CN103992392A - Solid-phase synthesis method of degarelix - Google Patents
Solid-phase synthesis method of degarelix Download PDFInfo
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- CN103992392A CN103992392A CN201410211655.3A CN201410211655A CN103992392A CN 103992392 A CN103992392 A CN 103992392A CN 201410211655 A CN201410211655 A CN 201410211655A CN 103992392 A CN103992392 A CN 103992392A
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Abstract
The invention discloses a solid-phase synthesis method of degarelix. The solid-phase synthesis method of degarelix comprises the following steps: on the basis of taking Fmoc-amino resin as a solid-phase carrier, replacing orotic acid fragments connected with an alanine benzene ring in the fifth site and an amino in the fourth site with ivdde, and performing sequential condensation reaction from the end C to the end N so as to connect 10 protected amino acids, thereby obtaining a full-protective peptide resin; then removing the Fmoc protecting group of end N D-Nal, acetylating by using acetic anhydride and pyridine, replacing ivDde with Hor, cutting the peptide resin by using a splitting agent, and precipitating by using frozen diethyl ether to obtain crude peptide. Besides, the invention also relates to a method for synthesizing a raw material Fmoc-Aph(ivDde)-OH by use of ivDde-OH and Fmoc-Aph-OH. The solid-phase synthesis method of degarelix has the advantages that the ivDde is introduced to synthesize decapeptide firstly and then the Hor is used as a substitute to avoid a rearrangement side reaction; the solid-phase synthesis method is simple in process steps, easy to control, low in influence on a human body and the environment, high in yield, and suitable for large-scale production.
Description
Technical field
The present invention relates to 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.
Background technology
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 (degarelix), commodity are called Firmagon, are the degarelix powder injection of Ferring Pharmaceuticals company exploitation, through U.S. FDA approval, in December, 2008 24 U.S. listing.Indication: FIRMAGON is that GnRH receptor antagonist is used for the treatment of advanced prostate cancer.Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is a gonadotropin releasing hormone (GnRH) acceptor inhibitor class medicine, and reversible inhibition hypophysis GnRH acceptor reduces the release that gonadotropin releasing hormone suppresses testosterone then.Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is by suppressing that prostate cancer is continued to growth and the deterioration that the vital testosterone of growth delays prostate cancer.The initial stage of reducing testosterone concentration with hormonotherapy prostate cancer but causes testosterone concentration to increase sharply, and this hormone receptor of this initial impulse can temporary promotion tumor growth rather than suppressed it, and ground degarelix can not.III phase clinical studies show, the effect that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 reduces testosterone concentration at least can compare favourably with Leuprolide depot controlled release injection (Lupron Depot), and it is significantly fast statistically to reduce testosterone concentration.At the 3rd day for the treatment of, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 group 96% reached gonadal testosterone concentration, and Leuprolide group effect is 0%.The 14th, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 group 99% reached gonadal testosterone concentration, and Leuprolide group is 18%.In clinical study, prostate specific antigen (PSA) concentration can be used as the 2nd curative effect judgement terminal of monitoring.Use degarelix after two weeks, to reduce PSA64%, after January 85%, after March 95%, in whole 1 year that treats, suppress all the time PSA.Side effect shows as: injection site reaction (as pain, erythema, swelling, or scleroma), and hectic fever, body weight increases, serum transaminase and gamma glutamyl transpeptidase (GGT increase).
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 seven alpha-non-natural amino acids, molecular weight is 1632.26, CAS registration number is 214766-78-6, and its aminoacid sequence is Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph (L-Hor)-D-4Aph (Cbm)-Leu-Ilys-Pro-D-Ala-NH
2.Structural formula is as follows.
Patent documentation WO9846634 has reported the method for the synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of a kind of Boc solid-phase synthesis; Boc-D-Ala-OH and mbha resin are carried out to coupling; and then washing; with acid, remove Boc protecting group; connect next amino acid; wherein the side chain of 5 and 6 s' 4Aph adopts Fmoc protection, then uses piperidines/DMF solution deprotection base, adds respectively L-Hor and Cbm group on side chain again.In addition; WO2011066386 and US5925730 adopt the synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of Boc solid-phase synthesis; Ser side chain in patent documentation WO2011066386 obtains Trt protection with easy acid dissociation; but because the steric hindrance of Trt protecting group is large and its hydrophobicity; cause that to connect peptide more difficult; time that need to be longer connects, and easily produces by product and racemization.In addition, Boc solid-phase synthesis all needs to carry out cracking with HF, to human body and environment, all can cause very large harm.
