Glucose-sensitive insulin derivatives

Abstract

The present invention relates to novel insulin derivatives and their use in the treatment or prevention of medical conditions associated with diabetes. The insulin derivatives are glucose sensitive and exhibit glucose sensitive albumin binding. The insulin derivative has a long half-life period. The invention also relates to novel intermediates. Finally, the invention provides pharmaceutical compositions comprising the insulin derivatives of the invention, and the use of such compositions in the treatment or prevention of medical conditions associated with diabetes.

Description

Technical Field

[0001] This invention relates to novel insulin derivatives and their pharmaceutical uses. Furthermore, this invention relates to pharmaceutical compositions comprising such insulin derivatives, and to the use of such compounds in the treatment or prevention of medical conditions related to diabetes. Incorporation by Reference of Sequence Listing sequence list The electronic sequence listing is filed with this application. The entire contents of the sequence listing are incorporated herein by reference. Background Technology

[0002] Insulin is the most effective drug for treating hyperglycemia, but due to the narrow physiological glucose window, insulin administration involves a delicate balance between too much and too little. Healthy individuals have blood glucose levels close to 5 mM during fasting, while diabetic patients attempt to achieve close to 5 mM with dietary and basal insulin preparations. However, blood glucose levels below approximately 3 mM (hypoglycemia) frequently occur during insulin therapy, and hypoglycemia can lead to discomfort, loss of consciousness, brain damage, or death. Therefore, diabetic patients hesitate to aggressively treat their high or moderately high blood glucose levels due to fear of hypoglycemia. Developing insulin drugs that are active or released from the reservoir only at higher blood glucose levels, and inactive or weakly active at lower blood glucose levels, could be beneficial in diabetes management.

[0003] WO2020201041 discloses glucose-sensitive insulin derivatives having an arylboron-containing moiety, which exhibit glucose-sensitive albumin binding. Therefore, these insulin derivatives exhibit glucose concentration-dependent insulin activity. To allow for less frequent dosing, glucose-sensitive insulin derivatives with longer half-lives are needed. Summary of the Invention

[0004] In its broadest sense, the present invention relates to insulin derivatives. These insulin derivatives activate insulin receptors based on glucose concentration, thereby lowering blood glucose levels, and thus act as glucose-sensitive insulin derivatives.

[0005] The insulin derivative of the present invention binds to both glucose and albumin (human serum albumin, HSA), and the HSA affinity is glucose-sensitive, meaning the HSA affinity is glucose-sensitive. Therefore, the apparent human insulin receptor (HIR) affinity in the presence of HSA also becomes glucose-sensitive. The portion of insulin bound to HSA is masked and cannot bind to the HIR, but glucose-induced release from HSA increases the free portion of insulin, thus glucose increases the apparent HIR affinity. Therefore, in the presence of HSA, such as in blood, the insulin derivative of the present invention exhibits a higher apparent insulin receptor affinity in the presence of glucose than in the absence of glucose. Therefore, the insulin derivative of the present invention is glucose-sensitive.

[0006] The insulin derivative of the present invention exhibits high glucose sensitivity and a long half-life.

[0007] In one aspect, the insulin derivative of the present invention comprises: an insulin peptide containing a Lys residue (B29K) at position 29 of the insulin peptide B chain; a peptide extension at the N-terminus of the insulin peptide B chain; and three modifying groups, one of which is attached to the Lys residue at position 29 of the insulin peptide B chain, and the other two modifying groups are attached to the Lys residue in the peptide extension.

[0008] In one aspect, the peptide extension has the following sequence Z-Lys-Y-Lys-aa1-aa2-(Gly)3-Ser-((Gly)4-Ser) p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Where aa1 either does not exist or is Pro, Where aa2 is Glu or Gly. Where p is 1, 2, or 3, and The # symbol represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues.

[0009] In one aspect, the modifying group is a group of formula A: , One of R1, R2, or R3 is an electron-withdrawing group, while the other two are hydrogen. Where m is 0, 1, 2 or 3; and where n is 0, 1, 2 or 3. Indicates the connection point.

[0010] In one aspect, the present invention relates to pharmaceutical compositions comprising an insulin derivative according to the invention. In one aspect, the present invention relates to an insulin derivative according to the invention used as a medicine. In one aspect, the present invention relates to an insulin derivative according to the invention for the treatment of diabetes. In one aspect, the present invention relates to the pharmaceutical use of an insulin derivative according to the invention.

[0011] In one aspect, the insulin derivative of the present invention exhibits high glucose sensitivity. In another aspect, the insulin derivative of the present invention has a long half-life. In another aspect, the insulin derivative of the present invention exhibits both high glucose sensitivity and a long half-life.

[0012] The present invention can also solve other problems that will become apparent from the disclosure of exemplary embodiments. Attached Figure Description

[0013] Figure 1 The concentration-response curves of insulin receptor phosphorylation in human primary hepatocytes at 3 mM and 20 mM D-glucose, respectively, were plotted for the insulin derivative of Example 4 and human insulin. Figure 1 The data in this example comes from Example 13.

[0014] Figure 2 The representative concentration-response curves for the insulin derivative of Example 4, obtained from adipogenesis assays (rFFC) in adipocytes of Sprague Dawley rats at 3 mM and 20 mM L-glucose were depicted. Figure 2 The data in this example comes from Example 14.

[0015] Figure 3 The concentration-response curves of glycogen accumulation in rat primary hepatocytes at 20 mM D-glucose for the insulin derivative of Example 4 and human insulin were depicted. Figure 3 The data in this example comes from Example 15. Detailed Implementation

[0016] In one aspect, the insulin derivative of the present invention comprises an insulin peptide, a peptide extension located at the N-terminus of the insulin peptide B chain, and three modifying groups.

[0017] Insulin derivative As used herein, the term "insulin derivative" refers to a modified insulin peptide, wherein the modification is in the form of connecting chemical parts and / or the presence of peptide extensions.

[0018] In one aspect, the modification is in the form of a modifying group covalently linked to form A. In another aspect, the modification is in the form of a peptide elongation present at the N-terminus of the insulin peptide B chain. In yet another aspect, the modification is in the form of a modifying group covalently linked to form A and a peptide elongation present at the N-terminus of the insulin peptide B chain.

[0019] Insulin peptide As used herein, the term "insulin peptide" refers to a peptide that is human insulin or a human insulin analogue. In one embodiment, as used herein, the term "insulin peptide" refers to a peptide that is human insulin or an analogue of insulin having insulin activity (i.e., the ability to activate the insulin receptor).

[0020] Human insulin As used herein, the term "human insulin" refers to the human insulin hormone, the structure and properties of which are well known. Human insulin has two polypeptide chains, designated as chain A and chain B. Chain A is a 21-amino acid peptide, while chain B is a 30-amino acid peptide. These two chains are linked by disulfide bonds: a first disulfide bond between cysteine ​​residues at positions 7 of chain A and 7 of chain B, and a second disulfide bond between cysteine ​​residues at positions 20 of chain A and 19 of chain B. A third disulfide bond exists between cysteine ​​residues at positions 6 and 11 of chain A. The human insulin chain A has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1), while the human insulin chain B has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).

[0021] Insulin analogue As used herein, the term "insulin analogue" means modified human insulin in which one or more amino acid residues of insulin have been replaced with other amino acid residues, and / or one or more amino acid residues have been deleted from insulin, and / or one or more amino acid residues have been added to and / or inserted into insulin. As used herein, the term "insulin analogue" means an insulin analogue that exhibits insulin activity (i.e., binds to and activates the insulin receptor).

[0022] Human insulin analogues contain fewer than 10 amino acid modifications (substitution, deletion, addition (i.e., extension), insertion, or any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin. In one aspect, human insulin analogues have fewer than 10 amino acid modifications (substitution, deletion, addition (i.e., extension), insertion, or any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin.

[0023] Modifications in the insulin molecule are indicated by specifying the chain (A or B) in which the amino acid residue that replaces a native amino acid residue, its position, and a single-letter or three-letter code. In this document, terms such as “A1,” “A2,” and “A3” represent amino acids at positions 1, 2, and 3 (counting from the N-terminus) in the insulin A chain, respectively. Similarly, terms such as B1, B2, and B3 represent amino acids at positions 1, 2, and 3 (counting from the N-terminus) in the insulin B chain, respectively. Using single-letter codes for amino acids, the term B29K indicates that the amino acid at position B29 is K. Using three-letter codes for amino acids, the corresponding designation is B29Lys. The term “desB30” refers to an insulin analog lacking the B30 amino acid.

[0024] In certain embodiments, an analog "has" or "contains" the specified changes. In other specific embodiments, an analog "composes of the changes." When the terms "composes" or "consisting of" are used in connection with an analog, such as when an analog is composed of a specified set of amino acid mutations, it should be understood that the specified amino acid mutations are the only amino acid mutations in the analog. Conversely, an analog "containing" a specified set of amino acid mutations may have additional mutations.

[0025] Examples of insulin analogs include: desB30 human insulin (chain A of SEQ ID NO:1 and chain B of SEQ ID NO:3).

[0026] Peptide extension As described above, the insulin analogues of the present invention contain fewer than 10 amino acid modifications (substitution, deletion, addition (i.e., extension), insertion, and any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin. In addition to these up to 9 modifications, the insulin peptide of the present invention also has a peptide extension at the N-terminus of the B chain of the insulin peptide.

[0027] Various peptide extensions or spacer peptides are known in the art and can be used in the insulin derivatives of the present invention. In one embodiment, the peptide extension is a peptide segment consisting of 28-58 amino acids linked by peptide bonds. The peptide extension of the present invention comprises one or more of the following amino acid residues: Gly (G), Glu (E), Ala (A), Ser (S), Thr (T), Pro (P), Gln (Q), and Lys (K).

[0028] The peptide extension in the insulin derivative of this invention contains two Lys(K) residues to allow the attachment of two modifying groups. The exact attachment positions of the modifying groups may vary, and some variation in the exact peptide sequence is allowed.

[0029] The peptide extension used in the insulin derivative of the present invention has the following peptide sequence: Z-Lys-Y-Lys-aa1-aa2-(Gly)3-Ser-((Gly)4-Ser) p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Where aa1 either does not exist or is Pro, Where aa2 is Glu or Gly. Where p is 1, 2, or 3, and The # symbol represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues.

