CN106589060A - N-(PAK)-2,3-dihydroxy isoquinoline-7-formyl-RGDV/F, synthesis, activity and application thereof - Google Patents
N-(PAK)-2,3-dihydroxy isoquinoline-7-formyl-RGDV/F, synthesis, activity and application thereof Download PDFInfo
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Abstract
The invention discloses (7S)-N-[Pro-Ala-Lys]-5,6,7,8-trtrahydro-2,3-dihydroxy isoquinoline-7-formyl]-Arg-Gly-Asp-AA as well as a preparation method, antithrombotic activity, thrombolytic activity and cerebral ischemia rat treating functions thereof, so that the invention discloses applications of (7S)-N-[Pro-Ala-Lys]-5,6,7,8-trtrahydro-2,3-dihydroxy isoquinoline-7-formyl]-Arg-Gly-Asp-AA to preparation of antithrombotic drugs, thrombolytic drugs and ischemic stroke treatment drugs.
Description
Technical field
The present invention relates to (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- first
Acyl]-Arg-Gly-Asp-AA, it is related to their preparation method, is related to their antithrombotic acitivity, is related to their thrombus dissolving
Activity, is related to the effect that they treat rats with cerebral ischemia, thus the present invention relates to they are preparing antithrombotic reagent, thrombus dissolving
Application in medicine and treatment cerebral infarction medicine.The invention belongs to biomedicine field.
Background technology
Cerebral infarction is a class more typically and endangers serious cerebrovascular disease, and feature is that sickness rate is high, case fatality rate is high, causes
Residual rate height and high recurrence rate.At present clinical treatment cerebral infarction faces the reality for not having active drug, especially apoplexy face 4h with
On patient it is non-extremely i.e. residual.Invention is clinical important need to the effective medicine of patient of more than apoplexy face 4h.Inventor was once
Jing discloses the imidazolinium compoundss of Formula II on the ischemia/reperfusion in rats apoplexy model of apoplexy face 24h, shows outstanding curative effect.It is i.e. continuous
The imidazolinium compoundss of 6 days Formula II of intravenous injection, once a day, initial dose is 5 μm of ol/kg, and the dosage of 5 times is 2 afterwards
μm ol/kg has outstanding curative effect.Aa in formula1And aa2Can be to exist simultaneously, aa1Exist but aa2Do not exist, or while do not exist；
Work as aa1And aa2In the presence of simultaneously, aa1For Arg, and aa2For Gly, Ala or Gln；Work as aa1Exist but aa2When not existing, aa1For
Arg；aa3Can be Ser, Val or Phe.As the 2- positions of the imidazolinium compoundss of Formula II are 4- oxygen acetyl-Lys, and the side of the Lys
Chain amino and main-chain carboxylic group are connected with RGD antithrombotics tetrapeptide and ARPAK thrombolytic peptides respectively, so the more complicated need of structure
Simplify.
Inventor has found to use (7S) -5,6,7,8- tetrahydrochysene -2, in the replacement of 3- dihydroxy isoquinolin -7- formoxyls through 3 years experimentatioies
1- substituted-phenyls imidazolinyl in formula can obtain simple structure and eutherapeutic dual unexpected technique effect.According to
This discovery, inventors herein proposes the present invention.
The content of the invention
One of present disclosure is to provide (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- first
Acyl]-Arg-Gly-Asp-AA
As AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val；The Arg-Gly-Asp-AA as AA=Phe
For Arg-Gly-Asp-Phe.
The two of present disclosure be to provide (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dioxies isoquinolin -7- formyls] -
The preparation method of Arg-Gly-Asp-Phe, the method are comprised the following steps:
(1) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- carboxylic acids are prepared；
(2) (7S)-N- tertbutyloxycarbonyl -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(3) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(4) Boc-Arg (NO are prepared2)-Gly-Asp(OBzl)-Phe-OBzl；
(5) Arg (NO are prepared2)-Gly-Asp(OBzl)-Phe-OBzl；
(6) prepare (7S)-N- [(Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-
(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(7) prepare (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-Gly
-Asp-Phe。
The three of present disclosure are to provide (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- first
Acyl]-Arg-Gly-Asp-Val preparation method, the method comprises the following steps:
(1) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- carboxylic acids are prepared；
(2) (7S)-N- tertbutyloxycarbonyl -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(3) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(4) Boc-Arg (NO are prepared2)-Gly-Asp(OBzl)-Val-OBzl；
(5) Arg (NO are prepared2)-Gly-Asp(OBzl)-Val-OBzl；
(6) prepare (7S)-N- [(Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-
(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(7) prepare (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-Gly-
Asp-Val。
The four of present disclosure are to evaluate (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- first
Acyl] (as AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val to-Arg-Gly-Asp-AA；As AA=Phe
Arg-Gly-Asp-AA is Arg-Gly-Asp-Phe) antithrombotic acitivity.
