CN110577576A - 1S-Methyl-β-tetrahydrocarbolinoyl-K(GRPAK)-RGDV, Its Synthesis, Activity and Application - Google Patents

Abstract

The invention discloses 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Lys of the following formula Asp-Val, discloses its preparation method, discloses its antithrombotic activity, discloses its thrombolytic activity and discloses its effect of treating stroke rats for 24 hours, thus the present invention discloses that it is used in the preparation of antithrombotic drug , thrombolytic drugs and the application of drugs for the treatment of ischemic stroke.

Description

1S-Methyl-β-tetrahydrocarbolinoyl-K(GRPAK)-RGDV, Its Synthesis, Activity and Application

technical field

The present invention relates to 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val, Related to its preparation method, related to its antithrombotic activity, related to its thrombolytic activity and related to its role in treating stroke rats for 24 hours, so the present invention relates to its preparation of antithrombotic drugs, thrombolytic drugs and treatment of ischemic Use in stroke medicine. The invention belongs to the field of biomedicine.

technical background

Ischemic stroke is a relatively common and serious cerebrovascular disease, characterized by high incidence, high mortality, high disability rate and high recurrence rate. At present, the clinical treatment of ischemic stroke faces the reality that there is no effective drug, especially for patients with stroke lasting more than 4 hours, either dying or being disabled. It is an important clinical need to invent drugs that are effective for patients with a stroke of more than 4 hours. The inventor once disclosed that the imidazoline of the following formula showed excellent curative effect on the ischemic stroke model in rats after 24 hours of stroke. That is, continuous intravenous injection of imidazoline for 6 days, once a day, with a dose of 100nmol/kg, has excellent curative effect. In the formula, AA is Ser, Val or Phe. Due to their structure, the imidazolines of the formula have two inevitable disadvantages. That is, the free radicals of the 1,3-dioximidazoline moiety are sensitive to reducing environments, making it difficult not only to prepare but also to preserve.

After 3 years of experimental research, the inventor found that the imidazoline part can be replaced by 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl to obtain a stable structure and easy storage. Unexpected technical effects. Based on this finding, the inventors have made the present invention.

Contents of the invention

The first content of the present invention is to provide 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl of the following formula

-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val.

The second content of the present invention is to provide 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl

-the synthetic method of Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val, the method comprising:

(1) Preparation of N-Boc-1S-methyl-1,2,3,4 tetrahydro-β-carboline-3S-carboxylic acid;

(2) Preparation of N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Boc);

(3) Preparation of HCl·Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl;

(4) Preparation of Boc-Gly-Arg(NO ₂ )-Pro-Ala-Lys(Cbz);

(5) Preparation of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl;

(6) Preparation of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl

-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val.

The third content of the present invention is to evaluate 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl

- Antithrombotic, thrombolytic and ischemic stroke activity of Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val.

Description of drawings

Fig. 1 Synthesis of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val route. i: CH ₃ CHO, H ₂ O; ii: TEA, DMF, di-tert-butyl dicarbonate (Boc) ₂ O; iii: Pb/C, H ₂ , tetrahydrofuran; iv: N,N-dicyclohexylcarbodi (DCC), N-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), tetrahydrofuran; v: 2N aqueous sodium hydroxide solution, CH ₃ OH; vi: hydrogen chloride in ethyl acetate ( 4M); vii: trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TMFA).

Detailed ways

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1 Preparation of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (1)

1 mL of concentrated sulfuric acid with a concentration of 98% was added dropwise to 800 mL of distilled water, and after stirring evenly, 10.0 g (34 mmol) of L-Trp-OBzl was added therein three times. Stir for five minutes to fully suspend L-Trp-OBzl and sulfuric acid aqueous solution. Afterwards, 10 mL of 40% acetaldehyde aqueous solution was added dropwise to the suspension. The reaction mixture was stirred for 12 h, and then 3 mL of concentrated ammonia water was added dropwise to adjust the pH of the reaction solution to 8. The reaction compound was allowed to stand for 1 h until the product was fully separated. The solid was filtered off and dried to yield 9.84 g (90%) of a light yellow solid as 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-carboxylate benzyl ester and 1S-methyl - Mixtures of benzyl 1,2,3,4-tetrahydro-β-carboline-3S-carboxylates. ESI-MS (m/e): 321 [M+H] ⁺ .

