CN113754663A - RR-heptacyclic aldehyde, its synthesis, antithrombotic activity and application - Google Patents
RR-heptacyclic aldehyde, its synthesis, antithrombotic activity and application Download PDFInfo
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- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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Abstract
The invention discloses (2 'S, 5' S) -tetrahydropyrazines [1 ', 2': 1, 6) of the formula]And bis { (1R, R) - [ 1-oxoethyl-2-yl]-2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ]]And indole } -1 ', 4' -dione (abbreviated as RR-heptacyclic aldehyde). Its preparing process, its activity for resisting vein thrombosis and artery thrombosis, and its advantage without bleeding side effect are disclosed. Also discloses the activity of reducing the contents of blood coagulation factors X, P selectin and GPIIb/IIIa. Therefore, the invention discloses the application of the derivative in preparing an anti-arterial thrombosis medicament without bleeding side effects, a blood coagulation factor X antagonist, a P-selectin antagonist and a GPIIb/IIIa antagonist.
Description
Technical Field
The invention relates to (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] bis { (1R, R) - [ 1-oxoethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indole } -1 ', 4' -dione (RR-heptacyclal for short). Relates to its preparation method, its activity for resisting venous thrombosis and arterial thrombosis, and its advantage without bleeding side effect. Simultaneously relates to the activity of reducing the content of blood coagulation factors X, P selectin and GPIIb/IIIa. Therefore, the invention relates to the application of the derivative in preparing an anti-arterial thrombosis medicament without bleeding side effects, a blood coagulation factor X antagonist, a P-selectin antagonist and a GPIIb/IIIa antagonist. The invention belongs to the field of biological medicine.
Background
Both arterial and venous thrombosis have become diseases with a high incidence of morbidity and mortality. In the diseases related to arterial thrombosis, coronary atherosclerotic heart disease is very serious, in order to treat coronary heart disease, a patient needs to be placed with a stent, the patient after stent placement needs to take an anti-platelet drug for a long time to perform anti-thrombosis treatment, and besides, the patient after heart mechanical valve replacement needs to take an anti-coagulation drug for life to prevent the generation of thrombus. Venous thrombotic diseases include deep vein thrombosis, which is the most common thrombotic disease, and pulmonary embolism, which has a high lethality rate. In 8 years from 2007 to 2016, the hospitalization rate of VTE patients in China is increased from 3.2/10 ten thousand to 17.5/10 ten thousand, the incidence rate of deep venous thrombosis continuously rises, and simultaneously, as the population in China ages increasingly seriously, the incidence rate of venous thrombosis also has the characteristic of exponential increase along with the age, so that the negative influence of the diseases on the people in China is particularly serious. Patients also need to take oral anticoagulants (warfarin or rivaroxaban) after discharge in order to treat venous thrombotic disease or to prevent recurrence of deep vein thrombosis. Whether they are antiplatelet or oral anticoagulants, they have a major problem in that they may increase the risk of bleeding in patients, the occurrence of which is frequently reported during the course of their administration. Therefore, the invention is a novel antithrombotic drug which has stable antithrombotic curative effect and can reduce or avoid side effects of bleeding, and has clinical importance.
The inventors have disclosed heptacyclic aldehydes of the formula (CN 110577530A). However, the heptacyclic aldehydes disclosed only inhibit venous thrombosis, but do not inhibit arterial thrombosis, blood coagulation factor X, P-selectin and GPIIb/IIIa. The inventors have conducted extensive studies and studies to overcome these disadvantages of the heptacyclic aldehydes disclosed, and have found that RR-heptacyclic aldehydes of the following formula inhibit not only venous thrombosis but also arterial thrombosis, blood coagulation factor X, P-selectin and GPIIb/IIIa. Based on these findings, the inventors have proposed the present invention.
Disclosure of Invention
In a first aspect of the invention there is provided (2 'S, 5' S) -tetrahydropyrazinyl [1 ', 2': 1,6] bis { (1R, R) - [ 1-oxoethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indolino } -1 ', 4' -dione (RR-heptacyclal for short) of the formula.
