CN103145810B - A kind of method preparing MFG - Google Patents
A kind of method preparing MFG Download PDFInfo
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- CN103145810B CN103145810B CN201210558336.0A CN201210558336A CN103145810B CN 103145810 B CN103145810 B CN 103145810B CN 201210558336 A CN201210558336 A CN 201210558336A CN 103145810 B CN103145810 B CN 103145810B
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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 present invention relates to the solid phase-liquid phase total synthesis method preparing MFG; described method comprises with Fmoc-P-CTC resin for carrier; solid-phase peptide synthesis is adopted to hold N to hold amino acid with Fmoc protecting group coupling one by one from C; again by cracking, intramolecular reaction, liquid phase condensations, deprotection base obtains MFG.High and the convenient post-treatment of method yield of the present invention, the industrial-scale production for MFG provides a kind of new thinking.
Description
Technical field
The present invention relates to a kind of method preparing echinocandin antifungal agent thing, be specifically related to a kind of method preparing MFG.
Background technology
Echinocandin class medicine is novel semi-synthetic antifungal drug, and it, by suppressing activity of β-1,3-glucan synthase thus the synthesis of Antifungi cell walls, has hypotoxicity and good kinetic property.MFG (Micafungin) wherein a kind ofly shows good echinocandin antifungal agent thing, developed by Japanese Teng Ze company, go on the market in December, 2002 in Japan, commodity are called Fungusrd, in March, 2005, by U.S. FDA certification, is approved for the prevention and therapy for the treatment of esophageal candidiasis, bone marrow transplantation and ADS patient's neutrophilic granulocytopenia at present.
The structure of MFG is as follows:
At present; MFG is prepared mainly through semisynthetic method: the acyl group being removed the outer acid amides of ring in echinocandin class microbiotic Coleophomaempetri structure by enzyme reaction, then on unhindered amina, connects side chain (see US5376634, US5569646, WO9611210 and WO9940108) by liquid phase reaction.The shortcoming of the method is dependence echinocandin class microbiotic Coleophomaempetri is raw material basis, needs the support of microbial culture technique.Up to now, still complete synthesis report is not carried out to MFG.
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide a kind of novel method preparing MFG, specifically provide a kind of solid phase-liquid phase total synthesis method of MFG, it comprises the steps:
(1) Fmoc-P-CTC resin is obtained by Fmoc-P-OH and 2-CTC resin reaction,
The alpha-non-natural amino acid with protecting group that wherein Fmoc-P-OH representative structure is following:
(2) with Fmoc-P-CTC resin for carrier, solid-phase peptide synthesis is adopted to hold N to hold amino acid with Fmoc protecting group coupling one by one from C, obtain full guard peptide resin, the amino acid of Fmoc protecting group is wherein with to be followed successively by (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline, (3R)-N-Fmoc-3-acetoxyl group-L-glutaminate, (4S)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group) phenyl-L-threonine, (4R)-N-Fmoc-4-acetoxyl group-L-PROLINE and (4R)-N-Fmoc-O-ethanoyl-L-threonine,
(3) cracking is carried out to the full guard peptide resin in step (2), obtain full guard peptide;
(4) intramolecular reaction is carried out to the full guard peptide in step (3), obtain full guard cyclic peptide;
(5) the Boc protecting group in full guard cyclic peptide on nitrogen in step (4) is removed;
(6) product of step (5) and side chain P1 are carried out liquid phase condensations, obtain the MFG being with protecting group, wherein the structure of P1 is:
(7) protecting group removed in the product of step (6) on hydroxyl obtains MFG;
The reaction of step of the present invention (1) is carried out in the basic conditions.Described alkali is DIPEA or TMP, preferred DIPEA.The substitution degree of CTC resin is 0.2-1.1mmol/g, preferred 0.4-1.0mmol/g, more preferably 0.7mmol/g.
Fmoc-P-OH in step of the present invention (1) is from known compd B oc-4-alkene-L-bird ammonia lactan (JournalofOrganometallicChemistry691 (2006) 5487 – 5496 of document; experimental section 4.3.3 compound 24) set out, by the well-known asymmetric dihydroxylation in organic synthesis field, lactan hydrolysis and the method preparation adding Acetyl Protecting Groups.Wherein lactan hydrolysis reaction and add the method for Acetyl Protecting Groups can the method that can realize this object that optionally prior art is known; The preferred Sharpless asymmetric dihydroxylation of asymmetric dihydroxylation.
In step of the present invention (2), described solid-phase peptide synthesis comprises: 1) remove Fmoc, then uses solvent wash resin, till detecting by detection method and removing Fmoc completely; 2) by appropriate amount after coupling amino acid and coupling agent dissolve in a solvent and activate, join in solid reaction post together, till reaction terminating being detected by detection method; 3) 1 is repeated) and 2).
