WO2023144240A1

WO2023144240A1 - Glucose sensitive insulin derivatives and uses thereof

Info

Publication number: WO2023144240A1
Application number: PCT/EP2023/051865
Authority: WO
Inventors: Thomas Hoeg-Jensen; Thomas Kruse; Alice Ravn MADSEN; Joakim Holck ANDERSEN; Tina Møller TAGMOSE; Kim Søholm HALSKOV; Per Sauerberg; Lennart LYKKE; Rita Slaaby; Anthony Peter DAVIS; Andrew Michael Chapman; Michael Roger TOMSETT
Original assignee: Novo Nordisk Research Centre Oxford Limited
Priority date: 2022-01-26
Filing date: 2023-01-26
Publication date: 2023-08-03

Abstract

The present invention relates to novel insulin derivatives and their use in the treatment or prevention of medical conditions relating to diabetes. The insulin derivatives are glucose sensitive and display glucose-sensitive insulin receptor binding. The invention also relates to novel intermediates. Finally, the invention provides a pharmaceutical composition comprising the insulin derivatives of the invention and the use of such a composition in the treatment or prevention of medical conditions relating to diabetes.

Description

TITLE: GLUCOSE SENSITIVE INSULIN DERIVATIVES AND USES THEREOF

TECHNICAL FIELD

The present invention relates to novel insulin derivatives, and their pharmaceutical use. Furthermore, the invention relates to pharmaceutical compositions comprising such insulin derivatives, and to the use of such compounds for the treatment or prevention of medical conditions relating to diabetes.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference.

BACKGROUND

Glucose is the most important component in human energy homeostasis, and uncontrolled blood glucose is the hallmark of diabetes. The major objective of diabetes treatment is to adjust blood glucose levels towards normal values, and insulin is the most effective drug for this purpose. Glucose adjustment using insulin is however a difficult balance between hyperglycaemia and hypoglycaemia. Even with modern blood glucose monitors, hypoglycaemia is commonly occurring, and various glucose-sensitive insulin delivery systems have been engineered in attempts to improve the situation, both in form of mechanical systems (pumps/sensors) or molecular delivery systems. It would therefore be advantageous to equip diabetes-related peptide and protein drugs with a glucose-regulated bioactivity, e.g. a weaker glucose-lowering activity of insulin at low blood glucose values.

Glucose-sensitive insulin bioactivity can be achieved by equipping insulin with a glucose-binding element plus a binding partner that binds the glucose-binding element in competition with blood glucose, and thus controls insulin folding in equilibria between active and inactive states (WO2016149222, WQ2010107520, WQ2020058322). When the glucose binding element on the insulin derivative binds the binding partner on the same insulin, the insulin attains an inactive or weakly active conformation. As glucose levels increase, the binding partner on the insulin derivative is displaced from the glucose binder, and the conformation of insulin changes to an active state. SUMMARY

The present invention provides glucose sensitive insulin derivatives. The insulin derivatives of the present invention comprise a macrocycle of Formula M, a glycoside and an insulin peptide.

The macrocycle is of Formula M:

wherein k is 0 or 1.

The macrocycle is attached to position 29 of the B-chain of the insulin peptide via a linker L1. The macrocycle is attached to the linker L1 via the attachment point marked with *1. The glycoside is attached to position 1 of the B-chain of the insulin peptide via a linker L2.

Without being bound by theory, it is believed that when the macrocycle on the insulin derivative binds the glycoside on the same insulin, the insulin attains an inactive or weakly active conformation. As glucose levels increase, the glycoside on the insulin derivative is displaced from the macrocycle, and the conformation of insulin changes to an active state. Figure 1 illustrates the principle in schematic form (the positions of substitution on insulin in Figure 1 should not be taken literally).

It is thus believed that the overall three-dimensional structure of the insulin derivative is essential for the glucose sensitivity of the insulin derivative. The correct length and orientation of the linker in combination with the points of attachment to the insulin peptide are thus needed to ensure that the glycoside is able to bind to the macrocycle and at the same time hinder or diminish the ability of the insulin derivative to bind to and activate the insulin receptor. In one aspect, the invention relates to the furnishing of insulin derivatives which, after administration, activate the insulin receptor as a function of the blood glucose concentration.

In one aspect, the invention relates to the furnishing of insulin derivatives having low or no activity/availability during situations of low blood glucose levels, for example at levels below about 3 mM glucose.

In one aspect, the invention relates to the furnishing of insulin derivatives having high activity/availability in response to high blood glucose levels, for example, above about 10 mM glucose.

The glucose sensitivity of the insulin derivatives can be measured by the increase in relative affinity for the insulin receptor from 0 to 20 mM glucose. The term ‘glucose factor’ used herein is the relative insulin receptor affinity for a given insulin derivative measured in the presence of 20 mM glucose divided by the relative insulin receptor affinity for the same insulin derivative measured in the presence of 0 mM glucose. The term relative affinity for the insulin receptor as used herein generally refers to the affinity for the insulin receptor relative to human insulin.

In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of above 1, when measured without the presence of human serum albumin (HSA). In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of at least 2, when measured without the presence of HSA.

To further mimic physiological conditions, the insulin receptor affinities were also measured in the presence of 1.5% human serum albumin (HSA).

In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of above 1 , when measured in the presence of 1.5% human serum albumin (HSA). In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of at least 9, when measured in the presence of 1.5% human serum albumin (HSA).

Conjugation of a macrocycle that comprises several carboxylic acids can be problematic for making homogeneous insulin conjugates in good yields. If there are several carboxylic acids on the macrocycle, then functionalization of carboxylate will give a mixture of mono- and multi-functionalized compounds, and such mixtures can be hard to separate at preparative scale in good yields. Functionalization and conjugation to insulin not using the carboxylic acids of the macrocycle is thus advantageous. The insulin derivatives of the present invention have been made by conjugation of the macrocycle to insulin via a handle built into the macrocycle comprising an azide functional group, enabling selective conjugation of the macrocycle resulting in a mono-functionalized product.

In one aspect, the invention provides an intermediate product in the form of novel macrocycle intermediates of Formula IM1 :

wherein k1 is 0 or 1, wherein W1 is (CH2)ni or (OCH2CH2)_PI , wherein n1 is an integer from 2 to 5, wherein p1 is an integer from 1 to 5; and wherein Y1 is absent or NH-C(O)-CH2.

In one aspect, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention. In another aspect, the invention relates to an insulin derivative according to the invention for use as a medicament. In another aspect, the invention relates to an insulin derivative according to the invention for use in the treatment of diabetes. In another aspect, the invention relates to medical use(s) of the insulin derivatives according to the invention.

The invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a schematic illustration of glucose sensitive insulin derivatives (Gm=glucose mimetic, G=glucose). Glucose-sensitive insulin bioactivity can be achieved by equipping insulin with a glucose-binding element plus a binding partner (a glucose mimetic, such as a glycoside) that binds the glucose-binding element in competition with blood glucose, and thus controls insulin folding in equilibria between active and inactive states. Fig. 2 shows representative glucose profiles with INS2 (Example 2) (▲ with solid line) versus insulin degludec (■ with dotted line) after stopping and restarting the glucose infusion in the hypoglycaemic study in LYD pigs (Example 34). Dosing of INS2 (Example 2) was 1.86 pmol/kg/min (n =7), insulin degludec was 0.9 pmol/kg/min (n = 8).

DESCRIPTION

The present invention relates to insulin derivatives. In one aspect, the invention relates to glucose sensitive insulin derivatives which binds to and activates the insulin receptor in a glucose dependent fashion.

In one aspect, the invention relates to a compound comprising human insulin or an analogue thereof, a glucose binder and a glucose mimetic. In one aspect, the glucose binder is a macrocycle of Formula M. In one aspect, the glucose mimetic is a glycoside. In one aspect, the invention relates to a compound comprising human insulin or an analogue thereof, a macrocycle, and a glycoside.

General definitions

The term “compound” is used herein to refer to a molecular entity, and “compounds” may thus have different structural elements besides the minimum element defined for each compound or group of compounds. It follows that a compound may be a fusion compound/peptide or a derivative thereof, as long as the compound comprises the defined structural and/or functional elements. The term “compound” is also meant to cover pharmaceutically relevant forms hereof, i.e. the invention relates to a compound as defined herein or a pharmaceutically acceptable salt thereof.

The term “peptide” or “polypeptide”, as e.g. used in the context of the invention, refers to a compound which comprises a series of amino acids interconnected by amide (or peptide) bonds. In a particular embodiment the peptide consists of amino acids interconnected by peptide bonds.

The term “analogue” generally refers to a peptide, the sequence of which has one or more amino acid changes when compared to a reference amino acid sequence. Analogues “comprising” certain specified changes may comprise further changes, when compared to their reference sequence. In particular embodiments, an analogue "has" or “comprises” specified changes. In other particular embodiments, an analogue “consists of’ the changes. When the term “consists” or “consisting” is used in relation to an analogue e.g. an analogue consists or consisting of a group of specified 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 group of specified amino acid mutations may have additional mutations. An “analogue” may also include amino acid elongations in the N-terminal and/or C-terminal positions and/or truncations in the N-terminal and/or C- terminal positions. In general, amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code. These three ways are fully equivalent.

The term “derivative” generally refers to a compound which may be prepared from a native peptide or an analogue thereof by chemical modification, in particular by covalent attachment of one or more substituents.

The term "amino acid" includes proteinogenic (or natural) amino acids (amongst those the 20 standard amino acids), as well as non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those which are naturally incorporated into proteins. The standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids are either not found in proteins, or not produced by standard cellular machinery (e.g., they may have been subject to post-translational modification).

In what follows, each amino acid of the peptides of the invention for which the optical isomer is not stated is to be understood to mean the L-isomer (unless otherwise specified). Amino acids are molecules containing an amino group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain. Herein, the term “amino acid residue” is an amino acid from which, formally, a hydroxy group has been removed from a carboxy group and/or from which, formally, a hydrogen atom has been removed from an amino group.

The term “non-basic nitrogen atoms” generally refers to nitrogen atoms, wherein the protonated form of the nitrogen atom has a pKa in the range of 5 to 14. Nitrogen atoms in amides and N-alkyl-triazoles are non-basic.

It is to be understood that certain macrocyclic compounds of the invention may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that are capable of saccharide recognition. It is also to be understood that certain compounds may exhibit polymorphism, and that the invention encompasses all such forms that are capable of saccharide recognition. The macrocyclic compounds of the invention may exist in a number of different tautomeric forms and references to compounds of specific formulas include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are nevertheless embraced by a given formula. Human insulin

The term “human insulin” as used herein means the human insulin hormone whose structure and properties are well-known. Human insulin has two polypeptide chains, named the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by disulphide bridges: a first bridge between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B- chain, and a second bridge between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain. A third bridge is present between the cysteines in position 6 and 11 of the A-chain.

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).

“An insulin” according to the invention is herein to be understood as human insulin or an insulin from another species such as porcine or bovine insulin.

The term “insulin peptide” as used herein means a peptide which is either human insulin or an analogue or a derivative thereof with insulin activity.

The term “parent insulin” as used herein is intended to mean an insulin before any modifications according to the invention have been applied thereto.

Insulin analogues

The term “insulin analogue” and ‘human insulin analogue’ as used herein means a modified human insulin wherein one or more amino acid residues of the insulin have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the insulin and/or wherein one or more amino acid residues have been added and/or inserted to the insulin.

In one embodiment an insulin analogue comprises less than 10 amino acid modifications (substitutions, deletions, additions (including insertions) and any combination thereof) relative to human insulin, alternatively less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification relative to human insulin.

Modifications in the insulin molecule are denoted stating the chain (A or B), the position, and the one or three letter code for the amino acid residue substituting the native amino acid residue.

By “desB30” or “B(1-29)” is meant a natural insulin B chain or an analogue thereof lacking the B30 amino acid and “A(1-21)” means the natural insulin A chain. Thus, e.g., desB30 human insulin is an analogue of human insulin where the amino acid in position 30 in the B chain is deleted. Likewise, B3E desB30 human insulin is an analogue of human insulin where the amino acid in position 3 in the B chain is substituted with glutamic acid, and the amino acid in position 30 in the B chain is deleted.

Herein terms like “A1”, “A2” and “A3” etc. indicates the amino acid in position 1, 2 and 3 etc., respectively, in the A chain of insulin (counted from the N-terminal end). Similarly, terms like B1 , B2 and B3 etc. indicates the amino acid in position 1 , 2 and 3 etc., respectively, in the B chain of insulin (counted from the N-terminal end). Using the one letter codes for amino acids, terms like A21A, A21G and A21Q designates that the amino acid in the A21 position is A, G and Q, respectively. Using the three letter codes for amino acids, the corresponding expressions are A21Ala, A21Gly and A21Gln, respectively.

Herein, the term “amino acid residue” is an amino acid from which, formally, a hydroxy group has been removed from a carboxy group and/or from which, formally, a hydrogen atom has been removed from an amino group.

In one aspect, an insulin analogue according to the invention is an insulin analogue which binds to the human insulin receptor. In one aspect, an insulin analogue according to the invention is an insulin analogue which binds to and activates the human insulin receptor.

Non-limiting examples of insulin analogues are such wherein Pro in position 28 of the B chain is substituted with Asp, Lys, Leu, Vai, or Ala and/or Lys at position B29 is substituted with Pro, Glu or Asp. Furthermore, Asn at position B3 may be substituted with Thr, Lys, Gin, Glu or Asp. The amino acid residue in position A21 may be substituted with Gly. Also one or more amino acids may be added to the C-terminal of the A-chain and/or B- chain such as , e.g., Lys. The amino acid in position B1 may be substituted with Glu. The amino acid in position B16 may be substituted with Glu or His. Further examples of insulin analogues are the deletion analogues, e.g., analogues where the B30 amino acid in human insulin has been deleted (des(B30) human insulin), insulin analogues wherein the B1 amino acid in human insulin has been deleted (des(B1) human insulin), des(B28-B30) human insulin and des(B27) human insulin. Insulin analogues wherein the A-chain and/or the B- chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B- chain have a C-terminal extension such as with two arginine residues added to the C- terminal of the B-chain are also examples of insulin analogues. Further examples are insulin analogues comprising combinations of the mentioned mutations. Insulin analogues wherein the amino acid in position A14 is Asn, Gin, Glu, Arg, Asp, Gly or His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations are further examples of insulin analogues. Insulin analogues of human insulin wherein the amino acid residue in position A21 is Gly and wherein the insulin analogue is further extended in the C-terminal with two arginine residues are also examples of insulin analogues.

Further non-limiting examples of insulin analogues include: desB30 human insulin; B28D human insulin; B28D desB30 human insulin; B3K B29E human insulin; B28K B29P human insulin; A21G B31 R B32R human insulin; A14E B25H human insulin; A14H B25H human insulin; A14E B25H desB30 human insulin; A14H B25H desB30 human insulin; A14E B25H desB27 desB28 desB29 desB30 human insulin; A14E B25H B27E desB30 human insulin; A14E B16H B25H desB30 human insulin; A14H B16H B25H desB30 human insulin; A8H A14E B25H B27E desB30 human insulin; A8H A14E B1 E B16E B25H B27E desB30 human insulin; and A8H A14E B16E B25H desB30 human insulin.

In one aspect, the insulin analogue comprises desB30 human insulin. In one aspect, the insulin analogue comprises B3E desB30 human insulin. In one aspect, the insulin analogue of the present invention is desB30 human insulin (A-chain of SEQ ID NO:1 and B- chain of SEQ ID NO:3). In one aspect, the insulin analogue of the present invention is B3E desB30 human insulin (A-chain of SEQ ID NO:1 and B-chain of SEQ ID NO:4).

Insulin peptides

The term “insulin peptide” as used herein means human insulin or a human insulin analogue, as defined above.

Insulin derivatives

The term “insulin derivative” as used herein means an insulin peptide (human insulin or an analogue thereof) to which a glycoside and a macrocycle are attached. In other words, an insulin derivative of the present invention comprises an insulin peptide, a macrocycle and a glycoside. In one aspect, the macrocycle and the glycoside, respectively, are each attached via a linker to the insulin peptide.

In one aspect, the macrocycle is attached via a linker to the B29 position of the insulin peptide. In one aspect, the macrocycle is attached via a linker to the epsilon amine or the alpha carboxylic acid of the lysine in the B29 position of insulin peptide.

In one aspect, the glycoside is attached via a linker to the B1 position of the human insulin or human insulin analogue. In one aspect, the glycoside is attached via a linker to the alpha amino acid of the amino acid residue in the B1 position of the human insulin or human insulin analogue. Macrocycle

In one aspect, the macrocycle of the invention is of Formula M:

wherein k is 0 or 1 , and wherein *¹ denotes the attachment point to the linker L1 .

Conjugation of the macrocycle via the linker L1 to insulin as illustrated in formula M is advantageous for the synthesis of building blocks. When there are several carboxylic acids on the macrocycle, functionalization of carboxylate will give a mixture of mono- and multifunctionalized compounds, and such mixtures can be hard to separate at preparative scale in good yields. It is thus advantageous to use macrocycles that are functionalized and conjugated to insulin as illustrated in formula M, where the carboxylic acids of macrocycle M are not used as conjugation handles. Instead, the macrocycle is conjugated to insulin via the conjugation handle *1 of formula M.

Linker L 1

In one aspect, the linker L1 connects the macrocycle of Formula M to the B29 position of the insulin peptide.

In one aspect, the linker L1 is a moiety having from 8 to 20 non-hydrogen atoms, wherein 3 to 10 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms. In another aspect, the linker L1 has from 10 to 18 non-hydrogen atoms, wherein 4 to 7 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms. In one aspect, the non-hydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms. In one aspect, the linker L1 comprises a triazol or a phenyl group. In one aspect, the linker L1 comprises a triazol. In one aspect, the linker L1 is of Formula L1a:

wherein m is an integer from 0 to 6;

W is (CH₂)_n or (OCH₂CH₂)_P, wherein n is an integer from 2 to 5, and p is an integer from 1 to 5;

Y is absent or NH-C(O)-CH₂;

X is C(O), O-C(O), or NH; wherein when X is C(O) or O-C(O), the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide, and when X is NH, the linker L1 is attached to the alpha carboxylic acid of the lysine in the B29 position of the insulin peptide; and wherein *² denotes the attachment point to the macrocycle and *³ denotes the attachment point to the insulin peptide.

Glycoside

A glycoside is a molecule in which a sugar is bound to another functional group via a glycosidic bond. The glycosidic bond can be an O-, S-, N- or C-glycosidic bond. In one aspect, the glycosidic bond is an O-, or N-glycosidic bond.

