CN107686506B - warfarin-4-O-acetyl-GPRP, synthesis, activity and application thereof - Google Patents
warfarin-4-O-acetyl-GPRP, synthesis, activity and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了华法林‑4‑O‑乙酰‑GPRP,公开了它的制备方法,公开了它的抗静脉血栓活性，公开了它抗三氯化铁诱导的小鼠动脉血栓活性，公开了它降低体内凝血因子Ⅸ的含量的活性，公开了它没有华法林样出血风险的优势。因而本发明公开了它在制备抗静脉血栓药物中的应用、在制备抗动脉血栓药物中的应用、在制备凝血因子Ⅸ拮抗剂中的应用。
The invention discloses warfarin-4-O-acetyl-GPRP, its preparation method, its anti-venous thrombosis activity, its anti-ferric chloride-induced mouse arterial thrombosis activity, and a Its activity in reducing the amount of coagulation factor IX in the body discloses the advantage that it does not have the risk of warfarin-like bleeding. Therefore, the present invention discloses its application in the preparation of an anti-venous thrombosis drug, an application in the preparation of an anti-arterial thrombosis drug, and an application in the preparation of a coagulation factor IX antagonist.
Description
Technical Field
The present invention relates to warfarin-4-O-acetyl-GPRP, its preparation process, its anti-thrombotic activity and its action of reducing the content of blood coagulation factor IX in vivo. The invention thus relates to its use in the preparation of a medicament for the treatment of venous thrombosis, in the preparation of a medicament for the treatment of arterial thrombosis and in the preparation of an antagonist of blood coagulation factor IX. 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, due to the narrow window of warfarin, its underdosage may cause pulmonary embolism, while overdosing has a risk of causing fatal hemorrhage. A great deal of structural modification is carried out on warfarin for more than 50 years, but analogs with strong anti-thrombus activity 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. After 5 years of exploration, the inventor finds that warfarin 4-position is modified by acetyl GPRP tetrapeptide, and the generated warfarin-4-O-acetyl-GPRP shows excellent activity in an in vivo anti-arteriovenous thrombosis model. Obviously, the compound is not a prodrug of warfarin. warfarin-4-O-acetyl-GPRP has the anti-arterial thrombosis activity which is 4.87 times stronger than that of warfarin and the anti-arterial thrombosis activity which is 240 times stronger than that of aspirin, and besides, the warfarin-4-O-acetyl-GPRP has no bleeding risk similar to 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-GPRP.
The second content of the invention is to provide a preparation method of warfarin-4-O-acetyl-GPRP, which comprises the following steps:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesizing HCl, Gly-Pro-Arg (NO) by adopting liquid phase condensation method with Dicyclohexylcarbodiimide (DCC) as condensing agent and 1-hydroxybenzotriazole (HOBt) as catalyst2)-Pro-OBzl；
4) Liquid phase condensation method using DCC as condensing agent and HOBt as catalyst, warfarin-4-O-BAcid with HCl-Gly-Pro-Arg (NO)2) -Pro-OBzl condensation;
5) synthesizing warfarin-4-O-acetyl-GPRP.
The third content of the invention is to evaluate the anti-venous thrombosis effect of warfarin-4-O-acetyl-GPRP.
The fourth aspect of the present invention is to evaluate the antithrombotic effect of warfarin-4-O-acetyl-GPRP.
The fifth aspect of the present invention was to evaluate the effect of warfarin-4-O-acetyl-GPRP in reducing the IX content of blood coagulation factor in vivo.
Drawings
FIG. 1. synthetic route for warfarin-4-O-acetyl-GPRP tetrapeptide (i) benzyl bromo-2-acetate, acetone, K2CO3,45℃；(ii)CH3OH,Pd/C,H2(ii) a (iii) DCC, HOBt, NMM, THF; (iv)4N hydrogen chloride in ethyl acetate; (v) trifluoroacetic acid, trifluoromethanesulfonic acid.
