Soluble hydrazino functionalized carrier as well as preparation method and application thereof

Abstract

The invention belongs to the field of polypeptide synthesis and relates to a soluble hydrazino functionalized carrier as well as a preparation method and application thereof. Wherein the structural formula of the soluble hydrazino-functionalized carrier is shown as a formula I. The soluble hydrazino-functionalized carrier is combined with a micro-channel modular reaction device to perform liquid-phase synthesis of Heterophyllin B through a hydrazide method, and compared with a conventional solid-phase synthesis route, the dosage of amino acid is reduced by 60% or above, the dosage of a condensation reagent is reduced by 50% or above, and the yield of the Heterophyllin B is improved. The use amount of the solvent is reduced by 85% or more, and the total yield of the final product Heterophyllin B is also improved by 45%. In conclusion, compared with a conventional solid-phase synthesis method and a large lactamization strategy depending on a coupling reagent, the hydrazide method disclosed by the invention for liquid-phase synthesis of the Heterophyllin B has obvious environmental protection advantages and cost advantages. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of polypeptide synthesis and relates to a soluble hydrazine functionalized carrier and a preparation method and application thereof. Background Art

[0002] In recent years, cyclic peptides have demonstrated unique advantages in clinical treatment. Typical examples include hormonal drugs (such as oxytocin and octreotide), antibiotics (such as vancomycin and daptomycin), and immunosuppressants (such as cyclosporine). The vast majority of clinically approved cyclic peptides are derived from natural products, such as terlipressin, conopeptide, and heterophyllin B. Cyclic peptides are easily synthesized chemically and easily modified. Most are composed of natural amino acids and are generally non-toxic. Cyclic peptides can be synthesized in various ways, the most common of which is amide bond cyclization, or direct end-to-end ligation of the N-terminal amino group and the C-terminal carboxyl group of the peptide chain to create a cyclic structure, which requires the use of coupling reagents. Traditional macrolactamization strategies, such as HATU / EDC, are prone to polymerizing side reactions when ring closure is induced, and require low concentrations for cyclization, resulting in low efficiency. This head-to-tail macrolactamization strategy, which relies on coupling reagents, has become an Achilles' heel for the chemical synthesis of cyclic peptides and urgently needs to be overcome. Summary of the Invention

[0003] The present invention addresses the shortcomings of existing technologies by providing a soluble hydrazine-functionalized carrier for modifying the C-terminus of a peptide to enhance its solubility in organic solvents while avoiding the use of solid-phase resins. This invention utilizes the Fmoc strategy to efficiently synthesize the natural cyclic peptide Heterophyllin B. This method reduces material and environmental costs by replacing the reproductively toxic solvent N,N-dimethylformamide (DMF) with the low-toxic, green solvent ethyl acetate.

[0004] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0005] The present invention discloses a soluble hydrazine functionalized carrier, the structural formula of the soluble hydrazine functionalized carrier is shown in Formula I:

[0006]

[0007] in,

[0008] R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 an alkyl group, or a substituted or unsubstituted C5-C8 aromatic group.

[0009] Among them, the above C1-C 22 The alkyl group includes a straight-chain alkyl group or a branched-chain alkyl group.

[0010] In some embodiments, preferably, R1=R1′; R2=R2′; R3=R3′; R1, R2, and R3 are independently selected from substituted or unsubstituted C1~C 22 an alkyl group, or a substituted or unsubstituted C5-C8 aromatic group.

[0011] In some embodiments, preferably, R1=R1′; R2=R2′; R3=R3′; R1, R2, and R3 are independently selected from unsubstituted C1 to C 18 Alkyl, or unsubstituted C6 aromatic group;

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In some embodiments, most preferably, the soluble hydrazine functionalized carrier is selected from any of the following structures:

[0017]

[0018] Among them, -C2H5, -C3H7, -C4H9, -C5H 11 、-C6H 13 、-C7H 15 、-C8H 17 、-C9H 19 、-C 10 H 21 、-C 12 H 25 、-C14 H 29 、-C 16 H 33 、-C 18 H 37 、-C 20 H 41 、-C 22 H 45 Unless otherwise specified, all are straight-chain saturated alkyl groups.

[0019] Furthermore, the present invention discloses a method for preparing the above-mentioned soluble hydrazine-functionalized carrier, and the synthesis route is as follows:

[0020]

[0021] Compound c undergoes a first reaction with 9-fluorenylmethyl carbamate to obtain compound d; compound d undergoes a second reaction under the action of a catalyst to obtain a soluble hydrazine-functionalized carrier I;

[0022] in,

[0023] X is selected from Br, Cl or I;

[0024] R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 an alkyl group, or a substituted or unsubstituted C5-C8 aromatic group.

[0025] Among them, the preparation of compound c refers to the existing technology (application number: 2025104202551; application date: April 3, 2025; applicant: Nanjing University of Science and Technology; invention name: a halogenated diphenylmethane compound and its preparation method and application).

[0026] In some embodiments, the catalyst is any one or a combination of tetrahydropyrrole, hexahydropyridine, DBU, tetramethylguanidine and triethylamine; and / or, the molar ratio of compound c to 9-fluorenylmethyl carbamate is 1.0: (1.0-3.0); and / or, the first reaction is carried out at room temperature; and / or, the first reaction, the reaction time is 0.2-2.0h; and / or, the solvent used in the first reaction is ethyl acetate, N,N-dimethylformamide, chloroform, toluene, chloroform, N-methylpyrrolidone, tetrahydrofuran, Any one or a combination of 2-methyltetrahydrofuran and dimethyl sulfoxide; and / or, the molar ratio of the compound d to the catalyst is 1.0:(10.0-15.0); and / or, the second reaction is carried out at room temperature; and / or, the reaction time of the second reaction is 0.2-2.0h; and / or, the solvent used in the second reaction is any one or a combination of ethyl acetate, N,N-dimethylformamide, chloroform, toluene, chloroform, N-methylpyrrolidone, tetrahydrofuran, 2-methyltetrahydrofuran and dimethyl sulfoxide.

[0027] In some embodiments, preferably, the catalyst is tetrahydropyrrole; and / or the molar ratio of the compound c to 9-fluorenylmethyl carbamate is 1.0:(1.0~2.0), further preferably 1.0:1.1; and / or the first reaction is carried out at room temperature; and / or the reaction time of the first reaction is 2.0h; and / or the solvent used in the first reaction is ethyl acetate; and / or the molar ratio of the compound d to the catalyst is 1.0:12.0; and / or the second reaction is carried out at room temperature; and / or the reaction time of the second reaction is 2.0h; and / or the solvent used in the second reaction is ethyl acetate.

[0028] Furthermore, the present invention discloses a protective agent for the C-terminus of an amino acid or a polypeptide, wherein the protective agent comprises the above-mentioned soluble hydrazine-functionalized carrier.

[0029] Furthermore, the present invention discloses a method for synthesizing Heterophyllin B using a microchannel modular reaction device, comprising the following steps:

[0030] (1) Fmoc-Gly-OH is mixed with a condensation reagent, an acid binding agent, and an organic solvent for activation to obtain a first Fmoc-Gly-OH activation solution; the above-mentioned soluble hydrazine functionalized carrier is dissolved in an organic solvent to obtain a soluble label solution; the first Fmoc-Gly-OH activation solution and the soluble label solution are respectively pumped into a first microstructured reactor at the same time for condensation reaction; then the effluent of the first microstructured reactor and the potassium carbonate aqueous solution are respectively pumped into a second microstructured reactor at the same time for washing, the effluent after washing is separated in a first liquid-liquid separator, and the organic phase containing Fmoc-Gly-TAG is retained; the retained organic phase containing Fmoc-Gly-TAG and the organic solvent solution containing the deprotection reagent are respectively pumped into a third microstructured reactor at the same time for deprotection reaction; then the effluent of the third microstructured reactor and the hydrochloric acid aqueous solution are respectively pumped into a fourth microstructured reactor at the same time for washing, the effluent after washing is separated in a second liquid-liquid separator, and the organic phase containing NH2-Gly-TAG is retained;

[0031] (2) Fmoc-Gly-OH is mixed with a condensation reagent, an acid binding agent, and an organic solvent for activation to obtain a second Fmoc-Gly-OH activation solution; the second Fmoc-Gly-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Gly-TAG obtained in step (1) is used to replace the soluble tag solution in step (1); the same reaction and treatment process as step (1) are carried out to obtain an organic phase containing NH2-Gly-Gly-TAG;

[0032] (3) mixing Fmoc-Leu-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain an Fmoc-Leu-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the Fmoc-Leu-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Gly-Gly-TAG obtained in step (2), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Leu-Gly-Gly-TAG;

[0033] (4) mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a first Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the first Fmoc-Pro-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Leu-Gly-Gly-TAG obtained in step (3), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Leu-Gly-Gly-TAG;

[0034] (5) Mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a second Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the second Fmoc-Pro-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Pro-Leu-Gly-Gly-TAG obtained in step (4), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Pro-Leu-Gly-Gly-TAG;

[0035] (6) Mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a third Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the third Fmoc-Pro-OH activation solution, replacing the soluble tag solution in step (1) with the organic phase containing NH2-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (5), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Pro-Pro-Leu-Gly-Gly-TAG;

[0036] (7) Fmoc-Ile-OH is mixed with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain an Fmoc-Ile-OH activation solution; the Fmoc-Ile-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Pro-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (6) is used to replace the soluble tag solution in step (1); the same reaction and treatment process as step (1) are carried out to obtain an organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG;

[0037] (8) Boc-Phe-OH is mixed with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a Boc-Phe-OH activation solution; the Boc-Phe-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (7) is used to replace the soluble tag solution in step (1); the Boc-Phe-OH activation solution and the organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG are mixed to obtain a Boc-Phe-OH activation solution; .... The organic phases of Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG were simultaneously pumped into the first microstructured reactor for condensation reaction; the effluent from the first microstructured reactor and the potassium carbonate aqueous solution were then simultaneously pumped into the second microstructured reactor for washing, and the washed effluent was separated in a first liquid-liquid separator, and the organic phase containing Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG was retained and spin-dried to obtain a fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus;

[0038] (9) The fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus obtained in step (8) was cleaved using a lysis buffer, precipitated, blown dry, and dried to obtain NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2;

[0039] (10) NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 is placed in a buffer solution, and an azidation reagent is added to carry out an oxidative azidation reaction; after the reaction is completed, an alkaline solution is added to adjust the pH, and an aminolysis cyclization reaction is carried out to obtain a heterophyllin B cyclization solution;

[0040] (11) The Heterophyllin B cyclization solution obtained in step (10) is purified by preparative chromatography to obtain Heterophyllin B.

