WO2018184316A1 - Nano-nemifitide liquid phase preparation method - Google Patents

Abstract

Provided is a nano-nemifitide liquid phase preparation method comprising the following steps: 1) preparing intermediate 1H-Trp(Boc)-NH-Dpm with Dpm-NH2 and Fmoc-Trp(Boc)-OH; 2) preparing intermediate 2H-Gly-Trp(Boc)-NH-Dpm; 3) preparing intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm; 4) preparing intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm; 5) preparing intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm; 6) preparing a crude product of nano-nemifitide; and 7) preparing nano-nemifitide by reversed phase chromatography.

Description

Nano non-peptide liquid preparation method Technical field

The invention relates to the field of medicine, in particular to a method for preparing a liquid phase of nano-nonpeptide.

Background technique

Single-phase depression is a common mental disorder caused by changes in chemical substances in the central nervous system and belongs to endogenous depression. The use of antidepressants to treat unipolar depression has been widely recognized in the medical community. Nano-peptide is an analog of the brain endogenous polypeptide MIF-1, which can exert anti-monophasic depression by 5-HT reuptake, which greatly changes the reuptake of 5-HT. Pass the blood-brain barrier and clear it slowly in the brain. At present, the US company InnapMarma is conducting Phase III clinical trials, and its antidepressant activity and safety have been clinically confirmed.

Nano-peptide, English name: nemifitide; molecular formula: C ₃₃ H ₄₃ FN ₁₀ O ₆ ; CAS: 173240-15-8; is a synthetic pentapeptide compound, the structure is 4-fluoro-L-phenylalanine Acyl-(trans) 4-hydroxy-L-prolyl-L-arginyl-glycyl-L-tryptophan, see below:

At present, there are few reports on the synthesis of nano-peptides at home and abroad. The original research patent reported a nanophase non-peptide solid phase synthesis method (US5589460, WO1995030430A1), which uses Fmoc-protected amino acids as the reaction raw materials, using amino resin in turn. Coupling, the crude peptide obtained after cleavage was purified by HPLC to obtain a product. According to the research of the present inventors, the original patented method can obtain qualified products, and the total yield is only 40%. The main reasons for the analysis are as follows: the crude peptide product after cleavage has low purity, and the fragment lacks peptide impurities, so the purification yield is not high and is not suitable for large-scale production.

Summary of the invention

The object of the present invention is to provide a liquid phase preparation method of nano-peptide, which has mild synthesis conditions, simple process and stable process, and is suitable for industrial continuous production.

The synthetic route map is as follows:

The preparation method of the nano-peptide of the invention comprises the following steps:

1) Preparation of intermediate 1H-Trp(Boc)-NH-Dpm with Dpm-NH ₂ and Fmoc-Trp(Boc)-OH;

2) Preparation of intermediate 2H-Gly-Trp(Boc)-NH-Dpm;

3) Preparation of intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

4) Preparation of intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

5) Preparation of intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

6) preparing a crude nanopeptide;

7) Reverse phase chromatography to prepare nanopeptide.

Specifically, an aspect of the present invention provides a method for preparing a liquid phase of nano-nonpeptide, which comprises the following steps:

1) Fmoc-Trp(Boc)-NH-Dpm was synthesized by Dpm-NH ₂ and Fmoc-Trp(Boc)-OH under the action of a condensing agent, and the Fmoc protecting group was removed to obtain the intermediate 1H-Trp(Boc)- NH-Dpm;

2) The intermediates 1H-Trp(Boc)-NH-Dpm and Fmoc-Gly-OH were used to synthesize Fmoc-Gly-Trp(Boc)-NH-Dpm under the action of a condensing agent, and the Fmoc protecting group was removed to obtain an intermediate. 2H-Gly-Trp(Boc)-NH-Dpm;

3) Synthesis of Fmoc-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 2 and Fmoc-Arg(Pbf)-OH under the action of a condensing agent, removing the Fmoc protecting group to obtain intermediate 3H -Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

4) Synthesis of Fmoc-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 3 and Fmoc-4-hydropro(OtBu)-OH under the action of a condensing agent In addition to the Fmoc protecting group, the intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm is obtained;

5) Synthesis of Fmoc-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 4 and Fmoc-4-Fphe-OH under the action of a condensing agent, Removal of the Fmoc protecting group to obtain the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

6) Removal of OtBu, Pbf and Boc protecting groups.

