CN107459556A - Left-handed Vc -2- oxygen acetyl-PAKPAK, it is synthesized, activity and application - Google Patents
Left-handed Vc -2- oxygen acetyl-PAKPAK, it is synthesized, activity and application Download PDFInfo
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- CN107459556A CN107459556A CN201610391350.4A CN201610391350A CN107459556A CN 107459556 A CN107459556 A CN 107459556A CN 201610391350 A CN201610391350 A CN 201610391350A CN 107459556 A CN107459556 A CN 107459556A
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Abstract
The invention discloses the oxygen acetyl Pro Ala Lys Pro Ala Lys of left-handed Vc 2 of following formula.Its preparation method is disclosed, its thrombus dissolving activity is disclosed, discloses it and treat the activity of cerebral thrombus and disclose it and remove NO free radical activities, thus the invention discloses it to prepare thrombus dissolving activity, treatment cerebral thrombus medicine and prepare the applications of NO free radical scavengers.
Description
Technical field
The present invention relates to left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys, it is related to its preparation method, it is related to its thrombus dissolving effect, is related to the effect that it treats the effect of cerebral thrombus and is related to its removing NO free radical, thus the present invention relates to it in the application in preparing thrombolytic agent, the application in treatment cerebral thrombus medicine is prepared and the application in NO free radical scavengers are prepared.The invention belongs to biomedicine field.
Background technology
Ishemic stroke is cranial vascular disease a kind of more typical and that harm is serious, and feature is that the incidence of disease is high, case fatality rate is high, disability rate is high and high recurrence rate.Clinical treatment ishemic stroke faces the reality of no active drug at present, and especially patient more than apoplexy face 4h is non-extremely i.e. residual.Invention is clinical important need to the apoplexy face effective medicine of the patient of more than 4 hours.It is ishemic stroke invention new compound that one of elementary object that the present invention is set, which is exactly, breaks through 4 hours gold treatment time windows of ishemic stroke, helps the clinical treatment of ishemic stroke to extricate oneself from a plight.In order to reach this target, inventor experienced arduous invention process.First invention of inventor is to be related to the related thrombolytic compounds of P6A, and they about obtained national patent mandate in 1996.Because thrombus dissolving activity is active not equal to treatment ishemic stroke, the thrombolytic compound related to P6A does not evaluate treatment ishemic stroke function in this patent.Second of inventor invention, which is related to the RGD- tetrapeptides thrombolytic compound related to P6A and be conjugated, forms antithrombotic compound.Although RGDF has antithrombotic acitivity under 2.5 μm of ol/kg dosage, P6A-RGDF and QP6A-RGDF does not have antithrombotic acitivity but under 2.5 μm of ol/kg dosage.Although there is no the RGDS of antithrombotic acitivity and RGDV and P6A and QP6A conjugate P6A-RGDS, P6A-RGDV, QP6A-RGDS and QP6A-RGDV but to have antithrombotic acitivity under 2.5 μm of ol/kg dosage under 5 μm of ol/kg dosage.Although 10 μm of ol/kg P6A and QP6A have thrombus dissolving activity, P6A-RGDF, QP6A-RGDF, P6A-RGDS, P6A-RGDV, QP6A-RGDS and QP6A-RGDV do not have thrombus dissolving activity substantially.It can be seen that pharmacophore combination is not that can cause desired invention.The 3rd invention of inventor is by PAK sequences (ARPAK, GRPAK, QRPAK) and 1, epoxide-the 2- of 3- bis- [(4- fluoroacetic acid) phenyl] -4,4,5,5- tetramethyl imidazolines are conjugated, and create with the conjugate for removing free radical and thrombus dissolving dual-use function.Epoxide-the 2- of 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4 under 10 μm of ol/kg dosage, 5,5- tetramethyl imidazolines-ARPAK, the epoxide -2- of 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4,5, epoxide-the 2- of 5- tetramethyls imidazoline-GRPAK and 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4,5,5- tetramethyl imidazolines-QRPAK shows thrombus dissolving activity.Thrombus dissolving activity of their activity significantly lower than the UK that dosage is 20000IU/kg, and it is only suitable with ARPAK, GRPAK and QRPAK activity.1, epoxide-the 2- of 3- bis- [(4- fluoroacetic acid) phenyl] -4, 4, 5, 5- tetramethyl imidazolines-ARPAK, 