Glucose-Sensitive Insulin and Its Uses

Abstract

The present invention provides glucose-sensitive insulin derivatives comprising a macrocycle, a glucose mimetic, and human insulin or an analogue thereof, and their pharmaceutical uses. In addition, the present invention relates to pharmaceutical compositions comprising such glucose-sensitive insulin derivatives, and to the use of such compounds in the treatment or prevention of medical conditions associated with diabetes.

Description

Background Art

[0001] Glucose is the most important component in human energy homeostasis, and uncontrolled blood glucose is a hallmark of diabetes. The main aim of diabetes treatment is to regulate blood glucose levels towards normal values, and insulin is the most effective drug for this purpose. However, it is difficult to achieve a balance between hyperglycemia and hypoglycemia when using insulin to regulate blood glucose. Even with modern blood glucose monitors, hypoglycemia often occurs, and various glucose-sensitive insulin delivery systems have been designed in an attempt to improve this situation, in the form of mechanical systems (pumps / sensors) or molecular delivery systems. Therefore, it is advantageous to endow diabetes-related peptide and protein drugs with glucose-regulated biological activity. For example, the hypoglycemic activity of insulin is weaker at low blood glucose values.

[0002] Glucose-sensitive insulin biological activity can be achieved as follows: Equip insulin with a glucose-binding element plus a binding partner that competes with blood glucose for binding to the glucose-binding element, thereby controlling the balance between the active and inactive states of insulin folding (WO2016149222, WO2010107520). When the glucose-binding element on the insulin derivative binds to the binding partner on the same insulin, the insulin acquires an inactive or weakly active conformation. As the glucose level increases, the binding partner on the insulin derivative moves away from the glucose conjugate, and the conformation of insulin changes to the active state.

[0003] Such systems must contain some form of glucose conjugate. This has led to attempts to de novo design small molecule glucose conjugates. However, due to the very subtle structural differences from other carbohydrates, it is a very difficult task to bind glucose in water with reasonable affinity and selectivity. Summary of the Invention

[0004] The present invention provides glucose-sensitive insulin derivatives. The insulin derivatives of the present invention comprise a macrocycle M, a glucose mimetic, and human insulin or an analogue thereof. The macrocycle M has the formula M1:

[0005]

[0006] wherein R1 and R2 are independently selected from –OH,

[0007]

[0008] wherein R4, R5, R6, R7, R8, and R9 are independently selected from hydrogen, halogen, (1-4C) alkyl, and (1-4C) alkoxy; and

[0009] wherein R3 represents the point of attachment of the macrocycle of formula M1.

[0010] The macrocycle contained in the insulin derivatives of the present invention exhibits an unusually high affinity for glucose in an aqueous medium. In addition, the macrocycle contained in the insulin derivatives of the present invention shows an unprecedented level of selectivity for glucose compared to other structurally similar sugars (such as mannose or fructose).

[0011] Without being bound by theory, it is believed that when the macrocycle M on the insulin derivative binds to the glucose mimic on the same insulin, the insulin acquires an inactive or weakly active conformation. As the glucose level increases, the glucose mimic on the insulin derivative moves away from the macrocycle M, and the conformation of the insulin changes to an active state. Figure 1 This principle is illustrated in schematic form ( Figure 1 the substitution positions on the insulin in

[0012] In one aspect, the present invention relates to providing insulin derivatives that activate insulin in a glucose-dependent manner after administration.

[0013] In one aspect, the present invention relates to providing insulin derivatives that have low or no activity / availability at low blood glucose levels, such as below about 3 mM glucose levels.

[0014] In one aspect, the present invention relates to providing insulin derivatives that have high activity / availability in response to high blood glucose levels, such as above about 10 mM glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram of a glucose-sensitive insulin derivative (Gm = glucose mimic, G = glucose). Glucose-sensitive insulin bioactivity can be achieved as follows: Equip insulin with a glucose-binding element plus a binding partner (glucose mimic, such as glucoside), which competes with blood glucose for binding to the glucose-binding element, thereby controlling the balance of insulin folding between the active state and the inactive state.

[0016] Figure 2 Shows the fluorescence titration (λex = 310 nm) of compound 6d (5 μM) titrated with D-glucose (10 mM) in 10 mM phosphate buffer.

[0017] Figure 3 : Open circles: Intensity measured at 380 nm relative to the D-glucose concentration. Dashed line: Isotherm fitting the experimental data (1:1 binding model). Calculated Ka = 14751.48 ± 637.3 M-1. DETAILED DESCRIPTION

[0018] The present invention relates to glucose-sensitive compounds that activate the insulin receptor in a glucose-dependent manner.

[0019] In one aspect of the present invention, the present invention relates to a glucose-sensitive insulin derivative comprising human insulin or a human insulin analogue, a glucose mimetic and a glucose conjugate.

[0020] In one aspect of the present invention, the glucose conjugate is macrocycle M. In one aspect of the present invention, the macrocycle M has formula M1:

[0021]

[0022] wherein R1 and R2 are independently selected from –OH,

[0023]

[0024] wherein R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen, halogen, (1-4C)alkyl and (1-4C)alkoxy; and

[0025] wherein R3 represents the point of attachment of the macrocycle of formula M1.

[0026] In another aspect, the present invention relates to a glucose-sensitive insulin derivative comprising human insulin or a human insulin analogue, two glucose mimetics and two glucose conjugates.

[0027] General Definitions

[0028] As used herein, the term "compound" refers to a molecular entity and thus a "compound" may have different structural elements in addition to the minimal elements defined for each compound or group of compounds. Thus, a compound may be a polypeptide or a derivative thereof, provided that the compound contains the defined structural and / or functional elements. The term "compound" is also intended to cover its pharmaceutically relevant forms, i.e., the present invention relates to the compounds defined herein or their pharmaceutically acceptable salts, amides or esters.

[0029] For example, the terms "peptide" or "polypeptide" as used in the context of the present invention refer to a compound comprising a series of amino acids linked to one another by amide (or peptide) bonds. In a particular embodiment, a peptide consists of amino acids linked by peptide bonds.

[0030] The term "analogue" generally refers to a peptide having one or more amino acid alterations in its sequence as compared to a reference amino acid sequence. An analogue "comprising" certain specified alterations may contain further alterations as compared to its reference sequence. In certain embodiments, an analogue "has" or "comprises" the specified alterations. In other certain embodiments, an analogue "consists of" the alterations. When the terms "consisting of" or "consists of" are used for an analogue, e.g., when an analogue consists of a specified set of amino acid mutations, it should be understood that the specified amino acid mutations are the only amino acid mutations in the analogue. In contrast, an analogue "comprising" a specified set of amino acid mutations may have additional mutations. "Analogue" may also include amino acid extensions and / or truncations at the N-terminal and / or C-terminal positions.

[0031] Generally, an amino acid residue may be represented by its full name, its single-letter code, and / or its three-letter code. These three ways are completely equivalent.

[0032] The term "derivative" generally refers to a compound that can be prepared from a native peptide or its analogue by chemical modification, particularly by covalent attachment of one or more substituents.

[0033] The term "amino acid" includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) and non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those that are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids either do not exist in proteins or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modification).

[0034] Hereinafter, each amino acid of the peptides of the present invention for which the optical isomer is not specified should be understood to mean the L-isomer (unless otherwise indicated). An amino acid is a molecule containing an amino group and a carboxylic acid group and optionally one or more additional groups commonly referred to as side chains.

[0035] Hereinafter, each carbohydrate for which the optical isomer is not specified should be understood to mean the D-isomer (unless otherwise indicated).

[0036] As used herein, the term "(1-4C)alkyl" encompasses straight-chain or branched-chain alkyl groups containing 1-4 carbon atoms.

[0037] As used herein, the term "(1-4C)alkoxy" encompasses straight-chain or branched-chain alkoxy groups containing 1-4 carbon atoms.

[0038] The solid and dashed lines used in the chemical formulae above are to be understood as being for illustrative purposes only (i.e., to show the relative orientation of the compounds of the invention). They do not relate to the absolute configuration (i.e., stereochemistry) of the compounds shown.

[0039] Compounds having the same molecular formula but different in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are called "isomers". Isomers that differ in the arrangement of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereoisomers", while stereoisomers that are non - superimposable mirror images of each other are called "enantiomers". When a compound has an asymmetric center, e.g., it is bonded to four different groups, a pair of enantiomers may exist. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and are described by the R and S ranking rules of Cahn and Prelog, or by the way the molecule rotates the plane of polarized light and are called dextrorotatory or levorotatory (i.e., (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0040] The compounds of the invention may have one or more asymmetric centers; thus, such compounds can exist as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless otherwise stated, the description or naming of a particular compound in the specification and claims is intended to include the individual enantiomers and their racemic or other forms of mixtures. Methods for determining stereochemistry and separating stereoisomers are well known in the art (see the discussion in Chapter 4 of "Advanced Organic Chemistry", 4th Edition, J. March, John Wiley and Sons, New York, 2001), e.g., by synthesis from optically active starting materials or by resolution of the racemic form. Certain compounds of the invention may have geometric isomeric centers (E - and Z - isomers). It is to be understood that the invention includes all optical, diastereoisomeric and geometric isomers of the compounds capable of recognizing sugars and their mixtures.

[0041] It is also to be understood that certain macrocyclic compounds of the invention can exist in solvated as well as non - solvated forms, e.g., in hydrated forms. It is to be understood that the invention includes all such solvated forms of the compounds capable of recognizing sugars. It is also to be understood that certain compounds may exhibit polymorphism, and the invention encompasses all such forms of the compounds capable of recognizing sugars.

[0042] The macrocyclic compounds of the present invention can exist in a variety of different tautomeric forms, and the mention of a compound of a specific general formula includes all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms and only one form is specifically described or shown, the given general formula still encompasses all other forms. Examples of tautomeric forms include keto, enol, and enolate forms, for example, in the following tautomeric pairs: keto / enol, imine / enamine, amide / imino alcohol, amidine / amidine, nitroso / oxime, thione / enethiol, and nitro / aci-nitro.

[0043] Compounds containing an amine functional group can also form N-oxides. The mention herein of a compound of a given general formula containing an amine functional group also includes the N-oxides. When a compound contains several amine functional groups, one or more than one nitrogen atom can be oxidized to form the N-oxide. Specific examples of N-oxides are N-oxides of the nitrogen atoms of tertiary amines or nitrogen-containing heterocycles. The N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid), see, for example, Jerry March, Advanced Organic Chemistry, 4th Edition, Wiley Interscience. More particularly, the N-oxides can be prepared by the procedure of L.W. Deady (Syn. Comm. 1977, 7, 509-514), in which the amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as dichloromethane.

[0044] Insulin

[0045] As used herein, the term "human insulin" means the human insulin hormone, the structure and properties of which are well known. Human insulin has two polypeptide chains, designated as the A chain and the B chain. The A chain is a peptide of 21 amino acids, while the B chain is a peptide of 30 amino acids, and the two chains are linked by the following disulfide bridges: a first bridge between cysteine at position 7 of the A chain and cysteine at position 7 of the B chain, and a second bridge between cysteine at position 20 of the A chain and cysteine at position 19 of the B chain. A third bridge exists between cysteines at positions 6 and 11 of the A chain.

[0046] The human insulin A chain has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1), while the B chain has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).

[0047] In the human body, the hormone is synthesized as a single-chain precursor, proinsulin (preproinsulin), which consists of a 24-amino acid prepeptide followed by proinsulin containing 86 amino acids, and has the configuration: prepeptide - B - Arg Arg - C - Lys Arg - A, where C is a 31-amino acid connecting peptide. Arg - Arg and Lys - Arg are the cleavage sites at which the connecting peptide is cleaved from the A and B chains.

[0048] "Insulin" according to the present invention is to be understood herein as human insulin or insulin from another species, such as porcine or bovine insulin.

[0049] As used herein, the terms "insulin peptide", "insulin compound" or "insulin" mean a peptide that is human insulin or an analogue or derivative thereof having insulin activity.

[0050] Insulin Analogue

[0051] As used herein, the term "insulin analogue" means a modified human insulin in which one or more amino acid residues of the insulin have been replaced by other amino acid residues, and / or in which one or more amino acid residues have been deleted from the insulin, and / or in which one or more amino acid residues have been added and / or inserted into the insulin.

[0052] As used herein, the term "modification" means replacement, deletion and addition (including insertion). As used herein, the term "mutation" means a replacement or deletion of an amino acid within the human insulin sequence. The term mutation does not include addition, extension or elongation to the human insulin sequence. Mutations in the insulin molecule are represented by stating the chain (A or B), its position, and the single-letter or three-letter code of the amino acid residue replacing the native amino acid residue.

[0053] In one embodiment, the insulin analogue contains fewer than 10 amino acid mutations relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation relative to human insulin. Any mutation of an insulin analogue as used herein refers to a mutation of the individual insulin peptide and does not include any linking groups attached to the insulin peptide / analogue.

[0054] "desB30" or "B(1 - 29)" means the native insulin B chain lacking the B30 amino acid or an analogue thereof, while "A(1 - 21)" means the native insulin A chain. Thus, for example, desB30 human insulin is an analogue of human insulin in which the amino acid at position 30 in the B chain is deleted.

[0055] In this text, terms such as "A1", "A2", and "A3" respectively represent the amino acids at positions 1, 2, and 3, etc. in the A-chain of insulin (counting from the N-terminus). Similarly, terms such as "B1", "B2", and "B3" respectively represent the amino acids at positions 1, 2, and 3, etc. in the B-chain of insulin (counting from the N-terminus). Using the single-letter codes of amino acids, terms such as A21A, A21G, and A21Q represent that the amino acids at position A21 are A, G, and Q respectively. Using the three-letter codes of amino acids, the corresponding representations are A21Ala, A21Gly, and A21Gln respectively.

[0056] In this text, the term "amino acid residue" refers to an amino acid from which the hydroxyl group has been formally removed from the carboxyl group, and / or an amino acid from which a hydrogen atom has been formally removed from the amino group.

[0057] In an example of an insulin derivative, a human insulin analogue containing desB30 was used. However, other tolerated mutations, combinations, numbers of mutations, sequence extensions / truncations, etc. of insulin analogues can also be used to illustrate the present invention. Examples of such insulin analogues are B28D human insulin, B28K B29P human insulin, and B3K B29E human insulin.

[0058] In one aspect, the insulin analogue of the present invention includes: desB30 human insulin (the A-chain of SEQ ID NO:1 and the B-chain of SEQ ID NO:3).

[0059] In one aspect, the insulin analogue of the present invention is desB30 human insulin (the A-chain of SEQ ID NO:1 and the B-chain of SEQ ID NO:3).

[0060] Insulin Derivative

[0061] As used herein, the term "insulin derivative" or "derivative of insulin analogue" refers to human insulin or its analogue (an "insulin analogue") to which a glucose mimetic and a macrocycle are attached. In other words, the insulin derivative of the present invention comprises human insulin or its analogue, a macrocycle, and a glucose mimetic.

[0062] In one aspect, the insulin derivative of the present invention comprises human insulin or its analogue and at least one macrocycle and at least one glucose mimetic.

[0063] In another aspect, the insulin derivative of the present invention comprises at least two macrocycles and at least two glucose mimetics.

[0064] In another aspect, the insulin derivative of the present invention comprises two macrocycles and two glucose mimetics.

[0065] In one aspect, the macrocycle and the glucose mimetic can each be linked to human insulin or a human insulin analogue via a linker. In one aspect, the point of attachment to human insulin or a human insulin analogue is selected from

[0066] a) the α-amino group of the amino acid residue at position 1 of the A-chain of human insulin or a human insulin analogue;

[0067] b) the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue; and

[0068] c) the ε-amino group or α-carboxylic acid group of the lysine at position 29 of the B-chain of the human insulin or human insulin analogue.

[0069] In one aspect, the macrocycle and the glucose mimetic are not linked to the same point of attachment on human insulin or a human insulin analogue.

[0070] Macrocycle

[0071] The macrocycle contained in the insulin derivative of the present invention shows a high affinity for glucose. The macrocycle contained in the insulin derivative of the present invention shows selectivity for glucose compared to other structurally similar sugars (such as mannose).

[0072] The macrocycle contained in the insulin derivative of the present invention is the macrocycle M of formula M1:

[0073]

[0074] wherein R1 and R2 are independently selected from –OH,

[0075]

[0076] wherein R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen, halogen, (1-4C)alkyl and (1-4C)alkoxy; and

[0077] wherein R3 represents the point of attachment of the macrocycle of formula M1.

[0078] In one aspect, R1 and R2 are the same. In one aspect, R1 and R2 are -OH. In another aspect, R1 and R2 are

[0079]

[0080] In a further aspect, R1 and R2 are

[0081]

[0082] In one aspect, R4, R5, R6, R7, R8 and R9 are the same. In a further aspect, R4, R5, R6, R7, R8 and R9 are ethyl.

[0083] Glucose Mimetic

[0084] As used herein, the term "glucose mimetic" refers to a compound that is structurally similar to glucose and binds to macrocycle M. In other words, the glucose mimetic has an affinity for macrocycle M.

