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WO2020019813A1 - 长效化胃泌酸调节素杂合肽及其制备方法和应用 - Google Patents

长效化胃泌酸调节素杂合肽及其制备方法和应用 Download PDF

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Publication number
WO2020019813A1
WO2020019813A1 PCT/CN2019/085593 CN2019085593W WO2020019813A1 WO 2020019813 A1 WO2020019813 A1 WO 2020019813A1 CN 2019085593 W CN2019085593 W CN 2019085593W WO 2020019813 A1 WO2020019813 A1 WO 2020019813A1
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acid
ser
gly
pro
gln
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English (en)
French (fr)
Inventor
钱海
黄文龙
蔡星光
李承业
刘春霞
戴雨轩
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China Pharmaceutical University
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China Pharmaceutical University
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Priority claimed from CN201810851571.4A external-priority patent/CN108948213B/zh
Priority claimed from CN201810851064.0A external-priority patent/CN109232743B/zh
Priority claimed from CN201810851509.5A external-priority patent/CN108822222B/zh
Priority claimed from CN201810851507.6A external-priority patent/CN108948212B/zh
Application filed by China Pharmaceutical University filed Critical China Pharmaceutical University
Priority to US17/262,489 priority Critical patent/US11970523B2/en
Publication of WO2020019813A1 publication Critical patent/WO2020019813A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • 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
    • 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
    • C07K14/605—Glucagons
    • 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 relates to the field of medicinal chemistry, in particular to a long-acting gastrin hybrid peptide, and a preparation method and application thereof.
  • the cause of the metabolic syndrome is the abnormal metabolism of a variety of substances such as proteins, fats and carbohydrates. Excessive nutrition and reduced physical activity can lead to obesity and obesity-related diseases, such as diabetes. In recent years, the incidence of type 2 diabetes and abnormal lipid metabolism has been increasing.
  • Oxyntomodulin is a polypeptide composed of 37 amino acids secreted by L cells of the small intestine. It contains the entire 29 amino acid sequence of glucagon and an 8 amino acid portion extended at the C-terminus.
  • Peptide-1 GLP-1
  • GLP-1R glucagon-like peptide-1 receptor
  • GCGR glucagon receptor
  • OXM activates GCGR, it can promote liver glycogen decomposition and gluconeogenesis, promote lipolysis and fatty acid oxidation; accelerate amino acids into liver cells, exert heat production, and have better weight loss and appetite suppression effects.
  • OXM has a better effect on intervention of body weight, regulation of lipid metabolism and improvement of glucose tolerance, but its blood glucose lowering activity is relatively weak, and its half-life is shorter.
  • GLP-1 is a glucose-dependent enterotropic hormone. It can stimulate GLP-1R and exert a hypoglycemic effect. The most significant function is to promote the regeneration and repair of ⁇ cells, increase the number of islet ⁇ cells, and also avoid the risk of hypoglycemia that often occurs in the treatment of diabetes. It has a broad application prospect in the field of diabetes treatment.
  • Exenatide is a typical short-acting GLP-1 receptor agonist that reduces the metabolism of DPP-IV enzymes. The introduction of a partial peptide sequence of exenatide in OXM can increase the compound's receptor agonistic activity on GLP-1R.
  • the present invention synthesizes a class of peptide sequence-modified OXM analogs.
  • the affinity of the peptide chain to GLP-1R is enhanced, and the GLP-1R is enhanced.
  • And maintained moderate GCGR agonistic activity and synthesized a class of peptide sequence engineered OXM analogs.
  • Coumarin and small molecules such as different types of fatty acids have high serum albumin binding rate.
  • Conjugating small molecules with OXM analogs greatly prolongs the duration of the hypoglycemic effect, which exceeds the existing marketed drugs liraglutide and exeter That peptide, to obtain a series of long-acting peptide drugs that are excellent in glucose-lowering activity and weight loss.
  • the present invention relates to a class of antidiabetic pharmaceutically acceptable salt thereof, or a polypeptide, which polypeptide is the amino acid sequence:
  • Xaa1 is Gly, Aib, D-Ser, Ser, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val;
  • Xaa2 is taken from
  • Y is -NH-CO- or -CO-NH-;
  • Xaa3 is Ser-OH, Ser-NH 2 .
  • a preferred solution of the present invention is characterized in that:
  • Xaa1 is taken from Gly or Aib;
  • Xaa2 is taken from
  • Y is -NH-CO- or -CO-NH-;
  • n is taken from the natural numbers 6,10,14,11,15;
  • n is taken from the natural number 10 or 11;
  • Xaa3 is taken from Ser-OH or Ser-NH 2 .
  • hypoglycemic polypeptide or a pharmaceutically acceptable salt thereof according to the present invention can also be expressed as:
  • a pharmaceutically acceptable salt is a compound with hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid ,
  • benzoic acid salicylic acid, 2- (4-hydroxybenzoic acid) Acyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, acetic acid, pectin ester acid
  • the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the above compounds and pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable carrier or diluent.
  • the present invention further provides the use of the above compound and a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable carrier or diluent in the preparation of a medicament for the treatment and prevention of diabetes.
  • such a hypoglycemic polypeptide has an increased agonistic activity on GLP-1R, maintains a moderate GCGR agonistic activity, and has an excellent hypoglycemic effect.
  • the blood glucose stabilization time exceeds 40 hours after a single administration.
  • such hypoglycemic peptides also have good weight-reducing activity.
  • hypoglycemic polypeptides compared with the OXM prototype, have an increased agonistic activity on GLP-1R, maintain a moderate GCGR agonistic activity, and have excellent hypoglycemic effects.
  • the blood glucose stabilization time exceeds 60 hours after one administration.
  • hypoglycemic peptides also have good weight-reducing activity.
  • the present invention also provides a method for preparing the compounds of the present invention by solid phase synthesis strategies quickly and efficiently synthesized above target compound.
  • the present invention also provides a method for preparing such a hypoglycemic polypeptide and an intermediate thereof.
  • the method for preparing such a hypoglycemic polypeptide provided by the present invention adopts a solid phase synthesis method to gradually couple each amino acid of the main chain of such a hypoglycemic polypeptide to obtain The peptide resin with the main chain connected to the L-lysine side chain of the 16-position fatty acid small molecule to obtain a hypoglycemic polypeptide.
  • the method has the advantages of simple synthesis steps, high coupling efficiency and easy purification, which is beneficial to the industrial production of such hypoglycemic peptides.
  • the invention provides a method for preparing a hypoglycemic polypeptide, which includes the following two strategies:
  • Step 1 Take the resin, and after activation, gradually couple amino acids to obtain the first peptide resin;
  • Step 2 Take the first peptide resin, lyse and purify it to obtain a pure peptide chain;
  • Step 3 Conjugating the thiol group of Cys in the pure peptide chain product with a fatty acid chain or a small coumarin molecule with a maleimide linking arm to obtain the compound;
  • Step 1 Take the resin, and after activation, gradually couple amino acids to obtain the first peptide resin;
  • Step 2 Take the first peptide resin and couple small molecules of fatty acid chains having Formula I or Formula II to the Lys side chain to obtain a second peptide resin;
  • Step 3 Take the second peptide resin, lyse and purify it to obtain the compound
  • R 1 is selected from tBu, Dmab, and Bzl;
  • the R 2 is selected from the group consisting of methyl, ethyl, t-butyl, and benzhydryl.
  • the Lys side chain protecting group coupled to the fatty acid chain small molecule of Formula I or Formula II is selected from the group consisting of Fmoc, Boc, Dde, and ivDde.
  • the resin in step 1 is Rink Amide AM Resin, Fmoc-Rink Amide-MBHA or Wang Resin.
  • the resin in step 1 is specifically Fmoc-Rinkamide-MBHA or Wang Resin.
  • the cracking reagent used for cracking is a mixture of TFA, anisole, anisole and EDT.
  • the volume ratio of TFA, anisole, anisole, and EDT in the reagent used in the cracking in step 3 is (85-92): (4- 6): (2 to 3): (2 to 6).
  • the volume ratio of TFA, anisole, anisole and EDT in the cleavage reagent used in step 3 is 90: 5: 3: 2 .
  • the method used for purification is a chromatographic separation method.
  • the chromatographic column used for purification is a C18 column.
  • the compound provided by the present invention has significant effects on reducing glucose and weight, has stable chemical properties, and has significantly better activity than the prototype peptide OXM.
  • hypoglycemic effect of some compounds provided by the present invention can be maintained for more than 40 hours, which is significantly improved compared to endogenous GLP-1 (half life 2 to 3 minutes) or the marketed drug exenatide (half life 2.4 hours).
  • Orthogonal protection strategy for the solid-phase synthesis of OXM hybrid peptides yields a crude peptide chain with a purity of greater than 85%, which is greatly improved compared with conventional synthesis methods and facilitates subsequent purification work.
  • This method uses solid-phase method to synthesize OXM hybrid peptide with low cost. Due to the high coupling efficiency, the protection of amino acids only requires a 2-fold excess on average, while conventional synthesis methods require 4- to 5-fold excess amino acids, which greatly saves costs.
  • the OXM hybrid peptide prepared by the solid-phase synthesis technology provided by the present invention has good activity in reducing glucose and slowing weight gain, long drug effect time, high yield, short synthesis cycle, easy crude purification, low production cost, and easy industrialization. Automated manufacturing.
  • the prepared OXM hybrid peptide is suitable as an active ingredient of a medicine for treating diabetes and obesity.
  • Figure 1 shows the results of the next-day glucose tolerance test of the OXM hybrid peptides SEQ. ID NO: 1 to 6.
  • Figure 2 shows the results of the next day glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 7-12.
  • Figure 3 shows the results of the next-day glucose tolerance test of the OXM hybrid peptides SEQ. ID NO: 13 to 18.
  • Figure 4 shows the results of the next day glucose tolerance test of the OXM hybrid peptides SEQ. ID NO: 19 to 24.
  • FIG. 5 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 19-21.
  • Figure 6 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 22-24.
  • FIG. 7 shows the TC detection results of the OXM hybrid peptides SEQ. ID NO: 1-12.
  • FIG. 8 shows the TC detection results of the OXM hybrid peptide SEQ. ID NO: 13-24.
  • FIG. 9 shows the results of TG detection of the OXM hybrid peptides SEQ. ID NO: 1-12.
  • Figure 10 shows the results of TG detection of the OXM hybrid peptides SEQ. ID NO: 13 to 24.
  • FIG. 11 shows the results of ALT detection of the OXM hybrid peptides SEQ. ID NO: 1-12.
  • FIG. 12 shows the ALT detection results of the OXM hybrid peptides SEQ. ID NO: 13-24.
  • Figure 13 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 25-27.
  • Figure 14 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 28-29.
  • FIG. 15 shows the TC detection results of the OXM hybrid peptides SEQ. ID NO: 25-29.
  • Figure 16 shows the results of TG detection of the OXM hybrid peptides SEQ. ID NO: 25-29.
  • FIG. 17 shows the ALT detection results of the OXM hybrid peptides SEQ. ID NO: 25-29.
  • FIG. 18 shows the results of the peritoneal glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 30-35.
  • FIG. 19 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 30 to 32.
  • FIG. 19 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 30 to 32.
  • Figure 20 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ.ID NO: 33-35.
  • FIG. 21 is a TC detection result of the OXM hybrid peptide SEQ. ID NO: 30-35.
  • Figure 22 shows the results of TG detection of the OXM hybrid peptides SEQ. ID NO: 30-35.
  • FIG. 23 shows the ALT detection results of the OXM hybrid peptide SEQ. ID NO: 30 to 35.
  • Figure 24 shows the results of the intraperitoneal glucose tolerance test of the OXM hybrid peptide SEQ.ID NO: 36 ⁇ 41
  • FIG. 25 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 36 to 38.
  • FIG. 25 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 36 to 38.
  • FIG. 26 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 39-41.
  • FIG. 27 is a TC detection result of the OXM hybrid peptide SEQ. ID NO: 36 to 41.
  • FIG. 27 is a TC detection result of the OXM hybrid peptide SEQ. ID NO: 36 to 41.
  • FIG. 28 shows the TG detection results of the OXM hybrid peptides SEQ. ID NO: 36 to 41.
  • FIG. 29 shows the ALT detection results of the OXM hybrid peptides SEQ. ID NO: 36 to 41.
  • FIG. 30 shows the results of the intraperitoneal glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 42-47.
  • FIG. 31 shows the results of the peritoneal glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 48 to 53.
  • FIG. 31 shows the results of the peritoneal glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 48 to 53.
  • FIG. 32 shows the results of the peritoneal glucose tolerance test of the OXM hybrid peptide SEQ. ID NO: 54-59.
  • Figure 33 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 42-47.
  • FIG. 34 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 48 to 53.
  • FIG. 34 shows the results of stable blood glucose experiments of the OXM hybrid peptide SEQ. ID NO: 48 to 53.
  • Figure 35 shows the results of stable blood glucose experiments of the OXM hybrid peptides SEQ. ID NO: 54-59.
  • FIG. 36 shows the TC detection results of the OXM hybrid peptide SEQ. ID NO: 42-59.
  • Figure 37 shows the results of TG detection of the OXM hybrid peptide SEQ. ID NO: 42-59.
  • FIG. 38 shows the ALT detection results of the OXM hybrid peptides SEQ. ID NO: 42 to 59.
  • FIG. 38 shows the ALT detection results of the OXM hybrid peptides SEQ. ID NO: 42 to 59.
  • Fmoc-Rink amide-MBHA Resin 50mg (substitution degree 0.4mmol / g) was weighed, swelled with 7mL of DCM for 30min, DCM was filtered off with suction, swelled with 10mL of NMP and swelled for 30min with NMP, and rinsed with 7mL of NMP and DCM.
  • Fmoc-Ser (tBu) -OH (15.4mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9 ⁇ L, 0.08mmol) were dissolved in NMP 10mL, and then This solution was added to the resin obtained in the previous step and reacted for 2 hours. After completion, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP.
  • the reaction was monitored using HPLC.
  • the chromatographic conditions were: C18 column (150 mm ⁇ 4.6 mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); Mobile phase gradient: Mobile phase B 35% -85%, 20min; flow rate 1mL / min; column temperature 40 ° C; detection wavelength 214nm.
  • purification was performed by preparative liquid chromatography.
  • the chromatographic conditions were: C18 column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate 6mL / min detection wavelength 214nm.
  • the collected solution was lyophilized to obtain 28.5 mg of pure product.
  • the theoretical relative molecular mass is 4273.7.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1425.6, [M + 4H] 4+ 1069.4; Found [M + 3H] 3+ 1425.1, [M + 4H] 4+ 1069.0.
  • the polypeptide chain numbered SEQ.ID NO.1 was dissolved in DMSO to prepare a solution of about 10 mg / mL, and N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred and monitored using LC-MS.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: C18 reverse phase column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 29.4 mg of pure product.
  • the theoretical relative molecular mass is 4483.0.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1495.3, [M + 4H] 4+ 1121.1; Found [M + 3H] 3+ 1495.8, [M + 4H] 4+ 1121.2.
  • the polypeptide chain numbered SEQ.ID NO.2 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 27.6 mg of a pure product. The theoretical relative molecular mass is 4479.0. ESI-MS m / z: Calcd. [M + 3H] 3+ 1494.0, [M + 4H] 4+ 1120.7; Found [M + 3H] 3+ 1494.0, [M + 4H] 4+ 1120.9.
  • the polypeptide chain numbered SEQ.ID NO.3 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.1 mg of a pure product. The theoretical relative molecular mass is 4437.9.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1480.3, [M + 4H] 4+ 1110.5; Found [M + 3H] 3+ 1480.0, [M + 4H] 4+ 1110.3.
  • the polypeptide chain numbered SEQ.ID NO.4 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.9 mg of a pure product. The theoretical relative molecular mass is 4493.0. ESI-MS m / z: Calcd. [M + 3H] 3+ 1498.7, [M + 4H] 4+ 1124.2; Found [M + 3H] 3+ 1498.9, [M + 4H] 4+ 1124.3.
  • the polypeptide chain numbered SEQ.ID NO.5 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.3 mg of a pure product. The theoretical relative molecular mass is 4463.0. ESI-MS m / z: Calcd. [M + 3H] 3+ 1488.7, [M + 4H] 4+ 1116.7; Found [M + 3H] 3+ 1488.0, [M + 4H] 4+ 1116.0.
  • the polypeptide chain numbered SEQ.ID NO.6 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-octyl maleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 29.4 mg of pure product. The theoretical relative molecular mass is 4465.9.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1489.6, [M + 4H] 4+ 1117.5; Found [M + 3H] 3+ 1489.6, [M + 4H] 4+ 1117.7.
  • the polypeptide chain numbered SEQ.ID NO.1 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-dodecylmaleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.7 mg of a pure product. The theoretical relative molecular mass is 4539.1.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1514.0, [M + 4H] 4+ 1135.8; Found [M + 3H] 3+ 1514.5, [M + 4H] 4+ 1135.5.
  • the polypeptide chain numbered SEQ.ID NO.2 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-dodecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 27.1 mg of a pure product. The theoretical relative molecular mass is 4535.1.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1512.7, [M + 4H] 4+ 1134.8; Found [M + 3H] 3+ 1512.5, [M + 4H] 4+ 1134.2.