1, epoxide-the 2- of 3- bis- [(4- fluoroacetic acid) phenyl] -4, 4, 5, 5- tetramethyls imidazoline-GRPAK and 1, epoxide-the 2- of 3- bis- [(4- fluoroacetic acid) phenyl] -4, 4, 5, 5- tetramethyls imidazoline-QRPAK can be measured on ESR instrument (i.e. EPR spectrometer) removes O free radicals, OH free radicals and NO free radical activities, but they are unable to the rat aortic article diastole of antagonism acetylcholine-induced on internationally recognized evaluation model (to the Inhibition test of the rat aortic article of acetylcholine diastole).Epoxide-the 2- of 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4,5,5- tetramethyl imidazolines-ARPAK, the epoxide -2- of 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4,5, epoxide-the 2- of 5- tetramethyls imidazoline-GRPAK and 1,3- bis- [(4- fluoroacetic acid) phenyl] -4,4, the least preferable properties of 5,5- tetramethyl imidazolines-QRPAK are not treat the effect of ishemic stroke.Inventor once disclosed the imidazolinium compounds of Formula II on apoplexy face 24h ischemia/reperfusion in rats apoplexy model, showed outstanding curative effect.That is the imidazolinium compounds of 6 days Formula II of continuous intravenous injection, 1 time a day, initial dose are 5 μm of ol/kg, and the dosage of latter 5 times has outstanding curative effect for 2 μm of ol/kg.Aa in formula1And aa2Can be to exist simultaneously, aa1In the presence of but aa2It is not present, or is not present simultaneously；Work as aa1And aa2In the presence of simultaneously, aa1For R (Arg), and aa2For G (Gly), A (Ala) or Q (Gln)；Work as aa1In the presence of but aa2In the absence of when, aa1For R (Arg)；aa3Can be S (Ser), V (Val) or F (Phe).Because the 2- positions of the imidazolinium compounds of Formula II are 4- oxygen acetyl-Lys.And the side-chain amino group and main-chain carboxylic group of the Lys is connected with RGD antithrombotics tetrapeptide and ARPAK thrombolysis peptides respectively, need to simplify so structure is more complicated.
Inventor passes through 3 years experimental studies again, finds 2 active drugs that treatment ishemic stroke can be obtained with oxygen acetyl-PAKPAK substitutions in left-handed Vc, minimum effective dose is 1 μm of ol/kg, hence it is evident that less than the imidazolinium compounds of Formula II.For the structural modification of left-handed Vc, this is unexpected technique effect.According to this discovery, the present invention is inventors herein proposed.
The content of the invention
First content of the present invention is to provide the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of following formula.
Second content of the present invention is to provide left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys synthetic method, and this method includes：
(1) left-handed Vc generates left-handed Vc -2- fluoroacetic acid ethyl esters under NaH catalysis with bromoacetate reaction；
(2) left-handed Vc -2- fluoroacetic acid ethyl ester is saponified into left-handed Vc -2- fluoroacetic acid in NaOH solution (2N)；
(3) Boc-Pro and active ester HOSu generations Boc-Pro-OSu；
(4) Boc-Pro-OSu and L-Ala is condensed into Boc-Pro-Ala under the conditions of weak base；
(5) Boc-Pro-Ala and Lys (Fmoc)-OBzl are condensed into Boc-Pro-Ala-Lys (Fmoc)-OBzl through DCC, HOBt；
(6) Boc-Pro-Ala-Lys (Fmoc)-OBzl de- Boc in the ethyl acetate solution of 4N hydrogen chloride obtain Pro-Ala-Lys (Fmoc)-OBzl；
(7) Pro-Ala-Lys (Fmoc)-OBzl and Boc-Lys (Fmoc) is condensed into Boc-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl through DCC, HOBt；
(8) Boc-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl obtain Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl in the ethyl acetate solution of 4N hydrogen chloride；
(9) Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl and Boc-Pro-Ala is condensed into Boc-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl through DCC, HOBt；
(10) Boc-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl obtain Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl in the ethyl acetate solution of 4N hydrogen chloride；
(11) Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are condensed into left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl with left-handed Vc -2- fluoroacetic acid through DCC, HOBt；
(12) left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are saponified into left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys in NaOH solution (2N)；
The 3rd content of the present invention is to evaluate left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys thrombus dissolving activity.
