Syntheses and bio-activities of the l-enantiomers of two potent transition state analogue inhibitors of purine nucleoside phosphorylases

Article abstract of DOI:10.1039/b517883e

(1R)-1-(9-Deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-l-ribitol [(+)-5] and (3S,4S)-1-[(9-deazahypoxanthin-9-yl)methyl]-4-(hydroxymethyl)pyrrolidin-3-ol [(-)-6] are the l-enantiomers of immucillin-H (d-ImmH) and DADMe-immucillin-H (d-DADMe-ImmH), respectively, these d-isomers being high affinity transition state analogue inhibitors of purine nucleoside phosphorylases (PNPases) developed as potential pharmaceuticals against diseases involving irregular activation of T-cells. The C-nucleoside hydrochloride d-ImmH [(-)-5)·HCl], now "Fodosine" is in phase II clinical trials as an anti-T-cell leukaemia agent, while d-DADMe-ImmH is a second generation inhibitor with extreme binding to the target enzyme and has entered the clinic for phase I testing as an anti-psoriasis drug. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the l-nucleoside analogues (+)-5·HCl and (-)-6, respectively, of d-ImmH and d-DADMe-ImmH, were prepared and their PNPase binding properties were studied. For the synthesis of compound (-)-6 suitable enzyme-based routes to the enantiomerically pure starting material (3S,4S)-4-(hydroxymethyl)pyrrolidin-3-ol [(-)-16] and its enantiomer were developed. The l-enantiomers (+)-5·HCl and (-)-6 bind to the PNPases approximately 5- to 600-times less well than do the d-compounds, but nevertheless remain powerful inhibitors with nanomolar dissociation constants. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b517883e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Syntheses and bio-activities of the L-enantiomers of two potent transition state
analogue inhibitors of purine nucleoside phosphorylases
Keith Clinch,*a Gary B. Evans,a George W. J. Fleet,*b Richard H. Furneaux,a Stephen W. Johnson,b
Dirk H. Lenz,a Simon P. H. Mee,a Peter R. Rands,b Vern L. Schramm,*c Erika A. Taylor Ringiac and
Peter C. Tylera
Received 16th December 2005, Accepted 12th January 2006
First published as an Advance Article on the web 26th January 2006
DOI: 10.1039/b517883e
(1R)-1-(9-Deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-L-ribitol [(+)-5] and
(3S,4S)-1-[(9-deazahypoxanthin-9-yl)methyl]-4-(hydroxymethyl)pyrrolidin-3-ol [(−)-6] are the
L-enantiomers of immucillin-H (D-ImmH) and DADMe-immucillin-H (D-DADMe–ImmH),
respectively, these D-isomers being high affinity transition state analogue inhibitors of purine nucleoside
phosphorylases (PNPases) developed as potential pharmaceuticals against diseases involving irregular
activation of T-cells. The C-nucleoside hydrochloride D-ImmH [(−)-5)·HCl], now “FodosineTM” is in
phase II clinical trials as an anti-T-cell leukaemia agent, while D-DADMe–ImmH is a second
generation inhibitor with extreme binding to the target enzyme and has entered the clinic for phase I
testing as an anti-psoriasis drug. Since the enantiomers of some pharmaceuticals have revealed
surprising biological activities, the L-nucleoside analogues (+)-5·HCl and (−)-6, respectively, of
D-ImmH and D-DADMe–ImmH, were prepared and their PNPase binding properties were studied. For
the synthesis of compound (−)-6 suitable enzyme-based routes to the enantiomerically pure starting
material (3S,4S)-4-(hydroxymethyl)pyrrolidin-3-ol [(−)-16] and its enantiomer were developed. The
L-enantiomers (+)-5·HCl and (−)-6 bind to the PNPases approximately 5- to 600-times less well than do
the D-compounds, but nevertheless remain powerful inhibitors with nanomolar dissociation constants.
R
Levovirinꢀ (2) have widely different anti-viral activities and,
Introduction
notably, the latter shows a much more favourable safety profile
R
than does the D-ribofuranose-based isomer.5 Lamivudineꢀ (3)
Chiral components of natural products occur predominantly in
one of their enantiomeric forms, these being, in particular, the L-
and D-modifications, respectively, of the a-amino acids and sugars.
was the first L-nucleoside derivative to be approved by the FDA
for use in combination therapy against HIV and hepatitis B
virus.4 In an entirely different sphere of fundamental biochemical
interest some L-sugars, particularly a-L-arabinopyranose, have
attracted attention since, when incorporated into (4ꢀ-2ꢀ)-linked
oligonucleotides, they lead to unusually strong Watson–Crick
base-pairing.6
Work in the area of synthetic drug discovery is usually restricted
to the investigation of compounds in their natural enantiomeric
forms and, in this context, it has been unusual until recently for
such substances as peptides and nucleosides to be made with D-
amino acids or L-sugars, respectively. It appears, however, from
many new observations, that this practice has been too restrictive.
Recent work has revealed that derivatives of the unusual L-
enantiomers of natural sugars and nucleosides can show con-
siderable potential as pharmaceuticals, and such is the perceived
value of L-nucleoside analogues that the chemistry and biological
activities of these compounds have been studied extensively and
reviewed on several occasions.1–3 2ꢀ-Deoxy-L-cytidine (1) and
several other L-nucleosides show potent and specific activity
against hepatitis B virus replication, and related unnatural nu-
cleosides have been tested as potential anti-viral and anti-cancer
In our laboratories we have turned attention to the bio-activities
of L-enantiomers of sugar-based specific inhibitors of glycoside-
and nucleoside-processing enzymes, and have encountered L-
compounds with inhibitory activities quite different from those
of the D-analogues. For example, the L-enantiomer 4 (L-DMDP)
of 2,5-dideoxy-2,5-imino-D-mannitol is several times more active
against some a-D-glucosidases than is its enantiomer, while it
is inactive against some b-D-glucosidases which are inhibited
by D-DMDP.7 These studies have been extended to compar-
isons of the inhibitory effects of the enantiomers of 5- and 6-
membered analogues of “nitrogen-in-the-ring compounds” on D-
glycohydrolases,8 and other workers have investigated a range
of both D- and L-isomers of pyranoid analogues, i.e. of 1-
deoxynojirimycin, as glycosidase inhibitors.9
R
compounds.1–4 More specifically, Ribavirinꢀ and its L-enantiomer
aCarbohydrate Chemistry Team, Industrial Research Limited, P.O. Box
31310, Lower Hutt, New Zealand. E-mail: k.clinch@irl.cri.nz
bChemistry Research Laboratory, Department of Chemistry, University
of Oxford, Mansfield Road, Oxford, UK OX1 3TA. E-mail: george.fleet@
chemistry.oxford.ac.uk
The structures of the family of immucillins, which are high
affinity transition state analogue inhibitors of PNPases, are based
on “nitrogen in the ring” D-ribofuranosyl C-glycosidic analogues
of natural nucleosides (and closely related compounds), and
cBiochemistry Department, Albert Einstein College of Medicine of Yeshiva
University, 1300 Morris Avenue, Bronx, New York, 10461, USA. E-mail:
vern@aecom.yu.edu
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their enzymic inhibitory actions have been studied in connection
with their development as pharmaceuticals, active in particular,
against T-cell-mediated disorders.10 We report now the synthesis
of the L-enantiomers (+)-5·HCl and (−)-6 of two of the most
biologically potent members of the group, D-ImmH11 and D-
DADMe-ImmH,12,13 and a brief comparison of the activities as
inhibitors of PNPases of these four compounds.
