A practical synthesis of (3R,4R)-N-tert-butoxycarbonyl-4- hydroxymethylpyrrolidin-3-ol

Article abstract of DOI:10.1039/b708796a

The title compound (+)-5, required for production of transition state analogue inhibitors of enzymes involved in T-cell-dependent disorders, was synthesized in five steps. A 1,3-dipolar cycloaddition of the nitrone formed from formaldehyde and N-benzylhydroxylamine to diethyl maleate gave the racemic cis-isoxazolidine (±)-7. Reduction of the N-O bond of this compound gave pyrrolidone (±)-8 in excellent yield. A very efficient enzymic resolution of this racemic product led to the title enantiomer (+)-5. This route employs only one chromatographic purification. The Royal Society of Chemistry.

Full text of DOI:10.1039/b708796a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A practical synthesis of (3R,4R)-N-tert-butoxycarbonyl-4-
hydroxymethylpyrrolidin-3-ol†
Keith Clinch,a Gary B. Evans,a Richard H. Furneaux,a,b Dirk H. Lenz,a Jennifer M. Mason,*a
Simon P. H. Mee,a Peter C. Tylera and Sarah J. Wilcoxb
Received 11th June 2007, Accepted 3rd July 2007
First published as an Advance Article on the web 20th July 2007
DOI: 10.1039/b708796a
The title compound (+)-5, required for production of transition state analogue inhibitors of enzymes
involved in T-cell-dependent disorders, was synthesized in five steps. A 1,3-dipolar cycloaddition of the
nitrone formed from formaldehyde and N-benzylhydroxylamine to diethyl maleate gave the racemic
cis-isoxazolidine ( )-7. Reduction of the N–O bond of this compound gave pyrrolidone ( )-8 in
excellent yield. A very efficient enzymic resolution of this racemic product led to the title enantiomer
(+)-5. This route employs only one chromatographic purification.
cyanide,10 and also by two routes involving incorporation of
Introduction
a chiral auxiliary and separation of diastereomers.11,12 The key
step of the more enantioselective of these last methods is a 1,3-
dipolar cycloaddition of an azomethine ylide to a 3-benzyloxy-
substituted alkenoylcamphorsultam,12 and this procedure has
been adapted to afford a large scale synthesis.13 Most recently, we
have obtained pyrrolidinol (+)-4 by enantioselective acylation of
racemic ethyl trans-N-benzyl-4-hydroxypyrrolidine-3-carboxylate
catalysed by Novozyme 435 (an immobilized form of lipase B of
Candida antarctica).14 We now report a more efficient synthesis
of pyrrolidine (+)-5 that mostly avoids chromatography and is
suitable for kilogram-scale preparations.
The nucleoside analogues 1–3, ‘DADMe-Immucillins’, are highly
potent transition state analogue inhibitors of N-ribosyl trans-
ferase enzymes. DADMe-Immucillins 1 and 2 inhibit purine
nucleoside phosphorylase (PNP), a therapeutic target for the
control of disorders involving proliferation of T-cells.1,2 Com-
pound 1 is currently in phase I clinical trials for treat-
ment of T-cell-mediated autoimmune diseases.3 Compound 3
inhibits 5ꢀ-methylthioadenosine/S-adenosylhomocysteine nucle-
osidase (MTAN)4 and 5ꢀ-methylthioadenosine phosphorylase
(MTAP).5 MTAN is a bacterial enzyme which has been iden-
tified and validated as a potential target for broad-spectrum
antimicrobials.4 MTAP functions solely in the polyamine pathway
of mammals. Inhibition of this pathway is a validated anti-cancer
target.5
Results and discussion
Our route to pyrrolidinol (+)-5 established the required trans
orientation of the ring substituents with a 1,3-dipolar cycload-
dition between diethyl maleate and the nitrone formed from
formaldehyde and N-benzylhydroxylamine, which gave racemic
cis-isoxazolidine ( )-7 (97%) (Scheme 1). When the N–O bond
of isoxazolidine ( )-7 was cleaved with zinc in acetic acid the
resulting aminodiester spontaneously formed the known15 trans-
lactam ( )-8 in 94% yield. These two steps were carried out
without any chromatographic purification. The purity of lactam
( )-8 obtained this way, and used in the next step, was estimated
by NMR to be 90–95%.
