Carbolinohexahydropyrazine-1, 4-dione-3-acetyl-Val-Val, and preparation and application thereof

Abstract

The invention discloses carboline-hexahydropyrazine-1, 4-dione-3-acetyl-Val-Val with a structure shown in the following formula, a preparation method of the carboline-hexahydropyrazine-1, 4-dione-3-acetyl-Val-Val, and venous thrombosis resisting activity of the carboline-hexahydropyrazine-1, 4-dione-3-acetyl-Val-Val. Therefore, the invention provides an effective technical means for inhibiting venous thrombosis. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val compound, a preparation method thereof, and its anti-venous thrombotic activity and application. The present invention belongs to the field of biomedicine. Background Art

[0002] The applicant has disclosed that the following carboline hexahydropyrazine-1,4-dione-3-acetic acid is 10 -8 M concentration could reduce the amount of P-selectin expressed by activated platelets in rats from 210.55±2.98 ng / mL to 110.12±3.96 ng / ml (p<0.001).

[0003]

[0004] The applicant has also disclosed carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide (the 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) show excellent anti-arterial thrombotic activity. On the one hand, the applicant has recognized that the expression level of P-selectin in the body is related to the pathology of arterial thrombosis, venous thrombosis, tumors and inflammation. In addition to showing anti-arterial thrombotic effects, carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide may also show thrombolytic effects, anti-venous thrombotic effects, anti-tumor effects and anti-inflammatory effects. On the other hand, the applicant found that shortening the oligopeptide sequence in carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide can reduce production costs and promote industrialization. Based on these findings, the applicant prepared carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val of the following formula.

[0005]

[0006] Through experiments, the applicant screened the anti-arterial thrombotic effect, thrombolytic effect, anti-venous thrombotic effect, anti-tumor effect and anti-inflammatory effect of the above-mentioned carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val. The screening found that the above-mentioned carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val showed excellent anti-venous thrombotic effect. Based on this discovery, the applicant proposed the present invention. Summary of the invention

[0007] The technical problem to be solved by the present invention is to propose a carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val compound and a method for preparing carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val, and further evaluate the anti-venous thrombotic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val. In order to achieve the above purpose, the present invention adopts the following technical means.

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

[0009]

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

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

[0012] 2. Coupling carboline hexahydropyrazine-1,4-dione-3-acetic acid with Val-Val-OBzl to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl;

[0013] 3. Carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl is debenzylated to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val.

[0014] The third technical approach is to evaluate the anti-venous thrombotic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val in a rat venous thrombosis model, as well as its application in the preparation of anti-venous thrombotic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0018] According to the conventional peptide preparation method, in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, Boc-Val was first coupled with Val-OBzl to prepare Boc-Val-Val-OBzl, and then Boc was removed in a hydrogen chloride ethyl acetate solution to prepare Val-Val-OBzl. The title compound was obtained with a purity of more than 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-Val-Val-OBzl

[0020] At 0°C, a solution of 313 mg (1 mmol) of carboline hexahydropyrazine-1,4-dione-3-acetic acid, 162 mg (1.2 mmol) of 1-hydroxybenzotriazole and 247 mg (1.2 mmol) of dicyclohexylcarbodiimide and 100 mL of anhydrous tetrahydrofuran was stirred for 10 minutes. Then, 306 mg (1 mmol) of Val-Val-OBzl was added thereto. After stirring for 30 minutes, the reaction mixture was adjusted to pH 9 with N-methylmorpholine. After stirring for 30 minutes, the reaction mixture was stirred at room temperature for 12 hours, and TLC (ethyl acetate / methanol = 30 / 1) showed that carboline hexahydropyrazine-1,4-dione-3-acetic acid disappeared. The reaction mixture was filtered to remove the generated dicyclohexylurea precipitate. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The obtained solution was washed with saturated NaHCO3 aqueous solution (30×3), saturated NaCl aqueous solution (30×3), 5% KHSO4 aqueous solution (30×3), saturated NaCl aqueous solution (30×3), saturated NaHCO3 aqueous solution (30×3) and saturated NaCl aqueous solution (30×3). The ethyl acetate was dried over anhydrous Na2SO4 for 8 hours. Filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain 614 mg (85%) of the title compound. FT-ICR-MS (m / e): 602.2979 [M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=11.63(s,1H),8.33(s,1H),8.32(s,1H),8. 21(s,1H),7.38(d,J=7.5Hz,1H),7.35(t,J=7.5Hz,1H),7.34(t,J=7.5Hz,1 H),7.31(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.32(d,J=7.5Hz,1H),7.3 1(d,J=7.5Hz,1H),7.21(d,J=7.5Hz,1H),6.95(t,J=7.5Hz,1H),6.91(t,J= 7.5Hz,1H),5.34(s,2H),5.12(t,J=6.5Hz,1H),5.01(t,J=6.5Hz,1H),4.35 (q,J=6.4Hz,1H),4.13(q,J=6.4Hz,1H),5.02(t,J=6.5Hz,1H),4.45(s,2H) ,4.47(d,J=6.5Hz,1H),2.91(d,J=6.5Hz,2H),2.75(m,1H),0.98(d,J=6.4H z, 3H), 0.97 (d, J = 6.4Hz, 3H), 0.96 (d, J = 6.4Hz, 3H), 0.95 (d, J = 6.4Hz, 3H). 13 CNMR (125MHz, DMSO-d6): δ / ppm=173.31,173.12,170.02,166.55,166.36,136.16,136.32,130.14,128.66,128. 41,127.69,127.51,127.40,127.33,121.55,119.76,111.23,105.27,73.73,66.43,62.42,61.72,60.69,55.38 37.64,35.86,31.32,30.48,21.79,18.86.18.53,18.45.18.44.

