Synthesis and application of novel non-classical solid-phase synthesis carrier

Abstract

The invention relates to a preparation method of a novel non-classical solid-phase synthesis carrier and application of the novel non-classical solid-phase synthesis carrier in synthesis of C-terminal amidation modified polypeptide drugs. The prepared non-classical solid-phase synthesis carrier not only realizes homogeneous coupling of a carrier or a peptide carrier and amino acid, but also can complete splitting decomposition in a splitting decomposition solution to obtain a C-terminal amidation modified polypeptide drug. Compared with the existing production technology, the industrial production of the C-terminal amidation modified polypeptide medicine can be realized, the production process is effectively simplified, the production cost is reduced, the production efficiency is improved, the environmental pollution is reduced, and the method is green and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly to the synthesis of a novel non-classical solid-phase synthesis carrier and its application in the synthesis of C-terminal amidated modified polypeptide drugs. Background Art

[0002] A polypeptide is a bioactive substance formed by dehydration condensation of two or more amino acids to form several peptide bonds and combined by peptide bonds in a certain arrangement order. It is a general term for bioactive substances involved in various cell functions in an organism and is often used in fields such as functional analysis, antibody research, and drug research and development.

[0003] The chemical synthesis methods of polypeptides can be mainly divided into two types: liquid-phase synthesis method and solid-phase synthesis method. Although humans have conducted research on the synthesis of polypeptides, the liquid-phase synthesis method of polypeptides has a small synthesis range and is generally only suitable for the synthesis of polypeptides within 10. Moreover, during the synthesis process, the intermediate products need to be purified, which takes a long time and involves a large amount of work. In 1963, Merrifield established the solid-phase peptide synthesis method (Solid Phase Peptide Synthesis, SPPS). With the development of polypeptide synthesis technology and amino protection technology, the solid-phase synthesis method of polypeptides quickly replaced the liquid-phase synthesis method and became the preferred method for polypeptide synthesis.

[0004] The solid-phase synthesis method of polypeptides can be divided into Boc solid-phase synthesis method and Fmoc solid-phase synthesis method. Among them, the Fmoc solid-phase synthesis method has mild reaction conditions and many advantages over the Boc solid-phase synthesis method, and it has been widely used quickly. By the 1980s, the Fmoc solid-phase synthesis method gradually replaced the Boc solid-phase synthesis method and became the commonly used synthesis method in solid-phase polypeptide synthesis. However, at present, there are still many problems to be solved in the Fmoc solid-phase synthesis method of polypeptides. (1) After swelling by the reaction medium, the volume of the solid-phase synthesis carrier of polypeptides can increase by 5-10 times, generally requiring a reactor with a larger volume, which limits the large-scale production of polypeptides. (2) The solid-phase synthesis of polypeptides requires the use of a solid-phase reactor with special designs such as a sieve plate configured at the bottom of the kettle, which increases the equipment cost. (3) In the solid-phase synthesis of polypeptides, an excessive amount of amino acids and condensation reagents often need to be added to the reaction system, which increases the production cost. (4) The amount of organic solvents used for washing in the solid-phase synthesis of polypeptides is huge, which not only increases the production cost but also goes against the current concept of green chemistry.

[0005] Among polypeptide drugs, there is a class of polypeptide drugs with C-terminal amidation modification, such as leuprorelin acetate, alarelin acetate, and buserelin, etc. This kind of modification is generally considered to be able to reduce the total charge of the peptide, lower the solubility of the polypeptide, be closer to the mimic of its natural protein, and improve the stability and biological activity of the peptide. However, the characteristics of the Fmoc-solid phase synthesis method, that is, the amino acid residue at the C-terminal of the polypeptide needs to be connected to the carrier first, creates an innate barrier for the synthesis of C-terminal amidated modified peptides and increases the difficulty of synthesis.

