Method for solid-phase synthesis of semeglutide dipeptide side chain

Abstract

The invention provides a method for solid-phase synthesis of a dipeptide side chain of semeglutide. Specifically, the method comprises the following steps: 1) in an alkaline environment, grafting Fmoc-Aib-OH to 2-CTC resin through a solid-phase reaction to obtain Fmoc-Aib-resin; (2) removing an Fmoc protecting group of the Fmoc-Aib-resin, so as to obtain Aib-resin; 3) in the presence of a condensation reagent, carrying out a coupling reaction on the Aib-resin and R-His (R ')-OH to obtain R-His (R')-Aib-resin; 4) in the presence of a cracking reagent, removing the resin from the R-His (R ')-Aib-resin to obtain R-His (R')-Aib-OH; 5) reacting the R-His (R ')-Aib-OH with the HR ''to obtain the semeglutide dipeptide side chain R-His (R')-Aib-R. The preparation method is simple to operate and easy to amplify production, and the semeglutide dipeptide side chain prepared according to the preparation method can meet the requirements of different semeglutide synthesis processes and is wide in market application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical synthesis, and particularly relates to a method for solid-phase synthesis of a dipeptide side chain of semaglutide. BACKGROUND

[0002] Semaglutide is a long-acting blood glucose-lowering drug developed by Novo Nordisk. It is based on the natural human glucagon-like peptide-1 (GLP-1) molecule, which is obtained by replacing the 8th (alanine to alpha-aminobutyric acid) and 34th (lysine to arginine) amino acids and simultaneously connecting a C18 fatty diacid side chain to the 26th lysine using a spacer. The semaglutide obtained by modifying the human GLP-1 molecule can resist dipeptidyl peptidase 4 (DPP-4) degradation and tightly bind to albumin, thereby significantly prolonging its half-life in the body, achieving once-a-week administration, and retaining up to 94% of the GLP-1 amino acid sequence homology, thereby providing good safety.

[0003] The 1st amino acid of semaglutide is L-histidine (His), and the 2nd amino acid is non-natural alpha-aminoisobutyric acid (Aib). The dipeptide chain formed by His and Aib is called semaglutide dipeptide, which is an indispensable part of semaglutide. Therefore, the compound containing His and Aib (also referred to as semaglutide dipeptide side chain) is an indispensable intermediate for the synthesis of semaglutide, and it is widely used in other biological and pharmaceutical fields, especially in the field of pharmaceutical intermediates.

[0004] Currently, the structure of semaglutide dipeptide side chain on the market is Fmoc-His-Aib-OH, Boc-His(Boc)-Aib-OH or Boc-His(Trt)-Aib-OH, which is mainly synthesized by solid-phase synthesis or liquid-phase synthesis. For example, Chinese Patent Application CN106928343A synthesizes semaglutide by solid-phase step-by-step coupling, wherein the dipeptide side chain is Boc-His(Boc)-Aib-OH. Chinese Patent Application CN113667006A synthesizes Fmoc-His-Aib-OH by liquid-phase synthesis. Chinese Patent Application CN109456401A mentions a liquid-phase synthesis of Boc-His(Trt)-Aib-OH.

[0005] However, the semaglutide dipeptide side chain synthesized by the above preparation methods has a single structure, which cannot meet the needs of different semaglutide synthesis processes, and is not synthesized into an active ester structure, resulting in a long semaglutide synthesis period, high production cost, and is not conducive to large-scale production. Therefore, it is currently urgent to obtain a semaglutide dipeptide side chain synthesis method with diverse product structures, stable products, low production cost, and conducive to large-scale promotion. SUMMARY

[0006] In order to solve the above problems, the application provides a method for solid-phase synthesis of a dipeptide side chain of semaglutide.

