CN113929735A - Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin, its synthesis, activity and application - Google Patents
Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin, its synthesis, activity and application Download PDFInfo
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Abstract
The invention discloses Gly-Pro-Arg-Pro-NHCH with the following formula2CH2NH-warfarin, discloses a preparation method thereof, discloses the anti-thrombus activity thereof, and further discloses that the anti-thrombus activity thereof has no bleeding side effectAnd discloses its activity in reducing factor II, reducing tissue factor/factor VII and reducing the content of soluble fibrin monomer complex. Therefore, the invention discloses the application of the derivative in the preparation of an anti-vein thrombosis medicament without bleeding side effects, the preparation of a blood coagulation factor II antagonist, the preparation of a tissue factor/blood coagulation factor VII antagonist and the preparation of a soluble fibrin monomer compound antagonist.
Description
Technical Field
The invention relates to Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin, to a process for its preparation, to its anti-thrombotic activity, further to its advantage of being free from bleeding side effects, and to its activity in reducing factor II, in reducing tissue factor/factor VII and in reducing the content of soluble fibrin monomer complex. The invention thus relates to its use in the preparation of an anti-thrombotic agent without bleeding side effects, of a factor II antagonist, of a tissue factor/factor VII antagonist and of a soluble fibrin monomer complex antagonist. The invention belongs to the field of biological medicine.
Background
Thrombotic diseases are diseases seriously threatening the life and health of human beings, and have the characteristics of high morbidity and high mortality. According to the location of the formation of thrombus mass, the thrombotic diseases can be divided into arterial thromboembolic diseases and venous thromboembolic diseases, wherein the venous thromboembolic diseases (VTE) are the most common thrombotic diseases, the main symptoms of the venous thromboembolic diseases comprise Deep Venous Thrombosis (DVT) and Pulmonary Embolism (PE), the death rate caused by PE is high, and the DVT can bring serious complications if the DVT is kept away. The main clinical treatment scheme of VTE is anticoagulation treatment using anticoagulation drugs, and vitamin K1 antagonists represented by warfarin are one of the most commonly used anticoagulation drugs, but warfarin has the disadvantages of large individual dosage difference, serious bleeding side effect, poor patient compliance, and the like. In response to the deficiencies of warfarin, the inventors have disclosed a class of modified warfarin (CN 107488157 a, CN 107488211 a, CN 107488214 a, CN 107488212A, CN 107488213A, CN 107488210 a, CN 107474030 a). However, they are all vitamin K inhibitors and have no effect on tissue factor/factor VII and soluble fibrin monomer complexes that play an important role in the process of thrombosis. To overcome such modified warfarinIn view of the shortcomings, the inventors have conducted studies and researches with different objectives. Later, Gly-Pro-Arg-Pro-NHCH was found2CH2NH-warfarin has no effect on vitamin K, but acts on the anti-venous thrombosis agent of tissue factor/coagulation factor VII and soluble fibrin monomer compound. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention is to provide Gly-Pro-Arg-Pro-NHCH of the following formula2CH2NH-warfarin.
In a second aspect of the invention, Gly-Pro-Arg-Pro-NHCH is provided2CH2A method for the preparation of NH-warfarin, the method comprising:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesis of Boc-NHCH2CH2NH-warfarin;
4) reacting Boc-NHCH2CH2Conversion of NH-warfarin to HCl & NH2CH2CH2NH-warfarin;
5) synthesis of Boc-Gly-Pro-Arg (NO)2)-Pro-OBzl；
6) Boc-Gly-Pro-Arg (NO)2) Conversion of-Pro-OBzl to Boc-Gly-Pro-Arg (NO)2)-Pro；
7) Adding HCl & NH2CH2CH2NH-warfarin and Boc-Gly-Pro-Arg (NO)2) Synthesis of Boc-Gly-Pro-Arg (NO) by-Pro reaction2)-Pro-NHCH2CH2NH-warfarin;
8) Boc-Gly-Pro-Arg (NO)2)-Pro-NHCH2CH2Conversion of NH-warfarin to Gly-Pro-Arg-Pro-NHCH of claim 12CH2NH-warfarin.
