CN104844694A - Ganirelix acetate preparation method - Google Patents
Ganirelix acetate preparation method Download PDFInfo
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- CN104844694A CN104844694A CN201410052402.6A CN201410052402A CN104844694A CN 104844694 A CN104844694 A CN 104844694A CN 201410052402 A CN201410052402 A CN 201410052402A CN 104844694 A CN104844694 A CN 104844694A
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Abstract
The present invention belongs to the technical field of drug synthesis, and discloses a ganirelix acetate preparation method, wherein Fmoc-Lys (Boc)-OH and Fmoc-D-Lys (Boc)-OH are adopted to respectively replace Fmoc-HArg (Et)2-OH and Fmoc-D-HArg (Et)2-OH, a ganirelix precursor I is previously synthesized, and the side chain amino of Lys and D-Lys in the precursor I is modified and treated so as to obtain the ganirelix acetate. According to the present invention, water is adopted to replace the organic solvent and is adopted as the reaction solvent, such that the reaction can be performed within the established pH value range, and the product content in the crude peptide is significantly improved; and the product yield can be significantly improved through the HPLC two-step purification method.
Description
Technical field
The present invention relates to technical field of medicine synthesis, be specifically related to a kind of preparation method of ganirelix acetate.
Background technology
Ganirelix acetate, English name is Ganirelix acetate; Chemical name is N-acetyl-3-(2-napthyl)-D-alanyl-4-chloro-D-phnylalanyl-3-(3-pyridyl)-D-alanyl-L-tyrosyl-N
9, N
10diethyl-D-homoarginyl-L-leucyl-N
9, N
10-diethyl-L-homoarginyl-L-prolyl-D-alanylamide acetate; Molecular formula is C
80h
113clN
18o
13; Relative molecular mass is 1570.3; CAS registration number is 123246-29-7.Its chemical structure is shown below:
Ganirelix acetate be a synthesis with endogenous gonadoliberin (GnRH) (also known as interstitialcellstimulating hormone (ICSH) liberin, LHRH) similar decapeptide compound, it is the antagonist of GnRH, the gonadotropic GnRH acceptor of its contestable antagonism, thus variation approach, cause fast, reversibly suppress gonad-stimulating hormone (lutropin LH and follicle stimulating hormone FSH) to be secreted.Ganirelix acetate suppresses pituitary LH more remarkable than secretion inhibitor FSH, thus reduces the generation of sexual hormoue.By cycle GnRH in suppressing to LH induced fluctuations, ganirelix acetate can ovulation inhibition, oocyte meiosis and luteinization.To trouble ovarian hyperstimulation women, ganirelix acetate can prevent LH to fluctuate and related stimulus, and improves implantation and pregnancy rate.Therefore, ganirelix acetate has very high pharmaceutical use and wide market outlook.
The synthetic method of current ganirelix acetate mainly contains traditional B oc solid-phase synthesis that US5767082 adopts and the Fmoc solid-phase synthesis that CN102584945A adopts.
The Boc solid-phase synthesis that US5767082 adopts needs to use HF to carry out cracking, and environmental pollution is very big, is unfavorable for producing.The Fmoc solid-phase synthesis that CN102584945A adopts needs to use Fmoc-HArg (Et)
2-OH and Fmoc-D-HArg (Et)
2-OH is raw material, and this two seed amino acid synthesis cost is higher, and be unfavorable for large-scale production, this two seed amino acid easily decomposes in addition, and it is connected to and also needs after on resin, through repeatedly linked reaction, to cause producing more impurity.
In addition, in the method for current acetic acid synthesized Ganirelix, all adopt organic solvent as solvent, accurately and in real time cannot monitor the pH value in reaction process, easily cause reaction not carry out or side reaction increases, thus the content of product in thick peptide is reduced.
Summary of the invention
The present invention is directed to the above-mentioned defect existed in prior art, provide one neither to need to use HF, also do not adopt Fmoc-HArg (Et)
2-OH and Fmoc-D-HArg (Et)
2-OH is the method preparing ganirelix acetate of raw material.Meanwhile, the present invention, using water to replace organic solvent as reaction solvent, makes reaction can carry out in set pH value range, significantly improves the content of product in thick peptide.
