CN107778351A - A kind of method of full synthesis in solid state Octreotide - Google Patents
A kind of method of full synthesis in solid state Octreotide Download PDFInfo
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- CN107778351A CN107778351A CN201610727254.2A CN201610727254A CN107778351A CN 107778351 A CN107778351 A CN 107778351A CN 201610727254 A CN201610727254 A CN 201610727254A CN 107778351 A CN107778351 A CN 107778351A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The present invention relates to medical synthesis field, discloses a kind of method for synthesizing Octreotide.The present invention is initial resin by peculiar Boc Thr (Bzl) OL Merrifield; the mode such as iodine oxidation method and the trifluoroacetic acid solution acidolysis resin of hydrogen bromide and protection group of cooperation realizes full solid phase and prepares Octreotide; finally obtaining Octreotide has higher purity and total recovery; greatly simplify preparation technology simultaneously, shorten preparation time, reduce production cost.
Description
Technical field
The present invention relates to medical synthesis field, and in particular to a kind of method of full synthesis in solid state Octreotide.
Background technology
Octreotide is a kind of artificial synthesized octapeptide cyclic compound, has the work similar with native endogenous growth hormone release inhibiting hormone
With, but act on relatively strong and lasting, half-life period more natural 30 times of chalone length.This product has a variety of physiologically actives, such as suppression growth hormone,
The pathological discharge of thyroid-stimulating hormone, intestines and stomach and pancreas endocrine hormone is excessive, to hydrochloric acid in gastric juice, pancreatin, hyperglycemic factor and pancreas islet
The secretion of element also has inhibitory action.Octreotide can reduce stomach motion and gall bladder emptying, suppress gall bladder emptying, suppress cholecystokinin-
The secretion of pancreatin secretin, pancreatic secretion is reduced, has direct protective action to pancreatic parenchmal cells film.It is compacted that this product can suppress stomach and intestine
It is dynamic, reduce visceral blood flow and reduce portal vein pressure, reduce enteron aisle excessive secretion, and absorption of the enteron aisle to water and Na can be strengthened.
Because Octreotide has a variety of physiologically actives, therefore have wide range of applications, clinic is mainly used in the following aspects, portal vein
Esophageal varices hemorrhage caused by high pressure, stress ulcer and hemorrhage of digestive tract, Severe Pancreatitis, alleviate by stomach, intestines and pancreas
Symptom caused by endocrine system carcinoma, exophthalmic goiter and acromegalia, intestines and stomach fistula.
There are many technologies for preparing Octreotide at present, these existing preparation methods are mostly with synthesis in solid state straight chain eight
Peptide, peptide (acidolysis/cracking) is then cut, carry out air oxidation in the liquid phase and complete intramolecular cyclization.As patent CN1810829A,
CN1569890A, CN1923849A, CN1837232A, but these existing patent total recoverys are not universal high, not less than 50%,
And the finished product purity having is also than relatively low.Patent CN101863961A discloses a kind of preparation method of Octreotide, and it mainly exists
Oxidation link is improved, by being aoxidized under the conditions of certain ph using the hydrogen peroxide of excess, final Octreotide
Total recovery 74%, purity has reached 98%；The fragment synthetic method that patent CN103102390A discloses a kind of 2+6 prepares Austria
Bent peptide, while in oxidation link by under the pH value in characteristic, being cyclized using the hydrogen peroxide or iodohydrin of excess, it is difficult to understand bent
For the total recovery of peptide 75% or so, purity has reached 99%.But both approaches are still not carried out the synthesis side of full solid phase
Case, because the technical reason country can not also realize high total recovery and height under the conditions of full synthesis in solid state in the prior art instantly
Purity, and the full synthesis in solid state of reality can greatly simplify preparation technology, shorten preparation time, reduce production cost.
The content of the invention
In view of this, it is an object of the invention to provide a kind of method of full synthesis in solid state Octreotide so that institute of the present invention
State method has higher purity and total recovery under full synthesis in solid state scheme.
