CN111718408A - Preparation method of Setmelanotide - Google Patents
Preparation method of Setmelanotide Download PDFInfo
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- CN111718408A CN111718408A CN202010643099.2A CN202010643099A CN111718408A CN 111718408 A CN111718408 A CN 111718408A CN 202010643099 A CN202010643099 A CN 202010643099A CN 111718408 A CN111718408 A CN 111718408A
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- resin
- setmelanotide
- setmelactone
- amino
- cys
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- 108700030852 setmelanotide Proteins 0.000 title claims description 45
- HDHDTKMUACZDAA-PHNIDTBTSA-N (4r,7s,10s,13r,16s,19r,22r)-22-[[(2s)-2-acetamido-5-(diaminomethylideneamino)pentanoyl]amino]-13-benzyl-10-[3-(diaminomethylideneamino)propyl]-16-(1h-imidazol-5-ylmethyl)-7-(1h-indol-3-ylmethyl)-19-methyl-6,9,12,15,18,21-hexaoxo-1,2-dithia-5,8,11,14,17,20 Chemical compound C([C@@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](C(N[C@@H](CC=2N=CNC=2)C(=O)N1)=O)C)NC(=O)[C@H](CCCNC(N)=N)NC(C)=O)C(N)=O)C1=CC=CC=C1 HDHDTKMUACZDAA-PHNIDTBTSA-N 0.000 title claims description 32
- 229950001912 setmelanotide Drugs 0.000 title claims description 32
- 238000002360 preparation method Methods 0.000 title description 13
- 229920005989 resin Polymers 0.000 claims abstract description 51
- 239000011347 resin Substances 0.000 claims abstract description 51
- 150000001413 amino acids Chemical class 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000012043 crude product Substances 0.000 claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 17
- 239000000047 product Substances 0.000 claims abstract description 17
- 229920003180 amino resin Polymers 0.000 claims abstract description 13
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 230000001681 protective effect Effects 0.000 claims abstract description 6
- 238000007243 oxidative cyclization reaction Methods 0.000 claims abstract description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 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 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 125000006239 protecting group Chemical group 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
- 125000003277 amino group Chemical group 0.000 claims description 2
- 230000003647 oxidation Effects 0.000 claims description 2
- 238000007254 oxidation reaction Methods 0.000 claims description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 238000011112 process operation Methods 0.000 abstract description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 38
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 33
- 239000000243 solution Substances 0.000 description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 29
- 239000012071 phase Substances 0.000 description 27
- 238000010828 elution Methods 0.000 description 15
- 238000000746 purification Methods 0.000 description 13
- 238000001914 filtration Methods 0.000 description 12
- 150000003839 salts Chemical group 0.000 description 12
- 238000003756 stirring Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000011068 loading method Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 238000012856 packing Methods 0.000 description 7
- 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 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 229910052740 iodine Inorganic materials 0.000 description 5
- 239000011630 iodine Substances 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000012046 mixed solvent Substances 0.000 description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000012982 microporous membrane Substances 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000007363 ring formation reaction Methods 0.000 description 3
- 239000012047 saturated solution Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- CSMYOORPUGPKAP-IBGZPJMESA-N (2r)-3-(acetamidomethylsulfanyl)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CSCNC(=O)C)C(O)=O)C3=CC=CC=C3C2=C1 CSMYOORPUGPKAP-IBGZPJMESA-N 0.000 description 2
- 102000001796 Melanocortin 4 receptors Human genes 0.000 description 2
- 108010021436 Type 4 Melanocortin Receptor Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000249 far-infrared magnetic resonance spectroscopy Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 102000005861 leptin receptors Human genes 0.000 description 1
- 108010019813 leptin receptors Proteins 0.000 description 1
- 231100001079 no serious adverse effect Toxicity 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000004448 titration Methods 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
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C07K14/68—Melanocyte-stimulating hormone [MSH]
- C07K14/685—Alpha-melanotropin
-
- 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)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for preparing Setmelonide, which comprises the steps of taking amino resin as starting resin, sequentially adding corresponding protective amino acids according to the reversed phase of an amino sequence of Setmelonide to obtain Setmelonide peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelonide protective peptide resin to obtain a Setmelonide crude product, and purifying and freeze-drying the Setmelonide crude product to obtain a pure Setmelonide product. The method has the advantages of simple process operation and high total product yield, and is suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a preparation method of Setmelanotide.
Background
Setmelactone is an oligopeptide melanocortin-4 receptor (MC4R) agonist, and research results show that subcutaneous daily injection of setelactone can reduce the excessive diet and body weight of three LEPR-deficient obesity patients. Setmelanotide was well tolerated and no serious adverse events were reported.
