Aromatic boron-containing compounds and related insulin analogs

Abstract

The present disclosure relates to novel compounds containing one or more aromatic boron-containing groups and methods for making the disclosed compounds. The present disclosure further relates to pharmaceutical compositions containing the disclosed compounds and their use in the prevention and treatment of diseases and disorders.

Description

Technical Field

[0001] (Cross-reference to related applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 364,893, filed May 18, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to novel compounds containing one or more aromatic boron-containing groups. The present disclosure relates to the use of novel compounds for binding to glucose. The present disclosure further relates to kits and the use of compounds and / or pharmaceutical compositions containing the disclosed compounds for the treatment of disorders characterized by elevated glucose levels, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, as well as obesity, metabolic syndrome, neuropathy, mood disorders, and mental disorders.

[0003] Sequence Listing This application has been filed together with a Sequence Listing in electronic form. The Sequence Listing is provided as a file entitled "02021_00153-00304_SL.xml", created on April 7, 2023, and is 6,324,718 bytes in size. The information in the electronic form of the Sequence Listing is hereby incorporated by reference in its entirety.

Background Art

[0004] Boric acid is generally regarded as a Lewis acid because it can form complexes with Lewis bases such as hydroxide anions, for example, and thus has a tendency to bind to hydroxyls. Therefore, molecules containing borate salts containing boric acid have a general tendency to bind to hydroxyl groups. This binding tendency can be used for the detection of hydroxyl-containing groups with borylated labeling reagents, where the boronic acid group binds to the hydroxyl group and, depending on the solvent and buffer conditions, the borate can form a hydrolyzable boronic acid ester bond to the hydroxyl group of a hydroxyl-containing molecule such as the hydroxyl groups present in a diol (e.g., glucose). Boron-containing compounds can bind to diol-containing molecules, but achieving selectivity using boron-containing compounds has been difficult because of their ability to bind to various diols containing cis-diols to varying degrees. There is a possibility of achieving an improved binding affinity of a boron-containing compound for a specific adjacent diol of interest, but this can result in a loss of selectivity.

[0005] Glucose is the body's main fuel, and the blood glucose levels of healthy individuals are tightly regulated. For example, between meals, blood glucose levels are around 5 mmol / L (mM), and when blood glucose concentrations rise after a meal, insulin action quickly returns blood glucose levels to 5 mM. The hormone insulin is secreted from pancreatic beta cells, and when insulin binds to insulin receptors on the body's cells (e.g., muscle and fat), the cells are stimulated to absorb glucose by the translocation of glucose transporters (GLUT4) from storage vesicles to the cell surface. (Huang, S.H. et al. (2007) Cell Metabolism 5:237-252.)

[0006] Diabetic patients may lose their ability to produce insulin due to autoimmunity against beta cells (type 1), or may have reduced sensitivity to insulin in combination with impaired insulin secretion (type 2). For example, patients with type 1 diabetes may rely on multiple daily insulin injections, both for a basic once-daily application range and in combination with meals (bolus administration) to control glucose levels. (Polonsky, K.S. et al. (1988) The Journal of Clinical Investigation 81:442 - 448). Since glucose values can vary unpredictably, achieving a complete daily insulin dosage is extremely difficult. In fact, despite many technological advancements in diabetes treatment, researchers are currently observing, in part due to lifestyle issues, a deterioration in long-term glucose control and / or overall metabolic health.

[0007] Glucose sensors and glucose-sensing insulin analogs are known in the art. See, for example, International Publication No. WO 2016 / 179568 (A1) and International Publication No. WO 2021 / 202802 (A1). However, there is a need in the art to develop glucose sensors and glucose-sensing insulins that have improved properties such as improved binding to the insulin receptor, respond proportionally to different glucose concentrations, and provide a stepwise and reversible response to changes in glucose levels under physiological conditions.

[0008] Accordingly, there is an unmet medical need for novel compounds such as those that can bind glucose and can be utilized in glucose-responsive insulin analogs / conjugates to control blood glucose levels.

[0009] Here, the inventors disclose glucose sensors and glucose-sensing insulin analogs having improved properties, such as improved affinity for relatively low concentrations of glucose, improved affinity for the insulin receptor, and improved activation of the insulin receptor. SUMMARY OF THE INVENTION

[0010] According to some embodiments, the present disclosure provides novel Z1c compounds (e.g., FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226) containing aromatic boron-containing functional groups (e.g., F2, F5, and F10). In some embodiments, the present disclosure provides an indirect linker represented by formula (X”), where X” is as defined herein and each n1 is 1, 2, 3, 4, or 5 (e.g., as exemplified in formulas FL3, FL5, FL5A, FL5B, and FL20 - FL75). In some embodiments, the present disclosure provides novel Z1c-linkers (e.g., formulas FFL-1 to FFL-101) containing aromatic boron-containing functional groups (e.g., F2, F5, and F10). n1 In some embodiments, the present disclosure provides a compound comprising a drug substance (e.g., X1) and at least one Z1c having an aromatic boron-containing functional group. In some embodiments, the disclosed compounds include one or more backbones (e.g., FF backbones and Z1c-linker backbones as exemplified in formulas FFL-1 to FFL-101). In some embodiments, the compound comprises a drug substance. In at least some embodiments, the drug substance is a polypeptide or a small molecule.

[0011]

[0012] ​In at least some embodiments, the disclosed compounds have at least two aromatic boron-containing functional groups. In some embodiments, the compounds are selective for a particular sugar, such as glucose, but show a reduced affinity for other sugars, and there are at least two aromatic boron-containing functional groups in the aromatic boron-containing moiety of the compound. In certain embodiments, the aromatic boron-containing moiety may be directly or indirectly covalently bonded to a drug substance containing an amine, a drug substance covalently bonded to an amine-containing linker, an amine covalently bonded to the drug substance, or NH2, or OH (e.g., when X1 is NH2 or OH). In some embodiments, the drug substance is covalently bonded to an amine-containing linker and the amine group is conjugated to the aromatic boron-containing moiety (Z1c). In certain embodiments, the aromatic boron-containing moiety (Z1c) has a structure that includes a tethering group (FF formula) and an aromatic boron-containing group that collectively forms the aromatic boron-containing moiety of the disclosed compound. In some embodiments, the aromatic boron-containing moiety (Z1c) is covalently bonded to an indirect linker (e.g., formulas FL3, FL5, FL5A, FL5B, and FL20 - FL75) and has a structure that includes a tethering group (FF formula) and an aromatic boron-containing group that collectively forms the aromatic boron-containing moiety of the compound of the present disclosure.

[0013] In some embodiments, one or more Z1c may be linked (e.g., conjugated, connected) to a prodrug (e.g., X1) via one or more indirect linkers (e.g., formulas FL3, FL5, FL5A, FL5B and FL20 - FL75, and / or one or more L - amino acids or D - amino acids linked to each other using an amide bond or a peptide bond). In at least some embodiments, the rotational restraint of the boron - functional group via a tethering group (e.g., FF formula) enhances the binding affinity of the compound (e.g., conjugate) for a particular diol (such as glucose) in the body and keeps it away from other diols in the body, thereby providing selectivity for the particular diol. In some embodiments, the compound may exhibit therapeutic pharmacokinetics and / or pharmacodynamics in response to endogenous and / or exogenous small molecules in the body such as glucose. Changes in the physiological concentration of glucose can result in the activation and / or release of the drug molecule due to the interaction of the boron - containing structure conjugated to the drug molecule with glucose, or conversely, inactivation and / or sequestration of the drug molecule (e.g., peptide hormone), and / or modulation of the activity of the drug molecule.

[0014] In some embodiments, the present disclosure provides a compound comprising X1 and one or more Z1c, or a tautomer, stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, or hydrate, or isotope derivative, wherein X1 is i. a prodrug containing an amine, ii. an amine configured to covalently bond to the prodrug, or iii. contains NH2 or OH (e.g., X1 is NH2 or OH), In the formula, each Z1c is independently selected from the formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, and each Z1c is covalently bonded directly or indirectly to an amine in X1, or NH2 when X1 is NH2, or OH when X1 is OH.

[0015] In some embodiments, the present disclosure provides a compound represented by formula I, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt,

Chemical formula

Chemical formula

[0016] In some embodiments, X1 is OH or NH2. In some embodiments, the compounds disclosed herein (e.g., formula I, formula II) are conjugated to a prodrug directly or via an optional linker.

[0017] In some embodiments, the compound of formula I is comprising at least one Z1c covalently bound to X1 or Z1a via an indirect linker, the indirect linker having the formula (X”) n1 represented by each n1 is independently selected from 1, 2, 3, 4, and 5, each X” is (i) an L or D-amino acid, wherein the amine functional group of the L or D-amino acid is directly or indirectly covalently bound to Z1c and the acid functional group of the L or D-amino acid is directly or indirectly conjugated to X1 or Z1a; and (ii) independently selected from formulae FL3, FL5, FL5A, FL5B, FL20 - FL68, and FL71 - FL75, which contain substituents R’’ and Z’’, and formulae FL69 and FL70, which contain substituents R’’, Z’’, A’, and A’’, and their stereoisomers, wherein R’’ represents a direct or indirect covalent bond to Z1c; Z’’ represents a direct or indirect covalent bond to X1 or Z1a; A’ is selected from H, an alkyl group (e.g., a C1 - C6 alkyl group), a saturated fatty acid, an unsaturated fatty acid, a cycloalkyl group (e.g., (C3 - C6 cycloalkyl)), a haloalkyl group (e.g., a C1 - C6 haloalkyl group), an aryl group, and a heteroaryl group; A’’ is selected from an alkyl group (e.g., a C1 - C6 alkyl group), a substituted acyl group, an acyl group terminated with an acid group, a saturated fatty acid, an unsaturated fatty acid, a cycloalkyl group (e.g., (C3 - C6 cycloalkyl)), a haloalkyl group (e.g., a C1 - C6 haloalkyl group), an aryl group, and a heteroaryl group; p is 1, 2, 3, 4, or 5, q is 1, 2, 3, 4, or 5, any primary amine is optionally acetylated or alkylated.

[0018] In some embodiments, n1 is 1.

[0019] In some embodiments of the compounds of formula I, m' = 0, n' = 0, o' = 0, and the compound is represented by formula IC:, [Chemical formula] wherein, each p' is 1, 2, 3, or 4, each q' is 1, 2, 3, 4, or 5.

[0020] In some embodiments, each of B1 and B2 is of formula F2.

[0021] In some embodiments, each Z1c is covalently bonded to an indirect linker, and each of Z1c and the indirect linker, in combination, is selected from formulae FFL-1 to FFL-101 containing substituent X, wherein X represents the amine of X1, or NH2 when X1 is NH2, or OH when X1 is OH, or an amine covalently bonded directly or indirectly to X1, or a direct covalent bond point with the amine of Z1a.

[0022] In some embodiments, X1 is a polypeptide comprising an insulin receptor agonist having an A chain and a B chain.

[0023] In some embodiments, at least one Z1c contains an alpha-methyl group or a beta-methyl group.

[0024] Therapeutic methods are also disclosed herein. The disclosed methods may include treating a subject in need thereof, where the subject has a disease characterized by elevated glucose levels. The method may include administering to the subject in need thereof a compound of formula I, a pharmaceutically acceptable salt of a compound of formula I, a pharmaceutical composition comprising a compound of formula I, or a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula I.

[0025] Also disclosed herein are methods of treating and / or preventing endocrine and / or metabolic diseases in a subject in need thereof, which comprise administering to the subject a compound of formula I or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) comprises a prodrug, which is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof.

[0026] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, is administered concurrently with a second agent or therapy. In some embodiments, the second agent comprises a drug substance, and the drug substance is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, is administered parallel to a second agent or therapy. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, is administered sequentially with a second agent or therapy (e.g., the compound of formula (I) is administered first and the second agent or therapy is administered thereafter, or the second agent or therapy is administered first and the compound of formula (I) is administered thereafter). In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, is administered in a different unit dosage form than the second agent or therapy.

[0027] In at least one embodiment, the pharmaceutical composition of the present disclosure can be for use in the treatment of a disease characterized by elevated glucose in a subject, such as diabetes (or in the manufacture of a medicament therefor). In at least one embodiment, a therapeutically effective amount of the pharmaceutical composition of the present disclosure can be administered to a subject diagnosed with diabetes or a metabolic disease. In at least one embodiment, the pharmaceutical composition of the present disclosure comprises at least one compound disclosed herein (e.g., Formula I, Z1c, Z1c-linker) and at least one additional component selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable vehicle, and a pharmaceutically acceptable excipient.

[0028] In some embodiments, the disclosed compounds comprise an insulin receptor agonist selected from insulin and insulin analogs (e.g., when X1 is insulin or an insulin analog). In some embodiments, X1 comprises insulin having an A chain and a B chain, the A chain comprises a sequence selected from SEQ ID NO: 1, 25, 24051, and 24052, and the B chain comprises a sequence selected from SEQ ID NO: 24060, 24061, 24062, 24063, 24064, and 25000-25397.

[0029] In some embodiments, X1 comprises insulin having an A chain and a B chain, the A chain comprises a sequence selected from SEQ ID NO: 24051 and 24052, the B chain comprises a sequence selected from SEQ ID NO: 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380-25397, each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116A, FF116B, FF116C, FF116D and is covalently bonded directly or via a linker to one or more lysine residues in X1, B1 and B2 are each F2; the indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL20-FL75, and their stereoisomers.

[0030] In some embodiments, the compound is selected from insulin comprising an A-chain and a B-chain, the A-chain comprises a sequence selected from SEQ ID NO: 24501 and 24502, and the B-chain comprises a sequence selected from SEQ ID NO: 25229, 25232, 25305, 25308, 25312, 25236, 25095, and SEQ ID NOs: 25380-25397.

[0031] In some embodiments, the present disclosure provides a compound represented by Formula II, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, Z1c-linker (Formula II) Wherein, Z1c-linker is selected from Formulas FFL-1 to FFL-101, Wherein, X is selected from a leaving group (e.g., halogen, N-hydroxysuccinimide (NHS) group, 2,3,5,6-tetrafluorophenol (TFP) group, pentafluorophenol (Pfp) group, or sulfonic acid ester), NH2, OH, and H, B1 and B2 may be the same or different and each independently represents an aromatic boron-containing group.

[0032] In some embodiments of the compound of Formula I, X1 comprises an insulin receptor agonist that is insulin or an insulin analog having an A-chain and a B-chain. In some embodiments, one or more lysine residues and / or the N-terminus of the A-chain or B-chain are covalently bonded as described by Formula I. In some embodiments, the insulin or insulin analog described herein is used as a compound (e.g., an intermediate compound) for the production of the conjugate described by Formula I. In some embodiments, the insulin or insulin analog described herein is used, for example, in a method for preventing and / or treating an endocrine and / or metabolic disease, including administering any compound (e.g., a modified insulin) of the embodiments described herein to a subject in need thereof, thereby treating the endocrine and / or metabolic disease.

Best Mode for Carrying Out the Invention

[0033] Aromatic boron-containing compounds (e.g., groups) can bind to diol-containing molecules, but achieving selectivity using aromatic boron-containing compounds that can function as molecular sensors is difficult because they have the ability to bind to various diols containing cis-diols to various degrees. The improved binding affinity of an aromatic boron-containing compound that can function as a sensor for a particular adjacent diol of interest can result in a loss of selectivity.

[0034] The backbone that positions the boron functional group of an aromatic boron-containing compound (e.g., a sensor) into a specific or specific-shaped aggregate can enhance selectivity for a particular vicinal diol while maintaining affinity for the diol of interest. According to some embodiments, the aromatic boron-containing compounds disclosed herein have different pendant groups on an aromatic boron-based backbone along with a specific backbone shape that affects binding to hydroxyl-containing molecules.

[0035] According to some embodiments, the compounds of the present disclosure include aromatic boron-containing compounds that orient the boron functional group in three-dimensional space, and the boron-containing compounds are spatially oriented to bind to hexoses containing adjacent diols, such that the boron group can properly bind to the hydroxyl groups of the adjacent diol molecule, providing enhanced selectivity. In some embodiments, the aromatic boron-containing compounds are modified with specific functional groups on the aromatic ring that, along with a suitable or preferred backbone, can provide higher selectivity and / or affinity for binding directed towards the adjacent diol of interest and away from other diols in the body.

[0036] In some embodiments, the aromatic boron-containing compound is conjugated to a drug substance (e.g., a small molecule, polypeptide), and the aromatic boron-containing compound provides intramolecular and / or intermolecular interactions with the drug substance and / or circulating proteins in the blood and / or plasma containing proteins in the body, such as albumin and / or globulin. In some embodiments, the aromatic boron-containing compound exhibits reversible binding to glycated proteins in the body such as glycated albumin, and this binding is reversibly affected by the level of blood glucose or plasma sugar molecules. In some embodiments, the selective binding of the sensor to specific adjacent diols changes the degree of their intramolecular and / or intermolecular binding, thereby regulating the pharmacokinetics and overall activity of the drug substance in the body. This effect can be controlled by the level of adjacent diols present.

[0037] In some embodiments, the drug substance is a peptide hormone. In some embodiments, the human peptide hormone, such as a peptide hormone, is insulin or an insulin analog, glucagon, or another incretin hormone. In some embodiments, the sensor is selective for adjacent diols in glucose, and this selectivity is enhanced while maintaining the affinity for glucose and at the same time reducing the affinity for other sugars in the blood. In some embodiments, the pendant groups on the backbone and the aromatic core of the boron-containing compound (e.g., in combination) enable the control of the overall activity and / or pharmacokinetics of the conjugated drug substance based on the level of glucose and / or other adjacent diols in the blood.

[0038] In some embodiments, the aromatic boron-containing compound includes specific backbone molecules having conjugated boron functional groups (i.e., F2, F5, and F10) (e.g., FF structure, FFL-1 to FFL-101, DSL-1 (dibronate backbone linker) to DSL-112), and the backbone is used to orient the boron functional groups in a three-dimensional shape such that the boron functional groups are oriented within a distance that helps them engage with each other in the vicinity and with a specific hydroxyl orientation of a selected hexose such as glucose.

[0039] Although not bound by theory, the aromatic boron-containing compounds (e.g., molecules) disclosed herein are thought to improve selectivity by at least one or more of the following three mechanisms: (1) The FF skeleton promotes the alignment of the hydroxyl group and / or alkoxy group on the boron group in the aromatic boron-containing compound with the hydroxyl group in the adjacent diol molecule, thereby improving selectivity. (2) For example, by identifying specific functional groups bonded to or near the aromatic core of the boron-containing compound that affect the electronic structure of the aromatic boron-containing compound and thereby favorably act on the reversible binding to the adjacent diol at physiological pH, a further increase in selectivity is obtained. (3) The functional group bonded to the aromatic boron-containing compound (e.g., the sensor skeleton) serves to provide steric hindrance to reduce binding to unwanted hexoses while maintaining binding to the target sugar such as glucose. In some embodiments, the FF skeleton provides a glucose binding. In some embodiments, the combination of the FF skeleton and the indirect linker provides an affinity for plasma proteins, such as, but not limited to, glycated proteins, and the combination of the FF skeleton and the indirect linker provides a reversible interaction with plasma proteins that is controlled through the binding of sugar molecules to the FF skeleton under physiological conditions. These effects combined together in the present disclosure (e.g., compounds of Formulas I and II) provide the desired or preferred selectivity of binding towards the target adjacent diol-containing molecule and away from other diols in the body.

[0040] In some embodiments, the aromatic boron-containing compound is conjugated to a prodrug that provides intramolecular and / or intermolecular interactions of the aromatic boron-containing compound with proteins in the body. Such proteins can include circulating proteins in blood and / or human plasma, such as albumin, glycosylated proteins, and / or glycated proteins such as glycated plasma proteins including immunoglobulins and glycated albumin. In some embodiments, the selective binding of the sensor to specific adjacent diols in the molecule of interest varies the degree of intramolecular and intermolecular binding, thereby modulating the pharmacokinetics and overall activity of the prodrug in the body. In some embodiments, the prodrug is a peptide hormone, and in certain embodiments thereof, the peptide hormone is an incretin hormone such as insulin, and the adjacent diol-containing molecule is glucose, but the present disclosure is not limited thereto.

[0041] Definitions Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning that coincides with their meaning in the context of the relevant art and / or this specification, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless explicitly so defined herein.

[0042] Unless specifically described herein, the functional groups, functional moieties, and reactions referred to herein are, for example, as described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rdEdition, Cambridge University Press, Cambridge, 1987; Smith and March, March’s Advanced Organic Chemistry, 5 th It is understood to have the meaning consistent with the standard description in organic chemistry and / or the general principles of organic chemistry as described in the 5th Edition, John Wiley & Sons, Inc., New York, 2001. General functional groups (such as alkyl, aryl, acetyl, etc.) include specific examples or species included in the categories of functional groups generally defined in the field of organic chemistry, and those skilled in the art can identify specific exemplary embodiments of the functional groups.

[0043] Unless otherwise specifically described herein, all chemical terms, functional groups, and general terms used throughout this specification are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., as identified on the inside cover. In certain embodiments, the terms “a,” “an,” and “the,” as well as similar referents used herein, should be construed to cover both the singular and plural forms unless the context of use indicates otherwise. The term “CAS#,” also referred to herein as CASRN or CAS number, is the unique numerical identifier assigned by the Chemical Abstracts Service (CAS) to all chemical substances described in the published scientific literature.

[0044] As used herein, the nomenclature of compounds, including organic compounds, can be given using the common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. Those skilled in the art can readily confirm the structure of a compound when it is named by systematic reduction of the compound structure using the naming rules or by any of the commercially available software such as TM (Cambridgesoft Corporation, U.S.A.).

[0045] The terms used in this specification are for the purpose of describing embodiments and are not intended to limit the present disclosure. As used herein, the terms "comprises," "comprising," "includes," and "including" identify the presence of the recited features, integers, acts, operations, elements, and / or components, but it will be further understood that they do not preclude the presence or addition of one or more other features, integers, acts, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. When preceding a list of elements, expressions such as "at least one of" modify the entire list of elements and not the individual elements of the list.

[0046] As used herein, the terms "substantially," "about," and similar terms are used as terms of approximation and not of degree, and are intended to account for inherent deviations in measured or calculated values that would be recognized by one of ordinary skill in the art. The term "about" is used throughout to describe and account for small variations. For example, "about" can mean that a numerical value can vary by up to ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, or ±0.05%. A numerical value modified by the term "about" includes the particular identified value. For example, "about 5.0" includes 5.0.

[0047] Further, the use of "may" when describing embodiments of the present disclosure refers to "one or more embodiments of the present disclosure." As used herein, the terms "use," "using," and "used" may each be considered synonymous with the terms "utilize," "utilizing," and "utilized," respectively. Also, the term "exemplary" is intended to refer to an example or illustration.

[0048] Any numerical range recited herein is also intended to include all sub-ranges of the same numerical precision that are included within the recited range. For example, the range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, i.e., having a minimum value of 1 or more and a maximum value of 10 or less, such as, for example, 2 to 7. The maximum numerical limitations recited herein are intended to include all lower numerical limitations subsumed therein, and any minimum numerical limitations recited herein are intended to include all upper numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-ranges that are subsumed within the ranges expressly recited herein.

[0049] As used herein, "aromatic boron-containing group" refers to a compound having at least one boron atom covalently bonded to an aromatic group and / or a compound having at least one boron atom covalently incorporated within an aromatic group. The term "aromatic" as used herein may include "heterocyclic", "heterocyclyl", or "heterocyclic". As used herein, the terms "heterocyclic", "heterocyclyl", or "heterocyclic" each refer to an unsaturated 3- to 18-membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. In some embodiments, the term "aromatic" may include "aryl". The term "aryl" as used herein refers to a monocyclic, bicyclic, or other polycarbocyclic aromatic ring system having 5 to 14 ring atoms. An aryl group can optionally be fused to one or more rings selected from aryl, cycloalkyl, heteroaryl, and heterocyclyl. Exemplary aryl groups include, but are not limited to, monocyclic aromatic ring systems in which the ring contains 6 carbon atoms.

[0050] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing one or more heteroatoms such as nitrogen, oxygen, and sulfur, for example 1 to 3 heteroatoms. Heteroaryl can be substituted with one or more substituents. Heteroaryl can also be fused to a non-aromatic ring. Exemplary heteroaryl groups include, but are not limited to, monocyclic aromatic rings, the ring containing 2 to 5 carbon atoms and 1 to 3 heteroatoms. In some embodiments, the aromatic boron-containing group can include, but is not limited to, aryl and heteroaryl boronic acids, aryl and heteroaryl boronic esters, and / or boroxoles. Exemplary aromatic boron-containing groups useful according to certain embodiments include, for example, those described herein as FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, FF226, F2, F5, F10, and FFL-1 to FFL-101, and further include, for example, those disclosed as compounds F1 to F9, F12 to F43, F500 to F520 in patent application PCT / US2021 / 025261 (filed Mar. 31, 2021), and those disclosed as compounds FF1 to FF224 and F1 to F10 in PCT / US2021 / 059802 (filed Nov. 18, 2021), the disclosures of which are hereby expressly incorporated by reference in their entirety.

[0051] As used herein, the term "small molecule linker" refers to a chemical group (e.g., backbone, moiety) that includes a first attachment point to X1 and a second attachment point to Z1b, Z1a, or Z1c. In some embodiments, the first attachment point is to X1 and the second attachment point is to Z1c. In some embodiments, the first attachment point is to X1 and the second attachment point is to Z1a. In some embodiments, the small molecule linker is a moiety / chemical group selected from Formulas IIa-IIai, IIIa-IIIai, and FL1-FL19 disclosed in Patent Application PCT / US2021 / 025261, the relevant disclosure of which is hereby expressly incorporated by reference herein.

[0052] As used herein, the term "indirect linker" refers to a chemical group (e.g., backbone, moiety) that includes a first attachment point to X1 and a second attachment point to Z1b, Z1a, or Z1c. In some embodiments, the first attachment point is to X1 and the second attachment point is to Z1c. In some embodiments, the indirect linker is a moiety / chemical group selected from (i) FL3, FL5, FL5A, FL5B, and FL20-FL75, their stereoisomers; and (ii) L or D-natural or unnatural amino acids. In some embodiments, at least one Z1c is covalently bonded to X1 or Z1a via one or more indirect linkers, and the indirect linker is of Formula (X”) n1 represented by, where each n1 is independently selected from 1, 2, 3, 4, and 5. For example, at least one Z1c is covalently bonded to X1 via an indirect linker, and the indirect linker is represented by Formula III: r”-(X1”) n1 -(X2”) n2 -(X3”) n3 -(X4”) n4 -(X5”) n5 -z” (Formula III) wherein z’’ represents the attachment point to the amine of X1, r’’ represents the attachment point to Z1c, Each of n1, n2, n3, n4, and n5 is independently 0 or 1, Each of X1'', X2'', X3'', X4'', and X5'' is independently selected from L- or D-amino acids, the amine functional group of the amino acid is covalently bonded towards r'' of Formula III, and the acid functional group of the amino acid is covalently bonded towards z'' of Formula III, FL3, FL5, FL5A, FL5B, and FL20 - FL75.

[0053] As used herein, "amino acid" includes proteinogenic (or natural) amino acids (among the 20 standard amino acids), as well as non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those that are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids are those not found in proteins or not produced by standard cellular machinery (e.g., they may have undergone post-translational modifications). Generally, amino acid residues (peptide / protein sequences) can be identified by their full names, their one-letter codes, and / or their three-letter codes. These three methods are completely equivalent. In the following, each amino acid of the compounds of the present disclosure for which no optical isomers are described should be understood to mean the L-isomer (unless otherwise specified). An amino acid is a molecule containing an amino group and a carboxylic acid group, and optionally one or more additional groups, often referred to as side chains.

[0054] As used herein, the term "amino acid residue" formally refers to an amino acid from which a hydroxy group has been removed from the carboxy group and / or an amino acid from which a hydrogen has been removed from the amino group. As is apparent from the following examples, amino acid residues can be identified by their full names, their one-letter codes, and / or their three-letter codes. These three methods are completely equivalent and interchangeable.

[0055] The term "alkyl" as used in the present invention herein is C 1~30It refers to a saturated straight-chain or branched-chain hydrocarbon such as a straight-chain or branched group of 1 to 30 carbon atoms called alkyl. In some embodiments, the alkyl group is C1-C 22 alkyl group. In some embodiments, the alkyl group is C1-C 20 alkyl group. In some embodiments, the alkyl group is C1-C 18 alkyl group. In some embodiments, the alkyl group is C1-C 16 alkyl group. In some embodiments, the alkyl group is C1-C 14 alkyl group. In some embodiments, the alkyl group is C1-C 12 alkyl group. In some embodiments, the alkyl group is C1-C 10 alkyl group. In some embodiments, the alkyl group is a C1-C8 alkyl group. In some embodiments, the alkyl group is a C1-C6 alkyl group. In some embodiments, the alkyl group is a C1-C4 alkyl group. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. In some embodiments, "alkyl" is a straight-chain hydrocarbon. In some embodiments, "alkyl" is a branched-chain hydrocarbon.

[0056] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated cyclic, bicyclic, or bridged bicyclic hydrocarbon group of 3 to 16 carbons, or 3 to 8 carbons, referred to herein as "(C3-C8) cycloalkyl" derived from cycloalkane. In some embodiments, the cycloalkyl is C3-C6 cycloalkyl. Examples of cycloalkyl groups include, but are not limited to, cyclohexane, cyclohexene, cyclopentane, and cyclopentene. The cycloalkyl group may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. The cycloalkyl group can condense with other cycloalkyl (saturated or partially unsaturated), aryl, or heterocyclyl groups to form bicyclic, tetracyclic, etc. The term "cycloalkyl" also includes bridged and spiro-condensed cyclic structures which may or may not contain heteroatoms.

[0057] As used herein, the term "acyl" refers to an R-C(O)- group such as, but not limited to, (alkyl)-C(O)-, (alkenyl)-C(O)-, (alkynyl)-C(O)-, (aryl)-C(O)-, (cycloalkyl)-C(O)-, (heteroaryl)-C(O)-, and (heterocyclyl)-C(O)-, and the group is bonded to the parent molecular structure via a carbonyl functional group. In some embodiments, it refers to, for example, the total number of chain or ring atoms of an alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or heteroaryl moiety plus the carbonyl carbon of the acyl. 1-10 An acyl radical. For example, a C4-acyl has a carbonyl in addition to the other three ring or chain atoms. In some embodiments, it is a C1-C 22 An acyl group. In some embodiments, it is a C1-C 20It is an acyl group. In some embodiments, it is a C1-C 18 It is an acyl group. In some embodiments, it is a C1-C 16 It is an acyl group. In some embodiments, it is a C1-C 14 It is an acyl group. In some embodiments, it is a C1-C 12 It is an acyl group. In some embodiments, it is a C1-C 10 It is an acyl group. In some embodiments, it is a C1-C8 acyl group.