[0030] In one embodiment, the peptide extension maintains overall polarity, for example by having at least five glutamic acid (Glu) amino acid residues in the peptide extension.

[0031] When recombinant insulin is produced, it is expressed as a single-chain insulin precursor, subsequently processed using proteases such as hydrolyzed Achromobacterium (…). Achromobacter lyticus Lysyl-specific endonuclease (ALP) or trypsin matures it into double-chain insulin. ALP specifically and exclusively cleaves the lysyl bond, including the lysyl-proline bond. However, since the peptide extension used in the insulin derivatives of the present invention contains two lysine (Lys) residues, ALP cleavage can lead to undesirable over-cleavage products. Unexpectedly, it was found that by having Pro-Glu after Lys, the cleavage rate of ALP was greatly reduced, thereby reducing over-cleavage and increasing the overall yield.

[0032] In one embodiment, the peptide extension used in the insulin derivative of the present invention has the following peptide sequence: Z-Lys-Y-Lys-aa1-aa2-(Gly)3-Ser-((Gly)4-Ser) p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Y consists of Pro-Gly and then 13 to 28 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Where aa1 is Pro, Where aa2 is Glu, Where p is 1, 2, or 3, and The # symbol represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues.

[0033] More specifically, the peptide elongation is linked to the insulin peptide through the carboxylic acid of the C-terminal serine residue of the peptide elongation and the amino acid of the N-terminal amino acid residue of the insulin peptide B chain, forming an amide bond between the two amino acid residues.

[0034] Examples of peptide extensions located at the N-terminus of the insulin peptide B chain include: GKPE(GEQP)4GEQGGKPEGGGS(G4S)2 (SEQ ID NO:4).

[0035] Modifying group A The insulin derivative of the present invention comprises three modifying groups A. In one embodiment, the insulin derivative of the present invention has exactly three modifying groups of formula A. These modifying groups may be the same or different. In one embodiment, these modifying groups are the same. The modifying groups are covalently linked to the ε-amino group of lysine residues in the insulin peptide and the peptide extension.

[0036] The modifying group of the insulin derivative of the present invention is a group of formula A: , One of R1, R2, or R3 is an electron-withdrawing group, while the other two are hydrogen. Where m is 0, 1, 2, or 3; and Where n is 0, 1, 2 or 3.

[0037] In all general formulas of this application, B represents a boron atom, H represents a hydrogen atom, N represents a nitrogen atom, and O represents an oxygen atom.

[0038] In other words, R1, R2, and R3 are independently selected from electron-withdrawing groups and hydrogen, provided that if R1 is an electron-withdrawing group, then R2 and R3 are hydrogen; if R2 is an electron-withdrawing group, then R1 and R3 are hydrogen; and if R3 is an electron-withdrawing group, then R1 and R2 are hydrogen.

[0039] In one embodiment, R1 is an electron-withdrawing group, while R2 and R3 are hydrogen atoms.

[0040] To adjust the pKa of boric acid and benzoxoboron pentane ring to promote glucose binding at neutral pH, electron-withdrawing groups are attached.

[0041] An electron-withdrawing group (EWG) is an atom or group that attracts electron density from neighboring atoms to itself. In one embodiment, the electron-withdrawing group is selected from CF3, F, NO2, CN, COX, SO2X, and POX2, wherein X is OR or NR2, and R is independently selected from H, alkyl, and aryl. In one embodiment, the electron-withdrawing group is selected from CF3, F, NO2, CN, COX, and SO2X, wherein X is OR or NR2, and R is independently selected from H, alkyl, and aryl. In one embodiment, the electron-withdrawing group is CF3, F, SO2NR2, or CN, wherein R is alkyl. In one embodiment, the electron-withdrawing group is CF3. In one embodiment, R1 is CF3, while R2 and R3 are hydrogen.

[0042] In the insulin derivative of the present invention, one modifying group is attached to a lysine (Lys) residue at position 29 of the B chain of the insulin peptide, while two other modifying groups are attached to each lysine (Lys) residue in the peptide extension. More specifically, each modifying group is attached to the ε-amino group of the lysine residue, forming an amide bond.

[0043] Formula A is obtained by using The connection point is covalently connected to Lys.

[0044] The modifying group of formula A has two chiral centers. Each chiral atom in the modifying group of formula A can be independently ( R )-type or ( S )-type. In one embodiment, both chiral atoms are ( S - type. In yet another embodiment, the chiral center is as shown in formula A1: R1, R2, R3, m, and n are defined above for equation A.

[0045] In a particular embodiment, the modifying group is a group of formula A, wherein R1 is CF3, R2 and R3 are hydrogen, m is 2 and n is 0.

[0046] In a particular embodiment, the modifying group is a group of formula A1, wherein R1 is CF3, R2 and R3 are hydrogen, m is 2 and n is 0.

[0047] Function of the insulin derivative of the invention Unexpectedly, the insulin derivative of the present invention exhibits both a long half-life and high glucose sensitivity in the presence of HSA.

[0048] The relative binding affinity of insulin analogs and insulin derivatives to the human insulin receptor (HIR) can be determined by competitive binding in a scintillation proximity assay (SPA) as described in Example 12.

[0049] In one embodiment, the insulin derivative of the present invention has the ability to bind to an insulin receptor. In one embodiment, the insulin derivative of the present invention exhibits a higher apparent insulin receptor affinity in the presence of HSA and 20 mM glucose compared to the absence of glucose. The increase in apparent relative affinity (HIR glucose factor) from 0 to 20 mM glucose in the presence of HSA reflects the glucose sensitivity of the insulin derivative. The glucose factor is greater than 1 when the relative insulin receptor affinity in the presence of 20 mM glucose is higher than that in the absence of glucose. In one embodiment, the glucose factor of the insulin derivative of the present invention is at least 20, 30, or 40 in the presence of 1.5% HSA.

[0050] As can be seen from the data shown in Example 12, the peptide extension located at the N-terminus of the insulin backbone B chain resulted in decreased glucose sensitivity in the presence of HSA, as measured by the fold change in affinity (glucose factor) from 0 to 20 mM glucose. Unexpectedly, the insulin derivative of the present invention exhibited high glucose sensitivity in the presence of HSA.

[0051] The insulin receptor phosphorylation assay described in Example 13, the lipogenesis assay described in Example 14, and the glycogen accumulation assay described in Example 15 can be used as measurements of the functional (agonistic) activity of the insulin derivative. Data from Examples 13, 14, and 15 show that the insulin derivative of the present invention activates the insulin receptor. The half-life (T½) after subcutaneous administration to LYD pigs can be determined using the method described in Example 16. Pharmacokinetic (PK) studies in LYD pigs in Example 16 show that the compounds of the present invention exhibit a long half-life (T½).

[0052] Intermediate product Furthermore, the present invention also provides an intermediate product in the form of an insulin analog having a peptide extension at the N-terminus of the insulin peptide.

[0053] Examples of insulin analogs with a peptide elongation at the N-terminus of the insulin peptide B chain include: GKPE(GEQP)4GEQGGKPEGGGS(G4S)2-B1 desB30 Human Insulin (A chain of SEQ ID NO:1 and B chain of SEQ ID NO:5).

[0054] GKPE(GEQP)4GEQGGKPEGGGS(G4S)2-B1 desB30 human insulin refers to desB30 human insulin derived from GKPE(GEQP)4GEQGGKPEGGGS(G4S)2, which is derived from B1. The C-terminal serine (S) is linked to the phenylalanine (F) at position B1 of desB30 human insulin.

[0055] General definitions The term "compound" is used herein to refer to a molecular entity; therefore, "compound" may have different structural elements in addition to the smallest element defined for each compound or group of compounds. The term "compound" is also intended to cover its pharmaceutically relevant form, namely, the present invention relates to compounds as defined herein or their pharmaceutically acceptable salts, amides, or esters.

[0056] For example, the term "peptide" or "polypeptide" as used in the context of this invention refers to a compound comprising a series of amino acids linked together by amide (or peptide) bonds. In a particular embodiment, a peptide consists of amino acids linked together by peptide bonds.

[0057] The term "amino acid" includes proteinogenic (or naturally occurring) amino acids (of which there are 20 standard amino acids) and non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids either do not exist in proteins or are not produced through standard cellular mechanisms (e.g., they may have undergone post-translational modifications).

[0058] Typically, amino acid residues (peptide / protein sequences) can be represented by their full name, their single-letter code, and / or their three-letter code. These three methods are completely equivalent. Throughout this application, the individual amino acids of the insulin peptide and peptide extension of the present invention, unless their optical isomers are specified, should be understood to refer to... L -Isomers (unless otherwise stated). Amino acids are molecules containing amino and carboxylic acid groups and optionally one or more additional groups, often referred to as side chains.

[0059] In this document, the term "amino acid residue" refers to an amino acid in which the hydroxyl group has been removed from the carboxyl group, and / or an amino acid in which the hydrogen atom has been removed from the amino group.

[0060] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and tetradecyl.

[0061] The term "aryl" refers to a cyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl.

[0062] The terms “including” or “contains” should be interpreted as meaning that the subject matter under discussion includes those specific features, but does not exclude the existence of other features.

[0063] Pharmaceutical composition The present invention also relates to pharmaceutical compositions comprising the insulin derivative of the present invention or a pharmaceutically acceptable salt, amide, or ester thereof, and one or more pharmaceutically acceptable excipients. Such compositions can be prepared as is known in the art.

[0064] The term “excipient” broadly refers to any component other than the active therapeutic ingredient. Excipients can be inert, inactive, and / or non-pharmaceutical active. Excipients can be used for a variety of purposes, such as as carriers, loading agents, diluents, and / or to improve administration and / or absorption of the active ingredient. Non-limiting examples of excipients include solvents, diluents, buffers, preservatives, tension modifiers, chelating agents, and stabilizers. Formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions).

[0065] The compositions of the present invention can be in the form of a liquid formulation, i.e., an aqueous formulation containing water. The liquid formulation can be a solution or a suspension. The compositions of the present invention can be used for parenteral administration, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0066] Arylboronic compounds, such as arylboronic acids, may exhibit low stability in aqueous solutions with a near-neutral pH. The insulin derivatives of this invention show improved stability in aqueous solutions. For example, stability can be assessed by placing the insulin derivative in an aqueous solution at a neutral pH at 25 or 37 degrees Celsius for an extended period, such as one week, followed by measuring the purity of the insulin derivative.

