CN105330726A - Leuprorelin synthesis method - Google Patents
Leuprorelin synthesis method Download PDFInfo
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- CN105330726A CN105330726A CN201510741266.6A CN201510741266A CN105330726A CN 105330726 A CN105330726 A CN 105330726A CN 201510741266 A CN201510741266 A CN 201510741266A CN 105330726 A CN105330726 A CN 105330726A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a leuprorelin synthesis method. According to the method, a dipeptide midbody is obtained through liquid-phase synthesis and then used for solid-phase synthesis to generate full-protection nonapeptide peptide resin, full-protection nonapeptide is cut off from the resin and then inoculated with ethylamine in the liquid phase to generate full-protection leuprorelin, and a leuprorelin crude product is obtained through splitting decomposition. The method specifically comprises the steps of obtaining Fmoc-Leu-OSU fat through condensation under the action of a condensing agent; reacting with H-Arg(pbf)-OH under the action of alkali to generate Fmoc-Leu-Arg(pbf)-OH; obtaining Fmoc-Pro-CTC Resin through reaction under the action of the alkali; removing Fmoc, and conducting amino acid coupling in sequence under the action of a condensing agent to generate full-protection nonapeptide peptide resin; cutting the full-protection nonapeptide peptide resin with a cutting reagent to generate full-protection nonapeptide; generating leuprorelin full-protection peptide by means of full-protection nonapeptide and ethylamine hydrochloride under the action of a condensing agent, and obtaining the leuprorelin crude product through splitting decomposition. By the adoption of the method, the yield and purity of leuprorelin are improved remarkably, ammonolysis is not needed, and reaction is easy and controllable. The method is suitable for industrial production.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical field, particularly the solid-liquid synthetic method of Leuprolide.
Background technology
The outer literary fame of Leuprolide or general Leuprorelin by name, have following structure:
L-pyroglutamyl-L-histidyl--L-tryptophyl-L-seryl-L-tyrasamine acyl-D-leucyl-L-leucyl-L-arginyl-L-prolyl ethamine
Leuprolide is the high reactivity derivative that short corpus luteum generates releasing hormone (LHRH), can produce transient hypophysis-Sexual gland system excitation (acute effect) after first administration immediately, then suppresses hypophysis to generate and release gonad-stimulating hormone.It can also suppress ovary and testis to the reaction of gonad-stimulating hormone further, thus reduces the generation (chronic effect) of estradiol and testosterone.So the function of hypophysis-Sexual gland system effectively can be suppressed.Leuprolide is widely used in the male patients such as female patient and prostate cancer such as treatment precocity, endometriosis, hysteromyoma or premenopausal breast cancer patients.
Beautiful
state is publicopen in patent documentation US4690916 the report having synthesis Leuprolide.Use Merrifield resin, adopt Boc method solid phase synthesis full guard nonapeptide fragment, use ethamine ammonia solution resin at low temperatures, then remove Side chain protective group through HF and obtain object product.This method ammonia solution yield is low, very easily causes the racemization of peptide chain.Patent CN101538315A and CN1865280A adopts Fmoc Solid phase synthesis full guard nonapeptide, then liquid phase connects ethamine modification.This method cannot avoid diketopiperazine side reaction completely, and when amplifying production, diketopiperazine side reaction causes peptide chain to come off, and significantly reduces yield.
Therefore research and develop that a kind of yield is high, reaction is simply controlled, the synthetic method being suitable for the Leuprolide of suitability for industrialized production new is significant.
Summary of the invention
Technical problem to be solved by this invention is to the deficiencies in the prior art, and provide a kind of solid liquid phase of Leuprolide in conjunction with synthetic method, the method improves yield and the purity of Leuprolide significantly, without the need to ammonia solution, reacts simply controlled, is applicable to industrial production.
