CN106554404B - Exenatide modifier and application thereof - Google Patents
Exenatide modifier and application thereof Download PDFInfo
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- CN106554404B CN106554404B CN201510619668.9A CN201510619668A CN106554404B CN 106554404 B CN106554404 B CN 106554404B CN 201510619668 A CN201510619668 A CN 201510619668A CN 106554404 B CN106554404 B CN 106554404B
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- C07—ORGANIC CHEMISTRY
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- 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
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- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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Abstract
The invention discloses an exenatide modifier and application thereof in preparing a medicament serving as a GLP-1 receptor agonist; the use for the manufacture of a medicament for the prevention and/or treatment of diseases and/or conditions associated with an impaired GLP-1 receptor activity; use in the manufacture of a medicament for a disease and/or condition associated with carbohydrate metabolism; use in the manufacture of a medicament for diabetes; the use in the manufacture of a medicament for the treatment of fatty liver; use in the manufacture of a medicament for use in weight loss.
Description
Technical Field
The invention relates to the field of therapeutic peptides, in particular to exenatide modifications, application and a preparation method thereof, a pharmaceutical composition containing the exenatide modifications and application of the exenatide modifications in treatment of diseases related to glycometabolism.
Background
Exenatide (also called Escissina peptide or Exenatide, or Exendin-4, trade name Byetta) is a polypeptide consisting of 39 amino acids, with molecular weight of 4186.6 and molecular formula C184H282N50O60S, the amino acid sequence is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2(ii) a Manufactured and sold by Amylin Pharmaceuticals and Eli Lilly Company (Eli Lillyand Company). Exenatide is approved by FDA to be marketed in 4 months of 2005, belongs to a drug subcutaneous injection preparation, has the effects of promoting glucose-dependent insulin secretion, restoring first-phase insulin secretion, inhibiting glucagon secretion, slowing down emptying of gastric contents, improving pancreatic beta cell function and the like, and is very suitable for treating type II diabetes, for example, for improving and controlling blood sugar of type II diabetes patients who are not ideal for treatment of metformin and sulfonylurea drugs.
Exenatide is a synthetic form of the hormone exendin-4 (J.biol.chem.1990,265, 20259-20262; J.biol.chem.1992,267,7402-7405) in the saliva of the Eremiatis (Gilamonter) grown in several states in the southwest U.S.A.), an analog of human glucagon-like peptide-1 (GLP-1) whose amino acid sequence partially overlaps with that of GLP-1, is a more potent GLP-1 receptor agonist, and is also called an incretin agonist because exenatide mimics the glucose-regulating effect of GLP-1. Unlike sulfonylureas and meglitinides, exenatide only increases insulin synthesis and secretion in the presence of glucose, reducing the risk of hypoglycemia. Byetta is also used by some physicians to treat insulin resistance.
Nevertheless, because protein/polypeptide drugs have the characteristics of short half-life in vivo, poor physical and chemical stability, and easy degradation by various proteases in vivo, these drugs usually need to be injected several times within a day, which brings much pain and inconvenience to patients. The pegylation technology that emerged in the last 70's of the century proved to be a more suitable technology in the current field of protein/polypeptide administration. However, the activity of the drug is generally reduced by the simple use of PEG.
A range of different approaches have been used to modify the structure of GLP-1 analogues in order to provide a longer duration of action in vivo. CN1384755 discloses a novel exendin agonist preparation and a method for administering the same, wherein a compound structure of exenatide and a preparation method thereof are disclosed. CN102532303 discloses a method for synthesizing polyethylene glycol-conjugated exenatide using methoxy polyethylene glycol residue conjugation with amino group of lysine residue or amino group of N-terminal histidine residue in exenatide molecule; CN101980725 discloses the structure of fatty acid-PEG-exenatide, and the modification site of PEG is on N-terminal His; WO2005028516 and WO2012035139 also disclose the structure of fatty acid-PEG-exenatide. CN101215324 discloses an exenatide short peptide mimic peptide obtained by structural modification of exenatide. CN101125207 reported PEG modification of Exendin-4. WO99/43708 discloses GLP-1(7-35) and GLP-1(7-36) derivatives with a lipophilic substituent attached to the C-terminal amino acid residue. WO2013059323 discloses PEG conjugated exenatide and a preparation method.
CN102397558 discloses that certain amino acids in Exendin-4 are substituted with cysteine, and then modified with PEG or PEG with methyl substituted end. CN102421796 discloses one or more polyethylene glycols polymerized to cysteine of Exendin variant, and discloses exenatide in which one amino acid is substituted with cysteine, and then the cysteine is modified with polyethylene glycol. CN102827270 discloses Exendin-4-Cys-PEG derivatives, specifically, 1 cysteine is introduced into the C end of the inactive area of an exenatide molecule, and maleamido polyethylene glycol is coupled, wherein the end of the polyethylene glycol is blocked by a methyl group.
Despite these various efforts, the currently available Exendin-4 or its variants and modifications have some disadvantages, including high administration frequency in vivo, large physical, psychological and economic burden on patients, limited patient compliance, and no wide application. There is still a great need for long-acting active GLP-1 analogs in the diabetic population.