A third aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin anti-venous thrombosisThe function of (1).
The fourth aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2The effect of NH-warfarin on bleeding time.
The fifth aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2The effect of NH-warfarin on clotting time.
The sixth aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2The effect of NH-warfarin on the internationally standardized ratio.
The seventh aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2The effect of NH-warfarin on plasma coagulation factor II levels.
The eighth aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin on plasma tissue factor/coagulation factor VII content.
The ninth aspect of the present invention is to evaluate Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin on plasma soluble fibrin monomer complex content.
Drawings
FIG. 1 Gly-Pro-Arg-Pro-NHCH2CH2(i) bromo-2-benzyl acetate, acetone, K2CO3,45℃；(ii)CH3OH,Pd/C,H2(ii) a (iii) Dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, N-methylmorpholine, tetrahydrofuran; (iv) hydrogen chloride in ethyl acetate (4N); (v) acetone, sodium hydroxide solution (2N); (vi) trifluoroacetic acid, trifluoromethanesulfonic acid.
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 (1)
Dispersing 26.48g (80.00mmol) of warfarin in 400mL of acetone, stirring at 45 ℃ until warfarin is dissolved, adding 12.1g (88.0mmol) of K to the obtained solution2CO3Then, then14mL (88mmol) of benzyl bromoacetate was added and the reaction was continued at 45 ℃ for 96 hours, TLC (Petroleum ether/ethyl acetate, 2/1) showed the disappearance of the starting material spot, filtration was carried out, the filtrate was concentrated under reduced pressure, and the pale yellow oil was purified by silica gel column chromatography (Petroleum ether/ethyl acetate, 8/1) to give 19.77g (54.2%) 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 (2)
19.77g (43.36mmol) of warfarin-4-O-benzyl acetate (1) was dissolved in 150mL of tetrahydrofuran, 4.94g of Pd/C was added, air was purged with stirring, hydrogen was introduced, and stirring was carried out at room temperature for 72 hours. TLC (petroleum ether/ethyl acetate, 2/1) showed the starting material spot to disappear, filtered and the filtrate was concentrated under reduced pressure to give 15.58g (98.2%) 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(dd,J1＝15.0Hz,J2＝30.0Hz,2H),3.54～3.47(m,2H),2.14(s,3H)。
EXAMPLE 3 preparation of Boc-NHCH2CH2NH-warfarin (3)
1.601g (4.374mmol) of warfarin-4-O-acetic acid (2) was dissolved in 20mL of tetrahydrofuran, 0.5906g (4.375mmol) of 1-hydroxybenzotriazole and 0.9831g (4.772mmol) of dicyclohexylcarbodiimide were added at 0 ℃ and activated with stirring in an ice bath for 30 min. Then 0.60mL (4.0mmol) of Boc-ethylenediamine was added to the reaction solution in ice bath, the pH of the reaction solution was adjusted to 9 with N-methylmorpholine, the reaction was stirred at room temperature for 14 hours, TLC (dichloromethane/methanol, 35/1 and 2d glacial acetic acid) showed disappearance of the starting material point, the filtrate was filtered, the filtrate was concentrated under reduced pressure, the residue was dissolved in 100mL of ethyl acetate and then saturated NaHCO respectively3Aqueous solution (40 mL. times.3), saturated aqueous NaCl solution (40 mL. times.3), 5% KHSO4Aqueous solution (40 mL. times.3), saturated aqueous NaCl solution (40 mL. times.3), saturated aqueous NaHCO solution3Washed with an aqueous solution (40 mL. times.3), washed with a saturated aqueous NaCl solution (40 mL. times.3), and combined with ethyl acetate and anhydrous Na2SO4Drying for 6 h, filtration and concentration of the filtrate under reduced pressure gave crude pale yellow solid which was purified by silica gel column chromatography (dichloromethane/methanol, 120/1) to give 1.57g (77%) of the title compound as a colorless solid.1H NMR(300MHz,DMSO-d6):δ/ppm＝8.39(s,1H),7.85(d,J＝7.8Hz,1H),7.65(t,J＝7.2Hz,1H),7.42(m,2H),7.36(d,J＝7.8Hz,2H),7.27(t,J＝7.2Hz,2H),7.19(m,1H),6.89(s,1H),4.90(t,J＝7.2Hz,1H),4.49(m,2H),3.46(d,J＝7.2Hz,2H),3.25(m,2H),3.10(m,2H),2.15(s,3H),1.38(s,9H)。