The four of present disclosure are to evaluate (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- first
Acyl] (as AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val to-Arg-Gly-Asp-AA；As AA=Phe
Arg-Gly-Asp-AA is Arg-Gly-Asp-Phe) thrombus dissolving activity.
The five of present disclosure are to evaluate (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dioxies isoquinolin -7- first
Acyl] (as AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val to-Arg-Gly-Asp-AA；As AA=Phe
Arg-Gly-Asp-AA is Arg-Gly-Asp-Phe) treatment cerebral infarction 24h activity.
Description of the drawings
Fig. 1 (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-Gly-Asp-AA (when
During AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val；As AA=Phe, Arg-Gly-Asp-AA is
Arg-Gly-Asp-Phe synthetic route). (i) formaldehyde, dilute hydrochloric acid；(ii) p-methyl benzenesulfonic acid, benzyl alcohol, normal hexane；(iii) N- hydroxyls
Base succimide, HOBt, THF, NMM；(iv) 4N hydrogen chloride-ethyl acetate solution；(v)Pd/C,methanol；(vi)
TFA,TFMSA。
Specific embodiment
In order to the present invention is expanded on further, a series of embodiments are given below.These embodiments be entirely it is illustrative, they
Only for being specifically described to the present invention, limitation of the present invention is not construed as.
Embodiment 1 prepares (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- carboxylic acids
To 988mg (0.005mmol), (S) -3,4- dihydroxy-Phe sequentially adds 5.9mL H232% hydrochloric acid of O, 0.7mL, stirs
Mix uniform, add 40% formalins of 1.2mL, room temperature reaction 6 hours until completely dissolved.TLC (dichloromethane:Methanol,
10:1) display (S) -3,4- dihydroxy-Phe disappears, and under ice bath, Deca saturated sodium bicarbonate makes pH be 5 and separate out colourless in a large number sinking
Form sediment.Filter to obtain 1.01g (95%).Mp 281-286℃,ESI-MS(m/z):210[M+H]+。
Embodiment 2 prepares (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates
By 1g (4.08mmol) (7S) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- carboxylic acids, 1.02g p-methyl benzenesulfonic acid, 8mL
The mixture of benzyl alcohol and 8mL normal hexane clarifies change in 90～95 DEG C of heated and stirred 36h.Question response mixed liquor is cooled to
Room temperature, adds a large amount of ether, makes a large amount of colorless solids of precipitation, filters, and the solid for obtaining is worn away with ether, is dried, obtains
1.08g (77%) title compound, is colourless powder.ESI-MS(m/z):300[M+H]+。
Embodiment 3 prepares Boc-Pro-Ala
1.075g (5.0mmol) Boc-Pro is dissolved in into 20mL anhydrous tetrahydro furans (THF), is added in solution under ice bath
0.637g (5mmol) N-hydroxy-succinamide (HOSu), and make to be completely dissolved.It is under ice bath, a small amount of anhydrous having been dissolved in
During dicyclohexyl carbonyl diimine (DCC) 1.236g (6.0mmol) of THF adds reactant liquor.7 hours are stirred at room temperature, TLC
(CHCl2：MeOH=10:1) monitoring reaction.1,3-Dicyclohexylurea (DCU) is filtered, filtrate reduced in volume removes THF.It is dense
Contracting thing ethyl acetate (EA) dissolves, and uses saturation NaHCO successively3Aqueous solution wash 3 times, saturation NaCl aqueous solution wash 3 times,
Then EA layers are evaporated to dry, the appropriate THF dissolvings of addition.Add the Ala 0.489g (5.5 for having been dissolved in a small amount of water
Mmol), use NaHCO3Solid adjusts pH to 8-9, normal-temperature reaction 12 hours, concentrating under reduced pressure to remove THF, adds 5mL
Water dissolution, uses saturation KHSO4Aqueous solution adjusts pH to 2, is repeatedly extracted with EA on a small quantity, merges EA layers, uses saturation NaCl
