Carbolinohexahydropyrazine-1, 4-dione-3-acetyl-Ser-Ser as well as preparation and application of carbolinohexahydropyrazine-1, 4-dione-3-acetyl-Ser-Ser

Abstract

The invention discloses a carboline-hexahydropyrazine-1, 4-diketone-3-acetyl-Ser-Ser compound as shown in the following formula and a method for preparing the carboline-hexahydropyrazine-1, 4-diketone-3-acetyl-Ser-Ser, and further evaluates the thrombolytic activity of the carboline-hexahydropyrazine-1, 4-diketone-3-acetyl-Ser-Ser, so that the thrombolytic activity of the carboline-hexahydropyrazine-1, 4-diketone-3-acetyl-Ser-Ser is improved, and the thrombolytic activity of the carboline-hexahydropyrazine-1, 4-diketone-3-acetyl-Ser-Ser is improved. Effective application of the compound in preparation of thrombolytic drugs is confirmed, so that the invention provides an effective technical means for preparation of thrombolytic drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser compound, its preparation method, and its thrombolytic activity. The present invention provides an effective technical means for thrombolysis. The present invention belongs to the field of biomedicine. Background Art

[0002] The applicant has previously disclosed the following formula carboline hexahydropyrazine-1,4-dione-3-acetic acid in 10 -8 The concentration of M could reduce the expression of P-selectin in rat activated platelets from 210.55±2.98ng / mL to 110.12±3.96

[0003] ng / ml(p<0.001).

[0004]

[0005] The applicant has also disclosed carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide (oligopeptide is Arg-Gly-Asp-Val, Arg-Gly-Asp-Phe, Arg-Gly-Asp-Ser, Lys-Arg-Gly-Asp-Val, Lys-Arg-Gly-Asp-Phe, Lys-Arg-Gly-Asp-Ser, Leu-Arg-Gly-Asp-Val, Leu-Arg-Gly-Asp-Phe, Leu- Arg-Gly-Asp-Ser, Thr-Arg-Gly-Asp-Val, Thr-Arg-Gly-Asp-Phe, Thr-Arg-Gly-Asp-Ser, Ala-Gly-Asp-Val, Leu-Ala-Gly-Asp-Val, Lys-Ala-Gly-Asp-Val, Thr-Ala-Gly-Asp-Val, and Leu-Arg-Gly-Asp-Val) exhibit excellent anti-arterial thrombotic activity. Applicants have recognized that P-selectin expression levels in vivo are correlated with the pathologies of arterial thrombosis, venous thrombosis, tumors, and inflammation. In addition to exhibiting anti-arterial thrombotic effects, carbolinohexahydropyrazine-1,4-dione-3-acetyl oligopeptide may also exhibit thrombolytic, anti-venous thrombotic, anti-tumor, and anti-inflammatory effects. The applicants also realized that shortening the oligopeptide sequence in carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide could reduce production costs and promote industrialization. Based on this knowledge, the applicants prepared the carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser of the present invention.

[0006] The applicants screened carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser for its anti-arterial thrombotic, thrombolytic, anti-venous thrombotic, anti-tumor, and anti-inflammatory effects. The screening revealed that carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser exhibited excellent thrombolytic activity. Based on this discovery, the applicants proposed the present invention. Summary of the Invention

[0007] The present invention addresses the following technical issues: A carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser compound and a method for preparing the compound are proposed. The thrombolytic activity of the compound is further evaluated. To achieve this objective, the present invention employs the following two technical approaches.

[0008] The first technical approach is to propose the following structure of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser,

[0009]

[0010] The second technical means is to propose a method for preparing carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser, which can be summarized as follows:

[0011] 1. Preparation of Ser-Ser-OBzl;

[0012] 2. Carboline hexahydropyrazine-1,4-dione-3-acetic acid is coupled with Ser-Ser-OBzl to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl;

[0013] 3. Carboline and hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl was debenzylated to prepare carboline and hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser.

[0014] The third technical approach was to evaluate the thrombolytic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser in a rat arteriovenous bypass catheter thrombolytic model, confirming the effective application of the compound in the preparation of thrombolytic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The synthetic route of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser: i) 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, N-methylpiperidin; ii) Pd / C, hydrogen. DETAILED DESCRIPTION

[0016] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention and should not be understood as limiting the present invention.

[0017] Example 1 Preparation of Ser-Ser-OBzl

[0018] Following conventional peptide preparation methods, Boc-Ser was first coupled with Ser-OBzl in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide to prepare Boc-Ser-Ser-OBzl. The Boc residue was then removed in hydrogen chloride in ethyl acetate to prepare Ser-Ser-OBzl. The title compound was obtained with a purity exceeding 96%. FT-ICR-MS (m / e): 307.2022 [M+H] + , directly used in the reaction of Example 2.

