Chemoenzymatic synthesis of the four diastereoisomers of 4-hydroxypipecolic acid from N-acetyl-(R,S)-allylglycine: Chiral scaffolds for drug discovery

Article abstract of DOI:10.1021/op020017x

All four diastereoisomers of 4-hydroxypipecolic acid were prepared in a form conveniently protected for drug discovery applications with the use of industrially scaleable methodology. Resolution of the racemic starting material using proprietary acylases followed by an acyliminium ion cyclisation gave diastereomeric mixtures of 4-formyloxypipecolic acid, which were differentiated using an enzyme-catalysed hydrolysis. The products were separated by partition, and by following a sequence of straightforward chemical steps, the individual stereoisomers of the protected 4-hydroxypipecolates were crystallized to optical purity in 100 g quantities.

Full text of DOI:10.1021/op020017x

Organic Process Research & Development 2002, 6, 762−766
Chemoenzymatic Synthesis of the Four Diastereoisomers of 4-Hydroxypipecolic
Acid from N-Acetyl-(R,S)-allylglycine: Chiral Scaffolds for Drug Discovery
Richard C. Lloyd,* Mark E. B. Smith, Dean Brick, Stephen J. C. Taylor, David A. Chaplin, and Raymond McCague
Chirotech Technology Ltd., Cambridge Science Park, Milton Road, Cambridge CB4 0WG, U.K.
Abstract:
All four diastereoisomers of 4-hydroxypipecolic acid were
prepared in a form conveniently protected for drug discovery
applications with the use of industrially scaleable methodology.
Resolution of the racemic starting material using proprietary
acylases followed by an acyliminium ion cyclisation gave
diastereomeric mixtures of 4-formyloxypipecolic acid, which
were differentiated using an enzyme-catalysed hydrolysis. The
products were separated by partition, and by following a
sequence of straightforward chemical steps, the individual
stereoisomers of the protected 4-hydroxypipecolates were
crystallized to optical purity in 100 g quantities.
Figure 1.
Introduction
activity; the naturally occurring sulphate 33 is an NMDA
Cyclic molecules bearing three functional groups, which
may be elaborated into a wide array of drug-like entities,
receptor agonist,4 and 1 is a constituent of the synthetic HIV
protease inhibitor, palinavir 4.5 Although many elegant
are potentially valuable as scaffolds in drug discovery. In
syntheses of the enantiomers of 1 exist,6 preparations of the
particular, where the presence of the functional groups creates
trans-diastereoisomer 2 are less common, involving either
stereocentres, there may be several stereoisomers of the
chromatographic separation of the cis- and trans-diastereo-
resultant scaffold. Thus, for example, we have recently
mers,4 or chemistry that is unfavourable on scale.7 Our
described the synthesis and potential utilization of a range
objective was thus to identify access to the individual
enantiomers of both the cis-(1) and trans-(2) 4-hydroxyp-
of enantiopure 4-(tert-butoxycarbonylamino)cyclopent-1-
enecarboxylic acid methyl ester scaffolds.1 In the event that
ipecolates, in suitably protected form for library synthesis,
one isomer of such a scaffold should prove valuable as a
via an easily scaleable route from readily available starting
materials.
component of a drug candidate, then it is essential that the
methods used in the synthesis are appropriate to large-scale
manufacture. In this respect, these cyclopentenecarboxylate
Results and Discussion
scaffolds are accessed through a combination of scaleable
bioresolution and catalytic diastereoselective hydrogenation
methods. In seeking to develop other useful trifunctional
cyclic scaffold structures, we were drawn to the 4-hydroxy-
pipecolic acids 1 and 2 (Figure 1). These are naturally
occurring nonproteinogenic amino acids that have been
isolated from the leaves of Calliandra pittieri, Strophantus
scandeus, and Acacia oswaldii.2 With their rigid structure
and multiple functionality they make ideal candidates for
use as scaffolds around which compound libraries may be
designed for drug discovery. Indeed, molecules derived from
both 1 and 2 have been demonstrated to possess biological
Piperidine Ring Synthesis. Preparation of the piperidine
ring system is outlined in Scheme 1. This route is based on
the known bioresolution of racemic N-acetyl amino acids
(3) Evans, S. V., Shing, T. K. M.; Aplin, R. T.; Fellows, L. E.; Fleet, G. W. J.
Phytochemistry 1985, 24, 2593.