Patent documentation WO2010121835 discloses the method for the synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of a kind of Fmoc solid-phase synthesis, uses respectively Fmoc-Aph (L-hor)-OH and Fmoc-Aph (tBuCbm)-OH to carry out coupling for 5 and 6.According to pertinent literature; hydroorotic acid fragment on 5 4-amino-benzene L-Ala side chains is unstable under alkaline environment, easily occurs to reset the by product that generates glycolylurea structure, and above-mentioned patent is after having connect 5 amino acids; need to use piperidines/DMF alkaline solution deprotection repeatedly, isomerization easily occurs.What patent documentation CN102329373A and CN102952174A synthesized Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is also synthetic with Fmoc strategy solid-phase synthesis, and their 5 4-amino-benzene L-Ala side chains first, with Trt or Alloc protection, have connect after decapeptide, and deprotection base adds upper Hor again.Trt protecting group on side chain need to remove with TFA, and in sour subtractive process, the Boc protecting group on ILys side chain easily comes off, and in addition, also easily rupture in peptide and resin joint place when TFA processes.When the Alloc protecting group on side chain removes, easily introduce heavy metal substance, be not suitable for medicine synthetic.
Summary of the invention
Technical problem to be solved by this invention is to provide that a kind of step is simplified and the solid phase synthesis process of the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 that yield is high.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A solid phase synthesis process for Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, the method comprises the steps:
(1) ivDde-OH and Fmoc-Aph-OH are dissolved in dehydrated alcohol, add trifluoroacetic acid, back flow reaction, TLC detection reaction process, synthetic Fmoc-Aph (ivDde)-OH, after having reacted, evaporate to dryness, dilute hydrochloric acid and saturated NaCl solution are respectively washed once, and purifying obtains two protected amino acids;
(2) adopt Fmoc solid phase synthesis strategy, utilize coupling agent successively by Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (X)-OH, Fmoc-4Aph (ivDde)-OH that step (1) makes, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH is connected on aminoresin according to the amino acid whose sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, obtain Fmoc-decapeptide resin, be Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala-aminoresin,
(3) Fmoc-decapeptide resin step (2) being obtained removes Fmoc protecting group, then the amino acid of acetylize N end prepares AC-decapeptide resin, i.e. AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala-aminoresin;
(4) AC-decapeptide resin step (3) being obtained removes ivDde protecting group, then with coupling agent, vitamin B13 is linked in C end the 5th amino acids, and cutting peptide resin, obtains the thick peptide of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
Wherein, X is Cbm protecting group or tBuCbm protecting group.
In step (1), the reaction molar equivalent of ivDde-OH, Fmoc-Aph-OH and trifluoroacetic acid is than being 1.1:1:0.1.
In step (2) and (3), described aminoresin is Rink-Amide-MBHA resin or Rink-Amide-AM resin.
In step (2) and (4), described coupling agent is choosing any one kind of them in following three kinds of mixtures: HBTU, Y reagent and DIEA are by the mixture of mole equivalence ratio 1:1:2; HATU, Y reagent and DIEA are by the mixture of mole equivalence ratio 1:1:2; DIC and Y reagent are by the mixture of mole equivalence ratio 1:1; Wherein, described Y reagent is HoBt or HoAt.
In step (2), by 1:(1～6): (1～6): (1～6): the mol ratio of (2～12) takes respectively the aminoresin after swelling, Fmoc protected amino acid, HoBt, HBTU and DIEA, by Fmoc protected amino acid, HoBt is dissolved in dimethyl formamide, activation in advance on ice, add HBTU and DIEA, and then add the aminoresin after swelling, room temperature concussion reaction connects Fmoc protected amino acid for 0.5～3 hour, connect next amino acid and use before the N of piperidines, the de-Fmoc protecting group of N dimethyl formamide solution 2 times, time is respectively 3～5 minutes and 15～25 minutes, after the upper amino acid of each connection and after removing Fmoc protecting group, all need with dimethyl formamide washing 3～10 times.
