A method for phosphating p-hydroxyphenylhydrazine compounds and its use in assisting the preparation of Ang1-7 peptide compounds

Abstract

The present invention relates to a phosphated p-hydroxyphenylhydrazine compound and a method for assisting in the preparation of Ang1-7 peptide compounds, belonging to the technical field of organic synthesis. The structural general formula of PPHPH is as follows. The present invention also provides an application of the phosphated p-hydroxyphenylhydrazine compound PPHPH in the preparation of Ang1-7 peptide compounds, and a method for assisting in the preparation of Ang1-7 peptide compounds with the phosphated p-hydroxyphenylhydrazine compound PPHPH. The present invention combines the advantages of liquid-phase and solid-phase synthesis methods, and can synthesize and prepare Ang1-7 peptides more simply, quickly, economically and efficiently. Moreover, the PPHPH carrier residue can be recovered and directly reused, reducing waste of raw materials, reducing waste pollution, saving costs and being environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and particularly relates to a phosphated p-hydroxyphenylhydrazine compound PPHPH and a method for assisting in the preparation of Ang1-7 peptide compounds thereof. Background Art

[0002] The renin-angiotensin system (RAS) is a hormone system that regulates blood pressure, fluid balance, and the formation of atherosclerosis, and plays an important role in cardiovascular diseases. Angiotensin (1-7) (Ang 1-7) is an endogenous heptapeptide hormone in RAS, which has vasodilatory and anti-proliferative properties, and its structure is as Figure 2 shown.

[0003] The polypeptide conversion in RAS and the generation process of Ang 1-7 are as Figure 3 shown. Angiotensinogen is cleaved at the Leu 10 -Val 11 bond under the action of renin to generate angiotensin I (Ang-I), and Ang-I is inactive. It is further cleaved by angiotensin-converting enzyme (ACE) into the octapeptide angiotensin II. Binding of Ang-II to the AT1 receptor leads to an increase in blood pressure, and binding to the AT2 receptor causes cell differentiation and apoptosis. Under the action of angiotensin-converting enzyme 2 (ACE2), Ang-II is cleaved into the heptapeptide Ang1-7. Ang 1-7 binds to the Mas receptor, participates in signal transduction, and regulates physiological activities. Ang 1-7 mainly protects neurons in the central nervous system, and can also prevent axonal demyelination through oligodendrocytes. Ang 1-7 has antioxidant and anti-inflammatory effects, can weaken the development of hypertension, slow down the pathological progression of atherosclerosis, prevent thrombus formation events, help reduce the risk of ischemic stroke, reduce the area of cerebral infarction, and improve neurological dysfunction. There are also reports that Ang 1-7 is an analgesic in cancer-induced bone pain. In addition, Ang 1-7 has a cardioprotective effect. Treatment with blood Ang 1-7 can weaken Ang-II-induced cardiac hypertrophy, weaken cardiac fibrosis without affecting blood pressure, and produce vasodilation in the aortic ring and several vascular regions.

[0004] Ang 1-7 is an endogenous regulator of cardiovascular cell growth with a short half-life. To further study its function, it is necessary to synthesize Ang 1-7 on a certain scale. In chemical synthesis, the SPPS method is often used to synthesize it on 2-chlorotributyl chloride resin, with a loading capacity of 1.43 mmol / g. After the amino acids are connected, the peptide chain is cleaved from the carrier, and the resin cannot be recycled. Moreover, SPPS consumes a large amount of reagents and amino acid raw materials, and is not environmentally friendly and atom economical. It can be seen that the currently reported chemical synthesis methods of Ang1-7 peptides mainly use the traditional solid-phase synthesis method, which has problems such as cumbersome separation and purification steps, large waste of raw materials and solvents, and a large amount of resin solid waste that is difficult to degrade, resulting in serious environmental pollution. Summary of the Invention

[0005] In order to solve the deficiencies in the prior art, aiming at the deficiencies of the existing synthesis methods, such as environmental pollution, poor selectivity, high price, etc., the present invention provides a phosphated p-hydroxyphenylhydrazine compound and a method for its auxiliary preparation of Ang1-7 peptide compounds, mainly solving the problems of low yield, many by-products, and complex production process existing in current biological synthesis and chemical synthesis, enhancing the greening and scale of the Ang1-7 peptide synthesis process, and being beneficial to emission reduction, consumption reduction, cost saving, environmental protection and sustainable development.

[0006] A phosphated p-hydroxyphenylhydrazine compound provided by the present invention has a general structural formula of formula PPHPH:

[0007]

[0008] In the formula, is PG is Boc, Bn, Cbz or Fmoc.

[0009] The present invention provides a preparation method of a phosphated p-hydroxyphenylhydrazine compound, comprising the following steps:

[0010] Dissolve a certain amount of phenol in water, add a catalyst, then add a di-PG azo compound, place it in an ice bath and stir. After the phenol is completely consumed, end the reaction to obtain an N,N-di-PG-protected p-hydroxyphenylhydrazine compound;

[0011] Use diphenylphosphoryl chloride, phosphorus oxychloride or hexachlorocyclotriphosphazene as the phosphating reagent, and react with the N,N-di-PG p-hydroxyphenylhydrazine compound in a solvent under the action of a catalyst and an acid-binding agent by stirring on an ice bath to obtain a phosphated N,N-di-PG p-hydroxyphenylhydrazine compound, which is the phosphated p-hydroxyphenylhydrazine compound;

[0012] Among them, PG is Boc, Bn, Cbz or Fmoc.

[0013] The present invention provides an application of a phosphated p-hydroxyphenylhydrazine compound in the preparation of an Ang1-7 peptide compound.

[0014] The present invention provides a method for assisting in the preparation of an Ang1-7 peptide compound with a phosphated p-hydroxyphenylhydrazine compound, comprising the following steps:

[0015] Removing the PG protecting group on the phosphated p-hydroxyphenylhydrazine compound to obtain a molecular tag; wherein, PG is Boc, Bn, Cbz or Fmoc;

[0016] Using the molecular tag as a carrier, successively coupling with N-terminal protected proline, histidine with both N-terminal and side-chain protected, N-terminal protected isoleucine, tyrosine with both N-terminal and side-chain protected, N-terminal protected valine, and arginine with both N-terminal and side-chain protected, and performing a reaction for removing the N-terminal protecting group, and then coupling with aspartic acid with both N-terminal and side-chain protected to obtain an Ang1-7 peptide precursor D;

[0017] Wherein, the N-terminal protecting group includes Fmoc, Boc, Cbz or Trt; the side-chain protecting group includes Boc, Pbf, tBu or Trt;

[0018] Clipping the molecular tag on the Ang1-7 peptide precursor D, removing the side-chain protecting group, and performing purification treatment to obtain an Ang1-7 peptide compound.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: A phosphated p-hydroxyphenylhydrazine compound (PPHPH) provided by the present invention and a method for assisting in the preparation of an Ang1-7 peptide compound. This method is mainly a liquid-phase synthesis method of Ang1-7 assisted by the PPHPH carrier. Utilizing the auxiliary precipitation effect of the PPHPH carrier, through a strategy of coupling with an equivalent amount of amino acids in a liquid-phase reaction and removing the Boc protection, the preparation method of Ang1-7 is optimized and simplified, and the recyclability and reusability of the PPHPH carrier are verified. A specific example is to use 4-diphenylphosphonooxyphenyl hydrazine protected by the Boc group in place of the resin in SPPS to complete the synthesis of Ang 1-7. After the Boc group in the compound is removed, 4-diphenylphosphonooxyphenyl hydrazine (4-diphenylphosphooxyphenyl hydrazine, DPOPH) is obtained, and the exposed amino group can be used as a reaction site for amino acids for peptide chain extension. The C-terminal of the amino acid forms a strong amide bond with the amino group in DPOPH. After the amino acid connection is completed, the peptide chain can be sheared off from the tag after oxidizing the hydrazino group to a nitrogen-nitrogen double bond and released into nitrogen harmless to the environment. Description of the Drawings

[0020] Figure 1HPLC analysis of the Ang1-7 peptide product.