Aqueous solution is washed till neutrality, anhydrous sodium sulfate drying.Filter, filtrate reduced in volume obtains 1.41g (98%) title compound to dry
Thing, is colourless powder.ESI-MS(m/e):285[M-H]-。
Embodiment 4 prepares Boc-Pro-Ala-Lys (Boc)-OBzl
314mg (1.1mmol) Boc-Pro-Ala is dissolved in into the anhydrous THF of 15mL, under ice bath, adds 149mg (1.1 inward
Mmol) the anhydrous THF solution of N- hydroxybenzotriazoles (HOBt) and 268mg (1.3mmol) DCC.Reaction mixing
Thing ice bath is stirred 20 minutes, obtains corresponding active ester solution.372mg (1.0mmol) HClLys (Boc)-OBzl is used
Anhydrous THF dissolvings, then miscible with the active ester solution for just obtaining, a small amount of N-methylmorpholine of Deca (NMM) is adjusted
PH 9, the reactant mixture room temperature reaction for obtaining 24 hours.1,3-Dicyclohexylurea (DCU) is filtered to remove, filtrate decompression is dense
Dry, residue with Ethyl acetate dissolving is reduced to, DCU is then filtered again, filtrate uses saturation NaHCO successively3Solution washes 3
Secondary, saturation NaCl solution washes 3 times, saturation KHSO4Solution wash 3 times, saturation NaCl solution wash 3 times, saturation NaHCO3
Solution wash 3 times, saturation NaCl solution wash 3 times, gained ethyl acetate layer anhydrous Na2SO4It is dried, filters, reduces pressure
It is concentrated to give yellow oil, Jing column chromatographies (petroleum ether/acetone system 6:1-1:1) 412mg (65%) title compound is obtained,
For colourless powder.ESI-MS(m/z)：605[M+H]+。
Embodiment 5 prepares Boc-Pro-Ala-Lys (Boc)
The Pd/C of 20mL methanol and 30mg is added to 604mg (1.0mmol) Boc-Pro-Ala-Lys (Boc)-OBzl.
Lead to hydrogen toward the suspension for obtaining, be stirred at room temperature 24 hours, TLC (CH2Cl2:MeOH=15:1) reaction raw materials are monitored
Point disappears, and reaction is completed.Pd/C is filtered, filtrate reduced in volume removes solvent, obtains 496mg (97%) title compound,
For colourless powder.ESI-MS(m/z)：513[M-H]-。
Embodiment 6 prepares (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates
564mg (1.1mmol) Boc-Pro-Ala-Lys (Boc) is dissolved in into the anhydrous THF of 15mL, under ice bath, Jia 149 inward
The anhydrous THF solution of mg (1.1mmol) HOBt and 468mg (1.3mmol) N- hydroxysuccinimides.Reactant mixture
Ice bath is stirred 20 minutes, obtains corresponding active ester solution.By 472mg (1.0mmol) (7S) -2,3 dihydroxies of -5,6,7,8- tetrahydrochysenes
Base isoquinolin -7- benzyl carboxylates are dissolved with anhydrous THF, then miscible with the active ester solution for just obtaining, the appropriate NMM of Deca
Adjust pH 8, room temperature reaction 24 hours.Filter, filtrate reduced in volume is to dry, residue with Ethyl acetate dissolving, filtrate
Saturation KHSO is used successively4Solution is washed 3 times, and saturation NaCl solution is washed 3 times, gained ethyl acetate layer anhydrous Na2SO4
It is dried, filters, filtrate reduced in volume, the yellow oil Jing column chromatography purification for obtaining obtains 190mg (19%) title compound,
For colourless powder.ESI-MS(m/z)：796[M+H]+。
Embodiment 7 prepares (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy-isoquinolin -7- carboxylic acids
To 795mg (1.0mmol) (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- carboxylic acids
20mL methanol and 30mgPd/C are added in benzyl ester.Lead to hydrogen toward the suspension for obtaining, be stirred at room temperature 24 hours,
TLC(CH2Cl2:MeOH,30:1) monitor reaction raw materials point to disappear, reaction is completed.Pd/C is filtered, filtrate reduced in volume is obtained
640mg (92%) title compound, is colourless powder.ESI-MS(m/z)：704[M-H]-。
Embodiment 8 prepares Boc-Arg (NO2)-Gly-OBzl
By 350mg (1.1mmol) Boc-Arg (NO2) the anhydrous THF of 15mL are dissolved in, add 149mg (1.1 under ice bath inward