In one aspect, the glycoside of the invention is selected from the group consisting of

Formula G1:

Formula G2:

Formula G3:

; and

Formula G4:

wherein Z is O or NH; wherein the glycoside is the D-isomer as shown or the corresponding L-isomer; wherein the glycoside is attached to the alpha amino group of the amino acid in the B1 position of the insulin peptide via a linker L2; and wherein *⁴ denotes the attachment point to the linker L2.

In one aspect, the glycoside is the D-isomer. In one aspect, the glycoside is of Formula G1. In one aspect, the glycoside is of Formula G2. In one aspect, the glycoside is of Formula G3. In one aspect, the glycoside is of Formula G4.

Linker L2

In one aspect, the linker L2 connects the glycoside to the B1 position of the insulin peptide.

In one aspect, the linker L2 is a moiety having from 2 to 15 non-hydrogen atoms, wherein 0 to 6 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms. In one aspect, the linker L2 is selected from the group consisting of:

Formula L2a: *⁵-R-CH₂-C(O)-*⁶, wherein R is absent, C(O)-CH₂O, C(O)-CH₂OCH₂CH₂O, CH₂-C(O)-NH, or (CH₂CH₂O)_q, wherein q is 1 or 2;

Formula L2b:

Formula L2c:

wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide.

of the invention

In one aspect, the compounds of the invention are insulin derivatives comprising an insulin peptide, a macrocycle, a linker L1 , a glycoside, and a linker L2, each as defined above.

Non-limiting examples of the insulin derivatives of the invention are the compounds INS1-INS31 of examples 1-31. The compounds of the invention are glucose sensitive insulin derivatives. In one aspect, the invention relates to the furnishing of insulin derivatives which, after administration, activate the insulin receptor as a function of the blood glucose concentration.

It is believed that the overall three-dimensional structure of the insulin derivative is essential for the glucose sensitivity of the insulin derivative. The correct length and orientation of the linker in combination with the points of attachment to the insulin peptide are thus needed to ensure that the glycoside is able to bind to the macrocycle and at the same time hinder or diminish the ability of the insulin derivative to bind to and activate the insulin receptor.

The relative binding affinity of insulin analogues for the human insulin receptor (HIR or hIR) can be determined by competition binding in a scintillation proximity assay (SPA) as described in Example 32.

The glucose sensitivity of the insulin derivatives can be measured by the increase in relative affinity for the insulin receptor from 0 to 20 mM glucose. The term ‘glucose factor’ used herein is the relative insulin receptor affinity for a given insulin derivative measured in the presence of 20 mM glucose divided by the relative insulin receptor affinity for the same insulin derivative measured in the presence of 0 mM glucose. The relative insulin receptor affinity for a given compound is the insulin receptor affinity for the given compound relative to that of human insulin.

In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of above 1, when measured without the presence of human serum albumin (HSA).

To further mimic physiological conditions, the insulin receptor affinities were also measured in the presence of 1.5% human serum albumin (HSA). In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of above 1 , when measured in the presence of 1.5% human serum albumin (HSA). In one aspect, the invention relates to the furnishing of insulin derivatives having a glucose factor of at least 9, when measured in the presence of 1.5% human serum albumin (HSA).

The lipogenesis assay described in Example 33 can be used as a measure of the functional (agonistic) activity of an insulin analogue.

The hypoglycaemic study in LYD pigs described in Example 34 shows that the compounds of the invention attenuates hypoglycemia. Intermediate products

The invention furthermore provides an intermediate product in the form of a novel macrocycle intermediate of Formula IM1 :

wherein k1 is 0 or 1 ,

W1 is (CH2)ni or (OCH2CH2)pi, wherein n1 is an integer from 2 to 5, and p1 is an integer from 1 to 5; and

Y1 is absent or NH-C(O)-CH2.

Non-limiting examples of intermediates of Formula IM1 includes Intermediate compound 3 (‘macrocycle propyl azide’), Intermediate compound 9 (‘macrocycle EG3 azide’), Intermediate compound 10 (‘macrocycle EG2 azide’), Intermediate compound 12 (‘macrocycle propyl-NHAc azide’), and Intermediate compound 17 (‘homo-macrocycle propyl azide’).

Production of human insulin and human insulin analogues

The production of polypeptides, e.g., insulins, is well known in the art. The insulin peptide may for instance be produced 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. The insulin peptide may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the insulin peptide in a suitable nutrient medium under conditions permitting the expression of the insulin peptide. Several recombinant methods may be used in the production of human insulin and human insulin analogues. Examples of methods which may be used in the production of insulin in microorganisms such as, e.g., Escherichia coli and Saccharomyces cerevisiae are, e.g., disclosed in W02008034881.

Typically, the insulin or insulin analogue is produced by expressing a DNA sequence encoding the insulin or insulin analogue in question or a precursor thereof in a suitable host cell by well-known techniques as disclosed in e.g. EP1246845 or W02008034881.

The insulin or insulin analogue may be expressed with an N-terminal extension as disclosed in EP1246845. After secretion to the culture medium and recovery, the insulin precursor will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and connecting peptide to give the insulin or insulin analogue. Such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin or insulin analogue into insulin or the insulin analogue by basic or acid hydrolysis as described in US patent specification No. 4,343,898 or 4,916,212.

Examples of N-terminal extensions of the type suitable in the present invention are disclosed in U.S. Patent No. 5,395,922 and EP patent No. 765395.

For insulin analogues comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue, for instance by use of tRNA mutants. Hence, briefly, the insulin analogues according to the invention are prepared analogously to the preparation of known insulin analogues.

Protein Purification

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

Pharmaceutical compositions

The invention also relates to pharmaceutical compositions comprising a compound of the invention, including e.g., an insulin derivative of the invention, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient(s). Such compositions may be prepared as is known in the art.

The term "excipient" broadly refers to any component other than the active therapeutic ingredient(s). The excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance. The excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, and/or to improve administration, and/or absorption of the active substance. Non-limiting examples of excipients are solvents, diluents, buffers, preservatives, tonicity regulating agents, chelating agents, and stabilisers. 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. 21st edition (2005), and any later editions).

A composition of the invention may be in the form of a liquid formulation, i.e. an aqueous formulation comprising water. A liquid formulation may be a solution, or a suspension. A composition of the invention may be for parenteral administration, e.g. performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

Injectable compositions containing an insulin derivative of this invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, an insulin derivative of this invention is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, and/or a preservative and/or a buffer is added as required and the pH value of the solution is adjusted, if necessary, using an acid, for example, hydrochloric acid, or a base, for example, aqueous sodium hydroxide, as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

Indications

Diabetes

The term “diabetes” or “diabetes mellitus” includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy) and other states that cause hyperglycaemia. The term is used for a metabolic disorder in which the pancreas produces insufficient amounts of insulin, or in which the cells of the body fail to respond appropriately to insulin thus preventing cells from absorbing glucose. As a result, glucose builds up in the blood.

Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is caused by beta cell destruction, usually leading to absolute insulin deficiency.

Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominant insulin resistance and thus relative insulin deficiency and/or a predominantly insulin secretory defect with insulin resistance.

Other Indications

In one embodiment, a compound according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes.

In another embodiment, a compound according to the invention is used as a medicament for delaying or preventing disease progression in type 2 diabetes.

In one embodiment of the invention, the compound is for use as a medicament for the treatment or prevention of hyperglycemia including stress induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes.

In a further embodiment the invention is related to a method for the treatment or prevention of hyperglycemia including 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 for such treatment of a compound according to the invention.

Mode of administration

The term “treatment” is meant to include both the prevention and minimization of the referenced disease, disorder, or condition (i.e., "treatment" refers to both prophylactic and therapeutic administration of an insulin derivative or composition comprising an insulin derivative unless otherwise indicated or clearly contradicted by context).

The route of administration may be any route which effectively transports a compound of this invention to the desired or appropriate place in the body, such as parenterally, for example, subcutaneously, intramuscularly or intravenously. For parenterally administration, a compound of this invention is formulated analogously with the formulation of known insulins. Furthermore, for parenterally administration, a compound of this invention is administered analogously with the administration of known insulins and the physicians are familiar with this procedure.

The amount of a compound of this invention to be administered, the determination of how frequently to administer a compound of this invention, and the election of which compound or compounds of this invention to administer, optionally together with another antidiabetic compound, is decided in consultation with a practitioner who is familiar with the treatment of diabetes.

Non-limiting embodiments

The invention is further described by the following non-limiting embodiments:

1. A compound comprising: i) an insulin peptide, wherein the insulin peptide is human insulin (SEQ ID NO:1 and SEQ ID NO:2) or a human insulin analogue comprising a lysine in position B29; ii) a macrocycle of Formula M:

wherein k is 0 or 1 , wherein the macrocycle of Formula M is attached to the lysine in the B29 position of the insulin peptide via a linker L1 ; wherein *¹ denotes the attachment point to the linker L1; and iii) a glycoside selected from the group consisting of

Formula G1 :

Formula G3:

; and

Formula G4:

wherein Z is O or NH; wherein the glycoside is the D-isomer as shown or the corresponding L-isomer; wherein the glycoside is attached to the alpha amino group of the amino acid in the B1 position of the insulin peptide via a linker L2; wherein *⁴ denotes the attachment point to the linker L2.

2. A compound consisting of: i) an insulin peptide, wherein the insulin peptide is human insulin (SEQ ID NO:1 and SEQ ID NO:2) or a human insulin analogue comprising a lysine in position B29; ii) a macrocycle of Formula M:

wherein k is 0 or 1 , wherein the macrocycle of Formula M is attached to the lysine in the B29 position of the insulin peptide via a linker L1 ; wherein *¹ denotes the attachment point to the linker L1 ; and iii) a glycoside selected from the group consisting of Formula G1 :

Formula G2:

Formula G3:

Formula G4:

3. The compound according to any one of embodiments 1 to 2, wherein k is 0.

4. The compound according to any one of embodiments 1 to 2, wherein k is 1.

5. The compound according to any one of embodiments 1 to 4, wherein the linker L1 is a moiety having from 8 to 20 non-hydrogen atoms, wherein 3 to 10 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms.

6. The compound according to any one of embodiments 1 to 5, wherein the linker L1 has from 10 to 18 non-hydrogen atoms, wherein 4 to 7 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms. 7. The compound according to any one of embodiments 1 to 6, wherein the linker L1 has from 10 to 18 non-hydrogen atoms, wherein 4 to 7 of the non-hydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms.

8. The compound according to any one of embodiments 1 to 7, wherein the linker L1 comprises a triazol or a phenyl group.

9. The compound according to any one of embodiments 1 to 8, wherein the linker L1 comprises a triazol.

10. The compound according to any one of embodiments 1 to 9, wherein the linker L1 is of Formula L1a:

wherein m is an integer from 0 to 6;

W is (CH2)n or (OCH₂CH₂)_P, wherein n is an integer from 2 to 5, and p is an integer from 1 to 5;

Y is absent or NH-C(O)-CH2;

11 . The compound according to embodiment 10, wherein m is an integer from 0 to 4.

12. The compound according to any one of embodiments 10 to 11 , wherein m is an integer from 2 to 4.

13. The compound according to any one of embodiments 10 to 12, wherein m is 3.

14. The compound according to any one of embodiments 10 to 13, wherein W is (CH₂)_n, wherein n is an integer from 2 to 5.

15. The compound according to any one of embodiments 10 to 14, wherein W is (CH₂)_n, wherein n is 3.

16. The compound according to any one of embodiments 10 to 13, wherein W is

(OCH2CH2)p, wherein p is an integer from 1 to 5. 17. The compound according to any one of embodiments 10 to 13, wherein W is (OCH₂CH₂)_P, wherein p is an integer from 2 to 3.

18. The compound according to any one of embodiments 10 to 17, wherein Y is absent.

19. The compound according to any one of embodiments 10 to 17, wherein Y is NH-C(O)- CH₂.

20. The compound according to any one of embodiments 10 to 19, wherein X is C(O) or O- C(O); wherein the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide.

21. The compound according to any one of embodiments 10 to 19, wherein X is C(O); wherein the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide.

22. The compound according to any one of embodiments 10 to 19, wherein X is O-C(O); wherein the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide.

23. The compound according to any one of embodiments 10 to 19, wherein X is NH; wherein the linker L1 is attached to the alpha carboxylic acid of the lysine in the B29 position of the insulin peptide.

24. The compound according to any one of embodiments 1 to 23, wherein the glycoside is of Formula G1:

wherein Z is O or NH.

25. The compound according to any one of embodiments 1 to 23, wherein the glycoside is of Formula G1:

wherein Z is O. 26. The compound according to any one of embodiments 1 to 23, wherein the glycoside is of Formula G1 :

wherein Z is NH.

27. The compound according to any one of embodiments 1 to 26, wherein the glycoside is the D-isomer.

28. The compound according to any one of embodiments 1 to 27, wherein the linker L2 is a moiety having from 2 to 15 non-hydrogen atoms, wherein 0 to 6 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms.

29. The compound according to any one of embodiments 1 to 28, wherein the linker L2 is selected from the group consisting of:

Formula L2b:

Formula L2c:

, wherein r is 0 or 1 ; and

Formula L2d:

30. The compound according to any one of embodiments 1 to 29, wherein the linker L2 is

L2a: *⁵-R-CH₂-C(O)-*⁶, wherein R is absent, C(O)-CH₂O, C(O)-CH₂OCH₂CH₂O, CH₂-C(O)-NH, or (CH₂CH₂O)_q, wherein q is 1 or 2; wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide.

31. The compound according to any one of embodiments 1 to 30, wherein the linker L2 is

L2a: *⁵-R-CH₂-C(O)-*⁶, wherein R is (CH₂CH₂O)_q, wherein q is 1 or 2; wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide.

32. The compound according to any one of embodiments 1 to 31 , wherein the linker L2 is

L2a: *⁵-R-CH₂-C(O)-*⁶, wherein R is (CH₂CH₂O)_q, wherein q is 1 ; wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide.

33. The compound according to any one of embodiments 1 to 32, wherein the linker L1 and the linker L2 in total have from 15 to 27 non-hydrogen atoms, wherein 5 to 11 of the nonhydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms.

34. The compound according to any one of embodiments 1 to 33, wherein the linker L1 and the linker L2 in total have from 20 to 22 non-hydrogen atoms, wherein 6 to 8 of the nonhydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms.

35. The compound according to any one of embodiments 1 to 34, wherein the linker L1 and the linker L2 in total have from 20 to 21 non-hydrogen atoms, wherein 6 to 8 of the nonhydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms.

36. The compound according to embodiment 1 , wherein the compound comprises i) an insulin peptide, wherein the insulin peptide is human insulin (SEQ ID N0:1 and SEQ ID N0:2) or a human insulin analogue comprising a lysine in position B29; ii) a macrocycle of Formula M:

wherein k is 0 or 1 , wherein the macrocycle of Formula M is attached to the lysine in the B29 position of the insulin peptide via a linker L1 ; wherein *¹ denotes the attachment point to the linker L1; wherein the linker L1 is of Formula L1a:

, wherein m is an integer from 0 to 6; W is (CH₂)_n or (OCH₂CH₂)_P, wherein n is an integer from 2 to 5, and p is an integer from 1 to 5;

Y is absent or NH-C(O)-CH₂;

X is C(O), O-C(O), or NH; wherein when X is C(O) or O-C(O), the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide, and when X is NH, the linker L1 is attached to the alpha carboxylic acid of the lysine in the B29 position of the insulin peptide; and wherein *² denotes the attachment point to the macrocycle and *³ denotes the attachment point to the insulin peptide; and iii) a glycoside, wherein the glycoside is of Formula G1:

wherein Z is O or NH; wherein the glycoside is attached to the alpha amino group of the amino acid in the B1 position of the insulin peptide via a linker L2; wherein *⁴ denotes the attachment point to the linker L2; wherein the linker L2 is selected from the group consisting of:

Formula L2b:

Formula L2c:

wherein r is 0 or 1 ; and

Formula L2d:

wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide. 37. The compound according to embodiment 1 , wherein the compound consists of i) an insulin peptide, wherein the insulin peptide is human insulin (SEQ ID NO:1 and SEQ ID NO:2) or a human insulin analogue comprising a lysine in position B29; ii) a macrocycle of Formula M:

wherein k is 0 or 1 , wherein the macrocycle of Formula M is attached to the lysine in the B29 position of the insulin peptide via a linker L1 ; wherein *¹ denotes the attachment point to the linker L1 ; wherein the linker L1 is of Formula L1a:

, wherein m is an integer from 0 to 6;

Y is absent or NH-C(O)-CH₂;

X is C(O), O-C(O), or NH; wherein when X is C(O) or O-C(O), the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide, and when X is NH, the linker L1 is attached to the alpha carboxylic acid of the lysine in the B29 position of the insulin peptide; and wherein *² denotes the attachment point to the macrocycle and *³ denotes the attachment point to the insulin peptide; and iii) a glycoside, wherein the glycoside is of Formula G1 :

Formula L2b:

Formula L2c:

wherein r is 0 or 1 ; and

Formula L2d:

and wherein *⁵ denotes the attachment point to the glycoside and *⁶ denotes the attachment point to the insulin peptide. ompound according to any one of embodiments 36 to 37, wherein k is 0; wherein m is an integer from 0 to 4; wherein W is (CH2)n, wherein n is an integer from 2 to 5; wherein X is C(O) or O-C(O); wherein the linker L1 is attached to the epsilon amino group of the lysine in the B29 position of the insulin peptide; and wherein the linker L2 is L2a: *⁵-R-CH2-C(O)-*⁶, wherein R is C(O)-CH₂O, C(O)-CH₂OCH₂CH₂O, CH₂-C(O)-NH, or (CH₂CH₂O)_q, wherein q is 1 or 2. ompound according to embodiment 38, wherein m is an integer from 2 to 4; wherein W is (CH₂)_n, wherein n is 3; wherein Z is O; wherein the linker L2 is L2a: *⁵-R-CH₂-C(O)-*⁶, wherein R is (CFhCFfeOJq, wherein q is 1 or 2. 40. The compound according to embodiment 39, wherein the linker L1 and the linker L2 in total have from 20 to 22 non-hydrogen atoms, wherein 6 to 8 of the non-hydrogen atoms are independently selected from non-basic nitrogen atoms and oxygen atoms.

41. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is human insulin.

42. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue.

43. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 10 amino acid modifications relative to human insulin.

44. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 9 amino acid modifications relative to human insulin.

45. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 8 amino acid modifications relative to human insulin.

46. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 7 amino acid modifications relative to human insulin.

47. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 6 amino acid modifications relative to human insulin.

48. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 5 amino acid modifications relative to human insulin.

49. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 4 amino acid modifications relative to human insulin.

50. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 3 amino acid modifications relative to human insulin.

51. The compound according to any one of embodiments 1 to 40, wherein the insulin peptide is a human insulin analogue comprising less than 2 amino acid modifications relative to human insulin. 52. The compound according to any one of embodiments 1 to 51, wherein the insulin peptide is a human insulin analogue comprising desB30.

53. The compound according to any one of embodiments 1 to 52, wherein the insulin peptide is desB30 human insulin.

54. The compound according to any one of embodiments 1 to 53 wherein the insulin peptide has the ability to bind to the insulin receptor.

55. The compound according to any one of embodiments 1 to 54, wherein the insulin peptide has the ability to bind to and activate the insulin receptor.

56. The compound according to embodiment 1, wherein the compound is selected from the group consisting of INS1 of Example 1; INS2 of Example 2; INS3 of Example 3; INS5 of Example 5; INS6 of Example 6; INS8 of Example 8; INS12 of Example 12; INS15 of Example 15; INS17 of Example 17; INS19 of Example 19; INS21 of Example 21; INS28 of Example 28; and INS31 of Example 31.

57. The compound according to embodiment 1, wherein the compound is selected from the group consisting of INS1 of Example 1; INS2 of Example 2; INS6 of Example 6; INS8 of Example 8; INS17 of Example 17; INS28 of Example 28; and INS31 of Example 31.

58. The compound according to embodiment 1, wherein the compound is selected from the group consisting of INS2 of Example 2; INS6 of Example 6; INS17 of Example 17; and INS31 of Example 31.

59. The compound according to embodiment 1, wherein the compound is selected from the group consisting of INS1 of Example 1; INS2 of Example 2; INS7 of Example 7; INS10 of Example 10; INS12 of Example 12; INS17 of Example 17; INS24 of Example 24; and INS28 of Example 28.

60. The compound according to embodiment 1, wherein the compound is INS1 of Example 1. 61. The compound according to embodiment 1, wherein the compound is INS2 of Example 2. 62. The compound according to embodiment 1, wherein the compound is INS3 of Example 3. 63. The compound according to embodiment 1, wherein the compound is INS4 of Example 4. 64. The compound according to embodiment 1 , wherein the compound is INS5 of Example 5. 65. The compound according to embodiment 1 , wherein the compound is INS6 of Example 6. 66. The compound according to embodiment 1 , wherein the compound is INS7 of Example 7. 67. The compound according to embodiment 1 , wherein the compound is INS8 of Example 8. 68. The compound according to embodiment 1, wherein the compound is INS9 of Example 9. 69. The compound according to embodiment 1, wherein the compound is INS10 of Example 10. 70. The compound according to embodiment 1 , wherein the compound is INS11 of Example 11. 71. The compound according to embodiment 1, wherein the compound is INS12 of Example 12. 72. The compound according to embodiment 1 , wherein the compound is INS13 of Example 13. 73. The compound according to embodiment 1, wherein the compound is INS14 of Example 14. 74. The compound according to embodiment 1 , wherein the compound is INS15 of Example 15. 75. The compound according to embodiment 1, wherein the compound is INS16 of Example 16. 76. The compound according to embodiment 1, wherein the compound is INS17 of Example 17. 77. The compound according to embodiment 1, wherein the compound is INS18 of Example 18. 78. The compound according to embodiment 1 , wherein the compound is INS19 of Example 19. 79. The compound according to embodiment 1, wherein the compound is INS20 of Example 20. 80. The compound according to embodiment 1 , wherein the compound is INS21 of Example 21. 81. The compound according to embodiment 1, wherein the compound is INS22 of Example 22. 82. The compound according to embodiment 1 , wherein the compound is INS23 of Example 23. 83. The compound according to embodiment 1, wherein the compound is INS24 of Example 24. 84. The compound according to embodiment 1 , wherein the compound is INS25 of Example 25. 85. The compound according to embodiment 1, wherein the compound is INS26 of Example 26. 86. The compound according to embodiment 1, wherein the compound is INS27 of Example 27. 87. The compound according to embodiment 1, wherein the compound is INS28 of Example 28. 88. The compound according to embodiment 1 , wherein the compound is INS29 of Example 29. 89. The compound according to embodiment 1, wherein the compound is INS30 of Example 30. 90. The compound according to embodiment 1 , wherein the compound is INS31 of Example 31.

91. The compound according to any one of embodiments 1 to 90, wherein the compound has the ability to bind to the insulin receptor.

92. The compound according to any one of embodiments 1 to 90, wherein the compound has the ability to bind to and activate the insulin receptor.

93. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 1% insulin receptor affinity relative to human insulin measured in the presence of 20 mM glucose and in the presence of 1.5% HSA.

94. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 5% insulin receptor affinity relative to human insulin measured in the presence of 20 mM glucose and in the presence of 1.5% HSA.

95. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 10% insulin receptor affinity relative to human insulin measured in the presence of 20 mM glucose and in the presence of 1.5% HSA. 96. The compound according to any one of embodiments 1 to 90, wherein the compound has higher insulin receptor affinity in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured without any HSA present.

97. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 2-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured without any HSA present.

98. The compound according to any one of embodiments 1 to 90, wherein the compound has higher insulin receptor affinity in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

99. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 2-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

100. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 5-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

101. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 8-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

102. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 9-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

103. The compound according to any one of embodiments 1 to 90, wherein the compound has at least 10-fold higher insulin receptor affinity relative to human insulin in presence of 20 mM glucose than when no glucose is present, wherein the human insulin receptor affinity is measured in the presence of 1.5% HSA.

104. The compound according to any one of embodiments 1 to 90, wherein the compound displays higher glucose sensitivity in the presence of 1.5% HSA, than when no HSA is present, when the glucose sensitivity is measured as the insulin receptor affinity relative to human insulin (%) in the presence of 20 mM glucose divided by the insulin receptor affinity relative to human insulin (%) when no glucose is present (also referred to as glucose factor in the present application).

105. The compound according to any one of embodiments 1 to 90, wherein the compound displays at least 1.5 times higher glucose sensitivity in the presence of 1.5% HSA, than when no HSA is present, when the glucose sensitivity is measured as the insulin receptor affinity relative to human insulin (%) in the presence of 20 mM glucose divided by the insulin receptor affinity relative to human insulin (%) when no glucose is present (also referred to as glucose factor in the present application).

106. The compound according to any one of embodiments 91 to 105, wherein the insulin receptor affinity is measured by the Insulin Receptor Scintillation Proximity Assay (SPA) binding assay described in Example 32.

107. An intermediate compound of Formula IM1 :

wherein k1 is 0 or 1,

W1 is (CH2)ni or (OCH2CH2)_PI, wherein n1 is an integer from 2 to 5, and p1 is an integer from 1 to 5; and

Y1 is absent or NH-C(O)-CH2.

108. The intermediate compound according to embodiment 107, wherein k1 is 0.

109. The intermediate compound according to any one of embodiments 107 to 108, wherein W1 is (CH₂)ni, wherein n1 is an integer from 2 to 5.

110. The intermediate compound according to any one of embodiments 107 to 109, wherein W1 is (CH₂)ni, wherein n1 is 3. 111. The intermediate compound according to any one of embodiments 107 to 108, wherein W1 is (OCH2CH2)_PI , wherein p1 is an integer from 1 to 5.

112. The intermediate compound according to any one of embodiments 107 to 108, wherein W1 is (OCH₂CH₂)_P, wherein p is an integer from 2 to 3.

113. The intermediate compound according to any one of embodiments 107 to 112, wherein Y1 is NH-C(O)-CH2.

114. The intermediate compound according to any one of embodiments 107 to 112, wherein Y1 is absent.

115. The intermediate compound according to embodiment 107, wherein the intermediate compound is selected from the group consisting of Intermediate compound 3 (‘macrocycle propyl azide’), Intermediate compound 9 (‘macrocycle EG3 azide’), Intermediate compound 10 (‘macrocycle EG2 azide’), Intermediate compound 12 (‘macrocycle propyl-NHAc azide’), and Intermediate compound 17 (‘homo-macrocycle propyl azide’).

116. The intermediate compound according to embodiment 107, wherein the intermediate compound is Intermediate compound 3 (‘macrocycle propyl azide’).

117. The intermediate compound according to embodiment 107, wherein the intermediate compound is Intermediate compound 9 (‘macrocycle EG3 azide’).

118. The intermediate compound according to embodiment 107, wherein the intermediate compound is Intermediate compound 10 (‘macrocycle EG2 azide’).

119. The intermediate compound according to embodiment 107, wherein the intermediate compound is Intermediate compound 12 (‘macrocycle propyl-NHAc azide’).

120. The intermediate compound according to embodiment 107, wherein the intermediate compound is Intermediate compound 17 (‘homo-macrocycle propyl azide’).

121. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 106.

122. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 106 and one or more pharmaceutically acceptable excipients.

123. A compound according to any one of embodiments 1 to 106 for use as a medicament.

124. A compound according to any one of embodiments 1 to 106 or a composition according to any one of embodiments 121 to 122 for use in the treatment and/or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

125. A compound according to any one of embodiments 1 to 106 or a composition according to any one of embodiments 121 to 122 for use in the treatment and/or prevention of diabetes, including diabetes of Type 1 and/or diabetes of Type 2. 126. A compound according to any one of embodiments 1 to 106 or a composition according to any one of embodiments 121 to 122 for use in the treatment and/or prevention of diabetes.

127. A compound according to any one of embodiments 1 to 106 or a composition according to any one of embodiments 121 to 122 for use in a method for treatment and/or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

128. A compound according to any one of embodiments 1 to 106 or a composition according to any one of embodiments 121 to 122 for use in a method for treatment and/or prevention of diabetes.

129. Use of a compound according to any one of embodiments 1 to 106 or the composition according to any one of embodiments 121 to 122, for the manufacture of a medicament for the treatment or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

130. Use of a compound according to any one of embodiments 1 to 106 or the composition according to any one of embodiments 121 to 122, for the manufacture of a medicament for the treatment or prevention of diabetes.

131. A method for treatment and/or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome) comprising administration of an effective amount of the insulin derivative according to any one of embodiments 1 to 106 or the composition according to any one of embodiments 121 to 122 to a patient in need thereof.

132. A method for treatment and/or prevention of diabetes comprising administration of an effective amount of the insulin derivative according to any one of embodiments 1 to 106 or the composition according to any one of embodiments 121 to 122 to a patient in need thereof.

EXAMPLES

List of Abbreviations

Ac Acetyl

Boc tert-butyloxycarbonyl

CV Column volume

DBU 1 ,8-Diazabicyclo(5.4.0)undec-7-en

DCC N,N’-dicyclohexylcarbodiimide

DCM Dichloromethane

DMAP N,N-dimethyl-4-aminopyridine

DMF N,N-dimethylformamide

EG Ethylene glycol

EDC N-(3-dimethylaminopropyl)-N-ethylcarbodiimide

EtOAc Ethyl acetate eq equivalents

DIPEA N,N-diisopropylethylamine

Fmoc-OSu 9-Fluorenylmethyl N-succinimidyl carbonate

HBTLI 2-(1 H-benzotriazol-1-yl)-1,1 ,3,3-tetramethyluronium hexafluorophosphate

HOBt 1 -Hydroxybenzotriazole

HRMS High resolution mass spectrometry

HSA Human serum albumin

LCMS Liquid chromatography mass spectrometry

MeCN Acetonitrile

MC Macrocycle

MS Mass spectrometry

MsCI methylsulfonyl chloride

NHS N-hydroxy-succinimide

NMR Nuclear magnetic resonance

NMP N-methyl-pyrrolidone

RP-HPLC Reverse-phase high performance liquid chromatography

TEA Triethylamine

THF Tetrahydrofuran

THPTA tris(3-hydroxypropyltriazolylmethyl)amine Preparation of intermediate compounds

Preparation of O-succinimidyl pentvn-1-oxycarbonyl (Intermediate compound 1)

Intermediate compound 1 :

A solution of bis(trichloromethyl)carbonate (52.9 g, 178 mmol) in dry tetrahydrofuran (215 mL) was added dropwise to a solution of pent-4-yn-1-ol (13.6 g, 162 mmol) in dry tetra hydrofuran (80 mL) over 15 minutes at 0 °C. The reaction mixture was stirred for 1 hour under cooling and then overnight at room temperature. The solvent was removed under reduced pressure and the residue (yellow oil) was purified by vacuum distillation (p= 0.2 Torr, t= 80-95 °C) to give pent-4-yn-1-yl carbonochloridate as off-white liquid. Yield: 16.3 g (69%). ¹H NMR spectrum (300 MHz, CDCI₃, d_H): 4.44 (q, J=6.2 Hz, 2 H); 2.41-2.31 (m, 2 H); 2.04- 1.90 (m, 3 H).

To a solution of /V-hydroxysuccinimide (12.8 g, 111 mmol) in dry tetrahydrofuran (450 mL) cooled at -5 °C was added triethylamine (15.5 mL, 111 mmol, with forming of fine precipitate). A solution of the above chloroformate (16.3 g, 111 mmol) in dry tetrahydrofuran (90 mL) was added dropwise over 10 minutes under intensive stirring (a precipitate triethylamine hydrochloride was gradually formed). The resulting suspension was stirred for 2 hours at -5 °C and then overnight at room temperature. The white solid was filtered off and solvent was removed in vacuo. The residue was dissolved in ethyl acetate (500 mL) and washed with 1 M aqueous solution of potassium hydrogen sulfate (2 x 300 mL), brine (3 x 300 mL) and dried over anhydrous sodium sulfate. After filtration the solvent was removed under reduced pressure and the crude product was purified by column chromatography (Silicagel 60, 0.040-0.063 mm; eluent: cyclohexane/ethyl acetate 3:1-1 :1) giving pure O- succinimidyl pentyn-1 -oxycarbonyl (Intermediate compound 1) as off-white liquid.

Yield: 9.41 g (38%). R_F (SiC>2, cyclohexane-ethyl acetate 1 :1): 0.40. ¹H NMR spectrum (300 MHz, CDCI₃, d_H): 4.46 (t, J=6.2 Hz, 2 H); 2.84 (s, 4 H); 2.40-2.32 (m, 2 H); 2.04-1.93 (m, 3 H). LC-MS: 226.4 (M+H)⁺.

Intermediate compound 2:

(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (4.71 g, 11.5 mmol) was dissolved in anhydrous 1,4-dioxane (6 mL) and diethylene glycol (7.60 mL). Silver carbonate (3.47 g, 12.6 mmol) was added in one portion (gas evolution after short induction period). The resulting mixture was stirred at ambient temperature for 3 hours. Afterwards, it was diluted with ethyl acetate (80 mL), filtered through a celite pad, washed with ethyl acetate (3 x 30 mL) and the filtrate was evaporated. The oily residue was dissolved in water (80 mL) and extracted with dichloromethane (4 x 60 mL). Combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The residue was purified by flash column chromatography (Silicagel 60, 0.040-0.063 mm; eluent: dichloromethane/ethyl acetate 1 :1 to 0:1) to give (2R,3R,4S,5R,6R)-2- (acetoxymethyl)-6-(2-(2-hydroxyethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a waxy white solid. Yield: 3.55 g (71%). ¹H NMR spectrum (300 MHz, CDCI3, dH): 5.22 (t, J=9.6 Hz, 1 H); 5.10 (t, J=9.6 Hz, 1 H); 5.00 (dd, J=9.4 and 7.9 Hz, 1 H); 4.62 (d, J=7.9 Hz, 1 H); 4.32-4.22 (m, 1 H); 4.20-4.11 (m, 1 H); 4.04-3.90 (m, 1 H); 3.80-3.55 (m, 8 H); 2.09 (s, 3 H); 2.06 (s, 3 H); 2.03 (s, 3 H); 2.01 (s, 3 H). LC-MS: 454.1 (M+H2O)⁺, 437.1 (M+Na)⁺.

To a well stirred solution of the given (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2- hydroxyethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3.54 g, 8.11 mmol) and bis(acetoxy)iodobenzene (PI DA, 6.27 g, 19.5 mmol) in acetonitrile (40 mL) and water (10 mL) was added (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (51.0 mg, 0.32 mmol) in one portion. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was treated with 10% aqueous solution of sodium sulfite (40 mL) and stirred for 30 minutes. Afterwards, pH of the solution was adjusted with concentrated hydrochloric acid to pH=2. The aqueous layer was extracted with dichloromethane (3 x 60 mL); the organic extracts were combined, dried over anhydrous magnesium sulfate, filtered, and evaporated. The residue was purified by flash column chromatography (Silicagel 60, 0.040-0.063 mm; eluent: acetonitrile/water 30:1 + 0.5% of formic acid). After freeze-drying, the residue was treated with dry diethyl ether (60 mL). A white solid was filtered, washed with dry diethyl ether (3 x 20 mL) and dried in vacuo affording 2-(2-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)acetic acid as a white solid.

Yield: 2.93 g (80%). ¹H NMR spectrum (300 MHz, CDCI3, dH): 5.22 (t, J=9.5 Hz, 1 H); 5.10 (t, J=9.6 Hz, 1 H); 5.01 (dd, J=9.5 and 8.0 Hz, 1 H); 4.63 (d, J=7.9 Hz, 1 H); 4.30-4.24 (m, 1 H); 4.22-4.12 (m, 3 H); 4.05-3.93 (m, 1 H); 3.85-3.65 (m, 4 H); 2.09 (s, 3 H); 2.06 (s, 3 H); 2.03 (s, 3 H); 2.01 (s, 3 H). LC-MS: 468.1 (M+H2O)⁺, 449.2 (M-H)’.

The given 2-(2-(((2R,3R,4S,5R,6R)-3,4,5-T riacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)ethoxy)acetic acid (3, 2.77 g, 6.15 mmol) was dissolved in dry dichloromethane (30 mL) and triethylamine (2.60 mL, 18.5 mmol) was added. A mixture was cooled to 0 °C and 5- bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (4, 2.50 g, 7.38 mmol) was added in three portions during 10 minutes. The mixture was stirred under cooling for 30 minutes, then at room temperature overnight. Solvent was evaporated in vacuo. To the residue ethyl acetate (40 mL) was added. Resulting suspension was filtered through celite pad and filtrate was evaporated. The residue was purified by flash column chromatography (Silicagel 60, 0.040-0.063 mm; eluent: dichloromethane/2-propanol 100:0 to 90:10 + 0.5% of triethylamine) giving Intermediate compound 2 as white foam.

