CN107474106B

CN107474106B - 4', 7-dioxyacetyl-APAK-isoflavone, its synthesis, activity and use

Info

Publication number: CN107474106B
Application number: CN201610405715.4A
Authority: CN
Inventors: 彭师奇; 赵明; 吴建辉; 王玉记; 张帅
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2016-06-08
Filing date: 2016-06-08
Publication date: 2020-11-27
Anticipated expiration: 2036-06-08
Also published as: CN107474106A

Abstract

The invention discloses 4', 7-dioxy acetyl-Ala-Pro-Ala-Lys-isoflavone, discloses a preparation method thereof, discloses antithrombotic activity thereof, discloses thrombolytic activity thereof, discloses activity for treating cerebral thrombosis, and discloses free radical scavenging activity thereof, and thus the invention discloses application thereof in preparing antithrombotic drugs, thrombolytic drugs, drugs for treating cerebral thrombosis and free radical scavenging drugs.

Description

4', 7-dioxyacetyl-APAK-isoflavone, its synthesis, activity and use

Technical Field

The invention relates to 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone, a preparation method thereof, antithrombotic activity thereof, thrombolytic activity thereof, and activity for treating cerebral thrombosis and scavenging free radicals, and thus relates to application thereof as an antithrombotic drug, a thrombolytic drug, a drug for treating cerebral thrombosis and a drug for scavenging free radicals. The invention belongs to the field of biological medicine.

Background

Ischemic stroke is a common and serious cerebrovascular disease, and is characterized by high morbidity, high fatality rate, high disability rate and high recurrence rate. At present, the clinical treatment of ischemic stroke faces the reality that no effective medicine exists, and especially, patients with stroke for more than 4 hours are not dead or are disabled. The invention of a drug effective in patients with stroke for more than 4 hours is an important clinical need. One of the basic targets set by the invention is to invent a novel compound for ischemic stroke, break through a 4-hour gold treatment time window of ischemic stroke and help the clinical treatment of ischemic stroke to go out of predicament. To achieve this goal, the inventors have undergone a laborious inventive process. The first invention of the inventor relates to P6A-related thrombolytic compounds, which were granted national patent grant in 1996. Since thrombolytic activity is not equal to that of treating ischemic stroke, the thrombolytic compound related to P6A in this patent was not evaluated for its function in treating ischemic stroke. Second inventorsThe invention relates to the conjugation of RGD-tetrapeptide with a thrombolytic compound related to P6A to form an antithrombotic compound. Although RGDF had antithrombotic activity at the 2.5. mu. mol/kg dose, P6A-RGDF and QP6A-RGDF did not have antithrombotic activity at the 2.5. mu. mol/kg dose. Although RGDS and RGDV conjugates with P6A and QP6A, P6A-RGDS, P6A-RGDV, QP6A-RGDS and QP6A-RGDV, which have no antithrombotic activity at 5. mu. mol/kg dose, have antithrombotic activity at 2.5. mu. mol/kg dose. Although 10. mu. mol/kg of P6A and QP6A both had thrombolytic activity, none of P6A-RGDF, QP6A-RGDF, P6A-RGDS, P6A-RGDV, QP6A-RGDS and QP6A-RGDV substantially had thrombolytic activity. It is seen that the combination of drug moieties does not always lead to the desired invention. The third invention of the inventor is to combine the PAK sequence (ARPAK, GRPAK, QRPAK) with 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]4,4,5, 5-tetramethyl imidazoline conjugate to create conjugate with dual functions of scavenging free radicals and dissolving thrombus. 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl group at a dose of 10. mu. mol/kg]-4,4,5, 5-tetramethylimidazoline-ARPAK, 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]-4,4,5, 5-tetramethylimidazoline-GRPAK and 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]The-4, 4,5, 5-tetramethylimidazoline-QRPAK showed thrombolytic activity. Their activity is significantly lower than the thrombolytic activity of UK at doses of 20000IU/kg and is only comparable to the activities of ARPAK, GRPAK and QRPAK. 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]-4,4,5, 5-tetramethylimidazoline-ARPAK, 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]-4,4,5, 5-tetramethylimidazoline-GRPAK and 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]The activities of-4, 4,5, 5-tetramethylimidazoline-QRPAK to scavenge O.free radical, OH free radical and NO.free radical were measured on ESR instrument (i.e. paramagnetic resonance instrument), but they were unable to antagonize acetylcholine-induced relaxation of rat aortic strips on internationally accepted evaluation models (inhibition experiment on acetylcholine-relaxed rat aortic strips). 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]-4,4,5, 5-tetramethylimidazoline-ARPAK, 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]-4,4,5, 5-tetramethylimidazoline-GRPAK and 1, 3-dioxy-2- [ (4-oxyacetic acid) phenyl]The most undesirable property of-4, 4,5, 5-tetramethylimidazoline-QRPAK is the lack of effect in treating ischemic stroke. The inventors have disclosed that imidazoline compounds of formula II are present in the rat in the stroke field for 24hThe blood stroke model shows excellent curative effect. That is, the imidazoline compound of formula II is continuously injected intravenously for 6 days, and has excellent curative effect 1 time per day, the first dosage is 5 mu mol/kg, and the last 5 dosages are 2 mu mol/kg. In the formula aa₁And aa₂Can be present simultaneously aa₁Exist but aa₂Absent, or both; when aa₁And aa₂In the meantime, aa₁Is R (Arg), and aa₂G (Gly), A (Ala) or Q (Gln); when aa₁Exist but aa₂In absence aa₁Is R (Arg); aa₃It may be S (Ser), V (Val) or F (Phe). Since the 2-position of the imidazoline compound of formula II is 4-oxyacetyl-Lys. The side chain amino group and the main chain carboxyl group of the Lys are respectively connected with the RGD antithrombotic tetrapeptide and the ARPAK thrombolytic peptide, so the structure is more complicated and needs to be simplified.

