CN102807604B - N alpha-(1,3-dioxy-4,4,5,5-tetramethyl imidazoline-2-phenyl-4'-oxo acetyl)-N omega-fatty acyl-Lys-Arg-Gly-Asp-Val and preparation method and application thereof - Google Patents
N alpha-(1,3-dioxy-4,4,5,5-tetramethyl imidazoline-2-phenyl-4'-oxo acetyl)-N omega-fatty acyl-Lys-Arg-Gly-Asp-Val and preparation method and application thereof Download PDFInfo
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
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Abstract
The invention provides six targeting anti-thrombosis compounds having a general formula I shown in the specification (in the formula, n=6, 8, 10, 12, 14 or 16), and further provides preparation method of the compounds. By studying the anti-thrombocyte aggregation activity, anti-thrombosis activity, NO free radical purging activity and self-assembly performance of the compounds, the six novel compounds not only have excellent anti-thrombosis action, but also have the activity of purging NO free radicals, namely, the compounds have the advantages of having NO purging action while resisting thrombosis, therefore, the six compounds having the general formula I have good clinical application prospects.
Description
Technical field
The present invention relates to a kind of compound of synthetic, particularly 6 kinds of compounds of general formula I representative, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg-Gly-Asp-Val (in formula, n is 6,8,10,12,14 or 16); also relate to their preparation method; further relate to their platelet aggregation inhibitory activities, antithrombotic acitivity, removing NO free radical activity and independently fill effect and the application of performance, the invention belongs to biomedicine field.
Background technology
Thrombotic diseases is one of major disease of harm people ' s health, can have targeting and anti thrombotic action with the platelet surface GPIIb/IIIa receptors bind of activation containing the compound of RGD sequence.NO is important information and effector molecule, too much NO can and superoxide anion react, generation has the Peroxynitrite of reactive behavior, and then the disease such as trigger cell toxicity, nerve injury and ischemical reperfusion injury.Nitronyl nitroxide is the imidazoles nitroxyl free radical of first stability, can remove rapidly the nitrogen protoxide in body, for molten/anti-bolt provides up-and-coming molecule of pharmaceutical building block.According to general understanding, containing the amphipathic molecule of polypeptide,, can there is self-assembly by noncovalent intermolecular interactions in the polypeptide that for example aliphatic alcohol chain is modified under suitable condition, forms nanostructure.Can improve conveying in vivo of polypeptide, delay degradation rate in vivo of polypeptide and improve the activity in vivo of polypeptide by nanostructure.According to these understanding, contriver has proposed the present invention.
Summary of the invention
First technical problem to be solved by this invention is to provide 6 kinds of compounds (4a-f represents with compound) with general formula I:
General formula I
In formula, n=6,8,10,12,14 or 16.
N=6 in compound 4a, n=8 in compound 4b, n=10 in compound 4c, n=12 in compound 4d, n=14 in compound 4e, n=16 in compound 4f.
Second technical problem to be solved by this invention is to provide the method for 6 kinds of compounds preparing general formula I representative, and the method comprises:
1) at bromine (Br
2) to have lower 2-nitropropane condensation in the 6N NaOH aqueous solution be 3-dimethyl-2,3-dinitrobutane;
2) at ammonium chloride (NH
4cl) and zinc powder (Zn) exist lower DMNB in moisture ethanolic soln, to be reduced to 2,3-dimethyl-2,3-dihydroxy amido butane;
3) in methyl alcohol 2,3-dimethyl-2,3-dihydroxy amido butane and paranitrobenzaldehyde condensation are 1,3-dihydroxyl-2-(4 '-hydroxy phenyl) phenyl-4,4,5,5-tetramethyl-imidazolidine;
4) at plumbic oxide (PbO
2) under existence, 3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine is oxidized to 2-(4 '-hydroxy phenyl)-4,4,5 in methyl alcohol, 5-tetramethyl--1,3-bis-Sinerols;
5) 2-(4 '-hydroxy phenyl)-4,4 under sodium hydride (NaH) exists, 5,5-tetramethyl--1,3-bis-Sinerols are 1,3-dioxy-4 with bromoethyl acetate condensation in anhydrous THF, 4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester;
6) in methyl alcohol 1,3-dioxy-4, the saponification of 4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester is 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid;
7) under DCC and HOBt exist 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid in anhydrous THF with HClH-Lys (Boc)-OCH
3condensation is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3;
8) N in the ethyl acetate of containing hydrogen chloride
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3remove Boc and generate N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3;
9) NN-Lys-OCH under DCC and HOBt existence
3in anhydrous THF, be N with saturated fatty acid condensation
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-OCH
3;
10) in methyl alcohol by N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-OCH
3saponification is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys;
11) under DCC and HOBt existence, Boc-Asp (OBzl) is Boc-Asp (OBzl)-Val-OBzl with Val-OBzl condensation in anhydrous THF;
12) in the ethyl acetate of containing hydrogen chloride, Boc-Asp (OBzl)-Val-OBzl removes Boc and generates Asp (OBzl)-Val-OBzl;
13) Boc-Arg (NO under DCC and HOBt existence
2)-Gly is Boc-Arg (NO with Asp (OBzl)-Val-OBzl condensation in anhydrous THF
2)-Gly-Asp (OBzl)-Val-OBzl;
14) Boc-Arg (NO in the ethyl acetate of containing hydrogen chloride
2)-Gly-Asp (OBzl)-Val-OBzl removes Boc and generates Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
15) N under DCC and HOBt existence
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys is Arg (NO in anhydrous THF
2)-Gly-Asp (OBzl)-Val-OBzl condensation is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
16) containing N in the trifluoroacetic acid solution of trifluoromethanesulfonic acid
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl removes OBzl and NO
2generate N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg-Gly-Asp-Val.