[0085] In one aspect, the glucose mimetic contained in the insulin derivative of the present invention is a glucoside. In a further aspect, the glucose mimetic contained in the insulin derivative of the present invention is a 1-substituted β-D-glucopyranoside.

[0086] Linker

[0087] The insulin derivative of the present invention may further comprise a linker between macrocycle M and the human insulin analogue and / or a linker between the human insulin analogue and the glucose mimetic.

[0088] In the context of the present invention, a linker is a chemical moiety or residue used to covalently attach the macrocycle and the glucose mimetic to human insulin or a human insulin analogue, respectively. Thus, the term "-linker-" is intended to denote a chemical unit of the insulin derivative that is covalently attached to an amino acid residue of human insulin or a human insulin analogue and to the macrocycle or the glucose mimetic, respectively.

[0089] Depending on the point of attachment, the reactivity of the linker terminus may vary. Depending on the product in question, the linker may have various forms.

[0090] The linker used to covalently attach the macrocycle to human insulin or a human insulin analogue may be different from the linker used to covalently attach the glucose mimetic to human insulin or a human insulin analogue.

[0091] In one embodiment, the linker comprises -(CH2CH2O) p -, where p is an integer from 1 to 5.

[0092] In one embodiment, the linker is -NH(CH2CH2O)3-CH2CH2-triazole-PhCH2. In one embodiment, the linker is -(CH2CH2O)2-CH2CH2C(O)-.

[0093] Alternative linkers of different lengths and compositions may be used to create linkages between human insulin or a human insulin analogue and the macrocycle and the glucose mimetic, respectively.

[0094] In one aspect, the macrocycle M is linked to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue via a linker of formula L1:

[0095]

[0096] wherein X is CH2- or (CH2CH2O-) p , where p is an integer from 2 to 4;

[0097] wherein Y is CH2-, (CH2CH2CO-) or -Ph-p-CH2-;

[0098] where * represents the point of attachment to the macrocycle M; and

[0099] where # represents the point of attachment to human insulin or a human insulin analogue, and

[0100] wherein the glucose mimetic is linked to the ε-amino group of lysine (K) at position 29 of the B-chain of human insulin or a human insulin analogue via a linker, wherein the linker is a linker of formula L2:

[0101] “-(CH2CH2O-) q -(CH2) r -C(O)-# Formula L2;

[0102] where q is 1 or 2;

[0103] where r is 1 or 2;

[0104] where “ represents the point of attachment to the glucose mimetic; and

[0105] where # represents the point of attachment to human insulin or a human insulin analogue.

[0106] In a second aspect, the insulin derivative comprises two glucose mimetics and two macrocycles M of formula M1, wherein the two macrocycles are linked to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue, and wherein the two glucose mimetics are linked to the α-carboxylic acid group and / or the ε-amino group of lysine (K) at position 29 of the B-chain of the human insulin or a human insulin analogue. The two glucose mimetics may be linked to the α-carboxylic acid group or the ε-amino group of lysine (K) at position 29 of the B-chain of human insulin or a human insulin analogue via a trivalent linker, or each of the two glucose mimetics may be linked to the α-carboxylic acid group and the ε-amino group of lysine (K) at position 29 of the B-chain of human insulin or a human insulin analogue via a divalent linker, respectively.

[0107] In one aspect, the trivalent linker connecting the α-amino group at position 1 of the two macrocycles M to the B chain of human insulin or a human insulin analogue is a linker of formula L3:

[0108]

[0109] where Z is CH2- or (CH2CH2O-)3;

[0110] where W is CH2-, (CH2CH2CO-), or -Ph-p-CH2-;

[0111] where * represents the point of attachment to the macrocycle M; and

[0112] where # represents the point of attachment to human insulin or a human insulin analogue.

[0113] In one aspect, the trivalent linker connecting the two glucose mimics to the α-carboxylic acid group of lysine (K) at position 29 of the B chain of human insulin or a human insulin analogue is a linker of formula L4:

[0114]

[0115] where “ represents the point of attachment to the glucose mimic; and

[0116] where # 1 represents the point of attachment to the α-carboxylic acid group of lysine (K) at position 29 of the B chain of human insulin or a human insulin analogue.

[0117] In one aspect, the trivalent linker connecting the two glucose mimics to the ε-amino group of lysine (K) at position 29 of the B chain of human insulin or a human insulin analogue is a linker of formula L5:

[0118]

[0119] where “ represents the point of attachment to the glucose mimic; and

[0120] where # 2 represents the point of attachment to the ε-amino group of lysine (K) at position 29 of the B chain of human insulin or a human insulin analogue.

[0121] In one aspect, the two divalent linkers connecting the two glucose mimics to the α-carboxylic acid group and the ε-amino group of lysine (K) at position 29 of the B chain of human insulin or a human insulin analogue are linkers of formula L6 and L7, respectively:

[0122]

[0123] and

[0124]

[0125] represents a point of attachment to said glucose mimetic; and

[0126] wherein # 1 represents a point of attachment to the α-carboxyl group of lysine (K) at position 29 of the B-chain of said human insulin or human insulin analogue; and

[0127] wherein # 2 represents a point of attachment to the ε-amino group of lysine (K) at position 29 of the B-chain of said human insulin or human insulin analogue.

[0128] Production of Human Insulin and Insulin Analogue

[0129] The production of polypeptides such as insulin is well known in the art. Insulin or insulin analogues can be produced, for example, by classical peptide synthesis, e.g., solid-phase peptide synthesis using t-Boc or Fmoc chemistry or other well-established techniques, see, e.g., Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999. Insulin or insulin analogues can also be produced by a method comprising culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the insulin analogue in a suitable nutrient medium under conditions permitting the expression of the insulin analogue. There are several recombinant methods available for the production of human insulin and human insulin analogues. Examples of methods that can be used to produce insulin in microorganisms such as Escherichia coli and Saccharomyces cerevisiae are disclosed, for example, in WO2008034881.

[0130] Generally, insulin or insulin analogues are produced by expressing a DNA sequence encoding the insulin analogue or its precursor in a suitable host cell by well-known techniques such as those disclosed in, for example, EP1246845 or WO2008034881.

[0131] Insulin or an insulin analogue is recovered from the cell culture medium and can be purified by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), solubility differences (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, edited by J.-C. Janson and Lars Ryden, VCH Publishers, New York, 1989). Preferably, insulin or an insulin analogue is purified by affinity chromatography on an anti-insulin analogue antibody column. Additional purification can be achieved by conventional chemical purification means, such as high performance liquid chromatography. Other purification methods, including barium citrate precipitation, are known in the art and can be applied to the purification of the insulin or insulin analogue described herein (see, e.g., Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982).

[0132] Pharmaceutical Composition

[0133] The present invention also relates to pharmaceutical compositions comprising a compound of the present invention or a pharmaceutically acceptable salt, amide, or ester thereof, and one or more pharmaceutically acceptable excipients. Such compositions can be prepared as is known in the art.

[0134] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. Excipients can be inert substances, inactive substances, and / or non-pharmaceutically active substances. Excipients can be used for various purposes, such as as carriers, vehicles, diluents, tablet adjuvants, and / or to improve the administration and / or absorption of the active substance. Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity regulators, chelating agents, and stabilizers. The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, e.g., Remington: The Science and Practice of Pharmacy (e.g., the 21st edition (2005) and any subsequent editions).

[0135] The compositions of the present invention can be in the form of liquid preparations, e.g., aqueous preparations containing water. Liquid preparations can be solutions or suspensions. Alternatively, it can be a solid preparation, e.g., a lyophilized or spray-dried composition.

[0136] The pharmaceutical compositions of the present invention can further comprise a second active ingredient, such as a therapeutic agent, which can simplify administration in the case of combination therapy.

[0137] Drug Indication

[0138] Diabetes

[0139] The term “diabetes” includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the amount of insulin produced by the pancreas is insufficient, or in which the body cells do not respond properly to insulin, preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.

[0140] Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by the destruction of B cells and usually results in absolute insulin deficiency.

[0141] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance and thus relative insulin deficiency, and / or with major insulin secretory defects accompanied by insulin resistance.

[0142] Other indications

[0143] In one embodiment, the compounds of the present invention are used for the preparation of a medicament for the treatment or prevention of hyperglycemia, which includes stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes.

[0144] In another embodiment, the compounds of the present invention are used as a medicament for delaying or preventing the disease progression of type 2 diabetes.

[0145] In one embodiment of the present invention, the compound is used as a medicament for the treatment or prevention of hyperglycemia, which includes stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes.

[0146] In a further embodiment, the present invention relates to a method for the treatment or prevention of hyperglycemia, which includes stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes, the method comprising administering to a patient in need of such treatment an effective amount of such treatment employing the compounds of the present invention.

[0147] Administration Route

[0148] The term “treatment” is intended to include prevention and minimization of the mentioned disease, disorder or condition (i.e., “treatment” means prophylactic and therapeutic administration of the compounds of the present invention or a composition comprising the compounds of the present invention, unless otherwise stated or clearly contradicted by the context).

[0149] The route of administration can be any route that effectively delivers the compounds of the present invention to the desired or appropriate location in the body, such as parenterally, for example, by subcutaneous, intramuscular or intravenous routes. Alternatively, the compounds of the present invention can be administered orally, via the lungs, rectally, transdermally, buccally, sublingually or nasally.

[0150] For parenteral administration, the compounds of the present invention are formulated similarly to the formulations of known insulins. In addition, for parenteral administration, the compounds of the present invention are administered similarly to the administration of known insulins, and the procedure is familiar to physicians.

[0151] The amount of the compounds of the present invention to be administered, the frequency of administration of the compounds of the present invention, and the selection of which one or more of the compounds of the present invention are optionally administered together with another antidiabetic compound are determined in consultation with a physician familiar with the treatment of diabetes.

[0152] Embodiment

[0153] The present invention is further described by the following non-limiting embodiments.

[0154] 1. An insulin derivative comprising human insulin or a human insulin analogue, a glucose mimetic, and a macrocycle M of formula M1:

[0155]

[0156] wherein R1 and R2 are independently selected from –OH,

[0157]

[0158] wherein R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen, halogen, (1-4C)alkyl and (1-4C)alkoxy; and

[0159] wherein R3 represents the point of attachment of the macrocycle of formula M1.

[0160] 2. The insulin derivative according to embodiment 1, wherein the insulin derivative comprises two glucose mimetics and two macrocycles M of formula M1.

[0161] 3. The insulin derivative according to any one of embodiments 1-2, wherein R1 and R2 are

[0162]

[0163] 4. The insulin derivative according to any one of embodiments 1-3, wherein R4, R5, R6, R7, R8 and R9 are each ethyl.

[0164] 5. An insulin derivative according to any one of embodiments 1-4, wherein the macrocycle and the glucose mimetic are each independently connected to the human insulin or human insulin analogue via an optional linker, and the point of attachment to the human insulin or human insulin analogue is selected from:

[0165] a) the α-amino group of the amino acid residue at position 1 of the A chain of human insulin or human insulin analogue;

[0166] b) the α-amino group at position 1 of the B chain of human insulin or human insulin analogue; and

[0167] c) the ε-amino group of lysine (K) at position 29 of the B chain of the human insulin or human insulin analogue;

[0168] provided that the macrocycle and the glucose mimetic are not attached to the same point of attachment on the human insulin or human insulin analogue.

[0169] 6. An insulin derivative according to any one of embodiments 1-5, wherein the macrocycle M is connected to the human insulin or human insulin analogue via a linker, and the linker is

[0170] *-NH(CH2CH2O) m -CH2CH2-triazole-PhCH2-#,

[0171] wherein m is an integer from 1 to 5;

[0172] * is the point of attachment to the macrocycle M; and

[0173] # is the point of attachment to the human insulin or human insulin analogue.

[0174] 7. An insulin derivative according to any one of embodiments 1-6, wherein the glucose mimetic is connected to the human insulin or human insulin analogue via a linker, and the linker is

[0175] “-(CH2CH2O) n -CH2CH2C(O)-#,

[0176] wherein n is an integer from 0 to 5;

[0177] “ is the point of attachment to the glucose mimetic; and

[0178] # is the point of attachment to the human insulin or human insulin analogue.

[0179] 8. An insulin derivative according to any one of embodiments 1-7, wherein the human insulin or human insulin analogue is the human insulin analogue desB30 human insulin.

[0180] 9. An insulin derivative according to any one of embodiments 1 - 8, wherein the glucose mimetic is β - D - glucopyranoside.

[0181] 10. An insulin derivative according to any one of embodiments 1 - 9, wherein the insulin derivative is selected from

[0182] B1 - macrocycle B29 - glycoside desB30 human insulin 8 of Example 1;

[0183] B1 - macrocycle A1 - glycoside desB30 human insulin 12 of Example 2;

[0184] B29 - macrocycle A1 - glycoside desB30 human insulin 13 of Example 3; and

[0185] B1 - bis - macrocycle B29 - bis - glycoside desB30 human insulin 14 of Example 4.

[0186] 11. A pharmaceutical composition for treating diabetes in a patient in need thereof, comprising a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 - 10, and a pharmaceutically acceptable excipient.

[0187] 12. The pharmaceutical composition of embodiment 11, which is used as a medicine.

[0188] 13. The pharmaceutical composition of embodiment 11, which is used for treating diabetic patients.

[0189] The present invention is further described by the following non - limiting embodiments.

[0190] 14. An insulin derivative comprising human insulin or a human insulin analogue, a glucose mimetic, and a macrocycle M of formula M1:

[0191]

[0192] wherein R1 and R2 are independently selected from –OH,

[0193]

[0194] wherein R4, R5, R6, R7, R8, and R9 are independently selected from hydrogen, halogen, (1 - 4C) alkyl, and (1 - 4C) alkoxy; and

[0195] wherein R3 represents the attachment point of the macrocycle of formula M1.

[0196] 15. The insulin derivative according to embodiment 14, wherein the glucose mimetic is a glucoside.

[0197] 16. The insulin derivative according to embodiment 15, wherein the glucose mimetic is β-D-glucopyranoside.

[0198] 17. The insulin derivative according to any one of embodiments 14 to 16, wherein R1 and R2 are -OH.

[0199] 18. The insulin derivative according to any one of embodiments 14 to 16, wherein R1 and R2 are

[0200]

[0201] 19. The insulin derivative according to any one of embodiments 14 to 16, wherein R1 and R2 are

[0202]

[0203] 20. The insulin derivative according to any one of embodiments 14 to 19, wherein R4, R5, R6, R7, R8 and R9 are each ethyl.

[0204] 21. The insulin derivative according to any one of embodiments 14 to 20, wherein the macrocycle and the glucose mimetic are each independently connected to the human insulin or human insulin analogue via an optional linker, and the points of attachment to the human insulin or human insulin analogue are selected from:

[0205] a) the α-amino group of the amino acid residue at position 1 of the A chain of human insulin or human insulin analogue;

[0206] b) the α-amino group at position 1 of the B chain of human insulin or human insulin analogue; and

[0207] c) the ε-amino group or α-carboxylic acid group of lysine (K) at position 29 of the B chain of the human insulin or human insulin analogue;

[0208] provided that the macrocycle and the glucose mimetic are not attached to the same point of attachment on the human insulin or human insulin analogue.

[0209] 22. The insulin derivative according to embodiment 21, wherein the macrocycle is attached to the α-amino group at position 1 of the B chain of the human insulin or human insulin analogue; and wherein the glucose mimetic is attached to the ε-amino group of lysine (K) at position 29 of the B chain of the human insulin or human insulin analogue.

[0210] 23. The insulin derivative according to embodiment 22, wherein the macrocycle M is connected to the human insulin or human insulin analogue via a linker, and the linker is a linker of formula L1

[0211]

[0212] wherein X is CH2- or (CH2CH2O-) p , where p is an integer from 2 to 4;

[0213] wherein Y is CH2-, (CH2CH2CO-), or -Ph-p-CH2-;

[0214] where * represents the point of attachment to the macrocycle M; and

[0215] where # represents the point of attachment to human insulin or a human insulin analogue.

[0216] 24. The insulin derivative according to embodiment 23, wherein the glucose mimetic is linked to the human insulin or human insulin analogue via a linker, and the linker is a linker of formula L2:

[0217] “-(CH2CH2O-) q -(CH2) r -C(O)-#;

[0218] where q is 1 or 2;

[0219] where r is 1 or 2;

[0220] where “ represents the point of attachment to the glucose mimetic; and

[0221] where # represents the point of attachment to human insulin or a human insulin analogue.

[0222] 25. The insulin derivative according to any one of embodiments 14 to 24, wherein the human insulin or human insulin analogue is the human insulin analogue desB30 human insulin.

[0223] 26. The insulin derivative according to embodiment 25, wherein the insulin derivative is selected from

[0224] INS1 of Example 1

[0225]

[0226] INS9 of Example 5;

[0227] INS18 of Example 8;

[0228] INS21 of Example 9;

[0229] INS28 of Example 12;

[0230] INS30 of Example 14;

[0231] INS34 of Example 18;

[0232] INS35 of Example 19;

[0233] INS36 of Example 20;

[0234] INS37 of Example 21; and

[0235] INS38 of Example 22.

