CN107022002B

CN107022002B - Method for preparing degarelix by solid-liquid combination

Info

Publication number: CN107022002B
Application number: CN201710389088.4A
Authority: CN
Inventors: 张颖; 李同金; 王仁友; 石鑫磊
Original assignee: JINAN KANGHE MEDICAL TECHNOLOGY CO LTD
Current assignee: JINAN KANGHE MEDICAL TECHNOLOGY CO LTD
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2020-04-14
Anticipated expiration: 2037-05-26
Also published as: CN107022002A

Abstract

The invention relates to the field of polypeptide synthesis, in particular to a method for preparing degarelix by adopting a solid-liquid combined chemical method, the invention synthesizes fragment peptide Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH for the solid phase synthesis of the degarelix for the first time, the synthesis process is simple, the used raw materials are cheap and easy to obtain, and the amplification production is easy; the invention avoids using virulent reagent HF, adopts TFA to carry out combined cracking, and obtains refined peptide with the purity of 99.5 percent through purification and freeze-drying, and the total yield reaches 65 percent.

Description

Method for preparing degarelix by solid-liquid combination

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a method for preparing degarelix by solid-liquid combination.

Technical Field

Degarelix (Degarelix) was developed by Ferring Pharmaceuticals, marketed in the united states on 24 days 12 months 2008 under the trade name firagon, for use in the treatment of advanced prostate cancer; degarelix is a gonadotropin releasing hormone (GnRH) receptor inhibitor drug, reversibly inhibits pituitary GnRH receptors to reduce gonadotropin release and consequently inhibit testosterone release, and delays growth and progression of prostate cancer by inhibiting testosterone, which is essential for continued growth of prostate cancer; the defect that the concentration of testosterone is increased suddenly when the concentration of testosterone is reduced by treating prostate cancer with hormone at the initial stage can be avoided, so that the phenomenon that the hormone receptor is stimulated initially to temporarily promote the growth of a tumor instead of inhibiting the tumor can be avoided.

The degarelix is a linear decapeptide containing seven unnatural amino acids, has the molecular weight of 1630.75, and has the amino acid sequence of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph (L-Hor) -D-4Aph (cbm) -Leu-Lys (iPr) -Pro-D-Ala-NH₂Wherein Ac is acetyl, 2Nal is 2-naphthylalanine (2-naphthylalanine), 4Cpa is 4-chlorophenylalanine (4-chlorophenylalanine), 3PaI is 3-pyridylalanine (3-pyridylalanine), 4Aph is 4-aminophenylalanine (4-aminophenylalanine), Hor is hydrogenated orotic acid, Cbm is carbamoyl (carbamoyl), Lys (iPr) is N6-isopropyllysine (N6-isopyryllysine); the structural formula is as follows:

boc and Fmoc two chemical synthesis degarelix methods are reported at home and abroad, and in the Boc method, a Boc chemical solid-phase synthesis method is disclosed in a patent US5925730, and degarelix is prepared firstly; wherein, the 6-position 4Aph (Cbm) adopts Boc-4Aph (Fmoc) -OH as a synthetic raw material to be connected into a peptide sequence, after coupling, Fmoc is removed, tert-butyl isocyanate is used for reacting with side chain amino to obtain amide which is protected by tert-butyl 4Aph (Cbm), and the tert-butyl is removed in the subsequent TFA Boc removal process; wherein, 5-position 4Aph (Hor) also adopts Boc-4Aph (Fmoc) -OH as a synthetic raw material to be connected into a peptide sequence, and after Fmoc is removed, the peptide sequence is subjected to coupling reaction with L-hydrogenated orotic acid to obtain 4Aph (L-Hor); the synthesis of degarelix is disclosed in patent US5977302 using a Boc chemical liquid phase synthesis process via a "4-2-4", "3-3-4" or "3-4-3" liquid phase synthesis strategy; the above synthesis method requires the use of highly toxic HF as a cracking reagent, so that mass production is limited; and the U.S. Pat. No. 5,90656 also adopts a liquid phase fragment strategy 3-4-3 of a Boc chemical method to prepare degarelix, and adopts a side chain protection strategy different from U.S. Pat. Nos. 5925730 and 5977302, so that the use of HF is effectively avoided, but the palladium-carbon deprotection group is repeatedly used in the synthesis process, so that the operation is complicated, the cost is high, and adverse factors such as excessive heavy metal and the like are easily caused.