Example 2 Preparation of N-Boc-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (2)

9.84g (30.8mmol) of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid benzyl ester obtained in Example 1 was mixed with 20mL N,N-dimethylformamide (DMF) dissolved. Add 6.98 g (32.0 mmol) (Boc) ₂ O to the solution at 0°C. 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 with 100 mL of ethyl acetate. The obtained ethyl acetate solution was washed successively with 5% potassium hydrogensulfate aqueous solution (50 mL×3) and saturated sodium chloride aqueous solution (50 mL×3). The separated ethyl acetate layer was dried over anhydrous sodium sulfate for 12 h, filtered, and the filtrate was concentrated under reduced pressure to obtain an oil. The oil was separated on a silica gel column (dichloromethane/methanol, 100/1) to obtain 5.02 g (38%) of N-Boc-1R-methyl-1,2,3,4-tetrahydro-β-carboline -benzyl 3S-carboxylate and 6.00 g (47%) of benzyl N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-carboxylate. Are colorless powder.

Example 3 Preparation of Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-carboxylic acid (3)

Add 100mg Pd/ C, Stir to make a homogeneous suspension. The air in the reaction system was extracted under reduced pressure, hydrogen gas was introduced, and stirred at room temperature for 10 h. TLC (dichloromethane/methanol, 40/1) showed that N-Boc-1S-methyl-1,2,3,4-tetrahydro- β-carboline-3S-carboxylate benzyl ester completely disappeared. Pd/C was removed by filtration, and the filtrate was concentrated under reduced pressure. The ESI-MS (m/e) of the obtained colorless powder was 329 [MH] ^- .

Example 4 Preparation of N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Boc)-OBzl(4)

Dissolve 4.950g (15.0mmol) of Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in 50mL of anhydrous tetrahydrofuran, and go to the obtained Add 2.025g (15.0mmol) N-hydroxybenzotriazole (HOBt) and 3.090g (15.0mmol) N,N-dicyclohexylcarbodiimide (DCC) solution dissolved in anhydrous THF in the solution, fully Stir and activate for 30 minutes to obtain solution A; then 5.066g (13.6mmol) HCl·Lys(Boc)-OBzl dissolved in 20mL of anhydrous THF was first adjusted to pH 8-9 with N-methylmorpholine. Then added to the reaction solution A. Removed from the ice bath and stirred at room temperature for 8 hours, TLC under UV (254 nm) (developing solvent dichloromethane/methanol = 40/1) showed that the raw material HCl·Lys(Boc)-OBzl completely disappeared. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The ethyl acetate layer was sequentially washed with saturated aqueous sodium bicarbonate (n=3), saturated aqueous sodium chloride (n=3), 5% aqueous potassium bisulfate (n=3), saturated sodium chloride Wash with aqueous solution (n=3), saturated aqueous sodium bicarbonate solution (n=3) and saturated aqueous sodium chloride solution (n=3), combine the upper ethyl acetate phase and dry with anhydrous sodium sulfate for 4 hours , filtered, and the obtained filtrate was concentrated to dryness under reduced pressure, and subjected to silica gel column chromatography to obtain 6.960 g (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)

Using the method of Example 3 to obtain the title compound from 1.296g (2.0mmol) N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-carboxylate benzyl ester, as color powder. ESI-MS (m/e): 557[MH] ^- .

Example 6 Preparation of Boc-Arg(NO ₂ )-Gly-OBzl

Using the method of Example 4, 5.560 g (84%) of the title compound was obtained as a colorless powder from 5.000 g (15.6 mmol) Boc-Arg(NO ₂ ) and 2.860 g (14.2 mmol) HCl·Gly-OBzl. ESI-MS (m/e): 467 [M+H] ⁺ .

