Synthesis of boronic ester and boronic acid compounds

Abstract

The invention relates to the synthesis of boronic ester and boronic acid compounds. More particularly, the invention provides improved synthetic processes for the large-scale production of boronic ester and boronic acid compounds, including the peptide boronic acid and the proteasome inhibitor bortezomib.

Description

SYNTHESIS OF BORONIC ESTER AND BORONIC ACID COMPOUNDS BACKGROUND OF THE INVENTION Field of the invention

[001] The present invention relates to the synthesis of boronic acid esters and boronic acid compounds. More particularly, the invention relates to synthesis processes for large-scale production of boronic acid ester and boronic acid compounds by Lewis acid promoted rearrangement of “boronate” complexes. Background to the invention

[002] Boronic acid and boronic ester compounds have a variety of pharmaceutical applicable biological effects. Shenvi et al., US Patent No. 4,499,082 (1985), states that peptide boronic acids are inhibitors of certain proteolytic enzymes. Kettner and Shenvi, US Patent No. 5,187,157 (1993), US Patent No. 5,242,904 (1993) and US- Patent No. 5,250,720 (1993), describes a class of peptide boronic acids that inhibit trypsin-like proteases. Kleeman et al., US Patent No. 5,169,841 (1992), describes N-terminally modified peptide boronic acids that inhibit the action of renin. Kinder et al., U.S. Patent No. 5,106,948 (1992), discloses that certain tripeptide- Boronic acid compounds inhibit the growth of cancer cells.

[003] Later, boronic acid and boronic acid ester compounds have been shown to be promising inhibitors of the proteasome, a multicatalytic protease responsible for that most of intracellular protein turnover. Ciechanover, Cell, 79: 13-21 (1994), states that the proteasome is the proteolytic component of the ubiquitin-proteasome- the pathway, where proteins are designated for degradation by conjugation to multiple molecules ubiquitin. Ciechanover also states that the ubiquitin-proteasome pathway plays a key role in a number of important physiological processes.

[004] Adams et al., US Patent No. 5,780,454 (1998), US Patent No. 6,066,730 (2000), US Patent No. 6,083,903 (2000), US Patent No. 6,297,217 (2001), US Patent No. 6,548,668 and U.S. Patent No. 6,617,317 (2003), which are hereby incorporated by reference. reference in its entirety, describes peptide boronic ester and boronic acid compounds which are useful as proteasome inhibitors. The references also describe the use of boronic ester and boronic acid compounds to reduce the rate of protein breakdown in the muscles, to reduce the activity of NF-κB in cells, to reduce the rate of degradation of p53 protein in cells, to inhibit cyclin degradation in cells, to inhibit the growth of cancer cells, to inhibit antigen- presentation in cells, to inhibit NF-κB-dependent cell adhesion and to inhibit HIV replication.

[005] Albanell and Adams, Drugs of the Future 27: 1079-1092 (2002), states that one such inhibitor of the peptide-boronic acid proteasome, bortezomib (N-2- pyrazinecarbonyl-L-phenylalanine-L-leucine-boronic acid), exhibits significant antitumor activity in human tumor xenograft models, and this is undergoing clinical evaluation. Richardson et al., New Engl. J. Med., 348:2609 (2003), report the results of a Phase 2 study of bortezomib, showing that it is effective in treating relapsed and refractory multiple myeloma.

[006] In developing chemistry for the preparation of functionalized boronic acid compounds, especially alpha-halogen and alpha-aminoboronic acids, great efforts have been made advances in studies of boronic acid protease inhibitors. Matteson and Majumdar, J. Am. Chem. Soc., 102:7590 (1980), describes a method for preparing alpha- chloroboronic acid esters by homologation of boronic acid esters, and Matteson and Ray, J. Am. Chem. Soc., 102:7591 (1980), reports that chiral control of homologation- the reaction can be achieved using pinanediol boronic acid esters. Preparation of alpha-aminoboronic acid and ester compounds from the corresponding alpha- chloroboronic acid esters have also been reported (Matteson et al., J. Am. Chem. Soc., 103:5241 (1981); Shenvi, US Patent No. 4,537,773 (1985)).

[007] Matteson and Sadhu, U.S. Patent No. 4,525,309 (1985), describe a improved procedure for homologation of boronic acid esters by rearrangement of the intermediate “boronate” complex in the presence of a Lewis acid catalyst. It is reported that the Lewis acid promotes the rearrangement reaction and minimizes epimerization at the alpha carbon atom. Strict exclusion of water and careful control of the Lewis acid stoichiometry is, however, necessary for optimal results. These the features make it difficult to achieve successful reaction at production scale and limits the availability of pharmaceutically important boronic acid esters and boronic acid compounds, such as bortezomib. Accordingly, there is still a need in the art for improved methods for large-scale production of boronic acid esters and boronic acid connections. DESCRIPTION OF THE INVENTION

[008] The present invention provides improved synthesis processes for large-scale production of boronic ester and boronic acid compounds. These the processes provide increased yield and purity, increased production capacity (“throughput”) and easier handling than previously known methods. The procedures that are described here, is particularly suitable for large-scale batch production (multikg), which is limited only by the size of the available production facility. The process the methods of the invention are particularly advantageous for synthesizing chiral boronic acid ester and boronic acid compounds, including alpha-aminoboronic acid ester and - boronic acid compounds. Regardless of the scale, the desired products are produced with very high chemical and stereochemical purity.

[009] The patents and scientific literature referred to herein show the knowledge available to experts. Unless otherwise stated, all technical and scientific terms used here, the usual significance to a person skilled in the art to which the present invention belongs. Methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, but the preferred methods and materials are described herein. Granted patents, applications and references that are mentioned herein, are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually stated to be incorporated by reference. By any discrepancies apply to this description, including The definitions. Otherwise, the materials, methods and examples are illustrative only. and is not intended to be limiting.

[010] The term “approx.” here means approximately, in the area, approximately or around. When the term “approx.” is used with a range of numbers, modifies the range by extending the upper and lower limits of the specified numerical values. Usually the term “approx.” is used here to modify a numerical value with a variance of 10% above and below the specified value.

[011] The term "includes" here means "includes, but is not limited to.”

[012] The term “aliphatic”, as used herein, means a straight-chain, branched or cyclic C1-12 hydrocarbon that is fully saturated or which contains one or more unsaturated units but is not aromatic. Suitable aliphatic groups include, for example, substituted or unsubstituted, linear, branched or cyclic alkyl, alkenyl, alkynyl groups and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. In various embodiments- forms, the aliphatic group has 1-12, 1-8, 1-6 or 1-4 carbon atoms.

[013] The terms “alkyl”, “alkenyl” and “alkynyl”, used alone or as a part of a larger group, refers to a linear and branched aliphatic group having from 1 to 12 carbon atoms, which are optionally substituted with one, two or three substituents. For purposes of the present invention, the term "alkyl" will be used when the carbon atom that connects the aliphatic group to the rest of the molecule is a saturated carbon atom. However, an alkyl group can have other unsaturated carbon atoms. Accordingly, alkyl groups include, without limitation, methyl, ethyl, propyl, allyl, propargyl, butyl, pentyl and hexyl.

[014] For purposes of the present invention, the term “alkenyl” is used when the carbon atom that connects the aliphatic group to the rest of the molecule, forms part of a carbon / carbon double bond. Alkenyl groups includes, without limitation, vinyl, 1-propenyl, 1-butenyl, 1-pentenyl and 1-hexenyl. For purposes of the present invention, the term "alkynyl" will be used when the carbon atom that connects the aliphatic group to the rest of the molecule forms part of a carbon-carbon triple bond. Alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl and 1-hexynyl.

[015] The terms “cycloalkyl”, “carbocyclic group”, “carbocyclyl”, “carbocyclo” or “carbocyclic”, used alone or as part of a larger group, means a saturated or partially unsaturated, cyclic, aliphatic ring system having from 3 to about 14 membered, where the aliphatic ring system is optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl and cyclooctadienyl. In some embodiments, cycloalkyl has 3-6 carbon atoms. The terms “cycloalkyl”, “carbocyclic group”, “carbocyclyl”, “carbocyclo” or “carbocyclic” also includes aliphatic rings fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydro- naphthyl, where the residue or point of attachment is on the aliphatic ring.

[016] The terms “haloalkyl”, “haloalkenyl” and “haloalkoxy” refers to an alkyl, alkenyl or alkoxy group optionally substituted with one or more halogen atoms. As used herein, “halogen” means F, C, Br or I. Unless otherwise specified, the terms “alkyl”, “alkenyl” and “alkoxy” include haloalkyl, haloalkenyl and haloalkoxy groups, including in particular those with 1-5 fluorine atoms.

[017] The terms “aryl” and “ar-”, used alone or as part of a larger group, e.g. "aralkyl", "araloxy" or "aryloxyalkyl", refers to a C 6-14 aromatic group comprising one to three aromatic rings, which optionally are substituted. The aryl group is preferably a C6-10 aryl group. Aryl groups include, without limitation, phenyl, naphthyl and anthracenyl. The term “aryl”, as it is as used herein also includes groups in which an aromatic ring is fused to one or several non-aromatic rings, such as indanyl, phenanthridinyl or tetrahydronaphthyl, where the residue or attachment point is on the aromatic ring. The designation "aryl" and The term “aryl ring” can be used interchangeably.

[018] An "aralkyl" or "arylalkyl" group includes an aryl group which is covalently bonded to an alkyl group, each of which is optionally and independently The aralkyl group is preferably C aryl(C )alkyl, which includes, without 6-10 1-6 limitation, benzyl, phenethyl and naphthylmethyl.

[019] The terms “heteroaryl” and “heteroar-”, used alone or as a part of a larger group, e.g. “heteroaralkyl” or “heteroaralkoxy”, refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; having 6, 10 or 14 shared π-electrons in a cyclic arrangement; and which in addition to carbon- the atoms have from one to four heteroatoms selected from the group consisting of N, O and S. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl and phenazinyl. The terms “heteroaryl” and “heteroar-”, as used here, also includes groups in which a heteroaromatic ring is fused to one or multiple non-aromatic rings, where the residue or point of attachment is on the hetero- aromatic ring. Non-limiting examples include tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[3,4-d]pyrimidinyl. The terms “heteroaryl”, “heteroaryl ring” or “heteroaromatic” can be used interchangeably, and all the designations include rings that are optionally substituted. The designation “heteroaralkyl” denotes an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl moieties are optionally and independently substituted.

[020] As used herein, the terms “heterocyclic group”, "heterocyclyl" or "heterocyclic residue" to a stable, 5- to 7-membered, monocyclic or a 7- to 10-membered, bicyclic, heterocyclic group that is either saturated or partially unsaturated and which in addition to the carbon atoms has one or more, preferably one to four, heteroatoms selected from the group consisting of N, O and S, where nitrogen and sulfur the heteroatoms are optionally oxidized and the nitrogen atoms are optionally quaternized. The heterocyclic ring may be attached to the attached group via any any heteroatom or carbon atom that results in a stable structure, and which any of the ring atoms may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic residues include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, tiazepinyl and morpholinyl. The terms “heterocyclic group”, “heterocyclyl” and “heterocyclic residue”, as they as used herein also includes groups in which a non-aromatic, heteroatom-containing ring is fused to one or more aromatic or non-aromatic rings, such as indolinyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl, where the residue or bond- the point is on the non-aromatic, heteroatom-containing ring. The designation “heterocyclylalkyl” denotes an alkyl group substituted with a heterocyclyl group, wherein the alkyl and heterocyclyl moieties are optionally and independently substituted.

[021] As used herein, the term “partially unsaturated” refers to a ring group comprising at least one double or triple bond between ring atoms. The term "partially unsaturated" is intended to encompass rings having one or more unsaturated sites, but are not intended to include aryl or heteroaryl groups, as they are defined here.

[022] The term “substituted,” as used herein, means that one or several hydrogen atoms in the indicated group are replaced, provided that the substitution results in a stable or chemically feasible compound. A stable compound or a A chemically feasible compound is a compound with a chemical structure that is not significantly changed when stored at a temperature of 40 °C or lower, for at least one week in the absence of moisture or other chemically reactive conditions, or a compound that retains its integrity long enough for it to be useful in synthesis the processes of the invention. The term "one or more substituents", as used herein, as used herein, refers to a number of substituents corresponding from one substituent to the maximum number of substituents possible, based on the number of available bonding sites seats, provided that the above conditions for stability and chemical permeability are met portability is fulfilled.

[023] An aryl group (including the aryl group in aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl group (including the heteroaryl group in heteroaralkyl and heteroarylalkoxy and the like) may contain one or more substituents. Examples on suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl + + group includes -halogen, -NO , -CN, -R*, -OR*, -SRº, -N(R ) , -NR C(O)R*, 2 2 + + + -NR C(O)N(R )2, -NR CO2Rº, -O-CO2R*, -OC(O)R*, -CO2R*, -C(O)R*, + + + + + -C(O)N(R )2, -OC(O)N(R )2, -S(O)2Rº, -SO2N(R )2, -S(O)Rº and -NR SO2N(R )2. + Each R is independently selected from the group consisting of R*, -C(O)R*, -CO R* and 2 + -SO R*, or two R on the same nitrogen atom together with the nitrogen atom form one 2 5- to 8-membered, aromatic or non-aromatic ring which, in addition to the nitrogen atom, has 0-2 ring heteroatoms selected from N, O and S. Each R* is independently selected from hydrogen or an optionally substituted, aliphatic, aryl, heteroaryl or heterocyclyl group. Each Rº is independently selected from an optionally substituted aliphatic group or a optionally substituted aryl group.

