CN110577575B - 1S-methyl-beta-tetrahydrocarboline acyl-K (ARPAK) -RGDV, and synthesis, activity and application thereof - Google Patents
1S-methyl-beta-tetrahydrocarboline acyl-K (ARPAK) -RGDV, and synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val with the following formula, discloses a preparation method thereof, discloses antithrombotic activity thereof, discloses thrombolytic activity thereof and discloses an effect of treating rats with 24 hours of apoplexy, thus the invention discloses application of the compound in preparing antithrombotic medicaments, thrombolytic medicaments and medicaments for treating ischemic stroke.
Description
Technical Field
The invention relates to 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val, a preparation method thereof, antithrombotic activity thereof, thrombolytic activity thereof and the effect of treating rats with 24 hours of stroke, thus the invention relates to the application thereof in preparing antithrombotic medicaments, thrombolytic medicaments and medicaments for treating ischemic stroke. The invention belongs to the field of biological medicine.
Technical Field
Ischemic stroke is a common and serious cerebrovascular disease, and is characterized by high morbidity, high fatality rate, high disability rate and high recurrence rate. At present, the clinical treatment of ischemic stroke faces the reality that no effective medicine exists, and especially, patients with stroke for more than 4 hours are not dead or are disabled. The invention is an important clinical demand for effective medicines for patients with stroke of more than 4 h. The inventors have disclosed that imidazolines of the following formula show excellent efficacy in a rat ischemic stroke model with stroke face of 24 h. Namely imidazoline of the following formula is continuously injected into vein for 6 days, 1 time per day, the dosage is 100nmol/kg, and the imidazoline has excellent curative effect. In the formula, AA is Ser, Val or Phe. For structural reasons, imidazolines of the formula below have two inevitable disadvantages. That is, the free radical of the 1, 3-dioxoimidazoline moiety is sensitive to the reductive environment, and is difficult not only to prepare but also to store.
After 3 years of experimental research, the inventor finds that the imidazoline part is replaced by 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl group, and double unexpected technical effects of stable structure and easy storage can be obtained. The inventors have made the present invention in light of this finding.
Disclosure of Invention
The first aspect of the present invention provides 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of the formula.
The second aspect of the present invention provides a method for synthesizing 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val, the method comprising:
(1) preparing N-Boc-1S-methyl-1, 2,3,4 tetrahydro-beta-carboline-3S-carboxylic acid;
(2) preparing N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Boc);
(3) preparing HCl, Arg (NO2) -Gly-Asp (OBzl) -Val-OBzl;
(4) preparation of Boc-Ala-Arg (NO2) -Pro-Ala-Lys (Cbz);
(5) preparing 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys-Arg (NO2) -Gly-Asp (OBzl) -Val-OBzl;
(6) preparation of 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl
–Lys(Ala-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val。
The third aspect of the present invention is to evaluate the antithrombotic activity, thrombolytic activity and ischemic stroke treating activity of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val.
Drawings
FIG. 11 synthetic route of S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val. i is CH3CHO，H2O; ii TEA, DMF, di-tert-butyl dicarbonate (Boc)2O；iii:Pb/C，H2Tetrahydrofuran; iv N, N-Dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), tetrahydrofuran; v 2N aqueous sodium hydroxide solution, CH3OH; vi, hydrogen chloride in ethyl acetate (4M); vii trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TMFA).