Example 7 Preparation of Boc-Arg(NO ₂ )-Gly

4.660 g (1 mmol) of Boc-Arg(NO ₂ )-Gly-OBzl were dissolved in 50 mL of methanol. Slowly add 2N NaOH aqueous solution dropwise to the obtained solution under ice bath and stirring, and stir in ice bath for 6 hours. TLC (developer dichloromethane/methanol=40/1) under UV (254 nm) showed that Boc-Arg(NO ₂ )-Gly-OBzl disappeared completely. Slowly add saturated KHSO ₄ aqueous solution dropwise under ice bath and stirring to adjust the pH to 7, and concentrate under reduced pressure to remove methanol. Adjust the pH to 2 with saturated potassium bisulfate aqueous solution, then repeatedly extract the aqueous solution 3 times with ethyl acetate (40mL×3), extract and wash with saturated sodium chloride aqueous solution 3 times, combine the ethyl acetate layers, and dry with anhydrous sodium sulfate for 2 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to afford 2.850 g (76%) of the title compound. ESI-MS (m/e): 375[MH] ^- .

Example 8 Preparation of Boc-Asp(OBzl)-Val-OBzl

The method of Example 4 was used to obtain 3.910 g (84%) of the title compound as a colorless solid from 3.230 g (10 mmol) of Boc-Asp(OBzl) and 3.450 g (9.1 mmol) of Tos.Val-OBzl. ESI-MS (m/e): 515 [M+H] ⁺ .

Example 9 Preparation of Asp(OBzl)-Val-OBzl

10.0 g of Boc-Asp(OBzl)-Val-OBzl was dissolved in 10 mL of anhydrous ethyl acetate. Add 100 mL of hydrogen chloride in ethyl acetate (4M) to the solution under ice bath and stirring, and stir for 2 h. TLC (dichloromethane/methanol, 40/1) under UV (254 nm) showed complete disappearance of the starting material Boc-Asp(OBzl)-Val-OBzl. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was diluted with 30 mL of anhydrous ethyl acetate, and then concentrated to dryness under reduced pressure. This operation was repeated 3 times. The resulting residue was diluted with 30 mL of anhydrous ether, and then concentrated to dryness under reduced pressure. This operation was repeated 3 times to obtain the title compound. ESI-MS (m/e): 413 [M+H] ⁺ .

Example 10 Preparation of Boc-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl

Using the method of Example 4, 5.190 g (74%) of the title compound were obtained from 3.750 g (10.0 mmol) Boc-Arg(NO ₂ )-Gly and 4.080 g (9.1 mmol) HCl·Asp(OBzl)-Val-OBzl, as Colorless solid. ESI-MS (m/e): 770 [M+H] ⁺ .

Example 11 Preparation of Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl

The method of Example 9 was used to obtain 4.27 g (98%) of the title compound as a colorless solid from 5 g (6.5 mmol) of Boc-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl. ESI-MS (m/e): 673 [M+H] ⁺ .

Embodiment 12 prepares Boc-Pro-Ala-OBzl

The method of Example 4 was used to obtain 3.040 g (90%) of the title compound as a colorless solid from 2.150 g (10 mmol) of Boc-Pro and 1.960 g (9.1 mmol) of HCl·Ala-OBzl. ESI-MS (m/e): 377 [M+H] ⁺ .

Embodiment 13 prepares Boc-Pro-Ala

The method of Example 3 was used to obtain 1.350 g (95%) of the title compound from 1.880 g (5 mmol) of Boc-Pro-Ala-OBzl as a colorless solid. ESI-MS (m/e): 285[MH] ⁺ .

Example 14 Preparation of Boc-Pro-Ala-Lys(Cbz)-OBzl

Adopt the method for embodiment 4 to obtain 14.5 from 10g Boc-Pro-Ala-OBzl and 14.2g HCl Lys (Cbz)-OBzl

g (65%) of the title compound as a colorless oily product. ESI-MS (m/e): 639 [M+H] ⁺ .

Example 15 Preparation of Pro-Ala-Lys(Cbz)-OBzl

Using the method of Example 9, 1.640 g (95%) of the title compound was obtained as a colorless oil from 1.901 g (3 mmol) of Boc-Pro-Ala-Lys(Cbz)-OBzl. 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, 1.701 g (75%) of the title compound was obtained from 0.957 g (3.0 mmol) Boc-Arg (NO ₂ ) and 1.550 g (2.7 mmol) HCl·Pro-Ala-Lys(Cbz)-OBzl, as Colorless oil. ESI-MS (m/e): 840 [M+H] ⁺ .

