Polyethylene glycol derivative and application thereof

Abstract

The invention relates to a polyethylene glycol derivative and application thereof. The present invention provides a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; processes for preparing the compounds; and the use of the compound in a method of preparing a physiologically active polypeptide linked to a polyethylene glycol compound, a method of preparing a conjugate in which the physiologically active polypeptide and a carrier protein are linked by a polyethylene glycol compound; and a physiologically active polypeptide, wherein the compound represented by Formula 1 is linked to the physiologically active polypeptide; and a conjugate in which each of the physiologically active polypeptide and the carrier protein is independently linked to a reactive group at both ends of the compound represented by Formula 1.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780028045.1 filed on March 7, 2017, with the invention name “Polyethylene glycol derivatives and uses thereof”. Technical Field

[0002] The present invention relates to polyethylene glycol derivatives and uses thereof. Background Art

[0003] Polyethylene glycol (PEG) is a material with a high in vivo half-life but no antigenicity. PEG is a representative biocompatible material that is widely used in pharmacy when combined with various physiologically active materials such as lipids, proteins, etc., and research on the pharmaceutical use of PEG itself is ongoing.

[0004] In particular, PEG is widely used in protein therapeutics because PEG can be bound to protein therapeutics, thereby increasing their blood half-life while reducing their antigenicity. In addition, it is reported that PEGylation can improve the stability of protein therapeutics, which refers to the covalent attachment of PEG molecules to proteins (Cantin et al., Am. J. 27: 659-665 (2002)).

[0005] At the same time, during the manufacturing process, improving the production yield of these drugs while maintaining the activity of the drugs requires complex manufacturing techniques. Therefore, there has been continuous research to develop methods for manufacturing protein therapeutics including PEG, and PEG used therein.

[0006] When preparing a physiologically active polypeptide conjugate using PEG, the present inventors used PEG having at least two reactive groups as a linker (Korean Patent Application Publication No. 10-2014-0018462).

[0007] Conventional PEG compounds are known to have aldehyde, succinimidyl, maleimide, vinyl sulfone, halogenated acetamide, orthopyridyl disulfide (OPSS), etc. as reactive groups.

[0008] However, conventionally used PEG compounds have an inconvenience in that, although they have a reactive group at the terminal, their reactivity varies depending on the structure of the PEG compound.

[0009] Therefore, there is a need to develop new polyethylene glycol derivatives that not only include reactive groups capable of binding to target materials but also react more easily with these materials. Summary of the invention

[0010] Technical issues

[0011] An object of the present invention is to provide polyethylene glycol, its stereoisomers or pharmaceutically acceptable salts thereof.

[0012] Another object of the present invention is to provide a method for preparing a physiologically active polypeptide linked to a polyethylene glycol compound, the method comprising reacting the polyethylene glycol compound with the physiologically active polypeptide.

[0013] Another object of the present invention is to provide a method for preparing a conjugate, wherein the physiologically active polypeptide and the carrier protein are linked via a polyethylene glycol compound.

[0014] Another object of the present invention is to provide a physiologically active polypeptide linked to a polyethylene glycol compound.

[0015] Another object of the present invention is to provide a conjugate, wherein each of the physiologically active polypeptide and the carrier protein is independently linked to reactive groups at both ends of the polyethylene glycol compound.

[0016] Another object of the present invention is to provide a method for preparing the polyethylene glycol compound.

[0017] Another object of the present invention is to provide a use of a polyethylene glycol compound in linking a carrier to a physiologically active polypeptide, wherein the carrier can increase the in vivo half-life of the physiologically active polypeptide.

[0018] Another object of the present invention is to provide a composition comprising a physiologically active polypeptide linked to a polyethylene glycol compound, or a conjugate.

[0019] Another object of the present invention is to provide a polyethylene glycol compound linker for connecting a carrier to a physiologically active polypeptide, wherein the carrier can increase the in vivo half-life of the physiologically active polypeptide.

[0020] Technical Solution

[0021] One aspect of the present invention provides a polyethylene glycol compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0022] In a specific embodiment, the compound is a compound represented by Formula 1:

[0023]

[0024] Among them, in formula 1,

[0025] R 1 Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, thiols, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives;

[0026] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0027] R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and

[0028] n is an integer from 10 to 2400.

[0029] In another specific embodiment, R 2 It is o-disulfide pyridine, sulfhydryl or iodine.

[0030] In another specific embodiment, R 1 It's aldehyde.

[0031] In another specific embodiment, R 1 and R 2 Have different functional groups.

[0032] In another specific embodiment, the compound is represented by Formula 2:

[0033] CHO-(CH 2 ) j -O-(CH 2 CH 2 O) n -(CH 2 ) m -NH(CO)-(CH 2 ) k -R 2

[0034]

[0035] Among them, in formula 2,

[0036] n is an integer from 10 to 2400;

[0037] j, m and k are each independently an integer from 1 to 6; and R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen.

[0038] In another specific embodiment, the compound is selected from Formula 3 to Formula 11:

[0039]

[0040]

[0041] In Formula 3 to Formula 11, n is an integer of 10-2400.

[0042] Another aspect of the present invention provides a method for preparing a physiologically active polypeptide linked to a polyethylene glycol compound, the method comprising reacting a polyethylene glycol compound with a physiologically active polypeptide to prepare the physiologically active polypeptide linked to the polyethylene glycol compound.

[0043] In a specific embodiment of the method, at R 2 The sulfhydryl group of the cysteine ​​residue in the physiologically active polypeptide is reacted with ortho-pyridine disulfide (OPSS), sulfhydryl group or halogen.

[0044] In another specific embodiment, the method further comprises purifying the physiologically active polypeptide linked to the polyethylene glycol compound.

[0045] In another specific embodiment, the physiologically active polypeptide is selected from hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, insulinotropic peptides, neuropeptides, pituitary hormones, anti-obesity peptides, antiviral peptides, non-natural peptide derivatives with physiological activity, structural proteins, ligand proteins and receptors.

[0046] In another specific embodiment, the physiologically active polypeptide is selected from glucagon; insulin; somatostatin; peptide YY (PYY); neuropeptide Y (NPY); glucagon-like peptides, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2); exendin-3; exendin-4; oxyntomodulin; peptides active at glucagon receptors, GLP-1 receptors and GIP receptors; fibroblast growth factor (FGF); factor); ghrelin; angiotensin; bradykinin; calcitonin; corticotropin; eledoisin; gastrin; leptin; oxytocin; vasopressin; luteinizing hormone; prolactin; follicle-stimulating hormone; parathyroid hormone; secretin; sermorelin; human growth hormone (hGH); growth hormone-releasing peptide; granulocyte colony-stimulating factor (GCSF); interferon (IFN); interleukin (Interleukin); prolactin-releasing peptide; orexin; thyroid-releasing peptide; cholecystokinin; gastrin-inhibiting peptide; calmodulin; gastric releasing peptide; motilin; vasoactive intestinal peptide; atrial natriuretic peptide peptide, ANP); B-type natriuretic peptide (BNP); C-type natriuretic peptide (CNP); Neurokinin A; Neuromedin; Renin; Endothelin; Sarafotoxin peptide; carsomorphin peptide; Dermorphin; Dynorphin; Endorphin; Enkepalin; T cell factor; Tumor necrosis factor; Tumor necrosis factor receptor; Urokinase receptor; Tumor suppressor factor; Collagenase inhibitor; Thymopoietin; Thymulin; Thymopentin; Tymosin;Thymic humoral factor; Adrenomodullin; Allatostatin; Amyloid beta-protein fragment fragment); antimicrobial peptides; antioxidant peptides; bombesin; osteocalcin; CART peptides; E-selectin; ICAM-1; VCAM-1; leucokine; kringle-5; laminin; inhibin; galanin; fibronectin; pancreas inhibitor; fuzeon; interferon receptor; G protein-coupled receptor; interleukin receptor; enzyme; interleukin binding protein; cytokine binding protein; macrophage activating factor; macrophage peptide; B cell factor; protein A; allergy inhibitor; necrosis glycoprotein; immunotoxin; lymphotoxin; tumor suppressor; metastatic growth factor; alpha-1-antitrypsin; albumin; alpha-lactalbumin; apolipoprotein-E; erythropoietin; hyperglycosylated erythropoietin; angiogenin ; hemoglobin; thrombin; thrombin receptor activating peptide; thrombomodulin; blood factors VII, VIIa, VIII, IX and XIII; plasminogen activator; fibrin-binding peptide; urokinase; streptokinase; hirudin; protein C; C-reactive protein; renin inhibitor; superoxide dismutase; platelet-derived growth factor; epidermal growth factor; epithelial cell growth factor; angiostatin; angiotensin; osteogenic growth factor; osteogenic stimulating protein; atrial natriuretic peptide; chondroitin; elcatonin; connective tissue Tissue activating factor; tissue factor pathway inhibitor; luteinizing hormone-releasing hormone; nerve growth factor; relaxin; somatomedin; insulin-like growth factor; adrenocortical hormone; pancreatic polypeptide; gastrin-releasing peptide; corticotropin-releasing factor; thyroid stimulating hormone; autotoxin; lactoferrin; myostatin; cell surface antigen; virus-derived vaccine antigen; monoclonal antibody; polyclonal antibody; antibody fragment; erythropoietic growth factor; leukopoietin; amylin; and their analogs. ;

[0047] Another aspect of the present invention provides a method for preparing a conjugate, wherein the physiologically active polypeptide and the carrier protein are linked via a polyethylene glycol compound.

[0048] In a specific embodiment, the method comprises:

[0049] (a) reacting a polyethylene glycol compound with any one of a physiologically active polypeptide or a carrier protein, thereby preparing a polyethylene glycol compound, wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive end group; and

[0050] (b) reacting the polyethylene glycol compound prepared in step (a), wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group, with the other of the physiologically active polypeptide or the carrier protein so as to link the carrier protein or the physiologically active polypeptide to the reactive terminal group of the polyethylene glycol compound, thereby preparing a conjugate in which the physiologically active polypeptide and the carrier protein are linked via the polyethylene glycol compound.

[0051] In another specific embodiment, the method comprises:

[0052] (a) reacting a polyethylene glycol compound with a physiologically active polypeptide to prepare a polyethylene glycol compound, wherein the physiologically active polypeptide is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group; and

[0053] (b) reacting the polyethylene glycol compound prepared in step (a), wherein the physiologically active polypeptide is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group, with a carrier protein to link the carrier protein to the reactive terminal group of the polyethylene glycol compound.

[0054] In another specific embodiment, the physiologically active polypeptide is selected from hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, insulinotropic peptides, neuropeptides, pituitary hormones, anti-obesity peptides, antiviral peptides, non-natural peptide derivatives with physiological activity, structural proteins, ligand proteins and receptors.

[0055] In another specific embodiment, the polyethylene glycol compound in step (a) has a structure of Formula 1.

[0056] In another specific embodiment, step (a) of the method is characterized in that R of the polyethylene glycol compound having the structure of Formula 1 2 Reacts with sulfhydryl groups on cysteine ​​residues of physiologically active polypeptides.

