CN102641506A - Bivalirudin-polyethylene glycol compound - Google Patents

Bivalirudin-polyethylene glycol compound Download PDF

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CN102641506A
CN102641506A CN2012101043274A CN201210104327A CN102641506A CN 102641506 A CN102641506 A CN 102641506A CN 2012101043274 A CN2012101043274 A CN 2012101043274A CN 201210104327 A CN201210104327 A CN 201210104327A CN 102641506 A CN102641506 A CN 102641506A
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bivalirudin
polyethylene glycol
cys
bvld
complex
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CN102641506B (en
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王良友
王小青
尹志峰
李云峰
高杨
赵红玲
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CHINESE MEDICINE INST CHENGDE MEDICAL COLLEGE
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CHINESE MEDICINE INST CHENGDE MEDICAL COLLEGE
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Abstract

The invention relates to a bivalirudin-polyethylene glycol compound and a preparation method thereof, a medicinal composition containing the compound and application. The bivalirudin-polyethylene glycol compound is a new structural compound which is obtained by cysteine substitution for any glycine from the 5th site to the 8th site in a bivalirudin sequence and polyethylene glycol modification, keeps the activity of bivalirudin and prolongs the half-life period of the bivalirudin. The application of the bivalirudin-polyethylene glycol compound in medicaments for treating or preventing thrombus diseases comprises application in medicaments for unstable angina pectoris, peripheral artery intervention therapy, hearth and lung transplantation and thrombus preventing treatment.

Description

Bivalirudin-Pegylation complex
[technical field]
The present invention relates to the Pegylation complex of bivalirudin; Its preparation method; The pharmaceutical composition that contains them with and application in the medicine of treatment or prevention thrombus disease, be included in the application in the medicine in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and the anti-bolt treatment.
[background technology]
The thrombotic disease of initiations such as atherosclerosis has become the first in the world cause of death, and its sickness rate is in rising trend in China, and the anticoagulant medicine is very important in the treatment of this disease.In at present numerous anticoagulant medicines, the bivalirudin (Angiomax of Medicines company TM) be a kind of anticoagulant new drug of FDA in approval listing in 2000, its anticoagulant composition is hirudin analog (fragment).This medicine is that thrombin is direct, special, reversible inhibitor; No matter thrombin is in the blood circulation still combines with thrombosis; It all can combine with its catalytic site and anion binding site (claiming the substrate recognition site again) generation specificity, thus the activity of direct Trombin inhibiting.It has overcome the shortcoming of heparin, Low molecular heparin and hirudin, in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt treatment, demonstrates good effect.
But bivalirudin is shorter to the inhibition persistent period in thrombin activity site, in the intravital half-life of people about 25 minutes.Realize long-time inhibition, need the repeated multiple times drug administration by injection sometimes the thrombin activity site.And bivalirudin costs an arm and a leg, and repeatedly medication has increased patient's the cost of seeking medical advice greatly repeatedly, and therefore, it is very necessary to develop long-acting, stable, economic similar medicine or preparation.
(enhancing water solublity and stability reduce immunogenicity and antigenicity to protein drug for polyethylene glycol, PEG) ability significant prolongation effect after the modification, and the protein drug of existing a plurality of PEG modifications goes on the market through Polyethylene Glycol.The present invention is through introducing cysteine in the bivalirudin structure, to realize fix a point method that Pegylation modifies and use and do not see bibliographical information as yet of bivalirudin.
[summary of the invention]
One of the object of the invention provides bivalirudin-Polyethylene Glycol complex.Chemically synthesized polypeptide medicine bivalirudin is the direct inhibitor of thrombin; It has overcome the shortcoming of heparin, Low molecular heparin and hirudin, in unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt thromboembolism preventing treatment, demonstrates good effect.But the single dose of bivalirudin is 250 milligrams, and price is about 1000 yuan of every grams at present.The PEGization modified outcome through synthetic bivalirudin is hoped in this research, can prolong the half-life of bivalirudin, reduces dosage or administration frequency, will bring more vast market for bivalirudin, creates bigger economic benefit and social benefit.Bivalirudin derives from the hirudin complex; The polypeptide of forming by 20 amino acid residues; Molecular weight is 2180, and its structural formula is D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH.Research shows that its N-end Pro-Arg-Pro and C-end Glu-Glu-Ile-Pro-Glu-Glu are bonded two critical sites of itself and thrombin; Therefore 4 glycine of 5 to 8 have been interconnect function, and selecting 5,6,7 or 8 s' glycine to modify is ideal site.
