CN114763354B - Heptacycloindole diketopiperazine non-covalent bond trimer, preparation, thrombolytic activity and application thereof - Google Patents
Heptacycloindole diketopiperazine non-covalent bond trimer, preparation, thrombolytic activity and application thereof Download PDFInfo
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Abstract
The invention discloses a heptacycloindole diketopiperazine of the following formula (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole } -1',4' -dione. Discloses a preparation method thereof, discloses thrombolysis activity of the composition on a rat artery and vein bypass cannula thrombolysis model, and further discloses activity of the composition for reducing the content of PAI-1 in rat serum. The invention thus discloses its use in the preparation of thrombolytic drugs and in the preparation of PAI-1 inhibitors.
Description
Technical Field
The present invention relates to a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzidine } -1',4' -dione, to a process for its preparation, to its thrombolytic activity, to its activity in reducing serum PAI-1 content. The invention thus relates to its use in the preparation of thrombolytic drugs and PAI-1 inhibitors. The invention belongs to the field of biological medicine.
Background
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor family. The physiological importance of PAI-1 is demonstrated in transgenic mice expressing high levels of human PAI-1 and suffering from severe venous thrombosis. Elevated plasma PAI-1 concentrations are hypothesized to be a risk factor for thrombotic disease. The target proteins for PAI-1 are tPA and uPA. The secretion of active PAI-1 from cells can form covalent complexes with tPA and uPA, with the direct consequence that tPA and uPA lose the ability to activate plasminogen. The indirect result is that tPA and uPA lose thrombolytic activity. Platelets are one of the primary cells that synthesize PAI-1. A reasonable premise of designing thrombolytic agents based on the inhibition of PAI-1 is that compounds with anti-venous thrombotic activity are able to bind to the platelet surface. The inventors disclose that (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione can effectively inhibit venous thrombosis in rats. Accordingly, the inventors evaluated its thrombolytic activity. However, (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione has no thrombolytic activity at an anti-venous thrombosis dose of 20 nmol/kg. Thus, the inventors contemplate that perhaps (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione does not bind strongly to platelets. It is thus further contemplated that the non-covalent multimer of (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione may have an increased degree of firmness in binding to platelets. According to this assumption, the inventors finally produced a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione. Evaluation shows that the noncovalent trimer exhibits definite thrombolytic activity at an anti-venous thrombosis dose of 20 nmol/kg. Based on these findings, the applicant has proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzidine } -1',4' -dione of the formula.
The second aspect of the present invention is to provide a process for preparing a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione according to claim 1, which comprises:
carrying out Pictet-Spengler condensation on L-tryptophan benzyl ester and 1, 3-dihydroxyacetone under the catalysis of trifluoroacetic acid to prepare 3S-1, 1-dimethylol tetrahydro-beta-carboline-3-benzyl carboxylate (1);
b.3S-1, 1-dimethylol-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (1) is reacted with t-butyldimethylsilyl chloride in N, N-dimethylformamide in the presence of imidazole to prepare 3S-1, 1-bis (t-butyldimethylsilyloxy) methyltetrahydro- β -carboline-3-carboxylic acid benzyl ester (2);
benzyl C.3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylate (2) is subjected to palladium-carbon catalytic hydrogenolysis in methanol to obtain 3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylic acid (3);
D. condensing two molecules of 3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylic acid (3) into (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-di (tert-butyldimethylsilyloxy) methyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (4) in anhydrous N, N-dimethylformamide with 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate as a condensing agent;
E. preparation of (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dimethylol-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (5) according to claim 1 from (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dimethylol-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (4) under the action of tetrabutylammonium fluoride in anhydrous tetrahydrofuran.
F. Suspending the compound 5 with deionized water, and performing ultrasonic treatment on the obtained suspension for 2 hours to dissolve the suspension, and performing freeze drying on the obtained solution to obtain the freeze-dried powder of the non-covalent trimer of the compound 5.
The third aspect of the present invention is to evaluate the thrombolytic activity of the non-covalent trimer of (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione.