[0041] Among them, the flow rate of each material pumped into the first microstructured reactor, the second microstructured reactor, the third microstructured reactor, and the fourth microstructured reactor is controlled by controlling the reaction molar ratio of the material, the concentration of the material in each feed liquid, and the reaction residence time.

[0042] In some embodiments, the condensation reagents used in each step are the same and are selected from any one or a combination of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-hydroxyphenyl-4,5-diphenylimidazole, N,N-diisopropylethylamine, O-benzotriazole-tetramethyluronium hexafluorophosphate and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; and / or the acid-binding agents used in each step are the same and are selected from N,N-diisopropylethylamine; and / or the organic solvents used in each step are the same and are selected from any one or a combination of dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, ethyl acetate and dimethyl sulfoxide; and / or the activation in each step is performed at room temperature for 15 min to 30 min.

[0043] In some embodiments, preferably, the condensation reagents used in each step are the same, selected from a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole, and the molar ratio of the two is preferably 1:1; and / or, the acid binding agents used in each step are the same, selected from N,N-diisopropylethylamine; and / or, the organic solvents used in each step are the same, selected from ethyl acetate; and / or, the activation in each step is performed at room temperature for 30 min.

[0044] In some embodiments, in step (1), the reaction molar ratio of the soluble hydrazine functionalized carrier to Fmoc-Gly-OH, the condensation reagent, and the acid binding agent is controlled to be 1.00: (1.05-1.10): (1.05-2.40): (0.10-0.50), and the replacement raw materials used in steps (2) to (8) are reacted according to the same molar ratio as in step (1); and / or, in step (1), the condensation reaction is performed at room temperature, and steps (2) to (8) are performed at room temperature. (8) is the same as the reaction temperature of step (1); and / or, in step (1), the condensation reaction has a reaction residence time of 9.0s to 18.0s, and the reaction residence time in steps (2) to (8) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of potassium carbonate in the potassium carbonate aqueous solution is 3% g / mL to 5% g / mL, and the potassium carbonate aqueous solution in steps (2) to (8) is the same as the potassium carbonate aqueous solution in step (1).

[0045] In some embodiments, preferably, in step (1), the reaction molar ratio of the soluble hydrazine functionalized carrier to Fmoc-Gly-OH, the condensation reagent, and the acid binding agent is controlled to be 1.00:1.05:2.40:0.10, and the replacement raw materials used in steps (2) to (8) are reacted according to the same molar ratio as in step (1); and / or, in step (1), the reaction temperature of the condensation reaction is room temperature, and the reaction temperature in steps (2) to (8) is the same as the reaction temperature in step (1); and / or, in step (1), the reaction residence time of the condensation reaction is 9.0s, and the reaction residence time in steps (2) to (8) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of potassium carbonate in the potassium carbonate aqueous solution is 5% g / mL, and the potassium carbonate aqueous solution in steps (2) to (8) is the same as the potassium carbonate aqueous solution in step (1).

[0046] In some embodiments, in step (1), the deprotecting agent in the organic solvent solution containing the deprotecting agent is tetrahydropyrrole or piperidine, and the concentration of the deprotecting agent in the solution is 10vt% to 15vt%, and the organic solvent solutions containing the deprotecting agent in steps (2) to (7) are the same as the organic solvent solution containing the deprotecting agent in step (1); and / or, in step (1), the reaction temperature of the deprotection reaction is room temperature, and the reaction temperature in steps (2) to (7) is the same as the reaction temperature in step (1); and / or, in step (1), the reaction residence time of the deprotection reaction is 9.0s to 18.0s, and the reaction residence time in steps (2) to (7) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 1vt% to 3vt%, and the hydrochloric acid aqueous solution in steps (2) to (7) is the same as the hydrochloric acid aqueous solution in step (1).

[0047] In some embodiments, preferably, in step (1), the deprotection reagent in the organic solvent solution containing the deprotection reagent is tetrahydropyrrole or piperidine, and the concentration of the deprotection reagent in the solution is 10vt%, and the organic solvent solutions containing the deprotection reagent in steps (2) to (7) are the same as the organic solvent solution containing the deprotection reagent in step (1); and / or, in step (1), the deprotection reaction has a reaction temperature of room temperature, and the reaction temperatures in steps (2) to (7) are the same as the reaction temperature in step (1); and / or, in step (1), the deprotection reaction has a reaction residence time of 9.0s, and the reaction residence time in steps (2) to (7) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 3vt%, and the hydrochloric acid aqueous solution in steps (2) to (7) is the same as the hydrochloric acid aqueous solution in step (1).

[0048] In some embodiments, in step (9), the lysis solution is a composition of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole, and the volume ratio of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole is 95%:2.5%:2.5%; and / or, the ratio of the fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus to the lysis solution is 1 g of solid crude product: 10 mL to 15 mL of lysis solution; and / or, the lysis reaction is carried out at 25°C to 40°C for 1 h to 2 h; and / or, the solvent used for the precipitation is ether, and the volume ratio of ether to the lysis solution is (5 to 10):1.

[0049] In some embodiments, preferably, in step (9), the lysis solution is a composition of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole, and the volume ratio of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole is 95%:2.5%:2.5%; and / or, the ratio of the fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus to the lysis solution is 1 g of solid crude product: 10 mL of lysis solution; and / or, the cleavage is reacted at 40°C for 2 h; and / or, the solvent used for the precipitation is ether, and the volume ratio of ether to the lysis solution is 5:1.

[0050] In some embodiments, in step (10), the buffer solution is prepared as follows: weigh 0.60 mol of guanidine hydrochloride, add 50 mL of deionized water, mix well, add 0.02 mol of sodium dihydrogen phosphate dihydrate, add concentrated hydrochloric acid to adjust the pH of the solution to 3.0-3.5, dilute to 100 mL with deionized water, mix well, and then vacuum filter through a filter membrane to obtain the solution;

[0051] and / or, the azidation reagent is sodium nitrite; and / or, the molar ratio of the NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 to the azidation reagent is 1:(15-20); and / or, the reaction temperature of the oxidative azidation reaction is -20°C to -15°C; and / or, the reaction time of the oxidative azidation reaction is 30-60 min;

[0052] And / or, the alkaline solution is a sodium bicarbonate solution; and / or, the pH is adjusted to 7.0-7.5; and / or, the reaction temperature of the aminolysis cyclization reaction is 0°C-5°C; and / or, the reaction time of the aminolysis cyclization reaction is 30-60 min.

[0053] There is no special requirement for the amount of the buffer solution, as long as the raw materials are dissolved or dispersed evenly and the viscosity is moderate.

[0054] In some embodiments, preferably, in step (10), the buffer solution is prepared as follows: 0.60 mol of guanidine hydrochloride is weighed, added to 50 mL of deionized water, mixed, 0.02 mol of sodium dihydrogen phosphate dihydrate is added, concentrated hydrochloric acid is added to adjust the pH of the solution to 3.0-3.5, the volume is made up to 100 mL with deionized water, mixed, and vacuum filtered through a filter membrane to obtain the buffer solution;

[0055] and / or, the azidation reagent is sodium nitrite; and / or, the molar ratio of NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 to the azidation reagent is 1:15; and / or, the reaction temperature of the oxidative azidation reaction is -15°C; and / or, the reaction time of the oxidative azidation reaction is 30 min;

[0056] And / or, the alkaline solution is a sodium bicarbonate solution; and / or, the pH is adjusted to 7.0-7.5; and / or, the reaction temperature of the aminolysis cyclization reaction is 0°C; and / or, the reaction time of the aminolysis cyclization reaction is 30 minutes.

[0057] In some embodiments, in step (11), the Heterophyllin B cyclization solution is purified by preparative chromatography, specifically by:

[0058] The Heterophyllin B cyclization solution was concentrated under reduced pressure, dissolved and diluted with deionized water, and filtered through a filter membrane. The filtrate was purified by reverse preparative chromatography using a C18 silica gel packing. The specific chromatographic parameters were as follows: chromatographic column: DAC-100 dynamic axial column, 600*600*2300 mm, packed with Bonnasil-BS C18 pro; column temperature: 35°C; flow rate: 200 mL / min; detection wavelength: 214 nm; injection volume: 4 L; mobile phase: A: 0.1% TFA aqueous solution, B: acetonitrile; gradient elution. The specific gradient elution conditions are shown in Table 1 below:

[0059] Table 1

[0060] Time (min) Flow rate (mL / min) Mobile phase A% Mobile phase B% 0 200 70 30 20 200 60 40 22 200 55 45 28 200 55 45 35 200 50 50 40 200 50 50 .