Through 3 years of experimental research, the inventor finds that the effective medicament for treating ischemic stroke can be obtained by simultaneously replacing the 4' position and the 7 position of isoflavone with acetyl-Ala-Pro-Ala-Lys, and the effective dose is 50 nmol/kg. This is an unexpected technical effect. In light of this finding, the inventors have devised the present invention.

Disclosure of Invention

The first aspect of the invention provides 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone.

A second aspect of the invention provides a process for the synthesis of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone comprising:

(1) at K₂CO₃Preparing 4', 7-dioxy acetic acid methyl ester-isoflavone (1) by a standard method under the catalysis of the (A);

(2) saponifying 4 ', 7-dioxyacetic acid methyl ester-isoflavone (1) into 4', 7-dioxyacetic acid-isoflavone (2) under the action of 2N NaOH;

(3) reacting 4 ', 7-dioxyacetic acid-isoflavone (2) with Ala-Pro-Ala-Lys (Z) -OBzl to prepare 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys (Z) -OBzl-isoflavone (3);

(4)4 ', 7-dioxyacetyl-Ala-Pro-Ala-Lys (Z) -OBzl-isoflavone (3) is prepared into 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) under the action of trifluoroacetic acid and trifluoromethanesulfonic acid.

A third aspect of the invention is the evaluation of the anti-arterial thrombotic effect of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone.

A fourth aspect of the invention is the evaluation of the anti-deep vein thrombosis activity of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone

The fifth aspect of the present invention was to evaluate the thrombolytic effect of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone.

A sixth aspect of the invention is the evaluation of the effect of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone on the treatment of cerebral thrombosis.

A seventh aspect of the invention is to evaluate the scavenging of free radicals by 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone.

