CN110191892A - Alanine-lysine-glutamic acid tripeptide derivative and its application - Google Patents
Alanine-lysine-glutamic acid tripeptide derivative and its application Download PDFInfo
- Publication number
- CN110191892A CN110191892A CN201880005352.2A CN201880005352A CN110191892A CN 110191892 A CN110191892 A CN 110191892A CN 201880005352 A CN201880005352 A CN 201880005352A CN 110191892 A CN110191892 A CN 110191892A
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- China
- Prior art keywords
- fmoc
- alanine
- lysine
- glu
- resin
- Prior art date
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- Granted
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- 239000004220 glutamic acid Substances 0.000 title claims description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 30
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 20
- 229920001184 polypeptide Polymers 0.000 claims abstract description 17
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 5
- 108010019598 Liraglutide Proteins 0.000 claims description 36
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 claims description 33
- 229960002701 liraglutide Drugs 0.000 claims description 33
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 15
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 239000001257 hydrogen Substances 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 238000010647 peptide synthesis reaction Methods 0.000 claims description 7
- 239000007790 solid phase Substances 0.000 claims description 7
- 206010012601 diabetes mellitus Diseases 0.000 claims description 6
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 125000006239 protecting group Chemical group 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 2
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 125000004185 ester group Chemical group 0.000 claims 3
- 125000001931 aliphatic group Chemical group 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 27
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- 229920005989 resin Polymers 0.000 description 68
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 64
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 36
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- 238000003756 stirring Methods 0.000 description 30
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- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 24
- 238000003746 solid phase reaction Methods 0.000 description 24
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- 230000008878 coupling Effects 0.000 description 21
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- 239000000706 filtrate Substances 0.000 description 19
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- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 18
- 229910052757 nitrogen Inorganic materials 0.000 description 18
- DBTMQODRSDEGRZ-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-(2-oxoethyl)carbamate Chemical compound C1=CC=C2C(COC(=O)NCC=O)C3=CC=CC=C3C2=C1 DBTMQODRSDEGRZ-UHFFFAOYSA-N 0.000 description 16
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 16
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 16
- REITVGIIZHFVGU-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[(2-methylpropan-2-yl)oxy]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](COC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 REITVGIIZHFVGU-IBGZPJMESA-N 0.000 description 12
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 12
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 12
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- 238000006467 substitution reaction Methods 0.000 description 12
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 11
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- ARBOVOVUTSQWSS-UHFFFAOYSA-N hexadecanoyl chloride Chemical compound CCCCCCCCCCCCCCCC(Cl)=O ARBOVOVUTSQWSS-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 125000002960 margaryl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 125000001196 nonadecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- BSCHIACBONPEOB-UHFFFAOYSA-N oxolane;hydrate Chemical compound O.C1CCOC1 BSCHIACBONPEOB-UHFFFAOYSA-N 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 description 1
- 125000002958 pentadecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000002889 tridecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- 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
- C07K14/605—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Emergency Medicine (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to compound of formula I and application of the compound in synthesis in solid state, compound of formula I is the polypeptide drugs intermediate for including peptide chain-Ala-Lys (X)-(X is the Glu- fatty alkyl side chain connected on Lys side chain) construction unit for synthesis in solid state.
Description
Technical Field
The invention relates to a tripeptide derivative, in particular to an alanine-lysine-glutamic acid tripeptide derivative, and also relates to an application thereof in polypeptide synthesis.
Background
At present, the polypeptide compound containing Lys (X) (X is Glu-fatty alkyl side chain) is mainly prepared by a gene recombination technology and a stepwise coupled solid-phase synthesis method. When the gene recombination technology is adopted to synthesize the polypeptide compound, the technical difficulty is high, the relative cost is high, and the main chain intermediate is purified by HPLC and then reacts with Glu-fatty alkyl side chain under the liquid phase condition, so that a plurality of impurities are generated and are difficult to purify. Therefore, the method of preparing lys (X) -containing polypeptide compounds (X is Glu-fatty alkyl side chain) which is currently widely used is a stepwise coupled solid-phase synthesis method. However, for the polypeptide containing side chains, the main chain is synthesized first, and then the side chains are coupled step by step, so that incomplete coupling exists, defective peptides which are difficult to remove are introduced, and the problem of incomplete coupling cannot be solved even if the feeding is increased and the reaction time is prolonged.
Incomplete coupling, more impurities and difficult purification, and more measures are necessarily taken to obtain the high-purity polypeptide medicament. This not only increases the production cost, but also brings more hidden troubles in the aspects of drug stability and safety. In the synthesis research of a polypeptide compound containing Lys (X) (X is Glu-fatty alkyl side chain), an intermediate substance capable of improving reaction efficiency and reducing impurity generation is searched, and the intermediate substance is very beneficial to improving the stability and safety of a medicament and reducing production cost.