CN103338790 discloses such exenatide modifications: cysteine was inserted at position 40, followed by succinimide-activated monomethoxy PEG modification. Although modified by the modification, compared with a medicament modified by PEG, the GLP-1 receptor binding property and the duration of hypoglycemia are improved to a certain extent. However, the improvement degree is not enough to meet the clinical requirement of GLP-1 receptor agonist drugs with excellent drug effect and pharmacokinetic property, and a new exenatide derivative is still urgently needed to be developed, so that the exenatide derivative has long in-vivo action time, good stability and good blood sugar reduction effect, and simultaneously keeps low toxicity and better activity.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of poor clinical use effect or frequent injection caused by low binding force of GLP-1 receptor and short hypoglycemic duration of the exenatide modifier disclosed at present.
It is known to those skilled in the art that in a biologically active molecule conjugated to a polymeric group, the biological activity of the conjugated biomolecule will decrease exponentially as the molecular weight of the conjugated group increases. It is also known to those skilled in the art that as the molecular weight of the polymer group increases, the biological half-life and/or plasma half-life of the conjugate molecule and systemic drug exposure gradually increase or increase.
The inventors of the present invention have unexpectedly found that modification of exenatide by the skilled person not only improves the pharmacokinetic properties, but also leads to an increased duration of hypoglycaemia. Compared with exenatide, the molecule still retains most GLP-1 receptor agonist activity, which means that the molecule not only has higher GLP-1 receptor agonist activity, but also has long hypoglycemia duration, and is likely to become a medicament with long in-vivo action time, good stability and good hypoglycemic effect in future clinic.
In one aspect, the present invention provides exenatide modifications or a pharmaceutically acceptable salt thereof, as shown in formula (i):
(Ex-4)-Cys-X-PEG-Y(Ⅰ)
wherein Ex-4 is Exendin-4; cys is cysteine-NH2The N end of the amino acid is connected with the C end of the serine at the Ex-439 position; x is ethyl succinimide
The ring is linked to the thiol group of Cys; PEG is modified by polyethylene glycol and is connected with the ethyl of X; y is an aliphatic hydrocarbon.
More particularly, the present invention provides exenatide modifications:
wherein m is any integer between 1 and 10; n is any integer between 6 and 20.
The exenatide modified substance has the following specific structure:
wherein m is any integer between 1 and 10; n is any integer between 6 and 20.
More specifically, the present invention provides exenatide modifications as follows:
TABLE 1 Compounds of the invention
| Compound (I) | m | n | Compound (I) | m | n |
| 1 | 1 | 20 | 8 | 8 | 6 |
| 2 | 4 | 6 | 9 | 8 | 8 |
| 3 | 4 | 8 | 10 | 8 | 10 |
| 4 | 4 | 10 | 11 | 8 | 12 |
| 5 | 4 | 12 | 12 | 8 | 14 |
| 6 | 4 | 14 | 13 | 8 | 16 |
| 7 | 4 | 16 | 14 | 10 | 18 |
。
In another aspect, the invention provides the use of the exenatide modification or a pharmaceutically acceptable salt thereof in the preparation of a medicament as a GLP-1 receptor agonist; the use for the manufacture of a medicament for the prevention and/or treatment of diseases and/or conditions associated with an impaired GLP-1 receptor activity; use in the manufacture of a medicament for a disease and/or condition associated with carbohydrate metabolism; use in the manufacture of a medicament for diabetes; the use in the manufacture of a medicament for the treatment of fatty liver; use in the manufacture of a medicament for use in weight loss.
In a third aspect, the invention provides a composition comprising the exenatide modification or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier.
In a fourth aspect, the invention provides the use of a composition as described above for the manufacture of a medicament as a GLP-1 receptor agonist; the use for the manufacture of a medicament for the prevention and/or treatment of diseases and/or conditions associated with an impaired GLP-1 receptor activity; use in the manufacture of a medicament for a disease and/or condition associated with carbohydrate metabolism; use in the manufacture of a medicament for diabetes; the use in the manufacture of a medicament for the treatment of fatty liver; use in the manufacture of a medicament for use in weight loss.
The exenatide modification compound provided by the invention not only has higher GLP-1 receptor agonism activity, but also has long hypoglycemia duration. The following is illustrated by pharmacological tests:
effect of Compounds on intracellular cAMP Activity