(4) Pellicciari, R.; Natalini, B.; Luneia, R.; Marinozzi, M.; Marinella, R.; Rosato,
G. C.; Sadeghpour, B. M.; Snyder, J. P.; Monahan, J. B.; Moroni, F. Med.
Chem. Res. 1992, 2, 491-496.
(5) (a) Anderson, P. C.; Soucy, F.; Yoakim, C.; Lavalle´e, P.; Beaulieu, P. L.
U.S. Patent 5,545,640, 1996. (b) Lamarre, D.; Croteau, G.; Bourgon, L.;
Thibeault, D.; Wardrop, E.; Clouette, C.; Vaillancourt, M.; Cohen. E.;
Pargellis, C.; Yoakim, C.; Anderson, P. C. Antimicrob. Agents Chemother.
1997, 41, 965.
(6) (a) Brooks, C. A.; Comins, D. L. Tetrahedron Lett. 2000, 41, 3551-3553.
(b) Di Nardo, C.; Varela, O. J. Org. Chem. 1999, 64, 6119-6125. (c)
Haddad, M.; Larcheveˆque, M. Tetrahedron: Asymmetry 1999, 10, 4231-
4237. (d) Bousquet, Y.; Anderson, P. C.; Bogri, T.; Duceppe, J.-S.; Grenier,
L.; Guse, I. Tetrahedron 1997, 53, 15671-15680. (e) Gillard, J.; Abraham,
A.; Anderson, P. C.; Beaulieu, P. L.; Bogri, T.; Bousquet, Y.; Grenier, L.;
Guse, I.; Lavalle´e, P. J. Org. Chem. 1996, 61, 2226-2231.
(7) (a) Sabat, M.; Johnson, C. R. Tetrahedron Lett. 2001, 42, 1209-1212. (b)
Golubev, A.; Sewald, N.; Burger, K. Tetrahedron Lett. 1995, 36, 2037-
2040.
* To whom correspondence may be addressed. Telephone: +44 1223 728010.
Fax: +44 1223 506701. E-mail: RLloyd@dow.com.
(1) (a) Smith, M. E. B.; Lloyd, M. C.; Derrien, N.; Lloyd, R. C.; Taylor, S. J.
C.; Chaplin, D. A.; Casy, G.; McCague, R. Tetrahedron: Asymmetry 2001,
12, 703-705. (b) McCague, R. Mod. Drug DiscoVery 2000, July/August,
29-32.
(2) (a) Romeo, J. T.; Swain, L. A.; Bleeker, A. B Phytochemistry 1983, 22,
1615-1618. (b) Schenk, V. W.; Schutte H. R. Flora 1963, 153, 426-443.
(c) Clark-Lewis, J. W.; Mortimer, P. I. J. Chem. Soc. 1961, 189-201.
762
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Vol. 6, No. 6, 2002 / Organic Process Research & Development
10.1021/op020017x CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/19/2002

Scheme 1a
Scheme 2
with D-aminoacylase at pH 8.0 followed by reaction with
benzyl chloroformate and extraction as above gave N-Cbz-
(R)-allylglycine (R)-7 (28% overall yield from racemate,
>99% ee). The protected amino acids were subsequently
esterified in thionyl chloride/methanol. Reaction of the ester
(S)-8 in a solution of paraformaldehyde in formic acid using
the procedure of Esch et al.9 gave the 4-formyloxy-(S)-
pipecolic acid methyl ester 9, as 1:1 mixture of diastereomers
at the 4-position of the piperidine ring, the stereochemistry
of the 2-position being previously fixed as that of the amino
acid starting material. Compound (R)-8 was elaborated in a
similar fashion to give the diastereoisomeric mixture 10.
