TW201946930A - Preparation method of liraglutide derivative - Google Patents

Abstract

Disclosed herein is a method for method of producing a liraglutide derivative of formula (I), in which, R1 is tert-butyl, methyl or benzyl, and R2 is a C12-18 acyl. The method includes steps of, (a) coupling a liraglutide precursor with a compound of formula (II) in a solvent system to produce the liraglutide derivative of formula (I), in which the coupling occurs at the lysine residue at position 26 of the liraglutide precursor via forming an amide linkage; (b) quenching the reaction of the step (a) with an addition of a glycine solution; and (c) collecting the liraglutide derivative of formula (I) from the quenched solution of the step (b); wherein, the solvent system is a mixture of water, a water-miscible solvent and an alcohol mixed in a volume ratio from about 1-5:1-5:1-5; the liraglutide precursor is Arg-34Lys26-GLP-1(7-37)-OH; and the compound of formula (II) has the structure of, (II) wherein R1 is tert-butyl, methyl or benzyl; and R2 is a C12-18 acyl.

Description

Method for preparing liraglutide derivative

The present disclosure relates to an improved method for preparing liraglutide derivatives, which can be converted into liraglutide by cutting off the protecting group on the liraglutide derivative.

Human glycopeptin-like peptide (GLP-1) is a hormone produced by the intestine and released into the body in response to food intake. GLP-1 can increase the secretion of insulin by the pancreas, slow down the absorption of glucose in the intestine, and reduce the effects of glucagon; all three of these effects can lower blood sugar levels. Liraglutide is an artificial hormone similar to GLP-1. Clinical studies have shown that liraglutide may lower blood glucose levels more than GLP-1.

However, current liraglutide preparation methods are limited by the low yield disadvantage caused by the partitioning of fatty acid moieties in the reactants. Therefore, there is an urgent need in the related technical field for an improved method for preparing liraglutide, which can significantly improve the yield of liraglutide.

The following summary is intended to provide a simplified summary of the disclosure so that readers may have a basic understanding of the disclosure. This summary is not a comprehensive overview of the disclosure, and it is not intended to indicate important / critical elements of the embodiments of the invention or to define the scope of the invention. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

Overall, the present disclosure is based on the unexpected discovery that the yield of liraglutide derivatives can be improved by coupling the liraglutide precursor with a glutamic acid-containing fatty chain in a mixed solvent, The desired product yield can be greatly increased to at least 90%.

A first aspect of the present disclosure is to provide a method for preparing a liraglutide derivative of formula (I);

Wherein R ₁ may be tert-butyl, methyl or benzyl, and R ₂ is a fluorenyl group having 12 to 18 carbons, the method includes the following steps:
(a) coupling a liraglutide precursor with a compound of formula (II) in a solvent system to produce a liraglutide derivative of formula (I), wherein said coupling is by a liraglutide precursor The lysine at position 26 of the compound is achieved by forming a monoamine bond with the compound of formula (II);
(b) adding a glycine solution to quench the reaction of step (a);
(c) collecting a liraglutide derivative of formula (I) from the quenching solution of step (b);
among them:
The solvent system is a mixture, which is composed of water, a solvent miscible with water, and alcohols in a volume ratio of 1-5: 1-5: 1-5;
The liraglutide precursor is Arg- ³⁴ Lys ²⁶ -GLP-1 (7-37) -OH; and the structure of the compound of formula (II) is as follows:
(II)
Among them, R ₁ may be tert-butyl, methyl or benzyl, and R ₂ is a fluorenyl group having 12 to 18 carbons.

According to an embodiment of the present disclosure, in the compound of formula (II), R ₂ may be composed of stearyl, palmityl, myristyl or lauryl. In a preferred embodiment, R ₁ of the compound of formula (II) is tert-butyl, and R ₂ is palmitoyl.

According to an embodiment of the present disclosure, in step (a), the amount of the compound of formula (II) needs to be at least 2 to 10 times greater than that of the liraglutide precursor.

According to a preferred embodiment of the present disclosure, the concentration of liraglutide precursor ranges from 1 to 4.5 millimolar concentrations (mM).

Examples of water-miscible solvents suitable for this method include, but are not limited to, acetonitrile (ACN), dimethylformamide (DMF), and dimethylformamide (dimethylformamide) sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF) and combinations thereof. Examples of alcohols suitable for the method include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, and combinations thereof.

