WO2013086785A1 - Compound and composition having hypoglycemic effect and use thereof - Google Patents

Compound and composition having hypoglycemic effect and use thereof Download PDF

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Publication number
WO2013086785A1
WO2013086785A1 PCT/CN2012/001699 CN2012001699W WO2013086785A1 WO 2013086785 A1 WO2013086785 A1 WO 2013086785A1 CN 2012001699 W CN2012001699 W CN 2012001699W WO 2013086785 A1 WO2013086785 A1 WO 2013086785A1
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chain
sequence
seq
deletion
formula
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PCT/CN2012/001699
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French (fr)
Chinese (zh)
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秦树林
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Qin Shulin
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Priority to CN201280061410.6A priority Critical patent/CN104011070A/en
Publication of WO2013086785A1 publication Critical patent/WO2013086785A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biopharmaceuticals, and particularly relates to compounds, compositions and uses thereof having hypoglycemic effects. Background technique
  • insulin has undergone three generations of historical evolution:
  • the first generation of products was extracted from the pancreas of animals such as pigs or cattle. Due to heterogeneous allergic reactions, these products are less effective.
  • the second generation of products is recombinant human insulin, which is obtained by extracting insulin genes from human cells, and then inserting them into yeast or E. coli for cultivation through complex modern biological gene technology.
  • the third generation is an insulin analogue obtained by structural modification of human insulin, including fast-acting insulin and long-acting insulin.
  • ADA American Diabetes Association
  • EASD European Diabetes Association
  • Basal insulin is used to maintain normal blood glucose secretion on an empty stomach. Many metabolic studies have found that maintaining a basic insulin level between meals and at night can reduce the breakdown of triglyceride, inhibit the liver's output of glucose, and stabilize the fasting blood glucose, thereby reducing overall blood sugar levels. Ideal basal insulins, such as long-acting insulin analogues, should be able to mimic physiological insulin secretion patterns, avoid hypoglycemia, especially nocturnal hypoglycemia, and do not increase body weight.
  • the most long-acting insulins currently used can be divided into three major categories.
  • the first type is a suspension of crystals formed by human insulin with zinc ions or protamine, such as NPH insulin, lente insulin, and the like. These insulin preparations are unstable and are being replaced by long-acting insulin analogues.
  • the second is detemir. It is an insulin analog in which myristic acid is linked to B29 lysine. Detemir is slowly absorbed after injection. The disappearance time T 50% at the injection site is approximately 10 hours. It binds to albumin in the blood through the fatty acid at position 29 and then slowly dissociates from the complex. Dihexamerization, hexamer and dimer binding to albumin prolongs the retention time of insulin detemir at the injection site. After the insulin enters the blood circulation, it binds to albumin, further prolonging the residence time in the body.
  • the third is insulin glargine.
  • the drug dissolves in the formulation at pH 3.0 and crystallizes when the pH rises to about 7.4 after injection.
  • the slow decomposition of the injection site brings about a delayed effect.
  • the absorption properties and pharmacokinetics vary widely in the population and in the body.
  • Insulin glargine and insulin detemir are the only two long-acting insulin analogues on the market, with a maximum duration of action of no more than 24 hours. Insulin glargine is much more active on the insulin-like growth factor-1 receptor (IGF-1R) than native human insulin. Because insulin-like growth factor-1 receptor is closely related to the occurrence of various cancers, it has been controversial for long-term use of ganjing pancreas Whether islands increase the risk of cancer in patients. The biological activity of insulin detemir in humans is about 20% of that of natural human insulin, so it is used at a dose five times that of conventional insulin, which significantly increases production and use costs.
  • IGF-1R insulin-like growth factor-1 receptor
  • Recombinant human insulin is difficult to meet the insulin requirements of the meal.
  • the human insulin molecule usually forms a hexamer structure, which is gradually depolymerized into a dimer after subcutaneous injection, and further dissociates into a monomer to enter the circulation through the capillaries, thereby exerting a hypoglycemic effect. Due to the depolymerization and absorption process, recombinant human insulin takes about 30 minutes after subcutaneous injection, and the peak time is long, and the effect lasts for about 6-7 hours. Moreover, due to individual differences, there is a significant difference in the amount of final influx after injection of the same dose of human insulin.
  • Rapid-acting insulin analogues such as insulin aspart, insulin lispro, etc.
  • Fast-acting insulin analogues absorb quickly, have a short peak time, have a higher peak value, and have a peak concentration of 1 to 3 hours.
  • the duration of action is 3 - 5 hours, which is significantly better than human insulin.
  • the onset time of insulin aspart and insulin lispro is about 20 minutes, which is still not convenient for diabetics, and there is still much room for improvement.
  • a first object of the present invention is to provide a compound having a hypoglycemic action, which comprises an A chain and a B chain, wherein
  • the amino acid sequence of the A chain is: X_ 1 GIVDX 5 C [3] C [ 4] X 8 X 9 X 10 C t5] X 12 LRRLEX 18 YC [6] X 21 X 22 , the B chain amino acid sequence is:
  • i is lysine, arginine or deletion; is lysine, arginine or deletion;
  • X 5 is glutamic acid, asparagine, glutamine or serine;
  • X 8 is histidine, arginine , phenylalanine or threonine;
  • X 9 is arginine or serine;
  • X 10 is serine or isoleucine;
  • X 12 is aspartic acid, serine, glutamine or asparagine;
  • X] 8 is asparagine, methionine or threonine;
  • X 21 is asparagine, alanine or glycine;
  • X 22 is lysine, arginine-lysine dipeptide or deletion;
  • X 2 3_26 Is a phenylalanine-valine-asparagine-glutamine tetrapeptide, or a glycine-valine-glutamate, a
  • [1]-[6] represents the number of a cysteine; in the compound, a three-paired double bond is formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains.
  • Sulfur bond connection a pair of intrachain disulfide bonds exist in the A chain, and the specific positions of the three pairs of disulfide bonds are: C ] and C [4] form a ⁇ bond, and C[ 2] and C [6] form two The sulfur bond, C [3] and C [5] form a disulfide bond.
  • the present invention provides a single-chain compound capable of binding to an insulin receptor and having hypoglycemic action, the amino acid sequence structure of which is:
  • Xioia is a glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine tetrapeptide or phenyldiacyl-proline-asparagine-glutamine-group a pentapeptide, or a glycine-valine-glutamic acid or phenylalanine-valine-asparagine-glutamine in the above tetrapeptide or pentapeptide by lysine or arginine Sequence after any one of the amino acid residues; Xioib is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 101c is phenylalanine or tryptophan; X 101d is phenylpropanoid leucine or tryptophan; X l () le is phenylalanine, tyrosine or histidine; X 102 is a
  • the present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further increase the solubility, stability, in vivo circulation time, and the like of the compound.
  • the modification is the attachment of the modified side chain to the ⁇ -amino group of the N-terminal amino acid residue of the B chain of the double-stranded compound of the present invention or the ⁇ -amino group of the ⁇ -terminal amino acid residue of the single-stranded compound, or The ⁇ -amino group of lysine present in the double-stranded or single-stranded compound of the invention.
  • the compound comprises an oxime chain and an oxime chain, wherein
  • amino acid sequence of the A chain is:
  • amino acid sequence of the B chain is:
  • C[,] It forms a disulfide bond with C [4] , C[ 2 ] and C [6] form a double bond, and C [3 ] and C [5] form a disulfide bond. among them,
  • X 3 99 is lysine, arginine or deletion;
  • X4CG is lysine, arginine or deletion;
  • 05 is glutamic acid, asparagine, glutamine or serine;
  • 0 8 is histidine, fine leucine, phenylalanine, threonine, or formula (I) structure;
  • 15 is arginine or formula (I) structure;
  • 17 Is glutamic acid or a structure of formula (I);
  • 18 is an asparagine or a structure of formula (I);
  • 21 is alanine, glycine or asparagine;
  • 22 is lysine, arginine-lysine
  • the compound is a single-stranded structure and the amino acid sequence structure is:
  • X201 a is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine, phenylalanine-valine
  • X 2Q ib is histidine, glutamic acid, glutamine, arginine or phenylalanine;
  • X 2 0k is phenylalanine Acid or tryptophan;
  • X 201 d is phenylalanine or tryptophan;
  • X 2 01e is phenylalanine, tyrosine or histidine;
  • X 2 o 2 is phenylalanine, tyrosine or Deletion;
  • ⁇ 3 is asparagine, threonine, aspartic acid, glutamic acid or deletion;
  • X 2Q4 is valine, lysine, arginine, aspartic acid, glutamic acid or deletion;
  • X 6 is threonine, lysine or deletion or a structure of formula (I);
  • X 2 Q 7 is serine, alanine, glycine, structure or deletion of formula (I);
  • X 207a is serine, alanine, Glycine, structure or deletion of formula (I);
  • X 2 .
  • a pharmaceutical composition which is obtained by mixing a hypoglycemic compound of the present invention and a pharmaceutically acceptable carrier, and the mixing ratio may be about 90/10%, about 80/20%. , about 70/30%, about 60/40%, about 50/50%, about 40/60%, about 30/70%, about 20/80%, or about 10/90%; preferably, the pharmaceutical combination Further comprising a fast-acting insulin analogue; the fast-acting insulin analog may be Asp B28 human insulin, Lys B28 P ro B29 human insulin or Lys B3 Glu B29 human insulin.
  • a fifth aspect of the invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of diabetes or hyperglycemia.
  • a sixth aspect of the invention provides a method of treating diabetes or hyperglycemia or the like comprising administering a compound or composition of the invention to a patient in need thereof.
  • the compound of the present invention Compared with the existing insulin and its analogs, the compound of the present invention has good water solubility, high activity in binding to the insulin receptor, low toxicity to an individual, and easy preparation. The cycle time of the modified compound in the body is significantly prolonged.
  • Figure 1 is a graph showing changes in blood glucose over time following subcutaneous injection of physiological saline, human insulin, and three different doses of a compound of the present invention
  • Figure 2 is a graph showing changes in blood glucose over time after subcutaneous injection of physiological saline, human insulin, and a compound of the present invention, III; 12;
  • Figure 3 is a hypoglycemia of a mouse subcutaneously injected with physiological saline, human insulin, and the ⁇ -7 compound of the present invention. Time change value.
  • Amino acid refers to any molecule that contains both amino and carboxyl functional groups, the amino and carboxyl groups of the ot-amino acid being attached to the same carbon atom (alpha carbon). The alpha carbon may have 1-2 organic substituents.
  • Amino acids contain L and D isomers and racemic mixtures. Unless otherwise specified, the amino acid residues in the polypeptide sequence of the present invention are L isomers, that is, L-amino acids, and D-amino acids are represented by a lowercase letter "d" before the amino acid name or abbreviation, such as dK.
  • encodeable amino acid or “encodeable amino acid residue” is used to mean an amino acid or amino acid residue which can be encoded by a nucleotide triplet.
  • hGlu is homoglutamic acid
  • ⁇ -hGlu is the L isomer of HNCH(CO-)CH 2 CH 2 CH 2 COOH;
  • ⁇ -hGlu is the L isomer of a HNCH(COOH)CH 2 CH 2 C3 ⁇ 4CO-;
  • a-Asp is the L isomer of HNCH(CO-)C3 ⁇ 4COOH
  • ⁇ -Asp is the L isomer of HNCH(COOH)CH 2 CO-;
  • a-Glu is the L isomer of a HNCH(CO-)CH 2 CH 2 COOH;
  • ⁇ -Glu is the L isomer of HNCH(COOH)CH 2 CH 2 CO-;
  • ⁇ -Ala is — HN-CH 2 —CH 2 —COOH
  • Sar is sarcosine.
  • Amino acid residues can be represented by three-letter amino acid codes or single-letter amino acid codes; the amino acid tables are as follows: Table 1: Amino acid names and shorthand
  • Natural insulin refers to mammalian insulin (such as human insulin, bovine insulin, porcine insulin, etc.) derived from natural, chemical synthesis, genetic engineering. Human insulin comprises an A chain consisting of 21 amino acids and a B chain consisting of 30 amino acids. The two chains are connected by three disulfide bonds: A7 and B7, A20 and B19, A6 and Al l. B7 and A7 refer to amino acid residues at position 7 (from the N-terminus) of the natural insulin B chain and amino acid residues at position 7 (from the N-terminus) of the insulin A chain.
  • Insulin analogs are generic terms for modified insulin polypeptides, including double-stranded molecules consisting of A and B chains with homologous sequences to native insulin, and single chain insulin analogs.
  • An "insulin analog” has a partial, total or potentiating activity of natural insulin, or can be converted in vivo or in vitro to a polypeptide having partial, total or enhanced activity of native insulin, for example increasing, decreasing or replacing one or more than native insulin A polypeptide of an amino acid residue.
  • Proinsulin, pro-proinsulin, insulin precursors, single-chain insulin precursors and the like of humans, animals and even non-mammals are referred to as "insulin analogs”. Many insulin analogs are found in the literature. "Insulin analogs" broadly include natural insulin and insulin analogs unless specifically stated otherwise.
  • IGF refers to insulin-like growth factor, including insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2).
  • IGF-1 The sequence of the A chain of human IGF-1 is the sequence shown in SEQ ID NO: 1, and the B chain sequence of human IGF-1 is the sequence shown in SEQ ID NO: 2.
  • IGF-1 analogs have one or more amino acid mutations, substitutions, deletions or additions relative to the native human IGF-1 molecule. IGF-1 analogs include double-stranded and single-chain IGF-1 analogs.
  • the IGF-2 analog has one or more amino acid residue mutations, substitutions, deletions or additions relative to the native human IGF-2 molecule.
  • IGF-2 analogs include both chain and single chain IGF-2 analogs.
  • the insulin referred to in the present application refers to human insulin
  • IGF-1 refers to human IGF-1.
  • the A chain of the compound starts from the number 1 (X! or) to the number 22 (X 22 or 2 2 ), except for the site changed in the present application, the first 21 amino acids of the compound A chain and the human IGF-1
  • the amino acid of the A chain corresponds, in particular, the 6th (X 6 or 06 ), 7 ( 7 or 07 ), 11 (X" or u ) and 20 ( ⁇ 2 or 20 ) positions of the A chain of the compound are cysteine. If an amino acid residue is added at the N-terminus of X, the number of the new amino acid residue is: 3 ⁇ 4 and; If an amino acid residue is added at the >1 end of 01 , the new amino acid residue is numbered X 399 and 00 .
  • the B chain of the compound begins with the number 23 (X 23 or 3 ⁇ 4 23 ), except for the site of variation in the present application, the amino acid number of the compound B chain from 28 (X 28 or 28 ) to 47 (X47 or 47) Corresponding to the amino acids 5 to 24 of the B chain of human IGF-1, in particular, the number 29 (X 29 or 29), 41 (X4i or X441) of the B chain of the compound is cysteine.
  • amino acid When referring to an amino acid alone, it can be represented by, for example, A1G, BIG or B9H, which means that the first amino acid of the A chain and the first and ninth amino acid residues of the B chain are 0, G, and H, respectively.
  • the numbering of the single-chain compound is based on the description of each compound.
  • a single-chain compound refers to a polypeptide sequence having a general structural B chain-C L -A chain or a modified polypeptide sequence, wherein the B chain is the B chain or analog of IGF-1, and the A chain is the A chain of IGF-1 or An analog, C L , is a peptide chain that links the C-terminal amino acid residue of the B chain to the N-terminus of the A chain.
  • amino acid indicated by the position of the A chain or the B chain in the present application such as A14, B28, etc., represents an amino acid corresponding to the position of the A chain or the B chain of IGF-1 or a change thereof, wherein A of IGF-1 The number of the chain or B chain starts at 1.
  • cysteines in each compound in the present invention are numbered, respectively, C n] ⁇ C [61 , which corresponds Is:
  • C n] ⁇ C [6] sequentially corresponds to the six cysteines of the single-stranded compound from the N-terminus to the C-terminus.
  • the compounds of the present invention are based on the structure of IGF-1, while IGF-1 and IGF-2 form disulfide bonds in the same manner as insulin.
  • the tertiary structure of any one of the compounds of the present invention contains a disulfide bond, and all single-chain IGF-1 analogs and double-stranded IGF-1 analogs have a three-pair disulfide bond in the same manner as insulin. Accordingly, those skilled in the art will fully understand and appreciate the disulfide bond positions of any of the compounds of the present invention based on the above explanations and common general knowledge.
  • the IGF-1 analog may comprise one or more modifying groups.
  • the modifying group is capable of providing the desired characteristics of the IGF-1 analog.
  • a modifying group can reduce the rate of degradation of an IGF-1 analog in various environments (e.g., digestive tract, blood).
  • Preferred modifying groups are those which allow the IGF-1 analog to retain comparable insulin receptor binding activity.
  • Preferred modifying groups include amphoteric groups, water soluble groups, or groups which render the IGF-1 analog less lipophilic, more water soluble than the unmodified analog.
  • the modifying group may comprise a degradable linking group, such as PAG; it may include a linking group that is susceptible to hydrolysis, such as lactide, glycolide, carbonic acid, esters, amino phthalates. This method can degrade the polymer into small molecular weight fragments.
  • the modifying group may include one or more hydrophilic groups, lipophilic groups, amphoteric groups, salt-forming groups, spacer groups, linking groups, capping groups, or a combination of these groups.
  • the various groups may be linked together by covalent bonds or by hydrolyzable or non-hydrolyzable bonds. Representative hydrophilic groups and lipophilic groups are described below.
  • hydrophilic group examples include a PAG group, a polysaccharide, a polysorbate, and a combination of these groups.
  • the polyalkylene Glycol (PAG) group consists of a plurality of alkylene glycol monomers. In one embodiment, all monomers are the same (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)). In another embodiment, the alkylene glycols are different.
  • the polymer may be a random copolymer such as a copolymer of ethylene oxide and propylene oxide, or a branched or graft copolymer.
  • PEG polyethylene glycol refers to any water soluble polyethylene glycol or polyethylene oxide.
  • the chemical formula of polyethylene glycol is -(CH 2 C3 ⁇ 40) n -, where n can be an integer from 2 to 2,000.
  • One end of the PEG is usually a relatively inactive functional group such as an alkyl group or an alkoxy group.
  • Alkyl groups include saturated straight or branched chain hydrocarbon groups. Representative examples of alkoxy are decyloxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy). And 2-mercapto-2-propoxy), pentyloxy, hexyloxy and the like.
  • PEG-terminated PEG is named mPEG, the structural formula CH 3 0(CH 2 CH 2 0) n -, but is still generally referred to as PEG.
  • PEG20K refers to a polyethylene glycol molecule having a molecular weight of 20,000.
  • the other end of the PEG is usually an activating functional group or a functional group which is liable to form a covalent bond, such as an amino group, a carboxyl group, a hydroxyl group, a thiol group, an aldehyde or the like.
  • PEG-maleimide, PEG-vinylsulfone and PEG-iodoacetyl can form a stable covalent bond with the thiol-SH of the cysteine side chain;
  • PEG -NHS succinimide
  • acylation the PEG-aldehyde and the amino group of the polypeptide are in a reducing agent (such as cyanoborohydride) Under sodium action, it can be joined by a reductive alkylation reaction.
  • the PEG molecule in the present invention may be a linear, branched, bifurcated or dumbbell-shaped PEG.
  • the branched PEG can be represented by the formula R(-PEG- n OH) m , wherein R (usually polyhydroxy) is a core group, such as Pentaerythritol, sugar, lysine or glycerol; m represents the number of branches, which may be the maximum number of attachment sites from 2 to the core group; n represents the number of PEG fragments, the number of PEG fragments per branch Can not wait. In general, n is an integer from 2 to 1800.
  • the branched PEG can be represented by the formula (CH 3 0-PEG- n ) p RZ, p is equal to 2 or 3, R is lysine or glycerol, and Z represents an activating functional group capable of undergoing a reaction.
  • the bifurcated PEG is represented by the general formula PEG(-LX) n , L is a linking group, and X is a terminal activating functional group.
  • PEG is generally polydisperse with a polydispersity index of less than 1.05.
  • the PEG group can also be a single ⁇ .
  • a single PEG has a single length (molecular weight) rather than a mixture of various lengths (molecular weight).
  • Representative sugar groups include, but are not limited to, glycerin, monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides such as starch, glycogen, cellulose, and/or polysaccharide gums.
  • Particular monosaccharides include C6 and above (especially C6 and C8) sugars such as glucose, fructose, mannose, galactose, nucleic acid sugar or sedose heptose; disaccharides and trisaccharides include two or three monosaccharide units ( In particular, groups of C5 to C8), such as sucrose, cellobiose, maltose, lactose and/or raffinose.
  • Biocompatible polycationic groups include polyamine groups having a plurality of amino groups on the backbone or side chain, such as polylysine and other natural or synthetic amino acids having a plurality of positively charged amino acid polymers, including poly birds.
  • Biocompatible polyanionic groups include groups having a plurality of carboxyl groups on the backbone or side chain, such as polyaspartic acid, polyglutamic acid, and the like.
  • Other hydrophilic groups include natural or synthetic polysaccharides such as chitosan, dextran, and the like.
  • Certain hydrophilic groups have potential bioadhesive properties. An example of this can be found in U.S. Patent 6,197,346. These polymers having a plurality of carboxyl groups exhibit bioadhesive properties. Rapid biodegradable polymers that exhibit multiple carboxyl groups upon degradation, such as poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, are also bioadhesives. These polymers can be used to administer IGF-1 analogs to the gastrointestinal tract. The carboxyl groups exposed during polymer degradation can be firmly attached to the gastrointestinal tract and assist in the administration of IGF-1 analogues.
  • the modifying group includes one or more lipophilic groups.
  • Lipophilic groups can be well known to those skilled in the art and include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, fatty acid, cholesterol, and lipophilic poly And oligomers.
  • the hydrocarbyl group can be a saturated, unsaturated, linear, branched or cyclic hydrocarbon having one or more carbon atoms.
  • the hydrocarbon group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms.
  • the hydrocarbyl group may be unsubstituted or have one or more substituents which preferably do not lose the biological activity of the conjugate.
  • the lipophilic group can also be a fatty acid such as a natural, synthetic, saturated, unsaturated, linear or branched fatty acid.
  • the fatty acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more carbon atoms.
  • an IGF-1 analog has one, two, three, four or more modifying groups after modification.
  • the binding site may include an amino acid residue, such as a lysine residue.
  • the IGF-1 conjugate is a single conjugate.
  • the IGF-1 conjugate is a multi-conjugate.
  • the IGF-1 conjugate is a mixture of monoconjugates, biconjugates, triple binders, tetraconjugates, and the like.
  • the modifying groups may be the same or different.
  • one or more modifying groups are preferably attached to IGF-1 via a hydrolyzable bond and the other one or more modifying groups are preferably passed through a non-hydrolyzable bond to the IGF- 1 combination is connected.
  • all of the modifying groups are linked to IGF-1 via a hydrolyzable bond, but the rate of hydrolysis of each modifying group in vivo is fast and slow.
  • binding sites include the B1-N terminus of the double-stranded polypeptide, the B chain lysine side chain amino group or the single-stranded N-terminus, the lysine side chain amino group, and the like.
  • the B1 single binder and B chain double binder are the most commonly used.
  • other binding sites can be created by introducing a natural or unnatural amino acid having an amino group or a thiol group in the ligated fragment of the single-stranded compound or the double-stranded polypeptide A chain or B chain.
  • the modifying group and the IGF-1 analog can be bound by a hydrolyzable bond (e.g., ester, carbonic acid, hydrolyzable amino phthalate).
  • the hydrolyzable bond gives the IGF-1 conjugate a prodrug effect.
  • a prodrug strategy is the preferred method if it is desired that the modifying group is inactive with the IGF-1 conjugate, such as where the binding site of the modifying group is in the IGF-1 analog to insulin receptor binding region.
  • the IGF-1 analog is linked to a modifying group by a non-hydrolyzable bond (eg, an amide bond, a hydrazone bond).
  • a non-hydrolyzable bond eg, an amide bond, a hydrazone bond.
  • non-hydrolyzed bonds help to prolong the circulation time of the IGF-1 conjugate in plasma.
  • IGF-1 homologs can be attached to the modifying group by various nucleophilic functional groups including, but not limited to, nucleophilic hydroxyl or amino groups.
  • nucleophilic hydroxyl or amino groups For example, serine, threonine, tyrosine have a nucleophilic hydroxyl group, a histidine, lysine or IGF-1 analog A chain, and a N-terminus of a B chain have a nucleophilic amino group.
  • the IGF-1 homologue can also be attached to the modifying group via a free thiol-SH, for example to form a sulphur ester, a thioether or a sulfonamide bond.
  • a common method is to form a hydrolyzable or non-hydrolyzable bond with a natural or synthetic macromolecule.
  • Biomacromolecules include albumin, polysaccharides, antibodies (such as IgG), and the like. 70% of the albumin in the blood vessels is mercaptalbumin, and the side chain thiol of cysteine-34 is the most active sulfhydryl group in plasma.
  • the IGF-1 analog can be reacted with a linker having an activating functional group such as maleimide at one end to form an IGF-1 - albumin conjugate.
  • the linker can be a long chain fatty acid or a PEG molecule. Specific examples can be found in Bioconjugate Chem. 2005, 16, 1000-1008. Synthetic macromolecules include polyethylene glycol and dextran. Another way is fatty acid acylation, which will be discussed in the acylation of the IGF-1 analog moiety.
  • Sortase is a transpeptidase that mediates the covalent binding of Gram-positive bacterial cell walls to surface proteins, mainly in Gram-positive bacteria.
  • SrtA or SrtA St aph Staphylococcus aureus sortase A
  • isoform isoform
  • SrtA recognizes a substrate comprising the LPXTG (Leu-Pro-X-Thr-Gly) motif, and the cysteine at position 184 acts as a nucleophilic group to attack the peptide bond Thr-Gly in the LPXTG motif, thereby producing a Acyl-enzyme intermediate.
  • the thioester intermediate of the threonine carboxyl group undergoes a nucleophilic reaction with the amino group of the oligoglycine of the substrate (penta-glycine (Gly 5 ) which is a bridge of the precursor of the branched lipid II in S. aureus) , creating new connection products.
  • sortase can accept a nucleophilic group consisting of two alanines, but the & aureus enzyme cannot.
  • This sortase (SrtA strep ) cleaves the LPXTA motif The peptide bond Thr-Ala, which allows an alanine-based nucleophilic group. SrtAstep also recognizes the LPXTG motif but has lower activity. The LPXTA motif is not cleaved by SrtA Staph .
  • the following method discusses Take SrtA Staph as an example, but the SrtA strep equivalent can also use the same or A similar approach.
  • SrtA is highly specific for the LPXTG motif and the glycine repeat (a-Gly n ) with a free amino group at the N-terminus.
  • the X position can be all natural amino acids other than cysteine and tryptophan (not yet tested).
  • a real face indicates that a polypeptide with a glycine at the N-terminus can participate in a sortase-catalyzed transpeptidation reaction, a substrate with two or more glycines at the N-terminus can achieve maximum reaction efficiency.
  • Sortase mediated ligation is the introduction of non-native functional groups into proteins or peptides.
  • the non-natural functional group can be a small molecule, a synthetic polypeptide or protein, a polymer, and the like. These functional groups can be combined with LPXTG or a-Gly n to form a substrate for SrtA.
  • Specific methods and reaction kits can be referred to related literature (eg, Tsukiji et al, "Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering", ChemBioChem, 2009, 10, 787-798; Popp et al, "Sortase-catalyzed transformations that Improve the properties of cytokines", PNAS, 2011, 108, 3169-3174).
  • SrtA is capable of introducing a non-native functional group on the IGF-1 analog, depending on the structure of the IGF-1 analog.
  • the binding site that generally less affects biological activity is the N-terminus of the B-chain or the C-terminus of the B-chain.
  • the N-terminus of the IGF-1 B chain preferably introduces multiple glycines, such as the GGGGG-IGF-1 B chain; and the modifying group to be introduced, such as PEG, long chain fatty acids or
  • the C-terminus of albumin or the like has an LPXTG motif such as PEG-LPATGGGG, albumin-LPETGGG or fatty acid LPGTGGGGG.
  • the C-terminal amino acid sequence of the A or B chain comprises an LPXTG motif, such as IGF-1 A-LPATGGGGG or IGF-1 B-LPGTGGGG, etc.
  • the introduced modified group such as PEG, long-chain fatty acid or albumin has one or more glycines at the N-terminus, such as GGG-PEG, GGGG-long-chain fatty acid, GGGGG-albumin and the like.
  • the easiest binding site is the N-terminus of the B-chain or the C-terminus of the A-chain, and the method is essentially identical to the double-stranded analog.
  • IGF-1 Insulin-like growth factor-1
  • IGF-1 Insulin-like growth factor-1
  • the A and B chains are homologous; the C domain is linked to the A and B domains, corresponding to the C-peptide of proinsulin; the D domain is the C-terminal extension of the A domain.
  • the three-dimensional structure of IGF-1 already has NMR and X A report of the radiant crystallization method.
  • the structure of the B domain and the A domain is very similar to the crystal structure of insulin and the NMR structure of proinsulin.
  • the insulin receptor family includes the insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF-1R), and insulin receptor-related receptors, all of which are receptor tyrosine kinases that act on specific tyrosines. Increase the phosphate molecule to deliver the signal.
  • Members of the insulin receptor family are composed of two receptor moieties, each comprising an extracellular alpha subunit and a transmembrane beta subunit, joined by a disulfide bond. When the ligand is not bound, the receptor exists as a dimer, forming an ⁇ 2 ⁇ 2 receptor that is held together by a disulfide bond between the two ⁇ subunits.
  • the sequence similarity between the insulin receptor and the IGF-1 receptor is 41-84% depending on the region to be compared.
  • IGF-1 insulin growth factor-1
  • the ability of IGF-1 to bind to the insulin receptor is 100-fold lower than that of insulin, which is inconsistent with the actual ability of IGF-1 to induce insulin receptor phosphorylation and hypoglycemia in vivo (IGF-1's ability to activate the insulin receptor is approximately 10 for insulin). %). Therefore, part of the signaling may be through the IGF-1 receptor/insulin receptor heterodimer.
  • IGF-1 Almost every cell in the human body is affected by IGF-1, especially in the muscles, cartilage, bones, liver, kidneys, nerves, skin and lungs. In addition to insulin-like effects, IGF-1 also regulates cell growth and development, particularly in neural cells, as well as in cellular DNA synthesis.
  • IGF-1-BP IGF-1 binding proteins
  • IGF-1 and insulin are similar: Both hormones increase glucose uptake and oxidation in a very similar manner, inhibiting glucose production, free fatty acid levels, and fat oxidation rates.
  • Insulin antagonism is composed of many common and several uncommon clinical symptoms. Patients with mutations in the insulin receptor gene or genes involved in the signaling pathway have different phenotypes, including lipodystrophy, partial lipodystrophy, insulin-resistant type A syndrome with insulin receptor gene mutation, and false sexual acromegaly, dwarfism syndrome and Rabson-Mendenhall syndrome. In many of these patients, hyperglycemia is quite difficult to treat because insulin is ineffective.
  • Insulin antagonism is also common in patients with non-insulin dependent diabetes.
  • the human body overcomes this problem by increasing insulin secretion, but it leads to a decrease in insulin receptor expression on target tissues, which worsens insulin antagonism.
  • Hypertriglyceridemia is a secondary metabolic abnormality in these patients. Therefore, the specific treatment for insulin antagonistic treatment can significantly improve the therapeutic effect of diabetes.
  • IGF-1 has been proposed for the treatment of severe insulin antagonism. Because its biological effects are similar to those of insulin, it is possible to avoid bodily defects that prevent insulin from functioning. Intravenous injection of recombinant IGF-1 has been found to reduce blood glucose and serum insulin concentrations in two patients with insulin-resistant type A syndrome and one child with Rabson-Mendenhall syndrome. In several studies of patients with different phenotypes of severe insulin antagonism, the use of IGF-1 reduced fasting and 24-hour mean serum insulin concentrations, increased glucose tolerance, increased insulin sensitivity and reduced fasting Serum triglyceride concentration.
  • IGF-1 may cause a cardiovascular response, including arrhythmia and hypotension. Some reactions may be due to the fact that IGF-1 causes a sharp decrease in blood phosphate. Long-term high-dose subcutaneous injection of IGF-1 can cause temporomandibular discomfort, facial and hand edema, weight gain, difficulty breathing, and sinus tachycardia. However, recent studies have shown that well tolerated and effective doses can be achieved. However, the best approach is to modify the molecular structure to increase the binding of the IGF-1 analogue to the insulin receptor while reducing its activity at the IGF-1 receptor.
  • IGF-1 analogs include, but are not limited to, better water solubility than human insulin, higher yields, the potential to develop dual receptors for insulin receptors and IGF-1 receptors, and the like.
  • the inventors have found through long-term studies that the A chain and the B chain of IGF-1 are linked by insulin, and the amino acid residue of B15 of the B chain is replaced by Q or W or F, such an IGF-1 analogue.
  • the ability to bind to the insulin receptor is comparable to that of human insulin.
  • the IGF-1 analog of the present invention which is highly bound to the insulin receptor is such that the B chain of IGF-1 is substituted at the two positions of B15 and B16.
  • the newly obtained IGF-1 analogue also exhibits an insulin receptor binding performance comparable to that of natural insulin.
  • IGF-1 analogs substituted with FF, WF or WW also retain some of their ability to bind to IGF-1 binding proteins. This ability of IGF-1 analogs to bind to IGF-1 binding proteins is believed to help prolong the time of circulation and action of IGF-1 analogs in serum.
  • Double-stranded analogs including the human IGF-1 A chain and the human insulin B chain show approximately 40% of insulin capacity in insulin-like activity assays (eg, lipogenesis), but in growth factor assays (eg, thymidine incorporation) Among them, the activity is significantly higher than insulin, which is equivalent to about 730%. However, this compound is an inefficient growth factor than IGF-1 itself, equivalent to IGF-1. Approximately 26.5% of the time.
  • IGF-1 IGF-1 receptor binding
  • double-stranded analogs including the human IGF-1 A chain and the human insulin B chain show attenuated but still Significant IGF-1 receptor binding indicates that the structural properties contained in the A domain of IGF-1 result in increased growth-promoting ability.
  • overactive IGF-1 receptors may cause cancer.
  • IR-A has a growth-promoting effect similar to that of the IGF-1 receptor, and the main function of IR-B is metabolic regulation (such as glucose metabolism).
  • Natural human insulin has essentially the same binding capacity as the two receptor subtypes. The binding ability of IGF-1 and IGF-2 to IR-A was significantly higher than that of IR-B. The IGF-1 analog still partially retains this selectivity. Ideal IGF-1 analogues should be able to bind highly to the insulin receptor but have a lower ability to bind to the IGF-1 receptor. In addition, IGF-1 analogues should have a balanced biological activity on the two insulin subtypes IR-A and IR-B.
  • the IGF-1 A chain and the insulin A chain have a high degree of sequence homology.
  • Two distinct differences in the sequences of the insulin A chain and the IGF-1 A chain are A5Gln-A5Glu and A12Ser-A12Asp. These residues are conserved in IGF-1 and IGF-2. These changes in the sequence involve changes between neutral residues in insulin and acidic residues in IGF-1.
  • the inventors have found that the A chain glutamic acid at the A chain and the aspartic acid side chain carboxyl group at the A12 position are critical for determining receptor selectivity.
  • the present invention provides a compound having a hypoglycemic action, the compound comprising an A chain and a B chain, wherein
  • the amino group of the A chain is: X ⁇ XoGrVDXsCCTCwXsXsXioCwXnLRRLEXwYCwX Xss ,
  • the B chain amino acid sequence is:
  • X 2 3- 2 6X27LC [1] GAX 32 LVDALX 38 X 39 VC [2] GDX 4 4GFX 47 X4 8 X49 X 50 X51 X52 X 53 , where, ! Is lysine, arginine or deletion; is lysine, arginine or deletion; X 5 is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine, Phenylalanine or threonine; X 9 is arginine or serine; X 10 is serine or isoleucine; X 12 is aspartic acid, serine, glutamine or asparagine; X 18 is day Asparagine, methionine or threonine; X 21 is asparagine, alanine or glycine; X 22 is lysine, arginine-lysine dipeptide or deletion; X23-26 is phenylprop
  • [1]-[6] represents the number of a cysteine; in the compound, three pairs of disulfide bonds are formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains. Sulfur-bonded, there is a pair of intrachain disulfide bonds in the A chain.
  • the specific positions of the three pairs of disulfide bonds are: C fl] and C [4] form disulfide bonds, C [2] and C [61 form disulfide The bonds, C [3 ] and C [5] form a disulfide bond.
  • amino acid residues of X 32 , 4 , 9 , X 50 , X 51 are related to whether the compound resembles human islets The same as self-association.
  • Human insulin is typically stored in islet beta cells by self-association to form hexamers. After subcutaneous injection of recombinant human insulin molecules, the hexamers are gradually depolymerized into dimers, and further dissociated into monomers to enter the circulation through the capillaries, and play a hypoglycemic effect.
  • Recombinant human insulin has a long-lasting effect after subcutaneous injection due to the presence of depolymerization and absorption processes (see Brange et al. "Monomeric insulins and their experimental and clinical implications" Diabetes Care, Vol 13 No.
  • X 32 is histidine, it is advantageous for the compound to form a hexamer structure with the aid of zinc ions. If X 32 is an amino acid residue such as aspartic acid, glutamic acid, phenylalanine, glutamine or arginine, a stable hexamer structure cannot be formed. If the amino acid residues of the 4 , X 5 o, and 51 sites are aspartic acid or glutamic acid, stable self-association is not easily formed.
  • X 32 is an unhistidine amino acid residue, or one or more of the amino acid residues of X44, X 5 0, X 51 and the like are aspartic acid or glutamic acid, the corresponding compound is more It is easy to exist in the form of dimer or monomer, and it can quickly enter the blood after subcutaneous injection, which can achieve the effect of lowering blood sugar in a short time.
  • the hypoglycemic compound comprises an A chain and a B chain, wherein the amino group of the A chain is: GIVDX 5 C[ 3] C [4] X 8 RSC [ 51 X 12 L RLEX 18 YC [6] X 21 X 22 ,
  • the B chain amino acid sequence is:
  • X 5 is glutamic acid, asparagine, glutamine or serine;
  • X 8 is histidine, arginine or phenylalanine;
  • 12 is aspartic acid, serine, glutamine or asparagine;
  • x 18 is asparagine, methionine or threonine;
  • x 21 is asparagine, alanine or glycine;
  • x 22 is lysine, arginine-lysine dipeptide or deletion;
  • X 2 6 is a glycine - proline - tripeptide glutamic acid or phenylalanine - valine - asparagine - glutamine tetrapeptide;
  • x 27 is histidine or threonine;
  • x 32 histidine , glutamic acid, glutamine, arginine or phenylalanine;
  • x 38 is phenylalanine or tryptophan;
  • x 39 is
  • [1]-[6] represents the number of a cysteine; in the compound, three pairs of disulfide bonds are formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains. The ⁇ bond is connected, there is a pair of intrachain disulfide bonds in the A chain, and the specific positions of the three pairs of disulfide bonds are: forming a disulfide bond with C [4 ], and forming a disulfide bond by C [2 ] and C [6] , C [3] and C [5] form a disulfide bond.
  • sequence of the B chain is:
  • GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFY-NH 2 ;
  • sequence of the B chain is:
  • GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFYFNKPT;
  • sequence of the B chain is:
  • GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFYdA-NH 2 ;
  • sequence of the B chain is:
  • sequence of the A chain is:
  • sequence of the A chain is:
  • X 5 in the A chain is glutamic acid, asparagine, glutamine or serine;
  • X 8 is histidine, arginine or phenylalanine;
  • X 12 is aspartame Acid, serine, glutamine or asparagine;
  • 18 is asparagine, methionine or threonine;
  • X 27 in the B chain is histidine or threonine;
  • X 32 is histidine, valley Amino acid, glutamine, Arginine or phenylalanine;
  • X 38 is phenylalanine or tryptophan;
  • X 39 is phenylalanine or tryptophan;
  • 4 is arginine, glutamic acid, aspartic acid or alanine acid.
  • a compound having high binding ability to the insulin receptor includes an A chain and a B chain, and the A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain.
  • C w and C [4] form a double bond
  • [ 6] forming a disulfide bond
  • C [3] and C [5] forming a disulfide bond
  • the compound is selected from the following double-stranded polypeptides:
  • a chain sequence is GIVDECCFRSCDLRRLEMYCA (SEQ ID NO: 1); the B chain sequence is GPETLCGAELVDALFFVCGDRGFY-NHz (SEQ ID NO: 4);
  • a chain sequence is the sequence of SEQ ID NO: 1; the B chain has the sequence GPETLCGAELVDALWFVCGDRGFY-NH 2 (SEQ ID NO: 5);
  • a chain sequence is the sequence shown in SEQ ID NO: 1; the sequence of the B chain is
  • a chain sequence is GIVDECCFRSCDLRRLEMYCN (SEQ ID NO: 7); the B chain has the sequence GPETLCGAELVDALFFVCGDRGFYFNKPT (SEQ ID NO: 3);
  • a chain sequence is GIVDECCFRSCDLR LENYCA (SEQ ID NO: 8); the sequence of the B chain is the sequence shown in SEQ ID NO:
  • a chain sequence is GIVDECCFRSCDLRRLETYCA (SEQ ID NO: 9); the sequence of the B chain is the sequence shown in SEQ ID NO:
  • a chain sequence is GIVDECCRRSCDLRRLENYCN (SEQ ID NO: 10); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDECCHRSCDLRRLENYCN (SEQ ID NO: 11); the sequence of the B chain is
  • a chain sequence is GIVDQCCFRSCDLRRLENYCA (SEQ ID NO: 12); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDNCCFRSCDLRRLENYCA (SEQ ID NO: 13); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDECCFRSCSLRRLENYCA (SEQ ID NO: 14); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDECCFRSCNLRRLENYCA (SEQ ID NO: 15); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDECCFRSCQLRRLENYCA (SEQ ID NO: 16); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is GIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 17); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
  • a chain sequence is the sequence of SEQ ID NO: 17; the B chain has the sequence GPETLCGAHLVDALFFVCGDRGFYF KPT (SEQ ID NO: 18);
  • a chain sequence is GIVDQCCFRSCSLRRLENYCAK (SEQ ID NO: 19); the sequence of the B chain is the sequence shown in SEQ ID NO: 18.
  • a chain sequence is GIVDQCCFRSCSLRRLENYCARK (SEQ ID NO: 20); the sequence of the B chain is the sequence shown as SEQ ID NO: 18.
  • a chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPETLCGAHLVDALFFVCGDRGFYdA-NH 2 (SEQ ID NO: 21);
  • a chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is GPEHLCGARLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 22);
  • a chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAFLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 23);
  • a chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAHLVDALFFVCGDEGFYFNKPT (SEQ ID NO: 24);
  • a chain sequence is the sequence shown in SEQ ID NO: 8; the sequence of the B chain is FVNQHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 25);
  • a chain sequence is GIVDQCCHRSCSLRRLENYCA (SEQ ID NO: 26); the sequence of the B chain is the sequence shown in SEQ ID NO: 25.
  • a chain sequence is the sequence of SEQ ID NO: 17; the B chain has the sequence of SEQ ID NO: 25;
  • a chain sequence is the sequence shown by SEQ ID NO: 17; the B chain sequence is GPEHLCGAHLVDALFFVCGDAGFYFNKPT (SEQ ID NO: 27);
  • a chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAQLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 28);
  • a chain sequence is KGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 151); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 152);
  • a chain sequence is RGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 153); the sequence of the B chain is the sequence shown as SEQ ID NO: 152;
  • a chain sequence is KGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 154); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNPKT (SEQ ID NO: 155);
  • sequence of the A chain is RGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 156); the sequence of the B chain is the sequence of SEQ ID NO: 155; the sequence of SEQ ID NO: 155;
  • a chain sequence is KKGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 158); the sequence of the B chain is the sequence shown in SEQ ID NO: 155;
  • a chain sequence is GIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 159); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNPKTE (SEQ ID NO: 160);
  • a chain sequence is the sequence shown in SEQ ID NO: 159; and the B chain sequence is the sequence shown in SEQ ID NO: 155.
  • Proinsulin is a single-stranded precursor of 86 amino acids constructed as: ⁇ chain -ArgArg-C peptide -LysArg-A chain.
  • the C peptide is a "linker peptide" composed of 31 amino acids.
  • Arg-Arg and Lys-Arg are the cleavage sites for proteolytic enzymes to cleave C-peptide from the A and B chains.
  • Proteolytic enzymes are known to be prohormone convertases (PC1 and PC2), and exoproteinase carboxypeptidases are £ . These changes in proinsulin removed the C peptide. The remaining B chain and A chain are bonded together by a disulfide bond.
  • the double-stranded structure of insulin allows insulin to have multiple conformations.
  • Insulin has the potential for considerable conformational changes, Limitations of these changes significantly reduce the affinity of the insulin receptor for the ligand. Blocking the amino terminus of GlyAl also impairs receptor binding ability. Proinsulin and insulin receptor affinity is only 1-2% of insulin.
  • C-peptide in proinsulin folding is still unclear.
  • the length of the C peptide varies between 26 and 38 amino acids in different animal species.
  • the dibasic amino acid residues at the junction of the B-chain-C peptide (B-C) and the C-peptide-A chain (C-A) are conserved and the need for insulin conservation is considered to be minimal.
  • the three-dimensional structure of insulin shows that the A chain and the B chain can be bound by a linker peptide which is much smaller than the 31 amino acid C peptide.
  • the inherent physical and chemical stability of the insulin molecule is a prerequisite for insulin therapy in diabetes, and is also the basis for insulin conformation, applicable insulin delivery methods, and shelf life and storage conditions for pharmaceutical formulations.
  • the use of solutions during insulin administration exposes insulin molecules to a variety of factors, such as elevated temperatures, gas-liquid-solid phase changes, and shear forces, which may result in irreversible conformational changes in insulin molecules, such as fibrillation. .
  • This is closely related to the insulin solution in the syringe pump, because insulin molecules are exposed to these factors, both externally and implanted, and from the shear forces generated during long-term pump movement. Therefore, fibrillation is a big problem when using a syringe pump as an insulin delivery system.
  • the solubility of insulin is affected by many factors and is significantly reduced in the range of pH 4.2-6.6.
  • the pH settling zone typically imposes limitations on the formulation.
  • the present invention addresses these problems by providing a stable single-stranded compound by introducing a C-peptide between the B and A chains to reduce molecular flexibility while reducing the fibrillation tendency, limiting or modifying the pH settling zone.
  • the main method of genetic engineering for the production of insulin is to first produce a single-chain insulin precursor which is linked end-to-end by a short peptide of the insulin B chain and the A chain, and then the insulin precursor is digested to form a double-stranded insulin. If the single-chain insulin analog is directly produced, the production process is greatly simplified and the cost is reduced.
  • insulin-like growth factor-1 is a single-chain peptide having 70 amino acid residues, including the A, B, C, and D domains.
  • the basic structure of the A and B domains of IGF-1 is highly similar to the A and B chains of insulin, with 52% and 45% homology, respectively. Their three-dimensional structure is also very similar.
  • the C domain of IGF-1 plays a minor role in insulin receptor binding. Removal of all IGF-1 C domains, replaced by a bridge of four glycines, resulted in a twofold increase in insulin receptor binding compared to wild type, while addition of the IGF-1 C domain to the C-terminus of the insulin B chain causes insulin Receptor affinity was reduced by a factor of 3.5 compared to wild type. The insulin binding capacity of the single-chain insulin/IGF-1 mixture consisting of the pancreatic and IGF-1 C domains was not significantly different from native human insulin. Interestingly, the IGF-1 CII hybrid has increased affinity for both IR-A and IR-B, while IGF-2 CI has a weaker affinity, indicating that the C domain determines IR binding specificity.
  • Tyr31 in IGF-1 is essential for maintaining high affinity for IGF-1 receptors, but it appears to block binding to the insulin receptor, as tyrosine is replaced by alanine, resulting in a small but distinct human placenta A double increase in insulin receptor binding.
  • the inventors further designed, synthesized and characterized single-chain IGF-1 analogs capable of binding to the insulin receptor and having hypoglycemic action.
  • a ligation fragment (C chain) of 6-12 or 8-12 amino acids is used in the IGF-1 analog to link the C-terminus of the B chain analog of IGF-1 to the A1 position of the IGF-1 A chain analog.
  • the resulting single-chain IGF-1 analog is represented as a B chain-C chain-A chain.
  • the ideal single-chain IGF-1 analogue should have a high binding capacity to the insulin receptor for electrostatic equilibrium; excellent thermodynamic stability and no self-assembly.
  • the A chain and the B chain are the A chain and B chain of IGF-1, or an analog thereof.
  • the B chain is a B chain remodeling of IGF-1 in which the amino acid Q (glutamine) at position B15 is replaced by F (phenylalanine) or W (tryptophan).
  • Double-chain IGF-1 analogues can enhance insulin receptor binding, improve receptor selectivity, and increase various amino acid changes in water solubility and stability of peptides, such as changes in A5, A12, A18, A22, B9, etc.
  • Single-chain IGF-1 analogues Used in double-stranded IGF-1 analogues
  • the selection and variation of amino acids that alter the self-association ability of a compound, such as a dimer or a monomer, are also applicable to single-chain IGF-1 analogs.
  • the ligation fragment C L is a 6-60 amino acid peptide sequence in which each amino acid residue is independently selected from the group consisting of glycine, alanine, serine, threonine, and valine.
  • a suitable connecting segment C L has a three-point feature: First, the connecting segment requires an appropriate length. When the B chain is 30 amino acids in length, the length of the ligated fragment is preferably not less than 6 amino acids; when the B chain is 25 amino acids, the length of the ligated fragment is preferably not less than 10 amino acids. When the length of the ligated fragment is shorter than the number of amino acids or longer than 60 amino acids, the insulin receptor binding ability of the single-chain analog has a decreasing tendency. Second, the ligated fragment preferably has no secondary structure, and the spatial conformation can be flexibly changed; The ligation fragment itself is not biologically active, but can provide polypeptide modification sites such as acylation, glycosylation and the like.
  • C L may be substituted by amino acid residues and comprising at least one insert or an aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine.
  • C L may include 1, 2, 3, 4 aspartic acid, glutamic acid, arginine or lysine to regulate the charge balance of the polypeptide sequence and improve solubility.
  • the sequence may comprise 1, 2, 3, 4, 5 asparagine and the same amount of serine or threonine to form the NXS/T consensus sequence required for N-glycosylation (X is a codeable natural amino acid) ).
  • the peptide may further comprise 1, 2, 3 or 4 lysine or cysteine, and the side chain amino group or sulfhydryl group may be a natural or synthetic modifying group such as a fatty acid, polyethylene glycol or albumin.
  • the modified IGF-1 molecules have different physical, chemical and biological properties by being linked by a hydrolysis bond or a non-hydrolysis bond.
  • the C-terminal amino acid of C L may be selected from the group consisting of glycine-lysine, glycine-arginine, arginine-arginine, lysine-lysine, arginine-lysine a group consisting of lysine-arginine, valine-glutamine-threonine, valine-glutamine-lysine, or valine-glutamine-arginine.
  • the C-terminal amino acid of C L is selected from the group consisting of lysine or arginine.
  • c L may be all or part of a sequence of a polypeptide fragment, or a difference of 1, 2 or 3 amino acid residues from the following polypeptide fragments, or 70%, 80% of the following polypeptide fragments, 90% similar, or 1, 2, 3, 4 or 5 repeats of all or part of the sequence of the following polypeptide fragments:
  • GASPGGSSGS (GASPGGSSGS)FortunatelyGR, where n is 1, 2, 3, 4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT;
  • GSGGAPSRSGSSR GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR;
  • GSGSSSRAPPSAPSPQR GSGSSSRAPPSAPSPQR; GSSSESPSGAPQT; GAGTPASGSAPGR; GSSPSGGSSAPQT; GSTSSTARSPGR; GAGPSGTASPSR; GSSTPSGAPQT; SSSSAPPPSAPSPSRAPQR;
  • GASPGTSSTSGR GASPGTSSTSGR ; GSGSSSAAAPQT; GSGSSSAAPQT; GSGSSSAPQT; GSGSSSRRA;
  • GSPAGSPTSTSR GSGPSSATPASR
  • GSGSSSRGR GSGGPSTRSAPQR
  • GPETPSGPSSAPQT GSPAGSPTSTSR
  • GSGPSSATPASR GSGSSSRGR
  • GGPSTRSAPQR GSGSSSRGR
  • GSGPSTRSAPQR GSGSSSRGR
  • GPETPSGPSSAPQT GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
  • GAGSSSRAPPPSAPSPSRAPGPSAPQR GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR;
  • GSSPSASTGTGR GAGSSSAPSAPSPSRAPGPSAPQR
  • GSGSGSGR GSPSSPTRGSAPQT
  • GASTSSRGAPSR GPSGTSTSAPGR
  • GAGSSSAPQT GAGSSSAPQT
  • SSSSAPSAPSPSRPQR SSSSAPSAPSPSRPQR
  • GSGASSPTSPQR GSSPATSATPQT ; GAGSSSAPPPSAPSPSRAPGPSAPQR ;
  • GASTGSSRPSGR GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;
  • GAGSATRGSASR GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQ; GSSPSGRSSSPGR;
  • GSPAGSPSSSAGSSASASPASPGR GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR; RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK; SSSAPPPSAPSPSRAPGPSPQR; SAASSSASSSSASSASAGR; GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR; GSGSSSRRAPQT; SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT; GAGPSSGAPPPSPQT; GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPS
  • the present invention further provides a single-chain compound having a hypoglycemic effect, the compound being modified based on the structure of human IGF-1, the structure of which is:
  • X 1()la is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine tetrapeptide or tyrosine
  • X101b is histidine, glutamic acid, glutamine, arginine or phenylalanine;
  • X 101c Is phenylalanine or tryptophan;
  • X 101d is phenylalanine or tryptophan;
  • Xnne is phenylalanine, tyrosine or histidine;
  • X 1() 2 is phenylalanine or deletion; 1()3 is asparagine or deletion;
  • X 104 is lysine, valine or deletion;
  • X 1Q5 is valine, lysine or deletion;
  • X 106 is threonine or deletion;
  • [1] - [6] represent the number of cysteine.
  • a disulfide bond in the chain is formed by the structure of insulin, specifically: C w and C [4] form a disulfide bond, and C [2] and C [ 6 ] are formed.
  • the disulfide bond, C [3 ] and C[ 5 ] form a two-stone charge.
  • the structure of the single chain compound is:
  • X 10 la is a glycine-valine-glutamate-threonine tetrapeptide or a strepyl-alanine-valine-asparagine-glutamine-histidine pentapeptide;
  • X 1 ( ) 2 is benzene Alanine or deletion;
  • X 1Q3 is asparagine or deletion;
  • X 1Q4 is lysine, valine or deletion;
  • ⁇ 05 is valine, lysine or deletion;
  • X 1Q6 is threonine or deletion;
  • X 1G9 is a lysine, arginine-lysine dipeptide or a deletion; it is a linking fragment selected from the above.
  • the structure of the single chain compound is:
  • the structure of the single chain compound is: : ⁇ - ⁇
  • V AN3TOnS0S3 ⁇ 4iICO0aAK 0 ⁇ ISS£)VOdO£)O ⁇ ⁇ ⁇ 3d0
  • GPETLCGAHLVDALFFVCGDRGFYSSSSPSAPSPSSPASPSPSSAPQRGIVDQCCFRSCSL RRLENYCA SEQ ID NO: 146
  • Polypeptide chemists have used several methods to solve the problem of rapid clearance of drug molecules with a molecular weight of less than 67 kDa in the kidney by plasma.
  • the injection site is built into a "depot"; 2. It is combined with non-covalent bonds in the plasma to prevent glomerular filtration; 3. Covalently linked to the carrier protein; 4. Large molecular weight Modification group binding, such as large molecular weight PEG, polysaccharides, etc. (as disclosed in the previous section of this application).
  • Hydrophobic depoting greatly increases the hydrophobicity of the peptide to reduce solubility and cause it to form a "storage" in the injection portion.
  • the polypeptide After the peptide compartment is slowly dissociated, the polypeptide binds to the cell membrane and/or systemic carrier proteins (such as albumin, etc.).
  • the molecular weight of the carrier protein is greater than the maximum molecular weight of the glomerular filtration, so it is not easily cleared by the kidneys and can be circulated in plasma for many days. Therefore, the polypeptide bound to the carrier protein is not easily filtered by the glomerulus or degraded by the protease on the inner membrane.
  • Fatty acids generally extend the time of action of the polypeptide in vivo in three ways.
  • First, fatty acids can bind non-covalently to albumin at the site of drug injection, resulting in slow release of the polypeptide-fatty acid-albumin macromolecule conjugate;
  • fatty acids reduce the immunogenicity of the polypeptide. The latter three features are similar to the long-chain PEG modification. Further mechanisms and experimental support can be found in Biochem. J.
  • modification of insulin with macromolecules such as polyethylene glycol (PEG, molecular weight not less than 20K) and human albumin can also achieve an effect of prolonging the in vivo action time similar to the above fatty acid modification. Therefore, all of the sites which can be acylated with fatty acids can be modified with macromolecules such as polyethylene glycol or human albumin.
  • the present invention is based on the recognition that the overall hydrophobicity of the hypoglycemic compound of the present invention plays an important role in the in vivo efficacy of the compound.
  • the present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further increase the in vivo circulation time of the compound.
  • the modification is an ⁇ -amino group which links the modified side chain to the N-terminal amino acid residue of the B chain of the silent chain compound of the present invention, or ⁇ -linked to the ⁇ -terminal amino acid residue of the single-chain compound of the present invention.
  • the compound is engineered based on an IGF-1 analog, the compound comprising an oxime chain and an oxime chain, wherein
  • [1]-[6] represents the number of cysteine; the compound passes 6 caspase
  • the amino acid forms a 3-pair disulfide bond, wherein the A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain.
  • the specific positions of the three pairs of disulfide bonds are: C tl] And C [4] form a disulfide bond.
  • [ 21 and. [6] Form a disulfide bond
  • [ 3] and. [ 51] forms a disulfide bond, wherein
  • X 3 99 is arginine, lysine or deletion;
  • X4 is arginine, lysine or deletion;
  • 0 5 is glutamic acid, asparagine, glutamine or serine;
  • 0 8 is histidine, Arginine, harmless alanine, threonine or a structure of the formula (I), the structure of the formula (I) is:
  • 09 is arginine, serine or the structure of formula (I); 10 is histidine, arginine, really alanine or the structure of formula (I); 12 is serine, isoleucine or formula (I) Structure; 14 is arginine or the structure of formula (I); 15 is arginine or structure of formula (I); 17 is glutamic acid or structure of formula (I); 18 is asparagine or formula (I) structure; 2 1 is alanine, glycine or asparagine; 22 is lysine, arginine-lysine dipeptide or deletion, or is of the formula (I); when 22 is a dipeptide When one of the amino acids is of the formula (I); 23 _4 26 is a glycine-valine-glutamic acid tripeptide, U L -glycine-valine-glutamic acid, phenylalanine-valine - asparagine-glutamine tetrapeptide or U L -phenylalanine
  • U L is -WXYZ structure, fatty acid, polyethylene glycol, albumin, Ln-M L structure, hydrogen atom or N a -( a -(HOOC(CH 2 ) n CO)-Y-Glu)- , N a -(N a -(CH 3 (CH 2 ) deliberatelyCO)-Y-Glu)- , where ⁇ is an integer from 8-20, such as 8, 10, 12, 14, 16, 20, ⁇ ⁇ represents an ⁇ -amino group of an amino acid or an amino acid residue, or a general formula ( ⁇ ) structure, wherein the general formula ( ⁇ ) structure is:
  • J is a -WXYZ structure, an L n -M L structure or a hydrogen atom.
  • M L is a modifying group including, but not limited to, -WXYZ, a fatty acid, polyethylene glycol, albumin, IgG Fc, a sugar group, and the like.
  • L Record is an optional linker, covalent bond or non-existent.
  • Optional linkers include, but are not limited to, polyethylene glycol, long chain fatty acids, or one or more polyethylene glycol molecules and long chain fatty acids The long chain formed by the covalent bond of the molecule. It can be -NH-(CH 2 ) n -£0-, -NH-(CH 2 CH 2 0) n -CH 2 -CO- , -NH-(CH 2 CH 2 0) n -(CH 2 ) r -CO- , n is an integer from 1 to 20, and r is an integer from 1 to 10.
  • 1 ⁇ is -> 3 ⁇ 4-(.3 ⁇ 4 ⁇ 3 ⁇ 40 2 - .3 ⁇ 4-.0 11-( ⁇ 01 2 0) 2 - CH 2 -C0- o
  • Nl and n2 are integers of 1-16, respectively.
  • L shadow forms an amide bond with the amino group of the polypeptide compound by a bond derived from the underlined fluorenyl carbon. The other end forms a covalent bond with M L.
  • the L noir is underlined by The bond of the base carbon forms an amide bond with the amino group of the polypeptide compound, and the other end forms an amide bond with -WXYZ.
  • the -W-X-Y-Z structure is:
  • W is an ⁇ -amino acid residue having a carboxyl group in a side chain which has a carboxyl group and an ⁇ -amino group of the ⁇ -terminal amino acid residue of the ⁇ chain of the double-stranded compound of the present invention or a ⁇ -terminal amino acid of the single-chain compound
  • the ⁇ -amino group of the residue or together with the ⁇ -amino group of the lysine residue on the single or double strand form an amide group;
  • W is a chain joined by an amide bond of 2, 3 or 4 ⁇ -amino acid residues, which chain is linked to the ⁇ -amino group of the ⁇ -terminal amino acid residue of the ⁇ chain of the double-stranded compound via an amide bond or is attached to The ⁇ -amino group of the ⁇ -terminal amino acid residue of the single-stranded compound or the ⁇ -amino group of the lysine residue attached to the single- or double-stranded compound, and the amino acid residue of W is selected from the amino acid residue having a neutral side chain
  • the base and side chains have an amino acid residue of a carboxyl group such that W contains at least one amino acid residue having a carboxyl group in the side chain;
  • W is the ⁇ -amino group of the ⁇ -terminal amino acid of the oxime chain from X to the double-stranded compound or the ⁇ -amino group of the ⁇ -terminal amino acid residue of the single-stranded compound or the lysine residue to the double-stranded or single-stranded compound a covalent bond of the epsilon-amino group;
  • X is -£0-, -CH(COOH)CO-, -N(CH 2 COOH)CH 2 CO- , -N(CH 2 COOH)CH 2 CON (CH 2 COOH)CH 2 CO- , -N( CH 2 CH 2 COOH)CH 2 CH 2 CO- , -N(CH 2 CH 2 COOH)CH 2 CH 2 CON(CH 2 CH 2 COOH)CH 2 CH 2 CO-, -NHCH(COOH)(CH 2 ) 4 NHCO-, -N(CH 2 CH 2 COOH)CH 2 £0- or -N (CH 2 COOH)CH 2 CH 2 CO- , wherein
  • ⁇ is -(CH 2 ) m , where m is an integer from 6 to 32;
  • V and w are in the range of 6-30;
  • Z is -COOH, -CO- Asp, -CO-Glu , -CO-Gly, -CO-Sar, -CH (COOH) 2, -N (CH 2 COOH) 2, -S0 3 H, -P0 3 H Or absent; condition is that when W is a covalent bond and X is -CO-, Z is not -COOH.
  • the middle W of the side chain -W-X-Y-Z can be a covalent bond.
  • W may be an ⁇ -amino acid residue having a carboxyl group in the side chain, including a total of 4 to 10 carbon atoms.
  • W may be an alpha-amino acid residue encoded by a genetic code.
  • W may be selected from a-Asp, ⁇ -Asp, ⁇ -Glu or ⁇ -Glu; other choices for W are, for example, a-hGlu or S-hGlu.
  • W is a chain consisting of two a-amino acid residues, wherein one a-amino acid residue has 4 to 10 carbon atoms and the side chain has a carboxyl group, and the other has 2 to 11 carbons. Atom but no free carboxyl group.
  • the a-amino acid residue having no free carboxyl group may be a neutral, codeable a-amino acid residue.
  • W examples are: a-Asp-Gly, Gly-a-Asp, ⁇ -Asp-Gly, Gly-p-Asp a-Glu-Gly, Gly-a-Glu, ⁇ -Glu- Gly, Gly-y-Glu, a-hGlu-Gly, Gly-a-hGlu, ⁇ -hGlu-Gly and Gly-S-hGlu.
  • W is a chain consisting of two a-amino acid residues, each having 4 to 10 carbon atoms and having a carboxyl group in the side chain.
  • a-amino acid residues may be a coding a-amino acid residue.
  • Examples of W according to this embodiment are: a-Asp-a-Asp, a-Asp-a-Glu.
  • a-Asp-a-hGlu > a-Asp--Asp, a-Asp-y-Glu, a-Asp-6-hGlu, ⁇ -Asp-a-Asp, ⁇ -Asp-a-Glu, ⁇ -Asp-a-hGlu, ⁇ -Asp-P-Asp, P-Asp-y-Glu, P- Asp-5-hGlu, a-Glu-a-Asp, a-Glu-a-Glu, a-Glu-a-hGlu, a-Glu-P-Asp, a-Glu-y-Glu, a-Glu- 5-hGlu, y-Glu-a-Asp, ⁇ -Glu-a-Glu» ⁇ -Glu-a-hGlu.
  • W is a chain consisting of three a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from residues having a neutral side chain and a side chain A residue having a carboxyl group such that the chain contains at least one residue having a carboxyl group in its side chain.
  • the amino acid residue is a codeable residue.
  • W is a chain consisting of four a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from the group consisting of a residue having a neutral side chain and a side chain having The residue of the carboxyl group is such that the chain contains at least one residue having a carboxyl group in its side chain.
  • the amino acid residue is a codeable residue.
  • W in -W-X-Y-Z can be attached to the ⁇ -amino group of the lysine residue by a urea derivative.
  • X in the side chain -WXYZ may be a group of the formula -£0-, forming an amide bond with an amino group in W by a bond from an underlined carbonyl carbon; or when W is a covalent bond, X is derived from The underlined carbonyl carbon bond with the ⁇ -terminal a-amino group of the ⁇ chain of the double-stranded compound or the a-amino group of the N-terminus of the single-stranded compound or the lysine residue in the single-stranded or double-stranded compound The ⁇ -amino group forms an amide bond.
  • X in the side chain -WXYZ may be a group of the formula -CH(COOH) £0-, which forms an amide bond with a group in W by a bond from an underlined carbonyl carbon.
  • W is a covalent bond
  • X passes through the bond from the underlined carbonyl carbon to the N-terminal a-amino group of the B chain of the double-stranded compound or to the N-terminal a-amino group of the single-stranded compound or The ⁇ -amino group of the lysine residue in the double-stranded or single-stranded compound forms an amide bond.
  • X in the side chain -WXYZ may be a group of the formula -N(CH 2 COOH)CH 2 £0-, which forms an amide with an amino group from W by a bond from an underlined carbonyl carbon.
  • W is a covalent bond
  • X passes through the bond from the underlined carbonyl carbon to the N-terminal ⁇ -amino group of the B chain of the double-stranded compound or to the ⁇ -terminal ⁇ -amino group of the single-stranded compound or An amide bond is formed with the ⁇ -amino group of the lysine residue in the double-stranded or single-stranded compound.
  • the X in the side chain -WXYZ may be a group of the formula -N(CH 2 CH 2 COOH)C3 ⁇ 4£0-, formed by a bond from an underlined carbonyl carbon and an amino group in W An amide bond; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal ⁇ -amino group of the B chain of the double-stranded compound or the ⁇ -terminal ⁇ -amino group of the single-stranded compound Or forming an amide bond with the ⁇ -amino group of a lysine residue in a double-stranded or single-stranded compound.
  • X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CH 2 £0-, which forms an amide with an amino group in W by a bond from an underlined carbonyl carbon.
  • W is a covalent bond
  • X passes through the bond from the underlined carbonyl carbon to the N-terminal ⁇ -amino group of the B chain of the double-stranded compound or to the ⁇ -terminal ⁇ -amino group of the single-stranded compound or An amide bond is formed with the ⁇ -amino group of the lysine residue in the double-stranded or single-stranded compound.
  • X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CON(CH 2 COOH)CH 2 £0-, by a bond from an underlined carbonyl carbon
  • the amino group in W forms an amide bond; or when W is a covalent bond, X passes through an N-terminal amino group of the B chain of the double-stranded compound or a single-chain compound from a bond derived from an underlined rebel carbon
  • the terminal ⁇ -amino group forms an amide bond with the ⁇ -amino group of the lysine residue in the double-stranded or single-stranded compound.
  • X in -WXYZ may be a group of the formula -N(CH 2 C3 ⁇ 4COOH) CH 2 CH 0-, which forms an amide bond with an amino group in W by a bond derived from an underlined carbonyl carbon;
  • W is a covalent bond
  • X passes through the N-terminal ⁇ -amino group of the B chain of the double-stranded compound from the underlined carbon-based bond or the ⁇ -terminal ⁇ -amino group of the single-stranded compound or The ⁇ -amino group of the lysine residue in the chain or single chain compound forms an amide bond.
  • X in -WXYZ may be a group of the formula -N(CH 2 C COOH) CH 2 CH 2 CON(CH 2 CH 2 COOH) C3 ⁇ 4CH 2 £0-, by underlining
  • the bond of the carbonyl carbon forms an amide bond with the amino group in W; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal ⁇ -amino group of the B chain of the double-stranded compound or
  • the ⁇ -terminal ⁇ -amino group of the chain compound forms an amide bond with the ⁇ -amino group of the lysine residue in the double-stranded or single-stranded compound.
  • the oxime in the side chain -WXYZ may be a group of the formula -(CH 2 ) m wherein m is an integer of 6-32, 8-20, 12-20 or 12-16.
  • Y in -WXYZ is a divalent hydrocarbon chain comprising 1, 2 or 3 -CH-CH- groups and a plurality of -CH 2 - groups, said plurality of -CH 2
  • the number of groups satisfying the total number of carbon atoms in the hydrocarbon chain is in the range of 6-32, 10-32, 12-20 or 12-16.
  • Y in -WXYZ is a divalent hydrocarbon chain of the formula -(CH 2 ) V C 6 H 4 (CH 2) W - wherein v and w are integers, or one of Zero, such that the sum of V and w ranges from 6-30, 10-20 or 12-16.
  • Z in the side chain -W-X-Y-Z is -COOH, provided that when W is a covalent bond and X is -CO-, Z is not -COOH.
  • Z in -WXYZ is -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH(COOH)2, -N(C3 ⁇ 4COOH) 2 , -S0 3 H or - P0 3 H.
  • W in -WXYZ is a-Asp, ⁇ -Asp, ⁇ -Glu or ⁇ -Glu;
  • X is -CO- or -CH(COOH)CO-;
  • Y is -(CH 2 ) m , wherein m is an integer from 12 to 18;
  • Z is -COOH -, -CH(COOH) 2 or is absent.
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-GIu or ⁇ -GIu;
  • -XYZ is -CO(CH 2 ) n , which is derived from underlined diff carbon The bond forms an amide bond with W, wherein n is an integer from 10-18.
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -XYZ is -CO(CH 2 ) 14 .
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu; -XYZ is -CO(CH 2 ) 16 .
  • W in -WXYZ is a-Asp, ⁇ - ⁇ > a-Glu or ⁇ -Glu; -XYZ is -CO(C3 ⁇ 4) 18 .
  • W in -WXYZ is a-Asp, ⁇ -Asp, a-Glu or ⁇ -Glu;
  • -XYZ is cholesterol, bile acid (such as cholic acid, chenodeoxycholic acid, hepatobiliary acid, Cow transverse acid, deoxycholic acid, lithocholic acid);
  • the compound comprises an A chain and a B chain, wherein
  • amino acid sequence of the A chain is:
  • the B chain amino acid sequence is:
  • [1]-[6] represents the number of a cysteine; the compound forms a 3-pair disulfide bond through 6 cysteines, wherein the A chain and the B chain pass two pairs of interchain disulfide The bond is connected, there is a pair of intrachain disulfide bonds in the A chain, and the specific positions of the three pairs of disulfide bonds are: C n] and . [ 4] Form a disulfide bond, C[ 2] and C[ 6 ] form a disulfide bond, [ 3] and . [5] forming a disulfide bond;
  • 12 is a serine or a structure of the formula (I); 14 is an arginine or a structure of the formula (I); 15 is an arginine or a structure of the formula (I); 17 is a glutamic acid or a structure of the formula (I) 18 is an asparagine or a structure of the formula (I); 22 is a lysine, arginine-lysine dipeptide or a deletion, or a structure of the formula (I); when 22 is a dipeptide, one of them
  • the amino acid is of the formula (I);
  • X42W26 is a glycine-valine-glutamic acid tripeptide, U L -glycine-valine-glutamic acid, phenylalanine-valine-asparagine-valley Aminoamide tetrapeptide or Ui phenylalanine-valine-asparagine-glutamine; 27 is histidine or threonine; 48 is -NH
  • the compound is based on IGF-1?
  • the single-stranded structure of the compound, the structure of the compound is:
  • X 2 oi a is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine, benzene 13 ⁇ 4-proline-asparagine-glutamine- Histidine, UL-glycine-valine-glutamate-threonine, UL-glycine-valine-glutamate-histidine or UL-phenylalanine-valine-asparagine - glutamine-histidine; X 2 .
  • Lb is histidine, glutamic acid, glutamine, arginine or phenylalanine;
  • X 2 o lc is phenylalanine or tryptophan;
  • X 2 oid is phenylalanine or tryptophan;
  • X 201e is phenylalanine, tyrosine or histidine;
  • X 2 o 2 is phenylalanine, tyrosine or deletion;
  • X 2 o 3 is asparagine, threonine, aspartic acid, Glutamate or deletion;
  • X 2 () 4 is valine, lysine, arginine, aspartic acid, glutamic acid or deletion;
  • X 2 Q5 is valine, lysine, arginine , aspartic acid, glutamic acid or deletion or structure of formula (I);
  • X 2 o 6 is threonine, lysine Acid or deletion or structure of formula (I
  • [1] - [6] represent the number of cysteine.
  • the single-chain compound of the present invention forms a diamole bond in the chain in the tertiary structure in the structure of insulin, specifically: C n] and C [4] form a disulfide bond, C [2] and C [6] A disulfide bond is formed, and C [3] and C [5] form a disulfide bond.