Disclosure of Invention
The object of the present invention is to provide a novel peptide intermediate carrying-Ala-Lys (X) - (X is Glu-fatty alkyl side chain attached to Lys side chain), and which can be easily inserted into SPPS.
The invention solves the technical problem and relates to the following compounds:
alanine-lysine-glutamic acid tripeptide derivative with a structure shown in a general formula I
Wherein,
R1and R2Is a hydrogen or an ester protecting group,
R1and R2Which may be the same or different from each other,
R3is hydrogen or an amino-protecting group,
R4is C10-20An alkyl group, a carboxyl group,
or enantiomers, diastereomers and salts thereof.
In some embodiments, R1And R2Is hydrogen or an ester protecting group, the ester protecting group is tert-butyl, methyl, ethyl, benzyl or allyl, R1And R2May be the same or different.
Said R1And R2In a particular embodiment, R1Especially tert-butyl, R2Especially hydrogen.
In some embodiments, R3Is hydrogen or an amino protecting group, and the amino protecting group is Fmoc, Dde, Alloc, Boc, Trt and Dmb.
Said R3In particular embodiments, Fmoc, Dde, Alloc groups are specifically mentioned.
In some embodiments, R4Is C10-20Alkyl radical, said C10-20Alkyl is straight-chain or branched saturated aliphatic alkyl of 10 to 20 carbon atoms, in particular straight-chain saturated aliphatic alkyl, and is selected from the group consisting of decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl.
In some embodiments, R4Is C14-16An alkyl group.
Said R4In particular embodiments, especially C15An alkyl group.
In some embodiments, the salt is a salt commonly used by those skilled in the art, including hydrochloride, acetate, formate, trifluoroacetate.
The alanine-lysine-glutamic acid tripeptide derivative is applied to solid phase peptide synthesis of peptides containing Glu-fatty alkyl side chain construction units connected with Lys-parts of peptide chains.
The use of (a), wherein the solid phase peptide synthesis is Fmoc solid phase peptide synthesis.
The preparation method of the alanine-lysine-glutamic acid tripeptide derivative comprises the following steps:
a) reacting a compound of formula II-R4-Glu-R1To obtain intermediate 1
Wherein R is1、R4The tripeptide derivative of alanine-lysine-glutamic acid has the structural formula shown in the specification, wherein R is1、R4The definitions are the same.
b) Reacting intermediate 1 with a compound R of formula III3-Ala-Lys-R2Coupling to obtain the compound shown in the formula I.
Wherein R is1、R2The tripeptide derivative of alanine-lysine-glutamic acid has the structural formula shown in the specification, wherein R is1、R2The definitions are the same.
The compounds of formula II and III may be obtained commercially or synthetically.
R in the intermediate 15OSU esters, OPfp esters and OBt esters are preferably OSU esters.
Dissolving the compound shown in the formula II in an organic solvent, and reacting under a condensing agent to obtain an intermediate 1.
The organic solvent is any one or a mixture of more of acetone, dichloromethane, methanol, dioxane, N dimethylformamide, tetrahydrofuran, ethyl acetate and acetonitrile, and particularly DCM.
The condensing agent is one of HOSU/DCC, HOSU/DIC, HOBt/DIC, HOPfp/DCC, HOPfp/DIC, TBTU/DIEA, TBTU/HOBt/DIEA and HBTU/HOBt/DIEA, and particularly the condensing agent is HOSU/DCC.
Dissolving the compound shown in the formula III in a solvent, and reacting with the intermediate 1 under the action of alkali to obtain the compound shown in the formula I.
The solvent is any one or more of acetone, dichloromethane, methanol, dioxane, N dimethylformamide, tetrahydrofuran, ethyl acetate, water and acetonitrile, and especially 20% tetrahydrofuran water solution.
The alkali is one or more of sodium bicarbonate, sodium carbonate, potassium bicarbonate, triethylamine, pyridine and DIEA, and especially the alkali is sodium bicarbonate.
The application of the alanine-lysine-glutamic acid tripeptide derivative in preparing antidiabetic polypeptide medicaments.
The alanine-lysine-glutamic acid tripeptide derivative is applied to the preparation of the anti-diabetes polypeptide medicament, and the diabetes polypeptide medicament is a GLP-1 analogue.
The GLP-1 analogue is liraglutide.
