CN107778350A - A kind of method for synthesizing romidepsin - Google Patents

A kind of method for synthesizing romidepsin Download PDF

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Publication number
CN107778350A
CN107778350A CN201610728784.9A CN201610728784A CN107778350A CN 107778350 A CN107778350 A CN 107778350A CN 201610728784 A CN201610728784 A CN 201610728784A CN 107778350 A CN107778350 A CN 107778350A
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romidepsin
protection
resin
peptide resin
val
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CN107778350B (en
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马中刚
郭德文
曾德志
文永均
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CHENGDU SHENGNUO BIOPHARM Co Ltd
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CHENGDU SHENGNUO BIOPHARM Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The present invention relates to medical synthesis field, discloses a kind of method for synthesizing romidepsin.The present invention synthesizes the linear peptide chain of romidepsin one by one with suitable protected amino acid; amido link cyclisation is successively completed by solid phase means and disulfide bond is cyclized; and details optimization has been carried out to synthesis in solid state scheme, the romidepsin finally obtained has higher purity and total recovery.

Description

A kind of method for synthesizing romidepsin
Technical field
The present invention relates to medical synthesis field, and in particular to a kind of method for synthesizing romidepsin.
Background technology
Romidepsin, the entitled Romidepsin of English, is a kind of 5 artificial synthesized peptide cyclic compounds, has stabilization Hydrophobic structure, distinctive disulfide bond is the crucial group for playing activity in its structure.2009, romidepsin obtained U.S.'s food Drug administration (FDA) ratifies, and for treating skin T cell lymphoma (CTCL), its amino acid structure composition is as follows：
[Val1-(R2-D-Val3-D-Cys4)-DH-Thr5]
Wherein, R is (3S, 4E) -3- hydroxyl -7- sulfenyl -4- heptenoic acids, the D-Cys formation disulfide bond with 4,1 The valine of position and the threonine of 5 form amido link.
Romidepsin is histon deacetylase (HDAC) (HDACs) inhibitor, enters cytoplasm through tumor cell membrane, thin Intracellular disulfide bond, into sulfydryl, is combined with the zinc in the HDACs that zinc relies on by glutathione reduction and is played the work for suppressing HDACs With so as to further induced tumor cell differentiation and apoptosis.
At present, two methods mainly prepare romidepsin, first, biological fermentation process, such as patent CN201310430220, but integrated artistic is more complicated；Second, prepared by chemical synthesis, such as patent CN201010133961, it first carries out the cyclisation of liquid phase acid amides ring, then carry out liquid phase disulfide bond ring by liquid phase synthesis linear peptides Change, cumbersome, product yield is low；Chinese patent CN201210579007, from (3S, 4E) -3- hydroxyls -7- [(triphenyl first Base) sulfenyl] -4- heptenoic acids start, using 2-Cl-Trt resins as initial resin, using Solid phase synthesis linear peptides, iodine oxidation method After cyclisation forms disulfide bond, removing resin carries out acid amides ring cyclisation in liquid phase, but its crude product purity highest only has 67.53%, Simultaneous purification and its total recovery is only up to 33.7% after turning acetate.The problem of yield and purity is always to influence romidepsin The principal element of production efficiency.
The content of the invention
In view of this, it is an object of the invention to provide a kind of method for synthesizing romidepsin so that side of the present invention Method has higher crude product purity, finished product purity and total recovery.
To achieve the above object, the present invention provides following technical scheme：
A kind of method for synthesizing romidepsin, comprises the following steps：
Step 1, the Thr-OAll of protection are coupled under organic base effect with resin, obtain peptide resin 1；
Step 2, from peptide resin 1, according to the polypeptide sequence of romidepsin amino acid sequence C-terminal to N-terminal, tried in condensation Under agent and activating reagent effect, successively by the D-Cys of protection, the D-Val of protection and protection (3S, 4E) -3- hydroxyl -7- sulphur Base -4- heptenoic acids carry out extension coupling one by one, obtain peptide resin 2；
Step 3, from peptide resin 2s, in the presence of condensation reagent, activating reagent and catalyst, access protection Val, obtain peptide resin 3；
Step 4, All protection group removing of the agent by Thr-OAll in peptide resin 3 is deprotected with All, then in condensation reagent Under being acted on activating reagent, carry out intramolecular amide cyclization and be coupled valine and threonine, then pass through iodine oxidation method Intramolecular disulfide bond cyclization is carried out, obtains romidepsin peptide precursor resin；
Step 5, romidepsin peptide resin obtain romidepsin precursor after acidolysis；
Step 6, romidepsin precursor obtain romidepsin crude product after dehydrating agent is dehydrated, and (precursor compares crude product in Thr structures More hydroxyls, dehydrating agent act as intramolecular dehydration)；
Step 7, romidepsin crude product be purified to turn after acetate to obtain romidepsin sterling.