The fifth position from the N-terminus in the degarelix structure is the unnatural amino acid 4Aph (L-Hor) with the structure (L-hydrogenated orotate) -4-amino-phenylalanine, in which under alkaline conditions dihydrouracil rearranges to hydantoin (Koedjikov, A.H.et. al., J.Chem.Soc.Perkin, trans.2,1984, pages 1077-1081; Kaneti, J.et. al., org.Biomol.Chem.,2004, pages 1098-1103) with the following rearrangement scheme:

under alkaline conditions, the structural formula I is rearranged into a structural formula II. The Fmoc chemical solid-phase synthesis strategy is simple to operate, avoids using virulent HF as a cracking reagent, and is widely applied to polypeptide synthesis; however, the design of the experimental protocol for solid-phase synthesis of degarelix using Fmoc chemistry is particularly important because of the inevitable repeated use of the basic deaminating protecting group Fmoc in Fmoc chemistry. To avoid rearrangement, US8828938 solid phase synthesis of degarelix using Fmoc chemistry and experiments on rearrangement indicated that no rearrangement by-product was found after multiple cycles of deprotection treatment with 20% piperidine/DMF; the sixth position from the N end is an unnatural amino acid 4Aph (Cbm), the situation is protected by adopting 4Aph (t-Bu-Cbm), the TFA is used for cracking and deprotection for 25h, and the time is too long, so that the degradation of a peptide chain is easily caused; WO2012055903 uses Fmoc chemical liquid phase fragmentation method to synthesize degarelix, and adopts 3-7 or 4-6; in patent CN102329373, when Fmoc chemical solid phase synthesis is adopted for degarelix, 4Aph (Trt) or 4Aph (Alloc) is firstly introduced, after the peptide chain synthesis is finished, the Trt or Alloc is removed, and then the coupling reaction is carried out with L-hydrogenated orotic acid; in patent CN201310531473, when Fmoc chemical solid phase synthesis is used for degarelix, a protecting group strategy with strong distribution, orthogonality and specificity is selected: introducing 4Aph (Teoc) from the fifth position of the N end and 4Aph (Dde) from the sixth position, after the peptide chain is synthesized, removing the Dde by using a DMF (dimethyl formamide) solution of hydrazine hydrate, and performing coupling reaction with trimethylsilyl isocyanate to obtain Cbm; then removing Teoc by using DMF (dimethyl formamide) solution of tetrabutylammonium fluoride, and performing coupling reaction with L-hydrogenated orotic acid; in patent CN103351428, when degarelix is synthesized by Fmoc chemical solid phase synthesis, 4aph (Mmt) or 4aph (Dmt) is introduced first, after the peptide chain synthesis is completed, Mmt or Dmt is removed, and then coupling reaction is performed with L-hydrogenated orotic acid; CN103992392 introduces 4Aph (ivDde) from the fifth position of the N end, removes ivDde, and then generates coupling reaction with L-hydrogenated orotic acid; the raw materials required in the 4Aph protection strategy adopted in the scheme are not easy to obtain, the post-treatment is more complicated, and the production cost is higher.

In conclusion, the Boc chemical solid-phase strategy needs virulent HF, the mass production is limited, the liquid-phase synthesis is complex to operate, the requirement on operators is high, and the industrial amplification is also restricted; in Fmoc chemistry, an alkaline condition is required to be repeatedly used to remove a deamination protecting group Fmoc, so that the generation of rearrangement impurities is caused, and in addition, the strategies adopted by Fmoc chemical solid-phase synthesis have the problems of troublesome post-treatment, difficult obtainment of used protected amino acid raw materials and high manufacturing cost; therefore, the inventors studied the synthesis method of degarelix, and obtained the technical solution of the present invention.