Compared with the prior art, the invention has the following beneficial effects:
when a polypeptide compound containing Lys (X) (such as a GLP-1 analogue polypeptide drug capable of resisting diabetes, liraglutide) is synthesized, the compound of the formula I provided by the invention can be inserted more easily in solid-phase peptide synthesis, so that the reaction rate of amino acid at the amino terminal of peptide resin and the compound I is improved, the coupling efficiency is effectively improved, the generation of impurities is reduced, the yield and purity of a crude liraglutide product are greatly improved, the purification of the crude liraglutide product to obtain a refined peptide is facilitated, and the production time is shortened.
Detailed Description
The synthesis of the compounds of formula I and the use of the compounds of formula I in the synthesis of liraglutide comprising Lys (X) polypeptide compounds of the present invention are described in further detail below with reference to specific examples to facilitate a further understanding of the present invention by those skilled in the art. The examples should not be construed as limiting the scope of protection.
The Chinese names corresponding to the English abbreviations related to the invention are shown in Table 1:
TABLE 1 specific meanings of English abbreviations used in the specification and claims
Example 1
Fmoc-Ala-Lys(Pal-Glu-OtBu)-OH
A mixture of Pal-Glu-OtBu (4.41g, 10.00mmol) DCM (100ml) and HOSU (1.27g, 11.00mmol) was placed in a 200ml three-necked flask, stirred at 0-5 ℃ for 10min, DCC (2.23g, 11.00mmol) was slowly added, stirred at room temperature for 3.0h, the reaction was filtered, the filtrate was concentrated to condensate droplets and dropped, transferred to a vacuum pump and dried to obtain 3.97g (7.38mmol) of Pal-Glu-OtBu-OSU as a white powder for later use.
Fmoc-Ala-Lys-OH (3.24g, 7.38mmol) and sodium carbonate (1.56g, 14.76mmol) were dissolved in 100ml of 50% aqueous tetrahydrofuran solution to obtain a mixed Fmoc-Ala-Lys-OH solution. Pal-Glu-OtBu-OSU (3.97g, 7.38mmol) was dissolved in 20ml of tetrahydrofuran, slowly added dropwise to the above Fmoc-Ala-Lys-OH mixed solution, stirred for reaction for 5.0h, and the reaction solution was concentrated and rotary evaporated to remove tetrahydrofuran. DCM was added to the concentrated solution and stirred for 5min, the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH5.26g. The characteristic peak relative time and peak area results are shown in table 2.
TABLE 2 example 1 characteristic peak relative retention time and peak area results for Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH
| Serial number | Relative retention time (min) | Peak area (AU. s) | Peak area ratio% |
| 1 | 6.384 | 599872 | 1.48 |
| 2 | 21.179 | 18341 | 0.05 |
| 3 | 21.683 | 55529 | 0.14 |
| 4 | 22.748 | 84909 | 0.21 |
| 5 | 23.148 | 31065 | 0.08 |
| 6 | 23.808 | 38468 | 0.09 |
| 7 | 24.262 | 107219 | 0.26 |
| 8 | 24.667 | 46422 | 0.11 |
| 9 | 25.293 | 538917 | 1.33 |
| 10 | 26.271 | 38897415 | 95.96 |
| 11 | 28.437 | 22255 | 0.05 |
| 12 | 31.302 | 94749 | 0.23 |
Example 2
Boc-Ala-Lys(Pal-Glu-OtBu)-OH
A mixture of ethyl acetate (100ml) and HOSU (1.27g, 11.00mmol) of Pal-Glu-OtBu (4.41g, 10.00mmol) was placed in a 250ml flask, stirred at 0-5 ℃ for 10min, DCC (2.27g, 11.00mmol) was slowly added, stirred at room temperature for 3.0h, the reaction was filtered, and the filtrate was concentrated to give Pal-Glu-OSU4.26g.
Boc-Ala-Lys-OH (2.28g, 7.2mmol) and sodium carbonate (2.30g, 21.60mmol) were dissolved in 100ml of 50% tetrahydrofuran aqueous solution to obtain a Boc-Ala-Lys-OH mixed solution. Pal-Glu-OtBu-OSU (4.26g, 7.92mmol) is dissolved in 20ml tetrahydrofuran and slowly dripped into the Boc-Ala-Lys-OH mixed solution, the mixture is stirred and reacted for 2.0h, 1.0mmol/L sodium hydroxide aqueous solution is added into the reaction solution to adjust the pH value to 8.0-11.0, a product is precipitated, the filter cake is collected, the filter cake is washed for 3-5 times by pure water, and 4.42g of Boc-Ala-Lys (Pal-Glu-OtBu) -OH is obtained by vacuum drying.