Example 17 Preparation of Arg(NO ₂ )-Pro-Ala-Lys(Cbz)-OBzl

The method of Example 9 was used to obtain 8.3 g (99%) of the title compound as a colorless solid from 9.1 g of Boc-Arg(NO ₂ )-Pro-Ala-Lys(Cbz)-OBzl. ESI-MS (m/e): 740 [M+H] ⁺ .

Example 18 Preparation of Boc-Gly-Arg(NO ₂ )-Pro-Ala-Lys(Cbz)-OBzl

Using the method of Example 4 to obtain 1.344g (75%) of the title compound from 0.385g (2.2mmol) Boc-Gly and 1.490g (2.0mmol) HCl Arg(NO ₂ )-Pro-Ala-Lys(Cbz)-OBzl , is a colorless solid. ESI-MS (m/e): 897 [M+H] ⁺ .

Example 19 Preparation of Boc-Gly-Arg(NO ₂ )-Pro-Ala-Lys(Cbz)

Using the method of Example 7, 1.449 g (90%) of the title compound was obtained as a colorless solid from 1.800 g (2.0 mmol) of Boc-Gly-Arg(NO ₂ )-Pro-Ala-Lys(Cbz)-OBzl. ESI-MS (m/e): 805[MH] ^- .

Example 20 Preparation of N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Boc)-Arg(NO ₂ )-Gly-Asp(OBzl )-Val-OBzl(6)

Using the method of Example 4 from 5.580g (10.0mmol) N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys (Boc) and 6.430g (9.1 mmol) HCl.Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl afforded 8.148 g (74%) of the title compound as a colorless solid. ESI-MS (m/e): 1211 [M+H] ⁺ .

Example 21 Preparation of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl(7 )

Adopt the method of embodiment 9 1.210g (1.0mmol) N-Boc-1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Boc)-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl afforded 0.96 g (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-Gly-Arg(NO ₂ )-Pro-Ala-Lys(Cbz) ]-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl(8)

Using the method of Example 4 from 1.612g (2.0mmol) Boc-Gly-Arg (NO ₂ )-Pro-Ala-Lys (Cbz) and 1.840g (1.82mmol) 1S-methyl-1,2,3,4 -Tetrahydro-β-carboline-3S-formyl-Lys-Arg(NO2)-Gly _- Asp(OBzl)-Val-OBzl afforded 425 mg (13%) of the title compound as a colorless solid. ESI-MS (m/e): 1799 [M+H] ⁺ . ¹ HNMR(DMSO-d ₆ ,300MHz)δ/ppm=11.28(s,1H),8.51(m,2H),8.32(d,J=8.1Hz,1H),8.19(m,3H),8.11(d ,J＝5.7Hz,1H),7.92(m,4H),7.71(m,1H),7.45(m,2H),7.36(m,15H),7.021(m,2H),5.04(m,6H) ,4.79(m,1H),4.53(m,1H),4.31(m,3H),4.18(m,3H),3.76(m,1H),3.57(m,3H),3.17(m,4H), 2.95(m,4H),2.74(m,1H),2.61(m,1H),2.05(m,2H),1.76(m,5H),1.62(m,11H),1.37(m,12H),1.19 (m, 5H), 0.85 (d, J = 5.1 Hz, 6H).

Example 23 Preparation of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys(Gly-Arg-Pro-Ala-Lys)-Arg-Gly-Asp-Val -OH(9)

