阅读说明：本技术 一种奥曲肽的合成方法 (Synthetic method of octreotide ) 是由 罗日朗 尹传龙 陶安进 余品香 于 2019-09-16 设计创作，主要内容包括：本发明公开了一种奥曲肽的合成方法,包括以下步骤：1)选择2-CTC树脂为合成起点,制备Fmoc-Thr(tBu)-OL-2CTC树脂；2)按照Fmoc/tBu策略依次偶联正交保护的胱氨酸、Fmoc-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-D-Trp(Boc)-OH和Fmoc-Phe-OH,然后用pd(pph3)4和苯硅烷脱去胱氨酸正交保护的Alloc保护基后偶联Boc-D-Phe-OH,然后进行分子内酰胺反应成环；3)裂解液切落肽片段,获得奥曲肽粗肽。本发明提供了一条避免氧化反应成环的固相合成方法,从而避免了现有方法带来的诸多问题,且本发明方法具有操作简单、工艺简化、环境友好等诸多优点。(The invention discloses a synthetic method of octreotide, which comprises the following steps: 1) selecting 2-CTC resin as a synthesis starting point, and preparing Fmoc-Thr (tBu) -OL-2CTC resin; 2) orthogonal protected cystine, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH and Fmoc-Phe-OH are coupled in sequence according to the Fmoc/tBu strategy, then the orthogonal protected Alloc protecting group of cystine is removed by pd (pph3)4 and phenylsilane, then Boc-D-Phe-OH is coupled, and then intramolecular amide reaction cyclization is carried out; 3) and (4) cutting off peptide fragments by the lysate to obtain crude octreotide peptides. The invention provides a solid-phase synthesis method for avoiding oxidation reaction cyclization, thereby avoiding a plurality of problems brought by the existing method, and the method has the advantages of simple operation, simplified process, environmental protection and the like.)

1. A synthetic method of octreotide is characterized by comprising the following steps:

1) selecting 2-CTC resin as a synthesis starting point, and preparing Fmoc-Thr (tBu) -OL-2CTC resin;

2) orthogonal protected cystine, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH and Fmoc-Phe-OH are coupled in sequence according to the Fmoc/tBu strategy, then the orthogonal protected Alloc protecting group of cystine is removed by pd (pph3)4 and phenylsilane, then Boc-D-Phe-OH is coupled, and then intramolecular amide reaction cyclization is carried out;

3) and (4) cutting off peptide fragments by the lysate to obtain crude octreotide peptides.

2. The method for synthesizing octreotide according to claim 1, wherein the Fmoc-Thr (tBu) -OL-2CTC resin is prepared by putting 2-CTC resin into a reaction vessel, dissolving Fmoc-Thr (tBu) -OL with a solvent, adding the solution into a solid phase synthesis carrier, slowly adding DIPEA dropwise, and stirring for reaction for a period of time to obtain Fmoc-Thr (tBu) -OL-2CTC resin.

3. The process for the synthesis of octreotide according to claim 1 or 2, wherein the 2-CTC resin is a 2-CTC resin having a resin substitution degree of greater than 1.0 mmol/g.

4. The method for synthesizing octreotide according to claim 1, wherein the orthogonally-protected cystine protection structure is as follows:

5. the method for synthesizing octreotide according to claim 1, wherein the step 2) specifically comprises: removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating a proper amount of orthogonally protected cystine and a coupling agent in a solvent, and adding the dissolved and activated amounts of orthogonally protected cystine and the coupling agent into a solid-phase reaction column together until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-D-Trp (Boc) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

after removal of the orthogonally protected Alloc protecting group of cystine by pd (pph3)4 and phenylsilane, an appropriate amount of Boc-D-Phe-OH was dissolved and activated in a solvent and then added to a solid phase reaction column together until the end of the reaction was detected by detection;

removing Fmoc, and washing the resin until the Fmoc is completely removed; HBTU and DIPEA are dissolved in a solvent and then put into a solid phase reaction column until the reaction is detected to be terminated by a detection method.

6. The method for synthesizing octreotide according to claim 5, wherein the reagent for removing Fmoc is 20% piperidine/DMF solution.

7. The method for synthesizing octreotide according to claim 5, wherein the coupling agent is DIC + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU and TBTU; preferably a combination of HBTU and compound A and DIPEA.

