CN106608904B - 1-hydroxy-2- (oxyacetyl decapeptide phenyl) -tetramethyl imidazoline, its synthesis, activity and application - Google Patents
1-hydroxy-2- (oxyacetyl decapeptide phenyl) -tetramethyl imidazoline, its synthesis, activity and application Download PDFInfo
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- CN106608904B CN106608904B CN201510690042.7A CN201510690042A CN106608904B CN 106608904 B CN106608904 B CN 106608904B CN 201510690042 A CN201510690042 A CN 201510690042A CN 106608904 B CN106608904 B CN 106608904B
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- 230000000694 effects Effects 0.000 title description 8
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- 238000000034 method Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims description 49
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- 239000003814 drug Substances 0.000 claims description 6
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Abstract
The present invention discloses 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val of the formula]Phenyl-4, 4,5, 5-tetramethyl imidazoline, its preparing process, its antithrombotic activity, its thrombolytic activity and its action to treat cerebral ischemia rat are disclosed.
Description
Technical Field
The invention relates to 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline, relates to a preparation method thereof, relates to antithrombotic activity thereof, relates to thrombolytic activity thereof, and relates to an effect thereof in treating cerebral ischemia rats, so that the invention relates to application thereof in preparing antithrombotic medicaments, thrombolytic medicaments and medicaments for treating ischemic stroke.
Background
Ischemic stroke is a common and serious cerebrovascular disease, and is characterized by high morbidity, high fatality rate, high disability rate and high recurrence rate. At present, the clinical treatment of ischemic stroke faces the reality that no effective medicine exists, and especially, patients with stroke for more than 4 hours are not dead or are disabled. The invention is an important clinical demand for effective medicines for patients with stroke of more than 4 h. The inventors have disclosed that imidazoline compounds of formula II show excellent efficacy in a rat ischemic stroke model with stroke duration of 24 h. That is, the imidazoline compound of formula II is continuously injected intravenously for 6 days, and has excellent curative effect 1 time per day, the first dosage is 5 mu mol/kg, and the last 5 dosages are 2 mu mol/kg. In the formula aa1And aa2Can be present simultaneously aa1Exist but aa2Absent, or both; when aa1And aa2In the meantime, aa1Is R (Arg), and aa2G (Gly), A (Ala) or Q (Gln); when aa1Exist but aa2In absence aa1Is R (Arg); aa3It may be S (Ser), V (Val) or F (Phe). Since the imidazoline of formula II is a NO radical, the stability is relatively poor and needs to be improved.
The inventor finds that unexpected technical effects with good stability can be obtained by replacing 1, 3-dioxyimidazoline in the formula with 1-hydroxyimidazoline through experimental research for 3 years. In light of this finding, the inventors have devised the present invention.
Disclosure of Invention
1. One aspect of the present invention is to provide 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline of the formula
2. The second aspect of the present invention provides a process for producing 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline, which comprises the steps of:
(1) preparing 2, 3-dimethyl-2, 3-dinitrobutane;
(2) preparing 2, 3-dimethyl-2, 3-dihydroxyaminobutane;
(3) preparing 1, 3-dihydroxy-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline;
(4) preparing 1, 3-dioxo-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline;
(5) preparation of Arg (NO) according to Standard procedure2)-Gly-Asp(OBzl)-Val-OBz；
(6) Preparation of Gly-Arg (NO) according to Standard procedure2)-Pro-Als-Lys(Z)-OBz；
(7) Preparing 1, 3-dioxo-2- (4' -oxyacetic acid ethyl ester-phenyl) -4,4,5, 5-tetramethylimidazoline;
(8) preparing 1, 3-dioxo-2- (4' -oxyacetic acid-phenyl) -4,4,5, 5-tetramethylimidazoline;
(9) preparing 1, 3-dioxo-2- [ 4' -oxoacetyl-L ys (Boc) -OMe-phenyl ] -4,4,5, 5-tetramethylimidazoline;
(10) preparing 1, 3-dioxy-2- [ (4' -oxyacetyl-L ys-OMe) phenyl ] -4,4,5, 5-tetramethyl imidazoline;
(11) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-L ys-OMe } phenyl-4, 4,5, 5-tetramethylimidazoline;
(12) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-L ys } phenyl-4, 4,5, 5-tetramethylimidazoline;
(13) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-Lys-Arg-(NO2) -Gly-asp (OBzl) -Val-OBzl } phenyl-4, 4,5, 5-tetramethylimidazoline;
(14) preparation of 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline.
3. The third aspect of the present invention is to evaluate the antithrombotic activity of 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline.
4. Fourth, the thrombolytic activity of 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline was evaluated.
5. The fifth aspect of the present invention was to evaluate the activity of 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline for treating cerebral ischemia 24h after cerebral ischemia.
