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CN102219834A - Method for preparing romurtide - Google Patents

Method for preparing romurtide Download PDF

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Publication number
CN102219834A
CN102219834A CN2011100942729A CN201110094272A CN102219834A CN 102219834 A CN102219834 A CN 102219834A CN 2011100942729 A CN2011100942729 A CN 2011100942729A CN 201110094272 A CN201110094272 A CN 201110094272A CN 102219834 A CN102219834 A CN 102219834A
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iso
lys
ala
gln
reaction
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高泽军
温守明
李必海
陈小平
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BEIJING FENGSHUO WEIKANG TECHNOLOGY DEVELOPMENT Co Ltd
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BEIJING FENGSHUO WEIKANG TECHNOLOGY DEVELOPMENT Co Ltd
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Priority to CN201210109448.8A priority patent/CN102731628B/en
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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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    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention relates to a method for preparing romurtide. The method adopts a full-liquid phase segment condensation method and can avoid possibility that muramic acid tripeptide reacts with stearic acid to product multiple products. The obtained final product has high purity, the problem that the complex prepared by the prior art is not easy to purify is solved, and consumption of valuable important intermediate muramyl tripeptide is avoided during direct reaction of muramic acid with stearoyl tripeptide, the yield is higher, and the cost is lower, so that the romurtide is suitable for industrial production.

Description

A kind of method for preparing romurtide
Technical field
The present invention relates to a kind of method for preparing romurtide, it is easy and simple to handle that application present method prepares romurtide, and raw material is easy to get, and products therefrom purity height is convenient to purifying, and yield is higher, and production cost is lower, is suitable for mass production.
Background technology
Romurtide (romurtide), chemistry N by name 2-(N-acetyl muramyl-L-alanyl-D-isoglutamine)-N 6-stearyl-L-Methionin (N 2-[N 2-[N-(N-acetylmuramoyl)-L-alanyl]-D-α glutaminyl]-N 6-(l-oxooctadecyl)-L-Lysine), be called DJ-7041 again, MDP-LYS-L18, Muroctasin, Nopia, the CA registration number is [78113-36-7], has following chemical structural formula:
Romurtide is a kind of Muramyl dipeptide (MDP) analog derivative, belongs to the G CFS agonist.Its main mechanism of action is, activity by cytokine network, induce the G CFS secretion, promote the propagation and the differentiation of hemopoietic stem cell in the marrow, thereby make white corpuscle and thrombocythemia in the peripheral blood, rising helper T cell activity activates neutrophil leucocyte and macrophage function, the nonspecific resistance of enhancing to infecting strengthens antibiotic curative effect.Romurtide went on the market in Japan in 1991, was used for the assisting therapy of tumour patient, because its effect is unique, application prospect is very wide.
Visible EP002367 of synthetic document and US4317771 about romurtide; it all adopts full liquid phase synthetic; its route is to be starting raw material with amino acid; at first use tertiary butyloxycarbonyl-L-L-Ala (1) and D-isoglutamine-r-benzyl ester (2) under N-hydroxy-succinamide (HOSu)/dicyclohexylcarbodiimide (DCC) effect, to generate protection dipeptides BOC-Ala-D-iso-Gln (OBzl) (3); get debenzylation protection dipeptides BOC-Ala-D-iso-Gln (OH) (4) with the Pd/C catalytic hydrogenation, with N 6The condensation under Vinyl chloroformate and triethylamine effect of-carbobenzoxy-(Cbz)-L-Methionin benzyl ester (5) generates protection tripeptides BOC-Ala-D-iso-Gln-Lys (Z)-OBzl (6), slough the BOC protecting group with trifluoroacetic acid and get intermediate A la-D-iso-Gln-Lys (Z)-OBzl (7), the latter and 1-benzyl-4,6-O-Ben Yajiaji--acetylmuramic acid [(B1, B4,6-NAcMur or protection teichoic acid (8)] under (HOSu)/(DCC) effect, generate and protect muramyl-tripeptide B1, B4,6-NAcMur-Ala-D-iso-Gln-Lys (Z)-OBzl (9), slough 4 with acetic acid treatment, the 6-O-Ben Yajiaji obtains part protection compound B-11-NAcMur-Ala-D-iso-Gln-Lys (Z)-OBzl (10), and then catalytic hydrogenation gets N-(N-acetyl muramyl-L-alanyl-D-isoglutamine base)-L-Methionin (11) (chemical formula NAcMur-Ala-D-iso-Gln-Lys); The latter and stearic N-hydroxyl-norbornylene-2,3-dicarboximide ester react romurtide.The synthetic method that CN200910155724 then adopts solid, liquid to combine; earlier with solid phase synthesis Fmoc-Ala-iso-Gln (Trt)-Lys (Boc)-Wang; muramyl-tripeptide must be protected with the condensation of protection teichoic acid (8) in the back, generates romurtide with K reagent cutting Wang resin products therefrom and stearic acid reaction.Above method all is that the preparation muramyl-tripeptide then reacts with stearic acid earlier when synthesizing romurtide, we press the repeatedly synthetic romurtide of literature method, all find since active reactive group except that amino, also have three hydroxyls, thereby by product is many in the product, gained end product purity not high (accompanying drawing 1-2) is difficult to purifying; And reaction generates the intermediate muramyl-tripeptide through tens steps, thereby rare and expensive, more with the stearic acid reaction consumes again, reduces with the teichoic acid rate that collects, and production cost is too high.
Therefore, prior art needs a kind of easy and simple to handle, and products therefrom purity height (accompanying drawing 3) is convenient to purifying, has avoided the consumption of important intermediate muramyl-tripeptide, and yield is higher, and cost is lower, is suitable for the method for preparing romurtide of suitability for industrialized production.
Summary of the invention
The present inventor provides a kind of method of synthetic romurtide newly through behind a large amount of creative works.The diverse synthesis strategy of this method and literature method, avoided bibliographical information because of the reaction of teichoic acid tripeptides and stearic acid the time produce the possibility of multiple product, gained end product purity height has thoroughly solved this compound of prior art for preparing and has been difficult for issues of purification; Simultaneously teichoic acid and stearyl tripeptides direct reaction have been avoided the consumption of rare valuable important intermediate muramyl-tripeptide, and yield is higher, and cost is lower, is suitable for suitability for industrialized production.