[0007] In a first aspect, the application provides a method for solid-phase synthesis of a dipeptide side chain of semaglutide, comprising the following steps:

[0008] 1) resin reaction of Fmoc-Aib-OH shown in formula (I) and 2-CTC resin under alkaline conditions to prepare Fmoc-Aib-resin shown in formula (II);

[0009]

[0010] 2) removal of Fmoc from the Fmoc-Aib-resin prepared in step 1) to prepare Aib-resin shown in formula (III);

[0011]

[0012] 3) coupling reaction of the Aib-resin prepared in step 2) and R-His(R’)-OH shown in formula (IV) in the presence of a condensation reagent to prepare R-His(R’)-Aib-resin shown in formula (V);

[0013]

[0014] 4) removal of resin from the R-His(R’)-Aib-resin prepared in step 3) in the presence of a cleavage reagent to prepare R-His(R’)-Aib-OH shown in formula (VI);

[0015]

[0016] 5) reaction of R-His(R’)-Aib-OH prepared in step 4) and HR” to prepare a dipeptide side chain R-His(R’)-Aib-R” of semaglutide shown in formula (VII)

[0017]

[0018] wherein R and R’ are each independently selected from the group consisting of: an amino protecting group;

[0019] R” is selected from the group consisting of:

[0020] In another preferred embodiment, the amino protecting group is selected from the group consisting of:

[0021] In a preferred embodiment, in step 1), the basic conditions are provided by a reagent selected from the group consisting of Et3N, DIEA, or a combination thereof.

[0022] In another preferred embodiment, the basic conditions refer to a pH of 8-14; preferably a pH of 9-14; more preferably a pH of 10.

[0023] In a preferred embodiment, in step 1), the degree of substitution of the 2-CTC resin is 0.9-1.8 mmol / g; preferably 1.1-1.5 mmol / g.

[0024] In another preferred embodiment, in step 1), the molar ratio of Fmoc-Aib-OH to the basic reagent is 1:(1-3).

[0025] In another preferred embodiment, in step 1), the solvent of the reaction is DCM.

[0026] In a preferred embodiment, in step 2), the reaction conditions are selected from the group consisting of 5% piperazine, 20% piperidine.

[0027] In a preferred embodiment, in step 3), the condensing reagent is selected from the group consisting of:

[0028] a. DIC, DCC, EDCI, HATU, DIEA and TBTU; and

[0029] b. HOBt or HOAt.

[0030] In another preferred embodiment, the molar ratio of reagent a to reagent b is (1-12):1; preferably (1-10):1.

[0031] In another preferred embodiment, in step 3), the solvent of the coupling reaction is DMF.

[0032] In a preferred embodiment, in step 4), the cleaving reagent is selected from the group consisting of:

[0033] A. TFE, TFA or HFIP; and

[0034] B. DCM.

[0035] In a preferred embodiment, the volume ratio of reagent A to reagent B is 1:(2-6); preferably 1:(3-5).

[0036] In a preferred embodiment, the method further comprises one or more of the following features:

[0037] i. In step 1), the molar ratio of Fmoc-Aib-OH to the 2-CTC resin is (1-5):1; preferably (1-3):1;

[0038] ii. In step 3), the molar ratio of R-His(R')-OH to Aib-resin is (1-10):1; preferably (1-7):1;

[0039] iii. In step 4), the mass-volume ratio of R-His(R')-Aib-resin to cleavage reagent is 0.05-0.4 g / ml; preferably 0.05-0.3 g / ml.

[0040] iv. In step 5), the molar ratio of R-His(R')-Aib-OH to HR" is 1:(1-3); preferably 1:(1-2).

[0041] In a preferred embodiment, the method further comprises one or more of the following features:

[0042] (1) In step 1), the temperature for resin reaction is 25-30°C.

[0043] (2) In step 2), the reaction temperature is 25-30°C.

[0044] (3) In step 3), the temperature for coupling reaction is 25-30°C.

[0045] (4) In step 4), the reaction temperature is 2-28°C.

[0046] (5) In step 5), the reaction temperature is 18-28°C.

[0047] In another preferred embodiment, in step 4), the reaction temperature is 2-8°C.

[0048] In another preferred embodiment, in step 4), the reaction temperature is 20-28°C.

[0049] In another preferred embodiment, in step 1), the reaction time is 1-4 h.

[0050] In another preferred embodiment, in step 3), the reaction time is 1-4 h.