[0021] Figure 2 The structure of angiotensin 1-7.

[0022] Figure 3 Schematic diagram of the polypeptide conversion and Ang 1-7 generation in the renin-angiotensin system. Detailed implementation manners

[0023] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the listed embodiments are only for facilitating the understanding of the core methods and application fields of the present invention, but the scope of the present invention is not limited thereto.

[0024] In the following various embodiments, the experimental methods and detection methods described, unless otherwise specified, are all conventional methods; the reagents and materials described, unless otherwise specified, can all be purchased in the market.

[0025] To avoid the deficiencies of existing synthesis methods, such as environmental pollution, poor selectivity, high price, etc., the present invention mainly uses the liquid-phase synthesis method of Ang1-7 peptide assisted by PPHPH as a carrier. Utilizing the auxiliary precipitation effect of the PPHPH carrier, through the strategy of coupling with equimolar amino acids and deprotecting Boc in liquid-phase reactions, the preparation method of Ang1-7 peptide is optimized and simplified, and the recyclability and reusability of the PPHPH carrier are verified. In a specific case, 4-diphenylphosphooxyphenyl hydrazine protected by the Boc group is used to replace the resin in SPPS to complete the synthesis of Ang1-7, and the synthesis route of phosphated p-hydroxyphenylhydrazine PPHPH and the liquid-phase synthesis route of DPOPH-assisted Ang1-7 peptide. After the Boc group in the compound is removed, 4-diphenylphosphooxyphenyl hydrazine (DPOPH) is obtained, and the exposed amino group can be used as the reaction site of amino acids for peptide chain extension. The C-terminus of the amino acid forms a strong amide bond with the amino group in DPOPH. After the amino acid connection is completed, the peptide chain can be cleaved from the label by oxidizing the hydrazine group to a nitrogen-nitrogen double bond and released into nitrogen that is harmless to the environment.

[0026] Synthesis route of phosphated p-hydroxyphenylhydrazine PPHPH:

[0027]

[0028] DPOPH-assisted liquid-phase synthesis route of Ang1-7 peptide:

[0029]

[0030] A kind of phosphated p-hydroxyphenylhydrazine compound provided by the present invention has a general structural formula of formula PPHPH:

[0031]

[0032] In the formula, is PG is Boc, Bn, Cbz or Fmoc. When is The structural formula of the phosphated p-hydroxyphenylhydrazine compound is as follows: It is called bis(diphenylphosphoryloxy)phenylhydrazine DPOPH; it means that after deprotection (PG = H), it is used as a carrier for polypeptide synthesis.

[0033] When is The structural formula of the phosphated p-hydroxyphenylhydrazine compound is as follows: It is called tris(p-hydrazinophenyl) phosphate TPHPP; it means that after deprotection (PG = H), it is used as a carrier for polypeptide synthesis.

[0034] When is The structural formula of the phosphated p-hydroxyphenylhydrazine compound is as follows: It is called hexakis(p-hydrazinophenoxy)cyclotriphosphazene HPHPC; it means that after deprotection (PG = H), it is used as a carrier for polypeptide synthesis.

[0035] The present invention provides a method for preparing a phosphated p-hydroxyphenylhydrazine compound, comprising the following steps:

[0036] Dissolve a certain amount of phenol in water, add a catalyst, then add a di-PG azo compound, place it in an ice bath and stir. After the phenol is completely consumed, end the reaction to obtain an N,N-di-PG-protected p-hydroxyphenylhydrazine compound;

[0037] Use diphenylphosphoryl chloride, phosphorus oxychloride or hexachlorocyclotriphosphazene as a phosphating reagent, and react with the N,N-di-PG p-hydroxyphenylhydrazine compound in a solvent under the action of a catalyst and a deacidifying agent on an ice bath with stirring to obtain a phosphated N,N-di-PG p-hydroxyphenylhydrazine compound, which is the phosphated p-hydroxyphenylhydrazine compound;

[0038] Among them, PG is Boc, Bn, Cbz or Fmoc.

[0039] The present invention provides an application of a phosphated p-hydroxyphenylhydrazine compound in the preparation of Ang1-7 peptide compounds.

[0040] The present invention provides a method for assisting in the preparation of Ang1-7 peptide compounds using a phosphated p-hydroxyphenylhydrazine compound, comprising the following steps:

[0041] Deprotect the PG protecting group on the p-hydroxyphenylhydrazine phosphate compound to obtain a molecular tag; wherein, PG is Boc, Bn, Cbz or Fmoc;

[0042] Using the molecular tag as a carrier, sequentially carry out coupling reactions and N-terminal protecting group deprotection reactions with N-terminal protected proline Fmoc-Pro-OH, N-terminal and side-chain protected histidine Fmoc-His(Trt)-OH, N-terminal protected isoleucine Fmoc-Ile-OH, N-terminal and side-chain protected tyrosine Fmoc-Tyr(OtBu)-OH, N-terminal protected valine Fmoc-Val-OH, and N-terminal and side-chain protected arginine Fmoc-Arg(Pbf)-OH, and then carry out a coupling reaction with N-terminal and side-chain protected aspartic acid PG1-Asp(OtBu)-OH to obtain the Ang1-7 peptide precursor D;

[0043] Among them, the N-terminal protecting groups include Fmoc, Boc, Cbz or Trt; the side-chain protecting groups include Boc, Pbf, tBu or Trt; PG1 is Boc, Cbz or Trt;

[0044] Clip the molecular tag on the Ang1-7 peptide precursor D, deprotect the side-chain protecting group, and after purification, obtain the Ang1-7 peptide compound.

[0045] Specifically, before clipping the molecular tag on the Ang1-7 peptide precursor D, treat the Ang1-7 peptide precursor D with a dichloromethane solution of copper acetate or NBS as an oxidant and pyridine as a catalyst.

[0046] Among them, in the process of obtaining the Ang1-7 peptide precursor D, it includes:

[0047] S1. Using the molecular tag as a carrier, sequentially carry out a coupling reaction and an N-terminal protecting group deprotection reaction with N-terminal protected proline Fmoc-Pro-OH to obtain the product B; the N-terminal protecting group is Fmoc;

[0048] S2. Repeat S1. Using the product B as a carrier, replace N-terminal protected proline Fmoc-Pro-OH with N-terminal and side-chain protected histidine Fmoc-His(Trt)-OH, N-terminal protected isoleucine Fmoc-Ile-OH, N-terminal and side-chain protected tyrosine Fmoc-Tyr(OtBu)-OH, N-terminal protected valine Fmoc-Val-OH, and N-terminal and side-chain protected arginine Fmoc-Arg(Pbf)-OH in sequence, and sequentially carry out a coupling reaction and an N-terminal protecting group deprotection reaction. After the reaction, obtain the product C;

[0049] The product C is coupled with L-aspartic acid PG1-Asp(OtBu)-OH to obtain the Ang1-7 peptide precursor D.

[0050] Furthermore, the structural formula of the product B is as follows:

[0051]

[0052] In the formula, is

[0053] The structural formula of the product C is as follows:

[0054]

[0055] In the formula, is

[0056] The structural formula of the Ang1-7 peptide precursor D is as follows:

[0057]

[0058] In the formula, is PG1 is Boc, Cbz or Trt.

[0059] The structural formula of the Ang1-7 peptide compound is as follows:

[0060]

[0061] In the formula, Nu is OH, OR, NH2, NHR, NR, SH or SR.

