A green chemical synthesis method of desipramine

Abstract

The present application belongs to the technical field of polypeptide synthesis, and particularly relates to a green chemical synthesis method of diferoline. The method is based on solid-phase synthesis, and uses a mixed solvent of 2-Me-THF / DMSO or a mixed solvent of DOL / DMSO as a synthesis solvent for a synthesis reaction, then uses a resin as a solid-phase carrier, sequentially couples amino acids to obtain a fully-protected polypeptide resin, and then acidolyzes the fully-protected polypeptide resin to obtain diferoline. The method does not need to use DMF, the synthesis solvent used is a green solvent, and thus has the advantage of small environmental pollution; and the synthesis solvent has a low boiling point and is easy to recycle, and thus the post-processing in the synthesis reaction is simple. The diferoline synthesized by the method has high yield and purity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide synthesis, and particularly relates to a green chemical synthesis method of difelikefalin. BACKGROUND

[0002] Difelikefalin is a kind of intravenous injection drug, which is a peripheral selective kappa opioid receptor (KOR) agonist. The compound is a polypeptide composed of five amino acids, and belongs to a peptide amide compound.

[0003] At present, the methods for synthesizing difelikefalin include solid-phase synthesis and liquid-phase synthesis.

[0004] Patent CN101627049B, US7402564B1 and US2009075907A1 report a solid-phase synthesis method of difelikefalin. In the method, CTC resin is used as a solid-phase carrier, and Fmoc-Pip(Boc)-OH, Fmoc-D-Lys(Dde)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH and Boc-D-Phe-OH are sequentially coupled on the resin. In the synthesis process, 25% piperidine / DMF is used to remove the Fmoc protecting group, HOBt / DIC is used for coupling, and DMF is used to wash the resin. After all the amino acid coupling is completed, 4% hydrazine / DMF is used to remove the Dde protecting group to obtain a fully protected peptide resin. Then, the fully protected peptide resin is treated by acid cleavage to obtain a crude difelikefalin peptide. In the solid-phase synthesis method, a large amount of DMF is required for deprotection, coupling and resin washing, which will produce a large amount of DMF waste liquid and cause environmental pollution.

[0005] Patent CN116239651A reports a liquid-phase synthesis method of difelikefalin. In the method, Boc-D-Phe-D-Phe-OH dipeptide fragment and NH2-D-Leu-D-Lys(Boc)-Pip(Boc)-OMe tripeptide fragment are first synthesized by a liquid-phase method, and then the two polypeptide fragments are coupled by a liquid-phase method. Subsequently, the difelikefalin crude peptide is obtained by saponification and acid cleavage. In the liquid-phase synthesis, the post-reaction treatment is complicated, the synthesis cycle is long, and the saponification reaction condition is prone to produce racemic impurities. SUMMARY

[0006] To solve the above technical problems, the application specifically provides the following technical scheme:

[0007] The purpose of the application is to provide a green chemical synthesis method of difelikefalin, which comprises the following steps:

[0008] 1) synthesizing Fmoc-Pip(Boc)-resin by mixing resin and Fmoc-Pip(Boc)-OH in a synthesis solvent;

[0009] 2) The Fmoc protecting group in the Fmoc-Pip(Boc)-resin is removed by using a deprotection solution prepared with a synthetic solvent to obtain a Fmoc-removed Pip(Boc)-resin;

[0010] 3) The Fmoc-D-Lys(Boc)-OH is activated with a coupling reagent in a synthetic solvent, and then the Fmoc-removed Pip(Boc)-resin is added to synthesize the Fmoc-Lys(Boc)-Pip(Boc)-resin;

[0011] 4) The steps of 2) and 3) are repeated, the Fmoc group is removed from the resin obtained in the last round, and then the activated Fmoc-D-Leu-OH, the activated Fmoc-D-Phe-OH, and the activated Boc-D-Phe-OH are sequentially coupled to finally synthesize the Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin;

[0012] 5) The Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin is cleaved using a cleavage solution to obtain H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH;

[0013] Preferably, the synthetic solvent includes at least one of 2-Me-THF, DMSO, DOL, and DMSO.

[0014] Preferably, the synthetic solvent includes a mixed solvent of 2-Me-THF / DMSO or a mixed solvent of DOL / DMSO.

[0015] Preferably, in the synthetic solvent, 2-Me-THF and DMSO are used in a volume ratio of 7:1-3; and DOL and DMSO are used in a volume ratio of 7:1-3.

[0016] Preferably, the deprotection solution includes piperidine and a synthetic solvent, and the piperidine and the synthetic solvent are mixed in a volume ratio of 1:1-5.

[0017] Preferably, the coupling reagent is a mixture of DIC and a condensing agent, or a mixture of an additive and a base.

[0018] Preferably, the condensing agent includes at least one of Oxymapure, HOBt, and HOAt.

[0019] Preferably, the additive includes at least one of PyAop, PyBop, HBTU, and HATU.