[0236] 27. An insulin derivative according to any one of embodiments 14 to 22, wherein the insulin derivative comprises two glucose mimics and two macrocycles M of formula M1.

[0237] 28. The insulin derivative according to embodiment 27, wherein the two macrocycles are attached to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue.

[0238] 29. The insulin derivative according to embodiment 28, wherein the two macrocycles M are attached to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue via a trivalent linker, wherein the linker is a linker of formula L3

[0239]

[0240] Formula L3;

[0241] wherein Z is CH2- or (CH2CH2O-)3;

[0242] wherein W is CH2-, (CH2CH2CO-) or -Ph-p-CH2-;

[0243] wherein * represents the point of attachment to the macrocycle M; and

[0244] wherein # represents the point of attachment to human insulin or a human insulin analogue.

[0245] 30. The insulin derivative according to embodiment 29, wherein the two glucose mimics are attached to the α-carboxyl group and / or the ε-amino group of lysine (K) at position 29 of the B-chain of the human insulin or human insulin analogue.

[0246] 31. The insulin derivative according to embodiment 30, wherein the two glucose mimics are attached to the α-carboxyl group or the ε-amino group of lysine (K) at position 29 of the B-chain of the human insulin or human insulin analogue via a trivalent linker;

[0247] wherein the linker is selected from

[0248] a)

[0249]

[0250] and

[0251] b)

[0252]

[0253] or each of the two glucose mimetics is separately attached via a bivalent linker to the α-carboxylic acid group and the ε-amino group of lysine (K) at position 29 of the B-chain of the human insulin or human insulin analogue;

[0254] wherein the linker is a linker of formula L6 and formula L7:

[0255]

[0256] and

[0257]

[0258] where “ represents the point of attachment to the glucose mimetic; and

[0259] where # 1 represents the point of attachment to the α-carboxylic acid group of lysine (K) at position 29 of the B-chain of the human insulin or human insulin analogue; and

[0260] where # 2 represents the point of attachment to the ε-amino group of lysine (K) at position 29 of the B-chain of the human insulin or human insulin analogue.

[0261] 32. The insulin derivative according to any one of embodiments 27 to 31, wherein the human insulin or human insulin analogue is the human insulin analogue desB30 human insulin.

[0262] 33. The insulin derivative according to embodiment 32, wherein the insulin derivative is selected from

[0263] INS22 of Example 10;

[0264] INS29 of Example 13;

[0265] INS32 of Example 16; and

[0266] INS33 of Example 17.

[0267] 34. The insulin derivative according to embodiment 14, wherein the insulin derivative is INS41 of Example 25.

[0268] 35. The insulin derivative according to embodiment 14, wherein the insulin derivative is INS5 of Example 3.

[0269] 36. The insulin derivative according to Embodiment 1, wherein the insulin derivative is INS4 of Example 2.

[0270] 37. The insulin derivative according to any one of Embodiments 14 to 36, wherein the insulin derivative has the ability to bind to an insulin receptor.

[0271] 38. The insulin derivative according to any one of Embodiments 14 to 36, wherein the insulin derivative has a higher insulin receptor affinity in the presence of 20 mM glucose as compared to the absence of glucose.

[0272] 39. The insulin derivative according to any one of Embodiments 14 to 36, wherein the insulin derivative has at least 2-fold insulin receptor affinity in the presence of 20 mM glucose as compared to the absence of glucose.

[0273] 40. A pharmaceutical composition for treating and / or preventing diabetes in a patient in need thereof, comprising a therapeutically effective amount of the insulin derivative according to any one of Embodiments 14 to 36, and a pharmaceutically acceptable excipient.

[0274] 41. The pharmaceutical composition of Embodiment 40, which is used as a drug.

[0275] 42. The pharmaceutical composition of Embodiment 40, which is used for treating and / or preventing diabetes.

[0276] 43. The insulin derivative according to any one of Embodiments 14 to 36, which is used as a drug.

[0277] 44. The insulin derivative according to any one of Embodiments 14 to 36, which is used for treating and / or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0278] 45. The insulin derivative according to any one of Embodiments 14 to 36, which is used in a method for treating and / or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0279] 46. A method for treating and / or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome), which comprises administering an effective amount of the insulin derivative according to any one of Embodiments 14 to 36 to a patient in need thereof.

[0280] Examples

[0281] Unless the chemical name is clear, the chemical structures of all end products and intermediate products are shown. The end products and some intermediates also have their chemical names listed together with their structures in the following sections; however, these chemical names are not the complete chemical names that clearly define the products, but are only for easy reading. The nomenclature used to number the PEG linkers varies in the literature; in this article, PEG3 represents a PEG linker with three ethylene groups; PEG4 represents a PEG linker with four ethylene groups, and so on.

[0282] Materials and Methods

[0283] List of Abbreviations

[0284] CV Column volume

[0285] DBU 1,8-Diazabicyclo(5.4.0)undec-7-ene

[0286] DCM Dichloromethane

[0287] DIC N,N-Diisopropylcarbodiimide

[0288] DMF N,N-Dimethylformamide

[0289] DMSO Dimethyl sulfoxide

[0290] EtOAc Ethyl acetate

[0291] DIPEA N,N-Diisopropylethylamine

[0292] Fmoc-OSu 9-Fluorenylmethyl N-succinimidyl carbonate

[0293] HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0294] HOBt 1-Hydroxybenzotriazole

[0295] HONSu M-Hydroxysuccinimide

[0296] HRMS High-resolution mass spectrometry

[0297] LCMS Liquid chromatography-mass spectrometry

[0298] MeCN Acetonitrile

[0299] NMR Nuclear magnetic resonance

[0300] NMP N-Methyl-pyrrolidone

[0301] RP-HPLC Reverse-phase high-performance liquid chromatography

[0302] TEMPO (2,2,6,6 - Tetramethylpiperidin - 1 - yloxy)

[0303] THF Tetrahydrofuran

[0304] THPTA Tris(3 - hydroxyphenyltriazolylmethyl)amine

[0305] Preparation of Structural Unit

[0306] Preparation of Compound 1

[0307]

[0308] 3,4 - Diaminobenzoic acid (41.0 g, 0.269 mol) was mixed with saturated NaHCO3 (0.40 L) and acetonitrile (0.40 L) to give a brown slurry. Next, solid Fmoc - OSu (99.99 g, 0.296 mol) was added in portions over 5 minutes. The heterogeneous suspension was stirred at room temperature for 16 h and then acidified with 1 M HCl (aqueous solution). The solid was collected on a frit and washed with cold ether (33 x 100 mL), hexane (3 x 100 mL), then with MeOH (3 x 50 mL), and then dried under vacuum. Brown solid (101 g, 0.269 mol, 100%). This intermediate (10.0 g, 0.027 mol), HOBt (8.181 g, 0.053 mol), and HBTU (20.259 g, 0.053 mol) were dissolved in THF (300 mL) and DIPEA (18.610 mL, 0.107 mol). The heterogeneous slurry was stirred at room temperature for 90 minutes, after which the solvent was removed in vacuo to give a viscous oil. The oil was dissolved in EtOAc (80 ml) and added to a rapidly stirred mixture of water (200 ml) and EtOAc (40 mL). A precipitate formed after about 2 minutes, and diethyl ether (100 mL) was added to the flask. After stirring for 10 minutes, solid 1 was collected by filtration, washed with water (3 x 10 mL) and diethyl ether (2 x 10 mL), and then dried under vacuum for 16 h.

[0309] 11H NMR: (500 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.14 (dd, J = 17.7, 8.4 Hz, 2H), 8.01–7.93 (m, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.87–7.68 (m, 6H), 7.68–7.61 (m, 1H), 7.54 (dt, J = 11.5, 7.5 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 1H), 6.46 (s, 2H), 4.44 (s, 2H), 4.31 (s, 1H), 3.40 (s, 7H), 3.03 (s, 7H), 2.50 (s, 4H).

[0310] Preparation of Compound 2a

[0311]

[0312] NaH (3.809 g, 0.095 mol, 60% in mineral oil) was added to a Schlenk flask (100 mL) and placed under nitrogen. The mineral oil was removed by washing the solid with 3 x 25 mL of petroleum ether at 60 - 80 °C. The washed NaH was suspended in anhydrous DMF (40 mL) and stirred vigorously for about 10 minutes while cooling in an ice bath. Solid trifluoroacetamide (16.147 g, 0.143 mol) was added in portions under a reverse nitrogen flow. After stirring for five minutes, the mixture was allowed to warm to room temperature. Once the evolution of gas had completely ceased (within 1 hour), solid 1,3,5-tris(bromomethyl)-2,4,6-triethylbenzene (7.00 g, 0.016 mol, Sigma - Aldrich) was added in portions under a reverse nitrogen flow and the resulting white suspension was stirred at room temperature. After 18 hours, the suspension was poured into 0.5 M HCl (150 mL) and the pale orange precipitate was collected on a sinter. The solid was washed with water (2 x 10 mL) and then dried under vacuum overnight (about 10 -2mbar). Off-white solid (7.910g, 0.015mol, 93%). Intermediate acetamide (4.90g, 0.009mol) was dissolved in methanol (38.6mL) and water (38.6mL). NaOH (1.05g, 3.150mol) was added and the reaction mixture was stirred at 65°C for about 18 hours. Solid Boc2O (7.287g, 0.033mol) and triethylamine (2.534mL, 0.026mol) were added and the reaction was stirred at ambient temperature for another 4 hours. The reaction mixture was diluted with DCM (200mL) and washed with saturated NaHCO3 (200mL), 1M NaOH (200mL) and brine (100mL). The organic phase was concentrated to dryness, and the resulting crude product was purified by MPLC (Biotage, using 0 to 50% MeOH in DCM). Colorless solid 2a (4.820 g, 0.009 mol, 96%).

[0313] 1 H NMR(450MHz,CDCl3)δm.br 4.33(9H,ArC H2 NHCO2C(CH3)3),N H ),q.2.71(6H, 3 J HH =7.5Hz,ArC H 2CH3),s.1.44(27H,CO2C(C H 3)3),t.1.19(9H, 3 J HH =7.5Hz,ArCH2C H 3).

[0314] 13 C NMR (125 MHz, CDCl3) δ 155.5 ( C O2C(CH3)3),143.9,132.6(Ar),79.7(CO2C( C H3)3),38.9Ar C H2NHCO2C(CH3)3),28.6(CO2C( C H3)3),23.0(Ar C H2CH3),16.7(ArCH2 C H3).

[0315] Preparation of Compound 2b

[0316]

[0317] A magnetic stirrer and compound 2a (1.266 g, 2.25 mmol) were placed into a pre-dried 200 mL Schlenk flask, and then it was placed under a nitrogen atmosphere. 2-Chloropyridine (1.7 mL, 20.21 mmol) and anhydrous DCM (70 mL) were added via syringe to obtain a colorless homogeneous solution. Trifluoromethanesulfonic anhydride (1.5 mL, 10.11 mmol) was added dropwise with stirring (400 rpm) over 2 minutes at ambient temperature. The reaction was stirred for 30 minutes, and then a small portion of the reaction mixture (ca. 50 mL) was removed and analyzed by TLC (SiO2, 50% Et2O in petrol), which showed complete consumption of the starting material (Rf = 0.24) and conversion to 2b (Rf = 0.5). The solvent was removed on a rotary evaporator to give an off-white solid. The solid was extracted with Et2O (x2 15 mL) and passed through an alumina plug (20 mm x 20 mm), and then eluted with 20 mL of Et2O. The colorless filtrate was evaporated to dryness, and the residue was recrystallized from hexane. Colorless crystalline solid 2b (0.435 g, 1.33 mmol, 59%).

[0318] 1 H NMR (400 MHz, toluene-d6) δ s. 3.93 (6H, ArC H 2NCO), m. 2.51 - 2.37 (6H, ArCH2CH3), m. 0.96 - 0.86 (9H, ArCH2C H 3).

[0319] 13 C NMR (100 MHz, toluene-d6) 143.2, 132.6 (Ar), 124.0 (N C O), 40.4 (Ar C H2NCO), 22.8 (ArC H 2CH3), 16.0 (ArCH2C H 3).

[0320] Preparation of Compound 3

[0321]

[0322] Compound 1 (3.308 g, 5.443 mmol) and anhydrous DIPEA (1.270 mL, 7.258 mmol) were dissolved in anhydrous THF (20 mL). 11-Azido-3,6,9-trioxaundecan-1-amine (1.000 mL, 4.536 mmol) was added dropwise. After stirring at room temperature for 16 h, the reaction mixture was concentrated in vacuo to give a brown residue. The crude product was purified by flash column chromatography, eluting with 50 to 100% EtOAc / DCM. The fractions containing the product were combined and evaporated to dryness to give colorless amorphous solid 3 (2.50 g, 4.35 mmol, 80%).

[0323] 1 1H NMR: (400 MHz, CDCl3) δ 7.74 (d, J = 7.5 Hz, 2H, ArH), 7.60 (d, J = 2.0 Hz, 2H, ArH), 7.45 (s, 1H, ArH), 7.38 (t, J = 7.5 Hz, 2H, ArH), 7.26 (d, J = 9.6 Hz, 2H, ArH), 7.05 (s, 1H, ArH), 6.84 (s, 1H ArH), 6.65 (d, J = 8.4 Hz, 1H C(O)NHCH2), 4.49 (s, 1H, Flu-CH2), 4.18 (s, 2H, Flu-OCH2), 3.68–3.45 (m, 14H, OCH2), 3.27 (t, J = 5.0 Hz, 2H C(O)NHCH2), 1.34 (d, J = 6.7 Hz, 2H, N3CH2).

[0324] Preparation of Compound 4

[0325]

[0326] N,N',N”-Tris[tris(2-tert-butoxycarbonyl-ethyl)methyl]-3,3',3”-(1-aminomethanetriyl)-tripropionamide (7.0 g, 5.0 mmol, Allichem (G2 amine)) and compound 1 (5.310 g, 10 mmol) were dissolved in anhydrous THF (210 mL). Triethylamine (2.23 mL, 16 mmol) was added and the reaction was stirred at room temperature for 18 h. The reaction mixture was diluted with Et2O (40 mL) and the resulting suspension was filtered. The resulting filtrate was concentrated in vacuo and the residue was loaded onto a 120 g C18 column in MeCN (10 mL) and purified by reverse-phase chromatography (70-100% aqueous acetone gradient). The fractions containing the product were combined and evaporated to dryness to give off-white amorphous solid 4 (7.61, 4.0 mmol, 81%).

[0327] 1 1H NMR: (400 MHz, (CDCl3)): δ 1.43 (s, 81H, C(26)H3), 1.95 (m, 18H, C(23)H2), 2.11 (t, J = 7.2 Hz, 6H, C(18)H2), 2.17 (m, 18H, C(22)H2), 2.25 (t, J = 7.2 Hz, 6H, C(19)H2), 4.27 (m, 3H, C(7)H and NH2), 4.47 (d, J = 7.4 Hz, 2H, C(8)H2), 6.08 (s, 3H, NH), 6.76 (d, J = 8.4 Hz, 1H, C(13)H), 7.26 - 7.31 (m, 2H, C(4)H), 7.38 (t, J = 7.4 Hz, 2H, C(3)H), 7.57 - 7.69 (m, 2H, C(5)H), 7.71 (d, J = 8.7 Hz, 1H, C(2)H), 7.75 (d, J = 7.6 Hz, 3H, C(2)H), 7.78 (d, J = 2.1 Hz, 1H, C(15)H), 8.54 (s, 1H, NH)

[0328] 13 13C NMR: (100 MHz, (CDCl3)): δ 28.0 (C26), 29.8 (C22), 29.9 (C23), 31.8 (C19), 32.2 (C18), 47.2 (C7), 53.4 (C17), 57.4 (C21), 67.3 (C8), 80.6 (C25), 116.6 (C12), 119.9 (C2), 122.6 (C10), 124.6 (C14), 125.3 (C4), 126.0 (C15), 126.8 (C13), 127.0 (C5), 127.6 (C3), 141.3 (C1), 143.8 (C6), 145.3 (C11), 154.9 (C9), 166.6 (C16), 172.7 (C24), 173.1 (C20);

[0329] IR: λ max (cm -1 ) 2977, 2963, 1752, 1723, 1689, 1637, 1535, 1367, 1242, 1151, 1098, 844;

[0330] HRMS: (ESI+) Found [M + 2Na] 2+ : 921.0252.

[0331] Preparation of Compound 5a

[0332]

[0333] 1-Amino-3,6,9-trioxadodecane-12-oic acid (Fluorochem) (10.0 g, 37.7 mmol) and MeOH (50 mL) were added to a flask. The mixture was cooled in an ice bath (0 °C). Thionyl chloride (7.25 mL, 99.4 mmol) was added dropwise. The mixture was heated to reflux (3 h), cooled to ambient temperature, and diluted with EtOAc (100 mL), yielding a white precipitate. The solid was separated by vacuum filtration and dried under vacuum to afford pure ester hydrochloride 1b (8.09 g, 25.6 mmol, 68% yield, white solid 5a), which was used directly in the next step.

