WO2019184088A1

WO2019184088A1 - Compound and preparation method therefor and application thereof

Info

Publication number: WO2019184088A1
Application number: PCT/CN2018/090204
Authority: WO
Inventors: 陈永汉; 李国弢; 宓鹏程; 陶安进; 袁建成
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2018-03-29
Filing date: 2018-06-07
Publication date: 2019-10-03
Also published as: CN110317130B; CN110317130A

Abstract

Disclosed are reaction carriers 1, 2 and 3 and a preparation method therefor and an application thereof. A reaction carrier 1/2/3 is used to synthesize angiotensin II, which can effectively increase the solubility of a full protective peptide in an organic solvent such as CHCl ₃, and reduce the solubility of a full protective peptide in an aqueous solution; by using the reaction carrier 1/2/3 as a raw material, a liquid-phase synthesis method can reduce the purification steps in each step of the conventional liquid-phase synthesis method and thus improve the production efficiency; the liquid-phase synthesis method using the reaction carrier 1/2/3 as a raw material can effectively reduce the dosage of organic solvent (DMF, DCM) in solid-phase synthesis method, and achieve the effect of reducing waste.

Description

Compound and preparation method and application thereof

The present application claims priority to Chinese Patent Application No. 201810273697.8, entitled "Compounds and Preparation Methods and Applications", filed on March 29, 2018, the entire contents of which are incorporated herein by reference. .

Technical field

The invention relates to the field of polypeptide synthesis, in particular to compounds and preparation methods and applications thereof.

Background technique

Angiotensin is a class of peptides that have a strong vasoconstriction and stimulate the secretion of aldosterone from the adrenal cortex. They are involved in the regulation of blood pressure and body fluids. Can be divided into angiotensin I-VII.

Angiotensin II is the most important component of angiotensin. Angiotensin receptors are present on the vascular smooth muscle of the human body, the spheroidal cells of the adrenal cortex, and some parts of the brain, cells of the heart and kidney organs. Angiotensin II binds to angiotensin receptors, causing a corresponding physiological effect. Angiotensin acts on vascular smooth muscle, which causes systemic arterioles to contract and arterial blood pressure to rise. Angiotensin II is one of the most known vasoconstrictor active substances. Acting on peripheral blood vessels, causing the veins to contract, increase the amount of blood returning to the center, causing thirst.

La Jolla Pharmaceutical's angiotensin II preparation LJPC-501 is being developed for the treatment of catecholamine-resistant hypotensive (CRH) patients.

The structure of angiotensin II is shown in formula I:

Regarding the preparation method of angiotensin II, at present, the synthesis method of the conventional short peptide can also adopt the method of Fmoc solid phase synthesis. Disadvantages of solid synthesis: it is necessary to use a solid carrier and ensure complete reaction by excessive feeding, and the cost is high; the resin needs repeated washing during the synthesis process, and requires a large amount of organic solvents such as DMF and DCM; the synthesis scale of the solid phase synthesis method is affected by the equipment. The influence is large and difficult to enlarge; the disadvantage of the traditional liquid phase synthesis: after the liquid phase reaction is completed, the required compounds and by-products are all together in the reaction mixture, and each step of synthesis requires purification and then the next synthesis is carried out, resulting in cumbersome operation. The yield is relatively low.

Therefore, it is of great practical significance to provide a method for preparing angiotensin II which is simple in operation and high in yield.

Summary of the invention

In view of this, the present invention provides compounds and methods for their preparation and use. The synthetic reaction carrier 1/2/3 of the present invention is used for liquid phase synthesis of angiotensin II. The reaction carrier can effectively increase the solubility of the full protective peptide in an organic solvent such as CHCl ₃ and reduce the solubility of the whole protective peptide in an aqueous solution while using the reaction carrier; the reaction carrier 1/2/3 is used as a raw material. The liquid phase synthesis method can reduce the steps of purification in each step of the conventional liquid phase synthesis, and improve the production efficiency; the synthesis method provided by the invention can effectively reduce the organic solvent (DMF, DCM) in the solid phase synthesis. The dosage can achieve the effect of reducing waste.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a compound characterized by the structure of Formula II:

Wherein R ₁ and R _{2 are} independently selected from H or

.

In some embodiments of the invention, the structure of the compound is as shown in Formula IV (Reaction Carrier 1), Formula V (Reaction Carrier 2), Formula VI (Reaction Carrier 3):

The invention also provides a preparation method of the compound, wherein the compound represented by the formula III is subjected to halogenation reaction, reacted with p-hydroxybenzene alkanoate to form an ether reaction, and then obtained by a reduction reaction.

Wherein R ₁ and R _{2 are} independently selected from H or

.

In some embodiments of the invention, the compound of Formula III comprises Formula VII (Raw 1, Preparation of Reaction Carrier 1), Formula VIII (Raw 2, Preparation of Reaction Carrier 2), Compound of Formula IX (Raw 3, Preparation) Reaction carrier 3).

In some embodiments of the invention, the reagent used in the halogenation reaction is one or both of HCl or HBr. HBr is preferred.