[0061] In some embodiments, the microchannel modular reaction device includes a connecting pipe, a first feeding tank, a second feeding tank, a third feeding tank, a fourth feeding tank, a fifth feeding tank, a sixth feeding tank, a first microstructured reactor, a second microstructured reactor, a third microstructured reactor, a fourth microstructured reactor, a metering pump, a first liquid-liquid separator and a second liquid-liquid separator; wherein, the first feeding tank and the second feeding tank are respectively connected in parallel to the feed port of the first microstructured reactor through a connecting pipe provided with a metering pump; wherein, the third feeding tank is respectively connected in parallel to the feed port of the second microstructured reactor through a connecting pipe provided with a metering pump and a discharge pipe of the first microstructured reactor; wherein, the second microstructured reactor is connected in parallel to the feed port of the second microstructured reactor; The discharge port of the reactor is connected to the feed port of the first liquid-liquid separator through a connecting pipe; the discharge port of the first liquid-liquid separator is connected to the feed port of the fourth feeding tank through a connecting pipe; wherein the fourth feeding tank and the fifth feeding tank are respectively connected in parallel to the feed port of the third microstructure reactor through a connecting pipe provided with a metering pump; wherein the sixth feeding tank is respectively connected in parallel to the feed port of the fourth microstructure reactor through a connecting pipe provided with a metering pump and the discharge port pipe of the third microstructure reactor; wherein the discharge port of the fourth microstructure reactor is connected to the feed port of the second liquid-liquid separator through a connecting pipe; the discharge port of the second liquid-liquid separator is connected to the feed port of the second feeding tank through a connecting pipe.

[0062] Beneficial effects:

[0063] (1) Compared with the prior art, the present invention is the first to synthesize Heterophyllin B by liquid phase synthesis using the hydrazide method, condensing the precursor of Heterophyllin B in solution to obtain a linear Heterophyllin B precursor, which is then deprotected, cyclized, and purified to obtain high-purity Heterophyllin B.

[0064] (2) The synthesis method provided by the present invention is carried out entirely in liquid phase, with higher reaction yield and purity, and greatly improved production efficiency; the reaction progress can be monitored by HPLC, and the production quality can be controlled; the amount of production materials and the discharge of three wastes are reduced, and the production cost is reduced, which has obvious environmental advantages and cost advantages.

[0065] (3) Compared with conventional solid-phase synthesis routes, the hydrazide liquid-phase synthesis route of Heterophyllin B of the present invention reduces the amount of amino acid used by more than 60%, the amount of condensation reagent used by more than 50%, and the amount of solvent used by more than 85%. The total yield of the final product, Heterophyllin B, is also increased by 45%. In summary, the hydrazide liquid-phase synthesis of Heterophyllin B disclosed in the present invention has significant environmental and cost advantages compared with conventional solid-phase synthesis methods and macrolactamization strategies relying on coupling reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0067] Figure 1 This is the synthetic route for the liquid phase synthesis of Heterophyllin B by the hydrazide method in Example 2.

[0068] Figure 2 Schematic diagram of the process flow of the microchannel modular reaction device used in Example 2.

[0069] Figure 3 This is the HPLC purity data of the white flocculent product Heterophyllin B prepared in Example 2.

[0070] Figure 4 This is the H NMR spectrum of compound CDMH.

[0071] Figure 5 This is the NMR carbon spectrum of compound CDMH.

[0072] Figure 6 This is the mass spectrum data of compound CDMH.

[0073] Figure 7 Mass spectrum of the white flocculent product Heterophyllin B.

[0074] Figure 8 This is the HPLC chart of Heterophyllin B in the Heterophyllin B cyclization solution in Comparative Example 1.

[0075] Figure 9 It is the HPLC spectrum of the organic phase in Comparative Example 2.

[0076] Figure 10 It is the HPLC spectrum of the aqueous phase in Comparative Example 2. DETAILED DESCRIPTION

[0077] The present invention provides a concept and method for the liquid-phase synthesis of heterophyllin B using the hydrazide method. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

[0078] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0079] The schematic flow diagram of the microchannel modular reaction device used in the embodiment of the present invention is as follows Figure 2 As shown, the microchannel modular reaction device includes connecting pipes, a first feeding tank, a second feeding tank, a third feeding tank, a fourth feeding tank, a fifth feeding tank, a sixth feeding tank, a first microstructure reactor, a second microstructure reactor, a third microstructure reactor, a fourth microstructure reactor, a metering pump, a first liquid-liquid separator and a second liquid-liquid separator.

[0080] The first feeding tank and the second feeding tank are respectively connected in parallel to the feed port of the first microstructured reactor through connecting pipes provided with metering pumps.

[0081] The third feeding tank is connected in parallel to the feed port of the second microstructure reactor through a connecting pipe provided with a metering pump and the discharge port pipe of the first microstructure reactor.

[0082] The discharge port of the second microstructured reactor is connected to the feed port of the first liquid-liquid separator through a connecting pipe; the discharge port of the first liquid-liquid separator is connected to the feed port of the fourth feeding tank through a connecting pipe.

[0083] The fourth feeding tank and the fifth feeding tank are respectively connected in parallel to the feed port of the third microstructured reactor through connecting pipes provided with metering pumps.

[0084] The sixth feeding tank is connected in parallel to the feed port of the fourth microstructure reactor through a connecting pipe provided with a metering pump and a discharge port pipe of the third microstructure reactor.

[0085] The discharge port of the fourth microstructured reactor is connected to the feed port of the second liquid-liquid separator through a connecting pipe; the discharge port of the second liquid-liquid separator is connected to the feed port of the second feeding tank through a connecting pipe.

[0086] The interior of the microstructure reactor is a pipe structure made of high borosilicate glass.

[0087] The liquid holding capacity of the microstructured reactor can be calculated by the flow rate of the pumped liquid and the reaction residence time inside the microstructured reactor. In the embodiment of the present invention, the liquid holding capacity of the microstructured reactor is 3 mL.

[0088] The liquid-liquid separator is a Zaiput SEP-10 liquid-liquid separator, manufactured by Zaiput Flow Technologies.

[0089] Example 1: Synthesis of soluble hydrazine functionalized tag BTPH

[0090]

[0091] The soluble tag BTPH was synthesized using 4,4-dihydroxybenzophenone and triisopropylsilyl chloride as starting materials, and a benzene ring structure with two triisopropylsilyl ether groups was introduced to increase its hydrophobicity.

[0092] (1) Synthesis of compound 1-a: 4,4-dihydroxybenzophenone (24.6 mmol, 1.0 eq.), triisopropylsilyl chloride (74.1 mmol, 3.0 eq.), and imidazole (103.3 mmol, 4.2 eq.) were weighed and placed in a round-bottom flask. 200 mL of dichloromethane was added to completely dissolve the mixture and the mixture was allowed to react at room temperature for 2 h. After the reaction was completed, the reaction solution was transferred to a separatory funnel and washed three times with an equal volume of 5% (w / v) K2CO3 aqueous solution. After each wash, the organic phase was collected in a separatory funnel. The organic phase was concentrated under reduced pressure using a rotary evaporator, slurried with 200 mL of anhydrous acetonitrile under ultrasonic conditions, filtered under reduced pressure using a Buchner funnel, and dried in a vacuum dryer to obtain a white solid product 1-a with an isolated yield of 97%.

[0093] The NMR and mass spectrometry data of product 1-a are: 1 H NMR (400MHz, CDCl3), δ: 7.72 (d, J = 8.6Hz, 4H), 6.95–6.90 (m, 4H), 1.32–1.26 (m, 6H), 1.12 (d, J = 7.4Hz, 36H). 13 C NMR (101MHz, CDCl3), δ: 195.09, 160.31, 132.58, 131.43, 119.83, 18.28, 13.10.MS (ESI-TOF): Calcd forC 31 H 50 O3Si2[M+H] + :527.33;Found:527.5.

[0094] (2) Synthesis of Compound 1-b: Compound 1-a (23.86 mmol, 1.0 eq.) obtained in the previous step was placed in a round-bottom flask, potassium borohydride (71.58 mmol, 3.0 eq.) was added, and 200 mL of a mixed solvent consisting of tetrahydrofuran (THF) and deionized water (v / v = 9:1) was added. The reactants were completely dissolved by ultrasonic treatment for 15 minutes until clear, with no obvious insoluble matter. The reaction was then allowed to react at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the water-miscible mixed solvent. 200 mL of ethyl acetate was added and ultrasonic treatment was performed for 15 minutes to dissolve the mixture to obtain a clear, homogeneous solution. The solution was transferred to a separatory funnel and washed three times with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected in a separatory funnel after each wash. Subsequently, the product 1-b was purified by silica gel column chromatography to obtain a colorless, transparent oily product with an isolated yield of 94%.