Drawings

FIG. 14, 7-dioxo-acetyl-Ala-Pro-Ala-Lys-isoflavone synthetic route i) BrCH₂COOCH₃,K₂CO₃,THF；ii)2N NaOH,MeOH；iii)DCC,DMAP,DMF,NMM；iv)TFA,TFMSA。

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 4', 7-Dioxomethyl acetate-isoflavone (1)

20.19g (79.5mmol) of daidzein was dissolved in 300mL of Tetrahydrofuran (THF), 87.8g (635.9mmol) of potassium carbonate was added, and after activation for 20 minutes, 43.59mL (320mmol) of methyl bromoacetate was added, and the reaction was carried out in an oil bath at 45 ℃ for 5 days. Monitoring by TLC: (dichloromethane: methanol ═ 20:1) the progress of the reaction was monitored, and after completion of the reaction, the reaction mixture was filtered by settling and filtrationThe solution was spun off THF under reduced pressure, triturated with a large amount of petroleum ether to remove excess methyl bromoacetate, and the resulting solid was dissolved with methanol and found to be slightly soluble, heated at 45 ℃, filtered while hot, the solid was air dried, the filtrate was recrystallized from cold heat, filtered, and the solid was collected to give 20.71g (61.2%) of the title compound. ESI^--MS(m/e):397[M-H]^-。

EXAMPLE 2 preparation of 4', 7-Dioxoacetic acid-isoflavone (2)

After 5.75g (13.5mmol) of 4', 7-dioxomethyl acetate-isoflavone (1) was dissolved in 100mL of methanol (slightly soluble), 2N NaOH was added to adjust the pH of the solution to 12, the solid was gradually dissolved, and the reaction was carried out at room temperature for 4 hours, TLC (dichloromethane: methanol: glacial acetic acid: 20: 1: 2 drops) was used to monitor the progress of the reaction, the starting material point disappeared, a saturated potassium hydrogen sulfate solution was added to adjust the pH of the solution to 1-2, a large amount of white solid was precipitated, a large amount of water was added to dissolve the salt, followed by filtration, and the cake was repeatedly washed with water and the solid was collected by air-drying to obtain 4.60g (92.2%) of the title compound. ESI^--MS(m/e):369[M-H]^-。

EXAMPLE 3 preparation of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys (Z) -OBzl isoflavone (3)

1.01g (2.74mmol) of 4', 7-dioxyacetic acid-isoflavone (2) was dissolved in 80mL of anhydrous N, N-Dimethylformamide (DMF), 100mg (0.98mmol) of 4-Dimethylaminopyridine (DMAP) and 0.85g (4.1mmol) of Dicyclohexylcarbodiimide (DCC) were added, respectively, after activation for 20 minutes, 2.80g (4.1mmol) of HCl. Ala-Pro-Ala-Lys (Z) -OBzl was dissolved in 40mL of anhydrous DMF, the pH was adjusted to 8-9 with NMM under ice bath, added to the reaction solution, and the pH was adjusted to 8-9 with NMM, after 3 days of reaction, DMF was blown dry, dichloromethane was dissolved, filtered, and purified by column chromatography (dichloromethane/methanol) to obtain 2.47g, (55.5%) of the title compound. ESI^--MS(m/e):1553[M-H]^—。

EXAMPLE 4 preparation of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys (Z) -OBzl-isoflavone (4)

Adding 412mg (0.25mmol) of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys (Z) -OBzl-isoflavone (3) into a 100mL eggplant flask, adding 4mL of trifluoroacetic acid into the reaction flask under ice bath to dissolve the trifluoroacetic acid, adding 1mL of trifluoromethanesulfonic acid, reacting for 30 minutes, and addingAdding a large amount of diethyl ether into a bottle, stirring for 20 minutes, standing for 15 minutes, pouring out supernatant, adding a large amount of diethyl ether, stirring, repeating the operation for 3 times, and finally pumping out the residual diethyl ether to form powder. Dissolving the powder in 10mL of triple distilled water, adjusting the pH value of the solution to 7 with ammonia water, centrifuging at 3000 rpm for 10 minutes, desalting the supernatant with Sephadex G15, freeze-drying the aqueous solution, separating the freeze-dried powder by preparative liquid phase separation, and purifying to obtain 54mg (19.3%) of the title compound. ESI^--MS(m/e):1103[M-H]^-；Mp:179.4～180.1℃；[α]_D ²⁵＝-86.6(c＝0.12,H₂O)；IR(cm^-1):3305,3071,2980,2879,1730,1624,1510,1446,1375,1243,1181,1051,886,834,638；¹H-NMR(800MHz,DMSO-d6):/ppm＝8.449(dd,J＝4.5Hz,2H),8.350(d,J＝7.2Hz,1H),8.197(d,J＝5.6Hz,1H),8.084～8.024(m,2H),7.917(s,1H),7.541(d,J＝4.0Hz,2H),7.163(d,J＝12.0Hz,2H),7.035(d,J＝12.0Hz,2H),4.881(s,1H),4.732(d,J＝4.0Hz,2H),4.626(m,2H),4.614(s,2H),4.570(m,1H),4.365(t,J＝2.4Hz,1H),4.149(t,J＝7.2Hz,2H)3.623(m,4H),2.038～1.522(m,12H),1.298(d,J＝1.6Hz,6H),1.238(d,J＝1.8Hz,6H)。

