A phosphated p-hydroxybenzaldehyde compound and a method for assisting preparation of octreotide therefrom

Abstract

The present application relates to a kind of phosphorus of p-hydroxybenzaldehyde PPHBA compound and its auxiliary preparation octreotide method, belong to organic synthesis technical field.The present application also provides a kind of phosphorus of p-hydroxybenzaldehyde PPHBA compound in preparation octreotide application, and a kind of phosphorus of p-hydroxybenzaldehyde compound auxiliary preparation octreotide method.The present application is with diphenylphosphine acyl oxy benzaldehyde DPOBA as carrier auxiliary octreotide phase synthesis method, using the auxiliary precipitation effect of DPOBA carrier, through liquid phase reaction with equivalent amino acid coupling and the strategy of Fmoc protection, optimize and simplify the preparation method of octreotide, verify the recyclability and reusability of DPOBA carrier.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organic synthesis, and particularly relates to a phosphorized p-hydroxybenzaldehyde compound and a method for assisting preparation of octreotide. BACKGROUND

[0002] Somatostatin is a cyclic peptide compound composed of 14 amino acids and mainly inhibiting secretion of growth hormone in the hypothalamus, but its half-life in the animal body is only 2-4 minutes. In 1982, Bauer synthesized an analog of somatostatin, named octreotide. Octreotide is a cyclic octapeptide containing a pair of disulfide bonds in the molecule, and has similar pharmacological effects to somatostatin, but has a longer duration of action. Octreotide has a definite therapeutic effect on various diseases and tumors, and was approved for marketing by the FDA of the United States in 1988.

[0003] Octreotide is first synthesized by using a liquid-phase fragment condensation method. The fragment is divided into two fragments of N-terminal dipeptide and C-terminal hexapeptide, and the peptide is connected by using an azide method with Boc / Bzl strategy protection, and desalting and salt conversion are performed by using a gel chromatography column. This method has problems of easy oxidation, alkylation and other side reactions of D-Trp when Boc protection is removed by using acid, harsh azide conditions, long time consumption and low yield in salt conversion by using a gel chromatography column. Liu Keliang et al. divide the fragment into N-terminal tripeptide and C-terminal pentapeptide, and perform coupling reaction under the condition of DCM / NMM / DCC / HOBt. After removing side chain protection, oxidation and salt conversion, octreotide acetate is obtained. This process has few side reactions and can be scaled up, but the overall yield is only about 6-10%. LPPS prepares octreotide by using a step-by-step condensation and fragment condensation method to obtain a straight-chain octapeptide with a protection group, and then removes the side chain protection group to perform oxidation to obtain a ring product. In this process, a complex chromatographic separation procedure needs to be established for each step, and organic solvents are wasted.

[0004] Octreotide is usually synthesized by Fmoc strategy in SPPS, and then the linear peptide is cleaved to get the intermediate, and the two cysteines in the sequence are oxidized to form disulfide bond, and finally octreotide is obtained by purification through liquid phase preparation. Yasush et al. used 2-chlorotrityl resin to synthesize octreotide, while Hsieh et al. ingeniously used the specific dihydroxy structure of threonine to form an acetal with p-formaldehyde as a linker to fix the C-terminal of Thr on the resin, and then extend the peptide chain, and finally the peptide chain is cut off for cyclization, but both methods have racemization problems in the reduction cleavage method. Alexei Iarov and Xu Hongyan et al. have successively invented a method for the solid-phase synthesis of octreotide using Fmoc / tBu strategy, which uses chloromethyl resin as a carrier to prepare 3,4-dihydropyranyl hydroxymethyl resin, and then Fmoc-Thr(tBu)-ol is connected to the resin, and then the amino acids are connected one by one according to the Fmoc / tBu solid-phase peptide synthesis method, and the crude product after the peptide chain is cut off is oxidized by air oxidation to form disulfide bond, and then purified by preparative HPLC to obtain octreotide by freezing. This method can avoid the racemization problem in the cleavage. SPPS can realize the automatic production of polypeptide coupling, but the loading rate of resin-based carrier is low, and there are problems such as complex preparation and difficult recovery, which is not environmentally friendly and is not conducive to green and sustainable development.

[0005] The improved and optimized solid-phase synthesis method has been gradually used in the industrial production of octreotide in China. At present, there are many process technologies for preparing octreotide. Most of these existing preparation methods are to synthesize a straight-chain octapeptide by solid-phase synthesis, and then cut the peptide (acidolysis / cleavage), and then perform air oxidation in liquid phase to complete intramolecular cyclization. However, the total yield of these existing patents is generally not high, and all of them are less than 50%, and the purity of some finished products is also relatively low. Patent CN101863961A discloses a preparation method of octreotide, which mainly improves the oxidation step by using excess hydrogen peroxide under specific pH conditions. The final total yield of octreotide is 74%, and the purity reaches 98%; patent CN103102390A discloses a 2+6 segment synthesis method for preparing octreotide, and in the oxidation step, excess hydrogen peroxide or iodine ethanol solution is used for cyclization under specific pH conditions. The total yield of octreotide is about 75%, and the purity reaches 99%. However, these two methods still do not realize the full solid-phase synthesis scheme. Due to technical reasons, the existing technology in China cannot realize high total yield and high purity under full solid-phase synthesis conditions, and the full solid-phase synthesis can greatly simplify the preparation process, shorten the preparation time, and reduce the production cost.

[0006] It can be seen that the current octreotide chemical production method mainly adopts traditional solution synthesis and solid phase synthesis method, and there are problems of complicated separation and purification steps, large waste of raw materials and solvents, and serious environmental pollution caused by difficult degradation of resin solid waste. SUMMARY

[0007] In order to solve the problems in the prior art, in view of the problems of the prior synthesis method, such as environmental pollution, poor selectivity, high price and the like, the application provides a phosphated p-hydroxybenzaldehyde compound and a method for assisting preparation of octreotide, and mainly solves the problems of the current nine-peptide chemical synthesis method, such as more liquid phase reaction steps, long time period, high content of by-product β-type isomer, difficult separation and impurity removal, low product purity, small purification scale, high production cost, small production scale of solid phase reaction, high price and large waste of raw materials, and serious environmental pollution caused by much resin waste.

[0008] The application provides a phosphated p-hydroxybenzaldehyde compound, and the general structure is as follows:

[0009]

[0010] In the formula, R represents is represented as

[0011] The application provides a preparation method of a phosphated p-hydroxybenzaldehyde compound, and the method comprises the following steps.

[0012] The p-hydroxybenzaldehyde is reacted with diphenylphosphoryl chloride, phosphorus oxychloride or hexachlorocyclotriphosphazene under alkaline conditions, after the reaction is completed, the phosphated p-hydroxybenzaldehyde compound is obtained through separation and purification.

[0013] The application provides an application of the phosphated p-hydroxybenzaldehyde compound in preparation of octreotide.

[0014] The application provides a method for assisting preparation of octreotide by using the phosphated p-hydroxybenzaldehyde compound, and the method comprises the following steps.

[0015] The phosphated p-hydroxybenzaldehyde compound is used as an auxiliary group to perform condensation reaction on dihydroxyl or hydroxyl mercaptan on an N-terminal protected aminodiol, after the reaction is completed, the product A is obtained through purification treatment;

[0016] After the N-terminal protected group in the purified product A is removed, the product B is obtained through purification;

[0017] The product B is used as a carrier to sequentially perform coupling reaction and N-terminal protection group removal reaction on N-terminal and side chain protected cysteine activated by an amino acid carboxyl terminal activator, and the product C is obtained through separation and purification;

[0018] Again, taking product C as a carrier, sequentially coupling with N-terminal and side chain both protected threonine, lysine, D-tryptophan, phenylalanine, cysteine, and removing N-terminal protecting group, after the reaction, coupling with N-terminal protected D-phenylalanine to obtain the precursor D of octreotide;

[0019] Among them, the N-terminal protecting group includes Fmoc, Boc, Cbz or Bn; the side chain protecting group includes Acm, Boc or tBu;

[0020] After the precursor D of octreotide is subjected to oxidative cyclization reaction, the auxiliary group is cut off, the protecting group on the side chain is removed, and then purification treatment is performed, to obtain octreotide.

[0021] Compared with the prior art, the beneficial effects of the present application are: the phosphonated p-hydroxybenzaldehyde compound (PPHBA) and the method for assisting preparation of octreotide provided by the present application have the advantages of both liquid phase and solid phase synthesis methods, and octreotide can be synthesized and prepared more simply, quickly, economically and efficiently, the PPHBA carrier can be recycled and directly reused, the waste of raw materials is reduced, waste pollution is reduced, costs are saved, and the environment is protected.

[0022] The method of the present application can be developed into a general liquid phase polypeptide synthesis strategy for preparing octreotide and its analogs, and 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.

[0023] The raw materials for synthesizing the PPHBA carrier are abundant and easy to obtain, the reaction conditions are mild, the time consumption is short, the yield is good, the separation is easy, and the purity is high. The PPHBA carrier replaces the high molecular resin in solid phase polypeptide synthesis, and the homogeneous reaction efficiency is high, saving raw material consumption. Due to the protection and auxiliary precipitation effect of the PPHBA carrier, the separation and purification steps of the intermediates in the polypeptide synthesis process are simplified, time is saved, and yield is improved. The PPHBA residual fragments are easily separated from octreotide after shearing, the PPHBA residual fragments can be recycled, waste emissions are reduced, costs are saved, the environment is protected, and the social and economic benefits are good. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The HPLC analysis results of the "one-pot" reaction in the examples: (a) compound 5r; (b) oxidation reaction liquid.

[0025] Figure 2 For Figure 1 (b) HPLC ① peak mass spectrum.

[0026] Figure 3 For the precipitation method for purifying the product. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. However, it should be understood that the listed embodiments are only for the purpose of understanding the core methods and application fields of the present invention, but the scope of the present invention is not limited thereto.

[0028] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0029] This invention provides a phosphorylated p-hydroxybenzaldehyde compound with the general structural formula PPHBA:

[0030]

[0031] In the formula, Represented as

[0032] when Represented as The structural formula of phosphorylated p-hydroxybenzaldehyde compounds is as follows:

[0033] It is called p-(diphenylphosphonobenzaldehyde)DPOBA;

[0034] when Represented as The structural formula of phosphorylated p-hydroxybenzaldehyde compounds is as follows:

[0035] It is called tris(p-formylphenyl) phosphate (TPFPP);

[0036] when Represented as The structural formula of phosphorylated p-hydroxybenzaldehyde compounds is as follows: It is called hexa(p-formylphenoxy)cyclotriphosphazene HPFPC.

