CN101168594B - Polyglycol active derivative with oligopeptide as framework, preparation method thereof and conjugate of with pharmaceutical molecule - Google Patents
Polyglycol active derivative with oligopeptide as framework, preparation method thereof and conjugate of with pharmaceutical molecule Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/021—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
Abstract
The invention relates to a compound formed by polyethylene glycol and oligopeptide and expressed by the general formula I, wherein, i, j, p, and q are integral numbers, i is equal to 1, 2, to j; j is larger than or equal to 2; p is equal to 1, 2, to q; q is larger than or equal to 1; PEGi is a polyethylene glycol chain; Xi is a linking group; A is derived from oligopeptide and composed of 2 to 20 amino acids, and two amino acids are different; Fp is an active group. The compound can link macromolecular protein or polypeptide or micromolecular active constituents of natural drugs through the Fp to improve the physiological action of drug molecular in the human body, or ensure the appropriate drug concentration and provide the slow-releasing function.
Description
Technical field
The present invention relates to polyethylene active derivatives, relate in particular to the polyethylene active derivatives that a kind of oligopeptides is a skeleton, and preparation method thereof with the binding substances of described derivative and drug molecule.
Background technology
Various protein and polypeptide natural and reorganization all have certain medical operability.Various goods to after its purifying, the separation can be used for various treatment indications through the gi tract administration by non-.Yet non-protein through the gi tract administration may have immunogenicity, also may be water-fast relatively, also may have the very short pharmacology transformation period.Therefore, how to improve and keep these drug molecules, have the practical significance of highly significant at the intravital effective blood drug concentration of patient.
Equally, various natural medicinal ingredients such as flavonoid, terpene, quinones, steroidal and various alkaloid that isolating protein is outer, the requirement that its performance is further improved is also arranged clinically, as pharmacology transformation period of improving medicine, strengthen its stability and arrive target site probability, improve water-soluble, change route of administration and improve bioavailability etc.
At present, polyethyleneglycol derivative is widely used for combining to prolong the physiology transformation period of described medicine with protein, polypeptide and other treatment medicine, reduces its immunogenicity and toxicity.In clinical use, PEG and derivative thereof have obtained using widely in a lot of commercial medicines as the carrier of making pharmaceutical preparation, and the trial that PEG is bonded to drug molecule has also been obtained significant progress in last decade, in many approval medicines, be widely used, as
, the binding substances of a kind of alpha-interferon and polyoxyethylene glycol has just shown longer circulating half-life and better result of treatment.The binding substances of taxol and polyoxyethylene glycol has also reduced toxicity accordingly and has prolonged biological activity.They are quite clear in the intravital metabolic process of people, are a kind of safe, the drug modified agent that has no side effect.
The derivative of many PEG is in the news, and for example No. 5672662 United States Patent (USP) reported linear PEG propionic acid and butyric acid and their NHS ester.In No. 5643575 United States Patent (USP), disclosed a kind of U-shaped ramose polyethylene glycol structures.The polyoxyethylene glycol component of two linearities is connected on a molecule or a kind of structure by two identical functional groups (such as two amino or two carboxyls) in this new polyethyleneglycol derivative.Among the embodiment disclosed herein, it is that PEG and Methionin by linearity makes that the contriver points out with ramose PEG, and Methionin is a kind of amino acid that has two amino.In Chinese patent (ZL03801105.0), showed a kind of Y shape ramose polyethyleneglycol derivative.
Though multiple polyethylene active derivatives based on polyoxyethylene glycol is provided in the prior art, when these derivatives are used in conjunction with some drugs,, has been difficult to play its due effect especially to the locator qualification aspect of drug molecule.Find that from practice part locator qualification protein is the chemical reaction inertia because the functional group of required modification is nonactive group.Existing modification exists modification degree low with polyethyleneglycol derivative, unfavorable factors such as combination stability difference.Therefore, the application's purpose is to overcome the deficiencies in the prior art, and a kind of novel polyethylene active derivatives is provided.This technology has increased modification degree and transformation efficiency, and the stability of product is increased.
In the prior art, known " oligopeptides " is amino acids formed peptide quasi-molecule more than two.When wherein to have two amino acid at least be inequality, just can form diversified oligopeptides molecule.The present inventor finds, when these oligopeptides molecules and plural polyoxyethylene glycol chain link company, also keeps at least one activable group simultaneously, has just formed a kind of polyethyleneglycol derivative of uniqueness.
Provided by the invention is exactly the reactive derivative with poly glycol chain that this novel polyoxyethylene glycol and oligopeptides form, its preparation method and with the binding substances of drug molecule.
