Synthetic anisomycin analogues activating the JNK/SAPK1 and p38/SAPK2 pathways

Article abstract of DOI:10.1039/b311242j

The synthesis of C(4)H and C(4)Me analogues of the JNK/p38 pathway activator anisomycin, based upon an aldol or Claisen construction of the C(3)-C(4) bond, has been demonstrated. The relative activation of the JNK/SAPK1 and p38/SAPK2 pathways in RAW macrophages by these analogues, and their synthetic precursors, has been assessed using immunoblot assays against phosphorylated c-Jun and MAPKAP-K2. These studies demonstrate that some of the synthetic C(4) analogues are also potent activators of these stress kinase pathways.

Full text of DOI:10.1039/b311242j

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Synthetic anisomycin analogues activating the JNK/SAPK1 and
p38/SAPK2 pathways
Edward M. Rosser,a Simon Morton,b Kate S. Ashton,a Philip Cohen*b and Alison N. Hulme*a
a School of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh,
UK EH9 3JJ. E-mail: Alison.Hulme@ed.ac.uk; Fax: ϩ44 131 650 4743;
Tel: ϩ44 131 650 4711
b MRC Protein Phosphorylation Unit, Division of Cell Signalling, School of Life Sciences,
University of Dundee, Dundee, UK DD1 5EH. E-mail: p.cohen@dundee.ac.uk;
Fax: ϩ44 1382 223 778; Tel: ϩ44 1382 344 238
Received 15th September 2003, Accepted 21st November 2003
First published as an Advance Article on the web 2nd December 2003
The synthesis of C(4)H and C(4)Me analogues of the JNK/p38 pathway activator anisomycin, based upon an aldol
or Claisen construction of the C(3)–C(4) bond, has been demonstrated. The relative activation of the JNK/SAPK1
and p38/SAPK2 pathways in RAW macrophages by these analogues, and their synthetic precursors, has been
assessed using immunoblot assays against phosphorylated c-Jun and MAPKAP-K2. These studies demonstrate
that some of the synthetic C(4) analogues are also potent activators of these stress kinase pathways.
Introduction
The pyrrolidine antibiotic anisomycin (1) has long been known
to act as a peptidyl transferase inhibitor, binding to the 60S
ribosomal subunit in eukaryotes, and has been shown to exhibit
selective action against protozoa and several strains of fungi.1
Since its introduction a little over 10 years ago2,3 as a tool for
the activation of stress kinase pathways at “sub-inhibitory”
levels,† the precise target(s) of the pyrrolidine antibiotic in
mammalian, insect, or yeast cells that triggers the activation of
these signalling pathways has not been elucidated. Biochemical
and genetic studies demonstrate that the stress-activated kinase
(SAPK) pathways regulate processes such as cellular responses
to stress and infection, as well as cell repair/apoptosis.3 Hence,
studies into the mechanism of initiation and regulation of these
pathways may provide new leads for therapeutic targets.
Whilst the cellular targets of anisomycin are not currently
known, biochemical and genetic studies have yielded inform-
ation as to its downstream effects on the stress-activated
mitogen-activated protein (MAP) kinases.4 Thus treatment of
mammalian cells with anisomycin at sub-inhibitory, as well as
inhibitory levels is known to strongly activate the protein
kinases JNK/SAPK1 (c-Jun NH2-terminal kinase/stress-
activated protein kinase) and p38/SAPK2.3
Following our successful synthesis of anisomycin (1) utilizing
a highly diastereoselective syn glycolate aldol reaction with a
tyrosine-derived α-dibenzylamino aldehyde,5 we targeted our
Fig. 1 Analogues of anisomycin, nectrisine and CYB-3.
efforts towards the production of a range of anisomycin deriv-
atives for biological testing. Previous reports have shown that
anisomycin’s activity against a range of tumour cell lines6 is
Results and discussion
largely unaffected by modest changes to the substitutent at the
C(4) position. We hypothesised that the C(4)Me (2) and C(4)H
(3) analogues of anisomycin (Fig. 1) might show enhanced levels
of activation of the JNK/SAPK1 and p38/SAPK2 pathways
due to their more lipophilic nature. Similarly, the C(4)Me and
C(4)H analogues of deacetyl anisomycin (4) (5 and 6 respect-
ively) might allow us to address the level to which the reduced
activity of deacetyl anisomycin (4)‡ is associated with the loss of
interactions with this ester functionality rather than changes in
the molecule’s polarity.
Analogue preparation
We have recently reported the synthesis of the C(4)Me deriv-
ative 7 of nectrisine (8) using boron mediated aldol chemistry,7
and the epimer 9 of the natural product CYB-3 (10) using
Claisen chemistry (Fig. 1).8 We were therefore confident that
similar synthetic approaches would allow us to access the
C(4)Me, and C(4)H analogues of anisomycin. Our previous
synthesis of anisomycin (1) itself, made use of a ‘mismatched’
glycolate aldol reaction for the key C(3)–C(4) bond construc-
† At “sub-inhibitory” levels (typically 25–50 ng mLϪ1) anisomycin acti-
vates the kinase pathways but does not inflict any toxic effects on cells
through protein synthesis inhibition and may even cause a slight stimu-
lation of protein synthesis, presumably due to its gene-inducing effect.
‡ Deacetylation of anisomycin markedly inhibits its ability to bind to
ribosomes (104-fold reduction in potency), arrest translation, activate
JNK/SAPK1 and p38/SAPK2 and induce apoptosis.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
142

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tion.5 Adaptation of this approach, with the propionate
derivative of Evans oxazolidinone 11 in place of the previous
protected glycolate equivalent, allowed the synthesis of aldol
adduct 12 from aldehyde 13§ in excellent yield (87%) and as
a single diastereomer (Scheme 1). Borohydride reduction of
aldol adduct 12 to give diol 14, and selective tosylation of the
primary alcohol resulted in spontaneous cyclisation to give the
pyrrolidinium tosylate salt. This salt was converted to its chlor-
ide counterpart 15 by purification through Dowex ClϪ exchange
resin in excellent yield (83%). Selective mono-deprotection5 of
the salt allowed facile acylation of the free hydroxyl in 16 to give
the benzyl protected precursor 17 of the C(4)Me analogue. Pro-
tected pyrrolidine 17 was subjected to hydrogenolysis in the
presence 1 M hydrochloric acid to allow the isolation of the
C(4)Me analogue 2 as its hydrochloride salt in quantitative
yield (13 steps, 36% overall yield from -tyrosine). The C(4)Me
analogue 5 of deacetyl anisomycin was obtained from the
pyrrolidinium salt 15 directly in quantitative yield by exhaustive
debenzylation and treatment with Dowex HOϪ exchange resin
(11 steps, 47% overall yield from -tyrosine).
Scheme 2 Reagents and conditions: (a) LiOH, THF–H2O, reflux, 18 h
(95%); (b) (i) CDI, THF, rt, 2.5 h, (ii) CH2᎐᎐C(OLi)OEt, Ϫ78 ЊC, 2 h
(82%); (c) NaCNBH3, AcOH, Et2O, CH3OH, 0 ЊC rt, 18 h (83 %);
(d) LiAlH4, THF, Ϫ78 ЊC, 6 h (95%); (e) (i) TIBSCl, DMAP, CH2Cl2,
0 ЊC, 18 h; (ii) Dowex ClϪ (85%); (f ) H2, 5% Pd/C, K2CO3, CH3OH, rt,
20 min (80%); (g) Ac2O, NEt3, CH2Cl2, rt, 18 h (87%); (h) H2, 20%
Pd(OH)2/C, 1 M HCl/–Et2O, CH3OH, rt, 20 h (100%).
diol was not possible using toluene sulfonyl chloride (TsCl)
due to competing reaction of the secondary hydroxyl which is
significantly less hindered in this case than in diol 14. To
counter this, the more sterically demanding triisopropylbenzene
sulfonyl chloride (TIBSCl) was employed. Using this reagent,
selective sulfonylation of the primary alcohol was achieved and
in situ cyclisation to the pyrrolidinium tosylate salt was
observed. Treatment with Dowex ClϪ exchange resin facilitated
purification of the pyrrolidinium salt 23 (81% from β-hydroxy
ester 22).|| Monodebenzylation to give alcohol 24, acylation to
give 25 and final benzyl deprotection were achieved as in the
C(4)Me series allowing the isolation of the C(4)H analogue (3)
as its hydrochloride salt (12 steps, 33% overall yield from
-tyrosine). Similarly, direct conversion of pyrrolidinium salt
23 to the C(4)H analogue 6 of deacetyl anisomycin was
achieved in high yield (95%) by exhaustive debenzylation and
treatment with Dowex HOϪ exchange resin (10 steps, 45%
overall yield from D-tyrosine).
