CN102898507B - Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof - Google Patents

Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof Download PDF

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CN102898507B
CN102898507B CN201210323952.8A CN201210323952A CN102898507B CN 102898507 B CN102898507 B CN 102898507B CN 201210323952 A CN201210323952 A CN 201210323952A CN 102898507 B CN102898507 B CN 102898507B
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thrombolysis
lys
ala
imidazolidine
oligopeptide
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CN102898507A (en
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彭师奇
赵明
蒋雪云
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Shanghai is bright along bio tech ltd
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Abstract

The present invention discloses a thrombolysis oligopeptide-imidazolidine binary conjugate, a preparation method and uses thereof. According to the present invention, L-Lys is adopted as a linking arm to link 1,3-dioxy-2-[(4-oxyacetic acid)phenyl]-4,4,5,5-tetramethylimidazoline, ARPAK having a thrombolysis effect, GRPAK, RPAK or PAK to obtain the thrombolysis oligopeptide-imidazolidine binary conjugate represented by a formula I or II, wherein the thrombolysis oligopeptide-imidazolidine binary conjugate integrally has characteristics of nanometer structure, BBB crossing, thrombolysis and NO removing. The thrombolysis oligopeptide-imidazolidine binary conjugate has high NO free radical clearance activity, excellent thrombolysis activity and excellent antithrombosis activity. In vivo anti-stroke activity test results show that: with the thrombolysis oligopeptide-imidazolidine binary conjugate, nerve functions of stroke rats can be effectively protected, brain infarct volume of the stroke rats can be reduced, excellent anti-stroke activity is provided, and the thrombolysis oligopeptide-imidazolidine binary conjugate can be prepared into a clinical drug for brain infarction treatment.

Description

Thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate and its production and use
Technical field
The present invention relates to binary conjugate; particularly relate to by 1-(4-oxygen ethanoyl-phenyl)-3; 3; 4; 4-tetramethyl--tetrahydroglyoxaline and ARPAK, GRPAK, RPAK or the PAK with thrombus dissolving effect to be linked together the binary conjugate obtained by L-Lys; the invention still further relates to the preparation method of this binary conjugate; the invention further relates to this binary conjugate removing the medicinal use in NO, thrombus dissolving and cerebral infarction therapy, belonging to thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate and field of medical application thereof.
Background technology
In the world, the M & M of thrombotic diseases all ranks first.Coronary artery thrombosis, causes the heart to obstruct.Cerebral vessels embolism, causes cerebral infarction.Heart stalk patient both can intravenous injection thrombolytic drug, also can impose bypass surgery.Must be pointed out, be ischemia-reperfusion to the positive result of heart stalk patient intravenous injection thrombolytic drug.Owing to producing a large amount of NO free radical in Ischemia-Reperfusion Injury, so thrombolysis process is associated with myocardial damage and death.This is the serious problems of current thromboembolism treatment heart stalk.At present, cerebral infarction treatment problems faced is more complicated.Such as existing thrombolytic drug all can not stride across m brain barrier effectively, to such an extent as to intravenous injection thrombolytic drug to the curative effect of cerebral infarction patient extremely/limited.Operation such as also unsuitable at present again can give treatment to cerebral infarction patient.Equally, even if having positive result to cerebral infarction patient intravenous injection thrombolytic drug, in Ischemia-Reperfusion Injury, also can produce a large amount of NO free radical, thrombolysis process is associated with brain tissue impairment and death.This is the serious problems of current thrombolytic therapy cerebral.Therefore, invention can stride across hemato encephalic barrier effectively, again can effective thrombolysis, and the medicine also effectively can removing the NO free radical produced in Ischemia-Reperfusion Injury is most important to cerebral infarction treatment.
Summary of the invention
One of the object of the invention is to provide thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate;
Two of the object of the invention is to provide the preparation method of a kind of thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate;
Three of the object of the invention is to provide the medicinal use of thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate, its structural formula is for shown in formula I or formula II:
Wherein, K represents Methionin; A represents L-Ala; P represents proline(Pro);
Aa 1-aa 2it is arginine; Or aa 1when being arginine, aa 2l-Ala or glycine;
Long-term thrombolysis research makes contriver recognize, 2-[(4-fluoroacetic acid) phenyl]-4,4,5,5-tetramethyl--1,3-dioxy base tetrahydroglyoxaline not only can remove the NO free radical produced in Ischemia-Reperfusion Injury effectively, and has suitable fat-soluble.Long-term thrombolysis research makes contriver recognize further, to there is ARPAK, GRPAK, RPAK or PAK of thrombus dissolving effect by L-Lys and 1,3-dioxy base-2-[(4-fluoroacetic acid) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxaline combines, and can obtain collection nano junction, act on the cerebral infarction therapeutical agent of one across hemato encephalic barrier, thrombus dissolving and removing NO.In order to realize this combination, need the connecting arm that suitable.After having attempted tens of kinds of connecting arms, confirm that L-Lys is best connecting arm.Use L-Lys as connecting arm, can nanostructure be obtained and make to stride across the NO free radical produced in hemato encephalic barrier, thrombus dissolving and removing Ischemia-Reperfusion Injury effect maximize.
Two of object of the present invention is to provide the preparation method of the thrombolysis oligopeptides shown in a kind of formula I or formula II-tetrahydroglyoxaline binary conjugate, and the method comprises:
(1) 1,3-dioxy base-2-(4-fluoroacetic acid base-phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxalines are prepared;
(2) 1,3-dioxy base-2-[(4-oxygen ethanoyl-N is prepared ω-Boc-Lys-OMe) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines;
(3) Bo c-Ala-Arg (NO is prepared 2)-Pro-Ala-Lys (Z), Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z), Boc-Arg (NO 2)-Pro-Ala-Lys (Z), Boc-Pro-Ala-Lys (Z);
(4) l is prepared, 3-dioxy base-2-{4-oxygen ethanoyl-{ N ω-[Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines, 1,3-dioxy base-2-{4-oxygen ethanoyl-{ N ω-[Boc-Gly-Arg-(NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines, 1,3-dioxy base-2-{4-oxygen ethanoyl-{ N ω-[Boc-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines, 1,3-dioxy base-2-{4-oxygen ethanoyl-{ N ω-[Boc-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines;
(5) compound prepared by step (4) is sloughed protecting group, prepare 1,3-dioxy base-2-{4-oxygen ethanoyl-[N ω-(Ala-Arg-Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines, l, 3-dioxy base-2-{4-oxygen ethanoyl-(N ω-(Gly-Arg-Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines, l, 3-dioxy base-2-{4-oxygen ethanoyl-(N ω-(Arg-Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines, 1,3-dioxy base-2-{4-oxygen ethanoyl-[N ω-(Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines.