mainly the product 12 by displacement of the primary ester
group (42%), together with the diazide 13 (15%), and the starting
material 10 (34%). Hydrogenation of compound 12 in 1,4-dioxane
in the presence of palladium black and sodium acetate gave the
iminoribitol derivative 11 (94%) required for the synthesis of
L-C-nucleoside analogues. That the ring-closure reaction 12 →
11 occurred with inversion of configuration is evidenced by the
structure of compound 11 which is the enantiomer of the D-
iminoribitol derivative used to make ImmH.11 All comparable
1H NMR signals of these two enantiomers resonated within
0.02 ppm of each other, and the specific rotations of the D- and
L-compounds were (+)- and (−)-15.5, respectively. The former
figure is apparently recorded for the first time.11,15,16 The structure
and absolute configuration of the D-enantiomer, and hence of
compound 11, follow from the X-ray diffraction analysis of D-
ImmH17 and also from the method of synthesis of this compound
from D-gulono-lactone.11
Conversion of the iminoribitol 11 to the imine 14 was effected,
as for its enantiomer, by N-chlorination with N-chlorosuccinimide
in pentane followed by elimination of HCl by treatment of the
chloro derivative with lithium tetramethylpiperidide in THF.11,15,16
Coupling of 14 with lithiated deazapurine derivative 15, and
modification of the product in the way described for mak-
ing D-ImmH,11b afforded the required L-enantiomer (+)-5·HCl
(Scheme 2). This product gave NMR spectral data that identified
it as the enantiomer of D-ImmH·HCl. While the L-salt (+)-5·HCl
had [a]2D5 +51.5, the literature value for the D-salt is recorded as
−0.50.11a Remeasurement of the latter figure during the present
work gave −53.6, the discrepancy being identified as being due to
a spurious factor of 100 mistakenly used in the calculation of the
first value.
Results and discussion
Preparation of (1R)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-
1,4-imino-L-ribitol hydrochloride [L-ImmH, (+)-5·HCl]
For the synthesis of L-ImmH [(+)-5·HCl] the iminoribitol deriva-
tive 11 (the enantiomer of the compound used for the preparation
of D-ImmH11) was made from 2,3-O-isopropylidene-D-lyxono-1,4-
lactone (7)14 as outlined in Scheme 1. Reduction of the silylated
lactone 8 with lithium borohydride in THF afforded the readily
crystallised diol 9 in 75% yield, whereas other reducing agents
gave several products arising from silyl group migration to which
diol 9 is particularly prone. Mesylation of 9 afforded diester 10
which, with one equivalent of sodium azide in hot DMF, gave
Scheme 2 Reagents and conditions: See ref. 11b.
Preparation of (3S,4S)-1-[(9-deazahypoxanthin-9-yl)methyl]-4-
(hydroxymethyl)pyrrolidin-3-ol [L-DADMe-Imm H, (−)-6]
Our extensive studies in the immucillin field10–13 have led to
the preparation of D-DADMe–ImmH [(+)-6] which is an even
more potent inhibitor of human PNPase than is D-ImmH [(−)-
5]·HCl.12 The L-enantiomer [(−)-6], therefore, was of interest
within the present study, and we prepared it using the second
method previously adopted for making the D-compound,13 i.e. by
Mannich coupling of amine (−)-16 with deazahypoxanthine 17
in the presence of formaldehyde as illustrated in Scheme 3. For
this purpose ready access to pyrrolidine (−)-16 was required, and
the opportunity was taken to develop efficient syntheses of both
the D- and L-isomers of this amine which were isolated as their
respective hydrochlorides [(+)- and (−)-16·HCl]. These salts have
been known as components of mixed stereoisomers since 1965,18
and for a few years, as the “carbohydrate-parts” of racemic N–Cꢀ19
and N–Nꢀ20 linked nucleoside analogues.
Scheme 1 Reagents and conditions: (a) TBDMSCl, imidazole, DMF,
N2, 20 ◦C, 4 h (91%); (b) LiBH4, THF, N2, −30 to 20 ◦C, 24 h, (75%);
(c) (MeSO2)2O, pyridine, N2, 0 to 20 ◦C, 18 h (88%); (d) NaN3, DMF,
N2, 100 ◦C, 2 h (42% 12, 15% 13); (e) H2, Pd black, NaOAc, 1,4-dioxane,
20 ◦C, 72 h, (94%).
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enantioselective work was commenced, however, the racemic
hydroxyester ( )-18 was converted by standard methods to diester
( )-19 and diol ( )-20 which were required as reference materials.
The enzyme-catalysed trans-acetylation of racemic hydroxyester
( )-18 from vinyl acetate was readily followed by 1H NMR
monitoring of the ring proton on the hydroxyl-bearing ring
carbon atom (C-4) which underwent deshielding from d 4.5
to 5.4 on esterification. When the acetylation was performed
in tert-butyl methyl ether at 40 ◦C, 50% conversion occurred
within 2 h, and after another 5 h insignificant further reaction
took place. Chromatographic separation gave the crystalline
unreacted alcohol (+)-18 and the ester (−)-19 in 85 and 97%
yields, with their specific rotations being +16.9 and −41.5,
respectively.
These products proved to be of surprisingly high enantiomeric
purity. Conversion of the alcohol (+)-18 to (+)-16·HCl was
achieved by LiAlH4 reduction of the ester to give diol (+)-20
followed by de-N-benzylation (Scheme 4), and afforded a hy-
drochloride salt with [a]2D1 +19.1. Alternatively, the enantiospecific
sequence (+)-18 → (+)-20 → (+)-23 → (+)-16·HCl was employed,
(+)-23 being useful for preparing D-DADMe–ImmH derivatives.
Made by this route compound (+)-23 had [a]2D1 +15.9, whereas
a sample prepared from 1,2:5,6-di-O-isopropylidene-a-D-glucose
via N-Boc-carbamate 22 (made in an analogous way to that used
previously to make the N-Fmoc carbamate 2126) had [a]D21 +16.2,
and that of the salt (+)-16·HCl derived from (+)-23 had [a]D21
+18.9. This figure is in good agreement with that given by (+)-
16·HCl derived directly from diol (+)-20, and also with literature
data.23 The specific rotation results show that the alcohol (+)-18
was of high enantiomeric purity, and attention turned to that of
the ester (−)-19. Conversion of this product of selective enzymic
acetylation to the corresponding hydrochloride (−)-16·HCl via
(−)-20 afforded the salt with [a]2D1 −18.9, indicating that it too was
of high enantiomeric purity. As expected, the 1H NMR spectra of
Scheme 3 Reagents and conditions: (a) HCHO (30%, aq.), H2O, 85 ◦C,
15 h (48%).