  • the polypeptide chain numbered SEQ.ID NO.3 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-dodecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.5 mg of pure product. The theoretical relative molecular mass is 4494.0.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1499.0, [M + 4H] 4+ 1124.5; Found [M + 3H] 3+ 1499.0, [M + 4H] 4+ 1124.0.
  • the polypeptide chain numbered SEQ.ID NO.4 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-dodecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 28.1 mg of a pure product. The theoretical relative molecular mass is 4549.1.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1517.4, [M + 4H] 4+ 1138.3; Found [M + 3H] 3+ 1517.2, [M + 4H] 4+ 1138.8.
  • the polypeptide chain numbered SEQ.ID NO.5 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-dodecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 27.8 mg of a pure product. The theoretical relative molecular mass is 4519.1. ESI-MS m / z: Calcd. [M + 3H] 3+ 1507.4, [M + 4H] 4+ 1130.8; Found [M + 3H] 3+ 1507.0, [M + 4H] 4+ 1130.2.
  • the polypeptide chain numbered SEQ.ID NO.6 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-dodecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 29.1 mg of a pure product. The theoretical relative molecular mass is 4522.0.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1508.3, [M + 4H] 4+ 1131.5; Found [M + 3H] 3+ 1508.6, [M + 4H] 4+ 1131.4.
  • the polypeptide chain numbered SEQ.ID NO.1 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-hexadecylmaleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 30.2 mg of pure product. The theoretical relative molecular mass is 4595.2. ESI-MS m / z: Calcd. [M + 3H] 3+ 1532.7, [M + 4H] 4+ 1149.8; Found [M + 3H] 3+ 1532.8, [M + 4H] 4+ 1149.2.
  • the polypeptide chain numbered SEQ.ID NO.2 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-hexadecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 30.4 mg of pure product. The theoretical relative molecular mass is 4591.2.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1531.4, [M + 4H] 4+ 1148.8; Found [M + 3H] 3+ 1531.5, [M + 4H] 4+ 1149.0.
  • the polypeptide chain numbered SEQ.ID NO.3 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-hexadecylmaleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 29.4 mg of pure product. The theoretical relative molecular mass is 4550.1.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1517.7, [M + 4H] 4+ 1138.5; Found [M + 3H] 3+ 1517.0, [M + 4H] 4+ 1138.7.
  • the polypeptide chain numbered SEQ.ID NO.4 was dissolved in DMSO to prepare a solution of about 10 mg / mL, N-n-hexadecylmaleimide was also dissolved in DMSO, and 20 ⁇ l of DIEPA was added after mixing the two. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 30.2 mg of pure product. The theoretical relative molecular mass is 4605.2.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1536.1, [M + 4H] 4+ 1152.3; Found [M + 3H] 3+ 1536.6, [M + 4H] 4+ 1152.2.
  • the polypeptide chain numbered SEQ.ID NO.5 was dissolved in DMSO to prepare a solution of about 10 mg / mL. N-n-hexadecylmaleimide was also dissolved in DMSO. After mixing the two, DIEPA 20 ⁇ l was added. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 31.2 mg of pure product. The theoretical relative molecular mass is 4575.2. ESI-MS m / z: Calcd. [M + 3H] 3+ 1526.1, [M + 4H] 4+ 1144.8; Found [M + 3H] 3+ 1526.0, [M + 4H] 4+ 1145.6.
  • the polypeptide chain numbered SEQ.ID NO.6 was dissolved in DMSO to prepare a solution of about 10 mg / mL, and N-n-hexadecylmaleimide was also dissolved in DMSO. After mixing the two, 20 ⁇ l of DIEPA was added. The reaction was stirred at room temperature. The detection and purification of the reaction were the same as in Example 7. The collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 30.8 mg of pure product. The theoretical relative molecular mass is 4578.1.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1527.0, [M + 4H] 4+ 1145.5; Found [M + 3H] 3+ 1527.2, [M + 4H] 4+ 1145.9.
  • Fmoc-Rink amide-MBHA Resin 50mg (substitution degree 0.4mmol / g) was weighed, swelled with 7mL of DCM for 30min, DCM was filtered off with suction, swelled with 10mL of NMP and swelled for 30min with NMP, and rinsed with 7mL of NMP and DCM.
  • Fmoc-Ser (tBu) -OH (15.4mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9 ⁇ L, 0.08mmol) were dissolved in NMP 10mL, and then This solution was added to the resin obtained in the previous step and reacted for 2 hours. After the end, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP.
  • the chromatographic conditions were: C18 column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate 6mL / min detection wavelength 214nm.
  • the collected solution was lyophilized to obtain 28.5 mg of pure product.
  • the theoretical relative molecular mass is 4256.6.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1419.9, [M + 4H] 4+ 1065.2; Found [M + 3H] 3+ 1420.1, [M + 4H] 4+ 1065.5.
  • the 12- (2,5-dihydro-2,5-dioxo-1H-pyrrole-1-yl) dodecanoic acid obtained in the previous step was dissolved in DMSO to prepare a solution of about 10 mg / mL, and the obtained SEQ .ID NO: 1 main chain peptide sequence was also dissolved in DMSO. After the two were mixed with ultrasound, 20 ⁇ l DIEPA was added, and the reaction was stirred at room temperature. The reaction was monitored by LC-MS.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 8.1 mg of pure product. The theoretical relative molecular mass is 4551.7.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1518.3, [M + 4H] 4+ 1138.9; Found [M + 3H] 3+ 1517.4, [M + 4H] 4+ 1138.8.
  • the 16- (2,5-dihydro-2,5-dioxo-1H-pyrrole-1-yl) hexadecanoic acid obtained in the previous step was dissolved in DMSO to prepare a solution of about 10 mg / mL, and the obtained SEQ .ID NO: 1 main chain peptide sequence was also dissolved in DMSO. After the two were mixed with ultrasound, 20 ⁇ l DIEPA was added, and the reaction was stirred at room temperature. The reaction was monitored by LC-MS.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: C18 reverse phase column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 8.7 mg of a pure product.
  • the theoretical relative molecular mass is 4607.8.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1536.9, [M + 4H] 4+ 1152.9; Found [M + 3H] 3+ 1537.6, [M + 4H] 4+ 1153.5.
  • N-Boc-dodecyldiamine (1.2g, 4mmol) and maleic anhydride (0.49g, 4.8mmol) were dissolved in glacial acetic acid, and the reaction was heated at 120 ° C for 6h. After the reaction was detected by the thin-layer plate, the reaction was completed. The solution was cooled to room temperature, and extracted with ethyl acetate (3 ⁇ 20 mL). The upper extracts were combined, and the extracts were washed three times with saturated brine, and dried over anhydrous Na 2 SO 4 overnight. The extract was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography to obtain 1.10 g of a pale yellow pure product with a yield of 72%. MS (ESI, m / z): 380.5 [M + H] + .
  • reaction solution was poured into water and extracted three times with ethyl acetate.
  • the extracts were combined and washed with saturated K 2 CO 3 solution, HCl 1M, and saturated brine three times.
  • the extract was dried by adding anhydrous Na 2 SO 4 overnight, concentrated under reduced pressure to obtain a crude product, and purified by column chromatography to obtain 0.93 g of a pure product with a yield of 65%.
  • the 4- (bis (4-hydroxy-2-oxo-2H-chromen-3-yl) methyl) -N- (12- (2,5-dioxo-2,5- Dihydro-1H-pyrrole-1-yl) dodecyl) benzamide was dissolved in DMSO to prepare a solution of about 10 mg / mL, and the peptide sequence of SEQ.ID NO: 1 was also dissolved in DMSO. Both were sonicated After mixing, add 20 ⁇ l DIEPA, stir the reaction at room temperature, and monitor the reaction using LC-MS.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: C18 reverse phase column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 7.9 mg of pure product. The theoretical relative molecular mass is 4974.9.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1659.3, [M + 4H] 4+ 1244.7; Found [M + 3H] 3+ 1659.3, [M + 4H] 4+ 1245.8.
  • 12-maleamide dodecanoic acid (294.1 mg, 1 mmol) was dissolved in tetrahydrofuran, DIC (17 ⁇ L, 1.1 mmol) and HOBt (148.5 mg, 1.1 mmol) were added, and the mixture was stirred at room temperature for 30 min.
  • a solution of 3 '-(4-aminobenzylidene) -di-4-hydroxycoumarin and DIPEA (17.4 ⁇ L, 0.1 mmol) in tetrahydrofuran was stirred at room temperature overnight. After the reaction of the thin layer plate was detected, the reaction solution was poured into water and extracted three times with ethyl acetate.
  • the extracts were combined and washed three times with K 2 CO 3 , HCl 1M, and saturated saline.
  • the extract was dried by adding anhydrous Na 2 SO 4 overnight, concentrated under reduced pressure to obtain a crude product, and purified by column chromatography to obtain a pure product.
  • the yield was 69%, mp 204-206 ° C.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: C18 reverse phase column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 8.6 mg of pure product. The theoretical relative molecular mass is 4960.9.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1654.6, [M + 4H] 4+ 1241.2; Found [M + 3H] 3+ 1654.2, [M + 4H] 4+ 1240.2.
  • 1,8-Diamino-3,6-dioxaoctane (10.7 g, 72.3 mmol) was dissolved in 70 ml of DCM, Boc anhydride (2.2 g, 10.1 mmol) was dissolved in 50 ml of DCM. At 0 ° C, Boc The anhydride is slowly added dropwise to a solution of 1,8-diamino-3,6-dioxaoctane. After the dropwise addition was completed, the reaction solution was returned to room temperature, and the reaction was continued for 4 hours. After the reaction was completed, column chromatography was performed using basic alumina, and 1.8 g of a colorless transparent oil was obtained with a yield of 72.0%.
  • the extracts were combined and washed with saturated K 2 CO 3 , HCl 1M, and saturated brine three times.
  • the extract was dried by adding anhydrous Na 2 SO 4 overnight, and then concentrated under reduced pressure to obtain a crude product, which was separated by column chromatography to obtain 132.8 mg of a white paste-like pure product with a yield of 45%.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 8.3 mg of pure product. The theoretical relative molecular mass is 4993.8.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1665.6, [M + 4H] 4+ 1249.5; Found [M + 3H] 3+ 1665.8, [M + 4H] 4+ 1249.1.
  • Fmoc-Rink amide-MBHA Resin 50mg (substitution degree 0.4mmol / g) was weighed, swelled with 7mL of DCM for 30min, DCM was filtered off with suction, swelled with 10mL of NMP and swelled for 30min with NMP, and washed with NMP and 7mL.
  • Fmoc-Ser (tBu) -OH (15.4 mg, 0.04 mmol), HBTU (15.1 mg, 0.04 mmol), HOBt (5.4 mg, 0.04 mmol) and DIPEA (13.9 ⁇ L, 0.08 mmol) were dissolved in 10 mL of NMP, and then This solution was added to the resin obtained in the previous step and reacted for 2 hours. After completion, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP.
  • a 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed.
  • the chromatographic conditions were: C18 column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate 6mL / min detection wavelength 214nm.
  • the collected solution was lyophilized to obtain 31.2 mg of pure product.
  • the theoretical relative molecular mass is 4666.6.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1556.5, [M + 4H] 4+ 1167.7; Found [M + 3H] 3+ 1556.9, [M + 4H] 4+ 1166.9.
  • a 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed. Fmoc-AEEA was coupled again in the same way. After the reaction was completed, the resin was washed 3 times with 7 mL each of DCM and NMP. A 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed.
  • Fmoc-Glu-OtBu (68.0 mg, 0.16 mmol), HBTU (60.6 mg, 0.16 mmol), DIEA (55.6 ⁇ L, 0.32 mmol), HOBt (21.6 mg, 0.16 mmol) were dissolved in 10 mL of NMP, and this solution was added to After 2 hours of reaction in the resin, the reaction solution was filtered off after washing, and the resin was washed 3 times with 7 mL each of DCM and NMP. A 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed.
  • Fmoc-Rinkamide-MBHA Resin 50mg (substitution degree 0.4mmol / g) was weighed, swelled with 7mL of DCM for 30min, DCM was filtered off with suction, swelled with 10mL of NMP for 30min, and finally washed with NMP and 7mL of DCM.
  • Fmoc-Ser (tBu) -OH (15.4mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9 ⁇ L, 0.08mmol) were dissolved in NMP 10mL, and then This solution was added to the resin obtained in the previous step and reacted for 2 hours. After completion, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP.
  • the above steps of deprotection and coupling are repeated, and the corresponding amino acids are sequentially connected, and the corresponding amino acids are sequentially connected until the peptide chain is synthesized to obtain a resin with a main chain peptide sequence.
  • a 2% hydrazine hydrate solution was added to the resin to which the main chain peptide sequence was attached to remove the side chain protecting group Dde at the 16-position Lys, and the reaction was washed with NMP after the reaction was completed.
  • the formula I (33.8 mg, 0.04 mmol), HBTU (15.1 mg, 0.04 mmol), HOBt (5.4 mg, 0.04 mmol) and DIPEA (13.9 ⁇ L, 0.08 mmol) were dissolved in NMP and 10 mL, and this solution was added to the resin to react After 2 hours, the reaction solution was filtered off, and the resin was washed three times with 7 mL each of DCM and NMP to obtain the peptide resin with SEQ. ID NO: 1.
  • Reagent K (TFA / anisole / water / phenol / EDT, 82.5: 5: 5: 5: 2.5, V / V) 10 mL, first shake at 0 ° C for 30 min, and then react at room temperature for 3 h. After the reaction, suction filtration was performed, a small amount of TFA and DCM were added for washing three times, and the filtrates were combined. The filtrate was added to a large amount of ice ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by freeze centrifugation. Finally, 92.5 mg of crude product was obtained with a yield of 92.1%.
  • the reaction was monitored using HPLC.
  • the chromatographic conditions were: C18 column (150 mm ⁇ 4.6 mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); Mobile phase gradient: Mobile phase B 35% -85%, 20min; flow rate 1mL / min; column temperature 40 ° C; detection wavelength 214nm.
  • purification was performed by preparative liquid chromatography.
  • the chromatographic conditions were: C18 column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate 6mL / min; detection wavelength 214nm.
  • the collected solution was lyophilized to obtain 24.1 mg of pure product.
  • the theoretical relative molecular mass is 5014.6.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1672.5, [M + 4H] 4+ 1254.7; Found [M + 3H] 3+ 1672.9, [M + 4H] 4+ 1254.1.
  • Fmoc-Rinkamide-MBHA Resin 50mg (substitution degree 0.4mmol / g) was weighed, swelled with 7mL of DCM for 30min, DCM was filtered off with suction, swelled with 10mL of NMP for 30min, and finally washed with NMP and 7mL of DCM.
  • Fmoc-Ser (tBu) -OH (15.4mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9 ⁇ L, 0.08mmol) were dissolved in NMP 10mL, and then This solution was added to the resin obtained in the previous step and reacted for 2 hours. After completion, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP.
  • the above steps of deprotection and coupling are repeated, and the corresponding amino acids are sequentially connected, and the corresponding amino acids are sequentially connected until the peptide chain is synthesized to obtain a resin with a main chain peptide sequence.
  • the polypeptide chain was dissolved in DMSO to prepare a solution of about 10 mg / mL, and N-n-hexadecylmaleimide was also dissolved in DMSO. After mixing the two, 20 ⁇ L of DIEPA was added, and the reaction was stirred at room temperature. LC-MS was used. Monitor the response.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 29.7 mg of a pure product.
  • the theoretical relative molecular mass is 4603.3.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1535.4, [M + 4H] 4+ 1151.8; Found [M + 3H] 3+ 1535.8, [M + 4H] 4+ 1151.2.
  • Example 44 Solid-phase synthesis of SEQ.ID NO: 44
  • the method of synthesizing the polypeptide main chain is the same as the method of synthesizing the polypeptide main chain in Example 42.
  • 12-maleamide dodecanoic acid (294.1 mg, 1 mmol) was dissolved in tetrahydrofuran, DIC (17 ⁇ L, 1.1 mmol) and HOBt (148.5 mg, 1.1 mmol) were added, and the mixture was stirred at room temperature for 30 min.
  • a solution of 3 '-(4-aminobenzylidene) -di-4-hydroxycoumarin and DIPEA (17.4 ⁇ L, 0.1 mmol) in tetrahydrofuran was stirred at room temperature overnight. After the reaction of the thin layer plate was detected, the reaction solution was poured into water and extracted three times with ethyl acetate.
  • the 3,3 '-(4- (12-maleimidododecanoyl) benzylidene) -di-4-hydroxycoumarin obtained in the previous step was dissolved in DMSO to prepare about 10 mg / mL.
  • the chromatographic conditions were: C18 reversed-phase column (1.7 ⁇ m 2.1 ⁇ 50mm, Waters); mobile phase A: 0.1% formic acid / water (V / V), mobile phase B: 0.1% formic acid / acetonitrile (V / V), mobile phase Gradient: mobile phase B 10% -90%, 2min, B 90% -90%, 3min; flow rate 0.3ml / min; ultraviolet detection wavelength 214nm.
