WO2008052428A1

WO2008052428A1 - Preparation method and conjugate with drug molecule thereof

Info

Publication number: WO2008052428A1
Application number: PCT/CN2007/003030
Authority: WO
Inventors: Xuan Zhao; Qiang Gu
Original assignee: Beijing Jenkem Technology Co. Ltd
Priority date: 2006-10-24
Filing date: 2007-10-24
Publication date: 2008-05-08
Also published as: CN101168594B; CN101168594A

Abstract

A class of polyethylene glycol-oligopeptide derivative is represented by the following general formula (I): (PEGi Xi )j A ( Fp)q (I), where: i, j, p and q are integers; i=1, 2,&mldr;, j; j≥=2; p=1, 2,&mldr;, q; q≥=1; PEGi is a polyethylene glycol chain; Xi is a linker group; A is a oligopeptide selected from Lys-Gly, Gly-Lys (Gly), Gly-Lys-Lys or Gly-Lys(Gly)-Lys; Fp is a active group. Their preparation methods and conjugates bonding medicine molecule by Fp are also provided. They may be useful for improving stability of medicine molecule in vivo or ensuring appropriate drug concentration and providing sustained-release function.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polyethylene glycol active derivative, and more particularly to an active derivative of a polyethylene glycol having an oligopeptide as a skeleton, And a preparation method thereof and a combination of the derivative and a drug molecule. BACKGROUND OF THE INVENTION A variety of natural and recombinant proteins and polypeptides have certain pharmaceutical availability. The various purified and isolated preparations can be administered by parenteral routes for various therapeutic indications. However, proteins that are administered parenterally may be immunogenic, may be relatively insoluble in water, or may have a short pharmacological half-life. Therefore, how to improve and maintain the effective blood concentration of these drug molecules in patients has a very significant practical significance. Similarly, various natural pharmaceutical ingredients other than protein, such as flavonoids, terpenoids, terpenoids, steroids, and various alkaloids, have clinically required further improvements in their properties, such as increasing the pharmacological half-life of the drug, Enhance its stability and chances of reaching the target site, improve water solubility, change the route of administration, and improve bioavailability. Currently, polyethylene glycol derivatives are widely used in combination with proteins, polypeptides, and other therapeutic agents to prolong the physiological half-life of the drug, reducing its immunogenicity and toxicity. In clinical use, PEG and its derivatives have been widely used as carriers for the preparation of pharmaceutical preparations, and attempts to bind PEG to drug molecules have also been greatly developed in the last decade. Widely used in many approved drugs, such as PEGasys®, a combination of alpha-interferon and polyethylene glycol, exhibits longer circulating half-lives and better therapeutic effects. The combination of paclitaxel and polyethylene glycol also correspondingly reduces toxicity and prolongs biological activity. Their metabolic processes in the human body are quite clear, and they are safe, no side effects drug modifiers. A number of derivatives of PEG have been reported. For example, U.S. Patent No. 5,672,662 discloses linear PEG propionic acid and butyric acid and their NHS esters. A U-branched polyethylene glycol structure is disclosed in U.S. Patent No. 5,643,575. In this new polyethylene glycol derivative, two linear polyethylene glycol components are linked to one molecule or one structure by two identical functional groups, such as two amino groups or two carboxyl groups. In one embodiment disclosed herein, the inventors have indicated that branched PEG is made from linear PEG and lysine, while lysine is an amino acid with two amino groups. In the Chinese patent (ZL03801105.0), a Y-branched polyethylene glycol derivative is shown. Although a variety of polyethylene glycol-based polyethylene glycol active derivatives have been provided in the prior art, these derivatives are difficult to use in combination with certain drugs, especially in the localization of drug molecules. The role it should have. It has been found in practice that a partially localized modified protein is chemically inert due to the functional group to be modified being an inactive group. The existing modified polyethylene glycol derivatives have disadvantages such as low degree of modification and poor binding stability. By separating and purifying the modified product and measuring the activity, the ideal content in the final product is generally no more than 30%, and most of them are unfavorable or not. A modified compound that significantly improves performance. The technology improves the structure of the active end portion of the polyethylene glycol active intermediate to achieve a certain spatial configuration, thereby improving the degree of localization modification and conversion. Accordingly, it is an object of the present application to overcome the deficiencies of the prior art and to provide a novel polyethylene glycol reactive derivative. This technology increases the degree of modification and conversion, and the stability of the product is improved. In the prior art, "oligopeptides" are known to be peptide molecules formed by two or more amino acids. When at least two of the amino acids are different, a wide variety of oligopeptide molecules can be formed. The inventors of the present application have discovered that when these oligopeptide molecules are bonded to more than two polyethylene glycol chains while retaining at least one activatable group, a unique polyethylene glycol derivative is formed. The invention provides a novel derivative of the polyethylene glycol chain formed by the novel polyethylene glycol and the oligopeptide, a preparation method thereof and a combination with a drug molecule. SUMMARY OF THE INVENTION One aspect of the present invention provides a compound formed from polyethylene glycol and an oligopeptide represented by Formula I -