Bivalirudin (bivalirudin of the present invention; BVLD)-arbitrary glycine that the Polyethylene Glycol complex cuts down in the Lu Ding sequence the 5th to the 8th (holding the C-end by N-) for contrast carries out cysteine and substitutes the back and carry out the novel compound that obtains after Pegylation is modified, the chemical compound of said complex for having formula (I) structure in cysteine side chain:
[Cys(PEG-M)] X-BVLD (I)
Wherein
X is the arbitrary integer among the 5-8, and the 5th to the 8th arbitrary glycine in the expression bivalirudin sequence substituted by cysteine; PEG is RO (CH 2CH 2O) n-CH 2CH 2, R=H or CH 3, n=5-1000; Cys is a cysteine, and it links to each other with M group covalency through its side chain sulphur atom.Particularly, the structure of this complex comprises [Cys (mPEG 2000-MAL)] 7-BVLD, [Cys (mPEG 5000-MAL)] 7-BVLD and [Cys (mPEG 10000-MAL)]-BVLD.
Another object of the present invention provides the method for preparing of bivalirudin Polyethylene Glycol complex, and described method for preparing comprises: with [Cys] X-BVLD is dissolved in the pure water; Regulate pH to 7～8 with sodium bicarbonate; The PEG that adds 2-3 times of molar equivalent; Stirring at room reaction 2-3 hour, with reversed-phase high-performance liquid chromatography (RP-HPLC) monitoring reaction process and separate targets product, target product gets bivalirudin-Polyethylene Glycol complex [Cys (PEG-MAL)] through lyophilization X-BVLD, wherein PEG is dimaleoyl imino Polyethylene Glycol, vinyl Polyethylene Glycol or iodo acetyl group Polyethylene Glycol.
The present invention substitutes glycine to introduce cysteine at 7, carries out the polyethyleneglycol modified example that is then: adopt general solid-phase polypeptide synthesis strategy synthetic 7 bivalirudins by the cysteine substituted glycinic acid, i.e. [Cys] earlier 7-bivalirudin, its aminoacid sequence does
D-Phe-Pro-Arg-Pro-Gly-Gly-Cys-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH is then with [Cys] 7Additive reaction takes place and obtains bivalirudin-Polyethylene Glycol complex [Cys (mPEG-MAL)] in-bivalirudin and mPEG-MAL 7BVLD.This bivalirudin Polyethylene Glycol complex is to obtain through sulfydryl and the mPEG-MAL prepared in reaction that is incorporated into the cysteine in the bivalirudin sequence, and the process of sulfydryl reaction is as follows in the structure of PEG and complex thereof and mPEG-MAL and the peptide chain:
Figure BDA0000152178940000031
[Cys] 7The concrete preparation process of-bivalirudin comprises that preparation, the mensuration of substitution degree, coupling amino acid, the peptide resin of amino-acid resin obtain [Cys] 7-bivalirudin bullion, through anti-phase-HPLC (RP-HPLC) purification, lyophilizing obtains [Cys] 7The pure article dry powder of-bivalirudin.
Among the present invention, earlier with [Cys] behind the purification 7-bivalirudin is soluble in water, then with saturated sodium bicarbonate solution pH regulator is arrived alkalescence, is between the 7-8 like pH, and the mean molecule quantity that adds the 2-3 molar equivalent then respectively is 2000,5000,10000 mPEG-MAL, at room temperature reacts 2-3 hour.Through RP-HPLC to reaction monitor, target product separates and purity analysis.
