WO2022217395A1 - 一种高纯的人促肾上腺皮质激素或其类似物及其规模化制备方法 - Google Patents

一种高纯的人促肾上腺皮质激素或其类似物及其规模化制备方法 Download PDF

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WO2022217395A1
WO2022217395A1 PCT/CN2021/086395 CN2021086395W WO2022217395A1 WO 2022217395 A1 WO2022217395 A1 WO 2022217395A1 CN 2021086395 W CN2021086395 W CN 2021086395W WO 2022217395 A1 WO2022217395 A1 WO 2022217395A1
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boc
lys
pro
val
arg
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PCT/CN2021/086395
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English (en)
French (fr)
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孟俊东
芮康宁
刘彬
韩园园
陈松
张昊宁
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南京汉欣医药科技有限公司
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Priority to PCT/CN2021/086395 priority Critical patent/WO2022217395A1/zh
Priority to CN202180088662.7A priority patent/CN116761809A/zh
Priority to US17/357,255 priority patent/US11419919B1/en
Priority to US17/891,946 priority patent/US20220401521A1/en
Publication of WO2022217395A1 publication Critical patent/WO2022217395A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • A61K38/35—Corticotropin [ACTH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/695—Corticotropin [ACTH]

Definitions

  • the invention relates to the technical field of polypeptide solid-phase synthesis and purification and preparation methods, in particular to a high-purity adrenocorticotropic hormone (human sequence) or an analog thereof and a large-scale preparation method thereof.
  • Adrenocorticotropin (English name corticotropin, ACTH for short) is a polypeptide hormone produced and secreted by the pituitary gland, containing 39 amino acid residues coupled in a linear sequence. The N-terminal 24 amino acid fragment is identical for all species and contains corticotropin activity.
  • ACTH stimulates the adrenal cortex and promotes the synthesis of corticosteroids, mainly glucocorticoids, but also sex hormones. It is used to treat certain neurological disorders, such as infantile spasms and multiple sclerosis, and to diagnose adrenal insufficiency. It has the function of a diagnostic reagent. It belongs to a polypeptide, a peptide hormone and a biological macromolecule.
  • Infantile spasms are an age-related epilepsy syndrome accompanied by regression of mental and motor development.
  • ACTH extracted from animals has been used as a first-line drug for the treatment of infantile spasms, but its mechanism of action is not clear.
  • ACTH preparation products sold on the market whose ACTH raw materials are all extracted from animals, are immunogenic and prone to adverse reactions, and the preparation of ACTH by chemical synthesis can avoid the problem of immunogenicity.
  • adrenocorticotropic hormone human sequence
  • the porcine-derived sequence of commercially available ACTH is SEQ ID NO.1 and/or SEQ ID NO.2:
  • Corticotropin (human sequence) sequence is as follows:
  • amino acid is abbreviated as SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF (SEQ ID NO. 3).
  • corticotropin human sequence
  • corticotropin human sequence
  • amino acid is abbreviated as SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF (SEQ ID NO. 4).
  • SEQ ID NO.3 because the N-25 position Asn is unstable under alkaline conditions, deamidation reaction can occur and become Asp, so part of adrenocorticotropic hormone (human sequence) (SEQ ID NO.3) can be converted It is in the form of N-25 deamidated adrenocorticotropic hormone (human sequence) SEQ ID NO.4.
  • SEQ ID NO.3 when SEQ ID NO.3 is synthesized in solid phase, there will be less than 1% of SEQ ID NO.4 impurities generated by deamidation reaction.
  • the prior art discloses that there is an Asn-Gly sequence in a polypeptide or protein, or when Gly is connected to Asn, a ⁇ -aspartyl transfer reaction is more likely to occur; the fragment Val-Tyr-Pro-Asn-Gly-Ala of ACTH is at pH Deamidation reaction is more likely to occur under the conditions of 5-12; adrenocorticotropic hormone also exists in two forms in vivo (H.Tonie Wright.Nonenzymatic Deamidation of Asparaginyl and Glutaminyl Residues in Proteins.Critical reviews in Biochemistry and Molecular Biology, 1991, 26(1):1-52.).
  • sequence of adrenocorticotropin human-derived sequence
  • the commercial porcine-derived adrenocorticotropic hormone mainly differs in the 31st amino acid.
  • the examples 1-6 of US4055524 adopt the Boc method solid-phase synthesis technology, but the amino acid protecting groups are different, and there are the following shortcomings.
  • acid is repeatedly used to deprotect, and trifluoroacetic acid is used to remove Boc every time.
  • the protective group is used, part of the peptide will fall off from the resin. The longer the synthesized peptide chain, the more serious the loss; acid treatment will cause some side reactions in the side chain.
  • the Boc synthesis method is especially not suitable for the synthesis of acids containing tryptophan Unstable peptides; the final deprotection adopts hydrofluoric acid method, which is extremely corrosive and highly toxic, and cannot be industrially produced.
  • Steps 1-14 of Example 4 of US3953415 disclose a liquid phase method (non-solid phase synthesis technology, steps 1-14 are all carried out homogeneous reactions in a solution system, and intermediates in each step need to be separated and purified by crystallization or column chromatography Remove excess unreacted raw materials and reagents) to prepare corticotropin (human sequence), obtain crude peptide of protective group by liquid phase fragment synthesis, deprotect with trifluoroacetic acid, and transfer the residue to ion exchange and freeze-dry to obtain crude product .
  • the crude product was purified by ion exchange, eluted with ammonium acetate solution in the purification process, and lyophilized to obtain the product containing ammonium acetate but not removed.
  • step 1 The total molar yield of this process is only 17% (89.9% in step 1, 99% in step 2, 97.2% in step 3, 92.7% in step 4, 93% in step 5, 95% in step 6 %, step 7 yield 86.2%, step 8 yield 82.6%, step 9 yield 96.9%, step 10 yield 88%, step 11 yield 86.1%, step 12 yield 90%, step 13 yield 85% , the yield in step 14 is 60%), the yield is low, and the specific purity is not specified.
  • adrenocorticotropin human-derived sequence
  • the preparation of adrenocorticotropin adopts the Boc method solid-phase synthesis technology or liquid-phase fragment synthesis method.
  • the present invention provides a high-purity adrenocorticotropic hormone (human sequence) or an analog thereof and a preparation method thereof.
  • the method adopts the Fmoc solid-phase synthesis technology to obtain the product, and synthesizes the C-15 peptide.
  • the synthesis conditions and post-processing conditions were optimized to solve the problems of incomplete reaction, many missing peptide impurities and difficult purification.
  • a composition comprising adrenocorticotropic hormone (human sequence), the purity of the synthetic adrenocorticotropic hormone (human sequence) is ⁇ 99%, and the maximum single hybrid The content is less than or equal to 0.5%, and the total impurity content is less than or equal to 1%.
  • the specific sequence of the adrenocorticotropic hormone from the N-terminus to the C-terminus is as follows:
  • the present invention provides the following another technical solution, a composition comprising an adrenocorticotropic hormone analog, characterized in that the adrenocorticotropic hormone analog has a purity of ⁇ 99% and a maximum single-impurity content. ⁇ 0.5%, total impurity content ⁇ 1%, the sequence of the adrenocorticotropin analog from N-terminus to C-terminus is as follows:
  • the high-purity corticotropin (human sequence) or analog thereof is at least 99.0% pure, or at least 99.5% pure, or at least 99.8% or higher.
  • the high-purity corticotropin (human sequence) or its analogs has a maximum single hetero ⁇ 0.5%, total hetero ⁇ 1%; further maximum single hetero ⁇ 0.4%, total hetero ⁇ 0.9%; further maximum single hetero ⁇ 0.9% 0.3%, total impurities ⁇ 0.8%; further maximum single impurities ⁇ 0.2%, total impurities ⁇ 0.7%; further maximum single impurities ⁇ 0.1%, total impurities ⁇ 0.5%; or less.
  • the purity of the adrenocorticotropic hormone or adrenocorticotropic hormone analog is greater than or equal to 99.5%, the maximum single impurity content is less than or equal to 0.1%, and the total impurity content is less than or equal to 0.5%.
  • composition comprising adrenocorticotropic hormone or its analog (purity ⁇ 99%, HPLC) according to the present invention is obtained by the following preparation method, that is, by Fmoc solid-phase synthesis method, respectively according to SEQ ID NO.3 or SEQ ID
  • the amino acid sequence shown in NO.4 is sequentially coupled with amino acids from the C-terminus to the N-terminus, and then purified to obtain a composition containing adrenocorticotropic hormone or a composition containing adrenocorticotropic hormone analogs.
  • the preparation method of the above-mentioned high-purity adrenocorticotropic hormone (human sequence) or its analogs comprises the following steps:
  • cleavage reagent to treat adrenocorticotropic hormone (human sequence) or its analog peptide chain-resin with a protective group, and cleave the adrenocorticotropic hormone (human sequence) or its analog peptide chain from the resin And remove all protective groups of the peptide chain to obtain a solution containing adrenocorticotropic hormone (human sequence) or its analogues;
  • the Fmoc solid-phase synthesis method refers to a synthesis method in which the reactants are connected to an insoluble solid-phase carrier.
  • the principle is to first introduce chlorine on the solid-phase carrier (such as a resin).
  • the desired polypeptide sequence is obtained by the reaction; during the synthesis process, the polypeptide is stably covalently bound to the surface of the solid-phase carrier, and after the reaction, the polypeptide sequence is cut off from the solid-phase carrier by using special chemical reagents.
  • Fmoc solid-phase synthesis method can greatly simplify the reaction procedure, simplify the post-processing process of the reaction, reduce the difficulty of product purification in each step, and reduce the loss of products in the post-processing stage; while the traditional liquid phase synthesis method has unreacted raw materials, reagents and targets. Intermediates or products need to be separated and purified by crystallization or even column chromatography. The operation is complicated, the time is long, the workload is large, and the liquid phase synthesis range is small, generally focusing on the synthesis of peptides within 10 amino acids.
  • the cleavage reagent refers to a chemical reagent that cleaves the synthesized polypeptide from the solid support and removes the protection of the side chain.
  • the sedimentation reagent refers to a chemical reagent for precipitating a synthesized polypeptide from a solution.
  • the coupled amino acids are coupled one by one or by fragments, and the N-terminal of the amino acid is protected by an Fmoc group.
  • the solid-phase synthetic resin in the step 1), can be selected from chloromethyl resin or Wang resin (polybenzyloxybenzyl alcohol resin) or 2-trityl methyl chloride resin or Resin such as Rink Amide AM Resin or Rink Amide MBHA Resin or Rink Amide Resin, preferably 2-trityl chloride resin.
  • the resin substitution degree is 0.1-1.0 mmol/g, preferably 0.2-0.8 mmol/g.
  • the method for coupling Fmoc-Phe-OH can be selected as follows: taking the solid phase synthetic resin and soaking it with an organic solvent such as DCM of 6-20L/Kg resin, adding Fmoc-Phe-OH Phe-OH and organic amines such as DIPEA react at room temperature for 2-4h to obtain Fmoc-Phe-resin, then add DIPEA/methanol (volume ratio 1:5-1:15) to block unreacted sites, and finally use 6-20L/Kg The resin is washed with organic solvents such as DCM, DMF, NMP, methanol, dried by suction, and dried under vacuum for use.