Disclosure of Invention

The invention aims to provide a method for synthesizing degarelix. The invention adopts a solid-liquid combination strategy and adopts a common side chain protecting group, thereby reducing the synthesis cost and improving the purity of the crude peptide, thereby reducing the purification difficulty, improving the yield, reducing the production cost and being beneficial to large-scale industrial production.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preparing degarelix by solid-liquid combination comprises the following steps:

(a) preparing fragment peptide I under a liquid phase condition, namely Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH;

(b) and (3) preparing a fragment peptide resin II by taking amino resin as a solid phase carrier: H-D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins;

(c) coupling the fragment peptide I and the fragment peptide resin II to obtain a peptide resin III: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins;

(d) and (3) treating the peptide resin III by using a deprotection reagent, removing a side chain protecting group Fmoc, and then performing coupling reaction with L-hydrogenated orotic acid to obtain a peptide resin IV:

Ac-D-2Nal-D-4Cpa-D-3Pal-Ser(tBu)-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(Boc,iPr)-Pro-D-Ala-NH-Resins；

(e) and (3) cracking, purifying and freeze-drying the peptide resin IV to obtain the degarelix refined peptide.

Wherein the synthesis method of the fragment peptide I in the step (a) of the technical scheme comprises the following steps:

dissolving H-4Aph (Fmoc) -OH and alkali A in water according to a molar ratio of 1: 1-2, adding an organic solvent B with the volume of 5-20% for assisting dissolution, and dropwise adding an organic solvent B solution of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu with the molar amount of 0.9-1.0 times (calculated by the amount of H-4Aph (Fmoc) -OH) while stirring after complete dissolution; and monitoring the reaction end by TLC, after the reaction is finished, decompressing and evaporating to remove the organic solvent, adding 10% citric acid aqueous solution to adjust the pH value of the solution to 2-3, extracting by ethyl acetate, and crystallizing to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH.

The synthesis method of the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu comprises the following steps:

dissolving Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH and HOSu in THF according to the molar ratio of 1: 1.0-1.2, dropwise adding 1.0-1.2 times (calculated by the amount of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH) of THF solution of DCC under ice bath, reacting for 3-4h, filtering out insoluble substances, concentrating the filtrate, adding petroleum ether, and precipitating a solid to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu.

Wherein the synthesis method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH comprises the following steps:

the method comprises the following steps: taking Ac-D-2Nal-OH and H-D-4Cpa-OH as raw materials, and coupling under a liquid phase condition to prepare Ac-D-2Nal-D-4 Cpa-OH; then coupling with H-D-3Pal-OH under the liquid phase condition to prepare Ac-D-2Nal-D-4Cpa-D-3 Pal-OH; then coupling with H-Ser (tBu) -OH under the liquid phase condition to prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH.

The second method comprises the following steps: taking CTC resin as a solid phase carrier, and sequentially carrying out coupling reaction with Fmoc-Ser (tBu) -OH, Fmoc-D-3Pal-OH, Fmoc-D-4Cpa-OH and Ac-D-2Nal-OH to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -CTCResins; then obtaining Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH through TFE cracking and crystallization.

In the above steps, the base A is one of sodium carbonate, sodium bicarbonate, potassium carbonate, triethylamine, diethylamine, N-ethyldiisopropylamine, N-diisopropylethylamine, etc.;

the organic solvent B is one or more of tetrahydrofuran, dioxane, N-dimethylformamide, acetone, N-methyl-2-pyrrolidone and acetonitrile.

Wherein the synthesis method of the fragment peptide resin II in the step (b) of the technical scheme comprises the following steps:

taking amino resin as a solid phase carrier, adding corresponding Fmoc protected amino acid to perform coupling reaction at a charge ratio (based on the mass of a synthetic scale) of 2-5 times, wherein each coupling reaction is a solid phase peptide grafting reaction performed in the presence of a condensing agent, removing Fmoc by using a deprotection reagent after the reaction is finished, and performing coupling reaction with the next Fmoc protected amino acid; sequentially and respectively coupling with Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (Boc, iPr) -OH, Fmoc-Leu-OH and Fmoc-D-4Aph (Cbm) -OH to prepare H-D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins; the amino resin in the step (b) is one of Rink amide resins, Rink amide-AM resins, Rink amide-MBHAresins and Siembe resins.

Wherein the synthesis method of the peptide resin III in the step (c) of the technical scheme comprises the following steps:

dissolving 2.0-4.0 times (by solid phase synthesis scale) of fragment peptide I in DMF, cooling in ice water bath for 5-20min after complete dissolution, adding the same amount of condensing agent, mixing uniformly, cooling in ice water bath for 3-5min, adding the activated solution into a polypeptide synthesis column filled with fragment peptide resin II, and carrying out nitrogen-blowing coupling reaction for 2-3 h.