Romidepsin backbone amino acid has 4 and 1 carboxylic acid, forms as follows：
[Val1-(R2-D-Val3-D-Cys4)-DH-Thr5]
Wherein R is (3S, 4E) -3- hydroxyl -7- sulfenyl -4- heptenoic acids, the D-Cys formation disulfide bond with 4,1 Valine and 5 threonine formed amido link；DH-Thr represents dehydroxylation threonine.
The present invention synthesizes the linear peptide chain of romidepsin one by one with suitable protected amino acid, is successively completed by solid phase means Amido link is cyclized and disulfide bond cyclisation, and has carried out details optimization to synthesis in solid state scheme, the romidepsin tool finally obtained There are higher purity and total recovery.
Protection group of the present invention be needed in Amino acid synthesis field amino on protected amino acid main chain and side chain, The blocking group of the group of the interference such as carboxylic, sulfydryl synthesis, prevents amino, carboxyl, sulfydryl etc. from being sent out during target product is prepared Raw reaction, generates impurity, and such as present invention uses the protection group that Trt or Acm protection groups are sulfydryl.The amino acid protected by protection group It is referred to as the amino acid (such as Thr-OAll of protection, the D-Cys of protection) of protection.Protected amino acid N-terminal α ammonia of the present invention Base is preferably protected by Fmoc protection groups.
Preferably, the amino acid of protection of the present invention is as follows：
Fmoc-Thr-OAll, Fomc-D-Cys (Trt) or Fomc-D-Cys (Acm), Fmoc-D-Val, (3S, 4E) -3- Hydroxyl -7- [(Trt) sulfenyl] -4- heptenoic acids or (3S, 4E) -3- hydroxyls -7- [(Acm) sulfenyl] -4- heptenoic acids, Fmoc-D- Val。
Preferably, the resin is trityl resin, more preferably Trt resins or 2-Cl-Trt resins.
Coupling one by one of the present invention refers to that after previous amino acid and the coupling of resin or peptide resin remaining amino acid is pressed Condensation reaction occurs with the amino acid of previous coupling one by one it is coupled according to the polypeptide sequence of romidepsin C-terminal to N-terminal.This hair During bright coupling, the mol ratio of the protected amino acid and resin or corresponding peptide resin is preferably 1-6 during coupling every time:1, more preferably For 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 romidepsin polypeptide sequence and tree Lipid phase connects the peptide resin to be formed, not only include peptide resin 1-3 among these, is additionally included in during synthesis peptide resin 2 and obtains Multiple peptide resins.Corresponding peptide resin for example, if peptide resin 1 is exactly corresponding peptide resin that the D-Cys protected is coupled, The peptide resin for being coupled the D-Cys of protection is that the D-Val of protection carries out corresponding peptide resin during extension coupling, by that analogy, is protected After (3S, 4E) -3- hydroxyl -7- sulfenyl -4- heptenoic acids of shield, the Val of protection are respectively and a protected amino acid is coupled thereon Peptide resin 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.
Preferably, the substitution value of the peptide resin 1 is 0.2-1.5mmol/g resins, more preferably 0.5-1.0mmol/g Resin.
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 to set 1~6 times of resin total mole number, more preferably 2.5~3.5 times in fat 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 resin or the peptide resin that has synthesized in 1~6 times of resin total mole number, more Preferably 2.5~3.5 times.
Preferably, the catalyst is DMAP (DMAP).
Preferably, the All deprotection agent is dichloromethane (DCM) solution of tetra-triphenylphosphine palladium and phenylsilane.Four Triphenylphosphine palladium is 1 with phenylsilane mol ratio：8~12, preferably 1：10.Tetra-triphenylphosphine palladium dosage is the 0.2 of All moles ~0.3 times, preferably 0.25 times.