[0067] Pharmaceutical indication Diabetes The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas produces insufficient amounts of insulin, or in which the body's cells do not respond properly to insulin, thus preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.

[0068] Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is caused by the destruction of beta cells and usually results in an absolute lack of insulin.

[0069] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance and is therefore associated with relative insulin deficiency and / or major insulin secretion defects with insulin resistance.

[0070] In one embodiment, the insulin derivative according to the invention is used as a medicament for treating or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes such as metabolic syndrome X or insulin resistance syndrome. In one embodiment, the insulin derivative according to the invention is used as a medicament for treating or preventing hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes. In another embodiment, the insulin derivative according to the invention is used as a medicament for delaying or preventing the progression of type 2 diabetes. The term "treatment" is intended to include prevention and minimization of the mentioned disease, condition, or status (i.e., "treatment" means the prophylactic and therapeutic administration of the insulin derivative of the invention or a composition containing the insulin derivative of the invention, unless otherwise stated or clearly contradicted by the context).

[0071] Mode of administration The route of administration can be any route by which the insulin derivative of the present invention is efficiently delivered to the desired or appropriate location in the body, such as parenteral, subcutaneous, intramuscular or intravenous.

[0072] For parenteral administration, the insulin derivative of the present invention is formulated in a manner similar to that of known insulins. Furthermore, for parenteral administration, the insulin derivative of the present invention can be administered in a manner similar to that of known insulins, and the physician is familiar with this procedure.

[0073] The dosage of the insulin derivative of the present invention to be administered is determined in consultation with a physician familiar with diabetes treatment, the frequency of administration of the insulin derivative of the present invention is determined, and which one or more compounds of the present invention may be optionally administered together with another antidiabetic compound.

[0074] Non-limiting embodiments The invention is further described through the following non-limiting embodiments: 1. An insulin derivative comprising A. An insulin peptide containing B29K; B. Peptide extension Z-Lys-Y-Lys-aa1-aa2-(Gly)3-Ser-((Gly)4-Ser) p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Where aa1 either does not exist or is Pro, Where aa2 is Glu or Gly. Where p is 1, 2, or 3, and Where # represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues; C. Modifying groups of the three formulas A: , One of R1, R2, or R3 is an electron-withdrawing group, while the other two are hydrogen. Where m is 0, 1, 2 or 3; Where n is 0, 1, 2 or 3; in Indicates the connection point, and One of the modifying groups is attached to the Lys residue at position 29 of the B chain of the insulin peptide, while the other two modifying groups are attached to each Lys residue in the peptide extension.

[0075] 2. The insulin derivative according to the aforementioned implementation scheme, wherein Z consists of 1 to 4 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0076] 3. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of 1 to 3 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0077] 4. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of one or two amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0078] 5. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of one amino acid residue independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0079] 6. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of 1 to 4 Gly amino acid residues.

[0080] 7. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of 1 to 3 Gly amino acid residues.

[0081] 8. The insulin derivative according to any one of the foregoing embodiments, wherein Z consists of 1 to 2 Gly amino acid residues.

[0082] 9. An insulin derivative according to any one of the foregoing embodiments, wherein Z is Gly.

[0083] 10. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0084] 11. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0085] 12. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0086] 13. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0087] 14. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0088] 15. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Ala, Pro and Gln.

[0089] 16. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Ala, Pro and Gln.

[0090] 17. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Ala, Pro and Gln.

[0091] 18. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 18 to 23 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0092] 19. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 20 to 22 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro and Gln.

[0093] 20. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 21 amino acid residues independently selected from Gly, Ala, Ser, Thr, Pro and Gln.

[0094] 21. The insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 18 to 23 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0095] 22. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 20 to 22 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0096] 23. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Pro-Gly and subsequently 21 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0097] 24. An insulin derivative according to any one of the foregoing embodiments, wherein Y is PE(GEQP)4GEQGG (SEQ ID NO:13).

[0098] 25. An insulin derivative according to any one of the foregoing embodiments, wherein aa1 is Pro.

[0099] 26. The insulin derivative according to any one of the foregoing embodiments, wherein aa2 is Glu.

[0100] 27. An insulin derivative according to any one of the foregoing embodiments, wherein p is 2.

[0101] 28. An insulin derivative according to any one of the foregoing embodiments, wherein the peptide extension is GKPE(GEQP)4GEQGGKPEGGGS(G4S)2 (SEQ ID NO:4).

[0102] 29. The insulin derivative according to any one of the foregoing embodiments, wherein the modifying group is a group of formula A1: One of R1, R2, or R3 is an electron-withdrawing group, while the other two are hydrogen. Where m is 0, 1, 2 or 3; Where n is 0, 1, 2, or 3; and in Indicates the connection point.

[0103] 30. An insulin derivative according to any one of the foregoing embodiments, wherein the electron-withdrawing group is selected from CF3, F, NO2, CN, COX, SO2X and POX2, wherein X is OR or NR2 and R is H, alkyl or aryl.

[0104] 31. The insulin derivative according to any one of the foregoing embodiments, wherein the electron-withdrawing group is selected from CF3, F, NO2, CN, COX and SO2X, wherein X is OR or NR2 and R is H, alkyl or aryl.

[0105] 32. An insulin derivative according to any one of the foregoing embodiments, wherein X is OR.

[0106] 33. An insulin derivative according to any one of the foregoing embodiments, wherein X is NR2.

[0107] 34. An insulin derivative according to any one of the foregoing embodiments, wherein R is H.

[0108] 35. The insulin derivative according to any one of the foregoing embodiments, wherein R is an alkyl group.

[0109] 36. The insulin derivative according to any one of the foregoing embodiments, wherein the alkyl group is selected from methyl, ethyl, n-propyl and isopropyl.

[0110] 37. The insulin derivative according to any one of the foregoing embodiments, wherein the alkyl group is selected from methyl and ethyl.

[0111] 38. The insulin derivative according to any one of the foregoing embodiments, wherein the alkyl group is methyl.

[0112] 39. An insulin derivative according to any one of the foregoing embodiments, wherein R is an aryl group.

[0113] 40. The insulin derivative according to any one of the foregoing embodiments, wherein the aryl group is phenyl.

[0114] 41. An insulin derivative according to any one of the foregoing embodiments, wherein the electron-withdrawing group is selected from CF3, F, SO2NR2 or CN, wherein R is an alkyl group.

[0115] 42. The insulin derivative according to any one of the foregoing embodiments, wherein the electron-withdrawing group is selected from CF3 and F.

[0116] 43. The insulin derivative according to any one of the foregoing embodiments, wherein the electron-withdrawing group is CF3.

[0117] 44. An insulin derivative according to any one of the foregoing embodiments, wherein R1 is an electron-withdrawing group and R2 and R3 are hydrogen.

[0118] 45. An insulin derivative according to any one of the foregoing embodiments, wherein R1 is CF3 and R2 and R3 are hydrogen.

[0119] 46. ​​An insulin derivative according to any one of the foregoing embodiments, wherein m is 0, 1, 2 or 3 and n is 0.

[0120] 47. An insulin derivative according to any one of the foregoing embodiments, wherein m is 1, 2 or 3 and n is 0.

[0121] 48. An insulin derivative according to any one of the foregoing embodiments, wherein m is 3 and n is 0.

[0122] 49. An insulin derivative according to any one of the foregoing embodiments, wherein m is 2 and n is 0.

[0123] 50. An insulin derivative according to any one of the foregoing embodiments, wherein m is 1 and n is 0.

[0124] 51. An insulin derivative according to any one of the foregoing embodiments, wherein m is 0 and n is 3.

[0125] 52. An insulin derivative according to any one of the foregoing embodiments, wherein m is 0 and n is 2.

[0126] 53. An insulin derivative according to any one of the foregoing embodiments, wherein m is 0 and n is 1.

[0127] 54. An insulin derivative according to any one of the foregoing embodiments, wherein both chiral atoms in formula A are of the (S)-type.

[0128] 55. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is human insulin.

[0129] 56. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog.

[0130] 57. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 10 amino acid modifications relative to human insulin.

[0131] 58. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 9 amino acid modifications relative to human insulin.

[0132] 59. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 8 amino acid modifications relative to human insulin.

[0133] 60. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 7 amino acid modifications relative to human insulin.

[0134] 61. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 6 amino acid modifications relative to human insulin.

[0135] 62. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 5 amino acid modifications relative to human insulin.

[0136] 63. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 4 amino acid modifications relative to human insulin.

[0137] 64. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than 3 amino acid modifications relative to human insulin.

[0138] 65. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing fewer than two amino acid modifications relative to human insulin.

[0139] 66. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is a human insulin analog containing desB30.

[0140] 67. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide is desB30 human insulin.

[0141] 68. An insulin derivative according to any one of the foregoing embodiments, The insulin peptide mentioned is desB30 human insulin. The peptide extension is Z-Lys-Y-Lys-Pro-Glu-(Gly)3-Ser-((Gly)4-Ser). p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Pro, and Gln. Y consists of Lys-Pro and the following 13 to 23 amino acid residues independently selected from Gly, Glu, Pro and Gln. Where p is 1, 2, or 3; Where # represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues; and Where R1 is CF3, R2 and R3 are hydrogen, m is 2 and n is 0.

[0142] 69. An insulin derivative according to any one of the foregoing embodiments, wherein Z consists of 1 to 3 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0143] 70. An insulin derivative according to any one of the foregoing embodiments, wherein Z consists of Gly and optionally one or two amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0144] 71. An insulin derivative according to any one of the foregoing embodiments, wherein Z is Gly.

[0145] 72. An insulin derivative according to any one of the foregoing embodiments, wherein Y consists of Lys-Pro and subsequently 20 to 25 amino acid residues independently selected from Gly, Glu, Pro and Gln.

[0146] 73. An insulin derivative according to any one of the foregoing embodiments, wherein p is 2.

[0147] 74. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide has the ability to bind to an insulin receptor.

[0148] 75. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin peptide has the ability to bind to and activate an insulin receptor.

[0149] 76. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has the ability to bind to an insulin receptor.

[0150] 77. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has the ability to activate an insulin receptor.

[0151] 78. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a higher apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) compared to the absence of glucose.