EXAMPLE 4 preparation of HCl. NH2CH2CH2NH-warfarin (4)
1.452g (2.857mmol) of Boc-NHCH2CH2NH-warfarin (3) was dissolved in 15mL of dry ethyl acetate, and 20mL of a 4N solution of hydrogen chloride in ethyl acetate was added under an ice salt bath, followed by stirring for 4 hours. TLC (dichloromethane/methanol, 10/1 and 3 drops of glacial acetic acid) showed the disappearance of the starting material spot, and the reaction was concentrated under reduced pressure to give a colorless viscous solid which was dispersed with anhydrous ether and then left to stand, after decanting the upper solvent and concentrating under reduced pressure to give 1.29g (100%) of the title compound as a colorless solid. ESI-MS (M/e) 409[ M + H]+。
EXAMPLE 5 preparation of Boc-Gly-Pro-OBzl
7.730g (44.15mmol) of Boc-Gly were dissolved in 100mL of dried tetrahydrofuran, and 5.940g (44.00mmol) of 1-hydroxybenzotriazole and 9.888g (48.00mmol) of dicyclohexylcarbodiimide were added at 0 ℃ and stirred for 30 minutes. 9.665g (40.00mmol) HCl Pro OBzl are then added, the pH of the reaction mixture is adjusted to 9 with N-methylmorpholine, the mixture is stirred for 17 hours at room temperature, TLC (dichloromethane/methanol, 20/1) shows disappearance of the starting material, the filtrate is filtered and concentrated under reduced pressure, the residue is dissolved in 150mL ethyl acetate, the insoluble colorless solid is filtered off, and the filtrate is separately diluted with saturated NaHCO3Aqueous solution (50 mL. times.3), saturated aqueous NaCl solution (50 mL. times.3), 5% KHSO4Solution (50 mL. times.3), saturated aqueous NaCl solution (50 mL. times.3), saturated NaHCO3Washing with an aqueous solution (50 mL. times.3) and a saturated aqueous NaCl solution (50 mL. times.3) to obtain an ethyl acetate phase, and adding anhydrous Na to the ethyl acetate phase2SO4Drying for 12 h, filtration and concentration of the filtrate under reduced pressure gave a pale yellow solid which was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 3/1) to give 13.42g (93%) of the title compound as a colorless solid. ESI-MS (M/e) 363[ M + H]+。
EXAMPLE 6 preparation of Boc-Gly-Pro
4.774g (13.18mmol) of Boc-Gly-Pro-OBzl were dissolved in 50mL of methanol, 1.0g of Pd/C was added, and stirring was carried out while removing air, introducing hydrogen gas and stirring was carried out at room temperature for 48 hours. TLC (dichloromethane/methanol, 20/1) showed the starting material spot to disappear, filtered and the filtrate concentrated under reduced pressure to give 3.411g (95%) of the title compound as 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-Arg (NO)2)-Pro-OBzl
Using the method of example 5 from 3.509g (11.00mmol) of Boc-Arg (NO)2) And 2.516g (10.41mmol) HCl Pro-OBzl gave 4.05g (80%) of the title compound as 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 8 preparation of HCl.Arg (NO)2)-Pro-OBzl
From 1.80g (3.56mmol) Boc-Arg (NO) using the method of example 42) Pro-OBzl gave 1.53g (97%) of the title compound as a colorless solid. ESI-MS (M/e):407[ M + H]+。
EXAMPLE 9 preparation of Boc-Gly-Pro-Arg (NO)2)-Pro-OBzl
From 1.755g (6.45mmol) Boc-Gly-Pro and 2.566g (5.80mmol) HCl Arg (NO)2) Pro-OBzl gave 2.31g (60%) of the title compound as a colorless solid. 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 10 preparation of Boc-Gly-Pro-Arg (NO)2)-Pro