The 3rd technical problem to be solved by this invention is by studying them at platelet aggregation inhibitory activity, antithrombotic acitivity, removing NO free radical activity and independently filling the effect in performance, show that these 6 kinds of novel cpds not only have outstanding anti thrombotic action, and there is the activity of removing NO free radical.
The 4th technical problem to be solved by this invention is to provide compound of the present invention in the purposes of preparing in medicine: one of use of a compound of the present invention is the application of preparing in antithrombotic reagent; Two of use of a compound of the present invention is to remove the application in NO free radical medicine in preparation; Three of use of a compound of the present invention is in the application of preparing in antithrombotic and removing NO free radical dual-use function medicine.
Compound in the present invention is at the antithrombotic Action advantage that has removing NO simultaneously, and therefore 6 kinds of compounds of general formula I representative have good application prospect in clinical.
Accompanying drawing explanation
Fig. 1 is the synthetic route of the compounds of this invention.;
I) DCC, HOBt, NMM; Ii) the NaOH aqueous solution; Iii) hydrogenchloride/ethyl acetate solution (4N); Iv) CF
3cOOH, CF
3sO
3h; V) Br
2, the NaOH aqueous solution; Vi) Zn, NH
4cl; Vii) CH
3oH; Viii) PbO
2; Ix) NaH; N=6 in compound 1a-4a, n=8 in compound 1b-4b, n=10 in compound 1c-4c, n=12 in compound 1d-4d, n=14 in compound 1e-4e, n=16 in compound 1f-4f;
Fig. 2 is the representative transmission electron microscope photo of the nanometer ball that forms in the aqueous solution of compound 4c.
Breviary term
Embodiment
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative completely, and they are only used for the present invention to be specifically described, and not should be understood to limitation of the present invention.
69g (0.78mol) 2-nitropropane is added in the 130ml 6N NaOH aqueous solution, under ice-water bath agitation condition, drip 20ml (0.38mol) Br
2, in 1hr, drip, then add 240ml ethanol,, there is sheet insolubles in 90 ℃ of stirring and refluxing 3hrs, takes advantage of heat to pour in 800ml frozen water reaction solution, suction filtration, obtains white plates crystallization 55g (81%), is the target compound of the present embodiment.ESI-MS(m/e)：177.1[M+H]
+.
Embodiment 2 prepares 2,3-dimethyl-2,3-dihydroxy amido butane
By 7g (40mmol) DMNB and 4g NH
4cl is suspended in the aqueous ethanolic solution of 80ml 50%, under ice bath, stirs, and adds 16g zinc powder in 3hr.After zinc powder adds, remove ice bath, continue stirring at room temperature reaction 3hr, then by reaction solution suction filtration.50% aqueous ethanolic solution repetitive scrubbing for filter cake, 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, and after extracting 6hrs, decompression is steamed extracting solution to a small amount of, separates out white crystals 2.60g (44%) after adding sherwood oil, is the target compound of the present embodiment.ESI-MS(m/e)：149.1[M+H]
+.
Embodiment 3 prepares 1,3-dihydroxyl-2-(4 '-hydroxy phenyl) phenyl-4,4,5,5-tetramethyl-imidazolidine
By 1.47g (10mmol) paranitrobenzaldehyde and 1.48g (10mmol) 2,3-dimethyl-2,3-dihydroxy amido butane is dissolved in 10ml methyl alcohol, after stirring at room temperature 16hrs, TLC shows that raw material point disappears, and leaches white solid 2g (85%) and is directly used in next step reaction.Sample obtains the target compound of the present embodiment after TLC purifying.ESI-MS(m/e)：253.1[M+H]
+.
