SK59294A3 - Method of making a prolineboronate ester - Google Patents

Abstract

A method for the preparation of esters of prolineboronic acid is described. An N-protected pyrrole (I) is lithiated at the 2-position. The lithiated species (II) is reacted with trialkylborate, to yield a protected pyrrole-2-boronic acid (III). This is reduced to form a protected prolineboronic acid (IV), which, in turn, is reacted with a diol to yield an ester (VI). With the boronic acid moiety protected by the ester group, the protecting group on the nitrogen is removed, yielding the desired prolineboronic acid ester (VII). In an alternative synthesis, a protected pyrrolidine (VIII) is lithiated at the 2-position to yield a protected 2-lithio-pyrrolidine (IX). This is reacted with trialkylborate to yield the intermediate IV. The prolineboronic acid esters so produced have a chiral center to the boron atom. Also disclosed are methods for resolving enantiomers. The final products can be coupled to activated carboxylic acids, to yield peptides having a prolineboronic acid ester, instead of an amino acid, at the C-terminus. These boronic acid peptide analogs are useful for inhibiting biologically important proteases. Several methods for removing pinanediol from pinanediol boronate esters are also disclosed.

Description

Technical field

The invention relates to a process for the preparation of optically active proline boronic esters. These are used as among the products in. preparing peptides that introduce proline boronic acid instead of proline. These peptides are used to inhibit various biologically important proteases.

BACKGROUND OF THE INVENTION

Interest in α-amino acid analogs with boric acid, as well as peptides incorporating boric acid as an α-amino acid analogue instead of the C-terminal residue, has been prompted by the work which listed these compounds as potent inhibitors for most serine proteases. See, for example, Matteson et al. [Soc. 103, 5241 (1981)]; Kettner et al. Chem., 259, 15106 (1984)]; and Kinder et al. [J. Med. Chem. 27, 1919 (1985)],

Several workers, including Koehler et al. [Biochem.i.st.ry, 10, 2477 (1971)] and Rawn et al [Biochemistry, 13, 3124 (1974)] suggested that an empty p-orbital located on the boron in these compounds interacts with the active site of the hydroxyl group enzyme to form a tetrahedral compound that mimics the transition state of enzymatic hydrolysis. The boronic acid analog is believed to bind more strongly to the enzyme than to the substrate itself, thereby inhibiting the enzyme. c to react to the substrate.

Boric acids as analogues of α-amino acids and the peptides incorporating them are currently under investigation because they are able to elucidate the biological functions of the enzymes they inhibit. Further, as explained below, they are also therapeutically useful.

'· · /; > «,, · ', ·') -, 'ι'ί' λ» ιΧΐ'Λ ', ί · ν, T .'- ί.'λ s' -ι ·> ι · ν:'t;. * · yr ^; π ¹ ·? · · · · · · · · · · ·. · ··. ··, - ··, ··. ·! · .- ...

'.......-. 3: · ^ λ /;,.=. ·: Λ.ό'.νν ^ Λ ^ -ϊ, ΐ-ίί ^^., Ιί'ίί ·

Peptides having an incorporated α-aminoboronic acid analogue of proline (BoroPro) on terminal carbon are of particular interest since they have been shown to be effective inhibitors of certain post-proline cleavage enzymes. For example, Bachovchin et al. [Chem. 265, 3738 (1990)] reported that such peptides are inhibitors of IgA proteinases from certain bacteria. These enzymes are closely related to bacterial virulence. Flentke et al. [Proc. USA 88, 1556 (1991)] reported that such peptides inhibit dipeptidyl peptidase IV (DP-IV), which in turn causes inhibition of antigen-induced proliferation by IL-2 production in T cells. These effects are known to lead to suppression of the immune response. Suppression of the immune response, for example, useful in the treatment of rejection of b.i.o organ transplants, in transplant versus host diseases, and in various autoimmune diseases.

The foregoing methods for the synthesis of α-amino boronic acids are based on the procedure published in 1981 by Matteson et al., Supra, which follows the process of hydroboration, (asymmetric) homology with chloromethyl lithium, and aminolysis. Matteson et al. [Organometallics 3, 1284 (1984)] have described the use of this technique for the synthesis of boric acid as analogs for N-acetylamine, N-acetylvaline, N-acetylleucine, and N-acetylphenylamine; some of them were obtained with a good diastereomer ratio (9: 1).

The use of the Matteson process for the synthesis of BoroPro has been demonstrated by Bachovachin, supra, and Flentk, supra, but the extensive modifications required to form the pyrolysis of the ring ring make it unattractive. In addition, the conditions for preparing the single enantiomer of BoroPro by either asymmetric synthesis or by decomposition have not been reported.

Efforts to further investigate the biochemistry of post-proline cleavage enzymes, in particular DP-IV, and the potential for therapeutic uses of BoroPro-based enzyme inhibitors have not been successful because of the obstacles to an effective pathway to proline horiric acid.

The need for a better source for BoroP.ro has led us to develop an alternative approach to this compound, especially in its optically active form, which has resulted in the present invention.

SUMMARY OF THE INVENTION

A first broad aspect of the invention encompasses three interrelated methods for the synthesis of proline boronic esters. Two of these syntheses start from pyro.l.u. The third method starts from pyrrolidone. Part of this first aspect of the invention are certain novel intermediates. Prolineboric acid has a chi-ral center and a boron atom. A second broad aspect of the invention comprises a method for resolving enantiomers of proline and others. According to this procedure, prolinehoric acid ester is formed through reaction with a chiral alcohol, using pinanediol, which is particularly preferred. Resolution of the enantiomers is facilitated by separation of the diastereomeric mixture resulting from the introduction of additional chiral centers. The resulting proline boronic acid esters can be readily bound to activated carboxylic acid groups, which are typically used in peptide synthesis, to yield peptides having proline boronic ester instead of the amino acid πει terminal carbon. The ester protecting group can be removed to give the free boronic acid peptide. When the ester protecting group is pinandio.1, it is not easy to remove it by known techniques. A third aspect of the invention involves several methods of removing a pinanediol protecting group.

First, the synthesis of the invention starts with a pyrrole that reacts with an activated carbonic acid derivative to protect the nitrogen atom with a group of the formula -COOR, wherein R is alkyl, cycloalkyl, benzyl, phenyl, substituted phenyl. with one or more C 1-4 alkyl groups, or trimethylslyl ethyl, to obtain a compound of formula I

ii (I)

R0 \)

Preferred protecting groups are R-tert-butyl, benzyl, trimethylsilylethyl, phenyl, methyl or ethyl. The most preferred protecting group is tert-butyloxycarbonyl, or Boe. The protecting group is attached by well known methods. The specific synthesis for 1-Boc-pyrrole has been described by Grehn et al. [Angnew. Chem. I nt. Ed. Engl. 23, 296 (1984)].

The compound of formula I is further treated with a lithium reagent to obtain a compound of formula II

Li (II)

RO where R is already defined.

The binding of lithium to the compound of formula I may be accomplished by treatment with lithium tetramethylpiperidide in a known manner as described by Hasan et al. [J. Org. Chem. 46,157 (1981)], or with other protected lithium. amides such as lithium diisopropyl amide or lithium dicyclohexyl amide, or with n-butyl lithium in the presence of tetramethyl ethylenediamine. This reaction takes place simply in an inert solvent, preferably an ether such as THF, diethyl ether, dimethoxyethane, or methyl t-butyl ether at a temperature between. -78 ° C and -40 ° C. Alternatively, the pyrrole may be brominated at the 2-position, in a manner known per se, as described by Chen et al. [O.rg. A son. , 70, 151 (1991)], and the resulting product can be protected and then lithiated using other less expensive casting agents, such as n-butyl lithium, using known per se techniques.

The intermediate of formula II which is not isolated is further reacted with a trialkyl borate in which each alkyl group may be linear, branched or cyclic having 1 to 6 carbon atoms, preferably trimethyl or triethyl boron, followed by acid catalyzed hydrolysis, using a weak acid such as is citric or acetic acid or potassium hydrogen sulphate, for the purpose of obtaining the protected pyrrole-2-boronic acid of formula III

III '(III)

OH

Where R is as defined above.

The intermediate of formula III is catalytic hydrogenation to further reduce the pyrrolidineboronic acid of formula IV using protected acid-

(IV) wherein R i is as defined above.

Catalytic hydrogenation of the intermediate of formula III can be carried out in an organic solvent such as ethyl acetate or tetrahydrofuran, using a catalyst such as 5% platinum on carbon, platinum oxide, rhodium on carbon, rhodium on aluminum palladium on carbon or Raney nickel, either atmospheric pressure or at about 50 psi.

An alternative synthesis of boric acid of formula IV uses a pyrrolidine which is treated with a suitable acylating agent to give the protected compound of formula VIII

RO (VIII) wherein R is as previously defined.

