WO2022111642A1 - Method for preparing acylated derivative of insulin or of analog thereof - Google Patents

Abstract

The present disclosure provides a method for preparing an acylated derivative of insulin or of an analog thereof, comprising the step of reacting insulin or an analog thereof with the compound of formula (I), and also provides the compound of formula (I) and a method for preparation thereof. The method of the present disclosure has a high yield and a stable process, can significantly reduce the cost of production of acylated derivatives of insulin or its analogs, and enables commercial production on a large scale.

Description

A kind of method for preparing acylated derivatives of insulin or its analogues

This application claims the priority of Chinese patent application 202011355289.0 with the filing date of 2020/11/27. This application cites the full text of the above Chinese patent application.

technical field

The present disclosure relates to the field of biomedicine, in particular to a method for preparing an acylated derivative of insulin or an analog thereof.

Background technique

Diabetes mellitus is a metabolic syndrome characterized by hyperglycemia caused by defective insulin secretion and/or insufficient insulin action. Diabetes itself is not terrible, but complications caused by poor blood sugar control or disease progression, such as myocardial infarction, cerebral hemorrhage, blindness, renal failure and lower limb amputation, can seriously reduce the quality of life of patients and even endanger their lives. It is true that diabetes has become the third largest disease besides cardiovascular and cerebrovascular diseases and malignant tumors. The World Health Organization (WHO) currently defines diabetes as: fasting blood glucose ≥ 7.0 mmol/L, or elevated blood glucose treated with medication, or a history of diabetes diagnosis. The 2013 edition of the Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes pointed out that the normal reference value of HbA _1c (glycated hemoglobin) in the normal population is 4.0%-6.0%, and HbA _1c ≥ 6.5% can be used as a reference for the diagnosis of diabetes.

A common treatment for diabetes is long-acting insulin injections. Long-acting insulin is an exogenous insulin modified from human insulin, which is characterized by stable onset of action and long duration of drug effect, and the risk of causing nocturnal hypoglycemia is significantly lower than that of neutral protamine zinc insulin (neutral protamine zinc insulin). protamine hagedorn, NPH). At present, there are three main types of long-acting insulins in clinical use, namely Detemir (Insulin Detemir, Novo Nordisk), Insulin Degludec (Insulin Degludec, Novo Nordisk) and Glargine (Insulin Glargine, Sanofi original research, Eli Lilly imitation) .

In view of the clinical shortcomings of the current long-acting insulin products on the market and the market demand for insulin products, how to increase product output to reduce production costs is directly related to product competitiveness. WO2018024186A discloses a novel long-acting insulin, the preparation process of which still needs to be improved in the following aspects: 1) the active ester compound used for acylation of insulin has poor stability and cannot be produced on a large scale; 2) the active ester compound is used for insulin acylation. The modification rate of acylation is too low, and the production cost is too high. In view of the above problems, there is an urgent need for active ester compounds suitable for scale-up production and methods for preparing acylated derivatives of insulin or its analogs in high yields.

SUMMARY OF THE INVENTION

The present disclosure carries out structural improvement design for the active ester compound, and obtains the active ester compound that can be produced in kilograms, with stable production process and controllable quality. In addition, the method for acylating and condensing an active ester compound with insulin or its analogs provided by the present disclosure not only has high yield, but also has a stable process, can greatly reduce the production cost of acylated derivatives of insulin or its analogs, and can realize large-scale production. commercialized production.

The present disclosure provides a method of preparing an acylated derivative of insulin or an insulin analog comprising the step of reacting a compound of formula (I) with insulin or an insulin analog:

R-W-X-Y-Z

(I)

where R is

R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4;

W is a diacyl structure with -OC(CH ₂ ) _n CO-, where n is an integer from 2 to 10; W is one of its acyl groups with the A-chain or B-chain N-terminal amino acid of insulin or insulin analogs The α-amino group of the residue or the ε-amino group of a lysine residue present on the B-chain forms an amide bond;

X is a diamino compound containing a carboxylic acid group, and X is connected with one of its amino groups to an acyl group in W to form an amide bond;

Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

Z is -COOH;

Wherein, insulin is natural insulin; insulin analog is a polypeptide molecule comprising 1 or more amino acid deletions, additions, substitutions or combinations thereof relative to natural insulin, for example, comprising 1 to 10 amino acid deletions, additions, substitutions or the like combination.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. Examples of _C1 - _C4 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl. Examples of _{C1 -} C6alkanoyl groups include, but are not limited to: acetyl (-C(=O) _CH3 ), propionyl (-C(=O) _{CH2CH3 )} , butyryl (-C(=O) )C( _CH3 ) ₃ ) and benzoyl (-C(=O)Ph). Examples of halogens include, but are not limited to, F, Cl, Br, I.

In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro.