  • the reaction solution was diluted with acetonitrile containing 1% TFA, centrifuged at high speed, filtered through a 0.45 ⁇ m microporous membrane filter, and then purified by preparative liquid chromatography.
  • the chromatographic conditions were: 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 80%, 30min; 80% 85% to 10min; 85% to 95% for 10min; 95% to 40% for 10min; flow rate is 5ml / min and detection wavelength is 214nm.
  • the collected solution was concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain 31.6 mg of pure product. The theoretical relative molecular mass is 4986.6.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1663.2, [M + 4H] 4+ 1247.7; Found [M + 3H] 3+ 1663.5, [M + 4H] 4+ 1247.2.
  • Example 46 SEQ.ID NO: 46 solid-phase synthesis
  • the method of synthesizing the polypeptide main chain is the same as the method of synthesizing the polypeptide main chain in Example 42.
  • a 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed. Fmoc-AEEA was coupled again in the same way. After the reaction was completed, the resin was washed 3 times with 7 mL each of DCM and NMP. A 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed.
  • Fmoc-Glu-OtBu (68.0 mg, 0.16 mmol), HBTU (60.6 mg, 0.16 mmol), DIEA (55.6 ⁇ L, 0.32 mmol), HOBt (21.6 mg, 0.16 mmol) were dissolved in 10 mL of NMP, and this solution was added to After 2 hours of reaction in the resin, the reaction solution was filtered off after washing, and the resin was washed 3 times with 7 mL each of DCM and NMP. A 25% piperidine / NMP (V / V) solution containing 0.1 M HOBt was added to the resin to remove Fmoc, and washed with NMP after the reaction was completed.
  • a 2% hydrazine hydrate solution was added to the resin to which the main chain peptide sequence was attached to remove the side chain protecting group Dde at the 16-position Lys, and the reaction was washed with NMP after the reaction was completed.
  • Small molecules in side chains (33.8 mg, 0.04 mmol), HBTU (15.1 mg, 0.04 mmol), HOBt (5.4 mg, 0.04 mmol) and DIPEA (13.9 ⁇ L, 0.08 mmol) were dissolved in 10 mL of NMP, and this solution was added to the resin The reaction was allowed to proceed for 2 hours. After the reaction, the reaction solution was filtered off, and the resin was washed 3 times with 7 mL each of DCM and NMP to obtain a peptide resin with SEQ. ID NO: 5.
  • Reagent K (TFA / anisole / water / phenol / EDT, 82.5: 5: 5: 5: 2.5, V / V) 10 mL
  • suction filtration was performed, a small amount of TFA and DCM were added for washing three times, and the filtrates were combined.
  • the filtrate was added to a large amount of ice ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by freeze centrifugation. 95.0 mg of crude product was finally obtained with a yield of 94.6%.
  • the reaction was monitored using HPLC.
  • the chromatographic conditions were: C18 column (150 mm ⁇ 4.6 mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); Mobile phase gradient: Mobile phase B 35% -85%, 20min; flow rate 1mL / min; column temperature 40 ° C; detection wavelength 214nm.
  • purification was performed by preparative liquid chromatography.
  • the chromatographic conditions were: C18 column (320mm ⁇ 28mm, 5 ⁇ m); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 40% to 90%, 20min; flow rate 6mL / min; detection wavelength 214nm.
  • the collected solution was lyophilized to obtain 32.7 mg of pure product. The theoretical relative molecular mass is 5022.7.
  • ESI-MS m / z Calcd. [M + 3H] 3+ 1675.2, [M + 4H] 4+ 1256.7; Found [M + 3H] 3+ 1675.9, [M + 4H] 4+ 1256.1.
  • Example 47 Solid-phase synthesis of SEQ. ID NO: 47
  • Example 55 SEQ.ID NO: 55 solid-phase synthesis
  • HEK293 cells were co-transfected with cDNA encoding GLP-1R or GCGR, respectively.
  • cells were seeded in a 96-well plate 2 hours in advance, the compound was dissolved in DMSO, diluted to different multiples using a medium containing 0.1% bovine serum protein, and added to the co-transfected cells. After the cells were incubated for 20 minutes, the fluorescence readings were measured using a ELISA kit from Cisbo and a microplate reader was used to establish a standard curve to convert the fluorescence readings to the corresponding cAMP values. The EC 50 values of the compounds were calculated using the non-linear regression of Graphpad Prism 5.0 software.
  • Results are expressed as mean ⁇ SD, * P ⁇ 0.05, ** P ⁇ 0.01 vs OXM, #P ⁇ 0.05, ## P ⁇ 0.01 vs Exenatide.
  • mice Normal Kunming mice were randomly divided into groups of 8 mice. The mice were housed in standardized animal rooms. Fasted for 12 hours before the experiment and given only drinking water. Before the OXM hybrid peptide was administered to each group of mice, the initial blood glucose level was measured and set to -30 min, and then 50 nmol / kg of the OXM hybrid peptide was intraperitoneally injected. After 30 min, an 18 mmol / kg glucose solution was injected intraperitoneally, which was set to 0 min. The control group was injected with the same volume of normal saline or 50 nmol / kg of exenatide. At 0, 15, 30, 45, 60, and 120 minutes, the blood glucose level was measured with a blood glucose meter, and the hypoglycemic activity of the OXM hybrid peptide was detected.
  • Results are expressed as mean ⁇ SD, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vssaline.
  • the results of the hypoglycemic experiment show that when the concentration of the OXM hybrid peptide involved in the present invention is 50 nmol / kg, the hypoglycemic effect is equivalent to that of exenatide.
  • mice in each group were intraperitoneally injected with a glucose solution of 18 mmol / kg, and the glucose injection time was set to 0 min. Blood glucose levels were measured with a blood glucose meter at 0, 15, 30, 45, 60 and 120 min.
  • the results of the alternate-day glucose tolerance test show that the OXM hybrid peptide conjugated with a fatty acid side chain according to the present invention still has a blood glucose-lowering effect after being metabolized in vivo for 24 hours. Senatide has long been inactive. This indicates that the modified OXM hybrid peptides have significantly lowered the hypoglycemic time, and the hypoglycemic effect can be maintained for nearly 30 hours.
  • the blood glucose of STZ-induced diabetic model mice was measured. Mice with a value higher than 20 mmol / L were randomly divided into groups of six, and the mice fed freely during the experiment.
  • the positive control group was injected intraperitoneally with exenatide or liraglutide at a dose of 50nmol / kg
  • the negative control group was intraperitoneally injected with saline
  • the administration group was injected with 50nmol / kg of OXM hybrid peptide.
  • Compounds were administered at 0 h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 60 h.
  • the evaluation index is the time after the compound is injected intraperitoneally, and the blood glucose value of the mouse is lower than 8.35 mmol / L.
  • the time for stabilizing blood glucose of exenatide is only 4.1h, and the time for stabilizing blood glucose of liraglutide is 10.7h.
  • the time for stabilizing blood glucose of the long-acting hypoglycemic peptides involved in the present invention are all Above 40h, some can exceed 50h. Stable blood glucose experiments show that OXM hybrid peptide has good long-acting hypoglycemic effect, can achieve better long-acting hypoglycemic effect, and has the potential to develop into a hypoglycemic drug administered once every two days.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. The fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • OXM hybrid peptide 50nmol / kg, 10mL / kg
  • the fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • Results are expressed as mean ⁇ SD.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the total cholesterol (TC) and triglyceride (TG) contents were measured.
  • TC total cholesterol
  • TG triglyceride
  • mice Male C57bl / 6 mice were fed a high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the content of alanine aminotransferase was measured.
  • ALT alanine aminotransferase
  • Results are expressed as mean ⁇ SD, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vssaline.
  • the results of the hypoglycemic experiment show that when the concentration of the OXM hybrid peptide involved in the present invention is 50 nmol / kg, the hypoglycemic effect is equivalent to that of exenatide.
  • the blood glucose of STZ-induced diabetic model mice was measured. Mice with a value higher than 20 mmol / L were randomly divided into groups of six, and the mice fed freely during the experiment.
  • the positive control group was injected intraperitoneally with exenatide or liraglutide at a dose of 50nmol / kg
  • the negative control group was intraperitoneally injected with saline
  • the administration group was injected with 50nmol / kg of OXM hybrid peptide.
  • Compounds were administered at 0 h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 60 h.
  • the evaluation index is the time after the compound is injected intraperitoneally, and the blood glucose value of the mouse is lower than 8.35 mmol / L.
  • the time for stabilizing blood glucose of exenatide is only 4.7 hours, and the time for stabilizing blood glucose of liraglutide is 12.3 hours.
  • the time for stabilizing blood glucose of the long-acting hypoglycemic polypeptide involved in the present invention may be Over 40h. Stable blood glucose experiments show that OXM hybrid peptide has good long-acting hypoglycemic effect, can achieve better long-acting hypoglycemic effect, and has the potential to develop into a hypoglycemic drug administered once every two days.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 7 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. The fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • OXM hybrid peptide 50nmol / kg, 10mL / kg
  • the fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • Results are expressed as mean ⁇ SD.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 7 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the total cholesterol (TC) and triglyceride (TG) contents were measured.
  • TC total cholesterol
  • TG triglyceride
  • mice Male C57bl / 6 mice were fed a high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly divided into 8 groups of 7 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the content of alanine aminotransferase was measured.
  • HEK293 cells were co-transfected with cDNA encoding GLP-1R or GCGR, respectively, and the cell line was expressed and the protein levels of GLP-1R or GCGR in the constructed HEK293 cells were detected by Western Blot to confirm whether a stable and highly expressing cell line HEK293 had been established.
  • cells were seeded in a 96-well plate 2 hours in advance, the compound was dissolved in DMSO, diluted to different times using a medium containing 0.1% bovine serum protein, and added to the co-transfected cells.
  • the fluorescence readings were measured using a ELISA kit from Cisbo and a microplate reader was used to establish a standard curve to convert the fluorescence readings to the corresponding cAMP values.
  • the EC 50 values of the compounds were calculated using the non-linear regression of Graphpad Prism 5.0 software.
  • Results are expressed as mean ⁇ SD, * P ⁇ 0.05, ** P ⁇ 0.01 vs OXM, #P ⁇ 0.05, ## P ⁇ 0.01 vs Exenatide.
  • mice Normal Kunming mice were randomly divided into groups of 8 mice. The mice were housed in standardized animal rooms. Fasted for 12 hours before the experiment and given only drinking water. Before the OXM hybrid peptide was administered to each group of mice, the initial blood glucose level was measured and set to -30 min, and then 50 nmol / kg of the OXM hybrid peptide was intraperitoneally injected. After 30 min, an 18 mmol / kg glucose solution was injected intraperitoneally, which was set to 0 min. The control group was injected with the same volume of normal saline or 50 nmol / kg of exenatide. At 0, 15, 30, 45, 60, and 120 minutes, the blood glucose level was measured with a blood glucose meter, and the hypoglycemic activity of the OXM hybrid peptide was detected.
  • the results of the hypoglycemic experiment show that when the concentration of the OXM hybrid peptide involved in the present invention is 50 nmol / kg, the hypoglycemic effect is equivalent to that of exenatide and liraglutide.
  • the blood glucose of STZ-induced diabetic model mice was measured. Mice with a value higher than 20 mmol / L were randomly divided into groups of six, and the mice fed freely during the experiment.
  • the positive control group was injected intraperitoneally with exenatide or liraglutide at a dose of 50nmol / kg
  • the negative control group was intraperitoneally injected with saline
  • the administration group was injected with 50nmol / kg of OXM hybrid peptide.
  • Compounds were administered at 0 h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 60 h.
  • the evaluation index is the time after the compound is injected intraperitoneally, and the blood glucose value of the mouse is lower than 8.35 mmol / L.
  • the time for stabilizing blood glucose of Exenatide is only 4.0h, and the time for stabilizing blood glucose of liraglutide is 12.1h.
  • the time for stabilizing blood glucose of the long-acting hypoglycemic polypeptide involved in the present invention may be Over 40h. Stable blood glucose experiments show that OXM hybrid peptide has good long-acting hypoglycemic effect, can achieve better long-acting hypoglycemic effect, and has the potential to develop into a hypoglycemic drug administered once every two days.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 8 groups.
  • the OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days.
  • the negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM.
  • the fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • Results are expressed as mean ⁇ SD.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 8 groups.
  • the OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days.
  • the negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the total cholesterol (TC) and triglyceride (TG) contents were measured.
  • TC total cholesterol
  • TG triglyceride
  • mice Male C57bl / 6 mice were fed a high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly divided into 8 groups of 8 groups. The OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the alanine aminotransferase (ALT) content was measured.
  • ALT alanine aminotransferase
  • HEK293 cells were co-transfected with cDNA encoding GLP-1R or GCGR, respectively, and the cell line was expressed and the protein levels of GLP-1R or GCGR in the constructed HEK293 cells were detected by Western Blot to confirm whether a stable and highly expressing cell line HEK293 had been established.
  • cells were seeded in a 96-well plate 2 hours in advance, the compound was dissolved in DMSO, diluted to different times using a medium containing 0.1% bovine serum protein, and added to the co-transfected cells.
  • the fluorescence readings were measured using a ELISA kit from Cisbo and a microplate reader was used to establish a standard curve to convert the fluorescence readings to the corresponding cAMP values.
  • the EC 50 values of the compounds were calculated using the non-linear regression of Graphpad Prism 5.0 software.
  • Results are expressed as mean ⁇ SD, * P ⁇ 0.05, ** P ⁇ 0.01 vs OXM, #P ⁇ 0.05, ## P ⁇ 0.01 vs Exenatide.
  • mice Normal Kunming mice were randomly divided into groups of 8 mice. The mice were housed in standardized animal rooms. Fasted for 12 hours before the experiment and given only drinking water. Before the OXM hybrid peptide was administered to each group of mice, the initial blood glucose level was measured and set to -30 min, and then 50 nmol / kg of the OXM hybrid peptide was intraperitoneally injected. After 30 min, an 18 mmol / kg glucose solution was injected intraperitoneally, which was set to 0 min. The control group was injected with the same volume of normal saline or 50 nmol / kg of exenatide. At 0, 15, 30, 45, 60, and 120 minutes, the blood glucose level was measured with a blood glucose meter, and the hypoglycemic activity of the OXM hybrid peptide was detected.
  • the results of the hypoglycemic experiment show that when the concentration of the OXM hybrid peptide involved in the present invention is 50 nmol / kg, the hypoglycemic effect is equivalent to that of exenatide and liraglutide.
  • the blood glucose of STZ-induced diabetic model mice was measured. Mice with a value higher than 20 mmol / L were randomly divided into groups of six, and the mice fed freely during the experiment.
  • the positive control group was injected intraperitoneally with exenatide or liraglutide at a dose of 50nmol / kg
  • the negative control group was intraperitoneally injected with saline
  • the administration group was injected with 50nmol / kg of OXM hybrid peptide.
  • Compounds were administered at 0h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 60, 72, and 84h.
  • the evaluation index is the time after the compound is injected intraperitoneally, and the blood glucose value of the mouse is lower than 8.35 mmol / L.
  • the time for stabilizing blood glucose of Exenatide is only 4.0h, and the time for stabilizing blood glucose of liraglutide is 12.3h.
  • the time for stabilizing blood glucose of the long-acting hypoglycemic polypeptide involved in the present invention may be Over 60h. Stable blood glucose experiments show that OXM hybrid peptide has good long-acting hypoglycemic effect, can achieve better long-acting hypoglycemic effect, and has the potential to develop into a hypoglycemic drug administered once every two days.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 8 groups.
  • the OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days.
  • the negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM.
  • the fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • Results are expressed as mean ⁇ SD.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 8 groups.
  • the OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days.
  • the negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the total cholesterol (TC) and triglyceride (TG) contents were measured.
  • TC total cholesterol
  • TG triglyceride
  • mice Male C57bl / 6 mice were fed a high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly divided into 8 groups of 8 groups. The OXM hybrid peptide (50 nmol / kg, 10 mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the alanine aminotransferase (ALT) content was measured.
  • ALT alanine aminotransferase
  • mice Normal Kunming mice were randomly divided into groups of 6 mice. The mice were housed in standardized animal rooms. Fasted for 12 hours before the experiment and given only drinking water. Before the OXM hybrid peptide was administered to each group of mice, the initial blood glucose level was measured and set to -30 min, and then 50 nmol / kg of the OXM hybrid peptide was intraperitoneally injected. After 30 min, an 18 mmol / kg glucose solution was injected intraperitoneally, which was set to 0 min. The control group was injected with the same volume of normal saline or 50 nmol / kg of exenatide. At 0, 15, 30, 45, 60, and 120 minutes, the blood glucose level was measured with a blood glucose meter, and the hypoglycemic activity of the OXM hybrid peptide was detected.
  • the results of hypoglycemic experiments show that when the concentration of OXM hybrid peptide involved in the present invention is 50nmol / kg, the hypoglycemic effect is equivalent to that of exenatide and liraglutide .
  • the blood glucose of STZ-induced diabetic model mice was measured. Mice with a value higher than 20 mmol / L were randomly divided into groups of six, and the mice fed freely during the experiment.