Example 5 evaluation of the anti-arterial Thrombus Activity of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

1) Drawing a polyethylene tube into a thin tube with one end being an oblique opening, wherein the fixed length is 10.0cm, and the thin tube is respectively used for inserting a right jugular vein (with a thicker tube diameter) and a left carotid artery (with a thinner tube diameter); the length of the middle section polyethylene tube is 8.0cm, the thrombus line is pressed in the carotid intubation direction, and the tube needs to be filled with heparin before intubation.

2) Male rats weighing 200 + -20 g were anesthetized with 20% urethane (7mL/kg) by intraperitoneal injection 30 minutes after oral administration of 167. mu. mol/kg aspirin or 3mL/kg physiological saline. Fixing a rat on a rat board in a supine position, cutting the skin of the neck, separating a right common carotid artery and a left jugular vein, pressing a line under a blood vessel, ligating a far-end heart, cutting a small opening at the position of the vein close to the far-end heart, inserting a vein end into the vein, injecting heparin, then taking down an injector for injecting the heparin, replacing the injector with 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4), setting the injection dose to be 50nmol/kg, tying the line and fixing, clamping the near-end heart end of the artery by an artery clamp, cutting a small opening at the direction close to the far-end heart end, ligating the artery end, loosening the artery clamp after tying the line and fixing, and establishing an extracorporeal circulation bypass. After 15 minutes of circulation, the venous end is cut off to observe whether the blood circulation is normal, if the blood circulation is normal, a thrombus line is taken out from the arterial end, dry floating blood is stained on paper, then weighing is carried out, the thrombus weight represents the activity of the compound, and the data are listed in table 1. The results show that the thrombus weight of the 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) treated rats is obviously smaller than that of the rats treated by normal saline, and the thrombus activity is exact. The effective dose is 50nmol/kg, and unexpected technical effects are obtained.

TABLE 14 anti-arterial Thrombus Activity of 7' -dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

n is 8; a) p <0.01 to saline.

Example 6 evaluation of the anti-deep vein Thrombus Activity of 4', 7-Dioxoacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

Male rats weighing 250 + -20 g were orally administered 1.5mg/kg warfarin or 3mL/kg physiological saline, and were anesthetized with 20% urethane (7mL/kg) by intraperitoneal injection 30 minutes later. Rat tail vein was anesthetized with 20% urethane normal saline solution with 50nmol/kg 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4), followed by surgery. Fixing a rat, cutting the rat along a leucorrhea line into the abdomen, covering the periphery of the incision with gauze wetted by normal saline, leaking the incision, pulling out the abdominal intestine and related organs of the rat, placing the rat on the gauze, viewing the inferior vena cava, separating the tissues on the inferior vena cava in a blunt manner, exposing the complete veins, separating the inferior vena cava, ligating the junction of the left renal vein and the inferior vena cava with surgical threads, repositioning the organs and suturing the abdominal wall, cutting the abdominal wall after 4 hours, separating three to four branch veins of the inferior vena cava respectively, ligating with the surgical threads, clamping the later part of the ligated part with an artery clamp, cutting off all branch vessels at the middle part of the ligated part and the artery clamp, clamping the branch of the distal end of the inferior vena cava with a hemostatic clamp, clamping the proximal end of the inferior vena cava with a hemostatic clamp, lifting up the ligation lines, cutting off the proximal end of the inferior vena cava with a curved clamp, slowly shearing towards the far-end to avoid shearing the inferior vena cava until the branch of the far-end, shearing the inferior vena cava blood vessel along a hemostatic forceps at the far-end, extruding thrombus from the near-center end of the inferior vena cava to the far-end by using a bending forceps, weighing and recording the wet weight of the thrombus, counting the mean value and the standard deviation (+ -SD) of the wet weight of the thrombus of each group, using a statistical method for t test, using normal saline (NS, 3mL/kg) as blank control, and using warfarin (1.5mg/kg, 3mL/kg) as positive control. The activity of the compounds is shown in bar code and the data are shown in Table 2. The results show that the thrombus weight of the 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) treated rats is obviously smaller than that of the rats treated by normal saline, and the compound shows exact deep vein thrombosis resistance. The effective dose is 50nmol/kg, and unexpected technical effects are obtained. The (4) activity of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone decreases with decreasing dose, and thus there is a dose and effect dependence.

