CN114014912B - Method for preparing leuprorelin by combining solid phase and liquid phase - Google Patents

Method for preparing leuprorelin by combining solid phase and liquid phase Download PDF

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CN114014912B
CN114014912B CN202210002734.8A CN202210002734A CN114014912B CN 114014912 B CN114014912 B CN 114014912B CN 202210002734 A CN202210002734 A CN 202210002734A CN 114014912 B CN114014912 B CN 114014912B
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liquid phase
pro
arg
phase carrier
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CN114014912A (en
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章砚东
涂仕前
魏祝宇
潘海良
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Zhejiang Paitide Biological Co ltd Nanjing Branch
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Zhejiang Pai Peptide Biology Co ltd Nanjing Branch
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    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides

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Abstract

The invention discloses a method for preparing leuprorelin by combining a solid phase and a liquid phase, belongs to the technical field of polypeptide synthesis, and particularly relates to a liquid phase carrier and application thereof, wherein the liquid phase carrier is obtained by reacting 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) bromoethane in DMF (dimethyl formamide) containing potassium carbonate, and the liquid phase carrier is adopted to carry out fragment synthesis in a liquid phase; synthesizing the fragment to obtain an Fmoc-Arg-Pro-O-liquid phase carrier; removing the liquid phase carrier from the product obtained in the step, combining the product with solid phase resin, and then coupling amino acid reagents according to the sequence of amino acid sequences in leuprorelin to obtain leuprorelin-based polypeptide resin; the leuprorelin-based polypeptide is separated after the leuprorelin-based polypeptide resin is cut, and then the leuprorelin is obtained after ethylation treatment.

Description

Method for preparing leuprorelin by combining solid phase and liquid phase
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for preparing leuprorelin by combining a solid phase and a liquid phase.
Background
Leuprorelin is a gonadotropin-releasing hormone analogue which is widely used in the treatment of female patients such as precocity, endometriosis, uterine fibroids or pre-menopausal breast cancer and male patients such as prostate cancer. Leuprorelin has the chemical name 5-oxo-prolyl-histidyl-tryptophyl-seryl-tyrosyl-D-leucyl-arginyl-N-ethyl-prolinamide, the peptide sequence: H-Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NH-C2H5Molecular weight 1209.41. In the existing solid-phase synthesis method for synthesizing leuprorelin, after all amino acids are connected from a carbon end, ethylamine liquid is used for cutting off from resin, however, the cutting off by using liquid ethylamine needs to be carried out at low temperature under pressure sealing, is inconvenient in industrial production, and is difficult to produce on a large scale. The development of a preparation method of leuprorelin still has important significance.
Disclosure of Invention
The invention aims to provide a liquid phase carrier for synthesizing leuprorelin.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a liquid carrier comprising: the compound is prepared by reacting 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) ethyl bromide in DMF (dimethyl formamide) containing potassium carbonate and then reducing by using a reducing agent, wherein the reducing agent is sodium borohydride.
Preferably, in the preparation of the liquid phase carrier, potassium carbonate is added into DMF, then 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) bromoethane are added, nitrogen protection is performed, stirring reaction is performed for 18-48h at the temperature of 60-80 ℃, deionized water is added to separate out a precipitate, and the precipitate is filtered, washed and dried to obtain a benzaldehyde compound; adding benzaldehyde compounds into tetrahydrofuran solution under the protection of nitrogen, adding sodium borohydride, stirring and reacting at the temperature of 30-50 ℃ for 2-10h, adding deionized water into the solution after the reaction is finished to separate out a precipitate, filtering, washing and drying to obtain the liquid-phase carrier.
More preferably, potassium carbonate is used in an amount of 0.6-3 wt% of DMF.
More preferably, 3-ethoxy-4-hydroxybenzaldehyde is added in an amount of 2-9 wt% of DMF.
More preferably, 2- (2-ethoxyphenoxy) bromoethane is added in an amount of 3 to 12 wt% of DMF.
More preferably, the tetrahydrofuran solution is formed by mixing tetrahydrofuran and methanol, and the content of methanol in the tetrahydrofuran solution is 4-16 wt%.
More preferably, the benzaldehyde compound is used in an amount of 1 to 4 wt% based on the tetrahydrofuran solution.
More preferably, sodium borohydride is used in an amount of 10 to 50 wt% of the benzaldehyde compound.
