Aromatic boron-containing compounds and related insulin analogs

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 364,893, filed May 18, 2022, which is incorporated herein by reference.

[0003] FIELD OF THE DISCLOSURE

[0004] The present disclosure relates to novel compounds comprising one or more aromatic boron-containing groups. The present disclosure relates to the use of the novel compounds for binding glucose. The present disclosure further relates to kits and the use of the compounds and / or pharmaceutical compositions comprising the disclosed compounds for treating disorders characterized by elevated glucose levels (such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, and type 1 diabetes), as well as obesity, metabolic syndrome, neurological diseases, mood disorders, and psychiatric disorders.

[0005] Sequence Listing

[0006] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file titled 02021_00153-00304_SL.xml, which was created on April 7, 2023 and is 6,324,718 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0007] Background of the Disclosure

[0008] Boric acid is generally considered to be a Lewis acid that tends to bind to hydroxyl groups because, as a Lewis acid, boric acid can form complexes with Lewis bases (e.g., hydroxide anions). Therefore, molecules containing borate esters (including boric acid) have a general tendency to bind to hydroxyl groups. This binding tendency can be used to detect hydroxyl-containing groups by boronated labeling reagents, wherein the borate ester group binds to the hydroxyl group, and depending on the solvent and buffer conditions, the borate ester can form a hydrolyzable borate ester-ester bond with the hydroxyl group of the hydroxyl-containing molecule (such as the hydroxyl group present in diols (e.g., glucose)). Although boron-containing compounds can bind to diol-containing molecules, it has always been challenging to achieve selectivity using boron-containing compounds because boron-containing compounds can bind to various diols, including cis-diols, to varying degrees. Although the binding affinity of boron-containing compounds to specific vicinal diols of interest can be improved, this can result in a loss of selectivity.

[0009] Glucose is the body's primary fuel, and blood sugar levels in healthy individuals are tightly regulated. For example, between meals, blood sugar levels are around 5 mmol / L (mM), and when blood sugar levels rise after a meal, they quickly return to 5 mM through the action of insulin. Insulin, a hormone secreted by pancreatic beta cells, stimulates the cells to absorb glucose by transferring it from storage vesicles to the cell surface via glucose transporters (GLUT4) (Huang, SH et al. (2007) Cell Metabolism 5:237-252).

[0010] Diabetic patients may lose their ability to produce insulin (type 1) due to autoimmunity to beta cells, or have low sensitivity to insulin and impaired insulin secretion (type 2). For example, type 1 diabetic patients may rely on multiple insulin injections per day, both for basic coverage (usually once a day) and also together with meals (bolus) to control their glucose levels. (Polonsky, KS et al. (1988) The Journal of Clinical Investigation 81: 442-448.). Because glucose values ​​can fluctuate unpredictably, it is extremely difficult to perfectly administer insulin day after day. In fact, although diabetes treatment has achieved many technological advances, researchers currently observe that, partly due to lifestyle issues, long-term glucose control and / or overall metabolic health status deteriorate.

[0011] Glucose sensors and glucose-sensing insulin analogs are known in the art. See, for example, WO 2016 / 179568 A1 and WO 2021 / 202802 A1. However, there is a need in the art to develop glucose sensors and glucose-sensing insulin analogs with improved performance, such as those that bind to insulin receptors and respond proportionally to varying glucose concentrations and provide a graded and reversible response to changes in glucose levels under physiological conditions.

[0012] Therefore, there is an unmet medical need for new compounds that can control blood glucose levels, such as compounds that bind glucose and can be used in glucose-responsive insulin analogs / conjugates.

[0013] Herein, we disclose glucose sensors and glucose-sensing insulin analogs with improved properties, such as increased affinity for glucose at relatively low concentrations, increased affinity for the insulin receptor, and increased activation of the insulin receptor.

[0014] Overview of the Disclosure

[0015] According to certain 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) comprising aromatic boron-containing functional groups (e.g., F2, F5, and F10). In certain embodiments, the present disclosure provides compounds of formula (X") n1 Indirect linkers represented by , wherein 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 certain embodiments, the present disclosure provides novel Z1c-linkers (e.g., Formulas FFL-1 to FFL-101) comprising aromatic boron-containing functional groups (e.g., F2, F5, and F10).

[0016] In certain embodiments, the present disclosure provides compounds comprising a drug substance (e.g., X1) and at least one Z1c having an aromatic boron-containing functional group. In certain embodiments, the disclosed compounds comprise one or more scaffolds (e.g., FF scaffolds and Z1c-linker scaffolds as exemplified in formulas FFL-1 to FFL-101). In certain embodiments, the compounds comprise a drug substance. In at least some embodiments, the drug substance is a polypeptide or a small molecule.

[0017] In at least some embodiments, disclosed compounds have at least two aromatic boron-containing functional groups. In certain embodiments, the compound is selective for specific sugars (such as glucose) while exhibiting reduced affinity for other sugars, and wherein at least two aromatic boron-containing functional groups are present in the aromatic boron-containing portion of the compound. In certain embodiments, the aromatic boron-containing portion can be covalently conjugated directly or indirectly to a bulk drug comprising an amine, a bulk drug covalently conjugated to an amine-containing linker, or NH2 or OH (for example, wherein X1 is NH2 or OH). In certain embodiments, the bulk drug is covalently conjugated to an amine-containing linker, and the amine group is conjugated to the aromatic boron-containing portion (Z1c). In certain embodiments, the aromatic boron-containing portion (Z1c) has a structure consisting of a tethering group (tethering group) (FF formula) and an aromatic boron-containing group, and the groups together constitute the aromatic boron-containing portion of the disclosed compound. In certain embodiments, the aromatic boron-containing moiety (Z1c) is covalently conjugated to an indirect linker (e.g., Formulas FL3, FL5, FL5A, FL5B, and FL20-FL75) and has a structure comprising a tethering group (Formula FF) and an aromatic boron-containing group, which together constitute the aromatic boron-containing moiety of the disclosed compounds.

[0018] In certain embodiments, one or more Z1c can be linked (e.g., conjugated, linked) to the drug substance (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- or D-amino acids linked together using amide or peptide bonds). In at least some embodiments, the rotational restriction of the boron functional group by the tethering group (e.g., formula FF) enhances the binding affinity of the compound (e.g., conjugate) to a specific diol (such as glucose) and away from other diols in the body, thereby providing selectivity for the specific diol. In certain embodiments, the compound can exhibit therapeutic pharmacokinetics and / or pharmacodynamics in response to endogenous and / or exogenous small molecules (such as glucose) in the body. Changes in the physiological concentration of glucose may result in activation and / or release of the drug molecule, or conversely, may result in inactivation and / or sequestration of the drug molecule (e.g., peptide hormone), and / or modulate the activity of the drug molecule through the interaction of glucose with the boron-containing structure conjugated to the drug molecule.

[0019] In certain embodiments, the present disclosure provides a compound comprising X1 and one or more Z1c, or a tautomer, stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt or hydrate or isotopic derivative thereof, wherein:

[0020] X1 includes:

[0021] i. APIs containing amines;

[0022] ii. is constructed to be covalently conjugated to an amine of the drug substance; or

[0023] iii. NH2 or OH (eg, X1 is NH2 or OH);

[0024] wherein each Z1c is independently selected from formula 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 wherein each Z1c is covalently conjugated directly or indirectly to an amine in X1 or to NH2 when X1 is NH2 or to OH when X1 is OH.

[0025] In certain embodiments, the present disclosure provides a compound represented by Formula I, or a stereoisomer or mixture of stereoisomers, or a pharmaceutically acceptable salt thereof:

[0026]

[0027] in:

[0028] X1 includes:

[0029] i. APIs containing amines;

[0030] ii. is constructed to be covalently conjugated to an amine of the drug substance; or

[0031] iii. NH2 or OH (eg, X1 is NH2 or OH);

[0032] Each Z1a independently comprises from 1 to 50 amino acids linked together using amide or peptide bonds;

[0033] Each Z1b is independently a small molecule linker;

[0034] each Zlc is independently selected from Formula FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, wherein each Zlc is covalently conjugated directly or via an indirect linker to an amine of Zla or to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH;

[0035] Each m' is 0 or 1;

[0036] Each n' is 0;

[0037] Each o' is 1;

[0038] each p' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0039] each q' is 1, 2, 3, 4 or 5, wherein when p' or q' is 2 or greater, each corresponding Z1c is independently selected and may be the same or different, and wherein when q' is 2 or greater, each corresponding Z1a is independently selected and may be the same or different;

[0040] when m′=0 and n′=0, each Z1c is covalently conjugated directly or via an indirect linker to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH, and for m′=1, each Z1a is covalently conjugated to X1 via an amide or peptide bond, and each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to X1;

[0041] and

[0042] Wherein 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 are:

[0043]

[0044] wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1; and wherein B1 and B2 may be the same or different and each independently represents an aromatic boron-containing group; and wherein one or more positions of the compound of Formula I may contain an isotope (e.g., 2 H or 14 C).

[0045] In certain embodiments, X 1 is OH or NH 2 . In certain embodiments, the compounds disclosed herein (eg, Formula I, Formula II) are conjugated to a drug substance directly or via an optional linker.

[0046] In certain embodiments, the compound of Formula I comprises:

[0047] At least one Z1c, which is covalently conjugated to X1 or to Z1a via an indirect linker, wherein the indirect linker is represented by formula (X") n1 express,

[0048] Each n1 is independently selected from 1, 2, 3, 4 and 5, and

[0049] Each X" is independently selected from:

[0050] (i) an L- or D-amino acid, wherein the amine function of the L- or D-amino acid is covalently conjugated directly or indirectly to Z1c, and the acid function of the L- or D-amino acid is directly or indirectly conjugated to X1 or to Z1a; and

[0051] (ii) Formulas FL3, FL5, FL5A, FL5B, FL20-FL68 and FL71-FL75 comprising the substituents R″ and Z″, and Formulas FL69 and FL70 comprising the substituents R″, Z″, A′ and A″, and stereoisomers thereof;

[0052] in:

[0053] R" represents a covalent bond directly or indirectly to Z1c;

[0054] Z" represents a covalent bond directly or indirectly to X1 or to Z1a;

[0055] A' is selected from H, alkyl (e.g., C1-C6 alkyl), saturated fatty acid, unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl; and

[0056] A" is selected from one of 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., a (C3-C6)cycloalkyl group), a haloalkyl group (e.g., a C1-C6 haloalkyl group), an aryl group, and a heteroaryl group;

[0057] p is 1, 2, 3, 4, or 5,

[0058] q is 1, 2, 3, 4, or 5, and

[0059] Any primary amines are optionally acetylated or alkylated.

[0060] In certain embodiments, n1 is 1.

[0061] In some embodiments of the compound of Formula I, m'=0, n'=0, o'=0, and the compound is represented by Formula IC:

[0062] in:

[0063] (IC)

[0064] each p' is 1, 2, 3, or 4; and

[0065] Each q' is 1, 2, 3, 4 or 5.

[0066] In certain embodiments, each of B1 and B2 is of formula F2.

[0067] In certain embodiments, each Z1c is covalently conjugated to an indirect linker, and each combination of Z1c and an indirect linker is selected from formulas FFL-1 to FFL-101, which comprise a substituent X; wherein X represents a covalent attachment point that is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine that is directly or indirectly covalently conjugated to X1 or to an amine of Z1a.

[0068] In certain embodiments, X1 is a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain.

[0069] In certain embodiments, at least one Z1c comprises an α-methyl group or a β-methyl group.

[0070] Also disclosed herein are methods of treatment. The disclosed methods may include treating a subject in need thereof, wherein the subject has a disease characterized by elevated glucose levels. The methods may include administering to a 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.

[0071] Also disclosed herein are methods for treating or preventing endocrine and / or metabolic diseases in a subject in need thereof, comprising 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 certain embodiments, the compound of Formula (I) comprises a drug substance, wherein the drug substance is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory peptide, a glucose-dependent insulin-releasing polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof.

[0072] In certain embodiments, 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 is administered concurrently with a second agent or therapy. In certain embodiments, the second agent is a bulk drug, wherein the bulk drug is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory peptide, glucose-dependent insulin-releasing polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof. In certain embodiments, 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 is administered concurrently with a second agent or therapy. In certain embodiments, 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 is administered sequentially with a second agent or therapy (e.g., wherein the compound of Formula (I) is administered first and the second agent or therapy is administered subsequently, or wherein the second agent or therapy is administered first and the compound of Formula (I) is administered subsequently). In certain embodiments, 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 is administered in the same unit dosage form as the second agent or therapy. In certain embodiments, 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 is administered in a different unit dosage form than the second agent or therapy.

[0073] In at least one embodiment, the pharmaceutical compositions of the present disclosure can be used to treat a disease characterized by elevated glucose in a subject, such as diabetes (or for the preparation of a medicament for such use). In at least one embodiment, a therapeutically effective amount of a 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.

[0074] In certain embodiments, the disclosed compounds comprise an insulin receptor agonist selected from insulin and insulin analogs (e.g., wherein X1 is insulin or an insulin analog). In certain embodiments, X1 comprises an insulin having an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from SEQ ID NOs 1, 25, 24051, and 24052, and the B-chain comprises a sequence selected from SEQ ID NOs 24060, 24061, 24062, 24063, 24064, and 25000-25397.

[0075] In certain embodiments, X1 comprises an insulin having an A-chain and a B-chain, wherein:

[0076] said A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24051 and 24052,

[0077] The B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380-25397;

[0078] Each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116A, FF116B, FF116C, FF116D, and is covalently conjugated directly or via an indirect linker to one or more lysine residues in X1;

[0079] B1 and B2 are each F2; and

[0080] The indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL20-FL75, and stereoisomers thereof.

[0081] In certain embodiments, the compound is selected from an insulin comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24501 and 24502; and wherein the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25229, 25232, 25305, 25308, 25312, 25236, 25095, and 25380-25397.

[0082] In certain embodiments, the present disclosure provides a compound represented by Formula II, or a stereoisomer or mixture of stereoisomers, or a pharmaceutically acceptable salt thereof:

[0083] Z1c-Connector

[0084] (Formula II)

[0085] wherein the Z1c-linker is selected from the group consisting of formulas FFL-1 to FFL-101;

[0086] wherein X is selected from a leaving group (e.g., a halogen, an N-hydroxysuccinimide (NHS) group, a 2,3,5,6-tetrafluorophenol (TFP) group, a pentafluorophenol (Pfp) group, or a sulfonate), NH2, OH, and H; and

[0087] wherein B1 and B2, which may be the same or different, each independently represent an aromatic boron-containing group.

[0088] In some embodiments of the compound of Formula I, X1 comprises an insulin receptor agonist, which is insulin or an insulin analog with an A-chain and a B-chain. In certain embodiments, one or more lysine residues and / or N-terminal of the A-chain or B-chain are covalently conjugated as described in Formula I. In certain embodiments, insulin or insulin analogs described herein are used as compounds (e.g., intermediate compounds) for preparing the conjugates described by Formula I. In certain embodiments, insulin or insulin analogs described herein are used in methods for preventing and / or treating endocrine diseases and / or metabolic diseases, for example, including administering to a subject in need thereof any compound of the embodiments described herein (e.g., modified insulin), thereby treating endocrine diseases and / or metabolic diseases.

[0089] Detailed Description of the Disclosure

[0090] While aromatic boron-containing compounds (e.g., groups) can bind to diol-containing molecules, achieving selectivity using aromatic boron-containing compounds that can act as molecular sensors is challenging because they can bind to various diols, including cis-diols, to varying degrees. The increased binding affinity of aromatic boron-containing compounds (that can act as sensors) for specific vicinal diols of interest can result in a loss of selectivity.

[0091] Positioning the boron functional group (e.g., sensor) of an aromatic boron-containing compound on a scaffold with a unique or specific set of geometries can increase selectivity for specific vicinal diols while maintaining affinity for the diol of interest. According to certain embodiments, the aromatic boron-containing compounds disclosed herein have different side groups on the aromatic boron-based scaffold and specific scaffold geometries that influence binding to hydroxyl-containing molecules.

[0092] According to certain embodiments, the compounds of the present disclosure comprise aromatic boron-containing compounds that orient the boron functional group in three-dimensional space such that the boron-containing compound is spatially oriented to bind to a hexose containing a vicinal diol, such that the boron group can appropriately bind to the hydroxyl group in the vicinal diol molecule and provide enhanced selectivity. In certain embodiments, the aromatic boron-containing compound is modified with a specific functional group on the aromatic ring that, together with an appropriate or suitable scaffold, can provide greater selectivity and / or affinity to bind to the vicinal diol of interest and away from other diols in the body.

[0093] In certain embodiments, the aromatic boron-containing compound is conjugated to a drug substance (e.g., a small molecule, a polypeptide), wherein the aromatic boron-containing compound provides intramolecular and / or intermolecular interactions with the drug substance and / or with proteins in the body (such as circulating proteins in blood and / or plasma, including albumin and / or globulins). In certain 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 certain embodiments, the selective binding of the sensor to specific vicinal diols changes the extent of those 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 vicinal diols present.

[0094] In certain embodiments, the drug substance is a peptide hormone. In certain embodiments, the peptide hormone is a human peptide hormone such as insulin or an insulin analog, glucagon, or another incretin hormone. In certain embodiments, the sensor is selective for vicinal diols in glucose, and this selectivity is enhanced while maintaining affinity for glucose and simultaneously reducing affinity for other sugars in the blood. In certain embodiments, the side groups on the aromatic core of the scaffold and (e.g., in combination with) the boron-containing compound can control the overall activity and / or pharmacokinetics of the conjugated drug substance based on the levels of glucose and / or other vicinal diols in the blood.

[0095] In certain embodiments, the aromatic boron-containing compound comprises a specific scaffold molecule (e.g., FF structure, FFL-1 to FFL-101, DSL-1 ( D iboronate S caffold L Inker) to DSL-112), wherein the scaffolds have been used to orient boron functional groups in a three-dimensional geometry so that the boron functional groups are close to each other and within a distance that facilitates oriented binding to specific hydroxyl groups of selected hexoses (such as glucose).

[0096] Without wishing to be bound by theory, it is believed that the aromatic boron-containing compounds (e.g., molecules) disclosed herein enhance selectivity through at least one or more of the following three mechanisms: (1) the FF scaffold helps match the orientation of the hydroxyl and / or alkoxy groups on the boron group in the aromatic boron-containing compound with the hydroxyl groups in the vicinal diol molecule, thereby enhancing selectivity; (2) further selectivity gains are obtained by identifying specific functional groups attached to or near, for example, the aromatic core of the boron-containing compound, which affect the electronic structure of the aromatic boron-containing compound, thereby favoring reversible binding to the vicinal diol at physiological pH; and (3) the functional groups attached to the aromatic boron-containing compound (e.g., sensor scaffold) help provide steric hindrance to reduce binding to undesirable hexoses while maintaining binding to sugars of interest (such as glucose). In certain embodiments, the FF scaffold provides glucose binding. In certain embodiments, the combination of the FF scaffold and the indirect linker provides affinity for plasma proteins (such as, but not limited to, glycated proteins), and the combination of the FF scaffold and the indirect linker provides reversible interactions with plasma proteins that are controlled by binding of sugar molecules to the FF scaffold under physiological conditions. In the present disclosure, these effects combined together (eg, compounds of Formulas I and II) provide the desired or appropriate selectivity for binding to vicinal diol-containing molecules of interest and away from other diols in vivo.

[0097] In certain embodiments, the aromatic boron-containing compound is conjugated to a bulk drug, wherein the aromatic boron-containing compound provides intramolecular and / or intermolecular interactions with proteins in the body. Such proteins may include circulating proteins such as albumin, glycosylated proteins and / or immunoglobulins, glycosylated proteins (including glycosylated plasma proteins such as glycosylated albumin) in blood and / or human plasma. In certain embodiments, the selective binding of the sensor to a specific vicinal diol in the molecule of interest changes the degree of intramolecular and intermolecular binding, thereby regulating the pharmacokinetics and overall activity of the bulk drug in vivo. In certain embodiments, the bulk drug is a peptide hormone, and in certain embodiments thereof, the peptide hormone is an incretin hormone such as insulin, and the molecule containing the vicinal diol is glucose, but the disclosure is not limited thereto.

[0098] definition

[0099] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. It is further understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology and / or this specification, and should not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0100] Unless specifically described herein, functional groups, functional moieties, and reactions referred to herein should be understood to have meanings consistent with standard descriptions and / or general principles in organic chemistry, 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, 3rd edition, Cambridge University Press, Cambridge, 1987; Smith and March, March's Advanced Organic Chemistry, 5th edition, John Wiley & Sons, Inc., New York, 2001. Generic functional groups (such as alkyl, aryl, acetyl, etc.) encompass specific examples or species that fall within the categories of those functional groups generally defined in the art of organic chemistry, and one of ordinary skill in the art will be able to recognize specific example embodiments of the functional groups.

[0101] Unless specifically described in this article, the chemical terms, functional groups and general terms used throughout this specification are determined according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover). In certain embodiments, the terms "a", "one" and "said" and similar designations used herein should be interpreted as covering the singular and plural, unless otherwise indicated in the context. The term "CAS#" used herein is also referred to as CASRN or CAS numbering, which is a unique numerical identifier assigned by Chemical Abstracts Service (CAS) to each chemical substance described in the open scientific literature.

[0102] The nomenclature of compounds (including organic compounds) used herein may be given using common names, IUPAC, IUBMB or CAS nomenclature recommendations. If a name is given, the compound structure is systematically reduced using naming conventions or by commercially available software such as CHEMDRAW TM(Cambridgesoft Corporation, USA), those skilled in the art can easily determine the structure of the compound.

[0103] The terms used herein are for describing embodiments and are not intended to limit the present disclosure. It is further understood that, when used in this specification, the terms "comprising" and "including" specify the presence of the features, integers, actions, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, actions, operations, elements, components and / or their sets. The terms "and / or" used herein include any and all combinations of one or more of the listed related items. Statements such as "at least one" when preceding a list of elements modify the entire list of elements, rather than modifying the individual elements in the list.

[0104] As used herein, the terms "substantially," "about," and similar terms are used as approximate terms rather than terms of degree and are intended to account for inherent deviations in measurements or calculations that one of ordinary skill in the art would recognize. The term "about" is used throughout to describe and account for small variations. For example, "about" can mean that a value can be modified by ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, or ±0.05%. Values ​​modified by the term "about" include the specifically specified value. For example, "about 5.0" includes 5.0.

[0105] Furthermore, 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" and "using" are considered synonymous with the terms "utilizing" and "utilizing," respectively. Furthermore, the term "exemplary" is intended to mean an example or illustration.

[0106] In addition, any numerical range listed herein is intended to include all subranges with the same numerical precision contained within the listed range. For example, a range of "1 to 10" is intended to include all subranges between the listed minimum value of 1 and the listed maximum value of 10 (including 1 and 10), that is, with a minimum value equal to or greater than 1 and a maximum value equal to or less than 10, such as 2 to 7. Any maximum numerical limit listed herein is intended to include all lower numerical limits contained therein, and any minimum numerical limit listed in this specification is intended to include all higher numerical limits contained therein. Therefore, the applicant reserves the right to amend this specification (including the claims) to explicitly list any subranges contained within the ranges explicitly listed herein.

[0107] As used herein, "aromatic boron-containing groups" refer to compounds having at least one boron atom covalently bonded to an aromatic group and / or compounds having at least one boron atom covalently incorporated into an aromatic group. The term "aromatic" as used herein may include "heterocycle," "heterocyclyl," or "heterocyclic." As used herein, the terms "heterocycle," "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 certain embodiments, the term "aromatic" may include "aryl." As used herein, the term "aryl" refers to a monocyclic, dicyclic, or other polycyclic aromatic ring system having 5 to 14 ring atoms. The aryl group may optionally be fused to one or more rings selected from aryl, cycloalkyl, heteroaryl, and heterocyclyl. Exemplary aryl groups also include, but are not limited to, monocyclic aromatic ring systems wherein the ring contains 6 carbon atoms.

[0108] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic or polycyclic aromatic ring system containing one or more heteroatoms (e.g., 1-3 heteroatoms, such as nitrogen, oxygen and sulfur). The heteroaryl group may be substituted with one or more substituents. The heteroaryl group may also be fused to a non-aromatic ring. Exemplary heteroaryl groups include, but are not limited to, monocyclic aromatic rings, wherein the ring contains 2-5 carbon atoms and 1-3 heteroatoms. In certain embodiments, the aromatic boron-containing group may 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, F227, F5, F10 and FFL-1 to FFL-101, and further include, for example, those disclosed as compounds F1-F9, F12-F43, F500-F520 in patent application PCT / US2021 / 025261 (filed on March 31, 2021) and those disclosed as compounds FF1-FF224 and F1-F10 in PCT / US2021 / 059802 (filed on November 18, 2021); the disclosures of which are expressly incorporated herein by reference in their entirety.

[0109] The term "small molecule linker" as used herein refers to a chemical group (e.g., a scaffold, a portion) comprising a first connection point toward X1 and a second connection point toward Z1b, Z1a, or Z1c. In certain embodiments, the first connection point is toward X1, and the second connection point is toward Z1c. In certain embodiments, the first connection point is toward X1, and the second connection point is toward Z1a. In certain embodiments, the small molecule linker is a portion / chemical group selected from Formula IIa-IIai, Formula IIIa-IIIai, and FL1-FL19 disclosed in patent application PCT / US2021 / 025261, the relevant disclosures of which are expressly incorporated herein by reference.

[0110] As used herein, the term "indirect linker" refers to a chemical group (e.g., a scaffold, a moiety) comprising a first point of attachment toward X1 and a second point of attachment toward Z1b, Z1a, or Z1c. In certain embodiments, the first point of attachment is toward X1, and the second point of attachment is toward Z1c. In certain embodiments, the indirect linker is a moiety / chemical group selected from the group consisting of formula (i) FL3, FL5, FL5A, FL5B, and FL20-FL75, stereoisomers thereof; and (ii) L- or D-natural or non-natural amino acids. In certain embodiments, at least one Z1c is covalently conjugated to X1 or to Z1a via one or more indirect linkers, wherein the indirect linker is of formula (X") n1 and each n1 is independently selected from 1, 2, 3, 4, and 5. For example, at least one Z1c is covalently conjugated to X1 via an indirect linker, wherein the indirect linker is represented by Formula III:

[0111] r”—(X1”) n1 —(X2”) n2 —(X3”) n3 —(X4”) n4 —(X5”) n5 —z”

[0112] (Formula III)

[0113] in

[0114] z" represents the point of attachment to the amine in X1;

[0115] r” indicates the connection point with Z1c;

[0116] n1, n2, n3, n4 and n5 are each independently 0 or 1;

[0117] Each X1", X2", X3", X4", X5" is independently selected from L- or D-amino acids, wherein the amine functional group of the amino acid is covalently conjugated to r" in Formula III, and the acid functional group of the amino acid is covalently conjugated to z" in Formula III, FL3, FL5, FL5A, FL5B and FL20-FL75.

[0118] As used herein, "amino acid" includes protein (or natural) amino acids (including the 20 standard amino acids) and non-protein (or non-natural) amino acids. Protein amino acids are amino acids naturally incorporated into proteins. Standard amino acids are amino acids encoded by the genetic code. Non-protein amino acids are either not present in proteins or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modification). In general, amino acid residues (peptide / protein sequences) can be identified by their full names, their single-letter codes, and / or their three-letter codes. These three methods are completely equivalent. Hereinafter, each amino acid of a compound of the present disclosure that does not specify its optical isomers should be understood to refer to the L-isomer (unless otherwise noted). Amino acids are molecules containing an amino group and a carboxylic acid group and optionally one or more other groups (often referred to as side chains).