TABLE 24', 7-Dioxoacetyl-Ala-Pro-Ala-Lys-isoflavone (4) for their activity against deep vein thrombosis

n is 8; a) p <0.01 compared to saline; b) p <0.01 compared to saline; p >0.05 compared to warfarin; c) p <0.01 compared to saline; p <0.01 compared to warfarin; d) p >0.05 compared to normal saline.

Example 7 evaluation of thrombolytic Activity of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

The rats are kept for one day at rest and are randomly grouped into 10 rats in each group, and the rats are fasted without water prohibition.

1) Male SD rats weighing 250 + -20 g were anesthetized with 20% urethane solution (7mL/kg abdominal cavity). The anesthetized rat is fixed on a rat board, the right common carotid artery is separated, an artery clamp is clamped at the proximal end, the proximal end and the distal end are respectively introduced into an operation line, a blood vessel is inserted at the distal end, and about 1mL of arterial blood is clamped by loosening the artery clamp. Arterial blood was rapidly injected into vertical embolismic tubes (16 mm long, 2.5mm inner diameter, 5mm outer diameter, 1mL EP tube base for tube bottom) (note that no air bubbles were present) and 0.1mL rat arterial blood was injected into each embolismic tube, a thrombus fixing bolt (20 mm long) was rapidly inserted into the tube, blood was coagulated for 40min, and the thrombus was taken out with an acupuncture needle and weighed.

2) The bypass cannula comprises triplex, and wherein the middle section is polyethylene rubber tube, long 60mm, internal diameter 3.5mm, and both ends are the same polyethylene pipe, long 100mm, internal diameter 1mm, outdoor scene 2mm, and one section of this pipe is drawn into the sharp pipe, and the outside of the other end overlaps one section long 7mm, outside diameter 3.5 mm's polyethylene pipe. The inner walls of the three sections of pipes are silanized.

3) And placing the thrombus fixing bolt wrapped by the thrombus into the polyethylene rubber tube at the middle section, sleeving two ends of the rubber tube with the thickened ends of the two polyethylenes respectively, and filling heparin normal saline solution into the rubber tube through the tip end by using an injector. One end of the normal saline solution filled with the heparin sodium is inserted into the left vein, the other end of the normal saline solution is added with the heparin sodium with an accurate amount by using an injector for anticoagulation, and then the injector of the heparin sodium is pulled out and inserted into the arterial end. Physiological saline (3mL/kg) or a physiological saline solution (20000IU/kg) of urokinase or a physiological saline solution (50nmol/kg) of 4 ', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) is passed through the middle section of the bypass tube, the proximal vein far from the thrombus fixing bolt is punctured, the artery clamp is opened, the time when the blood flows from the artery into the vein through the bypass tube is the circulation starting time, the liquid in the syringe is slowly injected into the blood (about 6min), and the physiological saline, urokinase, 4', 7-dioxyethyl-Ala-Pro-Ala-Lys-isoflavone (4) is applied to the thrombus in the order of vein-heart-artery through the blood circulation. After 60min, the bolt with thrombus was removed, and the weight was recorded by dipping in floating blood. Thrombolytic activity is represented by loss of thrombus weight. The data are shown in Table 3. The results show that the weight reduction of the thrombus of the rat treated by the 4', 7-dioxy acetyl-Ala-Pro-Ala-Lys-isoflavone (4) is obviously larger than that of the rat treated by the normal saline, the exact thrombolytic activity is shown, the effective dose is 50nmol/kg, and the unexpected technical effect is obtained.