Separation of Diastereomers and Synthesis of (2S,4R)-
15 and (2R,4S)-15. Although TLC distinguished the two
diastereomers in mixtures 9 and 10, on scale their separation
by chromatography was unfeasible. An enzyme screen of
eight commercial enzymes showed both Lipase AY30
(Candida rugosa, Amano) and Chirazyme L1 (Burkholderia
cepacia, Roche) selectively hydrolysed one of the formate
groups of mixture 9. Interestingly, it was noted these enzymes
hydrolysed different diastereoisomers and it was subsequently
determined that Lipase AY30 preferentially hydrolysed the
trans-diastereoisomer, whereas Chirazyme L1 hydrolysed the
cis-diastereoisomer. However, Lipase AY30 was the more
selective enzyme and the reaction was duly scaled up. Thus,
treatment of mixture 9 with Lipase AY30 at pH 7.0 yielded
the (2S,4S)-4-hydroxypipecolic acid methyl ester 11 (90%
de) and the (2S,4R)-4-formyloxypipecolic acid methyl ester
12 (81% de) (Scheme 2). Although these two compounds
were themselves inseparable, reaction of the mixture with
phthalic anhydride converted (2S,4S)-11 to the hemiphthalate
ester derivative (2S,4S)-13, which could be separated from
(2S,4R)-12 by partitioning between toluene and saturated
a Reagents used: (i) t-BuOK, allyl bromide; (ii) NaOH; (iii) HCl; (iv)
L-acylase, pH 8.0, 65 °C; (v) Cbz-Cl, pH 8.0; (vi) D-acylase, pH 8.0; (vii) SOCl2,
MeOH; (viii) paraformaldehyde, HCO2H.
with aminoacylases8 and the reported acyliminium ion
cyclisation of allylglycine.9 Condensation of allyl bromide
with the diethylacetamidomalonate anion,10 followed by
hydrolysis and decarboxylation, gave the N-acetyl amino acid
(R,S)-6. Small-scale experiments showed that (R,S)-6 was
a good substrate for both our recombinant Thermococcus
litoralis L-aminoacylase11 (extremophile) and Alcaligenes sp.
D-aminoacylase12 enzymes, and in the interests of cost it was
decided to use these enzymes in preference to commercially
available ones.13 Hence treatment with L-aminoacylase at 60
°C and pH 8.0 gave a mixture of (S)-allylglycine and
unreacted (R)-6. This mixture was reacted with benzyl
chloroformate at pH 8.0, and after acidification to pH 2.0,
N-Cbz-(S)-allylglycine (S)-7 was extracted cleanly into
toluene (36% yield from racemate, >99% ee). Concentration
of the aqueous fraction and extraction into EtOAc allowed
recovery of (R)-6 (49%, 80% ee). Treatment of this material
(8) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989,
111, 6354-6364.
(9) Esch, P. M.; de Boer, R. F.; Hiemstra, H.; Boska, I. M.; Speckamp, W. N.
Tetrahedron 1991, 47, 4063-4076.
(10) Brace, N. O. J. Org. Chem. 1967, 32, 430-434.
(11) Toogood, H. S.; Hollingsworth, E. J.; Brown, R. C.; Taylor, I. N.; Taylor,
S. J. C.; McCague, R.; Littlechild, J. A. Extremophiles 2002. In press.
(12) Taylor, S. J. C.; Brown, R. C. PCT Pat. Appl. WO 00/23598 A1, 2000.
(13) At the time, there was no commercially available D-acylase enzyme.
Vol. 6, No. 6, 2002 / Organic Process Research & Development
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763

Scheme 3a
Scheme 4a
a Reagents used: (i) K2CO3, MeOH; (ii) recrystallise; (iii) H2, 10% Pd/C,
Boc2O.
ammonium carbonate solution.14 Concentration of the toluene
layer gave (2S,4R)-12 (51% overall yield from input into
biotransformation). The hemiphthalate ester (2S,4S)-13 was
recovered by acidification of the aqueous layer and extraction
into toluene (38% yield from input into biotransformation).
The formate ester (2S,4R)-12 was elaborated into the
desired product, (2S,4R)-N-Boc-2-carbomethoxy-4-hydroxy-
piperidine 15 as outlined in Scheme 3. After deformylation
of (2S,4R)-12 (81% de) using K2CO3/MeOH, the resultant
(2S,4R)-14 (90%, 81% de) was recrystallised to optical purity
from EtOAc. Removal of the Cbz group followed by in situ
Boc protection gave (2S,4R)-15 in 83% yield (>98% ee,
>98% purity).
a Reagents used: (i) 2 M HCl, then Amberlite IRA-93; (ii) Boc2O, Et3N;
(iii) BnNH2, recrystallise.
should be scaleable for manufacture.