[0037] This invention provides a method for preparing phosphorylated p-hydroxybenzaldehyde compounds, comprising the following steps:

[0038] p-hydroxybenzaldehyde is reacted with diphenylphosphine chloride, phosphorus oxychloride, or hexachlorocyclotriphosphazene under alkaline conditions. After the reaction is completed, the p-hydroxybenzaldehyde compounds are obtained by separation and purification.

[0039] In an embodiment, a phosphorized p-hydroxybenzaldehyde PPHBA compound small molecule carrier is prepared: p-hydroxybenzaldehyde is reacted with diphenyl phosphoryl chloride, phosphorus oxychloride and hexachlorocyclotriphosphazene under alkaline conditions, respectively, and 4-(diphenyl phosphinyl) benzaldehyde DPOBA, tris(p-formylphenyl) phosphate TPFPP and hexakis(p-formylphenoxy) cyclotriphosphazene HPFPC are obtained in sequence after separation and purification.

[0040] The specific phosphorized p-hydroxybenzaldehyde PPHBA synthesis route is as follows:

[0041]

[0042] The application provides an application of a phosphorized p-hydroxybenzaldehyde compound in preparation of octreotide.

[0043] The application provides a method for assisting in preparation of octreotide by using a phosphorized p-hydroxybenzaldehyde compound, comprising the following steps:

[0044] The phosphorized p-hydroxybenzaldehyde compound is used as an auxiliary group to perform condensation reaction with dihydroxyl or hydroxyl thiol on an N-terminal protected aminodiol, and after reaction, the product A is obtained after purification treatment.

[0045] After the N-terminal protected group in the purified product A is removed, and after purification, the product B is obtained.

[0046] The product B is used as a carrier to perform coupling reaction and N-terminal protection group removal reaction with N-terminal and side chain protected cysteine activated by an amino acid carboxyl terminal activator in sequence, and the product C is obtained after separation and purification.

[0047] The product C is used as a carrier to perform coupling reaction and N-terminal protection group removal reaction with N-terminal and side chain protected threonine, lysine, D-tryptophan, phenylalanine and cysteine in sequence, respectively, after reaction, coupling reaction is performed with N-terminal protected D-phenylalanine, and a precursor D of octreotide is obtained.

[0048] The N-terminal protection group includes Fmoc, Boc, Cbz or Bn; and the side chain protection group includes Acm, Boc or tBu.

[0049] After the precursor D of octreotide is subjected to oxidation cyclization reaction, the auxiliary group is cut off, the side chain protection group is removed, and after purification treatment, octreotide is obtained.

[0050] The aminodiol includes threonine alcohol, serine alcohol or cysteine alcohol.

[0051] The amino acid carboxyl terminal activator includes: carbodiimide condensing agents such as dicyclohexyl carbodiimide DCC, diisopropyl carbodiimide DIC and 1-(3-dimethylamino propyl)-3-ethyl carbodiimide EDCI, which are usually used in combination with catalyst 4-(dimethylamino) pyridine DMAP and activator 1-hydroxybenzotriazole HOBt to promote coupling reaction; or carbonyl onium salt condensing agents such as commonly used 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyl urea hexafluorophosphate HATU, O-benzotriazole-tetramethyl urea hexafluorophosphate HBTU, 6-chlorobenzotriazole-1,1,3,3-tetramethyl urea hexafluorophosphate HCTU, O-benzotriazole-N,N,N',N'-tetramethyl urea tetrafluoroborate TBTU, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyl urea tetrafluoroborate quaternary ammonium salt TNTU, 2-succinimidyl-1,1,3,3-tetramethyl urea tetrafluoroborate TSTU, O-(7-azabenzotriazol-1-yl)-di(tetrahydropyrrolyl) carbonyl hexafluorophosphate HAPyU, O-(benzotriazol-1-yl)-di(tetrahydropyrrolyl) carbonyl hexafluorophosphate HBPyU and the like; or phosphonium salt condensing agents such as benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate PyBOP; carbonyl diimidazole CDI and the like.

[0052] The structure general formula of the product A is as follows:

[0053]

[0054] Among them, Indicated as

[0055] PG is Fmoc, Boc, Cbz, Bn or H;

[0056] R is H or CH3;

[0057] X is S or O.

[0058] Among them, in the process of preparing product C, Fmoc-Cys(Acm)-OH activated by amino acid carboxyl terminal activator is coupled with product B as carrier, and the structure general formula of the obtained intermediate compound is as follows:

[0059]

[0060] Among them, Indicated as

[0061] PG is Fmoc, Boc, Cbz, Bn or H;

[0062] R is H or CH3;

[0063] X represents S or O.

[0064] The structural general formula of the precursor D of octreotide is as follows:

[0065]

[0066] wherein, represents

[0067] PG represents Fmoc, Boc, Cbz, Bn or H;

[0068] R represents H or CH3;

[0069] X represents S or O.

[0070] The intermediate compound after the oxidation cyclization reaction of the precursor D of octreotide has the structural formula as follows:

[0071]

[0072] wherein, represents

[0073] PG represents Fmoc, Boc, Cbz, Bn or H;

[0074] R represents H or CH3;

[0075] X represents S or O.

[0076] In an embodiment, the method for assisting in the preparation of octreotide by using the phosphatized p-hydroxybenzaldehyde compound comprises the following steps:

[0077] The synthetic route is as follows:

[0078]

[0079] Step 1, coupling of the auxiliary group PPHBA with Fmoc-protected amino group of threonine Fmoc-Thr-OH, serine Fmoc-Ser-OH or cysteine Fmoc-Cys-OH: PPHBA is used to replace the resin in solid-phase polypeptide synthesis, and under the action of an acid catalyst, the aldehyde group on the carrier is reacted with the dihydroxyl group or the hydroxyl thiol in the threonine Fmoc-Thr-OH, serine Fmoc-Ser-OH or cysteine Fmoc-Cys-OH in chloroform at 80-90°C under reflux until PPHBA is consumed, to obtain an acetal product A after separation and purification; the molar ratio of the amino alcohol to PPHBA is 1-1.2:1; the acid catalyst is p-toluenesulfonic acid or a derivative thereof;

[0080] The auxiliary group PPHBA is one of p-(diphenylphosphinoyl)benzaldehyde (DPOBA), tris(p-formylphenyl)phosphate (TPFPP) and hexakis(p-formylphenoxy)cyclotriphosphazene (HPFPC);

[0081] The amino acid is one of threonine, serine or cysteine protected by a protecting group PG which is fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc);

[0082] The acetal product A is Fmoc-Xaa-TPFPP, wherein Xaa is one of threonine, serine or cysteine.

[0083] The synthesis routes of Fmoc-threonine, Fmoc-serine and Fmoc-cysteine are as follows:

[0084]

[0085] Step 2, separation and purification: the product A is added with an alkane or ether solvent with small polarity, and the product A is separated from other impurities by virtue of the property of the auxiliary group PPHBA that it is easy to crystallize and precipitate in the solvent system;

[0086] The separated product A is filtered and washed or recrystallized to obtain the purified product A;

[0087] Step 3, removal of the N-terminal protecting group PG: the purified product A is treated with a Fmoc-removing reagent, stirred at 10-50°C for 0.5-2 hours, or treated with a Boc-removing reagent, stirred at 10-50°C for 0.5-2 hours, to obtain the amino-protected product B, H-Xaa-PPHBA;

[0088] The product B is added with an alkane or ether solvent with small polarity, and the product B is separated from other impurities by virtue of the property of the auxiliary group PPHBA that it is easy to crystallize and precipitate in the solvent system;

[0089] The separated product B is filtered and washed or recrystallized to obtain the purified product B;

[0090] Step 4, coupling of the second amino acid Fmoc-Cys(Acm)-OH: after the amino acid carboxyl activator commonly used in polypeptide synthesis is used to activate Fmoc-Cys(Acm)-OH, an equal molar amount of B is added for coupling reaction, and the product C, Fmoc-Cys(Acm)-Xaa-PPHBA (Xaa=Cys, Ser, Thr), is obtained after separation and purification;

[0091] Step 5, peptide chain elongation on the support: repeat the above steps (3) and (4) to sequentially couple with N-terminal and side chain protected amino acids Fmoc-Thr(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc- D Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, PG- D Phe-OH to prepare the precursor D of octreotide and its analogues, PG- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA (wherein PG = Boc or Fmoc, Xaa = Cys, Ser, Thr). It should be noted that the repetition of the above steps (3) and (4) herein refers to the repetition of the Boc group removal reaction and the coupling reaction in steps (3) and (4);

[0092] Step 6, simultaneous oxidative cyclization, PPHBA support cleavage and side chain protection group removal by "one-pot" method: the precursor D of octreotide and its analogues prepared in the above step is first treated with 10-20% iodine in methanol as the oxidative cyclization agent, and stirred at room temperature for 3-5 hours, during which the Acm on the side chain of the two cysteine Cys in the peptide chain is removed, and at the same time the disulfide bond is formed to realize in-situ cyclization. Subsequently, 50% acetic acid in water is added, and stirring is continued for 5-8 hours, during which the PPHBA support is completely cleaved, and at the same time the Boc and tBu protection groups on the side chain are also completely removed.

[0093] Step 7, isolation and purification of octreotide or its analogues: the reaction mixture obtained by the "one-pot" method above is concentrated by rotary evaporation to about one third of the original volume, extracted with ethyl acetate, and after separation, the aqueous solution obtained is freeze-dried to obtain the crude product of octreotide or its analogues. After purification by preparative HPLC, the pure product of octreotide or its analogues is obtained.

[0094] Step 8, method for recycling the PPHBA auxiliary group: the ethyl acetate phase extract obtained in step 7 is combined, concentrated by rotary evaporation to about one third of the original volume, and a polar small alkane or ether solvent is added. By virtue of the characteristic that PPHBA is easily crystallized and precipitated in different solvent systems, PPHBA can be separated from other impurities. The separated PPHBA is filtered and washed or recrystallized to obtain purified PPHBA, which can be directly or regenerated for repeated use as an auxiliary group.