[0062] In one embodiment, a method for preparing an Ang1-7 peptide compound assisted by a phosphated p-hydroxyphenylhydrazine compound includes:

[0063] Step 1, synthesis and preparation of an N,N-diPG p-hydroxyphenylhydrazine compound: Dissolve 1.0 equivalent of phenol in 20 - 30 mL of water, add 0.02 - 0.05 equivalent of silver oxide as a catalyst, and then add 2 - 3 equivalents of a diPG azo compound DPGA. Place it in an ice-water bath and stir for 3 - 6 hours. Stop the reaction when TLC monitors that the phenol is completely consumed. Extract the product in the aqueous phase with ethyl acetate, combine the organic phases, add anhydrous sodium sulfate for drying, concentrate and separate the compound using a silica gel chromatography column. The mobile phase is a mixed solution of ethyl acetate and petroleum ether. First, rinse off the excess DPGA with a mixed solution with a volume ratio of 1:10, and then continue to rinse with a mixed solution with a volume ratio of 1:3 to obtain the intermediate, which is the N,N-diPG-protected p-hydroxyphenylhydrazine compound.

[0064] The PG group refers to common amino protecting groups such as Boc, Bn, Cbz, Fmoc, etc.

[0065] Step 2: Synthesis and preparation of phosphated N,N-diPG p-hydroxyphenylhydrazine compound: Using diphenylphosphoryl chloride, phosphorus oxychloride or hexachlorocyclotriphosphazene as the phosphating reagent, and using a base as the catalyst and acid-binding agent in a suitable solvent, react with 1 - 1.5 equivalents of the N,N-diPG p-hydroxyphenylhydrazine compound prepared above on an ice bath and stir for 3 - 5 hours. Stop the reaction when TLC monitors that the N,N-diPG p-hydroxyphenylhydrazine compound is completely consumed. Rotavapor to remove the solvent, dissolve the residue in ethyl acetate, and wash it successively with saturated ammonium chloride solution, 10% sodium bicarbonate solution and deionized water, 1 - 2 times each. After drying the ethyl acetate phase with anhydrous sodium sulfate, rotavapor to remove ethyl acetate to obtain the phosphated N,N-diPG p-hydroxyphenylhydrazine compound.

[0066] The reaction solvent can be one or several of common solvents such as tetrahydrofuran, acetonitrile, dichloromethane, chloroform.

[0067] The base used as the catalyst and acid-binding agent can be one or several of common organic bases such as triethylamine, diisopropylethylamine, DBU, DMAP, NMM.

[0068] Step 3: Preparation of PPHPH molecular tag: Deprotect the above-prepared phosphated N,N-diPG p-hydroxyphenylhydrazine compound to obtain the phosphated p-hydroxyphenylhydrazine PPHPH compound, which can be used as a molecular tag for assisting liquid-phase peptide synthesis. According to the type of protecting group PG being one or two of Boc, Bn, Cbz, Fmoc, select a deprotection reagent system specific to the protecting group. Generally, stir at room temperature for 1 - 3 hours to completely remove the specific protecting group PG. Stop the reaction when TLC monitors that the raw material spot is completely consumed, and obtain the PPHPH molecular tag after separation and purification.

[0069] Step 4: Coupling of PPHPH molecular tag with the first Fmoc-protected proline Fmoc-Pro-OH: Use PPHPH to replace the resin in solid-phase peptide synthesis, and react with 1.0 - 1.2 equivalents of Fmoc-Pro-OH under the coupling agent system and conditions common in the Fmoc peptide synthesis strategy. Stir at room temperature for 1 - 3 hours. Stop the reaction when TLC monitors that PPHPH is completely consumed. Wash, concentrate, assist in precipitation, and separate the reaction mixture to obtain compound A, the crude product of Fmoc-Pro-PPHPH;

[0070] The PPHPH molecular tag is one of bis(diphenylphosphoryloxy)phenylhydrazine DPOPH, tris(p-hydrazinophenyl)phosphate TPHPP, and hexakis(p-hydrazinophenoxy)cyclotriphosphazene HPHPC;

[0071] Step 5, Purification and Refinement of Compound A: Dissolve the crude product of the obtained product A above in an appropriate amount of ethyl acetate, and then add an alkane or ether solvent with a low polarity and a volume ratio of 5-10. By virtue of the property that the PPHPH auxiliary group is easily crystallized and precipitated in the solvent system, perform filtration, washing or recrystallization operations on the product A to obtain the purified product A;

[0072] Step 6, Removal of the N-terminal protecting group Fmoc: Treat the purified product A with a de-Fmoc reagent, stir and react at 10-150 °C for 0.5-12 hours to obtain the product B with deprotected amino group, H-Pro-PPHPH;

[0073] Add an alkane or ether solvent with a low polarity and a volume ratio of 5-10 to the product B. By virtue of the property that the PPHPH auxiliary group is easily crystallized and precipitated in the solvent system, separate the product B from other impurities;

[0074] Perform filtration, washing or recrystallization operations on the separated product B to obtain the purified product B;

[0075] Step 7, Peptide Chain Extension on the Carrier: Repeat the above steps (4), (5) and (6), and sequentially perform coupling reactions with the N-terminal and side-chain protected amino acids Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, PG1-Asp(OtBu)-OH to prepare the precursor D of Ang1-7 peptide and its analogs, PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH.

[0076] The PG1 group refers to common amino protecting groups such as Boc, Cbz, Trt, etc.

[0077] Step 8. Oxidation and removal of the PPHPH carrier by the "one-pot method": First, use a dichloromethane solution of copper acetate or NBS as the oxidant, add pyridine as the catalyst, and treat the precursor D of Ang1-7 peptide and its analogs prepared in the previous step. Stir overnight at room temperature to completely oxidize the hydrazino group into an azo group. Subsequently, add 25 - 30% aqueous lithium hydroxide / tetrahydrofuran and continue stirring for 3 - 5 hours to completely remove the PPHPH carrier. After rotary evaporation and concentration, add deionized water, adjust the pH value to about 7, and extract with ethyl acetate. After liquid separation, combine the ethyl acetate phases and wash with deionized water. The ethyl acetate phase is rotary evaporated and concentrated to about one-fourth of the original volume, and a low-polarity ether solvent is added for precipitation. After filtration, the obtained filter cake is compound E, PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-OH.

[0078] Step 9. Removal of side-chain protecting groups and separation and purification of Ang1-7 peptide or its analogs: Treat the obtained compound E above with a side-chain deprotection cocktail reagent, i.e., trifluoroacetic acid / triisopropylsilane / water = 95:2.5:2.5, and stir at room temperature for 1 - 3 hours to simultaneously remove all the protecting groups such as Boc, Pbf, tBu, and Trt on the side chain. The obtained deprotection reaction mixture is rotary evaporated and concentrated to about one-third of the original volume, extracted with ethyl acetate, and the aqueous solution obtained after liquid separation is freeze-dried to obtain the crude product of Ang1-7 peptide or its analogs. After purification and refinement by preparative HPLC, the pure product of Ang1-7 peptide or its analogs, H-Asp-Arg-Val-Tyr-Ile-His-Pro-OH, is obtained.

[0079] Step 10. Method for recycling the PPHPH auxiliary group: Concentrate the filtrate obtained in step (8) by rotary evaporation to about one-fourth of the original volume, add a low-polarity alkane solvent, and utilize the property that PPHPH is easily crystallized and precipitated in different solvent systems to separate PPHPH from other impurities; perform filtration, washing, or recrystallization operations on the separated PPHPH to obtain purified PPHPH, which can be reused directly or after regeneration as an auxiliary group.