Each test sample was dissolved in double distilled water to a final concentration of 1.0X 10-2mol/L, and storing at 4 ℃. PC12 cells cultured at 25cm2Placing in a culture flask in CO2Incubator (37 ℃, 95% air, 5% CO)2) The medium was DMEM (pH 7.4, high sugar), and 5% fetal bovine serum and 10% horse serum were added. PC12 cells in good growth state were digested with 0.25% pancreatin, the cell concentration was adjusted to 1.0X 105 cells/ml, and the cells were seeded in a 24-well plate, and when the cells grew to 60-70% density, the cells were washed twice with PBS, 1ml of 1% BSA in PBS was added, and each test drug was fractionated into 5 gradient concentrations (10% BSA in each case)-10、10-9、10-8、10-7、10- 6mol/L) was incubated with IBMX (100. mu. mol/L) for 30min, 3 replicates were performed per concentration of sampleAnd (4) hole operation. At the end of the drug intervention period, cells were immediately harvested, suspended in cold PBS and adjusted to a cell concentration of 1.0X 107At one/ml, 1 volume of IN HCl was immediately added to 9 volumes of cell suspension, incubated for 10min at room temperature and sonicated for 15s with a sonicator. Centrifuging at 4 deg.C and 1000rpm for 10min to remove cell debris, adding 1N NaOH with the same volume as 1N HCl into the supernatant for neutralization to obtain cAMP-containing sample solution, and storing at-20 deg.C for detection. And (3) detecting the concentration of the total protein in the sample by using a non-interference protein concentration determination kit. The content of cAMP in the cell lysate is detected by using an ELISA kit, the operation is carried out according to the kit instruction, and the OD value is measured by using a BIO-RAD 680 enzyme-linked immunosorbent assay instrument at 450 nm. And fitting a curve by using CurveExpert 1.3 software according to the OD value of the standard substance, calculating a standard curve formula, and calculating the concentration of each sample. Data processing and charting were performed using the computer programs Microsoft Excel and GraphPad Prism 5 software, and the EC for each test drug was calculated50。
TABLE 2 Effect of Compounds on intracellular cAMP Activity
| Test article | EC50 | Test article | EC50 |
| Exendin-4 | 5.096nmol/L | Compound 8 | 5.289nmol/L |
| Compound 1 | 5.662nmol/L | Compound 9 | 5.178nmol/L |
| Compound 2 | 5.227nmol/L | Compound 10 | 5.296nmol/L |
| Compound 3 | 5.098nmol/L | Compound 11 | 5.285nmol/L |
| Compound 4 | 5.155nmol/L | Compound 12 | 5.342nmol/L |
| Compound 5 | 5.329nmol/L | Compound 13 | 5.263nmol/L |
| Compound 6 | 5.312nmol/L | Compound 14 | 5.234nmol/L |
| Compound 7 | 5.363nmol/L |
GLP-1, when bound to GLP-1 receptor (G-coupled protein of the beta receptor family), activates the cyclic adenosine adenine (cAMP) and mitogen-activated protein (MAPK) pathways in the cell membrane. GLP-1 receptor coupled Gs from mature beta cells of pancreatic islets activates adenylyl cyclase, produces cAMP, which in concert with glucose stimulates insulin synthesis and secretion, stimulates insulin gene transcription and proinsulin biosynthesis, lowers glucagon concentration and inhibits glucagon secretion, increases sensitivity of cells to insulin, stimulates insulin-dependent glycogen synthesis, lowers postprandial blood glucose concentration. EC (EC)50The smaller, it indicates the higher the agonistic activity of the GLP-1 receptor of the drug.
The results in Table 2 show that the activity of the compounds of the invention is reduced considerably or only slightly compared to exenatide, which indicates that the modification of exenatide of the invention does not affect its GLP-1 receptor agonistic activity.
Has effect of reducing blood glucose of spontaneous type 2 diabetes db/db mice
5-6 week old C57BL/6db/db mice (male) were purchased from Nanjing university model animal research institute, and experimental animals were housed in SPF animal houses. The animal room is well ventilated, an air conditioner is arranged, the temperature is kept at 20-25 ℃, the humidity is kept at 40% -70%, the ventilation frequency is 10-15 times/h, the lighting is carried out in a dark place for 12 hours respectively, the experimental animals eat and drink water freely, and each mouse is marked with an ear tag. Mice were used for one experiment per week, and mice were used for no more than three weeks. After one week of adaptation, mouse tip capillary blood glucose was measured using a majoram glucometer. 96 mice with blood glucose level of more than 16.7mmol/L were selected, and randomly divided into 16 groups according to blood glucose level, model control group was subcutaneously injected with 5mL/kg of PBS (pH 7.4), positive control group 1 was subcutaneously injected with exenatide (10. mu.g/kg, 5mL/kg), administration groups were subcutaneously injected with compounds 1-15 (10. mu.g/kg, 5mL/kg), 0, 1, 2, 4, 8, 12, 18, 24, 30, 36, 42, 48, 72h of blood glucose was measured using a glucometer after administration, and all data were inputted into Graphpad Prism to calculate mean blood glucose. The maximum hypoglycemic effect (maximum rate of decrease compared to the model group), the maximum hypoglycemic time (last time point of significant decrease in blood glucose compared to the model group), and the area under the curve were calculated.
TABLE 3 hypoglycemic effects on spontaneous type 2 diabetic db/db mice
| Group of | Maximum hypoglycemic action (%) | Maximum blood sugar lowering time (h) |
| Exenatide group | 64.46％ | 4 |
| Compound 1 | 59.23％ | 36 |
| Compound 2 | 61.11％ | 24 |
| Compound 3 | 64.32％ | 30 |
| Compound 4 | 63.15％ | 36 |
| Compound 5 | 60.20％ | 36 |
| Compound 6 | 59.89％ | 42 |
| Compound 7 | 60.51％ | 42 |
| Compound 8 | 61.84％ | 30 |
| Compound 9 | 63.20％ | 30 |
| Compound 10 | 62.23％ | 36 |
| Compound 11 | 60.54％ | 42 |
| Compound 12 | 61.39％ | 48 |
| Compound 13 | 60.71％ | 48 |
| Compound 14 | 60.35％ | 48 |
The results in table 3 show that the compounds of the present invention have great advantages in maintaining the hypoglycemic time compared to exenatide, extending the maximum hypoglycemic time of 4h to 24h-48h, especially extending the maximum hypoglycemic time of more than 40 h for compounds 6, 7, 11, 12, 13, 14.
