Synthesis method of desmopressin acetate

Abstract

The invention relates to the technical field of polypeptide synthesis, in particular to a synthetic method of desmopressin acetate. The synthesis method of the desmopressin acetate comprises the following steps that Fmoc-protected amino acids are coupled on amino resin one by one according to the sequence of the desmopressin acetate to obtain peptide resin, and the Fmoc-protected amino acids comprise Fmoc-Gly-OH, Fmoc-D-Arg (Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr-OH and Mpa (Trt)-OH; and carrying out cracking, oxidation, purification and salt conversion on the peptide resin to obtain desmopressin acetate. According to the synthesis method disclosed by the invention, Fmoc-protected amino acid without side chain protection is adopted, so that the generation of a specific impurity Mpa (tBu)-Tyr-Ph-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2 can be effectively prevented, and the purity and the yield of the product are effectively improved.

Description

Technical Field

[0001] The present application relates to the technical field of polypeptide synthesis, and particularly to a method for synthesizing desmopressin acetate. Background Art

[0002] Desmopressin Acetate is a structural analogue of natural arginine vasopressin, which is obtained by making two modifications to the chemical structure of the natural hormone, namely, removing the amino group from 1-cysteine and replacing 8-L-arginine with 8-D-arginine. The molecular formula is C50H72N14O16S2*xC2H4O2, and the molecular weight is 1129.35*x60.02. The peptide sequence structure is c(Mpa-Tyr-Phe-Gln-Asn-Cys)-Pro-D-Arg-Gly-NH2*xCH3COOH.

[0003] Desmopressin acetate is mainly used for the treatment of central diabetes insipidus, nocturnal enuresis and hemophilia, etc., and is also used for the test of renal urine concentration function and can be used as postoperative hemostasis.

[0004] During the synthesis of desmopressin acetate, the amino acids used usually contain multiple reactive functional groups, and side chain protection is required to prevent the generation of various by-products, thereby resulting in low purity and yield of the target product.

[0005] Currently, a kind of impurity (hereinafter referred to as this impurity as a specific impurity) will be generated in all existing synthesis methods of desmopressin acetate. The peptide sequence of this specific impurity is Mpa(tBu)-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2. The main reason for its generation is that after the tBu protecting group on the Tyr side chain is acidolyzed during the peptide chain cleavage process, part of it fails to be captured and quenched by the deionizing agent in time, and then links to the Mpa side chain again. There is no specific literature reporting on its research currently.

[0006] Since this impurity stably exists in the crude product and has a high content, it increases the difficulty of subsequent purification. It is necessary to seek a new synthesis method to effectively prevent the generation of this specific impurity, improve the product purity and yield, and reduce the production cost. Summary of the Invention

[0007] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for synthesizing desmopressin acetate, which uses Fmoc-Tyr-OH to solve the specific impurity generated due to the presence of the tBu side chain protecting group, and improve the purity and yield of desmopressin acetate.

[0008] To achieve the above purpose and other related purposes, the present application provides a method for synthesizing desmopressin acetate, including the following steps:

[0009] 1) Couple Fmoc - protected amino acids one by one according to the desmopressin acetate sequence on the amino resin to obtain peptide resin. The Fmoc - protected amino acids include Fmoc - Gly - OH, Fmoc - D - Arg(Pbf)-OH, Fmoc - Pro - OH, Fmoc - Cys(Trt)-OH, Fmoc - Asn(Trt)-OH, Fmoc - Gln(Trt)-OH, Fmoc - Phe - OH, Fmoc - Tyr - OH, Mpa(Trt)-OH;

[0010] 2) Carry out cleavage, oxidation, purification and salt conversion on the peptide resin obtained in step 1) to obtain desmopressin acetate.

[0011] The present invention also provides the application of desmopressin acetate obtained by the synthesis method as described above in the preparation of drugs for treating central diabetes insipidus, nocturnal enuresis and hemophilia.

