WO2005033076A1

WO2005033076A1 - Method for preparing 3-hydroxy-4-hydroxymethyl-pyrrolidine compounds

Info

Publication number: WO2005033076A1
Application number: PCT/NZ2004/000239
Authority: WO
Inventors: Peter Charles Tyler; Keith Clinch
Original assignee: Industrial Research Limited
Priority date: 2003-10-03
Filing date: 2004-10-01
Publication date: 2005-04-14
Also published as: NZ528663A

Abstract

This invention relates to a method of preparing (3R,4R)-3-hydroxy-4-hydroxymethylpyrrolidine, a key intermediate compound for the synthesis of certain inhibitor compounds, including the step of enzyme catalysed enantioselective esterification of an hydroxy group of an hydroxypyrrolidine. The invention further relates to a method for preparing (3S,4S)-3-hydroxy-4-hydroxymethylpyrrolidine, which is the enantiomer of (3R,4R)-3-hydroxy-4-hydroxymethylpyrrolidine.

Description

METHOD FOR PREPARING 3-HYDROXY-4-HYDROXYMETHYLPYRROLIDINE COMPOUNDS

TECHNICAL FIELD

This invention relates to a method for preparing 3-hydroxy-4- hydroxymethylpyrrolidine compounds. In particular, the invention relates to a method of preparing (3ft,4R)-3-hydroxy-4-hydroxymethylpyrrolidine, including the steps of enzyme catalysed enantioselective esterification of an hydroxy group of an hydroxypyrrolidine, and separation of the diastereomers obtained. The invention further relates to a method for preparing (3S,4S)-3-hydroxy-4- hydroxymethylpyrrolidine, which is the enantiomer of (3 ,4f?)-3-hydroxy-4- hydroxymethylpyrrolidine.

BACKGROUND

The known compound of formula (A), (3ft,4R)-3-hydroxy-4- hydroxymethylpyrrolidine, is a key intermediate compound for the synthesis of certain of the applicant's inhibitor compounds, including potent purine nucleoside phosphorylase inhibitors (see for example WO 2004/018496).

(A)

Makino and lchikawa (K. Makino and Y. Ichikawa, Tetrahedon Letters (1998)

39, 8245) have reported a synthesis of compound (A). The requisite chirality of compound (A) is introduced using a Sharpless asymmetric epoxidation.

Karlsson and Hόgberg (S. Karlsson and H.-E. Hogberg. Tetrahedron: Asymmetry (2001) 12, 1977) describe an alternative synthesis method. In this method, chirality is introduced using a chiral sultam auxiliary.

However, synthetic methods that employ an achiral starting material suffer from the disadvantages associated with additional reaction steps necessary to introduce chirality. The disadvantages include a greater number of handling steps, lower product yields, scale-up difficulties, and costly reagents.

Filichev et al. (V. V. Filichev and E. B. Pedersen, Tetrahedron (2001) 57, 9163; V. V. Filichev, M. Brandt and E. B. Pedersen, Carbohydrate Research

(2001) 333, 115) have used chiral starting materials to produce compound (A). For example, compound (A) can be prepared from diacetone-D-glucose or from D-xylose. However, both synthetic procedures are complex and require many reaction steps.

An alternative method for introducing chirality involves the use of biological catalysts. For example, Hansen and Bols (S. U. Hansen and M. Bols, Ada Chemica Scandinavica (1998) 52, 1214) attempted the enzymatic resolution of the N-Boc derivative of racemic frans-3-hydroxy-4-hydroxymethylpyrrolidine using immobilised lipases from Candida antarctica and Mucor mihei. This method focuses on attempting to resolve the diol by enzymatic means. However, poor enantiomeric excesses were obtained in this way, resulting in only small amounts of compound (A) being made available for use as an intermediate in the preparation of other compounds. Low product yields mean considerable wastage and therefore high overall cost.

The published syntheses of compound (A) are therefore unsatisfactory as commercially viable routes to this valuable intermediate compound. There has been an ongoing need to overcome this problem by developing an improved method which employs only a few reaction steps and with an acceptable overall product yield.

It is known that lipase catalysed resolution of carbocyclic cis- and trans-β- hydroxy esters by O-acylation can provide enantiopure compounds in high yields (L. M. Levy, J. R. Dehli and V. Gotor, Tetrahedron: Asymmetry (2003) 14, 2053). However, it can be difficult to predict the reactivity of an enzyme to a potential substrate. Even when a particular compound is found to be an enzyme substrate there is often little certainty as to reaction yield and enantiomeric purity of the product.

However, in the search for new and improved methods for preparing compound (A), the applicant has surprisingly found that compound (A) can be prepared in high yield and in high enantiomeric excess, via lipase catalysed esterification of a racemic 1-Λ/-protected tra/7s-4-hydroxypyrrolidine-3- carboxylic acid alkyl ester. The applicant's new method also advantageously allows the preparation of compound (E), the enantiomer of compound (A).

It is therefore an object of the invention to provide an improved method for preparing 3-hydroxy-4-hydroxymethylpyrrolidine compounds, or at least to provide a useful choice.

STATEMENTS OF INVENTION

In a first aspect, the invention provides process for preparing a compound of formula (A) or a compound of formula (E), or salts thereof

(A) _(E)

including the steps of (a), (b) and (c): where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-fraπs-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

(B)

where R¹ is a straight or branched chain alkyl group; and R² is a protecting group;

to give either a mixture of a compound of formula (C) and a compound of formula (D)

(C) (D)

where R¹ and R² are as defined above; and R³ is acyl;

or a mixture of a compound of formula (C), and a compound of formula (D')

(C) (D') where R¹ and R² are as defined above; and R³ is acyl; where the enzyme-catalysed enantioselective esterification is carried out using either (1) an enzyme capable of producing an enantiomeric excess of compound (C); or (2) an enzyme capable of producing an enantiomeric excess of compound (D'); step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D') to the compound of formula (A); or transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E).

Preferably the enzyme-catalysed enantioselective esterification in step (a) gives a mixture of compounds of formulae (C) and (D), and the enantiomeric excess of compound (C) is at least about 80%, most preferably at least about

90%.

Alternatively it is preferred that the enzyme-catalysed enantioselective esterification in step (a) gives a mixture of compounds of formulae (C) and (D'), and the enantiomeric excess of compound (D') is at least about 80%, most preferably at least about 90%.

It is preferred that the enzyme used in step (a) is an enzyme capable of catalysing the formation of an ester bond, preferably a lipase, most preferably lipase from Candida antarctica. It is preferred that the transformation of the compound of formula (C) or the compound of formula (D') to the compound of formula (A) includes the step of reduction of the ester group of the compound of formula (C), or reduction of both ester groups of the compound of formula (D').

Preferably the transformation further includes the step of replacement of the R² group with hydrogen to give the compound of formula (A).

Alternatively, it is preferred that the transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E) includes the step of reduction of the ester group of the compound of formula (C), or reduction of both ester groups of the compound of formula (D). In that case, the transformation preferably further includes the step of replacement of the R² group with hydrogen to give the compound of formula (E).

It is preferred that the reduction is carried out using either LiAIH or LiBH₄.

It is further preferred that the replacement of the R² group with hydrogen is carried out either in the presence of HCI or with HCOOH/CH₃OH in the presence of Pd-C.

Preferably the separation of the compound of formula (C) from the compound of formula (D) or the separation of the compound of formula (C) from the compound of formula (D') is effected by chromatography or fractional crystallisation.

Preferably R is a straight or branched chain C-i-C₆ alkyl group, most preferably ethyl.