In conclusion, compared with exenatide, the exenatide modified compound has equivalent or slightly reduced activity, and the GLP-1 receptor agonistic activity of the exenatide modified compound is not influenced. Meanwhile, the exenatide modification substance has great advantages in maintaining hypoglycemia time, and the maximum hypoglycemia time is prolonged from 4 hours to 24-48 hours. The molecule of the invention not only has higher GLP-1 receptor agonist activity, but also has long hypoglycemia duration, and is likely to become a medicament with long in-vivo action time, good stability and good hypoglycemic effect in future clinic.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1 Exendin-4(1-39) -Cys (40) -NH2Preparation of
Amino acids and abbreviations and english abbreviation:
the solid-phase peptide synthesis of the target peptide adopts Fmoc method solid-phase synthesis, Fmoc-Rink MBHA Amide resin is utilized, 20% piperidine/DMF is adopted to remove Fmoc, HOBT/DIC is adopted as a coupling reagent, DMF is used as a reaction solvent, ninhydrin detection method is adopted for reaction monitoring, and the following protected amino acids are connected to the Rink MBHA Amide resin in sequence: Fmoc-Cys (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH, Fmoc-Gln-Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, F, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc protection is finally removed, DMF washing, dichloromethane washing, MeOH washing and drying are carried out to obtain resin containing full protection, the cracking of the resin adopts 82.5 percent TFA/5 percent phenol/5 percent water/2.5 percent EDT/5 percent phenylmethyl sulfide, the MTBE of ice is precipitated and washed, and the crude product is purified by reversed phase HPLC to obtain Exendin-4(1-39) -Cys (40) -NH2And (5) purifying.
MS(ESI+,m/e)：4290.04[M+H]+。
EXAMPLE 2 preparation of Compound 1
Preparation of BP103a01
Under the protection of nitrogen, 200mL of pyridine and 120g of BP103a00(1.0eq) are added into a 1000mL three-neck flask, stirred and cooled to 0 ℃, 151.8g of TsCl (1.0eq) is added in portions, stirred for 1h, then slowly warmed to room temperature, and stirred for 3-4 h. After the reaction is finished, pouring the reaction solution into an ice dilute hydrochloric acid solution to generate solid, adding EA for extraction, washing an EA layer once with dilute hydrochloric acid, washing with saturated sodium bicarbonate, washing with saturated salt water, and washing with anhydrous Na2SO4Drying, vacuum evaporating to remove solvent to obtain 119g crude product, and performing silica gel column chromatography to obtain 55g pure product BP103a 01.
Preparation of BP103a02
55g BP103a01(1.0eq) and 160mL DMSO were added to a 1000mL three-necked flask, stirred well, and then NaN was added323.52g (2.0eq), heating to 50 ℃ for reaction for 3 hours, cooling to room temperature, pouring the reaction solution into 1.2L of water, extracting with EA, combining organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain BP103a02 colorless liquid 29.2 g.
Preparation of BP103a03
29g of the compound BP103a02, 360mL of methanol and 5.0g of palladium carbon are added into a 1L hydrogenation reaction kettle, the mixture is stirred and replaced by nitrogen, hydrogen is introduced for reaction for 3 to 4 hours, after the TLC monitoring reaction is finished, the reaction solution is filtered, and the filtrate is concentrated to obtain 23.5g of oily matter of BP103a 03.
Preparation of BP103a04
A250 mL flask was charged with 5.2g BP103a03(1.0eq), 50mL water, 5.86g NaHCO3(2.0eq), stirring, dropwise adding a solution of 15.25g of an activated ester of n-docosanoic acid (1.0eq) in 50ml of DME (ethylene glycol dimethyl ether), adding 50ml of THF, stirring overnight, monitoring the reaction by TLC, evaporating the organic solvent, adjusting the pH to 4 with dilute hydrochloric acid, extracting with dichloromethane, drying over anhydrous sodium sulfate, concentrating, and carrying out column chromatography to obtain 11.5g of BP103a 04.
Preparation of BP103a05
Into a 100mL three-necked flask, 50mL of THF, 5.00g of BP103a04, 2.57g of maleimide (2.5eq) were added; 4.17g triphenylphosphine (1.5eq) and 3.70g DEAD (2.0eq) were dissolved in 10ml THF, slowly added dropwise into the reaction flask, monitored by TLC, the reaction solution was spin-dried, and column chromatography was performed to obtain BP103a 052.23g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 13mg of BP103a05(2.0eq) was added and stirred at 20 ℃ for 1 hour, and after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to obtain 34mg of the conjugate compound.
MS(ESI+,m/e)：4840.49[M+H]+
EXAMPLE 3 preparation of Compound 2
Step1
150mL of pyridine and 50g of BP103g00(1.0eq) are added into a 250mL three-neck flask under the protection of nitrogen, stirred and cooled to 0 ℃, 33.7g of TsCl (1.0eq) is added in batches, stirred for 1h, slowly heated to room temperature, and stirred for 3-4 h. TLC monitoring reaction is finished, the reaction liquid is poured into ice dilute hydrochloric acid solution, EA is added for extraction, organic phases are combined, EA layer is washed once by dilute hydrochloric acid, washed by saturated sodium bicarbonate, washed by saturated salt solution and anhydrous Na2SO4Drying, spin-drying to obtain 59g, and performing column chromatography to obtain 31g of pure BP103g 01.
Step2
A250 mL three-necked flask was charged with 33.5g of BP103g01(1.0eq) and 100mL of DMSO, stirred, and 10.0g of NaN was added3(2.0eq), heating to 50 ℃ for reaction for 3 hours, cooling to room temperature, monitoring by TLC after the reaction is finished, pouring the reaction liquid into 1.5L of water, extracting by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, and concentrating to obtain 24.0g of colorless liquid BP103g 02.