117 mg (0.065 mmol) of 1S-methyl-1,2,3,4-tetrahydro-β-carboline-3S-formyl-Lys[Boc-GlyArg(NO ₂ )-Pro-Ala-Lys(Cbz) ]-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl was dissolved in ice-salt bath by adding 1mL trifluoroacetic acid (TFA), and then adding 0.30mL trifluoromethanesulfonic acid (TFMSA), 1 Hours later, TLC under UV (254nm) (developer n-butanol: water: glacial acetic acid = 1:1:1) showed that the raw material point disappeared, and the reaction was stopped. Anhydrous ether was added to the reaction and stirred, and a solid was precipitated, left to stand, and discarded. Remove the supernatant, then add anhydrous ether, let it stand, discard the supernatant, repeat 3 times, dry it under reduced pressure, then dissolve it with a small amount of water, adjust the pH to 7 with 25% ammonia water, desalt with Sephadex G ₁₅ , C ₁₈ Column purification and lyophilization afforded 12 mg (14%) of the title compound as a colorless powder. ESI-MS(m/e):1295[M+H] ⁺ ; mp:221.1-221.7℃; IR (cm ^-1 ): 3334.19, 3079.09, 2960.49, 1650.39, 1532.191455.85, 1384.18, 1239.36, 1224.94, 1166.12, 1026.74, 761.18, 636.86; ¹ H NMR (DMSO-d ₆ ) δ/2 ,30 s,1H),8.707(m,1H),8.61(m,2H),8.41(m,3H),8.17(m,2H),8.10(m,2H),7.91(m,2H),7.78(m ,2H),7.43(d,J=7.2Hz,1H),7.35(d,J=7.8Hz,1H),7.04(m,3H),4.53(m,2H),4.27(m,1H),4.16 (m,2H),3.92(m,5H),3.62(m,2H),3.35(m,2H),3.03(m,6H),2.75(m,3H),2.63(m,2H),2.01( m, 3H), 1.75 (m, 5H), 1.53 (m, 7H), 1.46 (m, 4H), 1.26 (m, 9H), 0.79 (d, J=6.6Hz, 6H).

Experimental example 1 evaluates the antithrombotic activity of compound 9

Male SD rats (200±20g) were divided into random groups, 10 in each group, fed for 1 day, and stopped feeding overnight. After intragastric administration of a normal saline solution (dose 10nmol/kg) of compound 9 or aspirin (dose 167 μmol/kg) or normal saline (dose 3mL/kg) for 30min, rats were treated with 20% urea sugar in normal saline Aqueous anesthesia followed by surgery. The right carotid artery and left jugular vein of the rat were separated, and an accurately weighed silk thread was placed on a bypass cannula. One end of the tube was inserted into the left vein, and the other end of the tube was inserted into the right artery, and 0.2 mL of heparin sodium was injected for anticoagulation. The blood flow was made to flow from the right artery through the bypass cannula into the left vein. After 15 minutes, the silk thread with the thrombus was taken out and weighed, and the weight of the thread before and after blood circulation was calculated. Thrombotic activity, for t test. The data are included in Table 1. The results showed that oral administration of 10nmol/kg compound 9 could effectively inhibit thrombus formation.

The antithrombotic activity of compound 9 under the dose of 10nmol/kg in table 1

a) Compared with normal saline, P<0.01, n=10

Experimental example 2 evaluates the thrombolytic activity of compound 9

SD rats (male, 200±20g) were anesthetized by intraperitoneal injection of urethane saline solution at a dose of 1200mg/kg. After the rats were anesthetized, they were fixed in the supine position, and the right common carotid artery was separated. tube, loosen the arterial clamp, take out 1mL of arterial blood, and place it in a 1mL centrifuge tube. Inject 0.1mL of rat arterial blood into a vertically fixed rubber tube (length 5mm, inner diameter 2.5mm, outer diameter 5.0mm, the bottom of the tube is sealed with a rubber stopper, and the para membrane is tightly sealed), and then a stainless steel tube is quickly inserted into the tube. The thrombus fixing bolt (the thrombus fixing helix is made of stainless steel wire with a diameter of 0.2mm, the length of the helix is 10mm, and contains 15 coils, the diameter of the coil is 1.0mm, the handle is connected with the helix, and the length is about 7.0mm, in the form of a question mark).

The bypass intubation tube is composed of three parts. The middle section is a polyethylene rubber tube with a length of 60.0mm and an inner diameter of 3.5mm; Pulled into a pointed tube, about 10.0mm long (for inserting into rat carotid artery and vein), with an outer diameter of 1.0mm, the other end of the outer sleeve is a section of polyethylene tube with a length of 7.0mm and an outer diameter of 3.5mm (with In the polyethylene rubber tube inserted into the middle section), the inner walls of the three sections of the tube need to be silanized (1% silicone oil ether solution). Place the thrombus-wrapped thrombus fixation helix in the middle section of the polyethylene hose, and the other two ends of the hose are respectively fitted with the thickened ends of the two polyethylenes to ensure that no blood leaks during circulation. Use a syringe to fill the tube with heparin saline solution (50 IU/kg) through the tip of the tip, remove air bubbles, and set aside.