[0006] Chinese Patent CN 106146622 A uses PAM resin as the solid-phase synthesis carrier and combines Boc and Fmoc methods to synthesize leuprorelin. The first four peptides are synthesized by the Boc-solid phase synthesis method, and the last five peptides are synthesized by the Fmoc solid-phase synthesis method. Ethylamine is used for aminolysis of the resin in the liquid phase, the solvent is concentrated and dried, and then the crude peptide is obtained by cutting with the cutting solution. This method uses PAM as the solid-phase synthesis carrier and combines the Boc-solid phase synthesis method, which well solves the problem of the DKP side reaction in the process of peptide resin synthesis. However, a large amount of TFA is needed to remove the Boc protecting group multiple times during the synthesis process, which is very dangerous. Finally, ethylamine is used for aminolysis of the resin, and the use of ethylamine is very dangerous, with a relatively large production safety risk.

[0007] For example, Chinese Patent CN 102702327 B discloses a solid-liquid phase synthesis method of alarelin. This method first synthesizes a peptide resin with a fully protected side chain peptide chain by the solid-phase synthesis method using CTC resin as the solid-phase synthesis carrier, and obtains a peptide chain with a fully protected side chain by cutting with a low-concentration acid; then acetylates the C-terminal of the peptide chain with a fully protected side chain by the liquid-phase synthesis method to obtain a peptide chain of alarelin with a fully protected side chain; finally, cuts to remove the side chain protecting group to obtain the crude product of alarelin. However, this method is relatively cumbersome to operate, especially the cutting needs to be carried out in two steps to complete, which increases the production cycle and production cost.

[0008] Chinese Patent CN 112585153 A discloses a compound or its salt, its preparation method and application. The application examples of this patent involve the synthesis of leuprorelin and alarelin. The synthesized compound is used as a Linker, first coupled to the selected solid-phase synthesis resin, and then according to the amino acid sequence of the polypeptide, the Fmoc-solid phase synthesis method is used to couple successively to obtain a fully protected peptide chain-Linker-Resin peptide resin, and the target polypeptide crude product is obtained after one cleavage. This method solves some challenges in the synthesis process of C-terminal amidated modified polypeptide drugs such as leuprorelin and alarelin, but still cannot get rid of the inherent defects of solid-phase synthesis, such as multi-fold feeding and washing with a huge amount of organic solvents.

[0009] Starting from improving the solid-phase synthesis carrier, the present invention designed and synthesized a new type of non-classical solid-phase synthesis carrier, which can be applied to the synthesis of C-terminal amidated modified polypeptide drugs, and has obvious advantages over the traditional solid-phase synthesis carrier in terms of cost reduction, efficiency improvement, energy conservation, emission reduction, and environmental friendliness. Summary of the Invention

[0010] In order to achieve the large-scale production of polypeptide products, reduce production costs, improve production efficiency, and reduce environmental pollution, the present invention provides the synthesis of a new type of non-classical solid-phase synthesis carrier and its application in the synthesis of C-terminal amidated modified polypeptide drugs.

[0011] The present invention provides a new type of non-classical solid-phase synthesis carrier, which can be applied to the synthesis of C-terminal amidated modified polypeptide drugs. The biggest feature of this new type of carrier is that its solubility shows great differences in different solvents. Utilizing this feature, in the good solvent of the carrier or peptide-carrier, homogeneous coupling of the carrier or peptide-carrier with amino acids can be implemented; in the poor solvent of the carrier or peptide-carrier, washing and purification of the carrier or peptide-carrier can be implemented.

[0012] Compared with the prior art, when using the new type of non-classical solid-phase synthesis carrier to synthesize C-terminal amidated modified polypeptide drugs, the crude product of the target polypeptide product can be obtained through one cleavage, with simple operation and large-scale production capacity. For the same production scale, the reactor volume can be reduced by 30%-50%, the usage amounts of raw material amino acids and condensing agents can be reduced by 50%-60%, and the usage amounts of organic solvents for reaction and washing can be reduced by 80%-90%, ultimately achieving a 50%-70% reduction in production costs.

[0013] In order to achieve the above object, the present invention adopts the following technical solutions:

[0014] In the first aspect, the present invention provides a new type of non-classical solid-phase synthesis carrier, and its structure can be represented by the following formula:

[0015]

[0016] Among them, R1 is methyl or ethyl; R2 is

[0017]

[0018] The functional group of the carrier is imino;

[0019] The carrier contains 1, 2 or 3 side chains, and the side chains are located at the ortho, meta or para positions of the functional group;

[0020] The side chain of the carrier is a long-chain alkyl ether, which can be n-decyl ether, n-dodecyl ether, n-tetradecyl ether, n-hexadecyl ether, n-octadecyl ether, n-eicosyl ether, n-docosyl ether or straight-chain alkyl ether.

