CN110903352A

CN110903352A - Preparation method of cetrorelix

Info

Publication number: CN110903352A
Application number: CN201911376082.9A
Authority: CN
Inventors: 刘宝生; 张巍; 李湘; 徐琪
Original assignee: Chinese Peptide Co
Current assignee: Chinese Peptide Co
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-03-24

Abstract

The invention relates to a preparation method of cetrorelix, and particularly relates to a solid-liquid combined synthesis method. According to the sequence of the cetrorelix sequence from C end to N end, under the action of coupling reagent the 1-9 fragments are connected one by means of solid phase synthesis method, then the 1-9 fragments are fully protected and cut, and then the cut fragments are mixed with DAla-NH₂Subjecting HCl to fragment condensation in a liquid phase to obtain fully protected cetrorelixAnd then the crude cetrorelix peptide is obtained by using cracking solution for cutting, then the cutting solution is precipitated by using glacial ethyl ether, and finally the finished cetrorelix product is obtained by reverse-phase high performance liquid chromatography purification.

Description

Preparation method of cetrorelix

Technical Field

The invention belongs to the technical field of preparation methods of polypeptide medicaments, particularly relates to a preparation method of cetrorelix, and particularly relates to a solid-liquid combined synthesis method.

Background

Cetrorelix (trade name Cetrotide) is a potent progesterone-releasing hormone-inhibin (LH-RH) receptor antagonist that controls ovarian stimulation, prevents premature follicular discharge, and aids conception. And can be used for treating breast cancer, gynecological cancer, endometriosis, precocious puberty, hysteromyoma, ovarian hyperandrogenism and premenstrual syndrome.

Chinese name: cetrorelix

English name: cetrorelix acetate

Sequence Ac-D2Nal¹-D4Cpa²-D3Pal³-Ser⁴-Tyr⁵-DCit⁶-Leu⁷-Arg⁸-Pro⁹-DAla¹⁰-NH₂

The molecular formula is as follows: c₇₀H₉₂ClN₁₇O₁₄

The molecular weight is as follows: 1431.06

Among the methods for synthesizing cetrorelix known at present, solid-phase synthesis methods are mostly used, in which amino acids are sequentially bonded to a resin in sequence from the C-terminus:

patent CN101284863B discloses a solid phase synthesis method of cetrorelix: Fmoc-Linker-MBHA-Resin is used as a starting material, amino acids with Fmoc protective groups are sequentially connected according to a sequence to obtain protected decapeptide Resin, acetylation reaction is carried out, then cutting is carried out to obtain cetrorelix acetate, separation and purification are carried out through a C18 or C8 chromatographic column, and finally, freeze drying is carried out to obtain cetrorelix acetate or trifluoroacetate. The temperature of the amino acid coupling reaction is 35-50 ℃. The method can generate more racemic impurities, and the product quality is influenced.

Patent CN101863960B discloses a preparation method of cetrorelix. The method comprises adding C-terminal amino acid into Boc amino acid and Melamine resin, ammonolyzing with 10 wt% ammonia water, and hydrolyzing with H₂And Pd is used as a deprotection reagent to prepare the cetrorelix. The method adopts H₂Deprotection reaction is carried out for 24 hours with Pd, the reaction time is long, the yield of the crude peptide is only 60-70%, and H is₂Use comparativelyThe method is dangerous, the Pd metal is difficult to remove, the requirement on equipment is high, and the method is not suitable for large-scale production.

Patent CN104277093A discloses that the yield of pure cetrorelix acetate product is more than 30% by taking RinkAmide-AM Resin as a carrier and DIC/HOBt as a condensing agent and adopting a microwave reaction technology to obtain crude cetrorelix acetate product which is purified by reversed-phase high performance liquid chromatography and a special acetate-transferring technology. The microwave synthesis method adopted by the method has high requirements on equipment and is not suitable for large-scale industrial synthesis.

