CN112010930B - RGD-modified pentacyclic piperazine dione and its preparation and application - Google Patents

Abstract

The present invention discloses the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione represented by general formula I, discloses its synthesis method, discloses its in vitro anti-platelet aggregation activity, and further discloses its in vitro anti-platelet aggregation activity. The anti-arterial thrombosis activity of the rat jugular artery and vein bypass cannulation thrombosis model and the anti-venous thrombosis activity of the rat inferior vena cava ligation model, the activity of reducing serum GPIIb/IIIa content is disclosed, and it is disclosed that it reduces serum P- Selectin content activity. Therefore, the present invention discloses its application in the preparation of anti-arterial thrombosis drugs, its application in the preparation of anti-venous thrombosis drugs and its application in the preparation of drugs with both anti-arterial thrombosis and anti-venous thrombosis functions.

Description

RGD-modified pentacyclic piperazine dione and its preparation and application

technical field

The present invention relates to pentacyclic piperazine dione modified by CH ₂ CO-Arg-Gly-Asp-AA, to its synthesis method, to its anti-platelet aggregation activity, and to its thrombus in rat jugular arteriovenous bypass intubation. The anti-arterial thrombosis activity of the formed model relates to its anti-venous thrombosis activity in a rat inferior vena cava ligation model, to its activity to reduce serum GPIIb/IIIa content, and further to its activity to reduce serum P-selectin content. Therefore, the present invention relates to its application in the preparation of anti-arterial thrombosis drugs, its application in the preparation of anti-venous thrombosis drugs, and its application in the preparation of drugs with dual effects of anti-arterial thrombosis and anti-venous thrombosis. The invention belongs to the field of biomedicine.

Background technique

Arterial embolism has become one of the diseases with high morbidity and mortality. Arterial thrombosis is responsible for transient ischemic attack, acute coronary syndrome, myocardial infarction and atrial fibrillation. Coronary artery disease is present in 18%-47% of patients with atrial fibrillation, and approximately 20% of patients with atrial fibrillation associated with coronary artery disease receive percutaneous coronary intervention. Arterial thrombosis is also responsible for prosthetic heart valves, arteriovenous fistulas and other postoperative arterial thrombosis and unstable angina. For example, after liver transplantation, patients face the risk of hepatic arterial thrombosis. In addition, patients with antiphospholipid syndrome also face the risk of arterial thrombosis. Although the association between tumors and venous thrombosis is broader than that of arterial thrombosis, there is a growing understanding of the pathogenesis of arterial thrombosis, including peripheral arterial thrombosis, in specific malignancies and tumor treatment. Arterial cannulation and ischemic stroke have contributed to the increasing incidence of arterial thrombosis in children. The risk of arterial thrombosis associated with cocaine abuse was raised more than a decade ago.

Venous thrombosis has become one of the diseases with high morbidity and mortality. The most serious venous thrombosis are mainly cerebral venous thrombosis and lower extremity deep venous thrombosis. Cerebral venous thrombosis arises from dural venous sinus thrombosis and/or cerebral vein vein thrombosis, leading to cerebral venous blockage, increased intracranial pressure, cerebral ischemia or intracranial hemorrhage. The standard treatment strategy for cerebral venous embolism is oral anticoagulants. The incidence of upper/lower extremity deep vein thrombosis and pulmonary embolism is 0.1-0.2%. Upper/lower extremity deep vein thrombosis and pulmonary embolism share similar risk factors. Although pulmonary embolism is a common complication of upper/lower extremity deep vein thrombosis, pulmonary embolism is not easy to diagnose. Because there are no characteristic clinical signs of pulmonary embolism. A 20-year study of the medical conditions of more than 42 million people who died found that about 1.5 percent of them suffered from blood clots in the lungs. Pulmonary embolism is the cause of 200,000 deaths. Another study found that more than 50% of patients with deep vein thrombosis of the upper/lower extremities confirmed by angiography also had pulmonary embolism. Upper/lower extremity deep vein thrombosis can affect the elderly, young people and children. A large number of clinical studies have proved that cancer patients are commonly complicated by upper/lower extremity deep venous thrombosis, and patients undergoing artificial heart valve surgery, venous fistula surgery and other surgeries, as well as organ transplantation are at risk of venous thrombosis. In addition, venous cannulation and ischemic stroke have increased the incidence of venous thrombosis in children.

Because of different etiologies, venous thrombosis and arterial thrombosis are regarded as two different diseases in the traditional concept. Recent epidemiological studies have shown that the association between venous thrombosis and arterial thrombosis is difficult to disentangle. This situation can be attributed to their overlapping risk factors. As a result, more and more attention has been paid to the prevention and treatment of venous thrombosis.

Direct oral anticoagulants are the only strategy for clinical treatment of arterial and venous thrombosis. Although oral anticoagulants are effective against arterial and venous thrombosis, they all have bleeding side effects. For example, at effective oral doses, aspirin can induce gastrointestinal or intracranial hemorrhage. This risk greatly limits the benefits to patients. There is a clinical need for drugs that are comparable to aspirin in efficacy without the risk of aspirin-like gastrointestinal bleeding or intracranial hemorrhage. In response to this clinical demand, researchers at home and abroad have paid a lot of effort. However, no substantial progress has been made.

In the research of antithrombotic drugs, the inventors found that the pentacyclic piperazine dione on the left side of the following formula can inhibit arterial thrombosis and venous thrombosis. In the follow-up research, the inventors further found that the CH ₂ CO-Arg-Gly-Asp on the right side of the following formula was generated by introducing CH ₂ CO-Arg-Gly-Asp-AA on the pyrrole nitrogen of the pentacyclic piperazine dione on the left side of the following formula -AA-modified pentacyclic piperazine dione has more excellent anti-arterial thrombotic activity and anti-venous thrombotic activity. Based on these findings, the inventors have come up with the present invention.

SUMMARY OF THE INVENTION

The first aspect of the present invention is to provide a CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione of the following formula.

