CN111233980B - Fragment method synthesis method of goserelin - Google Patents
Fragment method synthesis method of goserelin Download PDFInfo
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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 and third fragments are easy to synthesize and purify, and have high purity; different fragments can be synthesized at the same time, so that the synthesis time of goserelin is effectively shortened, and the preparation efficiency is improved; finally, the liquid phase method is adopted to butt joint the two fragments to obtain goserelin precursor, so that low-cost coupling is realized, and the industrialized amplification preparation is facilitated.
Description
Technical Field
The invention belongs to the technical field of polypeptide medicine preparation methods, and particularly relates to a fragment method synthesis method of goserelin.
Background
Goserelin (Goserlin) is a synthetic, potent analog of the decapeptide gonadotropin-releasing hormone (GnRH). Can promote the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) from pituitary gland, and has 40-200 times stronger effect than natural hormone. Goserelin was developed by the company aslick, uk, and was marketed in france in 1987 and FDA approved for marketing in 12 th 1989.
The goserelin has the structural formula:
H-Pyr 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -D-Ser 4 (tBu)-Leu 3 -Arg 2 -Pro 1 -NHNHCONH 2 10
goserelin is a slow release implant, which is a synthetic, luteinizing hormone-releasing hormone analogue. Can inhibit secretion of luteinizing hormone of pituitary gland after long-term use, thereby causing decrease of male serum testosterone and female serum estradiol, and is reversible after stopping administration. Goserelin is injected subcutaneously through the abdomen, 3.6mg each time. Once every 28 days, is suitable for treating prostatic cancer, pre-menopausal and perimenopausal breast cancer, and endometriosis by hormone. Goserelin has almost complete bioavailability. An injection implant is used every 4 weeks to maintain effective blood concentration without tissue accumulation.
The liquid phase method for synthesizing goserelin is not reported for a while because of complicated operation and long synthesis period. The related patents on goserelin synthesis at present are as follows:
1) Fragment solid phase synthesis. Patent CN201810547594A discloses a 3+7 fragment synthesis method, which synthesizes two fragments, namely the first fragment H-Arg-Pro-Azaply-NH of tripeptide by solid phase method 2 And a second fragment of heptapeptide Pyr-His-Trp-Ser-Tyr-D-Ser (tBu) -Leu-OH, and then coupling the two fragments to give crude product. Because the active guanidine group at the Arg terminal is not protected, competitive side reactions exist, so that the impurity is difficult to control, and the reaction yield is affected.
2) And (3) synthesizing the solid phase step by step. In the patents CN201610485117A and CN201510005951A, goserelin is sequentially connected with all the protected amino acids one by one according to the amino acid sequence, and then condensed with semicarbazide hydrochloride to reduce nitro compounds to obtain crude products. The yield of the product is inevitably low due to the long reaction steps.
3) Solid-phase liquid phase synthesis. Patent CN106589072a discloses a solid-liquid phase synthesis of 4+6. Namely, solid-liquid phase combination is adopted to obtain tetrapeptide fragments of [1-3,10 ]; solid-liquid phase combination to obtain [4-9] hexapeptide fragment containing D-Ser (tBu); finally, condensing and coupling the hexapeptide fragment and the tetrapeptide fragment in a liquid phase to obtain a crude product. The technical disadvantage is that:
3-a) the semicarbazide is directly coupled into the tetrapeptide fragment by adopting a liquid phase method, so that the yield is low; in segment docking, side reactions are difficult to avoid and affect yield and product quality; 3-b) on the other hand, the final step of synthesizing the hexapeptide fragment adopts 1N sodium hydroxide aqueous solution, the alkaline reaction condition is too strong, the racemization of amino acid is difficult to avoid, the purification difficulty of the product is increased, and the quality of the finished product is reduced; 3-c) finally, active guanidine groups are arranged at the Arg terminal in the fragment in the coupling reaction, 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 increased, and the product yield is reduced.
