WO2015100876A1 - Method for preparing liraglutide - Google Patents

Method for preparing liraglutide Download PDF

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WO2015100876A1
WO2015100876A1 PCT/CN2014/075113 CN2014075113W WO2015100876A1 WO 2015100876 A1 WO2015100876 A1 WO 2015100876A1 CN 2014075113 W CN2014075113 W CN 2014075113W WO 2015100876 A1 WO2015100876 A1 WO 2015100876A1
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Prior art keywords
fmoc
oh
resin
otbu
glu
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PCT/CN2014/075113
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French (fr)
Chinese (zh)
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路杨
杨东晖
方晨
周亮
刘少华
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杭州阿德莱诺泰制药技术有限公司
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Priority to CN201410001671.XA priority patent/CN103980358B/en
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Publication of WO2015100876A1 publication Critical patent/WO2015100876A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/605Glucagons

Abstract

Disclosed is a method for synthesizing liraglutide. The method comprises the specific steps: A) synthesizing a segment Fmoc-Lys-(Glu(Nα-Palmitoyl)-OtBu)-OH by using a liquid phase manner; B) coupling a resin solid-phase carrier and Fmoc-Gly-OH, so as to obtain Fmoc-Gly-resin; C) sequentially coupling amino acids with N-end Fmoc protection and side chain protection according to a liraglutide main chain peptide sequence by using a solid-phase synthesis method, a lysine tripeptide segment using Fmoc-Lys-(Glu(Nα-Palmitoyl)-OtBu)-OH; and D) performing splitting, purification and freeze-drying to obtain liraglutide. The method needs a short synthesis period and low cost, produces high yield, and is suitable for large-scale production.

Description

 Method for preparing liraglutide

 The present invention relates to a method for preparing a polypeptide drug, which is a synthetic therapeutic agent for long-acting type II diabetes mellitus having a glucagon-like peptide-1 (GLP-1) receptor agonist-liuraglutide Preparation method. Background technique

 Liraglutide, English name: Liraglutide, the structural formula is as follows:

Figure imgf000003_0001
The monthly sequence is:

 H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Al a-Ala-Lys(Ns-(Na-Palmitoyl- Ly-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gl y-Arg-Gly-OH

Liraglutide is the first and only long-acting human GLP-1 analogue developed by Novo Nordisk of Denmark. It has a GLP-1 receptor agonist function in molecular structure, biological activity, and action. Targets and immunogenicity are similar to GLP-1. The molecular structure of liraglutide is 97% homologous to GLP-1 (7-37). The structural difference is that Lys 34 is replaced by Arg. Lys 26 undergoes palmitoylation via glutamate, fatty acid side chains. It can make liraglutide reversibly bind to albumin in the blood, prolong the action time of liraglutide, and enhance the resistance to DPP-4 enzyme degradation. The fatty acid side chain can also make The liraglutide molecule self-crosslinks into a heptamer at the injection site, thereby delaying its subcutaneous attraction, allowing it to last for up to 24 hours, once a day and at any time, with a low risk of hypoglycemia. . In addition, this product can also reduce the secretion of glucagon in a blood glucose-dependent manner and delay gastric emptying.

Novo Nordisk's liraglutide is prepared by biological methods such as genetic engineering, which is technically difficult and has high production cost, which is not conducive to large-scale production of liraglutide. US6268343B1 and US6458924B2 reported the synthesis of solid-liquid liraglutide, intermediate GLP-l (7-37) -OH require purified by Reverse Phase HPLC, then under liquid phase conditions with N a -Palmitoyl-Glu (OSu) -OtBu reaction, this method requires two purifications, a long synthesis cycle, a large amount of waste liquid, and is expensive, which is disadvantageous for the disadvantage of mass production.

 WO2013037266A1 discloses a preparation method of liraglutide, the specific steps are: sequentially, by Fmoc solid phase synthesis, according to the liraglutide main chain peptide sequence, an amino acid having N-terminal Fmoc protection and side chain protection, wherein For the guanidine, Fmoc-Lys (Alloc)-OH was used to remove Alloc, and Palmitoyl-Glu-Offiu was coupled to the amino group of the lysine side chain by solid phase synthesis, and the product was obtained after cleavage. This method, due to the use of tetrakis(triphenylphosphine)palladium to remove Alloc, not only makes the cost high, is not conducive to large-scale production, but also causes the metal residue to lead to excessive levels of heavy metals, resulting in low product quality and content.