The second content of the present invention is to provide the preparation method of the pentacyclic piperazine dione modified by CH ₂ CO-Arg-Gly-Asp-AA, the method comprising:

1) L-tryptophan is carried out Pictet-Spengler condensation with formaldehyde under vitriol oil catalysis, obtains (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid;

2) react (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid with di-tert-butyl dicarbonate to obtain N-tert-butoxycarbonyl-(3S)-1 ,2,3,4-tetrahydro-β-carboline-3-carboxylic acid;

3) L-hydroxyproline is reacted with thionyl chloride and methanol to obtain hydroxyproline methyl ester;

4) Synthesis of 3S-N-Boc-1,2,3,4-tetrahydro-β-carboline- 3-formyl-hydroxyproline methyl ester;

5) De-Boc of 3S-N-Boc-1,2,3,4-tetrahydro-β-carboline-3-formyl-hydroxyproline methyl ester in ethyl acetate solution of hydrogen chloride to obtain 3S-1 , 2,3,4-tetrahydro-β-carboline-3 acyl-hydroxyproline methyl ester;

6) Preparation of 2-hydroxyoctahydro from 3S-1,2,3,4-tetrahydro-β-carboline-3-formyl-hydroxyproline in the presence of N-methylmorpholine in methanol solvent Pyrrolopyrazinopyridoindolediones;

7) Preparation of 2-hydroxyoctahydropyrrolopyrazinopyrido by the reaction of 2-hydroxyoctahydropyrrolopyrazinopyridoindole with benzyl bromoacetate in dimethylformamide under the catalysis of sodium hydride benzyl indoledione-11-yl acetate;

8) Hydrogenolysis of benzyl 2-hydroxyoctahydropyrrolopyrazinopyridoindoledione-11-yl acetate to 2-hydroxyoctahydropyrrolopyrazino in methanol and dichloromethane with palladium-carbon catalysis pyridoindoldione-11-ylacetic acid;

9) Coupling of 2-hydroxyoctahydropyrrolopyrazinopyridoindoledione-11-ylacetic acid with Arg(NO ₂ )-Gly-Asp(OBzl)-AA-OBzl to prepare 2-hydroxyoctahydropyrrolo Pyrazinopyridoindoldione _- 11-yl- _CH2CO -Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl;

10) 2-Hydroxyoctahydropyrrolopyrazinopyridoindoldione-11-yl-CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-AA-Obzl acid was removed to prepare CH ₂ CO -Arg-Gly-Asp-AA modified pentacyclic piperazine dione.

The third aspect of the present invention is to evaluate the anti-platelet aggregation activity of the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione.

The fourth aspect of the present invention is to evaluate the anti-arterial thrombosis activity of the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione.

The fifth aspect of the present invention is to evaluate the anti-intravenous activity of the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione.

Description of drawings

Figure 1. Synthesis scheme of CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione, i: formaldehyde, 98% H ₂ SO ₄ ; ii: (Boc) ₂ O, dimethylformamide , triethylamine; iii: dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, tetrahydrofuran, N-methylmorpholine; iv: hydrogen chloride solution in ethyl acetate (4M), 0°C; v: methanol , N-methylmorpholine; vi: sodium hydride, dimethylformamide, benzyl bromoacetate; vii: hydrogen, Pd/C, methanol; viii: methanol, aqueous sodium hydroxide (2M); ix: bicyclic Hexylcarbodiimide, 1-hydroxybenzotriazole, dimethylformamide, N-methylmorpholine; x: trifluoroacetic acid, trifluoromethanesulfonic acid, diethyl ether.

Detailed ways

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

Example 1 Preparation of (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1)

0.2 mL of concentrated _H2SO4 was diluted with ₂₀₀ mL of distilled water at 0 °C. 2.04g (10mmol) of L-tryptophan was added inside, and the L-Trp was fully dissolved by stirring. To the obtained solution, 5 mL of aqueous formaldehyde solution (37%) was added dropwise, and the mixture was stirred at room temperature for 6 hours. TLC (ethyl acetate/H ₂ O/acetic acid=4/1/1) showed that L-Trp disappeared. 5 mL of concentrated ammonia water was added to the reaction mixture at 0° C. to adjust the pH to 7, and the mixture was allowed to stand still and filtered to obtain 2.05 g (95%) of the title compound as a yellow solid. ESI-MS (m/e): 217 [M+H] ⁺ .

Example 2 Preparation of Boc-(3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2)

2.16g (10mmol) of (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1) was dissolved in 25mL of dimethylformamide to obtain the dimethylformamide of compound 1. The formamide solution was adjusted to pH 10 with triethylamine at 0°C. 2.62 g (12 mmol) of (Boc) ₂ O was dissolved in 5 mL of dimethylformamide and added to a solution of compound 1 in dimethylformamide. The resulting mixed solution was adjusted to pH 10 with triethylamine and stirred at room temperature for 100 hours. TLC ( _CH2Cl2 /CH3OH ₌ 15/ ₁ ) showed that compound 1 disappeared. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 100 mL of ethyl acetate. The obtained solution was washed with 5% KHSO ₄ aqueous solution (50 mL×3) and saturated NaCl aqueous solution (50 mL×3) successively. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ for 12 h, filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in 30 mL of ethyl acetate and allowed to stand for 6 hours to allow the solid to separate out. Filtration gave 1.98 g (62%) of the title compound as a pale yellow solid. ESI-MS (m/e): 317 [M+H] ⁺ .

Example 3 Preparation of Hyp-OMe

1.3 mL of thionyl chloride was slowly added dropwise to 20 mL of methanol at 0 °C, and stirred for 30 min. Then, 655 mg (5 mmol) of Hyp was added thereto, and the mixture was stirred at room temperature for 50 hours. TLC ( _CH2Cl2 /CH3OH ₌ 15/ ₁ ) showed disappearance of Hyp. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 10 mL of methanol and concentrated under reduced pressure. This operation is repeated 3 times. The residue was sonicated in 5 mL of anhydrous ether to make the dispersion uniform. Set aside, discard ether. This operation is repeated 3 times. 0.90 g (99%) of the title compound was obtained as a colorless solid. ESI-MS (m/e): 146 [M+H] ⁺ .