4) Synthesizing by a liquid phase method. Patent US4024248 discloses a 7+3 fragment synthesis method, which is 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 a pentapeptide fragment D-Ser (tBu) -Leu-Arg-Pro-AZgly-NH2 and a pentapeptide fragment Pyr-His-Trp-Ser-Tyr-OH are directly condensed in the presence of a condensing agent. The invention has the advantages of saving the cost of solid phase materials; the method has the defects that a plurality of post-treatment operations are needed in the synthesis of the multi-step liquid phase method, and compared with the solid phase method, the method has the advantages of troublesome operation and low reaction speed; and the Arg terminal in the fragment is provided with active guanidine groups, so that the competing 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 summary, the existing synthesis method of goserelin has the problems of more impurities, racemization of amino acid, difficult purification, low yield and the like, and the technical scheme which is suitable for industrial production is not seen. 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, complicated operation steps, excessive waste liquid and adverse industrialized production in the existing synthesis process, and provides a goserelin synthesis method.
The technical scheme is as follows: the invention discloses a method for synthesizing goserelin, which comprises the following steps:
(1) Solid phase synthesizing 5-9 th amino acid fragment [5-9] peptide resin according to the amino acid sequence from the C end to the N end of the main chain, and cutting the resin to obtain a fragment I;
(2) Solid phase synthesizing 1-3 amino acid fragment [1-3] peptide resin according to the amino acid sequence from the C end to the N end of the main chain, and cutting the resin to obtain a fragment III;
(3) According to the amino acid sequence from the C end to the N end of the main chain, the 4 th amino acid is connected with the resin by solid phase synthesis, and the fragment two-joint, namely [4] peptide resin is completed;
(4) Condensing and connecting the segment two connector and the segment one by a solid phase method to obtain segment [4-9] peptide resin, and cutting the resin to obtain segment two;
(5) Coupling the second segment with the third segment 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 purifying, salifying and freeze-drying the goserelin crude product;
the [5-9] peptide resin and the His, trp, ser, tyr of the fragment [1-3] peptide resin are provided with protecting groups.
Further, the protective groups of Ser and Tyr are arbitrarily selected from one of TES, TMS, TBS, DHP or tBu; the His and Trp protecting groups are selected from one of Adoc, boc, mmt, mtt and Trt, and in the fragment III, fmoc-Arg (NO 2 )-OH。
Further, the structural formula of the segment two-joint is as follows: D-Ser 4 (tBu)-2-CTC Resin；
The structural formula of the fragment one is as follows: pyr (Pyr) 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -OH；
The structural formula of the segment II is as follows:
Pyr 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -D-Ser 4 (tBu)-OH；
the three structural formulas of the segments are as follows: leu (Leu) 3 -Arg 2 (NO 2 )-Pro 1 -NHNHCONH 2 10 ；
The goserelin precursor has the structural formula:
Pyr 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -D-Ser 4 (tBu)-Leu 3 -Arg 2 (NO 2 )-Pro 1 -NHNHCONH 2 10 。
further, the synthesis method of goserelin comprises the following specific steps:
(1) Adding Wang resin into a solid phase reactor, performing coupling reaction with Fmoc-Tyr (tBu) -OH under the action of a condensing agent, and then performing Fomc deprotection reaction; the following amino acids Fmoc-Ser (tBu) -OH, fmoc-Trp (Boc) -OH, fmoc-His (Trt) -OH and Pyr-OH are sequentially connected according to goserelin polypeptide sequences to obtain fragments [5-9]]A peptide resin; cutting the resin to obtain a segment Pyr after the resin is cracked under the action of a cracking agent 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -OH；
(2) Sequentially coupling MBHA amino resin with phenyl p-nitrochloroformate and hydrazine hydrate, and sequentially connecting the following amino acids Fmoc-Pro-OH and Fmoc-Arg (NO) according to goserelin polypeptide sequence 2 )-OH、Fmoc-Leu-OHNHNHCONH 2 Obtaining fragments [1-3]]Peptide resin, cleavage of the resin, i.e.cleavage of the resin under the action of a cleavage agent, gives the fragment trileu 3 -Arg 2 (NO 2 )-Pro 1 -NHNHCONH 2 10 ；
(3) Adding 2-chlorotrityl resin into a solid phase reactor, performing coupling reaction with amino acid Fmoc-D-Ser (tBu) -OH under the action of condensing agent, and performing Fmoc deprotection reaction to obtain fragment di-linker D-Ser 4 (tBu)-2-CTC Resin；
(4) Coupling the fragment two connector with the fragment one under the action of a condensing agent by a solid phase method to obtain fragment [4-9] peptide resin, and cutting the resin to obtain a fragment two after the resin is cracked under the action of a cracking agent;
(5) Coupling the second segment and the third segment by a liquid phase method under the action of a condensing agent to obtain 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 the steps of carrying out coupling reaction and Fomc deprotection reaction under the action of condensing agent.