What the method for preparing romurtide provided by the present invention adopted is full liquid-phase fragment condensation method, with protection R 1-D-iso-Gln-OR 2Be raw material, slough R 1With R 3The condensation in the presence of I-hydroxybenzotriazole (Hobt)/DCC of-L-L-Ala generates R 3-L-Ala-D-iso-Gln-(OR 2) (II), the latter carries out hydrogenation under Pd/C catalysis, obtain dipeptides R 3-L-Ala-D-iso-Gln-(OH) (III); With protection Methionin Lys (R 4)-OR 5Be raw material, generate CH with the stearic acid reaction 3(CH 2) 16CO-Lys (R 4)-OR 5(IV) two peptide fragment are sloughed R 4Get CH 3(CH 2) 16CO-Lys-OR 5(V); III and V condensation in the presence of Hobt/DCC generates tetrapeptide R 3-L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VI), (VI) slough protecting group R 3Get L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VII), (VII) generate the protection romurtide with the condensation of protection teichoic acid, slough protecting group and obtain romurtide.
Romurtide preparation method provided by the invention may further comprise the steps:
A) with R 1-D-iso-Gln (OR 2) be raw material, slough protecting group with acid or alkali and then get D-iso-Gln (OR 2) (I).
B) (I) and R 3The condensation in the presence of Hobt/DCC of-L-L-Ala generates R 3-L-Ala-D-iso-Gln-(OR 2) (II).
C) (II) under Pd/C catalysis, carry out hydrogenation or zinc-acetate reacts, slough protecting group R 2, obtain R 3-L-Ala-D-iso-Gln-(OH) (III).
D) with L-Lys (R 4)-OR 5Be raw material, with stearic acid in the DCC method, condensation generates CH under the existence of Hobt/DCC or thionyl chloride 3(CH 2) 16CO-Lys (R 4)-OR 5(IV).
E) CH 3(CH 2) 16CO-Lys (R 4)-OR 5(IV) in acid or alkaline solution, slough R 4, obtain CH 3(CH 2) 16CO-Lys-OR 5(V).
F) intermediate R 3-L-Ala-D-iso-Gln-(OH) is (III) in the presence of Hobt/DCC, with CH 3(CH 2) 16CO-Lys-OR 5(V) carry out condensation and generate R 3-L-Ala-D-iso-Gln-Lys (OR 5)-CO (CH 2) 16CH 3(VI).
G) R 3-L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VI) slough protecting group with acid or alkaline solution and get L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VII).
H) intermediate L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VII) in the presence of Hobt/DCC, carry out condensation with teichoic acid, generate protection romurtide (VIII).
B1, B4,6-NAcMur-Ala-D-iso-Glu-(Lys (OR 5)-CO (CH 2) 16CH 3) carrying out sloughing protecting group generation romurtide in the presence of hydrogenation or the Zn/ acetate under the Pd/C catalysis.
Wherein, R 1Be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 2Be Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.; R 3With R 1Identical or different, can be selected from Fmoc, Adoc Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 4With R 1, R 3Identical or different, can be selected from and be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.; R 5With R 2Identical or different, can be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
Among the preparation method of the present invention, described acid can be selected from but be not limited to the HCl/ trifluoroethanol, the CH of 2-50%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, FSO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; HCl dioxane solution preferably, cold trifluoroacetic acid; Described alkali is alkali commonly used, for example can be but is not limited to hydrazine hydrate, hexahydropyridine, sodium amide etc.; Be preferably hexahydropyridine, hydrazine hydrate.
In the reaction of step a), employed solvent is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane.Preferred solvent is N, dinethylformamide, methylene dichloride, dioxane.Acid wherein is the HCl/ trifluoroethanol, the CH of 2-5%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, FSO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide; Reaction times is 2-4 hour, and preferred 2.5-3.5 hour, temperature of reaction was-5 ℃-30 ℃, preferred 10-20 ℃, and most preferably 13-17 ℃.
B) solvent that uses in the reaction is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane equal solvent.Preferred solvent is N, dinethylformamide, methylene dichloride, dioxane.The mol ratio of amino acid: DCC: Hobt is 1: (1-2): (1-2), preferred 1: (1-1.5): (1-1.5), most preferably 1: (1-1.1): (1-1.3), temperature of reaction is-5 ℃-40 ℃, preferred 0-20 ℃, most preferably 5-10 ℃, reaction times 2-10 hour, preferred 5-9 hour, most preferably 6-8 hour.
C) solvent that uses in the reaction is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, acetate, methyl alcohol, ethanol etc.Preferred solvent is N, dinethylformamide, methyl alcohol, ethanol equal solvent.Temperature of reaction is 0-40 ℃, preferred 10-30 ℃, and most preferably 13-20 ℃, reaction times 2-10 hour, preferred 4-9 hour, most preferably 4-8 hour.
D) in the reaction, the solvent that uses is N, dinethylformamide, and N, the N-diethyl acetamide, methyl-sulphoxide, trichloromethane, methylene dichloride etc., preferred solvent is N, dinethylformamide, methylene dichloride; The mol ratio of stearic acid: DCC: Hobt is 1: (1-2): (1-2), preferred 1: (1-1.5): (1-1.5), most preferably 1: (1-1.2): (1-1.3), temperature of reaction is 0-40 ℃, preferred 5-25 ℃, most preferably 10-15 ℃, reaction times 4-10 hour, preferred 5-9 hour, most preferably 6-8 hour.
E) in the reaction, the solvent of use is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane equal solvent.Preferred solvent is N, dinethylformamide, methylene dichloride; Acid wherein is the HCl/ trifluoroethanol, the CH of 2-5%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, F SO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Be preferably the HCl dioxane solution, cold trifluoroacetic acid; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide; Be preferably hexahydropyridine, hydrazine hydrate; Reaction times is 2-4 hour, preferred 2.5-3.5 hour; Temperature of reaction is 0-30 ℃, preferred 10-20 ℃, and most preferably 10-15 ℃.
F) in the reaction, be 1 (III) with (V) molar ratio: (1-1.5), preferred 1: (1-1.2), most preferably 1: (1-1.05).(III): DCC: the mol ratio of Hobt is 1.5: (1-1.5): (1-1.5), and preferred 1.5: (1-1.3): (1-1.4), most preferably 1.5: (1-1.2): (1-1.3).Temperature of reaction is-5 ℃-30 ℃, preferred 0-20 ℃, and most preferably 10-17 ℃; Reaction solvent is N, dinethylformamide, and N, the N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, DMF, trichloromethane, tetrahydrofuran (THF) etc., preferred solvent is a methylene dichloride, DMF.Reaction times is 5-24 hour, preferred 12-22 hour, and most preferably 15-20 hour; Preferred temperature is 10-40 ℃, preferred 15-30 ℃, and most preferably 20-25 ℃.