  • the structure of the single chain compound is:
  • X 201a is glycine-valine-glutamate-threonine, phenylalanine-valine-asparagine-glutamine-histidine, 1-glycine-valine-glutamate- Threonine or U L -phenylalanine-valine-asparagine-glutamine-histidine;
  • X 2 o2 is phenylalanine or deletion;
  • X 2 Q3 is asparagine or deletion;
  • X 2Q4 is valine, lysine, arginine, aspartic acid or deletion;
  • X 2Q5 is valine, lysine or the structure of formula (I);
  • X 2Q6 is threonine, lysine or a structure of the formula (I);
  • X 2 o7 is a serine, alanine or a structure of the formula (I);
  • X 2 () 8 is a serine or a structure of the formula (I);
  • the modified compound of the invention is selected from the group consisting of:
  • a triple-stranded structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain sequence is G (N a -PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT;
  • III-2 a double-stranded structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17; and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK (N E -PEG 20K);
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is: SEQ ID NO: 17; B chain sequence is: G( a -CO(C3 ⁇ 4) 14 COOH) PETLCGAHLVDALFFVCGDRGFYFNPPT;
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -( a - (HOOC(CH 2 ) 14 CO )-y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N s -(N a - (HOOC(CH 2 ) 16 CO)-y-Glu)];
  • Double-stranded structure including an ⁇ chain and an ⁇ chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -(N a - (HOOC(CH 2 ) 12 CO)-y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK ⁇ N E -[N a - (HOOC(C3 ⁇ 4)iiNHCO( CH 2 ) 3 CO) - ⁇ -Glu] ⁇ ;
  • III-ll double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu -N-(Y-G1U)];
  • a loop-like structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK[N S - (N a -(HOOC (CH 2 ) 14 CO )-y-Glu)]; 111-20: Single-chain structure, the sequence is:
  • double-stranded structure including A chain and B chain, wherein the A chain sequence is: GIVDQCCFRSCSLK[N e -(N a -(HOOC(CH 2 ) 14 CO)-y-Glu)]RLENYCA ; B chain sequence Is FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 163);
  • a chain sequence is: GIVDQCCFRSCSLRK[N E -(N a -(HOOC(CH 2 )i 4 CO)-y-Glu)]LENYCA ;
  • B chain The sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 164);
  • III-23 a double - stranded structure comprising an A chain and a B chain , wherein the A chain sequence is
  • the B chain sequence is the sequence shown in SEQ ID NO: 164;
  • a chain sequence is GIVDQCCFRSCSLRRLENYCARK[N £ -(N a -(HOOC(CH 2 )i 4 CO)-Y-Glu)]
  • B chain sequence is SEQ ID NO: 164 the sequence shown;
  • double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[N e - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
  • Double-stranded structure including an ⁇ chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [N e -(N a -(HOOC(CH 2 )i 4 CO)-Y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[N ( a -(HOOC(CH 2 )i 4 CO)- Y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence ⁇ ' J is GPETLCGAHLVDALFFVCGDRGFYFNPK[N E - (N A - (HOOC(CH 2 ) 14 CO)-Y-G1U-N-
  • Double-stranded structure including an ⁇ chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is G[N a -(N a -(CH 3 (C3 ⁇ 4) 14 CO )-YL-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT;
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is G( a -dPEG 12 -maleimide-albumin) PETLCGAHLVDALFFVCGDRGFYFNKPT ;
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[N S - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTK[N E - (N a -(HOOC(CH 2 ) 14 CO )-y-Glu)];
  • Double-stranded structure including an ⁇ chain and an ⁇ chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17 and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N £ -(N a -(HOOC(CH 2 ) 14 CO) -Y-Glu)];
  • Double-stranded structure including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK[N (N a -(HOOC(CH 2 ) i 4 CO) -y-Glu)];
  • ⁇ -35 single-stranded structure G(N a -PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA;
  • Double-stranded structure including ⁇ chain and B chain, wherein the A chain sequence is GIVDQCCHRSCSLRRLENYCA, and the B chain sequence is GPEHLCGAHLVDALFFVCGDRGFYFNPK [N E -CO-(CH 2 CH 2 0) 5 -(CH 2 ) 2 - NH- (N"-(HOOC
  • N a represents an a-amino group of an amino acid or an amino acid residue
  • ⁇ ⁇ represents an ⁇ -amino group of an amino acid or an amino acid residue, such as an ⁇ -amino group of a lysine side chain.
  • the disulfide bond in the chain is formed by the structure of insulin, specifically: C[ u and C [ 41 forms a two-stone charge, C [2 ] and C [ 6 ] The two sparse bonds are formed, and C[ 3] and [ 5] form a disulfide bond.
  • the number of cysteine is as defined herein.
  • the hypoglycemic compound of the present invention can be provided in the form of a substantially zinc-free compound or a complex of zinc.
  • a zinc complex of the compound of the present invention wherein the compound of the present invention can form a hexamer, each hexamer can bind 2 Zn 2+ 3 Zn 2+ or 4 Zn 2+ pharmaceutical compositions and use
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the invention and a pharmaceutically acceptable carrier for the treatment of type 1 diabetes, type 2 Diabetes and other conditions that cause hyperglycemia.
  • the compounds according to the invention may be used in the preparation of a pharmaceutical composition for the treatment of type 1 diabetes, type 2 diabetes and other conditions which cause hyperglycemia.
  • a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other conditions which cause hyperglycemia comprising a therapeutically effective amount of a compound according to the invention, Infused with insulin or insulin analogs having a rapid action effect, and pharmaceutically acceptable carriers and additives.
  • compositions of the IGF-1 analogs of the invention can be prepared using conventional techniques of the pharmaceutical industry, including dissolving and mixing the appropriate components to provide the desired final product.
  • the IGF-1 analog of the present invention is dissolved in a quantity of water in a volume slightly lower than the final volume of the composition to be prepared, according to a set of procedures.
  • the volume of the solution is finally adjusted to the desired concentration with water.
  • the buffering agent is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, phosphoric acid Disodium hydrogen phosphate, sodium phosphate and tris(hydroxymethyl)-aminodecane, N-bis(hydroxyethyl)glycine, N-(hydroxymethyl)methylglycine, malic acid, succinate, maleic acid, Fumaric acid, tartaric acid, aspartic acid or a mixture thereof.
  • Each of these specific buffers constitutes an alternative embodiment of the invention.
  • the formulation comprises a pharmaceutically acceptable preservative selected from the group consisting of phenol, o- Indophenol, m-nonylphenol, p-cresol, decyl hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, Benzyl alcohol, chlorobutanol, thimerosal, bromide, benzoic acid, imidate, chlorhexidine, sodium dehydroacetate, chlorocresol, benzethonamine, clopidogrel or mixtures thereof.
  • a pharmaceutically acceptable preservative selected from the group consisting of phenol, o- Indophenol, m-nonylphenol, p-cresol, decyl hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, Benzyl alcohol
  • the concentration of the preservative is from 0.1 mg mL to 20 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 0.1 mg/mL to 5 mg/mL. In another embodiment of the present invention, the concentration of preservative was 5mg / mL-10mg / mL alternative embodiment of the present invention, each of these specific preservatives constitutes the o.
  • the use of preservatives in pharmaceutical combinations is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • the formulation further comprises an isotonicity agent selected from the group consisting of a salt (eg, sodium chloride), a sugar or sugar alcohol, an amino acid, an alditol (eg, glycerol, propylene glycol, 1,3-propanediol) , 1,3-butanediol), polyethylene glycol (eg PEG400) or a mixture thereof.
  • a salt eg, sodium chloride
  • a sugar or sugar alcohol an amino acid
  • an alditol eg, glycerol, propylene glycol, 1,3-propanediol
  • 1,3-butanediol 1,3-butanediol
  • polyethylene glycol eg PEG400
  • Any sugar such as a monosaccharide, disaccharide, polysaccharide or water-soluble glucan, including, for example, sugar, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, Dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose-Na.
  • the sugar additive is sucrose.
  • a sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group, including, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • the above saccharides or sugar alcohols may be used singly or in combination. There is no fixed limit on the amount of use, as long as the sugar or sugar alcohol is dissolved in the liquid preparation and does not adversely affect the stabilization obtained by the method of the present invention.
  • the concentration of the sugar or sugar alcohol is from 1 mg/mL to 150 mg/mL.
  • the concentration of the isotonic agent is from 1 mg/mL to 50 mg/mL. In another embodiment, the concentration of the isotonic agent is from 1 mg/mL to 7 mg mL. In another embodiment, the concentration of the isotonic agent is from 8 mg/mL to 24 mg/mL. In another embodiment, the concentration of the isotonic agent is from 25 mg/mL to 50 mg/mL.
  • concentration of the isotonic agent constitutes an alternative embodiment of the invention.
  • the use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art. Reference Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • Typical isotonic agents are sodium chloride, mannitol, disulfoxide and glycerin.
  • Typical preservatives are phenol, m-nonylphenol, decyl hydroxybenzoate and benzyl alcohol.
  • surfactant examples include sodium acetate, glycylglycine, hydroxyethylpiperazineethanesulfonic acid (HEPES), and sodium phosphate.
  • HEPES hydroxyethylpiperazineethanesulfonic acid
  • ACN acetonitrile: acetonitrile
  • BOP benzotriazol- 1 -yloxytris(dimethylamino) phosphonium hexafluorophosphate: benzotriazole-1-tris(trimethylamino)-hexafluorophosphate (Carter condensate);
  • DCC ⁇ , ⁇ '-Dicyclohexylcarbodiimide: dicyclohexylcarbodiimide;
  • DCM dichloromethane;
  • DEPBT 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one:3- (2-B Oxykolyloxy)-1,2,3-benzotrien.
  • TEA triethylamine: triethylamine
  • TFA trifluoroacetic acid trifluoroacetic acid
  • TFE 2,2,2-Trifluoroethanol trifluoroethanol
  • THF tetrahydrofuran tetrahydrofuran
  • TIS triisopropylsilane triisopropylsilane.
  • Linear polypeptides use Boc or Fmoc solid phase peptide synthesis. If Fmoc is used to synthesize a polypeptide having a C-terminal carboxyl group, Wang resin is generally used; and a C-terminal amide is usually a Rink amide resin. If Boc is used to synthesize a polypeptide having a C-terminal carboxyl group, Pam resin is generally used; and a polypeptide having a C-terminal amide is usually selected from MBHA resin.
  • the condensing agent and activator are DIC and HOBT, and other optional peptide bond condensing agents include BOP, HBTU, DEPBT and the like. Amino acid 5 times excess. The condensation time was 1 hour.
  • the Fmoc protecting group used 50% piper. ⁇ /DMF removal.
  • the Boc protecting group is removed with TFA.
  • the peptide bond condensation reaction was monitored with Ninhydrin (2,2-Dihydroxyindane-1,3-dione) reagent.
  • the side chain amino group of lysine can be protected with an allyloxycarbonyl group (aloe).
  • the allyloxycarbonyl group can be removed with tetrakis(triphenylphosphine)palladium(0) and 37:2:1 ratio of DCM, glacial acetic acid and NMM (15 mL/g resin) in an argon atmosphere. Stir at room temperature for 2 hours.
  • the resin was washed with 0.5% DIPEA/DMF (10 mL), 0.5% sodium diethyldithiocarbonate/DMF (3 X 10 mL), 1:1 DCM:DMF (5 ⁇ 10 mL).
  • the side chain amino group of lysine can also be protected with 4-methyltriphenylmethyl (Mtt).
  • the resin was suspended in DCM, added to TFA/TIS/DCM (1:2:97), and shaken for 10 minutes. After repeating 2 times, the resin was washed with DCM, DMF and isopropyl alcohol.
  • the commonly used cleavage reagent is TFA.
  • the dry resin was placed in a shake flask, and an appropriate amount of TFA/TIS/H 2 0 (95:2.5:2.5, 10-25 m! Jg resin) was added, and the lid was capped, and intermittent rotation was performed at room temperature. After 2 hours, the resin was filtered, and the resin was washed 2-3 times with a new TFA, and the filtrate was combined, and 8-10 volumes of ice acetaminophen were added dropwise. Finally, the precipitated polypeptide was collected by centrifugation.
  • Boc-Cys(4-MeBzl)-OH Boc-Asp(OcHx)-OH, Boc-Glu(OcHx)-OH, Boc-His(Bom)-OH, Boc-Lys(2-Cl-Z)-OH , Boc-Asn(Xan)-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Trp( CHO)-OH and Boc-Tyr(2-Br-Z)-OH.
  • the side chain amino group of lysine may be protected with an aloin or Fmoc. If the side chain carboxyl group of aspartic acid or glutamic acid is used for the acylation reaction, the carboxyl group should be converted to allyl ester. (allyl ester) or 9-mercaptopurine protection.
  • the reaction solvent composition is as follows:
  • IGF-1A chain or B chain 0.5 mM IGF-1A chain or B chain was dissolved in the above reaction solvent, and the pH was adjusted to 8.5-8.7. The mixture was vigorously stirred at room temperature for 1-1.5 hours and then desalted using a G10 or G25 column.
  • Buffer A was 0.05 M ammonium bicarbonate aqueous solution
  • buffer B was 0.05 M ammonium hydrogencarbonate / 50% acetonitrile aqueous solution. Pure product after desalting, low pressure freeze-drying (lyophilization).
  • the A chain and the B chain were synthesized by Fmoc chemistry.
  • the molecular weight of the A chain was calculated to be 2436.9, the molecular weight of the mass spectrum was 2437.6, the molecular weight of the B chain was 3175.7, and the molecular weight of the mass spectrometer was 3176.9.
  • the final product had a calculated molecular weight of 5606.5 and a mass spectrometry test molecular weight of 56,073.
  • the B chain is dissolved in DMF or DMSO and an equimolar amount of 2,2'-dithiobis(5-nitropyridine) is added.
  • the reaction was detected by HPLC and purified to give B-(S-Npys) 6 (S-Acm) 18 .
  • Equimolar A- (SH) 7 (S- Acm) 6 '20 and B- (S-Npys) 6 ( S-Acm) 18 was dissolved in DMSO, polypeptide concentration 15mg / mL. After the A7-B6 disulfide bond was formed, 80% aqueous acetic acid solution was added, and the polypeptide concentration was diluted to 1 mg/mL. Add 40 times more 1 2 . After the reaction was stirred at room temperature for 1 hour, the reaction was quenched by the addition of aqueous ascorbic acid. The mixture was purified by HPLC and the final product was confirmed in succinct.
  • the LC-MS can be used to compare the retention time and molecular weight of each sample of the sample and the standard to disulfide bond and Structure of the compound
  • This analytical method is generally applicable to single-stranded or double-stranded polypeptides synthesized by various methods in the present invention.
  • the double-stranded compounds were separately synthesized by the above-mentioned synthetic method of the present invention, and the compounds were tested and analyzed by sequencing and mass spectrometry.
  • the sequencing results showed that the amino acid sequence of the compound was correct, and the mass spectrometry results showed that the structure of the compound was consistent with the original design structure, that is, The synthesized compound is the desired compound.
  • the data results are as follows:
  • Mass transfer detection revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between Qi] and C [4 ] and C[ 2] and C [6 interchain disulfide bonds form between], C [3] and C [intrachain disulfide bond formation between 51; molecular weight calcd 5055.9, 5057.3 molecular mass spectrometry;
  • Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular diterpene bonds: between C m and C [4] and C [2 ] and C [6] An interchain disulfide bond is formed between the two, and an intrachain disulfide bond is formed between C[ 3 ] and C [5] ; the molecular weight is calculated to be 5614.4, and the molecular weight is 5615.9;
  • the enthalpy detection shows that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: Qi] and . [ 4] between and. An interchain disulfide bond is formed between [ 2] and C[ 6] , and an intrachain disulfide bond is formed between 0[ 3] and C[ 5] ; molecular weight calculated value is 5636.5, mass spectrometry molecular weight is 5636.7;
  • Mass spectrometry revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tI] and C [4] and C [2] and C [6 An interchain disulfide bond is formed between them, and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5612.4, and the molecular weight is 5614.1;
  • Shield detection shows that the A chain and the B chain are linked by two pairs of intermolecular dichotomous bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and C [2] and C An interchain disulfide bond is formed between [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; molecular weight calculated value is 5599.4, mass spectrometry molecular weight is 5601.7;
  • Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and C [2] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3] and C [5] ; molecular weight calculated 5652.6, mass spectrometry molecular weight 5652.9;
  • Mass spectrometry showed that the A chain and the B chain were linked by two pairs of intermolecular two bowl bonds and a pair of intramolecular disulfide bonds: C[ u and C [4 ] and C [2] and C [6 is formed between a] interchain disulfide bonds, the disulfide bond is formed between C [3] and C [5]; mass calculated 5876.8, 5877.6 molecular mass spectrometry;
  • Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C n] and C [4] and C [2] and .
  • An interchain disulfide bond is formed between [6]
  • an intrachain disulfide bond is formed between C [3] and C [5] ;
  • the molecular weight is calculated to be 5798.7, and the molecular weight is 5799.6;
  • Mass spectrometry revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C m and C [4] and C [2 ] and C [6] An interchain disulfide bond is formed between them, and an intrachain disulfide bond is formed between C [3] and C [5] ; a molecular weight calculated value of 5803.7, a mass spectrometric test molecular weight of 5805.3;
  • Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tI] and C [4] and C[ 2] and [ 6 The interchain two bonds are formed between each other, and an intrachain disulfide bond is formed between C CT and C [5 ]; the molecular weight is 5831.7, and the mass spectrum is 5832.8;
  • Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C[ 4] .
  • An interchain disulfide bond is formed between [2] and C [6]
  • an intrachain disulfide bond is formed between C [3] and C [51 ; molecular weight calculated value 5987.9, mass spectrometry test molecular weight 5990.4;
  • Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C t i] and C [4] and C [2] and C An interchain disulfide bond is formed between [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5675.5, and the molecular weight is 5676.8. Synthesis of single-chain compounds
  • the IGF-1 analog is synthesized in two fragments.
  • One is the synthesis of IGF-1 B chain [l-17]-COS-(CH 2 ) 2 CO-(Arg) 4 A by Boc chemistry.
  • S-triphenylmethyl-3-mercaptopropionic acid is used in the synthesis of the thioester residue.
  • the second paragraph includes from B18Cys (corresponding to the pass) Wherein X 41 or X44l ) functions as the entire amino acid of the C-terminus of the B chain, the C peptide and the A chain amino acid.
  • the two peptides are solid phase synthesized, cleaved and purified by a general method.
  • the natural chemical ligation reaction is carried out in a buffer.
  • the reaction was detected by HPLC and purified.
  • Purified IGF-1 (SH) 6 was dissolved in 0.5 M guanidine hydrochloride, 20 mM Tris, 8 mM cysteine, 1 mM cystine hydrochloride buffer, pH 7.8, peptide concentration 0.5 mg/mL . After HPLC showed that the folding was completed, the buffer was acidified to pH 3 with 0.1 N hydrochloric acid. The polypeptide was purified by preparative RP-HPLC.
  • the first fragment 25.7 mg (10 ⁇ ) and the second fragment 48.3 mg (10 ⁇ ) were dissolved in buffer (5 mL).
  • the buffer contained 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxymethylthiophenol, 20 mM tris(2-nonanoylethyl)phosphine, pH 6.9.
  • the reaction was completed after 10 hours.
  • the mixture was transferred to a size exclusion chromatography column with 0.5 M guanidine hydrochloride, 20 mM Tris, pH 7.8.
  • the fraction containing the correct molecular weight of the polypeptide was collected, and 8 mM cysteine, 1 mM cystine hydrochloride buffer was added, and the polypeptide was folded 2 hours later.
  • the buffer was acidified to pH 3 with 0.1 N hydrochloric acid and then purified by RP-HPLC.
  • the molecular weight calculated was 6590.5, the molecular weight of the test was 6591.6, and the sequencing result was consistent with SEQ ID NO:31.
  • the single-chain compound based on IGF-1 was prepared by the above method, and the molecular weight of the molecule was detected by a linguistic expression, and the structure of the prepared single-chain polypeptide was detected by sequencing to verify the synthesized compound, and the result was as follows:
  • the NMR data is 'H-NMR (CDC1 3 ) ⁇ : 6.25 (d, lH), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47(s, 9H), 1.22-1.43 (m, 18H).
  • tert-Butylhexadecandioyl-L-Glu-OtBu ( lg, 1.9 mmol) dissolved in anhydrous DMF/DCM (1 mL: 4 mL).
  • DCC (0.412 g, 2 mmol) and N-hydroxybutane Imide (0.23 g, 2 mmol).
  • the mixture was stirred overnight at room temperature.
  • the mixture was filtered, and the filtrate was diluted with ethyl acetate, washed with 0.1 N HCI and brine, and dried over magnesium sulfate, and evaporated under reduced pressure to give tert-butylhexadecandioyl-L-Glu ( OSu ) -OtBu.
  • the NMR data are: ⁇ -NMRiCDC) ⁇ : 6.17 (d, lH), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H).
  • the molecular weight was calculated to be 5746.7, and the molecular weight of the mass spectrum was 5747.8.
  • the polypeptide 55 mg was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature.
  • tert-Butylhexadecandioyl-L-Glu(OSu)-OtBu 6.6 mg was dissolved in acetonitrile (5 mL). After stirring for 30 minutes, it was acidified with 50% acetic acid and purified on a RP-HPLC C5 column.
  • Buffer A 0.1% TFA in water, 10% acetonitrile buffer B: 0.1% TFA in water, 80% acetonitrile.
  • the lyophilized polypeptide was initially purified by adding TFA/TIS/H 2 0 (95:2.5:2.5, 10 mL), and after 30 minutes, the solvent was evaporated in vacuo, and the crude product was dissolved in buffer A and lyophilized. Purified using an RP-HPLC C5 column. The molecular weight calculated was 6144.2 and the mass spectrum was tested to have a molecular weight of 6146.0. After the B chain was subjected to trypsin hydrolysis, the calculated molecular weight of the terminal fragment containing the fatty acid was 1269.5, and the molecular weight of the mass spectrum was 1270.4.
  • the B chain (0.1 mmol) was synthesized by Boc chemistry, and the lysine side chain amino group was protected with Fmoc. After the B chain is synthesized, the Boc at the N-terminus of the B chain should not be removed. 20% piperidine / DMF (6 mL) was added to the resin, and the mixture was shaken for 15 minutes, and the test solution was drained. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM.
  • tert-Butylhexadecandioyl-L-Glu (OSu)-OtBu (550 mg) and DIPEA (0.2 mL) were dissolved in DMF and added to the resin. After shaking for 3 hours, the solution was pumped and the resin was washed with DMF and DCM. After vacuum drying, the peptide on the resin was cut with HF, p-phenol reagent was added, and the mixture was stirred under ice bath for 1 hour. After HF vacuum extraction, the polypeptide was precipitated with ice acetonitrile, and the precipitate was collected by centrifugation. Molecular weight calculated 3601.2, mass spectrometry test molecular weight 3602.8.
  • the crude B chain is sulfonated by sulfhydrylation, desalted by chromatography, separated and purified, and freeze-dried to obtain the final product IGF-1 B.
  • Chain S sulfonate IGF-1 A (Q5, S12, N18) was synthesized by a general method with a molecular weight of 2436.9 and a molecular weight of 2437.6.
  • the IGF-1 A chain S sulfonate and the B chain S sulfonate are synthesized using the above-described general A chain and B chain linkage method.
  • the molecular weight calculated was 6032.0, and the mass spectrum was tested to be 6033.4.
  • the B chain is chemically synthesized by Boc, the lysine side chain amino group is protected with Fmoc, and the thiol group of B18Cys (corresponding to the formula or X44! is protected with Acm. After the synthesis is completed, the Boc at the N-terminus of the B chain is not removed. 20% piperidine DMF (6 mL) was added to the resin (0.1 mmol), the mixture was shaken for 15 minutes, and the reagent was drained. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM.
  • the A chain was synthesized as IGF-1 A-(Q5, S12, N18) (SH) 7 (S-Acm) 6 '20 to calculate the molecular weight of 2650.1 and the mass spectrometry to test the molecular weight of 2651.5.
  • the method for joining the A chain and the B chain is in accordance with the above A chain and B chain linkage method (2).
  • the molecular weight calculated was 6032.0, and the mass spectrum was tested to have a molecular weight of 6034.2.
  • Palmitic acid (256 mg, 1 mmol) > DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, 1 mmol) and DIPEA (0.2 ml, 1.15 mmol) were dissolved in DMF and added to the resin. After 2 hours, the solution was drained and the resin was washed with DMF and DCM. After vacuum drying, the polypeptide on the resin was cut with HF, and a hydrazine reagent was added. The mixture was stirred under ice bath for 1 hour, and after HF vacuum-dried, the polypeptide was precipitated with ice diethyl ether, and the precipitate was collected by centrifugation.
  • A was synthesized by the above method. The molecular weight calculated was 6432.3 and the mass spectrum was tested to be 6434.1.
  • the polypeptide (643 mg) was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature.
  • the lyophilized polypeptide was initially purified and added to TFA/TIS/H 2 0 (95:2.5:2.5, 10 mL). After 30 minutes, the solvent was evaporated in vacuo to dissolve the crude product. Wash A and freeze dry. Purified using an RP-HPLC C5 column. The molecular weight calculated was 6829.8 and the mass spectrum was tested to have a molecular weight of 6831.6. A small amount of product was reduced by DTT and trypsin degradation.
  • 12-aminododecanoate hydrochloride ( lg, 3.8 mmol) was suspended in THF (15 ml), glutaric acid ( 1.29 g, 3.8 mmol) and TEA (0.52 ml, 3.8 mmol) were added and stirred at room temperature overnight. . After adding water (75 ml) for 1 hour, it was filtered, washed with water and dried to give lg l2-(4-carboxybutyryl) decanoate.
  • the polypeptide was synthesized by the above A and B chain ligation methods. The molecular weight was calculated to be 5746.7, and the molecular weight of the mass spectrum was 5748.0.
  • the polypeptide (58 mg) was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature. (CH 3 OOC(CH 2 ) u NHCO(CH 2 ) 3 CO)-Glu(OSu)-OCH 3 (7 mg) was dissolved in acetonitrile (5 mL), and the peptide solution was added.
  • the molecular weight was calculated to be 1012.2 and the molecular weight was tested to be 1013.1.
  • the polypeptide is synthesized by the above-described hydrazone and hydrazone linkage methods. The molecular weight was calculated to be 5531.4, and the mass spectrum was tested to be 5533.7. The polypeptide (56 mg, 10 ⁇ ) was dissolved in 100 mM Na 2 CO 3 (1 ml, pH 10) at room temperature.
  • the polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH 3 CN) was dissolved in a pH 4.3 acetic acid solution (0.1 M NaCl, 0.2 M CH 3 COOH, 0.1 M Na 2 CO 3 ) in a ratio of 1:2:45.
  • the polypeptide concentration is 0.5-1 mg mL.
  • the reaction was detected and purified by HPLC. The yield is about 55%.
  • the reductive alkylation reaction can selectively bind polyethylene glycol to the B1 position.
  • a (Q5, S12, N18): B (H9, F15, P27) synthesis method is the same as above.
  • B1 reductive alkylation gives the product.
  • the molecular weight calculated was 25576.4, and the mass enthalpy test obtained a broad peak with an intermediate molecular weight of 25580.2.
  • a small amount of the product was reduced by DTT.
  • the polypeptide and mPEG20K-NHS molar ratio 1:1 was dissolved in 0.1N ⁇ , ⁇ -bis(2-hydroxyethyl)glycine solution (pH 8), and the peptide concentration was 0.5 mg/mL.
  • the reaction was carried out at room temperature for 2 hours and purified by HPLC. The yield is about 90%.
  • a (Q5, S12, N18): B (H9, F15, P27, K28, desT) was synthesized by the above A and B chain linkage methods.
  • the molecular weight was calculated to be 5505.4, and the molecular weight of the test was 5506.8.
  • the polypeptide is reacted with mPEG20K-NHS to give 111-2.
  • the molecular weight calculated was 25505.4, and the mass spectrum test gave a broad peak with an intermediate molecular weight of 25501.7.
  • a small amount of product was subjected to DTT reduction and trypsin degradation of the B chain.
  • GPETLCGAH LVDALFFVCGDR (molecular weight calculated value 2220.6, mass spectrometry molecular weight 2221.7) was observed by liquid chromatography-mass spectrometry. No GFYFNPK (molecular weight calculated value 872.0) was observed, but mass spectrometry was observed. A broad peak with an intermediate molecular weight of 2,087,1.9. Upon analysis, the obtained compound was 111-2.
  • the aqueous layer was vacuum freeze dried.
  • the crude product contains Boc monosubstituted, disubstituted and trisubstituted polypeptides.
  • the mixture was purified using an SP-Sephadex C-25 ion exchange column.
  • the ion exchange column was first equilibrated with 1.5 M acetic acid containing 6 M urea, the peptide elution flow rate was 48 mL/h, and the linear gradient was 0.04-0.4 M sodium chloride / 1000 mL 6 M urea in 1.5 M acetic acid.
  • the Di-t-Boc polypeptide was further purified using a DEAE-Sephadex A-25 column.
  • the color column was previously equilibrated with 0.01 M Tris buffer (pH 8.5) containing 7 M urea.
  • the elution flow rate was 35 mL/h, gradient 0.14-0.28 M sodium chloride/100 mL Tris buffer.
  • the molecular weight calculated was 5806.7, and the molecular weight was 5808.3.
  • a small amount of the polypeptide was dissolved in 0.05 M NH 4 HCO 3 /20% ACN, and subjected to mass spectrometry after reduction with DTT for 10 minutes.
  • A (Gl-N a -Boc, Q5, S12, N18)
  • the calculated molecular weight was 2537.0, and the molecular weight of the mass spectrum was 2537.9.
  • B (H9, F15, K27-N s -Boc)
  • the calculated molecular weight was 3275.8, and the molecular weight of the shield test was 3276.0.
  • the molecular weight of the Boc-containing fragment was calculated to be 1073.2, and the molecular weight of the mass spectrum was 1074.5.
  • the B1 amino group of the Al, B27-di-Boc polypeptide can be combined with polyethylene glycol, albumin, fatty acid or the like to form a long acting polypeptide.
  • A (Gl-N a -Boc, Q5, S12, N18): The synthesis method of B (H9, F15, K27-N s -Boc) is as above.
  • the polypeptide (58 mg) was dissolved in DMF (3 mL), and Mal-dPEG12-NHS (8.7 mg) (quanta Biodesign) and triethylamine (30 L) were added. The reaction was stirred at room temperature for 2 hours. After evaporating the solvent under reduced pressure, TFA (2 mL) and TIS ( ⁇ ) were added. After stirring for 10 minutes, TFA was evaporated.
  • the crude material was dissolved in H 2 0/ACN (3: 1) and purified using RP-HPLC.
  • the molecular weight was calculated to be 6556.5 and the mass spectrum was tested to have a molecular weight of 6558.0.
  • the maleimide polypeptide is dissolved in purified water at a polypeptide concentration of 10 mM.
  • Human albumin (665 mg) was added and incubated at 37 °C for 30 minutes. It was then diluted to 5% with a 20 mM sodium phosphate solution containing 5 mM sodium octoate and 750 mM ammonium sulfate.
  • the unreacted reagent was removed by gel filtration chromatography, and the eluent was a 0.05 M aqueous solution of ammonium hydrogencarbonate. Purely obtained after vacuum freeze drying.
  • the calculated molecular weight of the compound was 73028.7, and the mass spectrum was tested to have a molecular weight of 73030.2, which was analyzed as Compound 111-30.
  • IGF-1-based modified compounds including single-stranded compounds and double-stranded compounds, was synthesized according to the above method, and the structures of the respective compounds were examined by traits. The results were as follows:
  • the molecular weight of the test is 6242.4.
  • the obtained compound is ⁇ -31 111-32: molecular weight calculated value 6101.1 mass spectrometry test molecular weight 6101.8
  • the obtained compound is ⁇ -32 111- 33: Molecular weight calculated value 6230.2
  • the obtained compound is ⁇ -33 111-34: Molecular weight calculated value 6287.3
  • Qualitative test molecular weight 6288.2 After analysis, the obtained compound is ⁇ -34: ⁇ -35: Molecular weight calculated value 26462.3
  • Mass spectrometry test gave a broad peak with an intermediate molecular weight of 26470.1. Analysis, i.e., the resultant compound ⁇ -35;
  • EK Frandsen and RA Bacchus "New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells.” Diabetes, 1987, 36, 3: 335-340) or the following method One. Unless otherwise stated, the method of preparation of the receptor is also used as a literature method using a human placental membrane. In general, 0.025 mg of placental membrane was used for the insulin receptor binding assay; 0.2 mg of placental membrane was used for the IGF-1 receptor binding assay.
  • the insulin standard and the starting concentration of the compound of the present invention were both ⁇ , and then the insulin and the compound of the present invention were diluted 3 times to obtain 7 different concentrations of the control and compound solution (100 nM 33.33 nM, respectively). , l l.llnM, 3.70nM, 1.23nM, 0.41nM, 0.13nM, 0.04nM).
  • the starting concentration of the compound is 500 nM.
  • the initial concentration of the IGF-1 standard is ⁇
  • the starting concentration of the compound of the present invention is ⁇
  • IGF-1 is diluted 3 times with the compound of the present invention to obtain 7 different Concentration of control and compound solutions (1000 nM, 333.33 nM, lll.l lnM. 37.04 nM, 12.35 nM, 4.12 nM, 1.37 nM, 0.46 nM).
  • the activity of the receptor is less than 1% of the IGF-1 standard, and the initial concentration of the compound is 5000 nM.
  • IGF-1 or insulin receptor 125 I-IGF-1 (3-10 pM) or 125 1-insulin (3 pM) and a series of 3-fold diluted polypeptides added to buffer [100 mM Hepes, H 8.0, 100 mM NaCl , 10 mM MgCl 2 , 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100], total volume 20 ( ⁇ L, cultured at 4 ° C for 48 hours. Receptor and receptor binding The polypeptide and ligand were precipitated with 0.2% ⁇ -globulin and 50 (L 25 % (w/v) PEG 8000, and the radioactivity in the precipitate was measured. The concentration of the receptor was adjusted to 15-20 when no polypeptide was added. % of the receptor binds to the ligand.
  • the membrane-bound receptors used in the receptor binding assay were derived from BHK cells that highly expressed full length insulin or IGF-1 receptor. Equal amounts of transfected BHK cells (2000-5000) were evenly distributed in each well of a 96-well plate and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum for 24 hours. Receptor binding assays were performed. The cells were washed once with binding buffer (DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8) and added with 40 (L 125 I-IGF-1 (6.5 pM) or 125 1-insulin (6.5 pM) and dissolved.
  • binding buffer DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8
  • 40 L 125 I-IGF-1 (6.5 pM) or 125 1-insulin (6.5 pM) and dissolved.
  • a 3-fold dilution of the peptide in combination with buffer Cultured at 16 ° C for 3 hours, unbound polypeptide was aspirated with aspirator and washed once with 1.2 ml of binding buffer. Cells were lysed at 500 ⁇ L 1% (w/v SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8), then measured. The number of cells is adjusted to 16-28% of the receptor binding to the ligand when no polypeptide is added.
  • IGF-1 receptor [Thr 59 ] IGF-1 is used for tyrosine iodination. 125 I-IGF-1 (50-80 Ci/g, 50 fmol), human placental membrane (0.2 mg) and serial 3-fold diluted polypeptide were added to 0.2 ml of 0.1 M Hepes buffer, pH 8, containing 120 mM sodium chloride, 5 mM potassium chloride, 0.12 mM magnesium sulphate and 0.1% bovine albumin were incubated at 20 ° C for 1 hour. Samples were filtered using Whatman GF/F filters to isolate bound and unbound polypeptide compounds. The filter was previously treated with 0.1% polyethyleneimine.
  • the culture tubes and filters were washed 4 times with 2.5 ml of cold buffer without bovine albumin. In the absence of a placental membrane, less than 5% of the polypeptide compound is attached to the filter. In the absence of polypeptide competition, the placental membrane binds approximately 38% of the ligand. Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated [Thr 59 ] IGF-1 (0.3 ⁇ ) to the culture mixture. Non-specific binding typically accounts for 5% of the total binding of the ligand to the placental membrane.
  • Insulin receptor 125 1-insulin (30 nCi), serial 3-fold diluted polypeptide and placental membrane (0.025 mg) were incubated in 0.05 ml of the above buffer for 20 hours at 20 Torr. The sample was filtered through an EHWP filter, and the culture tube and filter were washed 4 times with 2.5 ml of cold buffer containing no bovine albumin. In the absence of a placental membrane, less than 5% of the polypeptide compound is attached to the filter. Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated insulin (1 ⁇ M) to the culture mixture. Non-specific binding typically accounts for less than 1% of the total binding of the ligand to the placental membrane.
  • Specific binding percentage (binding amount - non-specific binding amount I total binding amount - non-specific binding amount) ⁇ 100.
  • the total bound amount of radiation is the total amount of radiation measured when no polypeptide is added.
  • the combined amount of radiation is the amount of radiation measured after the addition of the polypeptide.
  • IC 50 polypeptide compound using Origin software (OriginLab, Northampton, ⁇ ) is calculated.
  • the activity of the polypeptide relative to human insulin or IGF-1 standard IC 5 . Insulin or IGF-1 standard / IC 5Q polypeptide.
  • mice C57BL/6 male mice, 7-9 weeks old, averaged 20-25 g, divided into 6 groups, and fasted 4 hours before the start of the experiment. Blood glucose was measured before the start of the experiment, and blood glucose was measured at various designated time points in the future.
  • the control group was physiological saline, and the polypeptide was dissolved in physiological saline and injected subcutaneously. Observe the response of the mice throughout the experiment and record any abnormal behavior.
  • the A and B chains of IGF-1 are highly homologous to the A and B chains of insulin, and the results of NMR and X-ray crystallography also show the three-dimensional conformation of the A and B chains of IGF-1 and insulin. Almost completely coincident, but the binding of the double-stranded IGF-1 A:B to the insulin receptor is very low, but the activity at the IGF-1 receptor is too high compared to insulin. Therefore, it must be that the amino acid residues at specific sites in the IGF-1 A chain and the B chain cause the above difference.
  • IGF-1 A:B analog When B15Q is replaced by really alanine or tryptophan, the binding capacity of the IGF-1 A:B analog at the insulin receptor is comparable to that of insulin, demonstrating that the amino acid residue at position B15 is critical for binding to the insulin receptor.
  • IGF-1 has a clear selectivity for IR-A, and double-stranded compounds 11-16 also show a lower degree, but the same receptor selectivity. Substitution at position A8 can simultaneously increase the binding of the polypeptide at the three receptors, but does not alter the selectivity for the receptor.
  • Ideal insulin drugs should have high and balanced IR-A and IR-B receptor binding, and the lowest possible IGF-1 receptor binding capacity, IGF-1. Replacing the side chain negative charge of A5 and A12 with a neutral side chain restores the balance of the polypeptide on IR-A and IR-B and significantly reduces the activity at the IGF-1 receptor.
  • B9E is an important amino acid residue in the growth-promoting effect of IGF-1, and it also causes IGF-1 to cause cancer. Replacing glutamate with histidine, arginine, glutamine or phenylalanine still maintains comparable insulin receptor activity.
  • a lysine residue at positions A22 and A23 has no negative effect on insulin receptor binding, but the lysine side chain amino group can provide a site for an acylation reaction.
  • IGF-1 3.66 0.7 100 1-18 333 197 0.9
  • IR-A and IR-B are insulin receptor subtypes A and B; IGF-1 R insulin-like growth factor-1 receptor; IGF-1A: IGF-1B indicates IGF-1A chain and IGF-1B chain double Chain structure.
  • Natural IGF-1 is a single-chain polypeptide that binds less than 5% of insulin to the insulin receptor. Since the amino acid substitution at position B15 allows the biological activity of the IGF-1 based double-stranded compound to reach the level of insulin, the same substitution can also increase the activity of the single-chain compound based on IGF-1. II-1 shows that only the B15 amino acid residue is changed, and the D domain, which is generally considered to have no significant effect on biological activity, is removed, and the insulin receptor activity of the compound is increased by more than 10 times.
  • C2Tyr is a prominent amino acid residue in the IGF-1 crystal structure and is shown to be the key to binding to the GF-1 receptor.
  • the insulin receptor activity is substantially retained, but the IGF-1 receptor activity is greatly reduced, which is the expected effect.
  • the results show that the length of the C peptide does not necessarily require 12 amino acid residues. Multiple amino acid residues of the original IGF-1 C can be deleted or replaced. In particular, the removal of three amino acid residues PQT and four residues of APQT at the C-terminus of IGF-1 not only had no negative effect on the activity of the insulin receptor, but also reduced the activity of IGF-1 receptor to the level of insulin.
  • the 5 amino acid residues at the C-terminus of the IGF-1 B chain may have one, two One, three, four or all are missing without reducing insulin receptor binding.
  • the IGF-1 C peptide is only one of a wide variety of ligation sequences. These data indicate that proper C-chain length, flexible C-strand conformation, and amino acid substitution at specific sites are important factors in increasing insulin receptor activity and reducing IGF-1 receptor binding.
  • PEGylation and fatty acid acylation are common methods for extending the duration of action of a polypeptide in vivo, but pegylation and fatty acid acylation generally greatly reduce biological activity. Therefore, it is sought to introduce a new acylation site having an amino group such as lysine, and the acylation product has sufficient biological activity for the development of a long-acting polypeptide.
  • Figure 1 shows the mean change in blood glucose over time after subcutaneous injection of normal saline, human insulin, and three different doses of III-1 in mice.
  • the polyethylene glycol modified polypeptide has a significantly longer duration of action than human insulin, which can reach 24 hours or even longer.
  • the polyethylene glycol-modified polypeptide has a mild hypoglycemic effect, and even if it is increased to a three-fold dose, it does not cause a hypoglycemia. Therefore, ⁇ -1 is superior to human insulin in controlling blood sugar and safety.
  • Figure 2 shows the mean change in blood glucose over time after subcutaneous injection of normal saline, human insulin, and ⁇ -12 (both 80 nanomoles/kg).
  • the onset time of sputum-12 was comparable to that of human insulin, but it still had a significant blood sugar-suppressing effect at 8 hours after administration. At this time, insulin had lost its blood sugar-suppressing effect.
  • Human insulin showed a typical V-shaped blood glucose lowering curve in the experiment.
  • the disadvantage of this blood glucose lowering curve is that the initial blood sugar drops too fast, which is likely to cause hypoglycemia, and later it is impossible to control blood sugar.
  • ⁇ -12 shows an L-type blood glucose lowering curve, blood sugar control is smooth and long-lasting, and its effect is better than human insulin.
  • Fig. 3 is a graph showing changes in blood glucose with time after subcutaneous injection of physiological saline, insulin detemir and the indole-7 compound of the present invention.
  • Detemir is one of the only two long-acting insulin analog drugs on the international market. It has a half-life of 14 hours in human body and is usually used once a day. III-7 can only achieve a longer duration of action and a more stable hypoglycemic effect with a dose of 1/3 of insulin.

Abstract

Provided in the present invention are a compound and composition having hypoglycemic effect, and an application of the compound or composition in treating diabetes and hyperglycemia. Also provided in the present invention is a method for treating diabetes and hyperglycemia, comprising administration to patients in need of the compound or composition of the present invention. Compared to existing insulin and other analogues, the compound of the present invention has great aqueous solubility, long in vivo cycle, high activity for combining with an insulin receptor, significantly reduced toxic effect on individuals, and is easy to prepare.

Description

具有 p条血糖作用的化合物、 组合物及其用途 技术领域  Compound, composition and use thereof having p-glycemic effect
本发明属于生物制药领域， 具体涉及具有降血糖作用的化合物、 组合物及它们的用途。 背景技术  The invention belongs to the field of biopharmaceuticals, and particularly relates to compounds, compositions and uses thereof having hypoglycemic effects. Background technique
胰岛素作为治疗糖尿病的特效和首选药物， 到目前为止已经历了三代的历史演变： 第一 代产品是从猪或牛等动物胰脏提取而来。 由于异源性过敏反应， 该类产品疗效较差。 第二代 产品为重组人胰岛素， 是从人类细胞中获取胰岛素基因， 然后将其插入酵母菌或大肠杆菌中 进行培养， 通过复杂的现代化生物基因技术生产而成。 第三代为胰岛素类似物， 是通过对人 的胰岛素进行结构修改而获得， 包括速效胰岛素和长效胰岛素。  As a special and preferred drug for the treatment of diabetes, insulin has undergone three generations of historical evolution: The first generation of products was extracted from the pancreas of animals such as pigs or cattle. Due to heterogeneous allergic reactions, these products are less effective. The second generation of products is recombinant human insulin, which is obtained by extracting insulin genes from human cells, and then inserting them into yeast or E. coli for cultivation through complex modern biological gene technology. The third generation is an insulin analogue obtained by structural modification of human insulin, including fast-acting insulin and long-acting insulin.
本世纪初美国的 NHANS研究、 欧洲的 CODE研究等大型临床研究表明， 对糖尿病及其 并发症越来越严格的控制， 使得糖尿病患者的高血压、 高血脂、 总胆固醇达标率越来越高， 而最根本的血糖达标率却不升反降， 主要问题在于现有的胰岛素药物仍然不能够很好地模拟 生理性胰岛素分泌模式。 因此， 开发在疗效、 抗免疫源、 治疗达标、 模拟生理分泌等方面都 有大幅度提高的新型胰岛素类似物将是改善糖尿病治疗效果的主要解决方法之一。  Large-scale clinical studies such as the NHANS study in the United States and the CODE study in Europe at the beginning of this century show that the increasingly strict control of diabetes and its complications has led to higher and higher compliance rates for hypertension, hyperlipidemia and total cholesterol in diabetic patients. The most fundamental rate of blood glucose compliance is not rising or falling. The main problem is that existing insulin drugs still do not mimic physiological insulin secretion patterns. Therefore, the development of new insulin analogues with significant improvement in curative effect, anti-immune source, therapeutic compliance, and simulated physiological secretion will be one of the main solutions to improve the therapeutic effect of diabetes.
美国糖尿病学会（ADA ) 与欧洲糖尿病学会（EASD )指南均建议， 在生活方式干预和 口服降糖药物治疗后， 如果糖尿病患者的血糖控制仍然不理想， 应尽早开始胰岛素治疗， 而 且首选基础胰岛素与口服降糖药合用。 若此疗法仍不能控制血糖， 建议在此基础上在就餐时 加用速效胰岛素。  Both the American Diabetes Association (ADA) and the European Diabetes Association (EASD) guidelines recommend that after lifestyle interventions and oral hypoglycemic agents, if glycemic control in diabetic patients remains unsatisfactory, insulin therapy should begin as soon as possible, and basal insulin is preferred. Oral hypoglycemic agents are used together. If this therapy still does not control blood sugar, it is recommended to add fast-acting insulin to the meal on this basis.
基础胰岛素用于维持空腹时的正常血糖分泌。很多代谢研究发现在正餐之间和夜晚时间， 保持基本胰岛素水平可以减少甘油三磷酯分解， 抑制肝脏输出葡萄糖， 使空腹血糖保持稳定， 从而降低整体血糖水平。 理想的基础胰岛素， 如长效胰岛素类似物， 应该能够模拟生理性胰 岛素分泌模式， 避免发生低血糖尤其是夜间低血糖， 不增加体重。  Basal insulin is used to maintain normal blood glucose secretion on an empty stomach. Many metabolic studies have found that maintaining a basic insulin level between meals and at night can reduce the breakdown of triglyceride, inhibit the liver's output of glucose, and stabilize the fasting blood glucose, thereby reducing overall blood sugar levels. Ideal basal insulins, such as long-acting insulin analogues, should be able to mimic physiological insulin secretion patterns, avoid hypoglycemia, especially nocturnal hypoglycemia, and do not increase body weight.
目前使用的最多的中长效胰岛素可以分为三大种类。 第一类是人胰岛素与锌离子或^出 鱼精蛋白形成的晶体的悬浮液， 例如 NPH胰岛素， lente胰岛素等。 这些胰岛素制剂的疗效 不稳定， 正逐渐被长效胰岛素类似物取代。  The most long-acting insulins currently used can be divided into three major categories. The first type is a suspension of crystals formed by human insulin with zinc ions or protamine, such as NPH insulin, lente insulin, and the like. These insulin preparations are unstable and are being replaced by long-acting insulin analogues.
第二种是地特胰岛素 （detemir )。 它是十四烷酸连接至 B29赖氨酸的胰岛素类似物。 地 特胰岛素在注射后吸收緩慢。注射处的消失时间 T50%大约是 10小时。 它与血液中的白蛋白通 过 Β29位的脂肪酸结合， 然后从复合体中緩慢解离。 双六聚体化（Dihexamerization ), 六聚 体和二聚体与白蛋白结合都延长了地特胰岛素在注射处的留存时间。 地特胰岛素进入血液循 环后， 与白蛋白结合， 进一步延长了体内停留时间。 The second is detemir. It is an insulin analog in which myristic acid is linked to B29 lysine. Detemir is slowly absorbed after injection. The disappearance time T 50% at the injection site is approximately 10 hours. It binds to albumin in the blood through the fatty acid at position 29 and then slowly dissociates from the complex. Dihexamerization, hexamer and dimer binding to albumin prolongs the retention time of insulin detemir at the injection site. After the insulin enters the blood circulation, it binds to albumin, further prolonging the residence time in the body.
第三种是甘精胰岛素。 药物在 pH 3.0时溶解在制剂中， 而注射后当 pH上升到大约 7.4 时结晶。 注射位点的緩慢分解带来了延迟的效果。 但是吸收性质和药物动力学在人群中和个 体内变化都 4艮大。  The third is insulin glargine. The drug dissolves in the formulation at pH 3.0 and crystallizes when the pH rises to about 7.4 after injection. The slow decomposition of the injection site brings about a delayed effect. However, the absorption properties and pharmacokinetics vary widely in the population and in the body.
甘精胰岛素和地特胰岛素是目前市场上仅有的两种长效胰岛素类似物， 最长作用时间不 超过 24小时。 甘精胰岛素在胰岛素样生长因子 -1受体 (IGF-1R)上活性远高于天然人胰岛素。 因为胰岛素样生长因子 -1受体与多种癌症的发生密切相关， 所以一直有争议长期使用甘精胰 岛素是否会增加病人患癌症的风险。 地特胰岛素在人体内的生物活性大约是天然人胰岛素的 20%, 因此它的使用剂量是常规胰岛素剂量的 5倍， 这显著增加了生产和使用成本。 Insulin glargine and insulin detemir are the only two long-acting insulin analogues on the market, with a maximum duration of action of no more than 24 hours. Insulin glargine is much more active on the insulin-like growth factor-1 receptor (IGF-1R) than native human insulin. Because insulin-like growth factor-1 receptor is closely related to the occurrence of various cancers, it has been controversial for long-term use of ganjing pancreas Whether islands increase the risk of cancer in patients. The biological activity of insulin detemir in humans is about 20% of that of natural human insulin, so it is used at a dose five times that of conventional insulin, which significantly increases production and use costs.
重组人胰岛素难以满足餐时胰岛素需求。 人胰岛素分子通常形成六聚体结构， 皮下注射 后逐渐解聚成二聚体， 进一步解离为单体才能透过毛细血管进入循环， 发挥降糖作用。 由于 存在解聚、 吸收过程， 重组人胰岛素在皮下注射后约 30分钟才起效， 达峰时间长， 作用持续 大约 6 - 7小时。 而且因个体差异， 注射相同剂量人胰岛素后最终进入循环的量也会有明显差 异。  Recombinant human insulin is difficult to meet the insulin requirements of the meal. The human insulin molecule usually forms a hexamer structure, which is gradually depolymerized into a dimer after subcutaneous injection, and further dissociates into a monomer to enter the circulation through the capillaries, thereby exerting a hypoglycemic effect. Due to the depolymerization and absorption process, recombinant human insulin takes about 30 minutes after subcutaneous injection, and the peak time is long, and the effect lasts for about 6-7 hours. Moreover, due to individual differences, there is a significant difference in the amount of final influx after injection of the same dose of human insulin.
人胰岛素的局限性带来两个不利后果。 一方面， 为保证降低餐后血糖， 糖尿病人必须在 餐前 30分钟皮下注射人胰岛素， 进餐时间提前易导致血糖控制不佳， 延后则易引起低血糖。 另一方面， 由于人胰岛素皮下注射后存在解聚和个体吸收差异， 最终进入循环的胰岛素量无 法精确预估， 容易造成胰岛素过量或不足。  The limitations of human insulin bring two negative consequences. On the one hand, in order to ensure the reduction of postprandial blood sugar, diabetics must inject human insulin subcutaneously 30 minutes before the meal. The early meal time is likely to lead to poor glycemic control, and delayed hypoglycemia. On the other hand, due to the difference in depolymerization and individual absorption after subcutaneous injection of human insulin, the amount of insulin that eventually enters the circulation cannot be accurately predicted, and it is easy to cause excess or deficiency of insulin.
速效胰岛素类似物 （如门冬胰岛素、 赖脯胰岛素等） 的研发正是为了弥补人胰岛素的不 足。 速效胰岛素类似物吸收快， 达峰时间短， 峰值更高， 峰浓度持续 1 ~ 3小时， 作用持续时 间为 3 - 5小时， 明显优于人胰岛素。但门冬胰岛素和赖脯胰岛素的起效时间大约是 20分钟， 对于糖尿病人来说仍然不够方便， 仍然有很大改进余地。  Rapid-acting insulin analogues (such as insulin aspart, insulin lispro, etc.) were developed to compensate for the lack of insulin in humans. Fast-acting insulin analogues absorb quickly, have a short peak time, have a higher peak value, and have a peak concentration of 1 to 3 hours. The duration of action is 3 - 5 hours, which is significantly better than human insulin. However, the onset time of insulin aspart and insulin lispro is about 20 minutes, which is still not convenient for diabetics, and there is still much room for improvement.
因此， 开发新型胰岛素类似物， 提高生物活性和生物利用度、 延长作用时间 （长效胰岛 素）或缩短起效时间 （速效胰岛素）、 文善水溶解度、 降低了用药时的个体差异、 更有效地防 止低血糖风险、 增加稳定性是胰岛素类药物开发的重要方向。 发明内容  Therefore, the development of new insulin analogues, improve biological activity and bioavailability, prolong the duration of action (long-acting insulin) or shorten the onset of action (rapid insulin), Wenshan water solubility, reduce individual differences in medication, more effectively Preventing the risk of hypoglycemia and increasing stability are important directions for the development of insulin drugs. Summary of the invention
本发明的目的是提供具有胰岛素活性、 能够与胰岛素受体高度结合、 具有降血糖作用的 化合物、 药物学可接受的组合物及其在降血糖中的应用。  It is an object of the present invention to provide a compound having a insulin activity, capable of binding to an insulin receptor, having a hypoglycemic effect, a pharmaceutically acceptable composition and its use in hypoglycemia.
本发明的第一个目的是提供一种具有降血糖作用的化合物，所述化合物包括 A链和 B链， 其中，  A first object of the present invention is to provide a compound having a hypoglycemic action, which comprises an A chain and a B chain, wherein
A链的氨基酸序列为： X_1 GIVDX5C[3]C[4]X8X9X10Ct5]X12LRRLEX18YC[6]X21X22, B链氨基酸序列为：The amino acid sequence of the A chain is: X_ 1 GIVDX 5 C [3] C [ 4] X 8 X 9 X 10 C t5] X 12 LRRLEX 18 YC [6] X 21 X 22 , the B chain amino acid sequence is:
23-26X27LC [！] GAX32LVDALX38X3 VC[2]GDX44GFX47X48X4 50X5lX52 53 ' 其中 ,23-26X27LC [! ] GAX3 2 LVDALX 38 X 3 VC[ 2 ]GDX44GFX4 7 X 4 8X4 5 0 X 5 lX 5 2 53 '
i是赖氨酸、 精氨酸或缺失； 是赖氨酸、 精氨酸或缺失； X5是谷氨酸、 天冬酰胺、 谷氨酰胺或丝氨酸； X8是组氨酸、 精氨酸、 苯丙氨酸或苏氨酸； X9是精氨酸或丝氨酸； X10 是丝氨酸或异亮氨酸； X12是天冬氨酸、 丝氨酸、 谷氨跣胺或天冬酰胺； X】8是天冬酰胺、 曱 硫氨酸或苏氨酸； X21是天冬酰胺、 丙氨酸或甘氨酸； X22是赖氨酸、 精氨酸-赖氨酸二肽或缺 失； X23_26是苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽， 或甘氨酸 -脯氨酸-谷氨酸、 缬氨酸- 天冬酰胺-谷氨酰胺三肽， 或脯氨酸-谷氨酸、 天冬酰胺-谷氨酰胺二肽， 或谷氨酸、 谷氨酰胺、 赖氨酸或精氨酸， 或是以赖氨酸或精氨酸取代上述二、 三、 四肽序列中任何一个氨基酸残基 后的序列， 或缺失； X27是组氨酸或苏氨酸； X32是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯 丙氨酸； X38是苯丙氨酸或色氨酸； X39是笨丙氨酸或色氨酸； 4是精氨酸、 谷氨酸、 天冬 氨酸或丙氨酸； 7是苯丙氨酸、 酪氨酸或组氨酸； 8是 -NH2、 dA-NH2、 笨丙氨酸或酪氨酸 或缺失； X49是天冬酰胺、 天冬氨酸、 谷氨酸、 苏氨酸或缺失； X5G是赖氨酸、 脯氨酸、 精氨 酸、 天冬氨酸、 谷氨酸或缺失； X51是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X52是苏氨酸或缺失； X53是谷氨酸、 天冬氨酸、 谷氨酸-谷氨酸、 天冬氨酸-天冬氨酸二肽或 缺失； i is lysine, arginine or deletion; is lysine, arginine or deletion; X 5 is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine , phenylalanine or threonine; X 9 is arginine or serine; X 10 is serine or isoleucine; X 12 is aspartic acid, serine, glutamine or asparagine; X] 8 is asparagine, methionine or threonine; X 21 is asparagine, alanine or glycine; X 22 is lysine, arginine-lysine dipeptide or deletion; X 2 3_26 Is a phenylalanine-valine-asparagine-glutamine tetrapeptide, or a glycine-valine-glutamate, a proline-asparagine-glutamine tripeptide, or a proline- Glutamate, asparagine-glutamine dipeptide, or glutamic acid, glutamine, lysine or arginine, or lysine or arginine to replace the above two, three, tetrapeptide sequences a sequence after any one of the amino acid residues, or a deletion; X 27 is histidine or threonine; X 32 is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 3 8 is phenylalanine or tryptophan; X 39 is stupid alanine or tryptophan; 4 is arginine, glutamic acid, aspartic acid or alanine; 7 is phenylalanine, cheese Or histidine; 8 is -NH 2 , dA-NH 2 , stupid alanine or tyrosine or deletion; X49 is asparagine, aspartic acid, glutamic acid, threonine or deletion; X 5G is lysine, valine, arginine, aspartic acid, glutamic acid or a deletion; X 51 is valine, lysine, arginine, aspartic acid, glutamic acid or Deletion; X 52 is threonine or deletion; X 53 is glutamic acid, aspartic acid, glutamate-glutamate, aspartic acid-aspartate dipeptide or Missing
所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物中通过 6个半胱氨酸形成 3对 二疏键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫 键的具体位置是： C ]和 C[4] 形成二^ ^键， C[2] 和 C[6] 形成二硫键， C[3] 和 C[5]形成二硫键。 In the compound, [1]-[6] represents the number of a cysteine; in the compound, a three-paired double bond is formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains. Sulfur bond connection, a pair of intrachain disulfide bonds exist in the A chain, and the specific positions of the three pairs of disulfide bonds are: C ] and C [4] form a ^^ bond, and C[ 2] and C [6] form two The sulfur bond, C [3] and C [5] form a disulfide bond.
在第二个方面， 本发明提供一种单链的、 能够与胰岛素受体结合、 具有降血糖作用的化 合物， 所述化合物的氨基酸序列结构为：  In a second aspect, the present invention provides a single-chain compound capable of binding to an insulin receptor and having hypoglycemic action, the amino acid sequence structure of which is:
XioiaLC [！] GAX1o1bL DALXlolcX1oid C[2]GDRGFX1oie io2Xio3Xio4X[05Xio6-CL-GIVDQC[3] CwX^RSCwSLRRLENYCwX X, 其中， XioiaLC [! ] GAX 1 o 1 bL DALX lolc X 1 oid C[ 2 ]GDRGFX 1 oi e io2Xio3Xio4X[05Xio6-CL-GIVDQC[ 3 ] CwX^RSCwSLRRLENYCwX X, where
Xioia是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 甘氨酸-脯氨酸-谷氨酸 -组氨酸四肽或者苯兩氨酸 -缬氨酸-天冬酰胺 -谷氨酰胺-组氨酸五肽， 或是以赖氨酸或精氨酸取代上述四肽或五肽中的甘 氨酸 -脯氨酸-谷氨酸或苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺中任一个氨基酸残基后的序列； Xioib是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨酸； X101c是苯丙氨酸或色氨酸； X101d 是苯丙氨酸或色氨酸； Xl()le是苯丙氨酸、 酪氨酸或组氨酸； X102是苯丙氨酸或缺失； X1M是 天冬酰胺或缺失； X1M是赖氨酸、 脯氨酸或缺失； X1Q5是脯氨酸、 赖氨酸或缺失； X1()6是苏 氨酸或缺失； X1()7是苯丙氨酸、 精氨酸或组氨酸； X1Q8是丙氨酸、 甘氨酸或天冬酰胺； X109 是赖氨酸、 精氨酸-赖氨酸二肽或缺失； CL是本文中定义的连接片段。 Xioia is a glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine tetrapeptide or phenyldiacyl-proline-asparagine-glutamine-group a pentapeptide, or a glycine-valine-glutamic acid or phenylalanine-valine-asparagine-glutamine in the above tetrapeptide or pentapeptide by lysine or arginine Sequence after any one of the amino acid residues; Xioib is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 101c is phenylalanine or tryptophan; X 101d is phenylpropanoid leucine or tryptophan; X l () le is phenylalanine, tyrosine or histidine; X 102 is a phenylalanine or deletion; X 1M asparagine or deleted; X 1M is lysyl Acid, proline or deletion; X 1Q5 is valine, lysine or deletion; X 1 () 6 is threonine or deletion; X 1 () 7 is phenylalanine, arginine or histamine Acid; X 1Q8 is alanine, glycine or asparagine; X 109 is a lysine, arginine-lysine dipeptide or a deletion; CL is a linking fragment as defined herein.
在第三个方面， 本发明进一步提供一种具有降血糖作用的、 在多肽基础上进行修饰的化 合物， 以进一步提高所述化合物的溶解度、 稳定性、 体内循环作用时间等。 所述修饰是将修 饰侧链连接至本发明的双链化合物的 B链的 N-末端氨基酸残基的 α-氨基或单链化合物的 Ν- 末端氨基酸残基的 α-氨基，或者连接至本发明的双链或单链化合物中存在的赖氨酸的 ε-氨基。  In a third aspect, the present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further increase the solubility, stability, in vivo circulation time, and the like of the compound. The modification is the attachment of the modified side chain to the α-amino group of the N-terminal amino acid residue of the B chain of the double-stranded compound of the present invention or the α-amino group of the Ν-terminal amino acid residue of the single-stranded compound, or The ε-amino group of lysine present in the double-stranded or single-stranded compound of the invention.
在一种实施方式中， 所述化合物包括 Α链和 Β链， 其中，  In one embodiment, the compound comprises an oxime chain and an oxime chain, wherein
A链的氨基酸序列为：  The amino acid sequence of the A chain is:
X399X400GIVDX405C[3]C[4]X408X409 410C[5]X412LX414X415LX417X418YC[6]X421X422) X399X400GIVDX4 0 5C[ 3 ]C[4]X408X409 410C[5]X 4 1 2 LX4 14 X41 5 LX4 17 X 41 8YC[ 6 ]X4 21 X 422 )
B链的氨基酸序列为：  The amino acid sequence of the B chain is:
X423-426 427LC[1]GAHLVDALX438X439 C[2]GDRGFX447X448X449X450X45lX452X453X454X45 ; 所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物通过 6个半胱氨酸形成 3对二 硫键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： C[,] 和 C[4] 形成二硫键， C[2] 和 C[6] 形成二疏键， C[3] 和 C[5]形成二硫鍵。 其中， X 42 3 -42 6 427LC[ 1 ]GAHLVDALX 438 X4 3 9 C[ 2 ]GDRGFX447X448X449X450X45lX452X453X454X45 ; of the compounds, [1]-[6] represent the number of cysteine; the compound passes 6 cysteine The acid forms a 3-pair disulfide bond, wherein the A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain. The specific position of the three pairs of disulfide bonds is: C[,] It forms a disulfide bond with C [4] , C[ 2 ] and C [6] form a double bond, and C [3 ] and C [5] form a disulfide bond. among them,
X399是赖氨酸、 精氨酸或缺失； X4CG是赖氨酸、 精氨酸或缺失； 05是谷氨酸、 天冬酰 胺、 谷氨酰胺或丝氨酸； 08是组氨酸、 精氨酸、 苯丙氨酸、 苏氨酸或通式（I )结构； 09 是精氨酸、 丝氨酸或通式（I )结构； 是丝氨酸、 异亮氨酸或通式（I )结构； 12是天冬 氨酸、 丝氨酸、 谷氨酰胺、 天冬酰胺或通式（I )结构； 14是精氨酸或通式（I )结构； 15 是精氨酸或通式（I ) 结构； 17是谷氨酸或通式（I )结构； 18是天冬酰胺或通式（I )结 构； 21是丙氨酸、 甘氨酸或天冬酰胺； 22是赖氨酸、 精氨酸-赖氨酸二肽或缺失， 或为通 式（I )结构； 当 22为二肽时， 其中一个氨基酸为通式（I )结构； 23_426是甘氨酸-脯氨酸X 3 99 is lysine, arginine or deletion; X4CG is lysine, arginine or deletion; 05 is glutamic acid, asparagine, glutamine or serine; 0 8 is histidine, fine leucine, phenylalanine, threonine, or formula (I) structure; structure 09 is arginine, serine or formula (I); is a serine, isoleucine or structural formula (I); 12 is It is aspartic acid, serine, glutamine, asparagine or formula (I) structure; 14 is arginine or formula (I) structure; 15 is arginine or formula (I) structure; 17 Is glutamic acid or a structure of formula (I); 18 is an asparagine or a structure of formula (I); 21 is alanine, glycine or asparagine; 22 is lysine, arginine-lysine a dipeptide or a deletion, or a structure of the formula (I); when 22 is a dipeptide, one of the amino acids is of the formula (I); 23 _426 is a glycine-valine
-谷氨酸三肽、 UL-甘氨酸 -脯氨酸-谷氨酸、 苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽或 UL-笨 丙氨酸 -缬氨酸-天冬酰胺 -谷氨酰胺； 27是组氨酸或苏氨酸； 38是苯丙氨酸或色氨酸； 39 是苯丙氨酸或色氨酸； 47是苯丙氨酸、 组氨酸或酪氨酸； 48是 -NH2、 苯丙氨酸、 酪氨酸 或缺失； 49是天冬酰胺、 苏氨酸、 谷氨酸、 天冬氨酸或缺失； 50是赖氨酸、 精氨酸、 谷 氨酸、 天冬氨酸、 脯氨酸或缺失； 51是脯氨酸、 赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸或缺 失， 或为通式（I)结构； 52是苏氨酸、 赖氨酸或缺失， 或为通式（I)结构； 53是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I)结构； 54是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为 通式（I)结构； 55是赖氨酸或缺失， 或为通式（I) 结构； UL和通式（I)结构如本发明下 文所定义的。 - glutamic acid tripeptide, U L - glycine-valine-glutamic acid, phenylalanine-valine-asparagine-glutamine tetrapeptide or U L - stupid alanine-valine - asparagine-glutamine; 27 is histidine or threonine; 38 is phenylalanine or tryptophan; 39 is phenylalanine or tryptophan; 47 is phenylalanine, histamine Acid or tyrosine; 48 is -NH 2 , phenylalanine, tyrosine or deletion; 49 is asparagine, threonine, glutamic acid, aspartic acid or deletion; 50 is lysine, Arginine, valley Acid, aspartic acid, proline, or deleted; 51 is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or the general formula (I) structure; 52 Threonine, lysine or deletion, or a structure of formula (I); 53 is glutamic acid, glycine, lysine or deletion, or is a structure of formula (I); 54 is glutamic acid, glycine, Lysine or deletion, or a structure of the formula (I); 55 is a lysine or a deletion, or a structure of the formula (I); U L and the structure of the formula (I) are as defined hereinafter.
在另一种实施方式中， 所述化合物为单链结构， 氨基酸序列结构为：  In another embodiment, the compound is a single-stranded structure and the amino acid sequence structure is:
X201aLC [！] GAX201 bLVDALX201 CX201 dVC[2]GDRGFX201 eX202 2o3X204 205 206GX207X2o7a 208XX 201a LC [! ] GAX 20 1 bLVDALX 20 1 C X 20 1 dVC[ 2 ]GDRGFX 20 1 eX 202 2 o 3 X 20 4 2 0 5 206GX 207 X 2 o 7 a 2 0 8X
209X210X2uX212 213X21 215 216GIVDQC[3]C[4]X2i7RSC[5]X2i8LX2i9X220LX22iX222YC[6]X223X224 ' 其中， 209X210X2uX212 213X21 215 216GIVDQC[3]C[ 4 ]X 2 i7RSC[5]X 2 i8LX2i9X 220 LX 22 iX22 2 YC[6]X223X224 '
X201a是甘氨酸-脯氨酸 -谷氨酸-苏氨睃、甘氨酸-脯氨酸 -谷氨酸-組氨酸、苯丙氨酸-缬氨酸X201 a is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine, phenylalanine-valine
-天冬酰胺-谷氨酰胺-组氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸- 组氨酸或 UL-苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸； X2Qib是组氨酸、 谷氨酸、 谷氨酰 胺、 精氨酸或苯丙氨酸； X20k是苯丙氨酸或色氨酸； X201d是苯丙氨酸或色氨酸； X201e是苯丙 氨酸、 酪氨酸或组氨酸； X2o2是苯丙氨酸、 酪氨酸或缺失； Χ 3是天冬酰胺、 苏氨酸、 天冬 氨酸、 谷氨酸或缺失； X2Q4是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X2Q5是 脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失或通式（I)结构； X2。6是苏氨酸、 赖氨 酸或缺失或通式（I) 结构； X2Q7是丝氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X207a 是丝氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X2。8是丝氨酸、 通式（I)结构或缺失； X209是丝氨酸、 通式（I)结构或缺失； Χ21()是丝氨酸、 通式（I)结构或缺失； X211是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X212是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或 缺失； X213是丙氨酸、 脯氨酸、 精氨酸、 甘氨酸、 通式（I)结构或缺失； x214是脯氨酸、 谷 氨酰胺、 甘氨酸、 通式（I)结构或缺失； X215是谷氨酰胺、 苏氨酸、 甘氨酸、 通式（I)结构 或缺失； X216是苏氨酸、 精氨酸、 赖氨酸、 通式（I)结构或缺失； x217是苯丙氨酸、 精氨酸、 组氨酸或通式（I)结构； X218是天冬氨酸、 丝氨酸、 谷氨酰胺、 天冬酰胺或通式（I)结构； X2i9是精氨酸或通式（I)结构； x22Q是精氨酸或通式（I)结构； x221是谷氨酸或通式（I) 结构； X222是天冬酰胺或通式（I)结构； X223是丙氨酸、 甘氨酸或天冬酰胺； X224是赖氨酸、 精氨酸-赖氨酸二肽或缺失， 或为通式（I) 结构； 当 22为二肽时， 其中一个氨基酸为通式 (I)结构； UL和通式（I)结构如本发明下文中所定义的。 - asparagine-glutamine-histidine, UL-glycine-valine-glutamate-threonine, UL-glycine-valine-glutamate-histidine or UL-phenylalanine Acid-valine-asparagine-glutamine-histidine; X 2Q ib is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 2 0k is phenylalanine Acid or tryptophan; X 201 d is phenylalanine or tryptophan; X 2 01e is phenylalanine, tyrosine or histidine; X 2 o 2 is phenylalanine, tyrosine or Deletion; Χ 3 is asparagine, threonine, aspartic acid, glutamic acid or deletion; X 2Q4 is valine, lysine, arginine, aspartic acid, glutamic acid or deletion; X 2Q5 is valine, lysine, arginine, aspartic acid, glutamic acid or a deletion or a structure of formula (I); X 2 . 6 is threonine, lysine or deletion or a structure of formula (I); X 2 Q 7 is serine, alanine, glycine, structure or deletion of formula (I); X 207a is serine, alanine, Glycine, structure or deletion of formula (I); X 2 . 8 is serine, structure or deletion of formula (I); X 209 is serine, structure or deletion of formula (I); Χ 21 () is serine, structure or deletion of formula (I); X 211 is arginine, Alanine, glycine, structure or deletion of formula (I); X 212 is arginine, alanine, glycine, structure or deletion of formula (I); X 213 is alanine, valine, spermine Acid, glycine, structure or deletion of formula (I); x 214 is valine, glutamine, glycine, structure or deletion of formula (I); X 215 is glutamine, threonine, glycine, general formula (I) structure or deletion; X 216 is threonine, arginine, lysine, structure or deletion of formula (I); x 217 is phenylalanine, arginine, histidine or formula ( I) structure; X 218 is aspartic acid, serine, glutamine, asparagine or the structure of formula (I); X 2 i9 is arginine or the structure of formula (I); x 22Q is arginine or the general formula (I) structure; x 221 is glutamic acid or formula (I) structure; X 222 is asparagine or formula (I) structure; X 223 is alanine, glycine, or asparagine Amine; X 2 24 is lysine, arginine - lysine dipeptide or deleted, or the general formula (I) structure; 22 when the dipeptide, wherein one amino acid of the general formula (I) structure; the U- L and the structure of the formula (I) are as defined hereinafter in the present invention.
本发明的第四个方面是提供一种药物组合物， 由本发明的具有降糖作用的化合物和药物 学可接受的载体混合而成， 混合比例可以是大约 90/10%、 大约 80/20%、 大约 70/30%、 大约 60/40%、 大约 50/50%、 大约 40/60%、 大约 30/70%、 大约 20/80%或者大约 10/90%; 优选地， 所述药物组合物进一步包括速效胰岛素类似物； 所述速效胰岛素类似物可以是 AspB28人胰岛 素、 LysB28Pro B29人胰岛素或 LysB3GluB29人胰岛素。 According to a fourth aspect of the present invention, there is provided a pharmaceutical composition which is obtained by mixing a hypoglycemic compound of the present invention and a pharmaceutically acceptable carrier, and the mixing ratio may be about 90/10%, about 80/20%. , about 70/30%, about 60/40%, about 50/50%, about 40/60%, about 30/70%, about 20/80%, or about 10/90%; preferably, the pharmaceutical combination Further comprising a fast-acting insulin analogue; the fast-acting insulin analog may be Asp B28 human insulin, Lys B28 P ro B29 human insulin or Lys B3 Glu B29 human insulin.
本发明的第五个方面是提供本发明的化合物在制备治疗糖尿病或高血糖症等药物中的应 用。  A fifth aspect of the invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of diabetes or hyperglycemia.
本发明的第六个方面是提供一种治疗糖尿病或高血糖症等的方法， 包括对需要的病患施 用本发明的化合物或组合物。  A sixth aspect of the invention provides a method of treating diabetes or hyperglycemia or the like comprising administering a compound or composition of the invention to a patient in need thereof.