The reagent wherein removing Fmoc can be any reagent realizing this object known in the art, preferably piperidines/DMF the solution (DBLK) of 20%, i.e. piperidines: DMF(volume ratio) be the mixing solutions of 1:4.
Coupling agent in step of the present invention (2) is the composition of DIPCDI and compd A or the composition of DIPEA and compd A and compd B, wherein compd A is HOBt or HOAt, compd B is PyBOP, PyAOP, HATU, HBTU or TBTU, is preferably the composition of DIPCDI and compd A.Further, in coupling agent, the ratio of each composition take molar ratio computing as DIPCDI:A=1.2:1.1, DIPEA:A:B=2.0:1.1:1.0.
The reaction of step of the present invention (2) is carried out in solid state reaction post.Solid state reaction post is not particularly limited, can be any solid state reaction post that can realize this object.In addition, the time that every seed amino acid carries out linked reaction is generally 1.5-4 hour, preferred 2-3 hour; Pressure is preferably normal pressure, also can carry out under the pressure suitably improving or reduce; Temperature is preferably room temperature (namely 20 ± 5 DEG C), also can carry out at the temperature suitably improving or reduce.
Resin preferably carries out swelling by the reaction of step of the present invention (2) before coupling, and described washing and swelling step this area can adopt any reagent realizing this object to carry out, preferred DMF.The detection method applied in described reaction is any means realizing this object known in the art; such as chromatography or Chemical Calibration; preferred use can judge the reagent of reaction end; preferred triketohydrindene hydrate; when using triketohydrindene hydrate; if resin develops the color, illustrate in polypeptide have free acid amides, i.e. unprotect base on amide nitrogen.
In step (2), the source of alpha-non-natural amino acid is: and (4S)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group base) phenyl-L-threonine is by synthesis preparation, and all the other alpha-non-natural amino acids are commercially or by the method using upper ethanoyl well-known in the art to protect prepare ethanoyl protection on the amino acid of commercially available unprotect base.Particularly, (2S, 3S, 4S) precursor-(the 2S of-N-Fmoc-3-acetoxyl group-4-methyl-proline, 3S, 4S)-N-Fmoc-4-methyl-3-Hydroxyproline is purchased from J & K, (3R)-N-Fmoc-3-acetoxyl group-L-glutaminate prepares (Synthesis by the method that document is known, (12), 1032-5, 1986, experimental section 1a), (4R) precursor-(4R)-N-Fmoc-L-4-oxyproline of-N-Fmoc-4-acetoxyl group-L-PROLINE is purchased from J & K and the precursor of (4R)-N-Fmoc-O-ethanoyl-L-threonine-(4R)-N-Fmoc-L-Threonine is purchased from gill biochemistry, (4S)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group base) phenyl-L-threonine is from known the compound 3 '-acetoxyl group-4 of document '-benzyloxy-phenyl aldehyde (Org.Biomol.Chem., 2010, 8, 5199 – 5211 experimental section compounds 29) set out, by the hydrolysis reaction of the condensation reaction of aldehyde known in the art and ylide, asymmetric dihydroxylation, lactan and the method preparation adding Acetyl Protecting Groups.Wherein respectively react any means that can realize this object that prior art can be taked known, wherein the preferred Sharpless asymmetric dihydroxylation of asymmetric dihydroxylation.
Step of the present invention (3) is carried out under the existence of lysate.Described lysate is the mixing solutions of AcOH and TFE and DCM, wherein AcOH:TFE:DCM=1:1:8(volume ratio).
Step of the present invention (4) is carried out under the existence of coupling agent.The range of choice of described coupling agent is identical with the coupling agent range of choice in step mentioned above (2), is preferably the composition of DIPEA and PyBOP and HOAt, further, and DIPEA:HOAt:PyBOP=2.0:1.1:1.0(mol ratio); Use detection method known in the art to judge the reaction end of this step, preferably use tlc.
In step of the present invention (5), remove the mixing solutions that Boc protecting group agents useful for same is TFA or HCl and PhOMe or DCM, the mixing solutions of preferred TFA and PhOMe, further, TFA:PhOMe=97:3(volume ratio).
The outer amide condensed reaction of ring of step of the present invention (6) carries out under the existence of coupling agent.Preferred DIPEA and HOAt of described coupling agent; Use detection method known in the art to judge the reaction end of this step, preferably use tlc.In this reaction, side chain P1 prepares (see OrganicProcessResearch & Development2005,9,179-184) according to the method that document is known.
In step of the present invention (7), the reaction of deprotection base can be carried out in the basic conditions.Described alkaline condition is NaOMe and MeOH, NaOEt and MeOH, NaOMe and EtOH, NaOEt and EtOH, preferred NaOMe and MeOH, and the content of NaOMe and MeOH is every 100mlMeOH containing 15-25gNaOMe, preferably containing 16-19gNaOMe, more preferably containing 17gNaOMe.