Target product [Cys (mPEG-MAL)] 7-BVLD structure obtains conclusive evidence through carrying out amino acid composition analysis, HPLC and mass spectrum (MALDI-TOF-MS) analyses.
The bivalirudin of new construction-Polyethylene Glycol complex is through the anticoagulating active evaluation, prothrombin time (PT), and thrombin time (TT) and activated partial thromboplastin time (APTT) experimental result show the anticoagulating active that it has kept bivalirudin.Can know the polyethyleneglycol modified of bivalirudin by inference based on the existing knowledge of polyethyleneglycol modified drug research and prolong its half-life again.
A further object of the invention provides institute's bivalirudin-Polyethylene Glycol complex; The pharmaceutical composition that contains them with and application in the medicine of treatment or prevention thrombus disease, and be used for treating the purposes in the medicine of unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt treatment.Pharmaceutical composition of the present invention contains at least a bivalirudin-Polyethylene Glycol complex and pharmaceutic adjuvant.
The present invention adopts simple and easy to do method, and the anticoagulating active that can keep bivalirudin for preparing new construction prolongs the bivalirudin Polyethylene Glycol complex of its anticoagulant time again.
[description of drawings]
Accompanying drawing 1 is [Cys] 7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 2 is [Cys] 7The ESI-MS of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 3 is [Cys (mPEG 2000-MAL)] 7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 4 is [Cys (mPEG 2000-MAL)] 7The MALDI-TOF-MS of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 5 is [Cys (mPEG 5000-MAL)] 7The HPLC of-BVLD analyzes collection of illustrative plates.
Accompanying drawing 6 is that the MALDI-TOF-MS of [Cys (mPEG5000-MAL)] 7-BVLD analyzes collection of illustrative plates.
Accompanying drawing 7 is that the HPLC of [Cys (mPEG10000-MAL)] 7-BVLD analyzes collection of illustrative plates.
Accompanying drawing 8 is [Cys (mPEG 10000-MAL)] 7The MALDI-TOF-MS of-BVLD analyzes collection of illustrative plates.
The specific embodiment
The used aminoacid configuration of the present invention is the L-type except that specified otherwise, the relevant abbreviation of use has following implication:
TBu: the tert-butyl group
2-CTC Resin:2-chlorine trityl chloride resin
DCM: dichloromethane
DIC:N, the N-DIC
DMF:N, dinethylformamide
EDT:1, the 2-dithioglycol
The Fmoc:9-fluorenylmethyloxycarbonyl
The HOBt:1-hydroxy benzo triazole
The NMP:N-methyl pyrrolidone
Pbf:2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulfonyl
TFA: trifluoroacetic acid
Ts-Cl: paratoluensulfonyl chloride
Fmoc-Arg (Pbf)-OH:N-fluorenes methoxy carbonyl acyl group-2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulphonyl-L-arginine
Fmoc-Asn (Trt)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-L-aspartic acid
Fmoc-Asp (OtBu)-OH:N-fluorenylmethyloxycarbonyl-trityl-altheine
Fmoc-Cys (Trt)-OH:N-fluorenylmethyloxycarbonyl-S-trityl-L-cysteine
Fmoc-Glu (OtBu)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-L-glutamic acid
Fmoc-Gly-OH:N-fluorenylmethyloxycarbonyl glycine
Fmoc-Ile-OH:N-fluorenylmethyloxycarbonyl-L-isoleucine
Fmoc-Leu-OH:N-fluorenylmethyloxycarbonyl-L-leucine
Fmoc-D-Phe-OH:N-fluorenylmethyloxycarbonyl-D-phenylalanine
Fmoc-Phe-OH:N-fluorenylmethyloxycarbonyl-L-phenylalanine
Fmoc-Pro-OH:N-fluorenylmethyloxycarbonyl-L-proline
Fmoc-Tyr (tBu)-OH:N-fluorenylmethoxycarb-nyl-nyl O-tert-butyl-L-tyrosine
MALDI-TOF-MS: substance assistant laser desorpted attach time-of-flight mass spectrometry
PEG-MAL: dimaleoyl imino Polyethylene Glycol
Among the present invention, the reagent that the Kaiser detection method mainly adopts is 1,2,3-indantrione monohydrate, and reaction is purple or blueness shows that free amino the existence arranged on the resin, shows that coupling is incomplete.