与现有的胰岛素及其类似物相比， 本发明的化合物水溶性好， 具有结合胰岛素受体的高 活性， 对个体的毒性作用低， 制备容易。 修饰后的化合物在体内的循环时间明显延长。 附图说明 Compared with the existing insulin and its analogs, the compound of the present invention has good water solubility, high activity in binding to the insulin receptor, low toxicity to an individual, and easy preparation. The cycle time of the modified compound in the body is significantly prolonged. DRAWINGS
图 1是小鼠皮下注射生理盐水、人胰岛素和三种不同剂量的本发明的 III-1化合物后血糖随 时间变化值；  Figure 1 is a graph showing changes in blood glucose over time following subcutaneous injection of physiological saline, human insulin, and three different doses of a compound of the present invention;
图 2是小鼠皮下注射生理盐水、 人胰岛素和本发明的 III - 12化合物后血糖随时间变化值； 图 3是小鼠皮下注射生理盐水、 人胰岛素和本发明的 ΠΙ -7化合物后血糖随时间变化值。 具体实施方式  Figure 2 is a graph showing changes in blood glucose over time after subcutaneous injection of physiological saline, human insulin, and a compound of the present invention, III; 12; Figure 3 is a hypoglycemia of a mouse subcutaneously injected with physiological saline, human insulin, and the ΠΙ-7 compound of the present invention. Time change value. Detailed ways
定 术语  Terminology
除非另外说明， 下述定义适用于本发明全文。 未定义的术语可以根据行业内约定俗成的 定义理解。  Unless otherwise stated, the following definitions apply to the full text of the present invention. Undefined terms can be understood in accordance with established conventions in the industry.
"氨基酸"指任何同时包含氨基和羧基官能团的分子， ot-氨基酸的氨基和羧基连接在同一 个碳原子上 （α碳)。 α碳可以有 1-2个有机取代基。 氨基酸包含 L和 D同分异构体和消旋混 合物。 如无特别说明， 本发明中多肽序列中的氨基酸残基都是 L同分异构体即 L-氨基酸， D- 氨基酸在氨基酸名称或缩写前加小写字母 "d"表示， 如 dK。  "Amino acid" refers to any molecule that contains both amino and carboxyl functional groups, the amino and carboxyl groups of the ot-amino acid being attached to the same carbon atom (alpha carbon). The alpha carbon may have 1-2 organic substituents. Amino acids contain L and D isomers and racemic mixtures. Unless otherwise specified, the amino acid residues in the polypeptide sequence of the present invention are L isomers, that is, L-amino acids, and D-amino acids are represented by a lowercase letter "d" before the amino acid name or abbreviation, such as dK.
表达方式"可编码氨基酸 "或"可编码氨基酸残基"用于表示可以由核苷酸三联体编码的氨 基酸或氨基酸残基。  The expression "encodeable amino acid" or "encodeable amino acid residue" is used to mean an amino acid or amino acid residue which can be encoded by a nucleotide triplet.
hGlu 为高谷氨酸；  hGlu is homoglutamic acid;
α-hGlu 为— HNCH(CO-)CH2CH2CH2COOH的 L同分异构体； α-hGlu is the L isomer of HNCH(CO-)CH 2 CH 2 CH 2 COOH;
δ-hGlu 为一 HNCH(COOH)CH2CH2C¾CO-的 L同分异构体； δ-hGlu is the L isomer of a HNCH(COOH)CH 2 CH 2 C3⁄4CO-;
a-Asp 为— HNCH(CO-)C¾COOH的 L同分异构体；  a-Asp is the L isomer of HNCH(CO-)C3⁄4COOH;
β-Asp 为— HNCH(COOH)CH2CO-的 L同分异构体； β-Asp is the L isomer of HNCH(COOH)CH 2 CO-;
a-Glu 为一 HNCH(CO-)CH2CH2COOH的 L同分异构体； a-Glu is the L isomer of a HNCH(CO-)CH 2 CH 2 COOH;
γ-Glu 为— HNCH(COOH)CH2CH2CO-的 L同分异构体； γ-Glu is the L isomer of HNCH(COOH)CH 2 CH 2 CO-;
β-Ala为— HN-CH2-CH2-COOH; β-Ala is — HN-CH 2 —CH 2 —COOH;
Sar为肌氨酸。  Sar is sarcosine.
氨基酸残基可以用三字母氨基酸编码或者单字母氨基酸编码表示； 氨基酸表如下所示： 表一： 氨基酸名称及简写  Amino acid residues can be represented by three-letter amino acid codes or single-letter amino acid codes; the amino acid tables are as follows: Table 1: Amino acid names and shorthand
Figure imgf000006_0001
Figure imgf000006_0001
"天然胰岛素 "指来源于天然、 化学合成、 基因工程生产的哺乳动物胰岛素 (如人胰岛素、 牛胰岛素、 猪胰岛素等)。 人胰岛素包含 21个氨基酸组成的 A链和 30个氨基酸组成的 B链。 两条链通过 3条二硫键相连： A7和 B7、 A20和 B19、 A6和 Al l。 B7、 A7指的是天然胰岛 素 B链位置 7 (从 N端数起 )的氨基酸残基以及胰岛素 A链位置 7 (从 N端数起 )的氨基酸 残基。 "Natural insulin" refers to mammalian insulin (such as human insulin, bovine insulin, porcine insulin, etc.) derived from natural, chemical synthesis, genetic engineering. Human insulin comprises an A chain consisting of 21 amino acids and a B chain consisting of 30 amino acids. The two chains are connected by three disulfide bonds: A7 and B7, A20 and B19, A6 and Al l. B7 and A7 refer to amino acid residues at position 7 (from the N-terminus) of the natural insulin B chain and amino acid residues at position 7 (from the N-terminus) of the insulin A chain.
"胰岛素类似物"是修改过的胰岛素多肽的通称， 包括与天然胰岛素有同源序列的由 A链 和 B链组成的双链分子， 以及单链胰岛素类似物。 "胰岛素类似物"具有天然胰岛素的部分、 全部或增强活性， 或者在体内或体外能够转化为具有天然胰岛素的部分、 全部或增强活性的 多肽， 例如比天然胰岛素增加、 减少或替换一个或多个氨基酸残基的多肽。 人、 动物乃至非 哺乳动物的胰岛素原、 前胰岛素原、 胰岛素前体、 单链胰岛素前体和类似物都称为"胰岛素类 似物"。 很多胰岛素类似物见诸于文献。 除非特别另外说明， "胰岛素类似物"广义包括天然胰 岛素和胰岛素类似物。  "Insulin analogs" are generic terms for modified insulin polypeptides, including double-stranded molecules consisting of A and B chains with homologous sequences to native insulin, and single chain insulin analogs. An "insulin analog" has a partial, total or potentiating activity of natural insulin, or can be converted in vivo or in vitro to a polypeptide having partial, total or enhanced activity of native insulin, for example increasing, decreasing or replacing one or more than native insulin A polypeptide of an amino acid residue. Proinsulin, pro-proinsulin, insulin precursors, single-chain insulin precursors and the like of humans, animals and even non-mammals are referred to as "insulin analogs". Many insulin analogs are found in the literature. "Insulin analogs" broadly include natural insulin and insulin analogs unless specifically stated otherwise.
IGF指胰岛素样生长因子（ insulin-like growth factor )， 包括胰岛素样生长因子 -1 ( IGF-1 ) 和胰岛素样生长因子 -2 ( IGF-2 )。  IGF refers to insulin-like growth factor, including insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2).
人 IGF-1的 A链的序列是 SEQ ID NO: 1所示序列，人 IGF-1的 B 链序列是 SEQ ID NO: 2所示序列。 IGF-1类似物相对于天然人 IGF-1分子有一个或者多个氨基酸突变、 取代、 缺失 或添加。 IGF-1类似物包括双链和单链 IGF-1类似物。  The sequence of the A chain of human IGF-1 is the sequence shown in SEQ ID NO: 1, and the B chain sequence of human IGF-1 is the sequence shown in SEQ ID NO: 2. IGF-1 analogs have one or more amino acid mutations, substitutions, deletions or additions relative to the native human IGF-1 molecule. IGF-1 analogs include double-stranded and single-chain IGF-1 analogs.
IGF-2类似物相对于天然人 IGF-2分子有一个或多个多个氛基酸残基突变、取代、缺失或 添加。 IGF-2类似物包括议链和单链 IGF-2类似物。  The IGF-2 analog has one or more amino acid residue mutations, substitutions, deletions or additions relative to the native human IGF-2 molecule. IGF-2 analogs include both chain and single chain IGF-2 analogs.
如无特殊说明， 本申请中涉及的胰岛素指人胰岛素， IGF-1指人的 IGF-1。  Unless otherwise stated, the insulin referred to in the present application refers to human insulin, and IGF-1 refers to human IGF-1.
化合物的氨基酸编号规则:  The amino acid numbering rules for compounds:
在本申请文件中， 除非特殊说明， 当涉及双链化合物时， 双链化合物的 A链和 B链的编 号遵从以下原则：  In the present specification, unless otherwise specified, when a double-stranded compound is involved, the A-chain and B-chain numbers of the double-stranded compound follow the following principles:
该化合物的 A链从编号 1 ( X！或 )开始至编号 22 ( X22或 22 ), 除本申请中变化的 位点外， 该化合物 A链的前 21位氨基酸与人 IGF-1的 A链的氨基酸对应， 特别是该化合物 A链第 6 ( X6或 06 )、 7 ( 7或 07 )、 11 ( X"或 u )和 20 ( Χ2。或 20 )位是半胱氨酸。 若在 X 的 N端添加氨基酸残基， 那么新氨基酸残基的编号是：¾和 ； 若在 01的>1端添 加氨基酸残基， 那么新氨基酸残基的编号是 X399和 00。 The A chain of the compound starts from the number 1 (X! or) to the number 22 (X 22 or 2 2 ), except for the site changed in the present application, the first 21 amino acids of the compound A chain and the human IGF-1 The amino acid of the A chain corresponds, in particular, the 6th (X 6 or 06 ), 7 ( 7 or 07 ), 11 (X" or u ) and 20 (Χ 2 or 20 ) positions of the A chain of the compound are cysteine. If an amino acid residue is added at the N-terminus of X, the number of the new amino acid residue is: 3⁄4 and; If an amino acid residue is added at the >1 end of 01 , the new amino acid residue is numbered X 399 and 00 .
该化合物的 B链从编号 23 ( X23或 ¾23 )开始， 除本申请中变化的位点外， 该化合物 B 链的编号 28 ( X28或 28 )至编号 47 ( X47或 47 ) 的氨基酸与人 IGF-1的 B链的 5至 24位 的氨基酸相对应， 特别是该化合物 B链的编号 29 ( X29或 29 )、 41 ( X4i或 X441 )位是半胱 氨酸。 The B chain of the compound begins with the number 23 (X 23 or 3⁄4 23 ), except for the site of variation in the present application, the amino acid number of the compound B chain from 28 (X 28 or 28 ) to 47 (X47 or 47) Corresponding to the amino acids 5 to 24 of the B chain of human IGF-1, in particular, the number 29 (X 29 or 29), 41 (X4i or X441) of the B chain of the compound is cysteine.
单独指某一个氨基酸时， 可以用例如 A1G、 BIG或 B9H表示， 其分别指在 A链的第一 个氨基酸、 B链的第一和第九个氨基酸残基分别是0、 G、 H。  When referring to an amino acid alone, it can be represented by, for example, A1G, BIG or B9H, which means that the first amino acid of the A chain and the first and ninth amino acid residues of the B chain are 0, G, and H, respectively.
单链化合物的编号按照各化合物的说明为准。  The numbering of the single-chain compound is based on the description of each compound.
单链化合物指的是具有一般结构 B链 -CL-A链的多肽序列或修饰的多肽序列， 其中 B链 是 IGF-1的 B链或类似物， A链是 IGF-1的 A链或类似物， C L是连接 B链 C末端氨基酸残 基与 A链 N末端的肽链。 A single-chain compound refers to a polypeptide sequence having a general structural B chain-C L -A chain or a modified polypeptide sequence, wherein the B chain is the B chain or analog of IGF-1, and the A chain is the A chain of IGF-1 or An analog, C L , is a peptide chain that links the C-terminal amino acid residue of the B chain to the N-terminus of the A chain.
如无特殊说明，本申请中以 A链或 B链位置说明的氨基酸，如 A14、 B28等表示与 IGF-1 的 A链或 B链相对应位置的氨基酸或其变化， 其中 IGF-1的 A链或 B链的编号从 1开始。  Unless otherwise specified, the amino acid indicated by the position of the A chain or the B chain in the present application, such as A14, B28, etc., represents an amino acid corresponding to the position of the A chain or the B chain of IGF-1 or a change thereof, wherein A of IGF-1 The number of the chain or B chain starts at 1.
化合物中半胱氨酸的编号:  The number of cysteine in the compound:
为方便描述， 对本发明中的各化合物中的半胱氨酸进行编号， 分别为 Cn] ~C[61， 其对应 的是： For convenience of description, the cysteines in each compound in the present invention are numbered, respectively, C n] ~ C [61 , which corresponds Is:
当所述化合物为双链时， Cn]和。[2]分别对应双链化合物的 B链中从 N端至 C端的两个 半胱氨酸； 3]~ [6]依次对应 A链中从 N端至 C端的 4个半胱氨酸， 即分别对应 A链编号为 6、 7、 11和 20位的半胱氨酸； When the compound is double-stranded, C n ] and . [2] corresponding to two cysteines from the N-terminus to the C-terminus in the B chain of the double-stranded compound; 3] ~ [ 6] sequentially correspond to four cysteines in the A chain from the N-terminus to the C-terminus, ie Corresponding to the cysteines numbered A, 7, 11, and 20, respectively;
当所述化合物为单链时， Cn]~ C[6]依次对应所述单链化合物从 N端至 C端的 6个半胱氨 酸。 When the compound is a single strand, C n] ~ C [6] sequentially corresponds to the six cysteines of the single-stranded compound from the N-terminus to the C-terminus.
本发明的化合物是基于 IGF-1的结构， 而 IGF-1和 IGF-2与胰岛素以相同的方式形成二 硫键。 本发明中任何一个化合物的三级结构中都包含二硫键， 所有单链 IGF-1类似物及双链 IGF-1 类似物具有和胰岛素同样构成方式的三对二硫键。 因此， 本领域技术人员基于上述解 释和公知常识完全可以理解并知晓本发明中任何一个化合物的二硫键位置。  The compounds of the present invention are based on the structure of IGF-1, while IGF-1 and IGF-2 form disulfide bonds in the same manner as insulin. The tertiary structure of any one of the compounds of the present invention contains a disulfide bond, and all single-chain IGF-1 analogs and double-stranded IGF-1 analogs have a three-pair disulfide bond in the same manner as insulin. Accordingly, those skilled in the art will fully understand and appreciate the disulfide bond positions of any of the compounds of the present invention based on the above explanations and common general knowledge.
"修饰基团" "modifying group"
IGF-1类似物可以包含 1个或多个修饰基团。修饰基团能够提供 IGF-1类似物需要的特征。 例如， 修饰基团可以降低 IGF-1类似物在各种环境下 （如消化道， 血液） 的降解速率。 优选 的修饰基团是那些允许 IGF-1类似物保留相当胰岛素受体结合活性的基团。 优选的修饰基团 包括两性基团， 水溶性基团， 或者使 IGF-1类似物比非修饰的类似物更低亲脂性、 更高水溶 性的基团。 修饰基团可以包含可降解连接基， 例如 PAG; 可以包括易于水解的连接基， 如丙 交酯、 乙交酯、 碳酸、 酯、 氨基曱酸酯。 这种方法可以使聚合物降解成小分子量片段。  The IGF-1 analog may comprise one or more modifying groups. The modifying group is capable of providing the desired characteristics of the IGF-1 analog. For example, a modifying group can reduce the rate of degradation of an IGF-1 analog in various environments (e.g., digestive tract, blood). Preferred modifying groups are those which allow the IGF-1 analog to retain comparable insulin receptor binding activity. Preferred modifying groups include amphoteric groups, water soluble groups, or groups which render the IGF-1 analog less lipophilic, more water soluble than the unmodified analog. The modifying group may comprise a degradable linking group, such as PAG; it may include a linking group that is susceptible to hydrolysis, such as lactide, glycolide, carbonic acid, esters, amino phthalates. This method can degrade the polymer into small molecular weight fragments.
修饰基团可以包括一个或多个亲水基团、 亲脂基团、 两性基团、 成盐基团、 间隔基团、 连接基团、 封端基团或这些基团的组合。 各种基团可以以共价键， 或以可水解或不可水解的 键连接在一起。 代表性亲水基团和亲脂基团介绍如下。  The modifying group may include one or more hydrophilic groups, lipophilic groups, amphoteric groups, salt-forming groups, spacer groups, linking groups, capping groups, or a combination of these groups. The various groups may be linked together by covalent bonds or by hydrolyzable or non-hydrolyzable bonds. Representative hydrophilic groups and lipophilic groups are described below.
亲水基团  Hydrophilic group
亲水基团的实例包括 PAG基团、 多糖、 聚山梨醇酯以及这些基团的组合物。  Examples of the hydrophilic group include a PAG group, a polysaccharide, a polysorbate, and a combination of these groups.
聚亚烷基二醇（Polyalkylene Glycol, PAG )基团由多个亚烷基二醇单体组成。 在一个实 施例中， 所有单体是相同的 （例如聚乙二醇 （PEG )或聚丙二醇（PPG ) )。 在另一个实施例 中， 亚烷基二醇是不同的。 聚合体可以是无规共聚物 （例如环氧乙烷和环氧丙烷的共聚物）， 或者分枝或接枝共聚物。  The polyalkylene Glycol (PAG) group consists of a plurality of alkylene glycol monomers. In one embodiment, all monomers are the same (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)). In another embodiment, the alkylene glycols are different. The polymer may be a random copolymer such as a copolymer of ethylene oxide and propylene oxide, or a branched or graft copolymer.
本文使用的" PEG"或聚乙二醇指任何水溶性聚乙二醇或聚氧化乙烯。 聚乙二醇的化学结 构式为 -(CH2C¾0)n-， 其中 n可以是从 2到 2000的整数。 PEG的一端通常是相对没有活性的 官能团， 如烷基或烷氧基等。 烷基包括饱和的直链或支链烃基。 烷氧基代表性的例子是曱氧 基、 乙氧基、 丙氧基（例如 1-丙氧基和 2-丙氧基）、 丁氧基（例如 1-丁氧基、 2-丁氧基和 2- 曱基 -2-丙氧基）、 戊氧基、 己氧基等。 使用曱氧基封端的 PEG 命名为 mPEG , 结构式 CH30(CH2CH20)n -， 但一般仍然称为 PEG。 PEG20K指分子量为 20,000的聚乙二醇分子。 As used herein, "PEG" or polyethylene glycol refers to any water soluble polyethylene glycol or polyethylene oxide. The chemical formula of polyethylene glycol is -(CH 2 C3⁄40) n -, where n can be an integer from 2 to 2,000. One end of the PEG is usually a relatively inactive functional group such as an alkyl group or an alkoxy group. Alkyl groups include saturated straight or branched chain hydrocarbon groups. Representative examples of alkoxy are decyloxy, ethoxy, propoxy (e.g., 1-propoxy and 2-propoxy), butoxy (e.g., 1-butoxy, 2-butoxy). And 2-mercapto-2-propoxy), pentyloxy, hexyloxy and the like. The PEG-terminated PEG is named mPEG, the structural formula CH 3 0(CH 2 CH 2 0) n -, but is still generally referred to as PEG. PEG20K refers to a polyethylene glycol molecule having a molecular weight of 20,000.
PEG另一端通常是活化官能团或者易于形成共价键的官能团， 例如氨基、 羧基、 羟基、 巯基、 醛等。 PEG-马来酰亚胺、 PEG-乙烯砜和 PEG-碘代乙酰基（CO-CH2-I )等可以与半胱 氨酸侧链的琉基 -SH反应形成稳定的共价键； PEG-NHS (琥珀酰亚胺）可以与多肽 N末端 α- 氨基或赖氨酸侧链氨基通过亲核取代反应 （酰化）接合； PEG-醛与多肽的氨基在还原剂 （如 氰基硼氢化钠）作用下可以通过还原性烷基化反应接合。 The other end of the PEG is usually an activating functional group or a functional group which is liable to form a covalent bond, such as an amino group, a carboxyl group, a hydroxyl group, a thiol group, an aldehyde or the like. PEG-maleimide, PEG-vinylsulfone and PEG-iodoacetyl (CO-CH 2 -I ) can form a stable covalent bond with the thiol-SH of the cysteine side chain; PEG -NHS (succinimide) can be bonded to the N-terminal α-amino or lysine side chain amino group of the polypeptide by a nucleophilic substitution reaction (acylation); the PEG-aldehyde and the amino group of the polypeptide are in a reducing agent (such as cyanoborohydride) Under sodium action, it can be joined by a reductive alkylation reaction.
本发明中的 PEG分子可以是直链的、支链的、分叉的或哑铃状的 PEG。在一个实施例中， 支链 PEG可以用通式 R(-PEG-nOH)m表示， 其中 R (通常是多羟基的）是核心基团， 例如季 戊四醇、 糖、 赖氨酸或甘油； m代表支链数， 可以是从 2起到核心基团附着位点最大数目； n代表 PEG片段的数量， 每个支链上的 PEG片段的数量可以不等。 一般情况下， n是 2-1800 的整数。 在另一个实施例中， 支链 PEG可以用通式 (CH30-PEG-n)pR-Z表示， p等于 2或 3 , R是赖氨酸或甘油， Z代表可以进行反应的活化官能团。 在一个实施例中， 分叉 PEG用通式 PEG(-L-X)n表示， L是连接基， X是末端活化官能团。 The PEG molecule in the present invention may be a linear, branched, bifurcated or dumbbell-shaped PEG. In one embodiment, the branched PEG can be represented by the formula R(-PEG- n OH) m , wherein R (usually polyhydroxy) is a core group, such as Pentaerythritol, sugar, lysine or glycerol; m represents the number of branches, which may be the maximum number of attachment sites from 2 to the core group; n represents the number of PEG fragments, the number of PEG fragments per branch Can not wait. In general, n is an integer from 2 to 1800. In another embodiment, the branched PEG can be represented by the formula (CH 3 0-PEG- n ) p RZ, p is equal to 2 or 3, R is lysine or glycerol, and Z represents an activating functional group capable of undergoing a reaction. In one embodiment, the bifurcated PEG is represented by the general formula PEG(-LX) n , L is a linking group, and X is a terminal activating functional group.
PEG一般是多分散的，多分散指数小于 1.05。 PEG基团也可以是单^ ^的。单^^指 PEG 具有单一的长度（分子量）， 而不是各种长度（分子量） 的混合物。  PEG is generally polydisperse with a polydispersity index of less than 1.05. The PEG group can also be a single ^. A single PEG has a single length (molecular weight) rather than a mixture of various lengths (molecular weight).
糖基团  Sugar group
代表性糖基团包括， 但不局限于， 甘油、 单糖、 二糖、 三糖、 寡糖和多糖如淀粉、 糖原、 纤维素和 /或多糖树胶。 特别的单糖包括 C6及以上（特别是 C6和 C8 )糖如葡萄糖、 果糖、 甘露糖、 半乳糖、 核酸糖或景天庚糖； 二糖和三糖包括含有二或三个单糖单元（特别是 C5 至 C8 )的基团、 例如蔗糖、 纤维二糖、 麦芽糖、 乳糖和 /或蜜三糖。  Representative sugar groups include, but are not limited to, glycerin, monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides such as starch, glycogen, cellulose, and/or polysaccharide gums. Particular monosaccharides include C6 and above (especially C6 and C8) sugars such as glucose, fructose, mannose, galactose, nucleic acid sugar or sedose heptose; disaccharides and trisaccharides include two or three monosaccharide units ( In particular, groups of C5 to C8), such as sucrose, cellobiose, maltose, lactose and/or raffinose.
其它亲水基团  Other hydrophilic groups
生物适合的聚阳离子基团包括骨架或侧链上具有多个氨基的聚胺基团， 例如聚赖氨酸和 其它天然或合成的氨基酸构成的具有多个正电荷的氨基酸聚合物， 包括聚鸟氨酸、 聚精氨酸、 聚组氨酸， 非多肽聚胺如聚氨基苯乙烯、 聚氨基丙烯酸酯、 聚 -N曱基氨基丙烯酸酯、 季胺聚 合物等。 生物适合的聚阴离子基团包括骨架或侧链上具有多个羧基的基团， 如聚天冬氨酸、 聚谷氨酸等。 其它亲水基团包括天然或合成多糖， 如壳聚糖、 葡聚糖等。  Biocompatible polycationic groups include polyamine groups having a plurality of amino groups on the backbone or side chain, such as polylysine and other natural or synthetic amino acids having a plurality of positively charged amino acid polymers, including poly birds. Amino acid, polyarginine, polyhistidine, non-polypeptide polyamine such as polyaminostyrene, polyamino acrylate, poly-N-decylamino acrylate, quaternary amine polymer, and the like. Biocompatible polyanionic groups include groups having a plurality of carboxyl groups on the backbone or side chain, such as polyaspartic acid, polyglutamic acid, and the like. Other hydrophilic groups include natural or synthetic polysaccharides such as chitosan, dextran, and the like.
聚阴离子生物粘附剂  Polyanionic bioadhesive
某些亲水基团有潜在的生物粘附特性。 这样的例子可见于美国专利 US 6,197,346。 这些 具有多个羧基的聚合物显示生物粘附特性。 降解时显露出多个羧基的快速生物降解聚合物， 如乳酸羟基乙酸共聚物 （ poly (lactide- Co- glycolide) )、 聚酐、 聚原酸酯也都是生物粘附剂。 这些聚合物可以把 IGF-1类似物投放到胃肠道。 聚合物降解时暴露出来的羧基可以牢固附着 在胃肠道， 协助投放 IGF-1类似物。  Certain hydrophilic groups have potential bioadhesive properties. An example of this can be found in U.S. Patent 6,197,346. These polymers having a plurality of carboxyl groups exhibit bioadhesive properties. Rapid biodegradable polymers that exhibit multiple carboxyl groups upon degradation, such as poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, are also bioadhesives. These polymers can be used to administer IGF-1 analogs to the gastrointestinal tract. The carboxyl groups exposed during polymer degradation can be firmly attached to the gastrointestinal tract and assist in the administration of IGF-1 analogues.
亲脂基团  Lipophilic group
在一个实施例中， 修饰基团包括一个或多个亲脂基团。 亲脂基团可以是本领域人员众所 周知的， 包括， 但不限于： 烷基、 链烯基、 炔基、 芳基、 芳基烷基、 烷基芳香基、 脂肪酸、 胆甾醇以及亲脂性多聚物和低聚物。  In one embodiment, the modifying group includes one or more lipophilic groups. Lipophilic groups can be well known to those skilled in the art and include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, fatty acid, cholesterol, and lipophilic poly And oligomers.
烃基可以是饱和、 非饱和、 直链的、 支链的或环烃， 具有一个或多个碳原子。 在一个实 施例中， 烃基有 1、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、 30或更多的碳原子。 烃基可以是无取代， 或有 一个或者多个取代基， 这些取代基最好不会使结合物失去生物活性。  The hydrocarbyl group can be a saturated, unsaturated, linear, branched or cyclic hydrocarbon having one or more carbon atoms. In one embodiment, the hydrocarbon group has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms. The hydrocarbyl group may be unsubstituted or have one or more substituents which preferably do not lose the biological activity of the conjugate.
亲脂基团也可以是脂肪酸， 如天然的、 合成的、 饱和的、 不饱和的、 直链的或支链的脂 肪酸。 在一个实施例中， 脂肪酸有 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 20、 21、 22、 23、 24或更多个碳原子。  The lipophilic group can also be a fatty acid such as a natural, synthetic, saturated, unsaturated, linear or branched fatty acid. In one embodiment, the fatty acids are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more carbon atoms.
结合（Conjugation ) 策略  Conjugation strategy
修饰基团与多肽的结合程度、 结合点的选择、 修饰基团的选择要根据需要而变化， 例如 使结合物在体内不易降解， 从而延长血浆半衰期。 例如 IGF-1类似物修饰后具有一个、 两个、 三个、 四个或更多修饰基团。 结合部位可能包括一个氨基酸残基， 比如赖氨酸残基。 在一个 实施例中， IGF-1结合物是单结合物。 在另一个实施例中， IGF-1结合物是多结合物。 在另一 个实施例中， IGF-1 结合物是单结合物、 双结合物、 三结合物、 四结合物等的混合物。 修饰 基团可以相同， 也可以不同。 当 IGF-1结合物具有多个修饰基团时， 一个或多个修饰基团最 好通过可水解键与 IGF-1相连而其它一个或多个修饰基团最好通过不可水解键与 IGF-1结合 物相连。 或者， 所有修饰基团都通过可水解键与 IGF-1相连， 但各个修饰基团在体内的水解 速率有快有慢。 The degree of binding of the modifying group to the polypeptide, the choice of binding site, and the choice of modifying group will vary as desired, for example, the conjugate will not readily degrade in vivo, thereby prolonging the plasma half-life. For example, an IGF-1 analog has one, two, three, four or more modifying groups after modification. The binding site may include an amino acid residue, such as a lysine residue. In one embodiment, the IGF-1 conjugate is a single conjugate. In another embodiment, the IGF-1 conjugate is a multi-conjugate. In another In one embodiment, the IGF-1 conjugate is a mixture of monoconjugates, biconjugates, triple binders, tetraconjugates, and the like. The modifying groups may be the same or different. When the IGF-1 conjugate has a plurality of modifying groups, one or more modifying groups are preferably attached to IGF-1 via a hydrolyzable bond and the other one or more modifying groups are preferably passed through a non-hydrolyzable bond to the IGF- 1 combination is connected. Alternatively, all of the modifying groups are linked to IGF-1 via a hydrolyzable bond, but the rate of hydrolysis of each modifying group in vivo is fast and slow.
理想的结合策略是使结合物具有原 IGF-1 类似物部分或全部生物活性。 优选的结合部位 包括双链多肽的 B 1-N末端、 B链赖氨酸侧链氨基或单链的 N末端、 赖氨酸侧链氨基等。 B1 单结合物和 B链双结合物是最常用的。 另外可以通过在单链化合物的连接片段或双链多肽 A 链、 B链引入具有氨基或琉基的天然或非天然氨基酸来创造其它结合位点。  An ideal binding strategy is to have the conjugate have some or all of the biological activity of the original IGF-1 analog. Preferred binding sites include the B1-N terminus of the double-stranded polypeptide, the B chain lysine side chain amino group or the single-stranded N-terminus, the lysine side chain amino group, and the like. The B1 single binder and B chain double binder are the most commonly used. Further, other binding sites can be created by introducing a natural or unnatural amino acid having an amino group or a thiol group in the ligated fragment of the single-stranded compound or the double-stranded polypeptide A chain or B chain.
修饰基团与 IGF-1类似物可以通过可水解键（如酯、 碳酸、 可水解氨基曱酸酯）结合。 可水解键使 IGF-1结合物具有前药的效果。 如果希望修饰基团与 IGF-1结合物没有活性， 比 如修饰基团的结合部位在 IGF-1类似物对胰岛素受体结合区， 前药策略就是优选方法。 当一 个或多个修饰基团在一段时间内从 IGF-1结合物脱离， 从而释放出活性 IGF-1类似物， 使用 可水解键能够提供延时释放或緩释的效果。  The modifying group and the IGF-1 analog can be bound by a hydrolyzable bond (e.g., ester, carbonic acid, hydrolyzable amino phthalate). The hydrolyzable bond gives the IGF-1 conjugate a prodrug effect. A prodrug strategy is the preferred method if it is desired that the modifying group is inactive with the IGF-1 conjugate, such as where the binding site of the modifying group is in the IGF-1 analog to insulin receptor binding region. When one or more of the modifying groups are detached from the IGF-1 conjugate over a period of time, thereby releasing the active IGF-1 analog, the use of a hydrolyzable bond can provide a delayed release or sustained release effect.
在一个实施例中， IGF-1 类似物通过非水解键（如酰胺键， 瞇键） 与修饰基团相连。 必 要时， 非水解键有助于延长 IGF-1结合物在血浆中的循环时间。  In one embodiment, the IGF-1 analog is linked to a modifying group by a non-hydrolyzable bond (eg, an amide bond, a hydrazone bond). When necessary, non-hydrolyzed bonds help to prolong the circulation time of the IGF-1 conjugate in plasma.
IGF-1 同系物可以通过各种亲核官能团与修饰基团相连， 包括但不局限于亲核羟基或氨 基。 例如丝氨酸、 苏氨酸、 酪氨酸具有亲核羟基， 组氨酸、 赖氨酸或 IGF-1类似物 A链、 B 链 N-末端都具有亲核氨基。 IGF-1 同系物也可以通过自由巯基 -SH与修饰基团相连， 例如形 成疏酯、 硫醚、 磺胺键。  IGF-1 homologs can be attached to the modifying group by various nucleophilic functional groups including, but not limited to, nucleophilic hydroxyl or amino groups. For example, serine, threonine, tyrosine have a nucleophilic hydroxyl group, a histidine, lysine or IGF-1 analog A chain, and a N-terminus of a B chain have a nucleophilic amino group. The IGF-1 homologue can also be attached to the modifying group via a free thiol-SH, for example to form a sulphur ester, a thioether or a sulfonamide bond.
分子量较小的多肽化合物在血浆中的循环时间短的一个重要因素就是肾清除。 增加多肽 化合物分子量直至超过肾清除临界点可以显著降低腎清除率， 延长多肽在体内作用时间。 常 用的方法是使多肽与天然或合成大分子形成可水解或不可水解键。 生物大分子包括白蛋白、 多糖、 抗体（如 IgG )等。 血管中 70%的白蛋白是琉基白蛋白 （ mercaptalbumin )， 其半胱氨 酸 -34的侧链巯基是血浆中活性最强的琉基。 IGF-1类似物可以通过一个一端带有马来酰亚胺 等活化官能团的连接基与其反应生成 IGF-1 -白蛋白结合物。 连接基本身可以是长链脂肪酸或 PEG分子。 具体实例可以参照 Bioconjugate Chem. 2005， 16, 1000-1008。 合成大分子包括聚乙 二醇和葡聚糖。 另一种方式是脂肪酸酰化， 将在酰化 IGF-1类似物部分讨论。  An important factor in the short cycle time of peptide compounds with lower molecular weight in plasma is renal clearance. Increasing the molecular weight of the polypeptide compound until it exceeds the critical point of renal clearance can significantly reduce renal clearance and prolong the time of action of the polypeptide in vivo. A common method is to form a hydrolyzable or non-hydrolyzable bond with a natural or synthetic macromolecule. Biomacromolecules include albumin, polysaccharides, antibodies (such as IgG), and the like. 70% of the albumin in the blood vessels is mercaptalbumin, and the side chain thiol of cysteine-34 is the most active sulfhydryl group in plasma. The IGF-1 analog can be reacted with a linker having an activating functional group such as maleimide at one end to form an IGF-1 - albumin conjugate. The linker can be a long chain fatty acid or a PEG molecule. Specific examples can be found in Bioconjugate Chem. 2005, 16, 1000-1008. Synthetic macromolecules include polyethylene glycol and dextran. Another way is fatty acid acylation, which will be discussed in the acylation of the IGF-1 analog moiety.
近年来出现了以蛋白酶 sortase催化使两个分子通过酰胺键结合的方法。 Sortase是一种介 导革兰阳性细菌细胞壁与表面蛋白共价结合的转肽酶， 主要存在于革兰阳性细菌中。 对 GeneBank CDS 和 NCBI资料库的蛋白序列分析显示， sortase家族包括 150多种蛋白序列， 其中 Staphylococcus aureus sortase A (SrtA或 SrtAStaph)是目前研究最多的同种型 （ isoform )。 有多篇文献揭示 SrtA催化的转肽反应的分子机制。 SrtA识别包含 LPXTG( Leu-Pro-X-Thr-Gly ) 基序的底物， 其 184位的半胱氨酸作为亲核基团攻击 LPXTG基序中的肽键 Thr-Gly, 由此产 生一个酰基 -酶中间体。 苏氨酸羧基的硫酯中间体通过与底物的低聚甘氨酸（在 S. aureus中是 支链脂质 II的前体交连桥的五甘氨酸 (Gly5))的氨基基团发生亲核反应， 产生新的连接产物。 In recent years, a method of catalyzing the binding of two molecules through an amide bond by protease sortase has appeared. Sortase is a transpeptidase that mediates the covalent binding of Gram-positive bacterial cell walls to surface proteins, mainly in Gram-positive bacteria. Analysis of the protein sequences of the GeneBank CDS and NCBI databases revealed that the sortase family includes more than 150 protein sequences, of which Staphylococcus aureus sortase A (SrtA or SrtA St aph) is the most studied isoform (isoform). A number of publications have revealed the molecular mechanism of SrtA-catalyzed transpeptidation. SrtA recognizes a substrate comprising the LPXTG (Leu-Pro-X-Thr-Gly) motif, and the cysteine at position 184 acts as a nucleophilic group to attack the peptide bond Thr-Gly in the LPXTG motif, thereby producing a Acyl-enzyme intermediate. The thioester intermediate of the threonine carboxyl group undergoes a nucleophilic reaction with the amino group of the oligoglycine of the substrate (penta-glycine (Gly 5 ) which is a bridge of the precursor of the branched lipid II in S. aureus) , creating new connection products.
另一相关的 <»re/?tococa« ;7j oge"es sortase可以接受由两个丙氨酸构成的亲核基团，但& aureus酶不能。 这种 sortase (SrtAstrep) 切断 LPXTA基序中的肽键 Thr-Ala， 允许以丙氨酸为 基础的亲核基团。 SrtAstep也可以识别 LPXTG基序，但活性较低。 LPXTA基序不会被 SrtAStaph 切断。 为简单起见， 以下方法论述均以 SrtAStaph为例， 但 SrtAstrep等同种型也可以用相同或 相似的方法。 Another related <»re/?tococa« ;7j oge"es sortase can accept a nucleophilic group consisting of two alanines, but the & aureus enzyme cannot. This sortase (SrtA strep ) cleaves the LPXTA motif The peptide bond Thr-Ala, which allows an alanine-based nucleophilic group. SrtAstep also recognizes the LPXTG motif but has lower activity. The LPXTA motif is not cleaved by SrtA Staph . For simplicity, the following method discusses Take SrtA Staph as an example, but the SrtA strep equivalent can also use the same or A similar approach.
SrtA对 LPXTG基序和 N端带有自由氨基的甘氨酸重复（ a-Glyn) 高度专一。 X位可以 是除半胱氨酸和色氨酸以外（尚未测试） 的所有天然氨基酸。 尽管有实脸表明 N端带有一个 甘氨酸的多肽能够参与 sortase催化的转肽反应， N端带有两个或两个以上的甘氨酸的底物可 以达到最大反应效率。 Sortase辅助连接 （ Sortase mediated ligation ) 的一项主要应用就是将 非天然功能团引入蛋白或多肽。 非天然功能团可以是小分子、 合成多肽或蛋白、 聚合物等。 这些功能团与 LPXTG或 a-Glyn 融合而成的分子都可以成为 SrtA的底物。具体方法和反应奈 件可以参照相关文献 (如 Tsukiji等， "Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering", ChemBioChem, 2009,10,787-798; Popp等, "Sortase-catalyzed transformations that improve the properties of cytokines", PNAS, 2011, 108, 3169-3174 )。 SrtA is highly specific for the LPXTG motif and the glycine repeat (a-Gly n ) with a free amino group at the N-terminus. The X position can be all natural amino acids other than cysteine and tryptophan (not yet tested). Although a real face indicates that a polypeptide with a glycine at the N-terminus can participate in a sortase-catalyzed transpeptidation reaction, a substrate with two or more glycines at the N-terminus can achieve maximum reaction efficiency. One of the main applications of Sortase mediated ligation is the introduction of non-native functional groups into proteins or peptides. The non-natural functional group can be a small molecule, a synthetic polypeptide or protein, a polymer, and the like. These functional groups can be combined with LPXTG or a-Gly n to form a substrate for SrtA. Specific methods and reaction kits can be referred to related literature (eg, Tsukiji et al, "Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering", ChemBioChem, 2009, 10, 787-798; Popp et al, "Sortase-catalyzed transformations that Improve the properties of cytokines", PNAS, 2011, 108, 3169-3174).
SrtA能够在 IGF-1类似物上引入非天然功能团， 具体策 居 IGF-1类似物的结构而 定。 对于双链 IGF-1类似物， 一般较少影响生物活性的结合位点是 B链的 N端或 A链、 B链 的 C端。 如果结合位点是 B链的 N端， 那么 IGF-1 B链的 N端最好引入多个甘氨酸， 如 GGGGG-IGF-1 B链；而要引入的修饰基团如 PEG、长链脂肪酸或白蛋白等的 C端要有 LPXTG 基序， 比如 PEG-LPATGGGG、 白蛋白 -LPETGGG或脂肪酸 LPGTGGGGG等。 如果结合位点 是 A或 B链的 C端， 那么 A或 B链的 C端氨基酸序列要包含 LPXTG基序， 比如可以变为 IGF-1 A-LPATGGGGG或 IGF-1 B-LPGTGGGG等， 而要引入的修饰基团如 PEG、长链脂肪酸 或白蛋白等的 N端要有一个或多个甘氨酸， 如 GGG-PEG、 GGGG-长链脂肪酸、 GGGGG-白 蛋白等。 对于单链 IGF-1类似物， 最容易的结合位点是 B链的 N端或 A链的 C末端， 方法 和双链类似物基本相同。 双链化合物  SrtA is capable of introducing a non-native functional group on the IGF-1 analog, depending on the structure of the IGF-1 analog. For double-stranded IGF-1 analogs, the binding site that generally less affects biological activity is the N-terminus of the B-chain or the C-terminus of the B-chain. If the binding site is the N-terminus of the B chain, then the N-terminus of the IGF-1 B chain preferably introduces multiple glycines, such as the GGGGG-IGF-1 B chain; and the modifying group to be introduced, such as PEG, long chain fatty acids or The C-terminus of albumin or the like has an LPXTG motif such as PEG-LPATGGGG, albumin-LPETGGG or fatty acid LPGTGGGGG. If the binding site is the C-terminus of the A or B chain, then the C-terminal amino acid sequence of the A or B chain comprises an LPXTG motif, such as IGF-1 A-LPATGGGGG or IGF-1 B-LPGTGGGG, etc. The introduced modified group such as PEG, long-chain fatty acid or albumin has one or more glycines at the N-terminus, such as GGG-PEG, GGGG-long-chain fatty acid, GGGGG-albumin and the like. For single-stranded IGF-1 analogs, the easiest binding site is the N-terminus of the B-chain or the C-terminus of the A-chain, and the method is essentially identical to the double-stranded analog. Double-stranded compound
胰岛素样生长因子 -1 ( IGF-1 )主要由肝脏生成， 是由 70个氨基酸组成的单链蛋白， 具 有四个域（有些文献中称"链，，：)。 其中 A和 B域与胰岛素的 A链和 B链结构同源； 而 C域连 接 A和 B域， 与胰岛素原的 C肽相对应； D域是 A域的 C末端延伸部分。 IGF-1的三维结构 已经有 NMR和 X-射线结晶方法的报导。其 B域和 A域的结构与胰岛素的结晶结构和胰岛素 原的 NMR结构非常相似。  Insulin-like growth factor-1 (IGF-1), which is mainly produced by the liver, is a single-chain protein consisting of 70 amino acids with four domains (some are called "chains," in some literature). Among them, A and B domains and insulin. The A and B chains are homologous; the C domain is linked to the A and B domains, corresponding to the C-peptide of proinsulin; the D domain is the C-terminal extension of the A domain. The three-dimensional structure of IGF-1 already has NMR and X A report of the radiant crystallization method. The structure of the B domain and the A domain is very similar to the crystal structure of insulin and the NMR structure of proinsulin.
胰岛素受体家族包括胰岛素受体（IR )、 胰岛素样生长因子 -1 受体（IGF-1R )和胰岛素 受体相关受体， 它们都是受体酪氨酸激酶， 通过在特定酪氨酸上增加磷酸分子而传递信号。 胰岛素受体家族的成员由两个受体部分组成， 每一个包括胞外的 α亚基和跨膜的 β亚基， 通 过二硫键连接。 不结合配基时， 该受体以二聚体形式存在， 形成通过两个 α亚基之间的二硫 键结合在一起的 α2β2受体。取决于进行比较的区域， 胰岛素受体和 IGF-1受体的序列相似度 为 41-84%。  The insulin receptor family includes the insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF-1R), and insulin receptor-related receptors, all of which are receptor tyrosine kinases that act on specific tyrosines. Increase the phosphate molecule to deliver the signal. Members of the insulin receptor family are composed of two receptor moieties, each comprising an extracellular alpha subunit and a transmembrane beta subunit, joined by a disulfide bond. When the ligand is not bound, the receptor exists as a dimer, forming an α2β2 receptor that is held together by a disulfide bond between the two α subunits. The sequence similarity between the insulin receptor and the IGF-1 receptor is 41-84% depending on the region to be compared.
IGF-1结合胰岛素受体的能力比胰岛素低 100倍，与 IGF-1在体内诱导胰岛素受体磷酸化 和低血糖症的实际能力不符（IGF-1激活胰岛素受体的能力大约是胰岛素的 10% )。 因此部分 信号传导可能是通过 IGF-1受体 /胰岛素受体异二聚体。  The ability of IGF-1 to bind to the insulin receptor is 100-fold lower than that of insulin, which is inconsistent with the actual ability of IGF-1 to induce insulin receptor phosphorylation and hypoglycemia in vivo (IGF-1's ability to activate the insulin receptor is approximately 10 for insulin). %). Therefore, part of the signaling may be through the IGF-1 receptor/insulin receptor heterodimer.
几乎人体中的每一个细胞都受 IGF-1影响， 特别是肌肉、 软骨、 骨骼、 肝脏、 腎脏、 神 经、 皮肤和肺中细胞。 除了胰岛素样作用， IGF-1 同样可以调节细胞生长和发育， 特别是神 经细胞， 以及细胞的 DNA合成。  Almost every cell in the human body is affected by IGF-1, especially in the muscles, cartilage, bones, liver, kidneys, nerves, skin and lungs. In addition to insulin-like effects, IGF-1 also regulates cell growth and development, particularly in neural cells, as well as in cellular DNA synthesis.
大约 98%的 IGF-1总是与六种 IGF-1结合蛋白 （ IGF-1-BP )之一结合。 其中 IGF-1 ΒΡ3 数量最大， 结合了 80%的 IGF-U IGF-1与 IGF-1 BP-3的结合摩尔比是 1 :1。 Approximately 98% of IGF-1 is always bound to one of the six IGF-1 binding proteins (IGF-1-BP). Where IGF-1 ΒΡ3 The largest amount, combined with 80% IGF-U IGF-1 and IGF-1 BP-3 binding molar ratio is 1:1.
IGF-1 和胰岛素的代谢反应类似： 两种激素以非常相似的方式增加葡萄糖吸收和氧化， 抑制葡萄糖生产、 自由脂肪酸水平和脂肪氧化速率。  The metabolic response of IGF-1 and insulin is similar: Both hormones increase glucose uptake and oxidation in a very similar manner, inhibiting glucose production, free fatty acid levels, and fat oxidation rates.
胰岛素拮抗性由许多常见和几种不常见的临床症状的构成。 胰岛素受体基因或与信号传 导途径相关的基因中有突变的病人具有不同的显型， 包括脂肪代谢障碍、部分脂肪代谢障碍、 具有胰岛素受体基因突变的胰岛素拮抗性的 A型综合症、 假性类肢端肥大症、 矮妖精貌综合 症和 Rabson-Mendenhall综合症。 在许多这样的病患中， 高血糖症相当难以治疗， 因为胰岛素 是无效的。  Insulin antagonism is composed of many common and several uncommon clinical symptoms. Patients with mutations in the insulin receptor gene or genes involved in the signaling pathway have different phenotypes, including lipodystrophy, partial lipodystrophy, insulin-resistant type A syndrome with insulin receptor gene mutation, and false Sexual acromegaly, dwarfism syndrome and Rabson-Mendenhall syndrome. In many of these patients, hyperglycemia is quite difficult to treat because insulin is ineffective.
胰岛素拮抗性同样在非胰岛素依赖性糖尿病的病患中常见。 人体通过增加胰岛素分泌克 服这个问题， 却导致靶组织上的胰岛素受体表达减少， 恶化了胰岛素拮抗性。 高甘油三酯血 症在这些病患中是次级代谢异常。 因此， 具体针对胰岛素拮抗性的治疗可以明显改善糖尿病 治疗效果。  Insulin antagonism is also common in patients with non-insulin dependent diabetes. The human body overcomes this problem by increasing insulin secretion, but it leads to a decrease in insulin receptor expression on target tissues, which worsens insulin antagonism. Hypertriglyceridemia is a secondary metabolic abnormality in these patients. Therefore, the specific treatment for insulin antagonistic treatment can significantly improve the therapeutic effect of diabetes.
IGF-1 已经被提议用于严重胰岛素拮抗性的治疗。 因为它的生物学作用与胰岛素类似， 所以可以避开阻碍胰岛素发挥作用的身体机能缺陷。 已发现静脉内注射重组 IGF-1减少了两 名具有胰岛素拮抗性的 A型综合症的病患和一名具有 Rabson-Mendenhall综合症的孩子的血 糖和血清胰岛素浓度。在几个具有不同显型的严重胰岛素拮抗性的病患的研究中，使用 IGF-1 减少了禁食和 24小时平均血清胰岛素浓度，提高葡萄糖耐受性， 增加胰岛素敏感度及减少了 禁食血清甘油三酸酯浓度。  IGF-1 has been proposed for the treatment of severe insulin antagonism. Because its biological effects are similar to those of insulin, it is possible to avoid bodily defects that prevent insulin from functioning. Intravenous injection of recombinant IGF-1 has been found to reduce blood glucose and serum insulin concentrations in two patients with insulin-resistant type A syndrome and one child with Rabson-Mendenhall syndrome. In several studies of patients with different phenotypes of severe insulin antagonism, the use of IGF-1 reduced fasting and 24-hour mean serum insulin concentrations, increased glucose tolerance, increased insulin sensitivity and reduced fasting Serum triglyceride concentration.
大约 60%的糖尿病患者在一生中会遭受神经损伤， 导致手或脚感觉麻木或有麻刺感。 类 似神经损伤可能会削弱人体对血压的控制， 造成失禁或阳痿， 或引发腹泻或便秘。 糖尿病大 鼠研究表明， 这类糖尿病引起的神经损伤可以通过 IGF-1治疗恢复。 据猜测， 糖尿病阻止神 经细胞不能正常发育， 而 IGF-1使之恢复正常。  About 60% of people with diabetes suffer from nerve damage during their lifetime, causing numbness or tingling in their hands or feet. Similar nerve damage may impair the body's control of blood pressure, cause incontinence or impotence, or cause diarrhea or constipation. Studies in diabetic rats have shown that nerve damage caused by this type of diabetes can be restored by IGF-1 treatment. It is suspected that diabetes prevents nerve cells from developing properly, and IGF-1 returns it to normal.
但是， IGF-1的有益效果是有代价的。 IGF-1可能导致心血管的剧烈反应， 包括心律停止 和低血压。 某些反应可能是因为 IGF-1 造成血磷酸盐急剧过少。 长期大剂量皮下注射 IGF-1 会产生颞下颌不适、 脸和手浮肿、 体重增加、 呼吸困难、 窦性心搏过速。 不过最近期的研究 显示， 能够很好耐受而且有效的剂量是可以做到的。 但最好的方法就是修改分子结构， 提高 IGF-1类似物在胰岛素受体的结合力， 同时降低其在 IGF-1受体上的活性。  However, the beneficial effects of IGF-1 come at a price. IGF-1 may cause a cardiovascular response, including arrhythmia and hypotension. Some reactions may be due to the fact that IGF-1 causes a sharp decrease in blood phosphate. Long-term high-dose subcutaneous injection of IGF-1 can cause temporomandibular discomfort, facial and hand edema, weight gain, difficulty breathing, and sinus tachycardia. However, recent studies have shown that well tolerated and effective doses can be achieved. However, the best approach is to modify the molecular structure to increase the binding of the IGF-1 analogue to the insulin receptor while reducing its activity at the IGF-1 receptor.
IGF-1 类似物的其它优点包括， 但不局限于， 比人胰岛素更好的水溶性， 更高的产率， 开发出胰岛素受体和 IGF-1受体的双激动剂的潜力等。  Other advantages of IGF-1 analogs include, but are not limited to, better water solubility than human insulin, higher yields, the potential to develop dual receptors for insulin receptors and IGF-1 receptors, and the like.
发明人经长期研究发现，将 IGF-1的 A链和 B链以胰岛素连接的方式连接， 且将 B链的 B15位氨基酸残基由 Q替换为 W或 F后，这样的 IGF-1类似物与胰岛素受体结合能力和人胰 岛素相当。  The inventors have found through long-term studies that the A chain and the B chain of IGF-1 are linked by insulin, and the amino acid residue of B15 of the B chain is replaced by Q or W or F, such an IGF-1 analogue. The ability to bind to the insulin receptor is comparable to that of human insulin.
进一步地，本发明的与胰岛素受体高度结合的 IGF-1类似物是 IGF-1的 B链在 B15和 B16 两个位置上的氨基酸残基被取代。 当原 B15和 B16位置上的 QF由 FF、 WF或 WW取代后， 新得到的 IGF-1类似物也表现出与天然胰岛素相当的与胰岛素受体结合的性能。 由 FF、 WF 或 WW取代的 IGF-1类似物还保持了部分与 IGF-1结合蛋白结合的能力。 IGF-1类似物结合 IGF-1结合蛋白的这种能力被认为有助于延长 IGF-1类似物在血清中循环和作用的时间。  Further, the IGF-1 analog of the present invention which is highly bound to the insulin receptor is such that the B chain of IGF-1 is substituted at the two positions of B15 and B16. When the QF at the original B15 and B16 positions is replaced by FF, WF or WW, the newly obtained IGF-1 analogue also exhibits an insulin receptor binding performance comparable to that of natural insulin. IGF-1 analogs substituted with FF, WF or WW also retain some of their ability to bind to IGF-1 binding proteins. This ability of IGF-1 analogs to bind to IGF-1 binding proteins is believed to help prolong the time of circulation and action of IGF-1 analogs in serum.
包括人 IGF-1 A链和人胰岛素 B链的双链类似物在胰岛素样活性实验（如脂肪生成） 中 显示出相当于胰岛素能力的大约 40%, 但是在生长因子实验（如胸苷掺入） 中， 活性明显比 胰岛素高，相当于大约 730%。但是，该化合物是比 IGF-1本身低效的生长因子，相当于 IGF-1 的大约 26.5%„尽管已经证实 IGF-1的 C域是 IGF-1受体结合力的主要因素，但包括人 IGF-1 A链和人胰岛素 B链的双链类似物表现出减弱的但仍然明显的 IGF-1受体结合力，显示 IGF-1 的 A域中包含的结构性质造成升高的促生长能力。 而多项研究表明， 过分活跃的 IGF-1受体 可能导致癌症。 Double-stranded analogs including the human IGF-1 A chain and the human insulin B chain show approximately 40% of insulin capacity in insulin-like activity assays (eg, lipogenesis), but in growth factor assays (eg, thymidine incorporation) Among them, the activity is significantly higher than insulin, which is equivalent to about 730%. However, this compound is an inefficient growth factor than IGF-1 itself, equivalent to IGF-1. Approximately 26.5% of the time. Although the C domain of IGF-1 has been shown to be a major factor in IGF-1 receptor binding, double-stranded analogs including the human IGF-1 A chain and the human insulin B chain show attenuated but still Significant IGF-1 receptor binding indicates that the structural properties contained in the A domain of IGF-1 result in increased growth-promoting ability. Many studies have shown that overactive IGF-1 receptors may cause cancer.
胰岛素受体有两个亚型： IR-A和 IR-B。 IR-A有类似于 IGF-1受体的促生长作用，而 IR-B 的主要功能是代谢调节（如糖代谢)。 天然人胰岛素与两个受体亚型的结合能力基本相当。 而 IGF-1和 IGF-2对 IR-A的结合能力显著高于对 IR-B的结合能力。 IGF-1类似物仍然部分保持 了这种选择性。 而理想的 IGF-1类似物应该能够与胰岛素受体高度结合， 但与 IGF-1受体结 合的能力较低。 另外， IGF-1类似物在胰岛素受体两个亚型 IR-A和 IR-B上应该有均衡的生 物活性。  There are two subtypes of insulin receptors: IR-A and IR-B. IR-A has a growth-promoting effect similar to that of the IGF-1 receptor, and the main function of IR-B is metabolic regulation (such as glucose metabolism). Natural human insulin has essentially the same binding capacity as the two receptor subtypes. The binding ability of IGF-1 and IGF-2 to IR-A was significantly higher than that of IR-B. The IGF-1 analog still partially retains this selectivity. Ideal IGF-1 analogues should be able to bind highly to the insulin receptor but have a lower ability to bind to the IGF-1 receptor. In addition, IGF-1 analogues should have a balanced biological activity on the two insulin subtypes IR-A and IR-B.
IGF-1 A链和胰岛素 A链具有高度的序列同源性。 胰岛素 A链和 IGF-1 A链的序列中的 两个明显区别是 A5Gln-A5Glu和 A12Ser-A12Asp。 这些残基在 IGF-1和 IGF-2中是保守的。 序列中的这些变化涉及胰岛素中的中性残基和 IGF-1中的酸性残基之间的变化。发明人发现， A链 A5位谷氨酸和 A12位的天冬氨酸侧链羧基是决定受体选择性的关键。将 A5位谷氨酸转 换为谷氨酰胺、 天冬酰胺或丝氨酸， 将 A12位天冬氨酸转换为丝氨酸、 谷氨酰胺或天冬酰胺 等有近似大小和极性， 但没有带负电荷官能团的侧链的氨基酸残基， 由此得到的类似物有更 好的受体选择性。 A5和 A10同时取代比二者之一的单一取代效果更明显。  The IGF-1 A chain and the insulin A chain have a high degree of sequence homology. Two distinct differences in the sequences of the insulin A chain and the IGF-1 A chain are A5Gln-A5Glu and A12Ser-A12Asp. These residues are conserved in IGF-1 and IGF-2. These changes in the sequence involve changes between neutral residues in insulin and acidic residues in IGF-1. The inventors have found that the A chain glutamic acid at the A chain and the aspartic acid side chain carboxyl group at the A12 position are critical for determining receptor selectivity. Conversion of glutamic acid at position A5 to glutamine, asparagine or serine, conversion of aspartic acid at position A12 to serine, glutamine or asparagine with similar size and polarity, but no negatively charged functional groups The amino acid residues of the side chain, the resulting analog has better receptor selectivity. The simultaneous substitution of A5 and A10 is more pronounced than the single substitution effect of either.
基于以上发明构思， 本发明提供一种具有降血糖作用的化合物， 所述化合物包括 A链和 B链， 其中，  Based on the above inventive concept, the present invention provides a compound having a hypoglycemic action, the compound comprising an A chain and a B chain, wherein
A链的氨基 列为： X^XoGrVDXsCCTCwXsXsXioCwXnLRRLEXwYCwX Xss ,  The amino group of the A chain is: X^XoGrVDXsCCTCwXsXsXioCwXnLRRLEXwYCwX Xss ,
B链氨基酸序列为：  The B chain amino acid sequence is:
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFX47X48 X49 X50 X51 X52 X53, 其中， ！是赖氨酸、 精氨酸或缺失； 是赖氨酸、 精氨酸或缺失； X5是谷氨酸、 天冬酰胺、 谷氨酰胺或丝氨酸； X8是组氨酸、 精氨酸、 苯丙氨酸或苏氨酸； X9是精氨酸或丝氨酸； X10 是丝氨酸或异亮氨酸； X12是天冬氨酸、 丝氨酸、 谷氨酰胺或天冬酰胺； X18是天冬酰胺、 曱 硫氨酸或苏氨酸； X21是天冬酰胺、 丙氨酸或甘氨酸； X22是赖氨酸、 精氨酸-赖氨酸二肽或缺 失； X23-26是苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽， 或甘氨酸 -脯氨酸-谷氨酸、 缬氨酸- 天冬酰胺-谷氨酰胺三肽， 或脯氨酸-谷氨酸、 天冬酰胺-谷氨酰胺二肽， 或谷氨酸、 谷氨酰胺、 赖氨酸或精氨酸， 或是以赖氨酸或精氨酸取代上述二、 三、 四肽序列中任何一个氨基酸残基 后的序列， 或缺失； X27是组氨酸或苏氨酸； X32是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯 丙氨酸； x38是苯丙氨酸或色氨酸； x39是苯丙氨酸或色氨酸； 4是精氨酸、 谷氨酸、 天冬 氨酸或丙氨酸； 7是苯丙氨酸、 酪氨酸或组氨酸； 8 -NH2、 dA-NH2、 苯丙氨酸或酪氨酸 或缺失； 9是天冬酰胺、 天冬氨酸、 谷氨酸、 苏氨酸或缺失； X5G是赖氨酸、 脯氨酸、 精氨 酸、 谷氨酸、 天冬氨酸或缺失； X51是脯氨酸、 赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸或缺失； X52是苏氨酸或缺失； X53是谷氨酸、 天冬氨酸、 谷氨酸-谷氨酸、 天冬氨酸-天冬氨酸二肽或 缺失； X 2 3- 2 6X27LC [1] GAX 32 LVDALX 38 X 39 VC [2] GDX 4 4GFX 47 X4 8 X49 X 50 X51 X52 X 53 , where, ! Is lysine, arginine or deletion; is lysine, arginine or deletion; X 5 is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine, Phenylalanine or threonine; X 9 is arginine or serine; X 10 is serine or isoleucine; X 12 is aspartic acid, serine, glutamine or asparagine; X 18 is day Asparagine, methionine or threonine; X 21 is asparagine, alanine or glycine; X 22 is lysine, arginine-lysine dipeptide or deletion; X23-26 is phenylpropanoid -proline-asparagine-glutamine tetrapeptide, or glycine-valine-glutamate, proline-asparagine-glutamine tripeptide, or proline-glutamic acid , asparagine-glutamine dipeptide, or glutamic acid, glutamine, lysine or arginine, or lysine or arginine to replace any of the above two, three, or tetrapeptide sequences the sequence of amino acid residues, or deleted; X 27 is histidine or threonine; X 32 is histidine, glutamic acid, glutamine, arginine or phenylalanine; x 38 is Or tryptophan; x 39 is phenylalanine or tryptophan; 4 is arginine, glutamic acid, aspartic acid or alanine; 7 is phenylalanine, tyrosine, or Histidine; 8 -NH2, dA-NH 2 , phenylalanine or tyrosine or deletion; 9 is asparagine, aspartic acid, glutamic acid, threonine or deletion; X 5G is lysine Acid, proline, arginine, glutamic acid, aspartic acid or deletion; X 51 is valine, lysine, arginine, glutamic acid, aspartic acid or deletion; X 52 is Threonine or deletion; X 53 is glutamic acid, aspartic acid, glutamic acid-glutamic acid, aspartic acid-aspartic acid dipeptide or deletion;
所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物中通过 6个半胱氨酸形成 3对 二硫键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫 键的具体位置是： Cfl]和 C[4]形成二硫键， C[2]和 C[61形成二硫键， C[3]和 C[5]形成二硫键。 In the compound, [1]-[6] represents the number of a cysteine; in the compound, three pairs of disulfide bonds are formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains. Sulfur-bonded, there is a pair of intrachain disulfide bonds in the A chain. The specific positions of the three pairs of disulfide bonds are: C fl] and C [4] form disulfide bonds, C [2] and C [61 form disulfide The bonds, C [3 ] and C [5] form a disulfide bond.
需要特别指出的是， X32、 4、 9、 X50、 X51的氨基酸残基关系到化合物是否像人胰岛 素一样产生自联（ self association )。 人胰岛素一般通过自联形成六聚体存储在胰岛 β细胞中。 重组人胰岛素分子皮下注射后逐渐由六聚体解聚成二聚体， 进一步解离为单体才能透过毛细 血管进入循环， 发挥降糖作用。 由于存在解聚、 吸收过程， 重组人胰岛素在皮下注射后起效 时间长 (参照 Brange等 "Monomeric insulins and their experimental and clinical implications" Diabetes Care, Vol 13 No.9, 923-54, 1990 )。 如果 X32是组氨酸， 那么有利于化合物在锌离子的 协助下形成六聚体结构。 如果 X32是天冬氨酸、 谷氨酸、 苯丙氨酸、 谷氨酰胺、 精氨酸等氨 基酸残基， 则无法形成稳定的六聚体结构。 如果 4、 X5o、 51等位点的氨基酸残基是天冬氨 酸或谷氨酸， 也不易形成稳定的自联。 因此， 如果 X32是非组氨酸氨基酸残基， 或 X44、 X50、 X51等位点的氨基酸残基之一或多个位点是天冬氨酸或谷氨酸，则相应化合物更易于以二聚体 或单体形式存在， 皮下注射后快速进入血液， 可以达到在短时间内降低血糖的效果。 It is important to note that the amino acid residues of X 32 , 4 , 9 , X 50 , X 51 are related to whether the compound resembles human islets The same as self-association. Human insulin is typically stored in islet beta cells by self-association to form hexamers. After subcutaneous injection of recombinant human insulin molecules, the hexamers are gradually depolymerized into dimers, and further dissociated into monomers to enter the circulation through the capillaries, and play a hypoglycemic effect. Recombinant human insulin has a long-lasting effect after subcutaneous injection due to the presence of depolymerization and absorption processes (see Brange et al. "Monomeric insulins and their experimental and clinical implications" Diabetes Care, Vol 13 No. 9, 923-54, 1990). If X 32 is histidine, it is advantageous for the compound to form a hexamer structure with the aid of zinc ions. If X 32 is an amino acid residue such as aspartic acid, glutamic acid, phenylalanine, glutamine or arginine, a stable hexamer structure cannot be formed. If the amino acid residues of the 4 , X 5 o, and 51 sites are aspartic acid or glutamic acid, stable self-association is not easily formed. Therefore, if X 32 is an unhistidine amino acid residue, or one or more of the amino acid residues of X44, X 5 0, X 51 and the like are aspartic acid or glutamic acid, the corresponding compound is more It is easy to exist in the form of dimer or monomer, and it can quickly enter the blood after subcutaneous injection, which can achieve the effect of lowering blood sugar in a short time.
在本方面的一个优选实施方式中，所述具有降血糖作用的化合物包括 A链和 B链，其中， A链的氨基 ^^列为： GIVDX5C[3]C[4]X8RSC[51X12L RLEX18YC[6]X21X22, In a preferred embodiment of the present invention, the hypoglycemic compound comprises an A chain and a B chain, wherein the amino group of the A chain is: GIVDX 5 C[ 3] C [4] X 8 RSC [ 51 X 12 L RLEX 18 YC [6] X 21 X 22 ,
B链氨基酸序列为：  The B chain amino acid sequence is:
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFYX48X49 X50 X51 X52， 其中， X23-2 6 X27LC [1 ]GAX 32 LVDALX 38 X 39 VC [2] GDX 4 4GFYX4 8 X 49 X 50 X 51 X 52 , where
X5是谷氨酸、 天冬酰胺、 谷氨酰胺或丝氨酸； X8是组氨酸、 精氨酸或苯丙氨酸； 12是 天冬氨酸、 丝氨酸、 谷氨酰胺或天冬酰胺； x18是天冬酰胺、 曱硫氨酸或苏氨酸； x21是天冬 酰胺、 丙氨酸或甘氨酸； x22是赖氨酸、 精氨酸-赖氨酸二肽或缺失； X2 6是甘氨酸 -脯氨酸- 谷氨酸三肽或苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽； x27是组氨酸或苏氨酸； x32是组氨 酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨酸； x38是苯丙氨酸或色氨酸； x39是苯丙氨酸或色 氨酸； 4是精氨酸、 谷氨酸、 天冬氨酸或丙氨酸； 8是 -NH2、 dA-N¾或苯丙氨酸； 9是 天冬酰胺或缺失； X5Q是赖氨酸、 脯氨酸或缺失； x51是脯氨酸、 赖氨酸或缺失； x52是苏氨 酸或缺失； X 5 is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine or phenylalanine; 12 is aspartic acid, serine, glutamine or asparagine; x 18 is asparagine, methionine or threonine; x 21 is asparagine, alanine or glycine; x 22 is lysine, arginine-lysine dipeptide or deletion; X 2 6 is a glycine - proline - tripeptide glutamic acid or phenylalanine - valine - asparagine - glutamine tetrapeptide; x 27 is histidine or threonine; x 32 histidine , glutamic acid, glutamine, arginine or phenylalanine; x 38 is phenylalanine or tryptophan; x 39 is phenylalanine or tryptophan; 4 is arginine, glutamine Acid, aspartic acid or alanine; 8 is -NH 2 , dA-N3⁄4 or phenylalanine; 9 is asparagine or deletion; X 5Q is lysine, valine or deletion; x 51 is Proline, lysine or deletion; x 52 is threonine or a deletion;
所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物中通过 6个半胱氨酸形成 3对 二硫键， 其中 A链和 B链通过两对链间二砥键连接， A链内存在一对链内二硫键， 三对二硫 键的具体位置是： 和 C[4]形成二硫键， C[2]和 C[6]形成二硫键， C[3]和 C[5]形成二硫键。 In the compound, [1]-[6] represents the number of a cysteine; in the compound, three pairs of disulfide bonds are formed by six cysteines, wherein the A chain and the B chain pass through two pairs of interchains. The 砥 bond is connected, there is a pair of intrachain disulfide bonds in the A chain, and the specific positions of the three pairs of disulfide bonds are: forming a disulfide bond with C [4 ], and forming a disulfide bond by C [2 ] and C [6] , C [3] and C [5] form a disulfide bond.
在进一步优选的实施方式中， 所述 B链的序列是：  In a further preferred embodiment, the sequence of the B chain is:
GPEX27LCGAX32LVDALX38X39VCGDX44GFY-NH2; GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFY-NH 2 ;
在进一步优选的实施方式中， 所述 B链的序列是：  In a further preferred embodiment, the sequence of the B chain is:
GPEX27LCGAX32LVDALX38X39VCGDX44GFYFNKPT; GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFYFNKPT;
在进一步优选的实施方式中， 所述 B链的序列是：  In a further preferred embodiment, the sequence of the B chain is:
GPEX27LCGAX32LVDALX38X39VCGDX44GFYdA-NH2; GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44GFYdA-NH 2 ;
在进一步优选的实施方式中， 所述 B链的序列是：  In a further preferred embodiment, the sequence of the B chain is:
FVNQX27LCGAX32LVDALX3gX39VCGDX44GFYFNKPT; FVNQX 27 LCGAX 32 LVDALX 3g X 39 VCGDX44GFYFNKPT;
在进一步优选的实施方式中， 所述 A链的序列是：  In a further preferred embodiment, the sequence of the A chain is:
GIYDX5CCX8RSCX12LRRLEX18YCA; GIYDX 5 CCX 8 RSCX 12 LRRLEX 18 YCA;
在进一步优选的实施方式中， 所述 A链的序列是：  In a further preferred embodiment, the sequence of the A chain is:
GIVDX5CCX8RSCXi2LRRLEXi8YCN; GIVDX 5 CCX 8 RSCXi 2 LRRLEXi 8 YCN;
在这些进一步施方式中， A链中的 X5是谷氨酸、 天冬酰胺、 谷氨酰胺或丝氨酸； X8是 组氨酸、 精氨酸或苯丙氨酸； X12是天冬氨酸、 丝氨酸、 谷氨酰胺或天冬酰胺； 18是天冬酰 胺、 曱硫氨酸或苏氨酸； B链中的 X27是组氨酸或苏氨酸； X32是组氨酸、 谷氨酸、 谷氨跣胺、 精氨酸或苯丙氨酸； X38是苯丙氨酸或色氨酸； X39是苯丙氨酸或色氨酸； 4是精氨酸、 谷 氨酸、 天冬氨酸或丙氨酸。 In these further embodiments, X 5 in the A chain is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine or phenylalanine; X 12 is aspartame Acid, serine, glutamine or asparagine; 18 is asparagine, methionine or threonine; X 27 in the B chain is histidine or threonine; X 32 is histidine, valley Amino acid, glutamine, Arginine or phenylalanine; X 38 is phenylalanine or tryptophan; X 39 is phenylalanine or tryptophan; 4 is arginine, glutamic acid, aspartic acid or alanine acid.
在这一方面， 与胰岛素受体具有高度结合能力的化合物包括 A链和 B链， A链和 B链通 过两对链间二硫键连接， A链内存在一对链内二硫键， 具体为： Cw 和 C[4] 形成二 υ键， C[2] 和。[6] 形成二硫键， C[3] 和 C[5]形成二石充键； 该化合物选自以下双链多肽： In this respect, a compound having high binding ability to the insulin receptor includes an A chain and a B chain, and the A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain. For: C w and C [4] form a double bond, C [2] and . [ 6] forming a disulfide bond, C [3] and C [5] forming a disulfide bond; the compound is selected from the following double-stranded polypeptides:
1-1 : 其中 A链序列为 GIVDECCFRSCDLRRLEMYCA ( SEQ ID NO: 1 ); B链的序列为 GPETLCGAELVDALFFVCGDRGFY-NHz ( SEQ ID NO:4 );  1-1: wherein the A chain sequence is GIVDECCFRSCDLRRLEMYCA (SEQ ID NO: 1); the B chain sequence is GPETLCGAELVDALFFVCGDRGFY-NHz (SEQ ID NO: 4);
1-2： 其 中 A 链序 列 为 SEQ ID NO: 1 所示序 列 ； B 链的序 列 为 GPETLCGAELVDALWFVCGDRGFY-NH2 ( SEQ ID NO:5 ); 1-2: wherein the A chain sequence is the sequence of SEQ ID NO: 1; the B chain has the sequence GPETLCGAELVDALWFVCGDRGFY-NH 2 (SEQ ID NO: 5);
1-3 ： 其 中 A 链序 列 为 SEQ ID NO: 1 所示序 列 ； B 链的序 列 为 1-3 : wherein the A chain sequence is the sequence shown in SEQ ID NO: 1; the sequence of the B chain is
GPETLCGAELVDALWWVCGDRGFY-NH2 ( SEQ ID NO:6 ); GPETLCGAELVDALWWVCGDRGFY-NH 2 (SEQ ID NO: 6);
1-4: 其中 A链序列为 GIVDECCFRSCDLRRLEMYCN ( SEQ ID NO: 7 )； B链的序列为 GPETLCGAELVDALFFVCGDRGFYFNKPT ( SEQ ID NO:3 );  1-4: wherein the A chain sequence is GIVDECCFRSCDLRRLEMYCN (SEQ ID NO: 7); the B chain has the sequence GPETLCGAELVDALFFVCGDRGFYFNKPT (SEQ ID NO: 3);
1-5:其中 A链序列为 GIVDECCFRSCDLR LENYCA( SEQ ID NO: 8 )； B链的序列为 SEQ ID NO:3所示序列；  1-5: wherein the A chain sequence is GIVDECCFRSCDLR LENYCA (SEQ ID NO: 8); the sequence of the B chain is the sequence shown in SEQ ID NO:
1-6: 其中 A链序列为 GIVDECCFRSCDLRRLETYCA ( SEQ ID NO:9 )； B链的序列为 SEQ ID NO:3所示序列；  1-6: wherein the A chain sequence is GIVDECCFRSCDLRRLETYCA (SEQ ID NO: 9); the sequence of the B chain is the sequence shown in SEQ ID NO:
1-7: 其中 A链序列为 GIVDECCRRSCDLRRLENYCN ( SEQ ID NO: 10 ); B链的序列为 SEQ ID NO:3所示序列；  1-7: wherein the A chain sequence is GIVDECCRRSCDLRRLENYCN (SEQ ID NO: 10); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-8: 其中 A链序列为 GIVDECCHRSCDLRRLENYCN ( SEQ ID NO: 11 ); B链的序列为 1-8: wherein the A chain sequence is GIVDECCHRSCDLRRLENYCN (SEQ ID NO: 11); the sequence of the B chain is
SEQ ID NO:3所示序列； SEQ ID NO: 3 sequence;
1-9: 其中 A链序列为 GIVDQCCFRSCDLRRLENYCA ( SEQ ID NO: 12 ); B链的序列为 SEQ ID NO:3所示序列；  1-9: wherein the A chain sequence is GIVDQCCFRSCDLRRLENYCA (SEQ ID NO: 12); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-10: 其中 A链序列为 GIVDNCCFRSCDLRRLENYCA ( SEQ ID NO: 13 ); B链的序列 为 SEQ ID NO:3所示序列；  1-10: wherein the A chain sequence is GIVDNCCFRSCDLRRLENYCA (SEQ ID NO: 13); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-11 : 其中 A链序列为 GIVDECCFRSCSLRRLENYCA ( SEQ ID NO: 14 ); B链的序列为 SEQ ID NO:3所示序列；  1-11: wherein the A chain sequence is GIVDECCFRSCSLRRLENYCA (SEQ ID NO: 14); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-12: 其中 A链序列为 GIVDECCFRSCNLRRLENYCA ( SEQ ID NO: 15 ); B链的序列 为 SEQ ID NO:3所示序列；  1-12: wherein the A chain sequence is GIVDECCFRSCNLRRLENYCA (SEQ ID NO: 15); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-13: 其中 A链序列为 GIVDECCFRSCQLRRLENYCA ( SEQ ID NO: 16 ); B链的序列 为 SEQ ID NO:3所示序列；  1-13: wherein the A chain sequence is GIVDECCFRSCQLRRLENYCA (SEQ ID NO: 16); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-14: 其中 A链序列为 GIVDQCCFRSCSLRRLENYCA ( SEQ ID NO: 17 )； B链的序列为 SEQ ID NO:3所示序列；  1-14: wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 17); the sequence of the B chain is the sequence shown in SEQ ID NO: 3;
1-15： 其中 A 链序列 为 SEQ ID NO: 17 所示序列 ； B 链的序列 为 GPETLCGAHLVDALFFVCGDRGFYF KPT ( SEQ ID NO: 18 );  1-15: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain has the sequence GPETLCGAHLVDALFFVCGDRGFYF KPT (SEQ ID NO: 18);
1-16: 其中 A链序列为 GIVDQCCFRSCSLRRLENYCAK ( SEQ ID NO: 19 ); B链的序列 为 SEQ ID NO: 18所示序列；  1-16: wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCAK (SEQ ID NO: 19); the sequence of the B chain is the sequence shown in SEQ ID NO: 18.