[024] An aliphatic group may also be substituted with one or more substituents. Examples of suitable substituents on the saturated carbon atom in a aliphatic group or in a non-aromatic, heterocyclic ring, includes, without limitation, those listed above for the unsaturated carbon atom in an aryl- or heteroaryl group.

[025] The present inventors have discovered that the requirement for completely dry equipment, solvents and reagents, which characterize previously described procedures for Lewis acid-promoted rearrangement of “boronate” complexes, can eliminated by using an ether solvent that has low miscibility with water. It is noteworthy that the use of such a solvent makes it possible to run the reaction on a multikg scale without negative effect on yield or purity. Actually, the scale of reaction is only limited by the size of the available production plant.

[026] In one aspect, therefore, the invention provides a large-scale process for preparing a boronic acid ester compound of formula (I): 1 R 2 R 4 OR 3 RB 5 OR (IN) where: 1 R is an optionally substituted, aliphatic, aromatic or heteroaromatic group; 2 R is hydrogen, a nucleofuge group or an optionally substituted aliphatic, aromatic or heteroaromatic group; 3 R is a nucleofuge group or an optionally substituted, aliphatic, aromatic or heteroaromatic group; and 4 5 each R and R are independently selected from an optionally substituted, aliphatic, 4 5 aromatic or heteroaromatic group, or R and R form together with the intervening oxygen and boron atoms, an optionally substituted, 5- to 10-membered ring having additional 0-2 ring heteroatoms selected from N, O or S.

[027] The method comprises the following steps: (a) providing a “boronate” complex of formula (II): 3 R 2 1 RR 4 OR YB - + M 5 OR (II) where Y is a nucleofugic group; + M is a cation; and 1 5 each R to R is as defined above; and (b) contacting the "boronate" complex of formula (II) with a Lewis acid under conditions that provide the boronic acid ester compound of formula (IN), wherein said contacting step is carried out in a reaction mixture comprising: (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent.

[028] In the previously reported methods for Lewis acid-promoted rearrangement of “boronate” complexes, tetrahydrofuran, an ether solvent, is used which is completely miscible with water. If not used thoroughly dried equipment, solvents and reagents in these procedures, it results in a dramatically reduced diastereomer ratio. It is typical that especially the hygroscopic The Lewis acids must be flame-dried immediately before they are used in the reaction. Although methods for running moisture-sensitive reactions are known to those skilled in the art and are routinely practiced on a laboratory scale, such reactions are expensive and difficult to scale up.

[029] Furthermore, attempts to scale up previously known methods result in ways often further weakening of the diastereomer ratio during work-up and isolation of the boronic acid ester product. Matteson and Erdiik, Organometallics, 2:1083 (1983), report that exposure to alpha-haloboronic acid ester products for free halide ion results in epimerization at the alpha carbon center. Without any desire Without wishing to be bound by theory, the present inventors believe that epimerization is particularly problematic during reaction work-up and / or subsequent steps. It is believed that epimerization occurs, for example, under concentration of reaction the mixture to remove the tetrahydrofuran solvent and replace it with a solvent that is not miscible with water. If you cannot remove the tetrahydrofuran completely, it will also negatively affect the diastereomer ratio during the subsequent washing with water. It is difficult to avoid exposure to the product for halide ion during these steps, especially when the reaction is carried out in large scale.

[030] The present inventors have discovered that it is advantageous to perform the rearrangement of “boronate” complexes in an ether solvent that has low miscibility with water. The use of such solvents eliminates the need for solvent exchange before washing with water, and the organic- soluble products are effectively shielded from aqueous halide ion during washing, even in large-scale production. The solubility of water in the ether solvent is preferably less than about 5% w / w, more preferably less than about 2% weight / weight. In various embodiments, the ether solvent having low miscibility with water, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least about 95% volume / volume of the reaction mixture.

[031] The ether solvent is preferably one that is suitable for routine application in large-scale production. As used here, the term indicates "large scale" to a reaction where at least about five moles of at least one starting material. In a large-scale process, preferably at least about 10, 20, 50 or 100 moles of at least one starting material.

[032] For purposes of the invention, the term “ether” refers to any any of a class of chemical compounds characterized by having a oxygen atom bonded to two carbon atoms. An “ether solvent” is an ether- compound that exists in liquid form at the desired reaction temperature and which can dissolve the starting material(s) and / or the product(s) of the reaction. Non- non-limiting examples of ether solvents suitable for use in the method according to the invention, comprises tert-butyl methyl ether, tert-butyl ethyl ether, tert-amyl methyl ether and isopropyl ether.

[033] In one embodiment, the reaction mixture further comprises a coordinating co-solvent. In another embodiment, the ether solvent is which has low miscibility with water, sufficiently coordinating that a coordinating co-solvent is not necessary. For purposes of the invention, the terms “coordinating co-solvent” and “coordinating solvent” to a solvent that can coordinate the Lewis acid and solvate the ionic components in the reaction. Hindered ether solvents, such as tert-butyl methyl ether, is poorly coordinating and is preferably used with a coordinating conjunction solvent. Non-limiting examples of coordinating co-solvents suitable for use in the practice of the invention include tetrahydro- furan, dioxane, water and mixtures thereof.

[034] In some embodiments, the reaction mixture comprises at least about 5% or at least about 10% volume / volume of a coordinating co-solvent. It is preferably, the amount of a water-miscible, coordinating co-solvent in the reaction mixture is not so large that it affects phase separation during the reaction or processing. In various embodiments, the coordinating co- the solvent not more than about 20%, about 15% or about 10% volume / volume of the reaction mixture.

[035] As used here, the term "nucleofuge" refers to which one any group capable of undergoing nucleophilic substitution during rearrangement conditions according to the present method. Such nucleofuges groups are known in the art. The nucleofuge group is preferably a halogen, more preferably chlorine or bromine. During the rearrangement reaction where the “boronate”- the complex of formula (II) is converted into the boronic acid ester compound of formula (I), - the nucleofuge group Y is released as Y . For example, when Y is chlorine, the chloride- ion released in step (b). +

[036] The variable M is any cationic motion for the negative charged, trivalent boron atom in the “boronate” complex of formula (II). In some + + + + In preferred embodiments, M is selected from the group consisting of Li, Na and K. A + - The skilled person will understand that the salt MY is formed as a by-product in the rearrangement reaction in step (b). 1

[037] The variable R is preferably a group with good migratory ability. In 1 some embodiments, R is C1-8 aliphatic, C6-10 aryl or (C6-10 aryl)(C1-6 aliphatic), wherein any of the groups are optionally substituted. In certain embodiments 1 R is C1-4 aliphatic, especially isobutyl. 2

[038] The variable R is preferably hydrogen, a nucleofugic group or a optionally substituted C aliphatic, C aryl or (C aryl)(C aliphatic) group. 1-8 6-10 6-10 1-6 3 The variable R is preferably a nucleofugic group or an optionally substituted C 1-8 aliphatic, C6-10 aryl or (C6-10 aryl)(C1-6 aliphatic) group. A professional in the field 1 2 will understand that functional substituents may be present on any of R , R 3 or R , provided that the functional substituent does not interfere with the formation of the "boronate" complex of formula (II).

[039] One embodiment of the invention relates to a method for 3 preparation of a boronic acid ester compound of formula (I), where R is a nucleofuge group. Such compounds are useful as intermediates for synthesizing alpha-substituted boronic acid ester and boronic acid compounds, including alpha-amino- boronic acid ester and -boronic acid compounds, as described below. In certain 3 2 preferred embodiments, R is a nucleofuge group and R is hydrogen. 4 5

[040] The variables R and R may be the same or different. In some embodiments 4 5 4 5 are R and R directly connected, so that R and R together with the intermediate oxygen and boron atoms, form an optionally substituted, 5- to 10-membered ring, which may have 0-2 additional ring heteroatoms selected from N, O or S. In some embodiments- forms, the ring is a 5- or 6-membered ring, preferably a 5-membered ring.

[041] The present invention is particularly advantageous for Lewis acid-promoted 4 5 rearrangement of “boronate” complexes of formula (II), where R and R are directly bonded and together are a chiral group. One embodiment of the invention relates to the rearrangement of such chiral “boronate” complexes to provide a boronic acid ester compound 1 2 3 of formula (I), wherein the carbon atom bearing R, R and R is a chiral center. The rearrangement reaction proceeds preferentially with a high degree of stereocontrol of the 4 5 the chiral R -R group to provide the boronic acid ester compound of formula (I) 1 2 3 which has a diastereomer ratio for the carbon atom bearing R, R and R, of at least 4 5 approximately 96:4 relative to a chiral center in the chiral R -R group. Diastereomer- the ratio is preferably at least about 97:3.

[042] The terms “stereoisomer”, “enantiomer”, “diastereomer”, “epimer” and "chiral center", are used herein in accordance with the usual meaning according to professionals in the field. Stereoisomers are therefore compounds in which the same atoms are connected but differ from each other in terms of spatial arrangement of the atoms. Enantiomers are stereoisomers that are mirror images of each other, i.e. the stereochemical configuration at all corresponding chiral centers, is opposite. Diastereomers are stereoisomers that have more than one chiral center and which differ from each other in that the stereochemical configuration at at least one, but not all, of the corresponding chiral centers are opposite. Epimers are diastereomers that differ from each other with respect to stereochemistry configuration at only one chiral center.

[043] As used herein, the term “diastereomer ratio” refers to the ratio of diastereomers that differ from each other with respect to stereochemical configuration at one chiral center, relative to another chiral center in the same molecule. A chemical structure with, for example, two chiral centers, gives four possible stereoisomers: R*R, R*S, S*R and S*S, where the asterisk means that corresponding chiral center in each stereoisomer. The diastereomer ratio for a such a mixture of stereoisomers is the ratio of one diastereomer to its enantiomer, and the other diastereomer and its enantiomer = (R*R + S*S) : (R*S + S*R).

[044] Those skilled in the art will understand that multiple stereoisomers are possible when the molecule has more than two chiral centers. For purposes of the present invention the term "diastereomer ratio" has the same meaning when referring to compounds with multiple chiral centers, as in referring to compounds having two chiral centers. The term “diastereomer ratio” therefore refers to the ratio between all compounds that have R*R or S*S configuration at the specified chiral centers, and all compounds having R*S or S*R configuration at the specified chiral centers. Here, for practical reasons, this relationship is called diastereomeric the ratio of the asterisked carbon atom to the other specified one chiral center.

[045] The diastereomer ratio can be measured by any analytical method. method suitable for distinguishing between diastereomeric compounds that have different relative stereochemical configuration at the specified chiral centers. Such methods include, without limitation, nuclear magnetic resonance (NMR) methods, gas chromatography (GC) and high-pressure liquid chromatography (HPLC).

[046] As described above, one embodiment of the invention relates to methods providing a boronic acid ester compound of formula (I) 1 2 3 which has a diastereomer ratio for the carbon atom bearing R, R and R, of at least 4 5 about 96:4 relative to a chiral center in the chiral R -R group. One skilled in the art 4 5 the field will understand that the chiral R -R group itself may contain more than one chiral 4 5 center. When R -R has more than one chiral center, it preferably has high diastereomer 1 2 3 purity, and the diastereomer ratio of the carbon atom carrying R, R and R, can 4 5 is measured relative to any of the chiral centers in R-R. 4 5

[047] In the methods of the invention, the chiral R -R group has preferably a high degree of enantiomeric purity. For purposes of the invention, the term “enantiomeric purity” means “enantiomeric excess”, which is the excess of the major enantiomer over the bienantiomer, expressed as a 4 5 The chiral R -R group preferably has an enantiomeric purity of at least about 98%, more preferably at least about 99%, even more preferably at least about 99.5% and most preferably at least about 99.9%. 4 5

[048] When the chiral R -R group has very high enantiomeric purity, 1 2 3 the diastereomer ratio for the carbon atom bearing R, R, R, approximates the epimeric- the ratio at this center, i.e. the diastereomer ratio  (R*R) : (S*R) or (R*S) : (S*S)  (R*) : (S*). As used here, the term “epimer ratio” refers to the relationship between the product having one absolute stereochemical configuration at a given chiral center, and the product having the opposite absolute stereochemistry configuration at the corresponding chiral center. The products preferably have the same stereochemical configuration at all other corresponding chiral centers. In one embodiment, the invention therefore relates to the rearrangement of a chiral “boronate”- complex of formula (II) to provide a boronic acid ester compound with 1 2 3 formula (I), where the epimer ratio of the carbon atom bearing R, R and R is at least about 96:4, more preferably at least about 97:3.

