CN107344960A - The preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 - Google Patents
The preparation method 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 provides a kind of preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.This method includes：Using solid-phase synthesis, the raw material including Fmoc 4Nph OH is subjected to the first condensation reaction by the amino acid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 successively, obtains the first intermediate product；Amino acid residue 4Nph in first intermediate product is reduced to 4Aph, obtains the second intermediate product；The side-chain amino group and the dihydrooratic acids of L 4,5 for making 4Aph carry out the second condensation reaction, obtain the 3rd intermediate product；And the 3rd intermediate product is cracked using decomposition agent, obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.Raw material Fmoc 4Nph OH are relatively stable under alkaline environment, thus need not introduce blocking group using above-mentioned raw materials, this effectively avoids side reaction is produced the introducing because of blocking group, so as to improve the purity of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product.Above-mentioned operation is simple, reaction rate is fast, is advantageous to that process costs and synthesis difficulty are greatly reduced.
Description
Technical field
The present invention relates to pharmaceutical synthesis field, in particular to a kind of preparation method 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) is developed by Ferring Pharmaceuticals companies, is ratified through U.S. FDA, in
On December 24th, 2008 lists in the U.S., for treating 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 kind of gonadotropin-releasing hormone (GRH) (GnRH) acceptor inhibitor class medicine, reversibly suppresses to hang down
Body GnRH acceptors reduce the release that gonadotropin releasing hormone then suppresses testosterone.Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is by suppressing to prostate cancer
Continued propagation vital testosterone delays the growth of prostate cancer and deterioration.Prostate cancer is treated with Hormonal methods to reduce
Testosterone concentration in the early stage when but cause testosterone concentration to increase sharply, this initial impulse hormone receptor can temporarily promote and non-inhibited
Tumour growth, and Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 does not have this phenomenon then.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 reduce the effect of testosterone concentration
Fruit can at least compare favourably with Leuprorelin depot controlled release injection, and reduce testosterone concentration markedly fast.The 3rd for the treatment of the
Day, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 group 96% reaches the testosterone concentration of gonad, and Leuprorelin group effect is 0%.14th day, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
Group 99% reaches the testosterone concentration of gonad, and Leuprorelin group is 18%.In clinical studies, prostate specific antigen
(PSA) concentration can judge terminal as the 2nd curative effect of monitoring.After Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 two weeks, reduce PSA 64%, one month
PSA 85% is reduced afterwards, PSA 95% is reduced after three months, and suppress PSA all the time in a year and a 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 exists
The most common adverse reaction reported in clinical research is：Injection site reaction (pain, red and swollen and swelling), hot flash, body weight
Increase, weak and some liver enzyme concentration rises.
Following chemical constitution be present in the decapeptide artificial synthesized as one, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2：
Hexatomic ring L-Hor in the chemical constitution on C-terminal five amino acid Aph (L-Hor) is easy in the basic conditions
Generation isomerization reaction, generates hydantoins -5- acetic acid, and mechanism is as follows：
Above-mentioned reaction can cause impurity A c-D-2Nal-D-Phe similar in one and target product property in product be present
(4Cl)-D-3Pal-Ser-Aph(Z)-D-4Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2, wherein Z represent hydantoins-
5- acetyl group, which increase purifying difficulty, and reduce product yield.
In order to prevent the hexatomic ring L-Hor on Aph (L-Hor) from occurring isomerization reaction in the basic conditions, in most literature
Selection substitutes Aph (L-Hor) using Aph (X), and wherein X represents L-Hor alternative protection group.Then closed using Fmoc solid phases
Into strategy successively by Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH,
Fmoc-4Aph (X)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH and Fmoc-
D-2Nal-OH is attached by the sequence of amino acid of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2；N-terminal Fmoc is removed, second is carried out with acetic anhydride and pyridine
It is acylated；The protection group X on the 6th amino acid residue -4Aph (X) of C-terminal is removed, by L-4,5-dihydrooroticacid connections
Onto the 6th amino acid residue -4Aph of C-terminal side-chain amino group.
The drawbacks of above-mentioned technical proposal is brought be：
, it is necessary to use TFA/DCM during the orotic acid for replacing 5 bit aminos of phenylalanine phenyl ring 4 to be connected using Mmt or Dmt
L-Hor is coupled after removing Z, TFA removing Mmt or Dmt groups are used on fat.This can influence the groups such as the Boc of acid-sensitive, and cause
Product quality and yield be not high.And the raw materials for production that this method uses are more expensive, it is higher to directly result in production cost, and fragment is closed
Into complex operation, it is unfavorable for large-scale production.
When replacing the connected orotic acid of 5 bit aminos of phenylalanine phenyl ring 4 using Teoc, used Material synthesis is stranded
Difficulty, and cost is higher.
When replacing the connected orotic acid of 5 bit aminos of phenylalanine phenyl ring 4 using ivDde, need to use in reaction
Fmoc-Aph (ivDde)-OH is used as raw material.But this Material synthesis is difficult, and complex process, is unsuitable for industrial-scale production.
