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CN101817870B - Preparation method of thyrotropin releasing hormone and pharmaceutically acceptable salt thereof - Google Patents

Preparation method of thyrotropin releasing hormone and pharmaceutically acceptable salt thereof Download PDF

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CN101817870B
CN101817870B CN2010101373275A CN201010137327A CN101817870B CN 101817870 B CN101817870 B CN 101817870B CN 2010101373275 A CN2010101373275 A CN 2010101373275A CN 201010137327 A CN201010137327 A CN 201010137327A CN 101817870 B CN101817870 B CN 101817870B
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pglu
acid
pro
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preparation
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CN101817870A (en
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辛军
韩震
张伟
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Zhang Wei
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Jinan Zhaode Biotechnology Co ltd
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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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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention relates to a preparation method of Thyrotropin Releasing Hormone (TRH) and a medicinal salt thereof. The method comprises reacting Z-L-pGlu-L-His with Carbonyldiimidazole (CDI); then adding L-Pro-NH2Carrying out reaction; then mixing with a poor solvent, standing, and removing supernatant to obtain a residue; adding a solvent to the residue to form a residue solution; mixing the residue solution with a poor solvent, filtering, and post-treating the filter cake to obtain Z-L-pGlu-L-His-L-Pro-NH2For the Z-L-pGlu-L-His-L-Pro-NH2Catalytic hydrogenation is carried out to obtain TRH, which is optionally salted. The method of the invention adopts CDI as the condensing agent, has fast reaction speed, does not need excessive feeding of reaction raw materials, does not need recrystallization step for product post-treatment, and can obtain Z-L-pGlu-L-His-L-Pro-NH only by adding insoluble solvent into the reaction mixture2And the yield is greatly improved.

Description

The preparation method of a kind of TSH and pharmacologically acceptable salt thereof
Technical field
The present invention relates to a kind of preparation method of polypeptide drugs of pharmaceutical field, particularly relate to the large-scale preparation method of a kind of TSH and pharmacologically acceptable salt thereof.
Background technology
TH (swashing) hormone-releasing hormone is called the plain releasing hormone of TH (swashing) again, and English abbreviates TRH or TRF as, and the chemical structure of TRH is L-pyroglutamyl-L-histidyl--L-prolineamide (L-pGlu-L-His-L-Pro-NH 2).TRH is a hypothalamus excretory hormone, is present in people and the multiple animal body, except that the synthetic and secretion with stimulation TTH (TSH), can also cause the release of prolactin antagonist.In addition; TRH also has good promoter action to cns; As promote Spinal injury to recover, quicken the recovery of local asphyxia anoxybiotic Cranial nerve injury as birth trauma, improve the recovery etc. that cerebral blood flow alleviates cerebral edema, promotes the neural function sexual dysfunction of brain injury, prevent epileptic seizures and short clear-headed effect in addition.TRH has been put into Japanese Pharmacopoeia (14 editions), European Pharmacopoeia (5.2 editions), British Pharmacopoeia (2003 editions) as the medicine that the mankind use.
Since the chemical structure of TRH is illustrated, a lot of about its synthetic report.In these synthetic reports; Remove U.S. Abbott (Abbott Laboratories) and Japanese military field (the Takeda Chemical Industries of chemical company; Ltd.) outside the compound method of report, other compound methods all need adopt the way of column chromatography for separation just can obtain high-quality TRH.Well-known is that the technology of use column chromatography can make production cost rise significantly on producing, and operation easier is increased, and is difficult to realize that industrialization prepares in a large number.
The Kurath of U.S. Abbott in 1974 and Thomas; The Masahiko Fujino of Japan military field chemical company etc.; Successively reported very similar synthetic route respectively; This route need not make and use column chromatography, but midbody is adopted the way of recrystallization, can prepare high-quality TRH.The compound method that they adopt is: with Z-L-pGlu is starting raw material, is prepared into active ester, and an alkali metal salt with L-His reacts again, obtains Z-L-pGlu-L-His, last and L-Pro-NH 2Under the effect of DCC, obtain Z-L-pGlu-L-His-L-Pro-NH 2Z-L-pGlu-L-His-L-Pro-NH 2Through palladium carbon catalytic hydrogenation, obtain high-quality TRH again.The difference of Abbott and Wu Tian company is carried out condensation reaction for the former adopts the DCC method, and the latter uses DCC/HONB (N-hydroxyl-5-norbornylene-2,3-dicarboximide) method to carry out condensation reaction, and this external reaction solvent aspect is slightly different.The compound method of nineteen ninety Zhang Jiajin report is identical with Wu Tian company, is only adopting Abbott's reported method aspect the detail of testing.
