CN111233980A - Method for synthesizing goserelin by fragment method - Google Patents
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Abstract
The invention adopts a synthesis method combining a solid phase method and a liquid phase method to realize goserelin synthesis, wherein the second fragment and the third fragment are easy to synthesize and purify and have high purity; different fragments can be synthesized simultaneously, so that the goserelin synthesis time is effectively shortened, and the preparation efficiency is improved; finally, the two fragments are butted by adopting a liquid phase method to obtain the goserelin precursor, so that the low-cost coupling is realized, and the industrial amplification preparation is facilitated.
Description
Technical Field
The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a method for synthesizing goserelin by a fragment method.
Background
Goserelin (Goserlin) is a synthetic decapeptide gonadotropin releasing hormone (GnRH) potent analog. Can promote pituitary gland to release Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH), which has 40-200 times stronger effect than natural hormone. Goserelin was developed by astekang company, uk and marketed in france in 1987 and approved by FDA in 12 months in 1989.
The structural formula of goserelin is:
H-Pyr9-His8-Trp7-Ser6-Tyr5-D-Ser4(tBu)-Leu3-Arg2-Pro1-NHNHCONH2 10
goserelin is a slow release implant, a synthetic analogue of luteinizing hormone releasing hormone. After long-term use, the secretion of luteinizing hormone of pituitary can be inhibited, so that the decline of male serum testosterone and female serum estradiol is caused, and the decline is reversible after drug withdrawal. Goserelin was injected subcutaneously via the abdomen 3.6mg each time. Once every 28 days, it is suitable for hormone-treated prostate cancer, breast cancer at premenopausal and perimenopausal period, and endometriosis. Goserelin has almost complete bioavailability. The implant is administered every 4 weeks to maintain effective blood levels without tissue accumulation.
The synthesis of goserelin by adopting a liquid phase method is not reported for a while because of complicated operation and long synthesis period. The patents related to the current goserelin synthesis are as follows:
1) and (3) synthesizing the fragment by a solid phase method. Patent CN201810547594A discloses a 3+7 fragment synthesis method, which adopts a solid phase method to synthesize two fragments, namely a first fragment H-Arg-Pro-Azagly-NH of tripeptide2And a second fragment Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-OH of the heptapeptide, and then coupling the two fragments to obtain a crude product. Because the active guanidyl on the Arg end is not protected, competitive side reactions exist, impurities are difficult to control, and the reaction yield is influenced.
2) Solid phase stepwise synthesis method. In patents CN201610485117A and CN201510005951A, goserelin is sequentially linked and cleaved with fully protected amino acids one by one according to amino acid sequence, and then condensed with semicarbazide hydrochloride to reduce nitro compound to obtain crude product. The yield of the product is inevitably low due to the long reaction steps.
3) Solid-phase liquid-phase synthesis method. Patent CN106589072A discloses a solid-liquid phase synthesis method of 4+ 6. Namely, solid-liquid phase combination is adopted to obtain the tetrapeptide fragment of [1-3,10 ]; solid-liquid combination to obtain a [4-9] hexapeptide fragment containing D-Ser (tBu); finally, in a liquid phase, the hexapeptide fragment and the tetrapeptide fragment are subjected to condensation coupling to obtain a crude product. The technical disadvantages are that:
3-a) the semicarbazide is coupled into the tetrapeptide fragment by directly adopting a liquid phase method, so that the yield is low; in the fragment docking, side reactions are difficult to avoid and influence the yield and the product quality; 3-b), on the other hand, the last step of alkaline hydrolysis for synthesizing the hexapeptide fragment adopts 1N sodium hydroxide aqueous solution, the alkaline reaction condition is too strong, racemization of amino acid is difficult to avoid, the difficulty of product purification is increased, and the quality of a finished product is reduced; 3-c) finally, active guanidino is arranged on the Arg terminal in the segment in the coupling reaction, so that the competitive influence on the condensation reaction is difficult to avoid, side reactions are increased, the purification difficulty of the product is increased, and the product yield is reduced.