According to a preferred embodiment of the present disclosure, the water, the water-miscible solvent and the alcohol are a mixture of 1-5: 1-5: 1-5 in a volume ratio. In a preferred embodiment, the solvent system of the present disclosure is composed of water, NMP and methanol with a volume ratio of 1: 1.

According to an optional embodiment of the present disclosure, the method further includes step (b-1), which adjusts the pH of the quenching solution to between about 4.0 and 6.5 in step (b). In a preferred embodiment, the pH is adjusted to about 5.0 by adding trifluoroacetic acid (TFA) to the quenching solution in step (b).

According to other optional embodiments of the present disclosure, the method further includes step (b-2), which comprises adding an antisolvent to the quenching solution of step (b-1) to precipitate the benefit of formula (I) Laluotide derivatives. Examples of anti-solvents suitable for this method include, but are not limited to, water, diethyl ether, methyl tert-butyl ether, and combinations thereof. In a preferred embodiment, water is added to precipitate the liraglutide derivative of formula (I).

According to an embodiment of the present disclosure, the liraglutide derivative of formula (I) is collected from the quenching solution of step (b) by filtration or centrifugation.

Many accompanying features and advantages of the present disclosure will be better understood with reference to the following detailed description and related drawings.

In order to make the description of this disclosure more detailed and complete, the following provides an illustrative description of the implementation mode and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The implementation mode covers the functions of the specific embodiments, as well as the method steps and sequences for constructing and operating the specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

1. Noun definition

For convenience, some terms used in the context of this disclosure are collected here. Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and used by those having ordinary knowledge in the technical field to which the present invention pertains.

The term "liraglutide derivative" refers to a synthetic peptide precursor of liraglutide, which is produced in Saccharomyces cerevisiae by genetic recombination technology. The amino acid is substituted with lysine, making it 97% homologous to natural human GLP-1. Liraglutide is prepared by linking a 16-carbon fatty acid (ie, palmitic acid) to a glutamate spacer on the glutamic acid at the 26th position of the liraglutide precursor. This peptide precursor or liraglutide precursor can be represented by ³⁴ GLP-1 (7-37) -OH or Arg ³⁴ Lys ²⁶ -GLP-1 (7-37) -OH, which can be according to US Patent No. 7,273,921 No. and U.S. Patent No. 6,451,974.

Although the numerical ranges and parameters listed in the scope of the invention are approximate values, the numerical values proposed in the specific embodiments are as accurate as possible. However, any numerical value is inherently subject to certain errors due to the standard deviation of individual test measurements. Likewise, as used herein, "about" generally means 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, in the opinion of a person skilled in the art, "about" means within an acceptable standard error of the mean. Except for experimental examples, or unless explicitly stated otherwise, all numerical ranges, quantities, values, and percentages, such as material quantity, duration, temperature, operating conditions, quantity ratios, and all other similar terms disclosed herein, should be applicable. It is understood that all numerical parameters used herein are modified by "about". At a minimum, each numerical parameter should be interpreted based on the number of significant digits reported and through ordinary rounding techniques.

Unless otherwise defined in this specification, the singular noun used in this specification covers the plural form of the noun; and the plural noun used also covers the singular form of the noun.

2. Preparation method of liraglutide derivative

This disclosure is based at least in part on an unexpected discovery. The liraglutide precursor and a carrier with a fatty acid side chain can be greatly improved by coupling liraglutide precursors to a carrier with a fatty acid side chain in a 3-component solvent system Yields of liraglutide derivatives.

Accordingly, the present disclosure provides a method for preparing a liraglutide derivative of formula (I);

Wherein R ₁ may be tert-butyl, methyl or benzyl, and R ₂ is a fluorenyl group having 12 to 18 carbons.

To prepare a liraglutide derivative of formula (I), in a three-component solvent system, the liraglutide precursor (ie, Arg ³⁴ Lys ²⁶ -GLP-1 ( 7-37) -OH), coupled to the compound of formula (II),
(II)
The coupling system is formed by making the lysine at the 26th position of the liraglutide precursor and the compound of formula (II) form an amidine bond.