The invention aims to provide a method for preparing leuprorelin by solid-liquid combination.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a process for preparing leuprolide comprising:
carrying out fragment synthesis in a liquid phase by adopting the liquid phase carrier; synthesizing the fragment to obtain an Fmoc-Arg-Pro-O-liquid phase carrier;
removing the liquid phase carrier from the Fmoc-Arg-Pro-O-liquid phase carrier, combining the Fmoc-Arg-Pro-O-liquid phase carrier with solid phase resin, and then coupling amino acid reagents according to the sequence of amino acid sequences in leuprorelin to obtain leuprorelin-based polypeptide resin;
the leuprorelin-based polypeptide is separated after the leuprorelin-based polypeptide resin is cut, and then the leuprorelin is obtained after ethylation treatment. According to the invention, 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) bromoethane react in DMF (dimethyl formamide) containing potassium carbonate, then are reduced by sodium borohydride to obtain a liquid phase carrier, an Arg-Pro fragment is synthesized by the liquid carrier, then the Arg-Pro fragment is coupled with a solid phase carrier, and then the polypeptide is obtained by continuously adopting a solid phase synthesis method, so that diketopiperazine side reaction can be avoided, the defect of long liquid phase synthesis time is combined with the advantage of rapid solid phase synthesis, leuprorelin is successfully prepared, a new method is provided for leuprorelin synthesis, and the yield of leuprorelin is ensured.
Preferably, the Fmoc-Arg-Pro-O-liquid carrier is prepared from Fmoc-Arg (Pbf) -OSu and Fmoc-Pro-O-liquid carrier.
Preferably, the solid phase resin is any one of Wang resin and Rink-Amide-AM resin.
Preferably, the amino acid reagent is activated for 3-30 min by DMF solution containing HOBt, DIC.
Preferably, the coupling sequence of the amino acid reagents is Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH, H-Pyr-OH.
Preferably, the cleavage is performed with TFA, EDT, H2O, TIS and a cleaving agent.
Preferably, the ethylation treatment is carried out by reacting ethylamine with the leuprolide-based polypeptide in the presence of HOBt and DIC.
Preferably, in the preparation of the Fmoc-Pro-O-liquid phase carrier, the liquid phase carrier is added into dichloromethane, stirred and dissolved, mixed uniformly, then Fmoc-Pro-OH, DIC and DMAP are added, stirred and reacted for 30-90min at the temperature of 20-40 ℃, acetonitrile is added for crystallization, and then the Fmoc-Pro-O-liquid phase carrier is obtained after filtration.
More preferably, in the preparation of Fmoc-Pro-O-liquid phase carrier, the liquid phase carrier is used in an amount of 10-20 wt% based on dichloromethane.
More preferably, Fmoc-Pro-O-OH is used in an amount of 10 to 40 wt% based on dichloromethane in the preparation of the Fmoc-Pro-O-liquid phase carrier.
More preferably, DIC is used in an amount of 3 to 10 wt% based on dichloromethane in the preparation of Fmoc-Pro-O-liquid phase carrier.
More preferably, DMAP is used in an amount of 5-15 wt% of dichloromethane in the preparation of the Fmoc-Pro-O-liquid phase support.
Preferably, in the deprotection treatment, the Fmoc-Pro-O-liquid phase carrier is added into THF, piperidine and DBU are added, stirring treatment is carried out for 5-20min at the temperature of 20-40 ℃, the pH is adjusted to be neutral, a product is precipitated and filtered, and the H-Pro-O-liquid phase carrier is obtained.
More preferably, the Fmoc-Pro-O-liquid phase carrier is used in an amount of 10 to 20 wt% of THF in the deprotection treatment.
More preferably, in the deprotection treatment, piperidine is used in an amount of 7 to 21 wt% based on THF.
More preferably, DBU is used in an amount of 3 to 12 wt% of THF in the deprotection treatment.
Preferably, in the preparation of Fmoc-Arg (Pbf) -OSu, tetrahydrofuran and dicyclohexylcarbodiimide are added into Fmoc-Arg (Pbf) -OH and HOSu for reaction, and the filtrate is obtained by solid-liquid separation, namely the product Fmoc-Arg (pbf) -OSu.
More preferably, Fmoc-Arg (Pbf) -OSu is prepared in which Fmoc-Arg (pbf) -OH is used in an amount of 4 to 21 wt% of tetrahydrofuran.
More preferably, HOSu is used in an amount of 3-21 wt% of tetrahydrofuran in the preparation of Fmoc-Arg (Pbf) -OSu.
More preferably, in the preparation of Fmoc-Arg (Pbf) -OSu, dicyclohexylcarbodiimide is used in an amount of 4 to 26 wt% based on tetrahydrofuran.
Preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier, the H-Pro-O-liquid phase carrier is added into dichloromethane, stirred and dissolved, mixed uniformly, then added with Fmoc-Arg (Pbf) -OSu, DIC and DMAP, stirred and reacted for 30-90min at the temperature of 20-40 ℃, added with acetonitrile for crystallization, and filtered to obtain Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier.
More preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier, H-Pro-O-liquid phase carrier is used in an amount of 5-20 wt% based on methylene chloride.
More preferably, Fmoc-Arg (Pbf) -Pro-O-liquid carrier is prepared in an amount of 80-120 wt% of H-Pro-O-liquid carrier.
More preferably, DIC is used in an amount of 3-10 wt% based on dichloromethane in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase support.
More preferably, DMAP is used in an amount of 5-15 wt% of dichloromethane in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase support.
Preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-OH, Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier is added into dichloromethane, stirred and dissolved, mixed uniformly, then TFE and TFA are added, stirring reaction is carried out for 20-60 min at the temperature of 20-40 ℃, diatomite is filtered, isopropyl ether is added into filtrate to precipitate, and then the precipitate is filtered, washed and dried to obtain Fmoc-Arg (Pbf) -Pro-OH.
More preferably, Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier is used in an amount of 6 to 20 wt% based on methylene chloride in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier.
More preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase support, TFE is used in an amount of 7-14 wt% based on methylene chloride.
More preferably, Fmoc-Arg (Pbf) -Pro-O-liquid support is prepared using TFA in an amount of 0.4 to 1.6 wt% of dichloromethane.
Preferably, in the Wang resin pretreatment, the Wang resin is put into a reaction column, added into DCM for treatment for 1-4 h, and drained to obtain the pretreated Wang resin.
More preferably, DCM is used in an amount of 200-500 wt.% of the Wang resin in the pretreatment of the Wang resin.
Preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin, Fmoc-Arg (Pbf) -Pro-OH and HOBt are added into DMF, stirred and dissolved, DIC is added under ice water bath for activation for 3-10 min to obtain an activated Fmoc-Arg (Pbf) -Pro-OH reagent, the activated Fmoc-Arg (Pbf) -Pro-OH reagent is added into a pretreated Wang resin reaction column, the reaction is carried out for 3-12 h at the temperature of 20-40 ℃, reaction liquid is pumped out after the reaction is finished, and the reaction liquid is washed by DMF, absolute methanol and DCM in sequence and pumped out to obtain the Fmoc-Arg (Pbf) -Pro-Wang resin.
More preferably, Fmoc-Arg (Pbf) -Pro-Wang resin is used in an amount of 10-20 wt% of DMF in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin.
More preferably, HOBt is used in an amount of 5-15 wt% of DMF in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin.
More preferably, DIC is used in an amount of 3-10 wt% based on DMF in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin.
More preferably, in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin, the activated Fmoc-Arg (Pbf) -Pro-OH reagent is used in an amount of 150-450 wt% of Wang resin.
More preferably, 2- [ (3-aminophenyl) sulfonyl) ethanol may be added in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin. 2- [ (3-aminophenyl) sulfonyl) ethanol was used in an amount of 0.6 to 2.4% by weight based on DMF. In the step of preparing Fmoc-Arg (Pbf) -Pro-Wang resin, using 2- [ (3-aminophenyl) sulfonyl) ethanol, it was found that a higher yield of leuprolide could be finally obtained.
Preferably, in the preparation of the H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin, adding a deprotection reagent into a reaction column of the Fmoc-Arg (Pbf) -Pro-Wang resin for Fmoc removal treatment for 5-30 min, draining, adding an activated Fmoc-Leu-OH reagent for coupling reaction for 2-6H, then draining the reaction liquid, washing with DMF, anhydrous methanol and DCM in sequence, and draining; Fmoc-D-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and H-Pyr-OH are coupled in sequence according to the deprotection operation and the activated amino acid coupling reaction to obtain H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin which is hereinafter referred to as peptide resin.
More preferably, in the preparation of the H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin, the deprotection reagent is DBLK solution, and the amount of the deprotection reagent is 150-300 wt% of the Wang resin. The DBLK solution is a mixed solution of piperidine and DMF, and the amount of the piperidine in the DBLK solution is 15-25 wt% of the DMF.
More preferably, in the preparation of the H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin, the amount of the activated Fmoc-Leu-OH reagent used is 150-450 wt% of the Wang resin.
Preferably, in the deprotection treatment, a deprotection reagent is added to the protecting group-peptide-Wang resin for treatment for 5 to 30 min.
Preferably, in the Fmoc-Leu-OH reagent activation treatment, Fmoc-Leu-OH and HOBt are added into DMF, and DIC is added to activate for 3-30 min under the ice-water bath condition.
More preferably, Fmoc-Leu-OH is used in an amount of 10-20 wt% of DMF in the treatment of activating Fmoc-Leu-OH reagent.
More preferably, HOBt is used in an amount of 5-15 wt% of DMF in the treatment of activating Fmoc-Leu-OH reagent.
More preferably, DIC is used in an amount of 3-10 wt% based on DMF in the step of activating Fmoc-Leu-OH reagent.
Preferably, the other amino acid reagents are activated in a manner comparable to that of the activated Fmoc-Leu-OH reagent, are used in the same or slightly higher molar amounts than the Fmoc-Leu-OH reagent, and are used in the same amounts as those used in the activated Fmoc-Leu-OH reagent.
Preferably, in the cleavage of the peptide resin, the peptide resin is transferred into a cleavage reagent under the condition of ice-water bath, the mixture is stirred for 0.5 to 3 hours at the temperature of 20 to 40 ℃, the resin is removed by filtration, cold ethyl ether is added into the filtrate for precipitation, and the polypeptide is obtained by centrifugal collection, washing and drying.