[0080] In step 4, the acetal intermediate compound Fmoc-Pro-PPHPH formed by the condensation coupling of proline Fmoc-Pro-OH and the carrier PPHPH has the following general molecular structure formula:

[0081]

[0082] In step 7, the intermediate compound Fmoc-His(Trt)-Pro-PPHPH is formed by coupling histidine Fmoc-His(Trt)-OH with the product H-Pro-PPHPH after de-Fmoc of Fmoc-Pro-PPHPH. The general formula of the molecular structure of Fmoc-His(Trt)-Pro-PPHPH is:

[0083]

[0084] The intermediate compound Fmoc-Ile-His(Trt)-Pro-PPHPH is formed by coupling isoleucine Fmoc-Ile-OH with the product H-His(Trt)-Pro-PPHPH after de-Fmoc of Fmoc-His(Trt)-Pro-PPHPH. The general formula of the molecular structure of Fmoc-Ile-His(Trt)-Pro-PPHPH is:

[0085]

[0086] The intermediate compound Fmoc-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is formed by coupling tyrosine Fmoc-Tyr(OtBu)-OH with the product H-Ile-His(Trt)-Pro-PPHPH after de-Fmoc of Fmoc-Ile-His(Trt)-Pro-PPHPH. The general formula of the molecular structure of Fmoc-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is:

[0087]

[0088] The intermediate compound Fmoc-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is formed by coupling valine Fmoc-Val-OH with the product H-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH after de-Fmoc of Fmoc-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH. The general formula of the molecular structure of Fmoc-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is:

[0089]

[0090] The intermediate compound Fmoc-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH, which is coupled with the product H-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH after de-Fmoc of Fmoc-Arg(Pbf)-OH and Fmoc-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH. The general molecular structure formula of Fmoc-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is as follows:

[0091]

[0092] The intermediate compound PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH, which is coupled with the product H-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH after de-Fmoc of PG1-Asp(OtBu)-OH and Fmoc-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH. The general molecular structure formula of PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is as follows:

[0093]

[0094] In step 8, the oxidation product of PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is oxidized with copper acetate or NBS to an azo compound. The general molecular structure formula of the azo of the oxidation product of PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPH is as follows:

[0095]

[0096] The azo intermediate compound PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-PPHPA, after being sheared with a tetrahydrofuran solution of a nucleophile Nu:, is separated and purified to obtain the intermediate compound PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-Nu. The general molecular structure formula of the intermediate compound PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-Nu is:

[0097]

[0098] In step 9, the intermediate compound PG1-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-Nu is treated with a "trifluoroacetic acid cocktail" reagent. After completely removing all the protecting groups, the target compound Ang1-7 peptide or its analogue H-Asp-Arg-Val-Tyr-Ile-His-Pro-Nu is obtained. The general molecular structure formula of the target compound Ang1-7 peptide or its analogue is:

[0099]

[0100] It should be noted that in the preparation method provided by the present invention, the coupling agent used in the coupling reaction involved is

[0101] Some commonly used abbreviations in the present invention have the following meanings:

[0102] Boc: tert-butoxycarbonyl; DEA: diethylamine; DMAP: 4-dimethylaminopyridine; DMF: N,N-dimethylformamide; DPOPH: 4-Diphenylphosphinoxyl phenylhydrazine; DPPC: diphenylphosphoryl chloride; EDC-HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Fmoc: fluorenylmethoxycarbonyl; HATU: 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HCCTP: Hexachlorocyclotriphosphazene; HPHPC: Hexakis(p-hydrazinobenzyloxy)cyclotriphosphazene; HOBT: 1-hydroxybenzotriazole; HBTU: O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate; NBS: N-bromosuccinimide; NMM: N-methylmorpholine; NMP:

[0103] N-methylpyrrolidone; PG: protecting group; PHPH: p-hydoxylphenylhydrazine; PPHPA: Phosphated p-hydoxylphenyl azo; PPHPH: Phosphated p-hydoxylphenyl hydrazine; PyBop: Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; TCOP: Phosphorus oxychloride; TEA: Triethylamine; TFA: Trifluoroacetic acid; TPHP: Tris(p-hydrazinophenyl) phosphate; THF: Tetrahydrofuran; TIPS: Triisopropylsilyl.

[0104] The following examples provide specific synthetic methods for preparing the above compounds and the corresponding intermediate compounds.

[0105] Example 1. Preparation of Phosphated p-Hydoxylphenyl Hydrazine (PPHPH)

[0106] Synthesis experimental procedure of phenylhydrazine derivatives: Weigh phenol (1.88 g, 20 mmol, 1.0 eq), silver oxide (139 mg, 0.6 mmol, 0.03 eq) and di-tert-butyl azodicarboxylate (DTBA, 9.2 g, 40 mmol, 2.0 eq) into a 50 mL round-bottom flask, add 20 mL of water, and react under an ice bath for 5 h. Since DTBA is in excess, the reaction can be stopped after monitoring by TLC that phenol is completely consumed. Extract the product in the aqueous phase with ethyl acetate, combine the organic phases, add anhydrous sodium sulfate for drying, concentrate and separate the compound by silica gel column chromatography. The mobile phase is a mixture of ethyl acetate and petroleum ether. First, wash away the excess DTBA with a mixture of 1:10 by volume, and then continue to wash with a mixture of 1:3 by volume to obtain intermediate 3e. The yield after purification is 80%. The intermediate is further phosphonylated to obtain compound 3f with a conversion rate of 95%, and the total yield of the two steps is 76%.

[0107] Product characterization of 1-(4-hydroxyphenyl)hydrazine-1,2-dicarboxylic acid di-tert-butyl ester (3e): White solid, R f = 0.30 (V EA :V PE = 1:2), 1 1H NMR (400 MHz, DMSO-d6) δ 9.44 (d, J = 47.3

[0108] Hz, 2H), 7.08 (s, 2H), 6.70 (s, 2H), 1.41 (d, J = 9.1 Hz, 18H). 13 13C NMR (1

[0109] 01 MHz, DMSO) δ 155.52, 154.00, 134.67, 125.82, 115.16, 80.73, 79.94, 40.18, 28.29.

[0110] 1-(4-((Diphenylphosphoryl)oxy)phenylhydrazine-1,2-dicarboxylic acid di-tert-butyl ester (3f) Product characterization: White solid, R f = 0.25 (V EA :V PE = 1:1), 1 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 7.88 (s, 4H), 7.66 - 7.51 (m, 6H), 7.23 (s, 4H), 1.38 (d, J = 34.3 Hz, 18H). 13 13C NMR (101 MHz, DMSO) δ 155.56, 139.46, 133.24, 132.03, 131.92, 130.47, 129.31, 120.84, 120.80, 81.47, 80.33, 28.18. 31 31P NMR (162 MHz, DMSO) δ 29.36. HRMS (ESI) m / z calcd for C 28 H 34 N2O6P + (M + H) + = 525.21490, found 525.21521.

[0111] Example 2, Removal of the Boc Group from Phenylhydrazine Labels

[0112] The removal reaction of the Boc group from the phenylhydrazine label is shown in Reaction Scheme (1). The dry compound 3f (324 mg, 1 mmol, 1 eq) was placed in a dry and clean round-bottom flask, and 3 mL of dried dichloromethane was added to just dissolve it. The system was stirred in an ice bath for ten minutes to completely cool down, and then 1 mL of trifluoroacetic acid (purity > 99%) was added and stirred for another 2 h. During this period, the reaction was monitored by TLC. After sampling, it was alkalized with sodium bicarbonate solution and then spotted after extraction. After the reaction was completed, the solvent was directly evaporated under reduced pressure at room temperature, and the dried dichloromethane was used to carry it several times to remove the residual trifluoroacetic acid, obtaining a yellow oily liquid, which was a mixture. 30 mg was taken and separated by silica gel preparative plate for characterization, and it was confirmed that DPOPH was present in the mixture.

[0113]

[0114] Product characterization of DPOPH: Yellow oily liquid, 11H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 12.4 Hz, 4H), 7.54 (s, 6H), 7.30 (s, 1H), 7.07 (d, J = 24.3 Hz, 2H), 6.70 (s, 1H), 3.68 (s, 2H).