Patent CN104086632A discloses a method for using AM resin as starting resin to sequentially couple amino acids with N-terminal Fmoc protection and side chain protection according to cetrorelix main chain peptide sequence, wherein the amino acid at position 6 of the peptide sequence adopts Fmoc-D-orn (Dde) -OH to replace Fmoc-Cit-OH without side group protection, although the synthesis method can control the toxicity [ D-Ci t (Ac) ] to be below 0.1% from the content of reye impurities, the toxic hydrazine hydrate reagent is adopted to remove the Dde protection in the synthesis process, the process is complex and the safety is low.

Patent CN 104610433A and patent CN 104892732A both disclose a preparation method of cetrorelix, which uses Fmoc-Linker amino resin as starting material, sequentially connects nine amino acids with Fmoc protecting group from C terminal to N terminal, and connects Ac-D-2-Nal-OH after removing Fmoc protecting group to obtain peptide resin with N terminal acetylation, namely Ac full-protection decapeptide resin. And (3) cutting the peptide resin by using a cracking reagent, precipitating the cutting solution by using glacial ethyl ether to obtain crude cetrorelix peptide, separating and purifying by using an HPLC (high performance liquid chromatography) preparative chromatographic column, and freeze-drying to obtain cetrorelix trifluoroacetate or cetrorelix acetate. The method needs an extra Ac-D-2-Nal-OH raw material, and the production cost is higher.

Patent CN 107778355 a discloses a method for synthesizing cetrorelix. The method comprises the steps of adopting brand new amino resin as a carrier, adopting protected D-Orn as a precursor of D-Cit, then removing a side chain protecting group to react with tert-radical iso-amino acid vinegar to generate D-Cit (tBu), and carrying out acidolysis on the cetrorelix peptide resin by using a special trifluoroacetic acid solution containing hydrogen bromide. The method for converting D-Orn into D-Cit (tBu) adds one-step reaction, is difficult to ensure complete conversion and uses toxic hydrogen bromide, and is not beneficial to large-scale industrial production.

The patent CN 107337717A adopts a fragment method 5+5 to synthesize cetrorelix, and two fragments can be synthesized simultaneously, so that the synthesis period is shortened. However, the solid-phase synthesis of the solid-phase synthesis fragment B after the method adopts expensive Sieber resin, and the cost is higher. And the fragment B has more full-protection cutting steps, so that the total process steps are more and complicated, and the risk is higher in large-scale production.

The method disclosed by the patent has the problems of complex method, unsuitability for industrial production, low production efficiency and the like. Therefore, there is an urgent need to develop a simple, efficient and suitable process for mass production.

Disclosure of Invention

In order to solve the technical problems pointed out above, the invention provides a method for synthesizing cetrorelix by a solid-liquid phase combination method, which comprises the following steps:

the preparation method of the cetrorelix comprises the following steps:

a) according to the sequence of the cetrorelix sequence from the C end to the N end, 1-9 fragments are connected one by a solid phase synthesis method under the action of a coupling reagent to obtain Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTC Resin;

b) cutting the peptide resin obtained in the step a) in a full-protection manner to obtain a fully-protected cetrorelix-A; Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-OH;

c) combining the cetrorelix-A and DAla-NH obtained in step b)₂Carrying out fragment condensation on HCl in a liquid phase to obtain fully-protected cetrorelix;

d) cutting the fully protected cetrorelix obtained in the step c) by using a lysis solution, and precipitating the cutting solution by using glacial ethyl ether to obtain crude cetrorelix peptide;

e) and d) purifying the crude cetrorelix peptide obtained in the step d) by using a reverse phase high performance liquid chromatography to obtain a finished cetrorelix product, and completing the synthesis of cetrorelix.

Preferably, in step (a), the resin is a CTC resin and has a degree of substitution of 0.3 to 0.8 mmol/g.

Preferably, in the step (a), the solid phase synthesis method adopts CTC resin as a solid phase carrier, the CTC resin and Fmoc-Pro-OH are subjected to coupling reaction to obtain Fmoc-Pro-CTC resin, then after Fmoc protection is removed, corresponding Fmoc-protected amino acids are sequentially coupled according to a feeding ratio of 1.5-4 times of molar weight, each amino acid combination is carried out in the presence of a condensation reagent, Kaiser test reagent is used for detecting the reaction end point in each coupling process, after each coupling reaction is finished, washing is carried out, then after Fmoc protection is removed by a deprotection reagent, coupling reaction is carried out with the next Fmoc-protected amino acid, Fmoc-Arg (pbf) -OH, Fmoc-Leu-OH, Fmoc-DCit-OH, Fmoc-Thr (tBu) -OH and Fmoc-Ser (tBu) -OH are sequentially and respectively coupled, Fmoc-D-3Pal-OH Fmoc-D-Phe (4Cl) -OH, Fmoc-D-2Nal-OH, thereby obtaining Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTCResin.