[0019] Example 2 Preparation of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl

[0020] A solution of 313 mg (1 mmol) of carbolinohexahydropyrazine-1,4-dione-3-acetic acid, 162 mg (1.2 mmol) of 1-hydroxybenzotriazole, and 247 mg (1.2 mmol) of dicyclohexylcarbodiimide in 100 mL of anhydrous tetrahydrofuran was stirred at 0°C for 10 minutes. Then, 281 mg (1 mmol) of Ser-Ser-OBzl was added. After stirring for 30 minutes, the pH of the reaction mixture was adjusted to 9 with N-methylmorpholine. After stirring for 30 minutes, the reaction mixture was stirred at room temperature for 12 hours. TLC (ethyl acetate / methanol = 30 / 1) indicated the disappearance of carbolinohexahydropyrazine-1,4-dione-3-acetic acid. The reaction mixture was filtered to remove the resulting dicyclohexylurea precipitate. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was washed sequentially with saturated aqueous NaHCO₃ (30 × 3), saturated aqueous NaCl (30 × 3), 5% aqueous KHSO₃ (30 × 3), saturated aqueous NaCl (30 × 3), saturated aqueous NaHCO₃ (30 × 3), and saturated aqueous NaCl (30 × 3). The ethyl acetate was dried over anhydrous Na₂SO₄ for 8 hours. The filtrate was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to yield 491 mg (85%) of the title compound. FT-ICR-MS (m / e): 578.2251 [M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=10.93(s,1H),8.32(s,1H),8.31(s,1H),8.11(s,1H),7.68(t,J=7.5Hz,1H),7.42(d,J=7.5Hz,1H) ,7.35(t,J=7.5Hz,1H),7.34(t,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.32(d,J=7.5Hz,1H),7.31(d,J=7.5Hz,1H),7.30(d,J=7.5Hz ,1H),6.95(t,J=7.5Hz,1H),5.42(d,J=6.5Hz,1H),5.31(s,2H),4.95(s,1H),4.93(s,1H),4.43(s,2H),4.17(t,J=6.0Hz,1H),4.03 (t,J=6.2Hz,1H),3.68(d,J=6.2Hz,2H),3.53(t,J=6.4Hz,1H),3.16(d,J=6.4Hz,2H),3.01(d,J=6.5Hz,2H),2.75(d,J=6.0Hz,2H). 13 CNMR (125MHz, DMSO-d6): δ / ppm=172.11,170.32,169.23,166.57,166.46,137.11,136.32,130.14,128.52,128.31,127.65,127.4 4,127.31,121.45,119.16,111.51,106.27,72.71,66.44,62.62,61.77,60.66,56.28,52.14,47.76,37.67,33.81,25.37,21.73.

[0021] Example 3 Preparation of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser

[0022] Dissolve carbolinohexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl (491 mg, 0.85 mmol) in methanol, add 75 mg of Pd / C, and introduce hydrogen gas for 48 hours. Remove the Pd / C by filtration, concentrate under reduced pressure to remove the methanol, and triturate the resulting brown-red powder with diethyl ether (30 mL x 3). 364 mg (88%) of the title compound is obtained as a colorless solid. Mp: 189-191°C. FT-ICR-MS(m / e):488.1781[M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=12.32(s,1H),10.95(s,1H),8.33(s,1H),8.32(s,1H),8.12(s,1H),7. 44(d,J=7.5Hz,1H),7.34(d,J=7.5Hz,1H),7.71(t,J=7.5Hz,1H),6.99(t,J=7.5Hz,1H),5.40(d,J=6.5 Hz,1H),4.97(s,1H),4.95(s,1H),4.45(s,2H),4.18(t,J=6.0Hz,1H),4.01(t,J=6.2Hz,1H),3.69(d,J = 6.2Hz, 2H), 3.52 (t, J = 6.4Hz, 1H), 3.16 (d, J = 6.4Hz, 2H), 3.04 (d, J = 6.5Hz, 2H), 2.77 (d, J = 6.0Hz, 2H). 13 CNMR (125MHz, DMSO-d6): δ / ppm=172.28,169.31,166.59,166.48,157.17,136.34,130.16,127.33,12 1.49,119.18,111.53,106.30,72.73,61.80,60.67,56.26,52.10,47.90,37.69,33.79,25.07,21.75.