Drawings
FIG. 11-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val]Synthetic route of phenyl-4, 4,5, 5-tetramethylimidazoline i) Br2,6N NaOH；ii)Zn,NH4Cl, 50% ethanol; iii) 4-hydroxybenzaldehyde, CH3OH；iv)PbO2,CH3OH；v)BrCH2CO2C2H5；vi)2N NaOH,CH3OH；vii)TFA/TFMSA,NaNO2。
Detailed Description
To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.
EXAMPLE 1 preparation of 2, 3-dimethyl-2, 3-dinitrobutane
69g (0.78mol) of 2-nitropropane were added to 130ml of aqueous NaOH (6N), 20ml (0.38mol) of Br2 were added dropwise with stirring in an ice-salt bath over 1 hour, then 240m of L ethanol was added, reflux was carried out at 90 ℃ for 3 hours, the reaction mixture was heated and poured into 800ml of ice-water, and 55g (81%) of the title compound was obtained as colorless flaky crystals, Mp 110 and 112 ℃.
EXAMPLE 2 preparation of 2, 3-dimethyl-2, 3-dihydroxyaminobutane
Suspending 7g (40mmol) of 2, 3-dimethyl-2, 3-dinitrobutane and 4g of NH4Cl in 80m L of ethanol aqueous solution (50%), stirring in an ice bath, adding 16g of zinc powder in 3h, removing the ice bath, continuing stirring at room temperature for reaction for 3h, then carrying out suction filtration on the reaction liquid, repeatedly washing a filter cake with the ethanol aqueous solution (50%), combining filtrate and washing liquid, adjusting the pH to 2 with concentrated hydrochloric acid, carrying out reduced pressure evaporation to a slurry state, adding a proper amount of potassium carbonate, stirring uniformly, using a Soxhlet extractor, using chloroform as an extracting agent, extracting for 6h, carrying out reduced pressure concentration on the extracting solution to a small amount, adding petroleum ether, precipitating 2.60g (44%) of the title compound as a colorless crystal, and carrying out Mp157-159 ℃.
EXAMPLE 3 preparation of 1, 3-dihydroxy-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazolidine
1.22g (10mmol) p-hydroxybenzaldehyde and 1.48g (10mmol)2, 3-dimethyl-2, 3-dihydroxyaminobutane were dissolved in 10M L methanol and after stirring for 8h at room temperature, T L C showed disappearance of the starting material spot, which was filtered off with suction to give 1.29g (51%) of the title compound as colorless crystals EI-MS (M/z)252[ M]+.1H-NMR(DMSO-d6)(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)。
EXAMPLE 4 preparation of 1, 3-dihydroxy-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline
504mg (2mmol) of 1, 3-dihydroxy-2- (4)' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazolidine was dissolved in 30M L methanol, 3g of PbO2 was added, the mixture was stirred at room temperature for 40min, T L C showed disappearance of the starting material spot, the solid was removed by suction filtration, the filtrate was evaporated at room temperature under reduced pressure, the residue was purified by column chromatography (chloroform elution), 260mg (52%) of the title compound was obtained as a blue solid, Mp134-135 ℃, EI-MS (M/z)249[ M-]+.IR(KBr)3250,1610,1500,1490,840。
EXAMPLE 5 preparation of 1, 3-dioxo-2- (4' -oxoethyl acetate-phenyl) -4,4,5, 5-tetramethylimidazoline
250mg (1mmol) of 1, 3-dihydroxy-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline, 0.32m L of ethyl bromoacetate and 32mg of sodium hydride were dissolved in 5m L of anhydrous THF, the mixture was stirred at 60 ℃ for 5 hours, T L C showed disappearance of the starting point, the mixture was filtered under reduced pressure at room temperature, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (ethyl acetate: petroleum ether: 1:5) to give 300mg (90%) of the title compound, Mp 107-.