The protecting group is selected to allow activation of the pyrrolidine against adjacent carbon lithium lithiation, and should contain a spatially bulky portion that protects the carbonyl from the casting agent. Preferably, a carbamoyl protecting group of the formula -COOR is used, for example a group wherein R is tert-butoxy or 2,4,6-tri-tert-butyl.phenoxy. However, certain acyl or aroyl groups may also be used, e.g. tert-butylcarbonyl or triphenylmethylcarbonyl. Other suitable activating groups are described in Beak et al. [Chem. Rev., 84, 471-523, (1984)]. The most preferred protecting group is tert-butyloxycarbonyl, or Boe. The protecting group may be attached to the pyrrolidine by well known methods.

The compound of formula VIII is further treated with a lithium reagent to give a compound of formula IX

L i (ix) wherein R is as defined above

The introduction of lithium into the compound of formula VIII can be inoculated with secondary butyl lithium and tertramethyl ethylenediamine in a known manner per se, as described by Beak et al. [Tet. 30, 1197 (1989)]. This reaction is suitably carried out in an inert organic solvent, preferably in ether such as portions. ether, methyl tert-butyl ether or THF at a temperature between about -78 ° C and 0 ° C, preferably -78 ° C to - 40 ° C. The introduction of the cheek can be achieved by reactive alkyl lithium such as sec. butyl, lithium or tert. butyl lithium, preferably in the presence of a coordinating additive such as tetramethyl ethylenediamine, hexamethyl phosphoramide or N, N'-dimethylpropyleneurea (DMPU).

Among the product of formula IX, which is not isolated, is further reacted with xialkylborate, wherein each alkyl group may be linear, branched or cyclic and contains 1 to 6 carbon atoms, preferably trimethyl or triethyl borate, followed by hydrolysis with water and extraction into an aqueous base solution such as sodium hydroxide or potassium hydroxide for pulverization. Acidification of the alkaline solution to a pH of about 3 and extraction affords the protected proline boronic acid of the formula

IV.

In order to form the boronic ester, the free boronic acid intermediate of formula IV is further esterified by reaction with a diol of formula V,

HO-X-OH wherein X is a linking group to form a compound of formula VI

wherein X is the same linking group as above and R is as previously defined. The ester group thus formed is intended only as a removable protecting group. The structures, syntheses and methods for attachment and removal of such aesthetic protecting groups are generally known in chemistry. Accordingly, those skilled in the art will appreciate that the structure of the linking group X is not critical. The linking group X may, as indicated by the non-limiting examples, be a saturated 2 to 3 membered hydrocarbon chain; a saturated 2 to 3 membered hydrocarbon chain which forms part of the C 5-12 carbo-cyclic system, which may optionally contain either unsystematic or ring linkages; A 2 to 3 membered hydrocarbon chain that forms part of the aromatic ring system; or a group of the formula - (C1-4) _n -NH- (C1-4), wherein n and m are each 2 or 3; wherein such groups may be substituted or substituted by one or more C 1-6 alkyl or phenyl groups.

Accordingly, suitable components of formula V are, for example, ethylene glycol, pinacol, catechol, pinanediol, butane-2,3-diol, 2,2-dimethylpropane 1,3-diol, diethanol amine and 1,2-di.f. enyletan-1,2diol.

With a portion of the boronic acid protected by an ester group, in a next step, the nitrogen protecting group is removed using known per se methods such as those described by Green in the Protective Group in Organic Synthesis (J.Viley & Sons, 1981), thereby to obtain the hydrochloride of the desired proline boronic ester of formula VII.

(VII)

For example, if the protecting group is Boe, it can be easily removed with dry hydrogen chloride in ethyl acetate.

It is preferred to carry out esterification of the compound of formula IV with a chiral, non-racemic diol such as (1S, 2S, 3R, 5S) - (+) pinanedio1, 1,2-diphenylethane-1,2-diol or butane-2-3-diol because in this way, the introduction of further chiral reagents into the molecule is effected. This allows cleavage of the chiral center and the boron atom, using known per se methods for separating diastereomers, such as HPLC or fractional crystallization. This is illustrated in the reaction scheme of Figure 1, where the isomers of the compound of formula VI in which the boric acid is protected with (1S, 2S, 3R, 5S) - (+) - pinanediol can be separated by IIPLC to obtain the compounds VIa and VIb. Similarly, isomers of the hydrochloride of a compound of formula VII with the same boron protecting group can be separated by fractional crystallization in a solvent such as ethyl acetate, or a mixture of dichloromethane / ethyl acetate, isopropanol, or ethanol to give compound VIIb as a single isomer with R the configuration of the carbon attached to the boron.

A further advantage of the use of pinanediol is that the borate esters thus formed are more stable than those derived from other diols, for example pinacol, in which significant loss of the protecting group is often observed during chromatography. This is useful in both purification and separation of isomers by silica gel chromatography, as a better yield of the desired compound is obtained.

In a minor modification of the synthesis described above, the pyrrole derivative of formula III can be directly esterified with a diol of formula V. The resulting ester of formula IIIA

\ 3 \ \

(ΙΠΑ) can be reduced in the same manner as the compound of formula III to give the protected proline boronic acid ester of formula VI. In other words, the order of steps in which the pyrrole ring is reduced and the boric acid estrified can be replaced.

Thus formed proraboronic acid esters are readily bound to activated carboxylic acids such as those commonly used in the art. peptide synthesis, for example, a nitrogen-protected amino acid to give a compound of formula X

R ', z in which -COOR' is an amino protecting group of the type commonly used with 9 in peptide synthesis, so R is, for example,

O-tert-butyl, benzyl or fluorenylmethyl and R is the side chain of a naturally occurring amino acid, optionally with suitable, protecting groups of the type commonly used in peptide synthesis.

Compounds of formula X contain both borate and amino acid protecting groups. It may be necessary to remove one or both protecting groups for biological activity or for further chemical manipulation. The protecting groups may be removed in any order. Various methods for removing these protecting groups are described below.

Removal of the protecting group on nitrogen can be achieved in a known manner to give a compound of formula XI.

Z a

\ \

X (XT)

The ester group protecting the boronic acid unit is stable in a neutral and acidic organic environment, but most borate esters are rapidly cleaved in a slightly basic aqueous medium (pH = 7.5) to give boric acid. In the case of pinanediol borate esters, hydrolysis is known to be difficult and special conditions for pinanediol removal are required. See, for example, Matteson et al., [J. Am. Chem. Soc., 102, 7590 (1980)] and Brown et al. [J.Organometallic Chem., 385,15 (1988)]. These methods are not suitable for removing pinanediol from a compound of formula VI, X or XI. Several methods have been found to remove pinanediol from borate such as compounds VI, X or XI. Such pinanediol removal can be achieved under mild conditions using an oxidizing agent capable of cleaving 1.2 diol to remove the pinanediol from equilibrium and shift in the direction of free boric acid. For example, treatment of compounds of formula VI and X, whose protecting group is pinanediol, with sodium periodate in aqueous ammonium acetate and acetone at room temperature provides compounds of formula IV and optionally XI I.

This reaction is suitably carried out in water with the optional addition of a buffer such as ammonium acetate or sodium hydrogen phosphate at a pH between 3 to 10, preferably 6 to 8 and a temperature of 0 to 80 ° C preferably 20 to 40 ° C in the presence of water miscible organic solvents such as acetone, methanol, ethanol, THF or acetonitrile. A suitable oxidizing agent is a non-nucleophilic oxidizing agent capable of cleaving 1,2-diols such as periodic acid or its salts or permanganate salts. Under these conditions, oxidative cleavage of the carbon boron bond is not observed. Because of this, this method is applicable to any pinanediol protected boric acid. In addition, it is also applicable to any borate protecting group which is 1,2-dio.l.om, although this is particularly useful for protecting groups where simple aqueous hydrolysis is slow or incomplete.

In the case of borane acid borane ester compounds containing an unprotected amine, such as a compound of formula XI, the method described above can also be used, but a second new method is preferred for compounds of this type. The method consists of using an aqueous solution of the compound of formula XI, at pH 4 or less, in a cation exchange resin column, and washing the column with water or dilute acetic acid to remove the pinanediol. This removes pinanediol from the equilibrium and thus controls the reaction in the direction of hydrolysis. The column is then washed with a dilute aqueous ammonium hydroxide solution to remove the product which, after evaporation and acidification, is obtained as a salt of the compound of formula XIII.