 In summary, in the solid phase synthesis process of the existing liraglutide, due to the long synthesis cycle, high cost, low yield, and many impurities, it is not suitable for industrial production. Summary of the invention

 The present inventors have prepared liraglutide by the existing synthesis method, and found that the technical problems existing in the prior art are: more synthetic steps, long synthesis cycle, low purity and yield, and unsuitable for industrial scale production. For this reason, the inventors have studied the synthesis method of liraglutide, thereby obtaining the technical scheme of the present invention.

It is an object of the present invention to provide a solid phase synthesis method of liraglutide. The synthetic route of the present invention is shown in Figure 1: First, the lysine tripeptide fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH is synthesized by a liquid phase method, followed by the presence of an activator system. The Fmoc-Gly-resin is obtained by coupling the resin solid phase carrier and Fmoc-Gly-OH, and then the N-terminal Fmoc-protected and side-chain protected amino acid is sequentially coupled by the solid phase synthesis method according to the liraglutide main chain peptide sequence. , wherein the lysine tripeptide fragment is lysed and purified by Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH, Lyophilized to obtain liraglutide, and the amino acid sequence of the liraglutide is shown in the Sequence Listing (SEQ No. 1). Some commonly used abbreviations in the present invention have the following meanings;

 Fmoc : 芴曱oxycarbonyl

 Fmoc-AA: Aminocarbonyl protected amino acid

 DIC : N, Ν'-diisopropylcarbodiimide

 DCC : Ν, Ν'-dicyclohexylcarbodiimide

 PyBOP : benzotriazole hexafluorophosphate-1-yl-oxytripyrrolidinyl phosphorus

 HATU : 2-(7-azobenzotriazole) -Ν,Ν,Ν',Ν'-tetradecylurea hexafluorophosphate

 HOBt : 1-hydroxybenzotriazole

 HOSu : N-hydroxysuccinimide

tBu : tert-butyl

 Trt : triphenyl fluorenyl

 Boc : Tert-butyloxy

 Palmitoyl : Palmitoyl

 Pbf : 2,2,4,6,7-pentamethyldihydrobenzofuran -5-sulfonyl

 Tyr : Tyrosine

Lie : isoleucine

 Gin : Glutamine

 Asn : Asparagine

 Cys : Cysteine

 Pro : Proline

 Leu : Leucine

 Gly : Glycine

 Arg : arginine

 Val : Proline

 Trp : tryptophan

 Ala : Alanine

 Phe : Phenylalanine

Glu : glutamic acid Lys : Lysine

Ser : Serine

Asp : Aspartic acid

Thr : Threonine

His : histidine

 DMF : N, Ν'-dimercaptoamide

 MeOH : sterol

 DCM : Dichlorodecane

 NMP : N-decylpyrrolidone

 DMSO : Dimercaptosulfoxide

 TFA: trifluoroacetic acid

 EDT : Ethylene

Piperidine: hexahydro p ratio

 DMAP : 4-diaminopyridine

 DIEA : N, Ν'-diisopropylethylamine

 ΤΜΡ : 2,4,6-tridecylpyridine.

 ΝΜΜ: Ν-mercaptomorpholine

 2-CTC: 2-chlorotriphenylphosphonium chloride

 To this end, the present invention provides a method for synthesizing liraglutide, the steps of which are as follows:

 Step 1 : synthesizing a lysine tripeptide fragment by a liquid phase method

Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH;

 Step 2: Fmoc-Gly-resin is obtained by coupling a resin solid phase carrier and Fmoc-Gly-OH in the presence of an activator system;

Step 3, by solid phase synthesis, according to the liraglutide main chain peptide sequence, the amino acid having N-terminal Fmoc protection and side chain protection is sequentially coupled, wherein the lysine tripeptide fragment is Fmoc-Lys-(Glu(N) a -Palmitoyl)-OtBu)-OH;

 Step 4, cleavage, purification, and lyophilization to obtain liraglutide.