[0095] In step 1, the acetal intermediate compound PG-Xaa-PPHBA generated by condensation coupling of a certain amino alcohol with the support PPHBA has the following general molecular structure:

[0096]

[0097] In step 4, the N-terminal and side chain protected cysteine is coupled with the intermediate compound PG-Cys(Acm)-Xaa-PPHBA obtained from the coupling of N-terminal and side chain protected cysteine with H-Xaa-PPHBA, the molecular structure of which is as follows:

[0098]

[0099] In step 5, the N-terminal and side chain protected threonine is coupled with the compound H-Cys(Acm)-Xaa-PPHBA obtained from the removal of the N-terminal protecting group of PG-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound PG-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, the molecular structure of which is as follows:

[0100]

[0101]

[0102] The N-terminal and side chain protected lysine is coupled with the compound H-Thr(tBu)-Cys(Acm)-Xaa-PPHBA obtained from the removal of the N-terminal protecting group of PG-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound PG-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, the molecular structure of which is as follows:

[0103]

[0104] The N-terminal and side chain protected D-tryptophan is coupled with the compound H-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA obtained from the removal of the N-terminal protecting group of PG-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound PG- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, the molecular structure of which is as follows: D

[0105] ​

[0106]

[0107] N-terminally and side chain protected phenylalanine with PG-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound H-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound PG-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, said PG-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, said PG-Cys(Acm)-Phe-

[0108]

[0109] N-terminally and side chain protected phenylalanine with PG-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound H-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound PG-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, said PG-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, said PG-Cys(Acm)-Phe-

[0110]

[0111] N-terminally and side chain protected phenylalanine with PG-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA, to obtain the intermediate compound H-Phe- DThe intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound D Phe-Cys(Acm)-Phe- D The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound D Phe-Cys(Acm)-Phe- D The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound

[0112]

[0113]

[0114] The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound D Phe-Cys(Acm)-Phe- D The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound

[0115]

[0116] The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound

[0117]

[0118] The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound The intermediate compound PG- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Xaa-PPHBA is coupled to obtain the intermediate compound

[0119]

[0120] It should be noted that in the preparation method, the coupling agent used in the coupling reaction is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI, which is usually used in combination with a catalyst 4-(dimethylamino)pyridine DMAP and an activator 1-hydroxybenzotriazole HOBt to promote the coupling reaction.

[0121] Some common abbreviations in the present application have the following meanings:

[0122] DCM: dichloromethane CH2Cl2; DEA: diethylamine; DMAP: 4-dimethylaminopyridine; DMF: N,N-dimethylformamide; DPOBA: 4-Diphenylphosphinoxyl benzaldehyde; DPPC: diphenylphosphonyl chloride; EDC-HCl: l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Fmoc: fluorenylmethoxycarbonyl; HATU: 2-(7-oxadiazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HCCTP: Hexachlorocyclotriphosphazene; HPFPC: Hexakis(p-formylphenoxy) cyclotriphosphazene; HOBT: 1-hydroxybenzotriazole; HBTU: O-benzotriazolyl-tetramethyluronium hexafluorophosphate; NMM: N-methylmorpholine; NMP: N-methylpyrrolidone; PHBA: p-hydoxylbenzaldehyde; PPHBA: Phosphated p-hydoxylbenzaldehyde; PyBop: Benzotriazol- l-yl-oxytripyrrolidinophosphonium hexafluorophosphate; TCOP: Phosphorus oxychloride; TEA: triethylamine; TFA: trifluoroacetic acid; TPFPP: Tris(p-formylphenyl)phosphate; THF: tetrahydrofuran; TIPS: triisopropylsilane.

[0123] The following examples provide specific synthetic procedures for preparing the above compounds and corresponding intermediate compounds.

[0124] Example 1

[0125] Synthetic procedure for 4-diphenylphosphinoxyl benzaldehyde (DPOBA): TEA (1.5 mL, 11 mmol, 1.1 eq) was added dropwise to a solution of p-hydroxybenzaldehyde (or p-hydroxybenzenesulfonamide) (10 mmol, 1.0 eq) in THF (50 mL) at 0 °C and the reaction mixture was stirred at this temperature for 10 min to allow it to cool down. Then, diphenylphosphonyl chloride (1.9 mL, 10 mmol, 1.0 eq) was added dropwise to the above mixture and white fumes were produced at the bottle mouth and the solution inside became turbid. After the addition was completed, the reaction mixture was allowed to warm to room temperature and the progress of the reaction was followed by TLC. The reaction was stopped after 1-2 h when the starting material was consumed. The reaction was quenched by the addition of 5 mL of saturated NH4Cl solution and the solution became clear again. The THF solvent was removed under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate and washed with 30 mL of saturated brine solution three times. The organic phase was dried over anhydrous Na2SO4. It was found that the conversion of p-hydroxybenzaldehyde was 100% and therefore the compound DPOBA was obtained in 99% yield after the organic phase was concentrated.

[0126] DPOBA product characterization: white solid, R f = 0.55 (V EA :V PE = 1 : 1), 1 H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.01 - 7.86 (m, 6H), 7.66 - 7.51 (m, 8H). 13 C NMR (101 MHz, DMSO) δ 192.13, 155.92, 133.49, 132.09, 131.40, 130.04, 129.56, 128.79, 121.63. 31 P NMR (162 MHz, DMSO) δ 30.37. HRMS (ESI) m / z calcd for C 19 H 16 O3P + (M+H) + 323.08316, found 323.08304.

[0127] Example 2

[0128] Synthesis of tris(4-formylphenyl)phosphonate TPFPP: TEA (4.58 mL, 33 mmol, 3.3 eq) was added dropwise to a solution of p-hydroxybenzaldehyde (3.48 g, 33 mmol, 3.3 eq) in THF (50 mL) at 0 °C, and the reaction mixture was stirred at this temperature for 10 min to completely cool the system. Then POCl3(0.92 mL, 10 mmol, 1.0 eq) was added dropwise to the above mixture, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, 2 mL of 0.1 mol L -1 The reaction was quenched with dilute H2SO4, and the THF solvent was removed by concentration under reduced pressure. The concentrated and dried residue was dissolved in 50 mL of ethyl acetate, and 50 mL of saturated sodium bicarbonate and saturated brine were added for washing twice, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated, and 10 mL of hot methanol was added to completely dissolve the mixture, which was cooled to room temperature to form a white precipitate. The precipitate was filtered and dried to obtain the purified product TPFPP with a yield of 87%.

[0129] TPFPP product characterization: white solid, R f = 0.40 (V EA :V PE = 1 : 1), 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 3H), 8.04 (d, J = 8.5 Hz, 6H), 7.58 (d, J = 8.3 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 192.29, 154.08, 134.53, 132.46, 121.21. 31 P NMR (162 MHz, DMSO) δ -19.28. HRMS (ESI) m / z calcd for C 21 H 16 O7P + (M+H) + 411.06282, found 411.06305.

[0130] Example 3

[0131] Synthesis of Hexakis(4-formylphenoxy)cyclotriphosphazene HFPCP: Accurately weigh p-hydroxybenzaldehyde 3.30 g (27 mmol, 9 eq) into a 250 mL three-necked flask, take 50 mL of tetrahydrofuran to make it completely dissolved, add 3.80 mL (27 mmol, 9 eq) of triethylamine to the reaction system, and stir at room temperature for 1 h. Weigh 1.04 g (3 mmol, 1 eq) of hexachlorocyclotriphosphazene, heat to 50°C to make it dissolved in tetrahydrofuran, after complete dissolution, slowly add dropwise to the reaction solution of p-hydroxybenzaldehyde. After the addition is completed, the reaction temperature is raised to 70°C, and refluxed for 36 h. White precipitate gradually separates out during the reaction. After the reaction is completed, filter, and remove the solvent under reduced pressure. Dissolve the concentrated and dried residue in 50 mL of ethyl acetate solvent, add 40 mL of saturated NaHC03solution and wash 3 times, and dry with anhydrous MgS04. Finally, concentrate under reduced pressure, and recrystallize from ethyl acetate to obtain the product 2.32 g as a white powder, with a yield of 90%. Developing agent: PE: EA = 1:1.

[0132] Structural characterization data of HFPCP: 1 H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 6H), 7.81 (d, J = 8.6 Hz, 12H), 7.19 (d, J = 8.5 Hz, 12H); 13 C NMR (101 MHz, DMSO-d6) δ 192.15, 154.13, 131.96, 121.53; 31 P NMR (162 MHz, DMSO-d6) δ 7.58; HRMS (ESI) m / z calcd for C 42 H 30 N3O 12 P3Na+ (M+Na) + 884.09345, found 884.09351. The infrared spectrum of HFPCP shows peaks at 3065 cm -1 nearby are the absorption peaks of the unsaturated hydrocarbon (=C-H) stretching vibration in the benzene ring, 2731, 2820 cm -1 The peak appearing at 1700 cm -1 is the absorption peak of the C=0 stretching vibration in the aldehyde group; 1593 cm -1 is the skeletal deformation vibration absorption peak of the benzene ring; 1264, 1150 cm -1 The peak at 950 cm -1 nearby is the absorption peak of P-O-Ph, which indicates that the -CHO structure exists in the structure of the compound, and the chlorine atom is replaced, in addition, 599, 518 cm -1 The absorption peaks of P-Cl bonds completely disappear, proving that the chlorine atom has been completely replaced by p-hydroxybenzaldehyde.

[0133] Case 4

[0134] Synthesis of Fmoc-Thr-OH: L-Threonine (1.19 g, 10 mmol, 1 eq) was dissolved in 50 mL of 10% Na2CO3 solution, stirred in an ice bath until completely dissolved, weighed 9-fluorenylmethoxycarbonyl chloride (2.58 g, 10 mmol, 1 eq) was dissolved in 20 mL of acetone and added dropwise to the above Na2CO3 solution, solid was precipitated constantly, the ice bath was removed, and the stirring was continued at room temperature for 3 h, TLC was used to track the reaction, when the raw material was consumed, the reaction solution was extracted with ethyl acetate to remove part of the impurities, the aqueous phase was adjusted to pH 2 with 1M hydrochloric acid, and then extracted with ethyl acetate again, the organic phase was dried with anhydrous sodium sulfate and evaporated to concentrate, 3.2 g of Fmoc-threonine was obtained as a colorless oily liquid, with a yield of 95%.