Below in conjunction with concrete instance the present invention is done further detailed description, but the invention is not restricted to following examples.
Embodiment 1 [Cys (mPEG 2000-MAL)] 7The preparation of-BVLD
Weighing m PEG 2000-OH 20g (10mmol) places reaction bulb, adds dichloromethane 50mL, and (Ts-Cl, 50mmol), stirring at room is reacted to add triethylamine 7.5mL (50mmol) and paratoluensulfonyl chloride 9.5g after the solid dissolving again.After the TLC detection reaction was complete, rotary evaporation removed and desolvates, and added the 50mL absolute ether and was settled out solid, adds water again solid is dissolved; Separatory divides water-yielding stratum, with absolute ether 50mL washing 2 times; Water reuse dichloromethane 75mL extraction 2 times is collected organic facies, dried overnight.Revolve and boil off solvent, add the absolute ether sedimentation and go out solid.Obtain mPEG after the drying 2000-OTs 14.2g, yield 71%.
MPEG-OTs 14g (7mmol) is dissolved in 30mL DMF, the potassium phthalimide 3.88g (21mmol) of adding, under the nitrogen protection, 120 ℃ of reaction 4h.Vacuum rotary steam removes and desolvates, and adds dehydrated alcohol, adds hydrazine hydrate 4.5mL, reflux 4h.Vacuum rotary steam removes and desolvates, and adds dichloromethane 100mL, the elimination insoluble matter, and it is inferior to give a baby a bath on the third day after its birth with saturated nacl aqueous solution 50mL, anhydrous Na 2SO 4Dry 12h, the elimination desiccant, vacuum rotary steam removes and desolvates, the ether sedimentation, sucking filtration gets mPEG after the drying 2000-NH 210.8g, yield 77%.
With mPEG 2000-NH 210g (5mmol) is dissolved among the 20mL DMF, adds maleic anhydride 4g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 9.5g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 4.9g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in reactant liquor, use the dichloromethane extraction water then three times, organic facies after saturated nacl aqueous solution is given a baby a bath on the third day after its birth time, anhydrous Na 2S0 4Dry 12h.Filter, vacuum rotary steam removes and desolvates, and the ether sedimentation is centrifugal, sucking filtration, the dry mPEG that gets 2000-MAL 6.8g, yield 68%.
Take by weighing 2-CTC Resin 3.8g (5mmol, 1.3mmol/g) to reaction vessel, add an amount of DMF washing after, take out solution, add the about 30min of an amount of DCM swelling.The Fmoc-Leu-OH that takes by weighing 2.9g adds an amount of DMF dissolving in beaker, the DIPEA (1.4mL) that adds 1/3 cumulative volume amount stirs ice-water bath 10～15min in above-mentioned Freamine.After treating that the resin swelling finishes, take out DCM, the protection aminoacid feed liquid that dissolving is good is poured in the reaction vessel, behind reaction 10～15min, adds the DIPEA (2.85mL) of 2/3 cumulative volume, room temperature reaction 3～4h.Reaction is taken out reactant liquor after finishing, and adds an amount of DMF washing 3 times, and the ratio that adds volume is the mixed solution sealing unreacted radical of 17: 2: 1 DCM, methanol, DIPEA, sealing twice, about at every turn 20min.After sealing finished, the DMF that adds an amount of volume washed 3 times, reuse absolute methanol wash shrinkage 2 times, each about 10 minutes.Vacuum drying obtains about Fmoc-Leu-CTC resin 4.6g, and recording substitution degree is 0.52mmol/g.