For this reason, the invention provides a kind of method preparing ganirelix acetate, comprise the steps:
1, prepare Ganirelix precursor I, it has structure as follows,
Ac-D-2-Nal-D-Phe(4-Cl)-D-3-Pal-Ser-Tyr-D-Lys-Leu-Lys-Pro-D-Ala-NH
2；
2, the side-chain amino group of Lys and D-Lys of the Ganirelix precursor I that step 1 obtains is modified, obtain Ganirelix crude product;
3, step 2 obtain Ganirelix crude product purified, turn salt and freeze-drying after obtain ganirelix acetate sterling;
Wherein, the modifying method described in step 2 is dissolved in aqueous solvent by Ganirelix precursor I and reagent ethylamino ethyliminum base methylsulfonic acid, adds adjusting PH with base to 7.5 ~ 10.5 and react.
In the present invention's preferred embodiment, the preparation method of this precursor I, comprises the steps:
1) Fmoc-D-Ala-OH and resin reaction, obtains Fmoc-D-Ala-resin;
2) Fmoc-D-Ala-resin adopts other amino acid of the mode coupling Fmoc blocking group of coupling one by one, obtains Ganirelix precursor I-peptide resin after acetylize;
3) Ganirelix precursor I-peptide resin is through cleavage reaction, obtains Ganirelix precursor I.
In the embodiment that the present invention is more preferably, above-mentioned resin is preferably Rink Amide resin, Rink Amide AM resin or Rink Amide MBHA resin.
In the preparation method of this precursor I, step 2) described in other amino acid of Fmoc blocking group be respectively Fmoc-Pro-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys (Boc)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH and Fmoc-D-Nal-OH.
In the preparation method of this precursor I, the cutting reagent that the cleavage reaction described in step 3) adopts is preferably TFA:H
2o=95:5(V/V).
In a preferred embodiment of the present invention, the alkali that step 2 adopts is selected from NaOH, LiOH, NaHCO
3, KOH, Na
2cO
3, K
2cO
3and KHCO
3.
In the present invention's preferred embodiment, step 2 is reacted after adding adjusting PH with base to 8.5 ~ 9.0.
Prepare in the method for ganirelix acetate in the present invention, in step 2, the feed ratio of reagent ethylamino ethyliminum base methylsulfonic acid is preferably 2 ~ 10 times, and the reaction times is preferably 4 ~ 48h.
The present invention, in the process preparing ganirelix acetate, adopts HPLC two-step purifying method to carry out purifying to Ganirelix crude product, specifically comprises the steps:
1) the first step purifying: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, mobile phase A is 0.1%TFA, B phase is mutually acetonitrile, and gradient elution is that 79%A+21%B is changed to 64%A+36%B;
2) second step purifying: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, mobile phase A mutually in the concentration of sodium perchlorate solution be 20mM, adjusting pH to be 2.5, B phase with phosphoric acid is acetonitrile, and gradient elution is that 65%A+35%B is changed to 55%A+45%B.
After HPLC two-step purifying, carry out turning Ficus caricaL, the chromatographic column that specifically to comprise the steps: with anti-phase C18 chromatograph packing material be stationary phase, mobile phase A
1be 0.2%(V/V mutually) aqueous solution of ammonium acetate, A
2be 0.1% Glacial acetic acid (V/V) solution mutually, B phase is trifluoroacetic acid aqueous solution, and gradient elution is 95%A
1+ 5%B is as moving phase wash-out after 20 minutes, and in 2 minutes, moving phase is by 95%A
1+ 5%B is changed to 95%A
2+ 5%B, then uses 95%A
2+ 5%B is as moving phase wash-out after 10 minutes, and in 2 minutes, moving phase is by 95%A
2+ 5%B is changed to 80%A
2+ 20%B, then uses 80%A
2+ 20%B after 30 minutes, uses 70%A as moving phase wash-out
2+ 30%B carries out wash-out as moving phase.