4) Liquid phase synthesis. The patent US4024248 discloses a 7+3 fragment synthesis method, which is specifically characterized in that a heptapeptide fragment Ser-Tyr-D-Ser (Tbu) -Leu-Arg-Pro-AZgly-NH2 and a tripeptide fragment Pyr-His-Trp-OH are directly condensed to form a crude product in the presence of a condensing agent; patent CN 201010039547A discloses a 5+5 fragment synthesis method, which is specifically characterized in that the fragment is formed by directly condensing a pentapeptide fragment D-Ser (tBu) -Leu-Arg-Pro-AZgly-NH2 and the pentapeptide fragment Pyr-His-Trp-Ser-Tyr-OH in the presence of a condensing agent. The invention has the advantages of saving the cost of solid phase materials; the method has the disadvantages that the synthesis of multiple steps by adopting a liquid phase method needs a plurality of post-treatment operations, and compared with a solid phase method, the operation is troublesome and the reaction speed is slow; and the active guanidyl on the Arg end of the fragment is also present, so that the competitive influence on the condensation reaction is difficult to avoid, the side reaction is increased, the purification difficulty of the product is improved, and the product yield is reduced.
In conclusion, the existing goserelin synthesis method has the problems of more impurities, racemization of amino acid, difficulty in purification, low yield and the like, and a technical scheme which is relatively suitable for industrial production is not available. Therefore, the invention researches the synthesis method of goserelin, thereby obtaining the technical scheme of the invention.
Disclosure of Invention
The invention aims to solve the technical problems of more impurities, low purity and yield, high cost, complex operation steps, excessive waste liquid and inconvenience for industrial production in the conventional synthesis process, and provides a goserelin synthesis method.
The technical scheme is as follows: the invention relates to a goserelin synthesis method, which comprises the following steps:
(1) according to the sequence of the amino acids from the C end to the N end of the main chain, solid-phase synthesizing a fragment [5-9] peptide resin of the 5 th-9 th amino acid, and cutting the resin to obtain a fragment I;
(2) according to the sequence of the amino acids from the C end to the N end of the main chain, carrying out solid phase synthesis on fragment [1-3] peptide resin of 1-3 amino acids, and cutting the resin to obtain a fragment III;
(3) according to the sequence of the amino acids from the C end to the N end of the main chain, the 4 th amino acid is synthesized by solid phase and is connected with resin to complete the fragment two-joint, namely [4] peptide resin;
(4) the fragment II joint is connected with the fragment I in a condensation way by a solid phase method to obtain fragment [4-9] peptide resin, and the resin is cut to obtain fragment II;
(5) condensing and coupling the second fragment and the third fragment by a liquid phase method to obtain a goserelin precursor;
(6) reducing the goserelin precursor to obtain a goserelin crude product;
(7) the goserelin acetate is obtained by the steps of purifying, salifying and freeze-drying the goserelin crude product;
protecting groups are arranged on His, Trp, Ser and Tyr of the [5-9] peptide resin and the fragment [1-3] peptide resin.