[0020] Preferably, the base includes at least one of diisopropylethylamine and N-methylmorpholine.

[0021] Preferably, the cleavage solution includes TFA, TIS and H2O, TFA and TIS are used in a volume ratio of 95:1-3; TFA and H2O are used in a volume ratio of 95:1-3.

[0022] Preferably, the crude peptide product of Diphemide is H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH.

[0023] Preferably, the crude peptide product of Diphemide is purified by reverse phase preparation and freeze-dried to obtain finished Diphemide.

[0024] Preferably, the resin includes CTC resin and silicon-containing resin.

[0025] Further preferably, in step 1), 1-3 equivalents of blank resin and 1-3 equivalents of Fmoc-Pip(Boc)-OH are mixed, then 1-3 equivalents of DIPEA are slowly added in a ratio of 1g:1-5mL of the mass of 1-3 equivalents of blank resin and synthesis solvent, followed by reaction at 10-40℃ for 2-4h, then 1-3 equivalents of methanol are added in a ratio of 1g:1-5mL of the mass of 1-3 equivalents of blank resin and methanol, and reacted for 25-35min to obtain Fmoc-Pip(Boc)-resin. The synthesis solvent is a mixed solvent of 2-Me-THF / DMSO, wherein 2-Me-THF and DMSO in the 2-Me-THF / DMSO mixed solvent are mixed in a volume ratio of 7:1-3. In the present application, 1 equivalent of blank resin = the mass of blank resin x the degree of substitution of blank resin.

[0026] Further preferably, in step 2), a deprotection solution equivalent to 1-3 equivalents of blank resin in volume is added to the Fmoc-Pip(Boc)-resin, stirred and reacted for 9-11min, and the deprotection solution is removed; a deprotection solution equivalent to 1-3 equivalents of blank resin in volume is added, and reacted for 19-21min under nitrogen, and the deprotection solution is removed; then a synthesis solvent equivalent to 1-3 equivalents of blank resin in volume is added, stirred and washed for 2-3min, the synthesis solvent is removed, and the washing is repeated 8-10 times to obtain Fmoc-removed Pip(Boc)-resin. The deprotection solution is a mixed solvent of PIP / synthesis solvent, wherein PIP and synthesis solvent in the PIP / synthesis solvent mixed solvent are mixed in a volume ratio of 1:1-5.

[0027] Further preferably, in step 3), 1-3 equivalents of Fmoc-D-Lys(Boc)-OH and 1-3 equivalents of HOBT are weighed and mixed, and then placed in an ice water bath, 1-3 equivalents of the mass of the blank resin and 1-5 mL of the synthetic solvent are added to the synthetic solvent in a ratio of 1 g:1-5 mL, and stirred to dissolve; 1-3 equivalents of DIC are slowly added at 0-10°C, activated for 10-30 min to obtain an activated reactant; the activated reactant and the Fmoc-removed Pip(Boc)-resin are mixed, and reacted at 20-40°C for 1.5-3.0 h, then 1-3 equivalents of the volume of the blank resin are added to the synthetic solvent, stirred for 2-3 min, the synthetic solvent is removed, and the washing is repeated 1-3 times.

[0028] Further preferably, in step 4), according to steps 2) and 3), the Fmoc protecting group of the Fmoc-Lys(Boc)-Pip(Boc)-resin is removed, 1-3 equivalents of Fmoc-D-Leu-OH are weighed and activated, then the Fmoc-removed Lys(Boc)-Pip(Boc)-resin and the activated Fmoc-D-Leu-OH are mixed to synthesize Fmoc-D-Leu-Lys(Boc)-Pip(Boc)-resin; the Fmoc protecting group of the Fmoc-D-Leu-Lys(Boc)-Pip(Boc)-resin is removed, 1-3 equivalents of Fmoc-D-Phe-OH are weighed and activated, then the Fmoc-removed D-Leu-Lys(Boc)-Pip(Boc)-resin and the activated Fmoc-D-Phe-OH are mixed to synthesize Fmoc-D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-resin; the Fmoc protecting group of the Fmoc-D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-resin is removed, 1-3 equivalents of Boc-D-Phe-OH are weighed and activated, then the Fmoc-removed D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-resin and the activated Boc-D-Phe-OH are mixed to synthesize Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin.

[0029] Further preferably, in step 5), the cleavage solution is prepared and stored at -10-10 ℃, Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin and the cleavage solution are mixed at a ratio of 1 g:7-20 mL, and reacted at 20-40 ℃ for 1-4 h, then the filtrate is collected by filtration, and the filter residue is washed with TFA for 1-3 times, the washing liquid and the filtrate are collected and combined, to obtain a combined liquid, the combined liquid and pre-cooled anhydrous ether are mixed at a volume ratio of 1:5-30, to obtain a mixed liquid, the temperature of the mixed liquid is controlled at -20-30 ℃, then the filter cake is collected by filtration, the filter cake is washed with 1-3 times equivalent of anhydrous ether for 1-3 times, and then transferred to a vacuum drying oven for drying until constant weight, to obtain H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH, i.e. the crude peptide product of denifelafiban. The cleavage solution comprises TFA, TIS and H2O, which are mixed at a volume ratio of 95:1-3:1-3.

