CN111995661B - Ethyl ARPAK modified bis-carbolino-piperazinediones, preparation, activity and application thereof - Google Patents
Ethyl ARPAK modified bis-carbolino-piperazinediones, preparation, activity and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses (2 'S, 5' S) -tetrahydropyrazines [1 ', 2': 1, 6) of the formula]And bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]Indole-indole]-1 ', 4' -dione, discloses a process for its preparation, discloses its thrombolytic activity, discloses its antithrombotic activity and discloses its feature of being effective against ischemic stroke attacks for 24 hours. Therefore, the invention discloses the application of the compound in preparing the thrombolytic medicine, discloses the application of the compound in preparing the antithrombotic medicine, discloses the application of the compound in preparing the medicine which is still effective for 24 hours of ischemic stroke attack, and discloses the application of the compound in preparing the medicine which has triple effects of thrombolysis, antithrombotic and 24 hours of ischemic stroke attack.
Description
Technical Field
The present invention relates to (2 'S, 5' S) -tetrahydropyrazino [1 ', 2': 1,6] bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione, to a process for its preparation, to its thrombolytic activity, to its antithrombotic activity and to its feature of being effective against ischemic stroke attacks for 24 hours. The invention relates to the application of the compound in preparing thrombus dissolving medicines, the application of the compound in preparing antithrombotic medicines, the application of the compound in preparing medicines still effective in 24 hours of ischemic stroke attack and the application of the compound in preparing medicines with triple effects of dissolving thrombus, resisting thrombus and still effective in 24 hours of ischemic stroke attack. The invention belongs to the field of biological medicine.
Technical Field
Ischemic stroke is a common, severely damaging cerebrovascular disease. Ischemic stroke is characterized by high morbidity, high disability rate, high recurrence rate and high mortality rate, and is one of the most serious fatal diseases for human beings. Currently, rtPA is the only clinically accepted effective drug for the treatment of ischemic stroke. However, rtPA has two difficult problems to overcome in treating ischemic stroke. The first problem is that rtPA is not effective in patients with ischemic stroke that has occurred for more than 4 hours. The second problem is that continued use of rtPA can cause bleeding in the brain, thorax and abdominal cavities. The invention is a hot spot and a leading edge of research on cerebrovascular drugs, and the drugs are effective on patients with stroke attack for more than 4 hours, particularly patients with stroke attack for 24 hours and have no bleeding side effect. After years of exploration, the inventor finds that (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6] bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole ] -1 ', 4' -dione which is generated by introducing dicarbazolinopiperazine dione into ethyl-Ala-Arg-Pro-Ala-Lys and has the advantages of thrombolytic activity, antithrombotic activity, curative effect on ischemic stroke for 24 hours and no bleeding side effect. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
In a first aspect of the invention there is provided (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] oxabis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline ] -1 ', 4' -dione of formula.
The second aspect of the present invention provides a process for the preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indoline ] -1 ', 4' -dione, which comprises the following 7 steps:
(1) preparing (3S) -1- (2, 2-dimethoxyethan-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;
(2) preparing (3S) -1- (2, 2-dimethoxyethan-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid;
(3) preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione;
(4) preparing (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] bis [1S,1R- (1-carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole ] -1 ', 4' -dione;
(5) liquid phase method for synthesizing Ala-Arg (NO) by using N, N' -dicyclohexylcarbodiimide as condensing agent and 1-hydroxybenzotriazole as catalyst2)-Pro-Ala-Lys(Cbz)-OBzl；
(6) Ammonifying and reducing (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis [1S,1R- (1-carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]Indole-indole]-1 ', 4' -dione and Ala-Arg (NO)2) Preparation of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1, 6) by reaction of-Pro-Ala-Lys (Cbz) -OBzl]And bis [1S,1R- (1-ethyl-Ala-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]Indole-indole]-1 ', 4' -diketones;
(7) preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline ] -1 ', 4' -dione.
The third aspect of the present invention is to evaluate the thrombolytic activity of (2 'S, 5' S) -tetrahydropyrazino [1 ', 2': 1,6] bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione.
A fourth aspect of the present invention is to evaluate the antithrombotic activity of (2 'S, 5' S) -tetrahydropyrazino [1 ', 2': 1,6] bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione.
A fifth aspect of the present invention is to evaluate the efficacy of (2 'S, 5' S) -tetrahydropyrazino [1 ', 2': 1,6] bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione in rats with 24 hours of ischemic stroke onset.