(1.82mmol 0.52mmol/g) in reaction vessel, adds the about 30min of an amount of DCM swelling, takes out to take by weighing Fmoc-Leu-CTC Resin 3.5g.Adding 20% an amount of piperidines/DMF solution deprotection 2 times, be 10min for the first time, be 20min for the second time, needs an amount of DMF of adding to wash between twice deprotection.Take off an amount of DMF washing of protection back reuse 5 times, washed 1min at every turn.The Kaiser method detects, and resin is navy blue, shows that Fmoc removes.Take by weighing Fmoc-Tyr (tBu)-OH 1.1g, HOBt 0.6g puts in the beaker, adds an amount of DMF dissolving, and beaker is put into the about 5min of ice bath ice bath, adds DIC 1.3mL again in solution, adds in the reaction vessel room temperature reaction 2～3h behind ice bath activation 10～15min.Behind the reaction 2h, the Kaiser method detects, if observe resin with solution all shows light yellow, shows that then reaction is complete, if behind the reaction 3h, Kaiser method detection resin still is blueness, then needs the repetition coupling.According to [Cys] 7The sequence of-bivalirudin is held the N end from C, repeats above operating procedure, and coupling obtains full guard [Cys] after finishing last aminoacid Fmoc-D-Phe-OH and removing the terminal Fmoc blocking group of N-successively 7-BVLD-peptide resin: D-Phe-Pro-Arg (Pbf)-Pro-Gly-Gly-Cys (Trt)-Gly-Asn (Trt)-Gly-Asp (OtBu)-Phe-Glu (OtBu)-Glu (OtBu)-Ile-Pro-Glu (OtBu)-Glu (OtBu)-Tyr (tBu)-Leu-CTC resin.With getting 16g after the peptide resin drying.Peptide resin is joined (TFA/ thioanisole/EDT/ methyl phenyl ethers anisole=90/5/3/2) in the freezing in advance 150mL lysate, and about 2.5 hours of room temperature cracking is filtered and sedimentation in refrigerated absolute ether then.Centrifugal, washing, the about 14h of vacuum drying obtains [Cys] 7The thick peptide 3.2g of-BVLD.The thick peptide of above-mentioned gained gets after the lyophilizing [Cys] through the HPLC purification 7-BVLD1.4g, purity 96%, total recovery 34%.
Take by weighing [Cys] 7-bivalirudin 220mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG 2000-MAL 600mg, room temperature reaction 2 hours is used the RP-HPLC separated product, gets [Cys (mPEG after the lyophilizing 2000-MAL)] 7-BVLD 294mg, purity is greater than 95%, yield 70%.
[Cys (mPEG 2000-MAL)] 7-BVLD analyzes through MALDI-TOF-MS, finds near 4232, to have a series of peaks, adjacent two peak molecular weight to differ about 44, has the typical structure characteristic of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.99 (2); Glu, 4.11 (4); Pro, 3.09 (3); Arg, 0.96 (1); Gly, 4.21 (4); Tyr, 1.07 (1); Phe, 1.92 (2); Ile, 0.99 (1); Leu, 0.98 (1).
Embodiment 2 [Cys (mPEG 5000-MAL)] 7The preparation of-BVLD
Weighing m PEG 5000-OH 25g (5mmol) places reaction bulb, adds dichloromethane 100mL, and (Ts-Cl, 50mmol), stirring at room is reacted to add triethylamine 7.5mL (5mmol) and paratoluensulfonyl chloride 9.5g after the solid dissolving again.After the TLC detection reaction was complete, rotary evaporation removed and desolvates, and added the 150mL absolute ether and was settled out solid, adds water again solid is dissolved; Separatory divides water-yielding stratum, with absolute ether 100mL washing 2 times; Water reuse dichloromethane 150mL extraction 2 times is collected organic facies, dried overnight.Revolve to steam to remove and desolvate, add the absolute ether sedimentation and go out solid, filter, obtain mPEG after the drying 5000-OTs 21.6g, yield 86%.
With mPEG 5000-OTs 20g (4mmol) is dissolved in 30mL DMF, the potassium phthalimide 2.2g (12mmol) of adding, and under the nitrogen protection, 120 ℃ of reaction 4h.Vacuum rotary steam removes and desolvates, and adds dehydrated alcohol, adds hydrazine hydrate 2.0mL, reflux 4h.Vacuum rotary steam removes and desolvates, and adds dichloromethane 100mL, the elimination insoluble matter, and it is inferior to give a baby a bath on the third day after its birth with saturated NaCl solution 50mL, anhydrous Na 2SO 4Dry 12h, the elimination desiccant, vacuum rotary steam removes and desolvates, the ether sedimentation, sucking filtration, drying gets mPEG 5000-NH 217.5g, yield 82%.