More preferably, the cleavage reagent is selected from TFA, EDT, H in the cleavage of the peptide resin2O, TIS, the amount of EDT in the cleavage reagent is 1.5-4.5 wt% of TFA, and the amount of H in the cleavage reagent is 1.5-4.5 wt%2O is used in an amount of 1-4 wt% based on TFA and TIS in the cleavage reagent is used in an amount of 0.03-0.09 wt% based on TFA.
Preferably, in the preparation of the leuprorelin, namely ethylation treatment, HOBt and ethylamine are added into deionized water, stirred and mixed, freeze-dried, polypeptide and DIC are added, stirring reaction is carried out at the temperature of 20-40 ℃ for 12-48 h, cold ethyl ether is added for precipitation, filtration and washing are carried out, a crude leuprorelin product is obtained, the crude leuprorelin product is dissolved in water to prepare a solution, the solution is purified by a C18 column, leuprorelin components are collected, and the leuprorelin is obtained after drying.
More preferably, in the preparation of leuprolide, HOBt is used in an amount of 0.5-2 wt% based on deionized water.
More preferably, in the preparation of leuprolide, ethylamine is used in an amount of 1-4 wt% of deionized water.
More preferably, in the preparation of leuprolide, the polypeptide is used in an amount of 0.4-2.0 wt% of ethylamine.
More preferably, in the preparation of leuprolide, DIC is used in an amount of 0.5 to 1.5 wt% of ethylamine.
The invention discloses application of the liquid phase carrier in liquid phase preparation of polypeptide and/or solid-liquid phase preparation of polypeptide.
According to the invention, 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) ethyl bromide are reacted in DMF (dimethyl formamide) containing potassium carbonate and then reduced by sodium borohydride to obtain a liquid phase carrier, then an Arg-Pro fragment is synthesized by the liquid phase carrier, the fragment is combined with solid phase resin, then amino acid reagents are sequentially coupled, after the coupling is completed, the synthesized polypeptide is cut off, and finally ethylation treatment is carried out to obtain the leuprorelin. Therefore, the following beneficial effects are achieved: provides a method for liquid phase synthesis of amino acid coupled fragments, and successfully prepares leuprorelin by solid-liquid phase combination; the problem of low synthesis yield caused by instability of products in all-solid-phase coupling is avoided. Therefore, the invention provides a method for preparing leuprorelin by combining solid phase and liquid phase with high synthesis yield.
Drawings
FIG. 1 is an infrared spectrum of a liquid phase carrier;
FIG. 2 is an HPLC chart of leuprolide;
FIG. 3 is a MS picture of leuprolide.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
a method for preparing leuprorelin by combining solid phase and liquid phase,
preparation of liquid phase carrier: adding potassium carbonate into DMF, adding 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) bromoethane, introducing nitrogen for protection, stirring and reacting at 70 ℃ for 24 hours, adding deionized water to precipitate, filtering, washing and drying to obtain a benzaldehyde compound; adding benzaldehyde compounds into a tetrahydrofuran solution under the protection of nitrogen, adding sodium borohydride, stirring and reacting at the temperature of 40 ℃ for 6 hours, adding deionized water into the solution after the reaction is finished to separate out a precipitate, filtering, washing and drying to obtain a liquid-phase carrier. The using amount of potassium carbonate is 1.2 wt% of DMF, the adding amount of 3-ethoxy-4-hydroxybenzaldehyde is 6wt% of DMF, the adding amount of 2- (2-ethoxyphenoxy) ethyl bromide is 9 wt% of DMF, a tetrahydrofuran solution is formed by mixing tetrahydrofuran and methanol, the content of methanol in the tetrahydrofuran solution is 8 wt%, the using amount of benzaldehyde compounds is 3 wt% of the tetrahydrofuran solution, and the using amount of sodium borohydride is 30 wt% of the benzaldehyde compounds.
Preparation of Fmoc-Pro-O-liquid phase carrier: adding the liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding Fmoc-Pro-OH, DIC and DMAP, stirring for reacting for 60 min at the temperature of 20-40 ℃, adding acetonitrile for crystallizing, and filtering to obtain the Fmoc-Pro-O-liquid phase carrier. The liquid phase carrier was used in an amount of 15 wt% of dichloromethane, Fmoc-Pro-OH was used in an amount of 20 wt% of dichloromethane, DIC was used in an amount of 7 wt% of dichloromethane, and DMAP was used in an amount of 10 wt% of dichloromethane.
Deprotection treatment: adding the Fmoc-Pro-O-liquid phase carrier into THF, adding piperidine and DBU, stirring at 30 deg.C for 10 min, adjusting pH to neutrality, precipitating product, and filtering to obtain H-Pro-O-liquid phase carrier. The Fmoc-Pro-O-liquid phase carrier was used at 15 wt% THF, piperidine at 14 wt% THF, and DBU at 8 wt% THF.
Preparation of Fmoc-Arg (Pbf) -OSu: tetrahydrofuran and dicyclohexylcarbodiimide are added into Fmoc-Arg (Pbf) -OH and HOSu for reaction, and filtrate is obtained through solid-liquid separation, namely the product Fmoc-Arg (pbf) -OSu. Fmoc-Arg (pbf) -OH was used at 12 wt% tetrahydrofuran, HOSu was used at 12 wt% tetrahydrofuran, and dicyclohexylcarbodiimide was used at 16wt% tetrahydrofuran.
Preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase Carrier: adding the H-Pro-O-liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding Fmoc-Arg (Pbf) -OSu, DIC and DMAP, stirring for reacting for 60 min at the temperature of 20-40 ℃, adding acetonitrile for crystallization, and filtering to obtain the Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier. The amount of H-Pro-O-liquid carrier used was 10 wt% of dichloromethane, the amount of Fmoc-Arg (Pbf) -OSu used was 100 wt% of H-Pro-O-liquid carrier, the amount of DIC used was 6wt% of dichloromethane, and the amount of DMAP used was 10 wt% of dichloromethane.
Preparation of Fmoc-Arg (Pbf) -Pro-OH: adding Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding TFE and TFA, stirring for reacting for 40 min at the temperature of 20-40 ℃, filtering by using kieselguhr, adding isopropyl ether into filtrate for precipitating, filtering, washing and drying to obtain Fmoc-Arg (Pbf) -Pro-OH. Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier was used in an amount of 12 wt% of dichloromethane, TFE was used in an amount of 10 wt% of dichloromethane, and TFA was used in an amount of 1.2 wt% of dichloromethane.
Pretreatment of Wang resin: putting the Wang resin into a reaction column, adding DCM for treatment for 3 h, and draining to obtain the pretreated Wang resin. DCM was used in an amount of 300 wt% of the Wang resin.
Preparation of Fmoc-Arg (Pbf) -Pro-Wang resin: adding Fmoc-Arg (Pbf) -Pro-OH and HOBt into DMF, stirring for dissolving, adding DIC for activating for 5 min in ice water bath to obtain activated Fmoc-Arg (Pbf) -Pro-OH reagent, adding into a pretreated Wang resin reaction column, reacting for 9 h at the temperature of 30 ℃, pumping out reaction liquid after the reaction is finished, washing with DMF, absolute methanol and DCM in sequence, and pumping out to obtain Fmoc-Arg (Pbf) -Pro-Wang resin. Fmoc-Arg (Pbf) -Pro-OH was used at 15 wt% of DMF, HOBt was used at 10 wt% of DMF, DIC was used at 7 wt% of DMF, and the activated Fmoc-Arg (Pbf) -Pro-OH reagent was used at 300 wt% of Wang resin.
Preparation of H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin: adding a deprotection reagent into a reaction column of Fmoc-Arg (Pbf) -Pro-Wang resin for Fmoc removal treatment for 20min, draining, adding an activated Fmoc-Leu-OH reagent for coupling reaction for 4 h, then draining reaction liquid, washing with DMF, anhydrous methanol and DCM in sequence, and draining; Fmoc-D-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and H-Pyr-OH are coupled in sequence according to the deprotection operation and the activated amino acid coupling reaction to obtain H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin which is hereinafter referred to as peptide resin. The deprotection reagent is DBLK solution, the DBLK solution is mixed liquid of piperidine and DMF, the amount of the piperidine in the DBLK solution is 20 wt% of the DMF, the usage amount of the deprotection reagent is 200 wt% of the Wang resin, and the usage amount of the activated Fmoc-Leu-OH reagent is 300 wt% of the Wang resin.
Deprotection treatment: the deprotection reagent was added to the protecting group-peptide-Wang resin for 20 min.
Activating Fmoc-Leu-OH reagent: adding Fmoc-Leu-OH and HOBt into DMF, and adding DIC to activate for 20min under the condition of ice water bath. The amount of Fmoc-Leu-OH used was 15 wt% of DMF, the amount of HOBt used was 10 wt% of DMF, and the amount of DIC used was 6wt% of DMF.
The other amino acid reagents are activated in a manner equivalent to that of the Fmoc-Leu-OH reagent, the other amino acid reagents are used in the same or slightly higher molar amount than the Fmoc-Leu-OH reagent, and the other reagents are used in the same amount as the activated Fmoc-Leu-OH reagent.
Cleavage of peptide resin: transferring the peptide resin into a cutting reagent under the condition of ice-water bath, stirring for 2 h at the temperature of 30 ℃, filtering to remove the resin, adding cold ether into filtrate for precipitation, centrifugally collecting, washing and drying to obtain the polypeptide. The cleavage reagent consists of TFA, EDT, H2O, TIS, the amount of EDT used in the cleavage reagent is 3.0 wt% of TFA, and the amount of H used in the cleavage reagent is 3.0 wt%2O was used in an amount of 2.5 wt% based on TFA and TIS in the cleavage reagent was used in an amount of 0.06 wt% based on TFA.
Preparation of leuprorelin: adding HOBt and ethylamine into deionized water, stirring and mixing, freeze-drying, adding polypeptide and DIC, stirring and reacting at the temperature of 30 ℃ for 24 hours, adding cold ether for precipitation, filtering, washing to obtain a crude leuprorelin product, dissolving in water to prepare a solution, purifying by a C18 column, collecting leuprorelin components, and drying to obtain the leuprorelin. The usage amount of HOBt is 1 wt% of deionized water, the usage amount of ethylamine is 2 wt% of deionized water, the usage amount of polypeptide is 1.2 wt% of ethylamine, and the usage amount of DIC is 0.9 wt% of ethylamine.