As for the individual enantiomers of pyrrolidine 16·HCl,
the D-compound (3R,4R) has been made in high enantiomeric
excess by methods involving application of the asymmetric
Sharpless epoxidation reaction, followed by cyanide opening
of the epoxide, applied to an unsymmetrically substituted but-
2-en-1,4-diol,21 and by addition of (S)-1-phenylethylamine to
an ethyl 2-silyloxy-3-methoxycarbonyl-but-3-enoate.22 Both enan-
tiomers have also been obtained by a key 1,3-dipolar cycload-
dition of chiral azomethine ylides to 3-benzyloxy-substituted
alkenoylcamphorsultams,23 and this procedure has been adapted
to afford a large scale synthesis of (+)-16.24 Otherwise, the D-
enantiomer has been obtained exclusively by multi-step, low
yielding routes from D-xylose25 and D-glucose.26
Encouraged by the appreciable success reported for the lipase-
catalysed enantioselective acetylation of tert-butyl trans-( )-3-
hydroxy-4-(hydroxymethyl)pyrrolidine-1-carboxylate,27 and espe-
cially by the highly efficient resolution of cyclopentyl, cyclo-
hexyl and cycloheptyl cis- and trans-b-hydroxy esters permitted
by enantioselective acylation catalysed by lipase B of Candida
R
antarctica (Novozymeꢀ 435),28 we have applied this lipase to the
resolution of racemic ethyl trans-1-benzyl-4-hydroxypyrrolidine-3-
carboxylate18 [( )-18, Scheme 4]. This approach to the preparation
of the enantiomers (+)- and (−)-16 is very efficient. Before this
Scheme 4 Reagents: (a) NovozymeꢀR 435, tert-BuOMe, CH2 CHOAc; (b) Ac2O, Py; (c) LiAlH4, THF, Et2O; (d) (i) HCO2H, MeOH, Pd/C; (ii) HCl,
MeOH, H2O; (e) (i) NaIO4, H2O; (ii) NaBH4; (f) HCl, MeOH, H2O; (g) (tert-BuOCO)2O, MeOH, H2, Pd/C; (h) as Scheme 3.
=
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the various samples of the enantiomeric hydrochlorides 16·HCl,
made by the different routes described above, were identical, and
were consistent with the spectrum described in the literature.23
It was not possible to determine the selectivity of the en-
zymic acetylation reaction by direct measurement. However, the
specific rotations of several samples of each enantiomer of the
hydrochlorides 16·HCl prepared during the present work, were
all 19.0 0.1. In consequence, it was concluded that the enzyme
had acted, when presented with racemate ( )-18, efficiently and
also specifically with the (−)-enantiomer to within 3%, i.e. the
estimated error of measurement of the specific rotation of the
products. To test this conclusion, a further enzymic acetylation
of the substrate was conducted and stopped before completion
when the 18 : 19 ratio was approximately 1.2 : 1.0 (1H NMR
determination). Consistent with expectations, the sample of the
ester 19 obtained by chromatographic separation in this case had
[a]2D1 −41.8, indicating it was the pure (−)-enantiomer. Further,
the alcohol 18 that remained had [a]2D1 +14.0 showing that it
contained mainly the unreacted (+)-isomer, but was contaminated
with roughly 10% of the unreacted (−)-enantiomer.
To ensure that the inhibition observed with the L-DADMe–
ImmH [(−)-6] was not due to small proportions of residual,
more active, D-compound, samples of the L-enantiomer were pre-
treated with 0.01 to 1.0 molar equivalents of human PNPase
and the mixtures were subjected to ultrafiltration. In this way,
L-DADMe ImmH [(+)-6] gave a sample that inhibited PNPases
with kinetic parameters unchanged relative to those of the original
preparation. Accordingly, based on the error limits of the kinetic
constant for inhibition, it was concluded that no more than 2% of
the D-enantiomer could have been present as a contaminant in the
initial inhibitor (−)-6.
The L-enantiomer (+)-5·HCl of ImmH is revealed to be a slow
onset tight binding inhibitor of the PNPases of human, bovine
and Plasmodium falciparum (the protozoan parasite responsible
for malaria) origins. While it is, approximately, 15- to 600-fold less
active than the D-enantiomer against the enzymes studied, it still
shows surprising potency, and it seems remarkable that it is more
active by factors of >10 as an inhibitor of the human enzyme
than are the 3ꢀ- and 5ꢀ-monodeoxy analogues of D-ImmH.30 The
L-DADMe–ImmH [(−)-6] shows similar tendencies being from
5- to 160-times less active than its D-enantiomer against the three
enzymes. Nevertheless, it is still a sub-nanomolar inhibitor against
both the human and bovine enzymes, and in other circumstances
could be considered potent.
Conversion of (+)-16·HCl to (+)-6 has been reported
previously;12,13 and the alternative synthesis of (+)-16 now de-
scribed opened an alternative pathway to this nucleoside analogue.
The sample of (+)-6 obtained in this way had [a]2D1 +16.9. Likewise,
the target compound (−)-6, derived from pure (−)-16 (Scheme 3),
gave a 1H NMR spectrum identical to that reported and observed
for the D-enantiomer,12,13 and had [a]D21 −16.8.
Experimental
General
Imidazole was recrystallised from CH2Cl2. All other reagents were
used as supplied; anhydrous solvents were obtained commercially.
Air sensitive reactions were carried out under argon unless
otherwise stated. Organic solutions were dried over MgSO4 and the
solvents were evaporated under reduced pressure. Chromatogra-
phy solvents were distilled prior to use. Thin layer chromatography
(t.l.c.) was performed on glass or aluminium sheets coated with
60 F254 silica. Organic compounds were visualised under uv light
or by use of a spray or dip of cerium(IV) sulfate (0.2%, w/v) and
ammonium molybdate (5%) in sulfuric acid (2 M), one of I2 (0.2%)
and KI (7%) in H2SO4 (1 M) or, for nitrogen-containing
compounds, p-(N,N-dimethylamino)benzaldehyde (1%) in HCl
Biodata of compounds
Kinetic studies of the interactions between compounds (+)-5·HCl,
(−)-5·HCl, (+)-6 and (−)-6 and human, plasmodial and bovine
PNPases were carried out by the methods previously reported,29,30
and the results are given in Table 1. The inhibition constants Ki
are the dissociation constants for the enzyme–inhibitor complex
measured from initial reaction rates. For many, but not all,
immucillin inhibitors, a slow onset of inhibition then occurs
consequent upon a time-dependent conformational change in the
enzyme that leads to tighter binding characterised by the constant
Ki*.31
Table 1 Kinetic data for the inhibition of human, plasmodial and bovine PNPases by the enantiomers of ImmH [(−)-5·HCl and (+)-5·HCl] and
DADMe–ImmH [(+)-6 and (−)-6]
Compound
Enzyme source
H. sapiens
Ki/nM
Ki*/nM
Reference
D-ImmH [(−)-5]·HCl
0.072 0.026
0.056 0.015
0.6 0.1
29
10, 30
32
3.3 0.2
29
Plasmodium falciparum
8
0.86
33
B. taurus
41
18
32
8
6
9
0.023 0.005
0.9 0.5
29
L-ImmH [(+)-5]·HCl
H. sapiens
This work
This work
This work
12
Plasmodium falciparum
B. taurus
H. sapiens
Plasmodium falciparum
B. taurus
H. sapiens
9
14
6
4
360 60
1.1 0.1
0.50 0.04
2.1 0.3
1.5 0.1
1700 300
D-DADMe–ImmH [(+)-6]
L-DADMe–ImmH [(−)-6]
0.016 0.001
Not observed
0.110 0.014
0.68 0.26
34
This work
This work
This work
This work
Plasmodium falciparum
B. taurus
80
7
19
5
0.5 0.1
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(37%)–MeOH, 1 : 3 (100 ml) (Erlich reagent). Flash column
chromatography was performed on Sorbsil C60 40/60 silica,
Scharlau or Merck silica gel 60 (40–60 lm). Melting points were
recorded on a Ko¨fler hot block or 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.