The 4th content of the present invention is the activity of the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys treatments cerebral thrombus of evaluation.
The 5th content of the present invention is that the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of evaluation removes NO free radical activities.
Brief description of the drawings
Left-handed Vc -2- oxygen acetyl-the Pro-Ala-Lys-Pro-Ala-Lys of Fig. 1 synthetic route .a) DCC, THF；b)NaHCO3；c)DCC,HOBt,THF；D) 4N hydrogen chloride-ethyl acetate solution；e)BrCH2COOC2H5,NaH,DMF；f)2N NaOH,CH3OH；g)DCC,HOBt,DMF；h)2N NaOH.
Embodiment
In order to which the present invention is expanded on further, a series of embodiments are given below.These embodiments are entirely illustrative, and they are only used for that the present invention is specifically described, and are not construed as limitation of the present invention.
Embodiment 1 prepares left-handed Vc -2- fluoroacetic acid ethyl ester (1)
At 0 DEG C into the solution of 1.14g (28mmol) NaH (60%) and 10mL anhydrous dimethyl formamides (DMF), in batches plus 5.00g (28mmol) left-handed Vc, there is bubble to emerge, after bubble-free, 5.42g (28mmol) bromoacetate is added dropwise inward, stirring reaction 12 hours, TLC, which is shown, completes reaction.Reactant mixture removal of solvent under reduced pressure, obtained residue are purified by silica gel column chromatography, obtain 3.49g (49%) title compound, are colorless syrup thing.ESI-MS(m/e):261[M-H]-；Mp:177-179℃；[α]D 25=-12.1 (c=0.1, MeOH), IR (KBr, cm-1):3333,1761,1676,1269,1247；1HNMR(300MHz,DMSO-d6):δ/ppm=4.94 (m, 2H), 4.85 (s, 1H), 4.15 (q, J=0.6Hz, 2H), 3.83 (m, 1H), 3.48 (m, 2H), 1.20 (t, J=0.6Hz, 3H).
Embodiment 2 prepares left-handed Vc -2- fluoroacetic acid (2)
Left-handed Vc -2- fluoroacetic acid the ethyl esters (1) of 3.49g (13.3mmol) are dissolved with 10mL methanol, ice bath stirring is lower to add 1mL water, and pH value 12 is adjusted with the 2M NaOH aqueous solution.Ice bath stirs 12h, and TLC monitoring reactions are completed.Reactant mixture 5%KHSO4The aqueous solution adjusts pH value 7, is concentrated under reduced pressure and removes solvent, obtains 2.52g (89%) title compound, be colourless powder.ESI-MS(m/e):233[M-H]-。
Embodiment 3 prepares Boc-Pro-OSu
5.50g (25.6mmol) Boc-Pro is dissolved in 30mL anhydrous tetrahydro furans (THF), 6.32g (30.6mmol) dicyclohexylcarbodiimide (DCC) is added under ice bath, stir 30min, add 3.24g (28.1mmol) n-hydroxysuccinimide (HOSu), 12h is stirred, TLC monitoring reactions are completed.It is filtered under diminished pressure and removes dicyclohexylurea (DCU) (DCU), filtrate decompression is concentrated to dryness, and obtained residue with Ethyl acetate dissolving, then uses saturation NaHCO respectively3The aqueous solution is washed three times, is washed three times with the saturation NaCl aqueous solution, organic phase anhydrous Na2SO48h is dried, is filtered under diminished pressure, filtrate decompression concentration removes solvent, obtains 6.64g (83.2%) title compound, is colourless powder.ESI-MS(m/e):313[M+H]+。
Embodiment 4 prepares Boc-Pro-Ala
6.64g (21.2mmol) the Boc-Pro-OSu anhydrous THF of 100ml are dissolved, the lower solution for adding 2.08g (23.3mmol) L-Ala and 50mL water of ice bath stirring.Reactant mixture solid NaHCO3PH value 9 is adjusted, reacts 12h, TLC monitoring reactions are completed.Reactant mixture 5%KHSO4The aqueous solution adjusts pH value 7, is concentrated under reduced pressure except using saturation KHSO after THF4The aqueous solution adjusts solution ph 2, and reactant mixture is extracted with ethyl acetate (50mL × 3) three times, and ester layer washes (30mL × 3) three times with the saturation NaCl aqueous solution, obtained organic phase anhydrous Na2SO48h is dried, is filtered under diminished pressure, filtrate decompression concentration removes solvent, obtains 5.22g (86%) title compound, is colourless powder.ESI-MS(m/e):287[M+H]+。
