CN110903355A - Preparation method of Tirzepatide - Google Patents
Preparation method of Tirzepatide Download PDFInfo
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- CN110903355A CN110903355A CN201911063432.6A CN201911063432A CN110903355A CN 110903355 A CN110903355 A CN 110903355A CN 201911063432 A CN201911063432 A CN 201911063432A CN 110903355 A CN110903355 A CN 110903355A
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- tirzepatide
- resin
- gly
- ser
- tbu
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- 108091004331 tirzepatide Proteins 0.000 title claims abstract description 65
- 229940121512 tirzepatide Drugs 0.000 title claims abstract description 59
- BTSOGEDATSQOAF-SMAAHMJQSA-N tirzepatide Chemical compound CC[C@H](C)[C@@H](C(N[C@@H](C)C(N[C@@H](CCC(N)=O)C(N[C@@H](CCCCNC(COCCOCCNC(COCCOCCNC(CC[C@H](C(O)=O)NC(CCCCCCCCCCCCCCCCCCC(O)=O)=O)=O)=O)=O)C(N[C@@H](C)C(N[C@@H](CC1=CC=CC=C1)C(N[C@@H](C(C)C)C(N[C@@H](CCC(N)=O)C(N[C@@H](CC1=CNC2=C1C=CC=C2)C(N[C@@H](CC(C)C)C(N[C@@H]([C@@H](C)CC)C(N[C@@H](C)C(NCC(NCC(N(CCC1)[C@@H]1C(N[C@@H](CO)C(N[C@@H](CO)C(NCC(N[C@@H](C)C(N(CCC1)[C@@H]1C(N(CCC1)[C@@H]1C(N(CCC1)[C@@H]1C(N[C@@H](CO)C(N)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)NC([C@H](CCCCN)NC([C@H](CC(O)=O)NC([C@H](CC(C)C)NC(C(C)(C)NC([C@H]([C@@H](C)CC)NC([C@H](CO)NC([C@H](CC(C=C1)=CC=C1O)NC([C@H](CC(O)=O)NC([C@H](CO)NC([C@H]([C@@H](C)O)NC([C@H](CC1=CC=CC=C1)NC([C@H]([C@@H](C)O)NC(CNC([C@H](CCC(O)=O)NC(C(C)(C)NC([C@H](CC(C=C1)=CC=C1O)N)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O BTSOGEDATSQOAF-SMAAHMJQSA-N 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 13
- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract description 4
- 229920005989 resin Polymers 0.000 claims description 37
- 239000011347 resin Substances 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 19
- 239000012043 crude product Substances 0.000 claims description 14
- 239000000047 product Substances 0.000 claims description 14
- 229920003180 amino resin Polymers 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 125000006239 protecting group Chemical group 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 238000001308 synthesis method Methods 0.000 claims description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims 1
- 230000001681 protective effect Effects 0.000 abstract 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000012071 phase Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000010828 elution Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 150000003839 salts Chemical group 0.000 description 6
- 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 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 4
- ZPGDWQNBZYOZTI-UHFFFAOYSA-N 1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)C1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-UHFFFAOYSA-N 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- 238000005336 cracking Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 1
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-RXMQYKEDSA-N D-histidine Chemical compound OC(=O)[C@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-RXMQYKEDSA-N 0.000 description 1
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 108010004460 Gastric Inhibitory Polypeptide Proteins 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound 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 description 1
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 208000031964 Other metabolic disease Diseases 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940125542 dual agonist Drugs 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- BBKFSSMUWOMYPI-UHFFFAOYSA-N gold palladium Chemical class [Pd].[Au] BBKFSSMUWOMYPI-UHFFFAOYSA-N 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- 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
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a preparation method of Tirzepatide, which adopts special protective amino acids Boc-Tyr (tBu) -Aib-Glu (OtBu) -Gly-OH and Fmoc-Lys (AEEA-AEEA-gamma Glu (α -OtBu) -Eicoside acid (mon-tBu)) -OH, and solves the problem of low product purity in a large-scale preparation method.
Description
Technical Field
The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a preparation method of Tirzepatide.