(C-3), 76.0 (C-2), 79.4 (C-4), 114.1 [C(CH3)2], 173.7 (C-1); HRMS
(CI+) m/z 303.1625; C14H27O5Si [(M + H)+] requires 303.1628.
(Found: C, 55.7; H, 8.2%; C14H26O5Si requires C, 55.6; H, 8.7%).
5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-D-lyxitol (9)
Lithium borohydride (4 ml, 2 M in THF, 8 mmol) was added
dropwise to a stirred solution of the TBDMS ether 8 (1.21 g,
4.00 mmol) in THF (4 ml) and the stirring was continued at −30 ◦C
under an atmosphere of nitrogen. After 5 h, t.l.c. analysis (EtOAc–
cyclohexane, 1 : 1) showed residual starting material as well as a
major product (Rf 0.35). More lithium borohydride (2 ml, 2 M
in THF 4 mmol) was added and the mixture was left stirring at
−30 ◦C for a further 2 h. The reaction mixture was then allowed
to assume room temperature. After a further 16 h, t.l.c. analysis
(EtOAc–cyclohexane, 1 : 2) indicated most of the starting material
had reacted. A solution of aqueous ammonium chloride (4 ml,
saturated) was then added dropwise to the reaction mixture which
was partitioned between EtOAc (20 ml) and brine (20 ml). The
organic layer was then collected, washed with water (3 × 10 ml) and
dried. The solvent was removed and the residue was crystallized
from THF to afford the diol 9 (910 mg, 75%), mp 67–68 ◦C,
[a]2D3 −9.2 (c, 0.08, CHCl3); mmax (thin film) 3400 (broad, O–H);
NMR dH (400 MHz; CDCl3) 0.08 (6 H, s, SiMe2), 0.91 (9 H, s,
SitBu), 1.38, 1.50 [2 × 3 H, 2s, C(CH3)2], 2.78–2.91 (2 H, br s,
NMR spectra were recorded on a Bruker AC300E or Bruker
DPX 400 spectrometer. 1H spectra at 300 or 400 MHz were
measured in CDCl3, CD3OD or CD3CN (internal reference
Me4Si, d 0), and 13C spectra at 75.5 or 100.6 MHz in CDCl3
(reference, solvent centre line, d 77.0), CD3OD (reference, solvent
centre line d 49.0) or CD3CN (reference, solvent centre line
d 118.7, CN). Assignments of 1H and 13C resonances were
1
based on 2D (1H–1H DQF-COSY, H–13C HSQC) spectra, and
DEPT experiments gave unambiguous data on the numbers of
protons bonded to each carbon atom. The assignments of the
13C resonances were consistent with the multiplicities observed.
Coupling constants (J) are quoted in Hz. Infrared spectra were
recorded on a Perkin-Elmer 1750 IR Fourier Transform, or Perkin-
Elmer Paragon 1000 spectrophotometer using thin films on NaCl
plates (thin film). Only characteristic absorptions are quoted.
Electrospray ionisation (ES) low resolution mass spectra (m/z)
were measured on a Micromass BioQ II-ZS mass spectrometer.
For high resolution mass spectra (HRMS), ES data were collected
on a Waters 2790-Micromass LCT mass spectrometer operated
at a resolution of 5000 full width half height. Positive ion
electrospray ionisation (ES+) spectra were calibrated relative to
PEG with tetraoctylammonium bromide as the internal lock
mass. Negative ion ES spectra were calibrated relative to poly-DL-
alanine with Leu-enkephalin as the internal lock mass. Chemical
ionization (CI, NH3) HRMS were recorded on a Micromass 500
OAT spectrometer. Positive ion fast atom bombardment (FAB+)
HRMS were measured on a VG 7070 instrument in a glycerol
matrix, and positive ion electron impact (EI+) HRMS were
measured on a VG 70SE instrument. Microanalyses were carried
out by the Campbell Microanalytical Laboratory, University of
Otago or in the Department of Chemistry, University of Oxford.
ꢀ
ꢀ
OH), 3.63 (1 H, dd, J5,5 10.0, J5,4 6.8, H-5), 3.73 (1 H, dd, J5 ,4
6.2, H-5ꢀ), 3.77–3.82 (3 H, m, H-1,1ꢀ,4), 4.82 (2 H, m, H-2,3); dC
(100.6 MHz; CDCl3) −5.5, −5.4 [Si(CH3)2], 18.3 [C(CH3)3], 25.0
[C(CH3)2], 25.8 [C(CH3)3], 27.1 [C(CH3)2], 61.4 (C-1), 64.6 (C-5),
69.2 (C-4), 75.7, 77.3 (C-2, C-3), 110.4 [C(CH3)2]; HRMS (ES–)
m/z 305.1796; C14H29O5Si ([M–H]−) requires 305.1784. (Found:
C, 54.75; H, 10.0; C14H30O5Si requires C, 54.9; H, 9.9%).
5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-1,4-di-O-
methanesulfonyl-D-lyxitol (10)
A solution of the diol 9 (2.40 g, 8.16 mmol) in CH2Cl2 (3.5 ml) was
added dropwise to a mixture of 4-(N,N-dimethylamino)pyridine
(0.50 g, 4.08 mmol) and methanesulfonic anhydride (6.36 g,
36.5 mmol) in dry pyridine (13 ml) at 0 ◦C under an atmosphere
of nitrogen. The reaction mixture was stirred for 30 min at 0 ◦C,
then at 20 ◦C for 18 h, after which time t.l.c. analysis (EtOAc–
cyclohexane, 1 : 1) indicated the presence of a major product (Rf
0.54). The reaction mixture was then diluted with CH2Cl2 (100 ml)
and aqueous HCl (0.1 M, 50 ml) was added dropwise. The organic
layer was washed with aqueous NaHCO3 (50 ml, saturated), water
(50 ml), dried and evaporated. The residue was purified by flash
chromatography (EtOAc–cyclohexane, 1 : 4) to give the dimesylate
10 (3.17 g, 88%) as a colourless oil, [a]2D4 +5.0 (c, 0.14, CHCl3); mmax
5-O-tert-Butyldimethylsilyl-2,3-O-isopropylidene-D-lyxono-1,4-
lactone (8)
The isopropylidene lactone 714 (3.84 g, 20.4 mmol), derived from
the unsubstituted lactone,35 was added to a solution of tert-
butyldimethylsilyl chloride (4.61 g, 30.6 mmol) and imidazole
(2.78 g, 30.6 mmol) in DMF (25 ml). The solution was stirred
at 20 ◦C under an atmosphere of nitrogen. After 4 h, t.l.c. analysis
(EtOAc–cyclohexane, 1 : 1) indicated complete conversion of
starting material (Rf 0.12) into a major product (Rf 0.52). The
reaction mixture was concentrated and coevaporated with toluene.