In the technical solution of the present invention, the condensing agent is selected from one or more of HOBt, EDC.HCl, HATU, DCC, DIC, HBTU, HOAt, HOBt, PyBOP, BTC, preferably HOBt and EDC.HCl The combination.

In the technical solution of the present invention, the reagent for removing the Fmoc protecting group is a mixture of DBU and diethylamine.

In the technical solution of the present invention, in the step 6), the deprotecting reagent is a mixture of TFA, H ₂ O, EDT, PhOMe, thioanisole; preferably, TFA: H ₂ O: EDT: PhOMe = 90 -95: 1-5: 1-5: 3-8, more preferably TFA: H ₂ O: EDT: PhOMe = 92: 2: 2: 4.

In the technical solution of the present invention, the method further comprises a purification step, wherein the purification step is 7),

7) Purification by reverse phase chromatography. Preferably, reverse phase high pressure liquid chromatography is carried out by using reverse phase octadecylsilane as a stationary phase and 0.1% aqueous acetic acid/acetonitrile as a mobile phase to collect a peak fraction of interest and lyophilizing. .

In a solution of the invention, the solvent in steps 1) to 5) is independently selected from one or more of dichloromethane, chloroform, toluene, preferably chloroform.

In the technical solution of the present invention, in the steps 1) to 5), the Fmoc protecting group is subjected to crystallization, filtration and washing before removal of the Fmoc protecting group, and the Fmoc protecting group is removed, followed by crystallization, filtration and washing with acetonitrile.

In the technical solution of the present invention, in step 6), after removing the protecting group, crystallization, centrifugation and washing are carried out with methanol.

In the technical solution of the present invention, the liquid non-peptide liquid preparation method comprises the following steps:

1) Dpm-NH ₂ and Fmoc-Trp(Boc)-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl is added, the reaction is completed to complete, the solvent is removed, methanol is added, the solid is precipitated, and the solid is washed with methanol and dissolved. Chloroform, DBU, diethylamine was added in sequence, the reaction was completed, the solvent was removed, acetonitrile was added to the residue, and the solid was precipitated, filtered, washed with acetonitrile and dried to give intermediate 1H-Trp(Boc)-NH-Dpm;

2) The intermediates 1H-Trp(Boc)-NH-Dpm and Fmoc-Gly-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl are added, the reaction is completed to complete, the solvent is removed, methanol is added, the solid is precipitated, and the solid is filtered. After washing with methanol, it is dissolved in chloroform, DBU and diethylamine are added successively, the reaction is completed to completeness, the solvent is removed, acetonitrile is added to the residue, solid is precipitated, filtered, washed with acetonitrile and dried to give intermediate 2H-Gly-Trp (Boc). -NH-Dpm;

3) The intermediate 2H-Gly-Trp(Boc)-NH-Dpm and Fmoc-Arg(Pbf)-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl is added, the reaction is completed, the solvent is removed, methanol is added, and solid is precipitated. After filtration, the solid is washed with methanol, dissolved in chloroform, and then DBU and diethylamine are added to the reaction. The solvent is completely removed. The solvent is removed and the residue is added with acetonitrile. The solid is precipitated, filtered, washed with acetonitrile and dried to give intermediate 3H-Arg. (Pbf)-Gly-Trp(Boc)-NH-Dpm;

4) Dissolve the intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm and Fmoc-4-hydropro(OtBu)-OH in CHCl ₃ , add HOBt, EDC.HCl, and react to complete Solvent, add methanol, precipitate solids, filter, solids are washed with methanol, dissolved in chloroform, DBU, diethylamine are added in sequence, the reaction is complete, solvent is removed, acetonitrile is added to the residue, solid is precipitated, filtered, washed with acetonitrile and dried , intermediate H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

5) The intermediates H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm and Fmoc-4-Fphe-OH are dissolved in CHCl ₃ , and HOBt, EDC.HCl are added to react. To complete, remove the solvent, add methanol, precipitate the solid, filter, wash the solid with methanol, dissolve in chloroform, add DBU, diethylamine in sequence, complete the reaction, remove the solvent, add acetonitrile to the residue, precipitate solid, filter, The acetonitrile is washed and dried to obtain the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

6) Dissolve the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm in TFA:H ₂ O:EDT:PhOMe=92:2:2 : 4 mixed solution, the reaction was completed, the filtrate was slowly added to ice diethyl ether precipitation, centrifugation, washed with ice diethyl ether and dried to obtain crude naphthopeptide;

7) The crude nano-peptide obtained in step 6) is prepared by reverse-phase high-performance liquid phase purification; the reverse phase octadecylsilane is used as a stationary phase, and the target peak fraction is collected by using 0.1% aqueous acetic acid/acetonitrile as a mobile phase, and concentrated. Freeze-dried, denapeptide.

