阅读说明：本技术 一种索马鲁肽二肽侧链的制备方法 (Preparation method of side chain of Somalutide dipeptide ) 是由 翟立海 王全龙 汪慧岩 王少林 于 2020-05-14 设计创作，主要内容包括：本发明属于医药化工领域,具体涉及一种索马鲁肽二肽侧链的制备方法。本发明的制备方法为Fmoc-His(Trt)-OH和氯化亚砜加热回流,所得产物进一步与2-氨基异丁酸即得索马鲁肽重要侧链Fmoc-His-Aib-OH,本发明提供的索马鲁肽二肽侧链合成方法简化了操作步骤,即在第一步骤既可脱掉Trt,又可将羧酸制备成酰氯,仅需两步反应即可得目标物,成本低,副产物少；同时下步酰化反应速率快,收率高,有利于索马鲁肽的大规模生产,满足工业化生产的需求。(The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a preparation method of a side chain of a somaglutide dipeptide. The preparation method provided by the invention comprises the steps of heating and refluxing Fmoc-His (Trt) -OH and thionyl chloride, and further mixing the obtained product with 2-aminoisobutyric acid to obtain the important side chain Fmoc-His-Aib-OH of the Somalou peptide; meanwhile, the next step of acylation reaction has high speed and high yield, is beneficial to the large-scale production of the Somaloutide, and meets the requirement of industrial production.)

1. A preparation method of a side chain of a Somalutide dipeptide is characterized by comprising the following steps:

(1) heating and refluxing the compound II, namely N-protection-L-histidine, thionyl chloride and an organic solvent, and after the reaction is finished, evaporating redundant thionyl chloride under reduced pressure to obtain an intermediate III;

(2) dissolving a compound IV, namely 2-aminoisobutyric acid in an organic solvent, adding a compound III and alkali at low temperature, and obtaining a Somalutide dipeptide basic side chain compound I after the reaction is finished, wherein the synthetic route is as follows;

2. the preparation method according to claim 1, wherein the compound II and the thionyl chloride in the step (1) are fed in a molar ratio of 1: 6.0-10.0.

3. The method according to claim 1, wherein the organic solvent in step (1) is one of N, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone, or a combination thereof.

4. The preparation method according to claim 1, wherein the mass/volume ratio of the compound II to the organic solvent fed in the step (1) is 1: 0.5-1.5, the mass is g, and the volume is mL.

5. The preparation method according to claim 1, wherein the base in step (2) is selected from one of triethylamine, N-diisopropylethylamine, pyridine, potassium carbonate, sodium bicarbonate, or a combination thereof.

6. The preparation method according to claim 2, wherein the compound III, the compound IV and the base are fed in the step (2) in the following molar ratio: 1: 1.0-1.8: 1.2-2.0.

7. The preparation method according to claim 1, wherein the organic solvent in step (2) is selected from one or a combination of dichloromethane, tetrahydrofuran, N-dimethylformamide and N, N-dimethylacetamide; the reaction temperature is-10 ℃.

Technical Field

The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a preparation method of a side chain of a somaglutide dipeptide.

Background

The Somarlu peptide (Sermaglutide) is a new long-acting glucagon-like peptide-1 (GLP-1) analogue developed by Novonide company, has obvious effect of reducing blood sugar and obvious effect of losing weight, and is the GLP-1 hypoglycemic agent (the Liraglutide is another) with the effects of reducing blood sugar and losing weight in the No. 2 Novonide diabetes pipeline. Compared with liraglutide, the fat of the soxhlet peptide is longer and the hydrophobicity is strong, but the hydrophilicity of the soxhlet peptide is greatly enhanced through short-chain PEG modification. After being modified by PEG, the modified PEG not only can be tightly combined with albumin to cover DPP-4 enzyme hydrolysis sites, but also can reduce renal excretion, prolong the biological half-life and achieve the effect of long circulation. The somaglutide has various effects of reducing blood sugar, losing weight, promoting islet cell regeneration, protecting a cardiovascular system and the like, and has a wide clinical application prospect, and the somaglutide CAS: 910463-68-2 sequence: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (Octadec and iodoic-Glu-PEG 2-PEG2) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