G) solvent that uses in the reaction selects N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane equal solvent.Preferred solvent is N, dinethylformamide, methylene dichloride, dioxane etc.; Acid wherein is the HCl/ trifluoroethanol, the CH of 2-5%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, F SO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Be preferably the CH of 2-5%TFA 2Cl, the HCl dioxane solution; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide etc.; Be preferably hydrazine hydrate, hexahydropyridine; Reaction times is 2-4 hour, preferred 2.5-3.5 hour; Temperature of reaction is 0-30 ℃, preferred 10-20 ℃, and most preferably 13-17 ℃.
H) in the reaction, (VII) and B1, B4,6-NAcMur molar ratio are 1: (1-1.5), and preferred 1: (1-1.2), most preferably 1: (1-1.05).(VII): DCC: the mol ratio of Hobt is 1.5: (1-1.5): (1-1.5), and preferred 1.5: (1-1.3): (1-1.4), most preferably 1.5: (1-1.2): (1-1.3); Temperature of reaction is 0-40 ℃, preferred 15-30 ℃, and most preferably 20-25 ℃; Reaction times 10-24 hour, preferred 12-22 hour, most preferably 15-20 hour.Teichoic acid is selected from different protecting group intermediates, and perhaps free teichoic acid also can direct reaction.
I) in the hydrogenation, catalyzer is RanyNi or Pd/C, and the amount of catalyzer is the 50%-100% of protection romurtide (VII), preferred 60%-90%, most preferably 75-85%.Reaction solvent is an acetate, methyl alcohol, and DMF is preferably acetate, methyl alcohol.The mole number of Zn was 10 times of the protection romurtide when there was to descend deprotection in Zn/ acetate.Solvent is an acetate methanol mixed solvent.
Concrete synthetic route chart of the present invention is as follows:
Figure BDA0000055426260000061
R wherein 1Be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 2Be D pm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.; R 3Be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; Acid wherein is the HCl/ trifluoroethanol, the CH of 2-50%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, FSO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide etc.
Figure BDA0000055426260000071
R wherein 4Be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.; R 5Be Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.; Acid wherein is the HGl/ trifluoroethanol, the CH of 2-5%TFA 2Cl 2Or dilute acetic acid solution, HB r/ acetic acid solution, BTFA, CF 3SO 3H, FSO 3H, HF, H 2-Pd is in liquefied ammonia, and Zn is in dilute acetic acid, and HCl oxygen six encircles solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide etc.
Figure BDA0000055426260000081
Acid wherein is the HCl/ trifluoroethanol, the CH of 2-5%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF 3SO 3H, FSO 3H, HF, H 2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid, rare methylsulfonic acid etc.; Alkali wherein is alkali commonly used, hydrazine hydrate, hexahydropyridine, sodium amide etc.
Figure BDA0000055426260000091
Further, the present invention also provides a series of new midbody compounds, and this midbody compound all adopts preparation method's process of preparing of the present invention to obtain.Comprise:
The intermediate of formula A structure, concrete structure is as follows:
Figure BDA0000055426260000092
Its synthetic route is:
Figure BDA0000055426260000101
R 2Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.; R 3Be Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.The compound of the preferred especially following structure of formula A:
Figure BDA0000055426260000102
The intermediate of formula B structure, concrete structure is as follows:
Its synthetic route is:
Figure BDA0000055426260000111
R wherein 3Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.
The special preferably compound of following structure of formula B:
Figure BDA0000055426260000112
The midbody compound of formula C-structure, concrete structure is as follows:
Figure BDA0000055426260000113
R wherein 4Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.; R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
The midbody compound of formula D structure has following structure
Its synthetic route is:
Figure BDA0000055426260000122
R wherein 4Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.; R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.The preferred especially following compound of formula D:
Figure BDA0000055426260000123
The midbody compound of formula E structure, concrete structure is as follows:
Figure BDA0000055426260000131
Its synthetic route is:
Figure BDA0000055426260000132
R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
The preferred especially following compound of formula E:
Figure BDA0000055426260000133
The midbody compound of formula F structure, concrete structure is as follows:
Formula F
Its synthetic route is:
Figure BDA0000055426260000141
R wherein 3Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps etc.; R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
The preferred especially following compound of formula F:
Figure BDA0000055426260000151
The midbody compound of formula G structure, concrete structure is as follows:
The synthetic route of formula G compound is:
Figure BDA0000055426260000161
R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
The preferred especially following compound of formula G:
Figure BDA0000055426260000162
The midbody compound of formula H structure, concrete structure is as follows:
Figure BDA0000055426260000171
The synthetic route of formula G compound is:
Figure BDA0000055426260000172
R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc etc.
The preferred especially following compound of formula H:
Figure BDA0000055426260000181
Term
In the present invention, related term has following implication.
Figure BDA0000055426260000182
Figure BDA0000055426260000191
Figure BDA0000055426260000201
Description of drawings
The evaluation result of [Fig. 1] prior art for preparing romurtide
The evaluation result of [Fig. 2] prior art for preparing romurtide
[Fig. 3] adopts method of the present invention to prepare the evaluation result of romurtide
Embodiment
Embodiment one
The preparation of Fmoc-Ala-D-iso-Gln (ONb)
Boc-D-iso-Glu (ONb) 11.7g (30.0mmol) is placed the 500ml eggplant-shape bottle, add 2N hydrochloric acid dioxane solution 80ml, the dissolving back is separated out solid, room temperature (23 ℃) stirring reaction 1h at once.Removal of solvent under reduced pressure adds methylene dichloride and drains repeatedly three times.Add the 100ml tetrahydrofuran (THF), solid is insoluble, adds MM (N-methylmorpholine) 3.43g, and is standby.