Wherein the synthesis method of the peptide resin IV in the step (d) of the technical scheme comprises the following steps:

deprotecting a peptide resin III by using a deprotection reagent, washing with DMF (dimethyl formamide) for 6 times, adding 2.0-4.0 times of activated L-hydrogenated orotic acid/condensing agent solution (calculated by solid phase synthesis scale), and reacting for 2-3h by using nitrogen.

Wherein the condensing agent in the technical scheme is one of DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA and HATU/HOAT/DIPEA; the deprotection reagent is 15-25% (volume content) piperidine/DMF solution.

Wherein the cracking reagent in the step (e) is a TFA solution added with 1-5% by volume of a scavenging agent, and the scavenging agent is one or more of anisole, dimethyl sulfide, ethanedithiol, mercaptoethanol, phenol, water and triisopropylsilane.

Compared with the prior art, the invention has the beneficial effects that: the invention synthesizes Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH for the first time and is used for solid phase synthesis of degarelix, thereby avoiding the difficult problem of 4Aph (L-Hor) rearrangement side reaction caused by repeated use of alkaline condition to remove Fmoc in the solid phase gradual synthesis process; the process has simple synthetic process, cheap and easily obtained raw materials and easy large-scale production; the invention avoids using virulent reagent HF, adopts TFA to carry out combined cracking, and obtains refined peptide with the purity of 99.5 percent through purification and freeze-drying, and the total yield reaches 65 percent.

Detailed Description

The present invention will be described in detail with reference to the following specific examples, which are not intended to limit the scope of the present invention; it is within the scope of the present invention to vary the raw material feed ratio, the reaction solvent, the condensing agent, etc. according to the present invention.

Abbreviations used in the specification and claims have the following meanings:

fmoc 9-fluorenylmethyloxycarbonyl

CTC resin 2-chlorotrityl chloride resin

tBu tert-butyl

2Nal 2-Naphthylalanine

Trt trityl radical

4Cpa 4-chlorophenylalanine

Boc tert-butyloxycarbonyl group

3Pal 3-pyridylalanine

4Aph 4-Aminophenylalanine

Hor hydrogenated orotic acid

Cbm carbamoyls

Lys (iPr) N6-isopropyllysine

NMP N-methyl-2-pyrrolidone

DCM dichloromethane

DMF N, N-dimethylformamide

DMAP 4-dimethylaminopyridine

DIPEA N, N-diisopropylethylamine

DIC N, N-diisopropylcarbodiimide

HBTU benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate

HATU 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

TBTU O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate

HOBT 1-hydroxybenzotriazole

HOAT 1-hydroxy-7-azobenzotriazol

TFA trifluoroacetic acid

TIS Triisopropylsilane

Example 1: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH

Accurately weighing 199.6g (1.0mol) of H-D-4Cpa-OH and 127.2g (1.2mol) of sodium carbonate, dissolving in 2400mL of water, slowly adding 298.3g (1.0mol) of tetrahydrofuran solution of Ac-D-2Nal-OSu at low temperature (2-8 ℃), stirring for reaction, monitoring the reaction endpoint by TLC, after the reaction is completed, evaporating off THF in a rotary manner, adding dilute hydrochloric acid in an ice water bath to adjust the pH value of the solution to 2-3, extracting for 3 times by 2000mL of ethyl acetate, combining organic phases, washing for 3 times by 400mL of saturated salt solution, drying with anhydrous sodium sulfate, evaporating in a rotary manner to concentrate to 2000mL, standing for crystallization, obtaining 390.3g of Ac-D-2Nal-D-4Cpa-OH, wherein the yield is 82.1%.

Accurately weighing 136.3g (0.82mol) of H-D-3Pal-OH and 106.0g (1.0mol) of sodium carbonate, dissolving in 2400mL of water, slowly adding a tetrahydrofuran solution (390.3g, 0.82mol) of Ac-D-2Nal-D-4Cpa-OSu at low temperature (2-8 ℃) to 2000mL of the tetrahydrofuran solution, stirring for reaction, monitoring the reaction end point by TLC, after the reaction is completed, performing rotary evaporation to remove THF, adding dilute hydrochloric acid to adjust the pH value of the solution to 2-3 in an ice water bath, extracting 3 times by 2000mL of ethyl acetate, combining organic phases, washing 3 times by 400mL of saturated salt solution, drying with anhydrous sodium sulfate, performing rotary evaporation, concentrating to 2000mL, standing and crystallizing to obtain 417.3g of Ac-D-2Nal-D-4 Cpa-3 Pal-OH, wherein the yield is 81.6%.