Technical problem to be solved by this invention is realized by following technical scheme.The present invention is a kind of synthetic method of Leuprolide, be characterized in: the method adopts liquid phase synthesis dipeptides intermediate to be used further to solid phase synthesis and obtains full guard nonapeptide peptide resin, full guard nonapeptide is cut down from resin and connects ethamine in the liquid phase and obtain full guard Leuprolide, obtain Leuprolide crude product through cracking;
Its concrete steps are as follows:
(1) under condensing agent effect, Fmoc-Leu-OSU fat is obtained by Fmoc-Leu-OH and HOSU condensation;
(2) Fmoc-Leu-OSU fat and H-Arg (pbf)-OH are obtained by reacting Fmoc-Leu-Arg (pbf)-OH under alkali effect;
(3) Fmoc-Pro-OH and CTCResin is obtained by reacting Fmoc-Pro-CTCResin under alkali effect;
(4) Fmoc is sloughed, under condensing agent effect, the following amino acid of coupling: Fmoc-Leu-Arg (pbf)-OH successively, Fmoc-D-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH, obtain full guard nonapeptide peptide resin;
(5) cutting reagent cutting full guard nonapeptide peptide resin is used to obtain full guard nonapeptide;
(6) full guard nonapeptide and ethylamine hydrochloride obtain Leuprolide full guard peptide under condensing agent effect, obtain Leuprolide crude product through cracking.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme be: the condensing agent described in step (1) is selected from DCC, in DIC, EDCHCl any one.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme be: the alkali described in step (2) is selected from DIEA, in NMM, Py, DMAP, NaOH, NaHCO3, Na2CO3 any one.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme is: in step (3), CTCResin substitution degree is 0.5-2.0mmol/g.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme is: in step (3), CTCResin substitution degree is 1.0-1.5mmol/g.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme be: the alkali described in (3) is selected from DIEA, in NMM, Py, DMAP any one.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme is: the concrete operations of (4) are as follows:
4.1 adopt DBLK to make described Fmoc-Pro-CTCResin remove Fmoc protecting group, and obtain H-Pro-CTCResin, described DBLK is the solution of PIP/DMF=1/4;
4.2 condensing agent system exist under H-Pro-CTCResin and Fmoc-Leu-Arg (pbf)-OH coupling obtain Fmoc-Leu-Arg (pbf)-Pro-CTCResin;
4.3 repeating steps 4.1 and 4.2 obtain full guard nonapeptide peptide resin.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme is: coupling described in step (4) uses condensing agent and reaction solvent, described condensing agent is selected from DIC/HOBt, HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM combine one of them, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; Described reaction solvent is selected from DMF, DCM, NMP, DMSO or the arbitrary combination between them.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme be: institute's consumption of throwing Fmoc-Leu-Arg (pbf)-OH by 1.2-6 times of throwing CTCResin total mole number, preferably 1.5-2.5; Coupling time is 40-180 minute under room temperature condition, is preferably 60-90 minute.
The synthetic method of a kind of Leuprolide of the present invention, its further preferred technical scheme is: the concrete grammar of described step (5) and (6) is as follows: cut peptide resin under using 1-2%TFA/DCM or the 20%TFE/DCM room temperature of full guard nonapeptide peptide resin weight (g) 10 times of volumes (mL) and obtain full guard nonapeptide: be dissolved in organic solvent by full guard nonapeptide, ethylamine hydrochloride, be cooled to less than 5 DEG C, add coupling agent, room temperature reaction; Wherein organic solvent is selected from DMF, THF; Coupling agent is selected from HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM, and one of them combines, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; The solvent that the solution of preparation coupling agent adopts is selected from DMF, THF; The time of reaction is 30-300 minute under room temperature condition, 30-60 minute under preferred room temperature condition; After completion of the reaction, Leuprolide crude product is obtained by conventional aftertreatment; A concrete post-treating method is: reaction solution is poured into crystallization in the saturated sodium bicarbonate solution of 8 times of volumes, gained solid water washs 4 times, dries, obtains Leuprolide full guard nonapeptide; Full guard nonapeptide adds in the lysate of 8 times of volumes after cracking 2h, and add crystallization in the ice ether of 6 times of volumes, gained solid ether grinds washes centrifugal 3 times, and vacuum decompression is dry, obtains Leuprolide crude product.