The residue was then purified by flash chromatography (EtOAc–
cyclohexane, 1 : 2) to give the fully protected lactone 8 (5.69 g,
91%) as a white crystalline solid, mp 90–91 ◦C, [a]D22 +54.9 (c, 1.03,
=
(thin film) 1360, 1178 (S O); NMR dH (400 MHz; CD3CN) 0.12
(6 H, s, SiMe2), 0.92 (9 H, s, SitBu), 1.37, 1.49 [2 × 3 H,
2s, C(CH3)2], 3.09, 3.14 (2 × 3 H, 2s, 2SO2Me), 3.86 (1 H,
dd, J5,5 11.4, J5,4 5.3, H-5), 3.93 (1 H, dd, J5 ,4 5.3, H-5ꢀ),
4.33–4.39 (2 H, m, H-1,1ꢀ), 4.42–4.46 (1 H, m, H-2), 4.47 (1
H, dd, J3,2 6.1, J3,4 9.7, H-3), 4.72 (1 H, dt, J4,3 9.7, H-4);
dC (100.6 MHz; CD3CN) −6.0 [Si(CH3)2], 18.3 [C(CH3)3], 25.1
[C(CH3)2], 25.6 [C(CH3)3], 26.9 [C(CH3)2], 37.0, 38.8 (2 × SO2Me),
63.4 (C-5), 69.2 (C-1), 74.7 (C-2), 75.5 (C-3), 80.1 (C-4), 109.6
[C(CH3)2]; HRMS (ES+) m/z 463.1490; C16H35O9S2Si (M + H)+
ꢀ
ꢀ
=
CHCl3); mmax (thin film) 1773 (C O); NMR dH (400 MHz; CDCl3)
0.10 (6 H, s, SiMe2), 0.91 (9 H, s, SitBu), 1.40, 1.47 [2 × 3 H,
ꢀ
2s, C(CH3)2], 3.94 (1 H, dd, J5,4 6.5, J5,5 10.4, H-5), 3.99 (1 H,
dd, J5 ,4 6.1, H-5ꢀ), 4.53 (1 H, m, H-4), 4.82 (2 H, m, H-2,3); dC
ꢀ
(100.6 MHz; CDCl3) −5.5, −5.4 [Si(CH3)2], 18.3 [C(CH3)3], 25.8
[C(CH3)3], 25.9 [C(CH3)2], 26.8 [2 × C(CH3)2], 60.9 (C-5), 75.7
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requires 463.1492. (Found C, 41.6; H, 7.4; C16H34O9S2Si requires
C, 41.5; H, 7.4%).
(9 H, s, SitBu), 1.34, 1.48 (2 × 3 H, 2s, [C(CH3)2], 2.54 (1 H, br s,
NH), 3.02 (2 H, d, J1,2 2.7, H-1,1ꢀ), 3.23 (1 H, dt, J4,5, J4,5 5.4, H-4),
ꢀ
3.56 (1 H, dd, J5,5 10.4 J5,4 5.8, H-5), 3.65 (1 H, dd, H-5ꢀ), 4.65
ꢀ
(1 H, d, J3,2 5.8, H-3), 4.70 (1 H, dt, H-2); dC (100.6 MHz, CDCl3)
−5.5 [Si(CH3)2], 18.1 [C(CH3)3], 24.1 [C(CH3)2], 25.9 [C(CH3)3],
26.4 [C(CH3)2], 53.4 (C-1), 63.8 (C-5), 66.3 (C-4), 82.2 (C-2), 83.4
(C-3), 110.9 [C(CH3)2]; HRMS (ES+) m/z 288.1995; C14H30NO3Si
([M + H]+) requires 288.1995.
1-Azido-5-O-tert-butyldimethylsilyl-1-deoxy-2,3-O-
isopropylidene-4-O-methanesulfonyl-D-lyxitol (12) and
1,4-diazido-5-O-tert-butyldimethylsilyl-1,4-dideoxy-2,3-O-
isopropylidene-L-ribitol (13)
Sodium azide (0.133 g, 2.04 mmol) was added to a solution of the
dimesylate 10 (0.942 g, 2.04 mmol) in DMF (5 ml) and the reaction
mixture stirred at 100 C under an atmosphere of nitrogen. The
(1R)-1-(9-Deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-L-ribitol
hydrochloride [(+)-5·HCl]
◦
formation of the monoazide 12 (Rf 0.58, EtOAc–cyclohexane, 1 :
2) after 20 min was indicated by t.l.c., but much of the starting
material 10 (Rf 0.27) remained. After 1 h, a minor second product
(13) (Rf 0.74) started to form. The mixture was stirred at 100 ◦C
for another hour, allowed to cool to room temperature and the
solvent was removed. The resulting yellow oil was purified by
flash chromatography (EtOAc–cyclohexane, 1 : 4) to give the
azido mesylate 12 (0.35 g, 42%), [a]2D5 +27.7 (c, 1.43, CHCl3); mmax
The L-iminoribitol derivative 11 (500 mg) was converted to the
imine 14 (186 mg, 39%) and hence to L-immucillin-H [(+)-5·HCl]
(75 mg, 29%), [a]2D5 +51.5 (c 0.8, H2O) as was described for the
analogues of the D-series.11 The enantiomeric products (+)- and
(−)-5·HCl gave 1H and 13C NMR spectra with corresponding
3
chemical shifts within 0.1 and 0.01 ppm of each other and JH,H
values within 0.2 Hz. The specific rotation of the D-enantiomer
was found in the present work to be −53.6 (c 2.7, H2O).
=
(thin film) 2104 (N3), 1358, 1174 (S O); NMR dH (400 MHz;
CDCl3) 0.09, 0.10 (2 × 3H, 2s, SiMe2), 0.90 (9 H, s, SitBu), 1.40,
Ethyl (R,S/S,R)-1-benzyl-4-hydroxypyrrolidine-3-carboxylate
[( )-18]
1.53 [2 × 3 H, 2s, C(CH3)2], 3.12 (3 H, s, SO2Me), 3.42 (1 H,
dd, J1,1 12.8, J1,2 3.6, H-1), 3.50 (1 H, dd, J1 ,2 7.6, H-1ꢀ), 3.82
ꢀ
ꢀ
(1 H, dd, J5,5 11.2, J5,4 6.4, H-5), 3.93 (1 H, dd, J5 ,4 4.8, H-5ꢀ),
4.31 (1 H, m, H-2), 4.36 (1 H, dd, J3,4 13.2, J3,2 6.0, H-3), 4.73
(1 H, ddd, H-4); dC (100.6 MHz; CDCl3) −5.6 [Si(CH3)2], 18.2
[C(CH3)3], 25.4 [C(CH3)2], 25.8 [C(CH3)3], 27.6 [C(CH3)2], 38.7
(SO2Me), 51.0 (C-1), 63.3 (C-5), 75.8 (C-3), 76.1 (C-2), 79.4 (C-4),
109.2 [C(CH3)2]; HRMS (CI+) m/z 427.2052; C15H35N4O6SSi
([M + NH4]+) requires 427.2047. (Found: C, 44.35; H, 7.4; N,
10.1; C15H31N3O6SSi requires C, 44.0; H, 7.6; N, 10.3%).