Experimental Section
An enzyme screen showed both Chirazyme L9 (Mucor
miehei, Roche) and Lipase AY30 differentiated the diaster-
eomers present in the mixture 10. Both enzymes preferen-
tially hydrolysed the trans-diastereoisomer, but Chirazyme
L9 was the most selective and hence was used in the large-
scale biotransformation. After this slow bioresolution, reac-
tion with phthalic anhydride and partitioning as before
allowed the separation of (2R,4S)-12 from the hemiphthalate
ester derivative (2R,4R)-13. (2R,4S)-12 was elaborated as
in Scheme 3 to give the desired product, (2R,4S)-15.
Elaboration of 13 to trans-4-Hydroxypipecolic Acid.
Attempts to purify and enhance the de of (2S,4S)-13 by
crystallisation as an amine salt were unsuccessful. Although
hydrolysis of both ester moieties was readily achieved,
separation of the Cbz-protected acid (2S,4S)-16 from phthalic
acid proved difficult. Acid-catalyzed hydrolysis of (2S,4S)-
13 in HCl, followed by neutralization with Amberlite IRA-
93 yielded the free amino acid (2S,4S)-2 in 79% yield
without any significant epimerisation of either chiral centre
(Scheme 4). Boc protection was carried out using triethyl-
amine as the base in a modified Schotten-Baumann
procedure, and conveniently, the Boc-acid (2S,4S)-17 was
crystallised from MeOH/MTBE to >98% de, >95% purity,
as the benzylamine salt 18 in which form it was stored.
(2R,4R)-18 was prepared from hemiphthalate (2R,4R)-13 in
identical fashion.
General. All reagents and solvents were from com-
mercially available sources and used as received. NMR
spectra were obtained on a Bruker Avance 400 spectrometer
operating at 400 MHz for proton (1H) and 100 MHz for
carbon (13C). Chemical shifts are reported in ppm using Me4-
Si or residual nondeuterated solvent as reference. Coupling
constants (J) are measured in Hz. GC-MS data were
obtained using a HP 5890 series 2 GC fitted with a J&W
Scientific DB5 column, attached to a HP 5972 series mass
selective detector. De and ee data of final compounds were
obtained using a Perkin-Elmer Autosystem Gas Chromato-
graph fitted with a Varian Chirasil-Dex CB column. All
samples were run as the 2-carbomethoxy-4-acetoxy deriva-
tive. HPLC data were obtained using a Gilson HPLC system
fitted with a Luna phenylhexyl column. Optical rotation data
were obtained using a Perkin-Elmer Polarimeter 341 instru-
ment.
N-Acetyl-(R,S)-allylglycine 6. Sodium methoxide (25 wt
% in methanol, 9.0 kg, 9.5 L, 41.5 mol) was added to a
suspension of diethylacetamidomalonate (8.0 kg, 36.8 mol)
in methanol (15 kg, 19 L), and the mixture was heated to
reflux for 30 min. The heat source was removed, and allyl
bromide (4.48 kg, 3.2 L, 37.0 mol) was added at a rate
sufficient to maintain a gentle reflux. This reflux was
maintained for 30 min, after which water (10 L) was added
until all solids had dissolved, and the solution was adjusted
to pH 10 with 40% aqueous NaOH (7.25 kg), keeping the
reaction temperature below 50 °C. After the mixture refluxed
for 2 h, the methanol was removed by vacuum distillation
and the resultant solution cooled to 40 °C. The pH was
adjusted to 3.5 with concentrated HCl (3.7 kg) and stirred
for 2 h, after which more concentrated HCl (1.5 L) was added
to reduce the solution pH to 2. This was extracted with
EtOAc (4 × 9 L), and the combined organic layers were
concentrated to yield N-acetyl-(R,S)-allylglycine, 6, a yellow
Conclusions
From a single racemic starting material, all four stereo-
isomers of 4-hydroxypipecolic acid have been prepared in
suitably protected forms for use as chiral scaffolds in drug
discovery. Individual chiral centres have been defined, and
optical purity obtained using a powerful combination of
biotransformation and crystallisation. The methods used
1
(14) Boaz, N. W. U.S. Patent 5,312,950, 1994.
semisolid (3.25 kg, 56%). H NMR (d6-DMSO) 8.09 (1H,
764
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Vol. 6, No. 6, 2002 / Organic Process Research & Development

d, J 8), 5.73 (1H, m), 5.08 (1H, dd, J 17, 2), 5.03, (1H, m),
4.21 (1H, m), 2.42 (1H, m), 2.30 (1H, m), 1.83 (3H, s). 13
NMR (d6-DMSO) 173.4, 169.5, 134.4, 118.0, 52.1, 35.8,
22.7.
