Carbolinohexahydropyrazine-1, 4-diketone-3-acetyl-Gly-Gly, and preparation and application of carbolinohexahydropyrazine-1, 4-diketone-3-acetyl-Gly-Gly

Abstract

The invention discloses a carboline and hexahydropyrazine-1, 4-diketone-3-acetyl-Gly-Gly compound with a structure shown in the following formula, discloses a preparation method of the compound, and further confirms that the carboline and hexahydropyrazine-1, 4-diketone-3-acetyl-Gly-Gly compound has excellent anti-inflammatory activity. Therefore, the invention provides an effective technical means for treating inflammation. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound, a preparation method thereof, and an anti-inflammatory activity thereof. 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 The concentration of M could reduce the amount of P-selectin expressed by activated platelets in rats 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 (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 has realized that shortening the oligopeptide sequence in the carboline hexahydropyrazine-1,4-dione-3-acetyl oligopeptide can reduce production costs and promote the industrialization process. Based on these understandings, the applicant prepared the carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound of the present invention.

[0006] The applicant screened the anti-arterial thrombotic effect, thrombolytic effect, anti-venous thrombotic effect, anti-tumor effect and anti-inflammatory effect of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly. The screening found that the carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly of the present invention showed excellent anti-inflammatory 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 identify a carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly compound and a method for preparing carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly, and further evaluate the anti-inflammatory activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly. In order to achieve the above purpose, the present invention adopts the following technical means.

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

[0009]

[0010] The second technical means is to propose a method for preparing carboline hexahydropyrazine-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 is coupled with Gly-Gly-OBzl to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly-OBzl;

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

[0014] The third technical means is to evaluate the anti-inflammatory activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly in the S180 mouse model, and confirm the effective application of the carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly in the preparation of anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The synthetic route of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly is as follows: i) 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, N-methylpiperidin; 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 Gly-Gly-OBzl

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

[0019] Example 2 Preparation of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly-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, 222 mg (1 mmol) of Gly-Gly-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 resulting 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 easily 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 486 mg (94%) of the title compound as a colorless solid. Mp: 191-193°C; =-92 (c = 0.5, methanol); Mp: 157-158 ° C; =-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 carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly

[0022] Dissolve carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly-OBzl (486 mg, 0.94 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). 369 mg (92%) of the title compound is 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 Evaluation of the anti-inflammatory activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly

[0024] The anti-inflammatory effect of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly was evaluated in the xylene-induced mouse ear swelling model. ICR male mice (weight 25±2g) were rested at room temperature 25℃ for 2 days with free access to water and food, and randomly divided into groups, with 10 mice in each group. The mice were orally administered with normal saline (0.3mL / 100g), aspirin suspension in normal saline (dose 167μmol / kg), or carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly in normal saline (dose 0.01nmol / kg). 30 minutes after oral administration, 30μL of xylene was evenly applied to the left auricle of the mouse. Two hours later, the mouse was anesthetized with ether and killed by breaking the cervical vertebra. The left and right ears were cut off, and a round ear piece was taken at the same position of the two ears with a 7mm diameter puncher. The weight difference between the two ears was used as the swelling degree. That is, the degree of swelling = the weight of the left ear disc – the weight of the right ear disc.

[0025] The data in Table 1 show that an oral dose of 0.01 nmol / kg of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly effectively inhibits the inflammatory response induced by xylene (P<0.01 compared with saline). The data in Table 1 also show that the anti-inflammatory activity of an oral dose of 0.01 nmol / kg of carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly is significantly stronger than the anti-inflammatory activity of an oral dose of 167 μmol / kg of aspirin (P<0.01 compared with aspirin). Carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly has an outstanding technical effect.

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

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

[0028] a) P<0.01 compared with normal saline and aspirin; n=10.

Claims

1. Carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly of the following structure, 2. The method for preparing carboline hexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly according to claim 1, characterized in that: The method comprises the steps of: (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. Use of the carbolinohexahydropyrazine-1,4-dione-3-acetyl-Gly-Gly according to claim 1 in the preparation of anti-inflammatory drugs.

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