With C(4) anisomycin analogues 2 and 3, and C(4) deacetyl
anisomycin analogues 5 and 6 in hand, and the corresponding
N-benzyl derivatives of these (17, 25, 16 and 24 respectively) we
were well-placed to begin investigation of the JNK/SAPK1 and
p38/SAPK2 pathway activation by each of these. To this collec-
tion were added synthetic samples of anisomycin 1, deacetyl
anisomycin 4, and their benzylated derivatives 26 and 27 (Fig. 2)
prepared by our previously reported methodology,5 as well as a
final synthetic sample of 3097-B1 (28) which has been isolated
from the fermentation broths of Streptomyces strain SA3097
along with anisomycin,9 and which was prepared by a similar
route.
Scheme 1 Reagents and conditions: (a) (i) Bu2BOTf, NEt3, CH2Cl2,
0 ЊC, 15 min; (ii) 13, Ϫ78 ЊC
THF, 0 ЊC
0 ЊC, 5 h (87%); (b) LiBH4, CH3OH,
rt, 18 h (82%); (c) (i) TsCl, DMAP, CH2Cl2, 0 ЊC, 18 h;
(ii) Dowex ClϪ (83%); (d) H2, 5% Pd/C, K2CO3, CH3OH, rt, 20 min
(93%); (e) Ac2O, NEt3, CH2Cl2, rt, 18 h (82%); (f ) H2, 20% Pd(OH)2/C,
1 M HCl–Et2O, CH3OH, rt, 18 h (100%).
In tackling the synthesis of the C(4)H analogue of aniso-
mycin using a Claisen approach, we were aware of the potential
for epimerisation at the α-stereocentre in ester 18,¶ during con-
version via acid 19 to the more reactive acylimidazole inter-
mediate 20 (Scheme 2).8 To our relief, chiral HPLC analysis of
the syn β-hydroxy ester diastereomer 22 (obtained after sodium
cyanoborohydride reduction of β-keto ester 21) and com-
parison with traces from the corresponding racemic series, indi-
cated that in the case of the tyrosine-derived ester there is no
epimerisation during this reaction sequence (alcohol 22 >98%
ee). Even more pleasingly, the cyanoborohydride conditions for
ketone reduction which had been shown to give optimum
diastereoselectivity in the synthesis of 9,8 resulted in even higher
selectivity in this case (>40 : 1 ratio syn : anti reduction prod-
ucts). Subsequent lithium aluminium hydride reduction of the
ester 22 to give the required diol precursor to the pyrrolidine
ring proceeded cleanly and in excellent yield (95%). However,
monoprotection of the primary alcohol functionality in this
In total this gave 13 synthetic samples for study in the activ-
ation of the stress activated protein kinase pathways. Using
these aldol- and Claisen-based approaches more than 20 mg of
each sample was available for the biological assays described.
|| Confirmation of the relative stereochemistry achieved during the
cyanoborohydride reduction of 21 was obtained at this juncture by
preparation of the hexafluorophosphate salt of 23 (Dowex PF6Ϫ, 70%
from the intermediate diol) and X-ray crystallographic analysis.
§ Aldehyde 13 is available in 7 steps (80% overall yield) from -tyrosine
(ref. 5).
¶ Ester 18 is available in 5 steps (91% overall yield) from -tyrosine.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
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transfer to a nitrocellulose membrane generated the appro-
priate protein displays for immunoblot assay. The immunoblot
assay was carried out with antibodies that recognise all forms
of the c-Jun protein (c-Jun), c-Jun phosphorylated at Ser63
(Ser63), all forms of MAPKAP-K2 and MAPKAP-K2
phosphorylated at Thr334 (Thr334) for each of the analogues.
The results of the immunoblot assays are shown in Figs 4 and
5. In each case the levels of pathway activation may be com-
pared against those of commercially available anisomycin
(Sigma) and a blank in which the analogues, which were (dis-
solved in DMSO) were replaced by the equivalent volume of
DMSO (see Experimental).
These assays indicate that the C(4)–H analogue 3 is a particu-
larly good activator showing similar levels of activation to the
Fig. 2 Further analogues of anisomycin and deacetyl anisomycin.
SAPK pathway activation
Although the cellular target(s) of anisomycin is(are) not
currently known, its downstream effects on the SAPKs have
been determined (Fig. 3). Thus, treatment of mammalian cells
with anisomycin at sub-inhibitory levels is known to strongly
activate JNK/SAPK1 and p38/SAPK2, resulting in phos-
phorylation of their substrates, such as the nuclear trans-
cription factor c-Jun and the protein kinase MAPKAP-K2
respectively.3 Commercial antibodies against both the phos-
phorylated and non-phosphorylated states of c-Jun and
MAPKAP-K2 have recently become available, making it simple
to assess the differential activation of each of these pathways by
the current analogue set.
Fig. 4 Effect of anisomycin and its analogues on the phosphorylation
of c-Jun in RAW macrophages. The cells were exposed to anisomycin
(Sigma) or the anisomycin analogues indicated, each dissolved in
DMSO, or to DMSO alone as a control. The cells were lysed and an
aliquot (20 µg of lysate protein) was denatured in SDS, subjected
to electrophoresis on a 10% polyacrylamide gel, transferred to a
nitrocellulose membrane and immunoblotted with an antibody that
recognises c-Jun phosphorylated at Ser63 (Ser63), or with an antibody
that recognises phosphorylated and unphosphorylated c-Jun equally
well (c-Jun). Further details are given in the Experimental.
Fig. 3 SAPK pathway activation by anisomycin.
In order to assess the relative levels of pathway activation by
each of the anisomycin analogues, murine RAW macrophage
cell cultures were stimulated with the analogues for 30 minutes
before cell lysis, and assessment of protein content by Bradford
assay (Scheme 3). SDS PAGE separation of the lysates and
Fig. 5 Effect of anisomycin and its analogues on the phosphorylation
of MAPKAP-K2 in RAW macrophages. The experiment was carried
out as described in the legend to Fig. 4, except that the gels were immuno-
blotted with an antibody that recognises MAPKAP-K2 phosphoryl-
ated at Thr334 (Thr334), or with an antibody that recognises the
phosphorylated and unphosphorylated forms of MAPKAP-K2 equally
well. Further details are given in the Experimental.
Scheme 3 Immunoblot assay of SAPK pathway activation.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
144

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natural product itself, whilst the C(4)–Me analogue 2 is less
effective. Acylation is essential for SAPK activation, although
both acetate [anisomycin (1)] and propionate [3097-B1 (28)]
esters show similar levels of activity. Deacetyl anisomycin 4
shows a dramatic reduction in both JNK/SAPK1 and p38/
SAPK2 pathway activation. O- or N-benzylation reduces activ-
ation further, and the only analogue to show any pathway
activation in this category is 27 (very weak activation of the
JNK/SAPK1 pathway).
dropwise, dibutylboron triflate (0.92 cm3, 1.0 M in CH2Cl2, 0.92
mmol) followed by triethylamine (0.10 g, 0.13 cm3, 0.95 mmol).
