Method For Synthesizing Degarelix

Abstract

The present invention relates to the field of medicinal synthesis, and discloses a method for synthesizing degarelix. The method of the present invention as a whole divides the synthesis of degarelix into two parts from amino acids at positions 5 and 6, employs proper protective groups in part of the protected amino acids therein, and finally uses in association with a specific acidolysis agent to complete the whole synthesis process. In the present invention, a proper synthesizing scheme is selected, and adaptive protective group and acidolysis agent are selected, so that the overall synthesis process is optimized, the purity of degarelix is significantly improved with a higer total yield, and the production of the toxic hydantoin degradation product is avoided.

Claims

1. A method for synthesizing degarelix, comprising the following steps: step 1: condensing a protected D-alanine with an amino resin under the action of a condensation reagent and an activation reagent to obtain a peptide resin 1; step 2: sequentially extending and coupling a protected Pro, protected Lys(ipr), protected Leu and Boc-D-Aph(Fmoc) by starting from the peptide resin 1 according to the order from C-terminus to N-terminus of the amino acid sequence of degarelix under the action of the condensation reagent and the activation reagent, and then removing the side chain Fmoc protective group in Aph(Fmoc) to generate D-Aph(NH.sub.2), to obtain a peptide resin 2, wherein the protected Lys(ipr) is a protected Lys(ipr, Z); step 3: reacting the side chain amino group in D-Aph(NH.sub.2) in the peptide resin 2 with tert-butyl isocyanate under the catalysis of an organic base to generate D-Aph(tBu-Cbm), to obtain a peptide resin 3; step 4: sequentially extending and coupling a protected Aph(Boc), protected Ser(Bzl), protected D-Pal, protected D-Cpa, protected D-Nal and Ac.sub.2O by starting from the peptide resin 3 according to the order from C-terminus to N-terminus of the amino acid sequence of degarelix under the action of the condensation reagent and the activation reagent, to obtain a peptide resin 4; step 5: removing the side chain Boc protective group in the protected Aph(Boc) in the peptide resin 4, and incorporating L-4,5-dihydroorotic acid under the action of the condensation reagent and the activation reagent to obtain a degarelix resin; step 6: acidolysing the degarelix resin by an acidolysis agent to obtain a crude degarelix, wherein the acidolysis agent is a solution of hydrogen bromide in trifluoroacetic acid; and step 7: purifying the crude degarelix to obtain a pure degarelix.

2. The method according to claim 1, wherein the protected D-alanine in step 1 is Fmoc-D-Ala or Boc-D-Ala.

3. The method according to claim 1, wherein the protected Pro, protected Lys(ipr) and protected Leu in step 2 is Fmoc-Pro, Fmoc-Lys(ipr, Z) and Fmoc-Leu; or Boc-Pro, Boc-Lys(ipr, Z) and Boc-Leu.

4. The method according to claim 1, wherein the amino resin is MBHA resin.

5. The method according to claim 1, wherein the molar ratio of the protected D-alanine to the amino resin having its amino group coupled to a protective group is 1-6:1.

6. The method according to claim 1, wherein the condensation reagent is one of N,N-diisopropyl carbodiimide, N,N-dicyclohexyl carbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate/organic base, benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate/organic base, and O-benzotriazole-N,N,N′,N′-tetramethylurea tetrafluoroborate/organic base.

7. The method according to claim 1, wherein the organic base is N,N-diisopropylethylamine, triethylamine or N-methylmorpholine.

8. The method according to claim 1, wherein the activation reagent is 1-hydroxybenzotrizole or N-hydroxy-7-azabenzotriazole.

9. The method according to claim 1, wherein the concentration in mass percentage of hydrogen bromide in the acidolysis agent is 5%40%.

10. The method according to claim 1, wherein the protected Aph(Boc), protected Ser(Bzl), protected D-Pal, protected D-Cpa and protected D-Nal are Fmoc-Aph(Boc), Fmoc-Ser(Bzl), Fmoc-D-Pal, Fmoc-D-Cpa and Fmoc-D-Nal.

