A method for synthesizing carbetocin using special amino acids

Abstract

The invention relates to a method for synthesizing carbetocin using special amino acids, which mainly solves the technical problem that the current synthesis method is not suitable for industrial production. The invention comprises the following steps: synthesizing a peptide fragment (Fmoc-Cys((CH2)3COOH)-Pro-Leu-Gly-NH2) and a peptide resin (NH2-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-CTC Resin) containing special cysteine ​​by a standard Fmoc solid phase peptide synthesis method; coupling the peptide fragment with the peptide resin to obtain a fully protected linear carbetocin peptide resin, and then removing Fmoc and fully protecting cleavage to obtain a linear carbetocin with a protecting group in the side chain; and obtaining carbetocin after intramolecular amide bond cyclization and deprotection cleavage. The method is conducive to the large-scale industrial production of carbetocin.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drug preparation, and particularly relates to a method for synthesizing carbetocin by using special amino acids. Background Art

[0002] Carbetocin is a synthetic, long-acting oxytocin cyclic nonapeptide analog with agonist properties. It is effective in preventing uterine atony and postpartum hemorrhage. It was developed by Ferring Inc. of Canada. The clinical pharmacological properties of carbetocin are similar to those of oxytocin. Pharmacological research results show that after entering the human body, carbetocin can bind to the oxytocin body of uterine smooth muscle, induce rhythmic contraction of the uterus, and strengthen the contraction frequency and uterine tension on the basis of the original contraction of the uterus, thereby improving the symptoms of postpartum hemorrhage in parturients.

[0003] Studies have shown that, compared with oxytocin, which can only promote the contraction of the upper part of the uterus but has a poor effect on the lower part, carbetocin can have a good contraction effect on the entire uterus. In addition, carbetocin also has the characteristics of long half-life, single administration, long maintenance time, fewer side effects, and better efficacy.

[0004] Carbetocin has the following structure:

[0005] The amino acid abbreviations of carbetocin are: .

[0006] Most commonly used synthesis methods currently form a ring structure by constructing a thioether bond under alkaline conditions in the last step. However, the synthesis of thioether bonds often requires the use of strong alkaline reagents and a long reaction time, which ultimately leads to a significant reduction in yield and purity, greatly limiting industrial large-scale production.

[0007] Chinese patent CN102260326A describes a fully solid phase preparation method of carbetocin, which uses a novel protected amino acid Fmoc-Cys ((CH2)3COOAll), overcoming the shortcomings of the prior art. However, when removing the All protecting group, the use of Pd[P(C6H5)3]4 and phenylsilane will cause catalyst poisoning, and the product will contain heavy metal substances that are difficult to remove, resulting in the actual yield of the product being much lower than the theoretical yield.

[0008] Chinese patent CN102796178A describes a method for preparing carbetocin, which uses a novel thiol-protected amino acid Fmoc-Cys ((CH2)3COOCH2CH2CN) as a raw material. The raw material is expensive, and the Fmoc-Cys ((CH2)3COOCH2CH2CN) protection has many side reactions, which is not conducive to large-scale production.

[0009] Chinese patent CN113801199A describes a method for preparing carbetocin in a solid phase, which uses 4-mercaptobutyric acid with a thiol group protected to obtain a linear peptide resin with a terminal thiol group protected. After removing the 4-mercaptobutyric acid thiol protecting group and the serine hydroxyl protecting group, under the action of PPh3 / DEAD, the terminal thiol of the nonapeptide fragment resin and the serine side chain hydroxyl undergo Mitsunobu reaction dehydration to form a ring to obtain a carbetocin peptide resin. The reaction can be carried out under very mild conditions, usually at a reaction temperature of 0°C to room temperature, and most groups will not affect the reaction. The DIAD and DEAD used in the Mitsunobu reaction are sensitive to light, heat and strong impact, and are prone to explosion. Therefore, it is generally required to be stored in a dry place at low temperature and away from light, which also greatly limits industrial large-scale preparation.

[0010] European patent ES2115543 describes a solid-liquid synthesis method, which mainly uses conventional solid-phase peptide synthesis to obtain linear carbetocin and then cyclizes it under liquid alkaline conditions. However, during the liquid-phase oxidation process, LiOH, NaHCO3 and DMAP raw materials are used, which will lead to the generation of many impurities, reducing the purity and use effect of the product. In addition, the method is difficult to form intramolecular sulfide bonds, but disulfide bonds between molecules are very easy to form, resulting in extremely low crude product yields, which is not conducive to industrial production. Summary of the invention

[0011] The present invention aims to overcome the above problems and provide a method for synthesizing carbetocin using special amino acids. The method has simple and easy-to-obtain raw materials, no racemization, high yield and high purity, short synthesis time, and is conducive to large-scale industrial production of carbetocin.