8. The method for synthesizing octreotide according to claim 7, wherein the molar ratio of each component in the coupling agent to Fmoc-aa-OH is DIC: a: Fmoc-aa-OH ═ 1.2: 1.1: 1 or DIPEA: a: b: Fmoc-aa-OH ═ 2.0: 1.1: 1: 0.9, wherein Fmoc-aa-OH is Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH.

9. The method for synthesizing octreotide according to claim 5, wherein the reaction in step 2) is performed by swelling the resin before coupling, and the reagent used in the washing and swelling steps is DMF, NMP or dichloromethane, preferably DMF.

10. The method for synthesizing octreotide according to claim 1, wherein the lysis solution in step 3) is TFA or H₂A mixture of O, indole, preferably TFA: h₂O: indole 90: 5: 5.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a synthetic method of octreotide.

Background

Octreotide Acetate (Octreotide Acetate) is an analogue of human somatostatin, has a molecular weight of 1019.3 (calculated as free alkali) and a CAS number of 79517-01-4, is a cyclic nonapeptide drug, the peptide sequence of which is shown in formula I, and the original manufacturers are Novartis (Novartis), and in China, the United states and Europe, the products on the market are Octreotide Acetate injection and Octreotide Acetate microspheres; meanwhile, suspension is marketed in part of European countries.

Coy and Saratakis et al in 1973 simultaneously chemically synthesized the somatostatin tetradecapeptide by the solid phase method. The Meinhofer laboratory adopts modified N-fluorenylmethyloxycarbonyl (Fmoc) amino acid to synthesize the reduced somatostatin by solid phase synthesis according to a one-by-one extension method. Bauer et al synthesized an analogue of somatostatin, named Octreotide (Octreotide), in 1982. The octreotide has short peptide chain, 6 amino acids in SS are removed compared with somatostatin, 8-site is amino L-threoninol, and the octreotide is not easy to hydrolyze quickly by protease and is easy to artificially synthesize. The half-life period in vivo is as long as 2 hours, and the effect of inhibiting hormone secretion is stronger than that of somatostatin, so that the octreotide is an analogue of long-acting somatostatin, also called somatostatin release inhibin (SRIF or SS), and is widely used for treating acromegaly, endocrine tumors of digestive tract, non-secretory tumors of digestive tract, upper gastrointestinal hemorrhage, acute pancreatitis, systemic sclerosis, irritable bowel syndrome, cancer tumor cachexia, dumping syndrome, psoriasis, postural hypotension, intraoperative hypotension and other diseases.

In general, the metabolic stability and bioavailability of cyclic peptides are much higher than that of linear peptides. In view of the many advantages of cyclic peptides, the focus of polypeptide research in recent years has shifted to the synthesis and biological evaluation of cyclic peptides.

So far, in terms of synthesis, octreotide has been mainly synthesized by solid phase synthesis methods (such as patents CN103351426 and CN1923849) and liquid phase synthesis methods (CN 1355173). The synthetic octreotide process consists of two parts: 1) the synthesis of the linear octapeptide mainly adopts solid phase to sequentially insert amino acid or fragment for splicing, 2) cyclizing the linear octapeptide by natural oxidation of the refined peptide or oxidation by using an oxidation reagent, or direct oxidation by using the oxidation reagent at the later stage of solid phase synthesis.

The prior patent protects or is used in the process of synthesizing octreotide, the process is not ideal in the cyclization stage, mainly the oxidation reagent is mostly used with certain chemical toxicity or dangerous in use, and is not environment-friendly; the oxidation sites cannot be well controlled by natural oxidation in air, and polymers are easily generated.

The invention aims to provide another synthetic method for preparing octreotide. The method avoids using oxidation reaction for cyclization, and is novel, mild in synthesis condition, simple in process and stable in process.

Disclosure of Invention

In order to solve the problems of the background art, the present invention provides a method for synthesizing octreotide.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a synthetic method of octreotide comprises the following steps:

1) selecting 2-CTC resin as a synthesis starting point, and preparing Fmoc-Thr (tBu) -OL-2CTC resin;

2) orthogonal protected cystine, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH and Fmoc-Phe-OH are coupled in sequence according to the Fmoc/tBu strategy, then the orthogonal protected Alloc protecting group of cystine is removed by pd (pph3)4 and phenylsilane, then Boc-D-Phe-OH is coupled, and then intramolecular amide reaction cyclization is carried out;

3) and (4) cutting off peptide fragments by the lysate to obtain crude octreotide peptides.