It is also preferred that R³ is COR⁴, where R⁴ is a straight or branched chain

Cι-C₆ alkyl group, and that R¹ is ethyl and R² is benzyl or t-butoxycarbonyl.

In a preferred embodiment of the invention, R¹ is ethyl, R² is benzyl and R³ is acetyl. In another preferred embodiment of the invention R¹ is ethyl, R² is t- butoxycarbonyl and R³ is acetyl.

Preferably the enantioselective esterification in step (a) is carried out using vinyl acetate as an acyl donor, to give a mixture of compounds of formulae (C) and (D) where R³ in the compound of formula (D) is acetyl; or a mixture of compounds of formulae (C) and (D') where R³ in the compound of formula (D') is acetyl.

In another aspect, the invention provides process for preparing a compound of formula (A), or a salt thereof

including the steps of (a), (b) and (c): where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-fra/7s-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

(B) where R¹ is a straight or branched chain alkyl group; and R² is a protecting group;

(C) (D)

where R¹ and R² are as defined above; and R³ is acyl;

or a mixture of a compound of formula (C), and a compound of formula (D')

(C) (D')

where R¹ and R² are as defined above; and R³ is acyl;

where the enzyme-catalysed enantioselective esterification is carried out using either

(1) an enzyme capable of producing an enantiomeric excess of compound (C); or (2) an enzyme capable of producing an enantiomeric excess of compound (D'); step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D') to the compound of formula (A).

In still another aspect, the invention provides process for preparing a compound of formula (E), or a salt thereof

(E)

including the steps of (a), (b) and (c): where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-traπs-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

(B)

(C) (D)

where R¹ and R² are as defined above; and R³ is acyl;

or a mixture of a compound of formula (C), and a compound of formula (D¹)

(C) (D') where R¹ and R² are as defined above; and R³ is acyl; wherein the enzyme-catalysed enantioselective esterification is carried out using either (1) an enzyme capable of producing an enantiomeric excess of compound (C); or (2) an enzyme capable of producing an enantiomeric excess of compound (D'); step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E).

The invention further provides a compound of formula (C)

(C)

where R¹ is a straight or branched chain alkyl group; and R² is a protecting group.

In addition, the invention provides a compound of formula (D)

(D) where R¹ is a straight or branched chain alkyl group; R² is a protecting group; and R³ is acyl.

The invention also provides a compound of formula (C)

(C)

The invention further provides a compound of formula (D')

(D¹) where R¹ is a straight or branched chain alkyl group; R² is a protecting group; and R³ is acetyl.

Preferred intermediate compounds include a compound of formula (C) as defined above where R¹ is ethyl and R² is benzyl and a compound of formula (C) as defined above where R¹ is ethyl and R² is f-butoxycarbonyl.

Preferred intermediate compounds include a compound of formula (D) as defined above where R¹ is ethyl, R² is benzyl and R³ is acetyl and a compound of formula (D) as defined above where R¹ is ethyl, R² is t- butoxycarbonyl and R³ is acetyl.

In a further aspect, the invention provides a compound of formula (A) as defined in claim 1 , when prepared by the above process.

In a final aspect, the invention provides a compound of formula (E) as defined in claim 1 , when prepared by the above process.

DETAILED DESCRIPTION

This invention relates to the lipase or acylase catalysed resolution of a (±)- trat7S-4-hydroxypyrrolidine-3-carboxylic acid alkyl ester. This provides a convenient route to compound (A). As noted above, this compound is useful in the preparation of the applicant's purine nucleoside phosphorylase inhibitor compounds, such as those described in WO 2004/018496.

The invention has the added advantage that the enantiomer of (A), a compound of formula (E), can also be produced. Compound (E) may find use in other applications. The invention therefore provides an improved route to a valuable starting material and its enantiomer.

Advantageously, compounds (C) and (D) (or compounds (C) and (D')) are produced in high chemical yield with high enantioselectivity. Furthermore, these compounds are readily separated and converted to the diols (A) and (E).

According to one preferred embodiment of the invention (Scheme 1), enantioselective acylation of the 4-hydroxy group of (±)-trans-1-Λ/-benzyl-4- hydroxypyrrolidine-3-carboxylic acid ethyl ester (1) is carried out using Novozyme® 435 lipase from Candida antarctica, and vinyl acetate as the acyl donor molecule. The two products are then readily separated by chromatography on silica gel. Compound (A) is obtained from compound (2) by reduction of the ester group using LiAIH₄ and removal of the benzyl protecting group using CH₃OH/HCOOH and Pd/C. Advantageously, compound (E) may also be obtained from compound (3).

Scheme 1

According to another preferred embodiment of the invention (Scheme 2) enantioselective acylation of the 4-hydroxy group of trans-(±)-A- hydroxypyrrolidine-1-Λ/-3-dicarboxylic acid-1-terf-butyl ester-3-ethyl ester (10) is also carried out using Novozyme® 435 lipase from Candida antarctica, and vinyl acetate as the acyl donor molecule. As Scheme 2 shows, compound (10) can be prepared in high yield from (Ε -(±)-1-Λ/-benzyl-4- benzyloxypyrrolidine-3-carboxylic acid ethyl ester (8) which may itself be prepared from commercially available Λ/-(methoxymethyl)-Λ/- (trimethylsilylmethyl)-benzylamine and trans-3-benzyloxyacrylic acid ethyl ester.

Compounds (12) and (13) are readily separated by chromatography. Compound (A) is obtained in two steps from compound (12) by reduction of the ester group using LiBH and removal of the t-butoxycarbonyl protecting group. Similarly, compound (E) may also be obtained from compound (13).

Scheme 2

(+XA), HC1 (-)-E, HC1 The compounds of formulae (A) and (E) may be converted to the salts thereof, using a suitable inorganic acid, such as HCI, or organic acid, such as p-toluenesulphonic acid.

It will be appreciated by a person skilled in the art that alternative acyl donor molecules may be employed in the method of the present invention. Suitable alternative donor molecules include methyl acetate, ethyl acetate, isopropenyl acetate, vinyl benzoate, vinyl propanoate, vinyl butyrate, vinyl laurate, 2,2,2- trichloroethyl acetate, 2,2,2-trichloroethyl butyrate, 2,2,2-trichloroethyl laurate, 2,2,2-trifluoroethyl butyrate, 2,2,2-trifluoroethyl laurate, and methyl propanoate.

Similarly, it will be appreciated that any suitable reducing agent may be used to reduce the ester group, following the enantioselective esterification. Preferably the reducing agent used is LiAIH₄ or lithium borohydride. However, other possible reducing agents include sodium borohydride with or without a Lewis acid catalyst such as AICI₃ or BF₃, L-Selectride, NaBH(OMe)₃, LiAIH(O- tBu)₃, LiAIH(OMe)₃, and AIH₃.

Although it is preferred that the compound of formula (B) incorporates a benzyl protecting group or a t-butoxycarbonyl protecting group, it will be clear to the skilled person that other N-protecting groups may be employed (see for example, "Protective Groups in Organic Synthesis" by Theodora W. Greene, Wiley-lnterscience, 3rd edition (May 15, 1999)). As used herein, the term "protecting group" means "a group that selectively protects an organic functional group, temporarily masking the chemistry of that functional group and allowing other sites in the molecule to be manipulated without affecting the functional group". Other suitable protecting groups include 4- methoxy benzyl, 2-(trimethylsilyl)ethyl, and diphenylmethyl.