[0152] 79. An insulin derivative according to any one of the foregoing embodiments, wherein, in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose, the insulin derivative has at least 20 times higher apparent insulin receptor affinity.

[0153] 80. An insulin derivative according to any one of the foregoing embodiments, wherein, in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose, the insulin derivative has at least 30 times higher apparent insulin receptor affinity.

[0154] 81. An insulin derivative according to any one of the foregoing embodiments, wherein, in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose, the insulin derivative has at least 40 times higher apparent insulin receptor affinity.

[0155] 82. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has an apparent insulin receptor affinity that is 20 to 80 times higher in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose.

[0156] 83. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has an apparent insulin receptor affinity that is 30 to 60 times higher in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose.

[0157] 84. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has an apparent insulin receptor affinity that is 40 to 60 times higher in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose.

[0158] 85. The insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has 50 to 60 times higher apparent insulin receptor affinity in the presence of 20 mM glucose and 1.5% human serum albumin (HSA) compared to the absence of glucose.

[0159] 86. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin receptor affinity is determined by the assay described in Example 12.

[0160] 87. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ of at least 15 hours after subcutaneous administration to LYD pigs.

[0161] 88. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ of at least 16 hours after subcutaneous administration to LYD pigs.

[0162] 89. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ of at least 17 hours after subcutaneous administration to LYD pigs.

[0163] 90. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ of at least 18 hours after subcutaneous administration to LYD pigs.

[0164] 91. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ of at least 19 hours after subcutaneous administration to LYD pigs.

[0165] 92. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ in the range of 15 to 25 hours after subcutaneous administration to LYD pigs.

[0166] 93. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ in the range of 17 to 23 hours after subcutaneous administration to LYD pigs.

[0167] 94. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative has a T½ in the range of 18 to 23 hours after subcutaneous administration to LYD pigs.

[0168] 95. An insulin derivative according to any one of the foregoing embodiments, wherein the T½ is determined by the method described in Example 16.

[0169] 96. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is the insulin derivative of Example 4.

[0170] 97. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is .

[0171] 98. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is .

[0172] 99. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is .

[0173] 100. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is .

[0174] 101. An insulin derivative according to any one of the foregoing embodiments, wherein the insulin derivative is .

[0175] 102. An insulin derivative, wherein the insulin derivative is the insulin derivative of Example 4.

[0176] 103. An insulin derivative, wherein the insulin derivative is .

[0177] 104. A composition comprising an insulin derivative according to any one of embodiments 1 to 103.

[0178] 105. An insulin derivative according to any one of embodiments 1-103, which is used as a medicine.

[0179] 106. An insulin derivative according to any one of embodiments 1-103, used for the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and metabolic syndromes such as metabolic syndrome X or insulin resistance syndrome.

[0180] 107. An insulin derivative according to any one of embodiments 1-103, used for the prevention or treatment of diabetes, including type 1 diabetes and type 2 diabetes.

[0181] 108. An insulin derivative according to any one of embodiments 1 to 103, used for the prevention or treatment of diabetes.

[0182] 109. An insulin derivative according to any one of embodiments 1 to 103, used for the treatment of diabetes.

[0183] 110. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 in the preparation of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes such as metabolic syndrome X or insulin resistance syndrome.

[0184] 111. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 in the preparation of a medicament for the treatment or prevention of diabetes, including type 1 diabetes and type 2 diabetes.

[0185] 112. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 in the preparation of a medicament for the treatment or prevention of diabetes.

[0186] 113. A method for treating or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes such as metabolic syndrome X or insulin resistance syndrome, the method comprising administering to a subject in need a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.

[0187] 114. A method for treating or preventing diabetes, including type 1 diabetes and type 2 diabetes, the method comprising administering to a subject in need a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.

[0188] 115. A method for treating or preventing diabetes, the method comprising administering to a subject in need a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.

[0189] Example Insulin conjugates in the examples are depicted using standard single-letter abbreviations for amino acids. The sulfur atom of the cysteine ​​residue is specifically drawn to illustrate the disulfide bond. Residues modified by coupling are drawn to accurately show the location of the modification in the relevant amino acid. Following peptide chemistry standards, the N-terminus of insulin is indicated by a small-font H-, and the C-terminus by a small-font -OH. When terminal residues are modified by coupling, H- and -OH are not used; in this case, the residues are drawn in larger size, as described above.

[0190] Materials and Methods List of abbreviations AA amino acid residues ALP hydrolyzed colorless bacteria ( Achromobacter lyticus Lysyl-specific endopeptidase BHK baby hamster kidney C18 octadecyl (HPLC column) cpm (counting per minute) CV column volume EDTA (ethylenediaminetetraacetic acid) Dap 2,3-diaminopropionic acid DCC N , N '-Dicyclohexylcarbodiimide DIC N , N '-Diisopropylcarbodiimide DMF N,N -Dimethylformamide Fmoc fluorenylmethoxycarbonyl HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate Hepes (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) HI Human Insulin HIR human insulin receptor HIR-A Human Insulin Receptor A Subtype hr hours HSA human serum albumin IV. Intravenous LCMS (Liquid Chromatography-Mass Spectrometry) LYD Landrace-Yorkshire-Duroc min minutes MeCN Acetonitrile mm mM millimolecular concentration NBS N - Bromosuccinimide NHS N -Hydroxysuccinimide nm nanometer nM nanomolar concentration NMR (Nuclear Magnetic Resonance) RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography) sc subcutaneous SPA scintillation proximity measurement tBu tert-butyl TFA (trifluoroacetic acid) THF Tetrahydrofuran UHPLC (Ultra-High Performance Liquid Chromatography) Preparation of insulin variants Example 1 : Expression of insulin variants in yeast and conversion using ALP etc. Using known techniques, such as those disclosed in WO2017 / 032798, in yeast (Saccharomyces cerevisiae) Saccharomyces cerevisiae Insulin analogs are expressed in [the following]. More specifically, insulin analogs are expressed as single-chain precursors, separated by ion exchange capture, and cleaved into double-chain insulin analogs by ALP treatment as described below. Capture of precursor on SP Sepharose BB: The yeast supernatant was loaded onto a column packed with SP Sepharose BB at a flow rate of 10–20 CV / hr. The sample was washed with 0.1 M citric acid (pH 3.5) and then with 40% EtOH. The analogue was eluted with 0.2 M sodium acetate (pH 5.5) / 35% EtOH.

[0192] ALP digestion: The solution of the single-chain precursor was adjusted to pH 9, and ALP enzyme was added at a ratio of 1:100 (w / w). The reaction was performed on UHPLC. The ALP lysis pool was adjusted to pH 2.5 and diluted 2-fold in preparation for RP-HPLC purification. RP-HPLC purification: The following is an example of purification using RP-HPLC C18: Column: 15µm C18 50x250mm 200Å Buffer solution: A: 0.2% formic acid, 5% EtOH, B: 0.2% formic acid, 50% EtOH Gradient: 20-55% B-buffer.

[0194] Gradient: 20 CV Flow rate 20 CV / hr Sample loading g ~ 5 g / L resin The fractions were analyzed by UHPLC, combined, and freeze-dried.

[0195] Insulin analogs prepared and used in the following examples: GKPE-(GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:5) (GQEP)4-GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:6) (GQEP) 24 -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:7) (GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin (SEQ ID NO:1 and SEQ ID NO:8) GKPE-(GEQP)4-GEQG-GKPEGGGSGGGGSGGGGS-B1 desB30 human insulin refers to desB30 human insulin derived from B1 by extending GKPE-GEQPGEQPGEQPGEQP-GEQG-GKPEGGGSGGGGSGGGGS (the C-terminus S is linked to B1F). Other scaffolds are similar.

[0196] Fabrication of structural units Example 2: (S)-4-((S)-2,3-bis(l-hydroxy-4-(trifluoromethyl)-l,3-dihydrobenzo[c][l,2]oxa- borolan-6-carboxamido)propionamido)-5-(tert-butoxy)-5-oxopentanoic acid N,N NBS (34.0 g, 191 mmol) was added to a solution of 3-trifluoromethyl-4-methylbenzoic acid (39.0 g, 191 mmol) in concentrated sulfuric acid (400 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then poured into ice water (2 L). The resulting precipitate was filtered off, washed with water (500 mL), and dissolved in ethyl acetate (400 mL); dried over anhydrous sodium sulfate, filtered, and evaporated to give 3-bromo-4-methyl-5-trifluoromethylbenzoic acid as a white solid. Yield: 53.4 g (98%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 13.71 (bs, 1 H); 8.35 (d, J=0.4 Hz, 1 H); 8.15 (d, J=0.9 Hz, 1 H); 2.56 (s, 3 H).

[0197] Concentrated sulfuric acid (24 mL) was added to a solution of 3-bromo-4-methyl-5-trifluoromethylbenzoic acid (35.0 g, 124 mmol) in methanol (500 mL). The reaction mixture was stirred under reflux for 4 hours and then stirred at room temperature for 16 hours. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (250 mL), and washed with water (2 x 100 mL) and a mixture of saturated potassium carbonate solution (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate as a white solid. Yield: 35.3 g (96%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 8.36 (d, J=1.1 Hz, 1 H); 8.13 (d, J=1.1 Hz, 1 H); 3.90 (s, 3 H); 2.55 (d, J=1.3 Hz, 3 H).

[0198] A suspension of NBS (31.7 g, 178 mmol) and methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate (35.3 g, 119 mmol) in water (300 mL) was stirred at 80 °C for 6 hours under a 100 W bulb. The reaction mixture was extracted with diethyl ether (2 x 200 mL). The organic layer was washed with brine (150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate as a yellow solid. Yield: 44.0 g (98%). 1H NMR spectrum (300 MHz, CDCl3, δH): 8.47 (d, J=1.5 Hz, 1 H); 8.31 (d, J=1.3 Hz, 1 H); 4.75 (s, 2 H); 3.98 (s, 3 H).

[0199] A solution of methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate (44.0 g, 117 mmol) and potassium acetate (22.9 g, 234 mmol) in MeCN (0.5 L) was stirred overnight at 75 °C. The suspension was filtered through filter paper and evaporated. The crude product was dissolved in dichloromethane and filtered again. Evaporation gave methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate as a white solid. Yield: 37.9 g (91%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.49 (d,J=1.3 Hz, 1 H); 8.34 (d, J=1.3 Hz, 1 H); 5.37 (s, 2 H); 3.99 (s, 3 H); 2.11 (s, 3 H).

