CN107488214B - warfarin-4-O-acetyl-LPNISKP and synthesis, activity and application thereof - Google Patents
warfarin-4-O-acetyl-LPNISKP and synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, discloses a preparation method thereof, discloses anti-arterial thrombosis activity thereof, discloses activity for reducing the content of platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa) in vivo, discloses activity for reducing the content of blood coagulation factor II (F II) in vivo, and discloses activity for inhibiting platelet aggregation. Therefore, the invention discloses the application of the compounds in preparing the anti-arterial thrombosis drugs, the anti-venous thrombosis drugs, the platelet aggregation inhibiting drugs, the GP IIb/IIIa antagonists and the blood coagulation factor II antagonists.
Description
Technical Field
The invention relates to warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, a preparation method thereof, anti-arterial thrombosis activity thereof, an effect of reducing the content of GP IIb/IIIa in vivo, an effect of reducing the content of blood coagulation factor II in vivo and an effect of inhibiting platelet aggregation. Therefore, the invention relates to the application of the compound in preparing the anti-arterial thrombosis medicine, the anti-venous thrombosis medicine, the platelet aggregation inhibiting medicine, the GP IIb/IIIa antagonist and the blood coagulation factor II antagonist. The invention belongs to the field of biological medicine.
Background
Both arterial and venous thrombosis have become diseases with a high incidence of morbidity and mortality. Wherein the venous thrombosis mainly comprises deep vein thrombosis and pulmonary embolism. The number of patients with deep vein thrombosis and pulmonary embolism exceeds the total number of patients with myocardial infarction and apoplexy, and is higher than the total number of deaths caused by breast cancer and AIDS. Because the incidence of arterial thrombosis and venous thrombosis increases exponentially with age, the threat of the two diseases to the health of people in the aging countries in China is particularly serious. If the population cardinality is considered, the absolute negative influence on the national civilization of China is particularly serious. Therefore, prevention and treatment of arterial thrombosis and venous thrombosis have been the focus of attention in the field of medicine. Although warfarin was used in clinical practice as a representative drug in 1941, its activity is manifested by a common complex role of its pharmacokinetic profile, coagulation factor metabolism, and availability of vitamin K. The dose-response relationship of warfarin shows great variability, which indicates that there is a great dose range for the anticoagulant therapy of warfarin and that there are great individual differences. Due to the narrow window of warfarin, under-dosing may lead to pulmonary embolism, while overdosing risks fatal bleeding. A great deal of structural modification is carried out on warfarin for more than 50 years in the past, but analogs with strong anti-thrombus activity and low bleeding side effect cannot be obtained. Contrary to the conventional thinking, the aim of the inventor for modifying the structure of warfarin is to convert warfarin into an analogue with double activities of resisting arterial thrombosis and resisting arterial thrombosis. However, it has not been satisfactory. Cell adhesion is involved in the development of various diseases in human. The adhesion between cells and the extracellular matrix, mediated by cell adhesion molecules, adhesion proteins, and their association with thrombosis has received attention from a wide range of researchers. The objective of the research on the polypeptide with the cell adhesion inhibiting activity of the inventor is to convert the polypeptide Leu-Pro-Asn-Ile-Ser-Lys-Pro with the cell adhesion inhibiting activity into an analogue with double activities of resisting arterial thrombosis and resisting venous thrombosis. However, it has not been satisfactory. After 5 years of exploration, the inventor finds that 4-bit of warfarin is modified by acetyl Leu-Pro-Asn-Ile-Ser-Lys-Pro, and the generated warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro shows excellent anti-platelet aggregation activity on an in vitro anti-platelet aggregation model. Obviously, they are not prodrugs of warfarin. The anti-arterial thrombus activity of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is 167 times stronger than that of aspirin, and the anti-arterial thrombus activity is 48.7 times stronger than that of warfarin. It can be seen that the present invention has significant technical advances. Thus, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro.
The second content of the invention is to provide a method for synthesizing warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, which comprises the following steps:
1. synthesizing warfarin-4-O-benzyl acetate;
2. synthesizing warfarin-4-O-acetic acid;
3. adopts a liquid phase condensation method with DCC as a condensing agent and HOBt as a catalyst to synthesize HCl, Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl
4. A liquid-phase condensation method using DCC as condensing agent and HOBt as catalyst for condensing warfarin-4-O-acetic acid with HCl, Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl
5. Synthesis of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro
The third aspect of the present invention is to evaluate the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on arterial thrombosis.
The fourth aspect of the present invention is to evaluate the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on venous thrombosis.
The fifth aspect of the present invention was to evaluate the inhibition of platelet aggregation induced by Platelet Activating Factor (PAF), Thrombin (TH) and Adenosine Diphosphate (ADP) by warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro.
The sixth aspect of the present invention is to evaluate the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro in reducing the level of GPIIb/IIIa in vivo.
The seventh aspect of the present invention is to evaluate the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on reducing the level of coagulation factor II in vivo.
Drawings
FIG. 1 synthetic route for warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro (i) bromo-2-benzyl acetate, acetone, K2CO3,45℃；(ii)CH3OH,Pd/C,H2(ii) a (iii) DCC, HOBt, NMM, THF; (iv)4N hydrogen chloride in ethyl acetate; (v)2N NaOH, CH3OH。
FIG. 2 the anti-arterial thrombotic activity of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, n ═ 10;
FIG. 3 warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro anti-venous thrombus activity, n ═ 10;
FIG. 4 inhibition of platelet aggregation induced by PAF, TH and ADP by warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, with n being 3.