2.656g (4.02mmol) Boc-Gly-Pro-Arg (NO)2) Pro-OBzl was dissolved in 35mL of acetone, and the pH of the reaction mixture was adjusted to 13.0 by dropping a 2N aqueous solution of sodium hydroxide under ice bath and stirred for 6 hours, and TLC (dichloromethane/methanol, 10/1 and 3 drops of glacial acetic acid) showed the disappearance of the starting material spots. Saturated KHSO is used under ice bath4The pH of the reaction mixture was adjusted to 7 with an aqueous solution, concentrated under reduced pressure, and the residue was taken up in 3mL of H2Dissolving O in saturated KHSO4The solution was adjusted to pH 2 with aqueous solution, then extracted with ethyl acetate (30 mL. times.4) and the ethyl acetate phase was taken up with anhydrous Na2SO4Drying for 10 h, filtration and concentration of the filtrate under reduced pressure gave 2.115g (92%) of the title compound as a pale yellow oil. ESI-MS (M/e):571[ M + H]+。
EXAMPLE 11 preparation of Boc-Gly-Pro-Arg (NO)2)-Pro-NHCH2CH2NH-warfarin (5)
From 1.141g (2.000mmol) Boc-Gly-Pro-Arg (NO) using the method of example 52) Pro and 0.8081g (1.817mmol) HCl. NH2CH2CH2NH-warfarin (4) gave 1.19g (68%) of the title compound as a colorless solid.1H NMR(300MHz,DMSO-d6)δ/ppm＝8.50(s,1H),8.39(s,1H),8.12(m,1H),7.93(s,1H),7.85(d,J＝7.2Hz,1H),7.65(m,1H),7.43(d,J＝7.8Hz,2H),7.34(m,2H),7.27(m,2H),7.19(m,1H),6.74(s,1H),4.90(m,1H),4.50(s,3H),4.35(m,1H),4.21(m,1H),3.74(m,2H),3.57(m,4H),3.47(d,J＝6.6Hz,2H),3.27(s,3H),3.16(s,3H),2.16(s,3H),2.00～1.57(m,12H),1.38(s,9H)。
EXAMPLE 12 preparation of Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin (6)
Adding 7.5mL trifluoroacetic acid dropwise into 250mg (0.26mmol) Boc-Gly-Pro-Arg (NO)2)-Pro-NHCH2CH2In NH-warfarin (5), 2.5mL of trifluoromethanesulfonic acid was added dropwise, and TLC (ethyl acetate/glacial acetic acid/water, 3/1/1) after 1 hour of reaction showed the disappearance of the starting material spot. The reaction mixture was immediately concentrated under reduced pressure in an ice bath, the residue was diluted with 30mL of ether, which had been cooled beforehand, and after standing for 10 minutes the supernatant solution was decanted off, the residue was further diluted with 30mL of ether, which had been cooled beforehand, and after standing for 10 minutes the supernatant solution was decanted off. This operation was repeated 3 times. The residue was finally concentrated under reduced pressure to give a brown oil which was taken up in 3mL of H2O, adjusting the pH of the solution to 7 with 10% ammonia water in ice bath, filtering, preliminarily purifying the obtained 5mL brown solution by Sephadex-G10 gel filtration chromatography column, collecting eluent with UV color development monitored by TLC (ethyl acetate/glacial acetic acid/water, 3/1/1), and freeze-drying to obtain 170mg yellow powder. The powder was dissolved in 2mL of 5% aqueous methanol solution using RP-C18Column chromatography purification (methanol/water, 1/1) and freeze drying of the collected eluate gave 113mg (53%) of the title compound as a colourless powder. ESI-MS (M/e):816[ M + H]+；
(c＝0.13,CH3OH)；1H NMR(300MHz,DMSO-d6):δ/ppm＝8.45(m,1H),8.28～8.00(m,5H),7.85(d,J＝7.8Hz,2H),7.64(t,J＝7.5Hz,1H),7.43(d,J＝9.0Hz,2H),7.35(m,3H),7.26(t,J＝7.5Hz,3H),7.19(m,2H),4.90(t,J＝7.2Hz,1H),4.51(m,2H),4.42(m,2H),4.21(m,1H),3.78(s,2H),3.58(m,4H),3.47(d,J＝7.2Hz,2H),3.26～3.14(m,6H),2.15(s,3H),2.02～1.57(m,12H)。
EXAMPLE 13 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Anti-venous thrombosis function of NH-warfarin (6)
Experimental Material
Uratan (Ethyl carbamate, CAS:51-79-6, national pharmaceutical group chemical Co., Ltd.), warfarin sodium (CAS:129-06-6, Bailingwei science and technology Co., Ltd.).