The test of NO scavenging capacity shows, binary conjugate of the present invention has higher removing NO free radical activity; Inside and outside thrombus dissolving activity shows, binary conjugate of the present invention has excellent thrombus dissolving activity.In body, anti-stroke activity proves that binary conjugate of the present invention can protect the neural function of rats with stroke effectively, has excellent anti-stroke active.Rats with stroke infarction of brain Volume Test shows, binary conjugate of the present invention can reduce rats with stroke infarction of brain volume effectively.
Thus, the invention provides the medicinal use of thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate in following several respects:
1, thrombolysis oligopeptides of the present invention-tetrahydroglyoxaline binary conjugate is prepared into thrombolytic agent;
2, thrombolysis oligopeptides of the present invention-tetrahydroglyoxaline binary conjugate is prepared into removing NO free radical medicine;
3, thrombolysis oligopeptides of the present invention-tetrahydroglyoxaline binary conjugate is prepared into the medicine for the treatment of apoplexy or cerebral infarction;
Further, the invention provides thrombolytic compositions, remove NO free radical pharmaceutical composition or treatment apoplexy or the pharmaceutical composition of cerebral infarction, described pharmaceutical composition is made up of with pharmaceutically acceptable carrier or auxiliary material the formula I of significant quantity or formula II compound; To can obtain described antithrombotic, thrombus dissolving after the formula I of significant quantity or formula II compound are together with pharmaceutically acceptable carrier or auxiliary material combination or treat the pharmaceutical composition of apoplexy or cerebral infarction.
By add in formula I or formula II compound prepare different dosage form time required various auxiliary material and pharmaceutically acceptable auxiliary material or carrier after, the clinical acceptable suitable formulations of any one is prepared into the drug formulation process of routine, such as, can be injection formulations (powder pin, freeze-dried powder, liquid drugs injection, transfusion etc.), tablet, oral liquid, granule, capsule, soft capsule or dripping pill etc.; Wherein, described auxiliary material can be antioxygen complexing agent, weighting agent, framework material etc.; Described pharmaceutically acceptable carrier is one or more in Xylitol, N.F,USP MANNITOL, lactose, fructose, dextran, glucose, polyvinylpyrrolidone, low molecular dextran, sodium-chlor, calglucon or calcium phosphate.
The breviary term arrived involved in the present invention:
DCC:1,3-dicyclohexylcarbodiimide
DMF: dimethyl formamide
HOBt:1-hydroxybenzotriazole
TLC: tlc
Accompanying drawing explanation
The structural formula of Fig. 1 thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate.
The synthetic route chart of Fig. 2 thrombolysis oligopeptides-tetrahydroglyoxaline binary conjugate; (i) Br 2, NaOH (6M); (ii) Zn, NH 4cl; (iii) HOC 6h 4cHO; (iv) PbO 2; (v) BrCH 2cO 2c 2h 5, NaH, THF; (vi) P-aa in NaOH (2M) .Ia 1-aa 2common designation P, aa in Ib 1-aa 2represent R, aa in Ic 1represent R/aa 2represent A, aa in Id 1represent R/aa 2represent G.
Fig. 3 Ia is at l × l0 -6m and 1 × 10 -9nanostructure in the M aqueous solution; Diameter is the nanometer ball of 37-55nm and 25-50nm.
Fig. 4 Ib is 1 × 10 -6m and 1 × 10 -9nanostructure .32-64nm in the M aqueous solution and the nanometer ball of 24-48nm.
Fig. 5 Ic is 1 × 10 -6m and 1 × 10 -9nanostructure .11-33nm in the M aqueous solution and the nanometer ball of 22-44nm.
Fig. 6 Id is 1 × 10 -6m and 1 × 10 -9nanostructure .73-153nm in the M aqueous solution and the nanometer ball of 7-58nm.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 prepares DMNB
69g (0.78mol) 2-nitropropane adds in 130ml NaOH (6N) aqueous solution, under cryosel bath is stirred, drip 20ml (0.38mol) Br 2, drip in 1h, then add 240ml ethanol, 90 DEG C of backflow 3h, taken advantage of by reaction solution heat to pour in 800ml frozen water, filtering to obtain 55g (81%) title compound, is colorless plate crystal, Mp110-112 DEG C.
Embodiment 2 prepares 2,3-dimethyl-2,3 dihydroxy amido butane
By 7g (40mmol) DMNB and 4g NH 4cl is suspended in 80ml aqueous ethanolic solution (50%), stirs under ice bath, adds 16g zinc powder in 3h.After zinc powder adds, remove ice bath, continue stirring at room temperature reaction 3h, then by reaction solution suction filtration.Filter cake aqueous ethanolic solution (50%) repetitive scrubbing, merging filtrate and washings, regulate pH=2 with concentrated hydrochloric acid, and decompression is steamed to muddy.Add appropriate salt of wormwood, after mixing thoroughly, use apparatus,Soxhlet's, chloroform is extraction agent, extracting 6h, and extracting solution is evaporated on a small quantity, separates out 2.60g (44%) title compound after adding sherwood oil, is clear crystal.Mp157-159℃。
Embodiment 3 prepares 1,3---hydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine
1.22g (10mmol) p-Hydroxybenzaldehyde and 1.48g (10mmol) 2,3-dimethyl-2,3-dihydroxy amido butane are dissolved in 10ml methyl alcohol, and after stirring at room temperature 8h, TLC shows raw material point and disappears.Suction filtration obtains 1.29g (51%) title compound, is clear crystal.EI-MS(m/z)252[M] +. 1H-NMR(DMSO-d 6)δ(ppm)＝1.03(s，6H)，1.05(s，6H)，4.39(s，1H)，6.70(d，J＝6.9Hz，2H)，7.23(d，J＝6.9Hz，2H)，7.63(s，1H)，7.85(s，2H)。
Embodiment 4 prepares 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxaline
By 504mg (2mmol) l, 3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine be dissolved in 30ml methyl alcohol, add 3g PbO 2, stirring at room temperature 40min, TLC show raw material point and disappear.Suction filtration removing solid, evaporated under reduced pressure under filtrate room temperature, residue column chromatography purification (chloroform wash-out), 260mg (52%) title compound is blue look solid.Mp134-135℃，EI-MS(m/z)249[M] +.IR(KBr)3250，1610，1500，1490，840。
Embodiment 5 prepares 1,3-dioxy base-2-(4 '-ethoxyacetic acid ethyl ester-phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 250mg (1mmol) 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxaline, 0.32ml bromoethyl acetate and 32mg sodium hydride are dissolved in the anhydrous THF of 5ml, and 60 DEG C, mixture stirs 5 hours, and TLC shows raw material point and disappears.Room temperature under reduced pressure filters, and filtrate reduced in volume is to dry, and (ethyl acetate: sherwood oil=1: 5) purifying obtains 300mg (90%) target compound .Mp107-109 DEG C to residue column chromatography.