FIG. 5 the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on the level of GPIIb/IIIa in vivo, n-5;
FIG. 6 the effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on the F II content in rats, n ═ 5.
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
EXAMPLE 1 preparation of warfarin-4-O-benzyl acetate
Placing 3.31g (10.00mmol) of warfarin in a 100mL eggplant bottle, adding about 40mL of acetone, not completely dissolving the acetone, heating in a 45 ℃ oil bath and stirring until the warfarin is dissolved, adding 1.73mL (11.00mmol) of benzyl bromide-2-acetate, continuing the reaction under the 45 ℃ oil bath, and after about 1h, finding a white solid attached to the bottle wall. After 48h of reaction, the progress of the reaction was monitored by thin layer chromatography (TLC, petroleum ether/ethyl acetate 2:1), warfarin disappeared, the colorless solid produced by the reaction was filtered off, acetone was removed under reduced pressure to give a pale yellow oil, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 8:1) 3.02g (65%) of the title compound are obtained as a colorless solid. ESI-MS (M/e) 457[ M + H]+；1H-NMR(300MHz,DMSO-d6)δ/ppm＝7.89(dd,J1＝3.0Hz,J2＝9.0Hz,1H),7.63(dt,J1＝3.0Hz,J2＝9.0Hz,1H),7.43～7.31(m,9H),7.24(t,J＝9.0Hz,2H),7.15(tt,J＝9.0Hz,1H),5.26(s,2H),5.61(s,1H),5.02(d,J＝15.0Hz,1H),4.85(d,J＝15.0Hz,1H),4.97(t,J＝9.0Hz,1H),3.45(dq,J1＝9.0Hz,J2＝18.0Hz,2H),2.11(s,3H)。
EXAMPLE 2 preparation of warfarin-4-O-acetic acid
2.26g (4.95mmol) of warfarin-4-O-benzyl acetate was dissolved in 20mL of methanol, 220mg of palladium on carbon (Pd/C) was added, air was pumped out of a water pump with stirring, hydrogen was introduced, and the operation was repeated 3 times with stirring at room temperature with hydrogen. The reaction was monitored by TLC for completion, Pd/C was removed by filtration, the filtrate was concentrated under reduced pressure to remove the solvent, and the residue was solidified with petroleum ether and triturated with anhydrous ether to give 1.72g (93%) of the title compound as a colorless solid. ESI-MS (M/e):367[ M + H]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝12.86(s,1H),7.90(d,J＝6.0Hz,1H),7.63(t,J＝6.0Hz,1H),7.43～7.34(m,4H),7.27(t,J＝9.0Hz,2H),7.17(t,J＝9.0Hz,1H),4.99(t,J＝9.0Hz,1H),4.75(q,J1＝15.0Hz,J2＝30.0Hz,2H),3.54～3.47(m,2H),2.14(s,3H)。
EXAMPLE 3 preparation of Boc-Asn-Ile-OBzl
5.05g (21.76mmol) of Boc-Asn was dissolved in a 250mL eggplant flask with 100mL of anhydrous tetrahydrofuran, and then 2.91g (21.55mmol) of HOBt and 5.33g (25.87mmol) of DCC were added thereto in an ice bath (0 ℃ C.) to activate for 30 min. A large amount of Dicyclohexylurea (DCU) precipitated. 8.47g (21.55mmol) of Tos. Ile-OBzl was dissolved in 100mL of tetrahydrofuran, added to the reaction mixture in an ice bath, adjusted to pH 8-9 with N-methylmorpholine (NMM), stirred at room temperature for 5h, followed by TLC (dichloromethane/methanol 20:1) to monitor the progress of the reaction, the starting material point disappeared, DCU was filtered off, the solvent was removed from the filtrate under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, the insoluble DCU was filtered off, and the filtrates were each separately dissolved in saturated Na2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3),saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3) was washed with a saturated NaCl solution (40 mL. times.3), and the ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, and the solvent was removed from the filtrate under reduced pressure to give 9.02g (96%) of a pale yellow solid. ESI-MS (M/e):436[ M + H]+。
EXAMPLE 4 preparation of HCl. Asn-Ile-OBzl
9.00g of Boc-Asn-Ile-OBzl was weighed into a 250mL eggplant flask, 15mL of anhydrous ethyl acetate was added thereto to completely dissolve the Boc-Asn-Ile-OBzl, 90mL of a 4N ethyl acetate solution of hydrogen chloride was added thereto in an ice salt bath (-10 ℃), and the reaction was continued with stirring in the ice bath. TLC (dichloromethane/methanol ═ 20:1) monitored the progress of the reaction, the starting material disappeared, the reaction mixture was concentrated under reduced pressure using a water pump, the residue was redissolved with anhydrous ethyl acetate and concentrated under reduced pressure, and this was repeated 3 times to give a yellow oil, which was soaked with anhydrous ether and concentrated under reduced pressure, and this was repeated 3 times to give 7.67g (99%) of a pale yellow oily residue.