[0200] A solution of methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate (37.9 g, 107 mmol), bis(pinacol)diboron (29.8 g, 117 mmol), potassium acetate (31.4 g, 294 mmol), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.57 g, 1.92 mmol) in anhydrous tetrahydrofuran (500 mL) was stirred at 75 °C for 13 days under an argon atmosphere. The reaction mixture was then cooled to room temperature, filtered, and evaporated. The crude product was filtered through a silica gel column (silica gel, 0.063–0.200 mm; eluent: cyclohexane / ethyl acetate 8:1) to give methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxoborhexacyclopentan-2-yl)-5-(trifluoromethyl)benzoate. Yield: 31.1 g (72%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.65 (s, 1 H); 8.43 (s, 1 H); 5.48 (s, 2H); 3.97 (s, 3 H); 2.05 (s, 3 H); 1.36 (s, 12 H).

[0201] A solution of methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-5-(trifluoromethyl)benzoate (31.0 g, 77.1 mmol) and sodium hydroxide (15.4 g, 386 mmol) in water (300 mL) was stirred at room temperature for 3 hours. Then, a solution of hydrochloric acid (35 mL) in water (100 mL) was added to lower the pH to 1. The reaction mixture was stirred overnight. The precipitate was filtered off and dried to give a white solid of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[ c [1,2]Oxaborpentane-6-carboxylic acid. Yield: 16.6 g (86%).

[0202] 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.47 (bs, 1 H); 9.66 (s, 1 H); 8.62 (s, 1 H); 8.24 (s, 1 H); 5.22 (s, 2 H).

[0203] Pentafluorophenol (7.48 g, 40.7 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[ c A solution of oxaborptyl-6-carboxylic acid (10.0 mg, 40.7 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 8.37 mg, 40.7 mmol) in MeCN (0.5 L) was stirred overnight at room temperature. The reaction mixture was filtered, evaporated, dissolved in MeCN, filtered again, and evaporated to give a white solid of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[ c [1,2]Oxaboronolane-6-carboxylic acid pentafluorophenyl ester.

[0204] Yield: 16.7 g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1 H); 8.86 (s, 1 H); 8.46 (s, 1 H); 5.30 (s, 2 H).

[0205] Allow 100-200 mesh 1.5 mmol / g (3, 4.47 g, 6.71 mmol) of 2-chlorotriphenylmethyl chloro resin to swell in anhydrous dichloromethane (30 mL) for 30 min. (2) S)-5-(tert-butoxy)-2-{[(9H-fluorene-9-ylmethoxy)carbonyl]amino}-5-oxopentanoic acid (Fmoc-Glu-OtBu, 1.90 g, 4.47 mmol) and N,N A solution of diisopropylethylamine (2.96 mL, 17.0 mmol) in anhydrous dichloromethane (30 mL) was added to the resin, and the mixture was shaken overnight. The resin was filtered and... Example 3: (S)-3-(2,3-bis(l-hydroxy-4-(trifluoromethyl)-l,3-dihydrobenzo[c][l,2]oxa- borolan-6-carboxamido)-L-diaminopropionic acid]-beta-alanineThe resin was treated with a solution of diisopropylethylamine (1.56 mL, 8.95 mmol) in a methanol / dichloromethane mixture (4:1, 2 x 5 min, 2 x 40 mL). The resin was then washed with DMF (2 x 30 mL), dichloromethane (2 x 40 mL), and DMF (3 x 40 mL). The Fmoc groups were removed by treatment with 20% piperidine in DMF (1 x 5 min, 1 x 20 min, 2 x 40 mL). The resin was then washed with DMF (3 x 40 mL), 2-propanol (2 x 40 mL), and dichloromethane (3 x 40 mL). A solution of (S)-2,3-bis((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-Dap(Fmoc)-OH, 3.68 g, 6.71 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 2.55 g, 6.71 mmol), and 2,4,6-trimethylpyridine (1.60 mL, 12.1 mmol) in DMF (40 mL) was added to the resin, and the mixture was shaken for 2 hours. The resin was filtered and washed with DMF (2 x 40 mL), dichloromethane (2 x 40 mL), and DMF (2 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in DMF (1 x 5 min, 1 x 30 min, 2 x 40 mL). The resin was washed with DMF (3 x 40 mL), 2-propanol (2 x 40 mL), and dichloromethane (3 x 40 mL). A solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborptyl-6-carboxylic acid pentafluorophenyl ester (5.53 g, 13.4 mmol) and triethylamine (4.99 mL, 35.8 mmol) in DMF (40 mL) was added to the resin, and the mixture was shaken overnight. The resin was filtered and washed with DMF (6 x 40 mL) and dichloromethane (10 x 50 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 hours. The resin was filtered off and washed with dichloromethane (4 x 50 mL). The crude product (4) was dried under vacuum and extracted with ethyl acetate (2 x 70 mL) and 1 M potassium hydrogen sulfate aqueous solution (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was then ground in diethyl ether (20 mL) to give (S)-4-((S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[ c[1,2]Oxadione-6-carbamoyl)propamido)-5-(tert-butoxy)-5-oxovalerate. Yield: 1.89 g (57%). 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 8.50 (s, 1 H); 8.46 (s, 1 H); 8.29 (s, 1 H); 8.26 (s, 1 H); 5.28 (d, J=2.6 Hz, 4 H); 5.20 (t, J=5.9 Hz, 1 H); 4.55 (dd, J=8.5 and 5.2Hz, 1 H); 4.08 (dd, J=6.0 and 2.1 Hz, 2 H); 2.57-2.42 (m, 2 H); 2.34-2.16 (m, 1H); 2.17-2.08 (m, 1 H); 1.47 (s, 9 H). LC-MS: 746.3 (M+H)+.

[0206] N,N Piperidin-4-yl)carbonyl)phenyl)amino]pyrimidine-5-carboxamide N alpha , N beta - 1 -hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzofuran-5-yl - 1 -hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzofuran-5-yl Example 4: A solution of L-diaminopropionate ((S)-2,3-diaminopropionate) (15.0 g, 107 mmol), di-tert-butyl dicarbonate (46.6 g, 214 mmol), and potassium bicarbonate (32.0 g, 320 mmol) in a mixture of MeCN (400 mL) and water (400 mL) was stirred overnight. The solvent was removed under reduced pressure, and the residue was acidified with a saturated aqueous solution of potassium bisulfate until pH 1 was reached. The reaction mixture was extracted with ethyl acetate (3 x 200 mL) and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give (S)-2,3-bis((tert-butyloxycarbonyl)amino)propionic acid as a grayish-white solid. Yield: 28.2 g (87%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 5.85 (bs, 1 H); 5.17 (bs, 1 H); 4.31 (bs, 1 H); 3.64-3.46 (m, 2 H); 1.46 (s, 18 H).

[0207] (S)-2,3-bis((tert-butyloxycarbonyl)amino)propionic acid (27.9 g, 91.7 mmol), tert-butyl 3-aminopropionate (16.7 g, 91.7 mmol), N -(3-Dimethylaminopropyl)- N-Ethylcarbodiimide hydrochloride (EDC.HCl, 21.1 g, 110 mmol), 1-hydroxy-7-azabenzotriazole (HOAt, 15.0 g, 110 mmol) and Example 5 (comparative compound): A solution of diisopropylethylamine (64.0 mL, 367 mmol) in dichloromethane (300 mL) was stirred overnight. The solvent was removed under reduced pressure; the residue was dissolved in ethyl acetate (600 mL), washed with 1 M hydrochloric acid aqueous solution (4 x 300 mL) and saturated sodium bicarbonate aqueous solution (4 x 300 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give tert-butyl (S)-3-(2,3-bis((tert-butoxycarbonyl)amino)propionamido)propionate as a grayish-white solid. Yield: 36.1 g (91%).

[0208] 1 H NMR spectrum (300 MHz, CDCl3, δH): 7.01 (bs, 1 H); 5.75 (bs, 1 H); 5.14 (bs, 1 H); 4.15 (bs, 1 H); 3.57-3.39 (m, 4 H); 2.43 (t, J=6.0 Hz, 2 H); 1.45(s,27H).

[0209] To a solution of (S)-3-(2,3-bis((tert-butyloxycarbonyl)amino)propamido)propionate (36.1 g, 83.7 mmol) in dichloromethane (50 mL), 300 mL of 95% trifluoroacetic acid aqueous solution was added, and the solution was stirred for 3 hours. The solvent was removed under reduced pressure, and the residue was co-evaporated with MeCN (3 x 300 mL) and treated with a 1 M solution (300 mL) of hydrogen chloride in anhydrous diethyl ether. The precipitate was filtered off and ground with MeCN (2 x 600 mL) to give (S)-3-((2-carboxyethyl)amino)-3-oxopropane-1,2-diammonium chloride as a white powder.

[0210] Yield: 22.2 g (100%). 1 H NMR spectrum (300 MHz, D2O, δH): 4.35 (t, J=5.8 Hz, 1 H); 3.63-3.46 (m, 4 H); 2.67 (t, J=6.6 Hz, 2 H).

[0211] To a solution of (S)-3-((2-carboxyethyl)amino)-3-oxopropane-1,2-diammonium chloride (6.41 g, 24.3 mmol) and triethylamine (33.8 mmol, 243 mmol) in water (50 mL), a solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborptyl-6-carboxylic acid pentafluorophenyl ester (from Example 2, 20.0 g, 48.6 mmol) in 1,4-dioxane (100 mL) was added, and the solution was stirred overnight. The reaction mixture was partitioned between ethyl acetate (300 mL) and 1 M potassium hydrogen sulfate aqueous solution (1500 mL). The organic layer was washed with 1 M potassium hydrogen sulfate aqueous solution (1 x 300 mL) and the solvent was removed under reduced pressure. The residue was ground with diethyl ether (2 x 150 mL) and filtered. The solid was dissolved in 70% MeCN aqueous solution (600 mL) and freeze-dried to give (S)-3-(2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborane-6-carbamate)propionamido)propionic acid as a white powder. Yield: 12.1 g (80%).