Setmelanotide has the following structure:
Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2
the preparation method of Setmelanotide is rarely reported, Chinese patent CN201080051643 reports a liquid phase fragment condensation method, the method is complex and cumbersome, and large-scale production is not convenient, and the invention provides an efficient preparation method of Setmelanotide to meet the medical application.
Disclosure of Invention
The invention provides an efficient preparation method, which adopts amino resin as initial resin, and has the advantages of simple preparation process operation, high product yield and high purity.
The invention provides a preparation method of Setmelanotide, which comprises the following steps: the method comprises the following steps of taking amino resin as starting resin, sequentially inoculating corresponding protective amino acids according to the reverse phase of an amino sequence of Setmelactone to obtain Setmelactone peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelactone protective peptide resin to obtain a Setmelactone crude product, and purifying and freeze-drying the Setmelactone crude product to obtain a pure Setmelactone product.
In the above method for producing Setmelanotide, the substitution value of the amino resin is 0.3 to 1.5mmol/g resin, and preferably 0.6 to 1.0mmol/g resin.
Further, the amino resin is selected from Rink Amide-MBHA resin, Rink Amide-BHA resin and Rink Amide resin, preferably Rink Amide-MBHA resin.
In the preparation method of Setmelanotide, the dosage of Fmoc-protected amino acid is 1.2-6 times of the total mole number of the amino groups of the initial resin; preferably 2.5 to 4.0 times.
In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.
The Setmelanotide peptide resin is:
ac-arg (pbf) -Cys (R1) -D-Ala-his (trt) -D-Phe-arg (pbf) -Trp (R2) -Cys (R1) -amino resin wherein: r1 is Trt or Acm
R2 is Boc or H
Preferably, the Setmelactone peptide resin is subjected to acidolysis, resin and side chain protecting groups are removed at the same time, and then a crude Setmelactone product is obtained after oxidative cyclization.
More preferably, the acidolysis agent used in acidolysis of the Setmelanotide peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water; wherein the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.
More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.
The dosage of the acidolysis agent is 4-15 mL per gram of Setmelanotide peptide resin; preferably, 9-11 mL of acidolysis agent is needed per gram of the Setmelanotide peptide resin. The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.
Further, oxidizing agent used in the oxidative cyclization is iodine and H2O2Or DMSO, preferably iodine. The oxidant is added in a titration mode, and the oxidant is stopped adding when the oxidation end point is reached.
Further, the method for purifying the crude Setmelactone by high performance liquid chromatography and freeze-drying to obtain the pure Setmelactone comprises the following steps:
purifying by adopting a high performance liquid chromatography, wherein a chromatographic filler 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 into 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 Setmelantotide 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 90 mL/min; adopting a gradient elution and circulation loading method, 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 Setmelanotide acetic acid aqueous solution, and freeze-drying to obtain a pure Setmelanotide product.
The method has the advantages of simple process operation and high total product yield, and is suitable for large-scale production.
Detailed Description
The invention discloses a method for synthesizing Setmelanotide, which can be realized by appropriately improving process parameters by referring 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 English abbreviations used in the application documents have the corresponding Chinese meanings as shown in the following table.
The invention is further illustrated by the following examples.
Example 1 Synthesis of Setmelanotide peptide resin
The Setmelanotide peptide resin is:
Ac-Arg (Pbf) -Cys (Trt) -D-Ala-His (Trt) -D-Phe-Arg (Pbf) -Trp (Boc) -Cys (Trt) -amino resin
Rink Amide-MBHA resin is used as starting resin, and is coupled with protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare the Setmelanotide peptide resin.
The protected amino acids used in this example correspond to the protected amino acids shown in the table below:
| the peptide sequence n ═ | Protected amino acids |
| 1 | Fmoc-Cys(Trt) |
| 2 | Fmoc-Trp(Boc) |
| 3 | Fmoc-Arg(Pbf) |
| 4 | Fmoc-D-Phe |
| 5 | Fmoc-His(Trt) |
| 6 | Fmoc-D-Ala |
| 7 | Fmoc-Cys(Trt) |
| 8 | Fmoc-Arg(Pbf) |
| 9 | Ac2O |
1. Introduction of the 1 st protected amino acid
Dissolving 0.03mol of the 1 st protected amino acid and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol 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.
0.01mol of Rink Amide-MBHA resin (the substitution value is about 0.4mmol/g) is taken, and is deprotected by 20 percent PIP/DMF solution for 25 minutes, and the resin after Fmoc removal is obtained by washing and filtering.