The second aspect of the present invention provides a process for producing RR-heptacyclaldehyde, which comprises:
1) performing Pictet-Spengler condensation on L-tryptophan benzyl ester and 1,1,3, 3-tetramethoxypropane to generate (3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;
2) (3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester is converted into (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid;
3) condensing (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid into RR-heptacyclic acetal serving as a precursor of RR-heptacyclic aldehyde;
4) the RR-heptacyclic acetal is converted to RR-heptacyclic aldehyde.
The third content of the present invention is to evaluate the anti-arterial thrombosis effect of RS-heptacyclic aldehyde.
The fourth aspect of the present invention is to evaluate the anti-venous thrombosis effect of RS-heptacyclic aldehyde.
The fifth aspect of the present invention is to evaluate bleeding risk of RS-heptacyclic aldehyde.
A sixth aspect of the present invention is to evaluate the effect of RS-heptacyclaldehyde on plasma P-selectin.
The seventh aspect of the present invention is to evaluate the effect of RS-heptacyclic aldehyde on plasma GPIIb/IIIa.
An eighth aspect of the present invention is to evaluate the effect of RS-heptacyclic aldehyde on plasma coagulation factor X.
Drawings
FIG. 1 is a synthetic route of RR-heptacyclic aldehyde. (i) Dichloromethane, trifluoroacetic acid, 1,1,3, 3-tetramethoxypropane; (ii) CH (CH)3OH,Pd/C,H2(ii) a (iii)2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate, triethylamine, anhydrous N, N-dimethylformamide; (iv) glacial acetic acid, H2O。
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 benzyl (3S) -1- (2, 2-Dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylate (1)
A mixed solution of 100mL of methylene chloride, 4.0mL of 1,1,3, 3-tetramethoxypropane and 3.0mL of trifluoroacetic acid was stirred for 40 minutes under ice bath, and then 4g (13.6mmol) of L-Trp-OBzl was added and stirred at 60 ℃ for 7 hours. At this time, thin layer chromatography (petroleum ether/ethyl acetate, 1/1) showed that L-Trp-OBzl disappeared, whereupon the reaction was terminated. The reaction mixture was concentrated under reduced pressure and the residual oil was dissolved in 80mL of ethyl acetate. The resulting solution was sequentially saturated with NaHCO3Aqueous solution (30 mL. times.3) and saturated aqueous NaCl solution (30 mL. times.3). The combined ethyl acetate layer was added with anhydrous Na2SO4Drying for 6 h, filtration, concentration of the filtrate under reduced pressure and purification of the residue by column chromatography on silica gel (petroleum ether/ethyl acetate, 2/1) gave 3.26g (61%) of the title compound as a yellow oil. ESI-MS (M/e)393[ M-H]-；1HNMR(300MHz,DMSO-d6)δ/ppm＝10.83(d,J＝14.4Hz,1H),7.43～7.22(m,7H),7.03(t,J＝6.9Hz,1H),6.95(t,J＝7.2Hz,1H),5.20(m,2H),4.69(m,1H),4.26(d,J＝9.6Hz,0.43H),4.16(m,0.56H),4.00(m,0.45H),3.75(m,0.57H),3.31(d,J＝5.1Hz,3H),3.26(d,J＝7.8Hz,3H),2.96(m,1H),2.85(m,0.49H),2.70(m,1H),2.42(m,0.53H),2.13(m,0.42H),1.82(m,1H)。
EXAMPLE 2 preparation of (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid (2)
4.33g (11.0mmol) of (3S) -benzyl-1- (2, 2-dimethoxyethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b]Indole-3-carboxylic acid (1) dissolved in 80mL CH3To OH, 734mg of Pd/C was added, followed by stirring, introduction of hydrogen gas, stirring at room temperature for 12 hours, and TLC (petroleum ether/ethyl acetate, 1/1) showed disappearance of Compound 1, whereupon the reaction was terminated. Filtration and concentration of the filtrate under reduced pressure, the residue was dissolved in 10mL of purified water and subjected to preparative high performance liquid chromatography (methanol/water, 1/4) to give 2.34g (70%) of the title compound as a pale yellow solid. ESI-FT-ICR-MS (M/e)305.14986[ M + H [ ]]+；1H NMR(800MHz,DMSO-d6)δ/ppm＝10.86(s,1H),8.30(s,1H),7.40(d,J＝8.0Hz,1H),7.30(d,J＝8.0Hz,1H),7.05(t,J＝7.2Hz,1H),6.96(t,J＝7.2Hz,1H),4.73(m,1H),4.47(d,J＝8.0Hz,1H),3.77(m,1H),3.33(s,3H),3.32(s,3H),2.99(dd,J1＝4.0Hz,J2＝15.2Hz,1H),2.82(dd,J1＝8.0Hz,J2＝15.2Hz,1H),2.16(d,J＝12.8Hz,1H),2.05(m,1H)；