Example 2:
a method for preparing leuprorelin by combining solid phase and liquid phase,
preparation of liquid phase carrier: adding potassium carbonate into DMF, adding 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) bromoethane, introducing nitrogen for protection, stirring and reacting at 70 ℃ for 24 hours, adding deionized water to precipitate, filtering, washing and drying to obtain a benzaldehyde compound; adding benzaldehyde compounds into a tetrahydrofuran solution under the protection of nitrogen, adding sodium borohydride, stirring and reacting at the temperature of 40 ℃ for 6 hours, adding deionized water into the solution after the reaction is finished to separate out a precipitate, filtering, washing and drying to obtain a liquid-phase carrier. The using amount of potassium carbonate is 1.2 wt% of DMF, the adding amount of 3-ethoxy-4-hydroxybenzaldehyde is 6wt% of DMF, the adding amount of 2- (2-ethoxyphenoxy) ethyl bromide is 9 wt% of DMF, a tetrahydrofuran solution is formed by mixing tetrahydrofuran and methanol, the content of methanol in the tetrahydrofuran solution is 8 wt%, the using amount of benzaldehyde compounds is 3 wt% of the tetrahydrofuran solution, and the using amount of sodium borohydride is 30 wt% of the benzaldehyde compounds.
Preparation of Fmoc-Pro-O-liquid phase carrier: adding the liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding Fmoc-Pro-OH, DIC and DMAP, stirring for reacting for 60 min at the temperature of 20-40 ℃, adding acetonitrile for crystallizing, and filtering to obtain the Fmoc-Pro-O-liquid phase carrier. The liquid phase carrier was used in an amount of 15 wt% of dichloromethane, Fmoc-Pro-OH was used in an amount of 20 wt% of dichloromethane, DIC was used in an amount of 7 wt% of dichloromethane, and DMAP was used in an amount of 10 wt% of dichloromethane.
Deprotection treatment: adding the Fmoc-Pro-O-liquid phase carrier into THF, adding piperidine and DBU, stirring at 30 deg.C for 10 min, adjusting pH to neutrality, precipitating product, and filtering to obtain H-Pro-O-liquid phase carrier. The Fmoc-Pro-O-liquid phase carrier was used at 15 wt% THF, piperidine at 14 wt% THF, and DBU at 8 wt% THF.
Preparation of Fmoc-Arg (Pbf) -OSu: tetrahydrofuran and dicyclohexylcarbodiimide are added into Fmoc-Arg (Pbf) -OH and HOSu for reaction, and filtrate is obtained through solid-liquid separation, namely the product Fmoc-Arg (pbf) -OSu. Fmoc-Arg (pbf) -OH was used at 12 wt% tetrahydrofuran, HOSu was used at 12 wt% tetrahydrofuran, and dicyclohexylcarbodiimide was used at 16wt% tetrahydrofuran.
Preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase Carrier: adding the H-Pro-O-liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding Fmoc-Arg (Pbf) -OSu, DIC and DMAP, stirring for reacting for 60 min at the temperature of 20-40 ℃, adding acetonitrile for crystallization, and filtering to obtain the Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier. The amount of H-Pro-O-liquid carrier used was 10 wt% of dichloromethane, the amount of Fmoc-Arg (Pbf) -OSu used was 100 wt% of H-Pro-O-liquid carrier, the amount of DIC used was 6wt% of dichloromethane, and the amount of DMAP used was 10 wt% of dichloromethane.
Preparation of Fmoc-Arg (Pbf) -Pro-OH: adding Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier into dichloromethane, stirring for dissolving, uniformly mixing, then adding TFE and TFA, stirring for reacting for 40 min at the temperature of 20-40 ℃, filtering by using kieselguhr, adding isopropyl ether into filtrate for precipitating, filtering, washing and drying to obtain Fmoc-Arg (Pbf) -Pro-OH. Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier was used in an amount of 12 wt% of dichloromethane, TFE was used in an amount of 10 wt% of dichloromethane, and TFA was used in an amount of 1.2 wt% of dichloromethane.
Pretreatment of Wang resin: putting the Wang resin into a reaction column, adding DCM for treatment for 3 h, and draining to obtain the pretreated Wang resin. DCM was used in an amount of 300 wt% of the Wang resin.