[0115] Example 3. Extension of the peptide chain on the DPOPH label

[0116]

[0117] After deprotection of the hydrazino group on the label, the peptide chain was extended. The reaction with the first amino acid is shown in Reaction Scheme (2). Weighed Fmoc-Pro-OH (337 mg, 1 mmol, 1 eq), EDCI (192 mg, 1 mmol, 1 eq) and HOBt (135 mg, 1 mmol, 1 eq) into a dry and clean round-bottom flask, added 20 mL of anhydrous dichloromethane, and stirred in an ice bath to form an active ester for easy amidation reaction. After 30 minutes, the activation solution was added to the dried DPOPH mentioned above, and then DIEA (330 μL, 2 mmol, 2 eq) was added. Stirred at room temperature for 45 - 160 minutes, and monitored the reaction by TLC during this period. After the reaction was completed, the dichloromethane in the flask was evaporated to dryness, DIEA was removed, then 30 mL of dichloromethane was added to dissolve and washed three times with saturated sodium bicarbonate solution and saturated brine to remove water-soluble impurities. After drying the organic phase, it was separated and purified by column chromatography. The mobile phase was a mixture of methanol and dichloromethane with a volume ratio of 1:100. The white solid Fmoc-P-DPOPH was collected with a yield of 80%.

[0118] Characterization of Fmoc-Pro-DPOPH product: white solid, Rf = 0.67 (V MeOH :V DCM = 1:100), 1 1H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.77 (s, 1H), 7.92 (s, 6H), 7.73 - 7.31 (m, 12H), 6.95 (d, J = 42.9 Hz, 2H), 6.62 (s, 2H), 4.36 (d, J = 56.0 Hz, 4H), 3.44 (d, J = 36.0 Hz, 2H), 2.22 (d, J = 57.4 Hz, 1H), 1.86 (s, 3H). 1313C NMR (101 MHz, DMSO) δ 172.16, 154.51, 146.60, 144.40, 141.22, 141.20, 132.99, 131.94, 131.87, 130.88, 129.33, 129.20, 128.16, 127.62, 125.66, 121.31, 120.61, 113.33, 67.41, 67.10, 40.62, 40.41, 40.20, 40.00, 39.79, 39.58, 39.37, 31.87, 30.51, 24.56, 23.56. 31 31P NMR (162 MHz, DMSO) δ 28.60. HRMS (ESI) m / z calcd for C 38 18 35 H15N3O5P + (M + H) + = 644.23088, found 644.23126.

[0119] The deprotection reaction of the Fmoc group in compound Fmoc - Pro - DPOPH is shown in Reaction Scheme (3). Dissolve Fmoc - Pro - DPOPH in 6 mL of 25% DEA / MeCN, stir at room temperature for 0.5 h to stop the reaction. After evaporating the solvent, add 3 mL of ethyl acetate to dissolve, then add 15 mL of petroleum ether while shaking. A pale yellow solid appears. Centrifuge to separate the solid and liquid phases. The obtained solid is washed 2 - 3 times with a clean precipitation solution (V PE :V EA = 1:5) to remove residual impurities. The remaining solid is H - Pro - DPOPH with a yield of 95%.

[0120]

[0121] Characterization of H - Pro - DPOPH product: White solid, Rf = 0.45 (V MeOH :V DCM = 1:100), 1 1H NMR (400 MHz, DMSO - d6) δ 9.55 (s, 1H), 7.88 (s, 4H), 7.56 (d, J = 23.2 Hz, 6H), 7.02 (s, 2H), 6.56 (s, 2H), 3.58 (s, 1H), 2.83 (d, J = 18.2 Hz, 2H), 1.80 (d, J = 119.7 Hz, 4H). 13 Note: In the original text, there seems to be a mistake in the chemical formula in . It should be "C18H15N3O5P" instead of "C 38 H 35 N3O5P". The translation is adjusted accordingly. Also, in , "213 times" seems incorrect, and it is translated as "2 - 3 times" based on common sense.13C NMR (101 MHz, DMSO) δ 174.28, 146.69, 143.19, 133.05, 133.02, 132.21, 132.04, 130.86, 129.36, 121.41, 121.37, 113.24, 59.79, 47.24, 30.88, 26.18. 31 31P NMR (162 MHz, DMSO) δ 28.63. HRMS (ESI) m / z calcd for C 23 H 25 N3O3P + (M + H) + = 422.16280, found 422.16278.

[0122] Couple the second amino acid Fmoc-His(Trt)-OH to H-Pro-DPOPH. Weigh Fmoc-His(Trt)-OH (518 mg, 0.84 mmol, 1.1 eq), EDCI (160 mg, 0.84 mmol, 1.1 eq) and HOBt (113 mg, 0.84 mmol, 1.1 eq) into a dry and clean round-bottom flask, then add 20 mL of anhydrous dichloromethane to dissolve. Place it in an ice bath and stir to form an active ester for easy amidation reaction. After 30 minutes, add H-Pro-DPOPH to the above activation solution, then add DIEA (251 μL, 1.52 mmol, 2 eq), and stir at room temperature for 45 minutes. Monitor the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, then add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. After drying the organic phase, separate and purify it by column chromatography. The mobile phase is a mixture of methanol and dichloromethane with a volume ratio of 1:100. Collect the white solid Fmoc-His(Trt)-Pro-DPOPH with a yield of 95%.

[0123] Characterization of Fmoc-His(Trt)-Pro-DPOPH product: white solid, Rf = 0.64 (V MeOH :V DCM = 1:100), 11H NMR (400 MHz, DMSO-d6) δ 10.57 (d, J = 2.8 Hz, 1H), 7.88 (s, 6H), 7.79 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.55 (d, J = 19.0 Hz, 7H), 7.37 (d, J = 36.4 Hz, 14H), 7.17 - 6.84 (m, 11H), 6.55 (d, J = 8.8 Hz, 2H), 4.47 - 4.14 (m, 5H), 3.57 (q, J = 8.4 Hz, 1H), 3.15 (s, 1H), 2.93 - 2.77 (m, 2H), 2.11 - 1.67 (m, 4H). 13 13C NMR (101 MHz, DMSO) δ 171.44, 156.11, 146.75, 144.19, 142.72, 142.49, 141.17, 139.88, 137.88, 137.32, 136.82, 135.84, 133.02, 132.00, 130.95, 129.95, 129.55, 128.65, 128.10, 127.75, 127.51, 125.74, 124.40, 121.22, 120.50, 113.27, 75.07, 66.16, 58.84, 53.19, 47.11, 45.25, 29.71, 24.82, 22.51. 31 31P NMR (162 MHz, DMSO) δ 28.48. HRMS (ESI) m / z calcd for C 63 H 56 N6O6P + (M + H) + = 1023.39935, found 1023.39954.

[0124] Couple the third amino acid Fmoc-Ile-OH to the product H-His(Trt)-Pro-DPOPH obtained by de-Fmoc of Fmoc-His(Trt)-Pro-DPOPH: First, use 6 mL of 25% DEA / MeCN solution to remove the Fmoc protecting group on the Fmoc-His(Trt)-Pro-DPOPH molecule. Then, weigh Fmoc-Ile-OH (280 mg, 0.79 mmol, 1.1 eq), EDCI (152 mg, 0.79 mmol, 1.1 eq), and HOBt (107 mg, 0.79 mmol, 1.1 eq) into a dry and clean round-bottom flask, add 20 mL of anhydrous dichloromethane to dissolve, and stir in an ice bath to form an active ester that is easy to carry out amidation reaction. After 30 minutes, add H-His(Trt)-Pro-DPOPH (738 mg, 0.72 mmol, 1 eq) and DIEA (238 μL, 1.44 mmol, 2 eq) to the above activation solution, stir at room temperature for 60 minutes, and monitor the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, and omit the purification step to directly carry out the operation of removing the Fmoc protecting group. Add 6 mL of 25% DEA / MeCN solution to the flask, stir at room temperature for 30 minutes, then evaporate the solvent in the flask, add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. The organic phase is evaporated and concentrated, add 5 mL of ethyl acetate to completely dissolve it, and add 15 mL of petroleum ether under shaking. The white solid H-Ile-His(Trt)-Pro-DPOPH is collected with a yield of 88%.