Embodiment 6 prepares 1,3-dioxy base-2-(4 '-fluoroacetic acid-phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxaline (II)
To 33mg (0.1mmol) 1,3-dioxy base-2-(4 '-ethoxyacetic acid ethyl ester-phenyl)-4,4,5, add 7 NaOH (2N) aqueous solution in the solution of 5-tetramethyl-tetrahydroglyoxaline and 3ml methyl alcohol, stirring at room temperature 30min, TLC show raw material point and disappear.By reaction solution concentrating under reduced pressure, residue first adds the dilution of 2ml saturated aqueous common salt, pH6 is adjusted again with 2N HCl, then be extracted with ethyl acetate 3 times (3ml × 3), the ethyl acetate layer anhydrous sodium sulfate drying merged, filters, is evaporated to dry under filtrate room temperature, obtaining 30mg (99%) title compound, is blue colored crystal.Mp155-157℃.EI-MS(m/z)307[M] +。
Embodiment 7 prepares 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys-OMe) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines
The solution of 307mg (1mmol) 1,3-dioxy base-2-(4 '-fluoroacetic acid-phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxaline and the anhydrous THF of 30ml is stirred under ice bath.Add 250mg (1.2mmol) DCC and 135mg (1mmol) HOBt to solution, under ice bath, stir 10min.The solution prepared by 300mg (1mmol) HCLLys (Boc)-OMe, 122mg (1mmol) N-methylmorpholine and the anhydrous THF of 6ml is added to this solution.The reaction mixture room temperature reaction obtained 24 hours.TLC (ethyl acetate: sherwood oil=2: 1) show HCLLys (Boc)-OMe and disappear.Reaction mixture is evaporated to dry, and residue with Ethyl acetate dissolves, filtering insolubles.Filtrate is washed with saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution successively.The ethyl acetate layer anhydrous sodium sulfate drying separated, filtration, filtrate 37 DEG C of concentrating under reduced pressure (these operate in and are hereinafter referred to as conventional processing), residue column chromatography purification (ethyl acetate: sherwood oil=2: 1), obtaining 433mg (65%) title compound, is blue solid.ESI-MS(m/z)550[M+H] +。
Embodiment 8 prepares 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 625mg (1mmol) 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys-OMe) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines are dissolved in 3ml methyl alcohol, add the NaOH aqueous solution (2N), stirred at ambient temperature 30min under ice bath.Keep pHl2, stir 10min under ice bath, TLC shows raw material point and disappears.Compound of reaction 2N HCl adjusts pH7.By reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt dilution, adjusts pH2 with 2N HCl, is extracted with ethyl acetate 3 times (5ml × 3).The ethyl acetate layer anhydrous sodium sulfate drying merged, concentrating under reduced pressure under filtration, filtrate room temperature, obtaining 490mg (92%) title compound, is blue colored crystal.EI-MS(m/z)534[M-H] -。
Embodiment 9 prepares Boc-Ala-Lys (Z)-OBzl
473mg (2.5mmol) Boc-Ala is dissolved in the anhydrous THF of 10ml.The solution prepared by the anhydrous THF of 338mg (2.5mmol) HOBt, 619mg (3mmol) DCC and l0ml is added inward under ice bath.Stir 20 minutes under reaction mixture ice bath.The solution prepared by 936mg (2.3mmol) HClLys (Z)-OBzl, 232mg (2.3mmol) N-methylmorpholine elder generation and the anhydrous THF of 6ml is added in this solution.The reaction mixture room temperature reaction obtained 24 hours, TLC (CHCl 3: MeOH=30: 1) show HClLys (Z)-OBzl and disappear.Reaction mixture processes to obtain 1.204g (97%) title compound routinely, is colorless solid.Mp88-90℃. ESI-MS(m/z)565[M+Na] +。
Embodiment 10 prepares HClAla-Lys (Z)-OBzl
1.354g (2.5mmol) Boc-Ala-Lys (Z)-OBzl is dissolved in about 10ml anhydrous hydrogen chloride-ethyl acetate solution (4N), stirring at room temperature 3 hours, TLC (CHCl 3: MeOH, 30: 1) disappearance of display raw material point.Reaction mixture is concentrating under reduced pressure at room temperature, and residue is concentrated with acetic acid ethyl dissolution and under room temperature again, so repeatedly for several times, until the hydrogenchloride (these operate in and are hereinafter referred to as conventional processing) that Ex-all is free.Residue anhydrous diethyl ether crystallization, obtains title compound, is directly used in the next step.
Embodiment 11 prepares Boc-Pro-Ala-Lys (Z)-OBzl
538mg (2.5mmol) Boc-Pro is dissolved in appropriate anhydrous THF, under ice bath, adds the anhydrous THF solution of 338mg (2.5mmol) HOBt, 619mg (3mmol) DCC, react 20 minutes.The solution prepared by 1.099g (2.3mmol) HClAla-Lys (Z)-OBzl, 232mg (2.3mmol) N-methylmorpholine and the anhydrous THF of 10ml is added, room temperature reaction 24 hours to this solution.TLC (CHCl 3: MeOH, 20: 1) disappearance of display raw material point.Compound of reaction processes routinely, obtains 2.847g (98%) title compound .Mp82-83 DEG C. eSI-MS (m/z) 661 [M+Na] +.
Embodiment 12 prepares Boc-Pro-Ala-Lys (Z)
638mg (1mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved in 3ml methyl alcohol, adds the NaOH aqueous solution (2N) under ice bath, stirred at ambient temperature 30min.Keep pH12, under ice bath, stir 10min.TLC shows raw material point and disappears, and adjusts pH7 with 2N HCl.By reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt solution dilution, pH2 is adjusted with 2N HCl, be extracted with ethyl acetate 3 times (5ml × 3), the ethyl acetate layer anhydrous sodium sulfate drying merged, filters, concentrating under reduced pressure under filtrate room temperature, obtaining 509mg (91.6%) title compound, is colorless solid .EI-MS (m/z) 547 [M-H] -.