[049] Lewis acids suitable for use in the practice of invention, are those which are capable of forming a complex with the nucleofuge 1 group and thereby facilitate its displacement by migration of R . Moreover, The Lewis acid is preferably coordinated to an oxygen atom that is bonded to boron. Non-limiting examples of suitable Lewis acids include zinc bromide, zinc chloride, ferric bromide and ferric chloride. In certain preferred embodiments, The Lewis acid zinc chloride.

[050] The contacting step is preferably carried out at low temperature, but may carried out at ambient or elevated temperature. The selection of a suitable The reaction temperature will depend mainly on the Lewis acid used, as well as 1 as the migratory ability of the R group. Professionals in the field will be able to choose a suitable temperature in light of the reaction conditions used.

[051] In some embodiments, the contacting step is performed by a reaction temperature of at least approx. -100 ºC, -78 ºC or -60 ºC. In some embodiments, the contacting step is carried out at a reaction temperature that is not higher than approximately 80 ºC, 40 ºC or 30 ºC. Any area that includes these high and low temperatures, are encompassed by the invention. The contacting step is preferably carried out at a reaction temperature in the range of about -100 ºC to about 80 ºC, about -70 ºC to approx. 40 ºC, approx. -60 ºC to approx. 30 ºC or approx. -50 ºC to approx. 30 ºC. In certain preferred embodiments start the contacting step at a low temperature, preferably in area approx. -70 ºC to approx. -30 ºC, and then the reaction mixture is allowed to heat up, preferably to ambient temperature.

[052] It is surprising that the method according to the present invention invention does not require any special precautions to avoid the presence of water during the actual rearrangement reaction. In some embodiments, moist Lewis acid used, with minimal effect on the diastereomer ratio. When the designation "moist" is used in reference to the Lewis acid, it means that the water content in The Lewis acid is higher than about 100, 200, 500 or 1000 ppm. It is noteworthy- worthy that the Lewis acid can be added to the reaction mixture, even in the form of a aqueous solution, without negative impact on the diastereomer ratio.

[053] In some embodiments, therefore, the method of the invention the following steps: (a) providing a solution comprising a “boronate” complex with formula (II) and (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent; and (b) adding a Lewis acid solution comprising water and a Lewis acid, to the solution in step (a).

[054] In other embodiments, the Lewis acid solution comprises tetrahydrofuran and a Lewis acid.

[055] In contrast to previously known methods, the method is thus the method according to the invention is well suited for large-scale production. In various embodiments will be at least about 5, 10, 20, 50, 100, 500 or 1000 moles of a "boronate" complex of formula (II) brought into contact with a Lewis acid under conditions such as provides the boronic acid ester compound of formula (I). The invention provides further a preparation comprising a boronic acid ester compound of formula (I), which described herein, and an ether solvent which has low miscibility with water. The composition preferably comprises at least about 5, 10, 20, 50, 100, 500 or 1000 moles of 4 5 the boronic acid ester compound of formula (I). In certain embodiments, R and R are together a chiral group, and the compound of formula (I) present in the preparation, 1 2 has a diastereomeric ratio of at least about 96:4 for the carbon atom bearing R, R and 3 4 5 R, relative to a chiral center in the chiral R-R group.

[056] Workup of the reaction preferably includes washing the reaction mixture. the mixture with an aqueous solution and concentrating the washed reaction mixture the mixture to remove solvents, yielding a residue comprising boronic acid the ester compound of formula (I). It is preferred that the residue preferably comprises at least about 5, 10, 20, 50, 100, 500 or 1000 moles of the boronic acid ester compound with 4 5 formula (I). In those embodiments where R -R is a chiral group, the boronic acid ester has the compound of formula (I) present in the residue, preferably a diastereomer- 1 2 3 ratio of at least about 96:4 for the carbon atom bearing R, R and R, relative to a 4 5 chiral center in the chiral R -R group. It is more preferred that the diastereomeric the ratio is at least about 97:3.

[057] The "boronate" complex of formula (II) can be prepared by which any known method, but is preferably prepared by reacting a Boronic acid ester of formula (III): 1 4 R OR B 5 OR (III) with a reagent of formula (IV): 3 R 2 R + - M Y (IV) + 1 5 where each M, Y and R to R are as defined above for the “boronate” complex with formula (II). In some embodiments, the reaction is carried out at a reaction temperature of at least approx. -100 ºC, -78 ºC or -60 ºC. In some embodiments, the reaction becomes carried out at a reaction temperature not higher than about 0 ºC, -20 ºC or -40 ºC. All areas that include these high and low temperatures are covered by scope of the invention. The reaction is preferably carried out in a reaction temperature in the range of approx. -100 ºC to approx. 0 ºC, approx. -78 ºC to approx. -20 ºC or approx. -60 ºC to about -40 ºC. In some embodiments, the “boronate” complex of formula (II) is prepared in a solution comprising an ether solvent having low miscibility with water, and the reaction mixture is treated directly with a Lewis acid to effect rearrangement to the boron ester compound of formula (I).

[058] In some embodiments, the reagent of formula (IV) is formed in situ. Such embodiments include the following steps: (i) providing a solution comprising a boronic acid ester of formula (III), as defined above, and a compound of formula (V): 2 3 RR H Y (L) 2 3 wherein R and R are as defined above for the reagent of formula (IV); and (ii) treat the solution with a strong, sterically hindered base to form the "boronate" complex of formula (II).

[059] In some embodiments, the sterically hindered base is an alkali metal 2 # 2 # dialkylamide base with formula MN(R ) , where M is Li, Na or K and each R is 2 independently selected from a branched or cyclic C3-6 aliphatic group. In situ formation of the reagent of formula (IV) is particularly advantageous in those embodiments where Y is a nucleofuge group, since the reagent of formula (IV) is unstable.

[060] The boronic acid ester of formula (III) can be prepared by which preferably known method, but is typically prepared by esterification of the corresponding boronic acid compound, e.g. by methods described in Brown et al., Organometallics, 2: 1311-1316 (1983). Cyclic boronic acid esters of formula (III) become preferably prepared by: (a) provide a solution that includes: 1 (i) a boronic acid compound of formula R -B(OH); 2 4 5 4 5 (ii) a compound of formula HO-R-R-OH, where R and R together is an optionally substituted linking chain comprising 2-5 carbon atoms and 0-2 heteroatoms selected from the group consisting of O, N and S; and (iii) an organic solvent that forms an azeotrope with water; and (b) heating the solution at reflux and removing water as an azeotrope. 4 5

[061] As it is used when referring to R and R , the designation shows 4 “linking chain” to the shortest chain of atoms connecting the oxygen atoms as R 5 and R is bonded to. The linking chain is optionally substituted by any chain atom, and one or more chain atoms may also form part of a ring system which is spiro to, fused to or bridges the linear linkage chain. As a 4 5 example, but without limitation, the compound of formula HO-R-R-OH is in some embodiments pinanediol, which has the structure: HO HO . 4 5 In such embodiments, the linking chain R -R comprises two carbon atoms which together form one side of the bicyclo[3.1.1]heptane ring system and one that is additionally substituted with a methyl group. 4 5

[062] In some embodiments, the compound of formula HO-R-R-OH is a chiral diol, preferably one that has high diastereomeric and enantiomeric purity. A One skilled in the art will understand that in such an embodiment the compound of formula 4 5 HO-R-R-OH used as a chiral auxiliary to control the stereochemistry 1 2 3 configuration at the carbon atom bearing R, R and R. Chiral diols that are useful as chiral auxiliaries in organic synthesis, are well known in the art. Non-limiting examples include 2,3-butanediol, preferably (2R,3R)-(-)-2,3- butanediol or (2S,3S)-(+)-2,3-butanediol; pinanediol, preferably (1R,2R,3R,5S)-(-)- pinanediol or (1S,2S,3S,5R)-(+)-pinanediol; 1,2-cyclopentanediol, preferably (1S,2S)- (+)-trans-1,2-cyclopentanediol or (1R,2R)-(-)-trans-1,2-cyclopentanediol; 2,5-hexanediol, preferably (2S,5S)-2,5-hexanediol or (2R,5R)-2,5-hexanediol; 1,2-dicyclohexyl-1,2-ethanediol, preferably (1R,2R)-1,2-dicyclohexyl-1,2-ethanediol or (1S,2S)-1,2-dicyclohexyl-1,2-ethanediol; hydrobenzoin, preferably (S,S)-(-)-hydrobenzoin or (R,R)-(+)-hydrobenzoin; 2,4-pentanediol, preferably (R,R)-(-)-2,4-pentanediol or (S,S,)-(+)-2,4-pentanediol; erythro-γ-lactone, preferably D-erythro-γ-lactone. Carbohydrates, e.g. a 1,2,5,6-symmetrically protected mannitol, can also be used as chiral diols.

[063] Non-limiting examples of organic solvents that are suitable for use in the esterification reaction include acetonitrile, toluene, hexane, heptane and mixtures thereof. In some embodiments, the organic solvent is an ether solvent, preferably an ether solvent that has low miscibility with water. In certain preferred embodiments, the esterification reaction is carried out in a ether solvent which has low miscibility with water, and the product solution which comprising the boronic acid ester of formula (III), is used directly in the next step, without isolation of the boronic acid ester.

[064] As indicated above, the method of the present invention enables invention for the first time the processing of large-scale reactions, without significant negative impact on the diastereomer ratio. In another aspect, therefore, provides the invention a composition comprising at least about 5, 10, 20, 50, 100, 500 or 1000 moles of a boronic acid ester compound of formula (I): 1 R 2 R 4 OR 3 RB 5 OR (IN) where: 1 R is an optionally substituted, aliphatic, aromatic or heteroaromatic group; 2 R is hydrogen, a nucleofuge group or an optionally substituted, aliphatic, aromatic or heteroaromatic group; 3 R is a nucleofuge group or an optionally substituted, aliphatic, aromatic or heteroaromatic group; and 4 5 R and R together with the intervening oxygen and boron atoms form a optionally substituted, 5- to 10-membered, chiral ring having 0-2 additional ring- heteroatoms selected from N, O or S; 1 2 3 where the carbon atom to which R, R and R are bonded is a chiral center having a diastereomer ratio of at least about 96:4, preferably at least about 97:3, in 4 5 relative to a chiral center in the chiral R-R group. 1 3

[065] Preferred values ​​for R to R are as described above. solution agents preferably constitute less than about 30% w / w, 20% w / w, 10% w / w or 5% w / w of the composition according to this aspect of the invention. In some embodiments, the boronic acid ester compound of formula (I) constitutes at least about 70% w / w, 80% w / w, 90% w / w or 95% w / w of the preparation.

[066] One embodiment relates to the composition described above, wherein at least one of the following features is present: 3 (a) R is chlorine; (b) the boronic acid ester compound (I) is 1 1 RR 2 2 RR O.O. 3 3 RBRB O.O. or ; 2 (c) R is hydrogen; and 1 (d) R is C1-4 aliphatic group.

[067] All the boronic acid ester compounds of formula (I) present in the preparation, can be prepared in a single batch run. For purposes of the invention shows the term “batch run” for the execution of a synthesis process where each step in the process is carried out only once. The boronic acid ester compound of formula (I) as present in the preparation, is preferably produced in a single batch run of the process according to the first aspect of the invention. One skilled in the art will understand that the preparation of a given amount of product in a single batch run of a large-scale process, is more efficient and provides a more homogeneous product than manufacturing the same amount of product by repeated execution of a small-scale process. 3

[068] The boronic acid ester compounds of formula (I) wherein R is a nucleofuge group, are useful as intermediates for synthesizing alpha-amino- boronic acid ester compounds. In another aspect, therefore, the invention provides a large-scale process for the preparation of an alpha-aminoboronic acid ester, preferably by a method comprising the following steps: (a) providing a “boronate” complex of formula (II): 3 R 2 1 RR 4 OR YB - + M 5 OR (II) where Y is a nucleofugic group; + M is a cation; 1 R is an optionally substituted, aliphatic, aromatic or heteroaromatic group; 2 R is hydrogen; 3 R is a nucleofugic group; and 4 5 each R and R are independently selected from an optionally substituted, aliphatic, 4 5 aromatic or heteroaromatic group, or R and R form together with the intervening oxygen and boron atoms, an optionally substituted, 5- to 10-membered ring having additional 0-2 ring heteroatoms selected from N, O or S; (b) contacting the "boronate" complex of formula (II) with a Lewis acid under conditions which provide the boronic acid ester compound of formula (I): 1 R 2 R 4 OR 3 RB 5 OR (IN) 1 5 wherein each R to R is as defined above, and wherein said contacting step is carried out in a reaction mixture comprising: (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent; and (c) treating the boronic acid ester compound of formula (I) with a reagent 1 1 of formula M -N(G)2, where M is an alkali metal and each G, individually or together, 1 3 is an amino group protecting group, to form a by-product of formula M -R and a compound of formula (VIII): 1 R 4 OR (G) NB 2 5 OR (VIII) 1 5 where each G and R to R are as defined above; and (d) removing the G groups to form a compound of formula (VII): 1 R 4 OR HNB 2 5 OR (VII) or an acid addition salt thereof.