  • organic solvents such as DCM, DMF, NMP, methanol
  • Fmoc-Phe-resin Take Fmoc-Phe-resin and measure the molar substitution degree to determine the feeding amount.
  • Fmoc-Phe-OH can also be directly purchased from commercial channels, and there is no need to prepare it by oneself.
  • step 1) the method for coupling other amino acids AA except Fmoc-Phe-OH is,
  • the Fmoc-AA n -resin and the second organic solvent are carried out decap reaction, to completely remove the Fmoc protecting group, finally wash with the third organic solvent to obtain H-AA n -resin;
  • step iii Activating Fmoc-AA m -OH in the first organic solvent and the activating reagent to obtain an activated Fmoc-AA m -OH solution, wherein m is the (n+1)th amino acid; optional, step iii) can be done before or after step i) or step ii);
  • the first organic solvent is an aprotic organic solvent, optionally one or more of DCM, DMF, NMP; or a mixture of any one of it and HOBt solution; preferably a mixed solution of any one or more of DCM, DMF, NMP and 0.1-10% HOBt (mass/volume); more preferably a DCM solution (mass/volume) containing 1% HOBt calculated by mass volume ratio vol), one of 1% HOBt in DMF (mass/vol), 1% HOBt in NMP (mass/vol).
  • a DMF solution mass/volume containing 1% HOBt is most preferred.
  • the volume-to-mass ratio of the first organic solvent to the Fmoc-Phe-resin is 6-20L:1Kg.
  • the second organic solvent is an aprotic organic solvent comprising an organic base, and the organic base is selected from one of piperidine, piperazine, diethylamine or triethylamine or more.
  • the second organic solvent is preferably a DCM solution containing 15-30% piperidine (volume ratio), a DMF solution (volume ratio) of 15-30% piperidine, and a NMP solution of 15-30% piperidine in a volume ratio.
  • the volume-to-mass ratio of the capping reagent to the Fmoc-Phe-resin is 6-20 L/Kg.
  • the capping reaction time is 1-30min, drained, and repeated 1-5 times until the capping reaction is complete.
  • the third organic solvent is the first organic solvent or an alcohol solvent, and can be optionally one or more of DCM, methanol, ethanol, DMF, NMP, or any one of them A mixed solution with HOBt; preferably a mixed solution of any one or more of DCM, methanol, ethanol, DMF, NMP and 0.1-10% HOBt (mass/volume); more preferably a 1% HOBt DCM solution, One or more of 1% HOBt in DMF solution and 1% HOBt in NMP solution. Most preferred is a DMF solution.
  • the volume-to-mass ratio of the third organic solvent used each time to the Fmoc-Phe-resin is 6-20L:1Kg.
  • the third organic solvent is used for washing 4-10 times, preferably 6-9 times, most preferably 8 times.
  • the third organic solvent for the final wash must not be a solvent that shrinks the resin.
  • the activating reagents in step iii are DIC, HBTU and Oxyma Pure composition, DIC and Oxyma Pure composition, DIC and HOBt composition, DIEA, TBTU and HOBt composition and DIEA and PyBop composition one of the.
  • the coupling agent is the composition of DIC and Oxyma Pure, because the composition can better inhibit the racemization in the condensation reaction and improve the condensation rate.
  • the amount of each component in the coupling agent is 3-10 molar equivalents, preferably 5 molar equivalents.
  • the temperature of the activation reaction is room temperature, and the reaction time is 5-60min.
  • the reaction temperature for coupling other amino acids is 10-35°C, and the reaction time is 0.5-5h.
  • the third organic solvent for the final wash must not be a solvent that shrinks the resin.
  • a ninhydrin detection method is used for detection and monitoring in the reaction process, and the specific detection method is referred to in the literature "A Practical Method for Quantitative Determination of Amino Acids-Ninhydrin Method". If the test result is negative, proceed to the next step; if the test result is positive, repeat condensation until the test result is negative.
  • the temperature of the condensation reaction in step iv is about 40-60°C, most preferably about 45-55°C, such as about 40°C, about 42°C, Numerical values of about 45°C, about 48°C, about 50°C, about 52°C, about 55°C, about 58°C, about 60°C, etc. Unless otherwise specified, "about” in the present invention refers to an appropriate approach to the stated value, such as plus or minus 10%. Urea or perchlorate can be optionally added to the condensation reaction system.
  • the urea of about 0.1-1kg/kg Fmoc-Phe-resin or the perchlorate of about 0.1-1kg/kg Fmoc-Phe-resin can optionally be added, that is, the urea or perchlorate
  • the mass ratio to Fmoc-Phe-resin is about 0.1:1-1:1.
  • the reaction time for coupling the C-15 peptide is about 0.5-16 h, preferably about 2-4 h.
  • increasing the reaction temperature may increase the reaction conversion, but at the same time promote the production of the D configuration isomer impurity. It was unexpectedly found that by increasing the reaction temperature to 40-60 °C, the optional addition of Urea or sodium perchlorate, the condensation rate in this step is significantly improved, and at the same time, no D-configuration isomer impurity is observed or only a very small amount of D-configuration isomer impurity is observed at about 60 °C.
  • the adrenocorticotropic hormone (human sequence) or its analog-resin with a protective group obtained in step 1) is subjected to a capping reaction with a second organic solvent, and then washed with a third organic solvent and finally washed with a fourth organic solvent, wherein the fourth organic solvent is one or more of methanol, ethanol and DCM, preferably methanol.
  • the cleavage reagent consists of trifluoroacetic acid and a cleavage peptide protection reagent.
  • Peptide protection reagent is composed of one or more of phenol, anisole, dimethyl sulfide, 1,2-ethanedithiol, triethylsilane, triisopropylsilane or water. Reagents can reduce the probability of amino acids being re-modified or oxidized.
  • the volume ratio of the peptide protection reagent to trifluoroacetic acid is 1:4-1:19, for example, the concentration of trifluoroacetic acid can be selected as 80%-95%, preferably 90%; the sum of the concentrations of the peptide protection reagent The optional range is 5%-20%, preferably 10%.
  • the sedimentation reagent is an ether solvent, more preferably diethyl ether or methyl tert-butyl ether, and most preferably methyl tert-butyl ether.
  • the volume ratio of the cleavage reagent to the sedimentation reagent in step 3) is 1:5-1:20.
  • the liquid chromatography is reversed-phase high pressure liquid chromatography, and the purification uses octaalkylsilane-bonded silica gel, octadecylsilane-bonded silica gel or PS as a fixed phase, using a dynamic axial pressure column, with 0.01-0.2 mol/L organic salt aqueous solution and organic solvent as mobile phases or 0.01-0.2 mol/L inorganic salt aqueous solution and organic solvent as mobile phases for purification.
  • a combination of basic and acidic conditions is used for elution, preferably 0.1 mol/L basic Tris aqueous solution/acetonitrile and 0.1 mol/L acidic ammonium sulfate aqueous solution/acetonitrile are used as mobile phases for elution.
  • step 4 salt transfer is required.
  • 0.01-0.2mol/L ammonium acetate aqueous solution/acetonitrile is used as the mobile phase for salt transfer, and then the mass concentration is 0.01-0.2%.
  • the samples were eluted with acetic acid in 20-80% acetonitrile in water.
  • polypeptide resins which have the following sequences:
  • the P is Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro- Leu-Glu(OtBu)-Phe (SEQ ID NO.5)
  • the resin is 2-trityl methyl chloride resin
  • the Q is none or is selected from the following combination:
  • polypeptide resin of the present invention wherein the polypeptide has the following sequence selected from SEQ ID NO.6 ⁇ SEQ ID NO.29:
  • the Fmoc solid-phase synthesis process described in the present invention is not only suitable for small-scale laboratory experiments, but also suitable for various pilot-scale experiments and scale-ups, such as single-batch production of large-scale high-purity corticotropin (human-derived sequence),
  • the yield is at least about 250g/batch, or at least about 300g/batch, or at least about 500g/batch, and even reaches the kilogram scale, indicating that the solid phase synthesis process of the present invention has good stability and is suitable for commercial production.
  • the "single batch" referred to in the present invention refers to the synthesis of a specified amount of product at one time. A single batch therefore does not include multiple preparations of compounds performed at separate times or in separate amounts, which are then combined.
  • the high-purity adrenocorticotropic hormone (human sequence) or its analogs described in the present invention can be used alone or in combination with other pharmaceutical excipients or other active components to form a composition for preparing preparation products, such as oral dosage forms, parenteral
  • suitable dosage forms for adrenocorticotropic hormone (human sequence) or its analogs such as dosage forms, rectal dosage forms or external dosage forms.
  • oral dosage forms include but are not limited to tablets, capsules, granules, pills, powders, sustained and controlled release formulations
  • parenteral dosage forms include but are not limited to sterile solutions, suspensions or emulsions
  • rectal dosage forms include but are not limited to Suppositories
  • external dosage forms include but are not limited to inhalants, patches, ointments and the like.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising adrenocorticotropic hormone and a pharmaceutically acceptable carrier, the purity of the adrenocorticotropic hormone or its analogs is greater than or equal to 99%, and the content of any single impurity is less than or equal to 0.5%, The total impurity content is less than or equal to 1%, and the sequence of the adrenocorticotropic hormone from the N-terminus to the C-terminus is as follows:
  • a pharmaceutical composition comprising an adrenocorticotropic hormone analog and a pharmaceutically acceptable carrier is characterized in that: the purity of the adrenocorticotropic hormone analog is greater than or equal to 99%, and the content of any single impurity is less than or equal to 99%. 0.5%, total impurity content ⁇ 1%, the sequence of the adrenocorticotropic hormone analog from N-terminal to C-terminal is as follows:
  • the high-purity adrenocorticotropic hormone (human sequence) or its analogs and related preparation products of the present invention can be used for the treatment of neonatal spasm, adult multiple sclerosis, rheumatism, allergy, edema and other immune diseases .
  • the present invention has the following advantages: (1) through the step-by-step condensation method of Fmoc solid-phase synthesis, the content and chromatographic purity of adrenocorticotropic hormone (human sequence) in the crude peptide are greatly improved; (2) Fmoc solid-phase synthesis is adopted.
  • the stepwise condensation technology of phase synthesis can simplify the post-reaction process, greatly simplify the reaction procedure, and reduce the loss of products in the post-processing stage.
  • the operation is complicated, the time is long, the workload is large, and the liquid phase synthesis range is small, generally focusing on the synthesis of peptides within 10 amino acids;
  • the condensation of the -15 peptide cannot make this step reaction complete, resulting in missing peptide impurities, reducing the yield of the target product, increasing the content of impurities and the difficulty of subsequent purification;
  • the present invention has been screened by various condensation systems and optimized process conditions.
  • the first-step reaction can be completed, and it is difficult to produce missing peptide impurities, which increases the yield of the product and reduces the difficulty of purification; (4) It is prepared by reverse-phase high-pressure purification, which has high separation degree, good purification effect, few impurities and simple operation.