^R3 (XIII)

Suitably, a strongly acidic cation exchange resin, for example a sulfonic acid type resin such as Dovex 50, is used. The pinanediol eluted from the resin can be recovered from the aqueous solution and reused. This is most conveniently achieved by passing the aqueous solution through a column with a non-ionic polymeric adsorbent such as Amber1: i.te.XAD-200, which adsorbs pinanediol almost quantitatively. Pinanediol is removed by washing with methanol or ethanol. The two ion exchange and pinandiol absorption operations can be combined in a single process in which water is recycled

The compound is unprotected amine.

The third way

- ¹³ from one column to another using a pump. This has the advantage of requiring much less water and allows the process to continue long enough to achieve a high product conversion.

It is known to those skilled in the art that the above described cation exchange resin scavenging process is suitable containing basic functional groups as is also applicable to esters of pinanediol compounds containing an unprotected amine such as the compound of formula XI. This process consists of transesterifying the pinanediol borate with another boronic acid of formula R ⁴ -B (OH) ₇ in a biphasic system. R ⁴ represents a C 1-6 hydrocarbon group which may consist of linear, branched or cyclic alkyl chains and phenyl rings. R ⁴ is preferably phenyl. One phase is water with a pH adjusted below 7, preferably pH 1-4 ct, the other is a hydrocarbon organ, petroleum ether or toluene. XI by phenylboronic acid followed by phase separation, other phenylboronic acid in organic regenerated by evaporation, and a solution of the free boronic acid of formula XIII in the aqueous phase, which was used using an ion exchange resin similar to that described above. In this system the only one soluble in the organic phase is pinanediol. thereby removing pinandiol from equilibrium. The compound and the compound of formula XIII that is formed in the aqueous layer, since none of them is soluble in hydrocarbon solvents. The reaction can be carried out with any boronic acid having a hydrocarbon side chain, provided that its pinanediol ester is soluble in hydrocarbon solvents.

Any solvent such as hexa. Treatment of a compound of the formula in a mixture of water at pH 1 and hexane produces the pinanediol ester acid phase. which can simply be swelled from the way that the phenylboron tan component, the devils on the formula remain both

A completely deprotected compound of formula XIII may also be prepared by removing a nitrogen protecting group from a compound of formula XII using known methods. Advantageously, compounds of formula X, XI, XII and XIII generally contain two chiral centers. One is adjacent to the boron atom and the other is present on the amino acid unit except when the unit is glycine. It will further be appreciated that the pure single diastereoisomers of these compounds are more suitable for biological use than a mixture of diastereoisomers. Accordingly, it is important to be able to produce these compounds as pure single isomers. Since amino acids are generally available as individual enantiomers, this can in principle be achieved by separating a mixture of diastereoisomers formed by coupling an optically pure amino acid with racemic proline boronic acid using known methods. However, it has been found that, apart from the special case of valine, such separation is often difficult and time consuming. Therefore, the form of proline boronic acid, which is the only isomer at the chiral center attached to the boron, is preferably used, since no isolation of the isomer is then required upon attachment to the optically pure amino acid. The present invention provides a simple procedure to distinguish enantiomers of proline boronic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates a reaction scheme that represents a preferred embodiment of the process of the invention.

The following examples further illustrate the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

1- (1,1'-Dimethylethoxycarbonyl) -pyrrole-2-boric acid

To a solution of tertramethylpiperidine in TUP (275 mL) at -78 ° C under argon (8.8 mL, 52 mmol) was added

M solution of butyllithium in hexane (26 mL, 52 mol). After 15 minutes, 1- (1,1-dimethylethoxycarbonyl) pyrrole (8.35 g, 50 mmol) in TUF (10 mL) was added and the solution was stirred at -78 ° C for 4 hours. Triethyl borate (30 mL, 176 mmol) was then added and the mixture was allowed to warm to room temperature for 3 hours. After an additional 12 hours, the reaction mixture was diluted with ether (500 mL) and washed with 1 M aqueous KHSO 4 (3 x 100 mL) followed by 1 M aqueous NaHCO ₃ (1 x 100 mL). Drying over MgSO ₄ and rotary evaporation produced a brown solid, which was purified by flash chromatography over silica gel (1: 9 EtOAc: Hexane) to yield 8.7g (82%) of a white crystalline solid (m.p. 1.01, 0-101). 5 ° C).

^L 11 H NMR (CDC1 ₃₎ δ 1.65 (s, 9 H), 6.26 (t, J = 3.3 Hz, 1H);

7.10 (dd, J = 1.6, 3.2 Hz, 1H); 13 C NMR (CDCl ₃ ) 27.9, 85.5, 112.0, 127.0, 128.7, 152.0; CIMS m / z (% rel int) 212 (MH +, 11), 156 (100), 138 (68); Anal. PDF. for C ^ HH- ^NONO, C, 51.23, H, 6.96, N, 6.64. Found: C, 51.22, H, 6.51, N, 6.67

Example 2

1- (1,1-Dimethylethoxycarbonyl) pyrrolidine-2-boronic acid

A solution of 6.15 g (24 mmol) of 1- (1,1-dimethylethoxycarbonyl) -pyrrole-2-one as in Example 1 in EtOAc (100 mL) was hydrogenated over 5% Pt / C (ca. 500 mg) at 50 psi for 24 to 48 hours. The resulting suspension was filtered through a celite pad here or concentrated. This material was chromatographed on silica gel using sequential elution with 9: 1 hexane: EtOAc, then acetone. The acetone fractions were concentrated to give 6.05 g (97%) of the desired compound, clear as a glass, which crystallized upon removal of the residual solvent (mp 100-101 ° C).

1 H NMR (CDCl ₃ ): δ 1.42 (s, 9H), 1.6-2.15 (m, 5H), 3.1-3.6 (m, 2H); ¹³ C NMR (CDCl ₃ ) δ 25.1, 25.7, 28.4, 45.6 46.2, 78.6, 154.5, CIMS m / z (% rel int) 116 (100), 70 (46);

A n a 1 y t i. Found: C, 50.52; H, C, 50.57;

8.44, N 6.51. 8.22, N 6.58

Example 3 (1S, 2S, 3R, 5S (-pinanediol 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2S-borate) and (1S, 2S, 3R, 5S) -pinanediol 1- (1,1,1-dimethylethoxycarbonyl) -pyrrolidine-2S). (dimethylethoxycarbonyl) -pyrrolidine-2R-borate.

A solution of 1- (1-, 1-dimethylethoxycarbonyl) -pyrrolidin-2-boronic acid, prepared as in Example 2, (1.52 g, 7.1 mmol) and (1S, 2S, 3R, 5S) - (+) - Pinanediol (1.36 g, 8.0 mmol) was stirred at room temperature in ether (25 mL) for 2 hours. Concentration a. flash chromatography over silica gel (85:15 hexane: EtOAc) afforded 2.1g (85%) of a 1: 1 mixture of the two diastereomers. These were separated by HPLC on a 300 X 3.9 mm microporasil A column eluting with methyl tert-butyl ether: hexane (1: 9) and using u.v. detection at 220 nm. The (1S, 2S, 3R, 5S) -pinanediol 1- (1,1'-dimethyl-ethoxycarbonyl) -pyrrolidine-2S-borane: isomer was excluded as the first isomer under these conditions.

with- i am e r 1 H NMR (C ₆ D ₆ ): δ 0,55 ( s, 3 H), 1 , 09 (s, 3 H), 1, 5 2 (s, 9 H), 1.6 (s, 3 H), 4, 11 (m, 0.3 H) , 4.33 (m, 07 H) ; ¹³ C NMR (C ₆ D ₆ ): δ 23 , 9, 26 6, 27.1, 2 7 3, 28.4, 28.8. 36 , 0, 38 , 2, 39.9, 46.1, 51 , 9, 78 3, 78.5; 85 , 7 , 154.9 R isomer ¹ H NMR (C ₆ D ₆ ): δ 0,52 ( s, 3 H), First , 08 (s, 3 H), 1, 52 (s, 9 H), 1.61 (s, 3 H), 1, 2 - 2.2 (m, 8 H), 3.1 - 3,6 (N, 3 1-1) 4.01 (m, 0.3 H) , 4.25 ( m, 0.7 H) » ¹³ C MNR < ^C 6 ^D 6) 23 , 9. 26.6, 27.1, 27, 3, 28 4 , 28.7 » 28,9, 35,8, 3 8 , 2, 3 9.6, 46.2, 51.8 , 78.1 78.5, 85 , 7 , 154.5

Obviously, (1R, 2R, 3S, 5R) -pinanediol-1- (1,1-dimethylcarbonyl) -pyrrolidine-2S-borate and (1R, 2R, 3S, 5R) -pinanediol -1 -1- ( 1,1-dimethylethoxycarbonyl) -pyrrolidine-2R-borate can be prepared in an analogous manner starting from (1R, 2R, 3S, 5R) - (-) - pinanediol.