Example 3
Dde-Ala-Lys(Pal-Glu-OtBu)-OH
A mixture of Pal-Glu-OtBu (5.84g, 13.25mmol) DCM (100ml) and HOSU (1.68g, 14.58mmol) was placed in a 200ml three-necked flask, stirred at 0-5 ℃ for 10min, DCC (3.00g, 14.58mmol) was slowly added, stirred at room temperature for 1.0h, the reaction solution was filtered, and the filtrate was concentrated to obtain 5.75g of Pal-Glu-OtBu-OSU.
Dde-Ala-Lys-OH (3.86g, 9.72mmol) and DIEA (3.76g, 29.16mmol) were dissolved in 100ml of 50% tetrahydrofuran aqueous solution to obtain a Dde-Ala-Lys-OH mixed solution. Pal-Glu-OtBu-OSU (5.75g, 10.69mmol) was dissolved in 20ml of tetrahydrofuran, slowly added dropwise to the Dde-Ala-Lys-OH mixed solution, stirred for reaction for 5.0h, and the reaction solution was concentrated and rotary evaporated to remove tetrahydrofuran. DCM was added to the concentrated solution and stirred for 5min, and the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give Dde-Ala-Lys (Pal-Glu-OtBu) -OH 5.40 g.
Example 4
Fmoc-Ala-Lys(Pal-Glu-OMe)-OH
A mixture of Pal-Glu-OMe (4.87g, 11.34mmol) DCM (100ml) and pentafluorophenol (2.30g, 12.47mmol) was placed in a 250ml three-neck flask, stirred at 0-5 ℃ for 10min, DCC (2.57g, 12.47mmol) was slowly added, stirred at room temperature for 3.0h, the reaction was filtered, the filtrate was concentrated to a condensate droplet, and transferred to a vacuum pump to dry to obtain 5.54g of Pal-Glu-OtBu-OPfp.
Fmoc-Ala-Lys-OH (3.51g, 8.00mmol) sodium carbonate (2.54g, 24.00mmol) was dissolved in 100ml of 20% acetonitrile in water to obtain a Fmoc-Ala-Lys-OH mixed solution. Pal-Glu-OtBu-OPfp (5.54g, 8.86mmol) is dissolved in 20ml tetrahydrofuran, slowly added dropwise to the Fmoc-Ala-Lys-OH mixed solution, stirred for reaction for 5.0h, 1.0mmol/L sodium hydroxide aqueous solution is added dropwise to the reaction solution to adjust the pH value to 8.0-11.5, a product is precipitated, filtered, a filter cake is collected, the filter cake is washed for 3-5 times by pure water, and vacuum drying is carried out to obtain 6.07g of Fmoc-Ala-Lys (Pal-Glu-OMe) -OH.
Example 5
Fmoc-Ala-Lys(Pal-Glu-OAll)-OH
A mixture of Pal-Glu-OAll (4.95g, 11.43mmol) DCM (100ml) and HOSU (1.45g, 12.57mmol) was placed in a 200ml three-necked flask, stirred at 0-5 ℃ for 10min, DCC (2.59g, 12.57mmol) was slowly added, stirred at room temperature for 1.0h, the reaction was filtered, and the filtrate was concentrated to give 5.18g of Pal-Glu-OtBu-OSU.
Fmoc-Ala-Lys-OH (3.92g, 8.92mmol) was dissolved in 100ml of 20% acetonitrile water to obtain a Fmoc-Ala-Lys-OH mixed solution. Pal-Glu-OAll-OSU (5.29g, 9.82mmol) was dissolved in 20ml of tetrahydrofuran, and slowly added dropwise to the above Fmoc-Ala-Lys-OH mixed solution, followed by stirring and reaction for 3.0 hours, and the reaction solution was concentrated and then subjected to rotary evaporation to remove tetrahydrofuran. DCM was added to the concentrated solution and stirred for 5min, the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give Fmoc-Ala-Lys (Pal-Glu-OAll) -OH 6.18 g.
Example 6
Fmoc-Ala-Lys(Pal-Glu-OtBu)-OH
A mixture of Pal-Glu-OtBu (5.84g, 13.25mmol) DCM (100ml) and HOSU (1.68g, 14.58mmol) was placed in a 200ml three-necked flask, stirred at 0-5 ℃ for 10min, DIC (1.84g, 14.58mmol) was slowly added, stirred at room temperature for 1.0h, the reaction was filtered, and the filtrate was concentrated to give 5.96g of Pal-Glu-OtBu-OSU.