Further, in any step from step (1) to step (4), the condensing agent is one or more selected from HATU/HOBt/DIEA, HBTU/HOBt/DIEA, pyBop/HOBt/DIEA, DIC/HOBt; the condensing agent in the step (5) is one or more selected from HATU/HOBt/DIEA, HBTU/HOBt/DIEA and PyBop/HOBt/DIEA. Specifically, the condensing agent HATU/HOBt/DIEA represents a system comprising three of HATU, HOBt and DIEA, and the remaining condensing agents also represent the same meaning.
Furthermore, the cracking agent adopted in the step (1) or the step (2) is trifluoroacetic acid mixed solution, and the volume of the cracking agent is 7-15 times of the mass of the peptide resin; the mixed solution of the trifluoroacetic acid 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: TIS: water=70 to 97:10 to 1:10 to 1:10 to 1, TFA: EDT: water=90 to 95:1 to 5:1 to 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 the TFE is: dcm=25:75 to 85.
Further, the substitution degree of the Wang resin is 0.4-1.5mmol/g, the substitution degree of the MBHA amino resin is 0.4-1.0mmol/g, and the substitution degree of the 2-chlorotrityl resin is 0.4-1.0mmol/g.
Further, the solvent adopted in the reaction of each step is selected from one or more of dichloromethane, N-dimethylformamide, N-methylpyrrolidone and dimethyl sulfoxide.
The beneficial effects are that:
the invention adopts a synthesis method combining a solid phase method and a liquid phase method to realize goserelin synthesis, wherein the second and third fragments are easy to synthesize and purify, and have high purity; different fragments can be synthesized at the same time, so that the synthesis time of goserelin is effectively shortened, and the preparation efficiency is improved; finally, the liquid phase method is adopted to butt joint the two fragments to obtain goserelin precursor, so that low-cost coupling is realized, and the industrialized amplification preparation is facilitated.
Aiming at the problems that the Arg terminal has active guanidine group which has competitive side reaction on condensation reaction and is difficult to control in the prior art, the method brings about more impurities, difficult purification, reduced yield and the likeThe present invention provides a solid-liquid phase binding fragment synthesis method for introducing Arg (NO) 2 ) As an amino acid source in the fragment III, the reaction condition is mild, the side reaction is effectively avoided, and the problem that the amino acid sequence of the missing peptide, impurities and main components is more different can be effectively reduced; the reduction of the isomerization and racemization problems of amino acids is facilitated by the shortened reaction time. The invention makes the product easier to purify, the post-treatment operation is simple, the three wastes are reduced, and the purity of each intermediate is higher;
the method disclosed by the invention is stable in process, the purity of the prepared goserelin can reach more than 99.5%, and the total yield is higher.
Drawings
Fig. 1 is a flow chart of the goserelin preparation of the present invention.
