CN103936828A - Preparation method of carfilzomib intermediate and carfilzomib - Google Patents
Preparation method of carfilzomib intermediate and carfilzomib Download PDFInfo
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- CN103936828A CN103936828A CN201410198158.4A CN201410198158A CN103936828A CN 103936828 A CN103936828 A CN 103936828A CN 201410198158 A CN201410198158 A CN 201410198158A CN 103936828 A CN103936828 A CN 103936828A
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- MIVQDKORYUSPTQ-QKDODKLFSA-N CC(C)C[C@@H](C(N[C@@H](Cc1ccccc1)C(O)=O)=O)NC([C@H](CCc1ccccc1)NC(CN1CCOCC1)=O)=O Chemical compound CC(C)C[C@@H](C(N[C@@H](Cc1ccccc1)C(O)=O)=O)NC([C@H](CCc1ccccc1)NC(CN1CCOCC1)=O)=O MIVQDKORYUSPTQ-QKDODKLFSA-N 0.000 description 1
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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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Abstract
The invention relates to a preparation method of a carfilzomib intermediate and carfilzomib. The method comprises the following steps: (1) enabling a solid-phase resin carrier to couple with Fmoc-Phe-OH in the presence of an activator to obtain Fmoc-Phe-resin; (2) enabling Fmoc-Phe-resin to sequentially couple with Fmoc-Phe-OH, Fmoc-HoPhe-OH and 4-morpholine acetic acid through a solid-phase synthesis method to obtain tetrapeptide intermediate resin, wherein the coupling is performed in the presence of a coupling agent system, the coupling agent system comprises a condensing agent and a reaction solvent, and the resin firstly removes Fmoc protecting group before coupling; (3) performing pyrolysis on the tetrapeptide intermediate resin by a pyrolysis agent, and removing the resin to obtain the carfilzomib tetrapeptide intermediate. The method provided by the invention has the advantages of high yield, low pollution and simple and controllable reaction, and is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation method of a kind of Ka Feizuo meter intermediate and Ka Feizuo meter.
Background technology
The outer literary fame of Ka Feizuo meter or general Carfilzomib by name, have following structure:
On July 20th, 2012, FDA Food and Drug Administration (FDA) has ratified the listing of the product Ka Feizuo meter of ONYXPHARMSINC company (Carfilzomib) freeze-dried powder injection, and Carfilzomib can be used for accepting the multiple myeloma patients that at least two kinds of medicines (comprising Velcade and immunomodulator) are treated before treatment.Multiple myeloma is a kind of plasmocyte malignant clone disease, and sickness rate increases year by year, has occupied the second of neoplastic hematologic disorder, and along with the raising for the treatment of level, although complete remission rate is higher, survival rate is still lower, and one of the main reasons is recurrence.Carfilzomib's is granted, for the multiple myeloma patients of those recurrences after current therapy treatment, provides one to treat selection.
Ka Feizuo meter is the pentapeptide of 3 amino acid and 2 amino acid derivative compositions, the synthetic route of report is mainly used the scheme of 3 amino acid and 1 glycine derivative (tetrapeptide, intermediate compound I)+1 leucine derivative (intermediate II) to synthesize.
About the synthetic report of Ka Feizuo meter such as US20050245435; WO2009045497A; CN103641890A etc. have all adopted full liquid phase synthesizing method; from the former building-up process of expecting intermediate compound I; intermediate needs repeatedly extracting and separating excessive response component; some protecting group also needs hydrogenation to remove, and the reversed-phase HPLC that also needs having is purified.These some problems of method ubiquity, as low in synthesis yield, each intermediate extracting and separating causes product loss, therefore consumes more starting raw material; And full liquid phase building-up reactions type is many, reaction conditions and solvent for use complexity are various, and poor controllability, is unfavorable for large-scale commercial production.
Summary of the invention
One of object of the present invention is to provide a kind of resin type Ka Feizuo meter intermediate (tetrapeptide intermediate resin), it is made up of resin solid phase carrier and the Ka Feizuo meter tetrapeptide intermediate being coupled on described solid-phase resin carrier, and wherein Ka Feizuo meter tetrapeptide intermediate has the structure shown in formula I:
prepare Ka Feizuo meter by this intermediate, reactions steps is few, reaction conditions gentleness, and preparation technology is simply controlled, and yield is high, workable.
Two of object of the present invention is to provide a kind of preparation method of above-mentioned resin type Ka Feizuo meter intermediate, and the anti-reactions steps of the method is few, reaction conditions gentleness, and preparation technology is simply controlled, and yield is high.This preparation method comprises the steps:
(1), make resin solid phase carrier activator exist under coupling Fmoc-Phe-OH obtain Fmoc-Phe-resin, described resin solid phase carrier has the functional group that can react with the carboxyl of described Fmoc-Phe-OH;
(2), pass through solid-phase synthesis; make described Fmoc-Phe-resin coupling Fmoc-Leu-OH successively; Fmoc-HoPhe-OH and 4-morpholine acetic acid; obtain described resin type Ka Feizuo meter intermediate; described in wherein making, be coupled under the existence of coupling agent system and carry out; described coupling agent system comprises condensing agent and reaction solvent, before carrying out described coupling, first makes resin remove Fmoc protecting group.
According to the present invention, described solid-phase resin carrier can be for being selected from one or more the combination in trityl chloride type of resin and hydroxyl type of resin.Described trityl chloride type of resin includes but not limited to trityl chloride resin, 4-methyl trityl chloride resin, 4-methoxyl group trityl chloride resin and 2-chlorine trityl chloride resin (CTC Resin); Described hydroxyl type of resin includes but not limited to king's resin (Wang Resin) and to hydroxymethyl phenoxy methyl polystyrene resin (HMP Resin).