Embodiment 5 prepares Boc-Pro-Ala-Lys (Fmoc)-OBzl
By 2.60g (12.3mmol) Boc-Pro-Ala, 1.23g (12.3mmol) HOBt, after the anhydrous THF of 2.05g (13.4mmol) DCC and 100mL solution ice bath stirring 30min, inward plus 4.40g (11.2mmol) HClLys (Fmoc)-OBzl, pH 9 is adjusted with N-methylmorpholine (NMM), react 12 hours at room temperature, TLC monitoring reactions are completed.It is filtered under diminished pressure and removes DCU, filtrate decompression concentration, obtained residue with Ethyl acetate dissolving, successively with saturation NaHCO3The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL), saturation KHSO4The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL), saturation NaHCO3The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL) are respectively washed three times, use anhydrous Na2SO4Dry 8 hours, be filtered under diminished pressure, filtrate decompression concentration, obtained residue is purified using silica gel column chromatography, obtains 1.78g (28%) title compound, be colourless powder.ESI-MS(m/e):727[M+H]+。
Embodiment 6 prepares HClPro-Ala-Lys (Fmoc)-OBzl
The ethyl acetate solution (4M) of 1.78g (1.14mmol) Boc-Pro-Ala-Lys (Fmoc)-OBzl and 10mL hydrogen chloride is stirred into 2h under ice bath, TLC monitoring reactions are completed.Decompressing and extracting ethyl acetate, the ethyl acetate that 30mL is dried is added, then drained to take hydrogen chloride gas out of, this operation is repeated twice, then is poured into 20mL absolute ethers and drained, in triplicate.1.32g title compounds are obtained, not purified be directly used in is reacted in next step.ESI-MS(m/e):627[M+H]+。
Embodiment 7 prepares Boc-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl
By 1.01g (2.5mmol) Boc-Lys (Fmoc) under ice bath, 0.27g (2.5mmol) HOBt, 0.45g (2.7mmol) the DCC and anhydrous THF of 150mL solution stirring 30min, afterwards plus 1.2g (2.3mmol) HClPro-Ala-Lys (Fmoc)-OBzl, it is 9 with NMM regulation pH value, ice bath is removed, is reacted at room temperature 12 hours, TLC monitoring reactions are completed.It is filtered under diminished pressure and removes DCU, filtrate decompression concentration removes solvent, with 200mL ethyl acetate dissolution residual substances, respectively with saturation NaHCO3The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL), saturation KHSO4The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL), saturation NaHCO3The aqueous solution (50mL), the saturation NaCl aqueous solution (50mL) are respectively washed three times, organic phase anhydrous Na2SO4Dry, be filtered under diminished pressure and remove Na2SO4, filtrate decompression concentration removes solvent, obtains residue 2.32g title compounds, not purified to be used to react in next step.ESI-MS(m/z):787[M+Na]+。
Embodiment 8 prepares HClLys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl
Using the operation of embodiment 6,3.18g title compounds are obtained by 2.32g (4.5mmol) Boc-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl, are colourless powder product, it is not purified to be used to react in next step.ESI-MS(m/e):998[M+H]+。
Embodiment 9 prepares Boc-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl
Using the operation of embodiment 7,1.02g (26.1%) title compound is obtained by 0.99g (3.4mmol) Boc-Pro-Ala and 3.4g (3.4mmol) Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl, is colourless powder.ESI-MS(m/e):1245[M+H]+。