To achieve the above object, the present invention provides following technical scheme：
A kind of method of full synthesis in solid state Octreotide, comprises the following steps：
Step 1, from initial resin Boc-Thr (Bzl)-OL-Merrifield resins, according to Octreotide straight chain octapeptide
C-terminal to N-terminal amino acid sequence, condensation reagent and activating reagent effect under, by the Cys of protection, protection Thr, protection
Lys, the D-Trp of protection, the Phe of protection, the Cys of protection, the D-Phe of protection carry out extension coupling one by one, obtain peptide resin 1；
Step 2, iodine oxidation method oxidation peptide resin 1, carry out intramolecular cyclization reaction, obtain Octreotide resin；
Step 3, Octreotide resin use the trifluoroacetic acid solution acidolysis of hydrogen bromide, butterfly Octreotide crude product；
Step 4, Octreotide purifying crude obtain sterling after turning salt.
Octreotide amino acid has 10, and composition is as follows：
D-Phe1-[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7]-Thr8-OL
The present invention passes through the modes such as the trifluoroacetic acid solution of distinctive initial resin, suitable method for oxidation and hydrogen bromide
To realize that full solid phase prepares Octreotide, finally obtaining Octreotide has higher purity and total recovery.
Protection group of the present invention is that amino, carboxylic on protected amino acid main chain and side chain are needed in Amino acid synthesis field
The blocking group of the group of the interference such as base synthesis, prevents amino, carboxyl etc. from being reacted during target product is prepared, and generates
Impurity, such as present invention protect Cys side chain by Trt or Acm protection groups, and Thr side chain is protected by tBu or Bzl protection groups；
Lys or Trp side chain is protected by Boc protection groups.In addition, in the amino acid for the protection that the method for the invention is related to, N-terminal
It is preferred that protected by Fmoc or Boc protection groups.The amino acid of protection is referred to as (as protected by the amino acid that protection group is protected
Cys etc.).Preferably, the protected amino acid is as follows：
Fmoc-Cys (Trt) or Fmoc-Cys (Acm), Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-Phe,
Fomc-D-Phe or Boc-D-Phe, Fmoc-D-Trp or Fmoc-D-Trp (Boc).
Preferably, step 2 is：
By 5%I2/ DMF solution is added to peptide resin 1, carries out intramolecular cyclization reaction, obtains Octreotide resin.Relative to normal
The iodohydrin of rule, present invention selection I2/ DMF, DMF can further increase cyclisation yield.
Preferably, above-mentioned each protected amino acid is when on the peptide resin for being each coupled to resin or having synthesized, respectively with
The mol ratio of resin is 1-6:1, more preferably 2.5-3.5:1, i.e., the inventory of described protected amino acid and the mol ratio of resin
Example relation.
Preferably, the substitution value of the initial resin is 0.2-1.5mmol/g amino resins, more preferably 0.5-
1.0mmol/g amino resins.
Preferably, the condensation reagent is preferably N, and N- DICs (DIC), N, N- dicyclohexyls carbon two
Imines (DCC), hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus/organic base (PyBOP/ organic bases), 2- (7- nitrogen
Miscellaneous -1H- BTAs -1- bases) -1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/organic base (HATU/ organic bases), benzo three
Nitrogen azoles-N, N, N', N'- tetramethylurea hexafluorophosphate/organic base (HBTU/ organic bases), O- BTAs-N, N, N', N'-
One kind in tetramethylurea tetrafluoro boric acid ester/organic base (TBTU/ organic bases).The mole dosage of the condensation reagent is preferably ammonia
1~6 times of the amino total mole number of amino resins, more preferably 2.5~3.5 times in base resin or the peptide resin synthesized.
It should be noted that the PyBOP/ organic bases, HATU/ organic bases, HBTU/ organic bases, TBTU/ organic bases,
Belong to the condensation reagent of four kinds of Dual systems in the present invention, i.e., PyBOP, HATU, HBTU need when in use respectively with organic base group
Used together into a kind of condensation reagent, wherein the organic base and PyBOP, HATU, HBTU, TBTU mol ratio are preferred
For for 1.3-3.0:1, more preferably 1.3-2:1.
Preferably, organic base is both preferably N, N- diisopropyls in organic base and step 2 in the condensation reagent
Ethamine (DIPEA), triethylamine (TEA) or N- methylmorpholines (NMM), more preferably DIPEA.
Preferably, the activating reagent is I-hydroxybenzotriazole (HOBt) or N- hydroxyl -7- azepine BTAs
(HOAt).The dosage of the activating reagent be preferably amino resins or the peptide resin that has synthesized in the middle amino of amino resins always rub
1~6 times, more preferably 2.5~3.5 times of that number.