Fmoc-Ala-Lys-OH (4.38g, 10mmol) and sodium carbonate (3.18g, 30.00mmol) were dissolved in 100ml of 50% aqueous tetrahydrofuran solution to obtain a mixed Fmoc-Ala-Lys-OH solution. Pal-Glu-OtBu-OSU (5.96g, 11.07mmol) was dissolved in 20ml of tetrahydrofuran, slowly added dropwise to the above Fmoc-Ala-Lys-OH mixed solution, stirred for reaction for 5.0h, and the reaction solution was concentrated and rotary evaporated to remove tetrahydrofuran. DCM was added to the concentrated solution and stirred for 5min, the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give 7.00g of Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH.
EXAMPLE 7 use of Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH, a Compound of formula I, in the Synthesis of liraglutide
10.0g of Wang resin (0.40mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swollen with DMF for 30 minutes. Dissolving Fmoc-Gly-OH 4.46g, HOBt 2.03g and DMAP 0.73g in DMF 100mL, adding DIC 1.94g under ice bath condition, activating for 5 minutes, adding the activated solution into the solid phase reaction column, stirring with nitrogen for reaction for 4.0 hours, blocking the Fmoc-Gly-Wang resin with acetic anhydride/DIEA for overnight, washing the resin with DMF and DCM, shrinking and draining methanol to obtain Fmoc-Gly-Wang resin, wherein the detection substitution degree is 0.17 mmol/g; adding 8.82g of Fmoc-Gly-Wang resin with the substitution value of 0.17mmol/g into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-Wang resin by DMF for 30min, then using 20% DBLK to carry out deprotection, washing the resin 3 times by DMF, washing the resin 3 times by DCM, detecting the color of the resin by ninhydrin, wherein the color of the resin indicates that the Fmoc is completely removed, dissolving Fmoc-Arg (pbf) -OH 4.87g, HBTU 2.84g and HOBt 1.01g in 50mL of DMF, adding DIEA 1.94g for activation for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring and reacting for 2h by nitrogen, detecting that the reaction is negative by ninhydrin, repeating the deprotection and amino acid coupling steps for finishing the reaction, and finishing Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, OH (Pbf) and amino acid coupling steps according to the sequence of liraglutide, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-ThrtBu- (OH, Fmoc-Phe-OH, Fmoc-Il-OH, Fmoc-Glu-Leu-OH, Fmoc, Coupling of Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.
Taking 16.11g of the obtained liraglutide resin, adding TFA: and Tis: water 95: 2.5: 2.5 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for 5 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 5.93g of crude liraglutide was obtained in 105.39% yield and 80.86% purity, and the results of relative retention time of characteristic peaks and peak area are shown in Table 3.
Table 3 characteristic peak relative retention time and peak area results for crude liraglutide peptide of example 7
EXAMPLE 8 use of Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH, a Compound of formula I, in the Synthesis of liraglutide
8.80g of CTC resin (0.68mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swelled with DMF for 30 min. Dissolving 3.57g of Fmoc-Gly-OH in 50mL of DMF, adding 1.16g of DIEA under an ice bath condition to activate for 5 minutes, adding the activated solution into the solid-phase reaction column, stirring and reacting for 4.0 hours by using nitrogen, sealing and standing the Fmoc-Gly-CTC resin overnight by using methanol/DIEA, washing the resin by using DMF and DCM, shrinking and draining the methanol to obtain the Fmoc-Gly-CTC resin, wherein the detection substitution degree is 0.37 mmol/g; taking 4.05g of Fmoc-Gly-CTC resin with the substitution value of 0.37mmol/g, adding the Fmoc-Gly-CTC resin into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-CTC resin by DMF for 30min, then deprotecting by 20% DBLK, washing the resin 3 times by DMF, washing the resin 3 times by DCM, detecting the color of the resin by ninhydrin, indicating that the Fmoc is completely removed, dissolving 4.89g of Fmoc-Arg (pbf) -OH and 1.52g of HOBt into 30mL of DMF, adding DIC 1.89g of DIC for activation for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring the solution by nitrogen, detecting by ninhydrin, repeating the deprotecting and amino acid coupling steps, and sequentially completing Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-Lys (Pal-Glu-OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Coupling of Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.
Taking 9.97g of the obtained liraglutide resin, adding TFA: and Tis: water 95: 2.5: 2.5 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for 5 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 4.95g of crude liraglutide was obtained in 87.97% yield and 79.97% purity.