[0334] Preparation of Compound 5b

[0335]

[0336] Compound 1 (1.100 g, 2.014 mmol) and anhydrous DIPEA (0.702 mL, 4.028 mmol) were dissolved in anhydrous THF (70 mL). Compound 5a (0.763 g, 2.417 mmol) dissolved in anhydrous THF (10 mL) was added dropwise. After stirring at room temperature for 16 h, 150 mL of saturated NH4Cl was added. (水溶液) The resulting suspension was extracted with DCM (3 x 30 mL). The combined organic extracts were dried over MgSO4 and evaporated to dryness. The resulting residue was recrystallized from minimally boiling EtOAc to afford a waxy solid 5b (1.03 g, 1.62 mmol, 80%).

[0337] Preparation of Compound 6a

[0338]

[0339] A magnetic stir bar, compound 4 (0.933 g, 0.519 mmol), compound 2b (0.100 g, 0.305 mmol), anhydrous THF (6 mL), and anhydrous pyridine (0.147 mL, 1.833 mmol) were added to a Schlenk flask. The mixture was then heated to 50 °C and maintained for 5 h. Compound 3 (0.228 g, 0.397 mmol) dissolved in anhydrous THF (1 mL) was added in one portion, and the reaction was stirred for an additional 12 h. The reaction mixture was concentrated under vacuum, and the crude residue was loaded onto a 120 g C18 column in 5 mL MeCN and purified by reverse-phase flash chromatography (1 CV 85% acetone / H2O, 10 CV 85 - 95% acetone / H2O, 2 CV 95% acetone). The fractions containing the product were combined and evaporated to dryness to afford an off-white amorphous solid (458 mg, 46%).

[0340] 1 1H NMR: (400 MHz, (CD3OD): δ 8.02 - 7.46 (19H, br.m, ArH), 7.46 - 7.11 (14H, br.m, ArH), 4.60 - 4.30 (12H, br.m, NHCH2Ph and FmocH), 4.19 (3H, br.s, NHCH2Ph and FmocH), 3.71 - 3.55 (14H, m, PEG CH2), 3.33 (2H, m, PEG CH2), 2.85 (6H, br.s, CH2), 2.35 - 1.86 (96H, m, dendrimer CH2), 1.42 (162H, s, CH3), 1.23 (9H, br.s, CH3);

[0341] HRMS: for C 244 H 352 N 21 O 57 Na3 2+ Calculated (ESI+): 1520.8407, found [M + 3Na] 3+ : 1520.8395.

[0342] Preparation of Compound 6b

[0343]

[0344] Compound 6a (2.400 g, 0.534 mmol) was dissolved in anhydrous DCM (26 mL) and treated with DBU (0.479 mL, 3.204 mmol) at room temperature under nitrogen. After 1 h, the reaction mixture was concentrated in vacuo to remove DCM, redissolved in EtOAc (50 mL), washed with HCl (50 mL, 1 M), the organic phase was dried over MgSO4, filtered, and the resulting filtrate was concentrated in vacuo. The crude residue was loaded onto a 120 g SNAP Ultra C18 135 column in 5 mL MeCN and purified by reverse-phase flash chromatography (70 to 100% MeOH:water gradient). The fractions containing the product were combined and evaporated to dryness to afford the pale yellow amorphous solid 6b (1.56 g, 0.408 mmol, 76%).

[0345] 1 H NMR (400 MHz, methanol-d4) δ 7.91 (s, 2H), 7.41 (s, 7H), 7.38 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.27 (d, J = 2.0 Hz, 2H), 7.23 (d, J = 2.0 Hz, 1H), 7.18 (dd, J = 8.4, 2.0 Hz, 2H), 7.10 (dd, J = 8.3, 2.1 Hz, 1H), 4.48 (s, 6H), 3.71–3.55 (m, 14H), 3.51 (t, J = 5.3 Hz, 2H), 2.96–2.72 (m, 6H), 2.28–2.13 (m, 48H), 2.13–2.03 (m, 12H), 2.01–1.83 (m, 36H), 1.42 (s, 162H), 1.31–1.15 (m, 9H).

[0346] 13 C NMR (101 MHz, methanol-d4) δ 175.5, 174.4, 170.2, 170.1, 158.0, 157.9, 145.1, 141.8, 141.4, 133.9, 132.8, 132.3, 129.9, 129.7, 124.9, 124.5, 118.9, 118.4, 117.3, 117.0, 81.6, 71.6, 71.5, 71.3, 71.1, 70.6, 59.3, 58.7, 51.7, 40.9, 39.3, 32.5, 32.2, 30.7, 30.4, 28.4, 23.9, 17.0.

[0347] For C 199 H 328 N 21 O 51 Calculated HRMS [M+3H]3+ Requirement: 1276.1239, measured value: 1276.1305.

[0348] Preparation of Compound 6c

[0349]

[0350] Dissolve compound 6b (1.057 g, 0.276 mmol) in anhydrous pyridine and heat to 50 °C under nitrogen. In another pear-shaped flask, dissolve compound 2b (0.108 g, 0.331 mmol) in anhydrous DCM (11.500 mL) and add it via an injection pump (1.2 mL / hr). After the addition is complete, concentrate the reaction mixture under vacuum to obtain an orange solid, which is azeotroped with toluene (50 mL) to remove residual pyridine. Load the crude product onto a 120 g C18 column in 5 mL MeCN and purify by reverse-phase flash chromatography (80 to 100% acetone:water gradient). Combine the fractions containing the product and evaporate to dryness to obtain an orange amorphous solid 6c (0.70 g, 0.169 mmol, 61%).

[0351] 1 H NMR (400 MHz, methanol-d4) δ 8.09–7.86 (m, 6H), 7.64 (dd, J = 8.6, 2.1 Hz, 2H), 7.58 (dd, J = 8.5, 2.1 Hz, 1H), 7.44 (s, 6H), 4.57–4.24 (m, 12H), 3.69–3.54 (m, 12H), 3.53–3.45 (m, 2H), 3.34–3.30 (m, 2H), 3.07–2.82 (m, 6H), 2.80–2.68 (m, 6H), 2.33–2.04 (m, 60H), 2.04–1.80 (m, 36H), 1.41 (s, 162H), 1.28–1.11 (m, 18H).

[0352] 1313C NMR (101 MHz, methanol-d4) δ 174.3, 174.2, 173.1, 168.3, 168.0, 157.1, 156.0, 155.9, 143.2, 143.2, 143.1, 135.8, 135.4, 133.0, 133.0, 132.7, 132.5, 129.9, 129.2, 128.8, 128.1, 124.7, 124.6, 121.0, 120.9, 80.3, 70.3, 70.3, 70.2, 70.0, 69.8, 69.3, 58.1, 57.5, 57.4, 50.4, 39.6, 37.6, 37.4, 31.1, 30.9, 30.9, 29.4, 29.1, 27.1, 22.3, 15.5, 15.4, 15.3.

[0353] For C 217 H 349 N 24 O 54 Calculated HRMS [M+3H] 3+ Required: 1385.1768, found: 1385.1824

[0354] Preparation of Compound 6d

[0355]

[0356] Compound 6c (334.0 mg, 0.080 mmol) was dissolved in DCM (8.00 mL) and TFA (5.50 mL, 72 mmol) and stirred at room temperature for 16 h. The reaction mixture was poured into water (90 mL) and the DCM was evaporated in vacuo. The resulting white solid was collected by centrifugation. The precipitate was resuspended in water (30 mL) and centrifuged. The supernatant was decanted and the resulting solid was dried under high vacuum to give an off-white solid 6d. 250 mg, 0.0796 mmol, 99%.

[0357] 1 1H NMR (400 MHz, deuterated phosphate buffer in D2O) δ 7.78–7.60 (m, 6H), 7.52–7.41 (m, 3H), 7.33 (s, 3H), 4.55–4.07 (m, 12H), 3.70–3.38 (m, 14H), 3.24 (t, J = 4.9 Hz, 1H), 3.02–2.94 (m, 1H), 2.81–2.46 (m, 12H), 2.26–2.11 (m, 12H), 2.10–1.86 (m, 48H), 1.86–1.63 (m, 36H), 1.23–

[0358] 0.79 (m, 18H).

[0359] Preparation of Compound 7a

[0360]

[0361] Compound 7a was prepared using compound 5b and compound 2b in a manner similar to compound 6a.

[0362] Preparation of Compound 7b

[0363]

[0364] Compound 7b was prepared using compound 7a in a manner similar to compound 6b.

[0365] Preparation of Compound 7c

[0366] Compound 7c was prepared using compound 7b in a manner similar to compound 6c.

[0367] Preparation of Compound 7d

[0368] Compound 7c (421.6 mg, 0.1 mmol) was dissolved in THF (10 mL) and 0.5 M NaOH (aqueous solution) (10 mL), and stirred for 2 hours. THF was removed under reduced pressure, and 0.1 M HCl (水溶液) The resulting suspension was adjusted to pH, then extracted with DCM (2 x 10 mL). The combined organic extracts were combined, dried over MgSO4, and then evaporated to dryness to give an off-white solid 7d (420.1 mg, 0.99 mmol, 99%).

[0369] Preparation of PEG-β-D-Glucopyranoside Carboxylic Acid O-Succinimide Ester 8

[0370]

[0371] Succinimidyl ester β-D-glucopyranoside PEG3 8 was prepared by converting the carboxylic acid (Sussex Research) dissolved in DMF with N-hydroxysuccinimide (1 equivalent) and N,N-diisopropylcarbodiimide (1 equivalent).

[0372] Preparation of O-2,3,4,6-Tetraacetyl-Diethylene Glycol-β-D-Glucopyranoside 9

[0373]

[0374] To O-peracetyl-α-bromo-D-glucopyranoside (3 g, 7.3 mmol) in 25 mL of dichloroethane was added diethylene glycol (8.9 g, 83.7 mmol) and 2 spoons of molecular sieves, then silver carbonate (3.6 g, 13.1 mmol) was added, and the mixture was stirred vigorously overnight. The mixture was diluted with 60 mL of toluene and filtered through Celite to remove the solids, then the solution was washed twice with brine, once with water, then dried over MgSO4 and concentrated in vacuo. The oily crude residue was triturated with ether to form white crystals. They were washed twice with ether and then dried in a desiccator to give 9.

[0375] Preparation of O-Peracetyl-Diethylene Glycol-β-Glucopyranoside Carboxylic Acid 10

[0376]

[0377] To a mixture of glucoside 9 (1.3 g, 2.979 mmol) in acetone (50 mL) was added saturated NaHCO3 solution (10 mL), and the mixture was cooled in an ice bath. Then TEMPO (9.3 mg, 0.06 mmol) and NaBr (30.384 mg, 0.298 mmol) were added over about 20 minutes, followed by a small portion of trichloroisocyanuric acid (1.38 g, 5.958 mmol). Then the reaction was stirred overnight (18 h). Then it was partially concentrated in vacuo, acidified with HCl, then extracted 5 times with DCM (some salt was added to aid phase separation), dried over MgSO4, and concentrated to dryness to give a white solid.

[0378] Preparation of O-Peracetyl-Diethylene Glycol-β-Glucopyranoside Carboxylic Acid O-Succinimide Ester 11

[0379]

[0380] The carboxylic acid 10 (400 mg, 0.888 mmol) in THF (3 mL) was treated with N-hydroxysuccinimide (143 mg, 1.243 mmol) and DIC (179 mg, 1.421 mmol), stirred overnight, and 11 was used in the acylation of insulin in crude form.

[0381] Preparation of D-Glucopyranoside-1-PEG4-Azide 12

[0382]

[0383] D-glucopyranoside-1-PEG4-azide 12 was prepared as described in J. Am. Chem. Soc. 2017, 139, 3528.

[0384] Preparation of Compound 13

[0385]

[0386] Charge a 50 mL flask with Compound 1 (500 mg, 1.017 mmol), di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (Frontier Scientific - NTN1963, 560 mg, 1.35 mmol), and anhydrous toluene (10 mL). Evaporate the slurry to dryness and redissolve the residue in anhydrous pyridine (5 mL) and DCM (3 mL). Stir the mixture at 50 °C for 16 h. Remove the solvent to give a viscous brown oil, which is partitioned between EtOAc and 1 M aqueous HCl. Wash the organic phase successively with water and brine. Concentrate the combined organic fractions, then adsorb onto silica gel and purify by flash chromatography (EtOAc:DCM (20 / 50%)), to give 13 (467 mg, 0.612 mmol, 60%). 1H NMR: (400 MHz, CDCl3) δ 7.79 (d, J = 7.6 Hz, 2H, ArH), 7.64 (d, J = 2.1 Hz, 1H, ArH), 7.43 (t, J = 7.5 Hz, 2H, ArH), 7.34 (s, 3H, ArH), 6.78 (d, J = 9.0 Hz, 1H, NH), 6.60 (s, 1H, ArH), 6.30 (s, 1H, ArH), 4.56 (s, 2H, Flu-CH2O), 4.28 (s, 1H, Flu-CH2), 4.08 (s, 2H), 2.30 (dd, J = 8.8, 6.7 Hz, 6H, CH2C(O)), 2.16–2.04 (m, 6H, CCH2), 1.44 (s, 24H, C(CH3)3)

[0387] Preparation of Compound 14

[0388]

[0389] Compound 13 (2.36 g, 3.06 mmol, 2.0 equiv) and compound 2b (0.50 g, 1.53 mmol, 1.0 equiv) were dissolved in anhydrous THF (20.6 mL) and heated to 50 °C for 2 h. Then compound 3 (1.32 g, 2.29 mmol, 1.5 equiv) was added as a solid and the reaction was left overnight at 50 °C. DBU (1.4 mL, 9.2 mmol, 6.0 equiv) was added to the heterogeneous reaction mixture. The solvent was removed and the residue was dried using DCM and loaded onto C18, then loaded onto a 120 g C18 column and purified by reverse-phase flash chromatography (50 to 100% MeOH:water gradient). The fractions containing the product were identified by TLC and concentrated in vacuo to give a pale pink solid. The impure fractions were combined and purified again to give 14 (859 mg, 32%).

[0390] 1 1H NMR (400 MHz, methanol-d4) δ 7.35 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 2.1 Hz, 1H), 7.17 (d, J = 2.0 Hz, 2H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 7.06 (dd, J = 8.2, 2.1 Hz, 2H), 4.48 (s, 6H), 3.67–3.55 (m, 12H), 3.51 (t, J = 5.4 Hz, 2H), 3.31 (t, J = 5.2 Hz, 2H), 2.86 (q, J = 7.4 Hz, 6H), 2.23 (dd, J = 9.5, 6.5 Hz, 12H), 2.05 (dd, J = 9.5, 6.5 Hz, 12H), 1.41 (s, 54H), 1.22 (t, J = 7.4 Hz, 9H).

[0391] 13 13C NMR (101 MHz, methanol-d4) δ 174.6, 170.4, 170.3, 158.1, 145.2, 142.0, 133.9, 133.5, 129.5, 125.0, 125.0, 118.5, 118.4, 117.1, 81.8, 71.6, 71.5, 71.3, 71.1, 70.6, 59.3, 51.7, 49.8, 40.9, 40.3, 39.3, 30.8, 30.6, 28.4, 23.9, 16.9.

[0392] For C 91 H 141 N 15 O 21 Calculated HRMS [M+2H] 2+Requirement: 890.0213, Measured value: 890.0206

[0393] Preparation of Compound 15

[0394]

[0395] Dissolve compound 14 (0.820 g, 0.461 mmol, 1.0 equiv) in anhydrous pyridine (192 mL) and heat to 45 °C under nitrogen. In another pear-shaped vial, dissolve compound 2b (0.181 g, 0.553 mmol, 1.2 equiv) in anhydrous CH2Cl2 (19.2 mL) and add it to the solution of compound 14 via an injection pump (rate: 2.0 mL / hr, added over 10 h). Concentrate the reaction mixture in vacuo to give an orange solid, which is azeotroped with toluene (100 mL). Dissolve the crude residue in MeCN (2 mL), load it onto a C18 column, and purify by reverse-phase flash chromatography (60 to 100% acetone:water gradient). Identify the fractions containing the product by TLC and concentrate in vacuo to give an orange / pink solid (0.372 g, 38%).

[0396] 1 H NMR (400 MHz, methanol-d4) δ 8.01 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.92 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 2.0 Hz, 2H), 7.62 (s, 2H), 7.58–7.51 (m, 3H), 4.55–4.30 (m, 12H), 3.64–3.52 (m, 12H), 3.53–3.48 (m, 2H), 3.46–3.36 (m, 2H), 2.92–2.81 (m, 6H), 2.78–2.66 (m, 6H), 2.29–2.20 (m, 12H), 2.11–2.04 (m, 12H), 1.43 (s, 54H), 1.18 (t, J = 7.1 Hz, 18H).