Preferably, the reaction dissolvent respectively reacted described in building-up process uses DMF.
Coupling one by one of the present invention refers to that after first amino acid and resin coupling remaining amino acid is according to Octreotide
The C-terminal of straight chain octapeptide to the order of N-terminal occur one by one with the amino acid of previous coupling condensation reaction (backbone amino and carboxyl
Condensation reaction) it is coupled.During present invention coupling, the mol ratio of the protected amino acid and corresponding peptide resin is excellent during coupling every time
Elect 1-6 as:1, more preferably 2.5-3.5:1；The coupling reaction time is preferably 60~300 minutes, more preferably 120~
180 minutes.It should be noted that peptide resin of the present invention refers to any number amino acid according to Octreotide straight chain octapeptide amino acid
Sequentially be connected the peptide resin to be formed with resin, is not only additionally included in during synthesis peptide resin 1 obtains including peptide resin 1 among these
The multiple peptide resins arrived.Peptide resin corresponding to described refers to that the Cys of protection and initial resin are coupled the peptide resin 2 to be formed, peptide tree
Fat 2 is exactly the corresponding peptide resin that the Thr protected is coupled, and the peptide resin for being coupled the Thr of protection is that the Lys of protection is prolonged
Corresponding peptide resin during coupling is stretched, peptide tree by that analogy, after a remaining amino acid protected amino acid coupling respectively and thereon
Fat forms corresponding relation during coupling.
In extension is coupled, because there is protection group at each amino acid N end, it is therefore desirable to it is even again first to remove N-terminal protection group
Connection, this is common knowledge for a person skilled in the art.The present invention preferably uses PIP/DMF (piperidines/N, N- dimethyl formyls
Amine) mixed solution removing N-terminal Fomc protection groups containing piperidines are 10~30% (V) in mixed solution, remaining is DMF.N-terminal is gone to protect
It is preferably 10~60 minutes to protect the base time, preferably 15~25 minutes.Go the dosage of N-terminal protection group reagent preferably every
10mL/g peptide resins；The present invention preferably removes N-terminal Boc protection groups with TFA/DCM (trifluoracetic acid/dichloromethane) mixed solution,
Containing trifluoracetic acid it is 20~60% (V/V), preferably 25~35% (V/V) in mixed solution, goes the N-terminal protection group time to be preferably
10~50 minutes, preferably 25~35 minutes, the dosage for removing N-terminal protection group reagent are preferably 10mL/ grams of peptide resin.
Preferably, in the trifluoroacetic acid solution of the hydrogen bromide, the mass percent concentration of hydrogen bromide is preferably 5~
10%wt, more preferably concentration are 6~7%wt；The dosage of the acidolysis agent is 5~15mL acidolysis agent/gram peptide resin, preferably acidolysis
The dosage of agent is 7~12mL acidolysis agent/gram peptide resin；The time of the acidolysis is 1~6 hour, preferably 3~4 hours.
Preferably, the purifying turns salt and is specially：
Octreotide crude product, 0.1%TFA/ aqueous dissolutions, 0.45 μm of filtering with microporous membrane of solution, purifying are standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Octreotide purifying intermediate concentrate is obtained；
Take Octreotide to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Octreotide aqueous acetic acid, is freeze-dried, obtains song difficult to understand
Peptide sterling.
The Octreotide synthesized by the method for the invention detects through HPLC, and crude product purity is more than 80%, and product purity is more than
99.5%, maximum single contaminant is less than 0.15%, and total recovery is more than 75%.
From above technical scheme, the present invention is initial tree by peculiar Boc-Thr (Bzl)-OL-Merrifield
Fat, the mode such as iodine oxidation method and the trifluoroacetic acid solution acidolysis resin of hydrogen bromide and protection group of cooperation realize full solid phase and prepared
Octreotide, the final Octreotide that obtains has higher purity and total recovery, while greatly simplifies preparation technology, shortens preparation
Time, reduce production cost.
Embodiment
The invention discloses a kind of method for synthesizing Octreotide, those skilled in the art can use for reference present disclosure, suitably
Modified technique parameter is realized.In particular, all similar replacements and change be for a person skilled in the art
It will be apparent that they are considered as being included in the present invention.The method of the present invention is described by preferred embodiment,
Related personnel can substantially not depart from present invention, methods described herein are being modified in spirit and scope or suitably change
With combining, to realize and using the technology of the present invention.