The fourth aspect of the present invention is to evaluate the activity of the non-covalent trimer of (2 'S,5' S) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione to reduce serum PAI-1 concentration in rats.
Drawings
FIG. 1 is a synthetic route diagram of a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione, labeled i) 1, 3-dihydroxyacetone, trifluoroacetic acid, respectively; ii) N, N-dimethylformamide, t-butyldimethylchlorosilane, imidazole; iii) Methanol, palladium on carbon, hydrogen; iv) N, N-dimethylformamide, 2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate, N-methylmorpholine; v) tetrahydrofuran, tetrabutylammonium fluoride; vi) deionized water suspension, and the obtained suspension is sonicated for 2 hours and freeze-dried.
FIG. 2 is an ESI-FT-ICR-MS pattern of a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzidine } -1',4' -dione. The inserted locally enlarged peak at mass 1583.59136 is the non-covalent dimer chlorination peak, at mass 1067.37941 is the non-covalent dimer chlorination peak, and at mass 551.17013 is the monomer chlorination peak.
FIG. 3 is a qCID pattern of a non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzidine } -1',4' -dione. qCID plots represent peak bombardment for non-covalent trimer chlorination occurring at mass number 1583.59998, with the result that the non-covalent trimer chlorination anion is cleaved into 1067.37071 mass non-covalent dimer chlorination anion and 551.17138 mass monomer chlorination anion. Thus, both the peak of non-covalent dimer chlorination and the peak of monomer chlorination in FIG. 2 are cleavage products of non-covalent trimers under assay conditions. It can be seen that the non-covalent trimer is the only form of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione present in its lyophilized powder.
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention.
EXAMPLE 1 preparation of 3S-1, 1-dihydroxymethyl-1, 2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester (1)
6.4g (21.7 mmol) of L-Trp-OBzl was dissolved in 300ml of methylene chloride, and 6ml of trifluoroacetic acid and 2.35g (26.1 mmol) of 1, 3-dihydroxyacetone were added to the resulting solution at 0℃and stirred at room temperature for 16 hours. TLC (CH) 2 Cl 2 Meoh=10/1) showed the disappearance of L-Trp-OBzl, terminating the reaction. Saturated NaHCO was added to the reaction mixture at 0deg.C 3 The pH of the solution was adjusted to neutral, dichloromethane was removed by spinning, and the aqueous layer was extracted with ethyl acetate (300 mL. Times.3). The combined dichloromethane layers were washed with saturated aqueous NaCl (100 mL. Times.3) and the dichloromethane layer was washed with anhydrous Na 2 SO 4 Drying for 12h. Filtration and concentration of the filtrate under reduced pressure gave 7.9g (99.2%) of the title compound as a yellow, foamy solid. ESI-MS (m/e): 365[ M-H ]] - 。
Example 2 preparation of 3S-1, 1-bis (t-butyldimethylsilyloxy) -1,2,3, 4-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (2) 3g (8.2 mmol) of 3S-1, 1-dimethylol-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (1) was dissolved in 20ml of anhydrous N, N-dimethylformamide, 3.7g (24.6 mmol) of t-butyldimethylchlorosilane (TBDMSCl) and 2g (29.5 mmol) of imidazole were added to the resulting solution at 0 ℃ and stirred at 0 ℃ for 10min, and stirred at room temperature for 10h, tlc (petroleum ether/ethyl acetate=20/1) showed disappearance of compound 1, and the reaction was terminated. The reaction mixture was added with 100mL of ice-water, followed by extraction with ethyl acetate (50 mL. Times.3). The ethyl acetate layer was first treated with saturated NaHCO 3 Washing with aqueous solution (50 mL. Times.3), washing with saturated aqueous NaCl solution (50 mL. Times.3), washing with anhydrous Na 2 SO 4 Drying for 12h. The filtrate was filtered, concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel, eluting with a gradient of petroleum ether and ethyl acetate (petroleum ether/ethyl acetate=50/1-20/1) to give 4.5g (92%) of the title compound as a colorless solid. ESI-MS (m/e): 595[ M+H ]] + ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝10.343(s,1H),7.956(s,1H),7.394(m,5H),7.283(d,J＝7.2Hz,1H),7.033(t,J 1 ＝J 2 ＝7.2Hz,1H),6.941(t,J 1 ＝J 2 ＝7.2Hz,1H),5.222(m,2H),4.058(m,1H),3.775(m,4H),3.002(dd,J 1 ＝15Hz,J 2 ＝3.6Hz,1H),2.609(m,1H),0.79(d,J＝1.9Hz,18H),-0.03(m,12H)。
