CN108929372B - 1R-Methyl-β-tetrahydrocarbolinyl-K(GRPAK)-RGDV, its synthesis, activity and application - Google Patents
1R-Methyl-β-tetrahydrocarbolinyl-K(GRPAK)-RGDV, its synthesis, activity and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了下式的1R‑甲基‑1,2,3,4‑四氢‑β‑咔啉‑3S‑酰基‑Lys(Gly‑Arg‑Pro‑Ala‑Lys)‑Arg‑Gly‑Asp‑Val,公开了它的制备方法,公开了它的抗血栓活性,公开了它的溶血栓活性以及公开了它治疗中风24小时大鼠的作用,因而本发明公开了它在制备抗血栓药物,溶血栓药物以及治疗缺血性中风药物中的应用。
The invention discloses 1R-methyl-1,2,3,4-tetrahydro-β-carboline-3S-acyl-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp of the following formula -Val, discloses its preparation method, discloses its antithrombotic activity, discloses its thrombolytic activity, and discloses its effect on treating apoplexy rats for 24 hours, thus the present invention discloses that it is used in the preparation of antithrombotic drugs, Application of thrombolytic drugs and drugs for the treatment of ischemic stroke.
Description
Technical Field
The invention relates to 1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val with the following formula, relates to a preparation method thereof, relates to antithrombotic activity thereof, relates to thrombolytic activity thereof and relates to the effect thereof in treating rats with 24 hours of stroke, and therefore, 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.
Background
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 replacement of the imidazoline part by 1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl can obtain double unexpected technical effects of stable structure and easy storage. The inventors have made the present invention in light of this finding.
Disclosure of Invention
The first aspect of the present invention provides 1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of the formula.
The second aspect of the present invention provides a method for synthesizing 1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val, the method comprising:
(1) preparing N-Boc-1R-methyl-1, 2,3,4 tetrahydro-beta-carboline-3S-carboxylic acid;
(2) preparation of Lys [ (Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(3) Preparation of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl-Lys [ (Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(4) Preparing N-Boc-1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl-Lys (Boc-Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val;
(5) preparation of 1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl-Lys (Gly-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 1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val.
Drawings
FIG. 11 is a synthetic route for R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val. i) N-hydroxybenzotriazole (HOBt), N, N-Dicyclohexylcarbodiimide (DCC), N-methylmorpholine (NMM); ii) aqueous NaOH (2M), dilute hydrochloric acid; iii) a solution of hydrogen chloride in ethyl acetate (4M); iv) piperidine in DMF (20%); v) dilute sulfuric acid; vi) Pd/C.
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-beta-carboline-3-carboxylate
1mL of concentrated sulfuric acid having a concentration of 98% was added dropwise to 800mL of distilled water at 60 ℃ and, after stirring 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 12 hours at 60 ℃ 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 1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester and 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester. 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 (1)
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).6.98g (32.0mmol) of Boc was added to the solution 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.
ESI-MS (M/e):421[ M + H ] for N-Boc-1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-benzyl carboxylate]+。1H NMR(DMSO-d6,800MHz)δ/ppm＝7.457(d,J＝8.0Hz,1H)；7.314(d,J＝8.0Hz,1H)；7.239(m,5H),7.080(t,J＝7.2Hz,1H)；7.000(t,J＝7.2Hz,1H)；5.377(m,1H)；5.095(m,2H)；0.499(m,1H)；3.421(m,1H)；2.906(m,1H)；1.454(m,12H)。