[0012] A method for synthesizing carbetocin using special amino acids, characterized in that it specifically comprises the following steps: (1) Using Rink amide MBHA resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of the peptide fragment and the special amino acid Fmoc-Cys((CH2)3COOtBu)-OH containing a thioether bond were sequentially connected through standard Fmoc solid-phase peptide synthesis, and finally the peptide fragment, Fmoc-Cys((CH2)3COOH)-Pro-Leu-Gly-NH2, was obtained by cleavage using a cleavage buffer.

[0013] (2) Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of the peptide resin are sequentially connected through standard Fmoc solid phase peptide synthesis, and finally the Fmoc protecting group is removed in a solvent to obtain a peptide resin with a side chain protecting group and a free amino group at the N-terminus, NH2-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-CTC resin, which is then coupled with the peptide fragment obtained in step (1) to obtain a fully protected linear carbetocin peptide resin, which is then removed from Fmoc and fully protected in a solvent. After concentration, linear carbetocin with a protecting group on the side chain is obtained.

[0014] (3) The crude linear carbetocin peptide containing a side chain protecting group is condensed and cyclized in a liquid phase, and the crude carbetocin peptide is obtained after deprotection, purification, and lyophilization to obtain carbetocin.

[0015] Preferably, the cleavage solution in step (1) and the deprotection reagent in step (3) are TFA (volume concentration of 90-98%), and the reagent used for full protection cleavage in step (2) is HFIP with a volume concentration of 20% or 1% TFA solution, preferably HFIP with a volume concentration of 20%, and the solvent is DCM.

[0016] Preferably, the solution used to remove Fmoc in step (2) is a piperidine solution with a volume concentration of 20%, and the solvent is DMF or DCM, preferably DMF.

[0017] Preferably, the condensation reagent used in the synthesis of the fragments in steps (1) and (2) and the condensation of the fragments in step (3) is one of DIC, DCC, EDCI, HATU, TBTU, HCTU, HBTU, PyOxim, PyAop or PyBop, the auxiliary agent used in the condensation is one of HOAt, HOBt, Cl-HOBt, DIEA, Oxyma or NMM, and the solvent used in the condensation is DMF; the amounts of the components used in the coupling are calculated on a molar ratio of protected amino acid / peptide fragment: condensation reagent: auxiliary agent = 1: 1: (1-2), the preferred condensation reagent / auxiliary agent for amino acid condensation is DIC / Oxyma, protected amino acid: DIC: Oxyma = 1: 1.2: 1, and the preferred condensation reagent / auxiliary agent for peptide fragment condensation is HATU / DIEA, peptide fragment: HATU: DIEA = 1: 1: 2.

[0018] The beneficial effects of the present invention include: using a special amino acid molecule containing a thioether bond and being economically available, avoiding the problems of low yield and long time of thioether bond cyclization in a liquid phase, avoiding the use of special cysteine ​​and expensive deprotection metal reagents, no amino acid racemization occurs during the synthesis process, shortening the synthesis time, improving the synthesis yield and purity, and facilitating the large-scale industrial production of carbetocin. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the liquid chromatogram of crude carbetocin peptide.

[0020] Figure 2 This is the mass spectrum of the crude carbetocin peptide. DETAILED DESCRIPTION

[0021] Below in conjunction with the embodiment of the present invention, a method for synthesizing carbetocin with special amino acids is described in detail. The synthetic route is as follows:

[0022] Example 1: Synthesis of peptide fragment (Fmoc-Cys((CH2)3COOH)-Pro-Leu-Gly-NH2) Weigh Rink amide MBHA resin (1 mmol, 0.68 mmol / g) and place it in a peptide solid phase synthesizer. Add an appropriate amount of DMF / DCM (1 / 1 volume) to swell. After the resin is completely swollen, remove the solvent, add 20% piperidine to remove the Fmoc protecting group, and react for 5 min (repeat twice). The peptide sequence was synthesized using Fmoc-SPPS, and the amino acids were coupled under the conditions of Fmoc-AA-OH (2mmol, 2equiv.), DIC (2.2 mmol, 2.2 equiv.) and Oxyma (2 mmol, 2 equiv.). Special amino acids were coupled using Fmoc-Cys((CH2)3COOtBu)-OH (2 mmol, 2 equiv.), DIC (2.4 mmol,2.4 equiv.) and Oxyma (2 mmol, 2 equiv.). After solid phase synthesis, the obtained peptide resin was cleaved with TFA / TIS / H2O (95 / 2.5 / 2.5 by volume) cleavage solution (15 mL) at room temperature for 2 h, precipitated with cold methyl tert-butyl ether, centrifuged, dissolved in 50% acetonitrile by volume, and freeze-dried to obtain a powdered crude peptide fragment, Fmoc-Cys((CH2)3COOH)-Pro-Leu-Gly-NH2.