[0397] 13 C NMR (101 MHz, methanol-d4) δ 174.6, 169.7, 158.4, 158.3, 157.2, 144.5, 136.8, 134.3, 133.9, 131.5, 130.2, 129.9, 129.5, 125.6, 125.5, 125.2, 122.3, 81.7, 71.7, 71.5, 71.4, 71.3, 71.1, 70.6, 59.5, 51.7, 40.9, 38.8, 38.7, 30.8, 30.6, 28.4, 24.2, 23.6, 16.6.

[0398] For C 109 H 162 N 18 O 24 Calculated HRMS [M+2H] 2+ Required: 1054.1021, Found: 1054.1028.

[0399] Preparation of Compound 16

[0400]

[0401] Compound 15 (190 mg, 0.090 mmol) was dissolved in CH2Cl2 (9 mL) and TFA (6.2 mL, 81 mmol) and stirred at room temperature for 16 h. The reaction mixture was poured into water (90 mL) and the suspension was bubbled with nitrogen for 30 min to remove any CH2Cl2. The resulting white solid was collected by centrifugation. The precipitate was resuspended in water (30 mL) and centrifuged. The supernatant was decanted, the resulting solid was resuspended in water (25 mL) and neutralized to pH 7.4 by addition of aqueous NaOH. Sonication was required between additions to ensure complete solubility of all materials. Once homogeneous at the desired pH, the solution was passed through a 0.22 μm PES syringe filter and then lyophilized to give 16 (170 mg, 99%).

[0402] 1 H NMR (400 MHz, D2O containing 100 mM phosphate buffer and 2 mM DMF) δ 7.91–7.22 (m, 9H), 4.32 (br.s, 12H), 3.72–3.32 (m, 14H), 3.21 (s, 2H), 2.62 (br.s, 12H), 2.04 (br.s, 12H), 1.91 (br.s, 12H), 1.04 (br.s, 18H).

[0403] Preparation of Compound 17

[0404]

[0405] Prepared using commercially available 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine as described for compound 3. White solid 17 (3.2 g, 71%).

[0406] 11H NMR (400 MHz, methanol-d4) δ 7.97 (t, J = 5.6 Hz, 1H), 7.87–7.56 (m, 4H), 7.56–7.17 (m, 5H), 6.79 (d, J = 8.4 Hz, 1H), 4.49 (s, 2H), 4.27 (s, 1H), 3.70–3.59 (m, 8H), 3.54 (q, J = 5.2 Hz, 2H), 3.35–3.25 (m, 2H).

[0407] For C 28 H 31 HRMS [M + H] calculated for C + Required: 531.2351, found: 531.2362.

[0408] Preparation of Compound 18

[0409]

[0410] Compound 17 was prepared as described for compound 14, white solid 18 (0.78 g, 86%)

[0411] 1 1H NMR (400 MHz, methanol-d4 + CDCl3) δ 7.36–7.29 (m, 3H), 7.22 (d, J = 2.1 Hz, 1H), 7.18 (d, J = 2.1 Hz, 2H), 7.10 (dd, J = 5.0, 2.0 Hz, 1H), 7.07 (dd, J = 5.0, 2.1 Hz, 2H), 4.48 (s, 6H), 3.69–3.60 (m, 8H), 3.54 (t, J = 5.4 Hz, 2H), 3.34 (t, J = 5.0 Hz, 2H), 2.85 (q, J = 7.2 Hz, 6H), 2.30–2.21 (m, 12H), 2.11–2.02 (m, 12H), 1.43 (s, 54H), 1.23 (t, J = 7.2 Hz, 9H).

[0412] 1313C NMR (101 MHz, methanol-d4 + CDCl3) δ 174.5, 170.1, 170.0, 169.9, 157.9, 157.8, 144.9, 141.6, 141.5, 133.6, 133.1, 133.0, 132.1, 129.5, 129.4, 125.0, 124.9, 118.5, 118.4, 116.9, 116.9, 81.8, 71.3, 71.2, 70.9, 70.5, 59.1, 59.0, 51.5, 40.7, 39.2, 30.8, 30.7, 30.5, 28.4, 23.7, 16.9。

[0413] For C 89 H 137 N 15 O 20 Calculated HRMS [M + 2H] 2+ Required: 868.0082, Found: 868.0085

[0414] Preparation of Compound 19

[0415]

[0416] Compound 18 was prepared as described for Compound 15 to give white solid 19 (449 mg, 48%).

[0417] 1 1H NMR (400 MHz, methanol-d4) δ 8.03 (d, J = 2.1 Hz, 1H), 7.99–7.91 (m, 3H), 7.89 (d, J = 2.1 Hz, 2H), 7.65 (s, 2H), 7.59 (dd, J = 8.6, 2.1 Hz, 1H), 7.55 (dd, J = 8.6, 2.1 Hz, 2H), 4.54–4.35 (m, 12H), 3.68–3.62 (m, 6H), 3.60 (t, J = 5.2 Hz, 2H), 3.49 (t, J = 5.6 Hz, 2H), 3.35 (t, J = 4.9 Hz, 2H), 2.94–2.83 (m, 6H), 2.83–2.71 (m, 6H), 2.33–2.23 (m, 12H), 2.12–2.06 (m, 12H), 1.45 (s, 54H), 1.23–1.16 (m, 18H).

[0418] 1313C NMR (101 MHz, methanol-d4) δ 174.6, 169.8, 169.7, 158.4, 158.3, 157.3, 144.5, 136.9, 134.3, 133.9, 133.9, 131.5, 130.4, 130.0, 129.6, 125.6, 125.5, 122.6, 122.4, 81.7, 71.5, 71.5, 71.1, 70.7, 59.5, 51.7, 40.9, 38.8, 38.7, 30.8, 30.6, 28.4, 23.6, 16.6, 16.6。

[0419] For C 107 H 158 N 18 O 23 Calculated HRMS [M+2H] 2+ Required: 1031.5874, found: 1031.5889

[0420] Preparation of Compound 20

[0421]

[0422] Compound 19 was prepared as described for compound 16 to give white solid 20 (380 mg, 94%). Using 1 1H NMR spectroscopy, the purity was determined to be approximately 75% (by weight) by relative integration with respect to the DMF internal standard.

[0423] 1 1H NMR (400 MHz, D2O containing 100 mM phosphate buffer and 2 mM DMF) δ 8.07–7.20 (m, 9H), 4.27 (s, 12H), 3.61–3.33 (m, 10H), 3.19 (s, 2H), 2.55 (s, 12H), 2.03 (s, 12H), 1.90 (s, 12H), 1.00 (s, 18H).

[0424] Preparation of Compound 21, G1 Macrocyclic Propyl Azide

[0425]

[0426] Step 1: Preparation of Compound 21a

[0427]

[0428] Compound 1 was dissolved in anhydrous THF (110.5 mL) and stirred while DIPEA (4.1 mL, 23.731 mmol) was added. 3-Azidopropylamine (0.750 g, 6.742 mmol) was added, and the homogeneous solution was stirred overnight. The solvent was removed to dryness to give a pink solid, which was suspended in DCM (10 mL) and MeOH (ca. 10 mL) and warmed to give a solution. The solution was concentrated on a rotary evaporator, which resulted in the precipitation of a white solid. The precipitate was collected, washed successively with DCM (2 x 3 mL) and gasoline (2 x 5 mL), and then dried under high vacuum to give an off-white solid 21a (1.8 g, 3.94 mmol, 59%). 1 H NMR (400 MHz, methanol-d4) δ 8.57 (d, J = 7.6 Hz, 2H), 8.48–8.40 (m, 2H), 8.21 (s, 1H), 8.13 (t, J = 7.4 Hz, 2H), 8.06 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 5.22–5.14 (m, 2H), 5.05–4.95 (m, 2H), 4.08 (dt, J = 10.3, 6.8 Hz, 4H), 4.00–3.91 (m, 1H), 2.52 (p, J = 6.8 Hz, 2H).

[0429] Step 2: Preparation of Compound 21b

[0430]

[0431] Prepared from compound 4 (2.358 g, 3.055 mmol), 21a (1.046 g, 2.291 mmol), and 2b (500 mg, 1.527 mmol) according to compound 6a. The product was isolated as an off-white amorphous solid 21b (1.17 g, 0.503 mmol, 33%). 1 H NMR (400 MHz, methanol-d4) δ 8.31–7.00 (m, 33H), 4.43–3.97 (m, 15H), 3.37 (t, J = 6 Hz, 2H), 3.30 (m, 2H), 2.76 (br.s, 6H), 2.25–2.19 (m, 12H), 2.10–2.03 (m, 12H), 1.79 (p, J = 6 Hz, 2H) 1.39 (s, 54H), 1.18–1.12 (m, 9H).

[0432] Step 3: Preparation of Compound 21c

[0433]

[0434] Prepared from compound 21b (1.17 g, 0.503 mmol) according to compound 6b. The product was isolated as a grayish-white amorphous solid compound 21c (0.702 g, 0.423 mmol, 84%). 1 H NMR (400 MHz, methanol-d4) δ 7.36 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 2.1 Hz, 1H), 7.19 (d, J = 2.1 Hz, 2H), 7.12 (dd, J = 8.3, 2.1 Hz, 1H), 7.08 (dd, J = 8.2, 2.1 Hz, 2H), 4.50 (s, 6H), 3.42 (t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.7 Hz, 2H), 2.88 (q, J = 7.0 Hz, 6H), 2.31–2.20 (m, 12H), 2.12–2.01 (m, 12H), 1.85 (p, J = 6.7 Hz, 2H), 1.43 (s, 54H), 1.24 (t, J = 7.4 Hz, 9H). 13 C NMR (101 MHz, methanol-d4) δ 174.6, 170.4, 170.4, 158.1, 158.1, 145.1, 142.0, 141.9, 133.9, 133.5, 132.4, 129.7, 129.5, 125.0, 125.0, 118.5, 118.3, 117.0, 81.8, 59.3, 50.2, 39.3, 38.3, 30.8, 30.6, 29.8, 28.3, 23.9, 16.9. For C 86 H 130 N 15 O 18 Calculated HRMS ([M + H] + Required: 1660.9718, found: 1660.9723.

[0435] Step 4: Preparation of Compound 21d

[0436]

[0437] Prepared from compound 21c (740 mg, 0.445 mmol) and 2b (175 mg, 0.535 mmol) according to compound 6c. 20% DMF was added to the solvent and heated to 45 °C. The product was isolated as a white amorphous solid compound 21d (0.254 g, 0.128 mmol, 29%). 11H NMR (400 MHz, methanol-d4) δ 8.05 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.95 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 2.1 Hz, 2H), 7.57 (dd, J = 8.6, 2.2 Hz, 1H), 7.53 (dd, J = 8.6, 2.1 Hz, 2H), 4.47 (s, 6H), 4.42 (s, 6H), 3.48–3.39 (m, 4H), 2.94–2.80 (m, 6H), 2.80–2.71 (m, 6H), 2.33–2.23 (m, 12H), 2.13–2.05 (m, 12H), 1.88 (p, J = 6.7 Hz, 2H), 1.45 (s, 54H), 1.20 (t, J = 7.3 Hz, 18H). 13 13C NMR (101 MHz, methanol-d4) δ 174.6, 169.8, 169.7, 158.3, 158.2, 157.3, 157.2, 144.5, 144.5, 136.5, 136.3, 134.2, 134.1, 133.8, 133.8, 131.3, 130.4, 130.0, 129.5, 125.4, 125.1, 124.9, 122.6, 122.4, 81.7, 59.4, 50.2, 38.8, 38.4, 30.8, 30.7, 30.6, 29.8, 28.4, 23.6, 16.6, 16.6. For C 104 H 150 N 18 O 21 Calculated HRMS [M+2H] 2+ Required: 994.5690, Found: 994.5684

[0438] Step 5: Preparation of Compound 21, G1 Macrocyclic Propyl Azide

[0439]

[0440] Prepared from compound 21d (240 mg, 0.121 mmol) according to compound 6d. The product was separated after neutralizing to pH 7.5 with NaOH and freeze-dried to give white amorphous solid 21 (187 mg, 0.105 mmol, 87%). 1 1H NMR (400 MHz, deuterated phosphate buffer in D2O) δ 8.12–7.12 (m, 9H), 4.41–3.92 (m, 12H), 3.33 (m, 4H), 2.66–2.26 (m, 12H), 2.20–1.52 (m, 26H), 1.31–0.74 (m, 18H).

[0441] Preparation of Compound 22, G2 Macrocyclic PEG3 Azide:

[0442]

[0443] Step 1: Preparation of Compound 22a

[0444]

[0445] Compound 1 (7.00 g, 8.55 mmol, 1.0 equiv) was dissolved in anhydrous THF (85.5 mL), and DIPEA (3.0 mL, 17 mmol, 2.0 equiv) was added. N3-(PEG)2-NH2 (1.94 g, 11.1 mmol, 1.3 equiv) was added and the mixture was stirred at room temperature. The reaction mixture was concentrated in vacuo and then dissolved in MeCN for RP MPLC. The fractions containing the product were combined and concentrated in vacuo. The residue was loaded onto silica gel and dried with EtOAc / MeOH and then purified by NP MPLC. The fractions containing the product were combined and concentrated in vacuo to give an orange oil. Gasoline / CH2Cl2 was added, and then EtOAc was added, resulting in precipitation. The precipitate was collected by filtration and washed with gasoline to give off-white solid compound 22a (3.210 g, 6.050 mmol, 71%). 1 1H NMR (400 MHz, methanol-d4) δ 7.97 (t, J = 5.6 Hz, 1H), 7.87–7.56 (m, 5H), 7.56–7.17 (m, 5H), 6.79 (d, J = 8.4 Hz, 1H), 4.49 (s, 2H), 4.27 (s, 1H), 3.70–3.59 (m, 8H), 3.54 (q, J = 5.2 Hz, 2H), 3.35–3.25 (m, 2H). HRMS [M+H] calculated for C 28 1 31 6H6O5: 531.2351, found: 531.2362. + Required: 531.2351, found: 531.2362.

[0446] Step 2: Preparation of Compound 22b

[0447]

[0448] Similar to compound 6a, prepared from compound 21a and compound 4. 11H NMR (400 MHz, methanol-d4) δ 7.99–7.44 (m, 17H), 7.44–7.14 (m, 16H), 4.44 (s, 6H), 4.33 (br.s, 6H), 4.13 (br.s, 3H), 3.63–3.55 (m, 8H), 3.51 (t, J = 5.1 Hz, 2H), 3.25 (t, J = 5.0 Hz, 2H), 2.81 (br.s, 6H), 2.24–2.03 (m, 60H), 1.94–1.82 (m, 36H), 1.38 (s, 162H), 1.19–1.10 (m, 9H).

[0449] Step 3: Preparation of Compound 22c

[0450]

[0451] Prepared from compound 22b (2.75 g, 0.62 mmol) according to compound 6b. The product was isolated as a grayish-white amorphous solid, compound 22c (1.52 g, 0.403 mmol, 65%).

[0452] 1 1H NMR (500 MHz, methanol-d4) δ 7.39 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 2.1 Hz, 2H), 7.27 (d, J = 2.1 Hz, 1H), 7.21 (dd, J = 8.3, 2.1 Hz, 2H), 7.14 (dd, J = 8.3, 2.1 Hz, 1H), 4.51 (s, 6H), 3.67–3.62 (m, 8H), 3.54 (t, J = 5.5 Hz, 2H), 3.34 (t, J = 5.3 Hz, 2H), 2.89 (q, J = 7.5 Hz, 6H), 2.27–2.22 (m, 12H), 2.21–2.15 (m, 36H), 2.12–2.07 (m, 12H), 1.99–1.88 (m, 36H), 1.43 (s, 162H), 1.25 (t, J = 7.5 Hz, 9H). 1313C NMR (126 MHz, methanol-d4) δ 175.5, 174.4, 170.3, 170.1, 158.0, 158.0, 145.2, 141.9, 141.5, 133.9, 132.9, 132.4, 129.9, 129.7, 124.9, 124.5, 118.9, 118.4, 117.4, 117.1, 81.7, 71.5, 71.4, 71.1, 70.7, 59.4, 58.7, 51.7, 40.9, 39.3, 32.5, 32.2, 30.7, 30.5, 28.4, 23.9, 17.0. For C 197 H 324 N 21 O 50 Calculated HRMS [M + 3H] 3+ Required: 1261.4485, found: 1261.4509.

