CN103992392B - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention discloses a kind of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 and the synthetic method of raw material; with Fmoc amino resins as solid phase carrier; the whey acid fragment for replacing 5 bit aminos of phenylalanine phenyl ring 4 connected with ivdde; condensation reaction connects 10 amino acid of protection successively from C-terminal to N-terminal, obtains full guard peptide resin, then takes off the Fmoc protection groups of N-terminal D Nal; with acetic anhydride and pyridine acetylation; Hor in ivDde replacements is taken off, peptide resin is cut with lytic reagent, ice ether precipitation obtains thick peptide.Additionally, this patent is also related to the method for ivDde OH and Fmoc Aph OH synthesis material Fmoc Aph (ivDde) OH.The present invention first introduces ivDde synthesis decapeptides, replaces Hor again afterwards and avoids the generation for resetting side reaction, and processing step is simple, it is easy to control, small to human body and ambient influnence, yield is high, is adapted to 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), trade name Firmagon is that Ferring Pharmaceuticals companies open
The degarelix powder-injection of hair, ratifies through U.S. FDA, and on December 24th, 2008 lists in the U.S..Indication：FIRMAGON is
GnRH receptor antagonists are used to treat advanced prostate cancer.Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 system one gonadotropin-releasing hormone (GRH) (GnRH) acceptor presses down
Preparation class medicine, reversible inhibition hypophysis GnRH acceptors reduce the release that gonadotropin releasing hormone then suppresses testosterone.Ground adds
Rake is by suppressing to delay prostate cancer continued propagation vital testosterone the growth and deterioration of prostate cancer.With hormone
Treatment prostate cancer but causes testosterone concentration to increase sharply reducing the initial stage of testosterone concentration, and this initial impulse hormone receptor can be temporary transient
Property promote tumour growth rather than suppress it, and ground degarelix then will not.III phase clinical studies show, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 reduction
The effect of testosterone concentration can at least compare favourably with Leuprorelin depot controlled release injection (Lupron Depot), and reduce testis
Ketone concentration is statistically significantly fast.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 the testosterone concentration of gonad, bright
Third Rayleigh group effect is 0%.14th, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 group 99% reached the testosterone concentration of gonad, and Leuprorelin group is
18%.In clinical studies, prostate specific antigen (PSA) concentration can be used as the 2nd Outcome measure terminal of monitoring.Use
Degarelix reduces PSA64% after two weeks, 85% after January, 95% after March, PSA is suppressed all the time in whole 1 year for the treatment of.
Side effect shows as：Injection site reaction (such as pain, erythema, swelling, or scleroma), hectic fever, increased weight, serum transaminase and
Gamma glutamyl transpeptidase (GGT increases).
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is that, containing seven linear decapeptides of alpha-non-natural amino acid, molecular weight is that 1632.26, CAS registration numbers are
214766-78-6, its amino acid sequence is Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph (L-Hor)-D-4Aph
(Cbm)-Leu-Ilys-Pro-D-Ala-NH2.Structural formula is as follows.
Patent document WO9846634 reports a kind of method that Boc solid-phase synthesis synthesize Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, by Boc-D-
Ala-OH and mbha resin are coupled, and are then washed again, with acid removing Boc protection groups, next amino acid are connected, wherein 5
The side chain of position and the 4Aph of 6 is protected using Fmoc, then with piperidines/DMF solution deprotection base, is added respectively again on side chain
L-Hor and Cbm groups.Additionally, WO2011066386 and US5925730 are also to synthesize Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 using Boc solid-phase synthesis,
Ser side chains in patent document WO2011066386 obtain Trt and protect with easy acid dissociation, but because the steric hindrance of Trt protection groups is big
With its hydrophobicity, cause to connect that peptide is relatively difficult, it is necessary to longer time is connected, easily produce accessory substance and racemization.Additionally,
Boc solid-phase synthesis are required for being cracked with HF, and very big harm can be all caused to human body and environment.