Embodiment 10 prepares HClPro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl
Using the operation of embodiment 6,0.235g title compounds are obtained by 0.350g (0.28mmol) Boc-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys- (Fmoc)-OBzl, it is not purified to be used in next step.ESI-MS(m/z):687[M+Na]+。
Embodiment 11 prepares C-2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl
Using the operation of embodiment 7,11mg (4%) title compound is obtained by the left-handed Vc -2- fluoroacetic acid (2) of 50mg (0.22mmol) and 235mg (0.20mmol) HClPro-Ala-Lys (Fmoc)-Pro-Ala-Lys- (Fmoc)-OBzl, is colourless powder.ESI-MS(m/e):1383[M+Na]+。
Embodiment 12 prepares C-2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3)
By left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys- (the Fmoc)-OBzl 10mL acetone solutions of 100mg (0.07mmol) under ice bath, add 1mL water, it is 12 with 2M NaOH aqueous solution regulation pH value, TLC monitoring reactions, after raw material disappears, 5%KHSO is used4Aqueous solution regulation pH value is 7, be concentrated under reduced pressure solvent evaporated, residue is worn away with methanol, the salt for removing residual is filtered under diminished pressure, filtrate decompression concentration removes solvent, and residue 3mL ultra-pure waters dissolve, salt remaining in solvent is removed using sephadex G 10 (Sephadex G10), collect sample and freeze, obtain 9mg (15%) title compound using liquid phase purification process is prepared, be colourless powder product.Preparation condition：XBridge Prep HILIC prepare post (19 × 100mm Column), 228nm Detection wavelengths, mobile phase：Acetonitrile/water (pH value=3.0, adding 0.2/1000th formic acid, 10mmol/L ammonium formates)=73/27, retention time：38min, flow velocity 9.0mL/min.ESI-MS (m/z):849[M+Na]+；Mp:222-224℃；[α]D 25=+111.8 (c=0.1, MeOH) .IR (KBr, cm-1):3330,3481,2927,2920,2872,1762,1689,1685,1680,1720,1644,1269,1247,721；1HNMR(300MHz,DMSO-d6):δ/ppm=8.606 (s, 1H), 8.512 (s, 1H), 8.309 (s, 2H), 7.443 (s, 2H), 4.810 (s, 2H), 4.801 (s, 1H), 4.350 (m, 2H), 4.336 (m, 2H), 4.138 (m, 2H), 3.768 (m, 1H), 3.455 (m, 2H), 3.375 (m, 4H), 2.713 (m, 4H), 1.880 (m, 4H), 1.870 (m, 4H), 1.775 (m, 4H), 1.631 (m, 4H), 1.511 (m, 4H), 1.153 (m, 6H).
Experimental example 1 evaluates left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys thrombus dissolving activity
The C-2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of the present invention is with normal saline into required concentration.
SD rats (male, 200 ± 20g) are anaesthetized by the 1200mg/kg dosage intraperitoneal injection urethane aqueous solution.Its dorsal position is fixed after anesthetized rat, its right common carotid artery is separated, artery clamp is clamped at proximal part, ligatures distal end with surgical thread, distal end intubation takes blood, artery clamp is unclamped, and takes out about 1mL arterial bloods.Toward vertically fixed rubber tube (long 17mm, internal diameter 2.5mm, external diameter 5.0mm, ttom of pipe are held with support, para films are tamping) in injection rat arterial blood, then be put into rapidly in pipe the thrombus of a stainless steel fixing bolt (thrombus fix spiral be coiled into a diameter of 0.2mm stainless steel wire, the long 10mm of spiral part, include 15 bung flanges, a diameter of 1.0mm of bung flange, support handle is connected with spiral, 7.0mm is about, in question mark type).Blood is taken out by the fixation spiral of thrombus parcel with acupuncture needle is careful from pipe after room temperature solidifies 45min, accurately claims its weight and record.
