Glucose sensitive insulin derivatives

Abstract

To provide insulin derivatives with increased glucose sensitivity within physiological blood glucose levels.SOLUTION: The present invention relates to novel insulin derivatives and their use in the treatment or prevention of medical conditions relating to diabetes. The insulin derivatives are glucose sensitive and display glucose-sensitive albumin binding. The invention also relates to novel intermediates. Finally, the invention provides a pharmaceutical composition comprising the insulin derivatives of the invention and the use of such a composition in the treatment or prevention of medical conditions relating to diabetes.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to novel insulin derivatives and their pharmaceutical uses. Further, the present invention relates to pharmaceutical compositions containing such insulin derivatives and the use of such compounds for the treatment or prevention of medical conditions related to diabetes.

Background Art

[0002] Insulin is the most effective drug for the treatment of hyperglycemia, but the insulin dosage is a delicate balance between too much and too little because the physiological glucose range is narrow. Healthy people have a glucose level of approximately 5 mM in the fasting state, and diabetic patients try to obtain approximately 5 mM by administering both a meal and a basal insulin preparation. However, blood glucose levels below about 3 mM (hypoglycemia) often occur during insulin treatment, and hypoglycemia can lead to discomfort, loss of conciseness, brain damage, or death. Therefore, diabetic patients are hesitant to actively treat high or moderately high blood glucose levels for fear of hypoglycemia. If insulin drugs that are only active or released from higher glucose depots and are inactive or weakly active at lower glucose levels were developed, it would assist in the treatment of diabetes. Such a goal has been proposed in many papers since the 1970s (Brownlee et al. Science 1979, 1190, Zaykov et al. Nature Rev. Drug Disc. 2016, 425), but in most cases, it is through glucose-sensitive polymers that encapsulate and release insulin in a glucose-dependent manner from subcutaneous depots. However, such systems are slow and are therefore not good for treatments that rapidly fluctuate postprandial blood glucose levels. As a result, subcutaneous glucose-sensitive release systems have never reached clinical trials.

[0003] It is better when the glucose-sensitivity regulation of insulin bioactivity occurs in the blood. One approach that can fulfill this wish could be glucose-sensitive albumin binding as previously described with fatty acid-monoboronate insulin derivatives where the fatty acid moiety causes albumin binding (Novo Nordisk WO2011 / 000823, WO2014 / 093696, Chou et al. Proc. Nat. Acad. Sci. 2015, 2401). The main driving force for albumin interaction in these systems originates from the fatty acid moiety of the fatty acid-monoboronate insulin derivative (not the boronate), and the influence of glucose on albumin affinity is weak. Therefore, in order to enhance the glucose-sensitivity of albumin binding, a glucose-sensitive albumin binding motif that directly moves by glucose is needed. Monoboronates are known to bind to glucose and other sugars with an affinity (Kd) in the high millimolar range in the medium (Hansen et al. Sensors Actuators B 2012, 45). However, a strong affinity for glucose is required to provide appropriate glucose-sensitivity at physiological glucose levels. Diboron compounds having two boronate / boroxole arranged in an appropriate shape with respect to the hydroxy groups on glucose can bring about an increase in glucose affinity for monoboronates, i.e., a low mM Kd or sub-mM Kd (Hansen et al. Sensors Actuators B 2012, 45). Since the aim of those studies was to fabricate optical glucose sensors, most such diboronates described in the literature contain fluorescent probes. Since these probes are light-sensitive, toxic, and can be colored, fluorescent probes are not desirable in drug candidates. Therefore, an insulin derivative with enhanced glucose-sensitivity within physiological blood glucose levels is needed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

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Non-Patent Document 6

Summary of the Invention

[0006] In its broadest aspect, the present invention relates to insulin derivatives.

[0007] The compounds of the present invention have surprisingly been found to bind to both albumin (HSA) and glucose, and the HSA affinity is glucose-sensitive. Thus, the human insulin receptor (HIR) affinity in the presence of HSA also becomes glucose-sensitive. The fraction of HSA-bound insulin is shielded from binding to HIR, but glucose-promoted release from HSA increases the free fraction of insulin, and thus glucose enhances the HIR affinity.

[0008] In contrast to the already disclosed insulin derivatives having suspected glucose-sensitive albumin binding, the compounds of the present invention contain an albumin-binding motif that does not depend on the fatty acid moiety of albumin binding but is directly translocated by glucose, resulting in an increase in the effect of glucose on albumin binding and thus an increase in the glucose sensitivity of insulin.

[0009] Albumin binding can generally extend the in vivo half-life of peptide and protein-based drugs. The long-term effect is achieved when the albumin-bound fraction is protected from enzymatic degradation and renal excretion, and only the free portion is biologically active, thus preventing receptor-mediated clearance of the albumin-bound fraction.

[0010] Thus, the compounds of the present invention exhibit insulin activity dependent on glucose concentration and thus function as glucose-sensitive insulin derivatives.

[0011] In one aspect, the compounds of the present invention comprise insulin or an analogue thereof, and one or more modifying groups.

[0012] In one aspect, the modifying group has an affinity for glucose and albumin.

[0013] In one aspect, the insulin peptide or an analogue thereof optionally contains a spacer.

[0014] In one aspect, the compounds of the present invention are i) human insulin or a human insulin analogue, and ii) one or more modifying groups M, each of the modifying groups M containing two aryl moieties, with a boron atom attached to each of the two aryl moieties, and each of the one or more modifying groups M is attached, optionally via a spacer, to the amino group of the N-terminal amino acid residue of the A chain or B chain of the aforementioned human insulin or human insulin analogue, or to the epsilon amino group of lysine in the aforementioned human insulin or human insulin analogue.

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

[0016] In one aspect, the compounds of the present invention are i) a human insulin or human insulin analog, and ii) two or more modifying groups M, each modifying group M containing two aryl moieties, with a boron atom attached to each of the two aryl moieties, and are attached to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of the aforementioned human insulin or human insulin analog, or to the epsilon amino group of lysine in the aforementioned human insulin or human insulin analog, optionally via a spacer.

[0017] As can be seen from the examples, compounds having two or more modifying groups M generally exhibit a higher degree of glucose sensitivity (higher glucose coefficient) than compounds having only one modifying group M.

[0018] In one aspect, the present invention provides an intermediate product in the form of a novel insulin analog, including a novel insulin analog containing a peptide spacer.

[0019] In one aspect, the compounds of the present invention activate insulin receptors as a function of glucose concentration in blood and tissues.

[0020] In one aspect, the compounds of the present invention have low availability (low unbound, plasma free fraction) and thus are less active or inactive during hypoglycemic situations, for example, having glucose levels of less than about 3 mM (hypoglycemia).

[0021] In one aspect, the compounds of the present invention have high availability (high unbound, plasma free fraction) and thus are highly active in response to hyperglycemia, for example, having glucose levels above about 10 mM (hyperglycemia).

[0022] In one aspect, the compounds of the present invention exhibit glucose-sensitive albumin binding.

[0023] In another aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the present invention. In another aspect, the present invention relates to a compound according to the present invention for use as a medicament. In another aspect, the present invention relates to a compound according to the present invention for use in the treatment of diabetes. In another aspect, the present invention relates to the medical use of a compound according to the present invention.

[0024] The present invention can also solve further problems that become apparent from the disclosure of the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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[0026] The present invention relates to insulin derivatives. In one aspect, the present invention relates to glucose - sensitive insulin derivatives.

[0027] In one embodiment, the present invention relates to a compound comprising human insulin or an analogue thereof and a modifying group, and the modifying group exhibits an affinity for both glucose and albumin.

[0028] In one embodiment, the modifying group exhibits glucose - sensitive albumin binding.

[0029] In one embodiment, the insulin analogue is an analogue of human insulin (SEQ ID NO: 1 and SEQ ID NO: 2).

[0030] In one embodiment, the human insulin or human insulin analog of the present invention may include a spacer.

[0031] In one embodiment, the present invention provides a compound comprising a human insulin or human insulin analog, and one or more modifying groups M, each of the modifying groups M comprising two aryl moieties, the boron atom being attached to each of the two aryl moieties. Each of the one or more modifying groups M is optionally attached via a spacer to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of the aforementioned human insulin or human insulin analog, or to the epsilon amino group of a lysine in the aforementioned human insulin or human insulin analog.

[0032] In one embodiment, the present invention provides a compound comprising a human insulin or human insulin analog, and two or more modifying groups M, each of the modifying groups M comprising two aryl moieties, the boron atom being attached to each of the two aryl moieties. Each of the two or more modifying groups M is optionally attached via a spacer to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of the aforementioned human insulin or human insulin analog, or to the epsilon amino group of a lysine in the aforementioned human insulin or human insulin analog. Each of the modifying groups M may also be optionally attached via a spacer to the sulfide of a free cysteine of the aforementioned human insulin or human insulin analog.

[0033] General Definitions The term "compound" is used herein to refer to a molecular entity, and thus a "compound" may have different structural elements other than the minimal elements defined for each compound or group of compounds. The term "compound" is also meant to encompass the pharmaceutically relevant forms herein, i.e., the present invention also relates to the compounds defined herein, or pharmaceutically acceptable salts, amides, or esters thereof.

[0034] The term "peptide" or "polypeptide", when used, for example, in the context of the present invention, refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds. In certain embodiments, the peptide consists of amino acids interconnected by peptide bonds.

[0035] The term "analog" generally refers to a peptide having one or more amino acid changes when its sequence is compared to a reference amino acid sequence. An analog that "comprises" certain changes may also contain additional changes when compared to those reference sequences. In certain embodiments, the analog "has" or "comprises" certain changes. In other certain embodiments, the analog "consists of" the changes. When the term "consisting of" or "consisting essentially of" is used in relation to an analog, for example, when an analog consists of or consists essentially of a group of specific amino acid mutations, it goes without saying that the specific amino acid mutations are the only amino acid mutations in the analog. In contrast, an analog that "comprises" a group of specific amino acid mutations may have additional mutations.

[0036] The term "derivative" generally refers to a compound that may be prepared from a native peptide or an analog thereof by chemical modification, particularly by covalent attachment of one or more substituents.

[0037] In the context of the present invention, the modifying group M is a covalently attached substituent.

[0038] The term "amino acid" includes not only proteinogenic (or natural) amino acids (among which are the 20 standard amino acids), but also non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those that are naturally incorporated into proteins. Standard amino acids are those encoded by the genetic code. Non-proteinogenic amino acids are either not found in proteins or are not produced by standard cellular machinery (for example, they may not have been subjected to post-translational modification).

[0039] Generally, amino acid residues (peptide / protein sequences) can be identified by their official names, their one-letter codes, and / or their three-letter codes. These three methods are completely equivalent. In the following text, each amino acid of the peptides of the present invention for which no optical isomers are described should be understood to mean the L-isomer (unless otherwise specified). An amino acid is a molecule containing an amino group and a carboxylic acid group, and optionally one or more additional groups, often referred to as side chains.

[0040] As used herein, the term "amino acid residue" formally refers to an amino acid in which the hydroxy group has been removed from the carboxy group and / or formally the hydrogen atom has been removed from the amino group.

[0041] As is apparent from the following examples, amino acid residues can be identified by their official names, their one-letter codes, and / or their three-letter codes. These three methods are completely equivalent and interchangeable.

[0042] As used herein, the term "aryl" means a cyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. The term "aryl" includes both monovalent, divalent, and polyvalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc. In certain embodiments, aryl is phenyl. As used herein, the term "aryl" also includes "heteroaryl". The term "heteroaryl" means an aromatic monocyclic, bicyclic, or polycyclic ring incorporating one or more (e.g., 1 to 4, particularly 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, or sulfur.

[0043] Insulin As used herein, the term "human insulin" means the human insulin hormone, the structure and properties of which are well-known. Human insulin has two polypeptide chains designated the A-chain and the B-chain. The A-chain is a peptide of 21 amino acids, the B-chain is a peptide of 30 amino acids, and the two chains are connected by disulfide bridges, the first bridge being between cysteine at position 7 of the A-chain and cysteine at position 7 of the B-chain, and the second bridge being between cysteine at position 20 of the A-chain and cysteine at position 19 of the B-chain. The third bridge is present between cysteine at position 6 of the A-chain and cysteine at position 11 of the A-chain.

[0044] 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).

[0045] As used herein, the terms "insulin peptide", "insulin compound", or "insulin" mean a peptide that has insulin activity, i.e., activates the insulin receptor, and is either human insulin or an analog or derivative thereof.

[0046] Insulin analog As used herein, the term "insulin analog" means a modified human insulin in which one or more amino acid residues of insulin have been replaced by other amino acid residues and / or one or more amino acid residues have been deleted from insulin and / or one or more amino acid residues have been added and / or inserted into insulin.

[0047] As used herein, the term "insulin analog" means an insulin analog that exhibits insulin activity, i.e., activates the insulin receptor.

[0048] An insulin analog contains less than 10 amino acid modifications (substitutions, deletions, additions (i.e., extensions), insertions, and any combination thereof) compared to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification compared to human insulin. In one aspect, an insulin analog has less than 10 amino acid modifications (substitutions, deletions, additions (i.e., extensions), insertions, and any combination thereof) compared to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification compared to human insulin.

[0049] Modifications within the insulin molecule are represented by stating the chain (A or B), the position, and the one-letter or three-letter code of the amino acid residue that replaces the native amino acid residue.

[0050] As used herein, terms such as "A1", "A2", and "A3" represent the amino acids at positions 1, 2, and 3, etc. (counting from the N-terminus) of the A-chain of insulin, respectively. Similarly, terms such as B1, B2, and B3 indicate the amino acids at positions 1, 2, and 3, etc. (counting from the N-terminus) of the B-chain of insulin. Using the one-letter code for amino acids, terms such as A21A, A21G, and A21Q specify that the amino acid at position A21 is A, G, and Q, respectively. Using the three-letter code for amino acids, the corresponding expressions are A21Ala, A21Gly, and A21Gln, respectively.

[0051] "desB30" means the native insulin B-chain lacking the B30 amino acid or an analog thereof.

[0052] As used herein, the terms "A-1" or "B-1" indicate the positions of the amino acids that are N-terminal to A1 or B1, respectively. The terms A-2 and B-2 indicate the positions of the first amino acids that are N-terminal to A-1 or B-1, respectively.

[0053] The terms "A22" and "B31" each indicate the position of an amino acid that is C-terminal to A21 or B30, respectively.

[0054] Thus, for example, A14E B1K B2P B25H desB27 desB30 human insulin is an analogue of human insulin, where the amino acid at position 14 of the A chain is replaced by glutamic acid, the amino acid at position 1 of the B chain is replaced by lysine, the amino acid at position 2 of the B chain is replaced by proline, the amino acid at position 25 of the B chain is replaced by histidine, and the amino acids at positions 27 and 30 of the B chain are deleted.

[0055] Examples of insulin analogues having substitutions are those in which Tyr at position A14 is replaced by Glu. Further, the amino acid at position B1 or B4 may be replaced by Lys. The amino acid at position B2 may be replaced by Pro. The amino acid at position B25 may be replaced by His.

[0056] Examples of insulin analogues with deletions are analogues in which the B30 amino acid of human insulin is deleted (desB30 human insulin), insulin analogues in which the B1 amino acid of human insulin is deleted (desB1 human insulin), insulin analogues in which the B1 and B2 amino acids of human insulin are deleted (desB1 desB2 human insulin), and desB27 human insulin.

[0057] Examples of insulin analogues in which the A chain and / or the B chain have an N-terminal extension (i.e., one or more amino acid residues are added to the N-terminus) include human insulin analogues containing A-2K and A-1P, i.e., analogues of human insulin in which the A chain is extended at the N-terminus by KP. Another example is a human insulin analogue in which one glycine residue is added to the N-terminus of the B chain, i.e., the human insulin analogue contains B-1G.

[0058] Examples of insulin analogs in which the A-chain and / or the B-chain have a C-terminal extension (i.e., one or more amino acid residues are added to the C-terminus) are human insulin analogs including A22K.

[0059] Further examples are insulin analogs comprising combinations of the above-described mutations.

[0060] Examples of insulin analogs include desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11), A21Q desB30 human insulin (SEQ ID NO: 3 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 A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14), A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15), 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), A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11), A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19), A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), and A-2K A-1P desB30 human insulin (SEQ ID NO: 7 and SEQ ID NO: 11) are included.

[0061] Spacer As described above, the insulin analog of the present invention contains less than 10 amino acid modifications (substitutions, deletions, extensions, and any combination thereof) compared to human insulin, or less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification compared to human insulin. In addition to a maximum of 9 modifications, the human insulin or human insulin analog of the present invention may contain a spacer at the C-terminus of the A-chain of human insulin or human insulin analog, or at the N-terminus of the B-chain of human insulin or human insulin analog.

[0062] In one embodiment, the spacer is a peptide, which is referred to herein as a spacer peptide or peptide spacer. In another embodiment, the spacer is a non-peptide linker L.

[0063] Peptide spacer 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 to 40 amino acids connected via peptide bonds. In one embodiment, the spacer is a peptide segment consisting of 4 to 24 amino acids connected via peptide bonds.

[0064] 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.

[0065] In one embodiment, the human insulin or human insulin analog of the present invention comprises a peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or the aforementioned human insulin analog. In one embodiment, the aforementioned peptide spacer is (GES) p contains K, where p is an integer from 3 to 12.

[0066] Examples of the peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or the aforementioned human insulin analog include (GES)3K (SEQ ID NO: 29), (GES)6K (SEQ ID NO: 30), and (GES) 12 K (SEQ ID NO: 31).

[0067] In one embodiment, the human insulin or human insulin analog of the present invention comprises a peptide spacer at the N-terminus of the B-chain of the aforementioned human insulin or the aforementioned human insulin analog. In one embodiment, the aforementioned peptide spacer is GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s , or TYFFGRKPD(G4S) tcomprising, wherein each of q, r, s, and t is independently selected from integers of 1 to 5. In another embodiment, the peptide spacer is GKPG, GKP(G4S) q , KP(G4S)3, GKPRGFFYTP(G4S)2, or TYFFGRKPD(G4S)3, wherein q is an integer of 1 to 3.

[0068] Examples of peptide spacers at the N-terminus of the B-chain of the aforementioned human insulin or the aforementioned human insulin analog include GKPG (SEQ ID NO: 32), GKPGGGGS (GKP(G4S)) (SEQ ID NO: 33), GKPGGGGSGGGGS (GKP(G4S)2) (SEQ ID NO: 34), GKPGGGGSGGGGSGGGGS (GKP(G4S)3) (SEQ ID NO: 35), KPGGGGSGGGGSGGGGS (KP(G4S)3) (SEQ ID NO: 36), GKPRGFFYTPGGGGSGGGGS (GKPRGFFYTP(G4S)2) (SEQ ID NO: 37), and TYFFGRKPDGGGGSGGGGSGGGGS (TYFFGRKPD(G)4S)3) (SEQ ID NO: 38) are included.

[0069] Examples of insulin analogs comprising a peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or the aforementioned human insulin analog include A21Q(GES)3K desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 11), A21Q(GES)6K desB30 human insulin (SEQ ID NO: 9 and SEQ ID NO: 11), and A21Q(GES) 12 K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11) are included.

[0070] Examples of insulin analogs comprising a peptide spacer at the N-terminus of the B-chain of the aforementioned human insulin or the aforementioned human insulin analog include B1-KPGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 21), B1-KPGGGGSGGGGS 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), B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25), B1-GKPG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26), B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27), and B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28) are included.

[0071] Linker L In one aspect, the spacer is non-peptide linker L. Various non-peptide linkers are known in the art and can be used in the compounds of the present invention.

[0072] In one embodiment, the human insulin or human insulin analog of the present invention comprises linker L at the N-terminus of the B-chain of the aforementioned human insulin or the aforementioned human insulin analog.

[0073] In one embodiment, the linker is of formula L1,

Chemical formula

[0074] In one embodiment, the linker is of formula L2, [Chemical formula] wherein *1 represents the attachment point to the modifying group A, *2 represents the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of human insulin or a human insulin analog, and u is 1, 2, or 3. In one embodiment, u is 2 or 3.

[0075] In one embodiment, the linker is of formula L3, [Chemical formula] wherein *1 represents the attachment point to the modifying group A, *2 represents the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of human insulin or a human insulin analog, and v is 2 or 3.

[0076] Insulin derivative As used herein, the term "insulin derivative" means a chemically modified insulin or an analog thereof, and the modification is in the form of attachment of one or more modifying groups M.

[0077] In one embodiment, each of the one or more modifying groups M is optionally attached via a spacer to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of human insulin or a human insulin analog, or to the epsilon amino group of lysine in human insulin or a human insulin analog.