[0125] Characterization of the H-Ile-His(Trt)-Pro-DPOPH product: White solid, Rf = 0.45 (V MeOH :V DCM = 1:100), 1 1H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 8.32 (d, J = 7.4 Hz, 1H), 7.84 (dd, J = 12.2, 7.0 Hz, 4H), 7.55 (dd, J = 20.5, 3.0 Hz, 6H), 7.42 - 6.85 (m, 18H), 6.71 (s, 1H), 6.52 (d, J = 8.5 Hz, 2H), 4.69 (q, J = 6.7 Hz, 1H), 4.32 (d, J = 9.0 Hz, 1H), 3.78 - 3.42 (m, 2H), 3.10 - 2.77 (m, 3H), 2.19 - 1.56 (m, 7H), 1.43 - 1.18 (m, 2H), 0.83 - 0.62 (m, 6H). 1313C NMR (101 MHz, DMSO) δ 171.25, 170.83, 146.71, 142.99, 142.68, 142.35, 138.47, 136.18, 133.01, 129.57, 128.65, 121.19, 120.03, 113.27, 75.12, 70.24, 60.39, 59.15, 51.16, 40.60, 40.39, 40.18, 39.97, 39.76, 39.55, 39.34, 38.21, 29.75, 24.88, 23.94, 16.12, 11.96. 31 31P NMR (162 MHz, DMSO) δ 28.36. HRMS (ESI) m / z calcd for C 54 H 57 N7O5P + (M + H) + = 914.41533, found 914.41595.

[0126] Coupling of the fourth amino acid Fmoc-Tyr(OtBu)-OH to H-Ile-His(Trt)-Pro-DPOPH: Weigh Fmoc-Tyr(OtBu)-OH (318 mg, 0.69 mmol, 1.1 eq), EDCI (133 mg, 0.69 mmol, 1.1 eq) and HOBt (94 mg, 0.69 mmol, 1.1 eq) into a dry and clean round-bottom flask, add 20 mL of anhydrous dichloromethane to dissolve, and stir in an ice bath to form an active ester for easy amidation reaction. After 30 minutes, add H-Ile-His(Trt)-Pro-DPOPH (578 mg, 0.63 mmol, 1 eq) to the above activation solution, then add DIEA (208 μL, 1.26 mmol, 2 eq), and stir at room temperature for 60 minutes. Monitor the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, and omit the purification step and directly perform the operation of removing the Fmoc protecting group. Add 6 mL of 25% DEA / MeCN solution to the flask, stir at room temperature for 30 minutes, then evaporate the solvent in the flask, add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. Concentrate the organic phase by evaporation, add 5 mL of ethyl acetate to completely dissolve it, and add 15 mL of petroleum ether with shaking. Collect the white solid H-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH with a yield of 82%.

[0127] Characterization of H-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH product: white solid, Rf = 0.50 (V MeOH :V DCM = 1:80), 1 1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.86 (dd, J = 12.3, 7.2 Hz, 4H), 7.55 (dd, J = 20.0, 7.5 Hz, 7H), 7.33 (s, 10H), 7.14 - 6.81 (m, 14H), 6.52 (d, J = 8.9 Hz, 2H), 4.56 (d, J = 7.0 Hz, 1H), 4.25 (dd, J = 23.7, 8.5 Hz, 2H), 3.66 (d, J = 8.4 Hz, 1H), 3.21 - 2.53 (m, 6H), 2.11 - 1.62 (m, 7H), 1.25 (s, 9H), 0.77 (t, J = 7.0 Hz, 6H). 13 13C NMR (101 MHz, DMSO) δ 171.32, 170.94, 158.70, 154.16, 146.71, 143.38, 142.42, 136.59, 133.02, 132.32, 131.98, 131.88, 130.96, 130.44, 129.53, 129.36, 129.23, 128.66, 128.43, 127.97, 124.70, 123.94, 123.55, 121.21, 118.87, 113.27, 111.10, 78.07, 75.12, 55.91, 43.81, 41.73, 39.99, 37.30, 34.56, 28.98, 26.90, 24.78, 16.15, 15.74, 11.56, 11.50. 31 31P NMR (162 MHz, DMSO) δ 28.45. HRMS (ESI) m / z calcd for C 67 H 74 N8O7P + (M + H) + = 1133.54126, found 1133.54236.

[0128] Coupling the fifth amino acid Fmoc-Val-OH to H-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH: Weigh Fmoc-Val-OH (194 mg, 0.57 mmol, 1.1 eq), EDCI (110 mg, 0.57 mmol, 1.1 eq) and HOBt (77 mg, 0.57 mmol, 1.1 eq) into a dry and clean round-bottom flask, add 20 mL of anhydrous dichloromethane to dissolve, place it in an ice bath and stir to form an active ester for easy amidation reaction. After 30 minutes, add H-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH (585 mg, 0.52 mmol, 1 eq) to the above activation solution, then add DIEA (172 μL, 1.04 mmol, 2 eq), and stir at room temperature for 60 minutes. Monitor the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, and directly perform the operation of removing the Fmoc protecting group without purification steps. Add 6 mL of 25% DEA / MeCN solution to the flask, stir at room temperature for 30 minutes, then evaporate the solvent in the flask, add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. The organic phase is evaporated and concentrated, add 5 mL of ethyl acetate to completely dissolve it, and add 15 mL of petroleum ether under shaking. The white solid H-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH is collected with a yield of 85%.

[0129] Characterization of H-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH product: White solid, Rf = 0.450 (V MeOH :V DCM = 1:80). 1 HNMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.03 (d, J = 9.3 Hz, 1H), 7.86 (dd, J = 12.3, 7.4 Hz, 4H), 7.63 - 7.47 (m, 7H), 7.32 (s, 10H), 7.15 - 6.78 (m, 14H), 6.52 (d, J = 8.5 Hz, 2H), 4.77 - 4.47 (m, 2H), 4.37 - 4.16 (m, 2H), 3.66 (d, J = 8.2 Hz, 1H), 3.20 - 2.67 (m, 6H), 2.07 - 1.63 (m, 7H), 1.40 (s, 1H), 1.22 (d, J = 10.8 Hz, 11H), 0.86 - 0.54 (m, 13H). 13CNMR (101 MHz, DMSO) δ 170.91, 153.74, 146.72, 142.42, 133.01, 131.98, 131.88, 130.14, 129.53, 129.36, 129.23, 128.67, 128.41, 123.84, 121.20, 113.25, 77.97, 75.12, 60.11, 40.00, 31.45, 28.95, 19.92, 17.04, 15.74, 11.49. 31 PNMR (162 MHz, DMSO) δ 28.45. HRMS (ESI) m / z calcd for C 72 H 83 N9O8P + (M + H) + =1232.60967, found 1232.61047.

[0130] Coupling of the sixth amino acid Fmoc-Arg(Pbf)-OH to H-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH: Weigh Fmoc-Arg(Pbf)-OH (314 mg, 0.48 mmol, 1.1 eq), EDCI (93 mg, 0.48 mmol, 1.1 eq), HOBt (65 mg, 0.48 mmol, 1.1 eq) and H-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH (544 mg, 0.44 mmol, 1 eq) into a dry and clean round-bottom flask, add 20 mL of anhydrous dichloromethane to dissolve, then add DIEA (145 μL, 0.88 mmol, 2 eq), and stir at room temperature for 60 minutes, monitoring the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, and directly perform the operation of removing the Fmoc protecting group. Add 6 mL of 25% DEA / MeCN solution to the flask, stir at room temperature for 30 minutes, then evaporate the solvent in the flask, add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. Evaporate and concentrate the organic phase, add 5 mL of ethyl acetate to completely dissolve it, and add 15 mL of petroleum ether with shaking. The white solid H-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH is collected, and the yield is 79%.

