Carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val, its preparation and application

Abstract

This invention discloses a carbazohexahydropyrazine-1,4-dione-3-acetyl-Val-Val with the following structure, its preparation method, and its anti-venous thrombotic activity. Therefore, this invention provides an effective technical means for inhibiting venous thrombosis.

Description

Technical Field

[0001] This invention relates to a carbazohexahydropyrazine-1,4-dione-3-acetyl-Val-Val compound, its preparation method, its anti-venous thrombotic activity, and its applications. This invention belongs to the field of biomedicine. Background Technology

[0002] The applicant previously disclosed the following formula: carbo(hexahydropyrazine-1,4-dione-3-acetic acid) in 10 -8 At concentration M, the amount of P-selectin expressed in activated platelets of rats decreased from 210.55±2.98 ng / mL to 110.12±3.96 ng / mL (p<0.001).

[0003]

[0004] The applicant has also previously disclosed carboline-hexahydropyrazine-1,4-dione-3-acetyl oligopeptides (the oligopeptides are 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. The applicant recognizes that the expression level of P-selectin in vivo is associated with the pathology of arterial thrombosis, venous thrombosis, tumors, and inflammation. In addition to its anti-arterial thrombotic activity, carbino-hexahydropyrazine-1,4-dione-3-acetyl oligopeptide may also exhibit thrombolytic, anti-venous thrombotic, anti-tumor, and anti-inflammatory effects. On the other hand, the applicant discovered 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, thrombolytic, anti-venous thrombotic, antitumor, and anti-inflammatory effects of the above-formulated carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Val-Val. The screening revealed that the above-formulated carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Val-Val exhibited excellent anti-venous thrombotic activity. Based on this finding, the applicant proposed this invention. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a carbazohexahydropyrazine-1,4-dione-3-acetyl-Val-Val compound, a method for preparing carbazohexahydropyrazine-1,4-dione-3-acetyl-Val-Val, and to further evaluate the anti-venous thrombotic activity of carbazohexahydropyrazine-1,4-dione-3-acetyl-Val-Val. To achieve the above objective, the present invention employs the following technical means.

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

[0009]

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

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

[0012] 2. Carboline-hexahydropyrazine-1,4-dione-3-acetic acid was prepared by coupling carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl;

[0013] 3. Carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val was prepared by debenzylation of carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val.

[0014] The third technical approach is to evaluate the anti-venous thrombotic activity of carbophenazine-1,4-dione-3-acetyl-Val-Val in a rat venous thrombosis model, and its application in the preparation of anti-venous thrombotic drugs. Attached Figure Description

[0015] Figure 1 Synthetic route for carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val: i) 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, N-methylmaloline; ii) Pd / C, hydrogen. Detailed Implementation

[0016] 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] Following conventional peptide preparation methods, Boc-Val was first coupled with Val-OBzl in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide to prepare Boc-Val-Val-OBzl. Then, the Boc was removed in an ethyl acetate solution of hydrogen chloride to prepare Val-Val-OBzl. The title compound was obtained with a purity higher than 96%, FT-ICR-MS (m / e): 307.2022 [M+H]. + It is used directly in the reaction of Example 2.

[0019] Example 2 Preparation of carbolinohexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl

[0020] At 0 °C, 313 mg (1 mmol) of carbinohexahydropyrazine-1,4-diketone-3-acetic acid, 162 mg (1.2 mmol) of 1-hydroxybenzotriazole, and 247 mg (1.2 mmol) of dicyclohexylcarbodiimide were stirred in 100 mL of anhydrous tetrahydrofuran for 10 minutes. Then, 306 mg (1 mmol) of Val-Val-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) showed the disappearance of carbinohexahydropyrazine-1,4-diketone-3-acetic acid. The reaction mixture was filtered to remove the formed dicyclohexylurea precipitate. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was washed successively 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 easily with 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 give 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 carbolinohexahydropyrazine-1,4-dione-3-acetyl-Val-Val

[0022] Carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val-OBzl (601 mg, 1.0 mmol) was dissolved in methanol, 75 mg of Pd / C was added, and hydrogen gas was introduced for hydrogenolysis for 48 hours. Pd / C was filtered off, and methanol was removed by concentration under reduced pressure. The resulting brownish-red powder was washed with diethyl ether (30 mL × 3). 419 mg (82%) of the standard extract was obtained as a colorless solid. Mp: 189-191 °C; =-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 evaluates the antivenous thrombotic activity of carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Val-Val.

[0024] This invention evaluates the antithrombotic activity of carboplatin-1,4-dione-3-acetyl-Val-Val in a rat inferior vena cava ligation model. Male SD rats (250±20g) were kept at rest and fasted for one day at 25-28℃. They were then randomly divided into a positive control group, a blank control group, and a carboplatin-1,4-dione-3-acetyl-Val-Val group. Warfarin, a clinically used antithrombotic drug, was selected as the positive control at an oral dose of 4.87 μmol / kg in 11 rats; physiological saline was selected as the blank control at an oral dose of 10 mL / kg in 11 rats; and carboplatin-1,4-dione-3-acetyl-Val-Val was administered at an oral dose of 0.01 nmol / kg in 11 rats.

[0025] During the evaluation, rats were administered the drug orally in a double-blind, sequential manner at the prescribed dose. Thirty minutes after drug administration, 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. The incision extended from the coagulation gland to expose a corner of the liver. Organs such as the small intestine were removed from the abdominal cavity and wrapped with gauze soaked in physiological saline. The perivascular connective tissue was bluntly dissected to expose the inferior vena cava and its branches. The abdominal aorta and inferior vena cava were separated below the renal vein, and the inferior vena cava was ligated at the junction of the inferior vena cava and the left renal vein using sutures soaked in physiological saline. Organs such as the intestines, which had been removed from the abdominal cavity and wrapped with gauze soaked in physiological saline, were returned to the abdominal cavity according to their anatomical positions, and the abdominal cavity was sutured layer by layer. Because warfarin-treated rats were at risk of death at any time, the abdominal cavity was opened 30 minutes after suturing. The branches were ligated one by one, and a 2 cm section of the inferior vena cava was removed from the ligation site at the junction of the inferior vena cava and the left renal vein. The thrombus was then removed from the inferior vena cava and weighed. The data in Table 1 show that the 0.01 nmol / kg oral dose of carboplatin-1,4-dione-3-acetyl-Val-Val had significantly stronger antithrombotic activity than normal saline (p<0.01). Table 1 also shows that there was no significant difference in the antithrombotic activity of the 0.01 nmol / kg oral dose of carboplatin-1,4-dione-3-acetyl-Val-Val compared to that of the 4.87 μmol / kg oral dose of warfarin (p>0.05). For venous thrombosis, carbophenazo-1,4-dione-3-acetyl-Val-Val has unexpected technical effects.

[0026] Table 1. Antivenous thrombotic activity of carbophenazopyrazine-1,4-dione-3-acetyl-Val-Val.

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

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

Claims

1. Carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val, with 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 undergoes debenzylation to generate carboline hexahydropyrazine-1,4-dione-3-acetyl-Val-Val.

3. The use of the carboline-hexahydropyrazine-1,4-dione-3-acetyl-Val-Val as described in claim 1 in the preparation of an anti-venous thrombotic drug.

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