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 9 th protected amino acid is inoculated
And sequentially inoculating the corresponding 2 nd to 9 th protected amino acids by adopting the same method to obtain the Setmelanotide peptide resin.
Example 2 Synthesis of Setmelanotide peptide resin
The Setmelanotide peptide resin is:
Ac-Arg (Pbf) -Cys (Trt) -D-Ala-His (Trt) -D-Phe-Arg (Pbf) -Trp-Cys (Trt) -amino resin
The peptide resin was prepared in the same manner as in example 1.
The protected amino acids used in this example correspond to the protected amino acids shown in the table below:
| the peptide sequence n ═ | Protected amino acids |
| 1 | Fmoc-Cys(Trt) |
| 2 | Fmoc-Trp |
| 3 | Fmoc-Arg(Pbf) |
| 4 | Fmoc-D-Phe |
| 5 | Fmoc-His(Trt) |
| 6 | Fmoc-D-Ala |
| 7 | Fmoc-Cys(Trt) |
| 8 | Fmoc-Arg(Pbf) |
| 9 | Ac2O |
Example 3 Synthesis of Setmelanotide peptide resin
The Setmelanotide peptide resin is:
Ac-Arg (Pbf) -Cys (Acm) -D-Ala-His (Trt) -D-Phe-Arg (Pbf) -Trp-Cys (Acm) -amino resin
The peptide resin was prepared in the same manner as in example 1.
The protected amino acids used in this example correspond to the protected amino acids shown in the table below:
| the peptide sequence n ═ | Protected amino acids |
| 1 | Fmoc-Cys(Acm) |
| 2 | Fmoc-Trp |
| 3 | Fmoc-Arg(Pbf) |
| 4 | Fmoc-D-Phe |
| 5 | Fmoc-His(Trt) |
| 6 | Fmoc-D-Ala |
| 7 | Fmoc-Cys(Acm) |
| 8 | Fmoc-Arg(Pbf) |
| 9 | Ac2O |
EXAMPLE 4 preparation of crude Setmelanotide
Taking the riteletanotide peptide resin prepared in example 1, adding a cleavage reagent (10 mL/g resin of the cleavage reagent) with the volume ratio of TFA, water and EDT (95: 5), uniformly stirring, stirring at room temperature for reacting for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting a filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, carrying out reduced pressure concentration, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying at 35-45 ℃ under reduced pressure to obtain white-like powder.
Dissolving the obtained off-white powder by using a 30% acetic acid solution to prepare a solution of about 3mg/ml, dropwise adding an iodine/ethanol saturated solution while stirring until complete cyclization is achieved, and concentrating under reduced pressure at 35-40 ℃ to obtain a concentrated solution of a crude Setmelactonide product, wherein the purity of the crude product is 88.3%.
EXAMPLE 5 preparation of crude Setmelanotide
Taking the riteletanotide peptide resin prepared in example 2, adding a cleavage reagent (10 mL/g resin of the cleavage reagent) with the volume ratio of TFA, water and EDT (95: 5), uniformly stirring, stirring at room temperature for reacting for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting a filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, carrying out reduced pressure concentration, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying at 35-45 ℃ under reduced pressure to obtain white-like powder.
Dissolving the obtained off-white powder by using a 30% acetic acid solution to prepare a solution of about 3mg/ml, dropwise adding an iodine/ethanol saturated solution while stirring until complete cyclization is achieved, and concentrating under reduced pressure at 35-40 ℃ to obtain a concentrated solution of a crude Setmelactonide product, wherein the purity of the crude product is 92.8%.
EXAMPLE 6 preparation of crude Setmelanotide
Taking the riteletanotide peptide resin prepared in example 3, adding a cleavage reagent (10 mL/g resin of the cleavage reagent) with the volume ratio of TFA, water and EDT (95: 5), uniformly stirring, stirring at room temperature for reacting for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting a filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, carrying out reduced pressure concentration, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying at 35-45 ℃ under reduced pressure to obtain white-like powder.
Dissolving the obtained white-like powder by using 30% acetic acid solution to prepare about 3mg/ml solution, dropwise adding iodine/ethanol saturated solution under stirring until complete cyclization, and concentrating under reduced pressure at 35-40 ℃ to obtain a concentrated solution of a crude Setmelactone product, wherein the purity of the crude product is 90.7%.