In some embodiments, R ₁ is propionyl.

In some embodiments, q is 1, 2, or 3; eg, q is 1.

In some embodiments, W forms an amide bond with the epsilon-amino group of a lysine residue present on the B-chain.

In some embodiments, n is an integer from 2 to 5, eg, n is 2.

In some embodiments, X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10.

In some embodiments, p is an integer from 2 to 6.

In some embodiments, p is an integer from 2 to 4, eg, p is 4.

In some embodiments, m is an integer from 10 to 16, eg, m is an integer from 12 to 14.

In some embodiments, R has a structure selected from the group consisting of:

In some embodiments, the compound of formula (I) is a compound of formula (Ia) or a compound of formula (Ib):

wherein R is as defined above.

In the chemical structure of the compound described in the present disclosure, the bond

Indicates an unspecified configuration, i.e. if a chiral isomer exists in the chemical structure, the bond

can be

or both

Two configurations.

In some embodiments, the compound of formula (I) is:

In some embodiments, the method includes the steps of:

(a) Mixing the compound of formula (I) with an organic solvent selected from methanol, ethanol, acetonitrile, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide or The mixed solvent of the above solvent, for example, the organic solvent is isopropanol or N,N-dimethylformamide;

(b) mixing said solution A with insulin or insulin analog; such that the equivalent ratio of compound of formula (I) to insulin or insulin analog is (1-4):1, eg, about 2:1; and

(c) in the presence of a catalyst, for example, the catalyst is 1,2,4-triazole; at a reaction temperature of 0-25° C., for example, about 0° C.; reaction for 1-8 hours, for example, for about 4 hours; the catalyst is reacted with The equivalence ratio of insulin or insulin analog is (1-12):1, eg, about 8:1.

In some specific embodiments, in step (a), the organic solvent is isopropanol.

In some specific embodiments, in step (a), the organic solvent is N,N-dimethylformamide.

In some embodiments, in step (b), the insulin or insulin analog is in solution, eg, in 50 mM Tris-HCl buffer (pH 8.5).

In some embodiments, in step (b), the insulin or insulin analog solution is pre-cooled to 0-25°C prior to the reaction, such as pre-cooled to 0-20°C, 0-15°C, 0-10°C or 0- 5°C. In some embodiments, the insulin or insulin analog solution is pre-cooled to about 0°C prior to the reaction.

In some embodiments, in step (b), the equivalent ratio of the compound of formula (I) to insulin or insulin analog is about 1:1, about 2:1, about 3:1, or about 4:1, such as about 2:1.

In some embodiments, in step (c), the catalyst is 1,2,4-triazole.

In some embodiments, in step (c), the equivalent ratio of catalyst to insulin or insulin analog is (1-12):1, eg, about 11:1, about 10:1, about 9:1, about 8:1 1. About 7:1, about 6:1, or about 5:1.

In some embodiments, in step (c), the equivalent ratio of catalyst to insulin or insulin analog is about 8:1.

In some embodiments, in step (c), the reaction temperature is 0-20°C, 0-15°C, 0-10°C, or 0-5°C.

In some embodiments, in step (c), the reaction temperature is about 0°C.

In some embodiments, in step (c), the reaction is carried out for 1-8 hours, 2-8 hours, 2-7 hours, 3-7 hours, 3-6 hours, 3-5 hours, 3-4 hours, or 4-5 hours. In some embodiments, in step (c), the reaction is carried out for about 4 hours.

In some specific embodiments, the method includes the steps of:

(a) the compound of formula (I) is mixed with an organic solvent to obtain solution A, and the organic solvent is isopropanol or N,N-dimethylformamide;

(b) mixing said solution A with insulin or insulin analog; such that the equivalence ratio of compound of formula (I) to insulin or insulin analog is about 2:1; and

(c) In the presence of the catalyst 1,2,4-triazole at a reaction temperature of about 0° C. for about 4 hours, the equivalent ratio of the catalyst to insulin or insulin analog is about 8:1.

Optionally, in step (b), the insulin or insulin analog is present in solution, for example in about 50 mM Tris-HCl buffer (about pH 8.5);

Optionally, in step (b), the insulin or insulin analog solution is pre-cooled to 0-25°C before the reaction, for example, pre-cooled to 0-20°C, 0-15°C, 0-10°C, 0-5°C , 0°C.

In some embodiments, the insulin is human insulin, porcine insulin, or bovine insulin.

In some embodiments, the insulin analog is a deletion, addition, Substituted polypeptide molecules or combinations thereof. Examples of insulin analogs include, but are not limited to: (i) insulin aspart, which differs from human insulin in that the B28 proline is replaced by aspartic acid; (ii) insulin lispro, which differs from human insulin in the The penultimate lysine and proline residues on the C-terminus are reversed; (iii) insulin lysine, which differs from human insulin in that the asparagine of B3 is replaced by lysine and the lysine of B29 is replaced by Glutamate replacement; (iv) insulin glargine, which differs from human insulin in that the asparagine of A21 is replaced by glycine and the B chain is extended by two arginines at the carboxy terminus.