Embodiment 13 prepares HClPro-Ala-Lys (Z)-OBzl
1.596g (2.5mmol) Boc-Pro-Ala-Lys (Z)-OBzl is dissolved in 15ml anhydrous hydrogen chloride-acetic acid ethyl fluid (4N), stirring at room temperature 3 hours, TLC (CHCl 3: MeOH, 20: 1) disappearance of display raw material point.Reaction mixture processes according to a conventional method, and residue anhydrous diethyl ether crystallization, obtains title compound, be directly used in the next step.
Embodiment 14 prepares Boc-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl
By 798mg (2.5mmol) Boc-Arg (NO under ice bath 2), the solution stirring of the anhydrous THF of 338mg (2.5mmol) HOBt, 619mg (3mmol) DCC and 10ml 20 minutes.The solution prepared by 1.322g (2.3mmol) HClPro-Ala-Lys (Z)-OBzl, 232mg (2.3mmol) N-methylmorpholine and the anhydrous THF of 5ml is added, room temperature reaction 24 hours in this solution.TLC (CHCl 3: MeOH, 20: 1) disappearance of display raw material point.Process routinely, obtain 1.642g (85%) title compound .Mp84-85 DEG C. eSI-MS (m/z) 864 [M+Na] +.
Embodiment 15 prepares Boc-Arg (NO 2)-Pro-Ala-Lys (Z)
By 850mg (1mmol) Boc-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl is dissolved in 3ml methyl alcohol, adds the NaOH aqueous solution (2N), stirring at room temperature 30min under ice bath, keeps PH12, ice bath stirs 10min, TLC and shows raw material point and disappear.PH7 is adjusted with 2N HCl, by reaction solution concentrating under reduced pressure, residue adds the dilution of 2ml saturated aqueous common salt, pH2 is adjusted with 2N HCl, be extracted with ethyl acetate 3 times (5ml × 3), the ethyl acetate layer anhydrous sodium sulfate drying merged, under filtration, filtrate room temperature, concentrating under reduced pressure obtains 742mg (92%) title compound, is colorless solid.EI-MS(m/z)849[M-H] -。
Embodiment 16 prepares HCl.Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl
By 2.099g (2.5mmol) Boc-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl is dissolved in 20ml anhydrous hydrogen chloride-acetic acid ethyl fluid (4N), stirring at room temperature 3 hours, TLC (CHCl 3: MeOH, 20: 1) disappearance of display raw material point.Reaction mixture processes according to a conventional method, and residue anhydrous diethyl ether crystallization, obtains title compound, be directly used in the next step.
Embodiment 17 prepares Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl
By the solution stirring 20 minutes of the anhydrous THF of 473mg (2.5mmol) Boc-Ala, 338mg (2.5mmol) HOBt, 619mg (3mmol) DCC and 10ml under ice bath.Add in this solution by 1.785g (2.3mmol) HClArg (NO 2) solution that-Pro-Ala-Lys (Z)-Obzl, 232mg (2.3mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, react 24 hours, obtain 1.802g (86%) title compound .Mp87-89 DEG C. eSI-MS (m/e) 934 [M+Na] +.
Embodiment 18 prepares Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)
By 921mg (1mmol) Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl is dissolved in 3ml methyl alcohol, adds the NaOH aqueous solution (2N), stirred at ambient temperature 30min under ice bath, keeps pH12, stirs 10min under ice bath, TLC shows raw material point and disappears.PH7 is adjusted with 2N HCl, by reaction solution concentrating under reduced pressure, residue adds the dilution of 2ml saturated aqueous common salt, pH2 is adjusted with 2N HCl, be extracted with ethyl acetate 3 times (5ml × 3), the ethyl acetate layer anhydrous sodium sulfate drying merged, room temperature under reduced pressure concentrates to obtain 767mg (80%) title compound, is colorless solid .EI-MS (m/z) 830 [M-H] -.
Embodiment 19 prepares Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl
By the solution stirring 20 minutes of the anhydrous THF of 438mg (2.5mmol) Boc-Gly, 338mg (2.5mmol) HOBt, 619mg (3mmol) DCC and 10ml under ice bath.Add in this solution by 1.785g (2.3mmol) HClArg (NO 2) solution that-Pro-Ala-Lys (Z)-OBzl, 232mg (2.3mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, react 24 hours, obtain 1.857g (90%) title compound .Mp85-87 DEG C. eSI-MS (m/e) 920 [M+Na] +.
Embodiment 20 prepares Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)
By 907mg (1mmol) Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)-OBzl is dissolved in 3ml methyl alcohol, adds the NaOH aqueous solution (2N), stirred at ambient temperature 30min under ice bath, keeps pH12, stirs 10min under ice bath, TLC shows raw material point and disappears.PH7 is adjusted with 2N HCl, by reaction solution concentrating under reduced pressure, residue adds the dilution of 2ml saturated aqueous common salt, pH2 is adjusted with 2N HCl, be extracted with ethyl acetate 3 times (5ml × 3), the ethyl acetate layer anhydrous sodium sulfate drying merged, room temperature under reduced pressure concentrates to obtain 785mg (82%) title compound, is colorless solid .El-MS (m/z) 816 [M-H] -.