Yield: 3.34 g (63%). ¹H NMR spectrum (300 MHz, DMSO-d6, dH): 8.83 (bs, 0.8 H); 8.11 (dd, J=2.6 and 0.6 Hz, 1 H); 7.99 (dd, J=2.6 and 0.6 Hz, 1 H); 5.26 (t, J=9.5 Hz, 1 H); 4.95-4.82 (m, 2 H); 4.81-4.72 (m, 1 H); 4.35 (d, J=2.4 Hz, 2 H); 4.23-4.12 (m, 1 H); 4.06-3.93 (m, 2 H); 3.88-3.77 (m, 1 H); 3.75-3.57 (m, 3 H); 3.09 (q, J=7.3 Hz, 4.8 H); 2.02 (s, 3 H); 1.99 (s, 3 H); 1.98 (s, 3 H); 1.93 (s, 3 H); 1.17 (t, J=7.2 Hz, 7.2 H). 19F NMR spectrum (282 MHz, DMSO- d6, dF): -59.98. LC-MS: 751.3 and 753.3 (M-H)-. Preparation of macrocycle propyl azide (intermediate compound 3)

Intermediate compound 3:

3-(3,5-dimethylphenyl)propanoic acid (5.00 g, 28.1 mmol, 1 eq.) was dissolved in HBr/AcOH (33%, 53 mL). Paraformaldehyde (8.84 g, 295 mmol, 10.5 eq.) and ZnBr2 (10.1 g,

44.9 mmol, 1.6 eq.) were added and the reaction mixture was heated to 100 °C for 18 h. The reaction was cooled and the precipitate isolated by filtration, washing with AcOH (50 mL) and water (800 mL) to give the trisbromomethyl acid (12.2 g, 26.6 mmol, 95%) as white crystals. ¹H NMR (400 MHz, DMSO-cfe) 64.75 (d, J = 1.5 Hz, 6H), 3.14 - 2.96 (m, 2H), 2.58 (d, J = 18.5 Hz, 2H), 2.40 (d, J = 1.1 Hz, 6H). Step 2: Synthesis of the carboxy tris-azide

Trisbromomethyl acid (14.0 g, 30.6 mmol, 1 eq.) was dissolved in anhydrous DMF (12.0 mL) and NaN₃ (11.9 g, 184 mmol, 6 eq.) was added in 4 portions over 20 minutes. The reaction mixture was left to stir for 40 h and then quenched with 1 M HCI (400 mL). The resultant suspension was extracted with EtOAc (3 x 1.5 L) and the combined organics washed with water (5 x 400 mL), brine (400 mL) and dried (Na2SO4). The solvents were evaporated under reduced pressure to an oil which crystallised upon standing to give the carboxy tris-azide (9.89 g, 28.8 mmol, 94%). ¹H NMR (400 MHz, Chloroform-d) 5 4.54 (s, 4H), 4.51 (s, 2H), 3.25 - 3.10 (m, 2H), 2.72 - 2.57 (m, 2H), 2.48 (d, J = 0.7 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d) 5 177.51 , 140.23, 139.05, 132.37, 130.94, 49.04, 48.79, 35.37, 25.18, 16.73.

Step 3: Synthesis of the alcohol tris-azide

The given carboxy tris-azide (9.89 g, 28.8 mmol, 1 eq.) was dissolved in anhydrous THF (30 mL) and 1 M BH3.THF in THF (58 mL, 58.0 mmol, 2 eq.) was added dropwise. After stirring for five hours, further 1 M BH3.THF in THF (20 mL, 20.0 mmol, 0.7 eq.) added and the reaction was stirred for 16 hours. The reaction was quenched with saturated aqueous NaHCOs (60 mL). The THF was removed under a stream of N₂ and the remnants extracted with EtOAc (100 mL). The organic layer was dried (Na₂SO4) and concentrated under reduced pressure. The crude product was purified by flash chromatography (petroleum ether 40/60 with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the alcohol tris-azide (4.42 g, 13.4 mmol, 47%) as white crystals.

¹H NMR (400 MHz, Chloroform-d) 5 4.53 (s, 4H), 4.50 (s, 2H), 3.77 (t, J = 5.9 Hz, 2H), 2.99 - 2.88 (m, 2H), 2.46 (s, 6H), 1.86 - 1.74 (m, 2H). Step 4: Synthesis of the alcohol tris-Boc

The given alcohol tris-azide (1.40 g, 4.25 mmol, 1 eq.), BOC2O (4.18 g, 19.1 mmol, 4.5 eq.) were dissolved in anhydrous THF (80 mL). A slurry of Pd/C (200 mg, 10% w/w) in DCM was added, followed by Et₃N (1.80 mL, 12.8 mmol, 3 eq.). The reaction was placed under an atmosphere of hydrogen and stirred overnight. The reaction mixture was centrifuged and the supernatant concentrated under reduced pressure, before being redissolved in EtOAc (100 mL) and washed with 5% aqueous KHSO4 (100 mL), saturated aqueous NaHCOs (100 mL) and brine (100 mL) then dried (Na₂SC>4). The organic solvents were removed in vacuo and the crude product was purified by flash chromatography (petroleum ether 40/60 with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the alcohol tris-Boc (2.02 g, 3.64 mmol, 86%) as a white solid.

¹H NMR (400 MHz, Chloroform-d) 54.37 (d, J = 11.0 Hz, 7H), 3.72 (t, J = 5.7 Hz, 2H), 2.94 - 2.77 (m, 2H), 2.36 (s, 6H), 1.76 - 1.65 (m, 2H), 1.44 (d, J = 2.4 Hz, 27H).

MS [2M+H-Boc]⁺ calculated for C53H91N6O12 requires: 1003.7, found: 1003.6.

Step 5: Synthesis of the O-mesylate tris-Boc

The given alcohol tris-Boc_ (4.41 g, 7.99 mmol, 1 eq.) was dissolved in anhydrous DCM (16 mL). EtsN (2.80 mL, 20.0 mmol, 2.5 eq.) was added and the reaction cooled to 0 °C, followed by addition of MsCI (0.62 mL, 8.00 mmol, 1 eq.) over a period of 10 minutes. The reaction was left to stir at room temperature for 3 hours, then cooled to 0 °C and MsCI (0.40 ml, 5.17 mmol, 0.65 eq.) was added. After 3 hours stirring at room temperature, the reaction mixture was diluted with DCM (60 mL) and partitioned with brine (60 mL). The organics were dried (Na₂SC>4) and concentrated under reduced pressure. The crude product was purified by flash chromatography (petroleum ether 40/60 with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the O-mesylate tris- Boc (4.85 g, 7.70 mmol, 96%) as a white solid. ¹H NMR (400 MHz, Chloroform-d) 5 4.38 - 4.29 (m, 9H), 3.11 (s, 3H), 2.99 - 2.78 (m, 2H), 2.36 (s, 6H), 1.89 (dq, J = 11.8, 5.9 Hz, 2H), 1.43 (s, 27H). MS (electrospray) [M+Na]⁺ calculated for CsoHsiNsNaOgS requires: 652.3239, found: 652.3.

The given mesylate tris-Boc_ (1.31 g, 2.08 mmol, 1 eq.) was dissolved in anhydrous DMF (21 mL), NalXh (676 mg, 10.4 mmol, 5 eq.) was added and the reaction stirred at room temperature for 22 hours. The reaction mixture was diluted with water (100 mL) and extracted with Et20 (5 x 50 mL). The organic layer was dried (MgSCL) and concentrated under reduced pressure. The crude oil was purified by reverse phase flash chromatography. The fractions containing the product were combined and the solvent removed under vacuum to yield the azide tris-Boc (800 mg, 1.39 mmol, 67%) as a white solid. ¹H NMR (400 MHz, Chloroform-d) 5 4.50 - 4.19 (m, 9H), 3.43 (t, J = 6.7 Hz, 2H), 2.86 - 2.72 (m, 2H), 2.37 (s, 6H), 1.79 - 1.68 (m, 2H), 1.45 (s, 27H). MS (electrospray) [2M+H-Boc]⁺ calculated for C53H89N12O10 requires: 1053.6820, found: 1053.6.

The given azide tris-Boc (350 mg, 0.617 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (6 mL), followed by addition of anhydrous 2-chloropyridine (0.51 mL, 5.46 mmol, 9 eq.). Triflic anhydride (0.46 mL, 2.73 mmol, 4.5 eq.) was added dropwise to the reaction mixture and the reaction stirred at room temperature for a further 30 minutes after addition was complete. The solvent was removed under vacuum to give a crude solid. The product was extracted with boiling petroleum ether 40/60 3 times, the extracts combined and concentrated under vacuum until a precipitate was observed. This suspension was then reheated to the boiling point until a solution was obtained, which was then cooled in a freezer at -18 °C overnight. The product crystals were then obtained by filtration, washing with cold petroleum ether 40/60 and drying under high vacuum to yield the propyl azide tris-isocyanate (170 mg, 0.480 mmol, 79%). ¹H NMR (400 MHz, Chloroform-d) 5 4.50 (s, 6H), 3.49 (t, J = 6.2 Hz, 2H), 2.92 - 2.76 (m, 2H), 2.48 (s, 6H), 1.79 (dq, J = 11.9, 6.1 Hz, 2H). Step 8: Synthesis of the macrocycle propyl azide (Intermediate compound 3)

The half macrocycle (Intermediate compound 19) (315 mg, 0.363 mmol, 1 eq.) was dissolved in anhydrous DMF (26 mL) and anhydrous pyridine (60 mL) and heated to 45 °C. The given propyl azide tris-isocyanate (from Step 7) (154 mg, 0.435 mmol, 1.2 eq.) was then added as a solution in anhydrous toluene (2.5 mL) over 10 hours and the reaction mixture left to stir for a further 6 hours. The reaction mixture was then concentrated under vacuum and the crude residue precipitated with 1M aqueous HCI (1000 mL), filtered, washed with water and dried. The filtered material was dissolved in EtOH (15 mL) and water (15 mL) and NaOH (144 mg, 3.60 mmol, 10 eq.) was added. The reaction was stirred at 40 °C for 6 hours. The reaction was cooled to room temperature and the organic solvent removed under vacuum. The crude product was then precipitated with 1M aqueous HCI (400 mL), filtered, washed with 1M aqueous HCI and dried. The crude solid was then dissolved in acetone/water, dry loaded onto C18 column and purified by reverse phase flash chromatography. The fractions containing the product were combined and the solvent removed under vacuum to yield the macrocycle propyl azide (Intermediate compound 3) (278 mg, 0.244 mmol, 68%) as a white solid.

¹H NMR (400 MHz, DMSO-d6) 5 8.14 - 8.11 (m, 6H), 8.10 - 8.03 (m, 3H), 7.91 (d, J = 13.4 Hz, 3H), 7.59 (dd, J = 8.6, 2.0 Hz, 3H), 7.47 (s, 3H), 6.58 (s, 1 H), 6.51 (d, J = 5.6 Hz, 2H), 6.42 (s, 1 H), 6.34 (t, J = 5.6 Hz, 2H), 4.59 - 4.08 (m, 12H), 2.89 (s, 2H), 2.73 - 2.59 (m, 8H), 2.39 (s, 6H), 1.67 (s, 2H), 1.23 - 1.04 (m, 9H). MS (electrospray), [M+H]⁺ calculated for C56H64N15O12 requires: 1138.4854, found: 1138.4.

Intermediate compound 4:

4-Pentynoic acid (1 , 3.40 g, 34.0 mmol) and N-hydroxysuccinimide (4.30 g, 37.4 mmol) were dissolved in dry dichloromethane (173 mL). The solution was cooled to 0 °C and N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCI, 8.47 g, 44.2 mmol) was added. The mixture was stirred at 0 °C for 45 minutes, then the cooling bath was removed and the solution was stirred at room temperature for additional 3 hours. 0.5 M aqueous hydrochloric acid (173 mL) was added; the phases were separated and the aqueous one was extracted with dichloromethane (2 x 80 mL). The organic fractions were combined, dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was crystallized from hot 2-propanol (34 mL). The resulting crystals were collected by filtration, washed with 2- propanol (2 x 40 mL) and n-hexane (4 x 40 mL) and dried in vacuo to give O-succinimidyl- pentynoate (Intermediate compound 4) as off-white crystalline solid. Yield: 5.94 g (90%). RF (SiO2, dichloromethane/ethyl acetate 9:1): 0.60. ¹H NMR spectrum (300 MHz, CDCI3, dH): 2.93-2.78 (m, 6 H); 2.67-2.57 (m, 2 H); 2.05 (t, J=2.7 Hz, 1 H). LC-MS: 196.4 (M+H)+.

Preparation of O-peracetyl-glucoside Ac active ester (Intermediate compound 5)

Intermediate compound 5:

4-Bromo-2-(trifluoromethyl)phenol (10.0 g, 41.5 mmol) was dissolved in 1 ,2-dichloroethane (70 mL). To the reaction mixture chlorosulfonic acid (14.0 mL, 210 mmmol) was added dropwise at 0 °C. Then the mixture was heated at 70 °C for 2 hours. Afterwards, the mixture was poured carefully onto crushed ice (300 g). The suspension was washed with dichloromethane/ethyl acetate mixture (8:1 , 1 x 500 mL) and dichloromethane (2 x 500 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered and removed in vacuo. A beige solid was dissolved in cyclohexane (300 mL) under reflux and undissolved solid was filtered off. Filtrate was concentrated under reduced pressure to yield

5-bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride as an off-white solid.

Yield: 9.73 g (69%). ¹H NMR spectrum (300 MHz, DMSO-d₆, d_H): 14.44 (bs, 1 H); 7.79 (d, J=2.5 Hz, 1 H); 7.73 (d, J=2.4 Hz, 1 H). ¹⁹F NMR spectrum (282 MHz, DMSO-d₆, d_F): -61.53.

(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-bromotetrahydro-2/7-pyran-3,4,5-triyl triacetate (18.4 g, 44.8 mmol) was dissolved in anhydrous 1 ,4-dioxane (30 mL) and ethylene glycol (27.0 mL). Silver carbonate (13.4 g, 48.8 mmol) was added in one portion (gas evolution after short induction period). The resulting mixture was stirred at ambient temperature for 4 hours. Afterwards, it was diluted with ethyl acetate (125 mL), filtered through a celite pad, washed with ethyl acetate (3 x 40 mL) and the filtrate was evaporated. The oily residue was dissolved in water (250 mL) and extracted with dichloromethane (4 x 150 mL). Combined organic extracts were dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. Residual oily liquid was triturated with diisopropyl ether (80 mL) and allowed to crystallize in freezer. The fine precipitate was collected by filtration, washed with chilled (0 °C) diisopropyl ether (2 x 30 mL), dried with suction in air and then in vacuo to give ((2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-hydroxyethoxy)tetrahydro-2/7-pyran-3,4,5-triyl triacetate as a colorless solid. Yield: 13.0 g (74%). ¹H NMR spectrum (300 MHz, CDCI3, d_H): 5.23 (t, J=9.5 Hz, 1 H); 5.13-4.97 (m, 2 H); 4.56 (d, J=7.9 Hz, 1 H); 4.20 (d, J=4.0 Hz, 2 H); 3.89-3.82 (m, 2 H); 3.82-3.66 (m, 3 H); 2.43 (dd, J=7.2 and 6.1 Hz, 1 H); 2.10 (s, 3 H); 2.06 (s, 3 H); 2.04 (s, 3 H); 2.02 (s, 3 H). LC-MS: 410.1 (M+H₂O)⁺, 415.0 (M+Na)⁺.

To a well stirred solution of the given ((2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(2- hydroxyethoxy)tetrahydro-2/7-pyran-3,4,5-triyl triacetate (12.9 g, 32.9 mmol) and bis(acetoxy)iodobenzene (25.4 g, 78.9 mmol) in acetonitrile (130 mL) and water (35 mL) was added (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (206 mg, 1.32 mmol) in one portion. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was treated with 10% aqueous solution of sodium sulfite (120 mL) and stirred for 30 minutes. Afterwards, pH of the solution was adjusted with concentrated hydrochloric acid to pH=2. The aqueous layer was extracted with dichloromethane (3 x 120 mL); the organic extracts were combined, dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was treated with dry diethyl ether (100 mL). A white solid was filtered, washed with dry diethyl ether (3 x 30 mL) and dried in vacuo affording 2-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6- (acetoxymethyl)tetrahydro-2/7-pyran-2-yl)oxy)acetic acid as a white solid.

Yield: 10.6 g (80%). ¹H NMR spectrum (300 MHz, CDCI₃, d_H): 5.26 (t, J=9.4 Hz, 1 H); 5.16- 5.01 (m, 2 H); 4.67 (d, J=7.9 Hz, 1 H); 4.36 (s, 2 H); 4.32-4.23 (m, 1 H); 4.21-4.11 (m, 1 H); 3.79-3.69 (m, 1 H); 2.10 (s, 3 H); 2.08 (s, 3 H); 2.04 (s, 3 H); 2.03 (s, 3 H). LC-MS: 424.0 (M+H₂O)⁺.

The given 2-(((2R,3R,4S,5R,6R)-3,4,5-T riacetoxy-6-(acetoxymethyl)tetrahydro-2/7-pyran-2- yl)oxy)acetic acid (2.15 g, 5.30 mmol) was dissolved in dry dichloromethane (27 mL) and triethylamine (2.22 mL, 15.9 mmol) was added. A mixture was cooled to 0 °C and 5-bromo-2- hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (2.34 g, 6.89 mmol) was added in three portions during 10 minutes. The mixture was stirred under cooling for 30 minutes, then at room temperature overnight. Solvent was evaporated in vacuo. To the residue ethyl acetate (80 mL) was added. Resulting suspension was filtered through celite pad and filtrate was evaporated. The residue was purified by flash column chromatography (Silicagel 60, 0.040- 0.063 mm; eluent: dichloromethane/2-propanol 100:0 to 95:5 + 0.5% of triethylamine) giving the O-peracetyl-glucoside Ac active ester (Intermediate compound 5) as yellowish foam. Yield: 4.13 g (96%). ¹H NMR spectrum (300 MHz, DMSO-d₆, 80 C, d_H): 8.72 (bs, 1 H); 8.17 (d, J=2.6 Hz, 1 H); 7.90 (d, J=2.6 Hz, 1 H); 5.19 (t, J=9.4 Hz, 1 H); 5.08 (d, J=7.2 Hz, 1 H); 4.95 (t, J=9.6 Hz, 1 H); 4.84 (dd, J=9.5 and 7.9 Hz, 1 H); 4.64-4.44 (m, 2 H); 4.26-4.05 (m, 2 H); 4.03-3.92 (m, 1 H); 3.12 (q, J=7.2 Hz, 8 H); 2.04 (s, 3 H); 1.99 (s, 3 H); 1.95 (s, 3 H); 1.94 (s, 3 H); 1.21 (t, J=7.3 Hz, 9 H). ¹⁹F NMR spectrum (282 MHz, CDCI₃, d_F): -59.78. LC-MS: 707.1

l-salidroside Ac active ester

com

Intermediate compound 6:

Salidroside tetraacetate (1.0 mg, 2.1 mmol, 1.0 eq) was dissolved in dry DMF (10 mL) and added solid potassium carbonate (650 mg, 4.7 mmol, 2.2 eq) followed by tert-butyl bromoacetate (0.35 mL, 2.3 mmol, 1.1 eq.). After stirring overnight most of the solvent was removed to leave a paste, which then was extracted with DCM and filtering. The solvent was removed, and the product was purified by on silica column eluting with a gradient of EtOAc in DCM. Product containing fractions were collected and evaporated to give an oil 1.09 g (87% yield).