[0030] In an embodiment of the present application, the preparation steps of the silicon-containing resin include the following steps:

[0031] The styrene, divinylbenzene and allyltrimethylsilane are mixed to obtain a mixture; then polyvinyl alcohol is added, and then a dibenzoyl peroxide is used as an initiator, and liquid paraffin is used as a pore-forming agent, to prepare a polystyrene resin; the polystyrene resin is reacted with 2-chlorobenzoyl chloride to obtain a 2-chlorobenzophenone resin; the 2-chlorobenzophenone resin is reacted with phenyllithium to obtain a 2-chlorotriphenylmethanol resin; and the 2-chlorotriphenylmethanol resin is reacted with trimethylchlorosilane to obtain the silicon-containing resin.

[0032] Preferably, the styrene and divinylbenzene are used at a mass ratio of 10.7:1-4.

[0033] Preferably, the styrene and divinylbenzene are used at a mass ratio of 10.7:1-4.

[0034] Preferably, the mixture and the polyvinyl alcohol are used at a ratio of 16.1 g:150-180 mL.

[0035] Preferably, the mixture and the dibenzoyl peroxide are used at a mass ratio of 16.1:0.1-0.5.

[0036] Preferably, the mixture and the liquid paraffin are used at a mass ratio of 16.1:2-5.

[0037] Preferably, the polystyrene resin and the 2-chlorobenzoyl chloride are used at a mass ratio of 4:1-2.

[0038] Preferably, the 2-chlorobenzophenone resin and the phenyllithium are used at a mass ratio of 25.5:15-25.

[0039] Preferably, 2-chlorobenzhydrol resin and trimethylchlorosilane are used in a ratio of 33.86g: 70-80mL.

[0040] More preferably, styrene, divinylbenzene, allyltrimethylsilane are mixed in a ratio of 10.7: 1-4: 1-4 by mass, to obtain a mixture; then polyvinyl alcohol is added in a ratio of 15-18g: 150-180mL of the mixture and polyvinyl alcohol, followed by adding dibenzoyl peroxide and liquid paraffin in a ratio of 15-18: 0.1-0.5: 2-5 by mass of the mixture, dibenzoyl peroxide and liquid paraffin, after stirring and dissolving, reaction is carried out at 60-70°C for 1-2h; at 70-80°C for 1-3h; at 80-90°C for 1-2h, to obtain a product; the product is extracted and purified in tetrahydrofuran for 24-48h, then transferred into N,N-dimethylformamide and shaken for 48-72h, followed by water washing and drying, to obtain polystyrene resin;

[0041] Polystyrene resin, carbon disulfide and aluminum chloride are mixed in a ratio of 20g: 150-250mL: 5-8g, heated to 40-50°C, after reaction for 30-60min, 2-chlorobenzoyl chloride is added in a ratio of 4: 1-2 by mass of polystyrene resin and 2-chlorobenzoyl chloride, refluxed for 4-6h, cooled and filtered, the filter residue 1 is collected, washed with dioxane / water / HCl mixture, N,N-dimethylformamide, dioxane / water mixture, methyl ethyl ketone, water, N,N-dimethylformamide, methyl ethyl ketone, methanol, diethyl ether in sequence, finally added with toluene, vacuum distilled for 3-5 times, to obtain 2-chlorobenzophenone resin. Dioxane / water / HCl mixture is mixed in a ratio of 5: 1-2: 1-2 by volume of dioxane: water: HCl, dioxane / water mixture is mixed in a ratio of 5: 1-2 by volume of dioxane: water.

[0042] The phenyllithium is prepared by mixing lithium and bromobenzene in a mass ratio of 3.4:30-40, adding ethyl ether in a lithium:ethyl ether ratio of 3.4g:150-250mL under an argon atmosphere; the 2-chlorobenzophenone resin is mixed with the phenyllithium in a mass ratio of 25.5:15-25, tetrahydrofuran is added in a 2-chlorobenzophenone resin: tetrahydrofuran ratio of 25.5g:150-250mL, refluxed for 1h, N,N-dimethylformamide is added in a 2-chlorobenzophenone resin:N,N-dimethylformamide ratio of 25.5g:80-120mL, the filter residue 2 is collected, washed with N,N-dimethylformamide / methanol mixture, N,N-dimethylformamide / water mixture, N,N-dimethylformamide, dichloromethane / acetic acid mixture, isopropyl alcohol, methanol and ethyl ether in sequence, and then vacuum dried to obtain the 2-chlorotriphenylmethanol resin. The N,N-dimethylformamide / methanol mixture is prepared by mixing N,N-dimethylformamide and methanol in a volume ratio of 3:1-3, the N,N-dimethylformamide / water mixture is prepared by mixing N,N-dimethylformamide and water in a volume ratio of 2:1-3, and the dichloromethane / acetic acid mixture is prepared by mixing dichloromethane and acetic acid in a volume ratio of 4:1-3.