[0012] As described above, compared with the prior art, the present application has the following beneficial effects:

[0013] The present application uses tyrosine without side - chain protection for the reaction. The probability of the side - chain phenolic hydroxyl group reacting under the condition of no strong catalyst during the condensation reaction is relatively low. And a specific coupling agent is used to avoid the esterification reaction between the side - chain phenolic hydroxyl group and the carboxyl group. A relatively low reaction temperature is adopted, which does not reach the effective temperature of the esterification reaction, facilitating the acylation reaction between the carboxyl group and the amino group. This synthesis method can effectively prevent the generation of specific impurity Mpa(tBu)-Tyr - Phe - Gln - Asn - Cys - Pro - D - Arg - Gly - NH2, improve the product purity and yield, and greatly reduce the production cost. Description of the Drawings

[0014] Figure 1 It is the HPLC detection chart of the crude product obtained in Example 1;

[0015] Figure 2 It is the HPLC detection chart of the combined sample of the crudely purified product obtained in Example 1;

[0016] Figure 3 It is the HPLC detection chart of the combined sample of the finely purified product obtained in Example 1;

[0017] Figure 4 It is the process flow chart of Example 1;

[0018] Figure 5 It is the HPLC detection chart of the crude product obtained in the cleavage reaction a of Comparative Example 1;

[0019] Figure 6 It is the HPLC detection chart of the crude product obtained in the cleavage reaction b of Comparative Example 1;

[0020] Figure 7 HPLC detection chart of the crude product obtained from cracking reaction c of Comparative Example 1;

[0021] Figure 8 HPLC detection chart of the combined sample of the roughly purified crude product obtained from cracking reaction a of Comparative Example 1;

[0022] Figure 9 HPLC detection chart of the combined sample of the finely purified crude product obtained from cracking reaction a of Comparative Example 1. Detailed implementation manners

[0023] In order to make the invention object, technical solutions and beneficial effects of the present application clearer, the present application will be further described below in conjunction with embodiments. It should be understood that the embodiments are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following embodiments are all conventional methods unless otherwise specified. Those familiar with this technology can easily understand other advantages and effects of the present application from the content disclosed in this description.

[0024] Through a large amount of exploration and research, the inventors of the present application have discovered a synthesis method of desmopressin acetate, and completed the present application on this basis.

[0025] The present application provides a synthesis method of desmopressin acetate, comprising the following steps:

[0026] 1) Sequentially couple Fmoc-protected amino acids according to the desmopressin acetate sequence on an amino resin to obtain a peptide resin, and the Fmoc-protected amino acids include Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr-OH, Mpa(Trt)-OH;

[0027] 2) Subject the peptide resin obtained in step 1) to cracking, oxidation, purification and salt conversion to obtain desmopressin acetate.

[0028] In a preferred embodiment, in step 1), the amino resin is selected from one of Rink amide AM resin, Rinkamide MBHA resin or Rink amide resin, and preferably Rink amide AM resin.

[0029] Preferably, the degree of substitution of the amino resin is 0.3-1.0 mmol / g, specifically, it can be 0.3 mmol / g, 0.4 mmol / g, 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, 0.9 mmol / g, 1.0 mmol / g, 1.1 mmol / g.

[0030] The degree of substitution of the above amino resin refers to the number of millimoles of active reaction sites per gram of the amino resin.

[0031] In a specific embodiment, the above amino resin is a swollen amino resin. The swollen amino resin is specifically prepared by swelling the resin with DMF at 5-10 times the volume of the amino resin for 10-30 min, and then removing the DMF.

[0032] Preferably, the above swollen amino resin needs to be deprotected before participating in the reaction. The deprotecting agent used for deprotection is any one of 10-30% PIP (piperidine) / DMF (N,N-dimethylformamide), 10-30% PIP / DCM (dichloromethane), 10-30% PIP / NMP (N-methylpyrrolidone), 10-30% PIP / DMAc (dimethylacetamide), preferably 20% PIP / DMF. Preferably, the reaction time for deprotection is 10-30 min. More preferably, the time can be 10-15 min, or 15-20 min, or 20-25 min, or 25-30 min; in a certain preferred embodiment, the reaction time for deprotection is 20 min.

[0033] In a preferred embodiment, the coupling of the Fmoc-protected amino acid in step 1) specifically includes the following steps:

[0034] A1. Coupling the amino resin with the Fmoc-protected amino acid to be coupled under the action of a coupling reagent, and adding an acetylation reagent to block after the reaction is completed to obtain the Fmoc-protected amino acid-resin;

[0035] A2. Deprotect the Fmoc-protected amino acid-resin, repeat the coupling step in a, and couple the protected amino acids of the remaining amino acids one by one to obtain the peptide resin.

[0036] Further, in step A1, the coupling reagent is selected from one or more of DIC (N,N'-diisopropylcarbodiimide), HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), and HOAT (1-hydroxy-7-azabenzotriazole), preferably the DIC / HOBT combination. In some specific embodiments of the present invention, the molar ratio of HOBT to DIC in the above coupling reagent is 1 to 3:1 to 3, which can be 1:1, 1:2, 1:3, 3:1, 3:2, and preferably the molar ratio of HOBT to DIC is 1:1.