¹H NMR (400 MHz, chloroform-d) 5 7.13 - 7.05 (m, 2H), 6.83 - 6.75 (m, 2H), 5.16 (t, J = 9.5 Hz, 1 H), 5.12 - 5.01 (m, 1 H), 4.98 (dd, J = 9.6, 8.0 Hz, 1H), 4.49 - 4.45 (m, 3H), 4.25 (dd, J = 12.3, 4.7 Hz, 1H), 4.14 - 4.03 (m, 2H), 3.69 - 3.64 (m, 1 H), 3.61 (dt, J = 9.5, 7.3 Hz, 1H), 2.81 (td, J = 6.8, 6.3, 3.0 Hz, 2H), 2.07 (s, 3H), 2.01 (s, 7H), 1.98 (s, 3H), 1.91 (s, 3H), 1.47 (s, 9H).

The given tert-butyl ester (1.09 g, 1.8 mmol) was dissolved in DCM (12 mL) and then added TFA (6 mL) with stirring. After 90 minutes the reaction mixture was evaporated to give a gum which was dissolving in DCM and purified on silica column eluting with a gradient of methanol in DCM, to give the O-peracetyl-salidroside Ac as a gum (960 mg, 1.8 mmol, 97 %).

¹H NMR (400 MHz, chloroform-d) 5 7.14 - 7.09 (m, 2H), 6.86 - 6.81 (m, 2H), 5.17 (t, J = 9.5 Hz, 1 H), 5.08 (t, J = 9.6 Hz, 1H), 4.98 (dd, J = 9.5, 8.0 Hz, 1H), 4.64 (s, 2H), 4.45 (d, J = 7.9 Hz, 1 H), 4.25 (dd, J = 12.3, 4.6 Hz, 1H), 4.15 - 4.04 (m, 2H), 3.69 - 3.59 (m, 2H), 2.83 (td, J = 6.8, 6.3, 3.4 Hz, 2H), 2.08 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.92 (s, 3H). O-peracetyl-salidroside Ac was transformed to the active ester (Intermediate compound 6) as described above for Intermediate compound 2 using 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride, and the active ester was used for insulin conjugation in crude form.

Preparation of O-peracetyl-qlucoside EG2 Ac active ester (Intermediate compound 7)

Intermediate 7:

Commercial (2S,3R,4S,5R,6R)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate (150.0 g, 128 mmol) was dissolved in ice cold dichloromethane (60 mL) with stirring and 33% solution of hydrogen bromide in acetic acid (66 mL) was added slowly with cooling (ice-bath). Three hours later, dichloromethane (300 mL) was added followed by ice- cold water (300 mL). Layers were separated; the organic layer was washed with ice-cold water (1 x 300 mL) and 10% aqueous solution of potassium bicarbonate (2 x 300 mL). Organic layer was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo. The resulting solid was triturated rapidly in hot diisopropyl ether (110 mL) and allowed to cool slowly to ambient temperature. When the crystallization was well advanced, the mixture was put in the fridge overnight. The fine crystals were collected by filtration, washed once with chilled (20 °C) diisopropyl ether (60 mL) and twice with n-hexane (2 x 100 mL). It was dried with suction in air and then in vacuo to give (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6- bromotetrahydro-2H-pyran-3,4,5-triyl triacetate as white solid.

Yield: 35.1 g (67%). ¹H NMR spectrum (300 MHz, CDCI3, dH): 6.61 (d, J=3.9 Hz, 1 H); 5.56 (t, J=9.7 Hz, 1 H); 5.24-5.05 (m, 1 H); 4.84 (dd, J=10.0 and 4.1 Hz, 1 H); 4.37-4.26 (m, 2 H); 4.17-4.08 (m, 1 H); 2.10 (s, 3 H); 2.09 (s, 3 H); 2.05 (s, 3 H); 2.02 (s, 3 H). The given (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (12.3 g, 30.0 mmol) was dissolved in a mixture of anhydrous 1 ,4-dioxane (15 mL) and triethylene glycol (30.0 mL, 225 mmol). Silver carbonate (9.00 g, 33.0 mmol) was added in one portion (gas evolution after short induction period). The resulting mixture was stirred at ambient temperature for three hours. Afterwards, it was diluted with ethyl acetate (120 mL), filtered through celite pad and washed with ethyl acetate (3 x 60 mL); the filtrate was evaporated. The residue was dissolved in water (200 mL) and extracted with dichloromethane (3 x 150 mL). Organic extracts were combined, dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. The residue was purified by flash column chromatography (Silicagel 60 (300 g), 0.040-0.063 mm; eluent: ethyl acetate) to give (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)tetrahydro-2H- pyran-3,4,5-triyl triacetate as white crystals. Yield: 11.0 g (76%). ¹H NMR spectrum (300 MHz, CDCI3, dH): 5.20 (t, J=9.4 Hz, 1 H); 5.13-4.93 (m, 2 H); 4.61 (d, J=7.9 Hz, 1 H); 4.25 (dd, J=12.3 and 4.7 Hz, 1 H); 4.13 (dd, J=12.3 and 2.4 Hz, 1 H); 3.94 (dt, J=10.8 and 4.0 Hz, 1 H); 3.79-3.56 (m, 12 H); 2.49 (t, J=5.7 Hz, 1 H); 2.07 (s, 3 H); 2.04 (s, 3 H); 2.01 (s, 3 H); 1.98 (s, 3 H). LC-MS: 481.0 (M+H)+

A well stirred solution of the given ((2R,3R,4S,5R,6R)- 2-(acetoxymethyl)-6-(2-(2-(2- hydroxyethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3, 9.20 g, 19.1 mmol) and bis(acetoxy)iodobenzene (14.9 g, 46.3 mmol) in a mixture of acetonitrile (95 mL) and water (25 mL) was added 2,2,6,6-tetramethylpiperidin-1-yl-oxyl (121 mg, 0.77 mmol) in one portion. The reaction mixture was stirred for 16 hours and concentrated in vacuo. The residue was treated with 10% aqueous solution of sodium sulfite (100 mL) and stirred for 30 minutes. Afterwards, pH of the solution was adjusted with concentrated hydrochloric acid to pH=2. The aqueous layer was extracted with dichloromethane (3 x 100 mL). The organic extracts were combined, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by flash column chromatography (Silicagel 60 (300 g), 0.040-0.063 mm; eluent: acetonitrile/water 30:1 + 0.5% of formic acid). The oily residue was freeze dried from 50% aqueous acetonitrile (60 mL) to give 2-(((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)acetic acid as yellowish oil.

Yield: 9.20 g (97%). ^1H NMR spectrum (300 MHz, CDCI3, dH): 6.12 (bs, 1 H); 5.24 (t, J=9.4 Hz, 1 H); 5.05 (dt, J=17.4 and 9.3 Hz, 2 H); 4.62 (d, J=7.9 Hz, 1 H); 4.29-4.13 (m, 4 H); 4.00- 3.94 (m, 1 H); 3.79-3.67 (m, 8 H); 2.09 (s, 3 H); 2.05 (s, 3 H); 2.03 (s, 3 H); 2.01 (s, 3 H). LC-MS: 495.0 (M+H)+. The given 2-(((2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2- yl)oxy)acetic acid (200 mg) was dissolved in dry DMF (4 ml). TEA (169 uL) was added followed by 5-bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (151 mg) and the mixture turned yellow. After 50 minutes, the reaction mixture was diluted with DCM, washed with 0.1M HCI (2x), brine(1x), dried (Na2SO4) and concentrated in vacuo to give a clear sirup, which was used as crude product, O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7).

Preparation of O-peracetyl-qlucoside-ethyl-triazole-carboxylate active ester (Intermediate compound 8)

Intermediate compound 8:

Azidoethyl-glucoside (1.0 g, 2.4 mmol) and tert-butyl propiolate (0.41 g, 3.3 mmol, 1.4 eq) were dissolved in THF (10.0 mL) and water (4.0 mL). Then ascorbic acid (0.19 g, 0.96 mmol, 0.400 eq) dissolved in water (3 mL) followed by copper sulfate (0.12 g, 0.48 mmol, 0.200 eq) in water (1 mL) was added dropwise. After stirring this two-phase system overnight, the reaction was worked up by partitioning between EtOAc and water, washed with more water then brine, passed the organic through a bed of sodium sulfate then evaporated to give an oil that was adsorbed onto silica gel 4 g and purified on silica column eluting with a gradient of EtOAc in DCM. Evaporation of the product containing fractions started to crystallise on standing which gave a crystalline solid (0.40 g, 0.74 mmol, 31 % yield).

¹H NMR (400 MHz, chloroform-d) 5 8.03 (s, 1 H), 5.16 (t, J = 9.4 Hz, 1 H), 5.06 (t, J = 9.6 Hz, 1H), 4.99 (dd, J = 9.7, 8.0 Hz, 1H), 4.67 (ddd, J = 14.5, 4.5, 3.0 Hz, 1 H), 4.52 (ddd, J = 14.6, 9.0, 3.3 Hz, 1H), 4.45 (d, J = 8.0 Hz, 1 H), 4.27 - 4.20 (m, 2H), 4.13 (dd, J = 12.4, 2.4 Hz, 1H), 3.91 (ddd, J = 10.6, 9.0, 3.0 Hz, 1H), 3.68 (ddd, J = 10.0, 4.7, 2.4 Hz, 1 H), 2.09 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.96 (s, 3H). The given tert-butyl ester (150 mg, 275 pmol) was dissolved in a mixture of TFA and DCM (1 :3, 2 mL). After 3 hours the reaction mixture was blown down. Redissolved in DCM then blown down three times more, then dissolved in EtOAc and loaded onto a silica gel column eluting with a gradient of MeOH in EtOAc. The product containing fractions were evaporated to give O-peracetyl-glucoside-ethyl-triazole-carboxylic acid as a solid (56 mg, 41% yield).

¹H NMR (400 MHz, chloroform-d) 5 8.23 (s, 1 H), 5.18 (t, J = 9.5 Hz, 1 H), 5.07 (t, J = 9.7 Hz, 1 H), 5.00 (dd, J = 9.6, 7.9 Hz, 1 H), 4.72 (d, J = 14.5 Hz, 1 H), 4.65 - 4.54 (m, 1 H), 4.49 (d, J = 7.9 Hz, 1 H), 4.31 - 4.19 (m, 3H), 4.13 (dd, J = 12.8, 2.7 Hz, 1 H), 3.94 (t, J = 9.2 Hz, 1H), 3.69 (ddd, J = 9.9, 4.7, 2.3 Hz, 1 H), 2.10 (s, 3H), 2.03 - 1.97 (m, 9H).

O-Peracetyl-glucoside-ethyl-triazole-carboxylic acid was transformed to the active ester as described above for Intermediate compound 2 using 5-bromo-2-hydroxy-3-

(trifluoromethyl)benzenesulfonyl chloride, and the active ester (Intermediate compound 8) was used for insulin conjugation in crude form.

Preparation of macrocycle EG3 azide (Intermediate compound 9)

Intermediate compound 9:

Step 1: Synthesis of the tris-trifluoroacetate phenol

3,5-dimethylphenol (112 mg, 0.917 mmol, 1 eq.) and 2,2,2-trifluoro-N-hydroxymethyl)- acetamide (517 mg, 3.616 mmol, 3.95 eq.) were dissolved in anhydrous DCM (3 mL). TFA (1 mL) and 1 mM BF3.OEt₂ in Et₂O (3.21 mL, 3.21 mmol, 3.5 eq.) were added and the reaction was stirred for 18 hours. The reaction was quenched by pouring onto ice. The resultant suspension was diluted with EtOAc (200 mL), this was washed with water (3 x 100 mL) and dried (Na₂SC>4). The organic solvents were removed under reduced pressure and the crude product was purified by flash chromatography (DCM with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the tris-trifluoroacetate phenol (216 mg, 0.434 mmol, 47%) as a white solid.

Step 2: Synthesis of the tris-trifluoroacetate EG3 azide

The given tris-trifluoroacetate phenol (2.00 g, 4.02 mmol, 1 eq.), K₂COs (1.22 g, 8.85 mmol, 2.2 eq.), Nal (0.600 g, 4.02 mmol, 1 eq.) and 2-(2-(2-Azidoethoxy)ethoxy)ethyl 4- methylbenzenesulfonate (1.66 g, 5.03 mmol, 1.25 eq.) were dissolved in anhydrous DMF (10 mL) and the reaction was stirred at 70 °C for 16 hours. Some of the solvent was removed under a stream of N2 and the remainder partitioned between EtOAc (50 mL) and water (50 mL). The organic layer was further washed with water (50 mL) and brine (50 mL) then dried (Na2SC>4) and concentrated under reduced pressure to give a brown oil. The crude product was purified by flash chromatography (DCM with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the tris- trifluoroacetate EG3 azide (2.35 g, 3.59 mmol, 89%) as a solid.

1 H NMR (400 MHz, Chloroform-d) 5 7.39 (d, J = 6.1 Hz, 2H), 6.28 (s, 1 H), 4.59 (t, J = 4.8 Hz, 6H), 4.03 - 3.99 (m, 2H), 3.93 - 3.89 (m, 2H), 3.81 - 3.77 (m, 2H), 3.71 - 3.67 (m, 2H), 3.65 (dd, J = 5.5, 4.4 Hz, 2H), 3.38 (dd, J = 5.5, 4.3 Hz, 2H), 2.43 (s, 6H).

Step 3: Synthesis of the tris-amine EG 3 azide

The given tris-trifluoroacetate EG3 azide (2.35 g, 3.59 mmol, 1 eq.) was dissolved in MeOH (30 mL) and concentrated aqueous ammonia (20 mL) was added. The reaction was stirred for 70 hours and the solvents were then removed under reduced pressure. The residue was redissolved in water and this was evaporated again to give the tris-amine EG3 azide (1.32 g, 3.59 mmol, 100%) as an oil, which was used without any purification.

Step 4: Synthesis of the tris-Boc EG3 azide

The given tris-amine EG3 azide (1.32 g, 3.59 mmol, 1 eq.) was dissolved in THF (36 mL). BOC2O (3.53 g, 16.2 mmol, 4.5 eq.) and K2CO3 (1.51 g, 10.9 mmol, 3 eq.) were added and the reaction stirred for 30 minutes. Et20 (50 mL) was added, and the reaction stirred for 10 minutes. The resultant suspension was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM with increasing Et20). The fractions containing the product were combined and the solvent removed under vacuum to yield the tris-Boc EG3 azide (1.10 g, 1.65 mmol, 46%) as a white solid. 1 H NMR (400 MHz, chloroform-d) 5 4.97 (s, 3H), 4.42 - 4.34 (m, 6H), 3.94 (dd, J = 5.5, 3.1 Hz, 2H), 3.85 (dd, J = 5.7, 2.9 Hz, 2H), 3.75 - 3.68 (m, 6H), 3.41 - 3.38 (m, 2H), 2.39 (s, 6H), 1.44 (d, J = 4.2 Hz, 27H).

Step 5: Synthesis of the tris-isocyanate EG3 azide

The given tris-Boc EG3 azide (134 mg, 0.201 mmol), 2-chloropyridine (0.17 mL, 1.809 mmol) and triflic anhydride (0.15 mL, 0.904 mmol) were reacted as described for propyl azide trisisocyanate (3). The crude extract was purified by passing through a silica plug and evaporating the solvent to give the tris-isocyanate EG3 azide (89 mg, 0.201 mmol, 100%) as a clear oil.

1 H NMR (400 MHz, chloroform-d) 5 4.59 (s, 4H), 4.47 (s, 2H), 4.04 - 4.01 (m, 2H), 3.90 - 3.87 (m, 2H), 3.78 - 3.71 (m, 6H), 3.44 - 3.40 (m, 2H), 2.45 (s, 6H).

Step 6: Synthesis of macrocycle EG3 azide (Intermediate compound 9)

Macrocycle EG3 azide (Intermediate compound 9) was made from the given tris-isocyanate EG3 azide (84 mg, 0.190 mmol), the half macrocycle (Intermediate 19)(130 mg, 0.150 mmol), and NaOH (54 mg, 1.35 mmol) similar to the synthesis of the macrocycle propyl azide (Intermediate compound 3). The macrocycle EG3 azide (Intermediate compound 9) was isolated as a white solid (50 mg, 0.041 mmol, 27%). ¹H NMR (400 MHz, deuterium oxide 5 7.96 (d, J = 2.7 Hz, 3H), 7.60 (d, J = 8.5 Hz, 3H), 7.45 (d, J = 8.5 Hz, 3H), 4.05 (s, 4H), 3.96 (s, 2H), 3.76 (s, 2H), 3.59 (s, 6H), 3.38 (s, 4H), 2.54 (s, 6H), 2.35 - 2.00 (m, 12H), 1.10 (t, J = 7.2 Hz, 9H). MS (electrospray) [M+H]+ calculated for C59H70N15O15 requires: 1228.5, found: 1228.4.

Preparation of macrocycle EG2 azide (Intermediate compound 10)

Intermediate compound 10:

Step 1: Synthesis of tris-trifluoroacetate EG2 azide

2-(2-Azidoethoxy)ethyl 4-methylbenzenesulfonate (749 mg, 2.63 mmol), and the tris- trifluoroacetate phenol (1.05 g, 2.10 mmol) prepared as under step 1 in the preparation of Intermediate compound 9, K2CO3 (639 mg, 4.62 mmol) and Nal (315 mg, 2.10 mmol) were reacted as described as for Intermediate compound 9. The tris-trifluoroacetate EG2 azide was isolated as a white solid (970 mg, 1.59 mmol, 76%).

1 H NMR (400 MHz, methanol-d4) 5 4.66 (s, 4H), 4.59 - 4.55 (m, 2H), 4.03 - 3.98 (m, 2H), 3.88 - 3.84 (m, 2H), 3.79 - 3.72 (m, 2H), 3.47 - 3.42 (m, 2H), 2.35 (s, 6H).