Laboratory animal
Male SD rats (250. + -.20 g) purchased from Experimental animals technology, Inc. of Weitongli, Beijing. For evaluation, the rat inferior vena cava ligation model was prepared.
Dosage to be administered
Gly-Pro-Arg-Pro-NHCH of the invention2CH2NH-warfarin (6) is dissolved in normal saline, and the dosage is 0.82 mu mol/kg; dissolving the positive control warfarin in normal saline, wherein the dosage is 0.82 mu mol/kg; the negative control was physiological saline.
Experimental procedures
Rats were acclimatized and fasted for one day prior to surgery, and were gavaged with a saline solution of compound 6 at a dose of 0.82 μmol/kg; or a normal saline solution of warfarin, the dosage is 0.82 mu mol/kg; or physiological saline. Rats were anesthetized with a 20% urethane solution by intraperitoneal administration 30 minutes after administration and 2 minutes before surgery. Then fixing on a rat fixing plate, preparing skin at the abdomen, sterilizing, opening the abdominal cavity along the leucorrhea line, and getting up to expose one corner of the liver, wherein the opening is about 4cm long. The organs such as small intestine in the abdominal cavity were removed and wrapped with gauze soaked with normal saline. Blunt separating connective tissues around blood vessels, exposing inferior vena cava and branches thereof, peeling off the abdominal aorta and the inferior vena cava below the left renal vein, ligating the inferior vena cava at the junction of the inferior vena cava and the left renal vein by using a suture soaked by normal saline, moving organs such as intestines back to the abdominal cavity according to anatomical positions, suturing the abdominal cavity layer by using the suture, and then placing the rat in an environment at 25-28 ℃ for circulation for 4 days. Then, the patient is subjected to ether anesthesia, the abdominal cavity is opened, the branch vessels of the inferior vena cava are ligated one by one, the inferior vena cava with the length of 2cm is extracted from the ligation site at the junction of the inferior vena cava and the left renal vein, and the thrombus is extracted from the inferior vena cava. Blood was reserved as a sample for the determination of factor II, tissue factor/factor VII and soluble fibrin monomer complexes. The thrombus was weighed and the results were counted using the T-test. The operation was performed alternately with four of each group. The thrombus weights are shown in Table 1.
TABLE 1 treatment of venous thrombosis in rats
a) The ratio of P to normal saline is less than 0.01; n is 8
The data in Table 1 show that compound 6 is effective in inhibiting venous thrombosis in rats at an oral dose of 0.82 μmol/kg for 4 consecutive days. Therefore, the invention has obvious technical effect.
EXAMPLE 14 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin (6) on bleeding time
The rats of example 13 were anesthetized before embolectomy, and then a 2mm deep wound was opened with a scalpel at 8cm from the rat tail, while timing was started, blood was wiped off with filter paper every 5 seconds, and timing was stopped when no blood stain was visible on the filter paper, and the time was recorded as the tail bleeding time of the rats. The bleeding time was counted by means of the T test. The operation was performed alternately with four of each group. The bleeding time is shown in table 2.
TABLE 2 treatment of rat tail bleeding time
a) The ratio of the water to the normal saline is less than 0.01; n is 11
The data in Table 2 show that compound 6 at an oral dose of 0.82 μmol/kg for 4 consecutive days showed no difference in tail bleeding time from saline treated rats, whereas warfarin at an oral dose of 0.82 μmol/kg for 4 consecutive days showed a significantly longer tail bleeding time than saline treated rats, indicating significant bleeding side effects. Compound 6 caused no bleeding events similar to warfarin and was safer than warfarin. It can be seen that the present invention has an unexpected technical effect.