[0043] The 2-chlorotriphenylmethanol resin, dichloromethane, chlorotrimethylsilane and DMSO are mixed in a ratio of 33.86g:650-750mL:70-80mL:2-10mL, stirred for 1-2h, filtered, the filter residue 3 is collected, washed with dichloromethane and benzene in sequence, then benzene and acetyl chloride are added in a ratio of 2-chlorotriphenylmethanol resin:benzene:acetyl chloride of 33.86g:300-400mL:50-60mL, refluxed for 30-60min, filtered, the filter residue 4 is collected, washed with toluene, dichloromethane, methyl ether, toluene and ethyl ether in sequence, and then vacuum dried to obtain the silicon-containing resin. The silicon-containing resin is used in the synthesis of dihydrexidine, and the silicon-containing resin can have better porosity, so that the synthetic substrate and the silicon-containing resin can be fully contacted, the synthesis reaction is promoted and the generation of the synthetic substrate due to the formation of hydrogen bonds is reduced, the generation of the main product of dihydrexidine is improved, and thus the purity of the crude dihydrexidine peptide product is improved.

[0044] In an embodiment of the present application, the silicon-containing resin is swelled with a swelling liquid before the Fmoc-Pip(Boc)-resin is synthesized in the green chemical synthesis of dihydrexidine.

[0045] Preferably, the swelling liquid is a synthetic solvent containing ethyl gallate.

[0046] Preferably, the swelling liquid contains 10-20%vol of ethyl gallate.

[0047] More preferably, the swelling liquid containing 10-20%vol ethyl gallate is added into the silicon-containing resin, the liquid level of the swelling liquid is higher than the top of the silicon-containing resin by 1-3cm, the swelling is carried out for 30-60min, the swelling liquid is removed, 1-3 equivalents of the blank resin and 1-3 equivalents of Fmoc-Pip(Boc)-OH are mixed, then 1-3 equivalents of the mass of the blank resin and 1-5mL of the synthetic solvent are added in the ratio of 1g:1-5mL, then 1-3 equivalents of DIPEA is slowly added, the reaction is carried out at 10-40℃ for 2-4h, then 1-3 equivalents of the mass of the blank resin and 1-5mL of methanol are added in the ratio of 1g:1-5mL, the reaction is carried out for 25-35min, and the Fmoc-Pip(Boc)-resin is obtained. The synthetic solvent is a mixed solvent of 2-Me-THF / DMSO, wherein 2-Me-THF and DMSO in the 2-Me-THF / DMSO mixed solvent are mixed in the ratio of 7:1-3 by volume. In the present application, 1 equivalent of the blank resin = the mass of the blank resin x the degree of substitution of the blank resin.

[0048] The ethyl gallate in the swelling liquid can promote the swelling of the silicon-containing resin by interacting with the silicon-containing resin, thereby further exposing the active sites of the silicon-containing resin, making the amino acid and the silicon-containing resin fully combined, thereby improving the connection rate of the silicon-containing resin and the amino acid, making the synthesis reaction of the dephenelfarin proceed smoothly and reducing the generation of the self-polymerization of the synthetic substrate, and improving the generation of the main product of the dephenelfarin, thereby improving the purity of the crude peptide product of the dephenelfarin.

[0049] The present application also provides the use of the synthetic solvent in the synthesis of polypeptides.

[0050] Preferably, the polypeptide comprises dephenelfarin.

[0051] Compared with the prior art, the present application provides a green chemical synthesis method of dephenelfarin, which uses a synthetic solvent to replace DMF, and thus has the advantage of less environmental pollution. The synthetic solvent is a mixed solvent of 2-Me-THF / DMSO or a mixed solvent of DOL / DMSO, which is a low-boiling-point component and has the advantage of easy recovery, and thus the post-processing in the synthesis reaction is simple. The present application further uses a silicon-containing resin, and swells the silicon-containing resin with a swelling liquid containing ethyl gallate. When the swollen silicon-containing resin is used for the synthesis of dephenelfarin, the silicon-containing resin itself has good pores, and the swelling liquid containing ethyl gallate promotes the swelling of the silicon-containing resin, so that the synthetic substrate has a good connection rate with the silicon-containing resin; and the synthesis reaction of dephenelfarin proceeds smoothly, and the generation of the self-polymerization of the synthetic substrate is reduced, the generation of the main product of dephenelfarin is improved, thereby improving the purity of the crude peptide product of dephenelfarin. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1Infrared spectrum of the silicon-containing resin. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present application will be apparently and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative work fall within the protection scope of the present application.

[0054] The experimental methods in the following embodiments are all conventional methods or the conditions suggested by manufacturers, unless otherwise specified. The materials, reagents and the like used in the following embodiments can be obtained from commercial channels, unless otherwise specified.