  • the positive control group was injected intraperitoneally with exenatide or liraglutide at a dose of 50nmol / kg
  • the negative control group was intraperitoneally injected with saline
  • the administration group was injected with 50nmol / kg of OXM hybrid peptide.
  • Compounds were administered at 0h, and blood glucose levels were measured using a blood glucose meter at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, 60, 72, and 84h.
  • the evaluation index is the time after the compound is injected intraperitoneally, and the blood glucose value of the mouse is lower than 8.35 mmol / L.
  • the time for stabilizing blood glucose of exenatide is only 4.0h and the time for stabilizing blood glucose of liraglutide is 12.3h.
  • the time for stabilizing blood glucose of some long-acting hypoglycemic polypeptides involved in the present invention may be Over 60h. Stable blood glucose experiments show that OXM hybrid peptide has good long-acting hypoglycemic effect, can achieve better long-acting hypoglycemic effect, and has the potential to develop into a hypoglycemic drug administered every three days.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. The fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • OXM hybrid peptide 50nmol / kg, 10mL / kg
  • the fasting weight of the mice in each group was tested on day 1 and 56, and the changes in the average weight of the mice in each group were examined.
  • Results are expressed as mean ⁇ SD.
  • mice Male C57bl / 6 mice were fed with high-fat diet for 4 weeks. Mice weighing more than 30 g were selected for experiments. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the total cholesterol (TC) and triglyceride (TG) contents were measured.
  • TC total cholesterol
  • TG triglyceride
  • mice Male C57bl / 6 mice were fed a high-fat diet for 8 weeks to establish a non-alcoholic fatty liver disease model. Mice were randomly divided into 8 groups of 26 groups. OXM hybrid peptide (50nmol / kg, 10mL / kg) was administered daily for 56 consecutive days. The negative control group was administered daily with normal saline and the positive control group was administered with Drug OXM. After the administration, the serum of the mice was taken and the alanine aminotransferase (ALT) content was measured.
  • OXM hybrid peptide 50nmol / kg, 10mL / kg

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Abstract

本发明公开了一种多肽及其应用，通过对OXM进行改造，与Exenatide的肽序杂合，包括通过氨基酸修饰使其耐DPP-4酶降解，同时缀合脂肪酸链得到具有更长药理作用时间和更好的减重效果的OXM杂合肽，目标多肽的合成是通过正交保护策略固相合成方法快速实现，粗品经纯化，冻干得到OXM杂合肽。

Description

长效化胃泌酸调节素杂合肽及其制备方法和应用 技术领域
本发明涉及药物化学领域，具体涉及一种长效化胃泌酸调节素杂合肽及其制备方法和应用。
背景技术
代谢综合征的病因是蛋白质、脂肪及碳水化合物等多种物质的代谢异常。营养过剩、体力活动减少等会导致肥胖以及肥胖相关疾病，如糖尿病等。近年来，2型糖尿病、血脂代谢异常的发病率日益增高。
胃泌素调节素(Oxyntomodulin，OXM)是小肠L细胞分泌的37个氨基酸组成的多肽，包含胰高血糖素的全部29个氨基酸序列和C端延伸的8个氨基酸部分，与胰高血糖素样肽-1(GLP-1)具有50％的同源性，肽序为：HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA。OXM能够同时激活胰高血糖素样肽-1受体(GLP-1R)和胰高血糖素受体(GCGR)，具有一定的减缓体重增加和降糖效果。OXM激活GCGR后，能促进肝糖原分解和糖异生作用，促进脂肪分解和脂肪酸氧化；加速氨基酸进入肝细胞，发挥产热作用，具有较好的体重减轻和食欲抑制作用。与单纯的GLP-1R激动剂相比，OXM干预体重、调节脂质代谢、改善糖耐量的效果较好，但降血糖活性相对较弱，且半衰期较短。
GLP-1是一种葡萄糖依赖性肠促胰岛素激素。它可以激动GLP-1R，发挥降糖作用。最显著的功能是促进β细胞的再生和修复，增加胰岛β细胞的数量，同时还能避免糖尿病治疗中经常发生的低血糖风险，在糖尿病治疗领域有广阔的应用前景。艾塞那肽是减少DPP-IV酶代谢的典型短效GLP-1受体激动剂，在OXM中引入艾塞那肽的部分肽序，可以提高化合物对GLP-1R的受体激动活性。
发明内容
发明目的：本发明合成了一类肽序改造的OXM类似物，通过杂合OXM与艾塞那肽(Exenatide)的部分肽序结构，增强肽链对GLP-1R亲合力，提高对GLP-1R的激动活性，并保持适度的GCGR激动活性，合成了一类肽序改造的OXM类似物。香豆素以及不同种类脂肪酸等小分子具有高血清白蛋白结合率，将小分子与OXM类似物缀合，大大地延长了降糖作用维持时间，超过现有上市药物利拉鲁肽与艾塞那肽，获得一系列降糖活性和减轻体重效果俱佳的长效化多肽药物。
技术方案：在 第一方面，本发明涉及一类降糖多肽或其药学上可接受的盐，其多肽氨基酸序列为：
Figure PCTCN2019085593-appb-000001
其中
Xaa1为Gly，Aib，D-Ser，Ser，Arg，Asn，Asp，Cys，Gln，Glu，His，Ile，Leu，Lys，Met， Phe，Pro，Thr，Trp，Tyr或Val；
Xaa2取自
Figure PCTCN2019085593-appb-000002
其中，X为-CH 3或-COOH；
Y为-NH-CO-或-CO-NH-；
n取自自然数0-20； m取自自然数1-20；
Xaa3为Ser-OH，Ser-NH 2。本发明的优选方案，其特征是，
Figure PCTCN2019085593-appb-000003
其中，
Xaa1取自Gly或Aib；
Xaa2取自
Figure PCTCN2019085593-appb-000004
Figure PCTCN2019085593-appb-000005
其中，X为-CH 3或-COOH；
Y为-NH-CO-或-CO-NH-；
n取自自然数6，10，14，11，15；
m取自自然数10或11；
Xaa3取自Ser-OH或Ser-NH 2。
本发明涉及的降糖多肽或其药学上可接受的盐也可以表示为：
Figure PCTCN2019085593-appb-000006
Figure PCTCN2019085593-appb-000007
Figure PCTCN2019085593-appb-000008
Figure PCTCN2019085593-appb-000009
Figure PCTCN2019085593-appb-000010
Figure PCTCN2019085593-appb-000011
Figure PCTCN2019085593-appb-000012
Figure PCTCN2019085593-appb-000013
Figure PCTCN2019085593-appb-000014
Figure PCTCN2019085593-appb-000015
Figure PCTCN2019085593-appb-000016
Figure PCTCN2019085593-appb-000017
一种药学上可接受的盐，所述盐为化合物与盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸所成的盐。
在 第二方面，本发明提供了一种药物组合物，包括治疗有效量的至少一种上述化合物和其药学上可接受的盐，或药学上可接受的载体或稀释剂。同时，本发明进一步提供了上述化合物和其药学上可接受的盐，或药学上可接受的载体或稀释剂在制备用于治疗和预防糖尿病的药物中的运用。
在上述第二方面的某些实施方案中，与OXM原型相比，此类降糖多肽对GLP-1R的激动活性 有所提高，并保持适度的GCGR激动活性，具有出色的降血糖效果。在糖尿病模型小鼠体内，一次给药后，稳定血糖时间超过40小时。同时，此类降糖肽还具有良好的减缓体重增加活性。
在上述第二方面的某些实施方案中，与OXM原型相比，此类降糖多肽对GLP-1R的激动活性有所提高，并保持适度的GCGR激动活性，具有出色的降血糖效果。在糖尿病模型小鼠体内，一次给药后，稳定血糖时间超过60小时。同时，此类降糖肽还具有良好的减缓体重增加活性。
在 第三方面，本发明还提供了上述化合物的制备方法，本发明采用固相合成策略高效快速地合成得到上述目标化合物。
本发明还提供了此类降糖多肽的制备方法及其中间体，本发明提供的此类降糖多肽的制备方法采用固相合成法逐步偶联此类降糖多肽主链的各个氨基酸，得到连有主链的肽树脂，在16位L-赖氨酸侧链偶联脂肪酸小分子得到降糖多肽。此方法合成步骤简便，耦合效高，易于纯化，有利于此类降糖多肽的工业化生产。
在上述第三方面中，本发明采用如下的技术方案：
本发明提供了一种降糖多肽的制备方法，其包括以下两种策略:
策略1：肽链中Cys与马来酰亚胺连接的化合物合成方式；
步骤1：取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
步骤2：取所述第一肽树脂，经裂解、纯化，得到纯肽链；
步骤3：将肽链纯品中Cys的巯基与连有马来酰亚胺连接臂的脂肪酸链或香豆素小分子缀合，得到所述化合物；
策略2：肽链中Lys与小分子脂肪酸链连接的合成方式；
步骤1：取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
步骤2：取所述第一肽树脂，在Lys侧链偶联具有式I或式II的脂肪酸链小分子，得到第二肽树脂；
步骤3：取所述第二肽树脂，经裂解、纯化，即得所述化合物；
Figure PCTCN2019085593-appb-000018
其中，所述R 1选自tBu、Dmab、Bzl；
所述R 2选自甲基、乙基、叔丁基和二苯甲基。
所述偶联式I或式II的脂肪酸链小分子的Lys侧链保护基选自Fmoc、Boc、Dde、ivDde。
优选地，本发明提供的制备方法中，步骤1中的树脂为Rink Amide AM Resin，Fmoc-Rink amide-MBHA或Wang Resin。在本发明的一些实施例中，本发明提供的制备方法中，步骤1中的树脂具体为Fmoc-Rink amide-MBHA或Wang Resin。
优选地，本发明提供的制备方法中，裂解所用裂解试剂为TFA、苯甲硫醚、苯甲醚和EDT的混合物。在本发明的一些实施例中，本发明提供的制备方法中，步骤3中裂解所用试剂中TFA、苯甲硫醚、苯甲醚和EDT的体积之比为(85～92)：(4～6)：(2～3)：(2～6)。在本发明的另外一些实施例中，本发明提供的制备方法中，步骤3中裂解所用裂解试剂中TFA、苯甲硫醚、苯甲醚和EDT的体积之比为90:5:3:2。
在本发明的另外一些实施例中，本发明提供的制备方法中，纯化所用方法为色谱分离法。在本发明的另外一些实施例中，本发明提供的制备方法中，纯化所用色谱柱为C18柱。
本发明的有益效果：
1.本发明提供的化合物具有显著的降糖、降低体重效果，化学性质稳定，且活性明显优于原型肽OXM。
2.本发明提供的部分化合物降血糖作用可维持40h以上，较内源性GLP-1(半衰期2～3min)或上市药物艾塞那肽(半衰期2.4h)有显著的提高。
3.采用正交保护策略的固相合成OXM杂合肽得到肽链的粗品的纯度大于85％，与常规合成方法相比大大提高，方便后续的纯化工作。
4.本法采用固相方法合成OXM杂合肽的成本低。由于偶合效率较高，所需要保护氨基酸平均只需要2倍过量，而常规合成方法中需要4到5倍过量的氨基酸，极大的节约了成本。
5.采用Fmoc/tBu正交保护固相合成策略合成OXM杂合肽的方法易于实现自动化、大规模化，这使其更适合工业化生产。
因此用本发明提供的通过固相合成技术制备的OXM杂合肽，降糖和减缓体重增加活性好，药效时间长，收率高、合成周期短、粗品纯化容易，生产成本低、易于工业自动化生产。制备得到的OXM杂合肽，适合作为治疗糖尿病、肥胖药物的活性成分。
附图说明
图1为OXM杂合肽SEQ.ID NO:1～6的隔日糖耐量实验结果。
图2为OXM杂合肽SEQ.ID NO:7～12的隔日糖耐量实验结果。
图3为OXM杂合肽SEQ.ID NO:13～18的隔日糖耐量实验结果。
图4为OXM杂合肽SEQ.ID NO:19～24的隔日糖耐量实验结果。
图5为OXM杂合肽SEQ.ID NO:19～21的稳定血糖实验结果。
图6为OXM杂合肽SEQ.ID NO:22～24的稳定血糖实验结果。
图7为OXM杂合肽SEQ.ID NO:1～12的TC检测结果。
图8为OXM杂合肽SEQ.ID NO:13～24的TC检测结果。
图9为OXM杂合肽SEQ.ID NO:1～12的TG检测结果。
图10为OXM杂合肽SEQ.ID NO:13～24的TG检测结果。
图11为OXM杂合肽SEQ.ID NO:1～12的ALT检测结果。
图12为OXM杂合肽SEQ.ID NO:13～24的ALT检测结果。
图13为OXM杂合肽SEQ.ID NO:25～27的稳定血糖实验结果。
图14为OXM杂合肽SEQ.ID NO:28～29的稳定血糖实验结果。
图15为OXM杂合肽SEQ.ID NO:25～29的TC检测结果。
图16为OXM杂合肽SEQ.ID NO:25～29的TG检测结果。
图17为OXM杂合肽SEQ.ID NO:25～29的ALT检测结果。
图18为OXM杂合肽SEQ.ID NO:30～35的腹腔糖耐量实验结果。
图19为OXM杂合肽SEQ.ID NO:30～32的稳定血糖实验结果。
图20为OXM杂合肽SEQ.ID NO:33～35的稳定血糖实验结果.