Description

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0047] The invention discloses a method for synthesizing degarelix, which can be achieved by those skilled in the art through properly modifying the process parameters in light of the present disclosure. It is specifically to be indicated that, all such similar substitutions and modifications are apparent to those skilled in the art and are deemed to be included in the present invention. The method of the present invention has been described by preferred examples, and it is apparent that the related personnel would achieve and apply the techniques of the present invention by alterations or proper modifications and combinations of the compound and the preparation method described herein, without departing from the content, spirit and scope of the preset invention.

[0048] In particular embodiments of the present invention, the amino acids therein are purchased from Chengdu Huirong Biotechnology Co., Ltd., and all the resins are purchased from Shangyu Pure Resin Co., Ltd. The corresponding meanings of the abbreviations used in the application document are listed in Table 1.

TABLE-US-00001 TABLE 1 Definitions for abbreviations Abbreviation Name Abbreviation Name Fmoc 9-fluorenyl- Aph 4-amino-phenylalanine methyloxy- carbonyl tBu tert-butyl D-Cpa 4-chloro-D-phenylalanine Boc tert-butoxy- D-Ala D-alanine carbonyl Bzl benzyl D-Aph 4-amino-D-phenylalanine Z carbobenzoxy Lys(iPr) N.sup.6-(1-methylethyl)lysine Ser serine D-Nal 3-(2-naphthyl)-D-alanine Leu leucine D-Pal 3-(3-pyridyl)-D-alanine Ac acetyl DIEA

[0049] Hereinafter, the present invention will be further explained in conjunction with Examples.

Example 1: Synthesis of the Peptide Resin 1

[0050] 0.15 mol Fmoc-D-Ala and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution for later use.

[0051] 0.05 mol MBHA resin (with a substitution value of about 0.6 mmol/g) was taken, swollen in DMF for 25 min, washed and filtered. The activated Fmoc-D-Ala solution was added and the reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for three times and then with DCM for 3 times (3 min for each wash) to obtain Fmoc-D-Ala-MBHA resin, which was deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to obtain the peptide resin 1 (D-Ala-MBHA resin).

Example 2: Synthesis of the Peptide Resin 1

[0052] 0.15 mol Boc-D-Ala and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 mm at room temperature to obtain the activated protected amino acid solution for later use.

[0053] 0.05 mol MBHA resin (with a substitution value of about 0.6 mmol/g) was taken, swollen in DMF for 25 min, washed and filtered. The activated Fmoc-D-Ala solution was added and the reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash) to obtain Boc-D-Ala-MBHA resin, which was deprotected with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to obtain the peptide resin 1 (D-Ala-MBHA resin).

Example 3: Synthesis of the Peptide Resin 2

[0054] 0.15 mol Fmoc-Pro and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 mm at room temperature to obtain the activated protected amino acid solution.

[0055] The activated protected amino acid solution described above was added to the peptide resin 1 prepared in Example 1, and the reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), deprotected again with 20% PIP/DMF solution for 25 min, washed and filtered to complete the incorporation of Pro.

[0056] Fmoc-Lys(ipr, Z), Fmoc-Leu and Boc-D-Aph(Fmoc) were incorporated by the same process, and then finally deprotected again with 20% PIP/DMF solution to form the peptide resin 2

[Boc-D-Aph(NH.sub.2)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 4: Synthesis of the Peptide Resin 2

[0057] 0.15 mol Boc-Pro and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution.

[0058] The activated protected amino acid solution described above was added to the peptide resin 1 prepared in Example 2, and the reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), deprotected again with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to complete the incorporation of Pro.

[0059] The incorporation of Boc-Lys(ipr, Z) and Boc-Leu was completed by the same process.

[0060] 0.15 mol Boc-D-Aph(Fmoc) and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution, which was added to the above resin which had completed the incorporation of Pro, Boc-Lys(ipr, Z) and Boc-Leu, and the reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), and deprotected again with 20% PIP/DMF solution to obtain the peptide resin 2 [Boc-D-Aph(NH.sub.2)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 5: Synthesis of the Peptide Resin 3

[0061] 0.5 mol tert-butyl isocyanate and 0.5 mol DIEA were taken, dissolved with an appropriate amount of DMF, and added to the peptide resin 2 prepared in Example 3. The reaction was stirred at room temperature overnight. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), and deprotected again with 30% TFA/DCM solution for 30 min, washed with DMF, DCM and filtered to obtain the peptide resin 3 [NH.sub.2-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 6: Synthesis of the Peptide Resin 3

[0062] 0.5 mol tert-butyl isocyanate and 0.5 mol DIEA were taken, dissolved with an appropriate amount of DMF, and added to the peptide resin 2 prepared in Example 4. The reaction was stirred at room temperature overnight. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), and deprotected again with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to obtain the peptide resin 3 [NH.sub.2-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 7: Synthesis of the Peptide Resin 4

[0063] 0.15 mol Fmoc-Aph(Boc) and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution.