[0055] Table 1 English abbreviation explanation

[0056]

[0057] The method of the present application is based on solid-phase synthesis, and uses a mixed solvent of 2-Me-THF / DMSO or a mixed solvent of DOL / DMSO as a synthesis solvent for the synthesis reaction, and then uses CTC resin as a solid-phase carrier to sequentially couple the synthesis substrates to obtain a fully-protected polypeptide resin, i.e. Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-CTC resin, and then performs acid cleavage on the fully-protected polypeptide resin to obtain denifelafiban. It should be noted that the above steps obtain a crude polypeptide product of denifelafiban, and the crude polypeptide product can be subjected to reverse-phase preparation purification and lyophilization to obtain a finished product of denifelafiban. The synthesis substrates include Fmoc-Pip(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and Boc-D-Phe-OH.

[0058] Example 1: Synthesis of denifelafiban

[0059] The synthesis of denifelafiban includes the following steps:

[0060] 1) Synthesis of Fmoc-Pip(Boc)-CTC resin. Take 1 equivalent of blank CTC resin, add 2 equivalents of Fmoc-Pip(Boc)-OH, then add synthesis solvent at a ratio of 1 mL:1 g of the mass of 1 equivalent of blank CTC resin, slowly add 1 equivalent of DIPEA, react at 20°C for 2 h, and finally add methanol at a ratio of 1 g:1 mL of the mass of 1 equivalent of blank CTC resin, continue to react for 30 min to obtain Fmoc-Pip(Boc)-CTC resin. The synthesis solvent is a mixed solvent of 2-Me-THF / DMSO, wherein 2-Me-THF and DMSO in the 2-Me-THF / DMSO mixed solvent are mixed at a volume ratio of 7:3. In the present application, 1 equivalent of CTC resin = the mass of blank CTC resin x the degree of substitution of blank CTC resin.

[0061] 2) Removal of Fmoc protecting group in Fmoc-Pip(Boc)-CTC resin. Add a deprotection solution equivalent to the volume of 1 equivalent of blank CTC resin in Fmoc-Pip(Boc)-CTC resin, stir for 10 min, remove the deprotection solution, add the same amount of deprotection solution, and react under nitrogen for 20 min, remove the deprotection solution, then add synthesis solvent equivalent to the volume of 1 equivalent of blank CTC resin, stir for 3 min, remove the synthesis solvent, and repeat the washing 8 times to obtain Pip(Boc)-CTC resin with Fmoc removed. The deprotection solution is a mixed solvent of PIP / synthesis solvent, wherein PIP and synthesis solvent in the PIP / synthesis solvent mixed solvent are mixed at a volume ratio of 1:4.

[0062] 3) Synthesis of Fmoc-Lys(Boc)-Pip(Boc)-CTC resin. Take 2.5 equivalents of Fmoc-D-Lys(Boc)-OH and 2.5 equivalents of HOBT and mix, then place in an ice water bath, add synthesis solvent at a ratio of 1 mL:1 g of the mass of 1 equivalent of blank CTC resin, and stir to dissolve, slowly add 2.5 equivalents of DIC at 0°C, activate for 30 min to obtain the activated reactant, mix the activated reactant and Pip(Boc)-CTC resin with Fmoc removed, react at 20°C for 2 h, then add synthesis solvent equivalent to the volume of 1 equivalent of blank CTC resin, stir for 3 min, remove the synthesis solvent, and repeat the washing 3 times to obtain Fmoc-Lys(Boc)-Pip(Boc)-CTC resin.

[0063] 4) Synthesis of Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-CTC resin. According to steps 2) and 3), remove the Fmoc protecting group of Fmoc-D-Leu-Lys(Boc)- Pip(Boc)-CTC resin, activate 2.5 equivalents of Fmoc-D-Phe-OH, then mix the Fmoc-removed D-Leu-Lys(Boc)-Pip(Boc)-CTC resin and the activated Fmoc-D-Phe-OH, to synthesize Fmoc-D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-CTC resin; remove the Fmoc protecting group of Fmoc-D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-CTC resin, activate 2.5 equivalents of Boc-D-Phe-OH, then mix the Fmoc-removed D-Phe-D-Leu-Lys(Boc)-Pip(Boc)-CTC resin and the activated Boc-D-Phe-OH, to synthesize Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-CTC resin.

[0064] 5) Synthesis of H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH. Prepare a cleavage solution with TFA: Tis: H2O in a volume ratio of 95:2.5:2.5, then control the temperature of the cleavage solution to be stable at 10°C. Mix Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-CTC resin and the cleavage solution in a ratio of 1 g: 15 mL, react at 20°C for 2 h, then filter to collect the filtrate, wash the filter cake with TFA twice, collect the washing liquid, combine the filtrate and the washing liquid to obtain a combined liquid, mix the combined liquid and pre-cooled anhydrous ether in a volume ratio of 10:1 to obtain a mixed liquid, control the temperature of the mixed liquid at -20°C, filter the mixed liquid to collect the filter cake, wash the filter cake with 1 equivalent of ether based on the volume of the filter cake, repeat the washing for 3 times, then transfer to a vacuum drying oven, dry to constant weight, to obtain H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH, which is the crude peptide product of difelikefalin.

