CN103145810A - Method for preparing micafungin - Google Patents
Method for preparing micafungin Download PDFInfo
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- CN103145810A CN103145810A CN2012105583360A CN201210558336A CN103145810A CN 103145810 A CN103145810 A CN 103145810A CN 2012105583360 A CN2012105583360 A CN 2012105583360A CN 201210558336 A CN201210558336 A CN 201210558336A CN 103145810 A CN103145810 A CN 103145810A
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Abstract
The invention relates to a solid phase-liquid phase full synthetic method for preparing micafungin. The method comprises that Fmoc-P-CTC resin serves as a carrier, a solid phase polypeptide synthetic method is adopted to gradually couple amino acid with an Fmoc protecting group from the C end to the N end, and then the protecting group is deprived through splitting, intramolecular reaction and liquid phase condensation to obtain the micafungin. The method is high in yield, brings convenience to aftertreatment and provides a new thinking way for industrial scale production of the micafungin.
Description
Technical field
The present invention relates to a kind of method for preparing the echinocandin antifungal agent thing, be specifically related to a kind of method for preparing MFG.
Background technology
The echinocandin class medicine is novel semi-synthetic antifungal drug, and it is by suppressing β-1, thereby synthesizing of the activity Antifungi cell walls of 3-glucan synthase has hypotoxicity and good kinetic property.MFG (Micafungin) is wherein a kind ofly to show good echinocandin antifungal agent thing, by the exploitation of Japanese rattan pool company, in December, 2002, in Japan, go on the market, commodity are called Fungusrd, authenticate in March, 2005 by U.S. FDA, is approved at present prevention and the treatment for the treatment of esophageal candidiasis, bone marrow transplantation and ADS patient's neutrophilic granulocytopenia.
The structure of MFG is as follows:
At present; MFG is mainly by semisynthetic method preparation: remove the acyl group of the outer acid amides of ring in echinocandin class microbiotic Coleophoma empetri structure by enzyme reaction, then connect side chain (referring to US5376634, US5569646, WO9611210 and WO9940108) by liquid phase reaction on unhindered amina.The shortcoming of the method is that dependence echinocandin class microbiotic Coleophomaempetri is the raw material basis, needs the support of microorganism culturing technology.Up to now, still MFG is not carried out to complete synthesis report.
Summary of the invention
For the deficiencies in the prior art, the purpose of this invention is to provide a kind of novel method for preparing MFG, a kind of solid phase of MFG-liquid phase total synthesis method specifically is provided, it comprises the steps:
(1) obtain the Fmoc-P-CTC resin by Fmoc-P-OH and 2-CTC resin reaction,
The following alpha-non-natural amino acid with protecting group of Fmoc-P-OH representative structure wherein:
(2) take the Fmoc-P-CTC resin as carrier, adopt solid-phase peptide synthesis to hold N to hold coupling one by one the amino acid with the Fmoc protecting group from C, obtain the full guard peptide resin, wherein the amino acid with the Fmoc protecting group is followed successively by (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline(Pro), (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) the full guard peptide resin in step (2) is carried out to cracking, obtain the full guard peptide;
(4) the full guard peptide in step (3) is carried out to intramolecular reaction, obtain the full guard cyclic peptide;
(5) remove the Boc protecting group on nitrogen in the middle full guard cyclic peptide of step (4);
(6) product of step (5) and side chain P1 are carried out to the liquid phase condensation, obtain the MFG with protecting group, wherein the structure of P1 is:
(7) remove the protecting group on hydroxyl in the product of step (6) and obtain MFG;
The reaction of step of the present invention (1) is carried out under alkaline condition.Described alkali is DIPEA or TMP, preferably DIPEA.The substitution degree of CTC resin is 0.2-1.1mmol/g, preferably 0.4-1.0mmol/g, more preferably 0.7mmol/g.
Fmoc-P-OH in step of the present invention (1) is the compd B oc-4-alkene known from document-L-bird ammonia lactan (Journal of Organometallic Chemistry 691 (2006) 5487 – 5496; experimental section 4.3.3 compound 24) set out, be hydrolyzed and add the method preparation of ethanoyl protecting group by the well-known asymmetric dihydroxylation in organic synthesis field, lactan.The optional known method that can realize this purpose of prior art of lactan hydrolysis reaction and the method that adds the ethanoyl protecting group wherein; 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 use the solvent wash resin, until detect and remove Fmoc fully by detection method; 2) by appropriate amount until coupling amino acid and coupling agent after dissolving in solvent and activating, join together in the solid reaction post, until reaction terminating detected by detection method; 3) repeat 1) and 2).