Embodiment 4 prepares 2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--1,3-bis-Sinerols
By 475mg (2mmol) 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine is dissolved in 50ml methyl alcohol, adds 2.5g PbO
2, after stirring at room temperature 30min, TLC shows that raw material point disappears.Suction filtration is removed solid, evaporated under reduced pressure under filtrate room temperature, and column chromatography for separation (chloroform is eluent), obtains black-and-blue crystal 370mg (80%), is the target compound of the present embodiment.ESI-MS(m/e)：250.1[M+H]
+。
Embodiment 5 prepares 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester
400mg (1.6mmol) 2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl--1,3-bis-Sinerols are dissolved in 30ml dry tetrahydrofuran, add NaH powder under ice bath, 2ml bromoethyl acetate, after 60 ℃ of stirring and refluxing 1h, TLC shows that raw material point disappears.Suction filtration is removed solid, evaporated under reduced pressure under filtrate room temperature, and column chromatography for separation (chloroform is eluent), obtains blue colored crystal 470mg (87%), is the target compound of the present embodiment.ESI-MS(m/e)：336.2[M+H]
+。
Embodiment 6 prepares 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid
By 400mg (1mmol) 1, 3-dioxy-4, 4, 5, 5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester is dissolved in 2ml methyl alcohol, in ice bath downhill reaction liquid, drip 2N aqueous sodium hydroxide solution, detect pH value to 12, ice bath stirs after 0.5h, TLC shows that raw material point disappears, to drip in reaction solution saturated aqueous potassium hydrogen sulfate to PH be 7, under reaction solution room temperature, remove most of solvent under reduced pressure, continue to adjust PH to 2 with saturated aqueous potassium hydrogen sulfate, ethyl acetate extraction, combined ethyl acetate layer, after anhydrous sodium sulfate drying, TLC detects as single point, filter, evaporated under reduced pressure, obtain blue crystalline thing 320mg (87%), be the target compound of the present embodiment.ESI-MS(m/e)：308.1[M+H]
+。
Embodiment 7 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3
By 550mg (1.79mmol) 1,3-dioxy base-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-ethoxyacetic acid is dissolved in 20ml dry THF solution, under ice-water bath, add after 242mg (1.79mmol) N-hydroxy benzo triazole (HOBt), in above-mentioned mixed solution, add 403mg (1.49mmol) dicyclohexyl carbonyl diimine (DCC).Obtain reaction solution (I), stir 30min, stand-by.By 424mg (1.63mmol) HCl H-Lys (Boc)-OCH
3be dissolved in the anhydrous THF of 20ml, obtain reaction solution (II).The lower reaction solution of ice bath (II) is added drop-wise in reaction solution (I), adds 1-2 to drip NMM and adjusts pH to 8-9, under first ice bath, stir 1 hour, then stirring at room temperature 12 hours, TLC shows HCl Lys (Boc)-OCH
3disappear, start aftertreatment.Remove by filter DCU, evaporated under reduced pressure under filtrate room temperature, adds 60ml EtOAc to carry out ultrasonic dissolution, again removes by filter after DCU, and filtrate is used 5%NaHCO successively
3extraction is washed three times, and saturated NaCl extraction is washed three times, 5%KHSO
4extraction is washed three times, and saturated NaCl extraction is washed three times, 5%NaHCO
3extraction is washed three times, and saturated NaCl extraction is washed three times.By ethyl acetate layer anhydrous Na
2sO
4dried overnight.Remove by filter Na
2sO
4, be spin-dried for except desolventizing, through column chromatographic isolation and purification, obtain sterling title compound, blue colored crystal 400mg,, being the target compound of the present embodiment, productive rate is 45%.ESI-MS(m/e)：550[M+H]
+。
Embodiment 8 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3
By 500mg N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3(0.91mmol) under condition of ice bath, be dissolved in 10ml 4N hydrogenchloride-ethyl acetate solution, put immediately cotton and CaCl are housed
2drying tube.After ice bath stirring reaction 0.5h, reaction solution becomes red-purple turbid solution, some plate monitoring raw material N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3after disappearance, start aftertreatment.Under warm water bath condition, drain solvent EtOAc, obtain red-purple powder.Use again a small amount of EtOAc twice, all drain.Adding ether 3 times again, all drain ether at every turn, obtain title compound, is red-purple pressed powder 396mg,, be the target compound of the present embodiment, productive rate 96.9%.ESI-MS(m/e)：450[M+H]
+。
Embodiment 9 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-OCH
3(1a)
By 155mg (1.08mmol) CH
3(CH
2)
8cO
2h is dissolved in 15ml dry THF solution, adds after 133mg HOBt (0.98mmol) under ice-water bath, in above-mentioned mixed solution, is added dropwise to 10ml DCC (221mg, dry THF solution 1.08mmol), obtain reaction solution (I), stir 30min, stand-by.By 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3be dissolved in the anhydrous THF of 20ml, obtain reaction solution (II).The lower reaction solution of ice bath (II) is added drop-wise in reaction solution (I), adds 1-2 to drip NMM and adjusts pH to 8-9, under first ice bath, stir 1 hour, then stirring at room temperature 12 hours, TLC shows NN-Lys-OCH