The hydrolytic resolution of racemic ester ( )-8 using
Novozyme 435‡ (10% w/w) in aqueous buffer at pH 7.5 was
superbly selective (E > 300).16 At the end of the reaction (50 2%
conversion)§ ester (−)-8 and all non-acidic organic impurities were
removed from the aqueous reaction medium by extraction with
chloroform. The aqueous phase was then acidified and multiple
extractions with ethyl acetate gave a moderate yield of crystalline
acid (+)-9 (32%, based on amount of ( )-8), pure by NMR and of
To supply our ongoing synthetic efforts in this area, we required
an efficient route to kilogram quantities of the key intermedi-
ate (3R,4R)-N-tert-butoxycarbonyl-4-hydroxymethylpyrrolidin-
3-ol [(+)-5]. The parent amine, 4-hydroxymethylpyrrolidin-3-ol,
was first synthesised as a component of a racemic cis,trans
mixture,6 and later as part of a trans racemic mixture.7 The
(3R,4R) enantiomer [(+)-4] has been prepared enantio-pure from
both D-glucose and D-xylose,8,9 and in very high enantiomeric
excess by a route involving opening of a Sharpless epoxide with
‡ A total of twenty-two commercially available lipases, proteases and
esterases were screened for efficacy in this hydrolysis. Only Novozyme
435 and pig liver esterase were able to hydrolyse ( )-8 efficiently, and the
former was more selective.
aCarbohydrate Chemistry Team, Industrial Research Ltd, P.O. Box 31310,
Lower Hutt, New Zealand. E-mail: j.mason@irl.cri.nz
bGlycoSynirl, P.O. Box 31310, Lower Hutt, New Zealand
† Electronic supplementary information (ESI) available: NMR spectra of
§ Inhomogeniety of the reaction mixture prevents precise determination of
(
)-7, ( )-8, (−)-8 and (+)-10. See DOI: 10.1039/b708796a
degree of conversion.
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Microanalyses were carried out by the Campbell Microanalytical
Laboratory, University of Otago.
( )-cis-Diethyl 2-benzylisoxazolidine-4,5-dicarboxylate [( )-7].
N-Benzylhydroxylamine hydrochloride (280.0 g, 1.75 mol) and
anhydrous NaOAc (157.8 g, 1.92 mol) were stirred together in
EtOH (1.2 L) at rt for 1 h, after which time aqueous formaldehyde
(37%, 260 mL, 3.5 mol) was added and stirring was continued for
1 h. Diethyl maleate (6, 260 mL, 1.61 mol) was added and the
mixture heated under reflux for 2.5 h. After cooling, the solvent
was evaporated and the residue partitioned between EtOAc (1.5 L)
and NaHCO3 (sat. aq., 1.6 L). The organic layer was dried and
concentrated to give the product ( )-7 as a yellow oil (523 g, 97%).
This material was pure by NMR and was used in the next step
without further treatment. NMR dH 7.40–7.20 (5 H, m, Ar), 4.76
(1H, br d, H-5), 4.29–4.08 (5 H, m, 2 × CH2CH3 and CHHPh),
4.02 (1H, br m, CHHPh), 3.77 (1 H, q, J 8.7, H-4), 3.60–3.00
(2 H, m, H-3, H-3), 1.28 (3H, t, J 7.2, CH3), 1.25 (3H, t, J 7.2,
CH3); dC 169.7 (CO), 169.1 (CO), 136.4 (Ar), 129.0 (ArH), 128.4
(ArH), 127.6 (ArH), 77.0 (C-5), 62.5 (PhCH2), 61.4 (CH2CH3),
61.3 (CH2CH3), 56.8 (C-3), 50.4 (C-4), 14.0 (2 × CH2CH3); HRMS
(EI) m/z 307.1418, C16H21NO5 (M+) requires 307.1420.