Embodiment 21 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By the solution stirring 20 minutes of the anhydrous THF of 548mg (1mmol) Boc-Pro-Ala-Lys (Z), 135mg (1mmol) HOBt, 250mg (1mmol) DCC and 10ml under ice bath.Add by 480mg (1mmol) 1 to this solution; 3-dioxy base-2-[(4 '-oxygen ethanoyl-Lys-OMe) phenyl]-4; 4; 5; the solution that 5-tetramethyl-tetrahydroglyoxaline and 100mg (1mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, room temperature reaction 24 hours.TLC (CHCl 3: MeOH, 40: 1) disappearance of display raw material point.Compound of reaction processes routinely, obtains 876mg (87%) title compound, is blue solid.Mp77-80℃. ESI-MS(m/z)1003[M+Na] +。IR(KBr)∶3315，3069，2937，1671，1531，1449，1394，1364，1302，1167，1132，1054，836，743，698，596，54l，458cm -1。
Embodiment 22 prepares 1,3--oxygen base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Arg (NO2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 760mg (1mmol) Boc-Arg (NO under ice bath 2) solution stirring 20 minutes of the anhydrous THF of-Pro-Ala-Lys (Z), 135mg (1mmol) HOBt, 250mg (1mmol) DCC and 10ml.Add by 480mg (1mmol) 1 to this solution; 3-dioxy base-2-[(4 '-oxygen ethanoyl-Lys-OMe) phenyl]-4; 4; 5; the solution that 5-tetramethyl-tetrahydroglyoxaline and 100mg (1mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, room temperature reaction 24 hours.TLC (CHCl 3: MeOH, 40: 1) disappearance of display raw material point.Compound of reaction processes routinely, obtains 920mg (83%) title compound, is blue solid.Mp72-76℃. ESl-MS(m/z)1204[M+Na] +。IR(KBr)3317，2937，1658，1531，1447，1362，1254，1168，1055，835，746，697，541，460Gm -1。
Embodiment 23 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 821mg (1mmol) Boc-Ala-Arg (NO under ice bath 2) solution stirring 20 minutes of the anhydrous THF of-Pro-Ala-Lys (Z), l35mg (1mmol) HOBt, 250mg (1mmol) DCC and 10ml.Add by 480mg (1mmol) 1 to this solution; 3-dioxy base-2-[(4 '-oxygen ethanoyl-Lys-OMe) phenyl]-4; 4; 5; the solution that 5-tetramethyl-tetrahydroglyoxaline and 100mg (1mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, room temperature reaction 24 hours.TLC (CHCl 3: MeOH, 40: 1) disappearance of display raw material point.Compound of reaction processes routinely, obtains 925mg (83%) title compound, is blue solid.Mp179-182℃. ESI-MS(m/z)1275[M+Na] +。IR(KBr)3319，2935，1658，1531，1448，1363，1254，1168，1053，835，749，540Gm -1。
Embodiment 24 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 817mg (1mmol) Boc-Gly-Arg (NO under ice bath 2) solution stirring 20 minutes of the anhydrous THF of-Pro-Ala-Lys (Z), 135mg (1mmol) HOBt, 250mg (1mmol) DCC and 10ml.Add by 480mg (1mmol) 1 to this solution; 3-dioxy base-2-[(4 '-oxygen ethanoyl-Lys-OMe) phenyl]-4; 4; 5; the solution that 5-tetramethyl-tetrahydroglyoxaline and 100mg (1mmol) N-methylmorpholine and the anhydrous THF of 5ml prepare, room temperature reaction 24 hours.TLC (CHCl 3: MeOH, 40: 1) disappearance of display raw material point.Compound of reaction processes routinely, obtains 680mg (52%) title compound, is blue solid.Mp79-82℃. ESI-MS(m/z)1261[M+Na] +。IR(KBr)3319，2935，1658，1531，1448，1363，1254，1168，1053，835，749，540cm -1。
Embodiment 25 prepares 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 625mg (1mmol) 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys-OMe) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines are dissolved in 3ml methyl alcohol, add the NaOH aqueous solution (2N), stirred at ambient temperature 30min under ice bath.Keep pHl2, stir 10min under ice bath, TLC shows raw material point and disappears.Compound of reaction 2N HCl adjusts pH7.By reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt dilution, adjusts pH2 with 2N HCl, is extracted with ethyl acetate 3 times (5ml × 3).The ethyl acetate layer anhydrous sodium sulfate drying merged, concentrating under reduced pressure under filtration, filtrate room temperature, obtaining 490mg (92%) title compound, is blue colored crystal.EI-MS(m/z)534[M-H] -。
Embodiment 26 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-[N ω-(Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines (Ia)
193mg (0.2mmol) 4; 4; 5; 5-tetramethyl--1; 3-dioxy base tetrahydroglyoxaline-2-phenyl-4 '-oxygen base ethanoyl Lys (Boc-Pro-Ala-Lys (Z))-OH adds in eggplant bottle; 6ml TFA and 1.5ml TFMSA is added, TCL (CHCl after 1 hour under ice bath 3: MeOH=1: 1) show raw material point and disappear, stopped reaction, uses anhydrous diethyl ether repetitive scrubbing, decompressing and extracting, then use water dissolution, adjust PH=8, with Sephadex G10 desalination with 25% ammoniacal liquor, C18 column purification, freeze-drying, obtains 145mg (84%) title compound, is blue solid, Mp103.6 ~ 104.9 DEG C eSI-MS (m/z) 732.6 [M+H] +, g=2.0078, IR (KBr) 3085,1672,1543,1449,1183,1032,1302,1167,837,801,723,639,578,519cm -1.
Embodiment 27 prepares 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 625mg (1mmol) 1,3-dioxy base-2-[(4 '-oxygen acetyl-N ω-Boc-Lys-OMe) phenyl]-4,4,5,5-tetramethyl-tetrahydroglyoxaline is dissolved in 3ml methyl alcohol, adds the NaOH aqueous solution (2N), stirred at ambient temperature 30min under ice bath.Keep pHl2, stir 10min under ice bath, TLC shows raw material point and disappears.Compound of reaction 2N HCl adjusts pH7.By reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt dilution, adjusts pH2 with 2N HCl, is extracted with ethyl acetate 3 times (5ml × 3).The ethyl acetate layer anhydrous sodium sulfate drying merged, concentrating under reduced pressure under filtration, filtrate room temperature, obtaining 490mg (92%) title compound, is blue colored crystal.EI-MS(m/z)534[M-H] -。
Embodiment 28 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-[N ω-(Arg-Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines (Ib)
233mg (0.2mmol) 4,4,5,5-tetramethyl--1,3-dioxy base tetrahydroglyoxaline-2-phenyl-4 '-oxygen base ethanoyl Lys (Boc-Arg (NO 2)-Pro-Ala-Lys (Z))-OH adds in eggplant bottle, adds 6ml TFA and l.5mlTFMSA, TCL (CHCl after 1 hour under ice bath 3: MeOH=1: 1) show raw material point and disappear, stopped reaction, uses anhydrous diethyl ether repetitive scrubbing, decompressing and extracting, then use water dissolution, adjust PH=8, with Sephadcx G10 desalination with 25% ammoniacal liquor, C18 column purification, freeze-drying, obtains 145mg (84%) title compound, is blue solid, Mp113.9 ~ 116.7 DEG C eSI-MS (m/z) 889.0 [M+H] +, g=2.00789, IR (KBr) 3213,2980,1659,1536,1262,1170,1031,639,542em -1.