3disappear, start aftertreatment.Remove by filter DCU, evaporated under reduced pressure under filtrate room temperature, adds 40ml EtOAc to carry out ultrasonic dissolution, again removes by filter after DCU, and filtrate is used 5%NaHCO successively
3extraction is washed three times, and saturated NaCl extraction is washed three times, 5%KHSO
4extraction is washed three times, and saturated NaCl extraction is washed three times, 5%NaHCO
3extraction is washed three times, and saturated NaCl extraction is washed three times.By ethyl acetate layer anhydrous Na
2sO
4dried overnight.Remove by filter Na
2sO
4, be spin-dried for except desolventizing, through column chromatographic isolation and purification, obtain sterling the present embodiment title compound, blue colored crystal 240mg, productive rate is 47%.ESI-MS(m/e)：576[M+H]
+。
Embodiment 10 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys-OCH
3(1b)
According to the method for embodiment 9 by 186mg (1.08mmol) CH
3(CH
2)
8cO
2h and 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3making 241mg (45%) the present embodiment title compound, is blue colored crystal.ESI-MS(m/e)：604[M+H]
+。
Embodiment 11 preparation preparation N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys-OCH
3(1c)
According to the method for embodiment 9 by 216mg (1.08mmol) CH
3(CH
2)
10cO
2h and 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3making 252mg (45%) the present embodiment title compound, is blue colored crystal.ESI-MS(m/e)：632[M+H]
+。
Embodiment 12 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys-OCH
3(1d)
According to the method for embodiment 9 by 246mg (1.08mmol) CH
3(CH
2)
12cO
2h and 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3making 270mg (46.0%) the present embodiment title compound, is blue colored crystal.ESI-MS(m/e)：660[M+H]
+。
Embodiment 13 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys-OCH
3(1e)
According to the method for embodiment 9 by 276mg (1.08mmol) CH
3(CH
2)
14cOOH and 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3making 257mg (42%) the present embodiment title compound, is blue colored crystal.ESI-MS(m/e)：688[M+H]
+。
Embodiment 14 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys-OCH
3(1f)
According to the method for embodiment 9 by 306mg (1.08mmol) CH
3(CH
2)
16cO
2h and 400mg (0.89mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3making 254mg (40%) the present embodiment title compound, is blue colored crystal.ESI-MS(m/e)：716[M+H]
+。
Embodiment 15 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys (2a)
By 500mg (0.87mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-OCH
3be dissolved in 10ml methyl alcohol, ice bath adds NaOH (2N) that the pH value of reaction solution is adjusted to 14 under stirring, ice bath stirring reaction 1 hour, and some plate, TLC shows raw material N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-OCH
3disappear, start aftertreatment.Use saturated KHSO
4reaction solution pH is adjusted to 7, is spin-dried for solvent methanol and water, in resistates, add 10ml distilled water, use saturated KHSO
4its pH is adjusted to 2, makes to be extracted with ethyl acetate, each 20ml, extracts three times.Ethyl acetate layer is merged, use saturated NaCl extraction to wash three times, ethyl acetate layer is used to anhydrous Na
2sO
4dried overnight.Remove by filter Na
2sO
4, be spin-dried for except desolventizing, obtain the present embodiment title compound, blue oily matter 478mg, productive rate is 98%.ESI-MS(m/e)：[M+H]
+。
Embodiment 16 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys (2b)
According to the method for embodiment 15, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys-OCH
3524mg (0.87mmol) saponification obtains 508mg (99%) the present embodiment title compound, is blue oily matter.ESI-MS(m/e)：590[M+H]
+。
Embodiment 17 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys (2c)
According to the method for embodiment 15, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys-OCH
3549mg (0.87mmol) saponification obtains 526mg (98%) the present embodiment title compound, is blue oily matter.ESI-MS(m/e)：618[M+H]
+。
According to the method for embodiment 15, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys-OCH
3573mg (0.87mmol) saponification obtains 550mg (98%) the present embodiment title compound, is blue oily matter.ESI-MS(m/e)：646[M+H]
+。
Embodiment 19 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys (2e)
According to the method for embodiment 15, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys-OCH
3597mg (0.87mmol) saponification obtains 568mg (97%) the present embodiment title compound, is blue oily matter.ESI-MS(m/e)：674[M+H]
+。
Embodiment 20 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys (2f)
According to the method for embodiment 15, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys-OCH
3622mg (0.87mmol) saponification obtains 562mg (98%) the present embodiment title compound, is blue oily matter.ESI-MS(m/e)：702[M+H]
+。
Embodiment 21 prepares Boc-Arg (NO
2)-Gly-OBzl
According to the method for embodiment 9, by 1.600g (5.0mmol) Boc-Arg (NO
2) and 2.026g (6.0 mmol) TosHH-Gly-OBzl make 2.24g (96%) the present embodiment title compound, be faint yellow oily body.ESI-MS(m/e)：467[M+H]
+。
Embodiment 22 prepares Boc-Arg (NO
2)-Gly
According to the method for embodiment 15, Boc-Arg (NO