Drawings
FIG. 1(2 ' S,5 ' S) -tetrahydropyrazine [1 ',2’:1,6]And bis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]Indole-indole]-1 ', 4' -dione synthesis route. i) Trifluoroacetic acid, dichloromethane, 40 ℃; ii) aqueous NaOH (2M), acetone, 0 ℃; iii)2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N-methylmorpholine, N, N-dimethylformamide; iv) glacial acetic acid, water; v) N, N' -dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; vi) aqueous NaOH (2M), methanol, 0 ℃; vii) hydrogen chloride in ethyl acetate (4M),0 ℃; viii) N-methylmorpholine, sodium cyanoborohydride, anhydrous magnesium sulfate, dichloromethane, methanol; ix) Palladium on carbon (Pd/C), H2And methanol.
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 (3S) -1- (2, 2-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylate (1)
5mL of 1,1,3, 3-tetramethoxypropane and 3.5mL of trifluoroacetic acid were added to 150mL of dichloromethane at 0 ℃ under stirring, and after reaction for 40 minutes, 5g (17.00mmol) of Trp-OBzl was added and the reaction was carried out at 40 ℃. After 4 hours of reaction, the reaction solution was adjusted to pH 7 with concentrated aqueous ammonia at 0 ℃ with stirring, concentrated under reduced pressure, the residue was dissolved in 100mL of dichloromethane, washed 3 times with saturated aqueous sodium bicarbonate solution, 3 times with saturated aqueous sodium chloride solution, the dichloromethane phase was dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated under reduced pressure to dryness to give a yellow oil, which was purified by silica gel column chromatography to give 5.50g (82%) of the title compound as a yellow oil. ESI-MS (M/e):395[ M + H]+。
EXAMPLE 2 preparation of (3S) -1- (2, 2-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid (2)
Dissolving 5.50g (13.96mmol) of (3S) -1- (2, 2-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester (1) in 50mL of acetone, adding aqueous NaOH solution (2M) at 0 ℃ under stirring, adjusting the pH value of the solution to 12, reacting for 3 hours, and keepingThe pH of the reaction mixture was 12. Then, the pH value was adjusted to neutral with a saturated aqueous potassium hydrogen sulfate solution, and the mixture was concentrated under reduced pressure. To the residue was added 50mL of acetone, the insoluble solid was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure to give 3.63g (86%) of the title compound as a yellow sticky substance. ESI-MS (M/e):303[ M-H]-。
EXAMPLE 3 preparation of (2 'S, 5' S) -Tetrahydropyrazine [1 ', 2': 1,6] Bis [1S,1R- (1-Dimethoxyethon-1-yl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol ] -1 ', 4' -dione (3)
Dissolving 3.63g (11.94mmol) of (3S) -1- (2, 2-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid (2) in 20mL of anhydrous N, N-dimethylformamide, adding 5.44g (14.32mmol) of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, adjusting the pH of the solution to 8 with N-methylmorpholine at 0 ℃ under stirring, stirring at room temperature for 16 hours, adding 150mL of distilled water to the reaction solution, extracting with dichloromethane (50 mL. times.3), combining dichloromethane phases, washing with saturated aqueous sodium chloride solution (50 mL. times.3), drying the dichloromethane phase with anhydrous sodium sulfate for 12 hours, filtering, concentrating the filtrate under reduced pressure to dryness, the resulting yellow oil was purified by silica gel column chromatography to give 0.73g (21%) of the title compound as a pale yellow solid. ESI-MS (M/e):573[ M + H]+。
EXAMPLE 4 preparation of (2 'S, 5' S) -Tetrahydropyrazine [1 ', 2': 1,6] Bis [1S,1R- (1-Carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole ] -1 ', 4' -dione (4)
0.30g (0.52mmol) of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis {1S,1R- (1-dimethoxyethan-1-yl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]And indole } -1 ', 4' -dione (3) was dissolved in 20mL of glacial acetic acid, 2mL of distilled water was added, the reaction was stirred at room temperature for 48 hours, and after filtration, the filter cake was washed with distilled water to remove the remaining glacial acetic acid and air-dried to obtain 0.22g (87%) of the title compound as a colorless solid. ESI-MS (M/e):479[ M-H]-。