Bypass intubation is made up of 3 parts, and interlude is long 60.0mm, internal diameter 3.5mm polyethylene rubber tube；Both ends are long 100.0mm, internal diameter 1.0mm, external diameter 2.0mm identical polyethylene pipe, the pipe one end pulls into spike tube, 10.0mm (being used to insert rat carotid artery and vein) is about, external diameter 1.0mm, one section of length of outer cover of its other end is 7.0mm, the polyethylene pipe (being used to insert in the polyethylene rubber tube in stage casing) that external diameter is 3.5mm, the inwall of 3 sections of pipes is intended to silanization (diethyl ether solution of 1% silicone oil).The fixation spiral that thrombus wraps up is placed in the polyethylene rubber tube in stage casing, the overstriking end of the other both ends of sebific duct respectively with two polyethylene is nested, and para films are sealed, and not occur the situation for revealing blood during circulation.With syringe heparin-saline solution (50IU/kg) will be filled by spike tube end in pipe, exclude bubble with standby.
The left vena jugularis externa of rat is separated, the blood vessel of distal end is ligatured with surgical thread, an osculum is cut on exposed left vena jugularis externa, the above-mentioned bypass duct spike tube prepared is inserted into left vena jugularis externa opening by osculum.The solution (50IU/kg) of the liquaemin of correct amount is injected by the spike tube of the other end with syringe, now syringe not withdraw polyethylene pipe, and the flexible pipe between syringe and polyethylene pipe is clamped with artery clamp.Stopped blooding in the proximal part of right common carotid artery with artery clamp, ligature distal end, right common carotid artery is nearby being cut into an osculum from artery clamp, extracted syringe from the tip of polyethylene pipe, the tip of polyethylene pipe is inserted to the proximal part of artery angle.Arteriovenous is fixed with sutures in the both ends of bypass duct.
With scalp acupuncture by physiological saline (3ml/kg), urokinase (UK) normal saline solution (20000IU/kg) and left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys normal saline solution (1 μm of ol/kg) pass through the stage casing of shunt valve, penetrate the nearly vein end that spiral is fixed away from thrombus, unclamp artery clamp, blood flow is set to flow to vein from artery by bypass duct, the model is that rat arteriovenous bypasses Thrombolysis Model, slowly the liquid in syringe is injected into blood, with physiological saline (blank control), UK (positive control) and left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys pass through blood circulation.After timing 1h when starting injection, the fixation spiral in bypass duct, its weight of accurate weighing are taken out.Calculate and the front and rear weight difference of spiral administration is fixed in every rat bypass duct, count the thrombus weight difference (mean value ± SD) of each group, and do t inspections.
Influence of the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) of table 1 to thrombus loss of weight
A) p is compared with physiological saline group<0.01, compare p with UK>0.05.
Experimental example 2 evaluates left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys treatments cerebral thrombus activity
Normal saline solution (400mg/kg) intraperitoneal injection of anesthesia of 10% chloraldurate of SD male rats (300 ± 20g), portion slightly to the right is hit exactly from neck and cuts an osculum vertically, and right common carotid artery (CCA), external carotid artery (ECA) and internal carotid (ICA) are separated to along nutator inner side edge.Blood clotting is pounded uniform tiny thrombi with steel shovel by preprepared thrombus addition 1mL physiological saline, then tiny thrombi suspension is transferred to standby in 1mL syringes.While rat ICA folder is unclamped, by the thrombi suspension in 1mL syringes slowly from rat external carotid artery to proximal part approach internal carotid injection to rat brain, then the proximal part of external carotid artery is ligatured, the artery clamp at internal carotid and arteria carotis communis is opened successively, recover blood flow, sew up a wound.After giving penicillin (40mg/10mL) 1mL revivals 24h, it is grouped after rat is carried out into Neurobiology scoring, ensure there is the rat of different scorings in every group, then caudal vein gives left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys, successive administration six days.
