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

Abstract

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

Description

Technical Field

[0001] The present invention relates to a carbazolohexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala compound, its preparation method, and its anti-tumor activity. The present invention belongs to the field of biomedicine. Background Art

[0002] The applicant has previously disclosed that carbazolohexahydropyrazine-1,4-dione-3-acetic acid of the following formula can reduce the amount of P-selectin expressed by activated platelets in rats from 210.55±2.98 ng / mL to 110.12±3.96 -8 ng / mL (p<0.001) at a concentration of 10

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

[0004]

[0005] The applicant has also previously disclosed that carbazolohexahydropyrazine-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 thrombosis activity. On the one hand, the applicant realizes that the in vivo P-selectin expression level is related to the pathology of arterial thrombosis, venous thrombosis, tumors and inflammation. In addition to showing anti-arterial thrombosis effects, carbazolohexahydropyrazine-1,4-dione-3-acetyl oligopeptides may also show thrombolytic effects, anti-venous thrombosis effects, anti-tumor effects and anti-inflammatory effects. On the other hand, the applicant realizes that shortening the oligopeptide sequence in carbazolohexahydropyrazine-1,4-dione-3-acetyl oligopeptides can reduce production costs and promote the industrialization process. Based on these understandings, the applicant has prepared the carbazolohexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala of the present invention.

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

[0008] The first technical means is to propose carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala having the following formula structure,

[0009]

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

[0011] 1 Coupling carboline hexahydropyrazine-1,4-dione-3-acetic acid with Ala-Ala-OBzl to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala-OBzl;

[0012] 2 Debenzylating carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala-OBzl to prepare carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala.

[0013] The third technical means is to evaluate the anti-tumor activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala on S180 mouse model, and confirm its effective application in the preparation of anti-tumor drugs. Description of the Drawings

[0014] Figure 1 Synthesis route for carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala: i) 1-hydroxybenzotriazole, dicyclohexylcarbodiimide, N-methylmorpholine; ii) Pd / C, hydrogen. Detailed Description of the Invention

[0015] 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.

[0016] Example 1 Preparation of Ala-Ala-OBzl

[0017] According to the conventional peptide preparation method, in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, Boc-Ala was first coupled with Ala-OBzl to prepare Boc-Ala-Ala-OBzl, and then Boc was removed in a hydrogen chloride ethyl acetate solution to prepare Ala-Ala-OBzl. The title compound was obtained with a purity of more than 96%, FT-ICR-MS (m / e): 251.1396 [M+H] + , directly used in the reaction of Example 2.

[0018] Example 2 Preparation of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala-OBzl

[0019] 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, 251 mg (1 mmol) of Ala-Ala-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 512 mg (94%) of the title compound as a colorless solid. Mp: 191-193°C; =-92 (c = 0.5, methanol); FT-ICR-MS (m / e): 546.2353 [M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=11.63(s,1H),8.33(s,1H),8.32(s,1H),8.21(s,1H),7.35(t,J=7.5Hz,1H),7.34(d,J=7.5H z,1H),7.33(t,J=7.5Hz,1H),7.32(t,J=7.5Hz,1H),7.31(d,J=7.5Hz,1H),7.30(d,J=7.5Hz,1H),7.29(d,J=7.5Hz,1H),7.0 2(t,J=7.5Hz,1H),6.99(t,J=7.5Hz,1H),5.33(s,2H),5.31(t,J=6.8Hz,1H),4.81(t,J=6.6Hz,1H),4.52(q,J=6.5Hz,1H),4 .51(q,J=6.4Hz,1H),4.46(s,2H),3.18(d,J=6.8Hz,2H),2.97(d,J=6.6Hz,2H),1.36(d,J=6.5Hz,3H),1.35(d,J=6.4Hz,3H). 13 CNMR (125MHz, DMSO-d6): δ / ppm=174.56,171.66,168.94,166.29,165.89,136.44,136.12,130.29,129.21,129.20,127.56,127.21 ,127.11,126.87,121.47,119.17,118.08,111.57,106.31,74.36,56.24,52.16,67.06,47.99,36.96,36.11,21.67,17.84,17.20.