[0095] The NMR and mass spectrometry data of product 1-b are: 1 H NMR (400MHz, CDCl3), δ: 7.20–7.12 (m, 4H), 6.85–6.77 (m, 4H), 5.71 (s, 1H), 1.27–1.19 (m, 6H), 1.09 (d, J = 7.4Hz, 36H). 13 C NMR (101MHz, CDCl3), δ: 155.18, 136.37, 128.40, 127.55, 119.50, 119.33, 75.27, 17.72, 12.44.MS (ESI-TOF): Calcd for C 31 H 52 O3Si2[M+K] + :528.35;Found:566.4.

[0096] (3) Synthesis of Compound 1-c: Compound 1-b (22.42 mmol, 1.0 eq.) obtained in the previous step was placed in a round-bottom flask. Compound 1-b was dissolved in 200 mL of dichloromethane by ultrasonic treatment for 15 minutes until clear and transparent. Thionyl chloride (24.66 mmol, 1.1 eq.) was slowly added dropwise and allowed to react at room temperature for 2 hours. After the reaction, the reaction solution was transferred to a separatory funnel and washed three times with an equal volume of saturated sodium chloride aqueous solution. After each wash, the organic phase was collected in a separatory funnel. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography to obtain a light yellow oily product 1-c with an isolated yield of 95%.

[0097] The NMR and mass spectrometry data of product 1-c are: 1H NMR (400MHz, CDCl3), δ7.15 (d, J = 7.8Hz, 4H), 6.77 (d, J = 7.6Hz, 4H), 5.09 (s, 1H), 1.26–1.19 (m, 6H), 1.08 (d, J = 7.5Hz, 36H). 13 C NMR (101MHz, CDCl3), δ: 154.82, 138.41, 127.82, 119.65, 58.53, 17.93, 12.65. HRMS (ESI-TOF): Calcd for C 31 H 52 O3Si2[M-Cl] + :511.3262;Found:511.2553.

[0098] (4) Synthesis of Compound 1-d: Compound 1-c (21.29 mmol, 1.0 eq.) obtained in the previous step was placed in a round-bottom flask, and 9-fluorenylmethyl carbamate (23.46 mmol, 1.1 eq.) was added. 200 mL of ethyl acetate was added, and the reactants were completely dissolved by ultrasonic treatment for 15 minutes. The reaction was then allowed to proceed at room temperature for 2 hours. After the reaction was completed, the organic phase was concentrated under reduced pressure using a rotary evaporator to obtain the reaction solution. The organic phase was first concentrated under reduced pressure and then purified by silica gel column chromatography to obtain the white solid product 1-d in an isolated yield of 94%.

[0099] The NMR and mass spectrometry data of product 1-d are: 1 H NMR (400MHz, CDCl3), δ: 7.76 (d, J = 7.6Hz, 2H), 7.51 (d, J = 7.2Hz, 2H), 7.40 (t, J = 7.4Hz, 2H), 7.29 (s, 2H), 7.24 (d, J = 12. 1Hz,4H),6.85–6.80(m,4H),5.19(s,1H),4.42(d,J=7.4Hz,2H),4.23(s,1H),1.26–1.21(m,6H),1.09(d,J=7.4Hz,36H). 13 C NMR (101MHz, CDCl3), δ: 157.25, 155.49, 143.77, 141.42, 133.91, 128.94, 127.8 7,127.20,125.15,120.13,119.96,67.10,18.05,12.76.HRMS(ESI-TOF):Calcd for C 46 H 64 N2O4Si2[M+H] + :765.4405;Found:765.5714.

[0100] (5) Synthesis of compound 1 (BTPH): Compound 1-d (20.02 mmol, 1.0 eq.) obtained in the previous step was placed in a round-bottom flask, 200 mL of ethyl acetate was added, and the mixture was completely dissolved by ultrasonic treatment for 15 minutes until it became clear and transparent. Tetrahydropyrrole (240.24 mmol, 12.0 eq.) was slowly added dropwise, and then reacted at room temperature for 2 h. After the reaction was completed, the reaction solution was transferred to a separatory funnel and washed three times with an equal volume of 3% (v / v) HCl aqueous solution. After each wash, the organic phase was collected in a separatory funnel. Subsequently, the organic phase was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain a white solid product 1 (denoted as BTPH) with an isolated yield of 96% and an overall yield of 78% (based on compound 4,4-dihydroxybenzophenone).

[0101] The NMR and mass spectrometry data of BTPH are: 1 H NMR (400MHz, CDCl3), δ: 7.33 (d, J = 8.3Hz, 4H), 6.82 (d, J = 8.4Hz, 4H), 5.30 (s, 1H), 1.24–1.17 (m, 6H), 1.05 (d, J = 7.5Hz, 36H). 13 C NMR (101MHz, CDCl3), δ: 154.82, 138.41, 127.82, 119.65, 58.53, 17.93, 12.65. HRMS (ESI-TOF): Calcd for C 31 H 54 N2O2Si2[MH] - :541.3724;Found:541.1208.

[0102] Example 2: Liquid Phase Synthesis of Heterophyllin B by the Hydrazide Method

[0103] The synthetic route of Heterophyllin B is as follows Figure 1 shown.

[0104] Amino acid preactivation: Fmoc-Gly-OH (10.5 mmol, 1.05 eq.), EDC·HCl (12 mmol, 1.2 eq.), HOBt (12 mmol, 1.2 eq.), and DIPEA (1 mmol, 0.1 eq.) were dissolved in 40 mL of ethyl acetate. The amino acid was preactivated at room temperature with magnetic stirring for 30 min to obtain an Fmoc-Gly-OH activation solution.

[0105] The remaining 7 amino acids in the heterophyllin B sequence (Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Boc-Phe-OH) were preactivated as described above.

[0106] 1. Synthesis of fully protected linear Heterophyllin B

[0107] (a) Preparation of Fmoc-Gly-BTPH

[0108] Prepare material 1 (placed in the first feeding tank): Fmoc-Gly-OH activation solution prepared according to the above method, wherein the concentration of Fmoc-Gly-OH in the activation solution is 0.26 mmol / mL.

[0109] Prepare material 2 (placed in the second feeding tank): dissolve BTPH (prepared in Example 1, 10 mmol, 1.0 eq.) in 40 mL of ethyl acetate, and the concentration of the soluble label BTPH is 0.25 mmol / mL.

[0110] The feed flow rates of material 1 and material 2 were adjusted by metering pumps and injected into the first microstructured reactor simultaneously for condensation reaction, wherein the flow rates of the two were equal to 10 mL / min, the reaction temperature was room temperature, and the residence time of the condensation reaction in the first microstructured reactor was 9.0 s to obtain a reaction solution containing intermediate 2 (Fmoc-Gly-BTPH).

[0111] Among them, the NMR and mass spectrometry data of compound 2 are: 1 H NMR (400MHz, CDCl3), δ: 7.75 (d, J = 7.5Hz, 2H), 7.66–7.46 (m, 3H), 7.40 (d, J = 7.5Hz, 2H),7.30(tt,J=7.5,1.5Hz,2H),7.25–7.17(m,3H),7.11(d,J=8.5Hz,1H),6.82(dd, J=19.8,8.3Hz,4H),5.07(s,1H),4.84(d,J=7.9Hz,1H),4.42–4.31(m,2H),4.27–4.0 0(m,2H),3.74(d,J=6.0Hz,1H),1.21(td,J=8.3,5.3Hz,6H),1.07(t,J=8.5Hz,36H). 13C NMR (101MHz, CDCl3), δ: 168.52, 155.52, 143.79, 141.38, 133.73, 128.81, 127.86, 127. 19,125.14,120.11,119.96,67.39,47.12,43.26,18.01,12.70.HRMS(ESI-TOF):Calcd for C 48 H 67 N3O5Si2[M+Na] + :844.4619;Found:844.4472.

[0112] (b) Purification of Fmoc-Gly-BTPH

[0113] Prepare material 3 (placed in the third feeding tank): 5% (g / mL) K2CO3 aqueous solution containing 2 times the volume of the reaction solution of intermediate 2.

[0114] The reaction solution containing intermediate 2 and material 3 were simultaneously injected into the second microstructured reactor for washing (removing excess amino acids and coupling reagents). The washing time was 12 s. After washing, the washing liquid flowed into the first liquid-liquid separator to separate the aqueous phase and the organic phase containing Fmoc-Gly-BTPH (the organic phase was placed in the fourth feeding tank). HPLC was used to monitor whether the organic phase was clean and impurities were removed. If there were still impurities, another round of washing could be performed. The feed flow rate of material 3 was adjusted by a metering pump, and the feed flow rate of material 3 was 10 mL / min.

[0115] (c) Deprotection process of Fmoc-Gly-BTPH

[0116] Material 4 (organic phase placed in the fourth feeding tank): concentrated under reduced pressure to obtain 40 mL of organic phase containing Fmoc-Gly-BTPH.

[0117] Prepare material 5 (placed in the fifth feeding tank): 40 mL of 10% (v / v) tetrahydropyrrole / ethyl acetate solution.

[0118] The feed flow rates of material 4 and material 5 were adjusted by metering pumps and injected into the third microstructured reactor simultaneously for deprotection reaction, wherein the flow rates of the two were equal to 10 mL / min, the reaction temperature was room temperature, and the residence time of the deprotection reaction in the third microstructured reactor was 9.0 s to obtain a reaction solution containing intermediate 3 (NH2-Gly-BTPH).