Fmoc-Ala 9.33g (30.0mmol) is placed the 500ml eggplant-shape bottle, add the 100ml methylene dichloride, dissolving, ice bath 10 minutes.Stir, add HOB 4.25g (31.5mmol) (being dissolved in 15mlDMF), add DCC 6.50g (31.6mmol), ice bath reaction 1h has solid to separate out.Reserve liquid is added, keep ice bath reaction 2h, remove ice bath, room temperature (23 ℃) reaction is spent the night.Filter, the filtering solid adds the 200ml methylene dichloride, and insolubles is arranged, and filtering is washed solid with methylene dichloride, merging filtrate, and with saturated brine, 10% citric acid, saturated sodium bicarbonate, saturated brine is respectively given a baby a bath on the third day after its birth inferior, dried over sodium sulfate, TLC detects.Condition: methylene dichloride: methyl alcohol=30: 1.The filtering siccative, the water-bath removal of solvent under reduced pressure adds sherwood oil, grinds, filter collection solid, sherwood oil is washed 1 time, and anhydrous diethyl ether is washed 1 time, dries, and gets off-white color solid Fmoc-Ala-D-iso-Gln (ONb) 16.65g, productive rate 96.5%.Measuring its fusing point is 150-152 ℃.MS：577.
6(M+1)，1HNMR(ppm)(CDCl 3)8.07(2H，d)，8.0(2H，s)，7.84(2H，d)，7.55(2H，d)，7.38(2H，t)，7.32(2H，d)，7.28(2H，d)，6.0(2H，s)，4.73.0(1H，s)，4.42(1H，m)，3.74(1H，m)，2.35(2H，s)，2.18(4H，m)，1.28(3H，t)。
TLC testing conditions: methylene dichloride: methyl alcohol=30: 1, Rf value are 0.5, (iodine colour developing, fluorescence developing 254nm).Principal spot purity is greater than 95%.
Embodiment two
The preparation of Boc-Ala-D-iso-Gln (OBZL)
Boc-D-iso-Gln (OBZL) 11.4g (30.Ommol) is placed the 500ml eggplant-shape bottle, add 2N hydrochloric acid dioxane solution 80ml, the dissolving back is separated out solid, room temperature (13 ℃) stirring reaction 1h at once.Removal of solvent under reduced pressure adds methylene dichloride and drains repeatedly three times.Add the 100ml methylene dichloride, solid is insoluble, adds MM (N-methylmorpholine) 3.43g, and is standby.
Boc-Ala 6.41g (33.8mmol) is placed the 500ml eggplant-shape bottle, add the 100ml methylene dichloride, dissolving, ice bath 10 minutes.Stir, add HOBt 4.8g (35.6mmol) (being dissolved in 15mlDMF), add DCC7.33g (35.6mmol), ice bath reaction 1h has solid to separate out.Reserve liquid is added, keep ice bath reaction 2h, remove ice bath, room temperature (13 ℃) reaction is spent the night.Removal of solvent under reduced pressure gets solid, adds the 400ml ethyl acetate, and insolubles is arranged, and filtering is washed solid with ethyl acetate, merging filtrate, and with saturated brine, 10% citric acid, saturated sodium bicarbonate, saturated brine is respectively given a baby a bath on the third day after its birth inferior, dried over sodium sulfate, TLC detects.(condition: methylene dichloride: methyl alcohol=19: 1).The filtering siccative, 30 ℃ of water-bath removal of solvent under reduced pressure have solid to separate out, when also having a small amount of solvent, stop, add sherwood oil, ice bath 1h, filter collection solid, sherwood oil is washed 1 time, anhydrous diethyl ether is washed 1 time, dries, and gets Boc-Ala-D-iso-Gln (OBZL), it is off-white color solid 12.65g, productive rate 91.6%.Fusing point 135-140 ℃.MS：430.24(M+Na)，1HNMR(ppm)(CDCl 3)：8.01(2H，s)，7.14(2H，t)，7.07(1H，t)，7.06(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.45(1H，m)，3.74(1H，m)，2.35(2H，s)，2.18(2H，m)，2.07(2H，t)，1.48(3H，t).1.40(9H，s)。
TLC testing conditions: methylene dichloride: methyl alcohol=19: 1, Rf value are 0.45, (iodine colour developing, fluorescence developing 254nm).TLC does not find the impurity spot.
Embodiment three
Fmoc-Lys (OBzl)-CO (CH 2) 16CH 3Preparation
Stearic acid 20.8g (0.245mol) is placed the 2L eggplant-shape bottle, add 200ml N, the N-diethyl acetamide adds HOBT 11.16g (0.083mol), and ice-water bath adds DCC 17.93g (0.083mol), ice-water bath reaction 2h.Add Fmoc-Lys (OBzl) tosilate 41g solution (using earlier 300mlN, the dissolving of N-diethyl acetamide, ice bath, adding NMM 6.85ml after 30 minutes), ice bath stirring reaction 2h rises to room temperature naturally, and reaction is spent the night.N is removed in decompression, and the N-diethyl acetamide adds the dissolving of 800ml methylene dichloride, the filtering insolubles, and organic phase washes with water 3 times, and saturated sodium bicarbonate, water, 10% citric acid, water are respectively given a baby a bath on the third day after its birth inferior, and dried over mgso is spent the night.
Methylene dichloride is removed in decompression, solid ethyl acetate 400ml heating for dissolving, and the filtering insolubles, the filtrate journey is frozen shape, filters, and washes 2 times with ethyl acetate, and drying gets 37g Fmoc-Lys (OBzl)-CO (CH 2) 16CH 3, productive rate: 80.7%.MS?725(M+1)；1HNMR(ppm)(CDCl 3)：8.0(2H，d)，8.0(1H，s)，7.84(2H，d)，7.55(2H，d)，7.38(2H，t)，7.32(2H，d)，7.28(2H，d)，7.19(5H，m)，6.0(2H，s)，5.34(2H，s)，4.81(1H，s)，4.42(1H，m)，3.74(1H，s)，2.45(2H，t)，1.90(2H，t)，1.57(2H，m)，1.55(2H，t)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
TLC condition: methylene dichloride: methyl alcohol=10: 0.18, Rf value are 0.65, (iodine colour developing, fluorescence developing 254nm).TLC does not find the impurity spot.