Wherein, in the solid phase synthesis method described in the step 1, the liquid phase synthesis of the fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH is: n-hexadecanoic acid, HOSu, Coupling with Palmitoyl-OSu activated ester by DCC, then reacting with H-Glu-OtBu to obtain the dipeptide fragment Palmitoyl-Glu-OtBu; Palmitoyl-Glu-OtBu, HOSu, DCC coupling to obtain Palmitoyl-Glu(OSu)-OtBu activated ester Then, it is reacted with Fmoc-Lys-OH to obtain a lysine tripeptide fragment Fmoc-Ly s-(Glu(N a -Palmitoyl)-OtBu)-OH.

 The solid phase synthesis method according to the second step, wherein the resin solid phase carrier is made of 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-Gly-resin is 0.10-0.35 mmol. /g substitution degree Fmoc-Gly-CTC resin.

 The solid phase synthesis method according to the second step, wherein the resin solid phase carrier is made of a king resin, the activator system is composed of DIC, HOBt and DMAP, and the Fmoc-Gly-king resin is 0.10 to 0.35 mmol/ Fmoc-Gly-king resin with degree of g substitution.

 In the present invention, the degree of substitution of the resin is the degree of substitution of the resin by ultraviolet spectrophotometry, and the Fmoc protecting group on the resin coupled with the Fmoc-protected amino acid is deprotected with a 20% piperidine/DMF solution. , the concentration is determined by ultraviolet spectrophotometry, and then the Fmoc molar value of the resin is calibrated with an amino acid standard compound containing Fmoc, such as Fmoc-Leu-OH, and the resin is used to determine the degree of substitution of the resin. Call it the degree of substitution.

 Wherein the solid phase synthesis method described in step 3,

 1) removing the Fmoc protecting group on the Fmoc-Gly-resin using a deprotecting solution consisting of piperidine and DMF in a volume ratio of 1:4 to obtain an H-Gly-resin;

 2) H-Gly-resin and Fmoc-protected and side-chain-protected arginine are coupled to obtain Fmoc-Arg(Pbf)-Gly-resin in the presence of a coupling agent system;

3) Repeat steps 1), 2), and sequentially perform amino acid coupling according to the liraglutide main chain peptide sequence, wherein lysine oxime is Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH, The amino acid sequence is: Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc- Ile-OH , Fmoc-Phe-OH , Fmoc-Glu(OtBu)-OH, Fmoc- Lys-(Glu(N a -Palmitoyl)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc -Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu ) -OH , Fmoc-Gly-OH , Fmoc-Glu(OtBu)-OH , Fmoc-Ala -OH, Boc-His(Trt)-OH;

 The coupling agent system comprises a condensing agent selected from the group consisting of DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA; and the reaction solvent is selected from DMF, DCM, NMP, DMSO or they Any combination between.

 Preferably, in step 3), during the amino acid coupling process, wherein when the condensing agent is selected

When HATU/HOBt/DIEA,

H-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin (hereinafter referred to as AA-resin): Fmoc-Lys-( The molar ratio of Glu(N a -Palmitoyl)-OtBu)-OH:HATU:HOBt:DIEA is preferably: 1:3:3:3:3-1:5:5:5:5, ie the Fmoc-Lys - (Glu(N a -Palmitoyl)-OtBu)-OH and the condensing agent HATU/HOBt/DIEA are equal in number of moles, and their respective molar ratios to the AA resin are 3/1 to 5/1. The reaction temperature is 25 to 35 ° C, and the reaction time is 2 to 3 hours; more preferably, the molar ratio of each of them is 5/1 with respect to the AA resin, the reaction temperature is 35 ° C, and the reaction time is 2 hours.

 The method of the present invention is obtained by screening, and the screening process is as follows:

1) Choice of molar ratio:

H-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-tree

The purpose of the month: Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH:HATU:HOBt:DIEA molar ratio is:

1:3:3:3:3 and 1:5:5:5:5;

 2) Selection of reaction temperature:

25 ° C and 35 0 C;

3) Choice of reaction time:

 2 hours and 3 hours.