Scheme 1 Reagents and conditions: (a) BnNOH, CH2O, EtOH, reflux,
2.5 h; (b) Zn, AcOH; (c) Novozyme 435, pH 7.5, 27 ◦C, 5.7 h; (d) BF3·Et2O,
NaBH4, THF, 72 h, then HCl; (e) H2, Pd/C; (f) (Boc)2O.
high enantiomeric purity (ee 95%). Reduction of (+)-9 with borane
gave N-benzyl amine (+)-10 in excellent yield (91%) and greater
than 97% purity (NMR) without chromatography. Benzyl amine
(+)-10 was hydrogenolysed to amine (+)-4 which was converted
to the N-tert-butoxycarbonyl- protected amine (+)-5 in excellent
yield (99%). Purified through a short column of silica gel, diol
(+)-5 was analytically pure and had 1H and 13C NMR spectra in
accordance with literature data.1
( )-trans-Ethyl 1-benzyl-4-hydroxy-5-oxopyrrolidine-3-carboxy-
late [( )-8]. Powdered zinc (210 g, 3.2 mol) was added portion-
wise to a solution of crude isoxazolidine ( )-7 (523 g, 1.7 mol)
in AcOH (1.5 L) cooled with an ice–water bath. The mixture was
stirred for 1 h and then filtered through Celite. The solvent was
evaporated and the residue was taken up in CH3Cl (1 L) and
washed with NaHCO3 (sat. aq., 2 × 2 L). The organic layer was
dried and concentrated to give the product ( )-8 as a yellowish
waxy solid (442 g, 99%). This material was 90–95% pure by NMR
and was used in the next step without further treatment. A small
amount was purified by column chromatography, eluting with
EtOAc–hexanes 1 : 1. Mp 65–66 ◦C (EtOAc–hexanes), lit. 62–
Conclusion
In summary, this synthesis of (+)-5 is short, efficient and robust.
It provides the target compound in excellent chemical and
enantiomeric purity in five steps and 26% overall yield from
commercially available starting materials. It has been undertaken
many times in our laboratories and has been used to manufacture
compound (+)-5 in kilogram quantities.
◦
63.5 C;15 NMR dH in agreement with literature data;15 dC 172.9
(CO), 171.4 (CO), 135.1 (Ar), 128.9 (ArH), 128.2 (ArH), 128.0
(ArH), 72.3 (C-4), 61.5 (CH2CH3), 47.0 (PhCH2), 46.1 (C-3),
45.1 (C-2), 14.1 (CH2CH3); HRMS (EI) m/z 263.1154, C14H17NO4
(M)+ requires 263.1158.
Experimental
(3R,4S)-Ethyl 1-benzyl-4-hydroxy-5-oxopyrrolidine-3-carboxy-
late [(−)-8] and (3S,4R)-1-Benzyl-4-hydroxy-5-oxopyrrolidine-3-
carboxylic acid [(+)-9]. A suspension of crude ester ( )-8 (345 g,
1.31 mol) in acetone (350 mL) and potassium phosphate buffer
(0.5 M, pH 7.5, 4 L) was stirred over Novozyme 435 (34.5 g) for
5.7 h at 27 ◦C. The enzyme was removed by filtration. The filtrate
was saturated with NaCl and extracted with CHCl3 (4 × 600 mL).
The combined extracts were dried (MgSO4) and concentrated
under reduced pressure to give (−)-8 (138 g, 40%) as a light
brown oil. Column chromatography of a small sample eluting
with EtOAc–hexanes (1 : 1) gave a pale yellow waxy solid. [a]D21
−46.8 (c 1.1, EtOH); NMR spectra in agreement with those of the
racemate; ee (HPLC) 98%. The aqueous phase was bought to pH 1
with HCl (6 M), resaturated with NaCl and extracted with EtOAc
(7 × 0.8 L). The combined extracts were dried and concentrated to
give a light brown solid that was purified by trituration with EtOAc
to give (+)-9 (109 g, 32%). This material was pure by NMR and
was used in the next step without further treatment. Crystallised
from water as colourless needles, it gave mp 146–147 ◦C; [a]D21 +62.3
General
All reagents, including anhydrous solvents, were used as supplied.
Organic solutions were dried over MgSO4 and the solvents were
evaporated under reduced pressure. Flash column chromatogra-
phy was performed on Scharlau or Merck silica gel 60 (40–60 lm)
and the solvents used were distilled prior to use or were of AR
grade. Melting points were recorded on a Reichert hot stage
microscope and are uncorrected. Optical rotations were recorded
on a Perkin-Elmer 241 polarimeter with a path length of 1 dm
and are in units of 10−1deg cm2 g−1; concentrations are in g
per 100 ml. NMR spectra were recorded on a Bruker AC300E
spectrometer in CDCl3. 1H spectra at 300 MHz are referenced to
internal Me4Si (d 0) and 13C spectra at 75.5 MHz are referenced
to the centre line of residual CHCl3 (d 77.0). Coupling constants
(J) are quoted in Hz. Positive ion electron impact (EI+) HRMS
were measured on a VG 70SE instrument. HPLC analysis was
preformed on a Chiralcel OD-H column connected to an HP Series
1100 chromatograph and eluted with 20% isopropanol in hexane.