Detailed Description
The present invention will be further described in detail with reference to the following examples and drawings for the purpose of enhancing the understanding of the present invention, which examples are provided for the purpose of illustrating the present invention only and are not to be construed as limiting the scope of the present invention.
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
Wang Reastin 60.00g and 600mLDCM are sequentially added into the solid phase reaction bottle, stirred for 15 minutes, and suction filtration is carried out after swelling to obtain a filter cake. To the flask were successively added DMF120mL, fmoc-Tyr (tBu) -OH (3 eq,41.40 g), DMAP (0.6 eq,2.19 g), DIC (3.0 eq,11.37 g) and HOBt (3.0 eq,12.17 g), and the mixture was stirred under nitrogen for 2 hours and suction filtration was carried out to obtain a cake. Washing the filter cake by using a proper amount of DMF, and carrying out suction filtration to obtain the filter cake. Fmoc deprotection reaction was performed using 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 applies below), stirring for 5 minutes, and carrying out suction filtration to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. Proper amount of filter cakeWashing with DMF and suction filtering to obtain filter cake. The amino acids Fmoc-Ser (tBu) -OH, fmoc-Trp (Boc) -OH, fmoc-His (Trt) -OH and Pyr-OH are condensed and connected in sequence and deprotected according to the method to obtain Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Wang Resin, namely the fragment-peptide Resin. 600ml of a solution of 90% TFA (TFA: water: EDT;90:5:5 by volume) was added as cleavage agent and stirred for 3 hours. And (5) carrying out suction filtration to obtain filtrate. Settling with proper amount of diethyl ether, centrifuging and washing to obtain solid. The solid was then subjected to settling with a suitable amount of diethyl ether, centrifugation, washing, and vacuum drying at 35℃to give 20.55g of fragment one, 89% (HPLC), mass spectrum [ M+H ]] + ＝703.5
Rink Amide MBHA Resin (45.00 g) and 600mLDCM are sequentially added into the solid phase reaction flask for synthesizing the third segment in the step (2), and filter cakes are obtained after swelling and suction filtration. Mixing the filter cake with 300ml of 20% pip/DMF solution, stirring for 5 minutes, and carrying out suction filtration to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. Washing the filter cake by a proper amount of DMF and DCM, and suction filtering to obtain the filter cake. To the flask was added phenyl p-nitrochloroformate (3 eq,12.00 g), DIEA (3 eq,9.00 g) and 300ml dcm in this order by filtration for 2 hours until the kaiser reagent detected colorless. Suction filtration, washing with a proper amount of DCM, and suction drying. Hydrazine hydrate (6 eq,15 mL), DIEA (4 eq,10.68 g) and DCM100mL were added sequentially and reacted for 8-12 hours. And (5) suction filtration. The filter cake was washed with an appropriate amount of DMF. Fmoc-Pro-OH (3 eq,20.40 g), pyBop (3 eq,33.31 g), HOBt (3 eq,8.70 g), DIEA (6 eq,16.13 g) and DMF (120 mL) were added sequentially to a 250mL reaction flask and stirred to give a solution. The new formulation was added to the solid phase reaction flask and stirred under nitrogen until the kaiser reagent was colorless. And (5) carrying out suction filtration to obtain a filter cake. Mixing the filter cake with 300ml of 20% pip/DMF solution, stirring for 5 minutes, and carrying out suction filtration to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. The filter cake was washed with an appropriate amount of DMF. And (5) carrying out suction filtration to obtain a filter cake. And condensing and connecting amino acids sequentially according to the method and deprotecting to obtain the fragment tripeptide resin. 600ml of a solution of 90% TFA (TFA: water: EDT;90:5:5, by volume) was added to the reaction mixture, and the mixture was stirred for 3 hours, followed by suction filtration to obtain a filtrate. Mixing the filtrate with proper amount of diethyl ether, settling, centrifuging and washing to obtain solid. Mixing the solid with proper amount of diethyl ether, settling, centrifuging, washing, and vacuum drying at 35 ℃ to obtain 10.68g of fragment III; 91% (HPLC), mass Spectrometry [ M+H] + ＝486.3