1-17: 其中 A链序列为 GIVDQCCFRSCSLRRLENYCARK ( SEQ ID NO: 20 ); B链的序 列为 SEQ ID NO: 18所示序列；  1-17: wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCARK (SEQ ID NO: 20); the sequence of the B chain is the sequence shown as SEQ ID NO: 18.
1-18： 其 中 A 链序列 为 SEQ ID NO: 17 所示序列 ； B 链的序列 为 GPETLCGAHLVDALFFVCGDRGFYdA-NH2 ( SEQ ID NO:21 ); 1-18: wherein the A chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPETLCGAHLVDALFFVCGDRGFYdA-NH 2 (SEQ ID NO: 21);
1-19:其 中 A 链序 列 为 SEQ ID NO: 17 所示序 列 ； B 链的序 列 为 GPEHLCGARLVDALFFVCGDRGFYFNKPT ( SEQ ID NO:22 );  1-19: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is GPEHLCGARLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 22);
1-20:其 中 A 链序 列 为 SEQ ID NO: 17 所示序 列 ； B 链的序 列 为 GPEHLCGAFLVDALFFVCGDRGFYFNKPT ( SEQ ID NO:23 );  1-20: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAFLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 23);
1-21 : 其 中 A 链序 列 为 SEQ ID NO: 17 所 示序 列 ； B 链的 序 列 为 GPEHLCGAHLVDALFFVCGDEGFYFNKPT ( SEQ ID NO:24 );  1-21: wherein the A chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAHLVDALFFVCGDEGFYFNKPT (SEQ ID NO: 24);
1-22: 其 中 A 链序 列 为 SEQ ID NO: 8 所 示序 列 ； B 链的 序 列 为 FVNQHLCGAHLVDALFFVCGDRGFYFNKPT ( SEQ ID NO:25 );  1-22: wherein the A chain sequence is the sequence shown in SEQ ID NO: 8; the sequence of the B chain is FVNQHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 25);
1-23:其中 A链序列为 GIVDQCCHRSCSLRRLENYCA (SEQ ID NO:26 ); B链的序列为 SEQ ID NO: 25所示序列；  1-23: wherein the A chain sequence is GIVDQCCHRSCSLRRLENYCA (SEQ ID NO: 26); the sequence of the B chain is the sequence shown in SEQ ID NO: 25.
1-24:其中 A链序列为 SEQ ID NO:17所示序列; B链的序列为 SEQ ID NO:25所示序列; 1-24: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain has the sequence of SEQ ID NO: 25;
1-25: 其 中 A 链序 列 为 SEQ ID NO: 17 所 示序 列 ； B 链的 序 列 为 GPEHLCGAHLVDALFFVCGDAGFYFNKPT ( SEQ ID NO:27 ); 1-25: wherein the A chain sequence is the sequence shown by SEQ ID NO: 17; the B chain sequence is GPEHLCGAHLVDALFFVCGDAGFYFNKPT (SEQ ID NO: 27);
1-26:其 中 A 链序 列 为 SEQ ID NO: 17 所示序 列 ； B 链的序 列 为 GPEHLCGAQLVDALFFVCGDRGFYFNKPT ( SEQ ID NO:28 );  1-26: wherein the A chain sequence is the sequence shown in SEQ ID NO: 17; the sequence of the B chain is GPEHLCGAQLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 28);
1-27:其中 A链序列为 KGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 151 ); B链的序列为 GPEHLCGAHLVDALFFVCGDRGFYFNKPT ( SEQ ID NO: 152 );  1-27: wherein the A chain sequence is KGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 151); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 152);
1-28:其中 A链序列为 RGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 153)； B链的序列为 SEQ ID NO: 152所示序列；  1-28: wherein the A chain sequence is RGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 153); the sequence of the B chain is the sequence shown as SEQ ID NO: 152;
1-29:其中 A链序列为 KGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:154); B链的序列为 GPEHLCGAHLVDALFFVCGDRGFYFNPKT ( SEQ ID NO: 155 );  1-29: wherein the A chain sequence is KGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 154); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNPKT (SEQ ID NO: 155);
1-30:其中 A链序列为 RGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 156 )； B链的序列 为 SEQ ID NO: 155所示序列； 为 SEQ ID NO: 155所示序列；  1-30: wherein the sequence of the A chain is RGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 156); the sequence of the B chain is the sequence of SEQ ID NO: 155; the sequence of SEQ ID NO: 155;
1-32:其中 A链序列为 KKGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 158); B链的序列 为 SEQ ID NO: 155所示序列；  1-32: wherein the A chain sequence is KKGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 158); the sequence of the B chain is the sequence shown in SEQ ID NO: 155;
1-33: 其中 A链序列为 GIVDQCCHRSCSLRRLENYCN(SEQ ID NO:159); B链的序列为 GPEHLCGAHLVDALFFVCGDRGFYFNPKTE ( SEQ ID NO: 160 );  1-33: wherein the A chain sequence is GIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 159); the B chain has the sequence GPEHLCGAHLVDALFFVCGDRGFYFNPKTE (SEQ ID NO: 160);
1-34: 其中 A链序列为 SEQ ID NO: 159所示序列； B链的序列为 SEQ ID NO:155所示序 列。 单链化合物  1-34: wherein the A chain sequence is the sequence shown in SEQ ID NO: 159; and the B chain sequence is the sequence shown in SEQ ID NO: 155. Single chain compound
在哺乳动物中，胰岛素在胰腺中的胰岛的 β细胞中合成。胰岛素原是含有 86个氨基酸的 的单链前体，构造为： Β链 -ArgArg-C肽 -LysArg-A链。 C肽是由 31个氨基酸组成的"连接肽"。 Arg-Arg和 Lys-Arg是蛋白水解酶作用使 C肽从 A和 B链中分裂的分裂点， 已知蛋白水解酶 是激素原转化酶（PC1和 PC2 ),以及外型蛋白酶羧肽酶£。胰岛素原的这些改变移去了 C肽。 剩余的 B链和 A链通过二硫键结合在一起。  In mammals, insulin is synthesized in beta cells of islets in the pancreas. Proinsulin is a single-stranded precursor of 86 amino acids constructed as: Β chain -ArgArg-C peptide -LysArg-A chain. The C peptide is a "linker peptide" composed of 31 amino acids. Arg-Arg and Lys-Arg are the cleavage sites for proteolytic enzymes to cleave C-peptide from the A and B chains. Proteolytic enzymes are known to be prohormone convertases (PC1 and PC2), and exoproteinase carboxypeptidases are £ . These changes in proinsulin removed the C peptide. The remaining B chain and A chain are bonded together by a disulfide bond.
胰岛素的双链结构使得胰岛素具有多种构象。 胰岛素具有相当大的构象变化的潜能， 对 这些变化的限制明显地降低胰岛素受体对配体的亲合力。封闭 GlyAl的氨基端同样削弱受体 结合能力。 胰岛素原与胰岛素受体亲和力只有胰岛素的 1-2%。 The double-stranded structure of insulin allows insulin to have multiple conformations. Insulin has the potential for considerable conformational changes, Limitations of these changes significantly reduce the affinity of the insulin receptor for the ligand. Blocking the amino terminus of GlyAl also impairs receptor binding ability. Proinsulin and insulin receptor affinity is only 1-2% of insulin.
目前还不清楚 C肽在胰岛素原折叠中的作用。 在不同动物种类中 C肽的长度在 26-38个 氨基酸之间变化。 在 B链 -C肽（ B-C )和 C肽 -A链（ C-A )连接处的二元氨基酸残基是保守 的， 并认为对于胰岛素保守性的需要是最小的。 胰岛素的三维结构显示 A链和 B链可以通过 比 31个氨基酸的 C肽小很多的连接肽结合。  The role of C-peptide in proinsulin folding is still unclear. The length of the C peptide varies between 26 and 38 amino acids in different animal species. The dibasic amino acid residues at the junction of the B-chain-C peptide (B-C) and the C-peptide-A chain (C-A) are conserved and the need for insulin conservation is considered to be minimal. The three-dimensional structure of insulin shows that the A chain and the B chain can be bound by a linker peptide which is much smaller than the 31 amino acid C peptide.
胰岛素分子内在的物理和化学稳定性是糖尿病的胰岛素疗法的先决条件， 也是胰岛素构 象、 可应用的胰岛素给药方法以及药物制剂的保存期限和保存条件的基础。 在胰岛素给药时 使用溶液使得胰岛素分子暴露于多种因素， 如升高的温度、 气-液-固相间变化和剪切力， 可 能导致胰岛素分子不可恢复的构象变化， 如原纤化作用。 这与注射泵中的胰岛素溶液关系密 切， 因为无论是外用还是植入， 都将胰岛素分子暴露于这些因素以及来自泵长期移动过程中 的产生剪切力中。 因此， 当使用注射泵作为胰岛素递送系统时， 原纤维化作用是一个很大的 问题。 此外， 胰岛素的溶解度受多种因素影响， 并在 PH4.2-6.6的范围内明显降低。 pH沉降 区通常给配方带来限制。  The inherent physical and chemical stability of the insulin molecule is a prerequisite for insulin therapy in diabetes, and is also the basis for insulin conformation, applicable insulin delivery methods, and shelf life and storage conditions for pharmaceutical formulations. The use of solutions during insulin administration exposes insulin molecules to a variety of factors, such as elevated temperatures, gas-liquid-solid phase changes, and shear forces, which may result in irreversible conformational changes in insulin molecules, such as fibrillation. . This is closely related to the insulin solution in the syringe pump, because insulin molecules are exposed to these factors, both externally and implanted, and from the shear forces generated during long-term pump movement. Therefore, fibrillation is a big problem when using a syringe pump as an insulin delivery system. In addition, the solubility of insulin is affected by many factors and is significantly reduced in the range of pH 4.2-6.6. The pH settling zone typically imposes limitations on the formulation.
因此， 胰岛素的稳定性和可溶性是目前胰岛素治疗的重要因素。 本发明致力于这些问题， 通过在 B和 A链之间引入 C肽提供稳定的单链化合物以降低分子柔性并同时减少原纤维化倾 向， 限制或修改 pH沉降区。 此外， 目前基因工程生产胰岛素的主要方法是首先生产由胰岛 素 B链和 A链通过短肽首尾连接的单链胰岛素前体， 然后胰岛素前体被酶切后生成双链胰岛 素。 如果直接生产单链胰岛素类似物， 则大大简化了生产过程， 降低了成本。  Therefore, insulin stability and solubility are currently important factors in insulin therapy. The present invention addresses these problems by providing a stable single-stranded compound by introducing a C-peptide between the B and A chains to reduce molecular flexibility while reducing the fibrillation tendency, limiting or modifying the pH settling zone. In addition, the main method of genetic engineering for the production of insulin is to first produce a single-chain insulin precursor which is linked end-to-end by a short peptide of the insulin B chain and the A chain, and then the insulin precursor is digested to form a double-stranded insulin. If the single-chain insulin analog is directly produced, the production process is greatly simplified and the cost is reduced.
作为胰岛素家族的成员， 胰岛素样生长因子 -1 ( IGF-1 )是具有 70个氨基酸残基的单链 肽， 包含 A、 B、 C和 D域。 IGF-1的 A域和 B域的基本结构与胰岛素的 A链和 B链高度相 似， 分别有 52%和 45%同源性。 它们的三维结构也非常相似。  As a member of the insulin family, insulin-like growth factor-1 (IGF-1) is a single-chain peptide having 70 amino acid residues, including the A, B, C, and D domains. The basic structure of the A and B domains of IGF-1 is highly similar to the A and B chains of insulin, with 52% and 45% homology, respectively. Their three-dimensional structure is also very similar.
IGF-1的 C域在胰岛素受体结合中作用很小。 去掉全部的 IGF-1 C域， 用 4个甘氨酸组 成的桥取代， 导致胰岛素受体结合力与野生型相比增加两倍， 而将 IGF-1 C域加至胰岛素 B 链的 C末端引起胰岛素受体亲合力与野生型相比减少 3.5倍。 由胰 素和 IGF-1 C域组成的 单链胰岛素 /IGF-1混合体的胰岛素结合力与天然人胰岛素没有显著差异。 有意思的是， IGF-1 CII混合体对 IR-A和 IR-B都具有增加的亲合力， 而 IGF-2 CI具有较弱的亲合力， 显示 C域 决定 IR结合特异性。  The C domain of IGF-1 plays a minor role in insulin receptor binding. Removal of all IGF-1 C domains, replaced by a bridge of four glycines, resulted in a twofold increase in insulin receptor binding compared to wild type, while addition of the IGF-1 C domain to the C-terminus of the insulin B chain causes insulin Receptor affinity was reduced by a factor of 3.5 compared to wild type. The insulin binding capacity of the single-chain insulin/IGF-1 mixture consisting of the pancreatic and IGF-1 C domains was not significantly different from native human insulin. Interestingly, the IGF-1 CII hybrid has increased affinity for both IR-A and IR-B, while IGF-2 CI has a weaker affinity, indicating that the C domain determines IR binding specificity.
IGF-1中 Tyr31对于保持 IGF-1的受体高亲合力至关重要，但是它似乎阻碍与胰岛素受体 结合， 因为当酪氨酸被丙氨酸取代， 导致了很小但明显的人胎盘胰岛素受体结合的双倍增加。  Tyr31 in IGF-1 is essential for maintaining high affinity for IGF-1 receptors, but it appears to block binding to the insulin receptor, as tyrosine is replaced by alanine, resulting in a small but distinct human placenta A double increase in insulin receptor binding.
在本发明中， 发明人进一步设计、 合成并表征能够与胰岛素受体结合、 具有降血糖作用 的单链 IGF-1类似物。 所述 IGF-1类似物中使用 6-12个或 8-12个氨基酸的连接片段（ C链） 将 IGF-1的 B链类似物的 C末端和 IGF-1 A链类似物的 A1位连接起来， 所得单链 IGF-1类 似物表示为 B链 -C链 -A链。 理想的单链 IGF-1类似物应该对胰岛素受体有高度结合力， 实 现静电平衡； 有优异的热力学稳定性， 无自组装。  In the present invention, the inventors further designed, synthesized and characterized single-chain IGF-1 analogs capable of binding to the insulin receptor and having hypoglycemic action. A ligation fragment (C chain) of 6-12 or 8-12 amino acids is used in the IGF-1 analog to link the C-terminus of the B chain analog of IGF-1 to the A1 position of the IGF-1 A chain analog. The resulting single-chain IGF-1 analog is represented as a B chain-C chain-A chain. The ideal single-chain IGF-1 analogue should have a high binding capacity to the insulin receptor for electrostatic equilibrium; excellent thermodynamic stability and no self-assembly.
进一步地， 在这些 IGF-1类似物中， 所述 A链和 B链是 IGF-1的 A链和 B链， 或者它 们的类似物。 基于本发明的第一部分， 所述 B链是 B15位的氨基酸 Q (谷氨酰胺）被替换为 F (苯丙氨酸）或 W (色氨酸） 的 IGF-1的 B链改造体。 双链 IGF-1类似物中可以增强胰岛 素受体结合力， 改善受体选择性， 增加多肽水溶性和稳定性的各种氨基酸变化， 例如 A5、 A12、 A18、 A22、 B9等位点的变化， 也适用于单链 IGF-1类似物。 双链 IGF-1类似物中用于 改变化合物自联能力， 使之主要以二聚体或单体存在的氨基酸的选择和变化， 也适用于单链 IGF-1类似物。 Further, in these IGF-1 analogs, the A chain and the B chain are the A chain and B chain of IGF-1, or an analog thereof. Based on the first part of the invention, the B chain is a B chain remodeling of IGF-1 in which the amino acid Q (glutamine) at position B15 is replaced by F (phenylalanine) or W (tryptophan). Double-chain IGF-1 analogues can enhance insulin receptor binding, improve receptor selectivity, and increase various amino acid changes in water solubility and stability of peptides, such as changes in A5, A12, A18, A22, B9, etc. Also suitable for single-chain IGF-1 analogues. Used in double-stranded IGF-1 analogues The selection and variation of amino acids that alter the self-association ability of a compound, such as a dimer or a monomer, are also applicable to single-chain IGF-1 analogs.
连接片段  Connection fragment
发明人在研究中发现， 通过连接片段将 IGF-1分子的双链连接形成的单链化合物， 同样 具有胰岛素活性， 并且具有易制备、 提供更多的多肽修饰位点等优势。  The inventors found in the study that a single-stranded compound formed by ligation of a double strand of an IGF-1 molecule by a ligation fragment is also insulin-active, and has the advantages of being easy to prepare, providing more polypeptide modification sites, and the like.
连接片段 CL是 6-60个氨基酸的肽序列， 其中每一个氨基酸残基都独立选自甘氨酸、 丙 氨酸、 丝氨酸、 苏氨酸、 脯氨酸。 适用的连接片段 CL具有三点特征： 第一， 连接片段需要适 当的长度。 当 B链为 30个氨基酸全长时， 连接片段长度最好不少于 6个氨基酸； 当 B链为 25个氨基酸时， 连接片段长度最好不少于 10个氨基酸。 连接片段长度短于上迷氨基酸数目 或者长于 60个氨基酸时， 单链类似物的胰岛素受体结合能力有降低趋势； 第二， 连接片段最 好没有二级结构， 空间构象可以灵活变化； 第三， 连接片段本身没有生物活性， 但可以提供 多肽修饰位点， 如酰化、 糖基化等。 The ligation fragment C L is a 6-60 amino acid peptide sequence in which each amino acid residue is independently selected from the group consisting of glycine, alanine, serine, threonine, and valine. A suitable connecting segment C L has a three-point feature: First, the connecting segment requires an appropriate length. When the B chain is 30 amino acids in length, the length of the ligated fragment is preferably not less than 6 amino acids; when the B chain is 25 amino acids, the length of the ligated fragment is preferably not less than 10 amino acids. When the length of the ligated fragment is shorter than the number of amino acids or longer than 60 amino acids, the insulin receptor binding ability of the single-chain analog has a decreasing tendency. Second, the ligated fragment preferably has no secondary structure, and the spatial conformation can be flexibly changed; The ligation fragment itself is not biologically active, but can provide polypeptide modification sites such as acylation, glycosylation and the like.
用以上方法设计的连接片段 CL可以通过氨基酸残基取代和插入包含 1个或 1个以上天冬 氨酸、 谷氨酸、 精氨酸、 赖氨酸、 半胱氨酸或天冬酰胺。 CL可以包括 1、 2、 3、 4个天冬氨 酸、 谷氨酸、 精氨酸或赖氨酸以调节多肽序列的电荷平衡， 改善溶解度。 该序列可以包括 1、 2、 3、 4、 5个天冬酰胺和相同数量的丝氨酸或苏氨酸，从而组成构成 N糖基化所需的 N-X-S/T 共有序列 （ X为可编码的天然氨基酸）。 进一步地， 该肽还可以包含 1、 2、 3或 4个赖氨酸或 半胱氨酸， 其侧链氨基或巯基可以与脂肪酸、 聚乙二醇、 白蛋白等天然或合成的修饰基团通 过水解键或非水解键相连， 从而使修饰后的 IGF-1分子具有不同的物理、 化学和生物特性。 Design methods above ligated fragments C L may be substituted by amino acid residues and comprising at least one insert or an aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine. C L may include 1, 2, 3, 4 aspartic acid, glutamic acid, arginine or lysine to regulate the charge balance of the polypeptide sequence and improve solubility. The sequence may comprise 1, 2, 3, 4, 5 asparagine and the same amount of serine or threonine to form the NXS/T consensus sequence required for N-glycosylation (X is a codeable natural amino acid) ). Further, the peptide may further comprise 1, 2, 3 or 4 lysine or cysteine, and the side chain amino group or sulfhydryl group may be a natural or synthetic modifying group such as a fatty acid, polyethylene glycol or albumin. The modified IGF-1 molecules have different physical, chemical and biological properties by being linked by a hydrolysis bond or a non-hydrolysis bond.
根据一种实施方式， CL的 C末端氨基酸可以选自由甘氨酸-赖氨酸、 甘氨酸-精氨酸、 精 氨酸-精氨酸、 赖氨酸-赖氨酸、 精氨酸-赖氨酸、 赖氨酸-精氨酸、 脯氨酸-谷氨酰胺-苏氨酸、 脯氨酸-谷氨酰胺-赖氨酸、 或脯氨酸-谷氨酰胺 -精氨酸组成的组。 根据一种实施方式， CL的 C 末端氨基酸选自赖氨酸或精氨酸。 According to one embodiment, the C-terminal amino acid of C L may be selected from the group consisting of glycine-lysine, glycine-arginine, arginine-arginine, lysine-lysine, arginine-lysine a group consisting of lysine-arginine, valine-glutamine-threonine, valine-glutamine-lysine, or valine-glutamine-arginine. According to one embodiment, the C-terminal amino acid of C L is selected from the group consisting of lysine or arginine.
在具体的实施方式中， cL可以是以下多肽片段的全部或部分序列， 或者与以下多肽片段 有 1、 2或 3个氨基酸残基的差异， 或者与以下多肽片段有 70%、 80%、 90%类似， 或者是以 下多肽片段的全部或部分序列的 1、 2、 3、 4或 5次重复序列： In a specific embodiment, c L may be all or part of a sequence of a polypeptide fragment, or a difference of 1, 2 or 3 amino acid residues from the following polypeptide fragments, or 70%, 80% of the following polypeptide fragments, 90% similar, or 1, 2, 3, 4 or 5 repeats of all or part of the sequence of the following polypeptide fragments:
(GASPGGSSGS)„GR, 其中 n是 1、 2、 3、 4或 5; GSSGSSGPGSSR; GSSGSGSSAPQT; (GASPGGSSGS) „GR, where n is 1, 2, 3, 4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT;
GSGGAPSRSGSSR; GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR;GSGGAPSRSGSSR; GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR;
GSGSSSRAPPSAPSPQR； GSSSESPSGAPQT； GAGTPASGSAPGR； GSSPSGGSSAPQT； GSTSSTARSPGR； GAGPSGTASPSR； GSSTPSGAPQT； SSSSAPPPSAPSPSRAPQR；GSGSSSRAPPSAPSPQR; GSSSESPSGAPQT; GAGTPASGSAPGR; GSSPSGGSSAPQT; GSTSSTARSPGR; GAGPSGTASPSR; GSSTPSGAPQT; SSSSAPPPSAPSPSRAPQR;
GASPGTSSTSGR ； GSGSSSAAAPQT; GSGSSSAAPQT; GSGSSSAPQT; GSGSSSRRA;GASPGTSSTSGR ; GSGSSSAAAPQT; GSGSSSAAPQT; GSGSSSAPQT; GSGSSSRRA;
GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR;GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR;
GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR ； GPSGTSTSAPGR ； GAGSSSAPQT; SSSSAPSAPSPSRPQR ；GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR; GPSGTSTSAPGR; GAGSSSAPQT; SSSSAPSAPSPSRPQR;
GSGASSPTSPQR ； GSSPATSATPQT ； GAGSSSAPPPSAPSPSRAPGPSAPQR ；GSGASSPTSPQR ; GSSPATSATPQT ; GAGSSSAPPPSAPSPSRAPGPSAPQR ;
GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;GASTGSSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;
GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQ ; GSSPSGRSSSPGR;GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQ; GSSPSGRSSSPGR;
GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR； RREAEDGGGPGAGSSQRK； GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK ； SSSAPPPSAPSPSRAPGPSPQR ； SAASSSASSSSASSASAGR ； GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR ; GSGSSSRRAPQT; SSSSAASAASASSSASGR； SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT ; GGPSSGAPPPSPQT ; SSSAPPPSAPSPSRAPQT； GAGPSSGAPPPSPQT； GGGGAPQT; GAGGPSSGAPPPQT； GGPSSGAPPPSPSPSRPGPSPQR ； SSASSASSSSAGR ； SSASSSAASSSASSSASGR ； SSSGAPPPSPSRAPGPSPQR； GSGSASRGR; SSSSAASSASGR； SASASASASSASSGR； SASSPSPSAPSSPSPAS ； GPSSPSPSAPSSPSPASPSSGR； SSSAPPPASPSPSRAPGPQR； SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; SSPSASPSSPASPSSGR; GAPASPAPSAPAPAAPSGR； GPSSPSPSAPSSPSPASPSSAPQT； SSASSASSSSSASAGR； SAPSSPSPSAPSSPSASPSGR ； SSSAPPPSAPSPSAPQR ； GASSPSPSAPSSPSPASGR ； SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT ； GGGGAPQR ； GSGSSSAAR ； GSGSSAAPQ ； SSSSRRAPQR ； SSSGSGSSAPQR; SSGSGSSSAPQR; GSGSSSRS; SSSSRAPQR; GASPGGSSGSGR。 GSPAGSPSSSAGSSASASPASPGR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR; RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK; SSSAPPPSAPSPSRAPGPSPQR; SAASSSASSSSASSASAGR; GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR; GSGSSSRRAPQT; SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT; GAGPSSGAPPPSPQT; GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPSRPGPSPQR; SSASSASSSSAGR; SSASSSAASSSASSSASGR; SSSGAPPPSPSRAPGPSPQR; GSGSASRGR; SSSSAASSASGR; SASASASASSASSGR; SASSPSPSAPSSPSPAS; GPSSPSPSAPSSPSPASPSSGR; SSSAPPPASPSPSRAPGPQR; SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; SSPSASPSSPASPSSGR; GAPASPAPSAPAPAAPSGR; GPSSPSPSAPSSPSPASPSSAPQT; SSASSASSSSSASAGR; SAPSSPSPSAPSSPSASPSGR; SSSAPPPSAPSPSAPQR; GASSPSPSAPSSPSPASGR; SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT; GGGGAPQR ; GSGSSSAAR ; GSGSSAAPQ ; SSSSRRAPQR ; SSSGSGSSAPQR; SSGSGSSSAPQ R; GSGSSSRS; SSSSRAPQR; GASPGGSSGSGR.
基于上述研究结果， 本发明进一步提供一种具有降血糖效果的单链的化合物， 所述化合 物基于人 IGF-1的结构进行改造， 所述化合物的结构为：  Based on the above findings, the present invention further provides a single-chain compound having a hypoglycemic effect, the compound being modified based on the structure of human IGF-1, the structure of which is:
XioiaLC [！] GAX101bLVDALXlolcX1o1d C[2]GDRGFX1oleXio2Xi03 i04 i05 io6-CL-Gr DQC[3] C[4]X107RSC[5]SLRRLENYC[6]X108 X109 , 其中， XioiaLC [! ] GAX 101 bLVDALX lolc X 1 o 1 d C[ 2 ]GDRGFX 1 o le Xio2Xi 0 3 i 0 4 i05 io6-CL-Gr DQC[ 3 ] C[ 4 ]X 107 RSC[5]SLRRLENYC[ 6 ]X 108 X 10 9 , where
X1()la是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 甘氨酸-脯氨酸-谷氨酸 -组氨酸四肽或者苯 氨酸X 1()la is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine tetrapeptide or tyrosine
-缬氨酸-天冬酰胺 -谷氨酰胺-组氨酸五肽， 或是以赖氨酸或精氨酸取代上述四肽或五肽中的甘 氨酸-脯氨酸-谷氨酸或苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺中任一个氨基酸残基后的序列； X101b是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨酸； X101c是苯丙氨酸或色氨酸； X101d 是苯丙氨酸或色氨酸； Xnne是苯丙氨酸、 酪氨酸或组氨酸； X1()2是苯丙氨酸或缺失； 1()3是 天冬酰胺或缺失； X104是赖氨酸、 脯氨酸或缺失； X1Q5是脯氨酸、 赖氨酸或缺失； X106是苏 氨酸或缺失； X1()7是苯丙氨酸、 精氨酸或组氨酸； X1()8是丙氨酸、 甘氨酸或天冬酰胺； X109 是赖氨酸、 精氨酸-赖氨酸二肽或缺失； 是选自上述的连接片段。 - valine-asparagine-glutamine-histidine pentapeptide, or lysine or arginine to replace glycine-valine-glutamic acid or phenylpropanoid in the above tetrapeptide or pentapeptide Sequence after any amino acid residue in lysine-valine-asparagine-glutamine; X101b is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 101c Is phenylalanine or tryptophan; X 101d is phenylalanine or tryptophan; Xnne is phenylalanine, tyrosine or histidine; X 1() 2 is phenylalanine or deletion; 1()3 is asparagine or deletion; X 104 is lysine, valine or deletion; X 1Q5 is valine, lysine or deletion; X 106 is threonine or deletion; X 1() 7 is phenylalanine, arginine or histidine; X 1 () 8 is alanine, glycine or asparagine; X 109 is lysine, arginine-lysine dipeptide or deletion; It is selected from the above-described linker fragments.
上述化合物结构中， [1]- [6]表示半胱氨酸的编号。 本发明的单链化合物在三级结构中， 以胰岛素的结构方式形成链内的二硫键， 具体为： Cw和 C[4]形成二硫键， C[2]和 C[6]形成二硫 键， C[3] 和 C[5]形成二石充键。 In the above compound structure, [1] - [6] represent the number of cysteine. In the tertiary structure of the single-chain compound of the present invention, a disulfide bond in the chain is formed by the structure of insulin, specifically: C w and C [4] form a disulfide bond, and C [2] and C [ 6 ] are formed. The disulfide bond, C [3 ] and C[ 5 ] form a two-stone charge.
在本方面的一个优选实施方式中， 所述单链化合物的结构为：  In a preferred embodiment of this aspect, the structure of the single chain compound is:
X!O!aLCnjGAHLVDALFFVC^!GDRGFYX!o^!oaXi^XiosXioe-CL-GIVDQC^jC^FRSCisjSL RRLENYC[6]A X109, 其中， X!O!aLCnjGAHLVDALFFVC^!GDRGFYX!o^!oaXi^XiosXioe-CL-GIVDQC^jC^FRSCisjSL RRLENYC [6] AX 109 , where
X10la是甘氨酸 -脯氨酸-谷氨酸-苏氨酸四肽或者笨丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组 氨酸五肽； X1()2是苯丙氨酸或缺失； X1Q3是天冬酰胺或缺失； X1Q4是赖氨酸、 脯氨酸或缺失； Χι05是脯氨酸、 赖氨酸或缺失； X1Q6是苏氨酸或缺失； X1G9是赖氨酸、 精氨酸-赖氨酸二肽或 缺失； 是选自上述的连接片段。 X 10 la is a glycine-valine-glutamate-threonine tetrapeptide or a strepyl-alanine-valine-asparagine-glutamine-histidine pentapeptide; X 1 ( ) 2 is benzene Alanine or deletion; X 1Q3 is asparagine or deletion; X 1Q4 is lysine, valine or deletion; Χι 05 is valine, lysine or deletion; X 1Q6 is threonine or deletion; X 1G9 is a lysine, arginine-lysine dipeptide or a deletion; it is a linking fragment selected from the above.
在进一步的实施方式中， 所述单链化合物的结构是：  In a further embodiment, the structure of the single chain compound is:
GPETLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFY-C L -GIVDQCCFRSCSLRRLENYCA;
在进一步的实施方式中， 所述单链化合物的结构是： :π-πIn a further embodiment, the structure of the single chain compound is: : π-π
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V0AN3TOnS S¾;ICO0aAI£a0<iVSSSOSE)SS入： ΙΟΉαθ Λ Ί ναΛΊΗνΕΌΊ丄 3dD S I V0AN3TOnS S3⁄4; ICO0aAI£a0<iVSSSOSE)SS into: ΙΟΉαθ Λ Ί ναΛΊΗνΕΌΊ丄 3dD S I
:63-11  :63-11
:( 95ΌΝ ai 03S ) :( 95ΌΝ ai 03S )
V 入 N31 lS S^H 0aAI£)¾0dVSS0S£)SSS入 dO aO AdiHV L IHVOCn丄 3d£ V into N31 lS S^H 0aAI£)3⁄40dVSS0S£)SSS into dO aO AdiHV L IHVOCn丄 3d£
:8 - Π :8 - Π
■( SS N ai 01S)V3AN3T¾TinS:DS¾j:O0aAIO 0dVa¾SSSS人^) ΉαΕ ΛΐΙίΠναΛΊΗνΕΌΊ丄 3dD ■( SS N ai 01S)V3AN3T3⁄4TinS:DS3⁄4j:O0aAIO 0dVa3⁄4SSSS人^) ΉαΕ ΛΐΙίΠναΛΊΗνΕΌΊ丄 3dD
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Figure imgf000022_0001
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:93- II S :93- II S
°(es-ON ai Das) VOAKS °(es-ON ai Das) VOAKS
■(ζζ-ON. αι 0as)v ■(ζζ-ON. αι 0as)v
669T00/ll0ZN3/X3d 80/ΠΟΖ ΟΛ\
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669T00/ll0ZN3/X3d 80/ΠΟΖ ΟΛ\
Figure imgf000023_0001
■( 8Ζ.ΌΝ αΐ 03S)V 入  ■( 8Ζ.ΌΝ αΐ 03S)V
:OS-II:OS-II
：( LL-OK ai HS ) N 入 N gi A S IS丄: 003AIiJai)(iV¾SdSdVScIdIVSSSS入 d£maE AJ;nvaAlHV£):n丄 3<K):( LL-OK ai HS ) N into N gi A S IS丄: 003AIiJai)(iV3⁄4SdSdVScIdIVSSSS into d£maE AJ; nvaAlHV£): n丄 3<K)
Figure imgf000023_0002
Figure imgf000023_0002
■(9L ON ai 3S)V3  ■(9L ON ai 3S)V3
HI HI
i(sz oNaii)3S)v 人 οε i(sz oNaii)3S)v person οε
Nai TS SWJ aAIOMSdSV丄 OSdOVDlcTHNLI入 JOa KOAJJlVaAlHVOCn丄 3<K Nai TS SWJ aAIOMSdSV丄 OSdOVDlcTHNLI into JOa KOAJJlVaAlHVOCn丄 3<K
HI  HI
'·( PL-O ai 03S)V 入  '·( PL-O ai 03S)V
■9 -ll SZ -(U-ON. ai 0HS)V3AN 3Ta¾lS S¾d 0aAI£)工 0dVSSDE)S<ISSE)丄 draNLLUETHaCOAJinVCLVIHVE l丄 3dO ■9 -ll SZ -(U-ON. ai 0HS)V3AN 3Ta3⁄4lS S3⁄4d 0aAI£)Work 0dVSSDE)S<ISSE)丄 draNLLUETHaCOAJinVCLVIHVE l丄 3dO
:( Zi項 ai 03S)V3AN3 :( Zi item ai 03S)V3AN3
■PP-II■PP-II
■ill ΌΝαΐ 0HS)V3AN 3 ffinS S¾ 0OAIE)丄 0dVOSdS3SSS£)丄 J¾«入 ££)Ήαθ ΛίΗΊναΛΊΗνο:Π丄 3dD ■ill ΌΝαΐ 0HS)V3AN 3 ffinS S3⁄4 0OAIE)丄 0dVOSdS3SSS£)丄 J3⁄4«入 ££)Ήαθ ΛίΗΊναΛΊΗνο:Π丄 3dD
■( ΟΖ,ΌΝ ai HS ) vo入 NSl^lSOS^aCO aAIDM dSdVSddV^SSSOSC)人 JD¾a£)3AJinVCLVlHV£ n丄 3dO ■( ΟΖ,ΌΝ ai HS ) vo into NSl^lSOS^aCO aAIDM dSdVSddV^SSSOSC) person JD3⁄4a£)3AJinVCLVlHV£ n丄 3dO
•HI  •HI
i(69ONai03S)V3A N31¾¾nS S dC 0aAI£))i0<iSd丄 VSSSVO丄 cTSfWAdCmaO AiiinVCL mVOCn丄 3<K)  i(69ONai03S)V3A N313⁄43⁄4nS S dC 0aAI£))i0<iSd丄 VSSSVO丄 cTSfWAdCmaO AiiinVCL mVOCn丄 3<K)
:(89·ΌΝ ai i)3S)V 入 :(89·ΌΝ ai i)3S)V into
■■OP- II■■OP- II
■(L9 :ONdli)3S)V:)入■(L9:ONdli)3S)V:)
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■6i-ll ■6i-ll
■(.99 OR ai HS)V3AN ■(.99 OR ai HS)V3AN
zz Zz
669100/Z10ZN3/X3J S8.980/£l0∑: OAV ■■ 9-ll 669100/Z10ZN3/X3J S8.980/£l0∑: OAV ■■ 9-ll
■( 16ONai0HS) YD ■( 16ONai0HS) YD
AN3TiHnS S¾;i:O0aAIO¾0iniSIS<IVSdVSSSS人 ίίΟΉαθ ΛίΗΊναΛΊΗΥΟ Ί丄 3dD AN3TiHnS S3⁄4;i:O0aAIO3⁄40iniSIS<IVSdVSSSS人 ίίΟΉαθ ΛίΗΊναΛΊΗΥΟ Ί丄 3dD
:£9-II  :£9-II
；( 06·環 ai as ) v 入  ; ( 06·环 ai as ) v
HI HI
；(68 :ON ai i)3S)V 人 ;(68 :ON ai i)3S)V people
N31¾¾lS SMd:O0aAIEraScIVCmSS丄 SVO丄 cTSNd人 dO¾a£OAdinvaAlHV£:n丄 3d£) N313⁄43⁄4lS SMd:O0aAIEraScIVCmSS丄 SVO丄 cTSNd person dO3⁄4a£OAdinvaAlHV£:n丄 3d£)
:19-11 οε ；(88 ON ai 03S)V0入  :19-11 οε ;(88 ON ai 03S)V0
:09-11 :09-11
'·( 8ΌΝ ai 0HS ) V3AN31¾¾1S  '·( 8ΌΝ ai 0HS ) V3AN313⁄43⁄41S
■■69-11 ■■69-11
ί(98 ON ai i)3S)V >入  ί(98 ON ai i)3S)V >in
:8S-II :8S-II
；( 58ΌΝ ai HS)VDAN Z  ;( 58ΌΝ ai HS)VDAN Z
••"-II••"-II
N ai 03S)V 入 N ai 03S)V into
N3TffaiS S¾ii: 0aAI£)¾£)<rHS丄 SdSSVf)丄 <T¾Nd入 JEmaE AdJlVCLVIHVDCn丄 3<K) N3TffaiS S3⁄4ii: 0aAI£)3⁄4£)<rHS丄 SdSSVf)丄 <T3⁄4Nd into JEmaE AdJlVCLVIHVDCn丄 3<K)
:95-II : 95-II
'·( £8ΌΚ ai HS ) 入 3™nS S¾d33i)a '·( £8ΌΚ ai HS ) into 3TMnS S3⁄4d33i)a
Aiaa dvsiDcivasdSiivsddiv^sssovaLc iNiLUEraaE AddivaAiHVOcn丄 a iD Aiaa dvsiDcivasdSiivsddiv^sssovaLc iNiLUEraaE AddivaAiHVOcn丄 a iD
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:(6Δ ON ai 03S)V3入:(6Δ ON ai 03S)V3
669画 Ϊ0 腿：) d S8L980/C10∑: OAV ■(SOI ΌΝ αΐ i)3S)V3人 N3TmnS S¾d i)aAI£a£)dSE) 669 drawing Ϊ 0 legs :) d S8L980/C10 ∑: OAV ■(SOI ΌΝ αΐ i)3S)V3 person N3TmnS S3⁄4d i)aAI£a£)dSE)
i(WH ΌΜ αΐ HS)V3ANai¾¾[lS3S¾J3 ) i)GAI£)"aE)ISVtISVSVSSE)VSSSdS{)VdSaLi¾NiI入 ίΙΟΉαθ ΛίίίΠναΛΊΗνΕ Ί丄 3dE) i(WH ΌΜ αΐ HS)V3ANai3⁄43⁄4[lS3S3⁄4J3 ) i)GAI£)"aE)ISVtISVSVSSE)VSSSdS{)VdSaLi3⁄4NiIin ίΙΟΉαθ ΛίίίΠναΛΊΗνΕ Ί丄 3dE)
HI S£ ;(£01 ON ai HS)V3AN 3 a¾lS )SM :)0aAI£yaDdSSS E)SdSSD丄 <T»Md入 ^ΟΉαΕΌΛίΙίΠναΛΊΗνΕ Ί丄 3d£)  HI S£ ;(£01 ON ai HS)V3AN 3 a3⁄4lS )SM :)0aAI£yaDdSSS E)SdSSD丄 <T»Md into ^ΟΉαΕΌΛίΙίΠναΛΊΗνΕ Ί丄 3d£)
■( 301 N ai HS ) V3AM31¾¾l■( 301 N ai HS ) V3AM313⁄43⁄4l
SDS o aAiaa dvsdOdVHScisdvsddcivssss入 30 αΕΌΛίΐ:πναΛΊΗνΕ):π丄 3d£) οε SDS o aAiaa dvsdOdVHScisdvsddcivssss into 30 αΕΌΛίΐ:πναΛΊΗνΕ):π丄 3d£) οε
HI  HI
:(101 ON ai 03S)V0人  :(101 ON ai 03S)V0 people
:ετΐι : ετΐι
；(001 ON ai 03S)V 人 N  ;(001 ON ai 03S)V person N
HI HI
'·( 66ΌΝ ai 03S)V0入 '·( 66ΌΝ ai 03S)V0
N31¾¾lS3S :i:):)0aAIE)¾E)SVS丄 VSSSVD丄 <TSMJ入 JEmaOOAJinVCLVlHVO n丄 3dD N313⁄43⁄4lS3S :i:):)0aAIE)3⁄4E)SVS丄 VSSSVD丄 <TSMJ into JEmaOOAJinVCLVlHVO n丄 3dD
： ΐΖ,-Π oz ■(86 ON ai 03S)V0入  : ΐΖ,-Π oz ■(86 ON ai 03S)V0
:0rII :0rII
■(16 ON ai 03S)V0入  ■(16 ON ai 03S)V0
:69- Π ■(96 ON ai 03S)V0入 :69- Π ■(96 ON ai 03S)V0
:89-11:89-11
■(S6 ON ai 3S)VDA 01 N31¾¾lS S¾dC i)aA )¾{)S<raSdS丄 SV£)丄 JHNiLUO aO AiHlVCL IHVD n丄 3<iO ■(S6 ON ai 3S)VDA 01 N313⁄43⁄4lS S3⁄4dC i)aA )3⁄4{)S<raSdS丄 SV£)丄 JHNiLUO aO AiHlVCL IHVD n丄 3<iO
:乙9-11  : B 9-11
:( P6-ON. ai Oas ) v人 N3i¾nsos :( P6-ON. ai Oas ) v people N3i3⁄4nsos
MdCO aAIO^ii VSdOdVaScIStlVSdddVSSSDVO入 JO aEOAdiHVLVIHVO n丄 3<K MdCO aAIO^ii VSdOdVaScIStlVSdddVSSSDVO into JO aEOAdiHVLVIHVO n丄 3<K
:99-11 5 ：(£6 -OK ai i)3S)V0入  :99-11 5 :(£6 -OK ai i)3S)V0
:S9-II :S9-II
:( Ζ6 Ν. ai i)3S)V0人 Z  :( Ζ6 Ν. ai i)3S)V0 people Z
669T00/Z10ZM3/X3d S8.980/CT0Z: OAV
Figure imgf000026_0001
669T00/Z10ZM3/X3d S8.980/CT0Z: OAV
Figure imgf000026_0001
ί(811ΌΝ αΐ 03S)V 人  ί(811ΌΝ αΐ 03S)V people
:06-11 ■(LU ON ai HS) :06-11 ■(LU ON ai HS)
:68-11 :68-11
;(911 ONai03S)V 入 M 31¾¾lS SW;i:)30aAID"a0SVSSSVSVVSVVSSSS人 JE^aEOAddlVaAlHVO n丄 3dO  ;(911 ONai03S)V into M 313⁄43⁄4lS SW;i:)30aAID"a0SVSSSVSVVSVVSSSS人 JE^aEOAddlVaAlHVO n丄 3dO
:88-11 ■(ζ\1 ONai 3S 0£ :88-11 ■(ζ\1 ONai 3S 0££
)V3入 NHTOnS S^HCO aAItya tlSdSdVSrVSSS人： ΙΕ) α£ΌΛ:Ι:ΠναΛΊΗνθ3Ί13<Κ) ) V3 into NHTOnS S^HCO aAItya tlSdSdVSrVSSS person: ΙΕ) α£ΌΛ:Ι:ΠναΛΊΗνθ3Ί13<Κ)
■LS-ll ■LS-ll
■(PU N ai 03S)V 入 N31 TS0S¾iiC 0aAI£)¾0<IScI£)<iS¾S<IS<IVSSS入 JEmaD AJinVCIAlHVD n丄 3<I ■(PU N ai 03S)V into N31 TS0S3⁄4iiC 0aAI£)3⁄40<IScI£)<iS3⁄4S<IS<IVSSSInto JEmaD AJinVCIAlHVD n丄 3<I
:98-11 ^ ί(£Π ON ai 3S)V3ANH1¾¾1S ； DS>M O0aAID¾t)VS<IWVdSVdVV<iSOVD丄 cTSNil入: ΚΤΗαΕΌΛΐΗΊναΛΊΗνθ Ί丄 3<I£)  :98-11 ^ ί(£Π ON ai 3S)V3ANH13⁄43⁄41S ; DS>M O0aAID3⁄4t)VS<IWVdSVdVV<iSOVD丄 cTSNil In: ΚΤΗαΕΌΛΐΗΊναΛΊΗνθ Ί丄 3<I£)
:S8-II :S8-II
■( ai 03S)V 入 ■( ai 03S)V into
HI HI
Kill ON ai C)3S)V 入 N3TH¾1S S^O 0aAI£) f)VSVSSVSSSSVSSSVVS丄DINil人 ίΙΟΉαΕΌΛίΙίΠναΛΊΗνθΟΊ丄 3dD  Kill ON ai C)3S)V into N3TH3⁄41S S^O 0aAI£) f)VSVSSVSSSSVSSSVVS丄DINil人 ίΙΟΉαΕΌΛίΙίΠναΛΊΗνθΟΊ丄 3dD
'•£8-11 ί(0ΙΙ N ai 03S)V 入 Μ3Ί SI lS S¾dC 0aAI£)¾0ciS<K)dV¾SISdVS(Id(iVSSS人： KmaOOAdiTIVCLVlHVEOl丄 3d£)  '•£8-11 ί(0ΙΙ N ai 03S)V Μ3Ί SI lS S3⁄4dC 0aAI£)3⁄40ciS<K)dV3⁄4SISdVS(Id(iVSSS人: KmaOOAdiTIVCLVlHVEOl丄 3d£)
HI  HI
;(601 ON ai 03S)VDA  ;(601 ON ai 03S)VDA
:18- II 01 ;(801 N ai HS:18- II 01 ;(801 N ai HS
)V AN3TOnS0S¾iICO0aAK) 0<ISS£)VOdO£)O人 ίΙΟΉαθ ΛϋΊναΛΊΗνθΟΊ丄 3d0 ) V AN3TOnS0S3⁄4iICO0aAK) 0<ISS£)VOdO£)O人 ίΙΟΉαθ ΛϋΊναΛΊΗνθΟΊ丄 3d0
:08-11 :08-11
■(L01 Μ ai 03S)VDAN31 ¾¾lSOS¾dC 0CIAID a0SSDVC)JC)£)D¾¾丄 人 dt aaO AJiHV LVIHVO n丄 3<K) S ■(L01 Μ ai 03S)VDAN31 3⁄43⁄4lSOS3⁄4dC 0CIAID a0SSDVC)JC)£)D3⁄43⁄4丄 person dt aaO AJiHV LVIHVO n丄 3<K) S
:6 -II  :6 -II
:(90i ON ai 03s)v3AKm¾rais Sm:O0aAIO¾¾i)SSOVOd£)O£)a3V3"a¾丄 人 ίΙΟ α£ΌΛ;ΙίΠναΛΊΗν{ :)Ί丄 3d )  :(90i ON ai 03s)v3AKm3⁄4rais Sm:O0aAIO3⁄43⁄4i)SSOVOd£)O£)a3V3"a3⁄4丄人 ίΙΟ α£ΌΛ;ΙίΠναΛΊΗν{ :)Ί丄 3d )
:8乙 -II  :8B-II
669T00/∑:i0lM3/13d S 0/ 0ί O ¾lS S dC 0aAIE)SVIS<ISSdVSdSdSSVS丄 traNiLUETa K AiHlVaAlHVOCn丄 3<I£) 669T00/∑: i0lM3/13d S 0/ 0ί O 3⁄4lS S dC 0aAIE)SVIS<ISSdVSdSdSSVS丄traNiLUETa K AiHlVaAlHVOCn丄3<I£)
： εοι-π : εοι-π
!(0£lONai 3S) HHdO  !(0£lONai 3S) HHdO
-II  -II
■(6Z\ ΌΜ QI HS)V3AK3  ■(6Z\ ΌΜ QI HS)V3AK3
：101-11 Ϊ N ai HS)V3AN3 l¾¾lS S¾dC 0aAI ¾OSVSSSVSSSVVSSSVSS人 ίΙΟΉαθ ΛίΜΊναΛΊΗνΕ 丄 3d£) :101-11 Ϊ N ai HS)V3AN3 l3⁄43⁄4lS S3⁄4dC 0aAI 3⁄4OSVSSSVSSSVVSSSVSS人 ίΙΟΉαθ ΛίΜΊναΛΊΗνΕ 丄 3d£)
•·οοι- II i( , 1OMai HS) :66- Π  •·οοι- II i( , 1OMai HS) :66- Π
:( 9ZVON. ai 03S) V ANHl^nSOS J330aAIO¾0dSdO<raS<IS<ISdd<IVOSSdOOIc SMJAJOHa03AiiinVaAlHVOD113<iO  :( 9ZVON. ai 03S) V ANHl^nSOS J330aAIO3⁄40dSdO<raS<IS<ISdd<IVOSSdOOIc SMJAJOHa03AiiinVaAlHVOD113<iO
:86-11 :86-11
； (SZl "ON ai HS)V3ANH 01 TH¾nS S¾d Oi)aAIE)丄 0d<IdV£)SS<I£)9VO丄 < II^IAJ£ma£OA:iinV LVlHVO:) 13<iD (SZl "ON ai HS)V3ANH 01 TH3⁄4nS S3⁄4d Oi)aAIE)丄 0d<IdV£)SS<I£)9VO丄 < II^IAJ£ma£OA:iinV LVlHVO:) 13<iD
■L6-U  ■L6-U
-(HI :O ai i)3S)V0AN31 FaiSDSMdCO aAID丄 C ScItldVOSSdDVOl INii入 ΐΙΟΉαθ ΛίΜΊναΛΊΗνθ Ί丄 3dO  -(HI :O ai i)3S)V0AN31 FaiSDSMdCO aAID丄 C ScItldVOSSdDVOl INiiIn ΐΙΟΉαθ ΛίΜΊναΛΊΗνθ Ί丄 3dO
:96- Π St :( £n:ONai03S)V 入 N3TiraiS3S :i: 0aAIE) VaSdSdVSdddVSSS入 ίΙΕ^αθ ΛϋΊναΛΊΗνΕ Ί丄 3dE)  :96- Π St :( £n:ONai03S)V into N3TiraiS3S :i: 0aAIE) VaSdSdVSdddVSSS into ίΙΕ^αθ ΛϋΊναΛΊΗνΕ Ί丄 3dE)
:S6-II  :S6-II
■( ZZV-OR ai 0HS)V3AN31 H¾lS S¾ii:O0aAI010dSdd<IVDSS<I£)E)丄 cTSN;LUO"aa£)C)AJ;nVaAlHVOCn丄 3dD 01 ■( ZZV-OR ai 0HS)V3AN31 H3⁄4lS S3⁄4ii:O0aAI010dSdd<IVDSS<I£)E)丄 cTSN;LUO"aa£)C)AJ;nVaAlHVOCn丄 3dD 01
6-11  6-11
■(ill ON ai HS)V  ■(ill ON ai HS)V
••£6-11 ••£6-11
:(03l Nai0HS)V S  :(03l Nai0HS)V S
HI HI
:(6U ON ai ό  :(6U ON ai ό
3S)V0入 N31 aiS0S¾d: 0aAIi^K <iiVE)SSSS人 £¾α£ΌΛ3ίΠναΛΊΗν£ΌΊ丄 3dO  3S)V0 into N31 aiS0S3⁄4d: 0aAIi^K <iiVE)SSSS people £3⁄4α£ΌΛ3ίΠναΛΊΗν£ΌΊ丄 3dO
91  91
669丽 ΙΟΖ謹： M S8 赚 10Z OAV ；( :ΟΜ αΐ 03S)V 入 N3TffHlS S¾d Ofr 669 丽ΙΟΖ谨: M S8 earn 10Z OAV ;( :ΟΜ αΐ 03S)V into N3TffHlS S3⁄4d Ofr
'•"I- II'•"I- II
■(tn ON αι i)3s)v 入 ■(tn ON αι i)3s)v
N3 iraiS S¾dC 0aAIO SS<ISWSSdSdVS<ISS入 JE^aD AddlVaAlHVD n丄  N3 iraiS S3⁄4dC 0aAIO SS<ISWSSdSdVS<ISS into JE^aD AddlVaAlHVD n丄
:911-11 S£ ； ( t^l ON QI 03S)V3AMH1¾¾1  :911-11 S£ ; ( t^l ON QI 03S)V3AMH13⁄43⁄41
••SI I- II ••SI I- II
■(Z l N ai 03S)v >人  ■(Z l N ai 03S)v >People
■(IPI ΌΜ ai 0as)VDAN31¾¾lS3 S dC C)aAI£yaOSdSVS<ISSdVSdS<ISSdVS丄 c INd人 30 αθ Λ:Ι^ΠναΛΊΗν£)ΟΊ丄 3d£) ■(IPI ΌΜ ai 0as)VDAN313⁄43⁄4lS3 S dC C)aAI£yaOSdSVS<ISSdVSdS<ISSdVS丄 c INd person 30 αθ Λ:Ι^ΠναΛΊΗν£)ΟΊ丄 3d£)
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••en-ii
Figure imgf000028_0001
入 Na^nrais sMii aAicmovsvsssssvssvss人 JD¾ao AddivaAiHvoo L3d£) Into Na^nrais sMii aAicmovsvssssssssvss people JD3⁄4ao AddivaAiHvoo L3d£)
••πι-ιι  ••πι-ιι
；( 6£1ΌΝ ai 03S)V3AN3TffinSDS¾J  ;( 6£1ΌΝ ai 03S)V3AN3TffinSDS3⁄4J
• m-ll oz; • m-ll oz;
:οπ-π : οπ-π
■(LZl ON ai 0HS)V■(LZl ON ai 0HS)V
A 31¾¾lSDS¾H33 aAIO"aOSSdSVdSSdSVSdSSAiiO¾a03Ad[JlVaAlHV031IHdO SI A 313⁄43⁄4lSDS3⁄4H33 aAIO"aOSSdSVdSSdSVSdSSAiiO3⁄4a03Ad[JlVaAlHV031IHdO SI
■•601-11 ■•601-11
■( 9£1ΌΝ QI 0HS)V3AN31¾¾1 ■( 9£1ΌΝQI 0HS)V3AN313⁄43⁄41
■•801-11 ■•801-11
■(SZ\ ON QI HS)V3ANH1 01  ■(SZ\ ON QI HS)V3ANH1 01
HI HI
： ( I ON ai 03S)V AN3  : ( I ON ai 03S)V AN3
•901-Π •901-Π
'·( eiON ai 03S)V AN3I¾¾IS:)S ^m aAiEraosscisvcisdSSdvsdScisScjaLj^NdAiiEmaD AJinvLviHvocn丄 3do  '·( eiON ai 03S)V AN3I3⁄43⁄4IS:)S ^m aAiEraosscisvcisdSSdvsdScisScjaLj^NdAiiEmaD AJinvLviHvocn丄 3do
"•SOI- II  "•SOI- II
■(Z£l Μ QI 03S)V3AN31¾  ■(Z£l Μ QI 03S)V3AN313⁄4
LZ  LZ
669l00/il0ZM3/X3d S8Z.980/£10∑; OAV II -118: 669l00/il0ZM3/X3d S8Z.980/£10∑; OAV II -118:
GPETLCGAHLVDALFFVCGDRGFYSSSSPSAPSPSSPASPSPSSAPQRGIVDQCCFRSCSL RRLENYCA(SEQ ID NO: 146);  GPETLCGAHLVDALFFVCGDRGFYSSSSPSAPSPSSPASPSPSSAPQRGIVDQCCFRSCSL RRLENYCA (SEQ ID NO: 146);
11 -119:  11 -119:
GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSRGIVDQCCFRSCSLRRLENYCA  GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSRGIVDQCCFRSCSLRRLENYCA
(SEQ ID NO: 147); (SEQ ID NO: 147);
11 -120:  11 -120:
GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSARGIVDQCCFRSCSLRRLENYCA  GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSARGIVDQCCFRSCSLRRLENYCA
(SEQ ID NO: 148 ); (SEQ ID NO: 148);
11 -121 :  11 -121 :
(SEQ ID NO: 149 )； (SEQ ID NO: 149);
11 -122: EQ ID NO: 150 )；  11 -122: EQ ID NO: 150 );
11 -123:  11 -123:
GPEHLCGAHLVDALFFVCGDRGFYFNKGGGGAPQTGIVDQCCHRSCSLRRLENYCN(S EQ ID NO: 161);  GPEHLCGAHLVDALFFVCGDRGFYFNKGGGGAPQTGIVDQCCHRSCSLRRLENYCN (S EQ ID NO: 161);
11 -124:  11 -124:
NYCN (SEQ ID NO: 162)。 修饰化合物 NYCN (SEQ ID NO: 162). Modified compound
多肽化学家为了解决血浆中分子量小于 67kDa的药物分子被腎脏快速清除的难题， 已经 使用了几种方法。 1、 注射部位建"仓"（ depot ); 2、 与血浆中的载体蛋白以非共价键联合以阻 止肾小球过滤； 3、 与栽体蛋白以共价键连接； 4、 与大分子量修饰基团结合， 例如大分子量 PEG、 多糖等（如在本申请文件之前章节中公开的）。 "疏水窖藏" (hydrophobic depoting)大幅 度增加肽的疏水性以减少可溶性， 并使它在注射部分形成"仓"。 肽仓緩慢解离后， 多肽结合 至细胞膜和 /或全身的载体蛋白 （如白蛋白等）。 载体蛋白分子量大于腎小球过滤最大分子量， 因此不易被腎脏清除， 可以在血浆中循环多日。 因此， 与载体蛋白结合的多肽不易被肾小球 过滤或被内膜上的蛋白酶降解。  Polypeptide chemists have used several methods to solve the problem of rapid clearance of drug molecules with a molecular weight of less than 67 kDa in the kidney by plasma. 1. The injection site is built into a "depot"; 2. It is combined with non-covalent bonds in the plasma to prevent glomerular filtration; 3. Covalently linked to the carrier protein; 4. Large molecular weight Modification group binding, such as large molecular weight PEG, polysaccharides, etc. (as disclosed in the previous section of this application). Hydrophobic depoting greatly increases the hydrophobicity of the peptide to reduce solubility and cause it to form a "storage" in the injection portion. After the peptide compartment is slowly dissociated, the polypeptide binds to the cell membrane and/or systemic carrier proteins (such as albumin, etc.). The molecular weight of the carrier protein is greater than the maximum molecular weight of the glomerular filtration, so it is not easily cleared by the kidneys and can be circulated in plasma for many days. Therefore, the polypeptide bound to the carrier protein is not easily filtered by the glomerulus or degraded by the protease on the inner membrane.
脂肪酸一般通过三种方式延长多肽在体内作用时间。 第一， 脂肪酸能够在药物注射部位 与白蛋白以非共价键结合， 形成的多肽-脂肪酸 -白蛋白大分子结合物释放緩慢； 第二， 多肽- 脂肪酸-白蛋白结合物的大分子量使肾清除率降低； 第三， 白蛋白为多肽提供保护， 不易被蛋 白酶降解。 第四， 脂肪酸减少多肽的免疫原性。 后三个特点与长链 PEG修饰的效果类似。 更 多的机理和实验支持可以参照 Biochem. J. (1995) 312, 725-731 ； Pharmaceutical Research, ( 2004 ), 21, 8, 1498-1504; Current Medicinal Chemistry ( 2009 ), 16, 4399-4418； WO95/07931; Diabetes, Obesity and Metabolism, 2007, 9, 290-299; Diabetes, 1997, 46， 637-642。  Fatty acids generally extend the time of action of the polypeptide in vivo in three ways. First, fatty acids can bind non-covalently to albumin at the site of drug injection, resulting in slow release of the polypeptide-fatty acid-albumin macromolecule conjugate; second, the large molecular weight of the peptide-fatty acid-albumin conjugate makes the kidney The clearance rate is reduced; third, albumin provides protection for the polypeptide and is not easily degraded by proteases. Fourth, fatty acids reduce the immunogenicity of the polypeptide. The latter three features are similar to the long-chain PEG modification. Further mechanisms and experimental support can be found in Biochem. J. (1995) 312, 725-731; Pharmaceutical Research, (2004), 21, 8, 1498-1504; Current Medicinal Chemistry (2009), 16, 4399-4418; WO 95/07931; Diabetes, Obesity and Metabolism, 2007, 9, 290-299; Diabetes, 1997, 46, 637-642.
典型例子是糖尿病治疗多肽药物地特胰岛素 (detemir)和利拉鲁肽（ liraglutide )。 它们利用 了基于脂肪酸修饰的疏水窖藏， 使体内作用时间延长（地特胰岛素 t1/2=14 小时）。 而未修饰 的胰岛素的作用时间仅有几个小时。 Typical examples are the diabetes treatment polypeptide drugs detemir and liraglutide. They utilize hydrophobic deposits based on fatty acid modifications to extend the duration of action in the body (tete insulin t 1/2 = 14 hours). Unmodified The action of insulin is only a few hours.
此外， 用聚乙二醇（PEG, 分子量不小于 20K)和人白蛋白等大分子修饰胰岛素， 也可 以达到和上述脂肪酸修饰类似的延长体内作用时间的效果。 因此， 在所有可以用脂肪酸酰化 的位点， 都可以用聚乙二醇或人白蛋白等大分子修饰。  In addition, modification of insulin with macromolecules such as polyethylene glycol (PEG, molecular weight not less than 20K) and human albumin can also achieve an effect of prolonging the in vivo action time similar to the above fatty acid modification. Therefore, all of the sites which can be acylated with fatty acids can be modified with macromolecules such as polyethylene glycol or human albumin.
本发明基于这样一种认识： 本发明的具有降血糖作用的化合物的总体疏水性在该化合物 的体内功效方面起重要作用。 本发明进一步提供一种具有降血糖作用的、 在多肽基础上进行 修饰的化合物， 以进一步提高所述化合物体内循环作用时间。 所述修饰是将修饰侧链连接至 本发明的默链化合物的 B链的 N-末端氨基酸残基的 α-氨基、或连接至本发明的单链化合物的 Ν-末端氨基酸残基的 α-氨基、或连接至本发明的双链或单链化合物中存在的赖氨酸的 ε-氨基。  The present invention is based on the recognition that the overall hydrophobicity of the hypoglycemic compound of the present invention plays an important role in the in vivo efficacy of the compound. The present invention further provides a compound having a hypoglycemic effect and modified on a polypeptide basis to further increase the in vivo circulation time of the compound. The modification is an α-amino group which links the modified side chain to the N-terminal amino acid residue of the B chain of the silent chain compound of the present invention, or α-linked to the Ν-terminal amino acid residue of the single-chain compound of the present invention. An amino group, or an ε-amino group attached to lysine present in the double-stranded or single-stranded compound of the present invention.
在一种实施方式中，所述化合物基于 IGF-1类似物进行改造，该化合物包括 Α链和 Β链， 其中，
Figure imgf000030_0001
In one embodiment, the compound is engineered based on an IGF-1 analog, the compound comprising an oxime chain and an oxime chain, wherein
Figure imgf000030_0001
X423-426X427LC[1]GAHLVDALX438X439 C[2]GDRGFX447X448X49 450X45lX452X453 54 55' 所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物通过 6个半胱氨酸形成 3对二 硫键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： Ctl]和 C[4]形成二硫键， 。[21和。[6] 形成二硫键， [3]和。[51形成二硫键， 其中，X423-426X427LC[ 1 ]GAHLVDALX4 3 8X4 3 9 C[ 2 ]GDRGFX447X448X49 4 50 X45lX452X453 54 55' In the compound, [1]-[6] represents the number of cysteine; the compound passes 6 caspase The amino acid forms a 3-pair disulfide bond, wherein the A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain. The specific positions of the three pairs of disulfide bonds are: C tl] And C [4] form a disulfide bond. [ 21 and. [6] Form a disulfide bond, [ 3] and. [ 51] forms a disulfide bond, wherein
X399是精氨酸、 赖氨酸或缺失； X4 是精氨酸、 赖氨酸或缺失； 05是谷氨酸、 天冬酰 胺、 谷氨酰胺或丝氨酸； 08是组氨酸、 精氨酸、 笨丙氨酸、 苏氨酸或通式（I)结构， 所述 通式（I)结构为： X 3 99 is arginine, lysine or deletion; X4 is arginine, lysine or deletion; 0 5 is glutamic acid, asparagine, glutamine or serine; 0 8 is histidine, Arginine, stupid alanine, threonine or a structure of the formula (I), the structure of the formula (I) is:
Figure imgf000030_0002
Figure imgf000030_0002
09是精氨酸、 丝氨酸或通式（I)结构； 10是组氨酸、 精氨酸、 笨丙氨酸或通式（I) 结构； 12是丝氨酸、 异亮氨酸或通式（I)结构； 14是精氨酸或通式（I)结构； 15是精 氨酸或通式（I)结构； 17是谷氨酸或通式（I)结构； 18是天冬酰胺或通式（I)结构； 21是丙氨酸、甘氨酸或天冬酰胺； 22是赖氨酸、精氨酸-赖氨酸二肽或缺失， 或为通式（I) 结构； 当 22为二肽时， 其中一个氨基酸为通式（I)结构； 23_426是甘氨酸-脯氨酸-谷氨酸 三肽、 UL-甘氨酸 -脯氨酸-谷氨酸、 苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽或 UL-苯丙氨酸- 缬氨酸-天冬酰胺 -谷氨酰胺； X427是组氨酸或苏氨酸； 38是苯丙氨酸或色氨酸； 39是苯丙 氨酸或色氨酸； 47是苯丙氨酸、 组氨酸或酪氨酸； X448是 -N¾、 苯丙氨酸、 酪氨酸或缺失； 49是天冬酰胺、 苏氨酸、 谷氨酸、 天冬氨酸或缺失； X45G是赖氨酸、 精氨酸、 谷氨酸、 天 冬氨酸、 脯氨酸或缺失； 51是脯氨酸、 赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸或缺失， 或为 通式（I)结构； 52是苏氨酸、 赖氨酸或缺失， 或为通式（I)结构； 53是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I)结构； 54是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I) 结构； 55是赖氨酸或缺失， 或为通式（I)结构； 09 is arginine, serine or the structure of formula (I); 10 is histidine, arginine, stupid alanine or the structure of formula (I); 12 is serine, isoleucine or formula (I) Structure; 14 is arginine or the structure of formula (I); 15 is arginine or structure of formula (I); 17 is glutamic acid or structure of formula (I); 18 is asparagine or formula (I) structure; 2 1 is alanine, glycine or asparagine; 22 is lysine, arginine-lysine dipeptide or deletion, or is of the formula (I); when 22 is a dipeptide When one of the amino acids is of the formula (I); 23 _4 26 is a glycine-valine-glutamic acid tripeptide, U L -glycine-valine-glutamic acid, phenylalanine-valine - asparagine-glutamine tetrapeptide or U L -phenylalanine - valine - asparagine - glutamine; X4 27 is histidine or threonine; 38 is phenylalanine or color 39 ; is phenylalanine or tryptophan; 47 is phenylalanine, histidine or tyrosine; X448 is -N3⁄4, phenylalanine, tyrosine or deletion; 49 is asparagine , threonine, glutamic acid, aspartic acid or deletion; X4 5 G is lysine, arginine, glutamic acid, aspartic acid, proline or deletion; 51 is valine, lysine, arginine, glutamic acid, aspartic acid or deletion, Or a structure of the formula (I); 52 is threonine, lysine or deletion, or a structure of the formula (I); 53 is glutamic acid, glycine, lysine or deletion, or is of the formula (I) a structure; 54 is glutamic acid, glycine, lysine or a deletion, or a structure of the formula (I); 55 is a lysine or a deletion, or a structure of the formula (I);
UL是 -W-X-Y-Z 结构、 脂肪酸、 聚乙二醇、 白蛋白、 Ln-ML结构、 氢原子或 Na-( a-(HOOC(CH2)nCO)-Y-Glu)- , Na-(Na-(CH3(CH2)„CO)-Y-Glu)- , 其中 η是 8-20的整数， 如 8、 10、 12、 14、 16、 20, Να表示氨基酸或氨基酸残基的 α-氨基， 或为通式（Π )结构， 所述 通式（Π )结构是： U L is -WXYZ structure, fatty acid, polyethylene glycol, albumin, Ln-M L structure, hydrogen atom or N a -( a -(HOOC(CH 2 ) n CO)-Y-Glu)- , N a -(N a -(CH 3 (CH 2 )„CO)-Y-Glu)- , where η is an integer from 8-20, such as 8, 10, 12, 14, 16, 20, Ν α represents an α-amino group of an amino acid or an amino acid residue, or a general formula (Π) structure, wherein the general formula (Π) structure is:
Figure imgf000031_0001
J是 -W-X-Y-Z 结构、 Ln-ML结构或氢原子。
Figure imgf000031_0001
J is a -WXYZ structure, an L n -M L structure or a hydrogen atom.
其中 Ln-ML结构中， ML是修饰基团， 包括但不局限于 -W-X-Y-Z、 脂肪酸、 聚乙二醇、 白 蛋白、 IgG Fc、 糖基团等。 In the Ln-M L structure, M L is a modifying group including, but not limited to, -WXYZ, a fatty acid, polyethylene glycol, albumin, IgG Fc, a sugar group, and the like.
L„是可选的连接基、 共价键或不存在。 可选的连接基包括但不局限于， 聚乙二醇、 长链 脂肪酸， 或一个或多个聚乙二醇分子和长链脂肪酸分子通过共价键连接形成的长链。 可以 是 -NH-(CH2)n-£0-、 -NH-(CH2CH20)n-CH2-CO- , -NH-(CH2CH20)n-(CH2)r-CO- , n是 1-20的整 数， r是 1-10的整数。 在一种实施方式中， 1^是->¾-(。¾〇¾0)2-。¾-。0 11-(〇 0120)2- CH2-C0-o 在一种实施方式中， 是 -NPHCH m-CKCi^CHzC nHCH na- O-, nl、 n2、 n3 分别是 1-16的整数。 在一种实施方式中，
Figure imgf000031_0002
nl、 n2分别是 1-16的整数。 在以上实施方式中， L„通过来自加下划线的狻基碳的键与多肽化合物的氨基形 成酰胺键。 另一端与 ML形成共价键。 在一种实施方式中， L„通过来自加下划线的叛基碳的 键与与多肽化合物的氨基形成酰胺键， 另一端与 -W-X-Y-Z形成酰胺键。 L„ is an optional linker, covalent bond or non-existent. Optional linkers include, but are not limited to, polyethylene glycol, long chain fatty acids, or one or more polyethylene glycol molecules and long chain fatty acids The long chain formed by the covalent bond of the molecule. It can be -NH-(CH 2 ) n -£0-, -NH-(CH 2 CH 2 0) n -CH 2 -CO- , -NH-(CH 2 CH 2 0) n -(CH 2 ) r -CO- , n is an integer from 1 to 20, and r is an integer from 1 to 10. In one embodiment, 1^ is -> 3⁄4-(.3⁄4〇3⁄40 2 - .3⁄4-.0 11-(〇01 2 0) 2 - CH 2 -C0- o In one embodiment, is -NPHCH m-CKCi^CHzC nHCH na- O-, nl, n2, n3 Each is an integer from 1 to 16. In one embodiment,
Figure imgf000031_0002
Nl and n2 are integers of 1-16, respectively. In the above embodiments, L„ forms an amide bond with the amino group of the polypeptide compound by a bond derived from the underlined fluorenyl carbon. The other end forms a covalent bond with M L. In one embodiment, the L „ is underlined by The bond of the base carbon forms an amide bond with the amino group of the polypeptide compound, and the other end forms an amide bond with -WXYZ.