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

[0022] Dissolve carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl (601 mg, 1.0 mmol) in methanol, add 75 mg Pd / C, pass hydrogen, and hydrogenolyze for 48 hours. Filter out Pd / C, concentrate under reduced pressure to remove methanol, and the brown-red powder obtained is washed with ether (30 mL×3). 419 mg (82%) of the standard compound is obtained as a colorless solid. Mp: 189-191℃; =-27 (c = 0.5, methanol); FT-ICR-MS (m / e): 512.2509 [M+H] + ; 1 HNMR (500MHz, DMSO-d6): δ / ppm=12.36(s,1H),11.56(s,1H),8.32(s,1H),8.31(s,1H),8.26(s,1H),7.44(d,J=7. 5Hz,1H),7.33(d,J=7.5Hz,1H),7.12(t,J=7.5Hz,1H),6.99(t,J=7.5Hz,1H),5.39(t,J=6.5Hz,1H),4.25(s,2H),4 .35(t,J=6.5Hz,1H),4.21(d,J=6.3Hz,1H),4.17(d,J=6.8Hz,1H),3.03(d,J=6.5Hz,2H),2.82(d,J=6.4Hz,2H),2. 75(m,1H),1.95(m,1H),0.99(d,J=6.3Hz,3H),0.98(d,J=6.3Hz,3H),0.97(d,J=6.5Hz,3H),0.96(d,J=6.5Hz,3H). 13 C NMR (125MHz, DMSO-d6): δ / ppm=174.31,173.33,169.36,166.57,166.35,136.41,130.11,126.82,121.48,119.17,1 18.11,111.51,106.30,58.25,57.46,57.14,56.22,33.77,30.87,30.26,30.04,21.22,19.34,19.31,18.64,18.51.

[0023] Example 4 Evaluation of the anti-venous thrombotic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val

[0024] The present invention evaluates the anti-venous thrombotic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val on a rat inferior vena cava ligation model. During the evaluation, male SD rats (250±20g) were rested and fasted for one day in an environment of 25-28°C. Afterwards, they were randomly divided into a positive control group, a blank control group and a carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val group. Warfarin is a clinical anti-venous thrombotic drug, which was selected as a positive control, with an oral dose of 4.87 μmol / kg, 11 rats; physiological saline was selected as a blank control, with an oral dose of 10 mL / kg, 11 rats; carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val, with an oral dose of 0.01 nmol / kg, 11 rats.

[0025] During the evaluation, the rats were given drugs orally in a double-blind, sequential and prescribed dosage manner. Thirty minutes after the administration, the rats were anesthetized by intraperitoneal injection of 20% urethane solution. The anesthetized rats were fixed in a supine position on a surgical board, and the skin was prepared and disinfected. Then, the abdominal cavity was opened along the linea alba of the rat's abdomen. The laparotomy incision was from the bottom to the coagulation gland and to the corner of the liver exposed. The small intestine and other organs were removed from the abdominal cavity and wrapped with gauze soaked in saline. The connective tissue around the blood vessels was bluntly separated to expose the inferior vena cava and its branches. The abdominal aorta and inferior vena cava were peeled off below the renal vein, and the inferior vena cava was ligated at the junction of the inferior vena cava and the left renal vein with sutures soaked in saline. The intestines and other organs removed from the abdominal cavity and wrapped with gauze soaked in saline were moved back into the abdominal cavity according to the anatomical position, and the abdominal cavity was sutured layer by layer with sutures. Since rats treated with warfarin may die at any time, the abdominal cavity was opened 30 minutes after the abdominal cavity was sutured, and the branches were ligated one by one. 2 cm of the inferior vena cava was removed from the ligature at the junction of the inferior vena cava and the left renal vein, and the thrombus was removed from the inferior vena cava and weighed. The data in Table 1 show that the activity of 0.01 nmol / kg oral dose of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val in inhibiting venous thrombosis is significantly stronger than the activity of saline in inhibiting venous thrombosis (p<0.01). The data in Table 1 also show that the activity of 0.01 nmol / kg oral dose of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val in inhibiting venous thrombosis is not significantly different from the activity of 4.87 μmol / kg oral dose of warfarin in inhibiting venous thrombosis (p>0.05). For venous thrombosis, carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val has unexpected technical effects.

[0026] Table 1 Anti-venous thrombotic activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val

[0027] Therapeutic agents dose Wet weight of venous thrombus: mean ± SD mg Normal saline 10mL / kg 16.9±3.1 Warfarin 4.87 μmol / kg 8.5±1.6 Carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val 0.01nmol / kg <![CDATA[8.7±1.5 a ]]>

[0028] a) P<0.01 compared with normal saline, P>0.05 compared with warfarin; n=11

Claims

1. Carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val of the following structure, 2. A method for preparing the carbolinohexahydropyrazine-1,4-dione-3-acetyl-Val-Val according to claim 1, the method comprising the following steps: (1) Preparation of Val-Val-OBzl; (2) Carboline hexahydropyrazine-1,4-dione-3-acetic acid is coupled with Val-Val-OBzl to generate carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl; (3) Carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl is debenzylated to generate carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val.

3. Use of the carbolinohexahydropyrazine-1,4-dione-3-acetyl-Val-Val according to claim 1 in the preparation of an anti-venous thrombotic drug.

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