[0119] As used herein, the term "amino acid residue" is an amino acid that has one hydroxyl group formally removed from the carboxyl group and / or one hydrogen atom formally removed from the amino group. As will be apparent from the following examples, an amino acid residue can be identified by its full name, its single-letter code, and / or its three-letter code. These three methods are fully equivalent and can be interchanged.

[0120] The term "alkyl" as used herein refers to a saturated straight or branched chain hydrocarbon, such as a straight or branched chain group of 1 to 30 carbon atoms, referred to herein as C 1-30 In certain embodiments, the alkyl group is C1-C 22 In certain embodiments, the alkyl group is C1-C 20 In certain embodiments, the alkyl group is C1-C 18 In certain embodiments, the alkyl group is C1-C 16 In certain embodiments, the alkyl group is C1-C 14 In certain embodiments, the alkyl group is C1-C 12 In certain embodiments, the alkyl group is C1-C 10Alkyl. In certain embodiments, the alkyl group is a C1-C8 alkyl group. In certain embodiments, the alkyl group is a C1-C6 alkyl group. In certain 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, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. In certain embodiments, "alkyl" is a straight chain hydrocarbon. In certain embodiments, "alkyl" is a branched chain hydrocarbon.

[0121] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated cyclic, bicyclic or bridged bicyclic hydrocarbon group of 3-16 carbons or 3-8 carbons (referred to herein as "(C3-C8)cycloalkyl") derived from a cycloalkane. In certain embodiments, the cycloalkyl is a (C3-C6)cycloalkyl. Exemplary cycloalkyls include, but are not limited to, cyclohexane, cyclohexene, cyclopentane, and cyclopentene. Cycloalkyls may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. Cycloalkyls may be fused with other cycloalkyls (saturated or partially unsaturated), aryl, or heterocyclic to form bicyclic, tetracyclic, and the like. The term "cycloalkyl" also includes bridged and spiro-fused ring structures which may or may not contain heteroatoms.

[0122] As used herein, the term "acyl" refers to a RC(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)-, wherein the group is attached to the parent molecular structure through a carbonyl functional group. In certain embodiments, it is C 1-10 Acyl groups, which refer to the total number of chain or ring atoms, such as alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or heteroaryl moieties, plus the carbonyl carbon of the acyl group. For example, a C4-acyl group has three other ring or chain atoms plus the carbonyl group. In certain embodiments, it is a C1-C 22 In certain embodiments, it is C1-C 20In certain embodiments, it is C1-C 18 In certain embodiments, it is C1-C 16 In certain embodiments, it is C1-C 14 In certain embodiments, it is C1-C 12 In certain embodiments, it is C1-C 10 In certain embodiments, it is a C1-C8 acyl group.

[0123] The term "haloalkyl" as used herein 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 certain embodiments, it is a C1-C 22 In certain embodiments, it is C1-C 20 In certain embodiments, it is C1-C 18 In certain embodiments, it is C1-C 16 In certain embodiments, it is C1-C 14 In certain embodiments, it is C1-C 12 In certain embodiments, it is C1-C 10 In certain embodiments, it is a C1-C8 haloalkyl group.

[0124] As used herein, the term "aryl" refers to an aromatic ring system having a monocyclic, dicyclic, or other polycyclic carbon ring with 5 to 14 ring atoms. The aryl group may optionally be fused to one or more rings selected from aryl, cycloalkyl, heteroaryl, and heterocyclic groups. The aryl groups of the present disclosure may be substituted with groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic group, 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, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups also include, but are not limited to, monocyclic aromatic ring systems wherein the ring contains 6 carbon atoms.

[0125] As used herein, a "leaving group" is an atom (or group of atoms) that can be replaced by a stable species and takes away a bonding electron. For example, a leaving group can be an anion (e.g., Cl -) or a neutral molecule (e.g., H 2 O). In certain 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 sulfonate.

[0126] "Isomers" refer to compounds that have the same number and nature of atoms, and therefore the same molecular weight, but differ with respect to the arrangement or configuration of the atoms in space.

[0127] "Stereoisomer" or "optical isomer" refers to a stable isomer that has at least one chiral atom or restricted rotation to produce a perpendicular asymmetric plane (e.g., certain biphenyls, allenes, and spirocyclic compounds) and can rotate plane-polarized light. Because there are asymmetric centers and other chemical structures in the compounds of the present disclosure, which may produce stereoisomerism, the present disclosure contemplates stereoisomers and mixtures thereof. The compounds of the present disclosure and their salts include asymmetric carbon atoms and therefore can exist as single stereoisomers, racemates, and mixtures of enantiomers and diastereomers. In certain embodiments, such compounds are prepared as racemic mixtures. In certain embodiments, such compounds can be prepared or separated into pure stereoisomers, such as individual enantiomers or diastereomers, or stereoisomer-enriched mixtures. As discussed in more detail below, each stereoisomer of a compound can be prepared by synthesis from optically active starting materials containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, such as conversion into a mixture of diastereomers followed by separation or recrystallization, chromatography techniques, use of a chiral resolving agent, or direct separation of enantiomers on a chiral chromatographic column. Starting compounds of specific stereochemistry are commercially available or prepared by the methods described below and resolved by techniques well known in the art.

[0128] 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 certain embodiments, the fatty acid is C3-C 26 In certain embodiments, the fatty acid is C4-C 20 Fatty acids. In certain embodiments, the fatty acids are selected from 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) 11COOH 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 In certain embodiments, the fatty acid is an unsaturated fatty acid selected from the group consisting of α-linoleic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, elaidic acid, γ-linolenic acid, oleic acid, elaidic acid, and gondoic acid.

[0129] The term "pharmaceutically acceptable salt" refers to salts of acidic or basic groups that may be present in the compounds used in the compositions of the present invention.

[0130] As used herein, "drug active ingredient" refers to small molecule compounds and / or polypeptide-containing compounds. According to certain embodiments, the drug active ingredients suitable for use in the compounds and methods described herein are therapeutically, prophylactically, and / or diagnostically active drug active ingredients.

[0131] It should be understood that although various elements (such as molecules, components, groups and / or parts) can be described in this article using terms such as "first", "second", "third", etc., those elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Therefore, the first element described below can be referred to as the second element, without departing from the spirit and scope of the present disclosure. It should be understood that when an element or group is referred to as "connected to", "with ... put together", "connected" or "coupled to" another element or group, the two elements can be directly connected, or one or more intermediate elements can be present. It should be understood that the conjugation described herein and connection have the option of directly conjugating or directly connecting, unless the context clearly excludes.

[0132] As used herein, the terms "directly" or "directly covalently conjugated" or "directly covalently conjugated" may be used interchangeably to indicate that a first group is "directly" or "directly covalently conjugated" or "directly covalently conjugated" to a second group, meaning that the first group and the second group are covalently bonded together without additional intervening groups.

[0133] As used herein, the terms "indirectly" or "indirectly covalently conjugated" or "indirectly covalently conjugated" may be used interchangeably to indicate that a first group is "indirectly" or "indirectly covalently conjugated" or "indirectly covalently conjugated" to a second group, meaning that the first group and the second group are covalently bonded together with 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).

[0134] The terms "insulin receptor agonist," "compound with agonist efficacy" in the context of a compound with agonist efficacy at the insulin receptor, and the term "agonist efficacy" in the context of a compound with agonist efficacy at the insulin receptor refer to compounds that bind to and / or activate the insulin receptor (e.g., compounds, proteins, fusion proteins of formula (I), as described, for example, in US 2016 / 0324932).

[0135] The terms "short-acting insulin receptor agonist" and "short-acting insulin" refer to insulins that have an onset of action equal to or faster than that of the insulins marketed under the trade names The insulin receptor agonist of oligosaccharide human insulin and human insulin that are sold.For example, short-acting insulin may start to take effect within 10 minutes after injection.As another example, short-acting insulin may start to take effect within 20 minutes after injection.In certain embodiments, short-acting insulin may have maximum effectiveness (peak) within 1-4 hour after injection.In certain embodiments, short-acting insulin can be used before or during meal and / or after meal soon.

[0136] The terms "long-acting insulin receptor agonist" and "long-acting insulin" refer to insulins that have a longer onset of action than In some embodiments, the protracted insulin may be administered in a dose of 100 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 300 mg / kg or more, or in a dose of 400 mg / kg or more, or in a dose of 500 mg / kg or more, or in a dose of 600 mg / kg or more, or in a dose of 800 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 300 mg / kg or more, or in a dose of 500 mg / kg or more, or in a dose of 500 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 300 mg / kg or more, or in a dose of 500 mg / kg or more, or in a dose of 500 mg / kg or more, or in a dose of 600 mg / kg or more, or in a dose of 100 mg / kg or more, or in a dose of 200 mg / kg or more, or in a dose of 300 mg / kg or more, or in a dose of 5 ...

[0137] The term "suitable for administration with meals" when used herein in connection with an insulin receptor agonist refers to a short-acting insulin receptor agonist that is suitable for use in controlling blood glucose levels during and / or shortly after a meal.

[0138] When used in connection with an insulin receptor agonist herein, the term "suitable for once-weekly administration" refers to an insulin receptor agonist that has prolonged pharmacokinetic and pharmacodynamic characteristics sufficient to control blood glucose levels throughout the day when administered no more than once a week. Examples of such molecules include fusion proteins described in US2016 / 0324932, including BIF. BIF, also known as insulin efsitora alfa, comprises a dimer of an insulin receptor agonist fused to a human IgG Fc region, wherein the insulin receptor agonist comprises an insulin B-chain analog fused to an insulin A-chain analog using a first peptide linker, and wherein the C-terminal residue of the insulin A-chain analog is directly fused to the N-terminal residue of a second peptide linker, and the C-terminal residue of the second peptide linker is directly fused to the N-terminal residue of the human IgG Fc region. BIF is identified by CAS Registry Number 2131038-11-2, which provides the following chemical names: (1) Insulin [16-Glu, 25-Histidine, 27-Gly, 28-Gly, 29-Gly, 30-Gly] (human B-chain) fusion protein-peptide (synthetic 7-amino acid linker) fusion protein-insulin [47-Th, 51-Aspartic acid, 58-Gly] (human A-chain) fusion protein-peptide (synthetic 20-amino acid linker) fusion protein-immunoglobulin G2 (human Fc fragment), dimer; and ( 2) Homo sapiens insulin B-chain [Y16>Y(16), F25>H(25), TPKT27-30>GGGG(27-30)](1-30) fusion protein-diglycylseryltetraglycyl(31-37) Insulin A-chain [I10>T(47), Y14>D(51), N21>G(58)](38-58) fusion protein-tris(tetraglycylglutamyl)pentaglycyl(59-78) Homo sapiens immunoglobulin heavy chain constant γ2 {del-CH1, hinge-(7-12), CH2, CH3[K 107 >del(300)]}(79-299), dimer (80-80':83-83')-bis-disulfide, expressed in CHO cells, alpha-glycosylated.

[0139] As used herein, "glucose-sensing insulin" refers to an insulin receptor agonist whose onset and / or activity level is dependent on blood glucose levels. Examples of such molecules include compounds of Formula (I) disclosed herein, such as 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.

[0140] The term "incretin-based therapy" includes any treatment that includes the administration of or promotes, enables, enhances, and / or mimics the effects of a group of metabolic hormones called incretins, which include, but are not limited to, GLP-1, gastric inhibitory peptide (GIP), and glucagon. Currently available incretin-based therapies include GLP-1R agonists.

[0141] "DPP-4 inhibitors" are compounds that block the DPP-4 enzyme, which is responsible for breaking down incretins. Currently available DPP-4 inhibitors include sitagliptin and linagliptin

[0142] "GLP-1R agonists" and "glucagon receptor agonists" are defined as compounds comprising the amino acid sequence of native human GLP-1 (SEQ ID NO: 25) or human glucagon, as well as compounds that retain full or partial activity at the GLP-1 receptor, i.e., GLP-1 analogs, GLP-1 derivatives, or GLP-1 fusion proteins. GLP-1R activity can be measured by methods known in the art, including in vivo assays and in vitro assays that measure GLP-1 receptor binding activity or receptor activation, for example, assays using pancreatic islet cells or insulinoma cells, as described in EP 619,322 and U.S. Pat. No. 5,120,712, respectively. GLP-1 analogs are molecules that have modifications compared to the amino acid sequence of native human GLP-1 (SEQ ID NO: 25), including one or more amino acid substitutions, deletions, inversions, or additions. A GLP-1 derivative is a molecule having the amino acid sequence of native human GLP-1 (SEQ ID NO: 25) or the amino acid sequence of a GLP-1 analog, but additionally having 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 comprising a GLP-1, a GLP-1 analog, or a GLP-1 derivative portion and a second polypeptide. Currently available GLP-1R agonists include exenatide ( and ), liraglutide Albiglutide and dulaglutide 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); US20140447 1 7 (albiglutide); and U.S. Patent No. 7,452,966 (dulaglutide).

[0143] The term "excipient" refers to any material that is added to the composition except that the fusion protein or any other other active ingredient. The example of such excipient that can be used for the compositions of the present invention comprises buffer, surfactant, isotonic agent and preservative. "Pharmaceutically acceptable excipient" refers to such excipient: it is compatible with other components in the composition, and it is suitable for contacting any tissue, organ or position of the health that it may encounter, which means that it must not have the risk of toxicity, stimulation, allergic response, immunogenicity or any other complication that far exceeds its therapeutic benefit. In certain embodiments, excipient can be used to stabilize the agonist in the solution or increase onset and maximum efficacy. Such excipient can comprise mannitol, sorbitol, metacresol, EDTA and citrate.

[0144] "Buffer" is a substance that resists pH changes through the action of its acid-base conjugate components. In certain embodiments, the composition of the present invention has a pH of about 5.5 to about 9.0, preferably about 7.0 to about 8.0, more preferably about 7.2 to 7.8. Buffers suitable for controlling the pH of the composition of the present invention within the desired range include, but are not limited to, reagents such as phosphates, acetates, 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 pharmacologically acceptable salt thereof. Free alkali and hydrochloride forms (i.e., TRIS-HCl) are two common forms of TRIS. TRIS is also known in the art as tris(hydroxymethyl)aminomethane, tromethamine and tris(hydroxymethyl)aminomethane. Preferred buffers in the composition of the present invention are citrates or citric acid, phosphates and TRIS.

[0145] Compound disclosed herein can include one or more protein components (for example, wherein X1 includes one or more protein components). In certain embodiments, compound disclosed herein includes two or more fusion protein components or two or more conjugated protein components. As used herein, the term "fusion protein" refers to a combination of two or more different proteins, wherein the protein is directly linked together via a peptide bond between the C-terminus of a protein and the N-terminus of another protein, or is indirectly linked together via a joint (i.e., by a chemical joint, such as a biofunctionalized PEG joint, or by a continuous amino acid chain), wherein the joint connects the C-terminus or N-terminus or side chain of a protein and the C-terminus or N-terminus or side chain of another protein. As used herein, the term "conjugated protein" refers to a combination of two or more different proteins, wherein the protein is chemically linked together directly or indirectly by a chemical joint by a chemical bond. In some cases, two or more proteins may act on identical or similar receptors (i.e., the fusion of two insulin agonist peptides), or may act on separate and different receptors (i.e., a protein is an insulin receptor agonist, and another protein is a glucagon receptor agonist). In some cases, the fusion protein may contain only one agonist protein, with the other protein being a human IgG Fc region or a single domain antibody (nanobody) or a variable heavy chain sequence (V H H).

[0146] The phrase "composition comprising fusion protein " encompasses monomers, homodimers, heterodimers or multimeric compositions comprising fusion protein. In certain embodiments, the pharmaceutical composition of the present disclosure is a composition comprising a fusion protein having 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 larger. In certain embodiments, the fusion protein exists at a concentration of 10-100 mg / mL. In certain embodiments, the fusion protein exists at a concentration of 15-75 mg / mL, and in certain embodiments, the fusion protein exists at a concentration of 20-65 mg / mL.

[0147] The pharmaceutical compositions of the present disclosure may also contain a "surfactant," a substance that reduces the surface tension of a liquid. Examples of surfactants used in pharmaceutical compositions and useful 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 ethers (e.g., BRIJ), polypropylene glycol, block copolymers (e.g., poloxamer, PLURONIC F68; poloxamer 407, PLURONIC F127; TETRONICS), sorbitan alkyl esters (e.g., SPAN), polyethoxylated castor oil (e.g., KOLLIPHOR, CREMOPHOR), and trehalose.

[0148] Pharmaceutical composition of the present invention can also contain preservative.Term " preservative " refers to the compound that adds in pharmaceutical preparation to serve as antimicrobial agent.It is known in the art that effective and acceptable preservative in parenteral preparation comprises benzalkonium chloride, benzethonium chloride (benzethonium), chlorhexidine, phenol, meta-cresol, benzyl alcohol, methyl or propyl parahydroxybenzoate, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid and its various mixture.Phenolic preservative comprises compound phenol, meta-cresol, o-cresol, p-cresol, chlorocresol, methyl parahydroxybenzoate, benzyl alcohol and its mixture.If preservative is necessary, the preservative used in the present composition is preferably phenolic preservative, preferably meta-cresol, phenol and / or benzyl alcohol.Known some phenolic preservatives (such as phenol and meta-cresol) can combine insulin and insulin hexamer, thereby stabilizing conformational change, and this can increase physical or chemical stability or both. However, in compositions containing other proteins, such preservatives may promote the formation of protein aggregates or high molecular weight polymers (HMWPs). See, for example, Maa YF and Hsu CC, Int J Pharm 140: 155-168 (1996); Fransson J, et al., Pharm. Res., 14: 606-612 (1997); Lam XM et al., Pharm. Res., 14: 725-729 (1997); Remmele RL Jr et al., Pharm Res 15: 200-208. (1998); Thirumangalathu R et al., J Pharm Sci 95: 1480-1497 (2006). In some cases, protein aggregates in therapeutic formulations may be undesirable because they tend to induce an immune response.

[0149] " conjunctive therapy " used herein or " combined " administration with one or more other therapeutic agents relates to the administration of two or more activating agents (for example, two or more pharmacological agents) intended to treat predetermined indications and / or conditions associated therewith. The administration can be simultaneous (in parallel) or continuous (in sequence) in any order. The two or more medicaments of " conjunctive therapy " can be formulated into independent compositions (for example, multiple preparations), or can be formulated into single compositions (a preparation). " conjunctive therapy " encompasses the administration of medicaments with different mechanisms of action or targeting different indications and / or conditions and medicaments with similar mechanisms of action or targeting similar indications and / or conditions. For example, because type 1 diabetes patients hardly produce or do not produce insulin, the effective insulin therapy of type 1 diabetes patients may relate to the use of two types of exogenously administered insulin: a fast-acting mealtime insulin provided by rapid bolus injection, and a long-acting basal insulin administered once or twice a day to control blood sugar levels between meals. Treatment for patients with type 2 diabetes typically begins with prescribed weight loss, exercise, and a diabetic diet; however, when these measures fail to control elevated blood sugar, oral medications and incretin-based therapies may be needed, such as administration of glucagon-like peptide-1 (GLP-1) receptor agonists and / or dipeptidyl peptidase 4 (DPP-4 inhibitors, which increase incretin levels. When these medications are still insufficient, treatment with insulin may be considered. For patients with type 2 diabetes whose disease has progressed to the point where insulin therapy is required, a single once-daily injection of long-acting basal insulin may also be initiated, although in some cases rapid-acting insulin may be injected with meals as needed. In certain embodiments, the present disclosure provides a combination therapy comprising administration of rapid-acting insulin and basal insulin, and / or a fusion protein comprising, for example, one or more diboronate sensors disclosed herein.

[0150] The term "administering" includes any method or behavior of delivering a pharmacological agent (e.g., a drug) to an intended subject (e.g., a patient). The pharmacological agent can be any suitable therapeutic agent, such as a biological agent, such as an antibody or its antigen-binding fragment (e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment), a peptide agent (e.g., a hormone or its modified analogs) or a low molecular weight agent (e.g., a structurally determined small molecule or chemical entity). The administration of a pharmacological agent can be systemic administration or topical administration. In certain embodiments, administration can involve one or more pharmacological agents that can be administered in parallel, simultaneously or sequentially.

[0151] The terms "simultaneously" and "concurrently" are used interchangeably and are used herein to refer to the administration of two or more therapeutic agents, wherein at least a portion of the administration overlaps in time, or wherein administration of a second therapeutic agent occurs within a short period of time after administration of a first therapeutic agent, no longer than the time required to initiate the subsequent administration. For example, concurrent administration includes administering a second agent after a first agent without additional delay beyond the time required to complete the first administration and initiate the second administration.

[0152] The terms "sequentially" and "continuously" are used interchangeably and are used herein to refer to the administration of two or more therapeutic agents with a delay period between the administration of one therapeutic agent and the administration of another therapeutic agent. For example, sequential administration includes administering two or more therapeutic agents at intervals of more than about 15 minutes, such as any of about 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, or 1 month or longer.

[0153] As used herein, the terms "basal insulin" and "basal insulins" may refer to several types of basal insulin (e.g., long-acting insulin). For example, in the commercial name Insulin glargine, marketed under the trade name Rezvoglar, comprises a modified insulin structure in which the asparagine at position 21 of the insulin A-chain is replaced by glycine and two arginines are added to the C-terminus of the B-chain. TM Insulin glargine, sold under the trade name glargine-algr, also comprises a modified insulin structure in which the asparagine at position 21 of the insulin A-chain is replaced by glycine and two arginines are added to the C-terminus of the B-chain. Insulin detemir marketed under the trade name ANTI-INSULIN comprises a modified insulin structure in which the threonine at position 30 of the B-chain has been deleted and the lysine at position 29 of the B-chain has been derivatized by covalent attachment of a 14-carbon myristoyl fatty acid to the E-amine group of the lysine at B29. The insulin degludec sold under the [preferred] label comprises a modified insulin structure in which the threonine at position 30 of the B-chain has been deleted and the ε-amino group of the 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 indicated for once-daily administration. In certain embodiments, the disclosure provides the use of one or more compounds (e.g., Formula I, fusion proteins) in the preparation of a medicament for the treatment of a disease (e.g., diabetes, obesity, dyslipidemia, or metabolic syndrome), wherein the medicament is to be administered simultaneously, separately, or sequentially in combination with another active ingredient. By administering a therapeutically effective amount of a compound and / or composition of the present disclosure to a patient in need thereof, the compounds and combinations disclosed herein (e.g., compounds of Formula I, fusion proteins) can effectively treat diseases and / or conditions of a subject in need thereof.

[0154] The phrases "therapeutically effective amount" and "prophylactically effective amount" used herein refer to an amount that provides a therapeutic benefit in treating, preventing or managing a disease or a significant symptom of a disease. The therapeutically effective amount can treat a disease or condition, a symptom of a disease or a disease tendency with the purpose of treating, curing, alleviating, alleviating, changing, remedying, improving, modifying or influencing a disease, a symptom of a disease or a disease tendency. The set amount or specific amount effective in treatment can be easily determined by a general medical practitioner and can vary according to factors known in the art (e.g., the type of disease, the patient's medical history and age, the stage of the disease and the administration of other therapeutic agents). In certain embodiments, the phrase "therapeutically effective amount" refers to an amount of a compound and / or pharmaceutical composition and / or a combination of active substances disclosed herein that is sufficient to regulate the patient's blood sugar without causing unacceptable side effects. The therapeutically effective amount of the compound and / or pharmaceutical composition and / or combination of active substances disclosed herein administered to a subject will depend on the type and severity of the disease and on the characteristics of the subject, such as overall health, age, sex, weight and tolerance to the drug. A skilled person will be able to determine an appropriate dosage based on these and other factors. For example, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 0.01 nmol / kg to about 100 nmol / kg. In certain embodiments, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 1 nmol / kg to about 50 nmol / kg. In certain embodiments, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 16 nmol / kg to about 25 nmol / kg. In certain embodiments, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 1 mg to about 200 mg. In certain embodiments, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 25 mg to about 175 mg. In certain embodiments, when administered once a week, the scope of the therapeutically effective amount of the fusion protein of the present disclosure is about 100 mg to about 160 mg.

[0155] The terms "subject" and "patient" are used interchangeably herein and refer to a party receiving therapy or treatment. In certain embodiments, the "subject" and "patient" are humans.

[0156] The terms "blood sugar level" and "blood glucose" 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 typically measured in mg of sugar per dL of blood (mg / dL). Blood glucose level can refer to the level of any sugar disclosed herein. For example, blood glucose level includes the concentration of glucose in the blood, also referred to as blood glucose level / concentration.

[0157] As used herein, the term "steady state" refers to a constant / fixed state in which the variable of interest does not increase or decrease or changes very little (i.e., less than 10% or less). By way of context, in certain embodiments, the insulin receptor agonist is infused intravenously (IV) as a fixed dose bolus, and blood glucose levels are maintained at a predetermined "steady state" level (e.g., 100 mg / dL for a normoglycemic state; 200 mg / dL or 400 mg / dL for a hyperglycemic state) by continuous infusion of glucose at a variable rate (mg / kg / min). The amount of glucose infused to maintain a "steady state" blood glucose level is equal to the total body glucose uptake and utilization.

[0158] The term "hyperglycemia" is used herein to refer to physiologically high blood sugar levels. Hyperglycemia refers to a state of physiologically high blood sugar levels. The blood sugar level considered hyperglycemic will vary based on the species of the subject. For example, in rats, hyperglycemia refers to a blood sugar level of ≥200 mg / dL. In humans, hyperglycemia refers to a blood sugar level of >180 mg / dL.

[0159] The term "normal blood sugar" is used herein to refer to physiologically normal blood sugar levels. Normal blood sugar refers to a state of normal blood sugar levels. The blood sugar level considered normal blood sugar will vary based on the species of the subject. For example, in rats, normal blood sugar refers to a blood sugar level of <200 mg / dL. In humans, normal blood sugar refers to a blood sugar level of 70-180 mg / dL.

[0160] 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 as follows in mg / kg / min: (infusion rate (mL / hr) x glucose concentration (g / dL) x 1000 (mg / g)) / weight (kg) x 60 (min / hr) x 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 glucose being infused, and "weight" refers to the weight of the subject. In certain embodiments, the GIR corresponds to the glucose infusion rate required to maintain a specific blood glucose level.

[0161] As used herein, phrase " relative glucose infusion rate difference " refers to the difference in the amount of glucose infused between two different conditions, experiments or measurements. In certain embodiments, it can be measured by taking the difference between the area under the curve (AUC) of the GIR for maintaining a specific blood glucose concentration recorded by (a) and the AUC of the GIR for maintaining different blood glucose concentrations recorded by (b) another. Typically, the AUC for maintaining a lower blood glucose concentration is subtracted from the AUC for maintaining a higher blood glucose concentration. For example, when the AUC of a first GIR (assuming that blood glucose concentration is 100mg / dL) is 600mg / kg / min.min and the AUC of a second GIR (assuming that blood glucose concentration is 200mg / dL) is 800mg / kg / min.min, the AUC of the first GIR is subtracted from the AUC of the second GIR to provide a relative glucose infusion rate difference of 200mg / kg / min.min.