[0051] In another preferred embodiment, in step 4), the reaction time is 1-5 h.

[0052] In another preferred embodiment, in step 5), the reaction time is 2-3 h.

[0053] In another preferred embodiment, in steps 1)-5), the reaction pressure is 100-102 kPa.

[0054] In a second aspect of the present application, a compound selected from the group consisting of:

[0055] In a second aspect of the present application, a compound selected from the group consisting of:

[0056]

[0057] In another preferred embodiment, the compound is prepared according to the method of the first aspect of the application.

[0058] It should be understood that, within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be repeated one by one again. DETAILED DESCRIPTION

[0059] The present inventors have found, through long-term and in-depth research, a method for solid-phase synthesis of the dipeptide side chain of semaglutide. Specifically, the method comprises the following steps: 1) Fmoc-Aib-OH is connected to 2-CTC resin through solid-phase reaction in an alkaline environment to obtain Fmoc-Aib-resin; 2) the Fmoc protecting group of the Fmoc-Aib-resin is removed to obtain Aib-resin; 3) in the presence of a condensing reagent, the Aib-resin is coupled with R-His(R’)-OH to obtain R-His(R’)-Aib-resin; 4) in the presence of a cleaving reagent, the R-His(R’)-Aib-resin is removed from the resin to obtain R-His(R’)-Aib-OH; 5) R-His(R’)-Aib-OH and HR” are reacted to obtain the dipeptide side chain R-His(R’)-Aib-R” of semaglutide. Based on this, the present inventors have completed the present application.

[0060] TERMS

[0061] As used herein, the term “resin substitution degree” refers to the number of active sites on the resin that can be used to connect amino acids. This number is usually expressed in millimoles per gram (mmol / g). The resin substitution degree directly affects the efficiency and yield of polypeptide synthesis.

[0062] As used herein, the term “active ester” refers to a class of organic compounds with active functions. They are formed by the reaction of an acid and an alcohol, usually by the reaction of an acid anhydride and an alcohol. Active esters contain ester groups in their structure, which can react with other compounds through the cleavage of ester bonds.

[0063] As used herein, the term “a plurality of” refers to a positive integer of 2, 3, 4, 5, or greater than 5.

[0064] As used herein, the term “Boc” is tert-butyloxycarbonyl, with the structure

[0065] As used herein, the term “Trt” is trityl, with the structure

[0066] As used herein, the term "Fmoc" is 9-fluorenylmethyloxycarbonyl, with the structure

[0067] As used herein, the term "OC6F5" is pentafluorophenyl ester, with the structure

[0068] As used herein, the term "OSu" is N-hydroxysuccinimidyl ester, with the structure

[0069] As used herein, the term "ONB" is N-hydroxy-5-norbornene-2,3-dicarboximide ester, with the structure

[0070]

[0071] The main advantages of the present application are:

[0072] 1. The method of the present application is simple to operate and easy to scale up compared to the prior art synthesis techniques.

[0073] 2. The dipeptide side chain of semaglutide prepared according to the method of the present application can meet the needs of different semaglutide synthesis processes and has wide market application.

[0074] Concise descriptors

[0075]

[0076]

[0077] Molecular weights of each peptide segment and compound

[0078] Peptide segment Molecular weight (Da) Compound Molecular weight (Da) Fmoc-Aib-OH 325.36 DIEA 129.24 Boc-His(Boc)-OH 355.39 HOSu 115.09 Boc-His(Boc)-Aib-OH 440.51 HOBt 212.25 Boc-His(Trt)-OH 497.58 HOAt 136.11 Boc-His(Trt)-Aib-OH 582.70 DIC 126.20 Trt-His(Trt)-OH 639.78 DCC 206.33 Trt-His(Trt)-Aib-OH 724.90 HONB 179.17 Fmoc-His(Boc)-OH 477.51 Pentafluorophenol 184.06 Fmoc-His(Boc)-Aib-OH 562.53 EDCI 155.24 Fmoc-His(Trt)-OH 619.71 HATU 380.23 Fmoc-His(Trt)-Aib-OH 704.83 TBTU 321.09 Fmoc-His(Fmoc)-OH 599.63 Fmoc-His(Fmoc)-Aib-OH 684.75