EXAMPLE 6 preparation of 1, 3-dioxo-2- (4' -oxoacetic acid-phenyl) -4,4,5, 5-tetramethylimidazoline
To a solution of 33mg (0.1mmol) of 1, 3-dioxy-2- (4' -ethoxyethyl acetate-phenyl) -4,4,5, 5-tetramethylimidazoline in 3M L methanol was added 7 drops of aqueous NaOH (2N) solution, and the mixture was stirred at room temperature for 30min, and T L C showed disappearance of starting material point, the reaction mixture was concentrated under reduced pressure, the residue was diluted with 2M L saturated saline, then adjusted to pH 6 with 2N HCl, and then extracted with ethyl acetate 3 times (3ml × 3), the combined ethyl acetate layers were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness at room temperature under reduced pressure to give 30mg (99%) of the title compound as blue crystals, Mp 155 ℃. EI-MS (M/z)307[ M/z ] M.M.M.M.M.M.]+。
EXAMPLE 7 preparation of 1, 3-dioxo-2- [ 4' -oxoacetyl-L ys (Boc) -OMe phenyl ] -4,4,5, 5-tetramethylimidazoline
A solution of 307mg (1mmol) of 1, 3-dioxo-2- (4' -oxoacetic acid-phenyl) -4,4,5, 5-tetramethylimidazoline in 30ml of anhydrous THF was stirred under ice bath, 250mg (1.2mmol) of DCC and 135mg (1mmol) of HOBt were added to the solution, and stirred under ice bath for 10min, a solution prepared from 300mg (1mmol) of HCl L ys (Boc) -OMe,122mg (1mmol) of N-methylmorpholine and 6M L anhydrous THF was added to the solution, the resulting reaction mixture was reacted at room temperature for 24 hours, T L C (ethyl acetate: petroleum ether ═ 2:1) showed disappearance of HCl L ys (Boc) -OMe, the reaction mixture was concentrated to dryness under reduced pressure, the residue was dissolved with ethyl acetate, the insoluble matter was filtered off, the filtrate was washed successively with a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution, the separated ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure at 37 ℃ and the residue was purified as a blue solid (title compound) (ESIm: 433).
EXAMPLE 8 preparation of 1, 3-dioxy-2- [ (4' -oxoacetyl-L ys-OMe) phenyl ] -4,4,5, 5-tetramethylimidazoline
625mg (1mmol) of 1, 3-dioxo-2- [ 4' -oxoacetyl-L ys (Boc) -OMe phenyl ] -4,4,5, 5-tetramethylimidazoline was dissolved in 15m L of anhydrous hydrogen chloride-ethyl acetate solution (4N), stirred at room temperature for 3 hours, T L C (CHCl3: MeOH,20:1) showed disappearance of the starting point, the reaction mixture was treated in a conventional manner, and the residue was crystallized from anhydrous ether to give the title compound.
EXAMPLE 9 preparation of Boc-Ala-L ys (Z) -OBzl
473mg (2.5mmol) of Boc-Ala are dissolved in 10ml of anhydrous THF, a solution of 338mg (2.5mmol) of HOBt,619mg (3mmol) of DCC and 10m L of anhydrous THF is added thereto under ice-bath stirring of the reaction mixture for 20 minutes under ice-bath, a solution of 936mg (2.3mmol) of HCl L ys (Z) -OBzl,232mg (2.3mmol) of N-methylmorpholine and 6m L of anhydrous THF is added to the solution, and the resulting reaction mixture is reacted at room temperature for 24 hours, T L C (CHCl)3MeOH ═ 30:1) showed disappearance of HCl L ys (z) -OBzl the reaction mixture was worked up as usual to give 1.204g (97%) of the title compound as a colorless solid.
Example 10 preparation of HCl. Ala-L ys (z) -OBzl:
1.354g (2.5mmol) of Boc-Ala-L ys (Z) -OBzl was dissolved in about 10ml of anhydrous hydrogen chloride-ethyl acetate solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,30:1) showed the disappearance of the starting point. The reaction mixture was concentrated under reduced pressure at room temperature, the residue was dissolved in ethyl acetate and concentrated at room temperature, and this was repeated several times until the free hydrogen chloride was removed (these operations are hereinafter summarized)Referred to as conventional processing). The residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 11 preparation of Boc-Pro-Ala-L ys (Z) -OBzl
538mg (2.5mmol) of Boc-Pro is dissolved in the appropriate amount of anhydrous THF, 338mg (2.5mmol) of HOBt,619mg (3mmol) of DCC in anhydrous THF is added under ice bath, and reaction is carried out for 20 minutes, to the solution is added a solution prepared from 1.099g (2.3mmol) of HCl Ala-L ys (Z) -OBzl,232mg (2.3mmol) of N-methylmorpholine and 10m L of anhydrous THF, and reaction is carried out at room temperature for 24 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up conventionally to give 2.847g (98%) of the title compound.
EXAMPLE 12 preparation of HCl Pro-Ala-L ys (Z) -OBzl
1.596g (2.5mmol) Boc-Pro-Ala-L ys (Z) -OBzl was dissolved in 15m L anhydrous hydrogen chloride-ethyl acetate solution (4N), and stirred at room temperature for 3 hours to obtain T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 13 preparation of Boc-Arg (NO)2)-Pro-Ala-Lys(Z)-OBzl
798mg (2.5mmol) of Boc-Arg (NO) was added under ice-bath2) 338mg (2.5mmol) of HOBt,619mg (3mmol) of DCC and 10m L of anhydrous THF were stirred for 20 minutes, to the solution was added a solution prepared from 1.322g (2.3mmol) of HCl Pro-Ala-L ys (Z) -OBzl,232mg (2.3mmol) of N-methylmorpholine and 5m L of anhydrous THF, and reacted at room temperature for 24 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. Work-up was carried out as usual to give 1.642g (85%) of the title compound.