Methods of removing such N-protecting groups are known to those skilled in the art, and it is envisaged that any suitable reagent may be used in the deprotection step, including: (a) in the case of a benzyl, 4-methoxybenzyl, or diphenylmethyl protecting group, catalytic hydrogenolysis using hydrogen (or a source of hydrogen such as formic acid) and a metal catalyst such as Pd/C; (b) in the case of a 2-(trimethylsilyl)ethyl group, a source of fluoride ion such as tetrabutylammonium fluoride; or (c) in the case of a 4-methoxybenzyl protecting group, an acidic catalyst such as trifluoroacetic acid or an oxidant such as eerie ammonium nitrate, (d) in the case of t-butoxycarbonyl, acidic cleavage using HCI. It will be clear to the person skilled in the art that any suitable separation method may be used to separate the compound of formula (C) from the compound of formula (D) or to separate the compound of formula (C) from the compound of formula (D'). However, it is preferred that the separation is effected either by chromatography or by fractional crystallisation.

The process of the invention contemplates the use, in the enzyme-catalysed enantioselective esterification step, of any enzyme capable of catalysing the formation of an ester bond. However, it is preferred that a lipase is used, most preferably lipase from Candida antarctica.

While it is preferred that the compound of formula (B) is one where R¹ is ethyl, it will be appreciated that R¹ may be other straight or branched chain alkyl substituents.

Compounds of formula (B) may be produced via known methods (see for example A. C. Pinto, R. V. Abdala, P. R. R. Costa, Tetrahedron Asymmetry

(2000) 1 1 , 4239; E. Jaeger and J. H. Biel, J. Org. Chem., (1965) 30, 740; M. N. Deshmukh, K. K. Gangakhedkar and U. S. Kumar, Synthetic Communications (1996) 26, 1657).

As used herein, the structural formulae showing the "wedge" notation, e.g.:

/ % are intended to represent pure enantiomeric forms. The structural formulae showing the "rectangular" notation e.g.:

are intended to represent racemic mixtures.

EXAMPLES

The invention is further described with reference to the following examples. It is to be appreciated that the invention is not limited by these examples. General Chemical Methods: Melting points were measured on a Reichert hot stage microscope and are uncorrected. Optical rotations were determined with a Perkin Elmer 241 polarimeter and are in units of 10^"1deg cm² g^"1 (conventionally °). TLC was performed on glass or aluminium backed silica gel 60 F254 (Merck) with detection by UV absorption and/or by heating after dipping in (NH₄)₆Mo₇O₂₄-6H₂O (5 g) and Ce(S0₄)₂ (100 mg) in 5% aq. H₂S0₄ (100 mL) solution or in a solution of l₂ (200 mg) and Kl (7 g) in 10% aq. H₂S0₄

(100 mL). Chromatography (flash column) was performed on Scharlau or Merck silica gel 60 (40-60 μm). Chromatography solvents were distilled prior to use. Anhydrous solvents were those commercially available. Organic solutions were dried over MgSO₄ and evaporated under reduced pressure. All air sensitive reactions were performed under argon. NMR spectra were recorded on a Bruker AC300E spectrometer at 300 MHz (¹H ) for solutions in CDCI₃, CD₃OD (internal Me₄Si, δ 0) or D₂O or at 75.5 MHz (¹³C) for solutions in CDCI₃ (centre line δ 77.0), or CD₃OD(centre line δ 49.0). Assignments of ¹H and ¹³C resonances were based on 2D (¹H-¹H DQF-COSY, ¹H-¹³C HSQC) and DEPT experiments. The ¹³C spectra gave unambiguous data on the numbers of protons bonded to each carbon atom; these are expressed as s, d, t and q being the multiplicities expected in C,H undecoupled spectra. High- resolution MS determinations were performed on a VG-7070 high resolution mass spectrometer. Example 1

Trans-(±)-1-N-benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester

(1). This compound was prepared by the method described by E. Jaeger and J.H.

Biel, J. Org. Chem., 1964, 30, 740, but used ethyl Λ/-benzyl-Λ/-(2- carbethoxyethyl)glycinate as prepared by A.C. Pinto, R.V. Abdala and P.R.R.

Costa, Tetrahedron: Asymm. , 2000, 11 , 4239 and also used the Dieckmann cyclization conditions described by M.N. Deshmukh, K.K. Gangakhedkar and U.S. Kumar, Synth. Commun., 1996, 26, 1657. It was purified by chromatography (eluant ethyl acetate-hexanes 1 :2 v/v →1 :1 v/v →ethyl acetate) and crystallized at -20 °C to a colourless solid.

Mp 52-53 °C (colourless needles, 40-60 petrol, -20 °C).

¹H NMR (CDCI₃) δ 7.37-7.22 (m, 5 H), 4.53-4.49 (m, 1 H, H-4), 4.16 (q, 2 H, J 7.1 Hz, CHaCHa), 3.64 (s, 2 H, PhChb), 3.12 (t, 1 H, J 9.0 Hz, H-2), 2.95 (dt, 1

H, J 7.5, 3.3 Hz, H-3), 2.76 (dd, 1 H, J 10.0, 2.8 Hz, H-5), 2.65 (dd, 1 H, J

10.0, 5.5 Hz, H-5'), 2.55 (dd, 1 H, J 9.4, 7.4 Hz, H-2'), 2.32 (br. s, 1 H, OH),

1.26 (t, 3 H, J 7.1 Hz, CtUCHa).

¹³C NMR (CDCI₃) δ 173.3 (s), 138.2 (s), 128.8 (d), 128.3 (d), 127.1 (d), 74.1 (d), 61.9 (t), 60.8 (t), 59.7 (t), 55.3 (t), 53.1 (d), 14.2 (q).

Example 2

Trans-(±)-1-N-benzyl-4-acetoxypyrrolidine-3-carboxylic acid ethyl ester (5).

7raπs-(±)-1-Λ/-benzyl-4-hyroxypyrrolidine-3-carboxylic acid ethyl ester from example 1 (1 , 100 mg, 0.4 mmol) was dissolved in a mixture of pyridine (4 mL) and acetic anhydride (2 mL) and left at ambient temperature overnight. The solvent was evaporated and the resulting oil dissolved in ethyl acetate and washed with sat. NaHCO₃, dried and evaporated. The residue was chromatographed (eluant ethyl acetate-hexanes 15:85 v/v) to afford trans-(±)- 1 -Λ/-benzyl-4-acetoxypyrrolidine-3-carboxylic acid ethyl ester (5) as a colourless oil (1 1 1 mg, 95%). It was stored at -20 °C. ¹H NMR (CDCI₃) δ 7.38-7.22 (m, 5 H), 5.42-5.38 (m, 1 H, H-4), 4.16 (q, 2 H, J 7.1 Hz, CH₂CH₃), 3.65 (d, 1 H, J 12.9 Hz, PhCHH), 3.59 (d 1 H, J 12.9 Hz, PhCHH), 3.15 (t, 1 H, J 8.5 Hz, H-2), 3.06 (dt, 1 H, J 8.0, 3.9 Hz, H-3), 2.87- 2.74 (m, 2 H, H-5, H-5'), 2.50 (br. t, 1 H, J ~ 8.3 Hz, H-2'), 2.04 (s, 3 H, COCH₃), 1.25 (t, 3 H, J 7.1 Hz, CH₂CH₃).

¹³C NMR (CDCI3) δ 172.3 (s), 170.5 (s), 138.0 (s), 128.7 (d), 128.3 (d), 127.2 (d), 76.0 (d, C-4), 61.0 (t, CH₂CH₃), 59.6 (t, PhCH₂ or C-5), 59.5 (t, PhCH₂ or

C-5), 56.0 (t, C-2), 50.1 (d, C-3), 21.0 (q, COCH₃), 14.1 (q, CHzCHs). +ve FABMS: m/z Calcd. for C₁₆H₂₂NO₄ (M+H)⁺ 292.154883. Found: 292.156263.