Mmol) the anhydrous THF solution of HOBt and 268mg (1.3mmol) dicyclohexyl carbonyl diimine (DCC), reaction mixing
Thing ice bath is stirred 20 minutes.337mg (1.0mmol) HClGly-OBzl are dissolved with anhydrous THF, then with just
The active ester solution for arriving is miscible, and Deca NMM adjusts pH 8, the reactant mixture room temperature reaction for obtaining 24 hours.Filter, filter
Liquid is evaporated to dry, residue with Ethyl acetate dissolving.Ethyl acetate solution uses saturation KHSO successively4Solution is washed 3 times,
Saturation NaCl solution washes 3 times, anhydrous Na2SO4It is dried, filters, filtrate reduced in volume.The yellow oil Jing post for obtaining
Chromatography purification, obtains 312mg (67%) title compound, is colourless powder.ESI-MS(m/z)：466[M+H]+。
Embodiment 9 prepares Boc-Arg (NO2)-Gly
By 466mg (1.0mmol) Boc-Arg (NO2)-Gly-OBzl 20mL methanol dissolvings, add 4N NaOH under ice bath
Solution adjusts pH to 12, ice bath reaction 3h, TLC (CHCl2:MeOH,30:1) show that raw material point disappears.Reactant mixture adds
5%KHSO4Aqueous solution adjusts pH to neutrality, and concentrating under reduced pressure removes methanol, adds saturation KHSO4Aqueous solution adjust pH to
2, it is extracted with ethyl acetate, anhydrous Na2SO4It is dried, filters, filtrate reduced in volume obtains 310mg (78%) title compound
Thing, is colourless powder.ESI-MS(m/z):375[M-H]-。
Embodiment 10 prepares Boc-Asp (OBzl)-Phe-OBzl
According to the method for embodiment 8 from 355mg (1.1mmol) Boc-Asp (OBzl) and 291mg (1.0mmol)
HClPhe-OBzl obtains 284mg (50%) title compound, is colourless powder.ESI-MS(m/z):561[M+H]+。
Embodiment 11 prepares HClAsp (OBzl)-Phe-OBzl
10mL 4M hydrogen chloride-ethyl acetate solution, room are added to 560mg (1mmol) Boc-Asp (OBzl)-Phe-OBzl
Temperature stirring 3 hours, TLC (CHCl2:MeOH,30:1) show that raw material point disappears.Reactant liquor is evaporated to dry, residue
Plus a small amount of anhydrous ethyl acetate dissolving, then be evaporated to dry.The operation is repeated 3 times.Residue adds a small amount of absolute ether dissolving,
It is evaporated to dry, then is evaporated to dry.The operation is repeated 3 times, and obtains 440mg (96%) title compound, is without toner
End.ESI-MS(m/z):461[M+H]+。
Embodiment 12 prepares Boc-Arg (NO2)-Gly-Asp(OBzl)-Phe-OBzl
According to the method for embodiment 8 from 413mg (1.1mmol) Boc-Arg (NO2)-Gly and 496mg (1.0mmol)
HClAsp (OBzl)-Phe-OBzl obtains 368mg (45%) title compound, is colourless powder.ESI-MS(m/z)：
819[M+H]+。
Embodiment 13 prepares HClArg (NO2)-Gly-Asp(OBzl)-Phe-OBzl
According to the method for embodiment 10 from 818mg (1mmol) Boc-Arg (NO2)-Gly-Asp (OBzl)-Phe-OBz obtains
690mg (93%) title compound, is colourless powder.ESI-MS(m/z)：719[M+H]+。
Embodiment 14 prepares (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- first
Acyl]-Arg (NO2)-Gly-Asp(OBzl)-Phe-OBzl
According to the method for embodiment 6 from 705mg (1.1mmol) (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3-
Dihydroxy-isoquinolin -7- carboxylic acids and 718mg (1.0mmol) HClArg (NO2)-Gly-Asp (OBzl)-Phe-OBzl obtains
279mg (17%) title compound, is colourless powder.ESI-MS(m/z)：1406[M+H]+。
Embodiment 15 prepares (7S)-N- (Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- formyls] -
Arg-Gly-Asp-Phe(Ia)
To 281mg (0.2mmol) (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin under ice bath
- 7- formyls]-Arg (NO2)-Gly-Asp (OBzl)-Phe-OBzl adds 3mL TFA and 1mL TFMSA successively, reacts 30min,
Reaction 30min, TLC (CHCl2:MeOH=15:1) monitor reaction to complete.A large amount of ether are added under ice bath in reactant liquor,
The a large amount of solids of precipitation are made, is stood, is toppled over upper strata ether, add ether.The operation 3 times repeatedly.It is evaporated to dry.