Example 3 preparation of 3S-1, 1-bis (t-butyldimethylsilyloxy-methyl) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (3)
1g (1.68 mol) of 3S-1, 1-bis (t-butyldimethylsilyloxy-methyl) -1,2,3, 4-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (2) was dissolved in 8ml of methanol, 100mg Pd/C was added to the solution, hydrogen was introduced and stirred at room temperature for 6 hours, and TLC (Petroleum ether/ethyl acetate=20/1) showed disappearance of Compound 2. Pd/C was filtered off and the filtrate concentrated under reduced pressure to give 821mg (97%) of the title compound as a colorless, foamy powder. ESI-MS (m/e): 505[ M+H ]] + 。
EXAMPLE 4 preparation of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-bis (t-butyldimethylsilyloxy-methyl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (4)
800mg (1.59 mmol) of 3S-1, 1-bis (t-butyldimethylsilyloxy-methyl) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (3) was dissolved in 8ml of anhydrous N, N-dimethylformamide, 724mg (1.90 mmol) of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate was added to the obtained solution at 0℃and stirred at 0℃for 5 minutes, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine and stirred at room temperature for 6 hours. TLC (CH) 2 Cl 2 /MeOH/CH 3 CO 2 H=10/1/0.1) showed that compound 3 disappeared, terminating the reaction. The reaction mixture was added with 60mL of ice-water, followed by extraction with ethyl acetate (20 mL. Times.3). Saturated NaHCO for ethyl acetate layer 3 Washing with the solution (30 mL. Times.3), washing with saturated aqueous NaCl solution (30 mL. Times.3), washing with anhydrous Na 2 SO 4 Drying for 12h. Filtration, concentration of the filtrate under reduced pressure, and purification of the residue by silica gel column chromatography were carried out by gradient elution with a petroleum ether-ethyl acetate elution system (petroleum ether/ethyl acetate=20/1-15/1) to give 650mg (84%) of the title compound as a colorless solid. ESI-MS (m/e): 973[ M+H ]] + ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝10.958(s,2H),7.321(t,J 1 ＝8Hz,J 2 ＝10Hz,4H),7.056(td,J 1 ＝7.5Hz,J 2 ＝1.1Hz,2H),6.963(td,J 1 ＝0.8Hz,J 2 ＝7.5Hz,2H),4.993(d,J＝9.5Hz,2H),4.541(d,J＝10.2Hz,2H),4.448(dd,J 1 ＝11.3Hz,J 2 ＝2.5Hz,2H),4.117(d,J＝10.1Hz,2H),3.797(d,J＝9.5Hz,2H),3.206(m,2H),2.857(m,2H),0.709(s,18H),0.598(s,18H),-0.484(s,6H),-0.131(s,6H),-0.223(s,6H),-0.462(s,6H)。
EXAMPLE 5 preparation of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] penta-indole } -1',4' -dione (5)
500mg (0.51 mmol) of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6]And bis {1, 1-bis (t-butyldimethylsilyloxy-methyl) -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b]And indole } -1',4' -dione (4) was dissolved in 1ml of tetrahydrofuran solution of tetrabutylammonium fluoride (4M) and 5ml of tetrahydrofuran, stirred at room temperature for 4h, TLC (CH) 2 Cl 2 Meoh=10/1) showed that compound 4 disappeared, terminating the reaction. Concentrating under reduced pressure, purifying the residue by silica gel column chromatography, and gradient eluting with dichloromethane-methanol (CH 2 Cl 2 /CH 3 Oh=100/1-10/1) to give 220mg (83%) of the title compound as a colorless solid. ESI-MS (m/e): 515[ M-H ]] - ； 1 H NMR(300MHz,DMSO-d 6 ):δ/ppm＝10.923(s,2H),7.449(d,J＝7.7Hz,2H),7.373(d,J＝7.9Hz,2H),7.085(t,J 1 ＝7.3Hz,J 2 ＝7Hz,2H),6.995(t,J 1 ＝7.3Hz,J 2 ＝7.1Hz,2H),5.075(t,J 1 ＝J 2 ＝5.6Hz,2H),4.650(t,J 1 ＝6.1,J 2 ＝5.2,2H),4.469(m,6H),4.013(m,2H),3.864(dd,J 1 ＝10.5Hz,J 2 ＝4.7Hz,2H),3.403(m,2H),2.897(m,2H)。 13 C NMR(75MHz,DMSO-d 6 ) Delta/ppm = 167.52,136.69,134.43,126.39,121.30,118.87,117.82,111.65,110.04,68.91,65.37,63.82,62.77,59.66,28.89,23.52,19.67,15.63,13.95.Hplc retention time was 30.5 minutes.