Example 3

Trans-(±)-1-N-benzyl-3-hydroxy-4-hydroxymethylpyrrolidine (7).

Ttat?s-(±)-1-Λ/-benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester from example 1 (1 , 500 mg, 2.01 mmol) was dissolved in a mixture of dry Et₂O (10 mL) and dry THF (5 mL) and cooled in an ice bath. Lithium aluminium hydride

(4.2 mL, 4.2 mmol, 1 M) was added and the mixture warmed to ambient temperature and stirred for 1 hr. The reaction mixture was cooled in an ice bath, quenched by the dropwise addition of water and extracted with ethyl acetate. The organic extract was washed with sat NaHCO₃, dried and evaporated to a residue that was chromatographed (eluant CH₂CI₂-MeOH- cNH₃ 95:5:0.5→90: 10:0.5 v/v) to give tra/7S-(±)-1-Λ/-benzyl-3-hydroxy-4- hydroxymethylpyrrolidine (7) as a colourless gum (364 mg, 88%). ¹H NMR (CD₃OD) δ 7.42-7.20 (m, 5 H), 4.04-3.95 (m, 1 H, H-3), 3.68-3.47 (m, 4 H, PhCJ±, & CϋO), 2.89 (br. t, 1 H, -8.8 Hz, H-5), 2.72 (dd, 1 H, J 10.0, 6.3 Hz, H-2), 2.55 (dd, 1 H, J 10.0, 4.1 Hz, H-2'), 2.34 (dd, 1 H, J 9.6, 6.6 Hz,

H-5'), 2.23-2.12 (m, 1 H, H-4).

¹³C NMR (CD₃OD) δ 139.4 (s), 130.2 (d), 129.3 (d), 128.3 (d), 74.1 (d, C-3), 64.2 (t, PhCH₂ or CH₂O), 63.1 (t, C-2), 61.5 (t, PhCH₂ or CH₂O), 57.3 (t, C-5), 51.2 (d, C-4). +ve FABMS: m/z Calcd. for C₁₂H₁₈NO₂ (M+H)⁺ 208.133754. Found:

208.134589. Example 4

(3S,4R)-1-N-Benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester (2) and (3R,4S)-4-acetoxy-1-N-benzylpyrrolidine-3-carboxylic acid ethyl ester (3).

Vinyl acetate (0.22 mL, 2.4 mmol) and Novozyme® 435 lipase from Candida antarctica (138 mg, product No L4777, 2002-2003 Sigma catalogue, batch 083K0739) were added sequentially to a solution of trans-(±)-1-Λ/-benzyl-4- hydroxypyrrolidine-3-carboxylic acid ethyl ester from example 1 (1, 200 mg, 0.8 mmol) in terf-butyl methyl ether (9.5 mL). The mixture was stirred at 25-30

°C for 16 h then diluted with CHCI₃ (10 mL) and filtered through Celite. After evaporating the solvent at reduced pressure, the residue was dissolved in CHCI₃, washed with sat. NaHCO₃, dried and evaporated. ¹H NMR (CDCI₃) analysis indicated a 1 :0.99 ratio of (3f?,4S)-4-acetoxy-1-Λ/-benzylpyrrolidine-3- carboxylic acid ethyl ester (3):(3S,4f?)-1-Λ/-benzyl-4-hydroxypyrrolidine-3- carboxylic acid ethyl ester (2). The residue was chromatographed (eluant EtOAc-hexanes 6:4 v/v) to give first (3R,4S)-4-acetoxy-1-Λ/-benzylpyrrolidine- 3-carboxylic acid ethyl ester (3) as a colourless gum (115 mg, 99%) that was stored at -20 °C. The ¹H NMR was identical to that for compound 5 in example 2.

[a]n -41° (c 0.36, CHCI3).

Further elution of the column with EtOAc gave (3S,4ft)-1-Λ/-benzyl-4- hydroxypyrrolidine-3-carboxylic acid ethyl ester (2) also as a colourless gum (93 mg, 93%). The ¹H NMR was identical to that for compound 1 in example 1.

[σjo +17 ° (c 0.295, CHCI3).

Example 5

(3S,4R)-1-N-Benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester (2) and (3R,4S)-4-acetoxy-1-N-benzylpyrrolidine-3-carboxylic acid ethyl ester (3). Vinyl acetate (6.66 mL, 72.21 mmol) and Novozyme® 435 lipase from Candida antarctica (4.2 g, Novozymes Australia Pty. Ltd, batch LC200207) were added sequentially to a solution of traπs-(±)-1-Λ/-benzyl-4- hydroxypyrrolidine-3-carboxylic acid ethyl ester from example 1 (1 , 6.00g, 24.1 mmol) in terf-butyl methyl ether (200 mL). The mixture was stirred at 40

°C for 2.5 h then filtered through Celite. The solids were washed with a little ethyl acetate and the combined filtrates were washed with sat. NaHCO₃, dried and evaporated. ¹H NMR (CDCI₃) analysis indicated a 1 :0.99 ratio of (3 4S)- 4-acetoxy-1-Λ/-benzylpyrrolidine-3-carboxylic acid ethyl ester (3):(3S,4 )-1 -Λ/- benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester (2). The residue was chromatographed (eluant EtOAc-hexanes 6:4 v/v) to give first (3ft,4S)-4- acetoxy-1 -Λ/-benzylpyrrolidine-3-carboxylic acid ethyl ester (3) as a colourless gum (3.44 g, 98%) that was stored at -20 °C. The ¹H NMR was identical to that for compound 5 in example 2.

[α] " -42° (c 0.74, CHCI₃).

Further elution of the column with EtOAc gave (3S,4 ?)-1 -Λ/-benzyl-4- hydroxypyrrolidine-3-carboxylic acid ethyl ester (2) also as a colourless gum which crystallized at -20 °C (2.53 g, 84%). The ¹H NMR was identical to that for compound 1 in example 1.

[α] o +17 ° (c 0.71 , CHCI₃).

MP 51 -52 °C.

Example 6

(3R,4R)-1-N-Benzyl-3-hydroxy-4-hydroxymethylpyrrolidine (4).

(3S,4f?)-1 -Λ/-Benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester from example 5 (2, 2.53 g, 10.15 mmol) was dissolved in THF (20 mL) and Et₂O (40 mL) and treated with lithium aluminium hydride (20.3 mL, 20.3 mmol, 1 M in ether) as described in example 3 to afford (3 4f?)-1 -Λ/-benzyl-3-hydroxy-4- hydroxymethylpyrrolidine (4) as a colourless gum (1.54 g, 73%). The ¹H NMR was identical to compound 7 from example 3.

[α] " +33° (c 0.745, MeOH). Example 7

(3R,4R)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride. [(+)-A, HCI] (3S,4R)-1 -Λ/-Benzyl-4-hydroxypyrrolidine-3-carboxylic acid ethyl ester from example 4 (2, 93 mg, 0.37 mmol) was dissolved in Et₂O (5 mL). A solution of lithium aluminium hydride in Et₂O (0.78 mL, 0.78 mmol, 1 M) was added and the mixture stirred at ambient temperature for 1 h. The excess hydride was quenched with H₂O (0.1 mL). Magnesium sulfate was added, the mixture filtered through Celite and the solvent evaporated. The residue was chromatographed (eluant CHCI₃-MeOH-Et₃N, 95:5:0.5 → 8:2:0.5 v/v) to give 52 mg of crude (3r?,4f?)-1 -Λ/-benzyl-3-hydroxy-4-hydroxymethylpyrrolidine (4) which was dissolved in MeOH-98% HCOOH (9:1 v/v, 8 mL) and 10% Pd-C (80 mg) added. The mixture was heated under reflux for 30 mins, filtered through Celite and the solvent evaporated. Chromatography (eluant CH₂CI₂-

MeOH-cNH₃-H₂0, 4:3:0.5:0.5 v/v) gave (3K,4R)-3-hydroxy-4- hydroxymethylpyrrolidine ((+)-A) as a colourless gum (16 mg, 37%) which began to darken on standing. The ¹H NMR (CD₃OD) was in agreement with the literature citing by V.V. Filichev, M. Brandt and E. Pedersen, Carbohydr. Res., 2001 , 333, 1 15.