The reagent that wherein removes Fmoc can be any reagent of realizing this purpose known in the art, preferably 20% piperidines/DMF solution (DBLK), i.e. piperidines: the DMF(volume ratio) be the mixing solutions of 1:4.
The composition of the composition that the coupling agent in step of the present invention (2) is DIPCDI and compd A or 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 be 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 the solid state reaction post.The solid state reaction post is not particularly limited, can be any solid state reaction post that can realize this purpose.In addition, the time that every seed amino acid carries out linked reaction is generally 1.5-4 hour, preferably 2-3 hour; Pressure is preferably normal pressure, also can under the pressure that suitably improves or reduce, carry out; Temperature is preferably room temperature (20 ± 5 ℃), also can at the temperature that suitably improves or reduce, carry out.
Swelling was preferably carried out resin in the reaction of step of the present invention (2) before coupling, and step this area of described washing and swelling can adopt any reagent of realizing this purpose to carry out, preferably DMF.The detection method of applying in described reaction is any means that realizes this purpose known in the art; for example chromatography or Chemical Calibration; preferably use can be judged the reagent of reaction end; preferred triketohydrindene hydrate; when using triketohydrindene hydrate; if the resin colour developing illustrates in polypeptide that free acid amides is arranged, i.e. unprotect base on amide nitrogen.
In step (2), the source of alpha-non-natural amino acid is: (4S)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group base) phenyl-L-threonine is by synthetic preparation, and all the other alpha-non-natural amino acids are commercially availablely obtain or by the method for protecting with upper ethanoyl well-known in the art prepared by ethanoyl protection on the amino acid of commercially available unprotect base.Particularly, the precursor of (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline(Pro)-(2S, 3S, 4S)-N-Fmoc-4-methyl-3-Hydroxyproline is purchased from J& Method preparation (the Synthesis that K, (3R)-N-Fmoc-3-acetoxyl group-L-glutaminate are known by document; (12); 1032-5; 1986, experimental section 1a), the precursor of (4R)-N-Fmoc-4-acetoxyl group-L-PROLINE-(4R)-N-Fmoc-L-4-oxyproline is purchased from J& The precursor of K and (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) compound 3 ' that phenyl-L-threonine is known from document-acetoxyl group-4 '-benzyloxy-phenyl aldehyde (Org.Biomol.Chem.; 2010; 8; 5199 – 5211 experimental section compounds 29) set out, the hydrolysis reaction of the condensation reaction by aldehyde known in the art and ylide, asymmetric dihydroxylation, lactan and add the method preparation of ethanoyl protecting group.Wherein each reaction can be taked the known any means that can realize this purpose of prior art, wherein the preferred Sharpless asymmetric dihydroxylation of asymmetric dihydroxylation.
Step of the present invention (3) is to carry out under the existence of lysate.The mixing solutions that described lysate is AcOH and TFE and DCM, wherein AcOH:TFE:DCM=1:1:8(volume ratio).
Step of the present invention (4) is to carry 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 the 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, the TFA:PhOMe=97:3(volume ratio).
The outer amide condensed reaction of the ring of step of the present invention (6) is to carry out under the existence of coupling agent.The preferred DIPEA of described coupling agent and HOAt; Use detection method known in the art to judge the reaction end of this step, preferably use tlc.The method preparation that in this reaction, side chain P1 is known according to document is (referring to Organic Process Research & Development 2005,9,179-184).
In step of the present invention (7), the reaction of deprotection base can be carried out under alkaline condition.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 that every 100ml MeOH is containing 15-25g NaOMe, preferably containing 16-19g NaOMe, more preferably containing 17g NaOMe.
The present invention preferably also comprises the purification step of MFG.Described purification step can adopt any peptide purification technology 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: take anti-phase octadecylsilane as stationary phase, take 0.1% aqueous acetic acid/acetonitrile as moving phase, collect purpose peak cut, and concentrated freeze-dried.
Terminological interpretation
This paper term " solid-phase peptide synthesis " in the whole text refers to the Fmoc solid phase synthesis process that Peptides Synthesis is known, for example be recorded in Fmoc Solid Phase Peptide Synthesis:A Practical Approach, W.C.Chan, Peter D.White work, March2,2000(ISBN-10:0199637245), Britain Oxford University Press.
This paper term " full guard peptide resin " in the whole text refers to that the C end of polypeptide is connected with resin, the N end is free amine group; The polypeptide resin that all the other free hydroxyl groups are protected by Boc by ethanoyl protection, free amine group.