Step3
24.0g BP103g02, 280mL methanol and 6.0g palladium carbon are added into a 1L hydrogenation reaction kettle, hydrogen is introduced for reaction for 3-4h, a point plate monitors that the reaction is finished, reaction liquid is filtered, and 21.0g oily matter BP103g03 is obtained by concentration.
Preparation of BP103g30
Step1
2.01g BP103g03(1.0eq), 20ml water, 0.66g sodium bicarbonate (1.1eq) were added into a 100ml three-necked flask, 1.89g of a solution of n-octanoic acid activated ester (1.1eq) dissolved in 20ml DME was added dropwise, 20ml THF was added, the mixture was stirred overnight to monitor completion of the reaction, DME and THF were evaporated, the pH of the aqueous phase was adjusted to 3, EA was extracted 3 times, the organic phases were combined, washed with a sodium bicarbonate aqueous solution, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain 103BPg292.15g.
Step2
A100 mL three-necked flask was charged with 10mL of THF, 1.0g of BP103g29, 0.60g of maleimide (2.5 eq); dissolving 0.97g of triphenylphosphine and 0.85g of DEAD in 10ml of THF, slowly dropping into a reaction bottle, monitoring the reaction by TLC, spin-drying the reaction solution, and carrying out column chromatography to obtain BP103g 300.53g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 11mg of BP103g30(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 36mg of the conjugated compound.
MS(ESI+,m/e)：4820.37[M+H]+
EXAMPLE 4 preparation of Compound 3
Step1
BP103g03 was prepared according to the method of example 3. Adding 2.00g BP103g03(1.0eq), 20ml water and 0.66g sodium bicarbonate (1.1eq) into a 100ml three-neck flask, adding 2.11g n-decanoic acid activated ester (1.1eq) dissolved in 20ml DME, supplementing 20ml THF, stirring overnight for TLC monitoring reaction, evaporating DME and THF, adjusting pH of aqueous phase to 3, extracting EA for 3 times, combining organic phases, washing with sodium bicarbonate solution, washing with water, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain 103BPg312.10g
Step2
A100 mL three-necked flask was charged with 10mL of THF, 1.00g of BP103g31, 0.56g of maleimide (2.5 eq); 0.90g of triphenylphosphine and 0.80g of DEAD are dissolved in 10ml of THF, the solution is slowly dropped into a reaction bottle, the reaction is monitored by TLC, the reaction solution is dried by spinning, and the column chromatography is carried out to obtain BP103g 320.45g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 11mg of BP103g32(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 32mg of the conjugate compound.
MS(ESI+,m/e)：4848.40[M+H]+
EXAMPLE 5 preparation of Compound 4
Step1
BP103g03 was prepared according to the method of example 3. . 2.00g BP103g03(1.0eq), 20ml water, 0.66g sodium bicarbonate (1.1eq) were added into a 100ml three-necked flask, 2.32g dodecanoic acid activated ester (1.1eq) was added dropwise in a solution of 20ml DME, 20ml THF was added, the mixture was stirred overnight to monitor the completion of the reaction, DME and THF were evaporated, the pH of the aqueous phase was adjusted to 3, EA was extracted 3 times, the organic phases were combined, washed with sodium bicarbonate solution, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain 103BPg332.34g.
Step2
A100 mL three-necked flask was charged with 10mL of THF, 1.00g of BP103g33, 0.52g of maleimide (2.5 eq); dissolving 0.85g of triphenylphosphine and 0.75g of DEAD in 10ml of THF, slowly and dropwise adding into a reaction bottle, monitoring the reaction by TLC, spin-drying the reaction solution, and carrying out column chromatography to obtain BP103g 340.36g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolving in 10ml 20mM sodium phosphate buffer solution (pH 6.5), adding 13mg BP103g34(2.0eq) and stirring at 20 deg.C for 1 hr, stopping reaction with excessive cysteine solution (0.5ml 0.5M cysteine solution) after HPLC monitoring reaction, and lyophilizing to obtain the final productConjugate 37 mg.
MS(ESI+,m/e)：4876.43[M+H]+
EXAMPLE 6 preparation of Compound 5
Step1
BP103g03 was prepared according to the method of example 3. Adding 2.00g BP103g03(1.0eq), 20ml water and 0.66g sodium bicarbonate (1.1eq) into a 100ml three-neck flask, dropwise adding a solution of 2.54g tetradecanoic acid activated ester (1.1eq) dissolved in 20ml DME, supplementing 20ml THF, stirring overnight for TLC monitoring reaction, evaporating DME and THF, adjusting pH of an aqueous phase to 3, extracting EA for 3 times, combining organic phases, washing with an aqueous solution of sodium bicarbonate, washing with water, washing with saturated saline, drying with anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain BP103g352.35gStep2
A100 mL three-necked flask was charged with 10mL of THF, 1.00g of BP103g35, 0.49g of maleimide (2.5 eq); dissolving 0.80g of triphenylphosphine and 0.71g of DEAD in 10ml of THF, slowly and dropwise adding into a reaction bottle, monitoring the reaction by TLC, spin-drying the reaction solution, and carrying out column chromatography to obtain BP103g of 360.31g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH 2 prepared according to the method of example 1 was dissolved in 10ml of 20mM sodium phosphate salt buffer (pH 6.5), 13mg of BP103g36(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to obtain 33mg of the conjugated compound.