EXAMPLE 14 preparation of HCl Arg (NO)2)-Pro-Ala-Lys(Z)-OBzl
2.099g (2.5mmol) of Boc-Arg (NO)2) -Pro-Ala-L ys (Z) -OBzl dissolved in 20m L anhydrous hydrogen chloride-ethyl acetate solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 15 preparation of Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)-OBzl
A solution of 438mg (2.5mmol) Boc-Gly,338mg (2.5mmol) HOBt,619mg (3mmol) DCC and 10m L anhydrous THF was stirred under ice-bath for 20 min, to which solution 1.785g (2.3mmol) HCl. Arg (NO: H) was added2) -Pro-Ala-L ys (Z) -OBzl,232mg (2.3mmol) of N-methylmorpholine in solution in 5m L anhydrous THF, and reacting for 24 hours, 1.857g (90%) of the title compound are obtained.
EXAMPLE 16 preparation of Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)
907mg (1mmol) of Boc-Gly-Arg (NO)2) -Pro-Ala-L ys (Z) -OBzl in 3M L methanol, NaOH aqueous solution (2N) added under ice bath, stirred at room temperature for 30min, maintaining pH 12, stirred at ice bath for 10min, T L C showing disappearance of the starting material point, pH7 adjusted with 2N HCl, reaction concentrated under reduced pressure, residue diluted with 2M L saturated saline, pH2 adjusted with 2N HCl, extracted 3 times with ethyl acetate (5M L× 3), combined ethyl acetate layers dried over anhydrous sodium sulfate, concentrated under reduced pressure at room temperature to 785mg (82%) of the title compound as a colorless solid]–。
EXAMPLE 17 preparation of Boc-Asp (OBzl) -Val-OBzl
A solution of 808mg (2.5mmol) Boc-Asp (OBzl),338mg (2.5mmol) HOBt,619mg (3mmol) DCC and 10ml anhydrous THF was stirred under ice-bath for 20 minutes, to this solution was added a solution of 558mg (2.3mmol) HCl Val-OBzl,232mg (2.3mmol) N-methylmorpholine and 5ml anhydrous THF, and reacted at room temperature for 24 hours T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up conventionally to give 1.129g (96%) of the title compound,as a colorless oily liquid. ESI-MS (M/z)512[ M + H [ ]]+。
EXAMPLE 18 preparation of HCl Asp (OBzl) -Val-OBzl
1.278g (2.5mmol) of Boc-Asp (OBzl) -Val-OBzl was dissolved in 15ml of anhydrous HCl-EtOAc solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 19 preparation of Boc-Gly-Asp (OBzl) -Val-OBzl
A solution of 438mg (2.5mmol) Boc-Gly,338mg (2.5mmol) HOBt,619mg (3mmol) DCC and 10m L anhydrous THF was stirred under ice-bath for 20 minutes, to the solution was added a solution prepared from 1.03g (2.3mmol) HCl. Asp (OBzl) -Val-Obzl,232mg (2.3mmol) N-methylmorpholine and 5m L anhydrous THF, and reacted at room temperature for 24 hours T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up as usual to give 1.242g (95%) of the title compound as a colourless solid.
EXAMPLE 20 preparation of HCl.Gly-Asp (OBzl) -Val-OBzl
1.421g (2.5mmol) of Boc-Gly-Asp (OBzl) -Val-OBzl was dissolved in 15m L anhydrous HCl-EtOAc solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 21 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
798mg (2.5mmol) of Boc-Arg (NO) was added under ice-bath2) A solution of 338mg (2.5mmol) of HOBt,619mg (3mmol) of DCC and 10m L of anhydrous THF was stirred for 20 minutes, a solution of 1.162g (2.3mmol) of HCl.Gly-Asp (OBzl) -Val-Obzl,232mg (2.3mmol) of N-methylmorpholine and 5m L of anhydrous THF was added to the solution and reacted at room temperature for 24 hours, T L C (CHCl)3:MeOH,20:1)Showing the disappearance of the material dots. The reaction mixture was worked up as usual to give 1.523g (86%) of the title compound as a colourless solid.
EXAMPLE 22 preparation of HCl Arg (NO)2)-Gly-Asp(OBzl)-Val-OBzl
1.925g (2.5mmol) of Boc-Arg (NO)2) -Gly-Asp (OBzl) -Val-OBzl was dissolved in 20m L anhydrous HCl-EtOAc solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up by conventional methods and the residue was crystallized from anhydrous ether to give the title compound.