In step (2); aminoresin after swelling prepares as follows: the aminoresin of Fmoc protection is encased in solid phase reactor to DCM swelling twice, each 1h; pump solvent; DMF washing 2～6 times, the de-Fmoc protecting group of DMF solution of piperidines 2 times; time is respectively 3～5 minutes and 15～25 minutes; preferably the time is respectively 5 minutes and 20 minutes, DMF washing 2～6 times, and triketohydrindene hydrate detection method detects removal effect.
In step (3), the method that Fmoc-decapeptide resin is removed to Fmoc protecting group is the de-Fmoc protecting group of DMF solution with piperidines.
In step (3), the method that the amino acid of acetylize N end prepares AC-decapeptide resin is, in having removed the decapeptide resin of Fmoc protecting group; the mixing solutions that adds acetic anhydride and DCM, reaction 30min, triketohydrindene hydrate detection method detects; drain solution, DMF washing 2～6 times.Wherein, acetic anhydride and DCM volume ratio are 1:3.
In step (4), the method that AC-decapeptide resin is removed to ivDde protecting group is, with the DMF solution of the hydrazine that contains 2 (v/v) %, removes ivDde protecting group, de-twice, each 30min, and triketohydrindene hydrate detection method detects, and drains solution, DMF washing 2～6 times.
In step (4), by the method that coupling agent is linked vitamin B13 in C end the 5th amino acids, be, L-4,5-dihydroorotate and HOBt are dissolved in DMF on ice, N
2under protection, add DIEA activation 3～10min; add again HBTU; then mixed solution is joined in the solid phase reactor that the AC-decapeptide resin that has removed ivDde protecting group is housed and react, stopped reaction while being negative to triketohydrindene hydrate detection method detection resin, DMF washing 2～6 times.
In step (4), the method of cutting peptide resin is by trifluoroacetic acid, thioanisole, methyl-phenoxide, 1,2-dithioglycol by volume 90:5:3:2 mixes, by having connected the AC-decapeptide resin after vitamin B13, add above-mentioned mixed solution, 40 ℃ of concussion reactions, after 0.5～4 hour, add reacting liquid filtering in cold diethyl ether precipitation, centrifugal rear collecting precipitation, obtains the thick peptide of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Beneficial effect: 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 the present invention; synthetic with Fmoc solid-phase synthesis; avoided the series of problems producing with Boc solid-phase synthesis: as repeatedly made while removing Boc protecting group the peptide of part split away off from branch with acid; the longer loss of peptide chain is more serious, and acid removes also can bring many side chain side reactions.In addition, avoided use HF gas, reduced the pollution of environment and the injury to human body; 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 the present invention, with ivDde, replace 5 vitamin B13 fragments that phenylalanine phenyl ring 4 bit aminos are connected, after decapeptide has connect, replace with again vitamin B13 fragment, avoided repeatedly removing the rearrangement reaction of the caused hydroorotic acid fragment of Fmoc process neutral and alkali environment; 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 the present invention, has avoided with in sour deprotection base process, and the Side chain protective group Boc of ILys easily comes off, and brings the situation of side reaction; 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 the present invention, processing step is simple, and mild condition is easy to control, and little to human body and environmental influence, productive rate is high, is applicable to scale operation.
Accompanying drawing explanation
The high-efficient liquid phase chromatogram of Fig. 1 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
The Mass Spectrometric Identification figure of Fig. 2 Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
The Mass Spectrometric Identification figure of Fig. 3 Fmoc-Aph (ivDde)-OH.
The nucleus magnetic hydrogen spectrum figure of Fig. 4 Fmoc-Aph (ivDde)-OH.
Fig. 5 is synthetic route chart of the present invention.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Specification sheets or right will be with regard to the abbreviation implication used in book in Table 1.
Table 1
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| DCM | Methylene dichloride |
| DMF | DMF |
| HoBt | I-hydroxybenzotriazole |
| HBTU | Benzotriazole-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester |
| DIEA | DIPEA |
| TFA | Trifluoroacetic acid |
| DIC | N, N'-DIC |
| HoAt | N-hydroxyl-7-azo benzotriazole |
| HATU | 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester |
The preparation of embodiment 1:Fmoc-D-Ala-Rink-Amide-MBHA.
