Halogenated diphenylmethane compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polypeptides, and relates to a halogenated diphenylmethane compound as well as a preparation method and application thereof. The hydrophobic label containing the halogenated diphenylmethane structure designed by the invention is compatible with Fmoc amino acid and Cbz amino acid, and the Fmoc amino acid and the Cbz amino acid are mounted at the C end of a peptide chain, so that rapid and efficient synthesis of peptidol can be realized in a green organic solvent. According to the liquid-phase synthesis method of the peptidol assisted by the soluble hydrophobic label, the advantages that the label is good in solubility in an organic solvent, easy to monitor and the like are utilized, and green, efficient, low-cost and large-scale synthesis of peptidol molecules is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and relates to a halogenated diphenylmethane compound, a preparation method thereof, and an application thereof. Background Art

[0002] Peptolide compounds have a hydroxyl group at the C-terminus of the polypeptide and have various biological activities and biomedical applications due to their specific physicochemical properties, such as peptolide antibiotics, coumermycin (A to D), gramacidin, moenomycin GA IV, and octreotide. Octreotide is an octapeptide cyclic compound with a C-terminal alcohol structure and is a homolog of natural somatostatin. It is used for the diagnosis and treatment of various tumors, gastrointestinal diseases, and chemotherapy-related conditions. The sales of octreotide and lutetium oxyoctreotide of Novartis Pharmaceuticals reached $2 billion in 2023. Currently, only a few effective methods for synthesizing peptolides have been reported, which limits their large-scale availability, research, and application.

[0003] The synthesis of peptidols is generally achieved by modifying the C-terminus of the peptide chain. However, in solid-phase peptide synthesis (SPPS), C-terminal modification is very difficult, and there are few commercially available resins that can be directly used for the synthesis of peptidols. Usually, it is necessary to modify the C-terminus of the resin in solid-phase synthesis, introduce a special linker, and then react with amino alcohol to introduce the first residue (Chem. Eur. J. 2020, 26, 379–383; ChemPlusChem 2020, 85, 641–652). This method has become a popular method for the synthesis of peptidols. However, the anchoring of the hydroxyl group requires a specific linker, and such linkers are generally unstable and have poor coupling efficiency with amino alcohols, which limits their commercial application scope. Edward et al. reported a method of introducing amino alcohol during cleavage through aminolysis reaction. However, the aminolysis reaction conditions are harsh, various by-products will be generated, and subsequent purification is difficult (J. Med. Chem. 1994, 37, 3749–3757); Tailhades et al. reported a method for the synthesis of peptidols by O-N acyl transfer reaction. Although this method is applicable to conventional commercial resins, its feasibility is limited by the low efficiency of the O-N acyl transfer reaction and it is difficult to scale up (Angew. Chem. Int. Ed. 2010, 49, 117–120); Crawhall and Elliott reported the use of LiAlH4 and NaBH4 to reduce the peptide sequence on the resin to obtain amino alcohol. However, the reduction reaction rate is slow and the conditions are harsh, and the obtained products are complex and difficult to purify (Reference: Biochem. J. 1955, 61, 264–275). To solve this problem, Kao et al. recently reported the application of diaminobenzoyl (Dbz) linker. This linker meets the requirements of reductive cleavage, and peptidols can be obtained by reducing the peptide sequence on the resin with NaBH4 (Adv. Synth. Catal. 2024, 366, 2534-2543), but this method requires complex modification of the resin, with cumbersome operations and high costs.

[0004] In addition to these methods for the synthesis of peptidols in solid phase, there is no report on the synthesis of peptidols in liquid phase system at present. Although solid-phase synthesis is the mainstream method for the chemical synthesis of polypeptides at present, it also has many limitations that are difficult to overcome by itself: it is necessary to use excessive amounts of amino acids and coupling reagents as well as expensive resins to ensure the completion of the condensation reaction, so the atom economy is extremely poor; a large amount of organic solvents are required to wash the resin, and the extensive use of harmful reagents and solvents causes irreversible damage to the environment; the reaction is a heterogeneous system, making it difficult to monitor and analyze the reaction process; the aggregation of the growing peptide chain will greatly increase the difficulty of synthesis.

[0005] Compared with solid-phase synthesis methods, liquid-phase peptide synthesis has several advantages, including that homogeneous reactions only require stoichiometric amounts of reactants, thus achieving higher atom economy and reducing costs. In addition, more environmentally friendly solvents are allowed during liquid-phase synthesis. For example, ethyl acetate can be used to replace N,N-dimethylformamide, a solvent that is unfriendly to the environment and human health in solid-phase synthesis. Despite these advantages, due to the reaction occurring in the liquid phase, during traditional liquid-phase peptide synthesis, the purification of intermediates usually involves multiple steps of recrystallization, extraction, or column chromatography operations. This is not only time-consuming and laborious but also reduces the overall synthesis efficiency. Especially in the synthesis of long peptide chains with multiple steps, the cumulative losses will significantly reduce the final yield. On the other hand, as the peptide chain length increases, the solubility problem of intermediates becomes more prominent. Especially in commonly used organic solvents, more hydrophobic peptide chains may precipitate during the reaction, resulting in a decrease in reaction efficiency. In recent years, significant progress has been made in liquid-phase peptide synthesis assisted by soluble hydrophobic tags. The basic principle of this method lies in the unique properties of soluble hydrophobic tags, which make peptide intermediates different from the reagents used in the synthesis reaction and the by-products generated. Therefore, unwanted substances can be effectively removed by simple methods such as precipitation, filtration, or extraction (Adv. Synth. Catal. 2024, 366, 2534–2543). This method combines the advantages of traditional liquid-phase synthesis and solid-phase synthesis, reduces the demand for excessive reagents and solvents, and conforms to the principles of green chemistry.

[0006] The structural design and property regulation of soluble hydrophobic tags are crucial. First, corresponding functional groups need to be designed according to the different structures at the C-terminus of the polypeptide chain. Second, according to the properties of the synthesized peptide chain, the hydrophilicity and hydrophobicity need to be adjusted by changing the structure of the tag substituents. By increasing the solubility of the intermediate in organic solvents, the efficiency of peptide chain coupling can be improved, thereby promoting the extension of the peptide chain in the liquid phase. Finally, the tag and side-chain protecting groups are cleaved to obtain the target peptide chain molecule. In the previously disclosed patent CN118561901A, the inventors designed soluble hydrophobic tags with core functional group structures of hydroxyl and amino, which can be used to synthesize conventional polypeptides with carboxylic acid and amide structures at the C-terminus, but cannot be used for peptide alcohols with a hydroxyl structure at the C-terminus. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a halogenated diphenylmethane compound, its preparation method and application in view of the deficiencies of the prior art.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0009] The present invention discloses a halogenated diphenylmethane compound, and the structural formula of the halogenated diphenylmethane compound is shown in Formula I:

[0010]

[0011] Among them,

[0012] X is selected from Cl, Br or I;

[0013] R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 alkyl, or substituted or unsubstituted C5-C8 aromatic groups.

[0014] In some embodiments, the structural formula of the halogenated diphenylmethane compound is shown in Formula I-1, I-2 or I-3:

[0015]

[0016] Among them,

[0017] R1 = R1'; R2 = R2'; R3 = R3';

[0018] R1, R2, R3 are independently selected from substituted or unsubstituted C1-C 22 alkyl, or substituted or unsubstituted C5-C8 aromatic groups.

[0019] Among them, the above-mentioned C1-C 22 alkyl includes straight-chain alkyl or branched-chain alkyl.

[0020] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1, R2, R3 are independently selected from unsubstituted C1-C 18 alkyl, or unsubstituted C6 aromatic groups.

[0021] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1 = R3; R1 is selected from unsubstituted C1-C3 alkyl or unsubstituted phenyl; R2 is selected from unsubstituted C2-C 18 alkyl or unsubstituted phenyl.

[0022] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1 ≠ R3; R1 is selected from unsubstituted C4 alkyl; R2 is selected from unsubstituted C2-C4 alkyl; R3 is selected from unsubstituted C1-C2 alkyl.

[0023] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1, R2, R3 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-octadecyl or phenyl.

[0024] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1 = R3; R1 is selected from methyl, ethyl, n-propyl, isopropyl or phenyl; R2 is selected from ethyl, n-octadecyl, n-propyl, isopropyl, tert-butyl, n-butyl, phenyl, n-pentyl, n-hexyl, n-octyl, n-decyl or n-dodecyl.

[0025] In some embodiments, preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1 ≠ R3; R1 is selected from n-butyl or tert-butyl; R2 is selected from ethyl or n-butyl; R3 is selected from methyl or ethyl.

[0026] In some embodiments, most preferably, the halogenated diphenylmethane compounds are selected from any of the following structures:

[0027]

[0028]

[0029] Among them, -C2H5, -C3H7, -C4H9, -C5H 11 , -C6H 13 , -C7H 15 , -C8H 17 , -C9H 19 , -C 10 H 21 , -C 12 H 25 , -C 14 H 29 , -C 16 H 33 , -C 18 H 37 , -C 20 H 41 , -C 22 H 45 Unless otherwise specified, they are all straight-chain saturated alkyl groups.

[0030] Furthermore, the present invention discloses a preparation method of the above-mentioned halogenated diphenylmethane compounds, which is selected from any one of the following synthetic routes:

[0031] Synthetic route 1:

[0032] Compound c reacts with a chlorinating reagent in a first reaction to obtain compound I-1;

[0033]

[0034] Synthetic route 2:

[0035] Compound c undergoes a second reaction with N-bromosuccinimide under the action of a first catalyst to obtain compound I-2;

[0036]

[0037] Synthetic Route Three:

[0038] Compound c undergoes a third reaction with iodine under the action of a second catalyst and a base to obtain compound I-3;

[0039]

[0040] Among them,

[0041] R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 alkyl groups, or substituted or unsubstituted C5-C8 aromatic groups.

[0042] In some embodiments, in Synthetic Route One, the chlorinating reagent is thionyl chloride or hydrogen chloride; the molar ratio of compound c to the chlorinating reagent is 1.0:(2.0-4.3); the solvent used in the first reaction is any one or a combination of dichloromethane, ethyl acetate, methyl tert-butyl ether, tetrahydrofuran, and carbon tetrachloride; the reaction temperature of the first reaction is the temperature at which the used solvent refluxes.

[0043] In some embodiments, preferably, in Synthetic Route One, the chlorinating reagent is thionyl chloride; the molar ratio of compound c to the chlorinating reagent is 1.0:(2.5-4.0), more preferably 1.0:2.2; the solvent used in the first reaction is dichloromethane; the reaction temperature of the first reaction is the temperature at which the used solvent refluxes.