Figure 2 warfarin-4-O-acetyl-GPRP anti-venous thrombosis activity, n ═ 6;
fig. 3 shows the results of evaluating the antithrombotic activity of warfarin-4-O-acetyl-GPRP, where n is 8;
FIG. 4 the effect of warfarin-4-O-acetyl-GPRP on FIX content in vivo, n ═ 3;
figure 5 effect of warfarin-4-O-acetyl-GPRP on rat tail bleeding time, n-10.
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 6.62g (20.00mmol) of warfarin in a 250mL eggplant flask, adding about 70mL of acetone, not completely dissolving the acetone, heating in a 45 ℃ oil bath and stirring until the warfarin is dissolved, adding 3.50mL (22.00mmol) of benzyl bromoacetate, and continuing the reaction under the 45 ℃ oil bath. After 72h, the progress of the reaction was monitored by thin layer chromatography (TLC, petroleum ether/ethyl acetate 2:1), warfarin had substantially disappeared and the colorless solid formed in the reaction was filtered offThe filtrate was evaporated under reduced pressure to remove acetone to give a pale yellow oil, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 8:1) to give 5.4g (59%) of the title compound 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
4.01g (8.79mmol) of warfarin-4-O-benzyl acetate was dissolved in 50mL of methanol, 405mg of Pd/C was added, air was pumped out of a water pump with stirring, hydrogen was introduced, and the operation was repeated 4 times with stirring at room temperature with hydrogen. The reaction was monitored by TLC for completion, palladium on carbon (Pd/C) was removed by filtration, and the filtrate was evaporated under reduced pressure to remove the solvent to give 2.74g (85.2%) 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-R (NO)2)-P-OBzl
3.509g (11.00mmol) of Boc-R (NO)2) -OH was added to a 250mL eggplant flask, dissolved in 80mL dry tetrahydrofuran, and 1.485g (11.00mmol) of HOBt was added under ice bath (0 ℃ C.), stirred for 10 minutes, 2.632g (12.19mmol) of DCC was added, and activated for 30 min. Colorless solids precipitated from the reaction solution. Adding 2.516g (10.41mmol) of HCl Pro-OBzl into the reaction solution under ice bath, adjusting pH value to 9 with N-methylmorpholine, stirring at room temperature for 12h, monitoring reaction progress by TLC (dichloromethane/methanol 20:1), filtering to remove colorless solid in the reaction solution, evaporating the filtrate under reduced pressure to remove solvent, dissolving residue with 100mL ethyl acetate, filtering to remove insoluble substance, and separating the filtrateRespectively using saturated NaHCO3Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3), 5% KHSO4Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3), saturated NaHCO3The solution (30 mL. times.3) was washed with a saturated NaCl solution (30 mL. times.3), and the resulting ethyl acetate phase was dried over anhydrous sodium sulfate for 12 hours or more. The sodium sulfate was filtered off, and the solvent was removed from the filtrate under reduced pressure to give a yellow oil, which was purified by silica gel column chromatography (dichloromethane/methanol 80:1) to give 4.05g (80.2%) of a colorless solid product. ESI-MS (M/e):507[ M + H]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝7.33(m,5H),6.98(d,J＝7.8Hz,1H),5.10(s,2H),4.40(m,1H),4.37(s,1H),3.92(m,1H),3.62(m,1H),3.10(s,2H),2.21(m,1H),1.94(t,J＝6.3Hz,2H),1.88(m,1H),1.56～1.45(m,4H),1.37(s,9H)。
EXAMPLE 4 preparation of HCl.Arg (NO)2)-Pro-OBzl
1.80g (3.56mmol) of Boc-Arg (NO) was weighed2) Pro-OBzl was added to a 100mL eggplant flask, 15mL of dry ethyl acetate was added to dissolve completely, 30mL of 4N ethyl acetate solution of hydrogen chloride was added under ice salt bath, and the reaction was stirred continuously under ice bath. TLC (dichloromethane/methanol ═ 20:1) showed the disappearance of the starting material spot, the reaction solution was concentrated under reduced pressure using a water pump, the residue was redissolved with dry ethyl acetate and concentrated under reduced pressure for 3 times to give a colorless solid, which was then infiltrated with anhydrous ether and concentrated under reduced pressure for 3 times to give 1.53g (97%) of the product as a colorless solid.