Further, the protecting groups of Ser and Tyr are selected from one of TES, TMS, TBS, DHP or tBu; the protecting groups of His and Trp are selected from one of Adoc, Boc, Mmt, Mtt or Trt, and in the third fragment, the amino acid adopts Fmoc-Arg (NO)2)-OH。
Further, the structural formula of the fragment di-linker is as follows: D-Ser4(tBu)-2-CTC Resin；
The structural formula of the fragment is as follows: pyr9-His8-Trp7-Ser6-Tyr5-OH；
The structural formula of the fragment II is as follows:
Pyr9-His8-Trp7-Ser6-Tyr5-D-Ser4(tBu)-OH；
the fragment has a three-structure formula: leu3-Arg2(NO2)-Pro1-NHNHCONH2 10；
The structural formula of the goserelin precursor is as follows:
Pyr9-His8-Trp7-Ser6-Tyr5-D-Ser4(tBu)-Leu3-Arg2(NO2)-Pro1-NHNHCONH2 10。
further, the goserelin synthesis method comprises the following specific steps:
(1) adding Wang resin into a solid phase reactor, carrying out coupling reaction with Fmoc-Tyr (tBu) -OH under the action of a condensing agent, and then carrying out Fomc deprotection reaction; sequentially connecting the following amino acids of Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and Pyr-OH according to a goserelin polypeptide sequence to obtain a fragment [5-9 [ ]]A peptide resin; cutting the resin, namely cracking the resin under the action of a cracking agent to obtain a fragment-Pyr9-His8-Trp7-Ser6-Tyr5-OH；
(2) Sequentially coupling MBHA amino resin with p-nitrophenyl chloroformate and hydrazine hydrate, and sequentially connecting the following amino acids Fmoc-Pro-OH and Fmoc-Arg (NO) according to a goserelin polypeptide sequence2)-OH、Fmoc-Leu-OHNHNHCONH2Obtaining the fragment [1-3]]Peptide resin, cleavage of the resin, i.e. cleavage of the resin under the action of a cleavage agent to give the fragment TriLeu3-Arg2(NO2)-Pro1-NHNHCONH2 10；
(3) Adding 2-chlorotrityl resin into a solid phase reactor, carrying out coupling reaction with amino acid Fmoc-D-Ser (tBu) -OH under the action of a condensing agent, and then carrying out FmCarrying out an oc deprotection reaction to obtain fragment di-linker D-Ser4(tBu)-2-CTCResin；
(4) Coupling the fragment II joint with the fragment I under the action of a condensing agent by a solid phase method to obtain a fragment [4-9] peptide resin, and cutting the resin, namely cracking the resin under the action of a cracking agent to obtain a fragment II;
(5) coupling the second fragment and the third fragment by a liquid phase method under the action of a condensing agent to obtain a goserelin precursor;
(6) carrying out reduction reaction on the goserelin precursor under the action of a reducing agent to obtain a goserelin crude product;
(7) purifying, salifying and freeze-drying the goserelin crude product to obtain goserelin acetate refined peptide;
the step of connecting amino acids in the step (1) and the step (2) comprises a coupling reaction under the action of a condensing agent and a Fomc deprotection reaction.
Further, in any step from the step (1) to the step (4), the condensing agent is selected from one or more of HATU/HOBt/DIEA, HBTU/HOBt/DIEA, PyBop/HOBt/DIEA and DIC/HOBt; the condensing agent in the step (5) is selected from one or more of HATU/HOBt/DIEA, HBTU/HOBt/DIEA and PyBop/HOBt/DIEA. In particular, the condensing agents HATU/HOBt/DIEA represent a system containing three substances, HATU, HOBt and DIEA, and the remaining condensing agents also have the same meaning.
Further, the cracking agent adopted in the step (1) or the step (2) is a trifluoroacetic acid mixed solution, and the volume of the cracking agent is 7-15 times of the mass of the peptide resin; the trifluoroacetic acid mixed solution is a mixed solution of TFA, PhSMe, TIS and water or a mixed solution of TFA, EDT and water, and the volume ratio is as follows: TFA: PhSMe: and (3) TIS: 70-97% of water: 10-1: 10-1: 10-1, TFA: EDT (electro-thermal transfer coating): water is 90-95: 1-5: 1-5; the cracking agent adopted in the step (4) is a trifluoroethanol mixed solution, the volume of the cracking agent is 7-15 times of the mass of the peptide resin, the trifluoroethanol mixed solution is prepared by mixing TFE and DCM easily, and the volume ratio of TFE: DCM is 25: 75-85.
Furthermore, the substitution degree of the Wang resin is 0.4 to 1.5mmol/g, the substitution degree of the MBHA amino resin is 0.4 to 1.0mmol/g, and the substitution degree of the 2-chlorotrityl resin is 0.4 to 1.0 mmol/g.
Further, the solvent adopted in the reactions of the steps is one or more of dichloromethane, N-dimethylformamide, N-methylpyrrolidone and dimethyl sulfoxide.
Has the advantages that:
the invention adopts a synthesis method combining a solid phase method and a liquid phase method to realize goserelin synthesis, wherein the second fragment and the third fragment are easy to synthesize and purify and have high purity; different fragments can be synthesized simultaneously, so that the goserelin synthesis time is effectively shortened, and the preparation efficiency is improved; finally, the two fragments are butted by adopting a liquid phase method to obtain the goserelin precursor, so that the low-cost coupling is realized, and the industrial amplification preparation is facilitated.