When replacing the connected orotic acid of 5 bit aminos of phenylalanine phenyl ring 4 using Trt, needed in this method on resin
Trityl is removed using TFA.This can influence the groups such as the Boc of acid-sensitive, so as to cause product quality and yield not high, and
And synthesis material is more expensive, it is higher to directly result in production cost.
The content of the invention
It is a primary object of the present invention to provide a kind of preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, to solve the system of existing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
In Preparation Method, the raw material used needs to be protected using blocking group, and Material synthesis can be caused difficult for this and synthetic route work
The problem of skill cost is high.
To achieve these goals, according to an aspect of the invention, there is provided a kind of preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, system
Preparation Method includes：Using solid-phase synthesis by Fmoc-D-Ala- resins, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH,
Fmoc-Leu-OH、Fmoc-D-4Aph(Cbm)-OH、Fmoc-4Nph-OH、Fmoc-Ser(tBu)-OH、 Fmoc-D-3Pal-
It is anti-that OH, Fmoc-D-Phe (4Cl)-OH and Ac-D-2Nal-OH carry out the first condensation by the amino acid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 successively
Should, obtain the first intermediate product；Amino acid residue 4Nph in first intermediate product is reduced to 4Aph, obtains producing among second
Thing；The side-chain amino group and L-4,5- dihydrooratic acid for making 4Aph carry out the second condensation reaction, obtain the 3rd intermediate product；And use
Decomposition agent cracks to the 3rd intermediate product, obtains Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Further, reduction process includes：The middle 4Nph residues of first intermediate product are reduced to by 4Aph using reducing agent,
Wherein reducing agent is CrCl2。
Further, preparation method also includes Fmoc-D- alanine and resin carrying out the obtained Fmoc- of the 3rd condensation reaction
The step of D-alanine-resin；Preferably, the dosage of reducing agent is 2.5~5.0 times of substitution degree of resin.
Further, resin is selected from Rink Amide resins, Rink Amide AM resins, Rink Amide mbha resins
Or Sieber amide resins, the substitution degree of preferred resin is 0.3~0.8mmol/g.
Further, the reaction temperature of reduction process is 65~75 DEG C, and the time is 1.5~2h.
Further, in cracking process, the volume ratio of decomposition agent and the 3rd intermediate product is 10~15:1.
Further, decomposition agent is selected from trifluoroacetic acid and/or water；Preferably 95vol% trifluoroacetic acid aqueous solutions.
Further, in cracking process, reaction temperature is 15~40 DEG C, and the reaction time is 2~3h.
Further, the precipitating reagent used in cracking process is selected from methyl tertiary butyl ether and/or ether.
Further, in the first condensation reaction, the second condensation reaction and the 3rd condensation reaction, the condensing agent difference used is only
N, N- diisopropyl carbon imines and Y mixture are on the spot selected from, wherein and Y is selected from 1- hydroxy benzos azoles and/or 1- hydroxyls -7- is folded
Nitrogen BTA；Or coupling agent is N- ethyl diisopropylamines and 2- (7- aoxidizes BTA)-N, N, N ', N '-tetramethyl
The mixed liquor of urea hexafluorophosphoric acid ester.
Apply the technical scheme of the present invention, using distinctive raw material Fmoc-4Nph-OH and Fmoc-D-Ala- resins,
Fmoc-Pro-OH、Fmoc-ILys(Boc)-OH、Fmoc-Leu-OH、Fmoc-D-4Aph(Cbm)-OH、Fmoc-Ser (tBu)-
OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH and Ac-D-2Nal-OH carry out solid phase synthesis, prepare ground and add
Rake product.Above-mentioned peculiar raw material Fmoc-4Nph-OH is relatively stable under alkaline environment, thus need not using above-mentioned raw materials
Blocking group is introduced, this effectively avoids side reaction is produced the introducing because of blocking group, so as to improve Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2
The purity of product.In addition, the simple to operate of above-mentioned solid phase synthetic reaction, reaction rate are fast, and raw material is cheap and easy to get, thus uses
The above method prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 also help be greatly reduced process costs and synthesis difficulty.In summary, using above-mentioned preparation
Method prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, not only contributes to improve the purity of product, also helps reduction synthesis difficulty and process costs.
Brief description of the drawings
The Figure of description for forming the part of the application is used for providing a further understanding of the present invention, and of the invention shows
Meaning property embodiment and its illustrate be used for explain the present invention, do not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 shows the mass spectrogram of the agent structure of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 standard specimen；
Fig. 2 shows the mass spectrogram of the agent structure of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 made from method according to an embodiment of the invention in 1；
Fig. 3 shows the mass spectrogram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 made from method according to an embodiment of the invention in 1.