[0162] 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 certain embodiments, it can be measured by taking the ratio between the area under the curve (AUC) of a recorded GIR for maintaining a blood glucose concentration and the AUC of another recorded GIR for maintaining different glucose concentrations. Typically, the AUC of the GIR for maintaining a higher blood glucose concentration is divided by the AUC of the GIR for maintaining a lower blood glucose concentration. For example, when the AUC of a first GIR (assuming that the blood glucose concentration is 100 mg / dL) is 600 mg / kg / min.min and the AUC of a second GIR (assuming that the blood glucose concentration is 200 mg / dL) is 800 mg / kg / min.min, the AUC of the second GIR divided by the AUC of the first GIR provides a relative glucose infusion rate ratio of 1.33.

[0163] As used herein, the phrase "area under the curve" refers to the area enclosed by the curve, the axis, and the two boundary points, whether drawn or mathematically represented. In certain embodiments, the curve used to calculate the area under the curve (AUC) is a measure of GIR over time, wherein the X-axis corresponds to time, the Y-axis corresponds to GIR, and the origin is located at 0 on the Y-axis (i.e., the X-axis and the 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 are the GIR at the beginning and end of the infusion in the experiment. The AUC is then calculated for the area between the drawn curve and the X-axis, and between the starting and ending boundary points. Mathematically, AUC can be calculated according to methods known to those skilled in the art, for example, as described in Tai M.M. (1994) Diabetes Care, 17 (2): 152-154 (the contents of which are hereby incorporated by reference in their entirety). In certain embodiments, the area under the curve (AUC) is calculated using the trapezoidal rule and applying baseline correction. In certain embodiments, AUC is calculated using GraphPad Prism v9. For baseline correction, the mean GIR value from 30 minutes before injection (x = -30 min) to the time of injection (x = 0) can be subtracted from each GIR value from time 0 (x = 0) to the time of the last time point measured (x = 300). The trapezoidal value is calculated as follows:

[0164]

[0165] Where a is the first time point (e.g., 0 min) and b is the last time point (e.g., 300 min). The area is calculated as follows using the subdivision of the small trapezoid:

[0166]

[0167] Let Δx be the difference between each time point (5 minute intervals).

[0168] The term "EC50" as used herein refers to the 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 the maximum possible effect due to exposure to a compound. EC50 can be calculated using methods known in the art. In certain embodiments, EC50 can be calculated using a four-parameter logistic regression curve using the following equation: Y = bottom + (X^Hillslope) * (top-bottom) / (X^Hillslope+EC50^Hillslope), where Hillslope refers to the slope of the S-shaped curve between the top and bottom platforms of the dose-response curve. In certain embodiments, EC50 can be calculated using GraphPad Prism v7, 8 or 9. When measuring the EC50 of a compound at two or more sugar concentrations (e.g., glucose), (a) the term "first sugar concentration" refers to the first lower concentration of sugar, for example, when the concentration is about 3 mM or about 5 mM, and (b) the term "second sugar concentration" refers to the second higher concentration of sugar, for example, when the concentration is about 10 mM, about 20 mM or about 30 mM. In certain embodiments, for example, the EC50 of the comparative dose-response curve is to assess the compound of exemplary Formula I from low (for example, about 3mM or about 4mM) to insulin receptor phosphorylation (IR phosphorylation) activity fold change of high glucose concentration (for example, about 10mM, about 20mM or about 30mM).The activity fold change is determined by dividing the EC50 of the compound (for example, Formula I compound) at "low" glucose concentration (for example, about 3mM) by the EC50 of the compound at "high" glucose concentration (for example, about 20mM), wherein all other conditions remain unchanged.Referring to the embodiment entitled "In vitro activity confirmation of compound of Formula I".

[0169] As used herein, the term "Kd" refers to the dissociation constant and reflects the binding affinity between a ligand (e.g., a diboronate sensor as described herein) and its target (e.g., a sugar, e.g., glucose). For example, "glucose Kd" and "average glucose Kd" in the context of the diboronate sensors described herein refer to the affinity of the diboronate sensor for glucose, wherein the Kd is measured before the diboronate sensor is conjugated to an insulin molecule to generate the compound described herein. The binding of the diboronate sensor described herein to glucose can be measured by an Alizarin Red S (ARS) displacement assay and recorded before the diboronate sensor is conjugated (e.g., covalently bound) to an insulin molecule (e.g., a compound of Formula I). ​​ARS displacement assays are known in the art, for example, in Springsteen and Wang (2001) Chem. Comm., 1608-1609, the contents of which are incorporated herein by reference in their entirety. In an ARS displacement assay, a diboronate sensor disclosed herein is incubated with ARS and fluorescence emission is recorded. The composition of the diborate sensor and ARS described herein is then titrated against serial dilutions of a sugar (e.g., glucose), and the fluorescence emission is then measured after incubation to determine the displacement of the ARS when the diborate sensor binds to the sugar. The change in intensity (fluorescence emission with and without sugar) can be plotted against the sugar concentration to generate an association constant for sugar binding. When the diborate sensor has a higher association constant for sugar, the Kd increases. In addition, the diborate sensors described herein include those that have selective affinity / binding for sugars. For example, the diborate sensors described herein can include sensors that have increased affinity (binding) for glucose but not for other sugars (such as lactate and / or fructose). See the example entitled "Procedure for Determining Glucose, Fructose, and Lactate Binding (Kd) Using an ARS Displacement Assay."

[0170] The terms "clamp", "clamp assay" and "clamped" used herein refer to a hyperglycemic / euglycemic clamp (glucose clamp), which has been recognized as a "gold standard" method for detecting insulin activity by glucose utilization in experimental animals and humans. In a hyperglycemic / euglycemic clamp, plasma (blood) insulin concentration rises sharply, and in certain embodiments, is maintained by continuous insulin infusion while plasma glucose concentration is kept constant at a predetermined hyperglycemic or normoglycemic level by a variable glucose infusion rate (GIR). When steady-state blood glucose levels are reached, the glucose infusion rate is equal to the glucose uptake rate stimulated by the insulin receptors of all tissues in the body, and is therefore a measure of insulin activity under a specific blood glucose level. For example, in certain embodiments, glucose is continuously infused into the subject during the study, and the glucose infusion rate is adjusted to maintain a constant blood glucose level in response to the administration of a compound (e.g., a compound of Formula I). ​​The changes in the sugar infusion rate that occur in response to the administration of a compound at a specific dose level or concentration are recorded and used to determine the sugar infusion rate. In certain embodiments, the sugar is glucose. See, e.g., Lautt WW, et al. (1998) Canadian Journal of Physiology and Pharmacology. 76(12): 1080-1086, the contents of which are hereby incorporated by reference in their entirety. The clamp techniques / assays mentioned and used herein are modified versions of techniques known and disclosed in the art. See the examples entitled "Demonstration of in vivo activity of compounds of Formula I."

[0171] In at least some embodiments, one or more groups (e.g., X1a, Z1a, Z1c) are covalently conjugated to each other directly or indirectly (e.g., via an indirect linker). For example, according to certain embodiments, Z1c is covalently conjugated to an amine in X1 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 conjugated to one or more amine-containing linkers. In certain embodiments, X represents a covalent attachment point that is directly linked to an amine in X1 or to an amine that is covalently conjugated directly or indirectly (e.g., via an indirect linker) to X1. In certain embodiments, each Z1c is independently directly or indirectly (e.g., via a group of formula (X") n1 In certain embodiments, at least one Z1c is covalently conjugated to an amine of Z1a or an amine of X1 via an indirect linker (e.g., formula (X") n1 ) is covalently conjugated to X1 or Z1a.

[0172] As used herein, terms such as "point of attachment toward [group]," "connected to," and "covalent bond toward [group]" mean that the indicated atom, connection, or bond is closer to the indicated group than other points of attachment or covalent bond variables in the structural formula. In certain embodiments, the point of attachment or covalent bond can be directly adjacent to the indicated group, and in certain embodiments, other atoms or groups can be present therebetween.

[0173] As used herein, the term "homology percentage" refers to the percentage of sequence identity between two sequences after optimal alignment. Identical sequences have a 100% homology percentage. Optimal alignment can be performed using the homology alignment algorithm described by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), the general method described for searching similarities by Neddleman and Wunsch, J. Mol. Biol. 48: 443 (1970), including implementations of these algorithms or visual comparisons. "Insulin A-chain" as used herein is the insulin chain with the highest homology percentage to the A-chain of wild-type human insulin. "Insulin B-chain" as used herein is the insulin chain with the highest homology percentage to the B-chain of wild-type human insulin.

[0174] In certain embodiments, the terms "covalently linked," "covalently conjugated," or "by covalent conjugation" may be used interchangeably to indicate that two or more atoms, groups, or chemical moieties are bonded or connected by a chemical bond. In certain embodiments, the chemical bond (which in certain embodiments may be referred to as a covalent bond) may be one or more shared electron pairs (e.g., in a single bond, a double bond, or a triple bond) between two atoms, groups, or chemical moieties (e.g., consisting of). In certain embodiments, the chemical (covalent) bond may further comprise one or more atoms or functional groups and may be referred to by the corresponding names of the functional groups in the art. For example, a covalent bond comprising an -SS- group may be referred to as a disulfide bond; a covalent bond comprising a -(C=O)- group may be referred to as a carbonyl bond; a covalent bond comprising a -(CF2)- group may be referred to as a difluoromethylene bond, and so on. The type of connection or functional group within a covalent bond is not limited unless explicitly stated, for example, when it is described as comprising or selected from certain groups. The type or kind of suitable covalent bond will be understood from the description and / or context.

[0175] In certain embodiments, the insulin receptor agonist can bind or associate with human serum albumin (HSA) prior to binding to the insulin receptor. For example, an insulin receptor agonist comprising any one of DSL-1 to DSL-112 can associate or bind to HSA via hydrogen bonding, ionic bonding, or hydrolyzable boron ester bonds. These interactions can occur on the surface of HSA, such as salt bridges with lysine residues, cleavable boron ester bonds formed with serine, threonine, and / or tyrosine hydroxyl groups, and / or occur at specific locations 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).

[0176] In certain embodiments, the side chain of amino acid can be covalently linked (for example, connected or cross-linked) by any number of chemical bonds (for example, bonding moiety), as generally described in Bioconjugate Techniques (3rd edition), Greg T. Hermanson compiles, Academic Press, Boston, 2013. For example, the side chain can be covalently linked by amides, esters, ethers, thioethers, isoureas, imines, triazoles or any suitable covalent conjugation chemistry available in the art to covalently link a peptide, protein or synthetic polymer to a second peptide, protein or synthetic polymer. The term polymer includes polypeptides. The term "covalent conjugation chemistry" can represent one or more functional groups included in the bonding moiety, and / or the chemical reaction for forming the bonding moiety.

[0177] The term "vicinal diol" refers to a group of molecules in which the two hydroxyl groups occupy adjacent positions, that is, they are attached to adjacent atoms. Such molecules can include, but are not limited to, sugars such as hexose, glucose, mannose, and fructose.

[0178] In certain embodiments, the term "albumin" refers to human serum albumin or a protein that has at least 60% homology to a human serum albumin protein. It should be understood that in certain embodiments, albumin can be further chemically modified for conjugation purposes. In certain embodiments, the modification can include one or more covalently attached linkers. In certain embodiments, the term "albumin" refers to human serum albumin or a protein that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a human serum albumin protein. In certain embodiments, the term "albumin" refers to human serum albumin or a protein that has at least 90% homology to a human serum albumin protein. In certain embodiments, the term "albumin" refers to human serum albumin or a protein that has at least 95% homology to a human serum albumin protein. In certain embodiments, the term "albumin" refers to human serum albumin or a protein that has at least 99% homology to a human serum albumin protein. In certain embodiments, the albumin is unmodified human serum albumin.

[0179] The term "treatment" is intended to include both prevention and minimization of the disease, disorder, or condition being mentioned (i.e., "treatment" refers to the prophylactic and therapeutic administration of a compound of the present invention or a composition comprising a compound of the present invention, unless otherwise indicated or clearly contradicted by the context). The route of administration can be any route that effectively transports the compound of the present disclosure to the desired or appropriate location in the body, such as parenteral, for example, subcutaneous, intramuscular, oral, or intravenous. For parenteral administration, the compound of the present disclosure is formulated similarly to the formulations of known insulins. In addition, for parenteral administration, the compound of the present disclosure is administered similarly to the administration of known insulins, and physicians are familiar with the procedure. The amount of the compound of the present disclosure to be administered, the determination of the frequency of administering the compound of the present disclosure, and the selection of which one or more compounds of the present disclosure to be administered (optionally together with another antidiabetic compound) are all determined in consultation with practitioners familiar with the treatment of the condition to be treated (e.g., diabetes).

[0180] In certain embodiments, "therapeutic composition" and "pharmaceutical composition" as used herein refer to compositions intended to have a therapeutic effect, such as pharmaceutical compositions, genetic materials, biological products and other substances. Pharmaceutical compositions can be configured to act with therapeutic properties in vivo; the concentration can be changed to reduce the frequency of supplementation, etc. In certain 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 a significant symptom of a disease. The therapeutically effective amount can treat a disease or condition, a symptom of a disease or a disease tendency, with the purpose of treating, curing, alleviating, alleviating, changing, remedying, improving, improving or influencing a disease, a symptom of a disease or a disease tendency. The set amount or specific amount that is effective in treatment can be easily determined by ordinary medical practitioners and can vary according to factors known in the art (e.g., the type of disease, the patient's medical history and age, the stage of the disease and the administration of other therapeutic agents). In certain embodiments, the modified insulin described herein is 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 certain embodiments, the modified insulins described herein are released from local reservoirs in the body or from a form bound to proteins in the serum (such as albumin) over an extended period of time. In certain embodiments, the release of the modified insulin is accelerated under elevated glucose levels, and in certain embodiments, such a rate of release may depend on blood glucose levels or the levels of other small molecules (including glycol-containing molecules) in the blood. In certain embodiments, the release, bioavailability, and / or solubility of the modified insulins described herein are controlled based on blood or serum glucose concentrations or the concentrations of other small molecules in the body.

[0181] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, a structure having the present invention but with hydrogen replaced by deuterium ( 2 H) or tritium ( 3 H) Replacement or carbon 13 C- or 14 Compounds with C-carbon atom substitutions are within the scope of the present disclosure. Such compounds are useful, for example, as analytical tools, probes in biological assays, or therapeutic agents.

[0182] In certain embodiments, the functional group can be covalently conjugated or connected via any suitable covalent conjugation chemistry (linker), which can be used to covalently conjugate a functional group or amino acid side chain to another functional group, non-limiting examples include amides, esters, ethers, thioethers, isoureas, imines and triazole linkers. In certain embodiments, the functional group is covalently conjugated by click chemistry reactions as defined in the art. These include, for example, cycloaddition reactions, including but not limited to 3+2 cycloadditions, stress-driven alkyne-nitrone cycloadditions, strained olefins, alkenes and tetrazines reverse demand Diels-Alder reactions, copper (I)-catalyzed azide-alkyne cycloadditions (CuAAC), thiol-maleimide additions, stress-driven azide-alkyne cycloadditions, Staudinger connections, nucleophilic ring-opening reactions and additions to carbon-carbon multiple bonds. Some of these reactions are described, for example, by HC Kolb, MG Finn, and KB 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, NJ; Baskin, JM; Prescher, JA; Lo, A.; Bertozzi, CRACS Chem. Biol. 2006, 1, 644. One skilled in the art will be able to select appropriate buffers, pH, and reaction conditions for such click reactions. In certain embodiments, the covalent conjugation is the result of a "bioorthogonal reaction," as defined in the art. Such reactions are described, for example, in 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.

[0183] In certain embodiments, functional groups can be linked using native chemical linkages, such as described, for example, by Dawson, PE; Muir, TW; Clark-Lewis, I.; Kent, SB (1994) Synthesis of proteins by native chemical ligation, Science 266(5186):776-778. Terms such as "linked," "covalently conjugated," and the like used herein may refer to any of the above chemistries in certain embodiments. The terms "amine," "amino," and / or "amine group" when used to describe a portion of a covalent bond or connectivity may be used interchangeably to indicate an amino group or an amine group to which the described elements are covalently linked. In certain embodiments, the amino group or amine group may be a primary amine, a secondary amine, or a fragment such as a amine group to which the conjugation is performed and described.

[0184] In certain embodiments, the amino or amine group can be the NH2 group at the N- end of a peptide or peptide chain or the NH2 group of a lysine side chain, but the embodiments of the present disclosure are not limited thereto. In certain embodiments, the connectivity of the first group and the second group is described by reference to the amine or amino group derived from the second group, and the amine or amino group is a part of the covalent bond between the first group and the second group. For example, the amine of the lysine side chain on X1 can be referred to as an amine, and can be described as being conjugated by an amide bond, so as to specify the structure and connectivity of the functional group constituting a covalent bond. If a covalent bond is via an amine bond (amine bond) or an amine bond (amine linkage), it is referred to as an amine bond. It should be understood that the carbonyl (for example, (C=O)-NH part) connected to an amine constitutes an amide bond, and therefore by definition, an amine bond is not directly connected to a carbonyl group. In other words, the terms "amide bond" and / or "amide linkage," when used to describe a covalent bond or connectivity, can be used interchangeably to refer to a carbonyl group (eg, a (C=O)-NH moiety) attached to an amine.

[0185] In certain embodiments, further modifications include the attachment of chemical entities (e.g., moieties or functional groups) such as 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, or linkers for conjugation, functionalization, or other modifications intended to affect the pharmacokinetic, pharmacodynamic, and / or biophysical solution characteristics of the insulin.

[0186] In certain embodiments, the compound includes a human peptide hormone (e.g., as X1). In certain embodiments, the peptide hormone is a polypeptide hormone of the human pancreas. In certain embodiments, the compound, such as a compound of Formula I, includes human insulin or a human insulin analog. In certain embodiments, the two different amine groups in insulin are covalently conjugated as shown in Formula I.

[0187] It should be understood that human peptide hormones, human pancreatic polypeptide hormones, insulin, human insulin, modified insulin, and human insulin analogs can be used interchangeably in some of the described embodiments; that is, for example, in certain embodiments, in the described embodiments using human insulin, "human insulin analogs" can be used alternatively. In certain embodiments, compounds, such as compounds of Formula I, include human insulin or human insulin analogs. In certain embodiments, compounds of Formula I include human insulin or human insulin analogs as described in Formula I (p'=1), wherein a single amino group in insulin is conjugated as described in Formula I. In certain embodiments, the amino group is the N-terminus of the B-chain of insulin or the amino group of a lysine side chain. In certain embodiments, two or more different amine groups in insulin are each independently covalently conjugated as described in Formula I. In certain embodiments, at least one amine group is the N-terminus of the B-chain of insulin. In certain embodiments, the amino group comprises the amino group of the side chain of a lysine residue in insulin.

[0188] Various suitable modifications of peptide hormones (e.g., human polypeptide hormones, e.g., insulin) known to those of ordinary skill in the art are included within the scope of the present disclosure. In certain embodiments, the polypeptide of Z1a or the optional extended polypeptide at the N-terminus of the B-chain of insulin or the C-terminus of the A-chain contains a sequence with up to 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the human polypeptide sequence. In certain embodiments, the polypeptide of Z1a or the optional extended polypeptide at the N-terminus of the B-chain of insulin or the C-terminus of the A-chain contains one or more lysine residues, which are optionally adjacent to a proline residue such that the proline is located at the C-terminus of the lysine. In certain embodiments, the amino groups of the lysine residues are each independently conjugated as described in Formula I.

[0189] In certain embodiments, insulin is further modified by conjugation to a molecule containing a sugar or glycol. In certain embodiments, the human polypeptide hormone is a dual or triple hybrid peptide comprising two or more human peptide hormone sequences and capable of acting through multiple receptors; for example, a glucose-dependent insulin-releasing polypeptide (GIP) and a GLP-1 receptor agonist or a GLP-1 / GIP / glucagon triple agonist. In certain embodiments, the human polypeptide hormone is an intestinal hormone. In certain 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, neuropeptide, the peptide hormone that affects cardiovascular health or appetite, one or more hybrids of these peptides and an analog of one of these peptides. In certain embodiments, the compound comprises a further modified human polypeptide hormone, for example, by being covalently conjugated to a polymer, XTEN protein sequence or aliphatic chain. In certain embodiments, the polymer-modified compound has a longer circulation time in the blood. In certain embodiments, the polymer-modified compounds have a circulation time in the blood that makes them suitable for daily, weekly, or monthly injections. In certain embodiments, the human polypeptide hormone or its analog comprises one or more L- or D-natural or unnatural amino acids, each of which is independently one of the twenty canonical amino acids or a non-canonical amino acid.

[0190] In certain embodiments, the insulin receptor agonist is an insulin analog comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 1, 25, 24051, and 24052, and the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24060, 24061, 24062, 24063, 24064, and 25000-25397. In certain embodiments, the insulin analog comprises an A-chain comprising sequence 24051 and a B-chain comprising a sequence selected from the group consisting of 25000, 25001, 25006-25009, 25076, 25077, 25082-25085, 25228, 25229, 25232, 25234-25237, 25304, 25305, 25308, and 25310-25313. In certain embodiments, the insulin analog comprises an A-chain comprising the sequence 24051 and a B-chain comprising a sequence selected from the group consisting of 25011, 25012, 25017-25020, 25087, 25088, 25093-25096, 25229, 25239, 25232, 25240, 25245-25248, 25305, 25308, 25315, 25316, and 25321-25324. In certain embodiments, the insulin analog comprises an A-chain comprising the sequence 24051 and a B-chain comprising a sequence selected from the group consisting of 25228, 25229, 25232, 25234-25237, 25304, 25305, 25308, and 25310-25313. In certain embodiments, the insulin analog comprises an A-chain comprising sequence 24051 and a B-chain comprising a sequence selected from 25011, 25012, 25017-25020, 25087, 25088, 25093-25096, 25229, 25232, 25305, and 25308. In certain embodiments, the insulin analog comprises human insulin having an A-chain and a B-chain, wherein the A-chain and / or the B-chain have each been mutated, deleted, or otherwise inserted with up to six residues. In certain embodiments thereof, the insulin analog comprises an insulin comprising an A-chain and a B-chain, wherein a connecting peptide connects the C-terminus of the B-chain to the N-terminus of the A-chain, and the connecting peptide comprises native proinsulin C-peptide and shorter versions of C-peptides known in the art, wherein the shorter version of the C-peptide allows the single-chain insulin to retain biological activity and / or potency.

[0191] The words analog and analogue are alternative spellings of the same word and are used interchangeably herein and have the same meaning. In the context of human hormones, endocrine hormones, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory peptide, glucose-dependent insulin-releasing polypeptide, hybrid peptides comprising sequences from two or more human polypeptide hormones, analogs refer to any related sequence derived from the parent sequence, which includes up to 8 amino acid mutations, deletions or insertions and / or additional chemical modifications. In certain embodiments, such analogs can enhance biological activity, pharmacokinetics, pharmacodynamics, efficacy, stability and / or chemical and physical characteristics, as known in the art.

[0192] In certain embodiments, human hormone analogs include one or more residues that are 2-aminoisobutyric acid and / or other artificial (ie, non-natural) amino acids.

[0193] In certain embodiments, in an insulin analog, the C-terminus of the B-chain of insulin is covalently conjugated to the N-terminus of the A-chain. In certain embodiments, the C-terminus of the B-chain of insulin is covalently conjugated to the N-terminus of the A-chain, and the connecting peptide is a C-peptide. In certain embodiments, the C-terminus of the B-chain of insulin is covalently conjugated to the N-terminus of the A-chain, and the connecting peptide is a C-peptide, and further include any intermediate compounds comprising the conjugate of Formula I.

[0194] In certain embodiments, described insulin analogue comprises insulin lispro, or the modification of insulin glargine type, or affects the pharmacokinetics of insulin in vivo or any suitable modification to human insulin of half-life.In certain embodiments, the insulin lispro of the insulin lispro compound for the preparation of Pegylated of the present invention can be prepared by any of multiple generally acknowledged peptide synthesis techniques, and described technology comprises solution phase method, solid phase method, semisynthetic method and recombinant DNA method.For example, U.S. Patent number 5,700,662 (Chance, et al.) and European Patent No. 214826 (Brange, et al.) disclose the preparation of various insulin analogues.Also can use recombinant DNA technology to prepare A-chain and the B-chain of insulin lispro by the original insulin sample precursor molecule.In certain embodiments, use the original insulin sample precursor to prepare insulin lispro, this insulin lispro is used to prepare the Pegylated insulin lispro of the present invention again.

[0195] In certain embodiments, the insulin portion of the compound of the present invention can be prepared by using recombinant DNA technology to produce a precursor protein molecule. The DNA (including cDNA and synthetic DNA) can be double-stranded or single-stranded. The coding sequence encoding the precursor protein molecule described herein may vary due to the redundancy or degeneracy of the genetic code. In order to produce the precursor protein of the present invention, the DNA can be introduced into a host cell. An appropriate host cell can be transiently or stably transfected or transformed with an expression system for producing the precursor protein. The host cell can be a bacterial cell such as K12 or B strain of Escherichia coli, a fungal cell such as a yeast cell, or a mammalian cell such as a Chinese hamster ovary ("CHO") cell. The expression vector can generally be replicated in a host organism as an episome or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selection marker, for example, tetracycline, neomycin, and dihydrofolate reductase, to allow selection of those cells transformed with the desired DNA sequence.

[0196] In certain embodiments, the polypeptide hormone is glucagon. In certain embodiments, glucagon has other mutations and modifications known to affect the solubility and solution stability of glucagon. In certain embodiments, compound (such as compound of Formula I) includes Z1a by peptide bond and the N- end of the B- chain of insulin conjugated, and at least one other conjugated with insulin described by Formula I. In certain embodiments, the other conjugated is conjugated with the lysine residue in insulin. In certain embodiments, at least one such lysine is the residue between position 15 and the C- end of the B- chain of insulin. In certain embodiments, the lysine residue is optionally adjacent to proline, glycine, arginine, threonine or serine. In certain embodiments, one or more amino acids in Formula I are D-amino acids. In certain embodiments, any secondary amine or primary amine in compound (such as compound represented by Formula I) is each independently optionally acetylated. In certain embodiments, the compound of Formula I has a polypeptide hormone X1 that is further conjugated to a drug molecule, an imaging agent, a chelating agent, a contrast agent, a radioisotope, or a molecule that binds to immune cells.

[0197] In certain embodiments, X1 is a polypeptide hormone comprising a peptide ligand that binds to an extracellular protein receptor. In certain embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone that has at least 50% homology to a native human polypeptide hormone. In certain embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a native human polypeptide hormone. In certain embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone that has at least 90% homology to a native human polypeptide hormone. In certain embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone that has at least 95% homology to a native human polypeptide hormone. In certain embodiments, X1 comprises a polypeptide analog of a human polypeptide hormone that has at least 99% homology to a native human polypeptide hormone. In certain embodiments, X1 is an analog of human insulin, wherein up to 10 additional residues are added to the A-chain or B-chain of insulin.