[0135] Characterization of Fmoc-Thr-OH product: colorless oily liquid, 1 H NMR (400 MHz, DMSO-d6) δ 12.51 (s, 1H), 7.89 (s, 2H), 7.75 (s, 2H), 7.38 (d, J = 36.2 Hz, 4H), 7.09 (s, 1H), 4.74 (s, 1H), 4.30 (s, 3H), 4.04 (s, 2H), 1.09 (s, 3H). 13C NMR (101 MHz, DMSO) δ 172.77, 156.88, 144.30, 141.17, 128.11, 127.54, 125.80, 120.58, 66.88, 66.26, 60.41, 47.10, 20.83. HRMS (ESI) m / z calcd for C 19 H 20 NO5 + (M+H) + 342.13360, found 342.13376.

[0136] Fmoc-Thr-ol synthesis procedure: Dry Fmoc-Threonine (1.7 g, 5 mmol) was dissolved in 50 mL dry tetrahydrofuran, 50 mL 1M BH3-THF complex was added dropwise under ice bath, TLC was used to track the reaction, after 5 h the starting material was consumed, 50 mL 1M hydrochloric acid was added dropwise to quench the excess borane, a large amount of gas was released, after the hydrochloric acid was added, the reaction was continued to stir at room temperature for 1 h, first extracted with a mixture of tetrahydrofuran and diethyl ether with a volume ratio of 1:1, the organic phase was combined and washed with saturated brine, 1M KOH solution, saturated brine, the organic phase was dried over anhydrous sodium sulfate, evaporated and concentrated, and purified by column chromatography, the mobile phase was a mixture of petroleum ether and ethyl acetate with a volume ratio of 1:5, 1.3 g of Fmoc-Thr-ol was obtained, white solid, yield 80%.

[0137] Fmoc-Thr-ol product characterization: white solid, R f = 0.35 (V EA :V PE = 1:1), 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.4 Hz, 2H), 7.84 (d, J = 7.5 Hz, 1H), 7.73 (s, 2H), 7.42 (s, 4H), 6.74 (s, 1H), 4.24 (s, 3H), 3.85 (dt, J = 8.5, 4.2 Hz, 1H), 3.54 - 3.45 (m, 1H), 3.40 (d, J = 5.4 Hz, 2H), 1.05 (s, 3H). 13CNMR(101MHz,DMSO)δ156.76,144.40,144.37,143.07,141.20,141.18,13 9.90,129.35,128.04,127.71,127.48,125.78,125.73,121.80,120.51,12 0.44,70.39,70.27,70.20,70.06,65.88,65.20,61.53,61.06,60.20,58. 38,58.03,47.27,40.61,40.40,40.19,39.99,39.78,39.57,39.36,20.38.

[0138] Implementation Case 5

[0139] The condensation reaction route of DPOBA support and Fmoc-threonine is as follows:

[0140]

[0141] The synthesis of Fmoc-Thr-DPOBA proceeded as follows: Fmoc-threonol (1.3 g, 4 mmol, 1 eq) and 4-diphenylphosphooxybenzaldehyde (DPOBA, 3.86 g, 12 mmol, 3 eq) were weighed and dissolved in 50 mL of chloroform. 5 mg of p-toluenesulfonic acid was added as a catalyst. A water separator was attached to the mouth of a round-bottom flask, followed by a spherical condenser. The reaction flask was refluxed overnight in an oil bath at 80 °C. The presence of water is detrimental to acetal formation; therefore, all reactants were dried before addition. After the reaction, the reaction solution was directly evaporated to dryness under reduced pressure and purified by column chromatography using a 1:1 (v / v) mixture of petroleum ether and ethyl acetate to yield 1.6 g of Fmoc-Thr-DPOBA as a white solid, with a yield of 65%.

[0142] Characterization of Fmoc-Thr-DPOBA product: White solid, R f =0.50(V) EA :V PE =1:1), 1 H NMR (400MHz, DMSO-d6) δ7.89 (s, 6H), 7.80 (s, 2H), 7.44 (d, J = 93.5Hz, 16H), 5.52 (s, 1H), 4.27 (d, J=29.4Hz,3H),4.14(s,1H),4.04(dd,J=11.9,2.0Hz,1H),3.95(s,1H),3.51(s,1H),1.08(s,3H). 13C NMR (101 MHz, DMSO) δ 156.99, 151.30, 144.31, 141.16, 135.51, 133.22, 132.07, 130.44, 129.44, 128.76, 128.10, 127.51, 125.93, 120.53, 120.38, 100.72, 74.49, 71.22, 70.18, 66.27, 47.22, 40.63, 40.43, 40.22, 40.01, 39.80, 39.59, 39.38, 17.75. 31 P NMR (162 MHz, DMSO) δ 29.19.

[0143] Fmoc group removal reaction step: the Fmoc-Thr-DPOBA obtained in the last step was placed in a round bottom flask, 6 mL of 25% DEA / MeCN solution was added, stirred at room temperature for 0.5 h, then the solvent was evaporated, 3 mL of ethyl acetate was added to dissolve the mixture in the bottle, 15 mL of petroleum ether was added dropwise while shaking, white solid precipitated during the process, after standing, the mixture was separated by glass funnel filtration, 1.02 g of white solid was obtained, which was the product H-Thr-DPOBA after removing Fmoc, the yield was 98%.

[0144] Characterization of Thr-DPOBA product: white solid, R f = 0.30 (V MeOH :V DCM = 1 :50), 1 H NMR (400 MHz, DMSO-d6) δ 7.91 (s, 4H), 7.59 (d, J = 23.2 Hz, 6H), 7.36 (s, 2H), 7.27 (d, J = 8.2 Hz, 2H), 5.45 (s, 1 H), 3.95 (d, J = 38.0 Hz, 3H), 2.43 (s, 1 H), 1.87 - 1.64 (m, 1 H), 1.10 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 151.27, 151.19, 135.77, 133.25, 133.22, 132.04, 131.94, 131.79, 130.43, 129.45, 129.32, 128.29, 120.57, 120.52, 100.57, 75.85, 74.07, 48.52, 40.62, 40.42, 40.21, 40.00, 39.79, 39.58, 39.37, 18.10. 31 P NMR (162 MHz, DMSO) δ 29.23. HRMS (ESI) m / z calcd for C23 H 24 NO4PNa + (M+Na) + 432.13352, found 432.13275.

[0145] Example 6

[0146] The route of synthesis of octreotide on DPOBA support is as follows:

[0147]

[0148] The second amino acid to be attached on the peptide chain is cysteine. The coupling reaction procedure is as follows: Fmoc-Cys(Acm)-OH (1.13 g, 2.74 mmol, 1.1 eq), EDCI (525 mg, 2.74 mmol, 1.1 eq) and HOBt (370 mg, 2.74 mmol, 1.1 eq) were weighed into a 100 mL round bottom flask, 25 mL dichloromethane was added to dissolve the solids completely, the flask was placed in an ice bath and stirred for 0.5 h to form the active ester of the amino acid which is more reactive, then H-Thr-DPOBA (1.02 g, 2.49 mmol, 1 eq) and DIEA (823 μL, 4.98 mmol, 2 eq) from the previous step were added and the reaction was continued for 1 h. TLC was used to monitor the reaction during the reaction. After the reaction was completed, the solvent was evaporated under reduced pressure, 30 mL ethyl acetate was added and washed with 30 mL saturated sodium bicarbonate solution three times to remove water-soluble impurities. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The appropriate amount of silica gel powder was added to obtain a uniform powder, and then the silica gel powder was packed in a chromatographic column with a height of 5 cm. The crude product was poured into the column and eluted with a mixture of methanol and dichloromethane (1:100, V / V). 1.90 g of product Fmoc-Cys(Acm)-Thr-DPOBA was collected as a white solid with a yield of 95%.

[0149] Characterization of Fmoc-Cys(Acm)-Thr-DPOBA product: white solid, Rf= 0.45 (V f = 1:50), MeOH :

[0150] V DCM = 1:50), 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (t, J = 6.5 Hz, 1H),

[0151] 7.95-7.85 (m, 6H), 7.75-7.21 (m, 16H), 5.52 (s, 1H), 4.49 (dq, J=9.1, 4.9, 4.4 Hz, 1H), 4.42-4.01 (m, 7H), 3.82 (dd, J=37.8, 10.3 Hz, 2H), 2.96-2.68 (m, 2H), 1.79 (s, 3H), 1.03 (d, J=6.3 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 171.30, 170.45, 156.57, 151.36, 151.28, 144.24, 144.20, 141.16, 135.43, 133.26, 133.23, 132.04, 131.94, 131.69, 130.33, 129.44, 129.31, 128.59, 128.12, 127.55, 125.78, 120.56, 120.46, 120.41, 100.69, 74.51, 71.05, 66.25, 54.60, 47.08, 41.37, 31.42, 22.88, 17.73. 31 P NMR (162 MHz, DMSO) δ 29.31. HRMS (ESI) m / z calcd for C 44 H 44 N3O8PSNa + (M+Na) + 828.24789, found 828.24731.

[0152] The removal reaction step of Fmoc group in compound Fmoc-Cys(Acm)-Thr-DPOBA, the Fmoc-Cys(Acm)-Thr-DPOBA obtained in the previous step was placed in a round bottom flask, 6 mL of 25% DEA / MeCN solution was added, and stirred at room temperature for 0.5 h, then the solvent was evaporated, then 4 mL of ethyl acetate was added to completely dissolve the material in the bottle, 20 mL of petroleum ether was added dropwise while shaking, and white solid was precipitated during the process. After standing, the mixture was separated by glass funnel filtration to obtain white solid, which was the product H-Cys(Acm)-Thr-DPOBA after removing Fmoc, with a yield of 98%.

[0153] Characterization of H-Cys(Acm)-Thr-DPOBA product: white solid, R f = 0.33 (V MeOH :V DCM = 1:50), 1HNMR (400 MHz, DMSO-d6) δ 8.55 (t, J = 6.3 Hz, 1H), 8.13 (d, J = 9.5 Hz, 1H), 7.97 - 7.83 (m, 4H), 7.66 - 7.26 (m, 10H), 5.56 (s, 1H), 4.28 - 3.71 (m, 6H), 3.53 (dd, J = 7.6, 4.5 Hz, 1H), 2.95 - 2.68 (m, 2H), 1.82 (s, 3H), 1.05 (d, J = 6.3 Hz, 3H). 13 C NMR (101 MHz, DMSO) δ 173.67, 169.81, 151.38, 151.30, 135.38, 133.27, 133.25, 132.05, 131.95, 131.74, 130.38, 129.46, 129.33, 128.33, 120.61, 120.56, 100.53, 74.60, 71.23, 60.23, 55.37, 54.72, 46.36, 41.12, 40.60, 40.39, 40.19, 39.98, 39.77, 39.56, 39.35, 36.90, 23.04. 31 P NMR (162 MHz, DMSO) δ 29.33. HRMS (ESI) m / z calcd for C 29 H 34 N3O6PSNa + (M+Na) + 606.17981, found 606.17938.