Residue Sephadex G15 desalinations, use C18 column purifications, lyophilizing to obtain 4mg (2%) title compound, be nothing
Color powder.ESI-MS(m/e):981[M+H]+ 1H-NMR(D2O, 800MHz):δ/ppm=7.255 (m, 2H),
7.205 (m, 1H), 7.190 (d, 2H), 6.792 (s, 1H), 6.723 (s, 1H), 4.601-4.544 (m, 3H), 4.476
(t, 1H), 4.355 (m, 1H), 4.309 (m, 1H), 3.740 (t, 2H), 3.399 (d, 2H), 3.117 (m, 2H),
3.034-3.008 (m, 3H), 2.956-2.915 (m, 4H), 2.688 (m, 1H), 2.540 (m, 1H), 2.014 (m, 2H),
1.950 (m, 1H), 1.721-1.638 (m, 4H), 1.582 (m, 1H), 1.460 (m, 1H), 1.457 (m, 1H), 1.376
(m, 1H), 1.328 (d, J=7.2Hz, 3H), 1.303-1.284 (m, 2H).
Embodiment 16 prepares Boc-Asp (OBzl)-Val-OBzl
According to the method for embodiment 8 from 355mg (1.1mmol) Boc-Asp (OBzl) and 243mg (1.0mmol)
HClVal-OBzl obtains 338mg (66%) title compound, is colourless powder.ESI-MS(m/z)：513[M+H]+。
Embodiment 17 prepares HClAsp (OBzl)-Val-OBzl
It is titled that method according to embodiment 10 obtains 400mg (94%) from 512mg (1mmol) Boc-Asp (OBzl)-Val-OBzl
Compound, is colourless powder.ESI-MS(m/z)：413[M+H]+。
Embodiment 18 prepares Boc-Arg (NO2)-Gly-Asp(OBzl)-Val-OBzl
According to the method for embodiment 8 from 413mg (1.1mmol) Boc-Arg (NO2)-Gly and 448mg (1.0mmol)
HClAsp (OBzl)-Val-OBzl obtains 533mg (69%) title compound, is colourless powder.ESI-MS(m/z)：
771[M+H]+。
Embodiment 19 prepares HClArg (NO2)-Gly-Asp(OBzl)-Val-OBzl
According to the method for embodiment 10 from 770mg (1mmol) Boc-Arg (NO2)-Gly-Asp(OBzl)-Val-
OBzl obtains 680mg (96%) title compound, is colourless powder.ESI-MS(m/z)：671[M+H]+。
Embodiment 20 prepares (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- first
Acyl]-Arg (NO2)-Gly-Asp(OBzl)-Val-OBzl
According to the method for embodiment 6 from 705mg (1.1mmol) (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3-
Dihydroxy-isoquinolin -7- carboxylic acids and 671mg (1.0mmol) HClArg (NO2)-Gly-Asp- (OBzl)-Val-OBzl obtains
217mg (16%) title compound, is colourless powder.ESI-MS(m/z)：1358[M+H]+。
Embodiment 21 prepares (7S)-N- (Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- formyls] -
Arg-Gly-Asp-Val(Ib)
To 271mg (0.2mmol) (7S)-N- (Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- under ice bath
Formyl]-Arg (NO2)-Gly-Asp (OBzl)-Val-OBzl adds 3mL TFA and 1mL TFMSA successively, reacts 30min,
Reaction 30min, TLC (CHCl2:MeOH=15:1) monitor reaction to complete.A large amount of ether are added under ice bath in reactant liquor,
The a large amount of solids of precipitation are made, is stood, is toppled over upper strata ether, add ether.The operation 3 times repeatedly.It is evaporated to dry.
Residue Sephadex G15 desalinations, use C18 column purifications, lyophilizing to obtain 4mg (2%) title compound, be nothing
Color powder.ESI-MS(m/e):933[M+H]+ 1H-NMR(D2O, 800MHz):δ/ppm=6.792 (s, 1H),
6.723 (s, 1H), 4.648-4.541 (m, 4H), 4.350 (m, 1H), 4.302 (m, 1H), 4.210 (m, 1H), 3.355
(m, 2H), 3.091 (m, 1H), 3.040 (m, 1H), 3.004 (m, 1H), 2.972-2.936 (m, 3H), 2.668 (m,
1H), 2.549 (m, 1H), 2.410 (m, 1H), 2.024 (m, 3H), 1.958 (m, 1H), 1.821 (m, 1H), 1.704-1.662
(m, 5H), 1.524 (m, 1H), 1.474 (m, 1H), 1.419 (m, 1H), 1.376 (m, 1H), 1.334 (d, J=4.8
Hz, 3H), 1.193-1.157 (m, 2H), 0.807 (d, J=23.6Hz, 6H).
Experimental example 1 evaluates compound Ia, the antithrombotic acitivity of b
It is by male SD rat (200 ± 20g), random to be grouped, 10 per group, raise 1 day, stop feeding overnight.