2)-Gly-OBzl 2.24g (4.8mmol) saponification obtains 1.713g (95%) the present embodiment title compound, is faint yellow oily matter.ESI-MS(m/e)：375[M-H]
-。
Embodiment 23 prepares Boc-Asp (OBzl)-Val-OBzl
According to the method for embodiment 9, making 2.324g (91%) the present embodiment title compound by 1.620g (5.0mmol) Boc-Asp (OBzl) and 2.275g (5.0mmol) TosHH-Val-OBzl, is colorless solid.ESI-MS(m/e)：513[M+H]
+。
Embodiment 24 prepares Asp (OBzl)-Val-OBzl
2.324g (4.5mmol) Boc-Asp (OBzl)-Val-OBzl is dissolved in 25ml 4N hydrogenchloride-ethyl acetate solution and stirring at room temperature 2 hours, TLC (chloroform/methanol, 30: 1) shows that Boc-Asp (OBzl)-Val-OBzl disappears.Concentrating under reduced pressure is removed ethyl acetate, and residue repeatedly adds a small amount of ether and carries out concentrating under reduced pressure to remove de-chlorine hydride.Finally adding a small amount of ether residue is ground to form to 1.704mg (92%) target compound, is colourless powder.ESI-MS(m/e)：413[M+H]
+。
Embodiment 25 prepares Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
According to the method for embodiment 9, by 1.715g (4.56mmol) Boc-Arg (NO
2)-Gly and 1.904g (4.14mmol) HClH-Asp (OBzl)-Val-OBzl reaction, through column chromatography purification, makes 1.930g (57%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：771[M+H]
+。
Embodiment 26 prepares Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl
According to the method for embodiment 24,1.930g (2.4mmol) Boc-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl makes 1.64g (95%) the present embodiment title compound, is colorless solid.ESI-MS(m/e)：671[M+H]
+。
Embodiment 27 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3a)
By 420mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys is dissolved in 20ml dry THF solution; under ice-water bath, add after 112mg (0.83mmol) HOBt; in above-mentioned mixed solution, be added dropwise to 10ml DCC (185mg; dry THF solution 0.90mmol); obtain reaction solution (I); stir 30min, stand-by.By 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl is dissolved in the anhydrous THF of 20ml, obtains reaction solution (II).The lower reaction solution of ice bath (II) is added drop-wise in reaction solution (I), adds 3-4 to drip NMM and adjusts pH to 8-9, under first ice bath, stir 1 hour, then stirring at room temperature 12 hours, TLC shows Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears, and solution is blue suspendible shape, starts aftertreatment.Evaporated under reduced pressure under room temperature, adds 50ml CH
2cl
2carry out ultrasonic dissolution, solution uses 5%NaHCO successively
3extraction is washed three times, and saturated NaCl extraction is washed three times, 5%KHSO
4extraction is washed three times, and saturated NaCl extraction is washed three times, 5%NaHCO
3extraction is washed three times, and saturated NaCl extraction is washed three times.By CH
2cl
2layer anhydrous Na
2sO
4dried overnight.Remove by filter Na
2sO
4, be spin-dried for except desolventizing, after column chromatography for separation, carry out after thin-layer chromatography separation and purification, obtain sterling the present embodiment title compound, blue colored crystal 400mg, productive rate is 47%.ESI-MS(m/e)：1214[M+H]
+.Mp：103-104℃.[α]
D 25＝-13.00(c＝1mg/ml，CH
3OH).IR(KBr)：3306，2928，2858，1739，1536，1451，1389，1369，1269，1186，999，835，746，835，746，698
Embodiment 28 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3b)
According to the method for embodiment 27 by 441mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys and 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl reaction, obtains 170mg (20%) the present embodiment title compound through column chromatography purification, is blue colored crystal.ESI-MS(m/e)：1242[M+H]
+.Mp：112-113℃.[α]
D 25＝-36.00(c＝1mg/ml，CH
3OH).IR(KBr)：3303，2929，2857，1739，1651，1535，1451，1389，1361，1259，1186，835，741，698，593.
Embodiment 29 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3c)
According to the method for embodiment 27 by 462mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys and 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl reaction, obtains 160mg (18%) the present embodiment title compound through column chromatography purification, is blue colored crystal.ESI-MS(m/e)：1270[M+H]
+.Mp：102-103℃.[α]
D 25＝-20.33(c＝1mg/ml，CH
3OH).IR(KBr)：3299，3084，2926，2854，1728，1634，1537，1451，1388，1256，1181，836，748，698，604.
Embodiment 30 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3d)
According to the method for embodiment 27 by 484mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys and 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl reaction, obtains 408mg (45%) the present embodiment title compound through column chromatography purification, is blue colored crystal.ESI-MS(m/e)：1298[M+H]
+.Mp：112-113℃.[α]
D 25＝-11.67(c＝1mg/ml，CH
3OH).IR(KBr)：3301，3068，2854，1739，1651，1534，1451，1389，1361，1259，1186，835，745，698，589.
Embodiment 31 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3e)
According to the method for embodiment 27 by 505mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys and 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl reaction, obtains 389mg (42%) the present embodiment title compound through column chromatography purification, is blue colored crystal.ESI-MS(m/e)：1326[M+H]
+.Mp：107-108℃.[α]
D 25＝-34.33(c＝1mg/ml，CH
3OH).IR(KBr)：3303，2925，2853，1738，1651，1536，1451，1389，1360，1260，1186，999，834，746，698，591.