In this method, the compound of formula (II) is used as the carrier of the fatty acid chain; therefore, once it is coupled with the liraglutide precursor, a fatty acid side chain (ie, R ₂ functional group) will be introduced into the The product is produced, thereby extending the duration of liraglutide in the body. To achieve this, the R ₂ functional group in the compound of formula (II) may be a fluorenyl group of different length. Preferably, the R ₂ functional group is a fluorenyl group having 12 to 18 carbons, such as stearyl fluorenyl, palmitoyl, myristyl, lauryl, and the like. As for the R ₁ functional group, as a protecting group for the carboxyl part of the compound of the formula (II), liraglutide can be produced when it can be cleaved. Examples of the R ₁ functional group include, but are not limited to, t-butyl, methyl, or benzyl. In a preferred embodiment, the R ₁ functional group in the compound of formula (II) is tert-butyl; and the R ₂ functional group is palmitoyl.

According to an embodiment of the present disclosure, the three-component solvent system is composed of water, a water-miscible solvent, and an alcohol. Examples of water-miscible solvents suitable for use in this method include, but are not limited to, acetonitrile, dimethylformamide, dimethylmethane, N-methylpyrrolidone, tetrahydrofuran, and combinations thereof. Examples of alcohols suitable for the method include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, and combinations thereof. Water and water-miscible solvents and alcohols are preferably mixed in a volume ratio of 1-5: 1-5: 1-5, such as 1: 1: 1, 1: 2: 1, 1: 3 : 1, 1: 4: 1, 1: 5: 1, 1: 2: 2, 1: 2: 3, 1: 2: 4, 1: 2: 5, 1: 3: 2, 1: 3: 3 1: 3: 4, 1: 3: 5, 1: 4: 2, 1: 4: 3, 1: 4: 4, 1: 4: 5, 1: 5: 2, 1: 5: 3, 1 : 5: 4, 1: 5: 5, 1: 1: 2, 1: 1: 3, 1: 1: 4, 1: 1: 5, 2: 1: 1, 2: 1: 2, 2: 1 : 3, 2: 1: 4, 2: 1: 5, 2: 2: 1, 2: 2: 3, 2: 2: 5, 2: 3: 1, 2: 3: 2, 2: 5: 2 3: 1: 1, 3: 2: 1, 3: 3: 1, 3: 4: 1, 3: 5: 1, 3: 2: 1, 3: 3: 1, 3: 4: 1, 3 : 5: 1, 3: 1: 2, 3: 1: 3, 3: 1: 4, 3: 1: 5, 3: 2: 2, 3: 2: 3, 3: 2: 4, 3: 2 : 5, 3: 3: 2, 3: 3: 4, 3: 3: 5, 3: 4: 2, 3: 4: 3, 3: 4: 4, 3: 4: 5, 3: 5: 2 3: 4: 3, 3: 5: 4, 3: 5: 5, 4: 1: 1, 4: 1: 2, 4: 1: 3, 4: 1: 5, 4: 2: 1, 4 : 2: 3, 4: 2: 5, 4: 3: 1, 4: 3: 2, 4: 3: 3, 4: 3: 4, 4: 3: 5, 4: 4: 1, 4: 4 : 3, 4: 4: 5, 4: 5: 1, 4: 5: 2, 4: 5: 3, 4: 5: 4, 4: 5: 5, 5: 1: 1, 5: 1: 2 5: 1: 3, 5: 1: 4, 5: 1: 5, 5: 2: 1, 5: 2: 2, 5: 2: 3, 5: 2: 4, 5: 2: 5, 5: 3: 1, 5: 3: 2, 5: 3: 4, 5: 3: 5, 5: 4: 1, 5: 4: 2, 5: 4: 3, 5: 4: 4, and 5: 4: 5. In a preferred embodiment, the solvent system is composed of water, N-methylpyrrolidone and methanol in a volume ratio of 1: 1: 1.

Generally, the stoichiometric liraglutide precursor is reacted with a compound of formula (II) in the three-component solvent system described above to produce a compound of formula (I), where the liraglutide precursor is through its 26th position The amino group on the side chain of lysine is coupled to the compound of formula (II) by forming an amine bond. Preferably, an excess amount of the compound of formula (II) is used in the coupling reaction, for example, the amount of the compound of formula (II) needs to be at least 2 to 10 times greater than the amount of liraglutide precursor. In one embodiment, the amount of the compound of formula (II) is 5 times greater than the amount of liraglutide precursor.