The latter product was dissolved in MeOH (2 mL), 5% HCI (1 mL) added and the solvent evaporated to give 21 mg of (3f?,4ft)-3-hydroxy-4- hydroxymethylpyrrolidine hydrochloride ((+)-A, HCI) as a colourless gum. The ¹H NMR (D₂O) was in agreement with the data in S. Karlsson and H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977.

[α] _D +19 ° (c 1.05, MeOH). Lit. [α] +19.0 ° (c 1.0, MeOH) (S. Karlsson

and H.-E. Hogberg, Tetrahedron: Asymm., 2001 , 12, 1977).

Example 8

(3S,4S)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride [(-)-E, HCI]

(3f?,4S)-4-Acetoxy-1-Λ/-benzylpyrrolidine-3-carboxylic acid ethyl ester from example 4 (3, 1 13 mg, 0.39 mmol) was dissolved in Et₂O (6 mL). A solution of lithium aluminium hydride in Et₂O (1 .6 mL, 1.6 mmol, 1 M) was added and the mixture stirred at ambient temperature for 1 h. The excess hydride was quenched with H₂O (0.18 mL). Magnesium sulfate was added, the mixture filtered through Celite and the solvent evaporated to give 55 mg of (3S,4S)-1 - Λ/-benzyl-3-hydroxy-4-hydroxymethylpyrrolidine (6). Without further purification the latter was dissolved in MeOH-98% HCOOH (9:1 v/v, 8 mL).

10% Pd-C (80 mg) was added and the mixture was heated under reflux for 30 mins, filtered through Celite and evaporated. Chromatography (eluant CH₂CI₂- MeOH-cNH₃-H₂O, 4:3:0.5:0.5 v/v) gave (3S,4S)-3-hydroxy-4- hydroxymethylpyrrolidine ((-)-E) as a colourless gum (18 mg, 39%) which began to darken on standing. The H NMR was identical to that described in example 7 for (3 4f?)-3-hydroxy-4-hydroxymethylpyrrolidine ((+)-A).

The latter ((-)-E) product was dissolved in 5% HCI (3 mL) and evaporated to give 21 mg of (3S,4S)-3-hydroxy-4-hydroxymethylpyrrolidine hydrochloride ((-)-E, HCI) as a colourless gum. The ¹H NMR (D₂O) was in agreement with the literature data (S. Karlsson and H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977).

[α] % -19 ° (c 1.05, MeOH). Lit.[α] -18.7 ° (c 1.2, MeOH) (S. Karlsson

and H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977).

Example 9

(3S,4S)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride [(-) -E, HCI].

(3f?,4S)-4-Acetoxy-1-/V-benzylpyrrolidine-3-carboxylic acid ethyl ester from example 5 (3, 1.2 g, 4.12 mmol) was dissolved in Et₂O (35 mL) and cooled in an ice bath. Lithium aluminium hydride (16.9 mL, 16.9 mmol, 1 M) was added and the mixture stirred 1 h at ambient temperature. After cooling in an ice bath water (3 mL) was added then the mixture extracted with ethyl acetate. The organic extract was washed with sat. NaHCO₃, dried and the solvent evaporated to give crude (3S,4S)-1-Λ/-benzyl-3-hydroxy-4- hydroxymethylpyrrolidine (6) as a gum (880 mg). The gum was dissolved in a mixture of MeOH-98% HCO₂H (4:1 v/v 50 mL), 10% Pd-C (500 mg) added and heated under reflux for 45 min. The mixture was cooled and filtered through Celite and the solvent evaporated. The residue was chromatographed (eluant CH₂Cl₂-MeOH-H₂O-cNH₃ 4:3:0.5:0.5 v/v) to give (3S,4S)-3-hydroxy-4- hydroxymethylpyrrolidine ((-)-E) as a colourless gum (345 mg). The ¹H NMR was identical to that described in example 7 for the (3R,4f?)-enantiomer. The latter ((-)-E) was dissolved in H₂O (10 mL) and MeOH (20 mL) and acidified with cHCI (0.2 mL) then evaporated to dryness, affording the hydrochloride as a colourless gum (449 mg). The ¹H NMR (D₂O) was in agreement with data reported in S. Karlsson and H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977.

[α] ²,¹ -18° (c 0.5, MeOH). Lit [α] ²,⁵ -18.7° (c 1.2, MeOH) (S. Karlsson and H.-

E. Hόgberg, Tetrahedron: Asymm., 2001, 12, 1977).

Example 10

Trans-(±)-1-N-benzyl-4-benzyloxypyrrolidine-3-carboxylic acid ethyl ester (S).

Λ/-(Methoxymethyl)- /-(trimethylsilylmethyl)-benzylamine (commercially available from Aldrich) (10.24 mL, 40.0 mmol) was added dropwise over 40 mins to a solution of 3-benzyloxyacrylic acid ethyl ester [6.35 g, 30.8 mmol, prepared as an ~ 12:1 mixture of E:Z isomers in 90% yield using the method described for the methyl ester in R. Hirsenkom and R.R. Schmidt, Liebigs Ann. Chem., 1990, 883. For the major E-isomer ¹H NMR (CDCI₃) δ 7.67 (d, 1 H, J 12.6 Hz), 7.41-7.32 (m, 5 H), 5.31 (d, 1 H, J 12.6 Hz), 4.90 (s, 2 H), 4.17 (q, 2 H, J 7.1 Hz), 1.27 (t, 3 H, J 7.1 Hz). The ¹³C NMR assignment for the major E-isomer was in agreement with data reported in S. Blaya, R. Chinchilla and C. Najera, Tetrahedron, 1995, 51 , 3617. Bp 110-115 °C/0.02 mmHg] in 5 mM TFA-CH₂CI₂ (150 mL) and the mixture stirred at ambient temperature for 1.5 hr. The mixture was washed with sat. NaHC0₃ solution, dried and the solvent evaporated. The residue was chromatographed (eluant toluene-ethyl acetate 97:3→94:6 v/v) to give trat7S-(±)-1-Λ/-benzyl-4-benzyloxypyrrolidine-3- carboxylic acid ethyl ester (8) as a colourless oil (7.41 g, 70%) which darkened slightly on standing. ¹H NMR (CDCI₃) δ 7.35-7.21 (m, 10 H), 4.57 (d, 1 H, J 12.0 Hz, OCHHPh), 4.50 (d, 1 H, J 12.0 Hz, OCHHPh), 4.38-4.35 (m, 1 H, H-4), 4.15 (q, 2 H, J 7.1 Hz, CH₂CH₃), 3.65 (d, 1 H, J 13.0 Hz, NCHHPh), 3.58 (d, 1 H, J 13.0 Hz, NCHHPh), 3.09-3.03 (m, 1 H, H-3), 2.98 (t, 1 H, J 8.6 Hz), 2.80-2.63 (m, 3 H), 1.25 (t, 3 H, 7.1 Hz, CH₂CH₃).