At present, the method for synthesizing the key dipeptide Fmoc-His-Aib-OH of the sommelutide is mainly a solid-phase synthesis method, such as the solid-phase stepwise coupling synthesis of the sommelutide by adopting an Fmoc strategy in Chinese patent application CN106928343A, Chinese patent application CN101133082A and Chinese patent application CN 106478806A. The chinese patent application CN109627317 mentions that the first main process is: preparing resin; the coupling of the amino acids one by one, finally cuts the entire amino acid chain from the resin. Because the solid phase synthesis uses sensitive resin, the cost is higher, the total yield is lower, and simultaneously, the impurities are more, and the purification is difficult.

The Chinese patent application CN109456401 mentions a liquid-phase synthesis method, Boc-His (Trt) -OH is used as a raw material, methyl 2-aminoisobutyrate hydrochloride is used as a raw material, HATU is used as a condensing agent to prepare Boc-His (Trt) -Aib-OH, and finally, the Boc protecting group is removed to obtain the product. The method uses a condensing agent HATU with high price, has high cost and complex operation, and the purity of the obtained dipeptide is not high, only 93.6 percent can not meet the medicinal requirement, thus being not suitable for large-scale production. .

In conclusion, in the existing preparation process of the soxhlet peptide side chain, the synthesis steps are more, the synthesis period is long, the purity and the yield are low, the production cost is high, and the large-scale production is not facilitated, so that the new synthesis method of the soxhlet peptide basic dipeptide side chain is a problem which needs to be solved urgently at present.

Disclosure of Invention

In order to solve the problems of more steps, long synthesis period, difficult product separation and the like in the process of synthesizing the Somalutide in the prior art, the invention provides the preparation method of the Somalutide basic dipeptide side chain, which has the advantages of short reaction route, simple and convenient operation, milder reaction, economy, environmental protection, high yield and suitability for industrial production.

The invention is realized by the following technical scheme:

a preparation method of a side chain of a Somalutide dipeptide comprises the following steps:

(1) heating and refluxing the compound I, namely N-protected-L-histidine, thionyl chloride and an organic solvent, and after the reaction is finished, evaporating redundant thionyl chloride under reduced pressure to obtain an intermediate III;

(2) dissolving a compound IV, namely 2-aminoisobutyric acid in an organic solvent, adding a compound III and alkali at low temperature, and obtaining the Somauyptidyl base side chain dipeptide compound I after the reaction is finished, wherein the synthetic route is as follows:

preferably, the above steps are described in further detail in the following sections:

step 1 preparation of Compound III

Adding the compound II, namely N-protection-L-histidine, thionyl chloride and an organic solvent into a reaction bottle, heating and refluxing, and carrying out reduced pressure distillation after the detection reaction is finished to obtain an intermediate compound III.

In a preferable embodiment, the feeding molar ratio of the compound II to the thionyl chloride is 1: 6.0-10.0, and particularly preferably 1: 8.0.

Preferably, the organic solvent is one of N, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone or a combination thereof, wherein N, N-dimethylformamide is particularly preferred.

Step 2 preparation of compound I:

and (3) placing the compound IV, namely 2-aminoisobutyric acid, into a reaction bottle, adding an organic solvent, adding the intermediate III at a low temperature, reacting with alkali at a constant temperature, adding water for quenching after the reaction is completed, and crystallizing to obtain the Maluyptidyl basic side chain dipeptide compound I.

Preferably, the organic solvent is selected from one or a combination of dichloromethane, trichloromethane, 1, 2-dichloroethane and N, N-dimethylacetamide, wherein dichloromethane is particularly preferred.

Preferably, the compound III, the compound IV and the alkali are fed in a molar ratio of: 1: 1.0-1.8: 1.2-2.0, especially preferably 1:1.2: 1.2.

Preferably, the base is selected from one or a combination of triethylamine, N-diisopropylethylamine, pyridine, potassium carbonate and sodium bicarbonate, wherein triethylamine is particularly preferred.

In a preferred scheme, the reaction temperature is-10 ℃, and particularly preferably 0 ℃.