Beneficial effect

The process of the present invention is suitable for industrial preparation, and the yield is improved as compared to the solid phase synthesis process efficiency and productivity.

detailed description

Example 1 Preparation of Intermediate 1H-Trp(Boc)-NH-Dpm

Dpm-NH ₂ (14.4 mmol), Fmoc-Trp (Boc)-OH (16.2 mmol) was added to the reactor, dissolved in CHCl ₃ (150 mL), then HOBt (14.4 mol), EDC.HCl (28.8 mmol) was added. ). The mixture was stirred for 3 hours, and the reaction was terminated by TLC dot plate. The solvent was evaporated under reduced pressure, and methanol (120 mL) was added, and the solid was precipitated, filtered, and the solid was washed three times with 200 mL of methanol. The solid was dissolved in 150 mL of chloroform, and DBU (14.4 mmol) and diethylamine 150 (mmol) were successively added, and the reaction was stirred for 1 hour, and the reaction was completed. The solvent was evaporated under reduced pressure, and EtOAc (50 mL). The solid was washed three times with 50 mL of acetonitrile, and dried under reduced pressure to give Intermediate 1H-Trp(Boc)-NH-D.

Example 2 Preparation of Intermediate 2H-Gly-Trp(Boc)-NH-Dpm

The intermediate 1H-Trp(Boc)-NH-Dpm14.0 mmol, Fmoc-Gly-OH14.0 mmol prepared in Example 1 was added to the reactor, dissolved in CHCl ₃ (150 mL), and then HOBt (14.0 mol) was added. , EDC.HCl (28.0 mmol). The mixture was stirred for 3 hours, and the reaction was terminated by TLC dot plate. The solvent was evaporated under reduced pressure, and methanol (120 mL) was added, and the solid was precipitated, filtered, and the solid was washed three times with 200 mL of methanol. The solid was dissolved in 150 mL of chloroform, and DBU (14.0 mmol) and diethylamine 150 (mmol) were successively added, and the reaction was stirred for 1 hour, and the reaction was completed. The solvent was evaporated under reduced pressure, and EtOAc (50 mL). The solid was washed three times with 50 mL of acetonitrile and dried under reduced pressure to give Intermediate 2H-Gly-Trp(Boc)-NH-Dpm 13.8 mmol.

Example 3 Preparation of Intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm

The intermediate 2H-Gly-Trp(Boc)-NH-Dpm 13.8 mmol, Fmoc-Arg(Pbf)-OH 13.8 mmol prepared in Example 2 was added to the reactor, dissolved in CHCl ₃ (150 mL), and then added. HOBt (13.8 mol), EDC.HCl (28.0 mmol). The mixture was stirred for 3 hours, and the reaction was terminated by TLC dot plate. The solvent was evaporated under reduced pressure, and methanol (120 mL) was added, and the solid was precipitated, filtered, and the solid was washed three times with 200 mL of methanol. The solid was dissolved in 150 mL of chloroform, and DBU (13.8 mmol) and diethylamine 150 (mmol) were successively added, and the reaction was stirred for 1 hour, and the reaction was completed. The solvent was evaporated under reduced pressure, and EtOAc (50 mL). The solid was washed three times with 50 mL of acetonitrile, and dried under reduced pressure to give Intermediate 3H- s(Pbf)-Gly-Trp(Boc)-NH-Dpm 13.6 mmol, yield 99%.

Example 4 Preparation of Intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm

The intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm13.6mmol, Fmoc-4-hydropro(OtBu)-OH13.6mmol was added to the reactor, dissolved in CHCl ₃ (130 mL), and then HOBt (13.6 mol), EDC.HCl (27.8 mmol) was added. The mixture was stirred for 3 hours, and the reaction was terminated by TLC dot plate. The solvent was evaporated under reduced pressure, and methanol (120 mL) was added, and the solid was precipitated, filtered, and the solid was washed three times with 200 mL of methanol. The solid was dissolved in 150 mL of chloroform, and DBU (13.6 mmol) and diethylamine 145 (mmol) were successively added, and the reaction was stirred for 1 hour, and the reaction was completed. The solvent was evaporated under reduced pressure, and EtOAc (50 mL). The solid was washed three times with 50 mL of acetonitrile, and dried under reduced pressure to give Intermediate 4H-4-hydropro(OtBu)- </RTI> (Pbf)-Gly-Trp(Boc)-NH-Dpm 13.0 mmol, yield 95%.