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-methyl-1, 2,3, 4-tetrahydro- β -carboline-3-carboxylate (1)
1mL of 98% concentrated sulfuric acid was added dropwise to 800mL of distilled water, and after stirring the mixture uniformly, 10.0g (34mmol) of L-Trp-OBzl was added thereto in three portions. Stirring for five minutes to fully suspend the L-Trp-OBzl and the aqueous solution of sulfuric acid. Thereafter, 10mL of a 40% aqueous solution of acetaldehyde was added dropwise to the suspension. The reaction mixture was stirred for 12h, and then 3mL of concentrated aqueous ammonia was added dropwise to adjust the pH of the reaction solution to 8. The reaction compound is kept stand for 1h until the product is fully separated out. The solid was filtered off and dried to give 9.84g (90%) of a pale yellow solid, which was a mixture of benzyl 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylate and benzyl 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylate. ESI-MS (M/e):321[ M + H]+。
EXAMPLE 2 preparation of N-Boc-1-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester (2)
9.84g (30.8mmol) of benzyl 1-methyl-1, 2,3, 4-tetrahydro- β -carboline-3-carboxylate obtained in example 1 were dissolved in 20ml of N-Dimethylformamide (DMF). To the solution was added 6.98g (32.0mmol) (Boc) at 0 deg.C2And O. The resulting solution was adjusted to pH 12 with triethylamine and stirred at room temperature for 48 h. The reaction mixture was concentrated under reduced pressure to remove DMF. The residue was dissolved in 100mL of ethyl acetate. The resulting ethyl acetate solution was washed with a 5% aqueous solution of potassium hydrogensulfate (50 mL. times.3) and a saturated aqueous solution of sodium chloride (50 mL. times.3) in this order. The separated ethyl acetate layer was dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated under reduced pressure to give an oil. This oil was separated on a silica gel column (dichloromethane/methanol, 100/1) to give 5.02g (38%) of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester and 6.00g (47%) of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester. All were colorless powders.
EXAMPLE 3 preparation of Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-carboxylic acid (3)
To a solution of 1.0g (2.38mmol) of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester and 40mL of anhydrous tetrahydrofuran was added 100mg of Pd/C, and the mixture was stirred to obtain a uniform suspension. Air in the reaction system was evacuated under reduced pressure, hydrogen was introduced, and the reaction system was stirred at room temperature for 10 hours, and TLC (dichloromethane/methanol, 40/1) showed complete disappearance of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-benzyl carboxylate. Filtering to remove Pd/C, concentrating the filtrate under reduced pressure to obtain colorless powder with ESI-MS (M/e) of 329[ M-H ]]-。
EXAMPLE 4 preparation of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Boc) -OBzl (4)
4.950g (15.0mmol) of Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid is dissolved in 50mL of anhydrous tetrahydrofuran, 2.025g (15.0mmol) of N-hydroxybenzotriazole (HOBt) and 3.090g (15.0mmol) of N, N-Dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF are sequentially added into the obtained solution under ice bath and stirring, and the mixture is fully stirred and activated for 30 minutes to obtain solution A; 5.066g (13.6mmol) of HCl-Lys (Boc) -OBzl dissolved in 20ml of anhydrous THF are then reacted with N-methylThe pH value of the morpholine is firstly adjusted to 8-9. Then, the mixture was added to the reaction solution A. The ice bath was removed and stirred at room temperature for 8 hours, and TLC (developing solvent dichloromethane/methanol-40/1) under UV (254nm) showed complete disappearance of the starting material HCl lys (boc) -OBzl. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The ethyl acetate layer was sequentially washed with a saturated aqueous sodium bicarbonate solution (n ═ 3), a saturated aqueous sodium chloride solution (n ═ 3), a 5% aqueous potassium hydrogensulfate solution (n ═ 3), a saturated aqueous sodium chloride solution (n ═ 3), a saturated aqueous sodium bicarbonate solution (n ═ 3), and a saturated aqueous sodium chloride solution (n ═ 3), and the ethyl acetate phases in the upper layer were combined, dried over anhydrous sodium sulfate for 4 hours, filtered, and the resulting filtrate was concentrated to dryness under reduced pressure, followed by silica gel column chromatography to give 6.960g (79%) of the title compound as a colorless powder. ESI-MS (M/e):649[ M + H]+。