Aiming at the problems that the competitive side reaction of the active guanidine group on the Arg end to the condensation reaction is difficult to control, the impurities are more, the purification is difficult, the yield is reduced and the like in the prior art, the fragment synthesis method adopting the solid-liquid phase combination introduces Arg (NO)2) As the source of amino acid in the third fragment, the reaction conditions are mild, side reactions are effectively avoided, and the problem that the amino acid sequences of the deletion peptide, impurities and the main component have more differences can be effectively solved; due to the shortened reaction time, the isomerization and amino acid racemization problems are reduced. The invention has the advantages of easier purification of the product, simple post-treatment operation, less three wastes and higher purity of each intermediate;
the method has stable process, the purity of the prepared goserelin can reach more than 99.5 percent, and the total yield is high.
Drawings
Fig. 1 is a flow chart of the preparation of goserelin according to the invention.
Detailed Description
For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
The abbreviations or english full names used in the claims and the specification of the present invention have the following meanings:
example 1
Step (1) Synthesis of fragment one
And sequentially adding Wang Reasin60.00g and 600mLDCM into a solid-phase reaction bottle, stirring for 15 minutes, swelling and performing suction filtration to obtain a filter cake. DMF120mL, Fmoc-Tyr (tBu) -OH (3eq, 41.40g), catalyst DMAP (0.6eq, 2.19g), DIC (3.0eq, 11.37g) and HOBt (3.0eq, 12.17g) were added to the flask in this order, stirred under nitrogen for 2 hours and filtered to obtain a cake. Washing the filter cake with DMF, and vacuum filtering to obtain the filter cake. Fmoc deprotection reaction adopts a Pip/DMF mixed solution: mixing the filter cake with 300mL of 20% Pip/DMF solution (the volume ratio of DMF to Pip is 80: 20, the same below), stirring for 5 minutes, and performing suction filtration to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. Washing the filter cake with proper amount of DMF, and vacuum filtering to obtain the filter cake. Amino acids Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and Pyr-OH are condensed and connected in sequence according to the method, and then the amino acids are deprotected to obtain Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Wang Resin, namely fragment-peptide Resin. Cleavage agent 600mL of 90% TFA (TFA: water: EDT; 90: 5: 5, vol.) solution was added and stirred for 3 hours. And (5) carrying out suction filtration to obtain filtrate. Settling with proper amount of ether, centrifuging and washing to obtain solid. The solid is settled by proper amount of ether, centrifuged, washed and dried in vacuum at 35 ℃ to obtain 20.55g of fragment I, 89 percent (HPLC) and mass spectrum of [ M + H ]]+＝703.5
And (3) sequentially adding Rink Amide MBHA Resin (45.00g) and 600ml of LPCM into a solid-phase reaction bottle for synthesizing the fragment III in the step (2), swelling and then performing suction filtration to obtain a filter cake. Mixing the filter cake with 300mL of 20% Pip/DMF solution, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. And washing the filter cake with a proper amount of DMF and DCM in sequence, and filtering to obtain the filter cake. To the flask were added phenyl p-nitrochloroformate (3eq, 12.00g), DIEA (3eq, 9.00g) and 300mL EDC in this order by filtration and reacted for 2 hours until the kaiser reagent was colorless. Suction filtration, washing with an appropriate amount of DCM and suction drying. Hydrazine hydrate (6eq, 15mL) and DIEA were added in sequence(4eq, 10.68g) and DCM100mL for 8-12 h. And (5) suction filtration. The filter cake was washed with appropriate amount of DMF. To a 250mL reaction flask were added Fmoc-Pro-OH (3eq, 20.40g), PyBop (3eq, 33.31g), HOBt (3eq, 8.70g), DIEA (6eq, 16.13g) and DMF120mL in that order, and the mixture was stirred to obtain a solution. Adding the new configuration solution into a solid-phase reaction bottle, and stirring under the protection of nitrogen until the kaiser reagent is