( ^PEG i― ^X i-) — ^A —^ ^F p) _q (I)

Wherein - PEGi is the same or different polyethylene glycol chain, and the polyethylene glycol chain has the following formula:

R_0 -4 , - CH ₂ CH ₂ 0―)— H

' n

among them:

R is H or d- ₁₂ alkyl;

n is any integer from 6 to 1300;

i is 1, 2, ..., j;

』 is an integer ≥ 2;

Is the same or different linking group selected from -(CH ₂ ) _m OCOO-, -(CH ₂ ) _m OCO H -, -(C3⁄4) _m NHCOO-, -(CH ₂ ) _m NHCONH -, -(CH ₂ ) _m COO-, -(CH ₂ ) _m CONH-, wherein m is an integer from 0 to 10;

A is derived from an oligopeptide, and the oligopeptide comprises 2-20 amino acids, wherein at least two amino acids are different, in particular, the oligopeptide is Lys-Gly, Gly-Lys (Gly), Gly-Lys-Lys or Gly-Lys (Gly)-Lys _;

F _p represents the same or different reactive groups selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, an acid chloride, a hydrazide group, a maleimide group, and a pyridine disulfide;

p is 1, 2, ..., q _;

Is an integer ≥1. In a preferred embodiment of the invention, R in the polyethylene glycol chain is selected from the group consisting of hydrogen, methyl, ethyl, and isopropyl. The group consisting of bases. The n in the polyethylene glycol chain is 60-800. A is derived from Gly-Lys, Gly-Lys (Gly), Glu-Lys or Gly-Lys (Gly)-Lys. Preferably, the present invention provides a compound formed from polyethylene glycol and an oligopeptide represented by Formula II:

Wherein: mPEG is CH ₃ 0-(CH ₂ CH ₂ 0) _n -. In a preferred embodiment of the formula ,, A is derived from Gly-Lys, Gly-Lys (Gly) or Glu-Lys. Preferably, the present invention provides a compound formed from polyethylene glycol and an oligopeptide represented by Formula III:

In a preferred embodiment, A in Formula III is derived from Gly-Lys, Gly-Lys (Gly), or Glu-Lys. Another aspect of the present invention provides a process for the preparation of a compound formed from polyethylene glycol and an oligopeptide represented by Formula I, which comprises: using polyethylene glycol under neutral or alkaline conditions The alcohol monomethyl ether ester is reacted with the oligopeptide, and the product is isolated and purified, and then subjected to a reactive group modification according to the type of F _p . Preferably, the polyethylene glycol monomethyl ether ester is polyethylene glycol monomethyl ether-acetic acid-N-hydroxysuccinimide ester or polyethylene glycol monomethyl ether-procarbonate-N-hydroxybutane Imidate ester. Still another aspect of the present invention is to provide a conjugate of a compound formed of polyethylene glycol and an oligopeptide through a reactive group _{Fp thereof} with a drug molecule. Preferably, the drug molecule is selected from the group consisting of amino acids, proteins, enzymes, nucleosides, sugars, organic acids, terpenoids, flavonoids, terpenoids, terpenoids, phenylpropanoid phenols, organisms and their steroids, organisms a group consisting of bases. The drug molecule may be a genetically engineered drug selected from the group consisting of α-, β- or γ-interferon, auxin, EPO, GCSF, interleukin, lysozyme, antibody and antibody fragments. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a GFC spectrum of a conjugate of a double-stranded polyethylene glycol active derivative (3) linked to lysozyme in Example 9. FIG. DETAILED DESCRIPTION OF THE INVENTION In the compounds provided by the present invention, the polyethylene glycol should include a homopolymer of ethylene glycol and a copolymer of ethylene glycol and propylene glycol or the like. Hereinafter, a method for preparing a reactive derivative having a polyglycol chain formed by a novel polyethylene glycol and an oligopeptide will be described by taking polyethylene glycol as an example. The structural formula of the polyethylene glycol chain polyethylene glycol (PEG) chain is as follows:

among them-

R is H or d. ₁₂ alkyl;

n can be any integer, characterizing its degree of polymerization. When R is a lower sulfhydryl group, R may be any lower alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl. Or just hexyl. A typical compound thereof is methoxypolyethylene glycol (mPEG), and other polyethylene glycol analogs or copolymers of ethylene glycol and epoxidizer may also be used in the invention. For polyethylene glycol, it is generally expressed by molecular weight as long as the molecular weight of the polyethylene glycol forming the conjugate is 300 to 60,000 Daltons, which corresponds to n being about 6 to 1300. More preferably, n is 78, 112 and 450, which correspond to molecular weights of 3,500, 5,000 and 20,000, respectively. The polyethylene glycol polymer is preferably characterized by molecular weight, rather than by the integer n, because of the potential heterogeneity of the starting PEG compound, which is generally defined by its average molecular weight rather than by repeating units. Starting PEG compounds of various molecular weights can be prepared by methods known in the art or can be obtained from commercial sources.

The so-called oligopeptide is a peptide molecule formed by two or more amino acids. The oligopeptide molecule may be an active molecule or an inactive molecule. Some active molecular oligopeptide molecules have been clinically applied, such as GHK (Gly-His- Lys), His Tag (His-His-His-His-His-His), KHG (Lys-His-Gly), RGD (Arg-Gly-As), TRH (Gly-His-Pro), GSH (Glu-Cys-Gly), TP-5 (Arg-Lys-Asp-Val-Tyr), etc., these small oligopeptides The unique physiological activity exhibited has always been the focus of attention. They play an important role in molecular recognition, signaling, protein integration, lymphocyte recognition, and cancer cell metastasis. For example, a polypeptide containing an RGD sequence can inhibit the adhesion of a variety of cells, and has an obvious anti-tumor effect. TP-5 (thym pentapeptide) is also prominent in lymphocyte recognition and activation. The oligopeptide used in the present invention is meant to include 2-20 amino acids, for example, the oligopeptide molecules listed below can be used in the present invention:

Dipeptide: GK (Gly-Lys), (Lys-Glu);

Tripeptides: (Gly-Lys (Gly)), GIIK (Gly-IIis-Lys), KHG (Lys-His-Gly), RGD CArg-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), and so on. The oligopeptides used in the present invention are preferably selected from inactive oligopeptides, and wherein at least two of the amino acids are different:

)

The reactive derivative having a polyethylene glycol chain formed by the polyethylene glycol and the oligopeptide according to the present invention can be PEGylated by reactive groups such as amino group, thiol group, hydroxyl group and carboxyl group in the oligopeptide. After getting it. Very common synthetic and preparative methods are available in the art. According to the structural compounds of the different claims, the corresponding synthesis and preparation methods are required, and the specific examples can be referred to the following examples and various technical literature and patent achievements in the field. Active group In the application of a polyglycol chain-forming active derivative formed by a polyethylene glycol and an oligopeptide according to the present invention, the reactive group F _p plays a decisive role, and derivatives of different reactive groups Has a different purpose. The introduction of these functional groups will determine the field of application and the applicable structure of the derivative. The most commonly used reactive group is N-hydroxysuccinimide ester, as shown by the formulae II and III. Similarly, it can also be modified by obtaining relevant synthetic methods in the art, thereby obtaining acid-based reactive groups: CH ₂ CH ₂ 0 -CH ₂ -CH ₂ -0 - C—NH CH ₂ CH ₂ 0 -CH ₂ - CH ₂ - OC——NH CH ₂ ― CH ₃

H"

H ₂ N—— H ₂ C― CH^

O CH ₂ —— CH ₃

Similarly, the maleimide functional group can be obtained by modifying the relevant synthetic methods readily available in the art.

