CN110551176B - LDV modified S, R-heptacyclic aldehyde, and synthesis, activity and application thereof - Google Patents
LDV modified S, R-heptacyclic aldehyde, and synthesis, activity and application thereof Download PDFInfo
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- CN110551176B CN110551176B CN201810561698.2A CN201810561698A CN110551176B CN 110551176 B CN110551176 B CN 110551176B CN 201810561698 A CN201810561698 A CN 201810561698A CN 110551176 B CN110551176 B CN 110551176B
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- tetrahydro
- dione
- val
- bis
- asp
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Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses the following formula S, R-heptacyclic aldehyde-LeuAsp-Val, its preparation process, its anti-thrombotic activity, its activity of inhibiting GPIIb/IIIa expression and its activity of inhibiting P-selectin expression. Therefore, the invention discloses the application of the derivative in preparing anti-arterial thrombosis medicaments, anti-venous thrombosis medicaments, GPIIb/IIIa antagonists and P-selectin antagonists.
Description
Technical Field
The present invention relates to S, R-heptacyclic aldehyde-Leu-Asp-Val, its preparation process, its anti-arterial thrombus activity, its anti-venous thrombus activity, the activity of inhibiting the expression of glycoprotein IIb/IIIa and the activity of inhibiting the expression of P-selectin. 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 in conjunction with Leu-Asp-Val. Based on this assumption, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide S, R-heptacyclic aldehyde-Leu-Asp-Val of the formula.
The second content of the invention is to provide a method for synthesizing S, R-heptacyclic aldehyde-Leu-Asp-Val, which comprises the following steps:
(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) catalyzing 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in methanol solution at Pd/CLower, with H2The 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] 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 (4);
(5) using dicyclohexylcarbodiimide and N-hydroxybenzotriazole as catalysts, and synthesizing HCl & Leu-Asp (OBzl) -Val-OBzl by adopting a liquid phase condensation method;
(6) ammoniation reduction of HCl & Leu-Asp (OBzl) -Val-OBzl and (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione in the presence of sodium cyanoborohydride, to obtain (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp (OBzl) -Val-OBzl ] -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole } -1, 4-dione (5);
(7) in methanol solution, (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp (OBzl) -Val-OBzl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione is produced under the condition that pH is 13, (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione is produced, namely S, R-heptacyclic aldehyde-Leu-Asp-Val (6).
The third content of the invention is to evaluate the antithrombotic activity of S, R-heptacyclic aldehyde-Leu-Asp-Val.
The fourth aspect of the present invention is to evaluate the antithrombotic activity of S, R-heptacyclic aldehyde-Leu-Asp-Val.
The fifth aspect of the present invention is to evaluate the activity of S, R-heptacyclic aldehyde-Leu-Asp-Val in inhibiting GPIIb/IIIa expression in vivo.
The sixth aspect of the present invention is to evaluate the activity of S, R-heptacyclic aldehyde-Leu-Asp-Val in inhibiting P-selectin expression in vivo.
Drawings
FIG. 1 is a synthetic route for (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino } -1, 4-dione.
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, ice bath; vii) sodium cyanoborohydride vii)4N NaOH, methanol, ice bath.
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. Then, a saturated aqueous solution of sodium chloride was added thereto and the mixture was washed with water for 3 times. The dichloromethane layer was collected, dried for 2 hours by adding anhydrous sodium sulfate. Filtration was performed under normal pressure, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (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-Asp (OBzl) -Val-OBzl
2.00g (6.19mmol) of Boc-Asp (OBzl) -OH was suspended in 30mL of anhydrous tetrahydrofuran, and 0.92g (6.81mmol) of N-hydroxybenzotriazole and 1.40g (6.81mmol) of dicyclohexylcarbodiimide were added to the suspension in this order under ice bath and stirred for 30 minutes in ice bath. 2.58g (6.81mmol) of Tos. Val-OBzl were added. And dropwise adding N-methylmorpholine into the reaction mixture to adjust the pH value to 8-9. The reaction was carried out at room temperature for 8 hours, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 60mL of an ethyl acetate solution. 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, which was separated by silica gel column chromatography (v: v; dichloromethane: methanol: 25:1) to give 3.10g (6.04mmol) of the objective compound as a colorless solid in a yield of 98%. ESI-MS (M/e):513[ M + H]+
EXAMPLE 6 preparation of HCl Asp (OBzl) -Val-OBzl