 Eight experimental conditions were proposed for this purpose:

Experimental conditions 1 : Take 3.43g

 H-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-resin

(1.0 mmol), 2.16 g Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH (3.0 mmol), 0.41 g HOBt (3.0 mmol) and 1.14 g HATU (3.0 mmol) were added to 20 ml DMF and stirred to dissolve. Cool to 0 ° C, add 0.5 ml DIEA (3.0 mmol) to the above solution, react at 25 ° C for 2 hours, and then couple the remaining amino acids in sequence. The amino acid sequence is: Fmoc-Ala-OH, Fmoc -Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr( tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH, cleavage, purification, lyophilization, to obtain liraglutide pedipeptide;

 Experimental conditions 2-8, experimental operations as shown in experimental conditions 1, different experimental conditions and their experimental results are shown in Table 1 below:

 Table 1

Figure imgf000009_0001

 The above results show that the purification effect of Experimental Condition 4 is optimal.

 The method of the invention has obvious advantages compared with the prior art, and the relevant comparative experiments are as shown in Table 2 below.

Figure imgf000009_0002
The beneficial effects of the invention are as follows: The direct solid phase synthesis of liraglutide by using the fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH solves the long synthesis cycle, high cost and purity of the prior art. Low, high impurity, not suitable for industrial production; The present invention provides a synthesis process of liraglutide which is short in synthesis cycle, low in cost and high in yield and suitable for large-scale production.

DRAWINGS

Figure 1 is a synthetic route of the liraglutide of the present invention;

Figure 2 HPLC chromatogram of a lysine tripeptide fragment;

Figure 3 is a HPLC chromatogram of the crude peptide of liraglutide;

Figure 4 HPLC chromatogram of liraglutide spermatine;

Figure 5 is a morphological language map of a lysine tripeptide fragment;

Figure 6 is a thirteenth diagram of liraglutide peptidic peptide. Detailed ways:

The invention is further illustrated by the following examples.

 Specifically, regarding the respective commercially available amino acids and amino acid fragments involved in the following examples, and each commercially available resin, the manufacturer and model number are as follows:

Fmoc protecting group amino acid raw materials, 2-CTC resin and Wang resin are all conventional commercial reagents (manufacturer: Jill Biochemical (Shanghai) Co., Ltd.; chemically pure); lysine tripeptide fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH is synthesized as described in this patent.

 The sources of organic solvents and other raw materials are commercially available (manufacturer: Sinopharm Chemical Reagent Co., Ltd.; chemically pure).

 In addition, the conditions of "spin evaporation" and "freeze-drying" as mentioned in the examples below, as well as the conditions for determining HPLC and mass spectrometry, and the type of equipment used and the manufacturer's description are as follows:

 Rotary distillation equipment: Rotary evaporator R-200/205 (Switzerland Buchi);

 Condensation and concentration conditions: under vacuum (-O.lMpa) at 30 ° C, the mixture is concentrated by steaming, and the volume after concentration is 75% or less before the steaming.

 Freeze-drying equipment: Freeze-drying machine FD-3 (Beijing Bo Yikang Experimental Instrument Co., Ltd.);

Freeze-drying conditions: Place the lyophilized tray in the freezer (-20 °C) and pre-freeze for 6 h. Turn on the freeze dryer, turn on the refrigeration, pre-cool for more than 30 min, and set the freeze-drying curve as follows: First stage: 16 h at -27 °C; second stage: 4 h at -5 °C; third stage: 2 h at 5 °C; fourth stage: 16 h at 30 °C.

 HPLC: Dionex high performance liquid chromatography; using octadecylsilane bonded silica gel (5μπι, 250 4.6mm) as a filler; 0.1% TFA solution as mobile phase A, gradient elution with acetonitrile as mobile phase; It is 1.0 ml per minute; the detection wavelength is 220 nm; the column temperature is 30 °C. Take 20 μl of the test solution and inject it into the liquid chromatograph to record the chromatogram.