According to a concrete aspect: described solid-phase resin carrier is trityl chloride type of resin, and described activator is to be selected from NMM, DIEA or the combination of the two.
According to another concrete aspect: described solid-phase resin carrier is hydroxyl type of resin, and described activator is made up of DIC, HOBt and DMAP.
Further, in step (1), the substitution value of prepared Fmoc-Phe-resin is 0.2～1.2mmol/g, is preferably 0.7～0.9mmol/g.
Further, step (2) comprises the following steps:
A, employing DBLK make Fmoc-Phe-resin remove Fmoc protecting group, obtain H-Phe-resin, and described DBLK is the solution that piperidines and DMF form according to volume ratio 1:3～5;
B, make H-Phe-resin and Fmoc-Leu-OH coupling under the existence of coupling agent system obtain Fmoc-Leu-Phe-resin;
C, employing DBLK make Fmoc-Leu-Phe-resin remove Fmoc protecting group, obtain H-Leu-Phe-resin;
D, make H-Leu-Phe-resin and Fmoc-HoPhe-OH coupling under the existence of coupling agent system obtain Fmoc-HoPhe-Leu-Phe-resin;
E, employing DBLK make Fmoc-HoPhe-Leu-Phe-resin remove Fmoc protecting group, obtain H-HoPhe-Leu-Phe-resin;
F, make H-HoPhe-Leu-Phe-resin and the coupling under the existence of coupling agent system of 4-morpholine acetic acid obtain described tetrapeptide intermediate resin.
Particularly, in step (2), the method that makes resin remove Fmoc protecting group is: in resin, add the solution being made up of according to volume ratio 1:3～5 piperidines and DMF, stir.
According to the present invention, in step (2), described condensing agent can be selected from: the combination of DIC and HOBt; The combination of the combination of HBTU, HCTU, PyBOP one of them or the two and HoBt and DIEA, NMM one of them or the two; The combination of HATU, one of them three's of PyAOP arbitrary combination and HOAt and DIEA, NMM one of them or the two.Coupling agent system has such as HoBt/DIC, HOBt+HBTU/DIEA, HOBt+PyBOP/NMM, HOBt+HCTU/NMM, HOAt+HATU/NMM, HOAt+HBTU/NMM, HOAt+PyBOP/NMM etc. more specifically.
The coupling agent system adopting in above-mentioned steps b, d, f can be identical or different.
Preferably, before the reaction of a～f of carrying out step (1), step (2), make resin in abundant swelling state.A concrete method is: resin is added in DCM or other lower boiling non-reactive solvents, and nitrogen brushes stirring, drains solvent after making resin fully swelling again.
Further, in step (2), described reaction solvent is selected from DMF, DCM, NMP, DMSO or the arbitrary combination between them.
Further, in step (2), can and preferably make coupling carry out at ambient temperature.
Further, in step (2), described Fmoc-Leu-OH, Fmoc-HoPhe-OH, the molar ratio of 4-morpholine acetic acid and their coupling resins is 1.2～6:1.Be preferably 2.5～3.5:1.
Further, in step (2), coupling time can be under room temperature condition 60～300 minutes.Preferably 100～140 minutes.
Another object of the present invention is to provide a kind of preparation method of Ka Feizuo meter tetrapeptide intermediate, and Ka Feizuo meter tetrapeptide intermediate has the structure shown in formula I:
described method is by making resin type Ka Feizuo meter intermediate of the present invention, under cracking agent exists, cracking occur, obtain Ka Feizuo meter tetrapeptide intermediate, described cracking agent is TFA or TFE or their combination, or be TFA or TFE or the combination of the two and the mixing of DCM, cracking agent is 4～15ml/g with the volume mass ratio of described resin type Ka Feizuo meter intermediate.
Further preferably, cracking agent is 7～9ml/g with the volume mass ratio of described resin type Ka Feizuo meter intermediate.
Further, cracking can be carried out at ambient temperature, and pyrolysis time can be 1～6 hour, preferably 2～3 hours.
According to a concrete aspect, described solid-phase resin carrier is CTC Resin resin, and described cracking agent is TFA and mixing of DCM or mixing of TFE and DCM.Preferably, cracking agent is 0.1%～0.2% TFA/DCM solution or 15%～25%TFE/DCM solution.
The another technical scheme that the present invention takes is: the preparation method of a kind of Ka Feizuo meter, it comprises makes Ka Feizuo meter tetrapeptide intermediate and intermediate II
iI reaction generates the step of Ka Feizuo meter, and particularly, described preparation method also comprises the step of taking the above-mentioned method of the present invention to prepare Ka Feizuo meter tetrapeptide intermediate.
Ka Feizuo meter tetrapeptide intermediate can be implemented with reference to prior art with reacting of intermediate II.A concrete grammar according to the present invention is as follows: tetrapeptide intermediate, intermediate II are dissolved in organic solvent, are cooled to below 5 DEG C, splash into the solution of coupling agent, room temperature reaction.Wherein organic solvent can be DCM, DMF or THF etc.; Coupling agent can be DCC/HOSu, EDCI/HOSu.The solvent that the solution of preparation coupling agent adopts can be DCM, DMF or THF etc.The time of reaction can be under room temperature condition 60～300 minutes, under preferred room temperature condition 100～140 minutes.After completion of the reaction, obtain Ka Feizuo meter crude product by the simple aftertreatment of routine.A concrete post-treating method is: filter reaction system, after adding purified water fully to wash, filter in filtrate, filtrate leaving standstill abandoned water.Organic phase is successively with saturated aqueous common salt, purified water washing, and gained organic phase anhydrous sodium sulfate drying, filters, and concentrated filtrate, pours filtrate in ice ether into, crystallization, and after solid washs with a small amount of ether, vacuum-drying.Get Ka Feizuo meter crude product, purity approximately 94%～99.2%.