¹³C NMR (CDCI₃) δ 173.4 (s), 138.4 (s), 138.1 (s), 128.7 (d), 128.3 (d), 128.2 (d), 127.7 (d), 127.6 (d), 127.0 (d), 80.8 (d, C-4), 71.4 (t, OCH₂Ph), 60.8 (t, CH₂CH₃), 59.8 (2t, NCH₂Ph & C-5 or C-2), 55.7 (t, C-5 or C-2), 50.6 (d, C-3), 14.2 (q, CH₂CH₃). +.ve FABMS: m/z Calcd. for C₂iH₂₆NO₃ (M+H)⁺ 340.191269. Found:

340.190784.

Example 11

Trans-(±)-4-benzyloxypyrrolidine-1-N-3-dicarboxylic acid-1-tert-bυtyl ester-3-ethyl ester (9) and trans-(±)-4-hydroxypyrrolidine-1-N-3- dicarboxylic acid-1-tert-butyl ester-3-ethyl ester (10).

To a solution of frans-(±)-1 -Λ/-benzyl-4-benzyloxypyrrolidine-3-carboxylic acid ethyl ester from example 10 (8, 4.90 g, 14.4 mmol) and di-terf-butyl dicarbonate (3.15 g, 14.4 mmol) in ethanol (100 mL) was added 10% Pd-C

(500 mg) then hydrogen was added from a balloon. After stirring for 20 h the mixture was filtered through Celite. A small aliquot was removed (consisted mainly of frans-(±)-4-benzyloxypyrrolidine-1-Λ/-3-dicarboxylic acid-1-terf-butyi ester-3-ethyl ester (9) by ¹H NMR) and chromatographed (eluant ethyl acetate-hexanes 1 :9 v/v) to give trat7S-(±)-4-benzyloxypyrrolidine-1 -Λ/-3- dicarboxylic acid-1 -terf-butyl ester-3-ethyl ester (9) as a colourless gum. ¹H NMR (CDCI₃) δ 7.48-7.27 (m, 5 H), 4.56 (s, 2 H, PhCFb) 4.38-4.29 (br. m, 1 H, H-4), 4.17 (q, 2 H, 7.1 Hz, CHsCHa), 3.77-3.53 (br.m, 3 H, H-5, H-2, H- 2'), 3.53-3.32 (br.m, 1 H, H-5'), 3.18-3.07 (br.m, 1 H, H-3), 1.45 (s, 9 H, C(CH₃)₃, 1.26 (t, 3 H, J 7.1 Hz, CH₂CH₃).

¹³C NMR (CDCI₃) δ 171.9 (s), 154.3 (s), 137.6 (s), 128.5 (d), 127.9 (d), 127.7 (d), 79.8 & 79.0 (d, C-4), 79.7 (s, C(CH₃)₃), 71.6 (t, PhCH₂), 61.2 (t, CH₂CH₃), 50.9 & 50.1 (t, C-5), 49.4 & 48.4 (d, C-3), 46.7 (t, C-2), 28.4 (q, C(CH₃)₃), 14.1 (q, CH₂CH₃). +ve EIMS: m/z Calcd. for C₁₉H₂₇N0₅ (M)⁺ 349.18892. Found: 349.18865. Fresh 10% Pd-C (500 mg) was added to the bulk of the ethanol solution from above and the mixture treated with hydrogen for a further 16 h. After filtering through Celite, the solvent was evaporated and the residue chromatographed (eluant ethyl acetate-hexanes 4:6 v/v) to afford frat?s-(±)-4-hydroxypyrrolidine- 1 -Λ/-3-dicarboxylic acid-1-te/t-butyl ester-3-ethyl ester (10) as a colourless oil

(3.66 g, 98%).

¹H NMR (CDCI₃) δ 4.59-4.51 (m, 1 H, after D₂O exchange became a q at δ 4.54, J 5.9 Hz), 4.19 (q, 2 H, J, 7.1 Hz), 3.83-3.65 (br.m, 2 H), 3.56 (dd, 1 H, J 11.0, 7.1 Hz), 3.33-3.22 (br.s, 1 H), 3.05-2.92 (br.s, 1 H), 2.35 (d, 1 H, J 3.9 Hz, exchanged to D₂0), 1.46 (s, 9 H), 1.28 (t, 3 H, J 7.1 Hz).

¹³C NMR (CDCI₃) δ 171.9 (s), 154.4 (s), 79.8 (s), 72.5 & 71.9 (d), 61.2 (t), 52.5

& 52.1 (t), 51.1 & 50.5 (d), 46.3 & 46.0 (t), 28.4 (q), 14.1 (q).

+ve FABMS: m/z Calcd. for C₁₂H₂₂NO₅ (M+H)⁺ 260.149798. Found:

260.149259.

Example 12

Trans-(±)-4-hydroxypyrrolidine-1-N-3-dicarboxylic acid-1-tert-butyl ester- 3-ethyl ester (10). rrat7S-(±)-1 -Λ/-benzyl-4-hyroxypyrrolidine-3-carboxylic acid ethyl ester from example 1 (1 , 1.92 g, 7.70 mmol) and di-tert-butyl dicarbonate (1.75 g, 8.00 mmol) were dissolved in EtOH (30 mL), 10% Pd-C was added and the mixture hydrogenolysed for 16 h with stirring. Purification as in example 1 1 gave trans-(±)-4-hydroxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -te/t-butyl ester-3-ethyl ester (10) as a colourless oil (1.92 g, 92%).

Example 13

Trans-(±)-4-acetoxypyrrolidine-1-N-3-dicarboxylic acid-1 -tert-butyl ester- 3-ethyl ester (11).

7^~rat7s-(±)-4-hydroxypyrrolidine-1 -Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3- ethyl ester from examples 11 or 12 (10, 100 mg, 0.39 mmol) was dissolved in a mixture of pyridine (4 mL) and acetic anhydride (2 mL) and left to stand at ambient temperature overnight. The solvent was evaporated and the residue dissolved in ethyl acetate, washed with sat. NaHCO₃, brine, dried and evaporated. The residue was chromatographed (eluant ethyl acetate-hexanes

2:8 v/v) to give traπs-(±)-4-acetoxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -tert- butyl ester-3-ethyl ester (11) as a colourless gum (118 mg, 100%).

¹H NMR (CDCI₃) δ 5.45 (br.s, 1 H), 4.19 (q, 2 H, 7.1 Hz), 3.84-3.61 (m, 3 H),

3.51-3.30 (m, 1 H), 3.14-3.04 (m, 1 H), 2.07 (s, 3 H), 1.46 (s, 9 H), 1.27 (t, 3

H, 7.1 Hz).

¹³C NMR (CDCI₃) δ 170.9 (s), 170.1 (s), 154.1 (s), 79.9 (s), 74.6 & 73.8 (d),

61.4 (t), 51.0 & 50.5 (t), 48.9 & 47.9 (d), 46.7 (t), 28.4 (q), 20.9 (q), 14.0 (q).

+ve CIMS: m/z Calcd. for C₁₄H₂₄NO₆ (M+H)⁺ 302.16036. Found: 302.16041.

Example 14

(3S,4R)-4-Hydroxypyrrolidine-1-N-3-dicarboxylic acid-1 -tert-butyl ester-3- ethyl ester (12) and (3R,4S)-4-acetoxypyrrolidine-1-N-3-dicarboxylic acid- 1 -tert-butyl ester-3-ethyl ester (13).