[0079] The present application is further described in conjunction with the following specific examples. It should be understood that these examples are merely for the purpose of illustration and are not intended to limit the scope of the present application. The experimental methods in the following examples, unless otherwise specified, were carried out under conventional conditions or under the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

[0080] Example 1: Synthesis of Aib-resin (Step 1 and Step 2)

[0081]

[0082] Take 100 g of 2-CTC resin (1.35 mmol / g), pour into the solid-phase reaction kettle. Take 88 g of Fmoc-Aib-OH into a beaker, add 300 mL of DCM and stir, ice bath for 10 min, then add 69.75 g of DIEA, ice bath for 5 min again, then pour the activated solution into the solid-phase reaction kettle, nitrogen blowing, 27°C reaction for 3h, then add 200 mL of anhydrous methanol to the solid-phase reaction kettle, blow for 30 min, then filter, the resin is washed with 500 mL of DMF for 3 times, filter, get 135 mmol of Fmoc-Aib-resin.

[0083]

[0084] Into the solid-phase reaction kettle, add 135 mmol of Fmoc-Aib-resin and 500 mL of 5% piperazine solution, 27°C reaction for 10 min, filter, the resin is washed with 500 mL of methanol, 600 mL of DCM, 550 mL of DMF respectively for 1 time, filter, then add 500 mL of 5% piperazine solution again, react at 27°C for 20 min, filter, the resin is washed with 550 mL of DMF for 6 times, filter, get 135 mmol of Aib-resin.

[0085] Example 2: Synthesis of Boc-His(Boc)-Aib-OSu (steps 3, 4 and 5)

[0086]

[0087] Take 240 g of Boc-His(Boc)-OH and 54.75 g of HOBt into a beaker and mix evenly, add 250 mL of DMF solution and stir evenly, ice bath for 10 min, then add 341 g of DIC pre-cooled slowly into the beaker and stir evenly, ice bath for 5 min again, pour the activated solution into the solid-phase reaction kettle containing 135 mmol of Aib-resin (prepared according to Example 1), nitrogen blowing, 27°C reaction for 3h. Filter, the peptide resin is washed with 550 mL of DMF for 3 times, filter, then use 500 mL of methanol, 600 mL of DCM, 500 mL of methanol respectively for 2 times, filter, put the peptide resin into the vacuum drying oven and dry to constant weight, then take out and weigh, get 135 mmol of Boc-His(Boc)-Aib-resin.

[0088]

[0089] Weigh 150g Boc-His(Boc)-Aib-resin into the solid-phase reactor, add 1.5L (TFE:DCM=1:4) (volume ratio) of cleavage solution, and react at 25°C for 4h. After the reaction is completed, filter, wash the resin with an appropriate amount of DCM, collect the filtrate and spin dry, then dissolve in 1.5L EA, extract twice with 500mL purified water and once with 500mL saturated brine, dry the organic phase with anhydrous sodium sulfate, spin to a volume of about 250mL, precipitate in 1.75L Hep, filter, wash the filter cake with an appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Boc-His(Boc)-Aib-OH with a purity of ≥95% and a yield of ≥50%.

[0090]

[0091] Add 50g Boc-L-His(Boc)-Aib-OH to the reactor, stir to dissolve in 500mL DCM, then add 15.75g HOSu and stir to dissolve, then add 32.75g EDCI, and react at 25°C for 2-3h. After the reaction is completed, add 175mL purified water to the reaction solution and extract twice, add 175mL saturated brine and extract once, collect the organic phase, dry with anhydrous sodium sulfate, spin dry to obtain an oil, dissolve in 150mL EA, add to 1L Hep to precipitate, filter, wash the filter cake with an appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Boc-His(Boc)-Aib-OSu with a purity of ≥90% and a yield of ≥80%, LC-MS: 538.82.