Background
Tirzepatide is a GIP and GLP-1 dual agonist, can improve β cell function and improve insulin sensitivity, thus proving curative effect, and shows double improvement of curative effect and tolerance to patients with lower initial dosage and smaller subsequent dosage increment, and after 8 weeks of treatment with Tirzepatide, the A1C and body weight of Japanese type 2 diabetes patients are obviously reduced, and the Tirzepatide can bring improvement to markers of nonalcoholic steatohepatitis (NASH, liver inflammation and cell damage caused by liver fat) of type 2 diabetes patients, so that the new Tirzepatide data are established on the positive results of the research carried out on type 2 diabetes patients so far, and the results provide additional evidence that the Tirzepatide can bring meaningful reduction of the A1C and body weight of type 2 diabetes patients, and can also treat other metabolic diseases.
Tirzepatide has the following structure:
Tyr1-Aib-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Tyr10-Ser-Ile-Aib-Leu-Asp15-Lys-Ile-Ala-Gln-Lys20(AEEA-AEEA-γGlu-Eicosanedioic acid)-Ala-Phe-Val-Gln-Trp25-Leu-Ile-Ala-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-Pro-Pro-Ser-NH2
the preparation method of the Tirzepatide has been reported, and the invention provides an efficient preparation method of the Tirzepatide to produce high-purity products so as to meet the medical application.
Disclosure of Invention
The invention provides a novel high-efficiency preparation method, which adopts special protected amino acid fragments and solves the problem of low product purity in a large-scale preparation method.
The invention provides a preparation method of Tirzepatide, which comprises the following steps: amino resin is used as starting resin, the starting resin is prepared by a solid phase polypeptide synthesis method, the Tirzepatide resin is obtained by the polypeptide solid phase synthesis method, the Tirzepatide resin is acidolyzed to obtain a crude Tirzepatide product, and finally the crude Tirzepatide product is purified to obtain a pure Tirzepatide product.
In addition to other conventional protected amino acids, the following special protected amino acids and fragments are used in the synthesis process of the Tirzepatide multi-resin:
(1)Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH
(2) Fmoc-Lys (AEEA-AEEA-gamma Glu (α -OtBu) -Eicosanedioic acid (mon-tBu)) -OHIRTIRzepatide peptide resin:
Boc-Tyr (tBu) -Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-Asp (OtBu) -Lys (Boc) -Ile-Ala-Gln (Trt) -Lys (AEEA-AEEA-gamma Glu-Eicosaedioic acid (mon-tBu)) -Ala-Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Ile-Ala-Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -amino resin
In the preparation method of the Tirzepatide, the amino resin has an amino substitution value of 0.3-1.0 mmol/g resin, and the preferable substitution value is 0.3-0.5 mmol/g resin.
In the preparation method of the Tirzepatide, the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, and Rink Amide MBHA resin is preferred.
In the preparation method of the Tirzepatide, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.
In a preferred embodiment of the present invention, the Tirzepatide resin is subjected to acidolysis while removing the resin and the side chain protecting groups to obtain a crude Tirzepatide linear peptide.
Further, an acidolysis agent adopted in the acid hydrolysis of the Tirzepatide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the mixture ratio of the mixed solvent is as follows: the TFA ratio is 80-95% (V/V), the EDT ratio is 1-10% (V/V), and the balance is water. The preferred formulation is 89-91% TFA, 4-6% EDT, and the balance water. Preferably, the mixture ratio is 90%, EDT 5% and the balance of water.
The dosage of the acidolysis agent is 4-15 ml of acidolysis agent per gram of Tirzepatide resin, and preferably 9-11 ml of acidolysis agent per gram of Tirzepatide resin. The time for cracking by using the acidolysis agent is 1-5 hours, preferably 2 hours at room temperature.
Further, the crude product of the Tirzepatide is purified by high performance liquid chromatography and freeze-dried to obtain a pure product of the Tirzepatide, and the specific method comprises the following steps:
adding water into the Tirzepatide crude product, stirring, adjusting the pH value to 8.5 by using ammonia water until the Tirzepatide crude product is completely dissolved, filtering the solution by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;
purifying by high performance liquid chromatography, wherein a chromatographic filler for purification is 10 mu m reverse phase C18, alternately purifying by two mobile phase systems, the first mobile phase system is 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the second mobile phase system is 50mmol ammonium acetate/aqueous solution-acetonitrile, the flow rate of a 77mm 250mm chromatographic column is 90mL/min, eluting by a gradient system, circularly injecting and purifying, sampling a crude product solution in the chromatographic column, starting mobile phase elution, collecting a main peak, evaporating acetonitrile, and filtering by a 0.45 mu m filter membrane to obtain a Tirzepatide purified intermediate concentrated solution;
performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); adopting gradient elution and circulation sample loading methods, loading the sample into a chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain a Tirzepatide acetic acid aqueous solution, and freeze-drying to obtain a Tirzepatide pure product.