Preferably, the configured condensation reagent in step (a) is selected from the group consisting of a condensation reagent selected from one of HBTU, HATU, PyAOP, PyBOP, EDC.HCl + HOBT, DIC + HOBT and the like, and a base selected from NMM, DIPEA, Et₃One of N; when mixed with HOBT, the ratio was 1: 1; the ratio of condensing agent to base is 1: 1.

preferably, the amount of amino acid charged in the solid phase synthesis reaction in step (a) is 1.5 to 4 times the molar number of the resin, and the reaction temperature is in the range of 5 to 40 ℃.

Preferably, in the step (b), the cutting fluid is selected from HFIP/DCM system, and the volume ratio of the two is 3:7, the concentration is 8-10ml/g, and the temperature is 10-35 ℃.

Preferably, the step (c) comprises fragment condensation in liquid phase, wherein the condensation reagent is selected from the group consisting of condensation reagents A + B, wherein the condensation reagent is selected from one of HBTU, HATU, PyAOP, PyBOP and NMM, DIPEA, Et₃One of N, or one of EDC, HCl, DIC and other reagents, and HOBT in a ratio of 1: 1.

Preferably, the step (c) comprises carrying out the fragment condensation in the liquid phase using a condensation agent, a base, DAla-NH₂HCl, cetrorelix-a in a ratio of 1:1:1:1 to 3:3:3: 1.

Preferably, TFA, H is used as the lysing solution in step (d)₂One or more combinations of O, EDT and TIS. More preferably TFA, H₂The ratio of O, EDT, TIS was, 90: 5: 2.5: 2.5.

preferably, in the step e), the reversed-phase high performance liquid chromatography is used for separation and purification, the mobile phase A is an aqueous solution of phosphoric acid and triethylamine, the phase B is a 20% acetonitrile solution of the mobile phase A, effluent liquids in different time periods are collected in a gradient elution mode, the purity of the sample is judged according to the detection result of HPLC, and a qualified sample can be subjected to salt exchange and freeze drying to obtain a finished product of cetrorelix.

The invention adopts a 9+1 fragment method to prepare the cetrorelix, and has the following advantages:

1. pro is selected from C end of cetrorelix fragment A⁹Amino acids in the formulae D-Ala-NH₂No racemization occurs during the liquid phase synthesis of HCl.

2. Cetrorelix fragments A and D-Ala-NH₂Compared with the common liquid phase synthesis, the post-treatment of the HCl liquid phase synthesis reaction is precipitation and filtration, the operation is simple, and the product purity is high.

3. Adopting a 9+1 solid-liquid phase mixing method, and adopting D-Ala-NH at the tail end₂HCl avoids the use of amino resin such as Seiber resin with higher price and reduces the production cost.

The liquid phase synthesis and the solid phase synthesis are mixed for use, thereby not only solving the defects that the liquid phase synthesis method is not suitable for connecting long chains and the liquid phase synthesis method has complex subsequent treatment, but also solving the characteristics of higher cost and lower crude product purity in the solid phase synthesis. A stable balance point is found by the two methods, and large-scale production is realized on the basis of realizing smooth reaction, cost control, impurity reduction and post-treatment reduction.