The method that Abbott and Wu Tian company prepare TRH exists a serious defective, i.e. Z-L-pGlu-L-His and L-Pro-NH 2Under the effect of DCC (NSC 57182), obtain Z-L-pGlu-L-His-L-Pro-NH 2In order to obtain specification product; The actual recovery of post-processing step after with ethanol or acetonitrile recrystallization is lower, has only 45-58%, and the by product DCU (NSC 30023), additive HONB or the HOSu that should the step reaction produce, superfluous DCC; Even, still possibly remain in the product through re-crystallization step.The product Z-L-pGlu-L-His-L-Pro-NH that this step obtains 2After Z (carbobenzoxy-(Cbz)) protection base is sloughed in hydrogenation, no longer carry out product purification, above-mentioned impurity (DCC, DCU, HONB, HOSu) all possibly remain in the finished product (TRH).Particularly residual DCC is because toxicity has very big harm greatly.Obviously, Abbott, Wu Tian company, the Zhang Jiajin method for preparing TRH can't satisfy the needs of economy of large scale ground preparation high quality TRH.
The preparation method of Z-L-pGlu-L-His can prepare according to following literature method:
P.Kurath，A.M.Thomas，Helv?Chimica?Acta，1973，56(5)：1656-61.
Chitoshi?Hatanaka，et?al.Biochem?Biophy?Res?Commun.1974，60(4)：1345-50.
Masahiko?Fujino，et?al.Chem?Pharm?Bull?1974，22(8)：1857-63.
Zhang Jiajin, etc. the journal .1990 of Capital Medical College, 11 (4): 276-8.
This shows that the preparation method of above-mentioned existing TSH still has inconvenience and defective, and demands urgently further improving.The present invention adopts carbonyl dimidazoles (CDI) to be used for synthetic Z-L-pGlu-L-His-L-Pro-NH as the polypeptide condensing agent 2, can overcome the deficiency of prior art scheme, advantage of the present invention is to improve yield greatly, reduce production costs, improve product purity, simplify post-processing step, making economically, mass preparation TRH becomes possibility.
Summary of the invention
Main purpose of the present invention is; Overcome the defective of preparing method's existence of existing TSH and pharmacologically acceptable salt thereof; And provide a kind of new TSH and the preparation method of pharmacologically acceptable salt thereof, technical problem to be solved to make it adopt CDI to be used for synthetic Z-L-pGlu-L-His-L-Pro-NH as the polypeptide condensing agent 2Thereby, but realize scale preparation TRH and pharmaceutically acceptable acid salt thereof.
The object of the invention and solve its technical problem and adopt following technical scheme to realize.The preparation method of a kind of TSH that proposes according to the present invention, this method comprises:
Step 1: with Z-L-pGlu-L-His is the reaction of raw material and carbonyl dimidazoles, forms Z-L-pGlu-L-His acylimidazole activated intermediate, and Z is a carbobenzoxy-(Cbz);
Step 2: in the product that step 1 obtains, add L-Pro-NH 2React, obtain Z-L-pGlu-L-His-L-Pro-NH 2
Step 3: to described Z-L-pGlu-L-His-L-Pro-NH 2Carry out catalytic hydrogenation, can obtain TSH (L-pGlu-L-His-L-Pro-NH 2).