[0065] Example 2: Synthesis of difelikefalin

[0066] Example 2 differs from Example 1 in that a silicon-containing resin is used in place of the CTC resin in Example 2.

[0067] The silicon-containing resin is prepared by the following steps:

[0068] 1) Polystyrene resin preparation. 10.7 g of styrene, 3.2 g of divinylbenzene, 1.2 g of allyltrimethylsilane and 0.3 g of dibenzoyl peroxide are mixed, then 2 g of liquid paraffin and 160 mL of polyvinyl alcohol are added, after stirring and dissolving, reaction is carried out at 65°C for 1 h, then the temperature is raised to 75°C for 3 h, followed by reaction at 90°C for 2 h, the product is obtained; the product is extracted and purified in tetrahydrofuran for 24 h, then transferred into 50 mL of N,N-dimethylformamide and shaken for 48 h, then washed with water, and dried to obtain the polystyrene resin.

[0069] 2) Silicon-containing resin preparation. 20 g of polystyrene resin is weighed, 200 mL of carbon disulfide is added, then 6.53 g of aluminum chloride is added, the temperature is raised to 45°C, and after reaction for 30 min, 8.75 g of 2-chlorobenzoyl chloride is added, refluxed for 4 h, then cooled, filtered, and the filter residue 1 is collected, then sequentially washed with dioxane / water / HCl mixture, N,N-dimethylformamide, dioxane / water mixture, methyl ethyl ketone, water, N,N-dimethylformamide, methyl ethyl ketone, methanol, ether, and finally with toluene, vacuum distilled for 3 times to obtain the 2-chlorobenzophenone resin. The dioxane / water / HCl mixture is mixed in a volume ratio of 5:1:1, and the dioxane / water mixture is mixed in a volume ratio of 5:1.

[0070] 3.4 g of lithium and 39 g of bromobenzene are mixed, 200 mL of ether is added, and phenyllithium is prepared under argon atmosphere, then 200 mL of tetrahydrofuran and 25.5 g of 2-chlorobenzophenone resin are added, refluxed for 1 h, 100 mL of N,N-dimethylformamide is added, then filtered, and the filter residue 2 is collected, sequentially washed with N,N-dimethylformamide / methanol mixture, N,N-dimethylformamide / water mixture, N,N-dimethylformamide, dichloromethane / acetic acid mixture, isopropyl alcohol, methanol and ether, and then vacuum dried to obtain the 2-chlorobenzhydrol resin. The N,N-dimethylformamide / methanol mixture is mixed in a volume ratio of 3:1, the N,N-dimethylformamide / water mixture is mixed in a volume ratio of 2:1, and the dichloromethane / acetic acid mixture is mixed in a volume ratio of 4:1.

[0071] To 33.86 g of 2-chlorotrityl alcohol resin was added 700 mL of dichloromethane, 70 mL of trimethylsilyl chloride and 7 mL of DMSO, stirred for 90 min, filtered, collected the residue 3, washed with dichloromethane and benzene in turn, then added 350 mL of benzene and 56 mL of acetyl chloride, refluxed for 30 min, filtered, collected the residue 4, washed with toluene, dichloromethane, methyl ether, toluene and diethyl ether in turn, and dried in vacuum to obtain the silicon-containing resin.

[0072] Example 3: Synthesis of denifelafiban

[0073] Example 3 is different from Example 2 in that the amount of allyl trimethylsilane used in the preparation of the silicon-containing resin is 3.2 g.

[0074] Polystyrene resin preparation. 10.7 g of styrene, 3.2 g of divinylbenzene, 3.2 g of allyl trimethylsilane and 0.3 g of dibenzoyl peroxide were mixed, then 2 g of liquid paraffin and 160 mL of polyvinyl alcohol were added, stirred and dissolved, then reacted at 65°C for 1 h, the temperature was raised to 75°C and reacted for 3 h, then reacted at 90°C for 2 h to obtain the product; the product was extracted and purified in tetrahydrofuran for 24 h, then transferred into 50 mL of N,N-dimethylformamide and shaken for 48 h, then washed with water and dried to obtain the polystyrene resin. The steps for preparing the silicon-containing resin from the polystyrene resin are the same as those in step 2) of Example 2.

[0075] Example 4: Synthesis of denifelafiban

[0076] Example 4 is different from Example 3 in that the silicon-containing resin is first swelled with a swelling liquid before the synthesis of denifelafiban in Example 4. The swelling liquid is a synthetic solvent containing 10%vol of ethyl gallate.