Seen from the above description, compared with prior art, the present invention adopts Fmoc-Lys (Boc)-OH and Fmoc-D-Lys (Boc)-OH to substitute Fmoc-HArg (Et) respectively
2-OH and Fmoc-D-HArg (Et)
2-OH, pre-synthesis ganirelix acetate precursor I, then the side-chain amino group of Lys and D-Lys in precursor I is modified, process and can obtain ganirelix acetate.Preparation method of the present invention does not need to use HF, considerably reduces the pollution to environment, does not need to adopt Fmoc-HArg (Et) yet
2-OH and Fmoc-D-HArg (Et)
2-OH, the Ganirelix purity of synthesis is high, and impurity is few, and advantage of lower cost, be applicable to large-scale production.
Simultaneously, the present invention passes through using water to replace organic solvent as reaction solvent, can monitor in real time pH value, thus reaction can be carried out in set pH value range, ensureing the generation that restrained effectively side reaction while reaction is carried out, significantly improve the content of product in thick peptide, be increased to 70% ~ 80% by original 40 ~ 50%, and finally make the yield of smart peptide improve more than 30% on the original basis.
In addition, the present invention can also adopt the method for HPLC two-step purifying, and by HPLC two-step purifying, significantly improve the yield of product, increase rate reaches 30%, and yield brings up to 34% by original 26%.
Accompanying drawing explanation
Fig. 1: the peptide spectrogram of the Ganirelix precursor I that embodiment 1 prepares;
Fig. 2: the smart peptide spectrogram of the ganirelix acetate sterling that embodiment 15 prepares.
Embodiment
Below by embodiment, the present invention is described in further detail, is intended to non-limiting the present invention for illustration of the present invention.It should be pointed out that to those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and fall into too within protection scope of the present invention.
The implication of abbreviation used in the present invention is listed in the following table.
| English abbreviation | Chinese implication |
| DIC | N, N '-DIC |
| DCM | Methylene dichloride |
| Et 2O | Anhydrous diethyl ether |
| MeOH | Methyl alcohol |
| EA | Ethyl acetate |
| H 2O | Water |
| NaOH | Sodium hydroxide |
| THF | Tetrahydrofuran (THF) |
| PyBOP | Benzotriazole-1-base-oxygen base tripyrrole alkyl hexafluorophosphate |
| DIPEA | DIPEA |
| HOBt | I-hydroxybenzotriazole |
| TFA | Trifluoroacetic acid |
| DMF | DMF |
| 20%DBLK | 20% hexahydropyridine (v)/DMF (v) |
Embodiment 1: the synthesis of Ganirelix precursor I
1, the preparation of Fmoc-D-Ala-Rink Amide resin
Be that in the Rink Amide resin loading solid state reaction post of 0.5mmol/g, DMF washs 2 times, swelling 30 minutes of DMF, and DBLK deprotection 2 times, be respectively 10 minutes and 15 minutes, DMF washs 6 times, triketohydrindene hydrate tests positive by 40g substitution degree.
By Fmoc-D-Ala-OH(18.68g, 60mmol) and HOBt(8.52g, 63mmol) be dissolved in 150ml DMF, DIC(9.9ml is added under condition of ice bath, 63mmol) activate 5 minutes, joined by the solution activated in solid state reaction post, nitrogen gas stirring reacts 2 hours, and triketohydrindene hydrate detects and is negative.Drain reaction solution, DMF washs 3 times, DBLK deprotection 2 times, and be respectively 5 minutes and 7 minutes, DMF washs 6 times, triketohydrindene hydrate tests positive.
2, other amino acid whose couplings of Fomc blocking group, obtain Ganirelix precursor I-peptide resin after acetylize
Repeat above step, adopt the mode of coupling one by one, complete the coupling of Fmoc-Pro-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys (Boc)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-Pal-OH and Fmoc-D-Cpa-OH successively.