EXAMPLE 6 preparation of non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] oxindole } -1',4' -dione (5)
The resulting 220mg of Compound 5 was suspended in 200ml of deionized water and sonicated for 2 hours to dissolve, and the solution was then lyophilized to give 220mg of lyophilized powder. ESI-FT-ICR-MS spectrum and qCID spectrum (see the attached drawings in the specification) show that the freeze-dried powder is the freeze-dried powder of the non-covalent bond trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole } -1',4' -dione (5).
Test example 1 evaluation of thrombolytic Activity of non-covalent trimer of Compound 5
1) Male SD rats (200+ -20 g) were randomly grouped, 12 per group, and kept at rest for one day. Rats were anesthetized by intraperitoneal injection with a 20% solution of uratam in physiological saline. The anesthetized rat is fixed in the supine position, the right common carotid artery is separated, an arterial clamp is clamped at the proximal end, the proximal end and the distal end respectively penetrate into operation wires, the operation wires at the distal end are clamped at fur by hemostatic forceps, the arterial clamp is loosened at the distal end, about 1mL of arterial blood is discharged, and the hemostatic forceps are arranged in a 1mLEP tube. A rubber tube (15 mm long, 2.5mm inner diameter, 5.0mm outer diameter, and sealed with rubber plug at the bottom of the tube) fixed vertically is filled with 0.1mL of rat arterial blood, and a thrombus fixing bolt made of stainless steel material is inserted into the tube rapidly. The thrombus fixing bolt is wound by stainless steel wires with the diameter of 0.2mm, the spiral part is 10mm long and comprises 15 coils, the diameter of each coil is 1.0mm, the support handle is connected with the spiral, and the length of each support handle is 7.0mm, so that the thrombus fixing bolt is question mark type. After 40 minutes of blood coagulation, the thrombus-fixing bolt wrapped with thrombus was carefully taken out of the rubber tube, and the initial weight of the thrombus-fixing bolt was precisely weighed.
2) 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 into a tip with the length of 10mm and the outer diameter of 1.0mm (used for being inserted into carotid artery and vein of a rat), and the outer part of the other end of the polyethylene tube is sleeved with a section of polyethylene tube with the length of 7mm and the outer diameter of 3.5mm (used for connecting the middle section of the polyethylene tube). The inner walls of the three-section tube are all required to be silanized (1% of silicone oil diethyl ether solution). And placing the thrombus fixing bolt wrapped by the thrombus with accurate weighing in the middle section polyethylene tube.
3) The trachea of the anesthetized rats was then isolated and tracheal intubation was performed. The left external jugular vein of the rat was isolated, and the proximal and distal ends were threaded into the surgical line, respectively, with a bevel carefully cut on the left exposed external jugular vein. The tip tube of the prepared bypass pipeline is inserted into the proximal end of the left external jugular vein opening from the bevel opening and is far away from the screw support handle for fixing thrombus in the middle section of the bypass pipeline. An accurate amount of saline solution (50 IU/kg) of heparin sodium was pushed through the tip of the other end with a syringe, at which time the syringe was not withdrawn from the polyethylene tube. The hose between the syringe and the polyethylene tube was clamped with a hemostat. The right common carotid artery was hemostatic with the arterial clip at the proximal end and carefully cut a bevel near the arterial clip. The syringe is pulled out from the tip of the polyethylene tube, and the tip of the polyethylene tube is inserted into the proximal end of the arterial bevel. Both ends of the bypass pipeline are fixed with the artery and vein by using a No. 4 operation suture.