  • the adrenocorticotropic hormone (human sequence) or its analog polypeptide prepared by the invention has a purity of more than 99%, good stability and a yield of ⁇ 63%.
  • Fig. 1 is the solid-phase synthesis step-by-step condensation flow chart of the present invention.
  • Fig. 2 is the liquid phase chromatogram of the reaction for 20h at 20°C when 15 peptides are condensed in Example 4, using DIC/Oxyma Pure/urea condensing agent.
  • FIG. 3 is the liquid phase spectrum of the condensation of 15 peptides in Example 4 at 30° C., using DIC/HOBt/NaClO 4 condensing agent for 20 h.
  • Fig. 4 is the liquid phase spectrum of the condensation of 15 peptides in Example 4 at 35°C using TBTU/DIPEA/HOBt condensing agent for 20 h.
  • Figure 5 is the liquid phase spectrum of the reaction for 3h at 40°C when the 15 peptide is condensed in Example 4, using DIC/Oxyma Pure condensing agent.
  • Fig. 6 is the liquid phase spectrum of the reaction for 3h at 45°C when the 15 peptide is condensed in Example 4, using DIC/HOBt condensing agent.
  • Fig. 7 is the liquid phase spectrum when the 15-peptide is condensed in Example 4 at 50°C and the DIC/HOBt condensing agent is used for the reaction for 3 h.
  • Fig. 8 is the liquid phase spectrum of the condensation of 15 peptides in Example 4 at 50°C using DIC/HOBt/urea condensing agent for 3 hours.
  • Fig. 9 is the liquid phase spectrum of the condensation of 15 peptides in Example 4 at 50°C using DIC/HOBt/NaClO 4 condensing agent for 3 hours.
  • FIG. 10 is the liquid phase spectrum of the reaction for 3 h at 60° C. using DIC/HOBt condensing agent when the 15 peptide is condensed in Example 4.
  • FIG. 10 is the liquid phase spectrum of the reaction for 3 h at 60° C. using DIC/HOBt condensing agent when the 15 peptide is condensed in Example 4.
  • Figure 11 is a liquid phase spectrum of the crude corticotropin (human sequence) peptide of Example 7.
  • Figure 12 is the liquid phase spectrum of the pure adrenocorticotropic hormone (human sequence) in Example 8.
  • Figure 13 is the liquid phase spectrum of the pure adrenocorticotropic hormone (human sequence) analog of Example 9.
  • Figure 14 is a liquid phase map of adrenocorticotropic hormone (human sequence) obtained according to the method of US 3953415A in the comparative example.
  • the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified, and the amino acids are all L-type amino acids unless otherwise specified.
  • Resin swelling get the Fmoc-Phe-resin that the molar substitution degree of 500g embodiment 1 preparation is 0.7mmol/g, namely the molar weight of Fmoc-Phe is 0.35mol, join in the reactor, add 5L containing 50g HOBt DMF solution to fully swell the Fmoc-Phe-resin.
  • step 2) Decap reaction: add 5L of decap reagent (20% piperidine/DMF solution by volume) to step 1), stir for 20min, drain the resin, repeat 3 times until the decap reaction is complete. After decapping reaction, wash with 5L DMF solution each time, and wash 8 times in total to obtain Phe-resin.
  • step 2 Condensation reaction: the activated solution of Fmoc-Glu(OtBu)-OH is added to the reactant Phe-resin obtained in step 2), the condensation reaction is carried out at 30°C for 3h, suction filtration, and then washed with solvent DMF for 8 times to obtain Fmoc-Glu (OtBu)-Phe-resin. The reaction is monitored by the ninhydrin detection method. If the detection result is negative, go to the next step. If the detection result is positive, repeat the above-mentioned steps 3) to 4) condensation reaction until the detection result is negative.
  • Example 2 To the Fmoc-Glu(OtBu)-Phe-resin prepared in Example 2, refer to the method described in steps 2)-4) in Example 2, that is, each time an amino acid is coupled, it undergoes decapping reaction, activation reaction and condensation reaction in turn. . And according to the adrenocorticotropin (human sequence) amino acid sequence from the C-terminal to the N-terminal 3-14 position sequence of amino acid coupling, the coupled amino acid sequence is:
  • Example 4b In the polypeptide reactor of Example 4b, refer to the method described in steps 2)-4) in Example 2, that is, each coupling of an amino acid undergoes decapping reaction, activation reaction and condensation reaction in sequence.
  • the coupled amino acid sequence is: Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Pro-OH
  • decapping reagent (20% piperidine/DMF solution) was added, the reaction was stirred for 20 min, the resin was drained, and repeated 3 times until the decapping reaction was complete. After decapping reaction, wash 8 times with 5L DMF solution each time. 5L methanol solution was added for washing 8 times, suction filtration, and vacuum drying at 40° C. to obtain 1.75Kg of adrenocorticotropin (human sequence) peptide chain-resin with N-terminal Fmoc protection removed.
  • the 1.75Kg peptide resin prepared in Example 6 was added to 17.5L of the cleavage reagent pre-cooled to -12°C for 2h (the cleavage reagent was protected by 90% volume of trifluoroacetic acid and the sum of the volume concentration was 10% of the cleavage peptide).
  • Reagent composition wherein the cleavage protection reagent is composed of 1% phenol, 1% anisole, 1% dimethyl sulfide, 1% 1,2-ethanedithiol, 1% triethylsilane, 1% by volume concentration % triisopropylsilane and 4% water), control the temperature of the whole reaction not to exceed 40 ° C, after the reaction is completed, filter, collect the filtrate, concentrate the filtrate under reduced pressure to remove part of the cleavage reagent, and then slowly add it to the pre-cooling to -12 Settling in 17.5L of methyl tertiary butyl ether at °C, centrifuged to collect the wet solid, washed the wet solid with 87.5L of methyl tert-butyl ether, the collected solid was dissolved in water, and 0.75Kg of solid crude peptide was obtained after lyophilization, The purity was 68.96%, and the target peptide content was 48% (ie
  • chromatographic column DAC-20 dynamic axial compression column with octadecylsilane-bonded silica gel as stationary phase, column diameter and packing length: 20*25cm.
  • Mobile phase A Tris aqueous solution with a molar concentration of 0.1 mol/L, adjusted to pH 8.0 by ammonia water; phase B: acetonitrile.
  • Flow rate 80ml/min.
  • Gradient B%: 30-60% (50min) The injection volume is 20g.
  • Purification process equilibrate the chromatographic column with mobile phase A and load the sample with a sample volume of 5L of the sample solution. Linear gradient elution was performed for 50 min, and the target peptide solution with a purity greater than 90% was collected. The recovery rate of target peptide components was 79%.
  • chromatographic column DAC-20 dynamic axial compression column with octadecylsilane-bonded silica gel as stationary phase, column diameter and packing length: 20*25cm.
  • Mobile phase A ammonium sulfate aqueous solution with molar concentration of 0.1 mol/L, pH adjusted to 3.0 by sulfuric acid; phase B: acetonitrile.
  • Flow rate 80ml/min.
  • Gradient B%: 30-60% (50min) The injection volume is 20g.
  • Salt transfer conditions chromatographic column: DAC-20 dynamic axial compression column with octadecylsilane-bonded silica gel as stationary phase, column diameter and packing length: 20*25cm.
  • Mobile phase A 0.1% acetic acid in water; phase B: acetonitrile.
  • Flow rate 80ml/min.
  • Salt transfer process equilibrate the chromatographic column with mobile phase A and load it with a sample volume of 1L of the sample solution. After linear gradient elution for 50 min, the target peptide solution was collected, and the recovery rate of the target peptide component was 95%.
  • Example 4b-Example 8 replace Asn(Trt) with Asp(Trt) at the 15th amino acid to obtain H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe- Pro-Leu-Glu-Phe-OH adrenocorticotropic hormone (human sequence) analog (SEQ ID NO.4) pure product, the purity is 99.58%, (specifically as shown in Figure 13, the main peak peak time is 28.213min , the largest single impurity is 0.16%, and the total impurity is 0.42%).
  • the collected target peptide was 255 g, the content was 89%, and the molar yield of the target peptide was 63%.
  • the exact molecular weight detected by high-resolution mass spectrometry was 4537.232 Da, which was consistent with the theoretical exact molecular weight of the corticotropin (human sequence) analog.
  • Adrenocorticotropin human sequence (SEQ ID NO.3) was prepared in strict accordance with the specific steps of step 1 to step 14 in Example 4 of the comparative document US3953415, and the total yield was 17%. ) was only 75.72% pure, as shown in Figure 14.
  • Example 8 Take the sample obtained in Example 8, pack it in a double-layer medicinal low-density polyethylene bag and a layer of aluminum-plastic composite bag, store it at a temperature of 2-8°C, and evaluate it at 1, 2, 3, and 6 months, respectively.
  • Table 2 The accelerated 6-month stability results are shown in Table 2 below:
  • Example 9 The samples obtained in Example 9 were taken, packed in double-layer medicinal low-density polyethylene bags and one-layer aluminum-plastic composite bags, stored at a temperature of 2-8°C, and evaluated at 1, 2, 3, and 6 months, respectively.
  • the accelerated 6-month stability results are shown in Table 3 below:
  • the polymer refers to an impurity with a molecular weight greater than that of the corticotropin analog (human sequence SEQ ID NO. 4).
  • Corticotropin is a melanocortin receptor 2 (MC2R) receptor agonist.
  • M2R melanocortin receptor 2
  • the combination of corticotropin and MC2R can stimulate the downstream signaling pathways of cells, resulting in changes in signaling factors such as calcium ions and cAMP.
  • the invention judges the binding ability of corticotropin and MC2R by detecting calcium ion signal, and then judges the in vitro cell activity of corticotropin.
  • Example 8 The samples of Example 8 and the samples of Example 10 were taken for in vitro activity studies.
  • MC2R-expressing CHO-K1 cells were cultured in 10 cm dishes and stored at 37 °C and 5% CO .
  • the present invention determines the magnitude of the cardiometabolic toxicity of the drug by detecting the affinity between the drug and the cardiac receptor protein (hERG) in vitro.
  • Example 8 Take the sample of Example 8, the sample of Example 10 and the positive control drug dofetilide for in vitro toxicity studies, the results show that the half-inhibitory concentration (IC 50 of high-purity adrenocorticotropic hormone and low-purity adrenocorticotropic hormone ) were greater than 10000nM, and the half-inhibitory concentration of the positive control was 2.09nM. Therefore, the toxic dose of high-purity adrenocorticotropin and low-purity adrenocorticotropic hormone was much higher than that of the positive control, and no cardiometabolic toxicity was shown.