[069] In some embodiments, the boronic acid ester compound of formula (I) is 1 6 1 in step (c) treated with a reagent of formula M -N(Si(R )3)2, where M is an alkali metal 6 metal and each R is independently selected from the group consisting of alkyl, aralkyl and aryl, wherein the aryl group or the aryl portion of the aralkyl group is optionally substituted.

[070] Reaction of the boronic acid ester compound of formula (I) with 1 the reagent of formula M -N(G)2, is preferably carried out at a reaction temperature of the range of about -100 ºC to about 50 ºC, preferably about -50 ºC to about 25 ºC and more 3 preferably about -30 ºC to about 0 ºC. In some embodiments, R is halogen, preferably 1 chlorine, and M is Li. To facilitate isolation of the product of formula (VIII), 1 3 preferably the reaction mixture is an organic solvent where the by-product M -R have low solubility. Non-limiting examples of suitable organic solvents are agents include methylcyclohexane, cyclohexane, heptane, hexane and toluene. In some embodiments include step (c) further filtering the reaction mixture to remove 1 3 M -R , and this gives a filtrate comprising the compound of formula (VIII). The filtrate is preferably used directly in step (d).

[071] In those embodiments where the reaction mixture comprises an organic 1 3 solvent where the by-product M -R has low solubility, the reaction can 1 3 the mixture also includes a solvent in which the by-product M -R has high 1 3 solubility. In such cases, the solvent where the by-product M -R has high solubility, preferably removed before filtering the reaction mixture. In some 1 6 In some embodiments, for example, a reagent of formula M -N(Si(R ) ) is added to 3 2 the reaction mixture as a solution comprising tetrahydrofuran. In such In embodiments, it is preferred that step (c) further comprises removing tetrahydro- the furan before filtering the reaction mixture.

[072] Those skilled in the art are aware of various methods that can be used to remove the protecting groups G in the compound of formula (VIII), including e.g. aqueous hydrolysis or treatment with acid. The alpha-aminoboronic acid ester product with formula (VII) has low stability and is preferably immediately derivatized (Matteson et al., J. Am. Chem. Soc., 103:5241 (1981)) or isolated as an acid addition salt. In some embodiments comprise step (d) treating the compound of formula (VIII) with an acid and isolating the compound of formula (VII) as the acid addition salt. In certain preferred embodiments, the acid is trifluoroacetic acid, and the compound with formula (VII) is isolated as the trifluoroacetic acid addition salt.

[073] As described above, the methods of the invention are particularly suitable for the preparation of alpha-aminoboronic acid ester compounds with formula (VII), wherein the alpha carbon atom is a chiral center. One embodiment of the invention therefore relates to a large-scale process for the production of an alpha- aminoboronic acid ester compound of formula (VIIa) or (VIIb): 1 1 RR 4 4 OR OR HNBHNB 2 2 5 5 OR OR (VIIa) (VIIb) or an acid addition salt thereof, wherein: 1 R is an optionally substituted, aliphatic, aromatic or heteroaromatic group; and 4 5 R and R together with the intervening oxygen and boron atoms, form a optionally substituted, chiral, cyclic boronic acid ester; wherein said method comprises: (a) providing a “boronate” complex of formula (IIa) or (IIb): 3 3 RR 2 1 2 1 RRRR 4 4 OR OR YBYB - - + + MM 5 5 OR OR (IIa) (IIb) where Y is a nucleofugic group; + M is a cation; 2 R is hydrogen; 3 R is a nucleofugic group; and 4 5 R and R are as defined above; (b) contacting the “boronate” complex of formula (IIa) or (IIb) with a Lewis acid under conditions which provide a boronic acid ester compound with formula (Ia) or (Ib): 1 1 RR 2 2 RR 4 4 OR OR 3 3 RBRB 5 5 OR OR (Ia) (Ib) 1 5 wherein each R to R is as defined above, wherein said contacting step is carried out in a reaction mixture comprising: (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent; and (c) treating the boronic acid ester compound of formula (Ia) or (Ib) with a 1 1 reagent with formula M -N(G) , where M is an alkali metal and G is an amino group- 2 protecting group, to form a compound of formula (VIIIa) or (VIIIb): 1 1 RR 4 4 OR OR (G) NB (G) NB 2 2 5 5 OR OR (VIIIa) (VIIIb) 1 5 where each G and R to R are as defined above; and (d) removing the G groups to form a compound of formula (VIIa) or (VIIb): 1 1 RR 4 4 OR OR HNBHNB 2 2 5 5 OR OR (VIIa) (VIIb) or an acid addition salt thereof. + 1 5

[074] Preferred meanings of Y, M, R to R and G are as described above. The compound of formula (VIIa) or (VIIb) preferably has a diastereomeric ratio for the alpha carbon atom of at least about 96:4, more preferably at least about 97:3, in 4 5 relative to a chiral center in the chiral R-R group.

[075] The alpha-aminoboronic acid ester compounds of formula (VII) are useful synthesis intermediates for the preparation of peptidyl boronic acid esters compounds. In some embodiments, therefore, the method of this invention comprises aspect of the invention, further coupling the compound of formula (VII) with a compound of formula (IX): 7 R X 1 PN H O (IX) where: 1 P is an amino group-blocking group; 7 R is selected from the group consisting of hydrogen, C aliphatic group, 1-10 8 optionally substituted C 6-10 aryl group or C 1-6 aliphatic-R group; and 8 R is selected from the group consisting of alkoxy, alkylthio, optionally substituted aryl, heteroaryl and heterocyclyl groups and optionally protected amino, hydroxy and guanidino groups; and X is OH or a leaving group; to form a compound of formula (X): 7 4 R OR H NB 1 5 OR PN H 1 OR (X) 1 1 4 5 7 where each P, R, R, R and R are as defined above.

[076] The outgoing group X is any group that is in capable of undergoing nucleophilic substitution at the alpha-amino group of the compound of formula (VII). In some embodiments, the group -C(O)-X is an activated ester, so as an O-(N-hydroxysuccinimide) ester. In some embodiments, an activated ester formed in situ by bringing a compound of formula (IX), where X is OH, i contact with a peptide coupling reagent. Examples of suitable peptide coupling reagents include, without limitation, carbodiimide reagents, e.g., dicyclohexyl- carbodiimide (DCC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); phosphonium reagents, e.g. benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent); and uronium reagents, e.g. O-(1H-benzotriazole-1- yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[077] Professionals are also aware of procedures that enable direct coupling of silyl-protected amines, without a prior deprotection step. In such procedures the silyl groups are removed in situ under the coupling reaction conditions. In some embodiments of the present invention therefore become a compound of formula (VIII) brought into contact with a compound of formula (IX), under conditions such as 6 removes the (R)Si groups in situ and forms a compound of formula (X). 3

[078] For purposes of the invention, the term “amino group- "blocking group" to any group that is used to derivatize an amino group, especially an N-terminal amino group with a peptide or an amino acid. The term "amino group-blocking group" includes, but is not limited to, protecting groups that are commonly used within organic synthesis, especially peptide synthesis. See for example Gross and Mienhoffer, ed., The Peptides, vol. 3, Academic Press, New York, 1981, pp. 3-88; Greene duck Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley and Sons, Inc., New York, 1999. Unless otherwise specified, however, it is not necessary that an amino group-blocking group is easy to cleave. Amino group- blocking groups include, e.g. alkyl-, acyl-, alkoxycarbonyl-, aminocarbonyl- and sulfonyl groups. In some embodiments, the amino group-blocking the group is an acyl group derived from an amino acid or a peptide or a derivative or an analogue thereof.

[079] As used herein, the term "amino acid" includes both naturally occurring and synthetic amino acids. For purposes of the invention, a “derivative” of an amino acid or a peptide one in which a functional group, e.g. a hydroxy, amino, carboxy or guanidino group at the N-terminus or on a side chain, is modified with a blocking group. As used herein, includes an “analog” of an amino acid or peptide one that includes a modified backbone or a modified side chain. The term “peptide analogue” shall include peptides where one or more stereocenters are inverted and one or more peptide bonds is replaced with a peptide isostere. 1

[080] In some embodiments, P is a leaving protecting group. Examples of leaving protecting groups include, without limitation, acyl- protecting groups, e.g. formyl, acetyl (Ac), succinyl (Suc) or methoxysuccinyl (MeOSuc), and urethane protecting groups, e.g. tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or fluorenylmethoxycarbonyl (Fmoc).

[081] In some such embodiments, the method further comprises

[082] According to this aspect of the invention the following steps: 1 (f) removing the protecting group P to form a compound of formula (XI): 7 4 R OR H NB 5 HN OR 2 1 OR (XI) 1 4 5 7 or an acid addition salt thereof, wherein each R 1 , R 2 , R 3 and R 4 are as defined above; and 2 (g) coupling the compound of formula (XI) with a reagent of formula P -X; 2 wherein P is any amino group-blocking group as described above, and X is a leaving group, to form a compound of formula (XII): 7 4 R OR H NB 2 5 PN OR H 1 OR (XII) 2 1 4 5 7 wherein each P, R, R, R and R are as defined above. One skilled in the art will understand that in the 2 embodiments where P is an acyl group, including e.g. an acyl group which is derived from an amino acid or a peptide or an analogue or derivative thereof, then the leaving group X is formed in situ, as described above for the compound with formula (IX).

[083] In both compound (X) and (XII) the boronic acid group is protected as one boronic acid ester. If desired, the boronic acid group can be deprotected with any by any method known in the art. The boronic acid group is deprotected preferably by transesterification in a biphasic mixture. It is more preferred that the boronic acid deprotection step comprises the following steps: (i) providing a biphasic mixture comprising boronic acid ester- the compound of formula (X) or (XII), an organic boronic acid acceptor, a lower alkanol, a C5-8 hydrocarbon solvent and aqueous mineral acid; (ii) stirring the biphasic mixture, giving the corresponding the deprotected boronic acid compound of formula (Xa) or (XIII): 7 7 R OH R OH HH NBNB 1 2 PN OH PN OH HH 1 1 OROR (Xa) (XIII); (iii) separating the solvent layers; and (iv) extracting the compound of formula (Xa), (XIII) or a boronic acid- anhydride thereof, into an organic solvent.

[084] The organic boronic acid acceptor in step (I) is preferably a aliphatic, aryl or ar(aliphatic) boronic acid. In some embodiments, the boronic acid is the acceptor selected from the group consisting of phenylboronic acid, benzylboronic acid, butylboronic acid, pentylboronic acid, hexylboronic acid and cyclohexylboronic acid. In certain embodiments, the boronic acid acceptor is isobutylboronic acid. In some embodiments, forms, the boronic acid acceptor is chosen so that the boronic acid ester compound of formula (III) is formed as a by-product in the deprotection reaction. Boronic acid ester- the compound of formula (III) can then be used in another batch run of the method described above. In such embodiments, the group is 4 5 4 5 R -R efficiently recovered, which can be particularly advantageous if R -R is a expensive chiral group.

[085] To improve the purity of the product, the aqueous layer is containing the compound of formula (Xa) or (XIII), preferably washed to remove neutral, organic impurities before extraction step (iv). In such embodiments step (iii) preferably comprises the following steps: (1) separate the solvent layers; (2) adjust the aqueous layer to basic pH; (3) washing the aqueous layer with an organic solvent; and (4) regulate the aqueous layer to a pH below approx. 6.

[086] In some embodiments, the invention relates to an improved method- method for preparing the proteasome inhibitor bortezomib. In one embodiment Accordingly, the invention provides a large-scale method for producing a compound of formula (XIV): Oh Oh H NNB N OH H O N (XIV) or a boronic anhydride thereof. The process comprises the following steps: (a) providing a “boronate” complex of formula (XV): 3 R O HB - YO + M (XV) where: 3 R is a nucleofugic group; Y is a nucleofugic group; and + M is an alkali metal; (b) contacting the "boronate" complex of formula (XV) with a Lewis acid, under conditions which provide a boronic acid ester compound of formula (XVI): O 3 RB O (XVI) wherein said contacting step is carried out in a reaction mixture comprising: (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent; (c) treating the boronic acid ester compound of formula (XVI) with a 1 1 reagent of formula M -N(G)2, where M is an alkali metal and each G, individually or together, is an amino group protecting group, to form a compound with formula (XVII): O (G) NB 2 O (XVII) (d) removing the G groups to form a compound of formula (XVIII): O B HN 2 O (XVIII) or an acid addition salt thereof; (e) coupling the compound of formula (XVIII) with a compound of formula (XIX); X 1 PN H O (XIX) where: 1 P is a cleavable amino group protecting group; and X is OH or a leaving group; to form a compound of formula (XX): O H 1 PNB O N H O (XX) 1 where P is as defined above; 1 (f) removing the protecting group P to form a compound of formula (XXI): O H NB O HN 2 O (XXI) or an acid addition salt thereof; (g) coupling the compound of formula (XXI) with a reagent of formula (XXII) N X N O (XXII) where X is OH or a leaving group, to form a compound of formula (XXIII): O.O. H NNB O N H O N (XXIII) ; and (h) deprotecting the boronic acid group to form the compound of formula (XIV) or a boronic anhydride thereof.