With mPEG 5000-NH 215g (3mmol) is dissolved among the 30mL DMF, adds maleic anhydride 1.25g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 13.8g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 5g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in reactant liquor, use the dichloromethane extraction water then three times, organic facies after saturated nacl aqueous solution is given a baby a bath on the third day after its birth time, anhydrous Na 2SO 4Dry 12h.Filter, vacuum rotary steam removes and desolvates, and the ether sedimentation is centrifugal, sucking filtration, the dry mPEG that gets 5000-MAL 12.6g, yield 80%.
Take by weighing [Cys] 7-bivalirudin 220mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG 5000-MAL 1.25g, room temperature reaction 3 hours is used the RP-HPLC separated product, gets [Cys (PEG after the lyophilizing 5000-MAL)] 7-BVLD 587mg, purity is greater than 95%, yield 81%.
[Cys (mPEG 5000-MAL)] 7-BVLD analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 7322, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure characteristic of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 2.00 (2); Glu, 4.31 (4); Pro, 3.18 (3); Arg, 1.03 (1); Gly, 4.13 (4); Tyr, 1.02 (1); Phe, 1.82 (2); Ile, 1.06 (1); Leu, 1.03 (1).
Embodiment 3 [Cys (mPEG 10000-MAL)] 7The preparation of-BVLD
Weighing m PEG 10000-OH 25g (2.5mmol) places reaction bulb, adds dichloromethane 100mL, and (Ts-Cl, 25mmol), stirring at room is reacted to add triethylamine 3.75mL (2.5mmol) and paratoluensulfonyl chloride 4.75g after the solid dissolving again.After the TLC detection reaction was complete, rotary evaporation removed and desolvates, and added the 150mL absolute ether and was settled out solid, adds water again solid is dissolved; Separatory divides water-yielding stratum, with absolute ether 100mL washing 2 times; Water reuse dichloromethane 150mL extraction 2 times is collected organic facies, dried overnight.Revolve and boil off solvent, add the absolute ether sedimentation and go out solid, filter, obtain mPEG after the drying 10000-OTs 24g, yield 92%.
With mPEG 10000-OTs 20g (2mmol) is dissolved in 30mL DMF, the potassium phthalimide 1.1g (6mmol) of adding, and under the nitrogen protection, 120 ℃ of reaction 4h.Vacuum rotary steam removes and desolvates, and adds dehydrated alcohol, adds hydrazine hydrate 1.0mL, reflux 4h.Vacuum rotary steam removes and desolvates, and adds dichloromethane 100mL, the elimination insoluble matter, and it is inferior to give a baby a bath on the third day after its birth with saturated NaCl solution 50mL, anhydrous Na 2SO 4Dry 12h, the elimination desiccant, vacuum rotary steam removes and desolvates, the ether sedimentation, sucking filtration, drying gets mPEG 10000-NH 217.2g, yield 86%.
With mPEG 10000-NH 215g (1.5mmol) is dissolved among the 30mL DMF, adds maleic anhydride 0.625g, 80 ℃ of stirring reaction 30min.The pressure reducing and steaming solvent, the ether sedimentation, filter collection solid gets 14.5g after the drying.Solid is dissolved in the 30mL acetic anhydride, adds anhydrous sodium acetate 2.5g, 100 ℃ of stirring reaction 45min.Solid in the filtering reactant liquor adds the water of 10 times of volumes in reactant liquor, use the dichloromethane extraction water then three times, organic facies after saturated NaCl solution is given a baby a bath on the third day after its birth time, anhydrous Na 2SO 4Dry 12h.Filter, vacuum rotary steam removes and desolvates, and the ether sedimentation is centrifugal, sucking filtration, the dry mPEG that gets 10000-MAL 13.6g, yield 90%.