[0037] In some specific embodiments of the present invention, the molar ratio of the above amino resin, Fmoc-protected amino acid, and coupling reagent is 1:1.5 to 2.5:1.8 to 3.

[0038] Further, after the coupling reagent activates the amino acid, it can effectively assist the acylation reaction between the carboxyl group of the amino acid and the exposed amino group on the resin. Further, in the presence of the coupling reagent, the esterification reaction between the phenolic hydroxyl group of the tyrosine side chain without side chain protection and the carboxyl group is avoided to produce by-products.

[0039] Specifically, the coupling reaction time is 90 to 180 min, and the reaction temperature is 20 to 40 °C. Preferably, the coupling reaction time can be 90 to 100 min, 100 to 110 min, 110 to 120 min, 120 to 130 min, 130 to 140 min, 140 to 150 min, 150 to 160 min, 160 to 170 min, 170 to 180 min, and preferably 90 min; the reaction temperature can be 20 - 25 °C, 25 - 30 °C, 30 - 35 °C, 35 - 40 °C, and preferably 30 to 35 °C.

[0040] Further, the coupling reaction temperature is relatively low and does not reach the effective temperature for the esterification reaction, further avoiding the generation of by-products.

[0041] In a preferred embodiment, in step A1, an acetylation reagent is used to block the amino acid that has not fully reacted. Preferably, the acetylation reagent is acetic anhydride / N-methylmorpholine reagent.

[0042] In a preferred embodiment, in step A1, after the coupling reaction, the Kaiser reagent is used to detect whether the reaction is complete.

[0043] In a preferred embodiment, the blocking reaction further includes post-treatment, and the post-treatment includes filtration, washing, and drying. Preferably, the washing liquid for washing is DMF reagent, and the number of washing times is 3 - 6 times.

[0044] In a preferred embodiment, in step A2, the deprotecting agent for deprotection is any one of 10-30% (v / v) PIP / DMF, 10-30% (v / v) PIP / DCM, 10-30% (v / v) PIP / NMP, 10-30% (v / v) PIP / DMAc, preferably 20% (v / v) PIP / DMF. Preferably, the reaction time for deprotection is 10-30 min. More preferably, the time can be 10-15 min, or 15-20 min, or 20-25 min, or 25-30 min; in a certain preferred embodiment, the reaction time for deprotection is 20 min.

[0045] According to the peptide sequence of desmopressin acetate (c(Mpa-Tyr-Phe-Gln-Asn-Cys)-Pro-D-Arg-Gly-NH2), each amino acid was coupled one by one on the resin by the above method of coupling Fmoc-protected amino acids to obtain c(Mpa-Tyr-Phe-Gln-Asn-Cys)-Pro-D-Arg-Gly-amino resin.

[0046] Furthermore, the Fmoc-Tyr-OH in the Fmoc-protected amino acids is selected as the Fmoc-protected amino acid without side-chain protection, so as to avoid the tBu protecting group of the Tyr side chain being acidolyzed during the peptide chain cleavage and part of it not being captured and quenched by the deionizing agent in time, and then linking to the Mpa side chain again.

[0047] In a preferred embodiment, in step 2), the cleavage solution used for cleavage includes TFA (trifluoroacetic acid) and a deionizing agent composition. Preferably, the deionizing agent includes one or more of Tis (triisopropylsilane), EDT (1,2-ethanedithiol), DTT (dithiothreitol), PhOH, H2O, methyl phenyl sulfide, 3-Mpa (3-mercaptopropionic acid). Specifically, the volume ratio of TFA to the deionizing agent in the cleavage solution is 75-95:5-25. More specifically, the volume ratio of TFA to the deionizing agent in the cleavage solution is 75:25, 80:20, 85:15, 90:10, 95:5, preferably 80:20, 90:10.

[0048] In some embodiments of the present invention, after cleavage, it further includes the steps of adding a precipitation solution to precipitate, wash and dry the cleavage product, and after the drying, the crude desmopressin peptide is obtained.

[0049] Preferably, the precipitation solution is any one or more of petroleum ether, diethyl ether, methyl tert-butyl ether, isopropyl ether, dipropyl ether, ethylene glycol dimethyl ether, preferably diethyl ether.