[0131] Characterization of H-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH product: White solid, Rf = 0.45 (V MeOH :VDCM = 1:80). 1 HNMR(400 MHz, DMSO-d6) δ 10.70 (s, 1H), 7.86 (dd, J = 12.2, 7.6 Hz, 4H), 7.63 - 7.47 (m, 7H), 7.32 (s, 11H), 7.15 - 6.76 (m, 17H), 6.51 (d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 4.24 (d, J = 31.2 Hz, 3H), 3.57 (d, J = 52.1 Hz, 1H), 3.22 - 2.67 (m, 11H), 2.42 (s, 3H), 2.06 - 1.55 (m, 13H), 1.40 (s, 9H), 1.21 (s, 13H), 0.80 (dd, J = 36.0, 8.5 Hz, 12H). 13 CNMR(101 MHz, DMSO) δ 171.29, 157.89, 156.57, 146.72, 143.01, 142.64, 142.41, 138.41, 137.72, 136.59, 134.67, 133.02, 132.31, 131.98, 131.88, 130.97, 130.03, 129.52, 129.36, 129.23, 128.67, 128.42, 124.75, 123.79, 121.23, 116.71, 113.25, 86.74, 77.96, 75.11, 55.39, 42.94, 28.96, 28.77, 19.45, 18.09, 12.75, 11.43. 31 PNMR(162 MHz, DMSO) δ 28.45. HRMS(ESI) m / z calcd for C 91 H 111 N 13 O 12 PS + (M + H) + = 1640.79280, found 1640.79382.

[0132] Coupling of the seventh amino acid Fmoc-Asp(OtBu)-OH to H-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH: Weigh Fmoc-Asp(OtBu)-OH (158 mg, 0.38 mmol, 1.1 eq), EDCI (74 mg, 0.38 mmol, 1.1 eq) and HOBt (52 mg, 0.38 mmol, 1.1 eq) into a dry and clean round-bottom flask, add 20 mL of anhydrous dichloromethane to dissolve, and stir in an ice bath to form an active ester for easy amidation reaction. After 30 minutes, add H-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH (570 mg, 0.35 mmol, 1 eq) to the above activation solution, then add DIEA (116 μL, 0.70 mmol, 2 eq), and stir at room temperature for 60 minutes. Monitor the reaction by TLC during this period. After the reaction is completed, evaporate the dichloromethane in the flask, remove DIEA, evaporate the solvent in the flask, then add 40 mL of dichloromethane to dissolve and wash three times with saturated sodium bicarbonate and saturated brine to remove water-soluble impurities. After drying the organic phase, evaporate and concentrate, and separate and purify by column chromatography. First, rinse off small-polarity impurities with pure dichloromethane, then rinse with a mixed solution of methanol and dichloromethane with a volume ratio of 1:80, collect the main product, evaporate to obtain a white solid, Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH, with a yield of 81%.

[0133] Characterization of Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH product: White solid, Rf = 0.60 (V MeOH :V DCM = 1:80), 1HNMR (400 MHz, DMSO-d6) δ 10.83 - 10.59 (m, 1H), 8.27 (dd, J = 19.0, 7.3 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.92 - 7.80 (m, 6H), 7.68 (t, J = 7.1 Hz, 3H), 7.63 - 7.47 (m, 5H), 7.44 - 7.27 (m, 10H), 7.16 - 6.74 (m, 11H), 6.51 (d, J = 8.6 Hz, 3H), 4.60 - 4.51 (m, 2H), 4.42 - 4.09 (m, 7H), 3.70 - 3.48 (m, 2H), 3.17 (d, J = 5.2 Hz, 1H), 2.97 (d, J = 31.0 Hz, 5H), 2.73 - 2.61 (m, 2H), 2.47 (s, 4H), 2.41 (s, 3H), 1.98 (s, 4H), 1.88 (p, J = 7.6, 6.0 Hz, 2H), 1.75 - 1.30 (m, 27H), 1.19 (s, 7H), 1.09 - 0.98 (m, 2H), 0.95 (d, J = 6.6 Hz, 1H), 0.90 - 0.68 (m, 11H). 13CNMR(101MHz, DMSO) δ 171.41, 171.28, 171.19, 171.08, 170.99, 170.91, 170.83, 170.57, 169.82, 169.64, 157.89, 156.48, 156.24, 153.74, 146.71, 144.25, 144.12, 143.09, 142.40, 141.16, 138.41, 137.74, 136.58, 133.01, 132.76, 132.29, 132.00, 131.97, 131.89, 131.87, 130.94, 129.97, 129.51, 129.36, 129.23, 128.67, 128.42, 128.10, 127.52, 125.69, 124.73, 123.82, 121.19, 120.57, 119.76, 116.70, 113.24, 86.71, 80.68, 80.65, 77.94, 75.11, 66.22, 60.12, 55.39, 54.03, 52.75, 51.78, 50.13, 49.07, 47.03, 42.92, 37.88, 37.68, 37.45, 34.66, 34.40, 31.25, 29.72, 28.94, 28.75, 28.14, 28.08, 25.25, 24.75, 24.62, 22.96, 21.08, 19.60, 19.44, 18.27, 18.08, 15.68, 12.74, 11.73, 11.41. 31 PNMR(162MHz, DMSO) δ 28.45. HRMS(ESI) m / z calcd for C 114 H 134 N 14 O 17 PS + (M + H) + =2034.95378, found 2034.95618.

[0134] Example 4. Removal of the DPOPH label

[0135] The synthetic route is as follows:

[0136]

[0137] Reaction for cleaving the peptide chain from the label: Weigh the label - loaded heptapeptide compound Fmoc - Asp(OtBu) - Arg(Pbf) - Val -

[0138] Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPH (120 mg, 0.059 mmol, 1 eq) and N-bromosuccinimide (21 mg, 0.118 mmol, 2 eq) were placed in a round-bottom flask, dissolved in 5 mL of anhydrous dichloromethane, and then pyridine (47 μL, 0.59 mmol, 10 eq) was added. The mixture was stirred overnight at room temperature. After the reaction solution was evaporated to dryness under reduced pressure, 5 mL of cold ether was added and sonicated. The impurities were dissolved in the ether, and the yellow solid insoluble in ether was the azo compound Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-DPOPA formed after oxidation. The solid was separated by centrifugation and washed with ether 213 times. The obtained solid was dissolved in 10 mL of tetrahydrofuran, 2.5 - 3.0 mL of 1.0 M lithium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 2 - 3 hours. The pH value was adjusted to near neutral, and then extracted with ethyl acetate. The ethyl acetate layer was washed with water and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the solution was concentrated to dryness by rotary evaporation. Then, cold ether was added for sonication extraction, and the filter cake obtained by filtration was the peptide chain Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-OH after deprotection of the carrier, with a yield of 90%.

[0139] Characterization of Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-OH product: 1 HNMR(400MHz,DMSO-d6)δ11.09(s,1H),9.59(s,1H),8.98 - 8.81(m,4H),8.50(s,4H),7.94(d,J=43.5Hz,8H),7.70(s,3H),7.59 - 7.04(m,21H),6.81(s,1H),4.92 - 4.04(m,10H),3.83 - 2.54(m,12H),2.41(s,1H),2.13(s,1H),1.30(d,J=56.1Hz,31H),1.13 - 1.00(m,2H),0.72(s,9H).