Accurately weighing 118.8g (0.74mol) of H-Ser (tBu) -OH and 94.3g (0.89mol) of sodium carbonate, dissolving in 2000mL of water, slowly adding a tetrahydrofuran solution (417.3g, 0.67mol) of Ac-D-2Nal-D-4Cpa-D-3Pal-OSu at low temperature (2-8 ℃), stirring for reaction, monitoring the reaction end point by TLC, after the reaction is completed, evaporating THF, adding 10% citric acid in an ice water bath to adjust the pH value of the solution to 2-3, extracting 3 times by 2000mL of ethyl acetate, combining organic phases, washing 3 times by 300mL of saturated common salt water, drying by anhydrous sodium sulfate, carrying out rotary evaporation and concentration to 2000mL, standing for crystallization to obtain 462.9g of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH (462.9 g), wherein the yield is 90.1%.

Accurately weighing H-4Aph (Fmoc) -OH 265.6g (0.66mol) and sodium carbonate 84.8g (0.80mol) and dissolving in 2000mL of water, slowly adding a tetrahydrofuran solution (462.9g, 0.60mol) of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu at low temperature (2-8 ℃), stirring for reaction, monitoring the reaction end by TLC, after the reaction is completed, evaporating off THF, adding 10% citric acid under an ice water bath to adjust the pH value of the solution to 2-3, extracting with 2000mL of ethyl acetate for 3 times, combining organic phases, washing with 300mL of saturated common salt solution for 3 times, drying with anhydrous sodium sulfate, carrying out rotary evaporation and concentration to 2000mL, standing and crystallizing to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser tBu) -4Aph (Fmoc) -OH 619.8g, the yield thereof was found to be 89.2%.

Example 2: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH

Weighing 500.0g (sub-1.0 mmol/g) of CTC resin, placing the CTC resin in a synthesis column, washing the CTC resin twice by 4000mL of DMF, adding 4000mL of DCM, and swelling for 30 min; after DCM was filtered off with suction, 1500ml of a DCM/DMF (3/1, vol.) solution containing 383.5g (100mmol) Fmoc-Ser (tBu) -OH was added, and after stirring, 330ml of DIPEA (2000mmol) and TuanN were added₂Reacting for 60min, draining the reaction solution, and adding DCM/CH₃3000ml of mixed solution of OH/DIPEA (volume ratio 17: 2: 1) is blocked for 3 times, 10min each time; then washed 6 times with DMF and a small sample was taken to determine the degree of substitution of 0.75mmol/g and the scale of synthesis of 0.5 mol.

Deprotecting the resin in the synthetic column twice with 3000ml of 20% piperidine/DMF solution, and washing with DMF 6 times; then adding activated Fmoc-D-3Pal-OH/HOBT/DIC solution, reacting for 3h at room temperature, filtering out reaction liquid, and washing for 6 times by using DMF; then recycling the operation, and respectively coupling reacting with Fmoc-D-4Cpa-OH and Ac-D-2Nal-OH to obtain 853.6g of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -CTC Resins after washing and drying;

adding the peptide resin into 900ml of cracking reagent TFE/AcOH/DCM (1/1/3) for reacting for 3h, concentrating, adding into petroleum ether for crystallization to obtain 381.2g of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH solid, wherein the yield is 99.4%.

Accurately weighing 241.4g (0.60mol) of H-4Aph (Fmoc) -OH and 84.8g (0.80mol) of sodium carbonate, dissolving in 2000mL of water, slowly adding 381.2g (0.50 mol) of tetrahydrofuran solution of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu at low temperature (2-8 ℃), stirring for reaction, monitoring the reaction end by TLC, after the reaction is completed, evaporating off THF in a rotary manner, adding 10% citric acid under an ice water bath to adjust the pH value of the solution to 2-3, extracting 3 times by 2000mL of ethyl acetate, combining organic phases, washing 3 times by 300mL of saturated saline, drying by anhydrous sodium sulfate, concentrating to 2000mL by rotary evaporation, standing for crystallization to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4 Apu (Fmoc) -4Aph (Fmoc) -OH 519.2g, the yield thereof was found to be 90.2%.