ESI-MS (M/e):421[ M + H ] for N-Boc-1S-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-benzyl carboxylate]+。1H NMR(DMSO-d6,800MHz)δ/ppm＝7.434(d,J＝7.2Hz,1H)；7.313(m,1H)；7.214(s,1H)；7.158(m,2H)；7.063(m,3H)；6.990(m,1H)；5.066(m,1H)；5.020(m,2H)；4.890(m,1H)；3.275(m,1H)；3.074(m,1H)；1.495(s,3H),1.438(s,3H)；1.331(m,6H)。
EXAMPLE 3 preparation of Boc-1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-carboxylic acid (2)
To a solution of 1g (2.38mmol) of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid benzyl ester in 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-1R-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 Boc-Arg (NO)2)-Gly-OBzl
10g of Boc-Arg (NO)2) Dissolving in 50mL of anhydrous tetrahydrofuran, adding a solution of 5.1g N-hydroxy benzotriazole (HOBt) and 7.5g N, N-dicyclohexyl carbodiimide (DCC) in the obtained solution in sequence under ice bath and stirring, and stirring for 0.5 h. To this solution was then added a solution of 11g HCl Gly-OBzl and 30mL dry tetrahydrofuran. The resulting reaction mixture was brought to pH 9 with N-methylmorpholine (NMM), stirred at room temperature for 6h and TLC (dichloromethane/methanol, 40/1) HCl Gly-OBzl disappeared completely. The precipitated Dicyclohexylurea (DCU) was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was sequentially saturated NaHCO3Three times washing with aqueous solution, three times washing with saturated NaCl aqueous solution and 5% KHSO4Three washes with aqueous solution, three washes with saturated aqueous NaCl solution, 5% NaHCO3Three washes with aqueous solution and three washes with saturated aqueous NaCl solution. Collecting ethyl acetate layer, and collecting anhydrous Na2SO4Drying for 12h, filtering, and concentrating the filtrate under reduced pressure to dryness. The resulting oil was dissolved in 150mL of dichloromethane and allowed to stand overnight at room temperature to allow a colorless solid to precipitate sufficiently. Filtration gave 12g (83%) of the title compound as a colorless powder. ESI-MS (M/e) 467[ M + H]+。
EXAMPLE 5 preparation of Boc-Arg (NO)2)-Gly
12g (2.58mmol) of Boc-Arg (NO)2) -Gly-OBzl in 50mL methanol. The resulting solution was slowly added dropwise with aqueous NaOH (2M) in ice bath with stirring, and stirred in ice bath for 6 h. TLC (dichloromethane/methanol, 40/1) 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. Dilute hydrochloric acid was slowly added dropwise to the remaining aqueous solution to adjust pH 2 with stirring in an ice bath. The resulting solution was extracted three times with 200mL of ethyl acetate. The combined ethyl acetate layers were washed three times with 200mL of saturated aqueous NaCl solution. Collecting ethyl acetate layer, and adding anhydrous Na2SO4Drying for 12 h. Filtration and concentration of the filtrate under reduced pressure gave 0.98g (98%) of the title compound. ESI-MS (M/e):359[ M-H]-。
EXAMPLE 6 preparation of Boc-Asp (OBzl) -Val-OBzl
14.5g of the title compound were obtained as light yellow blocks from 10g of Boc-Asp (OBzl) and 12g of Tos Val-OBzl using the method of example 3. ESI-MS (M/e) 515[ M + H]+。
EXAMPLE 7 preparation of Asp (OBzl) -Val-OBzl
10g of Boc-Asp (OBzl) -Val-OBzl was dissolved in 20mL 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) showed complete disappearance of 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 three times. The residue obtained is diluted with 30mL of anhydrous ether and concentrated to dryness under reduced pressure. This operation was repeated three times to give the title compound. ESI-MS (M/e) 415[ M + H]+。
EXAMPLE 8 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