The present invention preferably also comprises the purification step of MFG.Described purification step can adopt any polypeptide purification techniques known in the art to carry out, and preferably adopts reversed-phase high pressure liquid chromatography.Further, described reversed-phase high pressure liquid chromatography comprises: with anti-phase octadecylsilane for stationary phase, with 0.1% aqueous acetic acid/acetonitrile for moving phase, collects object peak cut, concentrated freeze-dried.
Terminological interpretation
Term " solid-phase peptide synthesis " in the whole text refers to the Fmoc solid phase synthesis process that Peptides Synthesis is known herein, such as be recorded in FmocSolidPhasePeptideSynthesis:APracticalApproach, W.C.Chan, PeterD.White work, March2,2000(ISBN-10:0199637245), Britain OxfordUniversityPress.
Term " full guard peptide resin " in the whole text refers to that the C end of polypeptide is connected with resin, N end is free amine group herein; The polypeptide resin that all the other free hydroxyl groups are protected by Boc by ethanoyl protection, free amine group.
Term " full guard peptide " in the whole text refers to that above-mentioned " the full guard peptide resin " mentioned removes C and hold resin to expose the polypeptide of free carboxy herein
Term " full guard cyclic peptide " in the whole text refers to and is connected with N Amino End Group by the C end carboxyl of above-mentioned " the full guard peptide " mentioned herein, to make in polypeptide containing free substituent circular polypeptides.
Compared with prior art, the present invention prepares the high and convenient post-treatment of the method yield of MFG, and the industrial-scale production for MFG provides a kind of new thinking.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of MFG preparation method of the present invention.
Embodiment
In order to understand the present invention further, below in conjunction with specific embodiment, the present invention is described in detail, should be understood that following embodiment is intended to illustrate, be not construed as limiting the present invention.
In the present invention, abbreviation used and implication thereof are listed in the table below:
Embodiment 1:Fmoc-P-OH alpha-non-natural amino acid is by following three step synthesis:
(1) by Sharpless asymmetric dihydroxylation synthesis o-dihydroxy.By 70g (217mmol) K
3fe (CN)
6, 29.3g (217mmol) K
2cO
3, 53.2mg (0.14mmol) K
2osO
2(OH)
4with 0.56g (0.7mmol) hydroquinidine Isosorbide-5-Nitrae-(2，3-benzodiazine) diether ((DHQD)
2-PHAL) be dissolved in the 350ml trimethyl carbinol and 350ml water, stirring at room temperature 10 minutes.Add 6.7g (70mmol) p-sulfonamidobenzoic acid, under 0 ° of C, by the Boc-4-alkene-L-bird ammonia lactan (JournalofOrganometallicChemistry691 (2006) prepared according to the method for known references, 5487 – 5496, experimental section 4.3.3, compound 24) 14.9g (70mmol) joins in system, keeps 0 ° of C to react 40h.Add 105g (833mmol) S-WAT subsequently, naturally rise to stirring at room temperature and react 1 hour and add ethyl acetate 700ml.Aqueous layer with ethyl acetate extraction (3 × 200ml).Merge organic phase and wash twice with the saturated common salt containing 2mol/LNaOH.After anhydrous sodium sulfate drying 2h, concentrating under reduced pressure is except desolventizing.Gained crude product, through Flash silica column chromatography (EA/PE2:1) purifying, obtains (1S, 3R, 4R)-Boc-3,4-dihydroxyl-bird ammonia lactan 15.4g (62.3mmol, yield 89%) of white solid;
1hNMR (500MHz, CDCl
3) δ: 5.41 (m, 1H), 5.07 (br, 2H), 4.53 (m, 1H), 3.98 (m, 1H), 2.09 ~ 1.96 (m, 4H), 1.39 (s, 9H);
13cNMR (125MHz, CDCl
3) δ: 168.44 (C=O), 156.33 (C=O), 81.64 (C5); 79.44 (Boc), 75.94 (C4), 52.06 (C2); 35.35 (C3), 28.36 (Boc); FAB-MS [M+H]
+: 246.3, calculated value 246.3.