In the specific embodiment of the invention, the protected amino acid in the present invention is limited purchased from Chengdu sunshine Rong's biotechnology
Company, resin used are purchased from Tianjin Nankai Hecheng S＆T Co., Ltd., Chinese corresponding to english abbreviation used in application documents
Implication is shown in Table 1.
The english abbreviation lexical or textual analysis of table 1
| English abbreviation | Chinese | English abbreviation | Chinese |
| Fmoc | 9-fluorenylmethyloxycarbonyl | Thr | Threonine |
| tBu | The tert-butyl group | Thr-OL | Soviet Union's ammonia alcohol |
| Boc | Tertiary butyloxycarbonyl acyl group | Phe | Phenylalanine |
| Trt | Trityl | D-Phe | D-phenylalanine |
| Cys | Cysteine | Tyr | Tyrosine |
| Lys | Lysine | D-Trp | D-trp |
With reference to embodiment, the present invention is expanded on further.
Embodiment 1：The synthesis of straight chain heptapeptide resin
0.15mol Fmoc-Cys (Trt) and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken,
It is slowly added under stirring into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the guarantor after being activated
Freamine Ⅲ is protected, it is standby.
0.05mol Boc-Thr (Bzl)-OL-Merrifield resins (substitution value about 0.5mmol/g) are taken, with 30%
TFA/DCM solution deprotects 30 minutes, is neutralized through DIEA/DCM solution, with DMF, DCM washing and filtering, adds after activating
Fmoc-Cys (Trt) solution, reaction 3 hours is stirred at room temperature, takes out reaction solution, after DMF is washed 3 times, DCM is washed 3 times, is washed every time
It is 3min to wash the time, then is deprotected 25 minutes with 20%PIP/DMF solution, washing and filtering, completes connecing for Fmoc-Cys (Trt)
Enter.
With method access Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-D-Trp, Fmoc-Phe and Fmoc-Cys
(Trt), then with 20%PIP/DMF solution deprotect, washing and filtering obtains obtaining straight chain heptapeptide resin (Cys (Trt)-Phe-D-
Trp-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(Bzl)-OL-Merrifield)。
Embodiment 2：The synthesis of straight chain heptapeptide resin
0.15mol Fmoc-Cys (Trt) and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken,
It is slowly added under stirring into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the guarantor after being activated
Freamine Ⅲ is protected, it is standby.
0.05mol Boc-Thr (Bzl)-OL-Merrifield resins (substitution value about 0.5mmol/g) are taken, with 30%
TFA/DCM solution deprotects 30 minutes, is neutralized through DIEA/DCM solution, with DMF, DCM washing and filtering, adds after activating
Fmoc-Cys (Trt) solution, reaction 3 hours is stirred at room temperature, takes out reaction solution, after DMF is washed 3 times, DCM is washed 3 times, is washed every time
It is 3min to wash the time, then is deprotected 25 minutes with 20%PIP/DMF solution, washing and filtering, completes connecing for Fmoc-Cys (Trt)
Enter.
With method access Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-D-Trp (Boc), Fmoc-Phe and Fmoc-
Cys (Trt), then deprotected with 20%PIP/DMF solution, washing and filtering obtains obtaining straight chain heptapeptide resin (Cys (Trt)-Phe-D-
Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(Bzl)-OL-Merrifield)。
Embodiment 3：The synthesis of straight chain heptapeptide resin
0.15molFmoc-Cys (Acm) and 0.15molHOBt is taken, is dissolved with appropriate DMF；0.15mol DIC separately are taken, are stirred
Mix down and be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection after being activated
Freamine Ⅲ, it is standby.
0.05mol Boc-Thr (Bzl)-OL-Merrifield resins (substitution value about 0.5mmol/g) are taken, with 30%
TFA/DCM solution deprotects 30 minutes, is neutralized through DIEA/DCM solution, with DMF, DCM washing and filtering, adds after activating
Fmoc-Cys (Trt) solution, reaction 3 hours is stirred at room temperature, takes out reaction solution, after DMF is washed 3 times, DCM is washed 3 times, is washed every time
It is 3min to wash the time, then is deprotected 25 minutes with 20%PIP/DMF solution, washing and filtering, completes connecing for Fmoc-Cys (Acm)
Enter.