Further, described preparation method also comprises that Ka Feizuo meter tetrapeptide intermediate is reacted to the Ka Feizuo meter crude product generating with intermediate II carry out the step of purifying.
Preferably, described purifying comprises following operation:
2.
"dMSO or DMF dissolve Ka Feizuo meter crude product, have dissolved filtration, retain filtrate;
2. adopt preparative liquid chromatography instrument by the separation and purification of Ka Feizuo meter crude product, described preparative liquid chromatography instrument adopts C18 or C8 chromatographic column, defining chromatographic detection wavelength is 230nm, and elution flow rate is 20～90ml/min, and condition of gradient elution is: time 0～40min; A phase volume ratio concentration becomes 30%～45%, B phase volume ratio concentration from 60%～65% and becomes from 40%～35% the TFA aqueous solution that 70%～55%, A is 0.1wt%～0.2wt% mutually, B is acetonitrile mutually, after being purified into peak, press the receiving flask of the number of finishing, collect sample, then according to HPLC analyzing and testing, press before peak, summit, behind peak respectively and sample, its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges, and purity is less than to 99% sample and carries out repeatedly purifying by above purification step and obtain salable product;
3. adopt the salable product that 2. preparative liquid chromatography instrument obtains operation to carry out desalination, described preparative liquid chromatography instrument adopts C18 or C8 chromatographic column, its filler is the alkyl linked silica gel of 10～50um, defining chromatographic detection wavelength is 230nm, elution flow rate is 20～90ml/min, and elution requirement is as follows:
Time 0～15min, A phase volume ratio concentration is between 75%～85%, and B phase volume ratio concentration is between 15%～25%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is ammonium acetate mutually, and B is acetonitrile mutually;
Time 16～25min, A phase volume ratio concentration is between 75%～85%, and B phase volume ratio concentration is between 15%～25%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is pure water mutually, and B is acetonitrile mutually;
Time 26～40min, A phase volume ratio concentration is between 15%～25%, and B phase volume ratio concentration is between 75%～85%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is pure water mutually, and B is acetonitrile mutually; After being purified into peak, collect main peak, be incorporated in receiving flask;
4. remove 3. acetonitrile and the water in gained solution of operation by concentrating under reduced pressure, adopt afterwards vacuum freeze drier freeze-drying to obtain white powder and be Ka Feizuo meter finished product.
Preferably, operation 2. in, establish purity and be greater than 99.2% for salable product.
Preferably, operation 4. in, concentrating under reduced pressure is at 30～40 DEG C, vacuum tightness 10mbar carries out below.
Particularly, vacuum lyophilization process is as follows:
1) pre-freeze: concentrated solution is placed on freeze drying box internal partition and carries out pre-freeze, and temperature drops to below-40 DEG C, maintains 1.5～2.5 hours;
2) sublimation drying: slowly heat by the heating system under dividing plate, temperature rises to-22 DEG C～-28 DEG C, maintains approximately 8～12 hours, and then temperature rises to-2 DEG C～2 DEG C, maintains 6～10 hours;
3) desorption: temperature rises to 28 DEG C～32 DEG C, maintains 3～7 hours, takes out.
Take above-mentioned method of purification of the present invention Ka Feizuo meter purity can be increased to more than 99.8%, and target product loss is little.
The corresponding Chinese of english abbreviation the present invention relates to sees the following form:
According to the present invention, raw material used, reagent and intermediate II all can be buied from market.
Due to the employing of above technical scheme, the present invention compared with prior art tool has the following advantages:
The present invention adopts solid-phase synthesis successfully to prepare Ka Feizuo meter tetrapeptide intermediate, and reactions steps is few, reaction conditions gentleness, and reaction yield high (can reach 82.3%～93.7%), workable, be beneficial to suitability for industrialized production.
The preparation method of Ka Feizuo meter provided by the invention, pollutes littlely, and technique is simply controlled, total recovery is high (can reach 76.8%～88.4%), is suitable for suitability for industrialized production Ka Feizuo meter.
Brief description of the drawings
Fig. 1 is the mass spectrum of the product of gained H-Leu-Phe-resin after cracking deresinate in embodiment 4 preparation process;
Fig. 2 is the mass spectrum of the product of gained H-Hophe-Leu-Phe resin after cracking deresinate in embodiment 4 preparation process;
Fig. 3 is the mass spectrum that embodiment 11 prepares gained tetrapeptide intermediate;
Fig. 4 is the nuclear magnetic spectrogram (DMSO) that embodiment 11 prepares gained tetrapeptide intermediate;
Fig. 5 is the HPLC spectrogram that embodiment 23 prepares gained Ka Feizuo meter.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further details.Should be understood that these embodiment are for ultimate principle of the present invention, principal character and advantage are described, and the present invention is not limited by the following examples.The implementation condition adopting in embodiment can be done further adjustment according to specific requirement, and not marked implementation condition is generally the condition in normal experiment.