This method is further comprising the steps of:
● in the reactant that step 2 obtains, add poor solvent and stirring; Leave standstill then; Remove supernatant; Obtain resistates, the reactant of step 2 and the volume ratio of poor solvent are 1: 20 to 1: 30, and described poor solvent is several kinds a mixture one of in ethers, halohydrocarbon, alkane, naphthenic hydrocarbon and the aromatic hydrocarbon or wherein;
● one of in resistates, add in alcohol, THF, dioxane, acetone and the nitrile or the mixture of several kinds of materials wherein, stir down at 60 ℃ to 75 ℃ and make resistates dissolving formation resistates solution;
● resistates solution is mixed with poor solvent, filter then, aftertreatment obtains product Z-L-pGlu-L-His-L-Pro-NH to filter cake 2, described poor solvent is the mixture of one or more materials of ether, isopropyl ether and t-butyl methyl ether;
The object of the invention and solve its technical problem and also can adopt following technical measures further to realize.The preparation method of aforesaid TSH; Wherein said step 1 comprises: at-20 ℃ under 0 ℃; In the solution of Z-L-pGlu-L-His or suspension liquid, add carbonyl dimidazoles, the mol ratio of Z-L-pGlu-L-His and carbonyl dimidazoles is 1: 1-1: 3; Under agitation carry out the reaction of step 1 then, temperature of reaction is-20 ℃ to 0 ℃, and the reaction times is 10 minutes to 1 hour.Preferred charge temperature is-15 ℃ to-10 ℃, and the mol ratio of preferred Z-L-pGlu-L-His and carbonyl dimidazoles is 1: 1-1: 2.5, and preferred temperature of reaction is-15 ℃ to-10 ℃, the preferred reaction times is 25 minutes to 45 minutes.The solution of described Z-L-pGlu-L-His or the solvent of suspension liquid are THF, DMF, DMSO, DCM, EtOAc, NMP, 1; 2-ethylene dichloride, 1, one of among 4-dioxane, acetone, chloroform, acetonitrile, DMA and the HMPA or the wherein mixture of several kinds of materials.Preferred solvent is the mixture of several kinds of materials one of among DMF, DMSO, the NMP or wherein.
Described step 2 comprises :-15 ℃ under 0 ℃, in the product that step 1 obtains, add L-Pro-NH 2, Z-L-pGlu-L-His and L-Pro-NH 2Mol ratio be 1: 1; Under agitation react then, temperature of reaction is-15 ℃ to 0 ℃, and the reaction times is 1 hour to 5 hours.Preferred charge temperature is-15 ℃ to-10 ℃, and preferred temperature of reaction is-15 ℃ to-10 ℃, and the preferred reaction times is 2 hours to 5 hours.
Described step 3 comprises: with Z-L-pGlu-L-His-L-Pro-NH 2Through palladium carbon catalytic hydrogenation (normal pressure, or a hydrogen-pressure 1-6 normal atmosphere), obtain L-pGlu-L-His-L-Pro-NH 2, i.e. TRH free alkali.The hydrogen-pressure of preferred catalytic hydrogenation is a 4-6 normal atmosphere, and the preferred hydrogen time is 6-8 hour, and preferred hydrogenation temperature is 20-25 ℃.
The present invention also proposes a kind of preparation method of TSH pharmacologically acceptable salt, and this method is at first to prepare L-pGlu-L-His-L-Pro-NH through aforesaid method 2, and then react with tartrate, fumaric acid, toxilic acid, acetic acid, oxalic acid, lactic acid, Citric Acid, oxysuccinic acid, phenylformic acid, methylsulfonic acid, Sorbic Acid, L-glutamic acid, aspartic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, Hydrogen bromide or hydroiodic acid HI.
By technique scheme, the present invention adopts CDI to prepare TSH as peptide synthetic condensing agent and pharmacologically acceptable salt has advantage at least:
The firstth, the by product of reaction is carbonic acid gas and imidazoles; Carbonic acid gas is a volatile gases; Imidazoles dissolves in multiple organic solvents such as ether, can remove through solvent washing at an easy rate, and the product aftertreatment no longer needs re-crystallization step; Only be employed in the reaction mixture and add the indissoluble solvent and can obtain product, yield is increased substantially;
The secondth, the specific activity DCC of the activating terephthalamide amine that CDI forms in reaction does the active height of the active intermediate of condensing agent formation, is swift in response, and yield is high, and product purity is high;
The 3rd is that mol ratio such as each reaction raw materials employing feeds intake, and reaction finishes each reaction raw materials of back and all participated in reaction and residue not, and aftertreatment is simplified.
As used herein, unless otherwise indicated, term has following implication commonly used in this area usually.
As used herein, term " TRH " refers to TH (swashing) hormone-releasing hormone.