Take 0.88g (0.3mmol) Fmoc-Rink-Amide-MBHA resin (substitution degree 0.34mmol/g) and be placed in the 20ml BD syringe (simple glass reactive polypeptide device also can) that adds sieve plate; by twice of the DCM swelling of 3 times of resin volumes; each 1 hour; by the time resin swelling is completely rear takes off Fmoc protecting group 2 times with 20% (v/v) piperidines/DMF solution; time is respectively 5min and 20min, DMF washing 3 times.With 10ml DMF, dissolve 373.56mg (1.2mmol) Fmoc-D-Ala-OH and 162.12mg (1.2mmol) HOBt, ice bath ten minutes, add 0.4ml (2.4mmol) DIEA, activation 3min, then add 455.04mg (1.2mmol) HBTU, mixing solutions is joined in solid phase reactor, stirring reaction 2h, triketohydrindene hydrate check is negative, and DMF washes three times, drains and obtains Fmoc-D-Ala-Rink-Amide-MBHA.
Embodiment 2:Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA's is synthetic.
Fmoc-D-Ala-Rink-Amide-MBHA (0.3mmol) is packed in solid phase reactor; DMF washes twice, with 20% (v/v) piperidines/DMF solution, takes off Fmoc protecting group 2 times, and the time is respectively 5min and 20min; DMF washing 3 times, ninhydrin method check.With 10ml DMF, dissolve 404.88mg (1.2mmol) Fmoc-Pro-OH and 162.12mg (1.2mmol) HOBt, ice bath ten minutes, add 0.4ml (2.4mmol) DIEA, activation 3min, add again 455.04mg (1.2mmol) HBTU, mixing solutions is joined in solid phase reactor to stirring reaction 1.5h, triketohydrindene hydrate check is negative, and DMF washes three times.
Repeat aforesaid operations, according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aminoacid sequence, connect successively Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (tBuCbm)-OH, Fmoc-4Aph (ivDde)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH, consumption is as table 2.
Consumption and the reaction times of the every seed amino acid of table 2, condensing agent
Embodiment 3:AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA's is synthetic.
Pack Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA into solid phase reactor; DMF washes twice; with 20% (v/v) piperidines/DMF solution, take off Fmoc protecting group 2 times; time is respectively 5min and 20min; DMF washing 3 times, ninhydrin method check.Then the mixing solutions that adds acetic anhydride (3ml) and DCM (9ml), stirring reaction 30min, DMF washing 3 times, triketohydrindene hydrate detection method detects.
Embodiment 4:AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA's is synthetic.
AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA is encased in solid phase reactor, hydrazine/DMF the solution that adds 10ml2%, reaction 30min, reaction repeated once, DMF washes 3 times, and triketohydrindene hydrate detects.
Embodiment 5:AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (Hor)-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA's is synthetic.
By being encased in solid phase reactor of AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph-D-4Aph (tBuCbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA, by 189.73mg L-4,5-dihydroorotate and 162.12mg HOBt are dissolved in 10ml DMF, N
2protection, ice bath ten minutes, adds 0.4ml DIEA, activation 3min, then add 455.04mg HBTU, and mixing solutions is joined in solid phase reactor, stirring reaction 3h, triketohydrindene hydrate check, DMF washes three times, drains.
Embodiment 6: the cracking of peptide resin.
By the lysate of 10ml (TFA: thioanisole: methyl-phenoxide: 1，2-ethandithiol=90:5:3:2) join in the round-bottomed flask that peptide resin is housed 40 ℃ of reaction 3h, after reaction finishes, after filtration, join in 100ml ice ether, 5 ℃, 4000rpm centrifugal ten minutes, repeat with ice ether washing three times, obtain thick peptide 439.53mg, thick productive rate is 95%, purified, make purity and be 99.6% Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 (HPLC detects and sees Fig. 1, and Mass Spectrometric Identification is shown in Fig. 2).
HPLC purification process:
Equipment: receive micro-Unips10-300:10 * 250mm;
Elutriant A:0.1% (v/v) TFA/H
2o;
Elutriant B:100% acetonitrile;
Flow velocity: 3ml/min;
Detect wavelength: 220nm;
Gradient: 20%-40%.
Embodiment 7: synthesis material Fmoc-4Aph (ivDde)-OH.