EXAMPLE 3 preparation of (2 'S, 5' S) -Tetrahydropyrazine [1 ', 2': 1,6] and bis { (1R, R) - [ 1-dimethoxyethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole } -1 ', 4' -dione (3, RR-heptacyclic acetal)
1.52g (5.00mmol) of (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid was dissolved in 60mL of anhydrous N, N-Dimethylformamide (DMF), 2.28g (6.00mmol) of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATu) and 0.68g (5.00mmol) of 1-hydroxybenzotriazole (HOBt) were added with stirring in an ice bath, then the pH of the reaction solution was adjusted to 9 with triethylamine, stirred at room temperature for 18 hours, TLC (petroleum ether/ethyl acetate, 1/1) showed disappearance of the starting material points, and the reaction was terminated. The reaction was concentrated under reduced pressure, the residue was taken up in 80mL ethyl acetate and successively saturated NaHCO3Aqueous solution (40 mL. times.3), saturated aqueous NaCl solution (40 mL. times.3), 5% KHSO4Aqueous wash (40 mL. times.3), saturated aqueous NaCl wash (40 mL. times.3), 5% NaHCO3Aqueous solution (40 mL. times.3) and saturated aqueous NaCl solution (40 mL. times.3). The combined ethyl acetate phases were treated with anhydrous Na2SO4Drying for 8 hours, filtering, and concentrating the filtrate under reduced pressure. The residue obtained is purified by column chromatography on silica gel (petroleum ether/ethyl acetate)Ester, 1/1) to yield 701mg (49%) of the title compound as a light yellow solid. ESI-MS (M/e):573[ M + H]+；
(c＝0.12,CH3OH)；m.p.134～135℃；IR(cm-1):3398.82,3300.26,2926.03,2831.12,2359.62,2341.99,1652.04,1451.45,1387.46,1328.93,1310.92,1285.79,1231.27,1188.07,1120.46,1057.27,1006.96,970.05,838.08,739.70,704.22,668.01；1H NMR(800MHz,DMSO-d6)δ/ppm＝11.07(s,2H),7.39(d,J＝7.8Hz,2H),7.34(d,J＝8.2Hz,2H),7.08(t,J＝7.6Hz,2H),6.97(t,J＝7.4Hz,2H),5.93(dd,J1＝13.8Hz,J2＝7.0Hz,2H),4.61(dd,J1＝11.8Hz,J2＝4.1Hz,2H),4.53(t,J＝5.4Hz,2H),3.34(s,6H),3.26(s,6H),3.22(m,2H),2.79(m,2H),2.25(m,4H)。
EXAMPLE 4 preparation of (2 'S, 5' S) -Tetrahydropyrazine [1 ', 2': 1,6] and bis { (1R, R) - [ 1-oxoethyl-2-yl ] -2,3,4, 9-tetrahydro-1H-pyridine [3,4-b ] indoline } -1 ', 4' -dione (4, RR-heptacyclal)
50mg (0.09mmol) of RR-heptacyclic acetal (3) was dissolved in 5mL of glacial acetic acid, 0.4mL of ultrapure water was added dropwise with stirring, and the reaction was stirred at room temperature for 48 hours, at which time no pale yellow solid precipitated from the reaction solution and disappearance of RR-heptacyclic acetal (3) was observed by TLC (Petroleum ether/ethyl acetate, 1/1). The reaction was terminated. The reaction was filtered to give 42mg (98%) of the title compound as a pale yellow solid. ESI-ICR-FT-MS (M/e):479.17141[ M-H]-；
(c＝0.10,CH3OH)；M.p.237～238℃；IR(cm-1):3396.62,2846.89,1707.59,1655.08,1452.36,1406.41,1328.55,1294.69,1261.21,1227.36,1157.20,1112.79,1058.04,1008.41,923.04,740.22,671.75；1H NMR(800MHz,DMSO-d6)δ/ppm＝10.95(s,2H),9.80(s,2H),7.39(m,4H),7.10(m,2H),6.99(m,2H),6.23(m,2H),4.67(m,2H),3.26(m,3H),3.15(m,2H),3.06(m,1H),2.82(m,2H)。
Example 5 evaluation of the anti-arterial thrombotic Effect of RR-heptacyclaldehyde
Experimental Material
Uratan (Ethyl carbamate, CAS:51-79-6, national pharmaceutical group Chemicals Co., Ltd.), Aspirin (CAS:50-78-2, Shanghai Allantin Biotech Co., Ltd.).