Example 3: preparation of H-D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

220g of RinkAmideAM resin (initial substitution degree 0.91mmol/g) is accurately weighed and placed in a peptide resin synthesis reactor, and 1.0LDCM is added for swelling for 2 h. After swelling, the column was washed 3 times with 1.0L DMF and then deprotected twice by addition of 2.0L 20% piperidine/DMF for 10min and 10min, respectively. Deprotection was complete and the resin was washed 6 times with DMF at 2.0L each time. 124.4g (0.40mol) of Fmoc-D-Ala-OH and 59.4g (0.44mol) of HOBt were dissolved in 0.8L of DMF, 68mL (0.44mol) of DIC was added for activation, the solution was added to the reactor and reacted for 2 hours, and Kaiser test was performed to monitor the progress of the reaction. At the end of the reaction, the resin was washed 6 times with DMF. Then repeating the operation, and sequentially carrying out coupling reaction with Fmoc-Pro-OH, Fmoc-Lys (Boc, iPr) -OH and Fmoc-Leu-OH to prepare H-Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins; then coupling with Fmoc-D-4Aph (cbm) -OSu to prepare H-D-4Aph (cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins.

Example 4: preparation of H-D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

110g of Rinkamide resin (initial substitution degree of 0.91mmol/g) is accurately weighed and placed in a peptide resin synthesis reactor, and 0.5LDCM is added for swelling for 2 h. After swelling, the column was washed 3 times with DMF, 0.5L each time, and then deprotected twice by adding 1.0L 20% piperidine/DMF solution for 10min and 10min, respectively. Deprotection was complete and the resin was washed 6 times with DMF at 2.0L each time. 62.2g (0.20mol) of Fmoc-D-Ala-OH and 29.7g (0.22mol) of HOBt were weighed out and dissolved in 0.8LDMF, 34mL (0.22mol) of DIC was added for activation, the solution was added to the reactor and reacted for 2h, and Kaiser test was performed to monitor the progress of the reaction. At the end of the reaction, the resin was washed 6 times with DMF. Then repeating the operation, and sequentially carrying out coupling reaction with Fmoc-Pro-OH, Fmoc-Lys (Boc, iPr) -OH and Fmoc-Leu-OH to prepare H-Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins; then coupling with Fmoc-D-4Aph (cbm) -OSu to prepare H-D-4Aph (cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins.

Example 5: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

Accurately weighing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH460.5g (0.40mol) and HOBt 59.4g (0.44mol) obtained in example 1, dissolving with 1.2L DMF, placing into an ice water bath for cooling for 20min, adding 68mL (0.44mol) DIC, mixing uniformly, placing into the ice water bath for cooling for 5min, adding the activated solution into the resin reactor of example 3, monitoring the reaction process by Kaiser test, and reacting for 3 h. The reaction solution was pumped into 5L of 10% citric acid aqueous solution to recover Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH 210.2g.

Example 6: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

Accurately weighing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH230.3g (0.20mol) and 29.7g (0.22mol) of HOBt obtained in example 2, dissolving with 0.6L of DMF, placing into an ice water bath for cooling for 20min, adding 34mL (0.22mol) of DIC, uniformly mixing, placing into the ice water bath for cooling for 5min, adding the activated solution into the resin reactor of example 4, monitoring the reaction process by Kaiser test, and reacting for 3 h. The reaction solution was pumped into 2.5L of 10% citric acid aqueous solution to recover Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH 105.2g.

Example 7: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (L-Hor) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

The peptide resin obtained in example 5 was deprotected twice with 2.0L 20% piperidine/DMF for 10min and 10min, respectively. Deprotection was complete and the resin was washed 6 times with DMF at 2.0L each time. 161.0g (0.40mol) of L-hydrogenated orotic acid and 59.4g (0.44mol) of HOBt are accurately weighed and dissolved by 1.2L of DMF, then the mixture is placed into an ice-water bath to be cooled for 20min, 68mL (0.44mol) of DIC is added to be uniformly mixed, then the mixture is placed into the ice-water bath to be cooled for 5min, the activated solution is added into a reactor, Kaiser test monitors the reaction process, and the reaction lasts for 3 h. After the reaction was complete, the reaction was filtered off and the resin was washed 6 times with DMF, 2.0L each time, then 3 times with 2.0L of LDCM and three times with 2.0L of methanol. The mixture was dried by suction and vacuum dried to obtain 572.6g of peptide resin.