Step (3) Synthesis of fragment-di-linker peptide resin
15.00g of 2-CTC resin and 600mLDCM are added into a solid phase reaction bottle, and filter cakes are obtained after swelling and suction filtration. Fmoc-D-Ser (tBu) -OH (1.5 eq,6.30 g), DIEA (3 eq,4.50 g) and 60mLDCM were added in sequence and reacted for 2h. MeOH (15 mL) was added and stirred for 30min, filtered off with suction, and the filter cake washed with appropriate amount of DMF. Mixing the filter cake with 100ml of 20% pip/DMF solution, stirring for 5 minutes, and carrying out suction filtration to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. The filter cake was washed with an appropriate amount of DMF. Filtering to obtain fragment di-linker peptide resin which is directly used in the next step
Step (4) Synthesis of fragment two
The reaction was completed by adding fragment one (7 mmol,15.00 g), HATU (7 mmol,7.80 g), DIEA (14 mmol,6.30 g), HOBt (0.95 g) and DMF (20 mL) to the solid-phase reaction flask and reacting for 2 hours. Filtering, washing the filter cake with a proper amount of DMF, and filtering to obtain the filter cake. 150mL of DCM, 30mL of acetic anhydride and 30mL of pyridine were added sequentially and stirred for 30min. Filtering, washing the filter cake with DCM and methanol alternately, and filtering to obtain filter cake. 600mL of a solution of cleavage agent 20% TFE in DCM (DCM: TFE;80:20, vol.) was added, stirred for 2 hours and suction filtered to give a filtrate. Precipitating the filtrate with diethyl ether, centrifuging, and washing to obtain solid. The solid is subjected to sedimentation, centrifugation and washing by proper amount of diethyl ether, and then is dried in vacuum at 35 ℃ to obtain 8.25g of fragment II, 87% (HPLC) and mass spectrum [ M+H ]] + ＝846.1
Step (5) Synthesis of goserelin precursor
To the liquid phase flask were successively added fragment II (3.00 g), DIEA (2.76 g), HOBt (0.51 g), DMF36mL and HBTU (1.40 g), and the mixture was stirred at room temperature for 1h. Fragment three (2.25 g) was added and stirred at room temperature until the reaction was complete. Slowly adding the mixture into ice-cooled diethyl ether 420mL, precipitating solid, and removing solvent; the residue was mixed with 30mL of DMF and slowly added to 360mL of ice-cold diethyl ether, and the solid was separated off and the solvent was removed. Vacuum drying at 35deg.C to give 4.50g of white solid with 93% yield, 92% (HPLC), mass spectrum [ M+H ]] + = 1315.3. Directly used in the next step.
Step (6) synthesis of goserelin crude product
Sequentially adding into a liquid phase reaction bottleGoserelin precursor (3.00 g), methanol 75mL, acetic acid 3mL, and the solution was stirred at room temperature. 0.30g of 10% palladium on carbon was added under nitrogen. The reaction system was replaced with hydrogen three times and stirred at room temperature until the reaction was completed. Filtering to remove insoluble substances, and concentrating the filtrate at 35 ℃ under reduced pressure to obtain goserelin crude products. The residue was dissolved in 20mL of methanol, slowly added to 450mL of ice-cold diethyl ether, and the solvent was removed. HPLC89%, mass Spectrometry [ M+H ]] + = 1270.2; directly used in the next step.