Take by weighing [Cys] 7-BVLD 110mg is dissolved in the pure water, regulates pH to 7～8 with sodium bicarbonate, adds mPEG 10000-MAL 1.0g, room temperature reaction 3 hours is used the RP-HPLC separated product, gets [Cys (PEG after the lyophilizing 10000-MAL)] 7-BVLD 1065mg, purity is greater than 95%, yield 89%.
[Cys (mPEG 10000-MAL)] 7-BVLD analyzes through MALDI-TOF-MS, and a series of peaks are arranged near 11973, and it is about 44 that adjacent two peak molecular weight differ, and has the typical structure characteristic of Polyethylene Glycol.The composition of amino acid analysis of acid hydrolysis (6N aqueous hydrochloric acid solution, 110 ℃, 22 hours) conforms to theoretical value: Asp, 1.95 (2); Glu, 4.06 (4); Pro, 2.92 (3); Arg, 0.96 (1); Gly, 4.21 (4); Tyr, 1.06 (1); Phe, 1.92 (2); Ile, 1.01 (1); Leu, 0.97 (1).
The anticoagulating active evaluation of embodiment 4 bivalirudins-Polyethylene Glycol complex
4.1 the mensuration of prothrombin time (PT)
With PT reagent with examined blood plasma, put preparatory temperature 5min in 37 ℃ of water-baths.Get 1 in test tube, add and examined blood plasma 0.1mL, preparatory temperature 30s in 37 ℃ of water-baths adds the PT reagent 0.2mL of temperature in advance again, and mixing starts manual time-keeping immediately.When test tube to the liquid flow that constantly tilts slowly is tending towards stopping, the record required time.Repeat 3 times, average, make normal control simultaneously.
With PT reagent with examined blood plasma, put preparatory temperature 5min in 37 ℃ of water-baths.Get 1 in test tube, add and to be examined blood plasma 0.1mL, and add a certain amount of bivalirudin or its trim makes its final concentration as shown in the table, preparatory temperature 30s in 37 ℃ of water-baths adds the PT reagent 0.2mL of temperature in advance again, and mixing starts manual time-keeping immediately.When test tube to the liquid flow that constantly tilts slowly is tending towards stopping, the record required time.Repeat 3 times, average.
The PT experimental result shows that bivalirudin-Polyethylene Glycol complex all has good anticoagulant effect, wherein [Cys (mPEG 5000-MAL)] 7-BVLD and [Cys (mPEG 10000-MAL)] 7-BVLD anticoagulant effect is superior to bivalirudin, and difference has statistical significance (table 1) more than 3s.
PT experimental result (the normal control meansigma methods: 27.4s) of table 1 bivalirudin-Polyethylene Glycol complex
Figure BDA0000152178940000091
4.2 thrombin time of blood plasma (TT) is measured
Get and examined blood plasma 0.1mL in test tube, add a certain amount of bivalirudin or its trim, put in 37 ℃ of water-baths; Preparatory temperature 5min adds the thrombin reagent of 25U/mL again, starts stopwatch record PCT simultaneously; Repeat 3 times, average, as normal control.
Get and examined blood plasma 0.1mL in test tube, add a certain amount of bivalirudin or its trim and make its final concentration as shown in the table, put in 37 ℃ of water-baths; Preparatory temperature 5min adds 0.1mL standardization " thrombin " reagent again, starts stopwatch record PCT simultaneously; Repeat 3 times, average.
The TT experimental result shows that bivalirudin-Polyethylene Glycol complex maintains good anticoagulant effect, wherein [Cys (mPEG 2000-MAL)] 7-BVLD and [Cys (mPEG 5000-MAL)] 7-BVLD anticoagulant effect is superior to bivalirudin, and difference has statistical significance (table 2) more than 3s.