The preparation of embodiment 1Fmoc-Phe-resin
Get Wang Resin (1.7mmol/g) 10g and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Take Fmoc-Phe-OH19.8g, HOBt8.3g, adds DMF80mL stirring and dissolving, and ice bath 5 minutes, adds DIC9.5mL, ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction.Get DMAP0.75g and add after 20mLDMF dissolving, splash in polypeptide reactor, react and drain reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.Get acetic anhydride 8.0mL, pyridine 6.8mL, DMF80mL adds polypeptide reactor, and nitrogen brushes stirring reaction 6 hours.Drain reaction solution, DMF washing 3 times, each 100mL, 1 minute.DCM washing 4 times, each 100mL, 10 minutes.Take out resin, dry.Obtain Fmoc-Phe-Wang Resin14.8g.Substitution value is 0.87mmol/g.
The preparation of embodiment 2Fmoc-Phe-resin
Get CTC Resin (1.5mmol/g) 8g and be placed in polypeptide reactor, add DCM80mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Take Fmoc-Phe-OH13.9g, add DMF80mL stirring and dissolving, add DIEA18.9mL, ice bath adds after 15 minutes in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 80mL, 1 minute.Add twice each 80mL of closed reagent (anhydrous methanol/DIEA/DCM=1/2/17 (volume ratio)) sealing, 10 minutes.DCM washing 4 times, each 80mL, 10 minutes.Take out resin, dry.Obtain Fmoc-Phe-CTC Resin11.7g.Substitution value is 0.76mmol/g.
The preparation of embodiment 3Fmoc-Phe-resin
Get CTC Resin (1.5mmol/g) 80g and be placed in polypeptide reactor, add DCM800mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Take Fmoc-Phe-OH139g, add DMF800mL stirring and dissolving, add DIEA189mL, ice bath adds after 15 minutes in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 800mL, 1 minute.Add twice each 800mL of closed reagent (anhydrous methanol/DIEA/DCM=1/2/17 (volume ratio)) sealing, 10 minutes.DCM washing 4 times, each 800mL, 10 minutes.Take out resin, dry.Obtain Fmoc-Phe-CTC Resin108g.Substitution value is 0.75mmol/g.
The preparation of embodiment 4 tetrapeptide intermediate resins
Get Fmoc-Phe-Wang Resin11.5g (10mmol) and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Add DBLK (by piperidines and DMF by volume 1:4 form) the de-Fmoc protection of 80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOBt4.9g (36mmol) adds DMF80mL stirring and dissolving, and ice bath 5 minutes adds DIC5.6mL (36mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-Wang Resin peptide resin 16.3g.In above-mentioned preparation process, respectively H-Leu-Phe-resin and H-Hophe-Leu-Phe resin are carried out to sampling analysis, after being sloughed to resin with cracking agent cracking respectively, they carry out mass spectroscopy, and result is shown in respectively Fig. 1 and Fig. 2.
The preparation of embodiment 5 tetrapeptide intermediate resins
Get Fmoc-Phe-Wang Resin11.5g (10mmol) and be placed in polypeptide reactor, add DCM100mL nitrogen to brush and stir 30 minutes, drain DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOBt4.1g (30mmol), HBTU10.2g (27mmol) adds DMF80mL stirring and dissolving, ice bath 5 minutes, add DIEA7.9mL (45mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-Wang Resin peptide resin 16.1g.
The preparation of embodiment 6 tetrapeptide intermediate resins
Get Fmoc-Phe-Wang Resin11.5g (10mmol) and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOBt4.1g (30mmol), PyBOP14.1g (27mmol) adds DMF80mL stirring and dissolving, ice bath 5 minutes, add NMM9.9mL (90mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-Wang Resin peptide resin 15.8g.
The preparation of embodiment 7 tetrapeptide intermediate resins
Get Fmoc-Phe-Wang Resin11.5g (10mmol) and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOAt4.1g (30mmol), HATU10.3g (27mmol) adds DMF80mL stirring and dissolving, ice bath 5 minutes, add NMM9.9mL (90mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-Wang Resin peptide resin 17.2g.
The preparation of embodiment 8 tetrapeptide intermediate resins
Get Fmoc-Phe-CTC Resin13.3g (10mmol) and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOBt4.9g (36mmol), adds DMF80mL stirring and dissolving, and ice bath 5 minutes adds DIC5.6mL (36mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-CTC Resin peptide resin 17.8g.
The preparation of embodiment 9 tetrapeptide intermediate resins
Get Fmoc-Phe-CTC Resin13.3g (10mmol) and be placed in polypeptide reactor, add DCM100mL, nitrogen brushes and stirs 30 minutes, drains DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOAt4.1g (30mmol), PyAOP14.1g (27mmol) adds DMF80mL stirring and dissolving, ice bath 5 minutes, add NMM9.9mL (90mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-CTC Resin peptide resin 18.7g.
The preparation of embodiment 10 tetrapeptide intermediate resins
Get Fmoc-Phe-CTC Resin13.3g (10mmol) and be placed in polypeptide reactor, add DCM100mL nitrogen to brush and stir 30 minutes, drain DCM after making resin fully swelling.Add the de-Fmoc protection of DBLK80mL 2 times, 5+15 minute, each 80mL.DMF washing 5 times, each 80mL, 1 minute.Take Fmoc-Leu-OH10.6g (30mmol), HOBt4.1g (30mmol), HCTU11.2g (27mmol) adds DMF80mL stirring and dissolving, ice bath 5 minutes, add NMM9.9mL (90mmol), ice bath activation 15 minutes.Filter, filtrate adds in polypeptide reactor, and nitrogen brushes stirring reaction and drains reaction solution after 2 hours, DMF washing 3 times, each 100mL, 1 minute.According to said method access successively Fmoc-HoPhe-OH, DMF washing 3 times after 4-morpholine acetic acid, each 80mL, 1 minute.DCM washing 4 times, each 80mL, 10 minutes.Take out peptide resin, dry.Obtain tetrapeptide intermediate-CTC Resin peptide resin 18.5g.
