Glucose-sensitive insulin derivative

Abstract

The present invention relates to novel insulin derivatives and their use in the treatment or prevention of medical conditions associated with diabetes. The insulin derivatives are glucose sensitive and show glucose sensitive albumin binding. The present invention also relates to novel intermediates. Finally, the present invention provides pharmaceutical compositions comprising the insulin derivatives of the present invention, and the use of such compositions in the treatment or prevention of medical conditions associated with diabetes.

Description

TECHNICAL FIELD

[0001] The present invention relates to novel insulin derivatives and their pharmaceutical use. Furthermore, the present invention relates to pharmaceutical compositions comprising such insulin derivatives, and to the use of such compounds in the treatment or prevention of medical conditions associated with diabetes. BACKGROUND

[0002] Insulin is the most effective drug for the treatment of hyperglycemia, but due to the narrow physiological glucose window, insulin dosing is a delicate balance between too much and too little. Healthy people have blood glucose levels close to 5 mM in the fasted state, and diabetic patients try to give meal and basal insulin preparations to approach 5 mM. However, blood glucose values below about 3 mM (hypoglycemia) occur frequently during insulin therapy, and hypoglycemia can lead to discomfort, loss of consciousness, brain damage, or death. Therefore, diabetic patients hesitate to treat their high or moderately high blood glucose values aggressively out of fear of hypoglycemia. If an insulin drug were developed that is active only at higher blood glucose values or releases from a depot, but is inactive or less active at lower blood glucose values, it could help in the treatment of diabetes. Since the 1970s, many papers have approached these goals (Brownlee et al., Science 1979, 1190; Zaykov et al., Nature Rev. Drug Disc. 2016, 425), but most commonly through glucose-sensitive polymers that capture and release insulin from a subcutaneous depot in a glucose-dependent manner. However, such systems are slow and therefore not suitable for the treatment of, for example, the rapid postprandial fluctuations in blood glucose values. Therefore, subcutaneous glucose-sensitive release systems have never reached clinical trials.

[0003] It would be even better if the glucose-sensitive regulation of the biological activity of insulin could be performed in the blood. One way to achieve this wish could be a glucose-sensitive albumin binding, as previously described for the fatty acid-monoboronic acid insulin derivative, where the fatty acid moiety causes the albumin binding (Novo Nordisk WO2011 / 000823; WO 2014 / 093696; Chou et al., Proc. Nat. Acad. Sci. 2015, 2401). The main driving force for the albumin interaction in these systems comes from the fatty acid moiety of the fatty acid-monoboronic acid insulin derivative (not the boronate), and the influence of glucose on the albumin affinity is weak. To increase the glucose sensitivity of the albumin binding, a glucose-sensitive albumin binding motif that is directly displaced by glucose is therefore needed. Monoboronic acids are known to bind glucose and other sugars with an affinity (Kd) in the medium to high millimolar range (Hansen et al., Sensors Actuators B 2012, 45). However, to provide sufficient glucose sensitivity at physiological glucose levels, a stronger affinity for glucose is needed. Diboronic compounds with two boronates / boroxoles placed in a suitable geometry relative to the hydroxyl groups on glucose can provide an increased glucose affinity relative to monoboronic compounds, i.e. low mM Kd or sub-mM Kd (Hansen et al., Sensors Actuators B 2012, 45). Most of the diboronic compounds described in the literature include fluorescent probes, as the purpose of these studies was to make optical glucose sensors. Fluorescent probes are not desirable in a drug candidate, as these probes can be light sensitive, toxic and coloured. There is therefore a need for insulin derivatives with increased glucose sensitivity within physiological blood glucose levels. SUMMARY

[0004] In its broadest aspect, the present invention relates to insulin derivatives.

[0005] It was surprisingly found that the compounds of the present invention bind both to albumin (HSA) and to glucose, and that the HSA affinity is glucose sensitive. The human insulin receptor (HIR) affinity in the presence of HSA is thus also made glucose sensitive. The part of the insulin that is bound to HSA does not bind to HIR, but the glucose facilitated release from HSA increases the free fraction of the insulin, and thus the HSA affinity is increased by glucose.

[0006] In contrast to previously disclosed insulin derivatives with so-called glucose-sensitive albumin binding, the compounds of the present invention do not rely on a fatty acid moiety for albumin binding, but comprise an albumin binding motif that is directly displaced by glucose, resulting in an increased effect of glucose on albumin binding, thereby increasing the glucose sensitivity of the insulin.

[0007] Albumin binding can generally prolong the in vivo half-life of peptide- and protein-based drugs. The prolonged effect is achieved because the albumin-bound fraction is protected from enzymatic degradation and kidney elimination, and only the free fraction is biologically active, thereby preventing receptor-mediated clearance of the albumin-bound fraction.

[0008] The compounds of the present invention thus exhibit an insulin activity that is dependent on the glucose concentration and are therefore useful as glucose-sensitive insulin derivatives.

[0009] In one aspect, the compounds of the present invention comprise an insulin or an analogue thereof, and one or more modification groups.

[0010] In one aspect, the modification groups have an affinity for glucose and albumin.

[0011] In one aspect, the insulin peptide or analogue thereof optionally comprises a spacer.

[0012] In one aspect, the compounds of the present invention comprise

[0013] i) a human insulin or a human insulin analogue; and

[0014] ii) one or more modification groups M, wherein each modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties. Each of the one or more modification groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, via a spacer.

[0015] In one embodiment, the one or more modification groups M are optionally attached to the sulfide of a free cysteine in the human insulin or human insulin analogue via a spacer.

[0016] In one aspect, the compounds of the present invention comprise

[0017] i) a human insulin or a human insulin analogue; and

[0018] ii) two or more modifying groups M, wherein each modifying group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties. Each of the two or more modifying groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, via a spacer.

[0019] As can be seen from the examples, compounds with two or more modifying groups M generally exhibit a higher degree of glucose sensitivity (higher glucose factor) than compounds with only one modifying group M.

[0020] In one aspect, the present application provides intermediate products in the form of novel insulin analogues, including novel insulin analogues comprising a peptide spacer.

[0021] In one aspect, the compounds of the present application activate the insulin receptor in response to glucose concentrations in blood and tissue.

[0022] In one aspect, the compounds of the present application have low availability (low non-bound, plasma free fraction) and thus low or no activity in case of hypoglycemia, e.g. at levels below about 3 mM glucose (hypoglycemia).

[0023] In one aspect, the compounds of the present application have high availability (high non-bound, plasma free fraction) and thus high activity in response to hyperglycemia, e.g. above about 10 mM glucose (hyperglycemia).

[0024] In one aspect, the compounds of the present application exhibit glucose-sensitive albumin binding.

[0025] In another aspect, the present application relates to a pharmaceutical composition comprising a compound according to the present application. In another aspect, the present application relates to a compound according to the present application for use as a medicament. In another aspect, the present application relates to a compound according to the present application for use in the treatment of diabetes. In another aspect, the present application relates to the medical use of a compound according to the present application.

[0026] The present application can also solve other problems that will be apparent from the disclosure of exemplary embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 PK profiles of intravenous bolus doses of insulin aspart are shown at 10 mM and 3.5-4 mM glucose (Example E).

[0028] Figure 2 PK profiles of intravenous bolus doses of insulin degludec are shown at 10 mM and 3.5-4 mM glucose (Example E).

[0029] Figure 3 PK profiles for intravenous bolus of Example No. 210 (triangles) and Example No. 211 (circles) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0030] Figure 4 PK profiles for intravenous bolus of Example No. 233 (triangles) and Example No. 234 (squares) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0031] Figure 5 PK profiles for intravenous bolus of Example No. 240 (triangles) and Example No. 227 (circles) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0032] Figure 6 PK profiles for intravenous bolus of Example No. 241 (triangles) and Example No. 181 (squares) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0033] Figure 7 PK profiles for intravenous bolus of Example No. 205 (triangles) and Example No. 239 (squares) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0034] Figure 8 PK profiles for intravenous bolus of Example No. 285 (triangles) and Example No. 273 (squares) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0035] Figure 9 PK profiles for intravenous bolus of Example No. 280 (triangles) and Example No. 272 (squares) at 10 mM (solid) and 3.5-4 mM (open) glucose are shown (Example E).

[0036] Figure 10 Comparison between the area under the curve of the baseline-adjusted glucose infusion rate for Example Nos. 205, 239, 272, and 280 for the clamp experiment at 3.5-4 mM glucose vs. 10 mM glucose is shown (Example E).

[0037] Description

[0038] The present invention relates to insulin derivatives. In one aspect, the present invention relates to glucose-sensitive insulin derivatives.

[0039] In one embodiment, the present application relates to a compound comprising human insulin or an analogue thereof and a modification group, which modification group exhibits affinity for both glucose and albumin.

[0040] In one embodiment, the modification group exhibits glucose-sensitive albumin binding.

[0041] In one embodiment, the insulin analogue is an analogue of human insulin (SEQ ID NO: 1 and SEQ ID NO: 2).

[0042] In one embodiment, the human insulin or human insulin analogue of the present application can comprise a spacer.

[0043] In one embodiment, the present application provides a compound comprising human insulin or a human insulin analogue; and one or more modification groups M, wherein each modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties. Each of the one or more modification groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, via a spacer.

[0044] In one embodiment, the present application provides a compound comprising human insulin or a human insulin analogue; and two or more modification groups M, wherein each modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties. Each of the two or more modification groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, via a spacer. The modification group M can also optionally be attached to the sulfide of a free cysteine in the human insulin or human insulin analogue by a spacer.

[0045] General definitions

[0046] The term "compound" is used herein to denote a molecular entity, and thus, a "compound" can have different structural elements in addition to the minimum element defined for each compound or group of compounds. The term "compound" is also intended to encompass its pharmaceutically relevant forms, i.e. the present application relates to a compound as defined herein or a pharmaceutically acceptable salt, amide or ester thereof.

[0047] The term "peptide" or "polypeptide" as used, for example, in the context of the present application refers to a compound comprising a series of amino acids which are linked to each other by amide (or peptide) bonds. In particular embodiments, a peptide consists of amino acids which are linked to each other by peptide bonds.

[0048] The term "analog" generally refers to a peptide whose sequence has one or more amino acid changes compared to a reference amino acid sequence. An analog "comprising" certain specified changes can comprise further changes compared to its reference sequence. In particular embodiments, an analog "has" or "comprises" the specified changes. In other particular embodiments, an analog "consists of" the changes. When the term "consists of" or "consisting of" is used in relation to an analog, for example, an analog consisting of a specified set of amino acid mutations, it is to be understood that the specified set of amino acid mutations are the only amino acid mutations in the analog. In contrast, an analog "comprising" a specified set of amino acid mutations can have additional mutations.

[0049] The term "derivative" generally refers to a compound which can be prepared from a natural peptide or an analog thereof by chemical modification, in particular by covalent attachment of one or more substituents.

[0050] In the context of the present application, the modification group M is a covalently attached substituent.

[0051] The term "amino acid" includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) as well as non-proteinogenic (or unnatural) amino acids. Proteinogenic amino acids are amino acids which are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids either do not occur in proteins or are not produced by standard cellular machinery (for example, they can have undergone post-translational modification).

[0052] Generally, amino acid residues (peptide / protein sequences) can be denoted by their full name, their one-letter code and / or their three-letter code. These three ways are fully equivalent. In the following, each amino acid of a peptide of the present application for which no optical isomer is specified is to be understood as meaning the L-isomer (unless specified otherwise). An amino acid is a molecule containing an amino group and a carboxylic acid group, and optionally one or more additional groups, often referred to as side chains.

[0053] Herein, the term "amino acid residue" is an amino acid which has formally lost the hydroxyl group from the carboxyl group, and / or which has formally lost the hydrogen atom from the amino group.

[0054] As will be apparent from the following examples, amino acid residues can be denoted by their full name, their one-letter code and / or their three-letter code. These three ways are fully equivalent and can be used interchangeably.

[0055] In the present context, the term "aryl" refers to cyclic or polycyclic aromatic rings having 5 to 12 carbon atoms. The term aryl includes monovalent, divalent and polyvalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthryl, and the like. In particular embodiments, the aryl group is phenyl. In the present context, the term "aryl" also includes "heteroaryl". The term "heteroaryl" refers to aromatic monocyclic, bicyclic or polycyclic rings incorporating one or more (e.g. 1-4, in particular 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulphur.

[0056] Insulin

[0057] The term "human insulin" as used herein means the human insulin hormone, the structure and properties of which are well known. Human insulin has two polypeptide chains, designated the A chain and the B chain. The A chain is a 21 amino acid peptide, while the B chain is a 30 amino acid peptide, the two chains being connected by the following disulfide bridges: a first bridge between the cysteine at position 7 on the A chain and the cysteine at position 7 on the B chain, and a second bridge between the cysteine at position 20 on the A chain and the cysteine at position 19 on the B chain. A third bridge exists between the cysteines at positions 6 and 11 on the A chain.

[0058] 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).

[0059] The term "insulin peptide", "insulin compound" or "insulin" as used herein means a peptide which is human insulin or an analogue or derivative thereof having insulin activity, i.e. activating the insulin receptor.

[0060] Insulin analogue

[0061] The term "insulin analogue" as used herein means a modified human insulin, wherein one or more amino acid residues of the insulin have been replaced 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 into the insulin.

[0062] The term "insulin analogue" as used herein means an insulin analogue which exhibits insulin activity, i.e. which activates the insulin receptor.

[0063] An insulin analogue comprises less than 10 amino acid modifications (substitutions, deletions, additions (i.e. extensions), insertions, and any combination thereof) relative to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modifications relative to human insulin. In one aspect, an insulin analogue has less than 10 amino acid modifications (substitutions, deletions, additions (i.e. extensions), insertions, and any combination thereof) relative to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modifications relative to human insulin.

[0064] Modifications in the insulin molecule are indicated by specifying the chain (A or B) in which the amino acid residue replacing the natural amino acid residue is located, its position, and the one-letter or three-letter code.

[0065] Herein, terms such as "A1", "A2", and "A3" refer to the amino acid at position 1, 2, and 3, respectively, in the A chain of insulin (counting from the N-terminus). Similarly, terms such as "B1", "B2", and "B3" refer to the amino acid at position 1, 2, and 3, respectively, in the B chain of insulin (counting from the N-terminus). Using the one-letter code for amino acids, terms such as A21A, A21G, and A21Q indicate that the amino acid at the A21 position is A, G, and Q, respectively. Using the three-letter code for amino acids, the corresponding is A21Ala, A21Gly, and A21Gln, respectively.

[0066] "desB30" refers to the natural B chain of insulin or an analogue thereof lacking the B30 amino acid.

[0067] Herein, the terms "A-1" or "B-1" refer to the position of the amino acid that is N-terminal to A1 or B1, respectively. The terms A-2 or B-2 refer to the position of the first amino acid that is N-terminal to A-1 or B-1, respectively.

[0068] The terms "A22" or "B31" refer to the position of the amino acid that is C-terminal to A21 or B30, respectively.

[0069] Thus, for example, A14E B1K B2P B25H desB27 desB30 human insulin is an analogue of human insulin in which the amino acid at position 14 in the A chain is replaced by glutamic acid, the amino acid at position 1 in the B chain is replaced by lysine, the amino acid at position 2 in the B chain is replaced by proline, the amino acid at position 25 in the B chain is replaced by histidine, and the amino acids at positions 27 and 30 in the B chain are deleted.

[0070] An example of an insulin analogue with a substitution is an insulin analogue wherein the Tyr at position A14 is substituted with Glu. In addition, the amino acid at position B1 or B4 can be substituted with Lys. The amino acid at position B2 can be substituted with Pro. The amino acid at position B25 can be substituted with His.

[0071] An example of an insulin analogue with a deletion is an analogue wherein the B30 amino acid in human insulin has been deleted (desB30 human insulin), an insulin analogue wherein the B1 amino acid in human insulin has been deleted (desB1 human insulin), an insulin analogue wherein the B1 and B2 amino acids in human insulin have been deleted (desB1 desB2 human insulin) and desB27 human insulin.

[0072] An example of an insulin analogue wherein the A chain and / or the B chain has an N-terminal extension (i.e. wherein one or more amino acid residues have been added to the N-terminus) is a human insulin analogue comprising A-2K and A-1P, i.e. a human insulin analogue wherein the A chain has been extended at the N-terminus with KP. Another example is a human insulin analogue wherein one glycine residue has been added to the N-terminus of the B chain, i.e. a human insulin analogue comprising B-1G.

[0073] An example of an insulin analogue wherein the A chain and / or the B chain has a C-terminal extension (i.e. wherein one or more amino acid residues have been added to the C-terminus) is a human insulin analogue comprising A22K.

[0074] A further example is an insulin analogue comprising a combination of the mentioned mutations.

[0075] Examples of insulin analogues include:

[0076] desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11);

[0077] A21Q desB30 human insulin (SEQ ID NO: 3 and SEQ ID NO: 11);

[0078] A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 12);

[0079] A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13);

[0080] A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14);

[0081] A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15);

[0082] A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 16);

[0083] A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 17);

[0084] A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 18);

[0085] A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11);

[0086] A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19);

[0087] A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20); and

[0088] A-2K A-1P desB30 human insulin (SEQ ID NO: 7 and SEQ ID NO: 11).

[0089] Spacer

[0090] As mentioned above, the insulin analogue of the present application comprises less than 10 amino acid modifications (substitutions, deletions, extensions and any combination thereof) relative to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 modifications relative to human insulin. In addition to these up to 9 modifications, the human insulin or human insulin analogue of the present application can comprise a spacer at the C-terminus of the A chain of the human insulin or human insulin analogue, or at the N-terminus of the B chain of the human insulin or human insulin analogue.

[0091] In one embodiment, the spacer is a peptide, which is referred to herein as a spacer peptide or a peptide spacer. In another embodiment, the spacer is a non-peptide linker L.

[0092] Peptide spacer

[0093] Various spacer peptides are known in the art and can be used in the compounds of the present invention. In one embodiment, the spacer is a peptide segment consisting of 4-40 amino acids linked by peptide bonds. In another embodiment, the spacer is a peptide segment consisting of 4-24 amino acids linked by peptide bonds.

[0094] In one embodiment, the spacer comprises one or more of the following amino acid residues: Gly(G), Glu(E), Ser(S), Pro(P), Arg(R), Phe(F), Tyr(Y), Asp(D), and Lys(K). In one embodiment, the spacer comprises one or more of the following amino acid residues: Gly(G), Glu(E), Ser(S), and Lys(K). In one embodiment, the spacer comprises one or more of the following amino acid residues: Gly(G), Ser(S), Pro(P), Arg(R), Phe(F), Tyr(Y), Asp(D), and Lys(K). In one embodiment, the spacer comprises one or more of the following amino acid residues: Gly(G), Ser(S), Pro(P), and Lys(K). In one embodiment, the spacer comprises at least one Lys(K) residue.

[0095] In one embodiment, the human insulin or human insulin analog of the present invention includes a peptide spacer at the C-terminus of the A chain of the human insulin or human insulin analog. In one embodiment, the peptide spacer comprises (GES). p K, where p is an integer from 3 to 12.

[0096] Examples of peptide spacers at the C-terminus of the A chain of the human insulin or the human insulin analog include: (GES)3K (SEQ ID NO:29); (GES)6K (SEQ ID NO:30); and (GES) 12 K(SEQ ID NO:31).

[0097] In one embodiment, the human insulin or human insulin analog of the present invention includes a peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analog. In one embodiment, the peptide spacer comprises GKPG or GKP (G4S). q KP (G4S) r GKPRGFFYTP(G4S) s Or TYFFGRKPD(G4S) t Where each of q, r, s, and t is independently selected from integers from 1 to 5. In another embodiment, the peptide spacer comprises GKPG, GKP (G4S).q , KP(G4S) r , GKPRGFFYTP(G4S) s or TYFFGRKPD(G4S) t wherein q is an integer from 1 to 3.

[0098] Examples of peptide spacers at the N-terminus of the B chain of the human insulin or the human insulin analog include:

[0099] GKPG (SEQ ID NO: 32);

[0100] GKPGGGGS(GKP(G4S)) (SEQ ID NO: 33);

[0101] GKPGGGGSGGGGS(GKP(G4S)2) (SEQ ID NO: 34);

[0102] GKPGGGGSGGGGSGGGGS(GKP(G4S)3) (SEQ ID NO: 35);

[0103] KPGGGGSGGGGSGGGGS(KP(G4S)3) (SEQ ID NO: 36);

[0104] GKPRGFFYTPGGGGSGGGGS(GKPRGFFYTP(G4S)2) (SEQ ID NO: 37); and

[0105] TYFFGRKPDGGGGSGGGGSGGGGS(TYFFGRKPD(G4S)3) (SEQ ID NO: 38).

[0106] Examples of insulin analogs comprising a peptide spacer at the C-terminus of the A chain of the human insulin or the human insulin analog include:

[0107] A21Q(GES)3K desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 11);

[0108] A21Q(GES)6K desB30 human insulin (SEQ ID NO: 9 and SEQ ID NO: 11); and

[0109] A21Q(GES) 12 K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11).

[0110] Examples of insulin analogs comprising a peptide spacer at the N-terminus of the B chain of the human insulin or the human insulin analog include:

[0111] B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 21);

[0112] B1-KPGGGGSGGGGSGGGGS A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 22);

[0113] B1-GKPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 23);

[0114] B1-GKPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 24);

[0115] B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25);

[0116] B1-GKPRG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26);

[0117] B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27); and

[0118] B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28).

[0119] Linker L

[0120] In one aspect, the spacer is a non-peptide linker L. Various non-peptide linkers are known in the art and can be used in the compounds of the present application.

[0121] In one embodiment, the human insulin or human insulin analog of the present application comprises a linker L at the N-terminus of the B chain of the human insulin or the human insulin analog.

[0122] In one embodiment, the linker is of the formula L1:

[0123]

[0124] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the N-terminal amino acid residue of the B chain of human insulin or a human insulin analogue.

[0125] In one embodiment, the linker is of formula L2:

[0126]

[0127] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the N-terminal amino acid residue of the B chain of human insulin or a human insulin analogue, and wherein u is 1, 2 or 3. In one embodiment, u is 2 or 3.

[0128] In one embodiment, the linker is of formula L3:

[0129]

[0130] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the N-terminal amino acid residue of the B chain of human insulin or a human insulin analogue, and wherein v is 2 or 3.

[0131] Insulin derivative

[0132] The term "insulin derivative" as used herein refers to a chemically modified insulin or an analogue thereof, wherein the modification is in the form of the attachment of one or more modification groups M.

[0133] In one embodiment, each of the one or more modification groups M is attached to the amino group of the N-terminal amino acid residue of the A chain or the B chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, optionally via a spacer.

[0134] In one embodiment, each modification group M is attached to an attachment point selected from one of the following groups:

[0135] a) the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue;

[0136] b) the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue;

[0137] or

[0138] the epsilon amino group of a lysine in the optional peptide spacer of the C-terminus of the A chain of the human insulin or human insulin analogue;

[0139] c) the amino group of the N-terminal amino acid residue of the B chain of the human insulin or human insulin analogue.

[0140] the epsilon amino group of a lysine residue at position 1 or position 4 of the B chain of the human insulin analogue;

[0141] the epsilon amino group of a lysine in the optional peptide spacer N-terminal of the B chain of the human insulin or human insulin analogue; or

[0142] the epsilon amino group of a lysine in the optional linker L N-terminal of the B chain of the human insulin or human insulin analogue; and

[0143] d) the epsilon amino group of a lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue.

[0144] In one embodiment, no more than one modification group M is attached to the attachment point within each of groups a), b), c) and d).

[0145] In one embodiment, the compound of the application comprises two modification groups M, wherein one modification group M is attached to the amino group of a lysine residue at position 1 or position 4 of the B chain of the human insulin analogue, or the epsilon amino group of a lysine in the optional peptide extension N-terminal of the B chain of the human insulin or human insulin analogue; and the other modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0146] In one embodiment, the compound of the application has exactly two modification groups M, wherein one modification group M is attached to the amino group of a lysine residue at position 1 or position 4 of the B chain of the human insulin analogue, or the epsilon amino group of a lysine in the optional peptide extension N-terminal of the B chain of the human insulin or human insulin analogue; and the other modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0147] In one embodiment, the compound of the application comprises two modification groups M, wherein one modification group M is attached to the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue; and the other modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0148] In one embodiment, the compound of the application has exactly two modification groups M, wherein one modification group M is attached to the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue; and the other modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0149] In one embodiment, the compound of the application comprises two modification groups M, wherein one modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of said human insulin analogue, or to the epsilon amino group of a lysine in an optional peptide spacer C-terminal of said human insulin or human insulin analogue A chain; and the other modification group M is attached to the epsilon amino group of the lysine at position 22 or position 29 of said human insulin or human insulin analogue B chain.

[0150] In one embodiment, the compound of the application has exactly two modification groups M, wherein one modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of said human insulin analogue, or to the epsilon amino group of a lysine in an optional peptide spacer C-terminal of said human insulin or human insulin analogue A chain; and the other modification group M is attached to the epsilon amino group of the lysine at position 22 or position 29 of said human insulin or human insulin analogue B chain.

[0151] In one embodiment, the compound of the application comprises one modification group M, wherein the modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of said human insulin analogue; or to the epsilon amino group of the lysine at position 29 of said human insulin or human insulin analogue B chain.

[0152] In one embodiment, the compound of the application has exactly one modification group M, wherein the modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of said human insulin analogue; or to the epsilon amino group of the lysine at position 29 of said human insulin or human insulin analogue B chain.

[0153] In one embodiment, the compound of the application comprises three or four modification groups M, wherein the first modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of said human insulin analogue; the second modification group M is attached to the epsilon amino group of the lysine at position 22 or position 29 of said human insulin or human insulin analogue B chain; and the remaining modification groups M are each attached to the amino group of the N-terminal amino acid residue of said human insulin or human insulin analogue A chain; to the epsilon amino group of the lysine at position 22 or position 29 of said human insulin or human insulin analogue B chain; or to the distal amino group marked with *1 in said optional linker L N-terminal of said human insulin or human insulin analogue B chain.

[0154] In one embodiment, the compound of the application has exactly three or four modification groups M, wherein the first modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue; the second modification group M is attached to the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue; and the remaining modification groups M are each attached to the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue; to the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue; or to the distal amino group in the optional linker L marked with *1 at the N-terminus of the B chain of the human insulin or human insulin analogue.

[0155] Modifying group M

[0156] The compound of the application comprises one or more modification groups M. In one embodiment, the compound of the application comprises one, two, three or four modification groups M. In one embodiment, the compound of the application comprises two or more modification groups M. In one embodiment, the compound of the application comprises two, three or four modification groups M. In one embodiment, the compound of the application comprises two modification groups M. In one embodiment, the compound of the application has exactly two modification groups M. The one or more modification groups can be the same or different. The two or more modification groups can be the same or different. In one embodiment, the modification groups are the same.

[0157] Some modification groups comprise one or more amino acid residues. Each of these amino acid residues can independently be the D- or L- form of the corresponding amino acid residue, i.e. each chiral atom in a modification group can independently be the (R)- or (S)- form. In one embodiment, the amino acid residues of a modification group are L-amino acid residues.

[0158] Each modification group M comprises a diboron moiety, wherein the diboron moiety (i.e. the modification group M) comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties. The boron atom can be part of a boronic acid (or boronic acid ester, depending on the pKa / pH), or it can be part of a boroxole (or boroxolate, depending on the pKa / pH).

[0159] The term "comprising" or "including" certain features should be interpreted as meaning that the subject-matter discussed includes the certain features, but does not exclude other features. Thus, the modifying group M can have more than two aryl moieties to which a boron atom is attached. In one embodiment, the modifying group has exactly two aryl moieties to which a boron atom is attached. In one embodiment, the modifying group has exactly four aryl moieties to which a boron atom is attached.

[0160] The binding of the present dibo rates to glucose is stronger than the mono-boronic acid ester, as shown in Example A. In addition, it is surprising that the present diboronic compound is able to bind to human serum albumin (HSA), thus having a dual action, as the HSA binding is also glucose sensitive (the HSA binding part of the diboronic peptide is inactive due to the blocking of the receptor binding site on the peptide) (data shown in Example B).

[0161] In one embodiment, the modifying group is of formula M1:

[0162]

[0163] which represents the D- or L-amino acid form, and

[0164] wherein n represents an integer in the range of 1 to 4;

[0165] wherein W1 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W1 represents

[0166] NH-CH2-C(=0)-*,

[0167] NH-CH2CH2-C(=0)-*,

[0168] the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*,

[0169] the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-NH-CH2CH2-C(=0)-*,

[0170] or

[0171] NH-CH2CH2-C(=0)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*,

[0172] wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0173] wherein R1 is selected from

[0174]

[0175]

[0176] wherein Y1, Y2, Y3, Y4, Y5, and Y6are independently selected from H, F, CI, CHF2, and CF3.

[0177] In another embodiment, the modifying group is of formula M1, wherein Y1and Y2are H, and Y3is F or CF3; Y4is H or F; and Y5is H and Y6is F.

[0178] In yet another embodiment, the modifying group is of formula M1, wherein n is 1;

[0179] W1represents the L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and R1is

[0180]

[0181] wherein Y1and Y2are H; and Y3is F or CF3.

[0182] In one embodiment, the modifying group is of formula M2:

[0183]

[0184] wherein W2is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W2represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, or NH-CH2CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0185] wherein R2is selected from

[0186]

[0187] wherein Y7, Y8, Y9, Y10, Y11, and Y12are independently selected from H, F, CI, CHF2, and CF3.

[0188] In another embodiment, the modifying group is of formula M2, wherein Y7is H; Y8is H, CI, CHF2, or CF3; Y9is H, F, or CF3; Y10is F; Y11is H; and Y12is F; provided that only one of Y8and Y9is H.

[0189] In yet another embodiment, the modifying group is of formula M2, wherein W2 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W2 represents the L-form of NH-CH(COOH)-CH2CH2-C(=O)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein R2 is

[0190]

[0191] wherein Y7 and Y8 are H; and Y9 is CI, CHF2 or CF3.

[0192] In one embodiment, the modifying group is of formula M3:

[0193]

[0194] which represents the R,R or S,S, or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y13 and Y14 are independently selected from H, F, CI, CHF2 and CF3.

[0195] In another embodiment, the modifying group is of formula M3, wherein Y13 is H or F; and Y14 is H or CF3; with the proviso that only one of Y13 and Y14 is H.

[0196] In one embodiment, the modifying group is of formula M4:

[0197]

[0198] wherein * represents the point of attachment to the human insulin or human insulin analogue, and wherein Y15 and Y16 are independently selected from H, F, CI, CHF2 and CF3.

[0199] In another embodiment, the modifying group is of formula M4, wherein Y15 and Y16 are independently selected from H and F.

[0200] In yet another embodiment, the modifying group is of formula M4, wherein Y15 is H, and Y16 is F.

[0201] In one embodiment, the modifying group is of formula M5:

[0202]

[0203] wherein each of said amino acid residues independently represents the D- or L- amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analogue.

[0204] In one embodiment, the modification group is of formula M6:

[0205]

[0206] wherein the a-amino acid residue represents a D- or L-amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y17and Y18are independently selected from H, F, CI, CHF2, and CF3.

[0207] In another embodiment, the modification group is of formula M6, wherein Y17is H or F; and Y18is H or F.

[0208] In one embodiment, the modification group is of formula M7:

[0209]

[0210] wherein W3is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W3represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue. In one embodiment, W3represents a L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue.

[0211] In one embodiment, the modification group is of formula M8:

[0212]

[0213] wherein W4is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W4represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19is H, F, CI, CHF2, and CF3or SF5.

[0214] In another embodiment, the modification group is of formula M8, wherein Y19is CF3or SF5.

[0215] In yet another embodiment, the modification group is of formula M8, wherein Y19is CF3.

[0216] In one embodiment, the modification group is of formula M9:

[0217]

[0218] wherein * represents the point of attachment to the human insulin or human insulin analog; and wherein each of Y20, Y21, and Y22 is independently selected from H, F, CI, CHF2, and CF3.

[0219] In another embodiment, the modifying group is of formula M9, wherein each of Y20, Y21, and Y22 is independently selected from H and F; provided that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F.

[0220] In one embodiment, the modifying group is of formula M10:

[0221]

[0222] wherein * represents the point of attachment to the human insulin or human insulin analog.

[0223] In one embodiment, the modifying group is of formula M11:

[0224]

[0225] wherein each of the amino acid residues represents a D- or L-amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analog.

[0226] Compounds of the invention

[0227] In one embodiment, the compound of the application comprises a human insulin or a human insulin analog; and one or more modifying groups M, wherein each modifying group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein each of the one or more modifying groups M is attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analog, or to the epsilon amino group of a lysine in the human insulin or human insulin analog, optionally via a spacer.

[0228] In another embodiment, the compound of the application comprises a human insulin or a human insulin analog; and two modifying groups M, wherein each modifying group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein a first modifying group M is attached to the epsilon amino group of a lysine residue at position 1 or position 4 of the B-chain of the human insulin analog, or to the epsilon amino group of a lysine in an optional peptide spacer at the N-terminus of the B-chain of the human insulin or human insulin analog; and a second modifying group is attached to the epsilon amino group of a lysine at position 22 or position 29 of the B-chain of the human insulin or human insulin analog.

[0229] In another embodiment, the compound of the application comprises a human insulin or a human insulin analogue; and 2 modification groups M, wherein each modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein the first modification group M is attached to the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue; and the second modification group is attached to the epsilon amino group of the lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0230] In another embodiment, the compound of the application comprises a human insulin or a human insulin analogue; and 2 modification groups M, wherein each modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein the first modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue, or to the epsilon amino group of a lysine in an optional peptide spacer C-terminal of the A chain of the human insulin or human insulin analogue; and the second modification group is attached to the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue.

[0231] In another embodiment, the compound of the application comprises a human insulin or a human insulin analogue; and 1 modification group M, wherein the modification group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein the modification group M is attached to the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue, or to the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue.

[0232] In one embodiment, the present application relates to a compound independently selected from the group consisting of the compounds of Examples 181, 205, 210, 211, 227, 233, 234, 239, 240, 241, 272, 273, 280, 284, 285, 288, 291, 300, 301, 324, 327, 331, 333, and 335.

[0233] In one embodiment, the present application relates to a compound independently selected from the group consisting of the compounds of Examples 181, 205, 210, 211, 227, 233, 234, 239, 240, 241, 272, 273, 280, 285, 288, 291, 300, 301, 327, 331, 333, and 335.

[0234] In one embodiment, the compound of the application is the compound of Example 181. In one embodiment, the compound of the application is the compound of 205. In one embodiment, the compound of the application is the compound of 210. In one embodiment, the compound of the application is the compound of 211. In one embodiment, the compound of the application is the compound of 227. In one embodiment, the compound of the application is the compound of 233. In one embodiment, the compound of the application is the compound of 234. In one embodiment, the compound of the application is the compound of 239. In one embodiment, the compound of the application is the compound of 240. In one embodiment, the compound of the application is the compound of 241. In one embodiment, the compound of the application is the compound of 272. In one embodiment, the compound of the application is the compound of 273. In one embodiment, the compound of the application is the compound of 280. In one embodiment, the compound of the application is the compound of 284. In one embodiment, the compound of the application is the compound of 285. In one embodiment, the compound of the application is the compound of 288. In one embodiment, the compound of the application is the compound of 291. In one embodiment, the compound of the application is the compound of 300. In one embodiment, the compound of the application is the compound of 301. In one embodiment, the compound of the application is the compound of 324. In one embodiment, the compound of the application is the compound of 327. In one embodiment, the compound of the application is the compound of 331. In one embodiment, the compound of the application is the compound of 333. In one embodiment, the compound of the application is the compound of 335.