[0058] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, and the like. In some embodiments, it is a C1-C 22 haloalkyl group. In some embodiments, it is a C1-C 20 haloalkyl group. In some embodiments, it is a C1-C 18 haloalkyl group. In some embodiments, it is a C1-C 16 haloalkyl group. In some embodiments, it is a C1-C 14 haloalkyl group. In some embodiments, it is a C1-C 12 haloalkyl group. In some embodiments, it is a C1-C 10 haloalkyl group. In some embodiments, it is a C1-C8 haloalkyl group.

[0059] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or other polycarbocyclic aromatic ring system having 5 to 14 ring atoms. An aryl group can optionally be fused to one or more rings selected from aryl, cycloalkyl, heteroaryl, and heterocyclyl. An aryl group of the present disclosure can be substituted with a group selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, and benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups include, but are not limited to, monocyclic aromatic ring systems in which the ring contains 6 carbon atoms.

[0060] As used herein, a "leaving group" is an atom (or group of atoms) that can be substituted as a stable species having bonding electrons. For example, a leaving group can be an anion (e.g., Cl - ) or a neutral molecule (e.g., H2O). In some embodiments, the leaving group is a halogen, an N-hydroxysuccinimide (NHS) group, a 2,3,5,6-tetrafluorophenol (TFP) group, a pentafluorophenol (Pfp) group, or a sulfonic acid ester.

[0061] "Isomer" means a compound that has the same number and kinds of atoms and thus the same molecular weight but differs in the arrangement or configuration of the atoms in space.

[0062] "Stereoisomers" or "optical isomers" mean stable isomers that have at least one chiral atom or have restricted rotation that gives rise to a perpendicular plane of asymmetry (e.g., certain biphenyls, allenes, and spiro compounds) and can rotate plane-polarized light. Since the compounds of the present disclosure have asymmetric centers and other chemical structures that can give rise to stereoisomerism, the present disclosure contemplates stereoisomers and mixtures thereof. The compounds of the present disclosure and their salts can exist as single stereoisomers, racemates, and mixtures of enantiomers and diastereomers because they contain asymmetric carbon atoms. In some embodiments, such compounds are prepared as racemic mixtures. In some embodiments, such compounds can be prepared or isolated as pure stereoisomers, e.g., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. As will be discussed in more detail below, the individual stereoisomers of a compound can be synthesized from an optically active starting material containing the desired chiral center or by the preparation of a mixture of enantiomeric products followed by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, use of a chiral resolving agent, or direct separation of enantiomers on a chiral chromatography column. Specific stereochemical starting compounds are either commercially available or prepared by the methods described below and are solved by techniques well known in the art.

[0063] As used herein, "fatty acid" is a carboxylic acid having an aliphatic chain. Saturated fatty acids have saturated aliphatic chains, while unsaturated fatty acids have unsaturated aliphatic chains. In some embodiments, the fatty acid is a C3-C 26 fatty acid. In some embodiments, the fatty acid is a C4-C 20It is a fatty acid. In some embodiments, the fatty acid is CH3CH2COOH, CH3(CH2)2COOH, CH3(CH2)3COOH, CH3(CH2)4COOH, CH3(CH2)5COOH, CH3(CH2)6COOH, CH3(CH2)7COOH, CH3(CH2)8COOH, CH3(CH2)9COOH, CH3(CH2) 10 COOH, CH3(CH2) 11 COOH, and CH3(CH2) 12 COOH, CH3(CH2) 13 COOH, CH3(CH2) 14 COOH, CH3(CH2) 15 COOH, CH3(CH2) 16 COOH, CH3(CH2) 17 COOH, and CH3(CH2) 18 COOH is a saturated fatty acid selected from. In some embodiments, the fatty acid is an unsaturated fatty acid selected from α-linolenic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linolenic acid, γ-linolenic acid, oleic acid, elaidic acid, and gondoic acid.

[0064] The term "pharmaceutically acceptable salt" refers to salts of acidic or basic groups that may be present in the compounds used in the present composition.

[0065] As used herein, "drug substance" refers to small molecule compounds and / or polypeptide-containing compounds. According to some embodiments, the drug substances suitable for use in the compounds and methods described herein are therapeutically, prophylactically and / or diagnostically active drug substances.

[0066] Terms such as "first", "second", "third", etc. may be used herein to describe various elements (such as molecules, components, groups, and / or moieties), but it will be understood that these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, the first element described below could be referred to as the second element without departing from the spirit and scope of the present disclosure. When an element or group is said to be "connected to", "conjugated to", "linked to", or "coupled to" another element or group, it will be understood that the two elements may be directly bonded or there may be one or more intervening elements. It will be understood that the bonds and linkages described herein have the option of being direct bonds or direct linkages, unless explicitly excluded or precluded by the context.

[0067] The term "direct" or "direct covalent bond" or "directly covalently bonded" as used herein can be used interchangeably to indicate that a first group is "direct" or "direct covalent bond" or "directly covalently bonded" to a second group, which means that the first and second groups are covalently bonded together without additional intervening groups.

[0068] The term "indirectly" or "indirectly covalently bonded" or "indirectly covalently linked" as used herein can be used interchangeably to indicate that a first group is "indirectly" or "indirectly covalently bonded" or "indirectly covalently linked" to a second group, which means that the first and second groups are covalently bonded through at least one additional intervening group (e.g., a small molecule, a linker, a spacer, a linear sequence of amino acids and / or a non-linear sequence of amino acids).

[0069] The terms "insulin receptor agonist", "compound having agonist efficacy", and "agonist efficacy" in the context of a compound having agonist efficacy against the insulin receptor refer to a compound that binds and / or activates the insulin receptor (e.g., a compound of formula (I), a protein, e.g., a fusion protein described in U.S. Patent No. 2016 / 0324932).

[0070] The terms "rapid-acting insulin receptor agonist" and "rapid-acting insulin" refer to an insulin receptor agonist having an onset of activity equal to or faster than that of insulin isophane human and insulin human, which are sold under the trade name Humulin®. For example, a rapid-acting insulin can have an onset of action occurring within 10 minutes of injection. As another example, a rapid-acting insulin can have an onset of action occurring within 20 minutes of injection. In some embodiments, a rapid-acting insulin can have a maximum efficacy (peak) within 1 to 4 hours after injection. In some embodiments, a rapid-acting insulin can be administered before, during, and / or immediately after a meal.

[0071] The terms "long-acting insulin receptor agonist" and "long-acting insulin" refer to an insulin receptor agonist having a slower onset of action than Humulin®. For example, a long-acting insulin can have an onset of action occurring 1 to 2 hours or later. In certain embodiments, a long-acting insulin can have a maximum efficacy (peak) within 6 to 20 hours or can have a prolonged action. In some embodiments, a long-acting insulin can be administered once daily or, for example, once a week.

[0072] As used herein in the context of an insulin receptor agonist, the term "suitable for meal administration" refers to a rapid-acting insulin receptor agonist suitable for controlling blood glucose levels during and / or immediately after a meal.

[0073] As used herein in connection with insulin receptor agonists, the term "suitable for once-weekly dosing" refers to an insulin receptor agonist having a pharmacokinetic and pharmacodynamic profile that is long enough to control blood glucose levels throughout the day when administered at a frequency of once-weekly or less. Examples of such molecules include the fusion proteins described in U.S. Patent Application Publication No. 2016 / 0324932, including BIF. BIF, also known as insulin efcirtoalfa, comprises a dimer of an insulin receptor agonist fused to the human IgG Fc region, the insulin receptor agonist comprising an insulin B-chain analog fused to an insulin A-chain analog by use of a first peptide linker, the C-terminal residue of the insulin A-chain analog being directly fused to the N-terminal residue of a second peptide linker, and the C-terminal residue of the second peptide linker being directly fused to the N-terminal residue of the human IgG Fc region. BIF is identified by the CAS Registry Number 2131038-11-2 and has the following chemical name: (1) Insulin [47-threonine, 51-aspartic acid, 58-glycine] (human A-chain) fusion protein having a peptide (synthetic 20-amino acid linker) fusion protein having a peptide (synthetic 7-amino acid linker) fusion protein having an immunoglobulin G2 (human Fc fragment), dimer having an insulin [16-glutamic acid, 25-histidine, 27-glycine, 28-glycine, 29-glycine, 30-glycine] (human B-chain) fusion protein; and (2) Tris(tetraglycylglutaminyl)pentaglycyl(59-78) homosapiens immunoglobulin heavy chain constant gamma2 {del-CH1, hinge-(7-12), CH2, CH3[K 107 >del(300)]}(79-299), diglycylseryl-tetraglycyl(31-37) insulin A-chain [I10>T(47), Y14>D(51), N21>G(58)](38-58) fusion protein having a dimer (80-80’:83-83’)-bisdisulfide having a homosapiens insulin B-chain [Y16>Y(16), F25>H(25), TPKT27-30>GGGG(27-30)](1-30) fusion protein.

[0074] As used herein, "glucose-sensing insulin" refers to an insulin receptor agonist having onset and / or level of activity that is dependent on blood glucose levels. Examples of such molecules include the compounds of formula (I) disclosed herein, for example in Examples 2, 5, 13, 22, 28, 32, 35, 50, 56, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 126, 128, 132, 134, 135, 150, and 154.

[0075] "Incretin-based therapy" includes the administration of a group including, but not limited to, GLP-1, gastric inhibitory polypeptide (GIP), and glucagon, which are metabolic hormones known as incretins, or any therapy that promotes, enables, enhances, and / or stimulates their effects. Currently available incretin-based therapies include GLP-1R agonists.

[0076] "DPP-4 inhibitor" is a compound that interferes with the DPP-4 enzyme involved in incretin degradation. Currently available DPP-4 inhibitors include sitagliptin (Januvia®) and linagliptin (Tradjenta®).

[0077] "GLP-1R agonists" and "glucagon receptor agonists" are defined as compounds that contain the amino acid sequence of native human GLP-1 (SEQ ID NO: 25) or human glucagon, and compounds that maintain full or partial activity at the GLP-1 receptor, such as GLP-1 analogs, GLP-1 derivatives, or GLP-1 fusion proteins. GLP-1R activity may be measured by methods known in the art, including in vivo experiments and in vitro assays that measure GLP-1 receptor binding activity or receptor activation, as described in European Patent No. 619,322 and U.S. Patent No. 5,120,712, respectively, for example, the use of assays using pancreatic islet cells or insulinoma cells. A GLP-1 analog is a molecule that has a modification that includes one or more amino acid substitutions, deletions, inversions, or additions when compared to the amino acid sequence of native human GLP-1 (SEQ ID NO: 25). A GLP-1 derivative is a molecule that has the amino acid sequence of native human GLP-1 (SEQ ID NO: 25) or a GLP-1 analog, but further has at least one chemical modification of one or more of its amino acid side groups, α-carbon atoms, terminal amino groups, or terminal carboxylic acid groups. A GLP-1 fusion protein is a heterologous protein that includes a GLP-1, GLP-1 analog, or GLP-1 derivative moiety, and a second polypeptide. Currently available GLP-1R acting drugs include (Byetta® and Bydureon®), liraglutide (Victoza®), albiglutide (Tanzeum®), and dulaglutide (Trulicity®), and their structures are known in the art. See, for example, U.S. Patent No. 5,424,286 (exenatide); U.S. Patent No. 6,268,343 (liraglutide); U.S. Patent Application Publication No. 2014044717 (albiglutide); and U.S. Patent No. 7,452,966 (dulaglutide).

[0078] The term "excipient" means any substance added to a composition other than the fusion protein or any other additional active ingredient(s). Examples of such excipients that can be used in the compositions of the present invention include buffers, surfactants, tonicity agents, and preservatives. "Pharmaceutically acceptable excipients" refer to excipients that are compatible with the other components in the composition and are suitable for contact with any tissue, organ, or part of the body that they may encounter, and which should not have a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs their therapeutic benefit. In some embodiments, excipients can be used to stabilize an agonist while in solution or to increase onset and maximal efficacy. Such excipients can include mannitol, sorbitol, m-cresol, EDTA, and citrate salts.

[0079] A "buffer" is a substance that resists changes in pH by the action of its acid-base conjugate components. In certain embodiments, the compositions of the present invention have a pH of from about 5.5 to about 9.0, preferably from about 7.0 to about 8.0, more preferably from about 7.2 to about 7.8. Suitable buffers for controlling the pH of the compositions of the present invention within the desired range include, but are not limited to, phosphates, succinates, citrates, or their acids, arginine, TRIS, HEPES, and histidine buffers, and combinations thereof. "TRIS" refers to 2-amino-2-hydroxymethyl-1,3-propanediol and any of its pharmaceutically acceptable salts. The free base and hydrochloride form (i.e., TRIS-HCl) are two common forms of TRIS. TRIS is also known in the art as trimethylolaminomethane, tromethamine, and tris(hydroxymethyl)aminomethane. Preferred buffers in the compositions of the present invention are citrate or citric acid, phosphate, and TRIS.

[0080] The compounds disclosed herein may contain one or more protein components (e.g., when X1 contains one or more protein components). In some embodiments, the compounds disclosed herein contain two or more fusion protein components or two or more conjugate protein components. As used herein, the term "fusion protein" refers to a combination of two or more distinct proteins that are directly linked via a peptide bond between the C-terminus of one protein and the N-terminus of another protein, or indirectly via a linker that connects the C-terminus or N-terminus or side chain of one protein to the C-terminus or N-terminus or side chain of another protein (i.e., via a chemical linker such as a bi-functionalized PEG linker), or indirectly via a continuous amino acid chain. As used herein, the term "conjugate protein" refers to a combination of two or more distinct proteins that are chemically linked to each other directly by a chemical bond or indirectly via a chemical linker. In some examples, two or more proteins may act on the same or similar receptors (i.e., a fusion of two insulin agonist peptides), or on distinct different receptors (i.e., one protein is an insulin receptor agonist and another protein is a glucagon receptor agonist). In some examples, the fusion protein may contain only one agonist protein, while the other protein is a human IgG Fc region or a single domain antibody (nanobody) or a variable heavy chain sequence (V H H).

[0081] The term "composition comprising a fusion protein" encompasses compositions comprising monomers, homodimers, heterodimers or multimers of the fusion protein. In certain embodiments, the pharmaceutical compositions of the present disclosure are compositions comprising a fusion protein at a concentration of at least 1 mg / mL, at least 2 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 20 mg / mL, at least 25 mg / mL, at least 30 mg / mL, at least 35 mg / mL, at least 50 mg / mL, at least 55 mg / mL, at least 50 mg / mL, at least 65 mg / mL, at least 75 mg / mL, at least 100 mg / mL, or higher. In some embodiments, the fusion protein is present at a concentration of 10 to 100 mg / mL. In some embodiments, the fusion protein is present at a concentration of 15 to 75 mg / mL, and in some embodiments, the fusion protein is present at a concentration of 20 to 65 mg / mL.

[0082] The pharmaceutical compositions of the present disclosure may also contain a "surfactant", which means a substance that reduces the surface tension of a liquid. Examples of surfactants that are used in pharmaceutical compositions and that may be used in certain compositions of the present disclosure include polysorbate 20, polysorbate 80, polyethylene glycol (e.g., PEG 400, PEG 3000, TRITON X-100), polyethylene glycol alkyl ether (e.g., BRIJ), polypropylene glycol copolymer (e.g., poloxamer, PLURONIC F68; poloxamer 407, PLURONIC F127; TETRONICS), sorbitan alkyl ester (e.g., SPAN), polyethoxylated castor oil (e.g., KOLLIPHOR, CREMOPHOR), and trehalose.

[0083] The pharmaceutical composition of the present invention may also contain a preservative. The term "preservative" refers to a compound added to a pharmaceutical formulation to act as an antibacterial agent. Preservatives known in the art as effective and acceptable in parenteral formulations include, among others, benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methyl- or propyl-paraben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. Phenolic preservatives include the compounds phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben, benzyl alcohol, and mixtures thereof. When a preservative is required, the preservative used in the composition of the present invention is preferably either m-cresol or phenol, and / or benzyl alcohol. Certain phenolic preservatives such as phenol and m-cresol are known to bind to insulin and insulin hexamers, stabilizing conformational changes, which increases either physical stability or chemical stability, or both. However, in compositions containing other proteins, such preservatives can contribute to the formation of protein aggregates or high molecular weight polymers (HMWPs). See, for example, Maa Y F and Hsu C C, Int J Pharm 140:155-168(1996); Fransson J, et al., Pharm.Res., 14: 606-612 (1997); Lam X M, et al., Pharm.Res., 14: 725-729 (1997); Remmele R L Jr, et al., Pharm Res 15: 200-208.(1998); Thirumangalathu R,et al.,J Pharm Sci 95:1480-1497(2006). In some instances, such protein aggregates in therapeutic formulations may be undesirable due to their tendency to induce an immune response.

[0084] As used herein, "combination therapy" or administration "in combination with" one or more additional therapeutic agents includes the administration of two or more active agents (e.g., two or more pharmacological agents) intended to treat a given indication and / or a condition associated therewith. The administration may be simultaneous (concurrent) or sequential (in any order). The two or more agents of a "combination therapy" may be formulated as separate compositions (e.g., formulations) or as a single composition (formulation). "Combination therapy" encompasses the administration of agents having different mechanisms of action or targeting different indications and / or conditions, as well as agents having similar mechanisms of action or targeting similar indications and / or conditions. For example, since patients with type 1 diabetes produce little or no insulin, insulin therapy effective for type 1 diabetes may involve the use of two types of externally administered insulin, i.e., rapid-acting mealtime insulin provided by bolus injection and long-acting basal insulin administered once or twice a day to control blood glucose levels between meals. Treatment of patients with type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet, but if these measures fail to control elevated blood glucose, oral medications and incretin-based therapies may be required, e.g., administration of glucagon-like peptide-1 (GLP-1) receptor agonists and / or dipeptidyl peptidase 4 (DPP-4) inhibitors that increase incretin levels. If these agents are still insufficient, treatment with insulin may be considered. Patients with type 2 diabetes whose disease has progressed to the point where insulin therapy is required may generally begin with a once-daily injection of long-acting basal insulin, but in some cases, may also appropriately include mealtime injections of rapid-acting insulin. In some embodiments, the present disclosure provides a combination therapy comprising administering rapid-acting insulin and basal insulin and / or a fusion protein comprising, for example, one or more diboronatesensors disclosed herein.

[0085] The terms "administer", "administering", or "administration" include any method or act of delivering a pharmacological agent (e.g., a pharmaceutical) to an intended subject (e.g., a patient). A pharmacological agent can be any suitable therapeutic agent, such as a biological agent, such as an antibody or an antigen-binding fragment thereof (e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment), a peptide agent (e.g., a hormone or a modified analog thereof), or a low molecular weight agent (e.g., a structurally defined small molecule or chemical substance). Administration of a pharmacological agent can be systemic or local. In some embodiments, administration can include one or more pharmacological agents that are administered in parallel, simultaneously, or sequentially.

[0086] The terms "simultaneously" and "in parallel" are used interchangeably and herein are used to refer to the administration of two or more therapeutic agents where at least a portion of the administrations overlap in time or the administration of a second therapeutic agent enters within a period shorter than the time required to initiate subsequent administrations after the administration of a first therapeutic agent. For example, simultaneous administration includes administering a second agent after a first agent without adding a delay beyond the time required to complete the first administration and initiate the second administration.

[0087] The terms "sequentially" and "continuously" are used interchangeably and are used herein to refer to the administration of two or more therapeutic agents where there is a delay period between the administration of one therapeutic agent and the administration of another agent. For example, continuous administration includes the administration of two or more therapeutic agents that are administered at intervals of greater than about 15 minutes, such as about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, or 1 month, or more.

[0088] As used herein, the term "basal insulin" can refer to several types of basal insulin (e.g., long-acting insulin). For example, insulin glargine sold under the brand name LANTUS(®) contains a modified insulin structure in which asparagine at position 21 in the A chain of insulin is replaced by glycine, and two arginines are added to the C-terminus of the B chain. In another example, insulin glargine-algr sold under the brand name LANTUS TM contains a modified insulin structure in which asparagine at position 21 in the A chain of insulin is replaced by glycine, and two arginines are added to the C-terminus of the B chain. In yet another example, insulin detemir sold under the brand name LEVEMIR(®) contains a modified insulin structure in which threonine at position 30 of the B chain is deleted and lysine at position 29 of the B chain is derivatized through a covalent bond between the amine group of lysine at B29 and a 14-carbon myristoyl fatty acid. Insulin degludec, available in Europe and Japan under the brand name TRESIBA(®), contains a modified insulin structure in which threonine at position 30 of the B chain is deleted and the ε-amino group of lysine at position 29 of the B chain is covalently derivatized with hexadecanedioic acid via a γ-L-glutamic acid linker. All of these insulins are adapted for once-daily administration. In some embodiments, the present disclosure provides the use of one or more compounds (e.g., a fusion protein of formula I) in the manufacture of a medicament for the treatment of a disease (e.g., type 1 diabetes, obesity, dyslipidemia, or metabolic syndrome), the medicament being administered in combination with another active ingredient, simultaneously, separately, or sequentially. The compounds and combinations (e.g., compounds of formula I, fusion proteins) disclosed herein are effective to treat a disease and / or condition in a subject in need thereof by administering a therapeutically effective amount of a compound and / or composition of the present disclosure to a subject in need thereof.

[0089] As used herein, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a disease or an apparent symptom of a disease. A therapeutically effective amount can treat, cure, alleviate, mitigate, modify, correct, improve, or affect a disease, a symptom of a disease, or a predisposition to a disease for the purpose of treating, curing, alleviating, mitigating, modifying, correcting, improving, or affecting the disease, the condition, the symptom of the disease, or the predisposition to the disease. A therapeutically effective set or specific amount can be readily determined by a person of ordinary skill in the medical art and can vary depending on factors known in the art, such as the type of disease, the patient's medical history and age, the stage of the disease, and the administration of other therapeutic agents. In some embodiments, the term "therapeutically effective amount" refers to an amount of a compound and / or a pharmaceutical composition and / or a combination of active substances disclosed herein that is sufficient to regulate the blood glucose of a patient without causing unacceptable side effects. The therapeutically effective amount of a compound and / or a pharmaceutical composition and / or a combination of active substances disclosed herein administered to a subject depends on the type and severity of the disease and the characteristics of the subject, such as general health status, age, gender, weight, and drug tolerance. One of ordinary skill in the art would be able to determine an appropriate dosage according to these and other factors. For example, the therapeutically effective amount of the fusion protein of the present disclosure when administered once a week ranges from about 0.01 nmol / kg to about 100 nmol / kg. In some embodiments, the therapeutically effective amount of the fusion protein of the present disclosure when administered once a week ranges from about 1 nmol / kg to about 50 nmol / kg. In some embodiments, the therapeutically effective amount of the fusion protein of the present disclosure when administered once a week ranges from about 16 nmol / kg to about 25 nmol / kg. In certain embodiments, the therapeutically effective amount of the fusion protein of the present disclosure when administered once a week ranges from about 1 mg to about 200 mg. In some embodiments, the therapeutically effective amount of the fusion protein of the present invention when administered once a week ranges from about 25 mg to about 175 mg. In some embodiments, the therapeutically effective amount of the fusion protein of the present disclosure when administered once a week ranges from about 100 mg to about 160 mg.

[0090] The terms "subject" and "patient" are used interchangeably herein and refer to the party receiving the therapy or treatment. In some embodiments, the "subject" and "patient" are human.

[0091] The terms "blood glucose level" and "blood sugar" are used interchangeably herein and refer to the concentration of sugar present in the blood. A blood glucose meter or glucometer can be used to measure the amount of sugar in a blood sample. Blood glucose levels are generally measured as mg of sugar per dL of blood (mg / dL). Blood glucose levels can refer to any level of sugar disclosed herein. For example, blood glucose levels include the glucose concentration in the blood, also known otherwise as blood glucose level / concentration.

[0092] As used herein, the term "steady state" refers to a constant / fixed state where there is no increase or decrease, or minimal variation (i.e., less than or equal to 10%), in the variable of interest. In context, in some embodiments, an insulin receptor agonist is intravenously (IV) infused as a fixed dose bolus and glucose is continuously infused at a variable rate (mg / kg / min) such that the blood glucose level is maintained at a predetermined "steady state" level (e.g., 100 mg / dL for normoglycemia; 200 mg / dL or 400 mg / dL for hyperglycemia). The amount of glucose infused to maintain the "steady state" blood glucose level is equal to the total body glucose uptake and utilization.

[0093] The term "hyperglycemia" is used herein to refer to a physiologically high blood glucose level. Hyperglycemia refers to the state of having a physiologically high blood glucose level. The blood glucose levels considered to be hyperglycemic vary based on the species of the subject. For example, in rats, hyperglycemia refers to a blood glucose level of 200 mg / dL or higher. In humans, hyperglycemia refers to a blood glucose level of greater than 180 mg / dL.

[0094] The term "euglycemia" is used herein to refer to physiologically normal blood glucose levels. Euglycemia refers to the state of normal blood glucose levels. The blood glucose levels considered to be euglycemic vary based on the species of the subject. For example, in rats, euglycemia refers to blood glucose levels of less than 200 mg / dL. In humans, euglycemia refers to blood glucose levels of 70 - 180 mg / dL.

[0095] The phrase "glucose infusion rate" is used herein to refer to the rate at which glucose is infused into a subject. The glucose infusion rate (GIR) is calculated in units of mg / kg / min as (infusion rate (mL / hour) × glucose concentration (g / dL) × 1000 (mg / g)) / weight (kg) × 60 (min / hour) × 100 (mL / dL), where "infusion rate" refers to the rate at which glucose is infused into the subject, "glucose concentration" refers to the concentration of the infused glucose, and "weight" refers to the body weight of the subject. In some embodiments, the GIR corresponds to the glucose infusion rate required to maintain a specific blood glucose level.

[0096] The phrase "relative glucose infusion rate difference" as used herein refers to the difference in the amount of glucose infused between two different conditions, experiments, or measurements. In some embodiments, it can be measured by taking the difference between (a) the area under the curve (AUC) for one recorded GIR to maintain a specific blood glucose concentration and (b) the AUC for another recorded GIR to maintain a different blood glucose concentration. Generally, the AUC of the GIR to maintain a lower blood glucose concentration is subtracted from the AUC of the GIR to maintain a higher blood glucose concentration. For example, if the AUC of a first GIR (providing a blood glucose concentration of 100 mg / dL) is 600 mg / kg / min.min and the AUC of a second GIR (providing a blood glucose concentration of 200 mg / dL) is 800 mg / kg / min.min, subtracting the AUC of the first GIR from the AUC of the second GIR gives a relative glucose infusion rate difference of 200 mg / kg / min.min.

[0097] As used herein, the phrase "relative glucose infusion rate ratio" refers to the ratio of the amount of glucose infused between two different conditions, experiments, or measurements. In some embodiments, it can be measured by taking the ratio between (a) the area under the curve (AUC) for one recorded GIR to maintain blood glucose concentration and (b) the AUC for another recorded GIR to maintain a different glucose concentration. Generally, the AUC for the GIR to maintain a higher blood glucose concentration is divided by the AUC for the GIR to maintain a lower blood glucose concentration. For example, if the AUC of the first GIR (providing a blood glucose concentration of 100 mg / dL) is 600 mg / kg / min.min and the AUC of the second GIR (providing a blood glucose concentration of 200 mg / dL) is 800 mg / kg / min.min, dividing the AUC of the second GIR by the AUC of the first GIR gives a relative glucose infusion rate ratio of 1.33.

[0098] As used herein, the term "area under the curve" refers to the area bounded by a curve, an axis, and two boundary points, whether plotted or represented mathematically. In some embodiments, the curve used to calculate the area under the curve (AUC) is a measure of GIR as a function of time where the X-axis corresponds to time, the Y-axis corresponds to GIR, and the origin is at 0 on the Y-axis (i.e., the X-axis and Y-axis intersect at 0 on the Y-axis). For example, the curve may be the GIR recorded over a period of time, and the boundary points may be the GIR at the start and end of the infusion in an experiment. The AUC is then calculated for the area between the plotted curve and the X-axis between the start boundary point and the end boundary point. Mathematically, the AUC can be calculated according to methods known to those skilled in the art, such as the method in Tai M.M. (1994) Diabetes Care, 17(2): 152-154, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the area under the curve (AUC) is calculated using the trapezoidal rule and applying baseline correction. In some embodiments, the AUC is calculated using GraphPad Prism v9. For baseline correction, the average GIR value from 30 minutes before injection (x = -30 minutes) to the time of injection (x = 0) can be subtracted from each GIR value from time 0 (x = 0) to the last time point measured (x = 300). The trapezoidal calculation is as follows.

Number

[0099] Here, a is the first time point (e.g., 0 minutes) and b is the last time point (e.g., 300 minutes). The area was calculated as a subdivision of small trapezoids as follows.

Number

[0100] Δx is the difference between each time point (5-minute intervals).

[0101] As used herein, the term "EC50" refers to the maximum half - maximal effective concentration of a compound in a dose - response assay. EC50 is a measure of the concentration of a compound required to produce half of the maximum possible effect as a result of exposure to the compound. EC50 can be calculated using methods known in the art. In some embodiments, EC50 can be calculated using the following equation: Y = bottom+(X^hill slope) * (top - bottom) / (X^hill slope+EC50^hill slope) to fit a four - parameter logistic regression curve, where the hill slope refers to the gradient of the sigmoid curve between the upper and lower plateaus of the dose - response curve. In some embodiments, EC50 can be calculated using GraphPad Prism v7, 8, or 9. If a compound has its EC50 measured at two or more concentrations of a sugar (e.g., glucose), (a) the term "first sugar concentration" refers to the first, lower sugar concentration, e.g., when the concentration is about 3 mM or about 5 mM; (b) the "second sugar concentration" refers to the second, higher sugar concentration, e.g., when the concentration is about 10 mM, about 20 mM, or about 30 mM. In some embodiments, for example, by comparing the EC50 of the dose - response curves, the fold - change in insulin receptor phosphorylation (IR phosphorylation) activity of an exemplary compound of Formula I from a low concentration of glucose (e.g., about 3 mM or about 4 mM) to a high concentration (e.g., about 10 mM, about 20 mM, or about 30 mM) was evaluated. This fold - change in activity was determined by dividing the EC50 of the compound (e.g., a compound of Formula I) at the "low" glucose concentration (e.g., about 3 mM) by the EC50 of that compound at the "high" glucose concentration (e.g., about 20 mM), keeping all other conditions constant. See the examples entitled "In Vitro Demonstration of the Activity of Compounds of Formula I".