Many pharmaceutical ingredients contain active functional groups such as amino groups, carboxyl groups, and hydroxyl groups, which are usually combined with monosaccharides, polysaccharides, nucleosides, polynucleosides, and phosphoryl groups in living organisms to form active species in organisms. Pharmacological structure. Therefore, the modified polyethylene glycol derivative of the functional group can be combined with these drug molecules in the same manner to replace the bioorganic molecule, thereby overcoming the short-term physiological half-life of the bio-organic molecule in the living body and the short duration of the drug effect. The reactive derivative having a polyethylene glycol chain formed by the polyethylene glycol of the present invention and an oligopeptide provides a conjugate with a drug molecule using a suitable reactive group, such a protein, polypeptide or other natural The free amino group, hydroxyl group, thiol group and the like in the drug are linked to the PEG derivative. For a macromolecular protein or polypeptide, one or more of the above-mentioned active derivatives may be bonded to improve the physiological action of the drug molecule in vivo; for a small molecule of the natural pharmaceutically active ingredient, the appropriate active group may be One or more drug molecules are linked to one of the active derivatives to ensure proper drug concentration and provide sustained release function. The above various application fields only provide a possible reference model for the pharmaceutical application of the PEG derivative, and the specific use and selection need to be confirmed according to the necessary links of animal pharmacology, toxicology and clinical. In the combination of the present invention, the drug molecule is selected from the group consisting of amino acids, proteins, enzymes, nucleosides, sugars, organic acids, terpenoids, flavonoids, terpenoids, terpenoids, phenylpropanoid phenols, steroids and steroids thereof. , a group consisting of alkaloids. Wherein, the protein drug molecule is selected from the group consisting of an interferon drug, an EPO drug, an auxin drug, and an antibody drug. The combination of the present invention can be administered in the form of a pure compound or a suitable pharmaceutical composition, and can be carried out by any acceptable mode of administration or reagents for similar uses. Therefore, the mode of administration may be selected by oral, intranasal, rectal, transdermal or injection, in the form of a solid, semi-solid, lyophilized powder or liquid medicament, for example, tablets, suppositories, Pills, soft and hard gelatin capsules, powders, solutions, suspensions or aerosols, and the like, are preferably employed in unit dosage forms for simple administration of precise dosages. The composition may comprise a conventional pharmaceutical carrier or excipient and a combination of the invention as the active ingredient(s), and may further comprise other agents, carriers, adjuvants and the like. Generally, a pharmaceutically acceptable composition will comprise from about 1 to about 99% by weight of a combination of the invention, and from 99 to 1% by weight of a suitable pharmaceutical excipient, depending on the mode of administration desired. Preferably, the compositions comprise from about 5 to 75% by weight of a combination of the invention, the balance being a suitable pharmaceutical excipient. The preferred route of administration is by injection, using a conventional daily dosage regimen which can be adjusted depending on the severity of the disease. The conjugate of the present invention or a pharmaceutically acceptable salt thereof may also be formulated as an injectable preparation, for example, by using from about 0.5 to about 50% of the active ingredient in a pharmaceutical adjuvant which can be administered in a liquid form, an example being water. , saline, aqueous glucose, glycerol, ethanol, etc., thereby forming a solution or suspension. The pharmaceutical composition which can be administered in liquid form can be dissolved or dispersed in a carrier by, for example, dissolving, dispersing or the like, and a pharmaceutically acceptable adjuvant of the present invention, wherein the carrier is dissolved or dispersed. Examples are water, saline, aqueous dextrose, glycerol, ethanol, and the like to form a solution or suspension. If desired, the pharmaceutical compositions of the present invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, for example: citric acid, sorbitan monolaurate, triethanolamine oil Acid ester, butylated hydroxytoluene, and the like. The actual preparation of such dosage forms is well known or apparent to those skilled in the art, for example see