Preferably, it is mixed acid that water forms for 95-100% TFA, surplus that the acidolysis, which is used by percent by volume, Solve liquid acidolysis.It is highly preferred that be 98% by percent by volume TFA, surplus be mixing acid hydrolysis solution acidolysis that water forms.It is described It is preferably to need 4~15mL, more preferably 9~11mL per the pungent precursor peptide resin of Crow meter to mix acid hydrolysis solution dosage.The acid The time of solution is preferably more preferably 3~4 hours 1~6 hour under room temperature condition.
Preferably, the dehydrating agent is the pyridine solution of paratoluensulfonyl chloride or the pyridine solution of methylsufonyl chloride, more The preferably pyridine solution of paratoluensulfonyl chloride.The dosage of dehydrating agent is 1.2~4 times of OH moles in Thr more preferably 2 Times.
Preferably, the purifying turns acetate and is specially：
Romidepsin 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, romidepsin purifying intermediate concentrate is obtained；
Take romidepsin to purify intermediate concentrate, filtered with 0.45 μm of filter membrane standby；
Change salt using high performance liquid chromatography, flow phase system is 10% acetic acid/water solution-acetonitrile, purifying color The anti-phase C18 that filler is 10 μm is composed, 77mm*250mm column flow rate is 90mL/min, using gradient elution, circulates loading Method, it is splined in chromatographic column, starts mobile phase elution, gather collection of illustrative plates, observe the change of trap, collection is changed salt main peak and is used in combination Liquid phase detection purity is analyzed, merging changes salt main peak solution, is concentrated under reduced pressure, obtains romidepsin aqueous acetic acid, is freeze-dried, obtains Romidepsin finished product.
The romidepsin synthesized by the method for the invention detects through HPLC, and crude product purity is more than 70%, and product purity is big In 99.5%, maximum single contaminant is less than 0.15%, and total recovery is more than 50%.
From above technical scheme, the present invention synthesizes the linear peptide chain of romidepsin one by one with suitable protected amino acid, Amido link cyclisation is successively completed by solid phase means and disulfide bond is cyclized, and details optimization has been carried out to synthesis in solid state scheme, most The romidepsin obtained eventually has higher purity and total recovery.
Embodiment
The invention discloses a kind of method for synthesizing romidepsin, those skilled in the art can use for reference present disclosure, fit When modified technique parameter is realized.In particular, all similar replacements and change for a person skilled in the art It is it will be apparent that they are considered as being included in the present invention.The method of the present invention is retouched by preferred embodiment State, related personnel can substantially not depart from present invention, methods described herein are being modified in spirit and scope or suitably Change is 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
With reference to embodiment, the present invention is expanded on further.
Embodiment 1：The synthesis (Fmoc-Thr-OAll coupling) of peptide resin 1
0.3mol Trt resins (substitution value about 0.6mmol/g) are taken, are washed 3 times with DMF；Separately take 0.6mol Fmoc- Thr-OAll, dissolved, be added in above-mentioned resin with appropriate DMF, stir lower addition 1.2molDIPEA, 65 DEG C of stirring reactions 6 are small When, reaction solution is taken out, after DMF is washed 3 times, 10%DIPEA/ methanol is washed 3 times, and DMF is washed 3 times, and each wash time is 3min, obtain Fmoc-Thr-OAll-Trt resins, i.e. peptide resin 1.
Embodiment 2：The synthesis of peptide resin 2
0.3mol Fmoc-D-Cys (Trt) and 0.3mol HOBt are taken, are dissolved with appropriate DMF；0.3mol 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.
Peptide resin 1 made from 0.1mol embodiments 1 is taken, is deprotected 25 minutes with 20%PIP/DMF solution, is washed with DMF, Filtering, Fmoc-D-Cys (Trt) solution added after activation, reaction 3 hours is stirred at room temperature, takes out reaction solution, DMF is washed 3 times Afterwards, DCM is washed 3 times, and each wash time is 3min, then is deprotected 25 minutes with 20%PIP/DMF solution, and washing and filtering is complete Into Fmoc-D-Cys (Trt) access.
Obtained with method access Fmoc-D-Val and (3S, 4E) -3- hydroxyls -7- [(Trt) sulfenyl] -4- heptenoic acids, washing and filtering To (3S, 4E) -3- hydroxyls -7- [(Trt) sulfenyl] -4- heptene acyls-D-Val-D-Cys (Trt)-Thr-OAll-Trt resins, i.e., Peptide resin 2.