Embodiment 32 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl (3f)
According to the method for embodiment 27 by 525mg (0.75mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys and 470mg (0.70mmol) Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl reaction, obtains 368mg (39%) the present embodiment title compound through column chromatography purification, is blue colored crystal.ESI-MS(m/e)：1354[M+H]
+.Mp：99-100℃.[α]
D 25＝-26.33(c＝1?mg/ml，CH
3OH).IR(KBr)：3300，3069，2925，2853，1740，1650，1536，1452，1389，1360，1260，1186，834，743，698，593.
Embodiment 33 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-Arg-Gly-Asp-Val (4a)
By 100mg (0.082mmol) N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl is dissolved in 4ml trifluoracetic acid and 1ml trifluoromethanesulfonic acid, under ice bath, reacts 0.5h, and TLC shows N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-capryloyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl disappears, and adds a large amount of anhydrous diethyl ethers to stir, and has brown solid material to separate out in reaction solution, stir after 10min, leave standstill, supernatant liquid is discarded, again add anhydrous diethyl ether to stir, leave standstill, pour out supernatant liquid, so the repeated multiple times ether that adds, topples over supernatant liquid, drain ether, obtain brown color pressed powder.Use a small amount of water dissolution, drip weak ammonia the pH value of water solution of sample is adjusted to 7, use SephdexG15 desalination, water is eluent, collect sample liquid freeze-drying and obtain 40mg (51%) the present embodiment title compound, and be blue powder.ESI-MS(m/e)：989[M+H]
+Mp：124-125℃.[α]
D 25＝-18.33(c＝1mg/ml，CH
3OH).IR(KBr)：3172，1670，1540，1400，1260，1173，1033，762，643，578，519.
Embodiment 34 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys-Arg-Gly-Asp-Val (4b)
According to the method for embodiment 33, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-decanoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl 100mg (0.081mmol) removes nitro and benzyl obtains 45mg (55%) the present embodiment title compound, is blue powder.ESI-MS(m/e)：1017[M+H]
+.Mp：123-124℃.[α]
D 25＝-32.00(c＝1mg/ml，CH
3OH).IR(KBr)：3207，1673，1542，1400，1359，1255，1177，1137，1031，838，602，723，640，576，518.
Embodiment 35 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys-Arg-Gly-Asp-Val (4c)
According to the method for embodiment 33, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-lauroyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl 100mg (0.076mmol) removes nitro and benzyl obtains 48mg (58%) the present embodiment title compound, is blue powder.ESI-MS(m/e)：1045[M?+H]
+.Mp：109-110℃.[α]
D 25＝-24.67(c＝1mg/ml，CH
3OH).IR(KBr)：3350，2931，1662，1540，1450，1359，1252，1170，1030，837，640，576，518.
Embodiment 36 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys-Arg-Gly-Asp-Val (4d)
According to the method for embodiment 33, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-myristoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl 100mg (0.078mmol) removes nitro and benzyl obtains 50mg (60%) the present embodiment title compound, is blue powder.ESI-MS(m/e)：1073[M+H]
+.Mp：122-123℃.[α]
D 25＝-17.00(c＝1mg/ml，CH
3OH).IR(KBr)：3335，2928，2856，1660，1540，1450，1359，1251，1170，1030，837，640，519.
Embodiment 37 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys-Arg-Gly-Asp-Val (4e)
According to the method for embodiment 33, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-palmitoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl 100mg (0.075mmol) removes nitro and benzyl obtains 45mg (54%) the present embodiment title compound, is blue powder.ESI-MS(m/e)：1101[M+H]
+.Mp：124-125℃.[α]
D 25＝-28.67(c＝1mg/ml，CH
3OH).IR(KBr)：3340，2927，2855，1660，1542，1448，1359，1251，1170，1030，837，640，518.
Embodiment 38 prepares N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys-Arg-Gly-Asp-Val (4f)
According to the method for embodiment 33, N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-stearoyl-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl 100mg (0.073mmol) removes nitro and benzyl obtains 40mg (48%) the present embodiment title compound, is blue powder.ESI-MS(m/e)：1129[M+H]
+.Mp：116-117℃.[α]
D 25＝-27.33(c＝1mg/ml，CH
3OH).IR(KBr)：3411，2924，1674，1563，1547，1446，1256，1184，1137，732，637.
The antithrombotic acitivity test of experimental example 14a-f intravenous administration
Intubate forms by 3 sections, middle segment length 80mm, and internal diameter 3.5mm, two ends are identical polyethylene tube, long 100mm, internal diameter 1mm, external diameter 2mm, one end of this pipe pulls into point pipe (for inserting rat carotid artery or vein), the equal silanization of inwall of 3 sections of pipes.The silk thread of the long 60mm weighing is in advance put into stage casing polyethylene extra heavy pipe, the two ends of extra heavy pipe be nested with the not drawing-down end of two polyethylene tubules respectively (wherein one section silk thread is pushed down to 0.5mm fix).For subsequent use by filling heparin-saline solution (50IU/kg) in pipe by sharp pipe end with syringe.