Embodiment
It should be noted that in the case where not conflicting, the feature in embodiment and embodiment in the application can phase
Mutually combination.The present invention is described in detail below in conjunction with embodiment.
Just as described in the prior art, in the preparation method for solving existing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, the raw material used needs to use
The problem of blocking group is protected, and this can cause Material synthesis difficulty and high synthetic route process costs.It is above-mentioned in order to solve
Technical problem, this application provides a kind of preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, the preparation method includes：Will using solid-phase synthesis
Fmoc-D-Ala- resins, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-
OH, Fmoc-4Nph-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH and Ac-D-
2Nal-OH carries out the first condensation reaction by the amino acid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 successively, obtains the first intermediate product；Among first
Amino acid residue 4Nph in product is reduced to 4Aph, obtains the second intermediate product；Make above-mentioned 4Aph side-chain amino group and L-4,
5- dihydrooratic acids carry out the second condensation reaction, obtain the 3rd intermediate product；And the 3rd intermediate product is carried out using decomposition agent
Cracking, obtains required Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
In the above-mentioned preparation method of the application, using distinctive raw material Fmoc-4Nph-OH, (Fmoc- is to nitro-L- phenylpropyl alcohol ammonia
Acid) and Fmoc-D-ALa- resins (N- (9-fluorenylmethyloxycarbonyl)-D-alanine resin), Fmoc-Pro-OH (N- (9- fluorenes methoxies
Carbonyl)-L- proline), (N- fluorenylmethyloxycarbonyl-N'- tertbutyloxycarbonyl-N'- isopropyls-L- relies Fmoc-ILys (Boc)-OH
Propylhomoserin), Fmoc-Leu-OH (fluorenylmethyloxycarbonyl-L-Leu), Fmoc-D-4Aph (Cbm)-OH (4- [(amino carbonyl) ammonia
Base]-N- [fluorenylmethyloxycarbonyl]-D-phenylalanine), Fmoc-Ser (tBu)-OH (N- [(9H- fluorenes -9- ylmethoxies) carbonyl]-O-
The tert-butyl group-Serine), Fmoc-D-3Pal-OH (fluorenylmethyloxycarbonyl -3- (3- pyridine radicals)-D-alanine), Fmoc-D-Phe
(4Cl)-OH (N- (9- fluorenes methoxy carbonyl acyl group)-D-4- chlorophenylalanines) and Ac-D-2Nal-OH (R-2- acetamidos-β-
Naphthyl phenylalanine) solid phase synthesis is carried out, obtain the first intermediate product (structural formula I).Then by the first intermediate product
Amino acid residue 4Nph be reduced to 4Aph, obtain the second intermediate product (structural formula II).Finally by above-mentioned 4Aph side chain ammonia
Base and L-4,5- dihydrooratic acid carry out the second condensation reaction, obtain the 3rd intermediate product (structural formula III).Due to the above-mentioned 3rd
Intermediate product usually contains multiple peptide chain structures, thus needs to be cut to obtain single peptide chain structure.Third product is through splitting
After solving agent cracking, required Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product is obtained.
Above-mentioned peculiar raw material Fmoc-4Nph-OH is relatively stable under alkaline environment, thus need not be drawn using above-mentioned raw materials
Enter blocking group, this effectively avoids side reaction is produced the introducing because of blocking group, so as to improve Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 production
The purity of product.In addition, carry out above-mentioned solid phase synthetic reaction operation is simple, reaction rate is fast, and raw material is cheap and easy to get,
Thus using above-mentioned raw materials prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 also help be greatly reduced process costs and synthesize difficulty.In summary, use
Above-mentioned preparation method prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, not only contributes to improve the purity of product, also helps reduction synthesis difficulty and technique
Cost.
The term " solid-phase synthesis " mentioned in the application is peptide synthesis technology known in the art.Specifically, on
The first condensation reaction stated in preparation method includes：In the presence of deprotecting agent, the blocking group Fmoc of amino is taken off, then will
Fmoc-D-Ala- resins are fixed on solid phase carrier.Then by after the activated carboxylic in second amino acid Fmoc-Pro-OH,
The Fmoc-Pro-OH activated is bonded on Fmoc-D-Ala- resins in the presence of coupling agent and forms peptide bond, obtains centre
Product 1.Then with same principle, Fmoc-ILys (Boc)-OH is bonded to by shape on above-mentioned intermediate product 1 by coupling reaction
Into peptide bond, intermediate product 2 is obtained.Fmoc-Leu-OH is bonded on above-mentioned intermediate product 2 by coupling reaction and forms peptide bond,
Obtain intermediate product 3.Fmoc-D-4Aph (Cbm)-OH is bonded on above-mentioned intermediate product 3 by coupling reaction and forms peptide bond,
Obtain intermediate product 4.Fmoc-4Nph-OH is bonded on above-mentioned intermediate product 4 by coupling reaction and forms peptide bond, in obtaining
Between product 5.Fmoc-Ser (tBu)-OH is bonded on above-mentioned intermediate product 5 by coupling reaction and forms peptide bond, obtains centre
Product 6.Fmoc-D-3Pal-OH is bonded on above-mentioned intermediate product 6 by coupling reaction and forms peptide bond, obtains intermediate product
7.Fmoc-D-Phe (4Cl)-OH is bonded on above-mentioned centre by coupling reaction and forms peptide bond, obtains intermediate product 8.Pass through
Ac-D-2Nal-OH is bonded on above-mentioned centre and forms peptide bond by coupling reaction, obtains the first intermediate product.Pass through said process
The growth of peptide chain is realized, finally gives the first required intermediate product.
