WO2021051766A1

WO2021051766A1 - Method for synthesizing octreotide

Info

Publication number: WO2021051766A1
Application number: PCT/CN2020/079949
Authority: WO
Inventors: 罗日朗; 尹传龙; 陶安进; 余品香
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2019-09-16
Filing date: 2020-03-18
Publication date: 2021-03-25
Also published as: CN112500450A

Abstract

Disclosed is a method for synthesizing octreotide, the method comprising the following steps: 1) selecting a 2-CTC resin as a starting point of synthesis to prepare an Fmoc-Thr(tBu)-OL-2CTC resin; 2) sequentially coupling orthogonally protected cystine, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-Phe -OH according to an Fmoc/tBu strategy, removing an Alloc protecting group for the orthogonal protection of cystine by using pd(pph3)4 and phenylsilane, then coupling Boc-D-Phe-OH, and then carrying out an intramolecular amidation reaction to form a ring; and 3) cleaving a peptide fragment by means of a lysis liquid to obtain a crude octreotide peptide. Provided is a solid-phase synthesis method which avoids an oxidation reaction that forms a ring, thereby avoiding many problems caused by existing methods; in addition, the method of the present invention has many advantages such as simple operation, a simplified process, and being environmentally friendly.

Description

A kind of synthetic method of octreotide

Technical field

The invention belongs to the field of pharmacy, and specifically relates to a method for synthesizing octreotide.

Background technique

Octreotide Acetate is an analog of human somatostatin, molecular weight 1019.3 (calculated as free base), CAS number 79517-01-4, is a cyclic nonapeptide drug, its peptide sequence is shown in formula I The original manufacturer is Novartis. In China, the United States and Europe, the marketed products are octreotide acetate injection and octreotide acetate microspheres; at the same time, there are suspensions on the market in some European countries.

Coy and Sarantakis et al. simultaneously synthesized the somatostatin myristin peptide by a solid-phase method in 1973. The Meinhofer laboratory used modified N-fluorenylmethyloxycarbonyl (Fmoc) amino acids to synthesize reduced somatostatin in solid phase according to the method of extension one by one. In 1982, Bauer et al. synthesized a somatostatin analog and named it Octreotide. Octreotide has a short peptide chain. Compared with somatostatin, 6 amino acids in SS have been removed. The 8th position is amino L-threonine, which is not easy to be hydrolyzed by protease and is easy to artificially synthesize. Its half-life in the body is up to 2 hours, and its effect of inhibiting hormone secretion is stronger than that of somatostatin. Therefore, octreotide is a long-acting analog of somatostatin, also called somatostatin release inhibitor (SRIF or SS). Octreotide is widely used in acromegaly, endocrine tumors of the digestive tract, nonsecretory tumors of the digestive tract, upper gastrointestinal bleeding, acute pancreatitis, systemic sclerosis, irritable bowel syndrome, cancer cachexia, dumping syndrome, silver Treatment of diseases such as scoria, orthostatic hypotension, and intraoperative hypotension.

Generally speaking, 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 hot spot of peptide research in recent years has shifted to the synthesis and biological evaluation of cyclic peptides.

So far, in terms of synthesis, there are mainly solid-phase synthesis methods (such as patents CN103351426 and CN1923849) and liquid-phase synthesis methods (CN1355173) to synthesize octreotide. The process of synthesis of octreotide consists of two parts: 1) The synthesis of linear octapeptide, which mainly uses solid phase to connect amino acids or fragments in sequence; 2) The linear octapeptide is cyclized, and the refined peptide is naturally oxidized or oxidized with an oxidizing reagent. , Or use oxidizing reagent to oxidize directly in the late stage of solid phase synthesis.

The existing patents are protected or are being used in the process of synthesizing octreotide. The process in the cyclization stage is not ideal. The main reason is that most of the oxidizing reagents are chemically toxic or dangerous when used, and are not environmentally friendly; natural air oxidation cannot be very environmentally friendly. The oxidation sites are well controlled and multimers are easily produced.

The purpose of the present invention is to provide another synthetic method for preparing octreotide. The method avoids the use of oxidation reaction to form a ring, and is novel, mild in synthesis conditions, simple in process and stable in process.

Summary of the invention

In order to solve the above-mentioned background art problems, the purpose of the present invention is to provide a method for synthesizing octreotide.