[0198] In certain embodiments, term " glucose responsiveness " refers to the change of activity under the difference of glucose (for example, 3mM glucose vs20mM glucose) in the presence and absence of glucose.In certain embodiments, the activity of the insulin putatively is assessed by inducing the required insulin concentration of half maximum response (EC50) (insulin of nanomole unit (nM)) in the mensuration based on cell.The insulin putatively of lower EC50 concentration has the higher activity (for example, EC50 is that the insulin of 3nM is higher than the insulin activity of 50nM EC50) of the insulin of higher EC50 concentration.When respectively in the absence and presence of glucose or under lower and higher levels of glucose, when insulin becomes the higher EC50 (lower nM) of activity from the lower EC50 (higher nM) of activity, observe " glucose responsiveness ".

[0199] In certain embodiments, the compound of Formula I comprises one or more L- or D-artificial amino acids that are not one of the 20 naturally occurring amino acids. In certain embodiments, the side chains of such artificial amino acids can be covalently conjugated by a variety of reactions, including bioorthogonal reactions, such as those described by Rostovtsev, VV, Green, LG, Fokin, VV, and Sharpless, KBA stepwise huisgen cycloaddition process: copper (I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596-2599 (2002), or Liang, Y., Mackey, JL, Lopez, SA, Liu, F., and Houk, KN Control and design of mutual orthogonality in bioorthogonal cycloadditions. J. Am. Chem. Soc. 134, 17904-17907 (2012). In certain embodiments, Z1a contains one or more L- or D-artificial amino acids that are not one of the 20 naturally occurring amino acids.In certain embodiments, the side chains of two amino acids in Z1a are covalently conjugated together via a triazole bond.

[0200] The hormone insulin is an important regulator of blood glucose (sugar) levels. In normal individuals, insulin is present and, when released by the pancreas, acts to lower blood glucose levels, for example, by binding to and activating insulin receptors, thereby triggering glucose uptake by liver, fat, and skeletal muscle cells. Diabetes mellitus (DM), commonly known as diabetes, is a group of metabolic diseases characterized by long-term, persistently high blood glucose levels.

[0201] As used herein, "insulin" encompasses wild-type and altered forms of insulin that are capable of binding to and activating the insulin receptor, or that are capable of causing a measurable lowering of blood glucose when administered in vivo, and encompasses wild-type and altered forms of human insulin that are capable of binding to and activating the human insulin receptor, or that are capable of causing a measurable lowering of blood glucose when administered to a human in vivo.

[0202] In certain embodiments, insulin comprises insulin from any species, no matter it is purified, synthetic or recombinant form, and comprises human insulin, porcine insulin, bovine insulin, ovine insulin and rabbit insulin.In certain embodiments, insulin has two chains: B-chain and A-chain.In certain embodiments, described chain is linked together by peptide (for example, c-peptide as known in the art, or shortened version of c-peptide), and in other embodiments, insulin can be provided as proinsulin (insulin precursor), which can be further processed into ripe insulin.The multiple altered forms of insulin are known in the art, and can be such as by adding chemical moieties (such as PEG groups or fatty acyl chains) and chemically altered.The insulin of change may undergo sudden change, comprise amino acid whose addition, disappearance or displacement.In certain embodiments, term " desB30 " refers to the insulin lacking B30 amino acid residue.

[0203] As used herein, the term "insulin analogue" refers to a modified human insulin with insulin receptor agonist activity, wherein relative to human insulin, 1 to 10 amino acid residues of insulin have been modified (e.g., substitution, deletion, addition (i.e., extension), insertion, and any combination thereof). In this context, the insertion or addition of one or more amino acids is considered a single modification. For example, an insulin analogue may have 1 to 9 modified amino acid residues. As another example, an insulin analogue may have 1 to 8 modified amino acid residues. As another example, an insulin analogue may have 1 to 7 modified amino acid residues. As another example, an insulin analogue may have 1 to 6 modified amino acid residues. As another example, an insulin analogue may have 1 to 5 modified amino acid residues. As another example, an insulin analogue may have 1 to 4 modified amino acid residues. As another example, an insulin analogue may have 1 to 3 modified amino acid residues. As another example, an insulin analogue may have 1 to 2 modified amino acid residues. In certain embodiments, an insulin analogue may have 2, 3, 4, 5, 6, 7, 8, or 9 modified amino acid residues.

[0204] Modification in insulin molecule is represented by indicating the single letter or three letter code of the amino acid residue of chain (A or B), position and replacement natural amino acid residue.Herein, term is as " A1 ", " A2 " and " A3 " etc. represent respectively in insulin A chain at the amino acid of position 1,2,3 etc. (counting from N-terminal end).Similarly, term is as B1, B2 and B3 etc. represent respectively in insulin B chain at the amino acid of position 1,2 and 3 etc. (counting from N-terminal end).Use amino acid whose single letter code, term is as A21A, A21G and A21Q represent respectively that the amino acid in A21 position is A, G and Q.Use amino acid whose three letter code, corresponding expression is respectively A21Ala, A21Gly and A21Gln.

[0205] Thus, for example, an insulin analog with 4 modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25737); and wherein the B-chain comprises the sequence GKGSHKFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25738), wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), the B chain has been extended at the N-terminus with GKGSHK (SEQ ID NO: 25726) (i.e., 6 amino acids have been added / appended to the N-terminus), wherein the amino acid at position 21 of the B chain (i.e., E) is substituted with K, the amino acid at position 29 of the B chain (i.e., K) is substituted 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, respectively, in order of appearance)).

[0206]

[0207] As another example, an insulin analog with 7 modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCGK (SEQ ID NO: 25739); and wherein the B-chain comprises the sequence KGSHKFVDQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25740), wherein the amino acid at position 21 in the A chain (i.e., N) is substituted with G, and the A chain is extended at the C-terminus with K (i.e., 1 amino acid has been added / appended to the C-terminus), and the B chain has been extended at the N-terminus with KGSHK (SEQ ID NO: 25741). NO: 25727) is extended (i.e., 5 amino acids have been added / appended to the N-terminus), wherein the amino acid at position 3 in the B chain (i.e., N) is substituted with D, wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K, the amino acid at position 29 in the B chain (i.e., K) is substituted with R, and the amino acid at position 30 in the B chain (i.e., T) is deleted (as shown below (in order of appearance, SEQ ID NOs: 25739 and 25740, respectively)).

[0208]

[0209] As another example, an insulin analog with two modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25731); and wherein the B-chain comprises the sequence KFVNQHLCGSHLVEALYLVCGKRGFFYTPKT (SEQ ID NO: 25741), wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), the B chain has been extended at the N-terminus with K (i.e., 1 amino acid has been added / appended to the N-terminus), and wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K (as shown below (SEQ ID NOs 25731 and 25741, respectively, in order of appearance)).

[0210]

[0211] As another example, an insulin analog with 6 modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCGK (SEQ ID NO: 25732); and wherein the B-chain comprises the sequence KGSHKFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25742), wherein the amino acid at position 21 in the A chain (i.e., N) is substituted with G, the A chain is extended at the C-terminus with K (i.e., 1 amino acid has been added / appended to the C-terminus), the B chain has been extended at the N-terminus with KGSHK (SEQ ID NO: 25727) (i.e., 5 amino acids have been added / appended to the N-terminus), wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K, the amino acid at position 29 in the B chain (i.e., K) is substituted with R, and the amino acid at position 30 in the B chain (i.e., T) is deleted (as shown below (in order of appearance, SEQ ID NOs: 25743, 2575, 2576, 2577, 2578, 2579, 2580, 2591, 2634, 2647, 2650, 2661, 2679, 2680, 2691, 2701, 2713, 2714, 2725, 2734, 2747, 2691, 2650, 2661, 2679, 2680, 2691, 2680, 2692, 2734, 2693, 2747, 2680, 2694, NO25732 and 25742)).

[0212]

[0213] As another example, an insulin analog with four modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25733);; and wherein the B-chain comprises the sequence KGSHFVNQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25743); wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), and the B chain has been extended at the N-terminus with KGSH (SEQ ID NO: 25728) (i.e., 4 amino acids have been added / appended to the N-terminus), wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K, the amino acid at position 29 in the B chain (i.e., K) is substituted with R, and the amino acid at position 30 in the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs: 25733 and 25743, respectively, in order of appearance)).

[0214]

[0215] As another example, an insulin analog with 5 modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25734); and wherein the B-chain comprises the sequence KGSHQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25744); wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), and the B chain has been extended at the N-terminus with KGSH (SEQ ID NO: 25728) (i.e., 4 amino acids have been added / appended to the N-terminus), the first 3 residues (FVN) have been deleted, and wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K, the amino acid at position 29 in the B chain (i.e., K) is substituted with R, and the amino acid at position 30 in the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs 25734 and 25744, respectively, in order of appearance)).

[0216]

[0217] As another example, an insulin analog with 5 modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25735); and wherein the B-chain comprises the sequence KGSHKQHLCGSHLVEALYLVCGKRGFFYTPR (SEQ ID NO: 25745); wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), and the B chain has been extended at the N-terminus with KGSHK (SEQ ID NO: 25727) (i.e., 5 amino acids have been added / appended to the N-terminus), and the first 3 residues (FVN) have been deleted, and wherein the amino acid at position 21 in the B chain (i.e., E) is substituted with K, the amino acid at position 29 in the B chain (i.e., K) is substituted with R, and the amino acid at position 30 in the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs 25735 and 25745, respectively, in order of appearance)).

[0218]

[0219] As another example, an insulin analog with two modifications comprises an A-chain and a B-chain, wherein the A-chain comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 25736); and wherein the B-chain comprises the sequence GKGGGGSGGGGSGGGGSFVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO: 25746), wherein the A chain has the wild-type sequence of chain A of human insulin (e.g., no mutations, deletions, additions), and the B chain has been extended at the N-terminus with GKGGGGSGGGGSGGGGS (SEQ ID NO: 25729) (i.e., 17 amino acids have been added / appended to the N-terminus), and the amino acid at position 30 in the B chain (i.e., Thr) is deleted (as shown below (SEQ ID NOs 25736 and 25746, respectively, in order of appearance)).

[0220]

[0221] In certain embodiments, insulin analogs comprise chemically altered insulin compared with wild-type human insulin, such as, but not limited to, by adding chemical moieties such as PEG groups or fatty acyl chains. In certain embodiments, the insulin / insulin analogs / analogs (which are used interchangeably in this article) that change can suddenly change, including amino acid whose addition, disappearance or displacement. These variations may produce different insulin protomers, and are incorporated into certain embodiments. In certain embodiments, the active form of insulin has fewer 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 of human insulin (A-chain and B-chain) has an A-chain with the amino acid sequence of GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and a B-chain with the amino acid sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2). In certain embodiments, an insulin analog has at least 70% sequence homology to wild-type human insulin. In certain embodiments, an insulin analog has at least 80% sequence homology to wild-type human insulin. In certain embodiments, an insulin analog has at least 90% sequence homology to wild-type human insulin. In certain embodiments, an insulin analog has at least 95%, 96%, 97%, or 95% sequence homology to wild-type human insulin. In certain embodiments, an insulin analog has at least 99% sequence homology to wild-type human insulin.

[0222] Human insulin differs from rabbit, porcine, bovine, and ovine insulin in the amino acids A8, A9, A10, and B30, in the following order: Thr, Ser, Ile, Thr for humans; Thr, Ser, Ile, Ser for rabbits; Thr, Ser, Ile, Ala for pigs; Ala, Gly, Val, Ala for sheep; and Ala, Ser, Val, Ala for cattle. In certain embodiments, the modified insulins may be mutated at positions B1, B2, B28, or B29 of the B-chain, or at positions B28 and B29. In certain embodiments, the modified insulins may be mutated at A1, A2, A21, or other positions of the A-chain. For example, insulin lispro is a fast-acting modified insulin in which the lysine and proline residues at the C-terminal end of the B-chain have been reversed. Insulin aspart is a fast-acting modified insulin in which the proline at position B28 has been replaced by aspartic acid. In certain embodiments of the present disclosure, consider that the insulin of sudden change at B28 and B29 places can further include other sudden change.For example, insulin glulisine is a kind of quick-acting modified insulin, wherein the aspartic acid at position B3 place has been replaced by lysine residues, and the lysine at position B29 place has been replaced by glutamic acid residues.In certain embodiments, the insulin analogs of longer action time and higher stability are covalently modified as described in formula I, and can contain sudden change such as the tyrosine at A14 place is replaced by glutamic acid, the tyrosine at B16 place is replaced by histidine and the phenylalanine at B25 place is replaced by histidine.

[0223] In certain embodiments, use any suitable method, for example, by adding or replacing suitable amino acid, the isoelectric point of insulin herein can be made to shift relative to wild-type human insulin. In certain embodiments, the isoelectric point of modified insulin can be regulated by glucose (for example, by interacting with glucose). For example, insulin glargine is a kind of basal insulin, wherein two arginine residues have been added to the C-end of B-peptide, and A21 has been replaced by glycine. In certain embodiments, insulin may not have one or more (for example, insulin may be a deletion mutant at one or more of the listed residues) in residue B1, B2, B3, B26, B27, B28, B29 and B30. In certain embodiments, the insulin molecules contain five other amino acid residues at the most on the N- or C-end of A-chain or B-chain. In certain embodiments, one or more amino acid residues are located at position A1, A21, B1, B29, B30 and / or B31 or lack. In certain embodiments, the insulin molecules of the present disclosure suddenly change so that one or more amino acid is replaced (displaced) by its acidic form. In certain embodiments, asparagine is replaced by aspartic acid or glutamic acid. In certain embodiments, glutamine is replaced by aspartic acid or glutamic acid. In certain embodiments, A21 can be aspartic acid, B3 can be aspartic acid, or two positions can contain aspartic acid. Those skilled in the art will recognize that any previously reported or widely accepted mutation or modification can be made to the insulin retaining biological activity, and such insulin analogs can be used in the embodiments of the present disclosure. In certain embodiments, insulin can be connected to fatty acids at any position, or be acylated at the α-amino place on the N-terminal of insulin with fatty acids included in those on the lysine side chain at any amino group, and fatty acids can include C8, C9, C10, C11, C12, C14, C15, C16, C17 or C18 chains. In certain embodiments, the fatty acid chain is 8-20 carbon long. In certain embodiments, it is insulin detemir, wherein myristic acid is covalently conjugated to the lysine at B29, and B30 lacks or does not exist. In certain embodiments, position B28 of the insulin molecule is a lysine and the epsilon (ε)-amino group of the lysine is conjugated to a fatty acid.

[0224] In certain embodiments, a peptide ligase is used to connect the N- or C-terminal ends of the A- or B-chain of the modified insulin. In certain embodiments, a protein ligase is used to add the polypeptide to the C-terminus of the insulin A- and / or B-chain or to the N-terminus of the insulin A- and / or B-chain, and in certain embodiments thereof, the ligase is selected from sortases, butelases, trypsin ligases, subtilisin, peptide ligases, or enzymes with at least 75% homology to these ligases. In certain embodiments, the connection is achieved by expressed protein ligation, as described in the following literature: Muir TW, Sondhi D, Cole PA. "Expressed protein ligation: a general method for protein engineering." Proc Natl Acad Sci USA. 1998; 95(12): 6705-6710. In certain embodiments, the polypeptide is linked to the modified insulin using a Staudinger reaction as described in, for example, Nilsson, BL; Kiessling, LL; Raines, RT (2000). "Staudinger ligation: A peptide from athioester and azide". Org. Lett. 2(13): 1939-1941. In certain embodiments, the polypeptide is conjugated to the modified insulin using a Ser / Thr linkage, 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 certain embodiments, the B-chain itself has less than 32 amino acids or 34 amino acids, and in certain embodiments, the insulin has 4 disulfide bonds instead of 3. Disulfide bonds exist in the A and B chains 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.

[0225] In certain embodiments, the modified insulins of the present disclosure comprise one or more mutations and / or chemical modifications, including, but not limited to, one of 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 A2 1 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI、N εB28 -Myristoyl-Arg A0 Gly A21 Asp B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI、N εB28 -Myristoyl-Lys B28 Pro B29 Arg B31 ArgB32 -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-lithocolyl-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 B31Arg 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 Arg B31 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 ArgB32 -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 -Valeryl-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 B31 Arg 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 -Valeryl-Gly A21 -HI、N αB1 -Hexanoyl-Gly A21 -HI、N αA1 -Heptanoyl-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 aspart), Lys B3 Glu B29 -HI (insulin glulisine), Arg B31 Arg B32 -HI (insulin glargine), N εB29 -myristoyl-des(B30)-HI (insulin detemir), AlaB26 -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 -HI, and wherein Z1 is tridecanoyl, Z2 is tetradecanoyl, Z3 is dodecanoyl, Z4 is decanoyl, and HI is human insulin.

[0226] In certain embodiments, insulin comprises one or more of 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、N αB1 -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、NεB28 -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 B28 Pro B29 -HI, wherein YYYYY is one of acetyl or formyl, and wherein XXXXX is one of propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl or decanoyl, and HI is human insulin.

[0227] In certain embodiments, insulin can be conjugated to reactive moieties naturally occurring in the insulin structure or added before conjugation, including moieties such as carboxyl or reactive esters, amines, hydroxyls, aldehydes, sulfhydryls, maleimido groups, alkynyls, azido groups. Insulin naturally includes reactive α-terminal amine and ε-amine lysine groups, to which NHS-esters, isocyanates or isothiocyanates can be covalently conjugated. In certain embodiments, modified insulin can be used, wherein suitable amino acids (e.g., lysine or non-natural amino acids) have been added or replaced in the amino acid sequence to provide alternative conjugation points except the modified amino acids of the embodiments described herein. In certain embodiments, conjugation process can be controlled by selectively blocking certain reactive moieties before conjugation. In certain embodiments, insulin can include any combination of modifications, and present disclosure also encompasses modified forms of non-human insulin (e.g., porcine insulin, bovine insulin, rabbit insulin, sheep insulin, etc.) comprising any of the above-mentioned modifications. It will be understood that certain 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, those described in US2015 / 0353619, including the unnatural amino acids described or cited herein and including such modifications to the non-human insulins described herein. It should also be understood that in certain embodiments, the insulin can be covalently conjugated to a polyethylene glycol polymer, such as a polyethylene glycol polymer of no more than Mn 60,000, or covalently conjugated to albumin via a permanent or reversible bond.

[0228] In certain embodiments, a compound of the present disclosure (e.g., Formula I) is conjugated to a chelator, and in certain embodiments, the chelator can be used to capture a radioactive payload such as gallium 68, copper 64, lutetium 177, or actinium 225. In certain embodiments, the chelator is based on DOTA, NOTA, TETA, or 4-arm DOTA, and in certain embodiments, the chelator can be linked to the peptide using a PEG linker through an amide bond formed with the chelator and with the peptide.

[0229] In certain embodiments, the activity, bioavailability, solubility, isoelectric point, charge and / or hydrophobicity of the modified insulin can be controlled by chemical modification and / or due to the interaction of small molecules (such as sugars) with compounds (such as the compounds described herein that are covalently linked to or mixed with insulin).

[0230] In certain 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), such as 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, and it will be understood that such omitted or excluded elements render these groups (structures) different and not equivalent. For example, if an alternative version (variant) of the general structure lacks a nitro group in R1 for B1 or B2, the variant is not equivalent (e.g., structurally and chemically not equivalent) to the structure containing the nitro group, at least because the nitro group changes the pKa of B1 and B2 under physiological conditions, thereby changing the overall affinity of Z1c for glucose.

[0231] Rotationally restricted bound boron conjugates.

[0232] In certain embodiments, the aromatic boron-containing compound and / or aromatic boron-containing group is a rotationally restricted constrained boron conjugate. In certain embodiments, the rotationally restricted constrained boron conjugate proposed in the present disclosure contains a scaffold that is rotationally hindered due to unfavorable steric interactions (e.g., distortion vs anti-interactions of substituents), hindered rotation caused by bond hybridization (e.g., cis- vs trans-amide rotations), or by rigid covalent bonds (e.g., (E) vs (Z) configurations of olefinic moieties). For example, Formula FF116 contains an alkyl functional group that is geminal (e.g., attached to the same atom) with an amine group, which is covalently conjugated to a boronic acid-functionalized portion. The alkyl functional group may limit accessible dihedral angles and rotational freedom around CC or CX bonds (commonly referred to as χ (chi) dihedral angles in amino acids). For example, the hydroxyl side chain on a serine residue can adopt dihedral angles of 60°, 180°, or 240° (-60°) in a nearly uniform distribution, whereas the hydroxyl side chain of threonine can only adopt dihedral angles of 180° or 240° (-60°). The presence of a methyl group in a geminal position with the hydroxyl group on threonine may provide steric bulk that can lead to unfavorable interactions when other bulky substituents are in a distorted conformation relative to the methyl group. Formula FF116 contains a geminal alkyl substituent, which can limit the accessible dihedral angles adopted by the boron-conjugated amine, thereby affecting the adopted dihedral angle and bringing the boronic acid functional groups closer together, resulting in increased binding of the conjugate to the target molecule (such as a protein or sugar).

[0233] In certain embodiments, the activity of the insulin agonist (glucose responsiveness) is controlled by the three-dimensional structure of the sensor. For example, FF116A, FF115A, FF225A are structural isomers (containing the same molecular formula, but the atoms are connected differently). In certain embodiments, 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)) in Example 23, Example 82, and Example 27, these compounds exhibit different responsiveness to differences in glucose, with Example 23 showing the largest fold change (responsiveness) to glucose relative to Example 82 and Example 27. In certain embodiments, the presence of a methyl group on the FF scaffold (e.g., FF116A, FF225A) has greater potency (e.g., lower EC) than the non-methylated FF scaffold (e.g., FF115A). 50Example 23 had a fold change of about 7 to 8 fold in response to a glucose change of 3 mM to 20 mM glucose, while Example 27 had a fold change of about 6 fold and Example 82 had a fold change of about 4 fold over the same range of glucose changes. Thus, the presence of methyl groups in scaffolds FF116A (with β methyl groups) and FF225A (with α methyl groups) provides improved glucose responsiveness over FF115A (without methyl groups on the FF scaffold), and the glucose responsiveness provided by scaffold FF116A is superior to that of FF225A. As another comparison, as shown in Example 156, a compound having an insulin backbone (i.e., A chain sequence: 24051 and B chain sequence: 24061) and scaffold FF116A and linker FL3 has a diboronate scaffold 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butanamido)propanoic acid, which provides a fold change of 4.0 in response to a glucose change from 3 mM to 20 mM. In contrast, if the diboronate sensor in the same Example 156 is replaced with (S)-3-(2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)propanoic acid, a fold change of 3.6 is observed in response to a glucose change from 3 mM to 20 mM. This indicates that the diboronate sensor 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)butyramido)propanoic acid (with a methyl group on the FF scaffold) provides better glucose responsiveness to insulin than the insulin containing sensor (S)-3-(2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propionamido)propanoic acid (without a methyl group on the FF scaffold).

[0234] In certain embodiments, the stereochemistry of the isomeric structure (e.g., the stereochemistry of the compound (e.g., within the Z1c moiety)) can selectively increase the affinity of the conjugate (e.g., the Z1c moiety) for a particular target diol (such as glucose). For example, in certain 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 (and the overall molecular structure or conjugate) for glucose. In certain embodiments, the cis forms of Formulas FF12, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF117, FF193A, FF203, FF225, and FF226 are used where applicable (e.g., Z1c includes structures having cis stereochemistry). In certain embodiments, the trans forms of Formulas FF12, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 are used where applicable, e.g., when these formulas include two stereocenters connected by a bond (e.g., Z1c includes structures having trans stereochemistry). In certain 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 these formulas include at least one stereocenter (e.g., Z1c includes structures having R stereochemistry). In certain embodiments, the S form of Formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 is used where applicable, for example, when Formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 include at least one stereocenter (e.g., Z1c includes a structure having S stereochemistry). In certain 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 include two stereogenic centers connected by a bond (e.g., Z1c includes a structure having S,S stereochemistry).In certain 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 include two stereogenic centers connected by a bond (e.g., Z1c includes a structure having S,R stereochemistry). In certain embodiments, the R,R form of Formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 is used where applicable, for example, when Formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225, and FF226 includes two stereogenic centers connected by a bond (e.g., Z1c includes a structure having R,R stereochemistry). In certain embodiments, the R, S forms of formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225 and FF226 are used where applicable, for example, when formula FF12, FF114, FF115, FF116, FF117, FF193A, FF203, FF225 and FF226 include two stereocenters connected by a bond (for example, Z1c includes a structure with R, S stereochemistry). In certain embodiments, the compound includes one or more tautomers of the compounds disclosed herein. In certain embodiments, the compound includes one or more stereoisomers or a mixture of stereoisomers of the compounds disclosed herein.

[0235] In certain embodiments, the compound is covalently conjugated to glucagon, GLP-1, GLP-2, or a variant of any of these (e.g., any variant having a deletion, insertion, and / or substitution of one or more amino acids). In certain embodiments, any suitable chemical modification made to insulin discussed herein can be made to glucagon. In certain embodiments, the conjugate is mixed with a second or bulk drug or one or more compounds selected from the group consisting of aminoethylglucose, aminoethyldimannose, aminoethyltrimannose, 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 Glucose, sucrose, maltose, mannose, derivatives of these (e.g., glucosamine, mannosamine, methylglucose, methylmannose, ethylglucose, ethylmannose, etc.), sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol and / or higher combinations of these (such as linear and / or branched dimannose, linear and / or branched trimannose), molecules containing cis-diols, catechol, tris(hydroxymethylaminomethane) (tris) and DOPA molecules such as L-DOPA or L-3,4-dihydroxyphenylalanine.

[0236] In addition, those skilled in the art will recognize that, in certain embodiments, one or more suitable protein artificial amino acids can be used (including) in Z1a. For example, in certain embodiments, one or more of the following artificial amino acids can be used based on the methods described and cited in the following documents and the amino acid lists provided in the following documents: Liu, CC; Schultz, PG (2010). "Adding new chemistries to the genetic code." Annual Review of Biochemistry 79: 413-44. Those skilled in the art will recognize that, in certain embodiments, artificial amino acids can be incorporated into Z1a by peptide synthesis and then covalently conjugated to drugs or insulin, and these include the amino acids cited herein as well as previously reported non-protein amino acids. In certain embodiments, artificial amino acids are present in (e.g., can be included in) insulin, and in certain embodiments thereof, artificial amino acids can be incorporated into proteins by recombinant protein expression using suitable methods and protocols, including those described in U.S. patents and patent applications, including: US2008 / 0044854, US 8518666, US 8980581, US2008 / 0044854, US20140045261, US 2004 / 0053390, US 7229634, US 8236344, US2005 / 0196427, US2010 / 0247433, US 7198915, US 7723070, US2002 / 0042097, US 2004 / 0058415, US2008 / 0026422, US2008 / 0160609, US2010 / 0184193, US2012 / 0077228, US2014 / 025599, US7198915, US 7632492, US 7723070, and other proteins artificial amino acids can be recombinantly introduced using the methods and protocols described in US 7736872, US 7816320, US 7829310, US7829659, US 7883866, US 8097702, US 8946148.

[0237] In certain 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-thiomorpholine, 4-oxaproline, pyroglutamic acid, 1,3-oxazolidine-4-carboxylic acid, 1,3-thiazolidine-4-carboxylic acid, 3-thiaproline, 4-thiaproline, 3-selenoproline, 4-selenoproline, 4-ketoproline, Proline, 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, α-methylproline, α-ethylproline, α-propylproline, α -allylproline, α-benzylproline, α-(4-fluorobenzyl)-proline, α-(2-chlorobenzyl)-proline, α-(3-chlorobenzyl)-proline, α-(2-bromobenzyl)-proline, α-(4-bromobenzyl)-proline, α-(4-methylbenzyl)-proline, α-(diphenylmethyl)-proline, α-(naphthylmethyl)-proline, D-proline or S-homoproline, (2S,4S)-4-fluoro-L-proline, (2S,4R)- 4-Fluoro-L-proline, (2S)-3,4-dehydro-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.