(2) lactan is hydrolyzed and upper Fmoc protecting group.By (1S, 3R, 4R)-Boc-3,4-dihydroxyl-bird ammonia lactan 15.4g (62.3mmol), 1.58g (8.4mmol) tosic acid reflux 16h in 20ml acetone and 500ml toluene.After reaction solution cooling, extract (2 × 300ml), brine It twice with 10% aqueous sodium carbonate.Gained solid is dissolved in 200mlTHF and 100ml water after revolving and desolventizing by decompression, adds 13g(310mmol) lithium hydroxide, stirring at room temperature 16h.Under ice cooling, 4, add the solution of 32g (216mmol) sodium pyrosulfate in 100ml water, finish stirring 10 minutes.21g (62mmol) fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) is joined in reaction system, naturally rises to room temperature reaction 5 hours.React complete, in system, add saturated aqueous citric acid solution 60ml, stirring at room temperature 16h.Decompression is revolved except THF, is extracted with ethyl acetate (3 × 200ml), merges organic phase, with salt washing twice.After anhydrous sodium sulfate drying 2h, concentrating under reduced pressure is except desolventizing.Gained crude product, through Flash silica column chromatography (EA/PE2:1) purifying, obtains white solid (1S, 3R, 4R)-N-Boc-3,4-dihydroxyl (N-Fmoc) ornithine 22.1g (45.5mmol, yield 73%);
1hNMR (500MHz, CDCl
3) δ: 10.78 (br, 1H), 7.91 ~ 7.25 (m, 8H); 5.41 (m, 1H), 5.07 (br, 2H); 4.70 (d, 2H), 4.53 (m; 2H), 3.98 (m, 1H); 2.02 (s, 2H), 1.96 (m; 2H), 1.39 (s, 9H);
13cNMR (125MHz, CDCl
3) δ: 174.44 (C=O); 156.33 (C=O); 155.25 (C=O); 143.81 (Fmoc); 141.03 (Fmoc), 128.81 (Fmoc), 128.43 (Fmoc); 128.22 (Fmoc); 126.83 (Fmoc), 81.64 (C5), 79.44 (Boc); 72.94 (C4); 67.45 (Fmoc), 50.36 (C2), 47.10 (Fmoc); 33.35 (C3), 28.36 (Boc); FAB-MS [M+H]
+: 486.4, calculated value 486.5.
(3) ethanoyl protection on hydroxyl: 22.1g (45.5mmol) (1S, 3R, 4R)-N-Boc-3,4-dihydroxyl (N-Fmoc) ornithine is dissolved in 400mlDCM.Add 43ml (455mmol) diacetyl oxide and 36ml (450mmol) pyridine, stirring at room temperature reaction 2h.In reaction mixture, add 400ml water, stir 10 minutes, aqueous phase DCM extraction (2 × 200ml).Merge organic phase, respectively wash twice with 10% citric acid water, saturated aqueous common salt.After anhydrous sodium sulfate drying 2h, concentrating under reduced pressure is except desolventizing.Gained crude product, through Flash silica column chromatography (EA/PE1:1) purifying, obtains title compound 24.1g (42.3mmol, yield 93%, total recovery 60%).
1hNMR (500MHz, CDCl
3) δ: 10.78 (br, 1H), 7.91 ~ 7.25 (m, 8H); 6.63 (m, 1H), 5.17 (m, 1H); 5.07 (br, 2H), 4.70 (d; 2H), 4.53 (m, 2H); 2.02 (s, 6H), 1.96 (m; 2H), 1.39 (s, 4H);
13cNMR (125MHz, CDCl
3) δ: 174.44 (C=O); 170.29 (C=OofAc); 156.33 (C=O); 155.25 (C=O); 143.81 (Fmoc); 141.03 (Fmoc), 128.81 (Fmoc), 128.43 (Fmoc); 128.22 (Fmoc); 126.83 (Fmoc), 81.45 (C5), 79.44 (Boc); 72.75 (C4); 67.45 (Fmoc), 50.36 (C2), 47.10 (Fmoc); 31.23 (C3); 28.36 (Boc), 21.11 (Ac), 20.78 (Ac); FAB-MS [M+H]
+: 570.7, calculated value 570.6.
The synthesis of embodiment 2:Fmoc-P-CTC resin
Take the 2-CTC resin 20.0g that substitution degree is 0.7mmol/g, join in solid state reaction post, wash 2 times with DMF.With DMF swellable resins after 30 minutes, get 24.0gFmoc-P-OH DMF and dissolve, add 14.6mLDIPEA under ice-water bath and activate 3 minutes, then join and be above-mentionedly equipped with in the reaction column of resin, react after 2 hours, add 20mL anhydrous methanol and close 1 hour.Wash 3 times with DMF, DCM washes 3 times, closes 30 minutes with anhydrous methanol, and methyl alcohol shrinks to be drained, and obtain Fmoc-P-CTC resin, detection substitution degree is 0.56mmol/g.