Cerebral ischemia operation is all made in all experiments of rat, and the rat of survival scores after 24 hours, and is grouped.Neurological functional deficit is evaluated according to Zealonga methods.0 point indicates without any neurological deficit sign, and 1 point represents not damaging side forelimb not tensible, and 2 points represent to walk to not damaging skidding, and 3 points represent to turn-take into shape walking of knocking into the back to not damaging side, and 4 points represent the disturbances of consciousness without autonomous, and 5 points represent dead.The rat of different scorings is evenly distributed in each group, forms degree of ischemia
Different sample groups, every group at least 10, each group rat was scored in continuous 7 days, the results are shown in Table 2 and table 3.
3 points of rat has 1 before physiological saline group treatment, accounts for the 11.11% of toatl proportion；2 points of rat has 3 before treatment, accounts for the 33.33% of toatl proportion；1 point of rat has 5 before treatment, accounts for the 55.56% of toatl proportion.It is 5 points that 7 days rats dead afterwards of continuous treatment, which have 1 scoring, accounts for the 11.11% of toatl proportion；The rat that shape walking of knocking into the back is turn-taked into side has 1 to score as 3 points, accounts for the 11.11% of toatl proportion；It is 2 points that the rat that skidding is walked, which has 2 scorings, accounts for the 22.22% of toatl proportion；It is 1 point that the rat that self-service walking recovers, which has 4 scorings, accounts for the 44.44% of toatl proportion.Cerebral ischemia integral status degenerates.
3 points of rat has 2 before left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) treatment, accounts for the 18.18% of toatl proportion；2 points of rat has 3 before treatment, accounts for the 27.27% of toatl proportion；1 point of rat has 6 before treatment, accounts for the 54.55% of toatl proportion；The self-service walking of 100% rat recovers after continuously treating 7 days, and Neurobiology scores as 0 point.Cerebral ischemia integral status improves.Obtain unexpected technique effect.
The continuous normal saline of table 2 treats the Neurobiology scoring of 6 days cerebral ischemias, 24 hours rats
N=10, dosage 3mL/kg
Left-handed Vc -2- oxygen acetyl-the Pro-Ala-Lys-Pro-Ala-Lys of table 3 continuously treats the rat biology scoring in 24 hours of 6 days cerebral ischemias
N=11, dosage are 1 μm of ol/kg
After Zealonga methods evaluation neurological functional deficit, broken end takes brain after being anaesthetized with urethane, after brain tissue is placed in into -20 DEG C of refrigerators 2 hours, brain is thoroughly freezed, from the coronal serial section of preceding antinion starting row about 2mm, totally 6, be subsequently placed in 2% 2,3, in 5- triphenyltetrazolium chlorides (TTC) solution after 37 DEG C of lucifuges are incubated 30min, the color change of brain section is observed, normal structure can be dyed red by TTC, and ischemic section tissue is white.With digital photo camera, initial data is recorded, is handled through SPSS statistical softwares, calculates Infarction volume and normal structure volume in coronal section, counts the Infarction volume percent value of each group rat brain, and does t inspections.Activity is represented with cerebral infarction volume (%), data are included in table 4.As a result show, the cerebral infarction volume (%) of left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) treatment rat is significantly less than the cerebral infarction volume (%) of saline therapy rat.Cerebral infarction brain volume ratio is remarkably decreased.Obtain unexpected technique effect.
The compound 3 of table 4 treats the cerebral infarction volume ratio of rat
A) p is compared with physiological saline<0.01.
Experimental example 3 evaluates left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) and removes NO free radical activities
Prepare N- methyl-glucamines dithiocarbonic acid (MGD) solution:7.3mg MGD is dissolved in 1mL distilled water.Prepare FeSO4Solution:3.5mg FeSO4·7H2In O dissolvings and 1mL distilled water.
Prepare nitroso N-acetylpenicillamine (SNAP) solution:2.5mg SNAP are dissolved in 1mL distilled water, dilute 100 times.Assay method:5.0μL MGD+5.0μL FeSO4Left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) solution of the μ of solution+5 L or the μ L SNAP solution of physiological saline+5.0.NO free radicals basis peak heights are first determined, as blank height, then determine the peak heights after adding left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3).