Example 4 Preparation of Boc-(3S)-1,2,3,4-tetrahydro-β-carboline-3-formyl-Hyp-OMe(3)

822 mg (2.6 mmol) of Boc-(3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2) were dissolved in 15 mL of anhydrous tetrahydrofuran. To the solution at 0° C., 324 mg (2.4 mmol) of 1-hydroxybenzotriazole and 494 mg (2.4 mmol) of dicyclohexylcarbodiimide were successively added. Stir for 30 min. After that, 363 mg (2 mmol) of Hyp-OMe was added and the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, stirred at room temperature for 10 h, TLC (CH ₂ Cl ₂ /CH ₃ OH=45/1) showed that Hyp-OMe disappeared. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in 50 mL of ethyl acetate. The obtained solution was washed with saturated NaHCO ₃ aqueous solution (30 mL×3), saturated NaCl aqueous solution (30 mL×3), 5% KHSO ₄ aqueous solution (30 mL×3), saturated NaCl aqueous solution (30 mL×3), saturated NaCl aqueous solution (30 mL×3), and saturated aqueous solution. Washed with NaHCO ₃ aqueous solution (30 mL×3) and saturated NaCl aqueous solution (30 mL×3). The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ for 12 h, filtered, and the filtrate was concentrated under reduced pressure. The obtained yellow oil was purified by silica gel column chromatography, eluted with a gradient of dichloromethane-methanol elution system (CH ₂ Cl ₂ /CH ₃ OH=150/1-45/1). Obtained 450 mg (51%) of the title compound as a yellow solid. ESI-MS (m/e): 444 [M+H] ⁺ .

Example 5 Preparation of (3S)-1,2,3,4-tetrahydro-β-carboline-3-formyl-Hyp-OMe(4)

350 mg (0.79 mmol) of Boc-(3S)-1,2,3,4-tetrahydro-β-carboline-3-formyl-Hyp-OMe(3) were dissolved in 4 mL of hydrogen chloride in ethyl acetate ( 4M) was dissolved, stirred for 1 h, TLC (CH ₂ Cl ₂ /CH ₃ OH=45/1) showed that 3 disappeared. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 10 mL of ethyl acetate. The solution was concentrated under reduced pressure, and the residue was dissolved in 10 mL of ethyl acetate. This operation is repeated 3 times. The residue was sonicated in 10 mL of anhydrous ether to make the dispersion uniform. Set aside, discard ether. This operation is repeated 3 times. Obtained 286 mg (95%) of the title compound as a yellow solid. ESI-MS (m/e): 344 [M+H] ⁺ .

Example 6 Preparation of (2S,5aS,14aS)-2-hydroxy-1,2,3,5a,6,11,12,14a-octahydro-5H,14H-pyrrolo[1",2":4' ,5']pyrazino[1',2':1,6]pyrido[3,4-b]indole-5,14-dione (5)

200 mg (0.53 mmol) of (3S)-1,2,3,4-tetrahydro-β-carboline- ₃ -formyl-Hyp-OMe (4) were dissolved in 4 mL of CH3OH at 0°C. The solution was adjusted to pH 9 with N-methylmorpholine and stirred at room temperature for 5h. During this period, a pale yellow solid gradually precipitated out of the reaction solution. Filtration gave 104 mg (67%) of the title compound. Mp 204-205°C; ESI-MS (m/e): 312[M+H] ⁺ ; ¹ H-NMR (300MHz, DMSO-d6): δ/ppm=10.948(s, 1H), 7.481(d, J=7.5Hz, 1H), 7.336(d, J=7.8Hz, 1H), 7.070(t, J=7.5Hz, 1H), 6.992(t, J=7.8Hz, 1H), 5.209(d, J= 2.7Hz, 1H), 4.498(m, 1H), 4.459-4.343(m, 4H), 3.807(dd, J ₁ = 4.8 Hz, J ₂ = 12.9 Hz, 1H), 3.401 (d, J = 4.5 Hz, 1H), 3.231(m, 1H), _2.744 (m, 1H), 2.155(dd, J1 ₌ 6.0Hz, J2=12.3Hz, 1H), 1.992(dd, J1 ₌ 4.5Hz, _J2 =12.3 Hz,1H);

Example 7 Preparation of (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrolo[1 ",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid benzyl ester (6)

300 mg (0.96 mmol) of (2S,5aS,14aS)-2-hydroxy-1,2,3,5a,6,11,12,14a-octahydro-5H,14H-pyrrolo[1",2": 4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indole-5,14-dione (5) with 13 mL of anhydrous dimethylformamide dissolve. Add 77 mg (1.93 mmol) of sodium hydride to the obtained solution at 0° C., and stir for 5 min. After that, add 200 μL of benzyl bromoacetate and stir at room temperature for 4 h. After that, 100 μL of benzyl bromoacetate was added and stirred at room temperature for 3 h. TLC (CH ₂ Cl ₂ /CH ₃ OH=20/1) showed disappearance of compound 5, terminating the reaction. The reaction solution was added with 15 mL of ice water, and then extracted with ethyl acetate (50 mL×3). The ethyl acetate layer was washed with saturated aqueous NaCl solution (50 mL×3), and dried over anhydrous Na ₂ SO ₄ for 12 h. The filtrate was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with a gradient of dichloromethane-methanol elution system (CH ₂ Cl ₂ /CH ₃ OH=150/1-45/1) to obtain 245 mg ( 55%) the title compound as a yellow solid. ESI-MS (m/e): 460[M+H] ⁺ ; ¹ H-NMR (300MHz, DMSO-d6): δ/ppm=7.535 (d, J=7.2Hz, 1H), 7.452-7.336 (m , 6H), 7.093(m, 2H), 5.212-5.021(m, 6H), 4.498-4.305(m, 4H), 3.800(d, J＝10.8Hz, 1H), 3.445-3.234(m, 2H), 2.767 (m, 1H), 2.165 (m, 1H), 1.971 (m, 1H).

Example 8 Preparation of (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrolo[1 ",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid (7)

100 mg (0.22 mmol) of (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrolo [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid benzyl ester (6) was used 5 mL of methanol and 3 mL of dichloromethane were dissolved. Add 10 mg of Pd/C to the solution, pass hydrogen and stir at room temperature for 2 h, TLC (CH ₂ Cl ₂ /CH ₃ OH=20/1) shows that compound 6 disappears. Pd/C was filtered off, and the filtrate was concentrated under reduced pressure to give 61 mg (76%) of the title compound as a yellow solid. Mp 134-135°C; ESI-MS (m/e): 368 [MH] ⁻ ; ¹ H-NMR (300 MHz, DMSO-d6): δ/ppm=7.501 (d, J=7.5 Hz, 1H), 7.423 (d, J=7.8Hz, 1H), 7.119(t, J=7.8Hz, 1H), 7.043(t, J=7.5Hz, 1H), 5.043-4.939(m, 3H), 4.516-4.305(m, 4H), 3.809(dd, J1 ₌ 4.8Hz, J2=12.6Hz, 1H), _3.413 (dd, J1 ₌ 4.2Hz, J2=11.7Hz, 1H), 3.272(m, 1H), _2.796 ( m, 1H), 2.185 (dd, J ₁ =6.0Hz, J ₂ =12.6Hz, 1H), 1.997 (dd, J ₁ =4.5Hz, J ₂ =12.3Hz, 1H);