In synthetic method provided by the invention, used raw material, reagent and intermediate II all can be reached by market.Chinese corresponding to the english abbreviation that the present invention relates to is shown in
following table:
Compared with prior art, the inventive method adopts liquid phase synthesis dipeptides intermediate to be used further to solid phase synthesis and obtains full guard nonapeptide peptide resin,
Full guard nonapeptide is cut down from resin and connects ethamine in the liquid phase and obtain full guard Leuprolide, obtain Leuprolide crude product through cracking.The method considerably improves yield and the purity of Leuprolide, without the need to ammonia solution, reacts simply controlled, is applicable to industrial production.
Accompanying drawing explanation
Fig. 1 is the MS figure of sintetics of the present invention;
Fig. 2 is the HPLC figure of sintetics of the present invention.
Embodiment
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1, a kind of synthetic method of Leuprolide: the method adopts liquid phase synthesis dipeptides intermediate to be used further to solid phase synthesis and obtains full guard nonapeptide peptide resin, full guard nonapeptide is cut down from resin and connects ethamine in the liquid phase and obtain full guard Leuprolide, obtain Leuprolide crude product through cracking;
Its concrete steps are as follows:
(1) under condensing agent effect, Fmoc-Leu-OSU fat is obtained by Fmoc-Leu-OH and HOSU condensation;
(2) Fmoc-Leu-OSU fat and H-Arg (pbf)-OH are obtained by reacting Fmoc-Leu-Arg (pbf)-OH under alkali effect;
(3) Fmoc-Pro-OH and CTCResin is obtained by reacting Fmoc-Pro-CTCResin under alkali effect;
(4) Fmoc is sloughed, under condensing agent effect, the following amino acid of coupling: Fmoc-Leu-Arg (pbf)-OH successively, Fmoc-D-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH, obtain full guard nonapeptide peptide resin;
(5) cutting reagent cutting full guard nonapeptide peptide resin is used to obtain full guard nonapeptide;
(6) full guard nonapeptide and ethylamine hydrochloride obtain Leuprolide full guard peptide under condensing agent effect, obtain Leuprolide crude product through cracking.
Embodiment 2, in the synthetic method of a kind of Leuprolide described in embodiment 1: the condensing agent described in step (1) is selected from DCC, in DIC, EDCHCl any one.
Embodiment 3, in the synthetic method of a kind of Leuprolide described in embodiment 1 or 2: the alkali described in step (2) is selected from DIEA, in NMM, Py, DMAP, NaOH, NaHCO3, Na2CO3 any one.
Embodiment 4, in the synthetic method of a kind of Leuprolide described in embodiment 1: in step (3), CTCResin substitution degree is 0.5-2.0mmol/g.
Embodiment 5, in the synthetic method of a kind of Leuprolide described in embodiment 1: in step (3), CTCResin substitution degree is 1.0-1.5mmol/g.
Embodiment 6, in the synthetic method of a kind of Leuprolide described in embodiment 1: in step (3), CTCResin substitution degree is 1.2mmol/g.
Embodiment 7, in the synthetic method of a kind of Leuprolide described in embodiment 1-6 any one: the alkali described in step (3) is selected from DIEA, in NMM, Py, DMAP any one.
Embodiment 8, in the synthetic method of a kind of Leuprolide described in embodiment 1-7 any one: the concrete operations of step (4) are as follows:
4.1 adopt DBLK to make described Fmoc-Pro-CTCResin remove Fmoc protecting group, and obtain H-Pro-CTCResin, described DBLK is the solution of PIP/DMF=1/4;
4.2 condensing agent system exist under H-Pro-CTCResin and Fmoc-Leu-Arg (pbf)-OH coupling obtain Fmoc-Leu-Arg (pbf)-Pro-CTCResin;
4.3 repeating steps 4.1 and 4.2 obtain full guard nonapeptide peptide resin.
Embodiment 9, in the synthetic method of a kind of Leuprolide described in embodiment 1-8 any one: coupling described in step (4) uses condensing agent and reaction solvent, described condensing agent is selected from DIC/HOBt, HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM combine one of them, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; Described reaction solvent is selected from DMF, DCM, NMP, DMSO or the arbitrary combination between them.