[0078] In one embodiment, each modifying group M is attached to an attachment point selected from one of the following groups. a) the amino group of the N-terminal amino acid residue of the A-chain of the aforementioned human insulin or human insulin analog, b) the epsilon amino group of lysine at position 22 of the A-chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in the aforementioned optional peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or human insulin analog c) the amino group of the N-terminal amino acid residue of the B-chain of the aforementioned human insulin or human insulin analog, the epsilon-amino group of the lysine residue at position 1 or 4 of the B-chain of the aforementioned human insulin analog, the epsilon-amino group of lysine in the aforementioned optional peptide spacer at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, or the terminal amino group marked with *1 of the aforementioned optional linker L at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, and d) the epsilon-amino group of lysine at position 22 or 29 of the B-chain of the aforementioned human insulin or human insulin analog.

[0079] In one embodiment, one or fewer modifying groups M are attached to the attachment points in each of groups a), b), c) and d).

[0080] In one embodiment, the compound of the present invention contains two modifying groups M, one modifying group M is attached to the amino group of the lysine residue at position 1 or 4 of the B-chain of the human insulin analog, or the epsilon-amino group of lysine in an optional peptide extension at the N-terminus of the B-chain of the human insulin or human insulin analog, and the other modifying group M is attached to the epsilon-amino group of lysine at position 29 of the B-chain of the human insulin or human insulin analog.

[0081] In one embodiment, the compound of the present invention has exactly two modifying groups M, one modifying group M is attached to the amino group of the lysine residue at position 1 or 4 of the B-chain of the human insulin analog, or the epsilon-amino group of lysine in an optional peptide extension at the N-terminus of the B-chain of the human insulin or human insulin analog, and the other modifying group M is attached to the epsilon-amino group of lysine at position 29 of the B-chain of the human insulin or human insulin analog.

[0082] In one embodiment, the compound of the present invention contains two modifying groups M, one of the modifying groups M is attached to the amino group of the N-terminal amino acid residue of the A chain of the aforementioned human insulin or human insulin analog, and the other modifying group M is attached to the epsilon amino group of lysine at position 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0083] In one embodiment, the compound of the present invention has exactly two modifying groups M, one of the modifying groups M is attached to the amino group of the N-terminal amino acid residue of the A chain of the aforementioned human insulin or human insulin analog, and the other modifying group M is attached to the epsilon amino group of lysine at position 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0084] In one embodiment, the compound of the present invention contains two modifying groups M, one of the modifying groups M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in an optional peptide spacer at the C-terminus of the A chain of the aforementioned human insulin or human insulin analog, and the other modifying group M is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0085] In one embodiment, the compound of the present invention has exactly two modifying groups M, one of the modifying groups M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in an optional peptide spacer at the C-terminus of the A chain of the aforementioned human insulin or human insulin analog, and the other modifying group M is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0086] In one embodiment, the compound of the present invention contains one modifying group M, and the modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analogs, or the epsilon amino group of lysine at position 29 of the B chain of the aforementioned human insulin or human insulin analogs.

[0087] In one embodiment, the compound of the present invention has exactly one modifying group M, and the modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analogs, or the epsilon amino group of lysine at position 29 of the B chain of the aforementioned human insulin or human insulin analogs.

[0088] In one embodiment, the compound of the present invention contains three or four modifying groups M. The first modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analogs. The second modifying group M is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analogs. Each of the remaining modifying groups M is attached to the amino acid of the N-terminal amino acid residue of the A chain of the aforementioned human insulin or human insulin analogs, the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analogs, or the terminal amino group marked with *1 of the aforementioned optional linker L at the N-terminus of the B chain of the aforementioned human insulin or human insulin analogs.

[0089] In one embodiment, the compound of the present invention has exactly three or four modifying groups M, wherein the first modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analog, the second modifying group M is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog, and each of the remaining modifying groups M is attached to the amino acid of the N-terminal amino acid residue of the A chain of the aforementioned human insulin or human insulin analog, the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog, or the terminal amino group marked with *1 of the aforementioned optional linker L at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog.

[0090] Modifying group M The compound of the present invention contains one or more modifying groups M. In one embodiment, the compound of the present invention contains one, two, three, or four modifying groups M. In one embodiment, the compound of the present invention contains two or more modifying groups M. In one embodiment, the compound of the present invention contains two, three, or four modifying groups M. In one embodiment, the compound of the present invention contains two modifying groups M. In one embodiment, the compound of the present invention has exactly two modifying groups M. One or more of the modifying groups may be the same or different. Two or more of the modifying groups may be the same or different. In one embodiment, the modifying groups are the same.

[0091] Some of the modifying groups contain one or more amino acid residues. Each of these amino acid residues can independently be in the D or L form of the respective amino acid residue, i.e., each of the chiral atoms within the modifying group can independently be in the (R) or (S) form. In one embodiment, the amino acid residues of the modifying group are L-amino acid residues.

[0092] Each modifying group M contains a diboron moiety, and the diboron moiety (i.e., the modifying group M) contains two aryl moieties, with the boron atoms attached to each of the two aryl moieties. The boron atoms may be part of a boronic acid (or boronate depending on pKa / pH), or may be part of a boroxol (or boroxolate depending on pKa / pH).

[0093] The term "comprising" or "comprises" with respect to a particular feature is to be construed to mean that the subject in question includes those particular features but does not exclude the presence of other features. Thus, the modifying group M may have two or more aryl moieties, and the boron atoms are attached to each of the aryl moieties. In one embodiment, the modifying group has exactly two aryl moieties, and the boron atoms are attached to each of the two aryl moieties. In one embodiment, the modifying group has exactly four aryl moieties, and the boron atoms are attached to each of the four aryl moieties.

[0094] The diboronates / boroxols of the present invention bind more strongly to glucose than the monoboronates, as shown in Example A. Furthermore, surprisingly, the diboron compounds of the present invention are capable of binding to human serum albumin (HSA) and thus have a dual action, because the HSA-binding is also glucose-sensitive (the HSA-binding fraction of the diboron peptide is inactivated by blocking the receptor-binding site on the peptide) (data shown in Example B).

[0095] In one embodiment, the modifying group is of formula M1, [Chemical formula] represents the D- or L-amino acid form, wherein n represents an integer in the range of 1 to 4, W1 is absent and represents the attachment point * to the aforementioned human insulin or human insulin analog, or W1 is, NH-CH2-C(=O)-*, NH-CH2CH2-C(=O)-*, the D or L form of NH-CH(COOH)-CH2CH2-C(=O)-*, the D or L form of NH-CH(COOH)-CH2CH2-C(=O)-NH-CH2CH2-C(=O)-*, or NH-CH2CH2-C(=O)-NH-(CH2)2-O-(CH2)2-O-CH2-CO-*, wherein * represents an attachment point to human insulin or a human insulin analog, R1 is

Chemical formula

[0096] In another embodiment, the modifying group is of formula M1, wherein Y1 and Y2 are H, Y3 is F or CF3, Y4 is H or F, Y5 is H, and Y6 is F.

[0097] In yet another embodiment, the modifying group is of formula M1, wherein n is 1, W1 represents NH-CH2CH2-C(=O)-* or the L form of NH-CH(COOH)-CH2CH2-C(=O)-*, wherein * represents the attachment point to the aforementioned human insulin or human insulin analog, and R1 is

Chemical formula

[0098] In one embodiment, the modifying group is of formula M2,

Chemical formula

Chemical formula

[0099] In another embodiment, the modifying group is of formula M2, wherein Y7 is H, Y8 is H, Cl, 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.

[0100] In yet another embodiment, the modifying group is of formula M2, wherein W2 does not exist and represents an attachment point * to the aforementioned human insulin or human insulin analog, or W2 represents the L form of NH-CH(COOH)-CH2CH2-C(=O)-*, where * represents an attachment point to the aforementioned human insulin or human insulin analog, and R2 is

Chemical formula

[0101] In one embodiment, the modifying group is of formula M3

Chemical formula

[0102] In another embodiment, the modifying group is of formula M3, where Y13 is H or F, Y14 is H or CF3, provided that only one of Y13 and Y14 is H.

[0103] In one embodiment, the modifying group is of formula M4,

Chemical formula

[0104] In another embodiment, the modifying group is of formula M4, where Y15 and Y16 are independently selected from H and F.

[0105] In yet another embodiment, the modifying group is of formula M4, where Y15 is H and Y16 is F.

[0106] In one embodiment, the modifying group is of formula M5,

Chemical formula

[0107] In one embodiment, the modifying group is of formula M6,

Chemical formula

[0108] In another embodiment, the modifying group has the formula M6, where Y17 is H or F and Y18 is H or F.

[0109] In one embodiment, the modifying group has the formula M7

Chemical formula

[0110] In one embodiment, the modifying group has the formula M8

Chemical formula

[0111] In another embodiment, the modifying group has the formula M8, where Y19 is CF3 or SF5.

[0112] In yet another embodiment, the modifying group has the formula M8, where Y19 is CF3.

[0113] In one embodiment, the modifying group is of formula M9,

Chemical formula

[0114] 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.

[0115] In one embodiment, the modifying group is of formula M10,

Chemical formula

[0116] In one embodiment, the modifying group is of formula M11,

Chemical formula

[0117] The compound of the present invention In one embodiment, the compound of the present invention comprises a human insulin or human insulin analog, and one or more modifying groups M, each of the modifying groups M comprising two aryl moieties, a boron atom being attached to each of the two aryl moieties, and each of the one or more modifying groups M being optionally attached via a spacer to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of the aforementioned human insulin or human insulin analog, or to the epsilon amino group of lysine of the aforementioned human insulin or human insulin analog.

[0118] In another embodiment, the compound of the present invention comprises human insulin or a human insulin analog, and two modifying groups M, each of the modifying groups M comprises two aryl moieties, the boron atom is attached to the two aryl moieties, the first modifying group M is attached to the epsilon amino group of lysine at position 1 or 4 of the B chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in an optional peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, and the second modifying group is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0119] In another embodiment, the compound of the present invention comprises human insulin or a human insulin analog, and two modifying groups M, each of the modifying groups M comprises two aryl moieties, the boron atom is attached to each of the two aryl moieties, the first modifying group M is attached to the amino group of the N-terminal amino acid residue of the A chain of the aforementioned human insulin or human insulin analog, and the second modifying group is attached to the epsilon amino group of lysine at position 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0120] In another embodiment, the compound of the present invention comprises human insulin or a human insulin analog, and two modifying groups M, each of the modifying groups M comprises two aryl moieties, the boron atom is attached to the two aryl moieties, the first modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in an optional peptide spacer at the C-terminus of the A chain of the aforementioned human insulin or human insulin analog, and the second modifying group is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0121] In another embodiment, the compound of the present invention comprises human insulin or a human insulin analog, and one modifying group M, wherein the modifying group M comprises two aryl moieties, a boron atom is attached to the two aryl moieties, and the modifying group M is attached to the epsilon amino group of lysine at position 22 of the A chain of the aforementioned human insulin analog, or the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog.

[0122] In one embodiment, the present invention relates to a compound independently selected from the group of 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.

[0123] In one embodiment, the present invention relates to a compound independently selected from the group of 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.

[0124] In one embodiment, the compound of the present invention is the compound of Example 181. In one embodiment, the compound of the present invention is the compound of 205. In one embodiment, the compound of the present invention is the compound of 210. In one embodiment, the compound of the present invention is the compound of 211. In one embodiment, the compound of the present invention is the compound of 227. In one embodiment, the compound of the present invention is the compound of 233. In one embodiment, the compound of the present invention is the compound of 234. In one embodiment, the compound of the present invention is the compound of 239. In one embodiment, the compound of the present invention is the compound of 240. In one embodiment, the compound of the present invention is the compound of 241. In one embodiment, the compound of the present invention is the compound of 272. In one embodiment, the compound of the present invention is the compound of 273. In one embodiment, the compound of the present invention is the compound of 280. In one embodiment, the compound of the present invention is the compound of 284. In one embodiment, the compound of the present invention is the compound of 285. In one embodiment, the compound of the present invention is the compound of 288. In one embodiment, the compound of the present invention is the compound of 291. In one embodiment, the compound of the present invention is the compound of 300. In one embodiment, the compound of the present invention is the compound of 301. In one embodiment, the compound of the present invention is the compound of 324. In one embodiment, the compound of the present invention is the compound of 327. In one embodiment, the compound of the present invention is the compound of 331. In one embodiment, the compound of the present invention is the compound of 333. In one embodiment, the compound of the present invention is the compound of 335.

[0125] Intermediate product The present invention further provides an intermediate product in the form of a novel insulin analogue or an insulin analogue containing a peptide spacer.

[0126] Accordingly, the present invention also relates to an intermediate product independently selected from the group consisting of the following. 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), A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), A21Q(GES)3K desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 11), A21Q(GES)6K desB30 human insulin (SEQ ID NO: 9 and SEQ ID NO: 11), A21Q(GES)12K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11), 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), B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25), B1-GKPG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26), B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27), and B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28) are included.

[0127] Insulin function The relative binding affinity of an insulin analogue for the human insulin receptor (IR) can be determined by competitive binding in a scintillation proximity assay (SPA) as described in Example B.

[0128] In one embodiment, a compound of the invention has the ability to bind to the insulin receptor. In one embodiment, a compound of the invention has a higher insulin receptor affinity in the presence of 20 mM glucose than in the absence of glucose.

[0129] The AKT phosphorylation assay described in Example C and the lipidogenesis assay described in Example D can be used as a measure of the functional (agonist) activity of an insulin analogue.

[0130] Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising, for example, an analogue of the invention, or a compound of the invention in the form of a pharmaceutically acceptable salt, amide, or ester thereof, and one or more pharmaceutically acceptable excipients. Such compositions may be prepared as is known in the art.

[0131] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. Excipients may be inert substances, inactive substances, and / or substances that are not pharmaceutically active. Excipients can serve various purposes, for example, as carriers, vehicles, diluents, and / or function to improve the administration and / or absorption of the active substance. Non-limiting examples of excipients are solvents, diluents, buffers, preservatives, isotonicity regulators, chelating agents, and stabilizers. The formulation of pharmaceutically active ingredients with various excipients is known in the art (see, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions)).

[0132] The composition of the present invention may be in the form of a liquid preparation, i.e., an aqueous preparation containing water. The liquid preparation may be a solution or a suspension. The composition of the present invention may be for parenteral administration, for example, by subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0133] Aryl boron compounds generally have low stability in aqueous solution at a pH near neutral value. The C-B bond can be hydrolyzed to give a phenyl residue and free borate, Ph-H + B(OH)3, or the compound can be oxidized to give a phenol residue + free borate, Ph-OH + B(OH)3. Certain preferred diboron compounds and diboron-insulin conjugates of the present invention have been found to be more stable than other aryl-boron and general aryl-boron of the present invention. The stability can be evaluated, for example, by measuring the purity of the insulin derivative after being in aqueous solution at neutral pH at 25 °C or 37 °C for a long period, for example, one week.

[0134] Pharmaceutical indication Diabetes The term "diabetes" or "diabetes mellitus" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. This term is used for metabolic disorders in which the amount of insulin produced by the pancreas is insufficient or the body's cells cannot respond properly to insulin, thus preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.

[0135] Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is usually caused by beta-cell destruction that results in absolute insulin deficiency.

[0136] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance and hence relative insulin deficiency and / or major insulin secretory failure with insulin resistance.

[0137] Other indications In one embodiment, the compounds according to the invention are used in the preparation of a medicament for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes.

[0138] In another embodiment, the compounds according to the invention are used as a medicament for delaying or preventing the progression of the disease in type 2 diabetes.

[0139] In one embodiment of the invention, the compound is for use as a medicament for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes.

[0140] In a further embodiment, the invention relates to a method for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to the invention for such treatment.

[0141] Route of administration The term "treatment" is meant to include both the prevention and the minimization of the referenced disease, disorder, or condition (i.e., "treatment" refers to both prophylactic and therapeutic administration of a compound of the invention or a composition comprising a compound of the invention, unless otherwise indicated by the context or clearly inconsistent).

[0142] The route of administration may be any route that effectively transports the compound of the invention to the desired or appropriate location in the body, for example parenterally (such as subcutaneously, intramuscularly, or intravenously).

[0143] For parenteral administration, the compounds of the invention are formulated similarly to known insulin formulations. Further, for parenteral administration, the compounds of the invention are administered similarly to known insulin administrations, and physicians are familiar with this procedure.

[0144] The amount of the compound of the invention to be administered, the determination of the frequency of administration of the compound of the invention, and the selection, optionally together with another antidiabetic compound, of which compound(s) of the invention to administer are determined in consultation with a physician skilled in the treatment of diabetes.

[0145] Certain features of the invention are illustrated and described herein, but many modifications, substitutions, changes, and equivalents will occur to those skilled in the art. Accordingly, it is to be understood that the appended embodiments are intended to cover all such modifications and changes as fall within the true spirit and scope of the invention.

[0146] Embodiments The present invention is further illustrated by the following non-limiting embodiments of the invention: 1. A compound comprising: i) human insulin or a human insulin analog, and ii) one or more modifying groups M, each of the modifying groups M comprising two aryl moieties, a boron atom being attached to each of the two aryl moieties, wherein each of the one or more modifying groups M is optionally attached via a spacer to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of the aforementioned human insulin or human insulin analog, or to the epsilon amino group of a lysine in the aforementioned human insulin or human insulin analog.

[0147] 2. Each of the modifying groups M is independently

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0148] 3. Each of the modifying groups M is independently

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0149] Each of the modifying groups M is independently

Chemical formula

Chemical formula

[0150] 5. The compound according to any one of Embodiments 1 to 4, wherein the modifying group M is the same.

[0151] 6. The aforementioned human insulin or human insulin analog is optionally a) a peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or human insulin analog, wherein the peptide spacer contains (GES) p K, where p is an integer from 3 to 12, the peptide spacer, or b) a peptide spacer or linker L at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, wherein the peptide spacer contains GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s , or TYFFGRKPD(G4S) t , and each of q, r, s, and t is independently selected from integers from 1 to 5, a peptide spacer or linker L, a spacer selected from the group, wherein the aforementioned linker L is [Chemical formula] being, wherein *1 indicates the attachment point to the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, formula L1, [Chemical formula] being, wherein *1 indicates the attachment point to the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, and u is 1, 2, or 3, formula L2, [Chemical formula] being, wherein *1 indicates the attachment point to the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, and v is 2 or 3, formula L3, selected from the compounds according to any one of Embodiments 1 to 5.

[0152] 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.

[0153] 8. The compound according to any one of Embodiments 1 to 7, wherein the chiral amino acid is in the L-form.

[0154] 9. Each modifying group M is from the following group, a) the amino group of the N-terminal amino acid residue of the A-chain of the aforementioned human insulin or human insulin analog, b) the epsilon amino group of lysine at position 22 of the A-chain of the aforementioned human insulin analog, or the epsilon amino group of lysine in the aforementioned optional peptide spacer at the C-terminus of the A-chain of the aforementioned human insulin or human insulin analog, c) the amino group of the N-terminal amino acid residue of the B chain of the aforementioned human insulin or human insulin analog, the epsilon amino group of the lysine residue at position 1 or 4 of the B chain of the aforementioned human insulin analog, the epsilon amino group of lysine in the aforementioned optional peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, or the terminal amino group marked with *1 of the aforementioned optional linker L at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, and d) the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog, attached to an attachment point selected from one of them, the compound according to any one of Embodiments 1 to 8.

[0155] 10. The compound according to Embodiment 9, wherein one or less of the following modifying groups M is attached to the attachment point in each of groups a), b), c) and d).

[0156] 11. The compound according to any one of Embodiments 1 to 10, having exactly one, two, three, or four modifying groups M.

[0157] 12. The compound according to any one of Embodiments 1 to 10, comprising at least two modifying groups M.

[0158] 13. The compound according to any one of Embodiments 1 to 10, having exactly two, three, or four modifying groups M.

[0159] 14. The compound according to any one of Embodiments 1 to 10, having exactly two modifying groups M.

[0160] 15. The compound according to any one of Embodiments 1 to 14, wherein the aforementioned human insulin or human insulin analog is a human insulin analog containing desB30.

[0161] 16. The aforementioned human insulin or human insulin analog is desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11), A21Q desB30 human insulin (SEQ ID NO: 3 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 A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14), A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15), 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), A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11), A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19), A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), and A-2K A-1P desB30 human insulin (SEQ ID NO: 7 and SEQ ID NO: 11), a human insulin selected from the group consisting of: the compound according to any one of Embodiments 1 to 15.

[0162] 17. The compound according to Embodiment 1, wherein i) is human insulin or a human insulin analog, optionally the aforementioned human insulin or human insulin analog, and comprises a spacer selected from a peptide spacer or linker L at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, the foregoing peptide spacer is GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s , or TYFFGRKPD(G4S) t and each of q, r, s, and t is independently selected from the integers 1 to 5, the foregoing linker L is [Chemical formula] wherein *1 indicates the attachment point for the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the foregoing human insulin or human insulin analog, formula L1 [Chemical formula] wherein *1 indicates the attachment point for the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the foregoing human insulin or human insulin analog, and u is 1, 2, or 3, formula L2, and [Chemical formula] wherein *1 indicates the attachment point for the modifying group M, and *2 indicates the attachment point to the amino group of the amino acid residue at the N-terminus of the B-chain of the foregoing human insulin or human insulin analog, and v is 2 or 3, formula L3, selected from a human insulin or human insulin analog, ii) two, three, or four modifying groups M, each of the modifying groups M being independently [Chemical formula] wherein represents the D- or L-amino acid form, wherein n represents an integer in the range of 1 to 4, W1 is absent and represents an attachment point* to the aforementioned human insulin or human insulin analog, or W1 is NH-CH2CH2-C(=O)-*, represents the D or L form of NH-CH(COOH)-CH2CH2-C(=O)-*, wherein * represents an attachment point to the aforementioned human insulin or human insulin analog, R1 is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0163] 18. The compound according to embodiment 17, wherein one or less modifying groups M are attached to the attachment points in each of groups a), b), c) and d).