[0092] Example 3: Synthesis of Boc-His(Trt)-Aib-ONB (steps 3, 4 and 5)

[0093]

[0094] Weigh 81g Boc-His(Trt)-OH and 55.25g HOAt into a beaker and mix evenly, add 250mL DMF solution and stir evenly, ice bath for 10min, then add 341g pre-cooled DIC to the beaker and stir evenly, ice bath for 5min again, pour the activated solution into a solid-phase reactor containing 135mmol Aib-resin (prepared according to Example 1), blow nitrogen, react at 27°C for 3h. Filter, wash the peptide resin with 550mL DMF for 3 times, filter, then wash with 500mL methanol, 600mL DCM and 500mL methanol in sequence, each for 2 times, filter, place the peptide resin in a vacuum drying oven, dry to constant weight, take out and weigh to obtain 135mmol Boc-His(Trt)-Aib-resin.

[0095]

[0096] Weigh 150g Boc-His(Trt)-Aib-resin into the solid-phase reactor, add 750mL (TFE:DCM=1:4) (volume ratio) cleavage solution, and react at 25°C for 4h. After the reaction is completed, filter, wash the resin with appropriate amount of DCM, collect the filtrate and spin dry, then dissolve in 1.5L EA, extract twice with 500mL purified water, and once with 500mL saturated brine. Dry the organic phase with anhydrous sodium sulfate, spin to a volume of about 250mL, precipitate in 1.75L Hep, filter, wash the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Boc-His(Trt)-Aib-OH with purity ≥98% and yield ≥45%.

[0097]

[0098] Add 50g Boc-His(Trt)-Aib-OH into the reactor, add 500mL DCM to stir and dissolve, then add 23.25g HONB to stir and dissolve, and then add 21.75g DIC, and react at 25°C for 2-3h. After the reaction is completed, extract the reaction solution twice with 175mL purified water and once with 175mL saturated brine, collect the organic phase, dry with anhydrous sodium sulfate, spin dry to obtain an oil, dissolve in 150mL EA, add to 1L Hep to precipitate, filter, wash the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Boc-His(Trt)-Aib-ONB with purity ≥95% and yield ≥85%, LC-MS: 607.63.

[0099] Example 4: Synthesis of Trt-His(Trt)-Aib-OC6F5 (steps 3, 4 and 5)

[0100]

[0101] Take 172.75g Trt-His(Trt)-OH and 54.75g HOBt in a beaker and mix evenly, add 500mL DMF solution and stir evenly in ice bath for 10min, then add 77.75g EDCI into the beaker and stir evenly, ice bath for 5min again, pour the activated solution into the solid-phase reactor containing 135mmol of Aib-resin (prepared according to Example 1), nitrogen blowing, reaction at 27℃ for 3h. Filter, wash the peptide resin with 500mL DMF for 3 times, filter, then use 500mL of methanol, 600mL of DCM, 500mL of methanol in turn for 2 times, filter, and then put the peptide resin into the vacuum drying oven and dry to constant weight, then take out and weigh to get 135mmol of Trt-His(Trt)-Aib-resin.

[0102]

[0103] Weigh 150g Trt-His(Trt)-Aib-resin into the solid-phase reactor, add 750mL (HFIP: DCM = 1:4) (volume ratio) of cleavage solution, and react at 5℃ for 4h. After the reaction is completed, filter, wash the resin with appropriate amount of DCM, collect the filtrate and spin dry, then add 1.5L EA to dissolve, extract 2 times with 500mL purified water and 1 time with 500mL saturated brine, dry the organic phase with anhydrous sodium sulfate, spin to a volume of about 250mL, precipitate in 1.75L Hep, filter the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Trt-His(Trt)-Aib-OH, purity ≥98%, yield ≥40%.

[0104]

[0105] Add 50g Trt-His(Trt)-Aib-OH to the reactor, add 500mL DCM and stir to dissolve, then add 15g pentafluorophenol and stir to dissolve, then add 19.5g EDCI and react at 20℃ for 2-3h. After the reaction is completed, add 175mL purified water to the reaction solution and extract 2 times, add 175mL saturated brine and extract 1 time, collect the organic phase, dry with anhydrous sodium sulfate, spin dry to obtain an oil, dissolve in 150mL EA and add to 1L Hep to precipitate, filter the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Trt-His(Trt)-Aib-OC6F5, purity ≥90%, yield ≥85%, LC-MS: 890.87.