[0453] Step 4: Preparation of Compound 22d

[0454]

[0455] Prepared from compound 22c (1.50 g, 0.396 mmol) and 2b (156 mg, 0.476 mmol) according to compound 6c, 20% DMF was added to the solvent and heated to 45 °C. The product was isolated as a white amorphous solid compound 22d (0.94 g, 0.229 mmol, 58%). 1 1H NMR (500 MHz, methanol-d4) δ 8.07 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 8.01–7.95 (m, 3H), 7.92 (d, J = 8.6 Hz, 1H), 7.66 (dd, J = 8.7, 2.0 Hz, 2H), 7.60 (dd, J = 8.5, 2.1 Hz, 1H), 7.44 (s, 4H, NH signal exchanges slowly so the integral changes), 4.57–4.33 (m, 12H), 3.71–3.59 (m, 8H), 3.53 (t, J = 5.5 Hz, 2H), 3.36 (t, J = 5.2 Hz, 2H), 2.87 (br.s, 6H), 2.78 (br.s, 6H), 2.29–2.23 (m, 12H), 2.23–2.17 (m, 36H), 2.15–2.09 (m, 12H), 2.00–1.90 (m, 36H), 1.44 (s, 162H), 1.24–1.14 (m, 18H). 1313C NMR (101 MHz, methanol-d4) δ 175.6, 175.5, 174.4, 169.7, 169.4, 169.3, 158.4, 158.3, 157.3, 157.2, 144.5, 144.5, 144.4, 137.1, 136.7, 134.3, 134.3, 134.0, 133.8, 131.2, 131.2, 130.5, 130.2, 129.4, 126.1, 125.9, 125.4, 125.2, 122.7, 122.2, 81.6, 71.5, 71.4, 71.1, 70.6, 59.5, 59.4, 58.8, 58.7, 51.7, 40.9, 38.9, 38.8, 38.7, 38.7, 32.4, 32.2, 30.7, 30.7, 30.4, 28.4, 23.6, 16.8, 16.7, 16.6. For C 215 H 345 N 24 O 53 Calculated HRMS [M + 3H] 3+ Required: 1371.1700, found: 1371.1720.

[0456] Step 5: Preparation of Compound 22

[0457]

[0458] Prepared from compound 22d (500 mg, 0.122 mmol) according to compound 6d. The product was isolated as a yellow amorphous solid compound 22 (376 mg, 0.122 mmol, 99%). 1 1H NMR (400 MHz, deuterated phosphate buffer in D2O) δ 8.01–7.67 (m, 6H), 7.67–7.49 (m, 2H), 7.43 (s, 1H), 4.55–4.21 (m, 12H), 3.92–3.47 (m, 8H), 3.47–3.29 (m, 2H), 2.82–2.55 (m, 12H), 2.43–2.18 (m, 12H), 2.18–2.00 (m, 48H), 2.00–1.72 (m, 36H), 1.36–0.80 (m, 18H).

[0459] Preparation of Compound 23, G2 Macrocyclic PEG5 Azide

[0460]

[0461] Step 1: Preparation of compound 23a

[0462]

[0463] Prepared from Compound 1 (3.975 g, 6.470 mmol) and 14-azido-3,6,9,12-tetraoxatetradecan-1-amine (2.546 g, 9.705 mmol) according to Compound 3a. Isolated as a brown oil (3.933 g, 6.357 mmol, 98%). 1 H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.6 Hz, 2H, ArH), 7.67 - 7.55 (m, 2H, ArH), 7.50 (d, J = 13.6 Hz, 1H, ArH), 7.39 (t, J = 7.5 Hz, 2H, ArH), 7.34 - 7.27 (m, 2H, ArH), 7.15 (m, 1H, ArH), 6.89 (m, 1H, ArH), 6.69 (d, J = 8.4 Hz, 1H, C(O)NHCH2), 4.52 (s, 1H, Flu-CH), 4.24 (s, 2H, Flu-OCH2), 3.70–3.46 (m, 16H, OCH2), 3.28 (t, J = 5.1 Hz, 2H, (C(O)NHCH2), 1.37 (dd, J = 11.8, 7.0 Hz, 2H, N3CH2). 13 C NMR (101 MHz, CDCl3) δ 167.26 (C(O)NH), 143.80, 141.45, 127.89, 127.26, 125.14, 120.10 (Ar), 77.36, 70.57, 70.49, 70.46, 70.43, 70.42, 70.22, 70.03, 69.92 (OCH2), 55.66 (Flu-OCH2), 50.66 (Flu-CH), 39.80 (N3CH2), 38.72 (NHCH2).

[0464] Step 2: Preparation of Compound 23b

[0465]

[0466] Prepared from Compound 4a (3.24 g, 1.80 mmol), 23a (836 mg, 1.35 mmol) and 2b (295 mg, 0.90 mmol) according to Compound 6a. The product was isolated as an off-white amorphous solid 23b (1.77 g, 0.387 mmol, 43%). 1HNMR (400 MHz, methanol-d4) δ 8.02–7.48 (m, 18H), 7.46–7.11 (m, 15H), 4.46 (br.s, 6H), 4.34 (br.s, 6H), 4.15 (br.s, 3H), 3.54–3.44 (m, 18H), 3.25 (dd, J=5.6, 4.4, 2H), 2.80 (br.s, 6H), 2.35–2.04 (m, 60H), 2.04–1.80 (m, 36H), 1.41 (s, 162H), 1.25–1.11 (m, 9H).

[0467] Step 3: Preparation of Compound 23c

[0468]

[0469] Prepared from compound 23b (1.76 g, 0.38 mmol) according to compound 6b. The product was isolated as an off-white amorphous solid 23c (1.14 g, 0.29, 76%). 1 H NMR (400 MHz, methanol-d4) δ 7.46–7.37 (m, 3H), 7.29 (d, J=2.1, 2H), 7.27 (d, J=2.0, 1H), 7.21 (dd, J=8.3, 2.1, 2H), 7.15 (dd, J=8.4, 2.0, 1H), 4.51 (s, 6H), 3.66–3.51 (m, 16H), 3.36–3.34 (m, 4H), 2.94 - 2.82 (m, 6H), 2.29–2.14 (m, 48H), 2.13–2.06 (m, 12H), 1.99–1.89 (m, 36H), 1.44 (s, 162H), 1.30 - 1.21 (m, 9H).

[0470] Step 4: Preparation of Compound 23d

[0471]

[0472] Prepared from compound 23c (500 mg, 0.27 mmol) and 2b (100 mg, 0.30 mmol) according to compound 6c. 20% DMF was added to the solvent and heated only to 45 °C. The product was isolated as a white amorphous solid 23d (0.86 g, 0.19 mmol, 70%). 11H NMR (400 MHz, methanol-d4) δ 8.11–7.87 (m, 6H), 7.66 (d, J = 8.5, 2H), 7.59 (d, J = 8.4, 1H), 7.45 (s, 6H, NH signal exchanges slowly so the integral changes), 4.56–4.34 (m, 12H), 3.64–3.56 (m, 16H), 3.52–3.46 (m, 2H), 3.35–3.32 (m, 2H), 2.90 (br.s, 6H), 2.78 (br.s, 6H), 2.29–2.17 (m, 48H), 2.15–2.08 (m, 12H), 1.99–1.91 (m, 36H), 1.43 (s, 162H), 1.24–1.17 (m, 18H).

[0473] For C 219 H 350 N 24 O 55 Calculated HRMS [M + 3H] 3+ Required: 1400.5209, found: 1400.5232.

[0474] Step 5: Preparation of Compound 23

[0475]

[0476] Prepared from Compound 23d (750 mg, 0.179 mmol) according to Compound 6d. The product was isolated as a white amorphous solid 23 (570 mg, 0.179 mmol, 99%). 1 1H NMR (400 MHz, deuterated phosphate buffer in D2O) δ 8.08–7.73 (m, 6H), 7.55 (d, J = 8.6, 2H), 7.50–7.43 (m, 1H), 4.59–4.19 (m, 12H), 3.75–3.42 (m, 16H), 3.36–3.21 (m, 2H), 3.17–3.05 (m, 2H), 2.82–2.53 (m, 12H), 2.35–2.21 (m, 12H), 2.15–2.00 (m, 48H), 1.98–1.76 (m, 36H), 1.30–0.90 (m, 18H).

[0477] Preparation of Compound 24, G1 Macrocyclic PEG5 Azide

[0478]

[0479] Step 1: Preparation of Compound 24a

[0480]

[0481] According to Compound 6a, it was prepared from Compound 23a (2.36 g, 3.06 mmol), G1 dendrimer amine = 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)di-tert-butyl heptanedioate, Frontier Scientific - NTN1963 (1.42 g, 2.29 mmol), and 2b (500 mg, 1.53 mmol). The product was isolated as an off-white amorphous solid (1.58 g, 0.64 mmol, 42%). 1 H NMR (400 MHz, methanol-d4) δ 8.01–7.40 (m, 21H), 7.30 (t, J = 7.5, 7H), 7.26–7.1 (m, 7H), 4.56–4.24 (m, 12H), 4.19–4.02 (m, 3H), 3.67–3.39 (m, 18H), 3.29–3.20 (m, 2H), 2.80 (d, J = 9.0, 6H), 2.24 (dd, J = 9.4, 6.4, 12H), 2.13–1.97 (m, 12H), 1.40 (s, 54H), 1.15 (t, J = 7.3, 9H).

[0482] Step 2: Preparation of Compound 24b

[0483]

[0484] According to Compound 6b, it was prepared from Compound 24a (1.55 g, 0.62 mmol). The product was isolated as an off-white amorphous solid 24b (1.11 g, 0.61, 98%). 1 H NMR (400 MHz, methanol-d4) δ 7.39 (s, 1H (partially exchanged NH)), 7.33 (d, J = 8.3, 3H), 7.23 (d, J = 2.0, 1H), 7.18 (d, J = 2.0, 2H), 7.07 (ddd, J = 8.1, 4.1, 1.9, 3H), 4.49 (s, 6H), 3.70–3.54 (m, 16H), 3.51 (d, J = 5.5, 2H), 3.35–3.25 (m, 2H), 2.99–2.72 (m, 6H), 2.39–2.16 (m, 12H), 2.08 (dd, J = 9.5, 6.1, 12H), 1.44 (s, 54H), 1.24 (t, J = 7.3, 9H).

[0485] Step 3: Preparation of Compound 24b

[0486]

[0487] Prepared from compound 24a (500 mg, 0.27 mmol) and 2b (100 mg, 0.30 mmol) according to compound 6c. 20% DMF was added to the solvent and heated to 45 °C. The product was isolated as a white amorphous solid 24b (0.33 g, 0.153 mmol, 58%). 1 H NMR (400 MHz, methanol-d4) δ 8.03–7.94 (m, 3H), 7.94–7.87 (m, 3H), 7.62 (s, 1H), 7.56 (dd, J = 8.7, 2.1, 3H), 4.48 (s, 4H), 4.43 (s, 8H), 3.60–3.33 (m, 18H), 3.30–3.28 (m, 2H), 2.96–2.83 (m, 6H), 2.77 (d, J = 7.8, 6H), 2.32–2.23 (m, 12H), 2.10 (td, J = 7.5, 4.7, 12H), 1.45 (s, 54H), 1.25–1.14 (m, 18H). For C 111 H 164 N 18 O 25 Calculated HRMS [M+H] + Required: 2151.5, found: 2151.5.

[0488] Step 4: Preparation of Compound 24, G1 Macrocyclic PEG5 Azide

[0489]

[0490] Prepared from compound 24b (330 mg, 0.153 mmol) according to compound 6d. The product was isolated as a white amorphous solid 24 (281 mg, 0.153 mmol, 99%). 1 H NMR (400 MHz, deuterated phosphate buffer in D2O) δ 7.90–7.56 (m, 6H), 7.45–7.25 (m, 3H), 4.48–4.07 (m, 12H), 3.71–3.13 (m, 18H), 3.13–3.01 (m, 2H), 2.70–2.44 (m, 12H), 2.35–2.21 (m, 12H), 2.08–2.01 (m, 12H), 2.01–

[0491] 1.84 (m, 12H), 1.12–0.91 (m, 18H).

[0492] Preparation of Compound 25, G0 Macrocyclic PEG4 Azide

[0493]

[0494] Step 1: Preparation of compound 25a

[0495]

[0496] Ethyl 3,4-diaminobenzoate (25.0 g, 0.139 mol) was dissolved in THF (280 mL). Triethylamine (23.2 mL, 0.166 mol) and Boc2O (33.3 g, 0.153 mol, 1.1 equiv) were added. The mixture was stirred at room temperature for 16 h. The solvent was removed under vacuum, and the crude solid was triturated in hot ether / petroleum ether 40 - 60 (5:95, 500 mL), filtered, washed with petroleum ether 40 - 60 (100 mL), and dried under high vacuum to give the product as a light brown solid (34.8 g, 89.5%).

[0497] 1 1H NMR (400 MHz, chloroform-d) δ 7.83 (d, J = 1.9 Hz, 1H), 7.72 (dd, J = 8.4, 2.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 6.15 (s, 1H), 4.30 (q, J = 7.1 Hz, 3H), 4.26 (s, 2H), 1.50 (s, 10H), 1.35 (t, J = 7.2 Hz, 4H).

[0498] Step 2: Preparation of Compound 25b

[0499]

[0500] Under a N2 atmosphere, 25a (17.0 g, 60 mmol) and 2b (3.3 g, 10 mmol) were dissolved in anhydrous DCM (100 mL) and anhydrous DMF (9.9 mL). Anhydrous pyridine (7.3 mL, 91 mmol, 9 equiv) was added, and the reaction was stirred at 40 °C for 18 h. The reaction was cooled to room temperature, and the solvent was removed under vacuum. Then the crude oil was precipitated with 1 M aqueous HCl (1000 mL), filtered, washed with Et2O, and dried under high vacuum to give the product 25b as a light brown solid (11.7 g, 10 mmol, 99%). 1 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 3H), 8.21 (d, J = 8.7 Hz, 3H), 8.18 (s, 3H), 7.74 (s, 3H), 7.70 (dd, J = 8.7, 2.1 Hz, 3H), 7.07 (t, J = 3.7 Hz, 3H), 4.37 (br.s, 6H), 4.27 (q, J = 7.1 Hz, 6H), 2.80 (q, J = 7.5 Hz, 6H), 1.39 (s, 27H), 1.29 (t, J = 7.1 Hz, 9H), 1.18 (t, J = 7.3 Hz, 9H).

[0501] Step 3: Preparation of Compound 25c

[0502]

[0503] Dissolve 25b (11.1 g, 9.50 mmol) in CH2Cl2 (62 mL) and DMF (15 mL). Add trifluoroacetic acid (77.1 mL, 1.0 mol) within 5 minutes, and stir the reaction at room temperature for 3 h. Concentrate the reaction mixture under a nitrogen stream, and precipitate the crude oil with saturated aqueous Na2CO3 (1500 mL), filter, and wash with water. Then wash the crude solid with Et2O and dry under high vacuum to obtain the product 25c (8.0 g, 9.2 mmol, 97%) as an off-white solid.

[0504] 1 1H NMR (400 MHz, DMSO-d6) δ 7.82–7.68 (m, 6H), 7.31 (d, J = 2.0 Hz, 3H), 7.18 (dd, J = 8.5, 2.0 Hz, 3H), 6.47 (s, 3H), 4.79 (s, 6H), 4.39–4.27 (m, 6H), 4.20 (q, J = 7.1 Hz, 6H), 2.87–2.66 (m, 6H), 1.24 (t, J = 7.1 Hz, 9H), 1.15 (t, J = 7.4 Hz, 9H). For C 45 1H 58 N9O9 + Calculated MS [M+H] + Found: 868.4, Calcd: 868.4.

[0505] Step 4: Preparation of Compound 25d

[0506]

[0507] Under an N2 atmosphere, dissolve 25c (5.00 g, 6 mmol, 1 equiv) in anhydrous DMF (417 mL) and anhydrous pyridine (960 mL), and heat to 45 °C. Then add 2b (2.26 g, 7 mmol,

[0508] 1.2 equivalents), and the reaction mixture was stirred at 45 °C for an additional 6 h. The reaction mixture was then concentrated in vacuo, and the crude residue was precipitated with 1 M aqueous HCl (1000 mL), filtered, washed with water and dried. The crude solid was then dissolved in MeOH / CH2Cl2, dried and loaded onto C18, and purified by reverse-phase flash chromatography (45%-80% MeCN:water, containing 0.1% formic acid). The fractions containing the product were combined and concentrated in vacuo to give the product as an off-white solid (4.2 g, 4 mmol, 61%). 1 1H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 2.1 Hz, 3H), 8.08 (d, J = 8.7 Hz, 3H), 7.89 (s, 3H), 7.60 (dd, J = 8.6, 2.1 Hz, 3H), 7.53 (s, 3H), 6.49 (t, J = 5.1 Hz, 3H), 6.35 (t, J = 5.4 Hz, 3H), 4.36–4.24 (m, 18H), 2.80 (d, J = 7.7 Hz, 6H), 2.67 (d, J = 7.8 Hz, 6H), 1.31 (t, J = 7.1 Hz, 9H), 1.19–1.10 (m, 18H). For C 63 1 79 N 12 O 12 + Calculated MS [M+H] + Found: 1195.5, calcd: 1195.5.