In some embodiments of the present invention, the halogenation reaction has a reaction temperature of 90 to 110 ° C, a reaction time of 12 to 24 hours, and the catalyst is a mixture of one or more of concentrated sulfuric acid and zinc chloride. The reaction solvent is one or a mixture of two or more of water or acetic acid.

In some embodiments of the invention, the p-hydroxyphenyl alkanoate is a mixture of one or more of methyl p-hydroxybenzoate, methyl p-hydroxyphenylacetate, methyl p-hydroxyphenylpropionate. . Methylparaben is preferred.

In some embodiments of the present invention, the reaction temperature for the ether reaction with the p-hydroxyphenyl acid ester is 90-110 ° C, the reaction time is 12-24 h, and the catalyst is potassium carbonate, sodium carbonate, potassium hydroxide or hydrogen. One or a mixture of two or more of sodium oxide, and the reaction solvent is one or a mixture of two or more of DMF, tetrahydrofuran or acetone.

In some embodiments of the invention, the reducing agent used in the reduction reaction is a mixture of one or more of diisobutylaluminum hydride, lithium tetrahydrogenate or sodium borohydride. Preference is given to diisobutylaluminum hydride.

In some embodiments of the present invention, the reduction reaction has a reaction temperature of -5 to 5 ° C, a reaction time of 12 to 24 hours, and the reaction solvent is one or both of tetrahydrofuran, dioxane, and toluene. The above mixture.

The invention also provides the use of the compound or the compound prepared by the preparation method in the synthesis of a polypeptide.

The invention also provides the use of the compound or the compound prepared by the preparation method for the preparation of angiotensin II.

The invention also provides a preparation method of angiotensin II, wherein the compound or the compound prepared by the preparation method is used as a raw material, and the whole protective peptide is synthesized in a liquid phase, which is obtained by cleavage and purification.

In some embodiments of the invention, the solvent used in the method of producing angiotensin II is one or both of chloroform or dichloromethane, preferably chloroform.

In some embodiments of the invention, the coupling agent in the method of producing angiotensin II is HOBt/DIC, HOBt/EDC.HCl, EDC.HCl, preferably HOBt/EDC.HCl.

In some embodiments of the present invention, in the preparation method of angiotensin II, the lysate is a mixed solvent of TFA and water, and the ratio of the mixed solvent to the TFA is 80-95%, and the volume ratio of water is 5-20%. The preferred ratio is TFA: H2O = 95:5.

In some embodiments of the invention, the method of producing angiotensin II -

The present invention provides a compound having the structure shown in Formula II and having the following beneficial effects:

1. The reaction carrier 1/2/3 synthesized in the present invention is used for liquid phase synthesis of angiotensin II. The reaction carrier can effectively increase the solubility of the full protective peptide in an organic solvent such as CHCl ₃ while protecting the C-terminus, and reduce the solubility of the full protective peptide in the aqueous solution;

2. The new liquid phase synthesis method can reduce the steps of purification in each step of the traditional liquid phase synthesis, and improve the production efficiency;

3. The new synthetic method can effectively reduce the amount of organic solvent (DMF, DCM) in solid phase synthesis, and achieve the effect of reducing waste.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art description will be briefly described below.

Figure 1 shows the synthetic route of the compound of Formula IV (Reaction Carrier 1);

Figure 2 shows the synthetic route of the compound of the formula V (reaction carrier 2);

Figure 3 shows the synthetic route of the compound of the formula VI (reaction carrier 3);

Figure 4 shows a reaction route for synthesizing a fully protected peptide using a compound of the formula IV (reaction carrier 1) as a starting material;

Figure 5 shows the reaction route of the full protective peptide cleavage to obtain the crude peptide;

Figure 6 shows the HPLC chromatogram of the angiotensin II crude peptide prepared in Example 13;

Figure 7 is a HPLC chart showing the angiotensin II crude peptide prepared in Example 14;

Figure 8 is a HPLC chart showing the angiotensin II crude peptide prepared in Example 15;

Figure 9 shows an HPLC chromatogram of angiotensin II arginine prepared in Example 16;

Figure 10 is a mass spectrum of angiotensin II arginine prepared in Example 16;

Figure 11 shows an HPLC chromatogram of angiotensin II sperm peptide prepared in Example 17;

Figure 12 shows an HPLC chromatogram of angiotensin II arginine prepared in Example 18;

Figure 13 shows an HPLC chromatogram of angiotensin II sperm peptide prepared in an enlarged experiment of Example 19;

Figure 14 is a view showing the mass spectrum of angiotensin II arginine prepared in the amplification experiment of Example 19;

Figure 15 is a HPLC chart showing the solid phase synthesis of angiotensin II phagetine in Comparative Example 1;

Figure 16 is a chart showing the HPLC spectrum of liquid phase synthetic angiotensin II sperm in Comparative Example 2.

detailed description

The invention discloses a compound and a preparation method and application thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The compounds and reagents and reagents used in the methods and applications of the present invention are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Example 1. Synthesis of Reaction Carrier 1

13.6 g of pentaerythritol (100 mmol) was weighed and added to 200 ml of hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 100 ° C for 18 hours. The reaction solution was cooled to room temperature, extracted with 200 ml of n-hexane, washed twice with 100 ml of 5% sodium hydrogen carbonate solution, and then washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness, and then purified to silica gel column to afford 25.85 g of Intermediate 1a.