Step 2: Synthesis oftris-Boc EG2 azide

The given tris-trifluoroacetate EG2 azide (970 mg, 1.59 mmol, 1 eq.) was dissolved in MeOH (10 mL) and NaOH (508 mg, 12.7 mmol, 8 eq.) was added as a solution in water (5 mL). The reaction was stirred for 17 hours. NaHCO3 (667 mg, 7.95 mmol, 5 eq.) and water (5 mL) were added and the MeOH removed under reduced pressure. Boc2O (1.56 g, 7.15 mmol, 4.5 eq.) was added, followed by THF (14 mL) and the reaction was stirred for 2 hours. EtOAc (60 mL) was added and the organics washed with water (2 x 60 mL) and brine (40 mL) then dried (Na2SO4). The solution was dry loaded onto silica and purified by flash chromatography (DCM:Et2O 4:1). The fractions containing the product were combined and the solvent removed under vacuum to yield the tris-Boc EG2 azide (838 mg, 1 .35 mmol, 85%) as a white solid. 1 H NMR (400 MHz, methanol-d4) 5 6.55 (s, 1 H), 4.36 (d, J = 4.0 Hz, 4H), 4.29 (s, 2H), 3.98 - 3.94 (m, 2H), 3.87 - 3.83 (m, 2H), 3.75 (dd, J = 5.5, 4.5 Hz, 2H), 3.46 (dd, J = 5.6, 4.4 Hz, 2H), 2.34 (s, 6H), 1.45 (d, J = 1.8 Hz, 27H). Step 3: Synthesis of tris-isocyanate EG2 azide

The given tris-Boc EG2 azide (292 mg, 0.469 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (3 mL), followed by addition of anhydrous 2-chloropyridine (0.31 mL, 3.28 mmol, 9 eq.). T riflic anhydride (0.26 mL, 1.55 mmol, 4.5 eq.) was added dropwise to the reaction mixture and the reaction stirred at room temperature for a further 10 minutes after addition was complete. Toluene (10 mL) was added to the stirring solution and the suspension was filtered. The filtrate was concentrated under reduced pressure and the residue passed through a silica plug, eluting Et20. The solvent was evaporated to give the tris-isocyanate EG2 azide (85 mg, 0.212 mmol, 45%) as an amorphous solid.

1 H NMR (400 MHz, Chloroform-d) 54.60 (s, 4H), 4.48 (s, 2H), 4.08 - 4.03 (m, 2H), 3.92 - 3.86 (m, 2H), 3.78 (dd, J = 5.4, 4.5 Hz, 2H), 3.50 (dd, J = 5.5, 4.4 Hz, 2H), 2.46 (s, 6H).

Step 4: Synthesis of the macrocycle EG2 azide (intermediate compound 10)

Intermediate compound 10 was made from the given tris-isocyanate EG2 azide (122 mg, 0.304 mmol), Intermediate compound 19 (220 mg, 0.253 mmol), and NaOH (92 mg, 2.23 mmol) similar to the synthesis of Intermediate compound 3. The macrocycle EG2 azide (Intermediate compound 10) was isolated as a white solid (64 mg, 0.054 mmol, 21%). 1 H NMR (400 MHz, deuterium oxide (with NaOH)) 5 7.71 - 7.51 (m, 9H), 4.38 - 4.23 (m, 6H), 4.21 - 4.06 (m, 6H), 3.77 - 3.63 (m, 2H), 3.54 - 3.41 (m, 2H), 3.31 - 3.20 (m, 2H), 3.16 - 3.08 (m, 2H), 2.73 - 2.53 (m, 6H), 2.11 - 1.84 (m, 6H), 1.04 - 0.94 (m, 9H). MS (electrospray), [M+H]+ calculated for C57H66N15O14 requires: 1184.5, found: 1184.4.

Preparation of O-succinimidyl-propynoate (Intermediate compound 11)

Intermediate compound 11 :

/V-Hydroxysuccinimide (16.5 g, 143 mmol) and propiolic acid (10.5 g, 150 mmol) were added to ethyl acetate (500 mL) at 0 °C. Then A/./V-dicyclohexylcarbodiimide (29.5 g, 143 mmol) was added and reaction mixture was stirred at 0 °C for 3 hours. Precipitated byproduct was removed by filtration. The filtrate was concentrated to ca. 200 mL and washed with brine (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated to 30 mL and cooled to 0 °C. Heptane (12 mL) was added and the mixture containing precipitated solid was filtered through short silica gel column to give the O-succinimidyl-propynoate (Intermediate compound 11) as white solid. Yield: 8.88 g (35%). RF (SiO2, cyclohexane/ethyl acetate 3:2): 0.40. ¹H NMR spectrum (300 MHz, CDCI₃, H): 3.30 (s, 1 H); 2.87 (s, 4 H). LC-MS: 168.2 (M+H)⁺.

Preparation of macrocycle propyl-NHAc azide (Intermediate compound 12)

Intermediate compound 12:

Step 1: Synthesis of the MC propyl-amine, hydrochloride

Macrocycle propyl azide (Intermediate compound 3) (31 mg, 0.027 mmol, 1 eq.) was dissolved in THF (1 mL) and water (1 mL) with a drop of 1 M HCI. A slurry of Pd/C (5 mg, 10% w/w) in THF/water was added and the reaction was placed under an atmosphere of hydrogen and stirred overnight. The reaction mixture was centrifuged, and the supernatant concentrated under reduced pressure to give the MC propyl-amine, hydrochloride (30 mg, 0.027 mmol, 99%) as a white solid. MS (electrospray), [M+H]+ calculated for C56H66N13O12 requires: 1112.5, found: 1112.4.

Step 2: Synthesis of macrocycle propyl-NHAc azide (Intermediate compound 12)

The given MC propyl-amine, hydrochloride (100 mg, 0.090 mmol, 1 eq.) and NaHCCh (30 mg, 0.360 mmol, 4 eq.) were suspended in anhydrous THF (5 mL). To the suspension was added azidoacetic acid NHS ester (53 mg, 0.270 mmol, 3 eq.) and the reaction was stirred for 24 hours. Further NaHCOs (30 mg, 0.360 mmol, 4 eq.) and azidoacetic acid NHS ester (53 mg, 0.270 mmol, 3 eq.) was added and the reaction was stirred for 60 hours. The solvents were removed under reduced pressure and the crude solid was then dissolved in acetone/water, dry loaded onto C18 column and purified by reverse phase flash chromatography. The fractions containing the product were combined and the solvent removed under vacuum to yield the product macrocycle propyl-NHAc azide (Intermediate compound 12) (70 mg, 0.059 mmol, 65%) as a white solid. 1H NMR (400 MHz, DMSO-d6) 5 8.57 - 7.79 (m, 9H), 7.76 - 7.23 (m, 6H), 6.69 - 6.29 (m, 6H), 4.53 - 4.12 (m, 12H), 3.78 (s, 2H), 2.84 (s, 4H), 2.66 (s, 6H), 2.39 (s, 6H), 1.58 (s, 2H), 1.29 - 1.05 (m, 9H). MS (electrospray), [M+H]+ calculated for C58H67N16O13 requires:1195.5, found: 1195.4. Preparation of O-peracetyl-glucoside Ac-Glv active ester (Intermediate compound 13)

Intermediate compound 13:

A mixture of 1-((dimethylamino)(dimethyliminio)methyl)-1/7-[1 ,2,3]triazolo[4,5-b]pyridine 3- oxide hexafluorophosphate(V) (HATLI, 1.20 g, 3.15 mmol) and 2-(((2R,3R,4S,5R,6R)-3,4,5- triacetoxy-6-(acetoxymethyl)tetrahydro-2/7-pyran-2-yl)oxy)acetic acid (1.22 g, 3.00 mmol) in dry dichloromethane (15 mL) was stirred at room temperature for 30 minutes. Then 2,4,6- collidine (1.98 mL, 15.0 mmol) was added to the reaction mixture, followed by the addition of 2-(tert-butoxy)-2-oxoethan-1-aminium chloride (603 mg, 3.60 mmol) in four portions over 10 minutes. The mixture was stirred at room temperature overnight. Solvent was removed in vacuo. The residue was partitioned between ethyl acetate (30 mL) and water (30 mL). Separated organic layer was washed with 1 M aqueous solution of potassium bisulfate (2 x 30 mL), 5% aqueous solution of lithium chloride (3 x 30 mL), water (2 x 30 mL) and brine (2 x 30 mL), and dried over anhydrous magnesium sulfate. After filtration, solvent was evaporated under reduced pressure to afford (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-((2-(ferf-butoxy)- 2-oxoethyl)amino)-2-oxoethoxy)tetrahydro-2/7-pyran-3,4,5-triyl triacetate as white foam. Yield: 1.46 g (94%).

¹H NMR spectrum (300 MHz, CDCI₃, d_H): 6.92 (t, J=5.3 Hz, 1 H); 5.24 (t, J=8.0 Hz, 1 H); 5.16-5.02 (m, 2 H); 4.58 (d, J=7.9 Hz, 1 H); 4.39-4.21 (m, 2 H); 4.20-4.10 (m, 2 H); 4.07-3.96 (m, 1 H); 3.96-3.85 (m, 1 H); 3.78-3.69 (m, 1 H); 2.11 (s, 3 H); 2.09 (s, 3 H); 2.04 (s, 3 H); 2.03 (s, 3 H); 1.47 (s, 9 H). LC-MS: 520.1 (M+H)⁺.

The given (2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(2-((2-(terf-butoxy)-2-oxoethyl)amino)-2- oxoethoxy)tetrahydro-2/7-pyran-3,4,5-triyl triacetate (1.46 g, 2.81 mmol) was dissolved in trifluoroacetic acid (10 mL). Resulting solution was stirred at room temperature for 45 minutes. A mixture was diluted with 1 ,2-dichloroethane (20 mL) and solvents were removed under reduced pressure. The residue was co-distilled with 1 ,2-dichloroethane (3 x 15 mL). Dry diethyl ether (40 mL) was added to the residue. The mixture was stored at -20 °C in the freezer for 1 hour. A solid was filtered, washed with dry diethyl ether (3 x 10 mL) and n- hexane (3 x 10 mL), and dried in vacuo to give (2-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6- (acetoxymethyl)tetrahydro-2/7-pyran-2-yl)oxy)acetyl)glycine as a white solid. Yield: 1.17 g (90%). ¹H NMR spectrum (300 MHz, DMSO-d₆, d_H): 12.67 (bs, 1 H); 7.73 (t, J=5.6 Hz, 1 H);

5.26 (t, J=9.2 Hz, 1 H); 5.00-4.82 (m, 3 H); 4.21-3.95 (m, 5 H); 3.80 (dd, J=5.8 and 1.9 Hz, 2 H); 2.02 (s, 6 H); 1.99 (s, 3 H); 1.94 (s, 3 H). LC-MS m/z: 464.0 (M+H)⁺

O-Peracetyl-glucoside Ac-Gly active ester (Intermediate compound 13) was made in situ by activation as described for O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) above.

Preparation of O-peracetyl-glucoside benzaldehyde (Intermediate compound 14)

Intermediate compound 14:

(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (8.22 g, 20.0 mmol) and 4-hydroxybenzaldehyde (2, 2.44 g, 20.0 mmol) were dissolved in dry acetonitrile (50 mL) and silver carbonate (6.00 g, 21.7 mmol) was added. The reaction mixture was stirred overnight. Then the solvent was removed in vacuo. The residue was purified by flash column chromatography (Silicagel 60, 0.040-0.060 mm; eluent: cyclohexane/ethyl acetate 1 :1) and subsequently triturated with 2-propanol (15 mL). The solid was filtered through sintered glass giving the O-peracetyl-glucoside benzaldehyde (Intermediate compound 14) as white powder.

Yield: 2.29 g (25%). RF (SiO2, cyclohexane/ethyl acetate 1 :1): 0.40. 1H NMR spectrum (300 MHz, CDCI3, dH): 9.94 (s, 1 H); 7.86 (d, J=8.6 Hz, 2 H); 7.11 (d, J=8.6 Hz, 2 H); 5.38-5.14 (m, 4 H); 4.35-4.15 (m, 2 H); 3.98-3.88 (m, 1 H); 2.09-2.04 (m, 12 H). LC-MS: 470.1 (M+H2O)+.

Preparation of O-peracetyl-glucoside benzylaldehyde (Intermediate compound 15)

Intermediate compound 15:

(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (6.15 g, 15.0 mmol) and 1 ,4-phenylenedimethanol (2, 8.30 g, 60.0 mmol) were dissolved in dry 1,4-dioxane (40 mL). Silver carbonate (4.15 g, 15.0 mmol) was then added to the reaction mixture. A well mixed suspension was stirred at room temperature for 20 hours. The mixture was diluted with ethyl acetate (100 mL) and filtered through the celite pad. The filtrate was concentrated in vacuo and dichloromethane (150 mL) was added. Mixture was stirred for 30 minutes. The undissolved excess of 1 ,4-phenylenedimethanol was removed by filtration and washed with dichloromethane (10 mL). Filtrate was evaporated, dissolved in acetonitrile (50 mL) and evaporated again. The residue was purified by HPLC (Gemini C18, 5 m, 250 mm x 50 mm, acetonitrile/water, 15:85 during 20 minutes, 15:85 to 45:55 during 90 minutes, 45:55 to 55:45 during 30 minutes + 0.05% AcOH). The product was triturated with dry diisopropyl ether (35 mL) and white crystals were formed. The mixture was evaporated giving (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((4-(hydroxymethyl)benzyl)oxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate.

Yield: 2.33 g (33%). 1H NMR spectrum (300 MHz, CDCI3, dH): 7.38 (d, 2 H); 7.30 (d, 2 H); 5.21-5.04 (m, 3 H); 4.90 (d, 1 H); 4.72 (d, 2 H); 4.64 (d, 1 H); 4.55 (d, 1 H); 4.30-4.15 (m, 2 H); 3.70-3.64 (m, 1 H); 2.12 (s, 3 H); 2.03 (s, 6 H); 2.01 (s, 3 H); 1.73-1.63 (bs, 1 H). LC-MS m/z: 486.0 (M+H2O)+. A mixture of the given (2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-((4- (hydroxymethyl)benzyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3, 3.08 g, 6.57 mmol) and manganese dioxide (11.4 g, 131 mmol) in dry dichloromethane (30 mL) was stirred at room temperature for 4 hours. Afterwards, the reaction mixture was diluted with ethyl acetate (30 mL), filtered through the paper filter with wadding and evaporated in vacuo. The residue was dissolved in ethyl acetate (30 mL) and filtered through short silicagel column topped with celite (Silicagel 60 (30 g), 0.040-0.063 mm; eluent: ethyl acetate). The product was triturated with dry diisopropyl ether (27 mL) and white crystals were formed. The mixture was evaporated giving the O-peracetyl-glucoside benzylaldehyde (Intermediate compound 15) as white crystalline solid.

Yield: 2.60 g (85%). 1H NMR spectrum (300 MHz, CDCI3, dH): 10.02 (s, 1 H); 7.87 (d, J=8.1 Hz, 2 H); 7.47 (d, J=8.1 Hz, 2 H); 5.26-5.09 (m, 3 H); 5.01 (d, J=13.2 Hz, 1 H); 4.72 (d, J=13.2 Hz, 1 H); 4.63 (d, J=7.9 Hz, 1 H); 4.33-4.16 (m, 2 H); 3.76-3.71 (m, 1 H); 2.10 (s, 3 H); 2.04 (s, 3 H); 2.03 (s, 3 H); 2.02 (s, 3 H). LC-MS m/z: 484.0 (M+H2O)+.

Preparation of O-peracetyl-glucoside-1-NHAc-EG-Ac active ester (Intermediate compound

16)

Intermediate compound 16:

Step 1: Synthesis of the Ac-EG-Ac mono durene ester

A mixture of 2,2'-[ethylenebis(oxy)] bisacetic acid (3.2 g, 18 mmol), potassium carbonate (2.7 g, 19.7 mmol, 1.1 eq) and sodium iodide in DMF (25.0 mL) was stirred at 60 °C to give a milky suspension. To this was added alpha-2-chloroisodurene (3.0 g, 17.9 mmol, 1.0 eq) and after 4 hours the reaction mixture became a gel. After 6 hours the reaction was removed from the heat and partitioned between EtOAc and dilute citric acid. The organic phase was washed with more dilute citric acid solution, then brine before drying with sodium sulfate and evaporating to give an oil (4.14 g). 1 g of this material was taken for purification on silica gel column using methanol in DCM. A pure sample of the bis ester [bis(2,4,6-trimethylbenzyl) 2,2'-(ethane-1 ,2-diylbis(oxy))diacetate] was isolated along with a little of the desired mono ester [2-(2-(2-oxo-2-((2,4,6-trimethylbenzyl)oxy)ethoxy)ethoxy)acetic acid]. The majority of the material from the column was the bis ester (-775 mg), which was partially hydrolysed as described below. To the bis ester (0.755 g, 1.706 mmol, 1.000 eq) dissolved in DCM (2.4 mL) was added TFA (0.200 mL). After 24 hours the reaction mixture was diluted with EtOAc and washed several times with water before drying the organic with sodium sulfate and adsorbing onto silica gel. Attempts to purify with silica gel column using methanol in DCM gave 330 mg of a 1:1 mixture of the Ac-EG-Ac mono durene ester (0.137 g, 0.440 mmol) and bis ester (0.195 g, 0.440 mmol) which was used in the next step without further characterisation.

Step 2: Synthesis of the glucoside- 1-NHAc-EG- Ac mono durene ester

A mixture of the given mono and bis esters, which contains 2-(2-(2-oxo-2-((2,4,6-trimethyl- benzyl)oxy)ethoxy)ethoxy)acetic acid (0.14 g, 0.44 mmol), was dissolved in DMSO (500 pL). Then, DIPEA (84 pL, 0.48 mmol, 2.0 eq) and HBTLI (101 mg, 0.27 mmol, 1.1 eq) were added to the solution. After 5 minutes alpha-D-glucosylamine (52.0 mg, 0.290 mmol, 1.200 eq) was dissolved in DMSO (1 mL) and added to the reaction mixture. After 2 hours most of the DMSO was distilled away and the remaining gum purified by reverse phase MPLC with a gradient of acetone in water to give the glucoside-1-NHAc-EG-Ac mono durene ester (53.0 mg, 0.112 mmol, 46.5%) as a gum. 1 H NMR (400 MHz, DMSO-d6) 5 8.05 (d, J = 9.3 Hz, 1 H), 6.86 (s, 2H), 5.16 (s, 2H), 5.01 (d, J = 4.2 Hz, 1 H), 4.93 (dd, J = 7.5, 5.2 Hz, 2H), 4.74 (t, J = 8.8 Hz, 1 H), 4.52 (t, J = 5.8 Hz, 1 H), 4.15 (s, 2H), 3.91 (s, 2H), 3.61 (m, 5H), 3.21 - 3.08 (m, 3H), 3.05 (m, 1 H), 2.28 (s, 6H), 2.21 (s, 3H).