[0021] In a second aspect, a method for synthesizing a novel non-classical solid-phase synthesis carrier is provided, and the synthesis steps are as follows.

[0022] Step 1: In a reaction medium, hydroxybenzaldehyde, haloalkane, a base, and a catalyst are added, and the mixture is heated for reaction. After the reaction is complete, the reaction solution is added to a poor solvent, and the product precipitates. The product is filtered, and the filter cake is washed, dried, and purified to obtain a benzaldehyde intermediate containing a long-chain alkylether side chain.

[0023] The reaction medium is one of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or N-methylpyrrolidone, or any mixed solvent, preferably N,N-dimethylformamide.

[0024] The haloalkane in the starting materials for the reaction is chloroalkane, bromoalkane, or iodoalkane, preferably bromoalkane.

[0025] The base in the reaction is one or any combination of potassium carbonate, sodium carbonate, sodium hydroxide, or potassium hydroxide, preferably potassium carbonate; the catalyst is preferably potassium iodide; the reaction temperature is preferably 50-100 °C, and more preferably, 70-90 °C is selected.

[0026] The poor solvent for the benzaldehyde intermediate containing a long-chain alkylether side chain is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate, or acetonitrile, preferably water and n-hexane.

[0027] Step 2: The benzaldehyde intermediate containing a long-chain alkylether side chain is added to a reaction medium, methylamine hydrochloride or ethylamine hydrochloride is added, and a reducing agent is added. After the reaction is complete, a poor solvent is added to the reaction solution, and the product precipitates. The product is filtered, washed, and dried to obtain the target non-classical solid-phase synthesis carrier.

[0028] The reaction medium is one or any combination of tetrahydrofuran, methanol, and ethanol, preferably a mixed solvent of tetrahydrofuran and methanol, and more preferably, the volume ratio of tetrahydrofuran to methanol is 90:10.

[0029] The reducing agent in the reaction is one or any combination of sodium borohydride, sodium cyanoborohydride, or sodium triacetoxyborohydride, preferably sodium borohydride.

[0030] The reaction temperature is 0-30 °C, and more preferably, 0-10 °C is selected.

[0031] The poor solvent for the target novel non-classical solid-phase synthesis carrier is one or any combination of water, n-hexane, n-heptane, ethyl acetate, and acetonitrile, preferably water.

[0032] In a third aspect, an application of a non-classical solid-phase synthesis carrier in polypeptide synthesis is provided, and the application steps are as follows.

[0033] Step 1: Coupling reaction of the first amino acid: In the reaction medium, the non-classical solid-phase synthesis carrier and Fmoc-amino acid undergo a coupling reaction under the action of a condensation reagent, and HPLC, TLC or NT detection methods are used to determine the end point of the reaction. Among them:

[0034] The reaction temperature is 20 - 50 °C, preferably 30 - 35 °C.

[0035] The coupling reaction medium is one or a combination of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone and dimethyl sulfoxide, and preferably the solvent or combination that can dissolve the coupling reaction system clearly.

[0036] The condensation reagent is any combination of DIC / DCC / EDCI + HOOBT / HOBT / HOAT + DIPEA / NMM + DMAP / HBTU / HATU / TBTU / PyBOP, and preferably the DMAP + DIC combination.

[0037] The feed ratio of the coupling reaction is carrier:Fmoc-amino acid:DMAP:DIC = 1:1 - 3:0.1 - 0.3:1 - 3, and preferably carrier:Fmoc-amino acid::DMAP:DIC = 1:2:0.2:2.

[0038] Step 2: After the amino acid coupling reaction is completed, the reaction solution is spread into a poor solvent, filtered, washed and dried to obtain the Fmoc-peptide carrier; the obtained Fmoc-peptide carrier is added to a deprotection reagent for deprotection reaction to obtain the starting peptide carrier. Among them:

[0039] The Fmoc deprotection reagent is a dichloromethane solution of diethylamine, preferably a dichloromethane solution of 20% diethylamine.

