CN113929739A - Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin, and synthesis, activity and application thereof - Google Patents
Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin, and synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin with the following formula, discloses a preparation method thereof, further discloses the anti-thrombus activity thereof, and discloses the advantage of no bleeding side effect thereof. Also discloses that it can reduce blood plasma coagulation factorActivity of seed II and soluble fibrin monomer complex content. Therefore, the invention discloses the application of the derivative in preparing an anti-vein thrombosis medicament without bleeding side effects, and discloses the application of the derivative in preparing a blood coagulation factor II antagonist and the application in preparing a soluble fibrin monomer compound antagonist.
Description
Technical Field
The invention relates to Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin, relates to a preparation method thereof, further relates to the anti-venous thrombosis activity thereof, and relates to the advantage of no bleeding side effect thereof. Also relates to the activity of the polypeptide in reducing the content of blood coagulation factor II and TF/FVIIa. The invention thus relates to its use for the preparation of an anti-thrombotic agent without bleeding side effects, to its use for the preparation of a factor II antagonist and to its use for the preparation of a TF/FVIIa 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 plasma TF/FVIIa levels. In order to overcome the shortcomings of such modified warfarin in this regard, the inventors have further developed studies and explorations with different objectives. It was later found that Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin had no effect on vitamin K, but rather was an anti-thrombotic agent acting on TF/FVIIa. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
The first aspect of the present invention provides the following Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin.
The second content of the invention is to provide a preparation method of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin, which comprises the following steps:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesis of Cbz-Ethanolamine
4) The Cbz-aminoethanol reacts with warfarin-4-O-acetic acid to synthesize Cbz-aminoethoxycarbonyl warfarin;
5) cbz-aminoethoxycarbonyl warfarin is converted into aminoethoxycarbonyl warfarin;
6) synthesis of Cbz-Gly-Pro-Arg (NO)2)-Pro-OBzl；
7) Reacting Cbz-Gly-Pro-Arg (NO)2) Conversion of-Pro-OBzl to Cbz-Gly-Pro-Arg (NO)2)-Pro；
8) Aminoethoxycarbonylwarfarin and Cbz-Gly-Pro-Arg (NO)2) -Pro reaction synthesis
Cbz-Gly-Pro-Arg(NO2) -Pro-NH ethoxycarbonyl warfarin;
9)Cbz-Gly-Pro-Arg(NO2) -Pro-NH ethoxycarbonyl warfarin is converted to Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin.
The third content of the invention is to evaluate the anti-vein thrombosis effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin.
A fourth aspect of the present invention is to evaluate the effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin on bleeding time.
The fifth aspect of the present invention was to evaluate the effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin on clotting time.
The sixth aspect of the present invention is to evaluate the effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin on the international normalized ratio.
The seventh aspect of the present invention is to evaluate the effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin on plasma coagulation factor II content.
An eighth aspect of the invention is to evaluate the effect of Gly-Pro-Arg-Pro-NH ethoxycarbonylwarfarin on plasma TF/FVIIa levels.
Drawings
FIG. 1A synthetic route to Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (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) water, dioxane, Cbz-OSu, sodium hydroxide solution (2N); (vii) dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N-methylmorpholine, tetrahydrofuran.