for 1 h and then was allowed to warm to room temperature
and was stirred overnight. The excess solvent was removed
in vacuo and the residue dissolved in MTBE (6 L). The
solution was washed with 1 M HCl (3.4 L) and saturated
NaHCO3, dried (Na2SO4), and evaporated to yield (S)-8 as
a yellow oil (0.86 kg, 73%). 1H NMR (d6-DMSO) 7.75 (1H,
d, J 8), 7.33 (5H, m), 5.75 (1H, m), 5.09 (1H, m), 5.05-
5.00 (3H, m), 4.10 (1H, m), 3.60, (3H, s), 2.45 (1H, m),
2.34 (1H, m). 13C NMR (d6-DMSO) 173.5, 156.3, 137.2,
134.2, 128.6, 128.2, 128.0, 118.0, 65.8, 54.0, 52.2, 35.5.
Using an identical procedure, (R)-7 (0.68 kg, 2.73 mol)
gave (R)-8 (0.72 kg, 100%).
(2S,4R,S)-N-Benzyloxycarbamoyl-2-carbomethoxy-4-
formyloxypiperidine 9.9 Paraformaldehyde (144.0 g, 4.8
mol) was dissolved in hot formic acid (6.5 L) and the
resultant solution cooled to 25 °C. (S)-8 (904.3 g, 3.4 mol)
was added and the solution stirred for 72 h, at which time
GC analysis showed no starting material remained. Excess
solvent was removed in vacuo, and the residual oil was dried
by azeotroping with toluene (4 × 750 mL) and passed
through a silica plug, eluting with EtOAc. Evaporation of
the solvent in vacuo left 9 as a yellow oil (1056.3 g, 96%).
GC retention times 26.9 min (trans-diastereoisomer) 27.6
min (cis-diastereoisomer) in a 1:1 ratio. m/z 321 (M+), 262,
216, 172, 140, 91.
C
N-Benzyloxycarbamoyl-(S)-allylglycine (S)-7. To a so-
lution of (R,S)-6 (3.2 kg, 20.4 mol) in pH 8.0 Tris-HCl (32
L) at 70 °C was added the cell paste containing the
L-aminoacylase (0.32 kg, 3 units/g substrate), and the reaction
mixture was stirred at this temperature until GC analysis
dictated the reaction was 44% complete. Celite (4.8 kg) was
added and the mixture filtered. THF (31 kg, 35 L) was added
to the filtrate and the solution cooled to 5 °C. Benzyl
chloroformate (1.25 kg, 7.34 mol) was added, keeping the
temperature below 10 °C, and using 40% aqueous NaOH,
the pH was kept in the 7.5-8.5 range. Stirring was
maintained at 10 °C until there was no further pH change,
at which point the THF was removed by distillation. The
aqueous layer was washed with MTBE (2 × 17 L), acidified
to pH 2 using concentrated HCl, and extracted with toluene
(3 × 21 L). These toluene layers were combined, washed
with 10% NaCl (9 L), and concentrated to minimum volume
to yield N-benzyloxycarbamoyl-(S)-allylglycine, (S)-7, (1.20
1
kg, 24%, >99% ee) as a waxy solid. H NMR (d6-DMSO)
major rotamer 7.55 (1H, d, J 8), 7.34 (5H, m), 5.76 (1H,
m), 5.12-5.00 (4H, m), 4.02 (1H, m), 2.44 (1H, m), 2.34
(1H, m). 13C NMR (d6-DMSO) 173.5, 156.4, 137.3, 134.6,
128.8, 128.4, 128.1, 118.3, 65.7, 54.2, 35.5.The aqueous layer
was concentrated to one-third volume and extracted with
EtOAc (4 × 11 L). These extracts were combined and
concentrated to give the residual (R)-6, a brown solid (1.17
kg, 36%, 80% ee).
Using an identical method, (R)-8 (713 g, 2.71 mol) gave
10 (722 g, 83%), which proved spectroscopically identical
to 9.
Separation of Mixture 9. A 10-L jacketed reaction vessel
equipped with an overhead stirrer was charged with 9 (1056.3
g), MTBE (3.6 L), and 50 mM potassium phosphate buffer
pH 7.0 (4.5 L). Stirring was started to achieve an emulsion
and Lipase AY30 (300 g) added. The pH was kept constant
at pH 7.0 by the addition of 5 M NaOH. After 4 days at 20
°C the reaction was stopped by filtration through Celite 521.