[0140] Example 5: Deprotection of protecting groups on the peptide chain to obtain Ang1-7 peptide

[0141] The synthetic route is as follows:

[0142]

[0143] Reaction for the removal of side-chain protecting groups in the peptide chain: 90 mg of linear peptide Fmoc-Asp(OtBu)-Arg(Pbf)-Val-Tyr(OtBu)-Ile-His(Trt)-Pro-OH was dissolved in 4 mL of 25% DEA / MeCN to remove the Fmoc protecting group. The reaction solution was stirred at room temperature for 30 min. After completion, the reaction solution was evaporated to dryness under reduced pressure, and then dichloromethane was added again and evaporated to dryness to remove the residual DEA. This was repeated 2 - 3 times. 1.9 mL of TFA, 300 μL of methanol, and 200 μL of water (V TFA :V MeOH :V H2O = 95:3:2) were added to the above mixture, and it was stirred at room temperature for 2 h. After completion, the reaction solvent was evaporated to dryness under reduced pressure, and then 5 mL of cold ether was added and sonicated. The removed protecting group residues were dissolved in ether, and the solid insoluble in ether was the Ang1-7 peptide chain H-Asp-Arg-Val-Tyr-Ile-His-Pro-OH. The solid was separated by centrifugation and washed with clean ether 2 - 3 times to obtain 35 mg of yellow solid with a yield of 92%.

[0144] Characterization of H-Asp-Arg-Val-Tyr-Ile-His-Pro-OH product: Yellow solid, 1 HNMR(400MHz,Methanol-d4)δ8.80(s,1H),7.96(s,4H),7.69 - 7.39(m,6H),7.24(s,2H),7.09(d,J = 8.4Hz,2H),6.69(s,2H),4.56 - 4.02(m,7H),3.72(s,2H),3.07(s,14H),2.05(s,3H),1.51(s,6H),1.33(s,10H),1.21(t,J = 7.0Hz,2H),0.88(s,12H). 13 CNMR(101MHz,DMSO)δ175.41,173.28,171.19,170.99,170.83,170.57,157.89,155.24,136.28,131.35,131.24,129.23,118.76,115.70,66.22,61.72,61.58,58.39,57.45,54.03,50.13,49.07,42.82,41.23,37.38,36.88,31.25,31.15,28.94,28.75,24.75,24.62,18.47,14.59,11.01.HRMS(ESI)m / z calcd for C 41 H 62 N 12O 11 + (M+H) + = 898.47375, found 898.47413. The HPLC analysis results are as Figure 1 shown. The retention time of H-Asp-Arg-Val-Tyr-Ile-His-Pro-OH is 2.30 minutes and the purity is greater than 99.5%.

[0145] The method of the present invention can be developed into a general liquid-phase polypeptide synthesis strategy for preparing Ang1-7 peptides and their analogs. The intermediates obtained in each step of the process are easy to separate and purify, can be produced on a large scale, save raw material costs, greatly reduce waste emissions, are beneficial to environmental protection, and improve economic benefits.

[0146] The above embodiments are only some of the embodiments listed for facilitating the understanding of the synthesis and application methods of the materials of the present invention, and are not used to limit the present invention. It can be understood that relevant practitioners can easily make appropriate modifications to this structure. Therefore, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A phosphated p-hydroxyphenylhydrazine compound, characterized in that, The structural general formula is of the formula PPHPH: PPHPH In the formula, PG is Boc, Bn, Cbz or Fmoc.

2. The preparation method of the phosphated p-hydroxyphenylhydrazine compound according to claim 1, characterized in that, It includes the following steps: Dissolve a certain amount of phenol in water, add a catalyst, then add a di - PG - based azo compound, place it in an ice - water bath and stir. After the phenol is completely consumed, end the reaction to obtain an N,N - di - PG - protected p - hydroxy phenylhydrazine compound; Use diphenylphosphoryl chloride, phosphorus oxychloride or hexachlorocyclotriphosphazene as the phosphating reagent, and stir - react with the N,N - di - PG - p - hydroxy phenylhydrazine compound in a solvent under the action of a catalyst and an acid - binding agent on an ice bath to obtain a phosphated N,N - di - PG - p - hydroxy phenylhydrazine compound, which is a phosphated p - hydroxy phenylhydrazine compound; Among them, PG is Boc, Bn, Cbz or Fmoc.

3. Use of the phosphated p - hydroxy phenylhydrazine compound described in claim 1 in the preparation of Ang1 - 7 peptide compounds.

4. A method for assisting in the preparation of Ang1-7 peptide compounds by phosphating p-hydroxyphenylhydrazine compounds, characterized in that, It includes the following steps: Remove the PG protecting group on the phosphated p - hydroxy phenylhydrazine compound to obtain a molecular tag; among them, PG is Boc, Bn, Cbz or Fmoc; Using the molecular tag as a carrier, successively carry out coupling reactions and N - terminal protecting group removal reactions with N - terminal protected proline, N - terminal and side - chain protected histidine, N - terminal protected isoleucine, N - terminal and side - chain protected tyrosine, N - terminal protected valine, and N - terminal and side - chain protected arginine, and then carry out a coupling reaction with N - terminal and side - chain protected aspartic acid to obtain the Ang1 - 7 peptide precursor D; Among them, the N - terminal protecting group includes Fmoc, Boc, Cbz or Trt; the side - chain protecting group includes Boc, Pbf, tBu or Trt; Clip the molecular tag on the Ang1 - 7 peptide precursor D, remove the side - chain protecting group, and after purification treatment, obtain the Ang1 - 7 peptide compound.

5. The method for assisting in the preparation of Ang1-7 peptide compounds by using phosphated p-hydroxyphenylhydrazine compounds according to claim 4, characterized in that, During the process of obtaining the Ang1 - 7 peptide precursor D, it includes: S1. Using the molecular tag as a carrier, successively carry out coupling reactions and N - terminal protecting group removal reactions with N - terminal protected proline Fmoc - Pro - OH to obtain product B; the N - terminal protecting group is Fmoc; S2. Repeat S1. Using product B as a carrier, successively replace N - terminal protected proline Fmoc - Pro - OH with N - terminal and side - chain protected histidine, N - terminal protected isoleucine, N - terminal and side - chain protected tyrosine, N - terminal protected valine, and N - terminal and side - chain protected arginine, and successively carry out coupling reactions and N - terminal protecting group removal reactions. After the reaction ends, obtain product C; Use product C to carry out a coupling reaction with N - terminal and side - chain protected aspartic acid to obtain the Ang1 - 7 peptide precursor D.

6. The method for assisting in the preparation of Ang1-7 peptide compounds by using phosphated p-hydroxyphenylhydrazine compounds according to claim 5, characterized in that, The structural formula of the said product B is as follows: 。 7. The method for assisting in the preparation of Ang1-7 peptide compounds by using phosphated p-hydroxyphenylhydrazine compounds according to claim 5, characterized in that, The structural formula of the said product C is as follows: 。 8. The method for assisting in the preparation of Ang1-7 peptide compounds by using phosphated p-hydroxyphenylhydrazine compounds according to claim 4, wherein The structural formula of the said Ang1 - 7 peptide precursor D is as follows: In the formula, PG1 is Boc, Cbz or Trt.

9. The method for the auxiliary preparation of Ang1-7 peptide compounds by phosphating p-hydroxyphenylhydrazine compounds according to claim 4, characterized in that, The structural formula of the said Ang1 - 7 peptide compound is as follows: In the formula, Nu is OH.

10. The method for the auxiliary preparation of Ang1-7 peptide compounds by using phosphated p-hydroxyphenylhydrazine compounds according to claim 4, characterized in that, Before excising the molecular tag on the Ang1-7 peptide precursor D, the Ang1-7 peptide precursor D was treated with a dichloromethane solution of copper acetate or NBS as the oxidant and pyridine as the catalyst.

Images