EXAMPLE 5 preparation of Boc-Pro-Asn-Ile-OBzl
4.45g (20.69mmol) of Boc-Pro was added to a 250mL eggplant flask and dissolved with 100mL of anhydrous tetrahydrofuran, and 2.79g (20.66mmol) of HOBt and 5.12g (24.85mmol) of DCC were added under ice bath (0 ℃ C.) and activated for 30 min. There was a significant amount of DCU evolved. Dissolving 7.66g (20.61mmol) of HCl & Asn-Ile-OBzl in 100mL of tetrahydrofuran, adding the solution into the reaction solution in ice bath, adjusting the pH value to 8-9 with NMM, stirring the solution at room temperature for 7h, then monitoring the reaction progress by TLC (dichloromethane/methanol 20:1), removing the raw material point, filtering DCU, removing the solvent from the filtrate under reduced pressure, dissolving the residue with 100mL of ethyl acetate, filtering out insoluble DCU, and respectively using saturated Na for the filtrate2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3) was washed with a saturated NaCl solution (40 mL. times.3), and the ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, and the solvent was removed from the filtrate under reduced pressure to give a pale yellow solid, which was purified by silica gel column chromatography (dichloromethane/methanol 80:1) to give 8.26g (75%) of a colorless solid. ESI-MS (m/e):533[ lambda ], [ solution ]M+H]+。
EXAMPLE 6 preparation of HCl Pro-Asn-Ile-OBzl
6.26g (11.76mmol) of Boc-Pro-Asn-Ile-OBzl was weighed into a 250mL eggplant flask, 10mL of anhydrous ethyl acetate was added thereto and dissolved completely, and 60mL of a 4N ethyl acetate solution of hydrogen chloride was added thereto in an ice salt bath (-10 ℃ C.), and the reaction was stirred further in the ice bath. TLC (dichloromethane/methanol ═ 20:1) monitored the progress of the reaction, the starting material disappeared, the reaction was concentrated under reduced pressure using a water pump, the residue was redissolved with anhydrous ethyl acetate and concentrated under reduced pressure, repeated 3 times to give a yellow oil, which was soaked with anhydrous ether and concentrated under reduced pressure, repeated 3 times to give 5.50g (98%) of a pale yellow oily residue.
EXAMPLE 7 preparation of Boc-Leu-Pro-Asn-Ile-OBzl
2.72g (11.78mmol) of Boc-Leu was added to a 250mL eggplant flask, which was dissolved with 100mL of anhydrous tetrahydrofuran, and 1.59g (11.78mmol) of HOBt and 2.91g (14.12mmol) of DCC were added thereto under ice bath (0 ℃ C.) to activate for 30 min. There was a significant amount of DCU evolved. Dissolving 5.49g (11.72mmol) of HCl & Pro-Asn-Ile-OBzl in 100mL of anhydrous tetrahydrofuran, adding the solution into the reaction solution in ice bath, adjusting the pH value to 8-9 with NMM, stirring the reaction solution at room temperature for 4h, monitoring the completion of the reaction by TLC (dichloromethane/methanol & 20:1), filtering DCU, removing the solvent from the filtrate under reduced pressure, dissolving the residue with 100mL of ethyl acetate, filtering out insoluble DCU, and respectively using saturated Na for the filtrate2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3) was washed, and the ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, the solvent was removed from the filtrate under reduced pressure to give a pale yellow solid, which was purified by column chromatography (dichloromethane/methanol ═ 100:1) to give 6.37g (84%) of a colorless solid. ESI-MS (M/e) 646[ M + H]+。
EXAMPLE 8 preparation of Boc-Leu-Pro-Asn-Ile
5.02g (7.78mmol) of Boc-Leu-Pro-Asn-Ile-OBzl were weighed into a 250mL eggplant flask, dissolved by adding 10mL of methanol, adjusted to pH 12 with 2N aqueous NaOH solution in ice bath (0 ℃), reacted for about 4h with stirring, TLC (two-step TLC)Methyl chloride/methanol 20:1) the progress of the reaction was monitored, the starting material point disappeared and saturated KHSO was used4The pH was adjusted to 7, concentrated under reduced pressure to remove methanol, adjusted to pH 3, extracted with ethyl acetate (50 mL. times.3), washed with saturated NaCl (40 mL. times.3), the ethyl acetate layer was dried over anhydrous sodium sulfate for 2h or more, the solvent was removed under reduced pressure, triturated with petroleum ether, and filtered to give 4.20g (97%) of a colorless oil.
EXAMPLE 9 preparation of Boc-Lys (Z) -Pro-OBzl
5.07g (13.34mmol) of Boc-Lys (Z) was dissolved in a 250mL eggplant flask with 100mL of anhydrous tetrahydrofuran, and 1.78g (13.18mmol) of HOBt and 3.25g (15.77mmol) of DCC were added under ice bath (0 ℃ C.) to activate for 30 min. There was a significant amount of DCU evolved. Dissolving 3.18g (13.16mmol) of HCl Pro-OBzl in 100mL of anhydrous tetrahydrofuran, adding the mixture into the reaction solution under ice bath, adjusting the pH value to 8-9 by NMM, stirring the mixture at room temperature for 4h, monitoring the completion of the reaction by TLC (dichloromethane/methanol 20:1), filtering DCU, removing the solvent from the filtrate under reduced pressure, dissolving the residue in 100mL of ethyl acetate, filtering out insoluble DCU, and dissolving the filtrate with saturated Na2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3) was washed, and the ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, and the solvent was removed from the filtrate under reduced pressure to give 7.02g (94%) of a pale yellow solid. ESI-MS (M/e):568[ M + H]+。
EXAMPLE 10 preparation of HCl Lys (Z) -Pro-OBzl
6.99g (12.32mmol) of Boc-Lys (Z) -Pro-OBzl was weighed into a 250mL eggplant flask, 30mL of anhydrous ethyl acetate was added thereto and dissolved completely, 70mL of a 4N ethyl acetate solution of hydrogen chloride was added to the flask in an ice salt bath (-10 ℃ C.), and the reaction was stirred in the ice bath. TLC (dichloromethane/methanol ═ 20:1) showed that the reaction was complete, the reaction solution was concentrated under reduced pressure using a water pump, the residue was redissolved with anhydrous ethyl acetate and concentrated under reduced pressure, and the process was repeated 3 times to give a yellow oil, which was then soaked with anhydrous ether and concentrated under reduced pressure, and repeated 3 times to give 6.15g (98%) of a pale yellow oily residue.