[0023] Example 4 Evaluation of the Thrombolytic Activity of Carbolinohexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser

[0024] The present invention evaluated the thrombolytic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser in a rat model. Male SD rats (230±20g) were rested for one day and randomly divided into groups of 10 rats per group. The rats were anesthetized with an intraperitoneal injection of 20% urethane in saline. The anesthetized rats were immobilized in the supine position, and the right common carotid artery was isolated. An artery clamp was placed on the proximal end of the artery. Surgical sutures were passed through both the proximal and distal ends of the artery. The distal sutures were clamped to the fur with hemostats, and a cannula was inserted at the distal end. The artery clamp was released, and approximately 1 mL of arterial blood was injected into a vertically fixed rubber tube (15 mm long, 2.5 mm inner diameter, 5.0 mm outer diameter, sealed at the bottom with a rubber stopper). A stainless steel thrombus-fixing bolt was quickly inserted into the tube. The thrombus-fixing bolt is made of 0.2mm diameter stainless steel wire. The spiral portion is 10mm long and contains 15 coils, each with a diameter of 1.0mm. The handle, connected to the coil, is 7.0mm long and shaped like a question mark. After the blood has coagulated for 40 minutes, the thrombus-encased thrombus-fixing bolt is carefully removed from the rubber tube and its initial weight accurately weighed.

[0025] The bypass cannula consists of three sections. The central section is a 60mm long, 3.5mm inner diameter polyethylene tube. Each side section is a 100mm long, 1.0mm inner diameter, 2.0mm outer diameter polyethylene tube. One end of the polyethylene tube is stretched to a 10mm long, 1.0mm outer diameter tip for insertion into the rat carotid artery and vein. The other end of the polyethylene tube is covered with a 7mm long, 3.5mm outer diameter polyethylene tube (for connection to the central polyethylene section). The inner walls of all three sections are silanized (1% silicone oil in ether). A precisely weighed thrombus-encapsulated thrombus-fixing bolt is placed within the central polyethylene section.

[0026] Continue isolating the anesthetized rat's trachea and perform endotracheal intubation. Isolate the rat's left external jugular vein, insert surgical sutures through both the proximal and distal ends, and carefully make a bevel cut in the exposed left external jugular vein. Insert the beveled tip of the prepared bypass tubing into the proximal end of the left external jugular vein opening, and secure the handle of the thrombus coil within the tubing distal to the midsection. Use a syringe to inject an accurate amount of sodium heparinized saline solution (50 IU / kg) through the tip of the other end. Do not remove the syringe from the polyethylene tubing at this time. Clamp the flexible tube between the syringe and the polyethylene tubing with a hemostat. Apply an artery clamp to control bleeding at the proximal end of the right common carotid artery. Carefully make a bevel cut in the right common carotid artery just distal to the clamp. Remove the syringe from the tip of the polyethylene tubing and insert the tip of the polyethylene tubing into the proximal end of the arterial bevel. Secure both ends of the bypass tubing to the artery and vein with No. 4 surgical sutures.

[0027] Normal saline (3 mL / kg) or a saline solution of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser (0.01 nmol / kg) was slowly injected into the rat's stomach via the esophagus. Alternatively, urokinase (20,000 IU / kg) was slowly injected into the rat's stomach via a scalp needle into the vein near the midsection of the bypass tubing, distal to the thrombus fixation coil. The solution then circulated through the arterial clamp, allowing blood flow from the artery to the vein through the bypass tubing. One hour after the start of the injection, the thrombus fixation screw was removed from the midsection of the bypass tubing and accurately weighed. The final weight of the thrombus fixation screw was subtracted from the initial weight to calculate the thrombus weight loss.

[0028] Table 1 shows that the thrombus weight reduction in rats treated with an oral dose of 0.01 nmol / kg of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser was significantly greater than that in rats treated with saline (P < 0.01 for saline). Table 1 also shows that the thrombus weight reduction in rats treated with an oral dose of 0.01 nmol / kg of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser was significantly greater than that in rats treated with an intravenous dose of 20,000 IU / kg of urokinase (P < 0.01 for urokinase). Carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser has a significant technical effect.

[0029] Table 1 Thrombolytic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser

[0030] therapeutic agents dose Thrombus weight loss, mean ± SD mg Normal saline 0.1mL / 10g 22.23±1.64 Carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser 0.01 nmol / kg <![CDATA[32.25±2.04 a ]]> Urokinase 20,000 IU / kg 28.12±2.01

[0031] a) Compared with normal saline and urokinase, P<0.01; n=10.

Claims

1. Carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser of the following structure, 2. The method for preparing carbolinohexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of Ser-Ser-OBzl; (2) Carboline hexahydropyrazine-1,4-dione-3-acetic acid is coupled with Ser-Ser-OBzl to form carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl; (3) Carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser-OBzl is debenzylated to form carboline hexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser.

3. Use of the carbolinohexahydropyrazine-1,4-dione-3-acetyl-Ser-Ser according to claim 1 in the preparation of thrombolytic drugs.

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