Laboratory animal
Male SD rats (200. + -.20 g) purchased from Experimental animals technology, Inc. of Weitongli, Beijing. The model is used for preparing a rat common carotid artery external jugular vein bypass circulation silk thread model during evaluation.
Dosage to be administered
The RR-heptacyclaldehyde of the invention is suspended in 0.5 percent of CMC-Na, and the dosage is 0.1 mu mol/kg; the positive control aspirin was suspended in 0.5% CMC-Na at a dose of 167. mu. mol/kg; the negative control was 0.5% CMC-Na.
Experimental procedures
Rats were acclimatized and fasted for one day prior to surgery, and were gavaged at a dose of 0.3mL/100g body weight 30min prior to surgery using a double-blind sequential method. The polyethylene pipe is filled with physiological saline solution of heparin sodium, 20% of urethane solution is injected according to the weight of a rat to be completely anesthetized, the anesthetized rat is fixed on a rat board, the right common carotid artery is separated, an operation line is respectively penetrated into the proximal end and the distal end of the common artery, and the distal end is ligated. Then, the left external jugular vein of the rat is separated, and an operation line is respectively penetrated into the proximal end and the distal end of the vein, and the distal end is ligated. Carefully cutting an oblique opening on the exposed left external jugular vein, inserting the prepared tip of a circulation bypass pipeline into the oblique opening, fixing the vein and a polyethylene tube by using a surgical thread, pushing an accurate amount of heparin sodium physiological saline solution into the syringe through the other end, stopping bleeding by using an artery clamp at the proximal end of the right common carotid artery, cutting an oblique opening at a position not far away from the artery clamp, inserting the tip of the polyethylene tube into the proximal end of the artery oblique opening for fixing and loosening the artery clamp for circulation for 15 minutes, observing the breathing condition of a rat during circulation, and monitoring the blood circulation condition. After 15 minutes of circulation, the polyethylene tube at the venous end was cut open, the silk was removed with straight forceps, the filter paper was blotted to remove floating blood and weighed, and the wet weight of the thrombus was recorded. And counting the results by using a T test mode. The operation was performed alternately with four of each group. The experimental data are shown in table 1.
TABLE 1 treatment of arterial thrombotic heavies in rats
a) The ratio of P to CMC-Na is less than 0.01; n is 11.
The data in Table 1 show that RR-heptacyclaldehyde is effective in inhibiting arterial thrombosis in rats at an oral dose of 0.1 μmol/kg. Therefore, the invention has obvious technical effect.
Example 6 evaluation of RR-heptacyclaldehyde anti-thrombogenic Effect
Experimental Material
Uratan (Ethyl carbamate, CAS:51-79-6, national pharmaceutical group chemical Co., Ltd.), warfarin (CAS:129-06-6, Bailingwei science and technology Co., Ltd.).