Example 5 Preparation of intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm

Add intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm 13.0 mmol, Fmoc-4-Fphe-OH 13.0 mmol to the reactor with CHCl ₃ (140 mL) Dissolved, then HOBt (13.0 mol), EDC.HCl (27.5 mmol) was added. The mixture was stirred for 3 hours, and the reaction was terminated by TLC dot plate. The solvent was evaporated under reduced pressure, and methanol (120 mL) was added, and the solid was precipitated, filtered, and the solid was washed three times with 200 mL of methanol. The solid was dissolved in 150 mL of chloroform, and DBU (13.0 mmol) and diethylamine 140 (mmol) were successively added, and the reaction was stirred for 1 hour, and the reaction was completed. The solvent was evaporated under reduced pressure, and EtOAc (50 mL). The solid was washed three times with 50 mL of acetonitrile and dried under reduced pressure to give Intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm 12.5 mmol.

Example 6 Preparation of crude nanopeptides

The intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm12.5 mmol prepared in Example 5 was dissolved in TFA:H ₂ O:EDT:PhOMe =92:2:2:4 In a mixed solution of 20 ml, the mixture was reacted at room temperature for 2 hours, and the filtrate was slowly added to 200 ml of ice diethyl ether to precipitate. The mixture was centrifuged, washed with ice diethyl ether for 5 times, and dried under reduced pressure to give 10.5 mmol of crude crude crude crystals.

Example 7 Preparation of Nano-Peptide

The crude peptide obtained in Example 6 was prepared by reverse phase high performance liquid phase purification. The reverse phase octadecylsilane was used as the stationary phase, and the target peak fraction was collected with 0.1% aqueous acetic acid/acetonitrile as the mobile phase, and concentrated to freeze to obtain 8.8 g (9.5 mmol) of pure product, the yield was 90%, and the purity was more than 99. %.

Abbreviation and English	meaning
Dpm	Bis(4-docosyloxyphenyl)methyl

Fmoc	9-fluorenylmethoxycarbonyl
HOBt	1-hydroxybenzotriazole
EDC·HCl	1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride
TFA	Trifluoroacetate
TA	Benzoyl sulfide
PhOMe	Anisole
EDT	Ethylenedithiol
tBu	Tert-butyl
OtBu	Tert-butyl ester

Claims (9)

1. A method for preparing a liquid phase of nano-nonpeptide, which comprises the following steps,

   1) Fmoc-Trp(Boc)-NH-Dpm was synthesized by Dpm-NH ₂ and Fmoc-Trp(Boc)-OH under the action of a condensing agent, and the Fmoc protecting group was removed to obtain the intermediate 1H-Trp(Boc)- NH-Dpm;

   2) The intermediates 1H-Trp(Boc)-NH-Dpm and Fmoc-Gly-OH were used to synthesize Fmoc-Gly-Trp(Boc)-NH-Dpm under the action of a condensing agent, and the Fmoc protecting group was removed to obtain an intermediate. 2H-Gly-Trp(Boc)-NH-Dpm;

   3) Synthesis of Fmoc-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 2 and Fmoc-Arg(Pbf)-OH under the action of a condensing agent, removing the Fmoc protecting group to obtain intermediate 3H -Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

   4) Synthesis of Fmoc-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 3 and Fmoc-4-hydropro(OtBu)-OH under the action of a condensing agent In addition to the Fmoc protecting group, the intermediate 4H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm is obtained;

   5) Synthesis of Fmoc-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm by intermediate 4 and Fmoc-4-Fphe-OH under the action of a condensing agent, Removal of the Fmoc protecting group to obtain the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

   6) Removal of OtBu, Pbf and Boc protecting groups.
2. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein the condensing agent is selected from one of HOBt, EDC.HCl, HATU, DCC, DIC, HBTU, HOAt, HOBt, PyBOP, BTC or A plurality, preferably a combination of HOBt and EDC.HCl.
3. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein the reagent for removing the Fmoc protecting group is a mixture of DBU and diethylamine.
4. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein in step 6), the deprotecting agent is a mixture of TFA, H ₂ O, EDT, PhOMe, thioanisole; preferably, TFA: H ₂ O: EDT: PhOMe = 90-95: 1-5: 1-5: 3-8, more preferably TFA: H ₂ O: EDT: PhOMe = 92: 2: 2: 4.
5. The method for preparing a liquid phase of nanopeptide according to claim 1, further comprising a purification step, wherein the purification step is 7),