EXAMPLE 5 preparation of Boc-Pro-Ala-OBzl
5.00g (23.25mmol) Boc-Pro was dissolved in 20mL dry tetrahydrofuran, and 2.86g (21.18mmol) 1-hydroxybenzotriazole, 5.23g (25.39mmol) N, N' -dicyclohexylcarbodiimide were added with stirring at 0 deg.CStirred at 0 ℃ for 30 minutes with stirring. Thereafter, 3.79g (21.17mmol) of Ala-OBzl was added thereto, the pH was adjusted to 8 with N-methylmorpholine, and the mixture was stirred at room temperature for 16 hours. The insoluble solids were removed by filtration, the filtrate was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, the insoluble solids were removed by filtration, the filtrate was washed with a saturated aqueous solution of sodium hydrogencarbonate (30 mL. times.3), saturated aqueous solution of sodium chloride (30 mL. times.3), 5% aqueous solution of potassium hydrogensulfate (30 mL. times.3), saturated aqueous solution of sodium chloride (30 mL. times.3), saturated aqueous solution of sodium hydrogencarbonate (30 mL. times.3) and saturated aqueous solution of sodium chloride (30 mL. times.3), the ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to give 6.93g (87%) of the title compound as a colorless solid. ESI-MS (M/e):377[ M + H]+。
EXAMPLE 6 preparation of Boc-Pro-Ala
6.93g (18.43mmol) of Boc-Pro-Ala-OBzl was dissolved in 30mL of methanol, and an aqueous NaOH solution (2M) was added thereto at 0 ℃ with stirring to adjust the pH to 12, followed by stirring for 3 hours while keeping the pH of the reaction solution at 12. Then, the pH value is adjusted to be neutral by saturated potassium bisulfate water solution, and the mixture is decompressed and concentrated. The residue was added with 100mL of distilled water, adjusted to pH 2 with saturated aqueous potassium hydrogensulfate solution, extracted with ethyl acetate (30 mL. times.3), the ethyl acetate phases were combined, washed with saturated aqueous sodium chloride solution (30 mL. times.3), the ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to give 4.80g (91%) of the title compound as a colorless oil. ESI-MS (M/e):285[ M-H]-。
EXAMPLE 7 preparation of Boc-Pro-Ala-Lys (Cbz) -OBzl
From 4.80g (16.78mmol) Boc-Pro-Ala and 5.64g (15.24mmol) Lys (Cbz) -OBzl 7.65g (79%) of the title compound were obtained as colorless solid by the method of example 5. ESI-MS (M/e) 639[ M + H ]]+。
EXAMPLE 8 preparation of Pro-Ala-Lys (Cbz) -OBzl
5.00g (7.84mmol) Boc-Pro-Ala-Lys (Cbz) -OBzl was dissolved with 50mL hydrogen chloride in ethyl acetate (4M) at 0 ℃ with stirring, after which stirring was continued for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 50mL of anhydrous ethyl acetate and concentrated under reduced pressure. This operation was repeated 3 times. The resulting colorless solid was thoroughly suspended in 50mL of anhydrous ether and free hydrogen chloride was washed off to give 4.13g (98%) of the title compound as a colorless solid which was used directly in the next reaction.
EXAMPLE 9 preparation of Boc-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Starting from 2.69g (8.43mmol) Boc-Arg (NO) by the method of example 52) And 4.13g (7.68mmol) Pro-Ala-Lys (Cbz) -OBzl to give 4.17g (65%) of the title compound as a colorless solid. ESI-MS (M/e):840[ M + H]+。
EXAMPLE 10 preparation of Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Starting from 2g (2.38mmol) of Boc-Arg (NO) by the method of example 82) -Pro-Ala-Lys (Cbz) -OBzl gave 1.73g (98%) of the title compound as a colorless solid, which was used directly in the next reaction.
EXAMPLE 11 preparation of Boc-Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Starting from 0.48g (2.54mmol) Boc-Ala and 1.73g (2.34mmol) Arg (NO) by the method of example 52) -Pro-Ala-Lys (Cbz) -OBzl gave 1.10g (52%) of the title compound as a colorless solid. ESI-MS (M/e) 911[ M + H ]]+。
EXAMPLE 12 preparation of Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Starting from 0.57g (0.63mmol) Boc-Ala-Arg (NO) by the method of example 82) Pro-Ala-Lys (Cbz) -OBzl gave 0.49g (96%) of the title compound as a colorless solid, which was used directly in the next reaction.