[0040] The poor solvent for the peptide carrier is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate or acetonitrile, and preferably water and acetonitrile.

[0041] Step 3: Peptide carrier elongation reaction: In the reaction medium, the starting peptide carrier and Fmoc-amino acid undergo a coupling reaction under the action of a condensation reagent, and HPLC, TLC or NT detection methods are used to determine the end point of the reaction; after the amino acid coupling reaction is completed, the reaction solution is spread into a poor solvent, filtered, washed and dried to obtain the Fmoc-peptide carrier; the obtained Fmoc-peptide carrier is added to a deprotection reagent for deprotection reaction to obtain the elongated peptide carrier. Among them:

[0042] The reaction temperature is 20 - 50 °C, preferably 30 - 35 °C.

[0043] The coupling reaction medium is one or a combination of more than one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide, and preferably a solvent or combination that can dissolve the coupling reaction system clearly.

[0044] The condensation reagent is any combination of DIC / DCC / EDCI + HOOBT / HOBT / HOAT + DIPEA / NMM + DMAP / HBTU / HATU / TBTU / PyBOP, and preferably the DMAP + DIC combination.

[0045] The feeding ratio of the coupling reaction is support:Fmoc-amino acid:HOBT:DIC = 1:1 - 3:1 - 3:1 - 3, and preferably support:Fmoc-amino acid::DMAP:DIC = 1:1.2:1.2:1.2.

[0046] The Fmoc deprotection reagent is a dichloromethane solution of diethylamine, and preferably a dichloromethane solution of diethylamine with a concentration of 20%.

[0047] The poor solvent for the peptide support is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate or acetonitrile, and preferably water and acetonitrile.

[0048] Step 4: Repeat Step 3 to finally obtain the fully protected product peptide support.

[0049] Step 5: Peptide support cleavage reaction. Add the peptide support to the cleavage solution to start the cleavage reaction. After the cleavage is completed, add the cleavage product to cold methyl tert-butyl ether. The product precipitates, is filtered, and the filter cake is washed and dried to obtain the crude target peptide.

[0050] Step 6: Purification of the crude peptide. The crude peptide obtained by cleavage is purified by reverse-phase liquid chromatography, salt conversion, concentration and lyophilization to obtain the refined target peptide.

[0051] The abbreviations used in the specification and claims of the present invention have the following meanings:

[0052]

[0053] Description of the drawings:

[0054] Appendix Figure 1 : HPLC chromatogram of the crude leuprorelin synthesized using the novel solid-phase synthesis support.

[0055] Appendix Figure 2 : LC-MS chromatogram of the refined leuprorelin synthesized using the novel solid-phase synthesis support.

[0056] Appendix Figure 3: HPLC chromatogram of the crude alarelin synthesized using a novel solid-phase synthesis support.

[0057] Attachment Figure 4 : LC-MS chromatogram of the pure alarelin synthesized using a novel solid-phase synthesis support. Detailed implementation methods

[0058] The present invention is further defined in the following examples. It should be understood that although these examples indicate the preferred embodiments of the present invention, they are given by way of illustration only and do not limit the claims of the present invention.

[0059] Synthesis route of a novel solid-phase synthesis support HZ-WSP-X:

[0060]

[0061] Example 1: Synthesis of a novel solid-phase synthesis support pre-intermediate HZ-WSP-X10.

[0062] In a 1000 mL three-necked flask, add 550 mL of DMF, 7.70 g (50.0 mmol) of 3,4,5-trihydroxybenzaldehyde, and 55.0 g (165.0 mmol) of octadecyl bromide, stir to dissolve, heat in an oil bath to T 内 = 40 - 50 °C until the reaction solution becomes clear. Continue to add 27.6 g (200 mmol) of potassium carbonate and 2.5 g (15.0 mmol) of potassium iodide to the reaction flask, and heat in an oil bath to T 内 = 70 - 90 °C to start the reaction, and monitor the reaction end point by TLC. After the reaction is completed, stop heating, let it cool back to room temperature, then pour the reaction solution into cold water, and a solid will precipitate. Filter, wash the filter cake with water, dry, and purify to obtain 38.9 g (42.7 mmol) of intermediate HZ-WSP-X10, with a yield of 85.3%.