[0164] 19. The compound has exactly two modifying groups M, one modifying group M is attached to the epsilon amino group of lysine at position 22 or 29 of the B chain of the aforementioned human insulin or human insulin analog, and one modifying group is the amino group of the N-terminal amino acid residue of the B chain of the aforementioned human insulin or human insulin analog, the epsilon amino group of the lysine residue at position 1 or 4 of the B chain of the aforementioned human insulin analog, the epsilon amino group of lysine in the aforementioned optional peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, or the *1-marked terminal amino group of the aforementioned optional linker L at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, and the compound according to embodiment 17 or 18.

[0165] 20. The compound according to any one of embodiments 17 to 19, i) a human insulin or human insulin analog, wherein the aforementioned human insulin or human insulin analog optionally contains a peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, the aforementioned peptide spacer contains GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s , or TYFFGRKPD(G4S) t , and in the formula, q is an integer from 1 to 3, r is 3, s is 2, t is 3, a human insulin or human insulin analog, and ii) two modifying groups M, each of the modifying groups M being independently

Chemical formula

[0166] 21. A compound according to any one of embodiments 17 to 20, i) a human insulin analog, wherein the aforementioned human insulin analog comprises a peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, and the aforementioned peptide spacer is GKP(G4S) q , or KP(G4S) r wherein q is an integer from 1 to 3 and r is 3, a human insulin analog, and ii) two modifying groups M, independently

Chemical formula

Chemical formula

[0167] 22. A compound according to any one of Embodiments 17 to 21, wherein i) a human insulin analog, wherein the aforementioned human insulin analog optionally comprises a peptide spacer at the N-terminus of the B chain of the aforementioned human insulin or human insulin analog, the aforementioned peptide spacer comprises GKPG, GKP(G4S) q , KP(G4S) r , GKPRGFFYTP(G4S) s , or TYFFGRKPD(G4S) t , and in the formula, q is an integer from 1 to 3, r is 3, s is 2, and t is 3, a human insulin analog, ii) two modifying groups M, each of the modifying groups M independently

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0168] 23. The compound according to any one of Embodiments 17 to 22, i) A human insulin analog, wherein the aforementioned human insulin analog has a peptide spacer at the N-terminus of the B-chain of the aforementioned human insulin or human insulin analog, and the aforementioned peptide spacer is GKP(G4S) q or KP(G4S) r wherein q is an integer from 1 to 3 and r is 3, a human insulin analog ii) Two modifying groups M, independently

Chemical formula

Chemical formula

[0169] 24. The compound according to any one of Embodiments 17 to 23, wherein the chiral amino acid is in the L form

[0170] 25. The compound according to any one of Embodiments 17 to 24, wherein the compound has exactly two modifying groups M

[0171] 26. The compound according to any one of Embodiments 17 to 25, wherein the modifying groups M are the same

[0172] 27. The compound according to any one of embodiments 17 to 26, wherein the human insulin analog is desB30.

[0173] 28. The above-mentioned human insulin or human insulin analog is 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), A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11), A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19), and A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), and is a human insulin analog selected from the group consisting of, the compound according to any one of embodiments 17 to 27.

[0174] 29. The above-mentioned human insulin analog containing the above-mentioned spacer is 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 a compound according to any one of embodiments 17 to 28, selected from the group consisting of B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25).

[0175] 30. The compound is a compound according to any one of embodiments 17 to 29, selected from the group consisting of 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.

[0176] 31. The compound is a compound according to any one of embodiments 17 to 30, selected from the group consisting of 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.

[0177] 32. The compound is the compound of embodiment 280, a compound according to any one of embodiments 17 to 31.

[0178] 33. The compound is the compound of embodiment 284, a compound according to any one of embodiments 17 to 31.

[0179] 34. The compound is the compound of embodiment 285, a compound according to any one of embodiments 17 to 31.

[0180] 35. The compound is the compound of embodiment 288, a compound according to any one of embodiments 17 to 31.

[0181] 36. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 291.

[0182] 37. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 300.

[0183] 38. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 301.

[0184] 39. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 324.

[0185] 40. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 327.

[0186] 41. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 331.

[0187] 42. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 333.

[0188] 43. The compound according to any one of embodiments 17 to 31, wherein the compound is the compound of embodiment 335.

[0189] 44. The compound according to embodiment 1, i) human insulin or a human insulin analog, and ii) two modifying groups M, independently,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0190] 45. The compound according to Embodiment 44, wherein the modifying groups M are identical.

[0191] 46. The compound according to Embodiment 44 or 45, wherein the aforementioned human insulin or human insulin analog is a human insulin analog containing desB30.

[0192] 47. The compound according to Embodiment 46, wherein the aforementioned human insulin analog is desB30 human insulin.

[0193] 48. The compound according to Embodiment 1, i) A human insulin or human insulin analog, wherein the aforementioned human insulin or human insulin analog optionally contains a peptide spacer at the C-terminus of the A chain of the aforementioned human insulin or human insulin analog, and the aforementioned peptide spacer contains (GES) p K, wherein p is an integer from 3 to 12, a human insulin or human insulin analog. ii) Two modifying groups M, independently,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0194] 49. The compound according to embodiment 48, wherein the chiral amino acid is in the L-form.

[0195] 50. The compound according to embodiment 48 or 49, wherein the modifying group M is the same.

[0196] 51. The compound according to any one of embodiments 48 to 51, wherein the aforementioned human insulin or human insulin analog is a human insulin analog containing desB30.

[0197] 52. The aforementioned human insulin or human insulin analog is A21Q desB30 human insulin (SEQ ID NO: 3 and SEQ ID NO: 11), A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14), A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15), A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11), and A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), and is selected from the group consisting of the compounds according to any one of embodiments 48 to 51.

[0198] 53. The compound is The compound selected from the group consisting of the compound of Example 227, the compound of Example 239, the compound of Example 240, the compound of Example 241, and the compound of Example 272, and is the compound according to any one of embodiments 48 to 52.

[0199] 54. The compound according to embodiment 1, wherein i) a human insulin or human insulin analog, and ii) one modifying group M, wherein

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0200] 55. The compound according to Embodiment 54, wherein the aforementioned human insulin or human insulin analog is a human insulin analog comprising A22K and desB30.

[0201] 56. The compound according to any one of Embodiments 1 to 55, wherein the compound has the ability to bind to the insulin receptor.

[0202] 57. The compound according to any one of Embodiments 1 to 55, wherein the compound has a higher insulin receptor affinity in the presence of 20 mM glucose than in the absence of glucose.

[0203] 58. The compound according to any one of Embodiments 1 to 55, wherein the compound has an insulin receptor affinity at least 3-fold higher in the presence of 20 mM glucose than in the absence of glucose.

[0204] 59. The compound according to any one of Embodiments 1 to 55, wherein the compound has an insulin receptor affinity at least 10-fold higher in the presence of 20 mM glucose than in the absence of glucose.

[0205] 60. The compound according to any one of Embodiments 1 to 55, wherein the compound has an insulin receptor affinity at least 15-fold higher in the presence of 20 mM glucose than in the absence of glucose.

[0206] 61. A composition comprising the compound according to any one of Embodiments 1 to 55.

[0207] 62. The compound according to any one of Embodiments 1 to 55 for use as a medicament.

[0208] 63. A compound according to any one of embodiments 1 to 55 for use in the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0209] 64. Use of a compound according to any one of embodiments 1 to 55 or a composition according to embodiment 61 for the manufacture of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

[0210] 65. A method for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, 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 to 55 or a composition according to embodiment 61.

Examples

[0211] Materials and Methods

[0212]

Table 1-1

Table 1-2

[0213] Preparation of Insulin Variants [Example 1] Expression of Insulin Variants in Yeast and Transformation with ALP, etc. The insulin analog was expressed in yeast using well-known techniques as disclosed, for example, in WO2017 / 032798. More specifically, the insulin analog was expressed as a single-chain precursor that was isolated by ion-exchange capture and cleaved to a two-chain insulin analog by treatment with ALP as described below.

[0214] Capture of the precursor on SP Sepharose BB: The yeast supernatant was loaded onto a column packed with SP Sepharose BB at a flow rate of 10 - 20 CV / hour. Washing was performed with 0.1 M citric acid, pH 3.5, and with 40% EtOH. The analog was eluted with 0.2 M sodium acetate pH 5.5 / 35% EtOH.

[0215] ALP digestion: 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 monitored by UPLC. To prepare for RP-HPLC purification, the ALP cleavage pool was adjusted to pH 2.5 and diluted two-fold.

[0216] RP-HPLC purification: Purification was performed by RP-HPLC C18 as follows: Column: 15um C18 50×250mm 200Å Buffers: A: 0.2% formic acid, 5% EtOH, B: 0.2% formic acid, 50% EtOH Gradient: 20 - 55% B buffer. Gradient: 20 CV Flow rate 20 CV / hour Loading g ~5 g / L resin Fractions were analyzed by UPLC, pooled, and lyophilized.

[0217] Insulin analogs were prepared and used in the following examples: desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 11), A14E B1K B2P B25H desB27 desB30 human insulin (SEQ ID NO: 4 and SEQ ID NO: 13), A14E A22K B25H desB27 desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 14), A14E A22K B25H B27P B28G desB30 human insulin (SEQ ID NO: 5 and SEQ ID NO: 15), 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), A22K desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 11), A22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 19), A22K B22K B29R desB30 human insulin (SEQ ID NO: 6 and SEQ ID NO: 20), A-2K A-1P desB30 human insulin (SEQ ID NO: 7 and SEQ ID NO: 11), A21Q(GES)3K desB30 human insulin (SEQ ID NO: 8 and SEQ ID NO: 11), A21Q(GES)6K desB30 human insulin (SEQ ID NO: 9 and SEQ ID NO: 11), A21Q(GES)12K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11), 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), B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25), B1-GKPG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26), B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27), and B1-TYFFGRKPDGGGGSGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28).

[0218] B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin means desB30 human insulin extended from B1 having GKPRGFFYTPGGGGSGGGGS (described from the new N-terminus G having the C-terminus S connected to B1 of desB30 human insulin). A21Q(GES)3K desB30 human insulin means insulin extended from A21Q having GESGESGESK (described from the new N-terminus G connected to the C-terminus A21Q). The same is true for other B1 and A21 extended insulin analogs. B-1 means the position at the N-terminus from B1. For example, B-1G means the N-terminal extension of insulin B1 having G.

[0219] Preparation of Building Blocks Intermediates and final products are given numbers within each example to make reading easier. The same numbers are used across examples, but the numbers are clear within each example.

[0220] [Example 2][O-Succinimidyl 3,5-bis[[[3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]amino]methyl]benzoate] [Chemical Structure] 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 an off-white powder. Yield: 10.4 g (85%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.93 - 7.80 (m, 2H); 7.75 (d, J = 9.4 Hz, 1H); 1.39 (s, 12H).

[0221] 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.HCl, 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 × 120 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (40 mL) and precipitated by adding cyclohexane (130 mL). The product was collected by filtration, washed with cyclohexane, and dried under vacuum to give succinimidyl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (3) as a white powder. Yield: 13.9 g (99%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.93 - 7.84 (m, 2H); 7.77 (d, J = 9.4 Hz, 1H); 2.92 (s, 4H); 1.39 (s, 12H).

[0222] 3,5-Dimethylbenzoic acid (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 × 300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain methyl 3,5-dimethylbenzoate (5) as a pale yellow oil. Yield: 29.3 g (97%). 1 H NMR spectrum (300 MHz, CDCl3, δH): 7.67 (s, 2H); 7.19 (s, 1H); 3.91 (s, 3H); 2.37 (s, 6H).

[0223] A mixture of methyl 3,5-dimethylbenzoate 5 (29.3 g, 178 mmol), N-bromosuccinimide (NBS, 111 g, 623 mmol), and 1 spatula of azobisisobutyronitrile in methyl formate (450 mL) was irradiated with visible light while heating under 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 sulfate (2 × 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: hexane / ethyl acetate 15:1). The product was crystallized from an ethyl acetate / cyclohexane mixture to obtain methyl 3,5-bis(bromomethyl)benzoate (6) as a white solid. Yield: 25.6 g (45%). RF (SiO2, hexane / ethyl acetate 9:1): 0.50. 1 H NMR spectrum (300 MHz, CDCl3, δH): 8.02 - 7.95 (m, 2H); 7.62 (s, 1H); 4.51 (s, 4H); 3.94 (s, 3H).

[0224] A suspension of the above bromide 6 (25.3 g, 78.6 mmol) and sodium diformylamide (20.9 g, 220 mmol) in dry acetonitrile (350 mL) was refluxed for 4 h. After removing the white solid by filtration, the solvent was evaporated. Recrystallization from an ethyl acetate / cyclohexane mixture gave 3,5-bis((N-formylformamido)methyl)benzoate (7) as a white powder. 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).

[0225] 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 h. After cooling to room temperature, a stream of air was passed through the solution. The product began 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(aminomethyl)benzoic acid dihydrochloride (8) as a white powder. Yield: 17.1 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.26 (bs, 1H); 8.65 (bs, 6H); 8.10 (s, 2H); 7.88 (s, 1H); 4.08 (s, 4H).

[0226] The dihydrochloride 8 (2.08 g, 8.20 mmol) was dissolved in water (20 mL). Then, N,N-diisopropylethylamine (5.73 mL, 32.9 mmol), N,N-dimethylformamide (40 mL), and the activated ester (3, 5.97 g, 16.4 mmol) were added. The mixture was stirred overnight at room temperature and then acidified with 1 M aqueous hydrochloric acid. The solvent was co-evaporated three 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 three times from toluene. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 × 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 began to precipitate. Cyclohexane was added (170 mL). 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)benzamide)methyl)benzoic acid (9) as a white powder. Yield: 4.18 g (75%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0227] The above acid 9 (4.17 g, 6.20 mmol) was dissolved in an acetonitrile / N,N-dimethylformamide mixture (4:1, 100 mL). N-Hydroxysuccinimide (HOSu, 0.85 g, 7.40 mmol) was added. The mixture was cooled to 0 °C, and then N,N-dicyclohexylcarbodiimide (DCC, 1.53 g, 7.40 mmol) was added. The mixture was stirred at 0 °C for 30 minutes and at room temperature overnight. The 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 × 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 began to precipitate. Cyclohexane was added (170 mL). The precipitate was collected by filtration, washed with cyclohexane, and dried under vacuum to give O-succinimidyl 3,5-bis[[[3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]amino]methyl]benzoate (10) as a white powder. Yield: 4.62 g (97%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 value 773.4.

[0228] [Example 3][O-Succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide-Lys-beta-Ala] [Chemical formula] 2-Chlorotrityl resin, 100 - 200 mesh, 1.8 mmol / g (53.3 g, 96.0 mmol), was allowed to swell in dry dichloromethane (350 mL) for 20 minutes. The resin was then filtered and washed with dichloromethane (300 mL). A solution of Fmoc-Ala-OH (24.9 g, 80.0 mmol) and N,N-diisopropylethylamine (55.7 mL, 320 mmol) in dry dichloromethane (250 mL) was added to the resin, and the mixture was shaken overnight. Thereafter, the resin was filtered and treated with a solution of N,N-diisopropylethylamine (50 mL) in a methanol / dichloromethane mixture (4:1, 2×5 minutes, 2×250 mL). The resin was then filtered and washed with N,N-dimethylformamide (2×250 mL), dichloromethane (2×250 mL), and N,N-dimethylformamide (2×250 mL). The Fmoc group was removed by treatment with a 20% piperidine solution in N,N-dimethylformamide (1×5 minutes, 1×30 minutes, 2×250 mL). Thereafter, the resin was filtered and washed with N,N-dimethylformamide (2×250 mL), dichloromethane (2×250 mL), and N,N-dimethylformamide (2×250 mL). Thereafter, a solution of Fmoc-L-Lys(Boc)-OH (56.2 g, 120 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)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 added to the resin, and the mixture was shaken for 3 hours. Thereafter, the resin was filtered and washed with N,N-dimethylformamide (2×250 mL), dichloromethane (2×250 mL), and N,N-dimethylformamide (2×250 mL). The Fmoc group was removed by treatment with a 20% piperidine solution in N,N-dimethylformamide (1×5 minutes, 1×30 minutes, 2×300 mL). The resin was then filtered and washed with N,N-dimethylformamide (2×300 mL), dichloromethane (2×300 mL), methanol (2×300 mL), and dichloromethane (10×300 mL).The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (300 mL) overnight. The resin was filtered and washed with dichloromethane (2 × 200 mL), 2-propanol (2 × 200 mL), and dichloromethane (2 × 200 mL). The solvent was removed under reduced pressure and the residue was triturated in diethyl ether (2 × 300 mL). After filtration and drying, we obtained L-Lys(Boc)-β-Ala(2) as an off-white powder. Yield: 13.3 g (56%). 1 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).

[0229] Aqueous 95% 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 h. The solvent was then removed under reduced pressure and the residue was dried under vacuum to give L-Lys-β-Ala TFA salt (3) as a brown oil. Yield: 18.5 g (100%). 1 1H 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).

[0230] Triethylamine (14.1 mL, 101 mmol) was added to a solution of 3 (15.0 g, 33.7 mmol) in acetonitrile to give an off-white precipitate. After filtration and drying, L-Lys-β-Ala(4) was obtained as a white hygroscopic powder. Yield: 7.30 g (100%). 1 1H 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).

[0231] Succinimidyl 3 - fluoro - 4-(4,4,5,5 - tetramethyl - 1,3,2 - dioxaborolan - 2 - yl)benzoate 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 three times with toluene. Thereafter, ethyl acetate (70 mL) was added and the mixture was washed with water (3 × 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)benzamide - Lys - beta - Ala (6) as a white powder. Yield: 2.31 g (47%). 1 1H 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).

[0232] 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, and then N,N-dicyclohexylcarbodiimide (DCC, 0.87 g, 4.20 mmol) was added. After 30 minutes, the reaction mixture was warmed to room temperature and stirred overnight. The insoluble by-products were 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 × 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 five times with pinacol in toluene. 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 vigorously stirred cyclohexane / diethyl ether (10:1, 110 mL). The precipitate was collected by filtration, washed with cyclohexane, and dried under vacuum. The 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)benzamide-Lys-beta-Ala (7) was obtained as an off-white foam. Yield: 1.12 g (48%). 1 1H NMR spectrum (300 MHz, CDCl3, 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); 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.

[0233] [Example 4][O-Succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide-Lys-Gly] [Chemical formula] 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 pad of Celite, 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 yellow powder. Yield: 7.06 g (98%). 1 1H NMR spectrum (300 MHz, CDCl3, δ H ): 7.93 - 7.80 (m, 2H); 7.76 (d, J = 9.4 Hz, 1H); 1.39 (s, 12H).

[0234] 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 × 100 mL) and brine (1 × 100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (20 mL) and precipitated by adding cyclohexane (120 mL). The product was collected by filtration, washed with cyclohexane, and dried under vacuum to give succinimidyl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (3) as a white powder. Yield: 9.60 g (99%). 11H 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). L-Lys-Gly TFA salt 4 (2.67 g, 6.20 mmol) was dissolved in water (20 mL). Then, 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 overnight at room temperature and then acidified with 1 M aqueous hydrochloric acid. The solvent was co-evaporated with toluene three 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 three times. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 × 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). Diethyl 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 an off-white solid. Yield: 1.60 g (37%). 1 1H NMR spectrum (300 MHz, CDCl3, δ H): 7.91 - 7.79 (m, 2H); 7.78 - 7.64 (m, 2H); 7.58 - 7.34 (m, 4H); 7.19 - 7.07 (m, 1H); 4.86 - 4.72 (m, 1H); 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 - 2 × pinacol) + .

[0235] 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 × 80 mL) and brine (1 × 80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain O-succinimidyl N,N-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide-Lys-Gly(6) as a beige solid. Yield: 1.29 g (70%). 1 1H 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, 1H); 4.52 - 4.40 (m, 1H); 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 value 796.4.