  • the specific metabolic toxicity data are shown in Table 5 below:

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Abstract

本发明属于多肽制备方法技术领域，特别涉及一种高纯的促肾上腺皮质激素(人源序列)或其类似物及其规模化制备方法，其主要步骤包括，先通过Fmoc固相合成法从C端到N端的顺序偶联氨基酸，获得带保护基团的促肾上腺皮质激素(人源序列)或其类似物粗肽-树脂，其中，C-15肽合成的反应温度为40-60℃，再经过切割、沉降得到促肾上腺皮质激素(人源序列)或其类似物粗品，然后通过采用液相色谱法分离纯化，获得高纯度的成品。本发明制备的促肾上腺皮质激素(人源序列)或其类似物样品色谱纯度达到99％以上，稳定性好，目标肽收率≥63％。

Description

一种高纯的人促肾上腺皮质激素或其类似物及其规模化制备方法 技术领域
本发明涉及多肽固相合成及纯化制备方法技术领域，具体涉及一种高纯的促肾上腺皮质激素(人源序列)或其类似物及其规模化制备方法。
背景技术
促肾上腺皮质激素(英文名corticotropin，简称ACTH)是一种由垂体产生和分泌的多肽激素，包含39个以线性序列偶联的氨基酸残基。所有物种的N端24氨基酸片段都是相同的，并含有促肾上腺皮质激素活性。促肾上腺皮质激素刺激肾上腺皮质，促进皮质类固醇的合成，主要是糖皮质激素，但也包括性激素。它可用于治疗某些神经疾病，如婴儿痉挛和多发性硬化，并用于诊断肾上腺皮质功能不全。它具有诊断试剂的作用。它属于一种多肽、一种肽激素和一种生物大分子。
婴儿痉挛是一种与年龄相关同时伴有智力运动发育倒退现象的癫痫综合征。长期以来，从动物中提取得到的ACTH作为婴儿痉挛治疗的一线药物，其作用机制并不清楚。
市场上销售的ACTH制剂产品，其ACTH原料均从动物中提取得到的，有免疫原性，容易发生不良反应，而通过化学合成法制备ACTH可以避免出现免疫原性问题。但是现有技术中暂无将促肾上腺皮质激素(人源序列)开发成制剂产品。
市售ACTH的猪源序列为SEQ ID NO.1和/或SEQ ID NO.2：
NH 2-Ser 1-Tyr 2-Ser 3-Met 4-Glu 5-His 6-Phe 7-Arg 8-Trp 9-Gly 10-Lys 11-Pro 12-Val 13-Gly 14-Lys 15-Lys 16-Arg 1 7-Arg 18-Pro 19-Val 20-Lys 21-Val 22-Tyr 23-Pro 24-Asn 25-Gly 26-Ala 27-Glu 28-Asp 29-Glu 30-Leu 31-Ala 32-Glu 33-Ala 34-Phe 35-Pro 36-Leu 37-Glu 38-Phe 39-COOH(SEQ ID NO.1)。
NH 2-Ser 1-Tyr 2-Ser 3-Met 4-Glu 5-His 6-Phe 7-Arg 8-Trp 9-Gly 10-Lys 11-Pro 12-Val 13-Gly 14-Lys 15-Lys 16-Arg 1 7-Arg 18-Pro 19-Val 20-Lys 21-Val 22-Tyr 23-Pro 24-Asp 25-Gly 26-Ala 27-Glu 28-Asp 29-Glu 30-Leu 31-Ala 32-Glu 33-Ala 34-Phe 35-Pro 36-Leu 37-Glu 38-Phe 39-COOH(SEQ ID NO.2)。
上述SEQ ID NO.1中由于N-25位Asn在碱性条件下不稳定，可发生脱酰胺反应变为Asp，因此部分猪源促肾上腺皮质激素(SEQ ID NO.1)以N-25脱酰胺促肾上腺皮质激素(SEQ ID NO.2)形式存在。
促肾上腺皮质激素(人源序列)序列如下：
NH 2-Ser 1-Tyr 2-Ser 3-Met 4-Glu 5-His 6-Phe 7-Arg 8-Trp 9-Gly 10-Lys 11-Pro 12-Val 13-Gly 14-Lys 15-Lys 16-Arg 1 7-Arg 18-Pro 19-Val 20-Lys 21-Val 22-Tyr 23-Pro 24-Asn 25-Gly 26-Ala 27-Glu 28-Asp 29-Glu 30-Ser 31-Ala 32-Glu 33-Ala 34-Phe 35-Pro 36-Leu 37-Glu 38-Phe 39-COOH。
氨基酸简写为SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF(SEQ ID NO.3)。
N-25脱酰胺促肾上腺皮质激素(人源序列)(或促肾上腺皮质激素(人源序列)类似物)的序列如下：
NH 2-Ser 1-Tyr 2-Ser 3-Met 4-Glu 5-His 6-Phe 7-Arg 8-Trp 9-Gly 10-Lys 11-Pro 12-Val 13-Gly 14-Lys 15-Lys 16-Arg 1 7-Arg 18-Pro 19-Val 20-Lys 21-Val 22-Tyr 23-Pro 24-Asp 25-Gly 26-Ala 27-Glu 28-Asp 29-Glu 30-Ser 31-Ala 32-Glu 33-Ala 34-Phe 35-Pro 36-Leu 37-Glu 38-Phe 39-COOH，
氨基酸简写为SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF(SEQ ID NO.4)。
上述SEQ ID NO.3中由于N-25位Asn在碱性条件下不稳定，可发生脱酰胺反应变为Asp，因此部分促肾上腺皮质激素(人源序列)(SEQ ID NO.3)会转化为N-25脱酰胺促肾上腺皮质激素(人源序列)SEQ ID NO.4形式。在本发明中，当固相合成SEQ ID NO.3时，会有＜1％的SEQ ID NO.4杂质通过脱酰胺反应生成。现有技术公开多肽或蛋白中存在Asn-Gly序列，或者当Gly与Asn连接时，更容易发生β-天冬氨酰转移反应；ACTH的片段Val-Tyr-Pro-Asn-Gly-Ala在pH为5-12的条件下更容易发生脱酰胺反应；生物体内促肾上腺皮质激素也以两种形式存在(H.Tonie Wright.Nonenzymatic Deamidation of Asparaginyl and Glutaminyl Residues in Proteins.Critical reviews in Biochemistry and Molecular Biology,1991,26(1):1-52.)。
因此促肾上腺皮质激素(人源序列)或其类似物与市售猪源促肾上腺皮质激素的序列主要在第31位氨基酸存在差异。
现有技术中，US4055524实施例1-6采用Boc法固相合成技术，但氨基酸保护基团不同，存在如下缺点，合成过程中反复地用酸来脱保护，每次用三氟乙酸脱Boc等保护基时，部分肽会从树脂上脱落，合成的肽链越长，这样的损失越严重；酸处理会引起侧链的一些副反应，Boc合成法尤其不适于合成含有色氨酸等对酸不稳定的肽类；最终脱保护采用氢氟酸法，其腐蚀性极强且毒性较高，无法工业化生产。
US3953415实施例4步骤1-14公开了一种液相法(非固相合成技术，步骤1-14均在溶液体系中进行均相反应，每步中间体均需要通过结晶或柱层析分离纯化除去多余未反应原料、试剂)制备促肾上腺皮质激素(人源序列)，通过液相法片段合成得到保护基的粗肽，用三氟乙酸脱保护，残留物用离子交换转盐冻干得到粗品。粗品经离子交换纯化，纯化过程中用醋酸铵溶液进行洗脱，冻干获得产品中含有醋酸铵未进行去除。该工艺摩尔总收率只有17％(步骤1收率89.9％，步骤2收率99％，步骤3收率97.2％，步骤4收率92.7％，步骤5收率93％，步骤6收率95％，步骤7收率86.2％，步骤8收率82.6％，步骤9收率96.9％，步骤10收率88％，步骤11收率86.1％，步骤12收率90％，步骤13收率85％，步骤14收率60％)，收率较低，且未明确具体的纯度。
Kálmán等人(Synthesis of Human ACTH and Its Biologically Active Fragments[M]The Chemistry of Polypeptides.Springer US,1973.)报道了通过液相法片段合成得到带保护基的粗肽，未提供纯化工艺，且未明确具体的纯度和收率。
目前现有技术中关于促肾上腺皮质激素(人源序列)的制备采用Boc法固相合成技术或液相片段合成法，普遍存在因反应不完全导致收率低，纯度低且杂质种类多，后处理困难等问题，因此急需开发一种能够有效克服上述缺陷的高纯的促肾上腺皮质激素(人源序列)或其类似物及其制备方法。
发明内容
本发明针对现有技术的不足，提供一种高纯的促肾上腺皮质激素(人源序列)或其类似物及其制备方法，该方法采用Fmoc固相合成技术获得产品，并对C-15肽合成条件以及后处理条件进行优化，解决了反应不完全，缺失肽杂质多和纯化困难等问题。
在本发明中，相关的名词或缩写解释参见下表：
缩合 含义
Boc 叔丁氧羰基
Cbz 苄氧羰基
DCM 二氯甲烷
DIC N,N'-二异丙基碳二亚胺
DIEA N,N-二异丙基乙胺
DIPEA N,N-二异丙基乙胺
DMF N,N-二甲基甲酰胺
Fmoc 9-芴甲氧羰基
HOBt 1-羟基苯并三氮唑
HBTU 苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
NMP N-甲基吡咯烷酮
OtBu O-叔丁基
Oxyma Pure 2-肟氰乙酸乙酯
Pbf 2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基
PyBop 六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷
PS 聚苯乙烯
TBTU O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸
tBu 叔丁基
Tris 三羟甲基氨基甲烷
Trt 三苯甲游基
如无特殊说明，本发明中涉及的相关的名词解释均采用现有技术中常规的解释。
为了实现上述目的，本发明提供如下的技术方案，一种包含促肾上腺皮质激素(人源序列)的组合物，所述合成促肾上腺皮质激素(人源序列)的纯度≥99％，最大单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素的具体序列从N端到C端如下所示：
Figure PCTCN2021086395-appb-000001
为了实现上述目的，本发明提供如下另一种技术方案，一种包含促肾上腺皮质激素类似物的组合物，其特征在于，所述促肾上腺皮质激素类似物的纯度≥99％，最大单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素类似物的序列从N端到C端如下所示：
Figure PCTCN2021086395-appb-000002
所述纯度优选通过高效液相色谱法(HPLC)测定。所述高纯度促肾上腺皮质激素(人源序列)或其类似物的纯度至少为99.0％，或至少为99.5％或至少为99.8％或更高。所述高纯度促肾上腺皮质激素(人源序列)或其类似物的最大单杂≤0.5％，总杂≤1％；进一步最大单杂≤0.4％，总杂≤0.9％；进一步最大单杂≤0.3％，总杂≤0.8％；进一步最大单杂≤0.2％，总杂≤0.7％；进一步最大单杂≤0.1％，总杂≤0.5％；或者更少。
作为本发明的进一步优选，所述促肾上腺皮质激素或促肾上腺皮质激素类似物的纯度≥99.5％，最大单杂含量≤0.1％，总杂含量≤0.5％。
本发明所述的包含促肾上腺皮质激素或其类似物(纯度≥99％，HPLC)的组合物，通过如下制备方法得到，即通过Fmoc固相合成法，分别按照SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列从C端到N端的顺序偶联氨基酸，再经过纯化，得到包含促肾上腺皮质激素的组合物或包含促肾上腺皮质激素类似物的组合物。作为本发明的进一步优选，上述高纯的促肾上腺皮质激素(人源序列)或其类似物的制备方法，包括以下步骤：
1)通过Fmoc固相合成法，按照SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列从C端到N端的顺序偶联氨基酸，获得带保护基团的促肾上腺皮质激素(人源序列)或其类似物肽链-树脂；