Preparation of Fmoc-Arg (Pbf) -Pro-Wang resin: adding Fmoc-Arg (Pbf) -Pro-OH, HOBt and 2- [ (3-aminophenyl) sulfonyl) ethanol into DMF, stirring for dissolving, adding DIC under ice water bath for activating for 5 min to obtain an activated Fmoc-Arg (Pbf) -Pro-OH reagent, adding the activated Fmoc-Arg (Pbf) -Pro-OH reagent into a pretreated Wang resin reaction column, reacting for 9 h at the temperature of 30 ℃, pumping out reaction liquid after the reaction is finished, washing with DMF, absolute methanol and DCM in sequence, and pumping out to obtain Fmoc-Arg (Pbf) -Pro-Wang resin. Fmoc-Arg (Pbf) -Pro-OH was used at 15 wt% of DMF, HOBt was used at 10 wt% of DMF, 2- [ (3-aminophenyl) sulfonyl) ethanol was used at 1.1 wt% of DMF, DIC was used at 7 wt% of DMF, and the activating Fmoc-Arg (Pbf) -Pro-OH reagent was used at 300 wt% of Wang resin.
Preparation of H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin: adding a deprotection reagent into a reaction column of Fmoc-Arg (Pbf) -Pro-Wang resin for Fmoc removal treatment for 20min, draining, adding an activated Fmoc-Leu-OH reagent for coupling reaction for 4 h, then draining reaction liquid, washing with DMF, anhydrous methanol and DCM in sequence, and draining; Fmoc-D-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and H-Pyr-OH are coupled in sequence according to the deprotection operation and the activated amino acid coupling reaction to obtain H-Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Leu-Leu-Arg (Pbf) -Pro-Wang resin which is hereinafter referred to as peptide resin. The deprotection reagent is DBLK solution, the DBLK solution is mixed liquid of piperidine and DMF, the amount of the piperidine in the DBLK solution is 20 wt% of the DMF, the usage amount of the deprotection reagent is 200 wt% of the Wang resin, and the usage amount of the activated Fmoc-Leu-OH reagent is 300 wt% of the Wang resin.
Deprotection treatment: the deprotection reagent was added to the protecting group-peptide-Wang resin for 20 min.
Activating Fmoc-Leu-OH reagent: adding Fmoc-Leu-OH and HOBt into DMF, and adding DIC to activate for 20min under the condition of ice water bath. The amount of Fmoc-Leu-OH used was 15 wt% of DMF, the amount of HOBt used was 10 wt% of DMF, and the amount of DIC used was 6wt% of DMF.
The other amino acid reagents are activated in a manner equivalent to that of the Fmoc-Leu-OH reagent, the other amino acid reagents are used in the same or slightly higher molar amount than the Fmoc-Leu-OH reagent, and the other reagents are used in the same amount as the activated Fmoc-Leu-OH reagent.
Cleavage of peptide resin: transferring the peptide resin into a cutting reagent under the condition of ice-water bath, stirring for 2 h at the temperature of 30 ℃, filtering to remove the resin, adding cold ether into filtrate for precipitation, centrifugally collecting, washing and drying to obtain the polypeptide. The cleavage reagent consists of TFA, EDT, H2O, TIS, the amount of EDT used in the cleavage reagent is 3.0 wt% of TFA, and the amount of H used in the cleavage reagent is 3.0 wt%2O was used in an amount of 2.5 wt% based on TFA and TIS in the cleavage reagent was used in an amount of 0.06 wt% based on TFA.
Preparation of leuprorelin: adding HOBt and ethylamine into deionized water, stirring and mixing, freeze-drying, adding polypeptide and DIC, stirring and reacting at the temperature of 30 ℃ for 24 hours, adding cold ether for precipitation, filtering, washing to obtain a crude leuprorelin product, dissolving in water to prepare a solution, purifying by a C18 column, collecting leuprorelin components, and drying to obtain the leuprorelin. The usage amount of HOBt is 1 wt% of deionized water, the usage amount of ethylamine is 2 wt% of deionized water, the usage amount of polypeptide is 1.2 wt% of ethylamine, and the usage amount of DIC is 0.9 wt% of ethylamine.
Example 3:
a method for preparing leuprorelin by combining solid phase and liquid phase,
this example is different from example 2 only in that 2- [ (3-aminophenyl) sulfonyl) ethanol was used in an amount of 1.9 wt% of DMF in the preparation of Fmoc-Arg (Pbf) -Pro-Wang resin.
Test example:
1. liquid phase carrier infrared analysis
Test samples: the liquid phase carrier prepared in example 1.
And tabletting the liquid phase carrier by adopting potassium bromide, and then carrying out infrared analysis.
The infrared spectrum of the liquid phase carrier is shown in FIG. 1, in which 3368 cm-12972 cm, infrared absorption peak of hydroxyl-1Infrared absorption peak at 2853 cm of methyl-1Infrared absorption peak at 1700 cm of methylene-1About 1601 cm without infrared absorption peak of carbon-oxygen double bond-1Is at the infrared absorption peak of benzene ring, 1086 cm-1The infrared absorption peak of carbon, nitrogen and carbon indicates that the liquid phase carrier is successfully obtained.
2. HPLC analysis of leuprorelin
The leuprorelin purified in example 1 has high purity by HPLC, and the detection result is shown in fig. 2.
HPLC detection conditions:
the mobile phase A is 0.2% triethylamine water solution with pH value of 2-3;
the mobile phase B is acetonitrile;
gradient elution, flow rate of 1 mL/min, detection wavelength of 230 nm, sample size of 10 μ L.