As used herein, term " Z " refers to carbobenzoxy-(Cbz).
As used herein, term " Z-L-pGlu-L-His-L-Pro-NH 2" refer to benzyloxy carbonyl acyl-L-pyroglutamyl-L-histidyl--L-prolineamide.
As used herein, term " L-pGlu-L-His-L-Pro-NH 2" refer to L-pyroglutamyl-L-histidyl--L-prolineamide.
As used herein, term " HA " refers to pharmaceutically acceptable acid.
As used herein, term " Z-L-pGlu-L-His " refers to benzyloxy carbonyl acyl-L-pyroglutamyl-L-Histidine.
As used herein, term " Z-L-pGlu " refers to benzyloxy carbonyl acyl-L-Pyrrolidonecarboxylic acid.
As used herein, term " L-His " refers to the L-Histidine.
As used herein, term " L-Pro-NH 2" refer to the L-prolineamide.
As used herein, term " DCC " refers to NSC 57182.
As used herein, term " HONSu " refers to N-hydroxy-succinamide.
As used herein, term " HONB " refers to N-hydroxyl-5-norbornylene-2,3-dicarboximide.
As used herein, term " CDI " refers to carbonyl dimidazoles.
As used herein, term " THF " refers to THF.
As used herein, term " DMF " refers to N, dinethylformamide.
As used herein, term " DMSO " refers to DMSO 99.8MIN..
As used herein, term " DCM " refers to methylene dichloride.
As used herein, term " EtOAc " refers to ETHYLE ACETATE.
As used herein, term " NMP " refers to the N-N-methyl-2-2-pyrrolidone N-.
As used herein, term " DMA " refers to DMAC N,N.
As used herein, term " HMPA " refers to hexametapol.
Above-mentioned explanation only is the general introduction of technical scheme of the present invention, understands technique means of the present invention in order can more to know, and can implement according to the content of specification sheets, below with preferred embodiment of the present invention specify as after.
Embodiment
Reach technique means and the effect that predetermined goal of the invention is taked for further setting forth the present invention, below in conjunction with preferred embodiment, to specifying as follows according to the TSH of the present invention's proposition and the preparation method of pharmacologically acceptable salt thereof.
Z-L-pGlu-L-His-L-Pro-NH 2The preparation method
Adopt the method for prior art to prepare Z-L-pGlu-L-His.
Step 1: in the solution or suspension liquid of Z-L-pGlu-L-His, add CDI, in above-mentioned reaction, the preferred molar ratio that Z-L-pGlu-L-His and CDI feed intake is 1: 1 to 1: 2.5.Owing to the crystal water that contains 1.5 molecules in the Z-L-pGlu-L-His molecule for preparing by document usually, CDI touches water molecules and can decompose rapidly, so the mol ratio of the add-on of CDI and Z-L-pGlu-L-His is 2.5: 1; For adopting anhydrous Z-L-pGlu-L-His to react, the add-on of CDI and the mol ratio of Z-L-pGlu-L-His are 1: 1.For the generation that reduces side reaction and suppress activation Z-L-pGlu-L-His racemization takes place, charge temperature and temperature of reaction are controlled at-15 ℃ to-10 ℃.Temperature of reaction should remain on more than-15 ℃, causes side reaction to take place slowly to avoid reacting.
Step 2: in the solution or suspension liquid of Z-L-pGlu-L-His, behind the adding CDI, the bubble of visible carbonic acid gas is emitted, and stops to bubble, is approximately 25 minutes to 45 minutes during this period of time, can add L-Pro-NH after bubble stops 2Generation and inhibition racemization in order to reduce side reaction equally, temperature of reaction should remain on-15 ℃ to-10 ℃.The preferred reaction times is 2 hours to 5 hours.
Aftertreatment in the step 2: the reaction mixture that step 2 is obtained mixes with poor solvent, stirs, and leaves standstill.Reaction product L-pGlu-L-His-L-Pro-NH 2Be insoluble to poor solvent and precipitate, the by product imidazoles of reaction can be dissolved in the poor solvent, and is removed.Preferred poor solvent is the mixture of one or more materials of ether, isopropyl ether and t-butyl methyl ether.