According to an alternative embodiment, a base such as N, N-diisopropylethylamine may be additionally added to the reaction mixture to facilitate the coupling reaction. After the reaction is completed, a glycine solution is added to stop or quench this coupling reaction.

Depending on the selectively implemented embodiment, the pH of the quenching solution can be additionally adjusted to be acidic, or between about 4.0 and 6.5; for example, the pH can be adjusted to about 4.0 using an acid, preferably trifluoroacetic acid (TFA) , 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5 According to the embodiment of the present disclosure, if hydrochloric acid, phosphoric acid, or acetic acid is used to adjust the pH value, the yield and purity of liraglutide derivative of formula (I) cannot be achieved to a satisfactory level. In a preferred embodiment, the pH value of the quenching solution is adjusted to 5.0 with trifluoroacetic acid.

According to the embodiment of the present disclosure, the prepared liraglutide derivative is precipitated from the quenching solution by adjusting the pH value and adding an antisolvent. Examples of anti-solvents include, but are not limited to, water, diethyl ether, methyl tert-butyl ether, and combinations thereof. In a preferred embodiment, water is added to the pH-adjusted quenching solution to precipitate a liraglutide derivative of formula (I). The liraglutide derivative of formula (I) produced is collected by filtration or centrifugation. In one embodiment, liraglutide derivatives of formula (I) are collected by filtration. In another embodiment, liraglutide derivatives of formula (I) are collected by centrifugation.

According to an embodiment of the present disclosure, in order to achieve a yield of more than 50%, the concentration of liraglutide precursor in the reaction mixture of the coupling reaction is preferably between about 1-4.5 millimolar concentration (mM), For example 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 and 4.5 millimolar concentrations; more preferably between about 1.2-4.3 millimolar concentrations, such as 1.2, 1.3, 1.4, 1.5 , 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 , 4.1, 4.2, 4.3 millimolar concentrations.

According to an embodiment of the present disclosure, the yield of liraglutide derivative of formula (I) can exceed 50%; preferably it can exceed 60%; more preferably it can exceed 75%; and most preferably it can exceed 94% .

A number of experimental examples are provided below to illustrate some aspects of the present invention, so that those with ordinary knowledge in the technical field to which the present invention pertains can implement the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that those skilled in the art, upon reading the description presented herein, can fully utilize and practice the present invention without undue interpretation.

Examples

Examples 1 preparation N- Palm cricket --L- Alanine - Tert-butyl liraglutide derivative

Dissolve liraglutide precursor (Arg ³⁴ Lys ²⁶ -GLP-1 (7-37) -OH) (466.2 mg) in water (22.4 ml), then add methanol (22.4 ml), N at room temperature -Methylpyrrolidone (22.4 ml) and N, N-diisopropylethylamine (1.345 ml) were stirred for 30 minutes. After the mixture was cooled to 0 to 5 ° C, stirring was continued for five minutes to prepare a liraglutide precursor solution.

Dissolve N-palmitamidine-L-glutamic acid 1-tert-butyl 5- (N-succinimide) ester (374.5 mg) in N-methylpyrrolidone (2.228 ml) and pour the solution Into liraglutide precursor solution, and continue stirring at 0-5 ° C for about an hour; and monitor the progress of the reaction by high performance liquid chromatography. Glycine solution (104.03 mg of glycine in 1.034 ml of water) was added to quench the reaction, and water (290 ml) was added to the reaction solution. The pH of the quenching solution was then adjusted to about 5.0 with 20% trifluoroacetic acid. The product was collected by filtration, and a white solid (746.1 mg) of liraglutide derivative was obtained after vacuum drying. The yield was about 94.10%, and the purity was about 89.34%.

Examples 2 Optimization of liraglutide derivatives

2.1 Solvent system and formula (II) Of compounds R ₁ Effects of functional groups on the yield of liraglutide derivatives

The liraglutide derivative in this example is constructed according to the steps described in Example 1, except that different types of solvents are used to mix at the specified ratio for the liraglutide precursor and N-palmidine-L -Coupling reaction of glutamic acid 1-tert-butyl 5- (N-succinimide) ester, and the yield of liraglutide derivative made from it was measured. The results are summarized in Tables 1-3.