EXAMPLE 23 preparation of Boc-Asp (OBzl) -Phe-OBzl
A solution of 808mg (2.5mmol) Boc-Asp (OBzl),338mg (2.5mmol) HOBt,619mg (3mmol) DCC and 10m L anhydrous THF was stirred under ice-bath for 20 min, to this solution was added a solution of 668mg (2.3mmol) HCl Phe-OBzl,232mg (2.3mmol) N-methylmorpholine and 5m L anhydrous THF, and reacted at room temperature for 24h T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. Work-up was carried out as usual to give the title compound 1.222g (95%) as a colourless solid.
EXAMPLE 24 preparation of HCl. Asp (OBzl) -Phe-OBzl
1.398g (2.5mmol) Boc-Asp (OBzl) -Phe-OBzl was dissolved in 15m L anhydrous hydrogen chloride-ethyl acetate solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 25 preparation of Boc-Gly-Asp (OBzl) -Phe-OBzl
A solution of 438mg (2.5mmol) Boc-Gly,338mg (2.5mmol) HOBt,619mg (3mmol) DCC in dry THF was stirred under ice-bath for 20 min. To this solution was added 1.141g (2.3mmol) of HCl. Asp (OBzl) -Phe-OBzl232mg (2.3mmol) of N-methylmorpholine in 5m L anhydrous THF are reacted at room temperature for 24h, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up as usual to give 1.29g (91%) of the title compound as a colourless solid.
EXAMPLE 26 preparation of HCl.Gly-Asp (OBzl) -Phe-OBzl
1.541g (2.5mmol) Boc-Gly-Asp (OBzl) -Phe-OBzl was dissolved in 15m L anhydrous HCl-EtOAc solution (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was treated by a conventional method, and the residue was crystallized from anhydrous ether to give the title compound, which was used directly in the next reaction.
EXAMPLE 27 preparation of Boc-Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl
798mg (2.5mmol) of Boc-Arg (NO) was added under ice-bath2) A solution of 338mg (2.5mmol) of HOBt,619mg (3mmol) of DCC and 10m L of anhydrous THF was stirred for 20 minutes, to the solution was added a solution prepared from 1.272g (2.3mmol) of HCl.Gly-Asp (OBzl) -Phe-OBzl,232mg (2.3mmol) of N-methylmorpholine and 5m L of anhydrous THF, and reacted at room temperature for 24 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up conventionally to give 1.637g (87%) of the title compound as a colourless solid.
EXAMPLE 28 preparation of HCl Arg (NO)2)-Gly-Asp(OBzl)-Phe-OBzl
2.045g (2.5mmol) of Boc-Arg (NO)2) -Gly-Asp (OBzl) -Phe-OBzl was dissolved in 15m L anhydrous HCl-EtOAc (4N) and stirred at room temperature for 3 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up by conventional methods and the residue was crystallized from anhydrous ether to give the title compound.
EXAMPLE 29 preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Z)]-L ys-OMe } phenyl-4, 4,5, 5-tetramethylimidazoline
817mg (1mmol) Boc-Gly-Arg (NO) under ice bath2) A solution of-Pro-Ala-L ys (Z),135mg (1mmol) of HOBt,250mg (1mmol) of DCC and 10m L of anhydrous THF was stirred for 20 minutes, 480mg (1mmol) of 1, 3-dioxo-2- [ (4' -oxyacetyl-L ys-OMe) phenyl]-4,4,5, 5-tetramethylimidazoline and a solution of 100mg (1mmol) of N-methylmorpholine in 5m L anhydrous THF at room temperature for 24h T L C (CHCl)3MeOH,40:1) showed the disappearance of the starting point. The reaction mixture was worked up conventionally to give 680mg (52%) of the title compound as a blue solid.
IR(KBr)3319,2935,1658,1531,1448,1363,1254,1168,1053,835,749,540cm-1.
EXAMPLE 30 preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-L ys } phenyl-4, 4,5, 5-tetramethylimidazoline
1260mg (1mmol) of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO) was added under ice bath2)-Pro-Ala-Lys(Z)]-L ys-OMe } phenyl-4, 4,5, 5-tetramethylimidazoline was dissolved in 3M L methanol, NaOH aqueous solution (2N) was added, stirring was performed at room temperature for 30min, pH 12 was maintained, stirring was performed in ice bath for 10min, T L C showed disappearance of starting material point, pH7 was adjusted with 2N HCl, the reaction solution was concentrated under reduced pressure, the residue was diluted with 2M L saturated saline, pH2 was adjusted with 2N HCl, 3 times (5M L× 3) extraction was performed with ethyl acetate, the combined ethyl acetate layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at room temperature to obtain 945mg (82%) of the title compound as a blue solid, EI-MS (M/z)1223[ M-H ] 1223]–。
EXAMPLE 31 preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-Lys-Arg-(NO2) -Gly-Asp (OBzl) -Val-OBzl } phenyl-4, 4,5, 5-tetramethylimidazoline
611mg (0.5mmol) of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO) was added under ice bath2)-Pro-Ala-Lys(Z)]-LyS } phenyl-4, 4,5, 5-tetramethylimidazoline, 69mg (0.5mmol) of HOBt,126mg (0.6mmol) of DCC and 20m L of anhydrous THF, was stirred for 20 minutes, to which was added a solution prepared from 421mg (0.5mmol) of HCl. Arg (NO)2) A solution of-Gly-Asp (OBzl) -Val-OBzl,50mg (0.5mmol) of N-methylmorpholine and 5m L of anhydrous THF, was reacted at room temperature for 24 hours, T L C (CHCl)3MeOH,20:1) showed the disappearance of the feed point. The reaction mixture was worked up conventionally to give 392mg (35%) of the title compound as a blue solid.