[0077] One equivalent of the silicon-containing resin was added to the swelling liquid, so that the liquid level of the swelling liquid was 3 cm higher than the top of the silicon-containing resin. After swelling for 30 min, the swelling liquid was removed, and then the synthesis of denifelafiban was carried out according to steps 1) to 5) of Example 3. The swelling liquid is a synthetic solvent containing 10%vol of ethyl gallate.

[0078] Example 5: Synthesis of denifelafiban

[0079] Example 5 is different from Example 4 in that the swelling liquid in Example 5 is a synthetic solvent containing 20%vol of ethyl gallate.

[0080] Comparative Example 1: Synthesis of denifelafiban

[0081] Comparative Example 1 is different from Example 1 in that DMF is used to replace the synthetic solvent in Comparative Example 1.

[0082] Comparative Example 2: Synthesis of Difenofylline

[0083] Comparative Example 2 and Example 1 are different in that, in the synthesis of difenofylline, the silicon-containing resin is first swelled with a swelling solution, and then the synthesis of difenofylline is carried out as in Example 1. The swelling solution is a synthetic solvent containing 20%vol ethyl gallate.

[0084] Take 1 equivalent of blank CTC resin, add a swelling solution, so that the liquid level of the swelling solution is higher than the top of the blank CTC resin by 3CM, swell for 30min, then remove the swelling solution, and then carry out the synthesis of difenofylline as in steps 1) -5) of Example 1. The swelling solution is a synthetic solvent containing 20%vol ethyl gallate.

[0085] Test Example 1: Infrared spectrum of silicon-containing resin

[0086] The results are shown in Table 1. Figure 1 As can be seen, the C-H stretching vibration peak of the benzene ring appears at 3022cm -1 -1, the C-H stretching vibration peak of CH3appears at 2919cm -1 -1, the Si-H stretching vibration peak appears at 2130cm -1 -1, the Si-C stretching vibration peak appears at 1129cm -1 -1.

[0087] Test Example 2: Coupling rate and purity of difenofylline

[0088] 1) The coupling rate of the resin and the first amino acid is determined by colorimetry.

[0089] The colorimetry operation steps are as follows:

[0090] Take 10mg 2,4,6-trinitrobenzenesulfonic acid and add 1mL dimethylformamide solution to stir and dissolve to obtain a detection reagent. Weigh 2mg Fmoc-Pip(Boc)-CTC resin, add 0.3mL glacial acetic acid and 1mL methanol for washing; then add 1mL methanol for washing, repeat washing 3 times; add 0.25mL detection reagent, and prepare CTC resin without coupling to add detection reagent as a blank; then boil in a water bath for 5min, take out and add anhydrous ethanol to 3mL, adjust to zero with anhydrous ethanol, and detect the absorbance at 570nm, and calculate the coupling rate.

[0091] -NH2(mmol / g)= (OD sample-OD blank) x 3mL x 10 6 / (15000 x 2mg)

[0092] Coupling rate = 1- (-NH2(mmol / g) / (1000 x resin loading))

[0093] 2) The dried crude dihyflofumine peptide product was weighed and then the purity of dihyflofumine was determined by HPLC.

[0094] The results are shown in Table 2. Compared with Example 1 and Comparative Example 1, it can be seen that the coupling rate of Example 1 and Comparative Example 1 is close, but the purity of Example 1 is better, which indicates that the synthetic solvent of the present application can improve the purity of the crude dihyflofumine peptide product when used in the synthesis of dihyflofumine. It is possible that the synthetic solvent of the present application can promote the dissolution of impurities during the synthesis and promote the generation of the main product dihyflofumine, thereby improving the purity of the crude dihyflofumine peptide product.

[0095] Example 1 uses CTC resin for the synthesis of dihyflofumine, while Examples 2 and 3 use silica-containing resin for the synthesis of dihyflofumine. Compared with Examples 1-3, it can be seen that although the use of silica-containing resin does not significantly improve the coupling rate of the resin and amino acids, it improves the purity of the synthesized dihyflofumine crude peptide product. When silica-containing resin is used in the synthesis of dihyflofumine, it is possible that the silica-containing resin has better pores, allowing the synthetic substrate and the silica-containing resin to fully contact, promoting the synthesis reaction and reducing the aggregation of the synthetic substrate due to the formation of hydrogen bonds, thereby improving the generation of the main product dihyflofumine and improving the purity of the crude dihyflofumine peptide product.