1.23g ivDde-OH (1.1eq) and 2g Fmoc-Aph-OH (1eq) are dissolved in dehydrated alcohol, add a small amount of TFA (0.1eq), back flow reaction, TLC detection reaction process, after having reacted, evaporate to dryness, dilute hydrochloric acid and saturated NaCl solution are respectively washed once, purifying obtains Fmoc-4Aph (ivDde)-OH2.9g (Mass Spectrometric Identification is shown in Fig. 3, nucleus magnetic hydrogen spectrum Fig. 4).
Fmoc-4Aph(ivDde)-OH，C
37H
40N
2O
6，
1H?NMR(CDCl
3，400HZ)δ：7.77(2H，d，J7.6H
Z，-CH)，7.57(2H，d，J6.8H
Z，-CH)，7.41(2H，t，J7.6H
Z，-CH)，7.32(2H，t，J7.2H
Z，-CH)，7.20(2H，d，J7.6H
Z，-CH)，7.01(2H，d，J7.6H
Z，-CH)，5.43(1H，d，J7.2H
Z，-NH)，4.70(1H，d，J6.8H
Z，-CH)，4.48(2H，m，-CH
2)，4.22(1H，t，J6.8H
Z，-CH)，3.25(2H，m，-CH
2)，2.46(4H，s，-CH
2)，1.82(1H，t，J6.4H
Z，-CH)，1.07(6H，s，-CH
3)，0.74(6H，d，J5.2H
Z，-CH
3)。
Claims (10)
1. a solid phase synthesis process for Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, is characterized in that, the method comprises the steps:
(1) ivDde-OH and Fmoc-Aph-OH are dissolved in dehydrated alcohol, add trifluoroacetic acid, back flow reaction, synthetic Fmoc-Aph (ivDde)-OH;
(2) adopt Fmoc solid phase synthesis strategy, utilize coupling agent successively by Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (X)-OH, Fmoc-4Aph (ivDde)-OH that step (1) makes, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH is connected on aminoresin according to the amino acid whose sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, obtain Fmoc-decapeptide resin, be Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala-aminoresin,
(3) Fmoc-decapeptide resin step (2) being obtained removes Fmoc protecting group, then the amino acid of acetylize N end prepares AC-decapeptide resin, i.e. AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala-aminoresin;
(4) AC-decapeptide resin step (3) being obtained removes ivDde protecting group, then with coupling agent, vitamin B13 is linked in C end the 5th amino acids, and cutting peptide resin, obtains the thick peptide of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2;
Wherein, X is Cbm protecting group or tBuCbm protecting group.
2. the 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 according to claim 1, is characterized in that, in step (1), the reaction molar equivalent of ivDde-OH, Fmoc-Aph-OH and trifluoroacetic acid is than being 1.1:1:0.1.
3. the 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 according to claim 1, is characterized in that, in step (2) and (3), described aminoresin is Rink-Amide-MBHA resin or Rink-Amide-AM resin.
4. the 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 according to claim 1, it is characterized in that, in step (2) and (4), described coupling agent is choosing any one kind of them in following three kinds of mixtures: HBTU, Y reagent and DIEA are by the mixture of mole equivalence ratio 1:1:2; HATU, Y reagent and DIEA are by the mixture of mole equivalence ratio 1:1:2; DIC and Y reagent are by the mixture of mole equivalence ratio 1:1; Wherein, described Y reagent is HoBt or HoAt.
5. the 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 according to claim 4, it is characterized in that, in step (2), by 1:(1～6): (1～6): (1～6): the mol ratio of (2～12) takes respectively the aminoresin after swelling, Fmoc protected amino acid, HoBt, HBTU and DIEA, by Fmoc protected amino acid, HoBt is dissolved in dimethyl formamide, activation in advance on ice, add HBTU and DIEA, and then add the aminoresin after swelling, room temperature concussion reaction connects Fmoc protected amino acid for 0.5～3 hour, connect next amino acid and use before the N of piperidines, the de-Fmoc protecting group of N dimethyl formamide solution 2 times, time is respectively 3～5 minutes and 15～25 minutes, after the upper amino acid of each connection and after removing Fmoc protecting group, all need with dimethyl formamide washing 3～10 times.
6. the 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 according to claim 1, is characterized in that, in step (3), the method that Fmoc-decapeptide resin is removed to Fmoc protecting group is the de-Fmoc protecting group of DMF solution with piperidines.