Embodiment 10, in the synthetic method of a kind of Leuprolide described in embodiment 1-9 any one: throw Fmoc-Leu-Arg (pbf)-OH consumption by throwing CTCResin total mole number 1.2-6 doubly; Coupling time is 40-180 minute under room temperature condition.
Embodiment 11, in the synthetic method of a kind of Leuprolide described in embodiment 1-9 any one: throw Fmoc-Leu-Arg (pbf)-OH consumption by throwing CTCResin total mole number 1.5-2.5 doubly; Coupling time is 60-90 minute under room temperature condition.
Embodiment 12, in the synthetic method of a kind of Leuprolide described in embodiment 1-9 any one: throw Fmoc-Leu-Arg (pbf)-OH consumption by 2 times of throwing CTCResin total mole number; Coupling time is lower 75 minutes of room temperature condition.
Embodiment 13, in the synthetic method of a kind of Leuprolide described in embodiment 1-12 any one: the concrete grammar of described step (5) and (6) is as follows: cut peptide resin under using 1-2%TFA/DCM or the 20%TFE/DCM room temperature of full guard nonapeptide peptide resin weight (g) 10 times of volumes (mL) and obtain full guard nonapeptide: full guard nonapeptide, ethylamine hydrochloride are dissolved in organic solvent, be cooled to less than 5 DEG C, add coupling agent, room temperature reaction; Wherein organic solvent is selected from DMF, THF; Coupling agent is selected from HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM, and one of them combines, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; The solvent that the solution of preparation coupling agent adopts is selected from DMF, THF; The time of reaction is 30-300 minute under room temperature condition, 30-60 minute under preferred room temperature condition; After completion of the reaction, Leuprolide crude product is obtained by conventional aftertreatment; A concrete post-treating method is: reaction solution is poured into crystallization in the saturated sodium bicarbonate solution of 8 times of volumes, gained solid water washs 4 times, dries, obtains Leuprolide full guard nonapeptide; Full guard nonapeptide adds in the lysate of 8 times of volumes after cracking 2h, and add crystallization in the ice ether of 6 times of volumes, gained solid ether grinds washes centrifugal 3 times, and vacuum decompression is dry, obtains Leuprolide crude product.
Embodiment 14, a kind of synthetic method experiment of Leuprolide:
1, the preparation of Fmoc-Leu-Arg (pbf)-OH
Take Fmoc-Leu-OH35.35g, HOSU12.66g is placed in there-necked flask, adds DCM200mL, stirring and dissolving, is cooled to 3 ± 2 DEG C.Take DCC23.31g, be dissolved in 100mLDCM, be added dropwise in above-mentioned flask, temperature of reaction 5 ± 2 DEG C, reaction 3h.Elimination solid, 35 DEG C of concentrated liquids, to oily, add 200mL acetic acid ethyl dissolution.Purified water washs 4 times, each 100mL, and gained organic phase adds anhydrous sodium sulfate drying, and elimination solid gained liquid 35 DEG C is concentrated into oily, adds 100mLTHF and dissolves, and obtains the refrigeration of Fmoc-Leu-OSU solution stand-by.
Take 51.18gH-Arg (pbf)-OH51.18g, 10.00gNaHCO3 and be dissolved in the 200mL50%THF/ aqueous solution, 1NNaOH (aq) adjusts pH value of solution to 8-8.5, is cooled to 5 DEG C.Drip Fmoc-Leu-OSU solution, 1NNaOH (aq) keeps pH value of solution 8-8.5,10 ± 2 DEG C of reaction 3h.Reaction terminates rear filtering solid, and add 200mL extraction into ethyl acetate, aqueous phase uses 100mL extraction into ethyl acetate once again, abandons aqueous phase and merges organic phase.Once, once, each 200mL water washing is to pH value 3.5-4.5 in 1NHCl200mL washing in organic phase 200mL water washing.Add anhydrous sodium sulfate drying, elimination solid gained liquid concentration is to dry.Obtain solid 66.75g, yield 87.6%.
