CN102952174A - Method for synthesizing degarelix - Google Patents

Method for synthesizing degarelix Download PDF

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Publication number
CN102952174A
CN102952174A CN2012104601959A CN201210460195A CN102952174A CN 102952174 A CN102952174 A CN 102952174A CN 2012104601959 A CN2012104601959 A CN 2012104601959A CN 201210460195 A CN201210460195 A CN 201210460195A CN 102952174 A CN102952174 A CN 102952174A
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4aph
fmoc
ala
resin
cbm
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厉保秋
袁强
陈明鲁
房世红
董欣
李娜
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JINAN HUANTAI PHARMACEUTICAL TECHNOLOGY Co Ltd
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JINAN HUANTAI PHARMACEUTICAL TECHNOLOGY Co Ltd
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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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention discloses a method for synthesizing degarelix, wherein an amino resin protected with Fmoc is used as a raw material; according to a solid-phase synthesis method, DIC/HOBt (N,N'-diisopropylcarbodiimide/1-hydroxybenzotrizole) is used as a coupling agent for transpeptidase reaction; 4-serine uses tert-butyl dimethyl to replace tertiary butyl for protection protecting hydroxyl; a hydroorotic acid fragment connected with 4-amino of 5-phenylalanine benzene ring is protected with triphenylmethyl at first, and then introduced, so that the rearrangement side reaction is prevented; and since D-4Aph (Cbm) is used for replacing D-4Aph (Cbm-tBu), a t-Bu removal difficulty is prevented and the occurrence of the side reaction is reduced. A synthesis technology of the method for synthesizing degarelix is simple in steps, easy to control, small in environment pollution and high in yield, thereby being applicable for industrial production.

Description

A kind of synthetic method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
Technical field
The present invention relates to a kind of synthetic method with polypeptide of physiologically active, specifically the artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Background technology
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, English popular name Degarelix, it is a kind of gonadotropin releasing hormone (GnRH) acceptor inhibitor class medicine, reversible inhibition hypophysis GnRH acceptor reduces the release that gonadotropin releasing hormone suppresses testosterone then, by suppressing that prostate cancer is continued growth and the deterioration that the vital testosterone of growth delays prostate cancer.It is present unique clinical GnRH receptor antagonist for advanced prostate cancer.Its aminoacid sequence is Ac-D-2Nal-D-4 Cpa-D-3Pal-Ser-4Aph (L-Hor)-D-4Aph (Cbm)-Leu-Ilys
-Pro-D-Ala-NH 2 ,Structural formula is as follows:
Figure 209090DEST_PATH_IMAGE001
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 begins just can reduce testosterone concentration from medication, can not produce such as GnRH agonist (such as goserelin, Leuprolide etc.) and bring " falling after rising " phenomenon.The effect that Degarelix reduces testosterone concentration is suitable with Leuprolide depot controlled release injection, and it is significantly fast statistically to reduce testosterone concentration, suppresses all the time prostate specific antigen in whole 1 year for the treatment of.
Quick, the lasting control testosterone levels of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 energy suppresses the development of prostate cancer, and tolerance is with marketed drug is suitable at present.Along with Chinese society's aging is accelerated, the morbidity of prostate cancer improves year by year, and the market potential of Degarelix is huge.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is the linear decapeptide that contains 7 alpha-non-natural amino acids, and wherein containing 5 amino acid is D configuration amino acid.
Patent documentation is at first by the Semple Graeme of Ferring company and Jiang Guangcheng R ﹠ D Cooperation ,The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solid phase synthesis process of patent documentation WO9846634 report adopts the Boc strategy, on resin, then removes the Boc protecting group with TFA with the N-Boc-D-Ala coupling, then connects reactive polypeptide according to amino acid whose order, deprotection.Wherein 5 and 6 s' 4-amino-benzene L-Ala ( Aph) need to adopt the Fmoc protecting group to protect, and then remove with piperidines/DMF solution, react with tertiary butyl isocyanic ester or L-hydroorotic acid again, and then next peptide bond in succession in turn.After the acetic anhydride acidylate, remove 4 Ser-OBn with HF, 6 Aph-Cbm-tBu and 8 s' Lys-NCbz.WO9846634 has also mentioned another method, and just the vitamin B13 on the 5 amino acids side chains is placed on whole peptide chain and finishes afterwards again introducing.