图21为OXM杂合肽SEQ.ID NO:30～35的TC检测结果。
图22为OXM杂合肽SEQ.ID NO:30～35的TG检测结果。
图23为OXM杂合肽SEQ.ID NO:30～35的ALT检测结果。
图24为OXM杂合肽SEQ.ID NO:36～41的腹腔糖耐量实验结果
图25为OXM杂合肽SEQ.ID NO:36～38的稳定血糖实验结果。
图26为OXM杂合肽SEQ.ID NO:39～41的稳定血糖实验结果。
图27为OXM杂合肽SEQ.ID NO:36～41的TC检测结果。
图28为OXM杂合肽SEQ.ID NO:36～41的TG检测结果。
图29为OXM杂合肽SEQ.ID NO:36～41的ALT检测结果。
图30为OXM杂合肽SEQ.ID NO:42～47的腹腔糖耐量实验结果。
图31为OXM杂合肽SEQ.ID NO:48～53的腹腔糖耐量实验结果。
图32为OXM杂合肽SEQ.ID NO:54～59的腹腔糖耐量实验结果。
图33为OXM杂合肽SEQ.ID NO:42～47的稳定血糖实验结果。
图34为OXM杂合肽SEQ.ID NO:48～53的稳定血糖实验结果。
图35为OXM杂合肽SEQ.ID NO:54～59的稳定血糖实验结果。
图36为OXM杂合肽SEQ.ID NO:42～59的TC检测结果。
图37为OXM杂合肽SEQ.ID NO:42～59的TG检测结果。
图38为OXM杂合肽SEQ.ID NO:42～59的ALT检测结果。
具体实施方式
在本说明书全文中采用以下缩写：
英文缩写 中文
DCM 二氯甲烷
NMP N-甲基吡咯烷酮
HBTU 苯并三氮唑-N，N，N'，N'-四甲基脲六氟磷酸酯
HOBt 1-羟基-苯并三氮唑
DIEA/DIPEA N，N'-二异丙基乙胺
Fmoc N-9-芴甲氧羰基
ESI-MS 电喷雾质谱
EDT 乙二硫醇
HPLC 高效液相色谱
TFA 三氟乙酸
tBu 叔丁基
Boc 叔丁氧羰基
EDC·HCl 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
DMAP 4-二甲氨基吡啶
DIC N,N-二异丙基碳二亚胺
Na 2SO 4 硫酸钠
DMSO 二甲基亚砜
K 2CO 3 碳酸钾
HCl 氯化氢
Pd/C 钯碳
Dde 1-(4，4-二甲基-2，6-二氧杂亚环己基亚甲基)-乙基
Fmoc-AEEA [2-[2-(芴甲氧羰基氨基)乙氧基]乙氧基]乙酸
OtBu 氧叔丁基
Dmab 4-{N-[1-(4,4-二甲基-2,6-二氧代环己基)-3-甲基丁基]氨基}苄基
Bzl 卞基
ivDde 1-(4,4-二甲基-2,6-二氧代环亚己基)-3-甲基丁基
本发明是通过下列实施例来进行说明的，但这些实施例不做任何限制本发明的解释。
实施例1
Figure PCTCN2019085593-appb-000019
的固相合成
(1)树脂的溶胀
称取Fmoc-Rink amide-MBHA Resin 50mg(取代度0.4mmol/g)，经DCM 7mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，分别用NMP，DCM 7mL冲洗干净。
(2)Fmoc保护基的脱除
将溶胀好的树脂放入反应器中，向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。得到脱去初始连接的Fmoc保护基的树脂。
(3)Fmoc-Ser(tBu)-Rink amide-MBHA Resin的合成
将Fmoc-Ser(tBu)-OH(15.4mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
(4)肽链的延长
按照肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有SEQ.ID NO:1的树脂。
(5)树脂上多肽的裂解
将上述得到的连有SEQ.ID NO:1的树脂放入反应瓶中，各加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品77.1mg，收率为90.2％。
使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min检测波长为214nm。收集的溶液冻干得纯品28.5mg。理论相对分子质量为4273.7。ESI-MS m/z:Calcd.[M+3H] 3+1425.6,[M+4H] 4+1069.4；Found[M+3H] 3+1425.1,[M+4H] 4+1069.0。
实施例2
Figure PCTCN2019085593-appb-000020
合成方法同实施例1，收集的溶液冻干得纯品29.2mg。理论相对分子质量为4269.7。ESI-MS m/z: Calcd.[M+3H] 3+1424.2,[M+4H] 4+1068.4；Found[M+3H] 3+1424.8,[M+4H] 4+1068.1。
实施例3
Figure PCTCN2019085593-appb-000021
合成方法同实施例1，收集的溶液冻干得纯品27.6mg。理论相对分子质量为4228.6。ESI-MS m/z:Calcd.[M+3H] 3+1410.5,[M+4H] 4+1058.2；Found[M+3H] 3+1410.0,[M+4H] 4+1058.2。
实施例4
Figure PCTCN2019085593-appb-000022
合成方法同实施例1，收集的溶液冻干得纯品29.4mg。理论相对分子质量为4283.7。ESI-MS m/z:Calcd.[M+3H] 3+1428.9,[M+4H] 4+1071.9；Found[M+3H] 3+1429.0,[M+4H] 4+1072.0。
实施例5
Figure PCTCN2019085593-appb-000023
合成方法同实施例1，收集的溶液冻干得纯品27.6mg。理论相对分子质量为4253.7。ESI-MS m/z:Calcd.[M+3H] 3+1418.9,[M+4H] 4+1064.4；Found[M+3H] 3+1419.5,[M+4H] 4+1064.5。
实施例6
Figure PCTCN2019085593-appb-000024
合成方法同实施例1，收集的溶液冻干得纯品28.9mg。理论相对分子质量为4256.6。ESI-MS m/z:Calcd.[M+3H] 3+1419.9,[M+4H] 4+1065.2；Found[M+3H] 3+1420.1,[M+4H] 4+1065.5。
实施例7
Figure PCTCN2019085593-appb-000025
的固相合成
将编号为SEQ.ID NO.1的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高 速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品29.4mg。理论相对分子质量为4483.0。ESI-MS m/z:Calcd.[M+3H] 3+1495.3,[M+4H] 4+1121.1；Found[M+3H] 3+1495.8,[M+4H] 4+1121.2。
实施例8
Figure PCTCN2019085593-appb-000026
的固相合成
将编号为SEQ.ID NO.2的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品27.6mg。理论相对分子质量为4479.0。ESI-MS m/z:Calcd.[M+3H] 3+1494.0,[M+4H] 4+1120.7；Found[M+3H] 3+1494.0,[M+4H] 4+1120.9。
实施例9
Figure PCTCN2019085593-appb-000027
的固相合成
将编号为SEQ.ID NO.3的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.1mg。理论相对分子质量为4437.9。ESI-MS m/z:Calcd.[M+3H] 3+1480.3,[M+4H] 4+1110.5；Found[M+3H] 3+1480.0,[M+4H] 4+1110.3。
实施例10
Figure PCTCN2019085593-appb-000028
的固相合成
将编号为SEQ.ID NO.4的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.9mg。理论相对分子质量为4493.0。ESI-MS m/z:Calcd.[M+3H] 3+1498.7,[M+4H] 4+1124.2；Found[M+3H] 3+1498.9,[M+4H] 4+1124.3。
实施例11
Figure PCTCN2019085593-appb-000029
的固相合成
将编号为SEQ.ID NO.5的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.3mg。理论相对分子质量为4463.0。ESI-MS m/z:Calcd.[M+3H] 3+1488.7,[M+4H] 4+1116.7；Found[M+3H] 3+1488.0,[M+4H] 4+1116.0。
实施例12
Figure PCTCN2019085593-appb-000030
的固相合成
将编号为SEQ.ID NO.6的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正辛烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品29.4mg。理论相对分子质量为4465.9。ESI-MS m/z:Calcd.[M+3H] 3+1489.6,[M+4H] 4+1117.5；Found[M+3H] 3+1489.6,[M+4H] 4+1117.7。
实施例13
Figure PCTCN2019085593-appb-000031
的固相合成
将编号为SEQ.ID NO.1的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.7mg。理论相对分子质量为4539.1。 ESI-MS m/z:Calcd.[M+3H] 3+1514.0,[M+4H] 4+1135.8；Found[M+3H] 3+1514.5,[M+4H] 4+1135.5。
实施例14
Figure PCTCN2019085593-appb-000032
的固相合成
将编号为SEQ.ID NO.2的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品27.1mg。理论相对分子质量为4535.1。ESI-MS m/z:Calcd.[M+3H] 3+1512.7,[M+4H] 4+1134.8；Found[M+3H] 3+1512.5,[M+4H] 4+1134.2。
实施例15
Figure PCTCN2019085593-appb-000033
的固相合成
将编号为SEQ.ID NO.3的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.5mg。理论相对分子质量为4494.0。ESI-MS m/z:Calcd.[M+3H] 3+1499.0,[M+4H] 4+1124.5；Found[M+3H] 3+1499.0,[M+4H] 4+1124.0。
实施例16
Figure PCTCN2019085593-appb-000034
的固相合成
将编号为SEQ.ID NO.4的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品28.1mg。理论相对分子质量为4549.1。ESI-MS m/z:Calcd.[M+3H] 3+1517.4,[M+4H] 4+1138.3；Found[M+3H] 3+1517.2,[M+4H] 4+1138.8。
实施例17
Figure PCTCN2019085593-appb-000035
的固相合成
将编号为SEQ.ID NO.5的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品27.8mg。理论相对分子质量为4519.1。ESI-MS m/z:Calcd.[M+3H] 3+1507.4,[M+4H] 4+1130.8；Found[M+3H] 3+1507.0,[M+4H] 4+1130.2。
实施例18
Figure PCTCN2019085593-appb-000036
的固相合成
将编号为SEQ.ID NO.6的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十二烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品29.1mg。理论相对分子质量为4522.0。ESI-MS m/z:Calcd.[M+3H] 3+1508.3,[M+4H] 4+1131.5；Found[M+3H] 3+1508.6,[M+4H] 4+1131.4。
实施例19
Figure PCTCN2019085593-appb-000037
的固相合成
将编号为SEQ.ID NO.1的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品30.2mg。理论相对分子质量为4595.2。ESI-MS m/z:Calcd.[M+3H] 3+1532.7,[M+4H] 4+1149.8；Found[M+3H] 3+1532.8,[M+4H] 4+1149.2。
实施例20
Figure PCTCN2019085593-appb-000038
的固相合成
将编号为SEQ.ID NO.2的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品30.4mg。理论相对分子质量为4591.2。ESI-MS m/z:Calcd.[M+3H] 3+1531.4,[M+4H] 4+1148.8；Found[M+3H] 3+1531.5,[M+4H] 4+1149.0。
实施例21
Figure PCTCN2019085593-appb-000039
的固相合成
将编号为SEQ.ID NO.3的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品29.4mg。理论相对分子质量为4550.1。ESI-MS m/z:Calcd.[M+3H] 3+1517.7,[M+4H] 4+1138.5；Found[M+3H] 3+1517.0,[M+4H] 4+1138.7。
实施例22
Figure PCTCN2019085593-appb-000040
的固相合成
将编号为SEQ.ID NO.4的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品30.2mg。理论相对分子质量为4605.2。ESI-MS m/z:Calcd.[M+3H] 3+1536.1,[M+4H] 4+1152.3；Found[M+3H] 3+1536.6,[M+4H] 4+1152.2。
实施例23
Figure PCTCN2019085593-appb-000041
的固相合成
将编号为SEQ.ID NO.5的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品31.2mg。理论相对分子质量为4575.2。ESI-MS m/z:Calcd.[M+3H] 3+1526.1,[M+4H] 4+1144.8；Found[M+3H] 3+1526.0,[M+4H] 4+1145.6。
实施例24
Figure PCTCN2019085593-appb-000042
的固相合成
将编号为SEQ.ID NO.6的多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μl，室温下搅拌反应。反应的检测和纯化方法与实施例7相同。将收集的溶液减压浓缩去除乙腈，冻干得纯品30.8mg。理论相对分子质量为4578.1。ESI-MS m/z:Calcd.[M+3H] 3+1527.0,[M+4H] 4+1145.5；Found[M+3H] 3+1527.2,[M+4H] 4+1145.9。
实施例25
Figure PCTCN2019085593-appb-000043
的的固相合成
1、肽链的合成
1.1树脂的溶胀
称取Fmoc-Rink amide-MBHA Resin 50mg(取代度0.4mmol/g)，经DCM 7mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，分别用NMP，DCM 7mL冲洗干净。
1.2 Fmoc保护基的脱除
将溶胀好的树脂放入反应器中，向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。得到脱去初始连接的Fmoc保护基的树脂。
1.3 Fmoc-Ser(tBu)-Rink amide-MBHA Resin的合成
将Fmoc-Ser(tBu)-OH(15.4mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中，反 应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
1.4肽链的延长
按照肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有SEQ.ID NO:1主链肽序的树脂。
1.6树脂上多肽的裂解
将上述得到的连有SEQ.ID NO:25主链肽序的树脂放入反应瓶中，各加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品77.1mg，收率为90.2％。使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min检测波长为214nm。收集的溶液冻干得纯品28.5mg。理论相对分子质量为4256.6。ESI-MS m/z:Calcd.[M+3H] 3+1419.9,[M+4H] 4+1065.2；Found[M+3H] 3+1420.1,[M+4H] 4+1065.5。
2、12-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)十二酸的合成
将12-氨基十二酸(0.86g，4mmol)与马来酸酐(0.47g，4.8mmol)溶于冰醋酸中，超声溶解后，120℃回流反应6h，薄层板检测反应完全后，将反应液冷却至室温，乙酸乙酯萃取三次(3×20mL)，合并上层萃取液，萃取液使用饱和食盐水洗3次，无水Na 2SO 4干燥过夜。萃取液真空旋干得到粗品，粗品柱层析(乙酸乙酯/石油醚)分离得淡黄色纯品0.89g，产率80％，mp 91-92℃。
1H-NMR(DMSO-d 6,300MHz):δppm:12.45(s,1H,-COO H),7.50(s,2H,-COC H＝C HCO-),3.88(t,2H,J＝7.0Hz,-NC H 2- ),2.68(t,J＝7.3Hz,2H,-C H 2 COOH),2.00-1.96(m,4H,-NCH 2C H 2 (CH 2) 7C H 2 ),1.73(s,14H,-NCH 2CH 2(C H 2) 7 CH 2).ESI-MS m/z:294.1[M+H] +。
3、化学修饰的OXM缀合物的合成与纯化
将上步得到的12-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)十二酸用DMSO溶解配成约10mg/mL的溶液，将得到的SEQ.ID NO:1主链肽序也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品8.1mg。理论相对分子质量为4551.7。ESI-MS m/z:Calcd.[M+3H] 3+1518.3,[M+4H] 4+1138.9；Found[M+3H] 3+1517.4,[M+4H] 4+1138.8。
实施例26
Figure PCTCN2019085593-appb-000044
的合成
1、16-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)十六酸的合成
将16-氨基十六酸(1.09g，4mmol)与马来酸酐(0.47g，4.8mmol)溶于冰醋酸中，超声溶解后，120℃回流反应6h，薄层板检测反应完全后，将反应液冷却至室温，乙酸乙酯萃取三次(3×20mL)，合并上层萃取液，萃取液使用饱和食盐水洗3次，无水Na 2SO 4干燥过夜。萃取液真空旋干得到粗品，粗品柱层析(乙酸乙酯/石油醚)分离得淡黄色纯品1.02g，产率72％。
1H-NMR(DMSO-d 6,300MHz):δppm:12.45(s,1H,-COO H),7.50(s,2H,-COC H＝C HCO-),3.88(t,2H,J＝7.0Hz,-NC H 2- ),2.68(t,J＝7.3Hz,2H,-C H 2 COOH),2.00-1.96(m,4H,-NCH 2C H 2 (CH 2) 7C H 2 ),1.76(s,22H,-NCH 2CH 2(C H 2) 11 CH 2).ESI-MS m/z:352.4[M+H] +。
2、化学修饰的OXM缀合物的合成与纯化
将上步得到的16-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)十六酸用DMSO溶解配成约10mg/mL的溶液，将得到的SEQ.ID NO:1主链肽序也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品8.7mg。理论相对分子质量为4607.8。ESI-MS m/z:Calcd.[M+3H] 3+1536.9,[M+4H] 4+1152.9；Found[M+3H] 3+1537.6,[M+4H] 4+1153.5。
实施例27
Figure PCTCN2019085593-appb-000045
1、化学修饰基的合成
3,3’-(4-羧基苯亚甲基)-二-4-羟基香豆素的合成
对羧基苯甲醛(0.45g，3mmol)溶解于无水乙醇20ml，然后加入4-羟基香豆素(0.98g，6mmol)。加热回流12h后，反应液冷却至室温后过滤，滤饼使用乙醇10ml洗3次，即得产品1.12g，收率82.1％，ESI-MS m/z:456.4[M+H] +.
(12-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)十二烷基)氨基甲酸叔丁酯的合成
将N-Boc-十二烷基二胺(1.2g，4mmol)与马来酸酐(0.49g，4.8mmol)溶于冰醋酸中，120℃加热反应6h，薄层板检测反应完全后，将反应液冷却至室温，用乙酸乙酯萃取(3×20mL)，合并上层萃取液，萃取液使用饱和食盐水洗3次，无水Na 2SO 4干燥过夜。将萃取液减压浓缩，得到的粗品经柱层析纯化得淡黄色纯品1.10g，产率72％，MS(ESI,m/z):380.5[M+H] +.
4-(双(4-羟基-2-氧代-2H-色烯-3-基)甲基)-N-(12-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)十二烷基)苯甲酰胺的合成
(12-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)十二烷基)氨基甲酸叔丁酯(0.76g，2mmol)用HCl饱和的乙酸乙酯溶解，搅拌3h后减压蒸馏去除溶剂，DCM复溶，加入3,3’-(4-羧基苯亚甲基)-二-4-羟基香豆素(0.91g，2mmol)、DIC(0.30g，2.4mmol)和HOBt(0.32g，2.4mmol)，室温搅拌过夜。薄层板检测反应结束后将反应液倒入水中并用乙酸乙酯萃取三次，合并萃取液，分别用饱和K 2CO 3溶液，HCl 1M，饱和食盐水洗三次。萃取液加入无水Na 2SO 4干燥过夜，减压浓缩，得粗品，柱层析纯化得纯品0.93g，收率65％。ESI-MS m/z:719.4[M+H] +.
2、化学修饰的OXM缀合物的合成与纯化
将上步得到的4-(双(4-羟基-2-氧代-2H-色烯-3-基)甲基)-N-(12-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)十二烷基)苯甲酰胺用DMSO溶解配成约10mg/mL的溶液，将SEQ.ID NO:1主链肽序也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品7.9mg。理论相对分子质量为4974.9。ESI-MS m/z:Calcd.[M+3H] 3+1659.3,[M+4H] 4+1244.7；Found[M+3H] 3+1659.3,[M+4H] 4+1245.8。
实施例28
Figure PCTCN2019085593-appb-000046
1、化学修饰基的合成
3,3’-(4-硝基苯亚甲基)-二-4-羟基香豆素的合成
称取对硝基苯甲醛(3.02g,0.02mol)，用无水乙醇35ml溶解；加入4-羟基香豆素(6.6g,0.041mol)，补加无水乙醇15ml使溶解完全。80℃反应4h，趁热过滤，滤饼用热乙醇10ml洗3次，得产品8.2g，收率90.0％，mp 227℃。
1H-NMR(CDCl 3,300MHz)δppm:6.13(s,H,-CH-),7.43(m,8H,Ar-H),7.68(m,2H,Ar-H),8.18(m,2H,Ar-H).ESI-MS m/z:456.0[M+H] +.
3,3’-(4-氨基苯亚甲基)-二-4-羟基香豆素的合成
称取3,3’-(4-硝基苯亚甲基)-二-4-羟基香豆素(1.14g,0.0025mol)，用醋酸30ml使之悬浮，加入5％Pd/C 0.3g，搅拌，氢气三通抽气3次，瓶口涂凡士林，常温氢化，反应过夜，抽滤，滤液蒸去部分溶剂，丙酮重结晶，得产品0.8g，收率75.1％，mp 220℃。
1H-NMR(DMSO-d 6,300MHz)δppm:6.27(s,H,-CH-),7.23(m,8H,Ar-H),7.49(m,2H,Ar-H),7.81(m,2H,Ar-H).ESI-MS m/z:426.0[M+H] +.