[0238] It will be appreciated that in certain embodiments, a specific orientation of amino acids is achieved using, for example, the methods of Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. page 848. In certain embodiments, compounds of the present disclosure (such as compounds of Formula I or Formula II) can be bound to a diol, catechol, a hexose, glucose, xylose, fucose, galactosamine, glucosamine, mannosamine, galactose, mannose, fructose, galacturonic acid, glucuronic acid, iduronic acid, mannuronic acid, acetylgalactosamine, acetylglucosamine, acetylmannosamine, acetylmuramic acid, 2-keto-3-deoxy-glycero-galacto-nononicacid, acetylneuraminic acid, glycolylneuraminic acid, a neurotransmitter, dopamine, or a disaccharide or a polymer of a sugar or diol.

[0239] In certain embodiments, modifications or intermediates can include the use of N-methyliminodiacetic acid (MIDA) groups to prepare MIDA-conjugated boronates or MIDA boronates; such modifications can be used in the process of preparing boronates toward a final usable structure (e.g., in embodiments of methods of preparing conjugates described herein). In certain embodiments, boronic acid pinacol esters are used to approach the final structure, wherein the pinacol group can be easily removed by one skilled in the art using standard techniques. MIDA-protected boronates are easy to handle, stable in air, compatible with chromatography, unreactive under standard anhydrous cross-coupling conditions, and readily deprotected at room temperature under mild alkaline aqueous conditions (such as 1 M NaOH, or even NaHCO3, or as described in Lee, SJ et al. (2008). J. Am. Chem. Soc. 130:466).

[0240] The biological mechanism of wild-type insulin binding to the insulin receptor was previously reported in Menting, JG et al. (2013). Nature 493, 241-245; and Menting, JG et al. (2014). "Protective hinge in insulin opens to enable its receptor engagement." Proc. Natl. Acad. Sci. USA 111, E3395-3404. The binding of wild-type insulin to the insulin receptor was investigated using any suitable technique, for example, by using in vitro insulin receptor binding with TyrA14- as a tracer. 125In some embodiments, the compound disclosed herein is expressed partially or completely with recombinant protein (such as insulin) interested. Methods for expressing insulin in E. coli are known and can be readily performed by those skilled in the art, for example by using the procedures outlined 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 method, the protein is expressed as a single-chain proinsulin construct with a fission protein or affinity tag. When Z1a is present, a compound comprising Z1a (e.g., a compound of Formula I) can be expressed as part of proinsulin and then chemically modified to be conjugated to the structure of interest via an amide bond. This approach provides good yields, reduces experimental complexity by reducing the number of processing steps, and allows refolding in native-like 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 Escherichia coli, proinsulin is typically present in inclusion bodies and can be easily purified by those skilled in the art.

[0241] In certain embodiments, standard IPTG (isopropylthio-β-galactoside) induction of an IPTG-inducible expression construct and a carrier can be used to express proinsulin in an E. coli strain (such as a B21 strain). As an example, the expression construct is composed 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag before being conjugated to a sensor. The expression of desired proinsulin can be performed by procedures known in the art. See, for example, U.S. Patents 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 1,040,0021.

[0242] In certain embodiments, the compound 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 certain embodiments, the composition can be a pharmaceutical composition, such as a sterile, injectable pharmaceutical composition. In certain embodiments, the composition can be formulated for subcutaneous injection. In certain embodiments, the composition is formulated for transdermal, intradermal, transmucosal, nasal, inhalable or intramuscular administration. In certain embodiments, the composition can be formulated into an oral dosage form or a pulmonary dosage form. Pharmaceutical compositions suitable for injection can include sterile aqueous solutions containing, for example, sugar, polyols (such as mannitol and sorbitol), phenol, metacresol, and sodium chloride, and dispersions can be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. The carrier can, for example, be a solvent or a dispersion medium containing, for example, water, sugar, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Those skilled in the art recognize that specific formulations can be developed to best adapt to the application and use of the molecular structure of the present invention. General considerations for formulating and preparing pharmaceutical compositions, routes of administration, and the inclusion of suitable pharmaceutically acceptable carriers can be found, for example, in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, Pa., 1995. In certain embodiments, if the compound (e.g., a compound of Formula I) comprises insulin, the pharmaceutical composition may include zinc (e.g., Zn 2+) and insulin. For example, such zinc preparations are described in U.S. Patent No. 9,034,818. For example, the pharmaceutical composition can include zinc in a molar ratio of about M:N to modified insulin, wherein M is 1-11 and N is 6-1. In certain embodiments, such modified insulin can be stored in a pump, and the pump releases modified insulin outside or inside the body. In certain embodiments, a pump can be used to release a constant amount of modified insulin, wherein the insulin is glucose responsive and can automatically adjust activity based on the glucose level in the blood and / or the release rate from the injection site. In certain embodiments, the composition can be formulated in dosage unit form for ease of administration and uniform dosage. In certain embodiments, in addition to the effect provided by the molecular structure, the pharmaceutical composition can further include a second insulin type to provide fast-acting or basal insulin. In certain embodiments, the compound of the present disclosure (e.g., a compound of Formula I) is injected separately from insulin, but the activity of insulin is regulated by being combined with insulin, and in certain embodiments, this activity change depends on glucose.

[0243] In certain embodiments, the pharmaceutical composition comprises one or more compounds disclosed herein and at least one additional component selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable vehicle, and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises a compound of Formula I and at least one additional component selected from a pharmaceutically acceptable carrier, a pharmaceutically acceptable vehicle, and a pharmaceutically acceptable excipient.

[0244] In certain embodiments, the present disclosure includes compounds that can be part of a test kit, wherein the test kit includes a compound of formula I comprising modified insulin and a pharmaceutically acceptable carrier, and for injections, a syringe or pen can be included. In certain embodiments, the test kit can include a syringe or pen pre-loaded with a pharmaceutical composition, wherein the pharmaceutical composition includes a compound of formula I and a liquid carrier. Alternatively, the test kit can include a separate container such as a vial containing a pharmaceutical composition and an empty syringe or pen, wherein the pharmaceutical composition includes a compound of formula I and a dry carrier. In certain embodiments, such a test kit can include a separate container with a liquid carrier, which can be used to reconstruct a given composition, which can then be placed in a syringe or pen. In certain embodiments, the test kit can include instructions. In certain embodiments, the test kit can include a blood glucose measuring device that calculates locally or remotely the appropriate dose of modified insulin to be injected at a given time point or at fixed intervals. Such a dosage regimen is unique for the patient and can be provided, for example, as an instruction for programming a pump by a person or computer. The kit may include an electronic device that transmits the blood glucose measurement to a second computer, either locally or elsewhere (e.g., in the cloud), which then calculates the correct amount of a compound of Formula I (which includes, for example, a modified insulin) that the patient needs to use at a specific time.

[0245] In certain embodiments, the present invention relates to a method for treating a disease or condition in a subject, comprising administering to the subject a composition comprising a compound described herein. In certain 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, MODY 1, MODY 2 or MODY 3 diabetes, mood disorders and psychiatric disorders. It should be understood that this combination regimen can also be used for insulin resistant patients who are receiving insulin sensitizers or diabetes second drugs (secondary drugs) (e.g., biguanides such as metformin, glitazones) or / and insulin secretagogues (e.g., sulfonylureas, GLP-1, exenatide-4, etc.) or amylin.

[0246] In certain embodiments, the compound of the present disclosure (e.g., compound of Formula I) can be administered to a patient who is receiving at least one other therapy or taking at least one other drug or therapeutic protein. In certain embodiments, the at least one other therapy is intended to treat the same disease or disorder as the compound (e.g., compound of Formula I) administered. In certain embodiments, the at least one other therapy is intended to treat the side effects of the compound (e.g., compound of Formula I) or as an adjuvant therapy. The time ranges of the two therapies may be different or identical; they can be administered according to the same or different schedules, as long as there is a time period in which the patient benefits from the two therapies. Two or more therapies can be administered in the same or different preparations, as long as there is a time period in which the patient benefits from the two therapies. Any of these schemes can be used to administer more than one antidiabetic drug to the subject.

[0247] In certain embodiments, a therapeutically effective amount of a compound (e.g., a compound of Formula I) will be used, which is an amount sufficient to treat a disease or condition (meaning, for example, to improve its symptoms, delay its progression, prevent its recurrence, or delay its onset) at a reasonable benefit-risk ratio. In certain embodiments, this may involve balancing efficacy with additional safety and toxicity. Additional safety means, for example, that a compound (e.g., a compound of Formula I) can respond to changes in blood glucose levels or other molecule levels even when the patient is not actively monitoring the levels of that molecule (such as blood glucose levels over a given timeframe, for example, during sleep). In certain embodiments, ED can be measured by standard pharmacological procedures in cell cultures or in vivo with experimental animals, and for example 50 and LD 50To determine the therapeutic index of the drug, to determine therapeutic efficacy and toxicity. In certain embodiments, the average daily dose of the insulin with the molecular structure is in the range of 5 to 400U (for example 30-150U, wherein 1 unit of insulin is about 0.04mg). In certain embodiments, a certain amount of compound (for example, compound of Formula I) with these insulin dosages is used based on once a day or once every two days or once every three days or once every four days. In certain embodiments, the basis is determined by an algorithm, and the algorithm can be calculated by a computer. In certain embodiments, the compound of 5 to 10 times the amount of these dosages is used weekly or at fixed intervals, such as the compound of Formula I. In certain embodiments, the conjugate of 10 to 20 times the amount of these dosages is used every two weeks or at fixed intervals. In certain embodiments, the compound (for example, compound of Formula I) of 20 to 40 times the amount of these dosages is used monthly or at fixed intervals. In certain embodiments, the C- end of the A- chain of insulin can be further extended with a peptide (amino acid sequence) comprising 1-20 amino acid residues. In certain embodiments, the insulin analog is desB30 insulin.

[0248] In certain embodiments, Z1a is an amino acid or peptide. In at least some embodiments, the Z1a includes (consisting of) 1-50 amino acid residues, for example, 1 residue, 50 residues or any intermediate number of residues (for example, 10, 15, 25, 30, 42 residues, etc.). In certain embodiments, the Z1a includes 1-15 amino acids. In at least some embodiments, the peptide Z1a includes 1-8 amino acids. In certain embodiments, Z1a includes 5 to 6 amino acids. In certain embodiments, Z1a includes at least one independently selected from the following amino acids: 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 one of 20 naturally occurring amino acids. In certain embodiments, Z1a can include diaminopropionic acid, diaminobutyric acid or ornithine.

[0249] In certain embodiments, Z1a comprises 1 to 5 lysine residues (K). In certain embodiments, Z1a comprises 1 to 3 K amino acids. In certain embodiments, Z1a comprises 5 to 6 amino acids and at least one or more amino acids are K. In certain embodiments, Z1a comprises 5 to 6 amino acids and 1 to 3 amino acids are K.

[0250] In certain embodiments, Z1a is linked to the N-terminus and / or C-terminus, and / or inserted into the sequence of the A-chain or B-chain of insulin.

[0251] Compound

[0252] In certain embodiments, provided herein are compounds of Formula I, or a stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt thereof:

[0253]

[0254] in:

[0255] X1 includes:

[0256] i. APIs containing amines;

[0257] ii. is constructed to be covalently conjugated to an amine of the drug substance; or

[0258] iii. NH2 or OH (eg, X1 is NH2 or OH);

[0259] Each Z1a independently comprises from 1 to 50 amino acids linked together using amide or peptide bonds;

[0260] Each Z1b is independently a small molecule linker;

[0261] each Zlc is independently selected from Formula FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, wherein each Zlc is covalently conjugated directly or via an indirect linker to an amine of Zla or to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH;

[0262] Each m' is 0 or 1;

[0263] Each n' is 0;

[0264] Each o' is 1;

[0265] each p' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0266] each q' is 1, 2, 3, 4, or 5, wherein when p' or q' is 2 or greater, each corresponding Zlc is independently selected and may be the same or different, and wherein when q' is 2 or greater, each corresponding Zla is independently selected and may be the same or different; and

[0267] When m'=0, each Z1c is covalently conjugated directly or via an indirect linker to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH, and for m'=1, each Z1a is covalently conjugated to X1 via an amide or peptide bond, and each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to X1.

[0268] In certain embodiments, the compound is selected from a compound represented by Formula I, a stereoisomer thereof, a mixture of stereoisomers thereof, and a pharmaceutically acceptable salt thereof, with the proviso that the compound is not any of Examples 1-880 disclosed in PCT / US2021 / 059802.

[0269] In certain embodiments, X1 is a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain.

[0270] In certain embodiments, the present disclosure relates to a compound of the formula: or a stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt thereof:

[0271]

[0272] in:

[0273] X1 comprises a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain;

[0274] Each Z1a independently comprises from 1 to 50 amino acids linked together using amide or peptide bonds;

[0275] each Z1c is independently selected from formula FF12A, FF12B, FF114A, FF115A, FF116A, and FF225A, wherein each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to an amine of X1 or to OH when X1 is OH;

[0276] Each m' is 0 or 1;

[0277] each p' is 1, 2, 3, 4, or 5; and

[0278] each q' is 1, 2, 3, 4 or 5, wherein when p' or q' is 2 or greater, each corresponding Z1c is independently selected and may be the same or different, and wherein when q' is 2 or greater, each corresponding Z1a is independently selected and may be the same or different;

[0279] when m'=0, each Z1c is covalently conjugated directly or via an indirect linker to an amine of X1 or to OH when X1 is OH, when m'=1, each Z1a is covalently conjugated to X1 via an amide or peptide bond, and each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a; and

[0280] The compounds may contain isotopes at one or more positions.

[0281] In certain embodiments, at least one Z1c comprises an α methyl group or a β methyl group. In certain embodiments, Z1c comprises an α methyl group. In certain embodiments, Z1c comprises a β methyl group.

[0282] In certain embodiments, the compound comprises:

[0283] at least one Z1c, which is covalently conjugated via an indirect linker to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine of Z1a,

[0284] The indirect linker is represented by formula (X") n1 Indicates that

[0285] Each n1 is independently selected from 1, 2, 3, 4 and 5, and

[0286] Each X" is independently selected from:

[0287] i L- or D-amino acid, wherein the amine function of the L- or D-amino acid is covalently conjugated directly or indirectly to Z1c, and the acid function of the L- or D-amino acid is directly or indirectly conjugated to X1 or to Z1a; and

[0288] ii. Formulas FL(IA), FL(IB), FL69 and FL70;

[0289] The formulae FL(IA) and FL(IB) are:

[0290] and its stereoisomers;

[0291] in:

[0292] G is selected from 3- to 6-membered cycloalkyl, 3- to 10-membered heterocyclyl, heteroaryl, and aryl, each of which is optionally substituted with 1-3 groups independently selected from hydroxy, amino, halogen, cycloalkyl (e.g., (C3-C6)cycloalkyl), alkoxy, and alkyl (e.g., C1-C6 alkyl);

[0293] E is absent or is alkylene optionally substituted with 1 to 3 groups independently selected from halogen, hydroxy and amino;

[0294] Q is absent or selected from hydrogen, alkyl (e.g., C1-C6 alkyl), halogen, cyano, alkoxy, carboxylic acid, amino, hydroxy, amide, haloalkyl (e.g., C1-C6 haloalkyl), cycloalkyl (e.g., (C3-C6)cycloalkyl), heterocycle, heteroaryl, and aryl, wherein each of the alkyl, alkoxy, cycloalkyl, heterocycle, heteroaryl, and aryl is optionally substituted with 1-5 groups independently selected from alkyl (e.g., C1-C6 alkyl), amino, amide, halogen, hydroxy, cyano, haloalkyl (e.g., C1-C6 haloalkyl), and alkoxy;

[0295] Q' is selected from hydrogen, alkyl (e.g., C1-C6 alkyl), and acyl;

[0296] Q and Q', together with the carbon and nitrogen atoms to which they are attached, optionally form a 4-membered heterocyclyl, a 5-membered heterocyclyl, a 6-membered heterocyclyl, a 9-membered bicyclic heterocyclyl, or a 10-membered bicyclic heterocyclyl, wherein each of the 4-membered heterocyclyl, the 5-membered heterocyclyl, the 6-membered heterocyclyl, the 9-membered bicyclic heterocyclyl, and the 10-membered bicyclic heterocyclyl is optionally substituted with 1-5 groups independently selected from alkyl (e.g., C1-C6 alkyl), amino, halogen, hydroxy, cyano, amide, haloalkyl (e.g., C1-C6 haloalkyl), and alkoxy;

[0297] p is 0, 1, 2, 3, 4, or 5;

[0298] q is 0, 1, 2, 3, 4, or 5;

[0299] R" represents a covalent bond directly or indirectly to Z1c;

[0300] Z" represents a covalent bond, directly or indirectly, to X1 or to Z1a; and

[0301] Any primary amines are optionally acetylated or alkylated;

[0302] The formulas FL69 and FL70 are:

[0303] and stereoisomers thereof; wherein:

[0304] R" represents a covalent bond directly or indirectly to Z1c;

[0305] Z" represents a covalent bond directly or indirectly to X1 or to Z1a;

[0306] A' is selected from H, alkyl (e.g., C1-C6 alkyl), saturated fatty acid, unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl; and

[0307] A" is selected from alkyl (e.g., C1-C6 alkyl), substituted acyl, acid-terminated acyl, saturated or unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl;

[0308] p is 1, 2, 3, 4, or 5,

[0309] q is 1, 2, 3, 4, or 5, and

[0310] Any primary amines are optionally acetylated or alkylated.

[0311] In certain embodiments, the compound comprises:

[0312] At least one Z1c, which is covalently conjugated to X1 or to Z1a via an indirect linker, wherein the indirect linker is represented by formula (X") n1 express,

[0313] Each n1 is independently selected from 1, 2, 3, 4 or 5, and

[0314] Each X" is independently selected from:

[0315] an L- or D-amino acid, wherein the amine function of the L- or D-amino acid is covalently conjugated directly or indirectly to Z1c, and the acid function of the L- or D-amino acid is directly or indirectly conjugated to X1 or to Z1a; and

[0316] Formulas FL(IA), FL(IB), FL69 and FL70, wherein Formulas FL(IA) and FL(IB) are selected from Formulas FL3, FL5, FL5A, FL5B, FL20-FL68 and FL71-FL75:

[0317]

[0318]

[0319] and its stereoisomers;

[0320] in:

[0321] p is 1, 2, 3, 4, or 5,

[0322] q is 1, 2, 3, 4, or 5, and

[0323] Any primary amines are optionally acetylated or alkylated.

[0324] The formulas FL69 and FL70 are:

[0325] and stereoisomers thereof; wherein:

[0326] R" represents a covalent bond directly or indirectly to Z1c;

[0327] Z" represents a covalent bond directly or indirectly to X1 or to Z1a;

[0328] A' is selected from H, alkyl (e.g., C1-C6 alkyl), saturated or unsaturated fatty acid, cycloalkyl (e.g., C3-C6 cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl; and

[0329] A" is selected from alkyl (e.g., C1-C6 alkyl), substituted acyl, acid-terminated acyl, saturated or unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl;

[0330] p is 1, 2, 3, 4, or 5,

[0331] q is 1, 2, 3, 4, or 5, and

[0332] Any primary amines are optionally acetylated or alkylated.

[0333] In certain embodiments, the stereochemistry of FL70 at the stereocenter carbon can be S or R. In certain embodiments, the stereochemistry of FL70 at the stereocenter carbon is S. In certain embodiments, the stereochemistry of FL70 at the stereocenter carbon is R.

[0334] In certain embodiments, n1 is 1.

[0335] In certain embodiments, the compound is represented by Formula IC:

[0336]

[0337] each p' is 1, 2, 3, or 4; and

[0338] Each q' is 1, 2, 3, 4 or 5.

[0339] In certain embodiments, each of B1 and B2 is independently selected from Formulas F2, F5, and F10,

[0340] Wherein formula F2, F5 and F10 are:

[0341]

[0342] in:

[0343] One R1 represents (C=O)---* or (CH2) m (C=O)---*, wherein---* represents the point of attachment to the rest of Z1c, and m is 1, 2, 3, 4, 5, 6 or 7; and

[0344] Each remaining R1 is independently selected from H, F, Cl, Br, I, OH, CH2-NH2, NH2, (C=O)-NH2, CH=O, SO2CH3, SO2CF3, CF3, CHF2, NO2, CH3, OCH3, O(CH2) m” CH3, (SO2)NH-CH3, (SO2)NH(CH2) m” CH3 and OCF3, wherein m″ is 1, 2, 3, 4, 5, 6 or 7;

[0345] Y8 is O, N, or NR, wherein R is alkyl (e.g., C1-C6 alkyl) or H;

[0346] Y9 is H, CH3 or alkyl (e.g., C1-C6 alkyl), provided that when Y8 is O, Y9 is CH3 or alkyl (e.g., C1-C6 alkyl); and

[0347] i is 1, 2, or 3.

[0348] In certain embodiments, B1 and B2 are selected from formula F2, and each remaining R1 is independently selected from H, CF3, and F; and each remaining R1 is H, or one or both of each remaining R1 are F or CF3.

[0349] In certain embodiments, at least one Z1c is selected from FF12, FF12A-D, FF116, and FF116A-D. In certain embodiments, both B1 and B2 are selected from F2, wherein all remaining R1s are H; and the indirect linker is selected from relatively hydrophobic indirect linkers. In certain 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 with hydrophobic side chains. In certain embodiments, both B1 and B2 are selected from F2, wherein one of the remaining R1s is CF3 and the remaining R1s are H; and the indirect linker is selected from relatively hydrophilic indirect linkers. In certain 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 with hydrophilic side chains.

[0350] In certain embodiments, each Zlc is covalently conjugated to an indirect linker, and each combination of Zlc and an indirect linker (ie, Zlc-linker) is selected from Formulas FFL-1 to FFL-101:

[0351]

[0352]

[0353]

[0354]

[0355] and its stereoisomers;

[0356] wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1 or to an amine of Z1a.

[0357] In certain 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 conjugated to an indirect linker, and each combination of Z1c and an indirect linker is selected from Formulas FFL-1-4, 6-7, 9, 13-16, 20-23, 28-29, 31-33, 39, 42-45, 48, 53, 57-58, 60-62, 65, and 67:

[0358]

[0359]

[0360] and its stereoisomers;

[0361] wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1 or to an amine of Z1a.

[0362] In certain embodiments, X1 is a polypeptide drug substance comprising one or more lysine amino acids, each of which is independently conjugated to Z1c directly or via an indirect linker.

[0363] In certain embodiments, X1 comprises a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory peptide, a glucose-dependent insulin-releasing polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog of any one thereof.

[0364] In certain embodiments, X1 comprises an insulin having an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 1, 25, 24051, and 24052, and the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24060, 24061, 24062, 24063, 24064, and 25000-25397.

[0365] In certain embodiments, X1 comprises an insulin having an A-chain and a B-chain, wherein:

[0366] said A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24051 and 24052,

[0367] The B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380-25397;

[0368] Each Z1c is independently selected from FF12A, FF12B, FF12D, FF116A, FF116B, FF116C, and FF116D, and is covalently conjugated directly or via an indirect linker to one or more lysine residues in X1;

[0369] B1 and B2 are each selected from F2; and

[0370] The indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL20-FL75, and stereoisomers thereof.

[0371] In certain embodiments, X1 is a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain, wherein:

[0372] said A-chain comprising a sequence selected from the group consisting of SEQ ID NOs 1, 25 and 24051,

[0373] The B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24060, 24063, 25228, 25313, 25393, 25396, and 25397;

[0374] Each Z1c is independently selected from FF12A, FF12B, FF114A, FF115A, FF116A, and FF225A, and is covalently conjugated directly or via an indirect linker to one or more lysine residues in X1;

[0375] B1 and B2 are each selected from F2; and

[0376] The indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL21, and stereoisomers thereof.