Using the procedure of example 3, from 12g Boc-Asp (NO)2) -Gly and 15.9g Asp (OBzl) -Val-OBzl gave 15g (61%) of the title compound as a colorless solid. ESI-MS (M/e):773[ M + H]+。1H NMR(DMSO-d6,300MHz)δ/ppm＝8.481(m,1H),8.234(d,J＝7.8Hz,1H),8.137(d,J＝7.8Hz,1H),8.047(m,1H),7.355(m,10H),6.951(m,1H),5.101(m,4H),4.768(m,1H),4.184(m,1H),3.932(s,1H),3.708(m,2H),3.120(m,2H),2.762(m,1H),2.581(m,1H),2.073(m,1H),1.577(m,4H),1.371(s,9H),0.850(d,J＝6.0Hz,6H)。
EXAMPLE 9 preparation of Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
Using the method of example 5, 15g of Boc-Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 12.8g (98%) of the title compound as a colourless solid. ESI-MS (M/e):673[ M + H]+。
EXAMPLE 10 preparation of Boc-Pro-Ala-OBzl
15g (86%) of the title compound was obtained as a light yellow solid from 10g Boc-Pro and 10g HCl. Ala-OBzl using the procedure of example 3. ESI-MS (M/e):377[ M + H]+。
EXAMPLE 11 preparation of Boc-Pro-Ala
Using the method of example 4 from15g (39.9mmol) Boc-Pro-Ala-OBzl gave 10.63g (99%) of the title compound as a pale yellow solid. ESI-MS (M/e):268[ M-H]-。
EXAMPLE 12 preparation of Boc-Pro-Ala-Lys (Cbz) -OBzl
The procedure of example 3 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 13 preparation of Pro-Ala-Lys (Cbz) -OBzl
The procedure of example 5 was used to obtain 11.93g (99%) of the title compound as a colorless solid from 14.5g Boc-Pro-Ala-Lys (Cbz) -OBzl. ESI-MS (M/e):539[ M + H]+。
EXAMPLE 14 preparation of Boc-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Using the method of example 3, from 6.1g Boc-Arg (NO)2) And 11g HCl Pro-Ala-Lys (Cbz) -OBzl gave 9.1g (56%) of the title compound as a colorless solid. ESI-MS (M/e):840[ M + H]+。
EXAMPLE 15 preparation of Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
Using the method of example 5, from 9.1g Boc-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl to give the title compound 8.3g (99%) as a colourless solid. ESI-MS (M/e):740[ M + H]+。
EXAMPLE 16 preparation of Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)-OBzl
6.2g (64%) of the title compound were obtained as colorless solid from 2.1g Boc-Gly and 8.4g HCl Arg-Pro-Ala-Lys (Cbz) -OBzl using the method of example 3. ESI-MS (M/e) 897[ M + H]+。
EXAMPLE 17 preparation of Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)
Using the method of example 4, 6.2g Boc-Gly-Arg (NO)2) -Pro-Ala-Lys (Cbz) -OBzl gives 6.0g (97%) of the title compound as a colorless solid. ESI-MS (M/e):806[ M-H]-。
EXAMPLE 18 preparation of Fmoc-Lys (Boc) -Arg (NO)2)-Gly-Asp(OBzl)Val-OBzl
Using example 3Method from 10g Fmoc-Lys (Boc) and 14.8g HCl.Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 16.79g (70%) of the title compound as a colourless solid. ESI-MS (M/e) 1123[ M + H]+。
EXAMPLE 19 preparation of Fmoc-Lys-Arg (NO)2)-Gly-Asp(OBzl)Val-OBzl
Using the method of example 5 from 8g Fmoc-Lys (Boc) -Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 7.14g (98%) of the title compound as a colourless solid. ESI-MS (M/e) 1023[ M + H ]]+。
EXAMPLE 20 preparation of Fmoc-Lys [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
Using the method of example 3, 1.92g Boc-Gly-Arg (NO)2) Pro-Ala-Lys (Cbz) and 2.53g Fmoc-Lys-Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl gives 2.74g (64%) of the title compound as a colourless solid. ESI-MS (M/e) 1810[ M + H]+。
EXAMPLE 21 preparation of Lys [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl
2.0g Fmoc-Lys (Boc-Gly-Arg (NO) was added under ice bath2)-Pro-Ala-Lys-(Cbz))-Arg(NO2) -Gly-Asp (OBzl) -Val-OBzl was stirred well with 2mL piperidine in DMF (20%) for 0.5 h. TLC (dichloromethane/methanol, 40/1) showed Fmoc-Lys (Boc-Gly-Arg (NO)2)-Pro-Ala-Lys-(Cbz))-Arg(NO2) -Gly-Asp (OBzl) -Val-OBzl disappears completely. 100mL of diethyl ether was added to the reaction mixture, and the mixture was allowed to stand to sufficiently separate out a colorless solid. The supernatant was aspirated, and 100mL of ether was added thereto, followed by standing to sufficiently separate out a colorless solid. This operation was repeated 5 times. The supernatant from the last aspiration was concentrated to dryness under reduced pressure to give 1.5g (86%) of the title compound as a colorless solid. ESI-MS (M/e):1587[ M + H]+。