2, the preparation of Fmoc-Pro-CTCResin
Get CTCResin (1.2mmol/g) 50.00g and be placed in polypeptide reactor, add DCM400mL nitrogen and brush stirring 30 minutes, after making resin fully swelling, drain DCM.Take Fmoc-Pro-OH60.75g, add DMF3400mL stirring and dissolving, add DIEA94.5mL, ice bath adds after 15 minutes in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, and DMF washs 3 times, each 400mL, 1 minute.Add closed reagent (anhydrous methanol/DIEA/DCM=1/2/17 (volume ratio)) and close twice each 400mL, 10 minutes.DCM washs 4 times, each 400mL, 10 minutes.Take out resin, dry.Obtain Fmoc-Pro-CTCResin82.35g.Substitution value is 0.73mmol/g.
3, the preparation of peptide resin
Get Fmoc-Pro-CTCResin13.70g (10mmol) and be placed in polypeptide reactor, add DCM100mL nitrogen and brush stirring 30 minutes, after making resin fully swelling, drain DCM.Add DBLK80mL to take off Fmoc and protect 2 times, 5+15 minute, each 80mL.DMF washs 5 times, each 80mL, 1 minute.Take Fmoc-Leu-Arg (pbf)-OH22.86g (30mmol), HOBt4.86g (36mmol), add DMF80mL stirring and dissolving, ice bath 5 minutes, adds DIC5.6mL (36mmol), and ice bath adds after activating 15 minutes in polypeptide reactor, nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washs 3 times, each 100mL, 1 minute.According to said method access Fmoc-D-Leu-OH successively, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH.Obtain full guard nonapeptide peptide resin 27.8g.
4, the preparation of peptide resin
Get Fmoc-Pro-CTCResin13.70g (10mmol) and be placed in polypeptide reactor, add DCM100mL nitrogen and brush stirring 30 minutes, after making resin fully swelling, drain DCM.Add DBLK80mL to take off Fmoc and protect 2 times, 5+15 minute, each 80mL.DMF washs 5 times, each 80mL, 1 minute.Take Fmoc-Leu-Arg (pbf)-OH22.86g (30mmol), HBTU10.24g (27mmol), HOBt4.05g (30mmol), adds DMF80mL stirring and dissolving, ice bath 5 minutes, add DIEA7.8mL (45mmol), ice bath adds after activating 15 minutes in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, and DMF washs 3 times, each 100mL, 1 minute.According to said method access Fmoc-D-Leu-OH successively, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH.Obtain full guard nonapeptide peptide resin 25.3g.
5, the preparation of peptide resin
Get Fmoc-Pro-CTCResin13.70g (10mmol) and be placed in polypeptide reactor, add DCM100mL nitrogen and brush stirring 30 minutes, after making resin fully swelling, drain DCM.Add DBLK80mL to take off Fmoc and protect 2 times, 5+15 minute, each 80mL.DMF washs 5 times, each 80mL, 1 minute.Take Fmoc-Leu-Arg (pbf)-OH22.86g (30mmol), HCTU11.17g (27mmol), HOBt4.05g (30mmol), adds DMF80mL stirring and dissolving, ice bath 5 minutes, add NMM10.0mL (90mmol), ice bath adds after activating 15 minutes in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, and DMF washs 3 times, each 100mL, 1 minute.According to said method access Fmoc-D-Leu-OH successively, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH.Obtain full guard nonapeptide peptide resin 26.7g.
6, cut
Get obtained full guard nonapeptide peptide resin 26.7g, to add in 215mL1%TFA/DCM solution stirring at room temperature cracking 2.0 hours.Elimination resin, adds Anhydrous potassium carbonate 5g dry 8 hours.Filter, concentrated filtrate, pours into filtrate in the normal hexane of 1290mL.Filter, gained solid is dried, obtains full guard nonapeptide 11.2g, yield 59.3%.