IR(KBr)3311,3068,2937,1661,1531,1451,1395,1254,1163,839,743,697,596cm-1。
EXAMPLE 32 preparation of 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline (5)
396mg (0.2mmol) of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO) under ice bath2)-Pro-Ala-Lys(Z)]-Lys-Arg-(NO2) dissolving-Gly-Asp (OBzl) -Val-OBzl } phenyl-4, 4,5, 5-tetramethyl imidazoline in 4m L trifluoroacetic acid, adding 1m L trifluoromethanesulfonic acid into the obtained solution, stirring and reacting for 30 minutes under ice bath condition, transferring the reaction solution into 100m L anhydrous ether after 30 minutes, precipitating a large amount of precipitate, standing, discarding the supernatant after the precipitate is completely precipitated, adding 50m L anhydrous ether into the residue, discarding the supernatant after the precipitate is completely precipitated, concentrating under reduced pressure to dryness, dissolving the residue in 5m L distilled water, adding 150mg NaNO2The reaction solution turned gradually blue to colorless (30min), the pH of the solution was adjusted to 7, desalted using Sephadex G10 and lyophilized to give 130mg (36%) of the title compound as a colorless powder. ESI-MS (M/e) 1357.7497[ M + H ]]+.1H NMR(800MHz,D2O):/ppm＝8.687(d,J＝6.4Hz,1H),8.552(d,J＝6.4Hz,1H),8.493(t,J＝6.4Hz,1H),8.373(d,J＝5.6Hz,1H),8.28(d,J＝7.2Hz,1H),8.170(d,J＝7.2Hz,1H),7.998(m,2H),7.115(m,2H),4.111(m,1H),3.943(m,1H),3.900(tm,1H),3.775(s,2H),3.560(m,1H),3.113(m,7H),2.908(m,2H),2.775(dd,J＝8Hz,J＝5.6Hz,2H),2.223(m,1H),2.195(m,2H),1.977(m,3H),1.800(m,6H),1.710(m,3H),1.612(m,10H),1.450(m,3H),1.318(m,10H)。
Experimental example 1 evaluation of antithrombotic Activity of Compound 5
Male SD rats (200. + -.20 g) were randomized into groups of 10 rats each, kept for 1 day and stopped overnight, after intragastric administration of Compound 7 in physiological saline (1. mu. mol/kg dose) or aspirin in physiological saline (167. mu. mol/kg dose) or physiological saline (10m L/kg dose) for 30min, the rats were anesthetized with 20% Uigosaccharide in physiological saline, after which the rats were surgically isolated in the right and left carotid arteries, accurately weighed silk threads were placed in bypass cannulas, one end of the tube was inserted in the left vein and the other end was inserted in the right artery and anticoagulated with 0.2m L sodium so that blood flow from the right artery through the bypass cannulas into the left vein was measured 15min later, the weight of the silk threads before and after blood circulation was calculated and the resulting thrombus weight was expressed as a mean SD. + -. mg and represents antithrombotic activity and was used as a t-test data set forth in Table 1.
TABLE 11. mu. mol/kg antithrombotic Activity of Compound 5
n is 10; a) p <0.01 compared to saline.
Experimental example 2 evaluation of thrombolytic Activity of Compound 5
SD rat (male, 200 + -20 g) is anesthetized by intraperitoneal injection of Uratan physiological saline solution at a dose of 1200mg/kg, after anesthesia, the rat is fixed in a supine position, the right common carotid artery is separated, an artery clamp is clamped at the proximal end, a surgical line is respectively penetrated at the proximal end and the distal end, the surgical line at the distal end is ligated, a cannula at the distal end is inserted, the artery clamp is loosened, about 1m L arterial blood is taken out, the rat arterial blood is placed in a 1m L centrifuge tube, a vertically fixed rubber tube (15 mm long, 2.5mm inner diameter, 5.0mm outer diameter, rubber plug is used for sealing the tube bottom, para membrane is tightly sealed) is injected with 0.1m L rat arterial blood, then a fixing bolt of a thrombus made of stainless steel material is rapidly inserted into the tube (the thrombus fixing screw is wound by a stainless steel wire with the diameter of 0.2mm, the screw part is 10mm long and contains 15 spiral rings, the diameter of the spiral ring is connected with the screw, the length is about 7.0mm, the thrombus is solidified after 45min, the heart thrombus is accurately taken out from the glass spiral tube, and the small thrombus is accurately wrapped by the glass tube.