3,3’-(4-(12-马来酰亚胺基十二酰胺基)苯亚甲基)-二-4-羟基香豆素的合成
12-马来酰胺十二酸(294.1mg，1mmol)溶于四氢呋喃中，加DIC(17μL，1.1mmol)和HOBt(148.5mg，1.1mmol)，室温搅拌30min，接着将上述溶液缓慢滴入3,3’-(4-氨基苯亚甲基)-二-4-羟基香豆素和DIPEA(17.4μL，0.1mmol)的四氢呋喃溶液，室温搅拌过夜。薄层板检测反应结束后将反应液倒入水中并用乙酸乙酯萃取三次，合并萃取液，分别用K 2CO 3，HCl 1M，饱和食盐水洗三次。萃取液加入无水Na 2SO 4干燥过夜，减压浓缩，得粗品，柱层析纯化得纯品，收率69％，mp 204-206℃。 1H-NMR(DMSO-d 6,300MHz):δppm:10.17(s,1H,-CONH-),8.31(d,J＝7.8Hz,2H,Ar-H),8.00(t,J＝7.2Hz,2H,Ar-H),7.84(d,J＝8.0Hz,2H,Ar-H),7.76-7.72(m,6H,Ar-H),7.49(s,2H,-COCH＝CHCO-),6.70(s,1H,-CH-),3.87(t,J＝7.0Hz,2H,-NCH 2-),2.74(t,J＝7.2Hz,2H,-COCH 2-),2.05-1.97(m,4H,-NCH 2CH 2(CH 2) 7CH 2-),1.70(s,14H,-NCH 2CH 2(CH 2) 7CH 2-).ESI-MS m/z:703.1[M+H] +.
2、化学修饰的OXM缀合物的合成与纯化
将上步得到的3,3’-(4-(12-马来酰亚胺基十二酰胺基)苯亚甲基)-二-4-羟基香豆素用DMSO溶解配成约10mg/mL的溶液，将SEQ.ID NO:1主链肽序也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品8.6mg。理论相对分子质量为4960.9。ESI-MS m/z:Calcd.[M+3H] 3+1654.6,[M+4H] 4+1241.2；Found[M+3H] 3+1654.2,[M+4H] 4+1240.2。
实施例29
Figure PCTCN2019085593-appb-000047
1、化学修饰基的合成
3,3’-(4-羧基苯亚甲基)-二-4-羟基香豆素的合成
对羧基苯甲醛(0.45g,3mmol)，溶解于无水乙醇20ml,然后加入4-羟基香豆素(0.98g,6mmol)。加热回流12h，反应液冷却至室温后过滤，滤饼使用乙醇10ml洗3次，即得产品1.12g，产率82.1％。
1H-NMR(DMSO-d 6,300MHz):δppm:8.37(d,J＝7.8Hz,2H,Ar- H),8.29(d,J＝8.0Hz,2H,Ar- H),8.06(t,J＝7.2Hz,2H,Ar- H),7.84-7.74(m,6H,Ar- H),6.86(s,1H,-C H-).ESI-MS m/z:456.4[M+H] +.
2-(2-(2-氨基乙氧基)乙氧基)乙基氨基甲酸叔丁酯的合成
1,8-二氨基-3,6-二氧杂辛烷(10.7g，72.3mmol)溶DCM 70ml中，Boc酸酐(2.2g，10.1mmol)溶于DCM 50ml中，在0℃条件下，Boc酸酐缓慢滴加至1,8-二氨基-3,6-二氧杂辛烷溶液中。滴加完毕后，反应液恢复至室温后，继续反应4h，反应完毕后，使用碱性氧化铝进行柱层析，分离纯化可得无色透明油状物1.8g，产率72.0％。
1H NMR(DMSO-d 6,300MHz):δ4.96(s,1H,-N H-),3.54(s,4H,-OC H 2 -),3.42(dt,J＝5.1,5.1Hz,4H,-OC H 2 CH 2O-),3.10(dt,J＝5.1,5.1Hz,2H,-C H 2 NH(Boc)),2.55(s,2H,-C H 2 NH 2),1.45(s,2H,-N H 2 ),1.42(s,9H,-t-Bu).ESI-MS m/z:249.0[M+H] +.
(2-(2-(2-(3-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)丙酰胺基)乙氧基)乙氧基)乙基)氨基甲酸叔丁酯的合成
称取2.2.1.2部分所述的3-(2,5-二氢-2,5-二氧代-1H-吡咯-1-基)丙酸(523.mg，3.1mmol)和2-(2-(2-氨基乙氧基)乙氧基)乙基氨基甲酸叔丁酯(843mg，3.4mmol)并溶于15ml二氯甲烷中，冰浴冷却后，接着加入EDC·HCl(680mg，3.6mmoL)和DMAP(75mg，0.6mmoL)。将反应液的温度从0℃慢慢升高至室温，反应6h，经柱层析纯化得白色软膏状纯品0.99g，产率80.5％。
1H NMR(DMSO-d 6,300MHz):δ8.03(s,1H,-CH 2CON H-),7.00(s,2H,-COC H＝C HCO-),6.76(s,1H,-OCON H-),5.75(t,J＝7.2Hz,2H,-NC H 2 CH 2-),3.59(t,J＝4.4Hz,4H,-OC H 2 CH 2NH-),3.48(s,4H,-CH 2O C H 2C H 2OCH 2-),3.15(t,2H,J＝5.6Hz,-CH 2CONH C H 2-),3.06(t,2H,J＝5.8Hz,-OCONHC H 2-),2.33(t,J＝6.8Hz,2H,-C H 2CONH-),1.36(s,9H,-C H 3).ESI-MS m/z:399.5[M+H] +.
4-(双(4-羟基-2-氧代-2H-苯并吡喃-3-基)甲基)-N-(2-(2-(2-(3-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)丙酰胺基)乙氧基)乙氧基)乙基)苯甲酰胺的合成
(2-(2-(2-(3-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)丙酰胺基)乙氧基)乙氧基)乙基)氨基甲酸叔丁酯(159.8mg，0.4mmol)溶于乙腈3ml，冷却至室温后，加入三氟醋酸1ml，反应完全后，减压蒸馏去除溶剂，得到淡黄色油状物，复溶于四氢呋喃3ml。将3,3’-(4-羧基苯亚甲基)-二-4-羟基香豆素(182.6mg，0.4mmol)溶于四氢呋喃5ml中，加入DIC(68μL,0.44mmol)和HOBt(59.4mg,0.44mmol)，室温搅拌30min来活化羧基，将上述溶液缓慢滴入上述脱Boc所得产物的四氢呋喃溶液，室温搅拌 过夜反应。反应结束后将反应液倒入冰水中并用20mL二氯甲烷萃取三次，合并萃取液，分别用饱和K 2CO 3，HCl 1M，饱和食盐水洗三次。萃取液加入无水Na 2SO 4干燥过夜后，减压浓缩得粗品，柱层析分离得白色膏状纯品132.8mg，收率45％。
1H-NMR(DMSO-d 6,300MHz):δppm:8.94(s,1H,-N HCO-Ar),8.38(s,1H,-N HCOCH 2-),8.37(d,J＝7.8Hz,2H,Ar- H),8.29(d,J＝8.25Hz,2H,Ar- H),8.06(t,J＝7.2Hz,2H,Ar- H),7.84-7.74(m,6H,Ar- H),6.98(s,2H,-COC H＝C HCO-),6.86(s,1H,-C H-).5.75(t,J＝7.2Hz,2H,-NC H 2CH 2-),3.59(t,J＝6.0Hz,4H,-OC H 2 CH 2NH-),3.35(s,4H,-OC H 2 C H 2 O-),3.15(t,4H,J＝6.0Hz,-CONH C H 2-),2.33(t,J＝7.2Hz,2H,-C H 2CONH-).ESI-MS m/z:738.4[M+H] +.
2、化学修饰的OXM缀合物的合成与纯化
将上步得到的4-(双(4-羟基-2-氧代-2H-苯并吡喃-3-基)甲基)-N-(2-(2-(2-(3-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)丙酰胺基)乙氧基)乙氧基)乙基)苯甲酰胺用DMSO溶解配成约10mg/mL的溶液，将SEQ.ID NO:1主链肽序也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品8.3mg。理论相对分子质量为4993.8。ESI-MS m/z:Calcd.[M+3H] 3+1665.6,[M+4H] 4+1249.5；Found[M+3H] 3+1665.8,[M+4H] 4+1249.1。
实施例30
Figure PCTCN2019085593-appb-000048
的固相合成
1.多肽主链的合成
1.1树脂的溶胀
称取Fmoc-Rink amide-MBHA Resin 50mg(取代度0.4mmol/g)，经DCM 7mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，分别用NMP，DCM 7mL冲洗干净。
1.2 Fmoc保护基的脱除
将溶胀好的树脂放入反应器中，向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。得到脱去初始连接的Fmoc保护基的树脂。
1.3 Fmoc-Ser(tBu)-Rink amide-MBHA Resin的合成
将Fmoc-Ser(tBu)-OH(15.4mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04 mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
1.4肽链的延长
按照肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有SEQ.ID NO:1主链氨基酸序列的树脂。
2.在肽树脂上连接式I
将连有SEQ.ID NO:1主链的树脂放入反应器中，加入2％水合肼溶液脱除16位Lys的侧链保护基Dde，反应结束后用NMP洗涤干净。将Fmoc-Glu-OtBu(17.0mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。将16烷酸(10.3mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。得到连有SEQ.ID NO:1完整结构的树脂。
3.树脂上多肽的裂解
将上述连有SEQ.ID NO:1完整结构的树脂放入反应瓶中，加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品86.3mg，收率为92.4％。使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min检测波长为214nm。收集的溶液冻干得纯品31.2mg。理论相对分子质量为4666.6。ESI-MS m/z:Calcd.[M+3H] 3+1556.5,[M+4H] 4+1167.7；Found[M+3H] 3+1556.9,[M+4H] 4+1166.9。
实施例31
Figure PCTCN2019085593-appb-000049
合成方法同实施例30，将收集的溶液冻干得纯品29.9mg。理论相对分子质量为4662.3。ESI-MS m/z:Calcd.[M+3H] 3+1555.1,[M+4H] 4+1166.6；Found[M+3H] 3+1555.7,[M+4H] 4+1166.1。
实施例32
Figure PCTCN2019085593-appb-000050
合成方法同实施例30，将收集的溶液冻干得纯品30.4mg。理论相对分子质量为4621.2。ESI-MS m/z:Calcd.[M+3H] 3+1541.4,[M+4H] 4+1156.3；Found[M+3H] 3+1541.9,[M+4H] 4+1156.7。
实施例33
Figure PCTCN2019085593-appb-000051
合成方法同实施例30，将收集的溶液冻干得纯品32.2mg。理论相对分子质量为4676.3。ESI-MS m/z:Calcd.[M+3H] 3+1559.8,[M+4H] 4+1170.1；Found[M+3H] 3+1560.4,[M+4H] 4+1170.5。
实施例34
Figure PCTCN2019085593-appb-000052
合成方法同实施例30，将收集的溶液冻干得纯品30.7mg。理论相对分子质量为4646.3。ESI-MS m/z:Calcd.[M+3H] 3+1549.8,[M+4H] 4+1162.6；Found[M+3H] 3+1550.4,[M+4H] 4+1162.
实施例35
Figure PCTCN2019085593-appb-000053
合成方法同实施例30，将收集的溶液冻干得纯品29.4mg。理论相对分子质量为4649.2。ESI-MS m/z:Calcd.[M+3H] 3+1550.7,[M+4H] 4+1163.3；Found[M+3H] 3+1550.8,[M+4H] 4+1163.7。
实施例36
Figure PCTCN2019085593-appb-000054
的固相合成
1、式I的合成
称取2-CTC树脂100mg(取代度0.8mmol/g)，经DCM溶胀30min，再用NMP 10mL溶胀30min，分别用NMP，DCM 7mL冲洗干净。将Fmoc-AEEA(61.6mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，将DCM：甲醇：DIEA为5：4：1的10mL溶液加入树脂封闭反应1小时，滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。同样方法再次偶联Fmoc-AEEA，反应结束后，用DCM和NMP各7mL洗涤树脂3次。向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。将Fmoc-Glu-OtBu(68.0mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。将十八烷二酸单叔丁酯(59.2mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
将上述得到的连有式I的树脂放入反应瓶中，加入裂解剂20％三氟乙醇/DCM 10ml，常温振摇30分钟，反应结束后抽滤，蒸干溶剂得到式I粗品42.4mg。理论相对分子质量为846.1，ESI-MS m/z:845.4[M-H +]。
2、多肽主链的合成
2.1树脂的溶胀
称取Fmoc-Rink amide-MBHA Resin 50mg(取代度0.4mmol/g)，经DCM 7mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，最后分别用NMP，DCM 7mL冲洗干净。
2.2 Fmoc保护基的脱除
将溶胀好的树脂放入反应器中，向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。得到脱去初始连接的Fmoc保护基的树脂。
2.3 Fmoc-Ser(tBu)-Rink amide-MBHA Resin的合成
将Fmoc-Ser(tBu)-OH(15.4mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
2.4肽链的延长
按照肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有主链肽序的树脂。
3、式I与多肽主链的缀合
向连有主链肽序的树脂加入2％水合肼溶液脱除16位Lys的侧链保护基Dde，反应结束后用NMP洗涤干净。将式I(33.8mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次，得到连有SEQ.ID NO:1的肽树脂。
4、树脂上多肽的裂解
将上述连有SEQ.ID NO:1的树脂放入反应瓶中，各加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品92.5mg，收率为92.1％。使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min；检测波长为214nm。将收集的溶液冻干得纯品24.1mg。理论相对分子质量为5014.6。ESI-MS m/z:Calcd.[M+3H] 3+1672.5,[M+4H] 4+1254.7；Found[M+3H] 3+1672.9,[M+4H] 4+1254.1。
实施例37
Figure PCTCN2019085593-appb-000055
的固相合成
合成方法同实施例36，将收集的溶液冻干得纯品26.4mg。理论相对分子质量为5010.6。ESI-MS m/z:Calcd.[M+3H] 3+1671.2,[M+4H] 4+1253.7；Found[M+3H] 3+1671.8,[M+4H] 4+1253.1。
实施例38
Figure PCTCN2019085593-appb-000056
合成方法同实施例36，将收集的溶液冻干得纯品24.2mg。理论相对分子质量为4969.5。ESI-MS m/z:Calcd.[M+3H] 3+1657.5,[M+4H] 4+1243.4；Found[M+3H] 3+1657.3,[M+4H] 4+1243.7。
实施例39
Figure PCTCN2019085593-appb-000057
合成方法同实施例36，将收集的溶液冻干得纯品22.2mg。理论相对分子质量为5024.6。ESI-MS m/z:Calcd.[M+3H] 3+1675.9,[M+4H] 4+1257.2；Found[M+3H] 3+1675.7,[M+4H] 4+1257.4。
实施例40
Figure PCTCN2019085593-appb-000058
合成方法同实施例36，将收集的溶液冻干得纯品22.4mg。理论相对分子质量为4994.7。ESI-MS m/z:Calcd.[M+3H] 3+1665.9,[M+4H] 4+1249.7；Found[M+3H] 3+1665.4,[M+4H] 4+1249.5。
实施例41
Figure PCTCN2019085593-appb-000059
合成方法同实施例36，将收集的溶液冻干得纯品21.8mg。理论相对分子质量为4997.6。ESI-MS m/z:Calcd.[M+3H] 3+1666.9,[M+4H] 4+1250.4；Found[M+3H] 3+1666.8,[M+4H] 4+1249.7。
实施例42 SEQ.ID NO:42的固相合成
1.多肽主链的合成
1.1树脂的溶胀
称取Fmoc-Rink amide-MBHA Resin 50mg(取代度0.4mmol/g)，经DCM 7mL溶胀30min，抽滤去DCM，再用NMP 10mL溶胀30min，最后分别用NMP，DCM 7mL冲洗干净。
1.2 Fmoc保护基的脱除
将溶胀好的树脂放入反应器中，向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。得到脱去初始连接的Fmoc保护基的树脂。
1.3 Fmoc-Ser(tBu)-Rink amide-MBHA Resin的合成
将Fmoc-Ser(tBu)-OH(15.4mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
1.4肽链的延长
按照肽链的序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到连有主链肽序的树脂。
1.5树脂上多肽的裂解
将上述连有主链肽序的树脂放入反应瓶中，加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品82.4mg，收率为96.3％。理论相对分子质量为4280.8。ESI-MS m/z:Calcd.[M+3H] 3+1427.9,[M+4H] 4+1071.2；Found.[M+3H] 3+1430.2,[M+4H] 4+1071.5。
2.多肽链与边链缀合
将多肽链用DMSO溶解配成约10mg/mL的溶液，将N-正十六烷基马来酰亚胺也溶解于DMSO，两者混合后加入DIEPA 20μL，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品29.7mg。理论相对分子质量为4603.3。ESI-MS m/z:Calcd.[M+3H] 3+1535.4,[M+4H] 4+1151.8；Found[M+3H] 3+1535.8,[M+4H] 4+1151.2。
实施例43 SEQ.ID NO:43的固相合成
合成方法同实施例42，将收集的溶液冻干得纯品27.4mg。理论相对分子质量为4606.2。ESI-MS m/z:Calcd.[M+3H] 3+1536.4,[M+4H] 4+1152.6；Found.[M+3H] 3+1536.0,[M+4H] 4+1152.5。
实施例44 SEQ.ID NO:44的固相合成
1.多肽主链的合成
多肽主链合成方法同实施例42中多肽主链的合成方法。
2.化学修饰基的合成
3,3’-(4-硝基苯亚甲基)-二-4-羟基香豆素的合成
称取对硝基苯甲醛(3.02g,0.02mol)，用无水乙醇35ml溶解；加入4-羟基香豆素(6.6g,0.041mol)，补加无水乙醇15ml使溶解完全。80℃反应4h，趁热过滤，滤饼用热乙醇10ml洗3次，得产品8.4g，收率92.1％，mp 227℃。
3,3’-(4-氨基苯亚甲基)-二-4-羟基香豆素的合成
称取3,3’-(4-硝基苯亚甲基)-二-4-羟基香豆素(1.14g,0.0025mol)，用醋酸30ml使之悬浮，加入5％Pd/C 0.3g，搅拌，氢气三通抽气3次，瓶口涂凡士林，常温氢化，反应过夜，抽滤，滤液蒸去部分溶剂，丙酮重结晶，得产品0.7g，收率65.5％，mp 220℃。
3,3’-(4-(12-马来酰亚胺基十二酰胺基)苯亚甲基)-二-4-羟基香豆素的合成
12-马来酰胺十二酸(294.1mg，1mmol)溶于四氢呋喃中，加DIC(17μL，1.1mmol)和HOBt(148.5mg，1.1mmol)，室温搅拌30min，接着将上述溶液缓慢滴入3,3’-(4-氨基苯亚甲基)-二-4-羟基香豆素和DIPEA(17.4μL，0.1mmol)的四氢呋喃溶液，室温搅拌过夜。薄层板检测反应结束后将反应液倒入水中并用乙酸乙酯萃取三次，合并萃取液，分别用K 2CO 3，HCl 1M，饱和食盐水洗三次。萃取液加入无水Na 2SO 4干燥过夜，减压浓缩，得粗品，柱层析纯化得纯品51.4mg，收率73％，mp204-206℃。
1H-NMR(DMSO-d 6,300MHz):δppm:10.17(s,1H,-CONH-),8.31(d,J＝7.8Hz,2H,Ar-H),8.00(t,J＝7.2Hz,2H,Ar-H),7.84(d,J＝8.0Hz,2H,Ar-H),7.76-7.72(m,6H,Ar-H),7.49(s,2H,-COCH＝CHCO-), 6.70(s,1H,-CH-),3.87(t,J＝7.0Hz,2H,-NCH 2-),2.74(t,J＝7.2Hz,2H,-COCH 2-),2.05-1.97(m,4H,-NCH 2CH 2(CH 2) 7CH 2-),1.70(s,14H,-NCH 2CH 2(CH 2) 7CH 2-).ESI-MS m/z:703.1[M+H] +.