Summary of the invention
One aspect of the present invention provides a kind of compound of being represented by general formula I that is formed by polyoxyethylene glycol and oligopeptides:
Wherein:
PEG
iBe identical or different polyglycol chain, described polyglycol chain has following general formula:
Wherein:
R is H or C
1-12Alkyl;
N is any integer of 6～1300;
I is 1,2 ..., j;
J is 〉=2 integer;
X
iBe identical or different linking groups, described linking group is selected from by-(CH
2)
mOCOO-,-(CH
2)
mOCONH-,-(CH
2)
mNHCOO-,-(CH
2)
mNHCONH-,-(CH
2)
mCOO-,-(CH
2)
mThe group that CONH-forms, wherein, m is the integer of 0-10;
A stems from oligopeptides, and described oligopeptides comprises 2-20 amino acid, and it is inequality wherein having two amino acid at least;
F
pRepresent identical or different active groups, described active group is selected from the group of being made up of hydroxyl, carboxyl, ester group, acyl chlorides, hydrazide group, dimaleoyl imino and pyridine disulphide;
P is 1,2 ..., q;
Q is 〉=1 integer.
In a preferred embodiment of the invention, the R in the described polyglycol chain is selected from the group of being made up of hydrogen, methyl, ethyl, sec.-propyl.N in the described polyglycol chain is 60-800.A stems from Gly-Lys, Gly-Lys (Gly), Glu-Lys or Gly-Lys (Gly)-Lys.
Preferably, the invention provides a kind of compound of representing by general formula I I that forms by polyoxyethylene glycol and oligopeptides:
Wherein: mPEG is CH
3O-(CH
2CH
2O)
n-.
In the preferred embodiment of general formula I I, A stems from Gly-Lys, Gly-Lys (Gly) or Glu-Lys.Preferably, the invention provides a kind of compound of representing by general formula III that forms by polyoxyethylene glycol and oligopeptides:
In preferred embodiments, the A in the general formula III stems from Gly-Lys, Gly-Lys (Gly), perhaps Glu-Lys.
Another aspect of the present invention provides the method for the compound that is formed by polyoxyethylene glycol and oligopeptides that a kind of preparation represented by general formula I, described method comprises: under neutrality or alkaline condition, with poly glycol monomethyl ether ester and the reaction of described oligopeptides, product carries out separation and purification, and then carries out according to F
pThe type active group transform.
Preferably, described poly glycol monomethyl ether ester is poly glycol monomethyl ether-acetate-N maloyl imines ester or is poly glycol monomethyl ether-orthocarbonic acid-N maloyl imines ester.
Another aspect of the present invention provides the compound that is formed by polyoxyethylene glycol and oligopeptides and passes through its active group F
pWith the formed binding substances of drug molecule.
Preferably, described drug molecule is selected from the group of being made up of amino acid, protein, enzyme, nucleosides, carbohydrate, organic acid, glucoside, flavonoid, quinones, terpene, the plain phenols of phenylpropyl alcohol, steroidal and glucoside thereof, alkaloid.Described drug molecule can be a genetically engineered drug, be selected from by α-, β-or gamma-interferon, growth hormone, EPO, GCSF, interleukin, N,O-Diacetylmuramidase, the group that antibody and antibody fragment are formed.
Description of drawings
The GFC spectrogram of Fig. 1 binding substances that to be embodiment 9 double center chain polyethylene active derivatives (3) link with N,O-Diacetylmuramidase.
Concrete implementation content
In compound provided by the present invention, described polyoxyethylene glycol should comprise the homopolymer of ethylene glycol and the multipolymer of ethylene glycol and propylene glycol etc.
Be example below, illustrate that the present invention has the preparation method of the reactive derivative with poly glycol chain of novel polyoxyethylene glycol and oligopeptides formation with polyoxyethylene glycol.
Polyglycol chain
The general structure of polyoxyethylene glycol (PEG) chain is as follows:
Wherein:
R is H or C
1-12Alkyl;
N can be any integer, characterizes its polymerization degree.
When R was low alkyl group, R can be any low alkyl group that contains 1-6 carbon atom, as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, n-pentyl or n-hexyl.Its exemplary compounds is methoxy poly (ethylene glycol) (mPEG), and the multipolymer of the similar thing of other polyoxyethylene glycol or ethylene glycol and propylene oxide etc. also can be used for this invention and uses.
For polyoxyethylene glycol, generally adopt molecular weight to be represented, be 300～60,000 dalton as long as make the molecular weight of the polyoxyethylene glycol that forms binding substances, this is equivalent to n is about 6～1300.More preferably, n is 78,112 and 450, and this is 3500,5000 and 20,000 corresponding to molecular weight respectively.Owing to, preferably use the molecular weight characterization polyethylene glycol polymer, rather than represent the unit of repetition certainly in the PEG polymkeric substance with Integer n usually by its molecular-weight average but not repeat the potential unhomogeneity of the initial PEG compound that the unit limits certainly.The initial PEG compound of various molecular weight can or can obtain from commercial source by the preparation of the currently known methods in this area.