Clearance rate=(the basic left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) of NO signals peak heights-addition afterwards NO signals peak heights)/basic NO signals peak heights × 100%.Calculate the effective clearance rate (EC of half50) NO free radicals are removed in vitro.Data are included in table 5, the results showed that, left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys (3) can effectively remove NO free radicals, it is shown that unexpected technique effect.
Left-handed Vc -2- oxygen acetyl-the Pro-Ala-Lys-Pro-Ala-Lys (3) of table 5 removes NO free radical activities
N=4；A) p is compared with left-handed Vc -2- fluoroacetic acid<0.05.
Claims (5)
1. the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of following formula,
2. the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of claim 1 preparation method, this method include：
(1) left-handed Vc generates left-handed Vc -2- fluoroacetic acid ethyl esters under NaH catalysis with bromoacetate reaction；
(2) left-handed Vc -2- fluoroacetic acid ethyl ester is saponified into left-handed Vc -2- fluoroacetic acid in NaOH solution (2N)；
(3) Boc-Pro and active ester HOSu generations Boc-Pro-OSu；
(4) Boc-Pro-OSu and L-Ala is condensed into Boc-Pro-Ala under the conditions of weak base；
(5) Boc-Pro-Ala and Lys (Fmoc)-OBzl are condensed into Boc-Pro-Ala-Lys (Fmoc)-OBzl through DCC, HOBt；
(6) Boc-Pro-Ala-Lys (Fmoc)-OBzl de- Boc in the ethyl acetate solution of 4N hydrogen chloride obtain Pro-Ala-
Lys(Fmoc)-OBzl；
(7) Pro-Ala-Lys (Fmoc)-OBzl and Boc-Lys (Fmoc) are condensed into through DCC, HOBt
Boc-Lys(Fmoc)-Pro-Ala-Lys(Fmoc)-OBzl；
(8) Boc-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are obtained in the ethyl acetate solution of 4N hydrogen chloride
Lys(Fmoc)-Pro-Ala-Lys(Fmoc)-OBzl；
(9) Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl and Boc-Pro-Ala are condensed into through DCC, HOBt
Boc-Pro-Ala-Lys(Fmoc)-Pro-Ala-Lys(Fmoc)-OBzl；
(10) Boc-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are obtained in the ethyl acetate solution of 4N hydrogen chloride
To Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl；
(11) Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are condensed with left-handed Vc -2- fluoroacetic acid through DCC, HOBt
Into left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl；
(12) left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys (Fmoc)-Pro-Ala-Lys (Fmoc)-OBzl are in NaOH solution (2N)
It is saponified into left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys.
3. applications of the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of claim 1 in thrombolytic agent is prepared.
4. the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of claim 1 is in treatment cerebral thrombus medicine is prepared
Using.
5. the left-handed Vc -2- oxygen acetyl-Pro-Ala-Lys-Pro-Ala-Lys of claim 1 is in NO free radical scavengers are prepared
Application.
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| JPH07206840A (en) * | 1994-01-14 | 1995-08-08 | Kanto Denka Kogyo Co Ltd | Ascorbic acid-hydroxycarboxylic acid combination product and method for producing the same |
| CN101190941A (en) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | Polypeptide with thrombus dissolving activity and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07206840A (en) * | 1994-01-14 | 1995-08-08 | Kanto Denka Kogyo Co Ltd | Ascorbic acid-hydroxycarboxylic acid combination product and method for producing the same |
| CN101190941A (en) * | 2006-11-30 | 2008-06-04 | 首都医科大学 | Polypeptide with thrombus dissolving activity and its preparation method and application |
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| Title |
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| MING ZHAO ET AL.: "Synthesis and Biological Activity of Nitronyl Nitroxide Containing Peptides", 《J. MED. CHEM.》 * |
| MORISAKI KAZUO ET AL.: "Syntheses of Novel Hybrid Vitamin C Derivatives: Stability and Biological Activity", 《BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN》 * |
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