Example 4

(1S, 2S, 3R, 5S) -Pinanediol pyrrolidine-2S- hydrochloride

The solution of (1S, 2S, 3R, 5S) -pinanediol-1- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2S-borate prepared as in Example 3 (28.5 mg, 0.08 minol) was mixed into the solution. of dry HCl in EtOAc (about 3 M). After 2 hours, the solution was concentrated twice from EtOAc to produce 21.2 mg (91%) of the desired hydrochloride as a white solid (mp. 204 ° C (dec)).

Hl NMR (Cl) C 1 -C _3): δ 0.83 (s, 3H), 1.14 (d, J = llhz, 1H), 1.29 (s, 3H), 1.45 (s, 3 II), 1.85-2.15 (m, 6H), 2.17-2.50 (m, 3H), 3.18-3.25 (m, 1H), 3.45 (bs, 2 H), 4.42 (dd, J = 1.8, 8.6 Hz, 1H), 8.80 (bs, 1H), 10.56 (bs, 1H); ¹³ C NMR (CDCl ₃ ) δ 23.9, 24.5, 26.5, 27.0, 27.2, 28.4,

34.9. , 38.2, 39.4, 45.8, 51.2, 79.0, 87.6; CIMS m / z (% .rel i n r) 250 (MM-γ-, 100); IIRMS (EI) for C44II94BNO2 calculated 249.1900 found 249.1899. .1 e r m e, that hydrochloride acid (1R, 2R, 3S, 5R) -pi- n and n rl .i i.o'.l. py role.dln-2R-bor i could be made similar

way.

Example 5 (1S, 2S, 3R, 5S) - Pinanediol Pyrrolidine-2S-Acid Dichloride (1S, 2S, 3R, 5S) -pinanediol-1- (1,1-dimethylethoxycarbones) 1-pyrrolidine-2R-borate, prepared as in Example 3 (18.3 mg, 0.05 inmol), was treated with dry HCl in EtOAc as before. The yield was 14.3 mg (96%) of the desired hydrochloride i.i. as a white solid (m.p. 248 ° C (dec)).

¹ H NMR (CDCl 3): δ 0.83 (s, 3H), 1.14 (d, J = 11hz, 1h), 1.29 (s, 3H), 1.45 (s, 3H) 1.85-2.15 (m, 6H), 2.17-2.50 (m, 3H), 3.18-3.25 (m, 1H), 3.45 (bs, 2H) 1.42 (dd, J = 1.8, 8.6 Hz, 1H), 8.80 (bs, 1H), 10.56 (bs, 1H); ¹³ C NMR (CDCl 3) δ 23.9, 24.5, 26.5, 27.0, 28.5, 34.9,

38.1, 39.4, 45.8, 51.2, 79.0, 87.8; CIMS m / z (% rel int) 250 (MH &lt; + &gt;,100);

HRMS (LI) calcd for C14H24BNO2 249.1900 found 249.1903.

It is understood that (1R, 2R, 3S, 5R) -pinanediolpyrrolidine-2R-borate hydrochloride could be produced in a similar manner.

Example 6 (1S, 2S, 3R, 5S) -pinanediol

1- (1,1-dimethylethoxycarbonyl) -pyrrole-2-borate

1- (1,1-Dimethylethoxycarbonyl) -pyrrole-2-boric acid solution prepared as in Example 1 (1.36 mg, 6.45 mmol) and (1S, 2S, 3R, 5S) - (+) -pinanediol (1.10 g, 6.45 mmol) was stirred in 20 mL of ether for 4 hours. Rotary evaporation followed by flash chromatography on silica gel (95: 5 hexane: EtOAc) produced 1.83 g (82%) of the desired product as a clear oil.

^T H NMR _(CDCl3): δ 0.90 (s, H), 1.30 ( s, 3 H) 1.41 (d, J = 11Hz, 1 H), 1.50 (s H, 1.50 (s , 3 H), 1.59 (s, 9 H). 96, 96 (m 2 H), 2.21 (t, J = 6Hz, 1H) , 2,16 - 2.40 (m, 2H) H), 4.45 (d d , J = 6Hz, 1 H), 6.20 (t, J = 3Hz, 1 H), 6.65 (B. J = 3Hz, 1H, 7.40 (d, J = 3 Hz, 1 H); ¹³ C NMR (CDC1 ₃₎ : δ 24,0, 26.4, 27.2 , 28.1, 28.6, 35,5, 38.3, 39, 8, 51.8, 79.8, 83.6 , 83.9, 111.6, 123,2, 124.7, 150.0; CIMS m / z % rel int 346 (MH &lt; + &gt;, 4), 246 (81), 153 (100), 135 (56). Is a Obviously that (1 R, 2 R, 3 S, 5 R) -pinandiol- 1- (1,1-dimethyl- ethoxycarbonylmethylaminomethyl bonyl). pyroJ. -2-borate could would be produced period-

financial way.

Example 7

1- (1,1-Dimethylethoxycarbonyl) -pyrrolidine-2-boronic acid from 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine

To a solution of 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine (17.1 g, 100 mol) in diethyl ether (200 mL) at -78 ° C under a nitrogen atmosphere was added 1.3 M sec-butyl lithium in cyclohexane (92.3 mL). , 120 mol) while maintaining the temperature below -60 ° C. After completion of the addition, the reaction mixture was stirred at -78 ° C for 4 hours. Trimethyl borate (31.1 g 300 mol) was added and the mixture was warmed to room temperature for 3 hours. After an additional 12 hours, water (150 mL) was added followed by 2M NaOH (200 mL). The aqueous phase was isolated and the organic phase was re-extracted with 2M NaOH (150 mL). The combined basic extracts were acidified to pH 3 using 2M HCl and extracted using EtOAC (5 x 200 mL). The combined organic extracts were. dried (Na 2 SO ₄ ) and concentrated to give the desired product as a white crystalline solid (15.49 g, 72%), identical to that produced in Example 2.

Example 8 (1S, 2S, 3R, 5S) -Pinanediol

- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2RS-borane

To a stirred solution of the material obtained in Example 7 (15.49 g, 71 mol) in chloroform (250 mL) was added (1S, 2S, 3R, 5S) - (+) - pinanediol (12.77 g, 75 mol). After stirring, at room temperature under a nitrogen atmosphere for 16 h, the solution was removed and the residue purified by flash chromatography on silica gel (hexane / EtOAc 0: 1, 4: 1) to obtain. the desired product as a 1: 1 mixture of diastereomers as an oil (23.62 g, 67.7% based on 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine). This was identical to the mixture of isomers produced in Example 3.

^ι : ι

- 20 ^L 1 H NMR (CDCl ₃ ): δ 0.85 (s, 3H), 1.12-1.21 (m, 1H), 1.29 (s, 3H), 1.41 (s, 3H) 1.45 (s, 9H), 1.81-2.20 (m, 8H).

2.28-2.39 (m, 1H), 3.04-3.18 (m, 1H), 3.34-3.35 (m, 2H),

4.28- 4.38 (m, 1H); ¹³ C NMR (CDCl ₃ ): δ 23.7, 26.2, 27.1,

28.5, 35.5, 38.2, 39.6, 46.1, 78.0, 78.8, 85.7, 85.5, 85.8,

154.7; C1MS m / z (% rel Int) 350 (MH &lt; + &gt;, 100), 294 (72), 250 (30).

Example 9

Analytical method for diastereoisomers of (IS, 2S, 3R, 5s) -pinanediol pyrrolidine-2-boronic acid hydrochloride.

A solution of 0.2 M phenylisocyanate reagent in dichloromethane-triethylamine (9: 1) was prepared. The sample to be analyzed (1-5 mg) was treated with 10 μΐ solution reagent per μπιο.Ι. and the clear solution was allowed to stand at room temperature for 15 min. A 1 μΐ sample solution was then diluted in 1.00 mL in acetonitrile for HPLC and 10 μΐ of this solution was analyzed by HPLC (column: YMC AQ-303 S-5 I20A, 4.6 x 250 mm; mobile phase: 65% MeCN - 35% 25 mM ammonium phosphate, pH 7.5; flow rate 1 ml / min; detection with UV at 254 nm). The phenylthiourea derivative R of the isoform of the boronic acid is eliminated at about 6.4 min, its opioid is eliminated at about 7.8 min, and unreacted phenyl isocyanate, which serves as an internal standard, is eliminated at ca. 12.2 min.

Example 10

(1S, 2S, 3R, 5S) - Pinanediol pyrrolide n-2RS-boric acid hydrochloride.

