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

Abstract

This invention discloses a carbazohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound with the following structure, discloses a method for preparing the compound, and further confirms that the carbazohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound possesses excellent anti-inflammatory activity. Therefore, this invention provides an effective technical means for treating inflammation.

Description

Technical Field

[0001] This invention relates to a carbazohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound, its preparation method, and its anti-inflammatory activity. 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 expression of P-selectin in activated rat platelets decreased from 210.55±2.98 ng / mL to 110.12±3.96 ng / mL.

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

[0004]

[0005] 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. The applicant, on the other hand, recognized that shortening the oligopeptide sequence in carboline-hexahydropyrazine-1,4-dione-3-acetyl oligopeptide could reduce production costs and promote industrialization. Based on these findings, the applicant prepared the carboline-hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound of the present invention.

[0006] The applicant screened carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly for its anti-arterial thrombotic, thrombolytic, anti-venous thrombotic, antitumor, and anti-inflammatory effects. Screening revealed that carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly of the present invention exhibits excellent anti-inflammatory activity. Based on this finding, the applicant has proposed this invention. Summary of the Invention

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

[0008] The first technical approach is to propose a carbazohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound with the following structure.

[0009]

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

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

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

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

[0014] The third technical approach involved evaluating the anti-inflammatory activity of carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly in the S180 mouse model. This confirmed the effective application of carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly in the preparation of anti-inflammatory drugs. Attached Figure Description

[0015] Figure 1 Synthetic route for carboline-hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly: 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 Gly-Gly-OBzl

[0018] Following conventional peptide preparation methods, Boc-Gly was first coupled with Gly-OBzl to prepare Boc-Gly-Gly-OBzl in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide. Then, the Boc was removed in an ethyl acetate solution of hydrogen chloride to prepare Gly-Gly-OBzl. The title compound was obtained with a purity higher than 96%, FT-ICR-MS (m / e): 223.1083 [M+H]. + It is used directly in the reaction of Example 2.

[0019] Example 2 Preparation of carbolinohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly-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, 222 mg (1 mmol) of Gly-Gly-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 486 mg (94%) of the title compound as a colorless solid. MP: 191-193℃; =-92 (c=0.5, methanol); Mp: 157-158℃; = -65 (c = 0.5, methanol); FT-ICR-MS (m / e): 518.2040 [M+H] + ; 1 H NMR (500MHz, DMSO-d6): δ / ppm=11.95(s,1H),9.05(s,1H),9.04(s,1H),8.46(s,1H),7.44(d,J=7.5Hz,1H), 7.35(t,J=7.5Hz,1H),7.34(t,J=7.5Hz,1H),7.33(d,J=7.5Hz,1H),7.32(d,J=7.5Hz,1H),7.31(d,J=7.5Hz, 1H),7.29(t,J=7.5Hz,1H),7.07(t,J=7.5Hz,1H),6.99(t,J=7.5Hz,1H),5.32(s,2H),5.40(t,J=6.5Hz,1H) ,5.08(s,2H),5.05(t,J=6.4Hz,1H),4.25(s,2H),4.17(s,2H),3.87(d,J=6.5Hz,2H),2.82(d,J=6.4Hz,2H); 13 CNMR (125MHz, DMSO-d6): δ / ppm=173.10,170.18,169.86,165.59,164.97,136.44,136.32,130.29,128.48,128.44,128.41, 127.68,127.23,127.02,121.45,119.15,118.07,111.55,106.32,75.21,66.30,56,20,52.10,48.21,37.52,35.68,22.11.

[0021] Example 3 Preparation of carbolinohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly

[0022] Carboline-hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly-OBzl (486 mg, 0.94 mmol) was dissolved in methanol, 75 mg of Pd / C was added, and hydrogen gas was bubbled through the solution for hydrogenolysis over 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). 369 mg (92%) of the title compound was obtained. Mp: 157-158 °C; = -65 (c = 0.5, methanol); FT-ICR-MS (m / e): 428.1570 [M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=13.11(s,1H),11.54(s,1H),9.04(s,1H),9.03(s,1 H),8.46(s,1H),8.229(m,1H),7.43(d,J=7.5Hz,1H),7.32(d,J=7.5Hz,1H),7.01(t ,J=7.5Hz,1H),6.93(t,J=7.5Hz,1H),5.41(t,J=6.5Hz,1H),5.08(t,J=6.4Hz,1H), 4.25 (s, 2H), 4.17 (s, 2H), 4.16 (s, 2H), 3.16 (d, J = 6.5Hz, 2H), 3.02 (d, J = 6.4Hz, 2H). 13 CNMR (125MHz, DMSO-d6): δ / ppm=172.49,170.18,169.86,166.59,165.97,136.44,130.29,128. 48,121.45,119.15,118.07,111.55,106.32,66.30,56,20,52.10,50.13,35.51,26.66,21.67.

[0023] Example 4 evaluates the anti-inflammatory activity of carboline-hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly.

[0024] The anti-inflammatory effect of carboplatin-1,4-dione-3-acetyl-Gly-Gly was evaluated in a xylene-induced mouse ear swelling model. Male ICR mice (weighing 25 ± 2 g) were kept at room temperature (25 °C) for 2 days with free access to water and food, and then randomly divided into groups of 10 mice each. Mice were administered oral saline (0.3 mL / 100 g), an aspirin and saline suspension (167 μmol / kg), or a carboplatin-1,4-dione-3-acetyl-Gly-Gly and saline solution (0.01 nmol / kg). Thirty minutes after oral administration, 30 μL of xylene was evenly applied to the left auricle of the mice. Two hours later, the mice were euthanized by ether anesthesia and cervical vertebrae were broken. Both ears were removed, and circular ear pieces were taken from the same location on both ears using a 7 mm diameter punch. The weight difference between the two ears was used as the degree of swelling. That is, the degree of swelling = weight of the left ear disc - weight of the right ear disc.

[0025] The data in Table 1 show that carborohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly, administered orally at a dose of 0.01 nmol / kg, effectively inhibited xylene-induced inflammatory responses (P<0.01 compared to saline). Table 1 also shows that the anti-inflammatory activity of carborohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly at an oral dose of 0.01 nmol / kg was significantly stronger than that of aspirin at an oral dose of 167 μmol / kg (P<0.01 compared to aspirin). Carborohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly demonstrates outstanding technical efficacy.

[0026] Table 1. Anti-inflammatory activity of carbophenazopyrazine-1,4-dione-3-acetyl-Gly-Gly.

[0027] Therapeutic agents dose Ear swelling degree, mean ± SD mg physiological saline 10mL / kg 9.99±1.23 aspirin 167 μmol / kg 6.65±1.10 Carbophyllohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly 0.01 nmol / kg <![CDATA[3.55±0.39 a ]]>

[0028] a) Compared with physiological saline and aspirin, P<0.01; n=10.

Claims

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

3. The use of the carboline-hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly as described in claim 1 in the preparation of anti-inflammatory drugs.

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