Embodiment 3：The synthesis of peptide resin 2
0.3mol Fmoc-D-Cys (Acm) and 0.3mol HOBt are taken, are dissolved with appropriate DMF；0.3mol 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.
Take 0.1mol to apply peptide resin 1 made from example 1, deprotected 25 minutes, washed with DMF, mistake with 20%PIP/DMF solution Filter, Fmoc-D-Cys (Acm) solution added after activation, reaction 3 hours is stirred at room temperature, takes out reaction solution, after DMF is washed 3 times, DCM is washed 3 times, and each wash time is 3min, then is deprotected 25 minutes with 20%PIP/DMF solution, washing and filtering, is completed Fmoc-D-Cys (Acm) access.
Obtained with method access Fmoc-D-Val and (3S, 4E) -3- hydroxyls -7- [(Acm) sulfenyl] -4- heptenoic acids, washing and filtering To (3S, 4E) -3- hydroxyls -7- [(Acm) sulfenyl] -4- heptene acyls-D-Val-D-Cys (Trt)-Thr-OAll-Trt resins, i.e., Peptide resin 2.
Embodiment 4：The synthesis of peptide resin 3
0.3mol Fmoc-Val and 0.3mol HOBt are taken, are dissolved with appropriate DMF；0.3mol DIC separately are taken, it is slow under stirring Slowly add into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protected amino acid after being activated Solution.
Protected amino acid solution after above-mentioned activation is added to peptide resin 2 made from embodiment 2, adds 0.1molDMAP (catalyst)/DMF solution, reaction 8 hours is stirred at room temperature, washing and filtering, obtains obtaining peptide resin 3.
Embodiment 5：The synthesis of peptide resin 3
0.3mol Fmoc-Val and 0.3mol HOBt are taken, are dissolved with appropriate DMF；0.3molDIC separately is taken, under stirring slowly Add into protected amino acid DMF solution, stirring reaction 30 minutes in room temperature environment, the protected amino acid after being activated is molten Liquid.
Protected amino acid solution after above-mentioned activation is added to peptide resin 2 made from embodiment 3, adds 0.1molDMAP (catalyst)/DMF solution, reaction 8 hours is stirred at room temperature, washing and filtering, obtains obtaining peptide resin 3.
Embodiment 6：The synthesis of romidepsin precursor peptide resin
1st, All protection groups are gone
0.025mol tetra-triphenylphosphine palladiums and 0.25mol phenylsilanes are taken, is dissolved with appropriate DCM, is added to the peptide of embodiment 4 In resin 3, it is more than hour that the reaction time 10 is stirred at room temperature, after the completion of reaction, is washed 3 times with DMF.
2nd, Fmoc protection groups are gone
Deprotected 25 minutes with 20%PIP/DMF solution, after the completion of reaction, washed 3 times with DMF again.
3rd, acid amides ring cyclization
Take 0.3mol DIC and 0.3molHOAt, with appropriate DMF dissolve, be slowly added under stirring it is above-mentioned go All and In the peptide resin of Fmoc protection groups, cyclization 240~300 minutes, after the completion of reaction, washed 3 times with DMF.
4th, two sulphur ring cyclization
Take 5%I2/ DMF solution (10mL/ grams of resin), is added in above-mentioned peptide resin, 40 DEG C of stirring reactions 4 hours, Reaction solution is taken out, after the completion of reaction, is washed 3 times with DMF, obtains romidepsin precursor peptide resin.
Embodiment 7：The synthesis of romidepsin precursor peptide resin
1st, All protection groups are gone
0.025mol tetra-triphenylphosphine palladiums and 0.25mol phenylsilanes are taken, is dissolved with appropriate DCM, is added to the peptide of embodiment 5 In resin 3, it is more than hour that the reaction time 10 is stirred at room temperature, after the completion of reaction, is washed 3 times with DMF.
2nd, Fmoc protection groups are gone
Deprotected 25 minutes with 20%PIP/DMF solution, after the completion of reaction, washed 3 times with DMF again.
3rd, acid amides ring cyclization
Take 0.3mol DIC and 0.3mol HOAt, with appropriate DMF dissolve, be slowly added under stirring it is above-mentioned go All and In the peptide resin of Fmoc protection groups, cyclization 240~300 minutes, after the completion of reaction, washed 3 times with DMF.