Example 9 Preparation of Boc-Arg(NO ₂ )-Gly-OBzl

1.595 g (5 mmol) of Boc-Arg (NO ₂ ) were dissolved in 15 mL of dry tetrahydrofuran. Add 0.75 g (5.56 mmol) of 1-hydroxybenzotriazole and 1.133 g (5.5 mmol) of dicyclohexylcarbodiimide to the obtained solution at 0° C., and stir for 30 min. After that, 1.86 g (6 mmol) of Gly-OBzl was added. The pH of the reaction solution was adjusted to 9 with N-methylmorpholine, and the mixture was stirred at room temperature for 10 h. TLC (dichloromethane/methanol=20/1) showed that the reaction was complete. Filter, and concentrate the filtrate under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate, and the resulting solution was washed with saturated aqueous NaHCO ₃ solution (30 mL × 3), saturated aqueous NaCl solution (20 mL × 3), 5% aqueous KHSO ₄ solution (20 mL × 3), saturated aqueous NaCl solution (20 mL × 3), and saturated aqueous NaCl solution. washed (20 mL×3), saturated NaHCO ₃ aqueous solution (20 mL×3) and saturated NaCl aqueous solution (20 mL×3). The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ for 12 h, filtered, the filtrate was concentrated under reduced pressure, the residue was dissolved in 30 mL of dichloromethane, and allowed to stand for 6 hours to allow solid separation. Filtration gave 1.98 g (85%) of the title compound as a yellow oil. ESI-MS (m/e): 467 [M+H] ⁺ .

Example 10 Preparation of Boc-Arg(NO ₂ )-Gly

Dissolve 1.86g (4mmol) Boc-Arg(NO ₂ )-Gly-OBzl in 45mL methanol, adjust the pH of the reaction solution to 12 with aqueous sodium hydroxide solution (2M) at 0°C, stir for 4h, TLC (dichloromethane/methanol) =20/1) indicates the end of the reaction. The pH was adjusted to 7 with a saturated aqueous KHSO ₄ solution at 0 °C, concentrated under reduced pressure, 15 mL of distilled water was added to the residue, and the pH was adjusted to 2 with a saturated aqueous KHSO ₄ solution at 0 °C. The aqueous layer was extracted with ethyl acetate (20 mL×3), and the ester layer was Extracted and washed with saturated aqueous sodium chloride solution (20 mL×3), the ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ for 12 h, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.27 g (85%) of the title compound as a yellow oil. ESI-MS (m/e): 377 [M+H] ⁺ .

Example 11 Preparation of Boc-Asp(OBzl)-Ser-OBzl

Using the procedure of Example 9, 2.28 g (91%) of the title compound was obtained as a yellow oil from 1.615 g (5 mmol) Boc-Asp(OBzl) and 1.273 g (6 mmol) Ser-OBzl. ESI-MS (m/e): 518 [M+H] ⁺ .

Example 12 Preparation of Asp(OBzl)-Ser-OBzl

1.98g (4mmol) of Boc-Asp(OBzl)-Ser-OBzl was dissolved in 20mL of hydrogen chloride in ethyl acetate solution (4M) by ultrasonication at 0°C and stirred for 1h. TLC (petroleum ether/ethyl acetate = 3/1) showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 15 mL of anhydrous ethyl acetate. The solution was concentrated under reduced pressure, and 10 mL of anhydrous ether was added to the residue for sonication to make a complete suspension. The supernatant was discarded to obtain 1.70 g (97%) of the title compound as a pale yellow solid. ESI-MS (m/e): 418 [M+H] ⁺ .

Example 13 Preparation of Boc-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of Example 9, 2.46 g (65%) of the title compound was obtained as a colorless solid from 1.88 g (5 mmol) Boc-Arg(NO2)-Gly and 2.40 _g (6 mmol) Asp(OBzl)-Ser-OBzl. ESI-MS (m/e): 759 [M+H] ⁺ .

Example 14 Preparation of Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of Example 12, 0.67 g (96%) of the title compound was obtained as a colorless solid from 0.76 g (1 mmol) Boc-Arg(NO2)-Gly _- Asp(OBzl)-Ser-OBzl. ESI-MS (m/e): 659 [M+H] ⁺ .

Example 15 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl(8a)

80 mg (0.22 mmol) of (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrolo [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid (7) was dissolved in 3 mL of Methylformamide dissolves. To the solution at 0° C., 32 mg (0.24 mmol) of 1-hydroxybenzotriazole and 49 mg (0.24 mmol) of dicyclohexylcarbodiimide were successively added. Stir for 30 min. After that, add 166 mg (0.24 mmol) Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl and adjust the pH of the reaction solution to 9 with N-methylmorpholine, stir at room temperature for 10 h, TLC (CH ₂ Cl ₂ /CH ₃ OH=10/1) showed the disappearance of compound 7. DMF was blown dry, and the residue was purified by silica gel column chromatography, eluted with a gradient of dichloromethane-methanol elution system (CH ₂ Cl ₂ /CH ₃ OH=150/1-10/1), to obtain 70 mg (31%) The title compound as a yellow solid. ESI-MS (m/e): 1010 [M+H] ^{+ .1} H-NMR (300 MHz, DMSO-d6): δ/pp=8.606 (d, J=7.5 Hz, 1H), 8.380-8.296 (m , 3H), 7.489 (d, J=7.5Hz, 1H), 7.435-7.345 (m, 1H), 7.110 (t, J=7.5Hz, 1H), 7.023 (t, J=6.9Hz, 1H), 5.117 -5.035(m, 5H), 4.8621(s, 2H), 4.797(m, 1H), 4.519(m, 1H), 4.483-4.398(m, 5H), 3.796-3.156(m, 6H), 3.425-3.233 (m, 5H), 2.798-2.712 (m, 2H), 2.182 (dd, J ₁ =6.0Hz, J ₂ =12.6Hz, 1H), 1.963 (m, 1H), 1.719 (m, 1H), 1.518 ( m, 3H).