Novozyme® 435 lipase from Candida antarctica (3 g, Novozymes Australia Pty. Ltd, batch LC200207) was added to a solution of trans-(±)-4- hydroxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester from examples 11 and 12 (10, 4.3 g, 16.58 mmol) and vinyl acetate (4.6 mL, 50.17 mmol) in tert-butyl methyl ether (180 mL) and the mixture stirred at 30 °C for

20 h. The solids were removed by filtration through Celite and washed with a little ethyl acetate. The combined filtrates were washed with sat. NaHCO₃, dried and evaporated to a pale yellow oil (4.4 g). ¹H NMR (CDCI₃) analysis indicated a 1:0.96 ratio of (3f?,4S)-4-acetoxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester (13):(3S,4R)-4-hydroxypyrrolidine-1-Λ/-3- dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester (12). The residue was chromatographed (eluant ethyl acetate-hexanes 3:7 v/v) to afford (3f?,4S)-4- acetoxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester (13) as a pale yellow oil (2.49 g, 99%). The ¹H NMR was identical to that described for compound 11 in example 13.

[α]²,¹ -19° (c 0.63, CHCI₃).

The column was further eluted with ethyl acetate-hexanes (6:4 v/v) to give (3S,4R)-4-hydroxypyrrolidine-1 -Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester (12) as a colourless oil (1.83 g, 85%). The ¹H NMR was identical to that described for compound 10 in example 11.

[α]²,¹ +19° (c 0.53, CHCI₃).

The equal and opposite rotations for 12 and 13 appear to be coincidental.

Example 15

(3R,4R)-3-Hydroxy-4-hydroxymethylpyrrolidin-1-N-carboxylic acid tert- butyl ester (14). Lithium borohydride (228 mg, 10.47 mmol) was added to a solution of

(3S,4f?)-4-hydroxypyrrolidine-1 - /-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester from example 14 (12, 1.81 g, 6.98 mmol) in anhydrous Et₂O (27 mL) and methanol (0.49 mL, 12.22 mmol) and the mixture heated under reflux with stirring for 30 mins (using the general method of K. Soai and A. Ookawa, J. Org. Chem., 1986, 51 , 4000). After cooling, methanol (10 mL) was added and the solvent evaporated. The residue was dissolved in ethyl acetate and washed with sat NaHC0₃, dried and evaporated to a colourless gum (1.48 g). It was chromatographed (eluant ethyl acetate-methanol 19:1 v/v) to afford (3f?,4f?)-3-hydroxy-4-hydroxymethylpyrrolidin-1 -Λ/-carboxylic acid tert-butyl ester (14) as a colourless gum (1.36 g, 90%). The ¹H and ¹³C NMR were in agreement with that reported by G.B. Evans, R.H. Furneaux, A. Lewandocwicz, V.L. Schramm and P.C. Tyler, J. Med. Chem., 2003, 46, 5271.

[α]²,¹ +15.5° (c 1.09, MeOH). A sample prepared as in G.B. Evans, R.H. Furneaux, A. Lewandocwicz, V.L. Schramm and P.C. Tyler, J. Med. Chem.,

2003, 46, 5271 , ultimately derived from D-xylose, had an [α]^¹ +16° (c 0.795,

MeOH). Example 16

(3R,4R)-3-Hydroxy-4-hydroxymethylpyrrolidin-1-N-carboxylic acid tert- butyl ester (14). 10% Pd-C (300 mg) was added to a stirred solution of (3ft,4R)-1-Λ/-benzyl-3- hydroxy-4-hydroxymethylpyrrolidine from example 6 (4, 1.49 g, 7.19 mmol) and di-tert-butyl dicarbonate (1.63 g, 7.47 mmol) in MeOH (30 mL) and hydrogen added from a balloon for 24 h. The mixture was filtered through Celite, evaporated and the residue chromatographed (eluant ethyl acetate- methanol19:1 , v/v) to afford (3R,4R)-3-hydroxy-4-hydroxymethylpyrrolidin-1-

Λ/-carboxylic acid tert-butyl ester (14) as a colourless gum (1.56 g, 100%).

[α]o +16° (c 1.09, MeOH). A sample prepared as in G.B. Evans, R.H.

Furneaux, A. Lewandocwicz, V.L. Schramm and P.C. Tyler, J. Med. Chem.,

2003, 46, 5271 , ultimately derived from D-xylose, had [α]^¹ +16° (c 0.795, MeOH).

Example 17

(3R,4R)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride. [(+)-A, HCI]

A 25 mg portion of (3fi,4R)-3-hydroxy-4-hydroxymethylpyrrolidin-1-Λ/- carboxylic acid tert-butyl ester (14) from example 15 was dissolved in methanol (2 mL), 5% HCI (1 mL) added and the solution left to stand at ambient temperature overnight. Evaporation of the solvent left (3R,4R)-3- hydroxy-4-hydroxymethylpyrrolidine hydrochloride [(+)-A, HCI] as a colourless gum. The ¹H NMR (D₂0) was in agreement with that reported in S. Karlsson and H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977.

[a] ²,¹ +18° (c 0.81 , MeOH). Lit [α] * +19.0° (c 1.0, MeOH) (S. Karlsson and

H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977). Example 18

(3R,4R)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride. [(+)-A, HCI] A 25 mg portion of (3R,4R)-3-hydroxy-4-hydroxymethylpyrrolidin-1-/V- carboxylic acid tert-butyl ester (14) from example 16 was dissolved in methanol (2 mL), 5% HCI (1 mL) added and the solution left to stand at ambient temperature overnight. Evaporation of the solvent left (3R,4R)-3- hydroxy-4-hydroxymethylpyrrolidine hydrochloride [(+)-A, HCI] as a colourless gum. The ¹H NMR was in agreement with that reported in S. Karlsson and H.-

E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977.

[α] ,¹ +19° (c 0.795, MeOH). Lit [α] ²,⁵ +19.0° (c 1.0, MeOH) (S. Karlsson and

H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977).

Example 19

(3S,4S)-3-Hydroxy-4-hydroxymethylpyrrolidin-1-N-carboxylic acid tert- butyl ester (15).

Lithium borohydride (87 mg, 3.99 mmol) was added to a solution of (3R,4S)-4- acetoxypyrrolidine-1-Λ/-3-dicarboxylic acid-1 -tert-butyl ester-3-ethyl ester from example 14 (13, 400 mg, 1.33 mmol) in anhydrous Et₂0 (6 mL) and methanol

(0.19 mL, 4.66 mmol) and the mixture heated under reflux with stirring for 30 mins (using the general method of K. Soai and A. Ookawa, J. Org. Chem.,

1986, 51 , 4000). After cooling, methanol (3 mL) was added and the solvent evaporated. The residue was dissolved in ethyl acetate and washed with sat

NaHC0₃, dried and evaporated to a colourless gum (300 mg). It was chromatographed (eluant ethyl acetate-methanol 19:1 v/v) to afford (3S,4S)-3- hydroxy-4-hydroxymethylpyrrolidin-1-Λ/-carboxylic acid tert-butyl ester as a colourless gum (205 mg, 71%). The ¹H and ¹³C NMR were in agreement with the data for the (3R,4R)-enantiomer in G.B. Evans, R.H. Furneaux, A.

Lewandocwicz, V.L. Schramm and P.C. Tyler, J. Med. Chem., 2003, 46,

5271.

[α]²,¹ -14° (c 0.93, MeOH). A sample of the (3R,4R) enantiomer prepared as in G.B. Evans, R.H. Furneaux, A. Lewandocwicz, V.L. Schramm and P.C. Tyler, J. Med. Chem., 2003, 46, 5271 , ultimately derived from D-xylose, had

[α] I 2n1 +16° (c 0.795, MeOH).