[0377] In certain embodiments, the compound is selected from:

[0378] Example 1 (in order of appearance, SEQ ID NOs 25398 and 25399, respectively):

[0379]

[0380] Example 2 (in order of appearance, SEQ ID NOs 25400 and 25401, respectively):

[0381]

[0382] Example 3 (in order of appearance, SEQ ID NOs 25402 and 25403, respectively):

[0383] Example 4 (in order of appearance, SEQ ID NOs 25404 and 25405, respectively):

[0384]

[0385] Example 5 (in order of appearance, SEQ ID NOs 25406 and 25407, respectively):

[0386]

[0387] Example 6 (in order of appearance, SEQ ID NOs 25408 and 25409, respectively):

[0388]

[0389] Example 7 (in order of appearance, SEQ ID NOs 25410 and 25411, respectively):

[0390]

[0391] Example 8 (in order of appearance, SEQ ID NOs 25412 and 25413, respectively):

[0392]

[0393] Example 9 (in order of appearance, SEQ ID NOs 25414 and 25415, respectively):

[0394]

[0395] Example 10 (in order of appearance, SEQ ID NOs 25416 and 25417, respectively):

[0396]

[0397] Example 11 (in order of appearance, SEQ ID NOs 25418 and 25419, respectively):

[0398]

[0399] Example 12 (in order of appearance, SEQ ID NOs 25420 and 25421, respectively):

[0400]

[0401] Example 13 (in order of appearance, SEQ ID NOs 25422 and 25423, respectively):

[0402]

[0403] Example 14 (in order of appearance, SEQ ID NOs 25424 and 25425, respectively):

[0404]

[0405] Example 15 (in order of appearance, SEQ ID NOs 25426 and 25427, respectively):

[0406]

[0407] Example 16 (in order of appearance, SEQ ID NOs 25428 and 25429, respectively):

[0408] Example 17 (in order of appearance, SEQ ID NOs 25430 and 25431, respectively):

[0409]

[0410] Example 18 (in order of appearance, SEQ ID NOs 25432 and 25433, respectively):

[0411]

[0412] Example 19 (in order of appearance, SEQ ID NOs 25434 and 25435, respectively):

[0413]

[0414] Example 20 (in order of appearance, SEQ ID NOs 25436 and 25437, respectively):

[0415]

[0416] Example 21 (in order of appearance, SEQ ID NOs 25438 and 25439, respectively):

[0417]

[0418] Example 22 (in order of appearance, SEQ ID NOs 25422 and 25423, respectively):

[0419]

[0420] Example 23 (in order of appearance, SEQ ID NOs 25440 and 25441, respectively):

[0421]

[0422] Example 24 (in order of appearance, SEQ ID NOs 25442 and 25443, respectively):

[0423]

[0424] Example 25 (in order of appearance, SEQ ID NOs 25444 and 25445, respectively):

[0425]

[0426] Example 26 (in order of appearance, SEQ ID NOs 25446 and 25447, respectively):

[0427] Example 27 (in order of appearance, SEQ ID NOs 25448 and 25449, respectively):

[0428]

[0429] Example 28 (in order of appearance, SEQ ID NOs 25450 and 25451, respectively):

[0430]

[0431] Example 29 (in order of appearance, SEQ ID NOs 25452 and 25453, respectively):

[0432]

[0433] Example 30 (in order of appearance, SEQ ID NOs 25454 and 25455, respectively):

[0434]

[0435] Example 31 (in order of appearance, SEQ ID NOs 25456 and 25457, respectively):

[0436]

[0437] Example 32 (in order of appearance, SEQ ID NOs 25458 and 25459, respectively):

[0438]

[0439] Example 33 (in order of appearance, SEQ ID NOs 25460 and 25461, respectively):

[0440]

[0441] Example 34 (in order of appearance, SEQ ID NOs 25462 and 25463, respectively):

[0442]

[0443] Example 35 (in order of appearance, SEQ ID NOs 25464 and 25465, respectively):

[0444]

[0445] Example 36 (in order of appearance, SEQ ID NOs 25466 and 25467, respectively):

[0446]

[0447] Example 37 (in order of appearance, SEQ ID NOs 25468 and 25469, respectively):

[0448]

[0449] Example 38 (in order of appearance, SEQ ID NOs 25470 and 25471, respectively):

[0450]

[0451] Example 39 (in order of appearance, SEQ ID NOs 25472 and 25473, respectively):

[0452]

[0453] Example 40 (in order of appearance, SEQ ID NOs 25474 and 25475, respectively):

[0454]

[0455] Example 41 (in order of appearance, SEQ ID NOs 25476 and 25477, respectively):

[0456] Example 42 (in order of appearance, SEQ ID NOs 25478 and 25479, respectively):

[0457]

[0458] Example 43 (in order of appearance, SEQ ID NOs 25480 and 25481, respectively):

[0459]

[0460] Example 44 (in order of appearance, SEQ ID NOs 25482 and 25483, respectively):

[0461]

[0462] Example 45 (in order of appearance, SEQ ID NOs 25484 and 25485, respectively):

[0463]

[0464] Example 46 (in order of appearance, SEQ ID NOs 25486 and 25487, respectively):

[0465]

[0466] Example 47 (in order of appearance, SEQ ID NOs 25488 and 25489, respectively):

[0467]

[0468] Example 48 (in order of appearance, SEQ ID NOs 25490 and 25491, respectively):

[0469]

[0470] Example 49 (in order of appearance, SEQ ID NOs 25492 and 25493, respectively):

[0471]

[0472] Example 50 (in order of appearance, SEQ ID NOs 25494 and 25495, respectively):

[0473]

[0474] Example 51 (in order of appearance, SEQ ID NOs 25496 and 25497, respectively)

[0475]

[0476] Example 52 (in order of appearance, SEQ ID NOs 25498 and 25499, respectively):

[0477]

[0478] Example 53 (in order of appearance, SEQ ID NOs 25500 and 25501, respectively):

[0479] Example 54 (in order of appearance, SEQ ID NOs 25502 and 25503, respectively):

[0480]

[0481] Example 55 (in order of appearance, SEQ ID NOs 25504 and 25505, respectively):

[0482]

[0483] Example 56 (in order of appearance, SEQ ID NOs 25506 and 25507, respectively):

[0484]

[0485] Example 57 (in order of appearance, SEQ ID NOs 25508 and 25509, respectively):

[0486] Example 58 (in order of appearance, SEQ ID NOs 25510 and 25511, respectively):

[0487]

[0488] Example 59 (in order of appearance, SEQ ID NOs 25512 and 25513, respectively):

[0489]

[0490] Example 60 (in order of appearance, SEQ ID NOs 25514 and 25515, respectively):

[0491]

[0492] Example 61 (in order of appearance, SEQ ID NOs 25480 and 25481, respectively):

[0493]

[0494] Example 62 (in order of appearance, SEQ ID NOs 25516 and 25517, respectively):

[0495] Example 63 (in order of appearance, SEQ ID NOs 25518 and 25519, respectively):

[0496] Example 64 (in order of appearance, SEQ ID NOs 25520 and 25521, respectively):

[0497] Example 65 (in order of appearance, SEQ ID NOs 25522 and 25523, respectively):

[0498]

[0499] Example 66 (in order of appearance, SEQ ID NOs 25524 and 25525, respectively):

[0500]

[0501] Example 67 (in order of appearance, SEQ ID NOs 25526 and 25527, respectively):

[0502]

[0503] Example 68 (in order of appearance, SEQ ID NOs 25528 and 25529, respectively):

[0504]

[0505] Example 69 (in order of appearance, SEQ ID NOs 25530 and 25531, respectively):

[0506] Example 70 (in order of appearance, SEQ ID NOs 25532 and 25533, respectively):

[0507]

[0508] Example 71 (in order of appearance, SEQ ID NOs 25534 and 25535, respectively):

[0509]

[0510] Example 72 (in order of appearance, SEQ ID NOs 25536 and 25537, respectively):

[0511]

[0512] Example 73 (in order of appearance, SEQ ID NOs 25538 and 25539, respectively):

[0513]

[0514] Example 74 (in order of appearance, SEQ ID NOs 25540 and 25541, respectively):

[0515]

[0516] Example 75 (in order of appearance, SEQ ID NOs 25542 and 25543, respectively):

[0517]

[0518] Example 76 (in order of appearance, SEQ ID NOs 25544 and 25545, respectively):

[0519]

[0520] Example 77 (in order of appearance, SEQ ID NOs 25546 and 25547, respectively):

[0521]

[0522] Example 78 (in order of appearance, SEQ ID NOs 25548 and 25549, respectively):

[0523]

[0524] Example 79 (in order of appearance, SEQ ID NOs 25550 and 25551, respectively):

[0525]

[0526] Example 80 (in order of appearance, SEQ ID NOs 25552 and 25553, respectively):

[0527]

[0528] Example 81 (in order of appearance, SEQ ID NOs 25554 and 25555, respectively):

[0529]

[0530] Example 82 (in order of appearance, SEQ ID NOs 25556 and 25557, respectively):

[0531]

[0532] Example 83 (in order of appearance, SEQ ID NOs 25558 and 25559, respectively):

[0533]

[0534] Example 84 (in order of appearance, SEQ ID NOs 25560 and 25561, respectively):

[0535]

[0536] Example 85 (in order of appearance, SEQ ID NOs 25562 and 25563, respectively):

[0537]

[0538] Example 86 (in order of appearance, SEQ ID NOs 25564 and 25565, respectively):

[0539]

[0540] Example 87 (in order of appearance, SEQ ID NOs 25566 and 25567, respectively):

[0541]

[0542] Example 88 (in order of appearance, SEQ ID NOs 25568 and 25569, respectively):

[0543]

[0544] Example 89 (in order of appearance, SEQ ID NOs 25570 and 25571, respectively):

[0545]

[0546] Example 90 (in order of appearance, SEQ ID NOs 25572 and 25573, respectively):

[0547]

[0548] Example 91 (in order of appearance, SEQ ID NOs 25574 and 25575, respectively):

[0549]

[0550] Example 92 (in order of appearance, SEQ ID NOs 25576 and 25577, respectively):

[0551]

[0552] Example 93 (in order of appearance, SEQ ID NOs 25578 and 25579, respectively):

[0553]

[0554] Example 94 (in order of appearance, SEQ ID NOs 25580 and 25581, respectively):

[0555]

[0556] Example 95 (in order of appearance, SEQ ID NOs 25582 and 25583, respectively):

[0557]

[0558] Example 96 (in order of appearance, SEQ ID NOs 25584 and 25585, respectively):

[0559]

[0560] Example 97 (in order of appearance, SEQ ID NOs 25586 and 25587, respectively):

[0561]

[0562] Example 98 (in order of appearance, SEQ ID NOs 25588 and 25589, respectively):

[0563] Example 99 (in order of appearance, SEQ ID NOs 25590 and 25591, respectively):

[0564] Example 100 (in order of appearance, SEQ ID NOs 25592 and 25593, respectively):

[0565] Example 101 (in order of appearance, SEQ ID NOs 25594 and 25595, respectively):

[0566]

[0567] Example 102 (in order of appearance, SEQ ID NOs 25596 and 25597, respectively):

[0568]

[0569] Example 103 (in order of appearance, SEQ ID NOs 25598 and 25599, respectively):

[0570]

[0571] Example 104 (in order of appearance, SEQ ID NOs 25600 and 25601, respectively):

[0572]

[0573] Example 105 (in order of appearance, SEQ ID NOs 25602 and 25603, respectively):

[0574]

[0575] Example 106 (in order of appearance, SEQ ID NOs 25604 and 25605, respectively):

[0576]

[0577] Example 107 (in order of appearance, SEQ ID NOs 25606 and 25607, respectively):

[0578]

[0579] Example 108 (in order of appearance, SEQ ID NOs 25608 and 25609, respectively):

[0580]

[0581] Example 109 (in order of appearance, SEQ ID NOs 25610 and 25611, respectively):

[0582] Example 110 (in order of appearance, SEQ ID NOs 25612 and 25613, respectively):

[0583]

[0584] Example 111 (in order of appearance, SEQ ID NOs 25614 and 25615, respectively):

[0585]

[0586] Example 112 (in order of appearance, SEQ ID NOs 25616 and 25617, respectively):

[0587]

[0588] Example 113 (in order of appearance, SEQ ID NOs 25618 and 25619, respectively):

[0589]

[0590] Example 114 (in order of appearance, SEQ ID NOs 25620 and 25621, respectively):

[0591]

[0592] Example 115 (in order of appearance, SEQ ID NOs 25622 and 25623, respectively):

[0593]

[0594] Example 116 (in order of appearance, SEQ ID NOs 25624 and 25625, respectively):

[0595]

[0596] Example 117 (in order of appearance, SEQ ID NOs 25626 and 25627, respectively):

[0597]

[0598] Example 118 (in order of appearance, SEQ ID NOs 25628 and 25629, respectively):

[0599]

[0600] Example 119 (in order of appearance, SEQ ID NOs 25630 and 25631, respectively):

[0601]

[0602] Example 120 (in order of appearance, SEQ ID NOs 25632 and 25633, respectively):

[0603]

[0604] Example 121 (in order of appearance, SEQ ID NOs 25634 and 25635, respectively):

[0605] Example 122 (in order of appearance, SEQ ID NOs 25636 and 25637, respectively):

[0606]

[0607] Example 123 (in order of appearance, SEQ ID NOs 25638 and 25639, respectively):

[0608]

[0609] Example 124 (in order of appearance, SEQ ID NOs 25640 and 25641, respectively):

[0610] Example 125 (in order of appearance, SEQ ID NOs 25642 and 25643, respectively):

[0611]

[0612] Example 126 (in order of appearance, SEQ ID NOs 25644 and 25645, respectively):

[0613]

[0614] Example 127 (in order of appearance, SEQ ID NOs 25646 and 25647, respectively):

[0615]

[0616] Example 128 (in order of appearance, SEQ ID NOs 25648 and 25649, respectively):

[0617]

[0618] Example 129 (in order of appearance, SEQ ID NOs 25650 and 25651, respectively):

[0619] Example 130 (in order of appearance, SEQ ID NOs 25652 and 25653, respectively):

[0620]

[0621] Example 131 (in order of appearance, SEQ ID NOs 25654 and 25655, respectively):

[0622]

[0623] Example 132 (in order of appearance, SEQ ID NOs 25656 and 25657, respectively):

[0624]

[0625] Example 133 (in order of appearance, SEQ ID NOs 25658 and 25659, respectively):

[0626]

[0627] Example 134 (in order of appearance, SEQ ID NOs 25660 and 25661, respectively):

[0628]

[0629] Example 135 (in order of appearance, SEQ ID NOs 25662 and 25663, respectively):

[0630]

[0631] Example 136 (in order of appearance, SEQ ID NOs 25664 and 25665, respectively):

[0632]

[0633] Example 137 (in order of appearance, SEQ ID NOs 25666 and 25667, respectively):

[0634]

[0635] Example 138 (in order of appearance, SEQ ID NOs 25668 and 25669, respectively):

[0636]

[0637] Example 139 (in order of appearance, SEQ ID NOs 25670 and 25671, respectively):

[0638]

[0639] Example 140 (in order of appearance, SEQ ID NOs 25672 and 25673, respectively):

[0640]

[0641] Example 141 (in order of appearance, SEQ ID NOs 25674 and 25675, respectively):

[0642] Example 142 (in order of appearance, SEQ ID NOs 25676 and 25677, respectively):

[0643]

[0644] Example 143 (in order of appearance, SEQ ID NOs 25678 and 25679, respectively):

[0645]

[0646] Example 144 (in order of appearance, SEQ ID NOs 25680 and 25681, respectively):

[0647] Example 145 (in order of appearance, SEQ ID NOs 25682 and 25683, respectively):

[0648]

[0649] Example 146 (in order of appearance, SEQ ID NOs 25684 and 25685, respectively):

[0650]

[0651] Example 147 (in order of appearance, SEQ ID NOs 25686 and 25687, respectively):

[0652] Example 148 (in order of appearance, SEQ ID NOs 25688 and 25689, respectively):

[0653]

[0654] Example 149 (in order of appearance, SEQ ID NOs 25690 and 25691, respectively):

[0655]

[0656] Example 150 (in order of appearance, SEQ ID NOs 25692 and 25693, respectively):

[0657]

[0658] Example 151 (in order of appearance, SEQ ID NOs 25694 and 25695, respectively):

[0659]

[0660] Example 152 (in order of appearance, SEQ ID NOs 25696 and 25697, respectively):

[0661]

[0662] Example 153 (in order of appearance, SEQ ID NOs 25698 and 25699, respectively):

[0663]

[0664] Example 154 (in order of appearance, SEQ ID NOs 25700 and 25701, respectively):

[0665]

[0666] Example 155 (in order of appearance, SEQ ID NOs 25702 and 25703, respectively):

[0667]

[0668] Example 156 (in order of appearance, SEQ ID NOs 25704 and 25705, respectively):

[0669]

[0670] Stereoisomers thereof, pharmaceutically acceptable salts thereof, isotopes thereof, and combinations thereof.

[0671] In certain embodiments, provided herein is a compound selected from an insulin comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24501 and 24502; and wherein the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25229, 25232, 25305, 25308, 25312, 25236, 25095, and 25380-25397.

[0672] In certain embodiments, each indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL32, FL41, FL62, FL63, and stereoisomers thereof.

[0673] In certain embodiments, B1 and B2 are selected from Formula F2, and each remaining R1 is independently selected from H, F, Cl, I, Br, and CF3.

[0674] In certain embodiments, B1 and B2 are selected from Formula F2, and each remaining R1 is H.

[0675] In certain 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, wherein each B1 and B2 is F2; ​​each Z1c is conjugated directly or via an indirect linker to the amine side chain of a lysine in X1 or to the N-terminus in X1; 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.

[0676] In certain embodiments, at least one Zlc is conjugated to the amine side chain of a lysine in the B-chain or to the N-terminus of the B chain via an indirect linker.

[0677] In certain embodiments, X1 comprises two or more lysine amino acids.

[0678] In certain embodiments, the B-chain comprises at least two lysine amino acids, each of which is independently conjugated to Zlc directly or via an indirect linker.

[0679] In certain embodiments, the B-chain comprises at least three lysine amino acids, each of which is independently covalently conjugated to Zlc via an indirect linker.

[0680] In certain embodiments, the A-chain comprises one or more lysine amino acids, each independently covalently conjugated to Zlc, either directly or via an indirect linker.

[0681] In certain embodiments, X1 comprises an A-chain having at least one lysine covalently conjugated to Z1c via an indirect linker.

[0682] In certain embodiments, the compound of Formula I is selected from:

[0683]

[0684]

[0685] In certain embodiments, at least one Z1c is covalently conjugated to X1 via an indirect linker, and wherein the indirect linker is independently selected from:

[0686]

[0687] Formula FL3,

[0688] Where p is 2 or 3;

[0689]

[0690] Formula FL5, FL5A and FL5B,

[0691] where p is 1 or 2; and

[0692] and its stereoisomers;

[0693] in:

[0694] R" represents a covalent bond directly or indirectly to Z1c;

[0695] Z" represents a covalent bond directly or indirectly to X1 or to Z1a;

[0696] A' is selected from H, alkyl (e.g., C1-C6 alkyl), saturated fatty acid, unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl; and

[0697] A″ is selected from alkyl (e.g., C1-C6 alkyl), substituted acyl, acyl terminated with an acid group, saturated or unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl;

[0698] p is 1, 2, 3, 4, or 5, and

[0699] Any primary amines are optionally acetylated or alkylated.

[0700] In certain embodiments, the indirect linker is selected from:

[0701]

[0702] In certain embodiments, X1 is insulin further comprising 1 to 5 residues substituted with, inserted into, appended to, or mutated to an amino acid having a free amine directly or indirectly conjugated to Z1c.

[0703] In certain embodiments, at least one Zlc is conjugated to the free amine side chain of an amino acid on insulin that has been substituted, inserted, or mutated.

[0704] In certain embodiments, the compound is covalently conjugated, directly or via a linker, to a molecule that can bind to at least one protein present in human plasma.

[0705] In certain embodiments, in each of Formulas F2, F5, and F10, one R1 is (C=O)---*.

[0706] In certain embodiments, the compound is represented by Formula II, or a stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt thereof:

[0707] Z1c-Connector

[0708] (Formula II)

[0709] wherein the Z1c-linker is selected from:

[0710]

[0711]

[0712]

[0713]

[0714]

[0715] wherein X is selected from a leaving group, NH2, OH, and H; and

[0716] B1 and B2, which may be the same or different, each independently represent an aromatic boron-containing group.

[0717] In certain embodiments, the compound represented by formula (II) comprises at least one B1 or B2 independently selected from formula F2, F5 and F10,

[0718] Wherein formula F2, F5 and F10 are:

[0719]

[0720] in:

[0721] One R1 represents (C=O)---* or (CH2) m (C=O)---*, wherein---* represents the point of attachment to the rest of Z1c, and m is 1, 2, 3, 4, 5, 6 or 7; and

[0722] Each remaining R1 is independently selected from H, F, Cl, Br, I, OH, CH2-NH2, NH2, (C=O)-NH2, CH=O, SO2CH3, SO2CF3, CF3, CHF2, NO2, CH3, OCH3, O(CH2) m” CH3, (SO2)NH-CH3, (SO2)NH(CH2) m” CH3 and OCF3, wherein m″ is 1, 2, 3, 4, 5, 6 or 7;

[0723] Y8 is O, N, and NR, wherein R is alkyl (e.g., C1-C6 alkyl) or H;

[0724] Y9 is H, CH3 or alkyl (e.g., C1-C6 alkyl), provided that when Y8 is O, Y9 is CH3 or alkyl (e.g., C1-C6 alkyl); and

[0725] i is 1, 2, or 3.

[0726] In certain embodiments, the Zlc-linker (ie, Formula II) is selected from:

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733] and

[0734] wherein X is a leaving group or represents a point of covalent attachment directly to X1 of Formula I.

[0735] In certain embodiments, when X is a leaving group, X is selected from N-oxysuccinimide, 2,3,5,6-tetrafluorophenoxy (TFP), pentafluorophenoxy (Pfp), OH, halogen, maleimide alkylamino, maleimide amido polyethylene glycol amino and maleimide polyethylene glycol amino. In certain embodiments, X is selected from N-oxysuccinimide and OH. In certain embodiments, X is OH. In certain embodiments, X is N-oxysuccinimide. In certain embodiments, the maleimide polyethylene glycol amino is selected from Mal-PEG2-amine, Mal-PEG4-amine and Mal-PEG5-amine or a pharmaceutically acceptable salt thereof. In certain embodiments, the maleimide alkylamino is selected from Mal-C6-amine and N-(2-aminoethyl)maleimide, or a pharmaceutically acceptable salt thereof. In certain embodiments, the maleimide amido polyethylene glycol amino group is selected from Mal-amido-PEG9-amine, Mal-amido-PEG11-amine, Mal-amido-PEG23-amine, 4-Mal-methyl-cyclohexanecarboxamide-methyl-[1,2,3]triazole-PEG8-amine, or a pharmaceutically acceptable salt thereof. In certain embodiments, the Z1c-linker is selected from:

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742] Its stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts and combinations thereof. In certain embodiments, the Z1c-linker is selected from:

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749] Its stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts and combinations thereof.

[0750] In certain embodiments, a therapeutically effective amount of a pharmaceutical composition of the present disclosure may be administered to a subject. In certain embodiments, the pharmaceutical composition comprises at least one compound disclosed herein (eg, Formula I) and a pharmaceutically acceptable carrier.

[0751] In certain embodiments, the compounds disclosed herein are used as pharmaceuticals.

[0752] In certain embodiments, the present disclosure provides methods for treating or preventing diabetes, impaired glucose tolerance, hyperglycemia, or metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.

[0753] In certain embodiments, the pharmaceutical composition comprises one or more polyols.

[0754] In certain embodiments, the polyol is selected from the group consisting of mannitol, sorbitol, erythritol, isomalt, lactitol, glucose, and maltitol.

[0755] In certain embodiments, the pharmaceutical composition comprises at least one compound disclosed herein for use as a medicament for treating diabetes or obesity, for controlling blood sugar levels, or for controlling drug release.

[0756] In certain embodiments, the present disclosure provides methods of administering a compound disclosed herein or a pharmaceutical composition disclosed herein to a subject as a therapeutic or prophylactic agent.

[0757] In certain embodiments, the present disclosure provides methods of preparing compounds disclosed herein comprising at least one alkylation and / or amidation step.

[0758] In certain embodiments, the present disclosure provides methods of treating a subject by administering a device or formulation comprising a compound as disclosed herein, such as Examples 1-156. For example, the device can be a fixed-dose syringe, a microsyringe, an internal or external patch.

[0759] In certain embodiments, the compounds of the present disclosure can be used as intermediates in the preparation of bulk drugs or therapeutic or prophylactic compounds.

[0760] In another aspect, the disclosure provides a human insulin analog comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24051 and 24052; and wherein the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25000-25397.

[0761] In certain embodiments, the A-chain comprises 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.

[0762] In certain embodiments, the A-chain comprises 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.

[0763] In certain embodiments, the A-chain comprises sequence 24051 and the B-chain comprises a sequence selected from 25228, 25229, 25232, 25234-25237, 25304, 25305, 25308, and 25310-25313.

[0764] In certain embodiments, the A-chain comprises sequence 24051 and the B-chain comprises a sequence selected from 25011, 25012, 25017-25020, 25087, 25088, 25093-25096, 25229, 25232, 25305, and 25308.

[0765] In certain embodiments, the agonist potency of a compound disclosed herein is determined by measuring the potency of the compound to activate a receptor (e.g., an insulin receptor). In certain embodiments, the disclosure provides a compound comprising at least one aromatic boron-containing group that has an agonist potency on the insulin receptor, the compound 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 compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 1.2 to about 14, about 1.2 to about 10, about 2 to about 9, about 2 to about 8, about 3 to about 7, or about 3 to about 6.

[0766] In certain embodiments, the present disclosure relates to a compound comprising at least one aromatic boron-containing group, the aromatic boron-containing group being attached to a FF scaffold comprising a methyl group (e.g., FF116A, FF225A), the compound having an agonist effect on the insulin receptor, the compound having a first EC50 effect of activating the insulin receptor at a first glucose concentration and a second EC50 effect of 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 compound has an insulin receptor agonist effect ratio of the first EC50 to the second EC50 of about 2.5 to about 3.7, about 2.5 to about 3.5, about 2.5 to about 3, or about 3 to about 3.5. In certain embodiments, the methyl group is a β methyl group, such as FF116A. In certain embodiments, the methyl group is an α methyl group, such as FF225A.

[0767] In certain embodiments, the present disclosure provides compounds comprising at least one aromatic boron-containing group that have agonist potency at the insulin receptor, the compound 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 compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 1 to about 15, about 1 to about 10, about 2 to about 9, about 2 to about 8, about 3 to about 7, or about 3 to about 6. In certain embodiments, when the first glucose concentration is 3 mM and the second glucose concentration is 10 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of at least or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0768] In certain embodiments, the present disclosure provides compounds comprising at least one aromatic boron-containing group that have agonist potency at 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 20 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 10, about 2 to about 9, or about 2 to about 8. In certain embodiments, when the first glucose concentration is 3 mM and the second glucose concentration is 20 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of at least or at most 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.

[0769] In certain embodiments, the present disclosure relates to compounds comprising at least one aromatic boron-containing group comprising a methyl group (e.g., an alpha methyl diboronate sensor or a beta methyl diboronate sensor, such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110, wherein X represents a covalent attachment point directly linked to the amine in X1 of Formula I) and having an agonist potency at the insulin receptor, the compound 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 20 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of 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 certain embodiments, the compound has an insulin receptor agonist potency ratio of a first EC50 to a second EC50 of at least or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 when the first glucose concentration is 3 mM and the second glucose concentration is 20 mM.

[0770] In certain embodiments, the present disclosure provides compounds comprising at least one aromatic boron-containing group that have agonist potency at the insulin receptor, the compound 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 20 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of 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.

[0771] In certain embodiments, the present disclosure provides compounds comprising at least one aromatic boron-containing group that have agonist potency at the insulin receptor, the compound 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 5.6 mM and the second glucose concentration is 11.1 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 1.2 to about 5, about 1.3 to about 5, about 1.4 to about 5, about 1.5 to about 5, about 2 to about 4, or about 3 to about 4. In certain 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 potency ratio of the first EC50 to the second EC50 of at least or at most about 1.2, 1.3, 1.4, 1.5, 2, 3, 4, or 5.

[0772] In certain embodiments, the present disclosure relates to compounds comprising at least one aromatic boron-containing group comprising a methyl group (e.g., an alpha methyl diboronate sensor or a beta methyl diboronate sensor, such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110) that have agonist potency at the insulin receptor, the compound having a first EC50 potency that activates the insulin receptor at a first glucose concentration and a second EC50 potency that activates 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 compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 1.5 to about 3.5, about 1.5 to about 3, about 2 to about 3, or about 2.5 to about 3. In certain embodiments, the compound has an insulin receptor agonist potency ratio of a first EC50 to a second EC50 of at least or at most about 1.2, 1.3, 1.4, 1.5, 2, or 3 when the first glucose concentration is 5.6 mM and the second glucose concentration is 11.1 mM.

[0773] In certain embodiments, the present disclosure provides compounds comprising at least one aromatic boron-containing group that have agonist potency at the insulin receptor, the compound 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 5.6 mM and the second glucose concentration is 11.1 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 1 to about 6, about 2 to about 6, about 3 to about 5, or about 3 to about 4. In certain 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 potency ratio of the first EC50 to the second EC50 of at least or at most about 1, 2, 3, 4, 5, or 6.

[0774] In certain embodiments, the present disclosure provides compounds comprising at least one diboronate sensor having binding affinity for glucose, wherein the compound has a Kd for glucose 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 certain embodiments, the compound has a Kd for glucose of less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM. In certain embodiments, the compound has a Kd for glucose of less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM and the compound has agonist activity at the insulin receptor.

[0775] In certain embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having a binding affinity for glucose, wherein when administered at a dose of 30 nmol / kg to a first group of rats with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, the compound 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 certain 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.

[0776] In certain embodiments, the present disclosure relates to compounds comprising at least one aromatic boron-containing group comprising a methyl group (e.g., an alpha methyl diboronate sensor or a beta methyl diboronate sensor such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110) and having agonist efficacy at the insulin receptor, wherein the first glucose infusion rate is administered at a dose of 30 nmol / kg. The compound 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 when administered to a first group of rats at a second glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats at a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL.