[0063] Example 2: Synthesis of a novel solid-phase synthesis support HZ-WSP-X.

[0064] In a 2000 mL three-necked flask, add 315 mL of THF, 15 mL of MeOH, 36.5 g (40.0 mmol) of HZ-WSP-X10, and 4.9 g (60.0 mmol) of ethylamine hydrochloride, stir until clear, and then cool to T 内 = 0 - 10 °C in an ice-water bath; separately dissolve 4.6 g (120.0 mmol) of sodium borohydride in 20 mL of MeOH, and slowly add it dropwise to the reaction flask. After the addition is complete, remove the ice-water bath and heat in a water bath, raising the temperature to T 内React at 20 - 30 °C. Monitor the reaction endpoint by TLC. After the reaction is completed, pour the reaction solution into cold water. Solids will precipitate. Filter, wash the filter cake with water, dry, and purify to obtain 31.0 g (32.9 mmol) of the novel non-classical solid-phase synthesis carrier HZ-WSP-X, with a yield of 82.4%.

[0065] Example 3: Application of the novel non-classical solid-phase synthesis carrier: Use HZ-WSP-X as the synthesis carrier to synthesize leuprorelin.

[0066] Loading reaction of the first amino acid of leuprorelin:

[0067] Loading the first amino acid Fmoc-Pro-OH H2O: Add 100 mL of THF, 9.4 g (10.0 mmol; MW: 940.67, Substitution: 1.06 mmol / g) of HZ-WSP-X, 7.1 g (20.0 mmol) of Fmoc-Pro-OH H2O, and 0.3 g (2.0 mmol) of DMAP to a 250 mL three-necked flask. Stir until clear, then add dropwise 3.1 mL (2.5 g, 20.0 mmol) of DIC. Heat in an oil bath to T 内 = 30 - 35 °C, start the reaction, and monitor the reaction endpoint by TLC.

[0068] Work-up after the coupling of Fmoc-Pro-OH: After the coupling reaction is completed, pour the reaction solution into 1000 mL of cold water. A large amount of white solids will precipitate. Filter, wash the filter cake successively with water and ethanol, and dry to obtain the crude product of Fmoc-Pro-HZ-WSP-X, which is a white solid.

[0069] Deprotection of the Fmoc protecting group and work-up: Add Fmoc-Pro-HZ-WSP-X to a 20% diethylamine dichloromethane deprotection solution. Stir until clear and heat in an oil bath to T 内 = 30 - 35 °C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, concentrate under reduced pressure. Pulverize the concentrated residue with methyl tert-butyl ether, filter, and dry to obtain 10.4 g of the crude product of the peptide carrier H-Pro-HZ-WSP-X, which is a white solid.

[0070] Extension reaction of the leuprorelin peptide carrier:

[0071] Coupling of Fmoc-Arg(Pbf)-OH: Add 100 mL of THF and 10.4 g of the crude product of H-Pro-HZ-WSP-X to a 250 mL three-necked flask. Heat in an oil bath to T 内= 30 - 35 °C, stir until dissolved and clear. Add 10 mL of N,N-dimethylformamide, 7.8 g (12.0 mmol) of Fmoc-Arg(Pbf)-OH and 1.6 g (12.0 mmol) of HOBT to the activation flask, stir until dissolved and clear, and pre-cool to T in a low-temperature bath 内 = 0 - 5 °C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 min. After the activation is completed, add the activation solution to the reaction flask to start the reaction, and monitor the reaction end point by TLC or NT detection.

[0072] Work-up after the coupling of Fmoc-Arg(Pbf)-OH: After the coupling reaction is completed, pour the reaction solution into 1000 mL of cold water. A large amount of white solid precipitates. Filter, and wash the filter cake with water and ethanol successively, and dry to obtain the crude product of Fmoc-Arg(Pbf)-Pro-HZ-WSP-X, which is a white solid.