Example 15 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin (6) on clotting time
The rats of example 13 were anesthetized before they had their abdomen laid down on a rat board, their rat tails were wiped with absorbent cotton balls dipped with medical alcohol to sterilize them, 8cm was measured from the rat tail tip with a ruler and marked, a blood vessel located at the center of the rat tail was found at a position about in the middle of the rat tail, a wound about 5mm long and about 2mm deep was cut with a surgical blade, and a drop of blood was immediately taken with a glass board and a timer was started when blood flowed out. And repeatedly picking up a blood drop by using the needle tip of the syringe, wherein the frequency is about 2 times/second, and the timing is stopped until the needle tip can pick up the filamentous thrombus, so that the obtained time is the blood coagulation time of the rat. The obtained clotting times were counted using the t-test. The operation was performed alternately with four of each group. The clotting times are shown in Table 3.
TABLE 3 clotting time in treated rats
a) The ratio of P to normal saline is less than 0.01; n-10
The data in table 3 show that compound 6 at an oral dose of 0.82 μmol/kg for 4 consecutive days showed no difference in rat tail clotting time from saline treated rats, whereas warfarin at an oral dose of 0.82 μmol/kg for 4 consecutive days showed a significantly longer tail clotting time than saline treated rats, showing significant bleeding side effects. Compound 6 caused no bleeding events similar to warfarin and was safer than warfarin. It can be seen that the present invention has an unexpected technical effect.
EXAMPLE 16 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Influence of NH-warfarin (6) on the International Standard ratio of rats the International Standard ratio reflects prothrombin time and can be automatically calculated by a semi-automatic coagulometer (model TS6000, MD Pacific). The specific test method is that 3.6 mL/rat whole blood collected in example 13 is placed in a centrifuge tube containing 0.4mL sodium citrate solution (3.8%), centrifuged at 2500g for 15 minutes, then platelet plasma (PPP) for upper layer anemia is sucked, 100. mu.L PPP is added into a test cup, a test magnetic bead is added, a sample to be tested is pre-warmed in a pre-warm area of the instrument for 180 seconds, 200. mu.L prothrombin time test solution (kit cat # 20-7011, MD Pacific) is added, the instrument starts to automatically detect to obtain prothrombin time, and automatically calculates the international standard ratio, and the result is counted by a T test mode, and the international standard ratio is shown in Table 4。
TABLE 4 International normalized ratio of treated rats
a) The ratio of P to normal saline is less than 0.01; n-10
The data in Table 4 show that compound 6 at an oral dose of 0.82 μmol/kg for 4 consecutive days showed no difference in the internationally standardized ratio for rats from saline treated rats, whereas warfarin at an oral dose of 0.82 μmol/kg for 4 consecutive days showed a significantly higher internationally standardized ratio for rats than saline treated rats, indicating significant bleeding side effects. Compound 6 caused no bleeding events similar to warfarin and was safer than warfarin. It can be seen that the present invention has an unexpected technical effect.
