin, the dosage of administration is 4870 nmol/kg; the administration dose of Compound 6c of the present invention was 100nmol/kg, 10nmol/kg, 1 nmol/kg.
3) Statistical method
The statistics of the experimental data are all performed by adopting a t test, and the suppository weight is expressed by (mean value +/-SD mg).
4) Results of the experiment
As can be seen from the results in Table 2, the ratio P of 100nmol/kg and 10nmol/kg6c to physiological saline is less than 0.01, indicating that 6c has good anti-venous thrombosis activity at doses of 100nmol/kg and 10 nmol/kg; 100nmol/kg6c is comparable to 4.87. mu. mol/kg warfarin in anti-venous thrombosis activity, and compound 6c of the present invention is 48.7 times lower than warfarin dosage under the same activity conditions. The present invention has an unexpected technical effect.
TABLE 2 anti-thrombotic Activity of Compound 6c at three doses
Figure GDA0002910106010000102
a) P <0.01 to saline, P <0.05 to 10nmol/kg6c, and P >0.05 to 4.87 μmol/kg warfarin;
b) p <0.01 to normal saline, P <0.05 to 1nmol/kg 6 c; c) p >0.05 to saline; n is 12.
Experimental example 26 evaluation of anti-arterial Thrombus Activity of Compounds 6a to c
1) Experimental methods
Healthy SD male rats weighing 180-220g, preoperatively fasting for 24 hours, and preoperatively preparing anticoagulant heparin sodium (2.4mg/mL), physiological saline solution and urethane (20g/100mL, anesthetic dose 7 mL/kg). Randomly grouping SD male rats of experimental animals, wherein n is 9, the volume of the administration of rats by intragastric administration is 3mL/kg, anesthetizing with urethane after intragastric administration for 30 minutes, separating the right carotid artery and the left jugular vein, ligating the far end, filling a polyethylene tube containing 6cm of silk threads precisely weighed in advance with heparin sodium physiological saline solution, inserting one end of the polyethylene tube into the left jugular vein, inserting one end of the polyethylene tube into the right carotid artery, and ligating the near end. Blood flow from the right artery into the left vein through a polyethylene tube, the silk was removed after 15 minutes and the wet weight of the thrombus was recorded.
2) Methods of administration and dosages
The administration mode is intragastric administration. Blank control: physiological saline with the administration dosage of 0.3mL/100 g; positive control: aspirin (administered at a dose of 167 μmol/kg); 4 (administration dose is 1. mu. mol/kg); the invention 6 a-c (administered at a dose of 0.1. mu. mol/kg).
3) Statistical method
The statistics of the experimental data are all performed by adopting a t test, and the suppository weight is expressed by (mean value +/-SD mg).
4) Results of the experiment
As can be seen from the results in Table 3, the ratio P of 6a to c to physiological saline is less than 0.05, which indicates that 6a to c have good anti-arterial thrombosis activity; 0.1. mu. mol/kg6 a-c is equivalent to 1. mu. mol/kg4 in anti-arterial thrombosis activity. The compounds 6a to c of the invention are reduced by 10 times compared to the 4 doses under the same activity conditions. The present invention has an unexpected technical effect.
TABLE 3 anti-arterial Thrombus Activity of Compounds 6a to c
Figure GDA0002910106010000111
a) P <0.05 to saline, P <0.05 to 167 μmol/kg aspirin, and P >0.05 to 1 μmol/kg 4; n is 9.
Experimental example 27 dose-effect relationship for evaluation of anti-arterial thrombotic Activity of Compound 6c
1) The experimental method comprises the following steps: same as Experimental example 26
2) Methods of administration and dosages
The administration mode is intragastric administration. Blank control: physiological saline with the administration dosage of 0.3mL/100 g; positive control: aspirin in a dosage of 167 μmol/kg; the administration dose of Compound 6c of the present invention was 100nmol/kg, 10nmol/kg, 1 nmol/kg.
3) Statistical method
The statistics of the experimental data are all performed by adopting a t test, and the suppository weight is expressed by (mean value +/-SD mg).
4) Results of the experiment
As can be seen from the results in Table 4, the ratio P of 100nmol/kg and 10nmol/kg6c to physiological saline was less than 0.01, indicating that 6c has good anti-arterial thrombotic activity at doses of 100nmol/kg and 10 nmol/kg; compound 6c of the present invention was still effective at 10 nmol/kg. The present invention has an unexpected technical effect.