[0235] Intermediate

[0236] Furthermore, the present application provides intermediates which are in the form of novel insulin analogues or insulin analogues comprising a peptide spacer.

[0237] Thus, the present application also relates to intermediates independently selected from the group consisting of:

[0238] A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 16);

[0239] A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 17);

[0240] A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO:4 and SEQ ID NO: 18);

[0241] A22K B22K B29R desB30 human insulin (SEQ ID NO:6 and SEQ ID NO:20);

[0242] A21Q(GES)3K desB30 human insulin (SEQ ID NO:8 and SEQ ID NO: 11);

[0243] A21Q(GES)6K desB30 human insulin (SEQ ID NO:9 and SEQ ID NO: 11);

[0244] A21Q(GES)12K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11);

[0245] B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:21);

[0246] B1-KPGGGGSGGGGSGGGGS A14E B25H desB30 human insulin (SEQ ID NO:4 and SEQ ID NO:22);

[0247] B1-GKPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:23);

[0248] B1-GKPGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:24);

[0249] B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:25);

[0250] B1-GKPRG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:26);

[0251] B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO:27); and

[0252] B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28).

[0253] Insulin function

[0254] The relative binding affinity of an insulin analogue to the human insulin receptor (IR) can be determined by competition binding in a flash proximity assay (SPA) as described in Example B.

[0255] In one embodiment, the compounds of the application have the ability to bind to the insulin receptor. In one embodiment, the compounds of the application have a higher affinity for the insulin receptor in the presence of 20 mM glucose compared to the absence of glucose.

[0256] The AKT phosphorylation assay described in Example C and the lipogenesis assay described in Example D can be used as a measure of the functional (agonistic) activity of an insulin analogue.

[0257] Pharmaceutical composition

[0258] The present application also relates to pharmaceutical compositions comprising a compound of the present application, including, for example, an analogue of the present application or a pharmaceutically acceptable salt, amide or ester thereof, and one or more pharmaceutically acceptable excipients. Such compositions can be prepared as known in the art.

[0259] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. An excipient can be an inert substance, an inactive substance and / or a non-pharmaceutically active substance. Excipients can be used for various purposes, for example, as carriers, vehicles, diluents and / or to improve the administration and / or absorption of the active substance. Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity modifiers, chelating agents and stabilizers. The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21stEdition (2005) and any subsequent editions).

[0260] The compositions of the present application can be in the form of a liquid formulation, i.e., an aqueous formulation comprising water. The liquid formulation can be a solution or a suspension. The compositions of the present application can be used for parenteral administration, e.g., by subcutaneous, intramuscular, intraperitoneal or intravenous injection.

[0261] Aryl boron compounds generally have low stability in aqueous solutions at pH near neutral. The C-B bond can hydrolyze to produce a phenyl residue and a free borate, Ph-H + B(OH)3, or the compound can be oxidized to produce a phenol residue + free borate, Ph-OH + B(OH)3. Certain preferred diboron compounds and diboron insulin conjugates of the present application were found to be more stable than other aryl-boron and aryl-boron in general of the present application. Stability can be assessed, for example, by placing in aqueous solution at neutral pH for an extended period of time, e.g., one week, at 25 or 37 degrees Celsius, followed by measuring the purity of the insulin derivative.

[0262] Pharmaceutical indication

[0263] Diabetes

[0264] The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other states that cause hyperglycemia. The term is used for a metabolic disorder in which the amount of insulin that the pancreas produces is insufficient, or where the body's cells do not react properly to insulin, preventing the cells from absorbing glucose.

[0265] Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by the destruction of beta-cells, usually resulting in absolute insulin deficiency.

[0266] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with predominantly insulin resistance, and thus with relative insulin deficiency, and / or with predominantly insulin secretion defect with insulin resistance.

[0267] Other indications

[0268] In one embodiment, the compounds according to the present application are used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes, including stress-induced hyperglycemia.

[0269] In another embodiment, the compounds according to the present application are used as a medicament for delaying or preventing disease progression of type 2 diabetes.

[0270] In one embodiment of the present application, the compounds are used as a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes, including stress-induced hyperglycemia.

[0271] In a further embodiment, the present application relates to a method of treating or preventing hyperglycemia, type 2 diabetes, impaired glucose tolerance or type 1 diabetes, including stress-induced hyperglycemia, which method comprises administering to a patient in need of such treatment an effective amount of such treatment with a compound of the present application.

[0272] Mode of administration

[0273] The term "treatment" is intended to include preventative and minimization of the mentioned disease, disorder or condition (i.e. "treatment" refers to both prophylactic and therapeutic administration of a compound of the present application or a composition comprising a compound of the present application, unless otherwise indicated or apparent from context).

[0274] The route of administration can be any route that is effective in delivering a compound of the present application to the desired or appropriate location in the body, such as parenteral, e.g. subcutaneous, intramuscular or intravenous routes.

[0275] For parenteral administration, the compounds of the present application are formulated similarly to formulations of known insulins. Furthermore, for parenteral administration, the compounds of the present application are administered similarly to the administration of known insulins, which procedure is familiar to physicians.

[0276] The amount of a compound of the present application to be administered is determined in consultation with a physician familiar with the treatment of diabetes, the frequency of administration of a compound of the present application is determined, and which compound or compounds of the present application are selected, optionally together with another anti-diabetic compound, is / are administered.

[0277] While certain features of the application have been illustrated and described, 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 embodiments are intended to cover all such modifications and changes as fall within the true scope of the application.

[0278] Embodiments

[0279] The present application is further described by the following non-limiting embodiments of the present application:

[0280] 1. A compound comprising:

[0281] i) a human insulin or a human insulin analogue; and

[0282] ii) one or more modifying groups M, wherein each modifying group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and

[0283] wherein each of the one or more modification groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue, via a spacer.

[0284] 2. The compound according to embodiment 1, wherein each of the modification groups M is independently selected from

[0285]

[0286] which represents the D- or L-amino acid form, and

[0287] wherein n represents an integer in the range of 1 to 4;

[0288] wherein W1 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W1 represents

[0289] NH-CH2-C(=0)-*,

[0290] NH-CH2CH2-C(=0)-*,

[0291] the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*,

[0292] the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-NH-CH2CH2-C(=0)-*,

[0293] NH-CH2CH2-C(=0)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*,

[0294] wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0295] wherein R1 is selected from

[0296]

[0297] wherein Y1, Y2, Y3, Y4, Y5 and Y6 are independently selected from H, F, Cl, CHF2 and CF3;

[0298]

[0299] wherein W2 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W2 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-* or NH-CH2CH2CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0300] wherein R2 is selected from

[0301]

[0302] wherein Y7, Y8, Y9, Y10, Y11 and Y12 are independently selected from H, F, Cl, CHF2 and CF3;

[0303]

[0304] which represents the R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y13 and Y14 are independently selected from H, F, Cl, CHF2 and CF3;

[0305]

[0306] wherein * represents the point of attachment to said human insulin or human insulin analogue, and wherein Y15 and Y16 are independently selected from H, F, Cl, CHF2 and CF3;

[0307]

[0308] wherein each of said amino acid residues independently represents a D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue;

[0309]

[0310] wherein the alpha-amino acid residue represents a D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y17 and Y18 are independently selected from H, F, Cl, CHF2 and CF3;

[0311]

[0312] wherein W3 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W3 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue;

[0313]

[0314] wherein W4 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19 is H, F, Cl, CHF2 and CF3 or SF5;

[0315]

[0316] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H, F, Cl, CHF2 and CF3;

[0317]

[0318] wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0319]

[0320] wherein each of the amino acid residues independently represents a D- or L-amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analogue.

[0321] 3. The compound according to any one of embodiments 1 to 2, wherein each of the modification groups M is independently selected from

[0322]

[0323] which represents a D- or L-amino acid form, and

[0324] wherein n represents an integer in the range of 1 to 4;

[0325] wherein W1 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W1 represents

[0326] NH-CH2-C(=0)-*,

[0327] NH-CH2CH2-C(=0)-*,

[0328] a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*,

[0329] a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-NH-CH2CH2-C(=0)-*, or

[0330] NH-CH2CH2-C(=0)-NH-(CH2)2-0-(CH2)2-0-CH2-CO-*

[0331] wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0332] wherein R1 is selected from

[0333]

[0334] wherein Y1 and Y2 are H and Y3 is F or CF3; Y4 is H or F; and Y5 is H and Y6 is F;

[0335]

[0336] wherein W2 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W2 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-* or NH-CH2CH2CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0337] wherein R2 is selected from

[0338]

[0339] wherein Y7 is H; Y8 is H, CI, CHF2 or CF3; Y9 is H, F or CF3; Y10 is F; Y11 is H; and Y12 is F; with the proviso that only one of Y8 and Y9 is H;

[0340]

[0341] which represents the R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y13 is H or F; and Y14 is H or CF3; with the proviso that only one of Y13 and Y14 is H;

[0342]

[0343] wherein * represents the point of attachment to said human insulin or human insulin analogue, and wherein Y15 and Y16 are independently selected from H and F;

[0344]

[0345] which represents the D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue;

[0346]

[0347] wherein the a-amino acid residue represents a D- or L-amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y17 is H or F; and Y18 is H or F;

[0348]

[0349] wherein W3 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W3 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue;

[0350]

[0351] wherein W4 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19 is CF3 or SF5;

[0352]

[0353] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H and F; with the proviso that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F;

[0354]

[0355] wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0356]

[0357] wherein each of the amino acid residues independently represents a D- or L-amino acid form, and wherein * represents the point of attachment to the human insulin or human insulin analogue.

[0358] 4. The compound according to any one of embodiments 1 to 3, wherein each of the modification groups M is independently selected from

[0359]

[0360] which represents a D- or L-amino acid form, and wherein n is 1 ;

[0361] W1represents the D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)- CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0362] R1is

[0363] wherein Y1and Y2are H; and Y3is F or CF3;

[0364]

[0365] wherein W2is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W2represents the D- or L-form of NH-CH(COOH)- CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0366] wherein R2is

[0367] wherein Y7and Y8are H; and Y9is CI, CHF2or CF3;

[0368]

[0369] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y15is H, and Y16is F;

[0370]

[0371] wherein W3is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W3represents the D- or L-form of NH-CH(COOH)- CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue;

[0372]

[0373] wherein W4is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W4represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19is CF3; and

[0374]

[0375] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21, and Y22is independently selected from H and F; provided that when Y21is F, Y20and Y22are H; and when Y21is H, Y20and Y22are F.

[0376] 5. The compound according to any one of embodiments 1 to 4, wherein the modification groups M are identical.

[0377] 6. The compound according to any one of embodiments 1 to 5, wherein the human insulin or human insulin analogue optionally comprises a spacer selected from the group consisting of

[0378] a) a peptide spacer at the C-terminal end of the A chain of the human insulin or human insulin analogue, wherein the peptide spacer comprises (GES) p K, wherein p is an integer from 3 to 12; or

[0379] b) a peptide spacer or linker L at the N-terminal end of the B chain of the human insulin or human insulin analogue;

[0380] wherein the peptide spacer comprises GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s or TYFFGRKPD(G4S) t wherein each of q, r, s, and t is independently selected from an integer from 1 to 5; and

[0381] wherein the linker L is selected from

[0382]

[0383] wherein *1 indicates the point of attachment to the modification group M, and *2 indicates the point of attachment to the amino group of the amino acid residue at the N-terminal end of the B chain of the human insulin or human insulin analogue;

[0384]

[0385] wherein *1 indicates the point of attachment to the modification group M, and *2 indicates the point of attachment to the amino group of the amino acid residue at the N-terminal end of the B chain of the human insulin or human insulin analogue, and wherein u is 1, 2, or 3; and

[0386]

[0387] wherein *1 indicates the point of attachment to the modification group M, and *2 indicates the point of attachment to the amino group of the amino acid residue at the N-terminal end of the B chain of the human insulin or human insulin analogue, and wherein v is 2 or 3.

[0388] 7. The compound according to embodiment 6, wherein q is an integer selected from 1 to 3; r is 3; s is 2; and t is 3.

[0389] 8. The compound according to any one of embodiments 1 to 7, wherein the chiral amino acid is in L-form.

[0390] 9. The compound according to any one of embodiments 1 to 8, wherein each modification group M is attached to an attachment point selected from one of the following groups:

[0391] a) the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue;

[0392] b) the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue;

[0393] or

[0394] the epsilon amino group of the lysine in the optional peptide spacer of the C-terminus of the A chain of the human insulin or human insulin analogue;

[0395] c) the amino group of the N-terminal amino acid residue of the B chain of the human insulin or human insulin analogue;

[0396] the epsilon amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analogue;

[0397] the epsilon amino group of the lysine in the optional peptide spacer of the N-terminus of the B chain of the human insulin or human insulin analogue; or

[0398] the distal amino group marked with *1 in the optional linker L of the N-terminus of the B chain of the human insulin or human insulin analogue; and

[0399] d) the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue.

[0400] 10. The compound according to embodiment 9, wherein no more than one modification group M is attached to the attachment point within each of groups a), b), c) and d).

[0401] 11. The compound according to any one of embodiments 1 to 10, having exactly one, two, three or four modification groups M.

[0402] 12. The compound according to any one of embodiments 1 to 10, comprising at least two modification groups M.

[0403] 13. The compound according to any one of embodiments 1 to 10, having exactly two, three or four modification groups M.

[0404] 14. The compound according to any one of embodiments 1 to 10, which has exactly two modifying groups M.

[0405] 15. The compound according to any one of embodiments 1 to 14, wherein the human insulin or human insulin analogue is a human insulin analogue comprising desB30.

[0406] 16. The compound according to any one of embodiments 1 to 15, wherein the human insulin or human insulin analogue is a human insulin analogue selected from the group consisting of

[0407] desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11);

[0408] A21Q desB30 human insulin (SEQ ID NO: 3 and SEQ ID NO: 11);

[0409] A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 12);

[0410] A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13);

[0411] A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14);

[0412] A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15);

[0413] A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 16);

[0414] A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 17);

[0415] A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 18);

[0416] A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11);

[0417] A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19);

[0418] A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20); and

[0419] A-2K A-1P desB30 human insulin (SEQ ID NO: 7 and SEQ ID NO: 11).

[0420] 17. The compound according to embodiment 1, comprising

[0421] i) human insulin or a human insulin analogue, wherein the human insulin or the human insulin analogue optionally comprises a spacer or a linker L selected from a peptide spacer in the N-terminal end of the B chain of the human insulin or the human insulin analogue;

[0422] wherein the peptide spacer comprises GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s or TYFFGRKPD(G4S) t wherein each of q, r, s and t is independently selected from an integer from 1 to 5; and

[0423] wherein the linker L is selected from

[0424]

[0425] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the amino acid residue in the N-terminal end of the B chain of the human insulin or the human insulin analogue;

[0426]

[0427] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the amino acid residue in the N-terminal end of the B chain of the human insulin or the human insulin analogue, and wherein u is 1, 2 or 3; and

[0428]

[0429] wherein *1 denotes the point of attachment to the modification group M, and *2 denotes the point of attachment to the amino group of the amino acid residue in the N-terminal end of the B chain of the human insulin or the human insulin analogue, and wherein v is 2 or 3;

[0430] ii) two, three or four modifying groups M, wherein each of said modifying groups M is independently selected from

[0431]

[0432] which represents a D- or L-amino acid form, and

[0433] wherein n represents an integer in the range of 1 to 4;

[0434] wherein W1 is absent and represents an attachment point * to said human insulin or human insulin analogue, or W1 represents

[0435] NH-CH2CH2-C(=0)-*,

[0436] a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*,

[0437] wherein * represents an attachment point to said human insulin or human insulin analogue; and

[0438] wherein R1 is selected from

[0439]

[0440]

[0441] wherein Y1 and Y2 are H, and Y3 is F or CF3; Y4 is F; and Y5 is H and Y6 is F;

[0442]

[0443] wherein W2 is absent and represents an attachment point * to said human insulin or human insulin analogue, or W2 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents an attachment point to said human insulin or human insulin analogue; and

[0444] wherein R2 is selected from

[0445]

[0446] wherein Y7 is H; Y8 is H, CI, CHF2 or CF3; Y9 is H, F or CF3; Y10 is F; Y11 is H; and Y12 is F; with the proviso that only one of Y8 and Y9 is H;

[0447]

[0448] which represents the R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y13 is H or F; and Y14 is H or CF3; with the proviso that only one of Y13 and Y14 is H;

[0449]

[0450] which represents the point of attachment to the human insulin or human insulin analogue; and wherein Y15 is H, and Y16 is F;

[0451]

[0452] which represents the point of attachment to the human insulin or human insulin analogue; and wherein Y17 is F; and Y18 is H;

[0453]

[0454] which represents the point of attachment to the human insulin or human insulin analogue; and wherein W3 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue;

[0455]

[0456] which represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19 is CF3 or SF5;

[0457]

[0458] which represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H and F; with the proviso that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F; and

[0459]

[0460] which represents the point of attachment to the human insulin or human insulin analogue; and

[0461] wherein each modifying group M is attached to a point of attachment selected from one of the following groups:

[0462] a) the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue;

[0463] b) the epsilon amino group of the lysine at position 22 of the A chain of the human insulin analogue;

[0464] or

[0465] the epsilon amino group of the lysine in the optional peptide spacer C-terminal of the A chain of the human insulin or human insulin analogue;

[0466] c) the amino group of the N-terminal amino acid residue of the B chain of the human insulin or human insulin analogue;

[0467] the epsilon amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analogue;

[0468] the epsilon amino group of the lysine in the optional peptide spacer N-terminal of the B chain of the human insulin or human insulin analogue; or

[0469] the distal amino group marked with *1 in the optional linker L N-terminal of the B chain of the human insulin or human insulin analogue; and

[0470] d) the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue,

[0471] wherein one modifying group M is attached to one of the attachment points c) and one modifying group M is attached to the attachment point d).

[0472] 18. The compound according to embodiment 17, wherein no more than one modifying group M is attached to the attachment point within each of the groups a), b), c) and d).

[0473] 19. The compound according to any one of embodiments 17 to 18, wherein the compound has exactly two modifying groups M, wherein one modifying group M is attached to the epsilon amino group of the lysine at position 22 or position 29 of the B chain of the human insulin or human insulin analogue; and one modifying group M is attached to

[0474] the amino group of the N-terminal amino acid residue of the B chain of the human insulin or human insulin analogue;

[0475] the epsilon amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analogue;

[0476] the epsilon amino group of the lysine in the optional peptide spacer N-terminal of the B chain of the human insulin or human insulin analogue; or

[0477] the distal amino group marked with *1 in the optional linker L of the N-terminus of the B chain of the human insulin or the human insulin analogue.

[0478] 20. The compound according to any one of embodiments 17 to 19, comprising

[0479] i) a human insulin or a human insulin analogue, wherein the human insulin or the human insulin analogue optionally comprises a peptide spacer at the N-terminus of the B chain of the human insulin or the human insulin analogue;

[0480] wherein the peptide spacer comprises GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s or TYFFGRKPD(G4S) t wherein q is an integer from 1 to 3; r is 3; s is 2 and t is 3;

[0481] ii) two modifying groups M, wherein each of the modifying groups M is independently selected from

[0482]

[0483] which represents a D- or L-amino acid form, and wherein n is 1 ; W1 represents

[0484] the D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or the human insulin analogue; and

[0485] wherein R1 is

[0486]

[0487] wherein Y1 and Y2 are H and Y3 is CF3;

[0488]

[0489] wherein W3 is absent and represents the point of attachment * to the human insulin or the human insulin analogue, or W3 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or the human insulin analogue;

[0490]

[0491] wherein W4 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19 is CF3;

[0492]

[0493] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H and F; with the proviso that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F; and

[0494] wherein one modification moiety M is attached to the epsilon amino group of the lysine at position 29 of the B chain of the human insulin or human insulin analogue; and one modification moiety M is attached to

[0495] the epsilon amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analogue; or

[0496] the epsilon amino group of the lysine in the optional peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue.

[0497] 21. The compound according to any one of embodiments 17 to 20, comprising

[0498] i) a human insulin analogue, wherein the human insulin analogue comprises a peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue; wherein the peptide spacer comprises GKP(G4S) q or KP(G4S) r wherein q is an integer from 1 to 3; and r is 3;

[0499] ii) two modification moieties M, independently selected from

[0500]

[0501] representing the D- or L-amino acid form, and wherein n is 1 ; W1 represents

[0502] NH-CH2CH2-C(=0)-* or the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0503] wherein R1 is

[0504]

[0505] wherein Y1and Y2are H and Y3is CF3;

[0506] wherein one modification group M is attached to the epsilon amino group of a lysine in the peptide spacer; and one modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0507] 22. The compound according to any one of embodiments 17-19, consisting of

[0508] i) a human insulin analogue, wherein the human insulin analogue optionally comprises a peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue;

[0509] wherein the peptide spacer comprises GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s or TYFFGRKPD(G4S) t wherein q is an integer from 1 to 3; r is 3; s is 2 and t is 3;

[0510] ii) two modification groups M, wherein each of the modification groups M is independently selected from

[0511]

[0512] which represents a D- or L-amino acid form, and wherein n is 1 ; W1represents

[0513] a D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0514] wherein R1is

[0515]

[0516] wherein Y1and Y2are H and Y3is CF3;

[0517]

[0518] wherein W3is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W3represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue;

[0519]

[0520] wherein W4 is absent and represents the point of attachment to the human insulin or human insulin analogue, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y19 is CF3;

[0521]

[0522] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H and F; with the proviso that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F; and

[0523] wherein one modification moiety M is attached to the epsilon amino group of the lysine at position 29 of the B chain of the human insulin or human insulin analogue; and

[0524] one modification moiety M is attached to:

[0525] the epsilon amino group of a lysine residue at position 1 or position 4 of the B chain of the human insulin analogue, or

[0526] the epsilon amino group of a lysine in the optional peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue.

[0527] 23. The compound according to any one of embodiments 17 to 22, consisting of

[0528] i) a human insulin analogue, wherein the human insulin analogue has a peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue; wherein the peptide spacer is GKP(G4S) q or KP(G4S) r wherein q is an integer from 1 to 3; and r is 3;

[0529] ii) two modification moieties M, independently selected from

[0530]

[0531] representing the D- or L-amino acid form, and wherein n is 1 ; W1 represents

[0532] NH-CH2CH2-C(=0)-* or the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and

[0533] wherein R1 is

[0534]

[0535] wherein Y1and Y2are H, and Y3is CF3;

[0536] wherein one modification group M is attached to the epsilon amino group of a lysine in the peptide spacer; and one modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analog.

[0537] 24. The compound according to any one of embodiments 17 to 23, wherein the chiral amino acid is in the L-form.

[0538] 25. The compound according to any one of embodiments 17 to 24, wherein the compound has exactly 2 modification groups M.

[0539] 26. The compound according to any one of embodiments 17 to 25, wherein the modification groups M are identical.

[0540] 27. The compound according to any one of embodiments 17 to 26, wherein the human insulin analog comprises desB30.

[0541] 28. The compound according to any one of embodiments 17 to 27, wherein the human insulin or human insulin analog is a human insulin analog selected from the group consisting of

[0542] desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11);

[0543] A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 12);

[0544] A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13);

[0545] A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 16);

[0546] A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 17);

[0547] A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 18);

[0548] A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11);

[0549] A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19); and

[0550] A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20).

[0551] 29. The compound according to any one of embodiments 17 to 28, wherein the human insulin analogue comprising the spacer is selected from

[0552] B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 21);

[0553] B1-KPGGGGSGGGGSGGGGS A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 22);

[0554] B1-GKPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 23);

[0555] B1-GKPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 24); and

[0556] B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25).

[0557] 30. The compound according to any one of embodiments 17 to 29, wherein the compound is selected from:

[0558] the compound of Example 280; the compound of Example 284; the compound of Example 285; the compound of Example 288; the compound of Example 291; the compound of Example 300; the compound of Example 301; the compound of Example 324; the compound of Example 327; the compound of Example 331; the compound of Example 333; and the compound of Example 335.

[0559] 31. The compound according to any one of embodiments 17 to 30, wherein the compound is selected from:

[0560] the compound of Example 280; the compound of Example 285; the compound of Example 288; the compound of Example 291 ; the compound of Example 300; the compound of Example 301 ; the compound of Example 327; the compound of Example 331 ; the compound of Example 333; and the compound of Example 335.

[0561] 32. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 280.

[0562] 33. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 284.

[0563] 34. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 285.

[0564] 35. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 288.

[0565] 36. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 291.

[0566] 37. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 300.

[0567] 38. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 301.

[0568] 39. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 324.

[0569] 40. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 327.

[0570] 41. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 331.

[0571] 42. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 333.

[0572] 43. The compound according to any one of embodiments 17-31, wherein the compound is the compound of Example 335.

[0573] 44. The compound according to embodiment 1, comprising

[0574] i) human insulin or a human insulin analogue;

[0575] ii) two modifying groups M, independently selected from

[0576]

[0577] which represents a D- or L-amino acid form, and

[0578] wherein n represents an integer in the range of 1 to 4;

[0579] wherein W1 is absent and represents an attachment point * to the human insulin or human insulin analogue, or W1 represents

[0580] NH-CH2-C(=0)-*,

[0581] NH-CH2CH2-C(=0)-*,

[0582] wherein * represents an attachment point to the human insulin or human insulin analogue; and

[0583] wherein R1 is selected from

[0584]

[0585] wherein Y1 and Y2 are H, and Y3 is F or CF3; Y4 is H or F; and Y5 is H and Y6 is F;

[0586]

[0587] wherein W2 is absent and represents an attachment point * to the human insulin or human insulin analogue; and

[0588] wherein R2 is selected from

[0589]

[0590] wherein Y7 is H; Y8 is H, CI, CHF2 or CF3; Y9 is H, F or CF3; Y10 is F; Y11 is H; and Y12 is F; with the proviso that only one of Y8 and Y9 is H;

[0591]

[0592] wherein the alpha-amino acid residue represents a D- or L-amino acid form, and wherein * represents an attachment point to the human insulin or human insulin analogue; and wherein Y17 is H or F; and Y18 is H or F; and

[0593]

[0594] wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein each of Y20, Y21, and Y22 is independently selected from H and F; provided that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F; and

[0595] wherein one modification group M is attached to the amino group of the N-terminal amino acid residue of the A chain of the human insulin or human insulin analogue; and one modification group M is attached to the epsilon amino group of the lysine at position 29 of the B chain of the human insulin or human insulin analogue.

[0596] 45. The compound according to embodiment 44, wherein the modification groups M are identical.

[0597] 46. The compound according to any one of embodiments 44 to 45, wherein the human insulin or human insulin analogue is a human insulin analogue comprising desB30.

[0598] 47. The compound according to embodiment 46, wherein the human insulin analogue is desB30 human insulin.

[0599] 48. The compound according to embodiment 1, comprising

[0600] i) a human insulin or human insulin analogue, wherein the human insulin or human insulin analogue optionally comprises a peptide spacer at the C-terminus of the A chain of the human insulin or human insulin analogue, wherein the peptide spacer comprises (GES) p K, wherein p is an integer from 3 to 12;

[0601] ii) two modification groups M, independently selected from

[0602]

[0603] which represents the D- or L-amino acid form, and

[0604] wherein n represents an integer in the range of 1 to 4;

[0605] wherein W1 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W1 represents

[0606] NH-CH2-C(=O)-*,

[0607] NH-CH2CH2-C(=O)-*,

[0608] NH-CH(COOH)-CH2CH2-C(=O)-* of the D- or L-form,

[0609] NH-CH(COOH)-CH2CH2-C(=0)-NH-CH2CH2-C(=0)-* in D- or L-form, or

[0610] NH-CH2CH2-C(=0)-NH-(CH2)2-0-(CH2)2-0-CH2-CO-*

[0611] wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0612] wherein R1 is selected from

[0613]

[0614] wherein Y1 and Y2 are H and Y3 is F or CF3; Y4 is H or F; and Y5 is H and Y6 is F;

[0615]

[0616] wherein W2 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W2 represents NH-CH(COOH)-CH2CH2-C(=0)-* in D- or L-form, or NH-CH2CH2CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0617] wherein R2 is selected from

[0618]

[0619] wherein Y7 is H; Y8 is H, CI, CHF2 or CF3; Y9 is H, F or CF3; Y10 is F; Y11 is H; and Y12 is F; with the proviso that only one of Y8 and Y9 is H;

[0620]

[0621] which represents the R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y13 is H or F; and Y14 is H or CF3; with the proviso that only one of Y13 and Y14 is H;

[0622]

[0623] wherein * represents the point of attachment to said human insulin or human insulin analogue, and wherein Y15 and Y16 are independently selected from H and F;

[0624]

[0625] wherein each of said amino acid residues represents a D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue;

[0626]

[0627] wherein the alpha-amino acid residue represents a D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y17 is H or F; and Y18 is H or F;

[0628]

[0629] wherein W3 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W3 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue;

[0630]

[0631] wherein W4 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein Y19 is CF3 or SF5;

[0632]

[0633] wherein * represents the point of attachment to said human insulin or human insulin analogue; and wherein each of Y20, Y21 and Y22 is independently selected from H and F; with the proviso that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F;

[0634]

[0635] wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0636]

[0637] wherein each of said amino acid residues represents a D- or L-amino acid form, and wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0638] wherein one modification group M is attached to the epsilon amino group of lysine at position 22 or position 29 of the B chain of said human insulin or human insulin analogue; and one modification group M is attached to:

[0639] the epsilon amino group of a lysine at position 22 of the A chain of said human insulin analogue; or

[0640] the epsilon amino group of a lysine in said optional peptide spacer of the C-terminus of the A chain of said human insulin or human insulin analogue.

[0641] 49. The compound according to embodiment 48, wherein the chiral amino acid is in L-form.

[0642] 50. The compound according to any one of embodiments 48 to 49, wherein said modification group M is the same.

[0643] 51. The compound according to any one of embodiments 48 to 51, wherein said human insulin or human insulin analogue is a human insulin analogue comprising desB30.

[0644] 52. The compound according to any one of embodiments 48 to 51, wherein said human insulin or human insulin analogue is selected from the group consisting of:

[0645] A21Q desB30 human insulin (SEQ ID NO: 3 and SEQ ID NO: 11);

[0646] A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14);

[0647] A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15);

[0648] A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11); and

[0649] A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20).

[0650] 53. The compound according to any one of embodiments 48 to 52, wherein said compound is selected from the group consisting of:

[0651] the compound of embodiment 227; the compound of embodiment 239; the compound of embodiment 240; the compound of embodiment 241; and the compound of embodiment 272.

[0652] 54. The compound according to embodiment 1, comprising

[0653] i) a human insulin or a human insulin analogue;

[0654] ii) a modification group M selected from

[0655]

[0656] which represents the D- or L-amino acid form, and

[0657] wherein n represents an integer in the range of 1 to 4;

[0658] wherein W1 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W1 represents

[0659] NH-CH2-C(=O)-*,

[0660] NH-CH2CH2-C(=O)-*,

[0661] a D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-*,

[0662] a D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-NH-CH2CH2-C(=O)-*, or

[0663] NH-CH2CH2-C(=O)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*,

[0664] wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0665] wherein R1 is selected from

[0666]

[0667] wherein Y1 and Y2 are H, and Y3 is F or CF3; Y4 is H or F; and Y5 is H and Y6 is F;

[0668]

[0669] wherein W2 is absent and represents the point of attachment * to said human insulin or human insulin analogue, or W2 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-*, or NH-CH2CH2CH2-C(=O)-*, wherein * represents the point of attachment to said human insulin or human insulin analogue; and

[0670] wherein R2 is selected from

[0671]

[0672] wherein Y7 is H; Y8 is H, CI, CHF2, or CF3; Y9 is H, F, or CF3; Y10 is F; Y11 is H; and Y12 is F; provided that only one of Y8 and Y9 is H;

[0673]

[0674] wherein W3 is absent and represents the point of attachment * to the human insulin or human insulin analog, or W3 represents the D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analog;

[0675]

[0676] wherein W4 is absent and represents the point of attachment * to the human insulin or human insulin analog, or W4 represents NH-CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analog; and wherein Y19 is CF3 or SF5;

[0677]

[0678] wherein * represents the point of attachment to the human insulin or human insulin analog; and wherein each of Y20, Y21, and Y22 is independently selected from H and F; provided that when Y21 is F, Y20 and Y22 are H; and when Y21 is H, Y20 and Y22 are F;

[0679]

[0680] wherein * represents the point of attachment to the human insulin or human insulin analog; and

[0681] wherein the modifying group M is attached to the epsilon amino group of a lysine at position 22 of the human insulin analog A chain, or to the epsilon amino group of a lysine at position 22 or position 29 of the human insulin or human insulin analog B chain.

[0682] 55. The compound according to embodiment 54, wherein the human insulin or human insulin analog is a human insulin analog comprising A22K and desB30.

[0683] 56. The compound according to any one of embodiments 1 to 55, wherein the compound has the ability to bind to an insulin receptor.

[0684] 57. The compound according to any one of embodiments 1 to 55, wherein the compound has a higher affinity for an insulin receptor in the presence of 20 mM glucose compared to in the absence of glucose.

[0685] 58. The compound according to any one of embodiments 1 to 55, wherein the compound has at least 3-fold higher affinity for the insulin receptor in the presence of 20 mM glucose compared to the absence of glucose.

[0686] 59. The compound according to any one of embodiments 1 to 55, wherein the compound has at least 10-fold higher affinity for the insulin receptor in the presence of 20 mM glucose compared to the absence of glucose.

[0687] 60. The compound according to any one of embodiments 1 to 55, wherein the compound has at least 15-fold higher affinity for the insulin receptor in the presence of 20 mM glucose compared to the absence of glucose.

[0688] 61. A composition comprising a compound according to any one of embodiments 1-55.

[0689] 62. A compound according to any one of embodiments 1-55 for use as a medicament.