Oligopeptides
General alleged oligopeptides is an amino acids formed peptide quasi-molecule more than two.The oligopeptides molecule can be a bioactive molecule, also can be inactive molecule.There is the bioactive molecule oligopeptides molecule of part to obtain application clinically, as GHK (Gly-His-Lys), His Tag (His-His-His-His-His-His), KHG (Lys-His-Gly), RGD (Arg-Gly-Asp), TRH (Gly-His-Pro), GSH (Glu-Cys-Gly), TP-5 (Arg-Lys-Asp-Val-Tyr) etc., the physiologically active of the uniqueness that these small molecules oligopeptides are shown is the emphasis that people paid close attention to always.They all play an important role at aspects such as molecular recognition, signal transmission, albumen integration, lymphocyte identification, cancer metastasis.As: contain the adhesive effect that the RGD polypeptide of sequence can suppress various kinds of cell, have the effect of tangible anti metastasis.And TP-5 (thymopeptide-5) also has outstanding performance in lymphocyte identification and activation.
Employed oligopeptides is meant and comprises 2-20 amino acid among the present invention, and for example, following listed oligopeptides molecule can be used for the present invention:
Dipeptides: GK (Gly-Lys), (Lys-Glu);
Tripeptides: (Gly-Lys (Gly)), GHK (Gly-His-Lys), KHG (Lys-His-Gly), RGD (Arg-Gly-Asp), TRH (Gly-His-Pro), GSH (Glu-Cys-Gly);
Tetrapeptide: (Gly-Lys (Gly)-Lys);
Pentapeptide: TP-5 (Arg-Lys-Asp-Val-Tyr);
Other: His Tag (His-His-His-His-His-His), or the like.
Employed in the present invention oligopeptides is preferably from nonactive oligopeptides, and wherein to have two amino acid at least be inequality:
The reactive derivative with poly glycol chain according to polyoxyethylene glycol of the present invention and oligopeptides formation can pass through the active group in the oligopeptides, as: amino, sulfydryl, hydroxyl and carboxyl, carry out obtaining behind the Pegylation.Can adopt very general synthetic and preparation method to obtain in the art.According to the structural compounds of different claims, need to adopt corresponding synthetic and preparation method, particular case can be with reference to the embodiment of back and the various technical literature data and the patent achievement of this area.
Active group
In the application of the reactive derivative that the polyoxyethylene glycol according to the present invention and oligopeptides form with poly glycol chain, active group F
pDecisive role, the derivative of different activities group has different purposes.The introducing of these functional groups is with the Application Areas and the suitable structures of this derivative of decision.The most frequently used active group is a N-maloyl imines ester, shown in general formula I I and III formula.
Same, also can carry out modification, and then obtain the aldehyde radical active group by the synthetic method that can obtain easily in this area to be correlated with:
Same, also can carry out modification, and then obtain the maleimide functional group by the synthetic method that can obtain easily in this area to be correlated with:
All contain functional groups such as activated amino, carboxyl, hydroxyl in many pharmaceutical cpds, they all combine with compositions such as monose, polysaccharide, nucleosides, polymerized nucleoside, phosphoryls in vivo usually, to be formed on activated pharmacology structure in the organism.
Therefore the polyethyleneglycol derivative after functional group improves can to substitute biological organic molecule, overcome the biological organic molecule defect that the physiology transformation period is short in vivo, duration of efficacy is short by identical mode and these drug molecule combinations.
The reactive derivative with poly glycol chain that polyoxyethylene glycol of the present invention and oligopeptides form uses suitable active group to provide binding substances with drug molecule, described active group that free amine group in protein, polypeptide or other natural drugs, hydroxyl, thiol etc. are coupled together with the PEG derivative.To macromolecular protein or polypeptide, can bonding on one to a plurality of described reactive derivatives, to improve drug molecule physiological action in vivo; To micromolecular natural drug activeconstituents, can in a described reactive derivative, connect one to a plurality of drug molecules by suitable active group, to guarantee suitable drug level and slow-release function is provided.
More than various Application Areass just to the medical applications of this PEG derivative provide one may reference pattern, concrete use and select and need add their confirmation according to animal pharmacology, toxicity and necessary link such as clinical.