[0073] Removal of the Fmoc protecting group and work-up: Add the crude product of Fmoc-Arg(Pbf)-Pro-HZ-WSP-X to the deprotection solution of 20% diethylamine in dichloromethane, stir until dissolved and clear, heat in an oil bath to T 内 = 30 - 35 °C to start the reaction, and monitor the reaction end point by TLC detection. After the deprotection reaction is completed, concentrate under reduced pressure. Pulverize the concentrated residue with methyl tert-butyl ether, filter, and dry to obtain 14.5 g of the crude product of the peptide carrier H-Arg(Pbf)-Pro-HZ-WSP-X, which is a white solid.

[0074] Using HZ-WSP-X as the synthesis carrier, extend the reaction of the leuprorelin peptide carrier to synthesize Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-HZ-WSP-X. The coupling sequence and dosage of each amino acid are shown in Table 2.

[0075] Table 2 Coupling sequence and dosage of each amino acid

[0076] Coupling sequence Amino acid or peptide segment name MW. Moles / mmol Mass / g 1 <![CDATA[Fmoc-Pro-OH H2O]]> 355.37 20 7.1 2 Fmoc-Arg(Pbf)-OH 648.77 12 7.8 3 Fmoc-Leu-OH 353.41 12 4.2 4 Fmoc-D-Leu-OH 353.41 12 4.2 5 Fmoc-Tyr(tBu)-OH 459.53 12 5.5 6 Fmoc-Ser(tBu)-OH 383.44 12 4.6 7 Fmoc-Trp(Boc)-OH 526.58 12 6.3 8 Fmoc-His(Trt)-OH 619.71 12 7.4 9 Pyr-OH 129.11 12 1.6

[0077] Synthesize 28.2 g of the crude product of the leuprorelin peptide carrier Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-HZ-WSP-X, which is a white solid with a yield of 100%.

[0078] Cleavage reaction of the leuprorelin peptide carrier:

[0079] Prepare a lysis solution with TFA:TIS:EDT:H2O = 85:5:5:5 and pre-cool it to -5 - 5°C. Add 280 mL of the cold lysis solution and 28.2 g (10.0 mmol) of leuprorelin peptide carrier Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-HZ-WSP-X to a 500 mL lysis flask, and heat it in an oil bath to T 内 = 30 - 35°C and lyse for 2 - 3 h. After the lysis is completed, add the lysis solution to 2800 mL of cold methyl tert-butyl ether, centrifuge, wash, and dry to obtain 12.8 g of crude leuprorelin (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt). The crude product synthesis yield is 105.8%, and the HPLC purity of the crude product is 92.46%. The HPLC chromatogram of the crude leuprorelin is shown in the appendix Figure 1 .

[0080] Purification of crude leuprorelin:

[0081] 12.8 g of crude leuprorelin (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt) is purified by reverse preparative chromatography, salt conversion, concentration, and lyophilization to obtain 8.25 g of high-quality leuprorelin acetate, which is a white powder; the HPLC purity is ≥99.0%, the peptide content is 87.3%, and the total yield is 59.6%. The LC-MS chromatogram of the high-quality leuprorelin acetate is shown in the appendix Figure 2 .

[0082] Example 4: Application of a novel non-classical solid-phase synthesis carrier: Use HZ-WSP-X as the synthesis carrier to synthesize alarelin.

[0083] Loading reaction of the first amino acid of alarelin:

[0084] Load the first amino acid Fmoc-Pro-OH H2O: Add 100 mL of THF, 9.4 g (10.0 mmol; MW: 940.67, Substitution: 1.06 mmol / g) of HZ-WSP-X, 7.1 g (20.0 mmol) of Fmoc-Pro-OH H2O, and 0.3 g (2.0 mmol) of DMAP to a 250 mL three-necked flask, stir to dissolve clearly, add dropwise 3.1 mL (2.5 g, 20.0 mmol) of DIC, and heat it in an oil bath to T 内 = 30 - 35°C to start the reaction, and monitor the reaction end point by TLC.

[0085] Work-up after coupling of Fmoc-Pro-OH: After the coupling reaction was completed, the reaction solution was poured into 1000 mL of cold water. A large amount of white solid precipitated out. It was filtered, and the filter cake was rinsed successively with water and ethanol and then dried to obtain the crude product of Fmoc-Pro-HZ-WSP-X, which was a white solid.