[0102] As used herein, the term "Kd" refers to the dissociation constant and reflects the binding affinity between a ligand (e.g., a boronate sensor as described herein) and its target (e.g., a sugar, such as glucose). For example, "glucose Kd" and "average glucose Kd" in the context of the boronate sensors described herein refer to the affinity of the boronate sensor for glucose, where the Kd is measured before the boronate sensor is conjugated to an insulin molecule to produce the compounds described herein. The binding of the boronate sensors described herein to glucose can be measured by an alizarin red S (ARS) displacement assay and is recorded before the boronate sensor is conjugated (e.g., covalently) to an insulin molecule (e.g., a compound of formula I). The ARS displacement assay is known in the art, e.g., Springsteen and Wang (2001) Chem. 1608-1609, which is hereby incorporated by reference in its entirety. In the ARS displacement assay, the boronate sensors disclosed herein are incubated with ARS and the fluorescence emission is recorded. The composition of the boronate sensors and ARS described herein is then titrated against serial dilutions of a sugar (e.g., glucose), and the fluorescence emission after incubation is then measured to determine the displacement of ARS when the boronate sensor binds to the sugar. The change in intensity (fluorescence emission with and without the sugar) can be plotted against the concentration of the sugar to generate an association constant for sugar binding. When the boronate sensor has a higher binding constant for the sugar, the Kd increases. Further, the boronate sensors described herein include those having a selective affinity / binding for sugars. For example, the boronate sensors described herein can include sensors that have an increased affinity (binding) for glucose but not for other sugars such as lactate and / or fructose. See the Examples entitled "Procedure for Measuring Glucose, Fructose, and Lactate Binding (Kd) Using the ARS Displacement Assay."

[0103] As used herein, the terms "clamp", "clamp assay", and "clamped" refer to a hyperglycemic / euglycemic clamp (glucose clamp), which is recognized as the "gold standard" method for detecting insulin activity through glucose utilization in experimental animals and humans. In a hyperglycemic / euglycemic clamp, in certain embodiments, plasma (blood) insulin concentration rises rapidly and is maintained by continuous infusion of insulin, while plasma glucose concentration is held constant at a predetermined hyperglycemic or euglycemic level by a variable glucose infusion rate (GIR). Once a steady-state blood glucose value is achieved, the glucose infusion rate is equal to insulin receptor-stimulated glucose uptake by all tissues in the body and is thus a measure of insulin activity at a particular blood glucose value. For example, in some embodiments, glucose is continuously infused into the subject over the course of the study, and the glucose infusion rate is adjusted to maintain a constant blood glucose value in response to administration of a compound (e.g., a compound of formula I). The change in glucose infusion rate in response to administration of a compound at a particular dose level or concentration is recorded and used to determine the glucose infusion rate. In some embodiments, the sugar is glucose. See, for example, Lautt W.W., et al. (1998) Canadian Journal of Physiology and Pharmacology. 76 (12):1080-1086 (the entire contents of which are incorporated herein by reference). The clamp techniques / assays referred to and used herein are known in the art and are modified versions of the disclosed techniques. See the examples entitled "In Vivo Demonstration of Activity of Compounds of Formula I".

[0104] In at least some embodiments, one or more groups (e.g., X1a, Z1a, Z1c) are covalently bonded to each other, either directly or indirectly (e.g., via an indirect linker). For example, according to certain embodiments, Z1c is covalently bonded to the amine in X1, either directly or indirectly (e.g., via one or more indirect linkers). As another example, according to certain embodiments, one or more drug substances (X1) are covalently bonded to one or more amine-containing linkers. In some embodiments, X represents a point of covalent attachment directly to the amine of X1, or to an amine that is covalently bonded directly or indirectly (e.g., via an indirect linker) to X1. In certain embodiments, each Z1c is independently covalently bonded to the amine of Z1a, or to the amine of X1, either directly or indirectly (e.g., via an indirect linker of formula (X”) n1 ). In some embodiments, at least one Z1c is covalently bonded to X1 or Z1a via an indirect linker (e.g., of formula (X”) n1 ).

[0105] As used herein, terms such as “point of attachment to [group],” “attachment to,” and “covalent attachment to [group]” indicate that the indicated atom, bond, or linkage is closer to the indicated group than to other points of attachment or covalent bond variables within the structural formula. In some embodiments, the point of attachment or covalent bond may be directly adjacent to the indicated group, and in some embodiments, other atoms or groups may be present therebetween.

[0106] As used herein, the term "percent homology" refers to the percentage of sequence identity between two sequences after optimal alignment. The homology of identical sequences has a percent homology of 100%. Optimal alignment can be performed by homology alignment algorithms as described by Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by the general methods described for similarity search by Neddleman and Wunsch, J. Mol. Biol. 48:443 (1970), including execution of these algorithms or visual comparison. As used herein, the "insulin A chain" is the chain of insulin having the highest percentage of homology to the A chain of wild-type human insulin. As used herein, the "insulin B chain" is the chain of insulin having the highest percentage of homology to the B chain of wild-type human insulin.

[0107] In some embodiments, the terms "covalently connected," "covalently conjugated," or "covalently linked" may be used interchangeably to indicate that two or more atoms, groups, or chemical moieties are bonded or connected via a chemical bond. In some embodiments, a chemical bond (which may be referred to as a covalent bond in some embodiments) can be (e.g., can consist of) one or more shared electron pairs between two atoms, groups, or chemical moieties (e.g., in a single bond, double bond, or triple bond). In some embodiments, a chemical (covalent) bond can further include one or more atoms or functional groups and can be referred to using the corresponding name of the functional group in the art. For example, a covalent bond containing an -S-S- group can be called a disulfide bond, a covalent bond containing a -(C=O)- group can be called a carbonyl bond, a covalent bond containing a -(CF2- group can be called a difluoromethylene bond, among others. The type of bond or functional group within a covalent bond is not limited, unless explicitly stated, e.g., described as including a particular group or selected from a particular group. The suitable type or kind of covalent bond will be understood from the description and / or context.

[0108] In some embodiments, the insulin receptor agonist may be bound or associated with human serum albumin (HSA) before binding to the insulin receptor. For example, an insulin receptor agonist comprising any one of DSL-1 to DSL-112 may associate or bind to HSA via any of a hydrogen bond, an ionic association, or a hydrolyzable boron ester bond. These interactions may occur along the surface of HSA, such as salt bridges to lysine residues, cleavable boron ester bonds formed by serine, threonine, and / or tyrosine hydroxyl groups, and / or at specific positions on HSA, such as site I or site II small molecule drug binding sites, or one or more of the seven fatty acid binding sites (Yamasaki, K. et. al. (2013)Biochimica et Biophysica Acta 12:5435-5443).

[0109] In some embodiments, the side chain of an amino acid can be covalently bound (e.g., linked or cross-linked) via any number of chemical bonds (e.g., linker moieties), as generally described in Bioconjugate Techniques (Third edition), edited by Greg T. Hermanson, Academic Press, Boston, 2013. For example, the side chain can be covalently bound via an amide, ester, ether, thioether, isourea, imine, triazole, or any suitable covalent bonding chemistry available in the art for covalently bonding one peptide, protein, or synthetic polymer to a second peptide, protein, or synthetic polymer. The term polymer includes polypeptides. The term "covalent bonding chemistry" can refer to one or more functional groups contained in the linker moiety and / or the chemical reaction used to form the linker moiety.

[0110] The term "vicinal diol" refers to a group of molecules in which two hydroxyl groups occupy adjacent positions, i.e., they are bonded to adjacent atoms. Such molecules can include, but are not limited to, sugars such as hexose, glucose, mannose, and fructose.

[0111] In some embodiments, the term "albumin" means a protein having at least 60% homology to human serum albumin, or a human serum albumin protein. It should be understood that in some embodiments, albumin can be further chemically modified for the purpose of conjugation. In some embodiments, such modification can include one or more covalently attached linkers. In some embodiments, the term "albumin" means a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology to human serum albumin, or a human serum albumin protein. In some embodiments, the term "albumin" means a protein having at least 90% homology to human serum albumin, or a human serum albumin protein. In some embodiments, the term "albumin" means a protein having at least 95% homology to human serum albumin, or a human serum albumin protein. In some embodiments, the term "albumin" means a protein having at least 99% homology to human serum albumin, or a human serum albumin protein. In some embodiments, albumin is unmodified human serum albumin.

[0112] The term "treatment" is meant to include both the prevention and minimization of the recited disease, disorder or condition (i.e., unless otherwise indicated or inconsistent with the apparent context, "treatment" refers to both prophylactic and therapeutic administration of a compound of the invention or a composition comprising a compound of the invention).

[0113] The route of administration can be any route that effectively transports the compounds of the present disclosure to a desired or appropriate location in the body, such as parenterally, for example, subcutaneously, intramuscularly, orally, or intravenously. In the case of parenteral administration, the compounds of the present disclosure are formulated in the same manner as known insulin formulations. Further, in the case of parenteral administration, the compounds of the present disclosure are administered in the same manner as known insulin administration, and physicians are familiar with this procedure. The determination of the amount of the compounds of the present disclosure to be administered, the frequency of administering the compounds of the present disclosure, and optionally the selection of the group of compounds of the present disclosure to be administered together with another antidiabetic compound are determined in consultation with a practicing physician who is familiar with the treatment of the condition to be treated (e.g., diabetes).

[0114] In some embodiments, as used herein, "therapeutic composition" and "pharmaceutical composition" mean compositions intended to have a therapeutic effect, such as pharmaceutical compositions, genetic materials, biologics, and other substances. A pharmaceutical composition can be configured to function in the body with therapeutic quality. The concentration may be changed, for example, to reduce the replenishment frequency. In some embodiments, "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a disease or an apparent symptom of a disease. A therapeutically effective amount is an amount that can treat, cure, alleviate, mitigate, modify, correct, improve, or affect a disease, disease symptom, or predisposition to a disease for the purpose of treating, curing, alleviating, mitigating, modifying, correcting, improving, or affecting the disease, disease state, disease symptom, or predisposition to the disease. A therapeutically effective set or specific amount can be readily determined by a person of ordinary skill in the art and can vary depending on factors known in the art, such as the type of disease, the patient's medical history and age, the stage of the disease, and the administration of other therapeutic agents. In some embodiments, the modified insulin described herein can be delivered to the body by injection or inhalation, or by other routes, and can reversibly bind to soluble glucose in a non-depot form. In some embodiments, the modified insulin described herein is released over a long period of time from a local depot in the body or from a bound form to a protein in serum, such as albumin. In some embodiments, the release of the modified insulin is accelerated when the glucose level increases, and such release rate can depend on the blood glucose level or the level of other small molecules in the blood containing diol-containing molecules. In some embodiments, the release, bioavailability, and / or solubility of the modified insulin described herein are controlled as a function of the blood or serum glucose concentration or the concentration of other small molecules in the body.

[0115] Unless otherwise specified, the structures described herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, substitution of hydrogen by deuterium ( 2 H) or tritium ( 3 H), or 13 C- or 14Compounds having the current structure, except for substitution of carbon by C-carbon atoms, are within the scope of the present disclosure. Such compounds can be useful, for example, as analytical tools, probes in biological assays, or therapeutic agents.

[0116] In some embodiments, the functional group can be covalently bonded or linked via any suitable covalent chemistry (linker) that can be used to covalently bond one functional group or amino acid side chain to another, non-limiting examples of which include amide, ester, ether, thioether, isourea, imine, and triazole linkers. In some embodiments, the functional group is covalently bonded by click chemistry reactions as defined in the art. These include, for example, cycloaddition reactions, 3+2 cycloadditions, strain-promoted alkyne-nitrone cycloadditions, strained alkenes, alkenes, and tetrazine inverse-demand Diels-Alder reactions, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), thiol-maleimide addition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation, nucleophilic ring-opening reactions, and addition to carbon-carbon multiple bonds, but are not limited thereto. Some of these reactions are described, for example, by H.C. Kolb, M.G. Finn and K.B. Sharpless (2001); Click Chemistry: Diverse Chemical Function from a Few Good Reactions, Angewandte Chemie International Edition 40(11):2004-2021; Kolb and Sharpless, Drug Discovery Today 8:1128-1137, 2003; Huisgen, R. Angew.Chem.Int.Ed.Engl. 1963, 2, 565; Agard, N.J.; Baskin, J.M.; Prescher, J.A.; Lo, A.; Bertozzi, C.R. ACS Chem.Biol. 2006, 1, 644. Those skilled in the art can select suitable buffers, pH, and reaction conditions for such click reactions. In some embodiments, the covalent bond is the result of a "bioorthogonal reaction" as defined in the art.Such reactions are described, for example, by Sletten, Ellen M.; Bertozzi, Carolyn R. (2009). Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality, Angewandte Chemie International Edition 48(38):6974-98.; Prescher, Jennifer A; Bertozzi, Carolyn R(2005). Chemistry in living systems, Nature Chemical Biology 1(1):13-21.

[0117] In some embodiments, functional groups can be joined using, for example, native chemical ligation as described by Dawson, P.E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. (1994) Synthesis of proteins by native chemical ligation, Science 266(5186):776-778. As used herein, terms such as "ligation," "covalent bond," etc. can, in some embodiments, refer to any of the chemistries described above. The terms "amine," "amino group," and / or "amine group," when used to describe a part of a covalent bond or linkage, can be used interchangeably to denote an amino or amine group to which the described element is covalently bonded. In some embodiments, the amino or amine group is a primary amine, secondary amine, or

Chemical formula

[0118] In some embodiments, further modifications include carbohydrate groups, one or more cis-diol-containing groups, one or more phosphate groups, one or more catechol groups, farnesyl groups, isofarnesyl groups, fatty acid groups, and other chemical entities (e.g., moieties or functional groups) such as, or linkers attached for conjugation, functionalization, or other modifications intended to affect the pharmacokinetics, pharmacodynamics, and / or biophysical solution properties of insulin.

[0119] In some embodiments, the compound includes a human peptide hormone (e.g., as X1). In some embodiments, the peptide hormone is a polypeptide hormone of the human pancreas. In some embodiments, the compound of formula I includes human insulin or a human insulin analog. In some embodiments, two different amine groups in insulin are covalently bonded as described by formula I.

[0120] It will be understood that human peptide hormones, human pancreatic polypeptide hormones, insulin, human insulin, modified insulin, and human insulin analogs may be used interchangeably in some of the described embodiments. That is, for example, in certain embodiments, a "human insulin analog" may be used in place of an embodiment described as using human insulin. In some embodiments, the compound of Formula I comprises human insulin or a human insulin analog. In some embodiments, the compound of Formula I comprises human insulin or a human insulin analog described by Formula I for p' = 1, with a single amino group in insulin bonded as described by Formula I. In some embodiments, the amino group is the amino group at the N-terminus of the B-chain of insulin or the amino group of the side chain of lysine. In some embodiments, two or more different amine groups of insulin are each independently covalently bonded as described by Formula I. In some embodiments, at least one amine group is at the N-terminus of the B-chain of insulin. In some embodiments, the amino group comprises the amino group of the side chain of a lysine residue of insulin.

[0121] Various suitable modifications of peptide hormones known to those of skill in the art (e.g., human polypeptide hormones, e.g., insulin) are included within the scope of the present disclosure. In some embodiments, the polypeptide of Z1a, or the polypeptide optionally extended at the N-terminus of the B-chain or the C-terminus of the A-chain of insulin, comprises a sequence having from about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a human polypeptide sequence. In some embodiments, the polypeptide of Z1a, or the polypeptide optionally extended at the N-terminus of the B-chain or the C-terminus of the A-chain of insulin, comprises one or more lysine residues optionally adjacent to a proline residue, where the proline is at the C-terminus of the lysine. In some embodiments, the amino groups of the lysine residues are each independently bonded as described by Formula I.

[0122] In some embodiments, insulin is further modified by attachment to a sugar-containing molecule or a diol-containing molecule. In some embodiments, a human polypeptide hormone is a dual or triple hybrid peptide comprising the sequences of two or more human peptide hormones and can act via multiple receptors; for example, glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonists or GLP-1 / GIP / glucagon triple agonists. In some embodiments, the human polypeptide hormone is an intestinal hormone. In some embodiments, the human polypeptide hormone is selected from c-peptide, adrenocorticotropic hormone (ACTH), amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormone, follicle-stimulating hormone (FSH), insulin, leptin, melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, vasoactive intestinal peptide, neuropeptides, peptide hormones that affect cardiovascular health or appetite, one or more hybrids of these peptides, and analogs of one of these peptides. In some embodiments, the compound comprises a human polypeptide hormone further modified by covalent attachment to, for example, a polymer, an XTEN protein sequence, or an aliphatic chain. In some embodiments, the polymer-modified compound has a longer circulation time in the blood. In some embodiments, the polymer-modified compound has a blood circulation time suitable for once-daily injection, once-weekly injection, or once-monthly injection. In some embodiments, the human polypeptide hormone or an analog thereof comprises one or more L- or D-amino acids, each independently being one of 20 standard or non-standard amino acids.

[0123] In some embodiments, the insulin receptor agonist is an insulin analog comprising an A-chain and a B-chain, the A-chain comprising a sequence selected from SEQ ID NO: 1, 25, 24051, and 24052, and the B-chain comprising a sequence selected from SEQ ID NO: 24060, 24061, 24062, 24063, 24064, and 25000 - 25397. In some embodiments, the insulin analog comprises an A-chain comprising SEQ ID NO: 24051 and a B-chain comprising a sequence selected from 25000, 25001, 25006 - 25009, 25076, 25077, 25082 - 25085, 25228, 25229, 25232, 25234 - 25237, 25304, 25305, 25308, and 25310 - 25313. In some embodiments, the insulin analog comprises an A-chain comprising SEQ ID NO: 24051, and the B-chain comprises a sequence selected from 25011, 25012, 25017 - 25020, 25087, 25088, 25093 - 25096, 25229, 25239, 25232, 25240, 25245 - 25248, 25305, 25308, 25315, 25316, and 25321 - 25324. In some embodiments, the insulin analog comprises an A-chain comprising SEQ ID NO: 24051 and a B-chain comprising a sequence selected from 25228, 25229, 25232, 25234 - 25237, 25304, 25305, 25308, and 25310 - 25313. In some embodiments, the insulin analog comprises an A-chain comprising SEQ ID NO: 24051 and a B-chain comprising a sequence selected from 25011, 25012, 25017 - 25020, 25087, 25088, 25093 - 25096, 25229, 25232, 25305, and 25308. In some embodiments, the insulin analog comprises human insulin having an A-chain and a B-chain, with up to 6 residues being mutated, deleted, or additionally inserted into each of the A-chain and / or the B-chain. In some of those forms, the insulin analog comprises insulin containing an A-chain and a B-chain having a connecting peptide that connects the C-terminus of the B-chain to the N-terminus of the A-chain, the connecting peptide comprising the native proinsulin C-peptide as well as shorter versions of C-peptides known in the art, the shorter versions of the C-peptide enabling the single-chain insulin to retain biological activity and / or efficacy.

[0124] The terms "analog" and "analogue" are alternative notations for the same word and are used interchangeably herein and have the same meaning. In the context of a hybrid peptide comprising sequences derived from two or more human polypeptide hormones, an analog means any related sequence resulting from the parent sequence and containing up to eight mutations, deletions or insertions of amino acids and / or additional chemical modifications. In some embodiments, as is known in the art, such analogs may enhance biological activity, pharmacokinetics, pharmacodynamics, efficacy, stability and / or chemical and physical properties.

[0125] In some embodiments, the human hormone analogs contain one or more residues that are 2-aminoisobutyric acid and / or other artificial (i.e., non-natural) amino acids.

[0126] In some embodiments, in an insulin analog, the C-terminus of the B-chain of insulin is covalently bound to the N-terminus of the A-chain. In some embodiments, the C-terminus of the B-chain of insulin is covalently bound to the N-terminus of the A-chain, and the linking peptide is C-peptide. In some embodiments, the C-terminus of the B-chain of insulin is covalently bound to the N-terminus of the A-chain, the linking peptide is C-peptide, and further comprises any intermediate compound comprising a conjugate of formula I.

[0127] In some embodiments, the insulin analog includes insulin lispro, or glargine-type modifications, or any suitable modifications to human insulin that affect the pharmacokinetics or half-life of insulin in the body. In some embodiments, the insulin lispro used to prepare the PEGylated insulin lispro compounds of the invention can be prepared by any of various recognized peptide synthesis techniques, including solution-phase methods, solid-phase methods, semi-synthetic methods, and recombinant DNA methods. For example, U.S. Patent No. 5,700,662 (Chance, et al.) and European Patent No. 214 826 (Brange, et al.) disclose the preparation of various insulin analogs. The A and B chains of insulin lispro can also be prepared via proinsulin-like precursor molecules using recombinant DNA technology. In some embodiments, a proinsulin-like precursor is used to prepare the insulin lispro used to make the PEGylated insulin lispro of the invention.

[0128] In some embodiments, the insulin portion of the compounds of the invention can be prepared via the production of precursor protein molecules using recombinant DNA technology. The DNA, including cDNA and synthetic DNA, can be double-stranded or single-stranded. The coding sequences encoding the precursor protein molecules described herein can vary as a result of the redundancy or degeneracy of the genetic code. To produce the precursor proteins of the invention, the DNA can be introduced into host cells. Suitable host cells are transiently or stably transfected or transformed in an expression system for producing the precursor protein. The host cells can be bacterial cells such as the K12 or B strains of E. coli, fungal cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (「CHO」) cells. The expression vector is typically replicable in the host organism, either as an episome or as an integrated part of the host chromosomal DNA. Generally, the expression vector includes selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the selection of those cells transformed with the desired DNA sequence.

[0129] In some embodiments, the polypeptide hormone is glucagon. In some embodiments, glucagon has additional mutations and modifications known to affect the solubility and solution stability of glucagon. In some embodiments, a compound, such as a compound of formula I, comprises a linkage of Z1a to the N-terminus of the B-chain of insulin via a peptide bond, and at least one additional linkage to insulin as described by formula I. In some embodiments, the additional linkage is to a lysine residue of insulin. In some embodiments, at least one such lysine is a residue between position 15 of the B-chain of insulin and the C-terminus. In some embodiments, the lysine residue is optionally adjacent to proline, glycine, arginine, threonine, or serine. In some embodiments, one or more amino acids in formula I are D-amino acids. In some embodiments, any secondary or primary amines in a compound such as a compound represented by formula I are each independently optionally acetylated. In some embodiments, the compound of formula I further has a polypeptide hormone X1 conjugated to a drug molecule, a contrast agent, a chelating agent, a contrast agent, a radioisotope, or a molecule involved in immune cells.

[0130] In some embodiments, X1 is a polypeptide hormone comprising a peptide ligand that binds to an extracellular protein receptor. In some embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone having at least 50% homology to a native human polypeptide hormone. In some embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a native human polypeptide hormone. In some embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone having at least 90% homology to a native human polypeptide hormone. In some embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone having at least 95% homology to a native human polypeptide hormone. In some embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone having at least 99% homology to a native human polypeptide hormone. In some embodiments, X1 is an analog of human insulin having up to 10 additional residues added to the A or B chain of insulin.

[0131] In some embodiments, the term "glucose-responsive" refers to a change in activity in the presence and absence of glucose, or the difference between low and high levels of glucose (e.g., 3 mM glucose vs. 20 mM glucose). In some embodiments, the activity of the conjugate insulin is evaluated by the concentration of insulin (in nanomolar units (nM) of insulin) required to induce a half-maximal response (EC50) in a cell-based assay. A conjugate insulin with a low EC50 concentration is more active than an insulin with a high EC50 concentration (e.g., an insulin with an EC50 of 3 nM is more active than an insulin with an EC50 of 50 nM). "Glucose response" is observed when insulin changes from a less active EC50 (higher nM) to a more active EC50 (lower nM) in the absence and presence of glucose, or at low and high levels of glucose, respectively.

[0132] In some embodiments, the compound of Formula I comprises one or more L- or D-artificial amino acids that are not one of the 20 natural amino acids. In some embodiments, the side chains of such artificial amino acids can be covalently linked by some reactions including bioorthogonal reactions, such as those described by Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596-2599 (2002), or by: Liang, Y., Mackey, J.L., Lopez, S.A., Liu, F. & Houk, K.N. Control and design of mutual orthogonality in bioorthogonal cycloadditions. J. Am. Chem. Soc. 134, 17904-17907 (2012). In some embodiments, Z1a contains one or more L- or D-artificial amino acids that are not one of the 20 natural amino acids. In some embodiments, the side chains of two amino acids of Z1a are covalently bonded together via a triazole linkage.

[0133] The insulin hormone is an important regulator of blood glucose (sugar) levels. In normal individuals, insulin is present and, when released from the pancreas, acts, for example, by binding to and activating insulin receptors and inducing glucose uptake by liver, fat, and skeletal muscle cells, thereby lowering blood glucose levels. Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic disorders characterized by persistently high blood glucose levels over a long period.

[0134] As used herein, "insulin" includes both wild-type and modified forms of insulin that can bind to and activate the insulin receptor or cause a measurable decrease in blood glucose when administered in vivo, including wild-type and modified forms of human insulin that can bind to and activate the human insulin receptor or cause a measurable decrease in blood glucose when administered to a human in vivo.

[0135] In some embodiments, insulin can be in any form of purified, synthetic, or recombinant form, including insulin from any species, including human insulin, porcine insulin, bovine insulin, ovine insulin, and rabbit insulin. In some embodiments, insulin has two chains, a B-chain and an A-chain. In some embodiments, the chains are joined together via a peptide, such as c-peptide, or a shortened version of c-peptide, known in the art. In other embodiments, insulin can be provided as proinsulin (insulin precursor) that can be further processed into mature insulin. Various modified forms of insulin are known in the art and can be chemically modified, such as by the addition of chemical moieties such as PEG groups or fatty acyl chains. Modified insulin can be mutated and can include amino acid additions, deletions, or substitutions. In some embodiments, the term "desB30" refers to insulin lacking the B30 amino acid residue.

[0136] As used herein, the term "insulin analog" means a modified human insulin having insulin receptor agonist activity, in which 1 to 10 amino acid residues of insulin are modified (e.g., substituted, deleted, added (i.e., extended), inserted, and any combination thereof) as compared to human insulin. In this regard, an insertion or addition of one or more amino acids is considered a single modification. For example, an insulin analog may have 1 to 9 modified amino acid residues. As yet another example, an insulin analog may have 1 to 8 modified amino acid residues. As yet another example, an insulin analog may have 1 to 7 modified amino acid residues. As yet another example, an insulin analog may have 1 to 6 modified amino acid residues. As yet another example, an insulin analog may have 1 to 5 modified amino acid residues. As yet another example, an insulin analog may have 1 to 4 modified amino acid residues. As yet another example, an insulin analog may have 1 to 3 modified amino acid residues. As yet another example, an insulin analog may have 1 to 2 modified amino acid residues. In some embodiments, an insulin analog may have 2, 3, 4, 5, 6, 7, 8, or 9 modified amino acid residues.

[0137] Modifications in the insulin molecule are indicated by describing the chain (A or B), position, and the one-letter or three-letter code of the amino acid residue substituting the native amino acid residue. As used herein, terms such as "A1", "A2", and "A3" etc. indicate the amino acids at positions 1, 2, 3, etc. of the A chain of insulin (counting from the N-terminus). Similarly, terms such as "B1", "B2", and "B3" etc. indicate the amino acids at positions 1, 2, 3, etc. of the B chain of insulin (counting from the N-terminus). Terms such as A21A, A21G, and A21Q using the one-letter code of amino acids indicate that the amino acid at position A21 is A, G, and Q, respectively. Using the three-letter code of amino acids, the corresponding expressions are A21Ala, A21 Gly, and A21 Gln, respectively.

[0138] Thus, for example, an insulin analog having four modifications comprises an A chain and a B chain, the A chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25737), the B chain comprises the sequence GKGSHKFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25738), the A chain has the wild-type sequence of the A chain of human insulin (i.e., without mutations, deletions, or additions), the B chain is extended at the N-terminus with GKGSHK (SEQ ID NO: 25726) (i.e., six amino acids are added / appended at the N-terminus), the amino acid at position 21 of the B chain (i.e., E) is replaced with K, the amino acid at position 29 of the B chain (i.e., K) is replaced with R, and the amino acid at position 30 of the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs: 25737 and 25738, in the order of appearance)).

Number

[0139] As yet another example, for example, an insulin analog having seven modifications comprises an A chain and a B chain, the A chain comprises the sequence GIVEQCCTSICSLYQLENYCGK (SEQ ID NO: 25739), the B chain comprises the sequence KGSHKFVDQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25740), in the A chain, the amino acid at position 21 (i.e., N) is replaced with G, the A chain is extended at the C-terminus with K (i.e., one amino acid is added / appended at the C-terminus), the B chain is extended at the N-terminus with KGSHK (SEQ ID NO: 25727) (i.e., five amino acids are added / appended at the N-terminus), in the B chain, the amino acid at position 3 (i.e., N) is replaced with D, in the B chain, the amino acid at position 21 (i.e., E) is replaced with K, in the B chain, the amino acid at position 29 (i.e., K) is replaced with R, and in the B chain, the amino acid at position 30 (i.e., T) is deleted (as shown below (SEQ ID NOs: 25739 and 25740, in the order of appearance)).

Number

[0140] As yet another example, for instance, an insulin analog having two modifications includes an A chain and a B chain, the A chain includes the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25731), the B chain includes the sequence KFVNQHLCGSHLVEALYLVCGKRGFFYTPKT (SEQ ID NO: 25741), the A chain has the wild-type sequence of chain A of human insulin (e.g., without mutations, deletions, or additions), the B chain is extended at the N-terminus with K (i.e., one amino acid is added / appended at the N-terminus), and the 21st amino acid of the B chain (i.e., E) is replaced with K (as shown below (SEQ ID NO: 25731 and 25741, in the order of appearance respectively)).