In binding substances of the present invention, drug molecule is selected from the group of being made up of amino acid, protein, enzyme, nucleosides, carbohydrate, organic acid, glucoside, flavonoid, quinones, terpene, the plain phenols of phenylpropyl alcohol, steroidal and glucoside thereof, alkaloid.Wherein, described pharmaceutical grade protein molecule is selected from by the interferons medicine, EPO class medicine, auxins medicine, the group that the antibody class medicine is formed.
Binding substances of the present invention can the pure compound form or the appropriate drug composition carry out administration, the reagent that can adopt any acceptable administering mode or be used for similar applications carries out.Therefore, the administering mode that adopts can be selected in through port, the nose, rectum, transdermal or drug administration by injection mode, its form is solid, semisolid, lyophilized powder or liquid preparation form administration, for example, tablet, suppository, pill, soft hard-gelatin capsules, powder, solution, suspensoid or aerosol etc., the preferred presented in unit dosage form that adopts the simple administration that is applicable to exact dosage desired.Composition can comprise conventional pharmaceutical carrier or vehicle and as the binding substances of the present invention of activeconstituents (one or more), in addition, also can comprise other medicament, carrier, assistant agent etc.
Usually, according to required administering mode, pharmaceutically acceptable composition will comprise the suitable pharmaceutical excipient of about 1 to about 99 weight % binding substances of the present invention and 99 to 1 weight %.Preferred composition comprises the binding substances of the present invention of about 5 to 75 weight %, and all the other are suitable pharmaceutical excipient.
Preferred route of administration is a drug administration by injection, adopts conventional per daily dose scheme, and this scheme can be adjusted according to the severity of disease.Binding substances of the present invention or its pharmacy acceptable salt also can be mixed with the injection agent, for example use about 0.5 to about 50% activeconstituents to be scattered in the medicinal adjuvant that can adopt the liquid form administration, example is water, salt solution, dextrose hydrate, glycerine, ethanol etc., thereby forms solution or suspensoid.
Can adopt the pharmaceutical composition of liquid form administration for example can dissolve, be scattered in the carrier by the medicinal adjuvant of means such as dissolving, dispersion with binding substances of the present invention (about 0.5 to about 20%) and selectivity existence, the example of carrier is water, salt solution, dextrose hydrate, glycerine, ethanol etc., thereby forms solution or suspensoid.
If necessary, pharmaceutical composition of the present invention also can comprise a spot of auxiliary substance, as wetting agent or emulsifying agent, pH buffer reagent, antioxidant etc., for example: citric acid, Arlacel-20, Emulphor FM, Yoshinox BHT etc.
The actual fabrication method of such formulation is that those skilled in the art is known or conspicuous, for example can be referring to Remington ' s Pharmaceutical Sciences, the 18th edition, (Mack Publishing Company, Easton, Pennsylvania, 1990).In any case according to technology of the present invention, employed composition will contain the binding substances of the present invention for the treatment of significant quantity, to be used for the treatment of corresponding disease.
Embodiment
Below in conjunction with case description binding substances of the present invention and preparation method thereof, it does not limit the present invention, and scope of the present invention is defined by the claims.
Embodiment 1
The double-chain polyethylene glycol reactive derivative (1) that synthesizing polyethylene glycol acetate and Gly-Lys dipeptides form
With 20 gram molecular weights is that 20,000 poly glycol monomethyl ether acetate N-maloyl imines ester (mPEG-SCM) is dissolved in the phosphate buffered saline buffer that contains 1 gram Gly-Lys dipeptides, and pH=8.5 stirred three hours under the room temperature.Hydrochloric acid is transferred pH=3, and dichloromethane extraction three times merges organic phase, the organic phase anhydrous sodium sulfate drying, and vacuum steams solvent, adds 200 milliliters of isopropanol precipitatings.Sedimentation and filtration, vacuum-drying.The PEG acid product of branching type can further be used the ion-exchange chromatography purifying.Productive rate: 8 grams (40%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 3 hydrogen), 4.15 (t, 2 hydrogen).
0.5 the PEG acid of gram branching type (making by the last step), be dissolved in 5 milliliters of methylene dichloride, in solution, add 7 milligrams of N-maloyl imines (NHS) and 13 milligrams of dicyclohexylcarbodiimide (DCC), stirred 6 hours under the room temperature, remove by filter precipitation, vacuum steams solvent, and residuum adds in 20 milliliters of Virahols (IPA), filters collecting precipitation and dry in a vacuum.Productive rate: 0.48 gram (96%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 6 hydrogen) 2.81 (s, 4 hydrogen), 3.96 (s, 2 hydrogen), 4.07 (s, 2 hydrogen), 4.48 (t, 2 hydrogen).