 Mass spectrometry: MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry; instrument model is AUTO FLEX SPEED TOF-TOF. Example 1: Synthesis of Palmitoyl-OSu activated ester

 Weigh 256.42 g of n-hexadecanoic acid (1.0 mol), add 138.10 g of HOSu (1.2 mol) to 2000 ml of THF, and add 247.56 g of DCC (1.2 mol) under ice water bath, react for 1 hour, and warm to room temperature for 3 hours. The reaction solution was filtered, the mother liquor was spun dry, dissolved in DCM, filtered, washed with saturated sodium bicarbonate 3 times, purified water 2 times, stripped 2 times, combined organic phase, dried anhydrous sodium carbonate, spin dried, recrystallized from ice ethanol 3 The filter was filtered and the solid oil pump was dried to 314.62 g of Palmitoyl-OSu activated ester in a yield of 89%.

Example 2: Synthesis of Palmitoyl-Glu-OtBu

101.62 g of H-Glu-OtBu (0.5 mol) and 79.50 g of Na 2 C0 3 (0.75 mol) were weighed and dissolved in a mixed solution of 500 ml of water and 500 ml of THF, and 176.75 g of Palmitoyl-OSu (0.5 mol) was weighed and added. After dissolving, 500 ml of THF was added dropwise to the above mixed solution, and the reaction was allowed to proceed overnight at room temperature, and the pH was adjusted to 7 with 10% diluted hydrochloric acid, and then THF was evaporated to remove THF. A large amount of white precipitate was obtained and filtered. The resulting white precipitate was recrystallized from iced ethanol. The solid oil pump was dried to 192. llg Palmitoyl-Glu-OtBu, yield 87%.

Example 3: Synthesis of Palmitoyl-Glu(OSu)-OtBu

Weigh 88.33g of Palmitoyl-Glu-OtBu (0.2mol), 27.62g of HOSu (0.24mol), add it to 1000ml of THF, add 49.51g of DCC (0.24mol) under ice water bath, react for 1 hour, and warm to room temperature for 3 hours. Liquid filtration, mother liquor spin dry, add DCM to dissolve, filter, wash with saturated sodium bicarbonate 3 times, 2 times of pure water, stripping 2 times, combine organic phase, dry anhydrous sodium carbonate, spin dry, recrystallize ice ethanol 3 times , filtered, solid oil pump was pulled dry to 94.81 g of Palmitoyl-Glu(OSu)-OtBu activated ester, yield 88%. Example 4: Synthesis of Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH

36.74 g of Fmoc-Lys-OH (0.1 mol) and 15.90 g of Na 2 C0 3 (0.15 mol) were weighed and dissolved in a mixed solution of 100 ml of water and 100 ml of THF, and 53.87 g of Palmitoyl-Glu(OSu)-OtBu was weighed. (O.lmol) was added to 100 ml of THF, dissolved, and the above mixed solution was added dropwise thereto, and the reaction was allowed to proceed overnight at room temperature, and the pH was adjusted to 7 with 10% diluted hydrochloric acid, and then THF was removed by rotary evaporation, and then pH was adjusted to 3. A large amount of white precipitate was obtained and filtered. The resulting white precipitate was recrystallized from iced ethanol. The solid oil pump was dried to 67.24g Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH, and its HPLC spectrum is shown in Figure 2. The HPLC purity is 97.40%, the yield is 85%; As shown in Figure 5, [M+Na] + : 814.555, [M+K] + : 830.605, the theoretical precision of the lysine tripeptide fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH The molecular weight is: 791.5, and the mass spectrometry results of the sample are consistent with the theoretical molecular weight and the structure is correct.

Example 5: Synthesis of Fmoc-Gly-CTC resin with a degree of substitution of 0.10 mmol/g

 20 g of 2-CTC resin having a degree of substitution of 0.40 mmol/g was weighed, added to a solid phase reaction column, washed once with DMF, and swelled with DMF for 30 minutes, and then 13.37 g of Fmoc-Gly-OH (45 mmol) was used. The DMF was dissolved, and 7.5 ml of DIEA (45 mmol) was added to the ice water bath to be activated, and then added to the above reaction column containing the resin. After reacting for 2 hours, 100 ml of anhydrous methanol was added and blocked for 1 hour. The mixture was washed 3 times with DMF, washed 3 times with DCM, blocked with anhydrous decyl alcohol for 30 minutes, and sterol was shrink-dried to obtain 22.34 g of Fmoc-Gly-CTC resin, and the detection substitution was 0.10 mmol/g.