EXAMPLE 5 preparation of Boc-Gly-Pro-OBzl
3.862g (22.05mmol) of Boc-Gly were added to a 250mL eggplant flask, 90mL of dried tetrahydrofuran was added, and they were completely dissolved with stirring, and 2.971g (22.00mmol) of HOBt and 5.384g (24.92mmol) of DCC were added in an ice bath (0 ℃ C.), and activated with stirring in the ice bath for 30 min. Colorless solids were observed to precipitate. 4.833g (20.00mmol) HCl Pro OBzl was added to the reaction solution in ice bath, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, the reaction solution was stirred at room temperature for 17 hours, TLC (dichloromethane/methanol 20:1) was used to monitor the progress of the reaction, the starting material point disappeared, the insoluble matter in the reaction solution was filtered off, the solvent was evaporated from the filtrate under reduced pressure, the residue was dissolved in 100mL ethyl acetate, and the insoluble colorless solid was filtered offThe filtrate is respectively saturated NaHCO3Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), 5% KHSO4Solution (40 mL. times.3), saturated NaCl solution (40 mL. times.3), saturated NaHCO3The solution (40 mL. times.3) was washed with a saturated NaCl solution (40 mL. times.3), and the resulting ethyl acetate phase was dried over anhydrous sodium sulfate for 2 hours or more. The sodium sulfate was filtered off, and the solvent was evaporated from the filtrate under reduced pressure to give a pale yellow solid, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate 3:1) to give 7.02g (96.7%) of a colorless solid product. ESI-MS (M/e) 363[ M + H]+。
EXAMPLE 6 preparation of Boc-Gly-Pro
4.774g (13.18mmol) of Boc-Gly-Pro-OBzl were weighed into a 250mL eggplant flask, dissolved by adding 50mL of methanol, 1.0g of Pd/C was added, air was pumped out by a water pump with stirring, hydrogen was introduced, and the operation was repeated 3 times, and the reaction was stirred with hydrogen at room temperature for about 48 hours. TLC (dichloromethane/methanol ═ 20:1) monitored the progress of the reaction, the starting material point disappeared, Pd/C was filtered off, and the solvent was evaporated from the filtrate under reduced pressure to give 3.411g (95.1%) of a colorless solid. ESI-MS (M/e):273[ M + H]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝6.83(t,J＝6.0Hz,1H),4.22(dd,J1＝3.0Hz,J2＝9.0Hz,1H),3.80(dd,J1＝5.7Hz,J2＝17.1Hz,1H),3.67(dd,J1＝5.7Hz,J2＝16.8Hz,1H),3.48(m,1H),3.38(m,1H),2.10(m,1H),1.91(m,1H),1.83(m,1H),1.38(s,9H)。
EXAMPLE 7 preparation of Boc-Gly-Pro-Arg (NO)2)-Pro-OBzl
1.755g (6.45mmol) of Boc-Gly-Pro was charged into a 250mL eggplant flask, dissolved in 80mL of dry tetrahydrofuran, 0.8619g (6.38mmol) of HOBt was added under ice bath (0 ℃ C.), stirred for 5min, 1.5045g (6.96mmol) of DCC was added, and activated for 30 min. After a colorless solid had precipitated, 2.566g (5.80mmol) of HCl & Arg (NO) were added2) -Pro-OBzl is added into the reaction solution under ice bath, the pH value is adjusted to 9 by NMM, the reaction is stirred at room temperature for 10h, TLC (dichloromethane/methanol ═ 20:1) monitors the reaction progress, the raw material point disappears, colorless solid is filtered off, the filtrate is decompressed and evaporated to remove the solvent, the residue is dissolved by ethyl acetate, insoluble substances are filtered off, and the filtrate is respectively dissolved by saturated NaHCO3Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3) wash, 5% KHSO4Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3), saturated NaHCO3The solution (30 mL. times.3), the saturated NaCl solution (30 mL. times.3), and the ethyl acetate phase were dried over anhydrous sodium sulfate for 10 