[0119] Among them, the NMR and mass spectrometry data of compound 3 are: 1H NMR (400MHz, CDCl3), δ: 7.24 (d, J = 8.3Hz, 4H), 6.81 (d, J = 8.4Hz, 4H), 5.08 (s, 1H), 3.27 (s, 2H), 1.28–1.20 (m, 6H), 1.08 (d, J = 7.8Hz, 36H). 13 C NMR (101MHz, CDCl3), δ: 170.95, 155.29, 134.02, 128.75, 119.83, 67.31, 43.44, 17.90, 12.61. HRMS (ESI-TOF): Calcd for C 33 H 57 N3O3Si2[M+H] + :600.3938;Found:600.4018.

[0120] (d) Purification of NH2-Gly-BTPH

[0121] Prepare material 6 (placed in the sixth feeding tank): 3% (v / v) HCl aqueous solution with a volume twice that of the reaction solution of intermediate 3.

[0122] The reaction solution containing intermediate 3 and material 6 are respectively injected into the fourth microstructured reactor for washing (removing residual amino acids, coupling reagents, excess tetrahydropyrrole and tetrahydropyrrole addition base). The washing time is 12s. The washing liquid after washing flows into the second liquid-liquid separator to separate the aqueous phase and the organic phase containing NH2-Gly-BTPH (the organic phase is placed in the second feeding tank and concentrated under reduced pressure to an organic phase volume of 40mL). HPLC is used to monitor whether the organic phase is washed and impurities are clean. If there are still impurities, it can be washed again. The feed flow rate of material 6 is adjusted by a metering pump, and the feed flow rate of material 6 is 10mL / min.

[0123] (e) Microchannel modular reaction device

[0124] Similar to the treatment method of steps (a) to (d), Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Ile-OH are coupled in sequence, and purification and deprotection processes are performed after each coupling of an amino acid; finally, Boc-Phe-OH is coupled and purified according to the treatment method of steps (a) to (b). The concentration of amino acids in the activation solutions for preparing Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, and Boc-Phe-OH is 0.275 mmol / mL, and the solvent ethyl acetate is controlled at 40 mL. Finally, a reaction solution containing fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH with a soluble tag BTPH at the C-terminus (denoted as compound 4) was prepared and spin-dried to obtain a solid crude product of fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH with a soluble tag BTPH at the C-terminus, 9.24 g, with a purity of 87%.

[0125] Among them, the mass spectrometry data of compound 4 is: HRMS (ESI-TOF): Calcd for C 76 H 120 N 10 O 12 Si2[M-Boc+2K] 2+ :698.9313;Found:698.4469.

[0126] Among them, the NMR data of compound 4 is: 1H NMR(400MHz,Chloroform-d)δ8.65(d,J=70.1Hz,1H),7.85–7.37(m,3H),7.17(dq,J=24.3,7.7,6.8Hz ,10H),6.91–6.68(m,4H),5.10–4.77(m,2H),4.66–4.58(m,1H),4.49(t,J=17.4Hz,2H),4.33–4.18(m ,1H),4.15–3.69(m,6H),3.68–3.37(m,5H),3.26–2.78(m,2H),2.52–1.45(m,17H),1.37(d,J=11.4Hz ,6H),1.24(qd,J=14.8,9.1,7.3Hz,9H),1.07(d,J=7.8Hz,36H),0.98–0.85(m,8H),0.84–0.76(m,3H).

[0127] 2. Synthesis of Linear Heterophyllin B

[0128] Lysis buffer: TFA / EDT / AnisS lysis system, TFA (trifluoroacetic acid): EDT (1,2-ethanedithiol): AnisS (thioanisole) = 95%: 2.5%: 2.5% (v / v / v).

[0129] A crude solid product containing a fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH with a soluble C-terminal tag, BTPH, was mixed with 10 mL of lysis buffer per gram of crude solid. The reaction was incubated at 40°C for 2 hours to simultaneously remove the BTPH group and the N-terminal α-amino Boc protecting group. After the reaction, the reaction solution was cooled and precipitated with pre-cooled ether. The precipitate was collected by centrifugation at 3600 rpm (the volume ratio of ether to lysis buffer was 5:1), dried with ultrapure N2 gas, and finally dried in a vacuum dessicator to yield 4.94 g of the white powder product, NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 (designated as compound 5). The yield was 61% (based on the initial loading of the BTPH tag) and the HPLC purity was 91%.

[0130] Among them, the mass spectrometry data of compound 5 is: HRMS (ESI-TOF): Calcd for C 40 H 62 N 10 O8[M+H] + :811.4752;Found:811.4051.

[0131] Among them, the NMR data of compound 5 is: 13 C NMR(101MHz,DMSO-d6)δ172.83,172.15,171.06,170.12,169.41,169.17,169.0 5,167.59,158.82,158.47,134.66,129.70,128.47,127.23,117.62,114.70,59. 29,57.75,54.67,52.93,51.73,47.32,46.69,42.44,42.00,40.38,36.94,36.37,28.78,28.03,27.69,26.90,24.70,24.38,24.15,23.04,21.84,14.81,10.73. 1 H NMR(400MHz, DMSO-d6)δ8.28(d,J=14.8Hz,3H),8.13–8.03(m,2H),7.29(s,3H),7.18(dd,J=17.1,10.7Hz ,4H),4.57(dd,J=22.1,8.5Hz,2H),4.34(d,J=8.5Hz,2H),4.16(dd,J=14.3,7.0Hz,2H),3.66(qd,J=20.6, 7.8Hz,8H),3.52(dd,J=17.9,8.4Hz,2H),3.01(d,J=6.2Hz,2H),2.19–2.10(m,2H),1.97–1.77(m,9H),1.7 6–1.69(m,1H),1.61(s,1H),1.49(d,J=7.4Hz,2H),1.09(d,J=15.3Hz,2H),0.88(dd,J=20.9,6.8Hz,11H).

[0132] 3. Cyclization of Linear Heterophyllin B

[0133] Weigh guanidine hydrochloride (Gn·HCl, 0.60 mol) and add 50 mL of deionized water. Stir until completely dissolved. Add sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, 0.02 mol). Adjust the pH of the solution to 3.0 by adding concentrated hydrochloric acid (36-38%) dropwise in small amounts. Make up to 100 mL with deionized water. Mix thoroughly and vacuum filter through a 0.22 μm filter to remove trace insoluble matter. Obtain a clear solution and save for later use. Prepare the required amount of buffer solution according to this method.

[0134] Compound 5 (6.1 mmol) was added to 160 mL of the aforementioned buffer solution at -15°C, followed by the addition of sodium nitrite (91.5 mmol). The mixture was stirred for 30 minutes to allow for oxidative azidation. Saturated sodium bicarbonate solution was then added to adjust the pH to 7.0-7.5. The ice bath was removed, and an aminolysis cyclization reaction was carried out in an ice-water bath at 0°C for 30 minutes to obtain a cyclized solution of heterophyllin B. Liquid chromatography analysis confirmed that the linear heterophyllin B was completely cyclized, with a cyclization yield of 99.8%. The HPLC purity of the heterophyllin B in the reaction solution was 78%.

[0135] 4. Purification of Heterophyllin B

[0136] The heterophyllin B cyclization solution obtained in step 3 was concentrated under reduced pressure, dissolved in 6 L of deionized water, filtered through a 0.45 μm filter membrane, and the filtrate was purified by reverse preparative chromatography on C18 silica gel. Specific chromatographic parameters were as follows: Column: DAC-100 dynamic axial column, 600 x 600 x 2300 mm, packed with Bonnasil-BS C18 pro; column temperature: 35°C; flow rate: 200 mL / min; detection wavelength: 214 nm; injection volume: 4 L; mobile phase: A: 0.1% TFA in water, B: acetonitrile; gradient elution; separation time for heterophyllin B was approximately 24 minutes. The specific gradient elution conditions are as follows:

[0137] Table 2

[0138]

[0139] The collected fractions were analyzed by HPLC, and the fractions with a purity of more than 95% were combined, concentrated and lyophilized to obtain 2.76 g of white flocculent product Heterophyllin B (denoted as compound 6) with a purification yield of 81.94%; the total yield was 34.94% (calculated based on the initial loading amount of the labeled BTPH) and the HPLC purity was 98.49%.

[0140] The HPLC purity data of the white flocculent product Heterophyllin B is shown in the figure. Figure 3 Among them, the mass spectrum data of the product Heterophyllin B is: HRMS (ESI-TOF): Calcd for C 40 H 58 N8O8[M+Na] + :801.4378; Found:801.3464, see the specific mass spectrum Figure 7 shown.

[0141] Among them, the NMR data of the product Heterophyllin B is: 13 C NMR(101MHz,DMSO-d6)δ172.51,172.36,170.44,169.77,169.74,169.68,169.58 ,168.49,139.46,129.79,129.15,128.25,127.86,126.09,60.67,60.42,58.46, 58.13,56.67,48.88,47.22,47.15,46.11,44.67,41.81,38.19,34.30,32.80,30.61,27.81,26.68,25.44,25.15,25.11,24.91,23.20,21.78,21.16,15.94,9.90. 1 H NMR (400MHz, DMSO-d6) δ8.65(t,J=5.3Hz,1H),8.54(dd,J=8.3,4.5Hz,1H),8.14(d,J=8.6Hz,1H),7.28(d,J=6.8Hz,2H),7.11(dd,J=7.6,2.1H z,2H),5.96(d,J=6.9Hz,1H),4.60(t,J=10.8Hz,1H),4.38(t,J=8.5Hz, 1H),4.16–4.02(m,3H),3.77–3.66(m,2H),3.66–3.58(m,1H),3.58–3.3 0(m,10H),3.30–3.24(m,2H),3.24–3.11(m,2H),2.44(dd,J=12.1,6.2Hz,1H),2.26(ddt,J=15.2,8.0,3.9Hz,1H),2.08–1.93(m,2H),1.93–1. 89(m,1H),1.89–1.71(m,5H),1.56(ddt,J=14.9,11.1,5.4Hz,2H),1.48 –1.39(m,2H),1.39–1.26(m,2H),1.01–0.85(m,8H),0.85–0.70(m,4H).