[0050] In some specific embodiments, the steps of adding a sedimentation solution to the cracked product for precipitation, washing, and drying specifically include: pouring the cracked product into the sedimentation solution, mixing evenly, precipitating, and washing 4 to 6 times, and placing the washed product in a dryer for vacuum drying to obtain desmopressin crude peptide.

[0051] In a preferred embodiment, in step 2), the oxidant used for oxidation is a 20 - 30% (w / w) hydrogen peroxide aqueous solution, preferably a 30% (w / w) hydrogen peroxide aqueous solution. Specifically, the steps of the oxidation specifically include: dissolving the desmopressin crude peptide in purified water, adjusting the pH with NaOH, and then adding hydrogen peroxide for the oxidation reaction. After the reaction ends, acetic acid is added to terminate the reaction. Preferably, the concentration of the desmopressin crude peptide is 0.5 mg / ml - 5 mg / ml, and the preferred concentration is 1 mg / ml - 2 mg / ml; the pH range is 7.0 - 8.0, the oxidation reaction time is 30 - 90 min; the pH for terminating the reaction with acetic acid is 3.0 - 4.0.

[0052] The desmopressin crude peptide forms a disulfide bond between Mpa at the 1st position and Cys at the 6th position through oxidation to obtain a cyclized desmopressin crude product.

[0053] In a preferred embodiment, in step 2), the purification includes purification by reverse high - performance liquid chromatography. Further preferably, the mobile phase of the reverse high - performance liquid chromatography is acetic acid / aqueous solution, acetonitrile solution, and / or the mobile phase of the reverse high - performance liquid chromatography is ammonium acetate / aqueous solution, acetonitrile solution.

[0054] Specifically, the concentration of the ammonium acetate aqueous solution is 20 mmol / L, and the ammonium acetate aqueous solution also needs to be adjusted to pH 5.0 with acetic acid.

[0055] Through bold assumptions and careful verification, this application selects Fmoc - Tyr - OH without side - chain protection and synthesizes desmopressin acetate under specific temperature and coupling agent conditions, which can effectively prevent the generation of specific impurity Mpa(tBu) - Tyr - Phe - Gln - Asn - Cys - Pro - D - Arg - Gly - NH2.

[0056] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than for limiting the protection scope of the present invention; in the description and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0058] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0059] Example 1

[0060] A. Peptide resin synthesis

[0061] Weigh 2.183 g of Rink Amide-AM Resin and add it to the reaction kettle. Then add 20 ml of DMF to swell for 20 min. After the swelling is completed, drain and add DMF to wash 2 times. Add 20 ml of 20% Pip / DMF (DBLK) solution to deprotect for 20 min. After the reaction is completed, drain and add DMF to wash 6 times.

[0062] Weigh 1.213 g of Fmoc-Gly-OH and 0.664 g of HOBT. After completely dissolving them in 15 ml of DMF, cool to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction. Couple for 90 min (the reaction temperature is controlled at 30 °C, and the following condensation reaction temperatures are the same).

[0063] After the reaction is completed, drain and add DMF to wash 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0064] Add 20 ml of DMF, 1.0 ml of acetic anhydride, and 1.2 ml of NMM. Bubble nitrogen through the reaction for 20 min. After the reaction is completed, drain and wash with DMF 3 times.

[0065] Add 20 ml of 20% Pip / DMF (DBLK) solution to deprotect for 20 min. After the reaction is completed, drain and add DMF to wash 6 times.

[0066] Weigh 2.552 g of Fmoc-D-Arg(Pbf)-OH and 0.652 g of HOBT. After each is completely dissolved in 15 ml of DMF, cool to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction, and couple for 90 min.

[0067] After the reaction is completed, drain it, and wash it 3 times with DMF. Take the resin and detect it by the Kaiser method. The resin is colorless, and proceed to the next step of the reaction.

[0068] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction is completed, drain it, and wash it 6 times with DMF.

[0069] Weigh 1.442 g of Fmoc-Pro-OH and 0.679 g of HOBT. After adding and completely dissolving in 15 ml of DMF, cool to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction, and couple for 90 min.

[0070] After the reaction is completed, drain it, and wash it 3 times with DMF. Take the resin and detect it by the Kaiser method. The resin is colorless, and proceed to the next step of the reaction.

[0071] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction is completed, drain it, and wash it 6 times with DMF.

[0072] Weigh 2.416 g of Fmoc-Cys(Trt)-OH and 0.705 g of HOBT. After adding and completely dissolving in 15 ml of DMF, cool to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction, and couple for 90 min.