Embodiment 2
The double-chain polyethylene glycol reactive derivative (2) that synthesizing polyethylene glycol and Gly-Lys dipeptides form
With 20 gram molecular weights is that poly glycol monomethyl ether orthocarbonic acid-N-maloyl imines ester (mPEG-SC) of 20,000 is dissolved in the borate buffer solution that contains 1 gram Gly-Lys dipeptides, and pH=10.2 stirs under the room temperature and spends the night.Hydrochloric acid is transferred pH=3, and dichloromethane extraction three times merges organic phase, the organic phase anhydrous sodium sulfate drying, and vacuum steams solvent, adds 200 milliliters of isopropanol precipitatings.Sedimentation and filtration, vacuum-drying.The PEG acid product of branching type can further be used the ion-exchange chromatography purifying.Productive rate: 8 grams (40%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 3 hydrogen), 4.32 (t, 2 hydrogen).
0.5 the PEG acid of gram branching type (making by the last step), be dissolved in 5 milliliters of methylene dichloride, in solution, add 7 milligrams of N-maloyl imines (NHS) and 13 milligrams of dicyclohexylcarbodiimide (DCC), stirred 6 hours under the room temperature, remove by filter precipitation, vacuum steams solvent, and residuum adds in 20 milliliters of Virahols (IPA), filters collecting precipitation and dry in a vacuum.Productive rate: 0.48 gram (96%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 6 hydrogen) 2.81 (s, 4 hydrogen), 4.15 (s, 2 hydrogen), 4.07 (t, 2 hydrogen), 4.48 (t, 2 hydrogen).
The double-chain polyethylene glycol reactive derivative (3) that synthesizing polyethylene glycol and Gly-Lys (Gly) tripeptides forms
With 20 gram molecular weights is that poly glycol monomethyl ether orthocarbonic acid-N-maloyl imines ester (mPEG-SC) of 20,000 is dissolved in the borate buffer solution that contains 1.2 gram Gly-Lys (Gly) polypeptide, and pH=10.2 stirs under the room temperature and spends the night.Hydrochloric acid is transferred pH=3, and dichloromethane extraction three times merges organic phase, the organic phase anhydrous sodium sulfate drying, and vacuum steams solvent, adds 200 milliliters of isopropanol precipitatings.Sedimentation and filtration, vacuum-drying.The PEG acid product of branching type can further be used the ion-exchange chromatography purifying.Productive rate: 8 grams (40%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 3 hydrogen), 4.32 (t, 2 hydrogen).
0.5 the mPEG acid of gram branching type (making by the last step), be dissolved in 5 milliliters of methylene dichloride, in solution, add 7 milligrams of N-maloyl imines (NHS) and 13 milligrams of dicyclohexylcarbodiimide (DCC), stirred 6 hours under the room temperature, remove by filter precipitation, vacuum steams solvent, and residuum adds in 20 milliliters of Virahols (IPA), filters collecting precipitation and dry in a vacuum.Productive rate: 0.48 gram (96%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 6 hydrogen), 2.81 (s, 4 hydrogen), 4.15 (s, 2 hydrogen), 4.07 (t, 2 hydrogen), 4.48 (t, 2 hydrogen).
Embodiment 4
The three chain polyalkylene glycol reactive derivatives (4) that synthesizing polyethylene glycol and Gly-Lys-Lys tripeptides form
With 20 gram molecular weights is that poly glycol monomethyl ether orthocarbonic acid-N-maloyl imines ester (mPEG-SC) of 20,000 is dissolved in the borate buffer solution that contains 1.5 gram Gly-Lys-Lys polypeptide, and pH=10.2 stirs under the room temperature and spends the night.Hydrochloric acid is transferred pH=3, and dichloromethane extraction three times merges organic phase, the organic phase anhydrous sodium sulfate drying, and vacuum steams solvent, adds 200 milliliters of isopropanol precipitatings.Sedimentation and filtration, vacuum-drying.The PEG acid product of branching type can further be used the ion-exchange chromatography purifying.Productive rate: 8 grams (40%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 3 hydrogen), 4.32 (t, 2 hydrogen).
0.5 the PEG acid of gram branching type (making by the last step), be dissolved in 5 milliliters of methylene dichloride, in solution, add 7 milligrams of N-maloyl imines (NHS) and 13 milligrams of dicyclohexylcarbodiimide (DCC), stirred 6 hours under the room temperature, remove by filter precipitation, vacuum steams solvent, and residuum adds in 20 milliliters of Virahols (IPA), filters collecting precipitation and dry in a vacuum.Productive rate: 0.48 gram (96%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 6 hydrogen) 2.81 (s, 4 hydrogen), 4.15 (s, 2 hydrogen), 4.07 (t, 2 hydrogen), 4.48 (t, 2 hydrogen).