TT experimental result (the normal control meansigma methods: 18.0s) of table 2 bivalirudin-Polyethylene Glycol complex
Figure BDA0000152178940000101
4.2 blood plasma activated partial thromboplastin time (APTT) is measured
Get APTT reactant liquor 0.1mL and blood plasma 0.1mL, and add 5 μ L normal saline, put preparatory temperature 3min in 37 ℃ of water-baths.Add the APTT catalyst 0.1mL of temperature in advance again, mixing starts manual time-keeping immediately.Constantly tilt test tube when having flocculent deposit to generate, the record required time.Repeat 2 times, average, make normal control simultaneously.
Get APTT reactant liquor 0.1mL and examined blood plasma 0.1mL in plastic centrifuge tube, and bivalirudin and trim to its final concentration thereof of adding 5 μ L be shown in the following table, put preparatory temperature 3min in 37 ℃ of water-baths.Add the APTT catalyst 0.1mL of temperature in advance again, mixing starts manual time-keeping immediately.Constantly tilt test tube when having flocculent deposit to generate, the record required time.Repeat 2 times, average.
APTT experimental result (the normal control meansigma methods: 33.6s) of table 3 bivalirudin and trim thereof
Figure BDA0000152178940000111
All kept on the basis of anticoagulating active at bivalirudin-Polyethylene Glycol complex, can know the polyethyleneglycol modified of bivalirudin by inference based on the existing knowledge of polyethyleneglycol modified drug research can prolong its half-life.

Claims (6)

1. bivalirudin (bivalirudin; BVLD)-and the Polyethylene Glycol complex, it is characterized in that said complex is to contrast the arbitrary glycine that cuts down the 5th to the 8th (holding the C-end by N-) in the Lu Ding sequence to carry out the novel compound that the alternative back of cysteine carries out obtaining after the Pegylation modification in cysteine side chain.
2. bivalirudin according to claim 1-Polyethylene Glycol complex is characterized in that the chemical compound of said complex for having formula (I) structure:
[Cys(PEG-M)] X-BVLD (I)
Wherein
Figure FDA0000152178930000011
X is the arbitrary integer among the 5-8, and the 5th to the 8th arbitrary glycine in the expression bivalirudin sequence substituted by cysteine; PEG is RO (CH 2CH 2O) n-CH 2CH 2, R=H or CH 3, n=5-1000; Cys is a cysteine, and it links to each other with M group covalency through its side chain sulphur atom.
3. according to the said bivalirudin of claim 1-Polyethylene Glycol complex, wherein the structure of this complex is [Cys (mPEG 2000-MAL)] 7-BVLD, [Cys (mPEG 5000-MAL)] 7-BVLD and [Cys (mPEG 10000-MAL)]-BVLD.
4. a pharmaceutical composition is characterized in that containing at least a bivalirudin-Polyethylene Glycol complex and pharmaceutic adjuvant among the claim 1-3.
5. be used for treating or preventing the application of the medicine of thrombus disease according to the described bivalirudin of each claim among the claim 1-3-Polyethylene Glycol complex in preparation, it is characterized in that said will go to be used to treat unstable angina pectoris, peripheral arterial interventional therapy, heart-lung transplant operation and anti-bolt treatment.
6. the method for preparing of bivalirudin according to claim 2-Polyethylene Glycol complex is characterized in that comprising
With [Cys] X-BVLD is dissolved in the pure water; Regulate pH to 7～8 with sodium bicarbonate; The PEG that adds 2-3 times of molar equivalent; Stirring at room reaction 2-3 hour, with reversed-phase high-performance liquid chromatography (RP-HPLC) monitoring reaction process and separate targets product, target product gets bivalirudin-Polyethylene Glycol complex [Cys (PEG-MAL)] through lyophilization X-BVLD, PEG wherein are dimaleoyl imino Polyethylene Glycol, vinyl Polyethylene Glycol or iodo acetyl group Polyethylene Glycol.
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USRE46830E1 (en) 2004-10-19 2018-05-08 Polypeptide Laboratories Holding (Ppl) Ab Method for solid phase peptide synthesis
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CN113105639A (en) * 2021-04-30 2021-07-13 深圳市华创汇能技术有限公司 Humic acid-polyethylene glycol grafted polyacrylic acid composite material and preparation method thereof

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