[0044] Among them, in Synthetic Route One, there is no special requirement for the amount of the solvent used in the first reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0045] Among them, in Synthetic Route One, the first reaction is a reflux reaction, and the reaction temperature is determined by the boiling point of the solvent, and it is only necessary to make the used solvent reflux; the reaction time of the first reaction is 3-4 hours.

[0046] In some embodiments, in Synthetic Route II, the first catalyst is any one or a combination of several of N,N'-dimethylthiourea, diethyl phosphite, diisopropylethylamine, zinc bromide, iron bromide, and tetrabutylammonium bromide; the molar ratio of compound c to N-bromosuccinimide and the first catalyst is 1.0:(2.0 - 3.0):1.0; the solvent used in the second reaction is any one or a combination of several of dichloromethane, ethyl acetate, cyclohexane, methyl tert-butyl ether, tetrahydrofuran, and carbon tetrachloride; for the second reaction, the reaction temperature is 40°C - 60°C.

[0047] In some embodiments, preferably, in Synthetic Route II, the first catalyst is N,N'-dimethylthiourea; the molar ratio of compound c to N-bromosuccinimide and the first catalyst is 1.0:(2.3 - 2.6):1.0, and more preferably 1.0:2.5:1.0; the solvent used in the second reaction is dichloromethane; for the second reaction, the reaction temperature is 50°C.

[0048] Among them, in Synthetic Route II, there is no special requirement for the amount of the solvent used in the second reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0049] In some embodiments, in Synthetic Route III, the second catalyst is any one or a combination of several of triphenylphosphine, zinc chloride, iron chloride, and tetrabutylammonium bromide; the base is any one or a combination of several of imidazole, triethylamine, pyridine, sodium hydroxide, sodium bicarbonate, and sodium carbonate; the molar ratio of compound c to iodine is 1.0:(1.0 - 1.5); the molar ratio of compound c to the second catalyst and the base is 1.0:(0.8 - 1.2):(1.0 - 1.5); the solvent used in the third reaction is any one or a combination of several of dichloromethane, ethanol, methanol, ethyl acetate, dichloromethane, tetrahydrofuran, and carbon tetrachloride; for the third reaction, the reaction temperature is 25°C - 45°C; the third reaction is carried out under the protection of an inert gas.

[0050] In some embodiments, preferably, in Synthetic Route III, the second catalyst is triphenylphosphine; the base is imidazole; the molar ratio of compound c to iodine is 1.0:1.2; the molar ratio of compound c to the second catalyst and the base is 1.0:1.0:1.2; the solvent used in the third reaction is dichloromethane; for the third reaction, the reaction temperature is 35°C; the third reaction is carried out under the protection of an inert gas.

[0051] Among them, in Synthetic Route III, there is no special requirement for the amount of the solvent used in the third reaction, and it is only necessary to dissolve or disperse the raw materials evenly.

[0052] Among them, in Synthetic Route 3, preferably, the third reaction is carried out under the protection of nitrogen gas.

[0053] In some embodiments, the above compound c can be prepared according to the prior art (CN 118561901A), or can be prepared according to the following steps:

[0054]

[0055] (a) 4,4-Dihydroxybenzophenone and compound a undergo a fourth reaction under the action of a first base to obtain compound b;

[0056] (b) Compound b undergoes a reduction reaction under the action of a reducing agent to obtain compound c;

[0057] Among them, R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 alkyl, or substituted or unsubstituted C5-C8 aromatic groups.

[0058] In some embodiments, in step (a), the first base is imidazole or 4-dimethylaminopyridine; the molar ratio of 4,4-dihydroxybenzophenone to compound a is 1.0:(1.0 - 4.0); the molar ratio of 4,4-dihydroxybenzophenone to the first base is 1.0:(3.0 - 8.0); for the fourth reaction, the reaction temperature is room temperature.

[0059] In some embodiments, preferably, in step (a), the first base is imidazole or 4-dimethylaminopyridine; the molar ratio of 4,4-dihydroxybenzophenone to compound a is 1.0:2.5; the molar ratio of 4,4-dihydroxybenzophenone to the first base is 1.0:6.0; for the fourth reaction, the reaction temperature is room temperature.

[0060] Among them, in step (a), the solvent used in the fourth reaction is ethyl acetate, dichloromethane, tetrahydrofuran or dichloroethane, preferably dichloromethane. There is no special requirement for the amount of the solvent, and it is only necessary to dissolve or disperse the raw materials evenly.

[0061] In some embodiments, in step (b), the reducing agent is sodium borohydride, potassium borohydride or lithium aluminum hydride; the molar ratio of compound b to the reducing agent is 1.0:(1.5 - 3.0); for the reduction reaction, the reaction temperature is room temperature.

[0062] In some embodiments, preferably, in step (b), the reducing agent is sodium borohydride; the molar ratio of compound b to the reducing agent is 1.0:2.1; for the reduction reaction, the reaction temperature is room temperature.

[0063] Among them, in step (b), the solvents used in the reduction reaction are anhydrous diethylene glycol dimethyl ether, anhydrous ethanol, anhydrous methanol, anhydrous ethyl acetate, anhydrous dichloromethane, anhydrous tetrahydrofuran or anhydrous dichloroethane, preferably anhydrous ethanol. There are no special requirements for the amount of the solvent, and it is only necessary to dissolve or disperse the raw materials evenly.

[0064] The halogenated diphenylmethane compounds prepared by the above preparation method are also within the protection scope of the present invention.

[0065] Furthermore, the present invention discloses a protecting reagent for the C-terminus of an amino alcohol or a peptide alcohol, and the protecting reagent contains the above-mentioned halogenated diphenylmethane compounds.

[0066] The application of the above-mentioned halogenated diphenylmethane compounds or the protecting reagent for the C-terminus of the above-mentioned amino alcohol or peptide alcohol in the synthesis of peptide alcohol in a homogeneous solvent system is also within the protection scope of the present invention.

[0067] Furthermore, the present invention discloses a method for synthesizing peptide alcohol, using the above-mentioned halogenated diphenylmethane compounds; the method includes the following steps:

[0068] (1) Attachment of the first residue: Using the above-mentioned halogenated diphenylmethane compounds as a hydrophobic carrier, connecting with an N-protected amino alcohol or an N-protected peptide alcohol compound through a conventional condensation reaction to obtain an N-protected amino alcohol or an N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier;

[0069] (2) N-terminal deprotection: Mixing the N-protected amino alcohol or the N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier with an N-terminal protecting deprotecting reagent and a solvent, and performing N-terminal deprotection in an organic homogeneous system to obtain a solution of an N-deprotected amino alcohol or an N-deprotected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier;

[0070] (3) Peptide chain extension: Adding an N-protected amino alcohol or an N-protected peptide alcohol compound to the solution of the N-deprotected amino alcohol or the N-deprotected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier, then adding a condensation reagent to perform a coupling condensation reaction in a homogeneous system, and then performing extraction to remove impurities to obtain a solution of an N-protected amino alcohol or an N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier;

[0071] (4) Repeating steps (2) and (3) to attach the next amino alcohol until a complete peptide alcohol chain is obtained;

[0072] (5) Cleavage and precipitation: After deprotecting the N-terminus of the complete N-protected amino alcohol or N-protected peptide alcohol compound with a C-terminal protected halodiphenylmethane structure carrier, it is dissolved in a cleavage solution for a cleavage reaction. After the cleavage is completed, a precipitation solution is added for precipitation, and the peptide alcohol is obtained.

[0073] Specifically, the above includes but is not limited to octreotide and the antimicrobial peptide Trichogin GAⅣ.

[0074] Specifically, the principle of using the above-mentioned halodiphenylmethane compounds as carriers for the synthesis of peptide alcohols in a homogeneous solvent system is as follows: A hydrophobic protecting group containing a halodiphenylmethane structure is introduced at the C-terminus of the peptide alcohol, increasing the solubility of the peptide chain in the organic solvent and enabling the peptide coupling reaction to proceed in a homogeneous system.

[0075] In some embodiments, in step (1), in the conventional condensation reaction, any one or a combination of the condensation reagents 2,6-dimethylpyridine, pyridine, triethylamine, diisopropylethylamine, 4-dimethylaminopyridine, quinoline, and 1,8-diazabicycloundec-7-ene is used; the molar ratio of the halodiphenylmethane compound to the N-protected amino alcohol is 1.0:(1.0 - 3.0); the molar ratio of the halodiphenylmethane compound to the N-protected peptide alcohol compound is 1.0:(1.0 - 3.0); the molar ratio of the halodiphenylmethane compound to the condensation reagent is 1.0:(2.0 - 5.0); the solvent used in the conventional condensation reaction is any one or a combination of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, and chloroform, preferably ethyl acetate; the dosage of the solvent is not particularly limited, as long as the raw materials can be dissolved and the viscosity is moderate; the conventional condensation reaction is carried out at 20°C to 70°C; after the conventional condensation reaction is completed, the reaction solution is washed with an aqueous K2CO3 solution and extracted to remove impurities.

[0076] In some embodiments, in step (2), when the N-terminal protecting group is CBz, the deprotecting reagent used is Pd / C and hydrogen, and the solvent used is any one or a combination of ethyl acetate and methanol, preferably ethyl acetate. The dosage of the solvent is not particularly limited, as long as the raw materials can be dissolved and the viscosity is moderate; the dosage of Pd in the Pd / C is preferably 0.1 - 1.0 molar equivalents of the halodiphenylmethane compound; the pressure of the hydrogen is 1 standard atmosphere; the N-terminal deprotection is carried out at 25°C to 40°C; after the N-terminal deprotection is completed, the reaction solution is washed with an aqueous K2CO3 solution and extracted to remove impurities.

[0077] In some embodiments, in step (2), when the N-terminal protecting group is Fmoc-, the deprotecting reagent used is any one or a combination of piperidine, pyrrolidine, and palladium carbon, preferably pyrrolidine. The dosage of the deprotecting reagent is 5.0 - 16.0 times the molar equivalent of the halogenated diphenylmethane compound. The solvent used is any one or a combination of ethyl acetate, dichloromethane, cyclopentyl methyl ether, chloroform, and tetrahydrofuran. There is no special limitation on the dosage of the solvent, as long as the raw materials can be dissolved and the viscosity is moderate. The N-terminal deprotection is carried out at 25°C - 40°C. After the N-terminal deprotection is completed, the reaction solution is washed with an aqueous hydrochloric acid solution and then extracted to remove impurities. It is also possible to wash the reaction solution with an aqueous hydrochloric acid solution and then with an aqueous K2CO3 solution.