hours or more. The sodium sulfate was filtered off, and the solvent was distilled off from the filtrate under reduced pressure to give a pale yellow oil, which was purified by silica gel column chromatography (dichloromethane/methanol 50:1) to give 2.31g (60.3%) of a colorless solid product. ESI-MS (M/e):662[ M + H]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝8.49(s,1H),8.19(d,J＝7.5Hz,1H),7.35(m,5H),6.79(s,1H),5.10(t,J＝12.9Hz,1H),4.45～4.42(m,2H),4.37(dd,J1＝4.5Hz,J2＝8.1Hz,1H),3.83～3.66(m,2H),3.6～3.37(m,4H),3.11(s,2H),2.21(m,1H),1.94(t,J＝6.3Hz,2H),1.88(m,1H),1.56～1.45(m,4H),1.37(s,9H)。
EXAMPLE 8 preparation of HCl.Gly-Pro-Arg (NO)2)-Pro-OBzl
0.908g (1.38mmol) Boc-Gly-Pro-Arg (NO) was weighed2) Pro-OBzl in a 100mL eggplant flask, 5mL of dry ethyl acetate was added to dissolve completely, 15mL of 4N ethyl acetate solution of hydrogen chloride was added under ice salt bath, and the reaction was stirred continuously under ice bath. TLC (dichloromethane/methanol 15:1) detected the progress of the reaction, the starting material point disappeared, the reaction mixture was concentrated under reduced pressure using a water pump, the residue was redissolved with dry ethyl acetate and concentrated under reduced pressure for 3 times to give a colorless solid, which was further soaked with anhydrous ether and concentrated under reduced pressure for 3 times to give 0.81g (99%) of colorless solid product.
EXAMPLE 9 preparation of warfarin-4-O-acetyl-Gly-Pro-Arg (NO)2)-Pro-OBzl
0.5875g (1.61mmol) of warfarin-4-O-acetic acid was added to a 100mL eggplant flask, dissolved in 40mL of dry tetrahydrofuran, and activated for 30min by adding 0.2170g (1.61mmol) of HOBt and 0.3790g (1.75mmol) of DCC under ice bath (0 ℃). A colorless solid precipitated. 0.870g (1.46mmol) of HCl-Gly-Pro-Arg (NO)2) -Pro-OBzl was added to the reaction solution in ice bath, pH was adjusted to 9 with N-methylmorpholine, the reaction was stirred at room temperature for 12h, TLC (dichloromethane/methanol 20:1) was monitored for progress of the reaction, starting material point disappeared, colorless solid was filtered offThe solvent was evaporated from the filtrate under reduced pressure, the residue was dissolved in 40mL of ethyl acetate, the insoluble colorless solid was filtered off, and the filtrate was separately washed with saturated NaHCO3Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3), 5% KHSO4Solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3), saturated NaHCO3The solution (30 mL. times.3), saturated NaCl solution (30 mL. times.3) was washed, and the ethyl acetate phase was dried over anhydrous sodium sulfate for 5 hours. The sodium sulfate was filtered off, and the solvent was evaporated from the filtrate under reduced pressure to give a yellow oil, which was purified by silica gel column chromatography (dichloromethane/methanol ═ 40:1) to give 0.909g (68.6%) of a colorless solid. ESI-MS (M/e):909[ M + H]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝8.51～8.43(m,2H),8.22(d,J＝7.5Hz,1H),7.90(d,J＝7.8Hz,1H),7.63(t,J＝7.5Hz,1H),7.35(m,12H),5.10(s,2H),4.90(t,J＝7.2Hz,1H),4.58(s,2H),4.52～4.34(m,3H),4.06(m,2H),3.67～3.42(m,4H),3.47(d,J＝7.5Hz,2H),3.15(s,2H),2.20(m,1H),2.14(s,3H),1.97(m,1H),1.90(t,J＝6.3Hz,2H),1.86～1.70(m,4H),1.70～1.53(m,4H)。
EXAMPLE 10 preparation of warfarin-4-O-acetyl-Gly-Pro-Arg-Pro