Embodiment four
Boc-Lys (ONb)-CO (CH 2) 16CH 3Preparation
Stearic acid 12.4g (50mmol) is placed the 2L eggplant-shape bottle, add 700mlDMF, add HOBT 8.1g (60.0mmol), ice-water bath adds DCC 12.36g (60.0mmol), ice-water bath reaction 2h.Add Boc-Lys (ONb) 18.6g (50.0mmol) solution (dissolving with 500mlDMF earlier), ice bath stirring reaction 2h has a little solid to separate out in the reaction solution, and the gelation shape rises to room temperature naturally, and reaction is spent the night.DMF is removed in decompression, adds the dissolving of 800ml methylene dichloride, the filtering insolubles, and organic phase washes with water 3 times, and saturated sodium bicarbonate, water, 10% citric acid, water are respectively given a baby a bath on the third day after its birth inferior, and dried over mgso is spent the night.Methylene dichloride is removed in decompression, solid ethyl acetate 400ml heating for dissolving, and the filtering insolubles, the filtrate journey is frozen shape, filters, and washes 2 times with ethyl acetate, and drying gets 28.3g Boc-Lys (ONb)-CO (CH 2) 16CH 3, productive rate: 90.9%.MS?652.1(M+1)；1HNMR(ppm)(CDCl 3)：8.12(2H，d)，8.0(2H，s)，7.45(2H，d)，5.34(2H，s)，4.42(1H，m)，3.20(2H，t)，2.18(2H，m)，1.90(2H，m)，1.57(2H，m)，1.55(2H，t)，1.33(2H，m)，1.29(30H，m)，1.21(9H，s)0.96(3H，t)。
TLC condition: methylene dichloride: methyl alcohol=10: 0.18, Rf value are 0.6, (iodine colour developing, fluorescence developing 254nm).TLC does not find the impurity spot.
Embodiment five
Lys (ONb)-CO (CH 2) 16Preparation
Boc-Lys (ONb)-CO (CH2) 16CH324.57g (38.8mmol) is placed the 2L eggplant-shape bottle, add 50% trifluoroacetic acid/dichloromethane solution 300ml, molten entirely, stirring at room reaction 2h.Removal of solvent under reduced pressure gets solid, washs solid three times with ether.Dry, this solid suspension in the 200ml methylene dichloride, added the 40mlN-methylmorpholine, stir half an hour, filter, be concentrated into driedly, add petroleum ether and stirring, white solid, filter collection solid, vacuum-drying gets 19.1g Lys (ONb)-CO (CH 2) 16, productive rate 96.1%.MS?574(M+Na)；1HNMR(ppm)(CDCl 3)；8.12(2H，d)，8.0(1H，s)，7.45(2H，d)，5.34(2H，s)，4.42(1H，m)，3.20(2H，t)，2.18(2H，m)，2.01(2H，d)1.90(2H，m)，1.57(2H，m)，1.55(2H，t)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
TLC condition: methylene dichloride: methyl alcohol=10: 0.2, Rf value are 0.35, (iodine colour developing, fluorescence developing 254nm).Principal spot purity is greater than 95%
Embodiment six
Lys (OBzl)-CO (CH 2) 16CH 3Preparation
Fmoc-Lys (OBzl)-stearic acid 49.17g is placed the 2L eggplant-shape bottle, add 25% piperidines/dichloromethane solution 600ml, molten entirely, stirring at room reaction 2h.Removal of solvent under reduced pressure gets solid, is washed till no Fmoc with ethyl acetate.Dry, vacuum-drying gets 31.5g Lys (OBzl)-CO (CH 2) 16CH 3, productive rate 92.26%.MS?507(M+1)；1HNMR(ppm)(CDCl 3)；8.0(1H，s)，7.19(5H，m)，5.34(2H，s)，3.42(1H，m)，3.20(2H，t)，2.18(2H，m)，1.90(2H，m)，1.57(2H，m)，1.55(2H，t)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
TLC condition: methylene dichloride: methyl alcohol=10: 0.2, Rf value are 0.3, (iodine colour developing, fluorescence developing 254nm).Principal spot purity is greater than 95%.
Embodiment seven
The preparation of Fmoc-Ala-D-iso-Gln
Fmoc-Ala-D-iso-Glu (ONb) 29.8g (50.0mmol) places the 1L eggplant-shape bottle, adds acetate 400ml, fully dissolving, control reaction temperature is below 25, add Zn part 30g in batches, need 4 hours approximately, finish, stirring at room 4 hours, filter, acetate is removed in decompression, and residuum dissolves with the 300ML methylene dichloride, wash 3 times, anhydrous magnesium sulfate drying filters, and is evaporated to 100ML, add sherwood oil 50Ml, separate out solid, filter, filter collection solid, get white solid 20.8g Fmoc-Ala-D-iso-Gln, yield 94.5%.MS?441.7(M+1)；1HNMR(ppm)(CDCl 3)：11.0(1H，s)，8.07(2H，d)，8.0(2H，s)，7.84(2H，d)，7.55(2H，d)，7.38(2H，t)，7.28(2H，d)，6.0(2H，s)，4.81(1H，s)，4.71.0(1H，s)，4.46(1H，m)，3.74(1H，m)，2.18(2H，t)，2.06(2H，m)，1.48(3H，d)。
Methylene dichloride: methyl alcohol=19: 1, Rf value are 0.35, (iodine colour developing).Principal spot purity is greater than 95%.
Embodiment eight
The preparation of Boc-Ala-D-iso-Gln
Boc-Ala-D-iso-Glu (OBzl) 23.8g (58.3mmol) places the 1L eggplant-shape bottle, add methyl alcohol 300ml, dissolving fully, stirring at room 2 hours, DMF is removed in decompression, and residuum grinds with sherwood oil 200ML, filter, filter collection solid gets white solid 20.8gBoc-Ala-D-iso-Gln, yield 94.5%.MS317.7(M)；1HNMR(ppm)(CDCl 3)：11.0(1H，s)，8.0(2H，s)，6.0(2H，s)，.71.0(1H，s)，4.46(1H，m)，3.74(1H，m)，2.18(2H，t)，2.06(2H，m)，1.48(3H，d)；1.40(9H，s)。
Methylene dichloride: methyl alcohol=19: 1, Rf value are 0.15, (iodine colour developing).TCL does not find the impurity spot.
Embodiment nine
Fmoc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] preparation
Fmoc-Ala-D-iso-Gln6.22.2g (50.0mmol) is placed the 1L eggplant-shape bottle, and adding 300ml methylene dichloride and DMF 100ml are molten entirely, ice bath.Add HOBT 7.10g (52.5mmol), DCC11.08g (57.3mmol) ice bath reaction 2h.Add Lys (ONb)-CO (CH 2) 16CH 326.7g (50.1mmol), add the 100mlDMF dissolving, keep ice bath reaction 2h, rise to room temperature naturally, molten entirely, reaction is spent the night.Reaction solution is for freezing shape, and removal of solvent under reduced pressure gets solid, adds methylene dichloride, heats 5 minutes, place, filter, ethyl acetate is given a baby a bath on the third day after its birth inferior, methylene dichloride is given a baby a bath on the third day after its birth time, and sherwood oil is given a baby a bath on the third day after its birth time, dry white 44.6g Fmoc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3].Yield 92.0%.MS1014.6(M+Na)；1HNMR(ppm)(DMSO-d 6)：
8.07(2H，d)，8.0(4H，s)，7.84(2H，d)，7.55(2H，d)，7.38(2H，t)，7.32(2H，d)，7.28(2H，d)，6.0(2H，s)，4.81(1H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.71(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.65, (iodine colour developing, fluorescence developing 254nm).Principal spot purity is greater than 95%.