4) A scalp needle containing physiological saline (3 mL/kg) or urokinase physiological saline solution (20000 IU/kg) or a compound 5 non-covalent trimer physiological saline solution (20 nmol/kg) was inserted into the proximal vein of the middle section of the bypass tube, which was far from the thrombus-fixing helix, and the arterial clip was opened to allow blood flow from the artery to the vein through the bypass tube. Saline (blank control), urokinase (positive control) or a non-covalent trimer of compound 5 in the scalp needle was slowly injected into the middle section of the bypass tube to act on the thrombus through blood circulation in the order of vein-heart-artery. And (3) taking out the thrombus fixing bolt from the middle section of the bypass pipe after 1 hour from the timing of starting injection, and accurately weighing the final weight of the thrombus fixing bolt. And taking the whole blood to be tested for serum PAI-1 concentration. The difference between the initial weight and the final weight of the thrombus-fixing bolt is the weight reduction of the thrombus. The thrombus weight loss of each rat was calculated, and the thrombus weight loss of each group of rats was counted and t-test was performed. The data are shown in Table 1. It can be seen that the thrombus weight loss (22.84.+ -. 1.90 mg) of the non-covalent trimer treated rats of compound 5 at the dose of 20nmol/kg was significantly greater than the thrombus weight loss (17.89.+ -. 2.29mg, P < 0.01) of the normal saline treated rats. It is seen that the present invention has substantial technical features and creativity.
Table 1 thrombolytic Activity of non-covalent trimer of Compound 5
a) The ratio P of the polymer to physiological saline and the monomer is less than 0.01; b) The ratio P of the water to the normal saline is more than 0.05; n=12.
Experimental example 2 evaluation of the Effect of the non-covalent trimer of Compound 5 on the serum PAI-1 content of rats
1) Serum preparation
Whole blood from each group of rats collected in test example 1 was mixed with 3.8% sodium citrate. After 10 minutes, 1000g was centrifuged for 20 minutes to collect the supernatant for further use.
2) The PAI-1 content in the serum of the rat is determined by adopting a PAI-1 enzyme-linked immunoassay experiment of the rat. And setting blank holes, standard substance holes and sample holes to be tested in an experiment. Standard wells were each filled with 50 μl of standard and 100 μl of enzyme-labeled reagent at different concentrations. And (3) the sample to be tested is prepared by adding 40 mu L of sample diluent into the hole of the enzyme-labeled coating plate, and then adding 10 mu L of sample to be tested (the final dilution of the sample is 5 times). The blank control wells were not added with sample and enzyme-labeled reagent. Incubating at 37deg.C for 60min, diluting concentrated washing liquid with distilled water for 20 times, carefully removing sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 s, discarding, repeating for 5 times, and drying; adding 50 mu L of the color developing agent A and then 50 mu L of the color developing agent B into each hole, gently shaking and uniformly mixing, and developing for 15min at 37 ℃ in a dark place; add 50. Mu.L of stop solution per well to stop the reaction (blue turning yellow immediately at this time); zeroing by taking blank holes as a standard, and sequentially measuring absorbance values of all holes at a wavelength of 450 nm; and drawing a standard curve by taking the concentration of the standard substance as an abscissa and the absorbance value as an ordinate to obtain a linear regression equation, and substituting the absorbance value of the sample into the equation to calculate the concentration of the sample. Data results are expressed as mean.+ -. SD, pg/mL, and p <0.05 indicates statistical differences by t-test. The experimental results are shown in Table 2. It can be seen that the non-covalent trimer of compound 5 effectively reduces PAI-1 content in rat serum. It can be seen that the present invention has outstanding technical effects and creativity.