[0236] [Example 5][2-((23-(3,5-bis((3-(3-acetoxy-2,2-bis(acetoxymethyl)propoxy)propanamide)methyl)benzamide)-7,16-dioxo-3,9,12,18,21-pentaoxa-6,15-diazatricosyl)oxy)-N-(4-formylbenzyl)acetamide] [Chemical formula] 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 overnight until a clear solution was formed. tert-Butyl acrylate (2174 mL, 1.20 mol) was added, and the resulting mixture was heated at 80 °C for 24 hours, then cooled to room temperature, diluted with water (200 mL), and extracted with ethyl acetate (3 × 400 mL). The combined organic layers were washed with water (400 mL) and brine (100 mL). Since the aqueous washings contained the product (3), they were combined and re-extracted with ethyl acetate (2 × 200 mL). All the ethyl acetate fractions were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: dichloromethane / methanol 99:1 - 90:10) to obtain 3-(3-hydroxy-2,2-bis(hydroxymethyl)propoxy)propanoate (3) as a colorless oil. Yield: 39.7 g (15%). RF (SiO2, dichloromethane / methanol 9:1): 0.30. 1H NMR spectrum (300 MHz, CDCl3, 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)+.

[0237] Acetic anhydride (95.6 mL, 350 mmol) was added to the above 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) at 0 °C. The cooling bath was removed and the resulting solution was stirred at room temperature overnight. The volatile substances were removed under vacuum and 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 hydrogen carbonate solution (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 (Silicagel 60, 0.040 - 0.063 mm, eluent: 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 colorless oil. Yield: 86.7 g (79%). Rf (SiO2, hexane / ethyl acetate 3:2): 0.40. 1 1H NMR spectrum (300 MHz, 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)+.

[0238] Trifluoroacetic acid (300 mL) was added to a solution of the above 2-(acetoxymethyl)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl diacetate (4, 86.0 g, 220 mmol) in dichloromethane (100 mL). The resulting solution was stirred at room temperature for 2 hours and then evaporated to dryness, and the residue was evaporated from toluene (3 × 150 mL). The residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: dichloromethane / methanol 10:0 - 9:1), and the fractions containing the product were evaporated to give the title compound (5) as a pale brown oil. Yield: 70.4 g (96%). Rf (SiO2, hexane / ethyl acetate 1:1): 0.25. 1 1H NMR spectrum (300 MHz, 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)+.

[0239] 2-Chlorotrityl resin, 100 - 200 mesh, 1.5 mmol / g (10.7 g, 16.0 mmol) was left to swell in anhydrous dichloromethane (100 mL) for 20 minutes. 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 dry dichloromethane (20 mL) was added to the resin and the mixture was shaken for 16 hours. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (3.72 mL, 21.4 mmol) in a methanol / dichloromethane mixture (2:8, 2×5 minutes, 2×50 mL). The resin was then washed with N,N-dimethylformamide (2×50 mL), dichloromethane (2×50 mL), and N,N-dimethylformamide (2×50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 minutes, 1×10 minutes, 1×30 minutes, 3×50 mL). The resin was washed with N,N-dimethylformamide (2×50 mL), 2-propanol (2×50 mL), and dichloromethane (2×50 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-1-((dimethylamino)(dimethyliminio)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) was added to the resin and the mixture was shaken for 1 hour. The resin was then washed with N,N-dimethylformamide (2×50 mL), dichloromethane (2×50 mL), and N,N-dimethylformamide (2×50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 minutes, 1×10 minutes, 1×30 minutes, 3×50 mL). The resin was washed with N,N-dimethylformamide (2×50 mL), 2-propanol (2×50 mL), and dichloromethane (2×50 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-1-((dimethylamino)(dimethyliminio)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) was added to the resin and the mixture was shaken for 1 hour. The resin was then washed with N,N-dimethylformamide (2 × 50 mL), dichloromethane (2 × 50 mL), and N,N-dimethylformamide (2 × 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 min, 1 × 10 min, 1 × 30 min, 3 × 50 mL). The resin was washed with N,N-dimethylformamide (2 × 50 mL), 2-propanol (2 × 50 mL), and dichloromethane (2 × 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-1-((dimethylamino)(dimethyliminio)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) was added to the resin and the mixture was shaken for 1 hour. The resin was then washed with N,N-dimethylformamide (2 × 50 mL), dichloromethane (2 × 50 mL), and N,N-dimethylformamide (2 × 50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 min, 1 × 10 min, 1 × 30 min, 3 × 50 mL). The resin was washed with N,N-dimethylformamide (2 × 50 mL), 2-propanol (2 × 50 mL), and dichloromethane (2 × 50 mL).3-(3-acetoxy-2,2-bis(acetoxymethyl)propoxy)propionic acid (5, 10.7 g, 32.0 mmol), ethyl cyano-glyoxylate-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) were added to the resin in N,N-dimethylformamide (40 mL), and the mixture was shaken for 1 h. The resin was filtered and washed with N,N-dimethylformamide (3 × 50 mL), dichloromethane (4 × 50 mL), methanol (4 × 50 mL), and dichloromethane (7 × 50 mL). The product was cleaved from the resin by treatment with a trifluoroacetic acid / dichloromethane mixture (1:1, 50 mL) overnight. The resin was filtered and washed with dichloromethane (2 × 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 100:0–90:10) to give compound (8) contaminated with methyl ester and partially deacetylated product. 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 min, then neutralized with 1 M hydrochloric acid (80 mL) and lyophilized. 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 h, then evaporated from N,N-dimethylformamide several times to give the desired compound 8 as a brown oil. Yield: 13.2 g (99%). LC-MS: 1249 (M+H)+.

[0240] The above 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.HCl, 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 h and evaporated under vacuum. The residue was purified by HPLC (Deltapak, C18, 5 μm, 50×500 mm, acetonitrile / water, 15:85 to 25:75 for 30 min, 25:75 to 50:50 + 0.05% TFA for 170 min) to give the title compound 10 as a pale brown oil. Yield: 1.96 g (12%). 1 H NMR spectrum (300 MHz, CDCl3, 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)+.

[0241] [Example 6][Boc-Lys(Boc)-OEG3-benzaldehyde] [Chemical formula] The compound of Example 6 was prepared in the same manner as the compound of Example 5 from Boc-Lys(Boc).

[0242] [Example 7][Bis(bis(4-borono-3-fluorobenzoyl)-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)benzamide)methyl)benzamide)hexanamide)propanoate] [Chemical formula] 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 × 300 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain methyl 3,5-dimethylbenzoate (2) as a pale yellow oil. Yield: 46.8 g (95%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.67 (s, 2H); 7.19 (s, 1H); 3.91 (s, 3H); 2.37 (s, 6H).

[0243] A mixture of the above methyl 3,5-dimethylbenzoate (2, 46.7 g, 284 mmol), N-bromosuccinimide (NBS, 177 g, 994 mmol), and 1 spatula of azobisisobutyronitrile in methyl formate (550 mL) was irradiated with visible light while heating under 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 sulfate (2×250 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by flash column chromatography (Silicagel 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 1H NMR spectrum (300 MHz, CDCl3, δH): 8.03 - 7.97 (m, 2H); 7.62 (s, 1H); 4.50 (s, 4H); 3.94 (s, 3H).

[0244] A suspension of the above bromide (3, 35.2 g, 109 mmol) and sodium diformylamide (29.1 g, 306 mmol) in dry acetonitrile (200 mL) was refluxed for 4 h. After removing the white solid by filtration, the solvent was evaporated. Recrystallization from an ethyl acetate / cyclohexane mixture gave methyl 3,5-bis((N-formylformamido)methyl)benzoate (4) as a white powder. Yield: 32.7 g (98%). 1 1H 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).

[0245] 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, a stream of air was passed through the solution. The product began to precipitate. After 1 h, the solvent was evaporated and the product was recrystallized from a methanol / diethyl ether mixture (300 mL) to give 3,5-bis(aminomethyl)benzoic acid dihydrochloride (5) as a white powder. Yield: 22.2 g (82%). 1 1H NMR spectrum (300 MHz, D2O, δH): 8.08 (s, 2H); 7.72 (s, 1H); 4.26 (s, 4H).

[0246] The dihydrochloride (5, 6.33 g, 25.0 mmol) was dissolved in water (110 mL). Then 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 overnight at room temperature and then neutralized with 1 M aqueous hydrochloric acid. The solvent was co-evaporated with toluene three 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 three times. The residue was dissolved in ethyl acetate (250 mL) and washed with water (3 × 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 began to precipitate. Cyclohexane was added (170 mL). 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)benzamide)methyl)benzoic acid (7) as a white powder. Yield: 14.5 g (86%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 12.96 (bs, 1H); 9.33 - 9.23 (m, 2H); 7.83 - 7.67 (m, 6H); 7.64 - 7.57 (m, 2H); 7.54 (s, 1H); 4.55 - 4.46 (m, 4H); 1.31 (s, 24H). LC-MS: 512.0 (M + H - 2×pinacol)+.

[0247] The above acid (7, 14.4 g, 21.3 mmol) was dissolved in an acetonitrile / N,N-dimethylformamide mixture (4:1, 100 mL). Then, N-hydroxysuccinimide (HOSu, 2.95 g, 25.6 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 5.28 g, 25.6 mmol) were added. The mixture was stirred overnight at room temperature. The insoluble by-products were filtered off, and the filtrate was evaporated. The residue was dissolved in ethyl acetate (250 mL) and washed with water (2×150 mL) and brine (1×150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in acetonitrile (100 mL). The N,N-dicyclohexylurea of the residue 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 a molecular sieve 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 the addition of cyclohexane (150 mL). The precipitate was filtered, washed with cyclohexane and diethyl ether, and dried under vacuum to obtain 2,5-dioxopyrrolidin-1-yl 3,5-bis((3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)methyl)benzoate (8) as a white powder. Yield: 13.3 g (75%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2 × pinacol)+.

[0248] 2-Chlorotrityl resin, 100 - 200 mesh, 1.8 mmol / g (9.109 g, 19.7 mmol) was allowed to swell in dry dichloromethane (140 mL) for 20 minutes. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-bAla-OH, 4.08 g, 13.1 mmol) and N,N-diisopropylethylamine (8.68 mL, 49.9 mmol) in dry dichloromethane (120 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 (4.57 mL, 26.2 mmol) in a methanol / dichloromethane mixture (1:4, 10 minutes, 140 mL). The resin was then washed with dichloromethane (2 x 130 mL) and N,N-dimethylformamide (2 x 130 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 x 5 minutes, 1 x 15 minutes, 2 x 130 mL). The resin was washed with N,N-dimethylformamide (2 x 130 mL), 2-propanol (2 x 130 mL), dichloromethane (2 x 130 mL), and N,N-dimethylformamide (2 x 130 mL). A solution of N2,N6-bis(tert-butoxycarbonyl)-L-lysine (Boc-Lys(Boc)-OH, 9.09 g, 26.2 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 9.33 g, 26.2 mmol), and N,N-diisopropylethylamine (8.23 mL, 47.2 mmol) in N,N-dimethylformamide (110 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 130 mL) and dichloromethane (10 x 130 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (220 mL) overnight. The resin was filtered and washed with dichloromethane (2 x 200 mL).The solutions were combined, the solvent was evaporated, and the residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: dichloromethane / methanol 90:10) to obtain (S)-3-(2,6-bis((tert-butoxycarbonyl)amino)hexanamido)propanoic acid (10) as a white solid. Yield: 4.30 g (78%). RF (SiO2, dichloromethane / methanol 90:10): 0.40. 1 1H NMR spectrum (300 MHz, AcOD-d4, δH): 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)+.

[0249] The above compound (10, 4.30 g, 10.3 mmol) was dissolved in trifluoroacetic acid (50 mL) and left for 1.5 h. The solvent was evaporated. Diethyl ether (100 mL) was added and the mixture was stirred overnight. The solvent was decanted and the residue was dried under vacuum to obtain (S)-6-((2-carboxyethyl)amino)-6-oxohexane-1,5-diaminium 2,2,2-trifluoroacetate (11) as a solid oil. Yield: 4.50 g (100%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0250] The above salt (11, 2.70 g, 6.06 mmol) was dissolved in N,N-dimethylformamide (100 mL). Then, 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 overnight at room temperature and then neutralized with 1 M aqueous hydrochloric acid. The solvent was co-evaporated three times with toluene. 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 three times from toluene. The residue was dissolved in ethyl acetate (250 mL) and washed with water (1 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. Partial pinacol ester cleavage was observed by NMR analysis. The material was treated overnight with pinacol (0.04 g, 0.34 mmol) and magnesium sulfate (20.0 g) in tetrahydrofuran (110 mL). 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)benzamide)methyl)benzamide)hexaneamide)propionic acid (12) as a pale brown powder. Yield: 5.86 g (63%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0251] 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 overnight at room temperature. The by-product was removed by filtration. The filtrate was evaporated. The residue was dissolved in ethyl acetate (150 mL) and washed with water (1×100 mL) and brine (1×100 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 a molecular sieve overnight. The mixture was filtered and the filtrate was evaporated. The residue was dissolved in ethyl acetate (10 mL) and precipitated after the addition of diethyl ether (90 mL). The product was collected by filtration, washed with diethyl ether, and dried under vacuum to obtain the title compound (13) as a pale brown powder. The product contains a trace amount of N,N-dicyclohexylurea. Yield: 1.55 g (27%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×H2O - 2×pinacol / 2 + H)+, 1384.5 (M - 3×pinacol + H)+, 1302.3 (M - 4×pinacol + H)+.

[0252] [Example 8][(7S,18S)-18-(3-((S)-2,6-bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)hexanamide)propanamide)-7-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)-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] [Chemical formula] 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 over the weekend. The suspension was filtered through a Celite pad, the filtrate was evaporated, and dried under vacuum to obtain 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid as a pale yellow powder (2). Yield: 21.5 g (98%). 1 1H NMR spectrum (400 MHz, DMSO-d6, δH): 7.95 - 7.42 (m, 3H); 1.30 (s, 12H).

[0253] 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.HCl, 31.3 g, 163 mmol) were added. The resulting mixture was stirred overnight at room temperature. The reaction mixture was washed with 0.5 M aqueous hydrochloric acid (1×200 mL), water (1×200 mL), and brine (1×200 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (60 mL) and precipitated by adding cyclohexane (250 mL). The product was collected by filtration, washed with cyclohexane, and dried under vacuum to obtain 2,5-dioxopyrrolidin-1-yl 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (3) as an off-white powder. Yield: 27.8 g (93%). 1 1H NMR spectrum (400 MHz, DMSO-d6, δH): 7.98 - 7.87 (m, 2H); 7.80 (dd, J = 9.2 Hz, 1H); 2.90 (s, 4H); 1.33 (s, 12H).

[0254] 2-Chlorotrityl resin, 100 - 200 mesh, 1.8 mmol / g (4, 16.4 g, 29.5 mmol) was left to swell in dry dichloromethane (230 mL) for 20 minutes. A solution of 3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-bAla-OH, 6.13 g, 19.7 mmol) and N,N-diisopropylethylamine (13.0 mL, 74.8 mmol) in dry dichloromethane (180 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 (6.86 mL, 39.4 mmol) in a methanol / dichloromethane mixture (1:4, 10 minutes, 200 mL). The resin was then washed with 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 minutes, 1 x 15 minutes, 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(((9H-Fluoren-9-yl)methoxy)carbonyl)-L-lysine (Fmoc-Lys(Fmoc)-OH, 23.3 g, 39.4 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 14.0 g, 39.4 mmol), and N,N-diisopropylethylamine (12.3 mL, 70.9 mmol) in N,N-dimethylformamide (180 mL) was added to the resin and the mixture was shaken for 2.5 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 minutes, 1 x 15 minutes, 2 x 200 mL).The resin was washed with N,N-dimethylformamide (2 × 200 mL), 2-propanol (2 × 200 mL), dichloromethane (2 × 200 mL), and N,N-dimethylformamide (2 × 200 mL). A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-bAla-OH, 24.5 g, 78.7 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]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 h. The resin was filtered and washed with N,N-dimethylformamide (2 × 200 mL), dichloromethane (2 × 200 mL), and N,N-dimethylformamide (2 × 200 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 min, 1 × 15 min, 2 × 200 mL). The resin was washed with N,N-dimethylformamide (2 × 200 mL), 2-propanol (2 × 200 mL), dichloromethane (2 × 200 mL), and N,N-dimethylformamide (2 × 200 mL). A solution of N2,N6-bis(tert-butoxycarbonyl)-L-lysine (Boc-Lys(Boc)-OH, 27.3 g, 78.7 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]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 h. The resin was filtered and washed with N,N-dimethylformamide (2 × 200 mL), dichloromethane (2 × 200 mL), and N,N-dimethylformamide (2 × 200 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 min, 1 × 15 min, 2 × 200 mL).The resin was washed with N,N-dimethylformamide (2 × 200 mL), 2-propanol (2 × 200 mL), dichloromethane (2 × 200 mL), and N,N-dimethylformamide (2 × 200 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (350 mL) overnight. The resin was filtered and washed with dichloromethane (2 × 300 mL). The solutions were combined, the solvent was evaporated, and the residue was purified by flash column chromatography (Silicagel 60, 0.040 - 063 mm, eluent: dichloromethane / methanol 85:15) to give (10S,21S)-21-(3-((S)-2,6-bis(tert-butoxycarbonyl)amino)hexanamido)propanamide)-10-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,15,22-tetraoxo-3-oxa-5,12,16,23-tetraazapentacos-26-oic acid (5) as a white solid. Yield: 11.3 g (56%). 1 1H 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)+.

[0255] The above compound (5, 11.3 g, 11.1 mmol) was dissolved in trifluoroacetic acid (200 mL) and left for 1.5 h. The mixture was then concentrated, 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-tetraazapentacosane-1,5,23,27-tetraaminium 2,2,2-trifluoroacetate (6) as a white powder. Yield: 9.25 g (99%). 11H 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).

[0256] The above salt (6, 7.91 g, 9.37 mmol) was dissolved in N,N-dimethylformamide (170 mL). Then, N,N-diisopropylethylamine (14.7 mL, 84.3 mmol), water (0.50 mL), and the activated ester (3, 13.6 g, 37.5 mmol) were added. The mixture was stirred overnight at room temperature and then acidified with 1 M aqueous hydrochloric acid. The solvent was co-evaporated three times with toluene. 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 three times from toluene. The residue was dissolved in ethyl acetate (150 mL) and washed with water (1 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to 1 / 3 of its volume. Cyclohexane (150 mL) was added, the precipitate was filtered, and washed with cyclohexane. The solid was suspended in an acetonitrile / diethyl ether mixture (1:1, 150 mL). The precipitate was filtered, washed with acetonitrile, and dried under vacuum to obtain a white solid of the title compound (7). Yield: 4.10 g (27%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 3×H2O - 4×pinacol + H)+.

[0257] [Example 9][(2,5-Dioxopyrrolidin-1-yl) N-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)ethyl)-N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)glycinate] [Chemical Structure] 3-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (2, 8.85 g, 33.3 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of 1-((dimethylamino)(dimethylimino)-1H-[1,2,3]triazololo[4,5-b]pyridine 3-oxide hexafluorophosphate(V) (HATU, 12.3 g, 32.4 mmol), N,N-diisopropylethylamine (14.5 mL, 83.2 mmol), and tert-butyl (2-aminoethyl)glycinate 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 (2 × 100 mL), water (1 × 100 mL), and brine (1 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product was dissolved in dry 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 (Silicagel 60, 0.063~0.200 mm, eluent: dichloromethane / ethyl acetate 5:2) to give tert-butyl N-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)ethyl)-N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)glycinate (3) as a white foam. Yield: 8.13 g (73%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0258] The above-prepared compound (3, 8.13 g, 12.1 mmol) was dissolved in trifluoroacetic acid (100 mL) and left for 2.5 hours. Then, the solvent was 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 obtain 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)benzoyl)glycine (4) as a white powder. Yield: 6.91 g (93%). 1 1H NMR spectrum (300 MHz, DMSO-d6, 80 °C, 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 - 2×pinacol + H)+, 532.1 (M - pinacol + H)+, 614.2 (M + H)+.

[0259] The acid (4,6.90 g, 11.2 mmol) was dissolved in a dichloromethane / tetrahydrofuran mixture (1:1, 100 mL), and subsequently N-hydroxysuccinimide (1.36 g, 11.8 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (2.26 g, 11.8 mmol) were added. The mixture was stirred overnight at room temperature. The solvent was evaporated. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 × 100 mL) and brine (1 × 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 obtain the title compound (5) as a white powder. Yield: 7.62 g (96%). 1 1H NMR spectrum (300 MHz, DMSO-d6, 80 °C, δH): 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 - 2×pinacol + H)+, 629.1 (M - pinacol + H)+, 711.3 (M + H)+.

[0260] [Example 10][N-(6-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbonyl)-N-(2-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxamido)ethyl)glycine]

Chemical Structure

[0261] (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]oxaborole-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 an additional 72 h. The volatiles were then evaporated under reduced pressure, and the residue was redissolved in ethyl acetate (400 mL) and washed with 1 M aqueous hydrochloric acid (100 mL). The organic portion was dried over anhydrous sodium sulfate. The volatiles were evaporated under reduced pressure, and the product was precipitated from an acetonitrile / water mixture, collected by centrifuge, and lyophilized to give N-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbonyl)-N-(2-(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxamido)ethyl)glycine 4 as an off-white solid. Yield: 1.99 g (27%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0262] [Example 11] [2,5-Dioxopyrrolidin-1-yl (S)-3-(2,6-bis(3,5-bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)methyl)benzamido)hexanamido)propanoate]

Chem.