Step 3: Synthesis of the O-peracetyl-glucoside-1-NHAc-EG-Ac mono durene ester

To the given glucoside-1-NHAc-EG-Ac mono durene ester (50.0 mg, 0.106 mmol, 1.0 eq) dissolved in pyridine (1.0 mL) was added acetic anhydride (50 pL, 0.530 mmol, 5.0 eq). After 18 hours the reaction mixture was blown down to a gum, then partitioned between EtOAc and 1M HCI, washed with water then brine, dried with sodium sulfate and evaporated to give the O-peracetyl-glucoside-1-NHAc-EG-Ac mono durene ester (68.0 mg, 0.106 mmol, 100 %) as a gum. 1 H NMR (400 MHz, Chloroform-d) 5 7.68 (d, J = 9.4 Hz, 1 H), 6.87 (s, 2H), 5.32 - 5.23 (m, 4H), 5.13 - 5.07 (m, 1 H), 5.03 (t, J = 9.5 Hz, 1 H), 4.28 (dd, J = 12.4, 4.5 Hz, 1 H), 4.20 - 3.94 (m, 6H), 3.82 (ddd, J = 10.0, 4.5, 2.2 Hz, 1 H), 3.75 - 3.62 (m, 4H), 2.34 (s, 6H), 2.26 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.99 (d, J = 4.7 Hz, 6H).

Step 4: Synthesis of the O-peracetyl-glucoside-1-NHAc-EG-Ac

The given O-peracetyl-glucoside- 1-NHAc-EG-Ac mono durene ester (250.0 mg, 0.391 mmol, 1.000 eq) was dissolved in a mixture of DCM and TFA (2:1 , 3mL). After 30 minutes the solvent was evaporated to give a gum which was purified by reverse phase MPLC using a gradient of acetone in water. The organic solvent was evaporated, and the aqueous solution freeze dried to give O-peracetyl-glucoside-1-NHAc-EG-Ac (60.0 mg, 0.118 mmol, 30.3%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) 5 7.68 (d, J = 9.4 Hz, 1 H), 5.29 (td, J = 9.4, 5.4 Hz, 2H), 5.08 (dt, J = 18.8, 9.5 Hz, 2H), 4.32 (dd, J = 12.5, 4.5 Hz, 1 H), 4.26 - 4.15 (m, 2H), 4.10 (dd, J = 12.5, 2.1 Hz, 1 H), 4.07 - 3.97 (m, 2H), 3.83 (ddd, J = 10.1 , 4.5, 2.1 Hz, 1 H), 3.77 (q, J = 3.8 Hz, 2H), 3.73 - 3.65 (m, 2H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H).

Step 5: Synthesis of the active ester 16

The active ester 16 was made in situ by activation as described for O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) above.

Intermediate compound 17:

Step 1: Synthesis of the ethyl nitro Boc-amino phenylacetate

A solution of BOC2O (3.75 g, 17.2 mmol, 1.1 eq.) in DCM (25 mL) was added, by way of a dropping funnel, to a solution of ethyl 2-(4-amino-3-nitrophenyl)acetate (3.5 g, 15.6 mmol, 1 eq.), Et₃N (2.2 mL, 15.6 mmol, 1 eq.) and DMAP (1.05 g, 8.59 mmol, 0.55 eq.) in DCM (30 mL). After stirring for an hour, the reaction mixture was concentrated under reduced pressure and the residue partitioned between EtOAc (100 mL) and 1 M HCI (100 mL). The organic layer was concentrated under reduced pressure and the crude product was purified by flash chromatography (DCM with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the ethyl nitro Boc-amino phenylacetate (2.5 g, 7.71 mmol, 49%) as a yellow oil. 1H NMR (400 MHz, Chloroform-d) 5 9.62 (s, 1 H), 8.52 (d, J = 8.8 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1 H), 7.53 (dd, J = 8.8, 2.2 Hz, 1 H), 4.16 (q, J = 7.1 Hz, 2H), 3.62 (s, 2H), 1.54 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H).

Step 2: Synthesis of the ethyl amino Boc-amino phenylacetate

The given ethyl nitro Boc-amino phenylacate (295 mg, 0.91 mmol, 1 eq.) was dissolved in

EtOH (10 mL) and a slurry of Pd/C (37 mg, 10% w/w) in DCM was added and the reaction was placed under an atmosphere of hydrogen and stirred overnight. The reaction mixture was filtered through a bed of celite and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (petroleum ether 40/60 with increasing EtOAc). The fractions containing the product were combined and the solvent removed under vacuum to yield the ethyl amino Boc-amino phenylacetate (236 mg, 0.802 mmol, 88%) as an oil.

1 H NMR (400 MHz, Chloroform-d) 5 7.20 (d, J = 8.0 Hz, 1 H), 6.74 - 6.67 (m, 2H), 6.22 (s, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.49 (s, 2H), 1.50 (s, 10H), 1.24 (t, J = 7.1 Hz, 3H).

Step 3: Synthesis of the tris-Boc-amino homo half macrocycle

Boc

The given ethyl amino Boc-amino phenylacetate (226 mg, 0.769 mmol, 3.4 eq.) was dissolved in anhydrous DCM (4 mL) and the tris-isocyanate triethyl-benzene as synthesised according to WO2018167503 (compound 103, pages 114-115) (74 mg, 0.226 mmol, 1 eq.) was added. Pyridine (0.1 mL) was added, and the reaction stirred for 18 hours. The resultant suspension was treated with Et20 (20 mL) and sonicated, then stirred for 10 minutes and filtered. The solid was washed with Et20 and dried to give the tris-Boc-amino homo half macrocycle (265 mg, 0.219 mmol, 97%) as a white powder. 1 H NMR (400 MHz, DMSO-d6) 5 8.30 (s, 1 H), 7.88 (s, 1 H), 7.72 (s, 1 H), 7.18 (d, J = 8.2 Hz, 1H), 6.83 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (s, 1H), 4.36 (s, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.57 (s, 2H), 2.80 (d, J = 7.9 Hz, 2H), 1.40 (s, 8H), 1.18 (t, J = 7.1 Hz, 4H).

Step 4: Synthesis of the tris-amino homo half macrocycle

The given tris-Boc-amino homo half macrocycle (260 mg, 0.215 mmol, 1 eq.) was dissolved in DCM (3 mL) and TFA (3 mL) and stirred for 2 hours. The solution was partially concentrated under a flow of N2 and then the product precipitated with the addition of Et20 (20 mL). The suspension was sonicated, then stirred for 10 minutes and filtered. The solid was washed with Et20 and dried to give the tris-amino homo half macrocycle (252 mg, 0.207 mmol, 97%) as a white powder. 1 H NMR (400 MHz, DMSO-d6) 5 7.94 (s, 3H), 7.23 (d, J = 1.9 Hz, 3H), 6.94 (d, J = 8.1 Hz, 3H), 6.87 (dd, J = 8.1, 2.0 Hz, 3H), 6.44 (t, J = 4.7 Hz, 3H), 4.38 (d, J = 4.7 Hz, 6H), 4.05 (q, J = 7.1 Hz, 6H), 2.81 (q, J = 7.6 Hz, 6H), 1.18 (td, J = 7.2, 4.0 Hz, 18H).

Step 5: Synthesis of the homo-macrocycle propyl azide (Intermediate compound 17)

The given tris-amino homo half macrocycle (148 mg, 0.163 mmol), and propyl azide trisisocyanate as prepared under 3 (69 mg, 0.195 mmol) and NaOH (119 mg, 2.970 mmol) were reacted as described for compound 3. The homo-macrocycle propyl azide (Intermediate compound 17) was isolated as a white solid (61 mg, 0.052 mmol, 32%). 1 H NMR (400 MHz, DMSO-d6) 5 7.73 (d, J = 2.2 Hz, 3H), 7.60 - 7.55 (m, 6H), 7.47 (d, J = 18.3 Hz, 3H), 6.93 - 6.75 (m, 3H), 6.47 - 6.19 (m, 6H), 4.39 - 4.22 (m, 12H), 3.49 - 3.41 (m, 6H), 2.82 (s, 2H),

2.71 (s, 6H), 2.39 - 2.31 (m, 8H), 1.66 (s, 2H), 1.20 - 1.10 (m, 9H). MS (electrospray), [M+H]+ calculated for C59H70N15O12 requires: 1180.5, found: 1180.5.

Preparation of O-succinimidyl-heptynoate (Intermediate compound 18)

Intermediate 18:

Heptynoic acid (200 mg, 1.6 mmol) was dissolved in acetonitrile (4 mL). NHS was added (200 mg, 1.7 mmol) followed by DCC (360 mg, 1.7 mmol). The mixture was stirred 1 h, filtered and the crude O-succinimidyl-heptynoate (Intermediate 18) was used directly.

Preparation of half macrocycle (Intermediate compound 19)

Intermediate compound 19:

The half macrocycle (Intermediate compound 19) was synthesised according to W02020058322 (compound 25c, pages 65-67).

Preparation of insulin derivatives of the invention

Notably, B1 conjugates of the invention were prepared from insulin using no protection group on A1 by reaction with active esters of glycosides made from 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonate by conjugation in aqueous buffer near neutral pH over 1 to 3 days (W02022/090448, and New Phenol Esters for Efficient pH-Controlled Amine Acylation of Peptides, Proteins, and Sepharose Beads in Aqueous Media. Kim B. Jensen et al, Bioconjugate Chem. 2022, 33, 1 , 172-179).

Example 1: Preparation of B1-glucoside-EG-Ac B29-MC-propyl-triazole-propanoyl desB30 human insulin (INS1)

INS1 :

INS1 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle propyl azide (Intermediate compound 3). INS1 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1798.0 and 1438.6, calculated 1798.2 for [M+4H]⁴⁺ and 1438.8 for [M+5H]⁵⁺. Example 2: Preparation of B1-glucoside-EG-Ac B29-MC-propyl-triazole-propanoxycarbonyl desB30 human insulin (INS2)

INS2:

DesB30 human insulin (1.96 g, 343 mmol) was dissolved in 150 mM K2CO3 buffer (50 mL) and pH was checked to be 10.5. O-Succinimidyl pentyn-1 -oxycarbonyl (Intermediate compound 1) (116 mg, 515 mmol) was dissolved in DMSO (1 mL), and added to the insulin solution. After 1 h, the reaction was acidified using TFA, and the B29-alkyne insulin was purified by RP-HPLC on C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The product was isolated by lyophilisation. LCMS measured 1454.9, calculated 1455.1 for [M+4H]⁴⁺.

The given B29-alkyne insulin (255 mg, 44 mmol) was dissolved in 40 mM phosphate buffer (30 mL) at pH 7.5, and mixed with a solution of O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) (66 mg, 88 mmol) in DMSO (1 mL). 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonate active esters (such as 2) have been found to mainly give the B1-conjugates when reacted with insulin in aqueous buffer near neutral pH. The mixture was gently stirred 2 days, while the reaction was monitored by LCMS analysis. The reaction mixture was then acidified using TFA, and the B1-O-peracetyl-glycoside B29-alkyne insulin was purified by RP-HPLC on C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The product was isolated by lyophilisation. LCMS measured 1563.1 , calculated 1563.2 for [M+4H]⁴⁺.

The given B1-O-peracetyl-glycoside B29-alkyne insulin (78 mg, 12 mmol) was dissolved in 400 mM Na2CC>3 buffer (20 mL) at pH 10.5. Saponification of the O-acetyl esters were monitored by LCMS analysis. After 6 h, the reaction mixture was acidified using TFA, and the B1-glycoside B29-alkyne insulin was purified by RP-HPLC on C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The product was isolated by lyophilisation. LCMS measured 1520.9, calculated 1521.2 for [M+4H]⁴⁺.

The given B1 -glycoside B29-alkyne insulin (20 mg, 3 mmol) and macrocycle propyl azide (Intermediate compound 3) was dissolved in a mixture of DMSO (1.5 mL) and 2 M triethanolamine acetate (1.5 mL). THTPA was added (1 mg, 1.9 umol), and the reaction mixture was degassed, flushed with argon, and kept under argon atmosphere. Cui was added (1 mg, 5.2 umol), and click reaction was monitored by LCMS analysis. After 2 h, the reaction mixture was acidified using TFA, and the B1-glycoside B29-macrocyle insulin INS2 was purified by RP-HPLC on C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in acetonitrile as buffer B. The product was isolated by lyophilisation. LCMS measured 1805.5 and 1444.8, calculated 1805.8 for [M+4H]⁴⁺ and 1444.8 for [M+5H]⁵⁺.

Example 3: Preparation of B1-glucoside-Ac B29-MC-propyl-triazole-propanoyl desB30 human insulin (INS3)

INS3:

INS3 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac active ester (Intermediate compound 5) and macrocycle propyl azide (Intermediate compound 3). INS3 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1786.6 and 1429.5, calculated 1787.2 for [M+4H]⁴⁺ and 1430.0 for [M+5H]⁵⁺. Example 4: Preparation of B1-salidroside-Ac B29-MC-propyl-triazole-propanoyl desB30 human insulin (INS4)

INS4:

INS4 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-salidroside Ac active ester (Intermediate compound 6) and macrocycle propyl azide (Intermediate compound 3). INS4 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1817.0 and 1453.8, calculated 1817.3 for [M+4H]⁴⁺ and 1454.0 for [M+5H]⁵⁺.

Example 5: Preparation of B1-glucoside-Ac B29-MC-propyl-triazole-propanoyl B3E desB30 human insulin (INS5)

INS5:

INS5 was prepared similar to INS2 from B3E desB30 human insulin, O-succinimidyl- pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac active ester (Intermediate compound 5) and macrocycle propyl azide (Intermediate compound 3). INS5 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1790.8 and 1432.9, calculated 1791.0 for [M+4H]⁴⁺ and 1433.0 for [M+5H]⁵⁺. Example 6: Preparation of B1-glucoside-EG2-Ac B29-MC-propyl-triazole-propanoyl desB30 human insulin (INS6)

INS6:

INS6 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) and macrocycle propyl azide (Intermediate compound 3). INS6 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1809.1 and 1447.5, calculated 1809.3 for [M+4H]⁴⁺ and 1447.6 for [M+5H]⁵⁺. Example 7: Preparation of B1-alucoside-ethyl-triazole-CO B29-MC-EG3-triazole-propanoyl desB30 human insulin (INS7)

INS7:

INS7 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate

(Intermediate compound 4), O-peracetyl-glucoside-ethyl-triazole-carboxylate active ester

(Intermediate compound 8) and macrocycle EG3 azide (Intermediate compound 9). INS7 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1829.9 and 1464.1, calculated 1830.0 for [M+4H]⁴⁺ and 1464.2 for [M+5H]⁵⁺.

Example 8: Preparation of B1-glucoside-EG-Ac B29-MC-propyl-triazole-propanoyl B3E desB30 human insulin (INS8)

INS8:

INS8 was prepared similar to INS2 from B3E desB30 human insulin, O-succinimidyl- pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester 2 and macrocycle propyl azide (Intermediate compound 3). INS8 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1801.9 and 1441.7, calculated 1802.0 for [M+4H]⁴⁺ and 1441.8 for [M+5H]⁵⁺. Example 9: Preparation of B1-glucoside-EG-Ac B29-MC-EG3-triazole-propanoyl B3E desB30 human insulin (INS9)

INS9:

INS9 was prepared similar to INS2 from B3E desB30 human insulin, O-succinimidyl- pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester 2 and macrocycle EG3 azide (Intermediate compound 9). INS9 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1824.4 and 1459.7, calculated 1802.0 for [M+4H]⁴⁺ and 1441.8 for [M+5H]⁵⁺. Example 10: Preparation of B1-glucoside-EG2-Ac B29-MC-EG3-triazole-propanoyl desB30 human insulin (INS10)

INS10:

INS10 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) and macrocycle EG3 azide (Intermediate compound 9). INS10 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1831.7 and 1465.7, calculated 1831.8 for [M+4H]⁴⁺ and 1454.6 for [M+5H]⁵⁺. Example 11 : Preparation of B1-glucoside-Ac B29-C^alpha-MC-EG3-triazole-MeNH desB30 human insulin (INS11)

INS11 :

DesB30 human insulin (120 mg, 21 umol) and propynamine (115 mg, 2.1 mmol) were dissolved in DMSO (1 mL), DMF (1 mL), ethanol (1 mL) and 0.1 M phosphate buffer (1 mL), with pH adjusted to 7.1. Achromobacter lyticus protease was added (40 uL, 6.3 mg/mL), and the reaction was monitored by LCMS analysis. After 18 h, the reaction mixture was acidified and the product B29-C^alpha-propyne amide was isolated by RP-HPLC similar to the description in Example 2. The given B29 insulin derivative was reacted with O-peracetyl- glucoside Ac active ester (Intermediate compound 5) and macrocycle EG3 azide (Intermediate compound 9) similar to the description in Example 2. INS11 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1797.1 and 1439.2, calculated 1799.0 for [M+4H]⁴⁺ and 1439.4 for [M+5H]⁵⁺.

Example 12: Preparation of B1-glucoside-EG-Ac B29-C^alpha-MC-propyl-triazole-MeNH desB30 human insulin (INS12)

INS12:

INS12 was prepared similar to the description in Example 11 from desB30 human insulin, propynamine, O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle propyl azide (Intermediate compound 3). INS12 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1787.3 and 1430.1 , calculated 1787.5 for [M+4H]⁴⁺ and 1430.2 for [M+5H]⁵⁺. Example 13: Preparation of B1-glucoside-Ac B29-MC-EG3-triazole-CO desB30 human insulin (INS13)

INS13:

INS13 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-propynoate (Intermediate compound 11), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle EG3 azide (Intermediate compound 9). INS13 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1802.6 and 1442.3, calculated 1802.7 for [M+4H]⁴⁺ and 1442.4 for [M+5H]⁵⁺. Example 14: Preparation of B1-glucoside-EG-Ac B29-MC-EG2-triazole-CO desB30 human insulin (INS14)

INS14:

INS14 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-propynoate (Intermediate compound 11), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle EG2 azide (Intermediate compound 10). INS14 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1802.6 and 1442.2, calculated 1802.7 for [M+4H]⁴⁺ and 1442.4 for [M+5H]⁵⁺.