The specific implementation mode is as follows:

the abbreviations used in the description and claims of the present invention have the following meanings:

d-2Nal D-2-naphthylalanine

D-4Cl-Phe D-4-chloro-phenylalanine

D-3Pal D-3-pyridylalanine

Ser is serine

Tyr tyrosine

D-Cit D-citrulline

Leu leucine

Arg is arginine

Pro proline

D-Ala: D-alanine

DIC N, N-diisopropylcarbodiimide

HOBt 1-hydroxybenzotriazole

CTC 2-Triphenylmethyl chloride

HBTU: o-benzotriazole-tetramethylurea hexafluorophosphate

HATU 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

PyAOP (3H-1,2, 3-triazolo [4,5-b ] pyridin-3-yloxy) tris-1-pyrrolidinophosphonium hexafluorophosphate

PyBOP Benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate

NMM N-methylmorpholine

DIPEA: n-diisopropylethylenediamine

Et₃N: triethylamine

Edc.hcl: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

TFA trifluoroacetic acid

EDT (electro-thermal transfer coating): ethanedithiol

And (3) TIS: tri-isopropyl silane

Example 1:

preparation of Fmoc-Pro-CTC Resin

Accurately weighing 170g of CTC Resin (Sub: 0.96mmol/g) and Fmoc-Pro-OH (60.5g), adding 1500mL of LPCM, slowly dropwise adding 85mL of DIPEA under the stirring state after amino acid is completely dissolved, stirring at room temperature for 2-3 hours, adding 170mL of methanol to terminate for 30 minutes after the reaction is finished, filtering reaction liquid, washing the Resin with DMF for 3 times, washing with methanol for 1 time, washing with DCM for 2 times, washing with methanol for 3 times, drying in vacuum to obtain 239g of Fmoc-Pro-CTC Resin, and measuring the substitution degree to be 0.62 mmol/g. Preparation of Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTC Resin

Accurately weighing 64.5g of Fmoc-Pro-CTC Resin with the substitution degree of 0.62mmol/g into a solid phase reactor, adding DMF with the volume 2-3 times of the volume of the Resin for swelling for 2h, adding 20% PIP/DMF solution after draining the DMF, stirring for 30min to remove the Fmoc protecting group, washing with DMF for 6 times, washing for 2 min each time, adding activated amino acid solution (Fmoc-Arg (Pbf) -OH 51.9g and HOBt 10.94 g) into a beaker to dissolve after ninhydrin detects positive, adding DIC12.54mL under ice bath to activate for 15 min), reacting for 1 h at 20 ℃, adding 9mLNMM for reacting for 1.5-2h, draining the reaction solution after ninhydrin detects negative, washing for 4 times with DMF, and draining.

And then sequentially carrying out the operations of deprotection, washing, coupling and washing. Wherein the coupled Fmoc-protected amino acids are Fmoc-Leu-OH, Fmoc-DCit-OH, Fmoc-Thr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl) -OH and Fmoc-D-2Nal-OH in sequence. The coupling temperature is 20 ℃, and the reaction end point of each amino acid is detected by Kaiser test, so as to obtain Fmoc-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTC Resin.

20% PIP/DMF solution was added and stirred for 30 minutes, after the reaction was completed, DMF was washed 6 times, and 1600mL (Ac) was added₂O, NMM, DMF (10: 6:84), reacting for 30min, draining reaction liquid after ninhydrin detection is negative, washing with DMF for 4 times, washing with methanol for 2 times, washing with DCM for 1 time, washing with MeOH for 3 times, and draining. The obtained Resin is dried in vacuum to obtain Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTC Resin. Cleavage of Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTC Resin

The hexafluoroisopropanol and DCM are prepared into 850mL of cutting fluid according to the volume ratio of 3:7, and the cutting fluid is cooled to below 10 ℃ for standby. Weighing the cooled cutting fluid according to the ratio of 8 mL-10 mL/g of cutting fluid/peptide resin, adding the cutting fluid into a reactor, and starting a stirrer; 106g of peptide resin was slowly added to the cleavage solution, and when the temperature was returned to 10-35 ℃, the reaction time was recorded for 1 hour. Filtering the resin cutting fluid mixture after the reaction is finished, and collecting filtrate; the resin was washed once with 212mL of cleavage solution (2mL/g peptide resin) and once with 212mL of DCM (2mL/g peptide resin) and the combined filtrates were collected. Controlling the temperature of the water bath below 35 ℃, concentrating the filtrate by using a water pump/rotary evaporator, and removing the low-boiling-point solvent to obtain an oily substance; and controlling the temperature of the water bath at 40 +/-2 ℃, concentrating the filtrate by using an oil pump/rotary evaporator, removing the high-boiling-point solvent, gradually changing the product from oily matter into solid, continuously mashing the large solid into small blocks during the process, and continuously performing rotary evaporation for 0.5 hour by using an oil pump when the product properties are completely changed into small solid particles. The resulting small solid particles were dried under reduced pressure in vacuo to constant weight (during which time they were ground and then dried by suction) to give 77.02g of crude cetrorelix-a.