[0690] 63. A compound according to any one of embodiments 1-55 for use in the prevention or treatment of diabetes, diabetes mellitus type 1, diabetes mellitus type 2, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0691] 64. Use of a compound according to any one of embodiments 1-55 or a composition according to embodiment 61 for the manufacture of a medicament for the treatment or prevention of diabetes, diabetes mellitus type 1, diabetes mellitus type 2, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0692] 65. A method of treatment or prevention of diabetes, diabetes mellitus type 1, diabetes mellitus type 2, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome), the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of embodiments 1-55 or a composition according to embodiment 61. Examples

[0693] Materials and Methods

[0694] List of abbreviations

[0695] AIBN 2,2’-azobis-isobutyronitrile

[0696] AKT Alias PKB, protein kinase B (PKB)

[0697] ALP Achromobactor lyticus protease

[0698] Ar aryl

[0699] ARS alizarin red S

[0700] C18 octadecyl (HPLC column)

[0701] CV column volume

[0702] DAST diethylaminosulfur trifluoride

[0703] DBU 1,8-diazabicyclo(5.4.0)undec-7-ene

[0704] DCM dichloromethane

[0705] DIC N,N-diisopropylcarbodiimide

[0706] DMF N,N-dimethylformamide

[0707] DIPEA N,N-diisopropylethylamine

[0708] EDC.HCl N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride

[0709] EtOAc ethyl acetate

[0710] FFC free fat cells (r, rat)

[0711] Fmoc-OSu 9-fluorenylmethyl N-succinimidyl carbonate

[0712] HATU 1-((dimethylamino)(dimethylimino)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0713] HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0714] HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

[0715] HIR human insulin receptor (A = A isoform, B = B isoform)

[0716] HOBt 1-hydroxybenzotriazole

[0717] HONSU N-hydroxysuccinimide

[0718] HRMS high resolution mass spectrometry

[0719] HSA human serum albumin

[0720] Kd dissociation constant

[0721] LCMS liquid chromatography mass spectrometry

[0722] MeCN acetonitrile

[0723] mM millimolar

[0724] NBS N-bromosuccinimide

[0725] N.D. not detectable

[0726] NMM N-methyl-morpholine

[0727] NMR nuclear magnetic resonance

[0728] NMP N-methyl-pyrrolidone

[0729] OEG 2-(2-aminoethoxy)ethoxy-acetic acid (oligoethylene glycol amino acid)

[0730] OXYMA ethyl cyano hydroxy imino acetate

[0731] RP-HPLC reverse phase high performance liquid chromatography

[0732] Ph phenyl

[0733] SPA scintillation proximity assay

[0734] TFA trifluoroacetic acid

[0735] THF tetrahydrofuran

[0736] THPTA tris(3-hydroxypropyl triazolylmethyl)amine

[0737] TSTU succinimidyl-tetramethyluronium tetrafluoroborate

[0738] UPLC ultra performance liquid chromatography

[0739] WGA wheat germ agglutinin

[0740] Preparation of insulin variants

[0741] Example 1 : Expression of insulin variants in yeast and transformation with ALP etc.

[0742] Insulin analogues are expressed in yeast using well-known techniques, for example as disclosed in WO2017 / 032798. More specifically, the insulin analogue is expressed as a single chain precursor, which is isolated by ion exchange capture and cleaved into a 2-chain insulin analogue by ALP treatment as described below.

[0743] Capture of precursors on SP Sepharose BB:

[0744] The yeast supernatant was loaded onto a column packed with SP Sepharose BB at a flow rate of 10-20 CV / h. It was washed with 0.1 M citric acid pH 3.5 and washed with 40% EtOH. The analogue was eluted with 0.2 M sodium acetate pH 5.5 / 35% EtOH.

[0745] ALP digestion:

[0746] The solution of the single chain precursor was adjusted to pH 9 and ALP enzyme was added at 1 : 100 (w / w). The reaction was performed on UPLC. The ALP cleavage pool was adjusted to pH 2.5 and diluted 2-fold in preparation for RP-HPLC purification.

[0747] RP-HPLC purification:

[0748] Purification by RP-HPLC C18 was performed as follows:

[0749] Column: 15um C18 50x250mm

[0750] Buffer:

[0751] A: 0.2% formic acid, 5% EtOH,

[0752] B: 0,2% formic acid, 50% EtOH

[0753] Gradient: 20-55% B - buffer.

[0754] Gradient: 20 CV

[0755] Flow rate 20 CV / h

[0756] Load g ~ 5 g / l resin

[0757] Fractions were analysed by UPLC, pooled and freeze-dried.

[0758] The insulin analogues prepared and used in the following examples are:

[0759] desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11);

[0760] A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13);

[0761] A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO:5 and SEQ ID NO: 14);

[0762] A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO:5 and SEQ ID NO: 15);

[0763] A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO:4 and SEQ ID NO: 16);

[0764] A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO:4 and SEQ ID NO: 17);

[0765] A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO:4 and SEQ ID NO: 18);

[0766] A22K desB30 human insulin (SEQ ID NO:6 and SEQ ID NO: 11);

[0767] A22K B29R desB30 human insulin (SEQ ID NO:6 and SEQ ID NO: 19);

[0768] A22K B22K B29R desB30 human insulin (SEQ ID NO:6 and SEQ ID NO: 20);

[0769] A-2K A-1P desB30 human insulin (SEQ ID NO:7 and SEQ ID NO: 11);

[0770] A21Q(GES)3K desB30 human insulin (SEQ ID NO:8 and SEQ ID NO: 11);

[0771] A21Q(GES)6K desB30 human insulin (SEQ ID NO:9 and SEQ ID NO: 11);

[0772] A21Q(GES)12K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11);

[0773] B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 21);

[0774] B1-KPGGGGSGGGGSGGGGS A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 22);

[0775] B1-GKPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 23);

[0776] B1-GKPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 24);

[0777] B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25);

[0778] B1-GKPG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26);

[0779] B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27); and

[0780] B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28).

[0781] B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin refers to a desB30 human insulin extended from B1 with GKPRGFFYTPGGGGSGGGGS (written from the new N-terminal G, C-terminal S is attached to B1 of the desB30 human insulin). A21Q(GES)3K desB30 human insulin refers to an insulin extended from A21Q with GESGESGESK (written from the new N-terminal G attached to C-terminal A21Q). Other B1 and A21 extended insulin analogs are analogous. B-1 indicates a position on the N-terminal side of B1, for example, B-1G indicates an N-terminal extension of G from insulin B1.

[0782] Preparation of building blocks

[0783] In each example, intermediates and final products are given a number for ease of reading. The same numbers are used across examples, but the numbers in each example are explicit.

[0784] Example 2: 3,5-Bis[[3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido]methyl]benzoic acid O-succinimidyl ester Example 3: O-Succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzamido-Lys-beta-Ala

[0785]

[0786] A mixture of 2-fluoro-4-carboxyphenylboronic acid (1, 8.44 g, 45.9 mmol), pinacol (5.42 g, 45.9 mmol) and magnesium sulfate (60 g) in tetrahydrofuran (110 mL) was stirred at room temperature overnight. The suspension was filtered through a pad of celite and the filtrate was evaporated and dried under vacuum to give 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (2) as a beige powder. Yield: 10.4 g (85%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.93-7.80 (m, 2H); 7.75 (d, J = 9.4 Hz, 1H); 1.39 (s, 12H).

[0787] Carboxylic acid 2 (10.3 g, 38.6 mmol) was dissolved in dichloromethane (130 mL). 1-Hydroxy-pyrrolidine-2,5-dione (HOSu, 8.89 g, 77.2 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HC1, 14.8 g, 77.2 mmol) were added. The resulting mixture was stirred at room temperature overnight. The reaction mixture was partitioned between ethyl acetate (130 mL) and 0.5 M aqueous hydrochloric acid (130 mL). The organic layer was washed with 0.5 M aqueous hydrochloric acid (3 x 120 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (40 mL) and precipitated by the addition of cyclohexane (130 mL). The product was collected by filtration, washed with cyclohexane and dried under vacuum to give 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid succinimidyl ester (3) as a white powder. Yield: 13.9 g (99%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.93-7.84 (m, 2H); 7.77 (d, J = 9.4 Hz, 1H); 2.92 (s, 4H); 1.39 (s, 12H).

[0788] Methyl 3,5-dimethylbenzoate 4 (827.6 g, 18.4 mmol) was suspended in methanol (80 mL) and treated with concentrated sulfuric acid (8 mL). The mixture was refluxed for 2 days. After neutralization with sodium carbonate (50 g), the mixture was dissolved in water (250 mL) and extracted with diethyl ether (2 x 300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give methyl 3,5-dimethylbenzoate (5) as a light yellow oil. Yield: 29.3 g (97%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.67 (s, 2H); 7.19 (s, 1H); 3.91 (s, 3H); 2.37 (s, 6H).

[0789] A mixture of the above methyl 3,5-dimethylbenzoate 5 (29.3 g, 178 mmol), N- bromosuccinimide (NBS, 111 g, 623 mmol) and a spatula of azobisisobutyronitrile in methyl formate (450 mL) was irradiated with visible light while heating to reflux for 20 h. The solvent was evaporated and the residue was dissolved in dichloromethane (200 mL). The precipitated succinimide was filtered off and the filtrate was washed with saturated aqueous sodium sulfite solution (2 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluent: hexane / ethyl acetate 15:1). The product was crystallized from an ethyl acetate / cyclohexane mixture to give methyl 3,5-bis(bromomethyl)benzoate (6) as a white solid. Yield: 25.6 g (45%). RF(Si02, hexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.67 (s, 2H); 7.19 (s, 1H); 3.91 (s, 3H); 2.37 (s, 6H).

[0790] A suspension of the above bromide 6 (25.3 g, 78.6 mmol) and sodium formamide (20.9 g, 220 mmol) in anhydrous acetonitrile (350 mL) was refluxed for 4 h. After removal of the white solid by filtration, the solvent was evaporated. Crystallization from an ethyl acetate / cyclohexane mixture gave methyl 3,5-bis((N-formylformamidyl)methyl)benzoate (7) as a white powder.

[0791] Yield: 21.0 g (88%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.08 (s, 4H); 7.72 (s, 2H); 7.44 (s, 1H); 4.70 (s, 4H); 3.84 (s, 3H).

[0792] Benzoate 7 (20.9 g, 68.5 mmol) was dissolved in a mixture of 1,4-dioxane (220 mL) and concentrated hydrochloric acid (280 mL) and heated to reflux for 2 hours. After cooling to room temperature, air was bubbled through the solution. The product started to precipitate. After 1 hour, the solvent was evaporated and the product was recrystallized from a mixture of methanol / ethyl ether to give 3,5-bis(aminomethyl)benzoic acid dihydrochloride (8) as a white powder. Yield: 17.1 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 13.26 (bs, 1H); 8.65 (bs, 6H); 8.10 (s, 2H); 7.88 (s, 1H); 4.08 (s, 4H).

[0793] Di-hydrochloride 8 (2.08 g, 8.20 mmol) was dissolved in water (20 mL). N,N- diisopropylethylamine (5.73 mL, 32.9 mmol), N,N-dimethylformamide (40 mL) and activated ester (3, 5.97 g, 16.4 mmol) were subsequently added. The mixture was stirred at room temperature overnight; then acidified with 1 M aqueous hydrochloric acid. The solvent was co-evaporated 3 times with toluene. The residue was dissolved in a dichloromethane / toluene mixture (1:1, 100 mL) and treated with pinacol (1.40 g, 11.8 mmol). The mixture was evaporated 3 times from toluene. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (10 mL) and the product started to precipitate. Cyclohexane (170 mL) was added. The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoic acid (9) as a white powder. Yield: 4.18 g (75%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.96 (bs, 1H); 9.27 (t, J = 5.9 Hz, 2H); 7.82-7.67 (m, 6H); 7.64-7.56 (m, 2H); 7.53 (s, 1H); 4.58-4.44 (m, 4H); 1.31 (s, 24H).

[0794] Dissolve the above acid 9 (4.17 g, 6.20 mmol) in a mixture of acetonitrile / N,N- dimethylformamide (4:1, 100 mL). Add N-hydroxysuccinimide (HOSu, 0.85 g, 7.40 mmol). Cool the mixture to 0 °C, then add N,N-dicyclohexylcarbodiimide (DCC, 1.53 g, 7.40 mmol). Stir the mixture at 0 °C for 30 min and at room temperature overnight. Filter off the insoluble by-product and evaporate the filtrate. Dissolve the residue in ethyl acetate (250 mL) and wash with water (2 x 150 mL). Dry the organic layer over anhydrous sodium sulfate, filter and evaporate. Dissolve the residue in dichloromethane (10 mL) and the product starts to precipitate. Add cyclohexane (170 mL). Collect the precipitate by filtration, wash with cyclohexane and dry under vacuum to give 3,5-bis[[[3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]amino]methyl]benzoic acid O-succinimidyl ester (10) as a white powder. Yield: 4.62 g (97%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.31 (t, J = 5.7 Hz, 2H); 7.93 (s, 2H); 7.79-7.68 (m, 5H); 7.63-7.56 (m, 2H); 4.60-4.50 (m, 4H); 2.87 (s, 4H); 1.31 (s, 24H). LC-MS: 773.4 (M+H) + Calculated 773.4.

[0795] Example 4: O-Succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzamido-Lys-Gly Example 5: 2-((23-(3,5-Bis((3-(3-acetoxy-2,2-bis(acetyloxymethyl)propoxy)propionamido)methyl)benzamido)-7,16-dioxo-3,9,12,18,21 -pentoaoxa-6,15-diazatricosyl)oxy)-N-(4- formylbenzyl)acetamide

[0796]

[0797] Fmoc-Ala-OH (24.9 g, 80.0 mmol) and N,N-diisopropylethylamine (55.7 mL, 320 mmol) in dry dichloromethane (250 mL) were added to the resin and the mixture was shaken overnight. The resin was then filtered and treated with a solution of N,N-diisopropylethylamine (50 mL) in a methanol / dichloromethane mixture (4:1, 2 x 5 min, 2 x 250 mL). The resin was then filtered and washed with N,N-dimethylformamide (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). The Fmoc group was removed by treatment with a 20% solution of piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 30 min, 2 x 250 mL). The resin was then filtered and washed with N,N-dimethylformamide (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). A solution of Fmoc-L-Lys(Boc)-OH (56.2 g, 120 mmol), 5-chloro-1-((dimethylamino)(dimethylimino)methyl)-1H- benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 42.7 g, 120 mmol) and N,N- diisopropylethylamine (34.8 mL, 200 mmol) in N,N-dimethylformamide (180 mL) was then added to the resin and the mixture was shaken for 3 hours. The resin was then filtered and washed with N,N-dimethylformamide (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). The Fmoc group was removed by treatment with a 20% solution of piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 30 min, 2 x 300 mL). The resin was then filtered and washed with N,N-dimethylformamide (2 x 300 mL), dichloromethane (2 x 300 mL), methanol (2 x 300 mL) and dichloromethane (10 x 300 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (300 mL) overnight. The resin was filtered off and washed with dichloromethane (2 x 200 mL), 2-propanol (2 x 200 mL) and dichloromethane (2 x 200 mL). The solvent was removed under reduced pressure and the residue was triturated in diethyl ether (2 x 300 mL). After filtration and drying, we obtained L-Lys(Boc)-beta-Ala (2) as an off-white powder. Yield: 13.3 g (56%). 1H NMR spectrum (300 MHz, AcOD-d4, δ H ): 4.20 (t, J = 7.1 Hz, 1H); 3.66-3.46 (m, 2H); 3.17-3.00 (m, 2H); 2.65 (t, J = 6.4 Hz, 2H); 1.97-1.80 (m, 2H); 1.60-1.30 (m, 13H).

[0798] A 95% aqueous solution of trifluoroacetic acid (60 mL) was added to a suspension of 2 (13.2 g, 41.6 mmol) in dichloromethane (50 mL) and the whole mixture was stirred for 2 hours. Then the solvent was removed under reduced pressure and the residue was dried in vacuum to give L-Lys-beta-Ala TFA salt (3) as a brown oil. Yield: 18.5 g (100%). 1 H NMR spectrum (300 MHz, AcOD-d4, δ H ): 4.24 (t, J = 6.7 Hz, 1H); 3.71-3.46 (m, 2H); 3.09 (t, J = 7.5 Hz, 2H); 2.66 (t, J = 6.6 Hz, 2H); 2.01-1.89 (m, 2H); 1.85-1.68 (m, 2H); 1.60-1.46 (m, 2H).

[0799] Triethylamine (14.1 mL, 101 mmol) was added to a solution of 3 (15.0 g, 33.7 mmol) in acetonitrile, obtaining a grey-white precipitate. After filtration and drying, L-Lys-beta-Ala (4) was obtained as a white hygroscopic powder. Yield: 7.30 g (100%). 1 H NMR spectrum (300 MHz, AcOD-d4, δ H ): 4.21 (t, J = 6.4 Hz, 1H); 3.72-3.45 (m, 2H); 3.08 (t, J = 7.4 Hz, 2H); 2.65 (t, J = 6.0 Hz, 2H); 2.00-1.88 (m, 2H); 1.83-1.66 (m, 2H); 1.59-1.43 (m, 2H).

[0800] succinimidyl ester 5 (5.00 g, 13.8 mmol) was added to a suspension of 4 (3.00 g, 13.8 mmol) and triethylamine (7.74 mL, 55.5 mmol) in dry acetonitrile (80 mL) and the whole mixture was stirred overnight. Then the solvent was removed under reduced pressure and co-evaporated 3 times with toluene. After that ethyl acetate (70 mL) was added and the mixture was washed with water (3 x 50 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (2 mL) and added dropwise to vigorously stirred cyclohexane (100 mL). The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido-Lys-beta-Ala (6) as a white powder. Yield: 2.31 g (47%). 1 H NMR spectrum (300 MHz, AcOD-d4, δ H ): 7.83-7.73 (m, 2H); 7.71-7.45 (m, 4H); 4.77 (t, J = 7.2 Hz, 1H); 3.61-3.39 (m, 4H); 2.64 (t, J = 6.2 Hz, 2H); 2.00-1.80 (m, 2H); 1.77-1.47 (m, 4H); 1.37 (s, 24H).

[0801] N-hydroxysuccinimide (HOSu, 0.97 g, 8.41 mmol) was added to a solution of 6 (2.00 g, 2.80 mmol) in dry acetonitrile (70 mL). The mixture was cooled to 0 °C, then N,N-dicyclohexylcarbodiimide (DCC, 0.87 g, 4.20 mmol) was added. After 30 min, the reaction mixture was allowed to warm to room temperature and stirred overnight. The insoluble by-product was filtered off and the filtrate was evaporated. The residue was dissolved in ethyl acetate (100 mL) and washed with 1 M aqueous hydrochloric acid (3 x 70 mL), water (70 mL) and brine (70 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was co-evaporated with toluene five times with pinacol. The residue was then dissolved in ethyl acetate (100 mL) and washed with 0.1 M aqueous hydrochloric acid (70 mL), water (70 mL) and brine (70 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (3 mL) and added dropwise to a vigorously stirred mixture of cyclohexane / ethyl ether (10:1, 110 mL). The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum. Residual cyclohexane was removed by co-evaporation with dichloromethane five times. After drying, O-succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido-Lys-beta-Ala (7) was obtained as an off-white foam. Yield: 1.12 g (48%). 1 H NMR spectrum (300 MHz, CDC13, δ H ) 7.83-7.71 (m, 2H); 7.62-7.40 (m, 4H); 7.19 (d, J = 7.7 Hz, 1H); 7.02 (t, J = 6.1 Hz, 1H); 6.68 (t, J = 5.6 Hz, 1H); 4.67 (m, 1H); 3.73-3.62 (m, 2H);

[0802] 3.44 (q, J = 6.2 Hz, 2H); 2.90-2.78 (m, 6H); 2.07-1.60 (m, 4H); 1.53-1.30 (m, 26H). LC-MS: 810.5 (M+H) + , calculated 810.4.

[0803] Example 6: Boc-Lys(Boc)-OEG3-benzaldehyde Example 7: Bis(bis(4-boronic acid-3-fluorobenzamido)-3,5-aminomethylbenzoate-epsilon, alpha-Lys-N-beta-Ala-OSu = (S)-3-(2,6-bis(3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzamido)methyl)benzamido)hexanamido)propanoate

[0804]

[0805] A mixture of 2-fluoro-4-carboxyphenylboronic acid (1, 4.95 g, 27.0 mmol), pinacol (3.21 g, 27.2 mmol) and magnesium sulfate (450 g) in tetrahydrofuran (90 mL) was stirred at room temperature overnight. The suspension was filtered through a celite pad and the filtrate was evaporated and dried in vacuo to give 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (2) as a yellow powder. Yield: 7.06 g (98%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.93-7.80 (m, 2H); 7.76 (d, J = 9.4 Hz, 1H); 1.39 (s, 12H).

[0806] Carboxylic acid 2 (7.05 g, 26.5 mmol) was dissolved in dichloromethane (100 mL). N-hydroxysuccinimide (HOSu, 6.10 g, 53.0 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl, 10.2 g, 53.0 mmol) were added. The resulting mixture was stirred at room temperature overnight. The reaction mixture was partitioned between ethyl acetate (110 mL) and 0.1 M aqueous hydrochloric acid (110 mL). The organic layer was washed with 0.1 M aqueous hydrochloric acid (2 x 100 mL) and brine (1 x 100 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (20 mL) and precipitated by the addition of cyclohexane (120 mL). The product was collected by filtration, washed with cyclohexane and dried in vacuo to give 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid succinimidyl ester (3) as a white powder. Yield: 9.60 g (99%). 1 H NMR spectrum (300 MHz, DMSO-d6, δ H ): 7.96-7.89 (m, 2H); 7.79 (d, J = 9.4 Hz, 1H); 2.91 (s, 4H); 1.33 (s, 12H).

[0807] L-Lys-Gly TFA salt 4 (2.67 g, 6.20 mmol) was dissolved in water (20 mL). Subsequently, N,N-diisopropylethylamine (4.32 mL, 24.8 mmol), N,N-dimethylformamide (40 mL) and 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (3, 4.50 g, 12.4 mmol) were added. The mixture was stirred at room temperature overnight; then acidified with 1 M aqueous hydrochloric acid solution. The solvent was co-evaporated with toluene 3 times. The residue was dissolved in a dichloromethane / toluene mixture (1 :1, 100 mL) and treated with pinacol (1.00 g, 8.46 mmol). The mixture was evaporated from toluene 3 times. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (10 mL) and added dropwise to cold cyclohexane (200 mL). The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum. The solid was dissolved in dichloromethane (10 mL). Ethyl ether (10 mL) and cyclohexane (150 mL) were added. The solvent was decanted and the residue was dried under vacuum to give N,N'-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-L- lysylglycine (5) as a beige solid. Yield: 1.60 g (37%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.91 -7.79 (m, 2H); 7.78-7.64 (m, 2H); 7.58-7.34 (m, 4H); 7.19-7.07 (m, 1 H); 4.86-4.72 (m, 1 H); 4.15-3.88 (m, 2H); 3.47-3.25 (m, 2H); 2.00-1.74 (m, 2H); 1.66-1.52 (m, 2H); 1.50-1.37 (m, 2H); 1.34 (s, 24H). LC-MS: 699.3 (M+H) + , 617.2 (M+H-pinacol) + , 535.0 (M+H-2x pinacol) + .

[0808] The above acid 5 (1.59 g, 2.30 mmol) was dissolved in dichloromethane (70 mL). N-hydroxysuccinimide (HOSu, 0.31 g, 2.70 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl, 0.65 g, 3.40 mmol) were added. The reaction mixture was stirred at room temperature for 5 hours. The mixture was washed with 0.1 M aqueous hydrochloric acid (2 x 80 mL) and brine (1 x 80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give O-succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido-Lys-Gly (6) as a beige solid. Yield: 1.29 g (70%). 1 H NMR spectrum (300 MHz, DMSO-d6, δ H ): 8.73-8.53 (m, 3H); 7.78-7.61 (m, 5H); 7.54 (d, J = 10.4 Hz, 1 H); 4.52-4.40 (m, 1 H); 4.36-4.17 (m, 2H); 3.29-3.17 (m, 2H); 2.81 (s, 4H); 1.87-1.68 (m, 2H); 1.59-1.47 (m, 2H); 1.46-1.36 (m, 2H); 1.31 (s, 24H). LC-MS: 796.4 (M+H) + , calculated 796.4.

[0809] Example 8: (7S,18S)-18-(3-((S)-2,6-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzamido)hexanamido)propanamido)-7-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)benzamido)-1 -(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1,8,12,19- tetraoxo-2,9,13,20-tetraazatricosane-23-oic acid Example 9: 2,5-dioxopyrrolidin-1 -yl N-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzamido)ethyl)-N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide ​

[0810]

[0811] A mixture of pentaerythritol (136 g, 1.00 mol), sodium hydroxide (8.00 g, 200 mmol), dimethyl sulfoxide (200 mL) and water (18 mL) was heated at 80 °C until a clear solution was formed (overnight). Butyl acrylate (2, 174 mL, 1.20 mol) was added and the resulting mixture was heated at 80 °C for 24 h; it was then cooled to room temperature, diluted with water (200 mL) and extracted with ethyl acetate (3 x 400 mL). The combined organic layers were washed with water (400 mL) and brine (100 mL). As the aqueous washes contained product (3), they were combined and re-extracted with ethyl acetate (2 x 200 mL). All ethyl acetate fractions were combined, dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluents: dichloromethane / methanol 99:1-90:10) to give tert-butyl 3-(3-hydroxy-2,2-bis(hydroxymethyl)propoxy)propanoate (3) as a colourless oil.

[0812] Yield: 39.7 g (15%). RF (SiO2, dichloromethane / methanol 9:1): 0.30.

[0813] 1H NMR spectrum (300 MHz, CDC13, δH): 3.67 (t, J = 5.6 Hz, 2H); 3.65 (s, 6H); 3.52 (s, 2H); 2.73 (bs, 3H); 2.49 (t, J = 5.7 Hz, 2H); 1.46 (s, 9H). LC-MS: 287.2 (M+Na)+.

[0814] Acetic anhydride (95.6 mL, 350 mmol) was added at 0 °C to a solution of tert-butyl 3-(3-hydroxy-2,2-bis(hydroxymethyl)propoxy)propanoate (3, 74.5 g, 281 mmol) and N,N-diisopropylethylamine (88.1 mL, 506 mmol) in dry dichloromethane (600 mL). The cooling bath was removed and the resulting solution was stirred at room temperature overnight. The volatiles were removed in vacuo; the residue was redissolved in ethyl acetate (2 L) and washed with water (600 mL), 0.5 M aqueous hydrochloric acid (1.2 L), water (600 mL), 10% aqueous potassium bicarbonate (600 mL), water (600 mL) and brine (230 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluents: cyclohexane / ethyl acetate 9:1-8:2) to give 2-(acetoxymethyl)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl diacetate (4) as a colourless oil.

[0815] Yield: 86.7g (79%). RF (SiO2, hexane / ethyl acetate 3:2): 0.40. 1 HNMR spectrum (300MHz, CDCl3, δH): 4.11 (s, 6H); 3.65 (t, J = 6.2 Hz, 2H); 3.44 (s, 2H); 2.45 (t, J = 6.3 Hz, 2H); 2.06 (s, 9H); 1.46 (s, 9H). LC-MS: 413.2(M+Na)+.

[0816] Trifluoroacetic acid (300 mL) was added to the above solution of 2-(acetoxymethyl)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-dimethyldiacetate (4, 86.0 g, 220 mmol) in dichloromethane (100 mL). The resulting solution was stirred at room temperature for 2 hours, then evaporated to dryness, and the residue was evaporated from toluene (3 x 150 mL). The residue was purified by rapid column chromatography (silica gel 60, 0.040–0.063 mm; eluent: dichloromethane / methanol 10:0–9:1), and the fraction containing the product was evaporated to give the title compound (5) as a light brown oil.

[0817] Yield: 70.4g (96%). RF (SiO2, hexane / ethyl acetate 1:1): 0.25. 1 HNMR spectrum (300MHz, CDCl3, δH): 4.10 (s, 6H); 3.69 (t, J = 6.1 Hz, 2H); 3.46 (s, 2H); 2.60 (t, J = 6.1 Hz, 2H); 2.06 (s, 9H). LC-MS: 357.2(M+Na)+.

[0818] Let 2-chlorotrityl resin 100-200 mesh 1.5 mmol / g (10.7 g, 16.0 mmol) swell in anhydrous dichloromethane (100 mL) for 20 min. Add a solution of {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 4.12 g, 10.7 mmol) and N,N-diisopropylethylamine (7.07 mL, 40.6 mmol) in anhydrous dichloromethane (20 mL) to the resin and shake the mixture for 16 h. Filter the resin and treat with a solution of N,N-diisopropylethylamine (3.72 mL, 21.4 mmol) in a methanol / dichloromethane mixture (2:8, 2 x 5 min, 2 x 50 mL). Then wash the resin with N,N-dimethylformamide (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). Remove the Fmoc group by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 50 mL). Wash the resin with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL) and dichloromethane (2 x 50 mL). Add a solution of {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-OEG-OH, 6.17 g, 16.0 mmol), 5-chloro-1-((dimethylamino)(dimethylimino)methyl)-1H- benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 5.70 g, 16.0 mmol) and N,N- diisopropylethylamine (5.02 mL, 28.8 mmol) in N,N-dimethylformamide (50 mL) to the resin and shake the mixture for 1 h. Then wash the resin with N,N-dimethylformamide (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). Remove the Fmoc group by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 50 mL). Wash the resin with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL), dichloromethane (2 x 50 mL).A solution of {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]- ethoxy}-acetic acid (Fmoc-OEG-OH, 6.17 g, 16.0 mmol), 5-chloro-l-((dimethylamino)(dimethylimino)methyl)-lH-benzo[d][l,2,3]triazole 3-oxide tetrafluoroborate (TCTU, 5.70 g, 16.0 mmol) and N,N-diisopropylethylamine (5.02 mL, 28.8 mmol) in N,N-dimethylformamide (50 mL) was added to the resin and the mixture was shaken for 1 hour. The resin was then washed with N,N-dimethylformamide (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 50 mL). The resin was washed with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL), dichloromethane (2 x 50 mL). A solution of 3,5-bis(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)benzoic acid (10.0 g, 16.0 mmol), 5-chloro-l-((dimethylamino)(dimethylimino)methyl)-lH-benzo[d][l,2,3]triazole 3-oxide tetrafluoroborate (TCTU, 5.70 g, 16.0 mmol) and N,N-diisopropylethylamine (5.02 mL, 28.8 mmol) in N,N-dimethylformamide (50 mL) was added to the resin and the mixture was shaken for 1 hour. The resin was then washed with N,N-dimethylformamide (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 50 mL). The resin was washed with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL), dichloromethane (2 x 50 mL). A solution of 3-(3-acetyloxy-2,2-bis(acetyloxymethyl)propoxy)propanoic acid (5, 10.7 g, 32.0 mmol), cyano-acetaldehyde acid ethyl ester-2-oxime (OXYMA, 4.55 g, 32.0 mmol), 2,4,6-collidine (7.68 mL, 6.99 mmol) and N,N-diisopropylcarbodiimide (DIC, 4.96 g, 32.0 mmol) in N,N-dimethylformamide (40 mL) was added to the resin and the mixture was shaken for 1 hour. The resin was filtered and washed with N,N-dimethylformamide (3 x 50 mL), dichloromethane (4 x 50 mL), methanol (4 x 50 mL) and dichloromethane (7 x 50 mL).The product was cleaved from the resin by treatment with a mixture of trifluoroacetic acid / dichloromethane (1 :1, 50 mL) overnight. The resin was filtered off and washed with dichloromethane (2 x 50 mL). The solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel 60, 0.040-0.063 mm; eluent: dichloromethane / methanol 100:0 to 90:10) to give compound (8) contaminated with the methyl ester and partial deacetylation. Compound (8) was dissolved in dioxane and a solution of lithium hydroxide (3.42 g, 81.5 mmol) in water (160 mL) was added. The mixture was stirred for 30 minutes, then neutralized with 1 M hydrochloric acid (80 mL) and freeze-dried. Deacetylated 8 was dissolved in a mixture of dichloromethane (50 mL) and N,N-dimethylformamide (10 mL), then pyridine (50 mL) and acetic anhydride (30.5 mL) were added. The mixture was stirred for 72 hours, then evaporated from N,N-dimethylformamide several times to give the desired compound 8 as a brown oil.

[0819] Yield: 13.2 g (99%). LC-MS: 1249 (M+H)+.

[0820] Compound (8) (15.6 g, 12.5 mmol), 2,4,6-collidine (14.9 mL, 113 mmol), [1,2,3]triazolo[4,5-b]pyridin-1-ol (HOAt, 5.10 g, 37.6 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCI, 7.89 g, 41.3 mmol) were dissolved in dichloromethane (170 mL) and N,N-dimethylformamide (20 mL). 4-Formyl-benzyl- ammonium chloride (7.08 g, 41.3 mmol) was added. The mixture was stirred at room temperature for 48 hours and evaporated in vacuo. The residue was purified by HPLC (Deltapak, C18, 5 m, 50 x 500 mm, acetonitrile / water, 15:85 to 25:75 in 30 minutes, 25:75 to 50:50 + 0.05% TFA in 170 minutes) to give the title compound 10 as a brown oil. Yield: 1.96 g (12%). 1H NMR spectrum (300 MHz, CDC13, δH): 9.98 (s, 1H); 7.84 (d, J = 8.1 Hz, 2H); 7.56-7.41 (m, 3H); 7.39-7.33 (m, 1H); 7.25-7.14 (m, 2H); 7.09-7.00 (m, 1H); 4.56 (d, J = 6.2 Hz, 2H); 4.46-4.40 (m, 4H); 4.09-3.96 (m, 16H); 3.91 (s, 2H); 3.73-3.56 (m, 20H); 3.52 (t, J = 5.1 Hz, 4H); 3.45-3.32 (m, 8H); 2.49 (t, J = 5.8 Hz, 4H); 2.05 (s, 18H). LC-MS: 1366 (M+H)+.

[0821] ​

[0822]

[0823] The compound of Example 6 was prepared analogously to the compound of Example 5 from Boc-Lys(Boc).

[0824] ​ ​ ​

[0825]

[0826] The 3,5-dimethylbenzoic acid (1, 45.1 g, 18.4 mmol) was suspended in methanol (130 mL) and treated with concentrated sulfuric acid (13 mL). The mixture was refluxed for 2 days. After neutralization with sodium carbonate (80 g), the mixture was dissolved in water (250 mL) and extracted with diethyl ether (2 x 300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give methyl 3,5-dimethylbenzoate (2) as a light yellow oil. Yield: 46.8 g (95%). 1 H NMR spectrum (300 MHz, CDC13, δH): 9.98 (s, 1H); 7.84 (d, J = 8.1 Hz, 2H); 7.56-7.41 (m, 3H); 7.39-7.33 (m, 1H); 7.25-7.14 (m, 2H); 7.09-7.00 (m, 1H); 4.56 (d, J = 6.2 Hz, 2H); 4.46-4.40 (m, 4H); 4.09-3.96 (m, 16H); 3.91 (s, 2H); 3.73-3.56 (m, 20H); 3.52 (t, J = 5.1 Hz, 4H); 3.45-3.32 (m, 8H); 2.49 (t, J = 5.8 Hz, 4H); 2.05 (s, 18H). LC-MS: 1366 (M+H)+.

[0827] A mixture of the above methyl 3,5-dimethylbenzoate (2, 46.7 g, 284 mmol), N- bromosuccinimide (NBS, 177 g, 994 mmol) and one spatula of azobisisobutyronitrile in methyl formate (550 mL) was irradiated with visible light while heating to reflux for 20 h. The solvent was evaporated and the residue was dissolved in dichloromethane (300 mL). The precipitated succinimide was filtered off and the filtrate was washed with saturated aqueous sodium sulfite solution (2 x 250 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluent: hexane / ethyl acetate 15:1). The product was crystallized from an ethyl acetate / cyclohexane mixture (1:5, 360 mL) to give methyl 3,5-bis(bromomethyl)benzoate (3) as a white solid. Yield: 46.5 g (51%). RF(SiO2, hexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDC13, δH): 8.03-7.97 (m, 2H); 7.62 (s, 1H); 4.50 (s, 4H); 3.94 (s, 3H).

