CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application Serial No. 62/672,029 filed May 15, 2018, the entire content of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. FIELD OF THE INVENTION

[0003] The present disclosure in general relates to improved methods of producing a liraglutide derivative, which may be converted into liraglutide by cleaving off the protective group thereon.

[0004] 2. DESCRIPTION OF RELATED ART

[0005] Human glucagon-like peptide-l (GLP-l) is a hormone that is produced and released into the body by the intestine in response to the ingestion of food. GLP-l increases the secretion of insulin from pancreas, slows the absorption of glucose from the gut, and reduces the action of glucagon. All three of these actions reduce the level of glucose in the blood. Liraglutide is a man-made hormone that resembles the action of GLP-l, in clinical studies, liraglutide may reduce blood glucose level even further than GLP-L

[0006] However, the current production method of liraglutide suffers from a drawback of low production yield due to the segmentation of the fatty acid moiety in the reactant. Accordingly, there exists in the related art, a need of an improved method for the production of liraglutide, in which the yield of the liraglutide is substantially improved.

SUMMARY

[0007] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0008] In general, the present disclosure relates to the unexpected discovery that the yield of liraglutide derivative may be improved by allowing the coupling reaction between a liraglutide precursor and the fatty acid side chain carrier (e.g., fatty acid glutamate) in a mixed solvent, in which the production yield of the desired product is greatly improved to at least 90%. [0009] Accordingly, the first aspect of the present disclosure aims at providing a method of producing a liraglutide derivative of formula (I),

wherein Ri is /er/-butyl, methyl or benzyl, and R ₂ is a Ci _2-i6 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 of about 1-5: 1-5: 1-5;

the liraglutide precursor is Arg- ³⁴ Lys ²⁶ -GLP-l(7-37)-OH; and

the compound of formula (II) has the structure of,

wherein Ri is /er/-butyl, methyl or benzyl; and R ₂ is a C12-18 acyl.

[0010] According to embodiments of the present disclosure, in the compound of formula (II), R ₂ may be selected from the group consisting of stearioyl, palmitoyl, myristoyl, and laurioyl. In one preferred embodiment, in the compound of formula (II), Ri is tert-butyl, and R ₂ is palmitoyl.

[0011] According to embodiments of the present disclosure, in the step (a), the compound of formula (II) is at least 2-10 folds in excess to the amount of the liraglutide precursor.

[0012] According to preferred embodiments of the present disclosure, the liraglutide precursor has a concentration ranging from 1 mM to 4.5 mM. [0013] Examples of the water-miscible solvent suitable for use in the present method include, but are not limited to, acetonitrile (ACN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and a combination thereof. Examples of the alcohol suitable for use in the present method include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, «-butanol, and a combination thereof.

[0014] According to preferred embodiments of the present disclosure, water, the water-miscible solvent and the alcohol is mixed in a volume ratio from about 1-5: 1-5: 1-5. In one preferred embodiment, the solvent system of the present disclosure is composed of water, NMP and methanol respectively mixed in the volume ratio of 1 : 1 : 1.

[0015] According to optional embodiments of the present disclosure, the method further comprises adjusting the pH value of the quenched solution of the step (b) to 4.0 to 6.5. In one preferred embodiment, the pH value of the quenched solution of the step (b) is adjusted to about 5.0 by the addition of trifluoroacetic acid (TFA).

[0016] According to other optional embodiments of the present disclosure, the method further comprises adding an anti-solvent to the quenched solution of the step (b-l) to precipitate the liraglutide derivative of formula (I). Examples of the anti-solvent suitable for use in the present method include, but are not limited to, water, ether, methyl /cvV-butyl ether and a combination thereof. In one preferred embodiment, water is added to precipitate the liraglutide derivative of formula (I).

[0017] According to embodiments of the present disclosure, the precipitated liraglutide derivative of formula (I) in the step (b-l) is collected by filtration or centrifugation.

[0018] Many of the attendant features and advantages of the present disclosure will become better understood with reference to the following detailed description considered in connection with the accompanied drawings. DESCRIPTION

[0019] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples. [0020] 1. Definition

[0021] For convenience, certain terms employed in the context of the present disclosure are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs.