Table 1: Effect of solvent system on the yield of liraglutide derivatives

Table 2: Effect of reaction solvent ratio on the yield of liraglutide derivatives

Table 3: Effects of different alcohols on the yield of liraglutide derivatives

2.2 formula (II) Of compounds R ₁ Effects of functional groups on the yield of liraglutide derivatives

The liraglutide derivative in this example is constructed according to the procedure described in Example 1, except that a compound of formula (II) with a different structure (where R ₂ functional group is palmitoyl) is used to make liraglutide The peptide derivatives have different R ₁ functional groups in the compounds of formula (II). The results are summarized in Table 4.

Effects of formula (II) R ₁ functional group of the compound (where R ₂ is palmitic acyl functional group) to yield derivative liraglutide: Table IV

2.2 Effect of liraru precursor concentration on the preparation of liraglutide derivatives

The liraglutide derivative in this example was constructed in a similar procedure as described in Example 1, except that the concentration of the liraglutide precursor was in the range of 1.37 to 2.05 millimolar, or 4.11 millimolar. Variety. The results are summarized in Table 5.

Table 5: Effect of liraglutide precursor concentration on liraglutide derivative yield

Although the embodiments are only used as examples in the above description, those with ordinary knowledge in the technical field to which the present invention pertains can make various modifications thereto. The above description, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although the above text has described the different embodiments of the present invention with a certain degree of particularity, or refers to one or more separate embodiments, those with ordinary knowledge in the technical field to which the present invention pertains, without departing from the principles and spirit of the present invention In the circumstances, when various changes and modifications can be made to it, the scope of protection of the present invention shall be defined by the scope of the accompanying patent application.

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Claims (12)

A method for preparing a liraglutide derivative of formula (I), comprising: (a) coupling a liraglutide precursor to a compound of formula (II) in a solvent system to produce a liraglutide derivative of formula (I); wherein the coupling is by letting formula (II) The compound is formed by a monoamine bond with the lysine at position 26 of the liraglutide precursor; (b) adding a glycine solution to quench the reaction of step (a); and (c) by step (b The liraglutide derivative of formula (I) is collected in a quenching solution in); wherein: the solvent system is composed of water, a solvent miscible with water, and an alcohol in a range of 1-5: 1-5: 1 A mixture of -5 by volume ratio; the liraglutide precursor is Arg- ³⁴ Lys ²⁶ -GLP-1 (7-37) -OH; and the structure of the compound of formula (II) is as follows: (II) wherein, R ₁ is tert-butyl, methyl or benzyl, and R ₂ having 12-18 carbon acyl. The method according to claim 1, wherein in step (a), The water-miscible solvent is selected from the group consisting of acetonitrile, dimethylformamide, dimethylarsine, N-methylpyrrolidone, tetrahydrofuran, and combinations thereof; The alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, and combinations thereof. The method according to claim 2, wherein the solvent system is a mixture of water, the water-miscible solvent, and the alcohol mixed in a volume ratio of 1: 1. The method described in claim 2, further comprising: (b-1) Adjust the pH of the quenching solution in step (b) to between about 4.0 and 6.5. The method according to claim 4, wherein the pH is adjusted to about 5.0 by adding trifluoroacetic acid. The method described in claim 4, further comprising: (b-2) Adding an antisolvent to the quenching solution in step (b-1) to precipitate the liraglutide derivative of formula (I) therein. The method of claim 6, wherein the antisolvent is selected from the group consisting of water, diethyl ether, methyl tert-butyl ether, and combinations thereof. The method according to claim 7, wherein the antisolvent is water. The method according to claim 1, wherein in the compound of formula (II), R ₂ is selected from the group consisting of stearyl, palmityl, myristyl, and lauryl And R ₁ is selected from the group consisting of tert-butyl, methyl, or benzyl. The method according to claim 9, wherein R ₁ of the compound of formula (II) is tert-butyl; and R ₂ is palmitoyl. The method according to claim 1, wherein in step (a), the amount of the compound of formula (II) is about 2 to 10 times the liraglutide precursor. The method according to claim 11, wherein the amount of the compound of formula (II) is about 5 times the liraglutide precursor.