   7) Purification by reverse phase chromatography. Preferably, reverse phase high pressure liquid chromatography is carried out by using reverse phase octadecylsilane as a stationary phase and 0.1% aqueous acetic acid/acetonitrile as a mobile phase to collect a peak fraction of interest and lyophilizing. .
6. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein the solvent in steps 1) to 5) is independently selected from one or more of dichloromethane, chloroform and toluene, preferably trichlorobenzene. Methane.
7. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein in the steps 1) to 5), the Fmoc protecting group is removed by crystallization, filtration and washing, and the Fmoc protecting group is removed and then analyzed by acetonitrile. Crystal, filtration and cleaning.
8. The method for preparing a liquid phase of nano-peptide according to claim 1, wherein in step 6), after removing the protecting group, crystallization, centrifugation and washing are carried out with methanol.
9. The method for preparing a liquid phase of nano-peptide according to claim 1,

   1) Dpm-NH ₂ and Fmoc-Trp(Boc)-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl is added, the reaction is completed to complete, the solvent is removed, methanol is added, the solid is precipitated, and the solid is washed with methanol and dissolved. Chloroform, DBU, diethylamine was added in sequence, the reaction was completed, the solvent was removed, acetonitrile was added to the residue, and the solid was precipitated, filtered, washed with acetonitrile and dried to give intermediate 1H-Trp(Boc)-NH-Dpm;

   2) The intermediates 1H-Trp(Boc)-NH-Dpm and Fmoc-Gly-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl are added, the reaction is completed to complete, the solvent is removed, methanol is added, the solid is precipitated, and the solid is filtered. After washing with methanol, it is dissolved in chloroform, DBU and diethylamine are added successively, the reaction is completed to completeness, the solvent is removed, acetonitrile is added to the residue, solid is precipitated, filtered, washed with acetonitrile and dried to give intermediate 2H-Gly-Trp (Boc). -NH-Dpm;

   3) The intermediate 2H-Gly-Trp(Boc)-NH-Dpm and Fmoc-Arg(Pbf)-OH are dissolved in CHCl ₃ , HOBt, EDC.HCl is added, the reaction is completed, the solvent is removed, methanol is added, and solid is precipitated. After filtration, the solid is washed with methanol, dissolved in chloroform, and then DBU and diethylamine are added to the reaction. The solvent is completely removed. The solvent is removed and the residue is added with acetonitrile. The solid is precipitated, filtered, washed with acetonitrile and dried to give intermediate 3H-Arg. (Pbf)-Gly-Trp(Boc)-NH-Dpm;

   4) Dissolve the intermediate 3H-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm and Fmoc-4-hydropro(OtBu)-OH in CHCl ₃ , add HOBt, EDC.HCl, and react to complete Solvent, add methanol, precipitate solids, filter, solids are washed with methanol, dissolved in chloroform, DBU, diethylamine are added in sequence, the reaction is complete, solvent is removed, acetonitrile is added to the residue, solid is precipitated, filtered, washed with acetonitrile and dried , intermediate H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

   5) The intermediates H-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm and Fmoc-4-Fphe-OH are dissolved in CHCl ₃ , and HOBt, EDC.HCl are added to react. To complete, remove the solvent, add methanol, precipitate the solid, filter, wash the solid with methanol, dissolve in chloroform, add DBU, diethylamine in sequence, complete the reaction, remove the solvent, add acetonitrile to the residue, precipitate solid, filter, The acetonitrile is washed and dried to obtain the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm;

   6) Dissolve the intermediate 5H-4-Fphe-4-hydropro(OtBu)-Arg(Pbf)-Gly-Trp(Boc)-NH-Dpm in TFA:H ₂ O:EDT:PhOMe=92:2:2 : 4 mixed solution, the reaction was completed, the filtrate was slowly added to ice diethyl ether precipitation, centrifugation, washed with ice diethyl ether and dried to obtain a crude nanopeptide;

   7) The crude nano-peptide obtained in step 6) is prepared by reverse-phase high-performance liquid phase purification; the reverse phase octadecylsilane is used as a stationary phase, and the target peak fraction is collected by using 0.1% aqueous acetic acid/acetonitrile as a mobile phase, and concentrated. Freeze-dried, denapeptide.