The two layers in the filtrate were separated, and the organic
layer was reserved. The Celite was slurried with acetone (500
mL) and filtered. This filtrate was concentrated in vacuo until
only aqueous material remained, when it was extracted with
MTBE (2 × 500 mL). The organic layers were combined,
dried (MgSO4), and concentrated in vacuo to yield a viscous,
cloudy yellow oil (903 g) that was a mixture of (2S,4S)-N-
benzyloxycarbamoyl-2-carbomethoxy-4-hydroxypiperi-
dine, 11, of 90% de and (2S,4R)-N-benzyloxycarbamoyl-2-
carbomethoxy-4-formyloxypiperidine, 12, of 83% de, in an
approximate 1:1 ratio. This mixture and DMAP (17.9 g, 0.14
mol) was dissolved in CH2Cl2 (6 L) at 20 °C. Et3N (450
mL, 3.22 mol) was added using a pressure-equalising
dropping funnel over a 10-min period. Solid phthalic
anhydride (239 g, 1.61 mol) was added batch-wise and
stirring continued for 18 h. The reaction mixture was washed
with 1 M HCl (3.5 L), and the organic layer concentrated in
vacuo. The residue was redissolved in toluene (4 L) and
extracted with saturated (NH4)2CO3 (3 L). This aqueous layer
was washed with toluene (1 L), and the combined organic
extracts were dried (MgSO4) and concentrated in vacuo to
yield (2S,4R)-12 (545 g, 81% de, 52%). The aqueous layer
was acidified to pH 1 with concentrated HCl and extracted
with toluene (2 L). The layers were separated, and the
N-Benzyloxycarbamoyl-(R)-allylglycine (R)-7. A solu-
tion of 80% ee (R)-6 (1.17 kg, 7.45 mol) in Tris-HCl pH
8.0 (11.7 L) was heated to 40 °C and D-Aminoacylase
enzyme (0.64 L, 50 units/g substrate) added. The mixture
was stirred at 40 °C until GC analysis showed the reaction
to be 86% complete (24 h), when Celite (1.8 kg) was added
and the mixture filtered. THF (14 kg, 16 L) was added to
the filtrate and the solution cooled to 5 °C. Benzyl chloro-
formate (1.05 kg, 6.15 mol) was added, keeping the tem-
perature below 10 °C, and using 40% aqueous NaOH the
pH was kept in the 7.5-8.5 range. Stirring was maintained
at 10 °C until there was no further pH change, at which point
the THF was removed by distillation. The aqueous layer was
washed with MTBE (2 × 7.5 L), acidified to pH 2 using
concentrated HCl, and extracted with toluene (3 × 10 L).
These toluene layers were combined, washed with 10% NaCl
(5 L), and concentrated to minimum volume to yield
N-benzyloxycarbamoyl-(R)-allylglycine (R)-7 (0.68 kg, 37%,
1
>99% ee) as a waxy solid. H NMR (d6-DMSO) major
rotamer 7.55 (1H, d, J 8), 7.34 (5H, m), 5.76 (1H, m), 5.12-
5.00 (4H, m), 4.02 (1H, m), 2.44 (1H, m), 2.34 (1H, m). 13
C
NMR (d6-DMSO) 173.5, 156.4, 137.3, 134.6, 128.8, 128.4,
128.1, 118.3, 65.7, 54.2, 35.5.
N-Benzyloxycarbamoyl-(S)-allylglycine Methyl Ester
(S)-8. A solution of (S)-7 (1.13 kg, 4.53 mol) in methanol
(6 L) was cooled to 0 °C, and thionyl chloride (2.83 kg,
1.74 L, 23.8 mol) was added dropwise, keeping the temper-
ature below 5 °C. The reaction was stirred at this temperature
Vol. 6, No. 6, 2002 / Organic Process Research & Development
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765

aqueous was extracted once more with toluene (1 L). These
two organic layers were combined, dried (MgSO4), and
concentrated in vacuo to yield (2S,4S)-N-benzyloxycarbam-
oyl-2-carbomethoxy-4-hydroxypiperidine, 4-hemiphthalate
derivative, 13, (553 g, 38%). Both products were used
directly in the next steps.