2)使用切割试剂处理带保护基团的促肾上腺皮质激素(人源序列)或其类似物肽链-树脂，将促肾上腺皮质激素(人源序列)或其类似物肽链从树脂上裂解下来并去除肽链所有保护基团，得到包含促肾上腺皮质激素(人源序列)或其类似物的溶液；
3)使用沉降试剂处理上述包含促肾上腺皮质激素(人源序列)或其类似物的溶液，得到促肾上腺皮质激素(人源序列)或其类似物粗品；
4)将上述促肾上腺皮质激素(人源序列)或其类似物粗品利用液相色谱法纯化得到包含促肾上腺皮质激素(人源序列)或其类似物的组合物。
在本发明的技术方案中，所述Fmoc固相合成法是指将反应物连接在一个不溶性的固相载体上的一种合成方法，其原理为首先在固相载体(如树脂)上引入氯甲基(-CH 2Cl)或其他能与羧基反应的基团，使其与氨基被保护的氨基酸反应，将第一个氨基酸固载至树脂上，然后按照设计序列分别按顺序与氨基酸单体反应得到所需要的多肽序列；在合成的过程中，多肽稳定地共价结合于固相载体表面，反应结束后利用特殊化学试剂将多肽序列从固相载体上切下。Fmoc固相合成法可大大简化反应程序，简化反应的后处理过程，减轻了每步产品纯化的难度，降低产物在后处理阶段的损失；而传统液相合成法中未反应原料、试剂与目标中间体或产品需要通过结晶、甚至柱层析进行分离提纯，操作复杂，时间长工作量大，而且液相合成范围小，一般集中在10个氨基酸以内的多肽合成。
在本发明的技术方案中，所述切割试剂是指将合成的多肽由固相载体上切割下来并脱去侧链保护的化学试剂。
在本发明的技术方案中，所述沉降试剂是指将合成的多肽从溶液中析出的化学试剂。
在本发明的技术方案中，所述步骤1)中，偶联的氨基酸采用逐一偶联或者片段偶联，氨基酸N端采用Fmoc基团保护。
在本发明的技术方案中，所述步骤1)中，固相合成树脂可选为氯甲基树脂或Wang树脂(聚苄氧基苄醇树脂)或2-三苯甲基氯甲烷树脂树脂或Rink Amide AM Resin或Rink Amide MBHA Resin或Rink Amide Resin等树脂，优选为2-三苯甲基氯甲烷树脂。树脂替代度为0.1-1.0mmol/g，优选0.2-0.8mmol/g。
在本发明的技术方案中，所述步骤1)中，偶联Fmoc-Phe-OH的方法可选为：取固相合成树脂用6-20L/Kg树脂的有机溶剂如DCM浸泡，加入Fmoc-Phe-OH和有机胺如DIPEA在室温反应2-4h得到Fmoc-Phe-树脂，再加入DIPEA/甲醇(体积比1:5-1:15)封闭未反应位点，最后用6-20L/Kg树脂的有机溶剂如DCM、DMF、NMP、甲醇洗涤，抽干，真空干燥后备用。取Fmoc-Phe-树脂，测定摩尔取代度，以确定投料量。在本发明的技术方案中，Fmoc-Phe-OH也可直接从商业途径购买，无需自己制备。
在本发明的技术方案中，所述步骤1)中，偶联除Fmoc-Phe-OH以外的其它氨基酸AA的方法为，
i.使用第一有机溶剂溶胀Fmoc-AA n-树脂，其中AA n表示连接有n个氨基酸，n为1-38中的自然数；可选的，氨基酸AA的结构相同或不同，氨基酸AA的N端采用Fmoc或Boc或Cbz基团保护，氨基酸AA的侧链有保护基团或无保护基团；
ii.将Fmoc-AA n-树脂与第二有机溶剂进行脱帽反应，至完全脱除Fmoc保护基团，最后用第三有机溶剂洗涤，得到H-AA n-树脂；
iii.将Fmoc-AA m-OH在第一有机溶剂和活化试剂中进行活化，得到活化后的Fmoc-AA m-OH溶液，其中m为第(n+1)个氨基酸；可选的，步骤iii)可在步骤i)或步骤ii)之前或之后完成；
iv.将上述活化后的Fmoc-AA m-OH溶液与H-AA n-树脂进行偶联反应，得到Fmoc-AA (n+1)-树脂，最后用第三有机溶剂洗涤；
v.将上述洗涤后的Fmoc-AA (n+1)-树脂循环使用步骤ii)-步骤iv)的方法，最终得到带保护基团的促肾上腺皮质激素(人源序列)或其类似物肽链-树脂。
在本发明的技术方案中，步骤i和步骤iii中第一有机溶剂为非质子性有机溶剂，可选为DCM，DMF，NMP中的一种或几种；或其任意一种与HOBt的混合溶液；优选为DCM，DMF，NMP中的任意一种或几种与0.1-10％HOBt(质量/体积)的混合溶液；进一步优选为以质量体积比计算含有1％HOBt的DCM溶液(质量/体积)，1％HOBt的DMF溶液(质量/体积)，1％HOBt的NMP溶液(质量/体积)中的一种。因为适量的HOBt可以抑制氨基酸在缩合反应时的消旋，最优选为含有1％HOBt的DMF溶液(质量/体积)。第一有机溶剂与Fmoc-Phe-树脂的体积质量比为6-20L:1Kg。
在本发明的技术方案中，步骤ii中第二有机溶剂为包含有机碱的非质子性有机溶剂，所述有机碱选自哌啶、哌嗪、二乙胺或三乙胺中的其中一种或多种。所述第二有机溶剂优选为以体积比计算含有15-30％哌啶的DCM溶液(体积比)，15-30％哌啶的DMF溶液(体积比)，15-30％哌啶的NMP溶液(体积比)中的一种，进一步优选为含有20％哌啶的DCM溶液(体积比)，20％哌啶的DMF溶液(体积比)，20％哌啶的NMP溶液(体积比)中的一种。最优选为20％哌啶的DMF溶液。脱帽试剂与Fmoc-Phe-树脂的体积质量比为6-20L/Kg。脱帽反应的时间为1-30min，抽干，重复1-5次至脱帽反应完全。
在本发明的技术方案中，步骤ii中第三有机溶剂为第一有机溶剂或醇类溶剂，可选为DCM，甲醇，乙醇，DMF，NMP中的一种或几种，或其任意一种与HOBt的混合溶液；优选为DCM，甲醇，乙醇，DMF，NMP中的任意一种或几种与0.1-10％HOBt(质量/体积)的混合溶液；进一步优选为1％HOBt的DCM溶液，1％HOBt的DMF溶液，1％HOBt的NMP溶液中的一种或几种。最优选为DMF溶液。第三有机溶剂每次的使用量与Fmoc-Phe-树脂的体积质量比为6-20L:1Kg。使用第三有机溶剂洗涤4-10次，优选6-9次，最优选8次。最后一次洗涤的第三有机溶剂不得为可使树脂收缩的溶剂。
在本发明的技术方案中，步骤iii中的活化试剂为DIC、HBTU和Oxyma Pure组合物，DIC和Oxyma Pure组合物，DIC和HOBt组合物，DIEA、TBTU和HOBt组合物以及DIEA和PyBop组合物中的一种。优选地，偶联剂为DIC和Oxyma Pure组合物，因为该组合物可以更佳地抑制缩合反应中的消旋，提高缩合率。其中，偶联剂中各成分的量为3-10摩尔当量， 优选为5摩尔当量。活化反应的温度为室温，反应时间为5-60min。
在本发明的技术方案中，所述步骤iv中除偶联C-15位的氨基酸外，偶联其它氨基酸的反应温度为10-35℃，反应时间为0.5-5h。反应结束后抽滤，然后用第三有机溶剂洗涤4-10次。最后一次洗涤的第三有机溶剂不得为可使树脂收缩的溶剂。
在本发明的技术方案中，反应过程中使用茚三酮检测方法进行检测监控，具体的检测方法参考文献《一种比较实用的氨基酸定量测定方法-茚三酮法》。如检测结果呈阴性进入下一步反应，如检测结果呈阳性，则进行重复缩合至检测结果呈阴性。
在本发明的技术方案中，偶联C-15位的氨基酸时，步骤iv的缩合反应的温度为约40-60℃，最优选约为45-55℃，例如约40℃、约42℃、约45℃、约48℃、约50℃、约52℃、约55℃、约58℃、约60℃等数值。如无特殊说明，本发明所述的“约”指与所述数值适当的接近，如加减10％。缩合反应体系中可选加入尿素或高氯酸盐。作为本发明的进一步改进，可选加入约0.1-1kg/kg Fmoc-Phe-树脂的尿素或约0.1-1kg/kg Fmoc-Phe-树脂的高氯酸盐，即所述尿素或高氯酸盐与Fmoc-Phe-树脂的质量比约为0.1:1-1:1。偶联C-15肽反应时间约为0.5-16h，优选约为2-4h。通过研究发现，在10-30℃条件下，采用各种缩合试剂(如DIC/HOBt组合、TBTU/DIPEA组合、TBTU/DIPEA/HOBt组合、Oxyma Pure、PyBop/DIPEA组合、DIC/HOBt/尿素组合、DIC/HOBt/NaClO 4组合)，即使加入尿素和高氯酸钠，会离散困难位点的β-折叠结构，茚三酮检测仍然为阳性，即使增加重复缩合次数和延长缩合时间也未能解决难缩合的问题。通常情况下，升高反应温度可能会提高反应转化率，但同时会促进D构型异构体杂质的生产，令人出乎意料的发现，通过提高反应温度至40-60℃，可选加入尿素或高氯酸钠，该步骤缩合率显著提高了，同时未观察到D构型异构体杂质的生成或在约60℃仅有极少量的D构型异构体杂质。
在本发明的技术方案中，将步骤1)得到的带保护基团的促肾上腺皮质激素(人源序列)或其类似物-树脂与第二有机溶剂进行脱帽反应，再用第三有机溶剂洗涤，最后用第四有机溶剂洗涤，所述第四有机溶剂为甲醇、乙醇、DCM中的一种或几种，优选为甲醇。
在本发明的技术方案中，所述步骤2)中，切割试剂由三氟乙酸和切肽保护试剂组成。切肽保护试剂由苯酚、苯甲硫醚，二甲硫醚，1，2-乙二硫醇，三乙基硅烷，三异丙基硅烷或水中的一种或几种组成，加入切肽保护试剂可以降低氨基酸被重新修饰或者被氧化的概率。其中，切肽保护试剂与三氟乙酸的体积比为1:4-1:19，如三氟乙酸的浓度可选为80％-95％，优选为90％；切肽保护试剂的浓度之和可选为5％-20％，优选为10％。
在本发明的技术方案中，所述步骤3)中，所述沉降试剂为醚类溶剂，进一步优选为乙醚或甲基叔丁基醚，最优选为甲基叔丁基醚。
在本发明的技术方案中，切割试剂与步骤3)中沉降试剂的体积比为1:5-1:20。
在本发明的技术方案中，所述步骤4)中，液相色谱法为反相高压液相色谱法，纯化以八烷基硅烷键合硅胶、十八烷基硅烷键合硅胶或者PS为固定相，使用动态轴向加压柱，以0.01-0.2mol/L的有机盐水溶液与有机溶剂为流动相或者0.01-0.2mol/L的无机盐水溶液与有机溶剂为流动相进行纯化。采用碱性条件和酸性条件组合洗脱，优选为0.1mol/L的碱性Tris水溶液/乙腈和0.1mol/L的酸性硫酸铵水溶液/乙腈为流动相进行洗脱。
在本发明的技术方案中，步骤4)纯化后需要转盐，转盐过程先用0.01-0.2mol/L的乙酸铵水溶液/乙腈为流动相进行转盐，再用质量浓度为0.01-0.2％乙酸的20-80％乙腈水溶液洗脱 样品进行洗脱。
在本发明的技术方案中，采用如下多肽树脂，具有如下序列：
Q-P-树脂,所述P为Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.5)，所述树脂为2-三苯甲基氯甲烷树脂，所述Q无或选自下列的组合：
H-Pro-，
H-Tyr(tBu)-Pro-，
H-Val-Tyr(tBu)-Pro-，
H-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-。
即本发明所述的多肽树脂，其中多肽具有选自SEQ ID NO.6～SEQ ID NO.29的如下序列：
Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.6)；
Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.7)；
Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.8)；
Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.9)。
Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.10)。
Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.11)。
Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.12)。
Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.13)。
Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.14)。
Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.15)。
Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.16)。
Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.17)。
Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro- Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.18)。
Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.19)。
Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro- Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.20)。
Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)- Ala-Glu(OtBu)- Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.21)。
Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)- Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.22)。
Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)- Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.23)。
His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)- Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.24)。
Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)- Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.25)。
Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)- Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.26)。
Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro- Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.27)。
Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.28)。
Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.29)。
本发明所述的Fmoc固相合成工艺不仅适用于实验室小试规模，还能适用于各种中试、放大规模，如单批生产大规模的高纯度促肾上腺皮质激素(人源序列)，产量至少约250g/批，或至少约300g/批，或至少约500g/批，甚至达到公斤级规模，表明本发明的固相合成工艺稳定性好，适合商业化生产。本发明所述的“单批”是指一次合成指定量的产物。因此单批不包括在分开的时间或分开的量进行的化合物的多次制备，然后将其合并。
本发明所述的高纯的促肾上腺皮质激素(人源序列)或其类似物可单独或与其它药用辅料或其它活性组分构成组合物用于制备制剂产品，如口服剂型、胃肠外给药剂型、直肠给药剂型或外用剂型等其它适合促肾上腺皮质激素(人源序列)或其类似物的给药剂型。其中口服剂型包括但不限于片剂、胶囊、颗粒、丸剂、粉剂、缓控释制剂，胃肠外给药剂型包括但不限于无菌溶液、悬浮液或乳液，直肠给药剂型包括但不限于栓剂，外用剂型包括但不限于吸入剂、贴剂、膏剂等。
作为本发明的进一步优选，本发明涉及包含促肾上腺皮质激素与可药用载体的药用组合物，所述促肾上腺皮质激素或其类似物的纯度≥99％，任意单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素的序列从N端到C端如下所示：
Figure PCTCN2021086395-appb-000003
作为本发明的进一步优选，一种包含促肾上腺皮质激素类似物与可药用载体的药用组合物，其特征在于：所述促肾上腺皮质激素类似物的纯度≥99％，任意单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素类似物的序列从N端到C端如下所示：
Figure PCTCN2021086395-appb-000004
本发明所述的高纯的促肾上腺皮质激素(人源序列)或其类似物及其相关制剂产品可用于治疗新生儿痉挛症、成人多发性硬化症和风湿性、过敏、水肿等免疫性疾病。
本发明相对现有技术，具有以下优势：(1)通过Fmoc固相合成逐步缩合法，大大提高了粗肽中促肾上腺皮质激素(人源序列)的含量及色谱纯度；(2)采用Fmoc固相合成逐步缩合技术可简化反应后的后处理过程，大大简化反应程序，降低了产物在后处理阶段的损失，所需中间体或产物结合在固相载体上，过量的未反应原料和试剂可以直接通过过滤和洗涤去除，操作简单，容易实现自动化，有利于工业化放大，而且可以比较容易地合成到40个氨基酸左右的多肽；而液相合成法中未反应原料、试剂与目标中间体或产品需要通过结晶、甚至柱层析进行分离提纯，操作复杂，时间长工作量大，而且液相合成范围小，一般都集中在10个氨基酸以内的多肽合成；(3)常规的缩合反应工艺对C-15肽的缩合无法使得该步反应完全，产生缺失肽杂质，降低目标产物的产量，增加杂质的含量及后续纯化的难度；本发明经过多种 缩合体系的筛选和工艺条件的优化，使该步反应能够完全，不易产生缺失肽杂质，提高产物产量，降低了纯化的难度；(4)采用反相高压纯化制备，分离度高，纯化效果好，杂质少，操作简单。本发明制备的促肾上腺皮质激素(人源序列)或其类似物多肽纯度达到99％以上，稳定性好，收率≥63％。
附图说明
图1为本发明的固相合成逐步缩合流程图。
图2为实施例4中缩合15肽时在20℃，使用DIC/Oxyma Pure/尿素缩合剂，反应20h时的液相图谱。
图3为实施例4中缩合15肽时在30℃，使用DIC/HOBt/NaClO 4缩合剂，反应20h时的液相图谱。
图4为实施例4中缩合15肽时在35℃，使用TBTU/DIPEA/HOBt缩合剂，反应20h时的液相图谱。
图5为实施例4中缩合15肽时在40℃，使用DIC/Oxyma Pure缩合剂，反应3h时的液相图谱。
图6为实施例4中缩合15肽时在45℃，使用DIC/HOBt缩合剂，反应3h时的液相图谱。
图7为实施例4中缩合15肽时在50℃，使用DIC/HOBt缩合剂，反应3h时的液相图谱。
图8为实施例4中缩合15肽时在50℃，使用DIC/HOBt/尿素缩合剂，反应3h时的液相图谱。
图9为实施例4中缩合15肽时在50℃，使用DIC/HOBt/NaClO 4缩合剂，反应3h时的液相图谱。
图10为实施例4中缩合15肽时在60℃，使用DIC/HOBt缩合剂，反应3h时的液相图谱。
图11为实施例7促肾上腺皮质激素(人源序列)粗肽的液相图谱。
图12为实施例8促肾上腺皮质激素(人源序列)纯品的液相图谱。
图13为实施例9促肾上腺皮质激素(人源序列)类似物纯品的液相图谱。
图14为对比例中参照US 3953415A方法所得促肾上腺皮质激素(人源序列)的液相图谱。
具体实施方式
为便于本领域技术人员理解本发明内容，下面将结合具体实施例进一步描述本发明的技术方案，但以下内容不应以任何方式限制本发明权利要求书请求保护的范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到，氨基酸无特殊说明，均为L型氨基酸。
实施例1 Fmoc-Phe-树脂的制备
取500g 2-三苯甲基氯甲烷树脂加入到5L DCM中，浸泡溶胀1h，加入4.25mol DIPEA，0.75mol Fmoc-Phe-OH室温反应4h，得到Fmoc-Phe-树脂，再加入DIPEA/甲醇(体积比为1：9)溶液1.25L反应0.5h，封闭未反应位点；抽滤，Fmoc-Phe-树脂用5L DCM洗涤1次，抽干，然后用甲醇洗涤3次，每次洗涤甲醇用量为5L，Fmoc-Phe-树脂室温真空干燥至恒重，测定摩尔取代度为0.7mmol/g。
实施例2 Fmoc-Glu(OtBu)-Phe-树脂的制备
1)树脂溶胀：取500g实施例1制备的摩尔取代度为0.7mmol/g的Fmoc-Phe-树脂，即Fmoc-Phe的摩尔量为0.35mol，加入到反应器中，加入5L含有50g HOBt的DMF溶液，使Fmoc-Phe-树脂充分溶胀。
2)脱帽反应：往步骤1)中加入5L脱帽试剂(20％的哌啶/DMF溶液，以体积比计)，搅拌反应20min，抽干树脂，重复3次至脱帽反应完全。脱帽反应结束后，每次使用5L DMF溶液洗涤，总共洗涤8次，得到Phe-树脂。
3)活化：配制Fmoc-Glu(OtBu)-OH活化后溶液：将1.75摩尔量Fmoc-Glu(OtBu)-OH加入到5L的DMF溶液中，再分别加入1.75摩尔量Oxyma Pure和1.75摩尔量DIC，室温混合反应30min；
4)缩合反应：将Fmoc-Glu(OtBu)-OH活化后溶液加入步骤2)所得反应物Phe-树脂中，30℃缩合反应3h，抽滤，然后用溶剂DMF洗涤8次，得到Fmoc-Glu(OtBu)-Phe-树脂。反应以茚三酮检测方法监控，如检测结果呈阴性进入下一步，如检测结果呈阳性，则重复进行上述步骤3)至4)缩合反应至检测结果呈阴性。
实施例3 Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂的制备
向实施例2制备的Fmoc-Glu(OtBu)-Phe-树脂中参照实施例2中2)-4)步骤所述的方法，即每偶联一个氨基酸均依次经过脱帽反应、活化反应和缩合反应。并且按照促肾上腺皮质激素(人源序列)氨基酸序列从C端到N端的3-14位顺序依次进行氨基酸的偶联，偶联的氨基酸顺序为：
Fmoc-Leu-OH，Fmoc-Pro-OH，Fmoc-Phe-OH，Fmoc-Ala-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Ala-OH，Fmoc-Ser(tBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Ala-OH，Fmoc-Gly-OH。
实施例4a Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂的制备
1)取实施例3制备的Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂50g，加入500mL脱帽试剂(20％的哌啶/DMF溶液，以体积比计)，搅拌反应20min，抽干树脂，重复3次至脱帽反应完全。脱帽反应结束后，每次使用500mL DMF溶液洗涤，总共洗涤8次。得到反应物Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂，均匀分成21份，分别加入到独立的多肽反应器中。
2)配制Fmoc-Asn(Trt)-OH活化后溶液：分别配制多份活化后溶液，每份都将4.2mmol量的Fmoc-Asn(Trt)-OH加入到25mL的DMF溶液中，再分别加入4.2mmol量的表1所示缩合试剂，室温混合反应30min。
3)将每份Fmoc-Asn(Trt)-OH活化后溶液分别加入多肽反应器中，进行缩合反应，其中反应温度缩合试剂种类和反应时间如下表1所示：其中尿素或高氯酸钠的加入量为0.1kg。反应结束后抽滤，然后分别用溶剂DMF洗涤8次。反应过程中使用茚三酮方法直接定性检测反 应完成情况，或将Fmoc-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂使用85％的三氟乙酸溶液切割，溶液用乙醚沉淀，沉淀物加水溶解后使用高效液相色谱法(HPLC)进行定量检测15肽色谱纯度，底物14肽残留和D-Asn-15肽消旋杂质含量情况，具体如下表1所示：
表1 不同反应条件下的缩合反应
Figure PCTCN2021086395-appb-000005
上表中“15肽”即H-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH；“14肽”即H-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH；“D-Asn-15肽消旋杂质”指H-(D-Asn)-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH。