[0828] A suspension of the above bromide (3, 35.2 g, 109 mmol) and sodium formamide (29.1 g, 306 mmol) in dry acetonitrile (200 mL) was refluxed for 4 h. After removal of the white solid by filtration, the solvent was evaporated. Crystallization from an ethyl acetate / cyclohexane mixture gave methyl 3,5-bis((N-formylformamidyl)methyl)benzoate (4) as a white powder.

[0829] Yield: 32.7 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.08 (s, 4H); 7.72 (s, 2H); 7.44 (s, 1H); 4.70 (s, 4H); 3.84 (s, 3H).

[0830] The benzoate (4, 32.7 g, 107 mmol) was dissolved in a mixture of 1,4-dioxane (340 mL) and concentrated hydrochloric acid (430 mL) and heated to reflux for 2 h. After cooling to room temperature, air was bubbled through the solution. The product started to precipitate. After 1 h, the solvent was evaporated and the product was recrystallized from a methanol / ethyl ether mixture (300 mL) to give 3,5-bis(aminomethyl)benzoic acid dihydrochloride (5) as a white powder. Yield: 22.2 g (82%). 1 H NMR spectrum (300 MHz, D2O, δH): 8.08 (s, 2H); 7.72 (s, 1H); 4.26 (s, 4H).

[0831] The dihydrochloride salt (5, 6.33 g, 25.0 mmol) was dissolved in water (110 mL). Subsequently, N,N-diisopropylethylamine (17.4 mL, 100 mmol), N,N-dimethylformamide (110 mL) and 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (6, 18.2 g, 50.0 mmol) were added. The mixture was stirred at room temperature overnight; then neutralized with 1 M aqueous hydrochloric acid solution. The solvent was co-evaporated with toluene 3 times. The residue was dissolved in a dichloromethane / toluene mixture (1 :1, 100 mL) and treated with pinacol (0.60 g, 5.00 mmol). The mixture was evaporated from toluene 3 times. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (50 mL) and the product started to precipitate. Cyclohexane (170 mL) was added. The precipitate was collected by filtration, washed with cyclohexane and diethyl ether and dried under vacuum to give 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoic acid (7) as a white powder. Yield: 14.5 g (86%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.96 (bs, 1 H); 9.33-9.23 (m, 2H); 7.83-7.67 (m, 6H); 7.64-7.57 (m, 2H); 7.54 (s, 1 H); 4.55-4.46 (m, 4H); 1.31 (s, 24H). LC-MS: 512.0 (M+H-2xpinacol)+.

[0832] The above acid (7, 14.4 g, 21.3 mmol) was dissolved in a mixture of acetonitrile / N,N-dimethylformamide (4:1, 100 mL). N-hydroxysuccinimide (HOSu, 2.95 g, 25.6 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 5.28 g, 25.6 mmol) were added subsequently. The mixture was stirred at room temperature overnight. Insoluble by-products were filtered off and the filtrate was evaporated. The residue was dissolved in ethyl acetate (250 mL) and washed with water (2 x 150 mL) and brine (1 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in acetonitrile (100 mL). Residual N,N-dicyclohexylurea was filtered off and the filtrate was evaporated. The residue was dissolved in tetrahydrofuran (150 mL) and treated with pinacol (0.60 g, 5.00 mmol) and molecular sieves overnight. The mixture was filtered through a celite pad and the filtrate was evaporated. The residue was dissolved in dichloromethane (40 mL). The product was precipitated by addition of cyclohexane (150 mL). The precipitate was filtered off, washed with cyclohexane and diethyl ether and dried under vacuum to give 2,5-dioxopyrrolidin-1-yl 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoate (8) as a white powder. Yield: 13.3 g (75%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.34-9.21 (m, 2H); 7.94 (s, 2H); 7.79-7.66 (m, 5H); 7.65-7.56 (m, 2H); 4.62-4.50 (m, 4H); 2.88 (s, 4H); 1.31 (s, 24H). LC-MS: 773.4 (M+H)+, 691.2 (M+H-pinacol)+, 609.1 (M+H-2xpinacol)+.

[0833] Fluorenylmethoxycarbonyl chloride (Fmoc-Cl, 4.00 g, 14.7 mmol) was added to a solution of 2-aminobenzoic acid (2.00 g, 13.7 mmol) in N,N-dimethylformamide (100 mL) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered and the filtrate was evaporated. The residue was dissolved in dichloromethane (100 mL) and washed with water (2 x 100 mL). The organic phase was dried over sodium sulfate and the solvent was evaporated. The residue was purified by flash column chromatography (silica gel 60, 0.040-063 mm; eluent: dichloromethane / methanol 95:5) to give 2-(9H-fluoren-9-ylmethoxycarbonyl)amino)benzoic acid (8) as a white solid. Yield: 4.50 g (96%). RF(SiO2, dichloromethane / methanol 95:5): 0.40.

[0834] 1H NMR spectrum (300 MHz, AcOD-d4, δΗ): 4.27-3.99 (m, 1H); 3.65-3.44 (m, 2H); 3.17-3.00 (m, 2H); 2.70-2.56 (m, 2H); 1.86-1.58 (m, 2H); 1.57-1.26 (m, 22H). LC-MS: 417.5 (M+H)+.

[0835] The above compound (10, 4.30 g, 10.3 mmol) was dissolved in trifluoroacetic acid (50 mL) and left for 1.5 hours. The solvent was evaporated. Ethyl ether (100 mL) was added and the mixture was stirred overnight. The solvent was decanted and the residue was dried under vacuum to give (S)-6-((2-carboxyethyl)amino)-6-oxohexane-1,5-diamium 2,2,2- trifluoroacetate (11) as a tough oil. Yield: 4.50 g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 8.58 (t, J = 5.4 Hz, 1H); 8.18 (bs, 2H); 7.87 (bs, 2H); 3.77-3.62 (m, 1H); 3.34-3.18 (m, 2H); 2.83-2.65 (m, 2H); 1.74-1.60 (m, 2H); 1.60-1.44 (m, 2H); 1.37-1.19 (m, 2H). LC-MS: 217.2 (M+H)+.

[0836] The above salt (11, 2.70 g, 6.06 mmol) was dissolved in N,N-dimethylformamide (100 mL). Subsequently, N,N-diisopropylethylamine (5.30 mL, 30.3 mmol), water (50 mL) and the activated ester (8, 9.36 g, 12.1 mmol) were added. The mixture was stirred at room temperature overnight; then neutralized with 1 M aqueous hydrochloric acid solution. The solvent was co-evaporated with toluene 3 times. The residue was dissolved in a dichloromethane / toluene mixture (1 : 1, 100 mL) and treated with pinacol (0.50 g, 4.23 mmol). The mixture was evaporated from toluene 3 times. The residue was dissolved in ethyl acetate (250 mL) and washed with water (1 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. Partial cleavage of the pinacol ester was observed by NMR analysis. The material was treated with pinacol (0.04 g, 0.34 mmol) and magnesium sulfate (20.0 g) in tetrahydrofuran (110 mL) overnight. The mixture was filtered and the filtrate was evaporated. The product was crystallized from a dichloromethane / cyclohexane mixture (1 :5, 180 mL) to give 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzamide (12) as a light brown powder. Yield: 5.86 g (63%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.31-9.13 (m, 4H); 8.53-8.43 (m, 1H); 8.43-8.35 (m, 1H); 8.11-7.98 (m, 1H); 7.78-7.55 (m, 16H); 7.48-7.38 (m, 2H); 4.55-4.43 (m, 8H); 4.43-4.33 (m, 1H); 3.31-3.13 (m, 4H); 2.38 (t, J = 6.4 Hz, 2H); 1.79-1.64 (m, 2H); 1.57-1.44 (m, 2H); 1.42-1.21 (m, 50H).

[0837] The carboxylic acid (12, 5.46 g, 3.57 mmol) was dissolved in acetonitrile (50 mL). N-hydroxysuccinimide (HOSu, 0.70 g, 6.07 mmol) and N,N-dicyclohexylcarbodiimide (1.47 g, 7.14 mmol) were added. The resulting mixture was stirred at room temperature overnight. The byproduct was removed by filtration. The filtrate was evaporated. The residue was dissolved in ethyl acetate (150 mL) and washed with water (1 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (60 mL) and treated with pinacol (0.06 g, 0.50 mmol) and molecular sieves overnight. The mixture was filtered and the filtrate was evaporated. The residue was dissolved in ethyl acetate (10 mL) and precipitated after addition of diethyl ether (90 ml). The product was collected by filtration, washed with diethyl ether and dried under vacuum to give the title compound (13) as a light brown powder. The product contained a trace amount of N,N-dicyclohexylurea. Yield: 1.55 g (27%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.28-9.17 (m, 3H); 8.52-8.33 (m, 2H); 8.25-8.15 (m, 1H); 7.80-7.51 (m, 16H); 7.48-7.35 (m, 2H); 4.58-4.32 (m, 9H); 3.49-3.35 (m, 2H); 3.25-3.09 (m, 2H); 2.91-2.72 (m, 6H); 1.81-1.65 (m, 2H); 1.57-1.42 (m, 2H); 1.41-1.12 (m, 50H). LC-MS: 1631.9 (M+H)+, 1549.0 (M-pinacol+H)+, 715.0 (M-2xH2O-2xpinacol / 2+H)+, 1384.5 (M-3xpinacol+H)+, 1302.3 (M-4xpinacol+H)+.

[0838] ​ ​ ​ ​

[0839]

[0840] A mixture of 2-fluoro-4-carboxyphenylboronic acid (1, 15.1 g, 82.0 mmol), pinacol (9.81 g, 83.0 mmol) and magnesium sulfate (150 g) in tetrahydrofuran (400 mL) was stirred at room temperature for one weekend. The suspension was filtered through a celite pad and the filtrate was evaporated and dried under vacuum to give 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (2) as a light yellow powder. Yield: 21.5 g (98%). 1 H NMR spectrum (400 MHz, DMSO-d6, δΗ): 7.95-7.42 (m, 3H); 1.30 (s, 12H).

[0841] The carboxylic acid (2, 21.4 g, 81.9 mmol) was dissolved in dichloromethane (300 mL). N-hydroxysuccinimide (HOSu, 18.8 g, 163 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCI, 31.3 g, 163 mmol) were added. The resulting mixture was stirred at room temperature overnight. The reaction hydrate was washed with 0.5 M aqueous hydrochloric acid (1 x 200 mL), water (1 x 200 mL) and brine (1 x 200 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (60 mL) and precipitated by addition of cyclohexane (250 mL). The product was collected by filtration, washed with cyclohexane and dried under vacuum to give 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (3) as a beige powder. Yield: 27.8 g (93%). 1 H NMR spectrum (400 MHz, DMSO-d6, δΗ): 7.98-7.87 (m, 2H); 7.80 (dd, J = 9.2 Hz, 1H); 2.90 (s, 4H); 1.33 (s, 12H).

[0842] Fluorenylmethoxycarbonyl chloride (Fmoc-Cl, 6.0 g, 22.2 mmol) was added to a solution of 2-aminobenzoic acid (3.0 g, 19.7 mmol) in N,N-dimethylformamide (100 mL) and the mixture was stirred for 1 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed with water (2 x 200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 60-120 mesh, 10% ethyl acetate in hexanes) to give 2-(fluorenylmethoxycarbonylamino)benzoic acid (Fmoc-2-aminobenzoate, 6.0 g, 18.2 mmol, 94% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 7.9 Hz, 1H), 7.74 - 7.68 (m, 2H), 7.63 - 7.57 (m, 2H), 7.48 - 7.42 (m, 2H), 7.41 - 7.36 (m, 2H), 7.34 - 7.28 (m, 2H), 7.27 - 7.22 (m, 1H), 7.22 - 7.17 (m, 1H), 6.62 (s, 1H), 5.27 (s, 2H), 3.78 (s, 3H). 2-(Fluorenylmethoxycarbonylamino)benzoic acid (Fmoc-2-aminobenzoate, 6.0 g, 18.2 mmol) was dissolved in dichloromethane (200 mL) and treated with 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-bAla-OH, 6.13 g, 19.7 mmol) and N,N-diisopropyl ethylamine (13.0 mL, 74.8 mmol). The mixture was stirred for 2 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane (200 mL) and washed with water (2 x 200 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 60-120 mesh, 10% ethyl acetate in hexanes) to give 2-(fluorenylmethoxycarbonylamino)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-bAla-Fmoc-2-aminobenzoate, 9.0 g, 16.2 mmol, 87% yield) as a white solid.1H NMR (400 MHz, Chloroform-d) δ 7.82 (d, J = 7.9 Hz, 1H), 7.74 - 7.68 (m, 2H), 7.63 - 7.57 (m, 2H), 7.48 - 7.42 (m, 2H), 7.41 - 7.36 (m, 2H), 7.36 - 7.30 (m, 2H), 7.30 - 7.25 (m, 1H), 7.25 - 7.20 (m, 1H), 6.62 (s, 1H), 5.27 (s, 2H), 5.26 (s, 2H), 3.78 (s, 3H), 3.78 - 3.73 (m, 2H), 1.94 - 1.86 (m, 1H), 1.82 - 1.74 (m, 1H), 1.74 - 1.66 (m, 1H), 1.64 - 1.56 (m, 1H).A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-bAla-OH, 24.5 g, 78.7 mmol), 5-chloro-l-((dimethylamino)(dimethylimino)methyl)-lH- benzo[d][l,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 28.0 g, 78.7 mmol), and N,N- diisopropylethylamine (24.7 mL, 142 mmol) in N,N-dimethylformamide (230 mL) was added to the resin and the mixture was shaken for 3 hours. The resin was filtered and washed with N,N-dimethylformamide (2 x 200 mL), dichloromethane (2 x 200 mL), and N,N- dimethylformamide (2 x 200 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 15 min, 2 x 200 mL). The resin was washed with N,N-dimethylformamide (2 x 200 mL), 2-propanol (2 x 200 mL), dichloromethane (2 x 200 mL), and N,N-dimethylformamide (2 x 200 mL). A solution of N2,N6-bis(tert-butoxycarbonyl)-L-lysine (Boc-Lys(Boc)-OH, 27.3 g, 78.7 mmol), 5-chloro-l-((dimethylamino)(dimethylimino)methyl)-lH-benzo[d][l,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 28.0 g, 78.7 mmol), and N,N-diisopropylethylamine (24.7 mL, 142 mmol) in N,N-dimethylformamide (230 mL) was added to the resin and the mixture was shaken for 3 hours. The resin was filtered and washed with N,N-dimethylformamide (2 x 200 mL), dichloromethane (2 x 200 mL), and N,N-dimethylformamide (2 x 200 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 15 min, 2 x 200 mL). The resin was washed with N,N-dimethylformamide (2 x 200 mL), 2-propanol (2 x 200 mL), dichloromethane (2 x 200 mL), and N,N-dimethylformamide (2 x 200 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (350 mL) overnight. The resin was filtered off and washed with dichloromethane (2 x 300 mL).The combined solution was evaporated and the residue was purified by flash column chromatography (silica gel 60, 0.040-063 mm; eluent: dichloromethane / methanol 85:15) to give (10S,21S)-21-(3-((S)-2,6-bis((tert-butoxycarbonyl)amino)hexanoylamido)propanoylamido)-10-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,15,22-tetraoxo-3-oxa-5,12,16,23-tetraazahexacosan-26-oic acid (5) as a white solid. Yield: 11.3 g (56%). 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 4.52-4.43 (m, 1H); 4.22-3.98 (m, 2H); 3.64-3.44 (m, 6H); 3.27-3.16 (m, 2H); 3.15-3.03 (m, 4H); 2.69-2.48 (m, 6H); 1.84-1.59 (m, 6H); 1.58-1.28 (m, 48H). LC-MS: 1016.2 (M+H)+.

[0843] The above compound (5, 11.3 g, 11.1 mmol) was dissolved in trifluoroacetic acid (200 mL) and left for 1.5 hours. The mixture was then concentrated and diethyl ether (200 mL) was added. After stirring overnight, the precipitate was filtered, washed with diethyl ether and dried under vacuum to give (5S,12S,23S)-12-((2-carboxyethyl)carbamoyl)-6,10,18,22-tetraoxo-7,11,17,21-tetraazahexacosan-1,5,23,27-tetraammonium 2,2,2-trifluoroacetate (6) as a white powder. Yield: 9.25 g (99%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 8.56-8.44 (m, 2H); 8.27-7.72 (m, 11H); 4.22-4.08 (m, 1H); 3.78-3.60 (m, 2H); 3.39-3.17 (m, 6H); 3.07-2.92 (m, 2H); 2.82-2.66 (m, 4H); 2.42-2.19 (m, 6H); 1.77-1.43 (m, 10H); 1.42-1.14 (m, 8H).

[0844] The above salt (6, 7.91 g, 9.37 mmol) was dissolved in N,N-dimethylformamide (170 mL). Subsequently, N,N-diisopropylethylamine (14.7 mL, 84.3 mmol), water (0.50 mL) and activated ester (3, 13.6 g, 37.5 mmol) were added. The mixture was stirred at room temperature overnight; then acidified with 1 M aqueous hydrochloric acid solution. The solvent was co-evaporated with toluene 3 times. The residue was dissolved in a dichloromethane / toluene mixture (1 : 1, 100 mL) and treated with pinacol (1.00 g, 8.46 mmol). The mixture was evaporated from toluene 3 times. The residue was dissolved in ethyl acetate (150 mL) and washed with water (1 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to 1 / 3 volume. Cyclohexane (150 mL) was added; the precipitate was filtered and washed with cyclohexane. The solid was suspended in acetonitrile / diethyl ether mixture (1 : 1, 150 mL). The precipitate was filtered, washed with acetonitrile and dried under vacuum to yield the title compound (7) as a white solid. Yield: 4.10 g (27%).

[0845] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 8.67-8.43 (m, 4H); 8.05-7.83 (m, 4H); 7.82-7.47 (m, 13H); 4.46-4.27 (m, 2H); 4.20-4.05 (m, 1H); 3.42-3.13 (m, 10H); 3.05-2.90 (m, 2H); 2.42-2.27 (m, 4H); 2.27-2.17 (m, 2H); 1.84-1.66 (m, 4H); 1.63-1.10 (m, 62H). LC-MS: 1226.4 (M-3x H20-4x pinacol + H)+.

[0846] ​ ​ Alkyl-2-yl)benzoyl)glycinate

[0847]

[0848] (2,8.85 g, 33.3 mmol) was dissolved in dichloromethane (100 mL) followed by the addition of l-((dimethylamino)(dimethylimino)methyl)-lH-[l,2,3]triazolo[4,5- b]pyridine 3-oxide hexafluorophosphate (HATU, 12.3 g, 32.4 mmol), N,N- diisopropylethylamine (14.5 mL, 83.2 mmol) and (2-aminoethyl)glycine tert-butyl ester hydrochloride (1, 4.11 g, 16.6 mmol). The reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was extracted with 1 M aqueous hydrochloric acid solution (2 x 100 mL), water (1 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was dissolved in anhydrous tetrahydrofuran (50 mL) and 2,3-dimethyl-2,3-butanediol (3.70 g, 31.5 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then evaporated and the crude product was purified by flash chromatography (silica gel 60, 0.063-0.200 mm; eluent: dichloromethane / ethyl acetate 5:2) to give N-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzamido)ethyl)-N-(3-fluoro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoyl)glycine tert-butyl ester (3) as a white foam. Yield: 8.13 g (73%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 8.78-8.55 (m, 1H); 7.79-7.44 (m, 4H); 7.12-6.88 (m, 2H); 4.18-3.90 (m, 2H); 3.67-3.47 (m, 2H); 3.45-3.29 (m, 2H); 1.44 (s, 9H); 1.30 (s, 24H).

[0849] The compound prepared above (3, 8.13 g, 12.1 mmol) was dissolved in trifluoroacetic acid (100 mL) and left for 2.5 hours. The solvent was then evaporated and co-evaporated twice with toluene. The residue was dissolved in dichloromethane (30 mL) and cyclohexane (250 mL) was added. The product was collected by filtration, washed with cyclohexane and dried under vacuum to give N-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzamido)ethyl)-N-(3-fluoro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoyl)glycine (4) as a white powder. Yield: 6.91 g (93%). 1 HNMR spectrum (300 MHz, DMSO-d6, 80C, δH): 8.49-8.38 (m, 1H); 7.76-7.68 (m, 1H); 7.67-7.59 (m, 2H); 7.57-7.45 (m, 1H); 7.16-7.09 (m, 1H); 7.04-6.94 (m, 1H); 4.20-4.03 (m, 2H); 3.59-3.40 (m, 4H); 1.33 (s, 24H). LC-MS: 449.9 (M-2x pinacol + H)+, 532.1 (M-pinacol + H)+, 614.2 (M + H)+.

[0850] The acid (4, 6.90 g, 11.2 mmol) was dissolved in a dichloromethane / tetrahydrofuran mixture (1 : 1, 100 mL) followed by the addition of N-hydroxysuccinimide (1.36 g, 11.8 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (2.26 g, 11.8 mmol). The mixture was stirred at room temperature overnight. The solvent was evaporated. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The product was precipitated from a dichloromethane / cyclohexane mixture (25 mL / 250 mL). The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give the title compound (5) as a white powder. Yield: 7.62 g (96%). 1H NMR spectrum (300 MHz, DMSO-d6, 80C, δΗ): 8.51-8.38 (m, 1H); 7.77-7.57 (m, 3H); 7.55-7.45 (m, 1H); 7.18-7.10 (m, 1H); 7.06-6.97 (m, 1H); 4.62 (bs, 2H); 3.67-3.41 (m, 4H); 2.84 (s, 4H); 1.33 (s, 24H). LC-MS: 547.0 (M-2x pinacol + H)+, 629.1 (M-pinacol + H)+, 711.3 (M + H)+.

[0851] Example 10: N-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5- carbonyl)-N- (2-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5- carbonylamino)ethyl)glycinate (2-(6-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-carbonylamino)ethyl)glycinate

[0852]

[0853] Dissolve 6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-carboxylic acid (1, 10.0 g, 51.0 mmol) in tetrahydrofuran (100 mL). At room temperature, add N,N- dimethylformamide (15 mL), N-hydroxysuccinimide (6.46 g, 56.1 mmol) and N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (10.8 g, 56.1 mmol). After stirring for 2 hours, evaporate the volatiles under reduced pressure and re-dissolve the residue in ethyl acetate (400 mL) and wash with 1 M aqueous hydrochloric acid (2 x 100 mL). Dry the organic portion over anhydrous sodium sulfate. Evaporate the volatiles under reduced pressure to give 2,5-dioxopyrrolidin-1-yl 6-fluoro-1-hydroxy-1,3- dihydrobenzo[c][1,2]oxaborol-5-carboxylate (2) as a white solid. Yield: 13.8 g (92%).

[0854] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.65 (s, 1H); 8.11 (d, J = 5.9 Hz, 1H); 7.71 (d, J = 10.1 Hz, 1H); 5.08 (s, 2H); 2.90 (s, 4H). LC-MS: 294.4 (M + H)+.

[0855] (2-Aminoethyl)glycine (3, 1.81 g, 15.4 mmol) was dissolved in N,N- dimethylformamide (40 mL) and triethylamine (12.8 mL, 92.1 mmol) and 2,5- dioxopyrrolidin-1-yl 6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5- carboxylate (2, 9.00 g, 30.7 mmol) were added at room temperature. After stirring at room temperature for 16 h, the reaction mixture was heated to 40 °C and stirred for a further 72 h. The volatiles were evaporated under reduced pressure and the residue was re-dissolved in ethyl acetate (400 mL) and washed with 1 M aqueous hydrochloric acid (100 mL). The organic fraction was dried over anhydrous sodium sulfate. The volatiles were evaporated under reduced pressure, the product was precipitated from acetonitrile / water mixture, collected by centrifugation and freeze-dried to give N-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5- carbonyl)-N-(2-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5- carboxamido)ethyl)glycine 4 as an off-white solid.

[0856] Yield: 1.99 g (27%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.87 (bs, 1H); 9.50-9.37 (m, 2H); 8.52-8.22 (m, 1H); 7.69-7.30 (m, 4H); 5.08-4.70 (m, 4H); 4.27-3.96 (m, 2H); 3.74-3.35 (m, 4H). LC-MS: 475.5 (M+H)+.

[0857] Example 11: 2,5-Dioxopyrrolidin-1-yl (S)-3-(2,6-bis(3,5-bis((4-(4,4,5,5-tetra methyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzamido)hexanamido)propanoate

[0858]

[0859] 2-Chlorotriphenylmethyl resin (100-200 mesh, 1.5 mmol / g, 1, 21.0 g, 31.5 mmol) was swollen in anhydrous dichloromethane (300 mL) for 20 min. A solution of 3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-bAla-OH, 6.54 g, 21.0 mmol) and N,N-diisopropylethylamine (13.9 mL, 79.8 mmol) in anhydrous dichloromethane (250 mL) was added to the resin, and the mixture was shaken for one weekend. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (7.32 mL, 42.0 mmol) in a methanol / dichloromethane mixture (1:4, 1 x 15 min, 250 mL). The resin was then washed with dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). Fmoc groups were removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 10 min, 1 x 20 min, 2 x 250 mL). The resin was then washed with N,N-dimethylformamide (2 x 250 mL), 2-propanol (2 x 250 mL), dichloromethane (2 x 250 mL), and N,N-dimethylformamide (2 x 250 mL). A solution of N2,N6-bis(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH, 18.6 g, 31.5 mmol), 5-chloro-1-((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide tetrafluoroborate (TCTU, 11.2 g, 31.5 mmol), and N,N-diisopropylethylamine (9.87 mL, 56.7 mmol) in N,N-dimethylformamide (250 mL) was added to the resin, and the mixture was shaken overnight. The resin was filtered and washed with N,N-dimethylformamide (2 x 250 mL) and dichloromethane (3 x 250 mL).

[0860] Part of the resin was removed (2.00 mmol). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 30 mL). The resin was washed with N,N-dimethylformamide (4 x 30 mL), dichloromethane (4 x 30 mL) and N,N-dimethylformamide (4 x 30 mL). 2,5-dioxopyrrolidin-1-yl 3,5-bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoate (2, 3.65 g, 4.72 mmol) and N,N-diisopropylethylamine (1.40 mL, 8.00 mmol) in N,N-dimethylformamide (30 mL) were added to the resin and the mixture was shaken overnight. The resin was filtered and washed with N,N-dimethylformamide (4 x 30 mL), dichloromethane (4 x 30 mL), N,N-dimethylformamide (4 x 30 mL) and dichloromethane (10 x 30 mL).

[0861] The product was cleaved from the resin by treatment with a 1,1,1,3,3,3-hexafluoro-2-propanol / dichloromethane mixture (1:2, 30 mL) for 2 hours. The resin was filtered off and washed with dichloromethane (3 x 30 mL). The solutions were combined and the solvent was evaporated. The residue was dissolved in dichloromethane (5 mL) and precipitated after addition of cyclohexane (25 mL). The product was collected by filtration, washed with cyclohexane and dried under vacuum to give (S)-3-(2,6-bis(3,5-bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzamido)hexanamido)propanoic acid (3). Yield: 1.53 g (52%).

[0862] 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.20-9.03 (m, 4H); 8.52-8.42 (m, 1H); 8.39-8.32 (m, 1H); 8.06-7.99 (m, 1H); 7.94-7.80 (m, 10H); 7.78-7.65 (m, 10H); 7.48-7.39 (m, 2H); 4.56-4.43 (m, 8H); 4.43-4.32 (m, 1H); 3.27-3.14 (m, 4H); 2.40-2.29 (m, 2H); 1.78-1.64 (m, 2H); 1.56-1.430 (m, 3H) 1.37-1.21 (s, 49H).

[0863] The carboxylic acid (3, 1.53 g, 1.00 mmol) was dissolved in dichloromethane (40 mL). N-hydroxysuccinimide (HOSu, 148 mg, 1.30 mmol) and N-(3-dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (EDC.HCI, 242 mg, 1.30 mmol) were added. The resulting mixture was stirred at room temperature overnight. The solvent was evaporated. The residue was dissolved in ethyl acetate (100 mL) and washed with water (2 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (10 mL) and precipitated after addition of cyclohexane (50 mL). The product was collected by filtration, washed with cyclohexane and diethyl ether and dried under vacuum to give the title compound (4) as a white powder. Yield: 1.16 g (71%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.23-9.01 (m, 4H); 8.50-8.42 (m, 1H); 8.41-8.35 (m, 1H); 8.23-8.16 (m, 1H); 7.91-7.81 (m, 9H); 7.77-7.70 (m, 9H); 7.70-7.64 (m, 2H); 7.47-7.40 (m, 2H); 4.55-4.43 (m, 8H); 4.40-4.34 (m, 1H) 3.50-3.38 (m, 2H); 3.26-3.12 (m, 2H); 2.88-2.77 (m, 6H); 1.82-1.63 (m, 2H); 1.60-1.43 (m, 4H); 1.31 (s, 48H). LC-MS: 1631.9 (M+H)+, 1549.0 (M-pinacol+H)+, 715.0 (M-2x H20-2x pinacol / 2+H)+, 1384.5 (M-3x pinacol+H)+, 1302.3 (M-4x pinacol+H)+.

[0864] Example 12: (S)-3-(2,6-bis(3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxo borolan-2-yl)benzamido)methyl)benzamido)hexanamido)propanoic acid

[0865]

[0866] The 3,5-dimethylbenzoic acid (1, 300 g, 2.00 mol) was suspended in methanol (900 mL) and treated with concentrated sulfuric acid (90 mL). The mixture was stirred for 3 days. After neutralization with sodium carbonate (480 g), the solvent was evaporated. The residue was dissolved in water (1 L) and extracted with diethyl ether (3 x 1 L). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give methyl 3,5-dimethylbenzoate (2) as a light yellow oil. Yield: 309 g (94%). 1H NMR spectrum (300 MHz, CDC13, δH): 7.65 (s, 2H); 7.16 (s, 1H); 3.88 (s, 3H); 2.34 (s, 6H).

[0867] A mixture of the above methyl 3,5-dimethylbenzoate (2, 307 g, 1.87 mol), N- bromosuccinimide (1.17 kg, 6.55 mol) and a spatula of azobisisobutyronitrile in methyl formate (2.7 L) was irradiated with visible light while heating to reflux for 20 h. The solvent was evaporated and the residue was dissolved in dichloromethane (2 L). The precipitated succinimide was filtered off and the filtrate was washed with saturated aqueous sodium sulfite solution (2 x 1 L). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. Methyl 5-bis(bromomethyl)benzoate (3) was obtained as a white solid after several crystallizations from hot ethyl acetate / cyclohexane mixtures and washing with cyclohexane. The product was prepared in two batches. Yield: 243 g (40%). Rf (SiO2, hexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.65 (s, 2H); 7.16 (s, 1H); 3.88 (s, 3H); 2.34 (s, 6H).

[0868] A suspension of the above bromide (3, 122 g, 380 mmol) and sodium formamide (101 g, 1.06 mol) in dry acetonitrile (900 mL) was refluxed for 4 h. After removal of the white solid by filtration, the solvent was co-evaporated with ethyl acetate and dried in vacuo to give methyl 3,5-bis((N-formylcarboxamido)methyl)benzoate (4) as a light yellow solid. Yield: 116 g (100%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.65 (s, 2H); 7.16 (s, 1H); 3.88 (s, 3H); 2.34 (s, 6H).

[0869] The benzoate (4, 116 g, 380 mmol) was dissolved in a mixture of 1,4-dioxane (400 mL) and concentrated hydrochloric acid (600 mL) and heated to reflux for 3 h. After cooling to room temperature, air was bubbled through the solution. The product started to precipitate. After 1 h, the solvent was evaporated and the product was recrystallized from a methanol / diethyl ether mixture to give 3,5-bis(amino- methyl)benzoic acid dihydrochloride (5) as a white powder. Yield: 89.5 g (92%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.65 (s, 2H); 7.16 (s, 1H); 3.88 (s, 3H); 2.34 (s, 6H).

[0870] The dihydrochloride salt (5, 30.0 g, 118 mmol) and sodium hydroxide (14.2 g, 356 mmol) were dissolved in water (240 mL). Di-tert-butyl dicarbonate (77.6 g, 356 mmol) in 1,4-dioxane (480 mL) was added with stirring. The reaction mixture was stirred overnight, then diluted with ethyl acetate (400 mL) and 0.5 M aqueous hydrochloric acid (400 mL). The layers were separated, the organic layer was washed with water (2 x 350 mL), dried over anhydrous sodium sulfate and evaporated. The residue was dissolved in hot ethyl acetate (100 mL) and cyclohexane (400 mL) was added. The precipitate was collected by filtration and washed with cyclohexane to give 3,5-bis(((tert-butoxycarbonyl)amino)methyl)benzoic acid (6) as a white solid. Yield: 39.1 g (87%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 7.70 (s, 2H); 7.45-7.36 (m, 2H); 7.33 (s, 1H); 4.21-4.04 (m, 4H); 1.39 (s, 18H).

[0871] Let 2-chlorotrityl chloride resin 100-200 mesh 1.5 mmol / g (7, 21.2 g, 31.8 mmol) swell in anhydrous dichloromethane (280 mL) for 40 min. Add a solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Ala-OH, 6.61 g, 21.2 mmol) and N,N-diisopropylethylamine (14.1 mL, 80.7 mmol) in anhydrous dichloromethane (220 mL) to the resin and shake the mixture overnight. Filter the resin and treat with a solution of N,N-diisopropylethylamine (7.40 mL, 42.5 mmol) in a methanol / dichloromethane mixture (1:4, 1 x 20 min, 1 x 250 mL). Then wash the resin with dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). Remove the Fmoc group by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 220 mL). Wash the resin with N,N-dimethylformamide (2 x 250 mL), 2-propanol (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). Add a solution of N2,N6-bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH, 18.8 g, 31.8 mmol), 5-chloro-1- ((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 11.3 g, 31.8 mmol) and N,N-diisopropylethylamine (9.98 mL, 57.3 mmol) in N,N-dimethylformamide (220 mL) to the resin and shake the mixture for 2.5 h. Then wash the resin with N,N-dimethylformamide (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL). Remove the Fmoc group by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 220 mL). Wash the resin with N,N-dimethylformamide (2 x 250 mL), 2-propanol (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (2 x 250 mL).A solution of 3,5-bis(((tert-butoxycarbonyl)amino)methyl)benzoic acid (6, 24.2 g, 63.7 mmol), 5-chloro-l-((dimethylamino)(dimethylimino)methyl)-lH- benzo[d][l,2,3]triazole 3-oxide tetrafluoroborate (TCTU, 22.6 g, 63.7 mmol) and N,N- diisopropylethylamine (20.0 mL, 115 mmol) in N,N-dimethylformamide (220 mL) was added to the resin and the mixture was shaken for 2.5 h. The resin was washed with N,N- dimethylformamide (2 x 250 mL) and dichloromethane (10 x 250 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (400 mL) overnight. The resin was filtered off and washed with dichloromethane (2 x 200 mL). The solvent was evaporated and the residue was purified by flash column chromatography (silica gel 60, 0.063-0.200 mm; eluent: dichloromethane / methanol 90:10) to give (S)-3-(2,6-bis(3,5-bis(((tert- butoxycarbonyl)amino)methyl)benzamido)hexanamido)propanoic acid (8) as a white foam. Yield: 16.3 g (82%). RF(SiO2, dichloromethane / methanol 90:10): 0.30.