Separation of Mixture 10. The separation was carried
out in an identical manner, substituting Chirazyme L9 for
Lipase AY30. Hence, 10 (722 g, 2.25 mol) was separated
into (2R,4S)-12 (370 g, 51%) and (2R,4R)-11 (80% de, 426
g, 43%).
22.3 min (major diastereoisomer) in a ratio of 143:1. m/z
(as acetate) 301 (M+), 242, 182, 140, 126, 82, 57.
Using an identical method, >99% de (2R,4S)-14 (141 g,
0.48 mol) gave (2R,4S)-15 (100 g, 80%, >99% ee, >99%
de, purity (GC) >98%).
(2S,4S)-4-Hydroxypipecolic Acid 2. 11 (365 g, 0.82 mol)
was mixed with 2 M HCl (1.5 L) and heated to reflux for 5
days. The mixture was cooled and extracted with EtOAc (3
× 1 L). The aqueous layer was concentrated in vacuo to
leave a cloudy paste (170 g). This was redissolved in H2O
(500 mL) and the solution neutralised using Amberlite IRA-
93. The resin was filtered and washed with H2O (1.5 L),
and the filtrate was concentrated in vacuo and dried by
azeotroping with toluene (2 × 500 mL) to leave (2S,4S)-2,
a cream solid (94.5 g, 79%). 1H NMR (D2O) Major
diastereoisomer 4.21 (1H, m) 3.90 (1H, dd, J 11.5, 3.5) 3.28
(2H, m) 2.20 (1H, m) 1.97-1.84 (3H, m). Minor diastereo-
isomer 3.95 (1H, m) 3.63 (1H, dd, J 13.0, 3.0) 3.47 (1H,
ddd, J 13.0, 4.5, 2.5) 3.02 (1H, dt, J 13.5, 3.5) 2.47 (1H, m)
2.10 (1H, m), 1.58 (2H, m). 13C NMR (D2O) Major
diastereoisomer 174.6, 62.2, 54.6, 38.9, 33.1, 28.3.
(2S,4R)-N-Benzyloxycarbamoyl-2-carbomethoxy-4-hy-
droxypiperidine 14.15 (2S,4R)-12 (545 g, 81% de, 1.70 mol)
was dissolved in MeOH (1.5 L) and K2CO3 (23.5 g, 0.17
mol) added. The mixture was stirred for 2 h at room
temperature, by which time the reaction was complete.
MTBE (5 L) was added and the solution washed with H2O
(3 L). The organic phase was dried and concentrated in
vacuo. This residue was triturated with EtOAc (600 mL) and
heptane (75 mL) to yield (2S,4R)-N-benzyloxycarbamoyl-
2-carbomethoxy-4-hydroxypiperidine, 14, (444.1 g, 90%,
81% de) which was recrystallised from EtOAc (850 mL) to
yield optically pure (2S,4R)-14 (145.4 g, >99% de). Further
crops of identical quality crystals (58.2 g, 33.2 g, both >99%
de) were obtained from the liquors (overall yield 48% from
Using an identical method, 80% de (2R,4R)-11 (400 g,
0.91 mol) gave (2R,4R)-2 (130 g, 99%).
(2S,4S)-N-tert-Butyloxycarbamoyl-4-hydroxypiperidine-
2-carboxylic Acid 17. To a solution of (2S,4S)-2 (140 g,
0.97 mol) in H2O (1 L) and THF (500 mL), Et3N (135 mL,
0.97 mol) was added dropwise. Boc2O (317 g, 1.46 mol) in
THF (500 mL) was added in a steady stream. As the pH
started to drop, a further portion of Et3N (135 mL, 0.97 mol)
was added and the solution stirred at room temperature for
16 h. The THF was removed in vacuo, and the resultant
cloudy solution was acidifed to pH 4 with 6 M HCl and
then to pH 3 with 1 M HCl and extracted with EtOAc (4 ×
1 L). The combined organic extracts were washed with brine
(1 L), dried (MgSO4), and concentrated in vacuo to give
(2S,4S)-17, a viscous yellow oil (182 g, 76%, 80% de). This
was dissolved in EtOAc and the solution cooled on ice.