[0073] After the reaction is completed, drain it, and wash it 3 times with DMF. Take the resin and detect it by the tetrachlorobenzoquinone method. The resin is colorless, and proceed to the next step of the reaction.

[0074] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction is completed, drain it, and wash it 6 times with DMF.

[0075] Weigh 2.417 g of Fmoc-Asn(Trt)-OH and 0.656 g of HOBT. After adding and completely dissolving in 15 ml of DMF, cool to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction, and couple for 90 min.

[0076] After the reaction was completed, the solvent was drained, and the resin was washed three times with DMF. The resin was tested by Kaiser method and was colorless, then proceed to the next reaction step.

[0077] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction was completed, the solvent was drained, and the resin was washed six times with DMF.

[0078] Weigh 2.459 g of Fmoc-Gln(Trt)-OH and 0.652 g of HOBT, add 15 ml of DMF to completely dissolve, then cool to 0 - 15 °C, and then add 0.8 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for coupling reaction for 90 min.

[0079] After the reaction was completed, the solvent was drained, and the resin was washed three times with DMF. The resin was tested by Kaiser method and was colorless, then proceed to the next reaction step.

[0080] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction was completed, the solvent was drained, and the resin was washed six times with DMF.

[0081] Weigh 1.640 g of Fmoc-Phe-OH and 0.654 g of HOBT, add 15 ml of DMF to completely dissolve, then cool to 0 - 15 °C, and then add 0.8 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for coupling reaction for 90 min.

[0082] After the reaction was completed, the solvent was drained, and the resin was washed three times with DMF. The resin was tested by Kaiser method and was colorless, then proceed to the next reaction step.

[0083] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction was completed, the solvent was drained, and the resin was washed six times with DMF.

[0084] Weigh 1.626 g of Fmoc-Tyr-OH and 0.671 g of HOBT, add 15 ml of DMF to each to completely dissolve, then cool to 0 - 15 °C, and then add 0.8 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for coupling reaction for 90 min.

[0085] After the reaction was completed, the solvent was drained, and the resin was washed three times with DMF. The resin was tested by Kaiser method and was colorless, then proceed to the next reaction step.

[0086] Add 20 ml of DBLK solution for deprotection for 20 min. After the reaction was completed, the solvent was drained, and the resin was washed six times with DMF.

[0087] Weigh 1.655 g of Mpa(Trt)-OH and 0.652 g of HOBT. After completely dissolving them in 15 ml of DMF, cool the solution to 0 - 15 °C, and then add 0.8 ml of DIC to activate for 5 - 10 min. Add the activated solution to the reaction kettle, and additionally add 5 ml of DMF for the coupling reaction, and couple for 120 min.

[0088] After the reaction is completed, drain it, and wash it 3 times with DMF. Detect the resin by Kaiser method. The resin is colorless, and proceed to the next reaction. Wash it 3 times with ether and then drain it. Weigh the weight of the peptide resin after draining, which is 8.061 g.

[0089] B. Cleavage reaction

[0090] Add the above peptide resin to 100 ml of cleavage reagent (TFA / Tis / PhOH / H2O = 90 / 4 / 3 / 3 + DTT (2eq)) and react for 120 min. Control the reaction temperature at 25 °C. After the reaction is completed, filter out the liquid and add it to 500 ml of ice-cold ether to mix well and filter and wash. Wash the filter cake 5 times with ether. After washing, place the solid crude product in a dryer and dry it under vacuum to obtain 2.105 g of crude product. Detect the purity of the crude product by HPLC, as Figure 1 shown. The purity of the crude product is 89.406%, and no specific impurities are detected.

[0091] C. Oxidation reaction

[0092] Take 1.033 g of the above-obtained crude product, completely dissolve it in 1 L of purified water, adjust the pH to 8.0 with 1 M NaOH, add 18 ml of 30% hydrogen peroxide for oxidation reaction for 60 min. After the reaction is completed, add acetic acid to adjust the pH to 4.0 to terminate the reaction. Detect the purity of the oxidized peptide sample by HPLC, which is 88.541%.

[0093] D. Purification of crude product

[0094] Use a silica gel column bonded with octadecylsilyl (Unisil 10 - 120C18) for the oxidized peptide sample obtained from the above oxidation reaction. Mobile phase A is 1% acetic acid aqueous solution, and mobile phase B is acetonitrile. Perform preparative elution with a gradient of 0 min (95% A + 5% B) - 10 min (95% A + 5% B) - 15 min (90% A + 10% B) - 95 min (80% A + 20% B). Collect the target fractions and detect the purity by HPLC, as Figure 2 shown. The purity of the combined fraction samples is 99.25%, and the yield is 91.1%.