7 cuttings
Get above-mentioned " 3 " gained full guard nonapeptide peptide resin 27.8g, to add in 215mL20%TFE/DCM solution stirring at room temperature cracking 1.5 hours.Filtering resin, then to add in 215mL20%TFE/DCM solution stirring at room temperature cracking elimination resin after 1.5 hours, merging filtrate, concentrated, filtrate is poured in the normal hexane of 6 times of volumes.Filter, gained solid is dried, obtains full guard nonapeptide 14.6g.Yield 77.3%.
The synthesis of 8 Leuprolide full guard peptides
Get above-mentioned " 6 " gained full guard nonapeptide 11.2g (5.9mmol), HOSu0.75g (6.49mmol), is dissolved in 50mLDCM, is cooled to 5 DEG C.Get DCC1.34g (6.49mmol), be dissolved in 10mLDCM, to instill in above-mentioned solution of having lowered the temperature 5 DEG C of reactions 3 hours.Get ethylamine hydrochloride 0.72g (8.85mmol), DIEA1.5mL is dissolved in 50mLDCM, is cooled to 10 DEG C.Drip above-mentioned reaction solution, 10 DEG C of reaction 1h.Filter, filtrate is concentrated into oily, adds ethyl acetate 50mL and dissolves.Purified water 50mL fully shakes washing, filters, and filtrate leaving standstill abandons aqueous phase.Organic phase saturated common salt water washing 4 times, each 50mL; Purified water washs 4 each 50mL.Gained organic phase anhydrous sodium sulfate drying 8 hours, filters, and concentrated filtrate, to dry, obtains Leuprolide full guard peptide 10.6g, yield 93.3%.
9, the synthesis of Leuprolide full guard peptide
Get above-mentioned " 7 " gained full guard nonapeptide 14.6g (7.6mmol), HBTU4.32g (11.4mmol), HOBT1.54g (11.4mmol) ethylamine hydrochloride 0.93g (11.4mmol), is dissolved in 50mLDMF.Drip DIEA6.0mL room temperature reaction 1 hour.Reaction solution is poured into crystallization in 400mL saturated sodium bicarbonate solution, gained Purify water rinses 4 times, dry.Obtain Leuprolide full guard peptide 14.2g, yield 95.7%, total recovery 74.0%.
10, cracking
Preparation TFA: the lysate 85mL ice bath to 5 DEG C of thioanisole: EDT: methyl-phenoxide=90:5:3:2 (volume ratio).Getting above-mentioned " 6 " gained Leuprolide full guard peptide 10.6g adds in above-mentioned lysate, and lysis at room temperature 2h, adds crystallization in 510mL ice ether.Centrifugal, gained solid ether grinds washes centrifugal 3 times, and vacuum decompression is dry.Obtain thick peptide 11.4g, HPLC quantitatively containing object peptide g, yield 77.4%, total recovery 42.8%.
11, cracking
The lysate 115mL ice bath to 5 DEG C of preparation TFA:TIS:EDT: water=92.5:2.5:2.5:2.5 (volume ratio).Getting above-mentioned " 6 " gained Leuprolide full guard peptide 14.2g adds in above-mentioned lysate, and lysis at room temperature 2h, adds crystallization in 690mL ice ether.Centrifugal, gained solid ether grinds washes centrifugal 3 times, and vacuum decompression is dry.Obtain thick peptide 15.6g, HPLC quantitatively containing object peptide g, yield 84.7%, total recovery 62.7%.
Products obtained therefrom MS, HPLC is shown in
fig. 1with
fig. 2.
The above is only the preferred embodiment of the present invention, and those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art they be all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, apply and realize the technology of the present invention.