[0057] In another specific embodiment, step (b) of the method is characterized in that the aldehyde terminal group of the polyethylene glycol compound is reacted with an amine group of the immunoglobulin Fc fragment.

[0058] In another specific embodiment, the method further comprises purifying the conjugate, wherein the physiologically active polypeptide and the carrier protein are linked via a polyethylene glycol compound.

[0059] In another specific embodiment, the carrier protein is albumin and its fragments, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, FcRn-binding substances, fibronectin, transferrin, saccharide or elastin.

[0060] In another specific embodiment, the FcRn-binding substance is an immunoglobulin Fc fragment.

[0061] Another aspect of the present invention provides a physiologically active polypeptide linked to a polyethylene glycol compound.

[0062] In one embodiment, the physiologically active polypeptide linked to the above compound comprises a structure represented by any one of Formula 15 to Formula 17:

[0063] R 1 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -SSX

[0064] [Formula 15]

[0065] R 1 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-CH 2 -SX

[0066] [Formula 16]

[0067] X-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -R 2

[0068] [Formula 17]

[0069] In equations 15 to 17,

[0070] R 1Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, mercapto groups, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives;

[0071] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0072] n is an integer from 10 to 2400;

[0073] R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and

[0074] X is a physiologically active polypeptide moiety.

[0075] Another aspect of the present invention provides a conjugate, wherein each of the physiologically active polypeptide and the carrier protein is independently linked to reactive groups at both ends of a polyethylene glycol compound.

[0076] In a specific embodiment, the conjugate is a conjugate having a structure represented by Formula 18 or Formula 19:

[0077] Y-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -SSX

[0078] [Formula 18]

[0079] Y-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-CH 2 -SX

[0080] [Formula 19]

[0081] In equations 18 and 19,

[0082] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene,

[0083] n is an integer from 10 to 2400,

[0084] X is a physiologically active polypeptide moiety; and

[0085] Y is a carrier protein moiety.

[0086] In another specific embodiment, the carrier protein is albumin and its fragments, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, FcRn-binding substances, fibronectin, transferrin, sugars or elastin.

[0087] In another specific embodiment, the FcRn-binding substance is an immunoglobulin Fc fragment.

[0088] Another aspect of the present invention provides a method for preparing a polyethylene glycol compound.

[0089] In a specific embodiment, the method comprises:

[0090] (a) Introducing R 1 To one end of the polyethylene glycol, the R 1 Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, mercaptos, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives; and

[0091] (b) Introduction of -NH(CO)L 3 -R 2 to the other end of the polyethylene glycol, where R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen.

[0092] In another specific embodiment, the method comprises:

[0093] The first step is to prepare a compound represented by formula 21 from a compound represented by formula 20;

[0094] The second step is to prepare a compound represented by Formula 22 from the compound represented by Formula 21; and

[0095] In the third step, the diethoxymethyl group at the terminal of the compound represented by Formula 22 is converted into an aldehyde by treating the compound represented by Formula 22 with an acid solution:

[0096]

[0097] Wherein, in Formula 20, n' is n or n+1; and

[0098]

[0099] Where L 1 , L 2 , L 3 , n and R 2 Same as above.

[0100] In another specific embodiment, the compound represented by formula 20 in the first step is prepared by reacting the compound represented by formula 23 with methanesulfonyl chloride:

[0101]

[0102] [Formula 23].

[0103] In another specific embodiment, the first step is performed by reacting the compound represented by Formula 20 with aqueous ammonia solution and ammonium chloride.

[0104] In another specific embodiment, the first step comprises:

[0105] Step 1-1, reacting a compound represented by Formula 20 with a hydroxyalkyl tetrahydropyranyl ether to prepare a compound represented by Formula 24;

[0106] Step 1-2, reacting the compound represented by Formula 24 with p-toluenesulfonic acid to replace the terminal tetrahydropyranyloxy group with a hydroxyl group;

[0107] step 1-3, reacting the compound obtained in step 1-2 with methanesulfonyl chloride to replace the hydroxyl group with a methanesulfonic acid group; and

[0108] Step 1-4, reacting the compound obtained in step 1-3 with an aqueous ammonia solution and ammonium chloride.

[0109]

[0110] In another specific embodiment, the second step is performed by reacting a compound represented by Formula 21 with a compound represented by Formula 25:

[0111]

[0112] In another specific embodiment, the second step comprises: reacting a compound represented by formula 21 with a chloro-C 2-7 alkanoyl chloride, thereby synthesizing a compound containing a chlorine group at one end thereof, which is represented by Formula 26 as an intermediate product; and

[0113] The compound represented by Formula 26 is reacted with a halogen metal salt in the presence or absence of hydrogen sulfide to convert the chloro group into a mercapto group or a halogen:

[0114]

[0115] Another aspect of the present invention provides use of a polyethylene glycol compound in linking a carrier to a physiologically active polypeptide, wherein the carrier can increase the in vivo half-life of the physiologically active polypeptide.

[0116] Another aspect of the present invention provides a composition comprising a physiologically active polypeptide linked to a polyethylene glycol compound, or a conjugate.

[0117] Another aspect of the present invention provides a linker for connecting a carrier to a physiologically active polypeptide, wherein the carrier can increase the in vivo half-life of the physiologically active polypeptide, wherein the linker comprises a polyethylene glycol compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0118] Beneficial Effects

[0119] The polyethylene glycol derivatives of the present invention have the advantage of including a desired reactive terminal group at one end thereof and can easily react with a target substance (e.g., protein) to be connected. Therefore, the polyethylene glycol derivatives of the present invention can be effectively used in the pharmaceutical field of preparing conjugated drugs such as protein conjugates, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1a and 1b Illustrative examples of the novel polyethylene glycol compounds of the present invention are shown, illustrating the chemical structures of compounds having an aldehyde group and an o-dithiopyridine group (linkers #1, #4, and #7), 2) the chemical structures of compounds having an aldehyde group and an iodoacetamide group or an iodine group (linkers #2, #5, and #8), and 3) the chemical structures of compounds having an aldehyde group and a sulfhydryl group (linkers #3, #6, and #9). Figure 1b , connectors #10 to #12 correspond to the comparison group.

[0121] Figure 2 The results of nuclear magnetic resonance (hereinafter referred to as NMR) analysis are shown, confirming linker #1 after preparation.

[0122] Figure 3 Shown are the results of reverse phase chromatography (hereinafter referred to as RPC) analysis of linker #1 after preparation.

[0123] Figure 4 The results of NMR analysis are shown, confirming linker #2 after preparation.

[0124] Figure 5 Shown are the results of RPC analysis of linker #2 after preparation.

[0125] Figure 6 The results of NMR analysis are shown, confirming linker #3 after preparation.

[0126] Figure 7Shown are the results of RPC analysis of linker #3 after preparation.

[0127] Figure 8 The results of NMR analysis are shown, confirming linker #5 after preparation.

[0128] Fig. 9 Shown are the results of RPC analysis of linker #5 after preparation.

[0129] Fig.10 The results of MALDI-TOF analysis of the molecular weight of linker #5 after preparation are shown.

[0130] Fig.11 Shown are the results of RPC analysis of linker #7 after preparation.

[0131] Fig.12 The results of NMR analysis are shown, confirming linker #8 after preparation.

[0132] Fig.13 Shown are the results of RPC analysis of linker #8 after preparation.

[0133] Fig.14 The results of MALDI-TOF analysis of the molecular weight of linker #8 after preparation are shown.

[0134] Fig.15 Shown are the results of RPC analysis of linker #9 after preparation.

[0135] Fig.16 The results of NMR analysis are shown, confirming linker #11 after preparation.

[0136] Fig.17 Shown are the results of RPC analysis of linker #11 after preparation.

[0137] Fig.18 The results of comparative experiments on the reactivity of thiol reactive groups of the polyethylene glycol compounds of the present invention are shown.

[0138] Figure 19-21 The results of SDS-PAGE analysis of the triple agonist-PEG-Fc conjugate prepared using the polyethylene glycol compound of the present invention as a linker are shown, wherein Fig.19 The results of SDS-PAGE analysis using linker #7 of the present invention are shown. Fig. 20 Using connector #8, and Fig.21 Utilize connector #9. DETAILED DESCRIPTION

[0139] One aspect of the present invention provides a polyethylene glycol compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0140] Hereafter, the present invention is described in detail. Meanwhile, each description and embodiment disclosed in the present invention can be used to describe different descriptions and embodiments in this article. In other words, all combinations of the various components disclosed in the present invention are included within the scope of the present invention. In addition, the scope of the present invention should not be limited to the detailed description provided below.

[0141] As used herein, the term "polyethylene glycol compound" refers to a compound comprising a polyethylene glycol structure [-(OCH 2 CH 2 ) n More specifically, in the present invention, the polyethylene glycol compound may include two reactive terminal groups.

[0142] In particular, the two or more reactive end groups present in the polyethylene glycol compound may be identical or different from each other. More specifically, the polyethylene glycol compound may be a heterofunctional linker acting on reactive end groups that are different from each other. For example, one end of the compound may have functionality for an amine group, while the other end may have functionality for a thiol group, but the polyethylene glycol compound is not particularly limited thereto.

[0143] In addition, the polyethylene glycol compound can be used as a linker for connecting a carrier to a physiologically active polypeptide. Therefore, one end of a polyethylene glycol compound having two or more reactive terminal groups can be connected to a physiologically active polypeptide, while the other end of the polyethylene glycol compound can be connected to a carrier.

[0144] Meanwhile, polyethylene glycol compounds and polyethylene glycol derivatives may be used interchangeably in the present invention.

[0145] In a specific embodiment, the polyethylene glycol compound of the present invention may be a compound including an -NHCO- structure between a thiol-reactive group and a polyethylene glycol structure as shown below.

[0146] Specifically, the compound may be a compound represented by Formula 1:

[0147]

[0148] Among them, in formula 1,

[0149] R 1 Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, mercapto groups, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives;

[0150] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0151] R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and

[0152] n is an integer from 10 to 2400.

[0153] In formula 1, R 2 It may be o-dithiopyridine, thiol, F, Br, Cl or I, and more specifically, o-dithiopyridine or I, but is not particularly limited thereto.

[0154] In formula 1, R 1 It may be aldehyde, but is not particularly limited thereto.

[0155] In formula 1, R 1 It may be a succinimide derivative, and the types of the succinimide derivative may include succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate, but is not particularly limited thereto.

[0156] Furthermore, the compounds may be heterofunctional, acting on mutually different reactive end groups, and in particular, R 1 and R 2 The compounds may have functional groups different from each other, but are not particularly limited thereto.

[0157] Specifically, R 1 may be an aldehyde, and R 2 It may be o-pyridine disulfide (OPSS), a thiol group, or a halogen, but the compound is not particularly limited thereto.

[0158] In the above compounds, L 1 -L 3 Each independently may be a linear or branched C 1-6 Alkylene, and more specifically, C 1-4 An alkylene group is included but is not particularly limited thereto.