[0078] In some embodiments, in step (3), the condensing reagent is any one or a combination of DCC, DIC, EDC·HCl, DMAP, HOBt, HATU, and HBTU, preferably a mixed reagent of EDC·HCl and HOBt in any molar ratio, and further preferably a mixed reagent of EDC·HCl and HOBt with a molar ratio of 1:1. The usage amount of the condensing reagent is 2 - 5 times the molar equivalent of the halogenated diphenylmethane compound. The usage amount of the N-protected amino alcohol is 1 - 3 times the molar equivalent of the halogenated diphenylmethane compound. The usage amount of the N-protected peptide alcohol compound is 1 - 3 times the molar equivalent of the halogenated diphenylmethane compound. The solvent used in the coupling condensation reaction is any one or a combination of ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, and chloroform. There is no special limitation on the dosage of the solvent, as long as the raw materials can be dissolved and the viscosity is moderate. The coupling condensation reaction is carried out at 25°C - 40°C. After the coupling condensation reaction is completed, the reaction solution is washed with an aqueous K2CO3 solution and then extracted to remove impurities.

[0079] In some embodiments, in step (5), when the complete N-protected amino alcohol or N-protected peptide alcohol compound with C-terminal protection of the halogenated diphenylmethane structure carrier contains a side chain protecting group, the C-terminal carrier is removed using the method of step (2), and then a cleavage reaction is carried out in the cleavage solution. After the cleavage is completed, a sedimentation solution is added for sedimentation to obtain the peptide alcohol. Among them, the cleavage solution is a mixture of TFA, DODT, TIS, and pure water, and the volume ratio of the four is preferably: V TFA :V DODT :V TIS :V 纯水 = 92.5:2.5:2.5:2.5. The dosage of the cleavage solution is 10 - 15 times the volume of the complete N-protected amino alcohol or N-protected peptide alcohol compound with C-terminal protection of the halogenated diphenylmethane structure carrier.

[0080] In some embodiments, in step (5), the sedimentation liquid used is preferably a mixed liquid of methyl tert-butyl ether and n-hexane in any ratio, wherein the volume ratio of the two is preferably V 甲基叔丁基醚 :V 正己烷 = 1:1.

[0081] In some embodiments, it further includes step (6) of cyclizing the obtained chain-like peptide alcohol, and oxidizing the peptide alcohol into a cyclic disulfide bond; in the oxidation to form a ring, the solvent used is a mixture of aqueous ammonium acetate solution and ammonia water, so that the pH of the reaction solution is 7-8; the oxidant used is any one or a combination of two of air and dimethyl sulfoxide; when the oxidant contains dimethyl sulfoxide, the dosage of dimethyl sulfoxide is 1%-10% of the volume of the solvent used; when the oxidant is air, the oxidation to form a ring reaction is carried out under standard atmospheric pressure; the oxidation to form a ring is carried out at room temperature.

[0082] Beneficial effects:

[0083] (1) The hydrophobic tag containing a halogenated diphenylmethane structure designed by the present invention is compatible not only with Fmoc amino acids but also with Cbz amino acids (incompatible with solid-phase synthesis). Installing them at the C-terminus of the peptide chain can achieve rapid and efficient synthesis of peptide alcohol in green organic solvents.

[0084] (2) Compared with the solid-phase synthesis method, the beneficial effects of the label-assisted liquid-phase synthesis method of the present invention are as follows: it can achieve rapid on-line monitoring of the reaction process and avoid the occurrence of too many side reactions; it can use green organic solvents to replace N,N-dimethylformamide, which is an environmentally unfriendly and human health-unfriendly solvent used in solid-phase synthesis; it can use stoichiometric amounts of reactants to avoid the use of excessive reagents in solid-phase synthesis; the organic reaction system can be washed with an aqueous solution to achieve rapid and efficient impurity removal, avoiding the use of a large amount of organic solvents in solid-phase synthesis. Generally speaking, the hydrophobic label-assisted liquid-phase synthesis provided by the present invention is easy to achieve on-line monitoring, has high reaction efficiency, high atom economy, less three wastes, is green and environmentally friendly, has low cost, and can achieve large-scale production of peptide alcohol.

[0085] (3) The liquid-phase synthesis method of peptide alcohol assisted by a soluble hydrophobic label designed by the present invention utilizes the advantages of good solubility and easy monitoring of the label in organic solvents to achieve green, efficient, low-cost and large-scale synthesis of peptide alcohol molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0087] Figure 1 1H NMR spectrum of compound 5 in Example 1.

[0088] Figure 2 It is the mass spectrometry diagram of compound 28 in Example 2.

[0089] Figure 3 It is a representative HPLC diagram of the cleavage of the octapeptide synthesized in Example 7.

[0090] Figure 4 It is the mass spectrometry diagram after the cleavage of the octapeptide synthesized in Example 7

[0091] Figure 5 It is the mass spectrometry diagram after the cyclization of the octapeptide synthesized in Example 7.

[0092] Figure 6 It is the HPLC diagram of the cleavage of the 11-peptide synthesized in Example 13.

[0093] Figure 7 It is the mass spectrometry diagram of the cleavage of the 11-peptide synthesized in Example 13.

[0094] Figure 8 It is the 1H NMR spectrum diagram of compound 6 in Example 20. Detailed implementation manners

[0095] According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0096] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0097] The coupling reagent, 2,6-dimethylpyridine, M used in the examples of the present invention W: 107.153; 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, abbreviated as EDC·HCl, Mw = 191.70; 1-Hydroxybenzotriazole, abbreviated as HOBT, Mw = 135.13; Dicyclohexylcarbodiimide, abbreviated as DCC, Mw = 206.33; N,N'-Diisopropylcarbodiimide, abbreviated as DIC, Mw = 126.00; Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, abbreviated as BOP, Mw = 442.28; Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, abbreviated as PyBOP, Mw = 520.39; (1-Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinium hexafluorophosphate, abbreviated as COMU, Mw = 327.40; Ethyl 2-oximinoacetate, abbreviated as Oxyma, Mw = 142.11; 2-(7-Azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, abbreviated as HATU, Mw = 380.24; O-Benzotriazol-tetramethyluronium hexafluorophosphate, abbreviated as HBTU, Mw = 379.23; 4-Dimethylaminopyridine, abbreviated as DMAP, Mw = 122.17; Solvent, N,N-Diisopropylethylamine, abbreviated as DIPEA, Mw = 129.25; Cleavage reagent, Trifluoroacetic acid, abbreviated as TFA, Mw = 114.02; 1,2-Bis(2-ethoxymercapto)ethane (3,6-dioxa-1,8-octanedithiol), abbreviated as DODT, Mw = 182.30; Triisopropylchlorosilane, abbreviated as TIS, Mw = 192.80.

[0098] Example 1:

[0099]

[0100] (1) Preparation of Compound b-5

[0101] Dissolve tert-butyldimethylchlorosilane (25 mmol, 2.5 equiv.) and imidazole (60 mmol, 6.0 equiv.) in dichloromethane (200 mL) to obtain a mixed solution. Then add 4,4-dihydroxybenzophenone (10 mmol, 1.0 equiv.) to the mixed solution and stir overnight at room temperature. After the reaction is completed, add ethyl acetate (100 mL) to extract the reaction solution, and then wash it with 5 wt% K2CO3 aqueous solution (100 mL × 3). Dry the organic phase, concentrate the organic phase under reduced pressure, and purify and separate by column chromatography to obtain Compound b-5 with a yield of 95%.

[0102] (2) Preparation of Compound c-5

[0103] Dissolve the compound b-5 (9.5 mmol, 1.0 equiv.) obtained in step (1) in 250 mL of absolute ethanol, then add sodium borohydride (20 mmol, 2.1 equiv.), and carry out a reduction reaction at room temperature for 3 - 4 h. After the reaction is completed, transfer the reaction system to an ice bath and stir the reaction solution for 10 min. Slowly add 2 M HCl aqueous solution until no gas is generated, test the pH to be 6 - 7, remove the ice bath, transfer it to a stirrer at 80 °C until boiling stops, rotary evaporate the solvent, extract with 150 mL of ethyl acetate, wash three times with an equal amount of saturated brine, dry the organic phase over anhydrous magnesium sulfate, and rotary evaporate under vacuum to obtain compound c-5 with a yield of 90% (calculated based on 4,4-dihydroxybenzophenone).

[0104] (3) Preparation of Compound 5

[0105] Dissolve the compound c-5 (9 mmol, 1.0 equiv.) obtained in step (2) in 250 mL of dichloromethane, then add thionyl chloride (20 mmol, 2.2 equiv.), and carry out a halogenation reaction under heating reflux for 3 - 4 h. After the reaction is completed, wash three times with an equal amount of saturated brine, dry the organic phase over anhydrous magnesium sulfate, and rotary evaporate under vacuum to obtain compound 5 with a yield of 85% (calculated based on 4,4-dihydroxybenzophenone).

[0106] The 1H NMR spectrum of Compound 5 is shown in Figure 1 , and the 1H NMR data are as follows: δ 7.25–7.19 (m, 4H), 6.79 (d, J = 8.5 Hz, 4H), 5.74 (s, 1H), 0.97 (s, 15H), 0.91 (s, 3H), 0.18 (s, 10H), 0.10 (s, 2H).

[0107] Example 2:

[0108]

[0109] The preparation method of intermediate c-5 is the same as that in Example 1, except that the halogenating agent is N-bromosuccinimide.

[0110] Dissolve compound c-5 (9.5 mmol, 1.0 equiv.) in 250 mL of anhydrous dichloromethane, then add N,N'-dimethylthiourea (9.5 mmol, 1.0 equiv.) and N-bromosuccinimide (23.75 mmol, 2.5 equiv.), and then carry out a halogenation reaction at 50 °C for 3 - 4 h. After the reaction is completed, wash three times with an equal amount of saturated brine, dry the organic phase over anhydrous magnesium sulfate, and rotary evaporate under vacuum to obtain compound 28 with a yield of 83% (calculated based on 4,4-dihydroxybenzophenone).

[0111] The 1H NMR data of Compound 28 are as follows:1 1H NMR (400 MHz, CDCl3) δ 7.08 (d, J = 27.5 Hz, 4H), 6.84 (d, J = 8.6 Hz, 4H), 6.36 (s, 1H), 0.98 (d, J = 23.8 Hz, 18H), 0.21 (d, J = 34.5 Hz, 12H). The mass spectrometry data of Compound 28 is as Figure 2 shown, [M + H - Br] = 427.2492.

[0112] Example 3:

[0113]

[0114] The preparation method of Intermediate c - 5 is the same as that of Example 1, except that the halogenating reagent is iodine.

[0115] Dissolve Compound c - 5 (9.5 mmol, 1.0 equiv.) in 250 mL of anhydrous dichloromethane, add triphenylphosphine (9.5 mmol, 1.0 equiv.), imidazole (11.4 mmol, 1.2 equiv.) and I2 (11.4 mmol, 1.2 equiv.), and carry out the halogenation reaction at 35 °C under nitrogen protection for 3 h to synthesize Compound 51 with a yield of 80% (calculated based on 4,4 - dihydroxybenzophenone).