MS(ESI+,m/e)：4904.46[M+H]+。
EXAMPLE 7 preparation of Compound 6
Step1
BP103g03 was prepared according to the method of example 3. 2.00g BP103g03(1.0eq), 20ml water, 0.66g sodium bicarbonate (1.1eq) were added into a 100ml three-necked flask, 2.76g activated palmitate (1.1eq) dissolved in 20ml DME was added dropwise, 20ml THF was added, the reaction was monitored by stirring overnight, DME and THF were evaporated off, the pH of the aqueous phase was adjusted to 3, EA extraction was performed 3 times, the organic phases were combined, washed with sodium bicarbonate solution, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain BP103g 372.46g.
Step2
A100 mL three-necked flask was charged with 10mL of THF, 1.00g of BP103g37, 0.47g of maleimide (2.5 eq); dissolving 0.76g of triphenylphosphine and 0.67g of DEAD in 10ml of THF, slowly dropping into a reaction bottle, monitoring the reaction by TLC, spin-drying the reaction solution, and carrying out column chromatography to obtain BP103g 380.35g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 14mg of BP103g38(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 30mg of the conjugate compound.
MS(ESI+,m/e)：4932.49[M+H]+。
EXAMPLE 8 preparation of Compound 7
Step1
BP103g03 was prepared according to the method of example 3. 2.00g BP103g03(1.0eq), 20ml water, 0.66g sodium bicarbonate (1.1eq) were added into a 100ml three-necked flask, 2.98g octadecanoic acid activated ester (1.1eq) was added dropwise into the flask and dissolved in 20ml DME, 20ml THF was added, the mixture was stirred overnight for TLC monitoring reaction completion, DME and THF were evaporated off, the pH of the aqueous phase was adjusted to 3, EA was extracted 3 times, the organic phases were combined, washed with sodium bicarbonate solution, washed with water, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain BP103g392.75g.
Step2
A100 mL three-necked flask was charged with 10mL of THF, 1.00g of BP103g39, 0.44g of maleimide (2.5 eq); dissolving 0.72g of triphenylphosphine and 0.64g of DEAD in 10ml of THF, slowly dropping into a reaction bottle, monitoring the reaction by TLC, spin-drying the reaction solution, and carrying out column chromatography to obtain BP103g 400.36g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 15mg of BP103g40(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to obtain 27mg of the conjugate compound.
MS(ESI+,m/e)：4960.52[M+H]+
EXAMPLE 9 preparation of Compound 8
Step1
In a 250mL three-neck flask, under the protection of nitrogen, 800mL of pyridine and 200g of BP103k00(1.0eq) are added, stirred and cooled to 0 ℃, 83.2g of TsCl (1.0eq) are added in batches, stirred for 1h, slowly heated to room temperature, and stirred for 3-4 h. TLC monitoring reaction is finished, the reaction liquid is poured into ice dilute hydrochloric acid solution, EA is added for extraction, organic phases are combined, EA layer is washed once by dilute hydrochloric acid, washed by saturated sodium bicarbonate, washed by saturated salt solution and anhydrous Na2SO4Drying, spin-drying to obtain 120.5g, and performing column chromatography to obtain 70.3g of pure BP103k 01.
Step2
70.00g BP103k01(1.0eq), 210mL DMSO were added to a 250mL three-necked flask, stirred, and added14.81g of NaN3(2.0eq) was heated to 50 ℃ for reaction for 3 hours and cooled to room temperature, TLC monitored the completion of the reaction, the reaction solution was poured into 1200mL of water, extracted with EA several times, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give 25.20g of colorless liquid BP103k 02.
Step3
25.20g of BP103k02, 300mL of methanol and 4.2g of palladium on carbon are added into a 1L hydrogenation reaction kettle, hydrogen is introduced for reaction overnight, a point plate monitors that the reaction is finished, the reaction solution is filtered, and 25.5g of oily matter BP103k03 is obtained by concentration.
Preparation of BP103k29
Step1
A100 ml three-necked flask was charged with 3.00g of BP103k03(1.0eq), 60ml of water, and 0.61g of sodium bicarbonate (1.1eq), and 1.73g of a solution of activated ester of n-octanoic acid (1.1eq) in 60ml of DME was added dropwise, 30ml of THF was added thereto, and the mixture was stirred overnight to monitor completion of the reaction, DME and THF were distilled off, the pH of the aqueous phase was adjusted to 3, EA was extracted 3 times, the organic phases were combined, washed with an aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain 103BPk282.10g.
Step2
Into a 100mL three-necked flask, 20mL of THF, 2.00g of BP103k28, 0.83g of maleimide (2.5eq) were added; 1.35g of triphenylphosphine and 1.20g of DEAD are dissolved in 20ml of THF, the solution is slowly dropped into a reaction bottle, the TLC is used for monitoring the reaction, the reaction solution is dried in a spinning mode, and the column chromatography is carried out to obtain BP103k290.43g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolving in 10ml 20mM sodium phosphate buffer solution (pH 6.5), adding 15mg BP103k29(2.0eq) and stirring at 20 deg.C for 1 hr, stopping reaction with excess cysteine solution (0.5ml 0.5M cysteine solution) after HPLC monitoring reaction, and lyophilizing to obtain even solutionThe combination is 29 mg.