[0022] The term“liraglutide precursor” as used herein refers to a synthetic peptide precursor of liraglutide produced by recombinant DNA technology in Saccharomyces cerevisiae, in which the peptide has been engineered to be 97% homologous to native human GLP-l by substituting arginine for lysine at position 34. Liraglutide is made by attaching a C-16 fatty acid (i.e., palmitic acid) with a glutamic acid spacer on the remaining lysine at position 26 of the peptide precursor. The peptide precursor or liraglutide precursor is expressed as ³⁴ GLP-l(7-37)-OH or Arg ³⁴ Lys ²⁶ - GLP-l(7-37)-OH, and may be prepared in accordance with the process described in U.S. Patent Nos. 7,273,921 and 6,451,974.

[0023] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term“about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term“about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0024] The singular forms“a,”“and,” and“the” are used herein to include plural referents unless the context clearly dictates otherwise.

[0025] 2. Preperation methods of liraglutide derivative [0026] The present disclosure is based, at least in part, on the unexpected discovery that the production yield of liraglutide derivative of formula (I) is greatly improved by allowing the coupling of a liraglutide precursor and a fatty acid chain carrier to be proceeded in a 3 -components solvent system.

[0027] Accordingly, the present disclosure provides a method of producing a liraglutide derivative of formula (I),

in which, Ri is /er/-butyl, methyl or benzyl, and R ₂ is a Ci _2-i s acyl.

[0028] To produce the desired liraglutide derivative of formula (I), a liraglutide precursor as described in the section of“Definition” above (i.e., Arg ³⁴ Lys ²⁶ -GLP-l(7-37)-OH), is coupled to a compound of formula (II),

in a 3 -components solvent system, via forming an amide linkage at the lysine residue at position 26 of the liraglutide precursor.

[0029] In the present method, the compound of formula (II) serves as a fatty acid chain carrier, thus, upon coupling to the liraglutide precursor peptide, will introduce a fatty acid side chain (i.e., the R.2 group) into the produced product and thereby achieves the purpose of extending the life time of the liraglutide peptide in vivo. To this purpose, in the compound of formula (II), various length of acyl group may serve as the R ₂ group. Preferably, R ₂ may be a Ci _2-i8 acyl, such as stearioyl, palmitoyl, myristoyl, and laurioyl. As to the Ri group, it serves as a protecting group of the carboxy moiety of the compound of formula (II), which may be cleaved off to produce liraglutide. Examples of the Ri group include, but are not limited to, tert- butyl, methyl and benzyl. In one preferred embodiment, in the compound of formula (II), Ri is tert-butyl, and R ₂ is palmitoyl. [0030] According to embodiments of the present disclosure, the 3-components solvent system is composed of water, a water-miscible solvent and an alcohol. Examples of the water-miscible solvent suitable for use in the present method include, but are not limited to, acetonitrile (ACN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), and a combination thereof. Examples of the alcohol suitable for use in the present method include, but are not limited to, methanol, ethanol, propanol, isopropanol, n- butanol, and a combination thereof. Preferably, the water, the water-miscible solvent and the alcohol are mixed in a volume ratio from about 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 one preferred embodiment, the solvent system is composed of water, NMP and methanol, mixed in the volume ratio of 1 : 1 : 1.

[0031] In general, stoichiometric amounts of the liraglutide precursor and the compound of formula (II) are allowed to react in the 3 -components solvent system described above to produce the compound of formula (I), in which the liraglutide precursor and the compound of formula (II) are coupled via an amide linkage formed at the amino group on the side chain of the lysine residue at position 26 of the liraglutide precursor. Preferably, excess amount of the compound of formula (II) is used in the coupling reaction, for example, the amount of the compound of formula (II) is at least 2-10 folds in excess to that of the liraglutide precursor. In one example, the amount of the compound of formula (II) is 5-folds more of the stoichiometric amount of the liraglutide precursor.

[0032] Optionally or in addition, a base (e.g., N,N-diisopropylethylamine) is included in the reaction mixture to facilitate the coupling reaction. ETpon completion, the coupling reaction is quenched by use of a glycine solution.

[0033] Optionally or in addition, the pH value of the quenched solution is adjusted to an acidic value, or about 4.0 to 6.5, for example, about 4.0, 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, by use of an acid; preferably, by the addition of trifluoroacetic acid (TFA). According to embodiments of the present disclosure, the yield and the purity of the compound of formula (I) would not reach satisfactory levels if the pH value was adjusted by use of hydrochloric acid, phosphoric acid, or acetic acid. In one preferred embodiment, the pH value of the quenched solution is adjusted to pH 5.0 by use of TFA.