In a preferred embodiment, after the reaction is finished, a post-treatment operation is required, specifically: after the reaction is completed, adding water into the reaction system, stirring, collecting an organic phase, concentrating under reduced pressure, adding a crystallization solvent into the system, stirring and crystallizing at room temperature, and filtering to obtain a white solid, namely a side chain compound I, wherein the crystallization solvent is one or a combination of petroleum ether, ethyl acetate, methyl tert-butyl ether, dichloromethane, acetonitrile and acetone, and a dichloromethane/methyl tert-butyl ether mixed solvent is preferred.

Compared with the prior art, the invention has the following technical effects:

1. compared with the conventional solid phase synthesis method, the synthesis method does not cause resin polycondensation, enhances the degree of amino acid coupling, and improves the reaction activity and efficiency of coupling;

2. the liquid phase synthesis method provided by the invention simplifies the operation steps, namely the Trt can be removed in the step of heating and refluxing the N-protection-L-histidine and the thionyl chloride, the carboxylic acid can be prepared into acyl chloride, the target product can be obtained by only two steps of reaction, the cost is low, and the byproducts are few; meanwhile, the next step of acylation reaction has high speed and high yield, is beneficial to the large-scale production of the Somaloutide, and meets the requirement of industrial production.

The synthesis method provided by the invention simplifies the operation steps, has short synthesis period, low cost, few byproducts and high product yield, is beneficial to large-scale production of the Somalutide, and meets the requirements of industrial production.

Detailed Description

The invention is further illustrated by the following examples. It should be properly understood that: the examples of the invention are given solely for the purpose of illustration and are not to be construed as limitations of the invention, and therefore, simple modifications of the invention in the context of the methods of the invention

Preparation of Compound III

Example 1

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (1.21g, 12.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 98.2 percent, and the HPLC purity is 99.87 percent.

Example 2

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (7.14g, 0.06mmol) into a reaction flask, heating and refluxing, adding N, N-dimethylacetamide (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with a solvent and is not separated.

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (1.21g, 12.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) mixed solutionAnd stirring at room temperature for crystallization, and filtering to obtain a white solid, namely the compound I, wherein the yield is 95.5 percent, and the HPLC purity is 99.83 percent.

Example 3

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (11.9g, 0.1mmol) into a reaction flask, heating and refluxing, adding N, N-dimethylformamide (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain a solvent, namely the unseparated intermediate III (calculated according to the yield of 100%).

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (1.21g, 12.0mmol) and chloroform (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 94.3 percent, and the HPLC purity is 99.78 percent.

Example 4

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (5.95g, 0.05mmol) into a reaction flask, heating and refluxing, adding N-methylpyrrolidone (30mL), controlling the temperature and refluxing, and distilling under reduced pressure after the reaction is finished to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with a solvent and is not separated.

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (1.21g, 12.0mmol) and chloroform (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtration, the yield is 90.2 percent, and the HPLC purity is 99.71 percent.

Example 5

N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (13.09g, 0.11mmol) were added to a reaction flask and heated under reflux, N-methylpyrrolidone (30mL) was added thereto, reflux was controlled at a controlled temperature, and after the reaction, the intermediate III, which is mostly a solvent, was distilled under reduced pressure (calculated according to 100% yield).

2-aminoisobutyric acid (1.24g,12.0mmol), sodium bicarbonate (1.01g, 12.0mmol) and chloroform (20mL) were added to a reaction flask, the temperature was lowered to-10 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation reaction, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. To the system was added petroleum ether/ethyl acetate (30mL, V)_{Petroleum ether}：V_{Ethyl acetate}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtration, the yield is 88.5 percent, and the HPLC purity is 99.65 percent.

Example 6

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (1.01g, 10.0mmol) and 1, 2-dichloroethane (20mL) were charged into a reaction flask, the temperature was lowered to 10 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation reaction, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and stirred for 10 minutes, and the organic phase was collected and concentrated under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 95.8 percent, and the HPLC purity is 99.84 percent.