[0777] In certain embodiments, the present disclosure provides a compound having agonist potency for glucose comprising at least one aromatic boron-containing group with binding affinity for glucose, wherein the compound 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 when administered to a first group of rats at a dose of 30 nmol / kg with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL. In certain 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 at most about 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0778] In certain embodiments, the present disclosure provides a compound comprising at least one aromatic boron-containing group having a binding affinity for glucose, wherein when administered at a dose of 20 nmol / kg to a first group of rats with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, the compound 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 certain 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 at most about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0779] In certain embodiments, the present disclosure relates to compounds comprising at least one aromatic boron-containing group having binding affinity for glucose, wherein the aromatic boron-containing group comprises a methyl group (e.g., an alpha methyl diboronate sensor or a beta methyl diboronate sensor, such as DSL-9, DSL-14, DSL-18, DSL-66, DSL-69, DSL-72, DSL-75, DSL-109, DSL-110). In certain embodiments, the compound is administered to a first group of rats at a dose of 20 nmol / kg with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, the compound having 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, 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.

[0780] In certain embodiments, the present disclosure provides compounds having agonist potency towards glucose comprising at least one aromatic boron-containing group, wherein 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 when administered to a first group of rats at a dose of 20 nmol / kg with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL.

[0781] In certain embodiments, the present disclosure provides a compound having binding affinity for glucose, comprising at least one aromatic boron-containing group having binding affinity for glucose, wherein when administered at a dose of 10 nmol / kg to a first group of rats with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, the compound provides a relative glucose infusion rate difference (mg / kg / min.min) of 60 to 2400 and a relative glucose infusion rate ratio of 0.5 to about 4.

[0782] In certain embodiments, the present disclosure provides compounds having binding affinity for glucose comprising at least one aromatic boron-containing group comprising at least one diboronate sensor having affinity for glucose.

[0783] In certain embodiments, the present disclosure relates to compounds having agonist activity at the insulin receptor comprising at least one aromatic boron-containing group (e.g., diboronate sensors such as DSL-1 to DSL-112), wherein one or more aromatic boron-containing groups comprise a methyl group (e.g., a beta methyl group, an alpha methyl group). In certain embodiments, the compound is 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 68, Example 69, Example 70, Example 71, Example 72, Example 73, Example 74, Example 75, Example 76, Example 77, Example 78, Example 79, Example 80, Example 81, Example 82, Example 83, Example 84, Example 85, Example 86, Example 87, Example 88, Example 89, Example 90, Example 91 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 certain embodiments, at least one aromatic boron-containing group (e.g., a diboronate sensor) has a binding affinity for glucose. In certain embodiments, compounds comprising two or more diboronate sensors have a higher affinity for glucose. In certain embodiments, compounds comprising three or more diboronate sensors have a higher affinity for glucose.

[0784] In certain 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 certain embodiments, the DSL compounds disclosed herein have binding affinity for glucose with Kd values ​​ranging from about 0.01 mM to about 3 mM. Some exemplary DSL compounds disclosed herein have binding affinity for glucose with Kd values ​​ranging from about 0.01 mM to about 2.5 mM. Some exemplary DSL compounds disclosed herein have binding affinity for glucose with Kd values ​​ranging from about 0.5 mM to about 1.5 mM. In certain embodiments, exemplary DSL compounds have binding affinity for glucose with Kd values ​​less than about 3, 2.5, 2, 1.5, 1, 0.5, or 0.01 mM.

[0785] The present disclosure may also be defined according to any of the following numbered embodiments:

[0786] 1. A compound of the following formula or a stereoisomer or a mixture of stereoisomers or a pharmaceutically acceptable salt thereof:

[0787]

[0788] in:

[0789] X1 comprises a polypeptide comprising an insulin receptor agonist having an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 1, 25, and 24051, and the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24060, 24061, 24063, 25228, 25313, 25393, 25396, and 25397;

[0790] Each Z1a independently comprises from 1 to 50 amino acids linked together using amide or peptide bonds;

[0791] each Z1c is independently selected from formula FF12A, FF12B, FF114A, FF115A, FF116A, and FF225A, wherein each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to an amine of X1 or to OH when X1 is OH;

[0792] Each m' is 0 or 1;

[0793] each p' is 1, 2, 3, 4, or 5; and

[0794] each q' is 1, 2, 3, 4 or 5, wherein when p' or q' is 2 or greater, each corresponding Z1c is independently selected and may be the same or different, and wherein when q' is 2 or greater, each corresponding Z1a is independently selected and may be the same or different;

[0795] When m'=0, each Z1c is covalently conjugated to an amine of X1 or to OH when X1 is OH, directly or via an indirect linker, when m'=1, each Z1a is covalently conjugated to X1 via an amide or peptide bond, and each Z1c is covalently conjugated to an amine of Z1a, directly or via an indirect linker;

[0796] And wherein the formulas FF12A, FF12B, FF114A, FF115A, FF116A and FF225A are:

[0797]

[0798] wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1; and wherein B1 and B2, which may be the same or different, each independently represent an aromatic boron-containing group; and

[0799] The compounds may contain isotopes at one or more positions.

[0800] 2. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein at least one Z1c comprises a β-methyl group.

[0801] 3. The compound of embodiment 1, or a pharmaceutically acceptable salt thereof, wherein at least one Z1c comprises an α-methyl group.

[0802] 4. The compound of embodiment 1 or a pharmaceutically acceptable salt thereof,

[0803] wherein B1 and B2 are each independently of the formula F2,

[0804] The formula F2 is:

[0805]

[0806] in:

[0807] The first R1 represents (C=O)---* or (CH2) m (C=O)---*, wherein---* represents the point of attachment to the rest of Z1c, and m is 1, 2, 3, 4, 5, 6 or 7; and

[0808] Each remaining R1 is independently selected from H, F, Cl, Br, I, OH, CH2-NH2, NH2, (C=O)-NH2, CH=O, SO2CH3, SO2CF3, CF3, CHF2, NO2, CH3, OCH3, O(CH2) m” CH3, (SO2)NH-CH3, (SO2)NH(CH2) m”CH3 and OCF3, wherein m" is 1, 2, 3, 4, 5, 6 or 7.

[0809] 5. The compound of embodiment 4 or a pharmaceutically acceptable salt thereof,

[0810] wherein the first R1 is (C=O)---*.

[0811] 6. The compound of embodiment 4 or a pharmaceutically acceptable salt thereof,

[0812] wherein (a) each remaining R1 is independently selected from (i) H, CF3 and F; or (b) two remaining R1 are H and one remaining R1 is CF3 and F.

[0813] 7. The compound of embodiment 6 or a pharmaceutically acceptable salt thereof,

[0814] wherein each remaining R1 is H.

[0815] 8. The compound of embodiment 1 or a pharmaceutically acceptable salt thereof,

[0816] wherein one or more Z1c is covalently conjugated to X1 or to Z1a via an indirect linker, said indirect linker being of formula (X") n1 express,

[0817] Each n1 is independently selected from 1, 2, 3, 4 and 5, and

[0818] Each X" is independently selected from the formula FL3, FL5, FL5A, FL5B, FL21, FL25, FL33, FL41, FL59, FL60, FL64, FL65, FL68, FL69 and FL74:

[0819]

[0820] and its stereoisomers;

[0821] in:

[0822] R" represents a covalent bond directly or indirectly to Z1c;

[0823] Z" represents a covalent bond directly or indirectly to X1 or to Z1a;

[0824] A' is selected from H, alkyl (e.g., C1-C6 alkyl), saturated fatty acid, unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), C1-C6 haloalkyl, aryl, and heteroaryl; and

[0825] A″ is selected from alkyl (e.g., C1-C6 alkyl), substituted acyl, acyl terminated with an acid group, saturated or unsaturated fatty acid, cycloalkyl (e.g., (C3-C6)cycloalkyl), haloalkyl (e.g., C1-C6 haloalkyl), aryl, and heteroaryl;

[0826] p is 1, 2, 3, 4, or 5, and

[0827] Any primary amines are optionally acetylated or alkylated.

[0828] 9. The compound according to embodiment 8 or a pharmaceutically acceptable salt thereof,

[0829] Each of the indirect linkers is independently selected from FL3, FL5, FL5A, FL5B, FL33, FL64, FL65, FL69 and stereoisomers thereof.

[0830] 10. The compound according to embodiment 1 or a pharmaceutically acceptable salt thereof,

[0831] wherein one or more Z1c are covalently conjugated to X1 via an indirect linker, and wherein the indirect linker is independently selected from

[0832]

[0833] Formula FL3,

[0834] where p is 2 or 3; and

[0835]

[0836] Formula FL5, FL5A and FL5B,

[0837] where p is 1 or 2.

[0838] 11. The compound of embodiment 10 or a pharmaceutically acceptable salt thereof,

[0839] wherein the indirect linker is selected from:

[0840] and their stereoisomers.

[0841] 12. The compound of embodiment 1 or a pharmaceutically acceptable salt thereof,

[0842] wherein each of Z1c is covalently conjugated to an indirect linker to form a Z1c-linker independently selected from:

[0843]

[0844] wherein X represents a covalent attachment point, either directly to the amine in X1 or to the OH group when X1 is OH.

[0845] 13. The compound of embodiment 1, wherein the compound is selected from 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; pharmaceutically acceptable salts thereof, isotopes thereof, and combinations thereof.

[0846] 14. The compound of embodiment 1, wherein the compound is selected from 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; pharmaceutically acceptable salts thereof, isotopes thereof, and combinations thereof.

[0847] 15. A pharmaceutical composition comprising at least one compound according to embodiment 1 or a stereoisomer or a mixture of stereoisomers or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0848] 16. A pharmaceutical composition comprising at least one compound according to embodiment 13 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0849] 17. A pharmaceutical composition comprising at least one compound according to embodiment 14 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0850] 18. A method for treating diabetes, impaired glucose tolerance, hyperglycemia or metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of embodiment 1 or a stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt thereof.

[0851] 19. A method for treating diabetes, impaired glucose tolerance, hyperglycemia or metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to embodiment 15.

[0852] 20. A method of treating type 1 diabetes or type 2 diabetes comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to any one of embodiments 15-17.

[0853] 21. A method for treating type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to embodiment 16 or 17.

[0854] 22. A compound represented by formula II or a stereoisomer or mixture of stereoisomers or a pharmaceutically acceptable salt thereof:

[0855] Z1c-Connector

[0856] (Formula II)

[0857] wherein the Zlc-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; and wherein X is a leaving group, and

[0858] The compound of formula II may contain isotopes at one or more positions.

[0859] 23. The compound of embodiment 22, wherein X is selected from N-hydroxysuccinimide (NHS), 2,3,5,6-tetrafluorophenol (TFP), pentafluorophenol (Pfp), OH and halogen.

[0860] 24. The compound of embodiment 22, wherein the compound is 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, stereoisomers or mixtures of stereoisomers thereof, pharmaceutically acceptable salts, and combinations thereof.

[0861] 25. The compound of embodiment 22, wherein the compound is 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, DSL66B, 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, stereoisomers or mixtures of stereoisomers thereof, pharmaceutically acceptable salts, and combinations thereof.

[0862] Preparation method

[0863] In certain embodiments, the present disclosure provides methods of preparing compounds comprising aromatic boron-containing compounds and / or aromatic boron-containing groups (e.g., Zlc, Zlc-linker (Formula II), Formula I) or pharmaceutical products comprising one or more compounds of the present disclosure.

[0864] In certain embodiments, the present disclosure provides methods for preparing rotationally restricted, tethered boron conjugates containing a scaffold (Z1c, Z1c-linker) that is rotationally hindered due to unfavorable steric interactions (e.g., twisting vs anti-interactions of substituents), hindered rotation due to bond hybridization (e.g., cis- vs trans-amide rotation), or by rigid covalent bonds (e.g., (E) vs (Z) configuration of an olefin moiety). For example, Formulas FF116, FF116A, FF116B, FF116C, and FF116D contain an alkyl functional group that is geminal (e.g., attached to the same atom) with an amine group that is covalently conjugated to a boronic acid-functionalized moiety. As another example, one or more of Formulas FF116, FF116A, FF116B, FF116C, and FF116D contain geminal alkyl substituents that can restrict the accessible dihedral angles adopted by the boron-conjugated amine, thereby affecting the adopted dihedral angles and bringing the boronic acid functionalities closer together and allowing increased binding of the conjugate to a target molecule (such as a protein or carbohydrate).

[0865] In certain embodiments, the compounds disclosed herein are further modified by being connected (e.g., conjugated, fused, etc.) to a second agent or therapy to form a fusion protein. In certain embodiments, the second agent or therapy is a protein or peptide described herein. In certain embodiments, the second agent is a bulk drug, wherein the bulk drug is a polypeptide human hormone, an endocrine hormone, insulin, human insulin, glucagon or a glucagon analog, amylin, relaxin, GLP-1, oxyntomodulin, somatostatin, gastric inhibitory peptide, a glucose-dependent insulin-releasing polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog thereof. In certain embodiments, the fusion protein comprises one or more diboronate sensors described herein. In certain embodiments, two or more agents are connected (e.g., conjugated, fused, etc.) to produce a fusion protein comprising one or more diboronate sensors. In certain embodiments, the two or more agents are proteins or peptides described herein. According to methods known to those skilled in the art, the biological activity (e.g., agonist efficacy, bioavailability, etc.) or therapeutic methods of the compounds and compositions described herein can be evaluated. In certain embodiments, the biological activity of the compound is determined by evaluating the EC50 of the compound. In certain embodiments, the biological activity of the compound is determined using insulin receptor phosphorylation (IR phosphorylation) assay disclosed herein. In certain embodiments, the biological activity of the compound is determined by evaluating the binding affinity (Kd) of the compound to its target. In certain embodiments, the biological activity of the compound is determined by evaluating the relative glucose rate difference. In certain embodiments, the biological activity of the compound is determined by evaluating the relative glucose rate ratio.

[0866] Treatment

[0867] In certain embodiments, the present disclosure provides a method for treating a subject suffering from or susceptible to a disease that can be beneficially treated by a compound disclosed herein or a pharmaceutical product comprising one or more compounds disclosed herein. In certain embodiments, the method comprises administering an effective amount of a pharmaceutical product / composition of the present disclosure to a subject in need thereof. In at least one embodiment, the compound and / or pharmaceutical product of the present disclosure can be used to treat or prevent a disorder (or for the preparation of a medicament for treating or preventing a disorder), the disorder comprising hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome X or dyslipidemia, gestational diabetes, prediabetes, Alzheimer's disease, MODY 1, MODY 2 or MODY 3 diabetes, neurological diseases, mood disorders and psychiatric disorders. In at least one embodiment, a therapeutically effective amount of a compound and / or pharmaceutical product of the present disclosure is administered to a subject suffering from diabetes. In certain embodiments, the diabetes is type 1 diabetes or type 2 diabetes. In certain embodiments, the diabetes is type 1 diabetes.

[0868] In certain embodiments, when a first active agent is administered with a second (another) active agent, the dosage can be adjusted so that the combined activity of the two treatments is sufficient to regulate the patient's blood glucose level. Thus, in such a combination, the amount of the first or second active agent that can be administered to regulate blood glucose levels may be less than the amount required if the first or second active agent were administered as a monotherapy.

[0869] 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.

[0870] The following abbreviations have the following definitions:

[0871]

[0872] Example

[0873] A. Preparation of aromatic boron-containing compounds.

[0874] The disclosed compounds can be prepared according to the following schemes. The following schemes represent general methods for preparing these compounds. However, the synthesis of these compounds is not limited to these representative methods, as those skilled in the art of synthetic chemistry can also prepare them by various other methods.

[0875] DSL synthesis method 1:

[0876] On-resin synthesis of the diboronated sensor:

[0877] 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 (0.93 mg, 0.3 mmol) in DCM and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL), then DMF (3 x 5 mL), and treated with 20% piperidine in DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50°C for 30 min, washed with DMF (4 x 5 mL) to give 5. The resin was treated with 20% piperidine in DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 7. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL).

[0878] Cutting the diboronated sensor from the resin

[0879] Compound 7 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid 8 (DSL-66A), which was used in the next step without purification.

[0880] NHS-activation of diboronated sensors:

[0881] The crude 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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)propanoate 11 (DSL-66B) (20 mg) in approximately 33% yield. Calculated mass (M+H) + =607.2, observed mass (M+H) + =606.9.

[0882] Method 1

[0883]

[0884] The MS data for DSL-66B are listed in Table I. The other examples listed in Table I below were synthesized under similar conditions.

[0885] Table I

[0886] DSL-B Example <![CDATA[Calculated mass [M+H]+ 1 > <![CDATA[Quality of observation [M+H]+ 1 > DSL-9B 647.24 647 DSL-14B 643.19 643.07 DSL-66B 607.20 607.08 DSL-73B 607.20 DSL-112B 743.18 742.90

[0887] DSL synthesis method 2:

[0888] On-resin synthesis of the diboronated sensor:

[0889] 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swelled 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 (0.93 mg, 0.3 mmol) in DCM and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL) and then with DMF (3 x 5 mL). The resin was treated with 20% piperidine in DMF for 5 minutes (3 x 5 mL) and washed with DMF (4 x 5 mL). A solution of (2S,3S)-(Fmoc-amino)-3-azidobutanoic 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 min to produce 5. The resin was washed with DMF (4 x 5 mL) and THF (3 x 5 mL) and treated with 5 mL of a solution of 0.2 M SnCl2 (189 mg), 0.8 M thiophenol (440 μl), and 1 M DIEA (870 μl) in THF and gently mixed on a shaker for 1 hour. After 1 hour, the resin was washed with THF (3 x 5 mL) and DMF (3 x 5 mL). The resin was treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL) and washed with DMF (4 x 5 mL) to give 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 min to give 7. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL).

[0890] Cutting the diboronated sensor from the resin

[0891] Compound 7 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce diboronated sensor acid 8 (DSL-66A), which was used in the next step without purification. A similar procedure was followed to synthesize DSL-14A, DSL-15A, and DSL-68A.

[0892] NHS-activation of diboronated sensors:

[0893] The 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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), followed by gentle mixing overnight. The reaction progress was monitored by LCMS. Upon completion, 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 NaSO and dried under vacuum, dissolved in DMSO (100 μL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes, yielding the desired product as analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)propanoate 11 (DSL-66B) (17.4 mg) in 29% yield. Calculated mass (M+H) + =607.2, observed mass (M+H) + =606.9.

[0894] Similar procedures were followed to synthesize DSL-14B, DSL-15B, and DSL-68B.

[0895] method

[0896]

[0897] DSL synthesis method 3:

[0898] On-resin synthesis of the diboronated sensor:

[0899] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 41. The resin was washed with DCM (3 x 5 mL), then with DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50° C. for 30 min and washed with DMF (4 x 5 mL) to give 42. The resin was treated with 20% piperidine / DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 43. The resin was washed with DMF (4×5 mL) and DCM (3×5 mL).

[0900] Cutting the diboronated sensor from the resin

[0901] Compound 43 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-1A-OtBu, which was used in the next step without purification.

[0902] NHS-activation of diboronated sensors:

[0903] The 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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The reaction progress was monitored by LCMS. After the reaction was complete, 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 / DCM (50%), allowed to mix for 1 hour, reduced under vacuum, then dissolved in DMSO (100 uL) and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient of 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield DSL-1B (25 mg) as a white powder.

[0904]

[0905] The MS data for DSL-1B are listed in Table II. Other examples listed in Table II below were synthesized under similar conditions.

[0906] Table II

[0907] DSL-B Example <![CDATA[Calculated mass [M+H] +1 > <![CDATA[Quality of Observation [M+H] +1 > DSL-1B 648.20 DSL-2B 663.19 718.93 (with tBu group) DSL-70B 677.21 733.19 (with tBu group)

[0908] DSL synthesis method 4:

[0909] On-resin synthesis of the diboronated sensor:

[0910] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 44. The resin was washed with DCM (3 x 5 mL), then with DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50° C. for 30 min and washed with DMF (4 x 5 mL) to give 45. The resin was treated with 20% piperidine / DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 43. The resin was washed with DMF (4×5 mL) and DCM (3×5 mL).

[0911] Cutting the diboronated sensor from the resin

[0912] 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-15A-OtBu, which was used in the next step without purification.

[0913] NHS-activation of diboronated sensors:

[0914] The crude diboronated sensor acid DSL-15A-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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The reaction progress was monitored by LCMS. After the reaction was complete, 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 / DCM (50%), allowed to mix for 1 hour, reduced under vacuum, then dissolved in DMSO (100 uL) and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient of 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield DSL-15B (21 mg) as a white powder.

[0915] Method 1

[0916]

[0917] The MS data for DSL-15B are listed in Table III. The other examples listed in Table III below were synthesized under similar conditions.

[0918] Table III

[0919] DSL-B Example <![CDATA[Calculated mass [M+H]+ 1 > <![CDATA[Quality of observation [M+H]+ 1 > DSL-15B 636.20 DSL-51B 801.18 856.45 (with tBu group) DSL-75B 665.21 734.75 (with tBu group)

[0920] DSL synthesis method 5:

[0921] On-resin synthesis of the diboronated sensor:

[0922] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 13. The resin was washed with DCM (3 x 5 mL), then with DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50° C. for 30 min and washed with DMF (4×5 mL) to give 15. The resin was treated with 20% piperidine / DMF for 5 min (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 min to give 16. The resin was washed with DMF (4×5 mL) and DCM (3×5 mL).

[0923] Cutting the diboronated sensor from the resin

[0924] Compound 16 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-67A, which was used in the next step without purification.

[0925] 5b: NHS-activation of diboronated sensors:

[0926] The crude diboronated sensor acid DSL-67A 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 reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield (S)-3-((2S,4R)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pyrrolidine-2-carboxamido)-5-methylhexanoate 2,5-dioxopyrrolidin-1-yl ester DSL-67B in approximately 28% yield (18.7 mg). Calculated mass (M+H) + =675.2, observed mass (M+H) + =675.0.

[0927]

[0928] The MS data for DSL-67B are listed in Table IV. The other examples listed in Table IV below were synthesized under similar conditions.

[0929] Table IV

[0930] DSL-B Example <![CDATA[Calculated mass [M+H] +1 > <![CDATA[Quality of Observation [M+H] +1 > DSL-19B 619.20 DSL-37B 673.25 672.99 DSL-47B 727.24 727.88 DSL-48B 661.25 661.12 DSL-58B 739.26 738.95 DSL-65B 799.20 798.83 DSL-67B 675.27 675.07 DSL-105B 709.25 709.03 DSL-107B 723.27 722.93 DSL-111B 695.24 695.09

[0931] DSL synthesis method 6:

[0932] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 18. The resin was washed with DCM (3 x 5 mL), then with DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 (5 mL) was added to the resin and heated at 50 °C for 30 min and washed with DMF (4 x 5 mL) to give 20. The resin was treated with 20% piperidine / DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 21. The resin was washed with DMF (4×5 mL) and DCM (3×5 mL).

[0933] Cutting the diboronated sensor from the resin

[0934] Compound 21 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid (DSL-60A), which was used in the next step without purification.

[0935] The crude diboronated sensor acid DSL-60A 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 reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield 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)pyrrolidine-2-carboxamido)cyclopentyl)acetate DSL-60B (14.7 mg) in approximately 22% yield. Calculated mass (M+H) + =673.2, observed mass (M+H) + =673.0.

[0936] Similar procedures were followed to synthesize DSL-61B to DSL-77B and DSL-108B.

[0937]

[0938] The MS data for DSL-60B are listed in Table V. The other examples listed in Table V below were synthesized under similar conditions.

[0939] Table V

[0940] DSL-B Example <![CDATA[Calculated mass [M+H] +1 > <![CDATA[Quality of observation [M+H] +1 > DSL-60B 672.24 DSL-77B 619.20 619.11 DSL-108B 655.19

[0941] DSL synthesis method 7:

[0942] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL), then DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 5 mL). A solution of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((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) in DMF (5 mL) was added to the resin and heated at 50 °C for 30 min, washed with DMF (4 x 5 mL) to give 23, which was then treated with 20% piperidine in DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 and heated at 50° C. for 30 min and washed with DMF (4 x 5 mL) to give 24. The resin was treated with 20% piperidine / DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 25. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL) and treated with tetrakis(triphenylphosphine)palladium (20 mol%, 46 mg), and phenylsilane (493 ul, 4 mmol) in 6 mL of DCM was stirred at room temperature for 1 h and washed with DCM (3 x 5 mL) and DMF (3 x 5 mL).A solution of octanoic acid 26 (63 ul, 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 and heated at 50°C for 30 min, washed with DMF (4 x 5 mL) to give 27.

[0943] Cutting the diboronated sensor from the resin

[0944] Compound 27 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-68A, which was used in the next step without purification.

[0945] 7b: NHS-activation of diboronated sensors:

[0946] The 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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol) and gently mixed overnight. The reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were separated, combined, frozen, and lyophilized to yield 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-octanamidohexanamido)propanoate DSL-68B (26.1 mg) in approximately 30% yield. Calculated mass (M+H) + =873.4, observed mass (M+H) + =873.2.

[0947]

[0948] DSL synthesis method 8:

[0949] Synthesis of (R)-3,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyric acid

[0950] 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 h. After the reaction was complete, the solvent was removed under reduced pressure and further co-evaporated with DCM (15 mL x 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 h. The reaction mixture was diluted with ethyl acetate (35 mL) and acidified to pH ~3 with 1N HCl. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 x 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%).

[0951] Synthesis of (R)-3-(3,4-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butyramido)propanoic acid 2,5-dioxopyrrolidin-1-yl ester

[0952] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL), then DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50°C for 30 min and washed with DMF (4 x 5 mL) to give 31. The resin was treated with 20% piperidine in DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 32. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL).

[0953] Cutting the diboronated sensor from the resin

[0954] Compound 32 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-104A, which was used in the next step without purification.

[0955] 8b: NHS-activation of diboronated sensors:

[0956] The crude diboronated sensor acid DSL-104A 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 reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield DSL-104B(R)-2,5-dioxopyrrolidin-1-yl 3-(3,4-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)propanoate (13 mg) in approximately 22% yield. Calculated mass (M+H) + =607.2, observed mass (M+H) + =607.1.

[0957]

[0958] DSL synthesis method 9:

[0959] Synthesis of (R)-3-(4,5-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pentanamido)propanoic acid 2,5-dioxopyrrolidin-1-yl ester

[0960] 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 and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIPEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL), then DMF (3 x 5 mL), and treated with 20% piperidine / DMF for 5 minutes (3 x 5 mL), and washed with DMF (4 x 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 and heated at 50°C for 30 min and washed with DMF (4 x 5 mL) to give 34. The resin was treated with 20% piperidine in DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 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 min to give 35. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL).

[0961] Cutting the diboronated sensor from the resin

[0962] Compound 35 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-106A, which was used in the next step without purification.

[0963] 9b: NHS-activation of diboronated sensors:

[0964] The crude diboronated sensor acid DSL-106A 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 reaction progress was monitored by LCMS. After the reaction was complete, 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, dissolved in DMSO (100 uL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes and analyzed by LCMS. Pure fractions were isolated, combined, frozen, and lyophilized to yield DSL-106B(R)-2,5-dioxopyrrolidin-1-yl 3-(4,5-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pentanamido)propanoate (18.6 mg) in approximately 30% yield. Calculated mass (M+H) + =621.2, observed mass (M+H) + =621.1.