4th, two sulphur ring cyclization
Take 5%I2/ DMF solution (10mL/ grams of resin), is added in above-mentioned peptide resin, 40 DEG C of stirring reactions 4 hours, Reaction solution is taken out, after the completion of reaction, is washed 3 times with DMF, obtains romidepsin precursor peptide resin.
Embodiment 8：The preparation of romidepsin precursor
Romidepsin peptide precursor resin made from Example 6, add acidolysis solution (TFA:H2O=98:2, acid hydrolysis solution The pungent precursor peptide resin of 10mL/ Crows meter), stirring reaction 3 hours, filtrate is collected by filtration, resin is washed 3 times with a small amount of TFA again, Solvent evaporated is concentrated under reduced pressure into after merging filtrate, obtains romidepsin precursor.
Embodiment 9：The preparation of romidepsin precursor
Romidepsin peptide precursor resin made from Example 7, add acidolysis solution (TFA:H2O=98:2, acid hydrolysis solution The pungent precursor peptide resin of 10mL/ Crows meter), stirring reaction 3 hours, filtrate is collected by filtration, resin is washed 3 times with a small amount of TFA again, Solvent evaporated is concentrated under reduced pressure into after merging filtrate, obtains romidepsin precursor.
Embodiment 10：The preparation of romidepsin crude product
Romidepsin peptide precursor made from Example 8, with appropriate pyridinium dissolution, it is cooled to -5 DEG C, stirs lower add 0.2mol paratoluensulfonyl chlorides, and insulation reaction, after 12 hours, reactant mixture is poured into frozen water, it is 6 that pH is adjusted in stirring, and filtering is received Collect solid, washing obtains romidepsin crude product, and crude product purity is 75.2%.
Embodiment 11：The preparation of romidepsin crude product
Romidepsin peptide precursor made from Example 9, with appropriate pyridinium dissolution, it is cooled to -5 DEG C, stirs lower add 0.2mol paratoluensulfonyl chlorides, and insulation reaction, after 12 hours, reactant mixture is poured into frozen water, it is 6 that pH is adjusted in stirring, and filtering is received Collect solid, washing obtains romidepsin crude product, and crude product purity is 71.3%.
Embodiment 12：Romidepsin purifying crude
The gained romidepsin crude product of Example 10, dissolved with 30% acetum, 0.45 μm of miillpore filter mistake of solution Filter, 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, romidepsin purifying intermediate concentrate is obtained；
Take romidepsin 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 romidepsin aqueous acetic acid, is freeze-dried, obtains sieve The pungent sterling 30.6g of meter
Total recovery is 56.7%, molecular weight：541.6, purity：99.6%, maximum single contaminant 0.11%.
Embodiment 13：Romidepsin purifying crude
The gained romidepsin crude product of Example 11, dissolved with 30% acetum, 0.45 μm of miillpore filter mistake of solution Filter, 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, romidepsin purifying intermediate concentrate is obtained；
Take romidepsin 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 romidepsin aqueous acetic acid, is freeze-dried, obtains sieve The pungent sterling 28.5g of meter.
Total recovery is 52.8%, molecular weight：541.8, purity：99.7%, maximum single contaminant 0.09%.
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 (10)

  1. A kind of 1. method for synthesizing romidepsin, it is characterised in that comprise the following steps：
    Step 1, the Thr-OAll of protection are coupled under organic base effect with resin, obtain peptide resin 1；
    Step 2, from peptide resin 1, according to the polypeptide sequence of romidepsin amino acid sequence C-terminal to N-terminal, in condensation reagent and Under activating reagent effect, successively by the D-Cys of protection, the D-Val of protection and protection (3S, 4E) -3- hydroxyl -7- sulfenyls -4- Heptenoic acid carries out extension coupling one by one, obtains peptide resin 2；
    Step 3, from peptide resin 2s, in the presence of condensation reagent, activating reagent and catalyst, access the Val of protection, obtain Peptide resin 3；
    Step 4, All protection group removing of the agent by Thr-OAll in peptide resin 3 is deprotected with All, then in condensation reagent and work Change under reagent effect, carry out intramolecular amide cyclization and be coupled valine and threonine, then carried out by iodine oxidation method Intramolecular disulfide bond cyclization, obtain romidepsin peptide precursor resin；
    Step 5, romidepsin peptide resin obtain romidepsin precursor after acidolysis；
    Step 6, romidepsin precursor obtain romidepsin crude product after dehydrating agent is dehydrated；
    Step 7, romidepsin crude product be purified to turn after acetate to obtain romidepsin sterling.