Embodiment 5
The double-chain polyethylene glycol reactive derivative (5) that synthesizing polyethylene glycol and Lys-Glu dipeptides form
With 20 gram molecular weights is that poly glycol monomethyl ether orthocarbonic acid-N-maloyl imines ester (mPEG-SC) of 20,000 is dissolved in the borate buffer solution that contains 1.2 gram Lys-Glu dipeptides, and pH=10.2 stirs under the room temperature and spends the night.Hydrochloric acid is transferred pH=3, and dichloromethane extraction three times merges organic phase, the organic phase anhydrous sodium sulfate drying, and vacuum steams solvent, adds 200 milliliters of isopropanol precipitatings.Sedimentation and filtration, vacuum-drying.The PEG acid product of branching type can further be used the ion-exchange chromatography purifying.Productive rate: 8 grams (40%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 3 hydrogen), 4.32 (t, 2 hydrogen).
Embodiment 6
Synthetic polyoxyethylene glycol with aldehyde radical and the formation of Gly-Lys (Gly) tripeptides
Double-chain polyethylene glycol reactive derivative (6)
With 10 gram molecular weights is that the double-chain polyethylene glycol reactive derivative (3) that 40,000 polyoxyethylene glycol and Gly-Lys (Gly) tripeptides form is dissolved in 50 milliliters of methylene dichloride, adds 0.1 milliliter 2,2-diethoxy ethamine, and nitrogen protection is stirred down and is spent the night.Decompression concentrated solution adds 100 milliliters of ether.Filter collecting precipitation vacuum-drying, productive rate: 9.5 grams (95%).
9 gram double-chain polyethylene glycol diethyl acetals (being made by the last step) are dissolved in the phosphate buffer soln of pH=5, and reaction is 48 hours under the room temperature.Add 30 gram sodium-chlor dissolvings, dichloromethane extraction three times merges organic phase, concentrating under reduced pressure, and ether sedimentation filters collecting precipitation, the ether washing.Productive rate: 7.2 grams (80%).NMR (D
2O): 1.80 (q, 1 hydrogen), 2.72 (t, 1 hydrogen), 3.29 (s, 3 hydrogen), 3.61 (s, hydrogen among the PEG), 5.06 (t, 1 hydrogen).Fusing point: 56-58 ℃.
Embodiment 7
Synthetic have active polyoxyethylene glycol and the formation of Gly-Lys (Gly) tripeptides to thiol
Double-chain polyethylene glycol reactive derivative (7)
With 10 gram molecular weights is 40; the double-chain polyethylene glycol reactive derivative (3) that 000 polyoxyethylene glycol and Gly-Lys (Gly) tripeptides forms is dissolved in 50 milliliters of methylene dichloride; add 0.1 gram N-(2-amino-ethyl)-3-dimaleoyl imino-propionic acid amide, nitrogen protection is stirred down and is spent the night.Decompression concentrated solution adds 100 milliliters of ether.Filter collecting precipitation vacuum-drying, productive rate: 9.5 grams (95%).
Embodiment 8
Synthetic polyoxyethylene glycol with double-active group and Gly-Lys (Gly)-Lys tetrapeptide formation
Double-chain polyethylene glycol reactive derivative (8)
With 10 gram molecular weights is 40,000 polyoxyethylene glycol and NH
2The double-chain polyethylene glycol reactive derivative (3) that-Gly-Lys (Gly)-OH tripeptides forms is dissolved in 50 milliliters of methylene dichloride, adds 0.2 gram NH
2-Lys (Boc)-OH, stirring reaction is 18 hours under the room temperature, adds 20 milliliters of trifluoroacetic acids and handles organic phase saturated nacl aqueous solution washed twice 1 hour; anhydrous sodium sulfate drying filters, under the nitrogen protection, and concentrating under reduced pressure; add 200 milliliters of isopropanol precipitatings, sedimentation and filtration, vacuum-drying.
The previous step products therefrom is dissolved in 20 milliliters of methylene dichloride, adds 0.15 gram, 3-dimaleoyl imino-propionic acid-(N-hydroxyl-succimide) ester; reaction is spent the night under the room temperature, removes by filter precipitation, concentrating under reduced pressure under the nitrogen protection; add 100 milliliters of isopropanol precipitatings, sedimentation and filtration, vacuum-drying.