在本发明中， -W-X-Y-Z结构是：  In the present invention, the -W-X-Y-Z structure is:
W是侧链具有羧基的 α-氨基酸残基，该残基以一个羧基与本发明中双链化合物的 Β链的 Ν-末端氨基酸残基的 α-氨基或与单链化合物的 Ν-末端氨基酸残基的 α-氨基或与单链或双链 上的赖氨酸残基的 ε-氨基一起形成酰胺基；  W is an α-amino acid residue having a carboxyl group in a side chain which has a carboxyl group and an α-amino group of the Ν-terminal amino acid residue of the Β chain of the double-stranded compound of the present invention or a Ν-terminal amino acid of the single-chain compound The α-amino group of the residue or together with the ε-amino group of the lysine residue on the single or double strand form an amide group;
或者 W是由 2、 3或 4个 α-氨基酸残基通过酰胺键连接起来的链， 该链通过酰胺键连接 至双链化合物的 Β链的 Ν-末端氨基酸残基的 α-氨基或连接至单链化合物的 Ν-末端氨基酸残 基的 α-氨基或连接至单链或双链化合物上的赖氨酸残基的 ε-氨基， W的氨基酸残基选自具有 中性侧链的氨基酸残基和侧链具有羧基的氨基酸残基，使得 W含有至少一个在侧链具有羧基 的氨基酸残基；  Or W is a chain joined by an amide bond of 2, 3 or 4 α-amino acid residues, which chain is linked to the α-amino group of the Ν-terminal amino acid residue of the Β chain of the double-stranded compound via an amide bond or is attached to The α-amino group of the Ν-terminal amino acid residue of the single-stranded compound or the ε-amino group of the lysine residue attached to the single- or double-stranded compound, and the amino acid residue of W is selected from the amino acid residue having a neutral side chain The base and side chains have an amino acid residue of a carboxyl group such that W contains at least one amino acid residue having a carboxyl group in the side chain;
或者 W是从 X到双链化合物的 Β链的 Ν-末端氨基酸的 α-氨基或到单链化合物的 Ν-末端 氨基酸残基的 α-氨基或到双链或单链化合物的赖氨酸残基的 ε-氨基的共价键；  Or W is the α-amino group of the Ν-terminal amino acid of the oxime chain from X to the double-stranded compound or the α-amino group of the Ν-terminal amino acid residue of the single-stranded compound or the lysine residue to the double-stranded or single-stranded compound a covalent bond of the epsilon-amino group;
X 是 -£0- 、 -CH(COOH)CO- 、 -N(CH2COOH)CH2CO- 、 -N(CH2COOH)CH2CON (CH2COOH)CH2CO- 、 -N(CH2CH2COOH)CH2CH2CO- 、 -N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO-、 -NHCH(COOH)(CH2)4NHCO-、 -N(CH2CH2COOH)CH2£0-或者 -N (CH2COOH)CH2CH2CO- , 其中 X is -£0-, -CH(COOH)CO-, -N(CH 2 COOH)CH 2 CO- , -N(CH 2 COOH)CH 2 CON (CH 2 COOH)CH 2 CO- , -N( CH 2 CH 2 COOH)CH 2 CH 2 CO- , -N(CH 2 CH 2 COOH)CH 2 CH 2 CON(CH 2 CH 2 COOH)CH 2 CH 2 CO-, -NHCH(COOH)(CH 2 ) 4 NHCO-, -N(CH 2 CH 2 COOH)CH 2 £0- or -N (CH 2 COOH)CH 2 CH 2 CO- , wherein
a)当 W是氨基酸残基或氨基酸残基链时，上述 X通过由加下划线的羰基碳的键与 W中的 氨基形成酰胺键； 或者  a) when W is an amino acid residue or an amino acid residue chain, the above X forms an amide bond with an amino group in W by a bond of an underlined carbonyl carbon;
b)当 W是共价键时， 上述 X通过来自加下划线的羰基碳的键与双链化合物的 B链的 N- 末端 α-氨基或与单链化合物的 N-末端氨基酸残基的 α-氨基或与双链或单链化合物的赖氨酸残 基的 ε-氨基形成酰胺键； b) When W is a covalent bond, the above X is passed through the bond from the underlined carbonyl carbon to the N-terminal α-amino group of the B chain of the double-stranded compound or to the α-amino acid residue of the N-terminal amino acid residue of the single-stranded compound. Amino acid or lysine residue with double-stranded or single-stranded compounds The ε-amino group of the group forms an amide bond;
Υ是 -(CH2)m， 其中 m是 6-32的整数； Υ is -(CH 2 ) m , where m is an integer from 6 to 32;
或包含 1、 2或 3个 -CH=CH-基团和多个 -CH2-基团的二价烃链， 所述多个 -CH2-基团的个 数满足烃链中的碳原子总数范围是 10-32; Or a divalent hydrocarbon chain comprising 1, 2 or 3 -CH=CH- groups and a plurality of -CH 2 - groups, the number of the plurality of -CH 2 - groups satisfying carbon atoms in the hydrocarbon chain The total range is 10-32;
或通式 - (CH2)VC6H4 (CH2) W-的二价烃链， 其中 V和 w是整数， 或者它们之一是零， 使得Or a divalent hydrocarbon chain of the formula -(CH 2 ) V C 6 H 4 (CH 2 ) W - wherein V and w are integers, or one of them is zero, such that
V和 w总和的范围是 6-30; 且 The sum of V and w is in the range of 6-30;
Z是 -COOH、 -CO- Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH(COOH)2、 -N(CH2COOH)2, -S03H、 -P03H或不存在； 条件是当 W是共价键且 X是 -CO-时， Z不是 -COOH。 Z is -COOH, -CO- Asp, -CO-Glu , -CO-Gly, -CO-Sar, -CH (COOH) 2, -N (CH 2 COOH) 2, -S0 3 H, -P0 3 H Or absent; condition is that when W is a covalent bond and X is -CO-, Z is not -COOH.
侧链 -W-X-Y-Z的中 W可以是共价键。 另一方面， W可以是侧链具有羧基的 α-氨基酸残 基， 包括一共 4-10个碳原子。 W可以是由遗传密码子编码的 α-氛基酸残基。 例如， W可以 选自 a-Asp、 β-Asp, α-Glu或 γ-Glu; W的其它选择例如是 a-hGlu或者 S-hGlu。  The middle W of the side chain -W-X-Y-Z can be a covalent bond. On the other hand, W may be an α-amino acid residue having a carboxyl group in the side chain, including a total of 4 to 10 carbon atoms. W may be an alpha-amino acid residue encoded by a genetic code. For example, W may be selected from a-Asp, β-Asp, α-Glu or γ-Glu; other choices for W are, for example, a-hGlu or S-hGlu.
在另一个实施方式中， W是由两个 a-氨基酸残基组成的链， 其中一个 a-氨基酸残基具有 4-10个碳原子且侧链具有羧基， 而另一个具有 2-11个碳原子但没有自由羧基。 所述的没有自 由羧基的 a-氨基酸残基可以是中性的可编码的 a-氨基酸残基。根据这种实施方式的 W的例子 是： a-Asp-Gly、 Gly-a-Asp,卩-Asp-Gly、 Gly-p-Asp a-Glu-Gly、 Gly-a-Glu, γ-Glu-Gly, Gly-y-Glu, a-hGlu-Gly、 Gly-a-hGlu、 δ-hGlu-Gly和 Gly-S-hGlu。  In another embodiment, W is a chain consisting of two a-amino acid residues, wherein one a-amino acid residue has 4 to 10 carbon atoms and the side chain has a carboxyl group, and the other has 2 to 11 carbons. Atom but no free carboxyl group. The a-amino acid residue having no free carboxyl group may be a neutral, codeable a-amino acid residue. Examples of W according to this embodiment are: a-Asp-Gly, Gly-a-Asp, 卩-Asp-Gly, Gly-p-Asp a-Glu-Gly, Gly-a-Glu, γ-Glu- Gly, Gly-y-Glu, a-hGlu-Gly, Gly-a-hGlu, δ-hGlu-Gly and Gly-S-hGlu.
在另一个实施方式中， W是由两个 a-氨基酸残基组成的链， 两个 a-氨基酸残基分别具有 4-10个碳原子， 侧链上均具有羧基。 这些 a-氨基酸残基之一或两个可以是可编码的 a-氨基酸 残基。 据这一实施方式的 W 的例子是： a-Asp-a-Asp , a-Asp-a-Glu . a-Asp-a-hGlu > a-Asp- -Asp、 a-Asp-y-Glu、 a-Asp-6-hGlu、 β-Asp-a-Asp、 β-Asp-a-Glu、 β-Asp-a-hGlu、 β-Asp-P-Asp、 P-Asp-y-Glu、 P-Asp-5-hGlu、 a-Glu-a-Asp、 a-Glu-a-Glu、 a-Glu-a-hGlu、 a-Glu-P-Asp, a-Glu-y-Glu, a-Glu-5-hGlu、 y-Glu-a-Asp、 γ-Glu-a-Glu» γ-Glu-a-hGlu. y-Glu-P-Asp> y-Glu-y-Glu^ Y-Glu-6-hGlu» a-hGlu-a- Asp、 a-hGlu-a-Glu、 a-hGlu-a-hGlu> a- hGlu-P_Asp、 a-hGlu-y-Glu, a-hGlu-5-hGlu> S-hGlu-a-Asp、 δ-hGlu-a-Glu, δ-hGlu-a-hGlu, δ- hGlu-p-Asp, δ-hGlu-Y-Glu和 S-hGlu-5-hGlu。  In another embodiment, W is a chain consisting of two a-amino acid residues, each having 4 to 10 carbon atoms and having a carboxyl group in the side chain. One or both of these a-amino acid residues may be a coding a-amino acid residue. Examples of W according to this embodiment are: a-Asp-a-Asp, a-Asp-a-Glu. a-Asp-a-hGlu > a-Asp--Asp, a-Asp-y-Glu, a-Asp-6-hGlu, β-Asp-a-Asp, β-Asp-a-Glu, β-Asp-a-hGlu, β-Asp-P-Asp, P-Asp-y-Glu, P- Asp-5-hGlu, a-Glu-a-Asp, a-Glu-a-Glu, a-Glu-a-hGlu, a-Glu-P-Asp, a-Glu-y-Glu, a-Glu- 5-hGlu, y-Glu-a-Asp, γ-Glu-a-Glu» γ-Glu-a-hGlu. y-Glu-P-Asp> y-Glu-y-Glu^ Y-Glu-6- hGlu» a-hGlu-a-Asp, a-hGlu-a-Glu, a-hGlu-a-hGlu> a-hGlu-P_Asp, a-hGlu-y-Glu, a-hGlu-5-hGlu> S- hGlu-a-Asp, δ-hGlu-a-Glu, δ-hGlu-a-hGlu, δ-hGlu-p-Asp, δ-hGlu-Y-Glu and S-hGlu-5-hGlu.
在另一个实施方式中， W是由三个分别具有 4-10个碳原子的 a-氨基酸残基组成的链，， 该链的氨基酸残基选自具有中性侧链的残基和侧链具有羧基的残基， 使得该链含有至少一个 侧链具有羧基的残基。 在一个实施方式中， 所述氨基酸残基是可编码的残基。  In another embodiment, W is a chain consisting of three a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from residues having a neutral side chain and a side chain A residue having a carboxyl group such that the chain contains at least one residue having a carboxyl group in its side chain. In one embodiment, the amino acid residue is a codeable residue.
在另一个实施方式中， W是由四个分别具有 4-10个碳原子， a-氨基酸残基组成的链， 该 链的氨基酸残基选自具有中性侧链的残基和侧链具有羧基的残基， 使得该链含有至少一个侧 链具有羧基的残基。 在一个实施方式中， 所述氨基酸残基是可编码的残基。  In another embodiment, W is a chain consisting of four a-amino acid residues each having 4 to 10 carbon atoms, the amino acid residue of the chain being selected from the group consisting of a residue having a neutral side chain and a side chain having The residue of the carboxyl group is such that the chain contains at least one residue having a carboxyl group in its side chain. In one embodiment, the amino acid residue is a codeable residue.
在一个实施方式中， -W-X-Y-Z中的 W可以通过脲衍生物连接至赖氨酸残基的 ε-氨基。 侧链 -W-X-Y-Z中的 X可以是通式 -£0-的基团， 通过来自加下划线的羰基碳的键与 W中 的氨基形成酰胺键； 或当 W是共价键时， X通过来自加下划线的羰基碳的键与双链化合物的 Β链的 Ν-末端的 a-氨基或与单链化合物的 N-末端的 a-氨基或与单链或双链化合物中的赖氨 酸残基的 ε-氨基形成酰胺键。  In one embodiment, W in -W-X-Y-Z can be attached to the ε-amino group of the lysine residue by a urea derivative. X in the side chain -WXYZ may be a group of the formula -£0-, forming an amide bond with an amino group in W by a bond from an underlined carbonyl carbon; or when W is a covalent bond, X is derived from The underlined carbonyl carbon bond with the Ν-terminal a-amino group of the Β chain of the double-stranded compound or the a-amino group of the N-terminus of the single-stranded compound or the lysine residue in the single-stranded or double-stranded compound The ε-amino group forms an amide bond.
在进一步的实施方式中， 所述侧链 -W-X-Y-Z中的 X可以是通式 -CH(COOH)£0-的基团， 通过来自加下划线的羰基碳的键与 W中的象基形成酰胺键； 或当 W是共价键时， X通过来 自加下划线的羰基碳的键与双链化合物的 B链的 N-末端的 a-氨基或与单链化合物的 N-末端 的 a-氨基或与双链或单链化合物中的赖氨酸残基的 ε-氨基形成酰胺键。 在进一步的实施方式中，側链 -W-X-Y-Z中的 X可以是通式 -N(CH2COOH)CH2£0-的基团， 通过来自加下划线的羰基碳的键与 W中的氨基形成酰胺键； 或当 W是共价键时， X通过来 自加下划线的羰基碳的键与双链化合物的 B链的 N-末端的 α-氨基或与单链化合物的 Ν-末端 的 α-氨基或与双链或单链化合物中的赖氨酸残基的 ε-氨基形成酰胺键。 In a further embodiment, X in the side chain -WXYZ may be a group of the formula -CH(COOH) £0-, which forms an amide bond with a group in W by a bond from an underlined carbonyl carbon. Or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal a-amino group of the B chain of the double-stranded compound or to the N-terminal a-amino group of the single-stranded compound or The ε-amino group of the lysine residue in the double-stranded or single-stranded compound forms an amide bond. In a further embodiment, X in the side chain -WXYZ may be a group of the formula -N(CH 2 COOH)CH 2 £0-, which forms an amide with an amino group from W by a bond from an underlined carbonyl carbon. Key; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal α-amino group of the B chain of the double-stranded compound or to the Ν-terminal α-amino group of the single-stranded compound or An amide bond is formed with the ε-amino group of the lysine residue in the double-stranded or single-stranded compound.
在进一步的实施方式中， 侧链 -W-X-Y-Z中的 X可以是通式 -N(CH2CH2COOH)C¾£0-的 基团， 通过来自加下划线的羰基碳的键与 W中的氨基形成酰胺键； 或当 W是共价键时， X 通过来自加下划线的羰基碳的键与双链化合物的 B链的 N-末端的 α-氨基或与单链化合物的 Ν-末端的 α-氨基或与双链或单链化合物中的赖氨酸残基的 ε-氨基形成酰胺键。 In a further embodiment, the X in the side chain -WXYZ may be a group of the formula -N(CH 2 CH 2 COOH)C3⁄4£0-, formed by a bond from an underlined carbonyl carbon and an amino group in W An amide bond; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal α-amino group of the B chain of the double-stranded compound or the Ν-terminal α-amino group of the single-stranded compound Or forming an amide bond with the ε-amino group of a lysine residue in a double-stranded or single-stranded compound.
在进一步的实施方式中， -W-X-Y-Z中的 X可以是通式 -N(CH2COOH) CH2CH2£0-的基团， 通过来自加下划线的羰基碳的键与 W中的氨基形成酰胺键； 或当 W是共价键时， X通过来 自加下划线的羰基碳的键与双链化合物的 B链的 N-末端的 α-氨基或与单链化合物的 Ν-末端 的 α-氨基或与双链或单链化合物中的赖氨酸残基的 ε-氨基形成酰胺键。 In a further embodiment, X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CH 2 £0-, which forms an amide with an amino group in W by a bond from an underlined carbonyl carbon. Key; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal α-amino group of the B chain of the double-stranded compound or to the Ν-terminal α-amino group of the single-stranded compound or An amide bond is formed with the ε-amino group of the lysine residue in the double-stranded or single-stranded compound.
在进一步的 实施方式中 ， -W-X-Y-Z 中 的 X 可以是通式 -N(CH2COOH) CH2CON(CH2COOH)CH2£0-的基团， 通过来自加下划线的羰基碳的键与 W中的氨基形成酰 胺键； 或当 W是共价键时， X通过来自加下划线的叛基碳的键与双链化合物的 B链的 N-末 端的 α-氨基或与单链化合物的 Ν-末端的 α-氨基或与双链或单链化合物中的赖氨酸残基的 ε- 氨基形成酰胺键。 In a further embodiment, X in -WXYZ may be a group of the formula -N(CH 2 COOH) CH 2 CON(CH 2 COOH)CH 2 £0-, by a bond from an underlined carbonyl carbon The amino group in W forms an amide bond; or when W is a covalent bond, X passes through an N-terminal amino group of the B chain of the double-stranded compound or a single-chain compound from a bond derived from an underlined rebel carbon The terminal α-amino group forms an amide bond with the ε-amino group of the lysine residue in the double-stranded or single-stranded compound.
在进一步的实施方式中， -W-X-Y-Z中的 X可以是通式 -N(CH2C¾COOH) CH2CH 0-的 基团， 通过来自加下划线羰基碳的键与 W中的氨基形成酰胺键； 或当 W是共价键时， X通 过来自加下划线的談基碳的键与双链化合物的 B链的 N-末端的 α-氨基或与单链化合物的 Ν- 末端的 α-氨基或与双链或单链化合物中的赖氨酸残基的 ε-氨基形成酰胺键。 In a further embodiment, X in -WXYZ may be a group of the formula -N(CH 2 C3⁄4COOH) CH 2 CH 0-, which forms an amide bond with an amino group in W by a bond derived from an underlined carbonyl carbon; When W is a covalent bond, X passes through the N-terminal α-amino group of the B chain of the double-stranded compound from the underlined carbon-based bond or the Ν-terminal α-amino group of the single-stranded compound or The ε-amino group of the lysine residue in the chain or single chain compound forms an amide bond.
在进一步的实施方式中 ， -W-X-Y-Z 中的 X 可以是通式 -N(CH2C COOH) CH2CH2CON(CH2CH2COOH) C¾CH2£0-的基团，通过来自加下划线的羰基碳的键与 W中的 氨基形成酰胺键； 或当 W是共价键时， X通过来自加下划线的羰基碳的键与双链化合物的 B 链的 N-末端的 α-氨基或与单链化合物的 Ν-末端的 α-氨基或与双链或单链化合物中的赖氨酸 残基的 ε-氨基形成酰胺键。 In a further embodiment, X in -WXYZ may be a group of the formula -N(CH 2 C COOH) CH 2 CH 2 CON(CH 2 CH 2 COOH) C3⁄4CH 2 £0-, by underlining The bond of the carbonyl carbon forms an amide bond with the amino group in W; or when W is a covalent bond, X passes through the bond from the underlined carbonyl carbon to the N-terminal α-amino group of the B chain of the double-stranded compound or The Ν-terminal α-amino group of the chain compound forms an amide bond with the ε-amino group of the lysine residue in the double-stranded or single-stranded compound.
侧链 -W-X-Y-Z中的 Υ可以是通式 -(CH2)m基团， 其中 m是 6-32、 8-20、 12-20或 12-16 的整数。 The oxime in the side chain -WXYZ may be a group of the formula -(CH 2 ) m wherein m is an integer of 6-32, 8-20, 12-20 or 12-16.
在另一种实施方式中， -W-X-Y-Z中的 Y是包含 1、 2或 3个 -CH-CH-基团和多个 -CH2- 基团的二价烃链， 所述多个 -CH2-基团的个数满足烃链中的碳原子总数范围是 6-32、 10-32、 12-20或 12-16。 In another embodiment, Y in -WXYZ is a divalent hydrocarbon chain comprising 1, 2 or 3 -CH-CH- groups and a plurality of -CH 2 - groups, said plurality of -CH 2 The number of groups satisfying the total number of carbon atoms in the hydrocarbon chain is in the range of 6-32, 10-32, 12-20 or 12-16.
在另一种实施方式中， -W-X-Y-Z中的 Y是通式 - (CH2)VC6H4 (CH2) W-的二价烃链， 其中 v 和 w是整数， 或者其中之一是零， 使得 V和 w总和的范围是 6-30、 10-20或 12-16。 In another embodiment, Y in -WXYZ is a divalent hydrocarbon chain of the formula -(CH 2 ) V C 6 H 4 (CH 2) W - wherein v and w are integers, or one of Zero, such that the sum of V and w ranges from 6-30, 10-20 or 12-16.
在一种实施方式中， 侧链 -W-X-Y-Z中的 Z是- COOH, 条件是当 W是共价键而 X是 -CO- 时， Z不是 -COOH。  In one embodiment, Z in the side chain -W-X-Y-Z is -COOH, provided that when W is a covalent bond and X is -CO-, Z is not -COOH.
在另一种实施方式中， -W-X-Y-Z 中的 Z 是 -CO-Asp、 -CO-Glu, -CO-Gly、 -CO-Sar、 -CH(COOH)2、 -N(C¾COOH)2、 -S03H或- P03H。 In another embodiment, Z in -WXYZ is -CO-Asp, -CO-Glu, -CO-Gly, -CO-Sar, -CH(COOH)2, -N(C3⁄4COOH) 2 , -S0 3 H or - P0 3 H.
在进一步的实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Asp, α-Glu或 γ-Glu; X是 -CO- 或者 -CH(COOH)CO-; Y是 -(CH2)m, 其中 m是 12-18的整数； Z是 -COOH -、 -CH(COOH)2或 不存在。 在另一种实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Asp, a-GIu或 γ-GIu; -X-Y-Z是 -CO(CH2)n, 通过来自加下划线的叛基碳的键与 W中的 形成酰胺键， 其中 n是 10-18中 的整数。 In a further embodiment, W in -WXYZ is a-Asp, β-Asp, α-Glu or γ-Glu; X is -CO- or -CH(COOH)CO-; Y is -(CH 2 ) m , wherein m is an integer from 12 to 18; Z is -COOH -, -CH(COOH) 2 or is absent. In another embodiment, W in -WXYZ is a-Asp, β-Asp, a-GIu or γ-GIu; -XYZ is -CO(CH 2 ) n , which is derived from underlined rebel carbon The bond forms an amide bond with W, wherein n is an integer from 10-18.
在更具体的实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Asp, a-Glu或 γ-Glu; -X-Y-Z是 -CO(CH2)14。 In a more specific embodiment, W in -WXYZ is a-Asp, β-Asp, a-Glu or γ-Glu; -XYZ is -CO(CH 2 ) 14 .
在更具体的实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Asp, a-Glu或 γ-Glu; -X-Y-Z是 -CO(CH2)16。 In a more specific embodiment, W in -WXYZ is a-Asp, β-Asp, a-Glu or γ-Glu; -XYZ is -CO(CH 2 ) 16 .
在更具体的实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Αβρ> a-Glu或 γ-Glu; -X-Y-Z是 -CO(C¾)18。 In a more specific embodiment, W in -WXYZ is a-Asp, β-Αβρ> a-Glu or γ-Glu; -XYZ is -CO(C3⁄4) 18 .
在更具体的实施方式中， -W-X-Y-Z中的 W是 a-Asp、 β-Asp, a-Glu或 γ-Glu; -X-Y-Z是 胆固醇、 胆汁酸（如胆酸、 鹅脱氧胆酸、 肝胆酸、 牛横胆酸、 脱氧胆酸、 石胆酸）；  In a more specific embodiment, W in -WXYZ is a-Asp, β-Asp, a-Glu or γ-Glu; -XYZ is cholesterol, bile acid (such as cholic acid, chenodeoxycholic acid, hepatobiliary acid, Cow transverse acid, deoxycholic acid, lithocholic acid);
在一种优选的实施方案中， 该化合物包括 A链和 B链， 其中，  In a preferred embodiment, the compound comprises an A chain and a B chain, wherein
A链的氨基酸序列为：  The amino acid sequence of the A chain is:
GrVDQC[3]C[4]FRSC[5]X4i2LX414X415LX4i7X4i8Y [6]AX422) GrVDQC[3]C[4]FRSC[5]X 4 i 2 LX4 14 X4 15 LX4i 7 X4i 8 Y [ 6 ]AX4 22 )
B链氨基酸序列为：  The B chain amino acid sequence is:
X423-426 427LC[i]GAHLVDALFFVC[2]GDRGFYX448X44 X45oX45lX452¾53X454X455 ' X 42 3-4 26 427LC[i]GAHLVDALFFVC[ 2 ]GDRGFYX 448 X44 X4 5 oX45lX4 5 23⁄453X454X4 55 '
所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物通过 6个半胱氨酸形成 3对二 硫键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： Cn]和。[4]形成二硫键， C[2]和 C[6] 形成二硫键， [3]和。[5]形成二硫键； In the compound, [1]-[6] represents the number of a cysteine; the compound forms a 3-pair disulfide bond through 6 cysteines, wherein the A chain and the B chain pass two pairs of interchain disulfide The bond is connected, there is a pair of intrachain disulfide bonds in the A chain, and the specific positions of the three pairs of disulfide bonds are: C n] and . [ 4] Form a disulfide bond, C[ 2] and C[ 6 ] form a disulfide bond, [ 3] and . [5] forming a disulfide bond;
其中 12为丝氨酸或通式（I )结构； 14为精氨酸或通式（I )结构； 15是精氨酸或通 式（I )结构； 17为谷氨酸或通式（I ) 结构； 18为天冬酰胺或通式（I ) 结构； 22为赖 氨酸、 精氨酸-赖氨酸二肽或缺失， 或为通式（I )结构； 当 22为二肽时， 其中一个氨基酸 为通式（I )结构； X42W26是甘氨酸-脯氨酸-谷氨酸三肽、 UL-甘氨酸 -脯氨酸-谷氨酸、 苯丙氨 酸-纈氨酸-天冬酰胺-谷氨酰胺四肽或 Ui苯丙氨酸 -缬氨酸-天冬酰胺 -谷氨酰胺； 27是组氨酸 或苏氨酸； 48是 -NH2、 苯丙氨酸或缺失； X449是天冬酰胺或缺失； 50是赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸、 脯氨酸或缺失； 51是脯氨酸、 赖氨酸或缺失， 或为通式（I )结构； 52 是苏氨酸、 赖氨酸或缺失， 或为通式（I )结构； 53是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或 为通式（I )结构； 54是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I )结构； 55是赖氨 酸或缺失， 或为通式（I )结构； UL和通式（I ) 结构如本发明中所定义的。 Wherein 12 is a serine or a structure of the formula (I); 14 is an arginine or a structure of the formula (I); 15 is an arginine or a structure of the formula (I); 17 is a glutamic acid or a structure of the formula (I) 18 is an asparagine or a structure of the formula (I); 22 is a lysine, arginine-lysine dipeptide or a deletion, or a structure of the formula (I); when 22 is a dipeptide, one of them The amino acid is of the formula (I); X42W26 is a glycine-valine-glutamic acid tripeptide, U L -glycine-valine-glutamic acid, phenylalanine-valine-asparagine-valley Aminoamide tetrapeptide or Ui phenylalanine-valine-asparagine-glutamine; 27 is histidine or threonine; 48 is -NH 2 , phenylalanine or deletion; X449 is aspartic Amide or deletion; 50 is lysine, arginine, glutamic acid, aspartic acid, proline or deletion; 51 is valine, lysine or deletion, or is of the formula (I) 52 is threonine, lysine or deletion, or is of the formula (I); 53 is glutamic acid, glycine, lysine or deletion, or is of the formula (I); 54 is glutamic acid , glycine, lysine or Loss, or the general formula (I) structure; 55 is lysine or deletion, or the general formula (I) structure; U L general formula (I) in the structure of the present invention as defined herein.
在另一种实施方式中， 所述化合物是基于 IGF-1？丈造的单链结构， 该化合物的结构为： In another embodiment, the compound is based on IGF-1? The single-stranded structure of the compound, the structure of the compound is:
X2o 1 aLC[！ ]G AX2o njLVD ALX2o 10X201 d VC[2]GDRGFX201 eX202X2o3X204 205X206G¾07X207aX208X 2 o 1 a LC[! ]G AX 2 o njLVD ALX 2 o 10X201 d VC[ 2 ]GDRGFX 20 1 e X 202 X 2 o 3 X 204 2 0 5X206G3⁄4 0 7X207aX208
209X210X21l 212¾13X214X215X216GIVDQC[3]C[4]X2i7RSC[5]X218LX21 X220LX22iX222YC[6]X223X224， 其中， 209X210X21l 2123⁄413X214X215X216GIVDQC[3]C[4]X 2 i 7 RSC[5]X 218 LX 21 X 2 2 0 LX 22 iX 2 2 2 YC[6]X 223 X 224 , where
X2oia是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、甘氨酸-脯氨酸 -谷氨酸-组氨酸、苯 1¾氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸- 组氨酸或 UL-苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸； X2。lb是组氨酸、 谷氨酸、 谷氨酰 胺、 精氨酸或苯丙氨酸； X2olc是苯丙氨酸或色氨酸； X2oid是苯丙氨酸或色氨酸； X201e是苯丙 氨酸、 酪氨酸或组氨酸； X2o2是苯丙氨酸、 酪氨酸或缺失； X2o3是天冬酰胺、 苏氨酸、 天冬 氨酸、 谷氨酸或缺失； X2()4是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X2Q5是 脯氨酸、 赖氨酸、 精氨酸、 天冬氛酸、 谷氨酸或缺失或通式（I )结构； X2o6是苏氨酸、 赖氨 酸或缺失或通式 （I)结构； X2Q7是丝氨酸、 丙氨酸、 甘氨酸、 通式 （I) 结构或缺失； x2()7a 是丝氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X2G8是丝氨酸、 通式（I)结构或缺失； x209是丝氨酸、 通式（I)结构或缺失； x21Q是丝氨酸、 通式（I)结构或缺失； x211是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X212是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或 缺失； X213是丙氨酸、 脯氨酸、 精氨酸、 甘氨酸、 通式（I)结构或缺失； X214是脯氨酸、 谷 氨酰胺、 甘氨酸、 通式（I)结构或缺失； X215是谷氨酰胺、 苏氨酸、 甘氨酸、 通式（I)结构 或缺失； X216是苏氨酸、 精氨酸、 赖氨酸、 通式（I)结构或缺失； x217是苯丙氨酸、 精氨酸、 组氨酸或通式（I)结构； X218是天冬氨酸、 丝氨酸、 谷氨酰胺、 天冬酰胺或通式（I)结构； x219是精氨酸或通式（I) 结构； X22G是精氨酸或通式（I)结构； X221是谷氨酸或通式（I) 结构； X222是天冬酰胺或通式（I)结构； x223是丙氨酸、 甘氨酸或天冬酰胺； x224是赖氨酸、 精氛酸-赖氨酸二肽或缺失， 或为通式（I)结构， 当 X224为二肽时， 其中一个氨基酸为通式X 2 oi a is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine, benzene 13⁄4-proline-asparagine-glutamine- Histidine, UL-glycine-valine-glutamate-threonine, UL-glycine-valine-glutamate-histidine or UL-phenylalanine-valine-asparagine - glutamine-histidine; X 2 . Lb is histidine, glutamic acid, glutamine, arginine or phenylalanine; X 2 o lc is phenylalanine or tryptophan; X 2 oid is phenylalanine or tryptophan; X 201e is phenylalanine, tyrosine or histidine; X 2 o 2 is phenylalanine, tyrosine or deletion; X 2 o 3 is asparagine, threonine, aspartic acid, Glutamate or deletion; X 2 () 4 is valine, lysine, arginine, aspartic acid, glutamic acid or deletion; X 2 Q5 is valine, lysine, arginine , aspartic acid, glutamic acid or deletion or structure of formula (I); X 2 o 6 is threonine, lysine Acid or deletion or structure of formula (I); X 2Q7 is serine, alanine, glycine, structure or deletion of formula (I); x 2 () 7a is serine, alanine, glycine, formula (I) Structure or deletion; X 2G8 is serine, structure or deletion of formula (I); x 209 is serine, structure or deletion of formula (I); x 21Q is serine, structure or deletion of formula (I); x 211 is fine Acid, alanine, glycine, structure or deletion of formula (I); X 212 is arginine, alanine, glycine, structure or deletion of formula (I); X 213 is alanine, valine , arginine, glycine, structure or deletion of formula (I); X 214 is valine, glutamine, glycine, structure or deletion of formula (I); X 215 is glutamine, threonine, glycine , structure or deletion of formula (I); X 216 is threonine, arginine, lysine, structure or deletion of formula (I); x 217 is phenylalanine, arginine, histidine or formula (I) structure; X 218 is aspartic acid, serine, glutamine, asparagine or formula (I) structure; x 219 is fine Acid or the general formula (I) structure; X 22G is arginine or formula (I) structure; X 221 is glutamic acid or structural formula (I); X 222 is asparagine or formula (I) Structure x 223 is alanine, glycine or asparagine; x 224 is lysine, amic acid-lysine dipeptide or deletion, or is of the formula (I), when X 224 is a dipeptide, One of the amino acids is of the formula
(I)结构； UL和通式（I)结构如本发明中所定义的； (I) structure; U L and the structure of the formula (I) are as defined in the present invention;
上述化合物结构中， [1]- [6]表示半胱氨酸的编号。 本发明的单链化合物在三级结构中， 以胰岛素的结构方式形成链内的二疏键， 具体为： Cn] 和 C[4] 形成二硫键， C[2] 和 C[6] 形成 二硫键， C[3] 和 C[5]形成二硫键。 In the above compound structure, [1] - [6] represent the number of cysteine. The single-chain compound of the present invention forms a diamole bond in the chain in the tertiary structure in the structure of insulin, specifically: C n] and C [4] form a disulfide bond, C [2] and C [6] A disulfide bond is formed, and C [3] and C [5] form a disulfide bond.
在一种优选的实施方案中， 所述单链化合物的结构为：  In a preferred embodiment, the structure of the single chain compound is:
X201aLC[ I ] G AHLVD ALFF VC[2] GDRGFYX2o2X203 204X205 206GX2o7GX208X2o9X210X2 πΧ2ι2Χ 213X2"X2i5X2i6GrVDQC[3]C[4]FRSC[5]X218LX219X220LX221X222YC[6]A， 其中 , X 201a LC[ I ] G AHLVD ALFF VC[2] GDRGFYX2o 2 X203 204X2 05 206GX 2 o7GX 208 X 2 o9X 2 1 0 X 2 πΧ 2 ι 2 Χ 2 13 X2"X2i 5 X2i 6 GrVDQC[ 3 ]C[4 ]FRSC[ 5 ]X 218 LX 21 9X 220 LX 221 X 222 YC[ 6 ]A, where
X201a是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸、 1 -甘氨酸-脯氨酸 -谷氨酸-苏氨酸或 UL-苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸； X2o2是 苯丙氨酸或缺失； X2Q3是天冬酰胺或缺失； X2Q4是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸或缺 失； X2Q5是脯氨酸、 赖氨酸或通式（I)结构； X2Q6是苏氨酸、 赖氨酸或通式（I)结构； X2o7 是丝氨酸、 丙氨酸或通式（ I )结构； X2()8是丝氨酸或通式（ I )结构； X2Q9是丝氨酸或通式（ I ) 结构； X21D是丝氨酸或通式（I)结构； X2U是精氨酸、 丙氨酸、 通式（I)结构或缺失； x212 是精氨酸、 丙氨酸、 甘氨酸、 通式（I) 结构或缺失； X213是丙氨酸、 脯氨酸、 精氨酸、 通式 (I)结构或缺失； X2i4是脯氨酸、 谷氨酰胺、 通式（I) 结构或缺失； X215是谷氨酰胺、 苏氨 酸、 通式 （I) 结构或缺失； X216是苏氨酸、 精氨酸、 赖氨酸、 通式（I) 结构或缺失； x218 是丝氨酸或通式（I) 结构； 219是精氨酸或通式（I)结构； X22Q是精氨酸或通式（I)结构； 221是谷氨酸或通式（I)结构； X222是天冬酰胺或通式（I)结构； UL和通式（I)结构如本 文中所定义的。 X 201a is glycine-valine-glutamate-threonine, phenylalanine-valine-asparagine-glutamine-histidine, 1-glycine-valine-glutamate- Threonine or U L -phenylalanine-valine-asparagine-glutamine-histidine; X 2 o2 is phenylalanine or deletion; X 2 Q3 is asparagine or deletion; X 2Q4 is valine, lysine, arginine, aspartic acid or deletion; X 2Q5 is valine, lysine or the structure of formula (I); X 2Q6 is threonine, lysine or a structure of the formula (I); X 2 o7 is a serine, alanine or a structure of the formula (I); X 2 () 8 is a serine or a structure of the formula (I); X 2Q9 is a serine or a structure of the formula (I) X 21D is serine or the structure of formula (I); X 2U is arginine, alanine, structure or deletion of formula (I); x 212 is arginine, alanine, glycine, formula (I) Structure or deletion; X 213 is alanine, valine, arginine, structure or deletion of formula (I); X 2 i4 is valine, glutamine, structure or deletion of formula (I); X 215 is glutamine, threonine, of the general formula (I) junction Or deleted; X 216 is threonine, arginine, lysine, formula (I) structure, or deleted; x 218 serine or formula (I) structure; 219 is arginine or formula (I) Structure; X 22Q is arginine or the structure of formula (I); 2 21 is glutamic acid or the structure of formula (I); X 222 is asparagine or structure of formula (I); U L and formula ( I) The structure is as defined herein.
在基于 IGF-1改造的修饰的化合物方面， 本发明的修饰的化合物选自：  In terms of an IGF-1 engineered modified compound, the modified compound of the invention is selected from the group consisting of:
III- 1: 双链结构， 包括 A链和 B链， 其中， A链序列为 SEQ ID NO:17所示序列； B链 序列为 G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT; a triple-stranded structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain sequence is G (N a -PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT;
III-2: 双链结构， 包括 A链和 B链， 其中， A链序列为 SEQ ID NO:17所示序列； B链 序列为 GPETLCGAHLVDALFFVCGDRGFYFNPK(NE-PEG 20K); III-2: a double-stranded structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17; and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK (N E -PEG 20K);
III-3: 单链结构， 其序列为：  III-3: Single-stranded structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGK(Ne-PEG20K)GSSSR APQTGIVDQCCFR SCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGK(N e -PEG20K)GSSSR APQTGIVDQCCFR SCSLRRLENYCA;
III-4: 单链结构， 其序列为：  III-4: Single-stranded structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPPTGK(NE-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPPTGK(N E -PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
III-5: 单链结构， 其序列为：  III-5: Single-stranded structure, whose sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N£-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N £ -PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
111-6: 双链结构， 包括 A链和 B链， 其中， A链序列为： SEQ ID NO: 17所示序列； B链 序列为： G( a-CO(C¾)14COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT; 111-6: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is: SEQ ID NO: 17; B chain sequence is: G( a -CO(C3⁄4) 14 COOH) PETLCGAHLVDALFFVCGDRGFYFNPPT;
III-7: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-( a- (HOOC(CH2)14CO)-y-Glu)]； III-7: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -( a - (HOOC(CH 2 ) 14 CO )-y-Glu)];
III-8: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[Ns-(Na- (HOOC(CH2)16CO)-y-Glu)]; III-8: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N s -(N a - (HOOC(CH 2 ) 16 CO)-y-Glu)];
III-9: 双链结构， 包括 Α链和 Β链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)12CO)-y-Glu)]； III-9: Double-stranded structure, including an Α chain and an Β chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -(N a - (HOOC(CH 2 ) 12 CO)-y-Glu)];
111-10: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK{NE-[Na- (HOOC(C¾)iiNHCO(CH2)3CO) -γ-Glu]}; 111-10: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK{N E -[N a - (HOOC(C3⁄4)iiNHCO( CH 2 ) 3 CO) -γ-Glu]};
III-ll : 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列 为 ： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu -N-(Y-G1U)]; III-ll: double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu -N-(Y-G1U)];
III-12: 单链结构， 其序列为：  III-12: Single-stranded structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGK[N£-(Na-(HOOC(CH2)14CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGK[N £ -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA;
111-13: 单链结构， 其序列为：  111-13: Single-stranded structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[NE-(Na-(HOOC(CH2)14CO)-y-Glu)]SS AAPQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N E -(N a -(HOOC(CH 2 ) 14 CO)-y-Glu)]SS AAPQTGIVDQCCFRSCSLRRLENYCA;
III -14： 单链结构， 其序列为： GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK III -14: Single-stranded structure, the sequence is: GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK
[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)]AAPQTGIVDQCCFRSCSLRRLENYCA; [N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]AAPQTGIVDQCCFRSCSLRRLENYCA;
111-15: 单链结构， 其序列为：  111-15: Single-stranded structure, whose sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N E -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA;
111-16: 单链结构， 其序列为：  111-16: Single-chain structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[NE-( a-(HOOC(CH2)14 CO)-y-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N E -( a -(HOOC(CH 2 ) 14 CO)-y-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
III-17: 单链结构， 其序列为：  III-17: Single-stranded structure, whose sequence is:
GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[NE-(Na-(HOOC (CH2)14CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[N E -(N a -(HOOC (CH 2 ) 14 CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
111-18: 单链结构， 其序列为：  111-18: Single-chain structure, the sequence is:
GPETLCGAHLVDALFFVCGD GFYFNPTGSGK[Ne-(Na-(HOOC(CH2)I4CO)-y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGD GFYFNPTGSGK[N e -(N a -(HOOC(CH 2 ) I4 CO)-y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA;
III- 19: 欢链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPK[NS- (Na-(HOOC (CH2)14 CO)-y-Glu)]； 111-20: 单链结构， 其序列为： III- 19: A loop-like structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK[N S - (N a -(HOOC (CH 2 ) 14 CO )-y-Glu)]; 111-20: Single-chain structure, the sequence is:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA;
III -21 ： 双链 结 构 ， 包括 A 链 和 B 链 ， 其 中 A 链序 列 为 ： GIVDQCCFRSCSLK[Ne-(Na-(HOOC(CH2)14CO)-y-Glu)]RLENYCA ； B 链 序 列 为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPT(SEQ ID NO: 163 ); III -21 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is: GIVDQCCFRSCSLK[N e -(N a -(HOOC(CH 2 ) 14 CO)-y-Glu)]RLENYCA ; B chain sequence Is FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 163);
III -22 ： 双链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链序 列 为 ： GIVDQCCFRSCSLRK[NE-(Na-(HOOC(CH2)i4CO)-y-Glu)]LENYCA ； B 链 序 列 为 GPETLCGAHLVDALFFVCGDRGFYFNPPT ( SEQ ID NO: 164 ); III -22 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is: GIVDQCCFRSCSLRK[N E -(N a -(HOOC(CH 2 )i 4 CO)-y-Glu)]LENYCA ; B chain The sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 164);
III -23 ： 双 链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链 序 列 为 III-23 : a double - stranded structure comprising an A chain and a B chain , wherein the A chain sequence is
GIVDQCCFRSCSLRRLENYCAK[NE-( a-(HOOC(CH2)14CO)-Y-Glu)] ; B 链序列为 SEQ ID NO: 164所示序列； GIVDQCCFRSCSLRRLENYCAK[N E -( a -(HOOC(CH 2 ) 14 CO)-Y-Glu)] ; The B chain sequence is the sequence shown in SEQ ID NO: 164;
III -24 ： 双 链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链 序 列 为 GIVDQCCFRSCSLRRLENYCARK[N£-(Na-(HOOC(CH2)i4CO)-Y-Glu)] , B 链序列为 SEQ ID NO: 164所示序列； III -24 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCARK[N £ -(N a -(HOOC(CH 2 )i 4 CO)-Y-Glu)], and the B chain sequence is SEQ ID NO: 164 the sequence shown;
ΠΙ-25: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[Ne- (Na-(HOOC(CH2)14CO)-Y-Glu)]; ΠΙ-25: double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[N e - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
111-26: 双链结构， 包括 Α链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [Ne-(Na-(HOOC(CH2)i4CO)-Y-Glu)]; 111-26: Double-stranded structure, including an Α chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [N e -(N a -(HOOC(CH 2 )i 4 CO)-Y-Glu)];
111-27: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[N ( a-(HOOC(CH2)i4CO)-Y-Glu)]；111-27: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[N ( a -(HOOC(CH 2 )i 4 CO)- Y-Glu)];
111-28: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 歹' J 为 GPETLCGAHLVDALFFVCGDRGFYFNPK[NE- (NA- (HOOC(CH2)14CO)-Y-G1U-N-111-28: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence 歹 ' J is GPETLCGAHLVDALFFVCGDRGFYFNPK[N E - (N A - (HOOC(CH 2 ) 14 CO)-Y-G1U-N-
(γ-Qiu))]; (γ-Qiu))];
111-29: 双链结构， 包括 Α链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 G[Na-(Na-(CH3(C¾)14CO)-Y-L-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT; 111-29: Double-stranded structure, including an Α chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is G[N a -(N a -(CH 3 (C3⁄4) 14 CO )-YL-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT;
111-30: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 G( a-dPEG12-马来酰亚胺-白蛋白） PETLCGAHLVDALFFVCGDRGFYFNKPT; 111-30: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is G( a -dPEG 12 -maleimide-albumin) PETLCGAHLVDALFFVCGDRGFYFNKPT ;
111-31 : 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链 序列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[NS- (Na-(HOOC(CH2)14CO)-Y-Glu)]；111-31 : Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[N S - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
111-32: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTK[NE- (Na-(HOOC(CH2)14CO)-y-Glu)]; 111-32: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTK[N E - (N a -(HOOC(CH 2 ) 14 CO )-y-Glu)];
III-33 : 双链结构， 包括 Α链和 Β链， 其中 A链序列为 SEQ ID NO 17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N£-(Na-(HOOC(CH2)14CO)-Y-Glu)]； III-33: Double-stranded structure, including an Α chain and an Β chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17 and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N £ -(N a -(HOOC(CH 2 ) 14 CO) -Y-Glu)];
111-34: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK[N (Na-(HOOC(CH2) i4CO)-y-Glu)]；111-34: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK[N (N a -(HOOC(CH 2 ) i 4 CO) -y-Glu)];
ΠΙ-35:单链结构 G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA; ΠΙ-35: single-stranded structure G(N a -PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA;
III-36: 双链结构， 包括 Α链和 B链， 其中 A链序列为 GIVDQCCHRSCSLRRLENYCA, B 链序列为 GPEHLCGAHLVDALFFVCGDRGFYFNPK [NE-CO-(CH2CH20)5-(CH2)2- NH- (N"-(HOOC III-36: Double-stranded structure, including Α chain and B chain, wherein the A chain sequence is GIVDQCCHRSCSLRRLENYCA, and the B chain sequence is GPEHLCGAHLVDALFFVCGDRGFYFNPK [N E -CO-(CH 2 CH 2 0) 5 -(CH 2 ) 2 - NH- (N"-(HOOC
Figure imgf000038_0001
Figure imgf000038_0001
111-37: 单链结构 GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[N£-CO-(CH2CH20)5- (CH2)2-NH-(Na-(HOOC (CH2)16 CO)-y-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA; 111-37: Single-stranded structure GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[N £ -CO-(CH 2 CH 2 0) 5 - (CH 2 ) 2 -NH-(N a -(HOOC (CH 2 ) 16 CO)-y-Glu)] GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA;
Na表示氨基酸或氨基酸残基的 a-氨基； Νε表示氨基酸或氨基酸残基的 ε-氨基， 例如赖氨 酸侧链的 ε-氨基。 N a represents an a-amino group of an amino acid or an amino acid residue; Ν ε represents an ε-amino group of an amino acid or an amino acid residue, such as an ε-amino group of a lysine side chain.
上述的双链或单链化合物的三级结构中均以胰岛素的结构方式形成链内的二硫键， 具体 为： C[u和 C[41 形成二石充键， C[2] 和 C[6] 形成二疏键， C[3] 和 [5]形成二硫键„ 半胱氨酸的 编号如本文中所定义的。 In the tertiary structure of the above double-stranded or single-stranded compound, the disulfide bond in the chain is formed by the structure of insulin, specifically: C[ u and C [ 41 forms a two-stone charge, C [2 ] and C [ 6 ] The two sparse bonds are formed, and C[ 3] and [ 5] form a disulfide bond. The number of cysteine is as defined herein.
可以以基本不含锌的化合物形式或锌的复合物形式提供本发明的具有降血糖作用的化合 物。 当提供本发明的化合物的锌复合物时， 其中本发明的化合物可以形成六聚体， 每个六聚 体可以结合 2个 Zn2+ 3个 Zn2+或 4个 Zn2+ 药物组合物及用途 The hypoglycemic compound of the present invention can be provided in the form of a substantially zinc-free compound or a complex of zinc. When a zinc complex of the compound of the present invention is provided, wherein the compound of the present invention can form a hexamer, each hexamer can bind 2 Zn 2+ 3 Zn 2+ or 4 Zn 2+ pharmaceutical compositions and use
在本发明的另一方面中， 提供了一种药物组合物， 所述药物组合物包括治疗有效量的根 据本发明的化合物和制药学上可接受的载体， 用于治疗 1型糖尿病、 2型糖尿病和引起高血 糖症的其它情况。 根据本发明的化合物可以用于制备治疗 1 型糖尿病、 2型糖尿病和引起高 血糖症的其它情况的药物组合物。  In another aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the invention and a pharmaceutically acceptable carrier for the treatment of type 1 diabetes, type 2 Diabetes and other conditions that cause hyperglycemia. The compounds according to the invention may be used in the preparation of a pharmaceutical composition for the treatment of type 1 diabetes, type 2 diabetes and other conditions which cause hyperglycemia.
在本发明的另一方面中， 提供了一种治疗 1 型糖尿病、 2型糖尿病和引起高血糖症的其 它情况的药物组合物， 所述药物组合物包括治疗有效量的根据本发明的化合物， 混合有具有 快速作用效果的胰岛素或胰岛素类似物， 以及制药学上可接受的载体和添加剂。  In another aspect of the invention, there is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other conditions which cause hyperglycemia, said pharmaceutical composition comprising a therapeutically effective amount of a compound according to the invention, Infused with insulin or insulin analogs having a rapid action effect, and pharmaceutically acceptable carriers and additives.
可以使用制药工业的常规技术制备本发明 IGF-1类似物的可注射组合物， 包括溶解和混 合适当组分而得到所需终产品。 因此， 按照一套操作步骤， 将本发明的 IGF-1类似物溶于一 定量的水， 其体积稍低于待制备的组合物的最终体积。 如果需要， 加入防腐剂、 等渗剂和緩 冲剂。 如果有必要， 使用酸 (例如盐酸)或碱 (例如氢氧化钠)调节溶液的 pH。 最终用水将溶液 的体积调节到所需浓度。  Injectable compositions of the IGF-1 analogs of the invention can be prepared using conventional techniques of the pharmaceutical industry, including dissolving and mixing the appropriate components to provide the desired final product. Thus, the IGF-1 analog of the present invention is dissolved in a quantity of water in a volume slightly lower than the final volume of the composition to be prepared, according to a set of procedures. Add preservatives, isotonic agents and buffers if needed. If necessary, adjust the pH of the solution with an acid such as hydrochloric acid or a base such as sodium hydroxide. The volume of the solution is finally adjusted to the desired concentration with water.
在本发明的另一个实施方案中， 緩冲剂选自乙酸钠、 碳酸钠、 拧檬酸盐、 甘氨酰甘氨酸、 组氨酸、 甘氨酸、 赖氨酸、 精氨酸、 磷酸二氢钠、 磷酸氢二钠、 磷酸钠和三（羟甲基） -氨基 曱烷、 N-二（羟乙基）甘氨酸、 N- (羟曱基） 甲基甘氨酸、 苹果酸、 琥珀酸盐、 马来酸、 富 马酸、 酒石酸、 天冬氨酸或其混合物。 这些具体緩冲剂中的每一种构成了本发明的备选实施 方案。  In another embodiment of the invention, the buffering agent is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, phosphoric acid Disodium hydrogen phosphate, sodium phosphate and tris(hydroxymethyl)-aminodecane, N-bis(hydroxyethyl)glycine, N-(hydroxymethyl)methylglycine, malic acid, succinate, maleic acid, Fumaric acid, tartaric acid, aspartic acid or a mixture thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.
在本发明的另一个实施方案中， 所述制剂包含药学上可接受的防腐剂， 它选自苯酚、 邻- 曱酚、 间-曱酚、 对-甲酚、 对羟基苯曱酸曱酯、 对羟基苯甲酸乙酯、 对羟基苯曱酸丙酯、 对 羟基苯甲酸丁酯、 2-苯氧基乙醇、 苄醇、 氯丁醇、 硫柳汞、 溴硝丙二醇、 苯甲酸、 咪脲、 双 氯苯双胍己烷、 脱氢醋酸钠、 氯甲酚、 苄索氯胺、 氯笨甘瞇或其混合物。 在本发明的另一个 实施方案中， 防腐剂的浓度为 0.1mg mL-20mg/mL。 在本发明的另一个实施方案中， 防腐剂 的浓度为 0.1 mg/mL-5 mg/mL。 在本发明的另一个实施方案中， 防腐剂的浓度为 5mg/mL-10mg/mLo 这些具体防腐剂中的每一种构成了本发明的备选实施方案。 在药物组合 中应用防腐剂是本领域技术人员众所周知的。 参照 Remington: The Science and Practice of Pharmacy, 第 19版， 1995。 In another embodiment of the invention, the formulation comprises a pharmaceutically acceptable preservative selected from the group consisting of phenol, o- Indophenol, m-nonylphenol, p-cresol, decyl hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, 2-phenoxyethanol, Benzyl alcohol, chlorobutanol, thimerosal, bromide, benzoic acid, imidate, chlorhexidine, sodium dehydroacetate, chlorocresol, benzethonamine, clopidogrel or mixtures thereof. In another embodiment of the invention, the concentration of the preservative is from 0.1 mg mL to 20 mg/mL. In another embodiment of the invention, the concentration of the preservative is from 0.1 mg/mL to 5 mg/mL. In another embodiment of the present invention, the concentration of preservative was 5mg / mL-10mg / mL alternative embodiment of the present invention, each of these specific preservatives constitutes the o. The use of preservatives in pharmaceutical combinations is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
在本发明的另一个实施方案中， 所述制剂进一步包括等渗剂， 选自盐（例如氯化钠）、 糖 或糖醇、 氨基酸、 醛糖醇 （例如甘油、 丙二醇、 1,3-丙二醇、 1，3-丁二醇）、 聚乙二醇（例如 PEG400 )或其混合物。 任何糖， 如单糖、 二糖、 多糖或水溶性葡聚糖， 包括例如果糖、 葡萄 糖、 甘露糖、 山梨糖、 木糖、 麦芽糖、 乳糖、 蔗糖、 海藻糖、 葡聚糖、 普鲁蓝、 糊精、 环糊 精、 可溶性淀粉、 羟乙基淀粉和羧曱基纤维素 -Na。 在一个实施方案中， 糖添加剂为蔗糖。 将 糖醇定义为具有至少一个 -OH基团的 C4-C8烃， 包括例如甘露糖醇、 山梨醇、 肌醇、 半乳糖 醇、 卫矛醇、 木糖醇和阿拉伯糖醇。 在一个实施方案中， 该糖醇添加剂为甘露糖醇。 上述糖 类或糖醇类可以单独使用或组合使用。 对用量没有固定限制， 只要所述糖或糖醇溶于液体制 剂而且不会对使用本发明方法获得的稳定化作用产生不良影响即可。 在一个实施方案中， 糖 或糖醇的浓度为 1 mg/mL-150 mg/mL。 在另一个实施方案中， 等渗剂的浓度为 lmg/mL-50mg/mL。 在另一个实施方案中， 等渗剂的浓度为 1 mg/mL-7 mg mL. 在另一个实 施方案中， 等渗剂的浓度为 8 mg/mL-24 mg/mL。 在另一个实施方案中， 等渗剂的浓度为 25 mg/mL-50 mg/mL。 这些具体等渗剂中的每一种构成了本发明的备选实施方案。 在药物组合物 中应用等渗剂是本领域人员众所周知的。 参照 Remington: The Science and Practice of Pharmacy, 第 19版， 1995。  In another embodiment of the invention, the formulation further comprises an isotonicity agent selected from the group consisting of a salt (eg, sodium chloride), a sugar or sugar alcohol, an amino acid, an alditol (eg, glycerol, propylene glycol, 1,3-propanediol) , 1,3-butanediol), polyethylene glycol (eg PEG400) or a mixture thereof. Any sugar, such as a monosaccharide, disaccharide, polysaccharide or water-soluble glucan, including, for example, sugar, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, Dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethyl cellulose-Na. In one embodiment, the sugar additive is sucrose. A sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group, including, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol. The above saccharides or sugar alcohols may be used singly or in combination. There is no fixed limit on the amount of use, as long as the sugar or sugar alcohol is dissolved in the liquid preparation and does not adversely affect the stabilization obtained by the method of the present invention. In one embodiment, the concentration of the sugar or sugar alcohol is from 1 mg/mL to 150 mg/mL. In another embodiment, the concentration of the isotonic agent is from 1 mg/mL to 50 mg/mL. In another embodiment, the concentration of the isotonic agent is from 1 mg/mL to 7 mg mL. In another embodiment, the concentration of the isotonic agent is from 8 mg/mL to 24 mg/mL. In another embodiment, the concentration of the isotonic agent is from 25 mg/mL to 50 mg/mL. Each of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art. Reference Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
典型的等渗剂为氯化钠、 甘露糖醇、 二曱亚砜和甘油， 典型的防腐剂为苯酚、 间-曱酚、 对羟基苯曱酸曱酯和苄醇。  Typical isotonic agents are sodium chloride, mannitol, disulfoxide and glycerin. Typical preservatives are phenol, m-nonylphenol, decyl hydroxybenzoate and benzyl alcohol.
表面活性剂的实例包括乙酸钠、 甘氨酰甘氨酸、 羟乙基哌嗪乙磺酸（ HEPES )和磷酸钠。 实施例  Examples of the surfactant include sodium acetate, glycylglycine, hydroxyethylpiperazineethanesulfonic acid (HEPES), and sodium phosphate. Example
保护基：  Protection base:
Acm acetamidomethyl:乙醜胺甲基； Alloc 或 AOC allyloxycarbonyl:蛑丙氧叛基； Bom, benzyloxymethyl:苄氧甲基； 2-Br-Z, 2-bromobenzyloxycarbonyl:2-溴苄氧羰基； tBu, t-butyl:叔丁 基； Bz， benzoyl:苯曱跣基； Bzl， benzyl:苄基； Boc:叔丁氧羰基； CHO formyl:曱酰基； cHx, cyclohexyl:环己基； Cbz或 Z benzyloxycarbonyl:千氧叛基； Cl-Z, 2-chlorobenzyloxycarbonyl :2- 氯爷氧羰基； Fm, 9-fluorenylmethyl:9-药基曱基； Fmoc, 9-fluorenylmethoxycarbonyl:9-药曱氧叛 基； Mtt, 4-methyltrityl:4-曱基三苯曱基； Npys, 3-nitro-2-pyridinesulfenyl:3-硝基 -2-吡啶亚磺酰 基； Pmc, (2,2,5,7,8-pentametylchroman-6-sulphonyl:2,2,5,7,8-五 甲基 -6-羟基 色 满 ； Tos，4-toluenesulphonyl:对甲笨磺酰； Trt,tripheylmethyl:三苯曱基； Xan， xanthyl:吨基，氧 (杂）蒽基。  Acm acetamidomethyl: acetamidomethyl; Alloc or AOC allyloxycarbonyl: 蛑propoxyl; Bom, benzyloxymethyl: benzyloxymethyl; 2-Br-Z, 2-bromobenzyloxycarbonyl: 2-bromobenzyloxycarbonyl; tBu, t- Butyl: tert-butyl; Bz, benzoyl: benzoyl; Bzl, benzyl: benzyl; Boc: tert-butoxycarbonyl; CHO formyl: decanoyl; cHx, cyclohexyl: cyclohexyl; Cbz or Z benzyloxycarbonyl: Cl-Z, 2-chlorobenzyloxycarbonyl : 2-chlorooxycarbonyl; Fm, 9-fluorenylmethyl: 9-drug fluorenyl; Fmoc, 9-fluorenylmethoxycarbonyl: 9-drug oxime; Mtt, 4-methyltrityl: 4-mercaptotriphenylsulfonyl; Npys, 3-nitro-2-pyridinesulfenyl: 3-nitro-2-pyridinesulfinyl; Pmc, (2,2,5,7,8-pentametylchroman-6-sulphonyl: 2,2,5,7,8-pentamethyl-6-hydroxychroman; Tos, 4-toluenesulphonyl: p-methylsulfonyl; Trt, tripheylmethyl: triphenylsulfonyl; Xan, xanthyl: t-base, oxygen ( Miscellaneous) sulfhydryl.
试剂和溶剂：  Reagents and solvents:
ACN, acetonitrile： 乙腈； BOP, benzotriazol- 1 -yloxytris(dimethylamino) phosphonium hexafluorophosphate:苯并三唑 -1-三（三甲氨基） -六氟磷酸酯（卡特缩合剂）； DCC, Ν,Ν'-Dicyclohexylcarbodiimide:二环己基碳化二亚胺； DCM: 二氯甲垸； DEPBT, 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one:3- (二乙氧基邻酰氧基 )-1,2，3-苯并三。秦 -4-酮； DIC， N，N'-Diisopropylcarbodiimide:N，N'-二异丙基碳二亚胺； DIPEA (或 DIEA) , diisopropylethylamine: 二异丙基乙胺； DMAP, 4-N,N-dimethylaminopyridine: 4-Ν,Ν二曱氨基 吡啶； DMF: Ν,Ν-二曱基曱酰胺； DMSO: 二曱亚砜； DTT, dithiothreitol:二硫苏糖醇； EDC 或 EDCI, 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide: 1-乙基 -(3-二曱基氨基丙基)碳酰二亚 胺盐酸盐； EtOAc:乙酸乙酯； HBTU 0-(lH-benzotriazole-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate 苯 并 三 氮唑 -Ν,Ν,Ν',Ν'-四 甲 基脲六 氟磷 酸盐 ； Η0ΒΤ l-hydroxybenzotriazole:l-羟基-苯并-三氮唑； NMM， N-Methylmorpholine:N-曱基吗淋； NMP, N-methylpyrrolidinone: N-曱基吡咯烷酮； Piperidine： 啶； Su succinimide:琥珀酰亚胺；ACN, acetonitrile: acetonitrile; BOP, benzotriazol- 1 -yloxytris(dimethylamino) phosphonium hexafluorophosphate: benzotriazole-1-tris(trimethylamino)-hexafluorophosphate (Carter condensate); DCC, Ν,Ν'-Dicyclohexylcarbodiimide: dicyclohexylcarbodiimide; DCM: dichloromethane; DEPBT, 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one:3- (2-B Oxykolyloxy)-1,2,3-benzotrien. Qin-4-one; DIC, N, N'-Diisopropylcarbodiimide: N, N'-diisopropylcarbodiimide; DIPEA (or DIEA), diisopropylethylamine: diisopropylethylamine; DMAP, 4-N, N-dimethylaminopyridine: 4-indole, indole aminopyridine; DMF: hydrazine, hydrazine-dihydrazinamide; DMSO: disulfoxide; DTT, dithiothreitol: dithiothreitol; EDC or EDCI, 1 -ethyl -3-(3-dimethylaminopropyl)carbodiimide: 1-ethyl-(3-didecylaminopropyl)carbamoimide hydrochloride; EtOAc: ethyl acetate; HBTU 0-(lH-benzotriazole-l- Yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate benzotriazole-Ν,Ν,Ν',Ν'-tetramethylurea hexafluorophosphate; Η0ΒΤ l-hydroxybenzotriazole: 1-hydroxy-benzene And-triazole; NMM, N-Methylmorpholine: N-mercapto-collium; NMP, N-methylpyrrolidinone: N-decylpyrrolidone; Piperidine: pyridine; Su succinimide: succinimide;
TEA, triethylamine:三乙胺； TFA, trifluoroacetic acid三氟乙酸； TFE 2,2,2-Trifluoroethanol三 氟代乙醇； THF tetrahydrofuran 四氢呋喃； TIS triisopropylsilane三异丙基硅烷。 多肽化学合成方法 TEA, triethylamine: triethylamine; TFA, trifluoroacetic acid trifluoroacetic acid; TFE 2,2,2-Trifluoroethanol trifluoroethanol; THF tetrahydrofuran tetrahydrofuran; TIS triisopropylsilane triisopropylsilane. Polypeptide chemical synthesis method
线性多肽使用 Boc或 Fmoc固相多肽合成法。 如果使用 Fmoc化学合成 C-末端是羧基的 多肽， 一般选用 Wang树脂； C-末端是酰胺的多肽通常选用 Rink amide树脂。 如果使用 Boc 化学合成 C-末端是羧基的多肽， 一般选用 Pam树脂； C-末端是酰胺的多肽通常选用 MBHA 树脂。 缩合剂和活化剂是 DIC和 HOBT,其他可选肽键缩合剂包括 BOP、 HBTU, DEPBT等。 氨基酸 5倍过量。缩合时间为 1小时。 Fmoc保护基用 50%哌。^ /DMF脱除。 Boc保护基用 TFA 脱除。 肽键缩合反应用茚三酮 （Ninhydrin, 2,2-Dihydroxyindane- 1 ,3-dione)试剂监测。  Linear polypeptides use Boc or Fmoc solid phase peptide synthesis. If Fmoc is used to synthesize a polypeptide having a C-terminal carboxyl group, Wang resin is generally used; and a C-terminal amide is usually a Rink amide resin. If Boc is used to synthesize a polypeptide having a C-terminal carboxyl group, Pam resin is generally used; and a polypeptide having a C-terminal amide is usually selected from MBHA resin. The condensing agent and activator are DIC and HOBT, and other optional peptide bond condensing agents include BOP, HBTU, DEPBT and the like. Amino acid 5 times excess. The condensation time was 1 hour. The Fmoc protecting group used 50% piper. ^ /DMF removal. The Boc protecting group is removed with TFA. The peptide bond condensation reaction was monitored with Ninhydrin (2,2-Dihydroxyindane-1,3-dione) reagent.
使用 Fmoc固相多肽合成法时， 通用氨基酸及保护基如下：  When using Fmoc solid phase peptide synthesis, the general amino acids and protecting groups are as follows:
Fmoc-Cys(Trt)-OH、 Fmoc-Asp(OtBu)-OH、 Fmoc-Glu(OtBu)-OH、 Fmoc-His(Trt)-OH、 Fmoc-Lys(Boc)-OH 、 Fmoc-Asn(Trt)-OH 、 Fmoc-Gln(Trt)-OH 、 Fmoc-Arg(Pmc)-OH 、 Fmoc-Ser(tBu)-OH、 Fmoc-Thr(tBu)-OH、 Boc-Trp(Boc)-OH、 Fmoc-Tyr(tBu)-OH  Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt )-OH , Fmoc-Gln(Trt)-OH , Fmoc-Arg(Pmc)-OH , Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Boc-Trp(Boc)-OH, Fmoc -Tyr(tBu)-OH
如果赖氨酸的侧链氨基用于酰化反应， 赖氨酸的侧链氨基可以使用烯丙氧羰基（aloe)保 护。 肽链合成完毕， 脱除烯丙氧羰基可以用四 (三苯基膦)钯 (0)和 37:2:1 比例的 DCM、 冰醋 酸和 NMM ( 15 mL/g树脂）在氩气环境、 室温条件下搅拌 2小时。 反应后树脂需要用 0.5% DIPEA/DMF (lOmL), 0.5%三水合二乙基二硫代碳酸钠 /DMF (3 X 10 mL), 1:1 DCM:DMF(5 XlO mL)清洗。赖氨酸的侧链氨基也可以使用 4-甲基三苯曱基（Mtt )保护。树脂悬浮于 DCM, 加入 TFA/TIS/DCM(1 :2:97)， 震荡 10分钟。 重复 2遍后， 树脂用 DCM、 DMF和异丙醇洗涤。  If the side chain amino group of lysine is used for the acylation reaction, the side chain amino group of lysine can be protected with an allyloxycarbonyl group (aloe). After the peptide chain is synthesized, the allyloxycarbonyl group can be removed with tetrakis(triphenylphosphine)palladium(0) and 37:2:1 ratio of DCM, glacial acetic acid and NMM (15 mL/g resin) in an argon atmosphere. Stir at room temperature for 2 hours. After the reaction, the resin was washed with 0.5% DIPEA/DMF (10 mL), 0.5% sodium diethyldithiocarbonate/DMF (3 X 10 mL), 1:1 DCM:DMF (5×10 mL). The side chain amino group of lysine can also be protected with 4-methyltriphenylmethyl (Mtt). The resin was suspended in DCM, added to TFA/TIS/DCM (1:2:97), and shaken for 10 minutes. After repeating 2 times, the resin was washed with DCM, DMF and isopropyl alcohol.
固相 Fmoc化学合成多肽后， 常用的切割试剂是 TFA。 将干树脂放在一个摇瓶中， 加入 适当量 TFA/TIS/H20 ( 95:2.5:2.5, 10-25 m!Jg树脂）， 盖上盖子， 在室温下进行间歇式旋转 震荡。 2小时后抽滤树脂， 以新的 TFA清洗树脂 2-3次， 合并滤液， 滴加 8-10倍体积的冰乙 瞇。 最后， 离心收集沉淀出来的多肽粗品。 After solid phase Fmoc chemical synthesis of polypeptides, the commonly used cleavage reagent is TFA. The dry resin was placed in a shake flask, and an appropriate amount of TFA/TIS/H 2 0 (95:2.5:2.5, 10-25 m! Jg resin) was added, and the lid was capped, and intermittent rotation was performed at room temperature. After 2 hours, the resin was filtered, and the resin was washed 2-3 times with a new TFA, and the filtrate was combined, and 8-10 volumes of ice acetaminophen were added dropwise. Finally, the precipitated polypeptide was collected by centrifugation.
使用 Boc固相多肽合成法时， 通用氨基酸和保护基如下：  When using Boc solid phase peptide synthesis, the general amino acids and protecting groups are as follows:
Boc-Cys(4-MeBzl)-OH、 Boc-Asp(OcHx)-OH、 Boc-Glu(OcHx)-OH、 Boc-His(Bom)-OH、 Boc-Lys(2-Cl-Z)-OH, Boc-Asn(Xan)-OH 、 Boc-Gln(Xan)-OH 、 Boc-Arg(Tos)-OH 、 Boc-Ser(Bzl)-OH、 Boc-Thr(Bzl)-OH、 Boc-Trp(CHO)-OH和 Boc-Tyr(2-Br-Z)-OH。  Boc-Cys(4-MeBzl)-OH, Boc-Asp(OcHx)-OH, Boc-Glu(OcHx)-OH, Boc-His(Bom)-OH, Boc-Lys(2-Cl-Z)-OH , Boc-Asn(Xan)-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Trp( CHO)-OH and Boc-Tyr(2-Br-Z)-OH.
如果赖氨酸的侧链氨基用于酰化反应， 赖氨酸的侧链氨基可以使用烯丙氧痰基（aloe)保 护或 Fmoc保护。 如果天冬氨酸或谷氨酸的侧链羧基用于酰化反应， 羧基应该转化为烯丙酯 (allyl ester)或 9-芴基曱基保护。 If the side chain amino group of lysine is used for the acylation reaction, the side chain amino group of lysine may be protected with an aloin or Fmoc. If the side chain carboxyl group of aspartic acid or glutamic acid is used for the acylation reaction, the carboxyl group should be converted to allyl ester. (allyl ester) or 9-mercaptopurine protection.
固相 Boc化学合成多肽后， 对于 PAM、 MBHA树脂， 一般采用 HF切割， 每 0.1毫摩尔 树脂加 5毫升 HF, 同时加入对甲苯盼、 对巯基苯酚或笨曱醚等试剂， 混合物在冰浴条件下搅 拌 1小时。 HF真空抽干后， 多肽用;水乙瞇沉淀， 离心收集沉淀， 经过 HPLC分离纯化， 冷冻 千燥得到最后产品。  After solid phase Boc chemical synthesis of peptides, for PAM, MBHA resin, HF cutting is generally used, and 5 ml of HF is added per 0.1 mmol of resin, and reagents such as p-toluene, p-nonylphenol or clumsy ether are added, and the mixture is in an ice bath condition. Stir under 1 hour. After the HF vacuum was dried, the peptide was precipitated with water; the precipitate was collected by centrifugation, separated and purified by HPLC, and frozen to obtain the final product.
双链化合物的合成  Synthesis of double-stranded compounds
方法 1 :  method 1 :
巯基磺化反应 ( Sulfitolysis )  Sulfone sulfonation reaction
反应溶剂成分如下：  The reaction solvent composition is as follows:
6M胍 盐酸盐 （MW: 95.53 ) 86克 /150毫升  6M 盐 hydrochloride (MW: 95.53 ) 86 g / 150 ml
O.lM Tris ( MW: 121.1 ) 1.8克 /150毫升  O.lM Tris (MW: 121.1) 1.8 g / 150 ml
0.28 M Na2S03 (MW: 126) 5.2克 /150毫升 0.28 M Na 2 S0 3 (MW: 126) 5.2 g / 150 ml
0.082 M Na2S406 (MW: 306.2) 3.75克 /150毫升 0.082 M Na 2 S 4 0 6 (MW: 306.2) 3.75 g / 150 ml
0.5mM IGF-1A链或 B链溶解在上述反应溶剂中， pH值调节到 8.5-8.7。 混合物在室温剧 烈搅拌 1-1.5小时， 然后使用 G10或 G25柱脱盐。 緩冲液 A为 0.05M碳酸氢铵水溶液， 緩冲 液 B为 0.05M碳酸氢铵 /50%乙腈水溶液。 脱盐后的纯品低压冷冻干燥（ lyophilization )。  0.5 mM IGF-1A chain or B chain was dissolved in the above reaction solvent, and the pH was adjusted to 8.5-8.7. The mixture was vigorously stirred at room temperature for 1-1.5 hours and then desalted using a G10 or G25 column. Buffer A was 0.05 M ammonium bicarbonate aqueous solution, and buffer B was 0.05 M ammonium hydrogencarbonate / 50% acetonitrile aqueous solution. Pure product after desalting, low pressure freeze-drying (lyophilization).
A链与 B链的连接  A chain and B chain connection
文献方法 (Chance等, "The production of human insulin using recombinant DNA technology and a new chain combination procedure" ,Pept.: Synth., Struct., Funct., Proc. Am. Pept. Symp., 7th, 1981, Vol. 721, Issue 8， Page 721)。 经过琉基磺化反应得到的 A链 S磺酸酯和 B链 S磺酸酯按 2: 1重量比混合， 溶解在 0.1 M甘氨酸水溶液中 (pH 10.5), 多肽浓度为 5-10毫克 /毫升。 加入 DTT, 使 SH:S磺酸酯比例是 1.2。反应在 4°C下搅拌 12小时。反应所得混合物使用 RP-HPLC 进行纯化。  Literature method (Chance et al, "The production of human insulin using recombinant DNA technology and a new chain combination procedure", Pept.: Synth., Struct., Funct., Proc. Am. Pept. Symp., 7th, 1981, Vol 721, Issue 8, Page 721). The A chain S sulfonate and the B chain S sulfonate obtained by sulfhydryl sulfonation reaction are mixed in a weight ratio of 2:1, dissolved in 0.1 M glycine aqueous solution (pH 10.5), and the polypeptide concentration is 5-10 mg/ml. . DTT was added so that the SH:S sulfonate ratio was 1.2. The reaction was stirred at 4 ° C for 12 hours. The resulting mixture was purified using RP-HPLC.