ꢀ
ꢀ
This compound was prepared by the method described by Jaeger
and Biel,18 but ethyl-N-benzyl-N-(2-carbethoxyethyl)glycinate, as
prepared by the method of Pinto et al.,36 was used as well as
the Dieckmann cyclization conditions described by Deshmukh
et al.37 The racemic trans-isomer was purified by chromatography
(EtOAc–hexanes, 1 : 2 → 1 : 1 → EtOAc) and the resulting
gum crystallized at −20 ◦C (44% from the glycinate on the
5 mmol scale). A small sample was recrystallised at −20 ◦C from
EtOAc–hexanes to give colourless needles, mp 52–53 ◦C, NMR dH
(300 MHz; CDCl3): 1.26 (3 H, t, J 7.1, CH2CH3), 2.32 (1 H, br. s,
This was followed by the bis-azide (13), (0.11 g, 15%), [a]2D5 +87.1
(c, 0.56, CHCl3); mmax (thin film) 2102 (N3); NMR dH (400 MHz;
CD3CN) 0.15, 0.16 (2 × 3H, 2s, SiMe2), 0.94 (9 H, s, SitBu), 1.33,
1.46 [2 × 3 H, 2s, C(CH3)2], 3.47 (2 H, d, J1,2 13.2, H-1,1ꢀ), 3.53
ꢀ
OH, exchanged to D2O), 2.55 (1 H, dd, J2,2 9.4, J2,3 7.4, H-2), 2.65
(1 H, dd, J5,5 10.0, J5,4 5.5, H-5), 2.76 (1 H, dd, J5 ,4 2.8, H-5ꢀ),
ꢀ
ꢀ
ꢀ
(1 H, m, H-4), 3.84 (1 H, dd, J5,5 10.8, J5,4 6.0, H-5), 4.06 (1 H,
ꢀ
2.95 (1 H, dt, J3,2 = J3,2 8.0, J3,4 3.3, H-3), 3.12 (1 H, t, J 9.0,
dd, J5 ,4 2.8, H-5ꢀ), 4.11 (1 H, dd, J3,2 6.4, J3,4 11.6, H-3), 4.35 (1 H,
ꢀ
H-2ꢀ), 3.64 (2 H, s, PhCH2), 4.16 (2 H, q, J 7.1 CH2CH3), 4.51
(1 H, m, H-4), 7.22–7.37 (5 H, m, Ar); dC (75.5 MHz; CDCl3) 14.2
(Me), 53.1 (C-3), 55.3 (C-2), 59.7 (PhCH2), 60.8 (CH3CH2), 61.9
(C-5), 74.1 (C-4), 127.1 (ArH), 128.3 (ArH), 128.8 (ArH), 138.2
(Ar), 173.3 (CO); HRMS (EI+) m/z 249.1365; C14H19NO3 (M+)
requires 249.1365. (Found: C, 67.6; H, 7.5; N, 5.6; C14H19NO3
requires C, 67.5; H, 7.7; N, 5.6%).
dd, H-2); dC (100.6 MHz; CDCl3) −6.0 [Si(CH3)2], 18.2 [C(CH3)3],
25.0 [C(CH3)2], 25.5 [C(CH3)3], 25.7 [C(CH3)2], 51.0 (C-1), 61.0
(C-4), 64.5 (C-5), 74.4 (C-3), 76.7 (C-2), 109.4 [C(CH3)2]; (ES+)
m/z 329 ([M–N2 + H]+), (75%), 301 ([M − 2N2 + H]+), (20%). The
starting dimesylate (10) (0.317 g, 34%) was also recovered from
the reaction.
5-O-tert-Butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-
Ethyl (R,S/S,R)-4-(acetyloxy)-1-benzylpyrrolidine-3-carboxylate
isopropylidene-L-ribitol (11)
[( )-19]
The azidomesylate 12 (0.34 g, 0.84 mmol) in 1,4-dioxane (10 ml)
was stirred at 20 ◦C under an atmosphere of hydrogen in the
presence of palladium black (0.07 g) and sodium acetate (0.07 g,
0.825 mmol) for 3 d after which time t.l.c. analysis (EtOAc–
cyclohexane, 1 : 1) indicated complete conversion of starting
material (Rf 0.35) to a major product (Rf 0.17). The suspension was
Racemate 18 (100 mg, 0.4 mmol) was dissolved in a mixture of
pyridine (4 ml) and Ac2O (2 ml) and left at 20 ◦C overnight. The
solvent was evaporated and the resulting oil dissolved in EtOAc
and washed with aqueous NaHCO3 (saturated), dried and the
solvent was again evaporated. The residue was chromatographed
(EtOAc–hexanes, 15 : 85) to afford diester ( )-19 as a colourless
oil (111 mg, 95%) which was stored at −20 ◦C, NMR dH (300 MHz;
CDCl3) 1.25 (3 H, t, J 7.1, CH2CH3), 2.04 (3 H, s, COCH3), 2.50
R
then filtered through Celiteꢀ, and the 1,4-dioxane removed. The
resulting yellow oil was dissolved in CH2Cl2 (20 ml) and washed
with water (3 × 20 ml), dried and the solvent was removed to
afford the protected L-iminoribitol 11 (0.224 g, 94%) as a yellow
oil, [a]2D5 −15.5 (c, 0.56, CHCl3); mmax (thin film) 3332 (sharp, N–
H); NMR dH (400 MHz; CDCl3) 0.02, 0.03 (6 H, 2s, SiMe2), 0.91
(1 H, t, J2,2 = J2,3 8.3, H-2), 2.74–2.87 (2 H, m, H-5,5ꢀ), 3.06
ꢀ
ꢀ
(1 H, dt, J3,2 = J3,2 7.9, J3,4 3.9, H-3), 3.15 (1 H, t, J 8.5, H-
2ꢀ), 3.59 (1 H, d, J 12.9, PhCHH), 3.65 (1 H, d, PhCHH), 4.16
(2 H, q, J 7.1, CH2CH3) 5.40 (1 H, m, H-4), 7.22–7.38 (5 H,
1136 | Org. Biomol. Chem., 2006, 4, 1131–1139
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(1.54 g, 73%), [a]2D1 +33.0 (c 0.75, MeOH). The 1H NMR spectrum
was identical to that of compound ( )-20.
m, Ar); dC (75.5 MHz; CDCl3) 14.1 (CH2CH3), 21.0 (COCH3),
50.1 (C-3), 56.0 (C-2), 59.5 (PhCH2 or C-5), 59.6 (PhCH2 or C-
5), 61.0 (CH2CH3), 76.0 (C-4), 127.2 (ArH), 128.3 (ArH), 128.7
(ArH), 138.0 (Ar), 170.5 (CO), 172.3 (CO); HRMS (FAB+) m/z
292.1563; C16H22NO4 (M + H)+ requires 292.1549.
tert-Butyl (3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine-1-
carboxylate [(+)-23]
(R,R/S,S)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol [( )-20]
A. From diol (+)-20. Pd/C (300 mg, 10%) was added to a
stirred solution of the diol (+)-20 (1.49 g, 7.19 mmol) and di-
tert-butyl dicarbonate (1.63 g, 7.47 mmol) in MeOH (30 ml),
and hydrogen was added from a balloon over 24 h. The mixture
Racemate 18 (500 mg, 2.01 mmol) was dissolved in dry Et2O–
dry THF, (10 ml : 5 ml) and cooled in an ice bath. Lithium
aluminium hydride in Et2O (4.2 ml, 1 M, 4.2 mmol) was added,
and the mixture warmed to 20 ◦C and stirred for 1 h. After
cooling of the solution in an ice bath excess hydride was quenched
by the dropwise addition of water (0.50 ml) and the mixture
was extracted with EtOAc. The organic extract was washed with
aqueous NaHCO3 (saturated), dried and evaporated to give an
oily residue that was chromatographed [CH2Cl2–MeOH–NH4OH
(0.88), 95 : 5 : 0.5 → 90 : 10 : 0.5] to give racemic diol 20 as a
colourless gum (364 mg, 88%), NMR dH (300 MHz; CD3OD) 2.18
R
was filtered through Celiteꢀ, the solvent was evaporated and the
residue was chromatographed (EtOAc–MeOH, 19 : 1) to afford
the N-Boc protected pyrrolidine (+)-23 as a colourless gum (1.56 g,
100%), [a]2D1 +15.9 (c 1.09, MeOH), in good agreement with the
value derived from the sample made by method B.