Embodiment 3:(4S) synthesis of-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group) phenyl-L-threonine:
By 50g (185mmol) 3 '-acetoxyl group-4 '-benzyloxy-phenyl aldehyde (Org.Biomol.Chem., 2010,8,5199 – 5211 experimental section compounds 29) be dissolved in 250ml and newly steam in DCM, add 101.4g (185mmol) phosphonium ylide, stirring at room temperature reacts 4 hours, and reaction solution, through silica gel flash column chromatography, obtains white solid 80.1g.This solid is dissolved in 300ml newly steam in DCM, add the solution of 15g (277mmol) sodium methylate in 2300ml anhydrous methanol, after stirring at room temperature reacts 30 minutes, add the acidifying of 1500ml1mol/L hydrochloric acid soln, revolve except organic solvent, remaining liq DCM extraction (3 × 600ml).Merge organic phase, with saturated common salt washing twice, concentrating under reduced pressure is except desolventizing after with anhydrous sodium sulfate drying 2h.By residue and 152.3g (302mmol) 2; 3; 4-triacetyl-D-Glucopyranose methoxycarbonyl-(N-phenyl)-2; 2,2-trifluoroacetyl imines ester (J.Org.Chem., 2005; 70; compound 4 in 8884) be dissolved in 700ml and newly steam in DCM, add 6.38ml (51mmol) etherate of trifluoroboron, room temperature reaction 16 hours.In reaction solution, add 1000ml water, extraction, water layer is again with DCM extraction (2 × 300ml).Merge organic layer, with saturated common salt washing twice.Organic layer 500ml dissolve with methanol, adds the 500ml1mol/LNaOH aqueous solution, and stirring at room temperature reacts 4 hours.Gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o1:1) purifying.Obtain the 57.6g(108mmol of white solid (S)-N-Cbz-alpha-amino group-3-alkene-4-(3 '-sodium sulfonate-4 '-benzyloxy) phenylbutyric acid, two step yields 69%);
1hNMR (500MHz, DMSO-d
6) δ: 7.19 (m, 10H), 6.75 (d, 1H), 6.65 (d, 1H); (6.53 m, 2H), 6.19 (t, 1H), 5.34 (s; 2H), 5.20 (s, 2H), 5.15 (m, 1H);
13cNMR (125MHz, DMSO-d
6) δ: 174.44 (C=O), 156.33 (NH-C=O), 150.55 (C-OBn), 142.81 (C-OSO
3na), (141.13 Bn & Cbz), 129.81 (C4), 128.93 (Bn & Cbz), 128.82 (Ph), (127.63 Bn & Cbz), 127.13 (Bn & Cbz), 123.31 (C3), 119.98 (Ph), 115.23 (Ph), 113.16 (Ph), 70.75 (CH
2ofBn), 65.45 (CH
2ofCbz), 61.36 (C2).
By 108g (335mmol) K
3fe (CN)
6, 45.2g (335mmol) K
2cO
3, 83.6mg (0.22mmol) K
2osO
2(OH)
4with 0.88g (1.1mmol) hydroquinidine Isosorbide-5-Nitrae-(2，3-benzodiazine) diether ((DHQD)
2-PHAL) be dissolved in the 500ml trimethyl carbinol and 500ml water, stirring at room temperature 10 minutes.Add 10.3g (108mmol) p-sulfonamidobenzoic acid, under 0 ° of C, upper step gained intermediate 57.6g (108mmol) is added in system, keep 0 ° of C to react 40h.Add 157g (1.245mol) S-WAT, naturally rise to stirring at room temperature and react 1 hour.Gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o1:1) purifying.Obtain white solid 41.2g (72.4mmol, yield 67%).By this solid 500 methyl alcohol and 500ml water dissolution, add 5g5% palladium carbon, logical hydrogen hydrogenation 16 hours in autoclave pressure.Reaction terminates rear filtering palladium carbon.Filtrate adds 27.1g (80mmol) fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) and 7.6g (90mmol) sodium bicarbonate, stirring at room temperature reaction 5h.Gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o1:1) purifying.Obtain white solid (4S)-N-Fmoc-4-hydroxyl-4-(3 '-sodium sulfonate-4 '-hydroxyl) phenyl-L-threonine 37.8g (66.6mmol, two step yields 92%);
1hNMR (500MHz, DMSO-d
6) δ: 8.37 (s, 1H), 7.89-7.32 (m, 8H), 6.57 (d, 1H); (6.49 d, 1H), 6.43 (s, 1H), 4.70 (d; 2H), 4.63 ~ 4.46 (m, 4H), 2.02 (br, 2H);
13cNMR (125MHz, DMSO-d
6) δ: 174.83 (C=O), 156.06 (NH-C=O), 144.68 (Ph (C)-OH), 143.76 (C-OSO
3na), 143.53 (Fmoc), 141.03 (Fmoc), 132.61 (Ph), 128.83 (Fmoc), 128.43 (Fmoc), 128.22 (Fmoc), 126.63 (Fmoc), 122.23 (Ph), 117.38 (Ph), 115.23 (Ph), 77.75 (C3), 73.83 (C4), 67.36 (CH
2ofFmoc), 53.82 (C2), 47.0 (CHofFmoc).