The method of the invention directly uses the following special protected amino acids:
(1)Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH
(2)Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH
directly avoids the risk of introducing heavy gold palladium salt during Lys (alloc) deprotection or the genotoxic hydrazine during Lys (IVDde) deprotection, and simultaneously avoids [ D-His ]1]-Tirzepatide、[Des-Gly4]-Tirzepatide and [ Lys (AEEA-AEEA-D-gamma Glu-Eicosanedioic acid)20]The production of impurities such as Tirzepatide and the like improves the purity of a crude product, reduces the purification difficulty, improves the product yield, and has the purity of more than 99.0 percent.
Detailed Description
The invention discloses a method for synthesizing Tirzepatide, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.
In the specific embodiment of the present invention, the Chinese meanings corresponding to the English abbreviations used in the application documents are shown in Table 1.
TABLE 1
The invention is further illustrated by the following examples.
Example 1 Synthesis of Tirzepatide peptide resin
Tirzepatide peptide resin:
Boc-Tyr (tBu) -Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-Asp (OtBu) -Lys (Boc) -Ile-Ala-Gln (Trt) -Lys (AEEA-AEEA-gamma Glu-Eicosaedioic acid (mon-tBu)) -Ala-Phe-Val-Gln (Trt) -Trp (Boc) -Leu-Ile-Ala-Gly-Gly-Pro-Ser (tBu) -Gly-Ala-Pro-Pro-Ser (tBu) -amino resin
Rink Amide MBHA resin is used as initial resin, and is sequentially coupled with protected amino acids shown in table 2 through Fmoc protection removal and coupling reaction to prepare the Tirzepatide peptide resin. The protected amino acids or fragments corresponding to the protected amino acids used in this example are shown below:
TABLE 2
| The peptide sequence n ═ | Protected amino acids |
| 1 | Fmoc-Ser(tBu) |
| 2 | Fmoc-Pro |
| 3 | Fmoc-Pro |
| 4 | Fmoc-Pro |
| 5 | Fmoc-Ala |
| 6 | Fmoc-Gly |
| 7 | Fmoc-Ser(tBu) |
| 8 | Fmoc-Ser(tBu) |
| 9 | Fmoc-Pro |
| 10 | Fmoc-Gly |
| 11 | Fmoc-Gly |
| 12 | Fmoc-Ala |
| 13 | Fmoc-Ile |
| 14 | Fmoc-Leu |
| 15 | Fmoc-Trp(Boc) |
| 16 | Fmoc-Gln(Trt) |
| 17 | Fmoc-Val |
| 18 | Fmoc-Phe |
| 19 | Fmoc-Ala |
| 20 | Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)) |
| 21 | Fmoc-Gln(Trt) |
| 22 | Fmoc-Ala |
| 23 | Fmoc-Ile |
| 24 | Fmoc-Lys(Boc) |
| 25 | Fmoc-Asp(OtBu) |
| 26 | Fmoc-Leu |
| 27 | Fmoc-Aib |
| 28 | Fmoc-Ile |
| 29 | Fmoc-Ser(tBu) |
| 30 | Fmoc-Tyr(tBu) |
| 31 | Fmoc-Asp(OtBu) |
| 32 | Fmoc-Ser(tBu) |
| 33 | Fmoc-Thr(tBu) |
| 34 | Fmoc-Phe |
| 35 | Fmoc-Thr(tBu) |
| 36 | Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH |
1. Introduction of the 1 st protected amino acid
Dissolving 0.09mol of the 1 st protected amino acid and 0.09mol of HOBt in a proper amount of DMF; and adding 0.09mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.
0.03mol of Fmoc-Gly-resin (substitution value about 0.5mmol/g) was taken, deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.
And adding the activated 1 st protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 120-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.
2. 2 nd to 36 th protected amino acids or fragments are inoculated
And sequentially inoculating the corresponding 2 nd to 36 th protected amino acids or fragments by adopting the same method to obtain the Tirzepatide peptide resin.