Fully protected cetrorelix Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-DAla-NH₂And (4) synthesizing.

Measuring DMF 72mL (4mL/g LHRH-022-A crude product) and DIPEA 4.7mL (2.6eq.) and adding into a reactor, starting a stirrer, cooling the temperature of the solution in the reactor to 0-10 ℃ under the nitrogen atmosphere, and then adding H-DAla-NH₂HCl1.689g (1.3eq.) and cetrorelix-A18.0 g (1.0eq.) were added to the reactor and the temperature was returned to 10-35 ℃. The solution in the reactor gradually becomes clear with increasing temperature and continuous stirring, at this time, 5.14g (1.3eq.) of HBTU is added, and the mixture is stirred until the solution is clear, and then timing is started. The solution in the reactor reacts for 2-3 hours at the temperature of 10-35 ℃. The reaction was terminated by HPLC.

Preparing the diethyl ether and the anisole into a precipitation solution according to the volume ratio of 3: 1. The precipitation solution was weighed out in a volume 10 times the volume of DMF and added to the precipitator, and the solution in the reactor was slowly added with vigorous stirring, and a large amount of solid was precipitated. Transferring the solid-liquid mixture in the precipitator to a centrifuge cup, pouring out and removing supernatant liquor after centrifuging, washing the solid at the bottom of the centrifuge cup with diethyl ether, centrifuging, and pouring out and removing the supernatant liquor; this process was repeated twice. The obtained product is dried by a water pump for more than 1 hour in vacuum, and then is dried by an oil pump for more than 8 hours to constant weight, and 17.42g of the crude product of the fully protected cetrorelix is obtained.

Cetrorelix Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-NH₂Preparation of

Mixing TFA and H₂The O and the EDT are prepared into slices according to the volume ratio of 97:1:2The cut solution (126 mL) was stored at-5 ℃ for further use. The cutting fluid (7-9mL/g crude product) is added into the reactor, the stirrer is started, and the temperature of the solution in the reactor is cooled to be below 0 ℃ under the nitrogen atmosphere. Adding 18.0g of crude fully protected cetrorelix into the cutting fluid within 10-30 minutes, and controlling the feeding speed to ensure that the temperature of the mixture in the reactor does not exceed 10 ℃. After the completion of the addition, the temperature of the mixture in the reactor was controlled to 0 ℃ or lower, and the mixture was stirred for 10 minutes. The temperature was returned to 15-40 ℃ and the reaction time was recorded for 2.5 hours.

1260mL of ether, 10 times the volume of the cutting fluid, was weighed into the precipitator, stirred vigorously and the ether temperature was cooled to below 0 ℃. Slowly adding the mixture in the reactor into a precipitator, controlling the feeding speed to ensure that the temperature of the mixture in the precipitator is not more than 10 ℃ all the time, and after the mixture is added, stirring the mixture in the precipitator for 10 minutes at the temperature of below 0 ℃ to generate a large amount of solid precipitate. Transferring the solid-liquid mixture in the precipitator to a centrifuge cup, pouring out and removing supernatant liquor after centrifuging, washing the solid at the bottom of the centrifuge cup with diethyl ether, centrifuging, and pouring out and removing the supernatant liquor; this process was repeated three times. And (3) drying the obtained product for more than 1 hour in vacuum by using a water pump, and drying the product for more than 8 hours in vacuum by using an oil pump at room temperature until the weight is constant to obtain 14.2g of crude products of cetrorelix.