Embodiment ten
Boc-Ala-D-iso-Gln-[Lys (OBzl)-CO (CH 2) 16CH 3] preparation
Boc-Ala-D-iso-Gln 12.29g (39.0mmol) is placed the 1L eggplant-shape bottle, and 200mlDMF is molten entirely in adding, ice bath.Add HOBT 5.53g (40.95mmol), DCC8.88g (40.95mmol) ice bath reaction 2h.Add Lys (OBzl)-CO (CH 2) 16CH 319.66g (39.0mmol), add the dissolving of 300ml methylene dichloride, keep ice bath reaction 2h, rise to room temperature naturally, molten entirely, reaction is spent the night.Reaction solution is for freezing shape, and removal of solvent under reduced pressure gets solid, and the adding ethyl acetate heated 5 minutes, placed, and filtered, and ethyl acetate is given a baby a bath on the third day after its birth inferior, and methylene dichloride is given a baby a bath on the third day after its birth inferior, and sherwood oil is given a baby a bath on the third day after its birth inferior, dries.Get white solid 25.3g Boc-Ala-D-iso-Gln-[Lys (OBzl)-CO (CH 2) 16CH 3], yield 80.7%.MS?804.6(M+1)；1HNMR(ppm)(DMSO-d 6)：8.0(4H，s)，7.14(2H，d)，7.07(1H，dd)，7.06(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.71(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.40(9H，s)1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.4, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 11
Boc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] preparation of (protection tetrapeptide)
Boc-Ala-D-iso-Gln6.24.5g (78.0mmol) is placed the 1L eggplant-shape bottle, and 200mlDMF is molten entirely in adding, ice bath.Add HOBT 11.1g (81.1mmol), DCC 16.7g (81.1mmol) ice bath reaction 2h.Add Lys (ONb)-CO (CH 2) 16CH 342.2g (80.0mmol), add the 300mlDMF dissolving, keep ice bath reaction 2h, rise to room temperature naturally, molten entirely, reaction is spent the night.Reaction solution is for freezing shape, and removal of solvent under reduced pressure gets solid, adds methylene dichloride, heats 5 minutes, place, filter, methylene dichloride is given a baby a bath on the third day after its birth inferior, ethyl acetate is given a baby a bath on the third day after its birth time, and sherwood oil is given a baby a bath on the third day after its birth time, dry white solid 58.9g Boc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3], yield 88.9%.MS?891.6(M+Na)；1HNMR(ppm)(DMSO-d6)；8.07(2H，d)，8.0(4H，s)，7.32(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.71(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.40(9H，s)1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.6, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 12
Ala-D-iso-Gln-[Lys (OBzl)-CO (CH 2) 16CH 3] preparation
Boc-Ala-D-iso-Gln-[Lys (OBzl)-CO (CH 2) 16CH 3] 20g (24.9mmol) is dissolved in 300mL 50% trifluoroacetic acid dichloromethane solution, stirring at room 3 hours, removal of solvent under reduced pressure and residue trifluoroacetic acid, residuum adds methylene dichloride 200mL, drip N-methylmorpholine 26g, stir half an hour, filter, solid washed with dichloromethane three times get white Ala-D-iso-Gln-Lys (OBzl)-CO (CH 2) 16CH 3Solid 17.3g, yield 98.8%.MS?704.6(M+1)；1HNMR(ppm)(DMSO-d 6)；8.0(3H，s)，7.14(2H，d)，7.07(1H，dd)，7.06(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.74(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.01(2H，Br)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.55, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 13
Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] preparation
Boc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] 23.5g (27.7mmol) is dissolved in the 4MHCl/ dioxane solution, stirring at room 3 hours, removal of solvent under reduced pressure, residuum adds methylene dichloride 250mL, drip N-methylmorpholine 30g, stir half an hour, filter, solid washed with dichloromethane three times get white Ala-D-iso-Gln-[Lys (OBNb)-CO (CH 2) 16CH 3] solid 18.3g, yield 83.8%.MS?791.6(M+Na)；1HNMR(ppm)(DMSO-d 6)：8.07(2H，d)，8.0(3H，s)，7.32(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.74(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.01(2H，Br)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.35, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 14
Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] preparation
Fmoc-Ala-D-iso-Gln-[Lys (ONb)-CO (CH 2) 16CH 3] 26.5g (27.3mmol) is dissolved in 300mL 30% piperidines/DMF solution, stirring at room 3 hours, removal of solvent under reduced pressure, residuum adds sherwood oil 250mL and grinds, inclining sherwood oil, three times repeatedly, filters, solid petroleum ether three times get white Ala-D-iso-Gln-[Lys (OBNb)-CO (CH 2) 16CH 3] solid 19.3g, yield 94.4%.MS791.6(M+Na)；1HNMR(ppm)(DMSO-d 6)：8.07(2H，d)，8.0(3H，s)，7.32(2H，d)，6.0(2H，s)，4.71.0(1H，s)，4.53(1H，m)，4.45(1H，m)3.74(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.01(2H，Br)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.35, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 15
Protection romurtide B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (OBzl)-CO (CH 2) 16CH 3] preparation
With 2.83 (6.0mmol) gram protection teichoic acid (B1; B4; 6-NAcMur) in reaction flask; add dry DMF300 milliliter dissolving; the ice bath cooling adds HOBt 0.89 gram (6.6mmol), DCC 1.43g (6.6mmol); continue the ice bath reaction after 2 hours, with Ala-D-iso-Glu-[Lys (OBzl)-CO (CH 2) 16CH 3] 4.82 (6.0mmol) add in the reaction flask, continue the ice bath reaction and spend the night.Removal of solvent under reduced pressure adds ethyl acetate and separates out solid, filters to such an extent that white solid 6.3 restrains B 1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (OBzl)-CO (CH 2) 16CH 3], yield 81.8%.MS?1179.1.(M+Na)；1HNMR(ppm)(DMSO-d 6)：8.0(4H，s)，7.06-7.25(15H，m)，6.0(2H，s)，5.98(1H，m)，5.53(1H，m)，4.71.0(1H，s)，4.63(2H，s)，4.53(1H，m)，4.45(1H，m)4.43(1H，m)，4.34(1H，m)，4.03(1H，m)，3.96(1H，m)，3.89(1H，m)，3.02(1H，m)，3.81(1H，m)，3.74(2H，s)，3.