Embodiment 29 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 1260mg (1mmol) 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N under under ice bath ω-[Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines are dissolved in 3ml methyl alcohol, add the NaOH aqueous solution (2N), stirred at ambient temperature 30min.Keep pH12, stir 10min under ice bath, TLC shows raw material point and disappears.Adjust pH7 with 2N HCl, by reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt dilution, adjusts pH2 with 2N HCl, is extracted with ethyl acetate 3 times (5ml × 3).The ethyl acetate layer anhydrous sodium sulfate drying merged, concentrating under reduced pressure under filtration, filtrate room temperature, obtaining 945mg (82%) title compound, is blue solid.EI-MS(m/z)1238[M-H] -。
Embodiment 30 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-[N ω-(Ala-Arg-Pro-Ala-Lys)-Lysl phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines (Ic)
247mg (0.2mmol) 4,4,5,5-tetramethyl--l, 3-dioxy base tetrahydroglyoxaline-2-phenyl-4 '-oxygen base ethanoyl Lys (Boc-Ala-Arg (NO 2)-Pro-Ala-Lys (Z))-OH adds in eggplant bottle, adds 6ml TFA and 1.5ml TFMSA, TCL (CHCl after 1 hour under ice bath 3: MeOH=1: 1) show raw material point and disappear, stopped reaction, uses anhydrous diethyl ether repetitive scrubbing, decompressing and extracting, then use water dissolution, adjust PH=8, with Sephadex G10 desalination with 25% ammoniacal liquor, C18 column purification, freeze-drying, obtains 145mg (84%) title compound, is blue solid, Mp90.9 ~ 92.7 DEG C eSI-MS (m/z) 959.9 [M+H] +, g=2.00789, IR (KBr) 3228,3080,2930,1658,1540,1444,1267,1181,1029,641,542cm -1.
Embodiment 31 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N ω-[Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines
By 1260mg (1mmol) 1,3-dioxy base-2-{4 '-oxygen ethanoyl-{ N under under ice bath ω-[Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z)]-Lys-OMe} phenyl-4,4,5,5-tetramethyl-tetrahydroglyoxalines are dissolved in 3ml methyl alcohol, add the NaOH aqueous solution (2N), stirred at ambient temperature 30min.Keep pH l2, stir 10min under ice bath, TLC shows raw material point and disappears.Adjust pH7 with 2N HCl, by reaction solution concentrating under reduced pressure, residue 2ml saturated aqueous common salt dilution, adjusts pH2 with 2N HCl, is extracted with ethyl acetate 3 times (5ml × 3).The ethyl acetate layer anhydrous sodium sulfate drying merged, concentrating under reduced pressure under filtration, filtrate room temperature, obtaining 945mg (82%) title compound, is blue solid.EI-MS(m/z)1223[M-H] -。
Embodiment 32 prepares 1,3-dioxy base-2-{4 '-oxygen ethanoyl-[N ω-(Gly-Arg-Pro-Ala-Lys)-Lys] phenyl }-4,4,5,5-tetramethyl-tetrahydroglyoxalines (Id)
233mg (0.2mmol) 4,4,5,5-tetramethyl--1,3-dioxy base tetrahydroglyoxaline-2-phenyl-4 '-oxygen base ethanoyl Lys (Boc-Gly-Arg (NO 2)-Pro-Ala-Lys (Z))-OH adds in eggplant bottle, adds 6ml TFA and 1.5mlTFMSA, TCL (CHCl after 1 hour under ice bath 3: MeOH=1: 1) show raw material point and disappear, stopped reaction, uses anhydrous diethyl ether repetitive scrubbing, decompressing and extracting, then use water dissolution, adjust PH=8, with Sephadex G10 desalination with 25% ammoniacal liquor, C18 column purification, freeze-drying, obtains 145mg (84%) title compound, is blue solid, Mp96.9 ~ 98.7 DEG C eSI-MS (m/z) 945.8 [M+H] +, g=2.00789, IR (KBr) 3370,3230,3060,1668,1545,1451,1257,1171,1031,762,641,577,518cm -1.
Test example 1 conjugate NO of the present invention scavenging capacity is tested
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
Body weight 250-300g male Wistar rat, pre-operative anxiety 12 hours, freely drink water, dislocation of cervical vertebra is lethal, opens rapidly chest and wins thoracic aorta, peels off the reticular tissue of attachment, and arterial ring blood vessel being cut into 3-5mm long is placed in perfusion bath.Bath fills 15ml Krebs-Henseleit liquid, 37 DEG C of constant temperature, logical 95%O2-5%CO 2gas, the hook of fixing arterial ring connects tonotransducer, and dual-trace recorder is traced vasomotion curve, and chart speed is 1mm/min.Adjustment resting tension is 1.0g, and giving final concentration after balance 30min is 10 -8m norepinephrine makes arterial contraction to play pre-sharp effect.Wash away norepinephrine, balance 30min, it is 10 that bath adds final concentration -8m norepinephrine, until shrink tension continually and steadily after plateau levels, bath adds 20 μ l physiological saline (blank) or adds the normal saline solution of 20 μ l test compounds (Ia-d) that (final concentration is 5 × 10 -6or add the normal saline solution of 20 μ l Compound II per (1,3-dioxy base-2-(4 '-fluoroacetic acid-phenyl)-4,4,5,5-tetramethyl-tetrahydroglyoxaline) (final concentration is 5 × 10 M), -6m), after steadily, (final concentration is 10 to add the normal saline solution of 20 μ l vagusstoffs -6m).The ability that NO removed by medicine represents to suppress the per-cent of vagusstoff vasodilator bar.
3, test-results
Data are in Table l.Test-results shows, can suppress the vascular strip diastole effect of vagusstoff by removing NO test compound (Ia-d).Visible, by Lys by thrombus dissolving oligopeptides PAK, RPAK, ARPAK and GRPAK are connected on tetrahydroglyoxaline, the activity of the removing NO free radical of tetrahydroglyoxaline or reservation or enhancing.
Table 1 test compound (Ia-d) is to the percent inhibition of vagusstoff vasodilator bar
n＝6
Test example 2 conjugate ELA test of the present invention
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
Get pig blood to mix with 3.8% liquor sodii citratis saddle 9:1 part, immediately with the centrifugal 10min of 3000r/min, isolate the blood plasma of Platelet poor.Centrifuge tube to 50ml adds blood plasma and the 36ml ultrapure water of 2ml blows platelet.Often pipe adds 0.4ml acetic acid (1%), fully mixes, and puts into 4 DEG C of refrigerator freezing 10min, with the centrifugal 10min of 3000r/min.Be inverted centrifuge tube, after dried liquid stream, blot inside pipe wall with filter paper.By the euglobulin pellet frozen drying about 40min of centrifugal gained, scrape.Get about 35mg euglobulin and be dissolved in 7ml borate buffer solution (pH9.28).After 1 hour, euglobulin dissolves substantially, adds 0.7ml CaCl 2solution (25mM), is laid on the sheet glass of 10 × 10cm at once, thick about 1mm.After solidifying, with the normal saline solution (1 × 10 of liquid-transfering gun by 10 μ l physiological saline or 10 μ l test compounds (Ia-d) -3the normal saline solution (0.8mg/mL) of M) or 10 μ l urokinases is added drop-wise to and solidifies on flat board, and the interval between every 2 is greater than 1.5cm, each sample spot 3 times.The diameter of solusphere is measured after 4 hours.