Gavage gives compound Ia, the normal saline solution (dosage is 1nmol/kg) or the normal saline solution (dosage of aspirin of b
For 167 μm of ol/kg) or normal saline (dosage is 10mL/kg) 30min after, normal saline of the rat with 20% Ethylurethanm
Solution is anaesthetized, and is performed the operation afterwards.The right carotid and left neck vein of rat are separated, the silk thread of correct amount are placed in into bypass intubation,
Left vein is inserted in one end of pipe, and another end pipe insertion right artrial simultaneously injects 0.2mL heparin sodium anticoagulants.So that blood flow is from right side
Tremulous pulse flows through bypass intubation and enters left side vein, takes out the silk thread with thrombosis and weighs, before calculating blood circulation after 15min
The weight of silk thread, the thrombosis weight for obtaining, represent and represent antithrombotic acitivity with mean value ± SD mg afterwards, make t inspections.Data
List table 1 in.As a result show oral 1nmol/kg compounds Ia, b can effectively inhibition thrombosis.Illustrate present invention obtains
Unexpected technique effect.
1 1nmol/kg compound Ia of table, the antithrombotic acitivity of b
N=10；A) p compared with normal saline<0.01.
Experimental example 2 evaluates the thrombus dissolving activity of compound Ia-c
SD rats (male, 200 ± 20g) are carried out by the dosage lumbar injection urethane normal saline solution of 1200mg/kg
Anesthesia.Its dorsal position is fixed after anesthetized rat, separate its right common carotid artery, at proximal part, clamp bulldog clamp, by proximal part
And distal end respectively penetrates surgical thread, bulldog clamp is unclamped by the surgical thread ligation of distal end, distal end intubation, takes out about 1mL
Arterial blood, is placed in 1mL centrifuge tubes.Toward vertical fixed rubber tube (long 15mm, internal diameter 2.5mm, external diameter 5.0mm,
Ttom of pipe is sealed with plug, and para films are tamping) interior injection 0.1mL rat artery blood, subsequently one is rapidly inserted into not in pipe
The fixing bolt of the thrombosis of rust steel matter (fix spiral and be coiled into the stainless steel silk of a diameter of 0.2mm, spiral part by thrombosis
Long 10mm, includes 15 bung flanges, and a diameter of 1.0mm supports handle of bung flange is connected with spiral, is about 7.0mm, in asking
Number type).After blood coagulation 45min, from glass tubing, the careful thrombosis wrapped up by thrombosis that take out fix spiral, accurate to claim
Its weight.
Bypass intubation be made up of three parts, interlude be long 60.0mm, the polyethylene rubber tube of internal diameter 3.5mm；Two ends are length
The identical polyethylene tube of 100.0mm, internal diameter 1.0mm, external diameter 2.0mm, the pipe one end pull into spike tube, are about 10.0
Mm (for insert rat carotid artery and vein), external diameter is 1.0mm, and one section of the outer cover of its other end is long for 7.0mm,
Polyethylene tube (for insert the polyethylene rubber tube in stage casing in) of the external diameter for 3.5mm, the inwall of 3 sections of pipes is required to silanization
(1% silicone oil diethyl ether solution).The thrombosis that thrombosis are wrapped up are fixed spiral and are placed in the polyethylene rubber tube of stage casing, and sebific duct is in addition
Two ends are nested with the overstriking end of two polyethylene respectively, it is ensured that blood will not be leaked during circulation.Pass through spike tube with syringe
End will fill heparin-saline solution (50IU/kg) in pipe, exclude bubble, standby.
The left external jugular vein of rat is separated, proximal part and distal end respectively penetrate surgical thread, ligature the blood vessel of distal end, sudden and violent
An osculum is cut on the left external jugular vein of dew, the above-mentioned bypass duct spike tube for preparing is inserted into left external jugular vein opening by osculum
Place, while fixing spiral away from shunt valve stage casing (thrombosis containing accurate weighing fix spiral) interior thrombosis.Passed through with syringe
The spike tube of the other end injects the normal saline solution (50IU/kg) of the heparin sodium of correct amount, and now syringe should not withdraw poly- second
Alkene pipe, clamps the flexible pipe between syringe and polyethylene tube with bulldog clamp.Stopped blooding with bulldog clamp in the proximal part of right common carotid artery,
Right common carotid artery is nearby being cut an osculum from bulldog clamp, is extracting syringe from the tip of polyethylene tube by ligation distal end,
The proximal part of tremulous pulse angle is inserted in the tip of polyethylene tube.The two ends of bypass duct are solid by arteriovenous with No. 4 suturess
It is fixed.