Mixed (IS, 2S, 2R, 5S) -pinanediol solution. 1- (1,1-dimethylethoxycarbonyl) pyrrol.i. diethyl ether (224 g, 0.64 mol) in diethyl ether (900 ml) was added. ice-cooled and dry gaseous IIC1

- 21 was introduced into the solution for 35 min at 10-18 °. The solution was stirred at room temperature overnight, re-cooled in ice and the precipitate was filtered off. The solid was washed with cold ether (400 mL) followed by 9: 1 petroleum ether / diethyl ether (200 mL) and dried in vacuo to give the desired hydrochloride as a white solid (113 g, 62%) (bt 228-234θ (3)) HPLC analysis of Example 9 indicated that it was a 60:40 mixture of the R: S boronic acid isomers.

1 H NMR (CDCl ₃ ): δ 0.83 (s, 3 H), 1.14 (d, J = 11 Hz, 1 H), 1.29 (s, 3 H), 1.45 (s, 3 H) 1.85-2.15 (m, 6H), 2.17-2.50 (m, 3H), 3.18-3.25 (m, 1H), 3.45 (bs, 2H) H), 4.42 (dd, J = 1.8, 8.6 Hz, 1H), 8.80 (bs, 1H), 10.56 (bs, 1H); ¹³ C NMR (CDCl ₃ ) δ 23.9, 24.5, 26.5, 27.0, 27.2, 28.4, 34.9, 38.2, 39.4, 45.8, 51, 2, 79.0, 87.6; CIMS m / z (rel int) 250 (MH &lt; + &gt;,100); Anal.vyp. for IH4H24BNO2 · HCl: C, 58.87, H, 8.82, N, 4.90, Cl, 12.41. Found: C, 58.40, H, 8.86, N, 4.81, Cl, 12.39.

A similar reaction in 29 g, 101 mmoles, using ethereal HCl (approximately 4.4M, 200 mL) and EtOAc (150 mL) as solvent, gave the hydrochloride (11.1 g, 47%) as a mixture of 81: 1.9 R isomers: S.

Example 1 and d 11

(1S, 2S, 3R, 5S) -Pinanediol pyrrolidine-2R boronic acid hydrochloride by fractional crystallization

Method A:

The 60:40 isomer mixture obtained in Example 10 (1.18 g, 4.13 mmol) was dissolved in CH 2 Cl 2 (65 mL) with gentle heating and the solution was filtered. The filtrate was diluted with EtOAc (65 mL) and crystallization occurred within a minute. The supernatant was stirred for 1-2 hours at room temperature and the first crop of solid was collected and the diastereoineric ratio was determined as described in Example 9 (540 mg, 46%, R: S ratio 97.1: 2.9). ). The solvent was distilled off from the filtrate until most of the CH 2 Cl 2 was removed, then the remaining EtOAc solution was stirred at room temperature overnight to avoid a second crop of off-white solid (346 mg, 29%. R: S ratio 39.2: 60.8) ). The first crop was recrystallized from isopropyl alcohol (10 mL) to yield

sa 430 mg (80%) regeneration) > 99% of the 2-R isomer. (h. 269 až 272 ’C (dec) [α] ₂₅ D + 0.70 ° (c = 1.15, MeOH). 1 H NMR (CDC1 ₃ ) : δ 0.83 (p. 3 H), 1.14 (d, J = 11 Hz, 1H), 1.29 (s, 3 H), 1.45 (s, 3 H, 1.85-2.15 (m, 6H), 2.17 - 2,50 (111, 3 H), 3.18-3, 25 (m, 1H), 3.45 (bs, 2H), 4.42

(dd, J = 1.8, 8.6Hz, 1H), 8.80 (bs, 1H), 10.56 (bs, 1H); ¹³ C NMR (CDCl ₃ ) δ 23.9, 24.5, 26.5, 27.0, 27.2, 28.5,

34.9, 38.1, 39.4, 45.8, 51.2, 79.0, 87.8; CIMS m / z (rel int) 250 (MH &lt; + &gt;,100); Anál.vyp. for C 14 H 24BNO 2 · HCl: C, 58.87, H, 8.82, N, 4.90, Cl, 12.41. Found: C, 58.64, H, 8.79, N, 4.90, Cl, 12.66.

Method B:

Suspension of (1S, 2S, 3R, 5S) -pinanediol-pyrrolidine-2RS-boronic acid hydrochloride as a 1: 1 mixture of isomers (850 mg, 2.98 nimol) in EtOAc ( 60 mL) was heated under reflux with stirring for 4 hours. The mixture was hot filtered and the collected solid was dried to give a material enriched in the R isomer (541 mg 64%), R: S ratio 2: 1. The evaporated filtrate gave the enriched material of the S isomer (217 mg), Rf: S = 1: 4. The filtered solid was treated in the same way with EtOAc (45 mL) for 1.5 hours and filtered hot again. Obtained a simple solid yield (366 mg, 73%), ratio R: S = 7: 1. This material was treated again with EtOAc (38 inl) for 1.5 hours. The isolated solid (287 mg, 79%) had an R: S ratio of 97: 3. The spectral properties were the same as for the material obtained by method A.

- 23:. ...; ·

Example 12

1- (1,1-dimethyethoxycarbonyl) -pyrrolidine-2-boronic acid from (1S, 2S, 3R, 5S) pinanediol 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine. n - 2 - b o. r i t a n u

To a solution of (1S, 2S, 3R, 5S) pinanediol 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2RS-borate from Example 8 (1.9 g)

5.44 inmol) in acetone (80 mL) was added 0.114 ammonium acetate solution (80 mL) and sodium periodate (3.49 g, 16.33 mmol). The reaction mixture was stirred at room temperature for 40 hours, then the acetone was evaporated and the residue was treated with 214 NaOH solution. This aqueous phase was washed with CH 2 Cl 2 (2 x 80 mL), acidified with 2M HCl to pH 3 and extracted with CH 2 Cl 2 (4 x 80 mL). The combined organic extracts were dried and concentrated to give the desired product as a white foam solid (890 mg, 76%), identical to NMR with the material prepared in Example 2. Boric acid was derived with pinacoloin for analysis.

Example 13

Peptide 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2RS-borate

To a stirred solution of boric acid of Example 12 (890 mg, 4.14 mol) in chloroform was added pinacol (489 mg, 4.14 mol). After stirring for 16 hours p.r: i. at room temperature, the solvent was removed and the residue was purified by chromatography over silica gel (hexane / EtOAc, 4: 1) to give the desired product as a white solid (1.04 g, 85%) (mp.73-75 ° C).

1 H NMR (CDCl ₃ ): δ 1.18 (S, 6H), 1.21 (s, 6H), 1.38 (s, 9H),

1.57-2.00 (m, 41-1), 2.98 (br s, 1H), 3.27 (m, 2H); 13 C NMR (CDCl ₃ ): δ 24.3, 24.5, 24.7, 24.9, 25.3 ·, 27.0, 27.6, 28.4,

28.6, 43.6, 45.8, 46.3, 78.8, 83.2, 154.4, 154.8; CIMS m / z (% rel. Int) 298 (18), 242 (100, MH + -tBu), 198 (30, MH + - Boe); A n and Sticks γ calculated for C 18 H 18 N 2 O 2: C, 60.62, H 9.50, N 4.71. Found: C, 60.94, H 9.65, N 4.88.

Example 14 a. 18 petroleum ether to give (1.13.4 g) (b.

N- (1,1'-dimethylethoxycarbonyl) -L-valylpyrrolidine-2R-borate (1S, 2S, 3R, 5S) -pinanediol ester

A solution of T-BOC-L-valine (351.7 g, 1.62 mol) in CIT ₂ Cl ₂ (1.6 L) was cooled with an ice bath and a solution of dicyclohexylcarbodiimide (161.8 g, 0.784 mol) in CH ₂ CL ₂ (0.75 L) was added over 40 min at 0-2 ° C. After the addition, the solution was stirred for 3.5 hours at room temperature. 0-5 ° C. The white precipitate was filtered and washed with CH ₇ Cl ₂ (0.2 L). The resulting pure solution was weighed at 20 ° C (water bath cooling) to a solution of (1S, 2S, 3R, 5S) -pinanediol pyrrolidine-2RS-borate hydrochloride (210 g, 0.735 mol), prepared as in Example 10, in CH ₂ CL ₂ (2.0 L) containing N-methylmorpholine (164 g, 1.62 mol). The mixture was stirred overnight at room temperature. The cloudy solution was filtered through a layer of silica gel (200-425 mesh) with a diameter of 16 cm and a thickness of 2 cm and washed with CH ₂ Cl ₂ (1.5 L). The solvent was evaporated to give a high viscosity oil (542 g). This oil was dissolved in ethyl acetate (0.7 L) and the mixture was cooled in an ice bath. Crystals formed which were filtered at low temperature and washed with cold ethyl acetate (0.1 L). The wet filter cake was transferred to petroether (0.65 L) and stirred at room temperature for 1 hour. The white solid was filtered off, washed with cold (0.11), and dried to constant weight of the title compound as a white solid (128-130 ° C). All mother liquors were combined and concentrated to a volume of about 0.8 L. After standing for 2 days in the freezer, a solid precipitated which was filtered and treated with light petroleum as above to give a beige solid (50.4 g). This was a mixture of impurities and an undesired dlasteroisomer. The mother liquor from the above was i. concentrated and the residue purified over a silica gel column (14 cm diameter x 60 cm) using hexane / ethyl acetate (85.15) (1.41). Appropriate fractions were collected, treated with petroleum ether, a solid collected by filtration and dried to give the additional desired product (18.5 g). Another diastereoisomer is also obtained (7.5 g) (mp 82-83 ° C). The second column was designed for the combined mixture of fractions and mother liquors to give additional pure compound (13.5 g), total combined yield 145.4 g (44.3%) of the desired diastereomer (mp 128-130 ° C).