Furthermore, the preparation method of the Fmoc-Thr (tBu) -OL-2CTC resin comprises the steps of putting the 2-CTC resin into a reaction container, dissolving the Fmoc-Thr (tBu) -OL by using a solvent, adding the solution into a solid-phase synthesis carrier, slowly dropwise adding DIPEA, and stirring for reacting for a period of time to obtain the Fmoc-Thr (tBu) -OL-2CTC resin.

Further, the solvent for dissolving Fmoc-Thr (tBu) -OL can be any organic solvent which can dissolve the raw material and can be used for solid phase synthesis reaction, such as dichloromethane, DMF, NMP, DMSO, THF, etc., preferably dichloromethane.

Further, the 2-CTC resin is preferably a 2-CTC resin having a resin substitution degree of more than 1.0 mmol/g.

Further, the orthogonally protected cystine protection structure is as follows:

further, the step 2) specifically comprises: removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating a proper amount of orthogonally protected cystine and a coupling agent in a solvent, and adding the dissolved and activated amounts of orthogonally protected cystine and the coupling agent into a solid-phase reaction column together until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-D-Trp (Boc) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

after removal of the orthogonally protected Alloc protecting group of cystine by pd (pph3)4 and phenylsilane, an appropriate amount of Boc-D-Phe-OH was dissolved and activated in a solvent and then added to a solid phase reaction column together until the end of the reaction was detected by detection;

removing Fmoc, and washing the resin until the Fmoc is completely removed; HBTU and DIPEA are dissolved in a solvent and then put into a solid phase reaction column until the reaction is detected to be terminated by a detection method.

Further, the solvent for dissolving the substance in step 2) may be any solvent commonly used in solid phase synthesis, such as DMF, NMP, DMSO, etc.

Further, the reagents used to remove Fmoc were 20% piperidine/DMF solution (DBLK), i.e. piperidine: DMF (volume ratio) is 1: 4.

Further, the coupling agent is DIC + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU and TBTU; preferably a combination of HBTU and compound A and DIPEA.

Further, the molar ratio of each component in the coupling agent to Fmoc-aa-OH is DIC: a: Fmoc-aa-OH ═ 1.2: 1.1: 1 or DIPEA: a: b: Fmoc-aa-OH ═ 2.0: 1.1: 1: 0.9, wherein Fmoc-aa-OH is Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Phe-OH.

Further, the reaction of step 2) is to swell the resin before coupling, and the reagent used in the washing and swelling steps is DMF, NMP or dichloromethane, preferably DMF.

Further, the detection method is to select Kaiser reagent to determine the end point of the reaction, and if the resin is developed, it indicates that there is free amine in the polypeptide, i.e. there is no protecting group on the amine.

Further, the lysis solution in the step 3) is TFA or H₂A mixture of O, indole, preferably TFA: h₂O: indole 90: 5: 5.

the existing process for synthesizing octreotide is not ideal in the cyclization stage, mainly uses oxidation reagents mostly with certain chemical toxicity or dangerous in use, and is not environment-friendly; the oxidation sites cannot be well controlled by natural oxidation in air, and polymers are easily generated. The liquid phase synthesis is complex in process operation, and the purity of the obtained crude peptide is not high. The invention provides a solid-phase synthesis method for avoiding oxidation reaction cyclization, thereby avoiding a plurality of problems brought by the existing method, and the method has the advantages of simple operation, simplified process, environmental protection and the like.

Drawings

FIG. 1 is a chromatogram of crude octreotide;

fig. 2 is a mass spectrum of crude octreotide.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

The meanings of abbreviations used in the specification and claims are listed in the following table:

example 1: preparation of Fmoc-Thr (tBu) -OL-2CTC resin

20g (22.4mmol) of 2-CTC resin was weighed into a suitable round bottom flask, 8.61g (22.4mmol) of Fmoc-Thr (tBu) -OL was weighed out and dissolved in 200ml of dry dichloromethane, added to the round bottom flask of the existing resin, 19.5ml (112mmol) of DIPEA was slowly added and the reaction was stirred mechanically for 24 h. After the reaction, the reaction solution is transferred to a solid phase reaction column, the reaction solution is pumped to dry, the reaction solution is washed by DMF for 6 times, each time is 2min, methanol shrinks for 2 times (5min +10min), the dosage of methanol is 200ml each time, and the reaction solution is pumped to dry in vacuum.