MS(ESI+,m/e)：4996.47[M+H]+
EXAMPLE 10 preparation of Compound 9
Step1
BP103k03 was prepared according to the method of example 3. A100 ml three-neck flask is added with 3.00g BP103k03(1.0eq), 60ml water and 0.61g sodium bicarbonate (1.1eq), 1.94g n-decanoic acid activated ester (1.1eq) is added dropwise into a solution dissolved in 60ml DME, 30ml THF is added, the TLC is stirred overnight to monitor the completion of the reaction, DME and THF are evaporated, the pH of the aqueous phase is adjusted to 3, EA is extracted for 3 times, the organic phases are combined, washed by sodium bicarbonate aqueous solution, washed by water and saturated common salt water, dried by anhydrous sodium sulfate, concentrated and subjected to column chromatography to obtain BP103k302.33g.
Step2
Into a 100mL three-necked flask, 20mL of THF, 2.33g of BP103k30, 0.92g of maleimide (2.5eq) were added; 1.44g triphenylphosphine and 1.33g DEAD were dissolved in 20ml THF, slowly added into the reaction flask, TLC was used to monitor the reaction, the reaction solution was spun dry, and column chromatography was performed to obtain BP103k 310.54g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 16mg of BP103k31(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was stopped with an excess of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 33mg of the conjugate compound.
MS(ESI+,m/e)：5024.5[M+H]+
EXAMPLE 11 preparation of Compound 10
Step1
BP103k03 was prepared according to the method of example 3. . Adding 3.00g of BP103k03(1.0eq), 60ml of water and 0.61g of sodium bicarbonate (1.1eq) into a 100ml three-neck flask, dropwise adding a solution of 2.14g of dodecanoic acid activated ester (1.1eq) dissolved in 60ml of DME, supplementing 30ml of THF, stirring overnight for TLC monitoring reaction completion, evaporating DME and THF, adjusting the pH of an aqueous phase to 3, extracting EA for 3 times, combining organic phases, washing with an aqueous solution of sodium bicarbonate, washing with water, washing with saturated saline, drying with anhydrous sodium sulfate, concentrating, and carrying out column chromatography to obtain 103BPk321.97g
Step2
Into a 100mL three-necked flask, 20mL of THF, 1.97g of BP103k32, 0.75g of maleimide (2.5eq) were added; 1.21g of triphenylphosphine and 1.07g of DEAD are dissolved in 20ml of THF, the solution is slowly dropped into a reaction bottle, the reaction is monitored by TLC, the reaction solution is dried by spinning, and the column chromatography is carried out to obtain BP103k330.45g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 17mg of BP103k33(2.0eq) was added and stirred at 20 ℃ for 1 hour, and after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to obtain 27mg of the conjugate compound.
MS(ESI+,m/e)：5052.53[M+H]+
EXAMPLE 12 preparation of Compound 11
Step1
BP103k03 was prepared according to the method of example 3. A100 ml three-necked flask was charged with 3.00g of BP103k03(1.0eq), 60ml of water, and 0.61g of sodium bicarbonate (1.1eq), 2.45g of tetradecanoic acid activated ester (1.1eq) was added dropwise to the flask in 60ml of DME, 30ml of THF was added thereto, the mixture was stirred overnight for TLC monitoring, DME and THF were distilled off, the aqueous phase was adjusted to pH 3, EA was extracted 3 times, the organic phases were combined, washed with an aqueous sodium bicarbonate solution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to give BP103k 342.26g.
Step2
Into a 100mL three-necked flask, 20mL of THF, 2.26g of BP103k34, 0.82g of maleimide (2.5eq) were added; 1.31g of triphenylphosphine and 1.16g of DEAD were dissolved in 20ml of THF, and the solution was slowly added dropwise into the flask, followed by monitoring the reaction by TLC, spin-drying the reaction mixture, and column chromatography to obtain BP103k350.56g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 17mg of BP103k35(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 28mg of the conjugate compound.
MS(ESI+,m/e)：5080.56[M+H]+
EXAMPLE 13 preparation of Compound 12
Step1
BP103k03 was prepared according to the method of example 3. A100 ml three-necked flask was charged with 3.00g of BP103k03(1.0eq), 60ml of water, and 0.61g of sodium bicarbonate (1.1eq), and a solution of 2.55g of activated palmitate (1.1eq) in 60ml of DME was added dropwise, 30ml of THF was added thereto, and the mixture was stirred overnight to monitor completion of the reaction, DME and THF were distilled off, the pH of the aqueous phase was adjusted to 3, EA was extracted 3 times, the organic phases were combined, washed with a sodium bicarbonate solution, washed with water, washed with brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain 103K 362.10g of BPB.
Step2
Into a 100mL three-necked flask, 20mL of THF, 2.10g of BP103k36, 0.73g of maleimide (2.5eq) were added; 1.31g of triphenylphosphine and 1.19g of DEAD are dissolved in 20ml of THF, the solution is slowly dropped into a reaction bottle, TLC is used for monitoring the reaction, the reaction solution is dried in a spinning mode, and column chromatography is carried out to obtain BP103k370.42g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 18mg of BP103k37(2.0eq) was added and stirred at 20 ℃ for 1 hour, and after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to obtain 22mg of the conjugate compound.
MS(ESI+,m/e)：5108.59[M+H]+
EXAMPLE 14 preparation of Compound 13
Step1
BP103k03 was prepared according to the method of example 3. A100 ml three-necked flask was charged with 3.00g of BP103k03(1.0eq), 60ml of water, and 0.61g of sodium bicarbonate (1.1eq), 2.75g of activated palmitate (1.1eq) was added dropwise to 60ml of DME, 30ml of THF was added, the mixture was stirred overnight to monitor completion of the reaction, DME and THF were distilled off, the aqueous phase was adjusted to pH 3, EA was extracted 3 times, the organic phases were combined, washed with sodium bicarbonate solution, washed with water, washed with brine, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to obtain BP103k382.41g.