Separate the left external jugular vein of the rat, insert the proximal end and the distal end into the surgical thread respectively, ligate the blood vessels at the distal end, cut a small opening on the exposed left external jugular vein, and insert the prepared bypass pipe tip Insert it into the opening of the left external jugular vein through the small opening, and at the same time keep away from the internal thrombus fixation coil in the middle section of the bypass tube (including the accurately weighed thrombus fixation coil). Use a syringe to inject an accurate amount of heparin sodium saline solution (50IU/kg) through the pointed tube at the other end. At this time, do not withdraw the syringe from the polyethylene tube, and clamp the hose between the syringe and the polyethylene tube with an arterial clamp. Use an arterial clip to stop bleeding at the proximal end of the right common carotid artery, ligate the distal end, cut a small mouth of the right common carotid artery not far from the arterial clip, pull out the syringe from the tip of the polyethylene tube, and insert the polyethylene tube The tip of the needle is inserted into the proximal end of the beveled artery. Both ends of the bypass conduit were fixed with No. 4 surgical sutures.

Use a scalp needle to pass normal saline solution (3mL/kg) or urokinase normal saline solution (dose is 20000IU/kg) or compound 9 normal saline solution (dose is 10nmol/kg) through the middle section of the bypass tube (including accurately weighed The thrombus fixation helix) is inserted into the proximal venous end away from the thrombus fixation helix, and the arterial clamp is loosened to allow blood to flow from the artery to the vein through the bypass tubing. Slowly inject the solution in the syringe into the blood, and through the blood circulation, act on the helical thrombus in the order of vein-heart-artery. After the blood circulated for 1 hour, the helix for fixing the thrombus was taken out from the bypass pipe and weighed accurately. Calculate the weight difference of the thrombus before and after the helical blood circulation of the fixed thrombus in the bypass channel of each rat, that is, the weight loss of the thrombus. A t-test was performed on the data (mean ± SD mg). Thrombus weight loss represents thrombolytic activity. The results in Table 2 show that 10 nmol/kg compound 9 can effectively dissolve thrombus. The activity was not significantly different from 20000IU/kg urokinase. It shows that the technical effect is obvious.

Thrombolytic activity of compound 9 under the dose of 10nmol/kg in table 2

a) P<0.01 compared with normal saline, P>0.05 compared with urokinase; n=9.

Experimental example 3 evaluates the therapeutic effect of compound 9 on ischemic stroke 24h rats

A vertical incision about 2 cm long was made 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 separated along the inner edge of the sternocleidomastoid muscle. Clamp the opening of the internal carotid artery and the proximal end of the common carotid artery with a non-invasive arterial clip, ligate the distal end of the external carotid artery, cut a small hole in the external carotid artery, loosen the arterial clip at the proximal end of the common carotid artery, and take 10 μL Afterwards, the proximal end of the common carotid artery was clamped with a non-invasive arterial clip. Place 10 μL of blood obtained in a 1 mLEP tube at room temperature for 30 minutes to allow the blood to coagulate, and then transfer to a -20°C refrigerator for 1 hour to solidify the blood clot. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 400 mg/kg. Take out the blood clot, add 1mL of normal saline, use a steel spatula to smash the blood clot into fine thrombus of uniform size, prepare a suspension of fine thrombus and transfer it to a 1mL syringe. Loosen the arterial clamp at the proximal end of the common carotid artery, slowly inject 1mL of thrombus suspension from the external carotid artery to the proximal end through the internal carotid artery into the brain of the rat, then ligate the proximal end of the external carotid artery, and open the internal carotid artery. The artery and common carotid artery were clamped and blood flow was restored. Wait to wake up. The degree of neurological deficit was assessed by the Zealonga method 24 hours after the rats woke up. A score of 0 means no signs of neurological deficit, a score of 1 means that the forelimbs on the uninjured side cannot be stretched, a score of 2 means walking towards the uninjured side, a score of 3 means walking in a tail-like manner towards the uninjured side, and a score of 4 means that the consciousness is impaired and there is no voluntary walking , 5 points means death. Grouped by average score. Rats were injected with normal saline once a day through the tail vein at a dose of 3 mL/kg. Injections were given for 6 consecutive days and scored daily. The data in table 3 shows that the neurobiological score of rats before the administration of normal saline treatment is 2 points for 5, 3 points for 4, and 4 points for 2, and the neurobiological score of rats after 6 days of administration of normal saline is 5 dead, 0 points for 1, 1 point for 2, 2 points for 3. The overall performance showed a deterioration of the condition.