EXAMPLE 2 preparation of crude Tirzepatide
The Tirzepatide peptide resin prepared in the example 1 is taken, added with a cracking reagent (10 mL of the cracking reagent/g of the resin) with the volume ratio of TFA, water and EDT being 95: 5, evenly stirred and reacted for 3 hours at room temperature, a reaction mixture is filtered by a sand core funnel, filtrate is collected, the resin is washed for 3 times by a small amount of TFA, the filtrate is combined and concentrated under reduced pressure, anhydrous ether is added for precipitation, the anhydrous ether is used for washing and precipitating for 3 times, and the white-like powder is obtained by pumping drying, namely the Tirzepatide crude product, wherein the purity of the crude product is 72.7 percent.
EXAMPLE 3 purification of crude Tirzepatide
Taking the Tirzepatide crude product prepared in the example 2, adding water, stirring, adjusting the pH to 8.5 by using ammonia water until the Tirzepatide crude product is completely dissolved, filtering the solution by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;
purification was performed by high performance liquid chromatography using reverse phase C18 with 10 μm chromatography packing and alternating purification with two mobile phase systems, the first being 0.1% TFA/water-0.1% TFA/acetonitrile and the second being 50mmol ammonium acetate/water-acetonitrile. The flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, a gradient system is adopted for elution, the sample is circularly injected and purified, a crude product solution is taken to be loaded in the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the crude product solution is filtered by a 0.45-micrometer filter membrane to obtain a Tirzepatide purified intermediate concentrated solution;
performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); the method comprises the steps of adopting a gradient elution and circulation loading method, loading a sample into a chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain a Tirzepatide acetic acid aqueous solution, and freeze-drying to obtain a Tirzepatide pure product 37.9g, wherein the purity is 99.5%, the maximum single impurity is 0.09%, the total yield is 26.2%, and the molecular weight is 4813.6 (100% M + H).
The embodiment shows that the purity of the product obtained by the method provided by the invention is more than 99.0%, and the single impurity is less than 0.15%, so that the product quality is improved, and the method has wide practical value and application prospect.
Claims (7)
1. A preparation method of Tirzepatide comprises the following steps: amino resin is used as initial resin, Tirzepatide peptide resin is prepared by a solid phase polypeptide synthesis method, the Tirzepatide peptide resin is subjected to acidolysis to obtain a crude product of the Tirzepatide, and finally, the crude product of the Tirzepatide is purified and freeze-dried to obtain a pure product of the Tirzepatide:
Tyr1-Aib-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Tyr10-Ser-Ile-Aib-Leu-Asp15-
Lys-Ile-Ala-Gln-Lys20(AEEA-AEEA-γGlu-Eicosanedioic acid)-Ala-
Phe-Val-Gln-Trp25-Leu-Ile-Ala-Gly-Gly30-Pro-Ser-Ser-Gly-Ala35-Pro-
Pro-Pro-Ser-NH2。
2. the method of preparing Tirzepatide according to claim 1, wherein: when the His at the 1 st position to the Gly at the 4 th position are accessed together, the corresponding protected amino acid is Boc-Tyr (tBu) -Aib-Glu (OtBu) -Gly-OH.
3. The method for preparing Tirzepatide according to claim 1, wherein the corresponding protected amino acid is Fmoc-Lys (AEEA-AEEA-gamma Glu (α -OtBu) -Eicoside acid (mon-tBu)) -OH when Lys at position 20 is introduced.
4. The method of preparing Tirzepatide according to claim 1, wherein the amino resin has an amino substitution value of 0.3 to 1.0mmol/g resin, preferably a substitution value of 0.3 to 0.5mmol/g resin.
5. The method of preparing Tirzepatide according to claim 1, wherein the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, preferably Rink Amide MBHA resin.
6. The method for producing a Tirzepatide according to any one of claims 1 to 5, wherein: and (3) carrying out acidolysis on the Tirzepatide peptide resin, and simultaneously removing the resin and the side chain protecting groups to obtain a crude product of the Tirzepatide.
7. The method of preparing Tirzepatide according to claim 1, wherein: and purifying the crude Tirzepatide product by high performance liquid chromatography and freeze-drying to obtain a pure Tirzepatide product.
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| CN112661815A (en) * | 2020-12-30 | 2021-04-16 | 江苏诺泰澳赛诺生物制药股份有限公司 | Purification method of Tirzepatide |
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