[0095] The above-mentioned crudely purified sample was subjected to preparative elution on a polystyrene divinylbenzene polymer packing column (UniPS10-300) with mobile phase A being 20 mM ammonium acetate (pH adjusted to 5.0 with acetic acid) and mobile phase B being acetonitrile, with a gradient of 0 min (95% A + 5% B) - 10 min (95% A + 5% B) - 15 min (90% A + 10% B) - 135 min (75% A + 25% B). The target fractions were collected, and the purity was detected by HPLC. As Figure 3 shown, the purity of the combined fraction sample was 99.93% and the yield was 66.27%.

[0096] Experimental conclusion: In this example, using the preparative process flow as Figure 4 shown and using Fmoc-Tyr-OH without a side-chain protecting group to participate in the reaction can effectively avoid the appearance of specific impurities. Using the cleavage agent of Comparative Example 1 can achieve the effect of significantly improving the sample purity, and the sample yield is higher.

[0097] Comparative Example 1

[0098] A. Peptide resin synthesis

[0099] Weighed 25.816 g of Rink Amide AM Resin (substitution degree 0.93 mmol / g) and added it to the reaction kettle. Then added 200 ml of DMF to swell for 20 min. After the swelling ended, it was drained and washed twice with DMF. Added 200 ml of 20% Pip / DMF (DBLK) solution for deprotection for 20 min. After the reaction ended, it was drained and washed six times with DMF.

[0100] Weighed 14.281 g of Fmoc-Gly-OH and 7.793 g of HOBT, added 150 ml of DMF to completely dissolve, cooled to 0 - 15 °C, and then added 9.6 ml of DIC for activation for 5 - 10 min. Added the activated solution to the reaction kettle, and supplemented with 50 ml of DMF for coupling reaction for 90 min (the reaction temperature was controlled at 30 °C, and the following condensation reaction temperatures were the same). After the reaction ended, it was drained and washed three times with DMF. The resin was detected by the Kaiser method. If the resin was colorless, it entered the next reaction.

[0101] Added 200 ml of DMF, 12.0 ml of acetic anhydride, and 14.4 ml of NMM to the reaction kettle, reacted for 20 min. After the reaction ended, it was drained and washed three times with DMF.

[0102] Add 200 ml of DBLK solution for 20 min of deprotection reaction. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 31.177 g of Fmoc-D-Arg(Pbf)-OH and 7.773 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0103] Add 200 ml of DBLK solution for 20 min of deprotection reaction. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 17.241 g of Fmoc-Pro-OH and 7.770 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0104] Add 200 ml of DBLK solution for 20 min of deprotection reaction. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 28.177 g of Fmoc-Cys(Trt)-OH and 7.814 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for tetrachlorobenzoquinone method detection. The resin is colorless, and proceed to the next step of the reaction.

[0105] Add 200 ml of DBLK solution for 20 min of deprotection reaction. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 28.490 g of Fmoc-Asn(Trt)-OH and 7.763 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0106] Add 200 ml of DBLK solution for deprotection reaction for 20 min. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 29.333 g of Fmoc-Gln(Trt)-OH and 7.810 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0107] Add 200 ml of DBLK solution for deprotection reaction for 20 min. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 18.631 g of Fmoc-Phe-OH and 7.788 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take a little resin in a small test tube for Kaiser method detection. The resin is colorless, and proceed to the next coupling cycle.

[0108] Add 200 ml of DBLK solution for deprotection reaction for 20 min. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 22.132 g of Fmoc-Tyr(tBu)-OH and 7.814 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 90 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take the resin for Kaiser method detection. The resin is colorless, and proceed to the next step of the reaction.

[0109] Add 200 ml of DBLK solution for deprotection reaction for 20 min. After the reaction is completed, drain it, and add DMF for washing 6 times. Weigh 16.774 g of Mpa(Trt)-OH and 7.810 g of HOBT. After completely dissolving them in 150 ml of DMF, cool to 0 - 15 °C, and then add 9.6 ml of DIC for activation for 5 - 10 min. Add the activated solution to the reaction kettle, and then add an additional 50 ml of DMF for coupling reaction for 120 min. After the reaction is completed, drain it, and add DMF for washing 3 times. Take a little resin in a small test tube for Kaiser method detection. The resin is colorless. Add ether for washing 3 times. After washing, drain the resin, and the weight of the peptide resin obtained is 86.568 g.