The bypass cannula consists of three parts, wherein the middle section is a polyethylene rubber tube with the length of 60.0mm and the inner diameter of 3.5 mm; both ends are 100.0mm long, and internal diameter 1.0mm, the same polyethylene pipe of external diameter 2.0mm, and this pipe one end is drawn into the sharp pipe, and is about 10.0mm long (being used for inserting rat carotid artery and vein), and the external diameter is 1.0mm, and the outside cover section of its other end is long for 7.0mm, and the external diameter is 3.5 mm's polyethylene pipe (in being used for inserting the polyethylene rubber tube in middle section), and the inner wall of 3 sections pipes all needs silanization (1% silicon oil ether solution). The thrombus-wrapped thrombus fixing spiral is placed in the middle section polyethylene rubber tube, and the other two ends of the rubber tube are respectively sleeved with the thickened ends of the two polyethylenes, so that blood leakage can be avoided in the circulating process. The tube was filled with heparin normal saline solution (50IU/kg) through the tip end with a syringe to remove air bubbles for use.
The left external jugular vein of separation rat, proximal end and distal end penetrate the operation line respectively, and the blood vessel of ligature distal end cuts a osculum on the left external jugular vein that exposes, inserts the bypass pipeline taper pipe that has been prepared above-mentioned into left external jugular vein opening part by the osculum, keeps away from bypass pipe middle section (contains the thrombus fixed spiral of accurate weighing) internal thrombus fixed spiral simultaneously. An accurate amount of a physiological saline solution (50IU/kg) of heparin sodium was injected through the tip tube at the other end with a syringe, at which time the syringe was not removed from the polyethylene tube, and the flexible tube between the syringe and the polyethylene tube was clamped with an artery clamp. Stopping bleeding by an artery clamp at the proximal end of the right common carotid artery, ligating the distal end, cutting a small opening of the right common carotid artery at a position short of the artery clamp, pulling out the injector from the tip of the polyethylene tube, and inserting the tip of the polyethylene tube into the proximal end of the oblique opening of the artery. Both ends of the bypass pipeline are used for fixing the artery and the vein by using a No. 4 surgical suture.
Physiological saline (3m L/kg) or a physiological saline solution of urokinase (dosage is 20000IU/kg) or a physiological saline solution of compound 5 (dosage is 1 mu mol/kg) is passed through the middle section of the bypass tube (containing thrombus fixing spiral accurately weighed), the proximal venous end far away from the thrombus fixing spiral is inserted, the arterial clamp is loosened, blood flow flows from the artery to the vein through the bypass tube, the solution in the syringe is slowly injected into the blood and passes through the blood circulation to act on the thrombus of the spiral in the order of vein-heart-artery, after 1h of the blood circulation, the spiral fixing the thrombus is taken out from the bypass tube, accurately weighed, the weight difference of the thrombus before and after spiral blood circulation of the fixing the thrombus in the bypass tube of each rat is calculated, expressed by the mean value + -SD mg and represents the thrombolytic activity, a t test is performed, the data are listed in Table 2, the results show that 1 mu mol/kg of compound 5 can effectively dissolve the formed thrombus, and the unexpected technical effects of the present invention are obtained.
TABLE 21 μmol/kg thrombolytic Activity of Compound 5
n is 10; a) p <0.01 in comparison with normal saline and p >0.05 in comparison with urokinase.
Experimental example 3 evaluation of therapeutic Effect of Compound 5 on ischemic apoplexy rats
The rat brain function loss evaluation method comprises the steps of vertically opening a long incision with the length of about 2cm at the median neck of a male SD rat (the weight is 300 +/-20 g), separating a right common carotid artery, an external carotid artery and an internal carotid artery along the intramuscular side of a sternocleidomastoid process, clamping the opening of the internal carotid artery and the proximal end of the common carotid artery by using a noninvasive artery clamp, ligating the distal end of the external carotid artery, cutting a small opening of the external carotid artery by 1, loosening the artery clamp at the proximal end of the common carotid artery, taking 10 mu L blood, clamping the proximal end of the common carotid artery by using the noninvasive artery clamp, placing the obtained 10 mu L blood in a 1m L EP tube at normal temperature for 30 minutes to enable blood coagulation, transferring the blood to a-20 ℃ refrigerator for 1 hour to enable blood clot to be firm, carrying out the abdominal cavity by using 10% chloral hydrate for anesthesia, taking the blood out by using a dose of 400 mg/kg., adding 1m L physiological saline, smashing the blood into a small blood suspension with the uniform size, preparing a fine thrombus, transferring the small thrombus into a L in the small side of the small artery, transferring the small artery to the 1m syringe, transferring the small artery to the small artery, transferring the small artery, and transferring the small artery to the small artery, and transferring the small artery to the small artery, and transferring the small artery to the rat brain to the rat, and transferring the rat, and transferring the rat, and the rat, wherein the rat brain, the rat brain nerve to the rat brain, the rat brain, the.