detected to be colorless. And filtering to obtain a filter cake. Mixing the filter cake with 300mL of 20% Pip/DMF solution, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. The filter cake was washed with appropriate amount of DMF. And filtering to obtain a filter cake. And sequentially condensing and connecting amino acids and deprotecting to obtain the fragment tripeptide resin according to the method. Adding 600mL of 90% TFA (TFA: water: EDT; 90: 5: 5, volume ratio) solution of a cracking agent, stirring for 3 hours, and performing suction filtration to obtain a filtrate. Mixing the filtrate with appropriate amount of diethyl ether, settling, centrifuging, and washing to obtain solid. Mixing the solid with appropriate amount of diethyl ether, settling, centrifuging, washing, and vacuum drying at 35 deg.C to obtain fragment III 10.68 g; 91% (HPLC), Mass Spectrometry [ M + H ]]+＝486.3
Step (3) Synthesis of fragment-di-linker peptide resin
Adding 15.00g of 2-CTC resin and 600mLDCM into a solid phase reaction bottle, swelling and filtering to obtain a filter cake. Fmoc-D-Ser (tBu) -OH (1.5eq, 6.30g), DIEA (3eq, 4.50g) and 60mL of CCM were added in sequence for reaction for 2 h. MeOH (15mL) was added, stirred for 30min, filtered with suction, and the filter cake was washed with the appropriate amount of DMF. Mixing the filter cake with 100mL of 20% Pip/DMF solution, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. The filter cake was washed with appropriate amount of DMF. The fragment of the dipeptide resin was obtained by suction filtration and used directly in the next step
Step (4) Synthesis of fragment two
The solid phase reaction flask was charged with fragment one (7mmol,15.00g), HATU (7mmol, 7.80g), DIEA (14mmol, 6.30g), HOBt (0.95g), and DMF20mL and the reaction was detected to be complete for 2 h. And (4) carrying out suction filtration, washing a filter cake with a proper amount of DMF, and carrying out suction filtration to obtain the filter cake. DCM150mL, 30mL of acetic anhydride, and 30mL of pyridine were added in this order, and the mixture was stirred for 30 min. And (4) carrying out suction filtration, alternately washing the filter cake with a proper amount of DCM and methanol, and carrying out suction filtration to obtain the filter cake. 600mL of a DCM solution of 20% TFE as a cracking agent (DCM: TFE; 80: 20, vol.) was added thereto, and the mixture was stirred for 2 hoursAnd filtering to obtain filtrate. The filtrate is settled with a proper amount of ether, centrifuged and washed to obtain solid. The solid is settled by proper amount of ether, centrifuged, washed and dried in vacuum at 35 ℃ to obtain fragment two 8.25g, 87 percent (HPLC) and mass spectrum [ M + H ]]+＝846.1
Step (5) Synthesis of goserelin precursor
The second fragment (3.00g), DIEA (2.76g), HOBt (0.51g), DMF36mL and HBTU (1.40g) were added to a liquid phase reaction flask in this order and stirred at room temperature for 1 hour. Fragment three (2.25g) was added and stirred at room temperature until the reaction was complete. Slowly adding into ice-cold diethyl ether 420mL, precipitating solid, and removing solvent; the residue was mixed with DMF30mL, and slowly added to ice-cooled diethyl ether (360 mL) to precipitate a solid, and the solvent was removed. Vacuum drying at 35 deg.C to obtain white solid 4.50g, yield 93%, 92% (HPLC), and mass spectrum [ M + H ]]+1315.3. Used directly in the next step.
Step (6) synthesis of goserelin crude product
The goserelin precursor (3.00g), 75mL of methanol and 3mL of acetic acid were sequentially added to a liquid phase reaction flask, and the mixture was stirred at room temperature to dissolve. 0.30g of 10% palladium on carbon is added under the protection of nitrogen. The reaction system replaces hydrogen three times, and is stirred at room temperature until the reaction is finished. Filtering to remove insoluble substances, and concentrating the filtrate at 35 ℃ under reduced pressure to obtain the goserelin crude product. The residue was dissolved in 20mL of methanol, slowly added to 450mL of ice-cold diethyl ether, and the solvent was removed after precipitation of a solid. HPLC 89%, Mass Spectrometry [ M + H%]+1270.2; used directly in the next step.