Example 20

(3S,4S)-3-Hydroxy-4-hydroxymethylpyrrolidine hydrochloride. [(-)-E, HCI]

A portion of (3S,4S)-3-hydroxy-4-hydroxymethylpyrrolidin-1-Λ/-carboxylic acid tert-butyl ester (15) from example 19 (25 mg) was dissolved in MeOH (2 mL), cHCI (1 mL) added and the solvent evaporated to give (3S,4S)-3-hydroxy-4- hydroxymethylpyrrolidine hydrochloride [(-)-E, HCI] as a colourless gum. The

¹H NMR (D₂0) was in agreement with that reported by S. Karlsson and H.-E. Hόgberg, Tetrahedron: Asymm., 2001, 12, 1977.

[α]²¹ -15° (c , 0.855 MeOH). Lit [α]² -18.7° (c 1.2, MeOH) (S. Karlsson and

H.-E. Hόgberg, Tetrahedron: Asymm., 2001 , 12, 1977).

Although the invention has been described by way of example, it should be appreciated that variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.

INDUSTRIAL APPLICABILITY

This invention relates to a method for preparing 3-hydroxy-4- hydroxymethylpyrrolidine compounds. These compounds are key intermediates for the synthesis of certain potent inhibitor compounds such as the purine nucleoside phosphorylase inhibitors disclosed in WO 2004/018496, for example.

Claims

1. A process for preparing a compound of formula (A) or a compound of formula (E), or salts thereof

(A) (E) including the steps of (a), (b) and (c): where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-trat?s-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

(B) where R¹ is a straight or branched chain alkyl group; and R² is a protecting group; to give either a mixture of a compound of formula (C) and a compound of formula (D)

(C) (D)

where R¹ and R² are as defined above; and R³ is acyl;

or a mixture of a compound of formula (C), and a compound of formula (D')

where R¹ and R² are as defined above; and R³ is acyl;

where the enzyme-catalysed enantioselective esterification is carried out using either .

(1) an enzyme capable of producing an enantiomeric excess of compound (C); or

(2) an enzyme capable of producing an enantiomeric excess of compound (D');

step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D') to the compound of formula (A); or transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E).

2. A process according to claim 1 , where the enzyme-catalysed enantioselective esterification in step (a) gives a mixture of compounds of formulae (C) and (D), and the enantiomeric excess of compound (C) is at least about 80%.

3. A process according to claim 2, where the enantiomeric excess is at least about 90%.

4. A process according to claim 1 , where the enzyme-catalysed enantioselective esterification in step (a) gives a mixture of compounds of formulae (C) and (D'), and the enantiomeric excess of compound (D') is at least about 80%.

5. A process according to claim 4, where the enantiomeric excess is at least about 90%.

6. A process according to any one of claims 1 to 5, where the enzyme used in step (a) is an enzyme capable of catalysing the formation of an ester bond.

7. A process according to claim 6 where the enzyme used is a lipase.

8. A process according to claim 7 where the enzyme is lipase from Candida antarctica.

9. A process according to any one of claims 1 to 8 where the transformation of the compound of formula (C) or the compound of formula (D¹) to the compound of formula (A) includes the step of reduction of the ester group of the compound of formula (C); or reduction of both ester groups of the compound of formula (D').

10. A process according to claim 9 which further includes the step of replacement of the R² group with hydrogen to give the compound of formula (A).

1 1. A process according to any one of claims 1 to 8 where the transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E) includes the step of reduction of the ester group of the compound of formula (C); or reduction of both ester groups of the compound of formula (D).

12. A process according to claim 11 which further includes the step of replacement of the R² group with hydrogen to give the compound of formula (E).

13. A process according to claim 9 or claim 11 where the reduction of the ester group or groups is carried out using either LiAIH₄ or LiBH₄.

14. A process according to claim 10 or claim 12 where the replacement of the R² group with hydrogen is carried out either in the presence of HCI or with HCOOH/CH₃OH in the presence of Pd-C.

15. A process according to any one of claims 1 to 14 where the separation of the compound of formula (C) from the compound of formula (D) or the separation of the compound of formula (C) from the compound of formula (D') is effected by chromatography or fractional crystallisation.

16. A process according to any one of claims 1 to 15 where R is a straight or branched chain C C₆ alkyl group.

17. A process according to claim 16 where R¹ is ethyl.

18. A process according to any one of claims 1 to 17 where R³ is COR⁴, where R⁴ is a straight or branched chain Ci-Ce alkyl group.

19. A process according to any one of claims 1 to 18 where R¹ is ethyl and R² is benzyl or f-butoxycarbonyl.

20. A process according to claim 19 where R is ethyl, R² is benzyl and R³ is acetyl.

21. A process according to claim 19 where R¹ is ethyl, R² is t- butoxycarbonyl and R³ is acetyl.

22. A process according to any one of claims 1 to 15 where the enantioselective esterification in step (a) is carried out in the presence of vinyl acetate to give a mixture of compounds of formulae (C) and (D) where R³ in the compound of formula (D) is acetyl; or a mixture of compounds of formulae (C) and (D') where R³ in the compound of formula (D') is acetyl.

23. A process for preparing a compound of formula (A), or a salt thereof

(A) including the steps of (a), (b) and (c): where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-trat7s-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

(B) where R is a straight or branched chain alkyl group; and R² is a protecting group; to give either a mixture of a compound of formula (C) and a compound of formula (D)

(C) (D) where R and R are as defined above; and R³ is acyl; or a mixture of a compound of formula (C), and a compound of formula (D')

where R and R² are as defined above; and R³ is acyl; where the enzyme-catalysed enantioselective esterification is carried out using either (1) an enzyme capable of producing an enantiomeric excess of compound (C); or (2) an enzyme capable of producing an enantiomeric excess of compound (D'); step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D') to the compound of formula (A).

24. A process for preparing a compound of formula (E), or a salt thereof

(E)

including the steps of (a), (b) and (c):

where step (a) is enzyme-catalysed enantioselective esterification of the hydroxyl group of a racemic 3,4-trans-1-N-protected-4- hydroxypyrrolidine-3-carboxylic acid ester compound of formula (B)

where R and R are as defined above; and R³ is acyl,

or a mixture of a compound of formula (C), and a compound of formula (D")

(C) (D¹)

where R¹ and R² are as defined above; and R³ is acyl;

wherein the enzyme-catalysed enantioselective esterification is carried out using either

(1) an enzyme capable of producing an enantiomeric excess of compound (C); or

(2) an enzyme capable of producing an enantiomeric excess of compound (D¹); . - -

step (b) is separation of the compound of formula (C) from the compound of formula (D); or separation of the compound of formula (C) from the compound of formula (D'); and step (c) is transformation of the compound of formula (C) or the compound of formula (D) to the compound of formula (E).

25. A compound of formula (C)

(C) where R¹ is a straight or branched chain alkyl group; and R² is a protecting group.

26. A compound of formula (D)

27. A compound of formula (C)

28. A compound of formula (D¹)

29. A compound according to claim 25 where R¹ is ethyl and R² is benzyl.

30. A compound according to claim 26 where R¹ is ethyl, R² is benzyl and R³ is acetyl.

31. A compound according to claim 25 where R¹ is ethyl and R² is t- butoxycarbonyl.

32. A compound according to claim 26 where R¹ is ethyl, R² is t- butoxycarbonyl and R³ is acetyl.

33. A compound of formula (A) as defined in claim 1 , when prepared by the process of claim 1 or claim 23.

34. A compound of formula (E) as defined in claim 1 , when prepared by the process of claim 1 or claim 24.