[0212] 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 8.51 (s, 1 H); 8.47 (s, 1 H); 8.29 (s, 1 H); 8.27 (s, 1 H); 5.28 (s, 4 H); 5.15 (t, J=6.1 Hz, 1 H); 4.15-3.99 (m, 2H); 3.61 (t, J=6.4 Hz, 2 H); 2.67 (t, J=6.3 Hz, 2 H). LC-MS: 632.0 (M+H)+.

[0213] Preparation of insulin derivatives The structural unit in Example 2 was activated with NHS / DIC in THF, and the tBu protecting group was removed by treatment with TFA. The TFA was then evaporated before coupling with insulin.

[0214] The structural unit in Example 3 was activated using NHS / DIC in MeCN before being coupled with insulin.

[0215] LCMS analysis was performed using a C18 column with 0.1% TFA aqueous solution as buffer A and 0.1% TFA in MeCN solution as buffer B.

[0216] LCMS of boron-insulin derivatives typically shows various dehydrated components as the dominant peak; for example, for benzoxadiborone rings with ionization states of "n" and at most "m", it is [M + nH - 1x m M]. 水 ] n+ .

[0217] Example 6 (comparative compound): GKPE-(GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin (536 mg, 0.057 mmol) was dissolved in 0.1 M Na2HPO4 (5.6 mL) and DMSO (2.4 mL), and the pH was adjusted to 10.8 with 1.0 M NaOH aqueous solution. The structural unit (147 mg, 0.19 mmol) of Example 2 was activated with NHS and deprotected with tBu as described above, then dissolved in DMF (0.5 mL) and added dropwise over 10 minutes to the given insulin solution, while maintaining the pH near 10.8 by dropwise addition of 0.1 M NaOH. LCMS showed the formation of the desired product. The mixture was diluted with MeCN and water, and the pH was adjusted to 3.8 by dropwise addition of 1 M HCl. The product was purified by reversed-phase HPLC (RP-HPLC) on a C18 column, using 0.1% TFA aqueous solution as buffer A and 0.1% TFA in MeCN solution as buffer B. The product was then separated by lyophilization. For C... 480 H 666 B6F 18 N 122 O 169 S6, LCMS measurement value is 1894.1 [M + 6H - 5x water] 6+ The calculated values ​​are 1894.1 and 1623.6 [M + 7H - 5x water]. 7+ The calculated value is 1623.6; 1420.8 [M + 8H - 5x water] 8+ The calculated values ​​are 1420.8 and 1263.0 [M + 9H - 5x water]. 9+ The calculated value is 1263.0.

[0218] Example 7 (comparative compound): The insulin derivative of Example 5 was prepared similarly to the insulin derivative of Example 4, using (GQEP)4-GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and the structural unit from Example 2. The LCMS value of the product was 2049.0 [M + 5H - 3x water]. 5+ The calculated value is 2049.0.

[0219] Example 8 (comparative compound): The insulin derivative of Example 6 is similar to the insulin derivative of Example 4, and is derived from (GQEP). 24 -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and structural units from Example 2 were prepared. The LCMS value of the product was 1847.8 [M + 10H - 3x water]. 10+ The calculated value is 1847.8.

[0220] Example 9 (prior art compound): The insulin derivative of Example 7 was prepared similarly to the insulin derivative of Example 4, using (GQEP)4-GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and the structural unit of Example 3. The LCMS value of the product was 2029.2 [M + 5H - 2x water]. 5+ The calculated value is 2029.4.

[0221] Example 10 (prior art compound): The insulin derivative of Example 8 was prepared similarly to the insulin derivative of Example 4, using (GEQP)4-GEQGGKPEGGGSGGGGSGGGGS-B1 desB30 human insulin and the structural unit from Example 3. The LCMS value of the product was 2036.3 [M + 5H - 4x water]. 5+ The calculated value is 2036.6.

[0222] Example 11 : ALP cleavage rate The insulin derivative shown here is the compound of Example 324 in WO2020201041. It is a prior art compound, but its structure is shown here for ease of reference.

[0223] Achromobacter lyticus The insulin derivative shown here is the compound of Example 280 in WO2020201041. It is a prior art compound, but its structure is shown here for ease of reference.

[0224] Achromobacter When recombinant insulin is produced, it is expressed as a single-chain precursor, subsequently processed using proteases such as hydrolyzed Achromobacterium (…). Lyticus Lysine-specific endopeptidase EC 3.4.21.50 (ALP) or trypsin matures it into double-chain insulin. ALP specifically and exclusively cleaves the lysine bond, including the lysine-proline bond (Norioka & Sakiyama (1993) Lysine-specific serine protease from Achromobacter lyticus Achromobacter lyticus : Its substrate specificity and comparison with trypsin. Methods inProtein Sequence Analysis, pp. 101-106; Masaki et al. (1978) A New ProteolyticEnzyme from Achromobacter lyticus M497-I. Agricultural and BiologicalChemistry, pp. 1443-1445).

[0225] Peripheral location—P on the left hand side of the cleavage site n -P1 and P1'-P on the right side n ’ The amino acid residues (AA) of lysyl endopeptidase can vary within a wide range without affecting the enzyme's cleavage ability (Sakiyama & Masaki (1994) Lysyl Endopeptidase of Saccharomyces cerevisiae . Methods in Enzymology, Vol 244, pp.126-137).

[0226] However, the cleavage rate is affected by certain adjacent amino acids. For example, cleavage proceeds slowly when proline is located at position P1' or when there are one or two basic amino acids preceding the lysine residue (Tsusanawa et al. (1987), AminoAcid Sequence if Thermostable Direct Hemolysin Produced by Vibrioparahaemolyticus, Journal of Biochemistry, vol. 101, pp. 111-121; Sakiyama & Masaki (1994), Lysyl Endopeptidase of Example 12: Assay to determine affinity for human insulin receptor (HIR-A) in the absence or presence of glucose . Methods in Enzymology, Vol 244, pp. 126-137).

[0227] In this embodiment, the effect of glutamate (E) at the second position (P2') after lysine (K) on ALP cleavage rate was determined. The reference insulin precursor contains glycine (G) at P2'. In both cases, the amino acid at the first position after the cleavage site (P1') is proline (P).

[0228] To determine the ALP cleavage rate, the two insulin precursors in Table 1 were cleaved with ALP, and the formation of cleavage products was tracked over time by UPLC-MS (results are shown in Tables 2 and 3).

[0229] Table 1: Insulin prodrugs used to study the effect of E at position P2' on ALP cleavage rate. Both prodrugs contain three cleavage sites at positions A8, AA32, and AA77, denoted by '.

[0230] In brewer's yeast ( Insulin receptor preparationInsulin precursors were generated in the yeast. In short, the DNA fragment encoding the relevant insulin precursor was cloned into a vector designed to guide the recombinant protein into the secretory pathway of *Saccharomyces cerevisiae*. The construct was transformed into a *Saccharomyces cerevisiae* strain, which was then cultured in a specific glucose medium prepared as described by Verduyn et al. (1992). The supernatant containing the insulin precursor was adjusted to pH 8.5 with 1M NaHCO3 (pH 9.5) and diluted with water to a final concentration of 150 mg precursor / L. ALP was added to the pH-adjusted supernatant to a final concentration of 0.1 mg / mL, and the reaction was tracked over time by sampling at 0 min, 30 min, 60 min, 120 min, 240 min, 360 min, and 1440 min. To terminate the ALP reaction, the supernatant was diluted with 1 volume of 4M acetic acid. The lysis products in the ALP lysis samples were identified and quantified using UPLC-MS. The results are shown in Tables 2 and 3.

[0231] The desired cleavage product is designated as an "open insulin precursor" and is cleaved only at the Lys residue corresponding to position B29 of human insulin (the Lys residue at position 77 in SEQ ID NO:20 and SEQ ID NO:21, respectively, also designated as A77). Therefore, for open insulin precursor 1, the sequence of chain A is SDDMRGIVEQCCTSICSLYQLENYCN (SEQ ID NO:22), and the sequence of chain B is EEAEPRGKPEGEQPGEQPGEQPGEQPGEQGKPEGGGSGGGGSGGGSFVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO:23). The term "overly cleaved insulin precursor" is used to refer to a cleavage product that, in addition to cleavage at AA77, is also cleaved at the Lys residues at positions 8 (also designated as AA8) and / or 32 (also designated as AA32) in SEQ ID NO:20 and SEQ ID NO:21, respectively.

[0232] Open insulin precursors can be purified and chemically modified at lysine residues, and then fully matured by cleaving the N-terminal extensions of the B and A chains separately with trypsin (this concept is described in WO2005 / 047508A1). For the specific open insulin precursor 1 of this example, appropriate modifying groups are covalently coupled at three lysine residues, followed by cleavage of the N-terminal extensions EEAEPR and SDDMR with trypsin to obtain the insulin derivative of Example 4.

[0233] The ALP cleavage rate was found to be significantly influenced by the amino acid at position P2'. When P2' was glycine (G), cleavage at all three lysine residues proceeded rapidly, with over-cleavage observed after 60 minutes, well before AA77 was completely cleaved (Table 3). Conversely, when the AA at P2' was glutamic acid (E), cleavage proceeded very slowly. At the 60 and 120 minute time points, AA77 was almost 100% cleaved, while no cleavage was detected at residues AA8 and AA32 (Table 2). Cleavage at AA8 and AA32 was only observed after incubation times were extended to over 240 minutes, and in all cases, the extent of cleavage was limited.

[0234] In summary, having PE at the same positions can significantly reduce precursor loss due to excessive cleavage of ALP at lysine residues AA8 and AA32 compared to having PG at positions P1'-P2'.

[0235] Table 2: ALP cleavage of precursor 1.

[0236] Table 3: ALP cleavage of precursor 2 (comparative insulin precursor).

[0237] Functional data of insulin derivatives PLos One Insulin receptor scintillation proximity assay (SPA) binding assay Insulin derivative of the invention Young hamster kidney (BHK) cells overexpressing human insulin receptor A (HIR-A) were lysed in 50 mM Hepes pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 10% glycerol. Clarified cell lysates were fractionated for 90 min with wheat germ lectin (WGA)-agarose (lectin from Triticum vulgaris-Agarose, L1394, Sigma-Aldrich Steinheim, Germany). Receptors were washed with 20 volumes of 50 mM Hepes pH 8.0, 150 mM NaCl, and 0.1% Triton X-100, followed by elution with 50 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5 M acetylglucosamine, and 10% glycerol. All buffers contained the formulations described in Andersen et al. 2017. Insulin derivatives included for comparison The protease mixture described in 12 is Complete (Roche Diagnostic GmbH, Mannheim, Germany).