[0106] Example 5: Synthesis of Fmoc-His(Boc)-Aib-ONB (steps 3, 4 and 5)

[0107]

[0108] Weigh 193.5g Fmoc-His(Boc)-OH and 54.75g HOBt in a beaker and mix evenly, add 550mL DMF solution and stir evenly, then ice bath for 10min, 154g HATU is added to the beaker and stirred evenly, and ice bath for 5min again, pour the activated solution into the solid-phase reactor containing 135mmol of Aib-resin (prepared according to Example 1), and nitrogen blowing, reaction at 27℃ for 3h. Filter, wash the peptide resin with 500mL DMF for 3 times, filter, then use 500mL of methanol, 600mL of DCM, 500mL of methanol in turn for 2 times, filter, and then put the peptide resin into the vacuum drying oven to dry to constant weight, take out and weigh to get 135mmol of Fmoc-His(Boc)-Aib-resin.

[0109]

[0110] Weigh 150g Fmoc-His(Boc)-Aib-resin into a solid-phase reactor, add 1.5L (TFE:DCM=1:4) (volume ratio) cleavage solution, and react at 25℃ for 4h. After the reaction is completed, filter, wash the resin with appropriate amount of DCM, collect the filtrate and spin dry, then dissolve in 1.5L EA, extract twice with 500mL purified water and once with 500mL saturated brine, dry the organic phase with anhydrous sodium sulfate, spin to a volume of about 125mL, precipitate in 1.75L Hep, filter, wash the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Fmoc-His(Boc)-Aib-OH with purity ≥90% and yield ≥46%.

[0111]

[0112] Add 50g Fmoc-His(Boc)-Aib-OH to the reactor, add 500mL DCM and stir to dissolve, then add 24g HONB and stir to dissolve, then add 34.25g EDCI and react at 25℃ for 2-3h. After the reaction is completed, add 175mL purified water to the reaction solution and extract twice, add 175mL saturated brine and extract once, collect the organic phase, dry with anhydrous sodium sulfate, spin dry to obtain an oil, dissolve in 150mL EA and precipitate in 1L Hep, filter, wash the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Fmoc-His(Boc)-Aib-ONB with purity ≥90% and yield ≥90%, LC-MS: 587.85.

[0113] Example 6: Synthesis of Fmoc-His(Trt)-Aib-OC6F5 (steps 3, 4 and 5)

[0114]

[0115] Weigh 251 g of Fmoc-His(Trt)-OH and 55.25 g of HOAt in a beaker and mix evenly, add 500 mL of DMF solution and stir evenly, after ice bath for 10 min, add 104.75 g of DIEA and 130 g of TBTU to the beaker and stir evenly, ice bath for 5 min again, pour the activated solution into the solid-phase reactor containing 135 mmol of Aib-resin (prepared according to Example 1), nitrogen blowing, reaction at 27°C for 3h. Filter, wash the peptide resin with 550 mL of DMF for 3 times, filter, then use 500 mL of methanol, 600 mL of DCM, 500 mL of methanol in turn, each for 2 times, filter, put the peptide resin into the vacuum drying oven and dry to constant weight, then take out and weigh, get 135 mmol of Fmoc-His(Trt)-Aib-resin.

[0116]

[0117] Weigh 150 g of Fmoc-His(Trt)-Aib-resin into a solid-phase reactor, add 1.5 L of cleavage solution (HFIP: DCM = 1:4) (volume ratio), and react at 5°C for 4h. After the reaction is completed, filter, wash the resin with appropriate amount of DCM, collect the filtrate and spin dry, then add 1.5 L of EA to dissolve, extract twice with 500 mL of purified water and once with 500 mL of saturated brine, dry the organic phase with anhydrous sodium sulfate, spin to a volume of about 250 mL, precipitate in 1.75 L of Hep, filter, wash the filter cake with appropriate amount of Hep, and dry the filter cake in a vacuum drying oven to constant weight to obtain white solid Fmoc-His(Trt)-Aib-OH with purity ≥98% and yield ≥40%.