Embodiment 11 cracking
Get embodiment 4 gained tetrapeptide intermediate-Wang Resin10g, add in 80mL TFA stirring at room temperature cracking 2.5 hours.Elimination resin, adds filtrate in 640mL ice ether the centrifugal supernatant liquor of abandoning.Three final vacuums of gained solid ether washing are dry.Solid abrasive is become to powder, be dissolved in 500mL DCM, add Anhydrous potassium carbonate 5g dry 8 hours.Filtering, be spin-dried for filtrate, obtain solid 2.86g, is tetrapeptide intermediate (referring to Fig. 3 and Fig. 4), yield 82.3% through mass spectrum, nuclear-magnetism test confirmation.
Embodiment 12 cracking
Get embodiment 8 gained tetrapeptide intermediate-CTC Resin10g, add in 80mL TFA stirring at room temperature cracking 2.5 hours.Elimination resin, adds filtrate in 640mL ice ether the centrifugal supernatant liquor of abandoning.Three final vacuums of gained solid ether washing are dry.Solid abrasive is become to powder, be dissolved in 500mL DCM, add Anhydrous potassium carbonate 5g dry 8 hours.Filter, be spin-dried for filtrate, obtain tetrapeptide intermediate 2.63g, yield 82.7%.
Embodiment 13 cracking
Get embodiment 10 gained tetrapeptide intermediate-CTC Resin10g, add in 80mL0.1%TFA/DCM solution stirring at room temperature cracking 2.5 hours.Elimination resin, adds Anhydrous potassium carbonate 5g dry 8 hours.Filter, be spin-dried for filtrate, gained solid is dried, obtain tetrapeptide intermediate 2.71g, yield 88.7%.
Embodiment 14 cracking
Get embodiment 9 gained tetrapeptide intermediate-CTC Resin10g, add in 80mL20%TFE/DCM solution stirring at room temperature cracking 1.5 hours.Leach resin, then add in 80mL20%TFE/DCM solution stirring at room temperature cracking elimination resin after 1.5 hours, merging filtrate, adds 5g anhydrous sodium sulfate drying 8 hours.Filter, be spin-dried for filtrate, gained solid is dried, obtain tetrapeptide intermediate 2.84g, yield 93.7%.
Embodiment 15 Ka Feizuo meter 's is synthetic
Get embodiment 11 gained tetrapeptide intermediate 2.27g (4mmol), HOSu0.69g (6mmol), intermediate II 0.82g (4.8mmol) is dissolved in 100mL DCM, is cooled to 5 DEG C.Get DCC1.24g (6mmol), be dissolved in 10mLDCM, splash in the above-mentioned solution of having lowered the temperature room temperature reaction 2 hours.Filter, add purified water 50mL fully to shake washing in filtrate, filter, filtrate leaving standstill abandoned water.Organic phase saturated common salt water washing 4 times, each 50mL; 4 each 50mL of purified water washing.Gained organic phase anhydrous sodium sulfate drying 8 hours, filters, and concentrated filtrate, pours filtrate in the ice ether of 8 times of volumes, and solid washs after 3 times with a small amount of ether, vacuum-drying.Get Ka Feizuo meter 2.69g, yield 93.3%, purity 97.8%, total recovery 76.8%.
Embodiment 16 Ka Feizuo meter 's is synthetic
Get embodiment 13 gained tetrapeptide intermediate 2.27g (4mmol), HOSu0.69g (6mmol), intermediate II 0.82g (4.8mmol), is dissolved in 100mL DCM, is cooled to 5 DEG C.Get EDCI1.15g (6mmol), be dissolved in 10mLDCM, splash in the above-mentioned solution of having lowered the temperature room temperature reaction 2 hours.Filter, filtrate is used saturated common salt water washing 4 times, each 50mL; Purified water washing 4 times, each 50mL.After gained organic phase is concentrated, pour in 0 DEG C of purified water of 8 times of volumes, solid is with after a small amount of 0 DEG C of purified water washing 3 times, dry.Get Ka Feizuo meter 2.75g, yield 95.7%, purity 94.2%, total recovery 84.9%.
Embodiment 17 Ka Feizuo meter 's is synthetic
Get embodiment 11 gained tetrapeptide intermediate 2.27g (4mmol), HOSu0.69g (6mmol), intermediate II 0.82g (4.8mmol) is dissolved in 100mL50%TFH/DCM solution, is cooled to 5 DEG C.Get DCC1.24g (6mmol), be dissolved in 10mL DCM, splash in the above-mentioned solution of having lowered the temperature room temperature reaction 2 hours.Filter, add saturated aqueous common salt 50mL fully to shake washing in filtrate, filter, filtrate leaving standstill abandoned water.Organic phase saturated common salt water washing 4 times, each 50mL; 4 each 50mL of purified water washing.Gained organic phase anhydrous sodium sulfate drying 8 hours, filters, and concentrated filtrate, pours filtrate in the ice normal hexane of 8 times of volumes, and solid is with after a small amount of normal hexane washing 3 times, dry.Get Ka Feizuo meter 2.72g, yield 94.3%, purity 99.1%, total recovery 88.4%.
The purifying of embodiment 18 Ka Feizuo meter
1. sample dissolution: Ka Feizuo meter DMSO prepared by embodiment 15 methods dissolves by the concentration of 1g/20ml, has dissolved with filter paper filtering, obtains filtrate.