[087] In some embodiments, the method is characterized by at least one of the following features (1)-(5). In certain preferred embodiments, the method is characterized by all five traits (1)-(5) below. 3 (1) In the "boronate" complex of formula (XV) both R and Y are chlorine. (2) The linking step (e) comprises the following steps: (i) coupling the compound of formula (XVIII) with a compound with formula (XIX), where X is OH, in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) and a tertiary amine in dichloromethane; (ii) perform a solvent exchange to replace dichloromethane with ethyl acetate; and (iii) performing water washing of the ethyl acetate solution. (3) Step (f) for removing the protecting group comprises the following steps: (i) treating the compound of formula (XX) with HCl in ethyl acetate; (ii) adding heptane to the reaction mixture; and (iii) isolating the compound of formula (XXI) as the HCl addition salt by crystallization. (4) The coupling step (g) comprises the following steps: (i) coupling the compound of formula (XXI) with 2-pyrazine- carboxylic acid in the presence of TBTU and a tertiary amine in dichloromethane; (ii) perform a solvent exchange to replace dichloromethane with ethyl acetate; and (iii) performing water washing of the ethyl acetate solution. (5) The boronic acid deprotection in step (h) comprises the following steps: (i) providing a biphasic mixture comprising the compound with formula (XXIII), an organic boronic acid acceptor, a lower alkanol, et C5-8 hydrocarbon solvent and aqueous mineral acid; (ii) stirring the biphasic mixture, yielding the compound with formula (XIV); (iii) separating the solvent layers; and (iv) extracting the compound of formula (XIV) or et boronic anhydride thereof, into an organic solvent.

[088] Step (h)(iii) preferably comprises the following steps: (1) separate the solvent layers; (2) adjust the aqueous layer to basic pH; (3) washing the aqueous layer with an organic solvent; and (4) regulate the aqueous layer to a pH below approx. 6;

[089] In another embodiment, the invention relates to a large-scale process for preparing a compound of formula (XIV) Oh Oh H NNB N OH H O N (XIV) or a boronic anhydride thereof, comprising the following steps: (aa) coupling a compound of formula (XVIII): O HNB 2 O (XVIII) or an acid addition salt thereof, with a compound of formula (XIX): X 1 PN H O (XIX) where: 1 P is a cleavable amino group protecting group; and X is OH or a leaving group; to form a compound of formula (XX): O H 1 PN B O N H O (XX) 1 where P is as defined above, wherein said coupling step (aa) comprises the following steps: (i) coupling the compound of formula (XVIII) with a compound with formula (XIX), where X is OH, in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) and a tertiary amine in dichloromethane; (ii) perform a solvent exchange to replace dichloromethane with ethyl acetate; and (iii) performing water washing of the ethyl acetate solution; 1 (bb) removing the protecting group P to form a compound of formula (XXI): O H N B O HN 2 O (XXI) or an acid addition salt thereof, wherein said removal of the protecting group in Step (bb) includes the following steps: (i) treating the compound of formula (XX) with HCl in ethyl acetate; (ii) adding heptane to the reaction mixture; and (iii) isolating the compound of formula (XXI) as the HCl addition salt by crystallization; (cc) coupling the compound of formula (XXI) with a reagent of formula (XXII) N X N O (XXII) where X is OH or a leaving group, to form a compound of formula (XXIII): O.O. H N NB O N H O N (XXIII), wherein said coupling step (cc) comprises the following steps: (i) coupling the compound of formula (XXI) with 2-pyrazine- carboxylic acid, in the presence of TBTU and a tertiary amine in dichloromethane; (ii) perform a solvent exchange to replace dichloromethane with ethyl acetate; and (iii) performing water washing of the ethyl acetate solution; and (dd) deprotecting the boronic acid group to form the compound of formula (XIV) or a boronic anhydride thereof, wherein said deprotection in step (dd) includes the following steps: (i) providing a biphasic mixture comprising the compound with formula (XXIII), an organic boronic acid acceptor, a lower alkanol, et C5-8 hydrocarbon solvent and aqueous mineral acid; (ii) stirring the biphasic mixture, yielding the compound with formula (XIV); (iii) separating the solvent layers; and (iv) extracting the compound of formula (XIV) or et boronic anhydride thereof, into an organic solvent.

[090] Step (dd)(iii) preferably comprises the following steps: (1) separate the solvent layers; (2) adjust the aqueous layer to basic pH; (3) washing the aqueous layer with an organic solvent; and (4) regulate the aqueous layer to a pH below approx. 6;

[091] The effectiveness of the methods described above is further improved by telescopic steps, for example by transferring a reaction mixture or a prepared product solution from one reaction and directly to the next reaction, without isolating the intermediate. In some embodiments provides, for example, step (e)(iii) or (aa)(iii) an ethyl acetate solution which comprises a compound of formula (XX), and the ethyl acetate solution is in step (f) or 1 (bb) directly subjected to conditions which effectively remove the protecting group P . IN 1 In some such embodiments, the protecting group P is an acid-labile protecting group. group, for example tert-butoxycarbonyl (Boc), and the ethyl acetate solution from step (e)(iii) or (aa)(iii) is treated with acid. In certain preferred embodiments, the ethyl acetate solution from step (e)(iii) or (aa)(iii) dried by azeotroping and is then treated with gaseous HCl.

[092] When deprotection step (f) or (bb) is carried out under anhydrous conditions conditions, as described above, the product of formula (XXI) can be isolated by crystallization of the HCl addition salt from the reaction mixture. Crystallization of the product salt is promoted by the addition of a hydrocarbon solvent such as n- heptane. In some embodiments, the reaction mixture is partially concentrated before addition of the hydrocarbon solvent. The present inventors have discovered that crystallization of the compound of formula (XXI) in this manner effectively removes any tripeptide impurities that may have been formed during the coupling step(s) or (aa). Such impurities are difficult to remove in later steps of the synthesis.

[093] Furthermore, it is possible to shorten (“telescope”) the procedure by transfer the product mixture from the coupling step (g) or (cc) directly to the deprotection of the boronic acid group in step (h) or (dd). It is preferred that the organic the solvent from the coupling reaction is first replaced with ethyl acetate to facilitate water washing. A new solvent exchange to a hydrocarbon solvent then makes it possible to use the product solution from step (g) or (cc) directly in the biphasic boronic acid deprotection in step (h) or (dd), without isolation of the compound of formula (XXIII).

[094] Alternatively, a more convergent approach can be adapted synthesizing the compound of formula (XIV). In another embodiment Accordingly, the invention provides a large-scale method for producing a compound of formula (XIV) Oh Oh H NNB N OH H O N (XIV) or a boronic anhydride thereof. The process comprises the following steps: (a) providing a “boronate” complex of formula (XV): 3 R O HB - YO + M (XV) where: 3 R is a nucleofugic group; Y is a nucleofugic group; and + M is an alkali metal; (b) bringing the "boronate" complex of formula (XV) into contact with a Lewis acid under conditions which provide a boronic acid ester compound of formula (XVI): O 3 RB O (XVI) wherein said contacting step is carried out in a reaction mixture comprising: (i) a coordinating ether solvent that has low miscibility with water; or (ii) an ether solvent that has low miscibility with water, and a coordinating co-solvent; (c) treating the boronic acid ester compound of formula (XVI) with a 1 6 1 6 reagent with formula M -N(Si(R )3)2, where M is an alkali metal and each R is independently selected from the group consisting of alkyl, aralkyl and aryl, wherein the aryl group or the aryl portion of the aralkyl group is optionally substituted, to form a compound with formula (XVII): O 6 B ((R)Si)N 3 2 O (XVII) 6 (d) removing the (R)3Si groups to form a compound of formula (XVIII): O HNB 2 O (XVIII) or an acid addition salt thereof; (e) coupling the compound of formula (XVIII) with a compound of formula (XIXa): O NX N H O N (XIXa) where X is OH or a leaving group, to form a compound of formula (XXIII): O.O. H NNB O N H O N (XXIII) ; and (f') deprotecting the boronic acid group to form the compound of formula (XIV) or a boronic anhydride thereof.

[095] In some embodiments, the method is characterized by at least one of the following features (1)-(3). In certain preferred embodiments, the method is characterized by all three features (1)-(3) below. 3 (1) In the "boronate" complex of formula (XV) both R and Y are chlorine. (2) The coupling step (e') comprises the following steps: (i) coupling the compound of formula (XVIII) with a compound with formula (XIXa), where X is OH, in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) and a tertiary amine in dichloromethane; (ii) perform a solvent exchange to replace dichloromethane with ethyl acetate; and (iii) performing water washing of the ethyl acetate solution. (3) The boronic acid deprotection in step (f') comprises the following steps: (i) providing a biphasic mixture comprising the compound with formula (XXIII), an organic boronic acid acceptor, a lower alkanol, et C hydrocarbon solvent and aqueous mineral acid; 5-8 (ii) stirring the biphasic mixture, yielding the compound with formula (XIV); (iii) separating the solvent layers; and (iv) extracting the compound of formula (XIV) or et boronic anhydride thereof, into an organic solvent.

[096] step (f')(iii) preferably comprises the following steps: (1) separate the solvent layers; (2) adjust the aqueous layer to basic pH; (3) washing the aqueous layer with an organic solvent; and (4) regulate the aqueous layer to a pH below approx. 6;

[097] In steps (h)(iv), (dd)(iv) or (f')(iv) of the methods described above, the compound of formula (XIV) or a boronic anhydride thereof, becomes preferably extracted with ethyl acetate and crystallized by addition of hexane or heptane. In some embodiments, the method further comprises isolating a boronic anhydride of the compound of formula (XIV), preferably a trimer boronic anhydride of formula (XXIV): O.O. H NNB N 3 H O N (XXIV)