Example 6: Synthesis of Fmoc-Gly-CTC resin with a degree of substitution of 0.25 mmol/g

 10 g of 2-CTC resin having a degree of substitution of 0.95 mmol/g was weighed, added to a solid phase reaction column, washed once with DMF, and swollen with DMF for 30 minutes, and then taken with 14.11 g of Fmoc-Gly-OH (47 mmol). The DMF was dissolved, and 8.0 ml of DIEA (47 mmol) was added to the ice water bath to be activated, and then added to the above reaction column containing the resin. After reacting for 2 hours, 100 ml of anhydrous methanol was added and blocked for 1 hour. It was washed 3 times with DMF, washed 3 times with DCM, blocked with anhydrous decyl alcohol for 30 minutes, and sterol was shrink-dried to obtain Fmoc-Gly-CTC resin, and the detection substitution was 0.25 mmol/g.

Example 7: Synthesis of Fmoc-Gly-King Resin with a degree of substitution of 0.10 mmol/g

20 g of king resin having a degree of substitution of 0.45 mmol/g was weighed, added to a solid phase reaction column, washed once with DMF, and swelled with DMF for 30 minutes, and then 13.37 g of Fmoc-Gly-OH (45 mmol), 6.01 g. HOBt (45 mmol) was dissolved in DMF, activated by adding 7.0 ml of DIC (45 mmol) in an ice water bath, and then added to the reaction column containing the resin. After 5 minutes, 2.75 g of DMAP (22.5 mmol) was added, and after reacting for 2 hours, It was washed 3 times with DMF, washed 3 times with DCM, and capped with 100 ml of acetic anhydride/pyridine overnight, and the sterol was shrink-dried to obtain Fmoc-Gly-King resin, and the detection substitution was 0.10 mmol/g.

Example 8: Synthesis of Fmoc-Gly-King Resin with a Degree of Substitution of 0.25 mmol/g

 20 g of king resin having a degree of substitution of 0.75 mmol/g was weighed, added to a solid phase reaction column, washed once with DMF, and swollen with DMF for 30 minutes, and then 22.28 g of Fmoc-Gly-OH (75 mmol), 10.13 g was taken. HOBt (75 mmol) was dissolved in DMF, activated by adding 11.6 ml of DIC (75 mmol) in an ice water bath, and added to the reaction column containing the resin. After 5 minutes, 4.5 g of DMAP (37.5 mmol) was added, and after 2 hours of reaction, DMF was used. After washing 3 times, DCM was washed 3 times, capped with 100 ml of acetic anhydride/pyridine overnight, and sterol was shrink-dried to obtain 22.54 g of Fmoc-Gly-King resin, and the detection substitution was 0.25 mmol/g.

Example 9: Preparation of liraglutide CTC resin

Weigh 4.46 g of Fmoc-Gly-CTC resin (1 mmol) with a degree of substitution of 0.10 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-Gly-CTC resin with DMF for 30 minutes, then use DMF. The mixed solution having a pyridine volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3.24 g of Fmoc-Arg(Pbf)-OH (5 mmol) and 0.68 g of HOBt (5 mmol) were weighed and added to a volume ratio of 1. :1 mixed solution of DCM and DMF, activated by adding 0.8 ml of DIC (5 mmol) in an ice water bath, added to the reaction column containing the resin, and reacted at room temperature for 2 hours, and then judged by the ninhydrin method. If the resin is colorless and transparent, it means the reaction is complete; if the resin develops color, it means the reaction is incomplete and needs to be reacted for another hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, according to the liraglutide main chain peptide sequence, sequentially completing Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys-(Glu(N a - Palmitoyl)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH , Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Coupling of Trt)-OH. When Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is changed to: DMSO and DMF mixed solution with a volume ratio of 1:4; Fmoc-Asp(OtBu)-OH is coupled with the coupling reagent: PyBOP/HOBt/DIEA; The coupling reagent for Boc-His(Trt)-OH coupling was changed to: HATU/HOBt/DIEA. After coupling, the liraglutide CTC resin was washed 3 times with DMF, washed 3 times with DCM, and washed 3 times with MeOH. The DCM was washed 3 times, washed with MeOH 3 times, and dried to give 9.67 g of liraglutide CTC resin.