[0154] The third amino acid in the peptide chain is Fmoc-threonine, and the coupling reaction synthesis step is as follows: Fmoc-Thr(tBu)-OH (1.01 g, 2.55 mmol, 1.1 eq), EDCI (489 mg, 2.55 mmol, 1.1 eq) and HOBt (344 mg, 2.55 mmol, 1.1 eq) are weighed into a 100 mL round-bottom flask, 20 mL of dichloromethane is added to completely dissolve the solids, and the mixture is stirred in an ice bath for 0.5 h to form an active ester of the amino acid that is easy to react. Then 5f (1.35 g, 2.32 mmol, 1 eq) and DIEA (767 μL, 4.64 mmol, 2 eq) are added, and the reaction is continued for 1 h. During the reaction, TLC tracking is used for monitoring. After the reaction is completed, the solvent is evaporated, 40 mL of ethyl acetate is added, and the mixture is washed with 40 mL of saturated sodium bicarbonate solution three times to remove water-soluble impurities. The organic phase is dried over anhydrous sodium sulfate and evaporated. An appropriate amount of silica gel powder is added to the sample, and the sample is packed in a glass chromatographic column with silica gel powder to a height of 5 cm. The sample is eluted with a mixture of methanol and dichloromethane (1:100 by volume), and 2.14 g of the main product Fmoc-Thr(tBu)-Cys(Acm)-Thr-DPOBA is collected as a white solid with a yield of 96%.

[0155] Fmoc-Thr(tBu)-Cys(Acm)-Thr-DPOBA product characterization: R f = 0.57 (V MeOH :V DCM = 1:50), white solid, 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (t, J = 6.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.91 (dd, J = 12.8, 7.9 Hz, 6H), 7.75 (t, J = 7.1 Hz, 2H), 7.65 - 7.24 (m, 15H), 6.93 (d, J = 9.1 Hz, 1H), 5.53 (s, 1H), 4.83 (s, 1H), 4.35 - 4.03 (m, 8H), 3.91 - 3.74 (m, 3H), 2.82 (ddd, J = 51.9, 13.7, 6.8 Hz, 2H), 1.77 (s, 3H), 1.06 (d, J = 32.1 Hz, 15H). 13C NMR (101 MHz, DMSO) δ 170.65, 170.22, 156.28, 151.38, 144.39, 141.19, 135.44, 133.23, 133.21, 132.06, 131.78, 130.42, 129.43, 128.62, 128.10, 127.54, 127.51, 125.79, 125.75, 120.56, 120.45, 120.40, 100.68, 74.43, 74.21, 71.09, 68.16, 66.26, 52.21, 47.16, 46.73, 41.25, 40.68, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.37, 35.88, 34.66, 34.40, 33.88, 31.43, 28.95, 28.56, 27.55, 26.81, 25.85, 25.25, 22.95, 22.90, 22.53, 19.74, 19.62, 19.10, 17.73. 31 P NMR (162 MHz, DMSO) δ 29.21. HRMS (ESI) m / z calcd for C 52 H 59 N4O 10 PSNa + (M+Na) + 985.35817, found 985.35724.

[0156] Reaction step of removing Fmoc group in compound Fmoc-Thr(tBu)-Cys(Acm)-Thr-DPOBA: The Fmoc-Thr(tBu)-Cys(Acm)-Thr-DPOBA obtained in the previous step was placed in a round bottom flask, 9 mL of 25% DEA / MeCN solution was added, and after stirring at room temperature for 0.5 h, the solvent was evaporated, then 8 mL of ethyl acetate was added to completely dissolve the material in the bottle, 40 mL of petroleum ether was added dropwise while shaking, white solid precipitated during the process, after standing, the mixture was separated by centrifuge, and the solid was washed with clean precipitate (V EA :V PE = 1:5) for 2-3 times to completely remove the impurities, and the white solid obtained was the product H-Thr(tBu)-Cys(Acm)-Thr-DPOBA after removing Fmoc, with a yield of 97%.

[0157] Characterization of H-Thr(tBu)-Cys(Acm)-Thr-DPOBA product: white solid, R f = 0.32 (V MeOH :VDCM = 1 :50), 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (t, J = 6.4 Hz, 1H), 8.18 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 7.95 - 7.88 (m, 4H), 7.65 - 7.46 (m, 8H), 7.28 (d, J = 8.2 Hz, 2H), 5.52 (s, 1H), 4.78 (q, J = 6.3 Hz, 1H), 4.30 (dd, J = 13.6, 6.6 Hz, 1H), 4.24 - 4.12 (m, 2H), 4.08 - 4.01 (m, 1H), 3.92 - 3.74 (m, 3H), 3.01 (d, J = 3.8 Hz, 1H), 2.84 (qd, J = 13.6, 6.6 Hz, 2H), 1.78 (s, 3H), 1.12 - 1.00 (m, 15H). 13 C NMR (101 MHz, DMSO) δ 173.62, 170.91, 170.11, 151.39, 135.45, 133.25, 133.22, 132.06, 131.96, 131.77, 130.41, 129.44, 129.31, 128.64, 127.75, 121.85, 120.46, 120.41, 100.69, 74.47, 73.52, 71.05, 68.53, 60.35, 52.00, 46.72, 40.70, 40.62, 40.41, 40.20, 40.00, 39.79, 39.58, 39.37, 33.75, 28.87, 22.93, 17.74. 31 P NMR (162 MHz, DMSO) δ 29.22. HRMS (ESI) m / z calcd for C 37 H 49 N4O8PSNa + (M+Na) + 763.29009, found 763.29034.

[0158] The fourth amino acid of H-Thr(tBu)-Cys(Acm)-Thr-DPOBA was Fmoc-lysine, and the coupling reaction synthesis step was as follows: Fmoc-Lys(Boc)-OH (1.11 g, 2.37 mmol, 1.1 eq), EDCI (455 mg, 2.37 mmol, 1.1 eq) and HOBt (321 mg, 2.37 mmol, 1.1 eq) were weighed into a 100 mL round-bottom flask, 20 mL of dichloromethane was added to completely dissolve the solids, and the mixture was stirred in an ice bath for 0.5 h to form an active ester of the amino acid that was easy to react, then H-Thr(tBu)-Cys(Acm)-Thr-DPOBA (1.60 g, 2.16 mmol, 1 eq) and DIEA (714 μL, 4.32 mmol, 2 eq) obtained in the previous step were added, and the reaction was continued for 1 h. During the reaction, TLC was used for tracking and monitoring, and after the reaction was completed, the solvent was evaporated, 30 mL of dichloromethane was added, and the mixture was washed with 30 mL of saturated sodium bicarbonate solution three times to remove water-soluble impurities. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. 20 mL of ethyl acetate was added to the bottle to dissolve the mixture, then 100 mL of petroleum ether was added, and a white solid was precipitated. The solid-liquid phase was analyzed by centrifugation, and 2.39 g of white solid was obtained as Fmoc-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 93%.

[0159] Characterization of Fmoc-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product: white solid, R f = 0.55 (V MeOH :V DCM = 1:50), 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (t, J = 6.4 Hz, 1H), 7.98 (d, J = 9.1 Hz, 1H), 7.95-7.87 (m, 6H), 7.76-7.23 (m, 18H), 6.77 (t, J = 5.7 Hz, 1H), 5.53 (s, 1H), 4.82 (td, J = 8.0, 5.3 Hz, 1H), 4.41-3.73 (m, 12H), 3.00-2.72 (m, 4H), 1.78 (s, 5H), 1.45-0.86 (m, 30H). 13C NMR (101 MHz, DMSO) δ 172.29, 170.55, 170.20, 169.75, 156.55, 156.02, 151.37, 151.29, 144.32, 144.18, 141.17, 135.44, 133.23, 133.20, 132.05, 131.95, 131.77, 130.41, 129.43, 129.30, 128.60, 128.09, 127.56, 125.74, 120.56, 120.44, 120.39, 100.65, 77.79, 74.43, 74.24, 71.08, 67.37, 66.18, 57.60, 55.29, 52.23, 47.15, 46.76, 40.68, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.38, 33.83, 31.78, 29.71, 28.74, 28.48, 26.81, 23.39, 22.90, 19.25, 17.75. 31 P NMR (162 MHz, DMSO) δ 29.22. HRMS (ESI) m / z calcd for C 63 H 79 N6O 13 PSNa + (M+Na) + 1213.50556, found 1213.50427.

[0160] Removal of Fmoc group in compound Fmoc-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA: The Fmoc-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA obtained in the previous step was placed in a round bottom flask, dissolved in 9 mL of 25% DEA / MeCN, stirred at room temperature for 0.5 h, and then the solvent was evaporated. Subsequently, 10 mL of ethyl acetate was added to completely dissolve the contents of the flask, and 50 mL of petroleum ether was added dropwise while shaking, during which white solid precipitated. After standing, the mixture was separated by centrifuge, and the solid was washed with clean precipitate (V EA :V PE = 1:5) for 2-3 times to completely remove the impurities, and the white solid obtained was the product H-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA after removal of Fmoc, with a yield of 98%.

[0161] Characterization of H-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product: white solid, Rf = 0.33 (V MeOH :V DCM = 1 :50), 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.10 (s, 1H), 7.98 (dd, J = 8.6, 2.4 Hz, 2H), 7.95 - 7.86 (m, 4H), 7.67 - 7.46 (m, 8H), 7.28 (d, J = 8.3 Hz, 2H), 6.80 - 6.72 (m, 1H), 5.54 (d, J = 8.3 Hz, 1H), 4.82 (td, J = 8.2, 5.1 Hz, 1H), 4.37 - 3.74 (m, 8H), 3.19 (dd, J = 7.6, 4.4 Hz, 1H), 2.84 (ddd, J = 44.4, 12.7, 7.2 Hz, 4H), 2.07 - 1.91 (m, 2H), 1.79 (d, J = 8.0 Hz, 3H), 1.38 (d, J = 8.1 Hz, 13H), 1.15 - 0.94 (m, 15H). 13 C NMR (101 MHz, DMSO) δ 175.38, 170.62, 170.24, 170.22, 170.06, 156.03, 151.38, 151.30, 135.44, 133.23, 133.21, 132.06, 131.96, 131.78, 130.42, 129.43, 129.33, 129.30, 128.63, 120.44, 120.40, 100.68, 77.76, 74.43, 74.13, 71.09, 67.39, 57.34, 55.04, 52.30, 46.75, 40.64, 40.43, 40.22, 40.01, 39.80, 39.59, 39.38, 34.96, 33.76, 29.85, 28.75, 28.54, 23.01, 22.90, 19.46, 17.75. 31 PNMR (162 MHz, DMSO) δ 29.21. HRMS (ESI) m / z calcd for C 48 H 69 N6O 11 PSNa + (M+Na) + 991.43749, found 991.43732.