B. From 1,2:5,6-di-O-isopropylidine-a-D-glucose. 3-C-Azido-
methyl-3-deoxy-1,2:5,6-di-O-isopropylidene-a-D-glucose (42.6 g,
142 mmol), which was made from 1,2:5,6-di-O-isopropylidine-
a-D-glucose, hydrolysed and reduced as previously described,26
gave the unprotected pyrrolidine from which, in MeOH, (500 ml),
compound 22 was obtained by treatment with di-tert-butyl
dicarbonate (40 g, 185 mmol) and Et3N (25.7 ml, 185 mmol). The
volatiles were removed and the residue was adsorbed on silica gel
and chromatographed to give crude carbamate 22 (26.7 g, 68%).
The product was dissolved in EtOH (500 ml), cooled in an ice bath
and oxidised by the dropwise addition of NaIO4 (47 g, 0.22 mol)
in water (500 ml). After recooling of the products in an ice-bath
the product was reduced with NaBH4 (7.3 g, 0.19 mmol) added
portion-wise. The mixture was warmed to room temperature, the
solids were removed by filtration, the volatiles by evaporation and
the residue was purified by chromatography (CHCl3–MeOH, 9 :
1). Compound (+)-23 was obtained as a light yellow syrup (17 g,
81%) which gave 1H and 13C NMR data in agreement with those of
the sample made by method A and with literature data.12 A sample
of compound (+)-23 (50 mg), prepared in this way, in EtOAc was
further purified by washing with water and then brine to give a
colourless syrup (28 mg) after solvent evaporation, [a]2D1 +16.2 (c
0.795, MeOH).
ꢀ
(1 H, m, H-4), 2.34 (1 H, dd, J5,5 9.6, J5,4 6.6, H-5), 2.55 (1 H, dd,
J2,2 10.0, J2,3 4.1, H-2), 2.72 (1 H, dd, J2 ,3 6.3, H-2ꢀ), 2.89 (1 H, t,
ꢀ
ꢀ
J5 ,4 = J5 ,5 8.8, H-5ꢀ), 3.47–3.68 (4 H, m, PhCH2, CH2O), 4.00 (1 H,
m, H-3), 7.20–7.42 (5 H, m, Ar); dC (75.5 MHz; CD3OD) 51.2 (C-
4), 57.3 (C-5), 61.5 (PhCH2 or CH2O), 63.1 (C-2), 64.2 (PhCH2
or CH2O), 74.1 (C-3) 128.3 (ArH), 129.3 (ArH), 130.2 (ArH),
139.4 (Ar); HRMS (FAB+) m/z 208.1346; C12H18NO2 (M +
H)+ requires 208.1338.
ꢀ
ꢀ
Ethyl (3S,4R)-1-benzyl-4-hydroxypyrrolidine-3-carboxylate
[(+)-18] and ethyl (3R,4S)-4-(acetyloxy)-1-benzylpyrrolidine-3-
carboxylate [(−)-19]
R
Vinyl acetate (6.66 ml, 72.21 mmol) and Novozymeꢀ 435 lipase
from Candida antarctica (4.2 g, Novozymes Australia Pty. Ltd,
batch LC200207) were added sequentially to a solution of ( )-
18 (6.00 g, 24.1 mmol) in◦tert-butyl methyl ether (200 ml). The
R
mixture was stirred at 40 C for 2.5 h, filtered through Celiteꢀ,
the solids were washed with a little ethyl acetate and the combined
filtrates were washed with aqueous NaHCO3 (saturated), dried and
evaporated. 1H NMR analysis indicated that the residue consisted
of alcohol 18 and acetate 19 in equimolar proportions. It was
chromatographed (EtOAc–hexanes, 6 : 4) to give first (−)-19 as
◦
a colourless gum (3.44 g, 97%) that was stored at −20 C, [a]D21
(3R,4R)-4-(Hydroxymethyl)pyrrolidin-3-ol [(+)-16] and its
hydrochloride [(+)-16·HCl]
1
−41.5 (c 0.74, CHCl3). The H NMR spectrum was identical to
that for compound ( )-19 above. Further elution of the column
with Et◦OAc gave (+)-18 also as a colourless gum which crystallized
at −20 C (2.53 g, 85%), mp 51–52 ◦C, [a]D21 +16.9 (c 0.71, CHCl3).
The 1H NMR spectrum was identical to that for compound ( )-18
above.
A. From carbamate 23. A sample of compound (+)-23 (28 mg)
was dissolved in MeOH (2 ml) and HCl (37%, 1 ml) and after a few
mins the solvent was evaporated to give (+)-16·HCl, [a]2D1 +18.9 (c
0.92, MeOH). The 1H NMR spectrum was identical to that of the
sample made from diol (+)-20 (method B).
Repetition of the enzymic acetylation with ( )-18 (0.80 g,
3.21 mmol) under the same conditions, but for 100 min, gave a
mixture of 18 and 19 in the approximate ratio of 1.2 : 1 (1H NMR
determination). After chromatographic separation, pure (−)-19
(406 mg, 96%), [a]2D1 − 41.8 (c 0.895, CHCl3) and impure (+)-18
(0.393 g, 89%), [a]2D1 +14.0 (c 0.81 CHCl3) were isolated. The latter
contained about 10% of the unreacted (−)-enantiomer.
B. From (+)-20. Diol (+)-20 (52 mg, 0.25 mmol) was dissolved
in MeOH, HCOOH (98%) (9 : 1, 8 ml) and Pd/C (10%, 80 mg) was
added.38 The mixture was heated under reflux for 30 min, filtered
R
through Celiteꢀ and the solvent evaporated. Chromatography
[CH2Cl2–MeOH–NH4OH (0.88)–H2O, 4 : 3 : 0.5 : 0.5] gave the
unprotected pyrrolidine as a colourless gum (16 mg, 55%) which
1
darkened slowly on standing. The H NMR spectrum (CD3OD)
(3R,4R)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol [(+)-20]
was in agreement with literature spectral data.25 The product was
dissolved in MeOH (2 ml), HCl (5%, 1 ml) and the solvents were
evaporated to give the hydrochloride (+)-16·HCl (21 mg, 55%)
Compound (+)-18 (2.53 g, 10.15 mmol) was reduced, as indicated
for the racemic compound, to give (+)-20 as a colourless gum
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as a colourless gum, [a]D21 +19.1 (c 1.05, MeOH), lit.23 [a]2D5 +19.0
(c 1.0, MeOH). The 1H NMR spectrum (D2O) was in agreement
with the literature spectral data23 and was identical to that of the
compound made by method A.
4 M. L. Bryant, E. G. Bridges, L. Placidi, A. Faraj, A.-G. Loi, C. Pireerra,
D. Dukhan, G. Gosselin, J.-L. Imbach, B. Hernandez, A. Juodawlkis, B.
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Antiviral Chem. Chemother., 2003, 14, 1–21.
6 O. Jungmann, M. Beier, A. Luther, H. K. Huynh, M.-O. Ebert, B. Jaun,
R. Krishnamurthy and A. Eschenmoser, Helv. Chim. Acta, 2003, 86,
1259–1308.