200-220g male SD rat is anaesthetized with 20% urethane solution (6ml/kg, i.p.).Anesthetized rat dorsal position is fixed, isolate the left external jugular vein of rat, proximal part and distal end penetrate respectively surgical thread, ligation distal end, on the left external jugular vein exposing, cut carefully an angle, the not line ball end point pipe of the bypass duct preparing is inserted to the proximal part of left external jugular vein opening above by angle, push the heparin-saline (50IU/kg) of accurate amount by the sharp pipe of the other end with syringe, syringe is now with physiological saline is housed, the syringe of the normal saline solution of the normal saline solution of acetylsalicylic acid or different concns compound replaces, slowly push the liquid of accurate amount, separate subsequently right common carotid artery, in proximal part folder bulldog clamp, proximal part and distal end penetrate respectively surgical thread, ligation distal end, right common carotid artery is being cut to an angle carefully nearby from bulldog clamp.Extract syringe from the tip of polyethylene tube, the tip of polyethylene tube is inserted to the proximal part of artery angle.The two ends of bypass duct all use No. 4 sutures and arteriovenous to fix.Open bulldog clamp, make blood flow flow to vein by bypass duct from artery, this is the anti-bolt model of rat arteriovenous shut.From starting circulation time timing, after 15min, from bypass duct, take out the silk thread that hangs with thrombus, accurately weigh, the wet weight of thrombus that is of poor quality before and after silk thread, anti-thrombus activity in the body of statistics assessing compound.Result shows, 4a-f has outstanding antithrombotic acitivity (table 1).
Under table 1 1nmol/kg dosage, 4a-f is through the antithrombotic acitivity of intravenous administration
a
A) n=12; B) with physiological saline group ratio, P < 0.01
The antithrombotic dose-effect relationship of experimental example 24c intravenous administration
By the method for experimental example 1, choose the dose-effect relationship of 1nmol/kg, 0.1nmol/kg and 0.01nmol/kg Three doses investigation 4c.Result shows, anti-bolt effect show dose dependency (table 2) in the body of 4c.
The dose-effect relationship of table 24c intravenous administration
a
A) n=12; B) with physiological saline and 0.1nmol/kg 4c group ratio, p < 0.01; C) with physiological saline and 0.01nmol/kg 4c group ratio, p < 0.01; D) with physiological saline group ratio, p < 0.01.
Experimental example 34a-f platelet aggregation inhibitory activity is evaluated
Pig carotid artery is got blood, with 3.8% Sodium Citrate (V
sodium Citrate: V
whole blood=1: 9) anti-freezing.1000g/min obtains platelet rich plasma (PRP) for centrifugal 10 minutes.With ADP, PAF, AA, TH is that inductor (deriving from SIGMA company) induced platelet is assembled.Physiological saline solution containing 50%DMSO for 4a-f.The parallel survey of each data 3 times.Result is as shown in table 3.
Show 34a-f to ADP, PAF, AA, and the impact (n=3) of the platelet aggregation of TH induction
Table 3 data declaration compound 4a-f all has platelet aggregation inhibitory activity.
Experimental example 44a-f removes NO free radical activity and measures
Blank group: add 2.5 μ L MGD (7.325mg/mL) in 0.5ml EP pipe, then add 2.5 μ LFeSO
4 .7H
2o (3.475mg/mL), adds 10 μ L distilled water afterwards, finally adds after the violent mixing of 5 μ L SNAP (1.1 μ moL/mL), and mixed solution is sucked to quartz capillary, measures NO free radical after 1min, records spectrogram.
Experimental group: add 2.5 μ L MGD (7.325mg/mL) in 0.5ml EP pipe, then add 2.5 μ LFeSO
4 .7H
2o (3.475mg/mL), adds 10 μ L testing samples afterwards, finally adds after the violent mixing of 5 μ L SNAP (1.1 μ moL/mL), and mixed solution is sucked to quartz capillary, measures NO free radical after 1min, records spectrogram.
Under every kind of each concentration of compound, measure respectively 6 times.Record the clearance rate of each spectrogram peak height computerized compound under each concentration: clearance rate=(blank peak height-administration group peak height)/blank peak height.Result is as shown in table 4.
Table 410
-3the NO of M compound 4a-f removes active (n=6)
Table 4 data declaration compound 4a-f all has the effect of removing NO free radical.
The anti-angiogenic diastole activity rating of experimental example 5 compound 4a-f
Male Wistar rat disconnected neck in 250g left and right puts to death, cuts off breastbone, exposes heart, pick up heart, take off thoracic aorta along thoracic vertebrae with tweezers.Immediately in K-H damping fluid by thoracic aorta and other separate tissue, and keep blood vessel endothelium complete.In K-H damping fluid, thoracic aorta is cut into several sections of wide vascular circles of 2mm stand-by.
Open MD3000 System of organism signal, adjusting instrument, calibration.Thoracic aortic ring is hung on muscle tone transverter, immerses and continue logical oxygen (95%O
2+ 5%CO
2) bath in, regulate the tension force position 1g of thoracic aortic ring.