Example 15: Preparation of B1-qlucoside-EG-Ac B29-MC-propyl-NHAc-triazole-CO desB30 human insulin (INS15)

INS15:

INS15 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-propynoate (Intermediate compound 11), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS15 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1805.3 and 1444.6, calculated 1805.5 for [M+4H]⁴⁺ and 1444.6 for [M+5H]⁵⁺. Example 16: Preparation of B1-glucoside-EG-Ac B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS16)

INS16:

INS16 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle EG2 azide (Intermediate compound 10). INS16 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1809.6 and 1448.1, calculated 1809.7 for [M+4H]⁴⁺ and 1448.0 for [M+5H]⁵⁺. Example 17: Preparation of B1-glucoside-EG-Ac B29-MC-propyl-NHAc-triazole-propanoyl desB30 human insulin (INS17)

INS17:

INS17 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS17 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1812.3 and 1450.3, calculated 1812.5 for [M+4H]⁴⁺ and 1450.2 for [M+5H]⁵⁺. Example 18: Preparation of B1-glucoside-Ac B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS18)

INS18:

INS18 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac active ester (Intermediate compound 5) and macrocycle EG2 azide (Intermediate compound 10). INS18 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1798.5 and 1439.0, calculated 1798.7 for [M+4H]⁴⁺ and 1439.2 for [M+5H]⁵⁺. Example 19: Preparation of B1-glucoside-Ac B29-MC-propyl-NHAc-triazole-propanoyl desB30 human insulin (INS19)

INS19:

INS19 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac active ester (Intermediate compound 5) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS19 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1801.3 and 1441.4, calculated 1801.5 for [M+4H]⁴⁺ and 1441.4 for [M+5H]⁵⁺.

Example 20: Preparation of B1-glucoside-Ac B29-MC-EG3-triazole-propanoyl B3E desB30 human insulin (INS20)

INS20:

INS20 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac active ester (Intermediate compound

5) and macrocycle EG3 azide (Intermediate compound 9). INS20 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1813.3 and 1450.9, calculated 1813.5 for [M+4H]⁴⁺ and 1451.0 for [M+5H]⁵⁺.

Example 21 : Preparation of B1-glucoside-1-NHAc-EG-Ac B29-MC-propyl-NHAc-triazole- propanoyl desB30 human insulin (INS21)

INS21:

INS21 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside-1-NHAc-EG-Ac active ester (Intermediate compound 16) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS21 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1826.6 and 1461.7, calculated 1826.8 for [M+4H]⁴⁺ and 1461.6 for [M+5H]⁵⁺. Example 22: Preparation of B1-glucoside-Ac-Glv B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS22)

INS22:

INS22 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside Ac-Gly active ester (Intermediate compound 13) and macrocycle EG2 azide (Intermediate compound 10). INS22 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1813.3, calculated 1813.0 for [M+4H]⁴⁺. Example 23: Preparation of B1-glucoside-benzyl B29-MC-propyl-NHAc-triazole-propanoyl desB30 human insulin (INS23)

INS23:

INS23 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside benzaldehyde (Intermediate compound 14) and macrocycle propyl-NHAc azide (Intermediate compound 12). With regard to the reductive alkylation at B1 , B29-alkyne insulin from Example 2 (150 mg, 26 umol) was suspended in 0.1 M AcOH (3.5 mL) and pH was adjusted to 4.5. O-peracetyl-glucoside benzaldehyde (Intermediate compound 14) (14 mg, 31 umol) was dissolved in NMP (0.14 mL) and added to the insulin suspension. Upon stirring for 30 minutes, a solution of 2-methyl- pyridine borane (14 mg, 130 umol) in NMP (0.14 mL) was added, and pH adjusted to 4.5. After stirring 22 h, the mixture was diluted with 20% AcOH, and the product B1-glycoside- benzyl B29-pentynoate insulin was isolated by RP-HPLC similar to the description in Example 2

The given product was click reacted with macrocycle propyl-NHAc azide (Intermediate compound 12) similar to the description in Example 2. INS23 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1813.5, calculated 1813.5 for [M+4H]⁴⁺.

Example 24: Preparation of B1-glucoside-p-xylene B29-MC-propyl-NHAc-triazole-propanoyl desB30 human insulin (INS24)

INS24:

INS24 was prepared similar to INS23 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside benzylaldehyde (Intermediate compound 15) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS24 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1817.1, calculated 1817.0 for [M+4H]⁴⁺. Example 25: Preparation of B1-glucoside-benzyl B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS25)

INS25:

INS25 was prepared similar to INS23 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside benzaldehyde (Intermediate compound 14) and macrocycle EG2 azide (Intermediate compound 10). INS25 was isolated by RP- HPLC similar to the description in Example 2. LCMS measured 1810.8, calculated 1810.8 for [M+4H]⁴⁺. Example 26: Preparation of B1-glucoside-p-xylene B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS26)

INS26:

INS26 was prepared similar to INS23 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside benzylaldehyde (Intermediate compound 15) and macrocycle EG2 azide (Intermediate compound 10). INS26 was isolated by RP- HPLC similar to the description in Example 2. LCMS measured 1814.6, calculated 1814.3 for [M+4H]⁴⁺. Example 27: Preparation of B1-glucoside-EG2-Ac B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS27)

INS27:

INS27 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) and macrocycle EG2 azide (Intermediate compound 10). INS27 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1820.7, calculated 1820.8 for [M+4H]⁴⁺. Example 28: Preparation of B1-glucoside-EG2-Ac B29-MC-propyl-NHAc-triazole-propanoyl desB30 human insulin (INS28)

INS28:

INS28 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG2 Ac active ester (Intermediate compound 7) and macrocycle propyl-NHAc azide (Intermediate compound 12). INS28 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1823.8, calculated 1823.5 for [M+4H]⁴⁺.

Example 29: Preparation of B1-glucoside-1-NHAc-EG-Ac B29-MC-EG2-triazole-propanoyl desB30 human insulin (INS29)

INS29:

INS29 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside-1-NHAc-EG-Ac active ester (Intermediate compound 16) and macrocycle EG2 azide (Intermediate compound 10). INS29 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1824.1 and 1459.3, calculated 1824.0 for [M+4H]⁴⁺ and 1459.4 for [M+5H]⁵⁺. Example 30: Preparation of B1-glucoside-EG-Ac B29-homoMC-propyl-triazole-propanoyl desB30 human insulin (INS30)

INS30:

INS30 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-pentynoate (Intermediate compound 4), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and homo-macrocycle propyl azide (Intermediate compound 17). INS30 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1808.6 and 1447.1 , calculated 1808.8 for [M+4H]⁴⁺ and 1447.3 for [M+5H]⁵⁺. Example 31 : Preparation of B1-glucoside-EG-Ac B29-MC-propyl-triazole-pentanoyl desB30 human insulin (INS31)

INS31:

INS31 was prepared similar to INS2 from desB30 human insulin, O-succinimidyl-heptynoate (Intermediate 18), O-peracetyl-glucoside EG Ac active ester (Intermediate compound 2) and macrocycle propyl azide (Intermediate compound 3). INS31 was isolated by RP-HPLC similar to the description in Example 2. LCMS measured 1805.0 and 1444.2, calculated

1805.3 for [M+4H]⁴⁺ and 1444.4 for [M+5H]⁵⁺. Glucose sensitive insulin receptor activation

Example 32: Assay to determine affinity to the human insulin receptor (hIR-A) in absence or presence of glucose

To measure the glucose sensitivity of the insulin derivatives, the affinity of the insulin derivatives towards the human insulin receptor was measured in the presence of no glucose or 20 mM glucose. The experiments were done both in presence of 1 .5% HSA to mimic physiological conditions more and with no HSA present.

Insulin Receptor preparation

BHK cells over-expressing human Insulin Receptor A (hIR-A) were lysed in 50 mM Hepes pH 8.0, 150 mM NaCI, 1 % Triton X-100, 2 mM EDTA and 10% glycerol. The cleared 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 receptors were washed with 20 volumes 50 mM Hepes pH 8.0, 150 mM NaCI and 0.1 % Triton X-100, where after the receptors were eluted with 50 mM Hepes pH 8.0, 150 mM NaCI, 0.1 % Triton X-100, 0.5 M n-Acetyl Glucosamine and 10% glycerol. All buffers contained Complete (Roche Diagnostic GmbH, Mannheim, Germany) as described in Andersen et al. 2017 PLos One 12.

Insulin Receptor Scintillation Proximity Assay (SPA) binding assay

SPA PVT anti-mouse beads (Perkin Elmer) were diluted in SPA binding buffer, consisting of 100 mM Hepes, pH 7.4, 100 mM NaCI, 10 mM MgSO4, 0.025% (v/v) Tween-20. SPA beads were incubated with the IR-specific antibody 83-7 (Soos et al. 1986 Biochem J. 235, 199-208) and solubilized semi-purified HIR-A. Receptor concentrations were adjusted to achieve 10% binding of 5000 cpm ¹²⁵l-(Tyr31)-lnsulin (Novo Nordisk A/S). Dilution series of cold ligands were added to 96-well Optiplate, followed by tracer (¹²⁵l-l nsulin, 5000 cpm/well) and lastly receptor/SPA mix. In order to test the glucose sensitivity, the binding experiments were set up in absence or presence of 20 mM glucose. The plates were rocked gently for 22.5 hours at 22°C, centrifuged for 5 minutes at 1000 rpm and counted in TopCounter (Perkin Elmer). Data points were fitted to a four-parameter logistic model, whereby the relative affinity of the analogue compared to human insulin (within the same plate) was determined. The relative affinities for the analogues compared to human insulin were determined as fold change, and the increase in relative affinity from 0 to 20 mM glucose (HIR glucose factor) reflected the glucose sensitivity of the analogues in each experiment. The experiments were done both in presence of 1.5% HSA (w/w) to mimic physiological conditions more and with no HSA present. Data is shown in table 1 , n > 2.

The HIR (Human Insulin Receptor) glucose factor is calculated in the individual experiment as the relative HIR affinity at 20 mM glucose divided by the relative affinity in absence of glucose. The average glucose factor from several experiments is provided in table 1. If the glucose factor is calculated from the average HIR affinity in presence and absence of 20 mM glucose it may differ slightly from the average glucose factor calculated in the individual experiments.

Table 1. Relative insulin receptor binding affinity in the absence and presence of glucose under assay conditions with no HSA present and with 1.5% HSA, respectively.

The data in table 1 show that the compounds of the invention have higher insulin receptor affinity in presence of 20 mM glucose than when no glucose is present. This can readily be seen from the glucose factor, which is above 1 when the relative insulin receptor affinity is higher in the presence of 20 mM glucose, as compared to when no glucose is present. The compounds thus bind to the insulin receptor in a glucose sensitive fashion, and the compounds of the invention thus have the potential for glucose sensitive treatment of diabetes, where the compounds of the invention will be inactive or less active at low blood glucose levels and will bind to and activate the insulin receptor at higher blood glucose levels.

The insulin receptor affinity of the compounds of the invention are glucose sensitive both under assay conditions where no HSA is present and when 1.5% HSA is present.

Surprisingly, certain compounds have a lower relative affinity to the human insulin receptor when 1.5% HSA is present as compared to when no HSA is present. Surprisingly, certain compounds have a higher glucose factor when 1.5% HSA is present as compared to when no HSA is present.

When insulin binds to the insulin receptor it induces activation of downstream signaling pathways. One metabolic endpoint of insulin signaling is lipid metabolism, and the lipogenesis assay was used to measure an end point read-out because in presence of insulin, ³H-glucose uptake by the cells is stimulated and is incorporated into lipids.

Rat lipogenesis assay (rFFC)

Epidydimal fat pads from Sprague Dawley rat were degraded with collagenase in Hepes Krebs Ringer Buffer at 36.5 °C for 1-1.5 hours under vigorous shaking. The suspension was filtered through 2 layers of gauze. The phases were separated by 5 min standing at room temperature, allowing the adipocytes to collect in the upper phase. The lower phase was removed with a syringe. The adipocytes were washed twice with 20 ml Hepes Krebs Ringer Buffer. Cells were transferred to 96 well plates in Hepes Krebs Ringer buffer containing 1.5% HSA, 0.5 mM glucose, 0.1 pCi/well glucose (D-[3-³H] glucose (20.0 Ci/mmol) Perkin Elmer), and either 3 mM L-glucose or 20 mM L-glucose. Increasing amounts of insulin conjugates of the invention to generate concentration-response curves were added and incubated for 2 hours at 36.5 °C. The reactions were stopped by addition of 100 pL Microscient E (cat# 6013661 Perkin Elmer). The plates rested 3 hours before counting in Top counter. The EC50 at 3 mM L-glucose and at 20 mM L-glucose, respectively, was determined, and the glucose factor (fold change in EC50 from 20 to 3 mM L-glucose) was calculated and reflects the glucose sensitivity of the analogues.

The rFFC glucose factor is calculated in the individual experiment as the EC50 value at 3 mM L-glucose divided by the EC50 value at 20 mM L-glucose. The average glucose factor from several experiments are provided in table 2. If the glucose factor is calculated from the average EC50 values it may differ slightly from the average of glucose factors calculated in the individual experiments.

The lipogenesis data in table 2 show that the insulin conjugates of the invention give higher levels of lipogenesis (more glucose transport) in the presence of higher levels of sugar (20 mM L-glucose) compared to lower level of added sugar (3 mM L-glucose). The rFFC assay is itself sensitive to D-glucose levels, so L-glucose (which does not affect glucose transport in itself) was used as sugar to activate the insulin conjugates of the invention. Table 2

Example 34: Hypoglycaemic study in LYD pigs

To evaluate the effect of INS2 (Example 2) and to rule out effects of glucose fluctuations on endogenous hormone release on in vivo activity in healthy pigs, we used somatostatin infusion to suppress glucagon and insulin secretion, in combination with primed constant infusion of INS2 (Example 2) versus insulin degludec at different rates, glucagon replacement and constant glucose infusion. After 5 hours of infusion, when approximate steady state plasma exposure and plasma glucose was achieved, intervention by stopping the glucose infusion was made. This procedure resulted in a drop in plasma glucose (promoted by the continued insulin infusion), but the drop was observed to be less pronounced when studying INS2 (Example 2) compared to insulin degludec, thereby showing glucose sensitive switching of the insulin bioactivity. Evaluation of the full data set utilizing different insulin infusion rates of INS2 (Example 2) and insulin degludec shows that after glucose infusion is turned off, at any given plasma glucose concentration, plasma glucose drops less for INS2 (Example 2) than for insulin degludec. Data from two representative groups are shown in Fig. 2, where glucose dropped to about 4.5 mM with INS2 (Example 2) versus drop to about 3 mM glucose for insulin degludec, thus demonstrating attenuation of hypoglycaemia with INS2 (Example 2).

Female Landrace-Yorkshire-Duroc (LYD) pigs were used. Prior to the experiments, all animals were instrumented with two venous catheters, one for infusion and one for blood sampling. Animals were subjected to constant infusion of somatostatin (60 pg/kg/h) to suppress endogenous insulin and glucagon secretion, and glucagon (0.45 pmol/kg/min) was infused as basal replacement. In addition, primed infusions of either insulin degludec or INS2 (Example 2) were given as indicated below. Suitable priming doses were estimated based on the i.v. pharmacokinetics of the individual insulin analogue. The duration of the experiment was 9 hours. At time 0 when somatostatin, glucagon and glucose infusions were initiated, a primed infusion of either insulin degludec or INS2 (Example 2) was also started. To investigate a range of starting glucose concentrations, different insulin infusion rates were used (insulin degludec: 0.7, 0.9 and 1.1 pmol/kg/min; INS2 (Example 2): 1.30, 1.44, 1.58, 1.72, 1.86, 2.00, and 2.14 pmol/kg/min). During the study, the body weight of the pigs ranged from 74.5-110 kg.

While 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. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A compound comprising: i) an insulin peptide, wherein the insulin peptide is human insulin or a human insulin analogue comprising a lysine in position B29; ii) a macrocycle of Formula M:

Formula G1 :

Formula G2:

Formula G3:

; and

Formula G4:

2. The compound according claim 1, wherein the linker L1 is a moiety having from 8 to 20 non-hydrogen atoms, wherein 3 to 10 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms.

3. The compound according to any one of claims 1 to 2, wherein the linker L1 is of Formula L1a:

wherein m is an integer from 0 to 6;

Y is absent or NH-C(O)-CH₂;

4. The compound according to any one of claims 1 to 3, wherein the linker L2 is a moiety having from 2 to 15 non-hydrogen atoms, wherein 0 to 6 of the non-hydrogen atoms are independently selected from nitrogen atoms and oxygen atoms.

5. The compound according to any one of claims 1 to 4, wherein the linker L2 is selected from the group consisting of:

Formula L2b:

Formula L2c:

6. The compound according to claim 1, wherein the compound is selected from the group consisting of INS1 of Example 1 ; INS2 of Example 2; INS3 of Example 3; INS4 of Example 4; INS5 of Example 5; INS6 of Example 6; INS7 of Example 7; INS8 of Example 8; INS9 of Example 9; INS10 of Example 10; INS11 of Example 11; INS12 of Example 12; INS13 of Example 13; INS14 of Example 14, INS15 of Example 15; INS16 of Example 16; INS17 of Example 17; INS18 of Example 18; INS19 of Example 19; INS20 of Example 20; INS21 of Example 21 ; INS22 of Example 22, INS23 of Example 23; INS24 of Example 24; INS25 of Example 25; INS26 of Example 26; INS27 of Example 27; INS28 of Example 28; INS29 of Example 29; INS30 of Example 30; and INS31 of Example 31.

7. The compound according to claim 1, wherein the compound is INS2 of Example 2:

Example 2.

9. An intermediate compound of Formula IM 1 :

wherein k1 is 0 or 1 ,

W1 is (CH₂)ni or (OCH₂CH2)_PI, wherein n1 is an integer from 2 to 5, and p1 is an integer from 1 to 5; and

Y1 is absent or NH-C(O)-CH₂.

10. The intermediate compound according to claim 9, wherein the intermediate compound is selected from the group consisting of Intermediate compound 3 (‘macrocycle propyl azide’), Intermediate compound 9 (‘macrocycle EG3 azide’), Intermediate compound 10 (‘macrocycle EG2 azide’), Intermediate compound 12 (‘macrocycle propyl-NHAc azide’), and Intermediate compound 17 (‘homo-macrocycle propyl azide’).

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8.

12. A compound according to any one of claims 1 to 8 for use as a medicament.

13. A compound according to any one of claims 1 to 8 or a composition according to claim 10 for use in the treatment and/or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

14. Use of a compound according to any one of claims 1 to 8 or a composition according to claim 10, for the manufacture of a medicament for the treatment or prevention of diabetes, diabetes of Type 1 , diabetes of Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

15. A method for treatment and/or prevention of diabetes, diabetes of Type 1 , diabetes of

Type 2, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome) comprising administration of an effective amount of a compound according to any one of claims 1 to 8 or a composition according to claim 11 to a patient in need thereof.