With method access Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-D-Trp, Fmoc-Phe and Fmoc-Cys
(Trt), then with 20%PIP/DMF solution deprotect, washing and filtering obtains obtaining straight chain heptapeptide resin
(Cys(Acm)-Phe-D-Trp-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(Bzl)-OL-
Merrifield)。
Embodiment 4：The synthesis of straight chain heptapeptide resin
0.15mol Fmoc-Cys (Acm) and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken,
It is slowly added under stirring into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the guarantor after being activated
Freamine Ⅲ is protected, it is standby.
0.05mol Boc-Thr (Bzl)-OL-Merrifield resins (substitution value about 0.5mmol/g) are taken, with 30%
TFA/DCM solution deprotects 30 minutes, is neutralized through DIEA/DCM solution, with DMF, DCM washing and filtering, adds after activating
Fmoc-Cys (Trt) solution, reaction 3 hours is stirred at room temperature, takes out reaction solution, after DMF is washed 3 times, DCM is washed 3 times, is washed every time
It is 3min to wash the time, then is deprotected 25 minutes with 20%PIP/DMF solution, washing and filtering, completes connecing for Fmoc-Cys (Acm)
Enter.With method access Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-D-Trp (Boc), Fmoc-Phe and Fmoc-Cys
(Trt), then with 20%PIP/DMF solution deprotect, washing and filtering obtains obtaining straight chain heptapeptide resin
(Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(Bzl)-OL-
Merrifi eld)。
Embodiment 5：The synthesis of peptide resin 1
0.15molFmoc-D-Phe and 0.15molHOBt are taken, is dissolved with appropriate DMF；0.15mol DIC separately are taken, under stirring
It is slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection amino after being activated
Acid solution.
Protected amino acid solution after above-mentioned activation is added to straight chain heptapeptide resin made from embodiment 1, is stirred at room temperature
Reaction 3 hours, reaction solution is taken out, after DMF is washed 3 times, DCM is washed 3 times, and each wash time is 3min, then uses 20%PIP/
DMF solution deprotect 25 minutes, washing and filtering, obtain peptide resin 1 (D-Phe-Cys (Trt)-Phe-D-Trp-Lys (Boc)-
Thr(tBu)-Cys(Trt)-Thr(Bzl)-OL-Merrifiel d)。
Embodiment 6：The synthesis of peptide resin 1
0.15molFmoc-D-Phe and 0.15molHOBt are taken, is dissolved with appropriate DMF；0.15mol DIC separately are taken, under stirring
It is slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection amino after being activated
Acid solution.
Protected amino acid solution after above-mentioned activation is added to straight chain heptapeptide resin made from embodiment 2, is stirred at room temperature
Reaction 3 hours, reaction solution is taken out, after DMF is washed 3 times, DCM is washed 3 times, and each wash time is 3min, then uses 20%PIP/
DMF solution deprotects 25 minutes, washing and filtering, obtains obtaining (D-Phe-Cys (Trt)-Phe-D-Trp (the Boc)-Lys of peptide resin 1
(Boc)-Thr(tBu)-Cys(Trt)-Thr(Bzl)-OL-Merrifield)。
Embodiment 7：The synthesis of peptide resin 1
0.15molBoc-D-Phe and 0.15molHOBt are taken, is dissolved with appropriate DMF；0.15mol DIC separately are taken, under stirring
It is slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection amino after being activated
Acid solution.
Protected amino acid solution after above-mentioned activation is added to straight chain heptapeptide resin made from embodiment 3, is stirred at room temperature
Reaction 3 hours, reaction solution is taken out, after DMF is washed 3 times, DCM is washed 3 times, and each wash time is 3min, washing and filtering, is obtained
(Boc-D-Phe-Cys (Acm)-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys (Acm)-Thr of peptide resin 2 of Boc protections
(Bzl)-OL-Merrifield)。
Embodiment 8：The synthesis of peptide resin 1
0.15mol Boc-D-Phe and 0.15mol HOBt are taken, are dissolved with appropriate DMF；0.15mol DIC separately are taken, are stirred
Under be slowly added into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protection ammonia after being activated
Base acid solution.