2. purge process: adopt preparative liquid chromatography instrument by the separation and purification of Ka Feizuo meter crude product.The liquid chromatography instrument that this example adopts is the logical permanent P3000A preparative liquid chromatography instrument of Beijing innovation, chromatographic column is C18, this post is diameter 3cm, aperture is the reverse phase silica gel mesolow preparative column of 120A, its filler is the octadecylsilane chemically bonded silica of 50um, defining chromatographic detection wavelength is 230nm, and elution flow rate is 30ml/min, and applied sample amount is 2 grams.It is as shown in the table for condition of gradient elution.
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 60～30 | 40～70 |
In table, A, mutually for TFA volumetric concentration is 0.1% solution, makes by the following method: the TFA of 1ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 70% from 60% volumetric concentration that becomes 30%, B phase from 40%.
After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
3. desalination: the salable product desalination that adopts preparative liquid chromatography instrument that purifying is obtained, liquid chromatography instrument is the logical permanent LC6000 preparative liquid chromatography instrument of Beijing innovation, chromatographic column is C18, this post is diameter 3cm, aperture is the reverse phase silica gel mesolow preparative column of 120A, the octadecylsilane chemically bonded silica that its filler is 50um, and defining chromatographic detection wavelength is 230nm, elution flow rate is 30ml/min, and applied sample amount is 1.72 grams.Elution requirement is as shown in the table.
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～15 | 80 (50mmol ammonium acetates) | 20 (acetonitriles) |
| 15～25 | 80 (pure water) | 20 (acetonitriles) |
| 25～40 | 20 (pure water) | 80 (acetonitriles) |
In table, A is respectively ammonium acetate and pure water mutually.50mmol ammonium acetate solution makes by the following method: take 3.85 grams of ammonium acetate solids, add pure water dissolving and be settled to 1L, obtain 50mmol ammonium acetate solution.B is acetonitrile mutually
The main peak of collecting sample, is incorporated in receiving flask.
4. concentrated: qualified solution collected after desalination is at once through concentrating under reduced pressure place to go acetonitrile and part water.Concentrated condition is: 35 DEG C of temperature, and below vacuum tightness 10mbar.Concentrated solution leaves in glass culture dish, prepares freeze-drying;
5. freeze-drying: adopt vacuum freeze drier freeze-drying.The solution that upper step has been concentrated is put into freeze drier lyophilize and is obtained white powder, i.e. get Ka Feizuo meter.Concrete freeze-drying process is as follows:
1) pre-freeze: concentrated solution is placed on freeze drying box internal partition and carries out pre-freeze, and temperature drops to below-40 DEG C, maintains 2 hours.
2) sublimation drying: slowly heat by the heating system under dividing plate, temperature rises to-25 DEG C of left and right, maintains approximately 10 hours, and then temperature rises to 0 DEG C of left and right, maintains 10 hours.
3) desorption: temperature rises to 30 DEG C of left and right, maintains 6 hours, takes out, and weighs, and purification result is as follows:
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 2.00 gram | 1.82 gram | 1.72 gram | 99.4％ | 94.6％ |
The purifying of embodiment 19 Ka Feizuo meter
This example is substantially with embodiment 18, different: in this example, the liquid chromatography instrument that purification step adopts is the logical permanent LC6000 preparative liquid chromatography instrument of Beijing innovation, and chromatographic column is C18 mesolow chromatographic column, this post is diameter 5cm, the octadecylsilane chemically bonded silica that its filler is 50um.Setting elution flow rate is 80ml/min, and applied sample amount is 9 grams.Condition of gradient elution is as shown in the table.
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 60～30 | 40～70 |
In table, A, mutually for TFA volumetric concentration is 0.1% solution, makes by the following method: the TFA of 1ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 70% from 60% volumetric concentration that becomes 30%, B phase from 40%.After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
This routine purification result is as follows:
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 9.00 gram | 8.19 gram | 7.61 gram | 99.41％ | 92.9％ |
The purifying of embodiment 20 Ka Feizuo meter
This example is substantially with embodiment 18, different: purifying chromatographic column is diameter 3cm, the reverse phase silica gel high pressure chromatogram preparative column that aperture is 120A, and the octadecylsilane chemically bonded silica that its filler is 10um, definition elution flow rate is 25ml/min, applied sample amount is 2 grams.Condition of gradient elution is as shown in the table:
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 65～40 | 35～60 |
In table, A, mutually for TFA volumetric concentration is 0.2% solution, makes by the following method: the TFA of 2ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 60% from 65% volumetric concentration that becomes 40%, B phase from 35%.
After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
This routine purification result is as follows:
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 2.00 gram | 1.82 gram | 1.70 gram | 99.70％ | 93.4％ |
The purifying of embodiment 21 Ka Feizuo meter
This example is substantially with embodiment 18, different: different: purifying chromatographic column is C18, this post is diameter 3cm, aperture is the reverse phase silica gel mesolow preparative column of 120A, its filler is the octadecylsilane chemically bonded silica of 50um, defining chromatographic detection wavelength is 230nm, and elution flow rate is 30ml/min, and applied sample amount is 2 grams.Condition of gradient elution is as shown in the table:
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 65～40 | 35～60 |
In table, A, mutually for TFA volumetric concentration is 0.2% solution, makes by the following method: the TFA of 2ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 60% from 65% volumetric concentration that becomes 40%, B phase from 35%.