This paper term " full guard peptide " in the whole text refers to that above-mentioned " the full guard peptide resin " mentioned removes the polypeptide that C end resin exposes free carboxy
This paper term " full guard cyclic peptide " in the whole text refers to the C of above-mentioned " the full guard peptide " mentioned end carboxyl is connected with the N end is amino, so that do not contain free substituent annular polypeptide in polypeptide.
Compared with prior art, the present invention prepares the high and convenient post-treatment of the method yield of MFG, for the industrial-scale production of MFG provides a kind of new thinking.
The accompanying drawing explanation
The synthetic route chart that Fig. 1 is MFG preparation method of the present invention.
Embodiment
In order further to understand the present invention, below in conjunction with specific embodiment, the present invention is described in detail, should be understood that following embodiment is intended to explanation, is 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 synthetic by following three steps:
(1) by the synthetic o-dihydroxy of Sharpless asymmetric dihydroxylation.By 70g (217mmol) K
3fe (CN)
6, 29.3g (217mmol) K
2cO
3, 53.2mg (0.14mmol) K
2osO
2(OH)
4and 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 10 minutes.Add 6.7g (70mmol) p-sulfonamidobenzoic acid, under 0 ° of C, the Boc-4-alkene that will prepare according to the method for known references-L-bird ammonia lactan (Journal of OrganometallicChemistry 691 (2006), 5487 – 5496, experimental section 4.3.3, compound 24) 14.9g (70mmol) joins in system, keeps 0 ° of C reaction 40h.Add subsequently 105g (833mmol) S-WAT, naturally rise to stirring at room reaction 1 hour and add ethyl acetate 700ml.Water layer is extracted with ethyl acetate (3 * 200ml).Merge organic phase and wash twice with the saturated common salt containing 2mol/L NaOH.After anhydrous sodium sulfate drying 2h, concentrating under reduced pressure is except desolventizing.The gained crude product, through quick silica gel column chromatography (EA/PE 2:1) purifying, obtains (1S, 3R, the 4R)-Boc-3 of white solid, 4-dihydroxyl-bird ammonia lactan 15.4g (62.3mmol, yield 89%);
1h NMR (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);
13c NMR (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 hydrolysis 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 is cooling, by 10% aqueous sodium carbonate extraction (2 * 300ml), salt solution washed twice.Decompression is dissolved in the gained solid in 200ml THF and 100ml water after revolving and desolventizing, and adds 13g(310mmol) lithium hydroxide, stirring at room 16h.Under ice bath is cooling, add the solution of 32g (216mmol) sodium pyrosulfate in 100ml water, finish and stir 10 minutes.21g (62mmol) fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) is joined in reaction system, naturally rise to room temperature reaction 5 hours.React complete, in system, add saturated aqueous citric acid solution 60ml, stirring at room 16h.Decompression is revolved except THF, is extracted with ethyl acetate (3 * 200ml), merges organic phase, with twice of salt washing.After anhydrous sodium sulfate drying 2h, concentrating under reduced pressure is except desolventizing.The gained crude product, through quick silica gel column chromatography (EA/PE 2:1) purifying, obtains white solid (1S, 3R, 4R)-N-Boc-3,4-dihydroxyl (N-Fmoc) ornithine 22.1g (45.5mmol, yield 73%);
1h NMR (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);
13c NMR (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: by 22.1g (45.5mmol) (1S, 3R, 4R)-N-Boc-3,4-dihydroxyl (N-Fmoc) ornithine is dissolved in 400ml DCM.Add 43ml (455mmol) diacetyl oxide and 36ml (450mmol) pyridine, stirring at room reaction 2h.Add 400ml water in reaction mixture, stir 10 minutes, DCM extraction (2 * 200ml) for water.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.The gained crude product, through quick silica gel column chromatography (EA/PE 1:1) purifying, obtains title compound 24.1g (42.3mmol, yield 93%, total recovery 60%).
1h NMR (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);
13c NMR (125MHz, CDCl
3) δ: 174.44 (C=O); (170.29 C=O of Ac), 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.