If by Fmoc-D-Ala- resins, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-OH,
Fmoc-D-4Aph(Cbm)-OH、Fmoc-4Nph-OH、Fmoc-Ser(tBu)-OH、Fmoc-D-3Pal-OH、Fmoc-D-Phe
(4Cl)-OH and Ac-D-2Nal-OH is designated as amino acid/11,2 ..., N (N is 1~9) successively, will be produced in the first condensation reaction
Intermediate product be designated as intermediate product 1,2 ..., N (N be 1~8) successively.Preferably, in the first condensation reaction, amino acid 2 with
Amino acid/11 mole the ratio between be 2~3:1, intermediate product N and amino acid N+1 mole the ratio between be 2~3:1.
In the propagation process of above-mentioned peptide chain, it can detect whether coupling reaction reacts complete by ninhydrin.
It is above-mentioned " by amino acid residue 4Nph be reduced to 4Aph " refer to byIt is reduced to
In a preferred embodiment, reduction process includes：Using reducing agent by the amino in the first intermediate product
Sour residue 4Nph is reduced to 4Aph, and wherein reducing agent is CrCl2.In above-mentioned reduction reaction, reducing agent includes but is not limited to
CrCl2, and select it to be advantageous to the percent reduction of raising reduction reaction as reducing agent.
In a preferred embodiment, above-mentioned preparation method also includes：Fmoc-D- alanine and resin are carried out the
The step of Fmoc-D- alanine-resin is made in three condensation reactions, it is preferable that the dosage of reducing agent for the substitution degree of resin 2.5~
5.0. again.The dosage of reducing agent includes but is not limited to above range, and the dosage of reducing agent is defined into above range and is advantageous to
Improve the yield of Fmoc-D- alanine-resin in reduction reaction.
" resin substitution degree " refers to the reaction site of the amount containing how many mM (mmol) in every gram of resin in the application.
In a preferred embodiment, resin includes but is not limited to Rink Amide resins, Rink Amide AM trees
Fat, Rink Amide mbha resins or Sieber amide resins, the substitution degree of preferred resin is 0.3~0.8mmol/g.
In a preferred embodiment, the reaction temperature of reduction process is 65~70 DEG C, and the time is 1.5~2h.On
State in preparation method, the reaction temperature of reduction process and time include but is not limited to above range, and are limited to above-mentioned model
In enclosing, be advantageous to further improve the reaction rate of reduction reaction.
In above-mentioned preparation method, those skilled in the art can select the dosage of decomposition agent.In a kind of preferable embodiment party
In formula, in cracking process, the volume ratio of decomposition agent and the 3rd intermediate product is 10~15：1.Decomposition agent and the 3rd intermediate product
Volume ratio includes but is not limited to above range, and is limited the lytic effect for being advantageous to improve peptide chain within the above range.
In a preferred embodiment, decomposition agent includes but is not limited to trifluoroacetic acid (TFA) and/or water；Preferably
95vol% trifluoroacetic acid aqueous solutions.The effect of decomposition agent is to be cut peptide chain, and the acidity of above-mentioned several decomposition agents is stronger,
Be advantageous to improve the cutting effect of the peptide chain of cracking process from above-mentioned decomposition agent.
In above-mentioned preparation method, the reaction temperature of cracking reaction and reaction time can select reaction temperature commonly used in the art
Degree and reaction time.In a preferred embodiment, in cracking process, reaction temperature is 15~40 DEG C, the reaction time 2
~3h.The reaction temperature of cracking reaction and reaction time are limited within the above range, are advantageous to improve cutting in cracking process
Cut.
In a preferred embodiment, the precipitating reagent used in cracking process includes but is not limited to methyl tertiary butyl ether
And/or ether.Precipitating reagent can destroy the moisture film on peptide chain surface, and then the dissolution equilibrium of peptide chain is broken, and then
Be advantageous to improve the sedimentation effect of peptide chain.And above-mentioned several precipitating reagents have larger volume, and electric charge is easily carried, thus will be upper
Several materials are stated as precipitating reagent, are advantageous to the further settling rate for improving peptide chain.