EXAMPLE 11 preparation of Boc-Ser-Lys (Z) -Pro-OBzl
2.53g (12.34mmol) of Boc-Ser was dissolved in a 250mL eggplant flask with 100mL of anhydrous tetrahydrofuran, and 1.67g (12.37mmol) of HOBt and 3.05g (14.80mmol) of DCC were added under ice bath (0 ℃ C.) to activate for 30 min. There was a significant amount of DCU evolved. Dissolving 6.11g (12.13mmol) of HCl & Lys (Z) -Pro-OBzl in 100mL of anhydrous tetrahydrofuran, adding the mixture into the reaction solution under ice bath, adjusting the pH value to 8-9 by NMM, stirring the mixture at room temperature for 6h, monitoring the reaction progress by TLC (dichloromethane/methanol & gt 20:1), removing a raw material point, filtering DCU, removing the solvent from the filtrate under reduced pressure, dissolving the residue by 100mL of ethyl acetate, filtering insoluble DCU, and respectively using saturated Na for the filtrate2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3) was washed, and the ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, the solvent was removed from the filtrate under reduced pressure to give a pale yellow solid, which was purified by column chromatography (dichloromethane/methanol ═ 100:1) to give 5.17g (62%) of a colorless solid. ESI-MS (M/e):655[ M + H]+。
EXAMPLE 12 preparation of HCl Ser-Lys (Z) -Pro-OBzl
5.07g (7.75mmol) of Boc-Ser-Lys (Z) -Pro-OBzl was weighed into a 250mL eggplant flask, 15mL of anhydrous ethyl acetate was added thereto and dissolved completely, 50mL of a 4N ethyl acetate solution of hydrogen chloride was added to the ice salt bath (-10 ℃), and the reaction was stirred while cooling on ice. TLC (dichloromethane/methanol ═ 20:1) monitored the progress of the reaction, the starting material disappeared, the reaction was concentrated under reduced pressure using a water pump, the residue was redissolved with anhydrous ethyl acetate and concentrated under reduced pressure, repeated 3 times to give a yellow oil, which was soaked with anhydrous ether and concentrated under reduced pressure, repeated 3 times to give 4.50g (98%) of a pale yellow oily residue.
EXAMPLE 13 preparation of Boc-Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl
4.30g (7.75mmol) of Boc-Leu-Pro-Asn-Ile was dissolved in a 250mL eggplant flask with 100mL of anhydrous tetrahydrofuran, and 1.05g (7.78mmol) of HOBt and 1.92g (9.32mmol) of DCC were added under ice bath (0 ℃ C.) to activate for 30 min. There was a significant amount of DCU evolved. 4.59g (7.7)8mmol) HCl, Ser-Lys (Z) -Pro-OBzl was dissolved in 100mL anhydrous tetrahydrofuran, added to the reaction mixture in ice bath, the pH was adjusted to 8-9 with NMM, stirred at room temperature for 6h, then TLC (dichloromethane/methanol 15:1) monitored for completion of the reaction, DCU was filtered off, the solvent was removed from the filtrate under reduced pressure, the residue was dissolved in 100mL dichloromethane, insoluble DCU was filtered off, and the filtrate was each dissolved in saturated Na2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3) was washed, and the methylene chloride phase was dried over anhydrous sodium sulfate for 4 hours. The sodium sulfate was filtered off, the solvent was removed from the filtrate under reduced pressure to give a pale yellow solid, which was purified by column chromatography (dichloromethane/methanol ═ 20:1) to give 4.17g (49%) of a colorless solid. ESI-MS (M/e):1092[ M + H]+。
EXAMPLE 14 preparation of HCl Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl
0.91g (0.83mmol) of Boc-Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl was weighed into a 100mL eggplant flask, 3mL of anhydrous ethyl acetate was added thereto to dissolve completely, 10mL of a 4N ethyl acetate solution of hydrogen chloride was added to the flask in an ice bath (-10 ℃), and the reaction was stirred while continuing to be carried out in an ice bath. TLC (dichloromethane/methanol 15:1) monitored the progress of the reaction, the starting material point disappeared, the reaction was concentrated under reduced pressure using a water pump, the residue was redissolved with anhydrous ethyl acetate and concentrated under reduced pressure for 3 times to give a white solid, which was soaked with anhydrous ether and concentrated under reduced pressure for 3 times to give 0.84g (99%) of a white solid.