After the baseline of 1g pretension is walked to put down, in bath, add NE (10
-5mol/L, 20 μ L, final concentration 10
-8mol/L), vascular strip shrinks, and after 5～6min is stable, adds 20 μ L testers, until thoracic aorta reach stable after, in bath, add Ach (10
-3mol/L, 20 μ L, final concentration 10
-6mol/L), until thoracic aortic ring diastole reach stable after, the variation of recording tension force.With K-H damping fluid by aorta washing-round 3-5 time.Repeat experiment.Result is as shown in table 4.
Table 510
-6the diastole effect (n=6) of the anti-Wistar male rat of M compound 4a-f thoracic aortic ring
Table 5 data declaration compound 4a-f all has the vasorelaxation action that anti-Ach causes.
The autonomous dress performance evaluation of experimental example 64a-f
1) particle diameter of 4a-f nanometer ball in the aqueous solution
On Nano-ZS90 nano particle size determinator, measure 4a-f 1 × 10
-12the particle diameter of mg/ml.Result shows, 4a-f can be assembled into nanometer ball in the aqueous solution, and particle diameter is 185 to 274nm (table 6).
The particle diameter of the nanometer ball that table 64a-f forms in the aqueous solution
2) form of the nanometer ball of 4a-f
It is 1 × 10 that 4a-f is made into concentration
-11the aqueous solution of mg/ml, then drops in this solution on copper mesh, observes the form of nanometer ball after the dry solvent that volatilizees under JEM-1230 transmission electron microscope.Mensuration shows, the nanometer ball of 4a-f formation rule.The transmission electron microscope photo of 4c is as representative, describes with Fig. 2.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (5)
1. there is general formula
icompound:
In formula, n=6,8,10,12,14 or 16.
2. prepare the general formula that has claimed in claim 1
icompound method, it is characterized in that, formed by following steps:
1) under bromine exists, the condensation in the 6N NaOH aqueous solution of 2-nitropropane is DMNB;
2) under ammonium chloride and zinc powder existence, DMNB is reduced to 2,3-dimethyl-2,3-dihydroxy amido butane in moisture ethanolic soln;
3) in methyl alcohol 2,3-dimethyl-2,3-dihydroxy amido butane and paranitrobenzaldehyde condensation are 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine;
4) plumbic oxide exist under 1,3-dihydroxyl-2-(4 '-hydroxy phenyl)-4,4,5,5-tetramethyl-imidazolidine is oxidized to 2-(4 '-hydroxy phenyl)-4,4,5 in methyl alcohol, 5-tetramethyl--1,3-bis-Sinerols;
5) 2-(4 '-hydroxy phenyl)-4,4,5 under sodium hydride exists, 5-tetramethyl--1,3-bis-Sinerols are 1,3-dioxy-4 with bromoethyl acetate condensation in anhydrous THF, 4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester;
6) in methyl alcohol 1,3-dioxy-4, the saponification of 4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid ethyl ester is 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid;
7) under DCC and HOBt exist 1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-fluoroacetic acid in anhydrous THF with HClH-Lys (Boc)-OCH
3condensation is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3;
8) N in the ethyl acetate of containing hydrogen chloride
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-Boc-Lys-OCH
3remove Boc and generate N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3;
9) N under DCC and HOBt existence
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-Lys-OCH
3in anhydrous THF, be N with saturated fatty acid condensation
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-OCH
3;
10) in methyl alcohol by N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-OCH
3saponification is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys;
11) under DCC and HOBt existence, Boc-Asp (OBzl) is Boc-Asp (OBzl)-Val-OBzl with Val-OBzl condensation in anhydrous THF;
12) in the ethyl acetate of containing hydrogen chloride, Boc-Asp (OBzl)-Val-OBzl removes Boc and generates Asp (OBzl)-Val-OBzl;
13) Boc-Arg (NO under DCC and HOBt existence
2)-Gly is Boc-Arg (NO with Asp (OBzl)-Val-OBzl condensation in anhydrous THF
2)-Gly-Asp (OBzl)-Val-OBzl;
14) Boc-Arg (NO in the ethyl acetate of containing hydrogen chloride
2)-Gly-Asp (OBzl)-Val-OBzl removes Boc and generates Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
15) N under DCC and HOBt existence
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys in anhydrous THF with Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl condensation is N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl;
16) containing N in the trifluoroacetic acid solution of trifluoromethanesulfonic acid
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg (NO
2)-Gly-Asp (OBzl)-Val-OBzl removes OBzl and NO
2generate N
α-(1,3-dioxy-4,4,5,5-tetramethyl-tetrahydroglyoxaline-2-phenyl-4 '-oxygen ethanoyl)-N
ω-fatty acyl group-Lys-Arg-Gly-Asp-Val.
3. claimed in claim 1 have a general formula
icompound in the application of preparing in antithrombotic reagent.
4. claimed in claim 1 have a general formula
icompound remove the application in NO free radical medicine in preparation.
5. claimed in claim 1 have a general formula
icompound preparing antithrombotic and remove the application in NO free radical dual-use function medicine.
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