Step (7) preparation of goserelin acetate protamine
1.50g of goserelin crude product was weighed, mixed with 30mL of water containing 10% ammonia water, and dissolved. Purifying by Hanbang preparative HPLC system (wavelength is 210nm, chromatographic column is C8 reversed phase column, 20M ammonium dihydrogen phosphate solution, pH3.0/acetonitrile is mobile phase) at room temperature to obtain goserelin refined peptide solution with purity of more than 99.5%. Purifying the refined peptide solution by a preparative HPLC system (the chromatographic column is a C18 reverse phase column, and 0.2% acetic acid/acetonitrile is a mobile phase), collecting target components, concentrating under reduced pressure, and lyophilizing to obtain 1.20g of goserelin acetate, 99.85% (HPLC), with a purification yield of 80% and a total yield of 32%; mass spectrum of mass spectrometer [ M + H]+＝1270.4。
Example 2
The difference from example 1 is the synthesis of fragment one
And sequentially adding 60.00g of Wang resin and 600ml of LPCM into a solid-phase reaction bottle for synthesizing the fragment I, stirring for 15 minutes, and performing suction filtration to obtain a filter cake. DMF120mL, Fmoc-Tyr (tBu) -OH (3eq, 41.40g), catalyst DMAP (0.6eq, 2.19g), HBTU (3.0eq, 34.14g), HOBt (3.0eq, 12.17g), DIEA (3.0eq, 11.67g) were added to the flask in this order, stirred for 2 hours under nitrogen, and filtered to give a cake. Washing the filter cake with DMF, and vacuum filtering to obtain the filter cake. Mixing the filter cake with 300mL of 20% Pip/DMF solution, stirring for 5 minutes, and performing suction filtration to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. Washing the filter cake with a proper amount of DMF, and performing suction filtration to obtain a filter cake;
amino acids Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and Pyr-OH are condensed and connected in sequence according to the method and are deprotected to obtain Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -WangResin, namely a fragment-peptide resin. A solution of 90% TFA (TFA: water: EDT; 90: 5: 5 by volume) was added and stirred for 3 hours. And (5) carrying out suction filtration to obtain filtrate. Settling with proper amount of ether, centrifuging and washing to obtain solid. The solid is settled by proper amount of ether, centrifuged, washed and dried in vacuum at 35 ℃ to obtain 21.63g of fragment I, 91 percent (HPLC) and mass spectrum of [ M + H ]]+＝703.3
Example 3
The difference from example 1 is the synthesis of fragment one
Synthesis of fragment one
And sequentially adding 60.00g of Wang resin and 600ml of LPCM into a solid-phase reaction bottle, stirring for 15 minutes, and performing suction filtration to obtain a filter cake. DMF120mL, Fmoc-Tyr (tBu) -OH (3 equiv., 41.40g), catalyst DMAP (0.6 equiv., 2.19g), PyBop (3.0eq, 46.87g), HOBt (3.0eq, 12.17g) and DIEA (3.0eq, 11.67g) were added to the flask in this order, stirred under nitrogen for 2 hours, and filtered to obtain a cake. Washing the filter cake with DMF, and vacuum filtering to obtain the filter cake. Mixing the filter cake with 300mL of 20% Pip/DMF solution (DMF: Pip; 80: 20, volume ratio), stirring for 5 minutes, and filtering to obtain a filter cake; then 300mL of 20% Pip/DMF solution was added, stirred for 15 minutes and filtered with suction. Washing the filter cake with DMF, and vacuum filtering to obtain filter cake
Amino acids Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and Pyr-OH are condensed and connected in sequence according to the method and are deprotected to obtain Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -WangResin, namely a fragment-peptide resin. Cleavage agent 600mL of 85% TFA (TFA: PhSMe: TIS: water 85: 5: 5: 5; volume ratio) solution was added and stirred for 3 hours. And (5) carrying out suction filtration to obtain filtrate. Settling with proper amount of ether, centrifuging and washing to obtain solid. The solid is settled by proper amount of ether, centrifuged, washed and dried in vacuum at 35 ℃ to obtain 19.08g of fragment I, 92 percent (HPLC) and mass spectrum [ M + H ]]+＝703.5。