Benzylamine (81.2 mL, 0.74 mol) was added dropwise and
stirring maintained for 2 h. After overnight refrigeration, the
solid was collected by filtration and dried. This solid (154
g, 60% recovery) was recrystallised from MeOH (150 mL)
and MTBE (300 mL). Filtration yielded (2S,4S)-N-tert-
butyloxycarbamoyl-4-hydroxypiperidine-2-carboxylic acid,
benzylamine salt, 18, as a white solid (104 g, 31% from 2,
>98% ee, >98% de, purity (HPLC) >95%). 1H NMR (CD3-
OD) 7.40 (5H, m) 4.67 (0.4 H, minor rotamer, m) 4.59 (0.6H,
d, J 5.5, major rotamer) 4.10 (2H, s) 3.94 (1H, br d, J 13.0)
3.59 (1H, m) 3.17 (1H, m) 2.48 (1H, m) 1.80 (1H, m) 1.43
(10H, m) 1.25 (1H, m). 13C NMR (CD3OD) 178.1, 178.0
(rotamer pair) 157.6, 157.3 (rotamer pair) 135.0, 130.2,
130.0, 129.9, 80.8, 67.1, 67.0 (rotamer pair), 57.9, 56.9, 44.3,
41.6, 40.9 (rotamer pair), 37.6, 35.4, 35.2 (rotamer pair) 28.7.
1
(2S,4R)-12). H NMR (d6-DMSO) 7.37 (5H, m) 5.08 (2H,
m) 4.64 (2H, m) 3.90 (1H, br s) 3.70 (1H, dt, J 8.5, 3.5)
3.59 (3H, br s) 3.47-3.27 (1H, br m) 2.19 (1H, m) 1.82
(1H, dd, J 13.5, 6.5) 1.54 (2H, m). 13C NMR (d6-DMSO)
172.2, 172.0 (rotamer pair), 155.9, 155.5 (rotamer pair),
137.0, 128.6, 128.0, 127.6, 127.5, 66.5, 66.4 (rotamer pair),
51.8, 51.0, 50.8 (rotamer pair), 36.0, 35.9 (rotamer pair), 33.3,
31.0, 30.9 (rotamer pair). GC (material derivatised to acetate)
gave retention times 28.2 min (minor diastereoisomer), 29.0
min (major diastereoisomer) in a ratio 1:220. m/z (as acetate)
335 (M+), 276, 216, 172, 140, 91.
Using an identical method, 80% de (2R,4S)-12 (370 g,
1.15 mol) gave (2R,4S)-14 (>99% de, 144 g, 43%).
(2S,4R)-N-tert-Butyloxycarbamoyl-2-carbomethoxy-4-
hydroxypiperidine 15. (2S,4R)-14 (144.0 g, 0.49 mol) was
dissolved in EtOAc (2.8 L) and Boc2O (112.8 g, 0.52 mol)
added. 10% Pd/C (14 g, 10 wt %) was added and the
suspension placed in a 10-L pressure vessel. The vessel was
purged with N2 and charged to 2 bar with H2. Stirring was
maintained until no further H2 was taken up. The vessel was
purged with N2, and the contents were filtered through Celite
and concentrated in vacuo. The residue was purified on a
silica plug to yield (2S,4R)-15 (106 g, 83%, >98% ee, >99%
de, purity (GC) >98%) as a colourless glass. 1H NMR 4.59
(1H, br), 4.00 (1H, m) 3.73 (1H, m), 3.68 (3H, s), 3.41-
3.25 (1H br m), 2.37 (1H, br d, J 13), 1.87 (1H, dd, J 13, 6),
1.62 (2H, m) 1.43 (9H, app d, rotamer pair). 13C NMR (d6-
DMSO) 172.3, 172.2 (rotamer pair), 155.4, 155.0 (rotamer
pair), 79.2, 61.2, 51.7, 51.4, 50.2 (rotamer pair), 36.2, 35.3
[R]25 -14.6 (c 3.16, EtOH).
D
Using an identical method, (2R,4R)-2 (130 g, 0.89 mol)
gave (2R,4R)-18 (>98% ee, >98% de 111.4 g, 35%, purity
(HPLC) >95%).
(rotamer pair), 33.4, 31.2, 31.0 (rotamer pair) 28.1. [R]25
D
-24.1 (c 2.74, EtOH). GC (material derivatised to acetate)
gave retention times 21.8 min (minor diastereoisomer) and
Received for review January 30, 2002.
OP020017X
(15) Hays, S. J.; Malone, T. C.; Johnson, G. J. Org. Chem. 1991, 56, 4084-
4086.
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Vol. 6, No. 6, 2002 / Organic Process Research & Development