In order to achieve the above objective, the technical solution adopted by the present invention is: a method for synthesizing octreotide, which includes the following steps:

1) Choose 2-CTC resin as the starting point of synthesis to prepare Fmoc-Thr(tBu)-OL-2CTC resin;

2) Follow the Fmoc/tBu strategy to sequentially couple orthogonal protected cystine, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-Phe -OH, then use pd(pph3)4 and phenylsilane to remove the Alloc protecting group of cystine orthogonal protection, and then couple Boc-D-Phe-OH, and then carry out intramolecular lactam reaction to form a ring;

3) The lysate is cleaved off the peptide fragments to obtain crude octreotide peptide.

Further, the preparation method of Fmoc-Thr(tBu)-OL-2CTC resin is to put 2-CTC resin into a reaction vessel, dissolve Fmoc-Thr(tBu)-OL in a solvent and then add it to the solid phase synthesis carrier, slowly Add DIPEA dropwise, stir and react for a period of time to obtain Fmoc-Thr(tBu)-OL-2CTC resin.

Further, the solvent for dissolving Fmoc-Thr(tBu)-OL is any organic solvent that can dissolve the raw material and can be used in solid-phase synthesis reactions, such as dichloromethane, DMF, NMP, DMSO, THF, etc., preferably two Methyl chloride.

Further, the 2-CTC resin is preferably a 2-CTC resin with a resin substitution degree greater than 1.0 mmol/g.

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

Further, step 2) specifically includes: removing Fmoc, washing the resin until Fmoc is completely removed; dissolving and activating an appropriate amount of orthogonally protected cystine and coupling agent in the solvent, and then adding them to the solid Instead, it should be in the column until the termination of the reaction is detected by the detection method;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Thr(tBu)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected. The method detects the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Lys(Boc)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected. The method detects the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-D-Trp(Boc)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until Use the detection method to detect the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Phe-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected by the detection method Until the reaction is terminated;

After removing the Alloc protecting group of cystine orthogonal protection with pd(pph3)4 and phenylsilane, the appropriate amount of Boc-D-Phe-OH and coupling agent are dissolved and activated in the solvent, and then added to the solid Instead, it should be in the column until the termination of the reaction is detected by the detection method;

Remove Fmoc and wash the resin until Fmoc is completely removed; Dissolve HBTU and DIPEA in the solvent and put them into the solid phase reaction column until the end of the reaction is detected by the detection method.

Further, the solvent for dissolving the substance in step 2) can be any solvent used in general solid-phase synthesis, such as DMF, NMP, DMSO and the like.

Further, the reagent used to remove Fmoc is a 20% piperidine/DMF solution (DBLK), that is, a piperidine:DMF (volume ratio) 1:4 mixed solution.

Further, the coupling agent is DIC+A or DIPEA+A+B, where A is HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, and TBTU; preferably a combination of HBTU, 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, where 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) first swells 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 of the reaction. If the resin develops color, it indicates that there is free amine in the polypeptide, that is, there is no protecting group on the amine.

Further, the lysis solution in step 3) is a mixture of TFA, H ₂ O, and indole, preferably TFA: H ₂ O: indole = 90:5:5.

The existing process for synthesizing octreotide is not ideal in the cyclization stage. The main reason is that most of the oxidizing reagents used are chemically toxic or dangerous when used, and are not environmentally friendly; natural air oxidation cannot control the oxidation site well. Point, easy to produce multimers. However, the use of liquid phase synthesis is cumbersome in process operation, and the purity of the crude peptide obtained is not high. The invention provides a solid-phase synthesis method that avoids oxidation reaction to form a ring, thereby avoiding many problems caused by the existing method, and the method of the invention has many advantages such as simple operation, simplified process, and environmental friendliness.

Description of the drawings

Figure 1 is a chromatogram of octreotide crude peptide;

Figure 2 shows the mass spectrum of the crude octreotide peptide.

detailed description

In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

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

Example 1: Preparation of Fmoc-Thr(tBu)-OL-2CTC resin

Weigh 20g (22.4mmol) of 1.12mmol/g2-CTC resin into a suitable round-bottomed flask, weigh 8.61g (22.4mmol) of Fmoc-Thr(tBu)-OL and dissolve it with 200ml of dry dichloromethane, add the existing In the round bottom flask of the resin, 19.5ml (112mmol) of DIPEA was slowly added, and the reaction was mechanically stirred for 24h. After the reaction is completed, transfer to a solid phase reaction column to drain the reaction solution, wash 6 times with DMF, 2 min each time, shrink twice with methanol (5 min + 10 min), use 200 ml methanol each time, and vacuum dry.