Example 10: Preparation of Liraglutide King Resin

Weigh 4.57 g (lmmol) of Fmoc-Gly-Wang resin with a degree of substitution of 0.10 mmol/g, add to the solid phase reaction column, wash once with DMF, and swell Fmoc-Gly-Wang resin with DMF for 30 minutes, then use DMF. The mixed solution having a pyridine volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3.24 g of Fmoc-Arg(Pbf)-OH (5 mmol) and 0.68 g of HOBt (5 mmol) were weighed and added to a volume ratio of 1. :1 mixture of DCM and DMF, activated by adding 0.8 ml of DIC (5 mmol) in an ice water bath, and then added to the reaction column containing the resin, and reacted at room temperature for 2 hours, and then judged by the ninhydrin method. If the resin is colorless and transparent, it means the reaction is complete; if the resin develops color, it means the reaction is incomplete and needs to be reacted for another hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, according to the liraglutide main chain peptide sequence, sequentially completing Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys-(Glu(N a - Palmitoyl)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH , Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Coupling of Trt)-OH.

 In the case where Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is replaced by: a mixed solution of DMSO and DMF in a volume ratio of 1:4; and the coupling reagent of Fmoc-Asp(OtBu)-OH is converted to: PyBOP/HOBt/DIEA; Boc-His(Trt)-OH coupling reagent was changed to: HATU/HOBt/DIEA, after coupling, the liraglutide resin was washed 3 times with DMF and 3 times with DCM. The MeOH was washed 3 times, DCM was washed 3 times, MeOH was washed 3 times, and dried to give 9.78 g of liraglutide resin. Example 11: Large-scale preparation of liraglutide resin

Weigh 4570g (lmol) of Fmoc-Gly-Wang resin with a degree of substitution of 0.10mmol/g, add it to the solid phase reaction column, wash it once with DMF, and swell Fmoc-Gly-Wang resin with DMF for 30 minutes. DMF: The mixed solution with a pyridine volume ratio of 4:1 was deprotected by Fmoc, and then washed 6 times with DMF, and 3240 g of Fmoc-Arg(Pbf)-OH (5 mol) and 682 g of HOBt (5 mol) were weighed to a volume ratio of 1: The mixed solution of DCM and DMF of 1 is activated by adding 800 ml of DIC (5 mol) in an ice water bath, and then added to the reaction column containing the resin, and reacted at room temperature for 2 'J, and then the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, it means the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. Repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, according to the liraglutide main chain peptide sequence, sequentially completing Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Lys-(Glu(N a - Palmitoyl)-OtBu)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH , Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His( Coupling of Trt)-OH. When Fmoc-Leu-OH and Fmoc-Phe-OH are coupled, the solvent is changed to: DMSO and DMF mixed solution with a volume ratio of 1:4; Fmoc-Asp(OtBu)-OH is coupled with the coupling reagent: PyBOP/HOBt/DIEA; Boc-His(Trt)-OH coupling reagent was changed to: HATU/HOBt/DIEA, after coupling, the liraglutide resin was washed 3 times with DMF and 3 times with DCM. The MeOH was washed 3 times, DCM was washed 3 times, MeOH was washed 3 times, and dried to obtain 9975 g of liraglutide resin.

Example 12: Preparation of Liraglutide Crude Peptide

 Weigh 100g of fully protected liraglutide CTC resin or liraglutide resin, and add it to a 1000mL three-neck round bottom flask, according to TFA: phenyl sulfonyl ether: benzoquinone: EDT=90: 5: 3: The volume ratio of 2 is 10 L of the lysate, the lysate is added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin is washed 3 times with a small amount of TFA, the filtrate is combined, concentrated, and the concentrated liquid is added to ice diethyl ether. The mixture was precipitated for 1 hour, centrifuged, washed with diethyl ether for 6 times, and dried under vacuum to give 34.13 g of crude liraglutide. The HPLC chromatogram is shown in Fig. 3, the HPLC purity was 83.03%, and the crude peptide yield was 78%.