EXAMPLE 13 preparation of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis [1S,1R- (1-ethyl-Ala-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]Indole-indole]-1 ', 4' -dione (5)
0.49g (0.60mmol) of Ala-Arg (NO)2) Pro-Ala-Lys (Cbz) -OBzl was dissolved in 7mL of dichloromethane, and the pH was adjusted to 8 with N-methylmorpholine to obtain reaction solution A. 0.10g (0.21mmol) of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis [1S,1R- (1-carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]Indole-indole]The (4) -1 ', 4' -dione (4) was suspended in 5mL of dichloromethane, and the pH was adjusted to 8 with N-methylmorpholine, whereby a reaction solution B was obtained. Firstly, the reaction is carried outSolution B was added to reaction solution A, followed by addition of 50mg of anhydrous magnesium sulfate and 3mL of methanol in this order. The resulting reaction mixture was stirred at room temperature and sodium cyanoborohydride was added every 30 minutes 4 times for a total of 64mg (1.03mmol) of 16mg (0.26mmol) each time. The reaction mixture was stirred at room temperature for 16 hours. Thereafter, the reaction mixture was concentrated under reduced pressure, the residue was dissolved in 20mL of methylene chloride and 5mL of methanol, insoluble matter was filtered off, the filtrate was concentrated under reduced pressure, and the resulting pale yellow sticky solid was purified by silica gel column chromatography to give 0.10g (23%) of the title compound as a colorless solid. ESI-MS (M/e):2069[ M + H]+.1H NMR(300MHz,DMSO-d6):δ/ppm＝11.04～11.06(m,2H),7.79～8.50(m,12H),7.51(m,1H),7.45(m,1H),7.34(m,22H),7.22(m,2H),7.08(m,2H),7.00(m,2H),5.84(m,1H),5.28(m,1H),5.10(m,4H),5.00(m,4H),4.47(m,2H),4.35(m,2H),4.25(m,6H),3.51～3.61(m,4H),3.17(m,4H),3.11(m,4H),2.94～3.02(m,8H),2.03(m,6H),1.69～2.03(m,14H),1.49(m,4H),1.36(m,4H),1.25(m,8H),1.10～1.17(m,10H).13C NMR(125MHz,DMSO-d6):δ/ppm＝173.0,172.9,172.3,171.5,171.1,170.3,170.0,168.9,165.0,159.8,156.6,137.7,136.6,136.5,136.4,133.4,128.9,128.8,128.5,128.3,128.2,127.8,126.5,126.2,121.6,119.3,118.3,111.8,107.3,105.4,66.4,65.6,59.5,57.6,57.4,57.0,53.4,52.5,50.1,49.1,48.3,48.2,48.0,47.3,47.0,44.6,42.9,40.9,34.0,32.0,30.9,29.4,29.3,29.0,28.8,25.5,24.9,24.5,23.0,22.4,19.8,18.8,18.6,18.5。
EXAMPLE 14 preparation of (2 'S, 5' S) -Tetrahydropyrazine [1 ', 2': 1,6] Bis [1S,1R- (1-Ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indoline ] -1 ', 4' -dione (6)
0.10g (0.05mmol) of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis [1S,1R- (1-ethyl-Ala-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]Indole-indole]-1 ', 4' -dione (5) was dissolved in a mixed solvent of 5mL of methylene chloride and 15mL of methanol. Then 20mg of palladium on carbon (Pd/C) and 2 drops of formic acid were added thereto in this order. The reaction mixture was purged with hydrogen at room temperature and stirred for 16 hours. Then, Pd/C was filtered off. The filtrate was concentrated under reduced pressure, and the residue was dissolved in 5mL of methylene chloride and 15mL of methanol. To the resulting solution was added an additional 20mg Pd/C and 2 drops of formazanAcid, then hydrogen was passed through at room temperature and stirred for 16 hours. After that, Pd/C was filtered off, the filtrate was concentrated under reduced pressure, and the resulting colorless solid was purified by Sephadex to give 0.04g (54%) of the title compound as a colorless solid. Mp 230-231 deg.C;
ESI-MS(m/e):1531[M+H]+.1H NMR(300MHz,MeOD):δ/ppm＝7.56(m,2H),7.41(m,2H),7.17(m,2H),7.10(m,2H),6.04(m,1H),5.63(m,1H),4.62(m,2H),4.45(m,2H),4.21～4.33(m,6H),3.70(m,2H),3.56(m,2H),3.04～3.21(m,8H),2.92(m,6H),2.76(m,2H),2.00～2.30(m,8H),1.88(m,4H),1.70(m,14H),1.39～1.48(m,12H),1.31～1.35(m,8H).13C NMR(125MHz,MeOD):δ/ppm＝173.7,173.5,172.7,172.5,170.2,169.7,168.9,168.0,166.6,161.9,159.4,157.2,137.0,136.8,131.0,130.5,128.5,126.1,125.7,122.0,121.9,119.3,117.9,117.8,111.2,111.1,107.8,105.9,65.3,60.1,59.9,57.4,56.5,56.1,53.7,51.8,51.0,50.6,50.5,49.2,49.1,48.5,43.0,42.1,40.7,39.1,30.6,29.8,29.4,29.2,29.0,28.2,27.7,26.5,24.6,24.3,24.0,22.4,16.6,16.4,15.5,15.2.IR(cm-1):3176.31,3053.50,2954.48,2873.87,1633.06,1557.18,1447.66,1382.67,1330.99,1260.28,1201.11,1156.95,1009.15,744.54,674.24,598.57。