By Fmoc-D-Nal-OH(6.54g, 15mmol) and HOBt(2.13g, 15.75mmol) be dissolved in 40ml DMF, DIC(2.47ml is added under condition of ice bath, 15.75mmol) activate 5 minutes, joined by the solution activated in solid state reaction post, nitrogen gas stirring reacts 2 hours, and triketohydrindene hydrate detects and is negative.Drain reaction solution, DMF washs 3 times, DBLK deprotection 2 times, and be respectively 5 minutes and 7 minutes, DMF washs 6 times, and DCM washs 2 times, triketohydrindene hydrate tests positive.
Be dissolved in 30ml DCM by diacetyl oxide (10.2g, 100mmol) and pyridine (7.9g, 100mmol), add solid state reaction post under room temperature condition, nitrogen gas stirring reacts 2 hours, and triketohydrindene hydrate detects and is negative.Drain reaction solution, DCM washs 6 times, and MeOH shrinks 3 times, and the time is respectively 5 minutes, 5 minutes and 10 minutes, and vacuum-drying obtains 17.5g Ganirelix precursor I-peptide resin.
3, the cracking of the thick peptide of Ganirelix precursor I is standby, obtains the thick peptide of Ganirelix precursor I
80ml lysate (the TFA:H of freezing 2 hours is added in the round-bottomed flask that Ganirelix precursor I-peptide resin 7.94g is housed
2o=95:5), stirring at room temperature reacts 2 hours, and filter, filtrate joins in the freezing anhydrous diethyl ether of 800ml, collected by centrifugation solid, obtains the thick peptide of 31g Ganirelix precursor I, yield: 98.7%, purity: 93% after drying.
After testing, the MS data of Ganirelix precursor I are respectively: 1373.498(M+1); 1395.524 (M+23) and 1411.509 (M+39).As shown in Figure 1, wherein retention time T=15.682 minute place is product summary peak to peptide spectrogram, and purity is 94.92%.
Embodiment 2: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (0.72g, 4mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 7.5, mechanic whirl-nett reaction 8 hours, adds 10%NaOH between the reaction period, and maintain pH 7.5, in reaction solution, product purity is 35%.
Embodiment 3: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (0.72g, 4mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds 10%NaOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 41%.
Embodiment 4: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (0.72g, 4mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 10.5, mechanic whirl-nett reaction 8 hours, adds 10%NaOH between the reaction period, and maintain pH 10.5, in reaction solution, product purity is 31%.
Embodiment 5: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds 10%NaOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 72%.
Embodiment 6: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (3.6g, 20mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds 10%NaOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 76%.
Embodiment 7: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 4 hours, adds 10%NaOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 59%.
Embodiment 8: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, slowly drip 10%NaOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 48 hours, adds 10%NaOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 65%.
Embodiment 9: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, slowly drip 10%LiOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds 10%LiOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 67%.
Embodiment 10: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, slowly drip 10%KOH under room temperature condition and adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds 10%KOH between the reaction period, and maintain pH 9.0, in reaction solution, product purity is 70%.
Embodiment 11: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, under room temperature condition, slowly drip saturated Na
2cO
3adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds saturated Na between the reaction period
2cO
3maintain pH 9.0, in reaction solution, product purity is 59%.
Embodiment 12: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, under room temperature condition, slowly drip saturated NaHCO
3adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds saturated NaHCO between the reaction period
3maintain pH 9.0, in reaction solution, product purity is 59%.
Embodiment 13: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, under room temperature condition, slowly drip saturated K
2cO
3adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds saturated K between the reaction period
2cO
3maintain pH 9.0, in reaction solution, product purity is 61%.
Embodiment 14: the synthesis of Ganirelix crude product
The Ganirelix precursor I (3.1g, 2mmol) embodiment 1 obtained and ethylamino ethyliminum base methylsulfonic acid (2.16g, 12mmol) are dissolved in 20ml H
2in O, under room temperature condition, slowly drip saturated KHCO
3adjust pH to 9.0, mechanic whirl-nett reaction 8 hours, adds saturated KHCO between the reaction period
3maintain pH 9.0, in reaction solution, product purity is 57%.
Embodiment 15: the preparation of ganirelix acetate sterling
1, sample preparation
Liquid crude peptide embodiment 6 obtained 10% acetonitrile/25% acetic acid (V/V) dissolves, ultrasonic sample is dissolved completely after, with membrane filtration, collect filtrate for subsequent use.