With scalp acupuncture by the normal saline solution (dosage is 20000IU/kg) or compound of normal saline (3mL/kg) or urokinase
Stage casing (thrombosis containing accurate weighing fix spiral) of the normal saline solution (dosage is 1nmol/kg) of Ia, b by shunt valve,
The nearly vein end that spiral is fixed away from thrombosis is penetrated, bulldog clamp is unclamped, is made blood flow flow to vein from tremulous pulse by bypass duct.
Solution in syringe is slowly injected into into blood, by blood circulation, by the sequential action of vein-heart-tremulous pulse in spiral
On thrombosis.After blood circulation 1h, the spiral of fixed thrombosis, accurate weighing are taken out from bypass duct.Calculate every rat
The weight difference of thrombosis before and after the spiral blood circulation of thrombosis is fixed in bypass duct, is represented with mean value ± SD mg and is represented haemolysis
Thrombus activity, makees t inspections.Data list table 2 in.As a result 1nmol/kg compound Ia are shown, b can effectively lysigenous thrombosis.
Illustrate present invention obtains unexpected technique effect.
2 1nmol/kg compound Ia of table, the thrombus dissolving activity of b
N=10；A) compare p with normal saline<0.01, the p compared with urokinase>0.05.
Experimental example 3 evaluates compound Ia, therapeutical effect of the b to cerebral infarction rat
The long otch of about 2cm is opened vertically at the positive middle part of the cervical region of male SD rat (300 ± 20g of body weight), on the inside of sternocleidomastoid
Fate separates out right common carotid artery, external carotid artery and internal carotid artery.Pressed from both sides with noinvasive bulldog clamp respectively close at internal carotid artery opening and
Common carotid artery proximal part, ligatures the distal end of external carotid artery, cuts an osculum in external carotid artery, unclamps common carotid artery proximal part
Bulldog clamp, take 10 μ L blood, afterwards again with noinvasive bulldog clamp folder close Carotid proximal part.The 10 μ L blood for obtaining are put
Putting the room temperature in 1mL EP pipes makes blood coagulation in 30 minutes, places 1 hour, make blood in being then transferred to -20 DEG C of refrigerators
Liquid grumeleuse is solid.Rat is 400mg/kg with 10% chloral hydrate intraperitoneal injection of anesthesia, dosage.Blood clotting is taken out, plus
Enter 1mL normal saline, blood clotting is pounded uniform tiny thrombi with steel shovel, the suspension of tiny thrombosis is prepared
And be transferred in 1mL syringes.The bulldog clamp of common carotid artery proximal part is unclamped, by 1mL thrombosis suspension slowly from rat
External carotid artery through the brain of internal carotid injection rat, then ligatures external carotid artery proximal part, opens in neck to proximal part
Bulldog clamp is obtained at tremulous pulse and common carotid artery, recovers blood flow.Wait revival.Rat is commented by Zealonga methods after reviving 24 hours
Determine neurological functional deficit.0 point indicates that, without any neurological deficit sign, 1 point of expression does not damage side forelimb not tensible,
2 points represent, 3 points to represent and turn-take into shape walking of knocking into the back to not damaging side, and 4 points represent disturbance of consciousnesss without autonomous
Walking, 5 points represent dead.According to score average packet.Each group rat Jing tail veins inject 1 compound Ia, b, agent daily
Measure as 1nmol/kg.Continuous injection 6 days, is scored daily.As a result list table 3,4 in.The as shown by data of table 3, compound Ia are continuous
It is 0 point that treatment can make 4 cerebral ischemias rat scoring biology of 24 hours for 6 days, can make 7 cerebral ischemias rat of 24 hours
Neurobiology scoring is neurobiology scoring for 1 point, can make 1 cerebral ischemia rat scoring biology of 24 hours be
Neurobiology scoring is 2 points.The as shown by data of table 4, continuously treatment can make the big of 3 cerebral ischemias 24 hours for 6 days to compound Ib
The scoring of Mus neurobiology is 0 point, and 9 cerebral ischemias rat scoring biology of 24 hours can be made to score for neurobiology
For 1 point, 1 cerebral ischemia rat scoring biology of 24 hours can be made to be 2 points for neurobiology scoring.Because unlike
Compound initial dose disclosed in Jing needs 5 μm of ol/kg, afterwards 5 maintenance dosies 2 μm of ol/kg of needs, 6 times of compound Ia, b
Dosage is 1nmol/kg.So, initial dose and maintenance dose reduce 5000 times and 2000 times respectively.