4.68g (9.14mmol) of Boc-Asp (OBzl) -Val-OBzl was dissolved in 50mL of a solution of hydrogen chloride in ethyl acetate (4N) under ice-bath and reacted for 4 hours under ice-bath. The reaction solution was concentrated under reduced pressure, the residue was dissolved in anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. The operation is repeated3 times. The resulting yellow syrup sample was thoroughly triturated with anhydrous ether to give 3.92g (96%) of the title compound as a yellow solid. ESI-MS (M/e):413[ M + H]+。
EXAMPLE 7 preparation of Boc-Leu-Asp (OBzl) -Val-OBzl
Using the method of example 5, 2.10g (67.2%) of the title compound were obtained as a colorless powder from 1.25g (5mmol) of Boc-Leu and 2.69g (6mmol) of HCl. Asp (OBzl) -Val-OBzl. ESI-MS (M/e):626[ M + H]+。
EXAMPLE 8 preparation of HCl.Leu-Asp (OBzl) -Val-OBzl
From 2.00g (3.2mmol) of Boc-Leu-Asp (OBzl) -Val-OBzl 1.66g (92%) of the title compound was obtained as a colorless solid using the method of example 6. ESI-MS (M/e):526[ M + H ]]+
EXAMPLE 9 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] bis { (1S,1R) - [ 1-Ethyl-Leu-Asp (OBzl) -Val-OBzl ] -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indoline } -1, 4-dione (5)
206mg (0.43mmol) of Compound 4 was dispersed in 5mL of methanol. Adding 2 drops of triethylamine to adjust the pH value to 8 under ice bath to obtain a solution A; 724mg (1.29mmol) of HCl Leu-asp (OBzl) -Val-OBzl was dispersed in 5mL of methanol, and 2 drops of triethylamine was added to adjust pH 8 under 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. At intervals of 1 hour, 27mg (0.42mmol) of sodium cyanoborohydride as a reducing agent was added, 4 times in total, and 107mg (1.69mmol) 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 syrup was purified by silica gel column chromatography (v: v; dichloromethane: methanol 75:1) to give 586mg (0.39mmol) of the title compound as a yellow solid in 30% yield. ESI-MS (m/e): 1499[ M + H]+
1H-NMR(300MHz,CDCl3)δ/ppm：9.87(s,1H),9.59(s,0.5H),8.91(d,J＝6.9Hz,1H),8.11(d,J＝6.9Hz,1H),7.52(m,3H),7.35(m,20H),7.18(m,5H),6.15(s,1H),5.22(m,4H),5.12(m,4H),4.91(m,2H),4.55(m,2H),4.30(m,2H),3.63(m,3H),3.00(m,10H),2.76(m,3H),2.36(m,3H),2.22(m,3H),2.05(m,2H),1.85(m,4H),1.55(m,4H),1.01(m,2H),0.92(m,24H)
EXAMPLE 10 preparation of (2S,5S) -Tetrahydropyrazine [1,2:1,6] and bis { (1S,1R) - [ 1-Ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione (6)
300mg (0.20mmol) of Compound 5 are dissolved in 10mL of methanol and the pH is adjusted to 13 with 4N NaOH solution in an ice-water bath. The reaction was carried out in ice bath for 3 hours. The pH was adjusted to 7 with a saturated aqueous potassium hydrogensulfate solution under ice-cooling, and the solvent was concentrated under reduced pressure. The pH was adjusted to 2 with saturated aqueous potassium hydrogen sulfate in an ice bath. There was precipitation. Extracting the reaction solution with ethyl acetate, repeating the extraction process for 3 times, and collecting the ethyl acetate layer. And (3) carrying out extraction washing on the ethyl acetate layer by using a saturated sodium chloride solution, repeating the extraction washing process for 3 times, and collecting the ethyl acetate layer. The ethyl acetate layer was dried over anhydrous sodium sulfate for 2 hours, filtered under reduced pressure, and the reagent was concentrated under reduced pressure. 153mg (0.13mmol) of the title compound are obtained as a yellow solid in 67% yield. Melting range: 217.8-218.4 ℃; ESI-MS (m/e): 1140[ M + H]+IR(cm-1):3268.99,3060.27,3030.67,2960.20,2933.38,2872.62,1729.08,1637.84,1533.06,1496.55,1435.54,1390.21,1370.49,1329.74,1284.82,1260.16,1206.42,1156.31,1111.99,1079.82,1035.46,1007.34,925.10,844.20,816.49,794.23,781.90,739.96,696.81,671.74；1H-NMR(300MHz,DMSO-d6)δ/ppm：12.57(m,1.5H),11.25(m,1.5H),8.56(m,5H),7.31(m,8H),5.81(s,1H),5.21(s,1H),5.01(m,1H),4.72(s,1H),4.50(m,4H),4.15(m,2H),3.80(m,2H),3.00(m,5H),2.78(m,4H),2.22(m,2H),2.04(m,3H),1.59(m,5H),1.30(m,4H),0.92(m,25H)。
EXAMPLE 11 evaluation of anti-venous thrombosis Activity of Compound S, R-heptacyclaldehyde-Leu-Asp-Val
1) Experimental methods
SD rats (200 + -20 g) were acclimatized and fasted for one day before surgery, and were subjected to gavage administration at a dose of 0.3mL/100g body weight, and 30min after administration, to intraperitoneal administration for anesthesia with a 20% urethane solution 2min before surgery. 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); 4 (administration dose is 1. mu. mol/kg); invention 6 (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 6 to physiological saline is less than 0.01, indicating that 6 has good anti-venous thrombosis activity; 0.1 mu mol/kg6 is equivalent to 4.87 mu mol/kg warfarin in anti-thrombus activity (also equivalent to 1 mu mol/kg4 anti-thrombus activity). Under the same activity condition, the compound 6 of the invention is reduced by 48.7 times compared with warfarin dosage and 10 times compared with 4 dosages. The present invention has an unexpected technical effect.