The solution was stirred at 0 ЊC for 15 min, then cooled to
Ϫ78 ЊC and a Ϫ78 ЊC solution of aldehyde 13 (0.200 g, 0.557
mmol) in CH2Cl2 (3 cm3) was added dropwise via cannula. The
reaction mixture was stirred at Ϫ78 ЊC for 1 hour, and was
allowed to warm to 0 ЊC over a period of 4 h. The reaction was
quenched at 0 ЊC by the addition of methanol (5 cm3) followed
by pH 7 phosphate buffer (1 cm3). Hydrogen peroxide (30% aq.
solution; 5 cm3) in methanol (5 cm3) was added dropwise to the
solution and the mixture was stirred and allowed to warm to
room temperature over ca. 1 hour. The organic phase was
separated and the aqueous phase was extracted with CH2Cl2
(3 × 20 cm3); the combined organic phase was washed sequen-
tially with saturated aq. sodium bicarbonate (25 cm3) and brine
(25 cm3) then dried, and concentrated under reduced pressure.
The residue was chromatographed using preparative HPLC
[hexane : EtOAc (7 : 3)] to give 12 (0.144 g, 87%) as an oil, Rf
[hexane : EtOAc (7 : 3)] 0.40; Rt [hexane : EtOAc (7 : 3)] = 11.08
minutes; [α]D Ϫ48.7 (c 2.00, CHCl3); νmax (neat)/cmϪ1 3370,
2929, 1773, 1703, 1612, 1511; δH (200 MHz; CDCl3) 7.36–6.90
(17H, m), 6.87 (2H, d, J 8.7), 4.65–4.58 (2H, m), 4.22–4.09 (2H,
m), 3.91 (1H, dd, J 9.0, 2.5), 3.87 (2H, d, J 12.8), 3.81 (3H, s),
3.72 (1H, qd, J 6.9, 2.5), 3.40 (2H, d, J 12.8), 3.33 (1H, dd,
J 13.3, 3.0), 3.13–2.97 (2H, m), 2.85–2.76 (1H, m), 2.67 (1H, dd,
J 13.3, 10.0), 0.61 (3H, d, J 6.9); δC (62.9 MHz, CDCl3) 175.3,
158.5, 153.8, 139.0 (2C), 136.0, 132.3, 130.7 (2C), 129.9 (2C),
129.5 (4C), 129.3 (2C), 129.0 (4C), 127.8 (2C,), 127.7, 114.4
(2C), 70.5, 66.6, 61.6, 56.4, 55.7, 54.3 (2C), 40.6, 38.1, 31.2, 8.7;
m/z (FAB) 593 ([M ϩ H]ϩ, 59%), 307 (16), 154 (100), 136 (70),
121 (16), 91 (51); HRMS (FAB) (Found: [M ϩ H]ϩ, 593.3018.
C37H41N2O5 requires m/z, 593.3015).
The activation of the S6 kinase pathway,10 by these analogues
has also been tested using immmunoblot assays against ribo-
somal protein S6 phosphorylated at Ser235 and has been shown
to give similar results.
Conclusions
We have demonstrated rapid and efficient routes to the chemical
synthesis of C(4) analogues of anisomycin (1) and deacetyl
anisomycin (4). Several of these analogues show similar, or only
slightly reduced levels of activation of JNK/SAPK1 and p38/
SAPK2 pathways relative to the natural product itself. Since
differential acylation of the C(3) and C(4) hydroxyl groups has
led to lengthy syntheses of anisomycin analogues in the past,
the discovery of an active C(4)H analogue is of particular inter-
est for future studies. These results also suggest ways of coup-
ling anisomycin to insoluble supports to generate the matrices
required for affinity purification of the anisomycin receptors
from cell extracts.
Experimental
Analogue preparation
All reactions involving air- or water-sensitive reagents were
carried out under an atmosphere of argon using flame- or oven-
dried glassware. Unless otherwise noted, starting materials and
reagents were obtained from commercial suppliers and were used
without further purification. THF was distilled from Na-benzo-
phenone ketyl immediately prior to use. Toluene, CH2Cl2, and
Et3N were distilled from calcium hydride. Anhydrous methanol,
DMF, and acetonitrile were used as supplied by Acros. Unless
otherwise indicated, organic extracts were dried over anhydrous
sodium sulfate and concentrated under reduced pressure using a
rotary evaporator. Purification by flash column chromatography
was carried out using Merck Kieselgel 60 silica gel as the station-
ary phase. Chiral HPLC was performed using a Waters instru-
ment equipped with a UV detector and a Chiracel OD-H column
(internal diameter 4.6 mm, column length 250 mm, flow rate 0.5
cm3 minϪ1). Preparative HPLC was carried out using a Gilson
instrument equipped with an RI detector and a Spherisorb col-
umn (internal diameter 25 mm, column length 250 mm, flow rate
9 cm3 minϪ1). All solvents for use in HPLC analysis were vacuum
filtered and degassed prior to use. IR spectra were measured on a
Biorad FTS-7 or Perkin-Elmer Paragon 1000 FT-IR spectro-
(2S,3R,4R)-4-N,N-Dibenzylamino-3-hydroxy-5-(4-methoxy-
phenyl)-2-methylpentan-1-ol 14. To a solution of aldol adduct
12 (0.281 g, 0.475 mmol) in THF (20 cm3) at 0 ЊC was added
methanol (0.076 g, 0.096 cm3, 2.4 mmol) and LiBH4 (0.052 g,
1.1 mmol). The solution was warmed to room temperature and
stirred for 18 hours then re-cooled to 0 ЊC and quenched by the
addition of 1 M aqueous sodium hydroxide (2 cm3). The
organic phase was separated, and the aqueous phase was
extracted with CH2Cl2 (3 × 20 cm3); the combined organic
phase was washed with brine (30 cm3), dried, and concentrated
under reduced pressure. The residue was chromatographed on
silica gel [hexane : EtOAc (7 : 3)] to give the diol 14 (0.162 g,
82%) as an oil, Rf [hexane : EtOAc (7 : 3)] 0.10; [α]D Ϫ31.72
(c 1.95, CHCl3); νmax (neat)/cmϪ1 3397, 3027, 2933, 1611,
1583, 1512; δH (250 MHz; CDCl3) 7.34–7.10 (10H, m), 7.13
(2H, d, J 8.7), 6.88 (2H, d, J 8.7), 4.74 (1H, s), 3.90–3.78
(1H, m), 3.88 (2H, d, J 12.7), 3.82 (3H, s), 3.68 (1H, dd, J 10.6,
4.2), 3.58 (1H, dd, J 10.6, 6.0), 3.36 (2H, d, J 12.7), 3.07–2.97
(1H, m), 3.02 (1H, dd, J 17.2, 9.1), 2.67 (1H, s), 2.55 (1H, dd,
J 17.2, 8.4), 1.52 (1H, m), 0.39 (3H, d, J 6.8); δC (62.9 MHz,
CDCl3) 158.0, 138.4 (2C), 131.7, 130.0 (2C), 129.0 (4C),
128.3 (4C), 127.2 (2C), 113.9 (2C), 71.6, 67.6, 60.3, 55.1,
53.5 (2C), 35.8, 31.7, 8.2; m/z (FAB) 420 ([M ϩ H]ϩ, 67%), 330
(66), 298 (62), 240 (21), 208 (15), 121 (75), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 420.2538. C27H34NO3 requires m/z,
420.2539).
1
meter as thin films unless otherwise stated. H and 13C NMR
spectra were measured on a Varian Gemini 200, Bruker AC250,
Bruker AM360 or Varian Inova 600 spectrometer; J values are in
Hz. Melting points were determined on a Gallenkamp Electro-
thermal Melting Point apparatus and are uncorrected. Optical
rotations were measured on an AA-1000 polarimeter with a path
length of 1.0 dm, at the sodium D-line at room temperature.
Elemental analysis was carried out on a Perkin-Elmer 2400 CHN
Elemental Analyser. Fast atom bombardment (FAB) mass
spectra were obtained using a Kratos MS50TC mass spectro-
meter at The University of Edinburgh.