Number

[0141] As yet another example, an insulin analog having six modifications includes an A chain and a B chain, the A chain includes the sequence GIVEQCCTSICSLYQLENYCGK (SEQ ID NO: 25732), the B chain includes the sequence KGSHKFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25742), in the A chain, the 21st amino acid (i.e., N) is replaced with G, the A chain is extended at the C-terminus with K (i.e., one amino acid is added / appended at the C-terminus), the B chain is extended at the N-terminus with KGSHK (SEQ ID NO: 25727) (i.e., five amino acids are added / appended at the N-terminus), in the B chain, the 21st amino acid (i.e., E) is replaced with K, in the B chain, the 29th amino acid (i.e., K) is replaced with R, and in the B chain, the 30th amino acid (i.e., T) is deleted (as shown below (SEQ ID NO: 25732 and 25742, in the order of appearance respectively)).

Number

[0142] As yet another example, for instance, an insulin analog having four modifications includes an A chain and a B chain. The A chain includes the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25733), and the B chain includes the sequence KGSHFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25743). The A chain has the wild-type sequence of the A chain of human insulin (i.e., without mutations, deletions, or additions), and the B chain is extended at the N-terminus with KGSH (SEQ ID NO: 25728) (i.e., four amino acids are added / appended at the N-terminus). The 21st amino acid of the B chain (i.e., E) is replaced with K, the 29th amino acid of the B chain (i.e., K) is replaced with R, and the 30th amino acid of the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NO: 25733 and 25743, in the order of appearance)).

Number

[0143] As yet another example, for instance, an insulin analog having five modifications includes an A chain and a B chain. The A chain includes the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25734), and the B chain includes the sequence KGSHQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25744). The A chain has the wild-type sequence of the A chain of human insulin (i.e., without mutations, deletions, or additions), and the B chain is extended at the N-terminus with KGSH (SEQ ID NO: 25728) (i.e., four amino acids are added / appended at the N-terminus). The first three residues (FVN) are deleted. The 21st amino acid of the B chain (i.e., E) is replaced with K, the 29th amino acid of the B chain (i.e., K) is replaced with R, and the 30th amino acid of the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NO: 25734 and 25744, in the order of appearance)).

Number

[0144] As yet another example, for instance, an insulin analog having five modifications includes an A chain and a B chain. The A chain includes the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25735), and the B chain includes the sequence KGSHKQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25745). The A chain has the wild-type sequence of the A chain of human insulin (i.e., without mutations, deletions, or additions), and the B chain is extended at the N-terminus by KGSHK (SEQ ID NO: 25727) (i.e., five amino acids are added / appended at the N-terminus), the first three residues (FVN) are deleted, the 21st amino acid of the B chain (i.e., E) is replaced by K, the 29th amino acid of the B chain (i.e., K) is replaced by R, and the 30th amino acid of the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs: 25735 and 25745, in the order of appearance)).

Number

[0145] As yet another example, for instance, an insulin analog having two modifications includes an A chain and a B chain. The A chain includes the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25736), and the B chain includes the sequence GKGGGGSGGGGSGGGGSFVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO: 25746). The A chain has the wild-type sequence of chain A of human insulin (i.e., without mutations, deletions, or additions), and the B chain is extended at the N-terminus by GKGGGGSGGGGSGGGGS (SEQ ID NO: 25729) (i.e., 17 amino acids are added / appended at the N-terminus), and the 30th amino acid of the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs: 25736 and 25746, in the order of appearance)).

Number

[0146] In some embodiments, insulin analogs include, but are not limited to, insulin that has been chemically modified compared to wild-type human insulin, such as by the addition of chemical moieties such as PEG groups or fatty acyl chains. In some embodiments, the modified insulin / insulin analog / analog used interchangeably herein may have mutations including amino acid additions, deletions, or substitutions. Different protomers of insulin result from these changes and may be incorporated into some embodiments. In some embodiments, the active form of insulin has less than 11 such modifications (e.g., 1-4, 1-3, 1-9, 1-8, 1-7, 1-6, 2-6, 2-5, 2-4, 1-5, 1-2, 2-9, 2-8, 2-7, 2-3, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, 8-9, 9, 8, 7, 6, 5, 4, 3, 2, or 1). As used herein, the wild-type sequence (A-chain and B-chain) of human insulin has an A-chain having the amino acid sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and a B-chain having the amino acid sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2). In some embodiments, the insulin analog has at least 70% sequence homology to wild-type human insulin. In some embodiments, the insulin analog has at least 80% sequence homology to wild-type human insulin. In some embodiments, the insulin analog has at least 90% sequence homology to wild-type human insulin. In some embodiments, the insulin analog has at least 95%, 96%, 97%, or 98% sequence homology to wild-type human insulin. In some embodiments, the insulin analog has at least 99% sequence homology to wild-type human insulin.

[0147] Human insulin differs from rabbit, porcine, bovine, and ovine insulin at amino acids A8, A9, A10, and B30, which are as follows in order: human is Thr, Ser, Ile, Thr; rabbit is Thr, Ser, Ile, Ser; porcine is Thr, Ser, Ile, Ala; ovine is Ala, Gly, Val, Ala; bovine is Ala, Ser, Val, Ala. In some embodiments, the modified insulin can be mutated at position B1, B2, B28, or B29 of the B chain, or at positions B28 and B29. In some embodiments, the modified insulin can be mutated at A1, A2, A21 or other positions of the A chain. For example, insulin lispro is a rapid-acting modified insulin in which the lysine and proline residues on the C-terminus of the B chain are reversed. Insulin aspart is a rapid-acting modified insulin in which proline is substituted with aspartic acid at position B28. It is contemplated that in some embodiments of the present disclosure, insulin mutated at B28 and B29 may further include additional mutations. For example, insulin glulisine is a rapid-acting modified insulin in which aspartic acid is substituted with a lysine residue at position B3 and lysine is substituted with a glutamic acid residue at position B29. In some embodiments, long-acting and highly stable insulin analogs are covalently modified as described by formula I and may contain mutations such as tyrosine at A14 being substituted with glutamic acid, tyrosine at B16 being substituted with histidine, and phenylalanine at B25 being substituted with histidine.

[0148] In some embodiments, the isoelectric point of the insulin herein can be shifted relative to wild-type human insulin using any suitable method, e.g., by addition or substitution of suitable amino acids. In some embodiments, the isoelectric point of the modified insulin can be regulated by glucose (e.g., by interaction with glucose). For example, insulin glargine is a basal insulin in which two arginine residues are added to the C-terminus of the B peptide and A21 is substituted with glycine. In some embodiments, insulin may not have one or more of residues B1, B2, B3, B26, B27, B28, B29, and B30 (e.g., insulin can be a deletion mutant at one or more of the listed residues). In some embodiments, the insulin molecule contains up to 5 additional amino acid residues on the N-terminus or C-terminus of the A-chain or B-chain. In some embodiments, one or more amino acid residues are located at or missing at positions A1, A21, B1, B29, B30 and / or B31. In some embodiments, the insulin molecules of the present disclosure are mutated such that one or more amino acids are substituted in the acidic form. In some embodiments, asparagine is substituted with aspartic acid or glutamic acid. In some embodiments, glutamine is substituted with aspartic acid or glutamic acid. In some embodiments, A21 can be aspartic acid, B3 can be aspartic acid, or both positions can contain aspartic acid. Those skilled in the art will recognize that it is possible to make any previously reported or widely accepted mutations or modifications to insulin while retaining biological activity, and that such insulin analogs can be used in the embodiments of the present disclosure. In some embodiments, insulin can be conjugated to a fatty acid at any position, or acylated with a fatty acid at any amino group including those on the lysine side chain and the alpha-amino group on the N-terminus of insulin, and the fatty acid can include C8, C9, C10, C11, C12, C14, C15, C16, C17, or C18 chains. In some embodiments, the fatty acid chain is 8 to 20 carbons in length.In some embodiments, insulin detemir has myristic acid covalently bound to lysine at B29 and B30 is deleted or absent. In some embodiments, position B28 of the insulin molecule is lysine and the epsilon (ε)-amino group of this lysine is conjugated to a fatty acid.

[0149] In some embodiments, the N-terminus or C-terminus of the A-chain or B-chain of the modified insulin is ligated using a peptide ligase. In some embodiments, the polypeptide is added to the C-terminus of the insulin A and / or B-chain or the N-terminus of the insulin A and / or B-chain using a protein ligase, and in some of these embodiments, the ligase is selected from sortase, butelase, trypus ligase, subtilisin, peptidyl ligase, or an enzyme having at least 75% homology with these ligases. In some embodiments, the ligation is achieved by expressed protein ligation as described in Muir TW, Sondhi D, Cole PA. "Expressed protein ligation: a general method for protein engineering." Proc Natl Acad Sci U S A. 1998; 95(12):6705-6710. In some embodiments, the polypeptide is ligated to the modified insulin using Staudinger ligation, for example, as described in Nilsson, B.L.; Kiessling, L.L. Raines, R.T. (2000). "Staudinger ligation: A peptide from a thioester and azide". Org. Lett. 2 (13): 1939-1941., which utilizes the Staudinger reaction. In some embodiments, the polypeptide is conjugated to the modified insulin using Ser / Thr ligation, for example, as described in Zhang Y, Xu C, Kam HY, Lee CL, Li X. 2013, "Protein chemical synthesis by serine / threonine ligation." Proc. Natl. Acad. Sci. USA. 17:6657-6662. In some embodiments, the B-chain itself has less than 32 amino acids or 34 amino acids, and in some embodiments, the insulin has four disulfide bonds instead of three. Disulfide bonds are present in the A-chain and B-chain of insulin.For example, a disulfide bond exists between the cysteine at position 6 of SEQ ID NO: 1 and the cysteine at position 11 of SEQ ID NO: 1, a disulfide bond exists between the cysteine at position 7 of SEQ ID NO: 1 and the cysteine at position 7 of SEQ ID NO: 2, and a disulfide bond exists between the cysteine at position 20 of SEQ ID NO: 1 and the cysteine at position 19 of SEQ ID NO: 2.

[0150] In some embodiments, the modified insulin of the present disclosure includes, but is not limited to, the following insulin molecules: N εB29 -Octanoyl-Arg B0 Gly A21 Asp B3 Arg B31 Arg B32 -HI, N εB29 -Octanoyl-Arg B31 Arg B32 -HI, N εB29 -Octanoyl-Arg A0 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Gly A21 Gln B3 Lys B28 Pro B30 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Arg A0 Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Arg A0 Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Arg A0Gly A21 Asp B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Myristoyl-Arg A0 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Arg A0 Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB29 -Palmitoyl-HI, N εB29 -Myristoyl-HI, N εB28 -Palmitoyl-Lys B28 Pro B29 -HI, N εB28 -Myristoyl-Lys B28 Pro B29 -HI, N εB29 -Palmitoyl-des(B30)-HI, N εB30 -Myristoyl-Thr B29 Lys B30 -HI, N εB30 -Palmitoyl-Thr B29 Lys B30 -HI, N εB29-(N-Palmitoyl-γ-glutamyl)-des(B30)-HI,N εB29 -(N-Lithocholyl-γ-glutamyl)-des(B30)-HI,N εB29 -(ω-Carboxyheptadecanoyl)-des(B30)-HI,N εB29 -(ω-Carboxyheptadecanoyl)-HI,N εB29 -Octanoyl-HI,N εB29 -Myristoyl-Gly A21 Arg B31 Arg B31 -HI,N εB29 -Myristoyl-Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Myristoyl-Arg A0 Gly A21 Arg B31 Arg B32 -HI,N εB29 -Arg A0 Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Myristoyl-Arg A0 Gly A21 Asp B3 Arg B31 Arg B32 -HI,N εB29 -Myristoyl-Arg B31 Arg B32 -HI,N εB29 -Myristoyl-Arg A0 Arg B31 Arg B32 -HI,N εB29 -Octanoyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Octanoyl-Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Octanoyl-Arg A0 Gly A21 ArgB31 Arg B32 -HI, N εB29 -Octanoyl-Arg A0 Gly A21 Gln B3 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Arg A0 Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Arg A0 Gly A21 Asp B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -Octanoyl-Arg A0 Lys B28 Pro B29 Arg B31 Arg B32 -HI. N εB29 -Pentanoyl-Gly A21 Arg B31 Arg B32 -HI, N αB1 -Hexanoyl-Gly A21 Arg B31 Arg B32 -HI, N αA1 -Heptanoyl-Gly A21 Arg B31 Arg B32 -HI, N εB29 -Octanoyl-N αB1 -Octanoyl-Gly A21 Arg B31 Arg B32 -HI, N εB29 -Propionyl-N αA1 -Propionyl-Gly A21 Arg B31Arg B32 -HI,N αA1 -Acetyl-N αB1 -Acetyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Formyl-N αA1 -Formyl-N αB1 -Formyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Formyl-des(B26)-HI,N αB1 -Acetyl-Asp B28 -HI,N εB29 -Propionyl-N αA1 -Propionyl-N αB1 -Propionyl-Asp B1 Asp B3 Asp B21 -HI,N εB29 -Pentanoyl-Gly A21 -HI,N αB1 -Hexanoyl-Gly A21 -HI,N αA1 -He-ptanoyl-Gly A21 -HI,N εB29 -Octanoyl-N αB1 -Octanoyl-Gly A21 -HI,N εB29 -Propionyl-N αA1 -Propionyl-Gly A21 -HI,N αA1 -Acetyl-N αB1 -Acetyl-Gly A21 -HI,N εB29 -Formyl-N αA1 -Formyl-N αB1 -Formyl-Gly A21 -HI,N εB29 -Butyryl-des(B30)-HI,N αB31 -Butyryl-des(B30)-HI,N αA1 -Butyryl-des(B30)-HI,N εB29 -Butyryl-N αB31 -Butyryl-des(B30)-HI,N εB29 -Butyryl-N αA1-Butyryl-des(B30)-HI,N αA1 -Butyryl-N αB31 -Butyryl-des(B30)-HI,N εB29 -Butyryl-N αA1 -Butyryl-N αB31 -Butyryl-des(B30)-HI,Lys B28 Pro B29 -HI(Insulin Lispro), Asp B28 -HI(Insulin Asparto), Lys B3 Glu B29 -HI(Insulin Glulisine), Arg B31 Arg B32 -HI(Insulin Glulargin), N εB29 -Myristoyl-des(B30)-HI(Insulin Detemir), Ala B26 -HI, Asp B1 -HI, Arg A0 -HI, Asp B1 Glu B13 -HI, Gly A21 -HI, Gly A21 Arg B31 Arg B32 -HI, Arg A0 Arg B31 Arg B32 -HI, Arg A0 Gly A21 Arg B31 Arg B32 -HI, des(B30)-HI, des(B27)-HI, des(B28~B30)-HI, des(B1)-HI, des(B1~B3)-HIN εB29 -Tridecanoyl-des(B30)-HI,N εB29 -Tetradecanoyl-des(B30)-HI,N εB29 -Decanoyl-des(B30)-HI,N εB29 -Dodecanoyl-des(B30)-HI,N εB29 -Tridecanoyl-Gly A21 -des(B30)-HI,N εB29 -Tetradecanoyl-Gly A21 -des(B30)-HI,N εB29 -Decanoyl-Gly A21-des(B30)-HI,N εB29 -Dodecanoyl-Gly A21 -des(B30)-HI,N εB29 -Tridecanoyl-Gly A21 Gln B3 -des(B30 )-HI,N εB29 -Tetradecanoyl-Gly A21 Gln B3 -des(B30)-HI,N εB29 -Decanoyl-Gly A21 -Gln B3 -des(B30)-HI,N εB29 -Dodecanoyl-Gly A21 -Gln B3 -des(B30)-HI,N εB29 -Tridecanoyl-Ala A21 -des(B30)-HI,N εB29 -Tetradecanoyl-Ala A21 -des(B30)-HI,N εB29 -Decanoyl-Ala A21 -des(B30)-HI,N εB29 -Dodecanoyl-Ala A21 -des(B30)-HI,N εB29 -Tridecanoyl-Ala A21 -Gln B3 -des(B30)-HI,N εB29 -Tetradecanoyl-Ala A21 Gln B3 -des(B30)-HI,N εB29 -Decanoyl-Ala A21 Gln B3 -des(B30)-HI,N εB29 -Dodecanoyl-Ala A21 Gln B3 -des(B30)-HI,N εB29 -Tridecanoyl-Gln B3 -des(B30)-HI,N εB29 -Tetradecanoyl-Gln B3 -des(B30)-HI,N εB29 -Decanoyl-Gln B3 -des(B30)-HI,N εB29-Dodecanoyl-Gln B3 -des(B30)-HI,N εB29 -Z1-Gly A21 -HI,N εB29 -Z2-Gly A21 -HI,N εB29 -Z4-Gly A21 -HI,N εB29 -Z3-Gly A21 -HI,N εB29 -Z1-Ala A21 -HI,N εB29 -Z2-Ala A21 -HI,N εB29 -Z4-Ala A21 -HI,N εB29 -Z3-Ala A21 -HI,N εB29 -Z1-Gly A21 Gln B3 -HI,N εB29 -Z2-Gly A21 Gln B3 -HI,N εB29 -Z4-Gly A21 Gln B3 -HI,N εB29 -Z3-Gly A21 Gln B3 -HI,N εB29 -Z1-Ala A21 Gln B3 -HI,N εB29 -Z2-Ala A21 Gln B3 -HI,N εB29 -Z4-Ala A21 Gln B3 -HI,N εB29 -Z3-Ala A21 Gln B3 -HI,N εB29 -Z1-Gln B3 -HI,N εB29 -Z2-Gln B3 -HI,N εB29 -Z4-Gln B3 -HI,N εB29 -Z3-Gln B3 -HI,N εB29 -Z1-Glu B30 -HI,N εB29 -Z2-Glu B30-HI,N εB29 -Z4-Glu B30 -HI,N εB29 -Z3-Glu B30 -HI,N εB29 -Z1-Gly A21 Glu B30 -HI,N εB29 -Z2-Gly A21 Glu B30 -HI,N εB29 -Z4-Gly A21 Glu B30 -HI,N εB29 -Z3-Gly A21 Glu B30 -HI,N εB29 -Z1-Gly A21 Gln B3 Glu B30 -HI,N εB29 -Z2-Gly A21 Gln B3 Glu B30 -HI,N εB29 -Z4-Gly A21 Gln B3 Glu B30 -HI,N εB29 -Z3-Gly A21 Gln B3 Glu B30 -HI,N εB29 -Z1-Ala A21 Glu B30 -HI,N εB29 -Z2-Ala A21 Glu B30 -HI,N εB29 -Z4-Ala A21 Gln B30 -HI,N εB29 -Z3-Ala A21 Glu B30 -HI,N εB29 -Z1-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z2-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z4-Ala A21 Gln B3 Glu B30-HI,N εB29 -Z3-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z1-Gln B3 Glu B30 -HI,N εB29 -Z2-Gln B3 Glu B30 -HI,N εB29 -Z4-Gln B3 Glu B30 -HI,N εB29 -Z3-Gln B3 Glu B30 Comprising one or more mutations and / or chemical modifications, including one of the following -HI, wherein Z1 is tridecanoyl, Z2 is tetradecanoyl, Z3 is dodecanoyl, Z4 is decanoyl, and H1 is human insulin.

[0151] In some embodiments, insulin has the following mutations and / or chemical modifications: N εB28 -XXXXX-Lys B28 Pro B29 -HI, N αB1 -XXXXX-Lys B28 Pro B29 -HI, N αA1 -XXXXX-Lys B28 Pro B29 -HI, N εB28 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI, N εB28 -XXXXX-N αA1 -XXXXX-Lys B28 Pro B29 -HI, N αA1 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI, N εB28 -XXXXX-N αA1 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI, N εB29 -XXXXX-HI, NαB1 -XXXXX-HI, N αA1 -XXXXX-HI, N εB29 -XXXXX-N αB1 -XXXXX-HI, N εB29 -XXXXX-N αA1 -XXXXX-HI, N αA1 -XXXXX-N αB1 -XXXXX-HI, N εB29 -XXXXX-N αA1 -XXXXX-N αB1 -XXXXX-HI, N εB29 -YYYYY-HI, N αB1 -YYYYY-HI, N αA1 -YYYYY-HI, N εB29 -YYYYY-N αB1 -YYYYY-HI, N εB29 -YYYYY-N αA1 -YYYYY-HI, N αA1 -YYYYY-N αB1 -YYYYY-HI, N εB29 -YYYYY-N αA1 -YYYYY-N αB1 -YYYYY-HI, N εB28 -YYYYY-Lys B28 Pro B29 -HI, N εB21 -YYYYY-Lys B28 Pro B29 -HI, N αA1 -YYYYY-Lys B28 Pro B29 -HI, N εB28 -YYYYY-N αB1 -YYYYY-Lys B28 Pro B29 -HI, N εB28 -YYYYY-N αA1 -YYYYY-Lys B28 Pro B29 -HI, N αA1 -YYYYY-N αB1 -YYYYY-Lys B28 Pro B29 -HI, N εB28 -YYYYY-N αA1 -YYYYY-N αB1 -YYYYY-Lys B28Pro B29 including one or more of -HI, where YYYYY is one of acetyl or formyl, XXXXX is one of propionyl, butyryl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, or decanoyl, and HI is human insulin.

[0152] In some embodiments, insulin can be conjugated via a reactive moiety that naturally exists within the insulin structure and / or is added prior to conjugation, and that includes moieties such as carboxyl or reactive esters, amines, hydroxyls, aldehydes, sulfhydryls, maleimidyls, alkynyls, azides, etc. Insulin naturally contains reactive alpha-terminal amines and epsilon-amino lysine groups to which NHS-esters, isocyanates, or isothiocyanates can be covalently attached. In some embodiments, modified insulin is used in which suitable amino acids (e.g., lysine or non-natural amino acids) are added or substituted in the amino acid sequence to provide alternative attachment points in addition to the modified amino acids of the embodiments described herein. In some embodiments, the conjugation process can be controlled by selectively blocking specific reactive moieties prior to conjugation. In some embodiments, insulin can include any combination of modifications, and the present disclosure also encompasses modified forms of non-human insulin (e.g., porcine insulin, bovine insulin, rabbit insulin, ovine insulin, etc.) that include any one of the foregoing modifications. Some embodiments include these and other previously described modified insulins, such as those described in U.S. Patent Nos. 5,474,978; 5,461,031; 4,421,685; 7,387,996; 6,869,930; 6,174,856; 6,011,007; 5,866,538; 5,750,4976; 906,028; 6,551,992; 6,465,426; 6,444,641; 6,335,316; 6,268,335; 6,051,551; 6,034,054; 5,952,297; 5,922,675; 5,747,642; 5,693,609; 5,650,486; 5,547,929; 5,504,188; U.S. Patent Application Publication No. 2015 / 0353619, including non-natural amino acids described or referenced herein and such modifications to non-human insulin described herein.In some embodiments, it should also be understood that insulin can be covalently attached to a polyethylene glycol polymer, such as a polyethylene glycol polymer with a molecular weight of 60,000 or less of Mn, or can be covalently attached to albumin via either a permanent or reversible bond.

[0153] In some embodiments, a compound of the present disclosure (e.g., Formula I) is conjugated to a chelating agent, and in some embodiments, a chelating agent can be used to capture a radioactive payload such as gallium 68, copper 64, lutetium 177, or actinium 225. In some embodiments, the chelating agent is based on DOTA, NOTA, TETA, or tetra-arm DOTA, and in some embodiments, the chelating agent can be linked to a peptide using a PEG linker via an amide bond to the chelating agent and the peptide.

[0154] In some embodiments, the activity, bioavailability, solubility, isoelectric point, charge, and / or hydrophobicity of the modified insulin can be controlled as a result of chemical modification and / or interaction with small molecules such as sugars of a compound that is either covalently attached to or mixed with insulin, e.g., a compound described herein.

[0155] In some embodiments, one or more elements, functional groups, or atoms may be specifically omitted or excluded from the depicted structure (e.g., a terminal functional group may be replaced by a hydrogen atom, or a linking group may be replaced by a bond), e.g., in Formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, it will be understood that such omitted or excluded elements distinguish these groups (structures) from one another and are not equivalent. For example, if another version (variation) of a formula structure does not have a nitro group at R1 of B1 or B2, that variation is not equivalent to the structure containing the nitro group (e.g., is not structurally and chemically equivalent), at least because the nitro group changes the pKa of B1 and B2 under physiological conditions and thus changes the overall affinity of Z1c for glucose.

[0156] Rotationally constrained tethered boron conjugate. In some embodiments, the aromatic boron-containing compound and / or aromatic boron-containing group is a rotationally constrained tether boron conjugate. In some embodiments, the rotationally constrained tether boron conjugates presented in the present disclosure are those in which rotation is inhibited by unfavorable steric interactions (e.g., gauche - anti interactions of substituents), restricted rotation by bond hybridization (e.g., cis - to - trans - amide rotation), or via a rigid covalent bond (e.g., (E) vs. (Z) configuration of an alkene moiety), and contain a backbone. For example, Formula FF116 includes a geminal (e.g., bonded to the same atom) alkyl functional group relative to the amine group covalently bonded to the boronic acid functional moiety. The alkyl functional group can limit the accessible dihedral angles and the freedom of rotation around the C - C or C - X bond (commonly referred to as the χ (chi) dihedral angle of an amino acid). For example, the hydroxyl side chain of a serine residue can access dihedral angles of 60°, 180°, or 240° (-60°) with approximately equal distribution, while the hydroxyl side chain of threonine can only adopt dihedral angles of 180° or 240° (-60°). The presence of a geminal methyl group to the hydroxyl of threonine provides steric bulk and can create unfavorable interactions when other bulky substituents are in the gauche conformation relative to the methyl. Formula FF116 includes a geminal alkyl substituent that can limit the accessible dihedral angles adopted by the boron-conjugated amine, affect the dihedral angles adopted, place the boron functional group closer, and enable an increase in the binding of the conjugate to a target molecule such as a protein or a sugar.

[0157] In some embodiments, the activity (glucose responsiveness) of the insulin agonist is controlled by the three-dimensional structure of the sensor. For example, FF116A, FF115A, FF225A are structural isomers (contain the same molecular formula but have different bonds between atoms). In some embodiments, in Example 23, Example 82, and Example 27, when used with the same linker (FL3) and the same insulin backbone (i.e., the same A chain (i.e., sequence: 1) and the same B chain (i.e., sequence: 24060)), these compounds show different responsiveness to differences in glucose, and Example 23 shows the greatest fold change (responsiveness) to glucose compared to Example 82 and Example 27. In some embodiments, the presence of methyl groups on the FF backbone (e.g., FF116A, FF225A) results in higher potency (e.g., lower EC 50) has. Example 23 has a fold change of about 7 - 8 in response to a glucose change from 3 mM to 20 mM glucose, while for the same range of glucose change, Example 27 has a fold change of about 6 and Example 82 has a fold change of about 4. Thus, the presence of methyl groups in the skeletons FF116A (having beta-methyl) and FF225A (having alpha-methyl) results in an improvement in glucose responsiveness over FF115A (without methyl on the FF skeleton), and furthermore, the glucose responsiveness brought about by the skeleton FF116A is better than that of FF225A. As another comparison, as shown in Example 156, a compound having an insulin skeleton with the skeletons FF116A and the linker FL3 (i.e., A-chain sequence: 24051 and B-chain sequence: 24061) gives a fold change of 4.0 in response to a glucose change from 3 mM to 20 mM glucose and has diboronato skeleton 3 - ((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide)propanoic acid. On the other hand, in the same Example 156, when the diboronato sensor is changed to S-3-(2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanoamide)propanoic acid, the observed fold change is 3.6 in response to a glucose change from 3 mM to 20 mM glucose. This indicates that the diboronato sensor 3 - ((2S,3S-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide)propanoic acid (having methyl on the FF skeleton) provides better glucose responsiveness to insulin than insulin containing the sensor (S)-3-(2,3-bis(1-hydroxy-1,3-dihydrobenzo[b]thiophen-2-yl)methyl)-[c][1,2]oxaborole-6-carboxamido)propanoic acid (without methyl on the FF scaffold).

[0158] In some embodiments, the stereochemistry of the isomeric structure (e.g., the stereochemistry of a compound, (e.g., within the Z1c moiety)) can selectively increase the affinity of a conjugate (e.g., the Z1c moiety) for a specific target diol such as glucose. For example, in some embodiments, one or more stereoisomers of Z1c (e.g., cis or trans, (R) or (S), and (E) or (Z)) can be selected to increase or decrease the affinity of Z1c for glucose (and the overall molecular structure or conjugate). In some embodiments, where applicable, the cis forms of Formulas FF12, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF117, FF193A, FF203, FF225, and FF226 are used (e.g., Z1c comprises a structure having cis stereochemistry). In some embodiments, the trans forms of Formulas FF12, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used where applicable, e.g., when those formulas contain two stereocenters linked by a bond (e.g., Z1c comprises a structure having trans stereochemistry). In some embodiments, the R forms of Formulas FF12, FF114, FF114A, FF115, FF115A, FF116, FF117, FF193A, FF203, FF225, and FF226 are used where applicable, e.g., when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain at least one stereocenter (e.g., Z1c comprises a structure having R stereochemistry). In some embodiments, the S forms of Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used where applicable, e.g., when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain at least one stereocenter (e.g., Z1c comprises a structure having S stereochemistry).In some embodiments, the S,S forms of Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226 are used, where applicable, for example, when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain two stereocenters linked by a bond (e.g., Z1c includes a structure having S,S stereochemistry). In some embodiments, the S,R forms of Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used, where applicable, for example, when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain two stereocenters linked by a bond (e.g., Z1c includes a structure having S,R stereochemistry). In some embodiments, the R,R forms of Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used, where applicable, for example, when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain two stereocenters linked by a bond (e.g., Z1c includes a structure having R,R stereochemistry). In some embodiments, the R,S forms of Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used, where applicable, for example, when Formulas FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 contain two stereocenters linked by a bond (e.g., Z1c includes a structure having R,S stereochemistry). In some embodiments, the compounds include one or more tautomers of the compounds disclosed herein. In some embodiments, the compounds include one or more stereoisomers or a mixture of stereoisomers of the compounds disclosed herein.