Remington's Pharmaceutical Sciences, 18th edition, (Mack Publishing Company, Easton,

Pennsylvania, 1990). In any event, in accordance with the teachings of the present invention, the compositions employed will contain a therapeutically effective amount of a combination of the present invention for the treatment of a corresponding condition. EXAMPLES The following is a description of the combination of the present invention and the preparation thereof, which are not intended to limit the invention, and the scope of the invention is defined by the claims. Example 1

Synthesis of double-stranded polyethylene glycol active derivatives of polyethylene glycol acetic acid and Gly-Lys dipeptide (1)

20 g of polyethylene glycol monomethyl ether acetate-N-hydroxysuccinimide ester (mPEG-SCM) having a molecular weight of 20,000 was dissolved in a phosphate buffer containing 1 g of Gly-Lys dipeptide, pH = 8.5, stirred at room temperature for three hours. Hydrochloric acid was adjusted to pH = 3, and dichloromethane was extracted three times. The organic phase was combined, and then organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The precipitate was filtered and dried under vacuum. The branched PEG acidic product can be further purified by ion exchange chromatography. Yield: 8 g (40%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 3 hydrogens), 4.15 (t, 2 hydrogens).

0.5 g of branched PEG acid (prepared from the previous step), dissolved in 5 ml of dichloromethane, and added 7 mg of N-hydroxysuccinimide (NHS) and 13 mg of dicyclohexylcarbodiimide to the solution. (DCC), stirring at room temperature for 6 hours, the precipitate was removed by filtration, the solvent was evaporated in vacuo, and the residue was applied to 20 ml of isopropyl alcohol (IPA), and the precipitate was collected by filtration and dried in vacuo. Yield: 0.48 g (96%). MR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 6 hydrogens) 2.81 (s, 4 hydrogens), 3.96 (s, 2 hydrogens), 4.07 (s, 2 hydrogens), 4.48 (t, 2 hydrogens). Example 2

Synthesis of double-stranded polyethylene glycol active derivatives of polyethylene glycol and Gly-Lys dipeptide (2)

20 g of polyethylene glycol monomethyl ether carbonate-N-hydroxysuccinimide ester (mPEG-SC) having a molecular weight of 20,000 was dissolved in borate buffer containing 1 g of Gly-Lys dipeptide, pH = 10.2, stirred at room temperature overnight. Hydrochloric acid was adjusted to pH = 3, and dichloromethane was extracted three times. The organic phase was combined, and then organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The precipitate was filtered and dried under vacuum. The branched PEG acid product can be further purified by ion exchange chromatography. Yield: 8 g (40%). MVIR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 3 hydrogens), 4,32 (t, 2 wood *, _

0.5 g of branched PEG acid (prepared from the previous step), dissolved in 5 ml of dichloromethane, and added 7 mg of N-hydroxysuccinimide (NHS) and 13 g of dicyclohexyl carbon to the solution. The imine (DCC) was stirred at room temperature for 6 hours, the precipitate was removed by filtration, the solvent was evaporated in vacuo, and the residue was taken up in 20 liters of isopropyl alcohol (IPA), and the precipitate was collected by filtration and dried in vacuo. Yield: 0.48 g (96%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 6 hydrogens) 2.81 (s, 4 hydrogens), 4.15 (s, 2 hydrogens), 4.07 (t, 2 hydrogens), 4.48 (t, 2 hydrogens). Example 3

Synthesis of double-stranded polyethylene glycol active derivatives of polyethylene glycol and Gly-Lys (Gly) tripeptide (3)

20 g of polyethylene glycol monomethyl ether carbonate-N-hydroxysuccinimide ester (mPEG-SC) having a molecular weight of 20,000 The mixture was stirred at room temperature overnight in a borate buffer containing 1.2 g of Gly-Lys(Gly) polypeptide at pH = 10.2. The mixture was adjusted to pH 3 with hydrochloric acid and extracted three times with dichloromethane. The organic phase was combined and dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo. The precipitate was filtered and dried under vacuum. The branched PEG acidic product can be further purified by ion exchange chromatography. Yield: 8 g (40%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 3 hydrogens), 4.32 (t, 2 hydrogens).