Protected amino acid solution after above-mentioned activation is added to straight chain heptapeptide resin made from embodiment 4, is stirred at room temperature
Reaction 3 hours, reaction solution is taken out, after DMF is washed 3 times, DCM is washed 3 times, and each wash time is 3min, washing and filtering, is obtained
(Boc-D-Phe-Cys (Acm)-Phe-D-Trp (Boc)-Lys (Boc)-Thr (tBu)-Cys of peptide resin 1 of Boc protections
(Acm)-Thr(Bzl)-OL-Merrifield)。
Embodiment 9：The synthesis of Octreotide peptide resin
Take 5%I2/ DMF solution (10mL/ grams of resin), it is anti-to be added to 0 DEG C of stirring of peptide resin Isosorbide-5-Nitrae made from embodiment 5
Answer 4 hours, take out reaction solution, after DMF is washed 6 times, DCM is washed 3 times, and each wash time is 3min, obtains Octreotide peptide resin:
D-Phe- [Cys-Phe-D-Trp-Lys (Boc)-Thr (tBu)-Cys]-Thr (Bzl)-OL-Merrifield resins
Embodiment 10：The synthesis of Octreotide peptide resin
Take 5%I2/ DMF solution (10mL/ grams of resin), it is anti-to be added to 0 DEG C of stirring of peptide resin Isosorbide-5-Nitrae made from embodiment 6
Answer 4 hours, take out reaction solution, after DMF is washed 6 times, DCM is washed 3 times, and each wash time is 3min, obtains Octreotide peptide resin:
D-Phe- [Cys-Phe-D-Trp (Boc)-Lys (Boc)-Thr (tBu)-Cys]-Thr (Bzl)-OL-Merrifield resins
Embodiment 11：The synthesis of Octreotide peptide resin
Take 5%I2/ DMF solution (10mL/ grams of resin), it is added to the peptide resin Isosorbide-5-Nitrae 0 that Boc made from embodiment 7 is protected
DEG C stirring reaction 4 hours, reaction solution is taken out, after DMF is washed 6 times, DCM is washed 3 times, and each wash time be 3min, is obtained Boc and is protected
The Octreotide peptide resin of shield:D-Phe-[Cys-Phe-D-Trp-Lys(Boc)-Thr(tBu)-Cys]-Thr(Bzl)-OL-
Merrifield resins
Embodiment 12：The synthesis of Octreotide peptide resin
Take 5%I2/ DMF solution (10mL/ grams of resin), it is added to the peptide resin Isosorbide-5-Nitrae 0 that Boc made from embodiment 8 is protected
DEG C stirring reaction 4 hours, reaction solution is taken out, after DMF is washed 6 times, DCM is washed 3 times, and each wash time be 3min, is obtained Boc and is protected
The Octreotide peptide resin of shield:D-Phe-[Cys-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys]-Thr(Bzl)-
OL-Merrifield resins
Embodiment 13：The preparation of Octreotide crude product
Octreotide peptide resin made from Example 9, add 8% HBr/TFA solution (10mL/ grams of Octreotide of acid hydrolysis solution
Resin), filtrate is collected by filtration in stirring reaction 6 hours, and resin is washed 3 times with a small amount of TFA, is concentrated under reduced pressure, adds after merging filtrate again
Enter absolute ether precipitation, then precipitation washed 3 times with absolute ether, drain, off-white powder is Octreotide crude product, crude product purity
For 86.2%.
Embodiment 14：The preparation of Octreotide crude product
Octreotide peptide resin made from Example 10, add 8% HBr/TFA solution (10mL/ grams of Octreotide of acid hydrolysis solution
Resin), filtrate is collected by filtration in stirring reaction 6 hours, and resin is washed 3 times with a small amount of TFA, is concentrated under reduced pressure, adds after merging filtrate again
Enter absolute ether precipitation, then precipitation washed 3 times with absolute ether, drain, off-white powder is Octreotide crude product, crude product purity
For 85.0%.
Embodiment 15：The preparation of Octreotide crude product
Octreotide peptide resin made from Example 11, add 8% HBr/TFA solution (10mL/ grams of Octreotide of acid hydrolysis solution
Resin), filtrate is collected by filtration in stirring reaction 6 hours, and resin is washed 3 times with a small amount of TFA, is concentrated under reduced pressure, adds after merging filtrate again
Enter absolute ether precipitation, then precipitation washed 3 times with absolute ether, drain, off-white powder is Octreotide crude product, crude product purity
For 85.7%.