(2R,3R,4S )-1,1-Dibenzyl-3-hydroxy-2-(4-methoxybenzyl)-4-
methylpyrrolidinium chloride 15. To a solution of the diol 14
(0.125 g, 0.297 mmol) in CH2Cl2 (10 cm3) at 0 ЊC was added
DMAP (0.163 g, 1.34 mmol) and TsCl (0.170 g, 0.892 mmol).
The solution was stirred for 18 hours then diluted with CH2Cl2
(25 cm3) and water (25 cm3). The organic phase was separated
and washed with 1% aqueous hydrochloric acid (2 × 25 cm3)
then dried and concentrated under reduced pressure. The
residue was chromatographed on silica gel [CH2Cl2 : MeOH
(2ЈR,3ЈR,4R,4ЈR)-3-(4Ј-N,N-Dibenzylamino-3Ј-hydroxy-5Ј-
(4-methoxyphenyl)-2Ј-methyl-1Ј-oxopentyl)-4-phenylmethyl-
oxazolidin-2-one 12. To a solution of propionate equivalent 11
(0.202 g, 0.858 mmol) in CH2Cl2 (3 cm3) at 0 ЊC was added,
(100 : 0)
(95 : 5)] to give a white foam. The salt obtained was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
145

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subjected to ion exchange chromatography (Dowex ClϪ; pre-
pared by treatment of Dowex 1-X2 with 1% aqueous hydro-
chloric acid followed by flushing with methanol until the eluent
returned to pH 7) eluting with methanol to give the chloride salt
15 (0.108 g, 83%) as an amorphous solid, Rf [CH2Cl2 : MeOH
(95 : 5)] 0.05; [α]D Ϫ41.3 (c 3.08, CHCl3); νmax (neat)/cmϪ1 3182,
2960, 1612, 1513; δH (360 MHz; CDCl3) 7.76 (2H, d, J 7.0),
7.44–7.30 (10H, m), 6.83 (1H, d, J 3.6), 6.69 (2H, d, J 8.6), 5.78
(1H, d, J 13.4), 5.24 (1H, d, J 13.0), 5.09 (1H, d, J 13.0), 4.14
(1H, d, J 13.4), 3.95–3.92 (2H, m), 3.69 (3H, s), 3.66–3.56 (2H,
m), 3.31–3.28 (1H, m), 3.26–3.04 (1H, m), 2.56 (1H, br t,
J 11.1), 0.91 (3H, d, J 6.8); δC (62.9 MHz; CDCl3) 158.2, 133.4
(2C), 133.2 (2C), 130.5 (3C), 130.2, 129.1 (2C), 129.0 (2C),
127.8, 127.4, 126.8, 113.9 (2C), 74.4, 74.3, 61.2, 60.6, 59.9, 55.0,
38.7, 26.8, 17.0; m/z (FAB) 402 ([M]ϩ, 66%), 312 (65), 190 (63),
121 (76), 91 (100); HRMS (FAB) (Found: [M]ϩ, 402.2433.
[C27H32NO2]ϩClϪ requires m/z, 402.2433).
(2R,3R,4S )-3-Acetoxy-2-(4-methoxybenzyl)-4-methyl-
pyrrolidine hydrochloride 2. To a solution of the pyrrolidine 17
(0.0407 g, 0.115 mmol) and Pearlman’s catalyst [0.0407 g; 20%
Pd(OH)2/C] in methanol (5 cm3) was added hydrochloric acid
(1 M in ether; 0.23 cm3, 0.23 mmol). The solution was exposed
to a hydrogen atmosphere (1 atm) and stirred vigorously for
18 hours. The mixture was then filtered through a pad of Celite
and concentrated under reduced pressure to give the hydro-
chloride salt 2 (0.0341 g, 100%) as a colourless oil, [α]D ϩ20.0
(c 1.35, CH3OH); νmax (neat)/cmϪ1 3398, 2934, 1740, 1612, 1582,
1514; δH (250 MHz; CD3OD), 7.33 (2H, d, J 7.9), 7.01 (2H, d,
J 7.9), 5.05 (1H, br s), 4.15 (1H, br s), 3.87 (3H, s), 3.73 (1H, m),
3.19–3.04 (3H, m), 2.63 (1H, br s), 2.26 (3H, s), 1.26 (3H, d,
J 6.7); δc (62.9 MHz; CD3OD) 170.2, 159.4, 130.0 (2C), 127.8,
114.4 (2C), 78.5, 62.9, 54.7, 50.1, 39.3, 31.4, 19.8, 16.3; m/z
(FAB) 264 ([M ϩ H]ϩ, 100%), 204 (60), 142 (19), 121 (49);
HRMS (FAB) (Found: [M ϩ H]ϩ, 264.1593. [C15H22NO3]ϩClϪ
requires m/z 264.1600).
(2R,3R,4S )-1-Benzyl-3-hydroxy-2-(4-methoxybenzyl)-4-
methylpyrrolidine 16. To a solution of the chloride salt 15
(0.0782 g, 0.178 mmol) in methanol (5 cm3) was added 5% Pd/C
(0.0078 g) and potassium carbonate (0.074 g, 0.54 mmol). The
mixture was exposed to a hydrogen atmosphere (1 atm) and
stirred vigorously for 20 minutes. The suspension was filtered
through a pad of Celite and concentrated under reduced
pressure. To the residue was added CH2Cl2 (25 cm3) and water
(25 cm3). The organic phase was separated and the aqueous
phase was extracted with CH2Cl2 (3 × 20 cm3); the combined
organic phase was washed with saturated aq. sodium bicarb-
onate (50 cm3) then dried and concentrated under reduced pres-
sure. The residue was chromatographed on silica gel [hexane :
EtOAc (7 : 3)] to give pyrrolidine 16 (0.0518 g, 93%) as an oil,
Rf [hexane : EtOAc (1 : 1)] 0.35; [α]D Ϫ78.3 (c 1.5, CHCl3);
νmax (neat)/cmϪ1 3419, 2954, 1611, 1583, 1512, 1246; δH (250
MHz; CDCl3) 7.33–7.23 (5H, m), 7.26 (2H, d, J 8.7), 6.85
(2H, d, J 8.7), 4.10 (1H, d, J 12.7), 3.78 (3H, s), 3.50 (1H, dd,
J 4.7, 2.6), 3.16 (1H, d, J 12.7), 3.13 (1H, dd, J 9.4, 7.9), 2.94
(1H, dd, J 13.4, 8.9), 2.87 (1H, dd, J 13.4, 5.3), 2.57 (1H, dt,
J 8.9, 5.0), 2.35 (1H, br s), 2.03–1.94 (1H, m), 1.67 (1H, dd,
J 9.4, 7.9), 0.91 (3H, d, J 7.1); δc (62.9 MHz; CDCl3) 157.8,
138.6, 131.4, 130.1 (2C), 128.8 (2C), 128.1 (2C), 126.9, 113.7
(2C), 78.8, 69.3, 60.2, 58.2, 55.1, 40.1, 32.9, 18.0; m/z (FAB)
312 ([M ϩ H]ϩ, 73%), 190 (65), 121 (41), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 312.1963. C20H26NO2 requires m/z
312.1964).