0.5 g of branched mPEG acid (prepared from the previous step), dissolved in 5 ml of dichloromethane, and added 7 mg of N-hydroxysuccinimide (NHS) and 13 mg of dicyclohexylcarbodiimide to the solution. (DCC), stirring at room temperature for 6 hours, the precipitate was removed by filtration, the solvent was evaporated in vacuo, and the residue was taken in 20 ml of isopropyl alcohol (IPA), and the precipitate was collected by filtration and dried in vacuo. Yield: 0.48 g (%%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 6 hydrogens), 2.81 (s, 4 ft), 4.15 (s, 2 hydrogens), 4.07 (t, 2 hydrogens) , 4.48 (t, 2 hydrogens). Example 4

Synthesis of tri-chain polyethylene glycol active derivatives of polyethylene glycol and Gly-Lys-Lys tripeptide (4)

20 g of polyethylene glycol monomethyl ether carbonate-N-hydroxysuccinimide ester (mPEG-SC) having a molecular weight of 20,000 was dissolved in borate buffer containing 1.5 g of Gly-Lys-Lys polypeptide. pH = 10.2, stirred at room temperature overnight. The pH was adjusted to pH 3, and the dichloromethane was extracted three times. The organic phase was combined and dried and evaporated. The precipitate was filtered and dried under vacuum. The branched PEG acidic product can be further purified by ion exchange chromatography. Yield: 8 g (40%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 3 hydrogen), 4.32 (t, 2 hydrogen).

0.5 g of branched PEG acid (prepared from the previous step), dissolved in 5 ml of dichloromethane, and added 7 mg of N-hydroxysuccinimide (NHS) and 13 mg of dicyclohexylcarbazone to the solution. The amine (DCC) was stirred at room temperature for 6 hours, and the precipitate was removed by filtration. The solvent was evaporated in vacuo and the residue was taken in 20 ml of isopropyl alcohol (IPA), and the precipitate was collected by filtration and dried in vacuo. Yield: 0.4 S g (96%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 6 hydrogens) 2.81 (s, 4 hydrogens), 4.15 (s, 2 hydrogens), 4.07 (t, 2 hydrogens), 4.48 (t, 2 hydrogens). Example 5

Double-stranded polyethylene glycol active derivative formed by alcohol and Lys-Glu dipeptide (5)

20 g of polyethylene glycol monomethyl ether carbonate-N-hydroxysuccinimide ester (mPEG-SC) having a molecular weight of 20,000 was dissolved in borate buffer containing 1.2 g of Lys-Glu dipeptide, pH = 10.2, stirred at room temperature overnight. Hydrochloric acid was adjusted to pH = 3, and dichloromethane was extracted three times. The organic phase was combined, and then organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated in vacuo. The precipitate was filtered and dried under vacuum. The branched PEG acid product can be further purified by ion exchange chromatography. Yield: 8 g (40%). NMR (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 3 hydrogens), 4.32 (t, 2 ^ Example 6

Synthesis of double-stranded polyethylene glycol active derivatives of polyethylene glycol with aldehyde group and Gly-Lys (Gly) tripeptide (6)

10 g of a double-stranded polyethylene glycol active derivative (3) having a molecular weight of 40,000 polyethylene glycol and a Gly-Lys (Gly) tripeptide was dissolved in 50 ml of dichloromethane, and 0.1 ml of 2,2- was added. Diethoxyethylamine was stirred overnight under nitrogen. The solution was concentrated under reduced pressure and then diethyl ether (100 ml). The precipitate was collected by filtration and dried under vacuum, yield: 9.5 g (95%).