EXAMPLE 9 use of Dde-Ala-Lys (Pal-Glu-OtBu) -OH, a Compound of formula I, in the Synthesis of liraglutide
10.0g of Wang resin (0.40mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swollen with DMF for 30 minutes. Dissolving 4.43g of Fmoc-Gly-OH, 2.06g of HOBt and 0.72g of DMAP in 100mL of DMF, adding 1.94g of DIC under an ice bath condition to activate for 5 minutes, adding the activated solution into the solid phase reaction column, stirring nitrogen for reaction for 8.0 hours, blocking the Fmoc-Gly-Wang resin with acetic anhydride/DIEA for overnight, washing the resin with DMF and DCM, shrinking methanol and draining to obtain the Fmoc-Gly-Wang resin, wherein the detection substitution degree is 0.20 mmol/g; adding 7.50g of Fmoc-Gly-Wang resin with the substitution value of 0.20mmol/g into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-Wang resin by DMF for 30min, then using 20% DBLK to carry out deprotection, washing the resin 3 times by DMF, washing the resin 3 times by DCM, detecting the color of the resin by ninhydrin, wherein the color of the resin indicates that Fmoc is completely removed, dissolving Fmoc-Arg (pbf) -OH 4.85g, HBTU 2.83g and HOBt 1.00g in 50mL of DMF, adding DIEA 1.89g for activation for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring and reacting for 2h by nitrogen, finishing the reaction by ninhydrin detection of negative, repeating the deprotection and amino acid coupling steps, and finishing Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Pbf-OH, and amino acid coupling steps according to the sequence of liraglutide, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Dde-Ala-Lys (Pal-Glu-OtBu) -OH.
Coupling Dde-Ala-Lys (Pal-Glu-OtBu) -OH, washing the resin 4 times with DMF, removing Dde group with 5% hydrazine hydrate/DMF solution, finishing Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc, Coupling of Fmoc-Ala-OH, Boc-His (Trt) -OH.
Taking 16.50g of the obtained liraglutide resin, adding TFA: and Tis: mpr 95: 3.0: 2.0 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for 5 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 5.17g of crude liraglutide was obtained in 91.88% yield and 80.02% purity.
EXAMPLE 10 use of Fmoc-Ala-Lys (Pal-Glu-OAll) -OH, a compound of formula I, in the synthesis of liraglutide
10.0g of Wang resin (0.40mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swollen with DMF for 30 minutes. Dissolving 4.48g of Fmoc-Gly-OH, 2.05g of HOBt and 0.71g of DMAP in 100mL of DMF, adding 1.97g of DIC under an ice bath condition to activate for 5 minutes, adding the activated solution into the solid phase reaction column, stirring nitrogen for reaction for 6.0 hours, blocking the Fmoc-Gly-Wang resin with acetic anhydride/DIEA for overnight, washing the resin with DMF and DCM, shrinking methanol and draining to obtain Fmoc-Gly-Wang resin, wherein the detection substitution degree is 0.19 mmol/g; adding 7.89g of Fmoc-Gly-Wang resin with the substitution value of 0.19mmol/g into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-Wang resin by DMF for 30min, then deprotecting by 20% DBLK, washing the resin 6 times by DMF, detecting the color of the resin by ninhydrin, wherein the color of the resin indicates complete Fmoc removal, dissolving Fmoc-Arg (pbf) -OH 4.88g, TBTU 2.41g and HOBt 1.01g in 50mL DMF, adding DIEA 1.94g for activation for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring and reacting for 2h by nitrogen, finishing the reaction by detecting the negative of ninhydrin detection, repeating the deprotecting and amino acid coupling steps, and sequentially finishing Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH according to the sequence of Liragu peptide, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-Lys (Pal-Glu-OAll) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr-OH, Coupling of Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH. The peptide resin is allyl-removed by using tetrakis (triphenylphosphine) palladium.
15.31g of the liraglutide resin was taken, and added with TFA: and Tis: water 95: 2.5: 2.5 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for reaction for 3.0 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 5.74g of crude liraglutide was obtained in 102.01% yield and 77.34% purity.
Comparative example 1
10.0g of Wang resin (0.40mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swollen with DMF for 30 minutes. Dissolving 4.46g of Fmoc-Gly-OH, 2.03g of HOBt and 0.73g of DMAP in 100mL of DMF, adding 1.94g of DIC under an ice bath condition to activate for 5 minutes, adding the activated solution into the solid phase reaction column, stirring nitrogen for reaction for 4.0 hours, blocking the Fmoc-Gly-Wang resin with acetic anhydride/DIEA for overnight, washing the resin with DMF and DCM, shrinking methanol and draining to obtain the Fmoc-Gly-Wang resin, wherein the detection substitution degree is 0.17 mmol/g; adding 8.82g of Fmoc-Gly-Wang resin with the substitution value of 0.17mmol/g into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-Wang resin by DMF for 30min, then using 20% DBLK to carry out deprotection, washing the resin 3 times by DMF, washing the resin 3 times by DCM, detecting the color of the resin by ninhydrin, wherein the color of the resin indicates that Fmoc is completely removed, dissolving Fmoc-Arg (pbf) -OH 4.87g, HBTU 2.84g and HOBt 1.01g in 50mL of DMF, adding DIEA 1.94g for activation for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring and reacting for 2h by nitrogen, finishing the reaction by ninhydrin detection of negative, repeating the deprotection and amino acid coupling steps, and finishing Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Pbf-OH, and amino acid coupling steps according to the sequence of liraglutide peptide, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-ThrtBu (OH), Fmoc-Phe-OH, Fmoc-Il-OH, Fmoc-Glu-Leu-OH, Fmoc, Coupling of Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.