3 compound Ia of the table continuously treatment impacts to cerebral ischemia 24 hours rats scoring biology in 6 days
N=12
4 compound Ib of the table continuously treatment impacts to cerebral ischemia 24 hours rats scoring biology in 6 days
N=13.
Claims (6)
1. (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- the formyls]-Arg-Gly-Asp-AA of following formula,
As AA=Val, Arg-Gly-Asp-AA is Arg-Gly-Asp-Val；As AA=Phe, Arg-Gly-Asp-AA is
Arg-Gly-Asp-Phe
2. (7S)-N- [(Pro-Ala-Lys)-5,6,7,8- tetrahydrochysenes-2,3- dihydroxy isoquinolin-7- formyls] of claim 1-
The preparation method of Arg-Gly-Asp-Phe, the method are comprised the following steps:
(1) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- carboxylic acids are prepared；
(2) (7S)-N- tertbutyloxycarbonyl -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(3) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(4) Boc-Arg (NO are prepared2)-Gly-Asp(OBzl)-Phe-OBzl；
(5) Arg (NO are prepared2)-Gly-Asp(OBzl)-Phe-OBzl；
(6) prepare (7S)-N- [(Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dioxies isoquinolin -7- formyls] -
Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl；
(7) prepare (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-Gly
-Asp-Phe。
3. (7S)-N- [(Pro-Ala-Lys)-5,6,7,8- tetrahydrochysenes-2,3- dihydroxy isoquinolin-7- formyls] of claim 1-
The preparation method of Arg-Gly-Asp-Val, the method are comprised the following steps:
(1) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- carboxylic acids are prepared；
(2) (7S)-N- tertbutyloxycarbonyl -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(3) (7S) -5,6,7,8- tetrahydrochysene -2,3- dihydroxy isoquinolin -7- benzyl carboxylates are prepared；
(4) Boc-Arg (NO are prepared2)-Gly-Asp(OBzl)-Val-OBzl；
(5) Arg (NO are prepared2)-Gly-Asp(OBzl)-Val-OBzl；
(6) prepare (7S)-N- [(Boc-Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls] -
Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl；
(7) prepare (7S)-N- [(Pro-Ala-Lys) -5,6,7,8- tetrahydrochysenes -2,3- dihydroxy isoquinolin -7- formyls]-Arg-Gly
-Asp-Val。
4. (7S)-N- [(Pro-Ala-Lys)-5,6,7,8- tetrahydrochysenes-2,3- dihydroxy isoquinolin-7- formyls] of claim 1-
Applications of the Arg-Gly-Asp-AA in antithrombotic reagent is prepared.
5. (7S)-N- [(Pro-Ala-Lys)-5,6,7,8- tetrahydrochysenes-2,3- dihydroxy isoquinolin-7- formyls] of claim 1-
Applications of the Arg-Gly-Asp-AA in thrombolytic agent is prepared.
6. (7S)-N- [(Pro-Ala-Lys)-5,6,7,8- tetrahydrochysenes-2,3- dihydroxy isoquinolin-7- formyls] of claim 1-
Applications of the Arg-Gly-Asp-AA in treatment cerebral infarction medicine is prepared.
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| CN101318995A (en) * | 2007-06-04 | 2008-12-10 | 北京大学 | Substituted tetrahydro-isoquinoline isoquinolinium compound, preparation method and application thereof |
| CN101591376A (en) * | 2008-05-30 | 2009-12-02 | 首都医科大学 | Has oligopeptides of thrombolysis activity and its production and application |
| CN102241740A (en) * | 2008-01-30 | 2011-11-16 | 首都医科大学 | Compounds with thrombolytic activities and preparation methods and applications thereof |
| CN102477066A (en) * | 2010-11-30 | 2012-05-30 | 首都医科大学 | N-(S-1,2,3,4-tetrahydro-6,7-dihydroxyisoquinolyl-3-formyl)amino acid, synthetic method thereof, and application thereof |
| CN102485748A (en) * | 2010-12-01 | 2012-06-06 | 首都医科大学 | Oligopeptides with targeting thrombolytic activity, preparation method thereof, and application thereof |
| CN102485747A (en) * | 2010-12-01 | 2012-06-06 | 首都医科大学 | Oligopeptides with targeting thrombolytic activity, preparation method and application thereof |
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| 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 |
| CN101318995A (en) * | 2007-06-04 | 2008-12-10 | 北京大学 | Substituted tetrahydro-isoquinoline isoquinolinium compound, preparation method and application thereof |
| CN102241740A (en) * | 2008-01-30 | 2011-11-16 | 首都医科大学 | Compounds with thrombolytic activities and preparation methods and applications thereof |
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