Patent document WO2010121835 discloses a kind of method that Fmoc solid-phase synthesis synthesize Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2,5 and 6
Position is coupled with Fmoc-Aph (L-hor)-OH and Fmoc-Aph (tBuCbm)-OH respectively.According to pertinent literature, 5 4- ammonia
Hydroorotic acid fragment on base phenylalanine side-chain is unstable under alkaline environment, is susceptible to reset generation glycolylurea structure
Accessory substance, above-mentioned patent is after 5 amino acids have been connect, it is necessary to piperidines/DMF aqueous slkalis deprotection repeatedly, be susceptible to isomery
Change.Patent document CN102329373A and CN102952174A synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 are also to be closed with Fmoc strategy solid-phase synthesis
Into, their 5 4- amino phenylalanines side chains are first protected with Trt or Alloc, and after having connect decapeptide, deprotection base adds again
Plus Hor.Trt protection groups on side chain need to be removed with TFA, and in sour subtractive process, the Boc protection groups on ILys side chains are held
It is easy to fall off, additionally, being also easily broken when TFA is processed at peptide and resin joint.When Alloc protection groups on side chain are removed
Heavy metal substance is readily incorporated, is not suitable for pharmaceutical synthesis.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of step simplify and the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of high income solid phase close
Into method.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
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, the method comprises the following steps：
(1) ivDde-OH and Fmoc-Aph-OH are dissolved in absolute ethyl alcohol, add trifluoroacetic acid, back flow reaction, TLC inspections
Reaction process is surveyed, synthesizes Fmoc-Aph (ivDde)-OH, after the completion of reaction, be evaporated, watery hydrochloric acid and saturation NaCl solution respectively wash one
Secondary, purifying obtains double protected amino acids；
(2) use Fmoc synthesis in solid state strategies, using 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 obtained in step (1)
(ivDde)-OH、Fmoc-Ser(tBu)-OH、Fmoc-D-3Pal-OH、Fmoc-D-Phe(4Cl)-OH、Fmoc-D-2Nal-OH
Sequence according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid is connected on amino resins, obtains the peptide resins of Fmoc- ten, i.e. Fmoc-D-2Nal-D-
Phe (4Cl)-D-3Pal-Ser (tBu) -4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala- amino trees
Fat；
(3) peptide resins of Fmoc- ten for obtaining step (2) remove Fmoc protection groups, then the amino acid system of acetylation N-terminal
It is standby to obtain the peptide resins of AC- ten, i.e. AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu) -4Aph (ivDde)-D-4Aph
(X)-Leu-ILys (Boc)-Pro-D-Ala- amino resins；
(4), be connected to for orotic acid with coupling agent then by the peptide resins of the AC- ten removing ivDde protection groups for obtaining step (3)
In the amino acids of C-terminal the 5th, peptide resin is cut, obtain 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 protection groups or tBuCbm protection groups.
In step (1), the reaction molar equivalent ratio of ivDde-OH, Fmoc-Aph-OH and trifluoroacetic acid is 1.1：1：0.1.
In step (2) and (3), described amino resins is Rink-Amide-MBHA resins or Rink-Amide-AM trees
Fat.
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 presses mole equivalent proportion 1:1:2 mixture；HATU, Y reagent and DIEA press mole equivalent proportion 1:1:2 mixture；DIC with
Y reagents press mole equivalent proportion 1:1 mixture；Wherein, described Y reagents are HoBt or HoAt.
In step (2), by 1:(1~6):(1~6):(1~6):The mol ratio of (2~12) weigh respectively it is swelling after ammonia
Base resin, Fmoc protected amino acids, HoBt, HBTU and DIEA, dimethylformamide is dissolved in by Fmoc protected amino acids, HoBt,
Pre-activate on ice, adds HBTU and DIEA, then add it is swelling after amino resins, room temperature concussion reaction 0.5~3 hour is even
Fmoc protected amino acids are connect, with the de- Fmoc protection groups 2 of DMF solution of piperidines before connecting next amino acid
Secondary, the time is respectively 3~5 minutes and 15~25 minutes, connect every time after an amino acid and removing Fmoc protection groups it
After be required for being washed with dimethylformamide 3~10 times.
In step (2), it is swelling after amino resins be prepared as follows obtaining：The amino resins that Fmoc is protected loads
To in solid phase reactor, DCM is swelling twice, each 1h, pumps solvent, and DMF is washed 2~6 times, the N of piperidines, N dimethyl formyl
The de- Fmoc protection groups of amine aqueous solution 2 times, the time is respectively 3~5 minutes and 15~25 minutes, and the preferably time is respectively 5 minutes and 20
Minute, DMF is washed 2~6 times, ninhydrin detection method detection removal effect.
In step (3), the method by the peptide resins of Fmoc- ten removing Fmoc protection groups is with the N of piperidines, N dimethyl formyl
The de- Fmoc protection groups of amine aqueous solution.
In step (3), the method that the amino acid of acetylation N-terminal prepares the peptide resins of AC- ten is protected Fmoc is being stripped of
Protect in ten peptide resins of base, add the mixed solution of acetic anhydride and DCM, react 30min, the detection of ninhydrin detection method is drained molten
Liquid, DMF is washed 2~6 times.Wherein, acetic anhydride and DCM volume ratios are 1:3.