23.0 g of Intermediate 1a (60 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 100 ° C to react 12 . The reaction solution was cooled to room temperature, extracted with 400 ml of n-hexane, washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

31.5 g of intermediate 1b (51 mmol) was dissolved in 500 ml of tetrahydrofuran, the reaction solution was cooled to 0 ° C, and 245 ml of 1 M diisobutylaluminum hydride/toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 12 hours, then 100 ml of 0.2 M. The hydrochloric acid solution was quenched. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

^{1 H NMR (400MHz, CDCl3)} δ7.04-6.94 (m, 8H), 6.93-6.79 (m, 8H), 4.61 (s, 8H), 4.16 (s, 8H), 1.38 (s, 4H).

Example 2 Synthesis of Reaction Carrier 1

13.6 g of pentaerythritol (100 mmol) was weighed and added to 200 ml of a hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 90 ° C for 24 hours. The reaction solution was cooled to room temperature, extracted with 200 ml of n-hexane, washed twice with 100 ml of 5% sodium hydrogen carbonate solution, and then washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated and evaporated.

23.0 g of Intermediate 1a (60 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 90 ° C for 24 hours. The reaction solution was cooled to room temperature, extracted with 400 ml of n-hexane, washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

30.5g of intermediate 1b was dissolved in 500ml of tetrahydrofuran, the reaction solution was cooled to 5 ° C, and 245 ml of 1 M diisobutylaluminum hydride / toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 8 hours, then quenched with 100 ml of 0.2 M hydrochloric acid solution. Eliminate the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness to dryness to afford 24.9 g of the reaction carrier 1 in a yield of 87.2%, MS 560.58.

^{1 H NMR (400MHz, CDCl3)} δ7.05-6.93 (m, 8H), 6.94-6.79 (m, 8H), 4.61 (s, 8H), 4.17 (s, 8H), 1.39 (s, 4H).

Example 3 Synthesis of Reaction Carrier 1

13.6 g of pentaerythritol (100 mmol) was weighed and added to 200 ml of hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 110 ° C for 12 hours. The reaction solution was cooled to room temperature, extracted with 200 ml of n-hexane, washed twice with 100 ml of 5% sodium hydrogen carbonate solution, and then washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

23.0 g of Intermediate 1a (60 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 110 ° C for 8 hours. The reaction solution was cooled to room temperature, extracted with 400 ml of n-hexane, washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

30.9g of intermediate 1b was dissolved in 500ml of tetrahydrofuran, the reaction solution was cooled to -5 ° C, and 245 ml of 1 M diisobutylaluminum hydride / toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 24 hours, then 100 ml of 0.2 M hydrochloric acid solution was used. Quench the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

^{1 H NMR (400MHz, CDCl3)} δ7.04-6.93 (m, 8H), 6.92-6.78 (m, 8H), 4.62 (s, 8H), 4.18 (s, 8H), 1.38 (s, 4H).

Example 4 Synthesis of Reaction Carrier 2

10.6 g of 2-hydroxymethyl-1,3-propanediol (100 mmol) was weighed and added to 200 ml of a hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 100 ° C for 18 hours. The reaction solution was cooled to room temperature, and extracted with 200 ml of n-hexane. The organic phase was washed twice with 100 ml of 5% sodium hydrogencarbonate solution, and then washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solvent was purified by a silica gel column to give 27.0 g of Intermediate 1b.

23.5 g of Intermediate 2a (80 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 100 ° C for 18 hours. The reaction solution was cooled to room temperature, extracted by adding 400 ml of n-hexane, and the organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness to give 33.1 g of Intermediate 2b.

33.1 g of the intermediate 2b was dissolved in 500 ml of tetrahydrofuran, the reaction solution was cooled to 0 ° C, and 255 ml of 1 M diisobutylaluminum hydride/toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 18 hours, and then quenched with 100 ml of 0.2 M hydrochloric acid solution. Eliminate the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

1H NMR (400MHz, CDCl3) δ7.08–6.93 (m, 6H), 6.93–6.77 (m, 6H), 4.61 (s, 6H), 3.91 (d, J = 14.24 Hz, 6H), 3.09–3.04 ( m, 1H), 1.39 (s, 3H).

Example 5 Synthesis of Reaction Carrier 2

10.6 g of 2-hydroxymethyl-1,3-propanediol (100 mmol) was weighed and added to 200 ml of a hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 90 ° C for 24 hours. The reaction solution was cooled to room temperature, and extracted with 200 ml of n-hexane. The organic phase was washed twice with 100 ml of 5% sodium hydrogencarbonate solution, and then washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solvent was purified by a silica gel column to give 26.2 g of Intermediate 1b.