In some embodiments, the insulin analog is human insulin with a threonine deletion at position 30 of the B chain (also known as Des(B30) human insulin), and the amino acid sequences of the A and B chains of Des(B30) human insulin are shown below :

A chain: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1);

Chain B: FVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO: 2).

In some embodiments, the method for preparing acylated derivatives of insulin or insulin analogs further comprises the formula

The step that the compound of (I') removes the carboxyl protecting group to obtain the compound of formula (I):

Wherein, X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, such as C ₁ -C ₄ alkyl groups; R is as in formula (I) definition. Examples of _C1 - _C6 alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1, 1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl , 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2- Dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl or 2,3-dimethylbutyl base butyl. Examples of _C1 - _C4 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.

In some embodiments, the protecting group is methyl or tert-butyl.

In some embodiments, the method of preparing an acylated derivative of insulin or an insulin analog further comprises the step of reacting a compound of formula (III) with a compound of formula (IV) to obtain the compound of formula (I') ,

Wherein, W' is -OC(CH ₂ ) _n COOH, n is an integer from 2 to 10; X', Y, Z' are as defined above; R ₁ are independently electron withdrawing groups, and q is 1 to 4 the integer.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro. In some embodiments, R ₁ is propionyl. In some embodiments, q is 1, 2, or 3; eg, q is 1.

In some embodiments, the method of an acylated derivative of insulin or an insulin analog further comprises the step of reacting a compound of formula (III) with a compound of formula (IV) to provide said compound of formula (I'),

Wherein, W' is -OC(CH ₂ ) _n COOH, and n is an integer from 2 to 10;

Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, such as C ₁ -C ₄ alkyl groups; X is a dicarboxylic acid group containing a carboxylic acid group. Amino compound, X is connected with an acyl group in W to form an amide bond with one of its amino groups;

Z is -COOH;

R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro. In some embodiments, R ₁ is propionyl. In some embodiments, q is 1, 2, or 3; eg, q is 1.

In some embodiments, R has a structure selected from the group consisting of:

In some embodiments, n is an integer from 2 to 5, eg, n is 2. In some embodiments, X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10. In some embodiments, p is an integer from 2 to 6. In some embodiments, p is an integer from 2 to 4, eg, p is 4. In some embodiments, m is an integer from 10 to 16, eg, m is an integer from 12 to 14.

In some embodiments, the methods of the present disclosure achieve an acylation modification rate of insulin or insulin analog of at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% . The acylation modification rate can be determined by Phenomenex Gemini C18 liquid chromatography and calculated by the following formula:

In the formula,

is the average value of the ratio of the concentration of the reference solution to the main peak area;

is the average value of the main peak area in the test solution; C _i is the concentration of the test solution.

The present disclosure also provides a compound of formula (I):

R-W-X-Y-Z

(I)

where R is

R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4;

W is a diacyl structure with -OC( _CH2 )nCO-, wherein _n is an integer from 2 to 10;

X is a diamino compound containing a carboxylic acid group, and X is connected with one of its amino groups to an acyl group in W to form an amide bond;

Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

Z is -COOH.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. Examples of _{C1 -} C6alkanoyl groups include, but are not limited to: acetyl (-C(=O) _CH3 ), propionyl (-C(=O) _{CH2CH3 )} , butyryl (-C(=O) )C( _CH3 ) ₃ ) and benzoyl (-C(=O)Ph). Examples of _C1 - _C4 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl. Examples of halogens include, but are not limited to, F, Cl, Br, I.

In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro.

In some embodiments, R ₁ is propionyl.

In some embodiments, q is 1, 2, or 3; eg, q is 1.

In some embodiments, n is an integer from 2 to 5, eg, n is 2.

In some embodiments, X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10.

In some embodiments, p is an integer from 2 to 6.

In some embodiments, p is an integer from 2 to 4, eg, p is 4.

In some embodiments, m is an integer from 10 to 16, eg, m is an integer from 12 to 14.

In some embodiments, R has a structure selected from the group consisting of:

In some embodiments, the compound of formula (I) is a compound of formula (Ia) or a compound of formula (Ib):

wherein R is as defined above.

In some embodiments, the compound of formula (I) is:

In some embodiments, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% insulin or insulin analogs are achieved with compounds of formula (I) of the present disclosure acylation rate. The acylation modification rate can be determined by Phenomenex Gemini C18 liquid chromatography and calculated by the following formula:

In the formula,

is the average value of the ratio of the concentration of the reference solution to the main peak area;

is the average value of the main peak area in the test solution; C _i is the concentration of the test solution.