With 1 gram double-chain polyethylene glycol acid of previous step gained (making by the last step), be dissolved in 20 milliliters of methylene dichloride, in solution, add 20 milligrams of N-maloyl imines (NHS) and 0.2 gram dicyclohexylcarbodiimide (DCC), stirred 6 hours under the room temperature, remove by filter precipitation, vacuum steams solvent, and residuum adds in 20 milliliters of Virahols (IPA), filters collecting precipitation and dry in a vacuum.Productive rate: 0.98 gram (98%).NMR (DMSO) 3.50 (br m, the hydrogen among the PEG), 3.24 (s, 6 hydrogen), 2.81 (s, 4 hydrogen), 4.15 (s, 2 hydrogen), 4.07 (t, 2 hydrogen), 4.48 (t, 2 hydrogen).
Embodiment 9
The binding substances that double-chain polyethylene glycol reactive derivative (3) and N,O-Diacetylmuramidase (Lysozyme) link
With 1 milligram of N,O-Diacetylmuramidase be dissolved in 1 milliliter of 50 mmole/liter, in the phosphoric acid buffer of pH=7.0.27.8 milligrams of double-chain polyethylene glycol reactive derivatives (3) are added in the lysozyme soln.The molar ratio of protein and PEG is 1: 10.Room temperature concussion reaction 3 hours.GFC detects the replacement rate, and two PEG replace N,O-Diacetylmuramidase 4.8%, and single PEG replaces N,O-Diacetylmuramidase 48.0%, N,O-Diacetylmuramidase 47.2%.
The binding substances that double-chain polyethylene glycol reactive derivative (3) and growth hormone (hGH) link
Growth hormone with 50 mmoles/liter the pH=7.0 phosphoric acid buffer be diluted to 1 mg/ml.18.0 milligrams of double-chain polyethylene glycol reactive derivatives (3) are added in 1 milliliter of growth hormone solution.The molar ratio of protein and PEG is 1: 10.Room temperature concussion reaction 3 hours.The product that is not contained free hGH through the ion-exchange chromatography separation and purification.
The binding substances that double-chain polyethylene glycol reactive derivative (6) and GCSF link
GCSF with 50 mmoles/liter the pH=7.0 phosphoric acid buffer be diluted to 1 mg/ml.21.3 milligrams of double-chain polyethylene glycol reactive derivatives (3) are added in 1 milliliter of GCSF solution.The molar ratio of protein and PEG is 1: 10.Room temperature concussion reaction 3 hours.The product that is not contained free GCSF through the ion-exchange chromatography separation and purification.
Present embodiment illustrates the preparation of drug combination process of representative parenteral administration, and described composition comprises binding substances of the present invention.
Composition
Binding substances 2 grams of embodiment 8
0.9% salt brine solution to 100 milliliter
Binding substances 2 grams of embodiment 8 are dissolved in 0.9% salt brine solution, obtain 100 milliliters used for intravenous injection solution,, under aseptic condition, pack its membrane filtration filtration of material by 0.2 μ m.
Claims (14)
1. compound that forms by polyoxyethylene glycol and oligopeptides of representing by general formula I:
Wherein:
PEG
iBe identical or different polyglycol chain, described polyglycol chain has following general formula:
Wherein:
R is H or C
1-12Alkyl;
N is any integer of 6～1300;
I is 1,2 ..., j;
J is 〉=2 integer;
X
iBe identical or different linking groups, described linking group is selected from by-(CH
2)
mOCOO-,-(CH
2)
mOCONH-,-(CH
2)
mNHCOO-,-(CH
2)
mNHCONH-,-(CH
2)
mCOO-,-(CH
2)
mThe group that CONH-forms, wherein, m is the integer of 0-10;
A stems from oligopeptides, and described oligopeptides is selected from Gly-Lys, Gly-Lys (Gly), Glu-Lys or Gly-Lys (Gly)-Lys; F
pRepresent identical or different active groups, described active group is selected from the group of being made up of hydroxyl, carboxyl, ester group, acyl chlorides, hydrazide group, aldehyde radical, N-maloyl imido grpup, dimaleoyl imino and pyridine disulphide;
P is 1,2 ..., q;
Q is 〉=1 integer.
2. compound as claimed in claim 1, wherein, the described R in the described polyglycol chain is selected from the group of being made up of hydrogen, methyl, ethyl, sec.-propyl.
3. compound as claimed in claim 1, wherein, the described n in the described polyglycol chain is 60-800.
4. compound as claimed in claim 1, wherein, described compound is shown in general formula I I:
Wherein: mPEG is CH
3O-(CH
2CH
2O) n-.
5. compound as claimed in claim 4, wherein, described compound is shown in general formula III:
6. compound as claimed in claim 4, wherein, described compound is shown in general formula I V:
7. compound as claimed in claim 5, wherein, described compound is selected from one of group that the compound represented by following general formula forms:
8. compound as claimed in claim 1, wherein, described compound is selected from one of group that the compound represented by following general formula forms:
Wherein: k is the integer of 1-10.