Patent documentation US6214798B1 discloses a kind of liquid-phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and the liquid phase synthesis mode of proposition is more suitable for a large amount of preparing products.The purity of the finished product is greatly about 98%, yet point out at existing document (J.Med.Chem., 2005,48,4851-4860) to measure purity be that 98%, HPLC measures only is 96% by kapillary zone gel electrophoresis according to the product of the same manner preparation.And use at last the hazardous gases such as HF, be unfavorable for industrial production.
Patent documentation WO2010121835 discloses the technical scheme that a kind of Fmoc of employing method prepares Degarelix, by document (J.Chem.Soc.Perkin, Trans.2,1984, pg1077-1081; Org.Biomol. Chem., 2004, pg1098-1103) as can be known, the L-hydroorotic acid structure on the 5 amino acids side chains is unstable under alkaline condition, easily resets the by product that generates glycolylurea (Hydantoin) structure.And found through experiments at dense NaOH and regulate the by product that can generate 4.5% weight ratio when the pH value is processed, 2% DBU/DMF (1,8-diazabicylo [5.4.0] 11 carbon-7-alkene/yl) process degarelix and produce 1.8% glycolylurea structure product, if contain 5% water, by product then increases to 7%.But the piperidines 20%/DMF(dimethyl sulfoxide (DMSO)) in the solution degarelix without any degraded.Adopt at last this method can prepare degarelix less than the related substances of 0.3% weight percent, and the rearrangement product that has reduced the L-hydroorotic acid generates, avoided in addition above-mentioned Boc method to use in a large number TFA, reduced environmental hazard, reduced the TFA(trifluoroacetic acid to the high poison of human body) harm.
Patent documentation WO2011066386 points out to use in the above-mentioned Fmoc strategy hydroxyl of t-Bu protection Serine; the deprotection test shows; general condition can not remove the t-Bu protection fully; need 100% TFA(trifluoroacetic acid) process and just can finish deprotection in 25 hours; under this strong reaction condition, increasing of side reaction and by product can affect the quality of the finished product; if adopt 45 ℃ of reactions to remove fully, but still can produce the quality that degraded product affects the finished product.Adopt the hydroxyl of Trt protection Serine, can be at the 95%TFA(trifluoroacetic acid), the 2.5%TIS(tri isopropyl silane), 2.5EDT(1,2-dithioglycol) in the solution 45 ℃ removed in lower 1 ~ 2 hour.Adopt the Degarelix HPLC purity of this strategy preparation to reach more than 98.5%; the Trt protection of adopting acid easily to dissociate; because the space bit of Trt group is rented large and hydrophobicity; cause peptide bond to form difficulty, need the long reaction times and then produce more by product and sequence peptide disappearance and racemization.
Therefore, be badly in need of a kind of technique simple, be easy to the artificial chemistry synthesis technique controlling quality and be suitable for large industrial (S)-2-methyl-1,4,5,6-tetra-hydro pyrimidine-4-carboxylic acid.
Summary of the invention
For above-mentioned prior art, the invention provides Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 artificial synthesis.