1-15的合成：  Synthesis of 1-15:
A链和 B链用 Fmoc化学合成。 A链计算分子量 2436.9, 质谱测试分子量 2437.6; B链 计算分子量 3175.7, 质谱测试分子量 3176.9。 最终产物的计算分子量 5606.5, 质谱测试分子 量 56073。  The A chain and the B chain were synthesized by Fmoc chemistry. The molecular weight of the A chain was calculated to be 2436.9, the molecular weight of the mass spectrum was 2437.6, the molecular weight of the B chain was 3175.7, and the molecular weight of the mass spectrometer was 3176.9. The final product had a calculated molecular weight of 5606.5 and a mass spectrometry test molecular weight of 56,073.
方法 2:  Method 2:
A链与 B链的连接  A chain and B chain connection
文献方法 ( Han等， "Insulin chemical synthesis using a two-step orthogonal formation of the three
Figure imgf000041_0001
American Peptide Society Symposium, 2009 )。 A链和 B链用 Fmoc或 Boc 化学合成方法合成， A7、 B6 (对应于通式中 X29或 29 )半胱氨酸用通用保护基， 但 A6、 All、 A20和 B18 (对应于通式中 i或 41 )半胱氨酸的侧链琉基用 Acm保护。 合成好的 A 链和 B链从树脂切割下来后变成 A- ( SH ) 7 ( S-Acm ) 6' 20和 B- ( SH ) 6 ( S-Acm ) 18。 B 链溶于 DMF或 DMSO, 加入等摩尔 2，2'-二硫双 (5-硝基吡啶)。 反应用 HPLC检测和纯化， 得 到 B- ( S-Npys ) 6 ( S-Acm ) 18。 等摩尔 A- ( SH ) 7 ( S-Acm ) 6' 20和 B- ( S-Npys ) 6 ( S-Acm ) 18 溶于 DMSO， 多肽浓度 15mg/mL。 当 A7-B6二硫键形成后， 加入 80%醋酸水溶液， 多肽 浓度稀释到 l mg/mL。 再加入 40倍的 12。 反应在室温搅拌 1小时后， 加入抗坏血酸水溶液终 止反应。 混合物用 HPLC纯化， 终产物用质语确认。 Literature method ( Han et al, "Insulin chemical synthesis using a two-step orthogonal formation of the three
Figure imgf000041_0001
American Peptide Society Symposium, 2009). The A chain and the B chain are synthesized by Fmoc or Boc chemical synthesis, A7, B6 (corresponding to X 29 or 29 in the formula). The cysteine has a universal protecting group, but A6, All, A20 and B18 (corresponding to the formula The side chain thiol group of i or 41 ) cysteine is protected with Acm. The synthesized A chain and B chain are cut from the resin to become A-(SH) 7 (S-Acm) 6 '20 and B-(SH) 6 (S-Acm) 18 . The B chain is dissolved in DMF or DMSO and an equimolar amount of 2,2'-dithiobis(5-nitropyridine) is added. The reaction was detected by HPLC and purified to give B-(S-Npys) 6 (S-Acm) 18 . Equimolar A- (SH) 7 (S- Acm) 6 '20 and B- (S-Npys) 6 ( S-Acm) 18 was dissolved in DMSO, polypeptide concentration 15mg / mL. After the A7-B6 disulfide bond was formed, 80% aqueous acetic acid solution was added, and the polypeptide concentration was diluted to 1 mg/mL. Add 40 times more 1 2 . After the reaction was stirred at room temperature for 1 hour, the reaction was quenched by the addition of aqueous ascorbic acid. The mixture was purified by HPLC and the final product was confirmed in succinct.
1-24的合成 A链和 B链用 Fmoc化学合成。 A- ( SH ) 7 ( S-Acm ) 6' 20计算分子量 2650.1 , 质谱测 试分子量 2651.3; Β- ( SH ) 7 ( S-Acm ) 19计算分子量 3246.7, 质谱测试分子量 3247.5。 最终 产物的计算分子量 5847.8, 质旙测试分子量 5849.0。 Synthesis of 1-24 The A chain and the B chain were synthesized by Fmoc chemistry. A-(SH) 7 (S-Acm) 6 '20 Calculated molecular weight 2650.1, mass spectrometry test molecular weight 2651.3; Β-(SH) 7 (S-Acm) 19 Calculated molecular weight 3246.7, mass spectrometry test molecular weight 3247.5. The final product had a calculated molecular weight of 5847.8 and a molecular weight test of 5849.0.
用同样的方法合成其它双链化合物。  Other double-stranded compounds were synthesized in the same manner.
进一步确认化合物的结构， 尤其是三对二硫键的连接方式， 使用 Chance文献中的 HPLC Further confirm the structure of the compound, especially the connection of the three pairs of disulfide bonds, using HPLC in the Chance literature.
"指乡丈"分析法 (Chance等 , "The production of human insulin using recombinant DNA technology and a new chain combination procedure" ,Pept.: Synth., Struct., Funct., Proc. Am. Pept. Symp., 7th, 1981, Vol. 721, Issue 8, Page 721。筒而言之， 2 mg多肽样品溶于 0.2 ml 0.01N盐酸，加入 0.8 ml 含有 100 μg S. aureus V8蛋白醉的 0.05M NH4HC03, 使 H达到 7.9。 在 37°C培养 24小时。 小部分样品加入 DTT培养 30分钟。 使用 LC-MS对比样品和标准品的各个片段的保留时间 ( retention time )和分子量就可以二硫键和化合物的结构。 这个分析方法普遍适用于本发明中 各种方法合成的单链或双链多肽。 "Changxiangzhang", "The production of human insulin using recombinant DNA technology and a new chain combination procedure", Pept.: Synth., Struct., Funct., Proc. Am. Pept. Symp., 7th, 1981, Vol. 721, Issue 8, Page 721. In the case of a cartridge, 2 mg of the peptide sample is dissolved in 0.2 ml of 0.01 N hydrochloric acid, and 0.8 ml of 0.05 M NH 4 HC0 3 containing 100 μg of S. aureus V8 protein intoxication is added. , H is 7.9. Incubate for 24 hours at 37 ° C. A small portion of the sample is added to DTT for 30 minutes. The LC-MS can be used to compare the retention time and molecular weight of each sample of the sample and the standard to disulfide bond and Structure of the compound This analytical method is generally applicable to single-stranded or double-stranded polypeptides synthesized by various methods in the present invention.
双链化合物的合成结果:  Synthesis results of double-stranded compounds:
利用本发明上述的合成方法分别合成双链化合物， 利用测序和质谱测试的方法对这些化 合物进行检验分析， 测序结果显示化合物的氨基酸序列正确， 质谱结果显示出化合物的结构 与原设计结构一致， 即合成的化合物即是希望的化合物。 数据结果如下：  The double-stranded compounds were separately synthesized by the above-mentioned synthetic method of the present invention, and the compounds were tested and analyzed by sequencing and mass spectrometry. The sequencing results showed that the amino acid sequence of the compound was correct, and the mass spectrometry results showed that the structure of the compound was consistent with the original design structure, that is, The synthesized compound is the desired compound. The data results are as follows:
1-1 : 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Qi] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[51之间形成链内二硫键； 分子量计算 值 5055.9, 质谱测试分子量 5057.3; 1-1: Mass transfer detection revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between Qi] and C [4 ] and C[ 2] and C [6 interchain disulfide bonds form between], C [3] and C [intrachain disulfide bond formation between 51; molecular weight calcd 5055.9, 5057.3 molecular mass spectrometry;
1-2: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Ctl] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， 。[3]和 C[5j之间形成链内二硫键； 分子量计算 值 5094.9, 质谱测试分子量 5095.7; 1-2: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tl] and C [4] and C [2] and C [ 6] forms an interchain disulfide bond, . [ 3] and C [5 j form an intrachain disulfide bond; molecular weight calculated value 5094.9, mass spectrometry test molecular weight 5095.7;
1-3: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二疏键连接： Cm 和〇[4]之间以及。[2]和。[6]之间形成链间二硫键， 3]和。[5]之间形成链内二硫键； 分子量计算 值 5133.9， 质谱测试分子量 5135.1 ; 1-3: The mass transfer assay showed that the A chain and the B chain were linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular two sparse bonds: between C m and 〇 [ 4 ] . [ 2] and. [ 6] forms an interchain disulfide bond, 3] and. [ 5] formed an intrachain disulfide bond; molecular weight calculated value 5133.9, mass spectrometry test molecular weight 5135.1;
1-4: 质 ΐ瞽检测显示， Α链和 Β链通过两对分子间二硫键和一对分子内二砥键连接： Ctl] 和 C[4]之间以及。[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5687.6， 质谙测试分子量 5689.3; 1-4: The enthalpy detection showed that the Α chain and the Β chain were linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular bismuth bonds: between C tl] and C[ 4 ]. An interchain disulfide bond is formed between [ 2] and C [6] , and an intrachain disulfide bond is formed between C[ 3 ] and C [5] ; the molecular weight calculated is 5687.6, and the molecular weight is 5689.3;
1-5:质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： C[n 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， [3]和 C[5]之间形成链内二硫键； 分子量计算 值 5627.4, 质谱测试分子量 5628.5; 1-5: Quality transfer assay showed that the A chain and the B chain were linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C [n and C [4] and C [2 ] and C [ 6] formed an interchain disulfide bond, and formed an intrachain disulfide bond between [ 3] and C [5] ; molecular weight calculated value 5627.4, mass spectrometry test molecular weight 5628.5;
1-6: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硗键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5614.4， 质谱测试分子量 5615.9; 1-6: Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular diterpene bonds: between C m and C [4] and C [2 ] and C [6] An interchain disulfide bond is formed between the two, and an intrachain disulfide bond is formed between C[ 3 ] and C [5] ; the molecular weight is calculated to be 5614.4, and the molecular weight is 5615.9;
1-7: 质旙检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Qi] 和。[4]之间以及。[2]和 C[6]之间形成链间二硫键， 0[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5636.5, 质谱测试分子量 5636.7; 1-7: The enthalpy detection shows that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: Qi] and . [ 4] between and. An interchain disulfide bond is formed between [ 2] and C[ 6] , and an intrachain disulfide bond is formed between 0[ 3] and C[ 5] ; molecular weight calculated value is 5636.5, mass spectrometry molecular weight is 5636.7;
1-8: 质语检测显示， A链和 B链通过两对分子间二碗键和一对分子内二硫键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二^ «键， [3]和 C[5]之间形成链内二硫键； 分子量计算 值 5660.4, 质谱测试分子量 5661.2; 1-9: 质 i普检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5626.5， 质镨测试分子量 5627.6; 1-8: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular two bowl bonds and a pair of intramolecular disulfide bonds: between C m and C [4] and C [2] and C [6 is formed between a] between the two chains ^ «bonds, disulfide bonds formed between the inner [3] and C [5]; mass calculated 5660.4, 5661.2 molecular mass spectrometry; 1-9: Qualitative detection shows that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C m and C [4] and C [2] and C [ 6] form an interchain disulfide bond, and form an intrachain disulfide bond between C [3] and C [5] ; molecular weight calculated value 5626.5, mass test molecular weight 5627.6;
1-10: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： CtI] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5612.4, 质谙测试分子量 5614.1 ; 1-10: Mass spectrometry revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tI] and C [4] and C [2] and C [6 An interchain disulfide bond is formed between them, and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5612.4, and the molecular weight is 5614.1;
1-11 : 盾 i普检测显示， A链和 B链通过两对分子间二疏键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5599.4, 质谱测试分子量 5601.7; 1-11: Shield detection shows that the A chain and the B chain are linked by two pairs of intermolecular dichotomous bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and C [2] and C An interchain disulfide bond is formed between [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; molecular weight calculated value is 5599.4, mass spectrometry molecular weight is 5601.7;
1-12: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5626.5, 质谘测试分子量 5627.3; 1-12: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C m and C [4 ] and C [2] and C [6 interchain disulfide bonds form between], disulfide bonds are formed between the C [3] and C [5]; calc. 5626.5 molecular weight, molecular mass 5627.3 test consultation;
1-13: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和。[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5640.5， 质诸测试分子量 5641.2; 1-13: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C n] and . An interchain disulfide bond is formed between [4] and between C [2] and C [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; molecular weight calculated value 5640.5, quality test Molecular weight 5641.2;
1-14: 质诸检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Ctl] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5598.5, 质诰测试分子量 5599.1 ; 1-14: Qualitative tests show that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tl] and C [4] and C [2] and C [ 6] an interchain disulfide bond is formed, and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5598.5, and the molecular weight is 5599.1;
1-16: 质潘检测显示， A链和 B链通过两对分子间二硫键和一对分子内二疏键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5】之间形成链内二硫键； 分子量计算 值 5734.7, 质谱测试分子量 5735.4; 1-16: The mass detection showed that the A chain and the B chain were linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular two sparse bonds: between C m and C [4] and C [2] and C [6 interchain disulfide bonds form between], disulfide bonds are formed between the C [3] and C [5]; mass calculated 5734.7, 5735.4 molecular mass spectrometry;
1-17: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cfl] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5890.8， 质谱测试分子量 5992.2; 1-17: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C fl] and C [4 ] and C [2] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3] and C [5] ; molecular weight calculated value 5890.8, mass spectrometry test molecular weight 5992.2;
1-18: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[2]和 Cw之间形成链间二硫键， 。[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5088.9, 质谙测试分子量 5090.5; 1-18: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and between C[ 2 ] and C w An interchain disulfide bond is formed between. An intrachain disulfide bond is formed between [ 3] and C[ 5] ; the molecular weight is calculated to be 5088.9, and the molecular weight is 5090.5;
1-19: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二疏键连接： Cu] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5661.6, 质语测试分子量 5673.2; 1-19: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular dichotomous bonds: between C u] and C [4] and C [2] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3] and C [5] ; molecular weight calculated value 5661.6, molecular weight test molecular weight 5673.2;
1-20: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5652.6， 质谱测试分子量 5652.9; 1-20: Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and C [2] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3] and C [5] ; molecular weight calculated 5652.6, mass spectrometry molecular weight 5652.9;
1-21: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[21和 C[6]之间形成链间二硫键， C[3]^。[51之间形成链内二硫键； 分子量计算 值 5642.5, 质语测试分子量 5643.3; 1-21: Mass spectrometry revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C [4] and C [21 and C [6] An interchain disulfide bond is formed between C [3] ^. [Intrachain disulfide bond formed between 51 ; molecular weight calculated value 5642.5, molecular weight test 5643.3;
1-22: 质谱检测显示， A链和 B链通过两对分子间二碗键和一对分子内二硫键连接： C[u 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5876.8, 质谱测试分子量 5877.6; 1-22: Mass spectrometry showed that the A chain and the B chain were linked by two pairs of intermolecular two bowl bonds and a pair of intramolecular disulfide bonds: C[ u and C [4 ] and C [2] and C [6 is formed between a] interchain disulfide bonds, the disulfide bond is formed between C [3] and C [5]; mass calculated 5876.8, 5877.6 molecular mass spectrometry;
1-23: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Ctl] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5837.7, 质谱测试分子量 5839.0; 1-23: Quality transfer assays show that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C tl] An interchain disulfide bond is formed between C [4 ] and C [2] and C [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; molecular weight calculated 5837.7, mass spectrum Test molecular weight 5839.0;
1-25: 质借检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Ctl] 和 C[4〗之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5557.4, 质谙测试分子量 5558.7; 1-25: Qualitative detection shows that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C tl] and C [4 〗 and C[ 2 ] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3 ] and C [5] ; molecular weight calculated value 5557.4, mass test molecular weight 5558.7;
1-26: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二疏键连接： Cfl] 和 C[4]之间以及〇[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5633.5， 质讲测试分子量 5634.9; 1-26: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular dichotomous bonds: between C fl] and C [4] and 〇 [2] and C [ 6] form an interchain disulfide bond, and form an intrachain disulfide bond between C [3] and C [5] ; molecular weight calculated value 5633.5, qualitative test molecular weight 5634.9;
1-27: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cfl] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3】和 C[5]之间形成链内二硫键； 分子量计算 值 5770.7, 质谱测试分子量 5772.1 ; 1-27: The mass transfer assay showed that the A chain and the B chain were linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C fl] and C [4] and C [2] and C [ 6] formed an interchain disulfide bond, and an intrachain disulfide bond was formed between C [3 ] and C[ 5] ; molecular weight calculated value 5770.7, mass spectrometry test molecular weight 5772.1;
1-28: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[2]和。[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5798.7, 质谙测试分子量 5799.6; 1-28: Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: C n] and C [4] and C [2] and . An interchain disulfide bond is formed between [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5798.7, and the molecular weight is 5799.6;
1-29: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cm 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5803.7， 质谱测试分子量 5805.3; 1-29: Mass spectrometry revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C m and C [4] and C [2 ] and C [6] An interchain disulfide bond is formed between them, and an intrachain disulfide bond is formed between C [3] and C [5] ; a molecular weight calculated value of 5803.7, a mass spectrometric test molecular weight of 5805.3;
1-30: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： CtI] 和 C[4]之间以及 C[2]和 [6]之间形成链间二 键， CCT和 C[5】之间形成链内二硫键； 分子量计算 值 5831.7, 质谱测试分子量 5832.8; 1-30: Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C tI] and C [4] and C[ 2] and [ 6 The interchain two bonds are formed between each other, and an intrachain disulfide bond is formed between C CT and C [5 ]; the molecular weight is 5831.7, and the mass spectrum is 5832.8;
1-31: 质谱检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及。[2]和 C[6]之间形成链间二硫键， C[3]和 C[51之间形成链内二硫键； 分子量计算 值 5987.9, 质谱测试分子量 5990.4; 1-31: Mass spectrometry revealed that the A chain and the B chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C[ 4] . An interchain disulfide bond is formed between [2] and C [6] , and an intrachain disulfide bond is formed between C [3] and C [51 ; molecular weight calculated value 5987.9, mass spectrometry test molecular weight 5990.4;
1-32: 质语检测显示， A链和 B链通过两对分子间二硫键和一对分子内二碗键连接： C ] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， 。[3]和。[5]之间形成链内二 υ键； 分子量计算 值 5931.9, 质谙测试分子量 5932.5; 1-32: Qualitative detection revealed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular two bowls: C ] and C [4 ] and C [2] and C [6 interchain disulfide bonds form between]. [ 3] and. [5] The formation of intrachain diterpene bonds; molecular weight calculated value 5931.9, quality test molecular weight 5932.5;
1-33: 质语检测显示， Α链和 Β链通过两对分子间二硫键和一对分子内二硫键连接： Cn] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5804.6, 质谙测试分子量 5805.9; 1-33: Qualitative detection revealed that the Α chain and the Β chain are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C n] and C[ 4] and C [2] and C [ 6] an interchain disulfide bond is formed, and an intrachain disulfide bond is formed between C [3] and C [5] ; a molecular weight calculated value of 5804.6, a molecular weight test molecular weight of 5805.9;
1-34: 质傳检测显示， A链和 B链通过两对分子间二硫键和一对分子内二硫键连接： Cti] 和 C[4]之间以及 C[2]和 C[6]之间形成链间二硫键， C[3]和 C[5]之间形成链内二硫键； 分子量计算 值 5675.5, 质谱测试分子量 5676.8。 单链化合物的合成 1-34: Mass transfer assay showed that the A and B chains are linked by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds: between C t i] and C [4] and C [2] and C An interchain disulfide bond is formed between [6] , and an intrachain disulfide bond is formed between C [3] and C [5] ; the molecular weight is calculated to be 5675.5, and the molecular weight is 5676.8. Synthesis of single-chain compounds
自然化学连接 ( Native Chemical Ligation )  Natural Chemical Ligation
文献方法 (Kent, S.B.H.等， "Comparative Properties of Insulin-like Growth Factor 1 (IGF-1) and [Gly7D-Ala]IGF-l Prepared by Total Chemical Synthesis." Angew. Chem. Int. Ed. 2008, 47, 1102 -1106)。 根据氨基酸序列进行部分改进。  Literature Method (Kent, SBH, et al., "Comparative Properties of Insulin-like Growth Factor 1 (IGF-1) and [Gly7D-Ala] IGF-l Prepared by Total Chemical Synthesis." Angew. Chem. Int. Ed. 2008, 47 , 1102 -1106). Partial improvements were made based on the amino acid sequence.
IGF-1类似物分成两个片段合成。 一段是用 Boc化学合成 IGF-1 B链 [l-17]-COS- ( CH2 ) 2 CO-(Arg)4A。 硫酯残基合成时用 S-三苯曱基 -3-琉基丙酸。 第二段包括从 B18Cys (对应于通 式中 X41或X44l )起到 B链 C末端全部氨基酸， C肽和 A链氨基酸。 两段多肽用通用方法固 相合成， 切割， 纯化。 The IGF-1 analog is synthesized in two fragments. One is the synthesis of IGF-1 B chain [l-17]-COS-(CH 2 ) 2 CO-(Arg) 4 A by Boc chemistry. S-triphenylmethyl-3-mercaptopropionic acid is used in the synthesis of the thioester residue. The second paragraph includes from B18Cys (corresponding to the pass) Wherein X 41 or X44l ) functions as the entire amino acid of the C-terminus of the B chain, the C peptide and the A chain amino acid. The two peptides are solid phase synthesized, cleaved and purified by a general method.
自然化学连接反应在緩沖液中进行。緩冲液含 6 M胍盐酸盐， 200 mM磷酸盐， 200 mM 4- 羧曱基^^酚（MPAA )， 20 mM三 (2-曱酰乙基)膦（TCEP ), pH 6.9, 多肽按 1 : 1 摩尔比溶 解， 浓度 2 mM。 反应用 HPLC检测、 纯化。  The natural chemical ligation reaction is carried out in a buffer. Buffer containing 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxymethyl phenol (MPAA), 20 mM tris(2-decanoylethyl)phosphine (TCEP), pH 6.9, peptide Dissolved in a 1: 1 molar ratio at a concentration of 2 mM. The reaction was detected by HPLC and purified.
纯化的 IGF-1 (SH)6 溶解于 0.5 M胍盐酸盐， 20 mM Tris, 8 mM 半胱氨酸， 1 mM胱氨酸 盐酸盐緩冲液， pH 7.8， 多肽浓度 0.5 mg/mL。 当 HPLC显示折叠完成后， 緩冲液用 0.1N盐 酸酸化至 pH3。 多肽用制备 RP-HPLC纯化。 Purified IGF-1 (SH) 6 was dissolved in 0.5 M guanidine hydrochloride, 20 mM Tris, 8 mM cysteine, 1 mM cystine hydrochloride buffer, pH 7.8, peptide concentration 0.5 mg/mL . After HPLC showed that the folding was completed, the buffer was acidified to pH 3 with 0.1 N hydrochloric acid. The polypeptide was purified by preparative RP-HPLC.
II -3的制备。  Preparation of II-3.
RPUPLC纯化。 分子量计算值 2573.1， 质谱测试分子量 2573.8。 RPUPLC purification. Molecular weight calculated value 2573.1, mass spectrometry test molecular weight 2573.8.
第二片段 CGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA按通用方法 合成， 粗肽用 RP-HPLC纯化。 分子量计算值 4825.4， 质谱测试分子量 4827.0。  The second fragment, CGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA, was synthesized by a general method, and the crude peptide was purified by RP-HPLC. Molecular weight calculated value 4825.4, mass spectrometry test molecular weight 4827.0.
第一片段 25.7 mg ( 10 μηιοΐ )和第二片段 48.3 mg ( 10 μιηοΐ )溶解于緩沖液（5 mL )。 緩 冲液包含 6 M胍盐酸盐， 200 mM磷酸盐， 200 mM 4-羧曱基苯硫酚， 20 mM三 (2-曱酰乙基) 膦， pH 6.9。 10小时后反应完成。 分子量计算值 6596.5, 质谱测试分子量 6597.1。 将混合物 转移到尺寸排阻层析柱， 洗脱緩沖液是 0.5 M胍盐酸盐， 20 mM Tris, pH 7.8。 收取包含正确 多肽分子量的部分， 合并后加入 8 mM 半胱氨酸， 1 mM胱氨酸盐酸盐緩沖液， 2小时后多肽 折叠完毕。将緩冲液用 0.1N盐酸酸化至 pH 3 , 然后用 RP-HPLC纯化。分子量计算值 6590.5, 质语测试分子量 6591.6， 测序结果与 SEQ ID NO:31一致。  The first fragment 25.7 mg (10 μηιοΐ) and the second fragment 48.3 mg (10 μιηοΐ) were dissolved in buffer (5 mL). The buffer contained 6 M guanidine hydrochloride, 200 mM phosphate, 200 mM 4-carboxymethylthiophenol, 20 mM tris(2-nonanoylethyl)phosphine, pH 6.9. The reaction was completed after 10 hours. Molecular weight calculated value 6596.5, mass spectrometry test molecular weight 6597.1. The mixture was transferred to a size exclusion chromatography column with 0.5 M guanidine hydrochloride, 20 mM Tris, pH 7.8. The fraction containing the correct molecular weight of the polypeptide was collected, and 8 mM cysteine, 1 mM cystine hydrochloride buffer was added, and the polypeptide was folded 2 hours later. The buffer was acidified to pH 3 with 0.1 N hydrochloric acid and then purified by RP-HPLC. The molecular weight calculated was 6590.5, the molecular weight of the test was 6591.6, and the sequencing result was consistent with SEQ ID NO:31.
以上述方法制备基于 IGF-1的单链化合物， 利用质语检测分子的分子量， 通过测序检测制 备的单链多肽的结构以验证所合成的化合物， 其结果为：  The single-chain compound based on IGF-1 was prepared by the above method, and the molecular weight of the molecule was detected by a linguistic expression, and the structure of the prepared single-chain polypeptide was detected by sequencing to verify the synthesized compound, and the result was as follows:
1 -1 分子量计算值 6798.8, 质谱测试分子量 6799.3， 测序结果与 SEQ ID NO:29一致 1-1 molecular weight calculated value 6798.8, mass spectrometry molecular weight 6799.3, sequencing results consistent with SEQ ID NO:29
1 -2 分子量计算值 6760.7， 质谱测试分子量 6763.4， 测序结果与 SEQ ID NO:30—致1 -2 Molecular Weight Calculated 6760.7, Mass Spectrometry Test Molecular Weight 6763.4, Sequencing Results and SEQ ID NO: 30
1-4 分子量计算值 6519.4, 质谱测试分子量 6521.5， 测序结果与 SEQ ID NO:32一致1-4 Calculated molecular weight 6519.4, mass spectrometry molecular weight 6521.5, sequencing result is consistent with SEQ ID NO:32
1 -5 分子量计算值 6448.3 , 质谱测试分子量 6450.1， 测序结果与 SEQ ID NO:33—致1 -5 Molecular weight calculated value 6448.3 , Mass spectrometry test molecular weight 6450.1, Sequencing result and SEQ ID NO: 33
1 -6 分子量计算值 6434.3 , 质傳测试分子量 6436.0, 测序结果与 SEQ ID NO:34一致1 -6 molecular weight calculated value 6434.3 , mass transfer test molecular weight 6436.0, sequencing results consistent with SEQ ID NO: 34
1 -7 分子量计算值 6420.3， 质谱测试分子量 6421.9, 测序结果与 SEQ ID NO-.35一致1 -7 Calculated molecular weight 6420.3, mass spectrometry molecular weight 6421.9, sequencing results consistent with SEQ ID NO-.35
1 -8 分子量计算值 6404.3， 质谱测试分子量 6406.2, 测序结果与 SEQ ID NO:36一致1 -8 molecular weight calculated value 6404.3, mass spectrometry test molecular weight 6406.2, sequencing results consistent with SEQ ID NO: 36
1 -9 分子量计算值 6404.3 , 质谱测试分子量 6405.6, 测序结果与 SEQ ID NO:37一致1 -9 molecular weight calculated value 6404.3, mass spectrometry test molecular weight 6405.6, sequencing results consistent with SEQ ID NO:37
1 -10 分子量计算值 6248.1， 质谱测试分子量 6250.8， 测序结果与 SEQ ID NO:38—致1 -10 Molecular Weight Calculated 6248.1, Mass Spectrometry Tested Molecular Weight 6250.8, Sequencing Results and SEQ ID NO: 38
1 -11 分子量计算值 6234.1 , 质谱测试分子量 6235.7, 测序结果与 SEQ ID NO:39一致1 -11 Calculated molecular weight 6234.1 , mass spectrometry molecular weight 6235.7, sequencing results consistent with SEQ ID NO:39
1 -12 分子量计算值 6120.0, 质谱测试分子量 6121.4， 测序结果与 SEQ ID NO:40一致1 -12 Molecular weight calculated value 6120.0, mass spectrometry test molecular weight 6121.4, sequencing result is consistent with SEQ ID NO: 40
1 -13 分子量计算值 6207.1， 质谱测试分子量 6208.0, 测序结果与 SEQ ID NO:41—致1 -13 Molecular weight calculated value 6207.1, mass spectrometry test molecular weight 6208.0, sequencing result and SEQ ID NO: 41
1 -14 分子量计算值 6174.0, 质谱测试分子量 6175.5, 测序结果与 SEQ ID NO:42—致1 -14 calculated molecular weight 6174.0, mass spectrometry molecular weight 6175.5, sequencing result and SEQ ID NO: 42
1 -15 分子量计算值 6234.1 , 质诿测试分子量 6235.4， 测序结果与 SEQ ID NO:43—致1 -15 molecular weight calculated value 6234.1 , mass test molecular weight 6235.4, sequencing results and SEQ ID NO: 43
1 -16 分子量计算值 6261.1， 质谱测试分子量 6261.6， 测序结果与 SEQ ID NO:44一致1 -16 Calculated molecular weight 6261.1, mass spectrometry molecular weight 6261.6, sequencing result consistent with SEQ ID NO:44
1 -17 分子量计算值 6432.3 , 质谱测试分子量 6433.5， 测序结果与 SEQ ID NO:45一致1 -17 Calculated molecular weight 6432.3 , Mass spectrometry molecular weight 6433.5, sequencing results consistent with SEQ ID NO: 45
1 -18 分子量计算值 6002.8, 质语测试分子量 6004.1， 测序结果与 SEQ ID NO:46一致1 -18 Calculated molecular weight 6002.8, molecular weight test 6004.1, sequencing results consistent with SEQ ID NO:46
1 -19 分子量计算值 6078.9, 质谱测试分子量 6080.2, 测序结果与 SEQ ID NO:47一致 -20 分子量计算值 6121.9 质语测试分子量 6122.7 测序结果与 SEQ ID NO:48一致-21 分子量计算值 6163.0 质谋测试分子量 6164.4 测序结果与 SEQ ID NO:49一致 -22 分子量计算值 6071.0 质诰测试分子量 6072.8 测序结果与 SEQ ID NO:50一致 -23 分子量计算值 6590.5 质诰测试分子量 6592.3 测序结果与 SEQ ID NO:51一致 -24 分子量计算值 6505.4 质语测试分子量 6506.0 测序结果与 SEQ ID NO:52一致 -25 分子量计算值 6831.8 质 测试分子量 6833.7 测序结果与 SEQ ID NO:53一致 -26 分子量计算值 5899.7 质谙测试分子量 5900.6 测序结果与 SEQ ID N0.54一致 -27 分子量计算值 6114.0 质谙测试分子量 6115.9: 测序结果与 SEQ ID NO:55一致; -28 分子量计算值 6089.9 质语测试分子量 6091.5 测序结果与 SEQ ID N0.56一致 -29 分子量计算值 6089.9 质语测试分子量 6090.7 测序结果与 SEQ ID NO:57—致 -30 分子量计算值 6161.0 质诸测试分子量 6162.4 测序结果与 SEQ ID NO:58一致 -31 分子量计算值 6066.9 质 测试分子量 6068.1 测序结果与 SEQ ID N0.59一致 -32 分子量计算值 6093.9 质诸测试分子量 6095.6 测序结果与 SEQ ID NO:60一致 -33 分子量计算值 6334.2 质诸测试分子量 6336.0 测序结果与 SEQ ID N0.61一致 -34 分子量计算值 7159.1 质谙测试分子量 7161.2 测序结果与 SEQ ID NO:62一致 -35 分子量计算值 6546.4 质讲测试分子量 6546.0 测序结果与 SEQ ID NO:63一致 -36 分子量计算值 6592.5 质诸测试分子量 6593.8 测序结果与 SEQ ID NO:64一致 -37 分子量计算值 6592.4 质讲测试分子量 6594.1 测序结果与 SEQ ID NO:65一致 -38 分子量计算值 6732.6 质诸测试分子量 6733.4 测序结果与 SEQ ID NO:66一致 -39 分子量计算值 6644.6 质谱测试分子量 6646.7 测序结果与 SEQ ID O:67一致 -40 分子量计算值 6701.6 质谙测试分子量 6702.9 测序结果与 SEQ ID NO:68一致 -41 分子量计算值 6715.7 质潘测试分子量 6716.6 测序结果与 SEQ ID NO:69一致 -42 分子量计算值 6608.5 质诸测试分子量 6609.7 测序结果与 SEQ ID NO:70一致 -43 分子量计算值 6761.6 质谦测试分子量 6762.5 测序结果与 SEQ ID NO:71—致 -44 分子量计算值 6659.6 质诸测试分子量 6660.4 测序结果与 SEQ ID NO:72一致 -45 分子量计算值 6689.6 质诸测试分子量 6691.2 测序结果与 SEQ ID NO:73—致 -46 分子量计算值 6733.7 质谙测试分子量 6734.1 测序结果与 SEQ ID NO:74一致 -47 分子量计算值 6614.5 质谙测试分子量 6615.8 测序结果与 SEQ ID NO:75一致 -48 分子量计算值 6559.5 质谙测试分子量 6560.7 测序结果与 SEQ ID NO:76一致 -49 分子量计算值 6846.8 质诿测试分子量 6848.1 测序结果与 SEQ ID NO:77一致 -50 分子量计算值 6634.5 质语测试分子量 6636.0 测序结果与 SEQ ID NO:78—致 -51 分子量计算值 6774.6 质诿测试分子量 6675.9 测序结果与 SEQ ID NO:79一致 -52 分子量计算值 6644.6 质谙测试分子量 6645.3 测序结果与 SEQ ID NO:80一致 -53 分子量计算值 6770.7 质谦测试分子量 6772.2 测序结果与 SEQ ID NO-.81一致 -54 分子量计算值 6882.8 质谘测试分子量 6884.1 测序结果与 SEQ ID NO:82一致 -55 分子量计算值 8098.2 质谙测试分子量 8099.6 测序结果与 SEQ ID NO:83一致 -56 分子量计算值 6729.7 质谦测试分子量 6731.5 测序结果与 SEQ ID NO:84一致: -57 分子量计算值 6679.5 质谙测试分子量 6680.2 测序结果与 SEQ ID NO:85一致: -58 分子量计算值 6960.9 质谱测试分子量 6961.6 测序结果与 SEQ ID NO:86一致: -59 分子量计算值 7160.1 质谙测试分子量 7161.5 测序结果与 SEQ ID NO:87—致: -60 分子量计算值 6899.8, 质语测试分子量 6901.6， 测序结果与 SEQ ID NO:88一致; -61 分子量计算值 6703.6, 质诸测试分子量 6704.3， 测序结果与 SEQ ID NO:89一致;-62 分子量计算值 6644.6, 质语测试分子量 6645.8, 测序结果与 SEQ ID NO:90一致; -63 分子量计算值 6581.5, 质旙测试分子量 6583.3， 测序结果与 SEQ ID NO:91一致; -64 分子量计算值 6701.6， 质谙测试分子量 6702.4, 测序结果与 SEQ ID NO:92一致; -65 分子量计算值 6674.6, 质谱测试分子量 6675.7, 测序结果与 SEQ ID NO:93一致; -66 分子量计算值 7354.3， 质诸测试分子量 7356.2， 测序结果与 SEQ ID NO:94一致; -67 分子量计算值 6729.7, 质诸测试分子量 6730.2， 测序结果与 SEQ ID NO:95一致; -68 分子量计算值 6707.6, 质语测试分子量 6709.0, 测序结果与 SEQ ID NO:96一致; -69 分子量计算值 6774.7, 质诸测试分子量 6775.4, 测序结果与 SEQ ID NO:97一致; -70 分子量计算值 6620.5, 质语测试分子量 6621.1， 测序结果与 SEQ ID NO:98一致; -71 分子量计算值 6608.5, 质谱测试分子量 6609.7， 测序结果与 SEQ ID N0.99一致; -72 分子量计算值 6647.6, 质语测试分子量 6648.6, 测序结果与 SEQ ID NO: 100一致 -73 分子量计算值 6691.6, 质谱测试分子量 6693.0, 测序结果与 SEQ ID NO:101一致 -74 分子量计算值 7256.2，质谱测试分子量 7257.9， 测序结果与 SEQ ID NO: 102一致 -75 分子量计算值 6788.7, 质语测试分子量 6790.9, 测序结果与 SEQ ID NO: 103一致 -76 分子量计算值 7587.5, 质语测试分子量 7588.3 , 测序结果与 SEQ ID NO: 104一致 -77 分子量计算值 8530.5，质谱测试分子量 8532.8，测序结果与 SEQ ID NO: 105一致 -78 分子量计算值 7385.4, 质谱测试分子量 7386.0, 测序结果与 SEQ ID NO:106—致 -79 分子量计算值 6941.0, 质谱测试分子量 6941.5, 测序结果与 SEQ ID NO: 107一致 -80 分子量计算值 6009.8 , 质谱测试分子量 6011.4, 测序结果与 SEQ ID NO: 108一致 -81 分子量计算值 6555.5，质语测试分子量 6556.6, 测序结果与 SEQ ID NO:109—致 -82 分子量计算值 7098.1，质谱测试分子量 7100.2, 测序结果与 SEQ ID NO: 110—ft -83 分子量计算值 7186.0, 质谱测试分子量 7187.1， 测序结果与 SEQ ID NO:l ll—致 -84 分子量计算值 6934.9, 质谱测试分子量 6935.6， 测序结果与 SEQ ID NO: 112—致 -85 分子量计算值 7160.2，质语测试分子量 7161.9, 测序结果与 SEQ ID NO:113—致 -86 分子量计算值 6664.6, 质语测试分子量 6665.1 , 测序结果与 SEQ ID NO:114一致 -87 分子量计算值 6338.2，质谱测试分子量 6339.8, 测序结果与 SEQ ID NO: 115—致 -88 分子量计算值 6511.3 , 质谱测试分子量 6512.4, 测序结果与 SEQ ID NO:116—致 -89 分子量计算值 6407.3, 质谱测试分子量 6408.2， 测序结果与 SEQ ID NO:117—致 -90 分子量计算值 6636.6, 质谱测试分子量 6637.5, 测序结果与 SEQ ID NO: 118—致 -91 分子量计算值 6569.5，质谱测试分子量 6570.9, 测序结果与 SEQ ID NO:119一致 -92 分子量计算值 6385.3，质谱测试分子量 6387.0， 测序结果与 SEQ ID NO: 120一致 -93 分子量计算值 6371.3，质谱测试分子量 6372.3 , 测序结果与 SEQ ID NO:121—致 -94 分子量计算值 6806.8, 质谱测试分子量 6808.1 , 测序结果与 SEQ ID NO: 122一致 -95 分子量计算值 6704.6，质讲测试分子量 6705.9，测序结果与 SEQ ID NO: 123一致 -96 分子量计算值 6877.8，质谱测试分子量 6878.7, 测序结果与 SEQ ID NO: 124一致-97 分子量计算值 6750.7, 质谱测试分子量 6771.9, 测序结果与 SEQ ID NO: 125一致 -98 分子量计算值 7724.8，质谱测试分子量 7726.2, 测序结果与 SEQ ID NO:126—致 -99 分子量计算值 6123.9, 质语测试分子量 6125.8， 测序结果与 SEQ ID NO: 127一致: II -100: 分子量计算值 6614.4，质谱测试分子量 6615.3 测序结果与 SEQ ID NO 128一致;1 -19 molecular weight calculated value 6078.9, mass spectrometry test molecular weight 6080.2, sequencing results consistent with SEQ ID NO: 47 -20 Molecular weight calculated value 6121.9 Qualitative test molecular weight 6122.7 Sequencing result is consistent with SEQ ID NO: 48-21 Molecular weight calculated value 6163.0 Plot test molecular weight 6164.4 Sequencing result is consistent with SEQ ID NO: 49 -22 Molecular weight calculated value 6071.0 6072.8 Sequencing result is consistent with SEQ ID NO: 50-23 Molecular weight calculated 6590.5 诰 诰 分子量 659 6592.3 The sequencing result is identical to SEQ ID NO: 51-24 Molecular weight calculated value 6505.4 Qualitative test molecular weight 6506.0 Sequencing result is consistent with SEQ ID NO: 52 -25 Molecular weight calculated value 6831.8 Mass test molecular weight 6833.7 Sequencing result is consistent with SEQ ID NO: 53-26 Molecular weight calculated value 5899.7 谙 谙 test molecular weight 5900.6 Sequencing result is consistent with SEQ ID N0.54 -27 Molecular weight calculated value 6114.0 Quality test molecular weight 6115.9 : Sequencing result is consistent with SEQ ID NO: 55; -28 Molecular weight calculated value 6089.9 Qualitative test molecular weight 6091.5 Sequencing result is consistent with SEQ ID N0.56 -29 Molecular weight calculated value 6089.9 Qualitative test molecular weight 6090.7 Sequencing result and SEQ ID NO: 57 - -30 molecular weight calculated value 6161.0 quality test molecular weight 6162.4 sequencing results consistent with SEQ ID NO: 58 -31 molecules Calculated value 6066.9 Mass test molecular weight 6068.1 Sequencing result is consistent with SEQ ID N0.59 -32 Molecular weight calculated value 6093.9 Mass test molecular weight 6095.6 Sequencing result is consistent with SEQ ID NO: 60 -33 Molecular weight calculated value 6334.2 Mass test molecular weight 6336.0 Sequencing result Consistent with SEQ ID N0.61 -34 Molecular weight calculated 7159.1 谙 谙 test molecular weight 7161.2 Sequencing result is consistent with SEQ ID NO: 62 -35 Molecular weight calculated value 6546.4 Qualitative test molecular weight 6546.0 Sequencing result is consistent with SEQ ID NO: 63 - 36 Molecular weight Calculated value 6592.5 Mass tested molecular weight 6593.8 Sequencing result is consistent with SEQ ID NO: 64 -37 Molecular weight calculated value 6592.4 Qualitative test molecular weight 6594.1 Sequencing result is consistent with SEQ ID NO: 65 -38 Molecular weight calculated value 6732.6 Mass test molecular weight 6733.4 Sequencing result Consistent with SEQ ID NO: 66 - 39 Molecular Weight Calculated 6644.6 Mass Spectrometry Test Molecular Weight 6646.7 Sequencing Results Concord to SEQ ID O: 67 - 40 Molecular Weight Calculated Value 6701.6 谙 谙 Test Molecular Weight 6702.9 Sequencing Results Concord to SEQ ID NO: 68 - 41 Molecular Weight Calculation Value 6715.7 Quality Pan Test Molecular Weight 6716.6 Sequencing Results Concord to SEQ ID NO: 69 - 42 Molecular Weight Calculated Value 66 08.5 Qualitative test molecular weight 6609.7 Sequencing result is consistent with SEQ ID NO: 70 -43 Molecular weight calculated value 6761.6 Qualitative test molecular weight 6762.5 Sequencing result and SEQ ID NO: 71 - 44 - Molecular weight calculated value 6659.6 Qualitative test molecular weight 6660.4 Sequencing results and SEQ ID NO:72 unanimity -45 Molecular weight calculated 6689.6 Qualitative molecular weight 6691.2 Sequencing results and SEQ ID NO: 73 -46 - Molecular weight calculated 6733.7 谙 谙 分子量 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 673 Calculated value 6614.5 谙 test molecular weight 6615.8 Sequencing result is consistent with SEQ ID NO: 75 -48 Molecular weight calculated value 6559.5 谙 谙 test molecular weight 6560.7 Sequencing result is consistent with SEQ ID NO: 76 -49 Molecular weight calculated value 6846.8 诿 诿 test molecular weight 6848.1 Sequencing results Consistent with SEQ ID NO: 77 - 50 Molecular Weight Calculated 6634.5 Qualitative Test Molecular Weight 6636.0 Sequencing Results and SEQ ID NO: 78 - 51 - Molecular Weight Calculated Value 6774.6 诿 诿 Test Molecular Weight 6675.9 Sequencing Results Consistent with SEQ ID NO: 79 - 52 Molecular weight calculated 6644.6 谙 谙 test molecular weight 6645.3 Sequencing results are consistent with SEQ ID NO: 80 -53 Molecular weight calculated value 6770.7 Molecular weight test 6772.2 Sequencing result is consistent with SEQ ID NO-.81 -54 Molecular weight calculated value 6882.8 Quality control test molecular weight 6884.1 Sequencing result is consistent with SEQ ID NO: 82 -55 Molecular weight calculated value 8098.2 Quality test molecular weight 8099.6 Sequencing result and SEQ ID NO :83 unanimum-56 Molecular weight calculated value 6729.7 Qualitative test molecular weight 6731.5 Sequencing result is consistent with SEQ ID NO: 84: -57 Molecular weight calculated value 6679.5 谙 谙 test molecular weight 6680.2 Sequencing result is consistent with SEQ ID NO: 85: -58 Molecular weight calculated value 6960.9 Mass spectrometry molecular weight 6961.6 Sequencing results are consistent with SEQ ID NO: 86: -59 Molecular weight calculated 7160.1 谙 谙 test molecular weight 7161.5 Sequencing results with SEQ ID NO: 87: -60 molecular weight calculated value 6899.8, qualitative test molecular weight 6901.6, sequencing results consistent with SEQ ID NO: 88; -61 molecular weight calculated value 6703.6, quality test molecular weight 6704.3, sequencing results consistent with SEQ ID NO: 89; -62 molecular weight calculation The value was 6644.6, the molecular weight of the test was 6645.8, the sequencing result was consistent with SEQ ID NO: 90; -63 the molecular weight calculated value was 6581.5, the molecular weight test molecular weight was 6583.3, the sequencing result was consistent with SEQ ID NO: 91; -64 the molecular weight calculated value was 6701.6,谙 test molecular weight 6702.4, sequencing results consistent with SEQ ID NO: 92; -65 molecular weight calculated value 6674.6, mass spectrometry test molecular weight 6675.7, sequencing results consistent with SEQ ID NO: 93; -66 molecular weight calculated value 7354.3, quality test molecular weight 7356.2, The sequencing result was consistent with SEQ ID NO: 94; -67 molecular weight calculated value 6729.7, mass tested molecular weight 6730.2, sequencing result was consistent with SEQ ID NO: 95; -68 molecular weight calculated value 6707.6, qualitative test molecular weight 6709.0, sequencing result and SEQ ID NO: 96 consistent; -69 Molecular weight calculated value 6774.7, quality tested with molecular weight 6775.4, sequencing results consistent with SEQ ID NO: 97; -70 molecular weight calculated value 6620.5, scholastic test score The amount of 6621.1, the sequencing result is consistent with SEQ ID NO: 98; -71 The molecular weight calculated value is 6608.5, the mass spectrometric test molecular weight is 6609.7, the sequencing result is consistent with SEQ ID N0.99; -72 The molecular weight calculated value is 6647.6, the molecular weight test molecular weight is 6648.6, and the sequencing result is Consistent with SEQ ID NO: 100 - 73 molecular weight calculated 6691.6, mass spectrometric test molecular weight 6693.0, sequencing result consistent with SEQ ID NO: 101 - 74 molecular weight calculated 7256.2, mass spectrometric test molecular weight 7257.9, sequencing results consistent with SEQ ID NO: 102 - 75 molecular weight calculated value 6788.7, molecular weight test 6790.9, sequencing results consistent with SEQ ID NO: 103 -76 molecular weight calculated value 7587.5, qualitative test molecular weight 7588.3, sequencing results consistent with SEQ ID NO: 104 -77 molecular weight calculated value 8530.5, Mass spectrometry test molecular weight 8532.8, sequencing results consistent with SEQ ID NO: 105 -78 molecular weight calculated value 7385.4, mass spectrometry test molecular weight 7386.0, sequencing results and SEQ ID NO: 106 -79 molecular weight calculated value 6941.0, mass spectrometry molecular weight 6941.5, sequencing results Consistent with SEQ ID NO: 107 -80 molecular weight calculated value 6009.8, mass spectrometry test molecular weight 6011.4, sequencing result consistent with SEQ ID NO: 108 -81 The calculated molecular weight is 6555.5, the molecular weight of the test is 6556.6, the sequencing result is SEQ ID NO: 109, the molecular weight is 7098.1, the mass spectrometry is 7100.2, and the sequencing result is SEQ ID NO: 110-ft -83. Mass spectrometry test molecular weight 7187.1, sequencing results and SEQ ID NO: l ll--84 molecular weight calculated value 6934.9, mass spectrometry test molecular weight 6935.6, sequencing results and SEQ ID NO: 112 -85 molecular weight calculated value 7160.2, qualitative test molecular weight 7161.9 , sequencing results with SEQ ID NO: 113 - 86 molecular weight calculated value 6664.6, qualitative test molecular weight 6665.1, sequencing results consistent with SEQ ID NO: 114 -87 molecular weight calculated value 6338.2, mass spectrometry test molecular weight 6339.8, sequencing results and SEQ ID NO: 115-to-88 Molecular weight calculated value 6511.3, mass spectrometry test molecular weight 6512.4, sequencing result and SEQ ID NO: 116---89 molecular weight calculated value 6407.3, mass spectrometry test molecular weight 6408.2, sequencing result and SEQ ID NO: 117 - 90 molecular weight calculated value 6636.6, mass spectrometry test molecular weight 6637.5, sequencing results and SEQ ID NO: 118 -91 - molecular weight calculated value 6569.5, mass spectrometry test molecular weight 6570. 9, sequencing results consistent with SEQ ID NO: 119 -92 molecular weight calculated value 6385.3, mass spectrometry test molecular weight 6387.0, sequencing results consistent with SEQ ID NO: 120 -93 molecular weight calculated value 6371.3, mass spectrometry test molecular weight 6372.3, sequencing results and SEQ ID NO :121至致94 molecular weight calculated value 6806.8, mass spectrometry test molecular weight 6808.1, sequencing results consistent with SEQ ID NO: 122 -95 molecular weight calculated value 6704.6, qualitative test molecular weight 6705.9, sequencing results consistent with SEQ ID NO: 123 -96 molecular weight Calculated value 6877.8, mass spectrometry test molecular weight 6878.7, sequencing result consistent with SEQ ID NO: 124 -97 molecular weight calculated value 6750.7, mass spectrometry test molecular weight 6771.9, sequencing result consistent with SEQ ID NO: 125 -98 molecular weight calculated value 7724.8, mass spectrometry test molecular weight 7726.2 The sequencing result is the same as SEQ ID NO: 126, the molecular weight calculated value is 6123.9, the molecular weight test is 6125.8, and the sequencing result is consistent with SEQ ID NO: 127: II -100: molecular weight calculated value 6614.4, mass spectrometry test molecular weight 6615.3 The sequencing result is consistent with SEQ ID NO 128;
II -101: 分子量计算值 6899.8,质谱测试分子量 6901.4 测序结果与 SEQ ID NO 129一致;II -101: Calculated molecular weight 6899.8, mass spectrometry molecular weight 6901.4 The sequencing result is consistent with SEQ ID NO 129;
II -102: 分子量计算值 6036.8,质谱测试分子量 6038.0 测序结果与 SEQ ID NO 130一致;II -102: Calculated molecular weight 6036.8, mass spectrometry molecular weight 6038.0 The sequencing result is consistent with SEQ ID NO 130;
Π-103:分子量计算值 6853.7，质谙测试分子量 6854.5 测序结果与 SEQ ID NO 131一致;Π-103: molecular weight calculated value 6853.7, mass test molecular weight 6854.5 sequencing results consistent with SEQ ID NO 131;
II -104:分子量计算值 7284.2,质语测试分子量 7285.6 测序结果与 SEQ ID NO 132一致;II-104: Calculated molecular weight 7284.2, molecular weight of the test test 7285.6 The sequencing result is consistent with SEQ ID NO 132;
II -105:分子量计算值 7551.5,质语测试分子量 7552.9 测序结果与 SEQ ID NO 133一致;II-105: molecular weight calculated value 7551.5, molecular weight of molecular test 7552.9 The sequencing result is consistent with SEQ ID NO 133;
II -106:分子量计算值 6913.9,质讲测试分子量 6915.3 测序结果与 SEQ ID NO' 134一致;II -106: molecular weight calculated value 6913.9, qualitative test molecular weight 6915.3 The sequencing result is consistent with SEQ ID NO' 134;
II -107:分子量计算值 6837.7,质潘测试分子量 6839.3 测序结果与 SEQ ID NO 135一致;II-107: Calculated molecular weight 6837.7, mass test molecular weight 6839.3 The sequencing result is consistent with SEQ ID NO 135;
Π-108:分子量计算值 7063.1，质语测试分子量 7064.7 测序结果与 SEQ ID NO 136一致;Π-108: molecular weight calculated value 7063.1, molecular weight test molecular weight 7064.7 The sequencing result is consistent with SEQ ID NO 136;
II -109:分子量计算值 6528.4,质谱测试分子量 6529.8 测序结果与 SEQ ID NO 137一致;II-109: molecular weight calculated value 6528.4, mass spectrometry test molecular weight 6529.8 The sequencing result is consistent with SEQ ID NO 137;
Π -110:分子量计算值 7200.3,质谙测试分子量 7201.4 测序结果与 SEQ ID NO 138一致;Π -110: molecular weight calculated value 7200.3, mass 谙 test molecular weight 7201.4 sequencing results consistent with SEQ ID NO 138;
II -ll l :分子量计算值 7735.7,质旙测试分子量 7737.0 测序结果与 SEQ ID NO 139一致;II -ll l : molecular weight calculated value 7735.7, mass test molecular weight 7737.0 The sequencing result is consistent with SEQ ID NO 139;
Π -112:分子量计算值 6369.1 ,质谱测试分子量 6370.6 测序结果与 SEQ ID NO 140一致;Π -112: molecular weight calculated value 6369.1, mass spectrometry test molecular weight 6370.6 The sequencing result is consistent with SEQ ID NO 140;
II -113:分子量计算值 7468.4,质诲测试分子量 7470.2 测序结果与 SEQ ID NO 141一致;II-113: Calculated molecular weight 7468.4, 诲 test molecular weight 7470.2 The sequencing result is consistent with SEQ ID NO 141;
11 -114: 分子量计算值 6603.5,质谱测试分子量 6604.7 测序结果与 SEQ ID NO 142一致;11 -114: molecular weight calculated value 6603.5, mass spectrometry test molecular weight 6604.7 The sequencing result is consistent with SEQ ID NO 142;
Π -115: 分子量计算值 7254.2,质借测试分子量 7255.5 测序结果与 SEQ ID NO. 143一致;Π -115: molecular weight calculated value 7254.2, mass test molecular weight 7255.5 sequencing results consistent with SEQ ID NO.
II -116: 分子量计算值 6625.5,质讲测试分子量 6627.1 测序结果与 SEQ ID NO 144一致;II -116: Calculated molecular weight 6625.5, qualitative test molecular weight 6627.1 The sequencing result is consistent with SEQ ID NO 144;
Π -117:分子量计算值 7399.5，质谦测试分子量 7401.3 测序结果与 SEQ ID NO: 145一致;Π -117: molecular weight calculated value 7399.5, mass test molecular weight 7401.3 sequencing results consistent with SEQ ID NO: 145;
Π -118:分子量计算值 7223.1 ,质讲测试分子量 7224.4 测序结果与 SEQ ID NO: 146一致;Π -118: molecular weight calculated value 7223.1, qualitative test molecular weight 7224.4 sequencing results consistent with SEQ ID NO: 146;
II -119:分子量计算值 6120.0,质诰测试分子量 6121.6 测序结果与 SEQ ID NO- 147一致;II - 119: molecular weight calculated value 6120.0, mass test molecular weight 6121.6 The sequencing result is consistent with SEQ ID NO-147;
II -120:分子量计算值 6278.2,质诸测试分子量 6277.9 测序结果与 SEQ ID NO: 148一致;II - 120: Calculated molecular weight 6278.2, molecular weight of the test 6277.9 The sequencing result is consistent with SEQ ID NO: 148;
Π -121:分子量计算值 6264.1，质谱测试分子量 6265.8 测序结果与 SEQ ID NO- 149一致;Π -121: molecular weight calculated value 6264.1, mass spectrometry test molecular weight 6265.8 The sequencing result is consistent with SEQ ID NO-149;
II -122:分子量计算值 6242.2,质诸测试分子量 6243.4 测序结果与 SEQ ID NO: 150一致;II-122: Calculated molecular weight 6242.2, mass tested molecular weight 6243.4 Sequencing results are consistent with SEQ ID NO: 150;
II -123:分子量计算值 6084.9,质语测试分子量 6086.3 测序结果与 SEQ ID NO: 161一致;II -123: molecular weight calculated value 6084.9, molecular weight of test term 6086.3 The sequencing result is consistent with SEQ ID NO: 161;
II -124:分子量计算值 6659.5,质诿测试分子量 6660.7 测序结果与 SEQ ID NO: 162一致。 化合物的 II-124: Calculated molecular weight 6659.5, mass 诿 test molecular weight 6660.7 The sequencing result is identical to SEQ ID NO: 162. Compound
基于 IGF-1的化合物的修饰  Modification of IGF-1 based compounds
多肽的酰化  Acylation of peptides
叔丁基十六烷二酰基 -L-Glu(OSu)-OtBu制备  Preparation of tert-butylhexadecandioyl-L-Glu(OSu)-OtBu
十六烷二酸（5.72 g, 20 mmol)溶解于无水 DMF ( 240 mL ), 用冰浴冷却。 逐次添加 2- 甲基 -2-丙醇（ 1.48 g, 20mmol)、DIC ( 2.7 g， 2.25 mL, 21.4 mmol), HOBT ( 2.88 g, 21.4 mmol), NMM ( 2.16g, 2.34 mL, 21.4 mmol). DMAP ( 244 mg, 2 mmol). 混合物在室温下搅拌过 夜。 加入 80 mL水， 酸化到 pH 3 , 用乙酸乙酯萃取， 有机层用 0.1 N HC1和饱和食盐水洗，硫 酸镁干燥后， 溶剂减压蒸发得到十六烷二酸一叔丁酯 （3.32g， 产率 47%)。 核磁共振数据为 'H-NMRiCDC ) 5: 2.35 (t,2H), 1.56-1.66 (m,4H), 1.44(s,9H), 1.21-1.35 (m，20H)。  Hexadecandioic acid (5.72 g, 20 mmol) was dissolved in dry DMF (240 mL) and cooled with ice. Add 2-methyl-2-propanol ( 1.48 g, 20 mmol), DIC (2.7 g, 2.25 mL, 21.4 mmol), HOBT ( 2.88 g, 21.4 mmol), NMM ( 2.16 g, 2.34 mL, 21.4 mmol) DMAP (244 mg, 2 mmol). The mixture was stirred at room temperature overnight. After adding 80 mL of water, acidified to pH 3, and extracted with ethyl acetate. The organic layer was washed with 0.1 N EtOAc and brine. Yield 47%). The NMR data is 'H-NMRiCDC) 5: 2.35 (t, 2H), 1.56-1.66 (m, 4H), 1.44 (s, 9H), 1.21-1.35 (m, 20H).
Fmoc-Glu-OtBu ( 4.25g, 10 mmol) 溶解于 DCM ( 30 mL ), 加到 3 克 2-CTC树脂 ( 2-chlorotrityl chloride resin, sub. lmmol/g), 继续加入 DIPEA ( 1.29g, 10 mmol, 1.74 mL)。 混合物在摇动器振动 5分钟后， 再加入 DIPEA ( 1.93g, 15 mmol, 2.6 mL)。 混合物剧烈振动 1小时。 为了封住活性三苯甲游基， 树脂中加入 HPLC级曱醇 （2.4 mL ), 混合 15分钟。 树脂过 滤， 用 DCM (3 X 30 mL)、 DMF (2 X 30 mL)、 DCM (3 X 30 mL)、 曱醇 (3 X 30 mL)清洗后， 在真空中干燥。 Fmoc-Glu-OtBu ( 4.25 g, 10 mmol) dissolved in DCM (30 mL), added to 3 g of 2-chlorotrityl chloride resin (sub.lmmol/g), and then added to DIPEA ( 1.29 g, 10 M, 1.74 mL). After the mixture was shaken for 5 minutes in a shaker, DIPEA (1.93 g, 15 mmol, 2.6 mL) was added. The mixture was shaken vigorously for 1 hour. In order to block the active trityl group, HPLC grade sterol (2.4 mL) was added to the resin and mixed for 15 minutes. The resin was filtered, washed with DCM (3 X 30 mL), DMF (2 X 30 mL), DCM (3 X 30 mL), decyl alcohol (3 X 30 mL) and dried in vacuo.
用哌啶脱除 Fmoc后， 3 g树脂 (3mmol) 与十六烷二酸一叔丁酯（3.43 g, lO mmol)加入 无水 DMF( 50 mL ),逐次加入 DIC ( 1.35 g, 1.12 mL， 10.7 mmol)、 HOBT ( 1.44 g, 10.7 mmol)、 DIPEA ( 1.3 g, 10 mmol, 1.74 mL)。在室温振动过夜后 ,树脂用 DMF (2 X 30 mL) 和 DCM (2 X 30 mL)清洗。  After removing Fmoc with piperidine, 3 g of resin (3 mmol) and hexadecanedioic acid-tert-butyl ester (3.43 g, 10 mmol) were added to dry DMF (50 mL), and DIC ( 1.35 g, 1.12 mL, 10.7 mmol), HOBT ( 1.44 g, 10.7 mmol), DIPEA (1.3 g, 10 mmol, 1.74 mL). After shaking overnight at room temperature, the resin was washed with DMF (2 X 30 mL) and DCM (2 X 30 mL).
准备 AcOH/TFE/DCM ( 1 : 1 :8 ) 的切割液 (20 mL/g树脂）。 树脂悬浮在一半的切割液， 室 温下放置 30分钟。 过滤树脂， 用另一半切割液洗涤树脂三次。 混合滤液加入 15倍体积的正 己烷， 旋蒸去除多余醋酸， 得到叔丁基十六烷二酰基 -L-Glu-OtBu。 核磁共振数据为 'H-NMR(CDC13) δ: 6.25(d,lH), 4.53(m, 1H)， 2.42 (m， 2H), 2.21 (m,4H),1.92(m, 1H), 1.58 (m, 4H), 1.47(s, 9H)， 1.22-1.43 (m, 18H)。 Prepare a cutting solution (20 mL/g resin) of AcOH/TFE/DCM (1:1:8). The resin was suspended in half of the cutting solution and allowed to stand at room temperature for 30 minutes. The resin was filtered and the resin was washed three times with the other half of the cutting solution. The mixed filtrate was added to 15 volumes of n-hexane, and excess acetic acid was removed by rotary evaporation to obtain tert-butylhexadecandioyl-L-Glu-OtBu. The NMR data is 'H-NMR (CDC1 3 ) δ: 6.25 (d, lH), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47(s, 9H), 1.22-1.43 (m, 18H).
叔丁基十六烷二酰基 -L-Glu-OtBu ( l g, 1.9 mmol) 溶于无水 DMF/DCM (1 mL: 4 mL). 加 入 DCC ( 0.412 g, 2 mmol)和 N-羟基丁二酰亚胺（ 0.23 g, 2 mmol) 。 混合物在室温搅拌过 夜。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HCI和饱和食盐水洗涤， 石克酸镁干燥后， 减 压蒸发得到叔丁基十六烷二酰基 -L-Glu ( OSu ) -OtBu。 核磁共振数据为： ^-NMRiCDC ) δ: 6.17 (d,lH), 4.60 (m, 1H)， 2.84 (s, 4H)， 2.72 (m, 1H), 2.64 (m, 1H), 2.32(m, 1H), 2.20 (m, 4H)， 2.08 (m, 1H), 1.6 (m， 4H)， 1.47(s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H)。  tert-Butylhexadecandioyl-L-Glu-OtBu ( lg, 1.9 mmol) dissolved in anhydrous DMF/DCM (1 mL: 4 mL). DCC (0.412 g, 2 mmol) and N-hydroxybutane Imide (0.23 g, 2 mmol). The mixture was stirred overnight at room temperature. The mixture was filtered, and the filtrate was diluted with ethyl acetate, washed with 0.1 N HCI and brine, and dried over magnesium sulfate, and evaporated under reduced pressure to give tert-butylhexadecandioyl-L-Glu ( OSu ) -OtBu. The NMR data are: ^-NMRiCDC) δ: 6.17 (d, lH), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H).
III-7的合成。  Synthesis of III-7.
用上述 A、 B链连接方法合成。 计算分子量 5746.7， 质谱测试分子量 5747.8。 室温下将 多肽（ 55 mg)溶于 lOOmM Na2C03 (5 mL, pH 10)。叔丁基十六烷二酰基 -L-Glu( OSu )-OtBu (6.6 mg)溶于乙腈（5mL ), 加入多肽溶液。 搅拌 30分钟后， 用 50%醋酸酸化， 上 RP-HPLC C5 柱纯化。 緩冲液 A: 0.1%TFA水溶液， 10%乙腈 緩冲液 B:0.1%TFA水溶液， 80%乙腈。 初步 纯化冻干后的多肽加入 TFA/TIS/H20 ( 95:2.5:2.5,10mL ), 30分钟后真空蒸发溶剂， 将粗产品 溶于緩冲液 A并冻干。 使用 RP-HPLC C5柱纯化。 分子量计算值 6144.2 , 质谱测试分子量 6146.0。 B 链经胰蛋白酶水解后， 含有脂肪酸的末端片段的计算分子量 1269.5， 质谱测试分 子量 1270.4。 It was synthesized by the above A and B chain ligation methods. The molecular weight was calculated to be 5746.7, and the molecular weight of the mass spectrum was 5747.8. The polypeptide (55 mg) was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature. tert-Butylhexadecandioyl-L-Glu(OSu)-OtBu (6.6 mg) was dissolved in acetonitrile (5 mL). After stirring for 30 minutes, it was acidified with 50% acetic acid and purified on a RP-HPLC C5 column. Buffer A: 0.1% TFA in water, 10% acetonitrile buffer B: 0.1% TFA in water, 80% acetonitrile. The lyophilized polypeptide was initially purified by adding TFA/TIS/H 2 0 (95:2.5:2.5, 10 mL), and after 30 minutes, the solvent was evaporated in vacuo, and the crude product was dissolved in buffer A and lyophilized. Purified using an RP-HPLC C5 column. The molecular weight calculated was 6144.2 and the mass spectrum was tested to have a molecular weight of 6146.0. After the B chain was subjected to trypsin hydrolysis, the calculated molecular weight of the terminal fragment containing the fatty acid was 1269.5, and the molecular weight of the mass spectrum was 1270.4.
111-28的合成：  Synthesis of 111-28:
方法 1 :  method 1 :
B链（0.1 mmol )用 Boc化学合成， 赖氨酸侧链氨基用 Fmoc保护。 B链合成完毕后， B 链 N-末端的 Boc不要脱除。 20% 哌啶/ DMF (6 mL)加入树脂， 混合物振动 15分钟， 抽干试 剂。 经过两轮哌啶反应， 树脂用 DMF和 DCM洗涤 3遍。 Fmoc-L-Glu-OtBu (425 mg， 1 mmol)、 DIC (126 mg, 0.1 ml, 1 mmol) 、 HOBT (135 mg,l mmol)和 DIPEA ( 0.2 mL,1.15mmol )溶于 DMF, 加入树脂。 振动 2小时后， 溶液被抽干， 树脂用 DMF和 DCM洗涤。 20%哌啶/ DMF 加入树脂， 混合物振动 15分钟， 抽干试剂。 经过两轮哌啶反应， 树脂用 DMF和 DCM洗涤 3遍。 叔丁基十六烷二酰基 -L-Glu ( OSu ) -OtBu ( 550 mg )和 DIPEA(0.2 mL)溶于 DMF , 加 入树脂。 振动 3小时后， 溶液被抽千， 树脂用 DMF和 DCM洗涤。 真空干燥后， 树脂上的多 肽用 HF切割， 加入对曱酚试剂， 混合物在冰浴条件下搅拌 1小时， HF真空抽千后， 多肽用 冰乙瞇沉淀， 离心收集沉淀。 分子量计算值 3601.2， 质谱测试分子量 3602.8。  The B chain (0.1 mmol) was synthesized by Boc chemistry, and the lysine side chain amino group was protected with Fmoc. After the B chain is synthesized, the Boc at the N-terminus of the B chain should not be removed. 20% piperidine / DMF (6 mL) was added to the resin, and the mixture was shaken for 15 minutes, and the test solution was drained. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM. Fmoc-L-Glu-OtBu (425 mg, 1 mmol), DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, 1 mmol) and DIPEA (0.2 mL, 1.15 mmol) dissolved in DMF, added resin . After shaking for 2 hours, the solution was drained and the resin was washed with DMF and DCM. 20% piperidine / DMF was added to the resin, the mixture was shaken for 15 minutes, and the reagent was drained. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM. tert-Butylhexadecandioyl-L-Glu (OSu)-OtBu (550 mg) and DIPEA (0.2 mL) were dissolved in DMF and added to the resin. After shaking for 3 hours, the solution was pumped and the resin was washed with DMF and DCM. After vacuum drying, the peptide on the resin was cut with HF, p-phenol reagent was added, and the mixture was stirred under ice bath for 1 hour. After HF vacuum extraction, the polypeptide was precipitated with ice acetonitrile, and the precipitate was collected by centrifugation. Molecular weight calculated 3601.2, mass spectrometry test molecular weight 3602.8.
B链粗品经巯基磺化反应， 过色谱柱脱盐、 分离纯化， 冷冻干燥得到最后产品 IGF-1 B 链 S磺酸酯。 IGF-1 A (Q5, S12, N18)按通用方法合成， 分子量计算值 2436.9, 质谱测试分子 量 2437.6。 IGF-1 A链 S磺酸酯和 B链 S磺酸酯使用上述通用 A链与 B链的连接方法合成。 分子量计算值 6032.0, 质谱测试分子量 6033.4。 The crude B chain is sulfonated by sulfhydrylation, desalted by chromatography, separated and purified, and freeze-dried to obtain the final product IGF-1 B. Chain S sulfonate. IGF-1 A (Q5, S12, N18) was synthesized by a general method with a molecular weight of 2436.9 and a molecular weight of 2437.6. The IGF-1 A chain S sulfonate and the B chain S sulfonate are synthesized using the above-described general A chain and B chain linkage method. The molecular weight calculated was 6032.0, and the mass spectrum was tested to be 6033.4.
方法 2:  Method 2:
B链用 Boc化学合成，赖氨酸側链氨基用 Fmoc保护， B18Cys(对应于通式中 或 X44! ) 的巯基用 Acm保护。 合成完毕后， B链 N-末端的 Boc不要脱除。 20%哌 DMF(6 mL)加入 树脂（ 0.1 mmol ), 混合物振动 15分钟，抽干试剂。 经过两轮哌啶反应， 树脂用 DMF和 DCM 洗涤 3遍。 Fmoc-L-Glu-OtBu (425 mg)、 DIC (126 mg, 0.1 ml) 、 HOBT(135 mg)和 DIPEA ( 0.2 mL )加入树脂。 振动 2小时后， 溶液被抽千， 树脂用 DMF和 DCM洗涤后， 加入 20%哌啶 /DMF, 混合物振动 15分钟，抽干试剂。 经过两轮派啶反应，树脂用 DMF和 DCM洗涤 3遍。 叔丁基十六烷二酰基 -L-Glu-OtBu( 715 mg )、 DIC (126 mg, 0.1 ml) 、 HOBT(135 mg) 和 DIPEA ( 0.2 mL )溶于 DMF, 加入树脂， 振动 2小时后， 溶液被抽干， 树脂用 DMF和 DCM洗涤。 真空千燥后， 树脂上的多肽用 HF切割， 加入对甲酚试剂， 混合物在冰浴条件下搅拌 1小时， HF 真空抽干后， 多肽用冰乙醚沉淀， 离心收集沉淀。 计算分子量 3672.3， 质谱测试分子量 3673.5。  The B chain is chemically synthesized by Boc, the lysine side chain amino group is protected with Fmoc, and the thiol group of B18Cys (corresponding to the formula or X44!) is protected with Acm. After the synthesis is completed, the Boc at the N-terminus of the B chain is not removed. 20% piperidine DMF (6 mL) was added to the resin (0.1 mmol), the mixture was shaken for 15 minutes, and the reagent was drained. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM. Fmoc-L-Glu-OtBu (425 mg), DIC (126 mg, 0.1 ml), HOBT (135 mg) and DIPEA (0.2 mL) were added to the resin. After shaking for 2 hours, the solution was pumped, the resin was washed with DMF and DCM, 20% piperidine / DMF was added, the mixture was shaken for 15 minutes, and the reagent was drained. After two rounds of pyridine reaction, the resin was washed 3 times with DMF and DCM. tert-Butylhexadecandioyl-L-Glu-OtBu (715 mg), DIC (126 mg, 0.1 ml), HOBT (135 mg) and DIPEA (0.2 mL) were dissolved in DMF, added to the resin, and shaken for 2 hours. The solution was drained and the resin was washed with DMF and DCM. After the vacuum was dried, the polypeptide on the resin was cut with HF, p-cresol reagent was added, and the mixture was stirred under ice bath for 1 hour. After HF vacuum drying, the polypeptide was precipitated with ice diethyl ether, and the precipitate was collected by centrifugation. Calculated molecular weight 3672.3, mass spectrometry test molecular weight 3673.5.
Β链溶于 DMF或 DMSO, 加入等摩尔 2，2'-二硫双 (5-硝基吡啶)。 反应用 HPLC检测和纯 化， 得到 Β- ( S-Npys ) 6 ( S-Acm ) 18 [H9,F15,P27,K28-NeB28-(Na-(HOOC (CH2)14CO)-y- G1U-N-(Y-G1U))]„ 计算分子量 3826.4, 质谱测试分子量 3827.0。 The hydrazine chain is dissolved in DMF or DMSO, and an equimolar amount of 2,2'-dithiobis(5-nitropyridine) is added. The reaction was detected and purified by HPLC to obtain Β-(S-Npys) 6 (S-Acm) 18 [H9, F15, P27, K28-N eB28 - (N a -(HOOC (CH 2 ) 14 CO)-y- G1U-N-(Y-G1U))] „ Calculated molecular weight 3826.4, mass spectrometry test molecular weight 3827.0.
A链合成为 IGF-1 A-(Q5, S12, N18) ( SH ) 7 ( S-Acm ) 6' 20计算分子量 2650.1 , 质谱测 试分子量 2651.5。 A链和 B链的连接方法按照上述 A链和 B链连接方法（ 2 )。 分子量计算值 6032.0, 质谱测试分子量 6034.2。 The A chain was synthesized as IGF-1 A-(Q5, S12, N18) (SH) 7 (S-Acm) 6 '20 to calculate the molecular weight of 2650.1 and the mass spectrometry to test the molecular weight of 2651.5. The method for joining the A chain and the B chain is in accordance with the above A chain and B chain linkage method (2). The molecular weight calculated was 6032.0, and the mass spectrum was tested to have a molecular weight of 6034.2.