The present disclosure also provides a compound of formula (I'):

R-W-X’-Y-Z’

(I’)

Wherein, X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, such as C ₁ -C ₄ alkyl groups; R, W, X, Y and Z are as defined in formula (I).

In some embodiments, the protecting group is methyl or tert-butyl.

In some embodiments, the compound of formula (I') is:

The present disclosure also provides a method for preparing the compound of formula (I), which comprises the step of removing the carboxyl protecting group from the compound of formula (I') to obtain the compound of formula (I).

In some embodiments, the method of preparing a compound of formula (I) further comprises the step of reacting a compound of formula (III) with a compound of formula (IV) to obtain said compound of formula (I'),

Wherein, W' is -OC(CH ₂ ) _n COOH, n is an integer from 2 to 10; X', Y, Z' are as defined above; R ₁ are independently electron withdrawing groups, and q is 1 to 4 the integer.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro. In some embodiments, R ₁ is propionyl. In some embodiments, q is 1, 2, or 3; eg, q is 1.

The present disclosure also provides a method for preparing a compound of formula (I'), comprising the step of reacting a compound of formula (III) with a compound of formula (IV) to obtain said compound of formula (I'),

Wherein, W' is -OC(CH ₂ ) _n COOH, and n is an integer from 2 to 10;

Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, such as C ₁ -C ₄ alkyl groups; X is a dicarboxylic acid group containing a carboxylic acid group. Amino compound, X is connected with an acyl group in W to form an amide bond with one of its amino groups;

Z is -COOH;

R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4.

In some embodiments, R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, haloC ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid, or carboxyl. In some embodiments, R ₁ is selected from acetyl, propionyl, halo, or nitro. In some embodiments, R ₁ is selected from acetyl, propionyl, F, Cl, or nitro. In some embodiments, R ₁ is propionyl. In some embodiments, q is 1, 2, or 3; eg, q is 1.

In some embodiments, R has a structure selected from the group consisting of:

In some embodiments, n is an integer from 2 to 5, eg, n is 2. In some embodiments, X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10. In some embodiments, p is an integer from 2 to 6. In some embodiments, p is an integer from 2 to 4, eg, p is 4. In some embodiments, m is an integer from 10 to 16, eg, m is an integer from 12 to 14.

In some embodiments, acylated derivatives of insulin analogs of the present disclosure may be conventionally named Nα-(HOOC( _CH2 ) _{14CO ) -Nε-} ( ^OCCH2CH2CO- ( ^NεB29 - ^Des (B30) Human insulin))-Lys-OH, which has a specific structure of the following formula (IIa):

In some embodiments, acylated derivatives of insulin analogs of the present disclosure may be conventionally named lysine B29( ^Nε- ( ^Nα -hexadecanediacid-L-lysine- ^Nε- oxobutyryl)) des(B30) human insulin, which has the structure shown in formula (IIb):

In some embodiments, according to the preparation methods provided by the present disclosure, the yield of the acylated derivative of the insulin analog is not less than 55%, eg, the yield is about 55% to about 60%, about 55% to about 65% , about 55% to about 70%, about 57% to about 70%, about 55% to about 80%, about 55% to about 90%, about 55% to about 100%, about 60% to about 80%, about 60% to about 90%, about 65% to about 85%, about 65% to about 95%, about 70% to about 75%. In some specific embodiments, the yield of the acylated derivative of the insulin analog is from about 57% to about 70% according to the preparation methods provided by the present disclosure.

In some embodiments, according to the methods provided by the present disclosure, the acylation modification rate of the acylated derivative of the insulin analog is not less than 45%, eg, the modification rate is about 45% to about 55%, about 45% to about 60% %, about 45% to about 70%, about 45% to about 80%, about 45% to about 90%, about 45% to about 100%, about 50% to about 65%, about 50% to about 75%, About 50% to about 85%, about 50% to about 95%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%. In some specific embodiments, according to the methods provided by the present disclosure, the acylation modification rate of the acylated derivative of the insulin analog is from about 60% to about 70%.

In some embodiments, according to the methods provided by the present disclosure, the yield of the acylated derivative of the insulin analog is not less than 55% and the acylation modification rate is not less than 45%, eg, the yield is from about 55% to about 60%. %, about 55% to about 65%, about 55% to about 70%, about 57% to about 70%, about 55% to about 80%, about 55% to about 90%, about 55% to about 100%, About 60% to about 80%, about 60% to about 90%, about 65% to about 85%, about 65% to about 95%, about 70% to about 75%; acylation modification rate of about 45% to about 55%, about 45% to about 60%, about 45% to about 70%, about 45% to about 80%, about 45% to about 90%, about 45% to about 100%, about 50% to about 65% , about 50% to about 75%, about 50% to about 85%, about 50% to about 95%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%. In some embodiments, according to the methods provided by the present disclosure, the yield of the acylated derivative of the insulin analog is about 57% to about 70% and the acylation modification rate is about 60% to about 70%.