EXAMPLE 15 preparation of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl
1.02g (2.78mmol) of warfarin-4-O-acetic acid was added to a 100mL eggplant flask, and dissolved in 30mL of anhydrous tetrahydrofuran, and then 0.37g (2.74mmol) of HOBt and 0.68g (3.30mmol) of DCC were added thereto in an ice bath (0 ℃ C.) and activated for 30 min. There was a significant amount of DCU evolved. Dissolving 2.80g (2.73mmol) of HCl & Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl in 30mL of anhydrous tetrahydrofuran, adding the solution into the reaction solution in ice bath, adjusting the pH value to 8-9 by NMM, stirring the solution at room temperature for 8h, monitoring the reaction progress by TLC (dichloromethane/methanol & gt 20:1), and adding a raw materialAfter disappearance, DCU was filtered off, the filtrate was freed from the solvent under reduced pressure, the residue was dissolved in 50mL of dichloromethane, the insoluble DCU was filtered off, and the filtrates were each saturated with Na2CO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated Na2CO3The solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3) was washed, and the methylene chloride phase was dried over anhydrous sodium sulfate for 2 hours. The sodium sulfate was filtered off, the solvent was removed from the filtrate under reduced pressure to give a pale yellow solid, which was purified by silica gel column chromatography (dichloromethane/methanol ═ 8:1) to give 1.21g (67%) of a solid as a solid. ESI-MS (M/e):1362[ M + Na]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝8.42(d,J＝8.1Hz,1H),8.24(d,J＝7.2Hz,1H),8.13(d,J＝7.5Hz,1H),7.87(d,J＝7.8Hz,2H),7.63(td,J1＝1.2Hz,J2＝7.8Hz,1H),7.54(d,J＝8.7Hz,1H),7.42～7.14(m,18H),5.10(m,2H),5.00(s,2H),4.92(m,1H),4.64(s,2H),4.53～4.50(m,3H),4.39～4.30(m,3H),4.23(t,J＝2.1Hz,1H),3.65(m,1H),3.56(m,2H),3.46(m,4H),3.44(m,3H),2.92(m,2H),2.60(m,1H),2.42(m,1H),2.13(s,3H),1.92～1.79(m,10H),1.57～1.23(m,9H),1.10(m,2H),0.83(m,12H)。
EXAMPLE 16 preparation of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro (warfarin-4-O-acetyl-LPNISKP)
1.03g (0.76mmol) of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl was dissolved in 50mL of methanol, 100mg of Pd/C was added, air was pumped out of a water pump with stirring, hydrogen was introduced, and the operation was repeated 3 times, hydrogen was introduced, and the reaction was stirred at room temperature for about 10 hours. TLC (dichloromethane/methanol 15:1) monitored the progress of the reaction, the starting material point disappeared, Pd/C was filtered off, the filtrate was freed of solvent under reduced pressure, solidified with petroleum ether to a colorless solid using C18This was purified by column chromatography to give 231mg (27%) of the product as a colorless solid. Mp 165-167 deg.C;
(c＝0.09,H2O)；ESI-MS(m/e):1116[M+H]+；IR(cm-1):3306,3063,2939,1650,1609,1565,1494,1453,1396,1354,1272,1227,1198,1165,1146,1102,1070,1019,910,893,756,699,603；1H-NMR(300MHz,DMSO-d6):δ/ppm＝8.46(dd,J1＝7.5Hz,J2＝14.1Hz,1H),8.28(m,1H),8.10(d,J＝7.5Hz,1H),7.88(d,J＝7.8Hz,1H),7.80(m,1H),7.63(t,J＝7.5Hz,1H),7.55(d,J＝8.7Hz,1H),7.47～7.32(m,5H),7.22(t,J＝7.8Hz,2H),7.17(t,J＝7.2Hz,1H),7.10(m,1H),5.10(m,2H),4.91(m,1H),4.64～4.61(m,2H),4.51(m,2H),4.38(m,1H),4.30～4.24(m,2H),4.11(m,1H),3.62～3.34(m,10H),2.71(m,2H),2.60(m,1H),2.43(m,1H),2.13(s,3H),2.01～1.78(m,10H),1.69～1.62(m,2H),1.62～1.42(m,4H),1.42～1.37(m,3H),1.11～1.05(m,2H),0.93～0.76(m,12H)。
EXAMPLE 17 evaluation of the antithrombotic Effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro
Experimental materials:
urethane (CAS: 51-79-6, national drug group chemical Co., Ltd.), heparin sodium (CAS: 9041-08-1, Bailingwei science Co., Ltd.), and physiological saline (Shijiazhuang four drugs Co., Ltd.).
Experimental animals:
SD strain rats, male, 200 + -20 g, purchased from Experimental animals technologies, Inc. of Wei Tongli, Beijing.
The experimental method comprises the following steps: the test uses an arteriovenous bypass thrombosis model.
Preparation of bypass cannula:
the bypass intubation consists of three sections, wherein a polyethylene tube with the inner diameter of 1.0mm and the outer diameter of 2.0mm is heated and drawn into a thin tube with one end being an oblique opening, the length of the thin tube is 10.0cm, the thin tube is respectively a right carotid vein intubation and a left carotid artery intubation and is positioned at two ends of the bypass intubation; the middle section is composed of a polyethylene pipe with the inner diameter of 3.5mm, and the fixed length is 8.0 cm; the length of the silk thread with rough surface is fixed to 6.0cm, and the silk thread with 4.0 plus or minus 0.1mg weight and the same rough degree is selected.