[0086] Deprotection of Fmoc protecting group and work-up: To the deprotection solution of 20% diethylamine in dichloromethane, Fmoc-Pro-HZ-WSP-X was added and stirred until dissolved and clear. It was heated in an oil bath to T 内 = 30 - 35 °C, and the reaction was started. The reaction end point was monitored by TLC detection. After the deprotection reaction was completed, it was concentrated under reduced pressure. The concentrated residue was slurried with methyl tert-butyl ether, filtered, and dried to obtain 10.4 g of the crude product of the peptide carrier H-Pro-HZ-WSP-X, which was a white solid.

[0087] Elongation reaction of the alarelin peptide carrier:

[0088] Coupling of Fmoc-Arg(Pbf)-OH: To a 250 mL three-necked flask, 100 mL of THF and 10.4 g of the crude product of H-Pro-HZ-WSP-X were added. It was heated in an oil bath to T 内 = 30 - 35 °C and stirred until dissolved and clear. To the activation flask, 10 mL of N,N-dimethylformamide, 7.8 g (12.0 mmol) of Fmoc-Arg(Pbf)-OH and 1.6 g (12.0 mmol) of HOBT were added and stirred until dissolved and clear. It was pre-cooled to T 内 = 0 - 5 °C in a low-temperature bath; 1.9 mL (1.5 g, 12.0 mmol) of DIC was added to the activation flask, and the temperature was maintained for pre-activation for 5 min. After the activation was completed, the activation solution was added to the reaction flask, and the reaction was started. The reaction end point was monitored by TLC or NT detection.

[0089] Work-up after coupling of Fmoc-Arg(Pbf)-OH: After the coupling reaction was completed, the reaction solution was poured into 1000 mL of cold water. A large amount of white solid precipitated out. It was filtered, and the filter cake was rinsed successively with water and ethanol and then dried to obtain the crude product of Fmoc-Arg(Pbf)-Pro-HZ-WSP-X, which was a white solid.

[0090] Deprotection of Fmoc protecting group and work-up: To the deprotection solution of 20% diethylamine in dichloromethane, the crude product of Fmoc-Arg(Pbf)-Pro-HZ-WSP-X was added and stirred until dissolved and clear. It was heated in an oil bath to T 内 = 30 - 35 °C, and the reaction was started. The reaction end point was monitored by TLC detection. After the deprotection reaction was completed, it was concentrated under reduced pressure. The concentrated residue was slurried with methyl tert-butyl ether, filtered, and dried to obtain 14.5 g of the crude product of the peptide carrier H-Arg(Pbf)-Pro-HZ-WSP-X, which was a white solid.

[0091] Using HZ-WSP-X as a synthetic carrier, the reaction of the alarelin peptide carrier was extended, and Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(Pbf)-Pro-HZ-WSP-X was synthesized. The coupling sequence and dosage of each amino acid are shown in Table 3.

[0092] Table 3 Coupling sequence and dosage of each amino acid

[0093]

[0094]

[0095] 27.8 g of crude product of alarelin peptide carrier Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(Pbf)-Pro-HZ-WSP-X was synthesized, which was a white solid with a yield of 100%.

[0096] Cleavage reaction of alarelin peptide carrier:

[0097] Prepare a cleavage solution of TFA:TIS:EDT:H2O = 85:5:5:5 and pre-cool it to -5 - 5 °C. Add 280 mL of cold cleavage solution and 28.2 g (10.0 mmol) of alarelin peptide carrier Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(Pbf)-Pro-HZ-WSP-X to a 500 mL cleavage flask, and heat it in an oil bath to T 内 = 30 - 35 °C and cleave for 2 - 3 h. After the cleavage is completed, add the cleavage solution to 2800 mL of cold methyl tert-butyl ether, centrifuge, wash, and dry to obtain 12.1 g of crude product of alarelin (Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt). The crude product synthesis yield is 103.7%, and the crude product HPLC purity is 93.28%. The HPLC chromatogram of the crude alarelin product is shown in the appendix Figure 3 .

[0098] Purification of crude alarelin:

[0099] 12.1 g of crude alarelin (Pyr-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt) was purified by reverse preparative chromatography, followed by salt conversion, concentration, and freeze-drying to obtain 7.98 g of high-quality alarelin acetate, which is a white powder; HPLC purity ≥ 99.0%, peptide content 88.4%, and total yield 60.4%. The LC-MS spectrum of high-quality alarelin acetate is shown in the appendix Figure 4 .