[0116] The 1H NMR data of Compound 51 is as follows: 1 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 27.5 Hz, 4H), 6.88 (d, J = 8.6 Hz, 4H), 6.40 (s, 1H), 0.89 (d, J = 23.8 Hz, 18H), 0.24 (d, J = 34.5 Hz, 12H).

[0117] Example 4:

[0118]

[0119] The preparation method is the same as that of Example 1, except that tert - butyldimethylchlorosilane is replaced by dodecyldimethylchlorosilane, and finally Compound 23 is prepared with a yield of 86% (calculated based on 4,4 - dihydroxybenzophenone).

[0120] The 1H NMR data of Compound 23 is as follows: 1HNMR(400MHz, CDCl3) δ 7.08 (d, J = 7.8 Hz, 4H), 6.84 (d, J = 7.6 Hz, 4H), 6.04 (s, 1H), 1.33 - 1.23 (dd, J = 18.8, 7.5 Hz, 40H), 0.89 (dd, J = 17.8, 7.5 Hz, 6H), 0.61 (s, 4H), 0.21 (s, 12H).

[0121] Example 5:

[0122]

[0123] Using compound a - 23 as the raw material, the methods for sequentially synthesizing compound b - 23 and compound c - 23 are the same as in Example 4. The difference is that the method for synthesizing compound 46 from compound c - 23 is the same as the method in Example 2. Finally, compound 46 is prepared with a yield of 82% (calculated based on 4,4 - dihydroxybenzophenone).

[0124] The 1H - NMR data of compound 46 are as follows: 1 HNMR(400MHz, CDCl3) δ 7.20 (d, J = 7.8 Hz, 4H), 6.92 (d, J = 7.6 Hz, 4H), 6.36 (s, 1H), 1.43 - 1.30 (dd, J = 16.6, 7.2 Hz, 32Hz, 40H), 0.95 (dd, J = 16.7, 7.9 Hz, 6H), 0.70 (s, 4H), 0.26 (s, 12H).

[0125] Example 6:

[0126]

[0127] Using compound a - 23 as the raw material, the methods for sequentially synthesizing compound b - 23 and compound c - 23 are the same as in Example 4. The difference is that the method for synthesizing compound 68 from compound c - 23 is the same as the method in Example 3. Finally, compound 68 is prepared with a yield of 81% (calculated based on 4,4 - dihydroxybenzophenone).

[0128] The 1H - NMR data of compound 68 are as follows: 1 HNMR(400MHz, CDCl3) δ 7.14 (d, J = 7.8 Hz, 4H), 6.87 (d, J = 7.6 Hz, 4H), 6.62 (s, 1H), 1.39 - 1.21 (dd, J = 16.6, 7.2 Hz, 32Hz, 40H), 0.92 (dd, J = 16.7, 7.9 Hz, 6H), 0.66 (s, 4H), 0.24 (s, 12H).

[0129] Example 7: Liquid-phase synthesis of octreotide using compound 5 as a tag

[0130] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0131] (1) Synthesis of Fmoc-Thr(tBu)-TAG5

[0132] Compound 5 (1 mmol, 1.0 equiv.) (prepared in Example 1), Fmoc-Thr(tBu)-ol (2.1 mmol, 2.1 equiv.), and 2,6-dimethylpyridine (2.5 mmol, 2.5 equiv.) were added to 40 mL of ethyl acetate, and the reaction was carried out at 60 - 70 °C for 4 h. After the reaction, the reaction solution containing Fmoc-Thr(tBu)-TAG5 with the amino acid linked to compound 5 was obtained. The reaction solution was washed three times with 40 mL of 5 wt% K2CO3 to remove the excess amino alcohol and coupling reagent, the solvent was evaporated, and the product was purified by silica gel column chromatography (PE:EA = 5:1) to obtain Fmoc-Thr(tBu)-TAG5 with a yield of 95% (based on compound 5).

[0133] (2) Removal of Fmoc protection

[0134] Fmoc-Thr(tBu)-TAG5 (0.95 mmol, 1.0 equiv.) obtained in step (1) and pyrrolidine (16 mmol, 16.8 equiv.) were dissolved in 40 mL of ethyl acetate, and the deprotection reaction was stirred at room temperature to 40 °C for 1 h. After the reaction, the reaction solution was first extracted and washed three times with an equal volume of 40 mL of 3 wt% hydrochloric acid aqueous solution, and then extracted and washed three times with an equal volume of 40 mL of 5 wt% K2CO3. The upper organic phase solution was taken to obtain a washing solution containing NH2-Thr(tBu)-TAG5.

[0135] (3) Coupling of Fmoc-Cys(Trt)-OH

[0136] To the washing solution containing NH2-Thr(tBu)-TAG5 obtained in step (2), add Fmoc-Cys(Trt)-OH (the amount used is 1.0 - 2.0 equivalents of the tag compound 5), EDC·HCl (the amount used is 1.0 - 2.0 equivalents of the tag compound 5), and HOBT (the amount used is 1.0 - 2.0 equivalents of the tag compound 5), and react at a temperature of room temperature to 40 °C for 1 h. After the reaction is completed, extract and wash the reaction solution three times with 40 mL of 5 wt% K2CO3 to remove the excess amino acids and coupling reagents, take the upper organic phase to obtain a washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-TAG5.

[0137] (4) Fmoc deprotection

[0138] Add pyrrolidine (the amount used is 5.0 - 8.0 equivalents of the tag compound 5) and 40 mL of ethyl acetate to the washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-TAG5 obtained in step (3), stir and react at a temperature of room temperature to 40 °C for 1 h. After the reaction is completed, first extract and wash three times with an equal amount of 40 mL of 3 wt% hydrochloric acid aqueous solution, and then extract and wash three times with an equal amount of 40 mL of 5 wt% K2CO3, take the upper organic phase solution to obtain a washing solution containing NH2-Cys(Trt)-Thr(tBu)-TAG5.

[0139] (5) Coupling of Fmoc-Thr(tBu)-OH

[0140] Weigh Fmoc-Thr(tBu)-OH (the amount used is 1.2 - 1.3 equivalents of the tag compound 5), dissolve it in 20 mL of ethyl acetate, wash it twice with 3 wt% hydrochloric acid aqueous solution (20 mL × 2), then add it to the washing solution containing NH2-Cys(Trt)-Thr(tBu)-TAG5 obtained in step (4), and then add EDC·HCl (the amount used is 1.0 - 2.0 equivalents of the tag compound 5) and HOBT (the amount used is 1.0 - 2.0 equivalents of the tag compound 5), and react at a temperature of room temperature to 40 °C for 1 h. After the reaction is completed, wash the reaction solution three times with 40 mL of 5 wt% K2CO3 to remove the excess amino acids and coupling reagents, take the upper organic phase solution to obtain a washing solution containing Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5.

[0141] (6) Coupling of the remaining amino acids

[0142] Deprotect the Fmoc protecting group according to the method in step (4) above, and then continuously introduce Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH according to the methods in steps (3) and (4) and deprotect Fmoc to obtain a reaction solution containing NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5. Rotate the solvent to dryness under vacuum and separate and purify by column chromatography (DCM:MeOH = 20:1) to obtain NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5 with an HPLC purity of over 90%.

[0143] (7) Cleave side chains and label compounds

[0144] Dissolve the crude product of NH2–D–Phe–Cys(Trt)–Phe–D–Trp(Boc)-Lys(Boc)–Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5 obtained in step (6) in TFA:DODT:Tis:pure water (10 - 15 times the volume of the crude product, V TFA :V TIS :V DODT :V 纯水 =92.5:2.5:2.5:2.5), and cleave at a stable room temperature to 60 °C in a shaker for 1 - 3 h. After the cleavage is completed, add a sedimentation solution with a volume 10 - 15 times that of the cleavage solution (V 甲基叔丁基醚 :V 正己烷 =1:1) and sediment at 3000 r / min in a centrifuge. Repeat three times. Each time, pour off the liquid and retain the solid at the bottom. Centrifuge for three minutes each time when adding the sedimentation solution to obtain the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol with a yield of 78% - 82% (based on compound 5) and an HPLC purity of 82 - 84%. The liquid phase data is as Figure 3 shown.

[0145] The mass spectrometry data of the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol is as Figure 4 shown: [M + 2H] 2+ / 2: 511.2.

[0146] (8) Cyclization

[0147] Dissolve the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol obtained in step (7) in an aqueous solution of 0.2 M ammonium acetate, add ammonia dropwise to keep the pH of the solution at 7 - 8, and carry out a cyclization reaction by air oxidation for 36 h to obtain the cyclized NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) with a yield of 70% - 75% (based on compound 5) and an HPLC purity of 78% - 81%.

[0148] The mass spectrometry data of NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) is as Figure 5 shown: [M + 2H] 2+ / 2: 510.3.

[0149] Example 8: Liquid-phase synthesis of octreotide using compound 28 as a tag

[0150] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0151] (1) Synthesis of Fmoc-Thr(tBu)-TAG28

[0152] The experimental method is the same as step (1) in Example 7, except that compound 5 is replaced with compound 28 (prepared in Example 2), and the other material compositions and dosages are the same. Finally, Fmoc-Thr(tBu)-TAG28 is obtained with a yield of 93% (based on compound 28).

[0153] (2) Removal of the Fmoc protection

[0154] The experimental method is the same as step (2) in Example 7, and finally, a washing solution containing NH2-Thr(tBu)-TAG28 is obtained.

[0155] (3) Coupling of Fmoc-Cys(Trt)-OH

[0156] The experimental method is the same as step (3) in Example 7, and finally, a washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-TAG28 is obtained.

[0157] (4) Fmoc deprotection

[0158] The experimental method was the same as step (4) in Example 7, and finally a washing solution containing NH2-Cys(Trt)-Thr(tBu)-TAG28 was obtained.

[0159] (5) Coupling Fmoc-Thr(tBu)-OH

[0160] The experimental method was the same as step (5) in Example 7, and finally a washing solution containing Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG28 was obtained.

[0161] (6) Coupling the remaining amino acids

[0162] The experimental method was the same as step (6) in Example 7, and finally NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG28 was obtained, with an HPLC purity of over 85%.

[0163] (7) Cleaving the side chains and the tag compound

[0164] The experimental method was the same as step (7) in Example 7, and finally the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol was obtained, with a yield of 75% - 79% (based on compound 28), and an HPLC purity of 72% - 76%. The mass spectrometry data was the same as Figure 4 .

[0165] (8) Cyclization

[0166] The experimental method was the same as step (8) in Example 7, and finally NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) was obtained, with a yield of 70% - 72% (based on compound 28), and an HPLC purity of 70% - 73%. The mass spectrometry data was the same as Figure 5 .