Silanization is carried out on the inner walls of the three sections of polyethylene tubes by using 1% of silicon ether solution (1% of silicon oil in ether solution), after the polyethylene tubes are completely dried, silk threads are placed in the middle section of polyethylene tubes in the carotid artery intubation direction, the three sections of polyethylene tubes are assembled and fixed by using a sealing film, and the tubes are filled with heparin before intubation.
Grouping and administration dose:
the dose of the compound warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is 0.1 mu mol/kg; the positive control aspirin dose is 167 mu mol/kg and 16.7 mu mol/kg, and the negative control is physiological saline
Preparing used reagents:
the anesthetic is 20% urethane aqueous solution prepared by normal saline, and the anticoagulant is 42mg/100mL heparin sodium aqueous solution prepared by normal saline.
And (3) experimental operation:
rats were each gavaged at a dose of 0.3mL/100g body weight, and anesthetized 30min later by abdominal injection of a 20% urethane solution (0.7mL/100 g). Fixing a rat on a rat fixing plate in a supine position, cutting the skin of the neck, separating a right common carotid artery and a left external jugular vein, ligating the distal ends of the right common carotid artery and the left external jugular vein respectively by using an operation line, cutting a V-shaped small opening on the exposed left external jugular vein, inserting the inclined port of the vein end of the bypass cannula manufactured on the upper side into the proximal end of the opening of the left external jugular vein, fixing a blood vessel and a polyethylene tube at the cannula position by using the operation line, accurately injecting heparin sodium water solution through the bypass cannula at the dose of 0.1mL/100g of body weight, and ensuring that an injector does not withdraw from the polyethylene tube. Clamping the proximal end of the right common carotid artery by an artery clamp, cutting a V-shaped small opening on the exposed artery, taking the tip of the polyethylene tube down from the injector, inserting the tube into the proximal end of the right common carotid artery, fixing the artery blood vessel and the polyethylene tube by an operation line, loosening the artery clamp and establishing an extracorporeal circulation bypass.
The body temperature of a rat and the blood flow in a bypass cannula are kept smooth, after the body circulation is carried out for 15min, the venous end cannula is firstly cut to observe whether the blood circulation is smooth, a thrombus thread is taken out from the arterial end of the cannula, and after floating blood on the thread is absorbed on filter paper, the wet weight of the filter paper is weighed and recorded, which represents the anti-arterial thrombosis activity. Data were counted using t-test.
The experimental results are as follows:
the data are shown in FIG. 2. The results show that the thrombus weight of 0.1 mu mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro treated rat (23.15 +/-5.25 mg) is significantly less than the thrombus weight of normal saline treated rat (30.42 +/-3.49 mg, p <0.05), and the result shows that the compound 0.1 mu mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro shows the anti-arterial thrombus activity. And the aspirin under the dosage of 16.7 mu mol/kg does not have the activity of resisting the arterial thrombosis, which shows that the compound warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro has the activity of resisting the arterial thrombosis at least 167 times stronger than that of the aspirin. This is an unexpected technical effect.
EXAMPLE 18 evaluation of the anti-venous-thrombotic Effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro
Experimental materials:
uratan (ethyl carbamate, CAS: 51-79-6, national drug group chemical reagents Co., Ltd.), normal saline (Shijiazhuang four drugs Co., Ltd.) warfarin sodium (CAS: 129-06-6, Bailingwei science and technology Co., Ltd.);
experimental animals:
SD strain rats, male, 250 + -20 g, purchased from Experimental animals technologies, Inc. of Wei Tongli, Beijing.
The experimental method comprises the following steps: the experiment was performed using the rat inferior vena cava ligation model.
Grouping and administration dose:
the dose of the compound warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is 0.1 mu mol/kg, the dose of the positive control warfarin is 4.87 mu mol/kg, and the negative control is normal saline.
Preparing used reagents:
the anesthetic is a 20% urethane solution prepared from normal saline.
And (3) experimental operation:
the experimental rats were acclimatized and fasted for one day before surgery, and were administered with gavage at a dose of 0.3mL/100g body weight, administered in a gavage manner, and administered 30min later with a 20% urethane solution 2min before surgery for intraperitoneal anesthesia. Rats were fixed on a rat fixing plate, and 2mL of blood was taken from the carotid artery and used for measurement of blood-related indices. Preparing skin of abdomen of rat, sterilizing, opening abdominal cavity along the leucorrhea line, descending to coagulated gland, and ascending to expose one corner of liver. The organs such as small intestine in abdominal cavity are pulled out to expose inferior vena cava, and the pulled-out organs are wrapped with gauze soaked with normal saline. Blunt-separating connective tissue around blood vessel, exposing inferior vena cava and its branch, peeling off abdominal aorta and inferior vena cava below renal vein, ligating inferior vena cava at junction of inferior vena cava and left renal vein with suture soaked with physiological saline, placing organs such as intestine back into abdominal cavity according to anatomical position, and suturing abdominal cavity layer by layer with suture.
After the operation, the rat is placed in an environment with the temperature of 25-28 ℃ for circulation for 4h, after the abdominal cavity is opened, the branches of the rat are tied one by one, the 2cm inferior vena cava is taken out from the tying position of the junction of the inferior vena cava and the left renal vein, and the thrombus is taken out from the inferior vena cava. The blood weight was calculated and the results were counted using the t-test. The operation was performed alternately with two per group. The experimental data are shown in FIG. 3.