Synthesizing of embodiment 2:Fmoc-P-CTC resin
The 2-CTC resin 20.0g that to take substitution degree be 0.7mmol/g, join in the solid state reaction post, with DMF washing 2 times., get 24.0gFmoc-P-OH and dissolve with DMF after 30 minutes with DMF swelling resin, add 14.6mL DIPEA activation under ice-water bath 3 minutes, then join in the above-mentioned reaction column that resin is housed, after reacting 2 hours, add 20mL anhydrous methanol sealing 1 hour.With DMF washing 3 times, DCM washes 3 times, and with anhydrous methanol sealing 30 minutes, methyl alcohol shrinks to be drained, and obtains the Fmoc-P-CTC resin, and the detection substitution degree is 0.56mmol/g.
Embodiment 3:(4S) synthesizing 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) being dissolved in 250ml newly steams in DCM, add 101.4g (185mmol) phosphonium ylide, stirring at room reaction 4 hours, reaction solution, through silica gel rapid column chromatography purifying, obtains white solid 80.1g.This solid is dissolved in to 300ml newly to be steamed in DCM, add the solution of 15g (277mmol) sodium methylate in the 2300ml anhydrous methanol, the stirring at room reaction, after 30 minutes, adds the acidifying of 1500ml 1mol/L hydrochloric acid soln, revolve except organic solvent DCM extraction (3 * 600ml) for remaining liq.Merge organic phase, with saturated common salt washing twice, with concentrating under reduced pressure after anhydrous sodium sulfate drying 2h, removing desolventizing.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.Add 1000ml water in reaction solution, extraction, water layer is again with DCM extraction (2 * 300ml).Merge organic layer, with twice of saturated common salt washing.Organic layer 500ml dissolve with methanol, add the 500ml1mol/L NaOH aqueous solution, stirring at room reaction 4 hours.The gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o 1:1) 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%);
1h NMR (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);
13c NMR (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
2of Bn), 65.45 (CH
2of Cbz), 61.36 (C2).
By 108g (335mmol) K
3fe (CN)
6, 45.2g (335mmol) K
2cO
3, 83.6mg (0.22mmol) K
2osO
2(OH)
4and 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 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 reaction 40h.Add 157g (1.245mol) S-WAT, naturally rise to stirring at room reaction 1 hour.The gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o 1:1) purifying.Obtain white solid 41.2g (72.4mmol, yield 67%).By 500 methyl alcohol and 500ml water dissolution for this solid, add 5g 5% palladium carbon, in autoclave pressure, logical hydrogen hydrogenation is 16 hours.Filtering palladium carbon after reaction finishes.Filtrate adds 27.1g (80mmol) fluorenes methoxy carbonyl acyl succinimide (Fmoc-OSu) and 7.6g (90mmol) sodium bicarbonate, stirring at room reaction 5h.The gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o 1:1) 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%);
1h NMR (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
2of Fmoc), 53.82 (C2), 47.0 (CH of Fmoc).
Above-mentioned solid is dissolved in 400ml DMF.Add 43ml (455mmol) diacetyl oxide and 36ml (450mmol) pyridine, stirring at room reaction 2h.The reaction solution concentrating under reduced pressure is except desolventizing.The gained crude product is through reversed-phase silica gel column chromatography (MeOH/H
2o 1:1) purifying.Obtain title compound 27.9g (40.2mmol, yield 95%, total recovery 21.7%);
1h NMR (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);
13c NMR (125MHz, DMSO-d
6) δ: 174.83 (C=O), 170.31 (C=O of Ac), 169.02 (C=O of Ac), 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
2of Fmoc), 50.81 (C2), 47.0 (CH of Fmoc), 21.02 (Ac), 20.05 (Ac).
Embodiment 4: other alpha-non-natural amino acids synthetic.From commercially available, not with the amino acid of protecting group, use the method similar with the method for upper ethanoyl protection in embodiment 3 to embodiment 1 to prepare respectively the amino acid of protecting with ethanoyl on hydroxyl.
Embodiment 5: the preparation of full guard peptide resin
Fmoc-P-CTC resin 17.86 grams that to take substitution degree be 0.56mmol/g (synthetic scale 10mmol), join in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin 30 minutes, adding 20% piperidines/DMF(V/V) solution removes Fmoc twice, each time is respectively 5 minutes and 10 minutes, removes the complete DMF of using washing resin 6 times, and triketohydrindene hydrate detects resin color.Take 12.3 grams (30mmol) (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline(Pro), 4.5 gram (33mmol) HOBt, dissolve with 25mlDMF, under ice-water bath, add 6.1ml (36mmol) DIPCDI activation after 5 minutes, mixed solution is joined in reaction column, room temperature reaction 2 hours, with triketohydrindene hydrate detection reaction terminal (as resin water white transparency termination reaction; As the resin colour developing extends reaction 1 hour, lower same).