Step (7) preparation of goserelin acetate refined peptide
1.50g of crude goserelin is weighed, mixed with 30mL of water containing 10% ammonia water and dissolved. Purification using a hanbang preparative HPLC system (210 nm wavelength, C8 reverse phase column, 20M ammonium dihydrogen phosphate solution, ph 3.0/acetonitrile as mobile phase) gave goserelin peptide solution with a purity of greater than 99.5%. Purifying the refined peptide solution by a preparative HPLC system (chromatographic column is C18 reverse phase column, 0.2% acetic acid/acetonitrile is mobile phase), collecting target components, concentrating under reduced pressure, and lyophilizing to obtain goserelin acetate 1.20g,99.85% (HPLC), with purification yield of 80%, and total yield of 32%; mass Spectrometry [ M+H ]] + ＝1270.4。
Example 2
The difference from example 1 is the synthesis of fragment one
The Wang resin 60.00g,600mLDCM was added to the synthesized solid phase reaction flask of the first fragment, stirred for 15 minutes, and suction filtered to obtain a filter cake. To the flask were successively added DMF120mL, fmoc-Tyr (tBu) -OH (3 eq,41.40 g), catalyst DMAP (0.6 eq,2.19 g), HBTU (3.0 eq,34.14 g), HOBt (3.0 eq,12.17 g) and DIEA (3.0 eq,11.67 g) under nitrogen with stirring for 2 hours and suction filtration to give a filter cake. Washing the filter cake by using a proper amount of DMF, and carrying out suction filtration to obtain the filter cake. Mixing the filter cake with 300ml of 20% pip/DMF solution, stirring for 5 minutes, and carrying out suction filtration to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. Washing the filter cake with a proper amount of DMF, and carrying out suction filtration to obtain a filter cake;
the amino acids Fmoc-Ser (tBu) -OH, fmoc-Trp (Boc) -OH, fmoc-His (Trt) -OH, pyr-OH and deprotection were sequentially condensed and ligated as described above to give Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Wang Resin, i.e., fragment-peptideAnd (3) resin. 600ml of 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 diethyl ether, centrifuging and washing to obtain solid. The solid was then subjected to settling with a suitable amount of diethyl ether, centrifugation, washing and vacuum drying at 35℃to give 21.63g of fragment I, 91% (HPLC), mass spectrum [ M+H ]] + ＝703.3
Example 3
The difference from example 1 is the synthesis of fragment one
Synthesis of fragment one
Wang resin 60.00g and 600 mM LDCM are added into the solid phase reaction bottle in sequence, stirred for 15 minutes and filtered by suction to obtain a filter cake. To the flask were successively added DMF120mL, fmoc-Tyr (tBu) -OH (3 eq,41.40 g), catalyst DMAP (0.6 eq,2.19 g), pyBop (3.0 eq,46.87 g), HOBt (3.0 eq,12.17 g) and DIEA (3.0 eq,11.67 g) under nitrogen and stirred for 2 hours, followed by suction filtration to obtain a filter cake. Washing the filter cake by using a proper amount of DMF, and carrying out suction filtration 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 suction filtering to obtain a filter cake; 300ml of 20% pip/DMF solution was added, stirred for 15 minutes and suction filtered. Washing the filter cake with DMF, and suction filtering to obtain filter cake
The amino acids Fmoc-Ser (tBu) -OH, fmoc-Trp (Boc) -OH, fmoc-His (Trt) -OH and Pyr-OH are condensed and connected in sequence and deprotected according to the method to obtain Pyr-His (Trt) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -Wang Resin, namely the fragment-peptide Resin. A solution of 600ml of 85% TFA (TFA: phSMe: TIS: water=85:5:5; volume ratio) was added and stirred for 3 hours. And (5) carrying out suction filtration to obtain filtrate. Settling with proper amount of diethyl ether, centrifuging and washing to obtain solid. The solid was then subjected to settling with an appropriate amount of diethyl ether, centrifugation, washing, and vacuum drying at 35℃to give 19.08g of fragment one, 92% (HPLC), mass spectrum [ M+H ]] + ＝703.5。
Example 4