Example 17 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin (6) on rat plasma coagulation factor II (FIIa) levels
Example 13 Whole blood of rat collected 4.0 mL/body was put into a centrifuge tube containing 0.4mL of sodium citrate solution (3.8%), centrifuged at 1000g for 15 minutes, and then Platelet Poor Plasma (PPP) was aspirated. PPP samples of 6 rats after administration were taken from each experimental group, diluted 1:1 with the specimen diluent in the kit, and 50ul of the diluted sample was added to the reaction well. At the same time, 50. mu.L of the diluted standard was added to the reaction well. Immediately 50 μ L of biotin-labeled antibody was added. Cover the membrane plate, mix by gentle shaking, incubate for 1 hour at 37 ℃. And then throwing off liquid in the holes, filling the holes with the cleaning solution, oscillating for 30 seconds, throwing off the cleaning solution, and patting dry by using absorbent paper. This operation was repeated 3 times. Then 80. mu.L of streptavidin-HRP was added to each well, mixed by gentle shaking, and incubated at 37 ℃ for 30 minutes. And (3) throwing off liquid in the holes after the incubation is finished, filling washing liquid in each hole, oscillating for 30 seconds, throwing off the washing liquid, patting the holes dry by using absorbent paper, and repeating the operation for 3 times. 50 μ L of substrate A, B was added to each well, mixed by gentle shaking, and incubated at 37 ℃ for 10 minutes. Avoiding illumination, taking out the ELISA plate, quickly adding 50 mu L of stop solution, immediately measuring the result after adding the stop solution, and measuring the OD value of each hole at the wavelength of 450nm by using an ELISA reader. And subtracting the OD value read at 540nm from the OD value read at 450nm, taking the OD value as an ordinate, taking the concentration of the blood coagulation factor II (FIIa) standard product as an abscissa, and drawing a standard curve, wherein the FIIa content of the sample can be converted into the corresponding concentration according to the OD value of the sample from the standard curve. Differences in FIIa content among groups were analyzed using the one-way analysis of variance (ANOVA one-way, LSD) method in SPSS22.0, and FIIa content in rat plasma is shown in Table 5.
TABLE 5 FIIa content in plasma of treated rats
a) The ratio of P to normal saline is less than 0.01; n is 5
The data in Table 5 show that Compound 6 is effective in reducing plasma FIIa levels in rats after 4 consecutive oral administrations at an oral dose of 0.82 μmol/kg. Therefore, the invention has obvious technical effect.
EXAMPLE 18 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin (6) on plasma TF/FVIIa content in rats
Example 13 Whole blood of rat collected 4.0 mL/body was put into a centrifuge tube containing 0.4mL of sodium citrate solution (3.8%), centrifuged at 1000g for 15 minutes, and then Platelet Poor Plasma (PPP) was aspirated. PPP samples of 6 rats after administration were taken in each experimental group, 100. mu.L of each sample and standard were added to each well, incubated at 37 ℃ for 2 hours after being covered with a cover plate membrane, then all the liquid in the plate was poured off, the plate was not washed, 100. mu.L of Biotin solution was directly added to each well, and incubated at 37 ℃ for 1 hour after being covered with a new cover plate membrane. Then, the plate liquid was discarded, 200. mu.L of the washing solution was added to each well, and the washing solution was discarded after standing for 2min and the 96-well plate was patted dry, and this was repeated 3 times. Then 100. mu.L of HRP-avidin per well was added and incubated at 37 ℃ for another 1 hour after coating with a new coversheet membrane. After incubation, the plate washing operation was repeated 5 times to remove the liquid from each well. Adding 90 mu L of 3,3',5,5' -tetramethyl benzidine into each well, covering a new cover plate membrane, incubating for 20 minutes at 37 ℃ in the dark, adding 50 mu L of stop solution into each well, and rapidly reading the OD value of each well by using an enzyme-linked immunosorbent assay at the dual-wavelength of 450nm and 540 nm. And subtracting the OD value read at 540nm from the OD value read at 450nm, drawing a standard curve by taking the OD value as a vertical coordinate and the concentration of the TF/FVIIa standard substance as a horizontal coordinate, and converting the TF/FVIIa content of the sample into the corresponding concentration according to the OD value of the sample from the standard curve. The differences in TF/FVIIa content of each group were analyzed by one-way analysis of variance (ANOVA one-way, LSD) method in SPSS22.0, and the plasma TF/FVIIa content is shown in Table 6.
TABLE 6 treatment of TF/FVIIa content in rat plasma
a) P < 0.01.n ═ 4 in normal saline
The data in Table 6 show that compound 6, given at an oral dose of 0.82. mu. mol/kg for 4 consecutive days, is effective in reducing the TF/FVIIa levels in rat plasma. Therefore, the invention has obvious technical effect.