结果表明，控制反应温度在20-35℃条件时，使用多种缩合试剂、重复缩合次数、延长缩合时间，底物14肽残留量始终＞4％，表明反应缩合不完全。当反应温度在40-60℃条件下，底物14肽残留量控制在＜4％，特别是当反应温度在50℃时，DIC/HOBt缩合试剂中添加适量尿素或NaClO 4，可以明显提高缩合率，尤其是尿素的加入更有助于缩合率的提高，底物14肽残留量较好地控制在0.20％。
实施例4b
取实施例3制备的Fmoc-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala- Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂950g，以表1中最优条件(#19)，即与Fmoc-Asn(Trt)-OH在DIC/HOBt/尿素缩合试剂存在下进行缩合反应，所得中间体作为实施例5的上游原料。
实施例5 Fmoc-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂的制备
向实施例4b的多肽反应器中，参照实施例2中2)-4)步骤所述的方法，即每偶联一个氨基酸均依次经过脱帽反应、活化反应和缩合反应。并且按照促肾上腺皮质激素(人源序列)氨基酸序列从C端到N端的16-39位顺序依次进行氨基酸的偶联，偶联的氨基酸顺序为：Fmoc-Pro-OH，Fmoc-Tyr(tBu)-OH，Fmoc-Val-OH，Fmoc-Lys(Boc)-OH，Fmoc-Val-OH，Fmoc-Pro-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Lys(Boc)-OH，Fmoc-Gly-OH，Fmoc-Val-OH，Fmoc-Pro-OH，Fmoc-Lys(Boc)-OH，Fmoc-Gly-OH，Fmoc-Trp(Boc)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Phe-OH，Fmoc-His(Trt)-OH，Fmoc-Glu(OtBu)-OH，Fmoc-Met-OH，Fmoc-Ser(tBu)-OH，Fmoc-Tyr(tBu)-OH，Fmoc-Ser(tBu)-OH。偶联结束，得到带保护基团的促肾上腺皮质激素(人源序列)肽链-树脂。
实施例6 H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe-树脂的制备
加入5L脱帽试剂(20％的哌啶/DMF溶液)，搅拌反应20min，抽干树脂，重复3次至脱帽反应完全。脱帽反应结束后，每次使用5L DMF溶液洗涤8次。再加入5L甲醇溶液洗涤8次，抽滤，40℃真空干燥，得到1.75Kg脱去N端Fmoc保护的促肾上腺皮质激素(人源序列)肽链-树脂。
实施例7 H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH促肾上腺皮质激素(人源序列)粗肽制备
将实施例6制备的1.75Kg肽链树脂加入到17.5L预冷至-12℃的切割试剂中反应2h(切割试剂由90％体积的三氟乙酸和体积浓度之和为10％的切肽保护试剂组成，其中切肽保护试剂由以体积浓度计1％苯酚、1％苯甲硫醚，1％二甲硫醚，1％1，2-乙二硫醇，1％三乙基硅烷，1％三异丙基硅烷和4％水组成)，控制整个反应的温度不超过40℃，反应完成后，过滤，收集滤液，滤液减压浓缩去除部分切割试剂，然后缓慢加入到预冷至-12℃的17.5L的甲基叔丁基醚中进行沉降，离心收集湿固体，湿固体再用87.5L的甲基叔丁基醚洗涤，收集固体加水溶解，冷冻干燥后得到0.75Kg固体粗肽，纯度为68.96％，目标肽含量48％(即0.36Kg目标肽)，具体如图11所示，目标肽的出峰时间为28.176min。
实施例8 H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro- Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH促肾上腺皮质激素(人源序列)粗肽的纯化
1.样品处理：取实施例7所得的0.75Kg促肾上腺皮质激素(人源序列)粗肽溶解于37.5L体积比为乙腈：水＝30:70的乙腈水溶液中，搅拌至完全溶解，0.45μm滤膜过滤，收集滤液待用。
2.第1次纯化：
纯化条件：色谱柱：以十八烷基硅烷键合硅胶为固定相的DAC-20动态轴向加压柱，柱子直径和填装长度为：20*25cm。流动相A：摩尔浓度为0.1mol/L的Tris水溶液，氨水调pH为8.0；B相：乙腈。流速：80ml/min。检查波长280nm。梯度：B％：30-60％(50min)进样量为20g。纯化过程：将色谱柱用流动相A平衡后上样，上样量5L样品溶液。线性梯度洗脱50min，收集纯度大于90％的目标肽溶液。目标肽成分回收率79％。
3.第2次纯化：
纯化条件：色谱柱：以十八烷基硅烷键合硅胶为固定相的DAC-20动态轴向加压柱，柱子直径和填装长度为：20*25cm。流动相A：摩尔浓度为0.1mol/L的硫酸铵水溶液，硫酸调pH为3.0；B相：乙腈。流速：80ml/min。检查波长280nm。梯度：B％：30-60％(50min)进样量为20g。
纯化过程：将色谱柱用流动相A平衡后上样，上样量1L样品溶液。线性梯度洗脱50min，收集纯度大于99％的目标肽溶液。目标肽成分回收率86％。
4.转盐及离子控制
转盐条件：色谱柱：以十八烷基硅烷键合硅胶为固定相的DAC-20动态轴向加压柱，柱子直径和填装长度为：20*25cm。流动相A：浓度为0.1％的乙酸水溶液；B相：乙腈。流速：80ml/min。检查波长280nm。梯度：B％：30-60％(50min)进样量为20g。
转盐过程：将色谱柱用流动相A平衡后上样，上样量1L样品溶液。线性梯度洗脱50min，收集目标肽溶液，目标肽成分回收率95％。
5.冷冻干燥
将上步所得目标肽溶液转移至大小合适的不锈钢托盘中，冷冻干燥后进行HPLC检测，得到纯度为99.88％的醋酸促肾上腺皮质激素(人源序列，SEQ ID NO.3)纯品(如图12所示，主峰出峰时间为27.930min，最大单杂为0.07％，总杂为0.13％)。收集到的目的肽为258g，含量为90％，目标肽的摩尔收率为65％。高分辨率质谱检测出的精确分子量为4538.232Da，与促肾上腺皮质激素(人源序列)的理论精确分子量一致。
实施例9
参照上述实施例4b-实施例8的方法，将15位氨基酸用Asp(Trt)替换Asn(Trt)，得到H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH促肾上腺皮质激素(人源序列)类似物(SEQ ID NO.4)纯品，纯度为99.58％，(具体如图13所示，主峰出峰时间为28.213min，最大单杂为0.16％，总杂为0.42％)。收集到的目的肽为255g，含量为89％，目标肽的摩尔收率为63％。高分辨率质谱检测出的精确分子量为4537.232Da，与促肾上腺皮质激素(人源序列)类似物的理论精确分子量一致。
实施例10(对比例)
严格按照对比文献US3953415实施例4中step 1至step 14的具体步骤制备促肾上腺皮质激素(人源序列)(SEQ ID NO.3)，总收率为17％，促肾上腺皮质激素(人源序列)纯度仅75.72％，具体如图14所示。
实施例11稳定性研究
取实施例8所得样品，采用双层药用低密度聚乙烯袋和一层铝塑复合袋包装，贮存于温度2-8℃条件下，分别在1、2、3和6个月进行评估，加速6个月的稳定性结果如下表2所示：
表2 加速6个月的稳定性结果
Figure PCTCN2021086395-appb-000006
结果表明在温度2-8℃条件下，促肾上腺皮质激素(人源序列SEQ ID NO.3)性状、有关物质、色谱纯度、水分和聚合物没有明显的变化趋势。说明促肾上腺皮质激素(人源序列SEQ ID NO.3)在该储存条件下是稳定的。所述聚合物指分子量大于促肾上腺皮质激素(人源序列SEQ ID NO.3)的杂质。
取实施例9所得样品，采用双层药用低密度聚乙烯袋和一层铝塑复合袋包装，贮存于温度2-8℃条件下，分别在1、2、3和6个月进行评估，加速6个月的稳定性结果如下表3所示：
表3 加速6个月的稳定性结果
Figure PCTCN2021086395-appb-000007
Figure PCTCN2021086395-appb-000008
结果表明在温度2-8℃条件下，促肾上腺皮质激素类似物(人源序列SEQ ID NO.4)性状、有关物质、色谱纯度、水分和聚合物没有明显的变化趋势。说明促肾上腺皮质激素类似物(人源序列SEQ ID NO.4)在该储存条件下是稳定的。所述聚合物指分子量大于促肾上腺皮质激素类似物(人源序列SEQ ID NO.4)的杂质。
实施例12 安全性数据
(1)体外活性研究
促皮质素是黑素皮质素受体2(MC2R)受体激动剂，促皮质素与MC2R结合可以刺激细胞下游信号通路，发生钙离子、cAMP等信号因子的改变。本发明通过检测钙离子信号，判定促皮质素与MC2R的结合能力，进而判断促皮质素的体外细胞活性。
取实施例8的样品与实施例10的样品进行体外活性研究。表达MC2R的CHO-K1细胞在10cm器皿中培养并且在37℃和5％CO 2条件下保存。
结果表明高纯的促肾上腺皮质激素(人源序列SEQ ID NO.3，99.88％)与低纯度的促肾上腺皮质激素(人源序列SEQ ID NO.3，75.72％)，与黑素皮质素受体2(MC2R)均有结合能力且表现出生物活性，其中高纯促肾上腺皮质激素的半抑制浓度(EC 50)为180.2nM，低纯促肾上腺皮质激素的半抑制浓度为287.9nM，半抑制浓度越低则活性越高。因此高纯促肾上腺皮质激素的活性优于低纯促肾上腺皮质激素，且前者是后者活性的1.6倍。具体活性检测数据如下表4所示：
表4 活性检测数据
样品来源 Bottom Top HillSlope EC 50(nM)
实施例8 -2.739 100.9 0.9745 180.2
实施例10 -1.846 94.88 1.249 287.9
(2)体外毒性研究
本发明通过体外检测药物与心脏受体蛋白(hERG)的亲和力来判断药物心脏代谢毒性的大小。药物与hERG受体亲和力越高，即半抑制浓度越低，其对心脏产生毒性所需要的剂量越低，心脏代谢毒性越强。
取实施例8的样品与实施例10的样品以及阳性对照药物多非利特(dofetilide)进行体外毒性研究，结果表明高纯促肾上腺皮质激素与低纯促肾上腺皮质激素的半抑制浓度(IC 50)均大于10000nM，阳性对照的半抑制浓度为2.09nM。因此高纯促肾上腺皮质激素与低纯促肾上腺皮质激素的毒性剂量远高于阳性对照，没有呈现出心脏代谢毒性。具体代谢毒性数据如下表5所示：
表5 心脏代谢毒性数据
样品来源 IC 50(nM) MaxDose(nM) ％Inh@MaxDose
实施例8 ＞10000 10000 -1.28
实施例10 ＞10000 10000 12.47
阳性对照药 2.090 10000 104.47

Claims (24)

  1. 一种包含促肾上腺皮质激素的组合物，其特征在于，所述促肾上腺皮质激素的纯度≥99％，最大单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素的序列从N端到C端如下所示：
    SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF(SEQ ID NO.3)。
  2. 一种包含促肾上腺皮质激素类似物的组合物，其特征在于，所述促肾上腺皮质激素类似物的纯度≥99.0％，最大单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素类似物的序列从N端到C端如下所示：
    SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF(SEQ ID NO.4)。
  3. 根据权利要求1或2所述的组合物，其特征在于，所述促肾上腺皮质激素或促肾上腺皮质激素类似物的纯度≥99.5％，总杂含量≤0.5％。