Example 16 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg-Gly- Asp-Ser(9a)

0 °C, 100 mg (0.10 mmol) of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro- 1H,11H-pyrrolo[1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl -Arg(NO ₂ )-Gly-Asp(OBzl)-Ser-OBzl (8a) was dissolved with 1 mL of trifluoroacetic acid and 0.3 mL of trifluoromethanesulfonic acid, stirred for 0.5 h, and the reaction mixture was concentrated under reduced pressure. 10 mL of anhydrous ether was added to the residue, stirred for 10 minutes, a solid was precipitated, and the supernatant was discarded after standing for 10 minutes. This operation is repeated 3 times. The obtained solid was collected, dissolved with 50% water and 50% methanol, adjusted to pH 8 with 10% ammonia water, purified with a C18 column, the eluent was 75% water, the product was obtained at 25% methanol, and lyophilized to obtain 12 mg ( 16%) the title compound as a yellow solid. M _P 173-174°C, ESI-MS (m/e): 783 [MH] ^- . ¹ H-NMR (300 MHz, DMSO-d6): δ/ppm=9.997 (m, 1H), 8.607-8.320 ( m, 3H), 7.673-7.403 (m, 3H), 7.280-7.132 (m, 4H), 5.120 (m, 1H), 5.114 (m, 2H), 4.653 (m, 1H), 4.433-4.212 (m, 7H), 4.011 (m, 1H), 3.846-3.586 (m, 8H), 3.173-2.891 (m, 3H), 2.025-1.945 (m, 2H), 1.661-1.518 (m, 4H).

Example 17 Preparation of Boc-Asp(OBzl)-Phe-OBzl

Using the procedure of Example 9, 2.72 g (97%) of the title compound was obtained as a yellow oil from 1.615 g (5 mmol) Boc-Asp(OBzl) and 1.605 g (6 mmol) Phe-OBzl. ESI-MS (m/e): 561 [M+H] ⁺ .

Example 18 Preparation of Asp(OBzl)-Phe-OBzl

Using the procedure of Example 12, 1.53 g (93%) of the title compound was obtained from 2.24 g (4 mmol) of Boc-Asp(OBzl)-Phe-OBzl as a yellow oil. ESI-MS (m/e): 461 [M+H] ⁺ .

Example 19 Preparation of Boc-Arg(NO ₂ )-Gly-Asp(OBzl)-Phe-OBzl

Using the method of Example 9, 3.21 g (78%) of the title compound was obtained as a colorless solid from 1.88 g (5 mmol) Boc-Arg(NO2)-Gly and 2.83 _g (6 mmol) Asp(OBzl)-Phe-OBzl. ESI-MS (m/e): 819 [M+H] ⁺ .

Example 20 Preparation of Arg(NO ₂ )-Gly-Asp(OBzl)-Phe-OBzl

Using the procedure of Example 12, 0.74 g (99%) of the title compound was obtained as a colorless solid from 0.82 g (1 mmol) Boc-Arg(NO2)-Gly _- Asp(OBzl)-Phe-OBzl. ESI-MS (m/e): 719 [M+H] ⁺ .

Example 21 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg(NO ₂ )-Gly-Asp(OBzl)-Phe-OBzl(8b)

Using the method of Example 15, from 84 mg (0.23 mmol) (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro- 1H,11H-pyrrolo[1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid (7) and 190 mg (0.25 mmol) of Arg(NO2)-Gly _- Asp(OBzl)-Phe-OBzl to give 90 mg (37%) of the title compound as a yellow solid. ESI-MS (m/e): 1070[M+H] ⁺ , ¹ H-NMR (300MHz, DMSO-d6): δ/ppm=8.526(d, J=8.1Hz, 1H), 8.432(d, J =7.2Hz, 1H), 8.320(m, 1H), 8.250(d, J=7.8Hz, 1H), 7.451(d, J=7.5Hz, 1H), 7.357-7.197(m, 16H), 7.100(t , J=7.2Hz, 1H), 7.013 (t, J=6.9Hz, 1H), 5.166 (d, J=3.6Hz, 1H), 5.055 (d, J=6.6Hz, 1H), 4.836 (d, J =6.9Hz, 1H), 4.731(m, 1H), 4.456(d, J=6.9Hz, 1H), 4.391-4.264(m, 4H), 4.171-4.119(m, 2H), 3.793-3.739(m, 2H), 3.711-3.413(m, 2H), 3.177-2.922(m, 8H), 2.699(m, 1H), 2.017(m, 1H), 1.708(m, 1H), 1.532-1.510(m, 3H) .

Example 22 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg-Gly- Asp-Phe(9b)

Using the method of Example 16 from 100 mg (0.09 mmol) 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a- Octahydro-1H,11H-pyrrolo[1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indole-11- yl)acetyl-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl (8b) gave 15 mg (15%) of the title compound as a yellow solid. MP _184-185 °C. ESI-MS (m/e): 843[MH] ^- .1H-NMR (300MHz, DMSO-d6): δ/ppm=10.083 (m, 1H), 8.809-8.442 (m, 3H), 7.476-7.361 ( m, 2H), 7.296-6.770 (m, 10H), 5.158 (m, 1H), 4.857 (m, 1H), 4.394-4.198 (m, 7H), 3.880 (m, 1H), 3.616-3.348 (m, 3H), 3.169-2.897 (m, 8H), 2.436-2.312 (m, 2H), 2.167 (m, 1H), 1.909 (m, 1H), 1.632-1.466 (m, 4H).

Example 23 Preparation of Boc-Asp(OBzl)-Val-OBzl

Using the procedure of Example 9, 2.41 g (94%) of the title compound was obtained as a yellow solid from 1.615 g (5 mmol) Boc-Asp(OBzl) and 1.340 g (6 mmol) Val-OBzl. ESI-MS (m/e): 513 [M+H] ⁺ .

Example 24 Preparation of Asp(OBzl)-Val-OBzl

Using the procedure of Example 12, 1.67 g (93%) of the title compound was obtained from 2.05 g (4 mmol) of Boc-Asp(OBzl)-Val-OBzl as a yellow solid. ESI-MS (m/e): 413 [M+H] ⁺ .

Example 25 Preparation of Boc-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl

Using the procedure of Example 9, 2.51 g (65%) of the title compound was obtained as a colorless solid from 1.88 g (5 mmol) Boc-Arg(NO2)-Gly and 2.47 _g (6 mmol) Asp(OBzl)-Val-OBzl. ESI-MS (m/e): 771 [M+H] ⁺ .