1.39 (s, 3H), 1.41 (s, 9H),

2.26-2.36 (m, 1H), 3.15 (dd, J)

3.43-3.51 (m, 1H), 3.70-3.81 (m, 1H)

13n KTM

5.29 (d, J = 9.2 Hz, 1H);

24.0, 26.3, 27.1, 27.2, 27.4, 2

38.2, 39.6, 46.7, 51.2, 56.6

170.2; CIMS m / z (% rel int) Anal. Calcd for 2424 ^ 416 ^^ 5: C

77.8,

449

Z i s tené

C 64.58

(d, J = 6.7 Hz, 3 H) , 1.35-1.45 (m, 1 H) 72-2.14 (m, 9 H) 6.7, 10.1Hz, 1 H) 4.19-4.28 (m, 2 H) (CDC1 ₃ ) δ 17.3, 19.2 , 6, 31.4, 33.9, 35.5 79.2, 85.8, 155.9 (MH @ +, 100, 393) (50) H, 9.22; N, 6.25. H, 9.33; N, 6.52.

Example 15

Hydrogen maleate L-valpyrrolidine-2-R-borate (1S, 2S, 3R, 5S) -pinanediol ester

N - (1,1-dimethylethoxycarbonyl) - L -valylpyridine-2R-boronan (1S, 2S, 3R, 5S) -pinanediol ester (248 mg, 0. 553 mmol) was added to of a stirred solution of dry HCl in ethyl acetate. After 1.5 hours the solvent was evaporated to remove unbound hydrochloride. The residue was partitioned between CH 2 Cl 2 with sodium carbonate solution and the organic layer was dried over magnesium sulfate. The organic layer contains the free base of said compound, which exists in a cyclic form containing a nitrogen-boron bond, but is converted to the open form by addition of an acid. The organic solution was filtered into a solution of maleic acid (64 mg, 0.553 mmol) in methanol (5 mL), the solvent was evaporated to leave a crystalline residue (258 mg) which was recrystallized from ethyl acetate to give the title compound (193). mg, 75%) (mp 145-146 ° C) ^T N-NMR (CDC1 _3): δ 0.84 (s, 3H), 1.08 (d, J = 6.9 Hz. 3H), 1.13 ( d, j = 6.9 Hz, 3H), 1.26-1.31 (m, 2H), 1.29 (s, 3H), 1.38 (s, 3H), 1.72 -2.15 (m, 2H), 2.24-2.38 (m, 2H),

3.28 (dd, J = 6.9, 9.4 Hz, 1H), 3.38-3.47 (m, 1H),

3.73-3.78 (m, 1H), 4.14 (d, J = 5.1Hz, 1H), 4.26 (d, J =

7.11-1.2, 1H), 6.26 (s, 2H), 7.5-9.0 (v.br, 4H);

¹³ C NMR (CDCl ₃ ): δ 17.0, 18.4, 24.0, 26.3 27.0, 27.1, 28.7,

30.0, 35.4, 38.2, 39.5, 47.3, 51.2, 56.6, 78.1, 86.2, 135.6, 166.3, 169.6; CIMS m / z (% rel int) 349 (MH &lt; + &gt;, 100), 197 (18);

Calcd for C 23 H 37 N 2 O 4 207 C 59.49 H 8.03, N 6.03. Found: C 59.50, H 8.13, N 6.03.

Example 16

L-Valylpyrrolidine-2R-boronic acid methanesulfonate

a) cyclo-L-valylpyrrolidine-2R-boronic acid

A solution of the maleate salt obtained in Example 15 (5.0 g, 1.0.8 mmol) in dilute acetic acid (1.0%, 60 mL) was packed into a column (3.5 cm deep x 4 cm diameter) from Dowex 50x2- 200 ion exchange resin in the form of H ⁺ . The column was then washed with acetic acid (1.0%, 14 L), water (42

I.) and ammonium hydroxide solution (1: 100 dilution of a commercial 0.880 solution). Pinandiol could be recovered from the neutral and acidic fractions. The product was found in the first basic fractions which were combined and washed with CH 2 Cl 2 (2 x 100 mL). The organic extracts were dried and concentrated to restore the free base of the starting material (741 mg, 20%) by addition of pinanediol. The aqueous phase was lyophilized to give the title compound, which exists in cyclic form with nitrogen-boron bond as a white solid (1.52g, 66%) (mp 120-130 ° C).

1 H NMR (D ₂ O): δ 0.97 (d, J = 7.0 Hz, 3 H), 1.06 (d, J = 7.0 Hz, 3 H), 1.59-1.80 (m, 2 H), 1.95-2.03 (m, 2 H), 2.41-2.51 (m,

H), 2.62-2.69 (m, 1H), 3 (m with overlapping doublet, J = ¹³ C NMR _(D? 0): δ 19.0, 21.7, 170.3; IR (cm ^-1 ) 3400-3314

1452-1369; CIMS m / z (% rel (100, MH + -H ₂ O);

Calculations calculated for CqII ^ qBN ^^: C Found: C, 23-3.32 (m, 1H), 3.51-3.58

4.2 Hz, 2H);

27.3, 30.7, 29.9, 49.6, 61.0,

3221-3108, 2961-2872, 1637, int) 375 (90, M ² H + -3 H ₂ O), 197

50.50, H 8.95, N 13.09. H, 8.76; N, 12.93.

b) L-valylpyrrolidine-2R-boronic acid methanesulfonate

To a stirred suspension of the cyclized boric acid obtained above (5.17 g, 24.16 mmol) in acetonitrile (190 mL) under nitrogen was added a solution of raetanesulfonic acid (2.32 g, 24.16 mmol) in acetonitrile (10 mL) after dropwise over five minutes and the mixture was stirred at room temperature for 2 hours. The solid was collected by filtration, well washed with ace tons. tr i. refraction and diethyl ether and dried to give the title compound as a white solid (6.14, 82%) (mp. 179-180 ° C). Crystallization of this material from dimethylformainide / α-

ti c e t he i ľ. r I. Lu gave 70% material recovery in an individual collection (h . t. '1.8 1. until 182 “C). hi NMR (I) ₂ o, phosphate, pH2); δ 0.99 (d, J) , = 6.8Hz, 3h) 1.09 (No. 1 = 6, 9Hz, 3H), 1.69-1 75 (m, 1H), 1.90-1.99 (m, 1H), 9 10-2 , 1.4 (m (2H), 2.28-2.35 (m, 1H), 2.8 (s, 3H) 3.07 (Dd,

J = 7.0 and 11.2 Hz, 1H) , 3.46-3.51 (m, 11-1), 3.75 (t, J = 9.0) Hz, 1 h), 4.14 (d, J = 5.1 Hz, 1H); cis amide rotaineter (approx 3%) is also observed at 3.53-3.55 (m) and 3.83 (d, J = 6.2) Hz): ^I3 C NMR: δ 16.2, 18.4 . 26th 9, 27.1, 29.0, 38.8, 47.9, 57.2, 1 6 7 2; caused by C sharp amide rotamer are observed p i; and 16.8, 24.3, 29.9,. 57.8. 167.5; IR (cm ^-1 ) 3387, 3000 (Br) , 2972, 2655 1646 1370, 1197;

CIMS m / z (% rel int, ethylene glycol compound) 241 (MII + 100):

Calcd for C C ^H QQN BO ^S: C, 38.72; H, 7.47; N, 9.03.

Found: C 38.65, H 7.45, N 8.44.