Purifying in the step 2: the mixture of one or more materials in resistates adding ethanol, Virahol, THF, dioxane, acetone and acetonitrile, stir also heating, resistates is dissolved in forms resistates solution in the above-mentioned solvent.L-pGlu-L-His-L-Pro-NH 2Can react with methyl alcohol, so, L-pGlu-L-His-L-Pro-NH 2Should avoid contacting with methyl alcohol.The temperature of preferred heating is 65 ℃ to 70 ℃.Subsequently, resistates solution is mixed with poor solvent and stir.Preferred poor solvent is ether, isopropyl ether, t-butyl methyl ether, and their mixture.Preferred churning time is 15 minutes to 2 hours.Filter then, wash the product that obtains with poor solvent, drying under reduced pressure, the product that obtains are Z-L-pGlu-L-His-L-Pro-NH 2
L-pGlu-L-His-L-Pro-NH 2Preparation
Z-L-pGlu-L-His-L-Pro-NH 2Be dissolved in 1: 1 the THF/ water mixed liquid, at 20-25 ℃ through palladium carbon catalytic hydrogenation (normal pressure, or a hydrogen-pressure 1-6 normal atmosphere); Filter through zeyssatite or aluminum oxide; Removing THF under reduced pressure below 40 ℃, the aqueous solution lyophilize that obtains obtains TRH free alkali (L-pGlu-L-His-L-Pro-NH 2).
The preparation of TRH pharmacy acceptable salt
The TRH free alkali obtains pharmaceutically acceptable salt with acid-respons again.Described acid is tartrate, fumaric acid, toxilic acid, acetic acid, oxalic acid, lactic acid, Citric Acid, oxysuccinic acid, phenylformic acid, methylsulfonic acid, Sorbic Acid, L-glutamic acid, aspartic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, Hydrogen bromide or hydroiodic acid HI.For example, in ethanolic soln, react, obtain crystalline TRH tartrate with L-tartrate.
Instance 1 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
Z-L-pGlu-L-His under 60 ℃ and 0.1mmHg vacuum dry 24 hours makes anhydrous Z-L-pGlu-L-His.(11g 27.47mmol) is dissolved in the dry DMF of 60mL, is cooled to-20 ℃, and (4.5g 27.47mmol), keeps-15 ℃ and stirred 45 minutes to add solid CDI to get anhydrous Z-L-pGlu-L-His.Then, add L-Pro-NH 2(3.1g 27.47mmol), stirred 1 hour at-15 ℃, was warming up to room temperature then naturally, stirred 3 hours.Stir down reaction soln is poured in the 1.5L ether, left standstill 24 hours, supernatant is inclined; Add 2L THF and low-grade fever to 60 then and ℃ make the resistates dissolving on bottle wall, under fierce the stirring, this solution is poured in the 1.5L anhydrous diethyl ether again, produce a large amount of white powder solids; Continue under the room temperature to stir 15 minutes, suction filtration whitens toner end (each 200mL with anhydrous diethyl ether; 3 times), obtain 12.9g (yield 93%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 98.9%.
The HPLC testing conditions: normal phase silicagel column, moving phase: gradient elution, ethanol: chloroform (2: 3) to ethanol: chloroform (3: 2), detect wavelength 250nm, flow velocity 1ml/min.
Instance 2 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
(11g 25.74mmol) is dissolved in the dry DMF of 55mL Z-L-pGlu-L-His, is cooled to-15 ℃, and (10.4g 64.34mmol), keeps-10 ℃ and stirred 30 minutes to add solid CDI.Add L-Pro-NH then 2(2.9g, 25.74mmol) ,-10 ℃ were stirred 1 hour, were warming up to room temperature then naturally, stirred 5 hours.Stir down reaction soln is poured in the 2L ether, left standstill 48 hours, supernatant is inclined; Add 1.2L absolute ethyl alcohol and low-grade fever to 75 then and ℃ make the resistates dissolving on bottle wall, under fully stirring, the 2L isopropyl ether is slowly poured in this ethanolic soln again, produce a large amount of white powder solids; Continue under the room temperature to stir 2 hours, suction filtration whitens toner end (each 200mL with anhydrous diethyl ether; 3 times), obtain 12.6g (yield 97%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 98.5%.