100mL of 2.0% hydrazine hydrate solution in DMF is added into the solid phase reaction column, the mixture is stirred and reacted for 1.0h under nitrogen, the reaction mixture is pumped out, washed for 6 times by DCM, and the ninhydrin test shows positive. Dissolving Fmoc-Glu-OtBu (6.38g, 15mmol), HOBt (2.03g, 15mmol) and PyBOP (7.81g, 15mmol) in 50mL of DMF, adding DIPEA (1.94g, 15mmol) to activate for 3 minutes under ice bath condition, adding the activated solution to the solid phase reaction column, stirring and reacting for 2 hours under nitrogen, detecting negative ninhydrin, draining, washing 4 times with DMF, and washing 2 times with DCM; palmitic acid (3.84g, 15mmol), HOBt (2.03g, 15mmol) and PyBOP (7.81g, 15mmol) were dissolved in 50mL DCM/DMF ═ 1: 1, adding DIPEA (1.94g, 15mmol) into the solution under an ice bath condition for activation for 3 minutes, adding the activated solution into the solid phase reaction column, stirring and reacting for 2 hours under nitrogen, detecting ninhydrin to be negative, draining, and washing with DMF for 6 times; taking 14.68g of the obtained liraglutide resin, adding TFA: and Tis: water 90: 2.5: 2.5 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for 5 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 4.24g of crude liraglutide was obtained in 75.34% yield and 66.06% purity. The characteristic peak relative time and peak area results are shown in table 4.
Table 4 characteristic peak relative retention time and peak area results for the crude liraglutide peptide of comparative example 1
Comparative example 2
10g of CTC resin (0.7mmol/g) was loaded onto a solid phase reaction column, washed twice with DMF and swelled with DMF for 30 min. Dissolving 3.57g of Fmoc-Gly-OH in 50mL of DMF, adding 1.16g of DIEA under an ice bath condition to activate for 5 minutes, adding the activated solution into the solid-phase reaction column, stirring and reacting for 4.0 hours by using nitrogen, sealing and standing the Fmoc-Gly-CTC resin overnight by using methanol/DIEA, washing the resin by using DMF and DCM, shrinking and draining the methanol to obtain the Fmoc-Gly-CTC resin, wherein the detection substitution degree is 0.36 mmol/g; taking 4.17g of Fmoc-Gly-CTC resin with the substitution value of 0.36mmol/g, adding the Fmoc-Gly-CTC resin into a reaction vessel, washing the resin twice by DMF, swelling the Fmoc-Gly-CTC resin by DMF for 30min, then deprotecting by 20% DBLK, washing the resin 3 times by DMF, washing the resin 3 times by DCM, detecting the color of the resin by ninhydrin, wherein the color of the resin indicates that Fmoc is completely removed, dissolving Fmoc-Arg (pbf) -OH 4.89g and HOBt 1.52g in 30mL DMF, adding DIC 1.89g and activating for 5min under ice bath condition, adding the activated solution into the solid phase reaction column, stirring and reacting for 2h by nitrogen, finishing the reaction by detecting the ninhydrin to be negative, repeating the above steps of deprotection and amino acid coupling, and finishing Fmoc-Gly-OH, Fmoc-Arg-Pbf) -OH, Fmoc-Val-OH, Fmoc-Arg-Pbf, Fmoc-OH, Fmoc, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, (Fmoc-Ser tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-ThrtBu-OH, (Fmoc-Phe-OH, Fmoc-Thru) -OH, Fmoc-Thr-OH, Coupling of Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.