[0142] Comparative Example 1: Solid Phase Synthesis of Heterophyllin B Using Coupling Reagents

[0143] The resin used was 2-CTC resin with a substitution degree of 1.19 mmol / g. The following general SPPS procedure was followed for the synthesis of crude linear heterophyllin B using 2-CTC resin:

[0144] (1) 8.41 g of 2-CTC resin (1.19 mmol / g, 10 mmol) was weighed into a solid phase reactor using an electronic balance. 100 mL of DCM was added to swell the mixture for 1 h, and the mixture was washed with DCM (2 × 100 mL). Fmoc-Gly-OH (30 mmol, 3.0 eq.) was dissolved in 100 mL of DMF, and DIPEA (36 mmol, 3.6 eq.) was added to obtain amino acid solution A. Amino acid solution A was added to the solid phase reactor and reacted with 2-CTC resin at room temperature for 150 min. Coupling was completed, and the resulting product was recorded as Fmoc-Gly-Resin.

[0145] (2) The Fmoc-Gly-Resin obtained in step (1) was washed with DMF (4×100 mL), and then the Fmoc protecting group was removed with 20% pyrrolidine / DMF solution (30 mL). The reaction was carried out at room temperature for 8 min. After the reaction was completed, the solution was removed and an equal amount of 20% pyrrolidine / DMF solution (30 mL) was added to remove the Fmoc protecting group. The reaction was carried out at room temperature for 15 min. After the reaction was completed, a small amount of resin was placed in a test tube, and 6 drops of Kaiser reagent A and Kaiser reagent B were added to each test tube. The tube was then placed in a water bath at 100°C and heated for 30 s. The color of the resin was observed. If the resin was positive (the resin was black, purple or lavender), the deprotection was completed. The resin was washed with DMF (4×30 mL) to ensure that the residual deprotection reagent did not affect the next peptide grafting reaction.

[0146] (3) Repeat steps (1) to (2) to sequentially add Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, and Boc-Phe-OH until all amino acids are assembled. Wash the resin four times with DCM (100 mL) and finally with methanol (100 mL). Dry the resin to constant weight to obtain the fully protected peptide resin Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-Resin, 14.4 g, with a yield of 88.83%.

[0147] Among them, when coupling Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH and Boc-Phe-OH, DIPEA was not used, and condensation agents HOBT and DIC were used. The amount of HOBT used in each coupling process was 36 mmol, and the amount of DIC used was 36 mmol.

[0148] (4) Cracking solution: composed of trifluoroacetic acid, triisopropylsilyl chloride, thioanisole and H2O, with the volume ratio of trifluoroacetic acid, triisopropylsilyl chloride, thioanisole and H2O being 92.5:2.5:2.5:2.5.

[0149] The fully protected peptide resin Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-Resin obtained in step (3) was added to 144 mL of lysate (10 mL of lysate was required for each gram of fully protected peptide resin), and the reaction was stirred at room temperature for 30 minutes. After the reaction, the reaction solution was precipitated at low temperature with pre-cooled ether (720 mL). After sedimentation, the precipitate was collected by centrifugation (3600 rpm), dried with ultrapure N2 gas, and finally dried in a vacuum desiccant to obtain a white powder product NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-OH, 6.43 g, with a yield of 80.77% and an HPLC purity of 89%.

[0150] (5) The NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-OH obtained in step (4) was dissolved in anhydrous acetonitrile to prepare a linear heterophyllin B crude solution with a crude concentration of 1 mg / mL. 6.43 g of PyAoP was then added, and the reaction was stirred at room temperature for 12 hours. The reaction was completed by HPLC monitoring to obtain a cyclization solution with a cyclization yield of 99.7%. The HPLC purity of heterophyllin B in the heterophyllin B cyclization solution was 25.39%. The specific HPLC chart is shown in FIG. Figure 8 .

[0151] (6) The heterophyllin B cyclization solution obtained in step (5) was concentrated under reduced pressure, dissolved in 6 L of deionized water, filtered through a 0.45 μm filter membrane, and the filtrate was purified by reverse preparative chromatography using C18 silica gel packing. Specific chromatographic parameters were as follows: chromatographic column: DAC-100 dynamic axial column, 600*600*2300 mm, packed with Bonnasil-BS C18 pro; column temperature: 35°C; flow rate: 200 mL / min; detection wavelength: 214 nm; injection volume: 4 L; mobile phase: A: 0.1% TFA aqueous solution, B: acetonitrile; gradient elution; separation time for heterophyllin B was approximately 24 min; specific gradient elution conditions are the same as those in Table 2.

[0152] The collected fractions were analyzed by HPLC, and the fractions with a purity of more than 95% were combined, concentrated and freeze-dried to obtain 1.87 g of white flocculent product Heterophyllin B. The total yield was 24.01% (calculated based on 2-CTC resin) and the HPLC purity was 98.5%.

[0153] HRMS analysis of Heterophyllin B: The theoretical calculated value is 778.4378, and the measured value is [M+H] + =779.3645.

[0154] Table 3 Comparison of the effects of solid phase synthesis of Heterophyllin B vs. hydrazide liquid phase synthesis of Heterophyllin B (taking Comparative Example 1 and Example 2 as examples, the carrier group is 10 mmol)

[0155]

[0156]

[0157] As shown in Table 3, the hydrazide liquid-phase synthesis of Heterophyllin B of the present invention reduces the amount of amino acid used by over 60%, the amount of condensation reagent used by over 50%, and the amount of solvent used by over 85% compared to conventional solid-phase synthesis routes. Furthermore, the overall yield of the final product, Heterophyllin B, is also increased by 45%. In summary, the hydrazide liquid-phase synthesis of Heterophyllin B disclosed in the present invention offers significant environmental and cost advantages compared to conventional solid-phase synthesis methods.

[0158] Comparative Example 2: Synthesis of Heterophyllin B from CDMH, a Compound Without Silicon Oxygen Groups

[0159] 1. Synthesis of CDMH

[0160]

[0161] (1) Fmoc-CDMH was prepared with reference to the prior art (CN103755843A N-9-fluorenylmethyloxycarbonylhydrazine resin and its preparation method and application).

[0162] (2) The experimental method was the same as step (5) of Example 1, using Fmoc-CDMH as the raw material and tetrahydropyrrole as the deprotection reagent to prepare compound CDMH.

[0163] The NMR hydrogen spectrum of compound CDMH is shown in Figure 4 , NMR carbon spectrum see Figure 5 , mass spectrometry data see Figure 6 .

[0164] 2. Synthesis of Heterophyllin B using CDMH as a label compound

[0165]

[0166] (1) The silyl-free compound CDMH (2.27 mmol, 1.0 eq.) was placed in a reaction flask, and Fmoc-Gly-OH (2.5 mmol, 1.1 eq.), HATU (2.7 mmol, 1.2 eq.), and DIPEA (5.7 mmol, 2.5 eq.) were added and dissolved in 20 mL of ethyl acetate. The mixture was reacted at room temperature for 1 h and monitored by HPLC. After the reaction was complete, the mixture was washed three times with an equal volume of 5% (w / v) K2CO3 aqueous solution. After each wash, the organic phase was collected in a separatory funnel. The organic phase was concentrated, and the crude product was purified by column chromatography to obtain Fmoc-Gly-CDMH as a white solid, designated as compound 7, with a yield of 96%.

[0167] The H NMR spectrum data of compound 7 is 1 H NMR(400MHz,Chloroform-d)δ7.76(dd,J=7.7,4.1Hz,2H),7.60(d,J=7.5Hz,1H),7.55–7.45(m,3H),7.40(qd,J=7.5,5.2Hz,3H),7.36–7.11(m,14H) ,6.69(s,1H),5.86(s,1H),5.21(t,J=5.7Hz,1H),4.34(d,J=7.2Hz,1H),4 .23(dd,J=14.0,7.1Hz,1H),3.80(d,J=4.8Hz,1H),3.54(d,J=5.7Hz,1H).

[0168] (2) The Fmoc-Gly-CDMH (2.18 mmol, 1.0 eq.) obtained in step (1) was dissolved in 20 mL of ethyl acetate, and pyrrolidine (27.24 mmol, 12.5 eq.) was added. The reaction was allowed to react at room temperature for 30 min, and the reaction was monitored by HPLC. After the reaction was complete, an equal volume of 3% (v / v) HCl aqueous solution was used to remove residual amino acids, coupling reagents, excess tetrahydropyrrole and tetrahydropyrrole addition base. The organic phase was collected in a separatory funnel after each washing. HPLC detection revealed that the product NH2-Gly-CDMH was completely lost to the aqueous phase, and no product NH2-Gly-CDMH was present in the organic phase. Specifically, the HPLC liquid phase spectra of the organic phase and the aqueous phase are shown in FIG. Figure 9 、 Figure 10 shown.

[0169] The target product Heterophyllin B could not be obtained by liquid phase synthesis using the compound CDMH without siloxy groups.