The experimental results are as follows:
the results show that the thrombus weight (14.22 +/-3.90 mg) of a rat treated by 0.1 mu mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is obviously smaller than the thrombus weight (22.93 +/-5.03 mg, p is less than 0.01) of a rat treated by normal saline, and the warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro shows the anti-vein thrombus activity. And the plug weight of the compound warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro at the dose of 0.1 mu mol/kg does not differ significantly from the plug weight of the warfarin-treated rat at the dose of 4.87 mu mol/kg (12.12 +/-3.86 mg, p >0.05), indicating that the compound warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro has an anti-venous thrombosis activity at least 48.7 times stronger than that of warfarin. This is an unexpected technical effect.
EXAMPLE 19 evaluation of the inhibitory Effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on PAF, TH and ADP-induced platelet aggregation
Experimental materials:
sodium citrate (CAS: 68-04-2, national drug group chemical Co., Ltd.), physiological saline (Shijiazhuang Siyao Co., Ltd.), thrombin (TH, CAS:506-32-1, SIGMA reagent Co., Ltd.), adenosine diphosphate (ADP, CAS:58-64-0, SIGMA reagent Co., Ltd.), platelet activating factor (PAF, CAS:74389-68-7, SIGMA reagent Co., Ltd.).
An experimental instrument: platelet aggregation apparatus: MODEL 700, CHRONO-LOG
Blood for experiment: fresh pig carotid blood
Grouping experiments:
the compound of the invention is warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro, and the negative contrast is normal saline.
Preparing used reagents:
the anticoagulant is 3.8% sodium citrate solution prepared from normal saline, the platelet activator is 50U/mL TH solution prepared from normal saline, 1mM ADP solution prepared from normal saline, and 5 × 10 solution prepared from normal saline-5mM PAF solution. The final concentration of inducer was 1U/mL TH, 20. mu. ADP M, PAF 1X 10-6mM。
The experimental method comprises the following steps:
a blood collection bottle silanized and air dried one day in advance was charged with 50mL of an aqueous solution of sodium citrate (1: 9 ratio to blood). Collecting fresh pig carotid artery blood, shaking gently, rapidly centrifuging at 1000rpm for 10min, collecting supernatant to obtain Platelet Rich Plasma (PRP), centrifuging the rest blood at 3500rpm for 10min, and collecting supernatant to obtain Platelet Poor Plasma (PPP).
Add 500. mu.L of PPP to the cuvette on the platelet aggregation apparatus, to the PPP wells, add 480. mu.L of PRP to the cuvette, and to the PRP wells. The PRP used was incubated at 37 ℃ for 10min before measurement. After the instrument started running, the rotor was added and zeroed, 10 μ L of saline or compound aqueous solution was added to the PRP, followed by 10 μ L of platelet activation inducer. The change of the light transmittance is recorded, and the inhibition rate of the compound on platelet aggregation is calculated. The results of the experiment are shown in FIG. 4.
The experimental results are as follows:
experimental results show that warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro can inhibit platelet aggregation induced by TH and ADP. The inhibition rate of TH-induced platelet aggregation is 20.0%; the inhibition rate of ADP-induced platelet aggregation was 7.0%. warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro is not a prodrug because it inhibits platelet aggregation in vitro. This is an unexpected technical effect.
EXAMPLE 20 evaluation of the Effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro to reduce the level of GPIIb/IIIa in vivo
Experimental Material
Sodium citrate (CAS: 68-04-2, national drug group chemical Co., Ltd.), NS (Shijiazhuang Siyao Co., Ltd.), distilled water;
experimental sample
Example 17 warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro treatment of carotid artery blood in rats after systemic circulation
Experimental methods
The detection is carried out by adopting a rat platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa) enzyme linked immunosorbent assay kit.
Preparation of standard substance
Taking one standard substance out of the kit, centrifuging at 6000-10000rpm for 30S, dissolving with 1mL of sample diluent, repeatedly sucking and pumping 5 times by aligning a gun head to the bottom of a freezing tube to assist the dissolution, fully mixing to obtain a standard substance S7, sequentially arranging 7 centrifuge tubes of 1.5mL, adding 250 mu L of sample diluent into each centrifuge tube, sucking 250 mu L of the standard substance S7 into an S6 centrifuge tube, blowing and uniformly mixing to obtain S6-S1, and taking S0 as the sample diluent. The concentrations of the standard substances are respectively as follows: s7(400ng/mL), S6(200ng/mL), S5(100ng/mL), S4(50ng/mL), S3(25ng/mL), S2(12.5ng/mL), S1(6.25ng/mL), S0(0 ng/mL).
Collection of samples
Using 3.8% sodium citrate solution as anticoagulant, collecting warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro for treating carotid artery blood after anti-arterial thrombosis circulation of rats, centrifuging for 15min at 1000rpm at 4 ℃ within 30min, and taking supernatant (blood plasma) as a sample for detection.