TABLE 4 three doses of Compound 6c anti-arterial Thrombus Activity
Figure GDA0002910106010000121
a) P <0.01 to saline, P <0.05 to 10nmol/kg6c, and P <0.01 to 167000nmol/kg aspirin; b) p <0.01 to normal saline, P <0.05 to 1nmol/kg 6 c; c) p >0.05 to saline; n is 12.
EXAMPLE 28 evaluation of the Activity of Compounds 6a to c for inhibiting the expression of GPIIb/IIIa in vivo
1) The experimental method comprises the following steps:
serum: in vivo evaluation of arterial thrombosis SD rats were sacrificed, carotid artery blood was collected and centrifuged at 3000rpm/min at 4 ℃ for 15 minutes.
Enzyme linked immunoassay kit: rat platelet membrane glycoprotein IIb/IIIa kit (origin: enzyme linked biology; cat # m1003231)
Enzyme-linked immunosorbent assay (manufacturer: Molecular Devices; model: SpectraMax M3)
Oven (manufacturer: lester; model: 101-1AB)
Sample adding: and setting a standard hole, a sample hole to be detected and a blank hole. Standard wells, each well is added with 50 μ L of standard substance with different concentrations; mu.L of sample diluent was added to the sample well, followed by 10. mu.L of sample. Blank wells were not added.
Adding an enzyme: mu.L per well, plated, shaken and incubated in an oven at 37 ℃ for 1 hour. Blank wells were not added.
Washing the plate: diluting the concentrated washing liquid by 20 times with distilled water, removing the sealing plate membrane, spin-drying, filling the washing liquid in each hole, and standing for 30 seconds for spin-drying. This was repeated five times.
Adding a color developing agent: each well was first filled with 50. mu.L of developer A and then 50. mu.L of developer B. Shaking, incubating in oven at 37 deg.C in dark for 15min
Adding a termination solution: mu.L per well, optical density was measured using a microplate reader at a wavelength of 450nm over 15 minutes. The experimental data were set to blank wells with 0 standard concentration and zeroed.
2) Administration methods and dosages: same as Experimental example 26
3) Statistical method
The statistics of the experimental data are all represented by t test (mean value + -SD mg).
4) Results of the experiment
As can be seen from the results in Table 5, the ratio P of 0.1 mu mol/kg6 a-c to physiological saline is less than 0.01, which shows that 6 a-c has good activity of inhibiting the expression of GPIIb/IIIa in vivo; the ratio P of 0.1 mu mol/kg6 a-c to 1 mu mol/kg4 is less than 0.01, which shows that 0.1 mu mol/kg6 a-c is more effective than 1 mu mol/kg4 in inhibiting the expression of GPIIb/IIIa. The present invention has an unexpected technical effect.
TABLE 5 Effect of Compounds 6 a-c on GPIIb/IIIa expression
Figure GDA0002910106010000131
a) P <0.01 to saline, P <0.01 to 1 μmol/kg 4; n is 6.
Experimental example 29 evaluation of the Effect of Compounds 6a to c on P-selectin expression
1) The experimental method comprises the following steps:
1) the experimental method comprises the following steps:
serum: in vivo evaluation of arterial thrombosis SD rats were sacrificed, carotid artery blood was collected and centrifuged at 3000rpm/min at 4 ℃ for 15 minutes.
Enzyme linked immunoassay kit P-selectin: rat P-selectin enzyme linked immunoassay kit (source: CUSABIO company; product number: CSB-E07399r)
Enzyme-linked immunosorbent assay (manufacturer: Molecular Devices; model: SpectraMax M3)
Oven (manufacturer: lester; model: 101-1AB)
Preparing a reagent: according to the specification, a series of concentration gradient standards, a washing liquid working solution, a biotin-labeled antibody working solution and a horseradish peroxidase-labeled avidin working solution are prepared. Then allowed to equilibrate at room temperature for at least thirty minutes.
Sample adding: and setting a standard hole and a sample hole to be detected. Add 100 μ L of standard or test sample to each well, cover the plate, shake well, and incubate in 37 ℃ oven for 2 hours. And (5) spin-drying.
Adding biotin labeled antibody working solution: mu.L per well, plated, shaken and incubated in an oven at 37 ℃ for 1 hour. Spin-drying, soaking the plate in 200 μ L of plate washing liquid for 2min every time, and washing the plate three times.