[0263] Part of the resin (2.00 mmol) was removed. The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 min, 1×10 min, 1×30 min, 3×30 mL). The resin was washed with N,N-dimethylformamide (4×30 mL), dichloromethane (4×30 mL), and N,N-dimethylformamide (4×30 mL). 2,5-Dioxopyrrolidin-1-yl 3,5-bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)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 (4×30 mL), dichloromethane (4×30 mL), N,N-dimethylformamide (4×30 mL), and dichloromethane (10×30 mL).

[0264] 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 h. The resin was filtered and washed with dichloromethane (3×30 mL). The solutions were combined and the solvent was evaporated. The residue was dissolved in dichloromethane (5 mL) and precipitated after the 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)benzamide)methyl)benzamide)hexanamide)propionic acid (3). Yield: 1.53 g (52%). 11H 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).

[0265] 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·HCl, 242 mg, 1.30 mmol) were added. The resulting mixture was stirred overnight at room temperature. The solvent was evaporated. The residue was dissolved in ethyl acetate (100 mL) and washed with water (2 × 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 the 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%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×H2O - 2×pinacol / 2 + H)+, 1384.5 (M - 3×pinacol + H)+, 1302.3 (M - 4×pinacol + H)+.

[0266] [Example 12][(S)-3-(2,6-bis(3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)methyl)benzamide)hexanamide)propionic acid] [Chemical formula] 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 (3×1 L). The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain methyl 3,5-dimethylbenzoate (2) as a pale yellow oil. Yield: 309 g (94%) 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.65 (s, 2H); 7.16 (s, 1H); 3.88 (s, 3H); 2.34 (s, 6H).

[0267] A mixture of methyl 3,5-dimethylbenzoate (2,307 g, 1.87 mol), N-bromosuccinimide (1.17 kg, 6.55 mol), and 1 spatula of azobisisobutyronitrile in methyl formate (2.7 L) was irradiated with visible light while heating under 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 sulfate (2 × 1 L). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. Recrystallization from a hot ethyl acetate / cyclohexane mixture multiple times and washing with cyclohexane gave methyl 3,5-bis(bromomethyl)benzoate (3) as a white solid. The product was prepared in two batches. Yield: 243 g (40%) RF (SiO2, hexane / ethyl acetate 9:1): 0.50. 1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.00 (s, 2H); 7.62 (s, 1H); 4.51 (s, 4H); 3.94 (s, 3H).

[0268] A suspension of the above bromide (3, 122 g, 380 mmol) and sodium diformylamide (101 g, 1.06 mol) in dry acetonitrile (900 mL) was refluxed for 4 h. After removing the white solid by filtration, the solvent was co-evaporated with ethyl acetate and dried under vacuum to give methyl 3,5-bis((N-formylformamido)methyl)benzoate (4) as a pale yellow solid. Yield: 116 g (100%) 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.07 (s, 4H); 7.72 (s, 2H); 7.43 (s, 1H); 4.70 (s, 4H); 3.82 (s, 3H).

[0269] Benzoate (4, 116 g, 380 mmol) was dissolved in a mixture of 1,4-dioxane (400 mL) and concentrated hydrochloric acid (600 mL), and the mixture was heated to reflux for 3 hours. After cooling to room temperature, a stream of air was passed through the solution. The product began to precipitate. After 1 hour, the solvent was evaporated, and the product was recrystallized from a methanol / diethyl ether mixture to obtain 3,5-bis(aminomethyl)benzoic acid dihydrochloride (5) as a white powder. Yield: 89.5 g (92%). 1 H NMR spectrum (300 MHz, D2O, δH): 8.10 (s, 2H); 7.74 (s, 1H); 4.28 (s, 4H).

[0270] The hydrochloride (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 and 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 × 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 obtain 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, δH): 7.70 (s, 2H); 7.45 - 7.36 (m, 2H); 7.33 (s, 1H); 4.21 - 4.04 (m, 4H); 1.39 (s, 18H).

[0271] 2-Chlorotrityl chloride resin, 100 - 200 mesh, 1.5 mmol / g (7, 21.2 g, 31.8 mmol) was left to swell in dry dichloromethane (280 mL) for 40 minutes. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-Ala-OH, 6.61 g, 21.2 mmol) and N,N-diisopropylethylamine (14.1 mL, 80.7 mmol) in dry dichloromethane (220 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 (7.40 mL, 42.5 mmol) in a methanol / dichloromethane mixture (1:4, 1×20 min, 1×250 mL). The resin was then washed with dichloromethane (2×250 mL) and N,N-dimethylformamide (2×250 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 min, 1×20 min, 2×220 mL). The resin was washed with N,N-dimethylformamide (2×250 mL), 2-propanol (2×250 mL), dichloromethane (2×250 mL), and N,N-dimethylformamide (2×250 mL). 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)(dimethyliminio)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) was added to the resin and the mixture was shaken for 2.5 hours. The resin was washed with N,N-dimethylformamide (2×250 mL), dichloromethane (2×250 mL), and N,N-dimethylformamide (2×250 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 min, 1×20 min, 2×220 mL).The resin was washed with N,N-dimethylformamide (2 × 250 mL), 2-propanol (2 × 250 mL), dichloromethane (2 × 250 mL), and N,N-dimethylformamide (2 × 250 mL). A solution of 3,5-bis(((tert-butoxycarbonyl)amino)methyl)benzoic acid (6, 24.2 g, 63.7 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]triazol-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 × 250 mL) and dichloromethane (10 × 250 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (400 mL) overnight. The resin was filtered and washed with dichloromethane (2 × 200 mL). The solvent was evaporated, and the residue was purified by flash column chromatography (Silicagel 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)benzamide)hexanamido)propanoic acid (8) as a white foam. Yield: 16.3 g (82%). RF (SiO2, dichloromethane / methanol 90:10): 0.30. 1 1H NMR spectrum (300 MHz, CDCl3, 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). LC-MS: 942.1 (M + H)+.

[0272] The above compound (8, 16.1 g, 17.3 mmol) was dissolved in trifluoroacetic acid (80 mL) and left for 30 minutes. The solvent was concentrated to 1 / 3 of its volume, and a diethyl ether / cyclohexane mixture (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 obtain (S)-((((6-((2-carboxyethyl)amino)-6-oxohexane-1,5-diyl)bis(azanediyl))bis(carbonyl))bis(benzene-5,1,3-triyl))tetramethanaminium 2,2,2-trifluoroacetate (9) as a white powder. Yield: 16.5 g (96%). 1 1H NMR spectrum (300 MHz, AcOD-d4, δH): 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); 1.57 - 1.48 (m, 2H). LC-MS: 541.6 (M+H)+.

[0273] A suspension of 4-carboxy-3-fluorophenylboronic acid (10, 30.0 g, 163 mmol) and pinacol (21.2 g, 179 mmol) in a toluene / ethanol mixture (1:1, 480 mL) was refluxed for 24 hours. The solvent was then evaporated and co-evaporated with dichloromethane to obtain 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (11) as a white powder. Yield: 43.3 g (100%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0274] The acid (11, 35.2 g, 132 mmol) was dissolved in tetrahydrofuran (1:1, 600 mL), and then 1-hydroxy-pyrrolidine-2,5-dione (HOSu, 25.2 g, 219 mmol) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC.HCl, 42.0 g, 219 mmol) were added. 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 × 300 mL) and brine (1 × 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%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0275] (S)-(((6-((2-carboxyethyl)amino)-6-oxohexane-1,5-diyl)bis(azanediyl))bis(carbonyl))bis(benzene-5,1,3-triyl))tetramethanium 2,2,2-trifluoroacetate (9, 3.91 g, 3.92 mmol) was dissolved in a water / N,N-dimethylformamide mixture (1:1, 80 mL). Then, 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 and then acidified with 1 M aqueous hydrochloric acid. The solvent was co-evaporated three times with toluene. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 × 100 mL) and brine (1 × 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 three times from tetrahydrofuran. The residue was dried under vacuum to give the title compound (13) as an off-white solid. Yield: 5.82 g (95%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 4×pinacol - 4×H2O) / 2 + H)+.

[0276] [Example 13][2,5-dioxopyrrolidin-1-yl 3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)methyl)benzoate] [Chemical formula] 3,5-Bis(aminomethyl)benzoic acid dihydrochloride (2, 1.88 g, 7.43 mmol) was dissolved in water (20 mL). Then, 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) were added.

[0277] The mixture was stirred overnight at room temperature and then acidified with 1 M aqueous hydrochloric acid (200 mL). The solvent was co-evaporated with toluene three times. The residue was dissolved in a dichloromethane / toluene mixture (1:1, 100 mL) and treated with pinacol (1.24 g, 10.5 mmol). The mixture was evaporated from toluene three times. The residue was dissolved in ethyl acetate (150 mL) and washed with water (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (10 mL) and the product began to precipitate. Then, cyclohexane (190 mL) was added and 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)benzamide)methyl)benzoic acid (3) as a white powder. Yield: 4.38 g (87%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pinacol)+.

[0278] The above acid (3, 4.37 g, 6.48 mmol) was dissolved in an acetonitrile / N,N-dimethylformamide mixture (4:1, 100 mL), and N-hydroxysuccinimide (HOSu, 0.89 g, 7.77 mmol) was added. The mixture was cooled to 0 °C, and subsequently N,N-dicyclohexylcarbodiimide (DCC, 1.60 g, 7.77 mmol) was added. The mixture was stirred at 0 °C for 30 minutes and at room temperature overnight. The 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 × 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (10 mL), and cyclohexane was added thereto (170 mL). The precipitate was collected by filtration and 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 Celite pad, and the filtrate was evaporated. The residue was dissolved in dichloromethane (10 mL), and cyclohexane was added to the solution (170 mL). The precipitate was collected by filtration, washed with cyclohexane, and dried under vacuum to obtain the title compound (4) as a white powder. Yield: 3.99 g (80%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pinacol)+.

[0279] [Example 14] [Chemical formula] Prepared by solid-phase peptide synthesis from beta-Ala, Fmoc-Lys, and pinacol 4-carboxy-2-fluorophenylboronate

[0280] [Example 15][(R)-3-(2,4-Bis(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)butanamide)-2,5-dioxopyrrolidin-1-yl propanoate] [Chemical formula] 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 and then acidified with 5 M aqueous hydrochloric acid. 1,4-Dioxane was evaporated and the aqueous phase was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (3 × 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)butanoic acid (2) as a white powder. Yield: 13.3 g (94%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0281] 2-Chlorotrityl chloride resin, 100 - 200 mesh, 1.5 mmol / g (3, 5.84 g, 8.75 mmol) was left to swell in dry dichloromethane (70 mL) for 20 minutes. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic 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, 1×10 min, 1×50 mL). The resin was then washed with dichloromethane (2×50 mL) and N,N-dimethylformamide (2×50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 min, 1×20 min, 2×50 mL). The resin was washed with N,N-dimethylformamide (2×50 mL), 2-propanol (2×50 mL), dichloromethane (2×50 mL), and N,N-dimethylformamide (2×50 mL). (R)-2,4-Bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)butanoic acid (2, 6.57 g, 11.7 mmol), ethyl cyano-glyoxylate-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) were added to the resin and the mixture was shaken for 2.5 hours. The resin was filtered and washed with N,N-dimethylformamide (2×50 mL), dichloromethane (2×50 mL), and N,N-dimethylformamide (2×50 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1×5 min, 1×20 min, 2×50 mL). The resin was washed with N,N-dimethylformamide (2×50 mL), 2-propanol (2×50 mL), dichloromethane (2×50 mL), and N,N-dimethylformamide (2×50 mL).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) were added to the resin, and the mixture was shaken for 2 h. The resin was filtered and washed with N,N-dimethylformamide (3 × 60 mL) and dichloromethane (10 × 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 h. The resin was filtered and washed with dichloromethane (4 × 50 mL). The solvent was evaporated, and the residue was dissolved in ethyl acetate (100 mL) and washed with water (2 × 80 mL) and brine (1 × 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)benzamide)butanamide)propionic acid (5) as a beige solid. Yield: 3.25 g (81%). 1 1H NMR spectrum (300 MHz, CDCl3, 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 - 2×pinacol + H)+, 603.1 (M - pinacol + H)+, 685.3 (M + H)+.

[0282] The acid (5, 3.24 g, 4.73 mmol) was dissolved in dichloromethane (50 mL), and then N-hydroxysuccinimide (0.65 g, 5.67 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.09 g, 5.67 mmol) were added. The mixture was stirred overnight, then diluted with dichloromethane (50 mL) and washed with water (2 × 80 mL) and brine (1 × 80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the title compound (6) as a white solid. Yield: 3.42 g (92%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pinacol + H)+, 700.2 (M - pinacol + H)+, 782.4 (M + H)+.

[0283] [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] [Chemical formula] 3-Bromo-5-iodobenzoic acid (1, 16.4 g, 50.0 mmol) was suspended in methanol (100 mL), and methanesulfonic acid (1 mL) was added. The resulting mixture was stirred at 60 °C (oil bath) for 16 hours. The resulting clear solution was cooled to -20 °C in a freezer for 16 hours, and the resulting solid was collected by filtration, washed with cooled (-20 °C) methanol, and dried under vacuum to obtain methyl 3-bromo-5-iodobenzoate (2) as an off-white solid. Yield: 13.9 g (82%).1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.30 (s, 1H); 8.14 (s, 1H); 8.04 (s, 1H); 3.93 (s, 1H).

[0284] 1,3-Dibromo-5-fluorobenzene (3, 6.30 mL, 50.0 mmol) was dissolved in dry diethyl ether (150 mL) and cooled to -78 °C. 2.35 M n-butyllithium in hexane (22.0 mL, 52.5 mmol) was added dropwise with stirring. After 15 minutes, dry N,N-dimethylformamide (7.70 mL, 100 mmol) was added and the resulting mixture was stirred for 15 minutes and then warmed to ambient temperature. After 1 hour, the reaction mixture was quenched with 1 M aqueous hydrochloric acid (150 mL). The layers were separated, the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate and evaporated to give 3-bromo-5-fluorobenzaldehyde (4) as a yellowish oil which solidified on storage in the freezer. Yield: 10.2 g (100%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 9.92 (s, 1H); 7.80 (bs, 1H); 7.50 (bs, 2H).

[0285] Methyl 3-bromo-5-iodobenzoate (2.680 g, 20.0 mmol) was dissolved in dry tetrahydrofuran (50 mL) under a nitrogen atmosphere and cooled to -40 °C. A 1.3 M solution of 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 using dry tetrahydrofuran (5 mL). The resulting mixture was warmed to room temperature over 1 hour and stirred at ambient temperature for 1 hour. The reaction was quenched by the addition of 0.5 M aqueous hydrochloric acid (50 mL) and extracted with diethyl ether (1 × 200 mL). The organic layer was washed with brine (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 h) and then quenched with 2-propanol (3 mL). After stirring at room temperature for 1 hour, the reaction mixture was filtered through a silica gel plug (100 g) covered with Celite S and washed with dichloromethane (2 × 100 mL). The solvent was removed under vacuum and the residue was purified by flash column chromatography (Silicagel 60, 0.063 - 0.200 mm, eluent: cyclohexane / dichloromethane 6:1 - 2:1) to give methyl 3-bromo-5-(3-bromo-5-fluorobenzoyl)benzoate (6) as a colorless solid. Yield: 7.10 g (85%). 1 1H NMR spectrum (300 MHz, CDCl3, 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). LC-MS: Neither the molecular ion nor the fragment could be detected.

[0286] A 250 mL reaction flask was charged with potassium acetate (6.70 g, 68.4 mmol), and the salt was dried under vacuum at 110 °C for 1 hour. After cooling to room temperature, the reaction flask was refilled with nitrogen, and methyl 3-bromo-5-(3-bromo-5-fluorobenzoyl)benzoate (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) were added. The reaction flask was then emptied and refilled with nitrogen (this procedure was repeated twice), anhydrous tetrahydrofuran (3 mL) was added via syringe, the flask was sealed with a plastic stopper, and immersed in a preheated 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 (70 g) covered with celite S using dichloromethane (3 × 70 mL). The filtrate was concentrated under reduced pressure to afford the product as a yellowish waxy foam, which was triturated with ice-cold n-hexane (70 mL) to induce crystallization. The resulting solid was collected by filtration and dried under vacuum to give methyl 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)benzoate (7) as a white solid. Yield: 7.10 g (88%). 1 1H NMR spectrum (300 MHz, CDCl3, 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)+.

[0287] Methyl 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)benzoate (7.710 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 h. The reaction mixture was diluted with water (120 mL) and extracted with diethyl ether (70 mL). The ether layer was discarded, 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 (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 × 40 mL) to give the title 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 (8) was obtained as a colorless solid. The compound contained pinacol of the residue that could not be removed. Yield: 6.82 g (99%). 1 H NMR spectrum (300 MHz, CDCl3, 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)+.

[0288] [Example 17][(2,5-dioxopyrrolidin-1-yl) 3,5-bis[[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]amino]methyl]benzoate] [Chemical formula] 3,5-Bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)methyl)benzoic acid (1, 2.57 g, 4.00 mmol) was dissolved in an acetonitrile / N,N-dimethylformamide mixture (3:1, 100 mL). N-Hydroxysuccinimide (0.55 g, 4.80 mmol) was added. The mixture was cooled to 0 °C, and subsequently N,N-dicyclohexylcarbodiimide (0.99 g, 4.80 mmol) was added. The mixture was stirred at 0 °C for 30 minutes and at room temperature overnight. The 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 × 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in toluene (10 mL) and the product began to precipitate. Cyclohexane was added (170 mL). The precipitate was collected by filtration, washed with cyclohexane, and dried under vacuum to give the title compound (2) as a white powder. The product contains a trace amount of N,N-dicyclohexylurea. Yield: 2.85 g (97%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - pinacol)+, 573.1 (M + H - 2×pinacol

[0289] [Example 18][N2,N6-Bis(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbonyl)-L-lysine]

Chem.

[0290] L-Lysine hydrochloride (3, 1.56 g, 8.50 mmol) was dissolved in N,N-dimethylformamide (50 mL) and water (25 mL). N,N-Diisopropylethylamine (8.92 mL, 51.2 mmol) and 2,5-dioxopyrrolidin-1-yl 6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carboxylate (2, 5.00 g, 17.0 mmol) were added at room temperature. After stirring for 3 hours, the volatiles were evaporated under reduced pressure, and the residue was precipitated with 1 M aqueous hydrochloric acid. The precipitate was washed with water, purified by precipitation from an acetonitrile / water mixture, collected by centrifugation, and lyophilized to afford N2,N6-bis(6-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-5-carbonyl)-L-lysine (4) as a white solid. Yield: 3.25 g (76%).1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0291] [Example 19][(S)-2,3-Bis(4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanoic acid] [Chemical formula] 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. 6M aqueous hydrochloric acid (60 mL, 6M) was added to the reaction mixture to give a white precipitate. The flask with the precipitate was kept in the refrigerator for 1 hour. It was then filtered, and the filter cake was washed with water (200 mL) and lyophilized to give 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (2) as a white solid. Yield: 12.1 g (100%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 13.24 (bs, 1H), 9.58 (s, 1H), 8.20 (s, 1H), 7.73 (d, J = 9.9 Hz, 1H), 5.14 (s, 2H).

[0292] 4-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (2, 4.00 g, 20.4 mmol), N-hydroxysuccinimide (2.35 g, 20.4 mmol), and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (3.91 g, 20.4 mmol) were stirred in tetrahydrofuran (120 mL) and N,N-dimethylformamide (20 mL) at ambient temperature for 3.5 h. The reaction mixture was evaporated and extracted with acetic acid (3 × 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-1-yl 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (3) as a white solid. Yield: 5.68 g (97%). LC-MS: 294.3 (M+H)+.

[0293] A solution of 2,5-dioxopyrrolidin-1-yl 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-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 × 250 mL) and 1 M aqueous hydrochloric acid (150 mL), and 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-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propionic acid (5) as a white solid. Yield: 3.53 g (88%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0294] [Example 20] [2,5-Dioxopyrrolidin-1-yl 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] [Chemical formula] 3-Bromo-5-iodobenzoic acid (1, 5.00 g, 15.3 mmol) was dissolved in anhydrous 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 h. The reaction mixture was then washed with 1 M aqueous hydrochloric acid (2 × 50 mL) and brine (1 × 40 mL). The organic portion 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 1H NMR spectrum (300 MHz, CDCl3, δH): 8.22 (s, 1H); 8.06 (s, 1H); 8.00 (s, 1H); 1.58 (s, 9H).