[0159] In some embodiments, the compound is covalently bound to glucagon, GLP-1, GLP-2, or any variation thereof (e.g., any variation involving deletion, insertion, and / or substitution of one or more amino acids). In some embodiments, any suitable chemical modification performed on insulin as contemplated herein can be performed on glucagon. In some embodiments, the conjugate is mixed with one or more compounds selected from a second or prodrug or aminoethyl glucose, aminoethyl bimanose, aminoethyl trimannose, D-glucose, D-galactose, D-allose, D-mannose, D-gulose, D-idose, D-talose, N-azidomannosamine (ManNAz) or N-azidogalactosamine (GalNAz), or N-azidoglucosamine (GlcNAz), 2'-fluororibose, 2'-deoxyribose, glucose, sucrose, maltose, mannose, derivatives thereof (e.g., glucosamine, mannosamine, methyl glucose, methyl mannose, ethyl glucose, ethyl mannose, etc.), sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, and / or higher order combinations thereof (linear and / or branched bimanose, linear and / or branched trimannose, etc.), molecules containing cis diols, catechols, tris, and DOPA molecules, e.g., L-DOPA or L-3,4-dihydroxyphenylalanine.

[0160] Furthermore, one of ordinary skill in the art will recognize that in some embodiments, one or more suitable proteinogenic unnatural amino acids can be used (including) in Z1a. For example, in some embodiments, one or more of the following unnatural amino acids are described in Liu, C.C.; Schultz, P.G. (2010). "Adding new chemistries to the genetic code" Annual Review of Biochemistry 79:413-44, and can be used based on the methods described therein and the list of amino acids provided therein. One of ordinary skill in the art will recognize that in some embodiments, unnatural amino acids can be incorporated into Z1a by peptide synthesis and then covalently attached to a drug or insulin, and that the amino acids referenced herein and previously reported non-proteinogenic amino acids are included.In some embodiments, artificial amino acids are present in (e.g., may be included in) insulin, and in some of those embodiments, proteinogenic artificial amino acids can be introduced into recombinant protein expression using suitable methods and approaches including those described in U.S. Patent Application Publication No. 2008 / 0044854, U.S. Patent No. 8518666, U.S. Patent No. 8980581, U.S. Patent Application Publication No. 2008 / 0044854, U.S. Patent Application Publication No. 20140045261, U.S. Patent Application Publication No. 2004 / 0053390, U.S. Patent No. 7229634, U.S. Patent No. 8236344, U.S. Patent Application Publication No. 2005 / 0196427, U.S. Patent Application Publication No. 2010 / 0247433, U.S. Patent No. 7198915, U.S. Patent No. 7723070, U.S. Patent Application Publication No. 2002 / 0042097, U.S. Patent Application Publication No. 2004 / 0058415, U.S. Patent Application Publication No. 2008 / 0026422, U.S. Patent Application Publication No. 2008 / 0160609, U.S. Patent Application Publication No. 2010 / 0184193, U.S. Patent Application Publication No. 2012 / 0077228, U.S. Patent Application Publication No. 2014 / 025599, U.S. Patent No. 7198915, U.S. Patent No. 7632492, U.S. Patent No. 7723070, and other proteinogenic artificial amino acids can be recombinantly introduced using the methods and approaches described in U.S. Patent No. 7736872, U.S. Patent No. 7816320, U.S. Patent No. 7829310, U.S. Patent No. 7829659, U.S. Patent No. 7883866, U.S. Patent No. 8097702, U.S. Patent No. 8946148.

[0161] In some embodiments, cyclic amino acids such as 3-hydroxyproline, 4-hydroxyproline, aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, 3-carboxy-morpholine, 3-carboxy-thiamorpholine, 4-oxaproline, pyroglutamic acid, l,3-oxazolidine-4-carboxylic acid, l,3-thiazolidine-4-carboxylic acid, 3-thiaproline, 4-thiaproline, 3-selenoproline, 4-selenoproline, 4-ketoproline, 3,4-dehydroproline, 4-aminoproline, 4-fluoroproline, 4,4-difluoroproline, 4-chloroproline, 4,4-dichloroproline, 4-bromoproline, 4,4-dibromoproline, 4-methylproline, 4-ethylproline, 4-cyclohexylproline, 3-phenylproline, 4-phenylproline, 3,4-phenylproline, 4-azidoproline, 4-carboxyproline, a-methylproline, a-ethylproline, a-propylproline, a-allylproline, a-benzylproline, a-(4-fluorobenzyl)proline, a-(2-chlorobenzyl)proline, a-(3-chlorobenzyl)proline, a-(2-bromobenzyl)proline, a-(4-bromobenzyl)proline, a-(4-methylbenzyl)proline, a-(diphenylmethyl)proline, a-(naphthylmethyl)-proline, D-proline, or S-homoproline, (2S,4S)-4-fluoro-L-proline, (2S,4R)-4-fluoro-L-proline, (2S)-3,4-dihydro-L-proline, (2S,4S)-4-hydroxy-L-proline, (2S,4R)-4-hydroxy-L-proline, (2S,4S)-4-azido-L-proline, (2S)-4,4-difluoro-L-proline, (2S)-azetidine-2-carboxylic acid, (2S)-piperidine-2-carboxylic acid, or (4R)-1,3-thiazolidine-4-carboxylic acid can be used in the molecular structure conjugated to insulin.

[0162] In some embodiments, it should be understood that the specific orientation of the amino acids is achieved, for example, using the method of Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. p. 848. In some embodiments, the compounds of the present disclosure, such as the compounds of formula I or formula II, can be conjugated to diols, catechols, hexose sugars, glucose, xylose, fucose, galactosamine, glucosamine, mannosamine, galactose, mannose, fructose, galacturonic acid, glucuronic acid, iduronic acid, mannuronic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetylmuramic acid, 2-keto-3-deoxy-glycero-galacto-nononic acid, N-acetylneuraminic acid, glycolylneuraminic acid, neurotransmitters, dopamine, or disaccharides, or polymers of saccharides or diols.

[0163] In some embodiments, the modification or intermediate may involve using an N-methyliminodiacetic acid (MIDA) group to create a MIDA-conjugated boronate or MIDA boronate, and such modifications can be used during the preparation of the boronate towards the final structure used (e.g., in embodiments of the methods for preparing the conjugates described herein). In some embodiments, the boronic acid pinacol ester is used for the final structure, and the pinacol group can be readily removed by those skilled in the art using standard techniques. MIDA-protected boronic acid esters are easily handled, stable in air, compatible with chromatography, non-reactive under standard anhydrous cross-coupling conditions, and under mild aqueous basic conditions such as 1M NaOH, or even NaHCO3, or as described by Lee, S. J. et al. (2008). J. Am. Chem. Soc. 130:466.

[0164] The biological mechanism by which wild-type insulin binds to the insulin receptor has been previously reported in Menting, J.G. et al. (2013). Nature 493, 241-245; and Menting, J.G. et al. (2014). “Protective hinge in insulin opens to enable its receptor engagement.” Proc. Natl. Acad. Sci. U.S.A. 111, E3395-3404. Such insulin activity is, for example, TyrA14- 125Using an in vitro insulin receptor that binds to human insulin as a tracer and antibody-conjugated beads with an insulin receptor monoclonal antibody, it can be measured using any suitable technique. In some embodiments, the animal model can be used for in vivo evaluation of insulin activity during glucose administration using methods known to those skilled in the art. In some embodiments, the compounds disclosed herein are partially or fully expressed together with a recombinant protein of interest such as insulin. The process for the expression of insulin in E. coli is known, and those skilled in the art can easily perform it using procedures outlined, for example, in Jonasson (1996). Eur. J. Biochem. 236:656-661; Cowley (1997). FEBS Lett. 402:124:130; Cho (2001). Biotechnol. Bioprocess Eng 6:144-149; Tikhonov (2001). Protein Exp. Pur. 21:176-182; Malik (2007). Protein Exp. Pur. 55:100-111; and Min (2011). J. Biotech. 151:350-356. In the most common process, the protein is expressed as a split protein or a single-chain proinsulin construct with an affinity tag. A compound containing Z1a when Z1a is present (e.g., a compound of formula I) can be expressed as part of proinsulin and then chemically modified to bind to the structure of interest via an amide bond. This approach provides good yields, reduces the complexity of the experiment by reducing the number of processing steps, allows refolding with native insulin, see, for example, Jonasson, Eur. J. Biochem. 236:656-661 (1996); Cho, Biotechnol Bioprocess Eng. 6:144-149 (2001); Tikhonov, Protein Exp. Pur. 21:176-182 (2001); Min, J. Biotech. 151:350-356 (2011)).When expressed in E. coli, proinsulin is typically found in inclusion bodies and can be easily purified by those skilled in the art.

[0165] In some embodiments, proinsulin can be expressed using standard IPTG (isopropylthio-β-galactoside) induction of IPTG-inducible expression constructs and vectors in E. coli strains such as the B21 strain. As an example, the expression construct consists of a B chain, a C peptide, and an A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified, for example, via an existing his-tag prior to sensor conjugation. The expression of the desired proinsulin can be carried out by procedures known in the art. See, for example, U.S. Pat. Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0166] In some embodiments, the compounds of the present disclosure (e.g., Formula I, Formula II) can be formulated for injection. For example, it can be formulated for injection into a subject such as a human. In some embodiments, the composition can be a pharmaceutical composition such as a sterile injectable pharmaceutical composition. In some embodiments, the composition can be formulated for subcutaneous injection. In some embodiments, the composition is formulated for transdermal, intradermal, transmucosal, nasal, inhalation, or intramuscular administration. In some embodiments, the composition can be formulated in an oral dosage form or a pulmonary dosage form. Pharmaceutical compositions suitable for injection can include, for example, sterile aqueous solutions containing polyalcohols such as sugars, mannitol, and sorbitol, phenol, metacresol, and sodium chloride, and the dispersion can be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils, and the carrier can be, for example, a solvent or a dispersion medium containing water, saccharides, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Those skilled in the art will recognize that specific formulations can be developed to best suit the application of the molecular structure of the present invention and the method of use. General considerations in formulating and manufacturing pharmaceutical compositions, routes of administration, and including suitable pharmaceutically acceptable carriers can be found, for example, in Remington’s Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, Pa., 1995. In some embodiments, when the compound of the pharmaceutical composition (e.g., the compound of Formula I) contains insulin, the pharmaceutical composition contains zinc together with insulin, for example Zn 2+may be included. Such zinc formulations are described, for example, in U.S. Patent No. 9,034,818. For example, the pharmaceutical composition may contain zinc in a molar ratio to about M:N modified insulin where M is from 1 to 11 and N is from 6 to 1. In some embodiments, such modified insulin may be stored in a pump, and the pump, which is either outside or inside the body, releases the modified insulin. In some embodiments, the pump may be used to release a fixed amount of modified insulin, and the insulin is glucose-responsive and can automatically adjust its activity based on the level of glucose in the blood and / or the rate of release from the injection site. In some embodiments, the composition may be formulated in unit dosage form for ease of administration and uniformity of dosage. In some embodiments, the pharmaceutical composition may further contain a second insulin type that provides rapid-acting or basal insulin in addition to the effect brought about by the molecular structure. In some embodiments, the compounds of the present disclosure (e.g., compounds of formula I) are injected separately from insulin but modulate the activity of insulin by binding to insulin, and in some embodiments, this change in activity is glucose-dependent.

[0167] In some embodiments, the pharmaceutical composition contains at least one additional component selected from one or more of the compounds disclosed herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable vehicle, and pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains the compound of formula I and at least one additional component selected from a pharmaceutically acceptable carrier, pharmaceutically acceptable vehicle, and pharmaceutically acceptable excipient.

[0168] In some embodiments, the present disclosure includes a compound that can be part of a kit, the kit comprising a compound of Formula I comprising a modified insulin, and a pharmaceutically acceptable carrier, and may include a syringe or pen for injection. In some embodiments, the kit may include a syringe or pen pre-filled with a pharmaceutical composition comprising a compound of Formula I and a liquid carrier. Alternatively, the kit may include a separate container, such as a vial, containing a pharmaceutical composition comprising a compound of Formula I together with a dry carrier and an empty syringe or pen. In some embodiments, such a kit may include a separate container having a liquid carrier that can be used to reconstitute a given composition that can then be taken up into the syringe or pen. In some embodiments, the kit may include instructions. In some embodiments, the kit may include a blood glucose measurement device that calculates the appropriate dose of the modified insulin to be injected either locally or remotely at a given time or at regular intervals. Such a dosing regimen is specific to the patient and may be provided, for example, as instructions for programming a pump either by a human or by a computer. The kit may include, for example, an electronic device that transfers a blood glucose measurement to a second computer, either local or elsewhere (e.g., in the cloud), that then calculates the correct amount of a compound of Formula I comprising the modified insulin that needs to be used by the patient at a particular time.

[0169] In some embodiments, the present invention relates to a method of treating a disease or condition in a subject, comprising administering to the subject a composition comprising a compound described herein. In some embodiments, the disease or condition can be hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome X, or dyslipidemia, gestational diabetes, prediabetes, Alzheimer's disease, MODY1, MODY2, or MODY3 diabetes, mood disorder, and mental disorder. This combined approach can be used in insulin-resistant patients who are receiving insulin sensitizers or secondary drugs for diabetes (e.g., biguanides such as metformin, glitazones, etc.) or insulin secretagogues (e.g., sulfonylureas, GLP-1, exenatide-4, etc.) and / or amylin.

[0170] In some embodiments, the compounds of the present disclosure (e.g., compounds of formula I) can be administered to patients who are receiving at least one additional therapy or taking at least one additional drug or therapeutic protein. In some embodiments, the at least one additional therapy is intended to treat the same disease or disorder as the administered compound (e.g., a compound of formula I). In some embodiments, the at least one additional therapy is intended to treat the side effects of the compound (e.g., a compound of formula I) or as an adjuvant. The time frames of the two therapies can be different or the same, and they can be administered on the same or different schedules as long as there is a period during which the patient benefits from both therapies. Two or more therapies can be administered in the same or different formulations as long as there is a period during which the patient benefits from both therapies. Any of these approaches can be used to administer two or more anti-diabetic drugs to a subject.

[0171] In some embodiments, a therapeutically effective amount of a compound (e.g., a compound of formula I) is used that is sufficient to treat a disease or condition (e.g., meaning to improve its symptoms, delay its progression, or prevent its recurrence, delay its onset) with a reasonable benefit-to-risk ratio. In some embodiments, this may include balancing efficacy and additional safety against toxicity. By additional safety, it is meant that the compound (e.g., a compound of formula I) can be responsive to changes in blood glucose levels or levels of other molecules, even when the patient is not actively monitoring the levels of that molecule, for example, during sleep, within a given time frame. In some embodiments, therapeutic efficacy and toxicity are determined by standard pharmacological procedures using cell cultures or experimental animals in vivo, such as measuring the therapeutic index ED 50 and LD 50 In some embodiments, the average daily dose of insulin, including the molecular structure, ranges from 5 to 400 U (e.g., 30 to 150 U if 1 unit of insulin is about 0.04 mg). In some embodiments, the amount of the compound (e.g., a compound of formula I) and these insulin doses are administered daily, every other day, or every three or four days. In some embodiments, the criteria are determined by an algorithm that can be calculated by a computer. In some embodiments, a compound, such as a compound of formula I, in an amount 5 to 10 times these doses is administered weekly or at regular intervals. In some embodiments, a conjugate in an amount 10 to 20 times these doses is administered every other week or at regular intervals. In some embodiments, a compound (e.g., a compound of formula I) in an amount 20 to 40 times these doses is administered monthly or at regular intervals. In some embodiments, the C-terminus of the A-chain of insulin can be further extended with a peptide (amino acid sequence) containing 1 to 20 amino acid residues. In some embodiments, the insulin analog is desB30 insulin.

[0172] In some embodiments, Z1a is an amino acid or a peptide. In at least some embodiments, Z1a comprises (consists of) 1 to 50 amino acid residues, for example, 1 residue, 50 residues, or any intermediate number of residues (e.g., 10, 15, 25, 30, 42 residues, etc.). In some embodiments, Z1a comprises 1 to 15 amino acids. In at least some embodiments, the peptide Z1a comprises 1 to 8 amino acids. In some embodiments, Z1a comprises 5 to 6 amino acids. In some embodiments, Z1a comprises at least one amino acid independently selected from alanine (A), asparagine (N), glutamine (Q), threonine (T), methionine (M), histidine (H), cysteine (C), valine (V), isoleucine (I), lysine (K), and leucine (L), and the remaining amino acids are each independently selected from any of the 20 natural amino acids. In some embodiments, Z1a may comprise diaminopropionic acid, diaminobutyric acid, or ornithine. In some embodiments, Z1a comprises 1 to 5 lysine residues (K). In some embodiments, Z1a comprises 1 to 3 K amino acids. In some embodiments, Z1a comprises 5 to 6 amino acids and at least one or more of the amino acids is K. In some embodiments, Z1a comprises 5 to 6 amino acids and 1 to 3 of the amino acids are K.

[0173] In some embodiments, Z1a is added to the N-terminus and / or C-terminus and / or inserted into the sequence of the A-chain or B-chain of insulin.

[0174] Compound In some embodiments provided herein is a compound of formula I, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt,

Chemical formula

[0175] In some embodiments, the compound is selected from the group consisting of a compound represented by Formula I, its stereoisomers, mixtures of its stereoisomers, and pharmaceutically acceptable salts thereof, provided that the compound is not any of Examples 1-880 disclosed in PCT / US2021 / 059802.

[0176] In some embodiments, X1 is a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain.

[0177] In some embodiments, the present disclosure relates to a compound of the following formula, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt,

Chemical formula

[0178] In some embodiments, at least one Z1c contains an alpha-methyl group or a beta-methyl group. In some embodiments, Z1c contains an alpha-methyl group. In some embodiments, Z1c contains a beta-methyl group.

[0179] In some embodiments, the compound contains at least one Z1c covalently bonded to the amine of X1, or to NH2 when X1 is NH2, or to OH when X1 is OH, or to the amine of Z1a via an indirect linker, The indirect linker has the formula (X”) n1 as represented, wherein each n1 is independently selected from 1, 2, 3, 4, and 5, each X” is (i) an L or D-amino acid, wherein the amine functional group of the L or D-amino acid is covalently bonded directly or indirectly to Z1c and the acid functional group of the L or D-amino acid is conjugated directly or indirectly to X1 or Z1a; and (ii) independently selected from the formulas FL(IA), FL(IB), FL69, and FL70, The formulas FL(IA) and FL(IB) are

Chemical formula

Chemical Formula

[0180] In some embodiments, the compound comprises at least one Z1c covalently bonded to X1 or Z1a via an indirect linker, and the indirect linker has the formula (X”) n1 represented by each n1 is independently selected from 1, 2, 3, 4, or 5, each X” is an L or D-amino acid, wherein the amine functional group of the L or D-amino acid is directly or indirectly covalently bonded to Z1c, and the acid functional group of the L or D-amino acid is directly or indirectly conjugated to X1 or Z1a; and Formulas FL(IA), FL(IB), FL69 and FL70, wherein FL(IA) and FL(IB) are selected from FL3, FL5, FL5A, FL5B, FL20 - FL68 and FL71 - FL75, formulas FL(IA), FL(IB), FL69 and FL70; selected from,

Chemical formula

Chemical formula

[0181] Formulas FL69 and FL70 are,

Chemical formula

[0182] In some embodiments, the stereochemistry of FL70 can be S or R at the stereogenic carbon. In some embodiments, the stereochemistry of FL70 is S at the stereogenic carbon. In some embodiments, the stereochemistry of FL70 is R at the stereogenic carbon.

[0183] In some embodiments, n1 is 1.

[0184] In some embodiments, the compound is represented by Formula IC,

Chemical formula

[0185] In some embodiments, each of B1 and B2 is independently selected from Formulas F2, F5, and F10, wherein Formulas F2, F5, and F10 are

Chemical formula

[0186] In some embodiments, B1 and B2 are selected from Formula F2, and each remaining R1 is independently selected from H, CF3, and F; each remaining R1 is H, or one or two of each remaining R1 are F or CF3.

[0187] In some embodiments, at least one Z1c is selected from FF12, FF12A-D, FF116, and FF116A-D. In some embodiments, both B1 and B2 are selected from F2, where all remaining R1 are H; the indirect linker is selected from relatively hydrophobic indirect linkers. In some embodiments, the relatively hydrophobic indirect linker can be selected from FL3, FL20-22, FL25, FL27-28, FL30-33, FL35, FL37, FL41, FL43-70, and amino acids having hydrophobic side chains. In some embodiments, both B1 and B2 are selected from F2, where one of the remaining R1 is CF3 and the remaining R1 are H; the indirect linker is selected from relatively hydrophilic indirect linkers. In some embodiments, the relatively hydrophilic indirect linker can be selected from FL5, FL5A, FL5B, FL23-24, FL26, FL29, FL34, FL36, FL38-40, FL42, and amino acids having hydrophilic side chains.

[0188] In some embodiments, each Z1c is covalently bonded to an indirect linker, and each of the combined Z1c and indirect linker (i.e., Z1c-linker) has the formula FFL-1 to FFL-101:

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[0189] In some embodiments, B1 and B2 are selected from Formula F2, wherein each remaining R1 is independently selected from H, CF3 and F, wherein each Z1c is covalently bonded to an indirect linker, and each of the combination of Z1c and the indirect linker is of Formula FFL-1 to 4, 6 to 7, 9, 13 to 16, 20 to 23, 28 to 29, 31 to 33, 39, 42 to 45, 48, 53, 57 to 58, 60 to 62, 65, and 67:

Chem.

Chem.

[0190] In some embodiments, X1 is, independently of each other, a polypeptide prodrug comprising one or more lysine amino acids conjugated to Z1c directly or via an indirect linker.

[0191] In some embodiments, X1 comprises a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof.

[0192] In some embodiments, X1 comprises insulin having an A chain and a B chain, the A chain comprising a sequence selected from SEQ ID NOs: 1, 25, 24051, and 24052, and the B chain comprising a sequence selected from SEQ ID NOs: 24060, 24061, 24062, 24063, 24064, and 25000 - 25397.

[0193] In some embodiments, X1 comprises insulin having an A chain and a B chain, the A chain comprising a sequence selected from SEQ ID NOs: 24051 and 24052, the B chain comprising a sequence selected from SEQ ID NOs: 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380 - 25397; each Z1c is independently selected from FF12A, FF12B, FF12D, FF116A, FF116B, FF116C, and FF116D and is covalently bound directly or via a linker to one or more lysine residues in X1; B1 and B2 are each selected from F2 The indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL20 - FL75, and their stereoisomers.

[0194] In some aspects, X1 is a polypeptide comprising an insulin receptor agonist having an A chain and a B chain, wherein: the A chain comprises a sequence selected from SEQ ID NOs: 1, 25, and 24051, The B chain contains a sequence selected from SEQ ID NOs: 24060, 24063, 25228, 25313, 25393, 25396, and 25397, each Z1c is independently selected from FF12A, FF12B, FF114A, FF115A, FF116A, and FF225A and is covalently attached directly or via a linker to one or more lysine residues in X1; B1 and B2 are each selected from F2, the linker is independently selected from FL3, FL5, FL5A, FL5B, FL21, and their stereoisomers.

[0195] In some embodiments, the compound is Example 1 (SEQ ID NOs: 25398 and 25399, in the order of appearance, respectively):

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[0196] In some embodiments, a compound selected from the group consisting of insulin comprising an A chain and a B chain is provided herein, the A chain comprises a sequence selected from SEQ ID NO: 24501 and 24502, and the B chain comprises a sequence selected from SEQ ID NO: 25229, 25232, 25305, 25308, 25312, 25236, 25095, and 25380 - 25397.

[0197] In some embodiments, each indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL32, FL41, FL62, FL63, and stereoisomers thereof.

[0198] In some embodiments, B1 and B2 are selected from Formula F2, and each of the remaining R1s is independently selected from H, F, Cl, I, Br, and CF3.

[0199] In some embodiments, B1 and B2 are selected from Formula F2, and each of the remaining R1s is H.

[0200] In some embodiments, X1 comprises at least one lysine and an N-terminus, each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116, FF116A, FF116B, FF116C, FF116D, each B1 and B2 is F2 and each Z1c is conjugated to the amine side chain of the lysine of X1 or the N-terminus of X1, either directly or via an indirect linker, and each indirect linker is independently selected from FL3, FL5, FL5A, FL5B, and FL20 - FL75, and stereoisomers thereof, wherein in FL3 and FL5, each p is independently 1, 2, or 3.

[0201] In some embodiments, at least one Z1c is conjugated to the amine side chain in a lysine in the B chain or to the B chain N-terminus via an indirect linker.

[0202] In some embodiments, X1 comprises two or more lysine amino acids.

[0203] In some embodiments, the B chain comprises at least two lysine amino acids that are each independently conjugated to Z1c either directly or via an indirect linker.

[0204] In some embodiments, the B chain comprises at least three lysine amino acids that are each independently covalently bound to Z1c via an indirect linker.

[0205] In some embodiments, the A chain comprises one or more lysine amino acids that are each independently covalently bound to Z1c either directly or via an indirect linker.

[0206] In some embodiments, X1 comprises an A chain having at least one lysine covalently bound to Z1c via an indirect linker.

[0207] In some embodiments, the compound of formula I is

Chemical formula

[0208] In some embodiments, at least one Z1c is covalently bound to X1 via an indirect linker, and the indirect linker is

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[0209] In some embodiments, the indirect linker is selected from the following: [Chem.]

[0210] In some embodiments, X1 is insulin further comprising 1 to 5 residues substituted, inserted, appended, or mutated with an amino acid having a free amine conjugated directly or indirectly to Z1c.

[0211] In some embodiments, at least one Z1c is conjugated to the free amine side chain of an amino acid substituted, inserted, or mutated on insulin.

[0212] In some embodiments, the compound is covalently bonded directly or via a linker to a molecule that can bind to at least one protein present in human plasma.

[0213] In some embodiments, in each of Formulas F2, F5, and F10, one R1 is (C=O)--- * is.

[0214] In some embodiments, the compound is represented by Formula II, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, Z1c-linker (Formula II) wherein Z1c-linker is

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[0215] In some embodiments, the compound represented by Formula (II) comprises at least one B1 or B2 independently selected from Formulas F2, F5, and F10, wherein Formulas F2, F5, and F10 are

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[0216] In some embodiments, the Z1c-linker (i.e., Formula II) is

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[0217] In some embodiments, when X is a leaving group, X is selected from N -oxysuccinimide, 2,3,5,6 - tetrafluorophenoxy (TFP), pentafluorophenoxy (Pfp), OH, halogen, maleimidalkylamino, maleimidamidopolyethylene glycol amino, and maleimidopolyethylene glycol amino. In some embodiments, X is selected from N -oxysuccinimide and OH. In some embodiments, X is OH. In some embodiments, X is N -oxysuccinimide. In some embodiments, maleimidopolyethylene glycol amino is selected from Mal - PEG2 - amine, Mal - PEG4 - amine, and Mal - PEG5 - amine, or a pharmaceutically acceptable salt thereof. In some embodiments, maleimidalkylamino is selected from Mal - C6 - amine and N-(2 - aminoethyl)maleimide, or a pharmaceutically acceptable salt thereof. In some embodiments, maleimidamidopolyethylene glycol amino is selected from Mal - amide - PEG9 - amine, Mal - amide - PEG11 - amine, Mal - amide - PEG23 - amine, 4 - Mal - methyl - cyclohexanecarboxamide - methyl - [1,2,3]triazole - PEG8 - amine, or a pharmaceutically acceptable salt thereof. In some embodiments, the Z1c - linker is [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] It is selected from stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts thereof, and combinations thereof. In some embodiments, the Z1c-linker is

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[0218] In some embodiments, a therapeutically effective amount of the pharmaceutical composition of the present disclosure can be administered to a subject. In some embodiments, the pharmaceutical composition comprises at least one compound (e.g., Formula I) disclosed herein and a pharmaceutically acceptable carrier.

[0219] In some embodiments, the compounds disclosed herein are used as pharmaceuticals.

[0220] In some embodiments, the present disclosure provides a method for treating or preventing diabetes, impaired glucose tolerance, hyperglycemia, or metabolic syndrome, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.

[0221] In some embodiments, the pharmaceutical composition comprises one or more polyalcohols.

[0222] In some embodiments, the polyalcohol is selected from mannitol, sorbitol, erythritol, isomalt, lactitol, glucose, and maltitol.

[0223] In some embodiments, the pharmaceutical composition comprises at least one compound disclosed herein for use as a medicament for the treatment of diabetes or obesity, for the control of blood glucose levels, or for the control of drug release.

[0224] In some embodiments, the present disclosure provides a method of administering a compound disclosed herein or a pharmaceutical composition disclosed herein to a subject as a therapeutic or prophylactic agent.

[0225] In some embodiments, the present disclosure provides a method of making a compound disclosed herein, comprising at least one alkylation and / or amidation step.

[0226] In some embodiments, the present disclosure provides a method of treating a subject by administering a device or formulation comprising a compound disclosed herein, such as Examples 1 to 156. For example, the device can be a fixed-dose syringe, a microdosing syringe, an internal or external patch.

[0227] In some embodiments, the compounds of the present disclosure can be used as intermediates in the manufacture of therapeutic agents of prodrugs or prophylactic compounds.

[0228] In another aspect, the present disclosure provides a human insulin analog comprising an A chain and a B chain, wherein the A chain comprises a sequence selected from SEQ ID NO: 24051 and 24052, and the B chain comprises a sequence selected from SEQ ID NO: 25000 to 25397.

[0229] In some embodiments, the A chain comprises the sequence 24051, and the B chain comprises a sequence selected from 25000, 25001, 25006 - 25009, 25076, 25077, 25082 - 25085, 25228, 25229, 25232, 25234 - 25237, 25304, 25305, 25308, and 25310 - 25313.

[0230] In some embodiments, the A chain comprises the sequence 24051, and the B chain comprises a sequence selected from 25011, 25012, 25017 - 25020, 25087, 25088, 25093 - 25096, 25229, 25239, 25232, 25240, 25245 - 25248, 25305, 25308, 25315, 25316, and 25321 - 25324.