In addition, in order to further improve the purity of product, product can also further be purified.Preferably, it is pure
Changing step includes：(1) product is dissolved in organic solvent, obtains mixed liquor, then above-mentioned mixed liquor was carried out using filter membrane
Filter, obtains filtrate.(2) above-mentioned filtrate is repeatedly purified using liquid chromatogram, wherein the Detection wavelength in purge process is
280mm.(3) above-mentioned filtrate is subjected to desalination, concentration, freezed, obtain the higher Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product of purity.
In above-mentioned preparation method, in a preferred embodiment, the first condensation reaction, the second condensation reaction and the 3rd
In condensation reaction, the coupling agent used is separately selected from N, N- diisopropyl carbon imines (DIC) and Y mixture, or
N- ethyl diisopropylamines (DIPEA) and 2- (7- aoxidizes BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester
(HATU) mixed liquor；Wherein and Y is selected from 1- hydroxy benzos azoles (HOBt) and/or 1- hydroxyl -7- nitrine BTAs
(HOAt).Select DIC and (DIPEA is alkaline matter, and the carboxyl in amino acid is activated in the presence of above-mentioned alkaline matter, so
Afterwards in the presence of DIPEA, HATU or HOAt, amino acid carries out coupling reaction and forms peptide bond.Above-mentioned several couplings are selected simultaneously
Agent also advantageously improves coupling effect.
The title of involved material and structure are shown in Table 1 in the application.
Table 1
The application is described in further detail below in conjunction with specific embodiment, these embodiments are it is not intended that limit this
Apply for scope claimed.
Embodiment 1
1st, the synthesis of Fmoc-D-Ala-Rink resins
Rink amid AM resins (10mmol, substitution degree 0.65mmol/g) are fitted into solid phase reaction post, with DMF to upper
State solid phase reaction post to rinse twice, state addition 500mL DMF in solid phase reaction post then up, to make above-mentioned resin by immersion
Swelling, soak time 30min.It is rinsed using 500mL 20%DBLK to being added in above-mentioned solid phase reaction column, so that portion
Divide Fmoc removings, reaction time 10min.State then up and add 500mL 20%DBLK in solid phase reaction post again, so that
Remaining Fmoc removings, reaction time 10min, are then washed 6 times using DMF to above-mentioned solid phase reaction column.
9.34gFmoc-D-Ala-OH and 8.84gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and will be above-mentioned mixed
Close liquid and 10min is cooled down in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-D-Ala-OH is carried out in advance
Activation, the preactivated time is 2~5min.The mixed liquor activated is added in solid phase reaction post, it is anti-to carry out the 3rd condensation
Should, and be constantly stirred, the time of the 3rd condensation reaction is 2h, obtains the product of the 3rd condensation reaction.Using ninhydrin pair
The product of above-mentioned 3rd condensation reaction is detected, and testing result is negative.Finally the product by the 3rd condensation reaction is taken out
Filter, obtains filter cake.Then above-mentioned filter cake is rinsed 6 times using DMF, above-mentioned filter cake rinsed 3 times using DCM, using MeOH pairs
Above-mentioned filter cake is shunk (time is followed successively by 5min, 5min and 10min) three times, and after washing, above-mentioned filter cake is done
It is dry, Fmoc-D-Ala-Rink resins are obtained, substitution degree is 0.62mmol/g after testing.
2nd, the synthesis of the first intermediate product
Step 1：Fmoc-D-Ala-Rink resins (10mmol) are fitted into solid phase reaction post, with DMF to above-mentioned solid phase
Flushing 2 times is reacted, states addition 100mL DMF in solid phase chromatography post then up, to make above-mentioned resin swelling by immersion, is soaked
The bubble time is 30min.20%DBLK is set to rinse twice above-mentioned solid phase reaction column, so that (washing time is removing Fmoc
10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
6.74gFmoc-Pro-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and by above-mentioned mixing
Liquid cools down 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-Pro-OH carries out pre-activate,
The preactivated time is 2~5min.The mixed liquor activated is added in solid phase reaction post, carries out coupling reaction, and constantly
It is stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Product using ninhydrin to above-mentioned coupling reaction
Detected, testing result is negative.Finally the product of coupling reaction is filtered, obtains filter cake.Then using DMF to upper
State filter cake to rinse 4 times, above-mentioned solid phase reaction column is rinsed twice using 20%DBLK, so that (washing time is removing Fmoc
10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 2：10.28gFmoc-ILys (Boc)-OH and 4.26gHOBt are dissolved in 60mL DMF, mixed
Liquid, and above-mentioned mixed liquor is cooled down into 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-
ILys (Boc)-OH carries out pre-activate, and the preactivated time is 2~5min.The mixed liquor activated is added to solid phase reaction post
In, coupling reaction is carried out, and be constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using indenes three
Ketone detects to the product of above-mentioned coupling reaction, and testing result is negative.Finally the product of coupling reaction is filtered, obtained
To filter cake.Then above-mentioned filter cake is rinsed 4 times using DMF, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that