3, test-results
Reading lists table 2 in.Test-results shows that Ia-d has ELA.
The euglobulin lysis diameter of table 2Ia-d effect 4h
N=3; A) with physiological saline than p < 0.01.
Test example 3 conjugate hemolysis in vitro of the present invention thrombus activity is tested
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
SD rat (male, 350-400g) is anaesthetized by 1200mg/kg dosage abdominal injection urethane solution.Anesthetized rat dorsal position is fixed, be separated right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, the surgical thread of distal end is clamped in fur mosquito forceps, in distal end intubate, unclamps bulldog clamp, release whole arterial blood, be contained in the container of the prior silicone oil of 50ml.In vertical fixing Glass tubing (long 20mm, internal diameter 4mm, external diameter 5mm, seal with plug at the bottom of pipe), inject 0.8ml rat artery blood, in pipe, insert rapidly the thrombus standing bolt of a stainless steel material.It is that the Stainless Steel Wire of 0.2mm is coiled into that this thrombus fixes spiral diameter, the long 18mm of spiral part, and containing 15 bung flanges, the diameter of bung flange is 1.8mm, and holder handle is connected with spiral, and long 7.0mm, in question mark type.After blood coagulation 40min, open the plug bottom Glass tubing, fix with tweezers the holder handle that thrombus fixes spiral, from Glass tubing, the thrombus taken out carefully by thrombus wraps up fixes spiral.Suspended and be soaked in tri-distilled water, to remove the floating blood on surface.Take out after 1h, accurately weigh.Thrombus is suspended be immersed in 8mL physiological saline in the normal saline solution of Ia-d (concentration is 100nM) or the normal saline solution of the strongest oligopeptides GRPAK of activity (concentration is 100nM, oligopeptides contrasts) in or the normal saline solution (100IU/mL of urokinase, positive control) in, jolting in 37 DEG C of constant-temperature tables (63r/min), take out after 2h, accurate weighing bolt weight.Obtain thrombus before administration after of poor quality, add up the thrombus quality difference (X ± SD) of each group, and be t inspection.
3, test-results
Result lists table 3 in.Test-results shows, by Lys by thrombolysis oligopeptides PAK, RPAK, ARPAK or GRPAK are connected on tetrahydroglyoxaline, and the external thrombolysis activity of oligopeptides still exists.
The external thrombolysis activity of table 3Ia-d effect 2h
N=6; A) p < 0.01. compared with NS
Test example 4 the present invention puts together thrombus dissolving activity test in object
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
SD rat (male, 220 ~ 230g) is anaesthetized by 1200mg/kg dosage abdominal injection urethane solution.Anesthetized rat dorsal position is fixed, and is separated right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate surgical thread respectively, the surgical thread of distal end are clamped in fur mosquito forceps, in distal end intubate, unclamp bulldog clamp, release about 1ml arterial blood, be contained in lml sub warhead.In vertical fixing Glass tubing (long 15mm, internal diameter 2.5mm, external diameter 5.0mm, seal with plug at the bottom of pipe), inject 0.1ml rat artery blood, in pipe, insert rapidly the thrombus standing bolt of a stainless steel material.It is that the Stainless Steel Wire of 0.2mm is coiled into that this thrombus fixes spiral diameter, the long 12mm of spiral part, and containing 15 bung flanges, the diameter of bung flange is 1.0mm, and holder handle is connected with spiral, and long 7.0mm, in question mark type.After blood coagulation 15min, open the plug bottom Glass tubing, fix with tweezers the holder handle that thrombus fixes spiral, from Glass tubing, the thrombus taken out carefully by thrombus wraps up fixes spiral, accurately weighs.
Bypass intubate is formed by 3 sections, and stage casing is polyethylene rubber tube, long 60.0mm, internal diameter 3.5mm; Two ends are identical polyethylene tube, pipe range 100.0mm, internal diameter 1.0mm, one end of this pipe of external diameter 2.0mm pulls into point pipe (for inserting rat carotid artery or vein), external diameter is 1.0mm, the outer cover one segment length 7.0mm of the other end, external diameter is the equal silanization of inwall (the silicone oil diethyl ether solution of 1%) of polyethylene tube (overstriking, for inserting in the polyethylene rubber tube in stage casing) 3 sections of pipes of 3.5mm.The thrombus that thrombus wraps up is fixed spiral and puts into stage casing polyethylene rubber tube, the poly butt end that adds is nested with two respectively at the two ends of sebific duct.With syringe by sharp pipe end by filling heparin-saline solution (50IU/kg) in pipe, for subsequent use.
Continue the tracheae of dissociation anesthesia rat, and do trachea cannula.Be separated the left external jugular vein of rat, proximal part and distal end penetrate surgical thread respectively, the left external jugular vein exposed cuts an angle carefully, the sharp pipe of the bypass duct prepared is inserted the proximal part of left external jugular vein opening above by angle, fix the holder handle of spiral simultaneously away from bypass tube stage casing (fixing spiral containing the thrombus of accurately weighing) interior thrombus.Pushed the heparin-saline (50IU/kg) of correct amount by the sharp pipe of the other end with syringe, now syringe does not withdraw polyethylene tube, clamps the flexible pipe between syringe and polyethylene tube with mosquito forceps.Stop blooding at the proximal part bulldog clamp of right common carotid artery, right common carotid artery is being cut an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted the proximal part of artery angle.The two ends of bypass duct all use No. 4 sutures and arteriovenous to fix.
With scalp acupuncture by physiological saline (3ml/kg), normal saline solution (1 μm of ol/kg of the normal saline solution (20000IU/kg) of urokinase or the normal saline solution (0.1 μm of ol/kg) of Ia-d or the strongest oligopeptides GRPAK of activity, oligopeptides contrasts) by the stage casing of bypass tube (fixing spiral containing the thrombus of accurately weighing), thrust the nearly vein place fixing spiral away from thrombus, open bulldog clamp, blood flow is made to flow to vein by bypass duct from artery, this i.e. rat arteriovenous shut Thrombolysis Model, slowly the liquid in syringe is injected in blood, make physiological saline (blank), urokinase (positive control) or GRPAK (oligopeptides contrast) or Ia-d pass through blood circulation, press the sequential action of vein-heart-artery on thrombus.From inject time timing, after 1h, from bypass duct, removal of thromboses fixes spiral, accurately weighs.That asks thrombus in every rat bypass duct to fix before and after spiral administration is of poor quality, adds up the thrombus quality difference of each group and do t inspection.