Example 26 Preparation of Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl

Using the method of Example 12, 0.68 g (96%) of the title compound was obtained as a colorless solid from 0.77 g (1 mmol) Boc-Arg(NO2)-Gly _- Asp(OBzl)-Val-OBzl. ESI-MS (m/e): 671 [M+H] ⁺ .

Example 27 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg(NO ₂ )-Gly-Asp(OBzl)-Val-OBzl(8c)

Using the method of Example 15, from 450 mg (1.22 mmol) (2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro- 1H,11H-pyrrolo[1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl-acetic acid (7) and 915 mg (1.29 mmol) of Arg(NO2)-Gly _- Asp(OBzl)-Val-OBzl to give 240 mg (25%) of the title compound as a yellow solid. ESI-MS (m/e): 1022 [M+H] ^{+ .1} H-NMR (300MHz, DMSO-d6): δ/ppm=8.568 (d, J=7.2Hz, 1H), 8.336-8.258 (m , 2H), 8.191 (m, J=7.8Hz, 1H), 7.489 (d, J=7.8Hz, 1H), 7.437-7.349 (m, 11H), 7.109 (t, J=6.9Hz, 1H), 7.023 (t, J=7.2Hz, 1H), 5.206(d, J=1.2Hz, 1H), 5.159-5.078(m, 5H), 4.919(s, 2H), 4.778(m, 1H), 4.481(m, 1H), 4.481-4.200(m, 6H), 3.809-3.681(m, 2H), 3.274-3.144(m, 5H), 2.797-2.677(m, 2H), 2.209-1.929(m, 3H), 1.716( m, 1H), 1.540-1.511 (m, 3H), 0.944 (d, J=6.9Hz, 6H).

Example 28 Preparation of 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a-octahydro-1H,11H-pyrrole [1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indol-11-yl)acetyl-Arg-Gly- Asp-Val(9c)

Using the method of Example 16 from 100 mg (0.10 mmol) 2-((2S,5aS,14aS)-2-hydroxy-5,14-dione-2,3,5,5a,6,12,14,14a- Octahydro-1H,11H-pyrrolo[1",2":4',5']pyrazino[1',2':1,6]pyrido[3,4-b]indole-11- yl)acetyl-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl (8c) gave 10 mg (13%) of the title compound as a yellow solid. MP _176-177 °C. ESI-MS (m/e): 795[MH] ^- . ^1H -NMR (300MHz, DMSO-d6): δ/ppm=10.164 (m, 1H), 8.662 (d, J=7.8Hz, 1H), 8.592-8.489(m, 2H), 8.262(m, 1H), 8.184-7.419(m, 2H), 7.243(d, J=7.2Hz, 1H), 7.152-7.043(m, 3H), 5.157(m, 1H), 5.130(m, 2H), 4.878(m, 1H), 4.556-4.230(m, 6H), 3.685-3.275(m, 6H), 4.600(s, 2H), 4.418-4.256(m, 4H) , 4.185(m, 2H), 3.830-3.668(m, 4H), 3.600(m, 2H), 3.040(m, 2H), 2.856-2.616(m, 3H), 2.192(m, 1H), 2.169(m , 2H), 1.976-1.572 (m, 4H), 0.837-0.771 (m, 6H).

Test Example 1 Evaluation of the antiplatelet aggregation activity of compounds 7, 9a, 9b and 9c

Healthy male SD rats (200±20g) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., urethane (urethane, purchased from Sinopharm Chemical Reagent Co., Ltd.), trisodium citrate (purchased from Sinopharm Group) Chemical Reagent Co., Ltd.), physiological saline (purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), arachidonic acid (AA, purchased from Sinopharm Chemical Reagent Co., Ltd.). Compounds 7, 9a, 9b and 9c were prepared with physiological saline to a concentration of 10 μM, 5 μM and 1 μM; physiological saline was used as a negative control; AA was formulated into a solution with a concentration of 0.3 mg/mL in physiological saline; trisodium citrate was prepared with physiological The brine was formulated as a 3.8% solution.

Two healthy male SD rats (body weight 180-220 g) rested for one day and fasted for 12 hours before surgery. The rats were anesthetized with a single intraperitoneal injection of 20% urethane 0.7 mL/100 g according to the body weight of the rats. The anesthetized rat was fixed, the right common carotid artery was isolated, two surgical wires were passed under the artery, the distal end of the artery was ligated, the proximal end of the artery was clamped with an artery clip, and ophthalmic scissors were used to close the distal end of the artery. Cut the end of the tube, insert the polyethylene tube into the right artery, and fasten the interface with surgical thread. In a 15mL centrifuge tube, add 3.8% trisodium citrate aqueous solution for anticoagulation at a ratio of 1/9, loosen the arterial clamp, and collect 10mL of fresh blood from the rat. The fresh blood was centrifuged at 1500 rpm/min at 4°C for 10 min in a 15 mL centrifuge tube. The upper plasma was carefully aspirated and diluted 6 times with normal saline to obtain platelet rich plasma (PRP). Centrifuge the remaining blood components in the centrifuge tube at 3000 r/min for 10 min. The supernatant is platelet poor plasma (PPP).

Turn on the CHRONO-700 in vitro antiplatelet aggregator, preheat for 30 min, make the temperature of the preheating hole reach 37 °C, insert the glass vial equipped with the magnetic rotor into the preheating hole for 5-10 min, add 500 μL PPP to the glass vial and put it into the preheating hole. In the PPP position of the instrument, take another glass tube equipped with a magnetic rotor and add 480 μL of PRP to the PRP position, turn on the stirring, and after the instrument is zeroed, add 10 μL of physiological saline or the physiological salts of compounds 7, 9a, 9b and 9c into the PRP tube. After stirring evenly, add 10 μL of AA solution with a final concentration of 0.3 mg/mL. Observe the change of the curve, stop running when the curve becomes stable, calculate the platelet inhibition rate by the software, and calculate the IC ₅₀ value. The data in Table 1 demonstrate that the _IC50 values of compounds 9a, 9b and 9c for inhibiting AA-induced platelet aggregation were 1.47±0.89 μM, 8.03±1.34 μM and 6.54±2.21 μM, respectively, which were significantly lower _than those of compound 7 (26.48±1.26 μM). μM, p<0.01). The modification enhanced the activity of the compounds by 13-fold, 3-fold and 4-fold, respectively. The present invention has outstanding technical effects.