Fmoc (washing time is 10min) is removed, finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 3：7.10gFmoc-Leu-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and will
Above-mentioned mixed liquor cools down 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-Leu-OH enters
Row pre-activate, preactivated time are 2~5min.The mixed liquor activated is added in solid phase reaction post, be coupled instead
Should, and be constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using ninhydrin to above-mentioned coupling
The product of reaction is detected, and testing result is negative.Finally the product of coupling reaction is filtered, obtains filter cake.Then
Above-mentioned filter cake is rinsed 4 times using DMF, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that removing Fmoc
(washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 4：8.97gFmoc-D-4Aph (Cbm)-OH and 4.26gHOBt are dissolved in 60mL DMF, mixed
Liquid, and above-mentioned mixed liquor is cooled down into 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-
D-4Aph (Cbm)-OH carries out pre-activate, and the preactivated time is 2~5min.The mixed liquor activated is added to solid phase reaction
In post, coupling reaction is carried out, and is constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using indenes
Triketone detects to the product of above-mentioned coupling reaction, and testing result is negative.Finally the product of coupling reaction is filtered,
Obtain filter cake.Then using DMF to above-mentioned filter cake rinse 4 times, using 20%DBLK to above-mentioned solid phase reaction column rinse twice, with
Make removing Fmoc (washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 5：8.68gFmoc-4Nph-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and will
Above-mentioned mixed liquor cools down 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-4Nph-OH
Pre-activate is carried out, the preactivated time is 2~5min.The mixed liquor activated is added in solid phase reaction post, is coupled
Reaction, and be constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using ninhydrin to above-mentioned idol
The product of connection reaction is detected, and testing result is negative.Finally the product of coupling reaction is filtered, obtains filter cake.So
Above-mentioned filter cake is rinsed 4 times using DMF afterwards, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that removing Fmoc
(washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 6：7.69gFmoc-Ser (tBu)-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor,
And above-mentioned mixed liquor is cooled down into 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-Ser
(tBu)-OH carries out pre-activate, and the preactivated time is 2~5min.The mixed liquor activated is added in solid phase reaction post,
Coupling reaction is carried out, and is constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using ninhydrin
The product of above-mentioned coupling reaction is detected, testing result is negative.Finally the product of coupling reaction is filtered, obtained
Filter cake.Then above-mentioned filter cake is rinsed 4 times using DMF, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that de-
Except Fmoc (washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 7：7.86gFmoc-D-3Pal-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and
Above-mentioned mixed liquor is cooled down into 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-D-3Pal-
OH carries out pre-activate, and the preactivated time is 2~5min.The mixed liquor activated is added in solid phase reaction post, carried out even
Connection reaction, and be constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using ninhydrin to above-mentioned
The product of coupling reaction is detected, and testing result is negative.Finally the product of coupling reaction is filtered, obtains filter cake.
Then above-mentioned filter cake is rinsed 4 times using DMF, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that removing
Fmoc (washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 8：8.48gFmoc-D-Phe (4-Cl)-OH and 4.26gHOBt are dissolved in 60mL DMF, mixed
Liquid, and above-mentioned mixed liquor is cooled down into 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-
D-Phe (4-Cl)-OH carries out pre-activate, and the preactivated time is 2~5min.The mixed liquor activated is added to solid phase reaction
In post, coupling reaction is carried out, and is constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using indenes
Triketone detects to the product of above-mentioned coupling reaction, and testing result is negative.Finally the product of coupling reaction is filtered,
Obtain filter cake.Then using DMF to above-mentioned filter cake rinse 4 times, using 20%DBLK to above-mentioned solid phase reaction column rinse twice, with
Make removing Fmoc (washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
Step 9：5.41gAc-D-2Nal-OH and 4.26gHOBt are dissolved in 60mL DMF, obtain mixed liquor, and will
Above-mentioned mixed liquor cools down 10min in ice bath.State then up and 5.05gDIC is added in mixed liquor, so that Fmoc-Pro-OH enters
Row pre-activate, preactivated time are 2~5min.The mixed liquor activated is added in solid phase reaction post, be coupled instead
Should, and be constantly stirred, the time of coupling reaction is 2h, obtains the product of coupling reaction.Using ninhydrin to above-mentioned coupling
The product of reaction is detected, and testing result is negative.Finally the product of coupling reaction is filtered, obtains filter cake.Then
Above-mentioned filter cake is rinsed 4 times using DMF, above-mentioned solid phase reaction column rinsed twice using 20%DBLK, so that removing Fmoc
(washing time is 10min), finally above-mentioned solid phase reaction column is being washed 6 times using DMF.