In step (4), the method by the peptide resins of AC- ten removing ivDde protection groups is, with the hydrazine containing 2 (v/v) %
DMF solution removes ivDde protection groups, takes off twice, each 30min, the detection of ninhydrin detection method, drains solution, DMF washings 2~6
It is secondary.
In step (4), it is by the method that orotic acid is connected in the amino acids of C-terminal the 5th with coupling agent, L-4,5- dihydro whey
Acid and HOBt are dissolved in DMF on ice, N2Protection is lower to add DIEA to activate 3~10min, adds HBTU, then adds mixed liquor
Enter to reacting equipped with being stripped of in the solid phase reactor of the peptide resins of AC- ten of ivDde protection groups, detected to ninhydrin detection method and set
Stop reaction when fat is negative, DMF is washed 2~6 times.
In step (4), the method for cutting peptide resin is to press trifluoroacetic acid, thioanisole, methyl phenyl ethers anisole, 1,2- dithioglycols
Volume ratio 90:5:3:2 mixing, will be connected to the peptide resins of AC- ten after orotic acid and add above-mentioned mixed liquor, 40 DEG C of concussion reactions
After 0.5~4 hour, by reacting liquid filtering addition cold diethyl ether, precipitation, collected after centrifugation 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, is synthesized with Fmoc solid-phase synthesis, it is to avoid
With a series of problems produced by Boc solid-phase synthesis：As the peptide of part from branch can be made when removing Boc protection groups with acid repeatedly
On split away off, peptide chain it is more long loss it is more serious, acid removing can also bring many side chain side reactions.Additionally, avoid using HF
Gas, reduces the pollution to 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, uses
IvDde replaces the connected whey acid fragment of 5 bit aminos of phenylalanine phenyl ring 4, replaces with orotic acid piece again after decapeptide has connect
Section, it is to avoid the rearrangement reaction of the hydroorotic acid fragment during removing Fmoc caused by alkaline environment repeatedly；Institute of the present invention
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 stated, it is to avoid during with sour deprotection base, the Side chain protective group Boc of ILys easily takes off
Fall, bring 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, mild condition, easily
Small to human body and ambient influnence in control, yield is high, is adapted to large-scale production.
Brief description of the drawings
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-NH2s.
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-NH2s.
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 invention.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the specific material proportion described by example, process conditions and its result and be merely to illustrate the present invention, without that will not also should limit
The present invention described in detail in claims processed.
Specification or right will be shown in Table 1 with regard to the abbreviation implication used in book.
Table 1
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| DCM | Dichloromethane |
| DMF | N,N-dimethylformamide |
| HoBt | I-hydroxybenzotriazole |
| HBTU | BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester |
| DIEA | N, N- diisopropylethylamine |
| TFA | Trifluoroacetic acid |
| DIC | N, N'- DIC |
| HoAt | N- hydroxyl -7- azo BTAs |
| HATU | 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester |
Embodiment 1：The preparation of Fmoc-D-Ala-Rink-Amide-MBHA.
0.88g (0.3mmol) Fmoc-Rink-Amide-MBHA resins (substitution degree 0.34mmol/g) is weighed to be placed in plus sieve
The 20ml BD syringes (simple glass reactive polypeptide device can also) of plate, swelling twice with the DCM of 3 times of resin volumes, 1 is small every time
When, when resin swelling is completely afterwards with de- Fmoc protection groups of 20% (v/v) piperidines/DMF solution 2 times, the time be respectively 5min and
20min, DMF are washed 3 times.373.56mg (1.2mmol) Fmoc-D-Ala-OH and 162.12mg are dissolved with 10ml DMF
(1.2mmol) HOBt, ice bath ten minutes adds 0.4ml (2.4mmol) DIEA, activates 3min, adds 455.04mg
(1.2mmol) HBTU, mixed solution is added in solid phase reactor, stirring reaction 2h, and ninhydrin inspection is negative, and DMF is washed
Three times, drain and obtain Fmoc-D-Ala-Rink-Amide-MBHA.
Embodiment 2：Fmoc-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tBu)-4Aph(ivDde)-D-4Aph
(tBuCbm) synthesis of-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA.