[0159] For example, L 1 It can be an integer of 1, 2, 3, 4, 5 or 6; L 2 can be an integer of 1, 2, 3, 4, 5 or 6; and L 3 Can be an integer of 1, 2, 3, 4, 5 or 6.

[0160] In one embodiment, L 2 can be 2, 4 or 6, and L 1 and L3 It can be 1, 2, 3, 4, 5 or 6.

[0161] In the above compounds, R 1 -L 1 - may be an alkyl aldehyde, for example, C 2-6 Alkyl aldehyde, and specifically, propionaldehyde, butyraldehyde, etc., but not particularly limited thereto.

[0162] Furthermore, the polyethylene glycol compound of the present invention may have a molecular weight of about 100 Daltons to about 110,000 Daltons, specifically about 400 Daltons to about 110,000 Daltons, more specifically about 1,000 Daltons to about 100,000 Daltons, and even more specifically about 1,000 Daltons to about 20,000 Daltons, but is not particularly limited thereto.

[0163] In Formula 1, n may be an integer of 10-2400, and specifically an integer of 20-460, but is not particularly limited thereto.

[0164] Specifically, the compound may be a compound represented by Formula 2:

[0165] CHO-(CH 2 ) j -O-(CH 2 CH 2 O) n -(CH 2 ) m -NH(CO)-(CH 2 ) k -R 2

[0166] [Formula 2]

[0167] Among them, in formula 2,

[0168] n is an integer from 10 to 2400,

[0169] j, m and k are each independently an integer from 1 to 6, and

[0170] R 2 It is o-dithiopyridine, sulfhydryl or halogen.

[0171] More specifically, in Formula 2, n may be an integer of 10-2400, and even more specifically an integer of 20-460, but is not particularly limited thereto.

[0172] In a specific embodiment, j, m and k are each independently an integer from 1-6, and specifically an integer from 1-4.

[0173] For example, j can be an integer of 1, 2, 3, 4, 5, or 6; m can be an integer of 1, 2, 3, 4, 5, or 6; and k can be an integer of 1, 2, 3, 4, 5, or 6.

[0174] In one embodiment, j may be 2, 3, 4, 5, or 6; m may be 2, 4, or 6; and k may be 1, 2, 3, 4, 5, or 6.

[0175] In addition, R 2 It may be o-dithiopyridine, a thiol group, or a halogen; specifically o-dithiopyridine, a thiol group, or F, Br, Cl, or I; and more specifically o-dithiopyridine or I, but is not particularly limited thereto.

[0176] Specifically, the compound may be a compound selected from Formula 6 to Formula 11.

[0177]

[0178]

[0179] In Formula 6 to Formula 11, n is the same as defined above.

[0180] In an embodiment of the present invention, the compound represented by Formula 6 is named Linker #4; the compound represented by Formula 7 is named Linker #5; the compound represented by Formula 8 is named Linker #6; the compound represented by Formula 9 is named Linker #7; the compound represented by Formula 10 is named Linker #8; and the compound represented by Formula 11 is named Linker #9.

[0181] In one embodiment of the present invention, it is confirmed that the compound belonging to Formula 1 can show higher reactivity to a thiol group than the above-mentioned compound not including a -NHCO- structure. Therefore, the compound belonging to Formula 1 can be effectively used for connection to a substance including a thiol group.

[0182] Meanwhile, the compound may specifically have any structure selected from Formula 3 to Formula 5:

[0183]

[0184]

[0185] Where n is the same as defined above.

[0186] Meanwhile, the compound may exist in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed from a pharmaceutically acceptable free acid is available.

[0187] The kind of the salt is not particularly limited. However, the salt is preferably a salt that is safe and effective for an individual such as a mammal, but is not particularly limited thereto.

[0188] The term "pharmaceutically acceptable" refers to materials that are effective for their intended use within the scope of pharmaceutical-medical judgment without inducing undue toxicity, irritation, allergic response, and the like.

[0189] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid or base. Examples of suitable salts may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Examples of salts derived from suitable bases may include alkali metals such as sodium, potassium, and the like; alkaline earth metals such as magnesium; ammonium, and the like.

[0190] Acid addition salts can be prepared by conventional methods, for example, the compound is dissolved in an excess of aqueous acid solution and then a water-miscible organic solvent (for example, methanol, ethanol, acetone or acetonitrile) is used to precipitate the resulting salt. The equimolar amount of the compound in water and the acid or alcohol (for example, ethylene glycol monomethyl ether) can be heated, and subsequently, the mixture can be evaporated and dried, or the resulting salt can be filtered with suction.

[0191] In addition, pharmaceutically acceptable metal salts can be prepared using a base.Alkali metal salts or alkaline earth metal salts can be prepared, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating the filtrate, followed by drying.

[0192] Furthermore, the compound of the present invention and a pharmaceutically acceptable salt thereof are a concept including solvates that can be prepared therefrom.

[0193] As used herein, the term "solvate" refers to a complex formed between a compound of the present invention or a salt thereof and solvent molecules.

[0194] In addition, when the compounds of the present invention have asymmetric carbon centers in their substituents, the compounds may exist as (R) or (S) isomers, racemates, mixtures of diastereomers and individual diastereomers, and all such isomers and mixtures thereof fall within the scope of the present invention.

[0195] Another aspect of the present invention provides a method for preparing a physiologically active polypeptide linked to a polyethylene glycol compound, the method comprising reacting a polyethylene glycol compound with a physiologically active polypeptide to prepare the physiologically active polypeptide linked to the polyethylene glycol compound.

[0196] The polyethylene glycol compound is the same as explained above.

[0197] The above method may include connecting any reactive end group located at both ends of the polyethylene glycol compound to the physiologically active polypeptide. More specifically, the reactive end group located at R 1 The reactive end group of R 2 The reactive terminal group of the peptide is linked to a physiologically active polypeptide, but the method is not limited thereto.

[0198] More specifically, the method may include causing a 2 The method is to react o-disulfide pyridine, thiol or halogen with the thiol group of the cysteine ​​residue of the physiologically active polypeptide, but the method is not limited thereto.

[0199] The reaction between the polyethylene glycol compound and the physiologically active polypeptide can be appropriately determined by those skilled in the art, taking into account the characteristics of the reactive terminal group of the polyethylene glycol compound and the characteristics of the reactive group of the physiologically active polypeptide to which the polyethylene glycol compound is to be linked.

[0200] For example, the reaction can be carried out in citrate buffer or HEPES buffer or in an organic solvent such as C 1-6 The reaction may be carried out in the presence of an alcohol, but is not particularly limited thereto.

[0201] In addition, the method may further include purifying the physiologically active polypeptide linked to the polyethylene glycol compound.

[0202] For purification, any known method in the art can be used without limitation, and specifically chromatography can be used, but the purification method is not particularly limited thereto.

[0203] As used herein, the term "physiologically active polypeptide" refers to a concept including peptides or proteins capable of exhibiting physiological activity, and preferably refers to a substance that exhibits its physiological activity in an individual.

[0204] The physiologically active polypeptides are selected from hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, insulinotropic peptides, neuropeptides, pituitary hormones, anti-obesity peptides, antiviral peptides, non-natural peptide derivatives with physiological activity, structural proteins, ligand proteins and receptors.

[0205] Examples of physiologically active polypeptides may include GLP-1 receptor agonists, leptin receptor agonists, DPP-IV inhibitors, Y5 receptor antagonists, melanin-concentrating hormone (MCH) receptor antagonists, Y2 / 3 receptor agonists, MC3 / 4 receptor agonists, gastric / pancreatic lipase inhibitors, 5HT2c agonists, β3A receptor agonists, amylin receptor agonists, ghrelin antagonists, ghrelin receptor antagonists, etc., but are not particularly limited thereto.

[0206] Furthermore, the physiologically active polypeptide may be a peptide comprising an amino acid sequence represented by the following sequence, or consisting essentially of, or consisting of: The peptide may have activity on a glucagon receptor, a GLP-1 receptor, and a GIP receptor, and these peptides are named triple agonists.

[0207] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16 -Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R 1 (SEQ ID NO: 103)

[0208] In the above formula,

[0209] Xaa1 is histidine, 4-imidazoleacetyl or tyrosine,

[0210] Xaa2 is glycine, α-methyl-glutamate or Aib,

[0211] Xaa3 is glutamic acid or glutamine,

[0212] Xaa7 is threonine or isoleucine,

[0213] Xaa10 is leucine, tyrosine, lysine, cysteine ​​or valine,

[0214] Xaa12 is lysine, serine or isoleucine,

[0215] Xaa13 is glutamine, tyrosine, alanine or cysteine,

[0216] Xaa14 is leucine, methionine or tyrosine,

[0217] Xaa15 is cysteine, aspartic acid, glutamic acid or leucine,

[0218] Xaa16 is glycine, glutamic acid or serine,

[0219] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine ​​or lysine,

[0220] Xaa18 is alanine, glutamine, arginine or histidine,

[0221] Xaa19 is alanine, glutamine, cysteine ​​or valine,

[0222] Xaa20 is lysine, glutamine or arginine,

[0223] Xaa21 is glutamic acid, glutamine, leucine, cysteine ​​or aspartic acid,

[0224] Xaa23 is isoleucine or valine,

[0225] Xaa24 is alanine, glutamine, cysteine, asparagine, aspartic acid or glutamic acid,

[0226] Xaa27 is valine, leucine, lysine or methionine,

[0227] Xaa28 is cysteine, lysine, alanine, asparagine or aspartic acid,

[0228] Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid or histidine, Xaa30 is cysteine, glycine, lysine or histidine, or is absent, and

[0229] R 1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 104), m-SSGAPPPS-n (SEQ ID NO: 105) or m-SSGQPPPS-n (SEQ ID NO: 106), or is absent,

[0230] in:

[0231] m is -Cys-, -Pro- or -Gly-Pro-, and

[0232] n is -Cys-, -Gly-, -Ser- or -His-Gly-, or is absent.

[0233] Examples of the triple agonist may include a peptide including an amino acid sequence selected from SEQ ID NOs: 1-102, but are not particularly limited thereto.