7 C.-Y. Yu, N. Asano, K. Ikeda, M.-X. Wang, T. D. Butters, M. R.
Wormald, R. A. Dwek, A. L. Winters, R. J. Nash and G. W. J. Fleet,
Chem. Commun., 2004, 1936–1937.
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2005, 16, 223–229.
(3S,4S)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol [(−)-20]
Compound (−)-19 (400 mg, 1.37 mmol) was dissolved in Et2O
(9 ml) and THF (4 ml) and treated with lithium aluminium hydride
in Et2O (5.62 ml, 1 M, 5.62 mmol) as described for the preparation
of compound ( )-20 above to afford (−)-20 as a colourless gum
(190 mg, 67%), [a]2D1 −33.4 (c 0.805, MeOH). The 1H NMR
spectrum was identical to that of ( )-20.
9 A. Kato, N. Kato, E. Kano, I. Adachi, K. Ikeda, L. Yu, T. Okamoto,
Y. Banba, H. Ouchi, H. Takahata and N. Asano, J. Med. Chem., 2005,
48, 2036–2044.
(3S,4S)-4-(Hydroxymethyl)pyrrolidin-3-ol [(−)-16] and its
hydrochloride [(−)-16·HCl]
10 (a) G. B. Evans, Aust. J. Chem., 2004, 57, 837–854; (b) V. L. Schramm
and P. C. Tyler, Curr. Top. Med. Chem., 2003, 3, 525–540; (c) S.
Bantia, P. J. Miller, C. D. Parker, S. L. Anath, L. L. Horn, J. M.
Kilpatrick, P. E. Morris, T. L. Hutchison, J. A. Montgomery and J. S.
Sandhu, Int. Immunopharmacol., 2001, 1, 1199–1210; (d) S. Bantia, P. J.
Miller, C. D. Parker, S. L. Ananth, Y. S. Babu and J. S. Sandhu, Int.
Immunopharmacol., 2002, 2, 913–923; (e) S. Bantia, S. L. Ananth, C. D.
Parker, L. L. Horn and R. Upshaw, Int. Immunopharmacol., 2003, 3,
879–887.
11 (a) G. B. Evans, R. H. Furneaux, G. J. Gainsford, V. L. Schramm and
P. C. Tyler, Tetrahedron, 2000, 56, 3053–3062; (b) G. B. Evans, R. H.
Furneaux, T. Hutchison, H. S. Kezar, P. E. Morris, Jr., V. L. Schramm
and P. C. Tyler, J. Org. Chem., 2001, 66, 5723–5730.
12 G. B. Evans, R. H. Furneaux, A. Lewandowicz, V. L. Schramm and
P. C. Tyler, J. Med. Chem., 2003, 46, 5271–5276.
13 G. B. Evans, R. H. Furneaux, P. C. Tyler and V. L. Schramm, Org.
Lett., 2003, 5, 3639–3640.
14 G. W. J. Fleet, S. Petursson, A. L. Campbell, R. A. Mueller, J. R.
Behling, K. A. Babiak, J. S. Ng and M. G. Scaros, J. Chem. Soc., Perkin
Trans. 1, 1989, 665–666.
15 B. A. Horenstein, R. F. Zabinski and V. L. Schramm, Tetrahedron Lett.,
1993, 34, 7213–7216.
16 R. H. Furneaux, G. Limberg, P. C. Tyler and V. L. Schramm,
Tetrahedron, 1997, 53, 2915–2930.
Compound (−)-20 (189 mg, 0.91 mmol) was de-N-benzylated38
as for the (+)-enantiomer to give the unprotected amine (−)-16
as a colourless gum (107 mg, 100%), a portion of which (30 mg)
was converted to the hydrochloride salt (−)-16·HCl (39 mg), [a]D21
−18.9 (c 0.74, MeOH), lit.23 [a]2D5 −18.7 (c 1.2, MeOH). The H
1
NMR spectrum (D2O) was in agreement with the literature data23
and was identical to that of (+)-16·HCl.
(3S,4S)-1-[(9-Deazahypoxanthin-9-yl)methyl]-4-
(hydroxymethyl)pyrrolidin-3-ol [L-DADMe–ImmH, (−)-6]
To a solution of (3S,4S)-4-(hydroxymethyl)pyrrolidin-3-ol free
base (−)-16, (77 mg, 0.66 mmol) in H2O (1.5 ml) were added
9-deazahypoxanthine (17)39 (81 mg, 0.60 mmol) and aqueous
formaldehyde (53 ll, 12.3 M, 0.65 mmol). The mixture was heated
at 85 ◦C for 15 h (a small amount of precipitate formed), silica gel
was added to absorb the solvent, the solvent was evaporated and
the granular residue added to a column of silica gel and eluted
with CH2Cl2–MeOH–NH4OH (0.88), 5 : 4.5 : 0.5 to afford the
nucleoside analogue (−)-6 as a colourless solid (82 mg, 48%) after
washing with a little cold MeOH, [a]2D1 −16.8 (c 0.71, H2O). A
sample of the (3R,4R)-enantiomer (+)-6, prepared during the
present work, and ultimately derived from D-glucose via the
sequence (+)-22 → (+)-23 → (+)-16 → (+)-6, had [a]2D1 +16.9
(c 0.935, H2O). The 1H NMR spectrum of compound (−)-6 was in
agreementwith theliterature datafor (+)-612 and with the spectrum
of the latter isomer made during the present work.
17 A. Fedorov, W. Shi, G. Kicska, E. Fedorov, P. C. Tyler, R. H. Furneaux,
J. C. Hanson, G. J. Gainsford, J. Z. Larese, V. L. Schramm and S. C.
Almo, Biochemistry, 2001, 40, 853–860.
18 E. Jaeger and J. H. Biel, J. Org. Chem., 1965, 30, 740–744.
19 M. D. Sørensen, N. M. Khalifa and E. B. Pedersen, Synthesis, 1999,
1937–1943.
20 Y. H. Lee, H. K. Kim, I. K. Youn and Y. B. Chae, Bioorg. Med. Chem.
Lett., 1991, 1, 287–290.
21 K. Makino and Y. Ichikawa, Tetrahedron Lett., 1998, 39, 8245–
8248.
22 R. Galeazzi, G. Martelli, G. Mobbili, M. Orena and S. Rinaldi,
Tetrahedron: Asymmetry, 2004, 15, 3249–3256.
23 S. Karlsson and H.-E. Ho¨gberg, Tetrahedron: Asymmetry, 2001, 12,
1977–1982.
24 P. L. Kotian, T.-H. Lin, Y. El-Kattan and P. Chand, Org. Process Res.
Acknowledgements
Dev., 2005, 9, 193–197.
25 V. V. Filichev, M. Brandt and E. B. Pedersen, Carbohydr. Res., 2001,
333, 115–122.
Support from EPSRC [for S.W.J.] is gratefully acknowledged, and
Professor Robin Ferrier is thanked for helpful discussions and for
assistance with the preparation of the manuscript.
26 V. V. Filichev and E. B. Pedersen, Tetrahedron, 2001, 57, 9163–
9168.
27 S. U. Hansen and M. Bols, Acta Chem. Scand., 1998, 52, 1214–
1222.
28 L. M. Levy, J. R. Dehli and V. Gotor, Tetrahedron: Asymmetry, 2003,
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