Step2
Into a 100mL three-necked flask, 20mL of THF, 2.41g of BP103k38, 0.81g of maleimide (2.5eq) were added; 1.31g of triphenylphosphine and 1.16g of DEAD are dissolved in 20ml of THF, the solution is slowly dropped into a reaction bottle, the reaction is monitored by TLC, the reaction solution is dried by spinning, and the column chromatography is carried out to obtain BP103k390.51g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 19mg of BP103k39(2.0eq) was added and stirred at 20 ℃ for 1 hour, and after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 29mg of the conjugate compound.
MS(ESI+,m/e)：5136.62[M+H]+。
EXAMPLE 15 preparation of Compound 14
Preparation of BP103m01
Under the protection of nitrogen, 200mL of pyridine and 100.0g of BP103m00(1.0eq) are added into a 1000mL three-necked flask, stirred and cooled to 0 ℃, 34.9g of TsCl (1.0eq) is added in portions, stirred for 1h, then slowly warmed to room temperature, and stirred for 3-4 h. After the reaction is finished, pouring the reaction solution into an ice dilute hydrochloric acid solution to generate solid, adding EA for extraction, washing an EA layer once with dilute hydrochloric acid, washing with saturated sodium bicarbonate, washing with saturated salt water, and washing with anhydrous Na2SO4Drying, vacuum evaporating to remove solvent to obtain crude product 86g, and performing silica gel column chromatography to obtain pure product 36.3g of BP103m 01.
Preparation of BP103m02
Adding 36.0g of BP103m01(1.0eq) and 110mL of DMSO into a 250mL three-necked flask, stirring uniformly, then adding NaN36.7g (2.0eq), heating to 50 ℃ for reaction for 3 hours, cooling to room temperature, pouring the reaction liquid into 1.0L of water, extracting with EA, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, and concentrating to obtain 25.2g of BP103m02 colorless liquid.
Preparation of BP103m03
Adding 25.2g of compound BP103m02, 300mL of methanol and 4.3g of palladium carbon into a 1L hydrogenation reaction kettle, stirring, replacing with nitrogen, introducing hydrogen for reaction for 3-4h, filtering the reaction solution after TLC monitoring reaction, and concentrating the filtrate to obtain an oily compound BP103m0322.5g.
Preparation of BP103m04
A250 mL flask was charged with 12.0g of Compound BP103m03(1.0eq), 100mL of water, 3.7g of NaHCO3(2.0eq), stirring, dropwise adding a solution of 9.0g of an activated ester of n-docosanoic acid (1.0eq) in 100ml of DME (ethylene glycol dimethyl ether), supplementing 100ml of THF, stirring overnight, monitoring the reaction by TLC, evaporating the organic solvent, adjusting the pH to 4 with dilute hydrochloric acid, extracting with dichloromethane, drying over anhydrous sodium sulfate, concentrating, and carrying out column chromatography to obtain 13.4g of an oily compound BP103m 04.
Preparation of BP103m05
Into a 100mL three-necked flask, 50mL of THF, 5.00g of Compound BP103m04, 1.44g of maleimide (2.5eq) were added; 2.34g of triphenylphosphine (1.5eq) and 2.07g of DEAD (2.0eq) are dissolved in 10ml of THF, and the solution is slowly dropped into a reaction flask, TLC is used for monitoring the reaction, the reaction solution is dried by spinning, and column chromatography is carried out to obtain the compound BP103m052.43g.
50.0mg of Exendin-4(1-39) -Cys (40) -NH prepared according to the method of example 1 was taken2Dissolved in 10ml of 20mM sodium phosphate buffer (pH 6.5), 21mg of BP103M05(2.0eq) was added and stirred at 20 ℃ for 1 hour, after completion of the HPLC monitoring reaction, the reaction was terminated with an excess amount of cysteine solution (0.5ml of 0.5M cysteine solution), and lyophilized after the HPLC preparation to give 36mg of the conjugate compound.
MS(ESI+,m/e)：5164.65[M+H]+。
Claims (5)
1. An exenatide modification or a pharmaceutically acceptable salt thereof, as follows:
wherein, cysteine-NH2The N end of the amino acid is connected with the C end of the serine at the Ex-439 position; m is any integer between 1 and 10; n is any of 6 to 20An integer number.
2. The exenatide modification or a pharmaceutically acceptable salt thereof according to claim 1, comprising the following compounds:
3. use of exenatide modification or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2, which is: use in the manufacture of a medicament for diabetes; the use in the manufacture of a medicament for the treatment of fatty liver; use in the manufacture of a medicament for use in weight loss.
4. A pharmaceutical composition comprising the exenatide modification or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2, and optionally a pharmaceutically acceptable carrier.
5. Use of the composition of claim 4 which is: use in the manufacture of a medicament for diabetes; the use in the manufacture of a medicament for the treatment of fatty liver; use in the manufacture of a medicament for use in weight loss.
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| CN102781458A (en) * | 2009-11-23 | 2012-11-14 | 安米林药品公司 | Polypeptide conjugate |
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| CN102781458A (en) * | 2009-11-23 | 2012-11-14 | 安米林药品公司 | Polypeptide conjugate |
| CN102397558A (en) * | 2010-09-09 | 2012-04-04 | 中国人民解放军军事医学科学院毒物药物研究所 | Positioning pegylation modified compound of Exendin-4 analog and application thereof |
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