[0231] In some embodiments, the A chain comprises the sequence 24051, and the B chain comprises a sequence selected from 25228, 25229, 25232, 25234 - 25237, 25304, 25305, 25308, and 25310 - 25313.

[0232] In some embodiments, the A chain comprises the sequence 24051, and the B chain comprises a sequence selected from 25011, 25012, 25017 - 25020, 25087, 25088, 25093 - 25096, 25229, 25232, 25305, and 25308.

[0233] In some embodiments, the agonist potency of the compounds disclosed herein is determined by measuring the potency of the compounds for activation of a receptor (e.g., the insulin receptor). In some embodiments, the present disclosure provides compounds having agonist potency for the insulin receptor that include at least one aromatic boron-containing group, the compounds having a first EC50 potency for activating the insulin receptor at a first glucose concentration and a second EC50 potency for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 3 mM and the second glucose concentration is 10 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist potency of the compounds is from about 1.2 to about 14, from about 1.2 to about 10, from about 2 to about 9, from about 2 to about 8, from about 3 to about 7, or from about 3 to about 6.

[0234] In some embodiments, the present disclosure relates to compounds that include at least one aromatic boron-containing group attached to an FF backbone (e.g., FF116A, FF225A) that includes a methyl group, the compounds having agonist potency for the insulin receptor, the compounds having a first EC50 potency for activating the insulin receptor at a first glucose concentration and a second EC50 potency for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 3 mM and the second glucose concentration is 10 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist potency of the compounds is from about 2.5 to about 3.7, from about 2.5 to about 3.5, from about 2.5 to about 3, or from about 3 to about 3.5. In some embodiments, the methyl group is a beta-methyl group, such as FF116A. In some embodiments, the methyl group is an alpha-methyl group, such as FF225A.

[0235] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having agonist efficacy against the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 3 mM and the second glucose concentration is 10 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy of the compound is from about 1 to about 15, from about 1 to about 10, from about 2 to about 9, from about 2 to about 8, from about 3 to about 7, or from about 3 to about 6. In some embodiments, when the first glucose concentration is 3 mM and the second glucose concentration is 10 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy of the compound is at least or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0236] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having agonist efficacy against the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy of the compound is from about 2 to about 25, from about 2 to about 20, from about 2 to about 15, from about 2 to about 10, from about 2 to about 10, from about 2 to about 9, or from about 2 to about 8. In some embodiments, when the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the compound has an insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 of at least or up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0237] In some embodiments, the present disclosure relates to a compound comprising at least one aromatic boron-containing group comprising a methyl group (e.g., an alpha-methyl group diborononate sensor or a beta-methyl group diborononate sensor such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110, where X represents a direct covalent attachment point to the amine of X1 of Formula I), and having agonist efficacy against the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, where the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 is from about 1.5 to about 21, about 1.6 to about 21, about 1.7 to about 20, and about 3 to about 20, about 4 to about 15, or about 10 to about 15. In some embodiments, where the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the compound has an insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 of at least or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0238] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having agonist efficacy against the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, where the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the first EC50 to second EC50 ratio of the insulin receptor agonist efficacy of the compound is from about 1 to about 25, about 2 to about 24, about 2 to about 24, about 4 to about 20, about 5 to about 18, or about 10 to about 15.

[0239] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having agonist efficacy for the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy of the compound is from about 1.2 to about 5, from about 1.3 to about 5, from about 1.4 to about 5, from about 1.5 to about 5, from about 2 to about 4, or from about 3 to about 4. In some embodiments, when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the compound has an insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 of at least or up to about 1.2, 1.3, 1.4, 1.5, 2, 3, 4, or 5.

[0240] In some embodiments, the present disclosure relates to a compound comprising at least one aromatic boron-containing group comprising a methyl group (such as an alpha-methyl group diborononate sensor or a beta-methyl group diborononate sensor such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110, etc.) and having agonist efficacy for the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy is from about 1.5 to about 3.5, from about 1.5 to about 3, from about 2 to about 3, or from about 2.5 to about 3. In some embodiments, when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the compound has an insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 of at least or up to about 1.2, 1.3, 1.4, 1.5, 2, or 3.

[0241] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having agonist efficacy against the insulin receptor, the compound having a first EC50 efficacy for activating the insulin receptor at a first glucose concentration and a second EC50 efficacy for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist efficacy of the compound is from about 1 to about 6, from about 2 to about 6, from about 3 to about 5, or from about 3 to about 4. In some embodiments, when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM, the compound has an insulin receptor agonist efficacy ratio of the first EC50 to the second EC50 of at least or up to about 1, 2, 3, 4, 5, or 6.

[0242] In some embodiments, the present disclosure provides a compound comprising at least one diborononate sensor having binding affinity for glucose, the compound having a glucose Kd in the range of 0.01 mM to 3 mM, 0.01 mM to 2.5 mM, 0.01 mM to 2 mM, 0.01 mM to 1.5 mM, 0.01 mM to 1 mM, or 0.01 mM to 0.5 mM. In some embodiments, the compound has a glucose Kd of less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM. In some embodiments, the compound has a glucose Kd of less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM and the compound has agonist activity against the insulin receptor.

[0243] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having a binding affinity for glucose, wherein the compound, when administered at a dose of 30 nmol / kg, results in a blood glucose concentration of 100 mg / dL in a first group of rats having a first glucose infusion rate and is administered to a second group of rats having a second glucose infusion rate so as to result in a blood glucose concentration of 200 mg / dL, provides a relative glucose infusion rate difference (mg / kg / min·min) of 50 to 2500 and / or a relative glucose infusion rate ratio of 0.2 to 5. In some embodiments, the compound provides a relative glucose infusion rate difference (mg / kg / min·min) of at least about 50, 100, 200, 300, 400, 500, 1000, 1500, 2000, or 2500 and / or a relative glucose infusion rate ratio of at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0244] In some embodiments, the present disclosure relates to a compound comprising at least one aromatic boron-containing group comprising a methyl group (e.g., an alpha-methyl group diborononate sensor or a beta-methyl group diborononate sensor such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110, etc.) and having an agonist efficacy for the insulin receptor, wherein the compound, when administered at a dose of 30 nmol / kg, results in a blood glucose concentration of 100 mg / dL in a first group of rats having a first glucose infusion rate and is administered to a second group of rats having a second glucose infusion rate so as to result in a blood glucose concentration of 200 mg / dL, has a relative glucose infusion rate difference (mg / kg / min·min) of about 700 to about 2500, about 750 to about 2500, about 800 to about 2500, about 850 to about 2500, about 900 to about 2500, and a relative glucose infusion rate ratio of about 2 to about 4, about 2 to about 3, or about 3 to about 4.

[0245] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having binding affinity for glucose and having agonist efficacy for glucose, wherein the compound, when administered to a first group of rats having a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL at a dose of 30 nmol / kg, and a second group of rats having a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, provides a relative glucose infusion rate difference (mg / kg / min.min) of about 1 to about 2500, about 1 to about 2000, about 1 to about 1500, about 100 to about 1500, and about 1000 to about 1500, and a relative glucose infusion rate ratio of about 0.1 to about 5, about 0.2 to about 4.5, about 0.5 to about 4, about 0.5 to about 3.5, or about 1 to about 3. In some embodiments, the compound provides a relative glucose infusion rate difference (mg / kg / min.min) of at least about 50, 100, 200, 300, 400, 500, 1000, 1500, 2000, or 2500 and / or a relative glucose infusion rate ratio of at least or up to about 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0246] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having binding affinity for glucose, wherein the compound, when administered at a dose of 20 nmol / kg to a first group of rats having a first glucose infusion rate to result in a blood glucose concentration of 100 mg / dL and to a second group of rats having a second glucose infusion rate to result in a blood glucose concentration of 200 mg / dL, provides a relative glucose infusion rate difference (mg / kg / min.min) of 150 to 1000 and a relative glucose infusion rate ratio of about 1 to about 3. In some embodiments, the compound provides a relative glucose infusion rate difference (mg / kg / min.min) of at least about 150, 200, 250, 300, 450, 500, 600, 700, 800, 900, or 1000 and / or a relative glucose infusion rate ratio of at least or up to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0247] In some embodiments, the present disclosure relates to a compound comprising at least one aromatic boron-containing group having a binding affinity for glucose, the aromatic boron-containing group comprising a methyl group (e.g., an alpha-methyl group diborononate sensor or a beta-methyl group diborononate sensor such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110). In some embodiments, when the compound is administered to a first group of rats having a first glucose infusion rate for providing a blood glucose concentration of 100 mg / dL and a second group of rats having a second glucose infusion rate for providing a blood glucose concentration of 200 mg / dL at a dose of 20 nmol / kg, the compound has a relative glucose infusion rate difference (mg / kg / min.min) of about 10 to about 1000, about 10 to about 900, about 20 to about 800, about 50 to about 700, and about 100 to about 600, and about 200 to about 500, and a relative glucose infusion rate ratio of about 1 to about 2, about 1 to about 1.5, or about 2 to about 2.5.

[0248] In some embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having an agonist efficacy for glucose, and when the compound is administered to a first group of rats having a first glucose infusion rate for providing a blood glucose concentration of 100 mg / dL and a second group of rats having a second glucose infusion rate for providing a blood glucose concentration of 200 mg / dL at a dose of 20 nmol / kg, the compound provides a relative glucose infusion rate difference (mg / kg / min.min) of about 100 to about 2500, about 100 to about 1000, about 100 to about 900, about 200 to about 800, about 300 to about 700, about 400 to about 600, and about 400 to about 500, and a relative glucose infusion rate ratio of about 1 to about 2.5, about 1 to about 2, about 1 to about 1.5, about 0.5 to about 1.5, or about 1 to about 1.5.

[0249] In some embodiments, the present disclosure provides a compound having a binding affinity for glucose, comprising at least one aromatic boron-containing group having a binding affinity for glucose, wherein the compound, when administered at a dose of 10 nmol / kg, results in a blood glucose concentration of 100 mg / dL in a first group of rats having a first glucose infusion rate and a blood glucose concentration of 200 mg / dL in a second group of rats having a second glucose infusion rate, provides a relative glucose infusion rate difference (mg / kg / min / min) of 60 to 2400 and a relative glucose infusion rate ratio of about 0.5 to about 4.

[0250] In some embodiments, the present disclosure provides a compound having a binding affinity for glucose, comprising at least one diboronato sensor having an affinity for glucose and at least one aromatic boron-containing group.

[0251] In some embodiments, the present disclosure relates to compounds having agonist efficacy against the insulin receptor, comprising at least one aromatic boron-containing group (e.g., diboronatesensor, e.g., DSL-1 to DSL-112), wherein one or more aromatic boron-containing groups contain a methyl group (e.g., beta-methyl, alpha-methyl). In some embodiments, the compounds are selected from Example 1, Example 3, Example 4, Example 5, Example 6, Example 8, Example 9, Example 10, Example 11, Example 12, Example 15, Example 16, Example 18, Example 19, Example 23, Example 24, Example 25, Example 27, Example 30, Example 33, Example 34, Example 35, Example 37, Example 40, Example 41, Example 44, Example 46, Example 48, Example 49, Example 52, Example 54, Example 55, Example 56, Example 57, Example 59, Example 64, Example 65, Example 67, Example 69, Example 70, Example 75, Example 76, Example 80, Example 82, Example 83, Example 84, Example 85, Example 92, Example 94, Example 95, Example 96, Example 97, Example 100, Example 101, Example 102, Example 104, Example 107, Example 109, Example 112, Example 113, Example 114, Example 118, Example 119, Example 122, Example 123, Example 127, Example 129, Example 130, Example 131, Example 132, Example 133, Example 140, Example 152, and Example 154. In some embodiments, at least one aromatic boron-containing group (e.g., diboronatesensor) has a binding affinity for glucose. In some embodiments, compounds containing two or more diboronatesensors have a higher affinity for glucose. In some embodiments, compounds containing three or more diboronatesensors have a higher affinity for glucose.

[0252] In some embodiments, the binding constants of DSL compounds such as DSL-1A to DSL-112A for glucose, fructose, and / or lactate can be tested and calculated. In some embodiments, the DSL compounds disclosed herein have a binding affinity for glucose with a Kd value in the range of about 0.01 mM to about 3 mM. Some exemplary DSL compounds disclosed herein have a binding affinity for glucose with a Kd value in the range of about 0.01 mM to about 2.5 mM. Some exemplary DSL compounds disclosed herein have a binding affinity for glucose with a Kd value in the range of about 0.5 mM to about 1.5 mM. In some embodiments, the exemplary DSL compounds have a binding affinity for glucose with a Kd value of less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM.

[0253] The present disclosure can also be defined according to any one of the following numbered embodiments. 1. A compound of the following formula, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt:

Chemical formula

Chemical formula

[0254] 2. The compound according to Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein at least one Z1c contains a beta-methyl group.

[0255] 3. The compound according to Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein at least one Z1c contains an alpha-methyl group.

[0256] 4. The compound according to Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein in the formula, B1 and B2 are each independently Formula F2, Formula F2 is

Chemical formula

[0257] 5. The compound of embodiment 4 or a pharmaceutically acceptable salt thereof, wherein the first R1 is (C=O)--- *

[0258] 6. (a) The compound of embodiment 4 or a pharmaceutically acceptable salt thereof, wherein each remaining R1 is independently selected from (i) H, CF3, and F, or (b) two remaining R1s are H and one remaining R1 is CF3 and F.

[0259] 7. The compound according to embodiment 6 or a pharmaceutically acceptable salt thereof, wherein each remaining R1 is H.

[0260] 8. One or more Z1cs are covalently bonded to X1 or Z1a via an indirect linker, and the indirect linker is represented by the formula (X”) n1 and each n1 is independently selected from 1, 2, 3, 4, and 5, each X” is independently of the formula FL3, FL5, FL5A, FL5B, FL21, FL25, FL33, FL41, FL59, FL60, FL64, FL65, FL68, FL69, and FL74:

Chemical formula

[0261] 9. The compound according to embodiment 8 or a pharmaceutically acceptable salt thereof, wherein each of the indirect linkers is independently selected from FL3, FL5, FL5A, FL5B, FL33, FL64, FL65, FL69, and their stereoisomers.

[0262] 10. One or more Z1c are covalently bonded to X1 via an indirect linker, and the indirect linker is independently

Chemical formula

Chemical formula

[0263] 11. An indirect linker is

Chemical formula

[0264] 12. Each of Z1c covalently bonds with an indirect linker to form a Z1c-linker independently selected from the following:

Chemical formula

[0265] 13. The compound is Example 3, Example 19, Example 22, Example 23, Example 27, Example 41, Example 42, Example 44, Example 45, Example 48, Example 50, Example 55, Example 56, Example 69, Example 71, Example 76, Example 87, Example 93, Example 105, Example 131, Example 136, Example 140, Example 141, Example 142, Example 143, Example 144, Example 145, Example 146, Example 147, Example 148, Example 149, Example 151, Example 152, Example 153, Example 154, and Example 155; the compound according to Embodiment 1 selected from its pharmaceutically acceptable salts, its isotopes, and combinations thereof.

[0266] 14. The compound is Example 3, Example 19, Example 23, Example 27, Example 41, Example 44, Example 48, Example 55, Example 56, Example 69, Example 76, Example 131, Example 140, Example 152, Example 154, and Example 156; the compound according to Embodiment 1 selected from its pharmaceutically acceptable salts, its isotopes, and combinations thereof.

[0267] 15. A pharmaceutical composition comprising at least one compound according to Embodiment 1, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0268] 16. A pharmaceutical composition comprising at least one compound according to Embodiment 13 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0269] 17. A pharmaceutical composition comprising at least one compound according to Embodiment 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0270] 18. A method for treating diabetes, impaired glucose tolerance, hyperglycemia or metabolic syndrome, comprising administering a therapeutically effective amount of the compound according to Embodiment 1, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, to a subject in need thereof.

[0271] 19. A method for treating diabetes, impaired glucose tolerance, hyperglycemia or metabolic syndrome, comprising administering a therapeutically effective amount of the pharmaceutical composition according to Embodiment 15 to a subject in need thereof.

[0272] 20. A method for treating type 1 diabetes or type 2 diabetes, comprising administering a therapeutically effective amount of the pharmaceutical composition according to any one of Embodiments 15 to 17 to a subject in need thereof.

[0273] 21. A method for treating type 2 diabetes, comprising administering a therapeutically effective amount of the pharmaceutical composition according to Embodiment 16 or 17 to a subject in need thereof.

[0274] 22. A compound represented by Formula II, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt: Z1c - linker (Formula II) The Z1c-linker is selected from DSL-2, DSL-8, DSL-14, DSL-18, DSL-19, DSL-21, DSL-32, DSL-37, DSL-47, DSL-48, DSL-58, DSL-65, DSL-66, DSL-67, DSL-68, DSL-69, DSL-73, DSL-75, DSL-77, DSL-83, DSL-104, DSL-105, DSL-106, DSL-107, DSL-108, DSL-109, DSL-110, DSL-111, X is a leaving group, One or more positions of the compound of formula II may contain isotopes.

[0275] 23. When X is The compound according to embodiment 22, selected from N-hydroxysuccinimide (NHS), 2,3,5,6-tetrafluorophenol (TFP), pentafluorophenol (Pfp), OH, and halogen.

[0276] 24. The compound is The compound according to embodiment 22, selected from DSL-2A, DSL-9A, DSL-14A, DSL-18A, DSL-19A, DSL-21A, DSL-32A, DSL-37A, DSL-47A, DSL-48A, DSL-58A, DSL-65A, DSL-66A, DSL-67A, DSL-68A, DSL-69A, DSL-73A, DSL-75A, DSL-77A, DSL-83A, DSL-104A, DSL-105A, DSL-106A, DSL-107A, DSL-108A, DSL-109A, DSL-110A, DSL-111A, its stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts, and combinations thereof.

[0277] 25. The compound is The compound according to Embodiment 22, selected from DSL-2B, DSL-9B, DSL-14B, DSL-18B, DSL-19B, DSL-21B, DSL-32B, DSL-37B, DSL-47B, DSL-48B, DSL-49B, DSL-58B, DSL-65B, DSL-66B, DSL-67B, DSL-68B, DSL-69B, DSL-73B, DSL-75B, DSL-77B, DSL-83B, DSL-104B, DSL-105B, DSL-106B, DSL-107B, DSL-108B, DSL-109B, DSL-110B, DSL-111B, its stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts, and combinations thereof.

[0278] Preparation method In some embodiments, the present disclosure provides a method for preparing an aromatic boron-containing compound and / or a compound containing an aromatic boron-containing group (e.g., Z1c, Z1c-linker (Formula II), Formula I), or a pharmaceutical formulation comprising one or more compounds of the present disclosure.

[0279] In some embodiments, the present disclosure provides a method for preparing a rotationally constrained tether boron conjugate comprising a backbone (Z1c, Z1c-linker) whose rotation is inhibited by unfavorable steric interactions (e.g., gauche pair anti-interactions of substituents), bond hybridization-induced conformational rotation (e.g., cis- to trans-amide rotation), or a rigid covalent bond (e.g., (E) vs. (Z) configuration of an alkene moiety). For example, Formulas FF116, FF116A, FF116B, FF116C, and FF116D contain geminal (e.g., bonded to the same atom) alkyl functional groups relative to the amine group covalently bonded to the boronic acid functional moiety. As another example, one or more of Formulas FF116, FF116A, FF116B, FF116C, and FF116D contain geminal alkyl substituents that may limit the accessible dihedral angles adopted by the boron-conjugated amine, affect the dihedral angles adopted, place the boron functional group closer, and enable an increase in the binding of the conjugate to a target molecule such as a protein or a sugar.

[0280] In some embodiments, the compounds disclosed herein are further modified via connection (e.g., conjugation, fusion, etc.) to a second agent or therapy to form a fusion protein. In some embodiments, the second agent or therapy is a protein or peptide described herein. In some embodiments, the second agent comprises a prodrug, and the prodrug is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, a glucagon analog, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory polypeptide, a glucose-dependent insulinotropic polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof. In some embodiments, the fusion protein comprises one or more of the diboronates sensors described herein. In some embodiments, two or more agents are linked (e.g., conjugated, fused, etc.) to produce a fusion protein comprising one or more diboronates sensors. In some embodiments, the two or more agents are proteins or peptides described herein. The biological activity (e.g., agonist potency, bioavailability, etc.) of the compounds, compositions, or methods of treatment described herein can be evaluated according to methods known to those of skill in the art. In some embodiments, the biological activity of a compound is determined by evaluating the EC50 of the compound. In some embodiments, the biological activity of a compound is determined using the insulin receptor phosphorylation (IR phosphorylation) assay disclosed herein. In some embodiments, the biological activity of a compound is determined by evaluating the binding affinity (Kd) of the compound for its target. In some embodiments, the biological activity of a compound is determined by evaluating the relative glucose rate difference. In some embodiments, the biological activity of a compound is determined by evaluating the relative glucose rate ratio.

[0281] Treatment method In some embodiments, the present disclosure provides a method of treating a subject who has or is at risk of developing a disease that is beneficially treated by a compound disclosed herein or a pharmaceutical formulation comprising one or more of the compounds disclosed herein. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a pharmaceutical formulation / composition of the present disclosure. In at least one embodiment, the compounds and / or pharmaceutical formulations of the present disclosure are for use in the treatment or prevention of (or for the manufacture of a medicament for) disorders including hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome X, or dyslipidemia, gestational diabetes, prediabetes, Alzheimer's disease, MODY1, MODY2, or MODY3 diabetes, neuropathy, mood disorders, and mental disorders. In at least one embodiment, a therapeutically effective amount of the compounds and / or pharmaceutical formulations of the present disclosure is administered to a subject suffering from diabetes. In some embodiments, the diabetes is type 1 diabetes or type 2 diabetes. In some embodiments, the diabetes is type 1 diabetes.

[0282] In some embodiments, when a first active agent is administered with a second (different) active agent, the dosages can be adjusted such that the combined activities of the two treatments are sufficient to regulate the patient's blood glucose level. Thus, the amount of the first active agent or second active agent that can be administered to regulate blood glucose levels with such a combination may be less than that required when the first active agent or second active agent is administered as monotherapy.

[0283] The following examples and experimental data are provided for illustrative purposes only and do not limit the scope of the embodiments of the present disclosure.

[0284] The following abbreviations have the definitions set forth below:

Table 1

Examples

[0285] A. Preparation of aromatic boron-containing compounds. The disclosed compounds can be prepared according to the following scheme. The following scheme represents the general methods used in preparing these compounds. However, the synthesis of these compounds is not limited to these representative methods and can also be prepared by various other methods by those skilled in synthetic chemistry.

[0286] DSL synthesis method 1: On-resin synthesis of diboronated sensor: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen with dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (0.93 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then with DMF (3 × 5), treated with 20% piperidine in DMF (3 × 5 mL) for 5 minutes, and washed with DMF (4 × 5 mL). A solution of 2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid, 4 (0.224 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF (5 mL) was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 5. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 7. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0287] Cleavage of diboronated sensor from resin Compound 7 on the resin was treated with 20% 1,1,1,3,3,3 - hexafluoro - 2 - propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2×5 mL) and evaporated to obtain diboronated sensoric acid 8 (DSL - 66A), which was used in the next step without purification.

[0288] NHS activation of diboronated sensor: The crude diboronated sensoric acid 8 was dissolved in DMF (2 mL) and treated with 3 - (3 - dimethylaminopropyl) - 1 - ethyl - carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N - hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 uL), and fractionated over a C18 column by reverse - phase (RP) flash chromatography with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA over 10 minutes and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain 2,5 - dioxopyrrolidin - 1 - yl 3 - ((2S,3S) - 2,3 - bis(1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carboxamido)butanamide)propanoate 11 (DSL - 66B) in about 33% yield (20 mg). Mass calculated value (M + H) + = 607.2, mass found value (M + H) + = 606.9.

Chemical Structure

[0289] The MS data of DSL - 66B are shown in Table I. The other examples listed in Table I below were synthesized under similar conditions.

Table 2

[0290] DSL synthesis method 2: On-resin synthesis of diboronated sensor: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen with dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (0.93 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution in a solution of DCM and DIEA (1 M) and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then with DMF (3 × 5 mL). The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of (2S,3S)-(Fmoc-amino)-3-azidobutyric acid 4 (0.146 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol) and HATU (0.152 g, 0.4 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 5. The resin was washed with DMF (4 × 5 mL) and THF (3 × 5 mL), treated with 5 mL of a THF solution of 0.2 M SnCl2 (189 mg), 0.8 M thiophenol (440 μl) and 1 M DIEA (870 μl), and gently mixed on a shaker for 1 hour. After 1 hour, the resin was washed with THF (3 × 5 mL) and DMF (3 × 5 mL). The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL) to obtain 6a. A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol) and DIPEA (0.35 mL, 2 mmol) was added to the resin (6a) and heated at 50 °C for 30 minutes to obtain 7. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0291] Cleavage of diboronated sensor from resin Compound 7 on the resin was treated with 20% 1,1,1,3,3,3 - hexafluoro - 2 - propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2×5 mL) and evaporated to obtain diboronated sensor acid 8 (DSL - 66A), which was used in the next step without purification. The same procedure was followed for the synthesis of DSL - 14A, DSL - 15A, and DSL - 68A.

[0292] NHS activation of diboronated sensor: Diboronated sensor acid 8 was dissolved in DMF (2 mL) and treated with 3 - (3 - dimethylaminopropyl) - 1 - ethyl - carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol), N - hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and then gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4 and dried under vacuum, dissolved in DMSO (100 uL), and fractionated over a C18 column by reverse - phase (RP) flash chromatography with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA over 10 minutes to obtain the desired product analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain 2,5 - dioxopyrrolidin - 1 - yl 3 - ((2S,3S) - 2,3 - bis(1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carboxamido)butanamide)propanoate 11 (DSL - 66B) in 29% yield (17.4 mg). Mass calculated value (M + H) + = 607.2, mass found value (M + H) + = 606.9.

[0293] The same procedure was followed for the synthesis of DSL - 14B, DSL - 15B, and DSL - 68B.

Chemical formula

[0294] DSL synthesis method 3: On-resin synthesis of diboronated sensor: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen with dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)butanoic acid 40 (119 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was added immediately and mixed gently overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA, and mixed for 1 hour to obtain 41. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid, 14 (0.229 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 42. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 43. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0295] Cleavage of diboronated sensor from resin Compound 43 on the resin was treated with 20% 1,1,1,3,3,3 - hexafluoro - 2 - propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2×5 mL) and evaporated to obtain diboronated sensor acid DSL - 1A - OtBu, which was used in the next step without purification.

[0296] NHS activation of diboronated sensor: Crude diboronated sensor acid DSL - 1A - OtBu was dissolved in DMF (2 mL) and treated with 3 - (3 - dimethylaminopropyl) - 1 - ethyl - carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol), N - hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum. The tert - butyl protecting group was removed with TFA (50%) in DCM, mixed for 1 hour and reduced under vacuum, then dissolved in DMSO (100 μL) and fractionated over 10 minutes with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA on a C18 column by reverse - phase (RP) flash chromatography and analyzed by LCMS. The pure fractions were isolated, combined, frozen and lyophilized to obtain DSL - 1B as a white powder (25 mg).

Chemical formula

[0297] The MS data of DSL - 1B are listed in Table II. The other examples listed in Table II below were synthesized under similar conditions.

Table 3

[0298] DSL synthesis method 4: On-resin synthesis of diboronated sensor: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)butanoic acid 40 (119 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA and mixed for 1 hour to obtain 44. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid, 14 (0.229 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 45. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 43. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0299] Cleavage of diboronated sensor from resin Compound 45 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered, and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2 × 5 mL) and evaporated to obtain diboronated sensor acid DSL-15A-OtBu, which was used in the next step without purification.

[0300] NHS activation of diboronated sensor: The crude diboronated sensor acid DSL-15A-OtBu was dissolved in DMF (2 mL), treated with 3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum. The tert-butyl protecting group was removed with TFA (50%) in DCM, mixed for 1 hour, reduced under vacuum, then dissolved in DMSO (100 μL), fractionated over 10 minutes with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA by reverse phase (RP) flash chromatography on a C18 column, and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain DSL-15B as a white powder (21 mg). [Chemical formula]

[0301] The MS data of DSL-15B are listed in Table III. The other examples listed in Table III below were synthesized under similar conditions. [Table 4]

[0302] DSL synthesis method 5: On-resin synthesis of diboronated sensor: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of (R-3-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-methylhexanoic acid 12 (110 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was added immediately and mixed gently overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution in a solution of DCM and DIPEA (1 M), and mixed for 1 hour to obtain 13. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid, 14 (0.229 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol) and HATU (0.152 g, 0.4 mmol) in DMF was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 15. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol) and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 16. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0303] Cleavage of diboronated sensor from resin The compound 16 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM (7 mL) and mixed gently for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2 × 5 mL) and evaporated to obtain diboronated sensor acid DSL-67A, which was used in the next step without purification.

[0304] 5b: NHS activation of diboronated sensor: The crude diboronated sensor acid DSL-67A was dissolved in DMF (2 mL), treated with 3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA on a C18 column by reverse phase (RP) flash chromatography and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain 2,5-dioxopyrrolidin-1-yl (S-3-((2S,4R)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidine-2-carboxamido)-5-methylhexanoate DSL-67B in a yield of about 28% (18.7 mg). Mass calculated value (M+H) + = 675.2, mass found (M+H) + = 675.0.

Chemical formula

[0305] The MS data of DSL-67B are listed in Table IV. The other examples listed in Table IV below were synthesized under similar conditions.

Table 5

[0306] DSL synthesis method 6: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of 2-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)cyclopentyl)acetic acid 17 (109 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA and mixed for 1 hour to obtain 18. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (2S,4S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid, 19 (0.229 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 20. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 21. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0307] Cleavage of diboronated sensor from resin Compound 21 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered, and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2 × 5 mL) and evaporated to obtain diboronated sensor acid DSL-60A, which was used in the next step without purification.

[0308] The crude diboronated sensor acid DSL-60A was dissolved in DMF (2 mL), treated with 3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes on a C18 column by reverse-phase (RP) flash chromatography with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA, and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain 2,5-dioxopyrrolidin-1-yl 2-(1-((2S,4S)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidin-2-carboxamido)cyclopentyl)acetate DSL-60B in a yield of about 22% (14.7 mg). Mass calculated value (M+H) + = 673.2, mass found (M+H) + = 673.0.