The present invention is achieved by the following technical solutions:
A kind of solid phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 may further comprise the steps:
Take the aminoresin of Fmoc protection as starting raw material, according to the method for solid phase synthesis, use DIC/ HOBt
For coupling agent connects reactive polypeptide; the amino acid that connects successively the Fmoc protection; obtain the decapeptide resin of protection; the Fmoc group in the deaminize acid successively during this time; after obtaining the decapeptide resin of protection, remove amino acid whose Side chain protective group, cut peptide and obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product; then the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of purifying may further comprise the steps:
1) resin swelling and deprotection
The aminoresin of Fmoc protection is packed in the solid state reaction post, with DMF washing 1 ~ 4 time, then with
DCM swelling 20 ~ 40 minutes, suction filtration is removed DCM, adds the Fmoc protecting group that 20% DBLK solution removes aminoresin, then with DMF washing 4 ~ 6 times;
2) preparation Fmoc-D-Ala-resin:
Under the ice bath Fmoc-D-Ala-OH and HOBt are dissolved among the DMF, then add DIC activation 5 ~ 10
Minute, the solution weight that activation is good is added with in the resin of step 1) gained reacts, stopped reaction when detecting resin and be negative to the triketohydrindene hydrate detection method, and resin gets Fmoc-D-Ala-resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-
Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
With step 2) the Fmoc-D-Ala-resin of gained is with DMF washing 2 ~ 6 times, adds 20% DBLK
Solution removes the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (x)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-
D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove
Then the Fmoc protecting group adds diacetyl oxide and pyridine, detects with the triketohydrindene hydrate detection method, drains after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-
The D-4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-
D-3Pal-D-4-Cpa-D-2Nal-resin:
With L-4, the 5-dihydroorotate is dissolved among the DMF, adds HOBt, adds the DIC activation again, then will
The solution that activation is good add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, behind the stirring reaction 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-
D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-
In the solid state reaction post of D-3Pal-D-4-Cpa-D-2Nal-resin, add the DMF solution of TBAF, stirring reaction washed 3 ~ 8 times with DMF after 2 hours;
8) the standby thick peptide of cracking:
Freezing lysate is joined in the resin of oven dry, stirring reaction 2 hours adds ether with reaction solution
In, freezing 20 ~ 60 minutes, centrifugal, with DMF washing 6 times, be neutrality to pH till, separate, after the drying thick peptide;
9) purifying gets Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is got Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide through separation and purification;
Described aminoresin is selected from a kind of among Rink amide AM, the Rink amide MBHA; Step 2
The protecting group x of described Fmoc-Ser (x)-OH is selected from a kind of among TBDMS, the OBn; The described lysate of step 8) is comprised of TFA, TIS, phenol, methyl-phenoxide, and the volume ratio of TFA, TIS, phenol, methyl-phenoxide is 95:2:2:1.
The substitution degree of described aminoresin is 0.4 ~ 0.8mmol/g.
The volume ratio of the described diacetyl oxide of step 4) and pyridine is 1:1.
The described separation and purification of step 9) may further comprise the steps: thick peptide is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component.
The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, with the Fmoc solid phase synthesis, the problem of having avoided the Boc method repeatedly to come deprotection to bring with acid: such as the joint at peptide and resin, when the each 50%TFA(of using trifluoroacetic acid) when taking off the Boc base, 1.4% the peptide of having an appointment comes off from resin, synthetic peptide chain is longer, loses more serious; In addition, the acid treatment meeting causes some side reactions of side chain.Avoid HF gas to use, alleviated environmental protection pressure; Greatly reduce the TFA(trifluoroacetic acid) use, avoided in the building-up process by product to the pollution of environment; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, 4 Serines are with TBDMS(tertiary butyl dimethyl) replace the tertiary butyl to protect hydroxyl.Can use the TBAF(tetrabutyl ammonium fluoride at last) remove under the mild conditions, can not bring other side reactions, reduced the impurity generation; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention, 5 continuous hydroorotic acid fragments of phenylalanine phenyl ring 4 bit aminos are easily reset under alkaline condition, first with trityl as protecting group, introduce at last again, have avoided the rearrangement side reaction to occur; The artificial synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention replaces D-4Aph (Cbm-tBu) with D-4Aph (Cbm), has avoided t-Bu to remove difficulty, has reduced the side reaction generation.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, should be understood that, following examples only are used for explaining the present invention, rather than restriction protection scope of the present invention.
The solid phase synthesis of embodiment 1 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
1) Rink amide AM resin swelling and the deprotection of Fmoc protection
Rink amide AM(substitution degree 0.49mmol/g with the Fmoc protection; 0.82g; 0.4mmol) in the solid state reaction of packing into the post; with DMF washing 1 ~ 4 time; then with DCM swelling 20 ~ 40 minutes, suction filtration was removed DCM, adds 20% DBLK solution deprotection 5 minutes; 10 minutes respectively once, then with DMF washing 4 ~ 6 times.