0.385g (0.42mmol) of warfarin-4-O-acetyl-Gly-Pro-Arg (NO)2) Pro-OBzl was added to a 100mL eggplant flask, which was dissolved with 4.5mL trifluoroacetic acid under an ice-salt bath, then 1.5mL of trifluoromethanesulfonic acid was added, the reaction was stirred for 1h under ice salt bath with maintaining dry conditions, TLC (ethyl acetate/water/glacial acetic acid ═ 2:1:1) monitored the progress of the reaction, the starting material point disappeared, acid gas in the eggplant flask was removed by water pump under ice bath, then 50mL of anhydrous ether preserved in advance by refrigeration is added in the ice bath, orange solid is precipitated, the solution is stirred for 10min in the ice bath and then is kept stand, supernatant liquid is poured out, the steps are repeated for 4 times, then, the residue was subjected to removal of the remaining ether with a water pump to obtain an orange solid, the orange solid was dissolved in 2mL of distilled water, the pH was adjusted to 7 with 10% aqueous ammonia in ice bath, the solution was subjected to reduced pressure filtration of insoluble solids to obtain an orange filtrate, and the orange filtrate was subjected to RP-C.18Column chromatography purification (70% aqueous methanol) gave 0.111g (34%) of the product as a colourless solid ESI-MS (M/e):774[ M + H ]]+；1H-NMR(300MHz,DMSO-d6):δ/ppm＝8.46(m,1H),8.16～8.06(m,2H),7.90(d,J＝7.2Hz,1H),7.64(t,J＝7.2Hz,1H),7.45～7.35(m,5H),7.26(t,J＝7.2Hz,2H),7.16(d,J＝7.2Hz,1H),4.90(t,J＝7.2Hz,1H),4.58(s,2H),4.45(m,2H),4.14～4.02(m,3H),3.55(m,4H),3.47(d,J＝6.9Hz,2H),3.06(s,2H),2.14(s,3H),2.01～1.57(m,12H)。
EXAMPLE 11 evaluation of the anti-thrombotic Effect of warfarin-4-O-acetyl-GPRP tetrapeptide
Experimental Material
Uratan (ethyl carbamate, CAS: 51-79-6, chemical reagents of national drug group Co., Ltd.), and normal saline (Shijiazhuang four drugs Co., Ltd.) warfarin sodium (CAS: 129-06-6, Bailingwei science and technology Co., Ltd.).
Laboratory animal
SD strain rats, male, 250 + -20 g, purchased from Experimental animals technologies, Inc. of Wei Tongli, Beijing.
Experimental methods the rat inferior vena cava ligation model was used for the experiments.
Grouping and administration dose:
the dose of the warfarin-4-O-acetyl-GPRP tetrapeptide is 1.0 mu mol/kg, the dose of the positive control warfarin is 4.87 mu mol/kg, and the negative control is normal saline.
Reagent preparation
The anesthetic is a 20% urethane solution prepared from normal saline.
And (3) experimental operation:
rats were acclimatized and fasted for one day prior to surgery and were gavaged at a dose of 0.3mL/100g body weight. The administration is carried out 30min later and 2min before operation, and 20% urethane solution is used for abdominal cavity administration anesthesia. Rats were fixed on a rat fixing plate, and 5mL 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 leucorrhea, descending to coagulated gland, and ascending to expose one corner of liver. The organs such as small intestine in the abdominal cavity were removed and 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 with suture soaked with physiological saline at junction of inferior vena cava and left renal vein, moving intestine and other organs back to abdominal cavity according to anatomical position, and suturing abdominal cavity layer by layer with suture.
After operation, the rat is placed in an environment with the temperature of 25-28 ℃ for circulation for 4 hours, 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 thrombus was weighed and the results were counted using the t-test. The operation was performed alternately with two per group. The experimental data are shown in figure 2.
The experimental results are as follows:
the results show that the plug weight of warfarin-4-O-acetyl-GPRP treated rats at 1.0. mu. mol/kg dose (7.4. + -. 1.5mg) is significantly less than the plug weight of warfarin treated rats at 4.87. mu. mol/kg dose (15.4. + -. 3.2mg, p < 0.01). As can be seen, warfarin-4-O-acetyl-GPRP tetrapeptide has an anti-thrombus activity at least 4.87 times stronger than that of warfarin.