[0167] Example 9: Liquid-phase synthesis of octreotide using compound 51 as the tag

[0168] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0169] (1) Synthesis of Fmoc-Thr(tBu)-TAG51

[0170] The experimental method was the same as step (1) in Example 7, except that compound 5 was replaced with compound 51 (prepared in Example 3), and the other material compositions and material dosages were the same. Finally, Fmoc-Thr(tBu)-TAG51 was obtained with a yield of 90% (based on compound 51).

[0171] (2) Removal of Fmoc protection

[0172] The experimental method was the same as step (2) in Example 7, and finally a washing solution containing NH2-Thr(tBu)-TAG51 was obtained.

[0173] (3) Coupling of Fmoc-Cys(Trt)-OH

[0174] The experimental method was the same as step (3) in Example 7, and finally a washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-TAG51 was obtained.

[0175] (4) Fmoc deprotection

[0176] The experimental method was the same as step (4) in Example 7, and finally a washing solution containing NH2-Cys(Trt)-Thr(tBu)-TAG51 was obtained.

[0177] (5) Coupling of Fmoc-Thr(tBu)-OH

[0178] The experimental method was the same as step (5) in Example 7, and finally a washing solution containing Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG51 was obtained.

[0179] (6) Coupling of the remaining amino acids

[0180] The experimental method was the same as step (6) in Example 7, and finally NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG51 was obtained with an HPLC purity of over 85%.

[0181] (7) Cleavage of side chains and tag compounds

[0182] The experimental method was the same as step (7) in Example 7, and finally the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol was obtained with a yield of 73% - 76% (based on compound 51) and an HPLC purity of 70% - 74%. The mass spectrometry data was the same as Figure 4 .

[0183] (8) Cyclization

[0184] The experimental method was the same as step (8) in Example 7. Finally, NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) was obtained with a yield of 66% - 70% (based on compound 51) and an HPLC purity of 66% - 70%. The mass spectrometry data was the same as Figure 5 .

[0185] Example 10: Solid-phase synthesis of octreotide

[0186] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0187] (1) Synthesis of Fmoc-Thr(tBu)-CTC Resin

[0188] CTC Resin (1 mol, substitution degree 1.211 mmol / g) was soaked in dichloromethane (DCM) for 2 h to swell. After that, it was poured into a solid-phase reactor, rinsed three times with dichloromethane, and then Fmoc-Thr(tBu)-ol (3.5 mol, 3.5 equiv.), N,N-diisopropylethylamine (DIEA) (3.5 mol, 3.5 equiv.), 3 L DCM, and 3 L DMF were added. The reaction was carried out at 20 - 30 °C for 12 h. After the reaction, Fmoc-Thr(tBu)-CTC Resin with the amino acid linked to CTC Resin was obtained. The resin was washed three times with 6 L DCM to remove excess amino alcohol and coupling reagent, and then blocked three times with a blocking solution (V DCM :V MeOH :V DIEA = 17:2:1). After completion, the resin was washed three times with 6 L DMF to obtain Fmoc-Thr(tBu)-CTC Resin.

[0189] (2) Removal of Fmoc protection

[0190] Fmoc-Thr(tBu)-CTC Resin (0.95 mol, 1.0 equiv.) obtained in step (1) and pyrrolidine (16 mol, 16.8 equiv.) were dissolved in 6 L DMF, and the deprotection reaction was stirred at room temperature to 40 °C for 0.5 h. After the reaction, it was washed three times with 6 L DMF to obtain NH2-Thr(tBu)-CTC Resin.

[0191] (3) Coupling of Fmoc-Cys(Trt)-OH

[0192] Add Fmoc-Cys(Trt)-OH (the dosage is 3.0 - 4.0 equivalents of CTC Resin), DIC (the dosage of DIC is 3.0 - 4.0 equivalents of CTC Resin), HOBT (the dosage of HOBT is 3.0 - 4.0 equivalents of CTC Resin), and 6L of DMF to the NH2-Thr(tBu)-CTC Resin obtained in step (2), and react at a temperature of room temperature to 40 °C for 1 h. After the reaction is completed, wash three times with 6L of DMF to remove the excess amino acids and coupling reagents, and obtain Fmoc-Cys(Trt)-Thr(tBu)-CTC Resin.

[0193] (4) Fmoc deprotection

[0194] Add 6L of 20% pyrrolidine (dissolved in DMF) to the Fmoc-Cys(Trt)-Thr(tBu)-CTC Resin obtained in step (3), stir and react at a temperature of room temperature to 40 °C for 0.5 h. After the reaction is completed, wash three times with DMF to obtain NH2-Cys(Trt)-Thr(tBu)-CTC Resin.

[0195] (5) Couple Fmoc-Thr(tBu)-OH

[0196] Weigh Fmoc-Thr(tBu)-OH (the dosage is 3 - 4 equivalents of CTC Resin), then add it to the NH2-Cys(Trt)-Thr(tBu)-CTC Resin obtained in step (4), and then add DIC (the dosage of DIC is 3.0 - 4.0 equivalents of CTC Resin), HOBT (the dosage of HOBT is 3.0 - 4.0 equivalents of CTC Resin), and 6L of DMF, and react at a temperature of room temperature to 40 °C for 1 h. After the reaction is completed, wash three times with DMF to remove the excess amino acids and coupling reagents, and obtain Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-CTC Resin.

[0197] (6) Couple the remaining amino acids

[0198] Deprotect the Fmoc protecting group according to the method in step (4) above, and then successively couple Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH according to the methods in steps (3) and (4) and deprotect Fmoc. After obtaining NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-CTC Resin, wash the resin 3 times with DMF, then 3 times with DCM, and finally 3 times with methanol to shrink the resin, obtaining NH2–D–Phe–Cys(Trt)–Phe–D–Trp(Boc)–Lys(Boc)–Thr(tBu)–Cys(Trt)-Thr(tBu)-CTC Resin with an HPLC purity of over 65%.

[0199] (7) Cleave the side chain and label compounds

[0200] Dissolve the NH2–D–Phe–Cys(Trt)–Phe–D–Trp(Boc)–Lys(Boc)–Thr(tBu)–Cys(Trt)-Thr(tBu)-CTC Resin obtained in step (6) in TFA:DODT:Tis:pure water (10 - 15 times the volume of the crude product, V TFA :V TIS :V DODT :V 纯水 =92.5:2.5:2.5:2.5), and cleave at a stable room temperature to 60 °C in a shaker for 1 - 3 h. After the cleavage is completed, filter out the resin, add a sedimentation solution with a volume 10 - 15 times that of the cleavage solution (V 甲基叔丁基醚 :V 正己烷 =1:1), sediment at 3000 r / min in a centrifuge, repeat three times, each time pour out the liquid and retain the solid at the bottom. Centrifuge for three minutes each time when adding the sedimentation solution, obtaining the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol with a yield of 50% - 55% (based on CTC Resin) and an HPLC purity of 59% - 63%.

[0201] The mass spectrometry data of the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol is the same as Figure 4 shown.

[0202] (8) Cyclization

[0203] Dissolve the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol obtained in step (7) in an aqueous solution of 0.2 M ammonium acetate, add ammonia water dropwise to keep the pH of the solution at 7-8, and carry out a cyclization reaction by air oxidation for 36 h to obtain cyclized NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) with a yield of 40%-45% (based on CTC Resin) and an HPLC purity of 50%-55%.

[0204] The mass spectrometry data of NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) is the same as Figure 5 shown.

[0205] Example 11: Large-scale liquid-phase synthesis of octreotide using compound 5 (1 mol) as a tag

[0206] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0207] (1) Synthesize Fmoc-Thr(tBu)-TAG5

[0208] The experimental method is the same as step (1) in Example 7, except that 1 mmol of compound 5 is replaced by 1 mol of compound 5, and the other material compositions are the same. Finally, Fmoc-Thr(tBu)-TAG5 is obtained with a yield of 92% (based on compound 5).

[0209] (2) Remove the Fmoc protection

[0210] The experimental method is the same as step (2) in Example 7, and finally a washing solution containing NH2-Thr(tBu)-TAG5 is obtained.

[0211] (3) Couple Fmoc-Cys(Trt)-OH

[0212] The experimental method is the same as step (3) in Example 7, and finally a washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-TAG5 is obtained.

[0213] (4) Fmoc deprotection

[0214] The experimental method was the same as step (4) in Example 7, and finally a washing solution containing NH2-Cys(Trt)-Thr(tBu)-TAG5 was obtained.

[0215] (5) Coupling Fmoc-Thr(tBu)-OH

[0216] The experimental method was the same as step (5) in Example 7, and finally a washing solution containing Fmoc-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5 was obtained.

[0217] (6) Coupling the remaining amino acids

[0218] The experimental method was the same as step (6) in Example 7, and finally NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-TAG5 was obtained, with an HPLC purity of over 80%.

[0219] (7) Cleaving the side chains and the tag compound

[0220] The experimental method was the same as step (7) in Example 7, and finally the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol was obtained, with a yield of 65% - 70% (based on compound 5) and an HPLC purity of 71% - 75%. The mass spectrometry data was the same as Figure 4 .

[0221] (8) Cyclization

[0222] The experimental method was the same as step (8) in Example 7, and finally NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) was obtained, with a yield of 58% - 60% (based on compound 5) and an HPLC purity of 70% - 75%. The mass spectrometry data was the same as Figure 5 .

[0223] Example 12: Liquid-phase synthesis of octreotide using commercially available dibenzocycloheptenone-based chloride as a tag (comparative example)

[0224] Dibenzocycloheptenone-based chloride:

[0225] Octreotide: D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7)

[0226] (1) Synthesis of Fmoc-Thr(tBu)-Commercial Tag

[0227] The experimental method was the same as step (1) in Example 7, except that compound 5 was replaced with dibenzocycloheptenone chloride (commercially purchased), and the other material compositions and material dosages were the same. Finally, Fmoc-Thr(tBu)-Commercial Tag was obtained with a yield of 90% (calculated based on dibenzocycloheptenone chloride).

[0228] (2) Removal of Fmoc Protection

[0229] Dissolve the Fmoc-Thr(tBu)-Commercial Tag (0.90 mmol, 1.0 equiv.) obtained in step (1) and pyrrolidine (16 mmol, 17.8 equiv.) in 40 mL of ethyl acetate, and stir for 1 h at room temperature to 40 °C for the deprotection reaction. After the reaction, because the dibenzocycloheptenone chloride has poor hydrophobicity, most of the target product will dissolve in the aqueous phase after washing with water, resulting in huge losses. Therefore, the deprotected product obtained cannot be purified by washing with water to remove impurities and can only be purified and separated by silica gel column chromatography (PE:EA = 1:1) to obtain NH2-Thr(tBu)-Commercial Tag with a yield of 75% (calculated based on dibenzocycloheptenone chloride).