2) preparation Fmoc-D-Ala-Rink amide AM resin
Under the ice bath with F-D-Ala-OH(249mg, 0.8mmol), HOBt-Cl(164mg, 0.88mmol)
Be dissolved among the 2ml DMF, then adding 124 μ L DIC activation will activate in the resin that the solution weight of getting well is added with the step 1) gained and react in 5 ~ 10 minutes, stopped reaction when being negative to triketohydrindene hydrate detection method detection resin, resin gets Fmoc-D-Ala-Rink amide AM resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (TBDMS)-D-3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin
With step 2) the Fmoc-D-Ala-Rink amide AM of gained is with DMF washing 2 ~ 6 times, adds
20% DBLK solution removes the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (TBDMS)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (TBDMS)-D-
3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove
Then the Fmoc protecting group adds 2ml diacetyl oxide and 2ml pyridine, detects with the triketohydrindene hydrate detection method, drains after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (TBDMS)-D-3Pal-D-
4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-
Ser (TBDMS)-D-3Pal-D-Phe(4Cl)-D-2Nal-Rink amide AM resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (TBDMS)-D
-3Pal-D-4-Cpa-D-2Nal-Rink amide AM resin:
With L-4,5-dihydroorotate (190mg, 1.2mmol) is dissolved among the 1mlDMF, adding HOBt (183mg,
1.2mmol), add again DIC(124 μ L) activation, then will activate good solution add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (TBDMS)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, behind the stirring reaction 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-D-3Pal-D-4
-Cpa-D-2Nal-Rink amide AM resin:
Ac-Ala-Pro-ILys(Boc is being housed)-the solid state reaction post of Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (TBDMS)-D-3Pal-D-4-Cpa-D-2Nal-Rink amide AM resin in, add TBAF(314mg, 1.2mmol) DMF solution, stirring reaction after 2 hours with DMF washing 3 ~ 8 times;
8) the standby thick peptide of cracking:
Lysate (TFA:TIS: phenol: methyl-phenoxide=95:2:2:1,1ml) join with freezing (5 ℃)
In the resin of oven dry, stirring reaction 2h.Reaction solution is poured in the ether of 6ml freezing 20-60 minute.Centrifugal (4000rpm, 5min),, with DMF washing 6 times, be neutrality to pH till.Separate, get thick peptide 0.85g after the drying.
9 purifying get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component, get Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide 0.357g, yield 42%, purity 99.8%;
The solid phase synthesis of embodiment 2 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2s
The Rink amide AM resin of Fmoc protection among the embodiment 1 is changed into the Rink amide MBHA(substitution degree=0.73mmol/g of Fmoc protection, 0.55g, 0.4mmol).All the other operations get peptide resin 1.17g with embodiment 1 after the contraction, get thick peptide 0.52g, smart peptide 0.21g, yield 40% after the cracking.
The solid phase synthesis of embodiment 3 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2s
The Fmoc-Ser (TBDMS) of step 3) among the embodiment 1-OH is changed to Fmoc-Ser (OBn)-OH, and all the other operate with embodiment 1, and the reaction conditions that removes the Obn protecting group is identical in embodiment, gets smart peptide, and yield is 47%, purity 99.6%
The abbreviation that embodiment and aforementioned process adopt and the material of representative thereof are as follows:
Figure 20121046019591000021

Claims (4)

1. the solid phase synthesis process of an Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is characterized in that, may further comprise the steps:
Take the aminoresin of Fmoc protection as starting raw material; method according to solid phase synthesis; be that coupling agent connects reactive polypeptide with DIC/ HOBt, connect successively the amino acid of Fmoc protection, obtain the decapeptide resin of protection; the Fmoc group in the deaminize acid successively during this time; after obtaining the decapeptide resin of protection, remove amino acid whose Side chain protective group, cut peptide and obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product; then the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of purifying may further comprise the steps:
1) resin swelling and deprotection