Reference in this disclosure to one or more embodiments means that one or more of the particular features described in that embodiment are included in at least one embodiment of the disclosure, and in addition, the feature may also be included in one or more embodiments Combinations in any manner, ie combinations of features of different embodiments, are also included within the scope of this disclosure and constitute different embodiments.

Detailed ways

For an easier understanding of the present disclosure, some technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "insulin" refers to natural insulin, such as human insulin, porcine insulin or bovine insulin.

The term "insulin analog" refers to a polypeptide molecule comprising one or more amino acid deletions, additions, substitutions, or combinations thereof relative to native insulin.

The term "insulin derivative" or "derivative of an insulin analog" refers to a polypeptide molecule to which one or more organic substituents (e.g., fatty acids) are attached to one or more amino acids on insulin or insulin analogs.

The term "about" means that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±20%, eg, a pH of about 5.5 means pH 5.5 ± 1.1. Furthermore, particularly with respect to biological systems or processes, the term can mean at most one order of magnitude or at most five times the value.

The present disclosure is further described below in conjunction with the examples, but these examples do not limit the scope of the present disclosure. The experimental methods that do not specify specific conditions in the examples of the present disclosure generally follow conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1 Preparation of active ester compound

Step 1: in a 2L there-necked flask, add compound X12 (100.0g, 189.75mmol) and triethylamine (38.4g, 375mmol), then add ultra-dry tetrahydrofuran (Tetrahydrofuran, THF) (1L) to dissolve, cool down to -5 Stir at °C. Compound X12 can be synthesized according to the method described in patent publication WO2018024186A. Succinic anhydride (21.85 g, 218 mmol) was added to the reaction flask in batches. After the addition, the mixture was stirred at -5 °C to 5 °C for 30 min, and then transferred to room temperature and stirred for 16.5 h. The completion of the reaction was monitored by thin layer chromatography (TLC) [dichloromethane:methanol (DCM:MeOH) = 10:1, phosphomolybdic acid developed]. THF was concentrated under reduced pressure and spin-dried. The residue was dissolved in ethyl acetate (1L), washed twice with 0.5M hydrochloric acid solution (1L×2), washed twice with saturated NaCl (1L×2), and the organic phase was dried with anhydrous sodium sulfate (100.0 g). Concentrate under reduced pressure until no droplets drip down, and the crude product is weighed to obtain compound X13 (123.25 g), which is directly used in the next reaction without purification. The purity of the product was 93.89% by liquid phase detection.

Step 1 Reaction formula:

Step 2: In a 3L three-necked flask, add compound X13 (119.0g), add 4-hydroxypropiophenone (34.2g), add DCM (2.38L) to dissolve, replace with argon for protection, cool down to 0°C in a cold trap, add 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) 45.12 g, after the addition was completed, the reaction was continued to be stirred at 0° C. for 30 min. The reaction solution was raised to room temperature and continued to be stirred for 2 hours, and the plate was sampled, and the reaction of compound X13 was basically complete. The reaction solution was directly concentrated under reduced pressure to obtain a crude compound X17 (221.61 g) with a purity of 62.05%. Purified by normal silica gel column (n-hexane/ethyl acetate) to obtain pure compound X17 (107.88 g) with a purity of 97.30%.

Step 2 Reaction formula:

Step 3: In a 2L single-neck flask, add compound X17 (107.8g), DCM (107.8mL), turn on stirring, add trifluoroacetic acid (646.8mL) to dissolve, and replace with argon for protection. Stir at room temperature for 3.5h, after TLC monitoring the basic reaction is complete, add isopropyl ether (2156mL) at one time to wash out, stir vigorously, and a white solid is precipitated (obvious exothermic phenomenon occurs during the washout process). After filtering, the filter cake was washed with isopropyl ether (200 mL×3). The filter cake was vacuum dried overnight. Compound X18 (87.14 g) was obtained by weighing, and the purity was 97.25% by liquid phase detection.

Step 3 Reaction formula:

In addition to X18, other active ester compounds X15, X19, X20 and X21 were also synthesized in this example (Table 1). Except for the different raw material compounds used to introduce R groups, the preparation process is the same as that of X18.