3.化学修饰的OXM缀合物的合成与纯化
将上步得到的3,3’-(4-(12-马来酰亚胺基十二酰胺基)苯亚甲基)-二-4-羟基香豆素用DMSO溶解配成约10mg/mL的溶液，将多肽主链也溶解于DMSO，两者超声混合后加入20μl DIEPA，室温下搅拌反应，使用LC-MS监测反应情况。色谱条件为：C18反相柱(1.7μm 2.1×50mm,Waters)；流动相A：0.1％甲酸/水(V/V)，流动相B：0.1％甲酸/乙腈(V/V)，流动相梯度：流动相B 10％～90％，2min，B 90％～90％，3min；流速为0.3ml/min；紫外检测波长为214nm。反应结束后，反应液使用含有1％TFA的乙腈稀释后高速离心并使用0.45μm的微孔滤膜过滤后，使用制备液相色谱进行纯化，色谱条件为：C18反相柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～80％，30min；80％～85％，10min；85％～95％，10min；95％～40％，10min；流速为5ml/min，检测波长为214nm。将收集的溶液减压浓缩去除乙腈，冻干得纯品31.6mg。理论相对分子质量为4986.6。ESI-MS m/z:Calcd.[M+3H] 3+1663.2,[M+4H] 4+1247.7；Found[M+3H] 3+1663.5,[M+4H] 4+1247.2。
实施例45 SEQ.ID NO:45的固相合成
合成方法同实施例44，将收集的溶液冻干得纯品29.5mg。理论相对分子质量为4989.5。ESI-MS m/z:Calcd.[M+3H] 3+1664.2,[M+4H] 4+1248.4；Found[M+3H] 3+1664.5,[M+4H] 4+1248.3。
实施例46 SEQ.ID NO:46的固相合成
1.多肽主链的合成
多肽主链合成方法同实施例42中多肽主链的合成方法。
2.侧链的合成
称取2-CTC树脂100mg(取代度0.8mmol/g)，经DCM溶胀30min，再用NMP 10mL溶胀30min，分别用NMP，DCM 7mL冲洗干净。将Fmoc-AEEA(61.6mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，将DCM：甲醇：DIEA为5：4：1的10mL溶液加入树脂封闭反应1小时，滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。同样方法再次偶联Fmoc-AEEA，反应结束后，用DCM和NMP各7mL洗涤树脂3次。向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。将Fmoc-Glu-OtBu(68.0mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。向树脂中加入含0.1M HOBt的25％哌啶/NMP(V/V)溶液脱除Fmoc，反应结束后用NMP洗涤干净。将十八烷二酸单叔丁酯(59.2mg,0.16mmol)，HBTU(60.6mg,0.16mmol)，DIEA(55.6μL,0.32mmol)，HOBt(21.6mg,0.16mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次。
将上述得到的连有式I的树脂放入反应瓶中，加入裂解剂20％三氟乙醇/DCM 10ml，常温振摇30分钟，反应结束后抽滤，蒸干溶剂得到式I粗品41.6mg。理论相对分子质量为846.1，ESI-MS m/z:845.4[M-H +]。
3.多肽主链与侧链的缀合
向连有主链肽序的树脂加入2％水合肼溶液脱除16位Lys的侧链保护基Dde，反应结束后用NMP洗涤干净。将侧链小分子(33.8mg,0.04mmol)，HBTU(15.1mg,0.04mmol)，HOBt(5.4mg,0.04 mmol)和DIPEA(13.9μL,0.08mmol)溶于NMP 10mL中，将此溶液加入树脂中反应2小时，结束后滤去反应液，用DCM和NMP各7mL洗涤树脂3次，得到连有SEQ.ID NO:5的肽树脂。
4.树脂上多肽的裂解
将上述连有SEQ.ID NO:5的树脂放入反应瓶中，各加入裂解剂Reagent K(TFA/苯甲硫醚/水/苯酚/EDT,82.5:5:5:5:2.5,V/V)10mL，先在0℃下振摇30min，再在常温下反应3h。反应结束后抽滤，加少量TFA和DCM洗涤三次，合并滤液。将滤液加入大量的冰乙醚中析出白色絮状沉淀，冷冻离心得到目标多肽的粗品。最终得到粗品95.0mg，收率为94.6％。使用HPLC监测反应，色谱条件为：C18柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 35％～85％，20min；流速1mL/min；柱温40℃；检测波长214nm。反应结束后，采用制备液相色谱进行纯化，色谱条件为：C18柱(320mm×28mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 40％～90％，20min；流速为6mL/min；检测波长为214nm。将收集的溶液冻干得纯品32.7mg。理论相对分子质量为5022.7。ESI-MS m/z:Calcd.[M+3H] 3+1675.2,[M+4H] 4+1256.7；Found[M+3H] 3+1675.9,[M+4H] 4+1256.1。
实施例47 SEQ.ID NO:47的固相合成
合成方法同实施例46，将收集的溶液冻干得纯品26.1mg。理论相对分子质量为5025.6。ESI-MS m/z:Calcd.[M+3H] 3+1676.2,[M+4H] 4+1257.4；Found[M+3H] 3+1676.5,[M+4H] 4+1257.7。
实施例48 SEQ.ID NO:48的固相合成
合成方法同实施例42，将收集的溶液冻干得纯品30.1mg。理论相对分子质量为4605.3。ESI-MS m/z:Calcd.[M+3H] 3+1536.1,[M+4H] 4+1152.3；Found.[M+3H] 3+1536.5,[M+4H] 4+1152.1。
实施例49 SEQ.ID NO:49的固相合成
合成方法同实施例42，将收集的溶液冻干得纯品29.4mg。理论相对分子质量为4608.2。ESI-MS m/z:Calcd.[M+3H] 3+1537.1,[M+4H] 4+1153.1；Found.[M+3H] 3+1537.6,[M+4H] 4+1153.1。
实施例50 SEQ.ID NO:50的固相合成
合成方法同实施例44，将收集的溶液冻干得纯品31.2mg。理论相对分子质量为4988.5。ESI-MS m/z:Calcd.[M+3H] 3+1663.8,[M+4H] 4+1248.1；Found[M+3H] 3+1663.4,[M+4H] 4+1248.3。
实施例51 SEQ.ID NO:51的固相合成
合成方法同实施例44，将收集的溶液冻干得纯品32.5mg。理论相对分子质量为4991.5。ESI-MS m/z:Calcd.[M+3H] 3+1664.8,[M+4H] 4+1248.9；Found[M+3H] 3+1664.4,[M+4H] 4+1248.6。
实施例52 SEQ.ID NO:52的固相合成
合成方法同实施例46，将收集的溶液冻干得纯品32.3mg。理论相对分子质量为5024.7。ESI-MS m/z:Calcd.[M+3H] 3+1675.9,[M+4H] 4+1257.2；Found[M+3H] 3+1675.4,[M+4H] 4+1257.5。
实施例53 SEQ.ID NO:53的固相合成
合成方法同实施例46，将收集的溶液冻干得纯品31.1mg。理论相对分子质量为5027.6。ESI-MS  m/z:Calcd.[M+3H] 3+1676.9,[M+4H] 4+1257.9；Found[M+3H] 3+1676.7,[M+4H] 4+1257.8。
实施例54 SEQ.ID NO:54的固相合成
合成方法同实施例42，将收集的溶液冻干得纯品29.1mg。理论相对分子质量为4604.3。ESI-MS m/z:Calcd.[M+3H] 3+1535.7,[M+4H] 4+1152.1；Found.[M+3H] 3+1535.6,[M+4H] 4+1152.1。
实施例55 SEQ.ID NO:55的固相合成
合成方法同实施例42，将收集的溶液冻干得纯品24.8mg。理论相对分子质量为4607.2。ESI-MS m/z:Calcd.[M+3H] 3+1536.7,[M+4H] 4+1152.8；Found.[M+3H] 3+1536.5,[M+4H] 4+1152.6。
实施例56 SEQ.ID NO:56的固相合成
合成方法同实施例44，将收集的溶液冻干得纯品31.1mg。理论相对分子质量为4987.6。ESI-MS m/z:Calcd.[M+3H] 3+1663.5,[M+4H] 4+1247.9；Found[M+3H] 3+1663.4,[M+4H] 4+1247.6。
实施例57 SEQ.ID NO:57的固相合成
合成方法同实施例44，将收集的溶液冻干得纯品32.5mg。理论相对分子质量为4990.5。ESI-MS m/z:Calcd.[M+3H] 3+1664.5,[M+4H] 4+1248.6；Found[M+3H] 3+1664.4,[M+4H] 4+1248.3。
实施例58 SEQ.ID NO:58的固相合成
合成方法同实施例46，将收集的溶液冻干得纯品32.7mg。理论相对分子质量为5023.7。ESI-MS m/z:Calcd.[M+3H] 3+1675.6,[M+4H] 4+1256.9；Found[M+3H] 3+1675.4,[M+4H] 4+1256.6。
实施例59 SEQ.ID NO:59的固相合成
合成方法同实施例46，将收集的溶液冻干得纯品31.6mg。理论相对分子质量为5026.6。ESI-MS m/z:Calcd.[M+3H] 3+1676.5,[M+4H] 4+1257.7；Found[M+3H] 3+1676.2,[M+4H] 4+1257.5。
实验例：以下是本发明中涉及的SEQ.ID NO:1～SEQ.ID NO:24的OXM杂合肽的相关药理实验方法以及结果：
1、OXM类似物的GLP-1R和GCGR受体激动活性筛选
HEK293细胞分别共转染编码GLP-1R或GCGR的cDNA。测定化合物的试验中，提前2h将细胞种于96孔板中，化合物用DMSO溶解，使用含有0.1％牛血清蛋白的培养基稀释至不同倍数，加入共转染的细胞中。细胞孵化20min后，使用Cisbo公司的ELISA试剂盒，使用酶标仪测定荧光读数，建立标准曲线将荧光读数转化为相应的cAMP数值，使用Graphpad Prism 5.0软件的非线性回归计算化合物的EC 50数值。
如表1所示，所有化合物较原型胰高血糖素相比，对GLP-1R的激动活性都有明显提高，对GCGR的激动活性略有降低。化合物在缀合脂肪酸后，GLP-1R/GCGR受体激动活性都有不同程度的增强。其中，化合物SEQ.ID NO:6缀合脂肪酸后得到化合物SEQ.ID NO:24，GLP-1R受体激动活性提高了11.7倍，GCGR受体激动活性提高了4.4倍。
表1 OXM类似物对GLP-1R和GCGR的激动活性
Figure PCTCN2019085593-appb-000060
Figure PCTCN2019085593-appb-000061
Results are expressed as mean±SD,*P<0.05,**P<0.01vs OXM,#P<0.05,##P<0.01vs Exenatide.
2、OXM杂合肽的腹腔葡萄糖耐量实验
正常昆明小鼠，随机分组，每组8只，小鼠饲养在标准化动物房中。实验前12小时禁食，只给予饮水。每组小鼠在给药OXM杂合肽之前，测初始血糖值，定为-30min，然后腹腔注射50nmol/kg的OXM杂合肽。30min后，腹腔注射18mmol/kg的葡萄糖溶液，定为0min，对照组注射同体积的生理盐水或50nmol/kg的艾塞那肽。在0，15，30，45，60，120min用血糖仪测定血糖水平，检测OXM杂合肽的降糖活性。
表2 OXM杂合肽的腹腔葡萄糖耐量实验结果
Figure PCTCN2019085593-appb-000062
Figure PCTCN2019085593-appb-000063
Results are expressed as mean±SD,*P<0.05,**P<0.01,***P<0.001vs saline.
如表2所示，降血糖实验结果表明，本发明中涉及的OXM杂合肽给药浓度为50nmol/kg时，降血糖效果与艾塞那肽的降糖效果相当。
3、OXM杂合肽的隔日糖耐量实验
腹腔葡萄糖耐量实验结束后，立即正常饮食饮水10h，然后禁食12h，再次进行小鼠腹腔葡萄糖耐量实验。各组小鼠腹腔注射18mmol/kg的葡萄糖溶液，注射葡萄糖时间定为0min，在0、15、30、45、60和120min用血糖仪测定血糖水平。
如图1、图2、图3和图4所示，隔日糖耐量实验结果表明，本发明涉及的缀合有脂肪酸侧链的OXM杂合肽在体内代谢24h后仍然具有降低血糖作用，而艾塞那肽早已失去活性。说明修饰后得到的OXM杂合肽的降糖时间都显著延长，降血糖作用可维持近30h。
4、OXM杂合肽的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的OXM杂合肽。0h给予化合物，分别在0、0.5、1、2、3、4、6、8、10、12、16、24、36、48和60h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图5和图6可见，艾塞那肽的稳定血糖的时间仅为4.1h，利拉鲁肽的稳定血糖时间为10.7h，本发明中涉及的长效化降糖多肽的稳定血糖时间均在40h以上，部分可超过50h。稳定血糖实验表明，OXM杂合肽具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每两天给药一次的降糖药物的潜力。
5、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表3 OXM杂合肽的减缓体重增加实验
Figure PCTCN2019085593-appb-000064
Figure PCTCN2019085593-appb-000065
Results are expressed as mean±SD.
从表3可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果，控制体重效果明显优于OXM。
6、OXM杂合肽的降低血脂实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测总胆固醇(TC)、甘油三酯(TG)含量。
从图7-图10可以看出，生理盐水组小鼠的各项脂质参数含量均有所增加，而给药组小鼠的脂质参数含量有所降低，表明OXM类似物具有高血脂治疗效果。
7、OXM杂合肽的非酒精性脂肪肝病治疗实验
雄性C57bl/6小鼠，高脂饲料喂养8周，建立非酒精性脂肪肝病模型。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测谷丙转氨酶含量。
从图11-图12可以看出，生理盐水组小鼠的谷丙转氨酶(ALT)含量增加，符合非酒精性脂肪肝病的病理特征，而给药组小鼠的谷丙转氨酶含量降低，表明OXM类似物具有非酒精性脂肪肝病治疗效果。
以下是本发明中涉及的SEQ.ID NO:25～SEQ.ID NO:29的OXM杂合肽的相关药理实验方法以及结果：
表4 OXM杂合肽的腹腔葡萄糖耐量实验结果
Figure PCTCN2019085593-appb-000066
Figure PCTCN2019085593-appb-000067
Results are expressed as mean±SD,*P<0.05,**P<0.01,***P<0.001vs saline.