TABLE 2 Effect of non-covalent trimer of Compound 5 on PAI-1 content in rat serum
a) P <0.05 with physiological saline and monomer ratio; n=6.
Claims (4)
1. A non-covalent trimer of (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione of the formula,
2. a process for preparing a non-covalent trimer of (2's, 5's) -tetrahydropyrazine [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione according to claim 1, which process comprises:
carrying out Pictet-Spengler condensation on L-tryptophan benzyl ester and 1, 3-dihydroxyacetone under the catalysis of trifluoroacetic acid to prepare 3S-1, 1-dimethylol tetrahydro-beta-carboline-3-benzyl carboxylate (1);
b.3S-1, 1-dimethylol-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (1) is reacted with t-butyldimethylsilyl chloride in N, N-dimethylformamide in the presence of imidazole to prepare 3S-1, 1-bis (t-butyldimethylsilyloxy) methyltetrahydro- β -carboline-3-carboxylic acid benzyl ester (2);
benzyl C.3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylate (2) is subjected to palladium-carbon catalytic hydrogenolysis in methanol to obtain 3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylic acid (3);
D. condensing two molecules of 3S-1, 1-di (tert-butyldimethylsilyloxy) methyltetrahydro-beta-carboline-3-carboxylic acid (3) into (2 'S,5' S) -tetrahydropyrazine [1',2':1,6] bis {1, 1-di (tert-butyldimethylsilyloxy) methyl-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (4) in anhydrous N, N-dimethylformamide with 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate as a condensing agent;
E. preparing the (2's, 5's) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dimethylol-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (5) of claim 1 from (2's, 5's) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dimethylol-2, 3,4, 9-tetrahydro-1H-pyridine [3,4-b ] benzindole } -1',4' -dione (4) in anhydrous tetrahydrofuran under the action of tetrabutylammonium fluoride;
F. suspending the compound 5 by deionized water, carrying out ultrasonic treatment on the obtained suspension for 2 hours to dissolve the suspension, and freeze-drying the obtained solution to obtain the freeze-dried powder of the trimer of the compound 5.
3. Use of a non-covalent trimer of (2's, 5's) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione according to claim 1 in the manufacture of a thrombolytic medicament.
4. Use of a non-covalent trimer of (2's, 5's) -tetrahydropyrazin [1',2':1,6] bis {1, 1-dihydroxymethyl-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1',4' -dione according to claim 1 for the preparation of a PAI-1 inhibitor.
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| CN107686483A (en) * | 2016-08-05 | 2018-02-13 | 首都医科大学 | Seven cyclic ketals, it is prepared, anti-thrombus activity and application |
| CN109912597A (en) * | 2017-12-12 | 2019-06-21 | 首都医科大学 | Seven ring aldehyde, synthesis, anti-thrombus activity and application |
| CN110577530A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | heptacyclic aldehyde, its synthesis, antithrombotic activity and use |
| CN112010928A (en) * | 2019-05-30 | 2020-12-01 | 首都医科大学 | Ethyl PAK modified bis-carbolino-piperazinedione, preparation, activity and application thereof |
| CN112094318A (en) * | 2019-06-18 | 2020-12-18 | 首都医科大学 | Ethyl RPAK modified bis-carbolino-piperazinediones, preparation, activity and use thereof |
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| CN107686483A (en) * | 2016-08-05 | 2018-02-13 | 首都医科大学 | Seven cyclic ketals, it is prepared, anti-thrombus activity and application |
| CN109912597A (en) * | 2017-12-12 | 2019-06-21 | 首都医科大学 | Seven ring aldehyde, synthesis, anti-thrombus activity and application |
| CN110577530A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | heptacyclic aldehyde, its synthesis, antithrombotic activity and use |
| CN112010928A (en) * | 2019-05-30 | 2020-12-01 | 首都医科大学 | Ethyl PAK modified bis-carbolino-piperazinedione, preparation, activity and application thereof |
| CN112094318A (en) * | 2019-06-18 | 2020-12-18 | 首都医科大学 | Ethyl RPAK modified bis-carbolino-piperazinediones, preparation, activity and use thereof |
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