[0238] Comparative compounds and prior art compounds SPA PVT anti-mouse beads (Perkin Elmer) were diluted in SPA binding buffer, which consisted of 100 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MgSO4, and 0.025% (v / v) Tween-20. The SPA beads were incubated with IR-specific antibody 83-7 (Soos et al. 1986 Biochem J. 235, 199-208) and dissolved semi-purified HIR-A. The receptor concentration was adjusted to reach 5000 cpm. 125 10% binding of I-(Tyr31)-insulin (Novo Nordisk A / S). A series of cold ligand dilutions were added to 96-well Optiplates, followed by the addition of the tracer ( 125I-insulin (5000 cpm / well), with the receptor / SPA mixture added last. Binding experiments were established in the absence or presence of 20 mM glucose to test glucose sensitivity. Plates were gently shaken at 22°C for 22.5 h, centrifuged at 1000 rpm for 5 min, and counted in a TopCounter (Perkin Elmer). Data points were fitted to a four-parameter logistic model, thereby determining the relative affinity of the insulin derivative to human insulin (within the same plate). The relative affinity of the insulin derivative to human insulin was determined, and the increase in apparent relative affinity from 0 to 20 mM glucose in each experiment reflected the glucose sensitivity of the insulin derivative. Experiments were performed in the absence or presence of 1.5% HSA (w / w) to better simulate physiological conditions. Data are shown in Table 4a (without HSA) and Table 4b (1.5% HSA).

[0239] The insulin derivative of this invention binds to HSA; therefore, when HIR affinity is measured in the presence of HSA, a portion of the insulin derivative binds to HSA and cannot bind to the human insulin receptor. Therefore, the term "apparent affinity" is used to describe the affinity measured in the presence of 1.5% HSA.

[0240] In individual experiments, the HIR (human insulin receptor) glucose factor was determined as the apparent relative HIR affinity at 20 mM glucose divided by the relative affinity in the absence of glucose. Average glucose factors from several experiments are provided in Tables 4a and 4b. The glucose factors provided in Tables 4a and 4b are average glucose factors determined in individual experiments and may therefore differ slightly from the values ​​obtained by dividing the average HIR affinity in the presence of glucose by the average HIR affinity in the absence of glucose.

[0241] Example 13: Assay to determine glucose sensitive insulin receptor phosphorylation in human primary hepatocytes. The data in Tables 4a and 4b show albumin-dependent glucose sensitivity. The apparent insulin receptor affinity of the insulin derivative of Example 4 was reduced when measured in the presence of HSA compared to the absence of HSA (compare the data in Table 4a with those in Table 4b). Glucose can replace the binding of the insulin derivative of Example 4 to HSA, thereby increasing the apparent HIR in the presence of 20 mM glucose and 1.5% HSA compared to the absence of glucose (see data in Table 4b). This can be readily seen from the glucose factor, which is higher than 1 when the relative insulin receptor affinity is higher in the presence of 20 mM glucose than in the absence of glucose. Therefore, in the presence of HSA, this insulin derivative binds to the insulin receptor in a glucose-sensitive manner, thus possessing the potential for glucose-sensitive treatment of diabetes, where the insulin derivative will be inactive or less active at low blood glucose levels, but will bind and activate the insulin receptor at higher blood glucose levels. As can be seen from Table 4b, the insulin derivative of Example 4 has a high glucose factor of 51.9 in the presence of 1.5% HSA.

[0242] Table 4a. Relative HIR affinity (%) for human insulin (HI) in the absence of HSA and in the presence and absence of glucose.

[0243] Table 4b. Relative HIR affinity (%) for human insulin (HI) in the presence of 1.5% HSA and in the absence and presence of glucose.

[0244] Insulin receptor phosphorylation method: Table 5 shows data for three insulin derivatives, which are identical except for the peptide extension located at the N-terminus of the insulin B chain. The structures of these insulin derivatives are provided in Examples 5, 6, and 9, respectively, and the sequences of the peptide extensions are shown in Table 5 for reference. The modifying groups of these insulin derivatives are the same as those of the insulin derivative of Example 4. However, these insulin derivatives have only two modifying groups compared to the insulin derivative of Example 4 of this invention. The prior art compound of Example 324 in WO2020201041 (the structure is shown in Example 9 for reference) has a peptide extension GKP(G4S)3 at position B1 of the insulin backbone. It can be seen that further extending the peptide extension by 20 amino acid residues (as in the comparative insulin derivative of Example 5) results in a decrease in the glucose factor from 46.5 to 26.3. When the peptide extension is further extended, as in the comparative insulin derivative of Example 6 by 100 amino acid residues, the glucose factor further decreases to 18.6.

[0245] Surprisingly, although the insulin derivative of Example 4 of the present invention has a peptide extension of similar length to that of the insulin derivative of Example 5, it exhibits a glucose factor of 51.9.

[0246] Table 5. Comparison of the relative insulin receptor binding affinity of the compound and prior art compounds in the absence and presence of glucose, respectively.

[0247] Table 6 shows data for three insulin derivatives, which are identical except for the peptide extension located at the N-terminus of the insulin B chain. The modifying groups of these insulin derivatives differ from those of the insulin derivatives in Table 5. The structures of these insulin derivatives are provided in Examples 7, 8, and 10, respectively, while the sequences of the peptide extensions are provided in Table 6 for reference. The prior art compound of Example 280 in WO2020201041 (the structure of which is shown in Example 10 for reference) has a peptide extension GKP(G4S)3 at position B1 of the insulin backbone. It can be seen that further elongating the peptide extension by 20 amino acid residues in the comparative insulin derivatives of Examples 7 and 8 resulted in a decrease in glucose factor from 18.0 to 12.6 and 12.0, respectively. This further supports the finding that elongating the peptide extension in prior art compounds such as Examples 324 and 280 of WO2020201041 leads to a decrease in glucose factor.

[0248] Table 6. Results in the absence and presence of glucose, respectively. Figure 1 The relative affinity of insulin receptor binding.

[0249] Example 14: Assay to determine carbohydrate sensitive glucose uptake in cells (rat adipogenesis assay) Rat adipogenesis assay (rFFC)

[0250] When insulin binds to the insulin receptor, it induces the activation of the insulin receptor, which can be measured by tyrosine phosphorylation. This induces downstream signaling pathways, leading to mitotic and metabolic responses. The insulin derivative of the present invention binds to albumin and can be replaced by glucose. The glucose sensitivity of the insulin derivative of Example 4 can be measured at both low and high glucose concentrations, thereby detecting the glucose-dependent cellular response of the insulin derivative of Example 4.

[0251] Frozen-inoculated donor human hepatocytes were incubated for 15 minutes with gradually increasing concentrations of human insulin (HI) or the insulin derivative of Example 4 in the presence of 1.5% human serum albumin (HSA). The hepatocytes were then washed, lysed, and insulin receptor phosphorylation (p-Tyr1158) was revealed by ELISA using the Novo Nordisk-developed specific insulin receptor antibody D2 (Ørstrup et al., 2019, Journal of Immunological Methods, Vol. 465, February 2019, pp. 20-26) and the IR p-Tyr1158 antibody (ThermoFischer, catalog number 44-802G).

[0252] Figure 2 Human hepatocytes obtained from BioIVT (Liverpool cryopreserved hepatocytes, reference number X008001-P, batch number ACR; cryopreserved single donor hepatocytes #M00995P) were thawed and seeded at a density of 50,000 cells / well in 0.1 mL / well of InvitrogroCP medium supplemented with Torpedo antibiotic mixture (Z99000) according to the manufacturer's instructions. After 4 hours, the medium was replaced with M199 medium supplemented with 5.5 mM glucose, 100 units / ml penicillin and 100 mg / ml streptomycin, 4 mg / ml dexamethasone, 0.1% fetal bovine serum (FCS), and 1 nM human insulin (HI), and cultured overnight at 37°C. The day after seeding, the human hepatocytes were incubated for 15 minutes in assay medium corresponding to supplementation with 1.5% human serum albumin (HSA), 3 or 20 mM... Basal medium (M199, excluding phenol red, Tween 80, and adenosine-5-triphosphate) containing gradually increasing concentrations of D-glucose and HI or the insulin derivative of Example 4 was used. After stimulation, hepatocytes were washed twice with ice-cold PBS and then lysed at 4°C for 30 min by adding lysis buffer. Subsequently, insulin receptor phosphorylation (p-Tyr1158) was quantified by immunoassay ELISA using the IR-specific antibody IR (D2 (Ørstrup et al. (2019) J. Immunological Methods 465. 20-26), batch 0268-0000-0945-1B) and the pIR: pTyr1158 detection antibody (ThermoFischer, catalog number 44-802G). The absorbance at 450 nm was then read using a SpectraMax_1_190 (Molecular Devices).

[0253] The insulin derivative in Example 4 stimulated IR phosphorylation (pIR) in human primary hepatocytes in a concentration-dependent manner, reaching the same maximum level as HI, although EC 50 Higher. ECGs of the insulin derivative from Example 4 were found to be higher. 50 At a 20 mM D-glucose concentration (30.8 nM), the concentration was lower than at a 3 mM D-glucose concentration (58.1 nM), as revealed by the left shift of the concentration-response curve for pIR stimulation (n=6). Example 15: Assay to determine glycogen accumulation in rat primary hepatocytes. (As shown). Then, the EC50 of the insulin derivative of Example 4 at 3 mM D-glucose was calculated. 50 EC at 20 mM D-glucose 50The ratio between the two (IR pTyr human hepatocytes 1.5% HSA 3 mM vs 20 mM D-glucose factor) was 1.89 (P = 0.019) (Table 7). This indicates that the insulin derivative of Example 4 has higher activity at higher glucose levels than at lower glucose levels, and therefore the insulin derivative of Example 4 exhibits glucose-sensitive insulin activity.

[0254] Table 7. Insulin receptor phosphorylation data

[0255] Glycogen accumulation (PAS assay) method: Figure 3 When insulin binds to its receptor, it induces activation of downstream signaling pathways. One metabolic endpoint of insulin signaling is lipid metabolism, and lipogenesis assays are used to measure this endpoint because, in the presence of insulin, cells respond to... 3 The uptake of H-glucose is stimulated and incorporated into lipids.