Table 3 Effect of continuous treatment with normal saline for 6 days on the neurobiological score of rats with cerebral ischemia for 24 hours

Dose: 3mL/kg; n=11

Rats were injected with tPA once a day via the tail vein at a dose of 3 mg/kg. Rated daily. The data in table 4 shows that two of the 6 rats died within 24 hours. The dead rats were autopsied, and it was found that their organs had hemorrhage, especially severe pulmonary hemorrhage. Therefore, after administration of 2 Dosing was discontinued after that time. After 2 administrations, none of the rats improved and showed no signs of neurological deficit, 2 remained with slight signs of neurological deficit, 1 remained uninjured and turned into a tail-like walking sign, and 1 remained consciously impaired without autonomous walking signs .

Table 4 The curative effect of 3mg/kg tPA treatment after 6 hours of ischemia in rats

Dose: 3mg/kg; n=6

Rats were injected with compound 9 once a day via the tail vein at a dose of 10 nmol/kg. Injections were given for 6 consecutive days and scored daily. The data in Table 5 shows that the neurobiological score of the rats before compound 9 treatment was 2 points for 5, 3 points for 3, and 4 points for 3 rats, and the neurobiological score of the rats after 6 days of administration was 2 points for 2 , 3 1 point, 6 0 points. In addition, none of the surviving rats had orbital or tail bleeding. Overall, the curative effect is obvious.

Table 5 Effect of compound 9 continuous treatment for 6 days on the neurobiological score of rats with cerebral ischemia for 24 hours

Dose: 100nmol/kg; n=11

Experimental Example 4 Evaluation of the Effect of Compound 9 on Cerebral Infarction Volume in Rats with Ischemic Stroke for 24h

After accepting Experimental Example 3 to evaluate the degree of neurological deficit, they were anesthetized with urethane, and the brain was quickly decapitated and taken out, and placed in a -20°C refrigerator for 2 hours, and sliced continuously from the forehead pole to obtain 6 coronal brain slices with a thickness of 2 mm. The brain slices were incubated in 2% TTC solution for 30 min at 37°C in the dark, and the color changes of the brain slices were observed. After the normal brain tissue was stained red by TTC and the ischemic brain tissue was white, a digital camera was used to take pictures. The photos were processed by SPSS statistical software, the infarct volume and the volume of normal tissue in the coronal slices of the brain were calculated, and the percentage of infarct volume in each group was calculated. All experimental data were tested by t test. The data in Table 6 shows that the volume of cerebral infarction in rats with ischemic stroke for 24 hours treated with compound 9 was significantly smaller than that in rats with ischemic stroke treated with normal saline for 24 hours.

Table 6 compound 9 continuous treatment 6 days to the volume ratio of cerebral infarction in rats with cerebral ischemia for 24 hours

a) Data come from 6/11 surviving rat brain slices; b) Data come from 11/11 surviving rat brain slices, P<0.01 compared with normal saline; n=11

Summary: Compared with the previously disclosed compounds, the compounds of the present invention are stable and easy to prepare and preserve. One-time oral administration of 10 nmol/kg compound 9 can effectively inhibit thrombus formation, and one-time injection of 10 nmol/kg compound 9 can effectively dissolve thrombus. Intravenous administration of 100nmol/kg compound 9 once a day for 6 consecutive days can effectively restore the neurobiological behavior of rats with ischemic stroke for 24 hours, and can effectively reduce the volume of cerebral infarction in rats with ischemic stroke for 24 hours. That is to say, the present invention has obtained outstanding technical effects.

Claims (5)

1. 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of the formula,

2. The 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl of claim 1

A process for the preparation of-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val, the process 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-Gly-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 (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val.

3. Use of the 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Gly-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 (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1 in the manufacture of a thrombolytic drug.

5. Use of 1S-methyl-1, 2,3, 4-tetrahydro- β -carboline-3S-formyl-Lys (Gly-Arg-Pro-Ala-Lys) -Arg-Gly-Asp-Val of claim 1 for the manufacture of a medicament for the treatment of ischemic stroke.