After HPLC detection, the purified leuprorelin in the embodiment 1 of the invention has only a single main peak and no miscellaneous peak, and the purity of the obtained leuprorelin can be found to be very high.
3. Yield of leuprorelin
The yield of the leuprorelin in the invention is calculated by Fmoc-Arg (Pbf) -Pro-Wang resin 100 mmol, wherein, the yield of the leuprorelin prepared by the method of the embodiment 1 is 40.56 percent, and the purity is 99.72 percent; the yield of the leuprorelin prepared by the method of example 2 is 43.48 percent, and the purity is 99.81 percent; the leuprorelin prepared by the method of example 3 had a yield of 45.69 and a purity of 99.83%.
4. Leuprorelin mass spectrometry
The leuprorelin purified in example 1 was sampled and tested as required for MS testing, and the test results are shown in fig. 3. The molecular weight of the standard leuprorelin was about 1209.41, which combined with this data indicated that leuprorelin was obtained.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims (8)

1. A liquid carrier comprising: the compound is prepared by reacting 3-ethoxy-4-hydroxybenzaldehyde and 2- (2-ethoxyphenoxy) ethyl bromide in DMF (dimethyl formamide) containing potassium carbonate and then reducing by using a reducing agent, wherein the reducing agent is sodium borohydride;
the liquid phase carrier is prepared as follows:
mixing 3-ethoxy-4-hydroxybenzaldehyde, 2- (2-ethoxyphenoxy) ethyl bromide and potassium carbonate in DMF, stirring and reacting at 60-80 ℃ for 18-48h, separating to obtain a benzaldehyde compound, adding the benzaldehyde compound and sodium borohydride into a tetrahydrofuran solution, stirring and reacting at 30-50 ℃ for 2-10h, and separating to obtain a liquid phase carrier; the tetrahydrofuran solution is formed by mixing tetrahydrofuran and methanol, and the content of the methanol in the tetrahydrofuran solution is 4-16 wt%.
2. A process for preparing leuprolide comprising:
performing fragment synthesis in liquid phase using the liquid phase carrier of claim 1; synthesizing the fragment to obtain an Fmoc-Arg-Pro-O-liquid phase carrier;
removing the liquid phase carrier from the Fmoc-Arg-Pro-O-liquid phase carrier, combining the Fmoc-Arg-Pro-O-liquid phase carrier with solid phase resin, and then coupling amino acid reagents according to the sequence of amino acid sequences in leuprorelin to obtain leuprorelin-based polypeptide resin;
cutting the leuprorelin-based polypeptide resin, separating the leuprorelin-based polypeptide, and performing ethylation treatment to obtain leuprorelin;
the Fmoc-Arg-Pro-O-liquid phase carrier is prepared from Fmoc-Arg (Pbf) -OSu and Fmoc-Pro-O-liquid phase carrier;
in the preparation of the Fmoc-Pro-O-liquid phase carrier, the liquid phase carrier, dichloromethane, Fmoc-Pro-OH, DIC and DMAP are mixed, stirred and reacted for 30-90min at the temperature of 20-40 ℃, and separated to obtain the Fmoc-Pro-O-liquid phase carrier;
mixing the Fmoc-Pro-O-liquid phase carrier, THF, piperidine and DBU, stirring at 20-40 deg.C for 5-20min, and separating to obtain H-Pro-O-liquid phase carrier;
in the preparation of Fmoc-Arg (Pbf) -OSu, tetrahydrofuran and dicyclohexylcarbodiimide are added into Fmoc-Arg (Pbf) -OH and HOSu for reaction, and Fmoc-Arg (pbf) -OSu is obtained by separation treatment;
in the preparation of Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier, H-Pro-O-liquid phase carrier, dichloromethane, Fmoc-Arg (Pbf) -OSu, DIC and DMAP are mixed, stirred and reacted for 30-90min at the temperature of 20-40 ℃, and then separated to obtain Fmoc-Arg (Pbf) -Pro-O-liquid phase carrier.
3. The process for the preparation of leuprolide according to claim 2, wherein: the solid phase resin is any one of Wang resin and Rink-Amide-AM resin.
4. The process for the preparation of leuprolide according to claim 2, wherein: the amino acid reagent is activated by DMF solution containing HOBt and DIC for 3-30 min.
5. The process for the preparation of leuprolide according to claim 2, wherein: the coupling sequence of the amino acid reagent is Fmoc-Leu-OH, Fmoc-D-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and H-Pyr-OH.
6. The process for the preparation of leuprolide according to claim 2, wherein: the cleavage is performed by using TFA, EDT, H2O, TIS and a cleaving agent.
7. The process for the preparation of leuprolide according to claim 2, wherein: the ethylation treatment is carried out by reacting ethylamine with leuprolide-based polypeptide in the presence of HOBt and DIC.
8. Use of the liquid phase carrier of claim 1 for liquid phase production of a polypeptide and/or solid-liquid phase production of a polypeptide.
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