Cetrorelix Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-NH₂Purification of (2):

dissolving the crude cetrorelix peptide in a mixed solution of acetonitrile and water, carrying out suction filtration, carrying out sample loading and purification on a filtrate by adopting a C18 reversed-phase chromatographic column, collecting sample peaks, merging qualified samples, desalting, and freeze-drying to obtain the refined cetrorelix peptide, wherein the yield of the purified refined peptide is 48.9%, the purity is 98.0%, and the single impurity content is less than 0.5%.

The conditions and reaction steps for the following examples are the same as in example 1, except as shown in the following table:

the above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A preparation method of cetrorelix is characterized by comprising the following steps:

a) according to the sequence of the cetrorelix sequence from C end to N end, 1-9 segments are connected one by a solid phase synthesis method under the action of a coupling reagent to obtain Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTCResin peptide resin;

2. The process of claim 1, wherein in step (a), the resin is a CTC resin and the degree of substitution is from 0.3 to 0.8 mmol/g.

3. The preparation method of cetrorelix as claimed in claim 1, wherein in step (a), the solid phase synthesis method comprises the steps of connecting CTC resins one by one as solid phase carriers, performing coupling reaction on the CTC resins and Fmoc-Pro-OH to obtain Fmoc-Pro-CTC resins, sequentially coupling corresponding Fmoc-protected amino acids at a charge ratio of 1.5-4 times of the molar amount after Fmoc protection is removed, performing coupling reaction on each amino acid in the presence of a condensation reagent, detecting the reaction end point with a kaiser reagent in each coupling process, washing after each connecting reaction is finished, performing coupling reaction on the Fmoc-protected amino acids with a deprotection reagent after Fmoc protection is removed, sequentially coupling Fmoc-arg (pbf) -OH, Fmoc-Leu-OH and Fmoc-DCit-OH respectively, Fmoc-Thr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-D-3Pal-OHFmoc-D-Phe (4Cl) -OH, Fmoc-D-2Nal-OH, thereby obtaining Ac-D2Nal-D4Cpa-D3Pal-Ser (tBu) -Tyr (tbu) -DCit-Leu-Arg (Pbf) -Pro-CTCResin.

4. The process of claim 1,2 or 3, wherein the condensation reagent in step (a) is selected from the group consisting of HBTU, HATU, PyAOP, PyBOP, EDC.HCl + HOBT, DIC + HOBT and the like, and a base selected from NMM, DIPEA, Et and the like₃One of N; when mixed with HOBT, the ratio was 1: 1; the ratio of condensing agent to base is 1: 1.

5. the method for preparing cetrorelix according to claim 1,2 or 3, wherein the amount of the amino acid fed in the solid phase synthesis reaction in step (a) is 1.5 to 4 times the mole number of the resin, and the reaction temperature is in the range of 5 to 40 ℃.

6. The process for preparing cetrorelix according to claim 1,2 or 3, wherein in step (b) the cleavage is carried out in a solution selected from the group consisting of HFIP/DCM systems in a volume ratio of 3:7, the concentration is 8-10ml/g, and the temperature is 10-35 ℃.

7. The process of claim 1,2 or 3, wherein the step (c) comprises fragment condensation in liquid phase with a condensation reagent selected from the group consisting of A + B, HBTU, PyAOP, PyBOP and NMM, DIPEA, Et₃One of N, or one of EDC, HCl, DIC and other reagents, and HOBT in a ratio of 1: 1.

8. The process for the preparation of cetrorelix according to claim 7, characterized in that in step (c) the condensation of the fragments is carried out in the liquid phase, with a condensation agent, a base, DAla-NH₂HCl, cetrorelix-a in a ratio of 1:1:1:1 to 3:3:3: 1.

9. The method of claim 1, wherein TFA, H is used as the lysing solution in step (d)₂One or more combinations of O, EDT and TIS.

10. The preparation method of cetrorelix as claimed in claim 1, wherein in step e), the reverse-phase HPLC preparative chromatographic column is used for separation and purification, the mobile phase a is an aqueous solution of phosphoric acid and triethylamine, the phase B is a 20% acetonitrile solution of the mobile phase a, effluent liquids in different time periods are collected by adopting a gradient elution mode, the purity of the sample is judged by using a detection result of HPLC, and a finished product of cetrorelix can be obtained after a qualified sample is subjected to salt exchange and freeze drying.