Adding horseradish peroxidase labeled avidin working solution: mu.L per well, plated, shaken and incubated in an oven at 37 ℃ for 1 hour. Spin-drying, soaking the plate for 2 minutes in 200 mu L of plate washing liquid per hole, and washing the plate for five times.
Adding a substrate solution: each well is 90 mu L, and the mixture is incubated and developed for 15-30 min in an oven at 37 ℃ in the dark
Adding a termination solution: 50 μ L per well, and after 5 minutes, the optical density was measured using a microplate reader at a wavelength of 450nm over 15 minutes. The experimental data were set to blank wells with 0 standard concentration and zeroed.
2) Administration methods and dosages: same as Experimental example 26
3) Statistical method
Data statistics were performed using the t-test and expressed as (mean ± SD).
4) Results of the experiment
As can be seen from the results in Table 6, the ratio P of 0.1 mu mol/kg6 a-c to physiological saline is less than 0.01, which indicates that 6 a-c has good activity of inhibiting the expression of P-selectin in vivo; 0.1. mu. mol/kg6a is equivalent to 1. mu. mol/kg4 in inhibiting P-selectin expression activity in vivo. Compound 6a of the invention was reduced by 10-fold over the 4 dose under the same activity conditions. The ratio of 0.1. mu. mol/kg6b and 6c to 1. mu. mol/kg 4P < 0.05. Indicating that 0.1. mu. mol/kg6b and 6c inhibited P-selectin expression better in vivo than 1. mu. mol/kg 4. The present invention has an unexpected technical effect.
TABLE 6 Effect of Compounds 6 a-c on P-selectin expression
Figure GDA0002910106010000141
a) P <0.01 to normal saline, and P >0.05 to 1 μmol/kg 4; b) p <0.01 to saline, P <0.05 to 1. mu. mol/kg 4; n is 6.

Claims (6)

1.(2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione of formula,
Figure 661190DEST_PATH_IMAGE001
wherein AA is a Phe residue, a Val residue or a Ser residue.
2. A process for the preparation of (2S,5S) -tetrahydropyrazinyl [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione according to claim 1, which comprises:
(1) will be provided withLThe benzyl-tryptophan ester is subjected to Pictet-Spengler condensation with 1,1,3, 3-tetramethoxypropane under the catalysis of trifluoroacetic acid to obtain 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester;
(2) 1- (2, 2-Dimethoxyethyl) -2,3,4, 9-tetrahydro-βReacting (E) -carboline-3-carboxylic acid benzyl ester with H under the catalysis of Pd/C2The benzyl ester is removed by the reaction to obtain 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-β-carboline-3-carboxylic acid;
(3) 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-one in the presence of dicyclohexylcarbodiimide and N-hydroxybenzotriazoleβThe (2S,5S) -tetrahydropyrazine [1,2:1,6 is obtained by intermolecular condensation of (carboline) -3-carboxylic acid in anhydrous N, N-dimethylformamide]And bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione;
(4) adding (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione in the presence of glacial acetic acid, water and concentrated hydrochloric acid, and carrying out acidolysis reaction to obtain (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione;
(5) uses dicyclohexylcarbodiimide and N-hydroxybenzotriazole as catalysts to synthesize HCl & Arg (NO) by adopting a liquid phase condensation method2)-Gly-Asp(OBzl)-AA-OBzl；
(6) In the presence of sodium cyanoborohydride, HCl & Arg (NO)2) -Gly-Asp (OBzl) -AA-OBzl and (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-Carbonylmethyl group]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And then the indole } -1, 4-diketone is subjected to ammoniation reduction to obtain (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-AA-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione;
(7) (2S,5S) -tetrahydropyrazines [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg (NO)2)-Gly-Asp(OBzl)-AA-OBzl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione in the presence of trifluoroacetic acid and trifluoromethanesulfonic acid to obtain (2S,5S) -tetrahydropyrazine [1,2:1,6]And bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1, 4-dione.
3. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione according to claim 1 for the preparation of an anti-venous thrombosis medicament.
4. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione according to claim 1 for the preparation of an anti-arteriothrombotic medicament.
5. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione according to claim 1 for the preparation of GPIIb/IIIa antagonists.
6. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Arg-Gly-Asp-AA ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione according to claim 1 for the preparation of a P-selectin antagonist.
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