[0162] The fifth amino acid attached on H-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA was Fmoc-phenylalanine (D configuration), the coupling reaction synthesis step: Fmoc-D-Trp(Boc)-OH (1.14 g, 2.17 mmol, 1.1 eq), EDCI (415 mg, 2.17 mmol, 1.1 eq) and HOBt (293 mg, 2.17 mmol, 1.1 eq) were weighed into a 100 mL round-bottom flask, 20 mL of dichloromethane was added to completely dissolve the solid, and the mixture was stirred in an ice bath for 0.5 h to form an active ester of the amino acid that was easy to react, then 5j (1.91 g, 1.97 mmol, 1 eq) and DIEA (651 μL, 3.94 mmol, 2 eq) obtained in the previous step were added, and the reaction was continued for 1 h. During the reaction, TLC was used for tracking and monitoring, and after the reaction was completed, the solvent was evaporated, 30 mL of dichloromethane was added and washed with 30 mL of saturated sodium bicarbonate solution and saturated brine three times to remove water-soluble impurities, the organic phase was dried over anhydrous sodium sulfate, and after evaporation, 20 mL of ethyl acetate was added to dissolve it, then 100 mL of petroleum ether was added, and during the process, white solid was precipitated, and the solid-liquid phase was analyzed by centrifugation, and 2.71 g of white solid was obtained as Fmoc- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 93%.

[0163] Fmoc- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product characterization: white solid, R f = 0.50 (V MeOH :V DCM = 1:50), 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (t, J = 6.4 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.99-7.84 (m, 8H), 7.81-7.67 (m, 3H), 7.62-7.18 (m, 19H), 6.69 (t, J = 5.7 Hz, 1H), 5.52 (s, 1H), 4.82 (td, J = 7.9, 5.4 Hz, 1H), 4.48 (td, J = 9.2, 5.1 Hz, 1H), 4.40-3.73 (m, 13H), 3.12-2.74 (m, 6H), 1.78 (s, 3H), 1.57 (s, 11H), 1.34 (s, 11H), 1.19-0.92 (m, 17H). 13C NMR (101 MHz, DMSO) δ 171.91, 171.73, 170.56, 170.20, 169.69, 156.18, 155.95, 151.36, 151.28, 149.50, 144.20, 144.09, 141.08, 135.43, 135.08, 133.23, 132.05, 131.95, 131.77, 130.76, 130.41, 129.42, 129.29, 128.58, 128.03, 127.44, 125.68, 124.70, 122.84, 120.52, 120.43, 120.39, 120.07, 117.17, 115.07, 100.64, 83.90, 77.74, 74.44, 74.24, 71.08, 67.43, 66.24, 57.73, 55.03, 52.93, 52.26, 46.99, 46.76, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.38, 33.84, 31.84, 23.01, 22.90, 19.22, 17.74. 31 PNMR (162 MHz, DMSO) δ 29.20. HRMS (ESI) m / z calcd for C 79 H 97 N8O 16 PSNa + (M+Na) + 1499.63731, found 1499.63892.

[0164] Compound Fmoc- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA in a round bottom flask, 12 mL 25% DEA in MeCN was added, after stirring at room temperature for 0.5 h, the solvent was evaporated, then 12 mL ethyl acetate was added to dissolve the solid in the flask, 60 mL petroleum ether was added dropwise while shaking, white solid precipitated out during the process, after standing, the mixture was separated by centrifuge, the solid was washed with clean precipitate (V D :V EA =1:5) for 2-3 times, the impurities were removed completely, the product H- PE D ​Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA in 97% yield.

[0165] H- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product characterization: white solid, R f = 0.33 (V MeOH :V DCM = 1 :50), 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (t, J = 6.4 Hz, 1H), 8.23 (s, 1H), 8.06 - 7.87 (m, 6H), 7.73 - 7.43 (m, 10H), 7.28 (tt, J = 17.0, 7.5 Hz, 4H), 6.71 (q, J = 7.1, 5.8 Hz, 1H), 5.53 (s, 1H), 4.82 (td, J = 8.0, 5.1 Hz, 1H), 4.38 - 3.54 (m, 10H), 3.11 - 2.68 (m, 6H), 1.78 (s, 3H), 1.62 (s, 11H), 1.39 - 0.77 (m, 30H). 13 C NMR (101 MHz, DMSO) δ 174.87, 171.87, 170.56, 170.21, 169.75, 155.95, 151.36, 151.29, 149.55, 135.44, 135.18, 133.23, 133.20, 132.05, 131.95, 131.77, 130.88, 130.41, 129.43, 129.30, 128.60, 124.68, 124.41, 122.82, 120.43, 120.39, 119.88, 117.79, 115.12, 100.66, 83.90, 77.75, 74.44, 74.28, 71.07, 67.42, 60.21, 57.64, 55.10, 52.96, 52.22, 46.75, 40.62, 40.41, 40.21, 40.00, 39.79, 39.58, 39.37, 33.80, 32.13, 30.99, 29.75, 22.98, 22.89, 21.23, 19.32, 17.75, 14.55. 31 P NMR (162 MHz, DMSO) δ 29.21. HRMS (ESI) m / z calcd for C 64 H 87 N8O 14 PSNa +(M+Na) + 1277.56923, found 1277.56934.

[0166] H- D The sixth amino acid in the Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA peptide chain to be linked is Fmoc phenylalanine, and the coupling reaction synthesis step is as follows: Fmoc-Phe-OH (754 mg, 1.95 mmol, 1.1 eq), EDCI (373 mg, 1.95 mmol, 1.1 eq) and HOBt (263 mg, 1.95 mmol, 1.1 eq) are weighed into a 100 mL round-bottom flask, 30 mL of dichloromethane is added to completely dissolve the solids, and the flask is placed in an ice bath and stirred for 0.5 h to form an active ester of the amino acid that is easy to amidate, and then the H- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA (2.23 g, 1.77 mmol, 1 eq) and DIEA (585 μL, 3.54 mmol, 2 eq) are added, and the reaction is continued for 1 h. During the reaction, TLC tracking is used for monitoring, and after the reaction is completed, the solvent is evaporated, 30 mL of dichloromethane is added, and 30 mL of saturated sodium bicarbonate solution is used to wash three times to remove water-soluble impurities. The organic phase is dried with anhydrous sodium sulfate and evaporated, 20 mL of ethyl acetate is added to the bottle to dissolve it, and then 100 mL of petroleum ether is added, and a white solid is precipitated. The solid-liquid phase is analyzed by centrifugation, and 2.58 g of white solid is obtained, which is Fmoc-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 90%.

[0167] Fmoc-Phe- D Characterization of the Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product: white solid, R f = 0.45 (V MeOH :V DCM = 1:50), 1H NMR (400 MHz, DMSO-d6) δ 8.57 - 8.34 (m, 3H), 8.00 (dd, J = 15.4, 8.5 Hz, 2H), 7.94 - 7.79 (m, 8H), 7.74 (d, J = 8.4 Hz, 1H), 7.64 - 7.09 (m, 25H), 6.69 (t, J = 5.9 Hz, 1H), 5.52 (s, 1H), 4.90 - 4.74 (m, 2H), 4.46 - 4.01 (m, 10H), 3.91 - 3.70 (m, 3H), 3.15 - 2.63 (m, 8H), 1.77 (s, 3H), 1.73 - 1.60 (m, 2H), 1.49 (s, 9H), 1.33 (s, 11H), 1.22 - 0.91 (m, 17H). 13 C NMR (101 MHz, DMSO) δ 174.43, 171.81, 171.48, 170.55, 170.20, 169.69, 155.95, 151.37, 151.29, 149.43, 139.06, 135.43, 135.13, 133.23, 133.20, 132.05, 131.95, 131.77, 130.74, 130.41, 129.68, 129.43, 129.29, 128.59, 128.37, 126.40, 124.67, 122.81, 120.44, 120.39, 119.97, 116.77, 115.04, 100.65, 83.79, 77.74, 74.44, 74.28, 71.08, 67.43, 60.21, 57.69, 56.36, 53.04, 52.59, 52.24, 46.76, 41.36, 40.72, 40.62, 40.41, 40.20, 39.99, 39.79, 39.58, 39.37, 33.86, 31.89, 29.70, 26.81, 23.03, 22.90, 21.22, 19.24, 17.75, 14.55. 31 P NMR (162 MHz, DMSO) δ 29.21. HRMS (ESI) m / z calcd for C 88 H 106 N9O 17 PSNa + (M+Na) + 1646.70572, found 1646.70557.

[0168] Compound Fmoc-Phe- DRemoval of Fmoc group in Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA: The 5m of product from previous step was placed in a round bottom flask, 12mL of 25% DEA in MeCN was added, the flask was placed in a shaker at room temperature for 0.5h, then the solvent was evaporated, 15mL of ethyl acetate was added to dissolve the solid, 60mL of petroleum ether was added dropwise while shaking, white solid precipitated out, the mixture was separated by centrifuge, the solid was washed with fresh precipitate (V EA :V PE =1:5) for 2-3 times to remove the impurities, the white solid was the product H-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA after removal of Fmoc group, yield 97%.