Above-mentioned solid is dissolved in 400mlDMF.Add 43ml (455mmol) diacetyl oxide and 36ml (450mmol) pyridine, stirring at room temperature reaction 2h.Reaction solution concentrating under reduced pressure is except desolventizing.Gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o1:1) purifying.Obtain title compound 27.9g (40.2mmol, yield 95%, total recovery 21.7%);
1hNMR (500MHz, DMSO-d
6) δ: 8.37 (s, 1H), 7.89-7.32 (m, 8H); 6.87 (d, 1H), 6.72 (d, 1H); 6.63 (s, 1H), 5.88 (t, 1H); 5.83 (d, 1H), 5.05 (m, 1H); 4.72 (d, 2H), 4.46 (t, 1H); 2.09 (s, 3H), 2.01 (s, 6H);
13cNMR (125MHz, DMSO-d
6) δ: 174.83 (C=O), 170.31 (C=OofAc), 169.02 (C=OofAc), 156.05 (NH-C=O), 149.64 (C-OSO
3na), 143.53 (Fmoc), 141.03 (Fmoc), 137.71 (Ph), 136.83 (Ph), 128.83 (Fmoc), 128.43 (Fmoc), 128.22 (Fmoc), 126.63 (Fmoc), 123.23 (Ph), 121.18 (Ph), 114.23 (Ph), 77.75 (C3), 71.73 (C4), 67.36 (CH
2ofFmoc), 50.81 (C2), 47.0 (CHofFmoc), 21.02 (Ac), 20.05 (Ac).
Embodiment 4: the synthesis of other alpha-non-natural amino acids.From the commercially available amino acid not with protecting group, prepare respectively on hydroxyl with the amino acid that ethanoyl is protected by the method similar with the method that upper ethanoyl in embodiment 3 is protected to embodiment 1.
Embodiment 5: the preparation of full guard peptide resin
Take the Fmoc-P-CTC resin 17.86 grams (synthesis scale 10mmol) that substitution degree is 0.56mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins 30 minutes, add 20% piperidines/DMF(V/V) solution removal Fmoc twice, each time is respectively 5 minutes and 10 minutes, removes complete DMF washing resin 6 times, and triketohydrindene hydrate detects resin color.Take 12.3 grams of (30mmol) (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline, 4.5 grams of (33mmol) HOBt, dissolve with 25mlDMF, add after 6.1ml (36mmol) DIPCDI activates 5 minutes under ice-water bath, mixed solution is joined in reaction column, room temperature reaction 2 hours, with triketohydrindene hydrate detection reaction terminal (as resin water white transparency then termination reaction; As resin colour developing then extends reaction 1 hour, lower same).
Reaction terminates, and with DMF washing resin 3 times, add DBLK solution removal Fmoc twice, each time is respectively 5 minutes and 10 minutes, removes complete DMF washing resin 6 times, and triketohydrindene hydrate detection resin has color.Take 12.8 grams of (30mmol) (3R)-Fmoc-3-acetoxyl group-L-glutaminate, 4.5 grams of (33mmol) HOBt, dissolve with 25mlDMF, add 6.1ml (36mmol) DIPCDI under ice-water bath and activate 5 minutes, mixed solution is joined in reaction column, room temperature reaction 2 hours, with triketohydrindene hydrate detection reaction terminal.
Successively (the 4S)-Fmoc-4-acetoxyl group-4-prepared in advance (3 '-sodium sulfonate-4 '-acetoxyl group) phenyl-L-threonine, (4R)-4-acetoxyl group-L-PROLINE and (4R)-Fmoc-O-ethanoyl-L-threonine are carried out linked reaction after the same method; coupling terminates; remove Fmoc with DBLK and protect resin; contraction is drained; obtain full guard peptide resin 25.99 grams, resin rate of body weight gain 91.9%.
Embodiment 6: the preparation of full guard peptide
Full guard peptide resin embodiment 5 obtained 25.99 grams joins in 500ml single port bottle, adds pre-configured AcOH:TFE:DCM=1:1:8(V:V) 250ml, room temperature reaction 40 minutes, filters resin, collects filtrate.With a small amount of DCM washing resin, merging filtrate.Concentrated solution, to 50ml, slowly adds in 500ml ice ether and precipitates by concentrating under reduced pressure.Centrifugal, ice washed with diethylether 5 times, drying under reduced pressure obtains full guard peptide 12.15 grams, and detecting purity with HPLC is 91.3%.ESI, m/z, ([M+Na]
+) 1477.4(calculated value is 1477.4).