EXAMPLE 5 preparation of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Boc) (5)
The title compound was obtained as colorless powder from 1.296g (2.0mmol) of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester using the method of example 3. ESI-MS (M/e) of 557[ M-H ]]-。
EXAMPLE 6 preparation of Boc-Arg (NO)2)-Gly-OBzl
Using the method of example 4 from 5.000g (15.6mmol) Boc-Arg (NO)2) And 2.860g (14.2mmol) HCl Gly-OBzl gave 5.560g (84%) of the title compound as colorless powder. ESI-MS (M/e) 467[ M + H]+。
EXAMPLE 7 preparation of Boc-Arg (NO)2)-Gly
4.660g (1mmol) of Boc-Arg (NO)2) -Gly-OBzl in 50mL methanol. The resulting solution was slowly added dropwise with 2N NaOH aqueous solution under ice-bath and stirring, and stirred in ice-bath for 6 hours. TLC (developing solvent dichloromethane/methanol-40/1) under UV (254nm) showed Boc-Arg (NO)2) -Gly-OBzl disappeared completely. Slowly dropwise adding saturated KHSO under ice bath and stirring4The aqueous solution was adjusted to pH7 and concentrated under reduced pressure to remove methanol. Adjusting pH to 2 with saturated potassium hydrogen sulfate aqueous solution, extracting with ethyl acetate (40mL × 3) for 3 times, washing with saturated sodium chloride aqueous solution for 3 times, mixing ethyl acetate layers, drying with anhydrous sodium sulfate for 2 hr, and filteringFiltration and concentration of the filtrate to dryness under reduced pressure gave 2.850g (76%) of the title compound. ESI-MS (M/e) 375[ M-H]-。
EXAMPLE 8 preparation of Boc-Asp (OBzl) -Val-OBzl
3.910g (84%) of the title compound were obtained as a colorless solid by the method of example 4 from 3.230g (10mmol) of Boc-Asp (OBzl) and 3.450g (9.1mmol) of tos. Val-OBzl. ESI-MS (M/e) 515[ M + H]+。
EXAMPLE 9 preparation of Asp (OBzl) -Val-OBzl
10.0g of Boc-Asp (OBzl) -Val-OBzl was dissolved in 10mL of anhydrous ethyl acetate. To this solution was added 100mL of a solution of hydrogen chloride in ethyl acetate (4M) with stirring in an ice bath and stirred for 2 h. TLC (dichloromethane/methanol, 40/1) under UV (254nm) showed complete disappearance of the starting Boc-Asp (OBzl) -Val-OBzl. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was diluted with 30mL of anhydrous ethyl acetate and concentrated to dryness under reduced pressure. This operation was repeated 3 times. The residue obtained is diluted with 30mL of anhydrous ether and concentrated to dryness under reduced pressure. This operation was repeated 3 times to give the title compound. ESI-MS (M/e):413[ M + H]+。
EXAMPLE 10 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
From 3.750g (10.0mmol) of Boc-Arg (NO) by the method of example 42) -Gly and 4.080g (9.1mmol) HCl. Asp (OBzl) -Val-OBzl gave 5.190g (74%) of the title compound as a colorless solid. ESI-MS (M/e):770[ M + H]+。
EXAMPLE 11 preparation of Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
Starting from 5g (6.5mmol) of Boc-Arg (NO) by the method of example 92) -Gly-Asp (OBzl) -Val-OBzl gives 4.27g (98%) of the title compound as a colourless solid. ESI-MS (M/e):673[ M + H]+。
EXAMPLE 12 preparation of Boc-Pro-Ala-OBzl
The procedure of example 4 was used to obtain 3.040g (90%) of the title compound as a colorless solid from 2.150g (10mmol) Boc-Pro and 1.960g (9.1mmol) HCl. Ala-OBzl. ESI-MS (M/e):377[ M + H]+
EXAMPLE 13 preparation of Boc-Pro-Ala
By means ofThe procedure of example 3 gave 1.350g (95%) of the title compound as a colorless solid from 1.880g (5mmol) of Boc-Pro-Ala-OBzl. ESI-MS (M/e):285[ M-H]+。
EXAMPLE 14 preparation of Boc-Pro-Ala-Lys (Cbz) -OBzl
The procedure of example 4 was used to obtain 14.5g (65%) of the title compound as a colorless oily product from 10g Boc-Pro-Ala-OBzl and 14.2g HCl.Lys (Cbz) -OBzl. ESI-MS (M/e) 639[ M + H ]]+。
EXAMPLE 15 preparation of Pro-Ala-Lys (Cbz) -OBzl
From 1.901g (3mmol) Boc-Pro-Ala-Lys (Cbz) -OBzl, 1.640g (95%) of the title compound were obtained as colorless oil using the method of example 9. ESI-MS (m/e): 539[ M + H ]]+。
EXAMPLE 16 preparation of Boc-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Using the method of example 4 from 0.957g (3.0mmol) of Boc-Arg (NO)2) And 1.550g (2.7mmol) HCl Pro-Ala-Lys (Cbz) -OBzl gave 1.701g (75%) of the title compound as a colorless oil. ESI-MS (m/e): 840[ M + H]+。
EXAMPLE 17 preparation of Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
The procedure of example 9 was used to obtain 8.3g (99%) of the title compound as a colorless solid from 9.1g Boc-Arg (NO2) -Pro-Ala-Lys (Cbz) -OBzl. ESI-MS (M/e):740[ M + H]+。
EXAMPLE 18 preparation of Boc-Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