[0064] The activated protected amino acid solution above was added to the peptide resin 3 prepared in Example 5. The reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), deprotected again with 20% PIP/DMF solution for 25 min, washed and filtered to complete the incorporation of Fmoc-Aph(Boc).

[0065] Fmoc-Ser(Bzl), Fmoc-D-Pal, Fmoc-D-Cpa, Fmoc-D-Nal and Ac.sub.2O were incorporated by the same process, then deprotected again with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to obtain the peptide resin 4 [Ac-D-Nal-D-Cpa-D-Pal-Ser(Bzl)-Aph(Boc)-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 8: Synthesis of the Peptide Resin 4

[0066] 0.15 mol Fmoc-Aph(Boc) and 0.15 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.15 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution.

[0067] The activated protected amino acid solution described above was added to the peptide resin 3 prepared in Example 6. The reaction was stirred for 3 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash), deprotected again with 20% PIP/DMF solution for 25 min, washed and filtered to complete the incorporation of Fmoc-Aph(Boc).

[0068] Fmoc-Ser(Bzl), Fmoc-D-Pal, Fmoc-D-Cpa, Fmoc-D-Nal and Ac.sub.2O were incorporated by the same process, then deprotected again with 30% TFA/DCM solution for 30 min, neutralized with DIEA/DCM solution, washed with DMF, DCM and filtered to obtain the peptide resin 4 [Ac-D-Nal-D-Cpa-D-Pal-Ser(Bzl)-Aph(Boc)-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 9: Synthesis of the Degarelix Peptide Resin

[0069] 0.2 mol L-4,5-dihydrooratic acid (Hor) and 0.2 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.2 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution.

[0070] The activated protected amino acid solution described above was added to the peptide resin 4 prepared in Example 7. The reaction was stirred for 6 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash) to obtain the degarelix peptide resin [Ac-D-Nal-D-Cpa-D-Pal-Ser(Bzl)-Aph(Hor)-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 10: Synthesis of the Degarelix Peptide Resin

[0071] 0.2 mol L-4,5-dihydrooratic acid (Hor) and 0.2 mol HOBt were taken, and dissolved with an appropriate amount of DMF. 0.2 mol DIC was additionally taken, and slowly added to the solution of the protected amino acid in DMF under stirring. The reaction was stirred for 30 min at room temperature to obtain the activated protected amino acid solution.

[0072] The activated protected amino acid solution described above was added to the peptide resin 4 prepared in Example 8. The reaction was stirred for 6 h at room temperature. The reaction solution was sucked away, washed with DMF for 3 times and then with DCM for 3 times (3 min for each wash) to obtain the degarelix peptide resin [Ac-D-Nal-D-Cpa-D-Pal-Ser(Bzl)-Aph(Hor)-D-Aph(tBu-Cbm)-Leu-Lys(ipr, Z)-Pro-D-Ala-MBHA resin].

Example 11: Preparation of the Crude Degarelix

[0073] The degarelix peptide resin prepared in Example 9 was taken, and 8% of HBr/TFA solution (acidolysis solution 10 mL/g degarelix peptide resin) was added. The reaction was stirred for 6 h, and filtered to collect the filtrate. The resin was again washed with a small amount of TFA for 3 times. The filtrates were combined and concentrated under reduced pressure. Anhydrous diethyl ether was added to precipitate, and the precipitate was washed with anhydrous diethyl ether again for 3 times, and sucked dry to obtain an off-white power, i.e. the crude degarelix, which had a purity of 86.7%.