Example 2: Access to Cys-Cys

Weigh 10g of the 0.5mmol/g Fmoc-Thr(tBu)-OL-2CTC resin in Example 1 into the solid phase reaction column, add 100ml DMF, and swell with nitrogen bubbling for 60 minutes; then use 100mL DBLK to deprotect twice (5min +7min), DMF and DCM were washed alternately 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 15mmol Cys-Cys, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, then put it into the reaction column, bubbling with nitrogen for 2h, and wash with DMF and DCM 6 times alternately (Washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, it is required to be colorless, indicating that the reaction is complete; color development, indicating that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

Example 3: Access to Fmoc-Thr(tBu)-OH

After passing the Kaiser test in Example 2, deprotection with 100mL DBLK twice (5min+7min), washing with DMF and DCM alternately 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 15mmol Fmoc-Thr(tBu)-OH, 14.25mmolHBTU and 15mmol HOBT to dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, put it into the reaction column, bubbling with nitrogen for 2h, use DMF, DCM Alternate washing 6 times (washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, it is required to be colorless, indicating that the reaction is complete; color development, indicating that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

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

After passing the Kaiser test in Example 3, use 100 mL DBLK to deprotect twice (5min+7min), and alternately wash DMF and DCM 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 15mmol Fmoc-Lys(Boc)-OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, then put it into the reaction column, bubbling with nitrogen for 2h, use DMF, DCM was washed alternately 6 times (washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, if colorless is required, it means the reaction is complete; color development means the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

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

After passing the Kaiser test in Example 4, use 100 mL DBLK to deprotect twice (5min+7min), and wash DMF and DCM alternately 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 15mmol Fmoc-D-Trp(Boc)-OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, put it into the reaction column, bubbling with nitrogen for 2h, use DMF and DCM were washed alternately 6 times (washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, it is required to be colorless, indicating that the reaction is complete; color development, indicating that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

Example 6: Access to Fmoc-Phe-OH

After passing the Kaiser test in Example 5, 100 mL DBLK was used for deprotection twice (5min+7min), and DMF and DCM were alternately washed 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 15mmol Fmoc-Phe-OH, 14.25mmol HBTU and 15mmol HOBT to dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, put it into the reaction column, bubbling with nitrogen for reaction for 2h, washing with DMF and DCM alternately 6 times (washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, it is required to be colorless, indicating that the reaction is complete; color development, indicating that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

Example 7: Boc-D-Phe-OH

After passing the Kaiser test in Example 6, weigh 2.5mmol pd(pph3)4 and 100mmol phenylsilane and dissolve them in 40ml dichloromethane, add them to the reaction column, react for 1h, remove the Alloc protecting group, wash with DMF and DCM 6 times ( 3+3+3minDCM+3+3+3minDMF), weigh out 15mmol Boc-D-Phe-OH, 14.25mmol HBTU and 15mmol HOBT, dissolve in 50ml DMF, measure 30mmol DIPEA and add and stir until the material is completely dissolved, then put into the reaction In the column, nitrogen was bubbled to react for 2 hours, and washed with DMF and DCM alternately 6 times (washing sequence and time: 2+4minDMF+4minDCM+4minDMF+2minDCM+2minDMF). Sampling for Kaiser detection, it is required to be colorless, indicating that the reaction is complete; color development, indicating that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling.

Example 8: The reaction of molecular lactam to form a ring

After passing the Kaiser test in Example 7, deprotection with 100mL DBLK twice (5min+7min), and washing with DMF and DCM alternately 7 times (washing sequence and time: 2+4minDMF+4minDCM+4+4minDMF+2minDCM+2minDMF). Weigh 14.25mmol HBTU and 30mmol DIPEA to dissolve in NMP, put them into the reaction column, react for 2.5h, take samples for Kaiser detection, if colorless is required, it means the reaction is complete; color development means that the reaction is incomplete. If the color develops, prolong the reaction for 30 minutes. If the color still develops, repeat the coupling. After the reaction was completed, 200ml of methanol was contracted twice (5+10min), and 17.2g of peptide resin was collected.