EXAMPLE 12 evaluation of the antithrombotic Effect of warfarin-4-O-acetyl-GPRP
Experimental materials:
chloral hydrate (ethyl carbamate, CAS: 302-17-0, national pharmaceutical group chemical Co., Ltd.), ferric chloride (CAS: 7705-08-0, national pharmaceutical group chemical Co., Ltd.), and physiological saline (Shijiazhuang Siyao Co., Ltd.).
Experimental animals:
ICR strain mouse, male, 20 + -2 g, purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing.
The experimental method comprises the following steps: the experiment was carried out using a model of arterial thrombosis induced by ferric chloride.
Grouping and administration dose:
the dose of warfarin-4-O-acetyl-GPRP tetrapeptide of the compound is 1.0 mu mol/kg; the positive control aspirin dose was 240. mu. mol/kg, and the negative control was normal saline.
Preparing used reagents:
the anesthetic is a 5% chloral hydrate aqueous solution prepared by normal saline, and the ferric trichloride solution is a 20% ferric trichloride aqueous solution prepared by normal saline.
And (3) experimental operation:
after anesthetizing the mice by intraperitoneal injection with 5% chloral hydrate solution, the mice were fixed on a mouse fixing plate, the skin was cut along the median line of the abdomen, the muscle layer of the abdomen was cut along the white line of the abdomen, the organs such as small intestine in the abdomen were removed and wrapped with gauze soaked with normal saline. Blunt separating perivascular connective tissue, exposing abdominal aorta, peeling off it from mouse inferior vena cava, placing sealing membrane 0.6cm wide under abdominal aorta, and soaking with 20% FeCl3A strip of solution filter paper (1.0 cm. times.0.4 cm) was wrapped around the isolated abdominal aorta segment and the segment was wrapped with a sealing film to prevent FeCl3The solution touches other organs and blood vessels. After 15min, taking down the filter paper strip, ligating blood vessels at two ends of the filter paper strip, accurately cutting off a blood vessel section wrapped by the filter paper strip, sucking residual blood in the blood vessel by using clean filter paper, carefully sliding along the outside of the blood vessel by using an ophthalmic forceps, completely taking out the thrombus block, sucking floating blood on the surface of the thrombus block by using the filter paper, accurately weighing the wet weight of the thrombus block, and representing the anti-arterial thrombosis activity. Data were counted using t-test.
The experimental results are as follows:
the data are shown in FIG. 3. The result shows that the thrombus weight (0.52 plus or minus 0.19mg) of a warfarin-4-O-acetyl-GPRP treated mouse is obviously less than that of a normal saline treated rat thrombus (1.02 plus or minus 0.28mg, p is less than 0.01), and the warfarin-4-O-acetyl-GPRP tetrapeptide shows the anti-arterial thrombus activity. And the thrombus weight of warfarin-4-O-acetyl-GPRP treated mice at a dose of 1.0 mu mol/kg (0.52 +/-0.19 mg) was not significantly different from the thrombus weight of aspirin treated mice at a dose of 240 mu mol/kg (0.39 +/-0.16 mg), indicating that the compound warfarin-4-O-acetyl-GPRP had an anti-arterial thrombus activity at least 240 times stronger than aspirin. This is an unexpected technical effect.
EXAMPLE 13 evaluation of the Effect of warfarin-4-O-acetyl-GPRP on reducing the IX level of blood coagulation factor in rats
Experimental Material
Sodium citrate (CAS: 68-04-2, national drug group chemical reagents Co., Ltd.), and physiological saline (Shijiazhuang Siyao Co., Ltd.).
Experimental sample
Rat carotid blood after 4 days of continuous administration
Experimental methods
The experiment was performed using a rat FIX ELISA kit.