Adding 100mL of DCM and 3.24g of phenylsilane into the solid phase reaction column, stirring for 10 minutes in nitrogen, adding 0.87g of tetrakis (triphenylphosphine) palladium, reacting for 3.0 hours, draining, washing for 6 times with DCM, and detecting the result that ninhydrin is positive; dissolving Fmoc-Glu-OtBu (6.38g, 15mmol), HOBt (2.03g, 15mmol) and PyBOP (7.81g, 15mmol) in 50mL of DMF, adding DIPEA (1.94g, 15mmol) to activate for 3 minutes under ice bath condition, adding the activated solution to the solid phase reaction column, stirring and reacting for 2 hours under nitrogen, detecting negative ninhydrin, draining, washing 4 times with DMF, and washing 2 times with DCM; adding 50mL of DCM and DIPEA (1.94g, 15mmol) into the solid phase reaction column, stirring the mixture uniformly with nitrogen, slowly dropwise adding palmitoyl chloride (4.12g, 5mmol), continuing to react for 2 hours after dropwise adding, detecting ninhydrin to be negative, draining, washing with DCM for 6 times, shrinking MeOH, and drying in vacuum to obtain peptide resin; taking 13.94g of the obtained liraglutide resin, adding TFA: and Tis: water 95: 2.5: 2.5 (10 mL/g liraglutide resin), stirring uniformly, stirring at room temperature for 5 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin for 3 times by using a small amount of TFA, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate for 3 times by using the anhydrous ether, pumping to obtain white-like powder, and drying under reduced pressure in vacuum to constant weight. 4.47g of crude liraglutide was obtained in 79.44% yield and 57.39% purity.
From the results of the relative time and peak area of the characteristic peaks in example 7 and comparative example 1, it is understood that the number of impurities in example 1 is 35, the main peak purity is 80.86%, and the maximum impurity is 4.78%, the number of impurities in comparative example is 42, the main peak purity is 66.06%, and the maximum impurity is 8.54%, the number of impurities in comparative example is larger than that in example, the area ratio of the maximum impurity is also higher than that in example, and the main peak purity is also lower than that in example.
The compound of the formula I is adopted in the process of synthesizing liraglutide, so that the coupling efficiency of amino acid can be effectively improved, the generation of impurities with very similar properties to the product can be effectively reduced, and the purification is very facilitated. The compound of the formula I also shortens the production time and improves the production efficiency.
The method can greatly improve the purity of the crude liraglutide, and is beneficial to purifying the crude liraglutide to obtain the refined peptide.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (11)
1. Alanine-lysine-glutamic acid tripeptide derivative, characterized in that: has a structure shown as a general formula I
Wherein,
R1and R2Is a hydrogen or an ester protecting group,
R1and R2Which may be the same or different from each other,
R3is hydrogen or an amino-protecting group,
R4is C10-20An alkyl group, a carboxyl group,
or enantiomers, diastereomers and salts thereof.
2. The tripeptide derivative alanine-lysine-glutamate of claim 1 wherein: r1And R2Is hydrogen or an ester protecting group, the ester protecting group is tert-butyl, methyl, ethyl, benzyl or allyl, R1And R2May be the same or different.
3. The tripeptide derivative alanine-lysine-glutamate of claim 1 wherein: r3Is hydrogen or an amino protecting group, and the amino protecting group is Fmoc, Dde, Alloc, Boc, Trt and Dmb.
4. The tripeptide derivative alanine-lysine-glutamate of claim 1 wherein: r4Is C10-20Alkyl radical, said C10-20The alkyl group is a straight or branched chain saturated aliphatic alkyl group of 10 to 20 carbon atoms.
5. The tripeptide derivative alanine-lysine-glutamate of claim 1 wherein: r4Is C10-20Alkyl radical, said C10-20The alkyl is a straight-chain saturated aliphatic alkyl.
6. The tripeptide derivative alanine-lysine-glutamate of claim 1 wherein: r4Is C14-16An alkyl group.
7. Use of the alanine-lysine-glutamic acid tripeptide derivative according to claim 1 in solid phase peptide synthesis of peptides comprising a Glu-fatty alkyl side chain building block linked to a Lys-moiety of the peptide chain.
8. The use of claim 7, wherein the solid phase peptide synthesis is Fmoc solid phase peptide synthesis.
9. Use of the tripeptide derivative alanine-lysine-glutamic acid according to claim 1 for the manufacture of a medicament for a polypeptide for treating diabetes.
10. The use of the tripeptide derivative alanine-lysine-glutamate as claimed in claim 9, for the manufacture of a polypeptide drug for the treatment of diabetes mellitus: the diabetes polypeptide drug is GLP-1 analogue.
11. The GLP-1 analog of claim 10 which is liraglutide.
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| CN107827973A (en) * | 2017-09-15 | 2018-03-23 | 吴忠臣 | A kind of solid phase synthesis process of Liraglutide |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113135989A (en) * | 2020-01-20 | 2021-07-20 | 深圳市健元医药科技有限公司 | Method for preparing liraglutide |
| CN113135989B (en) * | 2020-01-20 | 2023-10-03 | 深圳市健元医药科技有限公司 | Method for preparing liraglutide |
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| CN110191892B (en) | 2022-04-26 |
| WO2020019128A1 (en) | 2020-01-30 |
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