[0110] B. Cleavage Reaction

[0111] Cleavage Reaction a: Take 7.193 g of the above peptide resin and add it to 100 ml of cleavage reagent (TFA / Tis / PhOH / H2O = 90 / 4 / 3 / 3 + DTT (2 eq)) for reaction for 120 min, and control the reaction temperature at 25°C. After the reaction, filter out the liquid, add it to 500 ml of ice-cold diethyl ether, mix well, filter and wash. Wash the filter cake with diethyl ether 5 times. After washing, place the solid crude product in a desiccator and dry it under vacuum to obtain 2.504 g of crude product. Detect the purity of the crude product by HPLC. As Figure 5 shown, the purity of the crude product is 74.967%, and the content of specific impurities is 9.122%.

[0112] Cleavage Reaction b: Take 4.142 g of the above peptide resin and add it to 60 ml of cleavage reagent (TFA / Tis / methylphenyl sulfide / PhOH / H2O = 80 / 8 / 4 / 4 / 4 + DTT (2 eq)) for reaction for 120 min, and control the reaction temperature at 25°C. After the reaction, filter out the liquid, add it to 300 ml of ice-cold diethyl ether, mix well, filter and wash. Wash the filter cake with diethyl ether 5 times. After washing, place the solid crude product in a desiccator and dry it under vacuum to obtain 1.366 g of crude product. Detect the purity of the crude product by HPLC. As Figure 6 shown, the purity of the crude product is 82.365%, and the content of specific impurities is 4.734%.

[0113] Cleavage Reaction c: Add 4.154 g of the above peptide resin to 60 ml of cleavage reagent (TFA / Tis / methylphenyl sulfide / EDT / PhOH / H2O = 80 / 8 / 3 / 3 / 3 / 3 + DTT (2 eq)) for reaction for 120 min, and control the reaction temperature at 25°C. After the reaction, filter out the liquid, add it to 300 ml of ice-cold diethyl ether, mix well, filter and wash. Wash the filter cake with diethyl ether 5 times. After washing, place the solid crude product in a desiccator and dry it under vacuum to obtain 1.313 g of crude product. Detect the purity of the crude product by HPLC. As Figure 7 shown, the purity of the crude product is 84.836%, and the content of specific impurities is 4.135%.

[0114] C. Oxidation Reaction

[0115] Oxidation Reaction a: Take 1.012 g of the crude product obtained from Cleavage Reaction a, add 1 L of purified water to completely dissolve it, add 1 M NaOH to adjust the pH to 8.0, add 18 ml of 30% hydrogen peroxide for oxidation reaction for 60 min. After the reaction, add acetic acid to adjust the pH to 4.0 to terminate the reaction. Detect the purity of the oxidized peptide sample by HPLC to be 74.058%.

[0116] Oxidation reaction b: Take 1.007 g of the crude product obtained from cleavage reaction b, dissolve it completely in 1 L of purified water, adjust the pH to 8.0 with 1 M NaOH, add 18 ml of 30% hydrogen peroxide for oxidation reaction for 60 min, and add acetic acid to adjust the pH to 4.0 to terminate the reaction after the reaction ends. The purity of the oxidized peptide sample detected by HPLC is 81.452%.

[0117] Oxidation reaction c: Take 1.056 g of the crude product obtained from cleavage reaction c, dissolve it completely in 1 L of purified water, adjust the pH to 8.0 with 1 M NaOH, add 18 ml of 30% hydrogen peroxide for oxidation reaction for 60 min, and add acetic acid to adjust the pH to 4.0 to terminate the reaction after the reaction ends. The purity of the oxidized peptide sample detected by HPLC is 84.550%.

[0118] D. Purification of the crude product

[0119] The oxidized peptide sample obtained from the above oxidation reaction a was subjected to preparative elution on an octadecylsilyl-bonded silica gel packing column (Unisil 10-120 C18) with mobile phase A being 1% aqueous acetic acid solution and mobile phase B being acetonitrile at a gradient of 0 min (95% A + 5% B) - 10 min (95% A + 5% B) - 15 min (90% A + 10% B) - 95 min (80% A + 20% B). The target fractions were collected and the purity was detected by HPLC. As Figure 8 shown, the purity of the combined fraction sample is 96.30% and the recovery rate is 71.0%.