[0309] The same procedure was followed for the synthesis of DSL-61B to DSL-77B and DSL-108B.

Chemical Structure

[0310] The MS data for DSL-60B are listed in Table V. The other examples listed in Table V below were synthesized under the same conditions.

Table 6

[0311] DSL synthesis method 7: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (94 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 6A solution of -((allyloxy)carbonyl)-L-lysine 22 (0.181 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol) and HATU (0.152 g, 0.4 mmol) was added to the resin, heated at 50 °C for 30 minutes, washed with DMF (4 × 5 mL) to obtain 23, then treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid, 14 (0.229 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol) and HATU (0.152 g, 0.4 mmol) in DMF (5 mL) was added to the resin, heated at 50 °C for 30 minutes, washed with DMF (4 × 5 mL) to obtain 24. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol) and DIPEA (0.35 mL, 2 mmol) was added to the resin, heated at 50 °C for 30 minutes to obtain 25. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL), treated with tetrakis(triphenylphosphine)palladium (20 mol%, 46 mg) and phenylsilane (493 μl, 4 mmol) in 6 mL of DCM, stirred at room temperature for 1 hour, and washed with DCM (3 × 5 mL) and DMF (3 × 5 mL). A solution of octanoic acid 26 (63 μl, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol) and HATU (0.152 g, 0.4 mmol) in DMF (5 mL) was added to the resin, heated at 50 °C for 30 minutes, washed with DMF (4 × 5 mL) to obtain 27.

[0312] Cleavage of diboronated sensor from resin Compound 27 on the resin was treated with 20% 1,1,1,3,3,3 - hexafluoro - 2 - propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2×5 mL) and evaporated to obtain diboronated sensor acid DSL - 68A, which was used in the next step without purification.

[0313] 7b: NHS activation of diboronated sensor: Crude diboronated sensor acid DSL - 68A was dissolved in DMF (2 mL) and treated with 3 - (3 - dimethylaminopropyl) - 1 - ethyl - carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol), N - hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 uL) and fractionated over 10 minutes on a C18 column by reverse - phase (RP) flash chromatography with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA and analyzed by LCMS. The pure fractions were isolated, combined, frozen and lyophilized to obtain 2,5 - dioxopyrrolidin - 1 - yl 3 - ((S - 2 - ((2R,4S) - 1 - (1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carbonyl) - 4 - (1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carboxamido)pyrrolidine - 2 - carboxamido) - 6 - octanamidehexanamide)propanoate DSL - 68B in about 30% yield (26.1 mg). Mass calculated value (M + H) + = 873.4, Mass found value (M + H) + = 873.2

Chemical Structure

[0314] DSL synthesis method 8: Synthesis of R-3,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid In a 100 mL round-bottom flask, (3R)-3-{[(tert-butoxy)carbonyl]amino}-4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid (1 g, 2.27 mmol) was treated with 1:1 TFA:DCM (15 mL) and stirred at room temperature for 1 hour. After completion of the reaction, the solvent was removed under reduced pressure and co-evaporated with DCM (15 mL × 3). The oily product was dissolved in 5:1 10% sodium carbonate and THF (50 mL), followed by the addition of Fmoc-OSu (0.76 g, 2.27 mmol) in THF (10 mL), and stirred for 15 hours. The reaction mixture was diluted with ethyl acetate (35 mL) and acidified to pH ~3 with 1N HCl. The organic layer was separated, the aqueous layer was extracted with ethyl acetate (2 × 15 mL), washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to give (R-3,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid (1.2 g, 95%).

[0315] Synthesis of 2,5-dioxopyrrolidin-1-yl (R-3-(3,4-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide)propanoate 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (94 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA, and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (R-3,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid 30 (0.224 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF (5 mL) was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 31. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 32. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0316] Cleavage of diboronated sensor from resin Compound 32 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered, and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2 × 5 mL) and evaporated to obtain diboronated sensor acid DSL-104A, which was used in the next step without purification.

[0317] 8b: NHS activation of diboronated sensor: The crude diboronated sensor acid DSL-104A was dissolved in DMF (2 mL), treated with 3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA on a C18 column by reverse-phase (RP) flash chromatography and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain DSL-104B 2,5-dioxopyrrolidin-1-yl (R-3-(3,4-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide)propanoate in a yield of about 22% (13 mg). Mass calculated value (M+H) + = 607.2, mass found (M+H) + = 607.1 [Chemical formula]

[0318] DSL synthesis method 9: Synthesis of 2,5-dioxopyrrolidin-1-yl (R-3-(4,5-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pentaamide)propanoate 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (94 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was added immediately and mixed gently overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution (1 M) in a solution of DCM and DIPEA, and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), and washed with DMF (4 × 5 mL). A solution of (R-4,5-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid 33 (0.224 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) in DMF (5 mL) was added to the resin, heated at 50 °C for 30 minutes, and washed with DMF (4 × 5 mL) to obtain 34. The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 35. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0319] Cleavage of diboronated sensor from resin The compound 35 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM (7 mL) and mixed gently for 1 hour. The resin was filtered, and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2 × 5 mL) and evaporated to obtain diboronated sensor acid DSL-106A, which was used in the next step without purification.

[0320] 9b: NHS activation of diboronated sensor: The crude diboronated sensor acid DSL-106A was dissolved in DMF (2 mL), treated with 3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA on a C18 column by reverse-phase (RP) flash chromatography and analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain DSL-106B 2,5-dioxopyrrolidin-1-yl (R-3-(4,5-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pentanamide)propanoate in a yield of approximately 30% (18.6 mg). Mass calculated value (M+H) + = 621.2, mass found (M+H) + = 621.1 [Chemical Structure]

[0321] DSL synthesis method 10: 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swollen with dry DCM (5 mL) for 30 minutes. The solvent was removed with nitrogen, and a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid 2 (0.93 mg, 0.3 mmol) in DCM containing DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and the unreacted sites were capped with a 20% MeOH solution in a solution of DCM and DIEA (1 M) and mixed for 1 hour to obtain 3. The resin was washed with DCM (3 × 5 mL), then DMF (3 × 5 mL). The resin was treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL) and washed with DMF (4 × 5 mL). A solution of (2S,3S)-(Fmoc-amino)-3-azidobutyric acid 4 (0.146 g, 0.4 mmol), DIPEA (0.14 mL, 0.8 mmol), and HATU (0.152 g, 0.4 mmol) was added to the resin and heated at 50 °C for 30 minutes to obtain 5. The resin was washed with DMF (4 × 5 mL) and THF (3 × 5 mL), treated with 5 mL of a THF solution of 0.2 M SnCl2 (189 mg), 0.8 M thiophenol (440 μl), and 1 M DIEA (870 μl), and gently mixed on a shaker for 1 hour. After 1 hour, the resin was washed with THF (3 × 5 mL) and DMF (3 × 5 mL) to obtain 36. A solution of 1-hydroxy-3H-2,1-benzoxaborole-6-carboxylic acid 6 (0.178 g, 1 mmol), HATU (0.380 g, 1 mmol), and DIPEA (0.35 mL, 2 mmol) was added to the resin (6a) and heated at 50 °C for 30 minutes to obtain 37.

[0322] The resin was washed with DMF (5 × 5 mL), treated with 20% piperidine in DMF for 5 minutes (3 × 5 mL), washed with DMF (4 × 5 mL), and subsequently coupled with 38 to obtain 39. The resin was washed with DMF (4 × 5 mL) and DCM (3 × 5 mL).

[0323] Cleavage of diboronated sensor from resin Compound 39 on the resin was treated with 20% 1,1,1,3,3,3 - hexafluoro - 2 - propanol (HFIP) in DCM (7 mL) and gently mixed for 1 hour. The resin was filtered and the filtrate was evaporated under reduced pressure. The residue was further suspended in DCM (2×5 mL) and evaporated to obtain diboronated sensor acid DSL - 109A, which was used in the next step without purification.

[0324] 10b: NHS activation of diboronated sensor: Diboronated sensor acid DSL - 109A was dissolved in DMF (2 mL) and treated with 3 - (3 - dimethylaminopropyl) - 1 - ethyl - carbodiimide hydrochloride 9 (EDC) (0.096 g, 0.5 mmol), N - hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), and then gently mixed overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 100 mmol HCl (10 mL). The organic layer was dried over anhydrous Na2SO4 and dried under vacuum, dissolved in DMSO (100 uL), and fractionated over 10 minutes on a C18 column by reverse - phase (RP) flash chromatography with a gradient of 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA to obtain the desired product analyzed by LCMS. The pure fractions were isolated, combined, frozen, and lyophilized to obtain 2,5 - dioxopyrrolidin - 1 - yl 3 - ((2S,3S) - 2 - (4 - fluoro - 1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carboxamido) - 3 - (1 - hydroxy - 1,3 - dihydrobenzo[c][1,2]oxaborole - 6 - carboxamido)butanamide)propanoate DSL - 109B in a yield of 20% (12.5 mg). Mass calculated value (M + H) + = 625.1, mass found (M + H) + = 625.1

Chemical Structure

[0325] List the MS data of DSL-109B in Table VI. The other examples listed in Table VI below were synthesized under similar conditions. [Table 7]

[0326] Summarize the chemical structures and IUPAC names of DSL-1A to DSL-112A in Table 1 below.

[0327] [Table 8-1]

[0328] [Table 8-2]

[0329] [Table 8-3]

[0330] [Table 8-4]

[0331] [Table 8-5]

[0332] [Table 8-6]

[0333] [Table 8-7]

[0334] [Table 8-8]

[0335]

Table 8-9

[0336]

Table 8-10

[0337]

Table 8-11

[0338]

Table 8-12

[0339]

Table 8-13

[0340]

Table 8-14

[0341]

Table 8-15

[0342]

Table 8-16

[0343]

Table 8-17

[0344]

Table 8-18

[0345]

Table 8-19

[0346]

Table 8-20

[0347]

Table 8-21

[0348]

Table 8-22

[0349]

Table 8-23

[0350]

Table 8-24

[0351]

Table 8-25

[0352]

Table 8-26

[0353]

Table 8-27

[0354]

Table 8-28

[0355]

Table 8-29

[0356]

Table 8-30

[0357]

Table 8-31

[0358]

Table 8-32

[0359]

Table 8-33

[0360]

Table 8-34

[0361]

Table 8-35

[0362]

Table 8-36

[0363]

Table 8-37

[0364] Synthesis of the compound of formula I Synthesis of the compound of formula I Exemplary synthesis protocols that can be used to synthesize the described examples are provided.

[0365] The lines connecting the cysteine residues are disulfide bonds. For clarity, the H at the N-terminus of the A and B chains of insulin is the hydrogen at the N-terminus, not histidine. The -OH shown at the C-terminus of the A and B chains is the C-terminus of the corresponding chain.

[0366] Insulin expression and conjugation method 1 1a. Expression of proinsulin Proinsulin can be expressed in E. coli strains such as B21 using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of the B chain, C peptide, and A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via an existing his-tag, for example, prior to sensor conjugation.

[0367] The expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, U.S. Pat. Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0368] 1b. Conjugation of proinsulin with diboronate sensor 11.

[0369] To a solution of single-chain proinsulin 12 (20 mg) in DMSO (200 uL), 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide) propanoate (11, DSL-66B, 4.4 mg) and diisopropylethylamine (DIPEA, 20 uL) in DMSO (50 uL) were added. The reaction mixture was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 uL) was added to the reaction mixture to precipitate proinsulin conjugate intermediate 13. The reaction mixture was centrifuged (11000 RPM, 5 minutes), decanted, and the supernatant was resuspended in a 100 μL solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl and 30% ACN. The solution was diluted with 500 uL of water, then 30 uL of 1M Tris pH 9 (50 mM final concentration) and 6 uL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5M NaOH (5 - 10 uL), and trypsin (1:500 - 1:2000 trypsin mass to insulin mass, about 10 uL of a 0.2 ug / mL stock solution) was added. The crude solution was stirred at room temperature for 16 hours. The crude conjugate insulin conjugate was precipitated with TFA (about 200 uL), centrifuged, and decanted. The crude precipitate was then washed with water (2 x 500 uL), then dissolved in DMSO (100 - 200 uL), diluted with 20% ACN / water (5 - 10 mL), and purified by reverse-phase HPLC. Fractions were collected, frozen, and lyophilized to obtain a white powder (5 - 10 mg) of 14 (Example 19).

[0370] Scheme 1. Conjugation of diborononate sensor (11) with proinsulin (12) (SEQ ID NOs: 25706 - 25707 and 25434 - 25435 (in order of appearance, respectively)).

Chemical formula

Chemical formula

[0371] The MS data of Example 19 (Compound 14) are listed in Table A. The other examples listed in Table A below were synthesized under similar conditions. [Table 9]

[0372] Insulin expression and conjugation method 2 2a. Expression of proinsulin Proinsulin can be expressed in E. coli strains such as B21 using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of a B chain, a C peptide, and an A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via an existing his-tag, for example, prior to sensor conjugation.

[0373] Expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0374] 2b. Conjugation of proinsulin with diboronate sensor DSL-51.

[0375] To a solution of single-chain proinsulin 15 (20 mg) in DMSO (200 uL), (S)-2-((2S,3S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamide)butanamide)-6-(2,5-dioxopyrrolidin-1-yl)-5-oxohexanoic acid (DSL-51B, 6.2 mg) in DMSO (50 uL) and diisopropylethylamine (DIPEA, 20 uL) was added. The reaction was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 uL) was added to the reaction mixture to precipitate proinsulin conjugate intermediate 15i. The reaction mixture was centrifuged (11000 RPM, 5 minutes), decanted, and the supernatant was resuspended in a 100 μL solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl and 30% ACN. The solution was diluted with 500 uL of water, then 30 uL of 1M Tris pH9 (50 mM final concentration), 6 uL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5M NaOH (5 - 10 uL), and trypsin (1:500 - 1:2000 trypsin mass to insulin mass, about 10 uL of a 0.2 ug / mL stock solution) and carboxypeptidase (CBP, 1:1000 carboxypeptidase to insulin mass) were added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugate insulin was precipitated with TFA (about 200 uL), the solution was centrifuged, decanted, the precipitate was dissolved in DMSO (100 - 200 uL), diluted with 20% ACN / water (5 - 10 mL), and purified by reverse-phase HPLC. The fractions were collected, frozen, and lyophilized to obtain the white powder (6 mg) of Example 11.

[0376] Alternatively, proinsulin 15 was treated with tert-butyl (S)-2-((2S,3S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide)-6-(2,5-dioxopyrrolidin-1-yl)-5-oxohexanoate (DSL-51-OtBu) under the same conditions as above. After digestion and precipitation, the crude conjugate was dissolved in TFA: H2O (95:5), stirred for 1 hour, concentrated under reduced pressure, dissolved in DMSO (100-200 uL), diluted with 20% ACN / water (5-10 mL), and purified by reverse-phase HPLC. The fractions were collected, frozen, and lyophilized to obtain Example 11.

[0377] Conjugation of Scheme 2. DSL-51B with proinsulin 15 (SEQ ID NOs: 25708-25709 and 25418-25419, in the order of appearance).

Chemical formula

Chemical formula

[0378] The MS data of Example 2 are listed in Table B. The other examples listed in Table B below were synthesized under the same conditions.

Table 10

[0379] Insulin Expression and Conjugation Method 3 3a. Expression of proinsulin Proinsulin can be expressed in E. coli strains such as B21 using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of the B chain, C peptide, and A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via existing his tags, for example, prior to sensor conjugation.

[0380] The expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, U.S. Pat. Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0381] 3b. Conjugation of the diboronated sensor DSL-58B with proinsulin

[0382] To a solution of single-chain proinsulin 16 (20 mg) in DMSO (200 μL) was added 2,5-dioxopyrrolidin-1-yl (S)-3-((2S,4R)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidine-2-carboxamido)-4-(4-methoxyphenyl)butanoate (DSL-58B, 5.3 mg) in DMSO (50 μL) and diisopropylethylamine (DIPEA, 20 μL). The reaction was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 μL) was added to the reaction mixture to precipitate the proinsulin conjugate intermediate 16i. The reaction mixture was centrifuged (11,000 RPM, 5 minutes), decanted, and the supernatant was resuspended in a 100 μL solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl, and 30% ACN. The solution was diluted with 500 μL of water, then 30 μL of 1 M Tris pH 9 (50 mM final concentration) and 6 μL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5 M NaOH (5 - 10 μL), and trypsin (1:500 - 1:2000 trypsin mass to insulin mass, approximately 10 μL of a 0.2 μg / mL stock solution) was added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugate insulin was precipitated with TFA (about 200 μL), the solution was centrifuged, decanted, the precipitate was dissolved in DMSO (100 - 200 μL), then diluted with 20% ACN / water (5 - 10 mL) and purified by reverse-phase HPLC. The fractions were collected, frozen, and lyophilized to obtain Example 141 as a white powder (8.2 mg).

[0383] Conjugation of Scheme 3.DSL-58B with proinsulin 16 (SEQ ID NOs: 25710 - 25711 and 25674 - 25675, in the order of appearance). [Chem.] [Chem.]

[0384] List the MS data of Example 141 in Table C. The other examples listed in Table C below were synthesized under similar conditions. [Table 11]

[0385] Insulin Expression and Conjugation Method 4 4a. Expression of proinsulin Proinsulin can be expressed in E. coli strains such as B21 using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of the B chain, C peptide, and A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via an existing his-tag, for example, before sensor conjugation.

[0386] Expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, U.S. Patent Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0387] 4b. Conjugation of the diboronated sensor DSL-66 with proinsulin 18 To a solution of single-chain proinsulin 18 (20 mg) in DMSO (200 μL), 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide) propanoate (DSL-66B, 6.2 mg) in DMSO (50 μL) and diisopropylethylamine (DIPEA, 20 μL) was added. The reaction mixture was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 uL) was added to the reaction mixture to precipitate the proinsulin conjugate intermediate 18i. The reaction mixture was centrifuged (11000 RPM, 5 minutes), decanted, and the supernatant was resuspended in 100 μL of a solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl and 30% ACN. The solution was diluted with 500 uL of water, then 30 uL of 1M Tris pH 9 (50 mM final concentration) and 6 uL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5M NaOH (5 - 10 uL), and trypsin (1:500 - 1:2000 trypsin mass to insulin mass, about 10 uL of a 0.2 μg / mL stock solution) was added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugate insulin was precipitated with TFA (about 200 uL), the solution was centrifuged, decanted, the precipitate was dissolved in DMSO (100 - 200 uL), then diluted with 20% ACN / water (5 - 10 mL) and purified by reverse-phase HPLC. The fractions were collected, frozen, and lyophilized to obtain Example 69 as a white powder (7 mg).

[0388] Scheme 4. Conjugation of DSL-66B with proinsulin 18 (SEQ ID NOs: 25712-25713 and 25530-25531, in the order of appearance). [Chemical Formula] [Chemical Formula]

[0389] The MS data of Example 69 are listed in Table D. The other examples listed in Table D below were synthesized under similar conditions. [Table 12]

[0390] Insulin Expression and Conjugation Method 5 5a. Expression of proinsulin Proinsulin can be expressed in Escherichia coli strains such as the B21 strain using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of the B chain, C peptide, and A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via an existing his-tag, for example, prior to sensor conjugation.

[0391] Expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, U.S. Pat. Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0392] 5b. Conjugation of the diboronated sensor DSL-58B with proinsulin 17 To a solution of single-chain proinsulin 17 (20 mg) in DMSO (200 μL), 2,5-dioxopyrrolidin-1-yl (S)-3-((2S,4R)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidine-2-carboxamido)-4-(4-methoxyphenyl)butanoate (DSL-58B, 5.8 mg) in DMSO (50 μL) and diisopropylethylamine (DIPEA, 20 μL) was added. The reaction mixture was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 μL) was added to the reaction mixture to precipitate proinsulin conjugate 17i. The reaction mixture was centrifuged (11000 RPM, 5 minutes), decanted, and the supernatant was resuspended in 100 μL of a solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl and 30% ACN. The solution was diluted with 500 μL of water, then 30 μL of 1M Tris pH 9 (50 mM final concentration) and 6 μL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5M NaOH (5 - 10 μL), and trypsin (1:500 - 1:2000 trypsin mass to insulin mass, about 10 μL of a 0.2 μg / mL stock solution) was added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugate insulin was precipitated with TFA (about 200 μL), the solution was centrifuged, decanted, the precipitate was dissolved in DMSO (100 - 200 μL), then diluted with 20% ACN / water (5 - 10 mL) and purified by reverse-phase HPLC. The fractions were collected, frozen, and lyophilized to obtain Example 153 as a white powder (11 mg).

[0393] Scheme 5. Conjugation of DSL-58 with proinsulin 17 (SEQ ID NOs: 25714-25715 and 25698-25699, in the order of appearance).

Chemical Structure

Chemical Structure

[0394] The MS data of Example 153 are listed in Table E. The other examples listed in Table E below were synthesized under similar conditions. [Table 13]

[0395] Insulin Expression and Conjugation Method 6 6a. Expression of proinsulin Proinsulin can be expressed in Escherichia coli strains such as B21 using standard IPTG induction of IPTG-inducible expression constructs and vectors. Briefly, the expression construct consists of the B chain, C peptide, and A chain. For example, the c-peptide sequence of EAEDLQVGQVELGGGPGAGSLQPLALEGSLQR (SEQ ID NO: 25730) can be used for the expression of proinsulin. Proinsulin is expressed in inclusion bodies (IBs), and the IBs are captured, washed, and further purified via an existing his-tag, for example, prior to sensor conjugation.

[0396] The expression of the desired single-chain proinsulin can be carried out via procedures known in the art. See, for example, U.S. Pat. Nos. 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[0397] 6b. Conjugation of the diboronated sensor DSL-69B with proinsulin 19 To a solution of single-chain proinsulin 19 (20 mg) in DMSO (200 μL), 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamide) propanoate (DSL-69B, 5 mg) in DMSO (50 μL) and diisopropylethylamine (DIPEA, 20 μL) was added. The reaction mixture was stirred at room temperature for 1 hour. Trifluoroacetic acid (TFA, 40 uL) was added to the reaction mixture to precipitate proinsulin conjugate intermediate 19i. The reaction mixture was centrifuged (11000 RPM, 5 minutes), decanted, and the supernatant was resuspended in a 100 μL solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl and 30% ACN. The solution was diluted with 500 uL of water, then 30 uL of 1M Tris pH9 (50 mM final concentration) and 6 uL of 500 mM CaCl2 (5 mM final concentration) were added. The pH was adjusted to about 9 - 9.5 with 0.5M NaOH (5 - 10 uL), then trypsin (1:500 - 1:2000 trypsin m...

Claims

1. A compound represented by formula I, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, wherein 【Chemical 1】 in the formula,[[]]END] X1 is i. A drug substance containing an amine, ii. An amine configured to covalently bond to the drug substance, or iii. NH ２ or contains OH, each Z1a independently contains 1 to 50 amino acids joined together using an amide bond or a peptide bond, each Z1b is independently a small molecule linker, Each Z1c is independently selected from the formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, and each Z1c is covalently bonded to the amine of Z1a, or the amine of X1, or to NH when X1 is NH ２ in the case of ２ or to OH when X1 is OH, each m' is 0 or 1, each n' is 0; each o' is 1; each p' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each q' is 1, 2, 3, 4, or 5, and when p' or q' is 2 or more, each corresponding Z1c is independently selected and may be the same or different, and when q' is 2 or more, each corresponding Z1a is independently selected and may be the same or different, When m' = 0 and n' = 0, each Z1c is covalently bonded to the amine of X1, or to NH when X1 is NH ２ or to NH when X1 is ２ or, when X1 is OH, to OH directly or via a linker. When m' = 1, each Z1a is covalently bonded to X1 via an amide bond or a peptide bond, and each Z1c is covalently bonded to the amine of Z1a or to X1 directly or via a linker and formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226 are as follows, 【Chemical Formula 2】 X is an amine of X1, or NH when X1 is NH ２ or OH when X1 is OH, or represents a direct covalent bond with either an amine directly or indirectly covalently bonded to X1, and B ２ and B １ and B ２ may be the same or different and each independently represents an aromatic boron-containing group A compound, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, wherein one or more positions of the compound of formula I may contain isotopes.

2. At least one Z1c is covalently bonded via an indirect linker to the amine of X1, or to NH if X1 is NH ２ or to NH if X1 is NH ２ or to OH if X1 is OH, or to the amine of Z1a wherein said indirect linker is of formula (X") ｎ１ as represented by, in which each n1 is independently selected from 1, 2, 3, 4, and 5, each X" is (i) an L or D-amino acid, wherein the amine functional group of the L or D-amino acid is directly or indirectly covalently bonded to Z1c, and the acid functional group of the L or D-amino acid is directly or indirectly conjugated to X1 or Z1a; and (ii) independently selected from formulas FL(IA), FL(IB), FL69, and FL70, Formulas FL(IA) and FL(IB) are [Chemical Formula 3] and their stereoisomers, in the formula,[[]]END] G is selected from a 3- to 6-membered cycloalkyl group, a 3- to 10-membered heterocyclyl group, a heteroaryl group, and an aryl group, where each group is hydroxy, amino, halogen, (C ３ -C ６ ) cycloalkyl, alkoxy, and C １ -C ６ alkyl, and may be substituted with 1 to 3 groups independently selected therefrom, E is an alkylene group which may be absent or substituted with 1 to 3 groups independently selected from halogen, hydroxy, and amino, Q is absent or is hydrogen, C １ ~C ６ alkyl, halo, cyano, alkoxy, carboxylic acid, amino, hydroxy, amide, C １ ~C ６ haloalkyl, (C ３ ~C ６ ) cycloalkyl, heterocycle, heteroaryl, and aryl, wherein the alkyl, alkoxy, cycloalkyl, heterocycle, heteroaryl, and aryl are each independently substituted with 1 to 5 groups selected from C １ ~C ６ alkyl, amino, amide, halo, hydroxy, cyano, C １ ~C ６ haloalkyl, and alkoxy, and may be substituted. Q' is hydrogen, C １ ~C ６ selected from alkyl and acyl groups, Q and Q' may together with the carbon and nitrogen atoms to which they are attached form a 4-membered heterocyclyl, 5-membered heterocyclyl, 6-membered heterocyclyl, 9-membered bicyclic heterocyclyl, or 10-membered bicyclic heterocyclyl, wherein the 4-membered heterocyclyl, 5-membered heterocyclyl, 6-membered heterocyclyl, 9-membered bicyclic heterocyclyl, and 10-membered bicyclic heterocyclyl are each independently substituted with 1 to 5 groups selected from C １ -C ６ alkyl, amino, halo, hydroxy, cyano, amido, C １ -C ６ haloalkyl, and alkoxy. p is 0, 1, 2, 3, 4, or 5, q is 0, 1, 2, 3, 4, or 5, R'' represents a direct or indirect covalent bond to Z1c, Z'' represents a direct or indirect covalent bond to X1 or Z1a, any primary amine is optionally acetylated or alkylated, The formulas FL69 and FL70 are, 【Chemical 4】 and their stereoisomers, wherein, R’’ represents a direct or indirect covalent bond to Z1c, Z’’ represents a direct or indirect covalent bond to X1 or Z1a, A' is H, C １ ~C ６ alkyl, saturated fatty acid, unsaturated fatty acid, (C ３ ~C ６ ), cycloalkyl, haloalkyl, aryl, and heteroaryl, and is selected from A'' is C １ to C ６ alkyl, substituted acyl, acyl terminated with an acid, saturated or unsaturated fatty acid, (C ３ to C ６ ) cycloalkyl, haloalkyl, aryl, and heteroaryl, selected from p is 1, 2, 3, 4, or 5, q is 1, 2, 3, 4, or 5, The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein any primary amine is optionally acetylated or alkylated.

3. The formulas FL(IA) and FL(IB) are the formulas FL3, FL5, FL5A, FL5B, FL20 - FL68 and FL71 - FL75: 【Chemical Formula 5】 【Chemical Formula 6】 and are selected from their stereoisomers, wherein, p is 1, 2, 3, 4, or 5, q is 1, 2, 3, 4, or 5, The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein any primary amine is optionally acetylated or alkylated.

4. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein n1 is 1.

5. The compound is represented by formula IC, 【Chemical Formula 7】 each p’ is 1, 2, 3, or 4, The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein each q’ is 1, 2, 3, 4, or 5.

6. Each B １ and B ２ is independently selected from formulas F2, F5, and F10, The formulas F2, F5, and F10 are, 【Chemical Formula 8】 and, wherein, One R １ is (C=O)--- ＊ or (CH ２ ) ｍ (C=O)--- ＊ represents,--- ＊ represents the bonding point to the rest of Z1c, m is 1, 2, 3, 4, 5, 6, or 7, Each remaining R １ is independently H, F, Cl, Br, I, OH, CH ２ -NH ２ , NH ２ , (C=O)-NH ２ , CH=O, SO ２ CH ３ , SO ２ CF ３ , CF ３ , CHF ２ , NO ２ , CH ３ , OCH ３ , O(CH ２ ) ｍ” CH ３ , (SO ２ )NH-CH ３ , (SO ２ )NH(CH ２ ) ｍ” CH ３ , and OCF ３ is selected from, and m" is 1, 2, 3, 4, 5, 6, or 7, Y8 is O, N, or NR, where R is C １ ~C ６ an alkyl group or H, Y9 is H, CH ３ , or C １ ~C ６ alkyl group, provided that when Y8 is O, the said Y9 is CH ３ or C １ ~C ６ alkyl group, and i is 1, 2, or 3, The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. (a) The remaining Rs １ are each independently (i) selected from H, CF ３ , and F, or (b) two of the remaining Rs １ are H and one of the remaining Rs １ is CF ３ or F, the compound according to claim 6, or a pharmaceutically acceptable salt thereof.