[0509] Step 5: Preparation of Compound 25e

[0510]

[0511] 25d (4.9 g, 4.10 mmol, 1 equiv) was suspended in ethanol (50 mL) and water (50 mL), and heated to 40 °C. Sodium hydroxide (0.246 g, 6.15 mmol, 1.5 equiv) was added, and the reaction was stirred at 40 °C for 16 h. The reaction was cooled to room temperature, and the organic solvents were removed in vacuo. The crude product was then precipitated with 1 M aqueous HCl (400 mL), filtered, washed with 1 M aqueous HCl and dried. The crude solid was then dissolved in acetone / water, dried and loaded onto C18, and purified by reverse-phase flash chromatography. The fractions containing the product were combined and the solvent was removed in vacuo to give product 25e as a white solid (1.7 g, 1.46 mmol, 36%).

[0512] 11H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 2.1 Hz, 2H), 8.17 (d, J = 2.1 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 8.6 Hz, 1H), 7.86 (s, 2H), 7.83 (s, 1H), 7.59–7.52 (m, 3H), 7.50 (s, 2H), 7.46 (s, 1H), 6.48–6.42 (m, 3H), 6.36–6.27 (m, 3H), 4.33–4.21 (m, 16H), 2.83–2.71 (m, 6H), 2.68–2.58 (m, 6H), 1.28 (t, J = 7.1 Hz, 6H), 1.11 (td, J = 7.3, 6.9, 2.4 Hz, 18H). For C 59 H 71 N 12 O 12 + Calculated MS [M+H] + Required: 1139.5, Found: 1139.5.

[0513] Step 6: Preparation of Compound 25f

[0514]

[0515] Under N2 atmosphere, 25e (300 mg, 0.231 mmol, 1.0 equiv), HBTU (97 mg, 0.254 mmol, 1.1 equiv) and HOBt·H2O (39 mg, 0.254 mmol, 1.1 equiv) were dissolved in anhydrous DMF (8.5 mL). DIPEA (80 mL, 0.460 mmol, 2 equiv) was added and the reaction mixture was stirred at room temperature for 15 min. 11-Azido-3,6,9-trioxaundecan-1-amine (100 μL, 0.463 mmol, 2 equiv) was added and the reaction was stirred at room temperature for 16 h. The reaction mixture was poured into water, the precipitate was filtered, washed with water and transferred to an RBF with acetone. The solvent was removed in vacuo and the crude residue was loaded onto a 30 g SNAP Ultra C18 column in acetone / water and purified by reverse phase flash chromatography (50 to 100% acetone:water gradient). The fractions containing the product were combined and the solvent was removed in vacuo to give a white solid which was further dried in vacuo to yield product 25f as a white solid (240 mg, 0.175 mmol, 76%). 1¹H NMR (400 MHz, methanol-d4) δ 8.23 (d, J = 2.0 Hz, 2H), 8.04 (d, J = 2.1 Hz, 1H), 8.01 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 1H), 7.79 (dd, J = 8.7, 2.0 Hz, 2H), 7.60 (dd, J = 8.6, 2.1 Hz, 1H), 4.49–4.40 (m, 12H), 4.35 (q, J = 7.1 Hz, 4H), 3.67–3.58 (m, 12H), 3.52 (t, J = 5.4 Hz, 2H), 2.91–2.71 (m, 12H), 1.39 (t, J = 7.1 Hz, 6H), 1.20 (t, J = 7.6 Hz, 18H).

[0516] Step 7: Preparation of Compound 25, G0 Macrocyclic PEG4 Azide

[0517]

[0518] 25f (0.22 g, 0.161 mmol, 1.0 equiv) was dissolved in ethanol (5 mL) and methanol (0.5 mL). 1M aqueous sodium hydroxide solution (5.5 mL) was added and the mixture was stirred at 40 °C for 3 h. The organic solvents were removed under vacuum to give an aqueous solution, which was then poured into 1M aqueous hydrochloric acid. The resulting suspension was centrifuged, the water was decanted, and the solid was washed with water. The solid was then centrifuged again, the water was decanted, and the solid was transferred to an RBF with acetone. The solvent was removed under vacuum and dried under high vacuum to give 25 as a white solid (190 mg, 0.111 mmol, 90%). 1 ¹H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 2H), 8.37 (d, J = 2.0 Hz, 2H), 8.31–8.23 (m, 2H), 8.21 (d, J = 2.1 Hz, 1H), 8.13 (s, 2H), 8.11 (d, J = 8.6 Hz, 2H), 8.01 (d, J = 8.7 Hz, 2H), 7.52 (dd, J = 8.6, 2.1 Hz, 2H), 7.43 (dd, J = 8.6, 2.1 Hz, 1H), 6.58–6.42 (m, 3H), 6.29 (d, J = 5.4 Hz, 3H), 4.32 (s,

[0519] 12H), 3.60–3.49 (m, 11H), 2.81–2.54 (m, 12H), 1.16 (dd, J = 9.2, 4.3 Hz, 18H). For C 65 H 83 N 16 O 14 + Calculated MS [M+H]+ Requirement: 1311.6, measured value: 1311.6.

[0520] Preparation of Insulin Derivative of the Present Invention

[0521] Example 1: Preparation of B1-Benzyl-4-Triazolyl-PEG4-G2 Macrocycle B29-PEG3-Glycoside desB30 Human Insulin INS1 Preparation

[0522]

[0523] The insulin derivative INS1 of Example 1 was prepared as described below.

[0524] Step 1: Preparation of B1-4-Ethynyl-Benzyl desB30 Human Insulin INS2 (Scheme 1)

[0525]

[0526] Scheme 1

[0527] DesB30 human insulin was dissolved in 0.1 M acetic acid, and the pH was adjusted to 4 using 8 M acetic acid. 4-Ethynyl-benzaldehyde (1.5 equivalents) in N-methyl-pyrrolidone (NMP) was added, and the mixture was stirred for 30 minutes. α-Methylpyridine borane (6 equivalents) in NMP was added, and the mixture was stirred for 2 hours while maintaining the pH near 4 by adding 8 M acetic acid. LCMS showed the formation of the desired product. The pH was adjusted to 2, and the product was purified by reverse-phase HPLC (RP-HPLC) on a C18 column, using an aqueous solution of 0.1% TFA as buffer A and an acetonitrile solution of 0.1% TFA as buffer B. The product (INS2 (Scheme 1)) was isolated by lyophilization.

[0528] Step 2: Preparation of INS3 (Scheme 2)

[0529]

[0530] Scheme 2

[0531] The macrocycle-PEG-azide 6d prepared as above was dissolved in N,N-dimethylformamide (DMF), and under an inert atmosphere (nitrogen), with CuI as a catalyst (0.1 equivalent) and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 2 equivalents) as a ligand, it was reacted with the alkyne insulin INS2 (chemical formula 1, 1 equivalent) dissolved in 2 M triethylamine adjusted to pH 7.8 using acetic acid. After this reaction, LCMS was performed, and the product INS3 (Scheme 2) was separated using RP-HPLC as described in Step 1 of Example 1.

[0532] Step 3: Preparation of Compound INS1

[0533]

[0534] Scheme 3

[0535] React the active ester 8 (1.2 equiv) with B1-macrocycle-insulin INS3 (Scheme 2) dissolved in 0.2 M sodium carbonate at pH 10.5. After this reaction, LCMS was performed and the B1-macrocycle B29-glycoside INS1 of Example 1 was separated using RP-HPLC as described in Step 1 of Example 1. LCMS measured value 1858.5 [M+5H]5+, calculated value 1858.6.

[0536] Example 2: Preparation of B1-benzyl-4-triazolyl-PEG4-G2 macrocycle A1-PEG3-glycoside desB30 human insulin INS4 Preparation

[0537]

[0538] React B1-macrocycle-insulin INS3 (Scheme 2, prepared as in Step 2 of Example 1) in 0.1 M sodium bicarbonate at pH 7.5 with the glucoside active ester 8. After this reaction, LCMS was performed and the B1-macrocycle A1-glucoside INS4 of Example 2 was separated using RP-HPLC as described in Step 1 of Example 1.

[0539] Example 3: Preparation of B29-PEG4-G2 macrocycle A1-PEG3-glycoside desB30 human insulin INS5

[0540]

[0541] The carboxyl macrocycle PEG 7d prepared as above was activated to the succinimide ester as described for glucoside 8 and reacted with desB30 human insulin at pH 10.5 as in Example 1. After this reaction, LCMS was performed and the B29-macrocycle derivative was separated using RP-HPLC as described above. As in Example 3, the B29-macrocycle derivative was reacted with the β-glycoside active ester 8 at pH 7.5. After this reaction, LCMS was performed and the crude product was deprotected by treatment with 95% TFA for 30 minutes. The B29-macrocycle A1-glucoside INS5 of Example 3 was separated using RP-HPLC as described in Step 1 of Example 1.

[0542] Example 4: Preparation of B1-benzyl-3,5-bis-triazolyl-PEG4-G2 macrocycle B29-benzoyl-3,5-methylamino- bis-PEG3-glycoside desB30 human insulin INS6

[0543]

[0544] Point of attachment of M on insulin INS6 = *

[0545]

[0546] Scheme 4, point of attachment of M on insulin INS8 = *

[0547] Similar to the preparation of compound INS2 (Scheme 4), B1-3,5-bis-ethynyl-benzyl desB30 human insulin was generated by reductive alkylation of 3,5-bis-ethynyl-benzaldehyde and desB30 human insulin. Under the conditions described for compound INS3, the bis-alkyne INS7 was reacted with the macrocyclic azide 6d. After this reaction, LCMS was performed and the intermediate INS8 was separated using RP-HPLC as described in Step 1 of Example 1. The active ester glucoside 8 was reacted with 3,5-bis-aminomethyl-benzoic acid. As described for glucoside 8, the purified product was activated to the succinimidyl ester and reacted with the insulin intermediate INS8 at pH 10.5 as described for INS3. After this reaction, LCMS was performed and the B1-bis-macrocycle B29-bis-glycoside desB30 human insulin INS6 of Example 4 was separated using RP-HPLC as described in Step 1 of Example 1.

[0548] Example 5: Preparation of B1-benzyl-4-triazolyl-PEG4-G2 macrocycle B29-acetyl ethylene glycol-D-glycoside desB30 human insulin INS9

[0549]

[0550] Step 1: Dissolve INS2 in 0.1 M Na2CO3 at pH 11 in an ice bath and treat with the active ester 11 in THF for 10 minutes. Similar to the description in Example 1, INS10 was separated by HPLC. LCMS measured value 1564.25 [M+4H] 4+ , calculated value 1564.24.

[0551]

[0552] Step 2: Dissolve INS10 in 0.2 M K2CO3 containing 33% MeOH and stir for 30 min. Similar to the description in Example 1, INS11 was separated by HPLC. LCMS measured value 1522.21 [M+4H] 4+ , calculated value 1522.21.

[0553]

[0554] Step 3: Dissolve INS11 (4.1 mg, 0.001 mmol) and macrocyclic azide 6d (3.579 mg, 0.001 mmol) in 2 M Et3N / CH3COOH aqueous buffer (0.6 mL) at pH 7 and DMSO (0.3 mL), further dilute with water (0.3 mL), and degas the mixture. Treat the mixture with THPTA (0.015 mg, 5 mol%) and a spatula tip of CuI, and let stand for 10 minutes. Similar to the description in Example 1, separate INS9 by HPLC, LCMS measured value 1846.8 [M+5H] 5+ , calculated value 1847.0.

[0555] Example 8: Preparation of B1-methyl-4-triazolyl-PEG4-G1 macrocycle B29-PEG4-glycoside desB30 human insulin INS18 Preparation

[0556]

[0557] Step 1: Dissolve DesB30 human insulin (1.71 g, 0.3 mmol) in 0.1 M AcOH / water (5 mL) + MeOH (5 mL). Add propargyl aldehyde (29 mg, 0.54 mmol), stir the mixture for 15 min, then treat with more propargyl aldehyde (15 mg) and stir for 30 min. Add α-methylpyridine borane (192 mg, 1.8 mmol) in DMF, and stir the mixture for 1 h. Similar to the description in Example 1, separate INS19 by HPLC. LCMS measured value 1436.8 [M+4H] 4+ , calculated value 1437.1.

[0558]

[0559] Step 2: Dissolve INS19 (150 mg, 0.026 mmol) in 0.1 M Na2HPO4 buffer at pH 11.5 (3 mL) and react with glucoside active ester 8 for 1 h. Similar to the description in Example 1, separate INS20 by HPLC. LCMS measured value 1517.4 [M+4H] 4+ , calculated value 1517.7.

[0560]

[0561] Step 3: Dissolve INS20 (11 mg, 1.8 μmol) and macrocyclic azide 6d (8.3 mg, 2.4 μmol) in 2 M triethanolamine pH 7.0 (0.4 mL) + DMSO (0.8 mL). Add THPTA (0.236 mg, 0.54 μmol), and carefully degas the mixture. Add CuI (0.003 mg, 0.018 μmol), and stir the mixture for 90 min. Separate INS18 by HPLC as described in Example 1. LCMS measured value 1843.5 [M+5H] 5+ , calculated value 1843.4.

[0562] Example 9: Preparation of B1-benzyl-4-triazolyl-PEG4-G2 macrocycle B29-acetyl diethylene glycol-glycoside desB30 human insulin INS21

[0563]

[0564] Prepare INS21 in a similar manner to INS9 using triethylene glycol and macrocyclic azide 6d for the preparation of glucosides. LCMS measured value 1855.7 [M+5H] 5+ , calculated value 1855.9.

[0565] Example 10: Preparation of B1-benzyl-3,5-bis-triazolyl-PEG4-G1 macrocycle B29-Nε-propionyl, Cα-aminomethyl- bis-triazolyl-PEG4-glycoside desB30 human insulin INS22

[0566]

[0567] Step 1: Dissolve DesB30 human insulin (120 mg, 0.21 mmol) and propargylamine (100 mg, 1.8 mmol) in a mixture of DMSO (1 mL), DMF (1 mL), ethanol (1 mL), and 0.1 M sodium phosphate pH 7 (1 mL) adjusted to pH 7.0 with TFA. Add a solution of achromobactor lyticus protease (40 μL, 6.3 mg / mL), and let the mixture stand overnight. Separate INS23 by HPLC as described in Example 1. LCMS measured value 1436.5 [M+4H] 4+ , calculated value 1436.9.

[0568]

[0569] Step 2: Dissolve INS23 (300 mg, 52 μmol) in 0.1 M Na2CO3 buffer (15 mL) at pH 11 and treat with O-succinimidyl pentynate (10.1 mg, 51 μmol) dissolved in DMSO (1 mL). Place the reaction for 1 h and separate INS24 by HPLC as described in Example 1. LCMS measured value 1456.6 [M+4H] 4+ , calculated value 1456.9.

[0570]

[0571] Step 3: React INS24 (97 mg, 17 μmol) with compound 12 (12.7 mg, 33 μmol) using THPTA and CuI as described for INS3. Separate INS25 by HPLC as described in Example 1. LCMS measured value 1647.4 [M+4H] 4+ , calculated value 1647.6.

[0572]

[0573] Step 4: Dissolve INS25 (85 mg, 13 μmol) in 50% acetonitrile / water (1.2 mL) and treat with 3,5-diethynylbenzaldehyde (3.1 mg, 21 μmol). Adjust the pH to 4 using 0.1 M Na2CO3. Add NaCNBH3 (4 mg, 65 μmol) and place the mixture overnight. Separate INS26 by HPLC similar to the description in Example 1. LCMS measured value 1345.7 [M+5H] 5+ , calculated value 1345.9.

[0574]

[0575] Step 5: Dissolve INS26 (24 mg, 4 μmol), compound 16 (20 mg, 11 μmol), THPTA (1 mg), and methyl-β-D-glucopyranoside (20 mg) in 2 M Et3N / AcOH (1 mL) + DMSO (1 mL) and degas. Treat with a spatula tip of CuI and place for 2 h. Separate INS22 by HPLC similar to the description in Example 1. LCMS measured value 1711.5 [M+6H] 6+ , calculated value 1711.7.

[0576] Example 11: Preparation of B1-benzyl-4-triazolyl-PEG4-G2 macrocycle B29-acetyl-glycoside desB30 human insulin INS27

[0577]

[0578] Using ethylene glycol and macrocyclic azide 6d for the preparation of glucosides, INS27 was prepared similarly to INS9. INS27 LCMS measurement 1838.2 [M+5H] 5+ , calculated value 1838.2.

[0579] Example 12: Preparation of B1-methyl-4-triazolyl-PEG3-G1 macrocycle B29-PEG3-glycoside desB30 human insulin INS28 Preparation

[0580]

[0581] INS20 (36.4 mg, 6 umol) and compound 20 (14.5 mg, 7.8 umol) were dissolved in DMSO (4 mL) + 2M triethylamine pH 7.0 (3 mL). THPTA (0.78 mg, 1.8 umol) was added and the flask was degassed. CuI (0.011 mg, 0.06 umol) was added and the mixture was left for 1 h. INS28 was separated by HPLC as described in Example 1. LCMS measurement 1559.2 [M+5H] 5+ , calculated value 1559.5.