23.5 g of Intermediate 2a (80 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 90 ° C for 24 hours. The reaction solution was cooled to room temperature, extracted by adding 400 ml of n-hexane, and the organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness to give 32.1 g of Intermediate 2b.

33.1 g of the intermediate 2b was dissolved in 500 ml of tetrahydrofuran, the reaction solution was cooled to 5 ° C, and 255 ml of 1 M diisobutylaluminum hydride/toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 12 hours, and then quenched with 100 ml of 0.2 M hydrochloric acid solution. Eliminate the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness to dryness to afford 26.6 g of reaction carrier 2, yield 88.4%, MS 424.48.

1H NMR (400MHz, CDCl3) δ7.09–6.94 (m, 6H), 6.93–6.78 (m, 6H), 4.62 (s, 6H), 3.912 (d, J = 14.3 Hz, 6H), 3.10–3.04 ( m, 1H), 1.38 (s, 3H).

Example 6 Synthesis of Reaction Carrier 2

10.6 g of 2-hydroxymethyl-1,3-propanediol (100 mmol) was weighed and added to 200 ml of a hydrobromic acid solution (40%), 0.5 ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 110 ° C for 12 hours. The reaction solution was cooled to room temperature, and extracted with 200 ml of n-hexane. The organic phase was washed twice with 100 ml of 5% sodium hydrogencarbonate solution, and then washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The solvent was purified by a silica gel column to give 26.1 g of Intermediate 1b.

23.5 g of Intermediate 2a (80 mmol), 44.1 g of methyl p-hydroxyphenylate (290 mmol) and 39.7 g of potassium carbonate (290 mmol) were dissolved in 200 ml of DMF solution and heated to 110 ° C for 12 hours. The reaction solution was cooled to room temperature, and extracted with 400 ml of n-hexane. The organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, and then washed twice with 200 ml of 5% sodium hydrogencarbonate solution; and then washed once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness to give 31.8 g of Intermediate 2b.

31.8g of intermediate 2b was dissolved in 500ml of tetrahydrofuran, the reaction solution was cooled to -5 ° C, and 255 ml of 1 M diisobutylaluminum hydride / toluene solution was slowly added dropwise under nitrogen atmosphere, and reacted at room temperature for 24 hours, then 100 ml of 0.2 M hydrochloric acid solution was used. Quench the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

1H NMR (400MHz, CDCl3) δ7.08–6.93 (m, 6H), 6.93–6.77 (m, 6H), 4.61 (s, 6H), 3.91 (d, J = 14.2 Hz, 6H), 3.09–3.04 ( m, 1H), 1.39 (s, 3H).

Example 7 Synthesis of Reaction Carrier 3

16.2 g of Intermediate 3a (80 mmol), 29.2 g of methyl p-hydroxyphenylate (192 mmol) and 26.5 g of potassium carbonate (192 mmol) were weighed and dissolved in 200 ml of DMF, and heated to 100 ° C for 18 hours. The reaction solution was cooled to room temperature, extracted by adding 400 ml of n-hexane, and the organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness, and then, then, 23.2 g of Intermediate 2b was obtained.

23.2g of intermediate 3b was dissolved in 500ml of tetrahydrofuran, the reaction solution was cooled to 0 ° C, and 175 ml of 1 M diisobutylaluminum hydride / toluene solution was slowly added dropwise under nitrogen atmosphere for 18 hours at room temperature, then quenched with 100 ml of 0.2 M hydrochloric acid solution. reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness to dryness to afford to afford 19.7 g of reaction carrier 3, yield 93.8%, MS 288.35.

1H NMR (400MHz, CDCl3) δ7.08–6.91 (m, 4H), 6.91–6.77 (m, 4H), 4.61 (s, 4H), 4.29 (s, 4H), 2.25 (s, 2H), 1.38 ( s, 2H).

Example 8 Synthesis of Reaction Carrier 3

16.2 g of Intermediate 3a (80 mmol), 29.2 g of methyl p-hydroxyphenylate (192 mmol) and 26.5 g of potassium carbonate (192 mmol) were weighed and dissolved in 200 ml of DMF and heated to 90 ° C for 24 hours. The reaction solution was cooled to room temperature, extracted by adding 400 ml of n-hexane, and the organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness to give 22.5 g of Intermediate 2b.

22.5g of intermediate 3b was dissolved in 500ml of tetrahydrofuran, the reaction solution was cooled to -5 ° C, and 175 ml of 1 M diisobutylaluminum hydride / toluene solution was slowly added dropwise under nitrogen atmosphere, room temperature for 24 hours, and then quenched with 100 ml of 0.2 M hydrochloric acid solution. Eliminate the reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

1H NMR (400MHz, CDCl3) δ7.08–6.92 (m, 4H), 6.91–6.76 (m, 4H), 4.61 (s, 4H), 4.28 (s, 4H), 2.26 (s, 2H), 1.39 ( s, 2H).

