CN110818790A - Preparation method of temeprelin - Google Patents
Preparation method of temeprelin Download PDFInfo
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- CN110818790A CN110818790A CN201911063506.6A CN201911063506A CN110818790A CN 110818790 A CN110818790 A CN 110818790A CN 201911063506 A CN201911063506 A CN 201911063506A CN 110818790 A CN110818790 A CN 110818790A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 150000001413 amino acids Chemical group 0.000 claims abstract description 19
- 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 12
- 108700002800 tesamorelin Proteins 0.000 claims abstract description 11
- QBEPNUQJQWDYKU-BMGKTWPMSA-N egrifta Chemical compound C([C@H](NC(=O)C/C=C/CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(N)=O)C1=CC=C(O)C=C1 QBEPNUQJQWDYKU-BMGKTWPMSA-N 0.000 claims abstract description 10
- 229960001874 tesamorelin Drugs 0.000 claims abstract description 10
- 230000001681 protective effect Effects 0.000 claims abstract 2
- 229920005989 resin Polymers 0.000 claims description 36
- 239000011347 resin Substances 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 20
- 239000000047 product Substances 0.000 claims description 20
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 14
- CBPNZQVSJQDFBE-FUXHJELOSA-N Temsirolimus Chemical compound C1C[C@@H](OC(=O)C(C)(CO)CO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 CBPNZQVSJQDFBE-FUXHJELOSA-N 0.000 claims description 12
- 229960000235 temsirolimus Drugs 0.000 claims description 12
- QFJCIRLUMZQUOT-UHFFFAOYSA-N temsirolimus Natural products C1CC(O)C(OC)CC1CC(C)C1OC(=O)C2CCCCN2C(=O)C(=O)C(O)(O2)C(C)CCC2CC(OC)C(C)=CC=CC=CC(C)CC(C)C(=O)C(OC)C(O)C(C)=CC(C)C(=O)C1 QFJCIRLUMZQUOT-UHFFFAOYSA-N 0.000 claims description 12
- 229920003180 amino resin Polymers 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 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
- 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
- 239000001602 (E)-hex-3-enoic acid Substances 0.000 claims 1
- XXHDAWYDNSXJQM-ONEGZZNKSA-N trans-hex-3-enoic acid Chemical compound CC\C=C\CC(O)=O XXHDAWYDNSXJQM-ONEGZZNKSA-N 0.000 claims 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 20
- 239000000243 solution Substances 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000012071 phase 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 6
- 150000003839 salts Chemical group 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 102000018997 Growth Hormone Human genes 0.000 description 4
- 108010051696 Growth Hormone Proteins 0.000 description 4
- 239000000122 growth hormone Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 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
- 206010049287 Lipodystrophy acquired Diseases 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
- 210000004027 cell Anatomy 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
- 239000000706 filtrate Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 208000006132 lipodystrophy Diseases 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
- 238000003786 synthesis reaction Methods 0.000 description 2
- 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
- 102100033365 Growth hormone-releasing hormone receptor Human genes 0.000 description 1
- 101710198286 Growth hormone-releasing hormone receptor Proteins 0.000 description 1
- 230000003187 abdominal effect Effects 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
- 230000003213 activating effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000002267 hypothalamic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 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
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
-
- 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)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a preparation method of tesamorelin, which adopts special protective amino acid fragments: (E) 3-Hexenolic acid-Tyr (tBu) -Ala-Asp (OtBu) -Ala-Ile-Phe-Thr (tBu) -Asn (Trt) -Ser (tBu) -Tyr (tBu) -Arg (Pbf) -Lys (Boc) -Val-Leu-OH, shortens the preparation process period and improves the purity and yield of products in large-scale preparation.
Description
Technical Field
The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a preparation method of temmorelin.
Background
Temorelin is a new therapeutic drug for abdominal adiposity in Human Immunodeficiency Virus (HIV) infected patients with lipodystrophy, is a GRF synthetic analogue. Similar to endogenous GRF action, it acts in vitro by binding to and activating GRF receptors. GRF is a hypothalamic regulatory polypeptide that stimulates pituitary growth hormone cells to synthesize and release endogenous Growth Hormone (GH). GH exerts pharmacological effects by interacting with specific receptors on target cells, including chondrocytes, osteoblasts, cardiomyocytes, hepatocytes and adipocytes.