[0234] In addition, the physiologically active polypeptide can be selected from glucagon; insulin; somatostatin; peptide YY (PYY); neuropeptide Y (NPY); glucagon-like peptides, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2); exendin-3; exendin-4; oxyntomodulin; peptides active at glucagon receptors, GLP-1 receptors and GIP receptors; fibroblast growth factor (FGF); factor); ghrelin; angiotensin; bradykinin; calcitonin; corticotropin; eledoisin; gastrin; leptin; oxytocin; vasopressin; luteinizing hormone; prolactin; follicle-stimulating hormone; parathyroid hormone; secretin; sermorelin; human growth hormone (hGH); growth hormone-releasing peptide; granulocyte colony-stimulating factor (GCSF); interferon (IFN); interleukin (Interleukin); prolactin-releasing peptide; orexin; thyroid-releasing peptide; cholecystokinin; gastrin-inhibiting peptide; calmodulin; gastric releasing peptide; motilin; vasoactive intestinal peptide; atrial natriuretic peptide peptide, ANP; B-type natriuretic peptide (BNP); C-type natriuretic peptide (CNP); Neurokinin A; Neuromedin; Renin; Endothelin; Sarafotoxin peptide; Carsomorphin peptide; Dermorphin; Dynorphin; Endorphin; Enkepalin; T cell factor; Tumor necrosis factor; Tumor necrosis factor receptor; Urokinase receptor; Tumor suppressor factor; Collagenase inhibitor; Thymopoietin; Thymulin; Thymopentin; Tymosin; Thymic humoral factor;Adrenomodullin; Allatostatin; Amyloid beta-protein fragment); antimicrobial peptides; antioxidant peptides; bombesin; osteocalcin; CART peptides; E-selectin; ICAM-1; VCAM-1; leucokine; kringle-5; laminin; inhibin; galanin; fibronectin; pancreas inhibitor; fuzeon; interferon receptor; G protein-coupled receptor; interleukin receptor; enzyme; interleukin binding protein; cytokine binding protein; macrophage activating factor; macrophage peptide; B cell factor; protein A; allergy inhibitor; necrosis glycoprotein; immunotoxin; lymphotoxin; tumor suppressor; metastatic growth factor; alpha-1-antitrypsin; albumin; alpha-lactalbumin; apolipoprotein-E; erythropoietin; hyperglycosylated erythropoietin; angiogenin; hemoglobin ; thrombin; thrombin receptor activating peptide; thrombomodulin; blood factors VII, VIIa, VIII, IX and XIII; plasminogen activator; fibrin-binding peptide; urokinase; streptokinase; hirudin; protein C; C-reactive protein; renin inhibitor; superoxide dismutase; platelet-derived growth factor; epidermal growth factor; epithelial cell growth factor; angiostatin; angiotensin; osteogenic growth factor; osteogenic stimulating protein; atrial natriuretic peptide; chondroitin; elcatonin; connective tissue activating factor; Tissue factor pathway inhibitor; luteinizing hormone-releasing hormone; nerve growth factor; relaxin; somatomedin; insulin-like growth factor; adrenocortical hormone; pancreatic polypeptide; gastrin-releasing peptide; corticotropin-releasing factor; thyroid stimulating hormone; autotoxin; lactoferrin; myostatin; cell surface antigen; virus-derived vaccine antigen; monoclonal antibody; polyclonal antibody; antibody fragment; erythropoietic growth factor; leukopoietin; amylin; and their analogs, but not particularly limited thereto. ;

[0235] Another aspect of the present invention provides a method for preparing a conjugate, wherein the physiologically active polypeptide and the carrier protein are linked via a polyethylene glycol compound.

[0236] The physiologically active polypeptide and polyethylene glycol compound are the same as explained above.

[0237] Specifically, the method may include:

[0238] (a) reacting a polyethylene glycol compound with any one of a physiologically active polypeptide or a carrier protein, thereby preparing a polyethylene glycol compound, wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group; and

[0239] (b) reacting the polyethylene glycol compound prepared in step (a), wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group, with the other of the physiologically active polypeptide or the carrier protein so as to link the carrier protein or the physiologically active polypeptide to the reactive terminal group of the polyethylene glycol compound, thereby preparing a conjugate in which the physiologically active polypeptide and the carrier protein are linked via the polyethylene glycol compound.

[0240] More specifically, the method may include:

[0241] (a) reacting a polyethylene glycol compound with a physiologically active polypeptide to prepare a polyethylene glycol compound, wherein the physiologically active polypeptide is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group; and

[0242] (b) reacting the polyethylene glycol compound prepared in step (a), wherein the physiologically active polypeptide is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group, with a carrier protein to link the carrier protein to the reactive terminal group of the polyethylene glycol compound.

[0243] Specifically, the polyethylene glycol compound in step (a) may have a structure of Formula 1.

[0244]

[0245] In formula 1,

[0246] R 1 It is aldehyde;

[0247] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0248] R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and

[0249] n is an integer from 10 to 2400.

[0250] In the above method, the reaction between the polyethylene glycol compound and the physiologically active polypeptide may include R 2The reaction with the thiol group of the cysteine ​​residue located in the physiologically active polypeptide and the reaction between the polyethylene glycol compound and the carrier protein may include a reaction between the aldehyde terminal group of the polyethylene glycol compound and the amine group of the carrier protein, but the reaction is not particularly limited thereto.

[0251] Specifically, in the above method, step (a) can make R of the polyethylene glycol compound having the structure of Formula 1 2 The step (b) can react with the sulfhydryl group of the cysteine ​​residue located in the physiologically active polypeptide, while the aldehyde terminal group of the polyethylene glycol compound can react with the amine group of the carrier protein.

[0252] The reaction between the polyethylene glycol compound and the physiologically active polypeptide or carrier protein can be appropriately determined by those skilled in the art, taking into account the characteristics of the reactive groups of the polyethylene glycol compound and those of the reactive groups of the physiologically active polypeptide or carrier protein to which the polyethylene glycol compound is to be linked.

[0253] For example, the PEGylation reaction can be carried out in an appropriate buffer such as citrate buffer or HEPES or an organic solvent such as C 1-6 The reaction is carried out in the presence of alcohol, but the buffer and the solvent are not particularly limited thereto.

[0254] Furthermore, the aldehyde reactive group selectively reacts with the amino terminus under low pH conditions and can form a covalent bond with a lysine residue under high pH conditions (eg, pH 9.0).

[0255] Meanwhile, the carrier protein may be a substance linked to the physiologically active polypeptide through a polyethylene glycol compound for the purpose of increasing the in vivo half-life of the physiologically active polypeptide.

[0256] The carrier protein may be albumin and its fragments, a polymer of repeating units of a specific amino acid sequence, an antibody, an antibody fragment, an FcRn-binding substance, fibronectin, transferrin, sugar or elastin, and the FcRn-binding substance may be an immunoglobulin Fc fragment, but is not particularly limited thereto.

[0257] In a specific embodiment, the terminal aldehyde group of the polyethylene glycol compound may be a group that reacts with an amine group of the immunoglobulin Fc fragment, particularly an amine group at the N-terminus, but is not particularly limited thereto.

[0258] In the present invention, "immunoglobulin Fc region" refers to a region including heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of immunoglobulins. The immunoglobulin Fc region can be a component of the protein conjugate of the present invention.

[0259] The immunoglobulin Fc region may include a hinge region in the heavy chain constant region, but is not limited thereto. In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region including part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), excluding the heavy chain and light chain variable regions of the immunoglobulin, as long as the immunoglobulin Fc region has a potency that is substantially the same as the natural type or improved compared to the natural type. In addition, the immunoglobulin Fc region of the present invention may be a region in which a considerable portion of the amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0260] For example, the immunoglobulin Fc region of the present invention can be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and one or two or more domains in an immunoglobulin hinge region (or a portion of a hinge region); and 6) a dimer between each domain of a heavy chain constant region and a light chain constant region, but is not limited thereto.

[0261] In addition, in a specific embodiment, the immunoglobulin Fc region can be in the form of a dimer, and one molecule of X can be covalently linked to one Fc region in the form of a dimer, wherein the immunoglobulin Fc and X can be linked to each other via a polyethylene glycol compound. At the same time, two molecules of X can also be symmetrically linked to one Fc region in the form of a dimer, wherein the immunoglobulin Fc and X can be linked to each other via a polyethylene glycol compound. However, the connection is not limited thereto.

[0262] In addition, the immunoglobulin Fc region of the present invention includes not only natural amino acid sequences but also sequence derivatives thereof. Amino acid sequence derivatives refer to amino acid sequences in which at least one amino acid residue has a difference due to deletion, insertion, non-conservative or conservative substitution or a combination thereof.

[0263] For example, amino acid residues at positions 214-238, 297-299, 318-322, or 327-331, which are known to be important in the conjugation of immunoglobulin Fc, can be used as suitable sites for modification.

[0264] In addition, various other derivatives are possible, including derivatives that delete regions capable of forming disulfide bonds, or delete some amino acid residues at the N-terminus of the native Fc or add methionine residues at the N-terminus of the native Fc. In addition, in order to remove effector functions, deletions can occur at complement binding sites, such as C1q binding sites and antibody dependent cell mediated cytotoxicity (ADCC) sites. The technology for preparing such sequence derivatives of immunoglobulin Fc regions is disclosed in International Patent Publication Nos. WO 97 / 34631, WO 96 / 32478, etc.

[0265] Amino acid exchanges in proteins and peptides that do not completely change the activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. Depending on the circumstances, the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0266] The above-mentioned Fc derivatives exhibit the same biological activities as the Fc region of the present invention, and they may have improved structural stability to heat, pH, and the like.

[0267] In addition, the immunoglobulin Fc region can be obtained from a natural form isolated in vivo from humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or can be a recombinant or derivative thereof, obtained from transformed animal cells or microorganisms. In this article, the Fc region can be obtained from natural immunoglobulins by isolating whole immunoglobulins from living human or animal bodies and treating the isolated immunoglobulins with proteases. When the whole immunoglobulin is treated with papain, it is cleaved into Fab and Fc regions, and when the whole immunoglobulin is treated with pepsin, it is cleaved into pF'c and F(ab). 2 Fc or pF'c can be separated by size exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0268] In addition, the immunoglobulin Fc region can be in the form of native glycans, glycans increased or decreased compared to the native type, or in a deglycosylated form. The increase, reduction or removal of immunoglobulin Fc glycans can be accomplished by conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. The immunoglobulin Fc region obtained by removing glycans from the Fc region exhibits a significant reduction in binding affinity to the C1q portion, as well as a reduction or loss of antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, so it does not induce an unnecessary immune response in the body. In this regard, the immunoglobulin Fc region in a deglycosylated or hypoglycosylated immunoglobulin Fc region may be a more suitable form of drug carrier, satisfying the original purpose of the present invention.

[0269] As used herein, the term "deglycosylation" refers to the enzymatic removal of carbohydrate moieties from an Fc region, while the term "aglycosylation" refers to an unglycosylated Fc region produced in a prokaryotic organism, in a more specific embodiment, E. coli.

[0270] Meanwhile, the immunoglobulin Fc region can be derived from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. In a more specific embodiment, it is of human origin.

[0271] In addition, the immunoglobulin (Ig) Fc region can be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is the most abundant protein in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to increase the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the IgG4 Fc region is a hypoglycosylated Fc region derived from human IgG4, but is not limited thereto.

[0272] In particular, as used herein, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same source is connected to a single-chain polypeptide of a different source to form a dimer or multimer. That is, the dimer or multimer can be formed from two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

[0273] In addition, the above method may further comprise purifying the conjugate, wherein the physiologically active polypeptide and the carrier protein are linked via a polyethylene glycol compound.

[0274] For purification, any known method in the art can be used without limitation, and specifically chromatography can be used, but the purification method is not particularly limited thereto.

[0275] Another aspect of the present invention provides a physiologically active polypeptide linked to a polyethylene glycol compound.

[0276] The polyethylene glycol compound and the physiologically active polypeptide are the same as explained above.