8. Each Z1c is covalently bonded to an indirect linker, and each of the Z1c and the indirect linker is combined to form FFL - 1 to FFL - 101; 【Chemical Formula 9】 【Chemical 10】 【Chemical 11】 【Chemical 12】 and their stereoisomers, In the formula, X is an amine of X1, or when X1 is NH ２ it is NH ２ , or when X1 is OH it is OH, or a direct covalent bond point with either an amine directly or indirectly covalently bonded to X1, or an amine of Z1a, the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. B １ and B ２ are each independently of the formula F2, and the remaining R １ are each independently H, CF ３ , and F; wherein each Z1c is covalently bonded to an indirect linker, and each of said Z1c and said indirect linker is combined to form the formulas FFL-1 to 4, 6 to 7, 9, 13 to 16, 20 to 23, 28 to 29, 31 to 33, 39, 42 to 45, 48, 53, 57 to 58, 60 to 62, 65, and 67: 【Chemical 13】 【Chemical 14】 and are selected from their stereoisomers, Wherein X is an amine of X1, or NH when X1 is NH ２ or OH when X1 is OH, or a direct covalent bond point with either an amine directly or indirectly covalently bonded to X1, or an amine of Z1a, the compound according to claim 6 or 7 or a pharmaceutically acceptable salt thereof. ２ ​

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, wherein X1 is a polypeptide prodrug containing one or more lysine amino acids conjugated independently directly or via an indirect linker to Z1c.

11. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein X1 comprises a polypeptide human hormone, an insulin receptor agonist, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide, a hybrid peptide comprising sequences derived from two or more human polypeptide hormones, or an analog thereof.

12. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein X1 comprises insulin having an A chain and a B chain, the A chain comprises a sequence selected from SEQ ID NO: 1, 25, 24051, and 24052, and the B chain comprises a sequence selected from SEQ ID NO: 24060, 24061, 24062, 24063, 24064, and 25000 to 25397.

13. X1 comprises insulin having an A chain and a B chain, the A chain comprises a sequence selected from SEQ ID NO: 24051 and 24052, the B chain comprises a sequence selected from SEQ ID NO: 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380 to 25397, each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116A, FF116B, FF116C, and FF116D and is covalently bonded directly or via a linker to one or more lysine residues in X1, B １ and B ２ each independently has the formula F2, the linker is independently selected from FL3, FL5, FL5A, FL5B, FL20 to FL75, and stereoisomers thereof, and the compound or a pharmaceutically acceptable salt thereof according to claim 12.

14. The compound is Example 1 (SEQ ID NO: 25398 and 25399, in the order of appearance): 【Chemical Formula 15】 Example 2 (SEQ ID NO: 25400 and 25401, in the order of appearance): 【Chemical 16】 Example 3 (SEQ ID NO: 25402 and 25403, in the order of appearance): 【Chemical 17】 Example 4 (SEQ ID NO: 25404 and 25405, in the order of appearance): 【Chemical Formula 18】 Example 5 (SEQ ID NO: 25406 and 25407, in the order of appearance): 【Chemical 19】 Example 6 (SEQ ID NO: 25408 and 25409, in the order of appearance): 【Chemical 20】 Example 7 (SEQ ID NO: 25410 and 25411, in the order of appearance): 【Chemical 21】 Example 8 (SEQ ID NO: 25412 and 25413, in the order of appearance): 【Chemical 22】 Example 9 (SEQ ID NOs: 25414 and 25415, in the order of appearance): 【Chemical 23】 Example 11 (SEQ ID NOs: 25418 and 25419, in the order of appearance): 【Chemical Formula 24】 Example 12 (SEQ ID NOs: 25420 and 25421, in the order of appearance): 【Chemical 25】 Example 13 (SEQ ID NOs: 25422 and 25423, in the order of appearance): 【Chemical 26】 Example 14 (SEQ ID NOs: 25424 and 25425, in the order of appearance): 【Chemical 27】 Example 15 (SEQ ID NOs: 25426 and 25427, in the order of appearance): 【Chemical formula 28】 Example 16 (SEQ ID NOs: 25428 and 25429, in the order of appearance): 【Chemical 29】 Example 17 (SEQ ID NOs: 25430 and 25431, in the order of appearance): 【Chemical 30】 Example 18 (SEQ ID NOs: 25432 and 25433, in the order of appearance): 【Chemical 31】 Example 19 (SEQ ID NOs: 25434 and 25435, in the order of appearance): 【Chemical 32】 Example 20 (SEQ ID NOs: 25436 and 25437, in the order of appearance): 【Chemical 33】 Example 21 (SEQ ID NOs: 25438 and 25439, in the order of appearance): 【Chemical 34】 Example 22 (SEQ ID NOs: 25422 and 25423, in the order of appearance): 【Chemical 35】 Example 23 (SEQ ID NOs: 25440 and 25441, in the order of appearance): 【Chemical 36】 Example 24 (SEQ ID NOs: 25442 and 25443, in the order of appearance): 【Chemical 37】 Example 26 (SEQ ID NOs: 25446 and 25447, in the order of appearance): 【Chemical 38】 Example 27 (SEQ ID NOs: 25448 and 25449, in the order of appearance): 【Chemical Formula 39】 Example 28 (SEQ ID NOs: 25450 and 25451, in the order of appearance): 【Chemical Formula 40】 Example 29 (SEQ ID NOs: 25452 and 25453, in the order of appearance): 【Chemical 41】 Example 30 (SEQ ID NOs: 25454 and 25455, in the order of appearance): 【Chemical 42】 Example 31 (SEQ ID NOs: 25456 and 25457, in the order of appearance): 【Chemical 43】 Example 32 (SEQ ID NOs: 25458 and 25459, in the order of appearance): 【Chemical 44】 Example 33 (SEQ ID NOs: 25460 and 25461, in the order of appearance): 【Chemical 45】 Example 34 (SEQ ID NOs: 25462 and 25463, in the order of appearance): 【Chemical 46】 Example 35 (SEQ ID NOs: 25464 and 25465, in the order of appearance): 【Chemical 47】 Example 36 (SEQ ID NOs: 25466 and 25467, in the order of appearance): 【Chemical 48】 Example 37 (SEQ ID NOs: 25468 and 25469, in the order of appearance): 【Chemical 49】 Example 38 (SEQ ID NOs: 25470 and 25471, in the order of appearance): 【Chemical Formula 50】 Example 39 (SEQ ID NOs: 25472 and 25473, in the order of appearance): 【Chemical 51】 Example 40 (SEQ ID NOs: 25474 and 25475, in the order of appearance): 【Chemical 52】 Example 41 (SEQ ID NOs: 25476 and 25477, in the order of appearance): 【Chemical 53】 Example 42 (SEQ ID NOs: 25478 and 25479, in the order of appearance respectively): 【Chemical 54】 Example 43 (SEQ ID NOs: 25480 and 25481, in the order of appearance respectively): 【Chemical Formula 55】 Example 44 (SEQ ID NOs: 25482 and 25483, in the order of appearance respectively): 【Chemical 56】 Example 45 (SEQ ID NOs: 25484 and 25485, in the order of appearance respectively): 【Chemical 57】 Example 46 (SEQ ID NOs: 25486 and 25487, in the order of appearance respectively): 【Chemical Formula 58】 Example 47 (SEQ ID NOs: 25488 and 25489, in the order of appearance respectively): 【Chemical Formula 59】 Example 48 (SEQ ID NOs: 25490 and 25491, in the order of appearance respectively): 【Chemical Formula 60】 Example 49 (SEQ ID NOs: 25492 and 25493, in the order of appearance respectively): 【Chemical Formula 61】 Example 50 (SEQ ID NOs: 25494 and 25495, in the order of appearance respectively): 【Chemical Formula 62】 Example 51 (SEQ ID NOs: 25496 and 25497, in the order of appearance respectively): 【Chemical Formula 63】 Example 52 (SEQ ID NOs: 25498 and 25499, in the order of appearance respectively): 【Chemical Formula 64】 Example 53 (SEQ ID NOs: 25500 and 25501, in the order of appearance respectively): 【Chemical 65】 Example 54 (SEQ ID NOs: 25502 and 25503, in the order of appearance respectively): 【Chemical Formula 66】 Example 55 (SEQ ID NOs: 25504 and 25505, in the order of appearance respectively): 【Chemical 67】 Example 56 (SEQ ID NOs: 25506 and 25507, in the order of appearance respectively): 【Chemical Formula 68】 Example 57 (SEQ ID NOs: 25508 and 25509, in the order of appearance respectively): 【Chemical Formula 69】 Example 58 (SEQ ID NOs: 25510 and 25511, in the order of appearance respectively): 【Chemical 70】 Example 59 (SEQ ID NOs: 25512 and 25513, in the order of appearance respectively): 【Chemical Formula 71】 Example 60 (SEQ ID NOs: 25514 and 25515, in the order of appearance respectively): 【Chemical 72】 Example 61 (SEQ ID NOs: 25480 and 25481, in the order of appearance respectively): 【Chemical 73】 Example 62 (SEQ ID NOs: 25516 and 25517, in the order of appearance respectively): 【Chemical 74】 Example 63 (SEQ ID NOs: 25518 and 25519, in the order of appearance respectively): 【Chemical Formula 75】 Example 64 (SEQ ID NOs: 25520 and 25521, in the order of appearance respectively): 【Chemical 76】 Example 65 (SEQ ID NOs: 25522 and 25523, in the order of appearance respectively): 【Chemical 77】 Example 66 (SEQ ID NOs: 25524 and 25525, in the order of appearance respectively): 【Chemical 78】 Example 67 (SEQ ID NOs: 25526 and 25527, in the order of appearance respectively): 【Chemical 79】 Example 68 (SEQ ID NOs: 25528 and 25529, in the order of appearance respectively): 【Chemical Formula 80】 Example 69 (SEQ ID NOs: 25530 and 25531, in the order of appearance respectively): 【Chemical 81】 Example 70 (SEQ ID NOs: 25532 and 25533, in the order of appearance respectively): 【Chemical 82】 Example 71 (SEQ ID NOs: 25534 and 25535, in the order of appearance respectively): 【Chemical 83】 Example 72 (SEQ ID NOs: 25536 and 25537, in the order of appearance respectively): 【Chemical 84】 Example 73 (SEQ ID NOs: 25538 and 25539, in the order of appearance): 【Chemical 85】 Example 74 (SEQ ID NOs: 25540 and 25541, in the order of appearance): 【Chemical 86】 Example 75 (SEQ ID NOs: 25542 and 25543, in the order of appearance): 【Chemical 87】 Example 76 (SEQ ID NOs: 25544 and 25545, in the order of appearance): 【Chemical 88】 Example 77 (SEQ ID NOs: 25546 and 25547, in the order of appearance): 【Chemical 89】 Example 78 (SEQ ID NOs: 25548 and 25549, in the order of appearance): 【Chemical Formula 90】 Example 79 (SEQ ID NOs: 25550 and 25551, in the order of appearance): 【Chemical Formula 91】 Example 80 (SEQ ID NOs: 25552 and 25553, in the order of appearance): 【Chemical Formula 92】 Example 81 (SEQ ID NOs: 25554 and 25555, in the order of appearance): 【Chemical Formula 93】 Example 82 (SEQ ID NOs: 25556 and 25557, in the order of appearance): 【Chemical Formula 94】 Example 83 (SEQ ID NOs: 25558 and 25559, in the order of appearance): 【Chemical Formula 95】 Example 84 (SEQ ID NOs: 25560 and 25561, in the order of appearance): 【Chemical Formula 96】 Example 85 (SEQ ID NOs: 25562 and 25563, in the order of appearance): 【Chemical Formula 97】 Example 86 (SEQ ID NOs: 25564 and 25565, in the order of appearance): 【Chemical Formula 98】 Example 87 (SEQ ID NOs: 25566 and 25567, in the order of appearance): 【Chemical Formula 99】 Example 88 (SEQ ID NOs: 25568 and 25569, in the order of appearance): 【Chemical 100】 Example 89 (SEQ ID NOs: 25570 and 25571, in the order of appearance): 【Chemical 101】 Example 90 (SEQ ID NOs: 25572 and 25573, in the order of appearance): 【Chemical 102】 Example 91 (SEQ ID NOs: 25574 and 25575, in the order of appearance): 【Chemical 103】 Example 92 (SEQ ID NOs: 25576 and 25577, in the order of appearance): 【Chemical 104】 Example 93 (SEQ ID NOs: 25578 and 25579, in the order of appearance): 【Chemical 105】 Example 94 (SEQ ID NOs: 25580 and 25581, in the order of appearance): 【Chemical 106】 Example 95 (SEQ ID NOs: 25582 and 25583, in the order of appearance): 【Chemical 107】 Example 96 (SEQ ID NOs: 25584 and 25585, in the order of appearance): 【Chemical 108】 Example 97 (SEQ ID NOs: 25586 and 25587, in the order of appearance): 【Chemical 109】 Example 98 (SEQ ID NOs: 25588 and 25589, in the order of appearance): 【Chemical 110】 Example 99 (SEQ ID NOs: 25590 and 25591, in the order of appearance): 【Chemical 111】 Example 100 (SEQ ID NOs: 25592 and 25593, in the order of appearance): 【Chemical 112】 Example 101 (SEQ ID NOs: 25594 and 25595, in the order of appearance): 【Chemical 113】 Example 102 (SEQ ID NOs: 25596 and 25597, in the order of appearance): 【Chemical 114】 Example 103 (SEQ ID NOs: 25598 and 25599, in the order of appearance): 【Chemical 115】 Example 104 (SEQ ID NOs: 25600 and 25601, in the order of appearance respectively): 【Chemical 116】 Example 105 (SEQ ID NOs: 25602 and 25603, in the order of appearance respectively): 【Chemical 117】 Example 106 (SEQ ID NOs: 25604 and 25605, in the order of appearance respectively): 【Chemical 118】 Example 107 (SEQ ID NOs: 25606 and 25607, in the order of appearance respectively): 【Chemical Formula 119】 Example 108 (SEQ ID NOs: 25608 and 25609, in the order of appearance respectively): 【Chemical 120】 Example 109 (SEQ ID NOs: 25610 and 25611, in the order of appearance respectively): 【Chemical 121】 Example 110 (SEQ ID NOs: 25612 and 25613, in the order of appearance respectively): 【Chemical 122】 Example 111 (SEQ ID NOs: 25614 and 25615, in the order of appearance respectively): 【Chemical 123】 Example 112 (SEQ ID NOs: 25616 and 25617, in the order of appearance respectively): 【Chemical 124】 Example 113 (SEQ ID NOs: 25618 and 25619, in the order of appearance respectively): 【Chemical 125】 Example 114 (SEQ ID NOs: 25620 and 25621, in the order of appearance respectively): 【Chemical 126】 Example 115 (SEQ ID NOs: 25622 and 25623, in the order of appearance respectively): 【Chemical 127】 Example 116 (SEQ ID NOs: 25624 and 25625, in the order of appearance respectively): 【Chemical 128】 Example 117 (SEQ ID NOs: 25626 and 25627, in the order of appearance respectively): 【Chemical 129】 Example 118 (SEQ ID NOs: 25628 and 25629, in the order of appearance respectively): 【Chemical 130】 Example 119 (SEQ ID NOs: 25630 and 25631, in the order of appearance respectively): 【Chemical 131】 Example 120 (SEQ ID NOs: 25632 and 25633, in the order of appearance respectively): 【Chemical 132】 Example 121 (SEQ ID NOs: 25634 and 25635, in the order of appearance respectively): 【Chemical 133】 Example 122 (SEQ ID NOs: 25636 and 25637, in the order of appearance respectively): 【Chemical 134】 Example 123 (SEQ ID NOs: 25638 and 25639, in the order of appearance respectively): 【Chemical 135】 Example 124 (SEQ ID NOs: 25640 and 25641, in the order of appearance respectively): 【Chemical 136】 Example 125 (SEQ ID NOs: 25642 and 25643, in the order of appearance respectively): 【Chemical 137】 Example 126 (SEQ ID NOs: 25644 and 25645, in the order of appearance respectively): 【Chemical 138】 Example 127 (SEQ ID NOs: 25646 and 25647, in the order of appearance respectively): 【Chemical 139】 Example 128 (SEQ ID NOs: 25648 and 25649, in the order of appearance respectively): 【Chemical 140】 Example 129 (SEQ ID NOs: 25650 and 25651, in the order of appearance respectively): 【Chemical 141】 Example 130 (SEQ ID NOs: 25652 and 25653, in the order of appearance respectively): 【Chemical 142】 Example 131 (SEQ ID NOs: 25654 and 25655, in the order of appearance respectively): 【Chemical 143】 Example 132 (SEQ ID NOs: 25656 and 25657, in the order of appearance respectively): 【Chemical 144】 Example 133 (SEQ ID NOs: 25658 and 25659, in the order of appearance respectively): 【Chemical 145】 Example 134 (SEQ ID NOs: 25660 and 25661, in the order of appearance): 【Chemical 146】 Example 135 (SEQ ID NOs: 25662 and 25663, in the order of appearance): 【Chemical 147】 Example 136 (SEQ ID NOs: 25664 and 25665, in the order of appearance): 【Chemical 148】 Example 137 (SEQ ID NOs: 25666 and 25667, in the order of appearance): 【Chemical 149】 Example 138 (SEQ ID NOs: 25668 and 25669, in the order of appearance): 【Chemical Formula 150】 Example 139 (SEQ ID NOs: 25670 and 25671, in the order of appearance): 【Chemical 151】 Example 140 (SEQ ID NOs: 25672 and 25673, in the order of appearance): 【Chemical 152】 Example 141 (SEQ ID NOs: 25674 and 25675, in the order of appearance): 【Chemical 153】 Example 142 (SEQ ID NOs: 25676 and 25677, in the order of appearance): 【Chemical 154】 Example 143 (SEQ ID NOs: 25678 and 25679, in the order of appearance): 【Chemical 155】 Example 144 (SEQ ID NOs: 25680 and 25681, in the order of appearance): 【Chemical 156】 Example 145 (SEQ ID NOs: 25682 and 25683, in the order of appearance): 【Chemical 157】 Example 146 (SEQ ID NOs: 25684 and 25685, in the order of appearance): 【Chemical 158】 Example 147 (SEQ ID NOs: 25686 and 25687, in the order of appearance): 【Chemical 159】 Example 148 (SEQ ID NOs: 25688 and 25689, in the order of appearance): 【Chemical 160】 Example 149 (SEQ ID NOs: 25690 and 25691, in the order of appearance): 【Chemical 161】 Example 150 (SEQ ID NOs: 25692 and 25693, in the order of appearance): 【Chemical 162】 Example 151 (SEQ ID NOs: 25694 and 25695, in the order of appearance): 【Chemical 163】 Example 152 (SEQ ID NOs: 25696 and 25697, in the order of appearance): 【Chemical 164】 Example 153 (SEQ ID NOs: 25698 and 25699, in the order of appearance): 【Chemical 165】 Example 154 (SEQ ID NOs: 25700 and 25701, in the order of appearance): 【Chemical 166】 Example 155 (SEQ ID NOs: 25702 and 25703, in the order of appearance): 【Chemical 167】 Example 156 (SEQ ID NOs: 25704 and 25705, in the order of appearance): 【Chemical 168】 The compound according to claim 11, selected from pharmaceutically acceptable salts thereof, isotopes thereof, and combinations thereof.

15. A compound selected from the group consisting of insulin containing an A chain and a B chain, wherein the A chain contains a sequence selected from 24501 and 24502, and the B chain contains a sequence selected from SEQ ID NO: 25229, 25232, 25305, 25308, 25312, 25236, 25095, and 25380-25397.

16. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

17. A compound according to any one of claims 1 to 15 for use as a medicament.

18. A method for treating or preventing diabetes, impaired glucose tolerance, hyperglycemia, or metabolic syndrome, the method comprising administering to a subject in need thereof a compound according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16.

19. The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein each of said indirect linkers is independently selected from FL3, FL5, FL5A, FL5B, FL32, FL41, FL62, FL63, and stereoisomers thereof.

20. Said B １ and said B ２ are each independently of the formula F2, and each of the remaining Rs １ is independently H, F, Cl, I, Br, and CF ３ selected from, the compound according to claim 6 or a pharmaceutically acceptable salt thereof.

21. said B １ and said B ２ are each independently of the formula F2, and each remaining R １ is H, a compound according to claim 6 or a pharmaceutically acceptable salt thereof.

22. X1 comprises at least one lysine and an N-terminus, Each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116, FF116A, FF116B, FF116C, and FF116D, where each B １ and B ２ is F2, each Z1c is conjugated directly or via said indirect linker to the amine side chain of said lysine in X1 or to the N-terminus in X1, each indirect linker is independently selected from FL3, FL5, FL5A, FL5B, and FL20 - FL75, and stereoisomers thereof, wherein in FL3 and FL5 each p is independently 1, 2, or 3, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 7.

23. The compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein at least one Z1c is conjugated via said indirect linker to the amine side chain of a lysine in the B chain or to the B chain N-terminus.

24. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, wherein X1 comprises two or more lysine amino acids.

25. The compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein the B chain comprises at least two lysine amino acids each independently conjugated directly or via an indirect linker to Z1c.

26. The compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein the B chain comprises at least three lysine amino acids each independently covalently bonded to Z1c via said indirect linker.

27. The compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein the A chain comprises one or more lysine amino acids each independently covalently bonded directly or via an indirect linker to Z1c.

28. The compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein X1 comprises an A chain having at least one lysine covalently bonded to Z1c via the indirect linker.

29. The compound as claimed in claim 1, selected from 【Chemical 169】 The compound or a pharmaceutically acceptable salt thereof according to claim 1.

30. At least one Z1c is covalently bonded to X1 via the indirect linker, and each indirect linker is 【Chemical 170】 Formula FL3 where p is 2 or 3, 【Chemical 171】 Formulas FL5, FL5A, and FL5B where p is 1 or 2, and 【Chemical 172】 independently selected from and its stereoisomers, wherein R’’ represents a direct or indirect covalent bond to Z1c, Z’’ represents a direct or indirect covalent bond to X1 or Z1a, A' is H, C １ ~C ６ an alkyl group, a saturated fatty acid, an unsaturated fatty acid, (C ３ ~C ６ ) cycloalkyl group, C １ ~C ６ selected from a haloalkyl group, an aryl group, and a heteroaryl group, A'' is C １ to C ６ an alkyl group, a substituted acyl group, an acyl group terminated with an acid group, a saturated or unsaturated fatty acid, (C ３ to C ６ ) cycloalkyl group, C １ to C ６ selected from a haloalkyl group, an aryl group, and a heteroaryl group, p is 1, 2, 3, 4, or 5, The compound according to claim 4 or 5, wherein any primary amine is optionally acetylated or alkylated.

31. Each indirect linker is as follows: 【Chemical 173】 The compound or a pharmaceutically acceptable salt thereof according to claim 30, selected from

32. Insulin further comprising 1 to 5 residues substituted, inserted, added, or mutated with an amino acid having a free amine conjugated directly or indirectly to Z1c, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

33. At least one Z1c is conjugated to the free amine side chain of an amino acid substituted, inserted, or mutated on insulin, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

34. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the compound is covalently bonded directly or via a linker to a molecule capable of binding to at least one protein present in human plasma.

35. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition comprises one or more polyalcohols.

36. The pharmaceutical composition according to claim 35, wherein the polyalcohol is selected from mannitol, sorbitol, erythritol, isomalt, lactitol, glucose, and maltitol.

37. A composition comprising at least one compound according to any one of claims 1 to 15 and 19 to 34 for use as a pharmaceutical for the treatment of diabetes or obesity, for the control of blood glucose levels, or for the control of drug release.

38. A method of administering to a subject a compound according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 or 35 to 36, the method comprising administering said compound or said composition to said subject as a therapeutic or prophylactic agent.

39. A method for preparing a compound according to any one of claims 1 to 15, comprising at least one alkylation and / or amidation step.

40. A method of treating a subject by administering a device or formulation comprising a compound according to any one of claims 1 to 15.

41. The compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, wherein the compound is used as an intermediate in the manufacture of a therapeutic agent of a prodrug or a prophylactic compound.

42. In each of F2, F5, and F10, one R １ is (C=O)--- ＊ is as defined in claim 6, or a pharmaceutically acceptable salt thereof.

43. A compound represented by formula II, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt, wherein Z1c - linker (Formula II) wherein said Z1c - linker is 【Chemical 174】 【Chemical 175】 【Chemical 176】 【Chemical 177】 selected from, and X is a leaving group, NH ２ , and selected from H, B １ and B ２ may be the same or different and each independently represents an aromatic boron-containing group, a compound, or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutically acceptable salt.

44. The compound according to claim 43, comprising at least one B independently selected from F2, F5, and F10 １ or B ２ and wherein formulae F2, F5, and F10 are 【Chemical 178】 and wherein One R １ is (C=O)--- ＊ or (CH ２ ) ｍ (C=O)--- ＊ represents,--- ＊ represents the bonding point to the remainder of Z1c, m is 1, 2, 3, 4, 5, 6, or 7, Each remaining R １ is, independently, H, F, Cl, Br, I, OH, CH ２ -NH ２ , NH ２ , (C=O)-NH ２ , CH=O, SO ２ CH ３ , SO ２ CF ３ , CF ３ , CHF ２ , NO ２ , CH ３ , OCH ３ , O(CH ２ ) ｍ” CH ３ , (SO ２ )NH-CH ３ , (SO ２ )NH(CH ２ ) ｍ” CH ３ , and OCF ３ is selected from, and m” is 1, 2, 3, 4, 5, 6, or 7, Y8 is O, N, and NR, where R is C １ ~C ６ an alkyl group or H, Y9 is H, CH ３ , or C １ ~C ６ alkyl group, provided that when Y8 is O, Y9 is CH ３ or C １ ~C ６ alkyl group, and i is 1, 2, or 3, the compound.

45. Said Z1c - linker is 【Chemical 179】 【Chemical 180】 【Chemical 181】 【Chemical 182】 【Chemical 183】 【Chemical 184】 selected from or a stereoisomer or mixture of stereoisomers thereof, a pharmaceutically acceptable salt, and combinations thereof, X is a leaving group, the compound according to claim 44.

46. X is selected from N -oxysuccinimide (NHS), 2,3,5,6 - tetrafluorophenoxy (TFP), pentafluorophenoxy (Pfp), OH, halogen, maleimidalkylamino, maleimidamidopolyethylene glycolamino, and maleimidopolyethylene glycolamino, the compound according to claim 45 or a pharmaceutically acceptable salt thereof.

47. Said compound is 【Chemical 185】 【Chemical 186】 【Chemical 187】 【Chemical 188】 【Chemical 189】 【Chemical 190】 selected from or a stereoisomer or mixture of stereoisomers thereof, a pharmaceutically acceptable salt, and combinations thereof, the compound according to claim 46.

48. An insulin analog comprising an A chain and a B chain, wherein said A chain comprises a sequence selected from 24051 and 24052, and said B chain comprises a sequence selected from 25000 to 25397.

49. Said A chain comprises sequence 24051, The insulin analog according to claim 48, wherein the B chain comprises a sequence selected from 25000, 25001, 25006 to 25009, 25076, 25077, 25082 to 25085, 25228, 25229, 25232, 25234 to 25237, 25304, 25305, 25308, and 25310 to 25313.

50. The A chain comprises sequence 24051, The insulin analog according to claim 48, wherein the B chain comprises a sequence selected from 25011, 25012, 25017 to 25020, 25087, 25088, 25093 to 25096, 25229, 25239, 25232, 25240, 25245 to 25248, 25305, 25308, 25315, 25316, and 25321 to 25324.

51. The A chain comprises sequence 24051, The insulin analog according to claim 48, wherein the B chain comprises a sequence selected from 25228, 25229, 25232, 25234 to 25237, 25304, 25305, 25308, and 25310 to 25313.

52. In at least one Z1c, both B1 and B2 are each independently of formula F2, all remaining R1 are H, and the indirect linker is selected from FL3, FL20 - 22, FL25, FL27 - 28, FL30 - 33, FL35, FL37, FL41, FL43 - 70, and amino acids having a hydrophobic side chain, the compound according to claim 6 or a pharmaceutically acceptable salt thereof.

53. In at least one Z1c, both B1 and B2 are each independently of formula F2, and one of the remaining R1 is CF ３ and the remaining R1 is H, and the indirect linker is selected from FL5, FL23-24, FL26, FL29, FL34, FL36, FL38-40, FL42, and amino acids having a hydrophilic side chain, the compound according to claim 6 or a pharmaceutically acceptable salt thereof.

54. The compound according to claim 52 or 53, or a pharmaceutically acceptable salt thereof, wherein the at least one Z1c is selected from FF12, FF12A - D, FF116, and FF116A - D.

55. A compound having agonist potency for the insulin receptor, comprising at least one aromatic boron - containing group having agonist potency for the insulin receptor, The compound has a first EC50 potency for activating the insulin receptor at a first glucose concentration and a second EC50 potency for activating the insulin receptor at a second glucose concentration, wherein when the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the ratio of the first EC50 to the second EC50 of the insulin receptor agonist potency of the compound is from about 2 to about 25, from about 2 to about 20, from about 2 to about 15, from about 2 to about 10, from about 2 to about 10, from about 2 to about 9, or from about 2 to about 8, a compound, wherein the at least one aromatic boron-containing group is attached to an FF skeleton, and the FF skeleton is selected from the formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226

56. A compound having agonist efficacy for glucose, comprising at least one aromatic boron-containing group having binding affinity for glucose, which, when administered to a first group of rats having a first glucose infusion rate for providing a blood glucose concentration of 100 mg / dL at a dose of 30 nmol / kg, and a second group of rats having a second glucose infusion rate for providing a blood glucose concentration of 200 mg / dL, provides a relative glucose infusion rate difference (mg / kg / min.min) of from about 1 to about 2500, from about 1 to about 2000, from about 1 to about 1500, from about 100 to about 1500, and from about 1000 to about 1500, and a relative glucose infusion rate ratio of from about 0.1 to about 5, from about 0.2 to about 4.5, from about 0.5 to about 4, from about 0.5 to about 3.5, or from about 1 to about 3, a compound, wherein the at least one aromatic boron-containing group is attached to an FF skeleton, and the FF skeleton is selected from the formulas FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226

57. The compound according to claim 55 or 56, wherein the FF skeleton is selected from the formulas FF116, FF116A, FF116B, FF116C, FF116D, FF225, FF225A, and FF225B.

58. A method of administering to a subject a compound according to any one of claims 55 to 57 or a pharmaceutical composition according to claim 16 or 35 to 36, the method comprising administering the compound or the composition to the subject as a therapeutic or prophylactic agent.

59. A method of preparing a compound according to any one of claims 55 to 57, the method comprising at least one alkylation and / or amidation step.

60. A method of treating a subject by administering a device or formulation comprising a compound according to any one of claims 55 to 57.

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