Embodiment 16：The preparation of Octreotide crude product
Octreotide peptide resin made from Example 12, add 8% HBr/TFA solution (10mL/ grams of Octreotide of acid hydrolysis solution
Resin), filtrate is collected by filtration in stirring reaction 6 hours, and resin is washed 3 times with a small amount of TFA, is concentrated under reduced pressure, adds after merging filtrate again
Enter absolute ether precipitation, then precipitation washed 3 times with absolute ether, drain, off-white powder is Octreotide crude product, crude product purity
For 86.8%.
Embodiment 17：Octreotide purifying crude
The gained Octreotide crude product of Example 13, dissolved with 20% acetum, 0.45 μm of filtering with microporous membrane of solution,
Purify standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Octreotide purifying intermediate concentrate is obtained；
Take Octreotide to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Octreotide aqueous acetic acid, is freeze-dried, obtains song difficult to understand
Peptide sterling 38.8g
Total recovery is 77.3%, molecular weight：1020.0 purity：99.6%, maximum single contaminant 0.10%.
Embodiment 18：Octreotide purifying crude turns salt
The gained Octreotide crude product of Example 14, dissolved with purifying mobile phase A, 0.45 μm of filtering with microporous membrane of solution,
Purify standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Octreotide purifying intermediate concentrate is obtained；
Take Octreotide to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Octreotide aqueous acetic acid, is freeze-dried, obtains song difficult to understand
Peptide sterling 39.4g.
Total recovery is 76.1%, molecular weight：1020.4 purity：99.5%, maximum single contaminant 0.12%.
Embodiment 19：Octreotide purifying crude turns salt
The gained Octreotide crude product of Example 15, dissolved with 20% acetum, 0.45 μm of filtering with microporous membrane of solution,
Purify standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Octreotide purifying intermediate concentrate is obtained；
Take Octreotide to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Octreotide aqueous acetic acid, is freeze-dried, obtains song difficult to understand
Peptide sterling 38.3g
Total recovery is 75.1%, molecular weight：1020.2 purity：99.7%, maximum single contaminant 0.09%.
Embodiment 20：Octreotide purifying crude turns salt
The gained Octreotide crude product of Example 16, dissolved with 20% acetum, 0.45 μm of filtering with microporous membrane of solution,
Purify standby；
Purified using high performance liquid chromatography, purifying chromatograph packing material is 10 μm of anti-phase C18, and flow phase system is
The 0.1%TFA/ aqueous solution -0.1%TFA/ acetonitrile solutions, 77mm*250mm column flow rate is 90mL/min, using gradient system
System elution, circulation sample introduction purifying, takes crude product solution to be splined in chromatographic column, starts mobile phase elution, collects main peak and boils off acetonitrile
Afterwards, Octreotide purifying intermediate concentrate is obtained；
Take Octreotide to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is 10 μm of anti-phase C18, and 77mm*250mm column flow rate is 90mL/min, using gradient elution, sample prescription in circulation
Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection changes salt main peak and with dividing
Liquid phase detection purity is analysed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains Octreotide aqueous acetic acid, is freeze-dried, obtains song difficult to understand
Peptide sterling 39.0g
Total recovery is 76.5%, molecular weight：1020.4 purity：99.5%, maximum single contaminant 0.12%.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (8)
- A kind of 1. method of full synthesis in solid state Octreotide, it is characterised in that comprise the following steps：Step 1, from initial resin Boc-Thr (Bzl)-OL-Merrifield resins, according to Octreotide straight chain octapeptide C-terminal To the amino acid sequence of N-terminal, under condensation reagent and activating reagent effect, by the Cys of protection, the Thr of protection, protection Lys, The D-Trp of protection, the Phe of protection, the Cys of protection, the D-Phe of protection carry out extension coupling one by one, obtain peptide resin 1；Step 2, iodine oxidation method oxidation peptide resin 1, carry out intramolecular cyclization reaction, obtain Octreotide resin；Step 3, Octreotide resin use the trifluoroacetic acid solution acidolysis of hydrogen bromide, butterfly Octreotide crude product；Step 4, Octreotide purifying crude obtain sterling after turning salt.
- 2. method according to claim 1, it is characterised in that the Cys of the protection, the Thr of protection, the Lys of protection, protection D-Trp, protection Phe, protection D-Phe be：Fmoc-Cys (Trt) or Fmoc-Cys (Acm), Fmoc-Thr (tBu), Fmoc-Lys (Boc), Fmoc-Phe, Fomc-D- Phe or Boc-D-Phe, Fmoc-D-Trp or Fmoc-D-Trp (Boc).