Example 7 purification of crude Setmelanotide
Filtering the Setmelanotide crude product concentrated solution prepared in the embodiment 4 by using a 0.45-micron mixed microporous 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 by 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 a filter membrane of 0.45 mu m is used for filtration, so that a concentrated solution of a purified intermediate of Setmelanottide is obtained;
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 90 mL/min; 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 Setmelanotide acetic acid aqueous solution, and freeze-drying to obtain a pure Setmelanotide product 6.1g, wherein the purity is 99.1%, the maximum single impurity is 0.11%, the total yield is 54.6%, and the molecular weight is 1117.8 (100% M + H).
EXAMPLE 8 purification of crude Setmelanotide
Taking the concentrated solution of the Setmelanotide crude product prepared in the embodiment 5, filtering the concentrated solution by using a 0.45-micron mixed microporous 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 by 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 a filter membrane of 0.45 mu m is used for filtration, so that a concentrated solution of a purified intermediate of Setmelanottide is obtained;
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 90 mL/min; 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 Setmelanotide acetic acid aqueous solution, and freeze-drying to obtain a pure Setmelanotide product 5.6g, wherein the purity is 99.5%, the maximum single impurity is 0.05%, the total yield is 50.1%, and the molecular weight is 1117.4 (100% M + H).
Example 9 purification of crude Setmelanotide
Filtering the Setmelanotide crude product concentrated solution prepared in the embodiment 6 by using a 0.45-micron mixed microporous 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 by 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 a filter membrane of 0.45 mu m is used for filtration, so that a concentrated solution of a purified intermediate of Setmelanottide is obtained;
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 90 mL/min; 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 Setmelanotide acetic acid aqueous solution, and freeze-drying to obtain a pure Setmelanotide product 5.8g, wherein the purity is 99.3%, the maximum single impurity is 0.10%, the total yield is 51.9%, and the molecular weight is 1117.6 (100% M + H).
The above examples show that the purity of the product obtained by the method provided by the invention is more than 99.0%, the total yield of the product is more than 50%, and the method has wide practical value and application prospect.
Claims (7)
1. A method for preparing Setmelanotide, comprising: taking amino resin as starting resin, sequentially inoculating corresponding protective amino acids according to the reverse phase of the amino sequence of Setmelactone to obtain Setmelactone peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelactone protective peptide resin to obtain a Setmelactone crude product, and purifying and freeze-drying the Setmelactone crude product to obtain a Setmelactone pure product:
Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2。
2. the method for producing Setmelantotide according to claim 1, characterized in that: the substitution value of the amino resin is 0.3-1.5 mmol/g resin, and the preferable substitution value is 0.6-1.0 mmol/g resin.
3. The amino resin according to claim 2 is selected from Rink Amide-MBHA resin, Rink Amide-BHA resin and Rink Amide resin, preferably Rink Amide-MBHA resin.
4. The method for producing Setmelantotide according to claim 1, characterized in that: the Setmelanotide peptide resin comprises the following components:
Ac-Arg (Pbf) -Cys (R1) -D-Ala-His (Trt) -D-Phe-Arg (Pbf) -Trp (R2) -Cys (R1) -amino resin
Wherein: r1 is Trt or Acm
R2 is Boc or H.
5. The method for producing Setmelantotide according to claim 1, characterized in that: the dosage of the Fmoc-protected amino acid is 1.2-6 times of the total mole number of the amino groups of the initial resin; preferably 2.5 to 4.0 times.
6. The method for producing Setmelanotide according to any one of claims 1 to 5, wherein:
the Setmelactone peptide resin is subjected to acidolysis, resin and side chain protecting groups are removed at the same time, and then a Setmelactone crude product is obtained after oxidation.
7. The method for producing Setmelantotide according to claim 1, characterized in that: and purifying the crude product of Setmelanotide by high performance liquid chromatography and freeze-drying to obtain a pure product of Setmelanotide.
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| CN113929763A (en) * | 2021-11-22 | 2022-01-14 | 申联生物医药（上海）股份有限公司 | Method for preparing semenotide by using soluble label as carrier |
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| US20200115416A1 (en) * | 2018-08-16 | 2020-04-16 | Regents Of The University Of Minnesota | Cyclic peptides and methods of use thereof |
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| CN1809372A (en) * | 2003-06-19 | 2006-07-26 | 伊莱利利公司 | Melanocortin recptor 4(MC4) agonists and their uses |
| CN105518021A (en) * | 2013-03-15 | 2016-04-20 | 节奏制药公司 | Pharmaceutical compositions |
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| US20200115416A1 (en) * | 2018-08-16 | 2020-04-16 | Regents Of The University Of Minnesota | Cyclic peptides and methods of use thereof |
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| CN113929763B (en) * | 2021-11-22 | 2024-01-30 | 申联生物医药（上海）股份有限公司 | Method for preparing semenopeptide by using soluble label as carrier |
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