EXAMPLE 22 preparation of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl(3)
From 365mg of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-carboxylic acid (2) and 1750mg Lys (L-Lys) using the method of example 3Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz))-Arg(NO2) -Gly-Asp (OBzl) -Val-OBzl gives 480mg (23%) of the title compound as a colourless solid. ESI-MS (M/e):1899[ M + H]+；1H NMR(DMSO-d6,300MHz)δ/ppm＝8.609(m,2H),8.302(m,1H),8.214(m,1H),8.078(m,3H),7.975(m,2H),7.881(m,2H),7.692(m,5H),7.354(m,12H),7.242(m,3H),6.953(m,4H),5.764(s,1H),5.125(m,4H),4.998(s,2H),4.743(s,1H),4.540(m,1H),4.324(m,1H),4.186(m,5H),3.748(m,1H),3.652(m,1H),3.561(m,4H),3.166(m,4H),2.954(m,9H),2.631(m,1H),2.026(m,2H),1.803(m,3H),1.523(s,8H),1.469(d,J＝5.7Hz,6H),1.376(m,28H),1.213(m,13H),1.010(m,2H),0.838(m,4H)。
EXAMPLE 23 preparation of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Boc-Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val (4)
To 100mg of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro-beta-carboline-3S-acyl-Lys [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Cbz)]-Arg(NO2) To a solution of-Gly-Asp (OBzl) -Val-Obzl (3) in 10mL of methanol was added 40mg of Pd/C and 2 drops of formic acid, and the mixture was stirred well to obtain a uniform suspension. Air in the reaction system is pumped out under reduced pressure, hydrogen is introduced, and the mixture is stirred for 24 hours at room temperature. TLC (dichloromethane/methanol, 40/1) showed complete disappearance of 3. Pb/C was removed by filtration, and the filtrate was concentrated under reduced pressure to give 62mg (66%) of the title compound as a colorless solid. ESI-MS (M/e):1496[ M-H]-。
EXAMPLE 24 preparation of 1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val (5)
From 60mg of N-Boc-1R-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-acyl-Lys (Boc-Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val (4) 51mg (98%) of the title compound was obtained as a colorless solid using the method of example 5. ESI-MS (M/e):1296[ M + H]+；Mp:214-215℃；
(c＝1.0,CH3OH)；IR(cm-1):3259.62,2956.97,2872.65,1724.37,1643.56,1536.45,1447.73,1367.57,1275.52,1072.68,743.88,672.29；1H NMR(D2O-d2,800MHz)δ/ppm＝7.527(m,1H),7.441(d,J＝7.2Hz,1H),7.233(t,J＝7.2Hz,1H),7.136(m,1H),5.018(m,1H),4.594(m,1H),4.599(s,1H),4.381(t,J＝7.2Hz,1H),4.356(m,1H),4.301(m,1H),4.275(m,1H),4.215(m,1H),4.116(m,3H),3.818(m,2H),3.626(m,1H),3.541(m,4H),3.350(m,2H),3.287(m,1H),3.125(m,6H),2.983(m,4H),2.822(m,3H),2.736(m,1H),2.671(m,1H),2.507(m,1H),2.223(t,J＝8Hz,1H),2.100(m,1H),2.026(m,3H),1.956(m,2H),1.680(m,6H),1.501(m,4H),1.331(m,4H),1.152(m,3H),0.839(m,4H)。
Experimental example 1 evaluation of antithrombotic Activity of Compound 5
Male SD rats (200. + -.20 g) were randomly divided into groups of 7 animals, kept for 1 day and stopped overnight. After 30min of gavage administration of a physiological saline solution of Compound 5 (dose of 10nmol/kg) or aspirin (dose of 167 or 16.7. mu. mol/kg) or a physiological saline (dose of 10mL/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 5 is effective in inhibiting thrombosis. The activity of 10nmol/kg compound 5 in inhibiting thrombosis was stronger than 16.7 μmol/kg aspirin, i.e. the antithrombotic activity of compound 5 was 1670 times stronger than aspirin.
TABLE 1 antithrombotic Activity of Compound 5 at 10nmol/kg dose
a) P is less than 0.01 compared with normal saline and aspirin; b) p >0.05 to normal saline; n is 7.