The aminoresin of Fmoc protection is packed in the solid state reaction post, and with DMF washing 1 ~ 4 time, then with DCM swelling 20 ~ 40 minutes, suction filtration was removed DCM, adds the Fmoc protecting group that 20% DBLK solution removes aminoresin, then with DMF washing 4 ~ 6 times;
2) preparation Fmoc-D-Ala-resin:
Under the ice bath Fmoc-D-Ala-OH and HOBt are dissolved among the DMF, then add DIC activation 5 ~ 10 minutes, the solution weight that activation is good is added with in the resin of step 1) gained reacts, stopped reaction when being negative to triketohydrindene hydrate detection method detection resin, resin gets Fmoc-D-Ala-resin with DMF washing 4 ~ 6 times;
3) preparation Fmoc-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
With step 2) the Fmoc-D-Ala-resin of gained is with DMF washing 2 ~ 6 times, add 20% DBLK solution and remove the Fmoc protecting group, with DMF washing 4 ~ 6 times, then according to step 2) operation connect reactive polypeptide, successively with Fmoc-Pro-OH, Fmoc-ILys(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-L-4Aph (Trt)-OH, Fmoc-Ser (x)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe(4Cl)-OH, Fmoc-D-2Nal-OH connects according to the aminoacid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
4) preparation Ac-D-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin:
After the step 3) reaction is complete resin is washed 2 ~ 6 times with DMF, add 20% DBLK solution and remove the Fmoc protecting group, then add diacetyl oxide and pyridine, detect with the triketohydrindene hydrate detection method, drain after reaction is finished, with DMF washing 3 ~ 8 times;
5) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-D-Ala-Pro-ILys(Boc is being housed)-Leu-D-4Aph (Cbm)-L-4Aph (Trt)-Ser (x)-D-3Pal-D-Phe(4Cl)-the D-2Nal-resin the solid state reaction post in to add volume ratio be 10% TFA/DCM solution, after the stirring at room reaction, drain, with DMF washing 3 ~ 8 times;
6) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin:
With L-4, the 5-dihydroorotate is dissolved among the DMF, add HOBt, add again the DIC activation, then will activate good solution add Ac-Ala-Pro-ILys(Boc is housed)-Leu-D-4Aph (Cbm)-L-4Aph-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin the solid state reaction post in, stirring reaction is after 2 hours, triketohydrindene hydrate detects and is negative, drain DMF washing 3 ~ 8 times;
7) preparation Ac-Ala-Pro-ILys(Boc)-Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser-D-3Pal-D-4-Cpa-D-2Nal-resin:
Ac-Ala-Pro-ILys(Boc is being housed)-the solid state reaction post of Leu-D-4Aph (Cbm)-L-4Aph(Hor)-Ser (x)-D-3Pal-D-4-Cpa-D-2Nal-resin in, the DMF solution that adds TBAF, stirring reaction washed 3 ~ 8 times with DMF after 2 hours;
8) the standby thick peptide of cracking:
Freezing lysate is joined in the resin of oven dry, stirring reaction 2 hours adds reaction solution in the ether, freezing 20 ~ 60 minutes, centrifugal, with DMF washing 6 times, be neutrality to pH till, separate, after the drying thick peptide;
9) purifying gets Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide:
The thick peptide of step 8) gained is got Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 essence peptide through separation and purification;
Described aminoresin is selected from a kind of among Rink amide AM, the Rink amide MBHA; The protecting group x of the described Fmoc-Ser of step 2 (x)-OH is selected from a kind of among TBDMS, the OBn; The described lysate of step 8) is comprised of TFA, TIS, phenol, methyl-phenoxide, and the volume ratio of TFA, TIS, phenol, methyl-phenoxide is 95:2:2:1.
2. solid phase synthesis process according to claim 1 is characterized in that, the substitution degree of described aminoresin is 0.4 ~ 0.8mmol/g.
3. solid phase synthesis process according to claim 1 is characterized in that, the volume ratio of the described diacetyl oxide of step 4) and pyridine is 1:1.
4. solid phase synthesis process according to claim 1, it is characterized in that, the described separation and purification of step 9) may further comprise the steps: thick peptide is dissolved in 5% aqueous acetic acid, behind 0.45 μ m filtering with microporous membrane, directly be loaded to C18 bonded silica gel filling chromatographic column, adopt gradient 6 ~ 9% methanol-water moving phases to carry out gradient elution separation, collect the purpose component.