[0118]

[0119] Into a reaction kettle, 50 g of Fmoc-His(Trt)-Aib-OH was added, and 500 mL of DCM was stirred to dissolve the solution, then 17 g of pentafluorophenol was stirred to dissolve the solution, then 29.25 g of DCC was added, and the reaction was carried out at 20°C for 2-3 h. After the reaction was completed, 175 mL of pure water was added to the reaction solution, and extracted twice with 175 mL of saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, and rotary evaporated to obtain an oil. After dissolving in 150 mL of EA and adding to 1 L of Hep, it was precipitated, filtered, and the filter cake was washed with an appropriate amount of Hep. The filter cake was dried in a vacuum drying oven to constant weight to obtain a white solid Fmoc-His(Trt)-Aib-OC6F5 with a purity of ≥90% and a yield of ≥80%. LC-MS: 872.02.

[0120] Example 7: Synthesis of Fmoc-His(Fmoc)-Aib-OSu (steps 3, 4 and 5)

[0121]

[0122] Into a beaker, 243 g of Fmoc-His(Fmoc)-OH and 54.75 g of HOBt were weighed and mixed uniformly, and 500 mL of DMF solution was added and stirred uniformly in an ice bath for 10 min. 111.5 g of DCC was added to the beaker and stirred uniformly, and the activated solution was poured into a solid-phase reaction kettle containing 135 mmol of Aib-resin (prepared according to Example 1) under nitrogen blowing. The reaction was carried out at 27°C for 3 h. After filtration, the peptide resin was washed with 500 mL of DMF for 3 times, and then filtered. The peptide resin was sequentially washed with 500 mL of methanol, 600 mL of DCM, and 500 mL of methanol for 2 times each, and then filtered. After the peptide resin was placed in a vacuum drying oven and dried to constant weight, it was taken out and weighed to obtain 135 mmol of Fmoc-His(Fmoc)-Aib-resin.

[0123]

[0124] Into a solid-phase reaction kettle, 150 g of Fmoc-His(Fmoc-Aib-resin was added, and 1.5 L of 5% TFA in DCM was added to cleave the solution at -5°C for 2 h. After the reaction was completed, the solution was filtered, and the filter cake was washed with an appropriate amount of DCM. The filtrate was collected and rotary evaporated, and then dissolved in 1.5 L of EA. The solution was extracted twice with 500 mL of purified water and once with 500 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate and rotary evaporated to a volume of about 250 mL. The solution was precipitated in 1.75 L of Hep, filtered, and the filter cake was washed with an appropriate amount of Hep. The filter cake was dried in a vacuum drying oven to constant weight to obtain a white solid Fmoc-His(Fmoc)-Aib-OH with a purity of ≥90% and a yield of ≥45%.

[0125]

[0126] Into a reaction kettle, 50 g of Fmoc-His(Boc)-Aib-OH was added, 500 mL of DCM was added to stir and dissolve, then 10.25 g of HOSu was added to stir and dissolve, then 22.75 g of DCC was added, and the reaction was carried out at 25°C for 2-3 h. After the reaction was completed, 175 mL of pure water was added to the reaction solution, extracted twice, 175 mL of saturated brine was added to extract once, the organic phase was collected, dried over anhydrous sodium sulfate, and rotary evaporated to obtain an oil. After being dissolved in 150 mL of EA and added to 1 L of Hep to precipitate, the filter cake was washed with an appropriate amount of Hep after filtration, and the filter cake was dried in a vacuum drying oven to constant weight to obtain white solid Fmoc-His(Fmoc)-Aib-OSu, purity ≥ 85%, yield ≥ 85%, LC-MS: 782.93.