[0023] Example 2: Synthesis of peptide resin (NH2-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-CTC resin) Weigh 2-Cl-CTC resin (1 mmol, 0.8 mmol / g) and place it in a peptide solid phase synthesizer. Add an appropriate amount of DMF / DCM (1 / 1 volume) to swell. After the resin is completely swollen, remove the solvent, and couple the first amino acid to the resin using Fmoc-Asn(Trt)-OH (2mmol, 2 equiv.) and DIEA (4 mmol, 4 equiv.). After 3 h of reaction, the resin is washed four times with DMF, and the unloaded resin is blocked with a DMF solution containing 5% MeOH and 10% DIEA by volume for 0.5 h. Other amino acids were coupled using Fmoc-AA-OH (2 mmol, 2 equiv.), DIC (2.2 mmol, 2.2 equiv.), and Oxyma (2 mmol, 2 equiv.) to obtain the peptide resin, NH2-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-CTC Resin.

[0024] Example 3: Referring to the synthetic route, the coupling agents for the condensation of amino acid (2.2 mmol, 2.2 equiv.) were HCTU (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest were the same as in Example 1 or Example 2.

[0025] Example 4: Referring to the synthetic route, the coupling agents for the condensation of amino acid (2.2 mmol, 2.2 equiv.) were PyBOP (2 mmol, 2 equiv.) and DIEA (3.2 mmol, 3.2 equiv.), and the rest were the same as in Example 1 or Example 2.

[0026] Example 5: Synthesis of Crude Linear Carbetocin Peptide The peptide fragment obtained in Example 1 (904 mg, 1.3 mmol, 1.3 equiv.) was coupled with the above-mentioned peptide resin in an appropriate amount of DMF at room temperature for 2 h in the presence of coupling agents HATU (1.3 mmol, 1.3 equiv.) and DIEA (2.6 equiv.), and washed three times with DMF and DCM respectively and dried in vacuo. The obtained crude peptide resin was fully protected and cleaved in DCM (20 mL) containing 20% ​​HFIP, and 1.39 g of powdered linear carbetocin crude peptide with protected groups was obtained after concentration.

[0027] Example 6: Referring to the synthetic route, the coupling agents between fragment I and the peptide resin were PyOxim (1.3 mmol, 1.3 equiv.) and DIEA (2.6 mmol, 2.6 equiv.), and the rest were the same as in Example 5.

[0028] Example 7: Referring to the synthetic route, the coupling agents between fragment I and the peptide resin were PyBOP (1.3 mmol, 1.3 equiv.) and DIEA (2.6 mmol, 2.6 equiv.), and the rest were the same as in Example 5.

[0029] Example 8: Synthesis of Crude Carbetocin Peptide The crude linear carbetocin peptide (1.39 g, 0.94 mmol) obtained in Example 5 containing a protective group was dissolved in DMF (15. mL, 0.06 M), and HATU (357 mg, 0.94 mmol, 1 equiv.) and DIEA (329 uL, 1.88 mmol, 2 equiv.) were added, and the mixture was allowed to react at room temperature. After the reaction was completed, the reaction solution was added to ice brine to obtain a white solid, which was centrifuged, and the upper clear brine was discarded. Clean water was added again for washing, precipitation, and centrifugation (repeated 2 times), and the crude carbetocin peptide with a side chain protective group was obtained after the solid was dried. The mixture was treated with 20 mL of lysis solution TFA / TIS / H2O (95 / 2.5 / 2.5 volume), and filtered after lysis for 1.5 h. The mixture was precipitated with methyl tert-butyl ether, centrifuged, purified, and freeze-dried to obtain 473 mg of crude carbetocin peptide with a purity greater than 99%. The total yield of Examples 5 and 8 was 47.9%. The final product was detected by HPLC-MS (solvent A: volume concentration 0.1% TFA / H2O, solvent B: volume concentration 0.1% TFA / MeCN, chromatographic column was C18 reverse phase column, gradient elution was used (method: 5-65% B, elution time 30 min, elution flow rate 1.0 mL / min, detection wavelength 214 nm, column temperature 25°C). Liquid chromatography of crude carbetocin peptide is shown in Figure 1 , mass spectrometry see Figure 2 .