20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.02(2H，s)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d).，1.39(2H，d)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.75, (iodine colour developing, fluorescence developing 254nm).TCL does not find the impurity spot.
Embodiment 16
Protection romurtide B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (ONb)-CO (CH 2) 16CH 3] preparation
Protect teichoic acids (BHBB S) in reaction flask 5.47 (11.60mmol) gram; add dry DMF500 milliliter dissolving; the ice bath cooling; add HOBt1.73 gram (12.76mmol); DCC2.63g (12.76mmol); continue the ice bath reaction after 2 hours, with Ala-D-iso-Glu-[Lys (ONb)-CO (CH 2) 16CH 3] 9.84 (11.60mmol) add in the reaction flask, continue the ice bath reaction and spend the night.Removal of solvent under reduced pressure adds ethyl acetate and separates out solid, filter white solid B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (ONb)-CO (CH 2) 16CH 3] 11.50 grams, yield 84.8%.MS?1224.1.(M+Na)；1HNMR(ppm)(DMSO-d 6)：8.12(2H，d)，8.0(4H，s)，7.45(2H，d)7.06-7.20(10H，m)，6.01(2H，s)，5.98(1H，m)，5.53(1H，m)，4.71.0(1H，s)，4.63(2H，s)，4.53(1H，m)，4.45(1H，m)4.43(1H，m)，4.34(1H，m)，4.03(1H，m)，3.96(1H，m)，3.89(1H，m)，3.02(1H，m)，3.81(1H，m)，3.74(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.02(2H，s)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d)，1.39(2H，d)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Methylene dichloride: methyl alcohol=10: 1, Rf value are 0.55, (iodine colour developing, fluorescence developing 254nm).Principal spot purity is greater than 95%.
Embodiment 17
The preparation of romurtide
With B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (ONb)-CO (CH 2) 16CH 3] 10.3 gram full guard romurtides are dissolved in the mixed solvent of 100mL glacial acetic acid and 200mL methyl alcohol; add Zn powder 33 grams in batches; reactionization is 6 hours under the normal temperature; the elimination solid; removal of solvent under reduced pressure, residue grinds with sherwood oil, filter collection solid; solid is given a baby a bath on the third day after its birth inferior with sherwood oil, dry to such an extent that romurtide 5.5 restrains.Yield 95%.MS?888.1.(M+1)；1HNMR(ppm)(DMSO-d 6)：8.12(2H，d)，8.0(4H，s)，7.45(2H，d)7.06-7.20(10H，m)，6.01(2H，s)，5.98(1H，m)，5.53(1H，m)，4.71.0(1H，s)，4.63(2H，s)，4.53(1H，m)，4.45(1H，m)4.43(1H，m)，4.34(1H，m)，4.03(1H，m)，3.96(1H，m)，3.89(1H，m)，3.02(1H，m)，3.81(1H，m)，3.74(2H，s)，3.20(2H，m)，2.18(4H，t)，2.07(2H，m)，2.02(2H，s)，1.79(2H，m)，1.57(2H，m)，1.48(3H，d)，1.39(2H，d)，1.33(2H，m)，1.29(30H，m)，0.96(3H，t)。
Propyl carbinol: water=4: 1, Rf value are 0.65, (iodine colour developing).Principal spot purity is greater than 95%.
Embodiment 19
The preparation of romurtide
With B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (OBzl)-CO (CH 2) 16CH 3] 8.3 gram full guard romurtides are dissolved in the mixed solvent of 100Ml acetate and 200mL methyl alcohol, add palladium black or Pd (OH) 23 gram normal pressure hydrogenations 8 hours, the elimination catalyzer, removal of solvent under reduced pressure, residue grinds with sherwood oil, filter collection solid, solid is given a baby a bath on the third day after its birth inferior with sherwood oil, dry to such an extent that romurtide 5.5 restrains.Yield 95%.MS?888.1.(M+1)；1HNMR(ppm)(DMSO-d6)5.19(1H，d)，4.28-4.46(4H，m)，3.88(1H，q)，3.86-3.74(3H，m)，3.66(1H，q)，3.51(1H，t)，3.19(2H，t)，2.38(2H，t)，2.18(2H，t)，1.96(3H，s)，1.92-1.23(38H，m)，1.42(3H，d).，1.39(3H，d)，，0.96(3H，t)。
Propyl carbinol: water=4: 1, Rf value are 0.65, (iodine colour developing).Principal spot purity is greater than 95%.The HPLC purity assay is consistent with TCL, and purity is 94.8%, and through HPLC purifying, the purity of romurtide can reach 98% medicinal standard even higher purity.
Embodiment 18
The preparation of romurtide
With B1, B4,6-NAcMur-Ala-D-iso-Glu-[Lys (OBzl)-CO (CH 2) 16CH 3] 8.3 gram full guard romurtides are dissolved in the mixed solvent of 100Ml DMF and 200mL methyl alcohol, add 10%Pd-C 3 gram normal pressure hydrogenations 6 hours, the elimination catalyzer; removal of solvent under reduced pressure, residue grinds with sherwood oil, filter collection solid; solid is given a baby a bath on the third day after its birth inferior with sherwood oil, dry to such an extent that romurtide 5.5 restrains.Yield 95%.MS?888.1.(M+1)；1HNMR(ppm)(DMSO-d6)5.19(1H，d)，4.28-4.46(4H，m)，3.88(1H，q)，3.86-3.74(3H，m)，3.66(1H，q)，3.51(1H，t)，3.19(2H，t)，2.38(2H，t)，2.18(2H，t)，1.96(3H，s)，1.92-1.23(38H，m)，1.42(3H，d).，1.39(3H，d)，，0.96(3H，t).
Propyl carbinol: water=4: 1, Rf value are 0.65, (iodine colour developing).Principal spot purity is greater than 95%.The HPLC purity assay is consistent with TCL, and purity is 95.90% (accompanying drawing 3), and through HPLC purifying, the purity of romurtide can reach 98% medicinal standard even higher purity.