9 g of double-stranded polyethylene glycol diethyl acetal (prepared from the above step) was dissolved in a phosphate buffer solution of pH = 5, and reacted at room temperature for 48 hours. It was dissolved in 30 g of sodium chloride and extracted three times with dichloromethane. EtOAc was evaporated. Yield: 7.2 g (80%). NMR (D ₂ 0): 1.80 (q, 1 hydrogen), 2.72 (t, 1 hydrogen), 3.29 (s, 3 hydrogen), 3.61 (s, hydrogen in PEG), 5.06 (t, 1 hydrogen) ). Melting point: 56-58 ° C. Example 7

Synthesis of double-stranded polyethylene glycol formed by polyethylene glycol active with thiol and Gly-Lys (Gly) tripeptide

Reactive derivatives ( 7 )

10 g of a double-stranded polyethylene glycol active derivative (3) having a molecular weight of 40,000 polyethylene glycol and a Gly-Lys (Gly) tripeptide was dissolved in 50 ml of dichloromethane, and 0.1 g of N- (2) was added. -Aminoethyl)-3-maleimido-propionamide, stirred under nitrogen overnight. The solution was concentrated under reduced pressure and then diethyl ether (100 mL). The precipitate was collected by filtration and dried under vacuum, yield: 9.5 g (95%). Example 8

Synthesis of double-stranded polyethylene glycol formed by polyethylene glycol with double active groups and Gly-Lys (Gly)-Lys tetrapeptide

Alcohol reactive derivatives (8)

10 g of a double-stranded polyethylene glycol active derivative (3) having a molecular weight of 40,000 polyethylene glycol and a H ₂ -Gly-Lys (Gly)-OH tripeptide was dissolved in 50 ml of dichloromethane, and 0.2 was added.克 NH ₂ - Lys(Boc)-OH, the reaction was stirred at room temperature for 18 hours, treated with 20 ml of trifluoroacetic acid for 1 hour, and the organic phase was washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and filtered. The mixture was concentrated under reduced pressure. The product obtained in the previous step was dissolved in 20 ml of dichloromethane, and 0.15 g of 3-maleimido-propionic acid-(N-hydroxy-succinimide) ester was added thereto, and the mixture was reacted overnight at room temperature. The precipitate was removed, concentrated under reduced pressure under nitrogen, and then filtered and evaporated. 1 gram of double-stranded polyglycolic acid obtained in the previous step (prepared from the above step) was dissolved in 20 ml of dichloromethane, and 20 mg of N-hydroxysuccinimide (NHS) and 0.2 g were added to the solution. Dicyclohexylcarbodiimide (DCC) was stirred at room temperature for 6 hours, the precipitate was removed by filtration, the solvent was evaporated in vacuo, and the residue was taken up in 20 ml of isopropyl alcohol (IPA), and the precipitate was collected by filtration and dried in vacuo. Yield: 0.98 g (98%). NM (DMSO) 3.50 (br m, hydrogen in PEG), 3.24 (s, 6 hydrogens), 2.81 (s, 4 hydrogens), 4.15 (s, 2 hydrogens), 4.07 (t, 2 hydrogens) , 4.48 (t, 2 hydrogens). Example 9

Double-stranded polyethylene glycol reactive derivative (3) Conjugate linked to Lysozyme 1 mg of lysozyme was dissolved in 1 ml of 50 mmol/L phosphate buffer at pH=7.0. 27.8 g of the double-stranded polyethylene glycol reactive derivative (3) was added to the lysozyme solution. The molar ratio of protein to PEG is 1:10. The reaction was shaken at room temperature for 3 hours. GFC detected the substitution rate, double PEG replaced lysozyme 4.8%, single PEG replaced lysozyme 48.0%, lysozyme 47.2%.

Compared with the modification degree of common polyethylene glycol active derivatives, the content of single PEG substituted lysozyme is greatly improved, and the content of double substitution is less. Example 10

Double-stranded polyethylene glycol reactive derivative (3) Binding to auxin (hGH) The auxin was diluted to 1 mg/ml with 50 mmol/L pH=7.0 phosphate buffer. 18.0 mg of the double-stranded polyethylene glycol reactive derivative (3) was added to 1 ml of the auxin solution. The molar ratio of protein to PEG is 1:10. The reaction was shaken at room temperature for 3 hours. Separation and purification by ion exchange chromatography gave a product which did not contain free hGH. Example 11

Double-stranded polyethylene glycol reactive derivative (6) conjugate linked to GCSF