[0872] 1 H NMR spectrum (300 MHz, CDC13, δH): 7.75-7.35 (m, 6H); 7.26-7.19 (m, 2H); 7.13 (bs, 1H); 5.61-5.35 (m, 4H); 4.76-4.61 (m, 1H); 4.25-4.08 (m, 8H); 3.60-3.26 (m, 4H); 2.60-2.45 (m, 2H); 2.02-1.85 (m, 1H); 1.85-1.69 (m, 1H); 1.62-1.51 (m, 2H); 1.46-1.39 (m, 38H).

[0873] LC-MS: 942.1 (M+H)+.

[0874] The above compound (8, 16.1 g, 17.3 mmol) was dissolved in trifluoroacetic acid (80 mL) and left for 30 min. The solvent was concentrated to 1 / 3 volume and a mixture of diethyl ether / cyclohexane (1:1, 300 mL) was added. The resulting mixture was stirred overnight. The precipitate was collected by filtration, washed with diethyl ether and dried under vacuum to give (S)-((((6-((2-carboxyethyl)amino)-6-oxohexane-l,5-diyl)bis(azanediyl))bis(carbonyl))bis(phen-5, 1,3- triyl))tetramethonium 2,2,2-trifluoroacetate (9) as a white powder. Yield: 16.5 g (96%). 1H NMR spectrum (300 MHz, AcOD-d4, δΗ): 8.09 (dd, J = 9.4 and 1.5 Hz, 4H); 7.84 (d, J = 10.5 Hz, 2H); 4.76 (dd, J = 8.2 and 6.1 Hz, 1H); 4.36 (s, 4H); 4.35 (s, 4H); 3.60-3.43 (m, 4H); 2.64 (t, J = 6.5 Hz, 2H); 2.00-1.80 (m, 2H); 1.77-1.65 (m, 2H);

[0875] 1.57-1.48 (m, 2H). LC-MS: 541.6 (M+H)+.

[0876] A suspension of 4-carboxy-3-fluorophenylboronic acid (10, 30.0 g, 163 mmol) and pinacol (21.2 g, 179 mmol) in a mixture of toluene / ethanol (1 : 1, 480 mL) was refluxed for 24 h. The solvent was then evaporated and co-evaporated 3 times with dichloromethane to give 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (11) as a white powder.

[0877] Yield: 43.3 g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 7.60 (t, J = 7.3 Hz, 1H); 7.39 (d, J = 7.5 Hz, 1H); 7.24 (d, J = 10.6 Hz, 1H); 1.29 (s, 12H).

[0878] The acid (11, 35.2 g, 132 mmol) was dissolved in tetrahydrofuran (1 : 1, 600 mL) followed by the addition of 1-hydroxy-pyrrolidine-2,5-dione (HOSu, 25.2 g, 219 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCI, 42.0 g, 219 mmol). The resulting mixture was stirred at room temperature overnight. The solvent was then evaporated. The residue was dissolved in ethyl acetate (400 mL) and washed with water (2 x 300 mL) and brine (1 x 300 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The product was precipitated from an ethyl acetate / cyclohexane mixture (1 :4, 600 mL). The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give 2,5-dioxopyrrolidin-1-yl 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (12) as a white powder. Yield: 45.2 g (94%). 1HNMR spectrum (300 MHz, DMSO-d6, δΗ): 8.07 (t, J = 7.3 Hz, 1H); 7.71 (d, J = 7.7 Hz, 1H); 7.60 (d, J = 10.8 Hz, 1H); 2.90 (s, 4H); 1.32 (s, 12H).

[0879] (S)-((((6-((2-carboxyethyl)amino)-6-oxohexane-1,5-diyl)bis(azanediyl))bis(carbonyl))bis(phen-5,1,3-triyl))tetramethonium 2,2,2-trifluoroacetate (9, 3.91 g, 3.92 mmol) was dissolved in a water / N,N-dimethylformamide mixture (1 :1, 80 mL). Subsequently, N,N-diisopropylethylamine (6.15 mL, 35.3 mmol) and the activated ester (12, 5.69 g, 15.7 mmol) were added. The mixture was stirred at room temperature overnight; then acidified with 1 M aqueous hydrochloric acid solution. The solvent was co-evaporated with toluene 3 times. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 x 100 mL) and brine (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was treated with pinacol (0.06 g, 0.49 mmol) in tetrahydrofuran (70 mL) and evaporated from tetrahydrofuran three times. The residue was dried in vacuo to give the title compound (13) as a beige solid. Yield: 5.82 g (95%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.01-8.83 (m, 4H); 8.51-8.42 (m, 1H); 8.39-8.30 (m, 1H); 8.10-8.00 (m, 1H); 7.80-7.31 (m, 18H); 4.59-4.33 (m, 9H); 3.30-3.19 (m, 4H); 2.39 (t, J = 6.7 Hz, 2H); 1.80-1.67 (m, 2H); 1.59-1.49 (m, 2H); 1.41-1.21 (m, 50H). LC-MS: 566.6 ((M-4x pinacol-4x H2O) / 2 + H)+.

[0880] Example 13: 2,5-Dioxopyrrolidin-1-yl 3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzamido)methyl)benzoate

[0881]

[0882] Dissolve 3,5-bis(aminomethyl)benzoic acid dihydrochloride (2, 1.88 g, 7.43 mmol) in water (20 mL). Subsequently add N,N-diisopropylethylamine (10.4 mL, 59.5 mmol), N,N-dimethylformamide (40 mL) and 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (1, 5.40 g, 14.8 mmol).

[0883] Stir the mixture at room temperature overnight; then acidify with 1 M aqueous hydrochloric acid (200 mL). Evaporate the solvent with toluene 3 times. Dissolve the residue in a dichloromethane / toluene mixture (1 : 1, 100 mL) and treat with pinacol (1.24 g, 10.5 mmol). Evaporate the mixture from toluene 3 times. Dissolve the residue in ethyl acetate (150 mL) and wash with water (3 x 100 mL). Dry the organic layer over anhydrous sodium sulfate, filter and evaporate. Dissolve the residue in dichloromethane (10 mL) and the product starts to precipitate. Then add cyclohexane (190 mL) and collect the precipitate by filtration, wash with cyclohexane and dry under vacuum to obtain 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoic acid (3) as a white powder.

[0884] Yield: 4.38 g (87%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.95 (bs, 1H); 9.05-8.97 (m, 2H); 7.82 (s, 2H); 7.64 (t, J = 7.3 Hz, 2H); 7.56-7.49 (m, 3H); 7.44-7.37 (m, 2H); 4.55-4.47 (m, 4H); 1.31 (s, 24H). LC-MS: 677.5 (M+H)+, 595.3 (M+H-pinacol)+, 513.3 (M+H-2x pinacol)+.

[0885] 7.56-7.49 (m, 3H); 7.44-7.37 (m, 2H); 4.55-4.47 (m, 4H); 1.31 (s, 24H). LC-MS: 677.5 (M+H)+, 595.3 (M+H-pinacol)+, 513.3 (M+H-2x pinacol)+.

[0886] The above acid (3, 4.37 g, 6.48 mmol) was dissolved in a mixture of acetonitrile / N,N-dimethylformamide (4:1, 100 mL) and N-hydroxysuccinimide (HOSu, 0.89 g, 7.77 mmol) was added. The mixture was cooled to 0 °C and then N,N-dicyclohexylcarbodiimide (DCC, 1.60 g, 7.77 mmol) was added. The mixture was stirred at 0 °C for 30 min and at room temperature overnight. The insoluble by-product was filtered off and the filtrate was evaporated. The residue was dissolved in ethyl acetate (250 mL) and washed with water (2 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (10 mL) and cyclohexane (170 mL) was added. The precipitate was collected by filtration, washed with cyclohexane. The white powder was dissolved in tetrahydrofuran (100 mL). Pinacol (0.19 g, 1.60 mmol) and magnesium sulfate (10 g) were added to the solution and the resulting mixture was stirred at room temperature overnight. The suspension was filtered through a pad of celite and the filtrate was evaporated. The residue was dissolved in dichloromethane (10 mL) and cyclohexane (170 mL) was added to the solution. The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give the title compound (4) as a white powder. Yield: 3.99 g (80%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.07 (t, J = 5.7 Hz, 2H); 7.96 (s, 2H); 7.75 (s, 1H); 7.65 (t, J = 7.2 Hz, 2H); 7.53 (d, J = 7.7 Hz, 2H); 7.41 (d, J = 10.4 Hz, 2H); 4.61-4.48 (m, 4H); 2.89 (s, 4H); 1.31 (s, 24H). LC-MS: 774.6 (M+H)+, 692.4 (M+H-pinacol)+, 610.3 (M+H-2x pinacol)+.

[0887] Example 14:

[0888]

[0889] Preparation by solid phase peptide synthesis from β-Ala, Fmoc-Lys and 4-carboxy-2- fluorophenylboronic acid pinacol ester

[0890] Example 15: 2,5-Dioxopyrrolidin-1-yl (R)-3-(2,4-bis(3-fluoro-4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)benzamido)butanamido)propanoate

[0891]

[0892] L-2,4-diaminobutyric acid dihydrochloride (1, 4.81 g, 25.2 mmol) was suspended in a solution of sodium bicarbonate (10.6 g, 126 mmol) in water (80 mL). The mixture was heated until a clear solution was formed. After cooling to room temperature, 1,4-dioxane (80 mL) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (20.4 g, 60.4 mmol) were added. The mixture was stirred at room temperature overnight, then acidified with 5 M aqueous hydrochloric acid solution. The 1,4-dioxane was evaporated, and the aqueous phase was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (3 x 100 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue was recrystallized twice from a hot ethyl acetate / cyclohexane mixture. The product was collected by filtration, washed with cyclohexane and dried under vacuum to give (R)-2,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butyric acid (2) as a white powder. Yield: 13.3 g (94%).

[0893] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.62 (bs, 1H); 7.94-7.83 (m, 4H); 7.79-7.55 (m, 5H); 7.46-7.26 (m, 9H); 4.34-4.13 (m, 6H); 4.08-3.94 (m, 1H); 3.14-3.02 (m, 2H); 1.98-1.84 (m, 1H); 1.84-1.65 (m, 1H). LC-MS: 562.6 (M+H)+.

[0894] Resin 100-200 mesh 1.5 mmol / g (3, 5.84 g, 8.75 mmol) was swelled in dry dichloromethane (70 mL) for 20 min. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Ala-OH, 1.82 g, 5.84 mmol) and N,N-diisopropylethylamine (3.86 mL, 22.2 mmol) in dry dichloromethane (50 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (2.03 mL, 11.7 mmol) in a methanol / dichloromethane mixture (1:4, 1 x 10 min, 1 x 50 mL). The resin was then washed with dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 50 mL). The resin was washed with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). A solution of (R)-2,4-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid (2, 6.57 g, 11.7 mmol), cyano-acetaldehyde acid ethyl ester-2-oxime (Oxyma, 1.66 g, 11.7 mmol), N,N-diisopropylcarbodiimide (DIC, 1.81 mL, 11.7 mmol) and 2,4,6-collidine (3.09 mL, 23.4 mmol) in N,N-dimethylformamide (50 mL) was added to the resin and the mixture was shaken for 2.5 h. The resin was filtered and washed with N,N-dimethylformamide (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 50 mL). The resin was washed with N,N-dimethylformamide (2 x 50 mL), 2-propanol (2 x 50 mL), dichloromethane (2 x 50 mL) and N,N-dimethylformamide (2 x 50 mL). A solution of 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (4, 8.48 g, 23.4 mmol) and N,N-diisopropylethylamine (7.32 mL, 42.0 mmol) in N,N-dimethylformamide (50 mL) was added to the resin and the mixture was shaken for 2 h. The resin was filtered and washed with N,N-dimethylformamide (3 x 60 mL) and dichloromethane (10 x 60 mL).The product was cleaved from the resin by treatment with a 1,1,1,3,3,3-hexafluoro-2-propanol / dichloromethane mixture (1:2, 90 mL) for 2 hours. The resin was filtered off and washed with dichloromethane (4 x 50 mL). The solvent was evaporated; the residue was dissolved in ethyl acetate (100 mL) and washed with water (2 x 80 mL) and brine (1 x 80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give (R)-3-(2,4-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)butanamido)propanoic acid (5) as a beige solid. Yield: 3.25 g (81%). 1 H NMR spectrum (300 MHz, CDC13, δH): 12.11 (bs, 1H); 8.69 (d, J = 7.9 Hz, 1H); 8.63-8.52 (m, 1H); 8.14-8.03 (m, 1H); 7.81-7.61 (m, 5H); 7.56 (d, J = 10.5 Hz, 1H); 4.54-4.39 (m, 1H); 3.44-3.17 (m, 4H); 2.43-2.33 (m, 2H); 2.14-1.99 (m, 1H); 1.99-1.85 (m, 1H); 1.31 (s, 24H). LC-MS: 521.0 (M-2x pinacol + H)+, 603.1 (M-pinacol + H)+, 685.3 (M + H)+.

[0895] The acid (5, 3.24 g, 4.73 mmol) was dissolved in dichloromethane (50 mL) followed by the addition of N-hydroxysuccinimide (0.65 g, 5.67 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.09 g, 5.67 mmol). The mixture was stirred overnight, then diluted with dichloromethane (50 mL) and washed with water (2 x 80 mL) and brine (1 x 80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give the title compound (6) as a white solid. Yield: 3.42 g (92%). 1H NMR spectrum (300 MHz, DMSO-d6, δΗ): 8.72 (d, J = 7.9 Hz, 1H); 8.57 (t, J = 5.4 Hz, 1H); 8.22 (t, J = 5.5 Hz, 1H); 7.77-7.62 (m, 5H); 7.56 (d, J = 10.1 Hz, 1H); 4.53-4.40 (m, 1H); 3.48-3.27 (m, 4H); 2.86 (t, J = 7.1 Hz, 2H); 2.80 (s, 4H); 2.15-2.02 (m, 1H); 2.02-1.88 (m, 1H); 1.31 (s, 24H). LC-MS: 618.1 (M-2x mesityl alcohol + H)+, 700.2 (M-mesityl alcohol + H)+, 782.4 (M + H)+.

[0896] Example 16: 3-(3-Fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid

[0897]

[0898] Dissolve 3-bromo-5-iodobenzoic acid (1, 16.4 g, 50.0 mmol) in methanol (100 mL) and add methanesulfonic acid (1 mL). Stir the resulting mixture at 60 °C (oil bath) for 16 hours. Cool the resulting clear solution to -20 °C in the freezer for 16 hours, collect the resulting solid by filtration, wash with cooled (-20 °C) methanol and dry under vacuum to give 3-bromo-5-iodobenzoic acid methyl ester (2) as an off-white solid.

[0899] Yield: 13.9 g (82%). 1 H NMR spectrum (300 MHz, CDCl3, δΗ): 8.30 (s, 1H); 8.14 (s, 1H); 8.04 (s, 1H); 3.93 (s, 1H).

[0900] Dissolve 1,3-dibromo-5-fluorobenzene (3, 6.30 mL, 50.0 mmol) in anhydrous diethyl ether (150 mL) and cool to -78 °C. Add 2.35 M n-butyllithium in hexanes (22.0 mL, 52.5 mmol) dropwise with stirring. After 15 minutes, add anhydrous N,N-dimethylformamide (7.70 mL, 100 mmol) and stir the resulting mixture for 15 minutes before warming to ambient temperature. After one hour, quench the reaction mixture with 1 M aqueous hydrochloric acid (150 mL). Separate the layers, wash the organic layer with brine (100 mL), dry over anhydrous magnesium sulfate and evaporate to give 3-bromo-5-fluorobenzaldehyde (4) as a slightly yellow oil which solidifies on storage in the freezer. Yield: 10.2 g (100%). 1H NMR spectrum (300 MHz, CDC13, δH): 9.92 (s, 1H); 7.80 (bs, 1H); 7.50 (bs, 2H).

[0901] Methyl 3-bromo-5-iodobenzoate (2, 6.80 g, 20.0 mmol) was dissolved in dry tetrahydrofuran (50 mL) under a nitrogen atmosphere and cooled to -40 °C. 1.3 M isopropylmagnesium chloride-lithium chloride complex in tetrahydrofuran (16.1 mL, 21.0 mmol) was added dropwise via an addition funnel. After 30 minutes, 3-bromo-5-fluorobenzaldehyde (4) (4.87 g, 24.0 mmol) was added with the aid of dry tetrahydrofuran (5 mL). The resulting mixture was allowed to warm to room temperature over one hour and stirred at ambient temperature for a further hour. The reaction was quenched by the addition of 0.5 M aqueous hydrochloric acid (50 mL) and extracted with diethyl ether (1 x 200 mL). The organic layer was washed with brine (3 x 100 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue 5 was dissolved in dry dichloromethane (100 mL) and pyridinium chlorochromate (PCC, 6.45 g, 30.0 mmol) was added. The reaction mixture was then stirred overnight (16 hours) after which time it was quenched with 2-propanol (3 mL). After stirring at room temperature for one hour, the reaction mixture was filtered through a plug of silica gel (100 g) with celite S on top and washed with dichloromethane (2 x 100 mL). The solvent was removed in vacuo and the residue purified by flash column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / dichloromethane 6:1 to 2:1) to give methyl 3-bromo-5-(3-bromo-5-fluorobenzoyl)benzoate (6) as a colourless solid.

[0902] Yield: 7.10 g (85%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.43 (s, 1H); 8.30 (s, 1H); 8.11 (s, 1H); 7.71 (s, 1H); 7.53 (d, J = 7.6 Hz, 1H); 7.41 (d, J = 8.3 Hz, 1H); 3.97 (s, 3H).

[0903] LC-MS: No molecular ion and fragments detected.

[0904] Potassium acetate (6.70 g, 68.4 mmol) was charged into a 250 mL reaction vessel and the salt was dried under vacuum at 110 °C for 1 hour. After cooling to room temperature, the reaction vessel was backfilled with nitrogen and charged with 3-bromo-5-(3-bromo-5-fluorobenzoyl)benzoic acid methyl ester (6, 7.10 g, 481 mol), palladium acetate (77.0 mg, 342 mol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 325 mg, 684 mol) and bis(pinacolato)diboron (9.53 mg, 37.6 mmol). The reaction vessel was then evacuated and backfilled with nitrogen (this process was repeated twice), anhydrous tetrahydrofuran (3 mL) was added using a syringe, the vessel was sealed with a plastic stopper and immersed in a pre-heated heating bath at 60 °C. After stirring at 400 rpm for 16 hours (overnight), the reaction mixture was cooled to ambient temperature, diluted with dichloromethane (100 mL) and filtered through a short plug of silica gel (70 g) with a celite S topped head using dichloromethane (3 x 70 mL). The filtrate was concentrated under reduced pressure to give the product as a yellowish waxy foam which was triturated with ice-cold n-hexane (70 mL) to induce crystallisation. The resulting solid was collected by filtration and dried under vacuum to give 3-(3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid methyl ester (7) as a white solid. Yield: 7.10 g (88%).

[0905] 1 H NMR spectrum (300 MHz, CDC13, δH): 8.69 (s, 1H); 8.48 (s, 1H); 8.38 (s, 1H); 7.97 (s, 1H); 7.72 (d, J = 8.5 Hz, 1H); 7.54 (d, J = 9.0 Hz, 1H); 3.95 (s, 3H); 1.36 (s, 12H); 1.35 (s, 12H). LC-MS: 511.6 (M+H)+.

[0906] Methyl 3-(3-fluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5- tetramethyl- 1,3,2-dioxaborolan-2-yl)benzoate (7, 7.10 g, 13.9 mmol) was suspended in methanol (42 mL) and water (13 mL). Lithium hydroxide (2.91 g, 69.5 mmol) was added and the resulting mixture was stirred vigorously at ambient temperature for 16 hours. The reaction mixture was diluted with water (120 mL) and extracted with diethyl ether (70 mL). The ether layer was discarded and the aqueous layer was acidified with concentrated hydrochloric acid (10 mL) and extracted with ethyl acetate (100 mL). The organic layer was washed with brine (3 x 100 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was dissolved in hot ethyl acetate (80 mL) and pinacol was added until a clear solution was obtained. The solution was evaporated to dryness and then evaporated twice from dichloromethane (2 x 40 mL) to give the title 3-(3-fluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5- tetramethyl- 1,3,2-dioxaborolan-2-yl)benzoic acid (8) as a colourless solid. This compound contained residual pinacol which could not be removed.

[0907] Yield: 6.82 g (99%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.77 (s, 1H); 8.54 (t, J = 1.8 Hz, 1H); 8.44 (d, J = 1.1 Hz, 1H); 7.98 (s, 1H); 7.80-7.68 (m, 1H); 7.63-7.50 (m, 1H); 1.37 (s, 12H); 1.35 (s, 12H). LC-MS: 497.5 (M+H)+, 415.4 (M-pinacol+H)+.

[0908] Example 17: (2,5-Dioxopyrrolidin-1-yl) 3,5-bis[[[4-(4,4,5,5-tetramethyl-1,3,2-dioxo borolan-2-yl)benzoyl]amino]methyl]benzoate

[0909]

[0910] Dissolve 3,5-bis((4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoic acid (1, 2.57 g, 4.00 mmol) in a mixture of acetonitrile / N,N-dimethylformamide (3:1, 100 mL). Add N-hydroxysuccinimide (0.55 g, 4.80 mmol). Cool the mixture to 0 °C, then add N,N-dicyclohexylcarbodiimide (0.99 g, 4.80 mmol). Stir the mixture at 0 °C for 30 min and at room temperature overnight. Filter off the insoluble by-product and evaporate the filtrate. Dissolve the residue in ethyl acetate (250 mL) and wash with water (2 x 150 mL). Dry the organic layer over anhydrous sodium sulfate, filter, and evaporate. Dissolve the residue in toluene (10 mL) and the product starts to precipitate. Add cyclohexane (170 mL). Collect the precipitate by filtration, wash with cyclohexane and dry under vacuum to give the title compound (2) as a white powder. The product contains traces of N,N-dicyclohexylurea.

[0911] Yield: 2.85 g (97%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.24 (t, J = 5.7 Hz, 2H); 7.95-7.83 (m, 6H); 7.79-7.70 (m, 5H); 4.60-4.52 (m, 4H); 2.87 (s, 4H); 1.31 (s, 24H). LC-MS: 737.4 (M+H)+, 655.2 (M+H-furanol)+, 573.1 (M+H-2x furanol)+.

[0912] Example 18: N2,N6-Bis(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-carbonyl)- L-lysine Example 19: (S)-2,3-Bis(4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonyl-

[0913]

[0914] Dissolve 6-fluoro-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-5-carboxylic acid (1, 6.00 g, 30.6 mmol) in tetrahydrofuran (80 mL). Add N,N-dimethylformamide (10 mL), N-hydroxysuccinimide (3.87 g, 33.7 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (6.46 g, 33.7 mmol) at room temperature. After stirring for 2 hours, evaporate the volatiles under reduced pressure and re-dissolve the residue in ethyl acetate (200 mL) and wash with 1 M aqueous hydrochloric acid (2 x 60 mL). Dry the organic portion using anhydrous sodium sulfate. Evaporate the volatiles under reduced pressure to give 2,5-dioxopyrrolidin-l-yl 6-fluoro-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-5-carboxylate (2) as a white solid. Yield: 8.35 g (93%).

[0915] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.65 (s, 1H); 8.11 (d, J = 5.9 Hz, 1H); 7.71 (d, J = 10.1 Hz, 1H); 5.08 (s, 2H); 2.90 (s, 4H). LC-MS: 294.4 (M+H)+.

[0916] Dissolve L-lysine hydrochloride (3, 1.56 g, 8.50 mmol) in N,N-dimethylformamide (50 mL) and water (25 mL). Add N,N-diisopropylethylamine (8.92 mL, 51.2 mmol) and 2,5-dioxopyrrolidin-l-yl 6-fluoro-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-5-carboxylate (2, 5.00 g, 17.0 mmol) at room temperature. After stirring for 3 hours, evaporate the volatiles under reduced pressure and precipitate the residue with 1 M aqueous hydrochloric acid. Wash the precipitate with water and purify by precipitation from acetonitrile / water mixture, collect by centrifugation and freeze-dry to give N2,N6-bis(6-fluoro-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-5-carbonyl)-L-lysine (4) as a white solid.

[0917] Yield: 3.25 g (76%). 1H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.66 (bs, 1H); 9.41 (d, J = 5.7 Hz, 2H); 8.59 (d, J = 7.2 Hz, 1H); 8.39 (t, J = 5.0 Hz, 1H); 7.61-7.53 (m, 2H); 7.53-7.44 (m, 2H); 4.97 (d, J = 5.7 Hz, 4H); 4.41-4.30 (m, 1H); 3.30-3.20 (m, 2H); 1.90-1.70 (m, 2H); 1.59-1.38 (m, 4H). LC-MS: 503.5 (M+H)+.

[0918] amino)propanoic acid Example 20: 2,5-Dioxopyrrolidin-1-yl 3-(2,4-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxo-

[0919]

[0920] A solution of methyl 4-(bromomethyl)-3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoate (1, 23.0 g, 61.7 mmol) and sodium hydroxide (12.3 g, 0.31 mol) in water (400 mL) was stirred at ambient temperature overnight. A 6M aqueous hydrochloric acid solution (60 mL, 6M) was added to the reaction mixture, resulting in a white precipitate. The flask with the precipitate was placed in the freezer for 1 hour. It was then filtered, the filter cake was washed with water (200 mL) and freeze-dried to give 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (2) as a white solid.

[0921] Yield: 12.1 g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 13.24 (bs, 1H), 9.58 (s, 1H), 8.20 (s, 1H), 7.73 (d, J = 9.9 Hz, 1H), 5.14 (s, 2H).

[0922] A solution of 2,5-dioxopyrrolidin-l-yl 4-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylate (3, 5.10 g, 17.4 mmol), (S)-2,3-diaminopropanoic acid hydrochloride (4, 1.22 g, 8.70 mmol) and N,N-diisopropylethylamine (9.28 mL, 52.2 mmol) in N,N-dimethylformamide (100 mL) and water (10 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated and extracted with ethyl acetate (2 x 250 mL) and 1 M aqueous hydrochloric acid (150 mL), the organic layer was washed with brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give (S)-2,3-bis(4-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6- carboxamido)propanoic acid (5) as a white solid. Yield: 3.53 g (88%).

[0923] LC-MS: 294.3 (M+H)+.

[0924] A solution of 2,5-dioxopyrrolidin-l-yl 4-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylate (3, 5.10 g, 17.4 mmol), (S)-2,3-diaminopropanoic acid hydrochloride (4, 1.22 g, 8.70 mmol) and N,N-diisopropylethylamine (9.28 mL, 52.2 mmol) in N,N-dimethylformamide (100 mL) and water (10 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated and extracted with ethyl acetate (2 x 250 mL) and 1 M aqueous hydrochloric acid (150 mL), the organic layer was washed with brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give (S)-2,3-bis(4-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6- carboxamido)propanoic acid (5) as a white solid. Yield: 3.53 g (88%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.82 (bs, 1H); 9.54 (d, J = 6.2 Hz, 2H); 8.86 (d, J = 7.7 Hz, 1H); 8.78 (t, J = 6.0 Hz, 1H); 8.10 (s, 1H); 8.04 (s, 1H); 7.80-7.63 (m, 2H); 5.13 (d, J = 6.2 Hz, 4H); 4.77-4.62 (m, 1H); 3.91-3.77 (m, 1H); 3.77-3.62 (m, 1H). LC-MS: 461.3 (M+H)+.

[0925] borolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate Example 21: 2,5-Dioxopyrrolidin-1-yl 3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxo- borolan-2-yl)benzamido)methyl)benzoate

[0926]

[0927] A solution of 3-bromo-5-iodobenzoic acid (1, 5.00 g, 15.3 mmol) was dissolved in dry dichloromethane (100 mL) and tert-butanol (1.52 mL, 16.1 mmol), N,N'-dicyclohexylcarbodiimide (3.31 mL, 16.1 mmol) and 4-(dimethylamino)pyridine (1.96 mL, 16.1 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then washed with 1 M aqueous hydrochloric acid solution (2 x 50 mL) and brine (1 x 40 mL). The organic fraction was dried over anhydrous sodium sulfate. The volatiles were evaporated under reduced pressure and the residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 10:1 ) to give tert-butyl 3-bromo-5-iodobenzoate (2) as a white solid. Yield: 4.67 g (80%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.22 (s, 1 H); 8.06 (s, 1 H); 8.00 (s, 1 H); 1.58 (s, 9H).

[0928] A solution of tert-butyl 3-bromo-5-iodobenzoate (2, 4.31 g, 11.3 mmol) was dissolved in dry tetrahydrofuran (50 mL) and cooled to -40 °C under a nitrogen atmosphere. 1.3 M isopropylmagnesium chloride-lithium chloride complex in tetrahydrofuran (9.52 mL, 12.4 mmol) was added dropwise slowly. After 40 minutes, 5-bromo-2,4-difluorobenzaldehyde (3, 2.86 g, 12.9 mmol) was added with the help of dry tetrahydrofuran (5 mL). The resulting mixture was allowed to warm to room temperature overnight (16 hours). The reaction was quenched by the addition of 0.5 M aqueous hydrochloric acid solution (15 mL) and extracted with ethyl acetate (2 x 100 mL). The organic layer was washed with brine (3 x 40 mL) and dried over anhydrous sodium sulfate. The volatiles were evaporated under reduced pressure and the residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 10:1 ) to give tert-butyl 3-bromo-5-((5-bromo-2,4-difluorophenyl)(hydroxy)methyl)benzoate (4) as a white solid. Yield: 4.38 g (81 %).

[0929] 1H NMR spectrum (300 MHz, CDC13, δH): 8.02 (t, J = 1.6 Hz, 1H); 7.92 (s, 1H); 7.77-7.65 (m, 2H); 6.89 (dd, J = 9.7 and 8.3, 1H); 6.09 (d, J = 3.9 Hz, 1H); 2.43 (d, J = 4.0 Hz, 1H); 1.68-1.58 (m, 9H).

[0930] tert-Butyl 3-bromo-5-((5-bromo-2,4-difluorophenyl)(hydroxy)methyl)benzoate (4) was dissolved in anhydrous dichloromethane (50 mL) and pyridinium chlorochromate (PCC, 2.96 g, 13.7 mmol) was added. The reaction mixture was then stirred overnight (16 hours) after which it was quenched with 2-propanol (1.5 mL). After stirring for one hour at room temperature, the reaction mixture was filtered through a short plug of celite (5 g) and washed with dichloromethane (50 mL). The volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 20:1) to give tert-butyl 3-bromo-5-(5-bromo-2,4-difluorobenzoyl)benzoate (5) as a colourless solid. Yield: 4.20 g (96%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.33 (t, J = 1.7 Hz, 1H); 8.28-8.24 (m, 1H); 8.10-8.07 (m, 1H); 7.86 (t, J = 7.3 Hz, 1H); 7.03 (dd, J = 9.3 and 8.1 Hz, 1H); 1.61 (s, 9H).

[0931] A reaction flask was charged with tert-butyl 3-bromo-5-(5-bromo-2,4-difluorobenzoyl)benzoate (5, 4.20 g, 8.82 mmol), palladium acetate (59.0 mg, 0.26 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 252 mg, 0.52 mmol), potassium acetate (3.46 g, 35.3 mmol) and bis(pinacolato)diboron (4.70 g, 18.5 mmol), the resulting mixture was evacuated and backfilled with argon (this process was repeated twice). Anhydrous tetrahydrofuran (60 mL) was added with a syringe, sealed with a rubber septum and immersed in a preheated heating bath at 60 °C. After stirring for 16 h, the reaction mixture was cooled to ambient temperature, diluted with cyclohexane (100 mL) and filtered short through celite with the aid of dichloromethane (100 mL). The volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 10:1) to give tert-butyl 3-(2,4-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (6) as a yellow solid. Yield: 4.78 g (95%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.61 (s, 1H); 8.41 (d, J = 1.5 Hz, 1H); 8.34 (s, 1H); 8.05 (dd, J = 8.3 and 6.7 Hz, 1H); 6.96-6.81 (m, 1H); 1.61 (s, 9H); 1.36 (d, J = 2.2 Hz, 24H).

[0932] Tert-butyl 3-(2,4-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (6, 4.78 g, 8.38 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (40 mL) was added at room temperature. The reaction mixture was stirred for 3 h. The volatiles were removed under reduced pressure and the residue was co-evaporated with dichloromethane (4 x 50 mL). The resulting 3-(2,4-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (7) was used in the next step without further purification.

[0933] Yield: 4.10 g (96%). 1H NMR spectrum (300 MHz, CDC13, δH): 8.75 (s, 1H); 8.50 (t, J = 1.7 Hz, 1H); 8.46 (s, 1H); 8.09 (dd, J = 8.4 and 6.8 Hz, 1H); 6.95-6.83 (m, 1H); 1.37 (d, J = 1.8 Hz, 24H).

[0934] (2,4-difluoro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5- tetramethyl- 1,3,2-dioxaborolan-2-yl)benzoic acid (7, 4.10 g, 8.00 mmol) was dissolved in dichloromethane (50 mL) and N-hydroxysuccinimide (1.29 g, 11.2 mmol) and N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (2.14 g, 11.2 mmol) were added at room temperature. After stirring for 6 h, the reaction mixture was washed with 10% aqueous potassium hydrogen sulfate solution (2 x 100 mL) and brine (30 mL). The organic fraction was dried using anhydrous sodium sulfate. The volatiles were evaporated under reduced pressure to give 2,5-dioxopyrrolidin-l-yl 3-(2,4-difluoro-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzoyl)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzoate (8) as a yellow solid.

[0935] Yield: 4.84 g (99%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.75 (s, 1H); 8.50 (t, J = 1.7 Hz, 1H); 8.46 (s, 1H); 8.09 (dd, J = 8.4 and 6.8 Hz, 1H); 6.95-6.83 (m, 1H); 1.37 (d, J = 1.8 Hz, 24H).

[0936] Example 22: (3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5, ​

[0937]

[0938] Dissolve 3,5-bis(aminomethyl)benzoic acid dihydrochloride (2, 1.88 g, 7.43 mmol) in water (20 mL). Subsequently add N,N-diisopropylethylamine (10.4 mL, 59.5 mmol), N,N-dimethylformamide (40 mL) and 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (1, 5.40 g, 14.8 mmol).

[0939] Stir the mixture at room temperature overnight; then acidify with 1 M aqueous hydrochloric acid (200 mL). Evaporate the solvent with toluene 3 times. Dissolve the residue in a dichloromethane / toluene mixture (1 : 1, 100 mL) and treat with pinacol (1.24 g, 10.5 mmol). Evaporate the mixture from toluene 3 times. Dissolve the residue in ethyl acetate (150 mL) and wash with water (3 x 100 mL). Dry the organic layer over anhydrous sodium sulfate, filter and evaporate. Dissolve the residue in dichloromethane (10 mL) and the product starts to precipitate. Then add cyclohexane (190 mL) and collect the precipitate by filtration, wash with cyclohexane and dry under vacuum to obtain 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzoic acid (3) as a white powder. Yield: 4.38 g (87%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.95 (bs, 1H); 9.05-8.97 (m, 2H); 7.82 (s, 2H); 7.64 (t, J = 7.3 Hz, 2H); 7.56-7.49 (m, 3H); 7.44-7.37 (m, 2H); 4.55-4.47 (m, 4H); 1.31 (s, 24H). LC-MS: 677.5 (M+H)+, 595.3 (M+H-pinacol)+, 513.3 (M+H-2x pinacol)+.