[0120] The above-mentioned roughly purified sample was subjected to preparative elution on a polystyrene divinylbenzene polymer packing column (UniPS10-300) with mobile phase A being 20 mM ammonium acetate (adjusted to pH 5.0 with acetic acid) and mobile phase B being acetonitrile at a gradient of 0 min (95% A + 5% B) - 10 min (95% A + 5% B) - 15 min (90% A + 10% B) - 135 min (75% A + 25% B). The target fractions were collected and the purity was detected by HPLC. As Figure 9 shown, the purity of the combined fraction sample is 99.79% and the recovery rate is 55.1%.

[0121] Experimental conclusion: Using Fmoc-Tyr-OH with side chain protecting groups to participate in the reaction, the content of specific impurities is relatively high. After using different cleavage agents for cleavage reactions, the purity of the samples is relatively low and the sample recovery rate is low.

[0122] Example 2

[0123] Optimization of the coupling reaction temperature

[0124] According to the method for synthesizing desmopressin acetate in Example 1, the coupling reaction temperature was optimized. The present invention compared five reaction temperatures: 20°C, 25°C, 30°C, 35°C, and 40°C. The results are shown in Table 1. By comparing the purity of the crude desmopressin acetate, within the range of 20-40°C, the purity of the crude product obtained in the experiment showed a parabolic trend, and the purity of the crude product was relatively high when the reaction temperature was between 30-35°C.

[0125] Table 1 Experimental results of the change in coupling reaction temperature

[0126]

[0127]

[0128] The above embodiments are intended to illustrate the embodiments disclosed in the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in connection with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for synthesizing desmopressin acetate, comprising the following steps: 1) coupling Fmoc-protected amino acids one by one on an amino resin according to the sequence of desmopressin acetate to obtain a peptide resin, wherein the Fmoc-protected amino acids include Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr-OH, and Mpa(Trt)-OH; 2) The peptide resin obtained in step 1) is cleaved, oxidized, purified and converted to salt to obtain desmopressin acetate.

2. The synthesis method according to claim 1, characterized in that In step 1), the amino resin is selected from one of Rinkamide AM resin, Rink amide MBHA resin or Rink amide resin, preferably, the amino resin is Rink amide AM resin; And / or, the degree of substitution of the amino resin is 0.3-1.0 mmol / g.

3. The synthesis method according to claim 1, characterized in that In step 1), the coupling of Fmoc-protected amino acids specifically comprises the following steps: A1. The amino resin and the Fmoc-protected amino acid to be coupled are subjected to a coupling reaction under the action of a coupling reagent, and an acetylation reagent is added after the reaction to obtain an Fmoc-protected amino acid-resin; A2. Deprotect the Fmoc-protected amino acid-resin, repeat the coupling steps of A1, and couple the Fmoc-protected amino acids of the remaining amino acids one by one to obtain a peptide resin.

4. The synthesis method according to claim 3, characterized in that In step A1, the coupling reagent is selected from one or more of DIC, HBTU, HATU, HOBT, and HOAT, preferably a combination of DIC and HOBT.

5. The synthesis method according to claim 4, characterized in that The molar ratio of HOBT to DIC in the coupling reagent is 1-3:1-3, and preferably the molar ratio of HOBT to DIC is 1:

1.

6. The synthesis method according to claim 3, characterized in that In step A1, the coupling reaction time is 90-180 min, preferably 90 min; And / or, the reaction temperature of the coupling reaction is 30-35°C.

7. The synthesis method according to claim 3, characterized in that In step A2, the deprotection removing agent is selected from any one of 10-30% (v / v) PIP / DMF, 10-30% (v / v) PIP / DCM, 10-30% (v / v) PIP / NMP, and 10-30% (v / v) PIP / DMAc. Preferably, the deprotection removing agent is 20% (v / v) PIP / DMF.

8. The synthesis method according to claim 1, characterized in that In step 2), the lysis solution used for the lysis includes TFA and a deionizer, and the deionizer includes one or more of Tis, EDT, DTT, PhOH, H2O, methyl phenyl sulfide, and 3-Mpa.

9. The synthesis method according to claim 8, characterized in that The volume ratio of TFA to deionizer is 75-95:5-25.

10. The synthesis method according to claim 1, characterized in that In step 2), the oxidant used in the oxidation is a 20-30% (w / w) hydrogen peroxide solution; And / or, the purification comprises purification by reverse phase high performance liquid chromatography.

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