The difference from example 1 is the synthesis of goserelin precursors
Synthesis of goserelin precursors
To the liquid phase flask were successively added fragment II (1.00 g), DIEA (0.92 g), HOBt (0.17 g), DMF12mL and HATU (0.46 g), followed by stirring at room temperature for 1h. Add fragment three (0.75 g), chamberStirred at the temperature for 8 hours until the reaction is finished. Slowly adding the reaction solvent into ice-cooled diethyl ether 140mL, separating out solid, and filtering the solvent; the residue was taken up in DMF100mL and slowly added to ice-cold diethyl ether 120mL, the solid was isolated and the solvent was filtered off. Vacuum drying at 35deg.C to obtain white solid 1.50g, 96% yield, 73% (HPLC), mass spectrum [ M+H ]] + ＝1315.0。
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (2)
1. The synthesis method of goserelin is characterized by comprising the following process steps:
(1) Adding Wang resin into a solid phase reactor, performing coupling reaction with Fmoc-Tyr (tBu) -OH under the action of condensing agents DIC/HOBt, HBTU/HOBt/DIEA or PyBop/HOBt/DIEA, and then performing Fomc deprotection reaction; the following amino acids Fmoc-Ser (tBu) -OH, fmoc-Trp (Boc) -OH, fmoc-His (Trt) -OH and Pyr-OH are sequentially connected according to goserelin polypeptide sequences to obtain fragments [5-9]]A peptide resin; cutting the resin to obtain a segment Pyr after the resin is cracked under the action of a cracking agent 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -OH；
(2) Sequentially coupling MBHA amino resin with p-nitro chloroformate and hydrazine hydrate, condensing agent PyBop/HOBt/DIEA, and sequentially connecting the following amino acids Fmoc-Pro-OH and Fmoc-Arg (NO) according to goserelin polypeptide sequence 2 )-OH、Fmoc-Leu-OHNHNHCONH 2 Obtaining fragments [1-3]]Peptide resin, cleavage of the resin, i.e.cleavage of the resin under the action of a cleavage agent, gives the fragment trileu 3 -Arg 2 (NO 2 )-Pro 1 -NHNHCONH 2 10 ；
(3) Adding 2-chlorotrityl resin into a solid phase reactor, performing coupling reaction with amino acid Fmoc-D-Ser (tBu) -OH under the action of condensing agent DIEA, and performing Fmoc deprotection reaction to obtain fragment di-linker D-Ser 4 (tBu)-2-CTC Resin；
(4) Coupling the fragment two joints with the fragment one under the action of a condensing agent HATU/HOBt/DIEA by a solid phase method to obtain a fragment [4-9] peptide resin, and cleaving the resin to obtain a fragment two after the resin is cleaved under the action of a cleaving agent, wherein the fragment two has the structural formula:
Pyr 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -D-Ser 4 (tBu)-OH
(5) Coupling the second segment and the third segment under the action of condensing agent HBTU/HOBt/DIEA or HATU/HOBt/DIEA by a liquid phase method to obtain goserelin precursor, wherein the goserelin precursor has the structural formula:
Pyr 9 -His 8 -Trp 7 -Ser 6 -Tyr 5 -D-Ser 4 (tBu)-Leu 3 -Arg 2 (NO 2 )-Pro 1 -NHNHCONH 2 10
(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 cracking agent adopted in the step (1) or the step (2) is trifluoroacetic acid mixed solution, and the volume of the cracking agent is 7-15 times of the mass of the peptide resin; the mixed solution of the trifluoroacetic acid 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: TIS: water=70 to 97:10 to 1:10 to 1:10 to 1, TFA: EDT: water=90 to 95:1 to 5:1 to 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 the TFE is: dcm=25:75 to 85;
the substitution degree of the Wang resin is 0.4-1.5mmol/g,
the substitution degree of the MBHA amino resin is 0.4-1.0mmol/g, and the substitution degree of the 2-chlorotrityl resin is 0.4-1.0mmol/g.
2. The process according to claim 1, wherein the solvent used in the reaction of each step is selected from DCM and/or DMF.
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| 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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