Example 2: introduction of Cys-Cys

Weighing 10g of Fmoc-Thr (tBu) -OL-2CTC resin of 0.5mmol/g in example 1, putting the resin into a solid-phase reaction column, adding 100ml of DMF, and carrying out nitrogen bubbling for swelling for 60 minutes; then deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 15mmol of Cys-Cys, 14.25mmol of HBTU and 15mmol of HOBT to dissolve in 50ml of DMF, weighing 30mmol of DIPEA to add, fully stirring until the materials are completely dissolved, putting into a reaction column, carrying out nitrogen bubbling reaction for 2h, and alternately washing 6 times with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4minDMF +2minDCM +2 minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 3: grafting of Fmoc-Thr (tBu) -OH

After passing Kaiser test in example 2, it was deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 15mmol Fmoc-Thr (tBu) -OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measuring 30mmol DIPEA to add, fully stirring until the materials are completely dissolved, putting into a reaction column, carrying out nitrogen bubbling reaction for 2h, and alternately washing 6 times with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4minDMF +2minDCM +2 minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 4: grafting Fmoc-Lys (Boc) -OH

After passing Kaiser test in example 3, it was deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 15mmol Fmoc-Lys (Boc) -OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measuring 30mmol DIPEA to add and fully stirring until the materials are completely dissolved, putting into a reaction column, carrying out nitrogen bubbling reaction for 2h, and alternately washing 6 times with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4minDMF +2minDCM +2 minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 5: grafting Fmoc-D-Trp (Boc) -OH

After passing Kaiser test in example 4, it was deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 15mmol Fmoc-D-Trp (Boc) -OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measuring 30mmol DIPEA to add and fully stirring until the materials are completely dissolved, putting into a reaction column, carrying out nitrogen bubbling reaction for 2h, and alternately washing 6 times with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4minDMF +2minDCM +2 minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 6: grafting Fmoc-Phe-OH

After passing Kaiser test in example 5, it was deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 15mmol Fmoc-Phe-OH, 14.25mmol HBTU and 15mmol HOBT, dissolving with 50ml DMF, weighing 30mmol DIPEA, adding into the mixture, stirring thoroughly until the materials are completely dissolved, putting into a reaction column, carrying out nitrogen bubbling reaction for 2h, and alternately washing 6 times with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4minDMF +2minDCM +2 minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 7: Boc-D-Phe-OH

After passing Kaiser test in example 6, 2.5mmol pd (pph3)4 and 100mmol of phenylsilane were weighed out and dissolved in 40ml of dichloromethane, added to a reaction column, reacted for 1h, the Alloc protecting groups were removed, DMF and DCM were washed 6 times (3+3+ 3. sup. minDCM +3+ 3. sup. minDMF), 15mmol Boc-D-Phe-OH, 14.25mmol HBTU and 15mmol HOBT were weighed out and dissolved in 50ml of DMF, 30mmol DIPEA was weighed out and added to the reaction column with stirring until the material was completely dissolved, nitrogen was bubbled for 2h, and washed 6 times alternately with DMF and DCM (washing sequence and time: 2+ 4. sup. minDMF + 4. sup. minDCM + 4. sup. minDMF + 2. sup. minDCM + 2. sup. minDMF). Sampling and carrying out Kaiser detection, and requiring no color to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed.

Example 8: cyclization of intramolecular amide

After passing the Kaiser test in example 7, it was deprotected twice with 100mL DBLK (5min +7min), washed 7 times alternately with DMF and DCM (washing sequence and time: 2+4minDMF +4minDCM +4+4minDMF +2minDCM +2 minDMF). Weighing 14.25mmol HBTU and 30mmol DIPEA, dissolving in NMP, putting into a reaction column, reacting for 2.5h, sampling, and performing Kaiser detection, wherein colorless is required to indicate complete reaction; color development indicated incomplete reaction. If color development occurred, the reaction was prolonged for 30 minutes. If color is still developed, repeat coupling is performed. After completion of the reaction, 200ml of methanol was contracted twice (5+10min), to yield 17.2g of a peptide resin.

Example 9: cleavage of peptide resins

17.2g of the resin obtained in example 8 was put into a 250ml three-necked flask, and a previously prepared lysis buffer (TFA: H) was added thereto₂O: indole 90: 5: 5(V: V))200mL, reacted at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the cleavage solution. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 2L of glacial ethyl ether for precipitation, centrifuged, washed 3 times with 1L of glacial ethyl ether, and dried under reduced pressure to give 4.65 g of crude peptide in 92% yield and 80.91% purity.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.