(2R,3R,4S )-3-Hydroxy-2-(4-methoxybenzyl)-4-methyl-
pyrrolidine 5. A solution of the chloride salt 15 (0.044 g, 0.10
mmol) and Pearlman’s catalyst [0.044 g; 20% Pd(OH)2/C] in
methanol (5 cm3) was exposed to a hydrogen atmosphere
(1 atm) and stirred vigorously for 24 hours. The mixture was
then filtered through a pad of Celite and concentrated under
reduced pressure to give (2R,3R,4S)-3-hydroxy-2-(4-methoxy-
benzyl)-4-methyl-pyrrolidine hydrochloride (0.026 g, 100%) as
a white solid, mp 191–192 ЊC; [α]D ϩ33.3 (c 0.27, CH3OH); νmax
(KBr)/cmϪ1 3356, 2956, 2920, 1614, 1577, 1516; δH (250 MHz;
CD3OD) 7.40 (2H, d, J 8.1), 7.03 (2H, d, J 8.1), 4.05 (1H, br s),
3.90 (3H, s), 3.84–3.83 (1H, m), 3.74 (1H, dd, J 11.4, 7.5), 3.27
(1H, dd, J 14.2, 6.1), 3.05 (1H, dd, J 14.2, 8.6), 2.97 (1H, dd,
J 11.4, 3.2), 2.24–2.12 (1H, m), 1.19 (3H, d, J 7.3); δc (62.9
MHz; CD3OD) 157.9, 128.7 (2C), 127.4, 112.9 (2C), 74.5, 63.1,
53.2, 48.6, 39.7, 30.1, 15.1; m/z (FAB) 222 ([M ϩ H]ϩ, 68%),
206 (47), 134 (52), 121 (40), 91 (68); HRMS (FAB) (Found:
[M ϩ H]ϩ, 222.1494. [C13H20NO2]ϩClϪ requires m/z 222.1494).
The HCl salt was subjected to ion-exchange chromatography
(Dowex OHϪ; prepared by treating Dowex 1-X2 with 1 M
aqueous NaOH, followed by methanol until the pH of the
eluent returned to 7). Eluting with methanol gave the free base
5 (quantitative recovery) as a white solid, Rf [CH2Cl2 : MeOH
(95 : 5)] 0.05; mp 108–110 ЊC; [α]D ϩ5.7 (c 0.35, CH3OH);
νmax (KBr)/cmϪ1 3420, 3260, 2951, 1612, 1584, 1512; δH (250
MHz; CD3OD) 7.28 (2H, d, J 8.7), 6.92 (2H, d, J 8.7), 3.84 (3H,
s), 3.71 (1H, dd, J 2.4, 1.7), 3.37 (1H, dd, J 11.1, 7.8), 3.19–3.11
(1H, m), 2.99 (1H, dd, J 13.6, 6.8), 2.74 (1H, dd, J 13.6, 7.6),
2.38 (1H, dd, J 11.1, 5.7), 2.24–2.15 (1H, m), 1.07 (3H, d, J 7.2);
δc (62.9 MHz; CD3OD) 157.7 (1C, Q), 131.2 (1C, Q), 129.1 (2C,
CH), 112.9 (2C, CH), 77.9 (1C, CH), 63.1 (1C, CH), 53.7 (1C,
CH3), 50.9 (1C, CH2), 41.7 (1C, CH), 33.3 (1C, CH2), 16.5 (1C,
CH3); m/z (FAB) 222 ([M ϩ H]ϩ, 100%), 121 (17), 91 (95), 57
(66); HRMS (FAB) (Found: [M ϩ H]ϩ, 222.1495. C13H20NO2
requires m/z 222.1494).
(2R,3R,4S )-3-Acetoxy-1-benzyl-2-(4-methoxybenzyl)-4-
methylpyrrolidine 17. To a solution of the alcohol 16 (0.0322 g,
0.104 mmol) in CH2Cl2 (5 cm3) was added freshly distilled acetic
anhydride (0.021 g, 0.020 cm3, 0.21 mmol), and triethylamine
(0.021 g, 0.030 cm3, 0.21 mmol). The solution was stirred for
18 hours before being diluted with CH2Cl2 (10 cm3) and satur-
ated aq. sodium bicarbonate (20 cm3). The organic phase was
separated and the aqueous phase was extracted with CH2Cl2
(3 × 10 cm3); the combined organic phase was dried and con-
centrated under reduced pressure. The residue was chromato-
graphed on silica gel [hexane : EtOAc (7 : 3)] to give the ester 17
(0.0300g, 82%) as a colourless oil, Rf [hexane : EtOAc (7 : 3)]
0.30; [α]D Ϫ73.96 (c 0.70, CHCl3); νmax (neat)/cmϪ1 2959, 2789,
1734, 1612, 1512; δH (250 MHz; CDCl3), 7.31–7.22 (5H, m),
7.10 (2H, d, J 8.6), 6.80 (2H, d, J 8.6), 4.62–4.58 (1H, m), 3.96
(1H, d, J 12.9), 3.77 (3H, s), 3.24 (1H, d, J 12.9), 3.12 (1H, dd,
J 9.3, 7.5), 2.94–2.80 (2H, m), 2.83 (1H, dd, J 14.2, 8.9), 2.11–
2.03 (1H, m), 2.06 (3H, s), 1.80 (1H, t, J 9.3), 0.99 (3H, d, J 7.0);
δc (62.9 Hz; CDCl3) 170.8, 157.9, 138.3, 131.1, 129.8 (2C),
129.1(2C), 128.2 (2C), 127.0, 113.8 (2C), 80.9, 67.1, 60.0, 58.8,
55.2, 38.3, 33.8, 21.2, 17.1; m/z (FAB) 354 ([M ϩ H]ϩ, 39%), 352
(52), 294 (34), 232 (63), 172 (28), 121 (75), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 354.2069. C22H28NO3 requires m/z
354.2069).
(2R)-2-Dibenzylamino-3-(4-methoxyphenyl)propanoic
acid
19. To a solution of methyl ester 18 (1.97 g, 5.06 mmol) in
tetrahydrofuran : water (30 cm3 [4 : 1]) was added lithium
hydroxide (1.06 g, 25.3 mmol). The solution was heated to
reflux and held there for 18 hours, and then acidified to pH 2.
The organic phase was separated, and the aqueous phase was
extracted with CH2Cl2 (3 × 25 cm3). The combined organics
were washed with water (20 cm3), dried, and then concentrated
under reduced pressure to give acid 19 (1.81 g, 95%) as a foam,
which was used in the Claisen condensation reaction without
further purification, Rf [hexane : EtOAc (7 : 3)] 0.30; [α]D ϩ25.8
(c 0.6, CHCl3) [lit.(ent-19),11 [α]D Ϫ31.4 (c 1.0, CHCl3)];
νmax (neat)/cmϪ1 3029, 2934, 2835, 1704, 1612, 1513; δH (360
MHz; CDCl3) 9.25 (1H, br s), 7.29–7.25 (10H, m), 7.11 (2H, d,
J 8.4), 6.82 (2H, d, J 8.4), 4.00–3.82 (4H, m), 3.86–3.83 (1H, m),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
146

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3.78 (3H, s), 3.30 (1H, dd, J 14.5, 6.3), 3.17 (1H, dd, J 14.5, 8.5);
δc (62.9 MHz; CDCl3) 175.5, 158.2, 137.3 (2C), 130.2 (2C),
129.8, 128.9 (4C), 128.4 (4C), 127.5 (2C), 113.7 (2C), 62.9, 55.2,
54.4 (2C), 33.3; m/z (FAB) 367 ([M ϩ H]ϩ, 83%), 330 (22), 254
(40), 154 (38), 91 (100); HRMS (FAB) (Found: [M ϩ H]ϩ,
376.1903. C24H26NO3 requires m/z 376.1913). All spectroscopic
data in good agreement with the literature data.11
cmϪ1 3501, 2934, 2833, 1728, 1612, 1584, 1512; δH (200 MHz;
CDCl3) 7.39–7.25 (10H, m), 7.20 (2H, d, J 8.8), 6.92 (2H, d,
J 8.8), 4.33–4.25 (1H, m), 4.18 (2H, q, J 7.0), 3.90 (3H, s), 3.82
(2H, d, J 13.7), 3.68 (2H, d, J 13.7), 3.19–2.97 (2H, m), 3.10
(1H, dd, J 9.0, 6.3), 2.80 (1H, dd, J 16.4, 2.7), 2.38 (1H, dd,
J 16.4, 9.8), 1.32 (3H, t, J 7.0); δc (62.9 MHz; CDCl3) 173.4,
158.0, 139.7 (2C), 133.1, 130.5 (2C), 128.9 (4C), 128.4 (4C),
127.1 (2C), 113.9 (2C), 69.2, 63.0, 60.8, 55.5, 54.8 (2C), 39.7,
31.5, 14.3; m/z (FAB) 448 ([M ϩ H]ϩ, 88%), 330 (96), 326 (96),
121 (78), 91 (100); HRMS (FAB) (Found: [M ϩ H]ϩ, 448.2489.