Example 9 Synthesis of Reaction Carrier 3

16.2 g of Intermediate 3a (80 mmol), 29.2 g of methyl p-hydroxyphenylate (192 mmol) and 26.5 g of potassium carbonate (192 mmol) were weighed and dissolved in 200 ml of DMF, and heated to 110 ° C for 12 hours. The reaction solution was cooled to room temperature, extracted by adding 400 ml of n-hexane, and the organic phase was washed once with 200 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated brine. After drying with anhydrous sodium sulfate, the organic solvent was evaporated to dryness to give 22.6 g of Intermediate 2b.

22.6 g of intermediate 3b was dissolved in 500 ml of tetrahydrofuran, the reaction solution was cooled to 5 ° C, and 175 ml of 1 M diisobutylaluminum hydride/toluene solution was slowly added dropwise under nitrogen atmosphere for 12 hours at room temperature, and then quenched with 100 ml of 0.2 M hydrochloric acid solution. reaction. The mixture was extracted twice by adding 800 ml of ethyl acetate, washed once with 300 ml of 1 M hydrochloric acid solution, twice with 200 ml of 5% sodium hydrogencarbonate solution, and once with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness.

1H NMR (400MHz, CDCl3) δ7.08–6.93 (m, 4H), 6.92–6.77 (m, 4H), 4.63 (s, 4H), 4.27 (s, 4H), 2.27 (s, 2H), 1.39 ( s, 2H).

Example 10 Synthesis of angiotensin II full protective peptide

5.60 g of Reaction Carrier 1 (10 mmol) in Example 1 was weighed and dissolved in 200 ml of chloroform. Under ice bath, 20.12 g of Fmoc-Phe-OH (52 mmol), 10.72 g of EDC.HCl (56 mmol) and 1.20 g of DMAP were added. (5.2 mmol), the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of a saturated aqueous solution of sodium chloride, and 15.6 g of 2-mercaptosuccinic acid (104 mmol) and 31.6 g of DBU (208 mmol) were added to the organic phase, and the mixture was stirred for 3 hours to obtain Intermediate 1c. Then, it was washed once with 50 ml of a 10% aqueous sodium hydrogencarbonate solution (containing 20% DMF), and washed twice with saturated brine to give Intermediate 1d.

17.52 g of Fmoc-Pro-OH (52 mmol), 10.72 g of EDC.HCl (56 mmol) and 7.02 g of HOBt (52 mmol) were added under ice-cooling, and the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of saturated brine, and the organic phase was added 15.6 g of 2-mercaptosuccinic acid (104 mmol) and 31.6 g of DBU (208 mmol) in an ice bath, and the reaction was stirred for 3 hours, and then 50 ml of 10% sodium hydrogencarbonate was used. The DMF/water solution (1/4) was washed once and washed twice with 10% NaCl in DMF/water solution (1/4). The subsequent methods were coupled to the following Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp. (OtBu)-OH, the loading ratio of all amino acids was 5.2 eq, and the reaction time was 3 hours. After all the amino acids were coupled, half of the solvent was distilled off under reduced pressure, then added to a mixture of 200 ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. The side chain fully protected angiotensin II crude peptide 53.0 g was obtained, and the yield was 75.2%.

Example 11 Synthesis of angiotensin II full protective peptide

4.24 g of Reaction Carrier 2 (10 mmol) in Example 2 was weighed and dissolved in 200 ml of chloroform. Under ice bath, 15.09 g of Fmoc-Phe-OH (39 mmol), 8.04 g of EDC.HCl (42 mmol) and 0.90 g of DMAP were added. (3.9 mmol), the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of a saturated aqueous solution of sodium chloride, and the organic phase was added 11.72 g of 2-mercaptosuccinic acid (78 mmol) and 23.72 g of DBU (156 mmol), and the mixture was stirred for 3 hours to obtain intermediate 1c. Then, it was washed once with 50 ml of a 10% aqueous sodium hydrogencarbonate solution (containing 20% DMF), and washed twice with saturated brine to give Intermediate 2d.

13.14 g of Fmoc-Pro-OH (39 mmol), 8.04 g of EDC.HCl (42 mmol) and 5.27 g of HOBt (39 mmol) were added under ice bath, and the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of saturated brine, and the organic phase was added 11.72 g of 2-mercaptosuccinic acid (78 mmol) and 23.72 g of DBU (156 mmol), and the reaction was stirred for 3 hours, then 50 ml of 10% sodium hydrogencarbonate was added. The DMF/water solution (1/4) was washed once and washed twice with 10% NaCl in DMF/water solution (1/4). The subsequent methods were coupled to the following Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp. (OtBu)-OH, the dosing ratio of all amino acids was 3.9 eq, and the reaction time was 3 hours. After all the amino acids were coupled, half of the solvent was distilled off under reduced pressure, then added to a mixture of 200 ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. The side chain fully protected angiotensin II crude peptide 42.2 g, the yield was 79.8%.