[0940] The above acid (3, 4.37 g, 6.48 mmol) was dissolved in a mixture of acetonitrile / N,N-dimethylformamide (4:1, 100 mL) and N-hydroxysuccinimide (HOSu, 0.89 g, 7.77 mmol) was added. The mixture was cooled to 0 °C and then N,N-dicyclohexylcarbodiimide (DCC, 1.60 g, 7.77 mmol) was added. The mixture was stirred at 0 °C for 30 min and at room temperature overnight. The insoluble by-product was filtered off and the filtrate was evaporated. The residue was dissolved in ethyl acetate (250 mL) and washed with water (2 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in dichloromethane (10 mL) and cyclohexane (170 mL) was added. The precipitate was collected by filtration, washed with cyclohexane. The white powder was dissolved in tetrahydrofuran (100 mL). Pinacol (0.19 g, 1.60 mmol) and magnesium sulfate (10 g) were added to the solution and the resulting mixture was stirred at room temperature overnight. The suspension was filtered through a pad of celite and the filtrate was evaporated. The residue was dissolved in dichloromethane (10 mL) and cyclohexane (170 mL) was added to the solution. The precipitate was collected by filtration, washed with cyclohexane and dried under vacuum to give the title compound (4) as a white powder. Yield: 3.99 g (80%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.07 (t, J = 5.7 Hz, 2H); 7.96 (s, 2H); 7.75 (s, 1H); 7.65 (t, J = 7.2 Hz, 2H); 7.53 (d, J = 7.7 Hz, 2H); 7.41 (d, J = 10.4 Hz, 2H); 4.61-4.48 (m, 4H); 2.89 (s, 4H); 1.31 (s, 24H). LC-MS: 774.6 (M+H)+, 692.4 (M+H-pinacol)+, 610.3 (M+H-2x pinacol)+.

[0941] ​ 5-Tetramethyl-1,3,2-dioxoboronyl-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine

[0942]

[0943] To a mixture of copper(II) sulfate pentahydrate (541 mg, 2.36 mmol) and potassium hydroxide (9.24 g, 216 mmol) in dimethyl sulfoxide / water mixture (10:1, 70 mL) was added 1,3-dibromo-5-(trifluoromethyl)benzene (1, 13.1 g, 43.1 mmol), the reaction flask was filled with nitrogen and finally 1,2-ethanedithiol (6.00 mL, 90.5 mmol) was added through a septum. The reaction mixture was heated to 110 °C overnight. Then the mixture was acidified to pH = 2 with 1 M aqueous hydrochloric acid solution and extracted with ethyl acetate. After drying over anhydrous sodium sulfate and filtration, the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane) to give 3-bromo-5-(trifluoromethyl)benzenethiol (2) as a white oil.

[0944] Yield: 5.76 g (52%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.61 (s, 1H); 7.56 (s, 1H); 7.46 (s, 1H); 3.66 (s, 1H).

[0945] To a mixture of copper(II) sulfate pentahydrate (541 mg, 2.36 mmol) and potassium hydroxide (9.24 g, 216 mmol) in dimethyl sulfoxide / water mixture (10:1, 70 mL) was added 1,3-dibromo-5-(trifluoromethyl)benzene (1, 13.1 g, 43.1 mmol), the reaction flask was filled with nitrogen and finally 1,2-ethanedithiol (6.00 mL, 90.5 mmol) was added through a septum. The reaction mixture was heated to 110 °C overnight. Then the mixture was acidified to pH = 2 with 1 M aqueous hydrochloric acid solution and extracted with ethyl acetate. After drying over anhydrous sodium sulfate and filtration, the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane) to give 3-bromo-5-(trifluoromethyl)benzenethiol (2) as a white oil. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.61 (s, 1H); 7.56 (s, 1H); 7.46 (s, 1H); 3.66 (s, 1H).

[0946] Methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoate (5, 5.46 g, 10.9 mmol) and lithium hydroxide monohydrate (1.33 g, 31.7 mmol) were dissolved in a mixture of methanol / water / tetrahydrofuran (4:2:5, 35 mL) and the reaction mixture was stirred at room temperature overnight. After this time the mixture was acidified to pH 2 with 1 M aqueous hydrochloric acid solution and extracted with ethyl acetate. Evaporation of all volatiles gave 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (6) as a white solid. Yield: 5.10 g (96%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.53 (m, 1H); 8.43 (m, 1H); 8.27 (m, 2H); 8.15 (m, 1H); 8.00 (m, 1H); 4.00 (s, 3H).

[0947] Methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoate (5, 5.46 g, 10.9 mmol) and lithium hydroxide monohydrate (1.33 g, 31.7 mmol) were dissolved in a mixture of methanol / water / tetrahydrofuran (4:2:5, 35 mL) and the reaction mixture was stirred at room temperature overnight. After this time the mixture was acidified to pH 2 with 1 M aqueous hydrochloric acid solution and extracted with ethyl acetate. Evaporation of all volatiles gave 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (6) as a white solid. Yield: 5.10 g (96%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.91 (bs, 1H); 8.68 (s, 2H); 8.50 (s, 1H); 8.45 (s, 1H); 8.41 (s, 1H); 8.32 (s, 1H).

[0948] A 100 mL reaction flask was charged with potassium acetate (5.13 g, 26.1 mmol) and the salt was dried under vacuum at 110 °C for 1 h. After cooling to room temperature, the reaction flask was backfilled with nitrogen and charged with (3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine tert-butyl ester (7, 6.30 g, 10.5 mmol), palladium acetate (120 mg, 0.52 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 500 mg, 1.04 mmol) and bis(pinacolato)diboron (5.9 g, 23.03 mmol). The reaction flask was then evacuated and backfilled with nitrogen (this process was repeated twice), anhydrous tetrahydrofuran (50 mL) was added using a syringe, the flask was sealed with a plastic stopper and heated to 60 °C. The reaction mixture was stirred overnight, then cooled to ambient temperature, diluted with dichloromethane (150 mL) and filtered through a short plug of silica gel with a celite top and washed with dichloromethane (3 x 50 mL). The filtrate was concentrated under reduced pressure to give (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine tert-butyl ester (8) as a black waxy foam. Yield: 6.70 g (92%). LC-MS: 640.5 (M+H-tBu)+, 558.4 (M-pinacol-tBu+H)+, 476.3 (M-2 pinacol-tBu+H)+.

[0949] Yield: 6.30 g (99%). LC-MS: 602.3 (M+H)+.

[0950] A 100 mL reaction flask was charged with potassium acetate (5.13 g, 26.1 mmol) and the salt was dried under vacuum at 110 °C for 1 h. After cooling to room temperature, the reaction flask was backfilled with nitrogen and charged with (3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine tert-butyl ester (7, 6.30 g, 10.5 mmol), palladium acetate (120 mg, 0.52 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 500 mg, 1.04 mmol) and bis(pinacolato)diboron (5.9 g, 23.03 mmol). The reaction flask was then evacuated and backfilled with nitrogen (this process was repeated twice), anhydrous tetrahydrofuran (50 mL) was added using a syringe, the flask was sealed with a plastic stopper and heated to 60 °C. The reaction mixture was stirred overnight, then cooled to ambient temperature, diluted with dichloromethane (150 mL) and filtered through a short plug of silica gel with a celite top and washed with dichloromethane (3 x 50 mL). The filtrate was concentrated under reduced pressure to give (3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine tert-butyl ester (8) as a black waxy foam. Yield: 6.70 g (92%). LC-MS: 640.5 (M+H-tBu)+, 558.4 (M-pinacol-tBu+H)+, 476.3 (M-2 pinacol-tBu+H)+.

[0951] tert-Butyl (3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycinate (8, 6.70 g, 10.5 mmol) was mixed with trifluoroacetic acid (25 mL) and stirred at room temperature for 1 hour after which all volatiles were evaporated under reduced pressure. The residue was then dissolved in ethyl acetate (50 mL) and filtered through a short plug of silica gel with celite on top. The filtrate was concentrated under reduced pressure to give an orange hard foam which was crushed. (3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycine (9) was obtained as a light orange solid. Yield: 5.50 g (83%).

[0952] 3.89 g (63%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.57 (m, 3H); 8.47 (s, 1H); 8.31 (s, 1H); 9.26 (s, 1H); 7.23 (t, 1H); 4.35 (d, 2H); 1.38 (s, 1H). 19 F NMR spectrum (282 MHz, CDC13, δF): -62.65 (s). LC-MS: 640.5 (M+H)+, 558.4 (M- pinacol + H)+, 476.3 (M- 2x pinacol + H)+.

[0953] Example 23: N-(5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborone-6-carbonyl)-N- (2-(5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborone-6-carboxamido)ethyl)glycine

[0954]

[0955] Chloroacetic acid (1, 13.0 g, 136 mmol) was added in small portions to pre-cooled (0 °C) ethylenediamine (2, 90 mL). After the addition was complete, the reaction mixture was allowed to reach room temperature overnight (16 hours). The ethylenediamine was evaporated in vacuo and the residue was triturated with dimethyl sulfoxide (140 mL) with stirring overnight. The precipitate was collected by filtration and washed with dimethyl sulfoxide (2 x 60 mL), acetonitrile (3 x 100 mL), and diethyl ether (3 x 100 mL) to give (2- aminoethyl)glycine (3) as a colorless solid. Yield: 13.2 g (83%). 1 H NMR spectrum (300 MHz, D20, δH): 3.27 (s, 2H); 3.05-3.01 (m, 2H); 2.92-2.88 (m, 2H).

[0956] A solution of 2,3,4,5,6-pentafluorophenol (9.39 g, 51.0 mmol), 5-fluoro-l- hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid (4, 10.0 g, 51.0 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 10.5 g, 51.0 mmol) in acetonitrile (300 mL) was stirred at ambient temperature overnight. The reaction mixture was filtered, washed with acetonitrile and evaporated. The crude product 5 was purified by crystallization from a mixture of dichloromethane / hexane (9:1, 500 mL) to give 5-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (5) as a white solid. Yield: 6.80 g (37%). LC-MS: 363.2 (M+H)+.

[0957] A solution of 5-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (5, 6.80 g, 18.8 mmol), (2-aminoethyl)glycine (3, 1.10 g, 9.39 mmol) and triethylamine (10.5 mL, 75.1 mmol) in N,N-dimethylformamide (80 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated and extracted with ethyl acetate (2 x 500 mL) and 1 M aqueous hydrochloric acid solution (400 mL), the organic layer was washed with brine (300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product 6 was purified by flash chromatography (silica gel, 0.063-0.200 mm; eluent: dichloromethane / methanol / formic acid 100:2:0.5 to 100:10:0.5) and freeze-dried to give N-(5-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carbonyl)-N-(2-(5-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-ylcarbonyl)ethyl)glycine (6) as a white solid.

[0958] 1,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxamide)ethyl)glycine (6).

[0959] Yield: 2.16 g (49%). RF(SiO2, dichloromethane / methanol / formic acid 100:2:0.5): 0.30.

[0960] 1H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.86 (bs, 1H); 9.45-9.17 (m, 2H); 8.48-8.11 (m, 1H); 8.11-7.88 (m, 1H); 7.65 (d, J = 7.0 Hz, 1H); 7.43-7.15 (m, 2H); 4.99 (d, J = 8.6 Hz, 4H); 4.36-3.90 (m, 2H); 3.80-3.34 (m, 4H). LC-MS: 475.4 (M+H)+.

[0961] Example 24: 4-((3S,4S)-3,4-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxy (Hyperborane-6-carbamoyl)pyrrolidine-1-yl)-4-oxobutyric acid

[0962]

[0963] 1-Hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (1, 13.5 g, 54.9 mmol), N-hydroxysuccinimide (6.31 g, 54.9 mmol) and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (10.5 g, 54.9 mmol) were stirred in tetrahydrofuran (270 mL) and N,N-dimethylformamide (40 mL) at ambient temperature for 4 hours. The reaction mixture was evaporated and extracted with ethyl acetate (3 x 300 mL) and 1M aqueous hydrochloric acid (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give 2,5-dioxopyrrolidin-1-yl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylate (2) as a white solid. Yield: 18.8 g (100%). LC-MS: 344.3 (M+H)+.

[0964] 4-((3S,4S)-3,4-diaminopyrrolidin-1-yl)-4-oxobutanoic acid dihydrochloride (3, 2.74 mg, 10.0 mmol), 2,5-dioxopyrrolidin-1-yl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylate (2, 6.86 mg, 20.0 mmol)

[0965] A solution of 4-((3S,4S)-3,4-bis(1-hydroxy-4-(trifluoromethyl)-1,3- dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid (4) and N,N-diisopropylethylamine (11.0 mL, 60.0 mmol) in N,N-dimethylformamide (240 mL) and water (60 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated, purified by column chromatography (silica gel, 0.063-0.200 mm; eluents: dichloromethane / methanol / formic acid 100:2:0.5 to 100:10:0.5) and freeze-dried to give 4-((3S,4S)-3,4-bis(1-hydroxy-4-(trifluoromethyl)-1,3- dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid (4) as a white solid.

[0966] Yield: 2.56 g (39%). Rf(SiO2, dichloromethane / methanol / formic acid 100:10:0.5): 0.3.

[0967] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ) 11.73 (bs, 1H); 9.62 (s, 2H); 9.01 (dd, J = 10.4 and 7.2 Hz, 2H); 8.49 (s, 2H); 8.24 (s, 2H); 5.20 (s, 4H); 4.94-4.51 (m, 2H); 4.16-3.94 (m, 1H); 3.95-3.80 (m, 1H); 3.56-3.44 (m, 1H); 3.41-3.34 (m, 1H); 2.49-2.40 (m, 4H). LC-MS: 658.7 (M+H)+.

[0968] Example 25: 2,5-Dioxopyrrolidone-1-yl 3-(difluoro(3-(4,4,5,5-tetramethyl-1,3,2-dioxoboron) Heterocyclopentane-2-yl)-5-(trifluoromethyl)phenyl)methyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxoborane- 2-yl)benzoate

[0969]

[0970] Methyl 3-bromo-5-iodobenzoate (1, 6.80 g, 20.0 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) and cooled to -30 °C. A 1.3 M solution of isopropylmagnesium chloride-lithium chloride complex in tetrahydrofuran (16.2 mL, 21.0 mmol) was added dropwise with stirring. After 30 minutes, 3-bromo-5-(trifluoromethyl)benzaldehyde (2, 6.00 g, 24.0 mmol) was added with the aid of tetrahydrofuran (3 mL). The resulting mixture was allowed to warm to ambient temperature and quenched after 1 hour by the addition of 1 M aqueous hydrochloric acid (40 mL). The reaction mixture was taken up in diethyl ether (150 mL), washed with water (150 mL) and brine (100 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The crude product (3) was dissolved in anhydrous dichloromethane (80 mL) and pyridinium chlorochromate (6.42 g, 30.0 mmol) was added with stirring. After 17 hours stirring, the reaction mixture was filtered through a plug of silica gel (80 g) with celite on top and the bed was washed with dichloromethane (3 x 120 mL). The yellowish solution was concentrated in vacuo and the residue was stirred in methanol (50 mL) for 16 hours. The precipitated solid was collected by filtration and air-dried to give methyl 3-bromo-5-(3-bromo-5-(trifluoromethyl)benzoyl)benzoate (4) as a colourless solid. Yield: 6.52 g (70%).

[0971] 1 H NMR spectrum (300 MHz, CDC13, δH): 8.45 (t, J = 1.4 Hz, 1H); 8.29 (m, 1H); 8.12 (t, J = 1.6 Hz, 1H); 8.08 (bs, 1H); 8.04 (bs, 1H); 7.95 (bs, 1H); 3.97 (s, 3H).

[0972] A 100 mL reaction vessel was charged with 3-bromo-5-(3-bromo-5-(trifluoromethyl)benzoyl)benzoic acid methyl ester (4, 6.50 g, 13.9 mmol) and Deoxo-Fluor (13.0 mL). The vessel was sealed with a bubbler (filled with silicone oil), purged with nitrogen and heated to 90 °C (oil bath) for 16 h. The reaction mixture was cooled to ambient temperature and diluted with dichloromethane (100 mL). The resulting solution was slowly added to a 1 M aqueous potassium carbonate solution (100 mL) and the biphasic mixture stirred for 1 h to decompose excess fluorinating reagent. The layers were separated and the organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluents: cyclohexane / ethyl acetate 30:1 to 15:1) to give 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)difluoromethyl)benzoic acid methyl ester (5) as a yellowish oil. Yield: 6902 mg (99%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.30 (s, 1H); 8.08 (s, 1H); 7.88 (s, 1H); 7.83 (s, 1H); 7.81 (s, 1H); 7.71 (s, 1H); 3.96 (s, 3H).

[0973] 19 F NMR spectrum (282 MHz, CDC13, δF): -62.87 (s, 3H); -90.00 (s, 2H).

[0974] Potassium acetate (6.83 g, 69.7 mmol) was charged into a 500 mL reaction vessel and the salt was dried under vacuum at 110 °C for 1 hour. After cooling to room temperature, the reaction vessel was backfilled with nitrogen and charged with methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)difluoromethyl)benzoate (5, 6.90 g, 13.9 mmol), palladium acetate (62.0 mg, 279 mol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 265 mg, 557 mol) and bis(pinacolato)diboron (838 mg, 30.7 mmol). The reaction vessel was then evacuated and backfilled with nitrogen (this process was repeated twice). Anhydrous tetrahydrofuran (50 mL) was added using a syringe, sealed with a plastic stopper and immersed in a pre-heated heating bath at 60 °C. After stirring at 400 rpm for 16 hours, the reaction mixture was cooled to ambient temperature, diluted with dichloromethane (200 mL) and filtered through a short plug of silica gel (90 g) with a celite S topped head via dichloromethane (3 x 120 mL). The filtrate was concentrated under reduced pressure to give methyl 3-(difluoro(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)methyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (6) as a light brown foam. This was suspended in methanol (50 mL) and water (15 mL), added lithium hydroxide monohydrate (2.94 g, 70.0 mmol) and the resulting mixture stirred at room temperature for 16 hours. The reaction mixture was taken up in water (150 mL) and washed with dichloromethane (2 x 30 mL) and diethyl ether (30 mL). The aqueous layer was acidified to pH = 2 with concentrated aqueous hydrochloric acid and extracted with ethyl acetate (100 mL). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a yellowish foam. To the foam was added pinacol (472 mg, 4.00 mmol) and stirred in acetonitrile (50 mL) overnight. The precipitated solid was collected by filtration, washed with ice-cold acetonitrile (2 x 20 mL) and air-dried to give the title 3-(difluoro(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)methyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (7) as a colourless solid. Yield: 5.90 g (77%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.64 (s, 1H); 8.28 (s, 1H); 8.22 (s, 1H); 8.15 (s, 2H); 7.85 (s, 1H); 1.38 (s, 12H); 1.37 (s, 12H). LC-MS: 569.7 (M+H)+.

[0975] (3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)methyl)-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzoic acid (7, 5.11 g, 9.00 mmol) and bis(succinimidyl) carbonate (3.22 g, 12.6 mmol) were suspended in anhydrous acetonitrile (45 mL) and pyridine (1.00 mL, 12.6 mmol) under nitrogen. The reaction mixture was gently heated with a heat gun to achieve dissolution. After stirring for 16 h, the reaction mixture was concentrated in vacuo, the residue taken up in ethyl acetate (100 mL), and washed with 0.5 M aqueous potassium bicarbonate (2 x 40 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield an off-white solid. Pinacol (354 mg, 3.00 mmol) was added and the mixture was stirred in acetonitrile (50 mL) overnight. The precipitated solid was collected by filtration, washed with ice-cold acetonitrile (2 x 20 mL), and air-dried to yield the title 2,5-dioxopyrrolidin-l-yl 3-(difluoro(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)methyl)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzoate (8) as a colorless solid. Yield: 5.36 g (90%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.67 (s, 1H); 8.29 (s, 1H); 8.26 (s, 1H); 8.15 (s, 1H); 8.10 (s, 1H); 7.86 (s, 1H); 2.92 (s, 4H); 1.36 (s, 24H). LC-MS: 646.8 (M-HF)+.

[0976] Example 26: (S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborane Cyclopenten-6-carbamate)propionic acid

[0977]

[0978] A solution of 2,5-dioxopyrrolidin-l-yl l-hydroxy-4-(trifluoromethyl)-l,3- dihydrobenzo[c] [l,2]oxaborol-6-carboxylate (1, 14.4 g, 42.0 mmol), (S)-2,3- diaminopropanoic acid hydrochloride (2, 2.81 g, 20.0 mmol) and N,N- diisopropylethylamine (21.4 mL, 120 mmol) in N,N-dimethylformamide (400 mL) and water (100 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated and purified by column chromatography (silica gel, 0.063-0.200 mm; eluent: dichloromethane / methanol / formic acid 100:2:0.5 to 100:10:0.5). Fractions containing the desired product were evaporated and washed with 1 M aqueous potassium hydrogen sulfate solution (400 mL). The precipitate was filtered off, dissolved in a mixture of acetonitrile and water (2:1) and freeze-dried to give (S)-2,3-bis(l-hydroxy-4-(trifluoromethyl)-l,3-dihydrobenzo[c] [l,2]oxaborol-6- carboxamido)propanoic acid (3) as a white solid. Yield: 4.32 g (39%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.59 (bs, 1H); 9.62 (d, J = 6.1 Hz, 2H); 9.09 (d, J = 7.9 Hz, 1H); 8.98 (t, J = 5.7 Hz, 1H); 8.50 (d, J = 14.5 Hz, 2H); 8.24 (d, J = 21.6 Hz, 2H); 5.20 (d, J = 5.7 Hz, 4H); 4.87-4.58 (m, 1H); 4.02-3.80 (m, 1H); 3.79-3.54 (m, 1H). LC-MS: 561.6 (M+H)+.

[0979] Example 27: (3-((3-(pentafluoro-6-thio)-5-(4,4,5,5-tetramethyl-1,3,2-dioxoborheptacyclopentane-) 2-yl)phenyl)sulfonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoyl)glycine

[0980]

[0981] A mixture of (3-bromo-5-((3-bromo-5-(pentafluoro-6-sulfido)phenyl)sulfonyl)benzoyl)glycine tert-butyl ester (1, 8.00 g, 12.1 mmol), palladium acetate (137 mg, 0.61 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 577 mg, 1.21 mol), bis(pinacolato)diboron (6.78 g, 26.7 mmol) and potassium acetate (5.95 g, 60.7 mmol) in dry tetrahydrofuran (450 mL) was heated at 60 °C under an argon atmosphere for 24 h. The mixture was cooled to room temperature and filtered through a short plug of celite. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluents: dichloromethane / ethyl acetate 10:0 to 6:4) to give (3-((3-(pentafluoro-6-sulfido)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)glycine tert-butyl ester (2) as an off-white foam. Yield: 6.70 g (72%).

[0982] 1 H NMR spectrum (300 MHz, CDC13, δH): 8.53-8.49 (m, 2H); 8.49-8.46 (m, 1H); 8.43 (t, J = 1.9 Hz, 1H); 8.39-8.36 (m, 1H); 8.32 (dd, J = 2.1 and 0.6 Hz, 1H); 6.76 (t, J = 5.0 Hz, 1H); 4.16 (d, J = 5.0 Hz, 2H); 1.51 (s, 9H); 1.36 (s, 24H). LC-MS: 754.9 (M+H)+.

[0983] A solution of (3-((3-(pentafluoro-6-sulfido)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)glycine tert-butyl ester (2, 6.68 g, 8.87 mmol) in dichloromethane (100 mL) was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The residue was evaporated ten times from dichloromethane (250 mL) before drying under vacuum. (3-((3-(pentafluoro-6-sulfido)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)glycine (3) was obtained as an off-white solid. Yield: 6.15 g (99%).1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.33 (t, J = 5.8 Hz, 1H); 8.65 (t, J = 1.8 Hz, 1H); 8.55 (t, J = 1.9 Hz, 1H); 8.47 (s, 1H); 8.41-8.29 (m, 2H); 8.25-8.16 (m, 1H); 3.96 (d, J = 5.9 Hz, 2H); 1.41-1.24 (m, 24H). LC-MS: 534.4 (M-2xpin+H)+.

[0984] Example 28: N-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborone-6- carbonyl)-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborone-cyclopentene-6-carboxamide ethyl glycine

[0985]

[0986] Bromine (1.0 mL, 19.7 mmol) was added to a solution of 3,5-dimethyl-4- methylbenzoic acid (1, 2.0 g, 11.9 mmol) in concentrated sulfuric acid (20 mL) and the reaction mixture was stirred at ambient temperature for 16 hours. The reaction mixture was then poured into ice water (200 mL). The resulting precipitate was filtered off, washed with water (100 mL) and dissolved in ethyl acetate (100 mL); dried over anhydrous sodium sulfate, filtered and evaporated to give 3-bromo-5-methyl-4-methylbenzoic acid (2) as a white solid. Yield: 2.8 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 13.71 (bs, 1H); 8.35 (d, J = 0.4 Hz, 1H); 8.15 (d, J = 0.9 Hz, 1H); 2.56 (s, 3H).

[0987] Concentrated sulfuric acid (24 mL) was added to a solution of 3-bromo-4-methyl-5- trifluoromethylbenzoic acid (2, 35.0 g, 124 mmol) in methanol (500 mL) and the reaction mixture was stirred at reflux for 4 hours and at ambient temperature for 16 hours. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (250 mL) and washed with a mixture of water (2 x 100 mL) and potassium carbonate saturated solution (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate (3) as a white solid. Yield: 35.3 g (96%). 1H NMR spectrum (300 MHz, DMSO-d6, δΗ): 8.36 (d, J = 1.1 Hz, 1H); 8.13 (d, J = 1.1 Hz, 1H); 3.90 (s, 3H); 2.55 (d, J = 1.3 Hz, 3H).

[0988] A suspension of 1-bromopyrrolidine-2,5-dione (NBS, 31.7 g, 178 mmol) and methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate (3, 35.3 g, 119 mmol) in water (300 mL) was stirred at 80 °C under a 100 W bulb for 6 hours. The reaction mixture was extracted with diethyl ether (2 x 200 mL). The organic layer was washed with brine (150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate (4) as a yellow solid. Yield: 44.0 g (98%). 1 H NMR spectrum (300 MHz, CDCl3, δΗ): 8.47 (d, J = 1.5 Hz, 1H); 8.31 (d, J = 1.3 Hz, 1H); 4.75 (s, 2H); 3.98 (s, 3H).

[0989] A solution of methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate (4, 44.0 g, 117 mmol) and potassium acetate (22.9 g, 234 mmol) in acetonitrile (0.5 L) was stirred at 75 °C overnight. The suspension was filtered through filter paper and evaporated. The crude product was dissolved in dichloromethane and filtered again. Evaporation gave methyl 3-bromo-4-(acetyloxymethyl)-5-(trifluoromethyl)benzoate (5) as a white solid. Yield: 37.9 g (91%). 1 H NMR spectrum (300 MHz, CDCl3, δΗ): 8.49 (d, J = 1.3 Hz, 1H); 8.34 (d, J = 1.3 Hz, 1H); 5.37 (s, 2H); 3.99 (s, 3H); 2.11 (s, 3H).

[0990] A solution of methyl 3-bromo-4-(acetyloxymethyl)-5-(trifluoromethyl)benzoate (5, 37.9 g, 107 mmol), bis(pinacolato)diboron (29.8 g, 117 mmol), potassium acetate (31.4 g, 294 mmol) and [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(ll) (1.57 g, 1.92 mmol) in dry tetrahydrofuran (500 mL) was stirred at 75 °C under an argon atmosphere for 13 days. The reaction mixture was then cooled to ambient temperature, filtered and evaporated. The crude product was filtered through a column of silica gel (silica gel, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 8:1) to give methyl 4-(acetyloxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)benzoate (6). Yield: 31.1 g (72%). Rf(SiO2, cyclohexane / ethyl acetate 8:1): 0.40. 1 H NMR spectrum (300 MHz, CDC13, δH): 8.65 (s, 1H); 8.43 (s, 1H); 5.48 (s, 2H); 3.97 (s, 3H); 2.05 (s, 3H); 1.36 (s, 12H).

[0991] A solution of methyl 4-(acetyloxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)benzoate (6, 31.0 g, 77.1 mmol) and sodium hydroxide (15.4 g, 386 mmol) in water (300 mL) was stirred at ambient temperature for 3 hours. A solution of hydrochloric acid (35 mL) in water (100 mL) was then added to reduce the pH to 1. The reaction mixture was stirred overnight. The precipitate was filtered and dried to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (7) as a white solid. Yield: 16.6 g (86%).

[0992] 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.47 (bs, 1H); 9.66 (s, 1H); 8.62 (s, 1H); 8.24 (s, 1H); 5.22 (s, 2H).

[0993] A solution of pentafluorophenol (7.48 g, 40.7 mmol), 1-hydroxy-4-(trifluoromethyl)- 1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (7, 10.0 mg, 40.7 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 8.37 mg, 40.7 mmol) in acetonitrile (0.5 L) was stirred at ambient temperature overnight. The reaction mixture was filtered, evaporated, dissolved in acetonitrile, filtered again and evaporated to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (8) as a white solid.

[0994] Yield: 16.7 g (100%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[0995] A solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (8, 16.7 g, 40.6 mmol), (2-aminoethyl)glycine (9, 2.40 g, 20.3 mmol) and triethylamine (28.4 mL, 203 mmol) in N,N-dimethylformamide (0.5 L) was stirred at ambient temperature for 3 days. The reaction mixture was then evaporated and the crude product 10 was purified by column chromatography (silica gel, eluent: dichloromethane / methanol / formic acid 100:2:0.5 to 100:10:0.5) to give N-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6- carbonyl)-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6- carboxamido)ethyl)glycine (10) as a white solid. Yield: 7.77 g (67%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.89 (bs, 1H); 9.68-9.48 (m, 2H); 9.00-8.67 (m, 1H); 8.56-7.36 (m, 4H); 5.27-5.03 (m, 4H); 4.30-3.95 (m, 2H); 3.77-3.48 (m, 4H). LC-MS: 575.5 (M+H)+.

[0996] Example 29: (2S)-3-(2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxa) Borolanyl-6-carboxamido)propionamido)propionic acid = N-[N α ,N β - Bis-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydro Benz[c][1,2]oxaborane-6-carbamate)-L-diaminopropionyl]-β-alanine

[0997]

[0998] A solution of L-diaminopropionic acid hydrochloride (alias (2S)-2,3- diaminopropionic acid hydrochloride) (1, 15.0 g, 107 mmol), di-tert-butyl dicarbonate (46.6 g, 214 mmol) and potassium bicarbonate (32.0 g, 320 mmol) in a mixture of acetonitrile (400 mL) and water (400 mL) was stirred overnight. The solvent was removed under reduced pressure and the residue was acidified with saturated aqueous potassium hydrogen sulphate until pH 1 was reached. The reaction mixture was extracted with ethyl acetate (3 x 200 mL) and dried over anhydrous sodium sulphate. The solvent was removed under reduced pressure to give (2S)-2,3-bis((tert-butoxycarbonyl)amino)propanoic acid (2) as an off-white solid. Yield: 28.2 g (87%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.01 (bs, 1H); 5.75 (bs, 1H); 5.14 (bs, 1H); 4.15 (bs, 1H); 3.57-3.39 (m, 4H); 2.43 (t, J = 6.0 Hz, 2H); 1.45 (s, 27H).

[0999] A solution of (2S)-2,3-bis((tert-butoxycarbonyl)amino)propanoic acid (2, 27.9 g, 91.7 mmol), tert-butyl 3-aminopropanoate (3, 16.7 g, 91.7 mmol), N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl, 21.1 g, 110 mmol), 1-hydroxy-7-azabenzotriazole (HOAt, 15.0 g, 110 mmol) and N,N- diisopropylethylamine (64.0 mL, 367 mmol) in dichloromethane (300 mL) was stirred overnight. The solvent was removed under reduced pressure; the residue was dissolved in ethyl acetate (600 mL), washed with 1 M aqueous hydrochloric acid (4 x 300 mL) and saturated aqueous sodium bicarbonate (4 x 300 mL) and dried over anhydrous sodium sulphate. The solvent was removed under reduced pressure to give tert-butyl (S)-3-(2,3-bis((tert-butoxycarbonyl)amino)propanamido)propanoate (4) as an off-white solid. Yield: 36.1 g (91%).

[1000] 1 H NMR spectrum (300 MHz, CDC13, δH): 7.01 (bs, 1H); 5.75 (bs, 1H); 5.14 (bs, 1H); 4.15 (bs, 1H); 3.57-3.39 (m, 4H); 2.43 (t, J = 6.0 Hz, 2H); 1.45 (s, 27H).

[1001] To a solution of (S)-tert-butyl 3-(2,3-bis((tert-butoxycarbonyl)amino) propanamido)propanoate (4, 36.1 g, 83.7 mmol) in dichloromethane (50 mL) was added 95% aqueous trifluoroacetic acid (300 mL) and the solution was stirred for 3 hours. The solvent was removed under reduced pressure, the residue was co-evaporated with acetonitrile (3 x 300 mL) and treated with 1 M hydrogen chloride in dry diethyl ether (300 mL). The precipitate was filtered off and triturated with acetonitrile (2 x 600 mL) to give (2S)-3-(2,3-diaminopropanamido)propanoic acid dihydrochloride (5) as a white powder.

[1002] Yield: 22.2 g (100%). 1 H NMR spectrum (300 MHz, D2O, δH): 4.35 (t, J = 5.8 Hz, 1H); 3.63-3.46 (m, 4H); 2.67 (t, J = 6.6 Hz, 2H).

[1003] A solution of pentafluorophenol (35.1 g, 191 mmol), 1-hydroxy-4-(trifluoromethyl)- 1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (1, 40.8 g, 166 mmol, prepared as described in Example 28) and N,N'-dicyclohexylcarbodiimide (DCC, 39.3 g, 191 mmol) in acetonitrile (1 L) was stirred at ambient temperature for 24 hours. The reaction mixture was filtered, evaporated, dissolved in acetonitrile, re-filtered and evaporated. The crude product was precipitated in dichloromethane (1 L) and filtered to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (6) as a white solid. Yield: 52.8 g (77%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[1004] To a solution of (2S)-3-(2,3-diaminopropionamido)propanoic acid dihydrochloride (5, 6.41 g, 24.3 mmol) and triethylamine (33.8 mmol, 243 mmol) in water (50 mL) was added a solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (6, 20.0 g, 48.6 mmol) in 1,4-dioxane (100 mL) and the solution was stirred overnight. The reaction mixture was partitioned between ethyl acetate (300 mL) and 1 M aqueous potassium hydrogen sulfate (1500 mL). The organic layer was washed with 1 M aqueous potassium hydrogen sulfate (1 x 300 mL) and the solvent was removed under reduced pressure. The residue was triturated with diethyl ether (2 x 150 mL) and filtered. The solid was dissolved in 70% aqueous acetonitrile (600 mL) and freeze-dried to give 3-(2(S),3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6- carboxamido)propionamido)propanoic acid (7) as a white powder. Yield: 12.1 g (80%).