[0170] Comparative Example 3: Tank Liquid Phase Synthesis of Heterophyllin B

[0171] (1) Synthesis of Fmoc-Gly-BTPH

[0172] BTPH (prepared in Example 1, 2 mmol, 1.0 eq.), Fmoc-Gly-OH (2.1 mmol, 1.05 eq.), EDC·HCl (2.4 mmol, 1.2 eq.), HOBT (2.4 mmol, 1.2 eq.), and DIPEA (0.2 mmol, 0.1 eq.) were added to 40 mL of ethyl acetate and the reaction was maintained at room temperature to 40° C. for 1 h. After the reaction, an Fmoc-Gly-BTPH reaction solution in which the amino acid was linked to BTPH was obtained. The reaction solution was washed three times with 40 mL of a 5 wt% K2CO3 aqueous solution to remove excess amino acids and coupling reagents. The solvent was then dried and purified by silica gel column chromatography (PE:EA=4:1) to obtain a slightly yellow solid compound, Fmoc-Gly-BTPH, with a purity of 96% and a yield of 91% (based on BTPH).

[0173] (2) Removal of Fmoc protection

[0174] The Fmoc-Gly-BTPH (1.82 mmol) obtained in step (1) and pyrrolidine (16 mmol, 8.0 eq. BTPH) were dissolved in 40 mL of ethyl acetate and stirred for deprotection reaction at room temperature to 40°C for 1 h. After the reaction, the mixture was extracted and washed three times with an equal amount of 40 mL of 5% hydrochloric acid aqueous solution. The upper organic phase solution was taken to obtain a washing solution containing NH2-Gly-BTPH.

[0175] (3) Coupling Fmoc-Gly-OH

[0176] To the washing solution containing NH2-Gly-BTPH obtained in step (2), Fmoc-Gly-OH (1.05 equivalents based on the support BTPH), EDC·HCl (1.2 equivalents based on the support BTPH), HOBT (1.2 equivalents based on the support BTPH), and DIPEA (0.1 equivalents based on the support BTPH) were added, and the reaction was maintained at room temperature to 40°C for 1 hour. After the reaction was completed, the reaction solution was washed three times with 40 mL of a 5 wt% K2CO3 aqueous solution to remove excess amino acids and coupling reagents. The upper organic phase was collected to obtain a washing solution containing Fmoc-Gly-Gly-BTPH.

[0177] (4) Fmoc deprotection

[0178] Pyrrolidine (the amount of pyrrolidine is 8.0 equivalents of the carrier BTPH) and 40 mL of ethyl acetate were added to the washing solution containing Fmoc-Gly-Gly-BTPH obtained in step (3), and the reaction was stirred at room temperature to 40°C for 1 h. After the reaction was completed, an equal amount of 40 mL of 5% hydrochloric acid aqueous solution was used for extraction and washing three times. The upper organic phase solution was taken to obtain a washing solution containing NH2-Gly-Gly-BTPH.

[0179] (5) According to the above steps (3) to (4), Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH and Boc-Phe-OH were continuously added to finally obtain a reaction solution containing fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH with a soluble tag BTPH at the C-terminus. The reaction solution was spin-dried to obtain a solid crude product of fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH with a soluble tag BTPH at the C-terminus. The HPLC purity was 84%, and the yield was 68% (based on BTPH).

[0180] (6) Lysis solution: TFA / EDT / AnisS lysis system, TFA (trifluoroacetic acid): EDT (1,2-ethanedithiol): AnisS (thioanisole) = 95%: 2.5%: 2.5% (v / v / v).

[0181] The fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-BTPH solid crude product with a soluble tag BTPH at the C-terminus obtained in step (5) was mixed with the lysis buffer, and 10 mL of lysis buffer was used per gram of the solid crude product. The reaction was carried out at 40°C for 2 hours to simultaneously remove the BTPH group and the N-terminal α-amino Boc protecting group. After the reaction was completed, the reaction solution was precipitated at low temperature with pre-cooled ether. After precipitation, the precipitate was collected by centrifugation (3600 rpm) (wherein the volume ratio of ether to lysis buffer was 5:1), dried with ultrapure N2 gas, and finally dried in a vacuum desiccant to obtain a white powder product NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 with an HPLC purity of 82% and a yield of 57% (based on BTPH).

[0182] (7) Weigh guanidine hydrochloride (Gn·HCl, 0.60 mol), add 50 mL of deionized water, and stir until completely dissolved. Add sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, 0.02 mol). Add concentrated hydrochloric acid (36-38%) dropwise in small amounts to adjust the pH of the solution to 3.0. Add deionized water to 100 mL. After mixing, vacuum filter through a 0.22 μm filter membrane to remove trace insoluble matter to obtain a clear solution, which is stored for future use. Prepare the required amount of buffer solution according to this method.

[0183] At -15°C, the linear polypeptide hydrazide NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 (0.9 mmol) obtained in step (6) was added to 24 mL of the above-prepared buffer solution, and sodium nitrite (13.5 mmol) was added. The mixture was stirred for 30 minutes to carry out an oxidative azidation reaction. Subsequently, a saturated sodium bicarbonate solution was added to adjust the pH to 7.0-7.5. The ice salt bath was removed, and an aminolysis cyclization reaction was carried out in an ice-water bath at 0°C for 30 minutes to obtain a cyclized solution of heterophyllin B. Liquid chromatography analysis showed that the linear heterophyllin B was completely cyclized, and the HPLC purity of the heterophyllin B in the reaction solution was 67%.

[0184] The present invention provides a soluble hydrazine-functionalized carrier and its preparation and application. While there are numerous methods and approaches for implementing this technical solution, the aforementioned are merely preferred embodiments of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A soluble hydrazine functionalized carrier, characterized in that The structural formula of the soluble hydrazine functionalized carrier is shown in Formula I: in, R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 an alkyl group, or a substituted or unsubstituted C5-C8 aromatic group.

2. The soluble hydrazine functionalized carrier according to claim 1, characterized in that R1=R1'; R2=R2'; R3=R3'; R1, R2, and R3 are independently selected from substituted or unsubstituted C1 to C 22 Alkyl, or substituted or unsubstituted C5-C8 aromatic group; Preferably, R1=R1'; R2=R2'; R3=R3'; R1, R2, and R3 are independently selected from unsubstituted C1 to C 18 Alkyl, or unsubstituted C6 aromatic group; More 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.

3. The soluble hydrazine functionalized carrier according to claim 1, characterized in that The soluble hydrazine functionalized carrier is selected from any of the following structures:

4. The method for preparing a soluble hydrazine-functionalized carrier according to any one of claims 1 to 3, characterized in that: The synthetic route is as follows: Compound c undergoes a first reaction with 9-fluorenylmethyl carbamate to obtain compound d; compound d undergoes a second reaction under the action of a catalyst to obtain a soluble hydrazine-functionalized carrier I; in, X is selected from Br, Cl or I; R1, R2, R3, R1', R2', R3' are independently selected from substituted or unsubstituted C1-C 22 an alkyl group, or a substituted or unsubstituted C5-C8 aromatic group.

5. The preparation method according to claim 4, characterized in that The catalyst is any one or a combination of tetrahydropyrrole, hexahydropyridine, DBU, tetramethylguanidine and triethylamine; and / or, the molar ratio of compound c to 9-fluorenylmethyl carbamate is 1.0:(1.0-3.0); and / or, the first reaction is carried out at room temperature; and / or, the first reaction has a reaction time of 0.2-2.0h; and / or, the solvent used in the first reaction is ethyl acetate, N,N-dimethylformamide, chloromethane, toluene, chloroform, N-methylpyrrolidone, tetrahydrofuran, 2-methyl Any one or a combination of tetrahydrofuran and dimethyl sulfoxide; and / or, the molar ratio of the compound d to the catalyst is 1.0:(10.0~15.0); and / or, the second reaction is carried out at room temperature; and / or, the reaction time of the second reaction is 0.2~2.0h; and / or, the solvent used in the second reaction is any one or a combination of ethyl acetate, N,N-dimethylformamide, chloroform, toluene, chloroform, N-methylpyrrolidone, tetrahydrofuran, 2-methyltetrahydrofuran and dimethyl sulfoxide.

6. A protective agent for the C-terminus of an amino acid or polypeptide, characterized in that: The protective agent comprises the soluble hydrazine functionalized carrier according to any one of claims 1 to 3.