Instance 3 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
(11g 25.74mmol) is suspended in the mixing solutions of 40mL analytical pure DMF and 40mL analytical pure THF Z-L-pGlu-L-His, is cooled to-10 ℃, and (12.5g 77.22mmol), keeps-5 ℃ and stirred 25 minutes to add solid CDI.Add L-Pro-NH then 2(2.9g 25.74mmol), stirred 1 hour at-5 ℃, was warming up to room temperature then naturally, stirred 5 hours.Stir down reaction soln is poured in the 2L ether, left standstill 72 hours, supernatant is inclined; Add 1.5L Virahol and low-grade fever to 70 then and ℃ make the resistates dissolving on bottle wall, under fierce the stirring, this ethanolic soln is poured in the 1.8L t-butyl methyl ether again, produce a large amount of white powder solids; Continue under the room temperature to stir 5 minutes, suction filtration whitens toner end (each 300mL with t-butyl methyl ether; 3 times), obtain 11.7g (yield 90%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 98.1%.
Instance 4 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
Z-L-pGlu-L-His under 60 ℃ and 0.1mmHg vacuum dry 24 hours makes anhydrous Z-L-pGlu-L-His.(11g 27.47mmol) is dissolved in the dry DMF of 60mL, is cooled to-5 ℃, and (4.5g 27.47mmol), keeps 0 ℃ and stirred 25 minutes to add solid CDI to get anhydrous Z-L-pGlu-L-His.Then, add L-Pro-NH 2(3.1g 27.47mmol), stirred 1 hour at 0 ℃, was warming up to room temperature then naturally, stirred 4 hours.Stir down reaction soln is poured in the 1.5L ether, left standstill 24 hours, supernatant is inclined; Add 1.5L acetonitrile and low-grade fever to 65 then and ℃ make the resistates dissolving on bottle wall, under fierce the stirring, this ethanolic soln is poured in the 1.5L anhydrous diethyl ether again, produce a large amount of white powder solids; Continue under the room temperature to stir 8 hours, suction filtration whitens toner end (each 200mL with anhydrous diethyl ether; 3 times), obtain 11.9g (yield 86%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 98.0%.
Instance 5 L-pGlu-L-His-L-Pro-NH 2Preparation
Z-L-pGlu-L-His-L-Pro-NH 2(12g 23.73mmol) is dissolved in the mixed solution of THF and 600mL water of 600mL, adds 2.4g palladium carbon (10%); Pressure hydration (4 normal atmosphere) 6 hours, through the zeyssatite suction filtration, filtering palladium carbon; With 1: 1 THF/ water mixed liquid filter wash layer 3 times, filtrate decompression concentrates and removes THF, and bath temperature is no more than 40 ℃; The obtained aqueous solution lyophilize obtains white amorphous solid 8.5g (yield 97%), is the TRH free alkali.HPLC detects purity 98.6% (according to the method check of British Pharmacopoeia 2003 editions).
Instance 6 L-pGlu-L-His-L-Pro-NH 2The preparation of tartrate
Instance 5 obtains the TRH free alkali, and (8g 21.56mmol) is dissolved in the 6.9mL water, and (3.2g 21.32mmol), after the dissolving, adds the 41mL absolute ethyl alcohol again, and room temperature was placed 2 days, crystallization occurred to add L-tartrate then.Add the 79mL absolute ethyl alcohol again, room temperature was placed 5 days again, mass crystallization occurred, and suction filtration obtains TRH tartrate 10.7g (yield 95%).HPLC detects purity 99.2% (according to the method check of Japanese Pharmacopoeia 14 editions).
Adopt fumaric acid, toxilic acid, acetic acid, oxalic acid, lactic acid, Citric Acid, oxysuccinic acid, phenylformic acid, methylsulfonic acid, Sorbic Acid, L-glutamic acid, aspartic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, Hydrogen bromide or hydroiodic acid HI to replace above-mentioned tartrate just can obtain the corresponding pharmacy acceptable salt of TRH.
Comparative Examples 1 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
With reference to P.Kurath, A.M.Thomas, Helv Chimica Acta, 1973,56 (5): 1656-61. experimentizes.