[0230] (3) Coupling of Fmoc-Cys(Trt)-OH

[0231] Add 40 mL of ethyl acetate, Fmoc-Cys(Trt)-OH (the dosage is 1.0 - 2.0 equivalents of the dibenzocycloheptenone chloride of the tag), EDC·HCl (the dosage of EDC·HCl is 1.0 - 2.0 equivalents of the dibenzocycloheptenone chloride of the tag), and HOBT (the dosage of HOBT is 1.0 - 2.0 equivalents of the dibenzocycloheptenone chloride of the tag) to the NH2-Thr(tBu)-Commercial Tag obtained in step (2), and keep the temperature at room temperature to 40 °C for 1 h. After the reaction, extract and wash the reaction solution three times with 40 mL of 5 wt% K2CO3 to remove excess amino acids and coupling reagents, take the upper organic phase to obtain a washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-Commercial Tag.

[0232] (4) Fmoc Deprotection

[0233] To the washing solution containing Fmoc-Cys(Trt)-Thr(tBu)-commercial tag obtained in step (3), pyrrolidine (the amount of pyrrolidine is 5.0 - 8.0 equivalents of the dibenzocycloheptenone-based chloride of the tag) and 40 mL of ethyl acetate were added, and the mixture was stirred at room temperature to 40 °C for 1 h. After the reaction, since the hydrophobicity of this commercial dibenzocycloheptenone-based chloride is poor, most of the target product will dissolve in the aqueous phase through water washing, resulting in huge losses. Therefore, the deprotected product obtained cannot be purified by water washing to remove impurities and can only be purified and separated by silica gel column chromatography (PE:EA = 1:1) to obtain NH2-Cys(Trt)-Thr(tBu)-commercial tag with a yield of 60% (based on the dibenzocycloheptenone-based chloride).

[0234] (5) Coupling of the remaining amino acids

[0235] According to the methods of step (3) and step (4), Fmoc-Thr(tBu)-OH (which needs to be washed with 3 wt% hydrochloric acid aqueous solution, and the method is the same as step 5 in Example 7), Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH were successively introduced and the Fmoc group was removed, obtaining a reaction solution containing NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-commercial tag. The solvent was evaporated under vacuum, and the product was separated and purified by column chromatography (DCM:MeOH = 20:1) to obtain NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-commercial tag with an HPLC purity of over 95%.

[0236] (6) Cleavage of side chains and tag compounds

[0237] According to the experimental method in step (7) of Example 1, the NH2-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-commercial tag obtained in step (5) of this example was subjected to side chain cleavage and tag compound cleavage, and finally the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol was obtained with a yield of 45% - 50% (based on the dibenzocycloheptenone-based chloride) and an HPLC purity of 88% - 90%. The mass spectrometry data of the naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol is the same asFigure 4 .

[0238] (7) Cyclization

[0239] The experimental method was the same as that in step 8 of Example 1. The naked peptide NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol obtained in step (6) of this example was subjected to an air oxidation cyclization reaction to finally obtain NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) with a yield of 40% - 43% (based on dibenzocycloheptenone chloride) and an HPLC purity of 83% - 85%. The mass spectrometry data of NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7) was the same as Figure 5 .

[0240] Conclusion: In Example 12, a commercial dibenzocycloheptenone chloride was used as the tag compound. Compared with Examples 7, 8, and 9 that used the tag compounds provided by the present invention, since the aromatic ring structure of the commercial dibenzocycloheptenone chloride was not modified, its hydrophobicity was relatively small, and the hydrophobic property difference between the peptide intermediate modified by it and the reaction substrate and the generated impurities was small. During the washing and impurity removal process, the loss of the peptide intermediate was large, and it was impossible to quickly remove impurities by washing with an aqueous solution. Only methods such as column chromatography could be used for separation and purification. The operation process was complex, the product loss was large, and the yield of each intermediate step and the final yield were significantly lower than those of the examples using the tags of the present invention.

[0241] Example 13: Liquid-phase synthesis of the antibacterial peptide Trichogin GAⅣ using compound 5 as the tag

[0242] Antibacterial peptide Trichogin GAⅣ: Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol

[0243] (1) Synthesis of Cbz-Leu-TAG5

[0244] Compound 5 (1 mmol, 1.0 equiv.) (prepared in Example 1), Cbz-Leu-ol (2.1 mmol, 2.1 equiv.), and 2,6-dimethylpyridine (2.5 mmol, 2.5 equiv.) were added to 40 mL of ethyl acetate, and the reaction was carried out at 60 - 80 °C for 1 h. After the reaction was completed, the reaction solution of Cbz-Leu-TAG5 with the amino acid attached to Compound 5 was obtained. The reaction solution was washed three times with 40 mL of 5 wt% K2CO3 to remove the excess amino acid and coupling reagent, the solvent was evaporated to dryness, and the product was purified by silica gel column chromatography (PE:EA = 5:1) to obtain Cbz-Leu-TAG5 with a yield of 95% (based on Compound 5).

[0245] (2) Removal of Cbz protection

[0246] Cbz-Leu-TAG5 obtained in step (1) was mixed with 5% Pd / C (the amount of palladium used was 0.1 - 1.0 equivalents of the labeled compound 5) and dissolved in 50 mL of ethyl acetate (the solvent could also be methanol). The air in the reaction flask was replaced with hydrogen using a water pump (a hydrogen balloon was inserted above the reaction flask), and the reaction was stirred in a hydrogen atmosphere at room temperature - 40 °C for 30 min. After the reaction was completed, Pd / C was filtered off, the solvent was evaporated to dryness, 40 mL of ethyl acetate was added, and the mixture was washed three times with 40 mL of 5 wt% K2CO3 to remove the excess amino acid and coupling reagent. The upper organic phase solution was taken to obtain a washing solution containing NH2-Leu-TAG5.

[0247] (3) Coupling of Cbz-Ile-OH·DCHA

[0248] Cbz-Ile-OH·DCHA (the amount used was 1.0 - 2.0 equivalents of the labeled compound 5), EDC·HCl (the amount of EDC·HCl used was 1.0 - 2.0 equivalents of the labeled compound 5), and HOBT (the amount of HOBT used was 1.0 - 2.0 equivalents of the labeled compound 5) were added to the washing solution containing NH2-Leu-TAG5 obtained in step (2), and the reaction was carried out at room temperature - 40 °C for 2.5 h. After the reaction was completed, the reaction solution was washed three times with 5 wt% K2CO3 to remove the excess amino acid and coupling reagent. The upper organic phase solution was taken to obtain a washing solution containing Cbz-Ile-Leu-TAG5.

[0249] (4) Removal of Cbz protection

[0250] The washing solution containing Cbz-Ile-Leu-TAG5 obtained in step (3) is mixed with 5% Pd / C (the amount of palladium used is 0.1 - 1.0 equivalents of the tag compound 5) and dissolved in 50 mL of ethyl acetate (the solvent can also be methanol). The air in the reaction flask is replaced with hydrogen using a water pump (a hydrogen balloon is inserted above the reaction flask), and the reaction is stirred for 30 min in a hydrogen atmosphere at room temperature to 40 °C. After the reaction is completed, Pd / C is filtered off, the solvent is rotary evaporated, 40 mL of ethyl acetate is added, and the mixture is washed three times with 40 mL of 5 wt% K2CO3 to remove the excess amino acids and coupling reagents. The upper organic phase solution is taken to obtain a washing solution containing NH2-Ile-Leu-TAG5.

[0251] (5) Couple Cbz-Gly-OH

[0252] Weigh Cbz-Gly-OH (the amount used is 1.2 - 1.5 equivalents of the tag compound 5), dissolve it in 10 mL of ethyl acetate, wash it twice with 3 wt% hydrochloric acid aqueous solution (10 mL × 2), then add it to the washing solution containing NH2-Ile-Leu-TAG5 obtained in step (4), and then add EDC·HCl (the amount of EDC·HCl used is 1.0 - 2.0 equivalents of the tag compound 5) and HOBT (the amount of HOBT used is 1.0 - 2.0 equivalents of the tag compound 5). The reaction is carried out at a temperature of room temperature to 40 °C for 2.5 h. After the reaction is completed, the reaction solution is washed three times with 5 wt% K2CO3 to remove the excess amino acids and coupling reagents. The upper organic phase solution is taken to obtain a washing solution containing Cbz-Gly-Ile-Leu-TAG5.

[0253] (6) Couple the remaining amino acids

[0254] Remove the Cbz protecting group according to the method of step (4) above, and then continuously introduce Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Gly-OH, Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Aib-OH according to the methods of steps (3) and (4). After removing Cbz, a reaction solution containing Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-TAG5 is obtained. The solvent is rotary evaporated under vacuum and separated and purified by a chromatography column (DCM:MeOH = 15:1) to obtain Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol-TAG5 with an HPLC purity of over 85%.

[0255] (7) Cleave the tag compound

[0256] Dissolve Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol-TAG5 obtained in step (6) in TFA:DODT:Tis:pure water (10 - 15 times the volume of the crude product, V TFA :V TIS :V DODT :V 纯水 =92.5:2.5:2.5:2.5), and cut at room temperature to 60 °C in a shaker for 1 - 3 h. After the cutting is completed, add a sedimentation solution with a volume 10 - 15 times that of the cutting solution (V 甲基叔丁基醚 :V 正己烷 =1:1) and centrifuge for sedimentation at 3000 r / min in a centrifuge. Repeat three times. Each time, pour out the liquid and retain the solid at the bottom. Centrifuge for three minutes each time when adding the sedimentation solution to obtain the naked peptide Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol, with a yield of 76% - 82% (based on compound 5) and an HPLC purity of 78% - 83%.

[0257] A representative HPLC chromatogram of Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol is as shown in Figure 6 ; the mass spectrometry data chart is as shown in Figure 7 ; [M+Na] + :963.4.

[0258] Example 14: Liquid-phase synthesis of the antimicrobial peptide Trichogin GAⅣ using compound 28 as a tag

[0259] Antimicrobial peptide Trichogin GAⅣ: Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol

[0260] (1) Synthesize Cbz-Leu-TAG28

[0261] The experimental method is the same as step (1) of Example 13. The difference is that compound 5 is replaced with compound 28 (obtained in Example 2), and the other materials and their dosages are the same. Finally, Cbz-Leu-TAG28 is prepared with a yield of 95% (based on compound 28).

[0262] (2) Remove the Cbz protection

[0263] The experimental method is the same as step (2) of Example 13. Deprotect Cbz-Leu-TAG28, and finally prepare a washing solution containing NH2-Leu-TAG28.

[0264] (3) Coupling of Cbz-Ile-OH·DCHA

[0265] The experimental method was the same as step (3) of Example 13. NH2-Leu-TAG28 was continuously coupled with Cbz-Ile-OH·DCHA, and finally a washing solution containing Cbz-Ile-Leu-TAG28 was prepared.