[0034] According to embodiments of the present disclosure, the thus produced liraglutide derivative is precipitated out of, the pH value adjusted, quenched solution by the addition of an anti-solvent. Examples of the anti-solvent include, but are not limited to, water, ether, methyl tert- buty ether and a combination thereof. In one preferred embodiment, water is added to the pH value adjusted, quenched solution to precipitate the liraglutide derivative of formula (I). The thus produced liraglutide derivative of formula (I) may then collected by filtration or centrifugation. In one example, the liraglutide derivative of formula (I) is collected by filtration. In another example, the liraglutide derivative of formula (I) is collected by centrifugation.

[0035] According to embodiments of the present disclosure, to achieve a production yield of more than 50%, the liraglutide precursor preferably has a concentration of about 1 mM to 4.5 mM in the reaction mixture of the coupling reaction, such as about 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 mM; more preferably, about 1.2 mM to 4.3 mM, such as about 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, and 4.3 mM.

[0036] According to embodiments of the present disclosure, the yield of the liraglutide derivative of formula (I) thus produced is over 50%, preferably over 60%, more preferably over 75%, and most preferably, over 94%.

[0037] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.

[0038] EXAMPLES

[0039] Example 1 Preparation of N-palmitolyl -L-glutamyl-OtBu liraglutide derivative

[0040] Liraglutide precursor (Arg- ³⁴ Lys ²⁶ -GLP-l(7-37)-OH) (466.2 mg) was dissolved in water (22.4 mL), then methanol (22.4 mL), N-methyl-2-pyrrolidone (NMP) (22.4 mL) and N,N- diisopropylethylamine (1.345 mL) were added at room temperature and stirred for 30 min. Allowed the mixture to cool to 0-5°C, and continued to stir for another 5 min to produce the liraglutide precursor solution.

[0041] l-tert-butyl 5-(N-succinimidyl) N-palmitoyl-L-glutamate (374.5 mg) was dissolved in NMP (2.228 mL), and the solution was poured into the liraglutide precursor solution with continuous stirring at 0-5°C for about 1 hr, the reaction was monitored by high performance liquid chromatography (HPLC). A glycine solution (104.3 mg glycine in 1.034 mL water) was added to quench the reaction; then water (290 mL) was added into solution. Then, pH of the quenched solution was adjusted to about 5.0 by the addition of 20% trifluoracetic acid (TFA). The product was collected by filtration, and vacuum dried to produce a white solid of the liraglutide derivative (746.1 mg), in which the yield was about 94.10%, and the purity was about 89.34%.

[0042] Example 2 Optimizing the preparation of liraglutide derivative

[0043] 2.1 Effects of solvent system and Ri of the compound of formula (II) on the production yield of liraglutide derivative

[0044] In this example, liraglutide derivative was prepared in according to procedures of Example 1, except various types of solvents at designated mixed ratios were used for the coupling reaction between liraglutide precursor and l-tert-butyl 5-(N-succinimidyl) N-palmitoyl-L-glutamate, the production yields of thus produced liraglutide derivatives were determined, and results are summarized in Tables 1 to 3.

[0045] Table 1. Effect of solvent system on the yield of liraglutide derivative

[0046] Table 2. Effect of the solvent ratio on the yield of liraglutide derivative

0047] Table 3. Effect of different types of alcohol on the yield of liraglutide derivative

[0048] 2.2 Effects of Ri of the compound of formula (II) on the production yield of liraglutide derivative

[0049] In this example, liraglutide derivative was prepared in according to procedures of Example 1, except various compounds of formula (II) (in which R ₂ = palmitoyl) were used for the production of liraglutide derivatives. Each compound of formula (II) differed from the other by the Ri group. Results are summarized in Table 4.

[0050] Table 4. Effect of Ri group of the compound of formula (II) (where R ₂ = palmitoyl) on the yield of liraglutide derivative

[0051] 2.3 Effect of the concentration of liraglutide precursor on the preparation of liraglutide derivative

[0052] In this example, liraglutide derivative was prepared in according to procedures of Example 1, except the concentration of liraglutide precursor was varied from 1.37 mM to 2.05 mM or 4.11 mM. Results are summarized in Table 5.

[0053] Table 5. Effect of the concentration of liraglutide precursor on the yield of liraglutide derivative

[0054] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.