[0169] H-Phe- D Characterization of H-Phe- f Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA: white solid, R MeOH :V DCM =1:50), 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (t, J = 6.4 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.26 (s, 1H), 8.06 - 7.87 (m, 7H), 7.72 (dd, J = 16.1, 8.0 Hz, 2H), 7.64 - 7.44 (m, 9H), 7.36 - 7.05 (m, 10H), 6.70 (q, J = 5.9, 5.4 Hz, 1H), 5.53 (s, 1H), 4.78 (dq, J = 30.1, 7.5 Hz, 2H), 4.38 - 3.75 (m, 10H), 3.42 (dd, J = 8.7, 4.5 Hz, 1H), 3.15 - 2.71 (m, 8H), 1.78 (s, 3H), 1.55 (s, 11H), 1.35 (s, 11H), 1.15 - 0.94 (m, 17H). 13C NMR (101MHz, DMSO) δ171.76,171.62,171.48,170.56,170.19,169.72,156.09,155.95,151.36,149.38,144.22,144.11,141.07,138.51,135.43 ,135.17,133.23,133.20,132.05,131.95,131.77,130.61,130.41,129. 71,129.43,129.29,128.60,128.21,128.03,127.45,126.52,125.78,12 5.69,124.96,124.68,122.77,120.48,120.44,120.39,120.15,116.80,114.99,100.65,83.70,77.73,74.44,74.24,71.08,67.45,66.10,57.74,56.46,52.86,52.51,52.20,46.96,46.76,40.63,40.42,40.21,40.00,39.79,39.58,39.38,38.00,33.87,29.72,23.05,22.90,19.17,17.74. 31 P NMR(162MHz,DMSO)δ29.21.HRMS(ESI)m / z calcd for C 73 H 96 N9O 15 PSNa + (M+Na) + 1424.63764, found 1424.63733.

[0170] H-Phe- D The seventh amino acid linked in the Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA peptide chain is Fmoc cysteine. The coupling reaction synthesis steps are as follows: Weigh Fmoc-Cys(Acm)-OH (702 mg, 1.69 mmol, 1.1 eq), EDCI (325 mg, 1.69 mmol, 1.1 eq), and HOBt (229 mg, 1.69 mmol, 1.1 eq) into a 100 mL round-bottom flask. Add 30 mL of dichloromethane to completely dissolve the solid. Stir in an ice bath for 0.5 h to allow the amino acid to form an easily reacting active ester. Then add the H-Phe- obtained in the previous step. DTrp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA (2.16 g, 1.54 mmol, 1 eq) and DIEA (509 μL, 3.08 mmol, 2 eq) were added and the reaction was continued for 1 h. The reaction was monitored by TLC during the period and after completion of the reaction, the solvent was evaporated, 30 mL of dichloromethane was added and washed with 30 mL of saturated sodium bicarbonate solution three times to remove water soluble impurities, the organic phase was dried over anhydrous sodium sulfate and evaporated, 20 mL of ethyl acetate was added to the flask to dissolve it, then 100 mL of petroleum ether was added, white solid was precipitated, the solid-liquid phase was analyzed by centrifugation, and 2.41 g of white solid was obtained, which was Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 87%.

[0171] Fmoc-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 87%.

[0172] Fmoc-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product characterization: white solid, R f = 0.43 (V MeOH :V DCM = 1 :50), 1 H NMR (400 MHz, DMSO-d6) δ 8.50 (q, J = 7.4 Hz, 4H), 8.01 (t, J = 7.8 Hz, 2H), 7.96 - 7.77 (m, 8H), 7.74 - 7.23 (m, 20H), 7.00 (dt, J = 25.2, 8.0 Hz, 4H), 6.71 (d, J = 6.2 Hz, 1H), 5.53 (d, J = 2.2 Hz, 1H), 4.82 (dt, J = 22.7, 6.5 Hz, 2H), 4.57 - 3.74 (m, 16H), 3.14 - 2.53 (m, 10H), 1.78 (d, J = 2.1 Hz, 6H), 1.73 - 1.59 (m, 2H), 1.56 - 1.45 (m, 9H), 1.43 - 1.29 (m, 11H), 1.27 - 0.93 (m, 17H). 13C NMR (101 MHz, DMSO) δ 171.90, 171.46, 170.60, 170.46, 170.29, 169.82, 155.99, 151.27, 149.39, 144.17, 143.02, 141.13, 139.87, 137.87, 137.70, 137.46, 135.43, 135.19, 133.25, 132.04, 131.94, 131.69, 130.58, 130.33, 129.63, 129.44, 129.31, 128.61, 128.10, 127.76, 127.53, 126.51, 125.76, 125.10, 124.72, 122.78, 121.84, 120.53, 120.48, 120.45, 120.40, 116.75, 114.99, 110.20, 100.67, 83.73, 77.79, 74.44, 74.29, 71.06, 67.52, 66.28, 57.75, 55.34, 54.74, 54.24, 52.92, 52.61, 52.23, 47.03, 46.77, 40.55, 40.34, 40.13, 39.92, 39.72, 39.51, 39.30, 33.85, 32.19, 29.70, 28.69, 28.49, 27.97, 23.02, 22.96, 22.89, 19.29, 17.75. 31 P NMR (162 MHz, DMSO) δ 29.22. HRMS (ESI) m / z calcd for C 94 H 116 N 11 O 19 PS2Na + (M+Na) + 1820.75202, found 1820.75256.

[0173] Compound Fmoc-Cys(Acm)-Phe- DReaction route for removal of Fmoc group in Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA Step: Put 5o from last step in a round bottom flask, add 12 mL of 25% DEA in MeCN, stir at room temperature for 0.5 h, then evaporate the solvent, add 12 mL of ethyl acetate to dissolve the solid in the flask, add 60 mL of petroleum ether drop by drop while shaking, white solid precipitates during the addition, separate the mixture by centrifuge, wash the solid with clean precipitate for 2-3 times, remove the impurities, the white solid is the product after removal of Fmoc, H-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, yield 98%.

[0174] H-Cys(Acm)-Phe- D Characterization of Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product: white solid, R f = 0.31 (V MeOH :V DCM = 1:50), 1 H NMR (400 MHz, DMSO-d6) δ 8.57 - 8.45 (m, 3H), 8.05 - 7.97 (m, 2H), 7.95 - 7.84 (m, 6H), 7.76 (d, J = 8.5 Hz, 1H), 7.65 - 7.45 (m, 9H), 7.37 - 6.96 (m, 8H), 6.83 (d, J = 7.4 Hz, 1H), 6.71 (s, 1H), 5.53 (s, 1H), 4.77 (dq, J = 37.3, 9.1, 8.4 Hz, 2H), 4.57 (q, J = 7.4 Hz, 1H), 4.44 - 4.10 (m, 7H), 4.05 (d, J = 11.2 Hz, 1H), 3.93 - 3.73 (m, 3H), 3.30 - 2.69 (m, 10H), 1.79 (d, J = 10.6 Hz, 6H), 1.65 (t, J = 11.0 Hz, 2H), 1.49 (s, 9H), 1.34 (s, 11H), 1.20 - 0.94 (m, 17H). 13C NMR (101 MHz, DMSO) δ 173.00, 171.89, 171.52, 170.70, 170.58, 170.22, 169.81, 169.77, 155.96, 151.36, 151.28, 149.39, 137.49, 135.43, 135.21, 133.23, 132.05, 131.95, 131.75, 130.58, 130.39, 129.71, 129.44, 129.30, 128.60, 128.08, 126.51, 125.14, 124.73, 122.79, 120.44, 120.40, 120.21, 116.83, 115.03, 100.66, 83.72, 77.75, 74.44, 74.27, 71.08, 67.51, 57.74, 54.84, 53.51, 52.91, 52.62, 52.21, 46.76, 40.61, 40.40, 40.20, 39.99, 39.78, 39.57, 39.36, 38.50, 33.86, 32.13, 29.72, 28.71, 28.51, 27.98, 23.03, 22.90, 19.29, 17.75. 31 P NMR (162 MHz, DMSO) δ 29.22. HRMS (ESI) m / z calcd for C 79 H 106 N 11 O 17 PS2Na + (M+Na) + 1598.68394, found 1598.68323.

[0175] H-Cys(Acm)-Phe- DThe eighth amino acid linked in the Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA peptide chain is Fmoc phenylalanine (D configuration), and the coupling reaction synthesis step is as follows: Fmoc-D-Phe-OH (558 mg, 1.44 mmol, 1.1 eq), EDCI (276 mg, 1.44 mmol, 1.1 eq) and HOBt (194 mg, 1.44 mmol, 1.1 eq) are weighed into a 100 mL round-bottom flask, 30 mL of dichloromethane is added to completely dissolve the solids, and the amino acid is stirred in an ice bath for 0.5 h to form an active ester that is easy to react, then 5p (2.07 g, 1.31 mmol, 1 eq) and DIEA (433 μL, 2.62 mmol, 2 eq) obtained in the previous step are added, and the reaction is continued for 1 h. During the reaction, TLC tracking is used for monitoring, and after the reaction is completed, the solvent is evaporated, 30 mL of dichloromethane is added, and 30 mL of saturated sodium bicarbonate solution is washed three times to remove water-soluble impurities. The organic phase is dried over anhydrous sodium sulfate and evaporated, 20 mL of ethyl acetate is added to the bottle to dissolve it, then 100 mL of petroleum ether is added, and white solid is precipitated. The solid-liquid phase is analyzed by centrifugation, and 2.19 g of white solid is obtained as Fmoc- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, with a yield of 86%.

[0176] Fmoc- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA product characterization: white solid, R f = 0.65 (V MeOH :V DCM = 1:30), 1 H NMR (500 MHz, DMSO-d6) δ 8.54-8.43 (m, 3H), 8.32 (d, J = 8.6 Hz, 1H), 8.04-7.98 (m, 2H), 7.95-7.79 (m, 8H), 7.74 (d, J = 8.2 Hz, 1H), 7.64-6.89 (m, 28H), 6.71 (t, J = 5.8 Hz, 1H), 5.52 (s, 1H), 4.89-3.74 (m, 19H), 3.12-2.55 (m, 10H), 2.46 (d, J = 10.4 Hz, 1H), 1.78 (s, 7H), 1.47 (s, 9H), 1.34 (s, 11H), 1.24-0.91 (m, 17H). 13C NMR (101 MHz, DMSO) δ 172.11, 171.92, 171.44, 170.77, 170.61, 170.41, 170.34, 170.27, 169.81, 156.23, 155.98, 151.35, 151.27, 149.40, 144.11, 143.02, 141.07, 139.87, 138.61, 137.88, 137.62, 135.43, 135.19, 133.25, 132.05, 131.95, 131.71, 130.59, 130.35, 129.76, 129.64, 129.44, 129.39, 129.31, 128.61, 128.41, 128.24, 128.05, 127.76, 127.48, 126.64, 125.76, 125.09, 124.70, 122.77, 121.84, 120.48, 120.45, 120.40, 116.74, 115.01, 110.20, 100.67, 83.72, 77.77, 74.44, 74.29, 71.06, 67.54, 57.74, 54.34, 52.20, 46.97, 46.77, 40.57, 40.36, 40.15, 39.94, 39.73, 39.52, 39.31, 33.88, 33.28, 29.70, 28.70, 28.50, 27.98, 27.96, 23.00, 22.89, 19.30, 17.75. 31 P NMR (162 MHz, DMSO) δ 29.30. HRMS (ESI) m / z calcd for C 103 H 125 N 12 O 20 PS2Na + (M+Na) + 1967.82043, found 1967.82104.