Collection of samples
Using 3.8% sodium citrate solution as anticoagulant, collecting 1.0 mu mol/kg warfarin-4-O-acetyl-GPRP tetrapeptide carotid artery blood of rats treated for 4 days continuously by a group to be tested, collecting rat carotid artery blood treated for 4 days continuously by normal saline by a control group, centrifuging at 1000rpm for 15min 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 standard concentrations were S5(200IU/L), S4(100IU/L), S3(50IU/L), S2(25IU/L), S1(12.5IU/L) and S0(0IU/L), 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. 4.
Results of the experiment
As can be seen from the data in FIG. 4, FIX content of 1.0. mu. mol/kg warfarin-4-O-acetyl-GPRP-treated rats was significantly lower than FIX content of saline-treated rats (p < 0.05), i.e., warfarin-4-O-acetyl-GPRP could decrease FIX content in rats at a dose of 1.0. mu. mol/kg.
EXAMPLE 14 evaluation of the Effect of warfarin-4-O-acetyl-GPRP on bleeding time in rats
Experimental Material
Normal saline (Shijiazhuang four drugs Co., Ltd.), warfarin sodium (CAS: 129-06-6, Bailingwei science Co., Ltd.).
Laboratory animal
SD strain rats, male, 250 + -20 g, purchased from Experimental animals technologies, Inc. of Wei Tongli, Beijing.
Experimental methods
Rats were evaluated using a rat tail bleeding model. After administration of 4.87. mu. mol/kg warfarin or 1.0. mu. mol/kg warfarin-4-O-acetyl-GPRP for 30min, a 2mm deep wound was opened with a scalpel at a distance of 4cm from the rat tail, and then timing was started, blood was wiped off with filter paper every 5s, and the timing was stopped when no blood stain could be seen on the filter paper, and the time counted was the bleeding time. The results of the experiment are shown in FIG. 5.
Results of the experiment
The results in FIG. 5 show that the bleeding time of rat tail after administration of warfarin-4-O-acetyl-GPRP at 1.0. mu. mol/kg was not significantly different from that of normal saline treated rat tail (p > 0.05). The bleeding time of the rat tail treated by warfarin at 4.87 mu mol/kg is obviously longer than that of the rat tail treated by normal saline, and the bleeding risk is obviously shown.
Claims (4)
1. The structural formula is warfarin-4-O-acetyl-GPRP,
2. a process for preparing warfarin-4-O-acetyl-GPRP of claim 1 comprising:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesizing HCl, Gly-Pro-Arg (NO) by adopting liquid phase condensation method with Dicyclohexylcarbodiimide (DCC) as condensing agent and 1-hydroxybenzotriazole (HOBt) as catalyst2)-Pro-OBzl；
4) A liquid-phase condensation method using DCC as condensing agent and HOBt as catalyst for preparing warfarin-4-O-acetic acid and HCl-Gly-Pro-Arg (NO)2) -Pro-OBzl condensation;
5) synthesizing warfarin-4-O-acetyl-GPRP.
3. Use of warfarin-4-O-acetyl-GPRP according to claim 1 in the preparation of an anti-venous thrombosis medicament.
4. The use of warfarin-4-O-acetyl-GPRP of claim 1 for the preparation of an anti-arterial thrombosis medicament.
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| CN113929736B (en) * | 2020-06-29 | 2024-01-26 | 首都医科大学 | Gly-Pro-Arg-Pro-oxaminocarbonyl warfarin, synthesis, activity and application thereof |
| CN113929741A (en) * | 2020-06-29 | 2022-01-14 | 首都医科大学 | Warfarin-4-O-acetyl-Gly-Pro-Arg-Pro-AA1 and its synthesis, activity and application |
| CN113929735B (en) * | 2020-06-29 | 2023-08-01 | 首都医科大学 | Gly-Pro-Arg-Pro-NHCH 2 CH 2 NH-warfarin, its synthesis, activity and use |
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| GB0516091D0 (en) * | 2005-08-04 | 2005-09-14 | Haemostatix Ltd | Therapeutic agent |
| KR101167285B1 (en) * | 2011-11-18 | 2012-07-23 | 에이앤펩주식회사 | Niacin-peptides having skin whitening effect and uses thereof |
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