2, HPLC the first step purifying
(1) purification condition
Chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are respectively: 50mm × 250mm.
Moving phase: A phase is 0.1%TFA; B phase is acetonitrile; Flow velocity is 80ml/min; Gradient elution is that 79%A+21%B is changed to 64%A+36%B; Determined wavelength is 280nm, and sample size is 15.7g(product content is 77%).
(2) purge process
Loading after chromatographic column being balanced 5 minutes, runs gradient-purified, monitoring and before swarming, behind summit, peak three sections collect object peak cuts.Before peak, peak after cut reclaims purifying after removing most of acetonitrile; Summit cut carries out second step purifying after removing most of acetonitrile.
3, HPLC second step purifying
(1) purification condition
Chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are respectively: 50mm × 250mm.
Moving phase: A phase is 20mM sodium perchlorate solution, adjusts pH to be 2.5 with phosphoric acid; B phase is acetonitrile; Flow velocity is 80ml/min; Gradient elution is that 65%A+35%B is changed to 55%A+45%B; Determined wavelength is 280nm.
(2) purge process
Loading after chromatographic column being balanced 5 minutes, runs gradient-purified, monitoring and before swarming, behind summit, peak three sections collect object peak cuts.Before peak, peak after cut reclaims purifying after removing most of acetonitrile; Summit cut turns round salt after removing most of acetonitrile.
4, salt purifying is turned
(1) purification condition
Chromatographic column: the chromatographic column being stationary phase with anti-phase C18 chromatograph packing material, pillar diameter and length are respectively: 50mm × 250mm.
Moving phase: A
1be 0.2%(V/V mutually) aqueous solution of ammonium acetate, A
2be 0.1% Glacial acetic acid (V/V) solution mutually; B phase is trifluoroacetic acid aqueous solution; Gradient elution is 95%A
1+ 5%B is as moving phase wash-out after 20 minutes, and in 2 minutes, moving phase is by 95%A
1+ 5%B is changed to 95%A
2+ 5%B, then uses 95%A
2+ 5%B is as moving phase wash-out after 10 minutes, and in 2 minutes, moving phase is by 95%A
2+ 5%B is changed to 80%A
2+ 20%B, then uses 80%A
2+ 20%B after 30 minutes, uses 70%A as moving phase wash-out
2+ 30%B carries out wash-out as moving phase; Determined wavelength is 280nm; Sampling volume is 200ml.
(2) purge process
Loading after chromatographic column being balanced 5 minutes, runs gradient-purified, monitors and collects object peak cut.Freeze-drying after object peak cut vacuum rotary steam is concentrated into 20 milliliters.White powdery solids essence peptide 5.2g is obtained after freeze-drying.Purity 99.77%, single impurity is all less than 0.1%.Purification yield 43%(is with Ganirelix cubage in crude product), total recovery 34%.
After testing, after ganirelix acetate purifying, the MS of smart peptide is 1570.862 (M+1).Essence peptide spectrogram as shown in Figure 2.Wherein, within retention time T=18.489 minute, place is product summary peak, and purity is 99.77%.
Claims (10)
1. prepare a method for ganirelix acetate, comprise the steps:
1) prepare Ganirelix precursor I, it has structure as follows,
Ac-D-2-Nal-D-Phe(4-Cl)-D-3-Pal-Ser-Tyr-D-Lys-Leu-Lys-Pro-D-Ala-NH
2；
2) side-chain amino group of Lys and D-Lys of the Ganirelix precursor I that step 1) obtains is modified, obtain Ganirelix crude product;
3) step 2) the Ganirelix crude product that obtains is purified, turn salt and freeze-drying after obtain ganirelix acetate sterling;
It is characterized in that, step 2) described in modifying method be that Ganirelix precursor I and reagent ethylamino ethyliminum base methylsulfonic acid are dissolved in aqueous solvent, add adjusting PH with base to 7.5 ~ 10.5 and react.