TABLE 3 Effect of Compound 5 on neurobiological scores in rats with 24h cerebral ischemia on 6 days of continuous treatment
n is 7, the target compound dose is 1 mu mol/kg, and the rat tail is administrated by intravenous injection.
Claims (5)
1. 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline of the formula,
2. a process for the preparation of 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline of claim 1, comprising the steps of:
(1) preparing 2, 3-dimethyl-2, 3-dinitrobutane;
(2) preparing 2, 3-dimethyl-2, 3-dihydroxyaminobutane;
(3) preparing 1, 3-dihydroxy-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline;
(4) preparing 1, 3-dioxo-2- (4' -hydroxyphenyl) -4,4,5, 5-tetramethylimidazoline;
(5) preparation of Arg (NO) according to Standard procedure2)-Gly-Asp(OBzl)-Val-OBz；
(6) Preparation of Gly-Arg (NO) according to Standard procedure2)-Pro-Als-Lys(Z)-OBz；
(7) Preparing 1, 3-dioxo-2- (4' -oxyacetic acid ethyl ester-phenyl) -4,4,5, 5-tetramethylimidazoline;
(8) preparing 1, 3-dioxo-2- (4' -oxyacetic acid-phenyl) -4,4,5, 5-tetramethylimidazoline;
(9) preparing 1, 3-dioxo-2- [ 4' -oxoacetyl-L ys (Boc) -OMe-phenyl ] -4,4,5, 5-tetramethylimidazoline;
(10) preparing 1, 3-dioxy-2- [ (4' -oxyacetyl-L ys-OMe) phenyl ] -4,4,5, 5-tetramethyl imidazoline;
(11) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-L ys-OMe } phenyl-4, 4,5, 5-tetramethylimidazoline;
(12) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-L ys } phenyl-4, 4,5, 5-tetramethylimidazoline;
(13) preparation of 1, 3-dioxo-2- { 4' -oxoacetyl- [ Boc-Gly-Arg (NO)2)-Pro-Ala-Lys(Z)]-Lys-Arg-(NO2) -Gly-asp (OBzl) -Val-OBzl } phenyl-4, 4,5, 5-tetramethylimidazoline;
(14) preparation of 1-hydroxy-2- [ 4' -oxoacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline.
3. Use of 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline according to claim 1 for the preparation of antithrombotic agents.
4. Use of 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline according to claim 1 for the preparation of a thrombolytic drug.
5. Use of 1-hydroxy-2- [ 4' -oxyacetyl-L ys (Gly-Arg-Pro-Ala-L ys) -Arg-Gly-Asp-Val ] phenyl-4, 4,5, 5-tetramethylimidazoline according to claim 1 for the preparation of a medicament for the treatment of ischemic stroke.
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| CN102875644A (en) * | 2012-09-05 | 2013-01-16 | 永光制药有限公司 | GRPAK/tetrahydroglyoxaline/RGD ternary conjugate as well as preparation method and application thereof |
| CN102898507A (en) * | 2012-09-05 | 2013-01-30 | 永光制药有限公司 | Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof |
| EP2894160A1 (en) * | 2012-09-05 | 2015-07-15 | Shanghai Lumosa Therapeutics Co., Ltd. | Novel compound with effects of thrombolysis, free radical scavenging and thrombus-targeting as well as preparation method and use thereof |
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| CN102807605A (en) * | 2011-06-03 | 2012-12-05 | 首都医科大学 | Nα-(1, 3-dioxo-4, 4,5, 5-tetramethylimidazolin-2-phenyl-4'-oxyacetyl)-Nω-fatty acyl-Lys-Arg-Gly-Asp-Phe, its preparation method and application |
| CN102875644A (en) * | 2012-09-05 | 2013-01-16 | 永光制药有限公司 | GRPAK/tetrahydroglyoxaline/RGD ternary conjugate as well as preparation method and application thereof |
| CN102898507A (en) * | 2012-09-05 | 2013-01-30 | 永光制药有限公司 | Thrombolysis oligopeptide-imidazolidine binary conjugate, preparation method and uses thereof |
| EP2894160A1 (en) * | 2012-09-05 | 2015-07-15 | Shanghai Lumosa Therapeutics Co., Ltd. | Novel compound with effects of thrombolysis, free radical scavenging and thrombus-targeting as well as preparation method and use thereof |
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