GCSF was diluted to 1 mg/ml with 50 mmol/L pH=7.0 phosphate buffer. 21.3 mg of the double-stranded polyethylene glycol reactive derivative (3) was added to 1 ml of the GCSF solution. The molar ratio of protein to PEG is 1:10. The reaction was shaken at room temperature for 3 hours. Separation and purification by ion exchange chromatography gave a product which did not contain free GCSF. EXAMPLE 12 This example illustrates the preparation of a representative parenterally administered pharmaceutical composition comprising a combination of the invention. ingredient

Combination of Example 8 2 g

0.9% saline solution to 100 ml 2 g of the conjugate of Example 8 was dissolved in 0.9% saline solution to obtain 100 ml of an intravenous solution, which was filtered through a 0.2 μm membrane filter and packaged under aseptic conditions.

Claims

Rights request

1. A compound formed from polyethylene glycol and an oligopeptide represented by the general formula I:

( ^PE °i― ^Χ ι)^~ ^Α F _P ) _q (I)

among them:

PEGi is the same or different polyethylene glycol chain, and the polyethylene glycol chain has the following formula:

R_0 - - CH ₂ CH ₂ 0

, - '- H

n

among them:

R is H or d- ₁₂ fluorenyl;

n is any integer from 6 to 1300;

i is 1, 2, ..., j;

:| is an integer ≥ 2;

Is the same or different linking group, the linking group is selected from -(CH ₂ ) _m OCOO-, -(CH ₂ ) _m OCO H -, -(CH ₂ ) _m HCOO-, -(CH ₂ a group consisting of _ra NHCONH -, -(CH ₂ ) _m COO-, -(CH ₂ ) _m CO H-, wherein m is an integer from 0 to 10;

A is derived from an oligopeptide, Lys-Gly, Gly-Lys (Gly), Gly-Lys-Lys or Gly-Lys (Gly)-Lys; F _p represents the same or different reactive groups The reactive group is selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, an acid chloride, a hydrazide group, a maleimide group, and a pyridine disulfide;

p is 1, 2, ..., q _;

9 is an integer ≥1.

The compound according to claim 1, wherein the R in the polyethylene glycol chain is selected from the group consisting of hydrogen, methyl, ethyl, and isopropyl.

The compound according to claim 1, wherein the n in the polyethylene glycol chain is from 60 to 800.

4. The compound according to claim 1, wherein the compound is as shown in the formula VI -

WO 2008/052428 PCT/feCT/CNIOOT/OOSOSO Q

The compound according to claim 1, wherein the compound is one selected from the group consisting of compounds represented by the following formula -

0 0 0 0

CH ₃ 0~^ CH ₂ CH ₂ 0 CH ₂ _ CH ₂ - O― C― NH— CH ₂ - C—NH— CH— C— NH—CH ₂ — C— H

Where: k is an integer from 1 to 10 _;

6. A method of preparing a compound formed of polyethylene glycol and an oligopeptide according to claim 1, the method comprising: using polyethylene glycol monomethyl ether ester under neutral or alkaline conditions The oligopeptide reaction, the product is isolated and purified, and then the reactive group modification according to the type of Fp.

7. The method according to claim 10, wherein the polyethylene glycol monomethyl ether ester is polyethylene glycol monomethyl ether-acetic acid-N-hydroxysuccinimide ester or polyethylene glycol single Methyl ether - procarbonate - N hydroxy succinimide ester.

8. A conjugate of a compound according to any one of claims 1 to 9 formed by a reactive group _Fp with a drug molecule.

9. The conjugate according to claim 12, wherein the drug molecule is selected from the group consisting of amino acids, proteins, enzymes, nucleosides, sugars, organic acids, terpenoids, flavonoids, terpenoids, terpenoids, styrene-acrylic acid. A group consisting of a phenol, a body, a steroid, and an alkaloid.

10. The conjugate according to claim 12, wherein the drug molecule is a genetic engineering drug.

The conjugate according to claim 14, wherein the genetic engineering drug is selected from the group consisting of α-, β- or γ-interferon, auxin, EPO, GCSF, interleukin, lysozyme, antibody and antibody fragment. Group.