Claims (10)
1. the synthetic method of a Leuprolide, it is characterized in that: the method adopts liquid phase synthesis dipeptides intermediate to be used further to solid phase synthesis and obtains full guard nonapeptide peptide resin, full guard nonapeptide is cut down from resin and connects ethamine in the liquid phase and obtain full guard Leuprolide, obtain Leuprolide crude product through cracking;
Its concrete steps are as follows:
(1) under condensing agent effect, Fmoc-Leu-OSU fat is obtained by Fmoc-Leu-OH and HOSU condensation;
(2) Fmoc-Leu-OSU fat and H-Arg (pbf)-OH are obtained by reacting Fmoc-Leu-Arg (pbf)-OH under alkali effect;
(3) Fmoc-Pro-OH and CTCResin is obtained by reacting Fmoc-Pro-CTCResin under alkali effect;
(4) Fmoc is sloughed, under condensing agent effect, the following amino acid of coupling: Fmoc-Leu-Arg (pbf)-OH successively, Fmoc-D-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Trp (Boc)-OH, Fmoc-His (Trt)-OH, Pyr-OH, obtain full guard nonapeptide peptide resin;
(5) cutting reagent cutting full guard nonapeptide peptide resin is used to obtain full guard nonapeptide;
(6) full guard nonapeptide and ethylamine hydrochloride obtain Leuprolide full guard peptide under condensing agent effect, obtain Leuprolide crude product through cracking.
2. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: the condensing agent described in step (1) is selected from DCC, in DIC, EDCHCl any one.
3. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: the alkali described in step (2) is selected from DIEA, NMM, Py, DMAP, NaOH, NaHCO
3, Na
2cO
3in any one.
4. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: in step (3), CTCResin substitution degree is 0.5-2.0mmol/g.
5. the synthetic method of a kind of Leuprolide according to claim 4, is characterized in that: in step (3), CTCResin substitution degree is 1.0-1.5mmol/g.
6. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: the alkali described in step (3) is selected from DIEA, in NMM, Py, DMAP any one.
7. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: the concrete operations of step (4) are as follows:
4.1 adopt DBLK to make described Fmoc-Pro-CTCResin remove Fmoc protecting group, and obtain H-Pro-CTCResin, described DBLK is the solution of PIP/DMF=1/4;
4.2 condensing agent system exist under H-Pro-CTCResin and Fmoc-Leu-Arg (pbf)-OH coupling obtain Fmoc-Leu-Arg (pbf)-Pro-CTCResin;
4.3 repeating steps 4.1 and 4.2 obtain full guard nonapeptide peptide resin.
8. the synthetic method of a kind of Leuprolide according to claim 1 or 7, it is characterized in that: coupling described in step (4) uses condensing agent and reaction solvent, described condensing agent is selected from DIC/HOBt, HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM combine one of them, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; Described reaction solvent is selected from DMF, DCM, NMP, DMSO or the arbitrary combination between them.
9. the synthetic method of a kind of Leuprolide according to claim 1, is characterized in that: throw Fmoc-Leu-Arg (pbf)-OH consumption by throwing CTCResin total mole number 1.2-6 doubly, be preferably 1.5-2.5; Coupling time is 40-180 minute under room temperature condition, is preferably 60-90 minute.
10. the synthetic method of a kind of Leuprolide according to claim 1, it is characterized in that: the concrete grammar of described step (5) and (6) is as follows: cut peptide resin under using 1-2%TFA/DCM or the 20%TFE/DCM room temperature of full guard nonapeptide peptide resin weight (g) 10 times of volumes (mL) and obtain full guard nonapeptide: full guard nonapeptide, ethylamine hydrochloride are dissolved in organic solvent, be cooled to less than 5 DEG C, add coupling agent, room temperature reaction; Wherein organic solvent is selected from DMF, THF; Coupling agent is selected from HBTU, HCTU, TBTU, PyBOP one of them and HOBt, DIEA, NMM, and one of them combines, or HATU, PyAOP one of them and HOAt, DIEA, NMM one of them combine; The solvent that the solution of preparation coupling agent adopts is selected from DMF, THF; The time of reaction is 30-300 minute under room temperature condition, 30-60 minute under preferred room temperature condition; After completion of the reaction, Leuprolide crude product is obtained by conventional aftertreatment; A concrete post-treating method is: reaction solution is poured into crystallization in the saturated sodium bicarbonate solution of 8 times of volumes, gained solid water washs 4 times, dries, obtains Leuprolide full guard nonapeptide; Full guard nonapeptide adds in the lysate of 8 times of volumes after cracking 2h, and add crystallization in the ice ether of 6 times of volumes, gained solid ether grinds washes centrifugal 3 times, and vacuum decompression is dry, obtains Leuprolide crude product.
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