[0295] tert-Butyl 3-bromo-5-iodobenzoate (2, 4.31 g, 11.3 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL) under a nitrogen atmosphere and cooled to -40 °C. A 1.3 M solution of isopropylmagnesium chloride-lithium chloride complex in tetrahydrofuran (9.52 mL, 12.4 mmol) was slowly added dropwise. After 40 minutes, 5-bromo-2,4-difluorobenzaldehyde (3, 2.86 g, 12.9 mmol) was added using dry tetrahydrofuran (5 mL). The resulting mixture was warmed to room temperature overnight (16 h). The reaction was quenched by the addition of 0.5 M aqueous hydrochloric acid (15 mL) and extracted with ethyl acetate (2 × 100 mL). The organic layer was washed with brine (40 mL) and dried over anhydrous sodium sulfate. The volatile materials were evaporated under reduced pressure and the residue was purified by column chromatography (Silicagel 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%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0296] tert-Butyl 3-bromo-5-((5-bromo-2,4-difluorophenyl)(hydroxy)methyl)benzoate (4) was dissolved in dry dichloromethane (50 mL), and pyridinium chlorochromate (PCC, 2.96 g, 13.7 mmol) was added. The reaction mixture was then stirred overnight (16 h), and then quenched with 2-propanol (1.5 mL). After stirring at room temperature for 1 h, 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 (Silicagel 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 colorless solid. Yield: 4.20 g (96%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0297] 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) were mixed in a reaction flask, and the resulting mixture was evacuated and refilled with argon (this procedure was repeated twice). Anhydrous tetrahydrofuran (60 mL) was added via syringe, the flask was sealed with a rubber septum, and immersed in a heating bath preheated to 60 °C. After stirring for 16 h, the reaction mixture was cooled to ambient temperature, diluted with cyclohexane (100 mL), and filtered through a short plug of celite using dichloromethane (100 mL). Volatiles were removed under reduced pressure, and the residue was purified by flash column chromatography (Silicagel 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 1H NMR spectrum (300 MHz, CDCl3, 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).

[0298] 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. Volatiles were removed under reduced pressure, and the residue was co-evaporated with dichloromethane (4 × 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. Yield: 4.10 g (96%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0299] 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, 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 hours, the reaction mixture was washed with 10% aqueous potassium persulfate solution (2 × 100 mL) and brine (30 mL). The organic portion was dried using anhydrous sodium sulfate. The volatile substances were evaporated under reduced pressure to obtain 2,5-dioxopyrrolidin-1-yl 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 (8) as a yellow solid. Yield: 4.84 g (99%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.81 - 8.75 (m, 1H); 8.57 - 8.51 (m, 1H); 8.47 (s, 1H); 8.08 (dd, J = 8.5 and 6.7 Hz, 1H); 6.89 (dd, J = 9.9 and 9.0 Hz, 1H); 2.92 (bs, 4H); 1.36 (s, 24H). LC-MS: 448.4 (M - 2 pinacol + H)+.

[0300] [Example 21] [2,5-Dioxopyrrolidin-1-yl 3,5-bis((2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide)methyl)benzoate] [Chemical formula] 3,5-Bis(aminomethyl)benzoic acid dihydrochloride (2, 1.88 g, 7.43 mmol) was dissolved in water (20 mL). Then, 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) were added.

[0301] The mixture was stirred overnight at room temperature and then acidified with 1 M aqueous hydrochloric acid (200 mL). The solvent was co-evaporated three times with toluene. The residue was dissolved in a dichloromethane / toluene mixture (1:1, 100 mL) and treated with pinacol (1.24 g, 10.5 mmol). The mixture was evaporated three times from toluene. The residue was dissolved in ethyl acetate (150 mL) and washed with water (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (10 mL) and the product began to precipitate. Then, cyclohexane (190 mL) was added and 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)benzamide)methyl)benzoic acid (3) as a white powder. Yield: 4.38 g (87%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pinacol)+.

[0302] The above acid (3, 4.37 g, 6.48 mmol) was dissolved in an acetonitrile / N,N-dimethylformamide mixture (4:1, 100 mL), and N-hydroxysuccinimide (HOSu, 0.89 g, 7.77 mmol) was added. The mixture was cooled to 0 °C, and subsequently N,N-dicyclohexylcarbodiimide (DCC, 1.60 g, 7.77 mmol) was added. The mixture was stirred at 0 °C for 30 minutes and at room temperature overnight. The 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 × 150 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in dichloromethane (10 mL), and cyclohexane was added thereto (170 mL). The precipitate was collected by filtration and 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 Celite pad, and the filtrate was evaporated. The residue was dissolved in dichloromethane (10 mL), and cyclohexane was added to the solution (170 mL). The precipitate was collected by filtration, washed with cyclohexane, and dried under vacuum to obtain the title compound (4) as a white powder. Yield: 3.99 g (80%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pinacol)+.

[0303] [Example 22][(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] [Chemistry] 1,3 - Dibromo - 5 - (trifluoromethyl)benzene (1, 13.1 g, 43.1 mmol) was added to a mixture of copper(II) sulfate pentahydrate (541 mg, 2.36 mmol) and potassium hydroxide (9.24 g, 216 mmol) in a dimethyl sulfoxide / water mixture (10:1, 70 mL). The reaction flask was purged with nitrogen and finally 1,2 - ethanedithiol (6.00 mL, 90.5 mmol) was added through a septum. The reaction mixture was heated at 110 °C overnight. The mixture was then acidified to pH = 2 with 1 M aqueous hydrochloric acid 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 (Silicagel 60, 0.063 - 0.200 mm, eluent: cyclohexane) to give 3 - bromo - 5 - (trifluoromethyl)benzenethiol (2) as a white oil. Yield: 5.76 g (52%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.61 (s, 1H); 7.56 (s, 1H); 7.46 (s, 1H); 3.66 (s, 1H).

[0304] 3 - Bromo - 5 - (trifluoromethyl)benzenethiol (2, 5.76 g, 22.4 mmol), methyl 3 - bromo - 5 - iodobenzoate (3, 5.09 g, 14.9 mmol), potassium carbonate (2.95 g, 24.8 mmol), and copper(I) iodide (410 mg, 2.49 mmol) were dissolved in dry dimethoxyethane (44 mL). The reaction flask was heated to 80 °C for 48 h. After this time, the mixture was diluted with ethyl acetate, filtered through celite, and then the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (Silicagel 60, 0.063 - 0.200 mm, eluent: cyclohexane / ethyl acetate 1:0 - 20:1) to give methyl 3 - bromo - 5 - ((3 - bromo - 5 - (trifluoromethyl)phenyl)thio)benzoate (4) as a yellow oil. Yield: 6.64 g (63%). 11H NMR spectrum (300 MHz, CDCl3, δH): 7.82 (m, 1H); 7.68 (m, 3H); 7.52 (m, 1H); 2.54 (s, 3H).

[0305] Methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)thio)benzoate (4, 6.64 g, 14.1 mmol) and oxone (8.20 g, 35.3 mmol) were suspended in methanol (30 mL), and water (10 mL) was added. The reaction mixture was stirred at room temperature overnight. It was then diluted with ethyl acetate (50 mL) and washed with water (1 L) and then brine (100 mL). The organic phase was evaporated under reduced pressure, and the residue was chromatographed by column chromatography (Silicagel 60, 0.063 - 0.200 mm, eluent: cyclohexane / ethyl acetate 9:1 - 3:1) to give methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoate (5) as a white solid. Yield: 5.46 g (77%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0306] 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. Thereafter, the mixture was acidified to pH 2 with 1 M aqueous hydrochloric acid and extracted with ethyl acetate. After evaporation of all volatile substances, 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (6) was obtained as a white solid. Yield: 5.10 g (96%). 1 1H 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).

[0307] 3-Bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoic acid (6, 5.10 g, 10.5 mmol) was mixed with 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate(V) (HATU, 4.40 g, 11.6 mmol) in dry N,N-dimethylformamide (130 mL) and stirred for 30 minutes, then triethylamine (7.5 mL, 52.3 mmol) was added, glycine tert-butyl ester hydrochloride (3.51 g, 20.9 mmol) was added, and the mixture was stirred overnight. After completion of the reaction, water was added, the reaction mixture was extracted with ethyl acetate (150 mL), all volatile substances were evaporated under reduced pressure, and the residue was purified by column chromatography (Silicagel 60, 0.063 - 0.200 mm; eluent: cyclohexane / ethyl acetate 3:1) to obtain tert-butyl (3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)glycinate (7) as a white solid. Yield: 6.30 g (99%). LC-MS: 602.3 (M + H)+.

[0308] 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 hour. After cooling to room temperature, the reaction flask was refilled with nitrogen, and tert-butyl (3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)sulfonyl)benzoyl)glycinate (7, 6.30 g, 10.5 mmol), palladium(II) 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) were added. The reaction flask was then emptied and refilled with nitrogen (this procedure was repeated twice), anhydrous tetrahydrofuran (50 mL) was added via 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), filtered through a short plug of silica gel covered with celite, and washed with dichloromethane (3 × 50 mL). The filtrate was concentrated under reduced pressure to afford tert-butyl (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)glycinate (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-2pinacol-tBu+H)+.

[0309] tert-Butyl (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)glycinate (8, 6.70 g, 10.5 mmol) was mixed with trifluoroacetic acid (25 mL) and stirred at room temperature for 1 hour. After this time, all volatile substances were evaporated under reduced pressure. The residue was then dissolved in ethyl acetate (50 mL) and filtered through a short plug of silica gel covered with celite. The filtrate was concentrated under reduced pressure to give an orange solid foam, which was crushed. (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 (9) was obtained as a pale orange solid. Yield: 3.89 g (63%). 1 1H NMR spectrum (300 MHz, CDCl3, 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 19F NMR spectrum (282 MHz, CDCl3, δF): -62.65 (s). LC-MS: 640.5 (M+H)+, 558.4 (M - pinacol + H)+, 476.3 (M - 2×pinacol + H)+.

[0310] [Example 23][N-(5-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine] [Chemical Structure] 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 h). Ethylenediamine was evaporated under vacuum, 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 × 60 mL), acetonitrile (3 × 100 mL), and diethyl ether (3 × 100 mL) to give (2-aminoethyl)glycine (3) as a colorless solid. Yield: 13.2 g (83%). 1 1H NMR spectrum (300 MHz, D2O, δH): 3.27 (s, 2H); 3.05 - 3.01 (m, 2H); 2.92 - 2.88 (m, 2H).

[0311] A solution of 2,3,4,5,6-pentafluorophenol (9.39 g, 51.0 mmol), 5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-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 pentafluorophenyl 5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (5) as a white solid. Yield: 6.80 g (37%). LC-MS: 363.2 (M+H)+.

[0312] A solution of pentafluorophenyl 5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (5, 6.80 g, 18.8 mmol), (2-aminoethyl)glycine (3, 1.10 g, 9.39 mmol), and triethylenediamine (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 × 500 mL) and 1 M aqueous hydrochloric acid (400 mL), and 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 (Silicagel, 0.063 - 0.200 mm, eluent: dichloromethane / methanol / formic acid 100:2:0.5 - 100:10:0.5) and lyophilized to give N-(5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine (6) as a white solid. Yield: 2.16 g (49%). Rf (SiO2, dichloromethane / methanol / formic acid 100:2:0.5): 0.30. 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0313] [Example 24][4-((3S,4S)-3,4-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid] [Chemical formula] 1-Hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-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 acetic acid (3 × 300 mL) and 1 M 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]oxaborole-6-carboxylate (2) as a white solid. Yield: 18.8 g (100%). LC-MS: 344.3 (M+H)+.

[0314] A solution of 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-hydroxybenzo[c][1,2]oxaborole-6-carboxylate (2, 6.86 mg, 20.0 mmol) 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 and purified by column chromatography (Silicagel, 0.063 - 0.200 mm, eluent: dichloromethane / methanol / formic acid 100:2:0.5 - 100:10:0.5) and lyophilized to give 4-((3S,4S)-3,4-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid (4) as a white solid. Yield: 2.56 g (39%). Rf (SiO2, dichloromethane / methanol / formic acid 100:10:0.5): 0.3. 1 1H NMR spectrum (300 MHz, DMSO-d6, δH) 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)+.

[0315] [Example 25] [2,5-Dioxopyrrolidin-1-yl 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]

Chemical Structure

[0316] Methyl 3-bromo-5-(3-bromo-5-(trifluoromethyl)benzoyl)benzoate (4.650 g, 13.9 mmol) and deoxo-fluor (13.0 mL) were charged into a 100 mL reaction vessel. The vessel was sealed with a bubbler (filled with silicone oil), purged with nitrogen, and heated at 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 was stirred for 1 h to decompose the excess fluorination reagent. The layers were separated, the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm; eluent: cyclohexane / ethyl acetate 30:1 - 15:1) to give methyl 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)difluoromethyl)benzoate (5) as a yellowish oil. Yield: 6902 mg (99%). 1 1H NMR spectrum (300 MHz, CDCl3, 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). 19 19F NMR spectrum (282 MHz, CDCl3, δF): -62.87 (s, 3H); -90.00 (s, 2H).

[0317] A 500 mL reaction vessel was charged with potassium acetate (6.83 g, 69.7 mmol), and the salt was dried under vacuum at 110 °C for 1 hour. After cooling to room temperature, the reaction vessel was refilled with nitrogen, and charged with 3-bromo-5-((3-bromo-5-(trifluoromethyl)phenyl)difluoromethyl)benzoate (5, 6.90 g, 13.9 mmol), palladium(II) acetate (62.0 mg, 279 μmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos, 265 mg, 557 μmol), and bis(pinacolato)diboron (838 mg, 30.7 mmol). The reaction vessel was then emptied and refilled with nitrogen (this procedure was repeated twice). Anhydrous tetrahydrofuran (50 mL) was added via syringe, the vessel was sealed with a plastic stopper, and submerged in a preheated 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 (90 g) of silica covered with Celite S using dichloromethane (3 × 120 mL). The filtrate was concentrated under reduced pressure to afford 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 brownish foam. It was suspended in methanol (50 mL) and water (15 mL), lithium hydroxide monohydrate (2.94 g, 70.0 mmol) was added, and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was taken up in water (150 mL) and washed with dichloromethane (2 × 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 afford a yellowish foam. Pinacol (472 mg, 4.00 mmol) was added to the foam, and the mixture was left to stir in acetonitrile (50 mL) overnight.The precipitated solid was collected by filtration, washed with ice-cold acetonitrile (2 × 20 mL), and dried in air 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 colorless solid. Yield: 5.90 g (77%). 1 1H NMR spectrum (300 MHz, CDCl3, 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)+.

[0318] 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, 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 effect dissolution. After stirring for 16 h, the reaction mixture was concentrated under vacuum and the residue was taken up in ethyl acetate (100 mL) and washed with 0.5 M aqueous potassium bicarbonate (2 × 40 mL) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford an off-white solid. Pinacol (354 mg, 3.00 mmol) was added and the mixture was left to stir in acetonitrile (50 mL) overnight. The precipitated solid was collected by filtration, washed with ice-cold acetonitrile (2 × 20 mL), and dried in air to give the title 2,5-dioxopyrrolidin-1-yl 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 (8) as a colorless solid. Yield: 5.36 g (90%). 1 1H NMR spectrum (300 MHz, CDCl3, 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)+.

[0319] [Example 26][(S)-2,3-Bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanoic acid] [Chemical formula] A solution of 2,5-dioxopyrrolidin-1-yl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (1, 14.4 g, 42.0 mmol), (S)-2,3-diaminopropanoate 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 (Silicagel, 0.063 - 0.200 mm, eluent: dichloromethane / methanol / formic acid 100:2:0.5 - 100:10:0.5). Fractions containing the desired product were evaporated and washed with 1 M aqueous potassium disulfate solution (400 mL). The precipitate was filtered, dissolved in a mixture of acetonitrile and water (2:1), and lyophilized to give (S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanoic acid (3) as a white solid. Yield: 4.32 g (39%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0320] [Example 27][(3-((3-(Pentafluoro-6-sulfanyl)-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] [Chemical Structure] A mixture of tert-butyl (3-bromo-5-((3-bromo-5-(pentafluoro-6-sulfanyl)phenyl)sulfonyl)benzoyl)glycinate (1, 8.00 g, 12.1 mmol), palladium(II) acetate (137 mg, 0.61 mmol), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos, 577 mg, 1.21 mmol), bis(pinacolato)diboron (6.78 g, 26.7 mmol), and potassium acetate (5.95 g, 60.7 mmol) in anhydrous tetrahydrofuran (450 mL) was heated at 60 °C for 24 h under an argon atmosphere. 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 (Silicagel 60, 0.040–0.063 mm, eluent: dichloromethane / ethyl acetate 10:0–6:4) to give tert-butyl (3-((3-(pentafluoro-6-sulfanyl)-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)glycinate (2) as an off-white foam. Yield: 6.70 g (72%). 1 1H NMR spectrum (300 MHz, CDCl3, 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)+.

[0321] A solution of tert-butyl (3-((3-(pentafluoro-6-sulfanyl)-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)glycinate (2, 6.68 g, 8.87 mmol) in dichloromethane (100 mL) and trifluoroacetic acid (200 mL) was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The residue was evaporated 10 times from dichloromethane (250 mL) before drying under vacuum. (3-((3-(Pentafluoro-6-sulfanyl)-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 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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 - 2×pin + H)+.

[0322] [Example 28][N-(1-Hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine] [Chemical Structure] 1-Bromopyrrolidine-2,5-dione (NBS, 34.0 g, 191 mmol) was added to a solution of 3-trifluoromethyl-4-methylbenzoic acid (1, 39.0 g, 191 mmol) in concentrated sulfuric acid (400 mL), and the reaction mixture was stirred at ambient temperature for 16 h. The reaction mixture was then poured into ice water (2 L). The resulting precipitate was filtered, washed with water (500 mL), dissolved in ethyl acetate (400 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to give 3-bromo-4-methyl-5-trifluoromethylbenzoic acid (2) as a white solid. Yield: 53.4 g (98%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 13.71 (bs, 1H); 8.35 (d, J = 0.4 Hz, 1H); 8.15 (d, J = 0.9 Hz, 1H); 2.56 (s, 3H).

[0323] 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 under reflux for 4 h and at ambient temperature for 16 h. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (250 mL), and washed with water (2 × 100 mL) and a mixture of saturated potassium carbonate 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%). 1 H NMR spectrum (300 MHz, DMSO-d6, δH): 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).

[0324] 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 light bulb for 6 h. The reaction mixture was extracted with diethyl ether (2 × 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 1H NMR spectrum (300 MHz, CDCl3, δH): 8.47 (d, J = 1.5 Hz, 1H); 8.31 (d, J = 1.3 Hz, 1H); 4.75 (s, 2H); 3.98 (s, 3H).

[0325] 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-(acetoxymethyl)-5-(trifluoromethyl)benzoate (5) as a white solid. Yield: 37.9 g (91%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 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).

[0326] A solution of methyl 3-bromo-4-(acetoxymethyl)-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(II) (1.57 g, 1.92 mmol) in dry tetrahydrofuran (500 mL) was stirred at 75 °C for 13 days under an argon atmosphere. The reaction mixture was then cooled to ambient temperature, filtered, and evaporated. The crude product was filtered through a silica gel column (Silicagel, 0.063 - 0.200 mm, eluent: cyclohexane / ethyl acetate 8:1) to give methyl 4-(acetoxymethyl)-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 1H NMR spectrum (300 MHz, CDCl3, 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).

[0327] A solution of methyl 4-(acetoxymethyl)-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 h. A solution of hydrochloric acid (35 mL) in water (100 mL) was then added to lower the pH to 1. The reaction mixture was stirred overnight. The precipitate was filtered off and dried to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (7) as a white solid. Yield: 16.6 g (86%). 1 1H 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).

[0328] A solution of pentafluorophenol (7.48 g, 40.7 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-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, refiltered, and evaporated to give pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (8) as a white solid. Yield: 16.7 g (100%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[0329] A solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 - 100:10:0.5) to give N-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine (10) as a white solid. Yield: 7.77 g (67%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0330] [Example 29][(2S)-3-(2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamide)propanoic acid = N-[N,N α ,N β -bis-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)-L-diaminopropionyl]-β-alanine] [Chemical formula] A solution of L-diaminopropionic acid hydrochloride, also known as (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 a saturated aqueous solution of potassium hydrogen sulfate until pH 1 was achieved. The reaction mixture was extracted with ethyl acetate (3 × 200 mL) and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain (2S)-2,3-bis((tert-butoxycarbonyl)amino)propanoic acid (2) as an off-white solid. Yield: 28.2 g (87%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 5.85 (bs, 1H); 5.17 (bs, 1H); 4.31 (bs, 1H); 3.64 - 3.46 (m, 2H); 1.46 (s, 18H).

[0331] 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, and the residue was dissolved in ethyl acetate (600 mL) and washed with 1 M aqueous hydrochloric acid (4 × 300 mL) and saturated aqueous sodium bicarbonate (4 × 300 mL), and dried over anhydrous sodium sulfate. 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%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0332] tert-Butyl (S)-3-(2,3-bis((tert-butoxycarbonyl)amino)propanamido)propanoate (4, 36.1 g, 83.7 mmol) in dichloromethane (50 mL) was added to 95% aqueous trifluoroacetic acid (300 mL), and the solution was stirred for 3 hours. The solvent was removed under reduced pressure, and the residue was co-evaporated with acetonitrile (3 × 300 mL) and treated with 1 M hydrogen chloride solution in dry diethyl ether (300 mL). The precipitate was filtered and triturated with acetonitrile (2 × 600 mL) to give (2S)-3-(2,3-diaminopropanamido)propanoic acid dihydrochloride (5) as a white powder. Yield: 22.2 g (100%). 11H 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).