2nd, reduction reaction
Above-mentioned peptide resin (the first intermediate product) is imported in 250mL four-hole bottles, and 3.07g is added into above-mentioned four-hole bottle
CrCl2(equivalent to 2.5 times of resin substitution degree) and 60mL DMF, obtain mixed liquor.At 70 DEG C, above-mentioned mixed liquor is entered
Row reduction reaction, reaction time are 3 hours, obtain the product of reduction reaction.The second intermediate product is transferred to after completion of the reaction
In Solid-phase Polypeptide reaction column, washed six times using DMF, DCM is washed twice, and obtains the second intermediate product.
3rd, the second condensation reaction
3.27g L-4,5- dihydrooratic acids and 4.26gHOBt are dissolved in 60mLDMF, obtain mixed liquor, and will be upper
State mixed liquor and 10min is cooled down in ice bath.State then up and 5.05gDIC is added in mixed liquor, make L-4,5- dihydrooratic acids enter
Row activation, soak time is 2~5min.
The above-mentioned mixed liquor activated is added in Solid-phase Polypeptide reaction column, makes L-4, among 5- dihydrooratic acids and second
Second condensation reaction occurs for product, and is stirred continuously, reaction time 2h.The product of the second condensation reaction is carried out with ninhydrin
Detection, testing result are negative.The product of second condensation reaction is filtered to obtain filter cake, then used above-mentioned filter cake
DMF is washed four times, and DCM is washed twice, and methanol three times, and shrinkage resin, is finally transferred to vacuum drying chamber drying, obtains peptide resin
Ac-D-2Nal-D-Phe (Cl)-D-Pal-Ser-4Nph-D-Aph (Cbm)-Leu-ILys (Boc)-Pro-D-Ala-Rink, i.e.,
3rd intermediate product, 28.17g.
4th, the cracking of the 3rd intermediate product
Above-mentioned 3rd intermediate product is transferred in round-bottomed flask, and 360mL is freezed into (- 5 DEG C) good lysate (trifluoro
Acetic acid：H2O volume ratio is 95:5) add in the round-bottomed flask, and the stirring reaction 2h under conditions of room temperature (25 DEG C), mistake
Filter, obtains filtrate.Then peptide chain precipitation will be carried out with 3.6L frost methyl tertiary butyl ether(MTBE)s in above-mentioned filtrate, obtains precipitated product.
Then after above-mentioned precipitated product being carried out into filtration drying, the thick peptides of 16.8g (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product) (yield is obtained:99.6wt%,
Purity：91.8wt%).
5th, the purifying of crude product
(1) dissolve
Thick peptide (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product) is weighed, pulverizes and puts into beaker, solvent is added with 30g/L dosage, wherein
Solvent is acetonitrile solution, and the volume ratio of acetonitrile and water is 40：60%.Then above-mentioned mixed liquor is surpassed using ultrasonic wave
Sound, so that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product is completely dissolved, obtain mixed liquor.Using 0.45 μm of nylon66 fiber miillpore filter to above-mentioned mixed liquor
Filtered.
(2) purify
A) purify for the first time：
After chromatographic column is rinsed well with 50wt% acetonitrile solutions, after 5wt% acetonitrile solution balance systems 5min,
HPLC purifying, Detection wavelength 280nm are carried out with the applied sample amount of 16~23g/ times.According to chromatographic peak Fractional Collections cut, and will
The cut of collection is detected with analytical liquid chromatograph, purity >=99.0wt% and single miscellaneous≤0.2wt% cuts are first
Walk qualified cut.Remaining is slop cut, and slop cut moderate purity is more than 80wt% parts and carries out recovery purifying, purity again
Make liquid waste processing less than 80wt%.
B) second of purifying：
After chromatographic column is rinsed well with 50wt% acetonitrile solutions, after 5wt% acetonitrile solution balance systems 5min,
HPLC purifying is carried out with the applied sample amount of 14~20g/ times, Detection wavelength is to be monitored at 280nm wavelength.It is segmented and is received according to chromatographic peak
Collect cut, and the cut of collection is detected with analytical liquid chromatograph, purity >=99.8wt% and single miscellaneous≤0.1wt% evaporates
It is the qualified cut of second step to divide.Remaining is slop cut, and slop cut moderate purity is more than 80wt% parts and returned again
Purifying is received, purity makees liquid waste processing less than 80wt%.
C) desalination, turn salt
The first eluted product b) obtained is rinsed using ammonium acetate, obtains intermediate product；Pass through acetate pair again
Intermediate product is rinsed, and the part trifluoroacetic acid root in the first product can be converted into acetate, and then first is eluted
Product is converted into acetate.
D) concentrate and lyophilized：
20mg/mL or so is concentrated into, 6.8g fine peptides (Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product) (yield 40wt%, purity is obtained after lyophilized：
99.8wt%).The structure of above-mentioned product is tested using mass spectrometer.Fig. 1 shows the main body of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 standard specimen
Structure mass spectrogram, Fig. 2 are the mass spectrograms of the agent structure of the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product obtained in embodiment 1, and Fig. 3 is shown to be added over the ground
Rake carries out mass spectrogram during mass spectrometric measurement.By comparing, can confirm that product made from embodiment 1 is Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Embodiment 2
Difference with embodiment 1 is：The dosage of reducing agent is 3.0 times of resin substitution degree.