[0266] (4) Removal of Cbz protection

[0267] The experimental method was the same as step (4) of Example 13. Cbz-Ile-Leu-TAG28 was deprotected, and finally a washing solution containing NH2-Ile-Leu-TAG28 was prepared.

[0268] (5) Coupling of Cbz-Gly-OH

[0269] The experimental method was the same as step (5) of Example 13. NH2-Ile-Leu-TAG28 was coupled with Cbz-Gly-OH, and finally a washing solution containing Cbz-Gly-Ile-Leu-TAG28 was prepared.

[0270] (6) Coupling of the remaining amino acids

[0271] The experimental method was the same as step (6) of Example 13. Cbz-Gly-Ile-Leu-TAG28 was continuously coupled with Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Gly-OH, Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Aib-OH in sequence, and then Cbz was removed, separated and purified to obtain Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol-TAG28 with an HPLC purity of over 85%.

[0272] (7) Cleavage of the tag compound

[0273] The experimental method was the same as step (7) of Example 13. The tag compound 28 was cleaved, and finally Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol was prepared with a yield of 74% - 80% (based on compound 28) and an HPLC purity of 75% - 81%.

[0274] Example 15: Liquid-phase synthesis of antibacterial peptide Trichogin GAⅣ using compound 51 as a tag

[0275] Antibacterial peptide Trichogin GAⅣ: Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol

[0276] (1) Synthesize Cbz-Leu-TAG51

[0277] The experimental method was the same as step (1) of Example 13, except that compound 5 was replaced with compound 51 (obtained from the preparation in Example 2), and the other materials and their dosages were the same. Finally, Cbz-Leu-TAG51 was prepared with a yield of 95% (based on compound 51).

[0278] (2) Remove the Cbz protection

[0279] The experimental method was the same as step (2) of Example 13. Cbz-Leu-TAG51 was deprotected, and finally a washing solution containing NH2-Leu-TAG51 was prepared.

[0280] (3) Couple Cbz-Ile-OH·DCHA

[0281] The experimental method was the same as step (3) of Example 13. NH2-Leu-TAG51 was further coupled with Cbz-Ile-OH·DCHA, and finally a washing solution containing Cbz-Ile-Leu-TAG51 was prepared.

[0282] (4) Remove the Cbz protection

[0283] The experimental method was the same as step (4) of Example 13. Cbz-Ile-Leu-TAG5 was deprotected, and finally a washing solution containing NH2-Ile-Leu-TAG51 was prepared.

[0284] (5) Couple Cbz-Gly-OH

[0285] The experimental method was the same as step (5) of Example 13. NH2-Ile-Leu-TAG51 was coupled with Cbz-Gly-OH, and finally a washing solution containing Cbz-Gly-Ile-Leu-TAG51 was prepared.

[0286] (6) Couple the remaining amino acids

[0287] The experimental method was the same as step (6) of Example 13. Cbz-Gly-Ile-Leu-TAG51 was successively coupled with Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Gly-OH, Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, and Cbz-Aib-OH, and then the Cbz group was removed and the product was separated and purified to obtain Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol-TAG51 with an HPLC purity of over 85%.

[0288] (7) Cleavage of the tag compound

[0289] The experimental method was the same as step (7) of Example 13. The tag compound 51 was cleaved, and finally Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol was prepared with a yield of 76% - 81% (based on compound 51) and an HPLC purity of 79% - 82%.

[0290] Example 16: Solid-phase synthesis of antibacterial peptide Trichogin GAⅣ

[0291] Antibacterial peptide Trichogin GAⅣ: Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol

[0292] (1) Synthesis of Fmoc-Leu-CTC Resin

[0293] CTC Resin (1 mol) was swollen in dichloromethane (DCM) for 2 h. After that, it was poured into a solid-phase reaction kettle, rinsed three times with dichloromethane, and then Fmoc-Leu-ol (3.5 mol, 3.5 equiv.), N,N-diisopropylethylamine (DIEA) (3.5 mol, 3.5 equiv.), 3 L of DCM, and 3 L of DMF were added. The reaction was carried out at a temperature of 20 °C - 30 °C for 12 h. After the reaction was completed, Fmoc-Leu-CTC Resin with an amino acid linked to CTC Resin was obtained. The resin was washed three times with 6 L of DCM to remove excess amino alcohol and coupling reagent, and then blocked three times with a blocking solution (V DCM :V MeOH :V DIEA = 17:2:1). After completion, the resin was washed three times with 6 L of DMF to obtain Fmoc-Leu-CTC Resin.

[0294] (2) Removal of the Fmoc protection

[0295] Dissolve Fmoc-Leu-CTC Resin (0.95 mol, 1.0 equiv.) obtained in step (1) and pyrrolidine (16 mol, 16.8 equiv.) in 6 L of DMF, and stir for deprotection reaction at room temperature to 40 °C for 0.5 h. After the reaction, wash three times with 6 L of DMF to obtain NH2-Leu-CTC Resin.

[0296] (3) Couple Fmoc-Ile-OH

[0297] Add Fmoc-Ile-OH (the dosage is 3.0 - 4.0 equivalents of CTC Resin), DIC (the dosage of DIC is 3.0 - 4.0 equivalents of CTC Resin), HOBT (the dosage of HOBT is 3.0 - 4.0 equivalents of CTC Resin), and 40 mL of DMF to NH2-Leu-CTC Resin obtained in step (2), and keep the temperature at room temperature to 40 °C for reaction for 1 h. After the reaction, remove the excess amino acid and coupling reagent with 6 L of DMF three times to obtain Fmoc-Ile-Leu-CTC Resin.

[0298] (4) Remove Fmoc protection

[0299] Dissolve Fmoc-Ile-Leu-CTC Resin containing the product obtained in step (3) and pyrrolidine (16 mmol, 16.8 equiv.) in 40 mL of DMF, and stir for deprotection reaction at room temperature to 40 °C for 0.5 h. After the reaction, wash three times with 6 L of DMF to obtain NH2-Ile-Leu-CTC Resin.

[0300] (5) Couple Fmoc-Gly-OH

[0301] Weigh Fmoc-Gly-OH (the dosage is 3.0 - 4.0 equivalents of CTC Resin), then add it to NH2-Ile-Leu-CTC Resin obtained in step (4), and then add DIC (the dosage of DIC is 3.0 - 4.0 equivalents of the labeled compound 5), HOBT (the dosage of HOBT is 3.0 - 4.0 equivalents of CTC Resin), and 6 L of DMF, and keep the temperature at room temperature to 40 °C for reaction for 2.5 h. After the reaction, wash three times with DMF to remove the excess amino acid and coupling reagent to obtain Fmoc-Gly-Ile-Leu-CTC Resin.

[0302] (6) Couple the remaining amino acids

[0303] Deprotect the Fmoc protecting group according to the method in step (4) above, and then continuously introduce Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Aib-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Aib-OH according to the methods in steps (3) and (4). After deprotecting Fmoc, Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-CTC Resin is obtained.

[0304] (7) Cleave the labeled compound

[0305] Dissolve the Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-CTC Resin obtained in step (6) in TFA:DODT:Tis:pure water (10 - 15 times the volume of the crude product, V TFA :V TIS :V DODT :V 纯水 =92.5:2.5:2.5:2.5), and cleave at room temperature to 60 °C in a shaker for 1 - 3 h. After the cleavage is completed, filter out the resin, add a sedimentation solution with a volume 10 - 15 times that of the cleavage solution (V 甲基叔丁基醚 :V 正己烷 =1:1), centrifuge and sediment at 3000 r / min in a centrifuge three times. Each time, the liquid needs to be poured out, and the bottom solid is retained. Each time the sedimentation solution is added, it needs to be centrifuged for three minutes to obtain the naked peptide Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol, with a yield of 54% - 60% (based on CTC Resin) and an HPLC purity of 55% - 61%.

[0306] Example 17: Using compound 5 (1 mol) as the label for liquid-phase scale-up synthesis of the antibacterial peptide Trichogin GAⅣ

[0307] Antibacterial peptide Trichogin GAⅣ: Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol

[0308] (1) Synthesize Cbz-Leu-TAG5

[0309] The experimental method is the same as step (1) of Example 13, except that 1 mmol of compound 5 is scaled up to 1 mol of compound 5, and the other materials are the same. Finally, Cbz-Leu-TAG5 is prepared with a yield of 92% (based on compound 5).

[0310] (2) Removal of Cbz protection

[0311] The experimental method was the same as that in step (2) of Example 13. Cbz-Leu-TAG5 was deprotected, and finally a washing solution containing NH2-Leu-TAG5 was prepared.

[0312] (3) Coupling of Cbz-Ile-OH·DCHA

[0313] The experimental method was the same as that in step (3) of Example 13. NH2-Leu-TAG5 was continuously coupled with Cbz-Ile-OH·DCHA, and finally a washing solution containing Cbz-Ile-Leu-TAG5 was prepared.

[0314] (4) Removal of Cbz protection

[0315] The experimental method was the same as that in step (4) of Example 13. Cbz-Ile-Leu-TAG5 was deprotected, and finally a washing solution containing NH2-Ile-Leu-TAG5 was prepared.

[0316] (5) Coupling of Cbz-Gly-OH

[0317] The experimental method was the same as that in step (5) of Example 13. NH2-Ile-Leu-TAG5 was coupled with Cbz-Gly-OH, and finally a washing solution containing Cbz-Gly-Ile-Leu-TAG5 was prepared.

[0318] (6) Coupling of the remaining amino acids

[0319] The experimental method was the same as that in step (6) of Example 13. Cbz-Gly-Ile-Leu-TAG5 was continuously coupled with Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Gly-OH, Cbz-Aib-OH, Cbz-Leu-OH·DCHA, Cbz-Gly-OH, Cbz-Aib-OH in sequence, and then Cbz was removed, separated and purified to obtain Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol-TAG5 with an HPLC purity of over 78%.

[0320] (7) Cleavage of the labeled compound

[0321] The experimental method was the same as that in step (7) of Example 13. The labeled compound 5 was cleaved, and finally Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-ol was prepared with a yield of 70% - 75% (based on compound 5) and an HPLC purity of 70% - 72%.