Example 9: Cleavage of peptide resin

Add 17.2g of the resin obtained in Example 8 into a 250ml three-necked flask, add _{200mL of pre-configured lysate (TFA: H 2} O: indole = 90: 5: 5 (V: V)), and react at room temperature for 2 hours , Filter the resin under reduced pressure, and collect the cutting fluid. The resin was washed with a small amount of TFA, and the filtrates were combined. The filtrate was slowly added to 2L of ice ether for precipitation, centrifuged, washed with 1L of ice ether for 3 times, and dried under reduced pressure to obtain 4.65 g of crude peptide with a yield of 92% and a purity of 80.91%.

The above are only specific implementations of the present invention, not all implementations. Any equivalent changes made to the technical solutions of the present invention by those of ordinary skill in the art by reading the specification of the present invention are covered by the claims of the present invention. .

Claims

A method for synthesizing octreotide, which is characterized in that it comprises the following steps:

1) Choose 2-CTC resin as the starting point of synthesis to prepare Fmoc-Thr(tBu)-OL-2CTC resin;

2) Follow the Fmoc/tBu strategy to sequentially couple orthogonal protected cystine, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-Phe -OH, then use pd(pph3)4 and phenylsilane to remove the Alloc protecting group of cystine orthogonal protection, and then couple Boc-D-Phe-OH, and then carry out intramolecular lactam reaction to form a ring;

3) The lysate is cleaved off the peptide fragments to obtain crude octreotide peptides.
The method for synthesizing octreotide according to claim 1, wherein the method for preparing the Fmoc-Thr(tBu)-OL-2CTC resin is to put 2-CTC resin into a reaction vessel, and put Fmoc-Thr(tBu) )-OL is dissolved in a solvent and added to the solid phase synthesis carrier. DIPEA is slowly added dropwise and stirred for a period of time to obtain Fmoc-Thr(tBu)-OL-2CTC resin.
The method for synthesizing octreotide according to claim 1 or 2, wherein the 2-CTC resin is a 2-CTC resin with a resin substitution degree greater than 1.0 mmol/g.
The method for synthesizing octreotide according to claim 1, wherein the protective structure of the orthogonally protected cystine is as follows:
The method for synthesizing octreotide according to claim 1, wherein the step 2) specifically includes: removing Fmoc, washing the resin until Fmoc is completely removed; adding an appropriate amount of orthogonally protected cystine and After the coupling agent is dissolved and activated in the solvent, it is added to the solid phase reaction column together until the termination of the reaction is detected by the detection method;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Thr(tBu)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected. The method detects the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Lys(Boc)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected. The method detects the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-D-Trp(Boc)-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until Use the detection method to detect the termination of the reaction;

Remove Fmoc and wash the resin until Fmoc is completely removed; After dissolving and activating the appropriate amount of Fmoc-Phe-OH and coupling agent in the solvent, they are added to the solid phase reaction column together until it is detected by the detection method Until the reaction is terminated;

After removing the Alloc protecting group of cystine orthogonal protection with pd(pph3)4 and phenylsilane, the appropriate amount of Boc-D-Phe-OH and coupling agent are dissolved and activated in the solvent, and then added to the solid Instead, it should be in the column until the termination of the reaction is detected by the detection method;

Remove Fmoc and wash the resin until Fmoc is completely removed; Dissolve HBTU and DIPEA in the solvent and put them into the solid phase reaction column until the end of the reaction is detected by the detection method.
The method for synthesizing octreotide according to claim 5, wherein the reagent for removing Fmoc is a 20% piperidine/DMF solution.
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 PyBOP, PyAOP, HATU, HBTU, TBTU One of them; preferably a combination of HBTU, compound A and DIPEA.
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, where Fmoc-aa-OH is Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp (Boc)-OH, Fmoc-Phe-OH.
The method for synthesizing octreotide according to claim 5, wherein the reaction in step 2) first swells the resin before coupling, and the reagent used in the washing and swelling steps is DMF, NMP or dichloromethane Methane, preferably DMF.
The method for synthesizing octreotide according to claim 1, wherein the lysate in step 3) is a mixture of TFA, H 2 O, and indole, preferably TFA: H 2 O: indole = 90:5 : 5.