[0277] In a specific embodiment, the physiologically active polypeptide may be a polypeptide having a structure represented by any one of Formula 15 to Formula 17:

[0278] R 1 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -SSX

[0279] [Formula 15]

[0280] R 1 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-CH 2 -SX

[0281] [Formula 16]

[0282] X-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -R 2

[0283] [Formula 17]

[0284] Among them, in formula 15-formula 17,

[0285] R 1 Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, mercapto groups, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives;

[0286] L 1-L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0287] n is an integer from 10 to 2400;

[0288] R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and

[0289] X corresponds to the physiologically active polypeptide portion.

[0290] All explanations provided above apply to the specific features and combinations of the above variables.

[0291] The "-SSX" in Formula 15 may be a structure formed by the reaction between a thiol group (located at X) and an o-disulfide pyridine or a thiol group; and the "-CH 2 -SX" may be a structure formed by the reaction between a thiol group (located at X) and a halogen, particularly iodoacetamide (IA); and "X-NHCH 2 -" may be a structure formed by reductive alkylation through a reaction between an amine group (located at X) and an aldehyde group, but the structure is not particularly limited thereto.

[0292] Another aspect of the present invention provides a conjugate, wherein each of the physiologically active polypeptide and the carrier protein is independently linked to reactive groups at both ends of a polyethylene glycol compound.

[0293] The polyethylene glycol compound, physiologically active polypeptide and carrier protein are the same as explained above.

[0294] In a specific embodiment, the conjugate has a structure represented by Formula 18 or Formula 19:

[0295] Y-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-L 3 -SSX

[0296] [Formula 18]

[0297] Y-NHCH 2 -L 1 -O-(CH 2 CH 2 O) n -L 2 -NH(CO)-CH 2 -SX

[0298] [Formula 19]

[0299] Among them, in equations 18 and 19,

[0300] L 1 -L 3 Each independently is a linear or branched C 1-6 Alkylene;

[0301] n is an integer from 10 to 2400;

[0302] X is a physiologically active polypeptide moiety; and

[0303] Y is a carrier protein moiety.

[0304] Another aspect of the present invention provides a method for preparing a polyethylene glycol compound.

[0305] The polyethylene glycol compound is the same as explained above.

[0306] Specifically, the method may include:

[0307] (a) Introducing R 1 To one end of the polyethylene glycol, the R 1 Selected from 2,5-dioxopyrrolidyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C 6-20 Aryl disulfide, C 5-20 Heteroaryl disulfides, vinyl sulfones, mercaptos, halogenated acetamides, succinimides, p-nitrophenyl carbonates and their derivatives; and

[0308] (b) Introduction of -NH(CO)L 3 -R 2 to the other end of the polyethylene glycol, where R 2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen.

[0309] As a non-limiting method, the method may include:

[0310] The first step is to prepare a compound represented by formula 21 from a compound represented by formula 20;

[0311] The second step is to prepare a compound represented by Formula 22 from the compound represented by Formula 21; and

[0312] In the third step, the diethoxymethyl group at the terminal of the compound represented by Formula 22 is converted into an aldehyde by treating the compound represented by Formula 22 with an acid solution:

[0313]

[0314] Wherein, in Formula 20, n' is n or n+1; and

[0315]

[0316] Where L 1 , L 2 , L 3 , n and R 2 Same as above.

[0317] In the case of polyethylene glycol compounds, when L 2 When n is 2, as in connector #5 (Formula 7), n' can be n+1.

[0318] In the above method, the compound represented by Formula 20 in the first step may be a compound prepared by reacting a compound represented by Formula 23 with methanesulfonyl chloride:

[0319]

[0320] In the case of polyethylene glycol compounds, when L 2 When is 2, as in linker #5 (Formula 7), the first step can be carried out by reacting the compound represented by Formula 20 with aqueous ammonia solution and ammonium chloride.

[0321] Meanwhile, in the case of polyethylene glycol compounds, when L 2 When is 3 or higher, such as in connectors #7-#9 (Formula 9-Formula 11), the method may include:

[0322] Step 1-1, reacting a compound represented by Formula 20 with a hydroxyalkyl tetrahydropyranyl ether to prepare a compound represented by Formula 24;

[0323] Step 1-2, reacting the compound represented by Formula 24 with p-toluenesulfonic acid to replace the terminal tetrahydropyranyloxy group with a hydroxyl group;

[0324] step 1-3, reacting the compound obtained in step 1-2 with methanesulfonyl chloride to replace the hydroxyl group with a methanesulfonic acid group; and

[0325] Step 1-4, reacting the compound obtained in step 1-3 with aqueous ammonia solution and ammonium chloride:

[0326]

[0327] Wherein step 1-1 can be carried out in the presence of potassium tert-amyl alcoholate.

[0328] In addition, in the case of introducing OPSS as the reactive terminal group, the second step can be performed by using

[0329] The compound represented by Formula 21 reacts with the compound represented by Formula 25.

[0330]

[0331] Meanwhile, in the case of introducing -I or -SH as the reactive terminal group, the second step can be carried out by reacting the compound represented by Formula 21 with chloro-C 2 -C 7 The alkanoyl chloride reaction is carried out to synthesize a compound containing a chlorine group at its end, which is represented by Formula 26, as an intermediate product; and the compound represented by Formula 26 is reacted with a halogen metal salt in the presence or absence of hydrogen sulfide to convert the chlorine group into a mercapto group or a halogen.

[0332]

[0333] Another aspect of the present invention provides use of a polyethylene glycol compound in linking a carrier to a physiologically active polypeptide, wherein the carrier is capable of increasing the in vivo half-life of the physiologically active polypeptide.

[0334] The physiologically active polypeptide, carrier and polyethylene glycol compound are the same as explained above.

[0335] Another aspect of the present invention provides a composition comprising a physiologically active polypeptide linked to a polyethylene glycol compound, or a conjugate.

[0336] The physiologically active polypeptide, conjugate and polyethylene glycol compound are the same as explained above.

[0337] The composition may be a pharmaceutically acceptable composition and may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include, for oral administration, adhesives, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc.; for injection, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., which may be used in combination; and for topical administration, substrates, excipients, lubricants, preservatives, etc. The formulation types of the pharmaceutical composition of the present invention may be prepared in various combinations with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the composition may be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, flakes, etc. For injection, the composition may be formulated as unit-dose ampoules or multiple dose forms. The composition may also be formulated as solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0338] Meanwhile, examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc.

[0339] Another aspect of the present invention provides a polyethylene glycol compound linker for connecting a carrier to a physiologically active polypeptide, wherein the carrier can increase the in vivo half-life of the physiologically active polypeptide.

[0340] The physiologically active polypeptide, carrier and polyethylene glycol compound are the same as explained above.

[0341] The compounds of the present invention can be synthesized by a series of reactions represented by the reaction scheme below. However, the formula below is only an illustrative example of the method for preparing the compounds of the present invention, so the compounds of the present invention should not be limited by these methods, and these compounds can be prepared by methods known in the art, or can be carried out after appropriate modification of the method.

[0342] Example

[0343] Reaction scheme

[0344]

[0345] Reaction Example 1: Preparation of Compound 2

[0346] Compound 1 and dichloromethane are added to the reaction vessel. While the reaction temperature is maintained at 10 ° C or less, triethylamine and methanesulfonyl chloride are added thereto and stirred at room temperature for 3 hours. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. After the organic layer obtained by extraction, dichloromethane is added to the obtained water layer again and extracted again. The organic layers are combined, washed with distilled water, dried over magnesium sulfate, filtered, and the obtained filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 2.

[0347] Reaction Example 2: Preparation of Compound 3

[0348] Toluene and compound 10 are added to a reaction vessel. After adding potassium tert-amyl alcohol thereto, the mixture is heated to about 50°C and stirred at 50°C for 1 hour (activation solution). Compound 2 and toluene are added to another container. The activation solution is cooled to room temperature and added dropwise to the mixed solution at 30°C for 1 hour. After stirring at 30°C for 3 hours, water is added to the reaction solution and extracted. After layer separation, dichloromethane is added to the resulting aqueous layer and the mixture is extracted. Dichloromethane is added to the aqueous layer again and the mixture is extracted again. The resulting organic layer is dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrate is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 3.

[0349] Reaction Example 3: Preparation of Compound 4

[0350] Compound 3, ethanol and dichloromethane are added in the reaction vessel. To it, p-toluenesulfonic acid (p-TsOH) is added and stirred at room temperature for 20 hours. To it, sodium hydroxide is added and the solvent is concentrated under reduced pressure. To it, dichloromethane and water are added and stirred for 5 minutes. By its extraction organic layer and washed with water. The organic layer magnesium sulfate is dried, filtered, and the resulting filtrate is concentrated under reduced pressure. By adding dichloromethane to it, the concentrated solution is dissolved, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 4.

[0351] Reaction Example 4: Preparation of Compound 5

[0352] Compound 4 and dichloromethane are added in the reaction vessel. While the reaction temperature is maintained at 10 DEG C or below, triethylamine and methanesulfonyl chloride are added thereto and stirred at room temperature for 3 hours. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. After the organic layer is extracted, dichloromethane is added to the obtained water layer and extracted again. The organic layers are combined, washed with distilled water (60mL), dried over magnesium sulfate, filtered, and the obtained filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 5.

[0353] Reaction Example 5: Preparation of Compound 6

[0354] Dimethylformamide and compound 5 are added to the reaction vessel. After the mixture is heated to about 30 ° C, potassium thioacetate is added thereto and stirred at 30 ° C for 5 hours. After the mixture is cooled to room temperature, dichloromethane and water are added thereto and extracted. After layer separation, the resulting water layer is extracted again with dichloromethane. The organic layers extracted after layer separation are combined and washed with 20% sodium chloride aqueous solution. After layer separation, sodium sulfate is added to the organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrate is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 6.

[0355] Reaction Example 6: Preparation of Compound 7

[0356] Water and compound 6 are added to the container. The reaction solution is adjusted to pH 14 by dropwise adding 0.1M sodium hydroxide solution thereto. After the mixture is stirred at room temperature for 12 hours, the mixture is adjusted to pH 6-7 using 1NHCl solution. After neutralization, dichloromethane is added thereto and the mixture is extracted. After layer separation, the resulting water layer is extracted again with dichloromethane. The organic layers extracted after layer separation are merged, sodium sulfate is added thereto and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added thereto dropwise, kept for 5 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 7.

[0357] Reaction Example 7: Preparation of Compound 8

[0358] Compound 7 and methanol are added to the reaction vessel. Compound 11 is added dropwise thereto and stirred at room temperature for 3 days. The reaction solvent is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 8.

[0359] Reaction Example 8: Preparation of Compound 9 [Linker #1]

[0360] Compound 8 and distilled water are added in the reaction vessel. Utilize 1N HCl solution that the reaction solution is adjusted to pH 1 and stir at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. Utilize 5% sodium bicarbonate that the mixture is adjusted to pH 6. Dichloromethane is added thereto and extracted. Sodium sulfate is added to the gained organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. By adding dichloromethane (1mL) to it, the concentrated solution is dissolved, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 9.