Table 1 Structures and raw material compounds of other active ester compounds

Example 2 Preparation of acylated derivatives of insulin analogs

In a 2L single-neck bottle, compound X18 (5g) was added, isopropanol (1L) was added, and the solution was dissolved by ultrasonic for 10min. Take another 3L three-necked bottle, add Des(B30) human insulin (28g/L, 787.50mL) in 50mM Tris-HCl buffer (pH 8.5), cool down to the external temperature of 0°C in a cold trap, use Na ₂ CO ₃ The solution (1.5 mol/L) was adjusted to pH 10.80 (130 mL of Na ₂ CO ₃ solution was added). Weigh 1,2,4-triazole (2.135g), add it to Des(B30) human insulin solution, and keep stirring at 0°C. The isopropanol solution of compound X18 was added to the Des (B30) human insulin solution with a peristaltic pump, and the flow rate was controlled to complete in 1 hour. After the addition was completed, the reaction was stirred at a temperature of 0°C in an ice-water bath for 1 hour. The reaction solution was transferred to room temperature and continued to stir for 2 hours. Take 347 μL (4 mg) of the reaction solution, add the prepared acetic acid solution (acetic acid: water = 1:10), adjust the pH to about 7.0 (the amount of acetic acid solution added is 70 μL), and the modification rate by liquid phase detection is 60.1%. Acetic acid was added dropwise to the reaction solution to adjust the pH to 7.0, and the reaction solution was transferred to a 3L glass bottle and sealed at -10°C for subsequent reverse silica preparation and purification.

Acylation reaction route of insulin analogs with compound X18:

In addition, a similar method to that described above was also adopted, except that the solvent was replaced by isopropanol with N,N-dimethylformamide, and the remaining steps were exactly the same to prepare Des(B30) insulin analog acylated derivatives. Among them, the liquid phase detection modification rate was 72%.

In this example, acylated derivatives of Des(B30) human insulin modified with active ester compounds X15, X19, X20 and X21 were also prepared, and the modification process was the same as that of X18.

Example 3 Examination of modification rate of acylated derivatives of insulin analogs

The modification rate was checked on the acylated derivatives of insulin analogs synthesized in Example 2, and the specific methods were as follows:

Chromatographic conditions

Column: Phenomenex Gemini C18

5μm 4.6×150mm column is suitable;

Flow rate: 1mL/min;

Mobile phase A: 0.02 mol/L anhydrous sodium sulfate-triethanolamine (100:1), pH 2.3 [weigh 2.84 g of anhydrous sodium sulfate, dissolve in water and dilute to 1000 mL, add 10 mL of triethanolamine and mix well, adjust pH with phosphoric acid value to 2.3];

Mobile phase B: acetonitrile;

Column temperature: 40℃;

Detection wavelength: 214nm;

Injection volume: 20μL; sample temperature control: 5°C;

Carry out gradient elution according to Table 2:

Table 2 Gradient elution conditions

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	75	25
10	65	35
35	60	40
45	20	80
55	20	80
60	75	25
68	75	25

Solution preparation

Diluent: acetonitrile-water (1:1.7);

System suitability solution: Take a aliquot and cryopreserved system suitability solution, and use it directly after thawing at room temperature;

Reference substance solution: take the reference substance (for example, lysine B29 prepared according to the method described in ^{WO2018024186A} ( ^Nε- (Nα-hexadecane fatty acid-L-lysine- ^Nε -oxobutyryl)) des (B30) human insulin) about 25mg, accurately weighed, placed in a 25mL volumetric flask, dissolved in a diluent and diluted to the mark, shaken to make a solution containing about 1mg per 1mL, as the reference solution (this solution Stable within 24h at 5℃±3℃).

The test solution in the control solution of the reaction solution: take an appropriate amount of the modified reaction solution, add the diluent [acetonitrile-water (1:1.7)] to dissolve and dilute to the mark, shake well, and prepare an acylated derivative containing insulin analogs per 1 mL The solution of about 1mg is used as the test solution (the solution is stable within 24h under the condition of 5℃±3℃).

test methods

Precisely measure 20 μL each of blank solution, system suitability solution, reference solution and test solution, inject them into a liquid chromatograph, and record the chromatogram. System suitability The resolution of the acylated derivatives of insulin analogs and the acylated derivatives of A ₂₁ deaminated insulin analogs (the relative retention time to the main peak is about 1.05) in the solution should meet the requirements.

calculation method

The modification rate of acylated derivatives of insulin analogs was calculated according to the following formula:

In the formula,

is the average value of the ratio of the concentration of the reference solution to the main peak area;

is the average value of the main peak area in the test solution; C _i is the concentration of the test solution.

Example 4 Long-term stability test of acylated derivatives of insulin analogs

In this example, a long-term stability test was carried out for X15 and X18, which was checked with reference to high performance liquid chromatography (General Chapter 0512 of the Fourth Part of the Chinese Pharmacopoeia, 2015 edition). The specific method is as follows:

1) Instrument: Agilent high performance liquid chromatograph;

2) Chromatographic conditions:

Mobile phase: use phosphoric acid water (pH=4.0, adjust pH to 4.0 with 5% phosphoric acid water) as mobile phase A, use acetonitrile as mobile phase B, and perform gradient elution according to Table 3.