Example 4
The difference from example 1 is the synthesis of goserelin precursor
Synthesis of goserelin precursor
The second fragment (1.00g), DIEA (0.92g), HOBt (0.17g), DMF12mL and HATU (0.46g) were added to a liquid phase reaction flask in this order and stirred at room temperature for 1 hour. Fragment three (0.75g) was added and stirred at room temperature for 8 hours until the reaction was complete. Slowly adding the reaction solvent into 140mL of ice-cold diethyl ether, separating out a solid, and filtering the solvent; the residue was taken up in DMF100mL and slowly added to ice-cold 120mL of diethyl ether to precipitate a solid which was filtered off the solvent. Vacuum drying at 35 deg.C to obtain white solid 1.50g, yield 96%, 73% (HPLC), and mass spectrum [ M + H ]]+＝1315.0。
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A goserelin synthesis method is characterized by comprising the following process steps:
(1) according to the sequence of the amino acids from the C end to the N end of the main chain, solid-phase synthesizing a fragment [5-9] peptide resin of the 5 th-9 th amino acid, and cutting the resin to obtain a fragment I;
(2) according to the sequence of the amino acids from the C end to the N end of the main chain, carrying out solid phase synthesis on fragment [1-3] peptide resin of 1-3 amino acids, and cutting the resin to obtain a fragment III;
(3) according to the sequence of the amino acids from the C end to the N end of the main chain, the 4 th amino acid is synthesized by solid phase and is connected with resin to complete the fragment two-joint, namely [4] peptide resin;
(4) the fragment II joint is connected with the fragment I in a condensation way by a solid phase method to obtain fragment [4-9] peptide resin, and the resin is cut to obtain fragment II;
(5) condensing and coupling the second fragment and the third fragment by a liquid phase method to obtain a goserelin precursor;
(6) reducing the goserelin precursor to obtain a goserelin crude product;
(7) the goserelin acetate is obtained by the steps of purifying, salifying and freeze-drying the goserelin crude product;
protecting groups are arranged on His, Trp, Ser and Tyr of the [5-9] peptide resin and the fragment [1-3] peptide resin.
2. The method according to claim 1, wherein the protecting groups for Ser and Tyr are optionally selected from one of TES, TMS, TBS, DHP or tBu; the protecting groups of His and Trp are selected from one of Adoc, Boc, Mmt, Mtt or Trt, and in the third fragment, the amino acid adopts Fmoc-Arg (NO)2)-OH。
3. The method of claim 1,
the structural formula of the fragment di-linker is as follows: D-Ser4(tBu)-2-CTC Resin；
The structural formula of the fragment is as follows: pyr9-His8-Trp7-Ser6-Tyr5-OH；
The structural formula of the fragment II is as follows:
Pyr9-His8-Trp7-Ser6-Tyr5-D-Ser4(tBu)-OH；
the fragment has a three-structure formula: leu3-Arg2(NO2)-Pro1-NHNHCONH2 10；
The structural formula of the goserelin precursor is as follows:
Pyr9-His8-Trp7-Ser6-Tyr5-D-Ser4(tBu)-Leu3-Arg2(NO2)-Pro1-NHNHCONH2 10。
4. the method according to claim 1, characterized by the following specific steps:
(1) adding Wang resin into a solid phase reactor, carrying out coupling reaction with Fmoc-Tyr (tBu) -OH under the action of a condensing agent, and then carrying out Fomc deprotection reaction; sequentially connecting the following amino acids of Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH and Pyr-OH according to a goserelin polypeptide sequence to obtain a fragment [5-9 [ ]]A peptide resin; cutting the resin, namely cracking the resin under the action of a cracking agent to obtain a fragment-Pyr9-His8-Trp7-Ser6-Tyr5-OH；