[0333] A solution of pentafluorophenol (35.1 g, 191 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (1, 40.8 g, 166 mmol, a preparation 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 h. The reaction mixture was filtered, evaporated, dissolved in acetonitrile, refiltered, and evaporated. The crude product was precipitated in dichloromethane (1 L), filtered to give pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (6) as a white solid. Yield: 52.8 g (77%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[0334] To a solution of (2S)-3-(2,3-diaminopropanamido)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 pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 solution (1500 mL). The organic layer was washed with 1 M aqueous potassium hydrogen sulfate solution (1 × 300 mL), and the solvent was removed under reduced pressure. The residue was triturated with diethyl ether (2 × 150 mL) and filtered. The solid was dissolved in 70% aqueous acetonitrile (600 mL) and lyophilized to give 3-(2(S),3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)propanoic acid (7) as a white powder. Yield: 12.1 g (80%). 1 1H 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)+.

[0335] [Example 30][(S)-3-(2,3-Bis(4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)propanoic acid] [Chemical formula] 4-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (1, 8.56 g, 43.7 mmol), N-hydroxysuccinimide (5.03 g, 43.7 mmol), and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (8.38 g, 43.7 mmol) were stirred in tetrahydrofuran (250 mL) and N,N-dimethylformamide (20 mL) at ambient temperature for 3.5 h. The reaction mixture was evaporated and extracted with ethyl acetate (3 × 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-1-yl 4-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (2) as a white solid. Yield: 10.2 g (79%). LC-MS: 294.3 (M+H)+.

[0336] 2-Chlorotrityl chloride resin, 100 - 200 mesh, 1.5 mmol / g (3, 10.5 g, 15.7 mmol), was allowed to swell in dry dichloromethane (80 mL) for 30 minutes. A solution of 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic 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 × 5 minutes, 2 × 80 mL). The resin was then washed with N,N-dimethylformamide (2 × 80 mL), dichloromethane (2 × 80 mL), and N,N-dimethylformamide (3 × 80 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 20 minutes, 2 × 80 mL). The resin was washed with N,N-dimethylformamide (3 × 80 mL), 2-propanol (2 × 80 mL), and dichloromethane (3 × 80 mL). A solution of (S)-2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionic acid (Fmoc-Dap(Fmoc)-OH, 8.61 g, 15.7 mmol), 5-chloro-1-((dimethylamino)(dimethyliminio)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 hours. The resin was filtered and washed with N,N-dimethylformamide (2 × 80 mL), dichloromethane (2 × 80 mL), and N,N-dimethylformamide (2 × 80 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 30 minutes, 2 × 80 mL). The resin was washed with N,N-dimethylformamide (3 × 80 mL), 2-propanol (2 × 80 mL), and dichloromethane (3 × 80 mL).A solution of 2,5-dioxopyrrolidin-1-yl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-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 × 80 mL) and dichloromethane (10 × 80 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (80 mL) for 16 h. The resin was filtered and washed with dichloromethane (4 × 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 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0337] [Example 31] [4-((3R,4R)-3,4-bis(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid] [Chemical formula] A solution of 4-((3R,4R)-3,4-diaminopyrrolidin-1-yl)-4-oxobutanoic acid dihydrochloride (2, 2.46 g, 12.2 mmol), pentafluorophenyl 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 μm, 250×50 mm, acetonitrile / water, 2:98 for 30 minutes, 2:98 to 30:0 for 180 minutes) and lyophilized to give the title compound 4-(3R,4R)-3,4-bis(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)pyrrolidin-1-yl)-4-oxobutanoic acid (3) as a white solid. Yield: 1.23 g (18%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH) 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)+.

[0338] [Example 32][N-(7-Fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine]

Chemical Structure

[0339] 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 h. After cooling to ambient temperature, the mixture was poured onto an ice / water mixture (500 g). The precipitated beige solid was filtered, washed with copious amounts of water and dried to give 2-fluoro-3-iodo-4-methylbenzoic acid (3) as a beige solid. Yield: 51.2 g (91%). 1 1H 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).

[0340] 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. Volatiles were removed under reduced pressure and the residue was taken up in ethyl acetate (1300 mL). After washing with saturated aqueous potassium bicarbonate (2 × 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 give methyl 2-fluoro-3-iodo-4-methylbenzoate (4) as a colourless solid. Yield: 67.6 g (72%). Rf (SiO2, cyclohexane / ethyl acetate 15:1): 0.40. 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0341] 2-Fluoro-3-iodo-4-methylbenzoic acid methyl ester (4, 35.0 g, 119 mmol), bis(pinacolato)diboron (5, 33.3 g, 131 mmol), potassium acetate anhydrous (35.0 g, 357 mmol), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex in anhydrous dimethyl sulfoxide (500 mL) containing dichloromethane (1.94 g, 2.38 mmol) were stirred at 110 °C over the weekend under an argon atmosphere. The reaction mixture was cooled to ambient temperature, the solvent was evaporated under vacuum, and the crude product 6 was extracted with ethyl acetate (4 × 500 mL) and water (1.0 L). The organic layers were combined, filtered through a Celite pad, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (Silicagel 60, 0.063 - 0.200 mm, eluent: 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 colorless solid. Yield: 29.4 g (84%). Rf (SiO2, cyclohexane / ethyl acetate 9:1): 0.30. 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.84 (t, J = 8.0 Hz, 1H); 7.00 (d, J = 8.1 Hz, 1H); 3.90 (s, 3H); 2.47 (s, 3H); 1.39 (s, 12H).

[0342] 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), N-bromosuccinimide (NBS, 18.3 g, 103 mmol), and 2,2'-azobis(2-methylpropionitrile) (AIBN, 0.77 g, 4.68 mmol) in benzotrifluoride (300 mL) were stirred at 85 °C for 16 h. The solvent was evaporated under vacuum, and the residue was extracted with diethyl ether (2 × 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%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.93 (t, J = 7.8 Hz, 1H); 7.21 (d, J = 8.1 Hz, 1H); 4.71 (s, 2H); 3.91 (s, 3H); 1.42 (s, 12H).

[0343] 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-(acetoxymethyl)-2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (8) as an off-white solid. Yield: 30.0 g (95%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0344] A solution of methyl 4-(acetoxymethyl)-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. Then, an aqueous 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 lyophilized to obtain 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (9) as an off-white solid. Yield: 9.76 g (58%). 1 1H 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)+.

[0345] A solution of 2,3,4,5,6-pentafluorophenol (9.61 g, 52.2 mmol), 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-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 over the weekend. The reaction mixture was filtered and evaporated under vacuum. The residue was dissolved in acetonitrile, filtered, and evaporated again under vacuum to obtain pentafluorophenyl 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (10) as a beige solid. Yield: 18.8 g (100%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.55 (bs, 1H); 8.32 - 8.20 (m, 1H); 7.51 (d, J = 8.1 Hz, 1H); 5.13 (s, 2H).

[0346] A solution of pentafluorophenyl 7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 h). 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. Therefore, it was precipitated from ethyl acetate (0.5 L) and the solid was collected by centrifugation. The first precipitate (A) was washed with 0.5 M hydrochloric acid solution (2 × 50 mL) to give a second precipitate (B), which was filtered and retained. The filtrate was lyophilized to give product 12 contaminated with salts. The salts were removed by dissolution and filtration in tetrahydrofuran. The remaining solution was evaporated under vacuum to give a first group of product 12. Precipitate (B) was dissolved in acetonitrile and water (3:1), filtered, and the remaining solution was lyophilized. The resulting solid was dissolved in tetrahydrofuran, the precipitated salts were filtered, and the filtrate was evaporated under vacuum to give a second portion of N-(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(7-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine (12) as an off-white solid. Yield: 2.09 g (34%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0347] [Example 33][2,5-Dioxopyrrolidin-1-yl 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanilidene)amino)acetate] [Chemical formula] tert-Butyl 2-((oxobis(3-(trifluoromethyl)phenyl)-λ 6 -sulfanilidene)amino)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-di-tert-butyl-2,2-dipyridyl (dtbpy, 82.0 mg, 0.31 mmol) were dissolved in degassed tetrahydrofuran (12 mL) under argon. The resulting mixture was warmed to 60 °C and heated at this temperature overnight. The mixture was evaporated to dryness and the residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: dichloromethane / ethyl acetate 10:0 - 4:1) to give tert-butyl 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanilidene)amino)acetate (2) as an off-white foam. Yield: 2.92 g (93%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 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). 19 19F NMR spectrum (282 MHz, CDCl3, δF): -62.55 (s). LC-MS: 556.6 (M - 2×pinacol + H)+, 638.8 (M - pinacol + H)+, 721.0 (M + H)+.

[0348] Trifluoroacetic acid (24 mL) was added to a solution of tert-butyl 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanylidene)amino)acetate (2, 2.91 g, 4.05 mmol) in dichloromethane (8 mL), and the mixture was stirred at room temperature for 2 h. The mixture was evaporated to dryness under vacuum, and the residue was evaporated from toluene (3 × 20 mL) and dichloromethane (3 × 20 mL). The residue was partitioned between dichloromethane (200 mL) and 0.5 M aqueous sodium hydroxide solution (250 mL). The separated aqueous phase was washed with dichloromethane (2 × 100 mL), acidified with 1 M hydrochloric acid (200 mL), and extracted with ethyl acetate (3 × 250 mL). The combined ethyl acetate extracts were dried over anhydrous sodium sulfate and evaporated under vacuum to give 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanylidene)amino)acetic acid (3) as an off-white foam. Yield: 2.30 g (86%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.55 (s, 2H); 8.33 (s, 2H); 8.29 (s, 2H); 3.85 (s, 2H); 1.39 (s, 24H). 19 19F NMR spectrum (282 MHz, CDCl3, δF): -62.69 (s). LC-MS: 500.5 (M - 2×pinacol + H)+, 582.6 (M - pinacol + H)+, 664.8 (M + H)+.

[0349] Dry acetonitrile (16.2 mL) was added to 2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6-Sulfaniliden)amino)acetic acid (3, 2.15 g, 3.24 mmol) and N,N-disuccinimidyl carbonate (DSC, 1.25 g, 4.86 mmol) were added under argon. Pyridine (392 mL, 4.86 mmol) was added and the mixture was sonicated to form a fine suspension. The resulting suspension was stirred for 4 hours to obtain a clear solution. An additional amount of N,N-disuccinimidyl carbonate (DSC, 415 mg, 1.62 mmol) and pyridine (131 mL, 1.62 mmol) were added and the mixture was stirred overnight at room temperature. LC / MS analysis showed complete conversion to the activated ester. The mixture was evaporated to dryness and the residue was partitioned between ethyl acetate (200 mL) and 0.1 M aqueous hydrochloric acid (100 mL). The phases were separated and the organic phase was washed with 0.1 M aqueous hydrochloric acid (2 × 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was dissolved in dichloromethane (40 mL) and subsequently pinacol (383 mg, 3.24 mmol) was added. The solution was evaporated and the residue was evaporated from dichloromethane (3 × 40 mL). The resulting foam was washed with cyclohexane (2 × 50 mL), redissolved in dichloromethane (40 mL), evaporated and dried under vacuum to obtain the title compound (4) as an off-white foam. Yield: 1.82 g (74%). 1 H NMR spectrum (300 MHz, CDCl3, 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, CDCl3, δF): -62.66 (s). LC-MS: 761.9 (M+H)+.

[0350] [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)methyl)sulfonyl)benzoate] [Chemical formula] Methyl 3-iodobenzoate (2, 10.5 g, 40.0 mmol), potassium carbonate anhydrous (11.0 g, 80.0 mmol), copper(I) iodide (1.52 g, 8.00 mmol), and 3-(trifluoromethyl)benzenethiol (1, 8.22 mL, 60.0 mmol) were suspended in dry 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) and filtered through a pad of silica gel covered with celite (125 g) (washed with ethyl acetate / cyclohexane 1:10, 3 × 200 mL) and evaporated in vacuo. The residue was dissolved in acetic acid (120 mL), and 30% aqueous hydrogen peroxide (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). Drying of the organic layer over anhydrous sodium sulfate, filtration, and evaporation in vacuo gave methyl ester 4 as a yellow oil, which was subjected to flash column chromatography (Silicagel 300, 0.063–0.200 mm, eluent: cyclohexane / ethyl acetate 4:1) to give methyl 3-((3-(trifluoromethyl)phenyl)sulfonyl)benzoate (4) as a colorless oil. Yield: 5.40 g (39%). LC-MS: 346.0 (M+H)+.

[0351] Methyl 3-((3-(trifluoromethyl)phenyl)sulfonyl)benzoate (4.540 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-di-tert-butyl-2,2-dipyridyl (dtbpy, 295 mg, 1.10 mmol) were dissolved in dry, 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 slowly added to decompose the pinacolborane (hydrogen gas evolution). After 30 min, lithium hydroxide monohydrate (6.59 g, 157 mmol) was added and the resulting mixture was stirred at ambient temperature for 3 h, then taken up in water (300 mL) and extracted with dichloromethane (3 × 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 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. 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 dried in a stream of air at the top 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)+.

[0352] 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 (5.59 g, 10.1 mmol) and bis(succinimidyl) carbonate (3.63 g, 14.2 mmol) were suspended in anhydrous acetonitrile (45 mL) and pyridine (1.14 mL, 14.2 mmol) under nitrogen. The reaction mixture was heated to effect dissolution. After stirring for 16 h, the reaction mixture was concentrated under vacuum, and the residue was taken up in ethyl acetate (200 mL) and washed with brine (3 × 200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford an off-white solid. Pinacol (473 mg, 4.00 mmol) was added, and the mixture was left to stir in acetonitrile (30 mL) for 1 h. The acetonitrile was evaporated under vacuum. The resulting white foam was dissolved in hexane (30 mL), and the solution was left to crystallize at ambient temperature overnight to afford 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%). 1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.76 - 8.72 (m, 2H); 8.67 (s, 1H); 8.55 (s, 1H); 8.32 (s, 1H); 8.27 (s, 1H); 2.92 (s, 4H); 1.37 (s, 12H) overlapping with 1.37 (s, 12H). 19 19F NMR spectrum (300 MHz, CDCl3, δF): 62.64 (s, 3H). LC-MS: 680.6 (M - H)+.

[0353] [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] [Chemical formula] 4-Methyl-2-(trifluoromethyl)benzoic acid (1, 25.0 g, 123 mmol) was dissolved in sulfuric acid (183 mL), and then N-iodosuccinimide (33.1 g, 147 mmol) was added. The resulting mixture was stirred overnight at room temperature and 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 5% aqueous sodium thiosulfate solution (2 × 250 mL) and water (1 × 250 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 5-iodo-4-methyl-2-(trifluoromethyl)benzoic acid (2) as an off-white powder. Yield: 37.7 g (93%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 13.68 (bs, 1H); 8.22 (s, 1H); 7.76 (s, 1H); 2.47 (s, 3H).

[0354] 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 overnight at 80 °C under a nitrogen atmosphere. The solvent was evaporated. The residue was dissolved in 5% aqueous sodium carbonate solution (200 mL) and extracted with ethyl acetate (3 × 250 mL). The combined organic layers were washed with water (1 × 300 mL) and brine (1 × 200 mL), dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was purified by flash column chromatography (Silicagel 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 1H NMR spectrum (300 MHz, CDCl3, δH): 8.26 (s, 1H); 7.57 (s, 1H); 3.93 (s, 3H); 2.53 (s, 3H).

[0355] A mixture of methyl 5-iodo-4-methyl-2-(trifluoromethyl)benzoate (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 benzotrifluoride (95 mL) was stirred at 85 °C overnight. Complete conversion was not achieved, but the reaction gradually increased. Dichloromethane (150 mL) was added, and the mixture was washed with water (3 × 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. The insoluble material was filtered off, and the filtrate was evaporated. The residue was purified by flash column chromatography (Silicagel 60, 0.040 - 0.063 mm, eluent: cyclohexane / dichloromethane 4:1 - 1:1.5) to give methyl 4-(acetoxymethyl)-5-iodo-2-(trifluoromethyl)benzoate (4) as a white powder. Yield: 17.5 g (42%). Rf (cyclohexane / ethyl acetate 9:1): 0.35. 1 1H NMR spectrum (300 MHz, CDCl3, δH): 8.28 (s, 1H); 7.70 (s, 1H); 5.16 (s, 2H); 3.95 (s, 3H); 2.20 (s, 3H). 19 19F NMR spectrum (282 MHz, CDCl3, δF): -59.96 (s).

[0356] A mixture of methyl 4-(acetoxymethyl)-5-iodo-2-(trifluoromethyl)benzoate (4, 17.5 g, 43.5 mmol), bis(pinacolato)diboron (14.3 g, 56.5 mmol), and dry potassium acetate (21.3 g, 217 mmol) in dry N,N-dimethyl sulfoxide (110 mL) was degassed and 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 a nitrogen atmosphere. After cooling, diethyl ether (500 mL) was added and the precipitate was filtered through a pad of celite. The filtrate was washed with 5% aqueous sodium chloride solution (3 × 500 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 4-(acetoxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethyl)benzoate (5) as a black oil. This oil was used in the next step without further purification. Yield: 22.5 g. 1 1H NMR spectrum (300 MHz, CDCl3, δH): 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 19F NMR spectrum (282 MHz, CDCl3, δF): -60.07 (s).

[0357] Methyl 4-(acetoxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-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 h and then extracted with diethyl ether (2 × 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)-1,3-dihydro-[c][1,2]oxaborole-6-carboxylic acid (6) as a grey powder. Yield: 7.62 g (71%). 11H NMR spectrum (300 MHz, DMSO-d6, δH): 13.50 (bs, 1H); 9.57 (s, 1H); 8.16 (s, 1H); 7.92 (s, 1H); 5.11 (s, 2H). 19 19F NMR spectrum (282 MHz, DMSO-d6, δF): -57.91 (s). LC-MS: 245.9 (M-H)-.

[0358] 1-Hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (6, 6.71 g, 27.3 mmol) was dissolved in a tetrahydrofuran / dichloromethane mixture (1:1, 50 mL), followed by the addition of 2,3,4,5,6-pentafluorophenol (5.03 g, 27.3 mmol) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (5.23 g, 27.3 mmol). The mixture was stirred overnight at room temperature. The solvent was evaporated. The residue was dissolved in ethyl acetate (150 mL) and washed with water (3 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was dissolved in diethyl ether (10 mL), and n-hexane (200 mL) was added. The precipitate was filtered, and the filtrate was evaporated. The same procedure was repeated twice with the precipitate. All the filtrates were combined and evaporated to dryness to give pentafluorophenyl 1-hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (7) as a yellow solid oil. Yield: 9.76 g (87%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.74 (s, 1H); 8.53 (s, 1H); 8.16 (s, 1H); 5.18 (s, 2H).

[0359] Pentafluorophenyl 1-hydroxy-5-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (7, 9.51 g, 23.1 mmol) was dissolved in N,N-dimethylformamide (30 mL). Subsequently, a solution of N,N-diisopropylethylamine (10.1 mL, 57.7 mmol) and (2-aminoethyl)glycine hydrochloride (8, 1.78 g, 11.5 mmol) in water (30 mL) was added. The resulting mixture was stirred overnight at room temperature. The solvent was then evaporated. The residue was dissolved in ethyl acetate (200 mL) and washed with aqueous hydrochloric acid (1 × 200 mL), water (2 × 200 mL), and brine (1 × 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 (Silicagel 60, 0.040 - 0.063 mm, eluent: dichloromethane / methanol / formic acid 10:1:0.05). The fractions containing the product were combined and evaporated. The residue was treated with cyclohexane. The precipitate was filtered, washed with cyclohexane, dissolved in acetonitrile (50 mL), and lyophilized to obtain the title compound (9) as an off-white powder. Yield: 3.63 g (55%). 1 1H NMR spectrum (300 MHz, AcOD-d4, 80 °C, δH): 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)+.

[0360] [Example 36][N-(4-chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(4-chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine]

Chemical Structure

[0361] Triethylamine (10.0 mL, 131.6 mmol) was added to a mixture of pentafluorophenyl 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (2, 8.29 g, 21.9 mmol) and N-2-aminoethylglycine (3, 1.30 g, 1.70 mmol) in N,N-dimethylformamide / water (2:1, 60 mL), and the resulting solution was stirred at room temperature overnight. Then, it was acidified with 1 M aqueous potassium persulfate solution (200 mL) and extracted with ethyl acetate (3 × 250 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was co-distilled with toluene (3 × 100 mL) and triturated with diethyl ether (60 mL). The precipitate was filtered, washed with diethyl ether (2 × 50 mL), and air-dried. The resulting powder was dissolved in an acetonitrile / water mixture (2:1, 20 mL) and lyophilized to give Compound 4 as a colorless solid. Yield: 1.50 g (15%). 1 1H NMR 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)+.