TABLE 1 anti-thrombotic Activity of Compound 6
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 12 evaluation of anti-arterial Thrombus Activity of Compound 6
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); invention 6 (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 2, the ratio P of 6 to physiological saline is less than 0.05, indicating that 6 has good anti-arterial thrombotic activity; 0.1. mu. mol/kg6 is comparable to 1. mu. mol/kg4 in anti-arterial thrombotic activity. Compound 6 of the invention was reduced by 10-fold over the 4 dose under the same activity conditions. The present invention has an unexpected technical effect.
TABLE 2 anti-arterial Thrombus Activity of Compound 6
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.
EXAMPLE 13 evaluation of the Activity of Compound 6 for inhibiting GPIIb/IIIa expression 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 (from ELISA: Bio/Rad; cat # M1003231), microplate reader (manufacturer: Molecular Devices; model: SpectraMax M3), oven (manufacturer: lester; model: 101-1 AB).
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 12
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 3, the ratio P of 0.1. mu. mol/kg6 to physiological saline is less than 0.01, indicating that 6 has good activity of inhibiting in vivo GPIIb/IIIa expression; the ratio of P to P of 0.1. mu. mol/kg6 to 1. mu. mol/kg4 was <0.01, indicating that 6 to 1. mu. mol/kg4 had better activity in inhibiting the expression of GPIIb/IIIa. The present invention has an unexpected technical effect.
TABLE 3 Effect of Compound 6 on GPIIb/IIIa expression
a) P <0.01 to saline, P <0.01 to 1 μmol/kg 4; n is 6
EXAMPLE 14 evaluation of the Effect of Compound 6 on P-selectin expression
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 ELISA kit (from CUSABIO, Inc.; product No. CSB-E07399r), microplate reader (manufacturer: Molecular Devices; model: SpectraMax M3) oven (manufacturer: lester; model: 101-1 AB).
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 12
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 4, the ratio P of 0.1. mu. mol/kg6 to physiological saline is less than 0.01, indicating that 6 has good activity of inhibiting the expression of P-selectin in vivo; 6 and 1 mu mol/kg4 inhibit the in vivo P-selectin expression activity. Compound 6 of the invention was reduced by 10-fold over the 4 dose under the same activity conditions. The present invention has an unexpected technical effect.
TABLE 4 Effect of Compound 6 on P-selectin expression
a) 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-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino } -1, 4-dione of formula,
2. a process for the preparation of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione as claimed in claim 1, 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;
(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/C2Removing benzyl ester by reaction to obtain 1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid;
(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;
(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) using dicyclohexylcarbodiimide and N-hydroxybenzotriazole as catalysts, and synthesizing HCl & Leu-Asp (OBzl) -Val-OBzl by adopting a liquid phase condensation method;
(6) subjecting HCl & Leu-Asp (OBzl) -Val-OBzl and (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-carbonylmethyl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino } -1, 4-dione to ammoniation reduction in the presence of sodium cyanoborohydride to give (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp (OBzl) -Val-OBzl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino } -1, 4-dione;
(7) in methanol solution, (2S,5S) -tetrahydropyrazine [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp (OBzl) -Val-OBzl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione is produced under the condition that pH is 13 (2S,5S) -tetrahydropyrazine [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1, 4-dione is produced.
3. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -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-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione as claimed in 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-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione as claimed in claim 1 for the preparation of GPIIb/IIIa antagonists.
6. Use of (2S,5S) -tetrahydropyrazino [1,2:1,6] bis { (1S,1R) - [ 1-ethyl-Leu-Asp-Val ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indoline } -1, 4-dione as claimed in claim 1 for the preparation of a P-selectin antagonist.
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