[0965]

[0966] DSL synthesis method 10:

[0967] 2-Chlorotrityl resin 1 (300 mg, 0.3 mmol) was swelled 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 (0.93 mg, 0.3 mmol) in DCM and DIPEA (0.3 mL, 1.7 mmol) was immediately added and gently mixed overnight. The mixture was washed with DCM, and unreacted sites were capped with 20% MeOH in DCM and DIEA (1 M) and mixed for 1 hour to produce 3. The resin was washed with DCM (3 x 5 mL) and then with DMF (3 x 5 mL). The resin was treated with 20% piperidine in DMF for 5 minutes (3 x 5 mL) and washed with DMF (4 x 5 mL). A solution of (2S,3S)-(Fmoc-amino)-3-azidobutanoic 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 min to produce 5. The resin was washed with DMF (4 x 5 mL) and THF (3 x 5 mL) and treated with 5 mL of a solution of 0.2 M SnCl (189 mg), 0.8 M thiophenol (440 μl) and 1 M DIEA (870 μl) in THF and gently mixed on a shaker for 1 hour. After 1 hour, the resin was washed with THF (3 x 5 mL) and DMF (3 x 5 mL) to produce 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 min to give 37.

[0968] The resin was washed with DMF (5 x 5 mL) and treated with 20% piperidine / DMF for 5 min (3 x 5 mL) and washed with DMF (4 x 5 mL) before coupling with 38 to give 39. The resin was washed with DMF (4 x 5 mL) and DCM (3 x 5 mL).

[0969] Cutting the diboronated sensor from the resin

[0970] Compound 39 on the resin was treated with 20% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) / 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 x 5 mL) and evaporated to produce the diboronated sensor acid DSL-109A, which was used in the next step without purification.

[0971] 10b: NHS-activation of diboronated sensors:

[0972] The 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) and N-hydroxysuccinimide 10 (NHS) (0.115 g, 1 mmol), then gently mixed overnight. The reaction progress 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 NaSO and dried under vacuum, dissolved in DMSO (100 μL), and fractionated by reverse phase (RP) flash chromatography on a C18 column using a gradient from 20% ACN / water containing 0.1% TFA to 60% ACN / water containing 0.1% TFA over 10 minutes, yielding the desired product as analyzed by LCMS. Pure fractions were isolated, combined, frozen and lyophilized to yield 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2-(4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)-3-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)propanoate DSL-109B (12.5 mg) in 20% yield. Calculated mass (M+H) + =625.1, observed mass (M+H) + =625.1.

[0973]

[0974] The MS data for DSL-109B are listed in Table VI. The examples listed in Table VI below were synthesized under similar conditions.

[0975] Table VI

[0976] DSL-B Example <![CDATA[Calculated mass [M+H] +1 > <![CDATA[Quality of Observation [M+H] +1 > DSL-109B 624.18 DSL-110B 675.19 675.07

[0977] The chemical structures and IUPAC names of DSL-1A to DSL-112A are summarized in Table 1 below.

[0978] Table 1

[0979]

[0980]

[0981]

[0982]

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

[0994]

[0995]

[0996]

[0997]

[0998]

[0999]

[1000]

[1001]

[1002]

[1003]

[1004]

[1005]

[1006]

[1007]

[1008]

[1009]

[1010]

[1011]

[1012]

[1013]

[1014]

[1015]

[1016]

[1017]

[1018]

[1019]

[1020]

[1021]

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

[1029]

[1030]

[1031]

[1032]

[1033]

[1034] Synthesis of compounds of formula I

[1035] Synthesis of compounds of formula I

[1036] Exemplary synthetic schemes are provided that can be used to synthesize the described examples.

[1037] 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 not histidine, but rather the N-terminal hydrogen. The -OH shown at the C-terminus of the A- and B-chains is the C-terminus of each chain.

[1038] Insulin expression and conjugation method 1

[1039] 1a. Expression of proinsulin

[1040] Proinsulin can be expressed in E. coli strains (such as B21 strains) 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag prior to sensor conjugation.

[1041] Expression of the desired single-chain proinsulin can be performed by 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.

[1042] 1b. Conjugation of diboronate sensor 11 to proinsulin.

[1043] To a solution of single-chain proinsulin 12 (20 mg) in DMSO (200 μL) was added 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butyramido)propanoate (11,DSL-66B, 4.4 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 13. The reaction mixture was centrifuged (11000 RPM, 5 min), decanted, and the supernatant resuspended in 100 μL of 50 mM acetic acid (pH 4.5), 100 mM NaCl, and 30% ACN solution. The solution is diluted with 500uL water, then with 30uL of 1M Tris pH 9 (50mM final concentration), and 6uL of 500mM CaCl2 (5mM final concentration) is added. With 0.5MNaOH (5-10uL), pH is adjusted to about 9-9.5 and trypsin (1:500 to 1:2000 trypsin mass: insulin mass, about 10uL of 0.2ug / mL stock solution) is added. The crude solution is stirred at room temperature for 16 hours. The crude conjugate of conjugated insulin is precipitated with TFA (~200uL), centrifuged, and decanted. The crude precipitate is then washed with water (2x 500uL), then dissolved in DMSO (100-200uL), diluted with 20% ACN / water (5-10mL), and purified by reversed-phase HPLC. The fractions were collected, frozen, and lyophilized to give 14 (Example 19) as a white powder (5-10 mg).

[1044] Scheme 1. Conjugation of the diboronate sensor (11) to proinsulin (12) (SEQ ID NOs 25706-25707 and 25434-25435, respectively, in order of appearance).

[1045]

[1046]

[1047] The MS data for Example 19 (Compound 14) are listed in Table A. Other Examples listed in Table A below were synthesized under similar conditions.

[1048] Table A

[1049]

[1050]

[1051]

[1052] Insulin expression and conjugation method 2

[1053] 2a. Expression of proinsulin

[1054] Proinsulin can be expressed in E. coli strains (such as B21 strains) 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag prior to sensor conjugation.

[1055] Expression of the desired single-chain proinsulin can be performed by procedures known in the art. See, for example, 5,457,066, 5,700,662, 5,514,646, 9,050,371, and 10,400,021.

[1056] 2b. Conjugation of the diboronate sensor DSL-51 to proinsulin.

[1057] To a solution of single-chain proinsulin 15 (20 mg) in DMSO (200 uL) was added (S)-2-((2S,3S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)-6-(2,5-dioxopyrrolidin-1-yl)-5-oxohexanoic acid (DSL-51B, 6.2 mg) in DMSO (50 uL) and diisopropylethylamine (DIPEA, 20 uL). The reaction 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 15i. The reaction mixture was centrifuged (11000 RPM, 5 min), decanted, and the supernatant resuspended in 100 uL 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 and then 30 uL of 1 M Tris pH 9 (50 mM final concentration), and 6 uL of 500 mM CaCl2 (5 mM final concentration) was added. The pH was adjusted to approximately 9-9.5 with 0.5 M NaOH (5-10 uL) and trypsin (1:500 to 1:2000 trypsin mass: insulin mass, approximately 10 uL of 0.2 ug / mL stock solution) and carboxypeptidase (CBP, 1:1000 carboxypeptidase: insulin mass) were added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugated insulin was precipitated with TFA (-200 uL) and the solution was centrifuged, decanted, and the precipitate was dissolved in DMSO (100-200 uL), diluted into 20% ACN / water (5-10 mL), and purified by reverse phase HPLC. The fractions were collected, frozen, and lyophilized to produce Example 11 as a white powder (6 mg).

[1058] Alternatively, proinsulin 15 was treated with (S)-tert-butyl 2-((2S,3S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butanamido)-6-(2,5-dioxopyrrolidin-1-yl)-5-oxohexanoate (DSL-51-OtBu) under similar conditions as described above. After digestion and precipitation, the crude conjugate was dissolved in TFA:HO (95:5), stirred for 1 hour, concentrated under reduced pressure, dissolved in DMSO (100-200 uL), diluted into 20% ACN / water (5-10 mL), and purified by reverse phase HPLC. Fractions were collected, frozen, and lyophilized to produce Example 11.

[1059] Scheme 2. Conjugation of DSL-51B to proinsulin 15 (SEQ ID NOs 25708-25709 and 25418-25419, respectively, in order of appearance).

[1060]

[1061]

[1062] The MS data for Example 2 are listed in Table B. The other examples listed in Table B below were synthesized under similar conditions.

[1063] Table B

[1064]

[1065] Insulin expression and conjugation method 3

[1066] 3a. Expression of proinsulin

[1067] Proinsulin can be expressed in E. coli strains (such as B21 strains) 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag prior to sensor conjugation.

[1068] Expression of the desired single-chain proinsulin can be performed by 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.

[1069] 3b. Conjugation of the diboronate sensor DSL-58B to proinsulin.

[1070] To a solution of single-chain proinsulin 16 (20 mg) in DMSO (200 uL) was added (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 uL) and diisopropylethylamine (DIPEA, 20 uL). The reaction 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 16i. The reaction mixture was centrifuged (11000 RPM, 5 min), decanted, and the supernatant resuspended in 100 uL 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 with 30 uL of 1 M Tris pH 9 (50 mM final concentration), and 6 uL of 500 mM CaCl2 (5 mM final concentration) was added. The pH was adjusted to approximately 9-9.5 with 0.5 M NaOH (5-10 uL) and trypsin (1:500 to 1:2000 trypsin mass: insulin mass, approximately 10 uL of 0.2 ug / mL stock solution) was added. The insulin mixture was stirred at room temperature for 16 hours. The crude conjugated insulin was precipitated with TFA (-200 uL) and the solution was centrifuged, decanted, and the precipitate was dissolved in DMSO (100-200 uL), then diluted into 20% ACN / water (5-10 mL) and purified by reverse phase HPLC. The fractions were collected, frozen, and lyophilized to yield Example 141 (8.2 mg) as a white powder.

[1071] Scheme 3. Conjugation of DSL-58B to proinsulin 16 (SEQ ID NOs 25710-25711 and 25674-25675, respectively, in order of appearance).

[1072]

[1073]

[1074] The MS data for Example 141 are listed in Table C. The other examples listed in Table C below were synthesized under similar conditions.

[1075] Table C

[1076]

[1077] Insulin expression and conjugation method 4

[1078] 4a. Expression of proinsulin

[1079] Proinsulin can be expressed in E. coli strains (such as B21 strains) 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag prior to sensor conjugation.

[1080] Expression of the desired single-chain proinsulin can be performed by 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.

[1081] 4b. Conjugation of the diboronate sensor DSL-66 to proinsulin 18.

[1082] To a solution of single-chain proinsulin 18i (20 mg) in DMSO (200 μL) was added 2,5-dioxopyrrolidin-1-yl 3-((2S,3S)-2,3-bis(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)butyramido)propanoate (DSL-66B, 6.2 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 18i. The reaction mixture was centrifuged (11,000 RPM, 5 min), decanted, and the supernatant resuspended in 100 μL of a solution of 50 mM acetic acid (pH 4.5), 100 mM NaCl, and 30% ACN. The solution is diluted with 500uL water, then with 30uL of 1M Tris pH 9 (50mM final concentration), and 6uL of 500mM CaCl (5mM final concentration) is added. With 0.5M NaOH (5-10uL), pH is adjusted to about 9-9.5 and trypsin (1:500 to 1:2000 trypsin mass: insulin mass, about 10uL of 0.2ug / mL stock solution) is added. The insulin mixture is stirred at room temperature for 16 hours. Crude conjugated insulin is precipitated with TFA (~200uL) and the solution is centrifuged, decant, and the precipitate is dissolved in DMSO (100-200uL), then diluted to 20% ACN / water (5-10mL) and purified by reversed-phase HPLC. The fractions are collected, frozen, and lyophilized to produce Example 69 (7mg) as a white powder.

[1083] Scheme 4. Conjugation of DSL-66B to proinsulin 18 (SEQ ID NOs 25712-25713 and 25530-25531, respectively, in order of appearance).

[1084]

[1085]

[1086] The MS data for Example 69 are listed in Table D. The other examples listed in Table D below were synthesized under similar conditions.

[1087] Table D

[1088]

[1089] Insulin expression and conjugation methods 5

[1090] 5a. Expression of proinsulin

[1091] Proinsulin can be expressed in E. coli strains (such as B21 strains) 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 to express proinsulin. Proinsulin is expressed in inclusion bodies (IBs), which are captured, washed, and further purified, for example, by an existing his-tag prior to sensor conjugation.

[1092] Expression of the desired single-chain proinsulin can be performed by procedures known in the art. See, for example, U.S. Patent Nos. 5,457,066, 5,700,662, 5,514,646,...

Claims

1. A compound represented by formula I or a stereoisomer or a mixture of stereoisomers or a pharmaceutically acceptable salt thereof: in: X1 Included i. APIs containing amines; ii. is constructed to be covalently conjugated to an amine of the drug substance; or iii.NH2 or OH; Each Z1a independently comprises from 1 to 50 amino acids linked together using amide or peptide bonds; Each Z1b is independently a small molecule linker; Each Z1c is independently selected from Formula FF12, FF12A, FF12B, FF12C, FF12D, FF114, FF114A, FF114B, FF115, FF115A, FF115B, FF116, FF116A, FF116B, FF116C, FF116D, FF117, FF193A, FF203, FF225, FF225A, FF225B, and FF226, wherein each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to an amine of X1 or to NH2 when X1 is NH2 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; and each q' is 1, 2, 3, 4 or 5, wherein when p' or q' is 2 or greater, each corresponding Z1c is independently selected and may be the same or different, and wherein when q' is 2 or greater, each corresponding Z1a is independently selected and may be the same or different; when m'=0 and n'=0, each Z1c is covalently conjugated directly or via an indirect linker to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH, and for m'=1, each Z1a is covalently conjugated to X1 via an amide or peptide bond, and each Z1c is covalently conjugated directly or via an indirect linker to an amine of Z1a or to X1; and Wherein 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 are: wherein X represents a covalent attachment point, which is directly attached to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1; and wherein B1 and B2 may be the same or different and each independently represents an aromatic boron-containing group; and The compounds of Formula I may contain isotopes at one or more positions.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein at least one Z1c is covalently conjugated to an amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine of Z1a via an indirect linker, Indirect joints are made of the formula (X") n1 Indicates that Each n1 is independently selected from 1, 2, 3, 4 and 5, and Each X" is independently selected from: iL- or D-amino acid, wherein the amine function of the L- or D-amino acid is covalently conjugated directly or indirectly to Z1c, and the acid function of the L- or D-amino acid is directly or indirectly conjugated to X1 or to Z1a; and ii. Formula FL(IA), FL(IB), FL69 and FL70; The formulas FL(IA) and FL(IB) are: and their stereoisomers; in: G is selected from 3- to 6-membered cycloalkyl, 3- to 10-membered heterocyclyl, heteroaryl and aryl, each of which is optionally substituted with 1-3 groups independently selected from hydroxy, amino, halogen, (C3-C6)cycloalkyl, alkoxy and C1-C6 alkyl; E is absent or is alkylene optionally substituted with 1 to 3 groups independently selected from halogen, hydroxyl and amino; Q is absent or selected from hydrogen, C1-C6 alkyl, halogen, cyano, alkoxy, carboxylic acid, amino, hydroxy, amide, C1-C6 haloalkyl, (C3-C6) cycloalkyl, heterocycle, heteroaryl and aryl, wherein said alkyl, alkoxy, cycloalkyl, heterocycle, heteroaryl and aryl are each optionally substituted with 1-5 groups independently selected from C1-C6 alkyl, amino, amide, halogen, hydroxy, cyano, C1-C6 haloalkyl and alkoxy; Q' is selected from hydrogen, C1-C6 alkyl and acyl; Q and Q' together with the carbon and nitrogen atoms to which they are attached optionally form a 4-membered heterocyclyl, a 5-membered heterocyclyl, a 6-membered heterocyclyl, a 9-membered bicyclic heterocyclyl, or a 10-membered bicyclic heterocyclyl, wherein said 4-membered heterocyclyl, 5-membered heterocyclyl, 6-membered heterocyclyl, 9-membered bicyclic heterocyclyl, and 10-membered bicyclic heterocyclyl are each optionally substituted with 1-5 groups independently selected from C1-C6 alkyl, amino, halogen, hydroxy, cyano, amide, C1-C6 haloalkyl, and alkoxy; p is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, 4, or 5; R" represents a covalent bond directly or indirectly to Z1c; Z" represents a covalent bond, directly or indirectly, to X1 or to Z1a; and Any primary amines are optionally acetylated or alkylated; The formulas FL69 and FL70 are: and stereoisomers thereof; wherein: R" represents a covalent bond directly or indirectly to Z1c; Z" represents a covalent bond directly or indirectly to X1 or to Z1a; A' is selected from H, C1-C6 alkyl, saturated fatty acid, unsaturated fatty acid, (C3-C6) cycloalkyl, haloalkyl, aryl and heteroaryl; and A" is selected from C1-C6 alkyl, substituted acyl, acid-terminated acyl, saturated or unsaturated fatty acid, (C3-C6) cycloalkyl, haloalkyl, aryl and heteroaryl; p is 1, 2, 3, 4, or 5, q is 1, 2, 3, 4, or 5, and Any primary amines are optionally acetylated or alkylated.

3. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein formula FL(IA) and FL(IB) are selected from formula FL3, FL5, FL5A, FL5B, FL20-FL68 and FL71-FL75: and their stereoisomers; in: p is 1, 2, 3, 4, or 5, q is 1, 2, 3, 4, or 5, and Any primary amines are optionally acetylated or alkylated.

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

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula IC: each p' is 1, 2, 3, or 4; and Each q' is 1, 2, 3, 4 or 5.

6. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 5, wherein each of B1 and B2 is independently selected from formula F2, F5 and F10, Wherein formula F2, F5 and F10 are: in: One R1 represents (C=O)---* or (CH2) m (C=O)---*, wherein---* represents the point of attachment to the rest of Z1c, and m is 1, 2, 3, 4, 5, 6 or 7; and Each remaining R1 is independently selected from H, F, Cl, Br, I, OH, CH2-NH2, NH2, (C=O)-NH2, CH=O, SO2CH3, SO2CF3, CF3, CHF2, NO2, CH3, OCH3, O(CH2) m” CH3, (SO2)NH-CH3, (SO2)NH(CH2) m” CH3 and OCF3, wherein m" is 1, 2, 3, 4, 5, 6 or 7; Y8 is O, N or NR, wherein R is C1-C6 alkyl or H; Y9 is H, CH3 or C1-C6 alkyl, provided that when Y8 is O, Y9 is CH3 or C1-C6 alkyl; and i is 1, 2, or 3.

7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein (a) each remaining R1 is independently selected from (i) H, CF3 and F; or (b) two remaining R1 are H and one remaining R1 is CF3 or F.

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein each Z1c is covalently conjugated to an indirect linker, and each combination of Z1c and an indirect linker is FFL-1 to FFL-101: and their stereoisomers; wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1 or to an amine of Z1a.

9. The compound of claim 6 or 7, or a pharmaceutically acceptable salt thereof, wherein B1 and B2 are each independently of formula F2, wherein each remaining R1 is independently selected from H, CF3, and F; wherein each Z1c is covalently conjugated to an indirect linker, and each combination of Z1c and an indirect linker is selected from formula FFL-1-4, 6-7, 9, 13-16, 20-23, 28-29, 31-33, 39, 42-45, 48, 53, 57-58, 60-62, 65, and 67: and their stereoisomers; wherein X represents a covalent attachment point, which is directly linked to the amine of X1 or to NH2 when X1 is NH2 or to OH when X1 is OH or to an amine covalently conjugated directly or indirectly to X1 or to an amine of Z1a.

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

11. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, 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 peptide, a glucose-dependent insulin-releasing polypeptide, a hybrid peptide comprising sequences from two or more human polypeptide hormones, or an analog of any one thereof.

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein: X1 comprises an insulin having an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 1, 25, 24051 and 24052, and the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24060, 24061, 24062, 24063, 24064 and 25000-25397.

13. The compound according to claim 12 or a pharmaceutically acceptable salt thereof, wherein: X1 comprises an insulin having an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of SEQ ID NOs 24051 and 24052, and wherein the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 25095, 25229, 25232, 25236, 25305, 25308, 25312, and 25380-25397; Each Z1c is independently selected from FF12A, FF12B, FF12C, FF12D, FF116A, FF116B, FF116C and FF116D, and is covalently conjugated directly or via an indirect linker to one or more lysine residues in X1; B1 and B2 are each independently of the formula F2; and The indirect linker is independently selected from FL3, FL5, FL5A, FL5B, FL20-FL75 and stereoisomers thereof.

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

15. A compound selected from an insulin comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of 24501 and 24502; and wherein the B-chain comprises a sequence selected from the group consisting of SEQ ID NOs 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, wherein the method comprises administering the compound of any one of claims 1 to 15 or the pharmaceutical composition of claim 16 to a subject in need thereof.

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

20. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein said B1 and B2 are each independently of the formula F2, and each remaining R1 is independently selected from H, F, Cl, I, Br and CF3.

21. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein said B1 and B2 are each independently of one another of formula F2, and each remaining R1 is H.

22. The compound or pharmaceutically acceptable salt thereof according to any one of claims 2 to 7, wherein: 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, wherein each B1 and B2 is F2; Each Z1c is conjugated directly or via an indirect linker to the amine side chain of a lysine in X1 or to the N-terminus in X1; 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.

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

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

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

26. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the B-chain comprises at least three lysine amino acids, each of which is independently covalently conjugated to Zlc via an indirect linker.

27. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the A-chain comprises one or more lysine amino acids, each of which is independently covalently conjugated to Zlc directly or via an indirect linker.

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

29. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

30. The compound of claim 4 or 5, wherein at least one Z1c is covalently conjugated to X1 via an indirect linker, and wherein each indirect linker is independently selected from Where p is 2 or 3; Type FL5, FL5A and FL5B, where p is 1 or 2, and and their stereoisomers; in: R" represents a covalent bond directly or indirectly to Z1c; Z" represents a covalent bond directly or indirectly to X1 or to Z1a; A' is selected from H, C1-C6 alkyl, saturated fatty acid, unsaturated fatty acid, (C3-C6) cycloalkyl, C1-C6 haloalkyl, aryl and heteroaryl; and A" is selected from C1-C6 alkyl, substituted acyl, acyl terminated with an acid group, saturated or unsaturated fatty acid, (C3-C6) cycloalkyl, C1-C6 haloalkyl, aryl and heteroaryl; p is 1, 2, 3, 4, or 5, and Any primary amines are optionally acetylated or alkylated.

31. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein each indirect linker is selected from:

32. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein X1 is insulin further comprising 1 to 5 residues substituted with, inserted into, appended to, or mutated to an amino acid having a free amine directly or indirectly conjugated to Z1c.

33. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein at least one Z1c is conjugated to a free amine side chain of an amino acid that has been substituted, inserted or mutated on insulin.

34. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein the compound is covalently conjugated directly or via a linker to a molecule that can bind to at least one protein present in human plasma.

35. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition comprises one or more polyols.

36. The pharmaceutical composition of claim 35, wherein the polyol is selected from the group consisting of 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 medicament for the treatment of diabetes or obesity, for controlling blood sugar levels or for controlled drug release.

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

39. A process for the preparation of a compound according to any one of claims 1 to 15, wherein the process comprises at least one alkylation and / or amidation step.

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

41. The compound of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein the compound is used as an intermediate in the preparation of a bulk drug or a therapeutic or prophylactic compound.

42. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein in each of formulas F2, F5 and F10, one R1 is (C=O)---*.

43. A compound represented by formula II or a stereoisomer or a mixture of stereoisomers or a pharmaceutically acceptable salt thereof: Z1c-Connector (Formula II) Wherein the Z1c-linker is selected from: wherein X is selected from a leaving group, NH2 and H; and B1 and B2 may be the same or different and each independently represents an aromatic boron-containing group.

44. The compound of claim 43, comprising at least one B1 or B2 independently selected from the group consisting of Formulas F2, F5 and F10, Wherein formula F2, F5 and F10 are: in: One R1 represents (C=O)---* or (CH2) m (C=O)---*, wherein---* represents the point of attachment to the rest of Z1c, and m is 1, 2, 3, 4, 5, 6 or 7; and Each remaining R1 is independently selected from H, F, Cl, Br, I, OH, CH2-NH2, NH2, (C=O)-NH2, CH=O, SO2CH3, SO2CF3, CF3, CHF2, NO2, CH3, OCH3, O(CH2) m” CH3, (SO2)NH-CH3, (SO2)NH(CH2) m” CH3 and OCF3, wherein m" is 1, 2, 3, 4, 5, 6 or 7; Y8 is O, N and NR, wherein R is C1-C6 alkyl or H; Y9 is H, CH3 or C1-C6 alkyl, provided that when Y8 is O, Y9 is CH3 or C1-C6 alkyl; and i is 1, 2, or 3.

45. The compound of claim 44, wherein the Z1c-linker is selected from: or a stereoisomer or a mixture of stereoisomers, a pharmaceutically acceptable salt thereof, and a combination thereof; Wherein X is a leaving group.

46. ​​The compound of claim 45 or a pharmaceutically acceptable salt thereof, wherein X is selected from N-oxysuccinimide (NHS), 2,3,5,6-tetrafluorophenoxy (TFP), pentafluorophenoxy (Pfp), OH, halogen, maleimidoalkylamino, maleimidoamidopolyethylene glycolamino, and maleimidopolyethylene glycolamino.

47. The compound of claim 46, wherein the compound is selected from: Its stereoisomers or mixtures of stereoisomers, pharmaceutically acceptable salts and combinations thereof.

48. An insulin analogue comprising an A-chain and a B-chain, wherein the A-chain comprises a sequence selected from the group consisting of 24051 and 24052; and wherein the B-chain comprises a sequence selected from the group consisting of 25000-25397.

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

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

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

52. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein in at least one Z1c, B1 and B2 are each independently of the formula F2, wherein 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 an amino acid with a hydrophobic side chain.

53. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein in at least one Z1c, B1 and B2 are each independently of the formula F2, wherein one of the remaining R1 is CF3 and the remaining R1 is H; and the indirect linker is selected from FL5, FL23-24, FL26, FL29, FL34, FL36, FL38-40, FL42 and an amino acid with a hydrophilic side chain.

54. The compound of 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 at the insulin receptor, comprising at least one aromatic boron-containing group having agonist potency at the insulin receptor, the compound 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 20 mM, the compound has an insulin receptor agonist potency ratio of the first EC50 to the second EC50 of about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, about 2 to about 10, about 2 to about 9, or about 2 to about 8, and wherein the at least one aromatic boron-containing group is attached to the FF scaffold, and wherein the FF scaffold is selected from the group consisting of formula 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, wherein when administered at a dose of 30 nmol / kg to a first group of rats with a first glucose infusion rate to provide a blood glucose concentration of 100 mg / dL and to a second group of rats with a second glucose infusion rate to provide a blood glucose concentration of 200 mg / dL, the compound 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, and wherein the at least one aromatic boron-containing group is attached to the FF scaffold, and wherein the FF scaffold is selected from the group consisting of formula 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 of claim 55 or 56, wherein the FF scaffold is selected from formula FF116, FF116A, FF116B, FF116C, FF116D, FF225, FF225A, and FF225B.

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

59. A process for the preparation of a compound according to any one of claims 55 to 57, wherein the process comprises at least one alkylation and / or amidation step.

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

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