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 into the reaction solution2CO3Then 14mL (88mmol) of benzyl bromoacetate was added and the reaction was continued at 45 ℃. After 96 hours of reaction, TLC (petroleum ether/ethyl acetate, 2/1) showed the disappearance of the warfarin point, whereupon the reaction was terminated, filtered, the filtrate was concentrated under reduced pressure, and the resulting pale yellow oil was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 8/1) to give 19.77g (54%) 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 the mixture was stirred at room temperature for 72 hours. TLC (petroleum ether/ethyl acetate, 2/1) showed the starting material spot to disappear whereupon the reaction was stopped, filtered and the filtrate was concentrated under reduced pressure to give 15.58g (98%) 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 Cbz-Ethanolamine
0.6108g (10.00mmol) ethanolamine was dissolved in 10mL H2O, then 2.770g (11.11mmol) of Cbz-OSu is weighed out and dissolved in 20mL dioxane, added dropwise to the above aqueous solution in ice bath, the pH of the reaction solution is adjusted to 8 by using sodium hydroxide solution (2N), after stirring and reacting for 48 hours, TLC (dichloromethane/methanol, 20/1 and 3 drops of glacial acetic acid) shows that the raw material point disappears, and saturated KHSO is used in ice bath4The reaction mixture was adjusted to pH 7, concentrated under reduced pressure, and the residue was taken up in 10mL of H2Dissolving O, and then using saturated KHSO4The reaction mixture was adjusted to pH 2, ethyl acetate was added thereto for extraction (30 mL. times.4), and the ethyl acetate phase was collected and extracted with anhydrous Na2SO4After drying for 6 hours, filtration and concentration of the filtrate under reduced pressure, the resulting colorless oil was recrystallized from petroleum ether-ethyl acetate to give 1.79g (92%) of the title compound as a colorless solid. ESI-MS (M/e):194[ M-H]-。
EXAMPLE 4 preparation of Cbz-aminoethoxycarbonyl warfarin (3)
1.208g (3.300mmol) of warfarin-4-O-acetic acid (2) were dissolved in 25mL of tetrahydrofuran, and 0.073g (0.60mmol) of 4-dimethylaminopyridine was added thereto at 0 ℃ and stirred for 10 minutes, and 0.742g (3.60mmol) of dicyclohexylcarbodiimide was added thereto and stirring was continued for 30 minutes. 0.585g (3.00mmol) of Cbz- (CH) at 0 deg.C2)2OH was added to the reaction mixture, the pH of the reaction mixture was adjusted to 9 with N-methylmorpholine, and the mixture was stirred at room temperature for 18 hours, after which TLC (petroleum ether/ethyl acetate, 2/1) was developedThe starting material point disappeared, filtration, the filtrate was concentrated under reduced pressure, the residue obtained was dissolved in 50mL ethyl acetate and filtered, the filtrate was saturated NaHCO3Aqueous 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 solution3Washing with aqueous solution (40 mL. times.3), washing with saturated aqueous NaCl solution (40 mL. times.3), and washing with anhydrous Na as ethyl acetate phase2SO4Drying for 6 h, filtration and concentration of the filtrate under reduced pressure gave crude product as a pale yellow oil which was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 4/1) to give 1.42g (87%) of the title compound as a colorless solid.1H NMR(300MHz,DMSO-d6)＝7.87(d,J＝7.8Hz,1H),7.61(t,J＝7.5Hz,1H),7.41(m,2H),7.32(m,7H),7.26(t,J＝7.5Hz,2H),7.16(m,1H),5.01(s,2H),4.96(t,J＝7.5Hz,1H),4.81(q,J＝15.6Hz,2H),4.20(m,2H),3.46(dd,J1＝7.5Hz,J2＝3.9Hz,2H),3.31(m,2H),2.13(s,3H)。
Example 5 aminoethoxycarbonyl warfarin (4)
498mg (0.915mmol) of Cbz-aminoethoxycarbonyl warfarin (3) are dissolved in 20mL of dichloromethane, 90mg of Pd/C are added to the solution and the solution is dispersed homogeneously, after removal of air and purging with hydrogen, TLC (dichloromethane/methanol, 20/1) is stirred at room temperature for 24 hours, showing disappearance of the starting material point, filtration is carried out and the filtrate is concentrated under reduced pressure to give 0.30g (80%) of the title compound as a colorless solid. ESI-MS (M/e):408[ M-H]-。
EXAMPLE 6 preparation of Cbz-Gly-Pro-OBzl
2.573g (12.31mmol) of Cbz-Gly was dissolved in 35mL of tetrahydrofuran, 1.660g (12.30mmol) of 1-hydroxybenzotriazole was added thereto at 0 ℃ and the mixture was stirred for 10 minutes, 2.770g (13.45mmol) of dicyclohexylcarbodiimide was added thereto and the mixture was stirred at 0 ℃ for 30 minutes. 2.705g (11.20mmol) HCl Pro OBzl are added to the reaction solution at 0 ℃, the pH value of the reaction solution is adjusted to 9 by N-methylmorpholine, the reaction solution is stirred for 18 hours at room temperature, TLC (dichloromethane/methanol, 20/1) shows that the raw material point disappears, the filtration is carried out, and the filtrate is added with saturated NaHCO3Aqueous 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 solution3Washing with aqueous solution (40 mL. times.3), washing with saturated aqueous NaCl solution (40 mL. times.3), and washing with anhydrous Na as the acid ethyl ester phase2SO4Dried 6 smallFiltration and concentration of the filtrate under reduced pressure gave 3.58g (81%) of the title compound as a pale yellow oil. ESI-MS (M/e):397[ M + H]+。