Table 1 Compounds 7, 9a, 9b and 9c inhibit AA-induced platelet aggregation activity

a) p<0.01 compared to compound 7; n=6.

Test Example 2 Evaluation of Oral Anti-arterial Thrombosis Activity of Compounds 5, 7, 9a, 9b and 9c

Male SD rats (200±20g) were randomly divided into groups, 12 in each group, raised for 1 day, and stopped feeding overnight. Compounds 5 (dose 1 μmol/kg), 7 (dose 1 μmol/kg), 9a (dose 0.1 μmol/kg), 9b (dose 0.1 μmol/kg) and 9c (dose 0.1 μmol/kg) were orally administered with aspirin and 5‰ Suspension of CMC-Na (dose 167 μmol/kg) or 5‰ CMC-Na (dose 0.3 mL/100 g). After 30 min, the rats were anesthetized with 20% ulose in normal saline (0.7 mL/kg), and then operated. The right carotid artery and left jugular vein of the rat were separated, and an accurately weighed silk thread was placed in the bypass cannula, one end of the tube was inserted into the left vein, the other end of the tube was inserted into the right artery, and 0.2 mL of heparin sodium was injected for anticoagulation. Make the blood flow from the right artery through the bypass cannula and enter the left vein. After 15 minutes, the silk thread with thrombus was taken out and weighed, and the weight of the silk thread before and after blood circulation was calculated. test. The data are listed in Table 2.

Table 2 Oral anti-arterial thrombotic activity of compounds 5, 7, 9a, 9b and 9c

a) p<0.01 versus CMC-Na, p>0.05 versus aspirin; b) p<0.01 versus CMC-Na, p<0.05 versus aspirin; n=12.

Test Example 3 Evaluation of Oral Anti-Venous Thrombosis Activity of Compounds 5, 7 and 9a, 9b and 9c

Healthy male SD rats (200±20g) were randomly divided into 12 groups; the oral dose of warfarin sodium for the positive control was 4.87 μmol/kg, the oral dose of compounds 5 and 7 was 1 μmol/kg, and the oral dose of compounds 9a, 9b and 9c The oral dose of CMC-Na was 0.1 μmol/kg, and the oral dose of CMC-Na of 5‰ of the blank control was 0.3 mL/100 g.

After 30 minutes of dose administration, rats were anesthetized by intraperitoneal injection of 20% urethane. The anesthetized rats were fixed, and the abdomen was prepared with skin, which was cut along the linea alba, up to the corner of the liver where the liver could be seen, down to the coagulation gland. The organs such as the small intestine were pulled out from the abdominal cavity, and the pulled organs were wrapped with gauze soaked in normal saline, and the remaining tissues were bluntly separated with curved forceps to expose the inferior vena cava. The inferior vena cava and its surrounding branches were bluntly dissected, and the abdominal aorta and inferior vena cava were carefully dissected starting below the renal vein. The junction of the inferior vena cava and the left renal vein was ligated with a suture infiltrated with normal saline, and the pulled out organ was put back in place according to the anatomical position. The abdominal cavity was closed layer by layer with sutures. After the operation, the rats were nursed at room temperature for 4 hours, and then the abdominal cavity was opened again, and the inferior vena cava and its branches were found again. Cut an opening with scissors, the thrombus will flow out of the opening with the blood, remove the thrombus with curved forceps, and check whether the thrombus in the inferior vena cava has been removed. After the thrombus was completely removed, the floating blood was dipped in filter paper, weighed with a balance and the weight of the thrombus was recorded. Data were tested with t test. The results are shown in Table 3.

Table 3 Oral anti-venous thrombotic activity of compounds 5, 7, 9a, 9b and 9c

a) p<0.01 versus CMC-Na, p>0.05 versus warfarin sodium; b) p<0.01 versus CMC-Na, p<0.05 versus compounds 7 and 9; n=12.

Test Example 4 Evaluation of the effects of compounds 5, 7, 9a, 9b and 9c on serum GPIIb/IIIa levels in rats

The whole blood of the rat collected in Test Example 3 was centrifuged at 1000 rpm for 10 min to collect the serum, and the GPIIb/IIIa content in the serum was determined by the rat GPIIb/IIIa enzyme-linked immunosorbent assay according to the operation described in the kit. The enzyme-labeled plate was equipped with GPIIb/IIIa standard wells, CMC-Na-treated rat serum wells, and compound 5, 7, 9a, 9b and 9c-treated rat serum wells. 50 [mu]L of various concentrations of GPIIb/IIIa standards (prepared by dilution of the standards from the kit) were added to the standard wells. In CMC-Na-treated rat serum wells, compound 5, 7, 9a, 9b and 9c-treated rat serum wells were firstly filled with 40 μL of sample diluent, followed by 10 μL of serum (final dilution was 5-fold). Add 100 μL of enzyme labeling reagent to each well, seal the plate with sealing film, and incubate at 37℃ for 60min. Except for the blank wells, the plates were sealed with a sealing film and incubated at 37°C for 60 min. The washing solution provided by the kit was diluted 20 times with distilled water for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, and discard the washing solution after standing for 30s. Repeat this operation 5 times and pat dry. To each well, add 50 μL of chromogenic reagent A provided by the kit first, then add 50 μL of chromogenic reagent B provided by the kit, shake gently to mix well, and develop color at 37°C for 15 min in the dark. After that, add 50 μL of the stop solution provided by the kit to each well to stop the color reaction (the blue turns to yellow at this time), zero-adjust the blank well as a reference, and measure the absorbance of each well at a wavelength of 450 nm. Blank control wells were set during measurement. In the blank control wells, the operations of other steps remained unchanged except that no samples and enzyme-labeling reagents were added. Take the concentration of GPIIb/IIIa standard as the abscissa and the absorbance value as the ordinate to draw the GPIIb/IIIa standard curve, and simulate the linear regression equation. The GPIIb/IIIa concentration in the serum samples was calculated by substituting the absorbances of CMC-Na-treated rat serum samples, and the absorbances of compounds 5, 7, 9a, 9b and 9c-treated rat serum samples into the equation. The data are expressed as mean±SD U/mL, after t-test, p<0.05 means statistical difference. The results are listed in Table 4. It can be seen that compounds 5, 7, 9a, 9b and 9c effectively reduce the level of GPIIb/IIIa in rat serum. It can be seen that GPIIb/IIIa is the target of compounds 5, 7, 9a, 9b and 9c showing anti-arterial thrombotic activity. This is also the outstanding technical effect of the present invention.