Claims

1. A novel non-classical solid-phase synthesis carrier, characterized in that, The structure of the carrier is represented by the following formula: wherein, R1 is methyl or ethyl; R2 is 2. The non-classical solid-phase synthesis support according to claim 1, wherein: The carrier contains 1, 2 or 3 side chains, and the side chains are located at the ortho, meta or para positions of the functional group.

3. The non-classical solid-phase synthesis carrier according to claim 1, wherein the preparation process comprises the following steps: A. In a reaction medium, hydroxybenzaldehyde, haloalkane, a base and a catalyst are added, and the reaction is carried out by heating. After the reaction is complete, the reaction solution is spread into a poor solvent to obtain a benzaldehyde intermediate containing a long-chain alkyl ether side chain; B. The benzaldehyde intermediate containing a long-chain alkyl ether side chain is added to the reaction medium, methylamine hydrochloride or ethylamine hydrochloride is added, and a reducing agent is added. After the reaction is complete, a poor solvent is added to the reaction solution to obtain the target non-classical solid-phase synthesis carrier.

4. The method for preparing a non-classical solid-phase synthesis carrier according to claim 3, wherein: In the step A, the reaction medium is one or any combination of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone; the haloalkane is chloroalkane, bromoalkane or iodoalkane; the base is one or any combination of potassium carbonate, sodium carbonate, sodium hydroxide and potassium hydroxide; the catalyst is selected from potassium iodide; the reaction temperature is 50-100 °C; the poor solvent is one or any combination of water, methanol, ethanol, n-hexane, n-heptane and acetonitrile.

5. The method for preparing a non-classical solid-phase synthesis carrier according to claim 3, wherein: In the step B, the reaction medium is one or any combination of tetrahydrofuran, methanol or ethanol; the reducing agent is one or any combination of sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride; the reaction temperature is 0-30 °C; the poor solvent is one or any combination of water, n-hexane, n-heptane or acetonitrile.

6. The non-classical solid-phase synthesis carrier according to claim 1, wherein its application feature is that the novel non-classical solid-phase synthesis carrier is applied to the synthesis of polypeptide drugs.

7. According to the application described in claim 6, in the synthesis application of polypeptide drugs, its features include the following steps: A. Amino acid coupling reaction: In a reaction medium, the non-classical solid-phase synthesis carrier and Fmoc-amino acid are coupled under the action of a condensing agent, and a detection method is used to determine the end point of the reaction; B. Post-treatment of amino acid coupling: After the amino acid coupling reaction is completed, the reaction solution is spread into a poor solvent to obtain an Fmoc-peptide carrier; C. Fmoc protecting group removal reaction: The Fmoc-peptide carrier obtained in step (A) is added to a dichloromethane solution of diethylamine with a volume concentration of 20%, and the deprotection reaction is carried out at 20-50 °C to obtain an extended peptide carrier; D. Repeat steps (A) and (B) until all amino acids in the polypeptide substance are connected to obtain a polypeptide substance carrier; E. The polypeptide substance carrier obtained in step (C) is added to a cleavage solution for cleavage to obtain a crude product of a C-terminal amidated modified polypeptide drug; F. The polypeptide crude product is analyzed and purified and separated from the target peptide by reverse-phase liquid chromatography.

8. The application according to claim 7, wherein: In the step A, the reaction medium is one or any combination of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide; the reaction temperature is 20 to 50 °C; the detection method for the amino acid coupling reaction to determine the end point is selected from HPLC, TLC or NT; the condensation reagent is selected from any combination of DIC / DCC / EDCI+HOOBT / HOBT / HOAT+DIPEA / NMM+DMAP / HBTU / HATU / TBTU / PyBOP.

9. The application according to claim 7, characterized in that: In the step A, the feeding ratio of the coupling reaction is carrier:Fmoc-amino acid:condensation reagent = 1:1:1 to 1:3:

3.

10. The application according to claim 7, characterized in that: In the step B, the poor solvent is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate or acetonitrile.

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