C28H34NO4 requires m/z 448.2488).
Ethyl
(4R)-4-dibenzylamino-5-(4-methoxyphenyl)-3-oxo-
pentanoate 21. To a solution of the acid 19 (1.81 g, 4.83 mmol)
in THF (20 cm3) was added carbonyldiimidazolide (1.56 g, 9.65
mmol). The mixture was stirred for 2.5 hours, and then cooled
to Ϫ78 ЊC. Meanwhile a separate flask containing ethyl acetate
(1.49 g, 1.65 cm3, 16.9 mmol) was cooled to Ϫ78 ЊC, and
lithium hexamethyldisilazide, (15.9 cm3, 1.06 M in THF, 16.9
mmol) was added. The solution was stirred at Ϫ78 ЊC for
20 minutes and then transferred to the flask containing the
imidazole via cannula. The combined solution was stirred at
Ϫ78 ЊC for 2 hours and then quenched by the addition of 1%
HCl (20 cm3) and diluted with CH2Cl2 (20 cm3). The organic
phase was separated and the aqueous phase was extracted with
CH2Cl2 (3 × 75 cm3); the combined organics were washed
sequentially with water (100 cm3) and brine (100 cm3) then
dried, and concentrated under reduced pressure. The residue
was chromatographed on silica gel [hexane : EtOAc (95 : 5)]
to give 21 (1.76 g, 82%) as a white solid, Rf [hexane : EtOAc
(10 : 1)] 0.30; mp 83–85 ЊC; [α]D ϩ75.3 (c 1.0, CHCl3);
νmax (KBr)/cmϪ1 2936, 1745, 1716, 1612, 1584, 1513; δH (360
MHz; CDCl3) 7.36–7.19 (10H, m), 7.05 (2H, d, J 8.7), 6.78 (2H,
d, J 8.7), 4.01 (2H, q, J 7.2), 3.83 (2H, d, J 13.5), 3.76 (3H, s),
3.65 (1H, d, J 15.8), 3.59 (1H, dd, J 9.3, 3.7), 3.54 (2H, d,
J 13.5), 3.34 (1H, d, J 15.8), 3.13 (1H, dd, J 13.6, 9.3), 2.89 (1H,
dd, J 13.6, 3.7), 1.09 (3H, t, J 7.2); δc (62.9 MHz; CDCl3) 202.2,
167.1, 157.8, 138.6 (2C), 130.9, 130.3 (2C), 128.9 (4C), 128.4
(4C), 127.3 (2C), 113.7 (2C), 68.3, 60.9, 55.1, 54.4 (2C), 46.6,
27.4, 13.8; (Found: C, 75.19; H, 6.83; N, 3.11. C28H31NO4
requires C, 75.51; H, 6.97; N, 3.15%).
(3R,4R)-4-Dibenzylamino-3-hydroxy-5-(4-methoxyphenyl)-
pentan-1-ol. A solution of ethyl ester 22 (0.697 g, 1.56 mmol) in
THF (30 cm3) was cooled to Ϫ78 ЊC and lithium aluminium
hydride (7.8 cm3, 1.0 M in THF, 7.8 mmol) was added. The
solution was stirred at Ϫ78 ЊC for 6 hours then quenched by the
addition of 1 M aqueous sodium hydroxide (10 cm3). The
resulting mixture was allowed to warm to room temperature
and diluted with CH2Cl2 (20 cm3). Saturated aq. sodium potas-
sium tartrate (40 cm3) was added and the biphasic mixture was
stirred vigorously for 15 hours by which time two clear phases
were apparent. The organic phase was separated and the aque-
ous phase was extracted with CH2Cl2 (3 × 50 cm3). The com-
bined organics were dried, and concentrated under reduced
pressure. The remaining residue was chromatographed on silica
gel [hexane : EtOAc] (7 : 3
1 : 1)] to give the title compound
(0.598 g, 95%) as an oil, Rf [hexane : EtOAc (3 : 1)] 0.35; [α]D
Ϫ38.3 (c 1.7, CHCl3); νmax (neat)/cmϪ1 3388, 3028, 2933, 2835,
1612, 1583, 1512; δH (250 MHz; CDCl3) 7.39–7.18 (10H, m),
7.12 (2H, d, J 8.7), 6.88 (2H, d, J 8.7), 4.80 (1H, s), 3.90 (2H, d,
J 13.2), 3.82 (3H, s), 3.77 (1H, dd, J 8.9, 2.7), 3.65 (2H, br s),
3.39 (2H,d, J 13.2), 3.08 (1H, dd, J 14.3, 6.4), 2.89 (1H, td,
J 6.4, 2.7), 2.60 (1H, dd, J 14.3, 6.4), 1.82 (1H, s), 1.65–155 (1H,
m), 1.35–1.23 (1H, m); δc (62.9 MHz; CDCl3) 158.0, 138.5 (2C),
131.8, 129.9 (2C), 128.9 (4C), 128.4 (4C), 127.2 (2C), 113.9
(2C), 70.5, 63.8, 61.2, 55.1, 53.6 (2C), 35.6, 31.1; m/z (FAB) 406
([M ϩ H]ϩ, 69%), 330 (71), 284 (69), 181 (45), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 406.2387. C26H32NO3 requires m/z
406.2382).
Ethyl
(3R,4R)-4-dibenzylamino-3-hydroxy-5-(4-methoxy-
phenyl)pentanoate 22. To a solution of β-keto ester 21 (1.54,
3.47 mmol) in ether (30 cm3) was added methanol (5.21 g, 6.59
cm3, 163 mmol). The solution was adjusted to pH 4 by the
addition of a few drops of acetic acid, and then cooled to 0 ЊC.
Sodium cyanoborohydride (2.15 g, 34.6 mmol) was added
cautiously and the mixture was allowed to warm to room tem-
perature. The solution was stirred for 18 hours and then
quenched by the addition of a saturated aqueous solution of
ammonium chloride (20 cm3). The organic phase was separated
and the aqueous phase was extracted with CH2Cl2 (3 × 75 cm3).
The combined organics were dried, and concentrated under
reduced pressure. The remaining residue was chromatographed
on silica gel [hexane : EtOAc (9 : 1)] to give the syn diastereomer
22 (1.28 g, 83%) and a small amount of the anti diastereomer
(0.0307 g, 2%) both as oils. Data for (3R,4R) major syn
diastereomer 22, Rf [hexane : EtOAc (10 : 1)] 0.40; [α]D Ϫ36.0
(c 2.2, CHCl3); νmax (neat)/cmϪ1 3591, 3025, 2933, 1731, 1611,
1581, 1511; δH (360 MHz; CDCl3) 7.35–7.25 (10H, m), 7.11
(2H, J 8.5), 6.88 (2H, d, J 8.5), 4.11–4.05 (2H, m), 4.08 (2H, q,
J 7.2), 4.01–3.98 (1H, m), 3.81 (3H, s), 3.44 (2H, d, J 13.4),
3.11–3.09 (1H, m), 2.82–2.74 (2H, m), 2.39 (1H, dd, J 15.7,
9.6), 2.12 (1H, br d, J 15.7), 1.21 (3H, t, J 7.2); δc (62.9 MHz;
CDCl3) 172.7, 157.9, 139.1 (2C), 131.7, 129.9 (2C), 128.9
(4C), 128.3 (4C), 127.1 (2C), 113.9 (2C), 67.8, 62.9, 60.4, 55.1,
54.3 (2C), 39.7, 29.9, 14.0; m/z (FAB) 448 ([M ϩ H]ϩ, 97%), 330
(88), 326 (89), 121 (37), 91 (100); HRMS (FAB) (Found: [M ϩ
H]ϩ, 448.2480. C28H34NO4 requires m/z 448.2488); Chiral
HPLC (R enantiomer) Rt = 16.0 minutes, (S enantiomer) Rt =
24.4 minutes [hexane–propan-2-ol (19 : 1)], >98% ee.