Experimental example 2 evaluation of thrombolytic Activity of Compound 5
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 about 1mL of arterial blood, and placing the arterial blood in a 1mL centrifuge tube. 0.1ml of rat arterial blood is injected into a vertically fixed rubber tube (15 mm in length, 2.5mm in inner diameter, 5.0mm in outer diameter, the tube bottom is sealed by a rubber plug, and the para membrane is tightly sealed), and then a fixing bolt of a stainless steel thrombus is rapidly inserted into the tube (the thrombus fixing spiral is wound by a stainless steel wire with the diameter of 0.2mm, the spiral part is 10mm long and internally contains 15 spiral rings, the diameter of the spiral rings is 1.0mm, and the support handle is connected with the spiral and is about 7.0mm long and in a question mark shape). After blood coagulation for 45min, the thrombus-immobilized spiral wrapped by the thrombus was carefully removed from the glass tube and weighed accurately.
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 5 (dosage of 10nmol/kg) is inserted into the proximal venous end far away from the thrombus-fixing spiral through the middle section of the bypass tube (containing the thrombus-fixing spiral accurately weighed) 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. Data (mean. + -. SD mg) were subjected to a t-test. Loss of thrombus represents thrombolytic activity. The results in Table 2 show that 10nmol/kg of Compound 5 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 5 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 5 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 1ml EP tube at normal temperature for 30 minutes to coagulate the blood, and then transferred to a-20 ℃ refrigerator and left for 1 hour to make the blood clot firm. 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 crushed with a steel spatula into fine thrombus pieces of uniform size, a suspension of the fine 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, and opening the internal carotid artery and the common carotid artery to obtain the artery clamp to restore 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 saline treatment were 3 points 1,3 points 2, 2 points 3. The neurobiological scores of the rats after saline treatment were 3 deaths, 41 and 10. The disease condition is worsened as a whole.
TABLE 3 Effect of continuous saline treatment for 6 days on neurobiological scores in rats with 24 hours cerebral ischemia
The dosage is 3 mL/kg; n is 8.
Rats were injected with tPA 1 time per day via the tail vein at a dose of 3 mg/kg. Injections were given continuously for 6 days, and scored daily. The data in table 4 demonstrate that the neurobiological scores of rats before tPA treatment were 3 points 1,3 points 2, 2 points 3. the neurobiological scores of rats after tPA treatment were 4 deaths, 1 score 1,3 scores 0. In addition, 4 surviving rats showed severe bleeding side effects on both the orbit and tail. The disease condition is worsened as a whole.
TABLE 4 Effect of 6 days of tPA continuous treatment on neurobiological scores in rats with 24h cerebral ischemia
The dosage is 3 mg/kg; 4 surviving rats had severe bleeding on both the orbit and tail; n is 8.
Rats were injected 1 time per day with compound 5 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 the rats before compound 5 treatment were 4 points 1,2 points 2, 1 point 3, 1 point 4. The neurobiological scores of rats treated with compound 5 were 3 deaths, 20 points, 3 points 2 points. In addition, no orbital and tail bleeding was seen in 5 surviving rats. The whole shows obvious curative effect.
TABLE 5 Effect of Compound 5 on neurobiological scores in rats with 24h cerebral ischemia on 6 days of continuous treatment
The dosage is 10 nmol/kg; n is 8.
Experimental example 4 evaluation of Effect of Compound 5 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 are placed in 2% TTC solution and incubated for 30min at 37 ℃ in the dark, and the color change of the brain slices is 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 test data adopts t test. The data in table 6 demonstrate that the cerebral infarct volume of compound 5 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 5 to rats with 24 hours cerebral ischemia after 6 consecutive days of treatment
a) Data were from 5/8 surviving rat brain sections; b) data from 4/8 brain sections of rats with severe bleeding on both the orbit and tail survived; c) data from 5/8 surviving rat brain sections, p <0.01 vs. saline; n is 8.
To summarize: the compounds of the present invention are stable and easy to prepare and store compared to the compounds once disclosed. The compound 5 can effectively inhibit the formation of thrombus by orally taking 10nmol/kg once, and the compound 5 can effectively dissolve the thrombus by orally taking 10nmol/kg once. Intravenous administration of 100nmol/kg of compound 5 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.
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