Example 17

N- (1,1-dimethylethoxycarbonyl) -L-valyipyrrolidine-2R-boronic acid

To a stirred solution of N- (1,1-dimethylethoxycarbonyl) -Lva-1-ylpyrrolidine-2R-borate (1S, 2S, 3R, 5S) -pinanediol ester, prepared as in Example 14, (1.0 g, 2.3 mmol) in acetone (75 mL) was added a solution of ammonium acetate (60 mL, 0.1 M) and sodium periodate (1.48 g, 6.91 mmol). The reaction mixture was stirred at room temperature for 48 hours. , then the acetone was evaporated. The residue was treated with 2M sodium hydroxide solution (100 mL), washed with CH 2 Cl 2 (2 ^x 50 mL). The aqueous layer was carefully acidified to pH 3 with 2M hydrochloric acid and extracted with CH 2 Cl 2 _. Cl 2 (4 x 70 mL). The combined organic extracts of the acid solution were dried over sodium sulfate and concentrated to afford the desired product as a white foam solid (700 mg, 97%). Further purification by chromatography over silica gel (CH 2 Cl 2 / methanol, 9: 1) gave boric acid again as a white solid (449 mg, 62%) (mp 82-92 ° C).

¹ H NMR (CDCl 3): δ 0.95 (d, J = 5.7Hz, 6H), 1.42 (s, 9H), 1.55-1.80 (m, 1H), 1.80-2 20 (m, 4H), 2.89-3.07 (m, 11H), 3.30-3.55 (m, 1H), 3.55-3.65 (m, 1H), 4.10 -4.30 (m, 1H); 5.34 (D, J = 9.2, 1H); ¹³ C NMR (CDCl 3): δ 18.0, 19.1, 26.3,

27.7, 28.3, 31.2, 46.1, 52.0, 55.7, 79.5, 155.6, 170.8; IR (cm ^-1 ) 3395-3319, 2971-2875, 1711, 1619, 1400, 1174; CIMS m / z (% rel int, ethylene glycol adduct) 341 (MH ⁺ , 100), 285 (MH ⁺ -tBu, 67), 241 (MH ⁺ -BOC, 21).

Example 18

L-valylpyrrolidine-2R-boronic acid hydrochloride

N- (1,1-dimethylethoxycarbonyl) -L-valylpyrrolidine-2R-boronic acid obtained in Example 17 (250 mg, 0.796 minol) was stirred with HCl / ether (4.5M, 20ml) at room temperature under a nitrogen atmosphere for 1.5 hours. The solution was then evaporated and the residue triturated with ether (x10ml) and decanted each time. The residue was then dried to yield the title compound as a white powdered solid (172 mg, 86%) (mp 211-213 ° C).

NMR (D 2 O, phosphate pH 2): δ (d, J = 6.9Hz, 3H), 1.09 (d, J = 7Hz, 3H), 1.67-1.76 (m, 1H) 1.87-2.01 (m, 1H),

2.09-2.15 (m, 2H), 2.28-2.35 (m, 1H), 3.07 (dd, J = 7 and 11.4 Hz, 1H), 3.48 (dt, J = 7.7 and 10.3 Hz, 1 H), 3.73 (dt, 1 = 1.7 and 10.2 Hz, 1 H), 4.14 (d, J = 5.2 Hz, 1H); ¹³ C NMR: δ 16.0, 18.3, 26.9, 27.1, 28.9, 47.9, 48.9, 57.2,

167.3; IR (cm ^-1 ) 3400-2800,3368, 2970/2880, 1635,

1475-1378, 1400; CIMS 111 / z (% rel int, ethylene glycol compound) 241 (MH ⁺ , 100).

Example 19

Eyclo-L-valylpyrrolidine-2R-boronic acid transesterified with phenylboronic acid.

A solution of L-valylpyrrolidine-2R-borate (1S, 2S, 3R, 5S) -pinanediol ester hydrochloride, prepared as in Example 15 (500mg, 1.3mmol) in 1M hydrochloric acid (10ml) containing hexane (20ml) and Phenylboronic acid (500 µL, 2.6 µmol) was stirred vigorously for 1 hour. at room temperature. The hexane was removed by decantation, then additional hexane (20 mL) was added and the mixture was stirred for an additional 30 min. The layers were separated and the combined hexane layers dried over sodium sulfate and concentrated to give pinanediol phenylborate

30 (331 ing, 99%) as a white crystalline solid. The aqueous layer was then passed through a column containing Dowex 50 ion exchange resin. The column was eluted with water (200 mL) followed by ammonium hydroxide solution (1: 100 dilution of a 0.880 commercial solution, 50 mL). Isolation of the basic fractions followed by lyophilization gave free boric acid (230 mg, 83%) as a white powder, identical to NMR with the material obtained in Example 16a.

Claims (42)