Example 12, Synthesis of angiotensin II full protective peptide

2.88 g of Reaction Support 3 (10 mmol) in Example 3 was weighed and dissolved in 200 ml of chloroform. Under an ice bath, 10.06 g of Fmoc-Phe-OH (26 mmol), 5.36 g of EDC.HCl (28 mmol) and 0.60 g of DMAP were added. (2.6 mmol), the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of a saturated aqueous solution of sodium chloride, and the organic phase was added to 7.80 g of 2-mercaptosuccinic acid (52 mmol) and 15.81 g of DBU (104 mmol), and the mixture was stirred for 3 hours to obtain intermediate 3c. Then, it was washed once with 50 ml of a 10% aqueous sodium hydrogencarbonate solution (containing 20% DMF), and washed twice with saturated brine to give Intermediate 3d.

8.76 g of Fmoc-Pro-OH (26 mmol), 5.36 g of EDC.HCl (28 mmol) and 3.51 g of HOBt (26 mmol) were added under ice-cooling, and the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 50 ml of a saturated aqueous solution of sodium chloride, and the organic phase was charged with 7.80 g of 2-mercaptosuccinic acid (52 mmol) and 15.81 g of DBU (104 mmol), and the reaction was stirred for 3 hours, and then 50 ml of 10% sodium hydrogencarbonate was used. The DMF/water solution (1/4) was washed once and washed twice with 10% NaCl in DMF/water solution (1/4). The subsequent methods were coupled to the following Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp. (OtBu)-OH, the loading ratio of all amino acids was 5.2 eq, and the reaction time was 3 hours. After all the amino acids were coupled, half of the solvent was distilled off under reduced pressure, then added to a mixture of 200 ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. The side chain fully protected angiotensin II crude peptide 30.1 g was obtained, and the yield was 84.9%.

Example 13, angiotensin II crude peptide cleavage

53.0 g of the side chain fully protected angiotensin II crude peptide in Example 4 was added to a 1000 ml bottle, and 530 mL of the prepared lysis reagent (TFA: H ₂ O = 9:1) was poured into the above reaction bottle. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into 5.3 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain angiotensin II crude peptide 39.8 g, yield 95.1%, HPLC purity 80.7% (see figure 6), MS 1046.50.

Example 14, angiotensin II crude peptide cleavage

42.2 g of the side chain fully protected angiotensin II crude peptide in Example 5 was added to a 500 ml bottle, and 425 mL of the prepared lysis reagent (TFA: H ₂ O = 9:1) was poured into the above reaction bottle. The reaction was carried out at room temperature for 2 hours. At the end of the reaction, the reaction solution was poured into 4.3 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain angiotensin II crude peptide 29.6 g, yield 94.5%, HPLC purity 81.2% (see figure 7), MS 1046.20.

Example 15, angiotensin II crude peptide cleavage

30.1 g of the side chain fully protected angiotensin II crude peptide in Example 6 was added to a 500 ml bottle, and 301 mL (TFA: H ₂ O = 9:1) of the prepared lysing reagent was poured into the above reaction bottle. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into 3.1 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, and dried to obtain 20.3 g of angiotensin II crude peptide, the yield was 96.1%, and the HPLC purity was 81.7% (see FIG. 8), MS 1046.18.

Example 16, Preparation of Angiotensin II

The 39.8 g angiotensin II crude peptide in Example 7 was purified by high performance liquid chromatography, concentrated, and lyophilized to obtain angiotensin II pedigree 21.2 g, HPLC purity of 99.5% (see Figure 9), total yield 50.7%, MS1046.45 (see Figure 10).

Example 17, Preparation of Angiotensin II

The 29.6 g angiotensin II crude peptide in Example 8 was purified by high performance liquid chromatography, concentrated, and lyophilized to obtain angiotensin II ectopeptide 16.7 g, HPLC purity was 99.5% (see Figure 11), total yield 53.4%, MS1046.18.

Example 18, Preparation of angiotensin II

The 20.3 g angiotensin II crude peptide in Example 9 was purified by high performance liquid chromatography, concentrated, and lyophilized to obtain 10.4 g of angiotensin II eptidin, and the HPLC purity was 99.6% (see Figure 12). 49.7%, MS1046.21.

Example 19, amplification experiment of angiotensin II

56.0 g of reaction carrier 1 (100 mmol), dissolved in 2 L of chloroform, and added 201.2 g of Fmoc-Phe-OH (520 mmol), 107.2 g of EDC.HCl (560 mmol) and 12.0 g of DMAP (52 mmol), and stirred at room temperature. Reaction for 3 hours. After the completion of the reaction, the mixture was washed once with 500 ml of a saturated aqueous sodium chloride solution, and 156.2 g of 2-mercaptosuccinic acid (1.04 mol) and 316.0 g of DBU (2.08 mol) were added to the organic phase, and the reaction was stirred for 3 hours to obtain Intermediate 1c. Then, it was washed once with 500 ml of 10% aqueous sodium hydrogencarbonate solution (containing 20% DMF), and washed twice with 500 ml of saturated brine to obtain Intermediate 1d.