7.5g (71%) of the title compound were obtained as a colorless solid by the method of example 4 from 2.6g Boc-Ala and 9.1g HCl Arg-Pro-Ala-Lys (Cbz) -OBzl. ESI-MS (M/e) 911[ M + H ]]+。
EXAMPLE 19 preparation of Boc-Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)
Using the method of example 7, 3.8g Boc-Gly-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl gave 3.6g (97%) of the title compound as a colorless solid. ESI-MS (M/e) 819[ M-H]-。
EXAMPLE 20 preparation of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl–Lys(Boc)-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(6)
From 5.580g (10.0mmol) N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Boc) and 6.430g (9.1mmol) HCl & Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gave 8.148g (74%) of the title compound as a colourless solid. ESI-MS (M/e):1211[ M + H]+。
EXAMPLE 21 preparation of 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl(7)
The method of example 9 was used to prepare 1.210g (1.0mmol) of N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Boc) -Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 0.96g (95%) of the title compound. ESI-MS (M/e) 1011[ M + H]+。
EXAMPLE 22 preparation of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys [ Boc-Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(8)
From 1.612g (2.0mmol) Boc-Ala-Arg (NO) using the method of example 42) -Pro-Ala-Lys (Cbz) -OH and 1.840g (1.82mmol) 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys-Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 396mg (12%) of the title compound as a colourless solid. ESI-MS (M/e):1813[ M + H]+。1H NMR(DMSO-d6,300MHz)δ/ppm＝10.79(s,1H),8.53(s,1H),8.28(d,J＝7.8Hz,1H),8.21(m,4H),8.10(d,J＝6.9Hz,1H),7.96(d,J＝7.5Hz,1H),7.91(d,J＝7.2Hz,1H),7.80(m,3H),7.39(m,2H),7.31(m,15H),7.21(t,J＝5.4Hz,1H),6.97(m,3H),5.06(m,6H),4.77(m,1H),4.50(m,1H),4.33(m,3H),4.15(m,4H),3.81(m,1H),3.73(m,2H),3.53(m,3H),3.16(m,6H),2.93(m,5H),2.73(m,1H),2.58(m,1H),2.05(m,2H),1.80(m,9H),1.53(m,8H),1.35(m,18H),1.15(m,10H),0.83(d,J＝6.6Hz,6H)。
EXAMPLE 23 preparation of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val-OH (9)
100mg (0.065mmol) of 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys [ Boc-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2) -Gly-Asp (OBzl) -Val-OBzl was dissolved completely by adding 1mL of trifluoroacetic acid (TFA) under ice salt bath, adding 0.30mL of trifluoromethanesulfonic acid (TFMSA), 1 hour later TLC (developing solvent n-butanol: water: glacial acetic acid 1:1:1) under UV (254nm) to show disappearance of starting material point, stopping the reaction, adding anhydrous ether to the reaction, stirring, precipitating solid, standing, discarding supernatant, adding anhydrous ether, standing, discarding supernatant, repeating 3 times, vacuum-pumping, dissolving with a small amount of water, adjusting pH to 7 with 25% ammonia, and Sephadex G15Desalting, C18Column purification and lyophilization gave 11mg (15%) of the title compound as a colorless powder. ESI-MS (M/e):1309[ M + H]+；mp:215.4-215.9℃；
(c＝0.1,CH3OH)；IR(cm-1):3219.04,3075.69,2954.87,1656.73,1536.63,1439.72,1240.22,1224.02,1164.47,1025.89,762.39,636.71；1H NMR(DMSO-d6,300MHz)δ/ppm＝11.09(s,1H),8.56(m,3H),8.36(m,1H),8.13(m,2H),8.02(m,1H),7.91(m,2H),7.78(m,1H),7.38(m,2H),7.09(m,5H),4.57(m,2H),4.35(m,3H),4.16(m,2H),4.08(m,1H),3.87(m,2H),3.66(m,2H),3.56(m,1H),3.04(m,7H),2.81(m,3H),2.27(m,1H),2.03(m,1H),1.85(m,4H),1.62(m,6H),1.50(m,9H),1.32(m,5H),1.21(m,5H),0.85(d,J＝6.0Hz,6H)。
Experimental example 1 evaluation of antithrombotic Activity of Compound 9
Male SD rats (200. + -.20 g) were randomly divided into groups of 10 animals each, kept for 1 day and stopped overnight. After 30min of gavage administration of a physiological saline solution of Compound 9 (dose 10nmol/kg) or aspirin (dose 167. mu. mol/kg) or physiological saline (dose 3mL/kg), rats were anesthetized with a physiological saline solution of 20% Ustilago and then operated. The right carotid artery and the left jugular vein of the rat were isolated, accurately weighed silk was placed in the bypass cannula, one end of the tube was inserted into the left vein and the other end was inserted into the right artery and injected with 0.2mL heparin sodium anticoagulation. Allowing blood flow to flow from the right artery through the bypass cannula into the left vein, taking out the thread with thrombus after 15min, weighing, calculating the weight of the thread before and after blood circulation, and performing t-test to obtain the weight of the thrombus represented by the average value + -SD mg and representing the antithrombotic activity. The data are shown in Table 1. The results show that oral administration of 10nmol/kg of compound 9 is effective in inhibiting thrombosis.