[1005] 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 8.51 (s, 1H); 8.47 (s, 1H); 8.29 (s, 1H); 8.27 (s, 1H); 5.28 (s, 4H); 5.15 (t, J = 6.1 Hz, 1H); 4.15-3.99 (m, 2H); 3.61 (t, J = 6.4 Hz, 2H); 2.67 (t, J = 6.3 Hz, 2H). LC-MS: 632.0 (M+H)+.

[1006] Example 30: (S)-3-(2,3-bis(4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaboranecyclopentane) olefin-6-carbamate)propionamide)propionic acid

[1007]

[1008] (1, 8.56 g, 43.7 mmol), N-hydroxysuccinimide (5.03 g, 43.7 mmol) and l-ethyl-3-(3'- dimethylaminopropyl)carbodiimide hydrochloride (8.38 g, 43.7 mmol) in tetrahydrofuran (250 mL) and N,N-dimethylformamide (20 mL) was stirred at ambient temperature for 3.5 hours. The reaction mixture was evaporated and extracted with ethyl acetate (3 x 150 mL) and 1 M aqueous hydrochloric acid (150 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give 2,5-dioxopyrrolidin-l-yl 4-fluoro-l-hydroxy-l,3- dihydrobenzo[c] [l,2]oxaborol-6-carboxylate (2) as a white solid. Yield: 10.2 g (79%). LC-MS: 294.3 (M+H)+.

[1009] Resin 100-200 mesh 1.5 mmol / g (3, 10.5 g, 15.7 mmol) was swelled in dry dichloromethane (80 mL) for 30 min. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Ala-OH, 3.26 g, 10.5 mmol) and N,N-diisopropylethylamine (6.93 mL, 39.8 mmol) in dry dichloromethane (50 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (3.65 mL, 20.9 mmol) in a methanol / dichloromethane mixture (4:1, 2 x 5 min, 2 x 80 mL). The resin was then washed with N,N-dimethylformamide (2 x 80 mL), dichloromethane (2 x 80 mL) and N,N-dimethylformamide (3 x 80 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 80 mL). The resin was washed with N,N-dimethylformamide (3 x 80 mL), 2-propanol (2 x 80 mL) and dichloromethane (3 x 80 mL). A solution of (S)-2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Dap(Fmoc)-OH, 8.61 g, 15.7 mmol), 5-chloro-1- ((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 5.58 g, 15.7 mmol) and N,N-diisopropylethylamine (4.92 mL, 28.2 mmol) in N,N-dimethylformamide (80 mL) was added to the resin and the mixture was shaken for 2 h. The resin was filtered and washed with N,N-dimethylformamide (2 x 80 mL), dichloromethane (2 x 80 mL) and N,N-dimethylformamide (2 x 80 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 30 min, 2 x 80 mL). The resin was washed with N,N-dimethylformamide (3 x 80 mL), 2-propanol (2 x 80 mL) and dichloromethane (3 x 80 mL). A solution of 2,5-dioxopyrrolidin-1-yl 1-hydroxy-4- (trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylate (2, 9.14 g, 31.4 mmol) and N,N-diisopropylethylamine (9.84 mL, 56.5 mmol) in N,N-dimethylformamide (80 mL) was added to the resin and the mixture was shaken for 1 day. The resin was filtered and washed with N,N-dimethylformamide (4 x 80 mL) and dichloromethane (10 x 80 mL).The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (80 mL) for 16 hours. The resin was filtered off and washed with dichloromethane (4 x 80 mL). The solvent was evaporated and the crude product (4) was washed with ethyl acetate (300 mL), filtered and dried under vacuum. The pure product (4) was obtained as an off-white solid. Yield: 4.10 g (74%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.57 (bs, 2H); 8.78-8.49 (m, 2H); 8.19-7.93 (m, 3H); 7.71 (dd, J = 30.8 and 10.8 Hz, 2H); 5.12 (d, J = 7.7 Hz, 4H); 4.74-4.55 (m, 1H); 3.72-3.61 (m, 2H); 3.29-3.15 (m, 2H); 2.36 (t, J = 6.9 Hz, 2H). LC-MS: 532.6 (M+H)+.

[1010] Example 31: 4-((3R,4R)-3,4-bis(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaboron heterocycle Penten-6-carbamoyl)pyrrolidine-1-yl)-4-oxobutyric acid

[1011]

[1012] A solution of 4-((3R,4R)-3,4-diaminopyrrolidin-1-yl)-4-oxobutanoic acid dihydrochloride (2, 2.46 g, 12.2 mmol), 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (1, 8.86 g, 24.5 mmol) and triethylamine (17.0 mL, 122 mmol) in N,N-dimethylformamide (300 mL) was stirred at ambient temperature overnight. The reaction mixture was evaporated and precipitated from ethyl acetate to give 6.40 g of crude compound 3 (6.4 g) which was purified by HPLC (YMC, C18, 5 m, 250 x 50 mm, acetonitrile / water, 2:98 in 30 min, 180 min 2:98 to 30:0) and freeze-dried to give the title compound 4-((3R,4R)-3,4-bis(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid (3) as a white solid. Yield: 1.23 g (18%).

[1013] 1H NMR spectrum (300 MHz, DMSO-d6, δΗ) 9.35 (bs, 2H); 8.68 (t, J = 8.4 Hz, 2H); 7.81-7.62 (m, 2H); 7.30 (d, J = 7.3 Hz, 2H); 5.02 (s, 4H); 4.66-4.45 (m, 2H); 3.94 (dd, J = 10.6 and 6.7 Hz, 1H); 3.77 (dd, J = 12.0 and 6.7 Hz, 1H); 3.54-3.41 (m, 1H); 3.27-3.18 (m, 1H); 2.47-2.34 (m, 4H). LC-MS: 558.6 (M+H)+.

[1014] Example 32: N-(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborane-6-carbonyl)-N- (2-(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborone-6-carboxamido)ethyl)glycine

[1015]

[1016] n-Butyllithium (2.38 M in hexanes, 107 mL, 255 mmol) was cannulated into a stirred and nitrogen purged solution of 2,2,6,6-tetramethylpiperidine (43.5 mL, 257 mmol) in anhydrous tetrahydrofuran (150 mL) at such a rate to maintain an internal temperature below -60 °C (ca. 20 min). The mixture was stirred for 60 min (internal temperature increased to -40 °C). The mixture was re-cooled to -78 °C and a solution of 2-fluoro-4-methylbenzonitrile (1, 30.0 g, 222 mmol) in anhydrous tetrahydrofuran (200 mL) was added dropwise to the vigorously stirred mixture at such a rate to maintain an internal temperature below -70 °C (ca. 40 min). The mixture was allowed to warm to -50 °C and maintained at this temperature for 45 min. The mixture was re-cooled to -78 °C and a solution of iodine (62.0 g, 244 mmol) in anhydrous tetrahydrofuran (150 mL) was added dropwise to the reaction mixture (using a peristaltic pump) while maintaining an internal temperature below -70 °C. The residual iodine was washed with anhydrous tetrahydrofuran (50 mL) and the mixture was stirred at -70 °C for 1 h. The stirred mixture was allowed to warm to room temperature overnight, then quenched by pouring into a stirred solution of sodium thiosulfate (20 g) in water (750 mL). The reaction mixture was stirred for 1 h, then extracted with ethyl acetate (3 x 300 mL). The combined organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluents: cyclohexane / ethyl acetate 10:1) then recrystallized from methanol to give 2-fluoro-3-iodo-4-methylbenzonitrile (2) as a colourless crystalline solid.

[1017] Yield: 29.6 g (51%). Rf (SiO2, cyclohexane / ethyl acetate 10:1): 0.35. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.48 (dd, J = 7.9 and 6.5 Hz, 1H); 7.17-7.12 (m, 1H), 2.56 (s, 3H).

[1018] 19 F NMR spectrum (282 MHz, CDC13, δF): -82.34 (s).

[1019] A slurry of 2-fluoro-3-iodo-4-methylbenzonitrile (2, 52.7 g, 202 mmol) in 75% sulfuric acid (65 mL) was stirred at 150 °C for 3 hours. After cooling to ambient temperature, the mixture was poured onto an ice / water mixture (500 g). The precipitated beige solid was filtered off, washed with copious amounts of water and dried to yield 2-fluoro-3-iodo-4-methylbenzoic acid (3) as a beige solid. Yield: 51.2 g (91%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.30 (s, 1H); 7.75 (t, J = 7.8 Hz, 1H); 7.27 (d, J = 8.0 Hz, 1H); 2.47 (s, 3H).

[1020] Acetyl chloride (23.0 mL, 321 mmol) was added dropwise to a stirred suspension of 2-fluoro-3-iodo-4-methylbenzoic acid (3, 90.0 g, 321 mmol) in dry methanol (350 mL) at 0 °C. The mixture was refluxed overnight. The volatiles were removed under reduced pressure and the residue was taken up in ethyl acetate (1300 mL). After washing with saturated aqueous potassium bicarbonate solution (2 x 1000 mL) and brine (1000 mL), the organic layer was dried over anhydrous magnesium sulfate and evaporated in vacuo. The residue was purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 30:1-15:1) to yield methyl 2-fluoro-3-iodo-4-methylbenzoate (4) as a colorless solid.

[1021] Yield: 67.6 g (72%). Rf (SiO2, cyclohexane / ethyl acetate 15:1): 0.40. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.80 (t, J = 7.7 Hz, 1H); 7.10 (d, J = 8.0 Hz, 1H); 3.93 (s, 3H); 2.52 (s, 3H).

[1022] A solution of 2-fluoro-3-iodo-4-methylbenzoate (4, 35.0 g, 119 mmol), bis(pinacolato)diboron (5, 33.3 g, 131 mmol), anhydrous potassium acetate (35.0 g, 357 mmol) and [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (1.94 g, 2.38 mmol) in anhydrous dimethyl sulfoxide (500 mL) was stirred under argon atmosphere at 1 10 °C for one weekend. The reaction mixture was cooled to ambient temperature, the solvent was evaporated in vacuo and the crude product 6 was extracted with ethyl acetate (4 x 500 mL) and water (1.0 L). The organic layers were combined, filtered through a pad of celite, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (silica gel 60, 0.063-0.200 mm; eluents: cyclohexane / ethyl acetate 9:1 ) to give methyl 2-fluoro-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (6) as a colourless solid. Yield: 29.4 g (84%). RF(SiO2, cyclohexane / ethyl acetate 9:1 ): 0.30. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.84 (t, J = 8.0 Hz, 1 H); 7.00 (d, J = 8.1 Hz, 1 H); 3.90 (s, 3H); 2.47 (s, 3H); 1.39 (s, 12H).

[1023] A solution of methyl 2-fluoro-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoate (6, 27.5 g, 93.5 mmol), 1 -bromopyrrolidine-2,5-dione (NBS, 18.3 g, 103 mmol) and 2,2-azobis(2-methylpropionitrile) (AIBN, 0.77 g, 4.68 mmol) in trifluorotoluene (300 mL) was stirred at 85 °C for 16 hours. The solvent was evaporated in vacuo and the residue was extracted with diethyl ether (2 x 150 mL). The organic layer was washed with water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give methyl 4-(bromomethyl)-2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (7) as a yellow solid. Yield: 33.5 g (96%).

[1024] 1 H NMR spectrum (300 MHz, CDC13, δH): 7.93 (t, J = 7.8 Hz, 1 H); 7.21 (d, J = 8.1 Hz, 1 H); 4.71 (s, 2H); 3.91 (s, 3H); 1.42 (s, 12H).

[1025] A solution of methyl 4-(bromomethyl)-2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoate (7, 33.5 g, 89.8 mmol) and potassium acetate (17.6 g, 180 mmol) in acetonitrile (1 L) was stirred at 75 °C overnight. The suspension was filtered through cotton wool and evaporated. The crude product was dissolved in dichloromethane and filtered again. The solvent was evaporated to give methyl 4-(acetyloxymethyl)-2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (8) as a beige solid. Yield: 30.0 g (95%). 1 H NMR spectrum (300 MHz, CDC13, δH): 7.96 (t, J = 7.8 Hz, 1H); 7.24 (d, J = 7.9 Hz, 1H); 5.25 (s, 2H); 3.92 (s, 3H); 2.11 (s, 3H); 1.39 (s, 12H).

[1026] A solution of methyl 4-(acetyloxymethyl)-2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoate (8, 30.0 g, 85.2 mmol) and sodium hydroxide (17.0 g, 426 mmol) in water (250 mL) was stirred at ambient temperature for 3 hours. After this time, a solution of hydrochloric acid (35% w / w, 45 mL) in water (50 mL) was added to lower the pH to 1. The reaction mixture was stirred for 16 hours. The resulting precipitate was filtered and freeze-dried to give 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (9) as an off-white solid. Yield: 9.76 g (58%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.17 (bs, 1H); 9.38 (bs, 1H); 8.29 (d, J = 7.7 Hz, 1H); 7.36 (d, J = 11.2 Hz, 1H); 5.02 (s, 2H). LC-MS: 197.3 (M+H)+.

[1027] A solution of 2,3,4,5,6-pentafluorophenol (9.61 g, 52.2 mmol), 7-fluoro-l- hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid (9, 10.2 g, 52.2 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 10.8 g, 52.2 mmol) in acetonitrile (300 mL) and dichloromethane (200 mL) was stirred at ambient temperature for one weekend. The reaction mixture was filtered and evaporated in vacuo. The residue was dissolved in acetonitrile, filtered and evaporated in vacuo again to give 7-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (10) as a beige solid. Yield: 18.8 g (100%).

[1028] 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.55 (bs, 1H); 8.32-8.20 (m, 1H); 7.51 (d, J = 8.1 Hz, 1H); 5.13 (s, 2H).

[1029] A solution of 7-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (10, 9.46 g, 26.1 mmol), (2-aminoethyl)glycine (11, 1.54 g, 13.1 mmol) and triethylamine (14.5 mL, 105 mmol) in N,N-dimethylformamide (200 mL) was stirred at ambient temperature overnight (16 hours). The reaction mixture was evaporated and an attempt was made to dissolve it in dichloromethane for TLC. The crude product was found to be insoluble in dichloromethane, ethyl acetate and acetonitrile. It was therefore precipitated from ethyl acetate (0.5 L) and the solid was collected by centrifugation. The first precipitate (A) was washed with 0.5 M hydrochloride salt solution (2 x 50 mL) to give a second precipitate (B) which was filtered off and retained. The filtrate was freeze-dried to give product 12 contaminated with salt. This was removed by dissolution in tetrahydrofuran and filtration. The remaining solution was evaporated in vacuo to give the first batch of product 12. Precipitate (B) was dissolved in acetonitrile and water (3: 1), filtered and the remaining solution freeze-dried. The resulting solid was dissolved in tetrahydrofuran, the precipitated salt was filtered off and the filtrate was evaporated in vacuo to give a second portion of N-(7-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6- carbonyl)-N-(2-(7-fluoro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxamido)ethyl)glycine (12) as a beige solid. Yield: 2.09 g (34%). 1HNMR spectrum (300 MHz, DMSO-d6, δΗ): 9.41-9.33 (m, 2H); 8.42-8.18 (m, 1H); 7.81-7.64 (m, 1H); 7.43-7.11 (m, 3H); 5.07-4.97 (m, 4H); 4.22 (s, 1H); 3.98 (s, 1H); 3.68 (t, J = 6.5 Hz, 1H); 3.60-3.40 (m, 3H). LC-MS: 475.5 (M+H)+.

[1030] Example 33: 2,5-dioxopyrrolidin-1-yl 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoate boran-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 sulfinyl)amino)acetate

[1031]

[1032] tert-Butyl 2-((oxobis(3-(trifluoromethyl)phenyl)-lambda 6 -ylidene)sulfanyl)acetate (1, 2.05 g, 4.38 mmol), bis(pinacolato)diboron (2.78 g, 11.0 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (87.0 mg, 0.13 mmol) and 4,4-ditert-butyl-2,2-dipyridyl (dtbpy, 82.0 mg, 0.31 mmol) were dissolved in degassed tetrahydrofuran (12 mL). The resulting mixture was warmed to 60 °C and heated at this temperature overnight. The mixture was evaporated to dryness; the residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluent: dichloromethane / ethyl acetate 10:0 to 4:1) to give tert-butyl 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 6 -ylidene)sulfanyl)acetate (2) as an off-white foam.

[1033] Yield: 2.92 g (93%). 1 H NMR spectrum (300 MHz, CDCl3, δΗ): 8.59 (s, 2H); 8.42 (s, 2H); 8.21 (s, 2H); 3.76 (s, 2H); 1.51 (s, 9H); 1.36 (s, 12H); 1.35 (s, 12H).

[1034] 19 F NMR spectrum (282 MHz, CDCl3, δF): -62.55 (s). LC-MS: 556.6 (M-2xpinacol + H)+, 638.8 (M-pinacol + H)+, 721.0 (M+H)+.

[1035] Trifluoroacetic acid (24 mL) was added to a solution of tert-butyl 2-((oxo bis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)- lambda 6 - sulfidyl)amino)acetate (2, 2.91 g, 4.05 mmol) in dichloromethane (8 mL) and the mixture was stirred at room temperature for 2 hours. The mixture was evaporated to dryness in vacuo and the residue was evaporated from toluene (3 x 20 mL) and dichloromethane (3 x 20 mL). The residue was partitioned between dichloromethane (200 mL) and 0.5 M aqueous sodium hydroxide (250 mL). The separated aqueous phase was washed with dichloromethane (2 x 100 mL), acidified with 1 M hydrochloric acid (200 mL) and extracted with ethyl acetate (3 x 250 mL). The combined ethyl acetate extracts were dried over anhydrous sodium sulfate and evaporated in vacuo to give 2-((oxo bis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 6 - sulfidyl)amino)acetic acid (3). Yield: 2.30 g (86%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.55 (s, 2H); 8.33 (s, 2H); 8.29 (s, 2H); 3.85 (s, 2H); 1.39 (s, 24H). 19 F NMR spectrum (282 MHz, CDC13, δF): -62.69 (s). LC-MS: 500.5 (M-2x pinacol + H)+, 582.6 (M-pinacol + H)+, 664.8 (M + H)+.

[1036] Anhydrous acetonitrile (16.2 mL) was added to 2-((oxo bis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)- lambda 6- Sulfhydryl)amino)acetic acid (3, 2.15 g, 3.24 mmol) and N,N-disuccinimidyl carbonate (DSC, 1.25 g, 4.86 mmol). Add pyridine (392 mL, 4.86 mmol) and sonicate the mixture to form a fine suspension. Stir the resulting suspension for 4 hours to give a clear solution. Add an additional amount of N,N-disuccinimidyl carbonate (DSC, 415 mg, 1.62 mmol) and pyridine (131 mL, 1.62 mmol) and stir the mixture at room temperature overnight. LC / MS analysis shows complete conversion to the activated ester. Evaporate the mixture to dryness and partition the residue between ethyl acetate (200 mL) and 0.1 M aqueous hydrochloric acid (100 mL). Separate the phases, wash the organic phase with 0.1 M aqueous hydrochloric acid (2 x 50 mL) and brine (50 mL), dry over anhydrous sodium sulfate and evaporate to dryness. Dissolve the residue in dichloromethane (40 mL) followed by the addition of pinacol (383 mg, 3.24 mmol). Evaporate the solution and evaporate the residue from dichloromethane (3 x 40 mL). Wash the resulting foam with cyclohexane (2 x 50 mL), redissolve in dichloromethane (40 mL), evaporate and dry under vacuum to give the title compound (4) as an off-white foam.

[1037] Yield: 1.82 g (74%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.57 (s, 2H); 8.37 (s, 2H); 8.24 (s, 2H); 4.20 (s, 2H); 2.83 (s, 4H); 1.36 (s, 24H). 19 F NMR spectrum (282 MHz, CDC13, δF): -62.66 (s). LC-MS: 761.9 (M+H)+.

[1038] Example 34: 2,5-dioxopyrrolidin-1-yl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)-5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoate Example 35: N-(1-hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6- carbonyl)-N-(2-(1-hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6- carboxamido)ethyl)glycine Example 36: N-(4-chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonyl)-N- (2-(4-chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)ethyl)glycine

[1039]

[1040] Methyl 3-iodobenzoate (2, 10.5 g, 40.0 mmol), anhydrous potassium carbonate (11.0 g, 80.0 mmol), copper iodide (1.52 g, 8.00 mmol) and 3-trifluoromethylbenzenethiol (1, 8.22 mL, 60.0 mmol) were suspended in anhydrous 1,2-dimethoxyethane (100 mL) and the resulting suspension was stirred at 80 °C for 48 h. After cooling to ambient temperature, the reaction mixture was diluted with cyclohexane (300 mL), filtered through a pad of silica gel (125 g) with celite on top (washed with ethyl acetate / cyclohexane 1:10, 3 x 200 mL) and evaporated in vacuo. The residue was dissolved in acetic acid (120 mL) and 30% aqueous hydrogen peroxide solution (16.0 mL, 156 mmol) was added portionwise (exothermic). After stirring at 80 °C (oil bath) for 16 h, the reaction mixture was evaporated in vacuo, taken up in ethyl acetate (400 mL) and washed with water (400 mL) and brine (400 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo to give methyl ester 4 as a yellow oil which was subjected to flash column chromatography (silica gel 300, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 4:1) to give methyl 3-((3-(trifluoromethyl)phenyl)sulfonyl)benzoate (4) as a colourless oil. Yield: 5.40 g (39%). LC-MS: 346.0 (M+H)+.

[1041] Methyl 3-((3-(trifluoromethyl)phenyl)sulfonyl)benzoate (4, 5.40 g, 15.7 mmol), bis(pinacolato)diboron (9.97 g, 39.0 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (310 mg, 0.47 mmol) and 4,4-ditert-butyl-2,2-dipyridyl (dtbpy, 295 mg, 1.10 mmol) were dissolved in anhydrous, degassed tetrahydrofuran (30 mL) under nitrogen. The reaction mixture was stirred at 50 °C (oil bath) for 16 h. After cooling to ambient temperature, ice-cold water (30 mL) was added slowly to decompose the generated pinacol borane (hydrogen gas evolved). After 30 min, lithium hydroxide monohydrate (6.59 g, 157 mmol) was added and the resulting mixture was stirred at ambient temperature for three hours, then it was taken up in water (300 mL) and extracted with dichloromethane (3 x 60 mL). The dichloromethane extracts were discarded and the aqueous layer was acidified to pH 2 with concentrated hydrochloric acid. The aqueous layer was extracted with ethyl acetate (50 mL) and discarded. The organic layer was washed with brine (3 x 50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting yellowish foam was treated with pinacol (118 mg, 1.00 mmol) and dissolved in warm acetonitrile (20 mL). The solution was left in the freezer to crystallize overnight. The precipitated product was collected by filtration, washed with cold acetonitrile and air-dried to give 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (5) as a colorless solid. Yield: 5.90 g (65%). LC-MS: 582.6 (M+H)+.

[1042] Dissolve 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (5, 5.90 g, 10.1 mmol) and bis(succinimidyl)carbonate (3.63 g, 14.2 mmol) in anhydrous acetonitrile (45 mL) and pyridine (1.14 mL, 14.2 mmol) under nitrogen. Heat the reaction mixture to achieve dissolution. After stirring for 16 h, concentrate the reaction mixture in vacuo, take up the residue in ethyl acetate (200 mL), and wash with brine (3 x 200 mL). Dry the organic layer over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to give an off-white solid. Add pinacol (473 mg, 4.00 mmol) and stir the mixture in acetonitrile (30 mL) for 1 h. Evaporate the acetonitrile in vacuo. Dissolve the resulting white foam in hexanes (30 mL) and crystallize the solution at ambient temperature overnight to give 2,5-dioxopyrrolidin-1-yl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoate (6) as a white solid. Yield: 6.50 g (94%).

[1043] 1 H NMR spectrum (300 MHz, CDC13, δH): 8.76-8.72 (m, 2H); 8.67 (s, 1 H); 8.55 (s, 1 H); 8.32 (s, 1 H); 8.27 (s, 1 H); 2.92 (s, 4H); 1.37 (s, 12H) overlapping with 1.37 (s, 12H).

[1044] 19 F NMR spectrum (300 MHz, CDC13, δF): 62.64 (s, 3H). LC-MS: 680.6 (M-H)+.

[1045] Example 37: (S)-4-((2S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2] oxaborol-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid Example 38: N-(4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6- carbonyl)-N-(2-(4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6- carboxamido)ethyl)glycine Example 39: 1-(tert-butyl)-5-(2,5-dioxopyrrolidin-1-yl)-(2-((oxobis(3-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)sulfonyl)benzoate

[1046]

[1047] A mixture of 4-methyl-2-(trifluoromethyl)benzoic acid (1, 25.0 g, 123 mmol) was dissolved in sulfuric acid (183 mL) followed by the addition of N-iodosuccinimide (33.1 g, 147 mmol). The resulting mixture was stirred at room temperature overnight, then poured onto ice. When the ice had completely melted, the mixture was extracted with ethyl acetate (500 mL). The organic layer was washed with a 5% aqueous sodium thiosulfate solution (2 x 250 mL) and water (1 x 250 mL), dried over anhydrous sodium sulfate, filtered and evaporated to dryness to give 5-iodo-4-methyl-2-(trifluoromethyl)benzoic acid (2) as a beige powder. Yield: 37.7 g (93%).

[1048] 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.68 (bs, 1H); 8.22 (s, 1H); 7.76 (s, 1H); 2.47 (s, 3H).

[1049] A mixture of 5-iodo-4-methyl-2-(trifluoromethyl)benzoic acid (2, 22.2 g, 67.2 mmol), trimethyl orthoformate (14.7 mL, 134 mmol) and methanesulfonic acid (2.8 mL) in methanol (135 mL) was refluxed under a nitrogen atmosphere at 80°C overnight. The solvent was evaporated. The residue was dissolved in a 5% aqueous sodium carbonate solution (200 mL) and extracted with ethyl acetate (3 x 250 mL). The combined organic layers were washed with water (1 x 300 mL) and brine (1 x 200 mL), dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by flash flash column chromatography (silica gel 60, 0.040-0.063 mm; eluent: cyclohexane / ethyl acetate 9:1) to give methyl 5-iodo-4-methyl-2-(trifluoromethyl)benzoate (3) as white crystals. Yield: 35.9 g (91%). RF(cyclohexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.26 (s, 1H); 7.57 (s, 1H); 3.93 (s, 3H); 2.53 (s, 3H).

[1050] A mixture of 5-iodo-4-methyl-2-(trifluoromethyl)benzoic acid methyl ester (3, 35.9 g, 104 mmol), N-bromosuccinimide (20.4 g, 114 mmol) and 2,2-azobis(2-methylpropionitrile) (AIBN, 5.12 g, 31.2 mmol) in trifluorotoluene (95 mL) was stirred at 85 °C overnight. No complete conversion was achieved, but the reaction had run. Dichloromethane (150 mL) was added and the mixture was washed with water (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was dissolved in acetonitrile (440 mL) and potassium acetate (10.2 g, 104 mmol) was added. The mixture was stirred at 75 °C overnight. Insoluble material was filtered off and the filtrate was evaporated. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluents: cyclohexane / dichloromethane 4:1 to 1:1.5) to give 4-(acetyloxymethyl)-5-iodo-2-(trifluoromethyl)benzoic acid methyl ester (4) as a white powder. Yield: 17.5 g (42%). RF(cyclohexane / ethyl acetate 9:1): 0.35. 1 H NMR spectrum (300 MHz, CDC13, δH): 8.28 (s, 1H); 7.70 (s, 1H); 5.16 (s, 2H); 3.95 (s, 3H); 2.20 (s, 3H). 19 F NMR spectrum (282 MHz, CDC13, δF): -59.96 (s).

[1051] A mixture of 4-(acetyloxymethyl)-5-iodo-2-(trifluoromethyl)benzoic acid methyl ester (4, 17.5 g, 43.5 mmol), bis(pinacolato)diboron (14.3 g, 56.5 mmol) and anhydrous potassium acetate (21.3 g, 217 mmol) in anhydrous N,N-dimethyl sulfoxide (110 mL) was degassed; then [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (1.59 g, 2.17 mmol) was added. The reaction mixture was stirred at 95 °C overnight under nitrogen atmosphere. After cooling, diethyl ether (500 mL) was added and the precipitate was filtered off through a pad of celite. The filtrate was washed with 5% aqueous sodium chloride solution (3 x 500 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to give 4-(acetyloxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2- (trifluoromethyl)benzoic acid methyl ester (5) as a black oil. This oil was used in the next step without further purification.

[1052] Yield: 22.5 g. 1H NMR spectrum (300 MHz, CDC13, δΗ): 8.22 (s, 1H); 7.74 (s, 1H); 5.44 (s, 2H); 3.94 (s, 3H); 2.14 (s, 3H); 1.36 (s, 12H). 19 F NMR spectrum (282 MHz, CDC13, δF): -60.07 (s).

[1053] Methyl 4-(acetoxymethyl)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-2- (trifluoromethyl)benzoate (5, 17.5 g, 43.5 mmol) was suspended in a solution of sodium hydroxide (8.70 g, 217 mmol) in water (150 mL). The mixture was stirred at room temperature for 6 hours, then extracted with diethyl ether (2 x 200 mL). The aqueous phase was acidified with concentrated hydrochloric acid (18.9 mL) and the resulting mixture was stirred at room temperature overnight. The precipitate was filtered, washed with water and dried to give 1-hydroxy-5-(trifluoromethyl)-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid (6) as a grey powder. Yield: 7.62 g (71%). 1 H NMR spectrum (300 MHz, CDC13, δΗ): 8.22 (s, 1H); 7.74 (s, 1H); 5.44 (s, 2H); 3.94 (s, 3H); 2.14 (s, 3H); 1.36 (s, 12H). 19 F NMR spectrum (282 MHz, CDC13, δF): -60.07 (s).

[1054] Dichloromethane / MeOH / Formic acid 10:1:0.05). Fractions containing product were combined and evaporated. The residue was treated with cyclohexane. The precipitate was filtered, washed with cyclohexane, dissolved in acetonitrile (50 mL) and freeze-dried to give the title compound (9) as a beige powder. Yield: 3.63 g (55%).

[1055] 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.74 (s, 1H); 8.53 (s, 1H); 8.16 (s, 1H); 5.18 (s, 2H).

[1056] Pentafluorophenyl 1-hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylate (7, 9.51 g, 23.1 mmol) was dissolved in N,N-dimethylformamide (30 mL). Subsequently, N,N-diisopropylethylamine (10.1 mL, 57.7 mmol) and a solution of (2-aminoethyl)glycine hydrochloride (8, 1.78 g, 11.5 mmol) in water (30 mL) were added. The resulting mixture was stirred at room temperature overnight. The solvent was then evaporated. The residue was dissolved in ethyl acetate (200 mL) and washed with 1 M aqueous hydrochloric acid solution (1 x 200 mL), water (2 x 200 mL) and brine (1 x 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was treated with cyclohexane. The precipitate was filtered, washed with cyclohexane and purified by flash column chromatography (silica gel 60, 0.040-0.063 mm; eluent: dichloromethane / methanol / formic acid 10:1:0.05). Fractions containing product were combined and evaporated. The residue was treated with cyclohexane. The precipitate was filtered, washed with cyclohexane, dissolved in acetonitrile (50 mL) and freeze-dried to give the title compound (9) as a beige powder. Yield: 3.63 g (55%).

[1057] 1H NMR spectrum (300 MHz, AcOD-d4, 80C, δΗ): 8.04-7.66 (m, 4H); 5.28-5.04 (m, 4H); 4.63-4.34 (m, 1H); 4.22-3.78 (m, 3H); 3.72-3.49 (m, 2H). LC-MS: 574.0 (M+H)+.

[1058] Example 40: (S)-5-(tert-butoxy)-4-(2-(1-hydroxy-N-(2-(1-hydroxy-4- (trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)ethyl)-4- (trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)acetylamino)-5- oxopentanoic acid Example 41: 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid

[1059]

[1060] N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HC1, 6.20 g, 23.1 mmol) was added to a suspension of 4-chloro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid (1, 4.90 g, 23.1 mmol) and pentafluorophenol (Pfp-OH, 5.53 g, 23.1 mmol) in dichloromethane (70 mL) and the mixture was stirred at room temperature overnight. The solvent was evaporated to dryness. The residue was partitioned between ethyl acetate (200 mL) and 10% aqueous potassium hydrogen sulfate solution (200 mL). The organic layer was separated and washed with water (2 x 100 mL), dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was dissolved in dichloromethane and left in the fridge overnight. The solid was filtered off and washed with ethyl acetate (2 x 20 mL). The filtrates were combined and evaporated to dryness. To the residue was added cyclohexane (100 mL) and the mixture was stirred at room temperature for 15 minutes. The mixture was decanted and the deposit was dried in vacuo to give 4-chloro-l-hydroxy-l,3-dihydrobenzo[c] [l,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (2) as an off-white solid. Yield: 8.29 g (95%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 9.83 (bs, 1H); 8.61 (s, 1H); 8.26 (s, 1H); 5.13 (s, 2H). LC-MS: 377.4 (M-H)-.

[1061] Triethylamine (10.0 mL, 131.6 mmol) was added to a mixture of 1-hydroxy-1,3- dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (2, 8.29 g, 21.9 mmol) and N-2-aminoethylglycine (3, 1.30 g, 1.70 mmol) in solution N,N-dimethylformamide / water (2:1, 60 mL) and the resulting solution was stirred at room temperature overnight. After that, it was acidified with 1 M aqueous potassium hydrogen sulfate solution (200 mL) and extracted with ethyl acetate (3 x 250 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was co-distilled with toluene (3 x 100 mL) and triturated with diethyl ether (60 mL). The precipitate was filtered, washed with diethyl ether (2 x 50 mL) and air-dried. The obtained powder was dissolved in acetonitrile / water mixture (2:1, 20 mL) and freeze-dried to give compound 4 as a colorless solid. Yield: 1.50 g (15%). 1 HNMR spectrum (300 MHz, DMSO-d6, δH): 12.87 (bs, 1H); 9.59-9.41 (m, 2H); 8.77-8.54 (m, 5H); 5.07-4.88 (m, 4H); 4.25-3.92 (m, 2H); 3.60-3.24 (m, 4H). LC-MS: 507.3 (M+H)+.