TABLE 34 thrombolytic Activity of', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

n is 8; a) p <0.01 to saline.

EXAMPLE 8 evaluation of the cerebral thrombosis treating Activity of 4', 7-Dioxoacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

SD male rats (300 + -20 g) were anesthetized by intraperitoneal injection with a 10% chloral hydrate in physiological saline solution (400mg/kg), a small opening was vertically cut from the center of the neck slightly to the right, and the right Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) were separated along the intramuscular side edge of the sternocleidomastoid. Adding 1mL of physiological saline into the prepared thrombus, smashing the blood clot into fine thrombus blocks with uniform size by using a steel shovel, and transferring the fine thrombus block suspension into a 1mL syringe for later use. While loosening the internal carotid artery clamp of the rat, slowly injecting thrombus mass suspension in a 1mL syringe from the external carotid artery of the rat to the internal carotid artery by the approach of the proximal end to the cerebrum of the rat, then ligating the proximal end of the external carotid artery, opening the artery clamps at the internal carotid artery and the common carotid artery in sequence, recovering the blood flow, and suturing the wound. After 1mL of awakening 24h after penicillin (40mg/10mL) administration, the rats were assigned to neurobiological scoring to ensure that each group had a different score, and then 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) was administered via the caudal vein for six consecutive days.

All rats were subjected to cerebral ischemia surgery and the surviving rats were scored and grouped after 24 hours. The degree of neurological deficit was assessed according to the Zealonga method. Score 0 indicates no signs of neurological deficit; score 1 indicates that the intact lateral forelimb is unable to stretch; score 2 indicates walking to the undamaged side; 3 points indicate that the robot turns to the undamaged side to walk in a rear-end collision shape; 4, identifying obstacle without autonomous walking; and score 5 indicates death. Rats with different scores (i.e., 1, 2, 3 and 4) were equally assigned to each group to constitute sample groups with different degrees of ischemia, at least 11 rats per group were scored for 7 consecutive days, and the results are shown in tables 4 to 5.

1 rat in 4 groups before the treatment of the normal saline group accounts for 7.69 percent of the total proportion; 1 rat in 3 points before treatment accounts for 7.69 percent of the total proportion; the number of 2 rats is 3, which accounts for 23.08 percent of the total proportion; 8 rats with 1 point account for 61.54% of the total proportion, 1 rat without autonomous walking of the disturbance of consciousness after continuously treating for 7 days is scored as 4 points, and accounts for 7.69% of the total proportion, 2 rats with side turning circle in rear-end-shaped walking are scored as 3 points, and account for 15.38% of the total proportion; 7 laterally walking rats with the score of 2 account for 53.85 percent of the total proportion; the number of rats recovered from self-walking is 3, the score is 1, and the rat recovery rate accounts for 23.08% of the total proportion. The overall condition of cerebral ischemia becomes worse.

4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) 1 rat in the first 4 th part of the treatment accounts for 9.09% of the total proportion; 2 rats divided by 3 before treatment account for 18.18 percent of the total proportion; 5 rats divided by 2 account for 45.45 percent of the total proportion; 3 rats accounting for 27.27% of the total proportion in 1 point, and 1 rat walking laterally after continuously treating for 7 days, with a score of 2, accounting for 12.5% of the total proportion; 87.5% of rats recover from self-walking, and 7 rats in 0 point account for 87.5% of the total proportion. The overall condition of cerebral ischemia becomes better. Unexpected technical effects are obtained.

TABLE 4 neurobiological scores for rats with 6 days of continuous saline treatment for 24 hours of cerebral ischemia

n is 13, dosage is 3 mL/kg.