EXAMPLE 7 preparation of Cbz-Gly-Pro
3.58g of Cbz-Gly-Pro-OBzl was dissolved in 20mL of acetone, a 2N aqueous solution of sodium hydroxide was added dropwise at 0 ℃ to adjust the pH of the reaction mixture to 13.0, and after stirring at 0 ℃ for 4 hours, TLC (dichloromethane/methanol, 20/1 and 3 drops of glacial acetic acid) showed the disappearance of the starting material spots. Saturated KHSO at 0 deg.C4The 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, extracted with ethyl acetate (30 mL. times.4) and the ethyl acetate phase was taken up with anhydrous Na2SO4After drying for 6 hours, insoluble matter was removed by filtration and the filtrate was concentrated under reduced pressure to give 2.62g (95%) of the title compound as a pale yellow oil. ESI-MS (M/e):305[ M-H]-。
EXAMPLE 8 preparation of Boc-Arg (NO)2)-Pro-OBzl
Using the method of example 6, 3.509g (11.00mmol) of Boc-Arg (NO)2) And 2.516g (10.41mmol) HCl Pro-OBzl gave 4.05g (80.2%) of the title compound as a colorless solid. 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 9 preparation of HCl Arg (NO)2)-Pro-OBzl
1.80g (3.56mmol) of Boc-Arg (NO)2) -Pro-OBzl was dissolved in 15mL of dry ethyl acetate, and 30mL of a 4N solution of hydrogen chloride in ethyl acetate was added at 0 ℃ and stirred for 4 hours. After TLC (dichloromethane/methanol, 20/1) showed the disappearance of the starting material, the reaction was concentrated under reduced pressure, the residue was redissolved with dry ethyl acetate and concentrated under reduced pressure, and the resulting colorless solid was washed repeatedly with anhydrous ether to give 1.53g (97%) of the title compound as a colorless solid. ESI-MS (M/e):407[ M + H]+。
EXAMPLE 10 preparation of Cbz-Gly-Pro-Arg (NO)2)-Pro-OBzl
Using the method of example 6, 2.00g of Cbz-Gly-Pro and 2.376g of HCl Arg (NO)2) Pro-OBzl yielded 2.36g (68%) of the title compound as a colorless solid.1H NMR(300MHz,DMSO-d6)＝8.49(m,1H),8.17(d,J＝7.5Hz,1H),7.39(m,10H),5.10(s,2H),5.03(s,2H),4.45(m,3H),3.81(m,2H),3.60(m,4H),3.11(s,2H),2.12～1.52(m,12H)。
EXAMPLE 11 preparation of Cbz-Gly-Pro-Arg (NO)2)-Pro
Using the method of example 7, starting from 0.698g (1.00mmol) of Cbz-Gly-Pro-Arg (NO)2) Pro-OBzl gave 0.604g (95%) of the title compound as a colorless oil. ESI-MS (M/e):603[ M-H]-。
EXAMPLE 12 preparation of Cbz-Gly-Pro-Arg (NO)2) -Pro-NH ethoxycarbonyl warfarin (5)
Using the method of example 6, starting from 0.377g (0.623mmol) of Cbz-Gly-Pro-Arg (NO)2) Pro and 0.232g (0.567mmol) aminoethoxycarbonyl warfarin (4) to give 0.20g (36%) of the title compound as a colorless solid.1H NMR(300MHz,DMSO-d6)δ/ppm＝8.48(m,1H),8.12(d,J＝4.5Hz,1H),7.80(dd,J1＝4.8Hz,J2＝0.6Hz,1H),7.61(m,1H),7.40(m,2H),7.32(m,5H),7.29(m,2H),7.24(t,J＝4.8Hz,2H),7.15(m,1H),5.00(s,2H),4.86(t,J＝4.5Hz,1H),4.49(m,2H),4.43(m,1H),4.30(m,2H),4.19(m,1H),4.08(m,1H),3.79(m,2H),3.60(m,2H),3.49～3.40(m,4H),3.44(d,J＝4.5Hz,2H),3.14(m,2H),2.12(s,3H),1.97～1.54(m,12H)。
EXAMPLE 13 preparation of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6)
150mg (0.15mmol) Cbz-Gly-Pro-Arg (NO)2) -Pro-NH Oxycarbonyl warfarin (5) dissolved in 20mL methanol, added with 30mg Pd/C and 2d formic acid and dispersed uniformly, pumped out air and introduced with hydrogen, stirred at room temperature for 7 days, TLC (ethyl acetate/glacial acetic acid/water, 3/1/1) showed disappearance of the starting material point, filtered, the filtrate was concentrated under reduced pressure to obtain a yellow oil which was dissolved in 5mL 5% methanol aqueous solution, and RP-C was used18Purification by column chromatography (methanol/water, 1/1) and freeze drying of the collected eluate yielded 42mg (34%) of the title compound as a colorless solid. ESI-MS (M/e):818[ M + H]+；
m.p.155.9～156.7℃；IR(cm-1):3289.27,2951.41,1715.30,1633.64,1567.27,1537.87,1445.89,1347.65,1279.12,1175.96,1098.60,915.42,827.09,756.63,699.71；1H NMR(500MHz,DMSO-d6):δ/ppm＝10.31(m,1H),8.52(m,1H),7.86(m,1H),7.80(m,1H),7.61(m,1H),7.49～7.38(m,4H),7.32(m,2H),7.24(m,3H),7.15(m,2H),4.87(t,J＝7.4Hz,0.5H),4.80(t,J＝7.8Hz,0.5H),4.50(m,2H),4.43～4.28(m,4H),4.20(m,2H),4.07(m,2H),3.78(m,1H),3.61(m,2H),3.53～3.49(m,4H),3.44(d,J＝7.4Hz,2H),3.05(m,4H),2.12(s,1.7H),2.05～1.81(m,6H),1.76(s,1.7H),1.72～1.45(m,6H)。