[0361]

[0362] Reaction Example 9: Preparation of Compound 13

[0363] Toluene and compound 12 are added to a reaction vessel. After potassium tert-amyl alcohol is added thereto, the mixture is heated to about 50°C and stirred at 50°C for 1 hour (activation solution). Compound 2 and toluene are added to another container. The activation solution is cooled to room temperature and added dropwise to the mixed solution at 30°C for 1 hour. After the mixture is stirred at 30°C for 3 hours, water is added to the reaction solution and extracted. After layer separation, dichloromethane is added to the water layer and extracted. Dichloromethane is added to the water layer again and the mixture is extracted again. The resulting organic layer is dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrate is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 13.

[0364] Reaction Example 10: Preparation of Compound 14

[0365] Compound 13, ethanol and dichloromethane are added to the reaction vessel. To it, p-toluenesulfonic acid (p-TsOH) is added and stirred at room temperature for 20 hours. To it, sodium hydroxide is added and the solvent is concentrated under reduced pressure. To it, dichloromethane and water are added and stirred for 5 minutes. The organic layer is extracted and washed with water. The organic layer is dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. By adding dichloromethane to it, the concentrated solution is dissolved, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 14.

[0366] Reaction Example 11: Preparation of Compound 15

[0367] Compound 14 and dichloromethane are added to the reaction vessel. While the reaction temperature is maintained at 10 ° C or less, triethylamine and methanesulfonyl chloride are added thereto and stirred at room temperature for 3 hours. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. After the organic layer is extracted, dichloromethane is added to the obtained water layer again and extracted again. The organic layers are combined, washed with distilled water, dried over magnesium sulfate, filtered, and the obtained filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 15.

[0368] Reaction Example 12: Preparation of Compound 16

[0369] Acetone and compound 15 are added to the reaction vessel. After the mixture is heated to about 30 ° C, potassium iodide is added thereto, heated to about 50 ° C and stirred at 50 ° C for 15 hours. The reaction solution is concentrated under reduced pressure and washed with dichloromethane and water. After layer separation, the obtained organic layer is washed with water again. After layer separation, sodium sulfate is added to the organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether (30mL) is added dropwise thereto, kept for 5 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 16.

[0370] Reaction Example 13: Preparation of Compound 17 [Linker #2]

[0371] Compound 16 and water are added to a container. The reaction solution is adjusted to pH 1.0 by adding 1N HCl solution dropwise thereto. After stirring at room temperature for 1 hour, the reaction solution is adjusted to pH 6-7 using 5% sodium bicarbonate solution. After neutralization, dichloromethane is added thereto and extracted. After layer separation, the aqueous layer is extracted again with dichloromethane. The organic layers extracted after layer separation are combined, sodium sulfate is added thereto and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added thereto dropwise, and kept for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 17.

[0372]

[0373] Reaction Example 14: Preparation of Compound 18

[0374] Dimethylformamide and compound 15 are added to a container. After the mixture is heated to about 30 ° C, potassium thioacetate is added thereto and stirred at 30 ° C for 5 hours. After the mixture is cooled to room temperature, dichloromethane and water are added thereto and extracted. After layer separation, the resulting water layer is extracted again with dichloromethane. The organic layers extracted after layer separation are combined and washed with 20% sodium chloride aqueous solution. After layer separation, sodium sulfate is added to the organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrate is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 18.

[0375] Reaction Example 15: Preparation of Compound 19

[0376] Compound 18 and water are added to the reaction vessel. The reaction solution is adjusted to pH 14 by adding 0.1M NaOH solution dropwise thereto. After stirring at room temperature for 12 hours, the reaction solution is adjusted to pH 6-7 using 1N HCl solution. After neutralization, dichloromethane is added thereto and extracted. After layer separation, the water layer is extracted again with dichloromethane. The organic layers extracted after layer separation are merged, sodium sulfate is added thereto and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 19.

[0377] Reaction Example 16: Preparation of Compound 20 [Linker #3]

[0378] Compound 19 and water are added to the reaction vessel. The reaction solution is adjusted to pH 1.0 by adding 1N HCl solution dropwise thereto. After stirring at room temperature for 1 hour, the reaction solution is adjusted to pH 6-7 using 5% sodium bicarbonate solution. After neutralization, dichloromethane is added thereto and extracted. After layer separation, the aqueous layer is extracted again with dichloromethane (5mL). The organic layers extracted after layer separation are merged, sodium sulfate is added thereto and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 20.

[0379]

[0380] Reaction Example 17: Preparation of Compound 21

[0381] Ammonia solution and ammonium chloride are added to the reaction vessel. Compound 5 is added thereto and stirred at room temperature for 4 days. Dichloromethane is added thereto and stirred for 5 minutes. After the organic layer is extracted, dichloromethane is added to the obtained water layer again and extracted again. The organic layers are combined, washed with distilled water, dried over magnesium sulfate, filtered, and the obtained filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 21.

[0382] Reaction Example 18: Preparation of Compound 22

[0383] Compound 21 and dichloromethane are added in a reaction vessel. Triethylamine and chloroacetyl chloride are added dropwise thereto and stirred at room temperature for 16 hours. When the reaction is complete, the reaction solution is washed with water, dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 22.

[0384] Reaction Example 19: Preparation of Compound 23

[0385] Compound 22 and acetone are added to the reaction vessel. KI is added thereto and stirred at 55 ° C for 6 hours. After the reaction solution is cooled to room temperature, the reaction solvent is concentrated under reduced pressure. Then, dichloromethane and water are added thereto and stirred for 5 minutes. After the organic layer is extracted, dichloromethane is added to the resulting water layer again and extracted again. The organic layer magnesium sulfate is dried, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 23.

[0386] Reaction Example 20: Preparation of Compound 24 [Linker #5]

[0387] Compound 23 and distilled water are added to the reaction vessel. The reaction solution is adjusted to pH 1.0 using 1N HCl solution and stirred at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. The resulting product is adjusted to pH 6 using 5% sodium bicarbonate. The resulting product is extracted by adding dichloromethane thereto, sodium sulfate is added to the resulting organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 24.

[0388]

[0389] Reaction Example 21: Preparation of Compound 25

[0390] Compound 5 and acetone are added to the reaction vessel. KI is added thereto and stirred at 55 ° C for 20 hours. After the reaction solution is cooled to room temperature, the reaction solvent is concentrated under reduced pressure. Then, dichloromethane and water are added thereto and stirred for 5 minutes. After the organic layer is extracted, dichloromethane is added to the resulting water layer again and extracted again. The organic layer magnesium sulfate is dried, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 25.

[0391] Reaction Example 22: Preparation of Compound 26 [Linker #11]

[0392] Compound 25 and distilled water are added to the reaction vessel. The reaction solution is adjusted to pH 1.0 using 1N HCl solution and stirred at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. The resulting product is adjusted to pH 6 using 5% sodium bicarbonate. The resulting product is extracted by adding dichloromethane thereto, sodium sulfate is added to the resulting organic layer and stirred for 30 minutes. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 26.

[0393]

[0394] Reaction Example 23: Preparation of Compound 27

[0395] Ammonia solution and ammonium chloride are added in the reaction vessel.Compound 5 is added thereto and stirred at room temperature for 4 days.Dichloromethane is added thereto, an organic layer is extracted therefrom, dichloromethane is added again and extracted again.The organic layer is combined, washed with distilled water, dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure.The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes.The resulting crystal is filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 27.

[0396] Reaction Example 24: Preparation of Compound 28

[0397] Compound 27, compound 30, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), hydroxybenzotriazole (HOBt), triethylamine and dimethylformamide are added to the reaction vessel and stirred at room temperature for 16 hours. Dichloromethane and water are added thereto and extracted. After layer separation, the obtained water layer is extracted again with dichloromethane. The organic layers extracted after layer separation are merged and washed with 20% sodium chloride aqueous solution. After layer separation, sodium sulfate is added to the organic layer and stirred. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 5 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 28.

[0398] Reaction Example 25: Preparation of Compound 29 [Linker #7]

[0399] Compound 28 and distilled water are added to the reaction vessel. The reaction solution is adjusted to pH 1.0 using 1N HCl solution and stirred at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred. The resulting product is adjusted to pH 6 using 5% sodium bicarbonate. The resulting product is extracted by adding dichloromethane thereto, and sodium sulfate is added to the resulting organic layer. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 29.

[0400]

[0401] Reaction Example 26: Preparation of Compound 31

[0402] Compound 27 and dichloromethane are added in the reaction vessel. Triethylamine and chloroacetyl chloride are added dropwise thereto and stirred at room temperature for 16 hours. When the reaction is complete, the reaction solution is washed with water, dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tertiary butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystal is filtered, washed with methyl tertiary butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 31.

[0403] Reaction Example 27: Preparation of Compound 32

[0404] Compound 31 and acetone are added to the reaction vessel. KI is added thereto and stirred at 55 ° C for 6 hours. After the reaction solution is cooled to room temperature, the reaction solvent is concentrated under reduced pressure. Then, dichloromethane and water are added thereto. After the organic layer is extracted, dichloromethane is added to the resulting water layer again and extracted again. The organic layer magnesium sulfate is dried, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrate is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 32.

[0405] Reaction Example 28: Preparation of Compound 33 [Linker #8]

[0406] Compound 32 and distilled water are added to the reaction vessel. The reaction solution is adjusted to pH 1 using 1N HCl solution and stirred at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. The resulting product is adjusted to pH 6 using 5% sodium bicarbonate. The resulting product is extracted by adding dichloromethane thereto, and sodium sulfate is added to the resulting organic layer. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 33.

[0407]

[0408] Reaction Example 29: Preparation of Compound 34

[0409] Compound 31 and methanol are added to the reaction vessel. KI and NaSH are added thereto and stirred at room temperature for 6 hours. After the reaction solution is cooled to room temperature, the reaction solvent is concentrated under reduced pressure. Then, dichloromethane and water are added thereto. After the organic layer is extracted, dichloromethane is added to the resulting water layer again and extracted again. The organic layer is dried over magnesium sulfate, filtered, and the resulting filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto, kept for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 34.

[0410] Reaction Example 30: Preparation of Compound 35 [Linker #9]

[0411] Compound 34 and distilled water are added to the reaction vessel. The reaction solution is adjusted to pH 1 using 1N HCl solution and stirred at room temperature for 1 hour. When the reaction is complete, water and dichloromethane are added thereto and stirred for 5 minutes. The resulting product is adjusted to pH 6 using 5% sodium bicarbonate. The resulting product is extracted by adding dichloromethane thereto, and sodium sulfate is added to the resulting organic layer. The mixed solution is filtered and the filtrate is concentrated under reduced pressure. The concentrated solution is dissolved by adding dichloromethane thereto, and methyl tert-butyl ether is added dropwise thereto for 20 minutes. The resulting crystals are filtered, washed with methyl tert-butyl ether, and dried under nitrogen at room temperature to obtain the target compound, compound 35.