[0177] Compound Fmoc- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA in Fmoc- Phe-Cys(Acm)-Phe- Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA: The reaction step of removing Fmoc group in Fmoc- D Phe-Cys(Acm)-Phe- DTrp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA was placed in a round bottom flask, dissolved with 9 mL 25% DEA / MeCN, stirred at room temperature for 0.5 h, then the solvent was evaporated, 20 mL ethyl acetate was added to dissolve the bottle contents completely, 100 mL petroleum ether was added dropwise while shaking, white solid precipitated, after standing, the mixture was separated by centrifuge, the clean precipitate (V EA :V PE =1:5) was used to wash the solid 2-3 times to remove impurities completely, white solid was obtained, which was the product after removing Fmoc, H- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, yield 98%.

[0178] H- D Phe-Cys(Acm)-Phe- D Product characterization of Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA: white solid, R f =0.40 (V MeOH :V DCM =1:30), 1 H NMR (500 MHz, DMSO-d6) δ 8.48 (dt, J = 29.2, 6.1 Hz, 4H), 8.17 - 7.97 (m, 3H), 7.94 - 7.76 (m, 8H), 7.64 - 7.43 (m, 9H), 7.33 - 7.14 (m, 9H), 7.03 (ddd, J = 43.9, 6.4, 2.9 Hz, 5H), 6.71 (t, J = 5.7 Hz, 1H), 5.52 (s, 1H), 4.88 - 4.74 (m, 2H), 4.53 - 4.03 (m, 10H), 3.94 - 3.74 (m, 3H), 3.53 - 3.39 (m, 1H), 3.13 - 2.69 (m, 10H), 2.56 (dd, J = 13.9, 9.3 Hz, 1H), 2.43 (d, J = 12.4 Hz, 1H), 1.78 (d, J = 4.0 Hz, 6H), 1.49 (s, 11H), 1.33 (s, 11H), 1.20 - 0.95 (m, 17H). 13C NMR (101 MHz, DMSO) δ 171.89, 171.42, 170.76, 170.60, 170.41, 170.27, 170.23, 169.79, 155.97, 151.36, 151.28, 149.39, 139.01, 137.87, 135.43, 135.19, 133.25, 133.22, 132.05, 131.95, 131.74, 130.59, 130.38, 129.75, 129.64, 129.44, 129.31, 128.61, 128.58, 128.23, 126.57, 125.10, 124.70, 122.77, 120.45, 120.40, 120.21, 116.75, 115.00, 100.66, 83.71, 77.75, 74.44, 74.29, 71.08, 67.55, 57.73, 56.33, 54.31, 52.90, 52.59, 52.16, 46.77, 40.60, 40.39, 40.18, 39.97, 39.76, 39.55, 39.35, 33.90, 33.26, 32.26, 29.71, 28.96, 28.70, 28.51, 27.98, 23.00, 22.90, 19.28, 17.76. 31 P NMR (162 MHz, DMSO) δ 29.25. HRMS (ESI) m / z calcd for C 88 H 115 N 12 O 18 PS2Na + (M+Na) + 1745.75235, found 1745.75232.

[0179] DPOBA tag loaded polypeptide chain cleavage and peptide chain cyclization "one pot method";

[0180] DPOBA tag loaded polypeptide chain cleavage and peptide chain cyclization "one pot method" is synchronously and synergistically carried out, the reaction steps are: weigh 100 mg (0.06 mmol, 1 eq) tag and its attached octapeptide H- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA is dissolved in 60 mL of 50% AcOH / H2O, prepared into 10 -3 mol·L -1 solution, 0.6 mL of 1 mol·L -1I2 / MeOH (10 eq), stirred at room temperature for one hour. A small amount of the oxidation reaction was taken for HPLC analysis, Figure 1 (a) is compound H- D Phe-Cys(Acm)-Phe- D HPLC analysis results of Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA, Figure 1 (b) is the HPLC analysis results of the reaction solution, compound H- D Phe-Cys(Acm)-Phe- D Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-DPOBA has been completely consumed, a small amount of peak ① (t = 3.94 min) substance was taken for mass spectrometry analysis, the test results are HRMS (ESI) m / z C 49 H 68 N 10 O 10 S2 2+ (M+2H) 2+ The calculated value is 510.22752, the measured value is 510.22769, multiply the result by 2 and subtract the mass of two protons to get 1018, which is the same as the molecular weight of octreotide, and the results are shown in Figure 2 According to the HPLC test results, the area ratio of each peak is analyzed, and the peak area ratio of peak ①, which is the final product, is 80.4%.

[0181] Purification of tag-assisted peptide chain intermediates

[0182] 4-diphenyl phosphoryl oxybenzaldehyde (DPOBA) has high synthesis yield, good solubility and precipitation performance, so it is suitable for the synthesis of auxiliary peptides. The aldehyde group has multiple reaction possibilities as an amino acid connection site, and the double hydroxyl group of threonine can react with the aldehyde group to form an acetal, and after dehydration, a stable six-membered ring is formed. This six-membered ring is unstable in acidic aqueous solution, and the removal of the protecting group in the Boc strategy needs to be carried out under acidic conditions, so it is more suitable to use the Fmoc strategy for peptide chain extension for the acid-labile ring. With the extension of the peptide chain, the solubility of the polypeptide product will change. The amount of ethyl acetate added before each precipitation is just enough to dissolve the bottle contents, and then 5 times the volume of petroleum ether is added to completely precipitate the carrier and the attached peptide chain, as shown in Figure 3 The solid-liquid separation can be replaced by centrifugation, and the solid in the tube can be washed with the precipitating liquid for 2-3 times to ensure that the impurities are completely removed.

[0183] Synthesis of octreotide: the total yield of the linear peptide precursor H- D Phe-Cys(Acm)-Phe- D The removal of the thiol protecting group Acm and the oxidation of the disulfide bond were carried out in 50% acetic acid / water as solvent and iodine as oxidant. The results of the reaction showed that the disulfide bond was oxidized and cyclized, the side chain protecting groups Boc and tBu in the amino acids were removed, and the tag was cut from the peptide chain without further reaction. However, according to the peak area ratio of the HPLC spectrum, the proportion of the product octreotide was 80.4%. After further HPLC preparation and purification, the refined octreotide product was obtained. The method of the present application can be developed into a general liquid-phase polypeptide synthesis strategy for preparing octreotide and its 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 conducive to environmental protection, and improve economic efficiency.

[0184] The above examples are only part of the examples listed to facilitate understanding of the synthesis and application method of the material of the present application, and are not intended to limit the present application. It can be understood that relevant practitioners can easily make appropriate modifications on this structure, and therefore any modifications, equivalent replacements and improvements, etc. made within the spirit and principles of the present application shall be included within the protection scope of the present application.

Claims

1. Use of a phosphonated p-hydroxybenzaldehyde compound for the preparation of octreotide, characterized in that, The structural general formula of the phosphated p-hydroxybenzaldehyde compound is formula PPHBA: The method for preparing octreotide by using the phosphated p-hydroxybenzaldehyde compound as an auxiliary group comprises the following steps: The phosphated p-hydroxybenzaldehyde compound is used as an auxiliary group to perform condensation reaction with dihydroxyl or hydroxyl thiol on the N-terminal protected aminodiol, and after the reaction is completed, the product A is obtained through purification treatment; After the N-terminal protected group in the purified product A is removed, the product B is obtained through purification; The product B is used as a carrier to sequentially perform coupling reaction and N-terminal protection group removal reaction with N-terminal and side chain protected cysteine activated by an amino acid carboxyl terminal activator, and the product C is obtained through separation and purification; The product C is used as a carrier to sequentially perform coupling reaction and N-terminal protection group removal reaction with N-terminal and side chain protected threonine, lysine, D-tryptophan, phenylalanine and cysteine, and after the reaction is completed, the precursor D of octreotide is obtained by performing coupling reaction with N-terminal protected D-phenylalanine; The protection group of the N-terminal includes Fmoc, Boc, Cbz or Bn; the protection group of the side chain includes Acm, Boc or tBu; After the precursor D of octreotide is subjected to oxidation cyclization reaction, the auxiliary group is cut off and the protection group on the side chain is removed, and then the octreotide is obtained through purification treatment; The aminodiol includes threonine, serine or cysteine; The amino acid carboxyl terminal activator includes a carbodiimide condensing agent, a carbonium salt condensing agent or a phosphonium salt condensing agent; The phosphated p-hydroxybenzaldehyde compound is prepared according to the following steps: The p-hydroxybenzaldehyde is reacted with diphenyl phosphoryl chloride under alkaline conditions, and after the reaction is completed, the phosphated p-hydroxybenzaldehyde compound is obtained through separation and purification.

2. Use of the phosphonated p-hydroxybenzaldehyde compound according to claim 1 for the preparation of octreotide, characterized in that, The structural general formula of the product A is as follows: PG represents Fmoc, Boc, Cbz, Bn or H; R represents H or CH3; X represents S or O.

3. Use of the phosphonated p-hydroxybenzaldehyde compound according to claim 1 for the preparation of octreotide, characterized in that, In the process of preparing the product C, the product B is used as a carrier to perform coupling reaction with Fmoc-Cys(Acm)-OH activated by an amino acid carboxyl terminal activator, and the structural general formula of the obtained intermediate compound is as follows: PG represents Fmoc, Boc, Cbz, Bn or H; R represents H or CH3; X represents S or O.

4. Use of the phosphonated p-hydroxybenzaldehyde compound according to claim 1 for the preparation of octreotide, characterized in that, The structural general formula of the precursor D of octreotide is as follows: PG represents Fmoc, Boc, Cbz, Bn or H; R represents H or CH3; X represents S or O.

5. Use of the phosphonated p-hydroxybenzaldehyde compound according to claim 1 for the preparation of octreotide, characterized in that, The structural formula of the intermediate compound after the precursor D of octreotide is subjected to oxidation cyclization reaction is as follows: PG represents Fmoc, Boc, Cbz, Bn or H; R represents H or CH3; X represents S or O.

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