EXAMPLE 14 evaluation of the anti-thrombotic Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl 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
Dissolving Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6) in normal saline, wherein 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 15 evaluation of the Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6) on bleeding time
The rats of example 14 were anesthetized before embolectomy, and then a 2mm deep wound was opened with a scalpel at 8cm from the rat tail, while the timing was started, blood was wiped off with filter paper every 5 seconds, and the 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 bleeding time in treated rats
a) The ratio of P to 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 the tail bleeding time of the rats 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 16 evaluation of the Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6) on clotting time in rats
The rats of example 14 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 17 evaluation of the Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6) on the International normalized ratio in rats
The international normalized ratio reflects prothrombin time and can be automatically calculated by a semi-automatic hemagglutination instrument (model TS6000, MD Pacific). The specific test method is that 3.6 mL/rat whole blood collected in example 14 is placed in a centrifuge tube filled with 0.4mL sodium citrate solution (3.8%), centrifuged for 15 minutes at 2500g, then Platelet Poor Plasma (PPP) at the upper layer 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 goods number: 20-7011, MD Pacific) is added, the instrument starts to automatically detect to obtain prothrombin time, and automatically calculates the international standardized ratio, and the statistical result by a T test mode 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 18 evaluation of the Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin (6) on activated blood coagulation factor II (FIIa) levels in rat plasma
Example 14 Whole blood of rats collected 4.0 mL/mouse was placed in 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 19 evaluation of the Effect of Gly-Pro-Arg-Pro-NH ethoxycarbonylwarfarin (6) on plasma TF/FVIIa content in rats
Example 14 Whole blood of rats collected 4.0 mL/mouse was placed in 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.
It should be noted that the above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention.
Claims (5)
1.Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin has the following structural formula:
2. a process for preparing Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin according to claim 1, comprising the steps of:
1) synthesizing warfarin-4-O-benzyl acetate;
2) synthesizing warfarin-4-O-acetic acid;
3) synthesizing Cbz-ethanolamine;
4) the Cbz-aminoethanol reacts with warfarin-4-O-acetic acid to synthesize Cbz-aminoethoxycarbonyl warfarin;
5) cbz-aminoethoxycarbonyl warfarin is converted into aminoethoxycarbonyl warfarin;
6) synthesis of Cbz-Gly-Pro-Arg (NO)2)-Pro-OBzl；
7) Reacting Cbz-Gly-Pro-Arg (NO)2) Conversion of-Pro-OBzl to Cbz-Gly-Pro-Arg (NO)2)-Pro；
8) Aminoethoxycarbonylwarfarin and Cbz-Gly-Pro-Arg (NO)2) Synthesis of Cbz-Gly-Pro-Arg (NO) by-Pro reaction2) -Pro-NH ethoxycarbonyl warfarin;
9)Cbz-Gly-Pro-Arg(NO2) -Pro-NH ethoxycarbonyl warfarin is converted to Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin according to claim 1.
3. Use of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin according to claim 1 for the preparation of an anti-venous thrombosis medicament without bleeding side effects.
4. Use of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin according to claim 1 for the preparation of a coagulation factor II antagonist.
5. Use of Gly-Pro-Arg-Pro-NH ethoxycarbonyl warfarin according to claim 1 for the preparation of soluble fibrin monomer complex antagonists.
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| EP2905614A1 (en) * | 2014-02-11 | 2015-08-12 | Siemens Healthcare Diagnostics Products GmbH | Method for determining the activity of the blood coagulation system |
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| CN108976284A (en) * | 2017-05-31 | 2018-12-11 | 首都医科大学 | The warfarin of pentapeptide modification containing Gly-Pro-Arg-Pro, synthesis, activity and application |
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