Example 8: synthesis of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (L-Hor) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins

The peptide resin obtained in example 6 was deprotected twice with 1.0L 20% piperidine/DMF for 10min and 10min, respectively. Deprotection was complete and the resin was washed 6 times with 1.0L each time of DMF. Accurately weighing 80.5g (0.20mol) of L-hydrogenated orotic acid and 29.7g (0.22mol) of HOBt, dissolving with 0.6L of DMF, placing into an ice-water bath, cooling for 20min, adding 34mL (0.22mol) of DIC, uniformly mixing, placing into the ice-water bath, cooling for 5min, adding the activated solution into a reactor, monitoring the reaction process by Kaiser test, and reacting for 3 h. After the reaction was complete, the reaction was filtered off and the resin was washed 6 times with DMF, 1.0L each time, then 3 times with 1.0L of LDCM and three times with 1.0L of methanol. The solution was then dried by suction and vacuum dried to obtain 286.9g of peptide resin.

Example 9: preparation of crude degarelix

572.6g of the peptide resin obtained in example 7 was slowly added to the cleavage reagent TFA/H in an ice bath₂Reacting in 3.5L of O/TIS (95/3/2, V/V) at low temperature for 30min, reacting at room temperature for 3h, filtering off resin, pouring lysate into 40L of frozen methyl tert-ether, precipitating solid, centrifuging, washing, and drying to obtain crude product 355.8g with purity of 92.6%.

Example 10: preparation of crude degarelix

286.9g of the peptide resin from example 8 were slowly added to the cleavage reagent TFA/phenol/thioanisole/H in ice bath₂Reacting O/TIS (82.5/5/5/5/2.5, V/V) in 1.8L at low temperature for 30min, reacting at room temperature for 3h, filtering off resin, pouring lysate into 20L of frozen methyl tert-ether, precipitating solid, centrifuging, washing, and drying to obtain crude product 176.2g with purity of 91.5%.

Example 11: refining of degarelix crude product

The crude product obtained in example 9 was taken, dissolved in purified mobile phase A and the solution was filtered through a 0.45um microfiltration membrane for use.

Purifying by liquid phase preparative chromatography, wherein the filler is C18 with particle size of 10um, the mobile phase is 0.1% TFA/water-0.1% TFA/acetonitrile, the inner diameter is 150mm, each sample loading is 40g, the components with the trapped central control purity of 99.5% are combined, and the components which do not meet the central control standard before and after the peak are respectively recycled and injected.

And (3) concentrating the qualified medium-control components under reduced pressure to remove most of acetonitrile, transferring salt through a liquid chromatographic column, intercepting the qualified components with the medium-control purity of 99.5%, concentrating and freeze-drying to obtain 212.8g of refined peptide, wherein the total yield is 65.2%.

Example 12: refining of degarelix crude product

The crude product obtained in example 10 was taken, dissolved in purified mobile phase A and the solution was filtered through a 0.45um microfiltration membrane for use.

Concentrating the qualified medium-control components under reduced pressure to remove most of acetonitrile, transferring salt through a liquid chromatographic column, intercepting the qualified medium-control components with the purity of 99.5%, concentrating and freeze-drying to obtain 106.0g of refined peptide, wherein the total yield is 65.0%.

Claims

1. A method for preparing degarelix by solid-liquid combination is characterized by comprising the following steps:

(a) preparation of fragment peptide i: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH:

dissolving H-4Aph (Fmoc) -OH and alkali A in water according to a molar ratio of 1: 1-2, adding 5-20% of organic solvent B for assisting dissolution, dropwise adding Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu organic solvent B solution in an amount which is 0.9-1.0 times of the molar amount by the molar amount of H-4Aph (Fmoc) -OH after complete dissolution, monitoring the reaction end point by TLC, removing the organic solvent by decompression and evaporation after the reaction is finished, adding 10% citric acid aqueous solution to adjust the pH value of the solution to 2-3, extracting ethyl acetate, and crystallizing to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -OH;

the alkali A is one of sodium carbonate, sodium bicarbonate, potassium carbonate, triethylamine, diethylamine, N-ethyldiisopropylamine and N, N-diisopropylethylamine; the organic solvent B is one or more of tetrahydrofuran, dioxane, N-dimethylformamide, acetone, N-methyl-2-pyrrolidone and acetonitrile;