(2) Sequentially coupling MBHA amino resin with p-nitrophenyl chloroformate and hydrazine hydrate, and sequentially connecting the following amino acids Fmoc-Pro-OH and Fmoc-Arg (NO) according to a goserelin polypeptide sequence2)-OH、Fmoc-Leu-OHNHNHCONH2Obtaining the fragment [1-3]]Peptide resin, cleavage of the resin, i.e. cleavage of the resin under the action of a cleavage agent to give the fragment TriLeu3-Arg2(NO2)-Pro1-NHNHCONH2 10；
(3) Adding 2-chlorotrityl resin into a solid phase reactor to perform coupling reaction with amino acid Fmoc-D-Ser (tBu) -OH under the action of a condensing agent, and then performing Fmoc deprotection reaction to obtain fragment di-linker D-Ser4(tBu)-2-CTCResin；
(4) Coupling the fragment II joint with the fragment I under the action of a condensing agent by a solid phase method to obtain a fragment [4-9] peptide resin, and cutting the resin, namely cracking the resin under the action of a cracking agent to obtain a fragment II;
(5) coupling the second fragment and the third fragment by a liquid phase method under the action of a condensing agent to obtain a goserelin precursor;
(6) carrying out reduction reaction on the goserelin precursor under the action of a reducing agent to obtain a goserelin crude product;
(7) purifying, salifying and freeze-drying the goserelin crude product to obtain goserelin acetate refined peptide;
the step of connecting amino acids in the step (1) and the step (2) comprises a coupling reaction under the action of a condensing agent and a Fomc deprotection reaction.
5. The method according to claim 4, wherein in any of the steps (1) to (4), the condensing agent is selected from one or more of HATU/HOBt/DIEA, HBTU/HOBt/DIEA, PyBop/HOBt/DIEA, DIC/HOBt; the condensing agent in the step (5) is selected from one or more of HATU/HOBt/DIEA, HBTU/HOBt/DIEA and PyBop/HOBt/DIEA.
6. The method according to claim 4, wherein the cracking agent used in step (1) or step (2) is a trifluoroacetic acid mixed solution, and the volume of the cracking agent is 7-15 times of the mass of the peptide resin; the trifluoroacetic acid mixed solution is a mixed solution of TFA, PhSMe, TIS and water or a mixed solution of TFA, EDT and water, and the volume ratio is as follows: TFA: PhSMe: and (3) TIS: 70-97% of water: 10-1: 10-1: 10-1, TFA: EDT (electro-thermal transfer coating): water is 90-95: 1-5: 1-5; the cracking agent adopted in the step (4) is a trifluoroethanol mixed solution, the volume of the cracking agent is 7-15 times of the mass of the peptide resin, the trifluoroethanol mixed solution is prepared by mixing TFE and DCM easily, and the volume ratio of TFE: DCM is 25: 75-85.
7. The method according to claim 4, wherein the Wang resin has a degree of substitution of 0.4 to 1.5mmol/g,
the degree of substitution of MBHA amino resin is 0.4-1.0mmol/g, and the degree of substitution of 2-chlorotrityl resin is 0.4-1.0 mmol/g.
8. The method according to claim 4, wherein the solvent used in each step of the reaction is selected from one or more of DCM, DMF, NMP, and DMSO.
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| CN106589072A (en) * | 2016-12-22 | 2017-04-26 | 江苏诺泰生物制药股份有限公司 | Synthesis method of goserelin |
| CN108383896A (en) * | 2018-05-31 | 2018-08-10 | 中肽生化有限公司 | A kind of method of segment method synthesis Goserelin |
| CN110128505A (en) * | 2019-05-21 | 2019-08-16 | 梯尔希（南京）药物研发有限公司 | A kind of synthetic method of Goserelin impurity |
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| CN106589072A (en) * | 2016-12-22 | 2017-04-26 | 江苏诺泰生物制药股份有限公司 | Synthesis method of goserelin |
| CN108383896A (en) * | 2018-05-31 | 2018-08-10 | 中肽生化有限公司 | A kind of method of segment method synthesis Goserelin |
| CN110128505A (en) * | 2019-05-21 | 2019-08-16 | 梯尔希（南京）药物研发有限公司 | A kind of synthetic method of Goserelin impurity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023030278A1 (en) * | 2021-08-30 | 2023-03-09 | 湖南三太药业有限公司 | Full-liquid-phase synthesis method for reelin drug |
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