Laboratory animal
Male SD rats (200. + -.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
The RR-heptacyclaldehyde of the invention is suspended in 0.5 percent of CMC-Na, and the dosage is 0.1 mu mol/kg; the positive control warfarin was suspended in 0.5% CMC-Na at a dose of 4.87. mu. mol/kg; the negative control was 0.5% CMC-Na.
Experimental procedures
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. Fixing the rat on a rat fixing plate, preparing skin on the abdomen, sterilizing, opening the abdominal cavity along the leucorrhea line, and opening to expose one corner of the liver until 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 hours. 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 coagulation factor X, P-selectin and GPIIb/IIIa. The thrombus was weighed and the results were counted using the T-test. The operation was performed alternately with four of each group. The experimental data are shown in table 2.
TABLE 2 treatment of venous thrombosis in rats
a) Compared with 0.5 percent of CMC-Na, P is less than 0.01; n is 9
The data in Table 2 show that RR-heptacyclaldehyde is effective in inhibiting venous thrombosis in rats at an oral dose of 0.1. mu. mol/kg. Therefore, the invention has obvious technical effect.
Example 7 evaluation of RR-heptacyclaldehyde for bleeding side Risk
The international normalized ratio was 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 6 is placed in a centrifuge tube filled with 0.4mL sodium citrate solution (3.8%), centrifuged for 15 minutes at 2500g, Platelet Poor Plasma (PPP) at the upper layer is sucked, 100 μ 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-warming area of the instrument for 180 seconds, 200 μ L plasma prothrombin time test solution (kit goods number: 20-7011, MD Pacific) is added, the instrument starts to automatically detect to obtain the plasma prothrombin time, the international standardized ratio is automatically calculated, the statistical result is carried out by using the T test mode, and the experimental data are shown in Table 3.
TABLE 3 International normalized ratio of treated rats
a) The ratio of P to CMC-Na is less than 0.01; n is 8
The data in Table 3 show that RR-heptacyclaldehyde effectively inhibits the internationally standardized ratio in rats at an oral dose of 0.1 μmol/kg. The RR-heptacyclic aldehyde has no bleeding risk, and the invention has obvious technical effect.
Example 8 evaluation of the Effect of RR-heptacyclaldehyde on P-selectin levels in the plasma of intravenous rats
Example 6 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 the supernatant Platelet Poor Plasma (PPP) was aspirated. Add 100. mu.L PPP sample and standard to each well of the assay plate, incubate at 37 ℃ for 2 hours after plating with coversheet membrane, then tilt all the liquid in the plate, do not wash the plate, add 100. mu.L Biotin solution directly to each well, incubate at 37 ℃ for 1 hour after plating with new coversheet 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 2 minutes 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 concentration of the P-selectin standard substance as a horizontal coordinate, and calculating the content of the P-selectin in the sample to be detected from the standard curve according to the OD value. The differences in P-selectin content of each group were analyzed using the one-way analysis of variance (ANOVA one-way, LSD) method in SPSS 22.0, and the experimental data are shown in Table 4.
TABLE 4 content of P-selectin in plasma of treated rats
a) The ratio of P to CMC-Na is less than 0.01; n is 5
The data in Table 4 show that RS-heptacyclaldehyde is effective in reducing plasma P-selectin levels in rats at an oral dose of 0.1. mu. mol/kg. Therefore, the invention has obvious technical effect.
Example 9 evaluation of the Effect of RR-heptacyclaldehyde on GPIIb/IIIa levels in venous thrombosed rat plasma
Example 6 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. Add 100. mu.L of LPPP sample and standard to each well of the assay plate, incubate at 37 ℃ for 2 hours after plating with coversheet membrane, then tilt all the liquid in the plate, do not wash the plate, add 100. mu.L of Biotin solution directly to each well, incubate at 37 ℃ for 1 hour after plating with new coversheet 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 2 minutes 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. mu.L of 3,3',5,5' -tetramethylbenzidine was added to each well, covered with a new cover film and incubated at 37 ℃ for 20 minutes in the dark. And then adding 50 mu L of stop solution into each hole, rapidly reading the OD value of each hole under the dual-wavelength of 450nm and 540nm by using an enzyme-labeling instrument, subtracting the OD value read at 540nm from the OD value read at 450nm, drawing a standard curve by taking the value as a vertical coordinate and the concentration of the GPIIb/IIIa standard substance as a horizontal coordinate, and calculating the content of GPIIb/IIIa in the sample to be detected from the standard curve according to the OD value. The difference of GPIIb/IIIa content of each group is analyzed by using a single-factor analysis of variance (ANOVA one-way, LSD) method in SPSS 22.0, and the experimental data are shown in Table 5.