[0582] Example 13: Preparation of B1-benzyl-3,5-bis-triazolyl-PEG4-G1 macrocycle B29-Cα-ethylenediamine-methyl triazolyl- PEG4-glycoside desB30 human insulin INS29

[0583]

[0584] As described for INS23, N,N-dipropynyl-ethylenediamine was conjugated to the Cα of B29 in desB30 human insulin by enzymatic coupling. As described for INS3, azide PEG4 glucoside 12 was conjugated to the alkyne doubly, and as described for INS26, B1 of the product was reductively alkylated with 3,5-diethynylbenzaldehyde. As described for the synthesis of INS22, the product was conjugated to G1 macrocyclic PEG4 azide (compound 16), and the product was purified by HPLC as described for INS1. LCMS measurement 1711.8 [M+6H] 6+ , calculated value 1711.9.

[0585] Example 14: Preparation of B1-methyl-4-triazolyl-PEG4-G1 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS30

[0586]

[0587] INS30 was prepared from INS20 and G1 macrocyclic PEG4 azide 16 as described for INS9, and purified by HPLC as described for INS1. LCMS measurement 1568.1

[0588] [M+5H] 5+ , the calculated value is 1568.3.

[0589] Example 15: Preparation of B1-methyl-4-triazolyl-propyl-G1 macrocycle B29Nε-acetyl-glycoside desB30 human insulin INS31

[0590]

[0591] As described in INS18, INS31 was prepared from 1-acetyl-glucoside and G1 macrocyclic propyl azide 21 and purified by HPLC as described for INS1. LCMS measured value 1524.2 [M+5H] 5+ , the calculated value is 1524.3.

[0592] Example 16: Preparation of B1-benzyl-3,5-triazolyl-PEG4-G1 macrocycle B29Nε-benzoyl-3,5-triazolyl- PEG4-glycoside desB30 human insulin INS32

[0593]

[0594] INS32 was prepared as follows: As described for INS23, desB30 human insulin in B29 was acylated with 3,5-diethynylbenzoic acid N-succinimidyl ester, then reacted with glucoside PEG4 azide 12 as described for INS3, and then reductively alkylated with 3,5-diethynylbenzaldehyde as described for INS26. As described for the synthesis of INS22, the product was conjugated with G1 macrocycle-PEG4-azide (Compound 16) and the product was purified by HPLC as described for INS1. LCMS measured value 1717.3 [M+6H] 6+ , the calculated value is 1717.6.

[0595] Example 17: Preparation of B1-benzyl-3,5-triazolyl-propyl-G1 macrocycle B29Nε-B29-benzoyl-3,5-triazolyl- PEG4-glycoside desB30 human insulin INS33

[0596]

[0597] As described for INS32, INS33 was prepared from desB30 human insulin and 3,5-diethynylbenzoic acid N-succinimidyl ester, subsequently reacted with glucoside PEG4 azide 12 as described for INS3, and then reductively alkylated with 3,5-diethynylbenzaldehyde as described for INS26. As described for the synthesis of INS32, the product was conjugated with G1 macrocycle-propyl azide (Compound 21) and the product was purified by HPLC as described for INS1. LCMS measured value 1677.9 [M+6H] 6+ , the calculated value is 1678.2.

[0598] Example 18: Preparation of B1-methyl-triazolyl-propyl-G1 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS34

[0599]

[0600] As described for INS9, INS34 was prepared from INS20 and G1 cyclopropyl azide 21. As described for INS1, the product was purified by HPLC. LCMS measured value 1930.7 [M+4H] 4+ , calculated value 1930.7.

[0601] Example 19: Preparation of B1-propionyl-triazolyl-PEG3-G2 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS35

[0602]

[0603] As described for INS20, A1-Boc desB30 human insulin (EP0132770) was reacted with glucoside PEG3 active ester 8. As described for INS1, the product was purified by HPLC, and B1 was acylated with pentynoic acid succinimidyl ester, and as described for INS1, the product was purified by HPLC. As described for INS9, the product was conjugated with G2-macrocyclic PEG3 azide 22. LCMS measured value 1843.1 [M+5H] 5+ , calculated value 1843.0.

[0604] Example 20: B1-propionyl-triazolyl-PEG5-G2 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS36

[0605]

[0606] As described for INS20, A1-Boc desB30 human insulin was reacted with glucoside PEG3 active ester 8. As described for INS1, the product was purified by HPLC, and B1 was acylated with pentynoic acid succinimidyl ester, and as described for INS1, the product was purified by HPLC. As described for INS9, the product was conjugated with G2-macrocyclic PEG5 azide 23. LCMS measured value 1860.73 [M+5H] 5+ , calculated value 1860.6.

[0607] Example 21: B1-methyl-triazolyl-PEG5-G1 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS37

[0608]

[0609] As described for INS9, INS37 was prepared from INS20 and G2 macrocyclic PEG5 azide 24. As described for INS1, the product was purified by HPLC. LCMS measured value 1971.3 [M+4H] 4+ , calculated value 1971.2.

[0610] Example 22: B1-methyl-triazolyl-PEG4-G0 macrocycle B29Nε-PEG3-glycoside desB30 human insulin INS38

[0611]

[0612] As described for INS9, INS38 was prepared from INS20 and G0 macrocyclic PEG4 azide 25. The product was purified by HPLC as described for INS1. LCMS measured value 1845.4 [M+4H] 4+ , calculated value 1845.6.

[0613] Example 23: B1-methyl-triazolyl-PEG4-G1 macrocycle B29Nε-acetyl-glycoside desB30 human insulin INS39

[0614]

[0615] INS39 was prepared using 1-acetyl-glycoside and G1 macrocyclic PEG4 azide 16 as described for INS18. The product was purified by HPLC as described for INS1. LCMS measured value 1934.4 [M+4H] 4+ , calculated value 1934.7.

[0616] Example 24: B1-methyl-triazolyl-PEG3-G1 macrocycle B29Nε-acetyl-glycoside desB30 human insulin INS40

[0617]

[0618] INS40 was prepared using 1-acetyl-glycoside and G1 macrocyclic PEG3 azide 20 as described for INS18. The product was purified by HPLC as described for INS1. LCMS measured value 1923.6 [M+4H] 4+ , calculated value 1923.6.

[0619] Example 25: B29-propionyl-triazolyl-propyl-G1 macrocycle A1-acetyl-glycoside desB30 human insulin INS41

[0620]

[0621] DesB30 human insulin was reacted with 4-pentynoic acid N-succinimidyl ester (pH 10.5) at B29 and with N-succinimidyl O-peracetyl-1-acetyl-glucoside (pH 8.5) at A1, and the product was purified by HPLC as described for INS1. The O-acetyl groups were removed by saponification using 1:1 MeOH + 100 mM aqueous sodium carbonate. The product was reacted with G1 macrocyclic propyl azide 21 as described for INS31. The product was purified by HPLC as described for INS1. LCMS measured value 1532.7 [M+5H] 5+ , calculated value 1532.7.

[0622] Glucose-Sensitive Insulin Receptor Activation

[0623] Example 26: Determination of the glucose affinity of the macrocycle by fluorescence titration

[0624] The optical properties (fluorescence) of a given macrocycle vary between the unbound state and the glucose-bound state. Thus, titrating the macrocycle with glucose within the relevant glucose concentration range and measuring the fluorescence change of the system can give a binding curve for glucose binding, from which the glucose binding constant Ka (and the displacement constant Kd) can be determined.

[0625] All solutions were maintained at pH 7.4 using 10 mM phosphate buffer. Titrations were performed using a Horiba Scientific Duetta fluorescence spectrometer with an excitation wavelength of 310 nm, and emission was monitored from 320 - 550 nm. The excitation and emission slit widths / band widths were set to 5 nm. All points were acquired using an integration time of 0.5 s and 5 accumulations.

[0626] Solution 1 - Stock solution of compound 6d:

[0627] 2 mg of compound 6d (Mw = 3540 g / mol) was dissolved in 5.6 mL of ultrapure H2O (pH 7.4, 10 mM PB). The purity of compound 6d was pre-determined by 1H NMR spectroscopy using a known concentration of DMF in D2O as an internal standard. The purity was calibrated to 80% (40 μM). It was then diluted four-fold to obtain a 10 μM solution of compound 6d.

[0628] Solution 2 - Stock solution of D-glucose and compound 6d:

[0629] D-glucose (180.16 mg) was dissolved in 1 mL of 10 mM PB and left overnight to equilibrate the α and β anomers. It was then diluted to obtain a 20 mM stock solution of D-glucose. 250 μL of this solution was combined with 250 μL of the compound 6d stock solution to give 10 mM D-glucose / 5 μM compound 6d.

[0630] In a 3 mL cuvette, 1500 μL of solution 1 was diluted with 1500 μL of 10 mM phosphate buffer to obtain a 5 μM solution. It was titrated with solution 2. For each addition of the titrant solution (solution 2), an equal volume was removed from the cuvette before the addition to keep the cell volume constant (3000 μL). Each addition was stirred for 1 minute and then stirring was stopped to allow the sample to equilibrate for 1 minute before acquisition. Additions (in μL): 0, 10, 10, 10, 10, 10, 20, 20, 50, 100, 100.

[0631] Data was in Figure 2 andFigure 3 is shown in

[0632] Calculate Ka = 14751.48 ± 637.3 M-1; Kd for compound 6d = 1 / 14751 M-1 = 68 uM.

[0633] Measure the glucose affinity similarly

[0634] For compound 21, Kd = 89 uM

[0635] For compound 22, Kd = 132 uM

[0636] For compound 23, Kd = 78 uM

[0637] For compound 24, Kd = 131 uM

[0638] The macrocycles bind glucose with an affinity of around 100 uM (Kd), regardless of which linker they have.

[0639] Example 27: Determination of the affinity for the human insulin receptor (hIR-A) in the absence or presence of glucose Measurement

[0640] To measure the glucose sensitivity of the insulin derivatives, the affinity of the insulin derivatives for the human insulin receptor was measured in the absence of glucose or in the presence of 20 mM glucose.

[0641] Insulin receptor preparation

[0642] BHK cells overexpressing human insulin receptor A (hIR-A) were lysed in 50 mM Hepes pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA and 10% glycerol. The clarified cell lysate was batch-absorbed with wheat germ agglutinin (WGA)-agarose (lectin from Triticum vulgaris - Agarose, L1394, Sigma - Aldrich Steinheim, Germany) for 90 minutes. The receptor was washed with 20 volumes of 50 mM Hepes pH 8.0, 150 mM NaCl and 0.1% Triton X-100, and then eluted with 50 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5 M N-acetylglucosamine and 10% glycerol. All buffers contained Complete (Roche Diagnostic GmbH, Mannheim, Germany).

[0643] Scintillation proximity assay (SPA) binding assay

[0644] Dilute SPA PVT anti-mouse beads (Perkin Elmer) in SPA binding buffer consisting of 100 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MgSO4, 0.025% (v / v) Tween-20. Incubate the SPA beads with the IR-specific antibody 83-7 and solubilized semi-purified hIR-A. Adjust the receptor concentration to achieve 5000 cpm 125 10% binding of I-(Tyr31)-insulin (Novo Nordisk A / S). Add a dilution series of cold ligand to a 96-well Optiplate, followed by the tracer ([[]] 125 I-insulin, 5000 cpm / well), and finally the receptor / SPA mixture. To test glucose sensitivity, establish the binding assay in the absence or presence of 20 mM glucose. Shake the plate gently at 22 °C for 22.5 h, centrifuge for 5 min at 1000 rpm, and count in a TopCounter (Perkin Elmer). Calculate the relative affinity of the analog compared to human insulin, and the increase in relative affinity from 0 to 20 mM glucose reflects the glucose sensitivity (glucose factor) of the analog. Experiments are conducted in the presence of 1.5% human serum albumin.

[0645] The data are shown in Table 1.

[0646] The results show that, compared to in the absence of glucose, in the presence of 20 mM glucose, the insulin derivatives of the present invention (insulins having a glucose-sensitive switch consisting of a glucose conjugate macrocycle at one position plus a glucoside at another position) have a higher insulin receptor affinity. INS27, INS31, INS39, and INS40 show the weakest glucose sensitivity, indicating that the length of the linker affects the glucose sensitivity of the insulin conjugate.

[0647] Table 1. Relative insulin receptor binding affinities in the absence and presence of glucose (20 mM)

[0648]

[0649] Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. Accordingly, it is to be understood that all such modifications and changes as fall within the true scope of the invention are intended to be covered by the appended claims.

Claims

1. An insulin derivative comprising human insulin or a human insulin analogue, a glucose mimetic, and a macrocycle M of formula M1: wherein R1 and R2 are independently selected from –OH, wherein R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen, halogen, C 1-4 alkyl and C 1-4 alkoxy; and wherein R3 represents the point of attachment of the macrocycle of formula M1; wherein the glucose mimetic is a glucoside; wherein the macrocycle is attached to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue; and wherein the glucose mimetic is attached to the ε-amino group of lysine at position 29 of the B-chain of the human insulin or human insulin analogue; wherein the macrocycle M is attached to the human insulin or human insulin analogue via a linker, wherein the linker is a linker of formula L1 wherein X is CH2- or (CH2CH2O-) p , wherein p is an integer from 2 to 4; wherein Y is CH2-, (CH2CH2CO-), or -Ph-p-CH2-; wherein * represents the point of attachment to the macrocycle M; and wherein # represents the point of attachment to the human insulin or human insulin analogue; wherein the glucose mimetic is attached to the human insulin or human insulin analogue via a linker, wherein the linker is a linker of formula L2: "-(CH2CH2O-) q -(CH2) r -C(O)-#; wherein q is 1 or 2; wherein r is 1 or 2; wherein " represents the point of attachment to the glucose mimetic; and wherein # represents the point of attachment to the human insulin or human insulin analogue.

2. The insulin derivative according to claim 1, wherein the glucose mimetic is β-D-glucopyranoside.

3. The insulin derivative according to any one of claims 1-2, wherein R1 and R2 are -OH.

4. The insulin derivative according to any one of claims 1-2, wherein R1 and R2 are 5. The insulin derivative according to any one of claims 1-2, wherein R1 and R2 are 6. The insulin derivative according to any one of claims 1-2, wherein R4, R5, R6, R7, R8, and R9 are ethyl.

7. The insulin derivative according to any one of claims 1-2, wherein the human insulin or human insulin analogue is the human insulin analogue desB30 human insulin.

8. An insulin derivative comprising human insulin or a human insulin analogue, a glucose mimetic, and a macrocycle M of formula M1: wherein R1 and R2 are independently selected from –OH and wherein R4, R5, R6, R7, R8 and R9 are independently selected from hydrogen, halogen, C 1-4 alkyl and C 1-4 alkoxy; and wherein R3 represents the point of attachment of the macrocycle of formula M1; wherein the insulin derivative comprises two glucose mimetics and two macrocycles M of formula M1; wherein the glucose mimetic is a glucoside; wherein the two macrocycles M are attached to the α-amino group at position 1 of the B-chain of human insulin or a human insulin analogue via a trivalent linker, wherein the linker is a linker of formula L3 wherein Z is CH2- or (CH2CH2O-)3; wherein W is CH2-, (CH2CH2CO-), or -Ph-p-CH2-; wherein * represents the point of attachment to the macrocycle M; and wherein # represents the point of attachment to the human insulin or human insulin analogue; and wherein the two glucose mimetics are attached to the α-carboxylic acid group or ε-amino group of lysine at position 29 of the B-chain of the human insulin or human insulin analogue via a trivalent linker; wherein the linker is selected from a) and b) or wherein the two glucose mimetics are each attached to the α-carboxylic acid group and ε-amino group of lysine at position 29 of the B-chain of the human insulin or human insulin analogue via a divalent linker respectively; wherein the linker is a linker of formula L6 and formula L7: where " represents the point of attachment to the glucose mimetic; and wherein# 1 represents the point of attachment to the α-carboxyl group of the lysine at position 29 of the B-chain of said human insulin or human insulin analogue; and wherein# 2 represents the point of attachment to the ε-amino group of the lysine at position 29 of the B-chain of said human insulin or human insulin analogue.

9. The insulin derivative according to claim 8, wherein the glucose mimetic is β-D-glucopyranoside.

10. The insulin derivative according to any one of claims 8-9, wherein R1 and R2 are -OH.

11. The insulin derivative according to any one of claims 8-9, wherein the R1 and R2 are 12. The insulin derivative according to any one of claims 8-9, wherein R4, R5, R6, R7, R8 and R9 are ethyl.

13. The insulin derivative according to any one of claims 8-9, wherein the human insulin or human insulin analogue is the human insulin analogue desB30 human insulin.

14. An insulin derivative, wherein the insulin derivative is selected from 15. A pharmaceutical composition for treating and / or preventing diabetes in a patient in need thereof, comprising a therapeutically effective amount of an insulin derivative according to any one of claims 1-14, and a pharmaceutically acceptable excipient.

16. The insulin derivative according to any one of claims 1-14, which is used as a drug.

17. Use of an insulin derivative according to any one of claims 1-14 for the preparation of a drug for treating and / or preventing diabetes and hyperglycemia.

18. The use according to claim 17, wherein the diabetes includes type 1 diabetes and type 2 diabetes.

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