TABLE 1 antithrombotic Activity of Compound 9 at 10nmol/kg dose
a) Compared with normal saline, P is less than 0.01, and n is 10
Experimental example 2 evaluation of thrombolytic Activity of Compound 9
SD rats (male, 200 + -20 g) were anesthetized by intraperitoneal injection of urethane normal saline solution at a dose of 1200 mg/kg. Fixing the rat in a supine position after anesthesia, separating the right common carotid artery of the rat, clamping an artery clamp at the proximal end, respectively penetrating the proximal end and the distal end into an operation line, ligating the operation line at the distal end, inserting a tube at the distal end, loosening the artery clamp, taking out 1mL of arterial blood, and placing the arterial blood in a 1mL centrifuge tube. A vertically fixed rubber tube (5mm long, 2.5mm inner diameter, 5.0mm outer diameter, tube bottom sealed with rubber plug, para membrane sealed) is injected with 0.1mL rat arterial blood, then a stainless steel thrombus fixing bolt (thrombus fixing spiral is wound by 0.2mm diameter stainless steel wire, the spiral part is 10mm long, 15 spiral rings are contained, the diameter of the spiral ring is 1.0mm, the support handle is connected with the spiral, the length is about 7.0mm, and the bolt is in question mark type).
The bypass cannula consists of three parts, wherein the middle section is a polyethylene rubber tube with the length of 60.0mm and the inner diameter of 3.5 mm; both ends are 100.0mm long, and internal diameter 1.0mm, the same polyethylene pipe of external diameter 2.0mm, and this pipe one end is drawn into the sharp pipe, and is about 10.0mm long (being used for inserting rat carotid artery and vein), and the external diameter is 1.0mm, and the outside cover section of its other end is long for 7.0mm, and the external diameter is 3.5 mm's polyethylene pipe (in being used for inserting the polyethylene rubber tube in middle section), and the inner wall of 3 sections pipes all needs silanization (1% silicon oil ether solution). The thrombus-wrapped thrombus fixing spiral is placed in the middle section polyethylene rubber tube, and the other two ends of the rubber tube are respectively sleeved with the thickened ends of the two polyethylenes, so that blood leakage can be avoided in the circulating process. The tube was filled with heparin normal saline solution (50IU/kg) through the tip end with a syringe to remove air bubbles for use.
The left external jugular vein of separation rat, proximal end and distal end penetrate the operation line respectively, and the blood vessel of ligature distal end cuts a osculum on the left external jugular vein that exposes, inserts the bypass pipeline taper pipe that has been prepared above-mentioned into left external jugular vein opening part by the osculum, keeps away from bypass pipe middle section (contains the thrombus fixed spiral of accurate weighing) internal thrombus fixed spiral simultaneously. An accurate amount of a physiological saline solution (50IU/kg) of heparin sodium was injected through the tip tube at the other end with a syringe, at which time the syringe was not removed from the polyethylene tube, and the flexible tube between the syringe and the polyethylene tube was clamped with an artery clamp. Stopping bleeding by an artery clamp at the proximal end of the right common carotid artery, ligating the distal end, cutting a small opening of the right common carotid artery at a position short of the artery clamp, pulling out the injector from the tip of the polyethylene tube, and inserting the tip of the polyethylene tube into the proximal end of the oblique opening of the artery. Both ends of the bypass pipeline are used for fixing the artery and the vein by using a No. 4 surgical suture.