TABLE 54', 7-Dioxoacetyl-Ala-Pro-Ala-Lys-isoflavone (4) neurobiological scores in 24-hour rats with 6-day continuous treatment of cerebral ischemia

n is 11, dosage is 50nmol/kg

After the neurological deficit degree is evaluated according to a Zealonga method, the brain is taken by cutting the head after the uratan is used for anesthesia, the brain tissue is placed in a refrigerator at the temperature of 20 ℃ below zero for 2 hours, the brain is completely frozen, about 2mm coronal continuous sections are taken from the forehead, the total number of the coronal continuous sections is 6, then the coronal continuous sections are placed in 2% 2, 3, 5-triphenyltetrazolium chloride (TTC) solution and incubated for 30 minutes in a dark place at the temperature of 37 ℃, the color change of the brain sections is observed, the normal tissue is stained red by the TTC, and the ischemic part of the tissue is white. Taking a picture by a digital camera, recording original data, processing by SPSS statistical software, calculating infarct volume and normal tissue area in a coronal section, calculating the percent of infarct volume of brains of rats of each group, and performing t test. The activity of the compounds was expressed as cerebral infarction volume (%) and the data are shown in Table 6. The results show that the cerebral infarction volume (%) of the rats treated by the 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) is obviously smaller than that of the rats treated by the normal saline, and the cerebral infarction brain volume ratio is obviously reduced. Unexpected technical effects are obtained.

TABLE 64', 7-Dioxoacetyl-Ala-Pro-Ala-Lys-isoflavone (4) cerebral infarct volume ratio in rats 24 hours after 6 days of ischemia

n is 8; a) p <0.01 in comparison to physiological saline

EXAMPLE 10 evaluation of in vitro free radical scavenging Activity of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

1) In vitro OH free radical scavenging

Preparation of lutidine N-oxide (DMPO) solution: 1.1mg of DMPO was dissolved in 1mL of distilled water.

FeSO₄Preparing a solution: 2.8mg FeSO₄·7H₂O was dissolved in 1mL of distilled water.

H₂O₂Preparing a solution: medical use 30% H₂O₂The solution was diluted to 1%.

The determination method comprises the following steps: 5 uL of FeSO₄Solution +5. mu.L DMPO solution +5. mu.L 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) or water +5. mu. L H₂O₂Sequentially adding

The height of the OH.radical base peak was determined as a blank control, and the height of the peak after addition of the test compound was determined. The OH.radical base peak height was measured as a blank, and the peak height after addition of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) solution was measured.

Clearance rate (basal OH. signal peak height-OH. signal peak height after addition of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4))/basal OH. signal peak height X100%

2) In vitro scavenging of NO free radical

Preparation of N-methyl-glucosamine dithioformic acid (MGD) solution: 7.3mg of MGD was dissolved in 1mL of distilled water.

FeSO₄Preparing a solution: 3.5mg FeSO₄·7H₂O dissolved in 1mL of distilled water.

Preparation of nitrosoacetylpenicillamine (SNAP) solution: 2.5mg of SNAP was dissolved in 1mL of distilled water and diluted 100-fold.

The determination method comprises the following steps: 5.0 uL MGD +5.0 uL FeSO₄Solution +5. mu.L 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) or Water + 5.0. mu.L SNAP solution

The NO & free radical base peak height was measured as a blank height, and the peak height after addition of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4) was measured.

Clearance rate (basal No. signal peak height-No. signal peak height after addition of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4))/basal No. signal peak height × 100%

Calculate half Effective Clearance (EC)₅₀)

TABLE 74 OH, NO free radical scavenging Activity of' 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone (4)

Claims

1. 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of the formula,

2. a process for the preparation of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone according to claim 1, which process comprises:

(1) at K₂CO₃Preparing 4', 7-dioxyacetic acid methyl ester-isoflavone (1) by a standard method under the catalysis of (1);

3. Use of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of claim 1 in the manufacture of an anti-thrombotic medicament.

4. Use of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of claim 1 in the manufacture of a medicament for the treatment of deep vein thrombosis.

5. Use of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of claim 1 in the preparation of a thrombolytic drug.

6. Use of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of claim 1 in the manufacture of a medicament for the treatment of cerebral thrombosis.

7. Use of 4', 7-dioxyacetyl-Ala-Pro-Ala-Lys-isoflavone of claim 1 for the preparation of a medicament for scavenging free radicals.