[098] The methods of the invention enable large-scale production of bortezomib with very high chemical and stereochemical purity. Previously known methods were limited in terms of scale and yielded products with lower purity overall. In yet another aspect, the invention therefore provides a composition which comprises at least one kg of a compound of formula (XXIV): O.O. H NNB N 3 H O N (XXIV) The compound of formula (XXIV) is preferably prepared according to the method described above, and preferably constitutes at least 99% weight / weight of the composition according to this aspect of the invention. EXAMPLES Abbreviations BOC tert-butoxycarbonyl you deionized DMF N,N-dimethylformamide GC gas chromatography GC-MS gas chromatography-mass spectrometry HDPE high density polyethylene HPLC high-performance liquid chromatography LDA lithium diisopropylamide LOD drying loss my minutes MTBE t-butyl methyl ether RP-HPLC Reversed Phase High Performance Liquid Chromatography RPM revolutions per minute TBTU O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium- tetrafluoroborate THF tetrahydrofuran Example 1: Process for the preparation of (1R)-(S)-pinanediol-1- ammonium trifluoroacetate-3-methylbutane-1-boronate (1S)-(S)-Pinanediol-1-chloro-3-methylbutane-1-boronate 1. (S)-Pinanediol-2-methylpropane-1-boronate (12.0 kg, 50.8 mol) was kept in a reaction vessel which was kept under a nitrogen atmosphere. 2. Tert-butyl methyl ether (53 kg) and dichloromethane (22.5 kg) were added, and the The resulting mixture was cooled to -57°C with stirring. 3. Diisopropylamine (6.7 kg) was kept in another reaction vessel which was kept under nitrogen atmosphere. 4. Tert-butyl methyl ether (27 kg) was added to the diisopropylamine, and the resulting the mixture was cooled to -10°C with stirring. 5. n-hexyllithium in hexane (33.2 wt% solution) (17.6 kg) was added to the diisopropylamine mixture over a period of 57 minutes, while the reaction temperature was maintained at -10°C to -7°C. 6. This mixture (LDA mixture) was stirred for 33 minutes at -9 °C to -7 °C before it was used. 7. Zinc chloride (12.1 kg) was added to a third reaction vessel which was kept under nitrogen atmosphere. 8. Tert-butyl methyl ether (16 kg) was added to the zinc chloride, and the resulting the mixture was heated to 30 °C with stirring. 9. Tetrahydrofuran (53 kg) was added to the zinc chloride suspension over a period of period of 18 minutes, while the reaction temperature was maintained at 35 °C until 40°C. 10. This mixture (ZnCl2 mixture) was stirred for 4 hours and 28 minutes at 38°C to 39°C until used. 11. The LDA mixture (from # 3 - 6) was added over a period of 60 minutes to the reaction vessel containing (S)-pinanediol-2-methylpropane-1-boronate, while the reaction temperature was maintained at -60 °C to -55 °C. 12. Washing with tert-butyl methyl ether (10 kg) was carried out to complete the addition. 13. The reaction mixture was stirred for an additional 20 minutes at -59°C to -55°C. 14. The reaction mixture was warmed to -50 °C over a period of 11 minutes. 15. The ZnCl2 mixture (from # 7 - 10) was added over a period of 48 minutes to the reaction vessel containing (S)-pinanediol-2-methylpropane-1-boronate and the LDA mixture, while maintaining the reaction temperature at -50 °C to -45 °C. 16. Washing with tert-butyl methyl ether (10 kg) was carried out to complete the addition. 17. The reaction mixture was stirred for an additional 30 minutes at -45 °C to -40 °C. and was then heated to 10 °C over a period of 81 minutes. 18. 10% sulfuric acid solution (72 kg) was added over a period of 40 minutes to the reaction vessel, while the reaction temperature was maintained at 10 °C to 21 °C. 19. The reaction mixture was stirred for 16 minutes at ambient temperature, before the aqueous phase was separated. 20. The organic phase was washed successively with deionized (di) water (32 kg) and 10% sodium chloride solution (26.7 kg), and each wash involved vigorous stirring for 15 to 17 minutes at ambient temperature. 21. The reaction mixture was concentrated under reduced pressure (pmin = 81 mbar), while maintaining an external temperature (heating jacket / bath) of 50 °C to 55 °C, giving a residue which was dissolved in methylcyclohexane (56 kg). 22. The reaction mixture was refluxed (in a Dean-Stark condenser) type for water separation) under reduced pressure (p = 67 mbar), while it was my maintained an external temperature (heating jacket / bath) of 50°C to 55°C for 2 hours and 7 minutes, until no more water was separated. 23. Approximately 35 L of the solvents were distilled off under reduced pressure (p = 81 my mbar), while maintaining an external temperature (heating jacket / bath) of 50°C to 55°C. 24. The resulting dry methylcyclohexane mixture containing (1S)-(S)- pinanediol-1-chloro-3-methylbutane-1-boronate, was cooled to 14 °C. (1R)-(S)-Pinanediol-1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate 1. Lithium bis(trimethylsilyl)amide in tetrahydrofuran (19.4 wt% solution) (41.8 kg) was added to a reaction vessel kept under a nitrogen atmosphere, and was cooled to -19 °C with stirring. 2. The methylcyclohexane mixture containing (1S)-(S)-pinanediol-1-chloro-3- methylbutane-1-boronate, was added over a period of 55 minutes, while the reaction temperature was maintained at -19°C to -13°C. 3. Washing with methylcyclohexane (5 kg) was carried out to complete the addition. 4. The reaction mixture was stirred for an additional 65 minutes at -13°C to -12°C. and was then heated to 25 °C over a period of 25 minutes. 5. A suspension of Celite (2.5 kg) in methylcyclohexane (22 kg) was added to the reaction mixture. 6. The reaction mixture was concentrated under reduced pressure (p = 25 mbar), my while maintaining an external temperature (heating jacket / bath) of 45 °C to 50 °C, giving a residue which was dissolved in methylcyclohexane (36 kg). 7. A sample was then taken for GC testing of the tetrahydrofuran content during the process. 8. The tetrahydrofuran content was 0.58%. 9. The solids were removed by filtration, and the filtrate was filtered through a bed of of silica gel (2.0 kg). 10. Both filter units were washed with isopropyl ether (30 kg). 11. The resulting methylcyclohexane / isopropyl ether mixture containing (1R)-(S)-Pinanediol-1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate, was stored in a container at ambient temperature until used in next step. (1R)-(S)-Pinanediol-1-ammonium-trifluoroacetate-3-methylbutane-1-boronate 1. Trifluoroacetic acid (12 kg) was added to another reaction vessel which was kept under nitrogen atmosphere. 2. Isopropyl ether (78 kg) was added to the trifluoroacetic acid, and the resulting the mixture was cooled to -10°C with stirring. 3. The methylcyclohexane / isopropyl ether mixture containing (1R)-(S)-pinanediol- 1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate, was added over a period of 53 minutes, which caused precipitation, while the reaction temperature was maintained at -10°C to -5°C. 4. Washing with isopropyl ether (5 kg) was carried out to complete the addition. 5. The reaction mixture was stirred for an additional 8 hours and 20 minutes at -9 °C. to -7 °C. 6. The solid was collected by filtration, washed with isopropyl ether (70 kg) in two portions and dried under reduced pressure (pmin = 56 mbar) at 41 °C to 42 °C for 2 hours and 15 minutes. 7. The solid was stirred with di water (60 kg) for 24 minutes at ambient temperature, before the di water was removed by filtration. 8. The solid was washed with di water (12 kg). 9. The solid was then dried under vacuum (pmin = 4 mbar) at 40 °C. to 44 °C for 9 hours and 22 minutes, during which time the drying loss was 0.51%, which meets the requirement of  1%. 10. The intermediate, crude (1R)-(S)-pinanediol-1-ammonium trifluoroacetate-3- methylbutane-1-boronate, was then packed in simple polyethylene bags in polypropylene barrel which was labeled. The yield was 72%. Recrystallization of (1R)-(S)-Pinanediol-1-ammonium-trifluoroacetate-3-methylbutane-1- boronate, crude 1. (1R)-(S)-Pinanediol-1-ammonium-trifluoroacetate-3-methylbutane-1-boronate, crude, (13 kg) was added to a reaction vessel held under nitrogen atmosphere. 2. Trifluoroacetic acid (31 kg) was added to the reaction vessel, and the resulting the mixture was cooled to 4 °C with stirring. 3. Once all the solids had dissolved and a slightly cloudy mixture had formed, isopropyl ether (29 kg) added over a period of 57 minutes, while the reaction temperature was maintained at 2 °C to 3 °C. 4. After the addition was complete, the mixture was filtered through a filter and into a container which was kept under a nitrogen atmosphere. 5. Reactor and filter were rinsed with a mixture of trifluoroacetic acid (3.8 kg) and isopropyl ether (5 kg). The washing volume was added to the filtrate. 6. Isopropyl ether (126 kg) was added over a period of 15 minutes, which caused product precipitation, while the reaction temperature was maintained at 16 °C to 18°C. 7. The mixture was stirred at 16 °C to 18 °C for 15 min, then cooled to -5 °C over a period of 67 minutes and was stirred at -3 °C to -5 °C under nitrogen atmosphere for 89 minutes. 8. The solid was then isolated by filtration, washed with isopropyl ether (48 kg) in two portions and dried under vacuum (pmin = 2 mbar) at 34 °C to 40 °C for 2 hours and 55 minutes, and after this time the drying loss was 0.32%, which meets the requirement of  0.5%. 9. The product, (1R)-(S)-pinanediol-1-ammonium trifluoroacetate-3-methylbutan-1- boronate, was then packed in double polyethylene bags in fiber drums which were labeled. The yield was 86%. Example 2: Process for the preparation of N-(2-pyrazinecarbonyl)-L- phenylalanine-L-leucine-boronic anhydride (1S,2S,3R,5S)-Pinanediol-N-BOC-L-phenylalanine-L-leucine-boronate 1. A three-necked glass reaction flask equipped with a Claisen condenser, temperature recorder and a mechanical stirrer, were flushed with nitrogen in a fume hood / exhaust cabinet. 2. (1R)-(S)-Pinanediol-1-ammonium-trifluoroacetate-3-methylbutane-1-boronate (2.0 kg) was added to the flask. 3. BOC-L-phenylalanine (1.398 kg) was added to the flask. 4. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (1.864 kg) was added to the flask. 5. Dichloromethane (15.8 L) was added to the flask. 6. The stirring motor was adjusted to a stirring speed of 260 RPM. 7. The reaction mixture was cooled to 1.0 °C using an ice / water cooling bath, while the nitrogen atmosphere was maintained. 8. N,N-diisopropylethylamine (2.778 L) was added to a glass flask and transferred to the reaction mixture over a period of 117 minutes using a peristaltic pump, while the reaction temperature was maintained in the range 0.7°C - 2.1°C. The total addition rate was 23.7 ml / min. 9. The addition was completed by washing the flask with dichloromethane (0.2 L), which was added to the reaction mixture. 10. The reaction mixture was stirred for an additional 35 minutes. At the start of stirring, the temperature was 1.8 °C, and at the end it was 2.5 °C. 11. A sample was then taken for testing by reverse phase high-performance liquid chromatography (RP-HPLC) during the process. Percentage conversion was found to be 99.3%. 12. The reaction mixture was transferred to two rotary evaporator flasks in two approximately equal volume. The reaction mixture was concentrated under reduced pressure at using a rotary evaporator, while maintaining an external bath temperature of 29-30 °C. 13. Ethyl acetate (4.0 L) was divided into two approximately equal volumes and was kept in the two rotary the evaporator flasks. 14. The mixtures in each flask were again concentrated under reduced pressure at using a rotary evaporator, while maintaining an external bath temperature of 29-30 °C. 15. The residues in each rotary evaporator flask were then returned to reaction flask using ethyl acetate (13.34 L). 16. A 1% aqueous phosphoric acid solution was prepared by mixing di water (13.18 L) and phosphoric acid (0.160 kg) in a glass flask equipped with a stirrer. 17. A 2% aqueous potassium carbonate solution (12.0 L) was prepared by mixing the water (11.76 L) and potassium carbonate (0.24 kg) in a glass flask equipped with a stirs. 18. A 10% aqueous sodium chloride solution (13.34 L) was prepared by mixing the water (13.34 L) and sodium chloride (1.334 kg) in a glass flask equipped with a stirs. 19. Distilled water (13.34 L) was added to the reaction flask containing ethyl acetate- the solution, and the mixture was stirred at 380 RPM for 7 minutes. The layers were separated, and the aqueous phase (lower layer) was transferred under vacuum to a suitable flask and was thrown away. 20. Distilled water (13.34 L) was again added to the reaction flask containing ethyl acetate- the solution, and the mixture was stirred at 385 RPM for 7 minutes. The layers were separated, and the aqueous phase (lower layer) was transferred under vacuum to a suitable flask and was thrown away. 21. The 1% phosphoric acid solution prepared in Step 16 was added to the reaction flask containing the ethyl acetate solution, and the mixture was stirred at 365 RPM for 7 minutes. The layers were allowed to separate, and the acidic, the aqueous phase (lower layer) was transferred to a suitable flask and discarded. 22. The 2% potassium carbonate solution prepared in Step 17 was added to the reaction flask containing the ethyl acetate solution, and the mixture was stirred at 367 RPM for 7 minutes. The layers were allowed to separate, and the basic, the aqueous phase (lower layer) was transferred to a suitable flask and discarded. 23. The 10% sodium chloride solution prepared in Step 18 was added to the reaction flask containing the ethyl acetate solution, and the mixture was stirred at 373 RPM for 6 minutes. The layers were allowed to separate, and the aqueous The phase (lower layer) was transferred to a suitable flask and discarded. 24. The ethyl acetate solution was transferred to a rotary evaporator flask and was concentrated under reduced pressure using a rotary evaporator, while maintaining a bath temperature of 29-30 °C, which gave a residue. 25. The residue was then dissolved in ethyl acetate (4.68 L). 26. The solution was concentrated under vacuum using a rotary evaporator, while maintaining a bath temperature of 29- 30 °C, which also gave a residue. 27. Again the residue was dissolved in ethyl acetate (4.68 L), and two samples were taken to determine the water content by Karl Fisher titration. The water content of the two the samples were found to be 0.216% and 0.207%. 28. Using more ethyl acetate (12.66 L), the mixture was transferred from rotary evaporator flask into a dry reaction flask that was equipped with a temperature recorder, a mechanical stirrer and a sintered gas dispersion tube glass, and was flushed with nitrogen. (1S,2S,3R,5S)-Pinanediol-L-Phenylalanine-L-Leucine Boronate, HCl Salt 1. The ethyl acetate solution containing (1S,2S,3R,5S)-pinanediol-N-BOC-L- phenylalanine-L-leucine boronate was cooled to -0.9 °C using a ice / water cooling bath. 2. Hydrogen chloride gas (1.115 kg) was bubbled into the reaction mixture during a period of 1.48 hours. The temperature at the start of the addition was -0.9 °C and at the end 6.8 °C. 3. The reaction was then allowed to warm to 14.4 °C over 50 minutes, while the nitrogen atmosphere was maintained. 4. A sample was taken for RP-HPLC testing during the process. Percentage Conversion was 68.9% (area percentage). 5. The reaction mixture was stirred for 35 minutes. The temperature at start-up was 14 °C and at the end 14.8 °C. 6. A sample was taken for RP-HPLC testing during the process. Percent conversion was 94.7% (area percentage). 7. The reaction mixture was stirred for about 50 more minutes, while maintained a temperature of 10 °C ± 5 °C. 8. A sample was taken for RP-HPLC testing during the process. Percent conversion was 97.3%. 9. The reaction mixture was stirred for about 50 more minutes, while maintained a temperature of 10 °C ± 5 °C. The final temperature was 14.6 °C. 10. A sample was taken for RP-HPLC testing during the process. The total reaction time after addition of hydrogen chloride gas was four (4) hours. 11. The percentage conversion was 99%. 12. A slurry was observed. 13. n-Heptane (8.8 L) was added to the reaction mixture. 14. The slurry was stirred for 2 hours. The temperature at the start of stirring was 12.7 °C and at the end 15.3 °C. 15. The solid was isolated by filtration on a Buchner funnel lined with with a filter pad made of polypropylene felt. 16. The solid was washed with n-heptane (4.68 L). 17. In a fume hood, the solid was transferred to three drying dishes that were not more than 25.4 mm, and was air dried for 1 hour. 18. The solid was then dried for 16 hours 28 minutes at <35 °C and under a vacuum of 687 mm Hg in a vacuum furnace equipped with a vacuum gauge and a temperature recorder. 19. Samples were taken from all drying trays to determine the percentage loss on drying. The LOD was found to be 0%, 0.02%, and 0.02% for the three samples. 20. (1S,2S,3R,5S)-Pinanediol-L-phenylalanine-L-leucine boronate, HCl salt, was then packed in double plastic bags in fiber drums and were labeled, and samples were taken. 21. The isolated yield was 1.87 kg, 79.1%. The intermediate was stored at 2-8 °C until used in further preparation. (1S,2S,3R,5S)-Pinanediol-N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronate 1. A three-necked glass reaction flask equipped with a Claisen condenser, temperature recorder and a mechanical stirrer, were flushed with nitrogen in a fume cupboard. 2. (1S,2S,3R,5S)-Pinanediol-L-phenylalanine-L-leucine boronate, HCl salt, (1.85 kg) was added to the flask. 3. 2-Pyrazinecarboxylic acid (0.564 kg) was added to the flask. 4. 2-(H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (1.460 kg) was added to the flask. 5. Dichloromethane (18.13 L) was added to the flask. 6. The stirring motor was adjusted to a stirring speed of 272 RPM. 7. The reaction mixture was cooled to -1.2°C using a cooling bath. 8. N,N-diisopropylethylamine (1.865 kg) was added to a glass flask and transferred to the reaction over a period of 50 minutes using a peristaltic pump, while maintaining a reaction temperature of the range -1.2 °C to 2.8 °C. 9. To complete the addition, the flask was washed with dichloromethane (0.37 L), which was hat in the reaction mixture. 10. The reaction mixture was allowed to warm and stirred for an additional 81 minutes. 11. The temperature at the start of the stirring was 15 °C and at the end 24.9°C. 12. A sample was then taken for RP-HPLC testing during the process. The percent conversion was found to be 99.9%. 13. The reaction mixture was transferred to two rotary evaporator flasks in approximately equal volume. The reaction mixture was concentrated under reduced pressure at using two rotary evaporators, while maintaining an external bath temperature of 33-34 °C. 14. Ethyl acetate (12.95 L) was divided into two approximately equal volumes which were kept in the two rotary the evaporator flasks. 15. The mixtures in each flask were then concentrated under reduced pressure at using a rotary evaporator, while maintaining an external bath temperature of 33-34 °C. 16. The residues in each rotary evaporator flask were then returned to reaction flask using ethyl acetate (12.95 L). 17. A 1% aqueous phosphoric acid solution (12.34 L) was prepared by mixing di water (12.19 L) and phosphoric acid (0.148 kg) in a glass flask equipped with a stirrer. 18. A 2% aqueous potassium carbonate solution (12.34 L) was prepared by mixing the following water (12.09 L) and potassium carbonate (0.247 kg) in a glass flask equipped with a stirs. 19. A 10% aqueous sodium chloride solution (12.34 L) was prepared by mixing the following water (12.34 L) and sodium chloride (1.234 kg) in a glass flask equipped with a stirs. 20. Distilled water (12.34 L) was added to the reaction flask containing ethyl acetate- the solution, and the mixture was stirred at 382 RPM for 7 minutes. The layers were separated, and the aqueous phase (lower layer) was transferred to a suitable flask and thrown. 21. Again, di water (12.34 L) was added to the reaction flask containing ethyl acetate- the solution, and the mixture was stirred at 398 RPM for 7 minutes. The layers were separated, and the aqueous phase (lower layer) was transferred to a suitable flask and thrown. 22. The 1% phosphoric acid solution prepared in Step 17 was added to the reaction the flask containing the ethyl acetate solution, and the mixture was stirred at 364 RPM for 8 minutes. The layers were allowed to separate, and the acidic aqueous phase (lower layer) was transferred to a suitable flask and discarded. 23. The 2% potassium carbonate solution prepared in Step 18 was added to the reaction flask containing the ethyl acetate solution, and the mixture was stirred at 367 RPM for 8 minutes. The layers were allowed to separate, and the basic, the aqueous phase (lower layer) was transferred to a suitable flask and discarded. 24. The 10% sodium chloride solution prepared in Step 19 was added to the reaction flask containing the ethyl acetate solution, and the mixture was stirred at 374 RPM for 8 minutes. The layers were allowed to separate, and the aqueous The phase (lower layer) was transferred to a suitable flask and discarded. 25. The ethyl acetate solution was transferred under vacuum and in two approximately equal volumes to two rotary evaporator flasks and was concentrated under reduced pressure at using a rotary evaporator, while maintaining an external bath temperature of 34 °C. 26. n-heptane (14.8 L) was divided into two approximately equal volumes and was kept in the two rotary The mixtures in each flask were then concentrated under reduced pressure using a rotary evaporator, while maintained an external bath temperature of 34 °C. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride, crude 1. A 1N solution of hydrochloric acid (22.2 L) was prepared by mixing di water (20.36 L) and hydrochloric acid (1.84 kg) in a glass flask equipped with a stirrer. 2. A 2N sodium hydroxide solution (12.03 L) was prepared by mixing di water (12.03 L) and sodium hydroxide (0.962 kg) in a glass flask equipped with a stirrer. 3. The residues in the rotary evaporator flasks containing (1S,2S,3R,5S)- pinanediol-N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronate, was then, using n-heptane (14.8 L) and methanol (14.8 L), transferred to a three-necked glass reaction flask equipped with a temperature recorder and a mechanical stirrer. 4. The stirring motor was adjusted to a stirring speed of 284 RPM. 5. 2-Methylpropaneboronic acid (0.672 kg) was added to the flask. 6. 1N hydrochloric acid, prepared in Step 1 (11.2 L), was added to the flask. 7. The stirring motor was adjusted to a stirring speed of 326 RPM. 8. The reaction mixture was stirred for 16.38 hours. The starting temperature of the batch was 28.6 °C and the final temperature was 21.6 °C. 9. A sample was then taken for RP-HPLC testing during the process. 10. The percentage conversion was found to be 100%. 11. Stirring was stopped and the biphasic mixture was allowed to separate. 12. The n-heptane layer (upper layer) was transferred to a suitable flask and discarded. 13. n-heptane (5.37 L) was added to the reaction flask, and the mixture was stirred at 381 RPM for 6 minutes. The layers were allowed to separate, and the n-heptane phase (upper layer) was transferred to a suitable flask and discarded. 14. Again, n-heptane (5.37 L) was added to the reaction flask, and the mixture was stirred at 340 RPM for 6 minutes. The layers were allowed to separate, and the n-heptane phase (upper layer) was transferred to a suitable flask and discarded. 15. The aqueous methanol solution was transferred to two rotary evaporator flasks in approximately equal volumes and were concentrated under reduced pressure using a rotary evaporator, while maintaining an external bath temperature of 33-34 °C. 15 L of methanol was collected. 16. Dichloromethane (5.37 L) was used to transfer the residue from the rotary the evaporator flasks and back to the reaction flask. 17. 2N sodium hydroxide (11.2 L), prepared in Step 2, was added to the flask. 18. The dichloromethane layer (lower layer) was transferred to a suitable flask and discarded. 19. Dichloromethane (5.37 L) was added to the flask, and the mixture was stirred at 374 RPM for 6 minutes. The phases were allowed to separate, and the dichloromethane layer (lower layer) was transferred to a suitable flask and discarded. 20. Again, dichloromethane (5.37 L) was added to the flask, and the mixture was stirred at 368 RPM for 8 minutes. The phases were allowed to separate, and the dichloromethane layer (lower layer) was transferred to a suitable flask and discarded. 21. Dichloromethane (5.37 L) was added to the flask. 22. 1N hydrochloric acid (10.7 L) was added to the flask with stirring. pH in the aqueous phase was found to be 6. 23. Stirring was stopped and the phases were allowed to separate. 24. The dichloromethane phase (lower layer) was transferred under vacuum to a collecting flask of glass. 25. Dichloromethane (5.37 L) was added to the flask, and the mixture was stirred at 330 RPM for 6 minutes. The phases were allowed to separate, and the dichloromethane layer (lower layer) was transferred to the glass collecting flask. 26. Again, dichloromethane (5.37 L) was added to the flask, and the mixture was stirred at 335 RPM for 6 minutes. The phases were allowed to separate, and the dichloromethane layer (lower layer) was transferred to the glass collecting flask. 27. The dichloromethane extracts were combined and transferred in two approximately equal volumes to two rotary evaporator flasks and was concentrated under reduced pressure at using a rotary evaporator, while maintaining an external bath temperature of 33-34 °C. 28. Ethyl acetate (12.95 L) was divided into two approximately equal volumes and kept in the two rotary The mixtures in each flask were then concentrated under reduced pressure using a rotary evaporator, while maintained an external bath temperature of 45-46 °C. 29. Again, ethyl acetate (12.95 L) was divided into two approximately equal volumes and kept in the two rotary The mixtures in each flask were then concentrated under reduced pressure using a rotary evaporator, while maintained an external bath temperature of 45-46 °C, up to about 10% of that original volume remained. 30. n-heptane (10.2 L) was divided into two approximately equal volumes and kept in the two rotary evaporator flasks, and the slurry was stirred under a nitrogen atmosphere for 2.67 hours at 22-23 °C. 31. The solid was isolated by filtration on a Buchner funnel lined with with a filter pad made of polypropylene felt. 32. The solid was washed with n-heptane (2.96 L). 33. In a fume hood, the solid was transferred to four drying dishes and air-dried in 1.25 hours. 34. The solid was then dried for 18 hours and 27 minutes at 36 - 50 °C and under a vacuum of 687 mm Hg in a vacuum furnace equipped with a vacuum gauge and a temperature recorder. 35. A sample of the solid was taken from each dish to determine percentage loss on drying (LOD). For the four samples taken, the LOD was found to be be 0.38%, 0.62%, 0.71% and 0.63%. 36. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride, crude, was obtained in two 5-liter, sealed, wide-necked HDPE bottles that were labeled. 37. The isolated yield was 1.314 kg, 83%. Recrystallization of N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine- boronic anhydride, crude 1. A glass reaction flask equipped with a mechanical stirrer, a reflux condenser and a temperature recorder were flushed with nitrogen in a fume cupboard. 2. Ethyl acetate (21 L) was added to the flask. 3. The ethyl acetate was heated to 66.8 °C under a nitrogen atmosphere, at use of a hot water / steam bath. 4. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride, crude, (1.311 kg) was slowly added to the reaction flask. The addition took place over a period of period of 3 minutes. 5. The mixture was stirred for 1 minute until all the solid had dissolved. The temperature of the solution was 64 °C. 6. The heat source was removed, and the mixture was slowly cooled to 60 °C at application of a cooling bath. 7. The hot ethyl acetate solution was pumped using a peristaltic pump transferred to a collecting flask via plastic tubing and an “in-line” filter capsule of polypropylene,. 8. The mixture was allowed to cool to 27.2 °C and placed under a nitrogen atmosphere. without stirring for 17.75 hours. The final temperature was 20.5 °C. 9. The mixture was cooled using an ice / water bath with stirring for 2.33 hours. The temperature at the start of the stirring was 3.8 °C and at the end -2.8°C. 10. The solid was isolated by filtration on a Buchner funnel lined with with a polypropylene felt filter pad. The filtrate was collected in a collecting flask. 11. The solid was washed with ethyl acetate (2.62 L) and cooled to 4.7 °C. 12. In a fume hood, the solid was transferred to two drying dishes. 13. The solid was then dried for 19 hours and 10 minutes at 51-65 °C and under a vacuum of 687 mm Hg in a vacuum furnace equipped with a vacuum gauge and a temperature recorder. 14. Samples of the solid were taken to determine the percent loss on drying (LOD). The LOD for the two samples taken was found to be 0.65% and 0.62%. 15. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride was had in four 1-litre, brown, wide-necked bottles, type 3, with Teflon-coated caps, which were marked. 16. The isolated yield was 1.132 kg, 86.3%. 17. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride was stored at - 25 to -15 °C. Example 3: Convergent synthesis of N-(2-pyrazinecarbonyl)-L-phenylalanine-L- leucine boronic anhydride (1S,2S,3R,5S)-Pinanediol-N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronate

[099] A solution of (1R)-(S)-pinanediol-1-ammonium trifluoroacetate-3- methylbutane-1-boronate (13.97 g) and N-hydroxysuccinimide (6.23 g) in 66 ml DMF, were cooled to -5 °C, followed by the addition of dicyclohexylcarbodiimide (10.83 g). The The resulting suspension was stirred for one hour at a temperature of -5 to 0 °C. A solution of N-(2-pyrazinecarbonyl)-L-phenylalanine (19.52 g; prepared by coupling it to pre-prepared succinimide esters of pyrazinecarboxylic acid with L-phenylalanine in dioxane-water) in 62 ml DMF, N-methylmorpholine (5.7 ml) was added at a temperature of 0 °C, and the resulting solution was added to the suspension. The suspension was adjusted to pH 7 by adding an additional 5.7 ml of N-methylmorpholine and was stirred overnight while the temperature slowly rose to 21 °C. After filtration, the filter cake was washed twice with MTBE, and the combined filtrates were diluted with 950 ml MTBE. The organic layer was washed with 20% aqueous citric acid (3 x 150 ml), 20% aqueous NaHCO3 (3 x 150 ml) and brine (2 x). The organic layer was dried with NaSO, filtered and concentrated, giving 25.5 g (95.5%) of the title compound as a 2 4 foam. TLC showed that this material contained small amounts of impurities, including approx. 2% cyclohexylurea. N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride

[0100] A solution of (1S,2S,3R,5S)-pinanediol N-(2-pyrazinecarbonyl)-L- phenylalanine-L-leucine boronate (25.2 g) in 207 ml MeOH and 190 ml hexane, was cooled to 15 °C, and 109.4 ml of 1N HCl was added portionwise, while the temperature was maintained between 15 and 25 °C. 2-Methylpropaneboronic acid (8.67 g) was then added under vigorous stirring, and stirring of the biphasic mixture was continued overnight. After separation of the two phases, the lower layer was extracted once with 75 ml hexane. The lower layer was then concentrated under vacuum until it became cloudy, followed by the addition of 109.4 ml 2N NaOH and 100 ml Et2O. The two phases were separated, and the lower layer was extracted with Et O (4 x 100 ml) and then adjusted to pH 6.0 2 by adding 109 ml of 1N HCl. After extraction with 100 ml of ethyl acetate, The lower layer was adjusted to pH 6.0 with 1N HCl and extracted once more with 75 ml ethyl acetate. The combined ethyl acetate layers were washed with half-saturated saline solution (2  25 ml) and saturated saline solution (2  25 ml) and was dried with Na2SO4, filtered and concentrated, yielding 15.3 g (81.8%) of crude N-(2-pyrazinecarbonyl)-L-phenylalanine-L- leucine boronic anhydride as a foam. The crude material was dissolved in 150 ml ethyl acetate and concentrated to a suspension under vacuum, followed by the addition of 150 ml MTBE. The suspension was stored between 2 and 8 °C overnight and was filtered, washed twice with MTBE and dried under high vacuum, yielding 10.69 g (57.2%) N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride as a white, solid substance. Example 4: Measurement of diastereomer ratio for (1R)-(1S,2S,3R,5S)-pinanediol-1- ammonium trifluoroacetate-3-methylbutane-1-boronate

[0101] The diastereomeric purity of (1R)-(1S,2S,3R,5S)-pinanediol-1- ammonium trifluoroacetate-3-methylbutane-1-boronate (compound 1) was determined by non- chiral gas chromatography (GC). Chemicals: Acetonitrile (for Bruker or equivalent) Tetradecane (internal standard) (Fluka puriss. or equivalent) Trifluoroacetic anhydride (TFAA) (from Merck or equivalent) Instrument: Trace-GC 2000 system or equivalent Mobile phase: H2 Solvent A Approximately 300 mg of tetradecane was weighed with an accuracy of (with internal of 0.1 mg in a 100 ml volumetric flask. 1.5 ml of TFAA was standard) was added, and the mixture was diluted with acetonitrile. Sample preparation: Approximately 150 mg of the sample was accurately weighed (within 0.1 mg) in a 10 ml volumetric flask. The flask was filled with solvent A to the desired volume. The solution was kept for 15 minutes before injection. GC parameters: Column: Rtx-200; 105m  0.25 mm id  0.25 μm film Mobile phase: H2 Temp. program: 130 ºC (0.5 min); 0.5 ºC / min to 200 ºC (0 min); 30 ºC / min to 300ºC (2 minutes) Average flow: 0.9 ml / min (const. flow) Injector temperature: 250 ºC Detector- 250 ºC (FID) temperature: Split ratio 1:50 Injection volume: 1 μl Substances Compound 1 (1R)-(1S,2S,3R,5S)-pinanediol-1-ammonium trifluoroacetate-3- methylbutane-1-boronate O B O NH 2 FC COOH 3 Compound 2 (1S)-(1S,2S,3R,5S)-pinanediol-1-ammonium trifluoroacetate-3- methylbutane-1-boronate O B O NH 2 FC COOH 3 Solution stability

[0102] A stock solution of compound 1 was prepared by weighing out 150.13 mg compound 1 in a 10 ml volumetric flask and dilute with solvent A to desired volume. The stability of this solution was tested at ambient temperature over 48 hours. The stock solution was filled into 6 separate GC vials. The GC system was were injected with solution from these ampoules after 0, 12, 24, 48 and 72 hours (double injection from each vial). Area percentage for compound 1 and compound 2 was determined. No change in area percentage was observed, indicating that the solution is stable for 72 hours at ambient temperature. Specificity

[0103] Approximately 150 mg of a sample containing compound 1 and compound 2, was dissolved in solvent A and injected into the GC chromatographic system. The peak for compound 1 was well separated from the peak for compound 2. A check of peak purity by GC-MS showed that no other components were co-eluted with compound 1 or compound 2. Detection limit

[0104] The limit of detection (LOD) was defined as the concentration where the signal for compound 1 showed a signal-to-noise ratio of at least 3:1. A blank measurement was first performed to ensure that no other peaks interfered with the signal. The signal-to-noise ratio was calculated using the equation: H (signal) S / N  H (baseline) S / N = signal to noise ratio (signal / noise) H(signal) = signal height for connection 1 [mm] H(baseline) = signal height for baseline [mm]

[0105] A sample concentration of 0.05% of the standard test sample concentration was injected and showed a signal to noise ratio of 4.3. The limit of detection is therefore 0.0075 mg / ml. Limit of quantification

[0106] The limit of quantification (LOQ) was defined as the concentration at which the signal for compound 1 showed a ratio of signal and noise of at least 10:1. The signal-to-noise ratio was calculated as described above. A sample concentration of 0.1% of the standard sample concentration was injected and showed a signal-to-noise ratio of 10.1. The limit of quantification is therefore 0.015 mg / ml. Example 5: Purity analysis of N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine- boronic anhydride

[0107] The purity of N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine- Boronic anhydride (compound 3) was analyzed by reverse phase HPLC. Reagents: Water, HPLC grade Acetonitrile, HPLC grade Formic acid, ACS grade  98% pure 3% hydrogen peroxide, ACS grade or equivalent Instrument High-pressure liquid autosampler capable of injecting 20 μl and maintaining a chromatography temperature of 5 ºC Pump capable of delivering a gradient at a rate of 1.0 ml / min UV detector that can monitor the effluent at 270 nm Column Symmetry C18 chromatographic column, 250 mm  4.6 mm ID, 5 μm, Waters, cat. no. WAT054275. Sample preparation: Approximately 50 mg of compound 3 was accurately weighed into a 50 ml volumetric flask. Mobile phase B (5 ml) was added, and the mixture was ultrasonicated to dissolve compound 3 (approx. 30-60 seconds). The solution has now reached room temperature, was diluted to the desired volume with mobile phase A and was well mixed. Each sample was prepared in duplicate and was stable for 7 days when stored at 2-8 ºC and protected from light. HPLC parameters: Mobile phase A: acetonitrile / water / formic acid, 30:70:0.1 (v / v / v), degassed Mobile phase B: acetonitrile / water / formic acid, 80:20:0.1 (v / v / v), degassed Average flow rate speed: 1.0 ml / min Detector: UV at 270 nm Injection volume: 20 μl Column temp: ambient temperature Sample dish temp: 5 ºC Gradient program: Time %A %B 0 100 0 15 100 0 30 0 100 45 0 100 47 100 0 55 100 0 Substances Compound 3 N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine- boronic anhydride O.O. H NNB N 3 H O N Compound 4 N-(2-pyrazinecarbonyl)-D-phenylalanine-L-leucine- boronic anhydride O.O. H NNB N 3 H O N Compound 5 N-(2-pyrazinecarbonyl)-L-phenylalanine-D-leucine- boronic anhydride O.O. H NNB N 3 H O N

[0108] The retention time for compound 3 was typically between 10 and 14 minutes. using an HPLC system with a 1.3 minute pause. Compound 4 and 5 were eluted together at a longer retention time, with a resolution of  2.0.

[0109] The relative retention of compound 3 in a sample chromatogram relative to to a standard chromatogram, was calculated according to the following equation: tsam Rr  tstd where: R = relative retention r tsam = retention time of the peak of compound 3 in sample chromatogram, minutes t = retention time of the peak of the drug substance standard in the nearest preceding standard chromatogram, minutes

[0110] Analysis results were calculated for each sample according to the following equation: Assam Wstd  P 1 %result    100 Astd Wsam 100  M     100   where: A = peak area response for compound 3 in together the sample preparation Astd = average peak area response for compound 3 in the standard preparation used W = weight of standard, mg standard P = specified purity of the standard (decimal format) Wsam = weight of the sample, mg M = moisture content of the sample, % 100 = conversion to percentage

[0111] Relative retention and amounts of impurities in each sample were calculated in according to the following equation: ten Rr  tds where: Rr = relative retention ti = retention time for individual impurity t = retention time of the peak of compound 3 ds Ai Wstd  P  DF  RFi %Ii  100 Astd, 1% Wsam where: I = individual impurity in A = peak area response for individual impurity in in the sample preparation Astd,1% = average peak area response for compound 3 in a 1% standard preparation Wstd = weight of standard, mg Wsam = weight of sample, mg P = specified purity of standard (decimal format) DF = dilution factor, 1 / 100 RFi = relative response factor for individual impurity 100 = factor for conversion to percentage

[0112] When N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucineboronic anhydride from Example 2 was analyzed according to this method, the total impurities of less than 1%.

[0113] Although the foregoing invention has been described in more detail with For the sake of clarity and understanding, these particular embodiments shall are considered illustrative and not limiting. When reading this description, skilled artisans will understand that various changes in form and detail may be carried out without departing from the true scope of the invention and the appended claims.

Claims

1. Large-scale process for the preparation of a compound of formula (XIV): or a boronic anhydride thereof, comprising the steps: (aa) coupling of a compound of formula (XVIII): or an acid addition salt thereof, with a compound of formula (XIX): where: 1 P is a cleavable amino protecting group; and X is OH or a leaving group; to form a compound of formula (XX): 1 where P is as defined above and where said coupling step (aa) comprises the steps: (i) coupling the compound of formula (XVIII) with a compound of formula (XIX) where X is OH in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate (TBTU) and a tertiary amine in dichloromethane; (ii) solvent exchange to replace dichloromethane with ethyl acetate; and (iii) aqueous washing of the ethyl acetate solution; 1 (bb) removing the protecting group P to form a compound of formula (XXI): or an acid addition salt thereof wherein said protecting group removal in step (bb) includes the steps: (i) treating the compound of formula (XX) with HCl in ethyl acetate; (ii) adding heptane to the reaction mixture; and (iii) isolating the compound of formula (XXI) as its HCl addition salt by crystallization; (cc) coupling the compound of formula (XXI) with a reagent of formula (XXII) where X is OH or a leaving group, to form a compound of formula (XXIII): wherein said coupling step (cc) comprises the steps: (i) coupling the compound of formula (XXI) with 2-pyrazinecarboxylic acid in presence of TBTU and a tertiary amine in dichloromethane; (ii) solvent exchange to replace dichloromethane with ethyl acetate; and (iii) aqueous washing of the ethyl acetate solution; and (dd) deprotecting the boronic acid group to form the compound of formula (XIV) or a boronic anhydride thereof wherein said deprotection in step (dd) comprises the steps: (i) providing a biphasic mixture comprising the compound of formula (XXIII), an organic boronic acid acceptor, a lower alkanol, a C 5-8 hydrocarbon solvent and aqueous mineral acid; (ii) stirring the biphasic mixture to give the compound of formula (XIV); (iii) separation of the solvent layers; and (iv) extracting the compound of formula (XIV) or a boronic anhydride thereof into an organic solvent. 1 2. A process according to claim 1, wherein P is tert-butoxycarbonyl.

3. The method of claim 1 or 2, wherein the ethyl acetate solution from step (aa)(iii) is dried azeotropically and then treated with gaseous HCl.

4. The method of claim 1, wherein step (dd)(iii) comprises the steps of: (1) separation of the solvent layers; (2) adjusting the aqueous layer to basic pH; (3) washing the aqueous layer with an organic solvent; and (4) adjusting the aqueous layer to a pH less than 8.

5. The method of claim 4, wherein the aqueous layer in step (dd)(iii)(4) is adjusted to a pH less than 6.

6. A process according to claim 4, wherein the compound of formula (XIV) or a boronic anhydride thereof in step (dd)(iv) is extracted into dichloromethane, the solvent is changed to ethyl acetate and the compound of formula (XIV) or et The boronic anhydride thereof is crystallized by adding hexane or heptane.

7. The method of claim 6, wherein the addition of hexane or heptane results in crystallization of a cyclic trimeric boronic anhydride of formula (XXIV):

8. The method according to any one of the preceding claims, wherein the process uses at least 5 moles of at least one starting material.