(2R,3R)-1,1-Dibenzyl-3-hydroxy-2-(4-methoxybenzyl)-pyrrol-
idinium chloride 23. To a solution of (3R,4R)-4-dibenzylamino-
3-hydroxy-5-(4-methoxyphenyl)pentan-1-ol (0.587 g, 1.45 mmol)
in CH2Cl2 (20 cm3) at 0 ЊC was added DMAP (0.292 g, 2.39
mmol) and triisopropylbenzene sulfonyl chloride (TIBSCl)
(0.483 g, 1.59 mmol). The solution was stirred for 18 hours
before being diluted with CH2Cl2 (20 cm3) and water (25 cm3).
The organic phase was separated and washed with 1% HCl
(2 × 30 cm3) then dried and concentrated under reduced pres-
sure. The residue was chromatographed on silica gel [CH2Cl2 :
MeOH (100 : 0)
(95 : 5)] to give a white foam. The salt
obtained was subjected to ion exchange chromatography
[Dowex ClϪ; prepared by treating Dowex 1-X2 with 1%
aqueous hydrochloric acid followed by flushing with methanol
until the eluent returned to pH 7] eluting with methanol to give
the chloride salt 23 (0.520 g, 85%) as an amorphous solid, Rf
[CH2Cl2 : CH3OH (95 : 5)] 0.05; [α]D Ϫ55.2 (c 0.75, CHCl3);
νmax (neat)/cmϪ1 3198, 1611, 1584, 1513; δH (360 MHz; CDCl3)
7.78–7.29 (10H, m), 7.33 (2H, d, J 8.5), 6.74 (1H, s), 6.67 (2H,
d, J 8.5), 5.72 (1H, br d, J 13.5), 5.12 (1H, d, J 13.1), 5.07 (1H,
d, J 13.1), 4.34 (1H, br s), 4.28 (1H, d, J 13.5), 3.97 (1H, br d,
J 12.0), 3.92–3.86 (1H, m), 3.69 (3H, s), 3.56 (1H, br t, J 12.0),
3.39 (1H, br t, J 10.5), 3.10 (1H, dt, 11.3, 8.3), 2.63 (1H, dt, 14.6,
7.3), 1.95–1.87 (1H, m); δc (62.9 MHz; CDCl3) 158.9, 134.1
(2C), 133.6 (2C), 131.2 (2C), 131.1, 130.8, 129.8 (2C), 129.6
(2C), 128.6, 128.0, 127.9, 114.5 (2C), 76.7, 68.2, 63.1, 62.4, 56.3,
55.5, 31.9, 28.1; m/z (FAB) 388 ([M]ϩ, 98%), 296 (48), 210 (24),
176 (71), 91 (100); HRMS (FAB) (Found: [M]ϩ, 338.2267.
[C26H30NO2]ϩClϪ requires m/z 388.2277).
Data for (3S,4R) minor anti diastereomer, Rf [hexane :
EtOAc (10 : 1)] 0.35; [α]D Ϫ25.9 (c 0.81, CHCl3); νmax (neat)/
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
147

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(2R,3R)-1-Benzyl-3-hydroxy-2-(4-methoxybenzyl)pyrrolidine
24. To a solution of the chloride salt 23 (0.269 g, 0.634 mmol)
in methanol (10 cm3) was added 5% Pd/C (0.027 g) and potas-
sium carbonate (0.263 g, 1.90 mmol). The mixture was exposed
to a hydrogen atmosphere (1 atm) and stirred vigorously for
20 minutes. The suspension was filtered through a pad of
Celite and concentrated under reduced pressure. To the residue
was added CH2Cl2 (25 cm3) and water (25 cm3). The organic
phase was separated and the aqueous phase was extracted with
CH2Cl2 (3 × 25 cm3); the combined organic phase were washed
with water (25 cm3) and then dried and concentrated under
reduced pressure. The residue was chromatographed on silica
gel [hexane : EtOAc (1 : 1)] to give pyrrolidine 24 (0.150 g,
80%) as an oil, Rf [hexane : EtOAc (1 : 1)] 0.35; [α]D Ϫ104.0
(c 1.18, CHCl3); νmax (neat)/cmϪ1 3418, 2934, 1611, 1583, 1512;
δH (250 MHz; CDCl3) 7.41–7.26 (7H, m), 6.89 (2H, d, J 8.7),
4.20 (1H, d, J 13.0), 3.96 (1H, br s), 3.83 (3H, s), 3.26
(1H, d, J 13.0), 3.05 (1H, dt, J 8.8, 3.7), 2.98–2.86 (2H, m),
2.49 (1H, ddd, J 8.5, 6.0, 3.7), 2.20–2.06 (1H, m), 1.99 (1H,
ddd, 13.7, 6.4, 3.7), 1.75–1.63 (1H, m); δc (62.9 MHz; CDCl3)
158.4, 139.4, 131.9, 130.7 (2C), 129.3 (2C), 128.7 (2C), 127.4,
114.2 (2C), 72.5, 71.3, 58.3, 55.7, 51.9, 33.4, 32.7; m/z (FAB)
298 ([M ϩ H]ϩ, 90%), 176 (93), 154 (62), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 298.1805. C19H24NO2 requires m/z
298.1805).
(2R,3R)-3-Hydroxy-2-(4-methoxybenzyl)pyrrolidine 6.
A
solution of the chloride salt 23 (0.243 g, 0.574 mmol) and
Pearlman’s catalyst [0.243 g; 20% Pd(OH)2/C] in methanol
(7 cm3) was exposed to a hydrogen atmosphere (1 atm) and
stirred vigorously for 24 hours. The mixture was then filtered
through a pad of Celite and concentrated under reduced pres-
sure to give (2R,3R)-3-hydroxy-2-(4-methoxybenzyl)pyrrolidine
hydrochloride (0.133 g, 95%) as a white solid, mp 261–263 ЊC;
[α]D ϩ15.2 (c 1.12, CH3OH); νmax (KBr)/cmϪ1 3330, 3302, 2955,
1614, 1556, 1516; δH (250 MHz; CD3OD) 7.35 (2H, d, J 8.7),
6.99 (2H, d, J 8.7), 4.39–4.36 (1H, m), 3.87 (3H, s), 3.67 (1H,
ddd, J 8.5, 6.6, 2.8), 3.55 (1H, ddd, J 11.5, 9.9, 8.2), 3.57–3.23
(1H, m), 3.24 (1H, dd, J 14.2, 6.6), 3.02 (1H, dd, J 14.2, 8.5),
2.32 Ϫ2.10 (2H, m); δc (62.9 MHz; CD3OD) 158.4, 129.2 (2C),
128.0, 113.4 (2C), 69.1, 66.0, 53.8, 42.3, 32.2, 30.8; m/z (FAB)
208 ([M ϩ H]ϩ, 97%), 154 (100), 136 (85), 121 (15); HRMS
(FAB) (Found: [M ϩ H]ϩ, 208.1340. [C12H18NO2]ϩClϪ requires
m/z 208.1338).