Fmoc-D-Ala-Rink-Amide-MBHA (0.3mmol) is fitted into solid phase reactor, DMF is washed twice, is used
The de- Fmoc protection groups of 20% (v/v) piperidines/DMF solution 2 times, the time is respectively 5min and 20min, DMF are washed 3 times, ninhydrin
Method is checked.404.88mg (1.2mmol) Fmoc-Pro-OH and 162.12mg (1.2mmol) HOBt, ice bath are dissolved with 10ml DMF
Ten minutes, 0.4ml (2.4mmol) DIEA is added, activate 3min, 455.04mg (1.2mmol) HBTU is added, by mixed solution
It is added in solid phase reactor, stirring reaction 1.5h, ninhydrin inspection is negative, and DMF is washed three times.
Aforesaid operations are repeated, according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 amino acid sequence, Fmoc-Pro-OH, Fmoc-ILys is sequentially connected
(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 such as table 2.
The every kind of amino acid of table 2, the consumption of condensing agent and reaction time
Embodiment 3：AC-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tBu)-4Aph(ivDde)-D-4Aph
(tBuCbm) synthesis of-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA.
By Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu) -4Aph (ivDde)-D-4Aph (tBuCbm) -
Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA loads solid phase reactor, and DMF is washed twice, with 20% (v/v) piperazine
The de- Fmoc protection groups of pyridine/DMF solution 2 times, the time is respectively 5min and 20min, DMF are washed 3 times, ninhydrin method inspection.Then
Add the mixed solution of acetic anhydride (3ml) and DCM (9ml), stirring reaction 30min, DMF washing 3 times, the inspection of ninhydrin detection method
Survey.
Embodiment 4：AC-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tBu)-4Aph-D-4Aph(tBuCbm)-Leu-
The synthesis of ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA.
By 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, adds the hydrazine/DMF of 10ml2%
Solution, reacts 30min, repeats reaction once, and DMF is washed 3 times, ninhydrin detection.
Embodiment 5：AC-D-2Nal-D-Phe(4Cl)-D-3Pal-Ser(tBu)-4Aph(Hor)-D-4Aph
(tBuCbm) synthesis of-Leu-ILys (Boc)-Pro-D-Ala-Rink-Amide-MBHA.
By AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu) -4Aph-D-4Aph (tBuCbm)-Leu-ILys
(Boc)-Pro-D-Ala-Rink-Amide-MBHA is encased in solid phase reactor, by 189.73mg L-4,5- dihydro wheys
Acid and 162.12mg HOBt are dissolved in 10ml DMF, N2Protection, ice bath ten minutes adds 0.4ml DIEA, activates 3min, then
455.04mg HBTU are added, mixed solution is added in solid phase reactor, stirring reaction 3h, ninhydrin inspection, DMF washes three
It is secondary, drain.
Embodiment 6：The cracking of peptide resin.
By the lysate (TFA of 10ml：Thioanisole：Methyl phenyl ethers anisole：1,2- dithioglycol=90:5:3:2) it is added to and is equipped with
In the round-bottomed flask of peptide resin, 40 DEG C of reaction 3h after reaction terminates, are added in 100ml ice ether, 5 DEG C after filtering,
4000rpm is centrifuged ten minutes, and repetition is washed three times with ice ether, obtains thick peptide 439.53mg, and thick yield is 95%, purified,
Prepared purity is 99.6% Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 (Fig. 1 is shown in HPLC detections, and Mass Spectrometric Identification is shown in Fig. 2).
HPLC purification process：
Equipment：Receive micro- Unips10-300：10×250mm；
Eluent A:0.1% (v/v) TFA/H2O；
Eluent B:100% acetonitrile；
Flow velocity：3ml/min；
Detection 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 absolute ethyl alcohol, are added a small amount of
TFA (0.1eq), back flow reaction, TLC detection reaction process after the completion of reaction, is evaporated, and watery hydrochloric acid and saturation 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, C37H40N2O6,1H NMR(CDCl3, 400HZ) and δ：7.77 (2H, d, J7.6HZ,-
CH), 7.57 (2H, d, J6.8HZ,-CH), 7.41 (2H, t, J7.6HZ,-CH), 7.32 (2H, t, J7.2HZ,-CH), 7.20 (2H,
D, J7.6HZ,-CH), 7.01 (2H, d, J7.6HZ,-CH), 5.43 (1H, d, J7.2HZ,-NH), 4.70 (1H, d, J6.8HZ,-
CH), 4.48 (2H, m ,-CH2), 4.22 (1H, t, J6.8HZ,-CH), 3.25 (2H, m ,-CH2), 2.46 (4H, s ,-CH2), 1.82
(1H, t, J6.4HZ,-CH), 1.07 (6H, s ,-CH3), 0.74 (6H, d, J5.2HZ,-CH3)。
Claims (8)
1. 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, it is characterised in that the method comprises the following steps：
(1) ivDde-OH and Fmoc-Aph-OH are dissolved in absolute ethyl alcohol, add trifluoroacetic acid, back flow reaction synthesizes Fmoc-
Aph(ivDde)-OH；
(2) Fmoc synthesis in solid state strategies are used, using 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 obtained in step (1),
Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH are according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
The sequence of amino acid is connected on amino resins, obtains the peptide resins of Fmoc- ten, i.e. Fmoc-D-2Nal-D-Phe (4Cl)-D-
3Pal-Ser (tBu) -4Aph (ivDde)-D-4Aph (X)-Leu-ILys (Boc)-Pro-D-Ala- amino resins；