The absolute bioavailability of healthy adults after subcutaneous injection of 2mg of tesamorelin is less than 4%. After a single dose of 2mg is injected into healthy people without lipodystrophy and HIV infected patients subcutaneously, the area under the curve (AUC) is respectively (634.6 +/-72.4) and (852.8 +/-91.9) pg.h.mL-1Blood peak concentration (C)max) Are respectively as(2874.6. + -. 43.9) and (2822.3. + -. 48.9) pg.mL-1The peak reaching time is 1.5h, and the apparent distribution volumes are (9.4 +/-3.1) and (10.5 +/-6.1) L.kg-1Average elimination half-life (t) after continuous application of 14d1/2) 26 and 38min, respectively.
Temorelin has the following structure:
(E)-3-Hexenoic acid-Tyr-Ala-Asp-Ala5-Ile-Phe-Thr-Asn-Ser10-Tyr-Arg-Lys-Val-Leu15-Gly-Gln-Leu-Ser-Ala20-Arg-Lys-Leu-Leu-Gln25-Asp-Ile-Met-Ser-Arg30-Gln-Gln-Gly-Glu-Ser35-Asn-Gln-Glu-Arg-Gly40-Ala-Arg-Ala-Arg-Leu45-NH2
the preparation method of the temorelin is reported, and the invention provides an efficient temorelin preparation method, which improves the purity and yield of high products so as to meet medical application.
Disclosure of Invention
The invention provides a novel high-efficiency preparation method, which adopts special protected amino acid, shortens the preparation process period and improves the product purity and yield in a large-scale preparation method.
The invention provides a preparation method of tesamorelin, which comprises the following steps: amino resin is adopted as initial resin, the starting resin is prepared by a solid-phase polypeptide synthesis method, the temmorelin resin is obtained by the polypeptide solid-phase synthesis method, the temmorelin resin is acidolyzed to obtain a crude temmorelin product, and finally the crude temmorelin product is purified to obtain a pure temmorelin product.
In addition to other conventional protected amino acids, the following special protected amino acid fragments are used in the synthesis process of the temorelin resinoid:
(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu-OH。
temsirolin peptide resin:
(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala5-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)10-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu15-Gly-Gln(Trt)-Leu-Ser(tBu)-Ala20-Arg(Pbf)-Lys(Boc)-Leu-Leu-Gln(Trt)25-Asp(OtBu)-Ile-Met-Ser(tBu)-Arg(Pbf)30-Gln(Trt)-Gln(Trt)-Gly-Glu(OtBu)-Ser(tBu)35-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Gly40-Ala-Arg(Pbf)-Ala-Arg(Pbf)-Leu45-amino resins
In the preparation method of the temsirolimus, the amino resin has an amino substitution value of 0.3 to 1.0mmol/g resin, and preferably a substitution value of 0.3 to 0.5mmol/g resin.
In the preparation method of the temsirorelin, 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 temorelin, 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 temorelin resin is subjected to acidolysis while removing the resin and the side chain protecting groups to obtain a temorelin linear peptide crude product.
Further, an acidolysis agent adopted in acidolysis of the temorelin 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 required by each gram of the tirmorelin resin, and preferably, 9-11 ml of acidolysis agent required by each gram of the tirmorelin resin. The time for cracking by using the acidolysis agent is 1-5 hours, preferably 2 hours at room temperature.
Further, the crude temsirolimus product is purified by high performance liquid chromatography and freeze-dried to obtain a pure temsirolimus product, and the specific method comprises the following steps:
adding water into the crude temeprelin product, stirring, adjusting the pH value to 8.5 by using ammonia water until the solution is completely dissolved, filtering the solution by using a 0.45-micrometer mixed microporous filter membrane, and purifying for later use;
purifying by adopting a high performance liquid chromatography, wherein a chromatographic filler for purification is 10 mu m reverse phase C18, alternately purifying by adopting 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 adopting a gradient system, circularly injecting and purifying, sampling a crude product solution in the chromatographic column, starting mobile phase elution, collecting a main peak to evaporate acetonitrile, and filtering by using a 0.45 mu m filter membrane to obtain a purified intermediate concentrated solution of the tesamorelin;
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 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 temorelin acetic acid aqueous solution, and freeze-drying to obtain a pure temorelin product.
The method of the invention directly uses the following special protected amino acids:
(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu-OH。
the production period is shortened, the purity of the crude product is greatly improved, the product yield is improved, the purity of the obtained product is more than 99.0 percent, and compared with the prior art, the process has wide practical value and application prospect.