[0256] The insulin derivative of Example 4 binds to albumin and can be replaced by glucose, resulting in glucose sensitivity. Both L-glucose and D-glucose can replace the derivative from albumin, but L-glucose is metabolically inert and can be used to alter glucose concentration in the culture medium without affecting metabolism. The fold change in potency of the insulin derivative of Example 4 (relative to human insulin) between 20 mM and 3 mM L-glucose concentrations was determined.

[0257] Example 16: Pharmacokinetic study in LYD pigs Epididymal fat pads from Sprague Dawley rats were degraded with collagenase in Hepes Krebs Ringer buffer at 36.5°C with vigorous shaking for 1–1.5 h. The suspension was filtered through two layers of gauze. The adipocytes were separated into phases by allowing them to stand at room temperature for 5 minutes, thus collecting them in the upper phase. The lower phase was removed with a syringe. The adipocytes were washed twice with 20 ml of Hepes Krebs Ringer buffer. Cells were transferred to 96-well plates containing 1.5% HSA, 0.5 mM glucose, and 0.1 µCi / well glucose (D-[3- 3[H] glucose (20.0 Ci / mmol) Perkin Elmer) and 3 mM or 20 mM L-glucose in Hepes Krebs Ringer buffer. Gradual increments of human insulin or the insulin derivative of this invention were added to generate a concentration-response curve, and the mixture was incubated at 36.5 °C for 2 h. The reaction was terminated by adding 100 µL Microscient E (catalog number 6013661 Perkin Elmer). The plate was allowed to stand for 3 h, and then counted in a Top counter. The EC50 of the insulin derivative at 3 mM L-glucose was determined. 50 EC at 20 mM L-glucose 50 The ratio between them (rFFC 1.5%HSA 3 mM vs. 20 mM L-glucose factor) (see Table 8).

[0258] Table 8. Data on rat lipogenesis assay.

[0259] Pharmacokinetic study in LYD pigs Representative concentration-response curves (n=1) are shown. The EC50 of the insulin derivative of Example 4 was found to be... 50 The concentration at 20 mM L-glucose was lower than that at 3 mM L-glucose, as revealed by the left shift in the concentration-response curve for lipogenesis. The data in Table 8 show that, compared to low L-glucose (3 mM), the insulin derivative of Example 4 resulted in higher levels of lipogenesis (i.e., more glucose transport) in the presence of higher levels of L-glucose (20 mM). This indicates that the insulin derivative of Example 4 exhibits higher activity at higher glucose levels than at lower glucose levels, and therefore demonstrates glucose-sensitive insulin activity.

[0260] Quantification of insulin analogue concentration in plasma samples

[0261] When insulin binds to the insulin receptor, it induces activation of downstream signaling pathways, leading to the activation of metabolic processes in insulin-sensitive tissues. Among other functions, insulin also induces glycogen accumulation in the liver. Therefore, the estimation of glycogen levels was used as the endpoint in primary rat hepatocytes over 24 hours in the presence of 20 mM glucose and 0.1% human serum albumin (HSA) in response to gradually increasing dose concentrations of human insulin or the insulin derivative of Example 4. Cellular glycogen content was quantified using a quantitative colorimetric assay based on the periodic acid-Schiff reagent (PAS) principle, with absorbance read at 550 nm.

[0262] ​ Rat hepatocytes (Lonza, RSCP01) were thawed and seeded at a density of 50,000 cells / well in basal M199 medium (5.5 mM glucose, 100 U / ml penicillin, 100 mg / ml streptomycin, and 4 mg / ml decatol) supplemented with 4% fetal bovine serum (FCS) and 1 nM human insulin (HI) in collagen-coated 96-well plates at 37°C. After approximately 4 hours, the medium was replaced with basal M199 medium supplemented with 0.1% FCS and 1 nM HI, and cultured overnight. The day after isolation, the rat hepatocytes were incubated at 37°C for 24 hours in assay medium corresponding to basal M199 medium supplemented with 100 U / ml penicillin, 100 mg / ml streptomycin, 4 mg / ml dexamethasone, 0.1% human serum albumin (HSA), 14.5 mM glucose, and gradually increasing concentrations of HI or a derivative of Example 4. Following stimulation, hepatocytes were washed three times in ice-cold PBS, thawed in liquid nitrogen, and stored at -80°C for subsequent analysis. To measure glycogen levels, the plates were thawed, and the cells were lysed in 1% Triton X100. Then, 100 µl of periodic acid (0.1% periodic acid in 7% acetic acid) was added to the plates, and the plates were incubated at 37°C in the dark for 1.5 hours to oxidize the hydroxyl groups in glycogen. Next, 85 µl of Schiff's reagent was added, and the plates were incubated for 5 minutes to react with the oxidized portions. The plates were then incubated at room temperature for another 10 minutes to develop the color. The absorbance was then read at 550 nm (spectraMax_1_190, Molecular Devices).

[0263] Table 9.

[0264] Data showed that the insulin derivative of Example 4 induced glycogen accumulation in rat primary hepatocytes in a concentration-dependent manner and exhibited a complete dose-response curve at a glucose concentration of 20 mM. ​ Compared to human insulin, the maximum response was slightly reduced (Emax = 86%, n = 5). Calculations showed that at 20 mM glucose, the in vitro EC50 of the insulin derivative of Example 4 was 20.4 nM, while the in vitro EC50 of human insulin was 2.8 nM (n = 5), suggesting a reduced potency relative to human insulin (Table 9).

[0265] ​ ​ The insulin derivative was administered intravenously (iv) or subcutaneously (sc) to female Landrace-Yorkshire-Duroc (LYD) pigs weighing approximately 70–110 kg. The intravenous dose was 0.3 nmol / kg, while the subcutaneous dose was 1 or 2 nmol / kg. Blood samples were collected at selected time points up to 72 hours later, plasma was prepared, and the insulin derivative concentration was analyzed (see below). The pigs were fasted overnight and fed 8 hours after insulin derivative administration.

[0266] Plasma concentration-time curves for each animal were analyzed using non-compartmental pharmacokinetic analysis (NCA) to validate WinNonlin, either using WinNonlin Professional (Certara, CA, USA) or a custom Rshiny application (Rstudio.com, Boston, MA, US).

[0267] ​ Insulin derivative dosing levels in plasma samples were analyzed using a luminescent oxygen channel immunoassay (LOCI), also known under the trademark AlphaLISA (Perkin Elmer, Waltham, Massachusetts, USA). The principle of the assay is briefly described below: a specific antibody against the target analyte is conjugated to a receptor bead. A second antibody, also specific to the analyte, is biotinylated. The two antibody conjugates are then incubated with a plasma sample containing the analyte to form an immune complex. Next, streptavidin donor beads are added and conjugated with the biotinylated antibody. By irradiating the donor beads at 680 nm, ambient oxygen is excited to form singlet oxygen. If the receptor bead is brought close, energy is transferred from the singlet oxygen to the receptor bead, resulting in the emission of light at 615 nm. The intensity of the emitted light signal is proportional to the concentration of the analyte. Calibration curves were constructed using plasma samples doped with known concentrations of the analyte. Unknown sample concentrations were then calculated from the calibration curves using a 5-parameter logistic regression. All insulin derivatives were tested using Novo Nordisk in-house antibodies. The results are shown in Table 10.

[0268] Table 10. PK Data

[0269] The PK data in Table 10 show that the insulin derivative of Example 4 of the present invention has a longer half-life than the prior art compounds of Examples 324 and 280 in WO2020201041.

[0270] While certain features of the invention have been set forth and described herein, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that all such modifications and alterations falling within the true scope of the invention are intended to be covered by the appended claims.

Claims

1. An insulin derivative comprising A. An insulin peptide containing B29K; B. Peptide extension Z-Lys-Y-Lys-aa1-aa2-(Gly)3-Ser-((Gly)4-Ser) p -#, Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln. Where aa1 either does not exist or is Pro, Where aa2 is Glu or Gly. Where p is 1, 2, or 3, and Where # represents the connection point to the N-terminus of the B chain of human insulin or human insulin analogues; C. Modifying groups of the three formulas A: ， One of R1, R2, or R3 is an electron-withdrawing group, while the other two are hydrogen. Where m is 0, 1, 2 or 3; Where n is 0, 1, 2 or 3; in Indicate the connection point; and One of the modifying groups is attached to the Lys residue at position 29 of the B chain of the insulin peptide, while the other two modifying groups are attached to each Lys residue in the peptide extension.

2. The insulin derivative according to claim 1, wherein Z is Gly.

3. The insulin derivative according to any one of claims 1 to 2, wherein Y is PE(GEQP)4GEQGG (SEQ ID NO: 13).

4. The insulin derivative according to any one of claims 1 to 3, wherein aa1 is Pro and aa2 is Glu.

5. The insulin derivative according to any one of claims 1 to 4, wherein p is 2.

6. The insulin derivative according to any one of claims 1 to 5, wherein the peptide extension is GKPE(GEQP)4GEQGGKPEGGGS(G4S)2 (SEQ ID NO:4).

7. The insulin derivative according to any one of claims 1 to 6, wherein the electron-withdrawing group is selected from CF3, F, NO2, CN, COX, SO2X and POX2, wherein X is OR or NR2 and R is H, alkyl or aryl.

8. The insulin derivative according to any one of claims 1 to 7, wherein R1 is an electron-withdrawing group, and R2 and R3 are hydrogen.

9. The insulin derivative according to any one of claims 1 to 8, wherein R1 is CF3 and R2 and R3 are hydrogen.

10. The insulin derivative according to any one of claims 1 to 9, wherein the insulin derivative is (Insulin derivative of Example 4).

11. An insulin derivative, wherein the insulin derivative is (Insulin derivative of Example 4).

12. A composition comprising an insulin derivative according to any one of claims 1 to 11.

13. The insulin derivative according to any one of claims 1 to 11 or the composition according to claim 12, used as a pharmaceutical.

14. An insulin derivative according to any one of claims 1 to 11 or a composition according to claim 12, for the prevention or treatment of diabetes, including type 1 diabetes and type 2 diabetes.

15. A method for treating or preventing diabetes, including type 1 diabetes and type 2 diabetes, the method comprising administering to a subject in need a therapeutically effective amount of an insulin derivative according to any one of claims 1 to 11 or a composition according to claim 12.

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