Table 4 Effects of compounds 5, 7, 9a, 9b and 9c on the content of GPIIb/IIIa in rat blood

a) p<0.01 vs. CMC-Na; n=6.

Test Example 5 Evaluation of the effects of compounds 5, 7, 9a, 9b and 9c on serum P-selectin levels in rats

The whole blood of rats collected in Test Example 3 was centrifuged at 1000 rpm for 10 min to collect serum, and the P-selectin content in serum was determined by rat P-selectin enzyme-linked immunosorbent assay according to the operation described in the kit. The enzyme-labeled plate was equipped with P-selectin standard wells, CMC-Na-treated rat serum wells, and compound 5, 7, 9a, 9b and 9c-treated rat serum wells. 50 [mu]L of different concentrations of P-selectin standards (prepared by dilution of the standard from the kit) were added to the standard wells. In CMC-Na-treated rat serum wells, compound 5, 7, 9a, 9b and 9c-treated rat serum wells were firstly filled with 40 μL of sample diluent and then 10 μL of serum (final dilution was 5 times). Add 100 μL of enzyme labeling reagent to each well, seal the plate with sealing film, and incubate at 37℃ for 60min. Except for the blank wells, the plates were sealed with a sealing film and incubated at 37°C for 60 min. The washing solution provided by the kit was diluted 20 times with distilled water for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, and discard the washing solution after standing for 30s. Repeat this operation 5 times and pat dry. Add 50 μL of chromogenic reagent A provided by the kit to each well first, then add 50 μL of chromogenic reagent B provided by the kit, shake gently to mix well, and develop color at 37°C for 15 min in the dark. After that, add 50 μL of the stop solution provided by the kit to each well to stop the color reaction (the blue turns to yellow at this time), zero-adjust the blank well as a reference, and measure the absorbance of each well at a wavelength of 450 nm. Blank control wells were set during measurement. In the blank control wells, the operations of other steps remained unchanged except that no samples and enzyme-labeling reagents were added. Take the concentration of P-selectin standard as the abscissa and the absorbance value as the ordinate to draw the P-selectin standard curve to simulate the linear regression equation. The P-selectin concentration in the serum samples was calculated by substituting the absorbances of CMC-Na-treated rat serum samples, and the absorbances of compounds 5, 7, 9a, 9b, and 9c-treated rat serum samples into the equation. The data are expressed as mean±SD ng/mL, after t test, p<0.05 means statistical difference. The results are listed in Table 5. It can be seen that compounds 5, 7, 9a, 9b and 9c effectively reduce the level of P-selectin in rat serum. It can be seen that P-selectin is the target of compounds 5, 7, 9a, 9b and 9c showing anti-arterial thrombotic activity. This is also the outstanding technical effect of the present invention.

Table 5 Effects of compounds 5, 7 and 9a, 9b and 9c on the content of P-selectin in rat blood

a) p<0.01 vs. CMC-Na; n=6.

Claims (6)

1. CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione represented by general formula I,

I where AA is selected from Ser, Phe or Val. 2. The method for preparing the modified pentacyclic piperazine dione of CH ₂ CO-Arg-Gly-Asp-AA of the general formula I according to claim 1, the method comprising: 1) Pictet-Spengler condensation of L -tryptophan with formaldehyde under the catalysis of concentrated sulfuric acid to obtain ( 3S )-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; 2) Reaction of (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid with di-tert-butyl dicarbonate to obtain N -tert-butoxycarbonyl-( 3S ) -1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid; 3) react L -hydroxyproline with thionyl chloride and methanol to obtain hydroxyproline methyl ester; 4) Synthesis of 3S-N-Boc-1,2,3,4-tetrahydro-β-carboline- 3-Formyl-hydroxyproline methyl ester; 5) De-Boc of 3S-N-Boc-1,2,3,4-tetrahydro-β-carboline-3-formyl-hydroxyproline methyl ester in ethyl acetate solution of hydrogen chloride to obtain 3S-1 ,2,3,4-Tetrahydro-β-carboline-3acyl-hydroxyproline methyl ester; 6) Preparation of 2-hydroxyoctahydro from 3S-1,2,3,4-tetrahydro-β-carboline-3-formyl-hydroxyproline in the presence of N-methylmorpholine in methanol solvent Pyrrolopyrazinopyridoindolediones; 7) Preparation of 2-hydroxyoctahydropyrrolopyrazinopyrido by the reaction of 2-hydroxyoctahydropyrrolopyrazinopyridoindole with benzyl bromoacetate in dimethylformamide catalyzed by sodium hydride benzyl indoledione-11-yl acetate; 8) Hydrogenolysis of benzyl 2-hydroxyoctahydropyrrolopyrazinopyridoindoledione-11-yl acetate to 2-hydroxyoctahydropyrrolopyrazino in methanol and dichloromethane with palladium-carbon catalysis Pyridinoindoldione-11-ylacetic acid; 9) Coupling of 2-hydroxyoctahydropyrrolopyrazinopyridoindoledione-11-ylacetic acid with Arg(NO ₂ )-Gly-Asp(OBzl)-AA-OBzl to prepare 2-hydroxyoctahydropyrrolo Pyrazinopyridoindoldione-11-yl-CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-AA-OBzl; 10) 2-Hydroxyoctahydropyrrolopyrazinopyridoindoldione-11-yl-CH ₂ CO-Arg(NO ₂ )-Gly-Asp(OBzl)-AA-OBzl acid dehydration to prepare claim 1 CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione. 3. The application of the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione of the general formula I of claim 1 in the preparation of an anti-platelet aggregation drug. 4. The application of the _CH2CO -Arg-Gly-Asp-AA modified pentacyclic piperazine dione of the general formula I of claim 1 in the preparation of an anti-arterial thrombosis drug. 5. The application of the _CH2CO -Arg-Gly-Asp-AA modified pentacyclic piperazine dione of the general formula I of claim 1 in the preparation of an anti-venous thrombosis drug. 6 . The application of the CH ₂ CO-Arg-Gly-Asp-AA modified pentacyclic piperazine dione of the general formula I according to claim 1 in the preparation of a drug with dual effects of anti-arterial thrombosis and anti-venous thrombosis.

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