- 3. method according to claim 1 or claim 2, it is characterised in that the inventory of the protected amino acid and initial resin Mol ratio is 1-6:1.
- 4. method according to claim 1, it is characterised in that step 2 is：By 5%I2/ DMF solution is added to peptide resin 1, carries out intramolecular cyclization reaction, obtains Octreotide resin.
- 5. method according to claim 1, it is characterised in that the condensation reagent is N, and N- diisopropyls carbon two is sub- Amine, N, N- dicyclohexylcarbodiimides, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus/organic base, 2- (7- nitrogen Miscellaneous -1H- BTAs -1- bases) -1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/organic base, BTA-N, N, N', N'- tetramethylureas hexafluorophosphate/organic base, O- BTAs-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester/organic base In one kind.
- 6. method according to claim 5, it is characterised in that the organic base be DIPEA, triethylamine or N- methylmorpholines.
- 7. method according to claim 1, it is characterised in that the activating reagent be I-hydroxybenzotriazole or N- hydroxyls- 7- azepine BTAs.
- 8. method according to claim 1, it is characterised in that hydrogen bromide quality hundred in the trifluoroacetic acid solution of the hydrogen bromide It is 5-10% to divide specific concentration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111378009A (en) * | 2018-12-27 | 2020-07-07 | 江苏金斯瑞生物科技有限公司 | Preparation method of octreotide |
| CN112778400A (en) * | 2021-02-05 | 2021-05-11 | 合肥科生景肽生物科技有限公司 | Preparation method of octreotide |
| CN114939190B (en) * | 2022-06-14 | 2024-01-12 | 健诺维(成都)生物科技有限公司 | Drainage tube material for glaucoma treatment and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837232A (en) * | 2005-03-21 | 2006-09-27 | 吉尔生化（上海）有限公司 | Process for solid phase synthesis of octreotide |
| CN1923849A (en) * | 2005-08-30 | 2007-03-07 | 上海子能制药有限公司 | Preparation method of synthesizing octriotide from solid phase polypeptide |
| CN102850438A (en) * | 2012-09-19 | 2013-01-02 | 上海昂博生物技术有限公司 | Solid phase preparation method of crude octrotide |
| CN103102390A (en) * | 2011-11-09 | 2013-05-15 | 杭州华津允上医药有限公司 | Preparation method for octreotide |
| CN103351426A (en) * | 2013-08-02 | 2013-10-16 | 上海苏豪逸明制药有限公司 | Polypeptide synthesis method for octreotide acetate |
| CN103965291A (en) * | 2014-05-27 | 2014-08-06 | 上海第一生化药业有限公司 | Method for preparing octreotide and octreotide acetate |
-
2016
- 2016-08-25 CN CN201610727254.2A patent/CN107778351B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837232A (en) * | 2005-03-21 | 2006-09-27 | 吉尔生化（上海）有限公司 | Process for solid phase synthesis of octreotide |
| CN1923849A (en) * | 2005-08-30 | 2007-03-07 | 上海子能制药有限公司 | Preparation method of synthesizing octriotide from solid phase polypeptide |
| CN103102390A (en) * | 2011-11-09 | 2013-05-15 | 杭州华津允上医药有限公司 | Preparation method for octreotide |
| CN102850438A (en) * | 2012-09-19 | 2013-01-02 | 上海昂博生物技术有限公司 | Solid phase preparation method of crude octrotide |
| CN103351426A (en) * | 2013-08-02 | 2013-10-16 | 上海苏豪逸明制药有限公司 | Polypeptide synthesis method for octreotide acetate |
| CN103965291A (en) * | 2014-05-27 | 2014-08-06 | 上海第一生化药业有限公司 | Method for preparing octreotide and octreotide acetate |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111378009A (en) * | 2018-12-27 | 2020-07-07 | 江苏金斯瑞生物科技有限公司 | Preparation method of octreotide |
| CN112778400A (en) * | 2021-02-05 | 2021-05-11 | 合肥科生景肽生物科技有限公司 | Preparation method of octreotide |
| CN114939190B (en) * | 2022-06-14 | 2024-01-12 | 健诺维(成都)生物科技有限公司 | Drainage tube material for glaucoma treatment and preparation method thereof |
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