7. the 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 according to claim 1; it is characterized in that; in step (3); the method that the amino acid of acetylize N end prepares AC-decapeptide resin is; in having removed the decapeptide resin of Fmoc protecting group, add the mixing solutions of acetic anhydride and DCM, reaction 30min; drain solution, DMF washing 2～6 times.
8. the 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 according to claim 1; it is characterized in that; in step (4); the method that AC-decapeptide resin is removed to ivDde protecting group is; with the DMF solution of the hydrazine that contains 2 (v/v) %, remove ivDde protecting group, take off twice, each 30min; drain solution, DMF washing 2～6 times.
9. the 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 according to claim 1, is characterized in that,, in step (4), by the method that coupling agent is linked vitamin B13 in C end the 5th amino acids, be, L-4,5-dihydroorotate and HOBt are dissolved in DMF on ice, N
2under protection, add DIEA activation 3～10min; add again HBTU; then mixed solution is joined in the solid phase reactor that the AC-decapeptide resin that has removed ivDde protecting group is housed and react, stopped reaction while being negative to triketohydrindene hydrate detection method detection resin, DMF washing 2～6 times.
10. the 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 according to claim 1, it is characterized in that, in step (4), the method for cutting peptide resin is by the 90:5:3:2 mixing by volume of trifluoroacetic acid, thioanisole, methyl-phenoxide, 1，2-ethandithiol, by having connected the AC-decapeptide resin after vitamin B13, add above-mentioned mixed solution, 40 ℃ of concussion reactions, after 0.5～4 hour, add reacting liquid filtering in cold diethyl ether precipitation, centrifugal rear collecting precipitation, obtains the thick peptide of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109929012A (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 tetrapeptide intermediate |
| CN110218243A (en) * | 2019-07-09 | 2019-09-10 | 江苏豪森药业集团有限公司 | A method of synthesis acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN110330552A (en) * | 2019-08-14 | 2019-10-15 | 凯莱英生命科学技术(天津)有限公司 | The synthetic method of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| US11168114B2 (en) | 2015-12-17 | 2021-11-09 | Fresenius Kabi iPSUM S.r.l | Process for the manufacture of degarelix and its intermediates |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121835A1 (en) * | 2009-04-24 | 2010-10-28 | Polypeptide Laboratories A/S | Method for the manufacture of degarelix |
| WO2011066386A1 (en) * | 2009-11-25 | 2011-06-03 | Novetide, Ltd. | Process for production of degarelix |
| CN102329373A (en) * | 2011-09-29 | 2012-01-25 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic process for degarelix |
| CN102952174A (en) * | 2012-11-16 | 2013-03-06 | 济南环肽医药科技有限公司 | Method for synthesizing degarelix |
-
2014
- 2014-05-19 CN CN201410211655.3A patent/CN103992392B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121835A1 (en) * | 2009-04-24 | 2010-10-28 | Polypeptide Laboratories A/S | Method for the manufacture of degarelix |
| WO2011066386A1 (en) * | 2009-11-25 | 2011-06-03 | Novetide, Ltd. | Process for production of degarelix |
| CN102329373A (en) * | 2011-09-29 | 2012-01-25 | 深圳翰宇药业股份有限公司 | Solid-phase synthetic process for degarelix |
| CN102952174A (en) * | 2012-11-16 | 2013-03-06 | 济南环肽医药科技有限公司 | Method for synthesizing degarelix |
Non-Patent Citations (1)
| Title |
|---|
| 李文曲等: "末端N-Ivdde保护的氨基酸的合成", 《合成化学》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109929012A (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 tetrapeptide 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 |
| CN109929012B (en) * | 2017-12-15 | 2023-07-28 | 连云港恒运药业有限公司 | Preparation method of degarelix key tetrapeptide intermediate |
| CN110218243A (en) * | 2019-07-09 | 2019-09-10 | 江苏豪森药业集团有限公司 | A method of synthesis acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN110218243B (en) * | 2019-07-09 | 2023-08-29 | 江苏豪森药业集团有限公司 | Method for synthesizing degarelix acetate |
| CN110330552A (en) * | 2019-08-14 | 2019-10-15 | 凯莱英生命科学技术(天津)有限公司 | The synthetic method of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
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