the synthesis method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu comprises the following steps: taking the amount of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH, dissolving Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH and HOSu in THF according to the molar ratio of 1: 1.0-1.2, dropwise adding 1.0-1.2 times of the molar amount of a THF solution of DCC under ice bath, reacting for 3-4h, filtering out insoluble substances, concentrating the filtrate, adding petroleum ether, and precipitating solids to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OSu;

the method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH comprises the following steps: taking Ac-D-2Nal-OH and H-D-4Cpa-OH as raw materials, and coupling under a liquid phase condition to prepare Ac-D-2Nal-D-4 Cpa-OH; then coupling with H-D-3Pal-OH under the liquid phase condition to prepare Ac-D-2Nal-D-4Cpa-D-3 Pal-OH; then coupling with H-Ser (tBu) -OH under the liquid phase condition to prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH;

(d) preparing 15-25% by volume of piperidine/DMF solution as a deprotection reagent, treating a peptide resin III with the deprotection reagent, removing a side chain protecting group Fmoc, and then performing coupling reaction with L-hydrogenated orotic acid to obtain a peptide resin IV: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (L-Hor) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins;

(e) cracking, purifying and freeze-drying the peptide resin IV to obtain degarelix; the cracking step is to add a TFA solution of a scavenging agent with the volume ratio of 1-5%, wherein the scavenging agent is one or more of anisole, dimethyl sulfide, dithioglycol, mercaptoethanol, phenol, water and triisopropylsilane.

2. A method for preparing degarelix by solid-liquid combination is characterized by comprising the following steps:

the method for synthesizing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH comprises the following steps: using CTC resin as a solid phase carrier, and sequentially carrying out coupling reaction with Fmoc-Ser (tBu) -OH, Fmoc-D-3Pal-OH, Fmoc-D-4Cpa-OH and Ac-D-2Nal-OH to obtain Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -CTC Resins; then obtaining Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -OH through TFE cracking and crystallization;

(c) coupling the fragment peptide I and the fragment peptide resin II to obtain a peptide resin III: Ac-D-2Nal-D-4Cpa-D-3Pal-Ser (tBu) -4Aph (Fmoc) -D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resi ns;

3. The method for preparing degarelix by solid-liquid combination according to claim 1 or 2, wherein the method for synthesizing the fragment peptide resin II in the step (b) comprises: taking amino resin as a solid phase carrier, adding corresponding Fmoc protected amino acid for coupling reaction according to the material amount ratio of 2-5 times of the synthetic scale, wherein each coupling reaction is a solid phase peptide grafting reaction carried out in the presence of a condensing agent, removing Fmoc by using a deprotection reagent after the reaction is finished, and then carrying out coupling reaction with the next Fmoc protected amino acid; sequentially and respectively coupling with Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (Boc, iPr) -OH, Fmoc-Leu-OH and Fmoc-D-4Aph (Cbm) -OH to prepare H-D-4Aph (Cbm) -Leu-Lys (Boc, iPr) -Pro-D-Ala-NH-Resins; the amino resin is one of Rink amide resins, Rink amide-AM resins, Rink amide-MBHA resins and Siembe resins.

4. The process for the preparation of degarelix by solid-liquid combination according to claim 3, wherein the condensing agent is one of DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA.

5. The method for preparing degarelix by solid-liquid combination according to claim 1 or 2, wherein the peptide resin III in step (c) is synthesized by: taking 2.0-4.0 times of the molar weight of the fragment peptide I to dissolve in DMF (dimethyl formamide), after the fragment peptide I is completely dissolved, putting the fragment peptide I into an ice water bath to be cooled for 5-20min, adding the same amount of condensing agent, uniformly mixing, putting the mixture into the ice water bath to be cooled for 3-5min, then adding the activated solution into a polypeptide synthetic column filled with the fragment peptide resin II, and carrying out a nitrogen-blowing coupling reaction for 2-3 h.

6. The method for preparing degarelix by solid-liquid combination according to claim 1 or 2, wherein the peptide resin IV in step (d) is synthesized by: and (3) deprotecting the peptide resin III by using a deprotection reagent, washing the peptide resin III for 6 times by using DMF (dimethyl formamide), adding an activated L-hydrogenated orotic acid/condensing agent solution with 2.0-4.0 times of molar weight, and reacting for 2-3h by using nitrogen.