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(c 1, EtOH); NMR dH 7.37–7.21 (5H, bm, Ar), 4.76 (3H, bs, OH
and H2O) 4.70 (1H, d, J 8.2, H-4), 4.49 (1H, d, J 14.6, CHHPh),
4.38 (1H, d, J 14.6, CHHPh) 3.44 (2H, m, H-2, H-2), 3.19 (1H, m,
H-3); dC 174.7 (CO), 173.8 (CO), 135.1 (Ar), 129.3 (ArH), 128.6
(ArH), 128.5 (ArH), 72.6 (C-4), 47.6(PhCH2), 46.1 (C-3), 45.5 (C-
2); found C 61.36, H 5.56, N 5.92, C12H13NO4 requires C 61.27,
H 5.57, N 5.95%; a sample that was converted to the methyl ester
using Me3SiCHN2 gave an ee of 95% by HPLC.
C 55.53, H 8.84, N 6.34, C10H19NO4 requires C 55.28, H 8.81, N
6.54%.
Acknowledgements
This work was supported by the New Zealand Foundation for
Research, Science and Technology. The authors thank Dr H. Wong
for NMR spectra and Prof. R. Ferrier for assistance with the
manuscript.
(3R,4R)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol
[(+)-10].
Freshly distilled BF3·OEt2 (258 mL, 2.06 mol) was added to a
suspension of acid (+)-9 (96.6 g, 411 mmol) and NaBH4 (62.5 g,
1.64 mol) in anhydrous THF (1.9 L) under argon at 0 ◦C, and
stirring was continued at rt for 72 h. The reaction was then
quenched with MeOH (100 mL) under ice-cooling and the solvent
evaporated. The residue was stirred with aqueous HCl (6 M,
200 mL) for 10 min and concentrated. This residue was slurried
with aqueous NaOH (15%, 200 mL) and again evaporated. The
residue was suspended in CHCl3 (1 L), filtered through Celite
and a plug of silica gel, and evaporated to dryness to give (+)-10
(77.8 g, 376 mmol, 91%) as a an oil that solidified on standing.
This material was pure by NMR and was used in the next step
without further treatment. A sm◦all quantity was recrystallised
from EtOAc–hexanes. Mp 60–62 C; [a]2D1 +36.0 (c 1.1, MeOH),
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1
lit.14 [a]2D1 +33.0 (c 0.75, MeOH). H and 13C NMR spectra were
identical to those reported in the literature.14
(3R,4R)-N-tert-Butoxycarbonyl-4-hydroxymethylpyrrolidin-3-ol
[(+)-5]. Crude amine (+)-10 (69.9 g, 338 mmol) was dissolved in
MeOH (440 mL), Pd/C (10%, 6 g) was added and the mixture was
stirred overnight under hydrogen. The catalyst was removed by
filtration through Celite and di-tert-butyl dicarbonate (81 g, 371
mmol) was added to the solution of amine (+)-4 at such a rate as
to maintain the temperature below 40 ◦C. The mixture was stirred
at rt for 1 h and the solvent was evaporated. The residue was
chromatographed on a column eluted with CH2Cl2–MeOH (9 : 1)
to give the title compound (+)-5 (72.4 g, 333 mmol, 99%) as an oil
12 S. Karlsson and H.-E. Ho¨gberg, Tetrahedron: Asymmetry, 2001, 12,
1977–1982.
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1
that formed an amorphous solid on standing. H and 13C NMR
spectra were identical to those reported in the literature.1 [a]D21 +16
(c 0.8, MeOH), lit.14 [a]D21 +15.9, +16.2 (c 0.8–1.1, MeOH); found
16 K. Faber, Biotransformations in Organic Chemistry, Springer-Verlag,
Berlin, 5th edn, 2004, ch. 2.
2802 | Org. Biomol. Chem., 2007, 5, 2800–2802
This journal is
The Royal Society of Chemistry 2007
©