  4. 一种权利要求1至3任意一项所述组合物的制备方法，其特征在于，通过Fmoc固相合成法，分别按照SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列从C端到N端的顺序偶联氨基酸，再经过纯化，得到包含促肾上腺皮质激素的组合物或包含促肾上腺皮质激素类似物的组合物。
  5. 根据权利要求4所述组合物的制备方法，其特征在于，进一步包括以下步骤：
    1)通过Fmoc固相合成法，按照SEQ ID NO.3或SEQ ID NO.4所示氨基酸序列从C端到N端的顺序偶联氨基酸，获得带保护基团的促肾上腺皮质激素或其类似物肽链-树脂；
    2)使用切割试剂处理带保护基团的促肾上腺皮质激素或其类似物肽链-树脂，将促肾上腺皮质激素或其类似物肽链从树脂上裂解下来并去除肽链所有保护基团，得到包含促肾上腺皮质激素或其类似物的溶液；
    3)使用沉降试剂处理上述包含促肾上腺皮质激素或其类似物的溶液，得到促肾上腺皮质激素或其类似物粗品；
    4)将上述促肾上腺皮质激素或其类似物粗品利用液相色谱法纯化得到包含促肾上腺皮质激素或其类似物的组合物。
  6. 根据权利要求4或5所述的制备方法，其特征在于，所述步骤1)中，偶联氨基酸采用逐一偶联或者片段偶联，氨基酸N端采用Fmoc基团保护。
  7. 根据权利要求4或5所述的制备方法，其特征在于：所述步骤1)中，固相合成法中所用树脂为Wang树脂或2-三苯甲基氯甲烷树脂或Rink Amide AM Resin或Rink Amide MBHA Resin或Rink Amide Resin或连接有Fmoc-Phe-OH的树脂，优选为2-三苯甲基氯甲烷树脂。
  8. 根据权利要求4或5所述的制备方法，其特征在于：所述步骤1)中，偶联除Fmoc-Phe-OH以外的其它氨基酸AA的方法为：
    i.使用第一有机溶剂溶胀Fmoc-AA n-树脂，其中AA n表示连接有n个氨基酸，n为1-38中的自然数；可选的，氨基酸AA的结构相同或不同，氨基酸AA的N端采用Fmoc或Boc或Cbz基团保护，氨基酸AA侧链有保护基团或无保护基团；
    ii.将Fmoc-AA n-树脂与第二有机溶剂进行脱帽反应，至完全脱除Fmoc保护基团，最后用第三有机溶剂洗涤，得到H-AA n-树脂；
    iii.将Fmoc-AA m-OH在第一有机溶剂和活化试剂中进行活化，得到活化后的Fmoc-AA m-OH溶液，其中m为第(n+1)个氨基酸；可选的，步骤iii)可在步骤i) 或步骤ii)之前或之后完成；
    iv.将上述活化后的Fmoc-AA m-OH溶液与H-AA n-树脂进行偶联反应，得到Fmoc-AA (n+1)-树脂，最后用第三有机溶剂洗涤；
    v.将上述洗涤后的Fmoc-AA (n+1)-树脂循环使用步骤ii)-步骤iv)的方法进行偶联，最终得到带保护基团的促肾上腺皮质激素或其类似物肽链-树脂。
  9. 根据权利要求8所述的制备方法，其特征在于：所述步骤i和步骤iii中第一有机溶剂为非质子性有机溶剂，优选为DCM，DMF，NMP中的一种或几种，或其任意一种与HOBt的混合溶液，最优选为以质量体积比计算含有1％HOBt的DMF溶液。
  10. 根据权利要求8所述的制备方法，其特征在于：所述步骤ii中第二有机溶剂为含有有机碱的非质子性有机溶剂，优选为以体积比计算含有15-30％哌啶的DCM溶液，15-30％哌啶的DMF溶液，15-30％哌啶的NMP溶液中的一种，最优选为含有20％哌啶的DMF溶液。
  11. 根据权利要求8所述的制备方法，其特征在于：所述步骤ii中第三有机溶剂为第一有机溶剂或醇类溶剂，优选为DCM，甲醇、乙醇，DMF，NMP中的一种或几种，或其任意一种与HOBt的混合溶液，最优选为DMF；最后一次洗涤的第三有机溶剂为第一有机溶剂。
  12. 根据权利要求8所述的制备方法，其特征在于：所述步骤iii中的活化试剂为DIC、HBTU和Oxyma Pure组合物，DIC和Oxyma Pure组合物，DIC和HOBt组合物，DIEA、TBTU和HOBt组合物以及DIEA和PyBop组合物中的一种，优选为DIC和Oxyma Pure的组合物。
  13. 根据权利要求8所述的制备方法，其特征在于：所述步骤iv中，除偶联C-15位的氨基酸外，偶联其它氨基酸的反应温度为10-35℃，反应时间为0.5-5h；偶联C-15位氨基酸时的反应温度为40-60℃，优选45-55℃；反应时间为0.5-16h，优选为2-4h。
  14. 根据权利要求13所述的制备方法，其特征在于：偶联C-15位的氨基酸时，所述步骤iv的反应体系中加入尿素或高氯酸盐。
  15. 根据权利要求14所述的制备方法，其特征在于：所述尿素或高氯酸盐与Fmoc-Phe-树脂的质量比为0.1:1-1:1。
  16. 根据权利要求5所述的制备方法，其特征在于：将步骤1)得到的带保护基团的促肾上腺皮质激素或其类似物肽链-树脂与第二有机溶剂进行脱帽反应，再用第三有机溶剂洗涤，最后用第四有机溶剂洗涤，所述第四有机溶剂为甲醇、乙醇、DCM中的一种或几种，优选为甲醇。
  17. 根据权利要求5所述的制备方法，其特征在于：所述步骤2)中，所述切割试剂由三氟乙酸和切肽保护试剂组成，,所述切肽保护试剂由苯酚、苯甲硫醚、二甲硫醚、1，2-乙二硫醇、三乙基硅烷、三异丙基硅烷或水中的一种或几种组成；所述切肽保护试剂与三氟乙酸的体积比为1:4-1:19。
  18. 根据权利要求5所述的制备方法，其特征在于：所述步骤3)中，所述沉降试剂为醚类溶剂，优选乙醚或甲基叔丁基醚。
  19. 根据权利要求5所述的制备方法，其特征在于：所述步骤2)中切割试剂与步骤3)中沉降试剂的体积比为1：5-1：20。
  20. 根据权利要求5所述的制备方法，其特征在于：所述步骤4)中液相色谱法为反相高压液相色谱法，纯化以八烷基硅烷键合硅胶、十八烷基硅烷键合硅胶或者PS反相聚合物色谱 填料为固定相，使用动态轴向加压柱，以0.01-0.2mol/L的有机盐水溶液与有机溶剂为流动相或者使用无机盐水溶液与有机溶剂为流动相进行洗脱，优选先使用碱性流动相，后使用酸性流动相进行洗脱；最优选0.1mol/L的碱性Tris水溶液/乙腈和0.1mol/L的硫酸铵酸性水溶液/乙腈为流动相进行洗脱。
  21. 一种多肽树脂，其特征在于，具有如下序列：
    Q-P-树脂,所述P为Asn(Trt)-Gly-Ala-Glu(OtBu)-Asp(OtBu)-Glu(OtBu)-Ser(tBu)-
    Ala-Glu(OtBu)-Ala-Phe-Pro-Leu-Glu(OtBu)-Phe(SEQ ID NO.5)，所述树脂为2-三苯甲基氯甲烷树脂，所述Q无或选自下列的组合：
    H-Pro-，
    H-Tyr(tBu)-Pro-，
    H-Val-Tyr(tBu)-Pro-，
    H-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-G ly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-，
    H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Met-Glu(OtBu)-His(Trt)-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Gly-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Pro-Val-Lys(Boc)-Val-Tyr(tBu)-Pro-。
  22. 一种包含促肾上腺皮质激素与可药用载体的药用组合物，其特征在于：所述促肾上腺皮质激素或其类似物的纯度≥99％，任意单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素的序列从N端到C端如下所示：
    SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF(SEQ ID NO.3)。
  23. 一种包含促肾上腺皮质激素类似物与可药用载体的药用组合物，其特征在于：所述促肾上腺皮质激素类似物的纯度≥99％，任意单杂含量≤0.5％，总杂含量≤1％，所述促肾上腺皮质激素类似物的序列从N端到C端如下所示：
    SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF(SEQ ID NO.4)。
  24. 权利要求1至3，22至23任意一项所述的组合物在制备用于治疗新生儿痉挛症、成人多发性硬化症和风湿性、过敏、水肿免疫性疾病药物中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4055524A (en) * 1974-02-12 1977-10-25 Armour Pharmaceutical Company Synthesis of peptides
GB1586974A (en) * 1977-07-04 1981-03-25 Armour Pharma Synthesis of peptide amides
CN102675453A (zh) * 2011-03-09 2012-09-19 杭州中肽生化有限公司 一种促肾上腺皮质激素的类似物及其制备方法和用途
WO2013098802A2 (en) * 2011-12-29 2013-07-04 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Solid phase peptide synthesis via side chain attachment

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH560182A5 (en) * 1971-09-07 1975-03-27 Ciba Geigy Ag D-Ser(1)-alpha(h)-corticotropin prepn. - by conventional peptide synthesis methods
HU166913B (zh) 1972-05-15 1975-06-28
KR101058467B1 (ko) * 2004-10-27 2011-08-24 유니버시티 오브 덴버 부신피질 자극 호르몬 유사체 및 관련 방법
MX2021010989A (es) 2019-03-13 2021-10-01 Adamis Pharmaceuticals Corp Formulacion que incluye una combinacion de la hormona beta-endorfina y adrenocorticotropica.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4055524A (en) * 1974-02-12 1977-10-25 Armour Pharmaceutical Company Synthesis of peptides
GB1586974A (en) * 1977-07-04 1981-03-25 Armour Pharma Synthesis of peptide amides
CN102675453A (zh) * 2011-03-09 2012-09-19 杭州中肽生化有限公司 一种促肾上腺皮质激素的类似物及其制备方法和用途
WO2013098802A2 (en) * 2011-12-29 2013-07-04 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Solid phase peptide synthesis via side chain attachment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OELOFSEN, W.: "The chemistry of the adrenocorticotropins and the melanotropins", PHARMACOLOGY & THERAPEUTICS, vol. 1, no. 3, 1 January 1975 (1975-01-01), GB , pages 459 - 500, XP023858293, ISSN: 0163-7258 *

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