Reaction finishes, and uses DMF washing resin 3 times, adds DBLK solution to remove Fmoc twice, and each time is respectively 5 minutes and 10 minutes, removes the complete DMF of using washing resin 6 times, and triketohydrindene hydrate detects resin color.Take 12.8 grams (30mmol) (3R)-Fmoc-3-acetoxyl group-L-glutaminate, 4.5 gram (33mmol) HOBt, with 25ml DMF, dissolve, add 6.1ml (36mmol) DIPCDI activation 5 minutes under ice-water bath, mixed solution is joined in reaction column, room temperature reaction 2 hours, with triketohydrindene hydrate detection reaction terminal.
(4S)-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group) phenyl-L-threonine, (4R)-4-acetoxyl group-L-PROLINE and (the 4R)-Fmoc-O-ethanoyl-L-threonine that will prepare in advance successively after the same method carry out linked reaction; coupling finishes; remove Fmoc protection resin with DBLK; 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 25.99 grams that embodiment 5 is obtained join in 500ml single port bottle, add pre-configured AcOH:TFE:DCM=1:1:8(V:V) 250ml, room temperature reaction 40 minutes, filter resin, collects filtrate.With a small amount of DCM washing resin, merging filtrate.Concentrating under reduced pressure, to 50ml, slowly adds concentrated solution in 500ml ice ether and precipitates.Centrifugal, ice ether washing 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]
+) the 1477.4(calculated value is 1477.4).
Embodiment 7: liquid phase is synthesized the full guard cyclic peptide
Full guard peptide 12.15 grams that embodiment 6 is obtained join in 250ml single port bottle, add 160ml DCM stirring and dissolving, then add 5.2 gram PyBOP and 1.5 gram HOAt.Ice-water bath is cooled to interior 5 ℃ of system, drips DIPEA solution (1.74ml DIPEA+10ml DCM), drips and finishes, and removes ice-water bath, naturally rises to stirring at room reaction 3 hours, thin-layer chromatography monitoring reaction terminal.With 150ml 10% citric acid solution extraction 2 times, saturated aqueous common salt extraction 3 times, organic phase anhydrous sodium sulfate drying 2 hours.Filter, spin off DCM, obtain after reaction finishes obtaining full guard cyclic peptide 11.81 grams, yield 96.6%.ESI, m/z, ([M+Na]
+) the 1436.4(calculated value is 1436.3).
Embodiment 8: remove the Boc protecting group
The 11.81 gram full guard cyclic peptide that embodiment 7 is obtained join in 250ml single port bottle, add pre-configured TFA+PhOMe(97ml+3ml), room temperature reaction 2.5 hours.Revolve except TFA, 50ml ether washing 3 times for the solid of institute, the vacuum steaming desolventizes, and obtains white solid 10.76g, yield 97.9%.ESI, m/z, ([M+Na]
+) the 1359.4(calculated value is 1359.3).
Embodiment 9: the outer acid amides of the synthetic ring of liquid phase
The white solid 10.76g that embodiment 8 is obtained and 1.2g HOAt, join in 250ml single port bottle, add 100ml DCM stirring and dissolving, add side chain P17.5g, ice-water bath is cooled to interior 5 ℃ of system, drips DIPEA solution (2.74ml DIPEA+10ml DCM) again, drip and finish, remove ice-water bath, naturally rise to stirring at room reaction 3 hours, thin-layer chromatography monitoring reaction terminal.With 150ml 10% citric acid solution extraction 2 times, saturated aqueous common salt extraction 3 times, organic phase anhydrous sodium sulfate drying 2 hours.Filter, spin off DCM, obtain encircling outer amide intermediate 10.92 grams, yield 81.3%.ESI, m/z, ([M+Na]
+) the 1950.7(calculated value is 1950.6).
Embodiment 10: the preparation of the thick peptide of MFG
By outer amide intermediate 10.92 grams of embodiment 9 gained rings, with 150ml MeOH, dissolve, then room temperature dropping 150ml 28%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 ether washing 3 times, collecting solid is the crude product MFG.Filtrate merges, and 100mlDCM extraction 3 times, merge DCM, saturated aqueous common salt extraction 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]
+) the 1314.5(calculated value is 1314.4).
Embodiment 11: the purifying MFG obtains the pure peptide of MFG through thick peptide
After the thick peptide of embodiment 10 gained 7.98g MFG is used to the 800ml deionized water dissolving, adopt the Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 * 250mm, take the 0.1%TFA aqueous solution as the A phase, acetonitrile is that B carries out purifying mutually, collects purpose peak cut, obtains the smart peptide that purity is greater than 98.5%.Cut merges, and rotation is concentrated, 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, in the situation that do not depart from purport of the present invention and scope, can be changed or be improved described embodiment, the scope of the invention limits by appended claims.