175.2 g of Fmoc-Pro-OH (520 mmol), 107.2 g of EDC.HCl (560 mmol) and 70.2 g of HOBt (520 mmol) were added under ice-cooling, and the reaction was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was washed once with 500 ml of saturated brine, and 156.2 g of 2-mercaptosuccinic acid (1.04 mol) and 316.2 g of DBU (2.08 mol) were added to the organic phase, and the reaction was stirred for 3 hours, and then 500 ml of 10% hydrogen carbonate was used. The sodium DMF/water solution (1/4) was washed once and washed twice with 500 ml of 10% NaCl in DMF/water solution (1/4). The subsequent methods were coupled to the following Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp. (OtBu)-OH, the loading ratio of all amino acids was 5.2 eq, and the reaction time was 3 hours. After all the amino acids were coupled, half of the solvent was evaporated under reduced pressure, then added to 2 L of acetonitrile to precipitate, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. The side chain fully protected angiotensin II crude peptide 509.1 g, the yield was 72.1%.

The above 509.1 g side chain fully protected angiotensin II crude peptide was added to a 6 L reaction flask, and 5.1 L of the prepared lysis reagent (TFA: H ₂ O = 9:1) was poured into the above reaction flask, and reacted at room temperature. 2 hours. After completion of the reaction, the reaction solution was poured into 51 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain angiotensin II crude peptide 397.8 g, yield 95.1%, HPLC purity 75.7%, MS 1046.50.

397.8 g of angiotensin II crude peptide in Example 7 was purified by high performance liquid chromatography, concentrated, and lyophilized to obtain 206.2 g of angiotensin II phage, HPLC purity was 99.5% (see Figure 13), total yield 49.3%, MS1046.62 (see Figure 14).

Comparative Example 1 Solid Phase Synthesis of Angiotensin II

200 g of Wang resin (100 mmol) with a degree of substitution of 0.5 mmol/g was used as the actual resin, washed twice with 1 L of DMF, and then swollen with 1 L of DMF for 30 minutes, and weighed 153.8 g of Fmoc-Phe-OH (400 mmol) and 145.9 g, respectively. HOBt (420 mmol) and 9.0 g of DMAP (40 mmol) were stirred at room temperature for 3 hours. It was blocked by adding 1 L of acetic anhydride/pyridine (1:1) solution for 6 hours. The blocking solution was extracted, washed 3 times with 1 L of DCM, and washed 3 times with 1 L of DMF. Fmoc was removed twice by adding 1 L of 20% piperidine/DMF (V/V) solution for 10 minutes each time, and the resin was washed 6 times with 1 L of DMF after removal. 168.7 g (500 mmol) of Fmoc-Pro-OH, 81.0 g (500 mmol) of HOBt were weighed, dissolved in 0.5 L of DMF and 0.5 L of DCM, and 108.3 ml (600 mmol) of DIC was added for 5 minutes in an ice water bath, and the mixture was added to the reaction. The column was reacted at room temperature for 2 hours. The subsequent methods were coupled to the following Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp. (OtBu)-OH, the dosing ratio of all amino acids was 500 mmol, the reaction time was 2 hours, and 1 L DMF was washed 6 times. Fmoc was removed twice by adding 1 L of 20% piperidine/DMF (V/V) solution for 10 minutes each time, and the resin was washed 6 times with 1 L of DMF after removal. After the peptide resin was coupled, it was shrunk 3 times with 1 L of methanol for 10 minutes each time. Then, it was dried under vacuum to obtain 365.1 g of a peptide resin.

The above 365.1 g of angiotensin II peptide resin was added to a 10 L reaction flask, and 3.65 L (TFA: H ₂ O = 9:1) of the prepared lysis reagent was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into 36.5 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, and dried to obtain angiotensin II crude peptide 98.0 g, yield 93.6%, HPLC purity 70.7%, MS 1046.18 .

The above 196 g angiotensin II crude peptide was purified by high performance liquid chromatography, concentrated and lyophilized to obtain 48.8 g of angiotensin II phage, HPLC purity was 99.6% (see Figure 15), total yield 46.7%, MS1046 .18.

Comparative example 2

21.6 g of Boc-Pro-OH (100 mmol) was dissolved in 200 ml of THF, followed by the addition of 36.3 g of H-Phe-OMe.HCl (100 mmol), 13.8 g of DIC (110 mmol), 14.9 g of HOBt (110 mmol), and 10.1 dropwise with stirring. g NMM (100 mmol), stirred at room temperature for 1.5 hours, TCL followed the reaction. After completion of the reaction, the solid was filtered off, and the THF was evaporated under reduced pressure. The solid was dissolved in 300 ml of ethyl acetate, and the ethyl acetate solution was washed with a 10% citric acid solution, a saturated sodium hydrogen carbonate solution and a saturated sodium chloride solution, and the organic layer was separated. It was dried over anhydrous sodium sulfate. The desiccant was filtered off, the ethyl acetate was evaporated under reduced pressure, 400 ml of petroleum ether was added, and the solid was lyophilized, and the solid was collected by filtration and dried under reduced pressure. 39.3 g of Boc-Pro-Phe-OMe.HCl were obtained in a yield of 75.1%. 39.3 g and 200 ml of 4N HCl/dioxane solution were reacted at room temperature for 1.5 hours, and a solid was precipitated. The solid was collected by filtration and dried in vacuo to give 31.5 g of H-Pro-Phe-OMe.HCl.