(3) peptide resins of Fmoc- ten for obtaining step (2) remove Fmoc protection groups, and then the amino acid of acetylation N-terminal is prepared into
To the peptide resins of AC- ten, i.e. AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu) -4Aph (ivDde)-D-4Aph (X) -
Leu-ILys (Boc)-Pro-D-Ala- amino resins；
(4) peptide resins of the AC- ten removing ivDde protection groups for obtaining step (3), are then connected to C-terminal with coupling agent by orotic acid
In 5th amino acids, peptide resin is cut, obtain 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 tBuCbm protection groups；
In step (4), the method by the peptide resins of AC- ten removing ivDde protection groups is, with the DMF solution of the hydrazine containing 2%v/v
Removing ivDde protection groups, are taken off twice, each 30min, drain solution, and DMF is washed 2~6 times；
In step (4), the method for cutting peptide resin is that trifluoroacetic acid, thioanisole, methyl phenyl ethers anisole, 1,2- dithioglycols are pressed into volume
Than 90:5:3:2 mixing, will be connected to the peptide resins of AC- ten after orotic acid and add above-mentioned mixed liquor, 40 DEG C of concussion reactions 0.5~4
After hour, by reacting liquid filtering addition cold diethyl ether, precipitation, collected after centrifugation 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.
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, it is characterised in that in step (1), ivDde-
The reaction molar equivalent ratio of OH, Fmoc-Aph-OH and trifluoroacetic acid is 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, it is characterised in that in step (2) and (3), institute
The amino resins stated is Rink-Amide-MBHA resins or Rink-Amide-AM resins.
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 characterised in that in step (2) and (4), institute
The coupling agent stated is choosing any one kind of them in following three kinds of mixtures：HBTU, Y reagent and DIEA press mole equivalent proportion 1:1:2 it is mixed
Compound；HATU, Y reagent and DIEA press mole equivalent proportion 1:1:2 mixture；DIC presses mole equivalent proportion 1 with Y reagents:1 it is mixed
Compound；Wherein, described Y reagents are 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 characterised in that in step (2), by 1:(1~
6):(1~6):(1~6):The mol ratio of (2~12) weigh respectively it is swelling after amino resins, Fmoc protected amino acids, HoBt,
HBTU and DIEA, is dissolved in dimethylformamide, on ice pre-activate by Fmoc protected amino acids, HoBt, adds HBTU and DIEA, so
Add afterwards it is swelling after amino resins, the connection Fmoc protected amino acids of room temperature concussion reaction 0.5~3 hour connect next
With de- Fmoc protection groups of the DMF solution of piperidines 2 times before amino acid, the time be respectively 3~5 minutes and 15~
25 minutes, connect every time after an amino acid and removing Fmoc protection groups after be required for washing 3 with dimethylformamide
~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, it is characterised in that in step (3), by Fmoc-
The method of ten peptide resins removing Fmoc protection groups is with the N of piperidines, the de- Fmoc protection groups of N-dimethylformamide solution.
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 characterised in that in step (3), acetylation N
The method that the amino acid at end prepares the peptide resins of AC- ten is, in ten peptide resins for being stripped of Fmoc protection groups, adds acetic acid
The mixed solution of acid anhydride and DCM, reacts 30min, drains solution, and DMF is washed 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 characterised in that in step (4), use coupling agent
It is that L-4,5- dihydrooratic acid and HOBt are dissolved in DMF on ice, N by the method that orotic acid is connected in the amino acids of C-terminal the 5th2Protect
Shield is lower to add DIEA to activate 3~10min, adds HBTU, and then mixed liquor is added to equipped with being stripped of ivDde protection groups
Reacted in the solid phase reactor of the peptide resins of AC- ten, reaction, DMF washings 2 are stopped when being negative to ninhydrin detection method detection resin
~6 times.
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| CN105085634A (en) * | 2015-08-29 | 2015-11-25 | 中肽生化有限公司 | Preparation method for degarelix |
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| 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 |
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| 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 |
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| 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 |
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| 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 |
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