[0096] Comparing Examples 3-5, it can be seen that swelling the silica-containing resin with the swelling solution can better improve the coupling rate of the resin and amino acids and the purity of the dihyflofumine crude peptide product. The swelling solution is the synthetic solvent containing ethyl gallate. It is possible that the ethyl gallate in the swelling solution interacts with the silica-containing resin, promoting the swelling of the silica-containing resin and further exposing the active sites of the silica-containing resin, allowing the amino acids and the silica-containing resin to fully combine, thereby improving the coupling rate of the silica-containing resin and the amino acids, allowing the synthesis reaction of dihyflofumine to proceed smoothly and reducing the aggregation of the synthetic substrate, improving the generation of the main product dihyflofumine, and thereby improving the purity of the crude dihyflofumine peptide product. Compared with Example 1 and Comparative Example 2, it can be seen that swelling the CTC resin does not significantly affect the coupling rate and purity, and it is possible that the swelling performance of the CTC resin is general and does not need to be swollen in advance. The CTC resin gradually swells and exposes the active sites during the synthesis reaction, allowing the amino acids to combine and allowing the synthesis reaction to proceed smoothly. The silica-containing resin has good swelling performance and needs to be swollen in advance to fully expose the active sites, thereby improving the coupling rate of the resin and the amino acids and the purity of the dihyflofumine crude peptide product.

[0097] Table 2 Coupling rate of resin and amino acids and purity of dihyflofumine

[0098]

[0099] The synthetic solvent in the embodiments of the present application can also be a mixed solvent of DOL / DMSO, wherein DOL and DMSO in the mixed solvent of DOL / DMSO are mixed in a volume ratio of 7:3.

[0100] Those skilled in the art can make improvements to the process parameters including the selection of mixed solvents and condensation conditions based on the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method of the present application has been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology. It should be pointed out that for ordinary skilled in the art, under the premise of not departing from the principles of the present application, some improvements and modifications can also be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A green chemical synthesis method of dephenylline, comprising the following steps: 1) synthesizing Fmoc-Pip(Boc)-resin from resin and Fmoc-Pip(Boc)-OH in a synthesis solvent; 2) removing the Fmoc protecting group in the Fmoc-Pip(Boc)-resin by using a deprotection solution prepared in the synthesis solvent to obtain Fmoc-removed Pip(Boc)-resin; 3) activating Fmoc-D-Lys(Boc)-OH in the synthesis solvent by using a coupling reagent, and then adding the Fmoc-removed Pip(Boc)-resin to synthesize Fmoc-Lys(Boc)-Pip(Boc)-resin; 4) repeating the operation steps of steps 2) and 3), removing the Fomc group from the resin obtained in the last round, and then sequentially coupling Fmoc-D-Leu-OH, Fmoc-D-Phe-OH and Boc-D-Phe-OH to finally synthesize Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin; 5) cutting Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Pip(Boc)-resin by using a cutting solution to obtain H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH; The synthesis solvent comprises a mixed solvent of 2-Me-THF / DMSO or a mixed solvent of DOL / DMSO, and 2-Me-THF and DMSO are used in a volume ratio of 7:1-3; and DOL and DMSO are used in a volume ratio of 7:1-3; The H-D-Phe-D-Phe-D-Leu-D-Lys-Pip-OH is a crude peptide product of dephenylline; the resin is a silicon-containing resin; when the silicon-containing resin is applied to the synthesis of dephenylline, the silicon-containing resin is first swelled by using a swelling liquid, and the swelling liquid is a synthesis solvent containing 10-20%vol ethyl gallate; The preparation steps of the silicon-containing resin comprise the following steps: stirring styrene, divinylbenzene and allyltrimethylsilane to obtain a mixture; then adding polyvinyl alcohol, and then using dibenzoyl peroxide as an initiator and liquid paraffin as a pore-forming agent to prepare polystyrene resin; reacting the polystyrene resin with 2-chlorobenzoyl chloride to obtain 2-chlorobenzophenone resin; reacting the 2-chlorobenzophenone resin with phenyllithium to obtain 2-chlorotrityl alcohol resin; and reacting the 2-chlorotrityl alcohol resin with trimethylsilyl chloride to obtain the silicon-containing resin; The styrene and divinylbenzene are used in a mass ratio of 10.7:1-4, and the styrene and allyltrimethylsilane are used in a mass ratio of 10.7:1-4; the polystyrene resin and 2-chlorobenzoyl chloride are used in a mass ratio of 20:5-10; the 2-chlorobenzophenone resin and phenyllithium are used in a mass ratio of 25.5:15-25; and the 2-chlorotrityl alcohol resin and trimethylsilyl chloride are used in a ratio of 33.86g:70-80mL.

2. The green chemical synthesis process as claimed in claim 1, wherein, The deprotection solution comprises piperidine and a synthesis solvent, and the piperidine and the synthesis solvent are mixed in a volume ratio of 1:1-5.

3. The green chemical synthesis process as claimed in claim 1, wherein, The coupling reagent is a mixture of DIC and a condensing agent, or a mixture of an additive and a base; the condensing agent comprises at least one of oxymapure, HOBt, HOAt; the additive comprises at least one of PyAop, PyBop, HBTU, HATU; and the base comprises at least one of diisopropylethylamine and N-methylmorpholine.

4. The green chemical synthesis process as claimed in claim 1, wherein, The cleavage solution comprises TFA, TIS and H2O, and the TFA and the TIS are mixed in a volume ratio of 95:1-3; and the TFA and the H2O are mixed in a volume ratio of 95:1-3.

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