Coupling the subsequent Boc-His(Trt)-OH, Boc-Ile-OH, Boc-Tyr(tBu)-OH, Boc-Val-OH, Boc-Arg(Pbf)-OH and Boc-Asp in the same way (OtBu)-OH, the feed size was 100 mmol, and 116.6 g of a fully protected peptide was obtained, and the yield was 64.8%.

The above 116.6 g angiotensin II full protective peptide was added to a 250 ml reactor bottle, and 1.2 L of the prepared lysis reagent (TFA: H ₂ O = 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. . After the reaction was completed, the reaction solution was poured into 12.0 L of ice diethyl ether, and a large amount of white precipitate was washed out, centrifuged, washed, and dried to obtain an angiotensin II crude peptide 67.9 g, yield 65.1%, HPLC purity 72.9%, MS 1046.28. .

The above 67.9 g angiotensin II crude peptide was purified by high performance liquid chromatography, concentrated and lyophilized to obtain 42.6 g of angiotensin II phage, HPLC purity was 99.6% (see Figure 16), and the total yield was 40.7%. MS1046.26.

Example 20

Experimental group 1: crude peptide and sperm peptide prepared in Example 19;

Control group 1: crude peptide and sperm peptide prepared in Comparative Example 1;

Control group 2: crude peptide and sperm peptide prepared in Comparative Example 2;

The comparison results are shown in Table 1.

Table 1

Note: ^* Compared with the control group 1, there is a significant difference (P <0.05); ^** shows a significant difference compared with the control group 1 (P <0.01);

^# shows a significant difference compared with the control group 2 (P<0.05);^## shows a significant difference compared with the control group 2 (P<0.01).

The results in Table 1 show that there are 2 to 4 reaction sites on the new carrier synthesized by the present invention, and the actual synthesis scale is higher under the same synthesis scale, so that more products are obtained; compared with the solid phase synthesis, the new one is adopted. The synthetic carrier synthetic peptide can effectively reduce the amount of organic solvent DMF and DCM; compared with the liquid phase synthesis, the synthesis of the polypeptide by the new carrier can reduce the synthesis step and reduce the amount of the organic solvent, thereby improving the total yield of the product.

The compounds provided by the present invention and their preparation methods and applications are described in detail above. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims

A compound characterized by having the structure shown in Formula II:

Wherein R 1 and R 2 are independently selected from H or
The method for preparing a compound according to claim 1, wherein the compound represented by the formula III is subjected to a halogenation reaction, an ether reaction with a p-hydroxybenzoic acid ester, and a reduction reaction;

Wherein R 1 and R 2 are independently selected from H or
The preparation method according to claim 2, wherein the reagent for the halogenation reaction is one or two of HCl or HBr;

The reaction temperature of the halogenation reaction is 90-110 ° C, the reaction time is 12-24 h, the catalyst is a mixture of one or more of concentrated sulfuric acid and zinc chloride, and the reaction solvent is one of water and acetic acid. Or a mixture of two or more.
The preparation method according to claim 2 or 3, wherein the p-hydroxyphenyl alkanoate is one of methyl p-hydroxybenzoate, methyl p-hydroxyphenylacetate or methyl p-hydroxyphenylpropionate. Or a mixture of two or more.
The preparation method according to any one of claims 2 to 4, wherein the reaction temperature of the ether-forming reaction is 90 to 110 ° C, the reaction time is 8 to 24 hours, and the catalyst is potassium carbonate, sodium carbonate, and hydroxide. One or a mixture of two or more of potassium or sodium hydroxide, and the reaction solvent is one or a mixture of two or more of DMF, tetrahydrofuran or acetone.
The preparation method according to any one of claims 2 to 5, wherein the reducing agent used in the reduction reaction is one or both of diisobutylaluminum hydride, lithium tetrahydrogenate or sodium borohydride. The above mixture.
The preparation method according to any one of claims 2 to 6, wherein the reaction temperature of the reduction reaction is -5 to 5 ° C, the reaction time is 12 to 24 hours, and the reaction solvent is tetrahydrofuran or dioxane. One or a mixture of two or more of toluene.
Use of a compound according to claim 1 or a compound prepared by the production method according to any one of claims 2 to 7 in the synthesis of a polypeptide.
Use of a compound according to claim 1 or a compound obtained by the production method according to any one of claims 2 to 7 for the preparation of angiotensin II.
A method for producing angiotensin II, which comprises a compound obtained according to claim 1 or a compound obtained by the production method according to any one of claims 2 to 7 as a raw material, and a fully protected peptide is synthesized in a liquid phase. It is obtained by cleavage and purification.