7. A method for synthesizing Heterophyllin B using a microchannel modular reaction device, characterized in that: The steps include: (1) Fmoc-Gly-OH is mixed with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a first Fmoc-Gly-OH activation solution; the soluble hydrazine-functionalized carrier according to any one of claims 1 to 3 is dissolved in an organic solvent to obtain a soluble tag solution; the first Fmoc-Gly-OH activation solution and the soluble tag solution are respectively and simultaneously pumped into a first microstructured reactor for condensation reaction; then the effluent of the first microstructured reactor and the potassium carbonate aqueous solution are respectively and simultaneously pumped into a second microstructured reactor for washing, and the effluent after washing is separated in a first liquid-liquid separator, and the organic phase containing Fmoc-Gly-TAG is retained; The retained organic phase containing Fmoc-Gly-TAG and the organic solvent solution containing the deprotection reagent are simultaneously pumped into the third microstructured reactor for deprotection reaction; then the effluent of the third microstructured reactor and the hydrochloric acid aqueous solution are simultaneously pumped into the fourth microstructured reactor for washing, and the washed effluent is separated in the second liquid-liquid separator, and the organic phase containing NH2-Gly-TAG is retained; (2) Fmoc-Gly-OH is mixed with a condensation reagent, an acid binding agent, and an organic solvent for activation to obtain a second Fmoc-Gly-OH activation solution; the second Fmoc-Gly-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Gly-TAG obtained in step (1) is used to replace the soluble tag solution in step (1); the same reaction and treatment process as step (1) are carried out to obtain an organic phase containing NH2-Gly-Gly-TAG; (3) mixing Fmoc-Leu-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain an Fmoc-Leu-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the Fmoc-Leu-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Gly-Gly-TAG obtained in step (2), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Leu-Gly-Gly-TAG; (4) mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a first Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the first Fmoc-Pro-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Leu-Gly-Gly-TAG obtained in step (3), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Leu-Gly-Gly-TAG; (5) Mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a second Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the second Fmoc-Pro-OH activation solution, replacing the soluble tag solution of step (1) with the organic phase containing NH2-Pro-Leu-Gly-Gly-TAG obtained in step (4), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Pro-Leu-Gly-Gly-TAG; (6) Mixing Fmoc-Pro-OH with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a third Fmoc-Pro-OH activation solution; replacing the first Fmoc-Gly-OH activation solution of step (1) with the third Fmoc-Pro-OH activation solution, replacing the soluble tag solution in step (1) with the organic phase containing NH2-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (5), and performing the same reaction and treatment process as step (1) to obtain an organic phase containing NH2-Pro-Pro-Pro-Leu-Gly-Gly-TAG; (7) Fmoc-Ile-OH is mixed with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain an Fmoc-Ile-OH activation solution; the Fmoc-Ile-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Pro-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (6) is used to replace the soluble tag solution in step (1); the same reaction and treatment process as step (1) are carried out to obtain an organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG; (8) Boc-Phe-OH is mixed with a condensation reagent, an acid-binding agent, and an organic solvent for activation to obtain a Boc-Phe-OH activation solution; the Boc-Phe-OH activation solution is used to replace the first Fmoc-Gly-OH activation solution of step (1); the organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG obtained in step (7) is used to replace the soluble tag solution in step (1); the Boc-Phe-OH activation solution and the organic phase containing NH2-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG are mixed to obtain a Boc-Phe-OH activation solution; .... The organic phases of Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG were simultaneously pumped into the first microstructured reactor for condensation reaction; the effluent from the first microstructured reactor and the potassium carbonate aqueous solution were then simultaneously pumped into the second microstructured reactor for washing, and the washed effluent was separated in a first liquid-liquid separator, and the organic phase containing Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG was retained and spin-dried to obtain a fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus; (9) The fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus obtained in step (8) was cleaved using a lysis buffer, precipitated, blown dry, and dried to obtain NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2; (10) NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 is placed in a buffer solution, and an azidation reagent is added to carry out an oxidative azidation reaction; after the reaction is completed, an alkaline solution is added to adjust the pH, and an aminolysis cyclization reaction is carried out to obtain a heterophyllin B cyclization solution; (11) The Heterophyllin B cyclization solution obtained in step (10) is purified by preparative chromatography to obtain Heterophyllin B.

8. The method according to claim 7, characterized in that The condensation reagents used in each step are the same and are selected from any one or a combination of N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, 1-hydroxyphenyl-4,5-diphenylimidazole, N,N-diisopropylethylamine, O-benzotriazole-tetramethyluronium hexafluorophosphate and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; and / or the acid-binding agents used in each step are the same and are selected from N,N-diisopropylethylamine; and / or the organic solvents used in each step are the same and are selected from any one or a combination of dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, ethyl acetate and dimethyl sulfoxide; and / or the activation in each step is carried out at room temperature for 15 to 30 minutes.

9. The method according to claim 7, characterized in that In step (1), the reaction molar ratio of the soluble hydrazine functionalized carrier and Fmoc-Gly-OH, condensation reagent, and acid binding agent is controlled to be 1.00: (1.05-1.10): (1.05-2.40): (0.10-0.50), and the replacement raw materials used in steps (2) to (8) are reacted according to the same molar ratio as in step (1); and / or, in step (1), the condensation reaction is carried out at room temperature, and in steps (2) to (8) The reaction temperature is the same as the reaction temperature of step (1); and / or, in step (1), the reaction residence time of the condensation reaction is 9.0s to 18.0s, and the reaction residence time in steps (2) to (8) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of potassium carbonate in the potassium carbonate aqueous solution is 3% g / mL to 5% g / mL, and the potassium carbonate aqueous solution in steps (2) to (8) is the same as the potassium carbonate aqueous solution in step (1).

10. The method according to claim 7, characterized in that In step (1), in the organic solvent solution containing a deprotecting agent, the deprotecting agent is tetrahydropyrrole or piperidine, the concentration of the deprotecting agent in the solution is 10vt% to 15vt%, and the organic solvent solutions containing the deprotecting agent in steps (2) to (7) are the same as the organic solvent solution containing the deprotecting agent in step (1); and / or, in step (1), the reaction temperature of the deprotection reaction is room temperature, and the reaction temperature in steps (2) to (7) is the same as the reaction temperature in step (1); and / or, in step (1), the reaction residence time of the deprotection reaction is 9.0s to 18.0s, and the reaction residence time in steps (2) to (7) is the same as the reaction residence time in step (1); and / or, in step (1), the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 1vt% to 3vt%, and the hydrochloric acid aqueous solution in steps (2) to (7) is the same as the hydrochloric acid aqueous solution in step (1).

11. The method according to claim 7, characterized in that In step (9), the lysis solution is a composition of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole, and the volume ratio of trifluoroacetic acid, 1,2-ethanedithiol and thioanisole is 95%:2.5%:2.5%; and / or, the dosage ratio of the fully protected Boc-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-TAG solid crude product with a soluble tag at the C-terminus to the lysis solution is 1 g of solid crude product: 10 mL to 15 mL of lysis solution; and / or, the lysis reaction is carried out at 25° C. to 40° C. for 1 h to 2 h; and / or, the solvent used for the precipitation is ether, and the volume ratio of ether to the lysis solution is (5 to 10):

1.

12. The method according to claim 7, characterized in that In step (10), the buffer solution is prepared as follows: weigh 0.60 mol of guanidine hydrochloride, add 50 mL of deionized water, mix well, add 0.02 mol of sodium dihydrogen phosphate dihydrate, add concentrated hydrochloric acid to adjust the pH of the solution to 3.0-3.5, dilute to 100 mL with deionized water, mix well, and then vacuum filter through a filter membrane to obtain the solution; and / or, the azidation reagent is sodium nitrite; and / or, the molar ratio of the NH2-Phe-Ile-Pro-Pro-Pro-Leu-Gly-Gly-NH-NH2 to the azidation reagent is 1:(15-20); and / or, the reaction temperature of the oxidative azidation reaction is -20°C to -15°C; and / or, the reaction time of the oxidative azidation reaction is 30-60 min; And / or, the alkaline solution is a sodium bicarbonate solution; and / or, the pH is adjusted to 7.0-7.5; and / or, the reaction temperature of the aminolysis cyclization reaction is 0°C-5°C; and / or, the reaction time of the aminolysis cyclization reaction is 30-60 min.

13. The method according to claim 7, characterized in that In step (11), the Heterophyllin B cyclization solution is purified by preparative chromatography, specifically by: The Heterophyllin B cyclization solution was concentrated under reduced pressure, dissolved and diluted with deionized water, and filtered through a filter membrane. The filtrate was purified by reverse preparative chromatography using a C18 silica gel packing. The specific chromatographic parameters were as follows: chromatographic column: DAC-100 dynamic axial column, 600*600*2300 mm, packed with Bonnasil-BS C18 pro; column temperature: 35°C; flow rate: 200 mL / min; detection wavelength: 214 nm; injection volume: 4 L; mobile phase: A: 0.1% TFA aqueous solution, B: acetonitrile; gradient elution. The specific gradient elution conditions are as follows: 。 14. The method according to claim 7, characterized in that The microchannel modular reaction device includes a connecting pipe, a first feeding tank, a second feeding tank, a third feeding tank, a fourth feeding tank, a fifth feeding tank, a sixth feeding tank, a first microstructured reactor, a second microstructured reactor, a third microstructured reactor, a fourth microstructured reactor, a metering pump, a first liquid-liquid separator and a second liquid-liquid separator; wherein the first feeding tank and the second feeding tank are respectively connected in parallel to the feed port of the first microstructured reactor through a connecting pipe provided with a metering pump; wherein the third feeding tank is connected to the discharge port of the first microstructured reactor through a connecting pipe provided with a metering pump. The first microstructured reactor is connected to the feed inlet of the second microstructured reactor in parallel; the discharge port of the second microstructured reactor is connected to the feed inlet of the first liquid-liquid separator through a connecting pipe; the discharge port of the first liquid-liquid separator is connected to the feed inlet of the fourth feeding tank through a connecting pipe; the fourth feeding tank and the fifth feeding tank are connected to the feed inlet of the third microstructured reactor in parallel through connecting pipes provided with a metering pump; the sixth feeding tank is connected to the feed inlet of the fourth microstructured reactor in parallel through connecting pipes provided with a metering pump and the discharge port pipe of the third microstructured reactor; The discharge port of the fourth microstructured reactor is connected to the feed port of the second liquid-liquid separator through a connecting pipe; the discharge port of the second liquid-liquid separator is connected to the feed port of the second feeding tank through a connecting pipe.

Images