[0030] The following are the full names of the chemicals involved in the present invention: Abbreviations Representative meaning Fmoc Fmoc tBu Tert-butyl Trt Trityl Me methyl DIC -Diisopropylcarbodiimide HFIP Hexafluoroisopropanol TFA Trifluoroacetic acid TIS Triisopropylsilane DCC Dicyclohexylcarbodiimide DIC -Diisopropylcarbodiimide EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HBTU -Benzotriazole-tetramethyluronium hexafluorophosphate TBTU 2-(1H-Benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate HCTU -(6-Chloro-1H-benzotriazol-1-yl)-tetramethyluronium hexafluorophosphate PyOxim Cyano(hydroxyimino)acetate ethyl-O2]tri-1-pyrrolidinyl hexafluorophosphate PyAop (7-Azabenzotriazole-1-oxy)tripyrrolylphosphonium hexafluorophosphate PyBop Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate HOAt 1-Hydroxy-7-azabenzotriazole HOB 1-Hydroxybenzotriazole Cl-HOBt 6-Chloro-1-hydroxybenzotriazole Oxyma Ethyl 2-oximecyanoacetate NMM N-Methylmorpholine DIEA Diisopropylethylamine DMAP 4-Dimethylaminopyridine MeCN Acetonitrile DCM Dichloromethane DMF N,N-Dimethylformamide DIAD Diisopropyl azodicarboxylate DEAD Diethyl azodicarboxylate The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for synthesizing carbetocin using special amino acids, characterized in that: The specific steps include: (1) Using Rink amide MBHA resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of the peptide fragment and the special amino acid Fmoc-Cys((CH2)3COOtBu)-OH containing a thioether bond were sequentially connected through standard Fmoc solid-phase peptide synthesis, and finally the peptide fragment, Fmoc-Cys((CH2)3COOH)-Pro-Leu-Gly-NH2, was obtained by cleavage with a cleavage buffer; (2) Using 2-Cl-CTC resin as a carrier, the corresponding Fmoc-protected amino acids in the amino acid sequence of the peptide resin are sequentially connected through standard Fmoc solid-phase peptide synthesis, and finally the Fmoc protecting group is removed to obtain a peptide resin with a side chain protecting group and a free amino group at the N-terminus, NH2-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-CTC resin, which is then coupled with the peptide fragment obtained in step (1) to obtain a fully protected linear carbetocin peptide resin, which is then removed from Fmoc and fully protected and cleaved, and concentrated to obtain a linear carbetocin with a side chain protecting group; (3) The crude linear carbetocin peptide containing a side chain protecting group is condensed and cyclized in a liquid phase, and the crude carbetocin peptide is obtained after deprotection, purification, and freeze-drying to obtain carbetocin.

2. A method for synthesizing carbetocin using special amino acids according to claim 1, characterized in that: The cleavage solution in step (1) and the deprotection reagent in step (3) are TFA with a volume concentration of 90-98%, and the reagent used for the full protection cleavage in step (2) is HFIP with a volume concentration of 20% or 1% TFA solution, and the solvent is DCM.

3. A method for synthesizing carbetocin using special amino acids according to claim 1, characterized in that: The solution used for removing Fmoc in step (2) is a piperidine solution with a volume concentration of 20%, and the solvent is DMF or DCM.

4. A method for synthesizing carbetocin using special amino acids according to claim 1, characterized in that: The condensation reagent used in the synthesis of the fragments in steps (1) and (2) and the condensation of the fragments in step (3) is one of DIC, DCC, EDCI, HATU, TBTU, HCTU, HBTU, PyOxim, PyAop or PyBop, the auxiliary agent used in the condensation is one of HOAt, HOBt, Cl-HOBt, DIEA, Oxyma or NMM, and the solvent is DMF.

5. The method for synthesizing carbetocin using special amino acids according to claim 1, characterized in that: During coupling, the amount of each component is calculated on a molar ratio of protected amino acid / peptide fragment: condensation reagent: auxiliary agent = 1: 1: (1~2).

6. The method for synthesizing carbetocin using special amino acids according to claim 5, characterized in that: The condensation reagent / auxiliary agent for amino acid condensation is DIC / Oxyma, and the protected amino acid: DIC:Oxyma=1:1.2:1; the condensation reagent / auxiliary agent for peptide fragment condensation is HATU / DIEA, and the peptide fragment: HATU:DIEA=1:1:2.

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