After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
This routine purification result is as follows:
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 2.00 gram | 1.82 gram | 1.69 gram | 99.6％ | 93.2％ |
The purifying of embodiment 22 Ka Feizuo meter
This example is substantially with embodiment 18, different: in this example, the liquid chromatography instrument that purification step adopts is the logical permanent LC6000 preparative liquid chromatography instrument of Beijing innovation, and chromatographic column is C18 mesolow chromatographic column, this post is diameter 5cm, the octadecylsilane chemically bonded silica that its filler is 50um.Setting elution flow rate is 80ml/min, and applied sample amount is 10 grams.Condition of gradient elution is as shown in the table:
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 65～40 | 35～60 |
In table, A, mutually for TFA volumetric concentration is 0.2% solution, makes by the following method: the TFA of 2ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 60% from 65% volumetric concentration that becomes 40%, B phase from 35%.
After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 10.00 grams | 9.1 gram | 8.64 gram | 99.5％ | 95.0％ |
The purifying of embodiment 23 Ka Feizuo meter
This example is substantially with embodiment 18, different: in this example, the liquid chromatography instrument that purification step adopts is the logical permanent LC6000 preparative liquid chromatography instrument of Beijing innovation, and chromatographic column is C8 mesolow chromatographic column, this post is diameter 5cm, the octadecylsilane chemically bonded silica that its filler is 50um.Setting elution flow rate is 80ml/min, and applied sample amount is 9 grams.Condition of gradient elution is as shown in the table:
| Time (min) | A phase (%, V/V) | B phase (%, V/V) |
| 0～40 | 64～42 | 36～58 |
In table, A, mutually for TFA volumetric concentration is 0.2% solution, makes by the following method: the TFA of 2ml is added in the water of 950ml, be settled to 1L, obtain TFA solution; B is acetonitrile mutually.The volumetric concentration of A phase becomes 58% from 64% volumetric concentration that becomes 42%, B phase from 36%.
After being purified into peak, press the receiving flask of the number of finishing, timely and appropriate discovery is collected sample, then according to HPLC analyzing and testing, and by peak before, summit, sample also respectively behind peak.Its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges.Purity being less than to 99% sample carries out repeatedly purifying by above purification step and obtains salable product.
The HPLC spectrogram of this routine gained Ka Feizuo meter is referring to Fig. 5.
This routine purification result is as follows:
| Product release amount | Theoretical yield | Actual output | Product purity | Yield |
| 9.00 gram | 8.19 gram | 7.70 gram | 99.8％ | 94.1％ |
Above the present invention is described in detail; the explanation of embodiment is just for helping to understand method of the present invention and core concept thereof; its object is to allow the personage who is familiar with this art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (14)
1. a resin type Ka Feizuo meter intermediate, is characterized in that: it is made up of resin solid phase carrier and the Ka Feizuo meter tetrapeptide intermediate being coupled on described resin solid phase carrier, and described Ka Feizuo meter tetrapeptide intermediate has the structure shown in formula I:
2. a preparation method for resin type Ka Feizuo meter intermediate claimed in claim 1, is characterized in that: comprise the steps:
(1), make resin solid phase carrier activator exist under coupling Fmoc-Phe-OH obtain Fmoc-Phe-resin, described resin solid phase carrier has the functional group that can react with the carboxyl of described Fmoc-Phe-OH;
(2), pass through solid-phase synthesis; make described Fmoc-Phe-resin coupling Fmoc-Leu-OH successively; Fmoc-HoPhe-OH and 4-morpholine acetic acid; obtain described resin type Ka Feizuo meter intermediate; described in wherein making, be coupled under the existence of coupling agent system and carry out; described coupling agent system comprises condensing agent and reaction solvent, before carrying out described coupling, first makes resin remove Fmoc protecting group.
3. the preparation method of resin type Ka Feizuo meter intermediate according to claim 2, is characterized in that: described resin solid phase carrier is one or more the combination being selected from trityl chloride type of resin and hydroxyl type of resin.
4. the preparation method of resin type Ka Feizuo meter intermediate according to claim 3, is characterized in that: described trityl chloride type of resin comprises trityl chloride resin, 4-methyl trityl chloride resin, 4-methoxyl group trityl chloride resin and 2-chlorine trityl chloride resin; Described hydroxyl type of resin comprises king's resin and to hydroxymethyl phenoxy methyl polystyrene resin.
5. according to the preparation method of the resin type Ka Feizuo meter intermediate described in claim 3 or 4, it is characterized in that: described resin solid phase carrier is trityl chloride type of resin, described activator is NMM, DIEA or the combination of the two; Or described resin solid phase carrier is hydroxyl type of resin, and described activator is made up of DIC, HOBt and DMAP.
6. the preparation method of resin type Ka Feizuo meter intermediate according to claim 2, is characterized in that: in step (1), the substitution value of described Fmoc-Phe-resin is 0.2～1.2mmol/g.
7. the preparation method of resin type Ka Feizuo meter intermediate according to claim 2; it is characterized in that: in step (2); the method that makes resin remove Fmoc protecting group is: in resin, add the solution being made up of according to volume ratio 1:3～5 piperidines and DMF, stir.
8. the preparation method of resin type Ka Feizuo meter intermediate according to claim 2, is characterized in that: in step (2), described condensing agent is selected from: the combination of DIC and HOBt; HBTU, HCTU, PyBOP one of them or three's arbitrary combination and the combination of HoBt and DIEA, NMM one of them or the two; The combination of the combination of HATU, PyAOP one of them or the two and HOAt and DIEA, NMM one of them or the two.
9. according to the preparation method of the resin type Ka Feizuo meter intermediate described in claim 2 or 8, it is characterized in that: in step (2), described reaction solvent is selected from DMF, DCM, NMP, DMSO or the arbitrary combination between them.
10. the preparation method of resin type Ka Feizuo meter intermediate according to claim 2, it is characterized in that: in step (2), coupling is carried out at ambient temperature, described Fmoc-Leu-OH, Fmoc-HoPhe-OH, the molar ratio of 4-morpholine acetic acid and their coupling resins is 1.2～6:1.