experimental example 1 evaluation of thrombolytic Activity of Compound 6
Male SD rats (200. + -.20 g) were randomly grouped into 8 per group and rested for one day. Rats were anesthetized by intraperitoneal injection with a 20% urethane physiological saline solution. The rat is fixed in a supine position, the right common carotid artery is separated, the artery clamp is clamped at the proximal end, the proximal end and the distal end respectively penetrate into an operation line, the operation line at the distal end is clamped by hemostatic forceps for fur, the artery clamp is loosened at the cannula at the distal end, about 1mL of arterial blood is discharged, and the rat is arranged in a 1mL bullet. 0.1mL of rat arterial blood was injected into a vertically fixed rubber tube (15 mm in length, 2.5mm in inner diameter, 5.0mm in outer diameter, sealed at the bottom with a rubber plug, and sealed with para membrane), and a stainless steel thrombus-fixing bolt was rapidly inserted into the tube. The thrombus fixing bolt is formed by winding a stainless steel wire with the diameter of 0.2mm, the length of a spiral part is 10mm, 15 spiral coils are contained, the diameter of each spiral coil is 1.0mm, and a support handle is connected with the spiral and is 7.0mm long, so that the thrombus fixing bolt is in a question mark shape. After 40 minutes of blood coagulation, the thrombus-coated thrombus-fixing bolt was carefully removed from the rubber tube, and the starting weight of the thrombus-fixing bolt was accurately weighed.
The bypass cannula is made up of three sections. The middle section is a polyethylene rubber tube with the length of 60mm and the inner diameter of 3.5 mm. The two side sections are polyethylene pipes with the length of 100mm, the inner diameter of 1.0mm and the outer diameter of 2.0 mm. One end of the polyethylene tube is pulled to a tip with the length of 10mm and the outer diameter of 1.0mm (used for inserting into the carotid artery and the vein of a rat), and a section of polyethylene tube with the length of 7mm and the outer diameter of 3.5mm (used for connecting the middle section of polyethylene tube) is sleeved outside the other end of the polyethylene tube. The inner walls of the 3-segment tubes need to be silanized (1% silicone oil ether solution). And placing the thrombus fixing bolt wrapped by the thrombus accurately weighed into the middle section polyethylene pipe.
The trachea of the anesthetized rat is continuously separated and the trachea cannula is made. The left external jugular vein of the rat was isolated, and surgical lines were threaded proximally and distally, respectively, and a bevel was carefully cut on the exposed left external jugular vein. The manufactured sharp tube of the bypass pipeline is inserted into the proximal end of the left external jugular vein opening from the oblique opening and is far away from a support handle for fixing the spiral thrombus in the middle section of the bypass tube. An accurate amount of sodium heparin in physiological saline solution (50IU/kg) was pushed in through the tip tube at the other end with the syringe without removing the syringe from the polyethylene tube. The hose between the syringe and the polyethylene tube was clamped with a hemostat. The right common carotid artery was stopped by an arterial clamp at the proximal end of the right common carotid artery and a beveled opening was carefully cut short of the arterial clamp. The syringe was pulled out from the tip of the polyethylene tube, and the tip of the polyethylene tube was inserted into the proximal end of the oblique orifice of the artery. Both ends of the bypass duct were secured to the artery and vein with a number 4 surgical suture.