TABLE 5 GPIIb/IIIa content in treated rat plasma
a) The ratio of P to CMC-Na is less than 0.01; n is 5
The data in Table 5 show that RR-heptacyclaldehyde is effective in reducing plasma GPIIb/IIIa in rats at 0.1. mu. mol/kg oral dose. Therefore, the invention has obvious technical effect.
Example 10 evaluation of the Effect of RR-heptacyclaldehyde on the level of blood coagulation factor X in the plasma of phlebothrombotic rat
Example 6 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 the supernatant Platelet Poor Plasma (PPP) was aspirated. The method comprises the following steps of taking a PPP sample of 6 rats after administration for each experimental group, diluting the PPP sample by 200 times with a sample diluent in a kit, adding 50 mu L of a sample to be detected or a standard substance into each hole in a test plate, simultaneously adding 50 mu L of HRP-avidin into each hole, covering a cover plate membrane, incubating at 37 ℃ for 1 hour, then discarding liquid in the plate, adding 200 mu L of washing liquid into each hole, standing for 2 minutes, discarding the washing liquid, drying a 96-hole plate, and repeating the steps for 5 times. Then, 90. mu.L of 3,3',5,5' -tetramethylbenzidine is added into each well, a new cover plate membrane is covered on the well, the well is incubated for 20 minutes in the dark at 37 ℃, 50. mu.L of stop solution is added into each well after incubation, and the OD value of each well is rapidly read 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 FXa standard substance concentration as a horizontal coordinate, and calculating the FXa content in the sample to be detected from the standard curve according to the OD value. The difference in FXa content among the groups was analyzed by the T Test (T-Test) method in SPSS 22.0, and the experimental data are shown in Table 6.
TABLE 6 treatment of FXa content in rat plasma
a) The ratio of P to 0.5 percent CMC-Na is less than 0.01, and the ratio of P to warfarin is more than 0.05; n is 5
The data in Table 6 show that RR-heptacyclaldehyde is effective in reducing plasma FXa levels in rats at 0.1. mu. mol/kg oral dose, which is not significantly different from warfarin at 4.87. mu. mol/kg oral dose, indicating that RR-heptacyclaldehyde is at least 48.7 times as active as warfarin in reducing plasma FXa levels in rats. 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 (7)
1. An RR-heptacyclic aldehyde having the formula:
2. a process for preparing the RR-heptacyclic aldehyde of claim 1, comprising the steps of:
1) performing Pictet-Spengler condensation on L-tryptophan benzyl ester and 1,1,3, 3-tetramethoxypropane to generate (3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;
2) (3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester is converted into (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid;
3) intermolecular condensation of (1R,3S) -1- (2, 2-dimethoxyethyl-2-yl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid into RR-heptacyclic acetal, a precursor of RR-heptacyclic aldehyde of claim 1;
4) conversion of the RR-heptacyclic acetal to the RR-heptacyclic aldehyde of claim 1.
3. Use of the RR-heptacyclic aldehyde of claim 1 for the preparation of an anti-arterial thrombosis medicament without bleeding side effects.
4. The use of the RR-heptacyclic aldehyde of claim 1 for the preparation of an anti-thrombotic drug without bleeding side effects.
5. Use of the RR-heptacyclic aldehyde of claim 1 for the preparation of a factor X antagonist.
6. Use of the RR-heptacyclic aldehyde of claim 1 for the preparation of a P-selectin antagonist.
7. Use of the RR-heptacyclic aldehyde of claim 1 for the preparation of a GPIIb/IIIa antagonist.
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