The preparation method of 11. Yi Zhong Ka Feizuo meter tetrapeptide intermediates, described Ka Feizuo meter tetrapeptide intermediate has the structure shown in formula I:
it is characterized in that: described method is by making resin type Ka Feizuo meter intermediate claimed in claim 1, under cracking agent exists, cracking occur, obtain described Ka Feizuo meter tetrapeptide intermediate, described cracking agent is TFA or TFE or their combination, or be TFA or TFE or the combination of the two and the mixing of DCM, cracking agent is 4～15ml/g with the volume mass ratio of described resin type Ka Feizuo meter intermediate.
The preparation method of 12. Ka Feizuo meter tetrapeptide intermediates according to claim 11, is characterized in that: described method also comprises takes the preparation method of the resin type Ka Feizuo meter intermediate described in any one claim in claim 2-10 to prepare described resin type Ka Feizuo meter intermediate.
The preparation method of 13. Yi Zhong Ka Feizuo meter, it comprises makes Ka Feizuo meter tetrapeptide intermediate and intermediate II
iI reaction generates the step of Ka Feizuo meter, it is characterized in that: described preparation method also comprises that the method for taking described in claim 11 or 12 prepares the step of described Ka Feizuo meter tetrapeptide intermediate.
The preparation method of 14. Ka Feizuo meter according to claim 13, it is characterized in that: described preparation method also comprises that Ka Feizuo meter tetrapeptide intermediate is reacted to the Ka Feizuo meter crude product generating with intermediate II carry out the step of purifying, and described purifying comprises following operation:
1. dissolve Ka Feizuo meter crude product with DMSO or DMF, dissolved filtration, retain filtrate;
2. adopt preparative liquid chromatography instrument by the separation and purification of Ka Feizuo meter crude product, described preparative liquid chromatography instrument adopts C18 or C8 chromatographic column, defining chromatographic detection wavelength is 230nm, and elution flow rate is 20～90ml/min, and condition of gradient elution is: time 0～40min; A phase volume ratio concentration becomes 30%～45%, B phase volume ratio concentration from 60%～65% and becomes from 40%～35% the TFA aqueous solution that 70%～55%, A is 0.1wt%～0.2wt% mutually, B is acetonitrile mutually, after being purified into peak, press the receiving flask of the number of finishing, collect sample, then according to HPLC analyzing and testing, press before peak, summit, behind peak respectively and sample, its moderate purity is greater than 99% for salable product, collects and merges; The sample that purity is less than 99%, collects and merges, and purity is less than to 99% sample and carries out repeatedly purifying by above purification step and obtain salable product;
3. adopt the salable product that 2. preparative liquid chromatography instrument obtains operation to carry out desalination, described preparative liquid chromatography instrument adopts C18 or C8 chromatographic column, its filler is the alkyl linked silica gel of 10～50um, defining chromatographic detection wavelength is 230nm, elution flow rate is 20～90ml/min, and elution requirement is as follows:
Time 0～15min, A phase volume ratio concentration is between 75%～85%, and B phase volume ratio concentration is between 15%～25%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is ammonium acetate mutually, and B is acetonitrile mutually;
Time 16～25min, A phase volume ratio concentration is between 75%～85%, and B phase volume ratio concentration is between 15%～25%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is pure water mutually, and B is acetonitrile mutually;
Time 26～40min, A phase volume ratio concentration is between 15%～25%, and B phase volume ratio concentration is between 75%～85%, and A phase volume ratio and B phase volume ratio sum are that 100%, A is pure water mutually, and B is acetonitrile mutually; After being purified into peak, collect main peak, be incorporated in receiving flask;
4. remove 3. acetonitrile and the part water in gained solution of operation by concentrating under reduced pressure, adopt afterwards vacuum freeze drier freeze-drying to obtain white powder and be Ka Feizuo meter finished product.
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| CN104557793A (en) * | 2015-01-08 | 2015-04-29 | 苏州齐力远医药科技有限公司 | Synthetic method of carfilzomib intermediate and carfilzomib intermediate |
| CN107402259A (en) * | 2016-05-18 | 2017-11-28 | 石药集团中奇制药技术（石家庄）有限公司 | The detection method of chiral isomer in Carfilzomib |
| CN110759967A (en) * | 2019-09-05 | 2020-02-07 | 雅本化学股份有限公司 | Preparation method of carfilzomib |
| CN111646958A (en) * | 2020-08-06 | 2020-09-11 | 上海翰森生物医药科技有限公司 | Preparation method of carfilzomib |
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| WO2013169282A1 (en) * | 2012-05-08 | 2013-11-14 | Onyx Therapeutics, Inc. | Cylodextrin complexation methods for formulating peptide proteasome inhibitors |
| CN104710507A (en) * | 2013-12-11 | 2015-06-17 | 深圳翰宇药业股份有限公司 | Preparation method of carfilzomib |
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| CN104402973A (en) * | 2014-11-24 | 2015-03-11 | 重庆泰濠制药有限公司 | Method for preparing carfilzomib amorphous crystal |
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| CN107402259B (en) * | 2016-05-18 | 2020-10-13 | 石药集团中奇制药技术（石家庄）有限公司 | Method for detecting chiral isomer in carfilzomib |
| CN110759967A (en) * | 2019-09-05 | 2020-02-07 | 雅本化学股份有限公司 | Preparation method of carfilzomib |
| CN111646958A (en) * | 2020-08-06 | 2020-09-11 | 上海翰森生物医药科技有限公司 | Preparation method of carfilzomib |
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