Table 3 Gradient elution conditions

time (min)	Mobile phase A (%)	Mobile phase B (%)
0	65	35
15	50	50
25	15	85
40	15	85
40.1	65	35
45	65	35

Chromatographic column: octadecylsilane bonded silica gel as filler (YMC-Pack ODS-AQ 5μm 150×4.6mm is suitable); flow rate is 1.2mL per minute; column temperature is 10℃; detection wavelength is 200nm; injection volume is 10 μL; the diluent is methanol-acetonitrile (1:9), and the sample temperature is controlled at 10 °C.

3) System applicability:

Take an appropriate amount of X15 (or X18), accurately weigh it, add diluent to dissolve and dilute to make a solution containing about 0.5 mg per 1 mL, as the system suitability solution. Precisely measure 10 μL of the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram. The degree of separation between X15 (or X18) and known impurity 1 (RRT=0.74) and impurity 2 (RRT=1.09) should meet the requirements.

4) Preparation of the test solution:

Take an appropriate amount of X15 (or X18), accurately weigh it, add diluent to dissolve and dilute to make a solution containing about 0.5 mg per 1 mL, as the test solution.

5) Calculation formula of relative retention time:

Relative retention time = RT _impurity /RT _{X15 (or X18)}

6) Detection:

Precisely measure 10μL of the system suitability solution and inject it into the liquid chromatograph, record the chromatogram, the degree of separation between X15/X18 and known impurity 1 (RRT=0.74) and impurity 2 (RRT=1.09) should meet the requirements; 10 μL of the test solution was injected into the liquid chromatograph, and the chromatogram was recorded.

7) Calculate:

The content of each impurity was calculated according to the peak area normalization method.

Table 4 shows the long-term stability test results of X15 and X18. It can be seen that from the beginning of the test to 6 months, the purity of the main peak of X18 is always more than 10% higher than that of X15, normalized total impurities; after 6 months of the stability test, the purity of the main peak of X15 decreased by 0.9% (normalized %). The normalized total impurity % increased by 0.9%); while the main peak purity % of X18 decreased by only 0.2% (normalized total impurity % increased by only 0.2%). X18 has better long-term stability than X15.

Table 4 Long-term stability test (-20℃±5℃) results of X15 and X18

Table 5 summarizes the comparison results of active compounds X15, X18, X19 and X20 in terms of synthesis process, quality, modification amount and modification rate, wherein X15 is the active ester described in patent publication WO2018024186A. It can be seen that the molecular structures of the active esters X18, X19 and X20 are relatively stable, the process stability is good, and they can be scaled up to kilogram production. The ester to insulin analog feed ratio was reduced by 50%. In addition, X18 has advantages in yield, purity and modification rate compared to X19 and X20.

Table 5 Comparison of synthesis process, quality, modification amount and modification rate of each active ester compound

Although the above disclosure has been described in detail with the aid of examples for the sake of clarity of understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (18)

1. A method of preparing acylated derivatives of insulin or insulin analogs comprising the step of reacting a compound of formula (I) with insulin or insulin analogs:

   R-W-X-Y-Z

   (I)

   where R is

   

   R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4;

   W is a diacyl structure with -OC(CH ₂ ) _n CO-, where n is an integer from 2 to 10; W is one of its acyl groups with the A-chain or B-chain N-terminal amino acid of insulin or insulin analogs The α-amino group of the residue or the ε-amino group of a lysine residue present on the B-chain forms an amide bond;

   X is a diamino compound containing a carboxylic acid group, and X is connected with one of its amino groups to an acyl group in W to form an amide bond;

   Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

   Z is -COOH;

   Wherein, the insulin is natural insulin; and the insulin analog is a polypeptide molecule comprising 1 to 10 amino acid deletions, additions, substitutions or combinations thereof relative to natural insulin.
2. The method of claim 1, wherein:

   R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, halogenated C ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid or carboxyl; preferably, R ₁ is selected from acetyl, propionyl, halogen or nitro; more preferably, R ₁ is propionyl;

   q is 1, 2 or 3, preferably q is 1;

   W forms an amide bond with the ε-amino group of a lysine residue present on the B-chain; n is an integer from 2 to 5, more preferably n is 2;

   X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10, preferably an integer from 2 to 6, particularly preferably an integer from 2 to 4, most preferably 4;

   and / or

   m is an integer of 10 to 16, preferably an integer of 12 to 14.
3. The method of claim 1 or 2, wherein R has a structure selected from the group consisting of:

   
4. The method according to any one of claims 1-3, wherein the compound of formula (I) is a compound of formula (Ia) or a compound of formula (Ib):