2. method according to claim 1, wherein step 1) is prepared Ganirelix precursor I and is comprised the steps:
(1) Fmoc-D-Ala-OH and resin reaction, obtain Fmoc-D-Ala-resin, described resin is preferably Rink Amide resin, Rink Amide AM resin or Rink Amide MBHA resin;
(2) Fmoc-D-Ala-resin adopts other amino acid of the mode coupling Fmoc blocking group of coupling one by one, obtains Ganirelix precursor I-peptide resin after acetylize;
(3) Ganirelix precursor I-peptide resin is through cleavage reaction, obtains Ganirelix precursor I.
3. method according to claim 2, other amino acid of the Fmoc blocking group wherein described in step (2) are respectively Fmoc-Pro-OH, Fmoc-Lys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Lys (Boc)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-Pal-OH, Fmoc-D-Cpa-OH and Fmoc-D-Nal-OH.
4. method according to claim 2, the cutting reagent that the cleavage reaction wherein described in step (3) adopts is TFA:H
2o=95:5(V/V).
5. method according to claim 1, wherein step 2) described in alkali be selected from NaOH, LiOH, NaHCO
3, KOH, Na
2cO
3, K
2cO
3and KHCO
3.
6. method according to claim 1, wherein step 2) add adjusting PH with base to 8.5 ~ 9.0 and react.
7. method according to claim 1, wherein step 2) in the feed ratio of reagent ethylamino ethyliminum base methylsulfonic acid be 2 ~ 10 times, the reaction times is 4 ~ 48h.
8. method according to claim 1, the purifying wherein described in step 3) is HPLC two-step purifying.
9. method according to claim 8, wherein the concrete steps of HPLC two-step purifying are:
(1) the first step purifying: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, mobile phase A is 0.1%TFA, B phase is mutually acetonitrile, and gradient elution is that 79%A+21%B is changed to 64%A+36%B;
(2) second step purifying: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, mobile phase A mutually in the concentration of sodium perchlorate solution be 20mM, adjusting pH to be 2.5, B phase with phosphoric acid is acetonitrile, and gradient elution is that 65%A+35%B is changed to 55%A+45%B.
10. method according to claim 1, the concrete steps turning salt wherein described in step 3) are: the chromatographic column being stationary phase with anti-phase C18 chromatograph packing material, mobile phase A
1be 0.2%(V/V mutually) aqueous solution of ammonium acetate, A
2be 0.1% Glacial acetic acid (V/V) solution mutually, B phase is trifluoroacetic acid aqueous solution, and gradient elution is 95%A
1+ 5%B is as moving phase wash-out after 20 minutes, and in 2 minutes, moving phase is by 95%A
1+ 5%B is changed to 95%A
2+ 5%B, then uses 95%A
2+ 5%B is as moving phase wash-out after 10 minutes, and in 2 minutes, moving phase is by 95%A
2+ 5%B is changed to 80%A
2+ 20%B, then uses 80%A
2+ 20%B after 30 minutes, uses 70%A as moving phase wash-out
2+ 30%B carries out wash-out as moving phase.
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| CN115260293A (en) * | 2022-08-22 | 2022-11-01 | 南京汉欣医药科技有限公司 | Purification method of ganirelix acetate |
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| US20170121371A1 (en) * | 2013-06-18 | 2017-05-04 | Hybio Pharmaceutical Co., Ltd. | Ganirelix precursor and method for preparing ganirelix acetate by using the same |
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| US10577394B2 (en) | 2013-06-18 | 2020-03-03 | Hybio Pharmaceutical Co., Ltd. | Ganirelix precursor and method for preparing ganirelix acetate by using the same |
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| WO2017114414A1 (en) * | 2015-12-29 | 2017-07-06 | 深圳翰宇药业股份有限公司 | Method for detecting ganirelix acetate |
| WO2023033017A1 (en) * | 2021-09-01 | 2023-03-09 | 積水メディカル株式会社 | Method for producing ganirelix or salt thereof |
| CN115260293A (en) * | 2022-08-22 | 2022-11-01 | 南京汉欣医药科技有限公司 | Purification method of ganirelix acetate |
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