Example 13: Preparation of liraglutide spermine acetate

Weigh 3413g of liraglutide crude peptide dissolved in 30% mixed solution of 50% acetonitrile + 50% water, then pass The target product was obtained after 2 purifications on a C18 or C8 column, salt transfer, and lyophilization. First purification conditions: The mobile phase is: Phase A: 0.1% TFA; Phase B: Acetonitrile, detection wavelength 220 nm, collection of target peaks. Second purification conditions: The mobile phase is: Phase A: 0.3% acetic acid; Phase B: acetonitrile. The detection wavelength was 220 nm, and the target peaks were collected. Salt transfer conditions: Mobile phase: Phase A: 20 mM ammonium acetate-water solution; Phase B: acetonitrile; detection wavelength 220 nm. The target peaks were collected, concentrated by steaming, and lyophilized to obtain 11.24 g of liraglutide acetate peptide. The HPLC diagram is shown in Figure 4. The HPLC purity was 99.75%, and the total purification yield was 40%. 31%. The mass spectrum is shown in Figure 6, [M] + : 3751.848, the theoretical exact molecular weight of liraglutide is: 3751.2, and the mass spectrometry results of the sample are consistent with the theoretical molecular weight. The above is a further detailed description of the present invention in connection with specific alternative embodiments, and it is not intended that the specific embodiments of the invention are limited to the description. For those skilled in the art to which the present invention pertains, a number of simple derivations or substitutions may be made without departing from the inventive concept.

Claims

Claim
A method for synthesizing liraglutide, the method steps being as follows:
Step 1, synthesizing a lysine tripeptide fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH by a liquid phase method;
 Step 2: Fmoc-Gly-resin is obtained by coupling a resin solid phase carrier and Fmoc-Gly-OH in the presence of an activator system;
Step 3, by solid phase synthesis, according to the liraglutide main chain peptide sequence, the amino acid having N-terminal Fmoc protection and side chain protection is sequentially coupled, wherein the lysine tripeptide fragment is Fmoc-Lys-(Glu(N) a -Palmitoyl)-OtBu)-OH;
 Step 4, cleavage, purification, and lyophilization to obtain liraglutide.
 2. The method of claim 1 wherein:
Wherein, in the solid phase synthesis method described in the step 1, the liquid phase synthesis step of the fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH is: n-hexadecanoic acid, HOSu and DCC coupling Palmitoyl-OSu is obtained, then Palmitoyl-OSu and H-Glu-OtBu are reacted to obtain the dipeptide fragment Palmitoyl-Glu-OtBu; Palmitoyl-Glu-OtBu, HOSu and DCC are coupled to obtain Palmitoyl-Glu(OSu)-OtBu, then Palmitoyl- Glu(OSu)-OtBu and Fmoc-Lys-OH are reacted to obtain a lysine tripeptide fragment Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu)-OH.
 3. The method of claim 1 wherein:
 The solid phase synthesis method according to the second step, wherein the resin solid phase carrier is made of 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-Gly-resin is 0.10-0.35 mmol. /g substitution degree Fmoc-Gly-CTC resin.
 4. The method of claim 1 wherein:
 The solid phase synthesis method according to the second step, wherein the resin solid phase carrier is made of a king resin, the activator system is composed of DIC, HOBt and DMAP, and the Fmoc-Gly-resin is 0.10 to 0.35 mmol/g. Degree of substitution of Fmoc-Gly-King resin.
 5. The method of claim 1 wherein:
The solid phase synthesis method described in the step 3 comprises the following steps: 1) removing the Fmoc protecting group on the Fmoc-Gly-resin by using a deprotecting solution consisting of piperidine and DMF in a volume ratio of 1:4. H-Gly-resin;
 2) H-Gly-resin and Fmoc-protected and side-chain-protected arginine are coupled to obtain Fmoc-Arg(Pbf)-Gly-resin in the presence of a coupling agent system;
3) Repeat steps 1) and 2) to sequence the amino acids according to the liraglutide main chain peptide sequence, wherein the lysine tripeptide fragment is Fmoc-Lys-(Glu(N a -Palmitoyl)-OtBu) -OH;
 6. The method of claim 5, wherein:
 The coupling agent system comprises a condensing agent selected from the group consisting of DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA; and the reaction solvent is selected from DMF, DCM, NMP, DMSO or they Any combination between.
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