A scalp needle containing physiological saline (3mL/kg) or a physiological saline solution of urokinase (20000IU/kg) or a physiological saline solution of compound 6 (100nmol/kg) was inserted into the proximal vein distal to the thrombus-anchoring helix in the middle of the bypass tube, and the arterial clamp was opened to allow blood flow from the artery to the vein through the bypass tube. Physiological saline (blank control), urokinase (positive control) or compound 6 in a scalp needle was slowly injected into the middle section of the bypass tube, and allowed to act on the thrombus through the blood circulation in the order of vein-heart-artery. And (4) timing from the beginning of injection, taking out the thrombus fixing bolt from the middle section of the bypass pipe after 1 hour, and accurately weighing the final weight of the thrombus fixing bolt. The difference between the initial weight and the final weight of the thrombus fixing bolt is the thrombus weight loss. Calculating the thrombus weight loss of each rat, counting the thrombus weight loss of each group of rats and performing t test. The experimental data are shown in table 1. It can be seen that the thrombus loss (28.44 + -3.44 mg) in compound 6 treated rats was significantly greater than that in physiological saline treated rats at a dose of 100nmol/kg (23.18 + -1.40 mg, P < 0.01). In addition, the thrombus loss in compound 6 treated rats at the 100nmol/kg dose was not significantly different from that in 20000IU/kg dose urokinase treated rats (27.61 ± 3.47mg, P > 0.05). The excellent thrombolytic activity of compound 6 shows the outstanding technical effect of the invention.
TABLE 1 thrombolytic Activity of Compound 6
a) P <0.01 to saline; b) p <0.01 to saline, P >0.05 to urokinase; n is 8.
Experimental example 2 evaluation of antithrombotic Activity of Compound 6
The same bypass cannula as in experimental example 1 was used for this experiment. Male SD rats (200. + -.20 g) were randomly divided into groups of 10 animals each, kept for 1 day and stopped overnight. Compound 6 was gavaged with either saline solution (dose 100nmol/kg) or aspirin (dose 167. mu. mol/kg) or saline (dose 3 mL/kg). After 30 minutes of administration, rats were anesthetized with an intraperitoneal injection of 20% urethane in a physiological saline solution. After anesthesia, the rat was fixed on a rat fixation plate, the right carotid artery and the left jugular vein of the rat were separated, the accurately weighed silk was placed in a bypass cannula, one end of the tube was inserted into the left vein, the other end was inserted into the right artery and 0.2mL heparin sodium anticoagulated was injected. The blood flow from the right artery was allowed to flow through the bypass cannula into the left vein, the thrombus-attached silk thread was taken out after 15 minutes and weighed, and the weight difference of the silk thread before and after blood circulation was calculated. The difference is the thrombus weight, representing the antithrombotic activity. The thrombus weight of each group of rats is counted and subjected to t test. The experimental data are shown in table 2. It can be seen that the plug weight of compound 6 treated rats (16.81 ± 4.19mg) at 100nmol/kg oral dose is significantly less than the plug weight of saline treated rats (26.55 ± 4.61mg, P < 0.01). Furthermore, the suppository weight of compound 6 treated rats at 100nmol/kg oral dose was not significantly different from that of 167 μmol/kg aspirin treated rats (17.08 ± 2.80mg, P > 0.05). That is, even at 1670 times lower doses compound 6 still had comparable anti-arterial thrombus activity to aspirin. The excellent antithrombotic activity of compound 6 indicates that the invention has outstanding technical effects.
TABLE 2 antithrombotic Activity of Compound 6
a) P <0.01 to saline; b) p <0.01 to saline, P >0.05 to aspirin; n is 10.