  2. 2. method according to claim 1, it is characterised in that the Thr-OAll of the protection, the D-Cys of protection, protection D-Val, (3S, 4E) -3- hydroxyl -7- sulfenyl -4- heptenoic acids of protection, the Val of protection are as follows：
    Fmoc-Thr-OAll, Fomc-D-Cys (Trt) or Fomc-D-Cys (Acm), Fmoc-D-Val, (3S, 4E) -3- hydroxyls - 7- [(Trt) sulfenyl] -4- heptenoic acids or (3S, 4E) -3- hydroxyls -7- [(Acm) sulfenyl] -4- heptenoic acids, Fmoc-D-Val.
  3. 3. method according to claim 1, it is characterised in that the resin is trityl resin.
  4. 4. method according to claim 1, it is characterised in that the condensation reagent is N, N- DICs, N, N- dicyclohexylcarbodiimides, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus/organic base, 2- (7- azepines - 1H- BTA -1- bases) -1,1,3,3- tetramethylureas hexafluorophosphoric acid ester/organic base, BTA-N, N, N', N'- tetra- In MU hexafluorophosphate/organic base, O- BTAs-N, N, N', N'- tetramethylurea tetrafluoro boric acid ester/organic base It is a kind of.
  5. 5. according to the methods described of claim 1 or 4, it is characterised in that the organic base is DIPEA, triethylamine Or N- methylmorpholines.
  6. 6. method according to claim 1, it is characterised in that the activating reagent be I-hydroxybenzotriazole or N- hydroxyls- 7- azepine BTAs.
  7. 7. method according to claim 1, it is characterised in that the catalyst is DMAP.
  8. 8. method according to claim 1, it is characterised in that the All deprotection agent is tetra-triphenylphosphine palladium and phenylsilane Dichloromethane solution.
  9. 9. method according to claim 1, it is characterised in that the dehydrating agent is the pyridine solution or first of paratoluensulfonyl chloride The pyridine solution of base sulfonic acid chloride.
  10. 10. method according to claim 1, it is characterised in that the acidolysis is used by percent by volume as 95-100%'s TFA, the mixing acid hydrolysis solution acidolysis that surplus is water composition.
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Cited By (2)

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CN109666062A (en) * 2018-12-20 2019-04-23 浙江昂利康制药股份有限公司 The synthetic method of natural products inhibitors of histone deacetylase FK228
WO2020125045A1 (en) * 2018-12-19 2020-06-25 深圳翰宇药业股份有限公司 Method for synthesizing romidepsin

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WO2013071138A1 (en) * 2011-11-11 2013-05-16 Sio2 Medical Products, Inc. PASSIVATION, pH PROTECTIVE OR LUBRICITY COATING FOR PHARMACEUTICAL PACKAGE, COATING PROCESS AND APPARATUS
CN103877010A (en) * 2012-12-21 2014-06-25 正大天晴药业集团股份有限公司 Preparation method of Romidepsin solution
CN103897029A (en) * 2012-12-27 2014-07-02 深圳翰宇药业股份有限公司 Preparation method for romidepsin

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WO2013071138A1 (en) * 2011-11-11 2013-05-16 Sio2 Medical Products, Inc. PASSIVATION, pH PROTECTIVE OR LUBRICITY COATING FOR PHARMACEUTICAL PACKAGE, COATING PROCESS AND APPARATUS
CN103877010A (en) * 2012-12-21 2014-06-25 正大天晴药业集团股份有限公司 Preparation method of Romidepsin solution
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Publication number Priority date Publication date Assignee Title
WO2020125045A1 (en) * 2018-12-19 2020-06-25 深圳翰宇药业股份有限公司 Method for synthesizing romidepsin
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CN111333697B (en) * 2018-12-19 2022-03-08 深圳翰宇药业股份有限公司 Synthesis method of romidepsin
CN109666062A (en) * 2018-12-20 2019-04-23 浙江昂利康制药股份有限公司 The synthetic method of natural products inhibitors of histone deacetylase FK228

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