Claims (10)
1. a method for preparing MFG, the method comprises the steps:
(1) obtain the Fmoc-P-CTC resin by Fmoc-P-OH and 2-CTC resin reaction,
The following alpha-non-natural amino acid with protecting group of Fmoc-P-OH representative structure wherein:
(2) take the Fmoc-P-CTC resin as carrier, adopt solid-phase peptide synthesis to hold from C
N holds in coupling one by one the amino acid with the Fmoc protecting group, obtain the full guard peptide resin, wherein the amino acid with the Fmoc protecting group is followed successively by (2S, 3S, 4S)-N-Fmoc-3-acetoxyl group-4-methyl-proline(Pro), (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) the full guard peptide resin in step (2) is carried out to cracking, obtain the full guard peptide;
(4) product in step (3) is carried out to intramolecular reaction, obtain the full guard cyclic peptide;
(5) remove the Boc protecting group on nitrogen in the middle full guard cyclic peptide of step (4);
(6) product of step (5) and side chain P1 are carried out to the liquid phase condensation, obtain the MFG with protecting group, wherein the structure of P1 is:
(7) remove the protecting group on hydroxyl in the product of step (6) and obtain MFG.
2. the process of claim 1 wherein that the Fmoc-P-CTC resin in step (1) is to be 0.2-1.1mmol/g by Fmoc-P-OH and substitution degree under alkaline condition, preferred 0.4-1.0mmol/g, more preferably prepared by the 2-CTC resin reaction of 0.7mmol/g; Wherein said alkali is DIPEA or TMP, preferably DIPEA.
3. 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 that adds the ethanoyl protecting group.
4. the method for claim 1, wherein step (2) and step (4) are carried out under the existence of coupling agent, the composition of the composition that described coupling agent is DIPCDI and compd A or DIPEA and compd A and compd B, wherein compd A is HOBt or HOAt, and compd B is PyBOP, PyAOP, HATU, HBTU or TBTU; The composition that coupling agent in step (2) is DIPCDI and A, wherein in coupling agent, the ratio of each composition be take molar ratio computing as DIPCDI:A=1.2:1.1; The composition that coupling agent in step (4) is DIPEA and PyBOP and HOAt, wherein in coupling agent, the ratio of each composition be take molar ratio computing as DIPEA:PyBOP:HOAt=2.0:1.1:1.0.
5. the process of claim 1 wherein alpha-non-natural amino acid (4S) in step (2)-N-Fmoc-4-acetoxyl group-4-(3 '-sodium sulfonate-4 '-acetoxyl group base) phenyl-L-threonine from compound 3 '-acetoxyl group-4 '-benzyloxy-phenyl aldehyde by condensation reaction, Sharpless asymmetric dihydroxylation, the lactan hydrolysis reaction of aldehyde and ylide and the method preparation that adds the ethanoyl protecting group.
6. the process of claim 1 wherein that step (3) carries out under the existence of lysate, the mixing solutions that described lysate is AcOH and TFE and DCM, wherein the ratio of each composition is counted AcOH:TFE:DCM=1:1:8 with volume ratio.
7. 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, the mixing solutions of preferred TFA and PhOMe, wherein the ratio of each composition is counted TFA:PhOMe=97:3 with volume ratio.
8. the method for claim 1; wherein in step (7), the reaction of deprotection base can be carried out under alkaline condition; described alkaline condition is NaOMe and MeOH, NaOEt and MeOH, NaOMe and EtOH, NaOEt and EtOH; preferred NaOMe and MeOH; wherein the content of NaOMe and MeOH is that every 100ml MeOH is containing 15-25g NaOMe; preferably containing 16-19gNaOMe, more preferably containing 17g NaOMe.
9. the method for any one in claim 1-8, also comprise the purification step of MFG.
10. the method for claim 9, wherein purification step adopts reversed-phase high pressure liquid chromatography, comprising: take anti-phase octadecylsilane as stationary phase, take 0.1% aqueous acetic acid/acetonitrile as moving phase, collect purpose peak cut, concentrated freeze-dried.
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| WO2012136498A1 (en) * | 2011-04-04 | 2012-10-11 | Xellia Pharmaceuticals Aps | Methods for manufacturing an antifungal agent |
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