[0412] Hereafter, the present invention will be described in more detail with reference to the following embodiments, etc., so as to assist in understanding the present invention. However, these embodiments can be modified into various other forms, and the scope of the present invention should not be understood to be limited by these embodiments. Embodiments are provided to more fully illustrate the present invention to those skilled in the art.

[0413] Example 1: Preparation of polyethylene glycol derivatives

[0414] The present inventors have prepared polyethylene glycol derivatives in which desired reactive groups are introduced at both ends. The method for preparing the derivatives is described in Reaction Examples 1-25.

[0415] Typically, heterofunctional PEG compounds are prepared in which a propionaldehyde group is added to one end of the polyethylene glycol backbone, while o-pyridinium disulfide (OPSS), iodoacetamide (IA), iodine, or a thiol group (SH-) is added to the other end [Figure 1].

[0416] The purity of each PEG compound thus prepared was analyzed by NMR and reverse phase chromatography (RPC), respectively.

[0417] Details of representative polyethylene glycol derivatives are as follows.

[0418] (1) Linker #1: pALD-PEG-o-dithiopyridine

[0419]

[0420] Here, n is an integer between 200 and 300.

[0421] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 80% by RPC analysis.

[0422] 1 H-NMR (CDCl 3 ,400MHz): δ9.79(t,1H,J=2.0Hz),8.50(d,1H,J=5.6Hz),7.71-7.64(m,2H),7.09-6.70(m ,1H),3.87-3.40(m,908H),2.82(t,2H,J=5.6Hz),2.68(t,2H,J=2.0Hz),1.86-1.66(m,4H)

[0423] (2) Linker #2: pALD-PEG-iodide

[0424]

[0425] Here, n is an integer between 200 and 300.

[0426] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 87% by RPC analysis.

[0427] 1 H-NMR (CDCl 3 ,400MHz): δ9.81(s,1H),3.84-3.47(m,910H),3.29(t,2H,J=6.8Hz),2.71-2.69(m,2H),2.10-2.06(m,2H)

[0428] (3) Linker #3: pALD-PEG-thiol group

[0429]

[0430] Here, n is an integer between 200 and 300.

[0431] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 76% by RPC analysis.

[0432] 1 H-NMR (CDCl 3 ,400MHz): δ9.79(s,1H),3.82-3.45(m,910H),2.74(t,2H,J=6.8Hz),2.69-2.67(m,2H),1.99-1.95(m,2H)

[0433] (4) Linker #5: pALD-PEG-iodide

[0434]

[0435] Here, n is an integer between 200 and 300.

[0436] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 88% by RPC analysis.

[0437] 1 H-NMR (CDCl 3 ,400MHz): δ9.78(t,1H,J=1.6Hz),3.82-3.41(m,912H),2.67(t,2H,J=2.0Hz)

[0438] (5) Linker #7: pALD-PEG-o-dithiopyridine

[0439]

[0440] Here, n is an integer between 200 and 300.

[0441] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and the purity was confirmed to be about 81% by RPC analysis.

[0442] (6) Linker #8: pALD-PEG-iodide

[0443]

[0444] Here, n is an integer between 200 and 300.

[0445] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 78% by RPC analysis.

[0446] 1 H-NMR (CDCl 3,400MHz): δ9.78(s,1H),3.82-3.41(m,912H),2.70(t,2H,J=2.0Hz),1.44-1.24(m,4H)

[0447] (7) Linker #9: pALD-PEG-thiol group

[0448]

[0449] Here, n is an integer between 200 and 300.

[0450] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and the purity was confirmed to be about 76% by RPC analysis.

[0451] (8) Linker #11: pALD-PEG-iodide (Comparative Example)

[0452]

[0453] Here, n is an integer between 200 and 300.

[0454] The molecular weight of the polyethylene glycol linker thus prepared was about 10 kDa, and its structure was confirmed by NMR analysis. The purity was confirmed to be about 89% by RPC analysis.

[0455] 1 H-NMR (CDCl 3 ,400MHz): δ9.79(t,1H,J=2.0Hz),3.82-3.41(m,908H),3.29-3.22(m,2H),2.69(t,2H,J=2.0Hz)

[0456] Example 2: Comparison of reactivity between new polyethylene glycol derivatives

[0457] In order to prepare a physiologically active polypeptide conjugate using the polyethylene glycol derivative prepared in Example 1, a GLP-1 / glucagon / GIP triple agonist peptide was used as a representative physiologically active peptide to prepare a conjugate. The triple agonist peptide consists of 30 amino acids, and it corresponds to a peptide including a cysteine ​​residue. Therefore, the triple agonist peptide is used to compare the reactivity of the thiol reactive group of the polyethylene glycol derivative of the present invention.

[0458] In addition, linkers #2, #3, #5, #8, #9, and #11 having a molecular weight of about 10K (10,000 Da) were used as polyethylene glycol derivatives. In particular, although linkers #3 and #9 correspond to 10K pALD-PEG-SH (PEG having a propionaldehyde group and a thiol group at both ends, respectively), linker #9 is characterized in that it has an amide structure in front of the thiol reactive group, unlike linker #3. In addition, linkers #2, #5, #8, and #11 correspond to 10K pALD-PEG-I (PEG having a propionaldehyde group and an iodine group at both ends, respectively), and linkers #5 and #8 are characterized in that they have an amide structure in front of the thiol reactive group, unlike linkers #2 and #11.

[0459] After the peptide powder was dissolved in 10 mM HCl, for the PEGylation of the cysteine ​​residues of the peptide with 10K pALD-PEG-I and 10K pALD-PEG-SH, the peptide and PEG were reacted at a molar ratio of 1:3 to 1:5 with a reaction concentration of 3 mg / mL at room temperature for about 2 hours. In particular, the reaction was carried out in the presence of 50 mM sodium citrate (pH 5.0) or 50 mM HEPES (pH 7.5) and 60% isopropanol (IPA). At the end of SDS-PAGE analysis, the reactivity of each thiol reactive group was compared by the band integration method ( Fig.18 ). When the reactivity was confirmed, it was confirmed that the reactivity of PEG characterized by having an amide structure in front of the thiol group was superior to that of PEG having no amide structure in front of the thiol group.

[0460] Example 3: Preparation of a conjugate of a physiologically active polypeptide and immunoglobulin Fc using a novel polyethylene glycol derivative

[0461] In order to prepare a conjugate of a physiologically active polypeptide and immunoglobulin Fc using the polyethylene glycol derivative prepared in Example 1, a conjugate was prepared using the peptide mentioned in Example 2.

[0462] First, a linker #7 having a molecular weight of about 10K (10,000 Da) was used as a polyethylene glycol derivative. In particular, linker #7 corresponds to pALD-PEG-OPSS (PEG having a propionaldehyde group and an ortho-disulfide pyridine group at both ends, respectively) having a molecular weight of about 10K. After the peptide powder was dissolved in 10 mM HCl, for the PEGylation of the cysteine ​​residues of the peptide by 10K pALD-PEG-OPSS, the peptide and PEG were reacted at a molar ratio of 1:1 to 1:3 with a reaction concentration of 1 mg / mL or 3 mg / mL at room temperature for about 2 hours. In particular, the reaction was carried out in the presence of 50 mM sodium citrate (pH 3.0-pH 5.0) or 50 mM Tris (pH 8.0) and 60% isopropanol.

[0463] In addition, linkers #8 and #9 having a molecular weight of about 10K (10,000 Da) were used as polyethylene glycol derivatives. In particular, linker #8 corresponds to 10K pALD-PEG-IA (PEG having a propionaldehyde group and iodoacetamide (IA) at both ends, respectively) having a molecular weight of about 10K, and linker #9 corresponds to pALD-PEG-SH (PEG having a propionaldehyde group and a thiol group at both ends, respectively) having a molecular weight of 10K.

[0464] For the preparation of conjugates using pALD-PEG-IA and pALD-PEG-SH as linkers, the reaction was carried out under the same conditions as in Example 2, and the reaction solution was purified using an SP-HP (GE Healthcare, USA) column using a buffer containing sodium citrate (pH 3.0) and a concentration gradient of 45% EtOH and KCI.

[0465] Then, after conjugation to each reactive group, the purified mono-PEGylated peptide and immunoglobulin Fc were reacted at a molar ratio of 1:5 with a total protein concentration of 20 mg / mL at 4° C. for 15 hours. Specifically, as a reducing agent, 20% isopropanol and 20 mM sodium cyanoborohydride were added to 100 mM potassium phosphate buffer as a reaction solution.

[0466] When the reaction was completed, the reaction solution was applied to a Source 15Q (GE Healthcare, USA) column using a NaCl concentration gradient in Bis-Tris (pH 6.5) buffer and then to a Source ISO (GE Healthcare, USA) column using (NH 4 ) 2 SO 4 The triple agonist-10K PEG-IgFc conjugate was purified by a concentration gradient of 1% PEG-10K and Tris (pH 7.5) buffer. The purity of the conjugate sample thus prepared was confirmed by SDS-PAGE analysis, and the molecular weight of the conjugate of the triple agonist and PEG was confirmed. Then, the molecular weight of the triple agonist-10K PEG-IgFc conjugate was confirmed under non-reducing conditions, wherein IgFc was linked to the PEG conjugated to the triple agonist. The experimental results are shown in Figure 19-21 Shown in.

[0467] Based on the above, the technical personnel of the field to which the present invention relates can understand that the present invention can be embodied in other specific forms without modifying the technical concept or essential features of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended not only to cover the exemplary embodiments, but also to cover various alternatives, modifications, equivalents and other embodiments that may be included in the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A compound represented by formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in, In formula 1, R1 is an aldehyde; L1 and L3 are each independently a linear or branched C 1-6 Alkylene; L2 is a linear or branched C 4-6 Alkylene; R2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and n is an integer from 200 to 300.

2. The compound of claim 1, its stereoisomer or pharmaceutically acceptable salt, wherein R2 is thiol or iodine.

3. The compound of claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof, wherein R1 is an aldehyde and R2 is o-dithiopyridine.

4. The compound according to claim 1, its stereoisomer or pharmaceutically acceptable salt, wherein R1 and R2 have functional groups different from each other.

5. The compound of claim 1, its stereoisomer or pharmaceutically acceptable salt, wherein the compound is represented by Formula 2: CHO-(CH2) j -O-(CH2CH2O) n -(CH2) m -NH(CO)-(CH2) k -R2 [Formula 2] in, In formula 2, n is an integer between 200 and 300; j and k are each independently an integer from 1 to 6; m is an integer from 4 to 6; and R2 is o-pyridine disulfide (OPSS), thiol or halogen.

6. The compound of claim 1, its stereoisomer or pharmaceutically acceptable salt, The compound is selected from the compounds represented by Formula 9 to Formula 11: in, In Formula 9 to Formula 11, n is an integer of 200-300.

7. A method for preparing a physiologically active polypeptide linked to a polyethylene glycol compound, the method comprising reacting the polyethylene glycol compound according to any one of claims 1 to 6 with a physiologically active polypeptide.