PATENT CLAIMS A process for the preparation of a proline boronic ester of the general formula VII (VII) wherein X is a linking group, characterized in that it comprises: a) treating the pyrrole with activated carbonic acid derivatives to form the protected-N pyrrole of Formula I RO wherein R is C ^ alkyl, C ₃ _ ₆ cycloalkyl, benzyl, phenyl, phenyl substituted by one or more C ^ _g alkyl groups, or trimethylsilylethyl, b) treating the protected intermediate of formula I with a lithium reagent to form the intermediate of formula II. (II) wherein R is as defined above, c) reacting an intermediate of formula II with a trialkyl borate, wherein each alkyl group may be linear, branched, or cyclic of from 1 to 6 carbon atoms, followed by acid-catalyzed hydrolysis to form an intermediate of formula III OH (III) RO where R is as defined above, d) reducing the compound of formula III, using catalytic hydrogenation, to obtain a proline intermediate of formula IV (IV) wherein R is as defined above. e) reacting a compound of formula IV with a diol of formula V HO-X-OH (V) wherein X is the same linking group mentioned above to form boric acid of formula VI (VI) Wherein X is the same linking group mentioned above and R is also defined above and f) deprotection of the nitrogen atom in the proline ring. Process according to claim 1, characterized in that in step (c), the intermediate of formula II is reacted with trimethyl or triethyl borate. 3. The method of claim 1, wherein the linking group X is a saturated 2-3 membered hydrocarbon chain which forms part of a C2-5 hydrocarbon ring, which may optionally contain unsaturated or cyclic bonds, or which consists of an aromatic cyclic moiety. or X is a group of the formula - (CH 2) _n -NH - (CH 2) wherein m and n are 2 or 3, which groups may be unsubstituted or substituted by one or more C _1-3 alkyl or phenyl groups. A process according to claim 1, wherein the esterification in step (e) is carried out with a diol selected from the group consisting of ethylene glycol, pinacol, catechol, pinanediol, butane-2,3-diol, diethanolamine and 1,2-diphenylethane-1,2-diol. A process according to claim 4, wherein the diol is an optically active pinanediol. 6. The method of claim 5 v y z n and čuj ú c i. s a t ý m, that pinanediol ester s protected N in step (e) according to claim 1 is further separated on the own d j Lastereoizoméry. 7. The method of claim 5 v y z n and. s a characterized in that the unprotected N pinanediol ester formed in step (f) according to claim 1 is further separated into its own diastereomers. 8. The process of claim 1 wherein R is tert-butyl, benzyl, trimethylsilylethyl, phenyl, methyl, or ethyl. 9. A process for the preparation of este.ru of proleic acid: i.e. H • B '(VII) wherein X is a linking group, characterized in that it comprises: a) treating pyrrole with an activated derivative of carbonic acid to obtain the N-protected pyrrole of the formula I, (i) wherein R is a C ^. ^ alkyl, C ₃ _gcykloalkyl, benzyl, phenyl, phenyl substituted by one or more C £ _g alkyl, or triinetylsilyletylom. b) treating the protected intermediate of formula I with a lithium reagent to form an intermediate of formula II (II) wherein R is as defined above, c) reacting the intermediate of formula II with a trialkylborate, wherein each alkyl group may be linear, branched or cyclic of 1 to 6 carbon atoms, followed by acid catalyzed hydrolysis, to form the intermediate of formula III / 7 'A S.:/ OH .Zx Ό \ CH (III) Where R is as defined above, d) reacting a compound of formula III with a diol of formula V, HO-X-OH (V) wherein X is a linking group to obtain es ver boric acid of formula IIIA -, Z where R is as defined above, (IIIA) e) reduction of the resulting ester to yield an intermediate of formula VI, O (VI) f) deprotection of nitrogen in the proline ring. A process according to claim 9, wherein in step (o), the intermediate of formula II is reacted with vinyl or tri-butane. 11. The method of claim 9, wherein the linking group X is a saturated 2 to 3 membered hydrocarbon chain which forms part of the ^C 5-12 hydrocarbon ring, which may optionally contain unsaturated or cyclic bonds; or A 2 to 3 membered hydrocarbon chain that forms part of the aromatic ring system; or X is a group of the formula - (CH ₂ ) _n -NH- (CH ₂ ) _m - wherein n and m are 2 or 3; wherein such groups may be unsubstituted or substituted with one or more C 1-3 alkyl or phenyl groups. 12. The process of claim 9 wherein the esterification in step (d) is carried out with a diol selected from the group consisting of ethylene glycol, pinacol, catechol, pinanediol, butane-2,3-diol, diethanolamine, and 1,2-di. phenylethan-1,2-diol. 13. A process according to claim 12, wherein the diol is an optically active pinanediol. 14. The process according to claim 13, wherein the N-protected pinanediol ester formed in step (e) of claim 9 is further separated into its own diatereoisomers. 15. The process of claim 13, wherein the unprotected N-ester of pinanediol formed in step (f) of claim 9 is further separated into its own di-isomers. 16. The method of claim 9, wherein R is tert-butyl, benzyl, trimethylsilylethyl, phenyl, methyl, or ethyl. 37 17. Process for the preparation of the intermediate of formula IV ^: Ω (IV) RG ^ O is characterized by comprising: (a) treating the pyrrole with activated carbonic acid derivative to form the pyrrole with N-protected formula I i (I) wherein R is C ₁ _ alkyl, C ₃ _gcykloalkyl, benzyl, phenyl, phenyl substituted by one or more of C £ _g alkyl, I .1. or ismethy 1 y 1 ethyl, h) treating the protected intermediate of formula I with a lithium reagent to form the intermediate of formula II (II) Where R is as defined above, c) reacting an intermediate of formula II with a trialkyiborite, wherein each alkyl group may be linear, branched or cyclic and contains 1 to 6 carbon atoms, followed by acid-catalyzed hydrolysis to give an intermediate of formula I 11 wherein R is as defined above, and d) reducing the compound of formula III, using catalytic hydrogenation, to produce the proline intermediate of formula IV. 18. A process for preparing an intermediate of formula IV (IV), wherein the process comprises: ti) treating the pyrrolidine with an acylating agent to provide the protected pydolidine of formula VIII Z i (VIII) wherein R is alkyl, _C3 _g cycloalkyl, benzyl, phenyl, phenyl substituted by one or more C ^ _g alkyl, or trimetyIsilyletylom, h) treating the compound of formula VII with a lithium reagent to form a compound of formula IX (ix) wherein R is as previously defined, c) reacting a compound of formula IX with a trialkyl borate, wherein each alkyl group may be linear, branched or cyclic and may contain 1 to 6 carbon atoms, and d) hydrolyzing the product of the preceding step to give a compound of formula IV. 19. The process of claim 18, wherein in step (c), the intermediate of formula IX is reacted with triinetyl or triethyl borate. An intermediate of formula III (III) wherein R is alkyl, C 3-6 cycloalkyl, benzyl, phenyl, phenyl substituted with one or more alkyl groups and amino groups. m e t y 1 s i. 1 y 1 e t y 1 om. 21. 1- (1,1-Dimethylethoxycarbonyl) -pyrrole-2-bromide. A compound selected from the group consisting of (1S, 2S, 3R, 5S) -pinanediol 1- (1,1-dimethylethoxycarbonyl) -pyrrolidine-2S-boronate; (1S, 2S, 3R, 5S) -pinanediol- L- (1,1-dimetyletoxycarbonyl) -pyrrolidine-2R-boronate; (R, 2 R, 3 S, 5 R) -pinanediol 1- (1,1-dimetyletoxycarbonyl) -pyrrolidine-2S-boronate; and (1R, 2R, 3S, 5R) -pinanediol 1- (1,1-dimethylethoxycarbonyl) pyrrolidine-2R-borate; A compound selected from the group consisting of (1S, 2S, 3R, 5S) -pinanediol pyrrolidine-2S-borate hydrochloride; (1S, 2S, 3R, 5S) -pinanediol pyrrolidine-2R-borate hydrochloride; (1R, 2R, 3S, 5R) -pinanediol pyrrolidine-2S-borate hydrochloride; (1R, 2R, 3S, 5R) -pinanediolpyrrolidine-2R-boronate hydrochloride; A compound selected from the group consisting of (1S, 2S, 3R, 5S) -pinanediol 1- (1,1-dimethylethoxycarbonyl) -pyrrole-2-borane; (1R, 2R, 3S, 5R) -pinanediol- (1,1-dimethylethoxycarbonyl) -pyrrole-2-borate; 25. A process for removing pinanediol as a protective ester group from a borate ester, characterized in that the borate ester is treated with an oxidizing agent capable of cleaving 1,2-diols. and sodium. este.ro 26. The method of claim 25, wherein the oxidizing agent is a periodate 27. A method for removing pinanediol as the protective Vej of a compound of formula X (X) wherein characterized in that R is a protecting group ^of T and R is the side chain of a naturally occurring amino acid, optionally with suitable protecting groups, or from a compound of formula XI) R '□ wherein R is as defined above wherein the method for the boronic ester is oxidative capable of cleaving the 1,2-diol. / \ (XI) includes only a reagent that is 28. The process of claim 27 wherein said oxidizing agent is sodium periodate. 29. A method for removing pinandiol as a protective ester group from a boronic ester having a free amino group, the method comprising applying an aqueous boronic ester solution to a cation exchange resin column, washing the column with water or dilute acid to remove pinanediol and finally washing the column. with an aqueous solution of a base to remove the free boric acid product. 30. The method of claim 29, wherein the acidic cationic protective esterosis is a material in the xoion that is sunlit. c o u. A method of removing pinandiol as an O group from a compound of Formula XI, wherein R ^{3 is a} naturally occurring amino acid, (xi) is a side chain optionally with appropriate protecting groups, the method comprising applying the aqueous boronic ester solution to a column containing cation exchange resin, washing the column with water or dilute aqueous acid to remove pinanediol and finally washing the column with dilute aqueous base to remove free boric acid product. 32. The method of claim 31, wherein a strongly acidic cation exchange resin is used in the column. 33. Intermediate of formula X (X) Where R 'is a protecting group and R is the side chain of a naturally occurring amino acid, optionally with appropriate protecting groups. * 34. N- (1,1-dimethylethoxycarbonyl) -L-valylpyrrolidine-2R-borate (1S, 2S, 3R, 5S) - pinanediol ester. An intermediate of formula XI (xi) wherein the -O-X-O- protecting group is derived from pinanediol and wherein Q R is the side chain of a naturally occurring amino acid, optionally with appropriate protecting groups. .- 15 čjýsX / - 43 36. L-Valylpyrrolidine-2R-boronate, 2S, 3R, 5S) -pinanediol ester or a salt thereof. 37. A process of formula VII for preparing proline boronic ester (VII) 4th wherein X is a bond that includes: group, (a) treating the pyrrolidine with an acylating agent to form pyro- 1 i d i n of formula VIII H 1 (VIII) where R is _Ci _g alkyl, C ₃ _g cycloalkyl, benzyl, phenyl, phenyl substituted with one or more C 1-6 alkyl groups, or trimethylsilylethyl, b) treating the compound of formula VIII with a lithium reagent to give a compound of formula IX (ix) wherein R is as previously defined, c) reacting a compound of formula IX with a trialkyl borate, wherein each alkyl group may be linear, branched or cyclic and may contain 1 to 6 atoms; and d) hydrolyzing the product of the preceding step to give the compound of formula IV OH RO ^ H OH (iv) e) reacting a compound of formula IV with a diol of formula V, HO-X-OH (V) wherein X is the same linking group mentioned above to form a borate ester of formula VI (VI). Wherein X is the same linking group mentioned above and R is as defined above, and f) deprotection of the nitrogen atom in the proline ring. 38. The process of claim 37, wherein in step (c), the intermediate of formula IX is reacted with trimethyl or triethyl borate. 39. The method of claim 37, wherein the linking group X. is a saturated 2 to 3 membered hydrocarbon chain; nasýteným2 to 3 of Article hydrocarbon chain which constitutes part of the C ₅ _ ₁₂ carbocyclic system which may optionally contain unsaturated bonds or ring; A 2- to 3-membered hydrocarbon chain that forms part of the aromatic ring system; or, X is a group of the formula - (CH ₉ ) _n -NH- (CH ₂ ) _m -, where n and m are each 2 or 3; which groups may be unsubstituted or substituted with one or more C ^ _ ₃ alkyl or a phenyl or miskupin am i. 40. The process of claim 37, wherein the esterification in step (e) is performed with a diol selected from the group consisting of ethylene glycol, pinacol, catechol, pinanediol, butane-2,3-diol, diethanolamine. 1,2-di-phenylethane-1,2-diol. 41. The method of claim 40 wherein the diol is an optically active pinanediol. 42. The method of claim 41, wherein the pinanediol. the N-protected ester formed in step (e) of claim 37 is further separated into its diastreoisomers. 43. The process of claim 41, wherein the unprotected N-pinanediol ester formed in step (f) of claim 37 is further separated into its diastereoisomers. 44. The process of claim 37 wherein R is tert-butyl, benzyl, trimethylsilylethyl, phenyl, methyl or ethyl.