Z-L-pGlu-L-His (11g, 25.74mmol) and L-Pro-NH 2(3.6g 31.14mmol) is dissolved in the dry DMF of 60mL, stirs, and ice bath is cooled to 0 ℃, and (6g 29.06mmol), adds the 20mL dry DMF again, keeps 0 ℃ and stirred 2 hours, is warming up to room temperature, stirred overnight then naturally to add solid DCC.Suction filtration is washed deposition (11mL X 3) with DMF.To filtrate under stirring reaction soln will be poured in the 1.5L ether, leave standstill 48 hours, supernatant will be inclined; Add 1.1L absolute ethyl alcohol and low-grade fever then and make the resistates dissolving on bottle wall; Under fierce the stirring, this ethanolic soln is poured in the 1.5L anhydrous diethyl ether again, produced a large amount of white powder solids, continue under the room temperature to stir 30 minutes; Suction filtration whitens toner end (200mL X 3) with anhydrous diethyl ether.White powder is used the absolute ethyl alcohol recrystallization again, is noted that especially this step product is very slow in the speed that absolute ethyl alcohol precipitates fully, to leave standstill usually 3 days or more than, otherwise yield can receive very big the influence.Obtain 5.8g (yield 45%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 93.4%.
Comparative Examples 2 Z-L-pGlu-L-His-L-Pro-NH 2Preparation
Experimentize with reference to following document:
Chitoshi?Hatanaka，et?al.Biochem?Biophy?Res?Commun.1974，60(4)：1345-50.
Masahiko?Fujino，et?al.Chem?Pharm?Bull?1974，22(8)：1857-63.
Zhang Jiajin, etc. the journal .1990 of Capital Medical College, 11 (4): 276-8.
Z-L-pGlu-L-His (11g, 25.74mmol) and L-Pro-NH 2(3.6g 31.14mmol) is dissolved in the dry DMF of 60mL, is cooled to 0 ℃ under stirring, and adds the 20mL dry DMF; (5.2g 29.12mmol), stirred 10 minutes to add HONB; (6g 29.06mmol), adds the 20mL dry DMF again to add solid DCC; Keep 0 ℃ and stirred 2 hours, be warming up to room temperature, stirred overnight then naturally.Suction filtration is washed deposition (11mL X 3) with DMF.To filtrate under stirring reaction soln will be poured in the 1.5L ether, leave standstill 48 hours, supernatant will be inclined; Add 1.1L absolute ethyl alcohol and low-grade fever then and make the resistates dissolving on bottle wall; Under fierce the stirring, this ethanolic soln is poured in the 1.5L anhydrous diethyl ether again, produced a large amount of white powder solids, continue under the room temperature to stir 30 minutes; Suction filtration whitens toner end (200mL X 3) with anhydrous diethyl ether.White powder is used the absolute ethyl alcohol recrystallization again, is noted that especially this step product is very slow in the speed that absolute ethyl alcohol precipitates fully, to leave standstill usually 3 days or more than, otherwise yield can receive very big the influence.Obtain 7.6g (yield 58%) Z-L-pGlu-L-His-L-Pro-NH 2, HPLC detects purity 91.2%.
Instance and Comparative Examples reaction conditions, product purity and yield synopsis
Experiment numbers The low-temp reaction time The room temperature reaction time Purity (%) Yield (%)
Instance 1 1 hour 45 minutes 3 hours 98.9 93
Instance 2 1 hour 30 minutes 5 hours 98.5 97
Instance 3 1 hour 25 minutes 5 hours 98.7 90
Instance 4 1 hour 25 minutes 4 hours 98.0 86
Comparative Examples 1 2 hours Spend the night 93.4 45
Comparative Examples 2 2 hours 10 minutes Spend the night 91.2 58
The above only is preferred embodiment of the present invention; The selection of materials such as the temperature of reaction of the foregoing description, reaction times and solvent, thinner has provided concrete scope; Those skilled in the art can make suitable selection with reference to the content of above-mentioned instance, and the contriver gives an example at this no longer one by one.Though the present invention discloses as above with preferred embodiment; Yet be not in order to limit the present invention; Anyly be familiar with the professional and technical personnel; In not breaking away from technical scheme scope of the present invention, make a little change or be modified to the equivalent embodiment of equivalent variations when the technology contents of above-mentioned announcement capable of using, be the content that does not break away from technical scheme of the present invention in every case;, all still belong in the scope of technical scheme of the present invention any simple modification, equivalent variations and modification that above embodiment did according to technical spirit of the present invention.