Detailed Description
The invention discloses a method for synthesizing tesamorelin, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the text. 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 temorelin peptide resin
Temsirolin peptide resin:
(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala5-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)10-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu15-Gly-Gln(Trt)-Leu-Ser(tBu)-Ala20-Arg(Pbf)-Lys(Boc)-Leu-Leu-Gln(Trt)25-Asp(OtBu)-Ile-Met-Ser(tBu)-Arg(Pbf)30-Gln(Trt)-Gln(Trt)-Gly-Glu(OtBu)-Ser(tBu)35-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Gly40-Ala-Arg(Pbf)-Ala-Arg(Pbf)-Leu45-amino resins
Rink Amide MBHA resin is used as initial resin, and is coupled with protected amino acid shown in table 2 in sequence through Fmoc protection removal and coupling reaction to prepare the temsirolin peptide resin. The protected amino acids or fragments corresponding to the protected amino acids used in this example are shown below:
TABLE 2
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. Inoculating 2-45 th protected amino acid or fragment
And sequentially inoculating the corresponding 2 nd to 45 th protected amino acids or fragments by adopting the same method to obtain the tesamorelin peptide resin.
Example 2 preparation of crude temeprelin
Taking the temsirolin peptide resin prepared in example 1, adding a cracking reagent (10 mL/g resin of the cracking reagent) with the volume ratio of TFA, water and EDT being 95: 5, stirring uniformly, stirring at room temperature for reaction for 3 hours, filtering the reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and pumping to dry to obtain white-like powder, namely a temsirolin crude product, wherein the purity of the crude product is 70.9%.
Example 3 purification of crude temeprelin
Taking the crude temsirolimus product prepared in the example 2, adding water, stirring, adjusting the pH to 8.5 by using ammonia water until the solution is completely dissolved, filtering the solution by using a 0.45 mu m 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 purified intermediate concentrated solution of the tesamorelin;
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 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 temorelin acetic acid aqueous solution, and freeze-drying to obtain a temorelin pure product 32.9g, wherein the purity is 99.2%, the maximum single impurity is 0.12%, the total yield is 27.4%, and the molecular weight is 5135.8 (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 (6)
1. A method for preparing temsirolimus, comprising: amino resin is adopted as initial resin, a solid-phase polypeptide synthesis method is used for preparing temsirolimus peptide resin, the temsirolimus peptide resin is subjected to acidolysis to obtain a crude temsirolimus product, and finally, the crude temsirolimus product is purified and freeze-dried to obtain a pure temsirolimus product:
(E)-3-Hexenoic acid-Tyr-Ala-Asp-Ala5-Ile-Phe-Thr-Asn-Ser10-
Tyr-Arg-Lys-Val-Leu15-Gly-Gln-Leu-Ser-Ala20-Arg-Lys-Leu-
Leu-Gln25-Asp-Ile-Met-Ser-Arg30-Gln-Gln-Gly-Glu-Ser35-Asn-
Gln-Glu-Arg-Gly40-Ala-Arg-Ala-Arg-Leu45-NH2。
2. the process for the preparation of tesamorelin according to claim 1, characterized in that: when the 1 st (E) -3-Hexenoic acid is connected to the 15 th Leu, the corresponding protective amino acid fragment is (E) -3-Hexenoic-Tyr (tBu) -Ala-Asp (OtBu) -Ala-Ile-Phe-Thr (tBu) -Asn (Trt) -Ser (tBu) -Tyr (tBu) -Arg (Pbf) -Lys (Boc) -Val-Leu-OH.
3. The method for preparing tesamorelin 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.
4. The preparation method of temsirorelin 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.
5. The process for the preparation of tesamorelin according to any of claims 1 to 4, characterized in that: and (3) carrying out acidolysis on the temorelin peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain a temorelin crude product.
6. The process for the preparation of tesamorelin according to claim 1, characterized in that: and purifying the crude temsirolimus product by high performance liquid chromatography and freeze-drying to obtain a pure temsirolimus product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518192A (en) * | 2020-05-26 | 2020-08-11 | 成都圣诺生物制药有限公司 | Preparation method of Apraglutide |
| CN111560062A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Elisiglilutide |
| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| CN111574594A (en) * | 2020-05-26 | 2020-08-25 | 成都圣诺生物制药有限公司 | Preparation method of Bulevirtide |
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| CN111560061A (en) * | 2020-05-26 | 2020-08-21 | 成都圣诺生物制药有限公司 | Preparation method of Gelpaglutide |
| CN111574594A (en) * | 2020-05-26 | 2020-08-25 | 成都圣诺生物制药有限公司 | Preparation method of Bulevirtide |
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