Example 12: Preparation of the Crude Degarelix

[0074] The degarelix peptide resin prepared in Example 10 was taken, and 8% of HBr/TFA solution (acidolysis solution 10 mL/g degarelix peptide resin) was added. The reaction was stirred for 6 h, and filtered to collect the filtrate. The resin was again washed with a small amount of TFA for 3 times. The filtrates were combined and concentrated under reduced pressure. Anhydrous diethyl ether was added to precipitate, and the precipitate was washed with anhydrous diethyl ether again for 3 times, and sucked dry to obtain an off-white power, i.e. the crude degarelix, which had a purity of 84.0%.

Example 13: Purification of the Crude Degarelix

[0075] The crude degarelix obtained in Example 11 was taken and dissolved with 20% acetic acid solution. The solution was filtered using a 0.45 μm microporous filter membrane and used for purification.

[0076] High performance liquid chromatography was employed for purification. The chromatographic packing material for purification was reverse phase C18 (10 μm), the mobile phase system was 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the flow rate of the 77 mm*250 mm chromatographic column was 90 mL/min, a gradient system was employed for elution and the loading was cycled for purification. The crude solution was loaded onto the chromatographic column, the mobile phase was initiated for elution, the main peak was collected, and the concentrate of the intermediate for degarelix purification was obtained after evaporating acetonitrile.

[0077] The concentrate of the intermediate for degarelix purification was taken and filtered using a 0.45 μm filter membrane, for later use.

[0078] High performance liquid chromatography was employed for salt exchange. The mobile phase system was 1% acetic acid/aqueous solution-acetonitrile, the chromatographic packing material for purification was reverse phase C18 (10 μm), the flow rate of the 77 mm*250 mm chromatographic column was 90 mL/min, the gradient elution was employed and the loading process was cycled. The crude solution was loaded onto the chromatographic column, the mobile phase was initiated for elution, the spectrum was collected, the change in absorbance was observed, the main peak for salt exchange was collected and the purity was detected by analyzing the liquid phase, the solutions of the main peak for salt exchange were combined and concentrated under reduced pressure to obtain aqueous degarelix acetate, which was freeze-dried to obtain the pure degarelix, 49.3 g.

[0079] The total yield was 60.4%, the molecular weight was 1633.0, the purity was 99.5%, the largest single impurity was 0.10%, and no toxic hydantoin degradation product was detected.

Example 14: Purification of the Crude Degarelix

[0080] The crude degarelix obtained in Example 12 was taken and dissolved with mobile phase A for purification. The solution was filtered using a 0.45 μm microporous filter membrane and purified, for later use.

[0081] High performance liquid chromatography was employed for purification. The chromatographic packing material for purification was reverse phase C18 (10 μm), the mobile phase system was 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the flow rate of the 77 mm*250 mm chromatographic column was 90 mL/min, a gradient system was employed for elution and the loading was cycled for purification. The crude solution was loaded onto the chromatographic column, the mobile phase was initiated for elution, the main peak was collected, and the concentrate of the intermediate for degarelix purification was obtained after evaporating acetonitrile.

[0082] The concentrate of the intermediate for degarelix purification was taken and filtered using a 0.45 μm filter membrane, for later use.

[0083] High performance liquid chromatography was employed for salt exchange. The mobile phase system was 1% acetic acid/aqueous solution-acetonitrile, the chromatographic packing material for purification was reverse phase C18 (10 μm), the flow rate of the 77 mm*250 mm chromatographic column was 90 mL/min, the gradient elution was employed and the loading process was cycled. The crude solution was loaded onto the chromatographic column, the mobile phase was initiated for elution, the spectrum was collected, the change in absorbance was observed, the main peak for salt exchange was collected and the purity was detected by analyzing the liquid phase, the solutions of the main peak for salt exchange were combined and concentrated under reduced pressure to obtain aqueous acetic acid solution of degarelix, which was freeze-dried to obtain the pure degarelix, 47.5 g.

[0084] The total yield was 58.2%, the molecular weight was 1633.4, the purity was 99.7%, the largest single impurity was 0.11%, and no toxic hydantoin degradation product was detected.

[0085] The above described is merely preferred embodiments of the present invention. It should be noted that, an ordinary skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications are also deemed to be within the protection scope of the present invention.