Embodiment 7: liquid phase synthesis full guard cyclic peptide
Full guard peptide embodiment 6 obtained 12.15 grams joins in 250ml single port bottle, adds 160mlDCM stirring and dissolving, then adds 5.2 grams of PyBOP and 1.5 gram HOAt.Ice-water bath to be cooled in system 5 DEG C, drips DIPEA solution (1.74mlDIPEA+10mlDCM), drips and finishes, remove ice-water bath, naturally rise to stirring at room temperature and react 3 hours, and thin-layer chromatography monitors reaction end.Extract 2 times with 150ml10% citric acid solution, saturated aqueous common salt extracts 3 times, organic phase anhydrous sodium sulfate drying 2 hours.Filter, spin off DCM, obtain obtaining full guard cyclic peptide 11.81 grams, yield 96.6% after reaction terminates.ESI, m/z, ([M+Na]
+) 1436.4(calculated value is 1436.3).
Embodiment 8: remove Boc protecting group
11.81 grams of full guard cyclic peptide embodiment 7 obtained join in 250ml single port bottle, add pre-configured TFA+PhOMe(97ml+3ml), room temperature reaction 2.5 hours.Revolve except TFA, solid 50ml washed with diethylether 3 times, solvent evaporation in vacuo, obtains white solid 10.76g, yield 97.9%.ESI, m/z, ([M+Na]
+) 1359.4(calculated value is 1359.3).
Embodiment 9: the outer acid amides of liquid phase synthesis ring
White solid 10.76g and 1.2gHOAt that embodiment 8 is obtained, join in 250ml single port bottle, add 100mlDCM stirring and dissolving, add side chain P17.5g again, ice-water bath to be cooled in system 5 DEG C, drips DIPEA solution (2.74mlDIPEA+10mlDCM), drip and finish, remove ice-water bath, naturally rise to stirring at room temperature and react 3 hours, thin-layer chromatography monitoring reaction end.Extract 2 times with 150ml10% citric acid solution, saturated aqueous common salt extracts 3 times, organic phase anhydrous sodium sulfate drying 2 hours.Filter, spin off DCM, obtain the outer amide intermediate 10.92 grams of ring, yield 81.3%.ESI, m/z, ([M+Na]
+) 1950.7(calculated value is 1950.6).
Embodiment 10: the preparation of the thick peptide of MFG
By outer for embodiment 9 gained ring amide intermediate 10.92 grams, dissolve with 150mlMeOH, then room temperature drips 150ml28%NaOMe/MeOH solution, dropwise and continue stirring reaction 20 minutes.With vinegar acid for adjusting pH to 8, revolve and desolventize.Solid 50ml deionized water wash 3 times, 50ml washed with diethylether 3 times, collecting solid is crude product MFG.Filtrate merges, and 100mlDCM extracts 3 times, and merge DCM, saturated aqueous common salt extracts 3 times, organic phase anhydrous sodium sulfate drying 2 hours.Filter, spin off DCM, solid be the thick peptide of MFG.The thick peptide of two portions MFG merges totally 7.98 grams, yield 94.4%.ESI, m/z, ([M+Na]
+) 1314.5(calculated value is 1314.4).
Embodiment 11: purifying MFG obtains the pure peptide of MFG through thick peptide
After embodiment 10 gained 7.98g MFG thick peptide 800ml deionized water dissolving, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, with the 0.1%TFA aqueous solution for A phase, acetonitrile is that B phase carries out purifying, collects object peak cut, obtains the smart peptide that purity is greater than 98.5%.Cut merges, spin concentration, and freeze-drying obtains MFG essence peptide 6.93g, HPLC purity 99.0%, total recovery 53%.
Although describe the present invention with reference to particular, but what those skilled in the art will recognize that is, when not departing from purport of the present invention and scope, can change described embodiment or improve, the scope of the invention be limited by appended claims.
Claims (18)
1. prepare a method for MFG, the method comprises the steps:
(1) Fmoc-P-CTC resin is obtained by Fmoc-P-OH and 2-CTC resin reaction,
The alpha-non-natural amino acid with protecting group that wherein Fmoc-P-OH representative structure is following:
(2) with Fmoc-P-CTC resin for carrier, solid-phase peptide synthesis is adopted to hold N to hold amino acid with Fmoc protecting group coupling one by one from C, obtain full guard peptide resin, the amino acid of Fmoc protecting group is wherein with to be followed successively by (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline, (3R)-N-Fmoc-3-acetoxyl group-L-glutaminate, (4S)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group) phenyl-L-threonine, (4R)-N-Fmoc-4-acetoxyl group-L-PROLINE and (4R)-N-Fmoc-O-ethanoyl-L-threonine,
(3) cracking is carried out to the full guard peptide resin in step (2), obtain full guard peptide;
(4) intramolecular reaction is carried out to product in step (3), obtain full guard cyclic peptide;
(5) the Boc protecting group in full guard cyclic peptide on nitrogen in step (4) is removed;
(6) product of step (5) and side chain P1 are carried out liquid phase condensations, obtain the MFG being with protecting group, wherein the structure of P1 is:
(7) protecting group removed in the product of step (6) on hydroxyl obtains MFG.