如表4所示，降血糖实验结果表明，本发明中涉及的OXM杂合肽给药浓度为50nmol/kg时，降血糖效果与艾塞那肽的降糖效果相当。
2、OXM杂合肽的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的OXM杂合肽。0h给予化合物，分别在0、0.5、1、2、3、4、6、8、10、12、16、24、36、48和60h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图13和图14可见，艾塞那肽的稳定血糖的时间仅为4.7h，利拉鲁肽的稳定血糖时间为12.3h，本发明中涉及的长效化降糖多肽的稳定血糖时间可达到40h以上。稳定血糖实验表明，OXM杂合肽具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每两天给药一次的降糖药物的潜力。
3、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共7组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表5 OXM杂合肽的减缓体重增加实验
Figure PCTCN2019085593-appb-000068
Results are expressed as mean±SD.
从表5可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果，控制体重效果明显优于OXM。
4、OXM杂合肽的降低血脂实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共7组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测总胆固醇(TC)、甘油三酯(TG)含量。
从图15-图16可以看出，生理盐水组小鼠的各项脂质参数含量均有所增加，而给药组小鼠的脂质参数含量有所降低，表明OXM类似物具有高血脂治疗效果。
5、OXM杂合肽的非酒精性脂肪肝病治疗实验
雄性C57bl/6小鼠，高脂饲料喂养8周，建立非酒精性脂肪肝病模型。小鼠随机分组，8只为一组，共7组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测谷丙转氨酶含量。
从图17可以看出，生理盐水组小鼠的谷丙转氨酶(ALT)含量增加，符合非酒精性脂肪肝病的病理特征，而给药组小鼠的谷丙转氨酶含量降低，表明OXM类似物具有非酒精性脂肪肝病治疗效果。
以下是本发明中涉及的SEQ.ID NO:30～SEQ.ID NO:35的OXM杂合肽的相关药理实验方法以及结果：
1、OXM类似物的GLP-1R和GCGR受体激动活性筛选
HEK293细胞分别共转染编码GLP-1R或GCGR的cDNA，细胞系表达并利用Western Blot检测已构建的HEK293细胞中GLP-1R或GCGR的蛋白水平，确认是否已建立了稳定高表达细胞株HEK293。测定化合物的试验中，提前2h将细胞种于96孔板中，化合物用DMSO溶解，使用含有0.1％牛血清蛋白的培养基稀释至不同倍数，加入共转染的细胞中。细胞孵化20min后，使用Cisbo公司的ELISA试剂盒，使用酶标仪测定荧光读数，建立标准曲线将荧光读数转化为相应的cAMP数值，使用Graphpad Prism 5.0软件的非线性回归计算化合物的EC 50数值。
表6 OXM类似物对GLP-1R和GCGR的激动活性
Figure PCTCN2019085593-appb-000069
Results are expressed as mean±SD,*P<0.05,**P<0.01vs OXM,#P<0.05,##P<0.01vs Exenatide.
2、OXM杂合肽的腹腔葡萄糖耐量实验
正常昆明小鼠，随机分组，每组8只，小鼠饲养在标准化动物房中。实验前12小时禁食，只给予饮水。每组小鼠在给药OXM杂合肽之前，测初始血糖值，定为-30min，然后腹腔注射50nmol/kg的OXM杂合肽。30min后，腹腔注射18mmol/kg的葡萄糖溶液，定为0min，对照组注射同体积的生理盐水或50nmol/kg的艾塞那肽。在0，15，30，45，60，120min用血糖仪测定血糖水平，检测OXM杂合肽的降糖活性。
如图18所示，降血糖实验结果表明，本发明中涉及的OXM杂合肽给药浓度为50nmol/kg时，降血糖效果与艾塞那肽、利拉鲁肽的降糖效果相当。
3、OXM杂合肽的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的OXM杂合肽。0h给予化合物，分别在 0、0.5、1、2、3、4、6、8、10、12、16、24、36、48和60h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图19和图20可见，艾塞那肽的稳定血糖的时间仅为4.0h，利拉鲁肽的稳定血糖时间为12.1h，本发明中涉及的长效化降糖多肽的稳定血糖时间可达到40h以上。稳定血糖实验表明，OXM杂合肽具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每两天给药一次的降糖药物的潜力。
4、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表7 OXM杂合肽的减重效应
Figure PCTCN2019085593-appb-000070
Results are expressed as mean±SD.
从表7可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果，控制体重效果明显优于OXM。
5、OXM杂合肽的降低血脂实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测总胆固醇(TC)、甘油三酯(TG)含量。
从图21-图22可以看出，生理盐水组小鼠的各项脂质参数含量均有所增加，而给药组小鼠的脂质参数含量有所降低，表明OXM类似物具有高血脂治疗效果。
6、OXM杂合肽的非酒精性脂肪肝病治疗实验
雄性C57bl/6小鼠，高脂饲料喂养8周，建立非酒精性脂肪肝病模型。小鼠随机分组，8只为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测谷丙转氨酶(ALT)含量。
从图23可以看出，生理盐水组小鼠的ALT含量增加，符合非酒精性脂肪肝病的病理特征，而给药组小鼠的谷丙转氨酶含量降低，表明OXM类似物具有非酒精性脂肪肝病治疗效果。
以下是本发明中涉及的SEQ.ID NO:36～SEQ.ID NO:41的OXM杂合肽的相关药理实验方法以及结果：
1、OXM类似物的GLP-1R和GCGR受体激动活性筛选
HEK293细胞分别共转染编码GLP-1R或GCGR的cDNA，细胞系表达并利用Western Blot检测已构建的HEK293细胞中GLP-1R或GCGR的蛋白水平，确认是否已建立了稳定高表达细胞株HEK293。测定化合物的试验中，提前2h将细胞种于96孔板中，化合物用DMSO溶解，使用含有0.1％牛血清蛋白的培养基稀释至不同倍数，加入共转染的细胞中。细胞孵化20min后，使用Cisbo公司的ELISA试剂盒，使用酶标仪测定荧光读数，建立标准曲线将荧光读数转化为相应的cAMP数值，使用Graphpad Prism 5.0软件的非线性回归计算化合物的EC 50数值。
表8 OXM类似物对GLP-1R和GCGR的激动活性
Figure PCTCN2019085593-appb-000071
Results are expressed as mean±SD,*P<0.05,**P<0.01vs OXM,#P<0.05,##P<0.01vs Exenatide.
2、OXM杂合肽的腹腔葡萄糖耐量实验
正常昆明小鼠，随机分组，每组8只，小鼠饲养在标准化动物房中。实验前12小时禁食，只给予饮水。每组小鼠在给药OXM杂合肽之前，测初始血糖值，定为-30min，然后腹腔注射50nmol/kg的OXM杂合肽。30min后，腹腔注射18mmol/kg的葡萄糖溶液，定为0min，对照组注射同体积的生理盐水或50nmol/kg的艾塞那肽。在0，15，30，45，60，120min用血糖仪测定血糖水平，检测OXM杂合肽的降糖活性。
如图24所示，降血糖实验结果表明，本发明中涉及的OXM杂合肽给药浓度为50nmol/kg时，降血糖效果与艾塞那肽、利拉鲁肽的降糖效果相当。
3、OXM杂合肽的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的OXM杂合肽。0h给予化合物，分别在0、0.5、1、2、3、4、6、8、10、12、16、24、36、48、60、72和84h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图25和图26可见，艾塞那肽的稳定血糖的时间仅为4.0h，利拉鲁肽的稳定血糖时间为12.3h，本发明中涉及的长效化降糖多肽的稳定血糖时间可达到60h以上。稳定血糖实验表明，OXM杂合肽具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每两天给药一次的降糖药物的潜力。
4、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只 为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表9 OXM杂合肽的减重效应
Figure PCTCN2019085593-appb-000072
Results are expressed as mean±SD.
从表9可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果，控制体重效果明显优于OXM。
5、OXM杂合肽的降低血脂实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测总胆固醇(TC)、甘油三酯(TG)含量。
从图27-图28可以看出，生理盐水组小鼠的各项脂质参数含量均有所增加，而给药组小鼠的脂质参数含量有所降低，表明OXM类似物具有高血脂治疗效果。
6、OXM杂合肽的非酒精性脂肪肝病治疗实验
雄性C57bl/6小鼠，高脂饲料喂养8周，建立非酒精性脂肪肝病模型。小鼠随机分组，8只为一组，共8组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测谷丙转氨酶(ALT)含量。
从图29可以看出，生理盐水组小鼠的ALT含量增加，符合非酒精性脂肪肝病的病理特征，而给药组小鼠的谷丙转氨酶含量降低，表明OXM类似物具有非酒精性脂肪肝病治疗效果。
以下是本发明中涉及的SEQ.ID NO:42～SEQ.ID NO:65的OXM杂合肽的相关药理实验方法以及结果：
1、OXM杂合肽对DPP-4酶稳定性实验
将OXM杂合肽5nmol和DPP-4酶5mU在浓度为50mM的Tris-HCL缓冲溶液200μL中，pH7.4条件下，37℃温孵8h。反应结束后，加入20％的乙腈/水溶液10μL终止反应。分别取0h，8h的温孵溶液处理后进行HPLC分析。分析采用C18反相柱(150mm×4.6mm，5μm)；流动相A：0.1％TFA/水(V/V)，流动相B：0.1％TFA/乙腈(V/V)；流动相梯度：流动相B 10％～45％，22min；流速为1mL/min；柱温为40℃；检测波长为214nm。OXM类似物对DPP-4的稳定情况通过8h温孵溶液的HPLC色谱峰面积与0h温孵溶液的HPLC色谱峰面积比值(％intact peptide after 8h)表示。
表10 OXM类似物对GLP-1R和GCGR的激动活性
Figure PCTCN2019085593-appb-000073
从表10可以得出，经过8h的酶降解稳定性实验，OXM杂合肽对于DPP-4酶的稳定性远好于OXM原型和GLP-1原型。
2、OXM杂合肽的腹腔葡萄糖耐量实验
正常昆明小鼠，随机分组，每组6只，小鼠饲养在标准化动物房中。实验前12小时禁食，只给予饮水。每组小鼠在给药OXM杂合肽之前，测初始血糖值，定为-30min，然后腹腔注射50nmol/kg的OXM杂合肽。30min后，腹腔注射18mmol/kg的葡萄糖溶液，定为0min，对照组注射同体积的生理盐水或50nmol/kg的艾塞那肽。在0，15，30，45，60，120min用血糖仪测定血糖水平，检测OXM杂合肽的降糖活性。
如图30-33所示，降血糖实验结果表明，本发明中涉及的OXM杂合肽给药浓度为50nmol/kg时，降血糖效果与艾塞那肽、利拉鲁肽的降糖效果相当。
3、OXM杂合肽的稳定血糖实验
测定STZ诱导的糖尿病模型小鼠的血糖，选择数值高于20mmol/L的小鼠进行随机分组，每组六只，实验期间小鼠自由采食。阳性对照组腹腔注射艾塞那肽或利拉鲁肽，剂量为50nmol/kg，阴性对照组腹腔注射生理盐水，给药组分别注射50nmol/kg的OXM杂合肽。0h给予化合物，分别在0、0.5、1、2、3、4、6、8、10、12、16、24、36、48、60、72和84h使用血糖仪测定血糖水平。评价指标为腹腔注射化合物后，小鼠血糖数值低于8.35mmol/L的时间。
由图34-37可见，艾塞那肽的稳定血糖的时间仅为4.0h，利拉鲁肽的稳定血糖时间为12.3h，本发明中涉及的部分长效化降糖多肽的稳定血糖时间可达到60h以上。稳定血糖实验表明，OXM杂合肽具有良好的长效化降糖效果，可以达到更优的长效化降糖效果，具有开发成为每三天给药一次的降糖药物的潜力。
4、OXM杂合肽的减缓体重增加实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。测试第1天和第56天各组小鼠的空腹体重，考察各组小鼠的平均体重变化。
表11 OXM杂合肽的减重效应
Figure PCTCN2019085593-appb-000074
Figure PCTCN2019085593-appb-000075
Results are expressed as mean±SD.
从表11可以得出，长期给药后，所有的化合物都表现出了较好的体重控制效果，控制体重效果明显优于OXM。
5、OXM杂合肽的降低血脂实验
雄性C57bl/6小鼠，高脂饲料喂养4周，选体重大于30g的小鼠进行实验。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测总胆固醇(TC)、甘油三酯(TG)含量。
从图38和图39可以看出，生理盐水组小鼠的各项脂质参数含量均有所增加，而给药组小鼠的脂质参数含量有所降低，表明OXM类似物具有高血脂治疗效果。
6、OXM杂合肽的非酒精性脂肪肝病治疗实验
雄性C57bl/6小鼠，高脂饲料喂养8周，建立非酒精性脂肪肝病模型。小鼠随机分组，8只为一组，共26组，连续56天每日给药OXM杂合肽(50nmol/kg,10mL/kg)，阴性对照组每日给药生理盐水，阳性对照组给药OXM。给药结束后，取小鼠血清，检测谷丙转氨酶(ALT)含量。
从图40可以看出，生理盐水组小鼠的ALT含量增加，符合非酒精性脂肪肝病的病理特征，而给药组小鼠的谷丙转氨酶含量降低，表明OXM类似物具有非酒精性脂肪肝病治疗效果。

Claims (11)

  1. 一种多肽，其特征是，多肽氨基酸序列为：
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Met-Asp-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Glu-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Glu-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Val-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Ala-Gln-Leu-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；
    其中
    Xaa1为Gly，Aib，D-Ser，Ser，Arg，Asn，Asp，Cys，Gln，Glu，His，Ile，Leu，Lys，Met，Phe，Pro，Thr，Trp，Tyr或Val；
    Xaa2取自
    Figure PCTCN2019085593-appb-100001
    Figure PCTCN2019085593-appb-100002
    其中，X为-CH 3或-COOH；
    Y为-NH-CO-或-CO-NH-；
    n取自自然数0-20；
    m取自自然数1-20；
    Xaa3为Ser-OH，Ser-NH 2。
  2. 根据权利要求1所述的多肽，其特征是，
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Met-Asp-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Glu-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Glu-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Val-Gln-Asp-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Ala-Gln-Leu-Phe-Val-Gln-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；或
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa2-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa3；
    其中
    Xaa1取自Gly或Aib；
    Xaa2取自
    Figure PCTCN2019085593-appb-100003
    其中，X为-CH 3或-COOH；
    Y为-NH-CO-或-CO-NH-；
    n取自自然数6，10，14，11，15；
    m取自自然数10或11；
    Xaa3取自Ser-OH或Ser-NH 2。
  3. 根据权利要求1至2项中任意一项所述的化合物，选自：
    Figure PCTCN2019085593-appb-100004
    Figure PCTCN2019085593-appb-100005
    Figure PCTCN2019085593-appb-100006
    Figure PCTCN2019085593-appb-100007
    Figure PCTCN2019085593-appb-100008
    Figure PCTCN2019085593-appb-100009
    Figure PCTCN2019085593-appb-100010
    Figure PCTCN2019085593-appb-100011
    Figure PCTCN2019085593-appb-100012
    Figure PCTCN2019085593-appb-100013
    Figure PCTCN2019085593-appb-100014
    Figure PCTCN2019085593-appb-100015
  4. 根据权利要求1所述的化合物的制备方法，其特征在于，包括以下两种合成策略：
    策略1：肽链中Cys与马来酰亚胺连接的化合物合成方式；
    步骤1：取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
    步骤2：取所述第一肽树脂，经裂解、纯化，得到纯肽链；
    步骤3：将纯肽链中Cys的巯基与连有马来酰亚胺连接臂的脂肪酸链或香豆素小分子缀合，得到权利要求1所述化合物；
    策略2：肽链中Lys与小分子脂肪酸链连接的合成方式；
    步骤1：取树脂，活化后，逐步偶联氨基酸，得到第一肽树脂；
    步骤2：取所述第一肽树脂，在Lys侧链偶联具有式I或式II的脂肪酸链小分子，得到第二肽树脂；
    步骤3：取所述第二肽树脂，经裂解、纯化，即得权利要求1中所述化合物；
    Figure PCTCN2019085593-appb-100016
    Figure PCTCN2019085593-appb-100017
    其中，所述R 1选自tBu、Dmab、Bzl；
    所述R 2选自甲基、乙基、叔丁基和二苯甲基。
    所述偶联式I或式II的脂肪酸链小分子的Lys侧链保护基选自Fmoc、Boc、Dde、ivDde。
  5. 一种药学上可接受的盐，其特征在于：所述盐为化合物与盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸形成的盐。
  6. 根据权利要求1至3项中任意一项的化合物所制备的药剂，所说的药剂是任何一种药剂学上所说的片剂、胶囊、酏剂、糖浆、锭剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、块剂、乳剂、栓剂、复方制剂。
  7. 一种药物组合物，包括治疗有效量的权利要求1中任一所述的化合物，和其药学上可接受的载体或稀释剂。
  8. 根据权利要求1至3项中任意一项的化合物，在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
  9. 根据权利要求1至3项中任意一项的化合物所制备的一种药学上可接受的盐，在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
  10. 根据权利要求1至3项中任意一项的化合物所制备的药剂，在制备治疗和/或预防糖尿病、肥胖症、高血脂症、非酒精性脂肪肝的药物中的应用。
  11. 根据权利要求1至3项中任意一项的化合物的制备方法，包括生物表达、液相合成和固相合成制备方法。
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