   
5. The method of any one of claims 1-4, wherein the compound of formula (I) is:

   
6. The method according to any one of claims 1-5, the method comprising the steps of:

   (a) mixing the compound of formula (I) with an organic solvent selected from methanol, ethanol, acetonitrile, isopropanol, dimethyl sulfoxide or N,N-dimethylformamide to obtain solution A, Preferably isopropanol or N,N-dimethylformamide;

   (b) mixing said solution A with insulin or insulin analog; such that the equivalent ratio of compound of formula (I) to insulin or insulin analog is (1-4):1, preferably about 2:1; and

   (c) in the presence of a catalyst, preferably the catalyst is 1,2,4-triazole; at a reaction temperature of 0-25°C, preferably about 0°C; react for 1-8 hours, preferably for about 4 hours; the catalyst and The equivalence ratio of insulin or insulin analog is (1-12):1, preferably about 8:1.
7. The method of any one of claims 1-6, wherein the insulin is human insulin, porcine insulin or bovine insulin; the insulin analog is a deletion, addition, A polypeptide molecule of substitution or a combination thereof, preferably Des(B30) human insulin having an A chain as shown in SEQ ID NO:1 and a B chain as shown in SEQ ID NO:2.
8. The method according to any one of claims 1-7, the method further comprises the step of removing the carboxyl protecting group from the compound of formula (I') to obtain the compound of formula (I):

   

   Wherein, X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, preferably C ₁ -C ₄ alkyl groups, particularly preferably methyl groups or tert-butyl; R, W, X, Y and Z are as defined in any one of claims 1-5.
9. The method of claim 8, further comprising the step of reacting the compound of formula (III) with the compound of formula (IV) to obtain the compound of formula (I'),

   

   Wherein, W' is -OC(CH ₂ ) _n COOH, n is an integer from 2 to 10; R ₁ is independently an electron withdrawing group, q is an integer from 1 to 4; R, W, X', Y and Z' is as defined in any one of claims 1-5.
10. A compound of formula (I):

    R-W-X-Y-Z

    (I)

    where R is

    

    R ₁ are independently electron withdrawing groups, and q is an integer from 1 to 4;

    W is a diacyl structure with -OC( _CH2 )nCO-, wherein _n is an integer from 2 to 10;

    X is a diamino compound containing a carboxylic acid group, and X is connected with one of its amino groups to an acyl group in W to form an amide bond;

    Y is -A( _CH2 ) _m- , wherein A is absent or CO-; m is an integer from 6 to 32;

    Z is -COOH.
11. The compound of claim 10, wherein:

    R ₁ is selected from C ₁ -C ₆ alkanoyl, C ₁ -C ₄ alkyl, halogenated C ₁ -C ₄ alkyl, halogen, nitro, cyano, sulfonic acid or carboxyl; preferably, R ₁ is selected from acetyl, propionyl, halogen or nitro; more preferably, R ₁ is propionyl;

    q is 1, 2 or 3, preferably q is 1;

    n is an integer from 2 to 5, more preferably n is 2;

    X is -HN( _CH2 )pCH(COOH)NH-, wherein _p is an integer from 2 to 10, preferably an integer from 2 to 6, particularly preferably an integer from 2 to 4, most preferably 4;

    and / or

    m is an integer of 10 to 16, preferably an integer of 12 to 14.
12. The compound of claim 10 or 11, wherein R has a structure selected from the group consisting of:

    
13. The compound according to any one of claims 10-12, wherein the compound of formula (I) is a compound of formula (Ia) or a compound of formula (Ib):

    
14. The compound of any one of claims 10-13, wherein the compound of formula (I) is:

    
15. A compound of formula (I'):

    R-W-X’-Y-Z’

    (I’)

    Wherein, X' and Z' respectively represent the form in which all carboxyl groups in X and Z are protected by protective groups, and the protective groups are C ₁ -C ₆ alkyl groups, preferably C ₁ -C ₄ alkyl groups, particularly preferably methyl groups or tert-butyl; R, W, X, Y and Z are as defined in any one of claims 10-14.
16. The compound of claim 15, wherein the compound of formula (I') is:

    
17. A method for preparing the compound of formula (I) according to any one of claims 10-14, comprising the compound of formula (I') as claimed in claim 15 or 16, removing the carboxyl protecting group to obtain formula The step of the compound of (I).
18. The method of claim 17, further comprising the step of reacting a compound of formula (III) with a compound of formula (IV) to obtain said compound of formula (I'),

    

    Wherein, W' is -OC(CH ₂ ) _n COOH, n is an integer from 2 to 10; R ₁ is independently an electron withdrawing group, q is an integer from 1 to 4; R, W, X', Y and Z' is as defined in any of claims 10-16.