Sample detection
Transferring the reagents to room temperature (18-25 deg.C) and balancing for at least 30min, and respectively setting standard pores and warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro pores. And respectively adding 100 mu L of standard substance or sample to be detected into each hole of the precoated enzyme label plate, slightly shaking and uniformly mixing, covering the plate and sticking, and incubating for 2h at 37 ℃. Discarding the liquid, spin-drying, adding 100 μ L of biotin-labeled antibody working solution into each well, covering with a new plate, and incubating at 37 deg.C for 1 h. Discarding the liquid, spin-drying, washing the plate for 3 times, soaking for 2min each time, 200 μ L per hole, and spin-drying. 100 mu L of horse radish peroxidase labeled avidin working solution is added into each hole, a new plate is covered, and the temperature is raised for 1h at 37 ℃. Discarding the liquid, spin-drying, washing the plate for 5 times, sequentially adding 90 μ L of the substrate solution to each well, developing at 37 deg.C in dark for 15-30min, sequentially adding 50 μ L of the termination solution to each well, and terminating the reaction. The optical density (OD value) of each well was measured sequentially at a wavelength of 450nm with a microplate reader within 5 min. And (5) drawing a standard curve according to the OD value of the standard substance, calculating the sample concentration of the sample, and listing the data in a figure 5.
Results of the experiment
As can be seen from the data in FIG. 5, the levels of GPIIb/IIIa in 0.1. mu. mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro treated rats were significantly lower (p <0.05) than in saline treated rats, i.e., warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro reduced levels of GPIIb/IIIa in rats at a dose of 0.1. mu. mol/kg and comparable to levels of GPIIb/IIIa in 167. mu. mol/kg aspirin treated rats. As can be seen, the activity of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro for reducing the content of GP IIb/IIIa in the body of a rat is 167 times stronger than that of aspirin. This is an unexpected technical effect.
EXAMPLE 21 evaluation of the Effect of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro on reducing the level of coagulation factor II in rats
Experimental Material
Sodium citrate (CAS: 68-04-2, national drug group chemical reagents, Inc.), physiological saline (Shijiazhuang Siyao, Inc.), pipette (Germany Prandde), distilled water;
experimental sample
EXAMPLE 18 carotid artery blood of rats after warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro
Experimental methods
The experiment was performed using a rat F II ELISA kit.
Collection of samples
Collecting 0.1 mu mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro treated carotid artery blood of a rat by using a 3.8% sodium citrate solution as an anticoagulant, centrifuging the blood for 15min at 1000rpm at 4 ℃ within 30min, and taking supernatant (blood plasma) as a sample for detection.
Preparation of standard substance
And (3) taking the standard substance out of the kit, diluting 150 mu L of original-time standard substance by using 150 mu L of standard substance diluent, fully mixing to obtain a standard substance S5, arranging 4 centrifuge tubes of 1.5mL in sequence, adding 150 mu L of sample diluent respectively, sucking 150 mu L of standard substance S5 into an S4 centrifuge tube, blowing and uniformly mixing, and sequentially preparing an S5-S1 standard substance. The concentrations of the standard were S5(6ng/mL), S4(3ng/mL), S3(1.5ng/mL), S2(0.75ng/mL), S1(0.375ng/mL) and S0(0ng/mL), respectively.
A blank hole, a standard hole and a serum hole to be detected are respectively arranged. Adding 50 mu L of standard substance into the enzyme-labeled coated plate, adding 40 mu L of sample diluent into the serum hole to be detected, adding 10 mu L of serum to be detected, and slightly shaking and uniformly mixing. The plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min. Carefully remove the coversheet membrane, discard the liquid, and wash the plate 5 times. Adding 50 mu L of enzyme labeling reagent into each hole except the blank hole, incubating for 30min at 37 ℃, washing, adding 50 mu L of color development agent A into each hole, adding 50 mu L of color development agent B, shaking gently, mixing uniformly, and developing for 10min at 37 ℃ in a dark place. The reaction was stopped by adding 50. mu.L of stop solution to each well, and the absorbance (OD value) of each well was measured at a wavelength of 450nm while the blank wells were zeroed within 15 min. A standard curve was drawn according to the OD value of the standard, and the concentration of the serum sample was calculated, and the data are shown in FIG. 6.
Results of the experiment
As can be seen from the data in FIG. 6, the FII content of 0.1. mu. mol/kg warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro treated rat is significantly lower than that of saline treated rat (p <0.05), i.e. warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro at a dose of 0.1. mu. mol/kg can decrease FII content in vivo in rat and is comparable to that of 4.87. mu. mol/kg warfarin treated rat. As can be seen, the activity of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro for reducing the FII content in the rat body is 48.7 times stronger than that of warfarin. This is an unexpected technical effect.
Claims (6)
1. The structure is warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro,
2. a method of making warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro according to claim 1, the method comprising:
(1) synthesizing warfarin-4-O-benzyl acetate;
(2) synthesizing warfarin-4-O-acetic acid;
(3) synthesizing HCl, Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl by adopting a liquid phase condensation method with Dicyclohexylcarbodiimide (DCC) as a condensing agent and 1-hydroxybenzotriazole (HOBt) as a catalyst;
(4) adopting a liquid phase condensation method with DCC as a condensing agent and HOBt as a catalyst, condensing warfarin-4-O-acetic acid with HCl, Leu-Pro-Asn-Ile-Ser-Lys (Z) -Pro-OBzl;
(5) synthesizing warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro.
3. The use of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro according to claim 1 in the preparation of an anti-arterial thrombosis medicament.
4. The use of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1 in the preparation of an anti-venous thrombosis medicament.
5. The use of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro of claim 1 in the preparation of a medicament for inhibiting platelet aggregation.
6. Use of warfarin-4-O-acetyl-Leu-Pro-Asn-Ile-Ser-Lys-Pro according to claim 1 for the preparation of a medicament for reducing the level of GPIIb/IIIa in vivo.
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