Embodiment 3
Difference with embodiment 1 is：The dosage of reducing agent is 1.5 times of resin substitution degree.
Embodiment 4
Difference with embodiment 1 is：The temperature of reduction reaction is 65 DEG C, reaction time 3h.
Embodiment 5
Difference with embodiment 1 is：The temperature of reduction reaction is 75 DEG C, reaction time 3h.
Embodiment 6
Difference with embodiment 1 is：The temperature of reduction reaction is 40 DEG C.
Embodiment 7
Difference with embodiment 1 is：Resin is Rink Amide mbha resins.
Embodiment 8
Difference with embodiment 1 is：Resin is Sieber amide resins.
Embodiment 9
Difference with embodiment 1 is：The volume ratio of decomposition agent and the 3rd intermediate product is 10：1.
Embodiment 10
Difference with embodiment 1 is：The volume ratio of decomposition agent and the 3rd intermediate product is 15：1.
Embodiment 11
Difference with embodiment 1 is：Decomposition agent is TFA.
Embodiment 12
Difference with embodiment 1 is：The reaction temperature of cracking reaction is 5 DEG C.
Embodiment 13
Difference with embodiment 1 is：Precipitating reagent is ether.
Embodiment 14
Difference with embodiment 1 is：Coupling agent is DIC and HOAt.
Embodiment 15
Difference with embodiment 1 is：Coupling agent is DIPEA and HATU.
Embodiment 16
Difference with embodiment 1 is：Coupling agent is HOBT and DIC.
The purity and yield of product in embodiment 1 to 16, are shown in Table 1.
Table 1
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (10)
1. a kind of preparation method 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 preparation method includes：
Using solid-phase synthesis by Fmoc-D-Ala- resins, Fmoc-Pro-OH, Fmoc-ILys (Boc)-OH, Fmoc-Leu-
OH、Fmoc-D-4Aph(Cbm)-OH、Fmoc-4Nph-OH、Fmoc-Ser(tBu)-OH、
Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH and Ac-D-2Nal-OH are suitable by the amino acid of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 successively
Sequence carries out the first condensation reaction, obtains the first intermediate product；
Amino acid residue 4Nph in first intermediate product is reduced to 4Aph, obtains the second intermediate product；
The side-chain amino group and L-4,5- dihydrooratic acid for making the 4Aph carry out the second condensation reaction, obtain the 3rd intermediate product；
And
The 3rd intermediate product is cracked using decomposition agent, obtains the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
2. preparation method according to claim 1, it is characterised in that the reduction process includes：Using reducing agent by institute
The middle 4Nph residues for stating the first intermediate product are reduced to 4Aph, wherein the reducing agent is CrCl2。
3. preparation method according to claim 2, it is characterised in that the preparation method is also included the ammonia of Fmoc-D- third
Acid carries out the step of Fmoc-D- alanine-resin is made in the 3rd condensation reaction with resin；Preferably, the reducing agent
Dosage is 2.5~5.0 times of the substitution degree of the resin.
4. preparation method according to claim 3, it is characterised in that the resin is selected from Rink Amide resins, Rink
Amide AM resins, Rink Amide mbha resins or Sieber amide resins, the substitution degree of preferably described resin for 0.3~
0.8mmol/g。
5. preparation method according to any one of claim 1 to 4, it is characterised in that the reaction temperature of the reduction process
Spend for 65~75 DEG C, the time is 1.5~2h.
6. preparation method according to any one of claim 1 to 4, it is characterised in that described to split in the cracking process
The volume ratio for solving agent and the 3rd intermediate product is 10~15:1.
7. preparation method according to claim 6, it is characterised in that the decomposition agent is selected from trifluoroacetic acid and/or water；It is excellent
Elect 95vol% trifluoroacetic acid aqueous solutions as.
8. preparation method according to claim 6, it is characterised in that in the cracking process, reaction temperature is 15~40
DEG C, the reaction time is 2~3h.
9. preparation method according to claim 6, it is characterised in that the precipitating reagent used in the cracking process is selected from first
The tertiary butyl ether of base and/or ether.
10. preparation method according to claim 3, it is characterised in that first condensation reaction, second condensation are anti-
N, N- diisopropyl carbon imines and Y mixing should be separately selected from the condensing agent in the 3rd condensation reaction, used
Thing, wherein and Y be selected from 1- hydroxy benzos azoles and/or 1- hydroxyl -7- nitrine BTAs；Or the coupling agent is N- ethyls two
Isopropylamine and 2- (7- aoxidizes BTA)-N, N, N ', the mixed liquor of N '-tetramethylurea hexafluorophosphoric acid ester.
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| CN109575109A (en) * | 2018-12-27 | 2019-04-05 | 兰州大学 | The method that fragment condensation prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
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