CN2012104601959A 2012-11-16 2012-11-16 Method for synthesizing degarelix Pending CN102952174A (en)

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CN103351428A (en) * 2013-08-05 2013-10-16 海南双成药业股份有限公司 Synthesis of degarelix by solid phase segment method
CN103992392A (en) * 2014-05-19 2014-08-20 泰州施美康多肽药物技术有限公司 Solid-phase synthesis method of degarelix
CN103992378A (en) * 2013-11-01 2014-08-20 杭州诺泰制药技术有限公司 Method for preparing Degarelix acetate
CN104177478A (en) * 2014-08-27 2014-12-03 成都圣诺生物科技股份有限公司 Method for synthesizing degarelix
CN105085634A (en) * 2015-08-29 2015-11-25 中肽生化有限公司 Preparation method for degarelix
CN107417771A (en) * 2017-05-04 2017-12-01 苏州强耀生物科技有限公司 A kind of preparation method of the cysteine polypeptide of farnesyl modification
CN109929007A (en) * 2017-12-15 2019-06-25 连云港恒运药业有限公司 The preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 key dipeptides intermediate
CN112125956A (en) * 2019-06-25 2020-12-25 深圳市健元医药科技有限公司 Preparation method of degarelix
US11168114B2 (en) 2015-12-17 2021-11-09 Fresenius Kabi iPSUM S.r.l Process for the manufacture of degarelix and its intermediates
US11332495B2 (en) 2019-09-21 2022-05-17 RK Pharma Solutions LLC Process for the preparation of Degarelix acetate and Degarelix acetate-mannitol premix
CN115141257A (en) * 2021-03-31 2022-10-04 浙江苏泊尔制药有限公司 Purification method of degarelix

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CN102413806A (en) * 2009-05-06 2012-04-11 辉凌公司 Kit and method for preparation of a degarelix solution
CN102428097A (en) * 2009-04-24 2012-04-25 多肽实验室联合股份有限公司 Method For The Manufacture Of Degarelix

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CN102428097A (en) * 2009-04-24 2012-04-25 多肽实验室联合股份有限公司 Method For The Manufacture Of Degarelix
CN102413806A (en) * 2009-05-06 2012-04-11 辉凌公司 Kit and method for preparation of a degarelix solution
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CN103351428A (en) * 2013-08-05 2013-10-16 海南双成药业股份有限公司 Synthesis of degarelix by solid phase segment method
CN103992378A (en) * 2013-11-01 2014-08-20 杭州诺泰制药技术有限公司 Method for preparing Degarelix acetate
CN103992392A (en) * 2014-05-19 2014-08-20 泰州施美康多肽药物技术有限公司 Solid-phase synthesis method of degarelix
CN103992392B (en) * 2014-05-19 2017-05-31 泰州启瑞医药科技有限公司 A kind of solid phase synthesis process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
CN104177478B (en) * 2014-08-27 2018-04-03 成都圣诺生物制药有限公司 A kind of method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
CN104177478A (en) * 2014-08-27 2014-12-03 成都圣诺生物科技股份有限公司 Method for synthesizing degarelix
CN105085634A (en) * 2015-08-29 2015-11-25 中肽生化有限公司 Preparation method for degarelix
US11168114B2 (en) 2015-12-17 2021-11-09 Fresenius Kabi iPSUM S.r.l Process for the manufacture of degarelix and its intermediates
EP3981781A1 (en) 2015-12-17 2022-04-13 Fresenius Kabi iPSUM S.r.l. Process for the manufacture of degarelix and its intermediates
CN107417771B (en) * 2017-05-04 2021-06-29 苏州强耀生物科技有限公司 Preparation method of farnesyl-modified cysteine polypeptide
CN107417771A (en) * 2017-05-04 2017-12-01 苏州强耀生物科技有限公司 A kind of preparation method of the cysteine polypeptide of farnesyl modification
CN109929007A (en) * 2017-12-15 2019-06-25 连云港恒运药业有限公司 The preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 key dipeptides intermediate
CN109929007B (en) * 2017-12-15 2023-07-28 连云港恒运药业有限公司 Preparation method of degarelix key dipeptide intermediate
CN112125956A (en) * 2019-06-25 2020-12-25 深圳市健元医药科技有限公司 Preparation method of degarelix
WO2020259714A1 (en) * 2019-06-25 2020-12-30 深圳市健元医药科技有限公司 Method for preparing degarelix
US11332495B2 (en) 2019-09-21 2022-05-17 RK Pharma Solutions LLC Process for the preparation of Degarelix acetate and Degarelix acetate-mannitol premix
CN115141257A (en) * 2021-03-31 2022-10-04 浙江苏泊尔制药有限公司 Purification method of degarelix

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