3, test is recorded a demerit
Result lists table 4 in.Test-results shows, by Lys by thrombus dissolving oligopeptides PAK, RPAK, ARPAK or GRPAK are connected on tetrahydroglyoxaline, and the thrombus dissolving activity of these oligopeptides strengthens 10 times.
Thrombolysis activity in the body of table 4Ia-d effect 1h
N=10; A) with physiological saline than p < 0.01.
Test example 5 the present invention puts together anti-stroke activity test in object
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
SD male rat (250-300g) 10% Chloral Hydrate (400mg/kg) intraperitoneal injection of anesthesia, hit exactly slightly biased right part from neck and vertically open the long otch of about 2cm, isolate right common carotid artery, external carotid artery and internal carotid artery along nutator medial margin.Internal carotid artery opening part and arteria carotis communis proximal part is closed with pressing from both sides respectively without wound bulldog clamp, cut an osculum at external carotid artery, ligation external carotid artery distal end, unclamps the bulldog clamp of arteria carotis communis proximal part, get 10 μ l blood, after getting blood, close arteria carotis communis proximal part with without wound bulldog clamp folder again.The 10 μ l blood obtained are loaded in 1ml EP pipe, place under present normal temperature and make blood coagulation in 30 minutes, be then transferred in-20 DEG C of refrigerators and place 1 hour, make blood clotting solid.Take out blood clotting after 1 hour, add 1ml physiological saline steel shovel and blood clotting is pounded the more homogeneous tiny thrombus of size, then tiny thrombus suspension is transferred in 1ml syringe for subsequent use.While unclamping rat ICA folder, by the thrombus suspension in 1ml syringe slowly from rat external carotid artery to proximal part through the brain of internal carotid injection rat, then ligation external carotid artery proximal part, opens internal carotid artery and arteria carotis communis place obtains bulldog clamp, recovers blood flow.Be separated the total vein of rat neck, inject medicine, ligation vein, wound drips 3 penicillin, sews up a wound, and waits for that animal revives.Laboratory animal is divided into sham operated rats at random, positive controls, physiological saline group and Ia-d group, the strongest active GRPAK group in oligopeptides.Blank is physiological saline (3ml/kg), the dosage of positive control urokinase is 20000IU/kg (being dissolved in physiological saline), the dosage of Ia-d is 100mol/kg (being dissolved in physiological saline), and the dosage of GR PAK is 1 μm of ol/kg (being dissolved in physiological saline).By Zealonga method (Wen Wang after rat revives 24 hours, Jingdong Xu, Lei Li, Peichang Wang, Xunming Ji, Houxi Ai, Li Zhang, Lin Li, Neuroprotective effect of morroniside on focal cerebral ischemia in rats.Brain Research Bulletin, 2010,83,196-201) evaluate neurological functional deficit.0 point indicates without any neurological deficit sign, and 1 point of expression does not damage side forelimb not tensible, and 2 points of expressions are walked to not damaging skidding, and 3 points of expressions turn-take into not damaging side shape walking of knocking into the back, and 4 submeters show that the disturbance of consciousness is without autonomous, and 5 points represent dead.Above each group appraisal result is carried out statistics compare, and do t inspection.
3, test-results
Appraisal result lists table 6 in.Test-results shows, by Lys by thrombolysis oligopeptides PAK, RPAK, ARPAK or GRPAK are connected on tetrahydroglyoxaline, and the conjugate of tetrahydroglyoxaline and ARPAK and GRPAK can protect the neural function of rats with stroke.
In the body of table 6Ia-c effect 24h, anti-stroke is active
N=10; A) p < 0.01 compared with physiological saline; B) p < 0.05. compared with physiological saline
Test example 6 conjugate of the present invention reduces rats with stroke infarction of brain Volume Test
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
After rat revives and evaluates neurological functional deficit in 24 hours, brain is got with breaking end rapidly after urethane anesthesia, cerebral tissue is placed in-20 DEG C of refrigerators after 2 hours, in the past antinion begin column is about the crown serial section of 2mm, totally 6, be then placed in 2%TTC solution 37 DEG C of lucifuges and hatch 30min, and observe the colour-change of brain section, normal cerebral tissue is dyed redness by TTC, and ischemic tissue of brain and infarcted cerebral constitution are in white.Then use digital photo camera, through the process of SPSS statistical software, calculate infarcted cerebral constitution volume and normal cerebral tissue's volume in coronal section, add up the Infarction volume percent value of each group, and do t inspection.
3, test-results
Result lists table 7 in.Test-results shows, by Lys by thrombus dissolving oligopeptides PAK, RPAK, ARPAK or GRPAK are connected on tetrahydroglyoxaline, can reduce rats with stroke infarction of brain volume.
Table 7Ia-d treats the infarction of brain volume percent of rat
N=10; A) p < 0.01. compared with physiological saline
Test example 7 conjugate nanostructure test of the present invention
1, test compound: the compound prepared by embodiment 26,28,30,32, is numbered Ia, Ib, Ic and Id respectively.
2, test method
Test compound (Ia-d) is made into 1 × 10 respectively with tri-distilled water -6m and 1 × 10 -9the solution of M, draws trace (about 10 μ l) and drips in copper mesh surface, serve as a contrast filter paper, naturally dry, observe form and particle diameter under transmission electron microscope transmission electron microscope (JEOL, JEM-1230), and use photo record below copper mesh.
3, test-results
Test-results is shown in Fig. 3-6.Result shows, 1 × 10 -6m and 1 × 10 -9the nanometer ball of the nanostructure of Ia to be diameter be respectively 37-55nm and 25-50nm in the M aqueous solution, the nanometer ball of the nanostructure of Ib to be diameter be respectively 32-64nm and 24-48nm, the nanometer ball of the nanostructure of Ic to be diameter be respectively 11-33nm and 22-44nm, the nanometer ball of the nanostructure of Id to be diameter be respectively 73-153nm and 7-58nm.

Claims (2)

1. compound described in formula I removes the purposes in the NO free radical medicine of Ischemia-Reperfusion Injury generation in preparation:
Wherein, K represents Methionin; A represents L-Ala; P represents proline(Pro); Aa1-aa2 is arginine; Or when aa1 is arginine, aa2 is L-Ala or glycine; Wherein, the compound shown in formula I has nanostructure.
2. compound described in formula I preparation treatment apoplexy after nervous function damage medicine in purposes:
Wherein, K represents Methionin; A represents L-Ala; P represents proline(Pro); Aa1-aa2 is arginine; Or when aa1 is arginine, aa2 is L-Ala or glycine; Wherein, the compound shown in formula I has nanostructure.
CN201210323952.8A 2012-09-05 2012-09-05 Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof Expired - Fee Related CN102898507B (en)

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