Example 7

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-Aminoisobutyric acid (1.24g,12.0mmol) and tris (hydroxymethyl) phosphonium chloride were addedEthylamine (2.02g, 20.0mmol) and dichloromethane (20mL) are added into a reaction bottle, the temperature is reduced to-15 ℃, the compound III (4.32g, 10.0mmol) is added for heat preservation reaction, after the TLC detection reaction is completed, water (20mL) is added into the reaction system for stirring for 10 minutes, an organic phase is collected, and the mixture is concentrated under reduced pressure until no liquid flows out. Dichloromethane/acetone (30mL, V) was added to the system_{Methylene dichloride}：V_{Acetone (II)}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtration, the yield is 94.4 percent, and the HPLC purity is 99.79 percent.

Example 8

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-aminoisobutyric acid (1.24g,12.0mmol), triethylamine (2.23g, 22.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 18 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Acetonitrile/acetone (30mL, V) was added to the system_Acetonitrile：V_{Acetone (II)}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtration, the yield is 89.5 percent, and the HPLC purity is 99.72 percent.

Example 9

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with a solvent and is not separated.

2-aminoisobutyric acid (1.03g,10.0mmol), N-diisopropylethylamine (1.56g, 12.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto to carry out an incubation reaction, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. To the system was added ethyl acetate/acetone (30mL, V)_{Acetic acid BEsters}：V_{Acetone (II)}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 95.2 percent, and the HPLC purity is 99.81 percent.

Example 10

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-Aminoisobutyric acid (1.86g,18.0mmol), pyridine (0.95g, 12.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto, followed by incubation, and after completion of the reaction by TLC, water (20mL) was added to the reaction system and the mixture was stirred for 10 minutes, followed by collection of the organic phase and concentration under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 94.6 percent, and the HPLC purity is 99.78 percent.

Example 11

Adding N-protected-L-histidine (6.20g, 0.01mol) and thionyl chloride (9.52g, 0.08mmol) into a reaction flask, heating and refluxing, adding DMF (30mL), controlling the temperature and refluxing, and after the reaction is finished, distilling under reduced pressure to obtain the intermediate III (calculated according to the yield of 100%) which is mostly mixed with the solvent and is not separated.

2-aminoisobutyric acid (2.06g, 20.0mmol), potassium carbonate (1.66g, 12.0mmol) and dichloromethane (20mL) were added to a reaction flask, the temperature was lowered to 0 ℃ and the above-mentioned compound III (4.32g, 10.0mmol) was added thereto to carry out a heat preservation reaction, after completion of the reaction was detected by TLC, water (20mL) was added to the reaction system and stirred for 10 minutes, and the organic phase was collected and concentrated under reduced pressure until no liquid flowed out. Methylene chloride/methyl tert-butyl ether (30mL, V) was added to the system_{Methylene dichloride}：V_{Methyl tert-butyl ether}1: 1) the mixed solution is stirred and crystallized at room temperature, and white solid, namely the compound I, is obtained by filtering, the yield is 88.6 percent, and the HPLC purity is 99.70 percent.

Comparative examples

Boc-His (Trt) -OH (5.01g, 10.07mmol) with side chain protection, HOBT (2.72g, 20.14mmol), DIE A (3.90g, 30.21mmol), methyl 2-aminoisobutyrate hydrochloride (1.55g, 10.07mmol) were dissolved in DMF (100mL), HATU (6.54g, 17.20mmol) was added, stirring was carried out at room temperature for 4 hours, the reaction mixture was poured into ice water to precipitate a white solid, a white solid was obtained by filtration, the mixture was washed with saturated brine by beating three times, finally the mixture was washed with clear water by beating once and dried in a vacuum drying oven to obtain 5.98g, the product obtained by filtration and concentration was dried was dissolved in THF (50mL), water (30mL) was added, lithium hydroxide monohydrate (0.85g, 20.26mmol) was added, stirring was carried out at room temperature for 2 hours, THF was removed by concentration, pH was adjusted to 6-7 with hydrochloric acid having a concentration of 1N, a solid was precipitated, and filtered, washed with water three times to obtain a white solid, vacuum drying to obtain Boc-His (Trt) -Aib-OH with yield of 87.1% and HPLC purity of 93.58%.