[0322] Example 18: Data Comparison between Liquid-Phase Synthesis of Octreotide with Labeling Compound 5 and Solid-Phase Synthesis of Octreotide

[0323] Table 1: Amounts of Materials Required for Solid-Phase Synthesis of Octreotide with CTC Resin or Liquid-Phase Synthesis of Octreotide with Labeling Compound 5

[0324] Solid-phase synthesis (Example 10) Liquid-phase synthesis (Example 11) Yield of octreotide 40％ 60％ Amino acid / Kg 14.70 3.97 Coupling reagent / Kg 8.09 2.87 Solvent / Kg 516.20 41.83 Label / Kg 0.83 0.46 PMI 1510.38 96.43

[0325] Example 19: Data Comparison between Liquid-Phase Synthesis of Antimicrobial Peptide Trichogin GAⅣ with Labeling Compound 5 and Solid-Phase Synthesis of Antimicrobial Peptide Trichogin GAⅣ

[0326] Table 2: Amounts of Materials Required for Solid-Phase and Liquid-Phase Synthesis of Antimicrobial Peptide Trichogin GAⅣ with Labeling Compound 5

[0327] Solid-phase synthesis (Example 16) Liquid-phase synthesis (Example 17) Yield of antimicrobial peptide Trichogin GA Ⅳ 55％ 71％ Amino acid / Kg 14.09 3.33 Coupling reagent / Kg 11.61 4.05 Solvent / Kg 640.20 47.28 Label / Kg 0.83 0.46 PMI 1447.57 97.07

[0328] Note: Process mass intensity (PMI) is an important indicator for evaluating the efficiency and sustainability of chemical manufacturing processes (including pharmaceutical processes). PMI refers to the ratio of the total mass of substances used throughout the production process of a product (unit: kg) to the mass of the target product (unit: kg). A lower PMI indicates that the process is more sustainable and more environmentally friendly, which is extremely important in pharmaceutical production. When comparing the PMI values of solid-phase and hydrophobic label-assisted liquid-phase synthesis of octreotide and antimicrobial peptide Trichogin GAⅣ, there are significant differences in the PMI values of the two methods (Tables 1 and 2). The PMI of hydrophobic label-assisted liquid-phase synthesis is significantly lower than that of solid-phase synthesis. The main reason for the high PMI value of solid-phase synthesis is that a large amount of solvent is required for multiple washes after each coupling and deprotection cycle. In contrast, hydrophobic label-assisted liquid-phase synthesis effectively removes by-products through liquid-liquid extraction, significantly reducing the required aqueous solution and thus greatly reducing the PMI.

[0329] Example 20:

[0330]

[0331] The preparation method was the same as that of Example 1, except that tert-butyldimethylchlorosilane was replaced by tert-butyldiphenylchlorosilane, and finally Compound 6 was prepared with a yield of 81% (calculated based on 4,4-dihydroxybenzophenone).

[0332] The 1H NMR data of Compound 6 are as follows: 11H NMR (400 MHz, Chloroform-d) δ 7.83–7.61 (m, 8H), 7.51–7.30 (m, 12H), 7.12–6.92 (m, 4H), 6.78–6.52 (m, 4H), 4.97 (s, 1H), 1.11 (s, 18H). The specific NMR spectrum is shown in Figure 8 。

[0333] Example 21:

[0334]

[0335] Using compound a-6 as the raw material, the methods for sequentially synthesizing compound b-6 and compound c-6 are the same as those in Example 20. The difference is that the method for synthesizing compound 29 from compound c-6 is the same as the method in Example 2. Finally, compound 29 is prepared with a yield of 86% (calculated based on 4,4-dihydroxybenzophenone).

[0336] The 1H NMR data of compound 29 are as follows: 1 1H NMR (400 MHz, Chloroform-d) δ 7.78–7.63 (m, 8H), 7.58–7.27 (m, 12H), 7.10–6.86 (m, 4H), 6.72–6.50 (m, 4H), 4.90 (s, 1H), 1.08 (s, 18H).

[0337] Example 22:

[0338]

[0339] Using compound a-6 as the raw material, the methods for sequentially synthesizing compound b-6 and compound c-6 are the same as those in Example 20. The difference is that the method for synthesizing compound 52 from compound c-6 is the same as the method in Example 3. Finally, compound 52 is prepared with a yield of 80% (calculated based on 4,4-dihydroxybenzophenone).

[0340] The 1H NMR data of compound 52 are as follows: 1 1H NMR (400 MHz, Chloroform-d) δ 7.71–7.54 (m, 8H), 7.41–7.22 (m, 12H), 7.09–6.88 (m, 4H), 6.69–6.47 (m, 4H), 4.86 (s, 1H), 1.03 (s, 18H).

[0341] Examples 23 to 25: Using compounds 6, 29, and 52 as labels for liquid-phase synthesis of octreotide

[0342] The experimental method was the same as that in Example 7, except that compound 5 was replaced with compound 6, compound 29, and compound 52 respectively, and finally octreotide was prepared. The yield and recovery rate of octreotide are shown in Table 3.

[0343] Table 3

[0344]

[0345] Among them, the naked peptide in the table is NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol; octreotide is NH2-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulfide bridge, Cys2-Cys7).

[0346] Examples 26 to 28: Using compound 6, compound 29, and compound 52 as tags for liquid-phase synthesis of the antimicrobial peptide Trichogin GA Ⅳ

[0347] The experimental method was the same as that in Example 13, except that compound 5 was replaced with compound 6, compound 29, and compound 52 respectively, and finally the antimicrobial peptide Trichogin GAⅣ was prepared. The yield and recovery rate of the antimicrobial peptide Trichogin GAⅣ are shown in Table 4.

[0348] Table 4

[0349]

[0350] The present invention provides an idea and method for a halogenated diphenylmethane compound, its preparation method and application. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A halogenated diphenylmethane compound, characterized in that, The structural formula of the halogenated diphenylmethane compound is shown in Formula I: Wherein, X is selected from Cl, Br or I; R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 alkyl groups, or substituted or unsubstituted C5-C8 aromatic groups.

2. The compound according to claim 1, wherein The structural formula of the halogenated diphenylmethane compound is shown in Formula I-1, I-2 or I-3: Wherein, R1 = R1'; R2 = R2'; R3 = R3'; R1, R2, and R3 are independently selected from substituted or unsubstituted C1-C 22 alkyl groups, or substituted or unsubstituted C5-C8 aromatic groups.

3. The compound according to claim 1 or 2, characterized in that, R1 = R1'; R2 = R2'; R3 = R3'; R1, R2, and R3 are independently selected from unsubstituted C1-C 18 alkyl groups or unsubstituted C6 aromatic groups; Preferably, R1 = R1'; R2 = R2'; R3 = R3'; R1, R2, and R3 are independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-octadecyl or phenyl.

4. The compound according to claim 1, wherein The halogenated diphenylmethane compound is selected from any of the following structures:

5. The method for preparing a halogenated diphenylmethane compound according to any one of claims 1 to 3, characterized in that, Selected from any one of the following synthetic routes: Synthetic Route 1: Compound c undergoes a first reaction with a chlorinating reagent to obtain Compound I-1; Synthetic Route 2: Compound c undergoes a second reaction with N-bromosuccinimide under the action of a first catalyst to obtain Compound I-2; Synthetic Route 3: Compound c undergoes a third reaction with iodine under the action of a second catalyst and a base to obtain Compound I-3; Wherein, R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 alkyl groups, or substituted or unsubstituted C5-C8 aromatic groups.

6. According to the preparation method described in claim 5, characterized in that, In Synthetic Route 1, the chlorinating reagent is thionyl chloride or hydrogen chloride; the molar ratio of Compound c to the chlorinating reagent is 1.0:(2.0 - 4.3); the solvent used in the first reaction is any one or a combination of several of dichloromethane, ethyl acetate, methyl tert-butyl ether, tetrahydrofuran and carbon tetrachloride; the reaction temperature of the first reaction is the temperature at which the used solvent refluxes; In Synthetic Route 2, the first catalyst is any one or a combination of several of N,N'-dimethylthiourea, diethyl phosphite, diisopropylethylamine, zinc bromide, iron bromide and tetrabutylammonium bromide; the molar ratio of Compound c to N-bromosuccinimide and the first catalyst is 1.0:(2.0 - 3.0):1.0; the solvent used in the second reaction is any one or a combination of several of dichloromethane, ethyl acetate, cyclohexane, methyl tert-butyl ether, tetrahydrofuran and carbon tetrachloride; for the second reaction, the reaction temperature is 40°C - 60°C; In Synthetic Route 3, the second catalyst is any one or a combination of several of triphenylphosphine, zinc chloride, iron chloride and tetrabutylammonium bromide; the base is any one or a combination of several of imidazole, triethylamine, pyridine, sodium hydroxide, sodium bicarbonate and sodium carbonate; the molar ratio of Compound c to iodine is 1.0:(1.0 - 1.5); the molar ratio of Compound c to the second catalyst and the base is 1.0:(0.8 - 1.2):(1.0 - 1.5); the solvent used in the third reaction is any one or a combination of several of dichloromethane, ethanol, methanol, ethyl acetate, dichloromethane, tetrahydrofuran and carbon tetrachloride; for the third reaction, the reaction temperature is 25°C - 45°C; the third reaction is carried out under inert gas protection.

7. The halogenated diphenylmethane compound prepared by the preparation method according to claims 5 to 6.

8. A protecting reagent for the C-terminus of an amino alcohol or peptide alcohol, characterized in that, The protecting reagent comprises the halogenated diphenylmethane compound according to any one of claims 1 to 4.

9. Use of the halogenated diphenylmethane compound according to any one of claims 1 to 4 or the protecting reagent for the C-terminus of the amino alcohol or peptide alcohol according to claim 8 in the synthesis of peptide alcohol in a homogeneous solvent system.

10. A method for synthesizing a peptide alcohol, characterized in that, Using the halogenated diphenylmethane compound according to any one of claims 1 to 4; comprising the following steps: (1) Attachment of the first residue: The halogenated diphenylmethane compound according to any one of claims 1 to 4 is used as a hydrophobic carrier and is linked to an N-protected amino alcohol or N-protected peptide alcohol compound through a conventional condensation reaction to obtain an N-protected amino alcohol or N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier. (2) N-terminal deprotection: The N-protected amino alcohol or N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier is mixed with an N-terminal deprotection reagent and a solvent, and N-terminal deprotection is carried out in an organic homogeneous system to obtain a solution of an N-deprotected amino alcohol or N-deprotected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier. (3) Peptide chain elongation: In the solution of the N-deprotected amino alcohol or N-deprotected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier, an N-protected amino alcohol or N-protected peptide alcohol compound is added, and then a condensation reagent is added to carry out a coupling condensation reaction in a homogeneous system, and then extraction and impurity removal are carried out to obtain a solution of an N-protected amino alcohol or N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier. (4) Repeat steps (2) and (3) to attach the next amino alcohol until a complete peptide alcohol chain is obtained. (5) Cleavage and precipitation: The complete N-protected amino alcohol or N-protected peptide alcohol compound with the C-terminus protected by a halogenated diphenylmethane structure carrier is deprotected at the N-terminus and dissolved in a cleavage solution for a cleavage reaction. After the cleavage is completed, a precipitation solution is added for precipitation to obtain the peptide alcohol.

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