[1062] Example 42: 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6- carboxylic acid Example 43: 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid

[1063]

[1064] A solution of pentafluorophenol (35.1 g, 191 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3- dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (1, 40.8 g, 166 mmol) and N,N'- dicyclohexylcarbodiimide (DCC, 39.3 g, 191 mmol) in acetonitrile (1 L) was stirred at ambient temperature for 24 hours. The reaction mixture was filtered, evaporated, dissolved in acetonitrile, re-filtered and evaporated. The crude product was precipitated in dichloromethane (1 L) and filtered to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (2) as a white solid. Yield: 52.8 g (77%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[1065] Resin 100-200 mesh 1.5 mmol / g (3, 4.47 g, 6.71 mmol) was swelled in dry dichloromethane (30 mL) for 30 min. A solution of (2S)-5-(tert-butoxy)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-5- oxopentanoic acid (Fmoc-Glu-OtBu, 1.90 g, 4.47 mmol) and N,N- diisopropylethylamine (2.96 mL, 17.0 mmol) in dry dichloromethane (30 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (1.56 mL, 8.95 mmol) in a methanol / dichloromethane mixture (4:1, 2 x 5 min, 2 x 40 mL). The resin was then washed with N,N-dimethylformamide (2 x 30 mL), dichloromethane (2 x 40 mL) and N,N-dimethylformamide (3 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 40 mL). The resin was washed with N,N-dimethylformamide (3 x 40 mL), 2-propanol (2 x 40 mL) and dichloromethane (3 x 40 mL). A solution of (2S)-2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Dap(Fmoc)-OH, 3.68 g, 6.71 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 2.55 g, 6.71 mmol) and 2,4,6-trimethylpyridine (1.60 mL, 12.1 mmol) in N,N-dimethylformamide (40 mL) was added to the resin and the mixture was shaken for 2 h. The resin was filtered and washed with N,N-dimethylformamide (2 x 40 mL), dichloromethane (2 x 40 mL) and N,N-dimethylformamide (2 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 30 min, 2 x 40 mL). The resin was washed with N,N-dimethylformamide (3 x 40 mL), 2-propanol (2 x 40 mL) and dichloromethane (3 x 40 mL). A solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (2, 5.53 g, 13.4 mmol) and triethylamine (4.99 mL, 35.8 mmol) in N,N-dimethylformamide (40 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and washed with N,N-dimethylformamide (6 x 40 mL) and dichloromethane (10 x 50 mL).The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 hours. The resin was filtered off and washed with dichloromethane (4 x 50 mL). The crude product (4) was dried under vacuum and extracted with ethyl acetate (2 x 70 mL) and 1 M aqueous potassium hydrogen sulphate solution (50 mL), the organic phase was dried over anhydrous sodium sulphate, filtered and the solvent evaporated. The crude product was then triturated in diethyl ether (20 mL) to give (S)-4-((2S)-2,3-bis(l-hydroxy-4-(trifluoromethyl)-l,3-dihydrobenzo[c][l,2]oxaborol-6- carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid (4) as a beige solid. Yield: 1.89 g (57%). 1 H NMR spectrum (300 MHz, AcOD-d4, δH): 8.50 (s, 1H); 8.46 (s, 1H); 8.29 (s, 1H); 8.26 (s, 1H); 5.28 (d, J = 2.6 Hz, 4H); 5.20 (t, J = 5.9 Hz, 1H); 4.55 (dd, J = 8.5 and 5.2 Hz, 1H); 4.08 (dd, J = 6.0 and 2.1 Hz, 2H); 2.57-2.42 (m, 2H); 2.34-2.16 (m, 1H); 2.17-2.08 (m, 1H); 1.47 (s, 9H). LC-MS: 746.3 (M+H)+.

[1066] Preparation of insulin derivatives Example 101: Example 102:

[1067]

[1068] Concentrated sulphuric acid (35 mL) was added to a solution of 3-bromo-5-iodo-4- methylbenzoic acid (1, 55.4 g, 162 mmol) in methanol (1.2 L) and the reaction mixture was stirred at reflux overnight. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (700 mL) and washed with water (2 x 300 mL) and saturated potassium carbonate solution (1 x 300 mL). The organic layer was separated, dried over anhydrous sodium sulphate, filtered and evaporated to give methyl 3-bromo-5-iodo-4-methylbenzoate (2) as a white solid. Yield: 50.0 g (87%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 8.32 (d, J = 1.7 Hz, 1H); 8.09 (d, J = 1.3 Hz, 1H); 3.86 (s, 3H); 2.65 (s, 3H).

[1069] To a solution of 3-bromo-5-iodo-4-methylbenzoic acid methyl ester (2, 37.3 g, 105 mmol) in dry tetrahydrofuran (250 mL) was added dropwise a 1.3 M solution of isopropylmagnesium chloride lithium chloride complex in tetrahydrofuran (89.0 mL, 115 mmol) at -30 °C under an inert atmosphere and stirred for 20 minutes. N,N-dimethylformamide (12.2 mL, 158 mmol) was then added at -30 °C. The reaction mixture was allowed to warm to ambient temperature and stirred for 16 hours. The reaction mixture was then evaporated under reduced pressure, dissolved in ethyl acetate (300 mL) and washed with water (2 x 200 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give 3-bromo-5-formyl-4-methylbenzoic acid methyl ester (3) as a white solid. Yield: 24.9 g (92%).

[1070] 1 H NMR spectrum (300 MHz, CDC13, δH): 10.27 (s, 1H); 8.53-8.34 (m, 2H); 3.97 (s, 3H); 2.82 (s, 3H).

[1071] A solution of 3-bromo-5-formyl-4-methylbenzoic acid methyl ester (3, 24.8 g, 96.5 mmol) and (diethylamino)sulfur trifluoride (DAST, 25.5 mL, 193 mmol) in dichloromethane (300 mL) was stirred at ambient temperature for 16 hours. The reaction was quenched by the addition of water (200 mL) and extracted with dichloromethane (2 x 200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and evaporated to give 3-bromo-5-(difluoromethyl)-4-methylbenzoic acid methyl ester (4) as a white solid. Yield: 23.3 g (87%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.35 (d, J = 1.1 Hz, 1H); 8.14 (d, J = 0.9 Hz, 1H); 6.78 (t, J = 54.8 Hz, 1H); 3.93 (s, 3H); 2.55 (t, J = 1.4 Hz, 3H).

[1072] A solution of N-bromosuccinimide (16.4 g, 91.9 mmol), methyl 3-bromo-5- (difluoromethyl)-4-methylbenzoate (4, 23.3 g, 83.5 mmol) and 2,2-azobis(2- methylpropionitrile) (AIBN, 1.36 g, 8.36 mmol) in a,a,a-trifluorotoluene (120 mL) was stirred at 85 °C overnight. The reaction mixture was evaporated and then extracted with diethyl ether (2 x 300 mL). The organic layer was washed with brine (1 x 150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give crude methyl 3-bromo-4-(bromomethyl)-5-(difluoromethyl)benzoate (5) which was stirred with potassium acetate (16.4 g, 167 mmol) in acetonitrile (300 mL) at 75 °C overnight. The suspension was filtered through a short pad of celite and evaporated. The crude product was dissolved in dichloromethane and filtered again. The filtrate was evaporated and purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 9:1) to give methyl 4-(acetyloxymethyl)-3-bromo-5-(difluoromethyl)benzoate (6) as a white solid. Yield: 17.1 g (61%). Rf(SiO2, cyclohexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDC13, δH): 8.40 (s, 1H); 8.26 (s, 1H); 7.02 (t, J = 54.7 Hz, 1H); 5.38 (s, 2H); 3.97 (s, 3H); 2.11 (s, 3H).

[1073] A solution of methyl 4-(acetyloxymethyl)-3-bromo-5-(difluoromethyl)benzoate (6, 17.1 g, 50.7 mmol), bis(pinacolato)diboron (14.2 g, 55.7 mmol), potassium acetate (14.9 g, 152 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.24 g, 1.52 mmol) in anhydrous dioxane (200 mL) was stirred at 75 °C under an argon atmosphere for 2 days. The reaction mixture was then cooled to ambient temperature, filtered and evaporated. The crude product was filtered through a column of silica gel (silica gel, 0.063-0.200 mm; eluent: cyclohexane / ethyl acetate 9:1) to give methyl 4-(acetyloxymethyl)-3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (7). Yield: 16.3 g (84%). Rf(SiO2, cyclohexane / ethyl acetate 9:1): 0.30. 1H NMR spectrum (300 MHz, CDC13, δH): 8.57 (s, 1H); 8.38 (s, 1H); 7.04 (t, J = 55.1 Hz, 1H); 5.54 (s, 2H); 3.97 (s, 3H); 2.06 (s, 3H); 1.39 (s, 12H).

[1074] A solution of methyl 4-(acetoxymethyl)-3-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoate (7, 16.3 g, 42.3 mmol) and sodium hydroxide (8.45 g, 212 mmol) in water (200 mL) was stirred at ambient temperature for 3 hours. A solution of concentrated hydrochloric acid (20 mL) in water (50 mL) was then added to reduce the pH to 1. The reaction mixture was left in the fridge overnight. The precipitate was filtered and dried to give 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6- carboxylic acid (8) as a white solid. Yield: 8.55 g (89%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.25 (bs, 1H); 9.54 (s, 1H); 8.51 (s, 1H); 8.20 (s, 1H); 7.22 (t, J = 55.1 Hz, 1H); 5.19 (s, 2H).

[1075] A solution of pentafluorophenol (8.28 g, 45.0 mmol), 4-(difluoromethyl)-1-hydroxy-1,3- dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid (8, 8.55 g, 37.5 mmol) and N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl, 10.1 g, 52.5 mmol) in dichloromethane (100 mL) was stirred at ambient temperature for 3 hours. The reaction mixture was then evaporated, dissolved in ethyl acetate (200 mL) and washed with 1M aqueous hydrochloric acid (3 x 200 mL) and brine (1 x 200 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated. The crude product 9 was recrystallized from hot cyclohexane (300 mL) and ethyl acetate (30 mL) to give 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorophenyl ester (9) as a white solid. Yield: 8.20 g (56%). LC-MS: 395.5 (M+H)+.

[1076] A solution of 4-(difluoromethyl)-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-6- carboxylic acid pentafluorophenyl ester (9, 8.20 g, 20.8 mmol), (2- aminoethyl)glycine (10, 1.23 g, 10.4 mmol) and triethylamine (14.5 mL, 104 mmol) in tetrahydrofuran (40 mL) and water (20 mL) was stirred at ambient temperature overnight. Tetrahydrofuran was then evaporated and to the residue was added 1 M aqueous potassium hydrogen sulfate solution (30 mL). The mixture was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and evaporated. The crude product 11 was dissolved in ethyl acetate (10 mL) and precipitated with cyclohexane (100 mL). The precipitate was filtered, washed with cyclohexane (50 mL) and freeze-dried to give N-(4-(difluoromethyl)-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-6- carbonyl)-N-(2-(4-(difluoromethyl)-l-hydroxy-l,3-dihydrobenzo[c][l,2]oxaborol-6- carboxamido)ethyl)glycine (11) as a white solid. Yield: 4.59 g (82%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.87 (bs, 1H); 9.66-9.33 (m, 2H); 8.95-6.68 (m, 7H); 5.15 (d, J = 11.9 Hz, 4H); 4.39-3.94 (m, 2H); 3.76-3.37 (m, 4H). LC-MS: 539.1 (M+H)+.

[1077] Example 103: 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -mercapto)amino)acetyl)-L- Example 104:

[1078]

[1079] Subsequently anhydrous dichloromethane (37 mL) and triethylamine (1.53 mL, 11.0 mmol) were added to 2,5-dioxopyrrolidin-l-yl-2-((oxobis(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 6- Sulfenyl) amino) acetic acid (1, 2.78 g, 3.66 mmol) and (S)-4-amino-5-(tert- butoxy)-5-oxopentanoic acid (2, H-Glu-OtBu, 891 mg, 4.39 mmol). The mixture was sonicated to give a solution which was stirred at room temperature for 6 hours. The volatiles were removed in vacuo and the residue was re-dissolved in ethyl acetate (200 mL). The resulting solution was washed with 0.5 M aqueous hydrochloric acid (3 x 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was re-dissolved in ethyl acetate (50 mL) and a solution of pinacol (432 mg, 3.66 mmol) in ethyl acetate (20 mL) was added. The resulting solution was evaporated in vacuo to give (S)-5-(tert-butoxy)-5-oxo-4-(2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 6 - Sulfenyl) amino) acetic acid (1, 2.78 g, 3.66 mmol) and (S)-4-amino-5-(tert- butoxy)-5-oxopentanoic acid (2, H-Glu-OtBu, 891 mg, 4.39 mmol). The mixture was sonicated to give a solution which was stirred at room temperature for 6 hours. The volatiles were removed in vacuo and the residue was re-dissolved in ethyl acetate (200 mL). The resulting solution was washed with 0.5 M aqueous hydrochloric acid (3 x 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was re-dissolved in ethyl acetate (50 mL) and a solution of pinacol (432 mg, 3.66 mmol) in ethyl acetate (20 mL) was added. The resulting solution was evaporated in vacuo to give (S)-5-(tert-butoxy)-5-oxo-4-(2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 1 H NMR spectrum (300 MHz, CDC13, δH): 8.57 (d, J = 12.7 Hz, 2H); 8.40 (dd, J = 8.3 and 0.7 Hz, 2H); 8.25 (s, 2H); 7.96 (d, J = 8.1 Hz, 1H); 4.57 (m, 1H); 3.71 (dd, J = 22.9 and 17.4 Hz, 2H); 2.53-2.43 (m, 2H); 2.37-2.24 (m, 1H); 2.15-2.02 (m, 1H); 1.47 (s, 9H); 1.37 (s, 24H). 19 F NMR spectrum (282 MHz, CDC13, δF): -62.64 (s). LC-MS: 683.4 (M-2x pinacol-H)-.

[1080] Subsequently N,N-disuccinimidyl carbonate (DSC, 1.84 g, 7.19 mmol) and pyridine (0.58 mL, 7.19 mmol) were added to (S)-5-(tert-butoxy)-5-oxo-4-(2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5- (trifluoromethyl)phenyl)-lambda 6A solution of (sulphido)amino)acetylamino)pentanoic acid (3, 3.05 g, 3.59 mmol) in dry acetonitrile (18 mL) was prepared and the mixture was then sonicated to form a fine suspension. The resulting suspension was stirred at room temperature overnight to give a clear solution. The solution was evaporated to dryness and the residue was partitioned between ethyl acetate (250 mL) and 0.5 M aqueous hydrochloric acid (100 mL). The phases were separated; the organic phase was washed with 0.5 M aqueous hydrochloric acid (4 x 100 mL) and brine (70 mL); dried over anhydrous sodium sulphate and evaporated to dryness. The residue was dissolved in dichloromethane (40 mL) followed by the addition of pinacol (636 mg, 5.39 mmol). The solvent was removed in vacuo and the residue was evaporated from dichloromethane (50 mL). The resulting foam was triturated with cyclohexane (3 x 50 mL); the resulting semi-solid was decanted, dissolved in dichloromethane (50 mL) and evaporated to dryness in vacuo. The residue was evaporated from dichloromethane (3 x 50 mL) and dried in vacuo to give the title compound (4) as a white foam. Yield: 2.82 g (83%). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.57 (s, 1H); 8.51 (s, 1H); 8.43 (s, 1H); 8.31 (s, 1H); 8.25 (s, 1H); 8.24 (s, 1H); 7.77 (d, J = 7.9 Hz, 1H); 4.60 (m, 1H); 3.73 (dd, J = 39.6 and 17.3 Hz, 2H); 2.82 (s, 4H); 2.79-2.62 (m, 2H); 2.43-2.30 (m, 1H); 2.20-2.06 (m, 1H); 1.49 (s, 9H); 1.36 (s, 24H).

[1081] 19 F NMR spectrum (282 MHz, CDC13, δF): -62.63 (s). LC-MS: 864.5 (M - pinacol + H)+, 946.7 (M + H)+.

[1082] Example 105: Example 106: Example 107:

[1083]

[1084] Resin 100-200 mesh 1.5 mmol / g (1, 4.39 g, 6.59 mmol) was swelled in dry dichloromethane (30 mL) for 30 min. A solution of (S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)pentanedioic acid 1-tert-butyl ester (Fmoc-Glu-OtBu, 1.87 g, 4.39 mmol) and N,N-diisopropylethylamine (2.91 mL, 16.7 mmol) in dry dichloromethane (30 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (1.53 mL, 8.78 mmol) in a methanol / dichloromethane mixture (4:1, 2 x 5 min, 2 x 40 mL). The resin was then washed with N,N-dimethylformamide (2 x 30 mL), dichloromethane (2 x 40 mL) and N,N-dimethylformamide (3 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 20 min, 2 x 40 mL). The resin was washed with N,N-dimethylformamide (3 x 40 mL), 2-propanol (2 x 40 mL) and dichloromethane (3 x 40 mL). A solution of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)glycine (Fmoc-AEG(Fmoc)-OH, 3.71 g, 6.59 mmol), 1-((dimethylamino)(dimethylimino)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridazine 3-oxide hexafluorophosphate (HATU, 2.50 g, 6.59 mmol) and 2,4,6-trimethylpyridine (1.57 mL, 11.9 mmol) in N,N-dimethylformamide (40 mL) was added to the resin and the mixture was shaken for 2 hours. The resin was filtered and washed with N,N-dimethylformamide (2 x 40 mL), dichloromethane (2 x 40 mL) and N,N-dimethylformamide (2 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 min, 1 x 30 min, 2 x 40 mL). The resin was washed with N,N-dimethylformamide (3 x 40 mL), 2-propanol (2 x 40 mL) and dichloromethane (3 x 40 mL). A solution of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid pentafluorobenzyl ester (2, 5.43 g, 13.2 mmol) and triethylamine (4.90 mL, 35.1 mmol) in N,N-dimethylformamide (40 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and washed with N,N-dimethylformamide (6 x 40 mL) and dichloromethane (10 x 50 mL).The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 hours. The resin was filtered off and washed with dichloromethane (4 x 50 mL). The solvent was evaporated; the residue was extracted with 1 M aqueous potassium hydrogen sulfate solution (50 mL) and ethyl acetate (2 x 70 mL), the organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was evaporated. The crude product was precipitated from ethyl acetate / cyclohexane (1 :10, 40 mL), purified by column chromatography (silica gel 60, 0.063-0.200 mm; eluent: acetonitrile / water 10:1) and lyophilized to give (S)-5-(tert-butoxy)-4-(2-(1-hydroxy-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)ethyl)-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxamido)acetylamino)-5-oxopentanoic acid (3) as a white solid. Yield: 1.50 g (45%). 1 H NMR spectrum (300 MHz, AcOD-d4, δΗ): 8.44 (s, 1H); 8.24 (s, 1H); 8.05 (s, 1H); 7.77 (s, 1H); 5.25 (d, J = 17.1 Hz, 4H); 4.70-4.25 (m, 3H); 4.03-3.67 (m, 4H); 2.49 (bs, 2H); 2.22 (bs, 1H); 1.49 (s, 9H).

[1085] LC-MS: 760.3 (M+H)+.

[1086] Example 108:

[1087]

[1088] N-bromosuccinimide (NBS, 88.1 g, 495 mmol) was added to a cold suspension (10 °C) of 4-methylbenzonitrile (58.6 g, 500 mmol) in 50% aqueous sulfuric acid (270 mL). The reaction mixture was stirred in the dark at 10 °C for 40 hours. After filtration of the suspension, the filter cake was washed with water (100 mL) and dissolved in ethyl acetate (800 mL). The solution of the crude product in ethyl acetate was washed with water (400 mL), saturated aqueous sodium bicarbonate solution (2 x 400 mL) and brine (200 mL). The organic solution was dried over anhydrous magnesium sulfate and evaporated to dryness to give the crude 3-bromo-4-methylbenzonitrile as yellow crystals. This product was used without purification in the next step. Yield: 90.70 g (92%). RF(SiO2, hexane / ethyl acetate 9:1): 0.45. 1H NMR spectrum (300 MHz, CDC13, δH): 7.82 (d, J = 1.5 Hz, 1 H); 7.50 (dd, J = 7.9 and 1.7 Hz, 1 H); 7.34 (d, J = 7.9, 1 H); 2.47 (s, 3 H).

[1089] Benzoic peroxide (1 g) and N-bromosuccinimide (NBS, 96.3 g, 541 mmol) were added to a solution of 3-bromo-4-methylbenzonitrile (90.7 g, 463 mmol) in tetrahydrofuran (1.00 L). The mixture was refluxed overnight. After that the reaction mixture was cooled, diluted with dichloromethane (500 mL) and extracted with water (2 x 500 mL). The organic solution was dried over anhydrous magnesium sulfate and evaporated to dryness to give the crude 3-bromo-4-(bromomethyl)benzonitrile as a brown oil. Yield: 135 g. Rf (SiO2, hexane / ethyl acetate 9:1): 0.45.

[1090] Potassium acetate (98.1 g, 1.00 mol) was added to a cold (4°C) solution of the above crude 3-bromo-4-(bromomethyl)benzonitrile (135 g) in acetonitrile (700 mL). The mixture was stirred at 70°C for 24 h. The mixture was evaporated and the residue was diluted with ethyl acetate (800 mL) and extracted with water (2 x 500 mL). The organic phase was dried over magnesium sulfate and evaporated to dryness. The residue was purified by flash column chromatography (silica gel 60, 0.040-0.060 mm; eluent: hexane / ethyl acetate 20:1 to 5:1) to give 2-bromo-4-cyanobenzyl acetate as white crystals. Yield: 60.90 g (52% over two steps). Rf (SiO2, hexane / ethyl acetate 4:1):.0.30. 1 H NMR spectrum (300 MHz, CDC13, δH): 7.87 (d, J = 1.5 Hz, 1 H); 7.64 (dd, J = 8.1 and 1.7 Hz, 1 H); 7.53 (d, J = 8.1 Hz, 1 H); 5.22 (s, 2 H); 2.19 (s, 3 H).

[1091] A solution of sodium hydroxide (13.1 g, 327 mmol) in methanol (300 mL) was added dropwise to a solution of 4-cyano-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzyl acetate (44.8 g, 149 mmol) in methanol (300 mL) at 30 °C. The reaction mixture was stirred for additional 2 h. The solvent was evaporated and the residue was dissolved in tetrahydrofuran (200 mL). 2 M aqueous hydrochloric acid (660 mL) was added and the resulting suspension was stirred for 10 min. The suspension was cooled to 10 °C and filtered. The filter cake was washed with water (100 mL) and n-hexane (100 mL) to give 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonitrile as a white powder. Yield: 20.15 g (85 %). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.13 (d, J = 1.7 Hz, 1 H); 7.71 (dd, J = 7.9 and 1.9 Hz, 1 H); 7.49 (d, J = 8.1 Hz, 1 H); 5.42 (s, 2 H); 2.13 (s, 3 H).

[1092] A solution of sodium hydroxide (13.1 g, 327 mmol) in methanol (300 mL) was added dropwise to a solution of 4-cyano-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzyl acetate (44.8 g, 149 mmol) in methanol (300 mL) at 30 °C. The reaction mixture was stirred for additional 2 h. The solvent was evaporated and the residue was dissolved in tetrahydrofuran (200 mL). 2 M aqueous hydrochloric acid (660 mL) was added and the resulting suspension was stirred for 10 min. The suspension was cooled to 10 °C and filtered. The filter cake was washed with water (100 mL) and n-hexane (100 mL) to give 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonitrile as a white powder. Yield: 20.15 g (85 %). 1 H NMR spectrum (300 MHz, CDC13, δH): 8.13 (d, J = 1.7 Hz, 1 H); 7.71 (dd, J = 7.9 and 1.9 Hz, 1 H); 7.49 (d, J = 8.1 Hz, 1 H); 5.42 (s, 2 H); 2.13 (s, 3 H).

[1093] A suspension of 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carbonitrile (20.15 g, 127 mmol) in concentrated hydrochloric acid (1.50 L) was refluxed for 24 h and cooled to 10 °C. The suspension was filtered and the filter cake was washed with water (300 mL). The filter cake was suspended in water (500 mL) and freeze-dried. The residue was suspended in dichloromethane (500 mL) and filtered. The filter cake was washed with dichloromethane (200 mL) and dried under vacuum to give 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid as a white powder. Yield: 12.30 g (55%). 1 H NMR spectrum (300 MHz, DMSO-d6, δΗ): 12.92 (s, 1 H); 9.36 (s, 1 H); 8.37 (s, 1 H); 8.04 (dd, J = 7.9 and 0.9 Hz, 1 H); 7.52 (d, J = 8.1 Hz, 1 H); 5.05 (s, 2H). LC-MS m / z: 178.2 (M+H).

[1094] Example 109: Example 110:

[1095]

[1096] 1-Hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborol-6-carboxylic acid was prepared as described in Example 28. [10...

Claims

1. A compound comprising: i) human insulin or a human insulin analogue, wherein the human insulin or human insulin analogue is a human insulin analogue selected from the group consisting of: desB30 human insulin; A21Q desB30 human insulin; A14E B25H desB30 human insulin; A14E B1K B2P B25H desB27 desB30 human insulin; A14E A22K B25H desB27 desB30 human insulin; A14E desB1-B2 B4K B5P desB30 human insulin; A14E desB1-B2 B3G B4K B5P desB30 human insulin; A14E B-1G B1K B2P desB30 human insulin; and A22K desB30 human insulin; and ii) two, three or four modifying groups M, wherein each modifying group M comprises two aryl moieties, wherein a boron atom is attached to each of the two aryl moieties; and wherein each of the modifying groups M is optionally attached to the amino group of the N-terminal amino acid residue of the A- or B-chain of the human insulin or human insulin analogue, or to the epsilon amino group of a lysine in the human insulin or human insulin analogue; and wherein each modifying group M is independently selected from Formula M1 which represents a D- or L-amino acid form, and wherein n represents an integer in the range of 1 to 4; wherein W1 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W1 represents NH-CH2CH2-C(=O)-*, a D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-*, or NH-CH2CH2-C(=O)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein R1 is selected from wherein Y1, Y2 and Y3 are independently selected from H, F, Cl, CHF2 and CF3; R1a wherein W2 is absent and represents the point of attachment * to the human insulin or human insulin analogue, or W2 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=O)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and Formula M2 wherein R2 is selected from wherein Y7, Y8 and Y9 are independently selected from H, F, Cl, CHF2 and CF3; and R2a which represents a R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein Y13 and Y14 are independently selected from H, F, Cl, CHF2 and CF3, Formula M3 wherein each modifying group M is attached to a point of attachment selected from one of the following groups: a) the amino group of the N-terminal amino acid residue of the A-chain of the human insulin or human insulin analogue; b) the epsilon amino group of the lysine at position 22 of the A-chain of the human insulin analogue; or c) the epsilon amino group of the lysine at position 29 of the A-chain of the human insulin analogue. The ε-amino group of lysine in the optional peptide spacer at the C-terminus of the A chain of the human insulin or human insulin analogue; c) The amino group of the N-terminal amino acid residue of the B chain of the human insulin or human insulin analogue; The ε-amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analog; The ε-amino group of the lysine residue in the optional peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue; or The distal amino group marked with *1 in the optional linker L at the N-terminus of the human insulin or human insulin analog B chain; and d) The ε-amino group of lysine residue at position 22 or 29 of the B chain of the human insulin or human insulin analogue. And no more than one of the modifying groups M is attached to the attachment point in each of groups a), b), c) and d).

2. The compound according to claim 1, wherein each of the modifying groups M is independently selected from... Formula M1 It represents the D- or L-amino acid form, and Where n represents an integer in the range of 1 to 4; Where W1 is absent and represents an attachment point with the human insulin or a human insulin analog*, or W1 represents NH-CH2CH2-C(=O)-*, The D- or L- form of NH-CH(COOH)-CH2CH2-C(=O)-*, or NH-CH2CH2-C(=O)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*, Where * represents an attachment point to the human insulin or human insulin analogue; and R1 is selected from Formula R1a Where Y1 and Y2 are H, and Y3 is F or CF3; Y4 is H or F; Formula M2 Wherein W2 is absent and represents an attachment site with the human insulin or human insulin analogue*, or W2 represents the D- or L- form of NH-CH(COOH)-CH2CH2-C(=O)-*, where * represents an attachment site with the human insulin or human insulin analogue; and R2 is selected from R2a Where Y7 is H; Y8 is H, Cl, CHF2, or CF3; Y9 is H, F, or CF3; Y10 is F; the condition is that only one of Y8 and Y9 is H; and Formula M3 It represents the R,R or S,S or R,S stereoisomer of 3,4-diamino-pyrrolidine; and wherein * represents the attachment site with said human insulin or human insulin analog; and wherein Y13 is H or F; and Y14 is H or CF3; provided that only one of Y13 and Y14 is H.

3. The compound according to claim 2, wherein each of the modifying groups M is independently selected from... Formula M1 It represents the D- or L-amino acid form, where n is 1; W1 represents the D- or L-form of NH-CH2CH2-C(=O)-*, or NH-CH(COOH)-CH2CH2-C(=O)-*, where * represents the attachment site with said human insulin or human insulin analogue; and R1is of the formula R1a Where Y1 and Y2 are H; and Y3 is F or CF3; and Formula M2 wherein W2 is absent and represents a point of attachment to the human insulin or human insulin analogue, or W2 represents a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-, wherein * represents a point of attachment to the human insulin or human insulin analogue; and wherein R2is of the formula R2a wherein Y7 and Y8 are H; and Y9 is CI, CHF2 or CF3.

4. The compound according to any one of claims 1-3, wherein the human insulin or human insulin analogue comprises a spacer, which is a peptide and which peptide comprises at least one Lys residue.

5. The compound according to any one of claims 1-3, wherein the human insulin or human insulin analogue comprises a spacer selected from the group consisting of a) a peptide spacer at the C-terminal end of the A chain of the human insulin or human insulin analogue, wherein the peptide spacer comprises (GES) p K, wherein p is an integer from 3 to 12; or b) a peptide spacer or linker L at the N-terminal end of the B chain of the human insulin or human insulin analogue; wherein the peptide spacer comprises GKPG, GKP(G4S) q , or KP(G4S) r wherein each of q and r is independently selected from an integer from 1 to 5; and wherein the linker L is selected from wherein *1 indicates a point of attachment to the modifying group M, and *2 indicates a point of attachment to the amino group of the amino acid residue at the N-terminal end of the B chain of the human insulin or human insulin analogue; and wherein *1 indicates a point of attachment to the modifying group M, and *2 indicates a point of attachment to the amino group of the amino acid residue at the N-terminal end of the B chain of the human insulin or human insulin analogue; and wherein u is 1, 2 or 3.

6. The compound according to claim 5, wherein q is an integer selected from 1 to 3; and r is 3.

7. The compound according to any one of claims 1-3 or 6, wherein the modifying groups M are identical.

8. The compound according to any one of claims 1-3 or 6, wherein the chiral amino acid is in L-form.

9. The compound according to any one of claims 1-3 or 6, wherein the human insulin or human insulin analogue is a human insulin analogue comprising desB30.

10. The compound according to claim 1, comprising i) a human insulin or human insulin analogue, wherein the human insulin or human insulin analogue optionally comprises a peptide spacer at the N-terminal end of the B chain of the human insulin or human insulin analogue; wherein the peptide spacer comprises GKPG, GKP(G4S) q , or KP(G4S) r wherein q is an integer from 1-3; and r is 3; ii) two modifying groups M, wherein each of the modifying groups M is independently selected from Formula M1 which represents a D- or L-amino acid form, and wherein n is 1; W1 represents NH-CH2CH2-C(=0)-*, or a D- or L-form of NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents a point of attachment to the human insulin or human insulin analogue; and wherein R1 is R1a wherein Y1 and Y2 are H, and Y3 is CF3; and wherein one modifying group M is attached to the epsilon amino group of the lysine at position 29 of the B chain of the human insulin or human insulin analogue; and one modifying group M is attached to: the epsilon amino group of the lysine residue at position 1 or position 4 of the B chain of the human insulin analogue; or the epsilon amino group of the lysine in the optional peptide spacer at the N-terminal end of the B chain of the human insulin or human insulin analogue.

11. The compound according to claim 10, consisting of i) a human insulin analogue, wherein said human insulin analogue comprises a peptide spacer at the N-terminal end of the B chain of said human insulin or human insulin analogue; wherein said peptide spacer comprises GKP(G4S) q or KP(G4S) r wherein q is an integer from 1-3; and r is 3; ii) two modification genes M, which are independently selected from Formula M1 which represents the D- or L-amino acid form, and wherein n is 1 ; W1 represents the D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein R1 is R1a wherein Y1 and Y2 are H, and Y3 is CF3; wherein one modification group M is attached to the epsilon amino group of a lysine in the peptide spacer; and one modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

12. The compound according to claim 10, consisting of i) a human insulin analogue, wherein the human insulin analogue optionally comprises a peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue; wherein the peptide spacer comprises GKPG, GKP(G4S) q , or KP(G4S) r wherein q is an integer from 1-3; and r is 3; ii) two modification genes M, wherein each of the modification groups M is independently selected from Formula M1 which represents the D- or L-amino acid form, and wherein n is 1 ; W1 represents the D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein R1 is R1a wherein Y1 and Y2 are H, and Y3 is CF3; and wherein one modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue. one modification group M is attached to: the epsilon amino group of a lysine residue at position 1 or position 4 of the B chain of the human insulin analogue, or the epsilon amino group of a lysine in the optional peptide spacer at the N-terminus of the B chain of the human insulin or human insulin analogue.

13. The compound according to claim 11, consisting of i) a human insulin analogue, wherein said human insulin analogue has a peptide spacer at the N-terminus of the B chain of said human insulin or human insulin analogue; wherein said peptide spacer is GKP(G4S) q or KP(G4S) r wherein q is an integer from 1 to 3; and r is 3; ii) two modification genes M, which are independently selected from Formula M1 which represents the D- or L-amino acid form, and wherein n is 1 ; W1 represents the D- or L-form of NH-CH2CH2-C(=0)-* or NH-CH(COOH)-CH2CH2-C(=0)-*, wherein * represents the point of attachment to the human insulin or human insulin analogue; and wherein R1 is R1a wherein Y1 and Y2 are H, and Y3 is CF3; wherein one modification group M is attached to the epsilon amino group of a lysine in the peptide spacer; and one modification group M is attached to the epsilon amino group of a lysine at position 29 of the B chain of the human insulin or human insulin analogue.

14. The compound according to any one of claims 10 to 13, wherein the chiral amino acid is in the L-form.

15. The compound according to any one of claims 10 to 13, wherein the modification groups M are identical.

16. The compound according to any one of claims 10 to 13, wherein the human insulin analogue comprises desB30.

17. The compound according to any one of claims 10 to 13, wherein the human insulin or human insulin analog is a human insulin analog selected from the group consisting of desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11); A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 12); A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13); A14E desB1-B2 B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 16); A14E desB1-B2 B3G B4K B5P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 17); A14E B-1G B1K B2P desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 18); and A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11).

18. The compound according to any one of claims 10 to 13, wherein the human insulin analog comprising the spacer is selected from B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 21); B1-KPGGGGSGGGGSGGGGS A14E B25H desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 22); B1-GKPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 23); B1-GKPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 24); and B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25).

19. The compound according to claim 1, wherein the compound is selected from: the compound of Example 280; the compound of Example 300; the compound of Example 301; the compound of Example 324; the compound of Example 327; the compound of Example 331; the compound of Example 333; and the compound of Example 339.

20. The compound according to claim 19, wherein the compound is the compound of Example 280.

21. The compound according to claim 19, wherein the compound is the compound of Example 300.

22. The compound according to claim 19, wherein the compound is the compound of Example 301.

23. The compound of claim 19, wherein the compound is the compound of Example 324.

24. The compound of claim 19, wherein the compound is the compound of Example 327.

25. The compound of claim 19, wherein the compound is the compound of Example 331.

26. The compound of claim 19, wherein the compound is the compound of Example 333.

27. The compound of claim 19, wherein the compound is the compound of Example 339.

28. A composition comprising a compound according to any one of claims 1-27.

29. Use of a compound according to any one of claims 1-27 or a composition according to claim 28 for the manufacture of a medicament for the treatment or prevention of diabetes, impaired glucose tolerance, and hyperglycemia.

30. The use of claim 29, wherein the diabetes is type 1 diabetes or type 2 diabetes.

31. A compound having the chemical structure of

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