The comparative example 1
Technical scheme according to embodiment among the synthetic document US 4317771 of romurtide and the EP002367, adopt full liquid phase synthetic, the purity that obtains the romurtide finished product is respectively 25.93% (accompanying drawing 1) and 14.08% (accompanying drawing 2), through three purifying of HPLC, purity is respectively 90% and 88% romurtide.As can be seen, it is low that prior art is produced the romurtide productive rate, and purity is low, the cost height.

Claims (8)

1. the preparation method of optical activity romurtide may further comprise the steps: with protection R 1-D-iso-Gln-(OR 2) be raw material, slough behind the R1 and R 3The condensation in the presence of Hobt/DCC of-L-L-Ala generates R 3-L-Ala-D-iso-Gln-(OR 2), the latter carries out hydrogenation under Pd/C catalysis, obtain dipeptides R 3-L-Ala-D-iso-Gln-(OH); With protection Methionin is raw material, with stearic acid at DCC, Hobt/DCC, perhaps thionyl chloride exists down that condensation generates CH 3(CH 2) 16CO-Lys (R 4)-OR 5Two peptide fragment are sloughed R 4After CH 3(CH 2) 16CO-Lys-OR 5R 3-L-Ala-D-iso-Gln-(OH) and CH 3(CH 2) 16CO-Lys-OR 5Condensation generates tetrapeptide R in the presence of Hobt/DCC 3-L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (III), slough protecting group R 3Get L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3), L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) generate B1, B4,6-NAcMur-Ala-D-iso-Glu-(Lys (OR with the condensation of protection teichoic acid 5)-CO (CH 2) 16CH 3), slough protecting group and generate romurtide, wherein R 1Be selected from Fmo c, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 2Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, But, Ptm, Am, Me, Ph, Pic, Pnc; R 3With R 1Identical or different, be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 4With R 1, R 3Identical or different, be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps; R 5With R 2Identical or different, be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, But, Ptm, Am, Me, Ph, Pic, Pnc.
2. according to the preparation method of claim 1, it adopts acid or alkali deprotection base.
3. according to the method for claim 2, described acid wherein is the HCl/ trifluoroethanol, the CH of 2-50%TFA 2Cl 2Or dilute acetic acid solution, HBr/ acetic acid solution, BTFA, CF3SO3H, FSO3H, HF, H2-Pd in liquefied ammonia, Zn in dilute acetic acid, HCl dioxane solution, cold trifluoroacetic acid or rare methylsulfonic acid; Alkali is hydrazine hydrate, hexahydropyridine, sodium amide.
4. according to the method for claim 2, may further comprise the steps:
A) with R 1-D-iso-Gln (OR 2) 2Be raw material, slough protecting group with acid or alkali and then get D-iso-Gln (OR 2) (I);
B) (I) and R 3The condensation in the presence of Hobt/DCC of-L-L-Ala generates R 3-L-Ala-D-iso-Gln-OR 2(II);
C) (II) under Pd/C catalysis or zinc-acetate react, slough protecting group R 2, obtain R 3-L-Ala-D-iso-Gln-OH (III);
D) with L-Lys (R 4)-OR 5Be raw material, generate CH with stearic acid condensation in the presence of DCC or Hobt/DCC 3(CH 2) 16CO-Lys (R 4)-OR 5(IV);
E) CH 3(CH 2) 16CO-Lys (R 4)-OR 5(IV) in acid or alkaline solution, slough R 4, obtain CH 3(CH 2) 16CO-Lys-OR 5(V);
F) intermediate R 3-L-Ala-D-iso-Gln-(OH) is (III) in the presence of Hobt/DCC, with CH 3(CH 2) 16CO-Lys-(R 4)-OR 5(V) carry out condensation and generate R 3-L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VI);
G) R 3-L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VI) slough protecting group with acid or alkaline solution and get L-Ala-D-iso-Gln-(Lys (OR 5)-CO (CH 2) 16CH 3) (VII);
H) intermediate L-Ala-D-i so-Gln-L-(Lys (OR 5)-CO (CH 2) 16CH 3) (VII) in the presence of Hobt/DCC, carry out condensation with teichoic acid, generate protection romurtide (VIII);
I) BHBBS-Ala-D-iso-Glu-(Lys (OR 5)-CO (CH 2) 16CH 3) in the presence of catalyzer or Zn/ acetate, slough protecting group and generate romurtide;
Figure FDA0000055426250000021
Figure FDA0000055426250000031
5. according to the method for claim 4, employed solvent is selected from N in the reaction of step a), dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane;
The solvent that uses in the step b) reaction is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, dichloromethane solvent;
The solvent that uses in the step c) reaction is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, acetate, methyl alcohol, ethanol;
The solvent that uses in the reaction of step d) is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride;
The solvent that uses in the reaction of step e) is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane solvent;
Reaction solvent is selected from N in the reaction of step f), dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, DMF, trichloromethane, tetrahydrofuran (THF);
The solvent that uses in the step g) reaction is selected from N, dinethylformamide, N, N-diethyl acetamide, methyl-sulphoxide, methylene dichloride, dioxane solvent;
Step h) in the reaction, reaction solvent is selected from DMF, methylene dichloride, tetrahydrofuran (THF);
Step I) catalyzer is RanyNi or Pd/C or palladium black or Pd (OH) in 2, reaction solvent is acetate and/or methyl alcohol, DMF.
6. according to the preparation method of above arbitrary claim, its synthetic route is as follows:
Figure FDA0000055426250000041
Figure FDA0000055426250000051
Figure FDA0000055426250000061
7. prepare the midbody compound of romurtide, be selected from following structure:
Figure FDA0000055426250000062
Figure FDA0000055426250000071
R 2Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, Bu t, Ptm, Am, Me, Ph, Pic, Pnc; R 3Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps;
R 3Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps;
R 4Be selected from Fmoc, Adoc, Aoc, Z, BOC, Trt, Bpoc, Ddz, Ibc, Dpp, Mz, Doc, Dpc, 2Cz, 4Cz, 4Nz, Paz, Dobz, Inc, Iec, Tcc, Msc, For, Tfa, Pht, Tos, Nps;
R 5Be selected from Dpm, Mob, Pmb, Tmb, Bzl, Nb, Tms, But, Ptm, Am, Me, Ph, Pic, Pnc.
8. according to the compound of claim 7, have following structure:
Figure FDA0000055426250000101
Figure FDA0000055426250000111
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