The HCl salt was subjected to ion-exchange chromatography
(Dowex OHϪ; prepared by treating Dowex 1-X2 with 1 M
aqueous NaOH, followed by methanol until the pH of the
eluent returned to 7). Eluting with methanol gave the free base
6 (quantitative recovery) as a white solid, Rf [CH2Cl2 : MeOH
(95 : 5)] 0.05; mp 127–128 ЊC; [α]D Ϫ30.9 (c 1.36, CH3OH);
νmax (KBr)/cmϪ1 3426, 3290, 2961, 1612, 1583, 1512; δH (250
MHz; CD3OD) 7.23 (2H, d, J 8.7), 6.87 (2H, d, J 8.7), 4.06 (1H,
ddd, J 4.9, 3.1, 1.5), 3.79 (3H, s), 3.17 (1H, ddd, J 11.1, 8.5, 7.5),
2.96 (1H, dd, J 7.1, 3.2), 2.90 (1H, dd, J 7.1, 2.5), 2.79 (1H, ddd,
J 11.1, 9.7, 4.7), 2.85–2.67 (1H, m), 2.01 (1H, dddd, J 13.7, 9.7,
7.5, 4.9), 1.83 (1H, dddd, J 13.7, 8.5, 4.7, 1.5); δc (62.9 MHz;
CD3OD) 158.6, 132.1, 130.0 (2C), 113.8 (2C), 71.9, 66.2, 54.6,
43.6, 35.0, 34.1; m/z (FAB) 208 ([M ϩ H]ϩ, 100%), 190 (11), 121
(27), 91 (69); HRMS (FAB) (Found: [M ϩ H]ϩ, 208.1333.
C12H18NO2 requires m/z 208.1338).
(2R,3R)-3-Acetoxy-1-benzyl-2-(4-methoxybenzyl)pyrrolidine
25. To a solution of the alcohol 24 (0.0643 g, 0.216 mmol) in
CH2Cl2 (5 cm3) was added freshly distilled acetic anhydride
(0.044 g, 0.041 cm3, 0.43 mmol), and triethylamine (0.044 g,
0.061 cm3, 0.43 mmol). The solution was stirred for 18 hours
and then quenched by the addition of saturated aq. sodium
bicarbonate (20 cm3). The organic phase was separated and the
aqueous phase was extracted with CH2Cl2 (3 × 20 cm3); the
combined organic phase was dried and concentrated under
reduced pressure. The residue was chromatographed on silica
gel [hexane : EtOAc (7 : 3)] to give the ester 25 (0.0638 g, 87%)
as a colourless oil, Rf [hexane : EtOAc (1 : 1)] 0.60; [α]D Ϫ123.1
(c 0.26, CHCl3); νmax (neat)/cmϪ1 2935, 2791, 1733, 1612, 1581,
1513; δH (250 MHz; CDCl3) 7.40–7.33 (5H, m), 7.22 (2H, d,
J 8.7), 6.86 (2H, d, J 8.7), 5.01 (1H, ddd, J 8.8, 5.1, 3.4), 4.12
(1H, d, J 13.1), 3.39 (3H, s), 3.36 (1H, d, J 13.1), 2.97 (1H, dd,
J 8.7, 1.8), 2.94 (1H, dd, J 13.3, 5.1), 2.78 (1H, dd, J 13.3,
9.5,), 2.66 (1H, dt, J 9.5, 5.1, C2H ), 2.17 (2H, m), 2.14 (3H, s),
1.69–1.54 (1H, m); δc (62.9 MHz; CDCl3) 171.1, 158.4, 138.8,
131.5, 130.2 (2C), 129.6 (2C), 128.7 (2C), 127.5, 114.3 (2C),
75.2, 68.8, 58.8, 55.7, 52.1, 33.8, 31.1, 21.7; m/z (FAB) 340
([M ϩ H]ϩ, 70%), 280 (28), 218 (52), 121 (43), 91 (100); HRMS
(FAB) (Found: [M ϩ H]ϩ, 340.1916. C21H26NO3 requires m/z
340.1913).
SAPK pathway activation
Cell culture and stimulation.3c Murine RAW 264.7 macro-
phages were purchased from the European Tissue Culture
Collection and maintained in a 95% air/5% CO2 37 ЊC atmos-
phere. Cells were cultured in Dulbecco’s Modified Eagles’
Medium (DMEM) containing 10% heat inactivated foetal calf
serum, 100 U mlϪ1 penicillin, 100 µg mlϪ1 streptomycin and
2 mM -glutamine. Anisomycin was dissolved at 10 mg mlϪ1 in
DMSO. Cells were stimulated for 30 min by the addition of 1 µl
of 10 mg mlϪ1 anisomycin or an anisomycin analogue in
DMSO, or 1 µl of DMSO as control, to each 1 ml of culture
medium. The final concentration of DMSO in the culture
medium was therefore always 0.1% (v/v).
Cell lysis.3c After stimulation, the media was aspirated, the
cells from each dish washed with ice cold PBS and then lysed in
250 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA,
0.5 mM sodium orthovanadate, 5 mM sodium β-glycero-
phosphate, 25 mM sodium fluoride, 2.5 mM sodium pyrophos-
phate, 0.135 M sucrose, 0.5% (v/v) Triton X-100, 5% (v/v)
glycerol, 0.05% (v/v) 2-mercaptoethanol, 0.5 µM microcystin-
LR and ‘Complete’ protease inhibitor cocktail (one tablet per
100 ml)] and 1% (w/v) SDS. Protein concentrations were
determined according to Bradford.
(2R,3R)-3-Acetoxy-2-(4-methoxybenzyl)pyrrolidine
hydro-
chloride 3. To a solution of the pyrrolidine 25 (0.0527 g, 0.155
mmol) and Pearlman’s catalyst [0.0527 g; 20% Pd(OH)2/C]
in methanol (5 cm3) was added hydrochloric acid (1 M in
ether; 0.31 cm3, 0.31 mmol]. The solution was exposed to a
hydrogen atmosphere (1 atm) and stirred vigorously for
20 hours. The mixture was then filtered through a pad of Celite
and concentrated under reduced pressure to give hydrochloride
3 (0.0450 g, 100%) as a pale brown oil, [α]D Ϫ46.3 (c 0.56,
CHCl3); νmax (neat)/cmϪ1 3398, 2934, 1741, 1613, 1581, 1515;
δH (250 MHz; CD3OD) 7.32 (2H, d, J 8.4), 7.00 (2H, d, J 8.4),
5.40 (1H, br s), 4.02 (1H, br s), 3.86 (3H, s), 3.54 (2H, br q,
J 8.6), 3.18 (1H, dd, J 14.1, 6.2), 3.06 (1H, dd, J 14.1, 8.4),
2.52–2.44 (1H, m), 2.31–2.18 (1H, m), 2.27 (3H, s); δc (62.9
MHz; CD3OD) 169.3, 158.6, 129.1 (2C), 127.1, 113.6 (2C),
72.4, 64.1), 53.8, 42.4, 30.7, 29.9, 19.0; m/z (FAB) 250 ([M ϩ
H]ϩ, 100%), 190 (36), 128 (22), 121 (32), 91 (41); HRMS (FAB)
(Found: [M ϩ H]ϩ, 250.1442. [C14H20NO3]ϩClϪ requires m/z
250.1443).
Immunoblotting.3c Samples were denatured in SDS, run on
polyacylamide gels and transferred to nitrocellulose mem-
branes. The membranes were then incubated for 1 h at room
temperature in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2%
(v/v) Tween and 5% (w/v) skimmed milk powder. Antibodies
that recognise c-Jun and MAPKAP-K2 phosphorylated at
Thr334 were purchased from Cell Signalling Technologies
(Hitchin, UK), while an antibody that recognises all forms of
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 4 2 – 1 4 9
148

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MAPKAP-K2 was raised in sheep against a peptide corre-
sponding to the C-terminus of MAPKAP-K2 (MTSALATM-
RVDYEQIK). Antibodies were added to 10 ml of the above
buffer except that the skimmed milk was replaced by 5% (w/v)
BSA. Antibodies were then incubated with the membranes on a
shaker at 4 ЊC overnight. The membranes were then washed
four times with buffer (five min per wash) to remove the pri-
mary antibodies, and secondary antibodies were incubated with
the membranes for 1 h at room temperature. After washing six
times with buffer to remove the secondary antibodies (5 min per
wash), immunoreactive proteins were visualised via enhanced
chemiluminescence (ECL) reagent according to the manu-
facturer’s instructions.
References and notes
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Acknowledgements
We thank the BBSRC (Studentship 99/A1/B/05153 to EMR),
Pfizer (Summer Studentship to KSA), the MRC (Studentship
to SM) and the MRC and The Royal Society (PC) for financial
support of this work.
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