2. the process of claim 1 wherein that the Fmoc-P-CTC resin in step (1) is prepared by the 2-CTC resin reaction being 0.2-1.1mmol/g by Fmoc-P-OH and substitution degree in the basic conditions; Wherein said alkali is DIPEA or TMP.
3. the method for claim 2, the Fmoc-P-CTC resin wherein in step (1) is prepared by the 2-CTC resin reaction being 0.4-1.0mmol/g by Fmoc-P-OH and substitution degree in the basic conditions.
4. the method for claim 2, the Fmoc-P-CTC resin wherein in step (1) is prepared by the 2-CTC resin reaction being 0.7mmol/g by Fmoc-P-OH and substitution degree in the basic conditions.
5. the method for claim 2, wherein said alkali is DIPEA.
6. the process of claim 1 wherein that Fmoc-P-OH is from compd B oc-4-alkene-L-bird ammonia lactan, by Sharpless asymmetric dihydroxylation, lactan hydrolysis reaction and the method preparation adding Acetyl Protecting Groups.
7. the method for claim 1, wherein step (2) and step (4) are carried out under the existence of coupling agent, described coupling agent is the composition of DIPCDI and compd A or the composition of DIPEA and compd A and compd B, wherein compd A is HOBt or HOAt, and compd B is PyBOP, PyAOP, HATU, HBTU or TBTU.
8. the method for claim 7, the coupling agent wherein in step (2) is the composition of DIPCDI and A, and wherein in coupling agent, the ratio of each composition take molar ratio computing as DIPCDI:A=1.2:1.1; Coupling agent in step (4) is the composition of DIPEA and PyBOP and HOAt, and wherein in coupling agent, the ratio of each composition take molar ratio computing as DIPEA:PyBOP:HOAt=2.0:1.1:1.0.
9. the process of claim 1 wherein that alpha-non-natural amino acid (4S)-N-Fmoc-4-acetoxyl group-4-in step (2) (3 '-sodium sulfonate-4 '-acetoxyl group base) phenyl-L-threonine is from compound 3 '-acetoxyl group-4 '-benzyloxy-phenyl aldehyde is by the condensation reaction of aldehyde and ylide, Sharpless asymmetric dihydroxylation, lactan hydrolysis reaction and the method preparation adding Acetyl Protecting Groups.
10. the process of claim 1 wherein that step (3) is carried out under the existence of lysate, described lysate is the mixing solutions of AcOH and TFE and DCM, and wherein the ratio of each composition take volume basis as AcOH:TFE:DCM=1:1:8.
11. the process of claim 1 wherein in step (5), remove the mixing solutions that Boc protecting group agents useful for same is TFA or HCl and PhOMe or DCM.
12. the process of claim 1 wherein in step (5), and removing Boc protecting group agents useful for same is the mixing solutions of TFA and PhOMe, and wherein the ratio of each composition take volume basis as TFA:PhOMe=97:3.
13. the process of claim 1 wherein that the reaction of the middle deprotection base of step (7) is carried out in the basic conditions, and described alkaline condition is NaOMe and MeOH, NaOEt and MeOH, NaOMe and EtOH, NaOEt and EtOH.
The method of 14. claims 13, wherein said alkaline condition is NaOMe and MeOH, and wherein the content of NaOMe and MeOH is that every 100mlMeOH is containing 15-25gNaOMe.
The method of 15. claims 13, wherein said alkaline condition is NaOMe and MeOH, and wherein the content of NaOMe and MeOH is that every 100mlMeOH is containing 16-19gNaOMe.
The method of 16. claims 13, wherein said alkaline condition is NaOMe and MeOH, and wherein the content of NaOMe and MeOH is that every 100mlMeOH is containing 17gNaOMe.
Method any one of 17. claim 1-16, also comprises the purification step of MFG.
The method of 18. claims 17, wherein purification step adopts reversed-phase high pressure liquid chromatography, comprising: with anti-phase octadecylsilane for stationary phase, with 0.1% aqueous acetic acid/acetonitrile for moving phase, collects object peak cut, concentrated freeze-dried.
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| CN102618604A (en) * | 2011-01-31 | 2012-08-01 | 上海天伟生物制药有限公司 | Preparation method of cyclic lipopeptide compound |
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| CN102618604A (en) * | 2011-01-31 | 2012-08-01 | 上海天伟生物制药有限公司 | Preparation method of cyclic lipopeptide compound |
| WO2012136498A1 (en) * | 2011-04-04 | 2012-10-11 | Xellia Pharmaceuticals Aps | Methods for manufacturing an antifungal agent |
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