EXAMPLE 19 evaluation of Gly-Pro-Arg-Pro-NHCH2CH2Effect of NH-warfarin (6) on rat plasma Soluble Fibrin Monomer Complex (SFMC) content
Example 13 Whole blood of rat collected 4.0 mL/body was put into a centrifuge tube containing 0.4mL of sodium citrate solution (3.8%), centrifuged at 1000g for 15 minutes, and then Platelet Poor Plasma (PPP) was aspirated. Each experimental group was prepared by taking PPP samples from 6 rats after administration, diluting the PPP samples 200-fold with the sample diluent in the kit, adding 100 μ L of each sample and standard substance to each well, incubating at 37 ℃ for 2 hours after coating the cover plate membrane, then pouring all the liquid in the plate, not washing the plate, directly adding 100 μ L of Biotin solution to each well, incubating at 37 ℃ for 1 hour after coating the new cover plate membrane. Then, the plate liquid was discarded, 200. mu.L of the washing solution was added to each well, and the washing solution was discarded after standing for 2min and the 96-well plate was patted dry, and this was repeated 3 times. Then 100. mu.L of HRP-avidin per well was added and incubated at 37 ℃ for another 1 hour after coating with a new coversheet membrane. After incubation, the plate washing operation was repeated 5 times to remove the liquid from each well. Adding 90 mu L of 3,3',5,5' -tetramethyl benzidine into each well, covering a new cover plate membrane, incubating for 20 minutes at 37 ℃ in the dark, adding 50 mu L of stop solution into each well, and rapidly reading the OD value of each well by using an enzyme-linked immunosorbent assay at the dual wavelengths of 450nm and 540 nm. And subtracting the OD value read at 540nm from the OD value read at 450nm, drawing a standard curve by taking the OD value as a vertical coordinate and the SFMC standard concentration as a horizontal coordinate, and converting the SFMC content of the sample into the corresponding concentration from the standard curve according to the OD value. The difference in SFMC content of each group was analyzed by one-way analysis of variance (anova-way, LSD) method in SPSS22.0, and the SFMC content in rat plasma is shown in table 7.
TABLE 7 SFMC content in plasma of treated rats
a) The ratio of P to normal saline is less than 0.05; n is 5
The data in Table 7 show that compound 6, administered orally for 4 consecutive days at an oral dose of 0.82. mu. mol/kg, is effective in reducing the SFMC levels in rat plasma. Therefore, the invention has obvious technical effect.
Claims (6)
1.Gly-Pro-Arg-Pro-NHCH2CH2NH-warfarin, structural formula as follows:
2. a method for preparing Gly-Pro-Arg-Pro-NHCH as defined in claim 12CH2A method of NH-warfarin, the method comprising the steps of:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesis of Boc-NHCH2CH2NH-warfarin;
4) reacting Boc-NHCH2CH2Conversion of NH-warfarin to HCl & NH2CH2CH2NH-warfarin;
5) synthesis of Boc-Gly-Pro-Arg (NO)2)-Pro-OBzl；
6) Boc-Gly-Pro-Arg (NO)2) Conversion of-Pro-OBzl to Boc-Gly-Pro-Arg (NO)2)-Pro；
7) Adding HCl & NH2CH2CH2NH-warfarin and Boc-Gly-Pro-Arg (NO)2) Synthesis of Boc-Gly-Pro-Arg (NO) by-Pro reaction2)-Pro-NHCH2CH2NH-warfarin;
8) Boc-Gly-Pro-Arg (NO)2)-Pro-NHCH2CH2Conversion of NH-warfarin to Gly-Pro-Arg-Pro-NHCH as claimed in claim 12CH2NH-warfarin.
3. The Gly-Pro-Arg-Pro-NHCH of claim 12CH2Application of NH-warfarin in preparation of anti-vein thrombosis medicine without bleeding side effect.
4. The Gly-Pro-Arg-Pro-NHCH of claim 12CH2Application of NH-warfarin in preparation of blood coagulation factor II antagonist.
5. The Gly-Pro-Arg-Pro-NHCH of claim 12CH2Use of NH-warfarin for the preparation of tissue factor/factor VII antagonists.
6. The Gly-Pro-Arg-Pro-NHCH of claim 12CH2Use of NH-warfarin in the preparation of soluble fibrin monomer complex antagonists.
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