Experimental example 3 evaluation of therapeutic Effect of Compound 6 on rats suffering from ischemic stroke for 24 hours
Male SD rats (280. + -.20 g) were anesthetized with a 10% chloral hydrate (4mL/kg) by intraperitoneal injection. A2 cm long incision was made vertically in the middle of the neck of the rat, and the right common carotid artery, external carotid artery and internal carotid artery were isolated along the intramuscular side of the sternocleidomastoid. Respectively clamping an opening of an internal carotid artery and a proximal end of a common carotid artery by using a noninvasive artery clamp, ligating a distal end of an external carotid artery, cutting a small opening on the external carotid artery, loosening the artery clamp at the proximal end of the common carotid artery, and taking 10 mu L of blood to enter a 1mL centrifuge tube. Thereafter, the proximal end of the common carotid artery is clamped with a non-invasive artery clamp. The blood in the centrifuge tube was allowed to stand at room temperature for 30 minutes to coagulate, and then allowed to clot at-20 ℃ overnight. The blood clot was removed, 1mL of physiological saline was added, the blood clot was pounded with a steel spatula into small thrombus blocks of uniform size, a suspension of the small thrombus was prepared and transferred to a 1mL syringe. The artery clamp at the proximal end of the common carotid artery of the rat is loosened, 1mL of thrombus suspension is slowly injected into the brain of the rat from the external carotid artery of the rat to the proximal end through the internal carotid artery, then the proximal end of the external carotid artery is ligated, and the artery clamp at the internal carotid artery and the common carotid artery is opened to restore the blood flow. Waiting for wakeup. The degree of neurological deficit was assessed by the Zealonga method 24 hours after the rats were awakened. 0 point indicates no sign of neurological deficit, 1 point indicates that the front limb on the uninjured side cannot stretch, 2 points indicates walking to the uninjured side, 3 points indicates turning to the uninjured side to form rear-end walking, 4 points indicates that the disorder is identified without autonomous walking, and 5 points indicates death. Rats were evenly grouped according to score. Each group of rats was injected daily with 1 second injection of saline (3mL/kg/d) or rtPA (3mg/kg/d) or Compound 6(10nmol/kg/d) via the tail vein. Injections were continued for 3 days, and scored daily. The mortality rate of each group of rats is calculated according to the score, namely death counts/total counts multiplied by 100%; number of cases/total cases with a score on the last day lower than the score before dosing but not 0 x 100%; the cure rate is 0 counts/total counts on the last day x 100%. The data are shown in tables 3,4 and 5. The results show that compound 6 treated rats had a lower mortality rate than saline treated rats and a much higher cure rate than saline treatment. Furthermore, mortality was lower in rats treated with compound 6 than in rats treated with rtPA and cure rate was higher than that of rtPA. rtPA treatment caused systemic bleeding in rats, compound 6 had no bleeding side effects. Therefore, the invention has outstanding technical effects.
TABLE 3 Effect of physiological saline on neurobiological scores in rats with 24 hours ischemic stroke onset
TABLE 4 Effect of rtPA on neurobiological scores in rats with 24 h ischemic stroke onset
TABLE 5 Effect of Compound 6 on neurobiological scores in rats with 24 h ischemic stroke onset
Claims (6)
1. (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] bis [1S,1R- (1-ethyl) S
-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole ] -1 ', 4' -dione,
2. a process for the preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline ] -1 ', 4' -dione of claim 1, which comprises the following 7 steps:
(1) preparing (3S) -1- (2, 2-dimethoxyethan-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;
(2) preparing (3S) -1- (2, 2-dimethoxyethan-1-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid;
(3) preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-dimethoxyeth-1-yl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione;
(4) preparing (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] bis [1S,1R- (1-carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole ] -1 ', 4' -dione;
(5) liquid phase method for synthesizing Ala-Arg (NO) by using N, N' -dicyclohexylcarbodiimide as condensing agent and 1-hydroxybenzotriazole as catalyst2)-Pro-Ala-Lys(Cbz)-OBzl；
(6) Ammonifying and reducing (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1,6]And bis [1S,1R- (1-carbonylmethyl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]Indole-indole]-1 ', 4' -dione and Ala-Arg (NO)2) Preparation of (2 'S, 5' S) -tetrahydropyrazine [1 ', 2': 1, 6) by reaction of-Pro-Ala-Lys (Cbz) -OBzl]And bis [1S,1R- (1-ethyl-Ala-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]Indole-indole]-1 ', 4' -diketones;
(7) preparation of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline ] -1 ', 4' -dione.
3. Use of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione of claim 1 for the preparation of a thrombolytic drug.
4. Use of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione according to claim 1 for the preparation of an antithrombotic medicament.
5. Use of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] obis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione of claim 1 for the preparation of a medicament which is still effective against ischemic stroke attacks for 24 hours.
6. Use of (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] orbis [1S,1R- (1-ethyl-Ala-Arg-Pro-Ala-Lys) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino ] -1 ', 4' -dione according to claim 1 for the preparation of a medicament having triple effects of thrombolysis, antithrombotic and remaining effective against ischemic stroke attacks for 24 hours.
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