8. The method of claim 7, wherein the ortho-disulfide pyridine (OPSS), sulfhydryl group or halogen at R2 reacts with a sulfhydryl group at a cysteine ​​residue of the physiologically active polypeptide.

9. The method of claim 7, further comprising purifying the physiologically active polypeptide linked to the polyethylene glycol compound.

10. The method of claim 7, wherein the physiologically active polypeptide is selected from hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, insulinotropic peptides, neuropeptides, pituitary hormones, anti-obesity peptides, antiviral peptides, physiologically active non-natural peptide derivatives, structural proteins, ligand proteins and receptors.

11. The method of claim 7, wherein the physiologically active polypeptide is selected from the group consisting of glucagon; insulin; somatostatin; peptide YY (PYY); neuropeptide Y (NPY); glucagon-like peptides, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2); exendin-3; exendin-4; oxyntomodulin; peptides active at the glucagon receptor, the GLP-1 receptor, and the GIP receptor; fibroblast growth factor; ghrelin; angiotensin; bradykinin; calcitonin; adrenocorticotropic hormone; octopus sialoprotein; gastrin; leptin; oxytocin; vasopressin; luteinizing hormone; prolactin; Follicle-stimulating hormone; Parathyroid hormone; Secretin; sermorelin; human growth hormone (hGH); growth hormone-releasing peptide; granulocyte colony-stimulating factor (GCSF); interferon (IFN); interleukin; prolactin-releasing peptide; orexin; thyroxine-releasing peptide; cholecystokinin; gastrin-inhibiting peptide; calmodulin; gastric releasing peptide; Motilin; vasoactive intestinal peptide; atrial natriuretic peptide (ANP); B-type natriuretic peptide (BNP); C-type natriuretic peptide (CNP); neurokinin A; neuromodulatory peptides; renin; endothelin; ceratotoxin peptide; carsomorphin peptide; dermomorphin; dynorphin; endorphin; enkepalin; T-cell factor; tumor necrosis factor; tumor necrosis factor receptor; urokinase receptor; tumor suppressor factor; collagenase inhibitor; thymopoietin; thymosin; thymopentin; thymosin; thymic humoral factor; adrenomedullin; pleurosporin; β-amyloid protein fragment; antimicrobial peptide; antioxidant peptide; bombesin; osteocalcin; CART peptide; E-selectin; ICAM-1; VCAM-1; leucokine; kringle-5; laminin; inhibin; galanin; fibronectin; pancreatic inhibin; fuzeon; interferon receptor; G protein-coupled receptor; interleukin receptor; enzyme; interleukin binding protein; cytokine binding protein; macrophage activating factor; macrophage peptide; B cell factor; protein A; allergy inhibitor; necrosis glycoprotein; immunotoxin; lymphotoxin; tumor suppressor; metastatic growth factor; alpha-1-antitrypsin; albumin; alpha-lactalbumin; apolipoprotein-E; Erythropoietin; hyperglycosylated erythropoietin; angiogenin; hemoglobin; thrombin; thrombin receptor activating peptide; thrombomodulin; blood factors VII, VIIa, VIII, IX and XIII; plasminogen activator; fibrin-binding peptide; urokinase; streptokinase; hirudin; protein C; C-reactive protein; renin inhibitor; superoxide dismutase; platelet-derived growth factor; epidermal growth factor; epithelial cell growth factor; angiostatin; angiotensin; osteogenic growth factor; osteogenic stimulating protein; atrial natriuretic peptide; chondroitin; elcatonin; Connective tissue activating factor; tissue factor pathway inhibitor; luteinizing hormone-releasing hormone; nerve growth factor; relaxin; somatomedin; Insulin-like growth factor; Adrenal cortical hormone; Pancreatic polypeptide; Gastrin-releasing peptide; Corticotropin-releasing factor; thyroid stimulating hormone; autotoxins; lactoferrin; myostatin; cell surface antigens; virus-derived vaccine antigens; Monoclonal antibody; Polyclonal antibody; Antibody fragment; Erythropoietic growth factor; Leukopoietin; Amylin; and their analogs.

12. A method for preparing a conjugate in which a physiologically active polypeptide and a carrier protein are linked via a polyethylene glycol compound, the method comprising: (a) reacting the polyethylene glycol compound according to any one of claims 1 to 6 with any one of a physiologically active polypeptide or a carrier protein, thereby preparing the polyethylene glycol compound, wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group; as well as (b) reacting the polyethylene glycol compound prepared in step (a), wherein the physiologically active polypeptide or the carrier protein is linked to one end of the polyethylene glycol compound and the other end of the polyethylene glycol compound has a reactive terminal group, with the other of the physiologically active polypeptide or the carrier protein so as to link the carrier protein or the physiologically active polypeptide to the reactive terminal group of the polyethylene glycol compound, thereby preparing a conjugate in which the physiologically active polypeptide and the carrier protein are linked via the polyethylene glycol compound.

13. The method of claim 12, wherein the physiologically active polypeptide is selected from hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, insulinotropic peptides, neuropeptides, pituitary hormones, anti-obesity peptides, antiviral peptides, physiologically active non-natural peptide derivatives, structural proteins, ligand proteins and receptors.

14. The method of claim 12, wherein the polyethylene glycol compound of step (a) has a structure of Formula 1: in, In formula 1, R1 is an aldehyde group; L1 and L3 are each independently a linear or branched C 1-6 Alkylene; L2 is a linear or branched C 4-6 Alkylene; R2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and n is an integer from 200 to 300.

15. The method of claim 14, wherein step (a) is to react R2 of the polyethylene glycol compound having the structure of Formula 1 with a sulfhydryl group located at a cysteine ​​residue of the physiologically active polypeptide.

16. The method of claim 15, wherein step (b) is to react the aldehyde terminal group of the polyethylene glycol compound with an amine group of the carrier protein.

17. The method of claim 12, further comprising purifying the conjugate, wherein the physiologically active polypeptide and the carrier protein are linked through a polyethylene glycol compound.

18. The method of claim 12, wherein the carrier protein is albumin and fragments thereof, a polymer of repeating units of a specific amino acid sequence, an antibody, an antibody fragment, an FcRn-binding substance, fibronectin, transferrin, a sugar or elastin.

19. The method of claim 18, wherein the FcRn binding substance is an immunoglobulin Fc fragment.

20. A physiologically active polypeptide, wherein the compound according to any one of claims 1 to 6 is linked to the physiologically active polypeptide.

21. The physiologically active polypeptide of claim 20, wherein the physiologically active polypeptide to which the compound is linked has any one of the structures represented by Formula 15 to Formula 17: R1-L1-O-(CH2CH2O) n -L2-NH(CO)-L3-S-S-X [Formula 15] R1-L1-O-(CH2CH2O) n -L2-NH(CO)-CH2-S-X [Formula 16] <h2 style=";text-align:left;direction:ltr">X-NHCH2-L1-O-(CH2CH2O)<h2 style=";text-align:left;direction:ltr"> n <h2 style=";text-align:left;direction:ltr"> -L2-NH(CO)-L3-R2 [Formula 17] in, In equations 15 to 17, R1 is an aldehyde; L1 and L3 are each independently a linear or branched C 1-6 Alkylene; L2 is a linear or branched C 4-6 Alkylene; n is an integer between 200 and 300; R2 is o-pyridine disulfide (OPSS), sulfhydryl or halogen; and X is a physiologically active polypeptide moiety.

22. A conjugate, wherein each of a physiologically active polypeptide and a carrier protein is independently linked to reactive groups at both ends of the compound of any one of claims 1 to 6.

23. The conjugate of claim 22, wherein the conjugate has a structure represented by Formula 18 or 19: <h2 style=";text-align:left;direction:ltr">Y-NHCH2-L1-O-(CH2CH2O)<h2 style=";text-align:left;direction:ltr"> n <h2 style=";text-align:left;direction:ltr"> -L2-NH(CO)-L3-SSX [Formula 18] <h2 style=";text-align:left;direction:ltr">Y-NHCH2-L1-O-(CH2CH2O)<h2 style=";text-align:left;direction:ltr"> n <h2 style=";text-align:left;direction:ltr"> -L2-NH(CO)-CH2-SX [Formula 19] in, In formula 18 or 19, L1 and L3 are each independently a linear or branched C 1-6 Alkylene; L2 is a linear or branched C 4-6 Alkylene; n is an integer between 200 and 300; X is a physiologically active polypeptide moiety; and Y is a carrier protein moiety.

24. The conjugate of claim 22, wherein the carrier protein is albumin and fragments thereof, a polymer of repeating units of a specific amino acid sequence, an antibody, an antibody fragment, an FcRn-binding substance, fibronectin, transferrin, a sugar or elastin.

25. The conjugate of claim 24, wherein the FcRn binding substance is an immunoglobulin Fc fragment.

26. A method for preparing the compound of claim 1, the method comprising: (a) introducing R1 to one end of polyethylene glycol, wherein R1 is an aldehyde; and (b) Introducing the structure of -NH(CO)L3-R2 to the other end of the polyethylene glycol, wherein R2 is o-pyridine disulfide (OPSS), thiol or halogen.

27. The method of claim 26, comprising: The first step is to prepare a compound represented by formula 21 from a compound represented by formula 20; The second step is to prepare a compound represented by formula 22 from the compound represented by formula 21; as well as The third step is to convert the diethoxymethyl group at the terminal of the compound represented by Formula 22 into an aldehyde by treating the compound represented by Formula 22 with an acid solution; Wherein, in Formula 20, n' is n or n+1; and Wherein L1, L2, L3, n and R2 are the same as those described in claim 1.

28. The method of claim 27, wherein the compound represented by formula 20 in the first step is prepared by reacting a compound represented by formula 23 with methanesulfonyl chloride:

29. The method of claim 27, wherein the first step is performed by reacting the compound represented by Formula 20 with an aqueous ammonia solution and ammonium chloride.

30. The method of claim 27, wherein the first step comprises: Step 1-1, reacting a compound represented by Formula 20 with a hydroxyalkyl tetrahydropyranyl ether to prepare a compound represented by Formula 24; Step 1-2, reacting the compound represented by Formula 24 with p-toluenesulfonic acid to replace the terminal tetrahydropyranyloxy group with a hydroxyl group; step 1-3, reacting the compound obtained in step 1-2 with methanesulfonyl chloride to replace the hydroxyl group with a methanesulfonic acid group; and Step 1-4, reacting the compound obtained in step 1-3 with aqueous ammonia solution and ammonium chloride:

31. The method of claim 27, wherein the second step is performed by reacting a compound represented by Formula 21 with a compound represented by Formula 25:

32. The method of claim 27, wherein the second step comprises: The compound represented by formula 21 is reacted with chloro-C 2-7 alkanoyl chloride, thereby synthesizing a compound containing a chlorine group at one end thereof, which is represented by Formula 26 as an intermediate product; and The compound represented by Formula 26 is reacted with a halogen metal salt in the presence or absence of hydrogen sulfide to convert the chlorine group into a mercapto group or a halogen:

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