[0362] [Example 37][(S)-4-((2S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamide)-5-tert-butoxy)-5-oxopentanoic acid] [Chemical formula] A solution of pentafluorophenol (35.1 g, 191 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-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 h. The reaction mixture was filtered, evaporated, dissolved in acetonitrile, filtered again, and evaporated. The crude product was precipitated in dichloromethane (1 L), filtered to give pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (2) as a white solid. Yield: 52.8 g (77%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

[0363] 2-Chlorotrityl chloride resin, 100 - 200 mesh, 1.5 mmol / g (3, 4.47 g, 6.71 mmol), was allowed to swell in dry dichloromethane (30 mL) for 30 minutes. 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 × 5 minutes, 2 × 40 mL). The resin was then washed with N,N-dimethylformamide (2 × 30 mL), dichloromethane (2 × 40 mL), and N,N-dimethylformamide (3 × 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 20 minutes, 2 × 40 mL). The resin was washed with N,N-dimethylformamide (3 × 40 mL), 2-propanol (2 × 40 mL), and dichloromethane (3 × 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 hours. The resin was filtered and washed with N,N-dimethylformamide (2 × 40 mL), dichloromethane (2 × 40 mL), and N,N-dimethylformamide (2 × 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 30 minutes, 2 × 40 mL).The resin was washed with N,N-dimethylformamide (3 × 40 mL), 2-propanol (2 × 40 mL), and dichloromethane (3 × 40 mL). A solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 × 40 mL) and dichloromethane (10 × 50 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 h. The resin was filtered and washed with dichloromethane (4 × 50 mL). The crude product (4) was dried under vacuum, extracted with ethyl acetate (2 × 70 mL) and 1 M aqueous potassium hydrogen sulfate solution (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was then triturated in diethyl ether (20 mL) to give (S)-4-((2S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid (4) as a beige solid. Yield: 1.89 g (57%). 1 1H 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)+.

[0364] [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] [Chemical formula] Concentrated sulfuric 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 under reflux overnight. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (700 mL), washed with water (2 × 300 mL), and washed with a saturated solution of potassium carbonate solution (1 × 300 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-5-iodo-4-methylbenzoate (2) as a white solid. Yield: 50.0 g (87%). 1 1H 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).

[0365] To a solution of methyl 3-bromo-5-iodo-4-methylbenzoate (2, 37.3 g, 105 mmol) in dry tetrahydrofuran (250 mL), a 1.3 M solution of isopropylmagnesium chloride lithium chloride complex in tetrahydrofuran (89.0 mL, 115 mmol) was added dropwise at -30 °C under an inert atmosphere and stirred for 20 minutes. Then, N,N-dimethylformamide (12.2 mL, 158 mmol) was added at -30 °C. The reaction mixture was warmed 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 × 200 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-5-formyl-4-methylbenzoate (3) as a white solid. Yield: 24.9 g (92%). 11H NMR spectrum (300 MHz, CDCl3, δH): 10.27 (s, 1H); 8.53 - 8.34 (m, 2H); 3.97 (s, 3H); 2.82 (s, 3H).

[0366] A solution of methyl 3-bromo-5-formyl-4-methylbenzoate (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 h. The reaction was quenched by the addition of water (200 mL) and extracted with dichloromethane (2 × 200 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to give methyl 3-bromo-5-(difluoromethyl)-4-methylbenzoate (4) as a white solid. Yield: 23.3 g (87%). 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0367] 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 trifluorotoluene (120 mL) were stirred at 85 °C overnight. The reaction mixture was evaporated and then extracted with diethyl ether (2 × 300 mL). The organic layer was washed with brine (1 × 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 (Silicagel 60, 0.063 - 0.200 mm, eluent: cyclohexane / ethyl acetate 9:1) to give methyl 4-(acetoxymethyl)-3-bromo-5-(difluoromethyl)benzoate (6) as a white solid. Yield: 17.1 g (61%). Rf (SiO2, hexane / ethyl acetate 9:1): 0.50. 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0368] A solution of methyl 4-(acetoxymethyl)-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 dry dioxane (200 mL) was stirred at 75 °C for 2 days under an argon atmosphere. The reaction mixture was then cooled to ambient temperature, filtered, and evaporated. The crude product was filtered through a silica gel column (Silicagel, 0.063 - 0.200 mm, eluent: cyclohexane / ethyl acetate 9:1) to give methyl 4-(acetoxymethyl)-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. 1 1H NMR spectrum (300 MHz, CDCl3, 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).

[0369] 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 h. A solution of concentrated hydrochloric acid (20 mL) in water (50 mL) was then added to lower the pH to 1. The reaction mixture was left in the refrigerator overnight. The precipitate was filtered off and dried to give 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (8) as a white solid. Yield: 8.55 g (89%). 11H 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).

[0370] Pentafluorophenol (8.28 g, 45.0 mmol), 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-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) were stirred at ambient temperature for 3 hours. The reaction mixture was evaporated, dissolved in ethyl acetate (200 mL), and washed with 1 M aqueous hydrochloric acid (3 × 200 mL) and brine (1 × 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 pentafluorophenyl 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (9) as a white solid. Yield: 8.20 g (56%). LC-MS: 395.5 (M+H)+.

[0371] A solution of perfluorophenyl 4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 1 M aqueous potassium bisulfate solution (30 mL) was added to the residue. The mixture was extracted with ethyl acetate (2 × 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 lyophilized to give N-(4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-N-(2-(4-(difluoromethyl)-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)glycine (11) as a white solid. Yield: 4.59 g (82%). 1 1H NMR spectrum (300 MHz, DMSO-d6, δH): 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)+.

[0372] [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)-λ 6 -sulfanilidene)amino)acetyl)-L-glutamate] [Chemical formula] Subsequently, dry dichloromethane (37 mL) and triethylamine (1.53 mL, 11.0 mmol) were added to 2,5-dioxopyrrolidin-1-yl-2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanylidene)amino)amino)acetate (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) prepared in Example 33. The mixture was sonicated to obtain a solution, which was stirred at room temperature for 6 hours. Volatiles were removed under vacuum, and the residue was redissolved in ethyl acetate (200 mL). The resulting solution was washed with 0.5 M aqueous hydrochloric acid (3 × 50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was redissolved 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 under vacuum to obtain (S)-5-(tert-butoxy)-5-oxo-4-(2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 -sulfanylidene)amino)acetamido)pentanoic acid (3) as a pale yellow foam. Yield: 3.07 g (99%). 1 H NMR spectrum (300 MHz, CDCl3, 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, CDCl3, δF): -62.64 (s). LC-MS: 683.4 (M - 2×pinacol - H)-.

[0373] Subsequently, N,N'-disuccinimidyl carbonate (DSC, 1.84 g, 7.19 mmol) and pyridine (0.58 mL, 7.19 mmol) were added to a solution of (S)-5-(tert-butoxy)-5-oxo-4-(2-((oxobis(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenyl)-λ 6 6 -sulfanylidene)amino)acetamido)pentanoic acid (3, 3.05 g, 3.59 mmol) in dry acetonitrile (18 mL), and the mixture was sonicated to form a fine suspension. The resulting suspension was stirred overnight at room temperature to obtain 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, and the organic phase was washed with 0.5 M aqueous hydrochloric acid (4 × 100 mL) and brine (70 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was dissolved in dichloromethane (40 mL), and subsequently, pinacol (636 mg, 5.39 mmol) was added. The solvent was removed in vacuo, and the residue was evaporated from dichloromethane (50 mL). The resulting foam was triturated with cyclohexane (3 × 50 mL), the resulting semi-solid was decanted, dissolved in dichloromethane (50 mL), and evaporated to dryness under vacuum. The residue was evaporated from dichloromethane (3 × 50 mL) and dried in vacuo to obtain the title compound (4) as a white foam. Yield: 2.82 g (83%). 1 1H NMR spectrum (300 MHz, CDCl3, 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). 19 19F NMR spectrum (282 MHz, CDCl3, δF): -62.63 (s). LC-MS: 864.5 (M - pinacol + H)+, 946.7 (M + H)+.

[0374] [Example 40][(S)-5-(tert-Butoxy)-4-(2-(1-hydroxy-N-(2-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)ethyl)-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)acetamido)-5-oxopentanoic acid] [Chemical formula] 2-Chlorotrityl chloride resin, 100 - 200 mesh, 1.5 mmol / g (1, 4.39 g, 6.59 mmol) was left to swell in dry dichloromethane (30 mL) for 30 minutes. 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 × 5 minutes, 2 × 40 mL). The resin was then washed with N,N-dimethylformamide (2 × 30 mL), dichloromethane (2 × 40 mL), and N,N-dimethylformamide (3 × 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 20 minutes, 2 × 40 mL). The resin was washed with N,N-dimethylformamide (3 × 40 mL), 2-propanol (2 × 40 mL), and dichloromethane (3 × 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)(dimethyliminio)methyl-1H-[1,2,3]triazolo[4,5-b]pyridine 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 × 40 mL), dichloromethane (2 × 40 mL), and N,N-dimethylformamide (2 × 40 mL). The Fmoc group was removed by treatment with 20% piperidine in N,N-dimethylformamide (1 × 5 minutes, 1 × 30 minutes, 2 × 40 mL).The resin was washed with N,N-dimethylformamide (3 × 40 mL), 2-propanol (2 × 40 mL), and dichloromethane (3 × 40 mL). A solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (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 × 40 mL) and dichloromethane (10 × 50 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 h. The resin was filtered and washed with dichloromethane (4 × 50 mL). The solvent was evaporated, and the residue was extracted with 1 M aqueous potassium hydrogen sulfate (50 mL) and ethyl acetate (2 × 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) and purified by column chromatography (Silicagel 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]oxaborole-6-carboxamido)ethyl)-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)acetamido)-5-oxopentanoic acid (3) as a white solid. Yield: 1.50 g (45%). 1 1H NMR spectrum (300 MHz, AcOD-d4, δH): 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). LC-MS: 760.3 (M + H)+.

[0375] [Example 41][1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid] [Chemical formula] 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 at 10 °C in the dark for 40 h. The suspension was then filtered, 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 hydrogen carbonate (2 × 400 mL), and brine (200 mL). The organic solution was dried over anhydrous magnesium sulfate and evaporated to dryness to give crude 3-bromo-4-methylbenzonitrile as yellow crystals. The product was used in the next step without purification. Yield: 90.70 g (92%). Rf (SiO2, hexane / ethyl acetate 9:1): 0.45. 1 1H NMR spectrum (300 MHz, CDCl3, δH): 7.82 (d, J = 1.5 Hz, 1H); 7.50 (dd, J = 7.9 and 1.7 Hz, 1H); 7.34 (d, J = 7.9, 1H); 2.47 (s, 3H).

[0376] Benzoyl 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 tetrachloromethane (1.00 L). The mixture was refluxed overnight. The reaction mixture was then cooled, diluted with dichloromethane (500 mL) and extracted with water (2 × 500 mL...

Claims

1. A compound comprising: i) human insulin or a human insulin analogue, ii) two or more modifying groups M, each of said modifying groups M comprising two aryl moieties and a boron atom attached to each of said two aryl moieties; each of said two or more modifying groups M is attached, optionally via a spacer, to the amino group of the N-terminal amino acid residue of the A-chain or B-chain of said human insulin or human insulin analog, or to the epsilon amino group of a lysine in said human insulin or human insulin analog, each of the modifying groups M independently represents 【Chemistry 1】 And, represents the D- or L-amino acid form, In the formula, n represents an integer ranging from 1 to 4; W1 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W1 is H ２ -C(=O)-*、 H ２ CH ２ -C(=O)-*、 NH-CH(COOH)-CH ２ CH ２ the D or L form of -C(=O)-*; NH-CH(COOH)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ the D or L form of -C(=O)-*; or NH-CH ２ CH ２ -C(=O)-NH-(CH ２ ) ２ -O-(CH ２ ) ２ -O-CH ２ -CO-* represents where * represents the point of attachment to the human insulin or human insulin analogue, R1 is, 【Chemistry 2】 is selected from In the formula, Y1, Y2, Y3, Y4, Y5, and Y6 are independently H, F, Cl, CHF ２ , and C.F. ３ Formula M1, 【Chemistry 3】 And, wherein W2 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W2 is NH-CH(COOH)-CH ２ CH ２ the D or L form of —C(═O)—*, or NH—CH ２ CH ２ CH ２ -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, R2 is, 【Chemistry 4】 is selected from In the formula, Y7, Y8, Y9, Y10, Y11, and Y12 are independently H, F, Cl, CHF ２ , and C.F. ３ Formula M2, 【Chemistry 5】 And, represents the R,R, or S,S, or R,S stereoisomer of 3,4-diamino-pyrrolidine, where * represents the point of attachment to the human insulin or human insulin analog, and where Y13 and Y14 are independently H, F, Cl, CHF ２ , and C.F. ３ Formula M3, 【Chemistry 6】 And, wherein * represents the point of attachment to the human insulin or human insulin analog, and Y15 and Y16 are independently H, F, Cl, CHF ２ , and C.F. ３ Formula M4, 【Chemistry 7】 And, Formula M5, wherein each of said amino acid residues represents the D- or L-amino acid form and * represents the point of attachment to said human insulin or human insulin analogue; 【Chemistry 8】 And, wherein the α-amino acid residue represents either the D- or L-amino acid form, * represents the point of attachment to the human insulin or human insulin analog, and Y17 and Y18 are independently H, F, Cl, CHF ２ , and C.F. ３ Formula M6, 【Chemistry 9】 And, wherein W3 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W3 is NH-CH(COOH)-CH ２ CH ２ Formula M7, which represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue; 【Chemistry 10】 And, wherein W4 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W4 is NH-CH ２ -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, and Y19 is H, F, Cl, CHF ２ , and C.F. ３ Or SF ５ Formula M8, 【Chemistry 11】 And, wherein * represents the point of attachment to the human insulin or human insulin analog, and each of Y20, Y21, and Y22 is independently H, F, Cl, CHF ２ , and C.F. ３ Formula M9, 【Chemistry 12】 And, Formula M10, in which * represents the point of attachment to the human insulin or human insulin analogue; and 【Chemistry 13】 And, and two or more modifying groups M selected from the group of formula M11, wherein each of said amino acid residues represents the D- or L-amino acid form and * represents said point of attachment to said human insulin or human insulin analogue.

2. each of the modifying groups M independently represents 【Chemistry 14】 And, represents the D- or L-amino acid form, where n is 1; W1 is NH-CH ２ CH ２ -C(=O)-*, or NH-CH(COOH)-CH ２ CH ２ represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, R1 is, 【Chemistry 15】 and In the formula, Y1 and Y2 are H, and Y3 is F or CF ３ Formula M1, 【Chemistry 16】 And, wherein W2 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W2 is NH-CH(COOH)-CH ２ CH ２ represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, In the formula, R2 is 【Chemistry 17】 and In the formula, Y7 and Y8 are H, and Y9 is Cl, CHF ２ or CF3, 【Chemistry 18】 And, Formula M4, wherein * represents the point of attachment to the human insulin or human insulin analog, Y15 is H and Y16 is F; 【Chemistry 19】 And, wherein W3 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W3 is NH-CH(COOH)-CH ２ CH ２ Formula M7, which represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue; 【Chemistry 20】 And, wherein W4 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W4 is NH-CH ２ Formula M8, which represents -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analog, and Y19 is CF3; and 【Chemistry 21】 And, 2. The compound of claim 1, selected from the group of formula M9, ​​wherein * represents the point of attachment to the human insulin or human insulin analog, and 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.

3. The human insulin or human insulin analogue optionally comprises a) a peptide spacer at the C-terminus of the A-chain of the human insulin or human insulin analogue, The peptide spacer is (GES) ｐ a peptide spacer comprising K, where p is an integer from 3 to 12; or b) a peptide spacer or linker L at the N-terminus of the B chain of the human insulin or human insulin analogue, The peptide spacer is GKPG, GKP(G ４ S) ｑ , K.P. (G ４ S) ｒ ,GKPRGFFYTP(G ４ S) ｓ , or TYFFGRKPD (G ４ S) ｔ wherein each of q, r, s, and t is independently selected from the integers 1 to 5; The linker L is 【Chemical 22】 And, Formula L1, wherein *1 indicates the point of attachment to the modifying group M and *2 indicates the point of attachment to the amino group of the amino acid residue at the N-terminus of the B chain of the human insulin or human insulin analogue; 【Chemistry 23】 And, wherein *1 denotes the point of attachment to the modifying group M, and *2 denotes the point of attachment to the amino group of the amino acid residue at the N-terminus of the B chain of the human insulin or human insulin analogue, Formula L2, in which u is 1, 2, or 3; and 【Chemistry 24】 And, wherein *1 denotes the point of attachment to the modifying group M, and *2 denotes the point of attachment to the amino group of the amino acid residue at the N-terminus of the B chain of the human insulin or human insulin analogue, 3. The compound of claim 1 or 2, selected from formula L3, wherein v is 2 or 3.

4. 4. The compound of claim 3, wherein q is an integer selected from 1 to 3, r is 3, s is 2, and t is 3.

5. Each modifying group M is selected from the group 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 the epsilon amino group of the 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 epsilon amino group of a lysine residue at position 1 or 4 of the B chain of the human insulin analogue, 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, or the terminal amino group, marked with *1, of the optional linker L at the N-terminus of the B-chain of the human insulin or human insulin analogue, and d) the epsilon amino group of lysine at position 22 or 29 of the B-chain of human insulin or a human insulin analogue.

6. The compound of any one of claims 1 to 5, having exactly two, three or four modifying groups M.

7. The human insulin or human insulin analogue is 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 A22K B25H B27P B28G 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, A22K desB30 human insulin, A22K B29R desB30 human insulin, A22K B22K B29R desB30 human insulin, and The compound according to any one of claims 1 to 6, which is a human insulin analogue selected from the group consisting of A-2K A-1P desB30 human insulin.

8. i) human insulin or a human insulin analogue, said human insulin or human insulin analogue optionally comprising a peptide spacer at the N-terminus of the B-chain of said human insulin or human insulin analogue; The peptide spacer is GKPG, GKP(G ４ S) ｑ , K.P. (G ４ S) ｒ ,GKPRGFFYTP(G ４ S) ｓ , or TYFFGRKPD (G ４ S) ｔ wherein q is an integer from 1 to 3, r is 3, s is 2, and t is 3; and ii) two modifying groups M, each of said modifying groups M independently being 【Chemistry 25】 And, represents the D- or L-amino acid form, where n is 1 and W1 is NH-CH ２ CH ２ -C(=O)-*, or NH-CH(COOH)-CH ２ CH ２ represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, In the formula, R1 is 【Chemistry 26】 and In the formula, Y1 and Y2 are H and Y3 is CF ３ Formula M1, 【Chemical 27】 And, wherein W3 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W3 is NH-CH(COOH)-CH ２ CH ２ Formula M7, which represents the D or L form of -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue; 【Chemistry 28】 And, wherein W4 is absent and represents the point of attachment to said human insulin or human insulin analogue *, or W4 is NH-CH ２ Formula M8, which represents -C(=O)-*, where * represents the point of attachment to the human insulin or human insulin analogue, and Y19 is CF3; 【Chemical 29】 And, wherein * represents the point of attachment to the human insulin or human insulin analogue, and 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 with the proviso that when Y21 is H, Y20 and Y22 are F, One modifying group M is attached to the epsilon amino group of lysine at position 29 of the B chain of the human insulin or human insulin analogue, and one modifying group M is the epsilon amino group of a lysine residue at position 1 or 4 of the B chain of the human insulin analogue, or and two modifying groups M attached to the epsilon amino group of a lysine of the optional peptide spacer at the N-terminus of the B-chain of the human insulin or human insulin analogue.

9. The compound is 【Chemistry 30】 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 , and 【Chemistry 41】 The compound according to any one of claims 1 to 8, which is selected from the group consisting of:

10. An intermediate product, 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); A22K B22K B29R desB30 human insulin (SEQ ID NO:6 and SEQ ID NO:20); A21Q(GES)3K desB30 human insulin (SEQ ID NO:8 and SEQ ID NO:11); A21Q(GES)6K desB30 human insulin (SEQ ID NO: 9 and SEQ ID NO: 11); A21Q(GES)12K desB30 human insulin (SEQ ID NO: 10 and SEQ ID NO: 11); 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-GKPGGGGSGGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 24); B1-GKPGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 25); B1-GKPG desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 26); B1-GKPRGFFYTPGGGGSGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 27), and B1-TYFFGRKPDGGGGSGGGGSGGGGGS desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 28).

11. A composition comprising a compound according to any one of claims 1 to 9.

12. A compound according to any one of claims 1 to 9 for use as a medicament.

13. A compound according to any one of claims 1 to 9 for use in the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome).

14. A method for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndrome (metabolic syndrome X, insulin resistance syndrome), comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 9 or a composition according to claim 11.

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