Physiological saline (3mL/kg) or a physiological saline solution of urokinase (dosage of 20000IU/kg) or a physiological saline solution of compound 9 (dosage of 10nmol/kg) is inserted through the middle section of the bypass tube (containing the thrombus fixing helix accurately weighed) into the proximal venous end far away from the thrombus fixing helix by using a scalp needle, and the arterial clamp is released to allow blood flow from the artery to the vein through the bypass tube. The solution in the syringe is slowly injected into the blood, and acts on the spiral thrombus through the blood circulation in the order of vein-heart-artery. After 1h of blood circulation, the thrombus-immobilizing helix was removed from the bypass tube and accurately weighed. Calculating the weight difference of the thrombus before and after spiral blood circulation of the immobilized thrombus in the bypass duct of each rat, namely the thrombus weight loss. The data (mean. + -. SDmg) were subjected to a t-test. Loss of thrombus represents thrombolytic activity. The results in Table 2 show that 10nmol/kg of Compound 9 is effective in dissolving thrombi. The activity of the compound has no significant difference with 20000IU/kg urokinase. The technical effect is obvious.
TABLE 2 thrombolytic Activity of Compound 9 at 10nmol/kg dose
a) P <0.01 to saline, P >0.05 to urokinase; n is 9.
Experimental example 3 evaluation of therapeutic Effect of Compound 9 on ischemic Stroke 24h rat
A2 cm long incision was made vertically in the middle of the neck of male SD rats (body weight 300. + -.20 g), and the right common carotid artery, external carotid artery and internal carotid artery were isolated along the intramuscular side edge of the sternocleidomastoid muscle. 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, taking 10 mu L of blood, and then clamping the proximal end of the common carotid artery by using the noninvasive artery clamp. The obtained 10. mu.L of blood was placed in a 1mLEP tube at normal temperature for 30 minutes to coagulate the blood, and then transferred to a-20 ℃ refrigerator and left for 1 hour to clot the blood. Rats were anesthetized with 10% chloral hydrate by intraperitoneal injection at a dose of 400 mg/kg. 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. Loosening the artery clamp at the proximal end of the common carotid artery, slowly injecting 1mL of thrombus suspension into the brain of a rat from the external carotid artery of the rat to the proximal end through the internal carotid artery, then ligating the proximal end of the external carotid artery, opening the internal carotid artery and the common carotid artery to obtain the artery clamp, and recovering blood flow. Waiting for wakeup. The degree of neurological deficit was assessed by the Zealonga method 24 hours after the rats were awakened. Score 0 indicates no sign of neurological deficit, score 1 indicates that the intact forelimb cannot stretch, score 2 indicates walking to the intact side, score 3 indicates turning to the intact side and walking in a tail-end-collision manner, score 4 indicates that the disorder is not self-walking, and score 5 indicates death. And grouping according to the score average. Rats were injected with 1 second saline daily via tail vein at a dose of 3 mL/kg. Injections were given continuously for 6 days, and scored daily. The data in Table 3 show that the neurobiological scores of the rats before the saline administration treatment were 5 points 2, 4 points 3, 2 points 4, and the neurobiological scores of the rats after the saline administration treatment for 6 days were 5 deaths, 1 point 0, 2 points 1, and 3 points 2. The overall manifestations of the disease are worsened.
TABLE 3 Effect of continuous saline treatment for 6 days on neurobiological scores in rats with 24 hours cerebral ischemia
Dosage: 3 mL/kg; n is 11
Rats were injected with tPA 1 time per day via the tail vein at a dose of 3 mg/kg. Scoring is done daily. The data in Table 4 show that 6 rats died within 24 hours, and that dead rats were necropsied and their organs were found to have bleeding, particularly severe pulmonary bleeding, and thus, the administration was terminated 2 times after the administration. No rat is well treated after 2 times of administration, no neurological function loss sign is observed, 2 patients have slight neurological function loss signs, 1 patient has no injury side and turns into tail-end-collision-shaped walking sign, and one patient has no self-walking sign of residual consciousness disorder.