111-29的合成。  Synthesis of 111-29.
B链 (O.lmmol)用 Boc化学合成完毕后， B链 N-末端的 Boc用 TFA脱除。 Fmoc-L-Glu-OtBu (425 mg, 1 mmol), DIC (126 mg, 0.1 ml, 1 mmol) 、 HOBT (135 mg， lmmol) 和 DIPEA ( 0.2 ml,1.15mmol )溶于 DMF, 加入树脂。 振动 2小时后， 溶液被抽干， 树脂用 DMF和 DCM洗 涤。 20% 哌啶/ DMF 加入树脂， 混合物振动 15分钟。 经过两轮哌啶反应， 树脂用 DMF和 DCM洗涤 3遍。棕榈酸 (256 mg, 1 mmol)> DIC (126 mg, 0.1ml, lmmol), HOBT(135 mg, lmmol) 和 DIPEA ( 0.2 ml， 1.15mmol )溶于 DMF, 加入树脂。 2小时后， 溶液被抽干， 树脂用 DMF 和 DCM洗涤。 真空干燥后， 树脂上的多肽用 HF切割， 加入对曱酴试剂， 混合物在冰浴条件 下搅拌 1小时， HF真空抽干后， 多肽用冰乙醚沉淀， 离心收集沉淀。 分子量计算值 3512.1 , 质谱测试分子量 3513.5。 B链粗品经兢基横化反应 ( Sulfitolysis ), 过色潘柱脱盐、 分离纯化， 冷冻干燥得到最后产品 B链 S磺酸酯。 A链 S磺酸酯和 B链 S磺酸酯使用上述通用 A链与 B 链的连接方法合成。 分子量计算值 5943.0, 质谱测试分子量 5943.6。  After the B chain (O.lmmol) was chemically synthesized by Boc, the Boc at the N-terminus of the B chain was removed by TFA. Fmoc-L-Glu-OtBu (425 mg, 1 mmol), DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, 1 mmol) and DIPEA (0.2 ml, 1.15 mmol) were dissolved in DMF and added to the resin. After shaking for 2 hours, the solution was drained and the resin was washed with DMF and DCM. 20% piperidine / DMF was added to the resin and the mixture was shaken for 15 minutes. After two rounds of piperidine reaction, the resin was washed 3 times with DMF and DCM. Palmitic acid (256 mg, 1 mmol) > DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, 1 mmol) and DIPEA (0.2 ml, 1.15 mmol) were dissolved in DMF and added to the resin. After 2 hours, the solution was drained and the resin was washed with DMF and DCM. After vacuum drying, the polypeptide on the resin was cut with HF, and a hydrazine reagent was added. The mixture was stirred under ice bath for 1 hour, and after HF vacuum-dried, the polypeptide was precipitated with ice diethyl ether, and the precipitate was collected by centrifugation. Molecular weight calculated value 3512.1, mass spectrometry test molecular weight 3513.5. The crude B chain is subjected to Sulfoid Hydration, desalting, separation and purification, and freeze-drying to obtain the final product B chain S sulfonate. The A chain S sulfonate and the B chain S sulfonate are synthesized by the above-described method of linking the general A chain to the B chain. Molecular weight calculated value 5943.0, mass spectrometry test molecular weight 5943.6.
III- 12合成：  III- 12 synthesis:
A用上述方法合成。 分子量计算值 6432.3,质谱测试分子量 6434.1。 室温下将多肽（643 mg ) 溶于 lOOmM Na2C03 (5 mL, pH 10)。 叔丁基十六烷二酰基 -L-Glu ( OSu ) -OtBu (68 mg)溶于 乙腈（5mL )， 加入多肽溶液。 搅拌 30分钟后， 用 50%醋酸酸化， 上 RP-HPLC C5柱纯化。 緩冲液 A: 0.1%TFA水溶液， 10%乙腈 緩冲液 B: 0.1%TFA水溶液， 80%乙腈。 初步纯化冻 干后的多肽加入 TFA/TIS/H20 ( 95:2.5:2.5, 10mL ), 30分钟后真空蒸发溶剂， 将粗产品溶于緩 冲液 A并冻干。 使用 RP-HPLC C5柱纯化。 分子量计算值 6829.8,质谱测试分子量 6831.6。 少 量产 物经 DTT 还原 和胰蛋 白 酶降解 ， 液相 色谱 - 质谱联用 观测 到 GPETLCGAHLVDALFFVCGDR (计算分子量 2220.6, 测试分子量 2221.4 )， GFYFNPTGK- [N8-(Na-(HOOC(CH2)14CO)-Y-Glu)] -GSSSAAPQT GIVDQCCFR (计算分子量 3236.7， 测试分 子量 3237.5 )。 因此， 确认脂肪酸结合在 C肽赖氨酸侧链。 经分析， 所得化合物即 111-12。 A was synthesized by the above method. The molecular weight calculated was 6432.3 and the mass spectrum was tested to be 6434.1. The polypeptide (643 mg) was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature. tert-Butylhexadecandioyl-L-Glu(OSu)-OtBu (68 mg) was dissolved in acetonitrile (5 mL) and added to the peptide solution. After stirring for 30 minutes, it was acidified with 50% acetic acid and purified on a RP-HPLC C5 column. Buffer A: 0.1% TFA in water, 10% acetonitrile buffer B: 0.1% TFA in water, 80% acetonitrile. The lyophilized polypeptide was initially purified and added to TFA/TIS/H 2 0 (95:2.5:2.5, 10 mL). After 30 minutes, the solvent was evaporated in vacuo to dissolve the crude product. Wash A and freeze dry. Purified using an RP-HPLC C5 column. The molecular weight calculated was 6829.8 and the mass spectrum was tested to have a molecular weight of 6831.6. A small amount of product was reduced by DTT and trypsin degradation. Liquid chromatography-mass spectrometry was used to observe GPETLCGAHLVDALFFVCGDR (calculated molecular weight 2220.6, molecular weight tested 2221.4), GFYFNPTGK- [N 8 -(N a -(HOOC(CH2) 14 CO)-Y -Glu)] -GSSSAAPQT GIVDQCCFR (calculated molecular weight 3236.7, tested molecular weight 3237.5). Therefore, it was confirmed that the fatty acid binds to the C-peptide lysine side chain. Upon analysis, the obtained compound was 111-12.
(CH300C(CH2)! iNHCO(CH2)3CO)-Glu(OSu)-OCH3合成 (CH300C(CH 2 )! iNHCO(CH 2 ) 3 CO)-Glu(OSu)-OCH 3 Synthesis
甲醇（ 40 ml )冰浴冷却， 滴加 S0C12 (4 ml)。 加入 12-氨基十二酸（ 3g, 13.9 mmol ), 搅 拌过夜。 过滤混合物并干燥， 得到 3g 12-氨基十二酸曱酯盐酸盐。 Methanol (40 ml) was cooled in an ice bath, and S0C1 2 (4 ml) was added dropwise. Add 12-aminododecanoic acid (3 g, 13.9 mmol) and stir overnight. The mixture was filtered and dried to give 3 g of 12-aminododecanoate hydrochloride.
1H-NMR ( DMSO-d6 ) δ: 7.97 (bs, 3H), 3.58(s,3H), 2.73(m,2H), 2.28 (t,2H)， 1.52 (m, 4H)， 1.25 (m， 14H)。 1H-NMR ( DMSO-d 6 ) δ: 7.97 (bs, 3H), 3.58 (s, 3H), 2.73 (m, 2H), 2.28 (t, 2H), 1.52 (m, 4H), 1.25 (m, 14H).
12-氨基十二酸曱酯盐酸盐（ lg， 3.8 mmol )悬浮于 THF(15 ml), 加入戊二酸肝 ( 1.29g, 3.8 mmol )和 TEA ( 0.52 ml, 3.8 mmol )， 室温搅拌过夜。 加水（ 75 ml )搅拌 1小时后过滤， 用 水洗涤， 干燥后得到 lg l2-(4-羧基丁酰胺基)十二酸曱酯。  12-aminododecanoate hydrochloride ( lg, 3.8 mmol) was suspended in THF (15 ml), glutaric acid ( 1.29 g, 3.8 mmol) and TEA (0.52 ml, 3.8 mmol) were added and stirred at room temperature overnight. . After adding water (75 ml) for 1 hour, it was filtered, washed with water and dried to give lg l2-(4-carboxybutyryl) decanoate.
1H-NMR ( DMSO-d6 ) δ: 12 (bs, 1H), 7.73 (t, 1H), 3.57 (s, 3H), 3.00 (q， 2H)， 2.28 (t, 2H), 2.18 (t, 2H)， 2.06 (t, 2H)， 1.69 (p, 2H), 1.50 (p, 2H), 1.36 (p, 2H), 1.23 (m， 14H)。 1H-NMR ( DMSO-d 6 ) δ: 12 (bs, 1H), 7.73 (t, 1H), 3.57 (s, 3H), 3.00 (q, 2H), 2.28 (t, 2H), 2.18 (t, 2H), 2.06 (t, 2H), 1.69 (p, 2H), 1.50 (p, 2H), 1.36 (p, 2H), 1.23 (m, 14H).
12-(4-羧基丁酰胺基)十二酸曱酯（ 0.33g, 0.95 mmol )溶于无水 DMF/DCM(0.5 ml:0.5 ml), 加入 DCC ( 0.2g， 1 mmol) 和 N-羟基丁二酰亚胺（0.115 g, 1 mmol)。 混合物在室温搅拌过 夜。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HC1和饱和食盐水洗涤， 硫酸 4美干燥后， 减 压蒸发得到 OSu活化的 12-(4-羧基丁酰胺基)十二酸甲酯。 该化合物溶于 DMF ( 1.5 ml ), 加 入 DIEA ( 0.19g, 1.5 mmol )和 H-Glu-OMe (0.168g, 1.04 mmol), 室温搅拌过夜。 减压蒸发溶 剂后加入乙酸乙酯， 用 0.1 N HC1 和饱和食盐水洗涤， 硫酸镁干燥后， 减压蒸发得到 (CH3OOC(CH2)1 !NHCOCCH^jCOi-Glu-OCHj。 12-(4-Carboxybutyryl) decanoate (0.33 g, 0.95 mmol) dissolved in anhydrous DMF / DCM (0.5 ml: 0.5 ml), DCC (0.2 g, 1 mmol) and N-hydroxyl Succinimide (0.115 g, 1 mmol). The mixture was stirred at room temperature overnight. The mixture was filtered, and the filtrate was diluted with ethyl acetate. EtOAc (EtOAc) This compound was dissolved in DMF (1.5 mL), EtOAc (EtOAc m. After the solvent was evaporated under reduced pressure and ethyl acetate was added, washed with 0.1 N HC1 and saturated brine, dried over magnesium sulfate, evaporated under reduced pressure to give (CH 3 OOC (CH 2) 1! NHCOCCH ^ jCOi-Glu-OCHj.
1H-NMR ( DMSO-d6 ) δ: 12 ( bs, 1Η ) , 8.22 (d, 1H), 7.73 (t, 1H), 4.24 (m, 1H), 3.61 (s， 3H)， 3.57 (s， 3H)， 3.00 (q， 2H)， 2.27 (m, 4H)， 2.10 (t, 2H), 2.04 (t, 2H), 1.9 (m, 1H), 1.8 (m, 1H)， 1.68 (t, 2H), 1.50 (m, 2H), 1.36 (m， 2H), 1.23 (m, 14H)。 1H-NMR ( DMSO-d 6 ) δ: 12 ( bs, 1 Η ) , 8.22 (d, 1H), 7.73 (t, 1H), 4.24 (m, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 3.00 (q, 2H), 2.27 (m, 4H), 2.10 (t, 2H), 2.04 (t, 2H), 1.9 (m, 1H), 1.8 (m, 1H), 1.68 (t, 2H) ), 1.50 (m, 2H), 1.36 (m, 2H), 1.23 (m, 14H).
(CH3OOC(CH2)nNHCO(CH2)3CO)-Glu-OCH3 ( 0.36g, 0.36 mmol )溶于无水 DMF/DCM (0.5 ml: 0.5 ml), 加入 DCC (0.08 g, 0.4 mmol) 和 N-羟基丁二酰亚胺 （0.046 g, 0.4 mmol)。 混合物 在室温搅拌 24小时。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HC1和饱和食盐水洗涤， 硫酸镁干燥后， 减压蒸发得到 (CHsO CH nNHCC CH^CC -Gh^OSuHDCH (CH 3 OOC(CH 2 )nNHCO(CH 2 ) 3 CO)-Glu-OCH 3 (0.36 g, 0.36 mmol) dissolved in anhydrous DMF/DCM (0.5 ml: 0.5 ml), DCC (0.08 g, 0.4 Methyl) and N-hydroxysuccinimide (0.046 g, 0.4 mmol). The mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filtrate was diluted with ethyl acetate, washed with EtOAc EtOAc EtOAc EtOAc EtOAc.
1H-NMR ( DMSO-d6 ) δ: 8.27(d, 1H), 7.72 (t, 1H)， 4.31 (m, 1H), 3.63 (s, 3H), 3.57 (s, 3H),1H-NMR ( DMSO-d 6 ) δ: 8.27 (d, 1H), 7.72 (t, 1H), 4.31 (m, 1H), 3.63 (s, 3H), 3.57 (s, 3H),
3.00 (q, 2H), 2.81 (s， 4H), 2.28 (t, 2H), 2.12 (t, 2H)， 2.05 (t, 2H), 1.70 (m, 2H), 1.50 (m, 2H)， 1.35 (m, 2H), 1.23 (m, 14H)。 3.00 (q, 2H), 2.81 (s, 4H), 2.28 (t, 2H), 2.12 (t, 2H), 2.05 (t, 2H), 1.70 (m, 2H), 1.50 (m, 2H), 1.35 (m, 2H), 1.23 (m, 14H).
III- 10合成  III- 10 synthesis
用上述 A、 B链连接方法合成多肽。 计算分子量 5746.7, 质谱测试分子量 5748.0。 室温 下将多肽（58 mg)溶于 lOOmM Na2C03 (5 mL， pH 10)。 （CH3OOC(CH2)uNHCO(CH2)3CO)- Glu(OSu)-OCH3 (7 mg)溶于乙腈（ 5mL ), 加入多肽溶液。 搅拌 30分钟后， 用冰浴冷却， 加入 0.1N NaOH,搅拌 1小时后用醋酸酸化，上 RP-HPLC C5柱纯化。緩沖液 A: 0.1%TFA水溶液， 10%乙腈 緩冲液 B: 0.1%TFA水溶液， 80%乙腈。 分子量计算值 6187.2, 质谱测试分子量 6188.7。 经分析， 所得化合物即 111-10。 The polypeptide was synthesized by the above A and B chain ligation methods. The molecular weight was calculated to be 5746.7, and the molecular weight of the mass spectrum was 5748.0. The polypeptide (58 mg) was dissolved in 100 mM Na 2 CO 3 (5 mL, pH 10) at room temperature. (CH 3 OOC(CH 2 ) u NHCO(CH 2 ) 3 CO)-Glu(OSu)-OCH 3 (7 mg) was dissolved in acetonitrile (5 mL), and the peptide solution was added. After stirring for 30 minutes, it was cooled with an ice bath, added with 0.1N NaOH, stirred for 1 hour, acidified with acetic acid, and purified on a RP-HPLC C5 column. Buffer A: 0.1% TFA in water, 10% acetonitrile buffer B: 0.1% TFA in water, 80% acetonitrile. The molecular weight calculated was 6187.2, and the mass spectrum was tested to have a molecular weight of 6188.7. Upon analysis, the obtained compound was 111-10.
OSu-CO-(CH2CH20)5-(CH2)2-NH-[Na-(HOOC (CH2)I6 CO)-Y-G1U]合成 十八烷二酸（ 2.5g 8.0 mmol ) 悬浮于 DCM ( 60 ml ), 加入三乙胺（ 1.16 ml, 8.3 mmol ) 并用冰浴冷却。在氮气环境中滴加氯曱酸苄基酯（ 1.14 ml )，再加入 DMAP(0.097g 0.8 mmol)。 搅拌 30分钟后， 减压蒸发溶剂， 将粗品用硅胶柱纯化（乙酸乙酯： 庚烷 1 :7-1 :1 ), 蒸发溶剂 后得到十八烷二酸一苄酯（1.12g, 35% )。 Synthesis of OSu-CO-(CH 2 CH 2 0) 5 -(CH 2 ) 2 -NH-[N a -(HOOC (CH 2 )I 6 CO)-Y-G1U] Octadecanedioic acid (2.5 g 8.0 mmol) was suspended in DCM (60 mL). Benzyl chlorate (1.14 ml) was added dropwise under a nitrogen atmosphere, followed by DMAP (0.097 g, 0.8 mmol). After stirring for 30 minutes, the solvent was evaporated.jjjjjjjjjjjjjjjjjjjjjjjjj ).
1H-NMR ( CDC13 ) δ: 7.35 (m, 5H), 5.11 (s, 2H), 2.35 (t, 4H)， 1.63 (t 4H), 1.30-1.22 (m, 24)。 十八烷二酸一苄酯 (0.8g, 2 mmol) 溶于 DMF ( 3 ml )和 DCM ( 3 ml ), 用冰浴冷却。 加入 DCC (0.408 g, 2 mmol) 和 N-羟基丁二酰亚胺 （0.23 g, 2 mmol)。 混合物在室温搅拌 24小时。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HC1和饱和食盐水洗涤， 硫酸镇干燥后， 减压蒸 发得到琥珀酰亚胺基一苄基十八烷二酸酯。 1H-NMR (CDC1 3 ) δ: 7.35 (m, 5H), 5.11 (s, 2H), 2.35 (t, 4H), 1.63 (t 4H), 1.30-1.22 (m, 24). Phenyl octadecanoate (0.8 g, 2 mmol) was dissolved in DMF (3 mL) and DCM (3 mL). DCC (0.408 g, 2 mmol) and N-hydroxysuccinimide (0.23 g, 2 mmol) were added. The mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filtrate was diluted with ethyl acetate, washed with EtOAc EtOAc EtOAc EtOAc.
Ή-NMR ( CDC13 ) 5: 7.35 (m 5H 5.11 (s, 2H 2.83 (s, 4H), 2.60 (t 2H), 2.35 (t 2H),Ή-NMR (CDC1 3 ) 5: 7.35 (m 5H 5.11 (s, 2H 2.83 (s, 4H), 2.60 (t 2H), 2.35 (t 2H),
1.80-1.60 (m, 4H)， 1.40-1.20 (m, 24H)„ 1.80-1.60 (m, 4H), 1.40-1.20 (m, 24H) „
琥珀酰亚胺基一苄基十八烷二酸酯 (95 mg, 0.19 mmol)溶于 DMF(1.5 ml),加入 L-Glu-OBzl ( 49 mg, 0.21 mmol )和 DIEA(52 μΐ, 0.3 mmol),搅拌 16小时。减压蒸发溶剂，加入乙酸乙酯， 用 0.1N HC1、 饱和食盐水洗涤， 硫酸镁千燥后减压蒸发溶剂， 得到 BzlO-十八烷二酰基 -L-Glu-OBzl。  Succinimidyl-benzyl octadecanedioate (95 mg, 0.19 mmol) was dissolved in DMF (1.5 ml). EtOAc (EtOAc, m. ), stirring for 16 hours. The solvent was evaporated under reduced pressure, and ethyl acetate was evaporated. EtOAcjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj
'H-NMR( CDC13 )δ: 7.35(m, 5H 6.22(d, 2H), 5.17(s, 2H), 5.11(s, 2H), 4.71 (m, 1H 2.37 (m, 4H), 2.22(m, 3H 1.98 (m, 1H), 1.63 (m, 4H), 1.31-1.20 (m, 24H 'H-NMR(CDC1 3 ) δ: 7.35 (m, 5H 6.22(d, 2H), 5.17(s, 2H), 5.11(s, 2H), 4.71 (m, 1H 2.37 (m, 4H), 2.22 ( m, 3H 1.98 (m, 1H), 1.63 (m, 4H), 1.31-1.20 (m, 24H
BzlO-十八烷二酰基 -L-Glu-OBzl(110 mg, 0.18 mmol)溶于 DMF ( 1 ml )和 DCM ( 1 ml ), 用水浴冷却。 加入 DCC (41 mg， 0.2 mmol) 和 N-羟基丁二酰亚胺（23 mg， 0.2 mmol)。 混合物 在室温搅拌 12小时。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HC1和饱和食盐水洗涤， 硫酸镁干燥后， 减压蒸发得到 BzlO-十八烷二酰基 -L-Glu(OSu)-OBzl。  BzlO-octadecandioyl-L-Glu-OBzl (110 mg, 0.18 mmol) was dissolved in DMF (1 ml) and DCM (1 ml). DCC (41 mg, 0.2 mmol) and N-hydroxysuccinimide (23 mg, 0.2 mmol) were added. The mixture was stirred at room temperature for 12 hours. The mixture was filtered, and the filtrate was diluted with ethyl acetate. EtOAc EtOAcjjjjjjjjj
1H-NMR ( CDC13 ) δ: 7.36(m, 5H 6.40(d, 2H 5.19(s, 2H 5.11(s 2H), 4.75(m, 1H 2.82(s, 4H), 2.68(m, 1H), 2.59(m, 1H), 2.35(t, 2H 2.19(t, 2H), 1.62(m, 4H 1.32-1.21(m, 24H 1H-NMR (CDC1 3 ) δ: 7.36 (m, 5H 6.40 (d, 2H 5.19 (s, 2H 5.11 (s 2H), 4.75 (m, 1H 2.82 (s, 4H), 2.68 (m, 1H), 2.59 (m, 1H), 2.35(t, 2H 2.19(t, 2H), 1.62(m, 4H 1.32-1.21(m, 24H
BzlO-十八烷二酰基 -L-Glu(OSu)-OBzl(72mg,0.1 ol)和 H2N-(CH2)2-(OCH2CH2)5COOH (31mg，0.1mmol)溶于 DMF/DCM(0.5 ml:1.5ml), 加入 DIEA(26 L， 0.15 mmol), 搅拌 16小时。 减压蒸发溶剂， 加入乙酸乙酯， 用 0.1N HC1、 饱和食盐水洗涤， 硫酸 4美干燥后减压蒸发溶剂， 得到 3-[2-[2-[2-[2-[2-[[5-苄氧基 -4-[(18-苄氧基 -18-氧代-十八烷酰基)氨基] -5-氧代- 戊酰基]氨 基 ] 乙 氧 基 ] 乙 氧 基 ] 乙 氧 基 ] 乙 氧 基 ] 乙 氧 基 ] 丙 酸 ( 3-[2-[2-[2-[2-[2-[[5-benzyloxy-4-[(18-benzyloxy-18-oxo-octadecanoyl)amino]-5-oxo- pentanoyl] amino] ethoxy] ethoxy] ethoxy]ethoxy] ethoxy]propanoic acid )。 计算分子量 915.2 , 测试分子量 916.5 ο BzlO-octadecandioyl-L-Glu(OSu)-OBzl (72 mg, 0.1 ol) and H 2 N-(CH 2 ) 2 -(OCH 2 CH 2 ) 5 COOH (31 mg, 0.1 mmol) were dissolved in DMF /DCM (0.5 ml: 1.5 ml), DIEA (26 L, 0.15 mmol). The solvent was evaporated under reduced pressure, ethyl acetate was added, washed with EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc 5-benzyloxy-4-[(18-benzyloxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy Ethoxy]ethoxy]propionic acid (3-[2-[2-[2-[2-[2-[[5-benzyloxy-4-[(18-benzyloxy-18-oxo-octadecanoyl)amino ]-5-oxo- pentanoyl] amino] ethoxy] ethoxy] ethoxy]ethoxy] ethoxy]propanoic acid ). Calculated molecular weight 915.2, tested molecular weight 916.5 ο
3-[2-[2-[2-[2-[2-[[5-苄氧基 -4-[(18-苄氧基 -18-氧代-十八烷酰基)氨基] -5-氧代- 戊酰基]氨基] 乙氧基]乙氧基]乙氧基]乙氧基]乙氧基]丙酸 (91mg, O.lmmol)溶于 DMF( 1 ml )和 DCM( 1 ml ), 用水浴冷却。 加入 DCC (24.7mg, 0.12 mmol) 和 N-羟基丁二酰亚胺 （13.8 mg 0.12 mmol)。 混 合物在室温搅拌 12小时。 过滤混合物， 滤液用乙酸乙酯稀释， 用 0.1 N HC1和饱和食盐水洗 涤， 硫酸 4美干燥后， 减压蒸发得到苯曱基 18-[[1-苄氧羰基- 4-[2- [2-[2- [2-[2-[3-(2,5-二氧代吡 咯烷 -1-yl)氧 -3-氧代-丙氧基]乙氧基]乙氧基]乙氧基]乙氧基]乙氨基 ]-4-氧代-丁基]氨基] -18-氧 代-硬脂酸盐（benzyl 18-[[l-benzyloxycarbonyl- 4-[2- [2-[2- [2-[2-[3-(2,5-dioxopyrrolidin-l-yl) oxy-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo-butyl]amino]-18-oxo-octadec anoate )。 计算分子量 1012.2， 测试分子量 1013.1。 苯甲基 18-[[1-苄氧羰基- 4-[2- [2-[2- [2-[2-[3-(2,5-二氧代吡咯烷 -1-yl)氧 -3-氧代-丙氧基] 乙 氧基] 乙氧基] 乙氧基] 乙氧基]乙氨基 ]-4-氧代-丁基]氨基] -18-氧代-硬脂酸盐 (51mg, 0.05mmol)溶于曱醇 /丙酮 /0.1% TFA,加入 Pd/C， 在氮气环境下室温搅拌 5小时， 通过硅藻土 过滤， 从庚烷中沉淀并蒸发残余溶剂， 得到 18-[[1-羧基 -4-[2-[2-[2-[2- [2-[3-(2，5-二氧代吡咯烷 -1-yl)氧 -3-氧代-丙氧基]乙氧基]乙氧基]乙氧基]乙氧基]乙氨基 ]_4-氧代-丁基]氨基] -I8-氧代-硬 脂酸（ 18-[[l-carboxy-4-[2-[2-[2-[2- [2-[3-(2,5- dioxopyrrolidin- 1 -yl)oxy-3-oxo-propoxy]ethoxy] ethoxy] ethoxy] ethoxy] ethylamino] -4-oxo-butyl] amino]- 18-oxo-octadecanoic acid )。 计算分子量 832.0, 测试分子量 833.4。 3-[2-[2-[2-[2-[2-[[5-Benzyloxy-4-[(18-benzyloxy-18-oxo-octadecanoyl)amino] -5- Oxo-pentanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propionic acid (91 mg, 0.1 mmol) dissolved in DMF (1 ml) and DCM (1 ml) , Cool in a water bath. DCC (24.7 mg, 0.12 mmol) and N-hydroxysuccinimide (13.8 mg 0.12 mmol) were added. The mixture was stirred at room temperature for 12 hours. The mixture was filtered, and the filtrate was diluted with ethyl acetate. EtOAc (EtOAc)EtOAc -[2-[2-[2-[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propoxy]ethoxy]ethoxy]ethoxy Ethoxy]ethylamino]-4-oxo-butyl]amino] -18-oxo-stearate (benzyl 18-[[l-benzyloxycarbonyl- 4-[2- [2-[2- [2-[2-[3-(2,5-dioxopyrrolidin-l-yl) oxy-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo-butyl]amino]-18- Oxo-octadec anoate ). The molecular weight was calculated to be 1012.2 and the molecular weight was tested to be 1013.1. Benzyl 18-[[1-benzyloxycarbonyl-4-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrolidin-1-yl)oxy-) 3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo-butyl]amino]-18-oxo-stearate 51 mg, 0.05 mmol) was dissolved in methanol/acetone/0.1% TFA, added to Pd/C, stirred at room temperature under nitrogen for 5 hours, filtered through celite, precipitated from heptane and evaporated. [1-carboxy-4-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxo-propoxy) Ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]_ 4 -oxo-butyl]amino] -I 8 -oxo-stearic acid ( 18-[[l-carboxy- 4-[2-[2-[2-[2-[2-[3-(2,5- dioxopyrrolidin- 1 -yl)oxy-3-oxo-propoxy]ethoxy] ethoxy] ethoxy] ethoxy] ethylamino] - 4-oxo-butyl] amino]- 18-oxo-octadecanoic acid ). The molecular weight was calculated to be 832.0 and the molecular weight was measured to be 833.4.
III-36合成  III-36 synthesis
用上述 Α、 Β链连接方法合成多肽。 计算分子量 5531.4， 质谱测试分子量 5533.7。 室温下 将多肽（ 56 mg, 10 μηιοΐ)溶于 lOOmM Na2C03 (1 ml, pH 10)。 18-[[1-羧基 -4- [2-[2-[2-[2-[2- [3- (2，5_二氧代吡咯烷 -1-yl)氧 -3_氧代-丙氧基]乙氧基]乙氧基]乙氧基]乙氧基]乙氨基 ]-4-氧代-丁 基]氨基] -18-氧代-硬脂酸 (9.2 mg, 11 μπιοΐ)溶于乙腈（ 0.5 ml )， 加入多肽溶液。 搅拌 30分钟后 用醋酸酸化， 上 RP-HPLC C5 柱纯化。 緩冲液 A: 0.1%TFA 水溶液， 10%乙腈 緩冲液 B:0.1%TFA水溶液， 80%乙腈。 分子量计算值 6428.3， 质谱测试分子量 6430.2。 经分析， 所 得化合物即 111-36。 多肽的聚乙二醇化 方法（PEGylation): The polypeptide is synthesized by the above-described hydrazone and hydrazone linkage methods. The molecular weight was calculated to be 5531.4, and the mass spectrum was tested to be 5533.7. The polypeptide (56 mg, 10 μηιοΐ) was dissolved in 100 mM Na 2 CO 3 (1 ml, pH 10) at room temperature. 18-[[1-carboxy-4-[2-[2-[2-[2-[2-[3-( 2 , 5 _dioxopyrrolidine-1-yl)oxy- 3 oxo-) Propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]- 4 -oxo-butyl]amino] -18-oxo-stearic acid (9.2 mg, 11 μπιοΐ) Dissolved in acetonitrile (0.5 ml) and added to the peptide solution. After stirring for 30 minutes, it was acidified with acetic acid and purified on a RP-HPLC C5 column. Buffer A: 0.1% TFA in water, 10% acetonitrile buffer B: 0.1% TFA in water, 80% acetonitrile. The molecular weight calculated was 6428.3, and the mass spectrum was tested to be 6430.2. Upon analysis, the obtained compound was 111-36. Pegylation of polypeptides (PEGylation):
a)还原坑基化 ( reductive alkylation )  a) reductive alkylation
多肽, mPEG20K-CHO，氰基硼氢化钠 (NaBH3CN)按 1:2:45比例溶于 pH 4.3醋酸溶液（ 0.1M NaCl，0.2 M CH3COOH, 0.1M Na2CO3 )。多肽浓度为 0.5-1 mg mL。反应用 HPLC检测和纯化。 产率约 55%。 还原烷基化反应可以将聚乙二醇选择性地结合在 B1位。 The polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH 3 CN) was dissolved in a pH 4.3 acetic acid solution (0.1 M NaCl, 0.2 M CH 3 COOH, 0.1 M Na 2 CO 3 ) in a ratio of 1:2:45. The polypeptide concentration is 0.5-1 mg mL. The reaction was detected and purified by HPLC. The yield is about 55%. The reductive alkylation reaction can selectively bind polyethylene glycol to the B1 position.
III- 1合成方法：  III-1 synthesis method:
A(Q5，S12,N18):B(H9，F15,P27)合成方法同上。 B1 还原烷基化得到产物。 分子量计算值 25576.4, 质谮测试得到一宽峰， 中间分子量 25580.2， 少量产物经 DTT还原， 液相色谱-质 谱联用观测到 A(Q5,S12，N18) (分子量计算值 2436.9，质谱测试分子量 2438.0 ) 和 G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT (分子量计算值 23144.6，质谱测试得 到一宽峰， 中间分子量 23140.5 )， 没有分子量 22436.9。 经分析， 所得化合物即 111-1。 A (Q5, S12, N18): B (H9, F15, P27) synthesis method is the same as above. B1 reductive alkylation gives the product. The molecular weight calculated was 25576.4, and the mass enthalpy test obtained a broad peak with an intermediate molecular weight of 25580.2. A small amount of the product was reduced by DTT. A (Q5, S12, N18) was observed by liquid chromatography-mass spectrometry (molecular weight calculated value 2436.9, mass spectrometry molecular weight 2438.0 And G(N a -PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT (molecular weight calculated value 23144.6, mass spectrometry test gave a broad peak, intermediate molecular weight 23140.5), no molecular weight 22436.9. Upon analysis, the obtained compound was 111-1.
b) NHS酯 (N-羟基琥珀酰亚胺)酰化  b) NHS ester (N-hydroxysuccinimide) acylation
多肽和 mPEG20K-NHS按摩尔比 1： 1溶于 0.1N Ν,Ν-双 (2-羟乙基)甘氨酸溶液（ pH 8 ), 多肽浓度 0.5mg/mL。 反应在室温进行 2小时， 用 HPLC纯化。 产率约 90%。  The polypeptide and mPEG20K-NHS molar ratio 1:1 was dissolved in 0.1N Ν, Ν-bis(2-hydroxyethyl)glycine solution (pH 8), and the peptide concentration was 0.5 mg/mL. The reaction was carried out at room temperature for 2 hours and purified by HPLC. The yield is about 90%.
III-2合成方法：  III-2 synthesis method:
A(Q5,S12,N18):B( H9,F15,P27,K28, desT )用上述 A、 B链连接方法合成。计算分子量 5505.4, 质语测试分子量 5506.8。 多肽与 mPEG20K-NHS反应后得到 111-2。 分子量计算值 25505.4, 质 谱测试得到一宽峰， 中间分子量 25501.7。 少量产物经 DTT还原和胰蛋白酶降解 B链， 液相 色谱 -质谱联用观测到 GPETLCGAH LVDALFFVCGDR (分子量计算值 2220.6, 质谱测试分 子量 2221.7 )， 没有观测到 GFYFNPK (分子量计算值 872.0 ), 但质谱观测到一宽峰， 中间分 子量 20871.9。 经分析， 所得化合物即 111-2。  A (Q5, S12, N18): B (H9, F15, P27, K28, desT) was synthesized by the above A and B chain linkage methods. The molecular weight was calculated to be 5505.4, and the molecular weight of the test was 5506.8. The polypeptide is reacted with mPEG20K-NHS to give 111-2. The molecular weight calculated was 25505.4, and the mass spectrum test gave a broad peak with an intermediate molecular weight of 25501.7. A small amount of product was subjected to DTT reduction and trypsin degradation of the B chain. GPETLCGAH LVDALFFVCGDR (molecular weight calculated value 2220.6, mass spectrometry molecular weight 2221.7) was observed by liquid chromatography-mass spectrometry. No GFYFNPK (molecular weight calculated value 872.0) was observed, but mass spectrometry was observed. A broad peak with an intermediate molecular weight of 2,087,1.9. Upon analysis, the obtained compound was 111-2.
III-4的合成方法：  Synthesis method of III-4:
与上述 ΙΠ-2基本相同， 只是 C2Lys的侧链氨基与 mPEG20K-NHS反应， 得到 111-4。 分子 量计算值 26600.5， 质谱测试得到一宽峰， 中间分子量 26604.5。 少量产物经 DTT还原和胰蛋 白酶降解 B链， 液相色谱 -质潘联用观测到 GPETLCGAHLVDALFFVCGDR (分子量计算值 2220.6,测试分子量 2220.8 )和 GFYFNPPT-GK(N£-PEG 20K)-GSSSAAAPQTGIVDQCCFR (分 子量计算值 23007.4， 测试得到一宽峰， 中间分子量 23010.0 )。 经分析， 所得化合物即 111-4。 Basically the same as the above ΙΠ-2, except that the side chain amino group of C2Lys reacts with mPEG20K-NHS to obtain 111-4. Molecule The calculated value was 26600.5, and the mass spectrometry gave a broad peak with an intermediate molecular weight of 26604.5. A small amount of product was subjected to DTT reduction and trypsin degradation of the B chain, and liquid chromatography-mass spectroscopic observation of GPETLCGAHLVDALFFVCGDR (molecular weight calculated value 2220.6, molecular weight 2220.8) and GFYFNPPT-GK (N £ -PEG 20K)-GSSSAAAPQTGIVDQCCFR (molecular weight calculated value) 23007.4, the test yielded a broad peak with an intermediate molecular weight of 23010.0). Upon analysis, the obtained compound was 111-4.
合成 Al， B27-diBoc-IGF-l类似物  Synthesis of Al, B27-diBoc-IGF-l analogues
A(Gl-Na-Boc， Q5,S12,N18): B(H9, F15, K27-NE-Boc) A(Gl-N a -Boc, Q5,S12,N18): B(H9, F15, K27-N E -Boc)
1-15 A(Q5,S12,N18): B(H9, F15) 用以上通用方法合成。 计算分子量 5606.5, 质谱测试分 子量 5607.3。 多肽（100 mg, 17.8μπιο1 )溶于水（1 mL )、 IN NaHC03 ( 0.3 mL )和 DMF ( 3 mL )。 加入 t-Boc-叠氮化物 （ 5.5mg， 37.5μπιο1) 。 混合物在 40。C搅拌 3小时， 加入 50% 醋酸（0.35 mL )终止反应。 未反应的 t-Boc-azide用乙瞇萃取（2 X 15 mL )。 水层真空冷冻 干燥。 粗品含有 Boc单取代， 双取代和三取代多肽。 混合物用 SP-Sephadex C-25离子交换柱 纯化。 离子交换柱先用含有 6M尿素的 1.5 M 醋酸平衡， 多肽洗提流速是 48 mL/h, 线性梯 度 0.04-0.4 M 氯化钠 / 1000 mL 6M尿素的 1.5 M 醋酸。 Di-t-Boc 多肽进一步用 DEAE- Sephadex A-25 柱纯化。 色谙柱预先用含有 7M尿素的 0.01 M Tris緩沖液（pH 8.5)平衡。洗提 流速 35 mL/h， 梯度 0.14-0.28 M 氯化钠 /100 mL Tris緩沖液。 分子量计算值 5806.7, 质谙测 试分子量 5808.3。少量多肽溶于 0.05M NH4HCO3/20%ACN, 用 DTT还原 10分钟后质谱分析。 A(Gl-Na-Boc, Q5,S12,N18) 分子量计算值 2537.0 , 质谱测试分子量 2537.9。 B(H9， F15 , K27-Ns-Boc) 分子量计算值 3275.8 , 盾谱测试分子量 3276.0。 胰蛋白酶解后， 含 Boc片段的 分子量计算值 1073.2, 质譜测试分子量 1074.5。 Al, B27-di-Boc多肽的 B1氨基可以与聚乙二 醇、 白蛋白、 脂肪酸等结合形成长效多肽。 1-15 A (Q5, S12, N18): B (H9, F15) was synthesized by the above general method. The molecular weight was calculated to be 5606.5, and the molecular weight of the mass spectrum was 5607.3. The polypeptide (100 mg, 17.8 μπιο1) was dissolved in water (1 mL), IN NaHC0 3 (0.3 mL) and DMF (3 mL). t-Boc-azide (5.5 mg, 37.5 μπιο1) was added. The mixture is at 40. C was stirred for 3 hours, and the reaction was quenched by the addition of 50% acetic acid (0.35 mL). Unreacted t-Boc-azide was extracted with acetonitrile (2 X 15 mL). The aqueous layer was vacuum freeze dried. The crude product contains Boc monosubstituted, disubstituted and trisubstituted polypeptides. The mixture was purified using an SP-Sephadex C-25 ion exchange column. The ion exchange column was first equilibrated with 1.5 M acetic acid containing 6 M urea, the peptide elution flow rate was 48 mL/h, and the linear gradient was 0.04-0.4 M sodium chloride / 1000 mL 6 M urea in 1.5 M acetic acid. The Di-t-Boc polypeptide was further purified using a DEAE-Sephadex A-25 column. The color column was previously equilibrated with 0.01 M Tris buffer (pH 8.5) containing 7 M urea. The elution flow rate was 35 mL/h, gradient 0.14-0.28 M sodium chloride/100 mL Tris buffer. The molecular weight calculated was 5806.7, and the molecular weight was 5808.3. A small amount of the polypeptide was dissolved in 0.05 M NH 4 HCO 3 /20% ACN, and subjected to mass spectrometry after reduction with DTT for 10 minutes. A (Gl-N a -Boc, Q5, S12, N18) The calculated molecular weight was 2537.0, and the molecular weight of the mass spectrum was 2537.9. B (H9, F15, K27-N s -Boc) The calculated molecular weight was 3275.8, and the molecular weight of the shield test was 3276.0. After trypsinization, the molecular weight of the Boc-containing fragment was calculated to be 1073.2, and the molecular weight of the mass spectrum was 1074.5. The B1 amino group of the Al, B27-di-Boc polypeptide can be combined with polyethylene glycol, albumin, fatty acid or the like to form a long acting polypeptide.
111-30的合成。  Synthesis of 111-30.
A(Gl-Na-Boc， Q5,S12,N18): B(H9, F15 , K27-Ns-Boc)合成方法如上。 多肽 (58 mg) 溶于 DMF (3 mL), 加入 Mal-dPEG12-NHS(8.7mg) ( Quanta Biodesign ) 和三乙胺 (30 L)。 反应在 室温下搅拌 2小时。 减压挥发溶剂后， 加入 TFA (2 mL)和 TIS ( ΙΟΟμΙ )。 搅拌 10分钟后， 挥 发 TFA。 粗品溶于 H20/ACN (3: 1)用 RP- HPLC纯化。 分子量计算值 6556.5 , 质谱测试分子量 6558.0。马来酰亚胺多肽溶于纯净水，多肽浓度 10 mM。加入人白蛋白 （665 mg),在 37 °C 培 养 30分钟。 然后用含有 5 mM辛酸钠和 750 mM硫酸铵的 20 mM磷酸钠溶液稀释到 5%。用 凝胶过滤层析法除去未反应的试剂， 洗脱液为 0.05M碳酸氢铵水溶液。 真空冷冻干燥后得到 纯品。 化合物分子量计算值 73028.7, 质谱测试分子量 73030.2, 经分析为化合物 111-30。 A (Gl-N a -Boc, Q5, S12, N18): The synthesis method of B (H9, F15, K27-N s -Boc) is as above. The polypeptide (58 mg) was dissolved in DMF (3 mL), and Mal-dPEG12-NHS (8.7 mg) (quanta Biodesign) and triethylamine (30 L) were added. The reaction was stirred at room temperature for 2 hours. After evaporating the solvent under reduced pressure, TFA (2 mL) and TIS ( ΙΟΟμΙ) were added. After stirring for 10 minutes, TFA was evaporated. The crude material was dissolved in H 2 0/ACN (3: 1) and purified using RP-HPLC. The molecular weight was calculated to be 6556.5 and the mass spectrum was tested to have a molecular weight of 6558.0. The maleimide polypeptide is dissolved in purified water at a polypeptide concentration of 10 mM. Human albumin (665 mg) was added and incubated at 37 °C for 30 minutes. It was then diluted to 5% with a 20 mM sodium phosphate solution containing 5 mM sodium octoate and 750 mM ammonium sulfate. The unreacted reagent was removed by gel filtration chromatography, and the eluent was a 0.05 M aqueous solution of ammonium hydrogencarbonate. Purely obtained after vacuum freeze drying. The calculated molecular weight of the compound was 73028.7, and the mass spectrum was tested to have a molecular weight of 73030.2, which was analyzed as Compound 111-30.
合成结果:  Synthesis result:
按照上述方法分别合成各基于 IGF-1的修饰的化合物， 包括单链化合物和双链化合物， 通 过质语检测各化合物的结构， 结果如下：  Each of the IGF-1-based modified compounds, including single-stranded compounds and double-stranded compounds, was synthesized according to the above method, and the structures of the respective compounds were examined by traits. The results were as follows:
III-3: 分子量计算值 26673.6, 质谱测试得一宽峰， 中间分子量 26680.5， 经分析， 所得化 合物即 ΠΙ-3;  III-3: The calculated molecular weight is 26673.6, and the mass spectrum is tested to obtain a broad peak with an intermediate molecular weight of 26680.5. After analysis, the obtained compound is ΠΙ-3;
III-5: 分子量计算值 26618.5， 质谱测试得一宽峰， 中间分子量 26624.9， 经分析， 所得化 合物即 ΠΙ-5;  III-5: The calculated molecular weight is 26618.5, and the mass spectrum is tested to obtain a broad peak with an intermediate molecular weight of 26624.9. After analysis, the obtained compound is ΠΙ-5;
III-6: 分子量计算值 5845.8， 盾谱测试分子量 5847.2， 经分析， 所得化合物即 ΠΙ-6;  III-6: molecular weight calculated value 5845.8, shield test molecular weight 5847.2, after analysis, the obtained compound is ΠΙ-6;
III-8: 分子量计算值 6172.2, 质谱测试分子量 6173.4, 经分析， 所得化合物即 ΠΙ-8;  III-8: molecular weight calculated value 6172.2, mass spectrometry test molecular weight 6173.4, after analysis, the obtained compound is ΠΙ-8;
III-9: 分子量计算值 6116.1 , 质谱测试分子量 6118.2, 经分析， 所得化合物即 ΠΙ-9;  III-9: molecular weight calculated value 6116.1, mass spectrometry test molecular weight 6118.2, after analysis, the obtained compound is ΠΙ-9;
111-11 : 分子量计算值 6273.3 , 质谱测试分子量 6274.5， 经分析， 所得化合物即 ΠΙ-11 ; 111-13: 分子量计算值 6829.8 质谱测试分子量 6830.4 经分析， 所得化合物即 III- 13 ΠΙ-14: 分子量计算值 6829.8 质谱测试分子量 6831.1 经分析， 所得化合物即 ΙΠ-14 111-15: 分子量计算值 6845.8 质錯测试分子量 6846.9 经分析， 所得化合物即 ΙΠ-15 111-16: 分子量计算值 6972.0 质谱测试分子量 6973.3 经分析， 所得化合物即 ΠΙ-16 111-17: 分子量计算值 6457.4 质语测试分子量 6458.0 经分析， 所得化合物即 ΙΠ-17 111-18: 分子量计算值 6730.7 质谱测试分子量 6732.2 经分析， 所得化合物即 ΠΙ-18 111-19: 分子量计算值 5902.9 质谱测试分子量 5903.4 经分析， 所得化合物即 ΙΠ-19 111-20: 分子量计算值 6661.6 质谱测试分子量 6662.7 经分析， 所得化合物即 ΠΙ-20 111-21 : 分子量计算值 6186.2 质谱测试分子量 6187.8 经分析， 所得化合物即 111-21 111-22: 分子量计算值 5944.9 质谱测试分子量 5946.0 经分析， 所得化合物即 ΠΙ-22 ΙΙΙ-23: 分子量计算值 6101.1 质谱测试分子量 6101.6 经分析， 所得化合物即 ΠΙ-23 111-24: 分子量计算值 6257.3 质谱测试分子量 6258.9 经分析， 所得化合物即 ΠΙ-24 111-25: 分子量计算值 6342.4 质谱测试分子量 6344.1 经分析， 所得化合物即 ΙΠ-25 111-26: 分子量计算值 6471.5 质谱测试分子量 6472.3 经分析， 所得化合物即 ΠΙ-26 111-27: 分子量计算值 6528.6 质谱测试分子量 6529.1 经分析， 所得化合物即 ΙΠ-27 111-31 : 分子量计算值 6241.3 质借测试分子量 6242.4 经分析， 所得化合物即 ΠΙ-31 111-32: 分子量计算值 6101.1 质谱测试分子量 6101.8 经分析， 所得化合物即 ΙΠ-32 111-33: 分子量计算值 6230.2 质谱测试分子量 6231.5 经分析， 所得化合物即 ΙΠ-33 111-34: 分子量计算值 6287.3 质语测试分子量 6288.2 经分析， 所得化合物即 ΙΠ-34: ΙΠ -35: 分子量计算值 26462.3 质谱测试得一宽峰， 中间分子量 26470.1， 经分析， 所得 化合物即 ΠΙ -35; 111-11: molecular weight calculated value 6273.3, mass spectrometry test molecular weight 6274.5, after analysis, the obtained compound is ΠΙ-11; 111-13: Molecular weight calculated value 6829.8 Mass spectrometry test molecular weight 6830.4 After analysis, the obtained compound is III- 13 ΠΙ-14: Molecular weight calculated value 6829.8 Mass spectrometry test molecular weight 6831.1 After analysis, the obtained compound is ΙΠ-14 111-15: Molecular weight calculated value 6845.8 Mass error test molecular weight 6846.9 After analysis, the obtained compound is ΙΠ-15 111-16: Molecular weight calculated value 6972.0 Mass spectrometry test molecular weight 6973.3 After analysis, the obtained compound is ΠΙ-16 111-17: Molecular weight calculated value 6457.4 Qualitative test molecular weight 6458.0 After analysis , the obtained compound is ΙΠ-17 111-18: Molecular weight calculated value 6730.7 Mass spectrometry test molecular weight 6732.2 After analysis, the obtained compound is ΠΙ-18 111-19: molecular weight calculated value 5902.9 mass spectrometry test molecular weight 5903.4 After analysis, the obtained compound is ΙΠ-19 111 -20: Molecular weight calculated 6661.6 Mass spectrometry test molecular weight 6662.7 After analysis, the obtained compound was ΠΙ-20 111-21 : Molecular weight calculated value 6186.2 Mass spectrometry test molecular weight 6187.8 After analysis, the obtained compound was 111-21 111-22: Molecular weight calculated value 5944.9 Mass spectrum Test molecular weight 5946.0 After analysis, the obtained compound is ΠΙ-22 ΙΙΙ-23: Calculated molecular weight 6101.1 Mass spectrometry Test molecular weight 6101.6 After analysis, the obtained compound is ΠΙ-23 111-24: Calculated molecular weight 6257.3 Mass spectrometry molecular weight 6258.9 After analysis, the obtained compound is ΠΙ-24 111-25: Molecular weight calculated value 6342.4 Mass Spectrometry Test Molecular Weight 6344.1 After analysis, the obtained compound was ΙΠ-25 111-26: Molecular weight calculated value 6471.5 Mass spectrometry test molecular weight 6472.3 After analysis, the obtained compound was ΠΙ-26 111-27: Molecular weight calculated value 6528.6 Mass spectrometry test molecular weight 6529.1 After analysis, obtained The compound is ΙΠ-27 111-31 : molecular weight calculated value 6241.3. The molecular weight of the test is 6242.4. The obtained compound is ΠΙ-31 111-32: molecular weight calculated value 6101.1 mass spectrometry test molecular weight 6101.8 After analysis, the obtained compound is ΙΠ-32 111- 33: Molecular weight calculated value 6230.2 Mass spectrometry test molecular weight 6231.5 After analysis, the obtained compound is ΙΠ-33 111-34: Molecular weight calculated value 6287.3 Qualitative test molecular weight 6288.2 After analysis, the obtained compound is ΙΠ-34: ΙΠ -35: Molecular weight calculated value 26462.3 Mass spectrometry test gave a broad peak with an intermediate molecular weight of 26470.1. Analysis, i.e., the resultant compound ΠΙ -35;
III -37: 分子量计算值 7246.3， 质谱测试分子量 7248.0， 经分析， 所得化合物即 111-37。 受体结合分析  III -37: Calculated molecular weight 7246.3, mass spectrometry molecular weight 7248.0, analyzed, the resulting compound is 111-37. Receptor binding assay
1、 125I-IGF-1 和1251-胰岛素的制备 1, 125 I-IGF-1 and 125 1-insulin preparation
文献方法 ( Cresto等, "Preparation of biologically active mono-125I-insulin of high specific activity"， Acta Physiol Lat Am. 1981 , 31 ( 1 ) : 13 -24 ) Literature method (Cresto et al., "Preparation of biologically active mono- 125 I-insulin of high specific activity", Acta Physiol Lat Am. 1981, 31 (1): 13 -24)
2、 化合物的受体结合分析  2. Receptor binding analysis of compounds
文献方法 ( E. K. Frandsen and R. A. Bacchus. "New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells." Diabetes, 1987, 36, 3:335-340 )或下述方法之一。 如无特别说明， 受体制备方法亦如文献方 法， 使用人胎盘膜。 一般情况下， 胰岛素受体结合实验使用 0.025毫克胎盘膜； IGF-1受体结 合实验使用 0.2毫克胎盘膜。  EK Frandsen and RA Bacchus. "New, simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells." Diabetes, 1987, 36, 3: 335-340) or the following method One. Unless otherwise stated, the method of preparation of the receptor is also used as a literature method using a human placental membrane. In general, 0.025 mg of placental membrane was used for the insulin receptor binding assay; 0.2 mg of placental membrane was used for the IGF-1 receptor binding assay.
在胰岛素受体结合分析实验中，胰岛素标准和本发明化合物的起始浓度均为 ΙΟΟηΜ,然后 将胰岛素和本发明化合物系列 3倍稀释,分别得到 7个不同浓度的对照和化合物溶液（ 100nM 33.33nM、 l l.llnM, 3.70nM、 1.23nM、 0.41nM、 0.13nM、 0.04nM )。 对于本发明中其胰岛 素受体的活性低于人胰岛素标准 10%的化合物， 化合物起始浓度为 500nM。 在 IGF-1受体结 合分析实验中， IGF-1标准的起始浓度为 ΙΟΟηΜ, 本发明化合物起始浓度为 ΙΟΟΟηΜ, 然后将 IGF-1与本发明化合物系列 3倍稀释，分别得到 7个不同浓度的对照和化合物溶液（ 1000nM、 333.33nM、 lll.l lnM. 37.04nM、 12.35nM、 4.12nM、 1.37nM、 0.46nM )。对于本发明中其 IGF-1 受体的活性低于 IGF-1标准 1%的化合物， 化合物起始浓度为 5000nM。 In the insulin receptor binding assay, the insulin standard and the starting concentration of the compound of the present invention were both ΙΟΟηΜ, and then the insulin and the compound of the present invention were diluted 3 times to obtain 7 different concentrations of the control and compound solution (100 nM 33.33 nM, respectively). , l l.llnM, 3.70nM, 1.23nM, 0.41nM, 0.13nM, 0.04nM). For compounds in the present invention whose insulin receptor activity is less than 10% of the human insulin standard, the starting concentration of the compound is 500 nM. In the IGF-1 receptor binding assay, the initial concentration of the IGF-1 standard is ΙΟΟηΜ, the starting concentration of the compound of the present invention is ΙΟΟΟηΜ, and then IGF-1 is diluted 3 times with the compound of the present invention to obtain 7 different Concentration of control and compound solutions (1000 nM, 333.33 nM, lll.l lnM. 37.04 nM, 12.35 nM, 4.12 nM, 1.37 nM, 0.46 nM). For the IGF-1 in the present invention The activity of the receptor is less than 1% of the IGF-1 standard, and the initial concentration of the compound is 5000 nM.
受体结合分析 ( 1 )  Receptor binding assays ( 1 )
截断的水溶性受体  Truncated water soluble receptor
IGF-1或胰岛素受体， 125I-IGF-1 (3-10 pM)或 1251-胰岛素 （3 pM) 和系列 3倍稀释的多肽 加入緩沖液 [100 mM Hepes, H 8.0, 100 mM NaCl, 10 mM MgCl2, 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X- 100] , 总体积 20(^L, 在 4°C培养 48小时。 受体及与受体结合的多肽和配体用 0.2 %γ-球蛋白和 50( L 25 % (w/v) PEG 8000沉淀， 测量沉淀中的放射性。 受体的浓度要调节 到在未添加多肽的时候有 15-20%的受体与配体结合。 IGF-1 or insulin receptor, 125 I-IGF-1 (3-10 pM) or 125 1-insulin (3 pM) and a series of 3-fold diluted polypeptides added to buffer [100 mM Hepes, H 8.0, 100 mM NaCl , 10 mM MgCl 2 , 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100], total volume 20 (^L, cultured at 4 ° C for 48 hours. Receptor and receptor binding The polypeptide and ligand were precipitated with 0.2% γ-globulin and 50 (L 25 % (w/v) PEG 8000, and the radioactivity in the precipitate was measured. The concentration of the receptor was adjusted to 15-20 when no polypeptide was added. % of the receptor binds to the ligand.
膜结合受体  Membrane-bound receptor
受体结合分析使用的膜结合受体来自高度表达全长胰岛素或 IGF-1受体的 BHK细胞。等 量的转染 BHK细胞 (2000-5000)均匀分布在 96孔板的每一孔， 在包含 10% (v/v) 胎牛血清的 Dulbecco's改良的 Eagle's培养基（DMEM)中培养 24小时后再进行受体结合分析。 细胞先用 结合緩冲液 (DMEM,含 0.50% BSA, 20 mM Hepes, pH 7.8) 洗一遍，加入 40( L125I-IGF-1 (6.5 pM)或 1251-胰岛素 (6.5 pM)和溶于结合緩冲液的系列 3倍稀释的多肽。 在 16°C 培养 3小时， 未结合的多肽用吸引器吸出，用 1.2 ml 结合緩沖液洗一遍。细胞溶解于 500 μL 1% (w/v) SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8)， 然后测量。 细胞数量要调整到未加多肽时有 16-28 % 的受体与配体结合。 The membrane-bound receptors used in the receptor binding assay were derived from BHK cells that highly expressed full length insulin or IGF-1 receptor. Equal amounts of transfected BHK cells (2000-5000) were evenly distributed in each well of a 96-well plate and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum for 24 hours. Receptor binding assays were performed. The cells were washed once with binding buffer (DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8) and added with 40 (L 125 I-IGF-1 (6.5 pM) or 125 1-insulin (6.5 pM) and dissolved. A 3-fold dilution of the peptide in combination with buffer. Cultured at 16 ° C for 3 hours, unbound polypeptide was aspirated with aspirator and washed once with 1.2 ml of binding buffer. Cells were lysed at 500 μL 1% (w/v SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8), then measured. The number of cells is adjusted to 16-28% of the receptor binding to the ligand when no polypeptide is added.
受体结合分析（ 2 )  Receptor binding assay ( 2 )
IGF-1受体： [Thr59]IGF-l用于酪氨酸碘化反应 （ iodination )。 125I-IGF-1 (50-80 Ci/g, 50 fmol), 人胎盘膜（0.2 mg)和系列 3倍稀释的多肽加入 0.2毫升 0.1 M Hepes緩沖液， pH 8 , 包 含 120mM 氯化钠、 5mM 氯化钾、 0.12mM 石克酸镁以及 0.1% 牛白蛋白， 在 20°C培养 1小 时。样品用 Whatman GF/F 过滤器过滤以分离结合与未结合的多肽化合物。过滤器预先用 0.1% 聚乙烯亚胺处理。 培养管和过滤器用 2.5 毫升不含牛白蛋白的冷緩沖液洗 4遍。 没有胎盘膜 的情况下， 少于 5% 的多肽化合物附着在过滤器上。 在没有多肽竟争的条件下， 胎盘膜结合 大约 38% 的配体。 对胎盘膜非特异结合可以通过添加过量的非碘化 [Thr59] IGF-1 ( 0.3 μ Μ) 到培养混合物来测量。 非特异结合通常占配体与胎盘膜结合总量的 5%。 IGF-1 receptor: [Thr 59 ] IGF-1 is used for tyrosine iodination. 125 I-IGF-1 (50-80 Ci/g, 50 fmol), human placental membrane (0.2 mg) and serial 3-fold diluted polypeptide were added to 0.2 ml of 0.1 M Hepes buffer, pH 8, containing 120 mM sodium chloride, 5 mM potassium chloride, 0.12 mM magnesium sulphate and 0.1% bovine albumin were incubated at 20 ° C for 1 hour. Samples were filtered using Whatman GF/F filters to isolate bound and unbound polypeptide compounds. The filter was previously treated with 0.1% polyethyleneimine. The culture tubes and filters were washed 4 times with 2.5 ml of cold buffer without bovine albumin. In the absence of a placental membrane, less than 5% of the polypeptide compound is attached to the filter. In the absence of polypeptide competition, the placental membrane binds approximately 38% of the ligand. Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated [Thr 59 ] IGF-1 (0.3 μΜ) to the culture mixture. Non-specific binding typically accounts for 5% of the total binding of the ligand to the placental membrane.
胰岛素受体： 1251-胰岛素（30 nCi)、 系列 3倍稀释的多肽和胎盘膜（0.025 mg)在 0.05 毫 升上述緩冲液中， 20Γ培养 1小时。 样品用 EHWP过滤器过滤， 培养管和过滤器用 2.5 毫升 不含牛白蛋白的冷緩冲液洗 4遍。 没有胎盘膜的情况下， 少于 5% 的多肽化合物附着在过滤 器上。对胎盘膜非特异结合可以通过添加过量的非碘化胰岛素（ 1 μ M )到培养混合物来测量。 非特异结合通常占配体与胎盘膜结合总量的 1%以下。 Insulin receptor: 125 1-insulin (30 nCi), serial 3-fold diluted polypeptide and placental membrane (0.025 mg) were incubated in 0.05 ml of the above buffer for 20 hours at 20 Torr. The sample was filtered through an EHWP filter, and the culture tube and filter were washed 4 times with 2.5 ml of cold buffer containing no bovine albumin. In the absence of a placental membrane, less than 5% of the polypeptide compound is attached to the filter. Non-specific binding to the placental membrane can be measured by adding an excess of non-iodinated insulin (1 μM) to the culture mixture. Non-specific binding typically accounts for less than 1% of the total binding of the ligand to the placental membrane.
特异结合百分比 = (结合放射量 -非特异结合放射量 I全部结合放射量-非特异结合放射量) χ 100。 全部结合放射量是未添加多肽时测得的放射总量。 结合放射量是添加多肽后测得的放 射量。 多肽化合物的 IC50使用 Origin软件 (OriginLab, Northampton, ΜΑ)计算。 多肽相对于人 胰岛素或 IGF-1标准的活性 =IC5。胰岛素或 IGF-1标准 /IC5Q多肽。 Specific binding percentage = (binding amount - non-specific binding amount I total binding amount - non-specific binding amount) χ 100. The total bound amount of radiation is the total amount of radiation measured when no polypeptide is added. The combined amount of radiation is the amount of radiation measured after the addition of the polypeptide. IC 50 polypeptide compound using Origin software (OriginLab, Northampton, ΜΑ) is calculated. The activity of the polypeptide relative to human insulin or IGF-1 standard = IC 5 . Insulin or IGF-1 standard / IC 5Q polypeptide.
3、 动物实验 3. Animal experiment
7-9周龄 C57BL/6雄性小鼠， 平均体重 20-25g， 6只分为一组， 在实验开始前 4小时禁 食。 实验开始前测量血糖， 并在以后各个指定时间点取样测量血糖。 对照组是生理盐水， 多 肽溶解在生理盐水中， 皮下注射。 观察小鼠在实验全过程的反应， 记录任何异常行为。 实验结果： C57BL/6 male mice, 7-9 weeks old, averaged 20-25 g, divided into 6 groups, and fasted 4 hours before the start of the experiment. Blood glucose was measured before the start of the experiment, and blood glucose was measured at various designated time points in the future. The control group was physiological saline, and the polypeptide was dissolved in physiological saline and injected subcutaneously. Observe the response of the mice throughout the experiment and record any abnormal behavior. Experimental results:
1、 基于 IGF-1的本发明的化合物与胰岛素受体和 IGF-1受体的结合能力实验  1. Experiment of binding ability of the compound of the present invention based on IGF-1 to insulin receptor and IGF-1 receptor
在这一部分中， 对具有双链结构的化合物的生物活性进行检测， 检验这些化合物与胰岛 素受体和 IGF-1受体结合能力。 使用天然胰岛素和 IGF-1分别作为与胰岛素受体结合力和与 IGF-1受体结合力的基准（ 100% )。 结果见表二。  In this section, the biological activity of a compound having a double-stranded structure was examined, and the ability of these compounds to bind to the insulin receptor and the IGF-1 receptor was examined. Natural insulin and IGF-1 were used as a reference (100%) for binding to the insulin receptor and binding to the IGF-1 receptor, respectively. The results are shown in Table 2.
尽管 IGF-1的 A链和 B链与胰岛素的 A链和 B链有高度同源性， 而且 NMR和 X射线 晶体衍射的结果也显示 IGF-1和胰岛素的 A链和 B链的三维构象也几乎完全重合， 但双链 IGF-1 A:B与胰岛素受体的结合力非常低， 但对 IGF-1受体的活性与胰岛素相比又过高。 因此 一定是 IGF-1 A链和 B链中的特定位点的氨基酸残基导致上述差异。 当 B15Q被笨丙氨酸或 色氨酸取代后， IGF-1 A:B类似物在胰岛素受体的结合力与胰岛素相当， 证明 B15位的氨基 酸残基是与胰岛素受体的结合的关键。 IGF-1对 IR-A有明显的选择性， 双链化合物 11~16也 显示了程度较低，但同样的受体选择性。 A8位的取代能够同时提高多肽在 3个受体的结合力， 但没有改变对受体的选择性。 理想的胰岛素类药物应该具有高而且均衡的 IR-A和 IR-B受体 结合力，和尽可能低的 IGF-1受体结合力 IGF-1。 以中性侧链取代 A5和 A12的侧链负电荷恢 复了多肽在 IR-A和 IR-B上的平衡， 并且显著降低了在 IGF-1受体的活性。  Although the A and B chains of IGF-1 are highly homologous to the A and B chains of insulin, and the results of NMR and X-ray crystallography also show the three-dimensional conformation of the A and B chains of IGF-1 and insulin. Almost completely coincident, but the binding of the double-stranded IGF-1 A:B to the insulin receptor is very low, but the activity at the IGF-1 receptor is too high compared to insulin. Therefore, it must be that the amino acid residues at specific sites in the IGF-1 A chain and the B chain cause the above difference. When B15Q is replaced by stupid alanine or tryptophan, the binding capacity of the IGF-1 A:B analog at the insulin receptor is comparable to that of insulin, demonstrating that the amino acid residue at position B15 is critical for binding to the insulin receptor. IGF-1 has a clear selectivity for IR-A, and double-stranded compounds 11-16 also show a lower degree, but the same receptor selectivity. Substitution at position A8 can simultaneously increase the binding of the polypeptide at the three receptors, but does not alter the selectivity for the receptor. Ideal insulin drugs should have high and balanced IR-A and IR-B receptor binding, and the lowest possible IGF-1 receptor binding capacity, IGF-1. Replacing the side chain negative charge of A5 and A12 with a neutral side chain restores the balance of the polypeptide on IR-A and IR-B and significantly reduces the activity at the IGF-1 receptor.
研究表明， B9E是 IGF-1促生长作用的重要氨基酸残基， 也使 IGF-1有导致癌症的危险。 用组氨酸、 精氨酸、 谷氨酰胺或苯丙氨酸取代谷氨酸， 仍然能够保持相当的胰岛素受体活性。  Studies have shown that B9E is an important amino acid residue in the growth-promoting effect of IGF-1, and it also causes IGF-1 to cause cancer. Replacing glutamate with histidine, arginine, glutamine or phenylalanine still maintains comparable insulin receptor activity.
在 A22、 A23位添加赖氨酸残基， 对胰岛素受体结合没有负面影响， 但赖氨酸側链氨基 可以提供一个酰化反应的位点。  The addition of a lysine residue at positions A22 and A23 has no negative effect on insulin receptor binding, but the lysine side chain amino group can provide a site for an acylation reaction.
表二： 双链结构的 IGF-1类似物的生物活性结果  Table 2: Bioactivity results of double-stranded IGF-1 analogues
化合物 IR-A (%) IR-B (%) IGF-1R (%) 1化合物 IR-A (%) IR-B (%) IGF-1R (%) 胰岛素 100 100 0.5 1-17 195 112 3.2 Compound IR-A (%) IR-B (%) IGF-1R (%) 1 compound IR-A (%) IR-B (%) IGF-1R (%) Insulin 100 100 0.5 1-17 195 112 3.2
IGF-1 3.66 0.7 100 1-18 333 197 0.9IGF-1 3.66 0.7 100 1-18 333 197 0.9
IGF-1A: IGF-1A:
14 4.7 6.3 1-19 149 80 0.9 IGF-1B  14 4.7 6.3 1-19 149 80 0.9 IGF-1B
1-1 158 63 5.7 1-20 96 73 0.8 1-1 158 63 5.7 1-20 96 73 0.8
1-2 102 52 4.2 1-21 105 102 0.71-2 102 52 4.2 1-21 105 102 0.7
1-3 87 49 2.8 1-22 98 71 2.31-3 87 49 2.8 1-22 98 71 2.3
1-4 150 61 3.9 1-23 163 155 0.81-4 150 61 3.9 1-23 163 155 0.8
1-5 139 73 3.5 1-24 97 101 0.51-5 139 73 3.5 1-24 97 101 0.5
1-6 164 99 3.6 1-25 89 92 0.41-6 164 99 3.6 1-25 89 92 0.4
1-7 330 173 10.6 1-26 83 86 0.51-7 330 173 10.6 1-26 83 86 0.5
1-8 305 162 8.2 1-27 81 82 0.21-8 305 162 8.2 1-27 81 82 0.2
1-9 101 119 0.9 1-28 83 80 0.51-9 101 119 0.9 1-28 83 80 0.5
1-10 93 89 0.7 1-29 145 134 0.41-10 93 89 0.7 1-29 145 134 0.4
1-11 80 85 1.1 1-30 133 127 0.71-11 80 85 1.1 1-30 133 127 0.7
1-12 72 69 1.0 1-31 129 120 0.11-12 72 69 1.0 1-31 129 120 0.1
1-13 84 76 1.2 1-32 118 115 0.1
Figure imgf000058_0001
1-13 84 76 1.2 1-32 118 115 0.1
Figure imgf000058_0001
注： IR-A和 IR-B是胰岛素受体亚型 A和 B; IGF-1 R胰岛素样生长因子 -1受体； IGF-1A: IGF-1B表示 IGF- 1A链和 IGF-1B链双链结构。  Note: IR-A and IR-B are insulin receptor subtypes A and B; IGF-1 R insulin-like growth factor-1 receptor; IGF-1A: IGF-1B indicates IGF-1A chain and IGF-1B chain double Chain structure.
2、 基于 IGF-1的未经修饰的单链化合物的实验结果 2. Experimental results of unmodified single-chain compounds based on IGF-1
在这一部分中， 对基于 IGF-1 的未经修饰的单链化合物的生物活性进行检测， 检验这些 单链化合物与胰岛素受体和 IGF-1受体结合能力。 使用天然胰岛素和 IGF-1分别作为与胰岛 素受体结合力和与 IGF-1受体结合力的基准（ 100% )。 结果见表三。  In this section, the biological activity of IGF-1 based unmodified single-stranded compounds was examined and the ability of these single-chain compounds to bind to the insulin receptor and IGF-1 receptor was examined. Native insulin and IGF-1 were used as a reference (100%) for binding to insulin receptors and binding to IGF-1 receptor, respectively. The results are shown in Table 3.
天然 IGF-1是单链多肽， 与胰岛素受体的结合力不到胰岛素的 5%。 既然 B15位氨基酸 取代可以使基于 IGF-1 的双链化合物的生物活性达到胰岛素的水平， 同样的取代也能够提高 基于 IGF-1的单链化合物的活性。 II -1显示仅仅改变 B15氨基酸残基， 去掉一般认为对生物 活性没有显著影响的 D域， 就提高化合物的胰岛素受体活性达 10倍以上。 C2Tyr在 IGF-1晶 体结构中是非常突出的一个氨基酸残基，显示可能是与 GF-1受体的结合的关键。酪氨酸被丝 氨酸或丙氨酸取代后， 胰岛素受体活性基本保留， 但大幅度降低了 IGF-1受体活性， 正是预 期的效果。 另外， 结果显示， C肽的长度不一定需要 12个氨基酸残基。 原 IGF-1 C的多个氨 基酸残基都是可以缺失或被替换的。 尤其是 IGF-1 C末端去除 3个氨基酸残基 PQT和 4个残 基 APQT不但对胰岛素受体的活性没有负面影响， 而且使其 IGF-1受体活性降低到胰岛素的 水平。 进一步的研究发现， 在保留 C肽的长度是 12个氨基酸残基的情况下， IGF-1 B链 C末 端的 5个氨基酸残基， 即 X^XK XKHX^X^是可以有 1个、 2个、 3个、 4个或全部缺失， 而不会降低胰岛素受体结合力。进一步研究表明， IGF-1 C肽仅仅是很多种连接片段中的一个 选择。 这些数据说明， 适当的 C链长度、 灵活的 C链空间构象和特定位点的氨基酸取代是提 高胰岛素受体活性， 降低 IGF-1受体结合力的重要因素。  Natural IGF-1 is a single-chain polypeptide that binds less than 5% of insulin to the insulin receptor. Since the amino acid substitution at position B15 allows the biological activity of the IGF-1 based double-stranded compound to reach the level of insulin, the same substitution can also increase the activity of the single-chain compound based on IGF-1. II-1 shows that only the B15 amino acid residue is changed, and the D domain, which is generally considered to have no significant effect on biological activity, is removed, and the insulin receptor activity of the compound is increased by more than 10 times. C2Tyr is a prominent amino acid residue in the IGF-1 crystal structure and is shown to be the key to binding to the GF-1 receptor. When tyrosine is substituted with serine or alanine, the insulin receptor activity is substantially retained, but the IGF-1 receptor activity is greatly reduced, which is the expected effect. In addition, the results show that the length of the C peptide does not necessarily require 12 amino acid residues. Multiple amino acid residues of the original IGF-1 C can be deleted or replaced. In particular, the removal of three amino acid residues PQT and four residues of APQT at the C-terminus of IGF-1 not only had no negative effect on the activity of the insulin receptor, but also reduced the activity of IGF-1 receptor to the level of insulin. Further studies have found that in the case where the length of the retained C-peptide is 12 amino acid residues, the 5 amino acid residues at the C-terminus of the IGF-1 B chain, ie X^XK XKHX^X^, may have one, two One, three, four or all are missing without reducing insulin receptor binding. Further studies have shown that the IGF-1 C peptide is only one of a wide variety of ligation sequences. These data indicate that proper C-chain length, flexible C-strand conformation, and amino acid substitution at specific sites are important factors in increasing insulin receptor activity and reducing IGF-1 receptor binding.