9. method for preparing the compound that forms by polyoxyethylene glycol and oligopeptides as claimed in claim 1, described method comprises: under neutrality or alkaline condition, with poly glycol monomethyl ether ester and the reaction of described oligopeptides, product carries out separation and purification, and then carries out the type active group transformation according to Fp.
10. method as claimed in claim 9, wherein, described poly glycol monomethyl ether ester is poly glycol monomethyl ether-acetate-N maloyl imines ester or is poly glycol monomethyl ether-orthocarbonic acid-N maloyl imines ester.
11. pass through its active group F as the described compound of one of claim 1-8
pWith the formed binding substances of drug molecule.
12. binding substances as claimed in claim 11, wherein, described drug molecule is selected from the group of being made up of amino acid, protein, enzyme, nucleosides, carbohydrate, organic acid, glucoside, flavonoid, quinones, terpene, the plain phenols of phenylpropyl alcohol, steroidal and glucoside thereof, alkaloid.
13. binding substances as claimed in claim 11, wherein, described drug molecule is a genetically engineered drug.
14. binding substances as claimed in claim 13, wherein, described genetically engineered drug be selected from by α-, β-or gamma-interferon, growth hormone, EPO, GCSF, interleukin, N,O-Diacetylmuramidase, the group that antibody and antibody fragment are formed.
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| CN2006101500113A CN101168594B (en) | 2006-10-24 | 2006-10-24 | Polyglycol active derivative with oligopeptide as framework, preparation method thereof and conjugate of with pharmaceutical molecule |
| PCT/CN2007/003030 WO2008052428A1 (en) | 2006-10-24 | 2007-10-24 | Preparation method and conjugate with drug molecule thereof |
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| CN102180807B (en) * | 2011-03-17 | 2014-07-16 | 中国科学院化学研究所 | Amino acid and preparation method thereof |
| CN105218635A (en) * | 2014-06-11 | 2016-01-06 | 首都医科大学 | The β-carboline that Trp-Trp-Trp pentapeptide is modified, its preparation, nanostructure, active and application |
| JP7205827B2 (en) * | 2018-03-29 | 2023-01-17 | 日油株式会社 | Degradable polyethylene glycol derivative |
| US20220362397A1 (en) * | 2019-09-26 | 2022-11-17 | Nof Corporation | Heterobifunctional monodispersed polyethylene glycol having peptide linker |
| CN113831529A (en) * | 2020-06-24 | 2021-12-24 | 江苏众红生物工程创药研究院有限公司 | Preparation method of monomethoxy polyethylene glycol carboxylic acid and functional derivative thereof |
| CN112280755B (en) * | 2020-11-06 | 2023-03-14 | 深圳瑞德林生物技术有限公司 | Mutant enzyme, application thereof and process for preparing sanshengtai by enzyme catalysis method |
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| US5643575A (en) * | 1993-10-27 | 1997-07-01 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
| CN1461762A (en) * | 2002-05-30 | 2003-12-17 | 中国科学院过程工程研究所 | Method of preparing branched polyethylene glycol |
| CN1511861A (en) * | 2002-12-26 | 2004-07-14 | 北京键凯科技有限公司 | Polyglycol fatty acid derivative and its medicinal combination |
| CN1706865A (en) * | 2004-06-11 | 2005-12-14 | 北京键凯科技有限公司 | Branched polyglycol-amino acid oligopeptide and its active derivative and medicinal composition |
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| CN100475269C (en) * | 2002-03-05 | 2009-04-08 | 北京键凯科技有限公司 | Binding agent of hydrophilic polymer-glutamic acid oligopeptide and medicinal molecular, composition containing said binding agent and use thereof |
| WO2003076490A1 (en) * | 2002-03-13 | 2003-09-18 | Beijing Jiankai Technology Co., Ltd. | Hydrophilic polymer derivate with y type branch and preparation method of it medical composite comprising above compound |
| CN1626549A (en) * | 2003-12-11 | 2005-06-15 | 中国人民解放军军事医学科学院基础医学研究所 | Carbowax alcoholized ramification of TimopEntin, combination of medication and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5643575A (en) * | 1993-10-27 | 1997-07-01 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
| CN1461762A (en) * | 2002-05-30 | 2003-12-17 | 中国科学院过程工程研究所 | Method of preparing branched polyethylene glycol |
| CN1511861A (en) * | 2002-12-26 | 2004-07-14 | 北京键凯科技有限公司 | Polyglycol fatty acid derivative and its medicinal combination |
| CN1706865A (en) * | 2004-06-11 | 2005-12-14 | 北京键凯科技有限公司 | Branched polyglycol-amino acid oligopeptide and its active derivative and medicinal composition |
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