Claims (8)

1. a method for preparing TSH (TRH) comprises preparation Z-L-pGlu-L-His-L-Pro-NH 2Step and to described Z-L-pGlu-L-His-L-Pro-NH 2Carry out the step of catalytic hydrogenation,
It is characterized in that described preparation Z-L-pGlu-L-His-L-Pro-NH 2Step comprise:
(1): Z-L-pGlu-L-His and carbonyl dimidazoles reaction, form Z-L-pGlu-L-His acylimidazole activated intermediate, Z is a carbobenzoxy-(Cbz);
(2): in the product that step (1) obtains, add L-Pro-NH 2React, obtain Z-L-pGlu-L-His-L-Pro-NH 2
(3): the product that step (2) obtains is sloughed Z protection base, obtain TRH.
2. the preparation method of TSH according to claim 1 is characterized in that, described preparation Z-L-pGlu-L-His-L-Pro-NH 2Step also comprise:
In the reaction product that step (2) obtains, add poor solvent and stirring, leave standstill, remove supernatant, obtain resistates, the reactant of step (2) and the volume ratio of poor solvent are 1: 20 to 1: 30, and described poor solvent is an ether;
In resistates, add ethanol or Virahol, stir down at 60 ℃ to 75 ℃ and make resistates dissolving formation resistates solution;
Resistates solution is mixed with poor solvent, stir, filter then, to the filter cake aftertreatment, described poor solvent is the mixture of one or more materials of ether, isopropyl ether and t-butyl methyl ether.
3. the preparation method of TSH according to claim 1 and 2 is characterized in that, described step (1) comprising:
-20 ℃ under 0 ℃, in the solution of Z-L-pGlu-L-His or suspension liquid, add carbonyl dimidazoles, the mol ratio of Z-L-pGlu-L-His and carbonyl dimidazoles is 1: 1-1: 3;
Under agitation carry out the reaction of step (1) then, temperature of reaction is-20 ℃ to 0 ℃, and the reaction times is 10 minutes to 1 hour.
4. the preparation method of TSH according to claim 3 is characterized in that,
Described charge temperature is-15 ℃ to-10 ℃; Perhaps
The mol ratio of Z-L-pGlu-L-His and carbonyl dimidazoles is 1: 1-1: 2.5; Perhaps
Described temperature of reaction is-15 ℃ to-10 ℃; Perhaps
The described reaction times is 25 minutes to 45 minutes.
5. the preparation method of TSH according to claim 1 is characterized in that, described step (2) comprising:
-15 ℃ under 0 ℃, in the product that step (1) obtains, add L-Pro-NH 2, Z-L-pGlu-L-His and L-Pro-NH 2Mol ratio be 1: 1;
Under agitation react then, temperature of reaction is-15 ℃ to 0 ℃, and the reaction times is 1 hour to 5 hours.
6. the preparation method of TSH according to claim 5 is characterized in that,
Described charge temperature is-15 ℃ to-10 ℃; Perhaps
Described temperature of reaction is-15 ℃ to-10 ℃; Perhaps
The described reaction times is 2 hours to 5 hours.
7. the preparation method of TSH according to claim 3 is characterized in that,
The solution of described Z-L-pGlu-L-His or the solvent of suspension liquid are THF, DMF, DMSO, DCM, EtOAc, NMP, 1; 2-ethylene dichloride, 1, one of among 4-dioxane, acetone, chloroform, acetonitrile, DMA and the HMPA or the wherein mixture of several kinds of materials.
8. the preparation method of a TSH pharmacologically acceptable salt; It is characterized in that; Through preparing TRH, and then react with tartrate, fumaric acid, toxilic acid, acetic acid, oxalic acid, lactic acid, Citric Acid, oxysuccinic acid, phenylformic acid, methylsulfonic acid, Sorbic Acid, L-glutamic acid, aspartic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, Hydrogen bromide or hydroiodic acid HI like each described method of claim 1-7.
CN2010101373275A 2010-04-01 2010-04-01 Preparation method of thyrotropin releasing hormone and pharmaceutically acceptable salt thereof Expired - Fee Related CN101817870B (en)

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