表三： 基于 IGF-1的未经修饰的单链化合物的生物活性结果  Table III: Biological Activity Results of Unmodified Single-Stranded Compounds Based on IGF-1
化 合 IR IGF-1R 化合 IR IGF-1 化 合 IR IGF-1 化 合 IR IGF-1 物 (%) ( % ) 物 (%) R ( % ) 物 (%) R (%) 物 (%) R (%) Compound IR IGF-1R Compound IR IGF-1 Compound IR IGF-1 Compound IR IGF-1 (%) (%) (%) R (%) (%) R (%) (%) R (%) )
II -1 107 5.9 II -32 83 0.4 II -63 86 0.2 II -94 65 0.4II -1 107 5.9 II -32 83 0.4 II -63 86 0.2 II -94 65 0.4
II -2 95 1.6 II -33 85 0.3 II -64 75 0.4 II -95 87 0.2II -2 95 1.6 II -33 85 0.3 II -64 75 0.4 II -95 87 0.2
II -3 87 0.9 II -34 78 0.3 11 -65 58 0.5 11 -96 76 0.3II -3 87 0.9 II -34 78 0.3 11 -65 58 0.5 11 -96 76 0.3
II -4 90 0.8 11 -35 80 0.4 II -66 89 0.1 II -97 72 0.5II -4 90 0.8 11 -35 80 0.4 II -66 89 0.1 II -97 72 0.5
II -5 89 0.7 II -36 84 0.5 II -67 72 0.2 II -98 68 <0.1II -5 89 0.7 II -36 84 0.5 II -67 72 0.2 II -98 68 <0.1
II -6 79 1.3 II -37 93 0.7 II -68 76 0.5 II -99 71 0.2II -6 79 1.3 II -37 93 0.7 II -68 76 0.5 II -99 71 0.2
II -7 102 1.1 II -38 79 0.6 11 -69 50 0.4 11 -100 83 <0.1II -7 102 1.1 II -38 79 0.6 11 -69 50 0.4 11 -100 83 <0.1
II -8 99 1.0 II -39 57 0.4 11 -70 57 0.6 11 -101 78 <0.1II -8 99 1.0 II -39 57 0.4 11 -70 57 0.6 11 -101 78 <0.1
II -9 92 1.4 11 -40 62 0.4 II -71 51 0.6 11 -102 80 <0.1II -9 92 1.4 11 -40 62 0.4 II -71 51 0.6 11 -102 80 <0.1
11 -10 78 0.9 11 -41 53 0.7 II -72 63 0.4 11 -103 70 <0.111 -10 78 0.9 11 -41 53 0.7 II -72 63 0.4 11 -103 70 <0.1
11 -11 80 0.7 II -42 66 0.6 II -73 49 0.3 11 -104 76 <0.111 -11 80 0.7 II -42 66 0.6 II -73 49 0.3 11 -104 76 <0.1
11 -12 77 0.6 II -43 50 0.6 II -74 87 <0.1 11 -105 57 <0.1 II -13 75 0.6 II -44 49 0.5 11 -75 71 0.3 11 -106 90 <0.111 -12 77 0.6 II -43 50 0.6 II -74 87 <0.1 11 -105 57 <0.1 II -13 75 0.6 II -44 49 0.5 11 -75 71 0.3 11 -106 90 <0.1
II -14 63 0.5 11 -45 51 0.6 II -76 67 <0.1 11 -107 73 <0.1II -14 63 0.5 11 -45 51 0.6 II -76 67 <0.1 11 -107 73 <0.1
II -15 82 0.7 II -46 43 0.4 II -77 59 <0.1 11 -108 62 <0.1II -15 82 0.7 II -46 43 0.4 II -77 59 <0.1 11 -108 62 <0.1
II -16 65 0.6 II -47 47 0.3 II -78 40 0.4 11 -109 44 <0.1II -16 65 0.6 II -47 47 0.3 II -78 40 0.4 11 -109 44 <0.1
II -17 92 1.0 II -48 45 0.6 II -79 58 0.5 11 -110 39 <0.1II -17 92 1.0 II -48 45 0.6 II -79 58 0.5 11 -110 39 <0.1
II -18 95 0.4 II -49 87 0.3 11 -80 46 0.3 11 -111 43 <0.1II -18 95 0.4 II -49 87 0.3 11 -80 46 0.3 11 -111 43 <0.1
11 -19 97 0.2 II -50 62 0.2 II -81 53 0.4 11 -112 81 <0.111 -19 97 0.2 II -50 62 0.2 II -81 53 0.4 11 -112 81 <0.1
II -20 88 0.6 II -51 44 88 <0.1 11 -113 56 <0.1II -20 88 0.6 II -51 44 88 <0.1 11 -113 56 <0.1
II -21 87 0.5 II -52 51 0.5 1 11 -83 75 <0.1 11 -114 87 <0.1II -21 87 0.5 II -52 51 0.5 1 11 -83 75 <0.1 11 -114 87 <0.1
II -22 62 0.6 II -53 47 0.5 11 -84 62 0.2 11 -115 79 <0.1II -22 62 0.6 II -53 47 0.5 11 -84 62 0.2 11 -115 79 <0.1
II -23 73 0.8 II -54 63 0.4 11 -85 55 <0.1 11 -116 68 <0.1II -23 73 0.8 II -54 63 0.4 11 -85 55 <0.1 11 -116 68 <0.1
II -24 102 0.9 II -55 85 0 o.1 11 -86 82 <0.1 11 -117 43 <0.1 II -24 102 0.9 II -55 85 0 o.1 11 -86 82 <0.1 11 -117 43 <0.1
寸  Inch
II -25 98 0.9 II -56 78 0.3 11 -87 91 0.2 11 -118 65 <0.1 II -25 98 0.9 II -56 78 0.3 11 -87 91 0.2 11 -118 65 <0.1
II -26 93 0.4 II -57 90 0.5 11 -88 79 <0.1 11 -119 82 0.8II -26 93 0.4 II -57 90 0.5 11 -88 79 <0.1 11 -119 82 0.8
II -27 91 0.3 II -58 69 0.4 11 -89 86 0.1 11 -120 91 0.7II -27 91 0.3 II -58 69 0.4 11 -89 86 0.1 11 -120 91 0.7
II -28 106 0.5 II -59 93 0.1 11 -90 56 0.2 11 -121 89 0.7II -28 106 0.5 II -59 93 0.1 11 -90 56 0.2 11 -121 89 0.7
II -29 85 0.4 II -60 48 0.4 11 -91 78 0.2 11 -122 73 0.6II -29 85 0.4 II -60 48 0.4 11 -91 78 0.2 11 -122 73 0.6
11 -30 82 0.5 II -61 44 0.3 II -92 49 0.3 11 -123 85 1.011 -30 82 0.5 II -61 44 0.3 II -92 49 0.3 11 -123 85 1.0
II -31 91 0.5 II -62 53 0.3 II -93 53 0.5 11 -124 89 0.9 II -31 91 0.5 II -62 53 0.3 II -93 53 0.5 11 -124 89 0.9
3、 PEG或脂肪酸修饰的基于 IGF-1的化合物的实验结果 3. Experimental results of PEG or fatty acid modified IGF-1 based compounds
在这一部分中， 对 PEG、 白蛋白或脂肪酸修饰的基于 IGF-1的化合物的生物活性进行检 测， 检验这些化合物与胰岛素受体和 IGF-1受体结合能力。 使用天然胰岛素和 IGF-1分别作 为与胰岛素受体结合力和与 IGF-1受体结合力的基准（ 100% )。 结果见表四。  In this section, the biological activities of PEG, albumin or fatty acid modified IGF-1 -based compounds were examined and tested for their ability to bind to the insulin receptor and IGF-1 receptor. Native insulin and IGF-1 were used as a reference (100%) for binding to the insulin receptor and binding to the IGF-1 receptor, respectively. The results are shown in Table 4.
聚乙二醇化和脂肪酸酰化是延长多肽体内作用时间的常用方法， 但聚乙二醇化和脂肪酸 酰化一般大幅度降低生物活性。 因此寻找新的能够引入赖氨酸等具有氨基的酰化位点， 而酰 化产物具有足够的生物活性对于开发长效多肽有艮重要的意义。  PEGylation and fatty acid acylation are common methods for extending the duration of action of a polypeptide in vivo, but pegylation and fatty acid acylation generally greatly reduce biological activity. Therefore, it is sought to introduce a new acylation site having an amino group such as lysine, and the acylation product has sufficient biological activity for the development of a long-acting polypeptide.
实验证明，对应于 IGF-1的 Bl、 B28、 A14、 A15、 A22、 B链的 C末端延伸至 B31、 B32、 单链 IGF-1类似物 C肽的多个位点， 修饰后都保留了相当的胰岛素受体活性。 由于在受体结 合实验中要使用牛白蛋白以减少蛋白非特异性结合， 但酰化多肽上的脂肪酸能够与白蛋白结 合（这也是脂肪酸延长作用时间的机理）， 从而降低了多肽在实验中的有效浓度， 因此酰化多 肽的受体结合数据显得较低。但过去 30年的大量动物实验表明， 胰岛素类似物的体外活性与 体内活性没有明确的对应关系。 体外活性显著低于人胰岛素的类似物在体内与人胰岛素的活 性基本相当 (参照 Volund等 "In vitro and in vivo potency of insulin analogs designed for clinical use", Diabetic Medicine, 1991, 8:839-47; Ribel等 "Equivalent in vivo biological activity of insulin analogs and human insulin despite different in vitro potencies", Diabetes, 39, 1033-39, 1990 )。发明 人的动物实验也表明聚乙二醇化和脂肪酸酰化多肽的体内实际活性要优于胰岛素对照。  Experiments have shown that the C-terminus of the Bl, B28, A14, A15, A22, and B chains corresponding to IGF-1 extends to multiple sites of B31, B32, and single-chain IGF-1 analog C peptide, and remains after modification. Equivalent insulin receptor activity. Since bovine albumin is used in receptor binding experiments to reduce protein non-specific binding, fatty acids on acylated polypeptides can bind to albumin (this is also the mechanism by which fatty acids prolong the action), thereby reducing the polypeptide in the experiment. The effective concentration, therefore, the receptor binding data for the acylated polypeptide appears to be lower. However, a large number of animal experiments over the past 30 years have shown that there is no clear correspondence between the in vitro activity of insulin analogs and the in vivo activity. Analogs with significantly lower in vitro activity than human insulin are substantially equivalent in vivo to human insulin (see Volund et al. "In vitro and in vivo potency of insulin analogs designed for clinical use", Diabetic Medicine, 1991, 8: 839-47; Ribel et al. "Equivalent in vivo biological activity of insulin analogs and human insulin despite different in vitro potencies", Diabetes, 39, 1033-39, 1990). Animal experiments by the inventors have also shown that the actual activity of PEGylated and fatty acid acylated polypeptides in vivo is superior to that of insulin controls.
表四： PEG和脂肪酸修饰的基于 IGF-1的化合物的生物活性结果  Table 4: Biological Activity Results of PEG and Fatty Acid Modified IGF-1 Based Compounds
化合物 IR ( % ) IGF-1R 1化合物 IR ( % ) IGF-1R 1化合物 IR ( % ) IGF-1R Compound IR ( % ) IGF-1R 1 Compound IR ( % ) IGF-1R 1 Compound IR ( % ) IGF-1R
Figure imgf000060_0001
本申请中另给出本发明的化合物具有延效性的数据。
Figure imgf000060_0001
Further information on the potency of the compounds of the invention is given in the present application.
图 1显示了小鼠皮下注射生理盐水、 人胰岛素和 3种不同剂量的 III-1后血糖随时间变化 的平均值。 在同等剂量下， 聚乙二醇修饰的多肽作用时间显著长于人胰岛素， 可以达到 24小 时乃至更长时间。 而且聚乙二醇修饰的多肽降血糖作用平緩， 即使加大到 3倍剂量， 也没有 造成血糖低谷。 因此， ΠΙ-1在控制血糖和安全性方面都优于人胰岛素。  Figure 1 shows the mean change in blood glucose over time after subcutaneous injection of normal saline, human insulin, and three different doses of III-1 in mice. At the same dose, the polyethylene glycol modified polypeptide has a significantly longer duration of action than human insulin, which can reach 24 hours or even longer. Moreover, the polyethylene glycol-modified polypeptide has a mild hypoglycemic effect, and even if it is increased to a three-fold dose, it does not cause a hypoglycemia. Therefore, ΠΙ-1 is superior to human insulin in controlling blood sugar and safety.
图 2显示了小鼠皮下注射生理盐水、 人胰岛素和 ΠΙ-12 (均为 80纳摩尔 /公斤）后血糖随 时间变化的平均值。 在同等剂量下， ΠΙ-12的起效时间与人胰岛素相当， 但在给药后的 8小时 依然具有明显的抑制血糖效果， 胰岛素这时已经失去抑制血糖效果。 人胰岛素在实验中表现 出典型的 V字型降血糖曲线。 这种降血糖曲线的缺点在于初期血糖下降过快， 容易造成低血 糖， 而后期又无法控制血糖。 而 ΙΠ-12显示出 L型降血糖曲线， 血糖控制平稳而且持久， 效 果优于人胰岛素  Figure 2 shows the mean change in blood glucose over time after subcutaneous injection of normal saline, human insulin, and ΠΙ-12 (both 80 nanomoles/kg). At the same dose, the onset time of sputum-12 was comparable to that of human insulin, but it still had a significant blood sugar-suppressing effect at 8 hours after administration. At this time, insulin had lost its blood sugar-suppressing effect. Human insulin showed a typical V-shaped blood glucose lowering curve in the experiment. The disadvantage of this blood glucose lowering curve is that the initial blood sugar drops too fast, which is likely to cause hypoglycemia, and later it is impossible to control blood sugar. ΙΠ-12 shows an L-type blood glucose lowering curve, blood sugar control is smooth and long-lasting, and its effect is better than human insulin.
图 3是小鼠皮下注射生理盐水、地特胰岛素和本发明的 ΠΙ-7化合物后血糖随时间变化值。 地特胰岛素是当今国际市场上仅有的两种长效胰岛素类似物药物之一， 在人体内半衰期是 14 小时,使用时一般是一天一针。 III-7只使用地特胰岛素 1/3的剂量， 就能够达到更长的作用时 间和更平稳的降血糖效果。  Fig. 3 is a graph showing changes in blood glucose with time after subcutaneous injection of physiological saline, insulin detemir and the indole-7 compound of the present invention. Detemir is one of the only two long-acting insulin analog drugs on the international market. It has a half-life of 14 hours in human body and is usually used once a day. III-7 can only achieve a longer duration of action and a more stable hypoglycemic effect with a dose of 1/3 of insulin.
以上三个实验证明， 以聚乙二醇或脂肪酸修饰本发明中的 IGF-1类似物， 能够改善 IGF-1 类似物的治疗效果和药物动力学特征。  The above three experiments have demonstrated that modification of the IGF-1 analog of the present invention with polyethylene glycol or a fatty acid can improve the therapeutic and pharmacokinetic characteristics of the IGF-1 analog.

Claims

权利要求书 Claim
1、 一种具有降血糖作用的化合物， 其特征在于， 所述化合物包括 A链和 B链， 其中， A链的氨基^ 列为： X^ GIVDXsCCTCwXsX^ioCwX LRRLEX YCwX X , B链氨基 列为：  A compound having hypoglycemic action, characterized in that the compound comprises an A chain and a B chain, wherein the amino group of the A chain is: X^ GIVDXsCCTCwXsX^ioCwX LRRLEX YCwX X , and the B chain amino group is:
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFX47X48X49X5oX5iX52X53 , 其中， X 23-26 X 27 LC [1] GAX 32 LVDALX 38 X3 9 VC [2] GDX 44 GFX 47 X4 8 X4 9 X 5 oX 5 iX 5 2X53 , where
是赖氨酸、 精氨酸或缺失； 是赖氨酸、 精氨酸或缺失； Χ5是谷氨酸、 天冬酰胺、 谷 氨酰胺或丝氨酸； Χ8是组氨酸、 精氨酸、 苯丙氨酸或苏氨酸； Χ9是精氨酸或丝氨酸； 10是 丝氨酸或异亮氨酸； Xl2是天冬氨酸、 丝氨酸、 谷氨酰胺或天冬酰胺； Χ18是天冬酰胺、 曱硫 氨酸或苏氨酸； Χ21是天冬酰胺、 丙氨酸或甘氨酸； Χ22是赖氨酸、精氨酸-赖氨酸二肽或缺失； χ23.26是苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽， 或甘氨酸 -脯氨酸-谷氨酸、 缬氨酸-天冬酰 胺-谷氨酰胺三肽， 或脯氨酸-谷氨酸、 天冬酰胺-谷氨酰胺二肽， 或谷氨酸、 谷氨酰胺、 赖氨 酸或精氨酸， 或是以赖氨酸或精氨酸取代上述二、 三、 四肽序列中任何一个氨基酸残基后的 序列， 或缺失； χ27是组氨酸或苏氨酸； χ32是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨 酸； Χ38是苯丙氨酸或色氨酸； Χ39是苯丙氨酸或色氨酸； 4是精氨酸、 谷氨酸、 天冬氨酸 或丙氨酸； 7是苯丙氨酸、 酪氨酸或组氨酸； 8是 -NH2、 dA-NH2、 苯丙氨酸或酪氨酸或缺 失； 9是天冬酰胺、 天冬氨酸、 谷氨酸、 苏氨酸或缺失； X5()是赖氨酸、 脯氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X51是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X52 是苏氨酸或缺失； X53是谷氨酸、 天冬氨酸、谷氨酸-谷氨酸、 天冬氨酸-天冬氨酸二肽或缺失； 所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物中通过 6个半胱氨酸形成 3对二 疏键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： Ctl] 和 C[4] 形成二疏键， C[2] 和 C[6] 形成二石克键， Ct3]和 C[5]形成二硫键。 Is lysine, arginine or deletion; is lysine, arginine or deletion; Χ 5 is glutamic acid, asparagine, glutamine or serine; Χ 8 is histidine, arginine, Phenylalanine or threonine; Χ 9 is arginine or serine; 10 is serine or isoleucine; Xl2 is aspartic acid, serine, glutamine or asparagine; Χ 18 is asparagine , 曱 methionine or threonine; Χ 21 is asparagine, alanine or glycine; Χ 22 is lysine, arginine-lysine dipeptide or deletion; χ 23 . 26 is phenylalanine Acid-valine-asparagine-glutamine tetrapeptide, or glycine-valine-glutamate, valine-asparagine-glutamine tripeptide, or valine-glutamic acid, An asparagine-glutamine dipeptide, or glutamic acid, glutamine, lysine or arginine, or any one of the above two, three, and tetrapeptide sequences in lysine or arginine sequence residues, or deleted; χ 27 is histidine or threonine; χ 32 is histidine, glutamic acid, glutamine, arginine or phenylalanine; 38 is a phenylalanine or tryptophan; [chi] 39 is a phenylalanine or tryptophan; 4 is arginine, glutamic acid, aspartic acid or alanine; 7 is phenylalanine, tyrosine Or histidine; 8 is -NH 2 , dA-NH 2 , phenylalanine or tyrosine or deletion; 9 is asparagine, aspartic acid, glutamic acid, threonine or deletion; X 5 () is lysine, valine, arginine, aspartic acid, glutamic acid or deletion; X 51 is valine, lysine, arginine, aspartic acid, glutamine Acid or deletion; X 52 is threonine or deletion; X 53 is glutamic acid, aspartic acid, glutamic acid-glutamic acid, aspartic acid-aspartic acid dipeptide or deletion; the compound Wherein, [1]-[6] represent the number of a cysteine; in the compound, a 3-paired double bond is formed by 6 cysteines, wherein the A chain and the B chain are linked by two pairs of interchain disulfide bonds. There are a pair of intrachain disulfide bonds in the A chain. The specific positions of the three pairs of disulfide bonds are: C tl] and C [4] form a double bond, C [2] and C [ 6] form a two-gram bond, C t 3] and C[ 5 ] form a disulfide bond.
2、 根据权利要求 1所述的化合物， 其特征在于， 所述化合物包括 A链和 B链， 其中， A链的氨基酸序列为： GIVDX5C[3]C[4]X8RSC[5]X12LRRLEX18YC[6]X21X22， The compound according to claim 1, wherein the compound comprises an A chain and a B chain, wherein the amino acid sequence of the A chain is: GIVDX 5 C [3] C [4] X 8 RSC [ 5 ] X 12 LRRLEX 18 YC [6] X 21 X 22 ,
B链氨基酸序列为：  The B chain amino acid sequence is:
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFYX48X49 X50 X51 X52> 其中， X 2 3-26X27LC [1] GAX3 2 LVDALX 38 X 3 9VC [ 2]GDX44GFYX4 8 X49 X 50 X51 X 52 > where
X5是谷氨酸、 天冬酰胺、 谷氨酰胺或丝氨酸； X8是组氨酸、 精氨酸或苯丙氨酸； 12是 天冬氨酸、 丝氨酸、 谷氨酰胺或天冬酰胺； X18是天冬酰胺、 曱硫氨酸或苏氨酸； Χ2ι是天冬 酰胺、 丙氨酸或甘氨酸； X22是赖氨酸、 精氨酸-赖氨酸二肽或缺失； X23.26是甘氨酸 -脯氨酸- 谷氨酸三肽或苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽； X27是组氨酸或苏氨酸； X32是组氨 酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨酸； X38是苯丙氨酸或色氨酸； X39是苯丙氨酸或色 氨酸； 4是精氨酸、 谷氨酸、 天冬氨酸或丙氨酸； 8是 -NH2、 dA-NH2或苯丙氨酸； 9是 天冬酰胺或缺失； Χ5。是赖氨酸、 脯氨酸或缺失； X51是脯氨酸、 赖氨酸或缺失； X52是苏氨 酸或缺失。 X 5 is glutamic acid, asparagine, glutamine or serine; X 8 is histidine, arginine or phenylalanine; 12 is aspartic acid, serine, glutamine or asparagine; X 18 is asparagine, methionine or threonine; Χ 2 ι is asparagine, alanine or glycine; X 22 is lysine, arginine-lysine dipeptide or deletion; X 23 .26 is a glycine-valine-glutamic acid tripeptide or phenylalanine-valine-asparagine-glutamine tetrapeptide; X 27 is histidine or threonine; X 32 is a group Acid, glutamic acid, glutamine, arginine or phenylalanine; X 38 is phenylalanine or tryptophan; X 39 is phenylalanine or tryptophan; 4 is arginine, Glutamate, aspartic acid or alanine; 8 is -NH 2 , dA-NH 2 or phenylalanine; 9 is asparagine or a deletion; Χ 5 . Is lysine, valine or deletion; X 51 is valine, lysine or deletion; X 52 is threonine or deletion.
3、 根据权利要求 2 所述的化合物， 其特征在于， 所述 B 链的序列是 GPEX27LCGAX32LVDALX3gX39VCGDX44GFY-NH2; GPEX27LCGAX32LVDALX38X39VCGDX44The compound according to claim 2, wherein the sequence of the B chain is GPEX 27 LCGAX 32 LVDALX 3g X 39 VCGDX44GFY-NH 2 ; GPEX 27 LCGAX 32 LVDALX 38 X 39 VCGDX44
GFYFNKPT; GPEX27LCGAX32LVDALX3gX39VCGDX44GFYdA-NH2; 或 FVNQX27LCGAX32L VDALX38X39VCGDX44GFYFNKPT , 其中 X27、 X32 X38 . X39 如权利要求 2中所定义。 GFYFNKPT; GPEX 27 LCGAX 32 LVDALX 3g X3 9 VCGDX44GFYdA-NH 2 ; or FVNQX 27 LCGAX 32 L VDALX 38 X 39 VCGDX4 4 GFYFNKPT , wherein X 2 7 , X 32 X 38 . X 39 is as defined in claim 2.
4、 根据权利要求 2 所述的化合物， 其特征在于， 所述 A 链序列是 GIVDX5CCX8RSCX12LRRLEX18YCA或 GrVDX5CCX8RSCX12LRRLEX18YCN, 其中 X5、 X8、 X12、 X18如权利要求 2中所定义。 4. The compound according to claim 2, wherein the A chain sequence is GIVDX 5 CCX 8 RSCX 12 LRRLEX 18 YCA or GrVDX 5 CCX 8 RSCX 12 LRRLEX 18 YCN, wherein X 5 , X 8 , X 12 X 18 is as defined in claim 2.
5、 根据权利要求 1-4任一项所述的化合物， 其特征在于， 所述化合物选自以下化合物： 1-1 ： 其 中 A 链序 列 为 SEQ ID NO: 1 所示序 列 ； B 链的序列 为The compound according to any one of claims 1 to 4, wherein the compound is selected from the group consisting of 1-1: wherein the A chain sequence is the sequence of SEQ ID NO: 1; the sequence of the B chain for
GPETLCGAELVDALFFVCGDRGFY-NH2; GPETLCGAELVDALFFVCGDRGFY-NH 2 ;
1-2： 其 中 A 链序 列 为 SEQ ID NO: 1 所示序 列 ； B 链的序 列 为 1-2: wherein the A chain sequence is the sequence shown in SEQ ID NO: 1; the sequence of the B chain is
GPETLCGAELVDALWFVCGDRGFY-NH2; GPETLCGAELVDALWFVCGDRGFY-NH 2 ;
1-3： 其 中 A 链序 列 为 SEQ ID NO: 1 所示序 列 ； B 链的序 列 为 1-3: wherein the A chain sequence is the sequence shown in SEQ ID NO: 1; the sequence of the B chain is
GPETLCGAELVDALWWVCGD GFY-NH2; GPETLCGAELVDALWWVCGD GFY-NH 2 ;
1-4: 其中 A链序列为 SEQ ID NO: 7所示序列； B链的序列为 SEQ ID NO:3所示序列； 1-5: 其中 A链序列为 SEQIDNO: 8所示序列； B链的序列为 SEQIDNO:3所示序列； 1-6: 其中 A链序列为 SEQIDNO:9所示序列； B链的序列为 SEQ ID NO:3所示序列； 1-7: 其中 A链序列为 SEQIDNO: 10所示序列; B链的序列为 SEQ ID NO:3所示序列； 1-8: 其中 A链序列为 SEQIDNO: 11所示序列； B链的序列为 SEQIDNO:3所示序列； 1-9: 其中 A链序列为 SEQIDNO: 12所示序列； B链的序列为 SEQ IDNO:3所示序列； 1-10: 其中 A链序列为 SEQIDNO: 13所示序列； B链的序列为 SEQ ID NO:3所示序列; 1-11: 其中 A链序列为 SEQIDNO: 14所示序列； B链的序列为 SEQ ID NO:3所示序歹' J； 1-12: 其中 A链序列为 SEQIDNO: 15所示序列； B链的序列为 SEQ ID NO:3所示序列； 1-13: 其中 A链序列为 SEQIDNO: 16所示序列; B链的序列为 SEQ ID NO:3所示序列; 1-14: 其中 A链序列为 SEQ ID NO: 17所示序列； B链的序列为 SEQ ID NO:3所示序列； 1-15:其中 A链序列为 SEQIDNO: 17所示序列； B链的序列为 SEQIDNO:18所示序列； 1-16: 其中 A链序列为 SEQIDNO: 19所示序列； B链的序列为 SEQ ID NO: 18所示序列; 1-17:其中 A链序列为 SEQ ID NO: 20所示序列； B链的序列为 SEQ ID NO: 18所示序列； 1-18： 其中 A 链序列 为 SEQ ID NO: 17 所示序 列 ； B 链的序列 为 1-4: wherein the A chain sequence is the sequence of SEQ ID NO: 7; the B chain sequence is the sequence of SEQ ID NO: 3; 1-5: wherein the A chain sequence is the sequence of SEQ ID NO: 8; The sequence of SEQ ID NO: 3; 1-6: wherein the A chain sequence is the sequence of SEQ ID NO: 9; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-7: wherein the A chain sequence is SEQ ID NO The sequence shown by 10; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-8: wherein the A chain sequence is the sequence of SEQ ID NO: 11; the sequence of the B chain is the sequence of SEQ ID NO: 3; 9: wherein the A chain sequence is the sequence of SEQ ID NO: 12; the B chain sequence is the sequence of SEQ ID NO: 3; 1-10: wherein the A chain sequence is the sequence of SEQ ID NO: 13; the B chain sequence is SEQ ID The sequence of NO:3; 1-11: wherein the sequence of the A chain is the sequence of SEQ ID NO: 14; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-12: wherein the sequence of the A chain is SEQ ID NO The sequence shown by 15; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-13: wherein the A chain sequence is the sequence of SEQ ID NO: 16; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-14: wherein the A chain sequence is SEQ ID NO: The sequence of 17; the sequence of the B chain is the sequence of SEQ ID NO: 3; 1-15: wherein the sequence of the A chain is the sequence of SEQ ID NO: 17; the sequence of the B chain is the sequence of SEQ ID NO: 18; Wherein the sequence of the A chain is the sequence of SEQ ID NO: 19; the sequence of the B chain is the sequence of SEQ ID NO: 18; 1-17: wherein the sequence of the A chain is the sequence of SEQ ID NO: 20; the sequence of the B chain is SEQ ID NO: 18; 1-18: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is
GPETLCG AHLVD ALFFVCGDRGFYdA-NH2； GPETLCG AHLVD ALFFVCGDRGFYdA-NH 2 ;
1-19:其中 A链序列为 SEQIDNO: 17所示序列; B链的序列为 SEQ ID NO:22所示序列; 1-20:其中 A链序列为 SEQIDNO: 17所示序列； B链的序列为 SEQ ID NO:23所示序列； 1-21:其中 A链序列为 SEQIDNO: 17所示序列； B链的序列为 SEQ ID NO:24所示序列； 1-22:其中 A链序列为 SEQ ID NO: 8所示序列； B链的序列为 SEQ ID NO:25所示序列； 1-23:其中 A链序列为 SEQ ID NO:26所示序列； B链的序列为 SEQ ID NO: 25所示序列； 1-24:其中 A链序列为 SEQIDNO: 17所示序列； B链的序列为 SEQ ID NO:25所示序列； 1-25:其中 A链序列为 SEQIDNO: 17所示序列; B链的序列为 SEQIDNO:27所示序列; 1-26:其中 A链序列为 SEQIDNO: 17所示序列； B链的序列为 SEQ ID NO:28所示序列； 1-27:其中 A链序列为 SEQ ID NO:151所示序列； B链的序列为 SEQ ID NO: 152所示序歹' J； 1-28:其中 A链序列为 SEQID NO: 153所示序列； B链的序列为 SEQ ID NO:152所示序列； 1-29:其中 A链序列为 SEQ ID NO: 154所示序列； B链的序列为 SEQ ID NO: 155所示序歹' J； 1-30:其中 A链序列为 SEQIDNO: 156所示序列； B链的序列为 SEQ ID NO: 155所示序歹' J； 1-31:其中 A链序列为 SEQ IDNO:157所示序列； B链的序列为 SEQ ID NO:155所示序列； 1-32:其中 A链序列为 SEQ IDNO:158所示序列； B链的序列为 SEQ ID NO: 155所示序列； 1-33:其中 A链序列为 SEQ IDNO:159所示序列； B链的序列为 SEQ ID NO:160所示序列； 1-34:其中 A链序列为 SEQ IDNO:159所示序列； B链的序列为 SEQ ID NO:155所示序列。 1-19: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain sequence is the sequence of SEQ ID NO: 22; 1-20: wherein the A chain sequence is the sequence of SEQ ID NO: 17; Is the sequence of SEQ ID NO: 23; 1-21: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is the sequence of SEQ ID NO: 24; 1-22: wherein the A chain sequence is SEQ The sequence of ID NO: 8; the sequence of the B chain is the sequence of SEQ ID NO: 25; 1-23: wherein the sequence of the A chain is the sequence of SEQ ID NO: 26; the sequence of the B chain is SEQ ID NO: 25 The sequence shown is: 1-24: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the B chain is the sequence of SEQ ID NO: 25; 1-25: wherein the A chain sequence is the sequence of SEQ ID NO: 17. The sequence of the B chain is the sequence of SEQ ID NO: 27; 1-26: wherein the A chain sequence is the sequence of SEQ ID NO: 17; the sequence of the B chain is the sequence of SEQ ID NO: 28; 1-27: wherein the A chain sequence Is the sequence shown in SEQ ID NO: 151; the sequence of the B chain is the sequence of SEQ ID NO: 152; 1-28: wherein the A chain sequence is the sequence shown in SEQ ID NO: 153; the sequence of the B chain is SEQ ID NO: 152 sequence; 1-29: where A chain The sequence shown as SEQ ID NO: 154; the sequence of the B chain is the sequence of SEQ ID NO: 155; 1-30: wherein the sequence of the A chain is the sequence of SEQ ID NO: 156; the sequence of the B chain is SEQ ID NO: 155 indicates a sequence of 'J; 1-31: wherein the A chain sequence is the sequence of SEQ ID NO: 157; the B chain has the sequence of SEQ ID NO: 155; 1-32: wherein the A chain sequence Is the sequence shown in SEQ ID NO: 158; the sequence of the B chain is the sequence of SEQ ID NO: 155; 1-33: wherein the A chain sequence is the sequence shown in SEQ ID NO: 159; the sequence of the B chain is SEQ ID NO: 160 The sequence shown; 1-34: wherein the A chain sequence is the sequence shown in SEQ ID NO: 159; and the B chain sequence is the sequence shown in SEQ ID NO: 155.
6、 一种具有 降血糖作 用 的 单链化合物 ， 所述化合物 结 构 为 ： Xi01ALC [！] GAX1oibLVDALXLOLCX101dVC[2]GDRGFX1oie i02Xio3Xi04Xio5 i06-CL-GIVDQC[3]C[4]X1 07RSC[5]SLRRLENYC[6]X108 X109, 其中， 6. A single-chain compound having hypoglycemic action, wherein the structure of the compound is: Xi 01A LC [! ] GAX 1 oibLVDALX LOLC X 101 dVC[ 2 ]GDRGFX 1 oie i 0 2Xio3Xi 0 4Xio 5 i 0 6-CL-GIVDQC[3]C[ 4 ]X 1 07 RSC [5] SLRRLENYC [6] X 108 X 10 9 , among them,
X10ia是甘氨酸 -脯氨酸-谷氨酸-苏氨酸、 甘氨酸-脯氨酸 -谷氨酸-组氨酸四肽或者苯丙氨酸 -缬氨酸-天冬酰胺 -谷氨酰胺-组氨酸五肽， 或是以赖氨酸或精氨酸取代上述四肽或五肽中的甘 氨酸-脯氨酸 -谷氨酸或苯丙氨酸 -纈氨酸-天冬酰胺 -谷氨酰胺中任一个氨基酸残基后的序列； XlOlb是组氨酸、 谷氨酸、 谷氨酰胺、 精氨酸或苯丙氨酸； XiOlc是苯丙氨酸或色氨酸； XlOld 是苯丙氨酸或色氨酸； X10ie是苯丙氨酸、 酪氨酸或组氨酸； X1()2是苯丙氨酸或缺失； X1()3是 天冬酰胺或缺失； X104是赖氨酸、 脯氨酸或缺失； X1G5是脯氨酸、 赖氨酸或缺失； XIQ6是苏 氨酸或缺失； x1()7是苯丙氨酸、 精氨酸或组氨酸； x1()8是丙氨酸、 甘氨酸或天冬酰胺； x109 是赖氨酸、 精氨酸-赖氨酸二肽或缺失； CL是连接片段。 X 10 ia is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine tetrapeptide or phenylalanine-valine-asparagine-glutamine a histidine pentapeptide, or a glycine-valine-glutamic acid or phenylalanine-valine-asparagine-valley in the above tetrapeptide or pentapeptide by lysine or arginine Sequence after any amino acid residue in the aminoamide; XlOlb is histidine, glutamic acid, glutamine, arginine or phenylalanine; XiOlc is phenylalanine or tryptophan; XlOld is styrene Or a tryptophan; X 10 i e is phenylalanine, tyrosine or histidine; X 1 () 2 is phenylalanine or a deletion; X 1 () 3 is asparagine or a deletion; X 104 is lysine, valine or deletion; X 1G5 is valine, lysine or deletion; XIQ 6 is threonine or deletion; x 1 () 7 is phenylalanine, arginine or Histidine; x 1 () 8 is alanine, glycine or asparagine; x 109 is lysine, arginine-lysine dipeptide or deletion; CL is a ligation fragment.
7、 根据权利要求 6所述的化合物， 其特征在于， 所迷化合物的结构为：  7. A compound according to claim 6 wherein the structure of the compound is:
X1o1aLC[1]GAHLVDALFFVC[2]GDRGFYX1o2Xio3Xio4Xio5Xi06-CL-GIVDQC[3]C[4]FRSC[5]SLX 1 o 1 aLC [1] GAHLVDALFFVC [ 2 ] GDRGFYX 1 o 2 Xio3Xio4Xio 5 Xi06-CL-GIVDQC [ 3 ] C [ 4 ] FRSC [ 5 ] SL
RRLENYC[6]A X109, 其中， RRLENYC [6] AX 109 , where
XlOia是甘氨酸 -脯氨酸-谷氨酸-苏氨酸四肽或者苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组 氨酸五肽； X,。2是苯丙氨酸或缺失； X1()3是天冬酰胺或缺失； X,。4是赖氨酸、 脯氨酸或缺失； X105是脯氨酸、 赖氨酸或缺失； XiD6是苏氨酸或缺失； X1Q9是赖氨酸、 精氨酸-赖氨酸二肽或 缺失； CL是连接片段。 XlOia is a glycine-valine-glutamate-threonine tetrapeptide or a phenylalanine-valine-asparagine-glutamine-histidine pentapeptide; X,. 2 is phenylalanine or a deletion; X 1 () 3 is asparagine or a deletion; X,. 4 is lysine, proline or deletion; X 105 is valine, lysine or deletion; Xi D6 is threonine or deletion; X 1Q9 is lysine, arginine-lysine dipeptide Or missing; CL is a connected fragment.
8、 根据权利要求 7所述的化合物， 其特征在于， 所述化合物的结构是：  8. The compound according to claim 7, wherein the structure of the compound is:
GPETLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;  GPETLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;
GPETLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA;  GPETLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA;
FVNQHLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;  FVNQHLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;
FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA; 或 FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCARK。 FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA; or FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-C L -GIVDQCCFRSCSLRRLENYCARK.
9、 根据权利要求 6-8任一项所述的化合物， 其特征在于， 所述连接片段 CL是 6-60个氨 基酸的肽序列， 其中每一个氨基酸残基都独立选自甘氨酸、 丙氨酸、 丝氨酸、 苏氨酸或脯氨 酸。 9. A compound according to any one of claims 6-8, wherein said connector C L fragment is a peptide sequence of 6-60 amino acids, wherein each amino acid residue independently selected from glycine, alanyl Acid, serine, threonine or valine.
10、 根据权利要求 9所述的化合物， 其特征在于， 所述连接片段 包含 1个或 1个以上 天冬氨酸、 谷氨酸、 精氨酸、 赖氨酸、 半胱氨酸或天冬酰胺。  The compound according to claim 9, wherein the ligated fragment comprises one or more aspartic acid, glutamic acid, arginine, lysine, cysteine or aspartate Amide.
11、 根据权利要求 10所述的化合物， 其特征在于， 所述连接片段 CL是以下多肽片段的 全部或部分序列， 或者与以下多肽片段有 1、 2或 3个氨基酸残基的差异， 或者与以下多肽片 段有 70%、 80%、 90%类似， 或者是以下多肽片段的全部或部分序列的 1、 2、 3、 4或 5次重 复序列： 11. The compound of claim 10, wherein said connector C L fragment is all or part of the following sequence of polypeptide fragments or polypeptide fragments have the following 1, 2 or 3 differences in amino acid residues, or 70%, 80%, 90% similar to the following polypeptide fragments, or 1, 2, 3, 4 or 5 repeats of all or part of the sequence of the following polypeptide fragments:
(GASPGGSSGS)„GR, 其中 n是 1、 2、 3、 4或 5; GSSGSSGPGSSR; GSSGSGSSAPQT; (GASPGGSSGS) „GR, where n is 1, 2, 3, 4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT;
GSGGAPSRSGSSR; GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR; GSGSSSRAPPSAPSPQR； GSSSESPSGAPQT； GAGTPASGSAPGR； GSSPSGGSSAPQT；GSGGAPSRSGSSR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR; GSGSSSRAPPSAPSPQR; GSSSESPSGAPQT; GAGTPASGSAPGR; GSSPSGGSSAPQT;
GSTSSTARSPGR； GAGPSGTASPSR； GSSTPSGAPQT； SSSSAPPPSAPSPSRAPQR；GSTSSTARSPGR; GAGPSGTASPSR; GSSTPSGAPQT; SSSSAPPPSAPSPSRAPQR;
GASPGTSSTSGR ; GSGSSSAAAPQT； GSGSSSAAPQT ; GSGSSSAPQT ; GSGSSSRRA ;GASPGTSSTSGR ; GSGSSSAAAPQT; GSGSSSAAPQT ; GSGSSSAPQT ; GSGSSSRRA ;
GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR; GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR ； GPSGTSTSAPGR ； GAGSSSAPQT; SSSSAPSAPSPSRPQR ； GSGASSPTSPQR ； GSSPATSATPQT ； GAGSSSAPPPSAPSPSRAPGPSAPQR ； GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR; GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSGRSSSPGR; GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR； RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK ； SSSAPPPSAPSPSRAPGPSPQR ； SAASSSASSSSASSASAGR ； GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR; GSGSSSRRAPQT; SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT ; GGPSSGAPPPSPQT ; SSSAPPPSAPSPSRAPQT； GAGPSSGAPPPSPQT； GGGGAPQT; GAGGPSSGAPPPQT； GGPSSGAPPPSPSPSRPGPSPQR ； SSASSASSSSAGR ; SSASSSAASSSASSSASGR ； SSSGAPPPSPSRAPGPSPQR； GSGSASRGR; SSSSAASSASGR ; SASASASASSASSGR； SASSPSPSAPSSPSPAS ； GPSSPSPSAPSSPSPASPSSGR； SSSAPPPASPSPSRAPGPQR； SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; SSPSASPSSPASPSSGR; GAPASPAPSAPAPAAPSGR； GPSSPSPSAPSSPSPASPSSAPQT； SSASSASSSSSASAGR； SAPSSPSPSAPSSPSASPSGR ； SSSAPPPSAPSPSAPQR ； GASSPSPSAPSSPSPASGR ； SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT ； GGGGAPQR ； GSGSSSAAR ； GSGSSAAPQR ； SSSSRRAPQR ； SSSGSGSSAPQR; SSGSGSSSAPQR; GSGSSSRS; SSSSRAPQR; GASPGGSSGSGR。 GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR; GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR; GPSGTSTSAPGR; GAGSSSAPQT; SSSSAPSAPSPSRPQR; GSGASSPTSPQR; GSSPATSATPQT; GAGSSSAPPPSAPSPSRAPGPSAPQR; GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR; GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSGRSSSPGR; GSPAGSPSSSAGSSASASPASPGR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR; RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK; SSSAPPPSAPSPSRAPGPSPQR; SAASSSASSSSASSASAGR; GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR; GSGSSSRRAPQT; SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT; GAGPSSGAPPPSPQT; GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPSRPGPSPQR; SSASSASSSSAGR; SSASSSAASSSASSSASGR; SSSGAPPPSPSRAPGPSPQR; GSGSASRGR; SSSSAASSASGR; SASASASASSASSGR; SASSPSPSAPSSPSPAS; GPSSPSPSAPSSPSPASPSSGR; SSSAPPPASPSPSRAPGPQR; SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; SSPS ASPSSPASPSSGR; GAPASPAPSAPAPAAPSGR; GPSSPSPSAPSSPSPASPSSAPQT; SSASSASSSSSASAGR; SAPSSPSPSAPSSPSASPSGR; SSSAPPPSAPSPSAPQR; GASSPSPSAPSSPSPASGR; SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT; GGGGAPQR; GSGSSSAAR; GSGSSAAPQR; SSSSRRAPQR; SSSGSGSSAPQR; SSGSGSSSAPQR; GSGSSSRS; SSSSRAPQR; GASPGGSSGSGR.
12、根据权利要求 6或 7任一项所述的化合物， 所述化合物选自 SEQ ID NO:29~ SEQ ID NO: 150, SEQ ID NO: 161、 SEQ ID NO: 162所示的化合物。  The compound according to any one of claims 6 or 7, which is selected from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 150, SEQ ID NO: 161, and SEQ ID NO: 162.
13、 一种具有降血糖作用的化合物， 所述化合物包括 A链和 B链， 其中，  13. A compound having hypoglycemic action, the compound comprising an A chain and a B chain, wherein
A链的氨基酸序列为：  The amino acid sequence of the A chain is:
X3 9X 00GIVDX405C[3]C[4]X408X409X410C[5]X412LX414¾ 15L 17 8 YC【6]X421 22， X3 9X 00GIVDX4 0 5C[3]C[4]X408X409X410C[5]X412LX4143⁄4 1 5 L 1 7 8 YC[ 6 ]X4 2 1 22,
B链氨基酸序列为：  The B chain amino acid sequence is:
X423-426X427LC[i]GAHLVDALX438X439 C[2]GDRGFX447 448X44 450X45lX452 453 54 55， 所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物通过 6个半胱氨酸形成 3对二 硫键， 其中 A链和 B链通过两对链间二^■键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： Cn]和 C[4]形成二石充键， C[2]和 C[6] 形成二硫键， C[3]和 C[5]形成二石充键， 其中，X423-426X427LC[i]GAHLVDALX438X439 C[ 2 ]GDRGFX447 448X44 4 50 X45lX4 5 2 4 5 3 54 55, among the compounds, [1]-[6] represent the number of cysteine; Cysteine forms a 3-pair disulfide bond, in which the A chain and the B chain are linked by two pairs of interchain bonds, and a pair of intrachain disulfide bonds exist in the A chain. The specific positions of the three pairs of disulfide bonds are: C n] and C [4] form a two-stone charge, C [2] and C[ 6] form a disulfide bond, and C [3] and C [5] form a two-stone charge, wherein
X399是精氨酸、 赖氨酸或缺失； 00是精氨酸、 赖氨酸或缺失； 05是谷氨酸、 天冬酰 胺、 谷氨酰胺或丝氨酸； 。8是组氨酸、 精氨酸、 苯丙氨酸、 苏氨酸或通式（I )结构； 09 是精氨酸、 丝氨酸或通式（I )结构； 1()是组氨酸、 精氨酸、 苯丙氨酸或通式（I ) 结构； 12是丝氨酸、 异亮氨酸或通式（I )结构； 14是精氨酸或通式（I )结构； 15是精氨酸或 通式（I )结构； 17是谷氨酸或通式（I )结构； 18是天冬酰胺或通式（I )结构； 21是 丙氨酸、甘氨酸或天冬酖胺； 22是赖氨酸、精氨酸-赖氨酸二肽或缺失， 或为通式（I )结构； 当 22为二肽时， 其中一个氨基酸为通式（I )结构； X423 6是甘氨酸-脯氨酸-谷氨酸三肽、X 3 99 is arginine, lysine or deletion; 00 is arginine, lysine or deletion; 0 5 is glutamic acid, asparagine, glutamine or serine; 8 is histidine, arginine, phenylalanine, threonine or the structure of formula (I); 09 is arginine, serine or the structure of formula (I); 1 () is histidine, fine Amino acid, phenylalanine or a structure of the formula (I); 1 2 is a serine, isoleucine or a structure of the formula (I); 14 is an arginine or a structure of the formula (I); 15 is an arginine or the general formula (I) structure; 17 is glutamic acid or formula (I) structure; asparagine 18 or the general formula (I) structure; 21 is alanine, glycine, or asparagine amine poison; 22 Lysine, arginine-lysine dipeptide or deletion, or a structure of formula (I); when 22 is a dipeptide, one of the amino acids is of the formula (I); X423 6 is glycine-ammonium Acid-glutamic acid tripeptide,
UL-甘氨酸 -脯氨酸-谷氨酸、 苯丙氨酸-缬氨酸-天冬酰胺-谷氨酰胺四肽或 苯两氨酸-缬氨酸 -天冬酰胺 -谷氨酰胺； 27是组氨酸或苏氨酸； 38是苯丙氨酸或色氨酸； 39是苯丙氨酸或 色氨酸； Xw是苯丙氨酸、 组氨酸或酪氨酸； 48是 -NH2、 苯丙氨酸、 酪氨酸或缺失； ¾49 是天冬酰胺、 苏氨酸、 谷氨酸、 天冬氨酸或缺失； X45Q是赖氨酸、 精氨酸、 谷氨酸、 天冬氨 酸、 脯氨酸或缺失； 51是脯氨酸、 赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸或缺失， 或为通式U L -glycine-valine-glutamic acid, phenylalanine-valine-asparagine-glutamine tetrapeptide or phenyl-bis-proline-asparagine-glutamine; 27 Is histidine or threonine; 38 is phenylalanine or tryptophan; 39 is phenylalanine or Tryptophan; Xw is phenylalanine, histidine or tyrosine; 48 is -NH 2 , phenylalanine, tyrosine or deletion; 3⁄449 is asparagine, threonine, glutamic acid, Aspartic acid or deletion; X45Q is lysine, arginine, glutamic acid, aspartic acid, proline or deletion; 51 is valine, lysine, arginine, glutamic acid, Aspartic acid or deletion, or general formula
(I)结构； 52是苏氨酸、 赖氨酸或缺失， 或为通式（I)结构； 53是谷氨酸、 甘氨酸、 赖 氨酸或缺失， 或为通式（I)结构； 54是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I)结 构； 55是赖氨酸或缺失， 或为通式（I)结构， Structure (I); 52 is threonine, lysine, or deletions, or the general formula (I) structure; 53 is glutamic acid, glycine, lysine, or deletions, or the general formula (I) structure; 54 Is a glutamic acid, glycine, lysine or deletion, or a structure of the formula (I); 55 is a lysine or a deletion, or is a structure of the formula (I),
所述通式（I) ：  The general formula (I):
Figure imgf000065_0001
Figure imgf000065_0001
UL是 -W-X-Y-Z 结构、 脂肪酸、 聚乙二醇、 白蛋白、 L„-ML 结构、 氢原子或 Na-( a-(HOOC(CH2)„CO)-Y-Glu)- , Na-(Na-(CH3(CH2)nCO)-y-Glu)- , 其中 η是 8-20的整数， 如 8、 10、 12、 14、 16、 20, Να表示氨基酸或氨基酸残基的 a-氨基， 或为通式（II)结构， 所述 通式（ II )结构是： U L is -WXYZ structure, fatty acid, polyethylene glycol, albumin, L„-M L structure, hydrogen atom or N a -( a -(HOOC(CH 2 )„CO)-Y-Glu)- , N a -(N a -(CH 3 (CH 2 ) n CO)-y-Glu)- , wherein η is an integer from 8 to 20, such as 8, 10, 12, 14, 16, 20, Ν α represents an amino acid or The a-amino group of the amino acid residue, or the structure of the formula (II), wherein the structure of the formula (II) is:
Figure imgf000065_0002
Figure imgf000065_0002
J是 -W-X-Y-Z 结构、 -ML结构或氢原子。 J is a -W-X-Y-Z structure, an -ML structure or a hydrogen atom.
14、 根据权利要求 13所述的化合物， 其特征在于， 所述化合物包括 A链和 B链， 其中， A链的氨基酸序列为：  The compound according to claim 13, wherein the compound comprises an A chain and a B chain, wherein the amino acid sequence of the A chain is:
GIVDQC[3]C[4]FRSC[5]X4i2LX414X415LX4i7X418YC[6]AX22) GIVDQC[ 3 ]C[ 4 ]FRSC[5]X4i 2 LX 414 X4 1 5LX 4 i 7 X4 18 YC[ 6 ]AX 22 )
B链氨基酸序列为：  The B chain amino acid sequence is:
X423^26¾27LC[i]GAHLVDALFFVC[2]GDRGFYX448X449X45oX45lX452X453X454¾55' 所述化合物中， [1]- [6]表示半胱氨酸的编号； 所述化合物通过 6个半胱氨酸形成 3对二 硫键， 其中 A链和 B链通过两对链间二硫键连接， A链内存在一对链内二硫键， 三对二硫键 的具体位置是： Ctl]和 C[4]形成二硫键， C[2]和 C[6] 形成二硫键， C[3]和 C[5]形成二硫键； 其中， 12为丝氨酸或通式（I)结构； 14为精氨酸或通式（I)结构； 15是精氨酸或通式（I) 结构； 17为谷氨酸或通式（I)结构； 18为天冬酰胺或通式（I)结构； 22为赖氨酸、 精 氨酸-赖氨酸二肽或缺失， 或为通式（I)结构； 当 22为二肽时， 其中一个氨基酸为通式（I) 结构； 23_426是甘氨酸-脯氨酸-谷氨酸三肽、 UL-甘氨酸 -脯氨酸-谷氨酸、 苯丙氨酸 -缬氨酸- 天冬酰胺-谷氨酰胺四肽或 UL-苯丙氨酸 -缬氨酸-天冬酰胺 -谷氨酰胺； 27是组氨酸或苏氨酸； 48是 -NH2、 苯丙氨酸或缺失； 49是天冬酰胺或缺失； 50是赖氨酸、 精氨酸、 谷氨酸、 天冬氨酸、 脯氨酸或缺失； 51是脯氨酸、 赖氨酸或缺失， 或为通式（I)结构； 52是苏氨 酸、 赖氨酸或缺失， 或为通式（I)结构； 53是谷氨酸、 甘象酸、 赖氨酸或缺失， 或为通式 (I) 结构； 54是谷氨酸、 甘氨酸、 赖氨酸或缺失， 或为通式（I) 结构； 55是赖氨酸或缺 失， 或为通式（I )结构， UL和通式（I )如权利要求 13中所定义。 X423^263⁄427LC[i]GAHLVDALFFVC[2]GDRGFYX448X449X45oX45lX452X453X4543⁄455' In the compound, [1]-[6] represent the number of cysteine; the compound forms a 3-pair disulfide bond through 6 cysteines, wherein The A chain and the B chain are linked by two pairs of interchain disulfide bonds, and a pair of intrachain disulfide bonds exist in the A chain. The specific positions of the three pairs of disulfide bonds are: C tl] and C [4] form a disulfide bond, C [2], and C [6] form a disulfide bond, C [3] and C [5] form a disulfide bond; wherein serine 12 or the general formula (I) structure; formula 14 is arginine or (I) structure; 15 is arginine or the structure of formula (I); 17 is glutamic acid or the structure of formula (I); 18 is asparagine or structure of formula (I); 22 is lysine, An arginine-lysine dipeptide or a deletion, or a structure of the formula (I); when 22 is a dipeptide, one of the amino acids is of the formula (I); 23 _426 is a glycine-valine-glutamine Acid tripeptide, UL-glycine-valine-glutamic acid, phenylalanine-valine-asparagine-glutamine tetrapeptide or U L -phenylalanine-valine-asparagine - glutamine; 27 is histidine or threonine 48 is -NH 2, phenylalanine or deletion; 49 asparagine or deleted; 50 is lysine, arginine, glutamic acid, aspartic acid, proline, or deleted; 51 is preserved Acid, lysine or deletion, or a structure of formula (I); 52 is threonine, lysine or deletion, or is a structure of formula (I); 53 is glutamic acid, glycoic acid, Lai Or a deletion, or a structure of formula (I); 54 is glutamic acid, glycine, lysine or deletion, or a structure of formula (I); 55 is lysine or deficiency Loss, or a structure of the formula (I), U L and formula (I) are as defined in claim 13.
15、 根据权利要求 13-14任一项所述的化合物， 其特征在于， 所述化合物选自：  The compound according to any one of claims 13 to 14, wherein the compound is selected from the group consisting of:
III- 1： 双链结构， 包括 A链和 B链， 其中， A链序列为 SEQ ID NO: 17所示序列； B链 序列为 G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT; III- 1: double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17; the B chain sequence is G (N a -PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT;
III-2: 双链结构， 包括 A链和 B链， 其中， A链序列为 SEQ ID NO: 17所示序列； B链 序列为 GPETLCGAHLVDALFFVCGDRGFYFNPK(Ne-PEG 20K); III-2: a double-stranded structure comprising an A chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17; and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK (N e -PEG 20K);
ΙΠ-6: 双链结构， 包括 A链和 B链， 其中， A链序列为： SEQ ID NO: 17所示序列； B链 序列为： G(Na-CO(CH2)14COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT; ΙΠ-6: double-stranded structure, including A chain and B chain, wherein the A chain sequence is: SEQ ID NO: 17; B chain sequence is: G(N a -CO(CH 2 ) 14 COOH) PETLCGAHLVDALFFVCGDRGFYFNPPT ;
ΠΙ-7: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)14CO)-y-Glu)]； ΠΙ-7: double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -(N a - (HOOC(CH 2 ) 14 CO)-y-Glu)];
III-8: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N (Na- (HOOC(CH2)16CO)-y-Glu)]; III-8: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N (N a - (HOOC(CH 2 ) 16 CO) -y-Glu)];
III-9: 双链结构， 包括 Α链和 Β链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为： FV QHLCGAHLVDALFFVCGDRGFYFNPK[Ne-(Na- (HOOC(CH2)12CO)-y-Glu)]； III-9: Double-stranded structure, including an Α chain and an Β chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FV QHLCGAHLVDALFFVCGDRGFYFNPK[N e -(N a - (HOOC(CH 2 ) 12 CO)-y-Glu)];
111-10: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为： FVNQHLCGAHLVDALFFVCGDRGFYFNPK{NE-[Na- (HOOC(C¾)„NHCO(CH2)3CO)111-10: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK{N E -[N a - (HOOC(C3⁄4)„NHCO (CH 2 ) 3 CO)
-γ-Glu]} ; -γ-Glu]} ;
III-11 : 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列 为 ： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)i4CO)-y-Glu -N-(Y-GIU)] ; III-11: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N E -(N a - (HOOC(CH 2 )i 4 CO)-y-Glu-N-(Y-GIU)];
111-19: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPK[NS- (Na-(HOOC (CH2)14 CO)-y-Glu)]; 111-19: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK[N S - (N a -(HOOC (CH 2 ) 14 CO )-y-Glu)];
III -21 ： 双链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链序 列 为 ： GIVDQCCFRSCSLK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]RLENYCA ； B 链 序 列 为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPT(SEQ ID NO: 274 ); III -21 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is: GIVDQCCFRSCSLK[N E -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]RLENYCA ; B chain sequence Is FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 274);
III -22 ： 双链 结 构 ， 包括 A 链 和 B 链 ， 其 中 A 链序 列 为 ： GIVDQCCFRSCSLRK[Ne-(Na-(HOOC(CH2),4CO)-y-Glu)]LENYCA ； B 链 序 列 为 GPETLCGAHLVDALFFVCGDRGFYFNPPT ( SEQ ID NO:275 ); III -22 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is: GIVDQCCFRSCSLRK[N e -(N a -(HOOC(CH 2 ), 4 CO)-y-Glu)]LENYCA ; B chain The sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 275);
III -23 ： 双 链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链 序 列 为 GIVDQCCFRSCSLRRLENYCAK[NE-( a-(HOOC(CH2)14CO)-y-Glu)] ; B 链序列为 SEQ ID NO:275所示序列； III-23: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCAK[N E -( a -(HOOC(CH 2 ) 14 CO)-y-Glu)]; the B chain sequence is SEQ ID NO: 275 sequence;
III -24 ： 双 链 结 构 ， 包 括 A 链 和 B 链 ， 其 中 A 链 序 列 为 GIVDQCCFRSCSLRRLENYCARK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)] , B 链序列为 SEQ ID NO: 275所示序列； III -24 : double-stranded structure, including A chain and B chain, wherein the A chain sequence is GIVDQCCFRSCSLRRLENYCARK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)], and the B chain sequence is SEQ ID NO: sequence indicated by 275;
111-25: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[NE- (Na-(HOOC(CH2)14CO)-y-Glu)]；111-25: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[N E - (N a -(HOOC(CH 2 ) 14 CO )-y-Glu)];
ΠΙ-26: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [NE-(Na-(HOOC(CH2)i4CO)-y-Glu)]； 111-27: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[NE- (Na-(HOOC(CH2)i4CO)-Y-Glu)]; ΠΙ-28: 双链结构， 包括 Α链和 Β链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列 为 GPETLCGAHLVDALFFVCGDRGFYFNPK[NE- (Να- (HOOC(CH2)14CO)-y-Glu-N-ΠΙ-26: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [N E -(N a -(HOOC(CH 2 )i 4 CO)-y-Glu)]; 111-27: double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[N E - (N a -(HOOC(CH 2 )i 4 CO)-Y-Glu)]; ΠΙ-28: Double-stranded structure, including Α chain and Β chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPK[N E - (Ν α - (HOOC(CH 2 ) 14 CO )-y-Glu-N-
(γ-Giu))]; (γ-Giu))];
111-29: 双链结构， 包括 Α链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 G[Na-(Na-(CH3(CH2)14CO)-y-L-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT; 111-29: Double-stranded structure, including an Α chain and a B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is G[N a -(N a -(CH 3 (CH 2 ) 14 CO)-yL-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT;
111-30: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链序 列为 G(Na-dPEG12-马来酰亚胺-白蛋白） PETLCGAHLVDALFFVCGDRGFYFNKPT; 111-30: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is G (N a -dPEG 12 -maleimide-albumin) PETLCGAHLVDALFFVCGDRGFYFNKPT;
111-31 : 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO: 17所示序列， B链 序列为 FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[NS- (Na-(HOOC(CH2),4CO)-Y-Glu)]; 111-31 : Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence of SEQ ID NO: 17, and the B chain sequence is FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[N S - (N a -(HOOC(CH 2 ), 4 CO)-Y-Glu)];
III-32: 双链结构， 包括 Α链和 Β链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTK[N£- (Na-(HOOC(CH2)14CO)-Y-Glu)]； III-32: Double-stranded structure, including an Α chain and an Β chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTK[N £ - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
111-33: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO 17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N (Na-(HOOC(CH2)14CO)-Y-Glu)]； 111-33: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17 and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N (N a -(HOOC(CH 2 ) 14 CO)-Y -Glu)];
111-34: 双链结构， 包括 A链和 B链， 其中 A链序列为 SEQ ID NO:17所示序列， B链序 列为 GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK[NE- (Na-(HOOC(CH2)14CO)-Y-Glu)]；111-34: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is the sequence shown in SEQ ID NO: 17, and the B chain sequence is GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK[N E - (N a -(HOOC(CH 2 ) 14 CO )-Y-Glu)];
111-36: 双链结构， 包括 A链和 B链， 其中 A链序列为 GIVDQCCHRSCSLRRLENYCA, B 链序列为 GPEHLCGAHLVDALFFVCGDRGFYFNPK [Ne-CO-(CH2CH20)5-(CH2)2- NH- (Na-(HOOC 111-36: Double-stranded structure, including A chain and B chain, wherein the A chain sequence is GIVDQCCHRSCSLRRLENYCA, and the B chain sequence is GPEHLCGAHLVDALFFVCGDRGFYFNPK [N e -CO-(CH 2 CH 2 0) 5 -(CH 2 ) 2 - NH- (N a - (HOOC
Figure imgf000067_0001
Figure imgf000067_0001
Να表示氨基酸或氨基酸残基的 a-氨基； Νε表示氨基酸或氨基酸残基的 ε-氨基， 例如赖氨 酸侧链的 ε-氨基。 Ν α represents an a-amino group of an amino acid or an amino acid residue; Ν ε represents an ε-amino group of an amino acid or an amino acid residue, such as an ε-amino group of a lysine side chain.
16、 一种具有降血糖作用的化合物， 所述化合物的结构为：  16. A compound having hypoglycemic action, the structure of said compound being:
X20iaLC [！] GAX201bLVDALX201cX201d C[2]GDRGFX201eX202 203 204X205X206GX207X207a 208X 209X210 21l 212X213X214X215 216GIVDQC[3]C[4]X2i7RSC[5]X2i8LX219X220LX221X222YC[6]X223X224， 其中， X 2 0i a LC [! ] GAX 2 0 1 bLVDALX 201c X201d C[ 2 ]GDRGFX20 1 eX202 2 0 3 2 0 4X20 5 X206GX 207 X2 0 7a 208X 2 0 9X21 0 21l 212X213X214X21 5 216GIVDQC[ 3 ]C[4]X 2 i 7 RSC[ 5 ]X2i8LX 21 9X 220 LX 221 X 2 2 2 YC[ 6 ]X 22 3X 224 , where
X2oia是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、甘氨酸-脯氨酸 -谷氨酸-组氨酸、苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 UL_-甘氨酸-脯氨酸 -谷氨酸- 组氨酸或 UL-苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸； X2。lb是组氨酸、 谷氨酸、 谷氨酰 胺、 精氨酸或苯丙氨酸； 201(；是苯丙氨酸或色氨酸； X2()ld是苯丙氨酸或色氨酸； X201e是笨丙 氨酸、 酪氨酸或组氨酸； X2Q2是苯丙氨酸、 酪氨酸或缺失； X2。3是天冬酰胺、 苏氨酸、 天冬 氨酸、 谷氨酸或缺失； X2G4是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失； X2Q5是 脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸、 谷氨酸或缺失或通式（I )结构； X2。6是苏氨酸、 赖氨 酸或缺失或通式（I) 结构； X2Q7是丝氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； x207a 是丝氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X2G8是丝氨酸、 通式（I)结构或缺失； X209是丝氨酸、 通式（I)结构或缺失； X21Q是丝氨酸、 通式（I)结构或缺失； x211是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X212是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或 缺失； X213是丙氨酸、 脯氨酸、 精氨酸、 甘氨酸、 通式（I)结构或缺失； X214是脯氨酸、 谷 氨酰胺、 甘氨酸、 通式（I)结构或缺失； X215是谷氨酰胺、 苏氨酸、 甘氨酸、 通式（I)结构 或缺失； X216是苏氨酸、 精氨酸、 赖氨酸、 通式（I)结构或缺失； x217是苯丙氨酸、 精氨酸、 组氨酸或通式（I)结构； X218是天冬氨酸、 丝氨酸、 谷氨跣胺、 天冬酰胺或通式（I)结构；X 2 oi a is glycine-valine-glutamate-threonine, glycine-valine-glutamate-histidine, phenylalanine-valine-asparagine-glutamine- Histidine, U L - glycine-valine-glutamate-threonine, UL_-glycine-valine-glutamate-histidine or UL-phenylalanine-valine-aspartate Amide-glutamine-histidine; X 2 . Lb is histidine, glutamic acid, glutamine, arginine or phenylalanine; 201 ( ; is phenylalanine or tryptophan; X 2 () ld is phenylalanine or tryptophan X 201e is stupid alanine, tyrosine or histidine; X 2Q2 is phenylalanine, tyrosine or deletion; X 2 . 3 is asparagine, threonine, aspartic acid, valley Or a deletion; X 2G4 is valine, lysine, arginine, aspartic acid, glutamic acid or a deletion; X 2Q5 is valine, lysine, arginine, aspartic acid , glutamic acid or deletion or structure of formula (I); X 2 . 6 is threonine, lysine Acid or deletion or structure of formula (I); X 2Q7 is serine, alanine, glycine, structure or deletion of formula (I); x 207a is serine, alanine, glycine, structure or deletion of formula (I) X 2G8 is serine, structure or deletion of formula (I); X 209 is serine, structure or deletion of formula (I); X 21Q is serine, structure or deletion of formula (I); x 211 is arginine, Alanine, glycine, structure or deletion of formula (I); X 212 is arginine, alanine, glycine, structure or deletion of formula (I); X 213 is alanine, valine, spermine Acid, glycine, structure or deletion of formula (I); X 214 is valine, glutamine, glycine, structure or deletion of formula (I); X 215 is glutamine, threonine, glycine, general formula (I) structure or deletion; X 216 is threonine, arginine, lysine, structure or deletion of formula (I); x 217 is phenylalanine, arginine, histidine or formula ( I) structure; X 218 is aspartic acid, serine, glutamine, asparagine or the structure of formula (I);
X219是精氨酸或通式（I)结构； X22Q是精氨酸或通式（I)结构； 221是谷氨酸或通式（I) 结构； X222是天冬酰胺或通式（I)结构； X223是丙氨酸、 甘氨酸或天冬酰胺； X224是赖氨酸、 精氨酸-赖氨酸二肽或缺失， 或为通式（I) 结构； 当 X224为二肽时， 其中一个氨基酸为通式X 21 9 is arginine or a structure of the formula (I); X22Q is arginine or a structure of the formula (I); 221 is glutamic acid or a structure of the formula (I); X 222 is an asparagine or a formula (I) structure; X 223 is alanine, glycine or asparagine; X 22 4 is lysine, arginine-lysine dipeptide or deletion, or is of the formula (I); when X 224 When it is a dipeptide, one of the amino acids is a general formula
(I)结构， 其中 UL和通式（I)如权利要求 13中所定义。 (I) Structure, wherein U L and formula (I) are as defined in claim 13.
17、 根据权利要求 16 所述的化合物， 其特征在于， 所述化合物的结构是： 17. A compound according to claim 16 wherein the structure of the compound is:
X201aLC[1]GAHLVDALFFVC[2]GDRGFYX2o2X2o3X204X2o5X206GX207GX2o8X209X2ioX2iiX2i2X2i3X 2i4 2i5 2i6GIVDQC[3]C[4]FRSC[5]X218LX219X220LX221X222YC[6]A, 其中， X 201 aLC[ 1 ]GAHLVDALFFVC[ 2 ]GDRGFYX 2 o 2 X 2 o 3 X 204 X 2 o 5 X 206 GX 207 GX 2 o 8 X 20 9X2ioX2iiX2i2X2i3X 2i4 2i 5 2i6GIVDQC[3]C[4]FRSC[ 5 ] X 218 LX 21 9X2 20 LX 221 X22 2 YC[ 6 ]A, where
X20la是甘氨酸-脯氨酸 -谷氨酸-苏氨酸、 苯丙氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸、 UL-甘氨酸-脯氨酸 -谷氨酸-苏氨酸或 UL-苯西氨酸-缬氨酸 -天冬酰胺-谷氨酰胺-组氨酸； X202是 笨丙氨酸或缺失； X2Q3是天冬酰胺或缺失； X2Q4是脯氨酸、 赖氨酸、 精氨酸、 天冬氨酸或缺 失； X205是脯氨酸、 赖氨酸或通式（I)结构； X206是苏氨酸、 赖氨酸或通式（I)结构； X207 是丝氨酸、 丙氨酸或通式（ I )结构； X2G8是丝氨酸或通式（ I )结构； X2o9是丝氨酸或通式（ I ) 结构； X21Q是丝氨酸或通式（I)结构； X211是精氨酸、 丙氨酸、 通式（I)结构或缺失； X212 是精氨酸、 丙氨酸、 甘氨酸、 通式（I)结构或缺失； X213是丙氨酸、 脯氨酸、 精氨酸、 通式 (I)结构或缺失； X2i4是脯氨酸、 谷氨酰胺、 通式（I)结构或缺失； X215是谷氨酰胺、 苏氨 酸、 通式（I) 结构或缺失； X216是苏氨酸、 精氨酸、 赖氨酸、 通式（I) 结构或缺失； X218 是丝氨酸或通式（I)结构； 219是精氨酸或通式（I)结构； X22Q是精氨酸或通式（I)结构； X22i是谷氨酸或通式（I)结构； X222是天冬酰胺或通式（I)结构， 其中 UL和通式（I)如权 利要求 13中所定义。 X 20 la is glycine-valine-glutamate-threonine, phenylalanine-valine-asparagine-glutamine-histidine, UL-glycine-valine-glutamic acid - threonine or UL-phenyl- valine-valine-asparagine-glutamine-histidine; X 2 02 is stupid alanine or a deletion; X 2 Q 3 is asparagine or a deletion; X 2Q4 is valine, lysine, arginine, aspartic acid or deletion; X205 is valine, lysine or the structure of formula (I); X206 is threonine, lysine or a structure of formula (I); X 207 is a serine, alanine or a structure of formula (I); X 2G8 is a serine or a structure of formula (I); X 2 o 9 is a serine or a structure of formula (I); X 21Q Is a serine or a structure of formula (I); X 211 is arginine, alanine, structure or deletion of formula (I); X 212 is arginine, alanine, glycine, structure of formula (I) or Deletion; X 213 is alanine, valine, arginine, structure or deletion of formula (I); X 2 i4 is valine, glutamine, structure or deletion of formula (I); X 215 is Glutamine, threonine, structure of formula (I) or Deletion; X 216 is threonine, arginine, lysine, structure or deletion of formula (I); X 218 is serine or structure of formula (I); 219 is arginine or structure of formula (I) X 22Q is arginine or a structure of the formula (I); X 22 i is glutamic acid or a structure of the formula (I); X 222 is an asparagine or a structure of the formula (I), wherein U L and a formula (I) as defined in claim 13.
18、 根据权利要求 16-17任一项所述的化合物， 其特征在于， 所述化合物选自：  The compound according to any one of claims 16-17, wherein the compound is selected from the group consisting of:
III-3:  III-3:
GPETLCGAHLVDALFFVCGDRGFYFNPTGK( E-PEG20K)GSSSRRAPQTGIVDQCCFR SCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGK( E -PEG20K)GSSSRRAPQTGIVDQCCFR SCSLRRLENYCA;
III-4:  III-4:
GPETLCGAHLVDALFFVCGDRGFYFNPPTG (N£-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPPTG (N £ -PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
ΙΠ-5:  ΙΠ-5:
GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(Ne-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N e -PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
III- 12:  III- 12:
GPETLCGAHLVDALFFVCGD GFYFNPTGK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA; ΠΙ-13: GPETLCGAHLVDALFFVCGD GFYFNPTGK[N E -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA; ΠΙ-13:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N£-(Na-(HOOC(CH2)14CO)- Y-Glu)]SSAAPQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N £ -(N a -(HOOC(CH 2 ) 14 CO)- Y-Glu)]SSAAPQTGIVDQCCFRSCSLRRLENYCA;
111-14:  111-14:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]A APQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK[N E -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]A APQTGIVDQCCFRSCSLRRLENYCA;
111-15:  111-15:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA;
III- 16:  III- 16:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N£-(Na-(HOOC(CH2)14 GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N £ -(N a -(HOOC(CH 2 ) 14
CO)-y-Glu)]AAPQT GIVDQCCFRSCSLRRLENYCA; CO)-y-Glu)]AAPQT GIVDQCCFRSCSLRRLENYCA;
111-17:  111-17:
GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[NE-(Na-(HOOC (CH2)14CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[N E -(N a -(HOOC (CH 2 ) 14 CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
111-18:  111-18:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N E -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA;
111-20:  111-20:
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA; GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N e -(N a -(HOOC(CH 2 ) 14 CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA;
111-35: G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA; 111-35: G(N a -PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA;
111-37: GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[Ne-CO-(CH2CH20)5- (CH2)2-NH-(Na-(HOOC (CH2)16 CO)-y-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA; 111-37: GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[N e -CO-(CH 2 CH 2 0) 5 - (CH 2 ) 2 -NH-(N a -(HOOC (CH 2 ) 16 CO)-y-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA;
Na表示氨基酸或氨基酸残基的 a-氨基； Νε表示氨基酸或氨基酸残基的 ε-氨基， 例如赖氨 酸侧链的 ε-氨基。 N a represents an a-amino group of an amino acid or an amino acid residue; Ν ε represents an ε-amino group of an amino acid or an amino acid residue, such as an ε-amino group of a lysine side chain.
19、一种药物组合物，包括权利要求 1-18任一项所述的化合物和制药学上可接受的载体。 19. A pharmaceutical composition comprising a compound of any of claims 1-18 and a pharmaceutically acceptable carrier.
20、 根据权利要求 19所述的药物组合物， 进一步包括速效胰岛素或胰岛素类似物和 /或 添加剂。 20. The pharmaceutical composition of claim 19, further comprising a fast acting insulin or insulin analog and/or an additive.
21、根据权利要求 1-18任一项所述的化合物在制备治疗糖尿病或高血糖症的药物中的应 用。  Use of a compound according to any one of claims 1 to 18 for the manufacture of a medicament for the treatment of diabetes or hyperglycemia.
22、一种治疗糖尿病或高血糖症的方法， 包括对需要的病患施用根据权利要求 1-18任一 项所述的化合物或权利要求 19-20任一项所述的组合物。  A method of treating diabetes or hyperglycemia, comprising administering a compound according to any one of claims 1 to 18 or a composition according to any one of claims 19 to 20 to a patient in need thereof.
PCT/CN2012/001699 2011-12-15 2012-12-14 Compound and composition having hypoglycemic effect and use thereof WO2013086785A1 (en)

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