[0127] Example 8: Preparation of semaglutide 29+2 peptide using Fmoc-His(Trt)-Aib-OC6F5 as raw material

[0128] Into a reaction kettle, 10 g of semaglutide main chain 29 peptide was added, 500 mL of DMF:H2O = 1:1 mixed solvent was added to stir and dissolve, then 1 mL of DIEA was added to stir and dissolve, then 5.32 g of Fmoc-His(Trt)-Aib-OC6F5 prepared according to Example 6 of the present application was added to stir and dissolve, and the reaction was carried out at 25°C for 2-3 h. After the reaction was completed, the reaction solution was adjusted to pH = 4-5 with 5M phosphoric acid, and then centrifuged. The solid was washed with 50 mL of EA three times, and the filter cake was dried in a vacuum drying oven to constant weight to obtain semaglutide 29+2 peptide, purity ≥ 90%, yield ≥ 88%.

[0129] 29+2 peptide is a key raw material for synthesizing semaglutide. The 29+2 peptide is further connected to the side chain of semaglutide and then deprotected to obtain semaglutide.

[0130] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is individually incorporated by reference. In addition, it should be understood that, after reading the above teachings of the present application, those skilled in the art can make various modifications or improvements to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A method for solid-phase synthesis of smegglutinin dipeptide side chains, characterized in that, Includes the following steps: 1) Under alkaline conditions, Fmoc-Aib-OH of formula (I) and 2-CTC resin undergo a resin grafting reaction to obtain Fmoc-Aib-resin of formula (II). 2) Remove Fmoc from the Fmoc-Aib-resin obtained in step 1) to obtain the Aib-resin shown in formula (III); 3) In the presence of a condensing agent, the Aib-resin obtained in step 2) undergoes a coupling reaction with R-His(R')-OH as shown in formula (IV) to obtain R-His(R')-Aib-resin as shown in formula (V). 4) In the presence of a pyrolysis reagent, the R-His(R')-Aib- resin obtained in step 3) is deresinated to obtain R-His(R')-Aib-OH as shown in formula (VI); 5) The R-His(R')-Aib-OH obtained in step 4) reacts with HR” to obtain the smegglutinin dipeptide side chain R-His(R')-Aib-R” shown in formula (VII). Wherein, R and R' are each independently selected from the following group: Boc Trt Fmoc Amino protecting group; The “R” is selected from the following group:

2. The method according to claim 1, characterized in that, In step 1), the alkaline conditions are provided by a reagent selected from the group consisting of Et3N, DIEA, or a combination thereof.

3. The method according to claim 1, characterized in that, In step 1), the degree of substitution of the 2-CTC resin is 0.9-1.8 mmol / g; preferably 1.1-1.5 mmol / g.

4. The method according to claim 1, characterized in that, In step 2), the reaction conditions are selected from the following group: 5% piperazine, 20% piperidine.

5. The method according to claim 1, characterized in that, In step 3), the condensing agent is selected from the following group: a. DIC, DCC, EDCI, HATU, DIEA, and TBTU; and b.HOBt or HOAt.

6. The method according to claim 1, characterized in that, In step 4), the lysis reagent is selected from the following group: A.TFE, TFA, or HFIP; and B.DCM.

7. The method according to claim 6, characterized in that, The volume ratio of reagent A to reagent B is 1:(2-6); preferably 1:(3-5).

8. The method according to claim 1, characterized in that, The method also includes one or more of the following features: i. In step 1), the molar ratio of Fmoc-Aib-OH to 2-CTC resin is (1-5):1; preferably (1-3):

1. ii. In step 3), the molar ratio of R-His(R')-OH to the Aib-resin is (1-10):1; preferably (1-7):1; iii. In step 4), the mass-to-volume ratio of the R-His(R')-Aib-resin to the lysis reagent is 0.05–0.4 g / ml; preferably 0.05–0.3 g / ml; iv. In step 5), the molar ratio of R-His(R')-Aib-OH to HR” is 1:(1-3); preferably 1:(1-2).

9. The method according to claim 1, characterized in that, The method also includes one or more of the following features: (1) In step 1), the temperature for the resin reaction is 25-30℃; (2) In step 2), the reaction temperature is 25-30℃; (3) In step 3), the temperature of the coupling reaction is 25-30℃; (4) In step 4), the reaction temperature is -2 to 28℃; (5) In step 5), the reaction temperature is 18-28℃.

10. A compound selected from the group consisting of:

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