[0020] Example 3 Preparation of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala

[0021] Dissolve carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala-OBzl (512 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). 402 mg (94%) of the target compound is a colorless solid. Mp: 181-182℃; FT-ICR-MS(m / e):456.1883[M+H] + ; 1HNMR (500MHz, DMSO-d6): δ / ppm=12.64(s,1H),10.93(s,1H),8.33(s,1H),8.32(s,1H),8.20(s, 1H),7.34(d,J=7.5Hz,1H),7.19(d,J=7.5Hz,1H),7.07(t,J=7.5Hz,1H),6.92(t,J=7.5Hz,1H),5 .39(t,J=6.8Hz,1H),5.01(t,J=6.6Hz,1H),4.91(q,J=6.5Hz,1H),4.52(q,J=6.4Hz,1H),4.19(s ,2H),3.18(d,J=6.8Hz,2H),2.97(d,J=6.6Hz,2H),1.56(d,J=6.5Hz,3H),1.54(d,J=6.4Hz,3H); 13 CNMR (125MHz, DMSO-d6): δ / ppm=174.63,174.56,168.94,166.19,165.89,136.44,130.29,126.87,121 .47,119.17,118.08,111.57,106.31,74.33,56.24,52.16,49.06,37.35,36.21,21.75,17.84,17.02.

[0022] Example 4 Evaluation of the antitumor activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala

[0023] ICR male mice, weighing 20±2g, were randomly divided into groups. There were 10 mice in each group. The mice were inoculated with S180 ascites tumor subcutaneously in the armpit. Under sterile conditions, the tumor fluid of the vigorously growing S180 ascites tumor was extracted, diluted with physiological saline to a 1:2 liquid and then fully mixed. The obtained tumor cell suspension was stained with freshly prepared 0.2% trypan blue, mixed and counted according to the white blood cell counting method. Those stained blue were dead cells, and those not stained were living cells. The cell concentration and cell survival rate were calculated according to the formula. That is, cell concentration = number of living cells in 4 large squares / 4×10 4 × dilution factor = number of cells / mL. Cell viability = number of live cells / (number of live cells + number of dead cells) × 100%. Tumor fluid with a viability greater than 90% was homogenized to prepare 2.0 × 10 7 The cell suspension of cells / mL was inoculated under the armpit skin of each mouse at a rate of 0.2 mL to create the S180 ascites solid tumor mouse model.

[0024] After mice were inoculated with S180 tumor cell suspension, they were given oral saline (dose 0.3 mL / 100 g / day, once a day for 11 consecutive days), or intraperitoneal injection of a solution of doxorubicin and saline (dose 2 μmol / kg / day, once a day for 11 consecutive days), or oral administration of a solution of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala and saline (dose 0.01 nmol / kg / day, once a day for 11 consecutive days). On the 12th day, the body weight of the mice was measured, and after anesthesia with ether, they were sacrificed by cervical dislocation. The right axillary tumor growth site of the mice was fixed with forceps, the skin was cut open, the tumor was exposed, and blunt dissection was performed to weigh it. The anti-tumor activity was expressed by the tumor weight.

[0025] The data in Table 1 showed that carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala at an oral dose of 0.01 nmol / kg / day effectively inhibited tumor growth (P < 0.01 compared with saline). The data in Table 1 also showed that the anti-tumor activity of carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala at an oral dose of 0.01 nmol / kg / day was significantly stronger than that of doxorubicin at an intraperitoneal injection dose of 2 μmol / kg (P < 0.01 compared with doxorubicin). Carboline hexahydropyrazine-1,4-dione-3-acetyl-Ala-Ala at an oral dose of 0.01 nmol / kg / day had outstanding technical effects.

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

[0027]

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

Claims

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

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

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