TABLE 4 therapeutic efficacy of rats treated with tPA at 3mg/kg 6 hours after ischemia
Dosage: 3 mg/kg; n is 6
Rats were injected 1 time per day with compound 9 via the tail vein at a dose of 10 nmol/kg. Injections were given continuously for 6 days, and scored daily. The data in table 5 show that the neurobiological scores of rats before compound 9 treatment were 4 points 2, 4 points 3, 3 points 4, and the neurobiological scores of rats 6 days after treatment were 3 deaths, 5 points 1,3 points 0. In addition, no orbital and tail bleeding was seen in the surviving rats. The whole shows obvious curative effect.
TABLE 5 Effect of Compound 9 on neurobiological scores in rats with 24h cerebral ischemia on 6 days of continuous treatment
Dosage: 100 nmol/kg; n is 11
Experimental example 4 evaluation of Effect of Compound 9 on cerebral infarction volume of rats with ischemic Stroke 24h
After receiving the evaluation of the neurological deficit degree in experimental example 3, the brain was anesthetized with urethane, rapidly cut off, frozen in a refrigerator at-20 ℃ for 2 hours, and continuously sliced from the frontal pole to obtain 6 coronal brain slices with a thickness of 2 mm. The brain slices were incubated in 2% TTC solution at 37 ℃ for 30min in the dark, and the color change of the brain slices was observed. After the normal brain tissue is stained red by TTC and the ischemic brain tissue is white, a digital camera is used for photographing. The pictures are processed by SPSS statistical software, the infarct volume and the normal tissue volume in the brain coronary section are calculated, and the infarct volume percentage value of each group is calculated. The experimental data are all tested by t test. The data in table 6 demonstrate that the cerebral infarct volume of compound 9 treated ischemic stroke 24h rats is significantly less than the cerebral infarct volume of saline treated ischemic stroke 24h rats.
TABLE 6 cerebral infarction volume ratio of Compound 9 to rats with 24 hours cerebral ischemia after 6 consecutive days of treatment
a) Data were from 6/11 surviving rat brain sections; b) data from 8/11 surviving rat brain sections, P <0.01 vs. saline; n is 11
To summarize: the compounds of the present invention are stable and easy to prepare and store compared to the compounds once disclosed. The compound 9 can effectively inhibit the formation of thrombus by orally taking 10nmol/kg once, and the compound 9 can effectively dissolve the thrombus by injecting 10nmol/kg once. Intravenous administration of 100nmol/kg of compound 9 once daily for 6 consecutive days was effective in restoring neurobiological behavior in rats with 24h ischemic stroke and in reducing cerebral infarction volume in rats with 24h ischemic stroke. That is, the present invention obtains a remarkable technical effect.
Claims (5)
1. 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of the formula,
2. a process for the preparation of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1, comprising:
(1) preparing N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-carboxylic acid;
(2) preparing N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Boc);
(3) preparation of HCl. Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl；
(4) Preparation of Boc-Ala-Arg (NO)2)-Pro-Ala-Lys(Cbz)；
(5) Preparation of 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl；
(6) Preparation of 1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val.
3. Use of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1 for the preparation of an antithrombotic medicament.
4. Use of the 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1 for the manufacture of a thrombolytic drug.
5. Use of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Ala-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1 for the manufacture of a medicament for the treatment of ischemic stroke.
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| WO2000015639A1 (en) * | 1998-09-16 | 2000-03-23 | Icos Corporation | Carboline derivatives as cgmp phosphodiesterase inhibitors |
| CN102234277A (en) * | 2010-05-07 | 2011-11-09 | 首都医科大学 | Amino-acid-modified 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids, and synthesis method and application thereof |
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| CN107686510A (en) * | 2016-08-05 | 2018-02-13 | 首都医科大学 | The formyl RGD peptide of methyl tetrahydro-beta-carboline 3, it is synthesized, anti thrombotic action and application |
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| WO2000015639A1 (en) * | 1998-09-16 | 2000-03-23 | Icos Corporation | Carboline derivatives as cgmp phosphodiesterase inhibitors |
| CN102234277A (en) * | 2010-05-07 | 2011-11-09 | 首都医科大学 | Amino-acid-modified 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids, and synthesis method and application thereof |
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